1
Introduction
Photoremovable (sometimes called photoreleasable, photocleavable
or photoactivatable) protecting groups (PPGs) provide spatial and
temporal control over the release of various chemicals such as bioagents
(neurotransmitters and cell-signaling molecules), acids, bases, Ca2+ ions, oxidants,
insecticides, pheromones, fragrances, etc.
Following early reports on PPGs for use in organic synthesis by Barltrop,
1
Barton,
2
Woodward,
3
Sheehan
4
and their
co-workers, applications to biology were sparked off by Engels and
Schlaeger
5
and Kaplan and co-workers,
6
who first achieved the photorelease of cyclic
adenosine monophosphate (cAMP) and ATP, respectively. The latter authors
introduced the convenient, if somewhat misleading, term “caged”
to designate a compound protected by a PPG. Two general perspectives
7
and many more specialized reviews covering applications
of PPGs in synthesis,
8
biochemistry and
neurobiology,
9
biomedicine,
10
volatiles release,
11
polymerization,
12
and fluorescence activation
13
have been published during the past decade,
and a journal issue themed on these topics has recently been published.
14
The present review covers recent developments
in the field, focusing on the scope, limitations, and applications
of PPGs, which are used to release organic molecules. Photoactivation
of small inorganic species and ions, such as NO,
15
CO,
16
Ca2+,
9h,17
Zn2+,
18
Cd2+,
19
or Cu+,
20
is not covered. Simplified basic structures of the photoremovable
protecting groups discussed in this review are listed in Table 1 (the leaving groups
are shown in red).
Table 1
Photoremovable Protecting Groups
The criteria for the design of a good PPG
will depend on the application.
No single system needs to fulfill all of the following requirements:
(i)
In general, the
PPG should have strong
absorption at wavelengths well above 300 nm, where irradiation is
less likely to be absorbed by (and possibly cause damage to) the biological
entity.
21
Moreover, the photoreaction should
be clean and should occur with a high quantum yield or efficiency for release, Φ
rel. The quantum yield Φ
rel is equal
to the amount of released substrate, n
rel/mol, divided by the amount of photons at the irradiation wavelength
λ, n
p/mol = n
p/einstein, that were absorbed by the caged compound: Φ
rel = n
rel/n
p. An important measure for the efficacy
of a PPG is the product of the quantum yield and the molar decadic
absorption coefficient ε of the PPG, Φ
relε(λirr), which is proportional
to the amount of release at the given excitation wavelength.
22
(ii)
Sensitive detection of the response
under study often depends not only on the product Φ
relε(λirr) but also on the background
level of activity of the caged compound prior to irradiation. Hence,
the PPGs must be pure, exhibit low intrinsic activity, and be stable
in the media prior to and during photolysis.
(iii)
The PPGs should be soluble in the
targeted media; they may further be required to pass through biological
barriers such as cell membranes and show affinity to specific target
components, for example, binding sites on cancer cells or the active
site of an enzyme.
(iv)
The photochemical byproducts accompanying
the released bioactive reagent should ideally be transparent at the
irradiation wavelength to avoid competitive absorption of the excitation
wavelengths. Moreover, they must be biocompatible, i.e., they should
not react with the system investigated.
(v)
To study the kinetics of rapid responses
to a released agent in samples such as brain tissue or single live
cells, the PPG must be excited by a short light pulse and the appearance rate constant
k
app of the desired
free substrate must exceed the rate constant of the response under
investigation. Commonly, there are several reaction steps involving
ground- and excited-state intermediates that precede the actual release
of the free substrate. Therefore, detailed knowledge of the reaction
mechanism is needed; in particular, the rate-determining step in the
reaction path and its lifetime τrd or k
app must be known, unless the appearance of the free substrate
(k
app) can be monitored directly by time-resolved
techniques.
Nitrobenzyl, nitrophenethyl
compounds, and their dimethoxy derivatives
(nitroveratryl) (section 3) are by far the
most commonly used PPGs. The decay of their primary quinonoid intermediates
on the microsecond time scale does not generally correspond to the
rate-determining step of the overall reaction, and the release of
the free substrate may be orders of magnitude slower. Moreover, photolysis
of these compounds forms potentially toxic and strongly absorbing
byproducts such as o-nitrosobenzaldehyde. Quite a
number of alternative PPGs have been developed that do not suffer
from these disadvantages.
The appearance rate constant k
app of
the desired product is equal to the inverse of the rate-determining
intermediate’s lifetime τrd, k
app = 1/τrd, which often depends on the
solvent as well as on the concentrations of acids and bases including
those of the general acids and bases contained in buffers. Release rate constants,
k
r =
ηr/τr, are sometimes quoted, where
ηr is the efficiency of the releasing reaction step,
ηr = k
r /Σk, and τr = 1/Σk is the lifetime of the intermediate that is assumed to release
the
substrate; Σk includes k
r and the rate constant of all competing reactions occurring
from that intermediate. Note that the release rate constant k
r may be higher or lower than the more relevant
appearance rate constant k
app of the desired
substrate. Case (a): k
r < k
app if Σk > k
r, i.e., if reactions other than substrate release contribute
to the decay rate of the releasing intermediate. A trivial example
of case (a) is shown in Scheme 1. Case (b): k
r > k
app if the
actual release is preceded by a slower, rate-determining step of the
reaction sequence.
Scheme 1
Simple Case Where the Release Rate Constant of the
Free Substrate
(Leaving Group) X, k
r, Is Smaller than
Its Appearance Rate Constant, k
app
The speed of release is an ambivalent expression;
it may refer to the efficiency of a PPG, Φ
relε(λirr), the amount released by a
given irradiation dose, or to the appearance rate constant in time-resolved
work. The absorption spectra of a number of chromophores frequently
encountered as PPGs are shown in Figure 1.
Figure 1
UV spectra
of selected chromophores.
23
(a) Benzophenone
(ethanol; solid, red),
24
acetophenone (ethanol;
dashed, blue);
24
(b) 1-acetyl-5-bromo-7-nitroindoline
(acetonitrile; solid, red),
2-nitrotoluene (dashed, blue), (3,4-dimethoxy-6-nitrophenyl)methyl
(nitroveratryl) derivative (acetonitrile, dashed, green);
25
(c) coumarin (acetonitrile, solid, red), p-hydroxyacetophenone (acetonitrile, dashed,
blue), and
benzoin (acetonitrile, dotted, black); (d) tris(bipyridyl)ruthenium(II)
chloride (water).
26
2
Arylcarbonylmethyl Groups
Aromatic ketones
are thermally stable and synthetically readily
accessible compounds; their photophysical and photochemical properties
are well understood. The lowest energy transition of simple carbonyl
compounds is typically a weak n,π* band (ε
≈ 10–100 M–1 cm–1).
27
The higher-energy π,π*
absorption bands are strong, and internal conversion to the S1 state is very fast
(e.g., ∼100–260 fs for acetophenone).
28
The electronic transitions of aromatic ketones
are sensitive to solvent polarity and to substitution on the phenyl
ring. Hydrogen bonding of protic solvents to the carbonyl oxygen stabilizes
the oxygen nonbonding orbital, giving rise to a hypsochromic shift
of the n,π* absorption band. Both electron-donating
groups and polar solvents tend to stabilize the π,π* states.
The strong bathochromic shift induced by para-amino
substituents is attributed to a CT interaction
29
(for example, λmax for p-aminobenzaldehyde is ∼325 nm (π,π*) in cyclohexane).
24
Aromatic ketones are highly phosphorescent and
only weakly fluorescent
30
due to their
fast (>1010 s–1), very efficient intersystem
crossing to the triplet state, for which two energetically close lying
states (n,π* and π,π*) seem to
play a crucial role, possibly due to a S1/T2/T1 intersection.
31
The singlet–triplet
energy gap is much larger for π,π* than for n,π* states. The lowest π,π* and n,π*
triplet states are nearly degenerate, and substitution
on the phenyl ring as well as polar solvents may lead to triplet-state
inversion.
27,32
Some of the important photophysical
properties of acetophenone, the parent aryl ketone, are summarized
in Table 2. Examples of the absorption spectra
of other representative PPG aryl ketones are provided in Figure 1.
Table 2
Photophysical Properties
of Acetophenonea
solvent
E
S/kJ mol–1
b
τS/psc
Φ
f
d
Φ
T
e
E
T/kJ mol–1
f
Φ
p
g
nonpolar
330
30b
25
33
<1 × 10–6
34
1
35
310
36
4 × 10–4
37
polar
338
36
39
33
1
35
311
36
a
Photophysical
properties of many
other aromatic ketones can be found in the Handbook of Photochemistry.
38
b
Lowest excited singlet state (S1) energy.
c
The lifetime of S1.
d
Fluorescence quantum yield.
e
Intersystem crossing (ISC) quantum
yield.
f
Lowest triplet state
(T1) energy.
g
Phosphorescence quantum yield (23
°C, isooctane).
The
carbonyl group of aromatic ketones is usually the center of
the photochemical reactivity. Scheme 2 shows
the most important photoreactions that lead to the liberation of a
leaving group (X) and are discussed in the following paragraphs. Ketones
with n,π* lowest triplets, possessing a half
vacant n orbital localized on the carbonyl oxygen,
are far more reactive than those with π,π* lowest triplets
with spins delocalized on the aromatic ring. The singlet or triplet n,π* states thus
readily abstract hydrogen atoms from
suitable donors (entry 1), whereas both n,π*
and π,π* states can be reduced in the presence of good
electron donors (entry 2). The reaction intermediates hereby formed
may subsequently release X– from the α-position.
Intramolecular H-transfer reactions in o-alkylacetophenones
result in the formation of ground-state photoenols that liberate X– from the α- (entry
3) or o-ethyl
(entry 4) positions. Entry 5 shows the p-hydroxyphenacyl
moiety, which undergoes a photo-Favorskii rearrangement to release
X–. Finally, the benzoin derivative in entry 6 releases
X– to form 2-phenylbenzofuran.
Scheme 2
Photochemistry of
Aromatic Ketones that Release a Leaving Group (X)
2.1
Phenacyl and Other Related Arylcarbonylmethyl
Groups
Using phenacyl compounds as PPGs has been a subject
of interest for several decades.
39
α-Substituted
esters of the phenacyl chromophore are typical of the PPG framework
for release of carboxylic acids, for example. Homolytic scission of
the ester C–O bond, which would result in the formation of
phenacyl and acyloxy radicals, has not been confirmed. Instead, a
mechanism that involves hydrogen abstraction from a hydrogen-atom
donor by the excited carbonyl group (photoreduction
27
) of phenacyl ester via a ketyl ester intermediate (entry
1, Scheme 2) has been established by laser
flash photolysis.
40
Excited phenacyl
and 3-pyridacyl esters of benzoic acid were reported to react with
an excess of aliphatic alcohols in a chain reaction process to give
benzoic acid in addition to acetophenone and 3-acetylpyridine, respectively,
as the byproducts.
41
Singh and co-workers
have reported that arylcarbonylmethyl groups, i.e., naphth-2-ylcarbonylmethyl
42
and pyren-1-ylcarbonylmethyl,
43
can release various carboxylic acids upon irradiation.
The photochemistry of the 4-methoxyphenacyl moiety is discussed in
section 2.3.
In the presence of an
electron donor, a mechanism involving electron
transfer from the donor to the carbonyl group, followed by release
of the leaving group, can also be accommodated (entry 2, Scheme 2). This PPG strategy
will be discussed in section 8.2.
When a relatively stable radical can
be released from the α-carbon
of the phenacyl group, phenacyl radicals are produced in the primary
homolytic step. This has been demonstrated in the reactions of phenacyl
halogenides
44
or azides.
45
An alternative mechanism, formation of the phenacylium
cation from phenacyl ammonium salts, which are used as photoinitiators
for cationic polymerization reactions, upon irradiation via a heterolytic
cleavage of the C–N bond, has been proposed.
46
Recently Klán and co-workers demonstrated that readily
accessible S-phenacyl xanthates undergo photoinitiated
homolytic scission of the C–S bond in the primary step, opening
their use as PPGs for alcohols in the presence of H-atom-donating
solvents, where the xanthate moiety represents a photolabile linker.
47
The 4-acetyl-2-nitrobenzyl (ANB, 1) moiety, substituted
in both the benzylic and the phenacyl positions with leaving groups,
has recently been proposed as a monochromophoric photocleavable linker
(Scheme 3).
48
This
linker thus combines the properties of two well-known photoremovable
groups, 2-nitrobenzyl (section 3.1) and
phenacyl moieties, in a single chromophore. Liberation of R′CO2H from the intermediate
2 requires the presence
of an H-atom donor. Depending on the presence or absence of H-atom
donors, the attached groups can be disconnected selectively and orthogonally
upon irradiation in high chemical yields (88–97%).
Scheme 3
Monochromophoric
Photocleavable Linker
48
2.2
o-Alkylphenacyl
Groups
2-Alkylphenyl ketones readily photoenolize to the
corresponding
dienols (photoenols, o-xylylenols). For example,
2-methylacetophenone (3) undergoes intramolecular 1,5-hydrogen
abstraction via the triplet state to form a triplet 1,4-biradical
(enol, 3
E) that yields two isomeric, (E)- and (Z)-, photoenols, whereas fast
direct enolization from the lowest excited singlet state produces
only the (Z)-isomer (Scheme 4).
49
This scheme may serve as a blueprint
for the reactions of related 2-alkylphenacyl compounds. The (Z)-isomer, having a lifetime
similar to that of the triplet
biradical, is generally converted efficiently back to the starting
molecule via a 1,5-sigmatropic hydrogen transfer. Its lifetime is
solvent-dependent because hydrogen bonding of the hydroxyl group to
a polar solvent strongly retards intramolecular hydrogen back-transfer.
49a
In contrast, reketonization of the (E)-dienols requires intermolecular proton transfer
that
may occur either by protonation of the methylene group by a general
acid or by proton transfer from the enol by the solvent or a general
base, followed by carbon protonation of the dienol anion.
49a
The resulting long lifetime of the (E)-isomers in dry solvents allows for thermal
conrotatory
ring closure to give benzocyclobutenols, or trapping by diverse dienophiles
such as alkenes, alkynes, or carbonyl compounds in a stereospecific
[4 + 2]-cycloaddition reaction.
50
However,
they may persist up to seconds in the absence of trapping agents.
Photoenolization reactions have been thoroughly reviewed by Sammes
in the 1970s,
51
recently by Klán
et al.,
52
and, to a modest extent, in several
other reviews and book chapters.
8d,27,32,50,53
Scheme 4
Photoenolization of 2-Methylacetophenone
49
When leaving groups are present
on the α-carbon of 2-alkylphenacyl
derivatives (4), they are released from the photoenol
intermediates (Scheme 5). In general, the indanone
54
(5) and benzocyclobutenol
55
(6) side-products are formed in
non-nucleophilic solvents, whereas acetophenone derivatives substituted
on the o-methyl group 7 are produced
in the presence of a nucleophile, such as methanol.
Scheme 5
Photochemistry of
2-Alkylphenacyl Compounds
54
This reaction, reported for the first time on
phenacyl chlorides
and bromides by Bergmark,
56
was shown by
Klán and co-workers to be useful for PPG applications.
54b
Klán and Wirz later demonstrated that
2,5-dimethylphenacyl (DMP) can serve as a PPG for carboxylic acids,
57
phosphates, sulfonates,
54a
alcohols (as carbonates),
58
and
amines (as carbamates).
59
It was recognized
that only moderately good or excellent leaving groups are released
efficiently within the photoenol lifetime. Studies by laser flash
photolysis showed that photolysis produces the anticipated reaction
intermediates, the short-lived triplet enol 3
E, and two longer-lived, ground-state photoenols assigned to the corresponding
(Z)- and (E)-photoenols.
54a,57b,58
For example, the (E)-photoenol
was found to have a sufficient lifetime (1–100 ms) to release
carboxylic acids and carbonates, while the (Z)-photoenol (τ = 0.5–10 μs) regenerated
the starting ketone.
57b,58
Irradiation of DMP esters in
methanol efficiently releases the corresponding free acid (HX) along
with indanone and 2-(methoxymethyl)-5-methylacetophenone as the major
coproducts, as shown in Scheme 5. The mechanism
of DMP benzoate (8) photolysis, determined by laser flash
spectroscopy (LFP) in degassed methanol, is displayed in Scheme 6.
57b
Three intermediates,
a short-lived one, λmax ≈ 340 nm (triplet
enol 3
E), and two longer-lived ones, λmax ≈ 390 nm (photoenols), were formed. In this case,
only the longer-lived (E)-photoenol
released benzoic acid via the triplet pathway with an appearance rate
constant for benzoate of k
app = 1/τ(E-enol) = 4.5 × 102 s–1.
Scheme 6
Photochemistry of DMP Esters
57b
Structurally constrained phenyl
ketones, such as 1-oxoindan-2-yl
and 1,3-dioxoindan-2-yl derivatives, which can form only the short-lived
(Z)-xylylenols, do not release carboxylic
acids upon irradiation.
60
Only the chloride
anion was found to be eliminated from the (Z)-xylylenol (τ = 23 μs in methanol) obtained
from 2,5-dimethylphenacyl chloride via the singlet pathway.
61
Poor leaving groups such as alcohols and amines
that are not efficiently eliminated from the short-lived photoenol
intermediates have been attached through a carbonate
58
or carbamate
59
linkage, respectively,
which have similar leaving group properties to that of a carboxylate.
For example, the galactopyranosyl carbonate 9 releases
a carbonate monoester in a high chemical yield that disintegrates
thermally into the corresponding alcohol 10 and CO2 (Scheme 7)
58
on the millisecond time scale.
62
Table 3 summarizes the photochemical data for the DMP chromophore
substituted by various leaving groups.
Scheme 7
Photochemistry of
a DMP Galactopyranosyl Carbonate
58
Table 3
2,5-Dimethylphenacyl
(8, DMP) Photoremovable Group
leaving group, X (protected
species)
solvent
quantum yield, Φ
chemical yield of HX release (%)
k
app/s–1 a
Cl
benzene
0.11
56a
(0.12)
54a
4.4 × 106
61
methanol
0.76
56a
(0.78)
54a
4.4 × 104
61
OC(=O)R (carboxylic acids)
benzene
0.18–0.25
54b,57b
85–95
54b
∼2
57b
methanol
0.09–0.14
57b
92
54b
4.5 × 102
57b
OP(=O)(OR)2 (phosphates)
benzene
0.09
54a
∼2
54a
methanol
0.71
54a
94
54a
5 × 104
54a
OS(=O)2R (sulfonic acids)
benzene
0.16–0.19
54a
methanol
0.68
54a
90–93
54a
4
× 104
54a
OC(=O)OR (alcohols)
cyclohexane
0.36–0.51
58
>70
58
b
methanol
0.09–0.20
58
b
OC(=O)NR2 (amines)
cyclohexane
0.054–0.089
59
b
acetonitrile
0.035–0.070
59
97
59
b
methanol
0.027–0.061
59
b
a
Appearance
rate constant of the
leaving group, calculated as k
app = 1/τenol.
b
Slow, presumably
<1 ms–1.
Until now, only a few applications of the o-methylphenacyl
moiety as a photoremovable protecting group have been reported. Wang
and co-workers used the DMP photoremovable group in polymer-supported
synthesis,
63
and Park and Lee showed that
this moiety can be part of new photoresponsive polymers.
64
On the other hand, the photochemistry
of α-substituted o-alkylphenacyl derivatives
was utilized for the photochemical
synthesis of interesting functionalized indan-1-ones. In such cases,
releasing the leaving group is not of primary interest; it is designed
to be a good leaving group and not to interfere with the course of
the synthesis. Wessig and co-workers used this concept to prepare
various synthetically interesting 1-indanone model derivatives
65
and later two sesquiterpene indane derivatives,
pterosine B or C (e.g., 11, Scheme 8), in which the key step is the photoenolization
reaction of 12.
66
Klán and co-workers
showed that photolysis of 4,5-dimethoxy-2-methylphenacyl benzoate
can lead to the corresponding indanone derivative that is a precursor
for the subsequent synthesis of donepezil, a centrally acting reversible
acetylcholinesterase inhibitor used to treat Alzheimer’s disease.
67
Park and collaborators have recently shown that
photolysis of 2,4,6-trialkylphenacyl benzoates can also lead to the
corresponding benzocyclobutenols (6 in Scheme 5) in addition to indanones,
55
whereas irradiation of α-dichloro-2-acetophenone yields a
mixture of various photoproducts.
68
Berkessel
and co-workers used the photoenolization reaction as a tool to study
the cyclization of 4′-benzophenone-substituted nucleoside derivatives
as models for ribonucleotide reductases.
69
Scheme 8
Photochemical Synthesis of Pterosines
66
Klán and co-workers
reported that the photolysis of 2-(alkoxymethyl)-5-methyl-α-chloroacetophenones
(13) is very sensitive to traces of water in the solvent
(Scheme 9).
70
Whereas
3-methoxy-6-methylindan-1-one (14) was a major product
in dry, non-nucleophilic solvents, the isobenzofuran-1(3H)-one 15 was obtained in
the presence of trace amounts
of water. The authors demonstrated that the photoenols produced by
photolysis of 13 add water as a nucleophile to yield
2-acetyl-4-methylbenzaldehyde (16), which subsequently
forms 17 via a second, singlet-state photoenolization
reaction. The same research group also reported that irradiation of
the 2,5-dimethylbenzoyl oxiranes 18 results in a relatively
efficient and high-yield formation of β-hydroxy-functionalized
indanones that structurally resemble biologically active pterosines
(Scheme 10).
71
In
this case, a ring-opening process, rather than release of a leaving
group, follows the photoenolization step. An electronic excited-state
switching strategy has been utilized to control the selectivity of
this reaction in the total synthesis of indanorine.
72
The excited-state character of the parent compound was
changed to create a productive 3
n,π*
state by a temporary structural modification selected on the basis
of quantum chemical calculations prior to the synthesis. In addition,
competition of a triplet-state photoenolization reaction with a photo-Favorskii
rearrangement for (o/p)-hydroxy-o-methylphenacyl esters was shown to depend on the
water
content of the solvent.
73
Scheme 9
Photochemistry
of 2-(Alkoxymethyl)-5-methyl-α-chloroacetophenones
70
Scheme 10
Photochemistry of 2,5-Dimethylbenzoyl Oxiranes
71
In the 1970s, Tseng
and Ullman proposed a new PPG based on (2-hydroxyethyl)benzophenone
derivatives (19, R = Ph, Scheme 11), having a leaving group attached in the benzophenone
ortho position
via an ethylene linker.
74
A recent methodical
investigation by Pirrung and his co-workers was carried out to elucidate
the scope and limitations of the deprotection reaction.
75
Alternatively, Wirz
7a
and later Banerjee and their co-workers
76
proposed a similar photoremovable protecting group based on the
1-[2-(2-hydroxyalkyl)phenyl]ethanone 19 (R = alkyl, Scheme 11). The leaving group
was reported to be released
with a low photochemical efficiency.
76
Interestingly,
irradiation of 2-acetylphenyl- or 2-benzoylphenylacetic acid results
in efficient release of CO2.
77
Scheme 11
Photochemistry of 1-[2-(2-Hydroxyalkyl)phenyl]ethanones
74
The same mechanism, photoenolization followed by heterolytic
elimination
of HX, was shown to operate in substituted 5-(ethylen-2-yl)-1,4-naphthoquinones
(20, X = Br, dialkyl phosphate, carboxylate), a photoremovable
protecting group that absorbs up to 405 nm and provides fast and efficient
release of bromide or diethyl phosphate (Φ =
0.7 in aqueous solution) (Scheme 12).
78
The blue photoenol is formed in the ground state
within 2 ps of excitation and with a quantum yield of unity.
79
Scheme 12
Photochemistry of 5-(Ethylen-2-yl)-1,4-naphthoquinones
78
Photoenolization reactions can also be used for releasing
protected
alcohols through intramolecular lactonization. Gudmundsdottir and
her collaborators reported that the corresponding (Z)- and (E)-photoenols are produced
by irradiation
of the 2-(2-isopropylbenzoyl)benzoate ester 21 via the
triplet excited state (Scheme 13).
8d,80
An alcohol, such as geraniol (in up to 90% chemical yield), and
the side-product 22 are formed in various solvents as
well as in thin films. 2-(2-Methylbenzoyl)benzoate esters are not
reactive under the same conditions.
80a
In
addition, the 4-oxo-4-o-tolylbutanoate 23 releases methanol by a photoenolization-induced
lactonization process
(Scheme 14).
81
Scheme 13
Photochemistry of 2-(2-Isopropylbenzoyl)benzoate Esters
8d,80
Scheme 14
Photochemistry of 4-Oxo-4-o-tolylbutanoate
81
2.3
p-Hydroxyphenacyl Groups
Among the known photoremovable
protecting groups, p-hydroxyphenacyl (24, pHP, Scheme 2 entry 5 and Scheme 15) has
emerged as a promising
candidate.
82
Since debuting a little over
a decade ago, the pHP chromophore has found application as a photoremovable
protecting group in neurobiology,
7,83
enzyme catalysis,
7b,9u,83b,83c
and synthetic organic chemistry.
84
The
intriguing features of this protecting group are the skeletal rearrangement
that accompanies the release of a substrate, the quantitative chemical
yield of released product, and the necessary role of water.
7,9u,82,83,85
Advantageous properties are the hydrophilicity
of the pHP ligand, the high quantum yields, and the unusually clean
reaction that yields only one significant byproduct.
Scheme 15
Photochemistry
of pHP as a Protecting Group
82
The absorption spectrum changes
drastically as the reaction progresses
from a conjugated phenyl ketone (Figure 1)
to a nonconjugated phenol, 4-hydroxyphenyl acetate (25, R = H, Figure 2). The purported
intermediate
(26) shown in Scheme 15 is reminiscent
of the cyclopropanone intermediates proposed for the Favorskii rearrangement;
86
thus this transformation has been termed the
photo-Favorskii rearrangement.
87
Figure 2
UV–vis
absorption spectra of p-hydroxyphenacyl
diethyl phosphate (24, X = OPO(OEt)2; pHP
DEP; dashed, red) and p-hydroxyphenylacetic acid
(25, R = H; black) in H2O/MeCN (1:1).
88
p-Hydroxyacetophenone (pHA, 24, X
= H) serves as a model for the pHP chromophore. Figure 3 displays the absorption spectra
of pHA in neutral water,
of pHA– in aqueous NaOH, and of protonated HpHA+ in aqueous HClO4.
89
Figure 3
Absorption
spectra of pHA (24, X = H; dashed, in red)
in neutral water (λmax = 278 nm), of pHA– in 0.05 M aqueous NaOH (λmax = 325 nm; solid,
in
yellow), and of HpHA+ in 70% aqueous HClO4 (λmax = 333 nm; dash–dot, in blue). The
triplet excited
state equilibria are shown in Scheme 18. The
pK
a of ground state pHA is 7.9 ±
0.1 (the concentration quotient at ionic strength I = 0.1 M, 25 °C). Adapted with
permission from ref (89). Copyright 2012 American
Chemical Society.
pHA (24,
X = H) is also the basic framework for the
synthesis of the parent pHP protecting group that accommodates an
expanding number of leaving groups (HX).
7,9u,82,83,85d,85e
Most of the leaving groups have
been introduced through a sequence of bromination of pHA followed
by its SN2 displacement with the conjugate base of the
leaving group (X–) under basic conditions
82,83c,85a−85c
(Scheme 16a–c). In some instances,
protection of the phenol group by benzylation, silylation, or acetylation
is required.
Scheme 16
Nucleophilic Substitution Routes for pHP (24) Functional
Group Protection
More complex syntheses are required for more reactive
or highly
functionalized leaving groups, such as protected nucleotides, i.e.,
pHP ATP (24, X = ATP),
82,83c,84,90
pHP GTP,
91
and 18O-labeled isotopomers of pHP
GTP.
91,92
p-Hydroxyphenacyl monophosphates
are available either through displacement of pHP Br (24, X = Br) or through esterification
of 2,4′-dihydroxyacetophenone.
85b
Dibenzyl, diphenyl, and diethyl phosphates,
for example, are sufficiently nucleophilic to undergo SN2 replacement when the reagents
and solvents are rigorously dried.
The benzyl groups can be removed by hydrogenolysis with H2/Pd after ketal protection
of the phenacyl carbonyl.
82,83c
p-Hydroxyphenacyl phosphoric acid then can be coupled
with ADP or GDP through their imidazolium salts to provide the protected
nucleotides pHP ATP
82,83c,90
and pHP GTP,
90,91
respectively (Scheme 16d). These protected nucleosides have found several
applications in studies on enzyme catalysis. An advantage of this
sequence is the ability to introduce site-specific 18O-labeled
isotopomers of GTP
90,91
that are used as probes for functional
group assignment and dynamic changes in time-resolved Fourier transform
infrared (TR-FTIR) studies. Of the leaving groups thus far explored,
sulfonates,
9u,85d,93
phosphates,
7,9u,82,83,85a−85d,90−93
and carboxylates,
7,9u,83,85a−85c,93
are the most
efficacious and therefore most commonly encountered.
Another,
less frequently encountered synthetic method uses addition
of α-diazo-p-hydroxyacetophenone (27) to the conjugate acid of the leaving group (HX)
under acidic conditions
(Scheme 17).
93
This
approach is particularly useful for protection of highly reactive
or base-sensitive leaving groups. Advantages of the diazoketone approach
include the ease of synthesis of a variety of substituted diazoacetophenones
and the mild conditions for the coupling reaction. The yields are
generally good, and the only byproduct is N2. Furthermore,
protection of the phenolic OH group or other, less acidic functional
groups on the leaving group is normally unnecessary. When the phenolic
OH does require protection, the acetate ester is either retained or
otherwise readily prepared and is later removed by mild hydrolysis.
Scheme 17
Strategies for α-Diazo-p-Hydroxyacetophenone
Coupling to Protect Acidic Leaving Groups
93
The excited-state equilibria
of p-hydroxyacetophenone
(24, X = H; pHA) reflect the important nonproductive
reactions and the photophysical properties of pHP. A recent, detailed
study
89
of the primary photophysical processes
of pHA and the ensuing proton transfer reactions in aqueous solution
by picosecond pump–probe spectroscopy and nanosecond laser
flash photolysis has provided a comprehensive reaction scheme (Scheme 18): Following
fast and
quantitative ISC of excited pHA, τ(1pHA*) = 3.4 ps,
to the triplet state, 3pHA*, spontaneous adiabatic ionization
of 3pHA* in aqueous solution occurs with a rate constant k
H
+ ≈ 1 × 108 s–1, yielding the triplet of the conjugate base
anion 3pHA–* and, simultaneously, the
quinoid triplet enol tautomer 3pQ*. The latter is formed
by in-cage capture of a proton at the more basic
carbonyl oxygen of 3pHA–*. The equilibrium 3pQ* ⇆ 3pHA–* + H+ is established subsequently
by diffusional processes on the nanosecond
time scale. The formation of 3pQ* from 3pHA*
is accelerated by strong acids (via the protonated species 3HpHA+*) and is suppressed
by buffer bases, which form 3pHA–* upon encounter with 3pHA*.
The triplet-state proton-transfer equilibria of 3pHA* are
summarized in Scheme 18.
89
Scheme 18
Triplet-State Proton Transfer Equilibria of pHA (24,
X = H) in Aqueous Solution (the Experimental Values for the pK
a's and Absorption Maxima Are in Black
and Calculated
Values Are in Red). Adapted with Permission from Ref (89). Copyright 2012 American
Chemical Society.
It has been suggested
89
that
similar
proton-transfer processes may account for the lower-than-unity quantum
yields found for most pHP PPGs, especially those carrying poor leaving
groups, as formation of the less-reactive pHP triplet anion and the
nonreactive triplet quinoid enol represent energy-wasting pathways.
85d,85e,94
Furthermore, earlier studies
on pHP phosphate and carboxylate esters had documented the importance
of aqueous solvents for the photochemical release of the leaving group,
the rearrangement of the chromophore, and the role of the triplet
state as the reactive excited state, i.e., a short-lived, quenchable
triplet (E
T = 71.2 kcal mol–1).
82,83c
Subsequent work by Wan and Corrie,
95
Phillips
85a,85b,96
and Givens and Wirz
7,9u,83b,83c,85d,94,97
and their co-workers added a rich compilation of spectroscopic and
kinetic information. Recently, the effect of ring size on the photo-Favorskii-induced
ring-contraction reaction of various hydroxybenzocycloalkanonyl acetate
and mesylate esters has provided new insight into the mechanism of
the rearrangement.
98
The Phillips
group assigned electronic configurations of the key
excited states, confirming the triplet state as the reactive excited
state, using a combination of time-resolved transient absorption,
fluorescence, and resonance Raman spectroscopy, as well as femtosecond
and picosecond Kerr-gated resonance Raman spectroscopy (KTRF). An
examination of the weak fluorescence from p-hydroxyphenacyl
acetate (pHP OAc; 24, X = OAc) in anhydrous CH3CN revealed that the excited singlet
manifold of the pHP chromophore
is composed of two fluorescing states, a 1π,π*
(334 nm) state and a lower lying 1
n,π*
(427 nm).
96a
The positions of the two emission
bands are influenced by the solvent: in more polar, aqueous media
(e.g., 90% aq. CH3CN), the two bands are shifted toward
one another to 356 and 392 nm, respectively, or to an energy difference
between the 1π,π* and 1
n,π* states of 7.4 kcal mol–1 from an energy
difference of 18.7 kcal mol–1 in anhydrous CH3CN. The shift enhances the overlap of
the two states, resulting
in increased vibronic coupling and consequently more efficient internal
conversion to the lower 1
n,π* state.
Density functional theory (DFT) calculations of the electronic states
of pHP OAc further suggest that the 3π,π* state
(E
T = 72.9 kcal mol–1) lies just below the 1
n,π* state
(E
S = 75 kcal mol–1)
and is sandwiched between the 1n,π* singlet and the
nearby 3
n,π* state (71.5 kcal mol–1).
85a,95,96,99
The authors suggest that the surfaces of
these three states
31
merge, resulting in
enhanced intersystem crossing (Φ
ST = 1.0) with a rate of k
isc = 5 ×
1011 s–1 to a nearly degenerate, “mixed 3
n,π*–3π,π*”
state. Their findings reaffirmed the important role of water on the
photophysical and photochemical processes of pHP.
96a
For instance, the picosecond (ps)-KTRF studies showed
that added
water made only a small difference in the growth rate of the triplet
(from 7 to 12 ps) but greatly influenced its decay rate, resulting
in second-order quenching of the triplet.
96a
A solvent change from anhydrous to 50% aqueous CH3CN
caused a 100-fold diminution in the 3π,π* triplet
lifetime. Phillips and co-workers attributed the large decrease in
the lifetime to a leaving group effect: pHP OAc, with the poorer leaving
group, had nearly the same triplet rate constant in neat, air-saturated
CH3CN as that of pHP diethyl phosphate (pHP DEP, 150 ns).
In the aqueous media, both triplet lifetimes (3τ
∼ 150 ns) decreased, but the pHP OAc lifetime (3τ = 2.13 ns) was five times longer
than the lifetime of the
more reactive pHP DEP (3τ ≈ 420 ps; 70% CH3CN).
96a
The most important
mechanistic information obtained by Phillips’
group was from the picosecond time-resolved resonance Raman (ps-TR-RR)
results of the 600–1600 cm–1 spectral region
measured during photolysis of pHP DEP (Figure 4). Scans taken in the first few ps
show only diffuse, weak absorption
signals attributable to the excited singlet and triplet states of
pHP DEP. At ∼300 ps, the scans show the emergence of four new
bands that become prominent after 0.7–1.0 ns and by 6 ns are
the only bands remaining. These four peaks precisely match those obtained
with an authentic sample of the photoproduct, p-hydroxyphenylacetic
acid (25, R = H). This TR-RR profile sets the reaction
time-constant for the rearrangement and, therefore, encompasses the
period for both the release of the leaving group and the rearrangement
of the chromophore. In fact, the rearrangement product is in full
bloom within just 1 or 2 ns, demonstrating both that the leaving group
has departed and, more strikingly, that the complex rearrangement
including any intermediates that may intervene between the excited
triplet state and 25 had silently formed and then expired
completely, escaping ps-TR-RR detection. On the basis of a kinetic
analysis of the appearance of 25, Phillips and co-workers
showed that such a silent intermediate or intermediates were necessary.
He assigned a candidate for the intermediate to “M”
to a p-quinone methide cation that was formed by
direct heterolysis of the leaving group from triplet pHP DEP. This
assignment was corrected in a later study (vide infra).
100
Figure 4
Picosecond time-resolved resonance Raman spectra of pHP
DEP (24, X = diethyl phosphate) obtained with a 267 nm
pump and
200 nm probe wavelengths in a H2O/CH3CN (1:1)
mixed solvent. The resonance Raman spectrum of an authentic sample
of p-hydroxyphenylacetic acid recorded with 200 nm
excitation is displayed at the top. Reprinted with permission from
ref (96a). Copyright
2005 American Chemical Society.
Another significant result came from the analyses of the
photolysis
products from a series of pHP-substituted acetate esters in the study
by Corrie, Wan, and co-workers.
95
The acetates
were chosen for their increasing propensity toward decarboxylation
when converted to carboxy radicals by photoinduced homolysis of the
corresponding arylmethyl esters. Photolysis of the pHP esters, i.e.,
acetate, phenylacetate, pivalate, and diphenylhydroxyacetate, however,
produced only carboxylic acids in >90% yield, free of any radical-derived
decarboxylation products.
101
This confirmed
the heterolytic pathway suggested by the groups of Givens,
85d,85e
Falvey,
40
and Phillips.
99,100
The next layer of evidence on the photo-Favorskii mechanism
arose
from time-resolved transient absorption (TR-TA) studies,
85d
which revealed two additional reactive intermediates:
an early, very short-lived transient appearing on the tail of the
triplet decay (Figure 5) and a later, long-lived
species. The critical evidence for the first transient was obtained
using pHP OTs (24, X = OTs), OMs, and DEP, all excellent
leaving groups that depart efficiently and rapidly. For example, the
transient formed from 3pHP DEP (lifetime, 3τ
= 63 ps; in water) appears as a weak set of absorptions on the tail
of the pHP triplet. These three maxima were assigned to an allyloxy–phenoxy
triplet biradical (3
28, Scheme 19): the decay profile of 3
24 (3τ = 100 ps in 87% aqueous CH3CN) transforms
into the profile of the slightly longer-lived transient 3
28 (τbirad = 500 ps).
Scheme 19
Refined
Mechanism Based on Time-Resolved Transient Absorption Analysis
85d
The three weak absorptions of 28 were detected
85d
only with the best leaving groups. The bands
at 340, 430, and 440 nm were taken as evidence of a phenoxy radical
intermediate and thus assigned to the biradical 28. As
noted earlier, kinetic analysis of the ps-TR-RR spectra by Phillips
and co-workers
85a,85b
had suggested the intervention
of an intermediate “M”, formed from the triplet state
that proceeded to the final product 25. The intermediate
“M” is now attributed
85d
to
the triplet biradical 3
28 that is assumed
to be formed adiabatically. The formation of 3
28 can be viewed as being extruded from 3
24, leaving behind the leaving group X– and a proton
in the ground state, thus obeying the Wigner spin rule. The fate of 3
28 is ISC and closure to an as yet undetected
spirodienedione 26. The resulting traditional Favorskii-like
intermediate very rapidly hydrolyzes to p-hydroxyphenylacetic
acid, completing the formal ground state events normally proposed
for the Favorskii rearrangement.
102
A further, long-lived intermediate was identified as the known p-quinone methide
103
29 (τ = 0.3 s) that hydrates yielding p-hydroxybenzyl
alcohol (30).
85d
The formation
of small amounts of 30 is also a signature of the elusive
spirodione intermediate 26, the lifetime of which appears
to be shorter than its rate of formation under the reaction conditions.
Thus, the validation of 26 is based solely on a requirement
for the carbon skeleton reorganization and a very facile CO extrusion
from 26 due to its strained bicyclic structure, and is
complemented by DFT calculations.
A summary of the photo-Favorskii
mechanism, as currently understood,
is outlined in Scheme 19.
104
The rearrangement proceeds from the chromophore’s
triplet excited state (3
24) by a concerted
departure of the leaving group and the phenolic proton generating
the triplet biradical 3
28. Intersystem crossing
of 3
28 gives an intermediate common to the
ground state Favorskii rearrangement,
102a−102c
the putative cyclopropanone,
86,98,102
that either hydrolyzes or decarbonylates
on its pathway to the final products. The evidence provided, however,
requires involvement of another intermediate, presumably the singlet
allyloxy-phenoxy species 1
28,
98,99,104b
to account for the complete
racemization of a p-hydroxypropiophenone analogue
where the leaving group is affixed to a stereogenic α-carbon.
104b
This mechanism also provides a pathway for
the minor photohydrolysis byproduct 24 (X = OH) that
becomes predominant for the ring-contraction photoreactions of hydroxybenzocycloalkanonyl
esters when ring strain discourages or prevents cyclopropanone formation.
98
Figure 5
Pump–probe spectra of pHP DEP (24,
X = diethyl
phosphate) in 87% aqueous CH3CN. The sample was excited
with a pulse from a Ti/Sa–NOPA laser system (266 nm, 150 fs
pulse width, pulse energy 1 μJ). The inset shows the species
spectra of 3pHP DEP and biradical 3
28 that were determined by global analysis of the spectra taken with
delays of 10–1800 ps using a biexponential fit. Reprinted with
permission from ref (85d). Copyright 2008 American Chemical Society.
Position and Requirement for a p-Hydroxy Group
Unsubstituted phenacyl, which is also a PPG (section 2.1), does not undergo a photo-Favorskii
rearrangement
but rather reacts through a photoreduction mechanism. The p-OH modification of the
phenacyl chromophore causes a profound
change in the photochemical behavior. The search for alternative functional
groups or other locations of the OH group on the phenacyl chromophore
that would accommodate a Favorskii rearrangement pathway has met with
very little success. Only 2-hydroxyphenacyl esters were shown to release
carboxylic acids.
73
m-Hydroxyphenacyl
acetate and a 5-hydroxy-1-naphthacyl analog were unreactive under
photo-Favorskii conditions.
105
Electron
donors such as p-methoxy and o-methoxyphenacyl
have been tested as early as the seminal report of the photo-Favorskii
rearrangement by Anderson and Reese.
87
For
these examples, the photo-Favorskii rearrangement competes with photoreduction,
forming mixtures of the corresponding methoxyacetophenones and phenyl
acetates. In the early 1970s Sheehan and Umezawa developed the p-methoxyphenacyl derivatives
as a PPG for photolysis in
dioxane or ethanol, producing reduction products.
39,106
Givens and co-workers later showed that the reaction in methanol
or t-butanol (hydroxylic solvents) did undergo the
Favorskii rearrangement as the major pathway, yielding p-methoxyphenylacetates. The
competing photoreduction pathway was also
evident from the significant proportion of reduction to p-methoxyacetophenone.
107
Phillips and
co-workers showed that p-methoxyphenacyl diethyl
phosphate undergoes a rapid heterolytic cleavage that results in deprotection
and formation of a solvolytic rearrangement product.
108
Other electron-donating substituents have met with
even less success
toward the rearrangement of the chromophore.
82
Although several p-methoxy and other p-alkoxy analogues have been successfully employed
as PPGs for the
release of carboxylates,
39,106,109
phosphates,
82,107
carbonates, and carbamates,
110
they do not lead to rearrangement of the chromophore.
Nature of the Leaving Group
The most efficacious leaving
groups are conjugate bases of moderate to strong acids, e.g., sulfates,
85d,93
phosphates
7,9u,82−85,99
(thiophosphate),
100,111
carboxylates,
7b,9u,85a−85c,93−95,97b,97c,112
phenolates,
9u,93
and thiolates.
111,113
In general, quantum yields monotonically
decrease with an increase in the acid leaving group’s pK
a (Table 4), conforming
to a Brønsted leaving group relationship (βLG), which correlates the pHP release rate
constant with the K
a of the leaving group. A correlation of the
log of the rate constants (log k
r), derived
from the quantum yields and the triplet lifetimes as k
r = Φ/3τ (see
Table 4 footnote b for
details), with the pK
a of the leaving
groups gave βLG = −0.24 ± 0.03.
9u,114
Table 4
Disappearance Quantum Yields, pK
a’s, and Rates of Release for Different
Leaving Groups (X) for pHP X (24) Arranged According
to the pK
a of HX
X (released substrate)
λmax (log ε)a (pHP
X)
pK
a (HX)
Φ
–X
b
k
r/108 s–1
c
ref
mesylate
–1.54
0.93
50
(85d, 93, 114)
tosylate
–0.43
1.00
100
(85d, 93, 114)
OPO3Et2
271 (4.18)
2.12
0.4
12.
(82, 85a−85c, 96a, 99, 114)
Glu
273 (3.94)
4.33
0.14
1.9
(83c, 97b, 97c)
Ala·Ala
282 (4.12)
3.4
0.27
1.8
(83c, 112a)
bradykinin
282 (4.07)
3.4
0.22
1.8e
(83c, 112a)
p-CF3C6H4CO2
–
3.69
0.2
3.2
(9u, 93, 114)
formate
3.75
0.94
14
(9u)
benzoate
4.21
0.32
2.8
(9u, 73, 85c, 93, 114)
acetate
279 (4.09)
4.76
0.4
N/Af
(85a−85c, 95, 96)
GABA
282 (4.16);
325
4.76
0.2
6.2
(83c, 85e, 94, 97b, 97c)
RPO3S–
d
5.3
0.21
N/A
(111)
OPO3
–2
280 (4.48)
7.19
0.38
N/A
(82)
GTP
7.4
N/A
N/A
(91, 92)
ATP
253 (4.3); 320 (2.70)
7.4
0.37
68
(82, 83c, 90)
p-CNC6H4O–
7.8
0.11
0.76
(9u, 93)
RS–
d
8.4
0.085
N/A
(111, 113, 115)
C6H5O–
9.89
0.04
1.0
(9u, 93)
HO–
15.7
<0.01
N/A
(95)
a
In H2O unless otherwise
noted.
b
Appearance efficiencies
were identical
within experimental error (±5%). See Table 5 for examples.
c
The rates are derived from several
sources and conditions vary. The H2O content was between
10% and 50%, causing small variations in the quantum yields/rates
(see text).
d
Decaging of
the catalytic subunit
C199A/C343A of PKA (protein kinase A) at Thr-197.
111
e
Assumed to be
the same as the model,
Ala·Ala.
f
N/A = not
available.
The good quantum
efficiencies and high appearance rate constants
of the free substrates (k
app = 1/3τ) make the pHP protecting group attractive for quantitative
and mechanistic studies in biology and physiology.
9u
Other beneficial features include good aqueous solubility
and stability, the ease of synthesis, the biologically benign quality
of the pHP group and its photoproducts, and the lack of quenching
by adventitious O2 in aqueous solvents.
ATP and GTP
release from the pHP-protected nucleotides has been
extensively investigated, resulting in pHP becoming the “phototrigger”
of choice for fast kinetic studies of the enzyme-catalyzed hydrolysis
by Ras and Rap GTPase activating proteins (GAP proteins).
90−92
The phototrigger methodology for activating hydrolysis by photodeprotection
of GTP or ATP is rapid (an appearance rate constant k
app = 1/3τ = 1.6 × 1010 s–1 as measured for pHP diethyl phosphate in water
99
) and was assumed by the authors to be sufficient
for measuring the kinetic rate constants for most subsequent binding
and hydrolysis steps for the nucleotide.
116
Thus, the pHP protecting group provides researchers with a powerful
arsenal for fast kinetic mechanistic investigations.
Kötting,
Gerwert, and co-workers, for example, compared
the release rates for pHP versus NPE (1-(2-nitrophenyl)ethyl; section 3.2) GTP esters
(Figure 6).
116
The rise time for photorelease of
GTP from pHP GTP was too fast to record by their TR-FTIR instrument
(τrise = 10 ms), whereas they were able to monitor
the GTP appearance rate constant (k
app = 2 ± 1 s–1, Figure 6). They then exploited the rate advantage of pHP GTP to study
the
catalytic GTP hydrolysis by Ras GTPase and other GAP-based catalytic
hydrolysis mechanisms.
Figure 6
Formation of GTP measured as its Mg2+ complex
at 1128
cm–1 from pHP-caged GTP (black) is already complete
at the first data point. Formation of GTP from NPE-caged GTP (green)
takes place more slowly with a rate constant of 2 s–1 because the rate-limiting step
is the release from a ground-state
hemiacetal intermediate (section 3.2), an
inherently slow process on the time scale necessary for the kinetic
measurements reported here.
116
Reprinted
with permission from ref (116). Copyright 2007 Wiley and Sons.
In pursuing the mechanistic pathway for Ras GTPase catalysis,
comparisons
of the FTIR spectra of individual α-, β-, and γ-18O-labeled and unlabeled phosphates
of GTP, inorganic phosphate
(Pi), and GDP hydrolysis products as well as 13C and 14N-labeled site-specific amino
acids provided detailed
information on both bonding and environmental changes at the enzyme
active site. TR-FTIR was then employed to monitor the changes in binding
and the evolution and decay of the intermediates during hydrolysis
as well as the product-release step and to determine the rate constants.
92
Figure 7 illustrates
the power of TR-FTIR to resolve the changes in structure and binding
at the labeled sites as a function of reaction time. Scheme 20 summarizes the key
steps for the hydrolysis, beginning
with initial binding of free GTP and ending with the release of inorganic
phosphate (Pi) from the enzyme “pocket”,
in the rate-limiting step that controls signal transduction. In contrast,
with NPE GTP as the phototrigger, only the (last) rate-limiting step
could be determined.
92
Figure 7
(a) Difference spectra
by TR-IR absorption of the intrinsic Ras-catalyzed
GTPase reaction. A single exponential function by global fit analysis
shows the change from Ras GTP to Ras GDP at 1143 cm–1. (b) TR-IR absorbance difference
spectra for the GAP-catalyzed GTPase
by Ras. Two intermediates are seen by the fit of three exponential
functions at 1143 and 1114 cm–1. The appearance
of GTP at 1143 cm–1 arises from pHP-caged GTP followed
by GTP hydrolysis. Protein bound Pi appears at 1114 cm–1, which is subsequently released
as the rate-limiting
step (Scheme 20). Reprinted with permission
from ref (92a). Copyright
2004 Elsevier B. V.
Scheme 20
Rate Constants and
Mechanism for Ras GTPase (GAP) Hydrolysis of GTP
Derived from the Initial Photorelease of GTP from pHP-Caged GTP (the
Nonsignaling “OFF” to the signaling “ON”
States Are Shown)
92b
For thiolates, the nucleophilicity of the leaving
group is especially
noteworthy because leaving groups can readily be protected through
in situ derivatization. pHP Br can be added directly to thiols and
thiophosphates, even in the presence of other nucleophilic groups
on the substrate or in the media. Direct derivatization of thiols
and thiophosphates has been exploited for peptides and proteins that
possess exposed cysteine and thiophosphate residues, an especially
useful feature when the thiol group is an integral part of the catalytic
center.
115
Model reactions where pHP Br
was reacted directly with 3′-thiodeoxythymidine, cysteine,
and glutathione produced the corresponding pHP thioethers in 80–90%
yields in buffered solutions (Scheme 21). Deprotection
by irradiation at 300–365 nm releases the thiol in 60–70%
yield, performing essentially as a protection–deprotection
switch.
Scheme 21
Reversible Protection–Deprotection of a Thiol
on 3′-Thiodeoxythymidine
with pHP Br
115
The switch sequence was employed by Pei and co-workers
113
and by Bayley and co-workers.
111
Pei and co-workers inhibited the phosphorylation of a cysteine
located at the active site of protein tyrosine phosphatases (PTK)
by direct addition of pHP Br. The phosphorylated cysteine turned “OFF”
PTK, a common type of suicide inhibition used with other phenacyl
halides. However, photolysis of the pHP thiolates freed the catalytic
cysteine unit, turning PTK back “ON”. Interestingly,
the protection step was regioselective for blocking only Cys 453,
the cysteine at the catalytic site, and none of the other three available
cysteine residues.
The protection–deprotection sequence
was also reported for
the C subunit of protein kinase A (PKA) at Thr-197 and for a thiophosphorylated
tyrosine (Y) on a model 11-aa peptide, EPQYEEIPILG, by
Bayley’s group.
111
Two PPGs were
compared: reacting thiophosphate with o-nitrobenzyl
bromide (75%) and protection with pHP Br (90%). Deprotection proved
more difficult with the o-nitrobenzyl (oNB, section 3.1) thioether because the nitrosobenzaldehyde
as a side-product reacted with the newly exposed thiol, causing inhibition.
The quantum yield for the reaction was modest (0.37). pHP deprotection
was more efficient (Φ = 0.56 to 0.65) and the
70% recovery of the activity was higher because there were no competing
reactions of the byproducts with the exposed thiophosphate. This methodology
was transferrable into in vitro cell machinery by simply importing
the pHP Br into human B cells.
111
Substituent
Effects on the Chromophore
Very recently,
a number of new ortho- and meta-substituted p-hydroxyphenacyl
PPGs were introduced to extend the versatility by absorbing at longer
wavelengths and by altering the solubility properties. The influence
of substituents on the chromophore’s physical, spectral, and
mechanistic capabilities, by necessity, became the target of several
studies. GABA was selected as the common leaving group because it
imparts good aqueous solubility and is biologically significant. Quantum
yields of a representative collection of 2- and 3-substituted pHP
GABA (31, Table 5) vary only modestly
for these substituents. meta-Electron donors such as 3-OCH3 generally display lowered
quantum yields whereas electron-withdrawing
groups such as 3-CF3 and 3-CN often give rise to slightly
increased yields. The rate constants for release are consistently
high, in the range of 109 s–1.
94
Certain groups, m-nitro, m-OH, and m-acetyl, when present on the
chromophore completely quench the photorearrangement reaction.
94
Table 5
Effects of Substituents
and pK
a on Quantum Yieldsa for the Substituted pHP GABA 31 in Unbuffered
H2O;b Entries Are Arranged in the
Order
of Decreasing pK
a of the Substituted pHP
Chromophore
94
31
pK
a
Φ
dis
c
Φ
GABA
Φ (25)d
Φ
dis (Ac or
DEP)d
3,5-CH3
8.2
0.15
0.14
0.13
3-CH3
8.1
0.15
0.14
0.13
2-CH3
8.0
0.11
0.1
0.1
3-OCH3
7.9
0.07
0.06
NDe
0.39 (DEP)
R1–R4 = H
7.8
0.20
0.19
0.16
0.30
(Ac)
0.40 (DEP)
3,5-OCH3
7.8
0.03
0.03
ND
0.44 (DEP)
2-F
7.2
0.28
0.27
0.26
2,6-F
6.8
0.16
0.16
0.15
3-F
6.7
0.16
0.15
0.15
3-OCF3
6.5
0.09
0.09
0.07
2,3-diF
5.9
0.24
0.24
0.22
2,5-diF
5.7
0.22
0.21
0.2
3-CF3
5.5
0.17
0.16
0.14
3,5-F
5.3
0.11
0.11
0.1
3-CN
5.2
0.42
0.35
0.39
0.17 (Ac)
2,3,5-triF
4.5
0.08
0.07
0.06
tetra-F
3.9
0.11
0.1
0.1
a
All runs were low
conversions to
products (<5%); standard deviations were < ±0.02.
b
Unbuffered 18 MΩ ultrapure
H2O.
c
Disappearance
quantum yield when
GABA is the leaving group.
d
Quantum yield for the substituted
phenylacetic acid (25).
e
Disappearance quantum yield when
Ac (acetate) or DEP (diethyl phosphate) is the leaving group.
e
ND = not determined.
94
In
addition to GABA, there are many other small-molecule and amino
acid neuroactive agonists and antagonist with carboxylic acid end-groups.
Although carboxylate release is generally less efficient than those
of phosphates and tosylates, caged carboxylates find useful applications
in neurobiology and neurophysiology, taking advantage of the rapid
rate of release and the unreactive, benign qualities of 25 photoproduct vis-à-vis
o-nitrobenzyl-based
PPGs (section 3.1).
92,97b,97c,117
Substituent modification of the pHP derivative 32,
such as the 3-CF3 and 3-OCH3 pHP GABA compounds shown in Scheme 22, expands
and extends the versatility of the PPG methodology. Here, the relative
efficacy of modified pHP GABA to stimulate the GABAA receptor
is documented in Figure 8.
Scheme 22
m-Electron-Donor and -Acceptor Group Compatibility
for Photorelease of GABA from m-Substituted pHP GABA
97b
Figure 8
Comparison of EC50’s for GABAA receptor
activation by rapid photolysis of pHP (24) GABA. Dose–response
curves for 3-CF3O-pHP GABA (blue, n =
7 neurons), 3-CF3-pHP GABA (red, n = 6
neurons), and 3-CH3O-pHP GABA (black, n = 6 neurons) with population data of peak
currents normalized to
the maximum peak response. EC50 and Hill’s coefficient
values were as follows: 3-CF3O-pHP GABA, 49.2 μM,
1.8, n = 7 neurons; 3-CH3O-pHP GABA, 93.4
μM, 1.9, n = 6; and 3-CF3-pHP GABA
119.8 μM, 2.73, n = 6. Reprinted with permission
from ref (97b). Copyright
2009 American Chemical Society.
The effect of m-methoxy, trifluoromethoxy,
and
trifluoromethyl groups on pHP-caged GABAs were tested for their
efficacy to release GABA in whole-cell patch-clamp studies on neurons
in cortical slices. Local photolysis with short UV light pulses (10–50
ms) delivered through a small-diameter optical fiber produced transient
whole-cell inward currents from released GABA.
97b
Effect of Media pH and pHP pK
a
Because the ionization of substituted pHP derivatives
to their
conjugate bases changes during irradiation in unbuffered media (Figure 3), the pH
effects on the pHP photochemistry were
examined (Table 6). The extent of quinone methide
formation is altered also by the pH and the substituents on the chromophore
(Scheme 18).
Table 6
Substituent Effects
on the Quantum
Yieldsa As a Function of pH for GABA Release
from 31 at 300 nm in Buffered CH3CN–H2O; Entries Are Arranged According to Decreasing
pK
a of the Substituted pHP GABA
94
pHP GABA
pK
a
Φ
dis pH 5.0b
Φ
dis pH 7.3c
Φ
dis pH 8.2c
3,5-(CH3)2
8.2
N/A
0.17
0.11
3-CH3
8.1
N/A
0.15
0.08
parent
8.0
0.21
0.21
0.09
2-F
7.2
0.24
0.21
0.06
3-F
6.7
0.15
0.12
0.02
3-OCF3
6.5
0.07
0.06
0.02
3-CF3
5.5
0.24
0.12
0.08
3,5-F2
5.3
0.08
0.05
0.02
3-CN
5.2
0.21
0.33d
0.19e
2,3,5,6-F4
3.9
0.08
0.10
0.09
a
Standard deviations were < ±0.02.
b
0.01 M ammonium acetate.
c
0.01 M HEPES, 0.1 M LiClO4, pH 7.3.
d
0.01 M ammonium acetate, pH
7.
e
0.01 M ammonium acetate,
pH 9.
Raising the pH above
8 lowers the quantum yields, reflecting the
lower reactivities of the conjugate bases (Table 6). In all cases, the quantum yields
were maximal in neutral
or slightly acidic conditions but dropped at higher pH. As shown for
the pK
a’s of the corresponding
acetophenones, there is a substantial substituent effect on the pK
a, which is manifested in a pronounced UV–vis
spectral change (see Figure 3). The prominent
π,π* transition at 260–280 nm for neutral p-hydroxyacetophenone is shifted to 320–340
nm, the
π,π* transition for the conjugate base, and the absorptivity
nearly doubles.
The resultant interplay of pK
a of the
substituted pHP derivative and the pH of the solution influence the
quantum yield as illustrated with 3-CF3 pHP GABA. The quantum
yields at pH 5 (Φ = 0.24) decrease to half
of their value when the pH is 7.3 (Φ = 0.12)
and a third at pH 9.2 (Φ = 0.08). Product yields
remain the same at all three pH values, demonstrating that the photo-Favorskii
rearrangement is still the major reaction pathway for the conjugate
base. These initial results on substituted pHP protecting groups show
promise for extending their use in chemistry, physiology, and biochemistry.
2.4
Benzoin Groups
In the course of their
ground-breaking studies on benzoin (desyl alcohol; Figure 1; Scheme 2, entry 6) acetates,
Sheehan and Wilson determined that the 3′,5′-dimethoxybenzoin
(DMB, 33, X = OAc) derivative performed best as a PPG
of acetate.
4,118
The reaction proceeded in an
extraordinarily smooth fashion as illustrated by the spectra shown
in Figure 9. The expected product 2-phenyl-5,7-dimethoxybenzofuran
(34, DMBF, Scheme 23) was formed
in quantitative yield, and the quantum yield was determined as 0.64
± 0.03. The authors noted that the photocyclization of DMB acetate
was not quenched by either naphthalene or neat piperylene, and they
concluded that the reaction proceeds from the excited singlet state
or an extremely short-lived triplet state.
Scheme 23
Photocyclization
of DMB Acetate
4,118
Figure 9
Course of the photolysis of DMB (33) acetate to DMBF
(34) in acetonitrile (Scheme 23); irradiated in a photochemical reactor at 360 nm.
Reprinted with
permission from ref (118). Copyright 1971 American Chemical Society.
It has taken substantial efforts to elucidate the detailed
mechanism
of this reaction, and a clear picture (Scheme 24) has emerged only in recent years.
Time-resolved work on DMB derivatives
was performed by the groups of Trentham,
119
Wan,
120
Simon,
121
Wirz,
122
and Phillips.
123
By ns-LFP of several DMB carboxylate esters (33, X = OCOR) in dry acetonitrile, Shi,
Corrie, and Wan
120
observed a strong transient absorption at 485
nm that was formed within the lifetime of their laser pulse (∼10
ns) and decayed with a lifetime of 1 μs. This transient was
assigned to the cyclohexadienyl cation (35, Scheme 24). An additional transient absorption
with μs
lifetime was observed at λmax = 330 and 420 nm. Introduction
of air resulted in a faster decay of this transient but did not affect
the lifetime of the 485-nm transient or the yield of the final product 34 (DMBF).
The (330, 420)-nm transient was therefore assigned
to the (nonreactive) triplet state of the DMB esters (3
33). These assignments have stood the test of time.
However, subsequent studies with better time resolution showed that
heterolytic cleavage of the excited singlet state is not, as claimed,
120
the primary photochemical step of DMB esters,
and that the cyclohexadienyl cation is not even an intermediate along
the predominant reaction path releasing the substrate HX.
122,123b
Scheme 24
Mechanism of the Photocyclization of 3′,5′-Dimethoxybenzoin
(DMB) Derivatives (X = OCOR, OPO(OEt)2, F)
122,123b
Pump–probe experiments
of DMB acetate and fluoride (33, X = OCOMe, F) with picosecond
time resolution revealed
a preoxetane biradical intermediate 36, λmax = 355 nm, that was formed from the singlet
state, τ = 17 ps,
and decayed with a lifetime of 1–2 ns.
122
The biradical 36 was shown to be the precursor
of the cyclohexadienyl cation 35, λmax = 485 nm, the lifetime of which was found to
be strongly reduced
by the addition of water to the acetonitrile solution, k
w = 6 × 106 M–1 s–1. This work proved that the reactive intermediate
is the biradical 36, which releases the DMB-caged ligands
HX on a time scale of 1–2 ns, largely independent of the leaving
group ability of X. Thus, DMB is an excellent PPG for the kinetic
investigation of fast processes such as protein folding.
124
The existence of a biradical intermediate 36 had been proposed earlier on the basis
of preparative work
by Rock and Chan, who also showed that a benzoin side-product (37) is formed in aqueous
solution.
125
Otherwise, the reaction proceeds cleanly and efficiently in both
polar and apolar solvents.
In the most recent time-resolved
study of DMB phosphate (X = OPO(OEt2), Phillips and co-workers
123b
investigated
the ultrafast primary photophysical processes by absorption spectroscopy
and provided evidence for the formation of an intramolecular charge-transfer
singlet state (S1, τ = 14 ps), as had been proposed
earlier.
120
Moreover, using nanosecond
time-resolved resonance Raman spectroscopy, they proved that the final
product 34 (DMBF) is largely formed within the time resolution
(∼10 ns) of their setup. It was postulated that DMBF is formed
by concerted HX elimination directly from the biradical 36, largely independent of
the solvent, and that the cyclohexadienyl
cation is not a precursor of DMBF. Nevertheless, earlier ns-LFP experiments
had shown that a second, minor wave of DMBF is also released from
the cyclohexadienyl cation on a time scale of 1 μs in dry acetonitrile.
122,126
Finally, Phillips and co-workers
127
reported
extensive CASPT2 calculations in support of the ultrafast primary
processes that they had observed by transient absorption.
123b
The photoreaction of parent benzoin
derivatives follows an entirely
different course (Scheme 25). Givens and Matuszewski
reported that irradiation of benzoin diethyl phosphate (38, X = OPO(OEt)2; BDP) in
acetonitrile proceeds cleanly
and quantitatively with a quantum yield of 0.28.
128
Stern–Volmer quenching studies with either piperylene
or naphthalene indicated that the reaction proceeds via the triplet
state with a lifetime of a few nanoseconds. A study using ps-pump–probe
and ns-LFP of BDP was reported by Rajesh et al.
129
The assignment of the observed transient intermediates
was assisted by DFT calculations. Two competing reaction paths of
diethyl phosphate elimination proceed from the triplet state of BDP:
Reaction via path (a) yielding 39 (R = H or CH2CF3) predominates in water and 2,2,2-trifluoroethanol
(TFE), while exclusively path (b) yielding 2-phenylbenzofuran (40) is followed in
acetonitrile. Path (a) proceeds via a transient
intermediate, λmax = 570 nm, that was attributed
to the triplet state of the carbocation formed adiabatically from 3
38 by heterolytic release of the phosphate anion.
A triplet multiplicity of the cation 3
41 was
indicated by the observation of oxygen quenching, k
q ≈ 1 × 109 M–1 s–1, and by the fact that its lifetime in water
(430 ns) is similar to that in the much less nucleophilic solvent
TFE (660 ns). This indicated that the rate-determining step for hydrolysis
of the triplet cation is ISC to the singlet ground state. For both
pathways, the reactive intermediate releasing diethyl phosphate is
thus the excited triplet state with a lifetime of about 10 ns.
Scheme 25
Mechanism of the Photorelease of Diethylphosphate from 38 (X = OPO(OEt)2) in Various
Solvents
129,130
The assignments of the observed
transients have been largely confirmed
and corroborated by Phillips and co-workers using fs-pump–probe
and ns-resonance Raman spectroscopies as well as DFT calculations.
130,131
The formation and decay of the cation intermediate in 75% aqueous
acetonitrile solution was measured by ns-time-resolved resonance Raman
spectroscopy. An essential difference from the previous study
129
is that two cationic species appeared in sequence:
The first of these (main feature at 1560 cm–1),
which was formed within the rise time of the instrument (∼10
ns), was attributed to the triplet cation 3
41 with optical absorption at 570 nm. The 1560 cm–1 signal decayed with a lifetime
of 100 ns, forming another transient
species (main feature 1626 cm–1), which decayed
with a lifetime of 200 ns and was attributed to the singlet ground
state of the cation (41). The lifetime of the short-lived
species (100 ns, 1560 cm–1) was reduced by purging
the solution with oxygen. It is hard to reconcile the observations
by optical LFP
129
and Raman spectroscopy,
130
and more work may be required to settle this
point.
Very soon after the initial reports on PPGs, Sheehan
published
the photoconversion of substituted benzoin esters into benzofurans
with the concomitant loss of acetate (38, X = OAc, Scheme 23).
4,132
Several years later he exploited
this reaction as a PPG for carboxylic acids.
118
The benzoin group remained remarkably underutilized for two
decades,
until Baldwin and co-workers used it for the photorelease of phosphates.
Thus, phosphate derivatives of 33, 38, and 42 (X = OPO3
2–) were investigated,
and all three released inorganic phosphate upon irradiation with a
laser at 308 or 355 nm with appreciable yields (35–55% under
continuous irradiation).
106a
Although,
as Sheehan and co-workers had already pointed out in
their initial studies, the 3′,5′-dimethoxybenzoin derivative 33 was the most reactive,
easier synthesis of the symmetrical
benzoin derivative 38 made it equally attractive. Doubts
on the purity of the substrates prompted another study, which provided
a reliable preparative method for the phosphate (X = OPO3
2–) and esters (X = OAc), rendering 33 an ideal candidate for a fast and clean release
of phosphates.
119
The same PPG was used to protect the 3′-phosphate
of nucleotides.
133
DMB phototriggers have been extensively used for applications in
drug delivery,
134
muscle relaxation studies,
119
lithography,
135
biochip
fabrication,
133,136
protein folding and unfolding,
124
or for masking a photochemical switch.
137
cAMP derivatives of 38 were also
able to release cAMP upon photolysis.
107b,107c,138
Likewise, glutamate and GABA were released from 38, with the PPG at the γ-carboxyl
group (Scheme 26). However, neither the α- nor the N-protected derivatives of 38 led
to a clean
photolysis.
139
Scheme 26
Release of Glutamate
and GABA from Benzoin Derivatives
107b,107c,138
Various types of leaving groups, such as amines, can be
released
from derivatives of 3′,5′-dimethoxybenzoin (33).
140
Although there are no photochemical
reasons preventing the use of a whole array of leaving groups, the
sometimes delicate preparation of certain derivatives has to be considered.
α-Ketol rearrangement can scramble the position of the methoxy
groups on both arenes, and an activated ester precursor
can react intramolecularly into an inert cyclic product. However,
carbamates could be prepared by the reaction of 33 (X
= OH) with cyclohexylisocyanate or by preparing the p-nitrophenyl mixed carbonate.
140c,141
They were
utilized for the photogeneration of bases in films.
142
In the solid state, 33 released cyclohexylamine
with Φ = 0.067 (254 nm), 0.08 (313 nm), 0.054
(336 nm), and 0.028 (365 nm). The liberated benzofuran side-product
absorbs at longer wavelengths; this photobleaching is crucial for
applications in thick films, allowing light to reach deeper layers.
Again, the methoxy groups on the benzylic side were found to be important
for the reactivity; other substituents on the benzoyl side also had
an impact, but less significant. An alternative preparative method
was devised by Pirrung and Huang, where the benzoin is allowed to
react with carbonyl diimidazole (preactivated by methyl triflate),
followed by the amine.
140b
Primary amines,
however, failed to give the desired carbamate and gave instead the
cyclic product mentioned above.
The activation procedure with
carbonyldiimidazole proved to be
efficient also for the preparation of carbonates, thus allowing 33 to release alcohols
(X = OCOOR). It was used to protect
the 5′ primary alcohol of nucleotides,
135b
various kinds of alcohols, or benzylthiol.
136
The protection of chiral molecules (such
as nucleotides) can be
problematic with DMB, because it also bears a stereogenic center.
Access to an enantiopure derivative of 33 would therefore
be highly desirable. Enantioselective syntheses of 33 have been published, using enantiopure
TMS-protected cyanohydrins
generated either by asymmetric catalysis or enzymatic resolution (Scheme 27).
143
Addition of an
aryl-Grignard reagent to the nitrile, followed by acidic hydrolysis,
leads to chiral unsymmetrical benzoin. When asymmetry is not required,
more straightforward routes are available, in particular by using
a dithiane as a benzoyl anion equivalent.
144
This method was used to prepare derivatives of DMB that could release
a phenol (ubiquinol),
145
or as a linker
for peptides.
144
This dithiane route was
cleverly exploited in a safety-catch strategy, where keeping the carbonyl
function masked prevented any photolytic activity, whereas hydrolysis
restored the initial sensitivity.
146
A
related strategy was recently proposed, where the carbonyl group is
masked as a dimethyl ketal, which can be smoothly hydrolyzed into
the photolabile benzoin derivative (3% trifluoroacetic acid (TFA)
in CH2Cl2, 5 min).
147
Scheme 27
Preparation of Racemic and Enantiopure 3′,5′-Dimethoxybenzoin
(DMB; 33, X = OH)
143,144
As will be mentioned in the next section on
nitrobenzyl derivatives
(section 3.1), water-insolubility is a major
issue when the release of bioactive material is sought under physiologic
conditions. Thus, the water-soluble derivative 43 was
prepared. The possibility of carrying out the photolysis in aqueous
solution gave an additional hint in favor of the cationic mechanism
(Scheme 24), as the free DMB alcohol was also
observed as a side-product, in addition to the expected benzofuran.
125
Attempts to improve the reactivity
by further modification of the
main core were proposed, such as the replacement of both aromatic
groups by the 2-furyl moiety. Dipeptide esters of this “furoin”
analogue 44 indeed could be deprotected, but in lower
yields than the parent structure.
148
On
the other hand, esters of the achiral 1,2,2-triphenylethanone 45 proved to be as reactive
as 33 (DMB) (Scheme 28).
149
The diphenylbenzofuran
side-product continues to react under irradiation to form a more conjugated
heterocycle.
Scheme 28
Photolysis of 1,2,2-Triphenylethanone Esters
149
Chirality in the PPG is not necessarily a problem and
can actually
be exploited. Klán and co-workers used enantiopure acrylate
derivatives as a photoremovable chiral auxiliary (PCA).
Thus, the acrylate 46 reacted in a highly enantioselective
manner with cylclopentadiene in the presence of a Lewis acid, and
the cycloadduct was photolyzed (Φ = 0.43) at
313 nm to give the Diels–Alder exo product
as the main diasteroisomer, with enantiomeric excessed (ee’s)
up to 96% (Scheme 29).
150
The photochemistry of several benzoin derivatives is summarized
in Table 7.
Scheme 29
Use of a Chiral
Benzoin as a Photoremovable Chiral Auxiliary
150
Table 7
Photolysis Quantum
Yields for Benzoin
Derivatives
PPG
X (leaving group)
medium (λirr/nm)
Φ
ref
33
RCO2
MeCN (366)
0.64
(118)
33
c-HexNHC(O)O
film (365)
0.028
(142)
38
RCO2
MeCN–H2O (350)
0.14
(139)
46
RCO2
MeOH (313)
0.43
(150)
3
Nitroaryl
Groups
3.1
o-Nitrobenzyl Groups
o-Nitrobenzylic derivatives have been widely used
despite their disadvantages. They were proposed as general PPGs in
1970,
3
but there were earlier reports on
their photochemistry,
151
including the
one on the photoisomerization of o-nitrobenzaldehyde
into the corresponding nitrosobenzoic acid.
152
Hydrogen transfer from the o-alkyl substituent
to the nitro group forming an aci-nitro tautomer
in the ground state is commonly taken to be the primary photoreaction
of o-alkylnitroarenes. Parent o-nitrotoluene
(47, oNT, Scheme 30) and several
derivatives have been studied by time-resolved spectroscopy.
153
The most recent investigation of oNT and the
analogous o-nitrobenzaldehyde by Gilch and co-workers,
154
who used femtosecond transient absorption and
stimulated Raman spectroscopy, has finally provided a convincing and
complete picture of the early events. The results reported by these
authors about oNT
154a
are summarized in
Scheme 30, which bears a striking similarity
to that for the photoenolization of o-methylacetophenone
(Scheme 4 in section 2.2).
Scheme 30
Reaction Mechanism for the Phototautomerization of oNT in THF
154a
In aqueous solution, equilibration between the (Z)- and (E)-aci-isomers
by proton
exchange between the oxygen atoms through solvent water is faster
than the intramolecular back-reaction (Z)-aci → oNT and the aci-decay obeys
a single exponential rate law.
153f
A detailed
investigation of the pH–rate profile provided the acidity constant
of the equilibrated aci-tautomers of oNT, pK
a = 3.57 ± 0.02, and kinetic isotope effects
on the aci-decay kinetics indicated that the dominant
rate-determing step for aci-decay switches from carbon
protonation by H+ below pH 6 to carbon protonation by water
above pH 6. In strongly acidic solutions, acid-catalyzed addition
of water to the methylene carbon followed by dehydration of the resulting
nitroso hydrate yields o-nitrosobenzyl alcohol.
153f
A total quantum yield (Φ
aci
= 0.08) for the formation of aci-nitro tautomers
from oNT in tetrahydrofuran (THF) was estimated on the basis of transient
absorbance intensities;
154a
this value
is an order of magnitude larger than previous estimates that were
obtained with aqueous solutions of oNT by photoinduced H/D exchange
153f
and by another comparison of transient absorbance
intensities.
155
The discrepancy was attributed
to fast retautomerization of the (Z)-aci-nitro isomer to oNT.
154a
However, this
explanation cannot hold as the (Z)- and (E)-isomers are rapidly equilibrated in aqueous
solution.
153f
An independent value of Φ
aci
for oNT might be obtained by measuring
the quantum yield of the irreversible reaction in strongly acidic
solutions.
153f
Fortunately, many derivatives
of oNT show much higher quantum yields Φ
aci
(vide infra).
o-Nitrobenzyl (oNB) and 1-(2-nitrophenyl)ethyl
(NPE; see section 3.2) derivatives that
carry a leaving group at the benzylic position release the protected
substrate upon irradiation. The reaction proceeds via aci-nitro intermediates that
are readily observed by flash photolysis
at λmax ≈ 400 nm. The decay of these aci-transients frequently follows a biexponential
rate law
(due, presumably, to the formation of both geometrical isomers at
the methylene group); the aci-decay rate constants
are on the order of 102–104 s–1 and vary strongly with substitution, solvent, and
pH in aqueous
solution. A detailed mechanistic study of the release of ATP from
“caged ATP”,
6
P
3-1-(2-nitrophenyl)ethyl ester of adenosine triphosphate 48 (Scheme 31), was reported
in 1988
by Trentham and co-workers.
156
Scheme 31
Photochemistry
of P
3-1-(2-Nitrophenyl)ethyl
Ester of Adenosine Triphosphate
156
The appearance rates of the
three products formed by irradiation
of caged ATP, namely, ATP2–(bioassay), 2-nitrosoacetophenone
(49, absorption at 740 nm), and H+ (using
an indicator dye), were each monitored and were found to coincide
with the decay of the aci-nitro intermediate. Later
work using time-resolved infrared detection and isotopic labeling
further established that the release of ATP occurs in a first-order
reaction that is synchronous with the decay of the aci-anion (k = 52 s–1 at pH 7,
10
°C).
157
The reaction mechanism proposed
by Trentham and co-workers
156
was unchallenged
for many years, and it was frequently taken for granted that aci-decays were synchronous
with substrate release. That
assumption is not warranted in general, however, especially with more
nucleophilic leaving groups.
Subsequent LFP and time-resolved
IR studies were used to study
the release of alcohols from oNB
158
and
NPE
159
(section 3.2) ethers at near-neutral pH. Detailed kinetic studies covering a
wide pH range (Figure 10)
158
indicated that the mechanism proposed by Trentham and co-workers,
156
although it had been fully consistent with
their results, needed to be revised on several counts (Scheme 32): (a) Cyclization
to the 1,3-dihydrobenz[c]isoxazol-1-ol intermediate (50) occurs from
the neutral aci-compounds (51), as predicted
by DFT calculations,
160
not from the conjugate
bases (51
–). This accounts for the
specific acid catalysis often observed for the decay rates of the aci-intermediates.
(b) The cyclization 51 → 50 is irreversible. (c) At pH values below 8, hydrolysis
of
the hemiacetal intermediate 52 formed by ring-opening
of 50 is rate-determining for the release of methanol
and may be rate-determining with other poor leaving groups. The intermediates 50 and
51 have also been identified by IR analysis
following irradiation of oNB methyl ether in Ar and N2 matrices
at 12 K.
161
Figure 10
pH–rate profiles
for the reaction steps 51/51
– → 50 (• and
o), 50 → 52 (+ and × ), and 52 → 53 (□) of oNB methyl ether
in aqueous solution. Reprinted with permission from ref (158). Copyright 2004 American
Chemical Society.
Scheme 32
Mechanism of 1-(Methoxymethyl)-2-nitrobenzene
Photoreaction
160
As can be seen from Figure 10,
the release
of methanol from oNB methyl ether in the final reaction step 52 → 53 is many orders
of magnitude slower
than the decay of the aci-intermediates 51/51
– at pH values near neutral. A
faster reaction path bypassing intermediates 50 that
was proposed by Corrie et al.
159
was subsequently
shown not to be operative.
158
The same
holds for the release of glycolic acid from oNB glycolic acid ether,
which also proceeds via a long-lived hemiacetal with a lifetime of
4 s at 20 °C in wholly aqueous solution at pH 7.
162
Moreover, buffers were found to intercept the aci-intermediates when used in high
concentrations, thereby
further retarding the desired release of alcohols from oNB ethers.
162
Nevertheless, Ellis-Davies and Barsotti
reported fast release of
Ca2+ ions from the ethylene glycol tetraacetic acid (EGTA)
chelator complexes that are attached through an ether linkage to 3-nitrodibenzofuran-2-yl
(54, NDBF EGTA) and several 6-nitroveratryl (55, NV EGTA)
163
derivatives. Rate constants
of Ca2+ ion release on the order of 104–105 s–1 were observed by monitoring the fluorescence
signal arising from the Ca2+-dyes Ca-Green-5N
164
or Ca-Orange-5N.
163
This enables relatively fast mobilization of intracellular Ca2+ by photolysis of NV-
and NDBF-caged IP3 (inositol
1,4,5-trisphosphate).
165
The authors assumed
that cleavage of the benzylic carbon–ether linkage is much
faster in derivatives of NDBF and NV than that of oNB, possibly bypassing
a hemiacetal intermediate. In our view, a more likely interpretation
of these observations is that the formation of the cyclic intermediates
of type 50 already reduces the binding constants of the
chelator complexes sufficiently to afford substantial Ca2+ ion release with rates
corresponding to the aci-decay rates. Indeed, biphasic release of Ca2+ from compound
55 (NV EGTA) with rate constants of 5 × 104 and 1.5 s–1 has been observed.
163
The formation of the reactive aci-nitro intermediates
may proceed from both the excited singlet and triplet states. Steiner
and co-workers have shown that the 2-(2-nitrophenyl)prop-1-oxycarbonyl
(NPPOC, section 3.2) PPGs used in photolithographic
DNA chip synthesis can be considerably enhanced by covalently linked
triplet sensitizers (section 8.1) such as
thioxanthone.
166
Moreover, the same group
recently reported that sensitization by diffusion in a thin film can
be more effective than intramolecular sensitization for sensitizer
concentrations higher than 5 mM.
167
On
the other hand, Görner showed that triplet states of CT character
are formed by direct excitation of NV derivatives and related compounds
that, however, do not participate in aci-nitro formation
or the release of the substrate.
168
Various substituents at the benzylic position of oNB were found
to increase their perfomance as PPGs. For example, Hess and co-workers
discovered that addition of a carboxylate to the benzylic carbon atom
of the oNB caging chromophore increased release rates.
169
However, decarboxylation is a significant reaction
pathway for photolabile calcium chelator derivatives of ethylenediaminetetraacetic
acid (EDTA) and EGTA.
170
Radical stabilization
energies computed by DFT methods were shown to be useful predictors
of the relative efficiency with which LGs are photoreleased from oNB
protecting groups.
171
The presence
of a hydroxy group at the benzylic carbon in 56 opens
a new pathway for the decay of aci-nitro intermediates
via a nitroso hydrate (Scheme 33, path b).
168a,172
pH–Rate profiles for
the reaction steps involved in paths (a) and (b) in aqueous solution
have been determined.
172
The caged Ca2+ chelator “nitr-5” is a BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic
acid) derivative of an o-nitrobenzyl alcohol reported
by Tsien and co-workers,
173
who proposed
that the relatively fast Ca2+ release (3000 s–1) from “nitr-5” may be attributable
to proton shuffling,
i.e., path b of Scheme 33.
Scheme 33
Photochemistry of
the Hydroxy Derivative 56(168a,172)
The parent oNB structure 57, although not the most
widely used, represents the generic form of this family of PPGs. The
leaving group can be directly attached to the benzylic site, as in 57, and this is
the typical mode for the release of carboxylic
acids,
3
thiols,
174
histidine,
175
and phosphates (Scheme 34, several examples shown).
5,107b
Although it is possible to directly attach alcohols and amines,
they are most frequently linked as carbonic acid derivatives 58 (X = OCO–X′), which
are better leaving groups
and make the synthesis more convenient, i.e., by the direct reaction
of the alcohol or amine with the readily available o-nitrobenzyl chloroformate. These
strategies are, however, inappropriate
when rapid release is needed (e.g., for electrophysiological applications),
as the postphotolytic fragmentation is the rate-limiting step for
release.
140a,159
This also does not prevent other
major drawbacks of this PPG for the release of alcohols, which is
slow (vide supra), and of amines, which undergo condensation with
the nitrosoaldehyde side-product, leading to a stable imine; however,
the latter reaction can be avoided by the addition of carbonyl scavengers,
such as semicarbazide hydrochloride.
3
Another
more serious problem is the formation of nitrosoaldehyde that yields
brown-colored degradation products that absorb the incident light,
thus creating an internal filter.
Scheme 34
Photolysis of Some o-Nitrobenzyl Derivatives
o-Nitrobenzylic PPGs have naturally led
to various
synthetic and biological applications, especially for caged biomolecules,
and only selected examples will be discussed here as many reviews
have been published on the field during the past decade.
9c,9f,9l,9m,12b,176
For example,
photolytic deprotection of PPGs under very mild conditions provides
a great advantage for automated DNA or RNA synthesis compared to the
traditionally acyl-protecting group used. In this aim, a pioneer application
was developed by Stutz and Pitsch using oNB (57) as a
protecting group for the 2′-oxygen of the four different bases
for oligoribonucleotide synthesis (Scheme 35).
177
Interestingly, whereas nucleobases
were protected via the usual carbamate function, the 2′-oxygen
and the 2-nitrobenzyl group were linked together via an oxymethyl
acetal.
Scheme 35
Protected Phosphoramidite Building Blocks for Automated
RNA Synthesis
177
2,4-Dinitrophenol was released in mitochondria, by first
linking
it to a cationic phosphonium targeting group via an o-nitrobenzyl ether. Incubation
in the cytoplasm allowed migration
of the assembly into the mitochondria and was followed by subsequent
photolysis (λ = 355 nm).
178
Ingenious
inclusion of nitrobenzylic units in vesicles allowed a dose-controlled
release of the encapsulated hydrophobic guests upon irradiation (λ
> 315 nm).
179
Other interesting applications
were developed in the preparation of otherwise inaccessible metal–organic
frameworks, by inclusion of o-nitrobenzyl ethers
and subsequent photolysis.
180
o-Nitrobenzylic PPGs have also been employed in
natural product syntheses. One well-known and impressive application
was reported by Nicolaou and co-workers in the total synthesis of
the Calicheamicin γ1 where the high compatibility of 57 with other classical PGs and
functions was demonstrated.
181
Another smart example is the use of 57 in the synthesis of an analogue of Leukotriene
C4 (LTC4, Scheme 36).
182
In the final steps,
deprotection by irradiation at 350 nm of the nitrobenzyl derivative
affords the secondary amine in 74% yield. It is noteworthy that no
isomerization of the triene part and no racemization were induced
by photolysis.
Scheme 36
Final Steps in the Total Synthesis of N-Methyl LTC4
182
Over the years, multiple modifications have been developed
to tune
the properties, mostly toward an increase in quantum yield, an increase
in the rate of release, and increasing absorbance at longer wavelengths.
Substitution at the benzylic site mainly affects quantum yields, whereas
modification on the aromatic moiety affects the absorbance.
Substitution
at the Benzylic Position
In addition to
providing an electronic effect at the benzylic site, a second hydrogen-abstracting
unit could, in principle, increase the efficiency. Already in the
1970s, Patchornik, Amit, and Woodward proposed this modification,
which worked up to a certain point.
3
It
should be kept in mind that most substitutions at this site create
a chiral center, which can become a drawback if chiral molecules have
to be protected (and that is the case for most of the relevant applications,
such as amino acids, carbohydrates, and oligonucleotides), unless
the added subsituent is identical to the aromatic core. A second o-nitrophenyl would
fit in this category. However, an increase
in photolysis quantum yields was not explicitly mentioned, and the
increase in chemical yield might just be the consequence of the formation
of a ketone instead of an aldehyde side-product, which is less prone
to the parasitic imine formation. Thus, 59 was able to
release carboxylic acids quantitatively and inorganic phosphate with
85% yield,
106a
whereas 60 released
amino acids in yields between 70% and 95% in the absence of a carbonyl
scavenger. Various derivatives have been prepared since, among them 61 and 62, which
liberated acetic acid in good
quantum yields (Φ = 0.2 for 61),
168b
or 63.
183
Despite the problem of chirality,
a phenyl group can have a beneficial
effect, as had been shown much earlier by Barltrop and co-workers
with esters of 64 and carbamates of 65,
151
and was later used in linkers for solid-phase
synthesis.
184
Simpler substituents, such as a methyl group, have a beneficial
impact on the quantum yield. Thus, pivalate esters of 57 have release quantum yields
in a polymeric matrix of 0.04 and 0.13
in acetonitrile solution, whereas they are much higher for pivalate
ester 66 (Φ = 0.09 in polymer
and 0.64 in acetonitrile).
185
Solution-phase
quantum yields cannot be directly extrapolated to those of the solid
phase, as the available conformations can be quite different.
186
Derivatives of 66 have many other
uses, such as the release of biologically active carboxylic acids
in plant cells,
187
or on solid support.
188
Specht and Goeldner proposed the introduction
of a strongly electron-withdrawing group at the benzylic carbon, which
had a massive effect on the quantum efficiency on choline and arseniocholine
ether derivatives (Φ = 0.7 for 67 and Φ = 0.43 for 68) and sugar
ethers (Φ = 0.62 for 67 and Φ = 0.52 for 68). Such derivatives were,
however, not widely used in organic synthesis, as they were assembled
by Mistunobu reactions, which were sluggish for secondary alcohols.
Nevertheless, interesting applications, such as the release of choline
or arsenocholine, were used as examples.
189
Jullien and co-workers prepared and evaluated the 6-nitroveratryl
(NV, or 4,5-dimethoxy-2-nitrobenzyl, DMNB) derivatives 69, 70, 71, and 72 with electron-withdrawing
substituents at the benzylic site; despite earlier claims, it was
found that the substituents at the benzylic site, while having an
unambiguously observable effect (up to a factor 3 between the most
and least efficient), did not increase the quantum efficiency by orders
of magnitude (Φ = 0.013 for −Br and Φ = 0.003 for −CN).
190
Interestingly, the cyano compound had been independently identified
by a clever combinatorial technique by Pirrung and co-workers as being
quite efficient.
191
Such apparent contradictions
have been reported in the field of PPGs for decades and could potentially
slow down future development of new or modified PPGs, as no clear
trend emerges from the huge amount of sometimes unreliable data accumulated.
The determination of quantum yields remains a delicate operation subject
to significant experimental error; furthermore, all these different
groups are frequently tested for the release of different leaving
groups (phenols, carboxylic acids, amine, aliphatic alcohols, and
carbamates) under different irradiation wavelengths, in different
solvents and buffers, and each with a different photochemical apparatus.
We have seen above the enormous importance that the effects of solvation
and proton transfer play on the reaction mechanism.
As mentioned for the benzoin PPG (see section 2.4), water-solubility is a crucial
point for biological applications,
and this property can be achieved by the introduction of a CO2H substituent. Hess
and co-workers designed the α-carboxynitrobenzyl
(α-CNB) derivative 73 for the release of phenolic
OH groups; there was a significant increase in the release rate, but
this was observed only at shorter wavelengths.
169,192
Bassani and co-workers recently reported that the α-carboxy-6-nitroveratryl
(α-CNV) analogue 74 released carboxylic acids,
in quantitative chemical yields with Φ = 0.17
and a rate of release 2–3 orders of magnitude larger than the
parent compound,
193
and capsaicin through
an ether bond.
194
A related strategy (based
on 75) had been devised by Schaper and co-workers.
195
Thiols could also be released with the derivative 76.
196
A time-resolved infrared
study of the photoreactivity of α-CNB 74 was performed
by Corrie and co-workers.
197
Related groups
were studied computationally by Schaper and co-workers.
198
Pirrung and co-workers designed
a clever way of dealing with the
nitroso side-product, responsible for the internal filter effect.
It was trapped in situ by a Diels–Alder reaction with a diene
function included at the benzylic site (Scheme 37).
199
These pentadienylnitrobenzyl PPG
esters, ethers, and carbamates 77 were used to release
carboxylic acids, alcohols, and amines (Φ =
0.22, 0.41, and 0.38, respectively); an analogue with a methylenedioxy
group on the aromatic ring was also discussed, with improved properties
(in this case, the increase in absorbance more than compensated for
the usual decrease in quantum yields).
Scheme 37
Trapping of the
Nitroso Byproduct in a Diels–Alder Cycloaddition
199
Substituents on the Aromatic Ring of the o-Nitrobenzyl
Chromophore
Because of the problems of chirality, the synthetic
issues, and the limited impact on the absorbance, considerably less
work was devoted on substitution at the benzylic site than the very
numerous modifications on the aromatic ring. Such modifications bring
three features: (a) the tuning of the absorbance with concomitant
effects on the quantum yield, (b) the possibility to anchor the group
on a solid support or on a linker (this will not be discussed here,
as a book chapter reviewed the field recently),
200
and (c) the possibility to modulate the solubility properties
of the group.
A second electron-withdrawing nitro group was
added to increase the hydrogen-abstraction capacity (reminiscent of 59 and 60), and
the release of amines from carbamates 78 was much more efficient than for 58 (Φ =
0.62 versus Φ = 0.13 in
the solid state).
186
Interestingly, in
the same study, the carbamate derivative of 66 showed
a quantum yield of release of 0.11, which differs from earlier data.
This was attributed to the influence of the matrix. The analogue 79 was also exploited
a few years later.
187
Various groups have been added to increase the hydrophilicity
of
the cage again to address the water-solubility issue. As an example,
the carboxylic acid 80 with a one-carbon spacer was reported
by Allan et al.
187
Similarly, 81 and 82 were also exploited.
169b,201
Early on, two methoxy groups were added to increase the
absorbance
at longer wavelengths (λ > 350 nm, and Rayonet lamps at 420
nm still provide photolysis at reasonable rates), leading to the 6-nitroveratryl
(NV, 83) and 6-nitroveratryloxycarbonyl (NVOC, 84) moieties. These are undoubtedly
the most frequently used
groups to date both in solution
3
and in
the solid phase (for two recent, among many other, examples, see ref (202)). Multiple
variants have
been evaluated, such as the introduction of an α-methyl group
at the benzylic site: the methyl-6-nitroveratryl (MeNV, 85) and methyl-6-nitroveratryloxycarbonyl
(MeNVOC, 86)
groups. Very close analogues, but with slightly altered properties,
presumably due to a restriction in the freedom in aligning the nonbonding
orbitals of the oxygen with the aromatic π system, are the methylenedioxy
derivatives 87 and 88. Further addition
of an α-methyl group led to α-methyl-(6-nitropiperonyloxymethyl)
(MeNPOM, 89) and the now famous analogous 3,4-(methylenedioxy)-6-nitrophenylethoxycarbonyl
(MeNPOC 90) groups. The latter moiety was used in the
automated synthesis of DNA chips.
203
A
solution-phase comparison of the quantum yield of release of thymidine
derivatives of NVOC (Φ = 0.0013) and MeNPOC
(Φ = 0.0075) was performed.
204
Caged nucleobases were used for the
transient disruption of DNA
hybridization to photochemically controlled DNAzyme activity.
205
This strategy is illustrated in Scheme 38 where the 89 (MeNPOM)-protected thymidine
phosphoramidite is incorporated into DNA using classical synthetic
procedures. The caged-DNAzyme thus obtained can be quantitatively
reactivated by irradiation at higher wavelengths. MeNPOM presents
several advantages compared to the parent compounds 73, 87, and 90: the very efficient
conversion
upon irradiation at 365 nm, the high stability of the caged molecules
in aqueous media, the less toxic acetophenone byproduct, and its easy
incorporation via the chloromethyl ether derivative.
206
Scheme 38
Preparation of Caged-DNAzyme and Activation by Nonphoto-Damaging
UV Light
205
Inactivation of fluorophores (see section 11) was effected by similar caging, for
example, by binding
nitroveratryl
groups to rhodamines and fluoresceins.
207
A recent computational study
198
was complemented
by an experimental systematic evaluation of substituents on the aromatic
ring and at the benzylic site
168b
and showed
surprisingly low influence of these groups (except for the strongly
electron-releasing amine of 91, an effect that was reported
earlier). Indeed, one of us reported a very significant lowering of
the deprotection efficiency by having electron-releasing groups para
to the nitro group, a manifestation of the CT-character of the excitation,
as mentioned earlier.
208
This was exploited
in a so-called safety-catch approach, where esters
of 92, 93, 94, and 95 were protected against photolysis in normal conditions. Upon
protonation
with a strong acid, the reactivity was restored, thus allowing normal
photolysis.
209
Similarly, the reactions of the compounds 96, 97, and 98, evaluated by Jullien and
co-workers,
were found to be less efficient than those of the nonsubstituted congeners
(in fact, they were too slow to be measured in the case of 96 and 97).
190
The same authors also examined the effect of other groups, in para-position
to the nitro group, with or without additional conjugation (99, 100, and 101).
190
As was the case for the benzylic substituent, these extra
groups had a limited impact on the quantum yield, although the highly
conjugated group in 101 decreased the reaction efficiency.
The authors also explicitly pointed out an inverse correlation between
the quantum yield and longer absorption wavelengths. Various other o-nitrobenzyl alcohol
(e.g., 102 and 103) derivatives substituted in the ortho-position to the
benzylic group, or meta-position to the nitro group, have been reported.
12b,185,208
The reaction efficiency of their
ethers was tested in terms of kinetics of release, quantum yields
in solution, or quantum yields in the solid state as a function of
the irradiation wavelength for some of them.
In an effort to increase the absorbance at longer wavelength, Singh
and Khade extended the aromatic core as a naphthalene
210
or 7-methoxynaphthalene.
211
The corresponding esters 104 were able to
release aromatic rings containing carboxylic acids at 380 nm, with Φ ranging between
0.08 and 0.16 (in acetonitrile–water).
210
The nitrodibenzofuran 105 also
had very interesting properties (such as efficient deprotection),
which were applied in recent examples.
164,165
The carbonyl group is among the groups most frequently in need
of facile protection–deprotection protocols, so it is surprising
that very few PPGs have been developed for it. Gravel and co-workers
were among the first to address this issue, by first introducing the
ketal 106 in 1974.
212
One
issue was the generation of a mixture of diastereoisomers if the ketone
to be protected was nonsymmetrical. The same group found later that
the even simpler ketal 107 could circumvent the problem.
213
A solid-support version of 106 was developed.
214
Another way of
dealing with the issue of chirality was proposed
by one of us with the ketal 108, derived from the very
easily prepared enantiopure (1R,2R)-bis(o-nitrophenyl)ethanediol,
215
which was used by Walsh and co-workers for the preparation
of the otherwise too sensitive intermediate.
216
The substituted version 109 with a red-shifted absorption
was proposed later.
217
On the other hand,
for the release of simple aldehydes, it is not necessary to consume
two aromatic-releasing units, and α-acetyl acetals have been
shown to release aldehydes efficiently upon irradiation (Scheme 39).
218
Scheme 39
Photolysis
of α-Acetyl Acetals
218
In a similar strategy, the o-nitrobenzaldehyde
derived acetals 110 or 111 were used to
protect 1,2- or 1,3-diols, principally for protection of saccharides.
219
However, in this case, only one alcohol can
be deprotected, leading to photostable 2-nitrosobenzoic acid derivatives.
Moderate to high regioselectivities were obtained during the deprotection,
mostly determined by steric and electronic effects of the nonsymmetrical
substrates (Scheme 40).
219a
The final deprotection of diols could be achieved under
basic conditions or by intracellular hydrolysis. A reverse strategy
was developed by Iwamura et al. with the treatment of 111 by boron trifluoride in
the first step to release an ether of 57, which was subsequently cleaved photochemically.
Scheme 40
Selectivity in the
Deprotection of 110-Protected 1,2-Diol
219a
Two-Photon Absorption
The two-photon irradiation technique
(see also section 9) enhances the efficient
temporal and spatial control of biomolecule delivery. Taking advantage
of this for a biological application, Kao and co-workers reported
that vanilloid derivatives of oNB (57) and NV (83) can be photoreleased in situ to
activate TRPV1 receptors
on nociceptive neurons with one-photon quantum efficiencies of 0.13
and 0.041 (Scheme 41).
220
In another example, retinoic acid derivatives of 72 and 83 were prepared and photolyzed,
first
at 365 nm.
221
The presence of a long-wavelength
tail in the absorption spectrum does not prevent liberation, although
isomerization of double bonds did occur. In the two-photon mode (excitation
at 750 nm; 25 mGM) retinoic acid was also liberated from cage 57, and dynamic studies
on zebrafish embryogenesis were carried
out (vide infra).
Scheme 41
Two-Photon Activation of Vanilloid Derivatives
220
An interesting alternative to the simultaneous absorption
of two
photons is the use of lanthanide-containing upconverting nanoparticles
(UCNPs; see section 8.3). In this case,
two near-infrared (NIR) photons are absorbed sequentially, and the
nanoparticle re-emits one higher-energy photon, in a spectral range
that can be exploited by PPGs. Such an example was recently shown
by Branda, Zhao, and co-workers, where nitroveratrylester-containing
micelles were dissociated upon irradiation with a continuous-wave
NIR laser.
222
A similar strategy was also
used to photolyze benzoin esters.
223
Isotopic Substitution
As the transfer of hydrogen from
the benzylic site to the nitro group is involved in the primary photochemical
step, it was found that the rupture of the stronger C–D bond
was less efficient.
224
The photolysis of
the esters 57 bearing a perdeuterated benzylic position
was 3.8–8.3-times less efficient (depending on the irradiation
wavelength). The substituents on the aromatic ring in the analogous
esters 83 diminished this isotope effect. The origin
of these observations has not yet been fully elucidated; so far the
phenomenon was utilized in the selective removal of two photolabile
(oNB and NV) groups (Scheme 42 a and b depicts
two different isotopically labeled derivatives).
225
The photochemistry of the oNB derivatives is summarized
in Table 8.
Scheme 42
Impact of Isotopic
Substitution on the Photoreactivity
225
Table 8
Photolysis Quantum
Yields for Nitrobenzylic
Derivatives
PPG
X (leaving group)
solvent
(λirr/nm)
Φ
ref
57 (oNB)
thymidine-OCO2
MeOH/H2O, 1:1 (365)
0.033
(226)
57 (oNB)
t-BuCO2
MeCN (254)
0.13
(185)
61
CH3CO2
MeCN (254)
0.2
(168b)
66
t-BuCO2
polymer (254)
0.09
(185)
66
t-BuCO2
MeCN (254)
0.64
(185)
66
c-Hex-NHCO2
polymer (254)
0.11
(186)
67
choline-O
buffer, pH
7 (364)
0.7
(189)
67
sugar-O
buffer, pH 7 (364)
0.62
(189)
69
arsenocholine-O
buffer, pH 7 (364)
0.43
(189)
69
sugar-O
buffer, pH 7
(364)
0.52
(189)
70
coumarin-CO2
MeCN/buffer, pH 7, 1:1
(325)
0.013
(190)
71
coumarin-CO2
MeCN/buffer, pH 7, 1:1
(325)
0.011
(190)
72
coumarin-CO2
MeCN/buffer, pH 7, 1:1
(325)
0.003
(190)
73
PhCO2
EtOH/H2O, 1:1 (355)
0.17
(193)
77
PhCO2
CDCl3 (350)
0.22
(199)
77
BnO
CDCl3 (350)
0.41
(199)
77
c-Hex-NHCO2
CDCl3 (350)
0.38
(199)
79
c-Hex-NHCO2
THF (254)
0.62
(186)
79
c-Hex-NHCO2
polymer (254)
0.13
(186)
83 (NV)
thymidine-OCO2
MeOH/H2O, 1:1 (365)
0.0013
(204)
83
coumarin-CO2
MeCN/buffer, pH 7, 1:1 (325)
0.006
(190)
90 (MeNPOC)
thymidine-OCO2
MeOH/H2O, 1:1 (365)
0.0075
(204)
104
RCO2
MeCN/H2O, 3:2 (>370)
0.08–0.16
(210)
3.2
o-Nitro-2-phenethyloxycarbonyl
Groups
In the late 1990s, Hasan and co-workers designed the
oNB one-carbon homologue 112 (1-(2-nitrophenyl)ethyloxycarbonyl,
NPEOC; “OC” stands for the −OC(=O)–
group) and its α-methylated analogue 113 (NPPOC).
226
Interestingly, despite the
structural similarity between 112/113 and 57, the release mechanism differs
markedly, and the observation of a nitrostyrene side product is compatible
with a photoinduced elimination (Scheme 43),
226
reminiscent to the photoelimination from ketones
by photoenolization mentioned earlier (Scheme 11). It turned out that 112 eliminated
a 5′-O-nucleoside
carbonate (Φ = 0.042) with a higher quantum
yield than that of its oNB analogue 57 (Φ = 0.033). The substitution of the benzylic
center with a methyl
considerably added to the efficiency (Φ = 0.35),
which made 113 a candidate of choice for oligonucleotide
synthesis.
227
This group was further modified
to increase the absorbance, such as in the 2-(3,4-methylenedioxy-6-nitrophenyl)propoxycarbonyl
(MNPPOC; 114)
204,228
or analogous 3-(4,5-dimethoxy-2-nitrophenyl)-2-butyl
(DMNPB, section 9)
229
derivatives, but 113 was applied in automated light-directed
olgonucleotide synthesis (DNA-chips).
230
Amino acids
231
and carbohydrates were
protected with NPPOC at the 6-position, including thiophenyl glycoside
donors.
232
NPPOC derivatives are themselves
relatively inefficient in two-photon deprotections; however, in the
presence of sensitizers (section 8) (it has
been shown by Wöll and co-workers that NPPOC can be sensitized),
166,233
the cross section can reach useful levels for several applications.
234
This is discussed in more detail in section 9. A very recent example of sensitization
was shown
by Winssinger and co-workers, where a thioxanthenone sensitizer is
bound to a nucleic acid sequence, which is able to promote the photolysis
only when hybridized with a NPPOC-containing sequence with the matching
complementary bases.
235
Scheme 43
Photolysis of 2-Nitro-2-Phenethyl
Derivatives
226
Further modification of the backbone may increase the
two-photon
absorption cross section (section 9), such
as in the biphenyl derivative 115(236) or highly conjugated systems 116 and 117.
237
This has also been recently
demonstrated by Goeldner and co-workers, who studied the photochemical
properties of biphenyl-containing photoremovable protecting groups
possessing the o-nitrobenzyl, phenacyl, and 2-(o-nitrophenyl)propyl functionalities.
238
Replacement of the methoxy substituent
of 115 by an
amino group increased significantly the two-photon cross section (section 9).
239
The high efficiency
of NPPOC was exploited in many applications.
166,204,228,230−234,236,240
3.3
o-Nitroanilides
Nitroanilide
derivatives, such as 118, were shown in
the 1970s to be photolabile and to release carboxylic acids (Scheme 44) and various
byproducts depending on the conditions.
241
The early works concluded that the reaction
is a rearrangement and not a solvolysis, as no isotopic incorporation
was observed when the photolysis was carried out in H2
18O. Likewise, derivatives of 119 are also photolabile,
but their cyclic nature prevents a direct H-abstraction on the amide
alkyl substituent.
242
Failure to provide
esters when the reaction is carried out in alcoholic solvents was
an additional argument against a simple solvolysis.
Scheme 44
Photoactive o-Nitroanilide Derivatives
241
Neither 118 nor 119 were widely exploited
in synthesis, to the exception of the carbamate derivatives of 118 (R = O–alkyl, R1
= Me, R2 = H) that were used as a PPG for alcohols (Scheme 45).
243
Scheme 45
Use of o-Nitroanilide Carbamates as PPGs for Alcohols
243
The size of the fused
nitrogen heterocycle proved to be crucial
for the reactivity, and the 5-membered analogues of 119 (Scheme 44), such as 120 and
121 (R = alkyl), released carboxylic acids very efficiently
upon photolysis.
244
Most remarkably, when
the reaction was carried out in nucleophilic solvents, such as methanol
or ammonia-containing dichloromethane, the primary amide was obtained.
Corrie, Bochet, Toscano, and co-workers succeeded in providing a comprehensive
picture of the reaction mechanism.
245
The
mechanistic studies concluded that a photoinduced acyl migration from
the indoline nitrogen atom of 122 to one of the nitro
group oxygens occurs, leading to a highly electrophilic N–O–acyl
intermediate 123 (Scheme 46).
245a,245b
Scheme 46
Diverging
Reaction Pathways in Organic Solvents and in Water
245a,245b
Interestingly, this intermediate
further reacts via two distinct
pathways depending on the solvent. In water, deprotonation releases
a carboxylic acid, with the concomitant formation of a nitrosoindole 124 (pathway
b, Scheme 46), whereas
in moist organic solvents, the acyl moiety undergoes a nucleophilic
attack either by the solvent (water or alcohol) or by an external
nucleophile (pathway a). Variation in the substitution pattern on
the indoline aromatic ring can to a certain extent influence the ratio
of the side-products.
246
These 7-nitroindolines
release their protected function efficiently,
247
and tuning of the aromatic substituents at the C-4 and
C-5 position has an impact on their reactivity. Thus, the acetamide 120 (R = Me) releases
acetic acid with quantum yields of 0.06
and 0.12 in water and MeCN/H2O 99:1, respectively.
245a
On the other hand, the reaction of 125 was about 2.5 times more efficient than that
of 121 or 126, and 5 times more efficient than that of 127; 128 and 129 were totally
photochemically
inert, possibly due to a low-lying charge transfer excited state.
246,248
Corrie and co-workers also designed a clever combination of a nitroindoline
core and an absorbing “antenna”, which will be discussed
later (section 8.1).
249
The compounds 130(250) and 131(251) have recently
been proposed as PPGs.
The bromonitroindoline 120 was used in an early example
of peptide synthesis as a protecting group for the C-terminus.
252
The solvolysis pathway was used to give the
carboxamide by using an ammonia-containing solvent, a feature frequently
sought in peptide synthesis. A few years later, a spectacular exploitation
of this nucleophilic attack of the reactive intermediate was published
by Pass, Amit, and Patchornik, with the use of a N-nucleophilic peptide fragment (Scheme
47).
253
Otherwise sensitive glycopeptides were assembled
using this strategy.
254
Scheme 47
Peptide Coupling
Using Acylated 5-Bromo-7-Nitroindolines (Bni)
253
More recently, the
derivatives 121 were used in regular
organic synthesis for preparing amides from amines,
255
but also by using weaker nucleophiles in the formation
of thioesters from thiols
256
and esters
from alcohols.
257
The preparation of more
complex derivatives of 121 can be difficult, as the nucleophilicity
of the indoline nitrogen is greatly reduced by delocalization through
the two strong electron-withdrawing groups, but indirect routes were
designed and the synthesis of a series of amino acids was recently
published.
258
The availability of such
photoactivable amino acids allowed the all-photochemical synthesis
of peptides, by exploiting the different-wavelengths sensitivity of
the indoline protecting group on the N-terminus.
259
For other application of this chromatic orthogonality,
see section 10.
Carbamates can also
be prepared by photoinduced transfer of fluorenylmethyloxycarbonyl
(Fmoc) 132 or benzyloxycarbonyl (Cbz) 133 derivatives. So far, the Boc analogues were
significantly inferior
(Scheme 48).
260
Scheme 48
Photochemical Introduction of the Fmoc and Cbz Groups
260
Other functional groups were released from nitroindoline
derivatives;
Hassner et al. published the release of alcohols from carbamates of 120 (R = OR),
or amines from ureas of 120 (R
= NR1R2).
261
The
7-nitroindolines found the applications in neuronal studies
262
and other physiological sciences. For example,
derivatives of 125 were found to be active in releasing
glutamate or GABA in two-photon absorption mode.
249e,263
In some instances, 7-nitroindolines proved to be inferior compared
to nitrobenzyl cages.
264
In general, photoinduced
acyl transfers are relatively rare,
255c
and recent attempts to design groups with similar properties were
only moderately successful.
265
4
Coumarin-4-ylmethyl Groups
Photolysis of 7-methoxycoumarinyl-4-methyl
derivatives, which established
coumarins as a photoactivatable phosphate-releasing group, was first
demonstrated by Givens with diethyl phosphate ester 134 (Scheme 49).
266
Scheme 49
Release of Phosphates from 7-Methoxycoumarin Derivatives
266
This discovery set the stage for the development of a
new class
of (coumarin-4-yl)methyl cages (Table 9). The
attractive features of the coumarin phototriggers, such as large molar
absorption coefficients at longer wavelength, and fast release rates,
taken together with improved stability and fluorescent properties
that provide a tag for convenient monitoring of the reaction course,
have raised researchers’ awareness to this relatively new class
of PPGs.
9f,9u,267
Clever modifications
of the substituents at C6 and C7 in the coumaryl group addressed low
water solubility, typical of the first-generation 7-alkoxycoumaryl
PPG (types 135 and 136; Table 9), and also helped extend the absorption maximum of
the group
into the biologically benign range (≥350 nm). Thus, installation
of an additional alkoxy group at C6 in the structure of parent 7-alkoxycoumarinylmethanol
produced a shift of the absorption maximum by 30 nm. The 6-bromo-derivative
was designed to lower the pK
a of the 7-hydroxy
group by two units to effect a complete deprotonation at physiological
pH, thereby enhancing water solubility and causing a λmax shift of 60 nm. Introduction
of the second-generation of coumarinylmethyl
PPGs with 7-amino substituents (type 137; Table 9) further improved the spectroscopic
and photochemical
properties of the cage, moving the absorption maxima to 350–400
nm and recording the highest quantum yields (0.21–0.28) among
the analogues. The problem of water solubility, which can be an obstacle
to biological application, has been addressed by appending polar groups
such as carboxylates to the aniline moiety. Polyaromatic analogues
were also synthesized to improve fluorophoric properties of the cage
for use both as fluorescent tags and as PPGs (type 136; Table 9).
Comprehensive reviews by
Furuta,
268
Dore,
269
and Loudwig and Bayley,
270
and more recently
one by Givens, Rubina, and Wirz,
9u
encompass
much of the groundwork on the photochemistry
of coumarylmethyl PPGs with an emphasis on biological applications.
Important mechanistic aspects, synthesis, and several selected recent
applications are given below.
Table 9
Typical Coumarin Chromophores Applied
as PPGs
a
Leaving groups are in red.
b
Solvent: HEPES/MeOH (i); HEPES buffer
(ii); EtOH (iii); PB or PBS buffer (iv); KMOPS buffer (v); KMOPS/MeOH
(vi); HEPES/MeCN (vii); and water (viii).
Synthetic Approaches to Coumarin-Caged Compounds
Coumarin-4-ylmethanol 138, easily available
from the corresponding halides, is the common precursor of coumarin-caged
esters, including phosphates, carboxylates, and sulfonates as well
as carbonate, carbamate, and anhydride derivatives (Scheme 50).
9a
Carboxylic acids,
including amino acids, can also be efficiently caged via SN2 substitution of coumarinylmethyl
bromide 139. Likewise,
coumarin-4-methylamines and -thiols can be directly obtained from
bromides 139 by standard alkylation. Coumarin-caged diols,
e.g., acetals 140, were prepared in two steps from aldehyde 141, which in turn is
available from the corresponding alcohol 138 by oxidation with manganese dioxide (Scheme
50).
295
Aldehyde 141 is also a convenient precursor to 4-diazomethylcoumarin 142, which has
proven to be effective in protecting complex
phosphates, particularly cyclic adenosine nucleotides, when standard
approaches employing coumarin-4-ylmethanol 138 or coumarin-4-yl
bromide 139 fail to provide the desired product.
271a,279a
Scheme 50
Synthetic Approaches to Coumarin-Caged Compounds
Mechanism of Photorelease
and Selected Applications
A general mechanism of photorelease
of phosphates, sulfonates, and
carboxylic acids, the most successfully applied leaving groups, is
summarized in Scheme 51.
276,296
After initial light absorption, relaxation takes place to the lowest 1(π,π*) excited
singlet state, which partitions
between unproductive radiationless decay and fluorescence, and heterolytic
C–X bond cleavage. The initially formed tight ion pair (coumarinylmethyl
cation and leaving group conjugate base) is the key intermediate.
The coumarinylmethyl cation either reacts directly with adventitious
nucleophiles or solvent to generate a new stable coumarylmethyl product.
Alternatively, the tight ion pair escapes the solvent cage and reacts
with available nucleophiles. Although there is evidence of intersystem
crossing occurring with 7-aminocoumarins,
284c
there are no indications of triplet reactivity. Recombination of
the tight ion pair regenerates the ground-state caged derivative,
an additional nonproductive pathway.
Time-resolved absorption
studies have demonstrated that the heterolytic bond cleavage is very
fast with rate constants reaching 2 × 1010 s–1 (measured for phosphate esters), among
the most rapid photorelease
rates for any caged compounds.
297
However,
ion pair recombination dominates the subsequent reactions and is about
10 times faster than nucleophilic trapping of the coumarinylmethyl
cation by solvent.
276,296
Evidence in support of SN1 cleavage at carbon, vis-à-vis a photosolvolysis pathway,
was obtained using 18O-labeled water, which afforded labeled
coumarinylmethanol and label-free phosphoric acid (Scheme 51).
297
Installation of
electron-donating substituents on the coumaryl moiety and choice of
a leaving group with low pK
a values facilitate
the reaction and, at the same time, impede ion pair recombination.
276
Scheme 51
Mechanism of Photorelease of Coumarin-Caged
Compounds
297
Poor leaving groups such as alcohols, phenols, and thiols
render
(coumarin-4-yl)methyl derivatives resistant to heterolysis. Such groups
can be more efficiently released when caged through a carbonate linkage
with the (6-bromo-7-hydroxy/alkoxycoumarin-4-yl)methyl moiety 143 (X = O, S, Scheme
52). The initially
liberated carbonic or thiocarbonic acid is unstable and undergoes
decarboxylation to give free alcohol or thiol.
196,272,275,289
The rates of these decarboxylation reactions are usually quite slow, k
–CO2 = 10–3 s–1,
298
and subject
to both acid and base catalysis.
Scheme 52
Decarboxylative Photorelease of Alcohols,
Thiols, and Amines
Analogous to alcohols, release of amines from coumarin-4-ylmethyl
carbamates 143 (X = NR′′) proceeds uneventfully,
although at a slower rate (Scheme 52).
272,288,290,299
As in the case of carbonates, the rate-limiting step is decarboxylation
of the released carbamate anion, which is more strongly dependent
on the pH and on the nature of the released amine or amino acid.
62,300
This could become a limitation for applications requiring a very
rapid substrate release.
Carbamate-linked coumaryl PPG was used
by Hayashi, Kiso, and co-workers
to mask the benzylamine moiety and suppress the pharmacophore activity
in isotaxel (Scheme 53).
283
The resulting photoresponsive prodrug phototaxel (144) was selectively activated using
visible light (430 nm)
to release isotaxel (145), which converted into paclitaxel
(146) by a spontaneous intramolecular O- to N-acyl migration.
283
To improve water solubility of the phototaxel prodrug, the N-ethyl groups of the coumarin
cage were replaced with N-(2-(dimethylamino)ethyl)acetamide groups.
301
It should be mentioned that an alternative O-protected paclitaxel prodrug with a carbonate-linked
coumarin
was unstable in aqueous solutions.
Scheme 53
Activation of Paclitaxel
by Visible Light
283
Among the most significant advances in the photochemistry
of coumarin-based
protecting groups has been the discovery and development of two-photon
activation (see section 9).
9h,302
Initially reported for the two-photon release from coumarin-caged
substrates by Furuta, Dore, Tsien, and co-workers
303
this approach for decaging has developed into a major area
of research and discovery.
9a,269
Block methylmethacrylate
copolymer 147 esterified
with the (7-diethylaminocoumarin-4-yl)methyl group exhibiting a large
two-photon absorption cross section was used by Morris, Zhao, and
co-workers to study light-responsive micelle disruption (Scheme 54).
304
UV irradiation
of the micelles loaded with the hydrophobic Nile red dye revealed
nonradiative energy transfer between the coumarin chromophore and
the dye. Micelles without dye showed significant fluorescence self-quenching
due to tight packing of the coumarin groups within a confined space;
however, photorelease of the chromophore was accompanied by micelle
disruption, which caused a dramatic increase in its fluorescence.
Experiments with dye-loaded micelles placed in a dialysis cap immersed
in water showed efficient diffusion of photoreleased coumarin into
water (measured by fluorescence emission), while hydrophobic guest
dye molecules remained within the cap. Multiphoton irradiation using
near-infrared light was less disruptive for micelles yet sufficient
to release loaded dye.
304
Scheme 54
Light-Responsive
Micelle Disruption
304
An efficient multiphoton uncaging permitted
exposure of the functional
groups (primary amines and sulfides) buried within modified aragose
gel 148, creating complex 3D sites ready for selective
immobilization of biologically relevant entities without affecting
the mechanical properties of the patterned material (Scheme 55).
299,305
Scheme 55
Selective Photoactivation
of Functional Groups within Aragose Gel
299,305
A single example of photodeprotection
of a coumarinylmethylamine
through a C–N bond cleavage has been reported. This reaction
proceeded efficiently only in the presence of an excess of a hydrogen-atom
donor, such as n-decanethiol or 1,4-cyclohexadiene
(Scheme 56).
306
A
radical mechanism has been proposed that involved electron transfer
between the amine and coumaryl moieties forming the intramolecular
radical ion pair 149. Subsequent cleavage of the C–N
bond generated an aminyl radical and the resonance-stabilized coumarinylmethyl
radical 150, both of which were trapped by hydrogen-atom
donors.
Scheme 56
Uncaging of Amines via Direct C–N Bond Cleavage
306
When the released group is the conjugate base of a very
strong
acid, the caged substrate becomes a “caged proton source”
that permits the spatial and temporal control of rapid change in pH.
Thus, the release of phosphoric acid, methanesulfonic acid, or sulfuric
acid derivatives from their coumarin-caged precursors 151 has been employed as a proton
trigger in studies of proton-dependent
cellular signal transduction (Scheme 57).
307
The release occurred within 2 ns, giving a
significant drop in the pH of up to three units. It was also noted
that hydrophobic phosphate derivatives penetrate cell membranes whereas
charged sulfate was membrane-impermeant.
Scheme 57
Caged Proton Source:
Photorelease of Strong Acids
307
Photorelease of diols from
cyclic (coumarin-4-yl)methyl acetals 152 proceeds via
an ion-pair intermediate 153, which then reacts with
water to produce the hemiacetal 154. The latter decomposes
to give free diol and coumaryl aldehyde 155 (Scheme 58).
308
Interestingly,
the photodegradation of coumaryl-caged diols
was found to be dependent on the size of the cyclic acetal. Thus,
1,2- and 1,4-diols were released equally efficiently (Φ = 0.005–0.03), whereas the
corresponding 1,3-diols appeared
to be completely inert to photolysis. Although different stereoelectronic
properties of the three acetals precluded direct comparison of the
propensity toward ring-opening,
308
it is
probable that the divergent quantum yields result from lack of recyclization
because of the increasing ring strain for 5- and 7-membered acetals
(by ca. 6 kcal mol–1) compared with recyclization
to the “strainless” 6-membered analogue.
309
Scheme 58
Photorelease of Coumarin-Caged Diols
308
A complementary approach for protecting carbonyl compounds
in the
form of a photolabile cyclic coumaryl acetal has also been demonstrated
(Scheme 59).
287,291
Remarkably,
both single- and two-photon uncaging (section 9) of 4-coumarin-4-yl-1,3-dioxolanes
156 were successfully
achieved under physiological conditions. In line with the above example,
the proposed mechanism involved photoinitiated heterolysis and subsequent
solvolysis of the zwitterionic species 157.
Scheme 59
Photorelease
of Coumarin-Caged Carbonyl Compounds
287,291
Caged carbonyl compounds 158 were obtained from (coumarin-4-yl)methyl
chloride (159) by Wittig olefination followed by dihydroxylation
of the resulting alkene with OsO4 and subsequent acetalization
or ketalization under standard conditions (Scheme 60).
291
Alternatively, caged progesterone 160 was synthesized by Hagen and co-workers via
ketalization
of the commercially available pregnenolone 161, followed
by Oppenauer oxidation of intermediate 162 (Scheme 61).
310
Photoactivation
of coumaryl-caged progesterone produced 163 in only 30%
yield, which, however, was sufficient to initiate progesterone-mediated
response in sperm. Two-photon excitation of 160 (755
nm) also caused release of progesterone, albeit with ca. 5 times lower
efficiency compared with photolysis of the analogous glutamate derivative.
301
Scheme 60
Synthesis of Coumarin-Caged Carbonyl Compounds
291
Scheme 61
Photoactivation of Coumaryl-Caged Progesterone
310
5
Arylmethyl Groups
5.1
Simple
Arylmethyl Groups
A large
family of photolabile protecting groups is based on photochemically
induced hydrolysis (or less commonly alcoholysis) of benzyl or heterobenzyl
esters or ethers 164 (Scheme 62).
Scheme 62
Photochemical Cleavage of Benzyl Protection
In fact, photochemical release of glycine from
its benzyloxycarbonyl
derivative was the first application of a “photosensitive protecting
group” in organic chemistry.
1
Barltrop
and Schofield reported that irradiation of N-benzyloxycarbonylglycine 165 with 254
nm light resulted in the release of glycine in
75% yield and a quantum yield of 0.15 (Scheme 63). Photolabile protection of amino
acids and dipeptides was further
demonstrated by Chamberlin, who used the 3,5-dimethoxybenzyloxycarbonyl
protecting group (166).
311
Substrates were released in up to 85% yield upon illumination with
a high-pressure mercury lamp for 1.5 h.
Scheme 63
Uncaging of Glycine
1,311
From a mechanistic point of
view, this photosolvolysis can proceed
via initial light-induced homolysis of the C–O bond, followed
by rapid electron transfer to give an ion pair (Scheme 64). The latter is then trapped
by solvent to give the final
products. Pincock and co-workers have shown that this mechanism is
predominant in the photolysis of simple benzyl derivatives.
101
In fact, photosolvolysis of benzyl esters is
often accompanied by the formation of radical products.
312
Scheme 64
Photochemistry of Arylmethyl Ethers
Calculations by Zimmerman and
co-workers
312a,313
suggested that heterolysis of
the C–O bond is energetically
preferred over the homolysis, especially in the presence of ortho
and meta substituents stabilizing the cation. While studying the photochemical
solvolysis of benzyl acetates, Zimmerman observed the now famous “meta-effect”:
a higher efficiency of photocleavage for meta-methoxy-substituted
benzyl esters.
Structural features that help to delocalize positive
charge in
the ground state enhance the efficiency of the electron-transfer step
(Scheme 64), whereas excited-state cation-stabilizing
moieties promote the direct photoheterolysis of the C–O bond.
The efficiency of the uncaging reaction depicted in Scheme 64 is defined by the competition
between solvent
capture of the ion pair to give the liberated hydroxyl compound ROH
and recombination leading to the starting material (polar protic solvents
enhance both the heterolytic cleavage and electron transfer). Therefore,
photodeprotection of carboxylates, carbonates, carbamates, phosphates,
and other good leaving groups is usually quite efficient, whereas
uncaging of alcohols is more challenging. Enhanced stability of the
benzyl cation and increased steric hindrance around the cationic center
also help to slow down the ion recombination. Thus, the design of
new PPGs of this class was mostly focused on the incorporation of
features helping to delocalize positive charge. However, there is
a pitfall associated with this approach: a very stable cation makes
the PPG susceptible to acid-catalyzed hydrolysis. In fact, the conventional
acid-labile trityl protecting group can be removed from caged nucleosides
(167) in 71–99% yield upon 254 nm irradiation
with ca. 10% quantum efficiency (Scheme 65).
314
Scheme 65
Photocleavage of the Trityl PPG
314
Wang and co-workers systematically explored various trityl
derivatives
and found that the 3-(dimethylamino)trityl group (168, DMATr, Scheme 65) is the most
efficient
alcohol-caging group in the trityl series (Φ = 0.2).
315
Introduction of a strong electron-donating
meta-substituent in one of the trityl rings improves the photochemical
reactivity due to the meta-effect, stabilizing the PPG derivative
to hydrolysis in the dark. This PPG has a relatively long absorption
wavelength (λmax = 309 nm), is stable in acidic media,
and releases alcohols with 0.12–0.20 quantum yield efficiency.
DMATr can be easily installed by simple solvent-free heating of the
DMATr acetate with the alcohol. Several alcohols, including sugars
and thymidine, have been protected and released upon irradiation with
a medium-pressure mercury lamp in methanol with 80–90% yields.
DMATr is also orthogonal to conventional methoxytrityl protecting
groups, as it can be removed by irradiation in methanol, whereas the
methoxytrityl protection is cleaved upon treatment with 80% acetic
acid.
A similar α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl
PPG (169, Ddz, Scheme 66) was
used by Cameron and Frechet for caging of amines.
316
Irradiation of these thermally stable carbamates with a
medium-pressure mercury lamp results in liberation of free amines
both in THF solution and in the solid state.
Scheme 66
Uncaging of Amines
316
The stability of 9-phenylxanthylium cation prompted the
development
of the 9-phenylxanthyl (170, pixyl, Px) PPG for primary
alcohols (Scheme 67).
317
Scheme 67
Protection and Photochemical Release of Primary Alcohols
317
The pixyl PPG is readily introduced in 65–74% yield
by treating
alcohols with commercially available pixyl chloride in the presence
of pyridine. Irradiation of the pixyl ethers at 254 or 300 nm results
in the release of the alcohol in 78–97% yield. On the basis
of deprotection of the nucleoside thymidine, the authors suggested
that Px could be an effective protecting group for the combinatorial
synthesis of oligoribonucleotides.
The 9-phenylthioxanthyl group
(171, S-pixyl, S-Px, Scheme 68) was found to be an
even more efficient cage for primary alcohols, as it absorbs at longer
wavelengths and has a higher quantum yield of deprotection.
318
It was also shown that i-butyryl
and benzoyl protection of amino groups in nucleobases is not affected
by the photolysis. The chemical yield of deprotection varied from
75% for cytidine to 97% for thymidine.
Scheme 68
Use of the S-Pixyl PPG for Caging and Release of
Nucleosides
318
Further studies of the substituent effects on the photochemical
release of thymidine from the S-Px cage showed that an electron-donating
group in the 3-position enhances the efficiency of the photolysis
by a factor of 3.
319
Moreover, some of
these compounds proved to be suitable for deprotection under irradiation
with 350 nm light. For example, 5′-O-(3-methoxy-9-phenylthioxanthyl)-2′-deoxythymidine
(172, Scheme 68) quantitatively
releases thymidine by irradiation at 300 or 350 nm.
Direct photosolvolysis
of ethers, such as 167, 168, and 170–172, has great
advantage for alcohol caging over the use of photolabile carbonates.
Substrates are released directly from the excited state of the photocage
within a few microseconds in the former case, whereas decarboxylation
of photochemically generated monocarbonate in the latter case is a
much slower process.
Photosolvolysis of arylmethanol derivatives
has also been used
for the deprotection of carbonyl compound components of photolabile
acetals. Wang and co-workers developed a photoremovable protecting
group for carbonyl compounds based on trityl photochemistry.
320
Ketones and aldehydes are converted into cyclic
acetals (173) by treatment with 5-methoxysalicylic alcohol
derivatives (Scheme 69). Upon irradiation,
the carbonyl compounds are released in 75–90% yield and good
quantum efficiency (Φ = 0.11). The authors
proposed that this reaction proceeds via a two-step mechanism involving
a zwitterionic intermediate. However, it is also possible that initial
photocleavage of the benzylic C–O bond produces a hemiacetal,
which undergoes further hydrolysis. This cage is remarkably stable
to a variety of reagents including metalloorganic (PhLi, LiAlH4, and t-BuOK), strong
acids (AcOH, TFA, and
concentrated HCl), and mild oxidants (DDQ). Besides, Thevenet and
Neier have recently studied photorelease of alcohols from the corresponding
naphthalen-2-ylmethylidene and arylethynylmethylidene acetals.
321
Scheme 69
Photolabile Acetals for the Protection
of Ketones and Aldehydes
320
The methoxy substituent in 173 was
strategically positioned
to exploit Zimmerman’s meta-effect.
312a,313
The second methoxy group in 174 makes photoheterolysis
of benzylic C–O even more efficient, as noted earlier. A series
of carbonyl compounds were protected in the presence of p-toluenesulfonic acid
322
or in the neat
state and with no added catalysts at 140 °C
323
and then released (Φ = 0.17–0.23)
320c,320e
from the photocage 174 upon irradiation with a 450
W medium-pressure mercury lamp in aqueous acetonitrile. Both protection
and uncaging proceeded in 74–99% yields. Introduction of electron-donating
or -withdrawing substituents in the para-position of the phenyl substituents
in 174 allows for the adjustment of λmax of the chromophore.
320c
The majority
of trityl- and benzyl-based PPGs require irradiation
with light of rather short wavelength to achieve efficient substrate
release. To shift the caging chromophore absorbance to longer wavelengths,
the photocleavage of several polyaromatic analogues of the benzyl
protecting group, including (2-naphthyl)methyl,
324
(anthracen-9-yl)methyl,
325
(pyren-1-yl)methyl
(175, Scheme 70),
326
(perylen-3-yl)methyl,
327
and (phenanthren-9-yl)methyl,
326a−326c
was explored.
Scheme 70
Photocleavage of the Fluorescent (Pyren-1-yl)methyl Protecting Group
326
These cages have significant absorbance at 350 nm and
show weak
to moderate fluorescence (Φ
fl =
0.01–0.2). Polyaromatic PPGs are well-suited for release of
very good leaving groups, such as phosphates (175a).
Uncaging of carboxylic acids, on the other hand, is less reliable,
and alcohols are released with <0.005 quantum efficiency. The common
drawback of polyaromatic PPGs is their poor aqueous solubility. Addressing
this problem, Furuta and co-workers have developed the anthraquinon-2-ylmethoxycarbonyl
cage (176, Aqmoc),
324,326d
which undergoes
fairly efficient photocleavage at 350 nm (Φ
350 nm = 0.10) and has good aqueous solubility (Scheme 71). The utility of Aqmoc group
for photoremovable
protection of carbohydrates and nucleosides has been demonstrated
with 68% and 91% chemical yields, respectively. 1,3-Pentadiene efficiently
quenches the photosolvolysis reaction, suggesting that cleavage of
Aqmoc protection proceeds via the triplet state of the chromophore.
Scheme 71
Aqmoc Caging of Primary Alcohols
324,326d
A variation of the anthraquinon-2-ylmethoxy
group with a methyl
substituent in the benzylic position (177, anthraquinon-2-yleth-2-yl,
Aqe) was tested for caging of carboxylic acids (Scheme 72).
328
Scheme 72
Aqe Cage for Carboxylic
Acids
328
This chromophore has a λmax at 325 nm
and significant
absorbance at 350 nm. Irradiation of 2-(1′-hydroxyethyl)anthraquinone
carboxylates 177a–c at the latter
wavelength resulted in relatively efficient (Φ ≈ 0.12) release of the corresponding
carboxylic acids in
good yields. Uncaging of p-nitrobenzoic acid and N-acetyl-l-tryptophan proceeds with
a low quantum
(Φ < 0.01) and poor chemical yield, apparently
due to intramolecular energy transfer from anthraquinone to the caged
acids.
328
On the basis of quenching studies
and product analysis, the authors proposed a two-step mechanism for
the photocleavage of esters 177 (Scheme 73). The initial photoreduction of anthraquinone,
which proceeds
via the triplet state, produces hydroquinone 178. For
this step to work, the reaction media must contain hydrogen donors
(e.g., methanol or THF). The second photochemical step resembles the
cleavage of o-hydroxybenzyl esters (section 5.2). The (anthraquinon-2-yl)methyl chromophore
has been used in the development of photolabile acetals 179 (Scheme 74).
329
Scheme 73
Mechanism of Carboxylic Acid Release from the Aqe Cage
328
Scheme 74
(Anthraquinon-2-yl)methyl-Based Photolabile Acetals
329
The caged carbonyl compounds 179a–d were prepared in 40–60% yield by the reaction
of
anthraquinon-2-ylethyl-1′,2′-diol
(Aqe-diol, 180) with ketones or aldehydes in the presence
pyridinium p-toluenesulfonate (PPTS) and a dehydrating
agent. Acetals 179a–d are stable
in the dark in neutral solution. Upon 350-nm irradiation in MeCN/aqueous
buffer (1:1), carbonyl compounds are liberated with 0.03–0.09
quantum efficiency in 60–90% chemical yield. Under these reaction
conditions, Aqe-diol 180 undergoes further decomposition,
producing undetermined byproducts.
329
Dore and co-workers developed heteroaryl-based PPG for carboxylic
acids, (8-bromo-7-hydroxyquinoline-2-yl)methyl (BHQ), which has a
strong band with λmax= 370 nm.
330
Photodeprotection of acetic acid from the hydroxyquinoline 181a (Scheme 75) was found
to be more
efficient compared to 4,5-dimethoxy-2-nitrobenzyl (DMNB, section 3.1) and (6-bromo-7-hydroxycoumarin-4-yl)methyl
(BHCM, section 4) acetates.
331
It is noteworthy that this group can be removed under two-photon
excitation conditions
332
(see also section 9). BHQ-caged carboxylates (181b),
phosphates (181c), and diols (182) are efficiently
released under simulated physiological conditions using single-photon
and two-photon activation.
331
In addition,
a (2-phenylquinolin-4-yl)methyl group
333
and the corresponding arylmethylsulfonyl analogue
334
have been proposed as PPGs for alcohols and amines, respectively.
Scheme 75
(8-Bromo-7-hydroxyquinoline-2-yl)methyl (BHQ)-Based PPGs
331
Photochemical properties of the quinoline-based PPGs could
be further
adjusted by varying the substituent in the aromatic ring.
335
For example, replacing the bromine substituent
in BHQ with a cyano group ((8-cyano-7-hydroxyquinoline-2-yl)methyl,
CyHQ) results in a significant red-shift of the chromophore. However,
none of the (quinoline-2-yl)methyl derivatives had higher sensitivity
toward two-photon absorption than the parent BHQ group.
Another
heterocyclic analogue of benzyl PPG, (benzoxazol-2-yl)methyl,
and its derivatives were tested for photolabile protection of the
carboxyl group in amino acids.
336
N-(Phenylmethoxy)carbonyl-d
-alanine
was released in moderate to quantitative yield upon rather lengthy
irradiation at 350 nm.
5.2
o-Hydroxyarylmethyl
Groups
Introduction of the hydroxy substituent ortho to the
benzylic position
dramatically changes the mechanism of C–O bond cleavage and
enhances the overall efficiency of the process. Irradiation of o-hydroxybenzyl ethers
(183) or their naphthyl
analogues (184, (3-hydroxynaphthalen-2-yl)methyl, NQMP)
results in the quantitative release of an alcohol and the formation
of o-quinonemethide (185) (Scheme 76).
337
In the presence
of water, the latter undergoes very rapid hydration to give the parent
diol or can be trapped with vinyl ether to give photostable chroman.
Scheme 76
Mechanism of Substrate Release from the o-Hydroxybenzyl/Naphthyl
Cage
337a,337b
The release of the substrate proceeds within 12 μs
after
excitation with 0.20–0.40 quantum efficiency and in high to
quantitative yield. The quantum and chemical yields of the uncaging
reaction show little dependence on the nature of the hydroxy compound.
Thus, alcohols, phenols, and carboxylic acids caged with the (3-hydroxy-2-naphthalenyl)methyl
group (184) are released in 91–98% yield upon
300 or 350 nm irradiation (Scheme 77).
338
Scheme 77
Photochemical Release of Alcohols, Phenols,
and Carboxylic Acid from
NQMP Cage
338
The common drawback of photolabile protecting groups described
in this section is that the chromophore of the cage is usually preserved
in the photoreaction, reducing the efficiency of the photolysis at
higher concentration of caged compounds due to the internal filter
effect. The 2,5-dihydroxybenzyl (186, DHB) cage alleviates
this problem. The quinone methide 187 formed upon the
release of a substrate undergoes tautomerization into methyl-p-quinone 188 (Scheme
78).
339
UV spectra of the caged compounds 186a–d show a characteristic absorption
band at 297 nm, whereas 188 has no absorbance at this
wavelength. The compound 189 incorporates a safety-catch
feature: p-quinone precursor is photochemically inert,
but mild in situ reduction (sodium dithionite, NADH, etc.) of this
compound produces the photoreactive hydroquinone 186.
339
Scheme 78
2,5-Dihydroxybenzyl Cage Incorporating
a “Safety-Catch”
Feature
339
A variation of the DHB group was adapted for caging 1,2-
and 1,3-glycols,
as well as for photolabile benzylidene protection of carbohydrates.
Thus, glucose was released with 0.30 quantum yield and 97% chemical
yield upon irradiation of 190 (Scheme 79).
340
Scheme 79
Photolabile Benzylidene
Protection of Carbohydrates
340
o-Hydroxybenzylidene
acetals, such as 190 or 191 (Scheme 80), undergo
slow dark hydrolysis in aqueous solutions with a half-life from 2
days to several weeks, whereas p-quinone precursors
are stable both in solution and in the solid state. When hydrolytic
stability and bleaching of the caging chromophore are not important,
glycols can be efficiently caged as photolabile acetals of 5-methoxysalicyl
aldehyde (Scheme 80).
339
Scheme 80
Photolabile Protection of Glycols
339
A dihydroxybenzyl-based group
(192) was also developed
for the protection of carbonyl compounds (Scheme 81).
341
Ketones and aldehydes are
converted into 2-(1,2-dihydroxyethyl)-1,2-benzoquinone acetals 193. The latter compounds
are stable in the dark but are quantitatively
converted to the corresponding photolabile acetals 192 using mild reducing agents.
Irradiation of the latter with 300-nm
light results in the efficient photocleavage of the acetals, releasing
carbonyl compounds in 88–100% chemical yields.
341
Scheme 81
Safety-Catch Photolabile Acetal for Carbonyl
Group Protection
341
In addition, a novel bichromophoric fluorescent
photolabile protecting
group, based on (3-hydroxynaphthalene-2-yl)methyl and dansyl moieties,
was recently shown to combine the photochemical release with fluorescent
imaging by the caged substrates.
342
Falvey
and Sundararajan have used an entirely different strategy for the
photocleavage of benzyl analogues, such as 4-picolyl and N-methyl-4-picolinium esters
using photosensitizers (see section 8.2).
8b,343
In addition, visible-light
photoredox catalysts have also been used to trigger deprotection of
benzyl ethers
344
or benzyl amines.
345
The properties of the most useful arylmethyl-based
PPGs from section 5 are summarized in Table 10.
Table 10
Arylmethyl- and
Heteroarylmethyl-Based
Photoremovable Protecting Groups
PPG
protected group
λmax/nm
λirr/nm
release yield/%
Φ
ref
164 (benzyl)
amine (as a carbamate)
254
75
0.15
(1)
166 (DMB)
amine (as a carbamate)
280
263–312
85
(311)
168 (DMATr)
alcohol
309
broadband
∼85
0.12
(315)
168b
alcohol
92–100
0.23
(346)
169 (Ddz)
amines (as a carbamate)
276, 282
broadband
(316)
170 (Px)
alcohol
254 or 300
∼90
(317)
171 (S-Px)
alcohol
300 or 350
∼90
(318,319)
174
carbonyl
297
>280
92
0.17
(315)
176 (Aqm)
carboxylate, alcohol (as a carbonate), carbonyl
330
350
∼80
∼0.1
(324,326d)
181 (BHQ)
carboxylate, phosphate,
carbonyl
369
365
∼90
0.04
(330a, 331, 335)
184 (NQMP)
alcohol, phenol, carboxylate
307
300 or 350
72–100
0.1–0.3
(338)
186 (DHB)
alcohol, phenol, carboxylate, carbonyl, glycol
297
300
60–100
0.1–0.3
(339, 340)
194
carboxylate, alcohol
438
>410
94–97
0.072–0.093
(327)
6
Metal-Containing Groups
Metal-containing photoremovable protecting groups upon irradiation
have been shown to release coordinated metal ions, gaseous inorganic
molecules, such as NO or CO, or organic molecules, often of biological
interest. This innovative research topic has recently been reviewed
by Haas and Franz,
347
Schatzschneider,
9o
Ciesienski and Franz,
9t
and Schiller and his co-workers;
10b
thus,
it is not covered comprehensively here.
These cages are metal
complexes that undergo a change in the metal
coordination environment upon single-photon or multiphoton excitation.
Several types of organic ligands, such as amines, nitriles, azides,
or thioethers, have been shown to be released. Upon irradiation with
visible light above 480 nm, the most common [Ru2+(bpy)2]2+ core (195; an amine is
a leaving
group in this case) liberates various nitrogen atom-containing ligands
(Scheme 82), such as 4-aminopyridine, serotonin,
butylamine, tryptamine, tyramine,
348
γ-amino
butyric acid,
349
glutamate,
350
or azide.
351
One
of the amino group-containing ligands has also been replaced by triphenylphosphine
to increase the release quantum yield.
349b
Etchenique and collaborators have reported that ruthenium–bipyridine-based
caged nicotine ([Ru(bpy)2(nic)2]2+ where nic = nicotine) is released with the quantum
yield Φ = 0.23 upon irradiation with blue (473 nm) or green
(532 nm) light.
352
Gobetto, Sadler, and
co-workers have used spectroscopic and DFT computational methods to
study the release mechanism.
353
They showed
that the photochemical activity is related to the presence of singlet
and triplet transitions involving σ-antibonding orbitals and
identified and characterized the triplet state responsible for the
photodissociation process.
353a
Excitation
of the metal–ligand-to-ligand charge-transfer (MLLCT) band
of the [Ru(bpy)(4AP)4]2+ complex (4AP = 4-aminopyridine)
was found to provide selective photodissociation of two 4AP moieties
in two consecutive steps with quantum yields of approximately 6 ×
10–3 and 2 × 10–4, respectively.
353c
The photorelease of an aliphatic amine from
a ruthenium–bipyridine-based PPG attached to silica surfaces
has recently been shown to occur upon single-photon (460 nm) and two-photon
(900 nm) excitation (section 9).
354
A caged glutamate can also be liberated upon
single- and two-photon activation from a Ru complex.
355
In addition, a photoactivatable fluorescent probe
356
as well as caging peptidomimetic inhibitors
357
are recent examples in which a ruthenium–bipyridine-based
PPG releases a nitrile ligand. Thioether ligands, such as thioether-cholestanol
hybrid,
358
N-acetylmethionine,
359
or biotin,
359
can
also be liberated from the [Ru2+(bpy)2]2+ core upon irradiation with visible light.
Scheme 82
Photorelease
from the [Ru2+(bpy)2]2+ Cage
7
Miscellaneous
Groups
7.1
Pivaloyl Group
Initially used as
part of a photolabile linker for solid-phase synthesis, pivaloyl derivatives
such as 196 fragment by a Norrish-type I mechanism equally
well in solution, releasing carboxylic acids or alcohols (Scheme 83).
360
The carbonyl group
might seem to be a nuisance due to cross-reactivity, but these pivaloyl
esters show remarkable stability under typical synthetic conditions
(such as acids, bases, or transition-metal catalysts). The side-products
are all volatile: carbon monoxide, isobutene, and acetone, and the
quantum yield of release is quite high (Φ =
0.56). They require, however, the use of a relatively short-wavelength
source (280–340 nm), which can be problematic when dealing
with biomolecules.
Scheme 83
Photolysis of Pivaloylglycol Derivatives
360
7.2
Esters of Carboxylic Acids
The 2-benzoylbenzoic
acid moiety (197) has been utilized as a PPG for primary
and secondary alcohols, or thiols (Scheme 84).
361
This chromophore exhibits photochemical
reactivity typical for benzophenone, such as photoinitiated H-atom
or electron transfer to the excited carbonyl group. Alcohol release
is accompanied by the formation of a dimeric side-product 198 in the presence of an
H-atom donor, whereas the isobenzofuranone
derivative 199 is obtained by photoreduction with, for
example, amines.
Scheme 84
Photochemistry of 2-Benzoylbenzoic Acid Esters
361
2-Benzoylbenzoic acid derivatives were utilized as photoreversible
inhibitors of serine proteases by Jones and Porter.
362
Various esters 200 served as efficient chymotrypsin
(a digestive enzyme) inhibitors (Scheme 85).
The synthesized acyl-chymotrypsins were found to be stable to hydrolysis
from hours to months; a sharp increase of enzyme activity was then
observed upon irradiation at 366 nm: up to 80% of the preinhibition
activity was recovered. The corresponding 2-aroylbenzoic acid was
formed as a side-product; Scheme 85 shows one
of the proposed reaction mechanisms.
Scheme 85
Chymotrypsin Photorelease
362
A photofragmentation reaction of xanthenoic esters has
been shown
to give lactones of various ring sizes.
363
The initial homolytic O–C bond cleavage of the esters 201 leads to xanthene and formyl
radicals, which rearrange
to lactones 202 and 203 in moderate to low
chemical yields (10–55%; Scheme 86).
The corresponding alcohols 204 and formates 205 were isolated as side-products.
Scheme 86
Photofragmentation of Xanthenoic
Esters
363
A water-soluble visible-light absorbing (ε400 >
9000 M–1 cm–1) PPG for carboxylic
acids based on the aminonitrophenyl chromophore 206 has
been demonstrated to photorelease β-alanine (207), which then activates the inhibitory
glycine receptor in the mammalian
central nervous system (Scheme 87).
364
β-Alanine was found to be released in
5 μs with rather low quantum yields of Φ = 0.03 and 0.002, when irradiated at λ =
360 or 450 nm, respectively.
Scheme 87
Liberation of β-Alanine from the Aminonitrophenyl Chromophore
364
7.3
Arylsulfonyl Group
The photochemical
generation of acids (photoacids) is an important strategy in the development
of coating and imaging technologies as well as the synthesis of polymers.
365
In such a process, the acids are typically
photochemically produced from relatively small amounts of an excited
initiator to start a chain reaction. However, this topic is beyond
the scope of this review; here we only cite a few recent reports
366
as examples.
Arylsulfonyl ester (Scheme 88) or amide photoacids are also frequently used
as PPGs (e.g., ref (367)). For example, this strategy was utilized for dopamine release
from
a photocleavable biotin linker
368
or for
reactivation of a benzenesulfonyl-caged Zn2+ tetraazacyclododecane
complex.
369
Acid (proton) photorelease
has been used to study proton binding in the sarcoplasmic reticulum.
370
Scheme 88
Photochemistry of Arylsulfonyl Esters
7.4
Ketones:
1,5- and 1,6-Hydrogen Abstraction
A considerable focus has
been devoted to the development of photochemically
releasable volatile odoriferous compounds—fragrances. Such
applications have been the subject of several recent reviews.
11,371
Aldehydes and ketones are important classes of fragrance molecules.
Herrmann and co-workers have shown that protected carbonyl compounds
can be photoreleased from α-keto esters (208) via
the Norrish Type II photofragmentation under mild conditions in the
presence of air (Scheme 89).
372
The initially formed 1,4-biradical reacts with air oxygen
followed by the release of the aldehyde or ketone 209, carbon dioxide, and the corresponding
carboxylic acid. In the absence
of oxygen, 208 can also undergo β-cleavage to give 209 and a ketene intermediate.
Scheme 89
Photochemistry of
α-Keto Esters
372
Photochemically triggered hydrolysis of protected
volatile aldehydes
or ketones has also been demonstrated using 1,5-diketones with abstractable
γ-hydrogen
373
or 1-alkoxy-9,10-anthraquinones,
e.g., 210 (Scheme 90).
374
The reaction starts with 1,6-hydrogen atom
abstraction followed by electron transfer to form the zwitterion 211, which is trapped
by an alcohol. The final fast hydrolytic
step liberates the protected compound 212.
Scheme 90
Photochemistry
of 1-Alkoxy-9,10-anthraquinones
374
7.5
Carbanion-Mediated
Groups
Another
type of PPG relies on the photochemical formation of a carbon-centered
anion, which drives the subsequent release of a leaving group via
β-elimination (Scheme 91). This strategy,
developed by Scaiano and co-workers,
375
is often based on the photochemical decarboxylation in the side-chain
of a chromophore and is compatible with an aqueous environment. Benzophenone
(LG = carboxylate; Φ < 0.7),
375a
xanthone (LG = carboxylate; Φ < 0.7
376
),
377
and phthalimide
378
moieties have been
used as the chromophores. In this context, a stereodifferentiation
process involving the release of the caged molecule has been demonstrated
on photoinduced decarboxylation of 2-phthalimido-3-hydroxy-propionate
derivatives in which different reaction efficiencies were found for
cages with threo or erythro configurations.
379
Griesbeck and co-workers have shown that methylthiomethyl esters
of ω-phthalimido carboxylic acids can liberate the carboxylic
acids upon irradiation.
380
Scheme 91
Carbanion-Mediated
Photocleavage
7.6
Sisyl
and Other Silicon-Based Groups
Silicon-based protecting groups
are often used in organic synthesis
to protect alcohols as silyl ethers due to their predictable and selective
deprotection and tolerance to many organic reagents. A bulky sisyl
(tris(trimethylsilyl)) group has been introduced as a photocleavable
protecting group for primary and secondary alcohols.
381
It was shown that it is stable to aqueous bases and Grignard
and Wittig reagents, as well as resistant to selected fluoride salts.
Irradiation of sisyl ethers at 254 nm leads to the deprotection of
the alcohols in good chemical yields (62–95%)
382
by a radical mechanism.
383
The Si–O bond of various trialkylsilyl esters can also be
photocleaved by in situ generation of HBr from a catalytic amount
of CBr4.
384
Such an approach
has been used for the deprotection of t-butyldimethylsilyl
and β-(trimethylsilyl)ethoxy methyl ethers.
385
It was shown that the primary silyl ethers on carbohydrate
molecules can be selectively liberated in the presence of secondary
silyl ethers. Photochemical protodesilylation of 213 in
the presence of hexafluoroisopropanol (HFIP) or isopropanol (IPA)
was reported to form an alkoxy intermediate, in which the C–Si
bond is oxidized in a subsequent step to release a triol (Scheme 92).
386
Scheme 92
Photochemistry
of Trialkylsilyl Esters
386
7.7
2-Hydroxycinnamyl Groups
Porter and
co-workers have been first to utilize a 2-hydroxycinnamyl moiety (214, Scheme 93)
as a PPG in 1987, in
this particular case, for the photochemical activation of thrombin,
a serine proteinase.
387
In general, this
system undergoes initial photoinitiated isomerization followed by
cyclization, which facilitates the release of caged substrates such
as alcohols. Hiramatsu and co-workers utilized a caged reagent for
a fluorimetric assay of peroxidase that led to uniform addition of
a reagent without stirring.
388
A trihydroxycinnamyl
ester-based photocleavable detergent was applied for enhancing the
water solubility of cell proteins followed by their MALDI-MS determination.
389
The 3,5-dibromo-2,4-dihydroxycinnamic caging
group was synthesized and used for 2-photon release of a biologically
active substrate in a specific region within the cell.
390
The fluorescence emission of the photocyclization
byproduct, 6,8-dibromo-7-hydroxycoumarin, then provided the information
about the concentration of the released species. Applications of this
chemistry to release of a fluorescent reporter and the desired caged
substrate
391
or mask bioactivity of complex
enzymes, such as thrombin, factor Xa, and trypsin, have been demonstrated.
392
A small library of the o-hydroxycinnamic
derivatives was recently synthesized to study and improve one- and
two-photon release.
393
Cinnamate esters
were also successfully coupled with a CdSe nanocrystal surface.
394
The corresponding coumarins were then released
upon irradiation of the CdSe nanocrystal by visible light. This protocol
also aided the understanding of fundamental nanocrystal–ligand
interactions.
Scheme 93
Photochemistry of o-Hydroxycinnamic
Derivatives
Silyl analogues
of 2-hydroxycinnamyl derivatives (215) as PPGs have been
introduced by Pirrung and co-workers (Scheme 94).
395
A clean and high-yield
(<92%) deprotection of primary and secondary alcohols was reported.
Scheme 94
Photochemistry of Silyl Analogues of 2-Hydroxycinnamyl Derivatives
395
7.8
α-Keto Amides, α,β-Unsaturated
Anilides, and Methyl(phenyl)thiocarbamic Acid
Steinmetz and
co-workers reported that the carboxylates (including GABA, BocAla,
and Glu) attached to the α-carbon of α-ketoamides (216) could be photochemically released,
possibly through a
zwitterionic intermediate 217, in very good chemical
(<93%) and quantum (0.28–0.37) yields, along with the formation
of a mixture of two diastereomeric hemiacetals 218 and
a small amount of oxazolidinone 219 as byproducts (Scheme 95).
396
The yield of 219 was found to depend strongly on the type of alkyl substituent
on the carbon adjacent to the amide nitrogen.
397
Time-resolved pH-jump experiments showed that the reaction
rate is on the microsecond time scale and that carboxylate release
occurs in the rate-determining step.
398
Phenolates can also be liberated from 216.
399
Laser flash photolysis experiments demonstrated
that p-substituted phenolic substituents undergo
photocleavage to give the corresponding phenol with good quantum yields
(0.2–0.3).
400
The authors proposed
a mechanism that involves H-atom transfer from an N-alkyl group to the carbonyl to
produce a zwitterionic intermediate
that eliminates the phenolate.
Scheme 95
α-Ketoamides as PPGs
396
α,β-Unsaturated anilides (220) have been
utilized as PPGs for carboxylates or phenolates (Scheme 96).
401
The chemical (<71%)
and quantum (<0.083) yields of LG release were reported. A zwitterionic
intermediate analogous to 217, responsible for LG extrusion,
is assumed to form.
Scheme 96
α,β-Unsaturated Anilides as
PPGs
401
The cysteinyl radical was shown to be photochemically
released
from the methyl(phenyl)thiocarbamic acid chromophore 221 (Scheme 97).
402
The S–S bond cleavage and the cysteinyl radicals’
recombination was studied by time-resolved IR spectroscopic techniques.
Scheme 97
Photochemistry of a Methyl(phenyl)thiocarbamic Acid Chromophore
402
7.9
Thiochromone S,S-Dioxide
Kakiuchi and co-workers recently introduced a novel
PPG based on the thiochromone S,S-dioxide chromophore 222 (Scheme 98).
403
The alcohols (as carbonates), amines
(as carbamates), and carboxylic acids were released in excellent chemical
yields (up to 99%) upon irradiation at λ = 280 nm in methanol.
The reaction progress was monitored by fluorescence spectroscopy.
Scheme 98
Photochemistry of a Thiochromone S,S-Dioxide Derivative
403
7.10
2-Pyrrolidino-1,4-Benzoquinone
Group
Chen and Steinmetz have demonstrated that 2-pyrrolidino-1,4-benzoquinone 223 gives,
upon irradiation with visible light (below ∼700
nm), an unstable benzoxazoline photoproduct, which expels a leaving
group, such as carboxylate or phenolate, in a subsequent dark elimination
reaction (Scheme 99).
404
The reaction proceeds with quantitative chemical but modest quantum
(Φ = 0.03–0.10) yields.
Scheme 99
Photochemistry
of 2-Pyrrolidino-1,4-Benzoquinone Derivatives
404
7.11
Triazine
and Arylmethyleneimino Groups
A triazine moiety can be used
as a (thermally) labile linker in
solid-phase synthesis.
405
Its applicability
as a photocleavable linker has also been reported.
406
Laser irradiation of 224 at 355 nm leads to
liberation of the protected amines or amino acid derivatives in moderate
to high chemical yields (77–100%; Scheme 100). A triazine linker has also been used
as part of a homopolymer
backbone to yield photoinduced backbone fragmentation.
407
Scheme 100
Triazine Moiety as a Photolabile Linker
406
It has been shown that the naphthylmethyleneimino group
releases
aliphatic or aromatic primary amines as well as α-amino acids
(NH2R) in good to excellent chemical yields (51–96%)
upon irradiation at 280 or 340 nm. The reaction proceeds via a homolytic
cleavage of the N–O(CO) bond. Such strategies are similar to
those utilized in the field of photobase generation, which is not
covered by this review.
12a
7.12
Xanthene and Pyronin Groups
Wirz
and co-workers have recently demonstrated that the (6-hydroxy-3-oxo-3H-xanthen-9-yl)methyl
derivatives 225 release
diethyl phosphate or carboxylic acid upon irradiation with visible
light (over 500 nm) with quantum yields of 0.005–0.04 (Scheme 101).
408
This reaction
is the subject of further investigation.
Scheme 101
Photorelease from
the Xanthene Derivatives
408
A novel class of pyronin analogues 226, which undergo
a photochemical cleavage of the C–C bond in the presence of
water both in solution and on a silica gel surface upon direct irradiation
with yellow light (Scheme 102), has been reported
by Klán and co-workers.
409
The final
chromophoric photoproduct was shown to be a stable compound absorbing
below 430 nm. The course of the reaction was monitored by the characteristic
fluorescence emissions of both the starting compound (λmax = 607 nm) and the final
product (λmax =
448 nm). As one of the rare examples of visible-light triggered caged
systems, it was suggested that the moiety could be used in the field
of photoremovable protecting groups or caged fluorophores (section 11).
Scheme 102
Photochemistry of Pyronine
409
7.13
Retro-Cycloaddition Reactions
A
photocleavable linker based on 7-hydroxy-1,1-dimethylnaphthalenone
(227) for drug attachment to a polymer support has recently
been introduced.
410
It undergoes a photochemical
[2 + 2]-cycloaddition with 5-fluoro-1-heptanoyl uracil (228), a well-known cytotoxic
agent derivative, to form a heterodimer
(229; Scheme 103). The linker–drug
conjugate is then photochemically polymerized at 470 nm with methyl
methacrylate (230), yielding a (hydroxyethyl)methacrylate
(HEMA)/methyl methacrylate (MMA) copolymer. Photoinduced release of 228 from the polymer
via single- (Φ = 0.01 at λ = 331 nm) or two-photon absorption was observed.
Scheme 103
[2 + 2]-Photocycloaddition of 7-Hydroxy-1,1-dimethylnaphthalenone
410
A different strategy—photosensitizer drug delivery
via an
optical fiber—has recently been designed by Greer and co-workers.
411
A singlet oxygen sensitizer (pheophorbide)
bound to a porous silica cap of a hollow optical fiber via an alkene
spacer (231) was shown to be released in an oxygen stream
(Scheme 104). A (Z)-enol ether
bridge of 231 reacts with 1O2 to
give the dioxetane intermediate 232, which cleaves to
liberate the photosensitizer at the probe tip in the proximity of
a tumor cell. Such a sensitizer delivery has the potential for photodynamic
therapy. In a different study, Wilson and co-workers described the
photorelease of o-quinones from pyrene dihydrodioxin
(233, Scheme 105).
412
Scheme 104
Photosensitizer Drug Delivery (a: Optical
Fiber Equipped with Porous
Vycor Glass)
411
Scheme 105
Photorelease of o-Quinones from Pyrene
Dihydrodioxin
412a
Utilization of a dithienylethene photochemical switch
413
for the thermal release of an electron-deficient
alkene has been reported.
414
The thermally
stable derivative 234 undergoes photochemically reversible
ring-opening reaction to give 235, which liberates ethylene
via reverse Diels–Alder reaction (Scheme 106). The reaction can be controlled by selection
of the irradiation
wavelength.
Scheme 106
Sequential Photorelease Using a Molecular Switch
414
Triggering of a retro Diels–Alder reaction through
a photothermal
effect was demonstrated by Gates and co-workers for the first time.
415
A nanoparticle attached to the caged compound
through a linker absorbs the incident light at a specific frequency
(of a plasmon resonance) and converts it into heat, which then promotes
the chemical reaction to liberate the attached chemical species.
415
An alternative approach that utilizes
coumarin dimers as photocleavable
linkers between the substrate and the polymeric carrier was investigated
by Hampp and co-workers.
416
The core dimeric
coumarin cage 236 was constructed by [2 + 2]-photocycloaddition
(Scheme 107). Deprotection of a t-butyldimethylsilyl (TBS) group, followed by esterification,
was
used to successively install a polymer tether (237) and
chlorambucil (238).
416b
Scheme 107
Immobilization of Chloroambucil on Polymer Support via a Photolabile
Linker
416
This type of caging presumes linking through the 7-hydroxy
group
of one coumarin moiety to the active substance while the other coumarin
is attached to a polymer support. The cleavage of the dimer occurs
via [2 + 2]-cycloreversion, which can be triggered both by single-
and two-photon absorption (Scheme 108).
417
Scheme 108
Photorelease via [2 + 2]-Cycloreversion
417
8
Sensitized Release
In most cases, a
PPG is the chromophore that is responsible for
both the light absorption and the primary photochemical step, resulting
in a specific bond-breaking process. There have been more or less
successful attempts to modify the structure of a PPG in order to enhance
its absorption properties and chemical reactivity. Recent research
efforts have focused on the development of the systems composed of
two, separated or linked, molecular components, in which the photorelease
occurs from the moiety that is indirectly activated by energy transfer
from or electron transfer from/to the excited auxiliary chromophore
(photosensitizer).
27
Such a sensitizer
moiety should absorb strongly in the region of interest.
8.1
Sensitized Release: Photoinduced Energy Transfer
Energy
transfer is a process by which the excitation energy of
an excited molecule (donor; sensitizer, S) is transferred (k
ET) to a neighboring molecule (acceptor; quencher,
Q, Scheme 109).
27
Photosensitization via energy transfer is one of the most common
and practical ways to generate excited states, particularly triplet
states, especially when direct excitation at a desired wavelength
cannot be achieved or when it does not lead to the desired excited
state. For efficient triplet–triplet transfer, the process
should be exergonic to prevent reverse transfer (k
d); the donor molecule has to undergo efficient intersystem
crossing and have a high molar absorption coefficient at the irradiation
wavelength as well as a sufficiently long triplet lifetime to enable
quantitative energy transfer.
27,418
If the acceptor molecule
is a PPG, the reaction (k
r) then leads
to release of a leaving group. Bimolecular sensitization is achieved
through diffusive encounters. When the donor is covalently bound to
the acceptor molecule, intramolecular energy transfer enables a more
specific control of the transfer;
419
in
such a case, only “equimolar” amounts of a sensitizer
are needed. This strategy, benefiting from an increased “uncaging
cross section” (Φ
relε(λirr); see ref (22) for discussion) of release using triplet sensitization, has
already
been demonstrated on several examples.
Scheme 109
Photoinduced Energy
Transfer (Blue Color Depicts an Electronic Excitation)
In 2004, Steiner, Green, and co-workers demonstrated
that triplet
sensitization of a known photoremovable protecting 2-(2-nitrophenyl)propyl
moiety (239, Scheme 110; see also
section 3.2) in homogeneous solutions or
on glass substrates (microarray chips) enhances its uncaging cross
section.
418
The leaving group (alcohol),
attached as a carbonate, is liberated in the course of the reaction
as a carbonate monoester that disintegrates thermally into the corresponding
alcohol and CO2. Acridin-9(10H)-one (240) and 9H-thioxanthen-9-one (241) were used
as sensitizers (sens) that were excited by both continuous
illumination and laser flash photolysis to study the reaction mechanism.
Transient absorption observed at ∼400 nm was assigned to the aci-nitro intermediate
242 (see section 3.1). The sensitization kinetics of this bimolecular
process were found to be nearly diffusion-controlled both in solution
(k
q = 1–4 × 109 M–1 s–1; in acetonitrile or
methanol) and on the chip. Four photolabile phosphoramidites were
synthesized for testing high-density oligonucleotide microarrays;
the average chemical yield for release of the bases by using 241 was 97%. The kinetic
analysis of the photosensitized cleavage
reaction of surface-bound photolabile chromophores with free diffusion
of sensitizer molecules from the bulk of a solution to the surface
has recently been reported.
167
Scheme 110
Intermolecular
Triplet Sensitization of the 2-(2-Nitrophenyl)propyl
Chromophore
418
In addition, Pirrung, Dore, and co-workers have recently
demonstrated
that, while the 2-(2-nitrophenyl)propyl group has a low sensitivity
to two-photon excitation (see also section 9), the presence of a sensitizer with a
large two-photon absorption
cross section, such as 241, in the solution improves
it considerably.
234
Steiner and co-workers
have also designed and studied covalently linked 9H-thioxanthen-9-one (the sensitizer)
and 2-(2-nitrophenyl)propoxycarbonyl
chromophores.
166,233,240b,240g
Scheme 111 shows an example of such a compound (243), in which
the sensitizer is attached to the chromophore via a saturated four-bond
aliphatic tether. After the sensitizer is excited and the intersystem
crosses to the triplet state, the energy is transferred through space
to the 2-(2-nitrophenyl)propyl group, which liberates thymidine.
Scheme 111
Intramolecular Triplet Sensitization of the 2-(2-Nitrophenyl)propyl
Chromophore
A variety of other
covalently linked 9H-thioxanthen-9-one
derivatives, including 244 (Scheme 111), in which the sensitizer is directly attached
to the photoactive
chromophore, were also prepared and studied to compare their photophysical
and photochemical properties using stationary fluorescence and nanosecond
and femtosecond time-resolved laser spectroscopy.
166
The authors proposed a dual mechanism of triplet–triplet
energy transfer. It was suggested that a slower transfer involves
the lowest triplet (T1; π,π*) state of 9H-thioxanthen-9-one in the case of long-tether
bichromophores.
When bichromophores are connected through a short linker, the energy
is believed to be transferred from the T2 (n,π*) state. These protecting groups were
tested under conditions
for lithographic DNA-chip synthesis.
233
Their speed of release was found to be 10 times higher than that
of the nonmodified 2-(2-nitrophenyl)propyl moiety.
Corrie and
co-workers have designed the benzophenone antenna-sensitized
1-acyl-7-nitroindolines 245,
249a−249d
which display a significantly enhanced extent of photochemical cleavage
in solution compared to their nonsensitized analogues
247,248
(Scheme 112; see also section 3.3). The 4,4′-dialkoxybenzophenone chromophore
(sens), which has substantially higher molar absorption coefficients
in the region 270–320 nm than those of the nitroindoline moiety,
249d
serves as a light-harvesting antenna that
transfers its triplet energy to form the reactive triplet state of
the acceptor (PPG). It subsequently undergoes rapid photocleavage
to liberate the 1-acyl substituent as the carboxylate in aqueous media.
The triplet energy of 4,4′-dialkoxybenzophenone (E
T = 70 kcal mol–1) is about 10 kcal
mol–1 higher than the estimated E
T of nitroindoline, which is one of the key conditions
for efficient transfer.
249d
The authors
then evaluated biological effects of released l-glutamate
from 245 (R = CH2CH2CH(NH3
+)COO–) using hippocampal neurons in
primary culture and cerebellar granule cells in an acute brain slice
preparation.
249c
The data suggested that
glutamate was completely released in the irradiated samples. Recently,
a more water-soluble analogue of 245 was synthesized
and tested.
249a
Scheme 112
Antenna-Sensitized
1-Acyl-7-nitroindoline System
247,248
8.2
Sensitized Release: Photoinduced
Electron
Transfer
The release of a leaving group can also be induced
by photoinduced electron transfer (PET). In general, the excited state
of a sensitizer (S) is quenched due to electron transfer to a quencher
(Q; k
eT; Scheme 113). S and Q can be either separated or interconnected via a tether.
As a result, a radical ion pair is formed provided that both S and
Q are neutral prior the reaction. The radical ion pair can undergo
a chemical reaction
420
to give the products
(here, the leaving group is released from one of the radical ions, k
r) or a reverse electron transfer to regenerate
S and Q (k
–eT). This represents
an important fork in the reaction sequence that determines the overall
release quantum yields. The strategy allows the light-absorption step
to be studied separately from the release step; therefore, both processes
can be independently optimized. In principle, two major mechanistic
strategies can be designed because either of the S or Q moieties can
represent a PPG bearing the leaving group. As a result, the PPG can
either be oxidized by loss of an electron or reduced by accepting
an electron in a direct (the protecting group is a chromophore) or
sensitized manner. Obviously, only the latter method has the benefits
of a tunable chromophore. Some of the PET-based PPGs and their applications
have been reviewed by Falvey and Sundararajan.
8b
Scheme 113
Photoinduced Electron Transfer (Blue Color Depicts
a Sensitizer)
The Gibbs free energy
of photoinduced electron transfer ΔeT
G° in an excited encounter complex
(D··A)* can be calculated from eq 1, where E°(D•+/D) is the
standard electrode potential of the donor radical cation, E°(A/A•–) is that of the
acceptor
A, ΔE
0–0 is the 0–0′
excitation energy of the corresponding excited molecule (D* or A*),
and w are the electrostatic work terms.
27,421
1
Photofragmentation
of tosylamides (246) in the presence
of an electron donor (sensitizer), such as 1,4-dimethoxybenzene, and
a reducing agent, such as ascorbic acid or sodium borohydride, resulting
in the release of a free amine, was demonstrated by Yonemitsu and
co-workers (Scheme 114) as the first example
of a PET-based deprotection.
422
The reductant
acts as the source of the hydrogen atom and an electron (thus restoring
the sensitizer). The release of simple amines occurs in high chemical
(75–91%) and quantum (<0.65) yields. A covalently linked
sensitizer approach was also explored.
423
Scheme 114
Photosensitized Fragmentation of Tosylamides
422
More recently, Corrie
and Papageorgiou reported on the photochemistry
of sulfonamide derivatives of amino acids.
424
Glycine was released from the parent compound in rather low chemical
yields unless a large excess of ascorbate was present in the solution.
The best yields for the amino acid derivatives were found to be below
30%.
425
The concurrent decarboxylation,
triggered by intramolecular hydrogen-atom or electron transfer from
the peptide bond, was believed to represent the major competing reaction.
426
The tosyl group has also been used as a protecting
group for near-quantitative thymidine liberation in the presence of
1,5-dimethoxynaphthalene as an electron donor in the synthesis of
5′-amino analogues of 3′-azido-3′-deoxythymidine
(AZT).
427
In contrast, methanesulfonyl
and pentafluorosulfonyl esters were shown to be less suitable PET-based
protection moieties.
428
In addition
to their intrinsic photoreactivity, phenacyl derivatives
(section 2.1) can also release the leaving
groups from the α-position upon one-electron reduction to the
corresponding phenacyl anion radicals (Scheme 115). The carboxylates, for example,
are liberated with a rate constant
of ∼108 s–1.
429
Falvey and co-workers demonstrated that these leaving groups
are readily released from phenacyl esters (247) upon
photosensitized electron reduction by light-absorbing amines (sens),
provided they possess excited-state oxidation potentials below or
equal to −2.2 V.
430
Quantitative
yields were reported for various derivatives. This approach was later
extended for sensitizers that absorb at wavelengths in the near-UV
region (<400 nm).
430b
Similarly, irradiation
of phenacyl esters in micelles,
57a
or 1-oxoindan-2-yl
and 1,3-dioxoindan-2-yl esters in acetonitrile solutions,
60
was shown to liberate the carboxylic acids in
high chemical yields. The release of other leaving groups has also
been examined. A high-yielding (68–100%) liberation of phosphates,
diacids, and alcohols (protected via carbonate linker) was reported.
431
Scheme 115
Photosensitized Release of Carboxylic
Acids from Phenacyl Esters
430
The electron-donor sensitizers
can also be covalently attached
to the phenacyl moiety. Laser flash photolysis studies revealed that
a charge-transfer state (τ ≈ 500 ns) between N,N-dimethylaniline and the phenacyl
chromophores
of 248 is formed upon irradiation (Scheme 116).
343a
The phenacyl
anion radical subsequently releases the leaving group (carboxylate)
or regenerates the ground state of the parent compound.
Scheme 116
Bimolecular
Sensitization of Phenacyl Esters
343a
Falvey and co-workers demonstrated
that the 4-pyridylmethyl (picolyl)
group, previously used as a conventional protecting group for carboxylates
in peptide synthesis, could be photochemically reduced in the presence
of N,N,N′,N′-tetramethylbenzidine.
343b
The low reduction potential of the picolyl group (E[pyridine/pyridine–•]= −2.62 V
vs
SCE)
38
precludes this group from being
reduced by most sensitizers; therefore, alkylation on the nitrogen
(249, Scheme 117) was chosen to
increase its reduction potential. Irradiation of a perchlorate salt
of 249, in the presence of, for example, the carbazole 250 as an electron donor, liberates
a leaving group, such
as a carboxylate.
343b
The addition of 1,4-cyclohexadiene
as a hydrogen atom donor (to suppress back electron transfer) was
found to result in increased chemical (86%) and quantum (0.39) yields
of carboxylic acid release. The N-methylpicolinium
esters can also serve as protecting moieties in the absence of any
external photosensitizer when the iodide counterion (an electron donor)
is exchanged for perchlorate.
343b
Scheme 117
4-Pyridylmethyl
Group
343b
Deprotection of carboxylic acids, amino acids, and phosphates
from 249 in the presence of visible-light absorbing photosensitizers
and 1,4-cyclohexadiene has been reported to take place in high chemical
yields (87–100%).
432
The pyrromethene
photosensitizers (PM 546 and 597) employed in this work are dyes originally
developed for use in lasers. Mediated electron transfer with benzophenone
or xanthone tethered to the picolinium moiety in the presence of a
photosensitizer was used to increase the quantum yield of carboxylic
or amino acids release to Φ = 0.72 at λ
= 380 (Scheme 118).
343c,433
Gold nanoparticles absorbing the visible light (>500 nm) were
shown
to mediate electron transfer between dithiothreitol, a good electron
donor, and an N-methylpicolinium ester in aqueous
solution with quantum yields in the range between 0.5 and 4.5, suggesting
involvement of a radical chain mechanism.
434
Ketocoumarin dyes were also used in a similar application.
435
Tris(bipyridyl)ruthenium(II) as both a photosensitizer
and a mediator for electron transfer between a good electron donor
and the newly synthesized water-soluble 2-cyanopicolinium protecting
group was recently demonstrated to result in the release of various
carboxylic acids.
436
Scheme 118
Photorelease via
Mediated Electron Transfer
Falvey and co-workers then exercised the same principle
to control
the viscosity of aqueous solutions of 251 and cetyl trimethylammonium
bromide by using visible light to initiate sensitized photorelease
from the picolinium group and UV light to control photoisomerization
of the attached cinnamic acid (Rubpy = tris(bipyridyl)ruthenium(II)
as a sensitizer; Scheme 119).
437
The irradiation protocols are thus conveniently orthogonal
to one another.
Scheme 119
Orthogonal Photorelease and Photoisomerization
437
Visible-light-absorbing tris(bipyridyl)ruthenium(II) has
also been
recently used to mediate electron transfer to N-methylpicolinium
carbamates (252) to give free amines in several steps
(Scheme 120).
438
Another application of a visible-light photoredox catalyst, such
as Ir[dF(CF3)ppy]2(dtbbpy)PF6, in
the oxidation of electron-rich arenes resulting in the selective deprotection
of p-methoxybenzyl ethers in 69–91% yields
has been demonstrated by Stephenson and collaborators.
344a
2,3-Dichloro-5,6-dicyano-p-benzoquinone as an electron acceptor has been used for
deprotection
of various benzyl ethers by Toshima and collaborators.
344b
In addition, Lechner and König have
shown that flavin photocatalysis can be used as visible-light-absorbing
sensitizers in the deprotection of benzyl amines.
345
Scheme 120
Visible-Light Deprotection of N-Methylpicolinium
Carbamates
438
Benzyl ethers can release alcohols through oxidatively
sensitized
irradiation;
439
however, no additional
reports have been published in the past decade. In a different strategy,
Tu and Floreancig demonstrated the protection of carbonyl and carboxyl
compounds, which can be released by intramolecular photoinduced electron
transfer from a phenethyl alcohol/ether group to covalently tethered
anthraquinone (as a sensitizer) in 253 according to Scheme 121.
440
The reported chemical
yields of the deprotection were between 6 and 97%.
Scheme 121
Intramolecular
Sensitization of the Phenethyl Alcohol/Ether Group
440
Cossy and Rakotoarisoa
have discovered that the 2-acetoxyethyl
group can be used to protect secondary amines.
441
The deprotection is facilitated by photoinduced electron
transfer from the acetoxyethyl derivative 254 to the
dimethoxybenzophenone sensitizer 255 via a cation radical
intermediate and the iminium salt 256, which subsequently
hydrolyzes to release the secondary amine 257 in 60–80%
chemical yields (Scheme 122).
Scheme 122
Sensitization
of Acetoxyethyl Derivatives
441
A blue-light triggered photorelease
system based on the riboflavin-binding
protein dodecin (a dodecameric hollow-spherical flavoprotein with
six flavin binding sites) has recently been developed by Noll and
co-workers.
442
The release of the reduced
flavin derivatives is reported to be triggered by irradiation with
blue light of the dodecin-bound flavin anchor.
Kutateladze and
co-workers have developed a novel strategy of aldehyde
and ketone (258) protection using the dithiane moiety,
which can be efficiently removed via oxidative photoinduced electron
transfer in the presence of a sensitizer (sens), such as benzophenone
(Scheme 123).
443
The starting compounds 259 are easily synthesized from
the lithium salts of 1,3-dithianes and the corresponding carbonyl
compounds. Upon irradiation, the carbonyl compounds are released in
excellent to mediocre chemical yields (e.g., 35–97%)
443
and with reasonable efficiency (Φ = 0.06–0.17).
444
Scheme 123
Photochemistry
of Dithiane Moiety
443
A simplified mechanism of the oxidative deprotection
is shown in
Scheme 124. The reaction of 260 is facilitated by electron transfer to the excited
state of benzophenone,
resulting in formation of the cation radical 261 and
the benzophenone radical anion 262. Depending on the
solvent polarity, 262 deprotonates 261 in
the solvent cage, or both species escape the solvent cage and form
the same compounds through the reaction with water. It was evident
from LFP studies that in-cage deprotonation occurs in dry acetonitrile
whereas the latter process is dominant in more polar acetonitrile/water
mixtures.
445
Deprotonation of the radical
cation of an adduct of dithiane and, for example, a t-butyl-substituted carbonyl compound
gives the zwitterionic intermediate 263, which may, following intramolecular electron
transfer,
lead to an alternative C–C bond scission to give a stable t-butyl radical.
446
However,
the detailed mechanism is still not completely clear.
447
Adducts of trithianes and trithiabicyclo[2.2.2]octanes,
analogous to those of 1,3-dithianes, have also been studied as protection
moieties.
448
In addition, the dithiane
moiety was successfully coupled with amino acids to form novel dithiazane
photocleavable linkers.
449
9H-Thioxanthen-9-one has also been introduced as a sensitizer in the
dithiane-, trithiane-, and dithiazane-based photolabile scaffolds.
450
Scheme 124
Mechanism of Oxidative Deprotection of
Dithiane Moiety
445,446
The dithiane-based linkers were explored for various applications,
such as photolabile phospholipids and amphiphiles,
451
calixarene-based rosette,
452
or barbiturate receptors.
453
Dithiane-spiro-crown
ethers (e.g., 264; Scheme 125)
were used as photolabile moieties, which allowed photochemical interruption
of transport through liquid membranes,
448a
or as photolabile linkers.
454
Scheme 125
Photochemistry
of Dithiane-spiro-crown Ethers
448a
Triggering of two-photon fluorescence
(see also section 9) as a reporting function
has been developed.
455
Kutateladze and
co-workers used dithianes to
mask the sensitizer molecules immobilized on beads, dendrimers, or
peptides to demonstrate that excitation of a single free sensitizer
leads to liberation of its own copy, thus leading to signal amplification.
456
Events, which occur on an attomolar concentration
scale, can now be detected via molecular recognition-triggered photoamplified
fluorescence quenching.
457
Dithiane-based
photolabile tags have also been used for encoding and direct screening
of solution-phase combinatorial libraries.
458
Dalko and co-workers have recently reported on a very interesting
bichromophoric system composed from hydroxymethyl quinolene-derived
probes, which are activated through interactions of X-ray or γ-irradiation
with gadolinium(III) complexes (Scheme 126).
459
These metal complexes act as intramolecular
antennae and convert part of the energy resulting in electron transfer
and subsequent fragmentation of the quinolines.
Scheme 126
Photo- and Radiolysis
of Caged Hydroxymethyl Quinolone Derivatives
459
8.3
Sensitized
Release: Light Upconverting Nanoparticles
In contrast to
multiphoton excitation, which comprises the simultaneous
absorption of two or more photons using pulses with femtosecond duration
(section 9), photon upconversion is a sequential
absorption of two or more photons.
460
Light
upconverting nanoparticles (UCNPs) have attracted considerable attention
as visible or near-IR (NIR) harvesting light antennae that, upon energy
or heat transfer (photothermal effect), can trigger a release of species,
such as drugs.
461
Branda and co-workers
used a dimethoxybenzoin PPG coupled to monodispersed core–shell
nanoparticles (NPs) composed of NaYF4 nanocrystals doped
with lanthanides to release carboxylates upon NIR light activation
(980 nm) followed by NP fluorescence, exciting the benzoin chromophore.
223
The wavelength of emission may be controlled
by the power density of lasers used for NPs excitation.
462
The same principle has also been demonstrated
on photoswitches.
463
Remotely triggered
release was achieved by heat transfer from gold
nanoparticles, which can be tethered to, encapsulated within, or suspended
freely outside liposomes or micelles that can serve as drug carriers.
222,464
In a similar study, gold NPs were enclosed in biodegradable and
biocompatible microspheres (1–15 μm) containing the antitumor
drug paclitaxel.
465
The NIR-activated photothermal
effect leads to efficient paclitaxel release.
A variety of target
compounds, such as oligonucleotides or si-RNA, can
also be adsorbed on the modified surface of
a gold NP
466
or coupled to the NP surface
via gold–thiol conjugation.
467
For
example, a high payload of doxorubicin was coated and successfully
released from both the outer and inner shells of polyethylene glycol
(PEG) hollow gold nanoparticles.
468
Chromatic
orthogonality (see also section 10) to release
two different DNA oligonucleotides has been demostrated on gold nanorods
differing in their aspect ratio,
469
which
is promising for future applications in cancer theranostics.
470
9
Two-Photon Excitation-Induced
Photorelease
The removal of the photolabile protecting groups
discussed in this
review usually requires UV irradiation. These conditions, however,
are not compatible with many biomedical applications, as UV–vis
light is efficiently absorbed by the tissue.
471
The major absorbing species in vertebrate tissues is oxyhemoglobin,
which filters practically all the light of wavelengths shorter than
650 nm.
471
On the other hand, water becomes
increasingly absorbant at wavelengths longer than 950 nm. These two
factors define the “phototherapeutic window”, the region
of relative tissue transparency between 650 and 950 nm.
472
The increased depth of tissue penetration of
radiation in this region is accompanied by additional advantages:
lower scattering and reduced phototoxic effects. However, red and
NIR photons have relatively low energy, limiting the range of processes
they can initiate. In addition, the majority of chromophores with
useful single-photon absorbance in the “phototherapeutic window”
are also sensitive to visible light, complicating the handling of
the caged substrates. One of the approaches that overcomes most of
these problems is to use nonresonant two-photon excitation (2PE).
At high light intensity, chromophores may simultaneously absorb two
red/NIR photons, producing higher-energy excited states, the same
as or similar to those accessible by direct excitation with UV photons
of about twice the frequency.
473
Additionally,
focusing the irradiation of 2PE on UV chromophores within UV-absorbing
materials provides an opportunity to control substrate release in
three dimensions. The probability of 2PE is proportional to radiant
intensity squared, which is many orders of magnitude higher in the
λ3 volume around the focal point than in other areas
of the laser beam.
474
This phenomenon has
made possible the development of 3-D applications in fluorescent imaging,
473a,474b,475
microscopy,
474b,476
photonics,
477
3-D fabrication,
478
and potentially even drug delivery
304,479
and photodynamic therapy.
480
Substantially
less data are available on two-photon induced photochemical reactions
473c,481
and their use in biochemistry.
164,476b,482
The 3-D capabilities of two-photon-induced uncaging
were successfully used in neuron mapping,
10d,483
investigation of intracellular processes,
484
and regulation of protein activity in vitro
221,485
and in vivo,
221,485b
as well as in the development
of 2PE-uncaging microscopy.
486
2PE
requires very high light intensities, which can be achieved
only using ultrafast pulsed lasers. Thus, a commercially available
mode-locked Ti:sapphire laser can provide a photon irradiance (E
p) of 1025 photons cm–2 s–1 (3 mm beam at 100 fs pulse duration, and repetition
frequency ν = 90 MHz) or more. The principal emission of the
most common Ti:sapphire lasers is at 800 nm, which almost perfectly
corresponds to the minimum of mammalian tissues absorbance. Lasers
that can be tuned to shorter wavelengths are usually more expensive.
The differential form of the Beer’s law for two-photon excitation
487
is shown below (eq 2),
where E
p is the photon irradiance (photons
cm–2 s–1), δ is the two-photon
cross section (cm4 s photon–1 molecule–1), N is the concentration (molecules
cm–3), and x is the sample thickness
(cm).
2
3
As light intensity virtually
does not change when passing through
the sample due to low two-photon absorption, we can write eq 2 in a simplified form
(eq 3). The rate of photoreaction with quantum yield Φ (molecule photon–1) is proportional
to photon
irradiance absorbed by the sample, which is in turn proportional to
squared photon irradiance (eq 4).
481f
The coefficient 1/2 in eq 4 reflects the two-photon nature of the process.
4
5
Integration of eq 4 and taking into account
the pulsed nature of the irradiation gives eq 5.
481f
N
0 and N
t represent starting and current concentration
of a substrate; E
p represents the averaged
photon irradiance during the pulse, t
pulse is pulse duration, ν is laser repetition rate, and t
irr is the time of exposure. Strictly speaking, E
p
2 should be integrated for the duration
of the pulse, but this approximation introduces insignificant error.
For low-conversion experiments that use the Ti:sapphire laser discussed
above, we can write a simplified eq 6, where
δ is expressed in Goeppert-Mayer units (1 GM = 10–50 cm4 s photon–1 molecule–1).
6
Thus, to achieve 1% release
of a substrate within the focal volume
of a laser beam using a PPG with a quantum yield of uncaging Φ = 0.5 and a 2PE cross-section
of δ = 1 GM,
the sample has to be irradiated for about 30 min. One should also
take into account the background one-photon absorbance of tissue materials,
which is about 10–3 per 1 cm at 750 nm,
476b,488
and the broadening of ultrashort pulses in dense media. For an efficient
two-photon photochemistry in tissues, the product δE
p should be at least 10–25 cm2 molecule–1.
487
This
means that, at light intensities produced by a Ti:sapphire laser,
the two-photon absorption cross section δ of the PPG should
be around 1 GM or higher.
The cross section of two-photon absorption
for most organic chromophores
is rather low (δ < 1 GM); however, some recently synthesized
fluorophores, which usually contain D−π–A−π–D
(D = donor, A = acceptor) or a similar structural motif, have δ
over 1000 GM.
477b,477c
Unfortunately, these advances
in the design of two-photon chromophores have not yet been applied
to photolabile protecting groups. The suitability of several conventional
(single-photon) PPGs for 2PE-induced substrate release has been explored
instead (Table 11). The efficiency of this
process is better characterized by the two-photon cross section of
uncaging (δunc), which is a product of the two-photon
absorption cross section (δ) and the 2PE quantum efficiency
(Φ
2PE),
489
δunc = Φ
2PEδ.
It should be noted that the value of δunc is strongly
dependent on the wavelength of irradiation. Many cages that show decent
efficiency of substrate release at 740–750 nm have very low
δunc at 800 nm.
The most widely used o-nitrobenzyl-based PPGs
have a rather low two-photon uncaging cross section, which varies,
depending on the substitution in the ring and the wavelength of excitation,
from 0.01 to 0.035 GM (Table 11). The NV cage
(83) shows the highest sensitivity to 2PE in this family,
with δunc = 0.035 GM at 730 nm.
490
When o-nitrobenzyl (oNB) PPGs are used
for caging of fluorescent dyes (section 11), the quantum yield and two-photon cross
section of uncaging is
often improved apparently due to the energy transfer from the dye
to the caging chromophore. Thus, coumarin is released from the oNB
cage (57) with 53% quantum efficiency in single-photon
excitation at 365 nm and with 2PE δ = 0.37–0.68 GM at
740 nm.
491
The analogous 2-(2-nitrophenyl)prop-1-yl-based
PPGs, such as NPPOC, NPEOC (section 3.2),
or DMNPB (265), possess the same o-nitrophenyl
chromophore but have 5–12 times higher δunc due to more efficient photochemistry.
492
The two-photon uncaging cross section of these groups can be also
improved by sensitization. Thus, in the presence of a triplet sensitizer
with a large 2PE cross section (i.e., thioxanthone), the two-photon
uncaging action cross section of NPEOC (section 3.2) is enhanced to 0.86 from 0.12
GM.
234
Extension of the conjugated π-system of the chromophore
[(4′-methoxy-4-nitrobiphenyl-3-yleth-2-yl)methyl, PMNB, 115; (4′-tris-ethoxymethoxy-4-nitrobiphenyl-3-yleth-2-yl)methyl,
PENB, 266],
236,237,493
especially when combined with a symmetrical structure (BNSF, 117),
237
brings the two-photon
uncaging cross sections into a respectable 3–5 GM range. However,
these groups are rather bulky and suffer from poor solubility.
(Coumarin-4-yl)methyl-based (section 4)
PPGs 267, 268, 269, 270, and 143 are usually more efficient two-photon cages
than simple 1-(2-nitrophenyl)ethyl (NPE) and 2-(2-nitrophenyl)prop-1-yl
(NPP) (section 3.2) analogues (Table 11). The uncaging cross sections of the popular
BHCM
group (143, section 4) range
from 0.35 to 2 GM at 740 nm depending on the caged substrate.
288,294
Incorporation of two additional bromine atoms in the benzene ring
of BHCM (271) results in a red-shift of the 2PE maximum
and further enhancement of the two-photon uncaging efficiency (Table 11).
303
Dore and
co-workers have systematically studied the effects of
electron-donating and electron-withdrawing substituents on the two-photon
uncaging cross sections of (quinoline-4-yl)methyl PPGs (section 5).
331
The most efficient
two-photon-induced substrate release in this family was reported for
the BHQ cage (181, δunc = 0.6–0.9
GM at 740 nm).
330a
Replacement of the bromo
substituent for the cyano- (272) or chloro- (273) groups resulted in a reduced δunc,
nitro group
largely suppressed the two-photon sensitivity.
335a
The 7-dimethylamino- (274 and 275) and mercapto- (276) (quinoline-4-yl)methyl derivatives
are also presented in the table.
The 2-hydroxycinnamyl cage
(section 7.7) developed by Porter’s
group
387a
was also found to be amenable
to 2PE-triggered substrate release.
Jullien and co-workers have demonstrated that, with the appropriate
choice of substituents in the aromatic ring (277, 278, 279, and 280), the photochemical
step of the uncaging process, trans–cis isomerization of the
(E)-cinnamate moiety, can be induced
under two-photon photolysis conditions.
393b
4-Amino substituted (E)-(o-hydroxyphenyl)acrylates possess the highest two-photon
activation cross section (281, δunc =
2.0 GM; 282, δunc = 4.7 GM at 740 nm).
393a
Although the substrate release from these
cages is relatively slow at a biological pH (section 7.7), the 2PE-sensitive (E)-cinnamates
are remarkably easy to synthesize. Despite the rather
low two-photon cross sections of nitroindolyl cages (125, 127, 283, and 130, Table
11; see also section 3.3), they have been successfully employed in several biochemical
applications.
10d,250,483,494
Ruthenium complexes that contain amino compounds in their
coordination
sphere can release these substrates (section 6) under two-photon irradiation in aqueous
solution. Thus, 800-nm
irradiation of Ru(bipy)2(4-aminopyridine)2
2+ or Ru(bpy)2(PMe3)(GluH2)2+ was found to result in the release of 4-aminopyridine
or glutamine with 0.01 to 0.1 GM cross sections.
495
Table 11
Single-Photon and Two-Photon Uncaging
Quantum Efficiencies of Photolabile Protecting Groups
PPG
Φ
a
λ/nm
δunc/GMb
λ/nm
ref
83 (4,5-dimethoxy-2-nitrobenzyl,
NV)
0.16
305
0.035c
730
(490)
0.006
365
0.03
740
(303)
0.08
>365
0.01
800
(496)
0.23c
740
(221, 485b)
0.004c–0.025
750
(221, 485b)
57 (o-nitrobenzyl,
oNB; R = Cl,
OH, MeO, NH2, p-MeOC6H4, etc.)
0.001–0.1
325
0.015–0.065
750
(190)
265 (3-(4,5-dimethoxy-2-nitrophenyl)-2-butyl,
DMNPB)
0.26
365
0.17
720
(492)
115 ((4′-methoxy-4-nitrobiphenyl-3-yleth-2-yl)methyl,
PMNB)
0.1
315
0.45
800
(236)
3.1
740
(237)
266 ((4′-tris-ethoxymethoxy-4-nitrobiphenyl-3-yleth-2-yl)methyl,
PENB)
0.1
315
3.7
740
(493)
117 (2,7-bis-{4-nitro-8-[3-(2-propyl)-styryl]}-9,9-bis-[1-(3,6-dioxaheptyl)]-fluorene,
BNSF)
0.25
354
5.0
800
(237)
267 ((7-hydroxycoumarin-4-yl)methyl)
0.025
365
1.07
740
(331)
0.13
800
(303)
268 ((6-chloro-7-hydroxycoumarin-4-yl)methyl)
0.01
365
1.07
740
(303)
0.34
800
(303)
143 ((6-bromo-7-hydroxycoumarin-4-yl)methyl,
BHCM)
0.084
350
0.35
740
(288)
0.019–0.037
365
0.51–1.99
740
(294, 303)
0.21
780
(330a)
0.37–0.42
800
(303)
269 ({7-[bis(carboxymethyl)-amino]coumarin-4-yl}methyl,
BCMACM)
0.29
330–430
ca. 1c
800
(286)
270 ({7-[4-(dimethylamino)styryl]coumarin-4-yl}methoxycarbonyl)
0.83 × 10–3
407
0.26
800
(497)
271 ((3,6,8-tribromo-7-hydroxycoumarin-4-yl)methyl)
0.065
365
0.96
740
(303)
3.1
800
(303)
181 ((8-bromo-7-hydroxyquinoline-2-yl)methyl,
BHQ)
0.29–0.39
365
0.59–0.90
740
(331)
0.087
780
(330a)
272 ((8-cyano-7-hydroxyquinoline-2-yl)methyl,
CyHQ)
0.31
365
0.32
740
(335a)
273 ((8-chloro-7-hydroxyquinoline-2-yl)methyl,
CHQ)
0.10
365
0.12
740
(335a)
274 ([7-(dimethylamino)quinoline-2-yl]methyl,
DMAQ)
0.046
365
0.13
740
(335a)
275 ([7-(dimethylamino)-4-chloroquinoline-2-yl]methyl,
DMAQ-Cl)
0.09
365
0.47
740
(335a)
276 ((7-mercaptoquinoline-2-yl)methyl,
TQ)
0.063
365
0.42
740
(335a)
277 ((Z)-butyl 3-(4-(diethylamino)-2-hydroxyphenyl)-2-acetamidoacrylate)
0.05
300–400
0.3
750
(393b)
278 (3,5-dibromo-2,4-dihydroxycinnamate)
0.05
369
1.6
750
(390)
0.05
300–400
0.6
750
(393a)
279 ((E)-3-(6-hydroxy-benzo(1,3)dioxo-5-yl)acrylate)
0.03
300–400
3.8
750
(393a)
280 ((E)-3-(4-diethylamino-2-hydroxy-phenyl)acrylate)
0.02
300–400
2.0
750
(393a)
281 ((Z)-butyl 2-acetamido-3-(5-hydroxybenzo[d][1,3]dioxol-6-yl)acrylate)
0.07
300–400
2.0
750
(393b)
282 ((E)-3-(8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido(3,2,1-ij)quinolin-9-yl)acrylate)
0.03
300–400
4.7
750
(393a)
125 (4-methoxy-7-nitroindolinyl,
MNI)
0.085
350
0.006
730
(10d, 483)
127 (4-methoxy-5,7-dinitroindolinyl,
MDNI)
0.47
350
0.06c
730
(494)
283 (4-carboxymethoxy-5,7-dinitroindolinyl, CDNI)
0.5c
334–364
0.06
720
(483a)
130 (5-methoxy-8-nitro-1,2-dihydroquinolinyl,
MNDQ)
0.05
365
<0.06c
720
(250)
a
The single-photon quantum yield
of substrate release.
b
The
two-photon cross section of
uncaging.
c
By comparison
with the literature
data.
10
Chromatic
Orthogonality
The diversity of different PPGs, operating
by different mechanisms
and bearing different types of chromophores, opens the door for wavelength-selective
deprotection. Indeed, the possibility of releasing on demand various
types of molecules (e.g., in a cell, or wherever access by conventional
injection techniques is not possible) is a very appealing prospect.
A first-order approximation would suggest that absorption maxima sufficiently
distant could work; however, fast energy transfer may thwart such
a strategy, in particular for cases where both chromophores are part
of the same molecular entity. Careful choice of PPG pairs made such
a wavelength discrimination possible, allowing for the selective release
of two different carboxylic acids, in what was an early example of
chromatic orthogonality (Scheme 127).
208,498
Scheme 127
Example of Intermolecular Chromatic Orthogonality
498
The same benzoin (section 2.4)/nitroveratryl
(NV, section 3.3) pair was then shown to
be equally orthogonal in the intramolecular case, where spatial proximity
is more critical than in separate molecules (Scheme 128).
499
Reactivity tuning by way
of a kinetic isotope effect was also exploited to make two derivatives
of o-nitrobenzylic esters (section 3.1) chromatically orthogonal.
225
Scheme 128
Example of Intramolecular Chromatic Orthogonality
499
Examples of this principle were shown soon after in the
solid-phase
synthesis of peptides
360c
and in the selective
release from resins by using the nitroveratry/pivaloylglycol pair.
500
del Campo and co-workers investigated a more
complex version of the wavelength-selective cleavage by putting seven
photolabile protecting groups on various functionalities immobilized
through an organosilane tether on a glass surface against each other.
240e,501
Systematic analysis of photolytic characteristics of common photolabile
groups helped establish several protecting strategies that permit
the simultaneous use of up to four orthogonal photoactivated groups,
such as benzoin (λex = 255 nm, section 2.4), p-hydroxyphenacyl (pHP,
λex = 275 nm, section 2.3), 5,7-dinitroindolinyl (DNI, λex = 360 nm, section 3.3),
and [7-(diethylamino)coumarin-4-yl]methyl
(DEACM, λex = 435 nm, section 4), that can be cleaved sequentially from the same solid
support under
different irradiation conditions.
240e
It
was also noted that two-photon excitation can potentially expand the
spectral window and increase the number of possible functional levels
for selective spatiotemporal activation.
Applications in biochemistry
are appearing more and more frequently,
such as the protection of cysteines (using variously substituted coumarins),
196
nucleotides (using a coumarin/nitrophenethyl
pair),
502
or both,
503
and the release of both substrates and inhibitors (also
using a coumarin/nitrophenethyl pair),
504
or the selective release of glutamate or GABA by two-colors/two-photon
excitation.
263b
The latter strategy was
also applied very recently with a NV/p-MeO-phenacyl
pair by Emptage, Conway, and co-workers.
106c
The same concept was shown to be valid in the solid phase, where
the wavelength-selective modification of surfaces was performed with
orthogonal pairs
135a,501
or quartets.
240e
11
Photoactivatable Fluorescent Dyes
Photoactivatable (caged) fluorophores
13b
are fluorogenic and release a fluorescent molecule upon irradiation.
They are obtained by coupling a fluorescent dye to a PPG that prevents
it from displaying fluorescence. Caged fluorescent dyes provide a
highly sensitive tool to monitor the flow of liquids (rheology) and
to follow the movements and the distribution of particular species
of interest at the single molecule level and with a spatial resolution
at the nanometer scale, i.e., beyond the diffraction limit of optical
microscopy (∼250 nm). Fluorescence imaging microscopy is among
the most powerful techniques for observing dynamic processes in living
cells. To monitor the movement of target molecules or species in real
time, it is necessary to create a local region within the cell where
the fluorescence intensity is higher than in the bulk. Imaging with
high target-to-background contrast requires a high activation ratio,
i.e., a high ratio of postactivation to preactivation signal intensity.
Thus, the signal intensity arising from the caged fluorophore should
be close to zero.
The classic method for creating a differentially
labeled region
has been fluorescence photobleaching, i.e, reducing fluorescence intensity
by irradiation, usually by reaction of a fluorescent dye with singlet
oxygen. Alternatively, photochemically labile fluorophores such as
the aminophthalimide–serine shown in Scheme 129 can be used.
378
Disadvantages
of photobleaching through singlet oxygen are that it may cause local
damage to proteins and membranes and that it is difficult to track
a region of reduced fluorescence within a background of higher fluorescence.
The use of a nonbleachable reference fluorophore has been proposed
to track the distribution of the bleached molecules by image differencing.
505
Scheme 129
“Armed” Phthalimide (λmax = 340 nm,
Exhibits Strong Fluorescence, λmax = 513 nm, in Aqueous
Media at pH = 7; Irradiation Liberates Acetate and CO2,
and the Fluorescence Decreases)
378
To overcome these limitations,
numerous photoactivatable fluorophores
have been developed. The design and use of caged fluorophores have
been reviewed.
9n,13a,13b,506
Desiderata for an effective
caged fluorescent probe are biostability and -compatibility; a high
affinity to or, preferentially, specificity for the target (e.g.,
cancer cells);
507
rapid and efficient photoactivation
providing a high ratio of pre- and postirradiation fluorescence; a
high fluorescence quantum yield; photostability of the uncaged fluorophore;
and practicable synthesis. Up to 1998,
506
caged fluorophores have all used variants of the oNB (section 3) caging group (e.g.,
284).
The full activation of such bis-caged lactone (leuco) forms of
rhodamine or fluorescein requires the removal of both PPGs. Removal
of one PPG is sufficient to restore the chromophore, but the remaining
nitroaromatic group often largely quenches the fluorescence emission
of the monocaged dye. Full deprotection therefore requires high doses
of UV irradiation. On the other hand, the non-negligible fluorescence
quantum yield of monoprotected fluorescein hampers its use as a caged
fluorophore, because the activation ratio upon deprotection is usually
insufficient. A monoprotected variant of fluorescein (TokyoGreen, 285, Scheme 130)
has, however, been
developed that exhibits very little fluorescence.
508
The fluorescence quantum yield of singly 2-(2-nitrophenyl)prop-1-yl
(NPP) (section 3.1) protected TokyoGreen
(286) is less than 1/100th that of protected fluorescein.
It liberates the highly fluorescent free form (Φ
fl = 0.96) by removal of the PPG with a quantum yield
of 0.03.
509
Scheme 130
Deprotection of
Mono-Caged Tokyo Green (Its Fluorescence Is Quenched
by Intramolecular Electron Transfer in the Excited Singlet State)
508
Several other examples of fluorescence activation by photochemical
removal of a quencher moiety attached near the fluorophore have been
reported. Other than by electron transfer, fluorescent singlet states
can be quenched by Förster resonance energy transfer (FRET,
somewhat inadequately called “fluorescence resonance energy
transfer” by most biochemists)
510
to an adjacent, nonfluorescent, but photoremovable chromophore.
For example, a synthetic route to incorporate a photocleavable 4-dimethylaminoazobenzene-4′-sulfonyl
(287, dabsyl)
511
moiety and
fluorescein at adjacent cytidines in the middle of a 25-mer oligodeoxynucleotide
has been reported.
512
UV irradiation removed
the dabsyl moiety, which increased the fluorescence intensity 51-fold
and restored the melting temperature of the nucleotide. Such caged
fluorescent oligodeoxynucleotides will allow many DNA processes to
be controlled with light.
The same principle was used
to regulate the DNA polymerase reaction
by the Klenow fragment of polymerase I with UV light. A 25-mer caged
fluorescent oligodeoxynucleotide as the template was functionalized
with a fluorescein reporter and the photoremovable dabsyl quencher
moiety (287). With this template, the Klenow fragment
was blocked from extending a complementary 12-mer primer. Removal
of the quencher by short UV photolysis partly restored the activity
of the Klenow fragment, and the reactivation could be monitored by
fluorescence.
511
A related approach
was used to provide for real-time monitoring
of Smad2, a key protein involved in the transforming growth factor
β (TGF-β) signaling pathway, spatial and temporal control
of its activity, and differentiation between its active and inactive
state.
513
The protein was labeled at neighboring
sites with a fluorescein chromophore and with a 4-dimethylaminoazobenzene-4′-carboxylic
(dabcyl) acid quencher through a photocleavable 4-[4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy]
butanoic acid linker. This both suppressed the protein activity and
quenched its fluorescence. Photocleavage of the caging group resulted
in the simultaneous restoration of protein activity and luminescence
of the fluorescent tag.
Numerous photocleavable (coumarin-4-yl)methyl
esters with fast
release have been described (section 4).
276,289,296
NPP-caged coumarins that display
more than 200-fold fluorescence enhancement upon UV irradiation were
developed (Scheme 131).
491
The combined advantages of robust fluorescence contrast
enhancement, high uncaging efficiency, noninvasive cellular delivery,
and flexible chemistry for bioconjugations are promising for the use
of these caged coumarins in biochemical and biological research. The
high uncaging efficiency is attributed to the antenna function of
the coumarin, i.e., light absorbed by the coumarin chromophore is
utilized to cleave the NPP cage. It should, however, be noted that
NPE-caged fluorophores are subject to the known disadvantages of o-nitrobenzyl photochemistry
(see section 3.1).
Scheme 131
Caged Coumarin
491
To prepare a caged coumarin
emitting a different color, the o-nitrobenzyl-caged
coumarin was linked to a water-soluble
fluorescein dye emitting at 520 nm.
514
The
caged dye can be localized in the sample prior to photolysis by directly
exciting the fluorescein moiety at 490 nm. After uncaging the coumarin
chromophore, the green emission can be excited at 410 nm due to efficient
FRET from the coumarin moiety to the fluorescein dye. This is especially
desirable for experiments demanding highly localized photoactivation
by two-photon uncaging, which requires knowing the distribution of
the label in three dimensions. These probes thus offer new opportunities
to image molecular and cellular dynamics.
A light-activated
fluorescent reporter of intracellular protein
kinase activity has been designed (Scheme 132) that furnishes a fluorescent readout.
515
The photolytically labile appendage affords control over both the
timing and the amount of active sensor release. The quantum yield
for photolytic conversion is 0.06. The caged fluorescent substrate
was introduced into HeLa cells via microinjection. Following in situ
illumination of the caged peptide, time-dependent changes of the fluorescence
intensity due to phosphorylation of the released hydroxyl function
provided a measure of protein kinase activity in the cells.
Scheme 132
Fluorescent
Reporter of Protein Kinase Activity
515
Azidomethyl-caged fluorescein
derivatives (288 and 289) were utilized
to monitor the dynamics of oligonucleotides
in living human cells. Both compounds were rapidly activated upon
brief irradiation and showed a strong increase in fluorescence intensity.
516
The addition of lithiated
dithianes to 2-amidothioxanthones disrupts
conjugation, resulting in a blue-shift of the absorption and a dramatic
decrease in fluorescence intensity. The product represents a new caging
system capable of fast quantification of released payloads such as
TentaGel beads by single- or two-photon fluorescence.
455
Irradiation induces homolytic C–C fragmentation
followed by disproportionation of the two radicals (Scheme 133). The half-life of
the radical intermediates
was 1.7 μs.
Scheme 133
Caged Thioxanthone
455
Rhodamine dyes having a 2-diazo
caging group incorporated into
a spiro-9H-xanthene fragment have been synthesized
by reaction of diazomethane with the acid chloride of rhodamine B.
517
The yellow crystalline diazoketone 290 was obtained in high yield (Scheme 134).
Caged rhodamines with an additional carboxy group in the benzoyl fragment
of the diazoketone that may be linked to an amino or a thiol reactive
site were also made. Application of a related system featuring a high
contrast ratio (1:140) and activation by visible light (λ <
420 nm) to high-resolution microscopy was recently reported.
518
Scheme 134
Photochemistry of Caged Rhodamine (a Wolff
Rearrangement)
517
A ruthenium-rhodamine complex 291 (see Section 6) has been evaluated as an activatable
fluorescent
probe (Scheme 135).
356
Upon irradiation, it releases rhodamine in a fast and clean reaction,
increasing its fluorescence nearly 6-fold.
Scheme 135
Photochemistry
of Caged Rhodamine
356
A photoactivatable nitrobenzyl-caged (section 3.1) green fluorescent protein (292,
GFP) that
is practically nonfluorescent prior to irradiation was synthesized
recently (Scheme 136).
519
The relative brightness at the wavelength of maximum emission
(486 nm) increased by at least 4 orders of magnitude during photoactivation
at 365 nm.
Scheme 136
o-Nitrobenzyl-Caged GFP
519
Photoactivatable fluorescent proteins have become an important
addition to the set of molecular probes used to understand cellular
function. Wild-type GFP
520
is in fact already
a caged fluorophore of sorts. The protein can adopt two conformations
with different absorption maxima, and irradiation shifts the molecules
toward the longer-wavelength absorbing form. Photoconversion in wild-type
GFP is thought to involve rotation of threonine 203 and decarboxylation
of glutamic acid 222. A mutant of wild-type GFP, in which the threonine
203 position is replaced by histidine, was shown to increase its fluorescence
intensity (λex = 488 nm, λem = 520
nm) 100 times upon intense irradiation at 413 nm.
521
The activated protein remains stable for days under aerobic
conditions. This photoactivatable variant of the Aequorea
victoria GFP was used both as a free protein to measure protein
diffusion across the nuclear envelope and as a chimera with a lysosomal
membrane protein to demonstrate rapid interlysosomal membrane exchange.
Optical microscopy provides spatial resolution down to the diffraction
limit of ∼250 nm, but single fluorescent particles can now
be localized down to ∼1 nm precision, and spectroscopic techniques,
such as FRET, can offer yet smaller distance changes. Super-resolution
(subdiffraction) imaging techniques based on sequential imaging of
sparse subsets of single molecules have used fluorophores whose emission
can be photoactivated or photoswitched.
522
The common principle is that only one pointlike light source is
active in a diffraction-limited area at any time. Imaging with a sensitive
camera localizes this fluorophore. Its exact location can then be
determined to well below the diffraction limit by fitting a point-spread
function. In the beginning of data acquisition, all except a few fluorophores
are prepared in the off-state and the number of active fluorophores
is kept constant by applying an activating light source that compensates
for the loss of fluorophores by photobleaching or switching off. A
super-resolution image of a labeled complex structure can then be
reconstructed from many successive rounds of weak photoactivation
and fitting. This method has been used to image intracellular proteins
at nanometer spatial resolution. As an alternative to caged fluorophores
for super-resolution microscopy, on- and off-states have been engineered
by controlling the photophysics of fluorophores by electron transfer
reactions.
523
Because good organic
fluorophores are more photochemically stable
than most fluorescent proteins, organic fluorophores have a potential
benefit in super-resolution imaging schemes, but targeting to specific
cellular proteins must be provided. The design and application of
target-specific an azido derivative of 2-dicyanomethylene-3-cyano-2,5-dihydrofuran,
a photoactivatable push–pull fluorogen that produces bright
fluorescent labels suitable for single-molecule super-resolution imaging
in live bacterial and fixed mammalian cells, were reported (293).
524
The same authors subsequently demonstrated that the azide-to-amine
photoactivation process is generally applicable to a variety of push–pull
chromophores, which provide a new class of photoactivatable single-molecule
probes for fluorescent labeling and super-resolution microscopy.
525
Moreover, these photoactivated push–pull
dyes can insert into bonds of nearby biomolecules, simultaneously
forming a covalent bond and becoming fluorescent (fluorogenic photoaffinity
labeling).
The design, synthesis, and photophysical characterization
of a
class of ligand-directed, photoactivatable, turn-on fluorescent probes
for the spatially controlled imaging of microtubules in live mammalian
cells was reported.
526
A series of taxoid–tetrazoles
was reported in which 7-β-alanyltaxol L was attached to a water-soluble
tetrazole bearing an o-allyloxy group at the N-phenyl ring via a flexible linker.
Upon irradiation at
302, 365, or even 405 nm, the tetrazoles 294 undergo
deazotization generating reactive nitrile imine dipoles that spontaneously
react with the prealigned allyl group to form pyrazoline fluorophores
(Scheme 137). Whereas none of the taxoid–tetrazoles
was fluorescent, the taxoid–pyrazolines exhibited strong fluorescence
emission in the range of 500–800 nm. With this photoactivatable
fluorescent probe, it should be possible to label microtubules asymmetrically
within a single cell and identify factors that break cellular symmetry
during cell division.
Scheme 137
Intramolecular Photoclick Reaction
526
12
Conclusion
Photoremovable protecting
groups (PPGs) release molecules such
as enzymes, neurotransmitters, signaling molecules, fluorophores,
insecticides, pheromones, and fragrances that thereby exhibit desirable
physical, chemical, or biological qualities upon photoactivation.
They are synthetically malleable and accessible, offering a wide range
of structures for designed applications. They offer excellent spatial
and temporal control for the substrate release. Their applications
span many scientific fields, from DNA chip technology, drug delivery,
and photoregulation of proteins, to rheology, solid-phase synthesis,
surface chemistry, and nanotechnology. PPGs are excellent, versatile
tools for time-resolved studies of chemical processes in living cells.
Multiphoton excitation can provide superior spatial resolution, and
the new chromophores included herein offer more precise temporal control
for addressing the dynamics of in vivo events in living organisms.
There has been an upsurge in interest in PPGs over the past decade
that has resulted in a dramatic increase in the number of new designs
and the development of both new and known PPGs to fulfill the demands
for better sensitivity, faster kinetics, and more demanding bioanalytical
applications. The many new discoveries have brought substantial improvements
and versatility to the synthesis, thermal stability, solubility, and
absorption properties of PPGs, as well as improved efficiencies and
rates of release. Nonetheless, there still remain many important research
goals, such as extending the wavelength coverage for activation of
PPGs with visible and infrared light and improving the absorption
properties for wavelength-selective, orthogonal activation, and melding
these features with the inherent advantages of photoactivation that
PPG possess as outlined at the beginning of this review. Our goal
has been to aid and assist researchers in applying these tools and
to attract and encourage their participation in this rapidly developing
field.