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69
Photoremovable
Protecting Groups
69.1 Introduction ..................................................................... 69-1
69.2 Historical Review.............................................................. 69-2
o-Nitrobenzyl • Benzoin • Phenacyl • Coumaryl and
Arylmethyl
69.3 Carboxylic Acids............................................................. 69-17
o-Nitrobenzyl • Coumaryl • Phenacyl • Benzoin • Other
Richard S. Givens
University of Kansas
Peter G. Conrad, II
University of Kansas
Abraham L. Yousef
University of Kansas
Jong-Ill Lee
University of Kansas
69.4 Phosphates and Phosphites ........................................... 69-23
o-Nitrobenzyl • Coumaryl • Phenacyl • Benzoin
69.5 Sulfates and Other Acids................................................ 69-26
69.6 Alcohols, Thiols, and N-Oxides .................................... 69-27
o-Nitrobenzyl • Thiopixyl and Coumaryl • Benzoin • Other
69.7 Phenols and Other Weak Acids..................................... 69-36
o-Nitrobenzyl • Benzoin
69.8 Amines ............................................................................ 69-37
o-Nitrobenzyl • Benzoin Derivatives • Arylsulfonamides
69.9 Conclusion...................................................................... 69-40
69.1 Introduction
Photoremovable protecting groups are enjoying a resurgence of interest since their introduction by
Kaplan1a and Engels1b in the late 1970s. A review of published work since 19932 is timely and will provide
information about several new groups that have been recently developed. The scope of this review is,
therefore, limited to recent developments in the field and will cover only the applications with major
functional groups that have been “protected” by a photoremovable chromophore. The review is not
intended to be comprehensive but focuses instead on a series of well-chosen examples of chromophores
that were deployed as protecting groups with a select group of representative functional groups. Because
the focus of this review is the application of photoremovable protecting groups, emphasis is placed on
synthesis of the protected functionality and on the procedures employed for deprotection, including the
protection and photodeprotection yields, the deprotection reaction rates, and the quantum efficiencies,
when available. An attempt has been made to list the advantages and disadvantages of each photoremovable protecting group as well as a brief discussion of the mechanism for the photodeprotection.
0-8493-1348-1/04/$0.00+$1.50
© 2004 by CRC Press LLC
69-1
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CRC Handbook of Organic Photochemistry and Photobiology, 2nd Edition
When the literature is insufficient for providing a comprehensive treatment of applications of a
photoprotecting group, then only a brief discussion is provided. An exhaustive list of applications for
any of the chromophores is not included; these may be found by consulting other reviews or the original
literature on a topic. Several good reviews on photoremovable protecting groups have appeared since
this topic was reviewed in 1993 (e.g., Adams and Tsien3 and Corrie and Trentham4). Notable among the
more recent reviews are those by Wirz,5 Bochet,6 and Givens.7 A volume of Methods in Enzymology devoted
entirely to the chemistry and applications of photoremovable protecting groups, also termed “caged”
compounds, that are employed in biochemistry and other biological studies has also appeared.
In general, photolysis reactions present a noteworthy and often ideal alternative to all other methods
for introducing reagents or substrates into reactions or biological media. The ability to control the spatial,
temporal, and concentration variables by using light to photochemically release a substrate provides the
researcher with the ability to design more precisely the experimental applications in synthesis, physiology,
and molecular biology. Among the many possible examples is the recently reported inhibition–reactivation of protein kinase A by photolysis of the dormant enzyme.8–10 In this demonstration, it is necessary
that the deprotection process be initiated by photolysis of the dominant chromophore of the protecting
group. Covalent blocking of the functional groups at the active site of an enzyme essentially suspends its
mode of action and virtually shuts down the catalytic cycle. It is this feature that has attracted biochemists
to the use of protecting groups for the investigation of biological mechanisms.
In synthesis, the protecting group serves as a mask that renders a functional group inert to subsequent
synthetic reaction conditions,11 except, of course, conditions that are required for the removal of the
protecting group. Construction of combinatorial platforms with photoremovable linkers is just one
example of the applications in synthesis. Photorelease is sometimes termed a traceless reagent process
because no reagents other than light are needed. The advantage of a process that requires no further
separation of spent reagents is attractive.
There are several limitations to the use of commonly employed protecting groups in synthesis and for
mechanistic studies of biological processes. The reactions for incorporating and subsequently removing
protecting groups often involve acid or base that may be too harsh and interfere with the normal processes
or otherwise be incompatible with the chemistry or biology under investigation. In mechanistic biochemistry, it is often the case that the typical hydrolysis deprotection reaction is far too slow to serve as
a means of investigating the initial rates of reaction for rapid biochemical processes.
An ideal remedy to these limitations is a protecting group that could be removed under neutral buffered
aqueous conditions, thus avoiding any alterations to the substrate or to the natural biological environment.12 The release should occur on a time scale fast enough for kinetic analysis of any subsequent rapid
biological processes. Such a group may be a photoremovable protecting group.
69.2 Historical Review
In 1962, Barltrop et al.13 were among the first to report a photochemical deprotection reaction of a
biologically significant substrate; here, glycine was released from N-benzyloxycarbonyl glycine:
O
O
N
H
OH
O
hν
CH3
+
OH
H2N
O
+ CO2
(69.1)
This seminal discovery prompted the development of several additional photoremovable protecting
groups. The success of many researchers in biology, particularly Kaplan,1a led to the description of the
photoactivatable group as a “cage” to describe its deactivating influence on the biological substrate to
which it is covalently attached.14–17 Ideally, the cage detaches only through the action of light.
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Photoremovable Protecting Groups
It is important that the photoremovable protecting group also possess several other desirable properties. The properties were originally compiled by several researchers in the field, including Sheehan and
Umezawa12 and Lester and Nerbonne,18 who provide a series of benchmarks for evaluating the efficacy
of a photoremovable group in a given circumstance or for evaluating the potential of a new cage
chromophore. A more useful adaptation of the Lester rules and Sheehan criteria includes the following:
1. The substrate, caged substrate, and photoproducts have good aqueous solubility for biological
studies. For synthetic applications, this requirement is relaxed.
2. The photochemical release must be efficient (e.g., Φ > 0.10).
3. The departure of the substrate from the protecting group should be a primary photochemical
process (i.e., occurring directly from the excited state of the cage chromophore).
4. All photoproducts should be stable to the photolysis environment.
5. Excitation wavelengths should be longer than 300 nm and must not be absorbed by the media,
photoproducts, or substrate.
6. The chromophore should have a reasonable absorptivity (a) to capture the incident light efficiently.
7. The caged compounds, as well as the photoproduct from the cage portion, should be inert or at
least benign with respect to the media, other reagents, and products.
8. A general, high-yielding synthetic procedure for attachment of the cage to the substrate must be
available.
9. In the synthesis of a caged substrate, the separation of caged and uncaged derivatives must be
quantitative. This is also necessary for the deprotection process for synthetic applications.
While these are the desirable guidelines for an ideal photoremovable protecting group, a potential cage
that lacks one or two of these properties may still be very useful; however, the absence of several of these
features may militate against the use of that group as a photoremovable protecting group for a specific
application.
Some representative examples of photoremovable protecting groups that qualify as meeting the Lester
and Sheehan criteria include α-substituted acetophenones, benzoins, benzyl groups, cinnamate esters,
coumaryl groups, and, the most popular of them all, the o-nitrobenzyl esters and their analogs.
o-Nitrobenzyl
It was also Barltrop et al.19 who first reported the use of an o-nitrobenzyl group to release benzoic acid
(see Eq. (69.2)). The poor yield stemmed from the subsequent conversion of 2-nitrosobenzaldehyde (3),
the initial photoproduct, into azobenzene-2,2′-dicarboxylic acid (4),20 which then competed for the
incident light. Yields were dramatically improved with the use of α-substituted nitrobenzyl esters (75 to
95% conversion), as seen from 5 in Eq. (69.3). The resulting photoproduct from o-nitrobenzyl ester 5
was a less reactive nitroso benzophenone derivative.
O
O
O
hν
HO2C
O
OH +
N
H
NO2
NO
3
2, 17%
1
N
CO2H
4
(69.2)
Ph
O
O
hν
O
2
Ph
+
NO2
5
NO
6
(69.3)
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CRC Handbook of Organic Photochemistry and Photobiology, 2nd Edition
The report of the release of ATP by this method appeared in a 1978 rate study reported by Kaplan
and co-workers.1a Inorganic phosphate (Pi) and ATP were released from their 1-(2-nitrophenyl)ethyl
(NPE) and 2-nitrobenzyl (NB) esters, respectively:
R
O
OP R'
O
NO2
7a
7b
7c
7d
O-
O
hn, 342 nm
R = H, R' =
R = H, R' = O-ADP
R = CH3, R' = OR = CH3, R' = O-ADP
R
O
OP R'
O
+
NO
3 R = H, CH3
P R' = OATP R' = O-ADP
i
(69.4)
The results of the release of Pi from NPE and NB showed very similar quantum efficiencies of 0.58 and
0.50, respectively; however, the release of ATP from the two cages gave very different rates of conversion.
NPE released 80% of the caged ATP in less than 60 s compared with 25% for release from the NB caged
ATP. These results further indicated that the α-substituted nitrobenzyl esters were better suited as
phototriggers.
Kaplan’s investigation also explored the potential use of photoprotecting groups in a physiological
environment. Na+,K+-ATPase, the enzyme responsible for sodium/potassium transport through cell walls,
served as the model for exploring the effect of the caged ATP (NPE-ATP) on the Na+:K+ transport
associated with enzymatic activity. The enzyme acquires ATP as the energy source through hydrolysis of
the terminal γ-phosphate. The hydrolytic activity of the enzyme can be monitored by the detection of
Pi generated from the free ATP consumed by the enzyme. In the absence of photolysis, NPE-ATP was
shown to be resistant to hydrolysis by the enzyme. Upon photolysis, the liberated ATP triggered the
response of the enzyme and Pi release was observed.
The successful introduction of o-nitrobenzyl caged ATP into physiological media instigated interest
in expanding the applications of caged release to a wide variety of biochemical systems. The list includes
the mechanism of release of Pi in skeletal muscle,21 the function of cAMP in the relaxation of distal
muscle,22 the ATP-induced mechanism of actomyosin in muscle contraction,23 and the activation of
antitumor antibiotics to highly reactive pyrrolic-type intermediates responsible for DNA crosslinking
reactions.24
Benzoin
Sheehan and Wilson25 were the first to explore the photochemical rearrangement of certain benzoin
derivatives to yield 2-phenylbenzofuran (9). These rearrangements occurred with concurrent loss of
groups attached α to the carbonyl just as in the case of the α-chloroacetophenones. They suggested that
benzoins, especially the 3′,5′-dimethoxybenzoin chromophore, could serve as a photoremovable protecting group for carboxylic acids. In 1984, Givens et al.26 showed that phosphates were quantitatively expelled
from the ungarnished benzoin cage, as shown in Eq. (69.5), thus extending the range of applications and
the nature of the parent chromophore. The only major product accompanying the released phosphate
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TABLE 69.1 Quantum Efficiencies for Benzoin Phosphate Esters 8a–c
Phosphate Ester
8a
8b
8b
8c
8c
Solvent
pH
Φdis
Φfuran
Φphosphate
C 6H 6
H2O/CH3CN
H2O/CH3CN
H2O/CH3CN
H2O/CH3CN
nd
2.0
7.0
2.0
7.0
0.28
0.37
nd
0.38
nd
0.26
0.20
0.07
0.14
0.08
nd
0.12
0.013
0.15
0.01
Note: All reactions were run in 60% aqueous acetonitrile, except 8a, as
indicated; phosphate esters were irradiated at 350 nm and monitored via
31P NMR; nd = not determined.
Source: Adapted from Givens et al.27
O
hn
O
3
H
ST
PO(OR)(OR')
3
8
<20 ns
3
O
O
+
H
O
PO(OR)(OR')
8a R = R' = Et
8b R = iPr, R' = Na+ or H +
8c R = R' = +Na or +H
310,
670 ns
ST
<5 ns
1
10
CF3CH2OH
O
Ph
9
O
OCH2CF3
+
O
P OR'
O OR
from laser flash studies
R = R' = Et
R = iPr, R' = +Na or +H
R = R' = +Na or +H, (Pi)
(69.5)
was 2-phenylbenzofuran 9. These reactions were quenched with naphthalene, piperylene, or, for aqueous
studies, sodium 2-naphthalenesulfonate, all well-known triplet quenchers; this established the short-lived
triplet (3 to 14 ns) as the reactive excited state for benzoin. Further information was revealed from
Stern–Volmer quenching analyses which provided the rate of release of phosphate from the benzoincaged ester. Extremely fast rates (kr > 108 s–1) were measured along with good efficiencies for the reaction,
ranging from 28 to 38% (Table 69.1).27,28 Phosphorescence spectra supported the multiplicity assignments
and also established the triplet energy at 73 ± 1 kcal/mol.
The efficiencies for the disappearance of the caged phosphates (Φdis), as well as the appearance of 2phenylbenzofuran (Φfuran) and phosphate (Φphosphate), were determined to be pH dependent with higher
efficiencies reported under acidic conditions. The greater efficiency at lower pH suggests that the protonated phosphate is a more favorable leaving group than its conjugate base. This study was extended to
nucleotide release from the benzoin cage through the synthesis and photolysis of benzoin cAMP. The
efficient release of cAMP as the exclusive product with quantum efficiencies on the same order of
magnitude as the model phosphate esters first demonstrated the application of benzoin as a cage for
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CRC Handbook of Organic Photochemistry and Photobiology, 2nd Edition
TABLE 69.2 Quantum Efficiencies for Photolysis of
Benzoin Adenosine Cyclic 3′,5′-Monophosphatea
Aqueous Buffer
pH
Φdis
Φfuran
ΦcAMP
Tris (D2O)
Tris (H2O)
Phosphate (D2O)
Phosphate (H2O)
Perchloric (D2O)
7.3
7.3
8.4
8.4
1.6
0.39
0.37
nd
nd
0.40
0.19
0.17
0.17
0.17
0.16
0.34
0.34
nd
nd
0.36
a
Irradiations were carried out in 1:1 buffer: 1,4-dioxane at 350 nm. Quantum efficiencies (Φ) were measured using 31P NMR, except where indicated (nd).
Source: Adapted from Givens et al.28
*
OMe
OMe
O
O
hν
OMe
O
OMe
O
R
R
O
O
*
MeO
H
MeO
H
OMe
O
demotion
H
O
H
R
O
O
R
O
H
OMe
OMe
O
O
O
OMe
O
+
O
R
OMe
OMe
11
SCHEME 1 Benzoin photorelease mechanism.
nucleotides (Table 69.2). 31P spectra of released cAMP demonstrated that cAMP was the only phosphate
present after release. As Table 69.2 indicates, the quantum efficiencies remained relatively constant
throughout the pH range examined.
It has been determined that carboxylate derivatives are released more readily when there are electrondonating substituents at the meta positions of the benzyl ring.29 The absorption spectra of the benzoin
esters along with the observations that cyclization was enhanced by meta electron donating groups led
observers to believe that reaction was taking place through an n,π* singlet state (see Scheme 1). It was
suggested that the excitation of the phenacyl group led to a short-lived 1,3-biradical, followed by demotion
to the ground state. The zwitterionic intermediate led directly to the loss of the leaving group. Aromatization through loss of a proton gave the benzofuran 11 as the principal photoproduct. The inability to
quench the reactions with high concentrations of piperylene suggested that the reaction originated from
the singlet excited state or, alternatively, from a very short-lived, unquenchable triplet.
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Photoremovable Protecting Groups
Based on these observations Corrie and Trentham4 re-examined the photochemistry of several substituted benzoin phosphates. They found that the 3′,5′-dimethoxybenzoin cage was best suited among
those investigated for the release of Pi. The formation of 3′,5′-dimethoxy-2-phenylbenzofuran (11)
occurred at a rate that exceeded 105 s–1 and a quantum efficiency of 0.78. While the rate of product
formation was lower than that reported by Givens and Matuszewski,26 the efficiency for the substituted
benzofuran analog was much higher. For the benzoin series, the primary photoproduct 11 is also a
strongly absorbing chromophore and thus competes for incident light and forms photodimers along
with several other unidentified products upon further irradiation. Yet another limitation of this system
is the presence of a chiral center alpha to the carbonyl, which engenders isolation and purification
problems in the synthesis of benzoin-protected chiral substrates.
More recently, Rajesh et al.30 examined the earliest events in the photolysis of benzoin diethyl phosphate
(8a). With nanosecond resolution, the LFP excitation of 8a in trifluoroethanol gave an immediate,
permanent 300-nm absorption identified as the benzofuran photoproduct (Eq. (69.5)). A second transient absorption at 570 nm was also observed which decayed with a first-order rate constant of ~2 × 106
s–1 in degassed acetonitrile or trifluoroethanol that was assigned to the triplet α-ketocation 10. The
intermediate could be trapped by trifluoroethanol, yielding trifluoroethyl benzoin ether. Evidence for
the intermediacy of the α-ketocation triplet came from experiments with added halide ion or azide in
which electron transfer quenching of the transient was observed. Oxygen and naphthalene quenching
experiments demonstrated that 310 was formed adiabatically on the triplet manifold from 38a. Temperature-dependent studies indicated an activation energy for decay of 310 of 8.6 kcal/mol to the singlet,
which then reacted with trifluoroethanol to form the trifluoroethyl ether. Stern–Volmer analysis of the
naphthalene quenching gave a triplet lifetime for 38a of τ3 = 18 ns.
The formation of 2-phenylbenzofuran during the nanosecond laser flash experiment was corroborated by
a picosecond study of 8a. A rise time of 2 to 4 ps was determined for the 340 nm transient. A rich fluorescence
emission obtained in the nanosecond study was shown to arise from 19 generated during the nanosecond
laser excitation pulse. Naphthalene also quenched the formation of 9 at the same rate as the formation of
310, establishing that the two primary photoproducts came from the same triplet (i.e., 38a). Thus, for the
unsubstituted benzoin phosphates, reaction proceeds exclusively through the triplet manifold.
Phenacyl
In a similar study, Sheehan and Umezawa12 employed a stripped-down version of the benzoin chromophore, the p-methoxyphenacyl group, for the release of benzoic acid, several amino acid derivatives,
and peptides, as shown in Eq. (69.6) and Table 69.3.
O
O
hν, pyrex
R
ethanol or
dioxane
MeO
12
+ free acid
MeO
13
(69.6)
In this case, the photoproduct was the p-methoxyacetophenone (13), a reduction product. The proposed
mechanism (Scheme 2) was a simple homolysis of the carbon-oxygen bond. Ethanol serves as a hydrogen
atom donor during this process, and in the presence of 1 M benzophenone or naphthalene the reaction
was completely quenched, indicating a triplet reaction pathway. Benzophenone and naphthalene are
known quenchers of acetophenones and have triplet energies of 68 and 62 kcal/mol, respectively.
Epstein and Garrossian31 reported the release of ethyl and phenyl phosphate esters from the corresponding p-methoxyphenacyl phosphates in 1,4-dioxane. The released phosphates were recovered in high
yields (Et, 86%; Ph, 74%) along with 13 (91–84%) as the only observed photoproducts. The absence of
any rearranged ester products contrasted with reports by Anderson and Reese34 for substituted αchloroacetophenones (vide infra). The rationale for the discrepancy advanced by Epstein was an altered
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TABLE 69.3 Percent Yield for the Release of Various Acidsa
from the Corresponding 4-Methoxyphenacyl Esters (12)
R
Solvent
Irradiation Time (hr)
Yield of ROOH (%)
PhCOO
PhCOO
Boc-L-Ala
Boc-L-Ala
Boc-Gly
Boc-L-Phe
Z-D, L-Ala
Phthaloyl-Gly
Tri-Gly
Z-L-Trp
Z-Gly-Gly
Z-L-Asp(OBz)-L-Ser
Dioxane
Ethanol
Dioxane
Ethanol
Ethanol
Dioxane
Dioxane
Dioxane
Dioxane
Ethanol
Ethanol
Dioxane
17
6
17
6
6
17
6
17
17
4
5.5
9
81
96
82
93
94
89
84
80
58
33
77
49
a
All reactions were carried out below room temperature at (5 × 10–3–10–2) M using
a Pyrex filter. Irradiations were complete in 6 hr in ethanol and 11–17 hr in
dioxane. Yields were determined from product isolation following photolysis.
Source: Adapted from Sheehan and Umezawa.12
SCHEME 2 Sheehan et al. mechanism
mechanism due to a change in solvent. The solvent 1,4-dioxane may not be sufficiently polar to support
the formation of the zwitterionic precursor to the Favorskii-like rearrangement that was proposed by
Anderson and Reese. This was required for the rearrangement to the spirodienedione intermediate.
Reinvestigation of the photolytic cleavage using polar solvents would have been an interesting test of this
hypothesis.
(69.7)
Givens et al.28 examined the photorelease of phosphate esters using t-butyl alcohol and methanol as
solvents, the former being a poor hydrogen atom donor and both exhibiting increased polarity compared
with 1,4-dioxane. The results correlated well with those of Anderson and Reese in that the major
photoproduct was the rearranged ester, not the photoreduction product (Eq. (69.7)). The amount of 13
was decreased to 21% in methanol and 14% in t-butyl alcohol. Further investigation of the solvent dependency
for the release of phosphates revealed a solvent isotope effect with deuterated vs. protiated methanol as the
solvent (Table 69.4). The formation of p-methoxyacetophenone was suppressed by a factor of five when
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Photoremovable Protecting Groups
TABLE 69.4 Quantum Efficiencies and Solvent Isotope Effects
(kH/kD) for Photolysis of 4-Methoxyphenacyl Diethyl Phosphate (14a)a
Φ14a
Φ15
kH/kD15
Φ13
kH/kD13
0.036
0.42
—
—
0.026
0.20
0.14
0.11
—
—
1.4
1.8
0.074
0.07
0.013
0.053
—
—
5.4
1.3
Solvent
C6H6, t-BuOH (3:1)
CH3OH
CD3OD
CH3OD
a
Irradiations were performed in the indicated solvent at 300 nm.
kH/kD is a relative efficiency for H vs. D abstraction. Error limits
are ±10%.
Source: Adapted from Givens et al.28
TABLE 69.5 Photolysis of α-Chloroacetophenones: Yields Obtained for
Photoproducts 17 to 20 Under Varying Conditionsa
Methanol
Acetone(aq)
Acetonitrile(aq)
16
Ar
17
19
17
18
20
17
18
20
a
b
c
d
Phenyl
4-Methyl phenyl
4-Methoxy phenyl
4-Chloro phenyl
60
62
35
60
00
06
34
00
37
24
16
10
24
47
43
14
29
23
14
31
14
14
09
06
47
70
54
35
18
11
08
18
a
Photolysis of 2% degassed solution of 16a–d in methanol, 95:5 acetone/
water, or 95:5 acetonitrile/water using 300-nm lamps was carried out in
the presence of propylene oxide as a halogen scavenger. Irradiations were
4 hr and yields are isolated products.
Source: Adapted from Dhavale et al.32
photolysis was carried out in CD3OD compared with either CH3OD or CH3OH, suggesting that a ratedetermining hydrogen abstraction occurs in the photoreduction process. Sheehan first suggested this
mechanistic pathway (vide supra).
Indeed, Dhavale et al.32 have shown that the ratio of rearrangement to reduction for substituted αchloroacetophenones is solvent dependent [Eq. (69.8) and Table 69.5]. Dhavale’s group reported that
irradiation of substituted α-choroacetophenones in methanol resulted in more photoreduction, whereas
in aqueous acetonitrile rearrangement to the phenylacetate esters became the major pathway. For a given
solvent, the ratio of rearrangement to reduction increased with the electron-donating power of the
substituent.
hν
O
Cl
Ar
16
solvent
O
Ar
CH3
17
+
O
OR
Ar
+
O
18 R = H
19 R = CH3
Ar
Ar
O
20
(69.8)
Scheme 3 outlines Dhavale’s proposed mechanism for the chloride loss and subsequent rearrangement,
beginning with carbon–chlorine bond homolysis. An electron transfer from the α-ketoradical to the
chlorine atom leads to the ion pair 21. The ion pair is more susceptible to Favorskii-like rearrangement
in polar solvents; therefore, the rearranged phenylacetic acid is favored in polar, protic solvents. Hydrogen
abstraction, resulting in the formation of 17, prevails in those solvents that are good hydrogen atom
donors.
Sonawane et al.33 investigated the photorearrangement of several para-substituted propiophenones as
a convenient entry for substituted α-arylpropionic acids, as shown in Eq. (69.9):
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O
O
O
Cl
hν
H-abstraction
X
CH3
Cl
X
+ HCl
X
16
17
hν
single
electron
transfer (SET)
O
H
X
H
OH
a Favorskii-like
O
Cl
1,2-aryl migration
Cl
O
X
X
21
18
SCHEME 3 The mechanism suggested by Dhavale et al.32
TABLE 69.6 Product Distribution from Photolysis
of 22a–i in Aqueous Acetone or Aqueous Methanola
Substrate
Acetone(aq)
Methanol(aq)
22
X
23
24
25
23
24
26
a
b
c
d
e
f
g
h
i
H
CH3
C 2H 5
n-C3H7
i-C4H9
t-C4H9
Cl
Ph
OCH3
58
84
82
84
74
78
45
40
32
25
5
6
5
10
7
25
25
10
—
—
—
—
—
—
20
35
50
39
76
74
77
65
69
30
18
80
30
8
9
9
15
8
51
26
12
—
—
—
—
—
—
30
35
70
a
Irradiations were carried out in 95:5 solvent/water
solutions employing a Hanovia 200-W, mediumpressure mercury vapor lamp with a Pyrex filter until
the complete disappearance of starting material. The
photoreaction was monitored and yields were determined with GLC and 1H NMR.
Source: Adapted from Sonawane et al.33
O
Cl
X
22a-i
solvent
propylene
oxide
O
O
hν, 300 nm
OR
OH
O
X
23
+
+
X
X
24
25 R = H
26 R = CH3
(69.9)
In almost every case, para-substitution promoted rearrangement (Table 69.6). Phenyl- and chloro-substitutions in the para position were the only cited examples where rearrangement did not dominate in
methanol. These cases also showed significant reduced and hydrolyzed products.
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Photoremovable Protecting Groups
TABLE 69.7 Product Formation from Photolysis
of Substituted Phenacyl Chloridesa
Aryl Substitution, X
% Ethyl Aryl Acetate
% Acetophenone
32
32
—
32
4
—
—
—
—
—
26
30
3
16
58
53
48
55
45
15
p-OH (27)
p-OMe (16c)
o-OH
o-OMe
p-Me (16b)
H
p-CO2Me
p-Cl (16d)
o-Cl
m-OMe
a
Photolyses were carried out in 1% alcoholic solutions using a
500-W Hanovia mercury arc lamp. Reactions were carried out for
1 to 2 hr. Products were isolated via vapor phase chromatography.
Source: Adapted from Anderson and Reese.34
While not purported to be a photoremovable protecting group, the study by Anderson and Reese34
on substituted phenacyl chlorides did reveal an interesting photochemical rearrangement for certain
members of the series, particularly the report that the Favorskii-like rearrangement of p-hydroxyphenacyl
chloride (27) in 1% aqueous ethanol gave two major photoproducts: p-hydroxyacetophenone (28) and
ethyl p-hydroxyphenyl acetate (29), as shown in Eq. (69.10). The authors proposed a spiro intermediate 30.
O
O
hν
Cl
OEt
+
1% aqueous
EtOH
HO
HO
28
27
O
HO
29
O
O
30
(69.10)
Further examination of the proposed aryl participation hypothesis led to the observation that electrondonating groups in the ortho or para positions were necessary for the rearrangement to occur (Table 69.7).
The results of Anderson and Reese coupled with the efficacy of the benzoin chromophore for cleavage
of α substituents attracted our interest in developing the p-hydroxyphenacyl group as a photoremovable
protecting group. We further rationalized that the introduction of a phenolic hydroxy group would
enhance the aqueous solubility. The absence of the attendant α phenyl substituents on benzoin alleviated
the stereogenic center problem present with benzoin derivatives. Furthermore, Anderson and Reese
reported a Favorskii-like rearrangement of the chromophore, e.g., p-hydroxyphenacyl to p-hydroxyphenylacetate for 27 → 29 and for 16b,c, suggesting a significant hypsochromic shift in the chromophore.
Thus the promise of p-hydroxyphenacyl as a possible phototrigger was too enticing to pass up.
In 1995, we began a comprehensive exploration of a variety of p-substituted phenacyl phosphates for
their efficacy toward releasing phosphate.2,28 Among the substituents examined, the p-acetamido, methyl
p-carbamoyl, and n-butyl p-carbamoyl groups proved untenable because they gave a plethora of products,
most of which resulted from coupling or reduction of an intermediate phenacyl radical [Eq. (69.11)]35,36
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CRC Handbook of Organic Photochemistry and Photobiology, 2nd Edition
TABLE 69.8 Disappearance and Product Efficiencies for Ammonium
Salts of p-Substituted Phenacyl Phosphate in pH 7.2 Tris Buffer at 300 nm
p-Substituent
31a NH2
31b CH3CONH
31c CH3OCONH
14c CH3Oa
33 HOb
35 HOc
a
b
c
Φdis
Φ34
<0.05
0.38
0.34
0.42
0.38
0.37
0.0
0.0
0.0
0.20
0.12
0.31
Φ32,13
Φother
<0.05
0.11
Not determined
0.07
0.0
0.0
Not available
Dimers
Two unknowns
Not available
0.0
0.0
Solvent was MeOH and diethyl phosphate was the leaving group.
The diammonium salt of the mono ester; 10% CH3CN was added to the
solvent.
The ATP derivative.
O
O
O
X
O
P O
hn, ROH
O
HO
+
X
31a-c, 14c
O
P O + other products
O
32a-c, 13
a) X = NH2, b) X = NHCOCH3, c) X = NHCO2CH3, 14c) X = OCH3
(69.11)
Table 69.8 gives the disappearance efficiencies for several p-substituted phenacyl phosphates from which
it is evident that release of phosphate does occur very efficiently for the acetamido and carbamoyl
derivatives; however, the large array of products of the phototrigger discouraged our further interest in
these three electron-donating groups.
The methoxy substituent (14c) showed a much cleaner behavior, yielding only two products from the
chromophore, p-methoxyacetophenone and the rearrangement product p-methoxyphenylacetic acid. The
p-hydroxyphenacyl phosphate (33) gave the rearranged p-hydroxyphenylacetic acid when photolyzed in
mixed aqueous organic solvents (Eq. (69.12)); in fact, of all of the groups examined, only p-hydroxy and
p-methoxy produced any rearranged phenylacetic acids.
O
HO
OR
O
P OR
O
33, R = Et
OH
hn, H2O
O
HO
+
HO
OR
P OR
O
34
(69.12)
The initial discovery that diethyl p-hydroxyphenacyl phosphate exclusively followed a rearrangement
pathway was followed by an extension of our study to p-hydroxyphenacyl ATP (35). Irradiation of 35 at
350 nm released ATP and p-hydroxyphenylacetic acid with a quantum efficiency of 0.37 ± 0.01 and a
rate constant for ATP appearance of 5.5 ± 1.0 × 108 s–1 [Eq. (69.13)].
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Photoremovable Protecting Groups
NH2
O
O O O
N
OPOPOPO
-O -O -O
O
N
NH2
N
N
hn
HO
- 34
O
ATP
. = 0.37
OH OH
35
N
O O O
HO POPOP O
-O -O -O
N
N
N
OH OH
kr = 5.5 x 108 s-1
(69.13)
While the mechanism of this process is unknown, the p-hydroxyphenacyl photorelease likely involves
an initial triplet state deprotonation of the phenolic hydrogen. In one scenario, the initially generated
triplet intermediate partitions between loss of a proton and C-O bond cleavage, as pictured in Scheme 4.
The exact course of the reaction depends greatly on the leaving group, solvent, and substituents attached
to the chromophore. Here, the triplet phenol undergoes the equivalent of a homolytic cleavage of the
bond to the substrate. In this scenario, it was envisaged that initial homolysis of the C-Y bond might be
followed by a rapid single-electron transfer process; that is, the triplet phenol is essentially converted to
its conjugate base before other competing processes for the radical pair can intervene. In this sequence,
a spirodienedione is eventually generated by electrocyclic closure of the intermediate zwitterion or possibly
the diradical.
The conjugate base formed by the proton loss undergoes bond reorganization to the putative spirodienedione 30 accompanied by release of ATP. Further hydration of the spirodienedione and bond
reorganization lead to the phenylacetic acid that is suggested for both pathways.
A second mechanistic scenario involves proton loss concomitant with direct neighboring group assistance for the release of the substrate and formation of the spirodienedione. The subsequent proposed
nucleophilic hydrolysis of the spirodienedione follows as above. Current evidence from subsequent
solvent and substituent studies favor the latter mechanism (vide infra).
O
HO
3
O
Y hν, 300 nm
Y
-H
ST
HO
Y = (R'O)2PO2-
khet
khom
HO
+Y
Y
O
pKa3
O
3
O
+
-Y
H2O
ket
O
O
29
kH
28
34
SCHEME 4 Proposed triplet state mechanisms for photorelease of substrates from the p-hydroxyphenacyl protecting
group.
The onset of the triplet-state phosphorescence emission of several p-hydroxyphenacyl esters indicated
triplet energies of 68.9 to 70.6 kcal/mol. The phosphorescence emissions were quenched by sodium 2naphthalenesulfonate or potassium sorbate. Quenching studies confirmed the reactivity of the triplet
state and further provided a lifetime of 5.5 ns for the triplet with a release rate of 1.82 × 108 s–1 in later
studies (vide infra).
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CRC Handbook of Organic Photochemistry and Photobiology, 2nd Edition
1
O
O
hn
OAc
OAc
ESIPT
(H2O)n
HO
HO
36
*
HO
O
CH3
O
O
O
O
HO
-HOAc
H2O
OH
37*
O
34
30
* = singlet or reactive ground state intermediate
SCHEME 5 ESIPT singlet state photorelease mechanism of p-hydroxyphenacyl acetate.37
HO
0.8
O
–
39
O
O
pH 7.05
pH 5.32
pH 5.05
pH 4.66
pH 4.38
pH 4.04
pH 2
38
0.4
0.0
350
400
450
500
/ nm
FIGURE 69.1 Transient absorption spectra obtained by LFP of p-hydroxyacetophenone in water (10% CH3CN)
with various buffers. (From Conrad II, P. G. et al., J. Am. Chem. Soc., 122, 9346–9347, 2000. With permission.)
The original proposal that the triplet state was the reactive state was challenged by Zhang et al.37 An
excited singlet state or possibly a tautomeric ground state (e.g., 37*) was proposed as the reaction
intermediate. In their studies, quenching by triplet quentchers was not observed during photolysis of phydroxyphenacylacetate (36), suggesting that the release of acetate occurred through an excited singlet
state possibly involving an intramolecular proton transfer. They postulated that the excited singlet state
intramolecular proton transfer (ESIPT) mechanism would form the quinone methide 37* that could
either continue on to the spirodienedione (30) or decay to ground state and subsequently undergo release
of acetate to form 34. Such a mechanism has precedence in the earlier work of Wan37,38a and Yates.38b
Laser flash photolysis (LFP) studies by Givens and Wirz39 with diethyl pHP phosphate (Eq. (69.12))
confirmed the intermediacy of the phenacyl triplet state. Energy transfer quenching to naphthalene gave
a rate of formation of the naphthalene triplet of 7.8 × 109 M–1 s–1. The presence of dioxygen increased
the decay rate of the pHP phosphate triplet (kq ≈ 3 × 109 M–1 s–1). It was estimated from this study that
pHP intersystem crosses with a rate constant of 3.1 × 1011 s–1 in aqueous acetonitrile. Quenching studies
of the photochemical release of substrates from a series of pHP derivatives employing potassium sorbate
gave excellent linear Stern–Volmer quenching results with lifetimes of 10–8 to 10–9 s for their pHP triplet
states. These combined results firmly established the triplet as the reactive excited state.
LFP studies on the parent chromophore proved revealing. The p-hydroxyacetophenone triplet undergoes a facile adiabatic proton tautomerization converting the phenol 28 into its conjugate base 38.
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Photoremovable Protecting Groups
O
OH
pKE = 16.4 (-1.0)
O
HO
39
28
pKa = -8.5 (4.6)
pKa = 7.9 (3.6)
O
O
38
SCHEME 6 Tautomerization of p-hydroxyacetophenone in the ground state and triplet excited state (in parentheses).
(From Conrad II, P.G. et al., J. Am. Chem. Soc., 122, 9346-9347, 2000. With permission.)
Evidence for this unusual adiabatic proton transfer to solvent from the triplet p-hydroxyacetophenone
came from analysis of the transient triplet absorption spectra of 28 as a function of the pH (Figure 69.1).
These results were further supported by DFT calculations that provided the pKE values for the equi39 in their ground and triplet excited states. A pKa3 for 283 of 3.6 was derived from
librium 28
these data and the triplet state pKa3 of 39 (4.6) and the known ground state pKa of 28 (7.9) (Scheme 6).
These studies revealed an increase in acidity of over four pKa units relative to the ground state. The proton
tautomer 39 is a non-productive intermediate because it is thermodynamically incapable of cleaving the
ester C–O bond.
Incorporating the rapid deprotonation that results from the large adiabatic decrease in pKa of 28 as a
feature of the p-hydroxyphenacyl mechanism suggests that the conjugate base 38 is an attractive precursor
to the rate-limiting release of the substrate. Because the triplet is formed with a ST rate constant of 3.1
× 1011 s–1, it is unlikely that there is any singlet state contribution to the deprotonation step. Rather, this
appears to be exclusively a triplet process occurring on the excited triplet surface; that is, the two
protonated species and the unprotonated ion undergo adiabatic proton tautomerizations (Scheme 6).
The electron-rich aromatic ring increases the potential for intramolecular neighboring group attack at
the α-carbon, leading to the release of the substrate and rearrangement of the chromophore. Thus, it
becomes prudent to carefully explore the change in the pKa of the phenolic protons transitioning between
the ground and excited triplet states as a key element in understanding the role played by aryl participation
in the release step.
Coumaryl and Arylmethyl
Zimmerman’s early studies40 on the photosolvolysis of benzyl acetates in 50% aqueous dioxane set the stage
for a variety of studies that employ m,m′-dimethoxybenzyl as a photoremovable protecting group (69.14).
In general, the photofragmentation reactions of benzyl acetates are quite rapid, with rate constants of 108 s–1
or higher and are primarily singlet-state processes. According to Zimmerman, meta activation of the excited
singlet state of benzyl acetates occurs through the approach of the excited- and ground-state energy surfaces,
funneling the excited state toward heterolysis of the benzyl–ester bond. Substituents, including electron
donors, in the para position lead primarily to homolytic fission and radical derived products.
Direct heterolytic fission of the substrate-photoprotecting group bond is the required course for
photorelease of most biologically important substrates. This process avoids the generation of destructive
radicals that could result in reactions such as decarboxylation, radical dimerization, or redox processes.
Thus, the effect of m-substitution on the photochemistry of benzyl, naphthyl, and other aromatic
chromophores has become the object of many studies in search of alternatives to the o-nitrobenzyl class
of protecting group.
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CRC Handbook of Organic Photochemistry and Photobiology, 2nd Edition
-CO2
hn
X
hn
40
H3COCO
SCHEME 7 Mechanistic scheme for arylmethyl ester photolysis in methanol.43
OCOCH3
hn ,
50% aq. dioxane
OCH3
H3CO
OH
. = 0.10
H3CO
40
OCH3
41, 79%
(69.14)
41
42,43
on the nature of the meta effect
Recent studies by both Zimmerman and DeCosta and Pincock
have raised concerns about whether heterolytic or homolytic cleavage should be considered the primary
photochemical process. According to Pincock, the mechanistic pathway for all substituted arylmethyl
substrates begins with homolysis of the C–O ester bond to the substrate followed by a competition
between electron transfer to an ion pair or typical ground-state radical reactions (Scheme 7).43 For those
arylmethyl derivatives substituted with a meta electron-donating group, such as methoxy, the electron
transfer occurs more rapidly than competing radical processes due to favorable redox properties of the
radical pair.
Normally, the details of the steps leading to the ion pair would not play a significant role in the outcome
of the photorelease process except in those circumstances where the radical pair precursors have rapid,
favorable divergent pathways available. Such could be the case for carboxylate esters, such as C-protected
amino acid, peptides, and protein derivatives, where decarboxylation of the initially generated carboxy radical
may compete with electron transfer to the ion pair. This deleterious process can become significant, leading
to destruction of a portion of the released substrate. By either of these mechanisms, however, the productdetermining process for meta- and especially the di-meta-substituted arylmethyl chromophores leads principally to an ion pair, an intermediate arylmethyl carbocation, and the conjugate base of the leaving group.
The coumaryl chromophore is essentially another arylmethyl analog, which has the attractive feature
of high yields of fluorescence emission, sometimes a useful property for following the course of substratechromophore processes. One of the earliest studies using coumarin as a chromophore was the photorelease of diethyl phosphate from coumarylmethyl diethyl phosphate.26 The resulting coumarylmethyl
cation covalently attaches to a wide variety of nucleophiles, as shown in Eq. (69.15):
O
OP(OEt)2
Nu
hν, 360 nm
Nu
H3CO
O
O
H3CO
O
O
42
43
Nu = CH3OH, piperidine, cysteine, tyrosine, α-chymotrypsin, HMT
(69.15)
Furuta et al.44 have reported the application of the coumaryl chromophore as a phototrigger for the
release of cAMP (Eq. (69.16)); as shown in Table 69.9, the methoxy and hydroxy methylcoumarins gave
the best conversions.
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Photoremovable Protecting Groups
TABLE 69.9 Percent Conversion of Coumarylmethyl cAMP After a 10 s Irradiation at 334 to 365 nm
Caged cAMP
R = Acetyl
R = Propionyl
R = Hydroxy
R = Methoxy
23
9
64
60
Conversion (%)
NH2
N
N
O
O
P
O
RO
O
NH2
N
N
N
O
O
N
OH
O
hν, 334 - 365 nm
O
HO
N
N
O
P
O
O
OH
(69.16)
44
The historical and mechanistic background for the most common photoprotecting groups were presented above. Examples that employ photoremovable protecting groups are given to illustrate the range
and variety of the applications in chemistry and biology. As noted earlier, these are a very limited set of
examples of the numerous published applications in biology and, to a lesser extent, in chemistry. Further
information can be obtained from the reviews listed in References 1 through 7.
69.3 Carboxylic Acids
o-Nitrobenzyl
Because o-nitrobenzyl derivatives have been the most widely applied photoremovable protecting groups,
modifications of this chromophore have received considerable attention. A recent study45 employing 2,2′dinitrobenzhydryl (DNB) for N-methyl-D-aspartate (NMDA) probed the NMDA receptor, which is one
of the general classes of known glutamate receptors. The carboxyl group of NMDA was esterified with
DNB, a stronger UV absorber (λmax 350 nm: ε = 1.69 × 104 M–1 cm–1) than typical o-nitrobenzyl analogs.
The poor aqueous solubility of DNB-NMDA, however, required addition of 20% DMSO to attain
complete dissolution. A single 308-nm laser pulse was sufficient to release NMDA within 4.2 µs, with a
quantum efficiency of 0.18, as shown in Eq. (69.17).
O2N
O
O
hν, 308 nm
O
HO
H3C
NH
O
NO2
pH 7
O
NO2
+
NO
OH
HO
H3C
NH
O
NMDA
β-O-DNB-NMDA
(69.17)
The time constant for the release of NMDA is pH dependent, occurring within 3.8 µs at pH 3.8 and 13.8
µs at pH 10.6, consistent with the rate of decay of the aci-nitro intermediate. The relatively rapid release
rate from DNB suggests that this protecting group could be useful for further studies of the NMDA
receptor.
The versatility of the o-nitrobenzyl group has also been demonstrated in solid-phase synthesis. The
phosphate group of the nucleotide tethered to a carboxylic acid through an alkyl chain provides a
convenient link to the o-nitrobenzyl group, which is attached to a solid support. Synthetic manipulation
of the oligonucleotide can be carried out under standard conditions and then release of the synthesized
1348_C69.fm Page 18 Monday, October 13, 2003 3:22 PM
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CRC Handbook of Organic Photochemistry and Photobiology, 2nd Edition
TABLE 69.10 Isolated Yields of Completely Deprotected Oligonucleotides
Protection (%)
Deprotection (%) 45
Deprotection (%) 45′′
96
89
97
90
70
91
92
69
77
80
71
70
a
b
c
d
oligonucleotide from the support is conducted photochemically.46 The carboxylate and substituted onitrobenzyl alcohol were coupled in 89 to 97% yields followed by the controlled pore glass (CPG) loading.
Standard oligonucleotide synthesis was then carried out at the 3′-terminus to obtain 45a–d and 45′′a–d
(Eq. (69.18).
DMTrO
1. std. oligonuceotide
synthesis
2. hn, 400 nm
O
n
NO2
O
H3CO
HO
H
N
O
O
P O (CH2)n+1CO2H
O
Oligo.
3. detritylation
4. NH4OH
n 1 2 3 4
a b c d
O
45a-d (n = 1-4) Oligo = T20
45'a-d (n = 1-4) Oligo = TAC GCA ATC CTA GAT CTA AT
DMTr = 4,4'-dimethoxytrityl
(69.18)
Upon completion of the synthesis, the oligonucleotides were severed from the CPG solid support photochemically using a 400-nm light source. The efficiency of the release process was as high as 92%, as
shown in Table 69.10. This protocol is useful for elaborating the 3′-terminus of oligopeptides using mild,
traceless reagent conditions at room temperature and neutral pH.
Coumaryl
The photochemical and photophysical behavior of 4-(hydroxymethyl)-7-methoxycoumarin (MCM)
caged acids was studied under physiological conditions by Bendig et al.47 (Eq. (69.19)).
O
OH
X
hn, 333 nm
MeO
O
+
MeO
O
O
HOX
O
R
CH3
X=
O
O
46a
46b: R = OCH3
46c: R = H
46d: R = CN
(69.19)
Photocleavage of the excited singlet state of MCM caged compounds is thought to proceed via a photoSN1 mechanism (solvent-assisted photoheterolysis). Evidence favoring this mechanism was found from
irradiation of MCM caged derivatives in 18O-labeled water, which exclusively incorporated the 18O-label
in the MCM–18OH product (see Scheme 8). The deprotection process of the MCM derivatives was
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Photoremovable Protecting Groups
TABLE 69.11 Data for 4-(Hydroxymethyl)-7-Methoxycoumarin
(MCM) Caged Acids
Acid
Protection (% Yield)
Deprotection (Φ)
46a
46b
46c
46d
82.6
90.0
98.0
83.8
0.0043
0.0045
0.0052
0.0064
CH3
1
MC CH2 OX
*
1
MC CH2
OX
*
R-H
H3CO
kset
-kset
hn
O
O
(trace)
k1
MC CH2
OX
k0
kesc
MC CH2 OX
MC = 7-methoxycoumarin-4-yl moiety
MC CH2
OH
(solvent)
MC CH2 OH
+
OX
H
(solvent)
HOX
SCHEME 8 Reaction pathways for photolytic cleavage of MCM esters.
dependent on the leaving group and the solvent polarity. The quantum efficiencies for the reactions of
46a–d were low, in the range of 0.0043 to 0.0064 as listed in Table 69.11. MCM–OH is a highly fluorescent
product that can be conveniently monitored during the course of the reaction. Furthermore, MCM caged
compounds are very stable to the hydrolysis.
Phenacyl
The rapid release (~108 s–1) of substrates from the p-hydroxyphenacyl (pHP) group enables fast biological
processes to be studied. p-Hydroxyphenylacetic acid (34) is generated with a quantum efficiency (Φrea)
of ~0.18. In contrast, the presence of added electron-donating substituents on the aromatic ring of the
pHP group makes the rearrangement a minor pathway for 47′′a–b and completely suppress it for 47′′′a–b
(Scheme 9).48 The quantum efficiencies for the disappearance of the various pHP esters, the appearance
of (34), and corresponding pHP-protected substrates are given in Table 69.12 .
A complication that could arise in such systems is the potential for decarboxylation of the released
carboxylate ion. However, no decarboxylation products were observed within the detection limits of 1HNMR and HPLC.
With the production of a biologically benign photoproduct, the pHP protecting group has proven to
be an efficient tool for investigations of fast biological processes. For example, Givens et al.49 applied the
pHP phototrigger to the investigation of the bradykinin BK2 receptor. It is known that bradykinin acts
as an active pain-transducer when released during tissue damage. A major difficulty in studying the
detailed physiological mechanism of the action of bradykinin is a concomitant rapid enzymatic degradation of the nonapeptide immediately after its release from its precursor protein. Therefore, the photorelease from pHP bradykinin (48), which protects bradykinin from degradation of the agonist prior
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CRC Handbook of Organic Photochemistry and Photobiology, 2nd Edition
TABLE 69.12 Quantum Yields of pHP Esters for Irradiation at 300 nma
Ester
47a
47b
47c
47d
47e
47f
48
Solvent (Water/AcCN)
Φdis
Φapp
Φrea
Water
Water
6:4
6:4
6:4
7:3
Water
0.21
0.14
~0.2b
0.18
~0.18b
0.24
0.21
0.21
0.14
—
0.17
—
0.23
0.22
0.19
0.08
—
0.14
—
0.17
0.19
pHP GABA
pHP glutamate
pHP cyclopropylacetate
pHP phenylacetate
pHP pivalate
pHP oleate
pHP bradykinin
An NMR tube was charged with ~10 mg (~40 µmol) of the appropriate photoprotected acid and 10 mol% of 1,2,3-benzenetricarboxylic acid as an internal standard
in 2 mL of solvent. The quantum efficiencies were determined at less than 20%
conversion of the starting ester.
By comparison with 47d.
a
b
Abbreviations: dis = disappearance, app = appearance of acid, rea = rearrangement
to phenylacetic acid.
O
HO
3
O
1. hn
2. ISC
3. deprotonation
OCOR'
OCOR'
O
R
47a-f
47 R = H 47' R = 3-OMe 47" R = 3,5-OMe
R
47*
reduction
rearrangement
H2O
O
O
HO
OH
+
OH
HO
R
+ R'CO2
O
HO
O
R
R
O
R
SCHEME 9 Photoreaction for the pHP esters.
to release, facilitates the investigation of its action during the transduction process by allowing precise
temporal and spatial release of bradykinin to activate the bradykinin BK2 receptor (Eq. (69.20)).
O
NH
H2N
N
H
N
N
O
O
N
H
H
N
O
HO
O
N
H
N
O
O
N
H
NH2
H2N
48
H
N
O
NH
O
CO
O
OH
N
H
hn, 337 or 300 nm
NH2-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-CO2 + 34
D2O, Pyrex
(69.20)
pHP Bradykinin 48 was obtained in an overall yield of 84% by derivatizing the partially protected
bradykinin, obtained by cleavage of the C-terminus from the resin after sold-phase Merrifield synthesis.49
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Photoremovable Protecting Groups
Reaction of the C-termnus with p-hydroxyphenacyl bromide followed by treatment with 1% TFA gave
pHP bradykinin 48, free of the other protecting groups employed during the solid phase synthesis.49
A single 337-nm flash (<1 ns) released sufficient bradykinin to excite the BK2 receptors on single rat
sensory neurons, which dramatically increased the intracellular calcium concentration measured with
Indo-1, a Ca2+-chelating fluorescent indicator. The quantum efficiency of bradykinin appearance was
independently determined to be 0.22, as shown in Table 69.12.
Benzoin
There are a number of examples of benzoin and substituted benzoin esters that have been employed as
photoremovable protecting groups for carboxylic acids. Among these, the application of benzoin as a
traceless linker for solid-phase synthesis of oligopeptides and introduction of Fmoc-protected amino
acids by Balasubramanian50 is instructive. As an example, the release of Fmoc-Ala shown in Eq. 69.21
occurs upon photolysis at 350. Balasubramanian found that the maximum yield was obtained after a 2h photolysis as determined by HPLC.
NHFmoc
HO
O
O
hν, 350 nm
O
Ph
+
NHFmoc
O
O
Ph
O
O
(69.21)
In order to avoid premature photolysis of the benzoin linker by adventitious room light during the course
of the synthesis, the dithiane-protected 3-alkoxybenzoin (49a) has been suggested as a “UV-inactive”
linker. Dithiane 49 released less than 3% of the product after irradiation at 350 nm. The photosensitivity
is restored by hydrolysis of the dithiane prior to photocleavage, as illustrated in Eq. (69.22). Photolysis
of the deprotected linker resulted in a 75% yield of the product.
O
NHFmoc
O
S
S
Ph
(i)
NHFmoc
O
O
O
O
Ph
O
49b
49a
(i) (a) bis[(trifluoroacetoxy)iodo]benzene, (b) mercury (II) perchlorate, or (c) periodic acid
(4 eq.), THF:water (10:1) (for a and c), THF (for b), ambient temperature, 18 h. >95%
conversion
(69.22)
Other
A series of 2,5-dimethylphenacyl (DMP) esters were photolyzed in benzene or methanol (Eq. (69.23)).
O
R
O
O
50
hν, >300 nm
O
O
+
benzene
or methanol
HO
R
85-95%
51
(69.23)
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TABLE 69.13 Data for 2,5-Dimethylphenacyl Esters
Protection
(%Yield)
R
CH3
C 6H 5
C 6H 5
C6H5CH2
n-Pentadecyl
N-(t-butoxycarbonyl)-L-phenylalanine
a
b
Photolysis Condition
(nm)
84
95
95
84
69
51
Deprotection
(% Yield)a
85b
86
92
91
95
90
Benzene, >280
Benzene, >280
Methanol, >254
Benzene, >280
Benzene, >280
Benzene, >280
Isolated yield of the crude acids (>95% purity).
Determined by gas chromatography.
TABLE 69.14 Quantum Yieldsa of DMP Esters
Protection
(% Yield)
X
–OCOPh
95
–OCOCH2Ph
84
–OCOCH3
84
a
Photolysis
Condition
Deprotection
(Φ)
Benzene
Methanol
Benzene
Methanol
Benzene
Methanol
0.23
0.09
0.18
0.11
0.25
0.14
Quantum yield for ester release; valerophenone was used as
an actinometer; irradiated at λ >300 nm. Error margins are
approximately 10%.
The formation of the corresponding carboxylic acids occurred with almost quantitative isolated yields,
as shown in Table 69.13.51 In contrast with the structurally related p-hydroxyphenacyl esters, the release
mechanism from 50 occurs through an efficient intramolecular γ-hydrogen abstraction via the (n,π*)
excited ketone.
MeO
O
X
hν, >300 nm
51
+
O
+
HX
(69.24)
Recently, Wirz and Klan52 reported LFP studies on several of the DMP esters in benzene and methanol
(Eq. (69.24)). Quantum efficiencies (Φ) in benzene are 0.18 to 0.25, while those in methanol fell to 0.09
to 0.14. Thus, this photoprotecting group appears to be better suited to applications in nonpolar media
such as benzene rather than methanol (Table 69.14).
A novel design of “orthogonal” protecting groups, i.e., the removal of protecting groups selectively
from a multi-protected substrate, has been reported by Bochet53 in which the irradiation wavelengths
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Photoremovable Protecting Groups
serve as the “orthogonal reagents”. A mixed diester of pimelic acid was capped with a dimethoxybenzoin
group at one end and an o-nitrobenzyl group at the other terminus. Despite the possibility that intramolecular energy transfer or equilibration could occur between the two chromophores, selective photolysis
led to the sequential removal of each with high chemical yield, as shown in Eq. (69.25). Upon photolysis
of 52 at 254 nm for 5 minutes, 92% of 53b was released, whereas irradiation at 420 nm for 24 hours
released 70% of 53a, as determined by 1H NMR.
O
O
MeO
O
NO2
OMe
52
1. hν, 420 nm
2. TMSCHN2
70%
O
O
O
1. hν , 254 nm
2. TMSCHN2
92%
MeO
MeO
OMe
O
OMe
53a
O
O
Ph
O
MeO
OMe
O
O
MeO
Ph
NO2
(69.25)
53b
OMe
69.4 Phosphates and Phosphites
o-Nitrobenzyl
The most notable example of o-nitrobenzyl (ONB) caged phosphates remains that reported by Trentham
and co-workers54 on the synthesis and photochemistry of caged ATP in the late 1980s. Caged ATP, P3-1(2-nitrophenyl)ethyladenosine 5′-triphosphate (54), was synthesized in nearly quantitative yield in three
steps starting with commercially available o-nitroacetophenone. Reaction with hydrazine to give the
corresponding hydrazone, followed by oxidation with MnO2 provided the aryldiazoethane precursor that
was used to alkylate ATP. The photolysis of caged ATP furnished ATP with an efficiency of 0.63, as
illustrated in Eq. (69.26):
NH2
N
O
O
O
O P O P O P O
OOONO2
54
N
O
OH OH
N
N
hν, 320 nm
TES buffer, KCl, MgCl2, pH 7.1
Φ app = 0.63
ATP
+
O
NO
(69.26)
The rate of release of ATP was found to be dependent on the pH and the relative concentration of
magnesium ion in solution. Pelliccioli and Wirz5 have shown that the rate-determining step is the decay
of the hemiacetal (or hemiketal) intermediate between pH 4 and 8. In this region, the slow hydrolysis of
the hemiacetal limits the mechanistic value of the o-nitrobenzyl protecting group to studies of relatively
slow reactions (e.g., kr < 103 s-1).
The nitroso byproduct has also proved problematic for spectroscopic analyses of ONB reactions due
to its reactivity with some substrates and with proteins. This problem was circumvented by conducting
the photolysis in the presence of dithiothreitol, a hydrophilic thiol and an excellent nucleophile that
readily sequesters the nitroso byproduct.
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TABLE 69.15 Results of Photolysis of cAMP and cGMP Coumaryl Esters
Coumaryl Derivative
55a
55b
55c
a
Φdisa
λmax (nm)
0.21 (0.25)
0.12 (0.16)
0.10 (0.14)
402
326
346
Solvent
80:20 HEPES-KCl buffer/MeOH
HEPES-KCl buffer, pH 7.2
HEPES-KCl buffer, pH 7.4
For the cAMP ester; quantum efficiency for the cGMP derivative is in parentheses.
Coumaryl
Coumarylmethyl esters have been used as photoprotecting groups by several groups (e.g., Furuta et al.55,56
and Hagen et al.57) for deprotection of cAMP and cGMP. To overcome the limited aqueous solubility of
these derivatives, Hagen has modified the coumaryl chromophore with carboxyl and amino substituents.57
Three new variants of the coumaryl system were synthesized and their photochemistry explored — (7diethylaminocoumarin-4-yl)methyl (DEACM), (7-carboxymethoxycoumarin-4-yl)methyl (CMCM),
and (6,7-bis[carboxymethoxy]coumarin-4-yl)methyl (BCMCM) as esters of cAMP and cGMP. Photolysis
resulted in liberation of the free cyclic phosphate along with the hydrolyzed chromophores, i.e., 56a-c
(Eq. (69.27)).
R1
R1
R2
R2
hν, 333 nm
O
A (or G)
O
O
O
P O
O
O
HO
cAMP or cGMP
+
OH
O
O
56
55a R1 = H, R2 = Et2N
55b R1 = H, R2 = OCH2CO2H
55c R1 = R2 = OCH2CO2H
(69.27)
The caged coumaryl compounds were synthesized in 11 to 34% yield, clearly a limitation with this
photoremovable protecting group. The addition of the carboxymethoxy groups in 55b and 55c dramatically enhanced the water solubility of these analogs as compared with the ester 55a, and their photorelease
occurred with good quantum efficiencies as seen in Table 69.15. On the other hand, the less water-soluble
55a had the best quantum efficiency, and its absorption maximum was the most red shifted in the series.
Phenacyl
In light of its inherent advantages over other chromophores, the p-hydroxyphenacyl group has received
recent attention as a promising photoprotecting group for phosphates. Recent reports on p-hydroxyphenacyl esters of phosphate, diethyl phosphate, and ATP by Givens et al.36 and on GTP by Du et al.58 indicate
that these phosphate esters undergo efficient photorelease of the phosphoric acid moiety along with the
rearranged p-hydroxyphenylacetic acid as the sole photoproducts of the reaction (Eq. (69.28)).
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Photoremovable Protecting Groups
O
O
OP OR1
OR2
HO
O
hν, 300 nm
-
10% CH3CN/TRIS buffer, pH 7.3
O-P OR1
OR2
+
34
57a R1 = R2 = Et
57b R1 = R2 = NH4+
57c R1 = GDP, R2 = NH4+
35 R1 = ADP, R2 = NH4+
(69.28)
TABLE 69.16 Data for pHP Caged Phosphates
Caged
Phosphatea
Protection
(% Yield)
Deprotection
(% Yield)
Φdis
57a
57b
35
57c
87
96
42
20
Not determined
Quant
Quant
Not determined
0.77
0.38
0.37 (0.30)b
Not determined
a
b
Results shown for 57c are from Du et al.;58 derivative 57c was
photolyzed at 308 nm.
The value in parentheses for 35 is the quantum efficiency for the
appearance of ATP.
Derivative 57a was synthesized by reacting the phosphate directly with p-hydroxyphenacyl bromide.
Derivative 57b was synthesized from hydrogenolysis of the pHP dibenzyl phosphate ester. For 35 and
57c, the pHP monophosphate (57b) was first protected as the corresponding ketal that was then coupled
with ADP or GDP, respectively. The caged ATP and GTP analogs were then obtained by hydrolysis of
the ketal. Yields and quantum efficiencies for the disappearance of the pHP phosphate esters are given
in Table 69.16.
pHP caged ATP was recently used as a probe in the study of Na,+ K+-ATPase, an enzyme involved in
the intracellular transport of sodium and potassium ions.59 Membrane samples possessing the ionchannel proteins were bathed in caged ATP which was activated by UV laser flash photolysis. Na,+ K+
channel transport was observed as a result of the activation of the enzyme by the released ATP. Direct
spectroscopic evidence of the release of ATP was obtained by time-resolved Fourier transform infrared
(FTIR) spectroscopy (Figure 69.2). The changes in the characteristic absorptions of the prominent functional groups of the reactant and product include the disappearance of the γ-PO2– ester band of the caged
ATP at 1270 cm–1 and the appearance of the free γ-PO3–2 band of the released ATP at 1129 cm–1.
Benzoin
In 1994, Pirrung and Shuey60 reported the protection of phosphates using dimethoxybenzoin. Resolved
(R)3′,5′-dimethoxybenzoin (optically active) was converted to the phosphoramidite by treatment with
diisopropylaminocyanoethoxychlorophosphine. Subsequent reaction with the appropriate primary or
secondary alcohol followed by oxidation led to the series of phosphate esters 58a-e in moderate to high
yields:
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0.015
CO2-of product
ATP PO32-
∆Abs.
0.010
0.005
0.000
-0.005
Caged ATP PO2-
-0.010
2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900
Wavenumber / 1/cm
FIGURE 69.2 Time-resolved FTIR difference spectrum for the photolysis of pHP caged ATP. The absorbance was
measured 10 ms to 11 s after the photolysis flash and subtracted from the absorbance prior to photolysis. (We thank
Professors Klaus Fendler and Andreas Barth for the TR-FTIR results with pHP ATP.)
MeO
O CH2CH2CN
O P OR
O
Ph
hν, 350 nm
O
O
O
P
OR
+
CN
-O
11
OMe
58
CO2Me
CO2Me
R=
NHBoc
NHBoc
Me
Me
TrO
O
O
T
Me Me
O
O
O
58a
58b
58c
58d
58e
(69.29)
Photolysis of 58a-e shown in Eq. (69.29) led to release of the phosphate derivative along with the
benzofuran byproduct 11. The overall sequence from the phosphoramidites to phosphate esters 58a–e
was accomplished in moderate yields, and the unprotected phosphates were obtained in good yields upon
photolysis, as seen from Table 69.17.
TABLE 69.17 Yields for the
Photolysis of DMB Phosphoramidites
R
Protection
(% Yield)
Deprotection
(% Yield)
58a
58b
58ca
58d
58e
45
82
58
60
55
85
86
85
83
87
a
The (R) enantiomer.
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Photoremovable Protecting Groups
69.5 Sulfates and Other Acids
Sulfonic acids have largely remained unexplored in terms of a functional group that is released from a
photoremovable protecting group. One recent example has been reported by Bendig et al.,47 in which
methanesulfonic acid was protected as the corresponding methoxycoumarin derivative. (7-Methoxycoumarin-4-yl)methylmethanesulfonate 59 was synthesized from reaction of methanesulfonic acid with 4(diazomethyl)-7-methoxycoumarin in refluxing chloroform. Photolysis at 333 nm resulted in the release
of methanesulfonic acid, as shown in Eq. (69.30).
O
S
CH3
O
O
OH
hν, 333 nm
+
H3CO
O
O
7:3 HEPES buffer/MeOH
pH 7.2
O
H3CO
59
O
O
HO S CH3
O
60
HEPES = N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)
(69.30)
While the protection yield for 59 was low (26%), the quantum efficiency of 0.081 is reasonable when
compared with other leaving groups that were reported in this study. The chemical yield of the deprotection was not provided; however, the photoproduct 60 was recovered in ≥95% yield, suggesting a high
yield of the released sulfonic acid.
69.6 Alcohols, Thiols, and N-Oxides
o-Nitrobenzyl
Among the most common photoprotecting groups used for alcohols are the o-nitrobenzyl derivatives.
Recently, Iwamura61 reported the synthesis and photochemistry of caged carbohydrates protected with
an o-nitrobenzyl group at one or more positions within the molecule. Equation (69.31) illustrates a
typical example in which photolysis of the caged glucopyranoside 61 at 350 nm in methanol led to the
release of the free methylglycoside 62 in 60% yield, along with the nitroso byproduct 3.
NO2
O
HO
HO
OH
O
hν, 350 nm
MeOH
OH
O
HO
HO
HO
+
OMe
OMe
61
3
62
(69.31)
Derivative 61 was synthesized in 71% yield by reductive bond cleavage of the corresponding 4,6-O-onitrobenzylidene acetal of methyl 3,4-acetyl-β-glucoside with triethylsilane and boron trifluoride etherate, followed by deacetylation of the 3,4-diacetates with sodium methoxide in methanol.
o-Nitrobenzyl chemistry was also extended to the release of thiols as demonstrated by Smith et al.,62
who demonstrated that a cysteine congener was protected as the corresponding thioether 63 in 89%
yield. Photocleavage occurred at 366 nm to give the liberated Boc-protected thiol 64 in 44% yield in the
presence of semicarbazide hydrochloride, a carbonyl scavenger (Eq. (69.32)).
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O
O
NHBoc
H2N
S
hν, 366 nm
NO2
NHBoc
H2N
+
1:1 CH3CN/0.05 M PBS
3
SH
pH 6
64
63
(69.32)
Finally, the addition of ascorbic acid as an antioxidant to the reaction mixture gave a quantitative yield
of 64. No thiol was recovered when the photolysis was carried out in the absence of ascorbic acid, despite
experimental evidence of the disappearance of 63.
While the o-nitrobenzyl system is well accepted as a photoremovable protecting group, it nevertheless
suffers the limitation of toxicity to the biological entity and a highly reactive nitroso functional group
formed as the byproduct. As was seen in the case of the cysteine cogener 63, the photodeprotection may
require a scavenging or reducing agent if thiols are to be generated in such a strategy.
In the late 1990s, Pfleiderer and co-workers63 developed a new, β-substituted variant of the o-nitrobenzyl chromophore, 2-(o-nitrophenyl)ethoxycarbonate. The 5′-O-2-(o-nitrophenyl)ethoxycarbonyl thymidines were obtained from 2-(o-nitrophenyl)ethanol carbonates by reaction with diphosgene under
basic conditions, followed by treatment with thymidine in anhydrous methylene chloride at reduced
temperature. The synthetic yields ranged from 41 to 81%.
Photolysis of a 0.1-mM solution of the photoprotected thymidine 65 in a 1:1 methanol/water mixture
at 365 nm resulted in the release of thymidine (66), carbon dioxide, and the photolabile o-nitrostyrene
derivative 67 (Eq. (69.33)).The photorelease is believed to occur through a β-elimination mechanism64,65
from the aci-nitro intermediate 67′′ shown in Scheme 10.
NO2
hν
O
O
OH
N
O
NO2
O
O
OR'
+
R
R
R
R'OH
-CO2
OR'
67'
67
SCHEME 10 β-Elimination mechanism proposed for 2-(o-nitrophenyl)ethoxycarbonyl thymidines.
O
O
HN
R3
HN
NO2
R2
O
O
R1
O
O
R
N
hν, 365 nm
O
1:1 CH3OH/H2O
O
HO
O
R3
N
+
-CO2
NO2
R2
R1
R
OH
OH
66
65
65a
65b
65c
65d
R = CH3, R1, R2,R3 = H
R = o-nitrophenyl, R1, R2,R3 = H
R = H, R1 = I, R2,R3 = H
R = H, R1 = Br, R2,R3 = H
65e
65f
65g
65h
67
R = H, R1 = Cl, R2,R3 = H
R = H, R1, R3 = Cl, R2 = H
R = H, R1 = F, R2,R3 = H
R = R1 = H, R2,R3 = OCH3
(69.33)
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Photoremovable Protecting Groups
TABLE 69.18 Yields for 2-(o-Nitrophenyl)ethoxycarbonyl Thymidine Derivatives
Derivative
Protection
(% Yield)
Φ
Deprotectiona
(% Yield)
65a
65b
65c
65d
65e
65f
65g
65h
71
70
68
73
80
81
70
41
0.35
0.20
0.10
0.076
0.070
0.072
0.037
0.0013
76
nd
nd
nd
80
nd
nd
nd
a
Based on the recovery of thymidine; Buhler, S., Giegrich, H.,
and Pfleiderer, W., Nucleosides & Nucleotides, 18, 1281-1283,
1999.
nd = not determined
Some advantages of this nitrobenzyl variant include the lack of the nitroso byproduct and release rates
that are relatively fast compared with the parent o-nitrobenzyloxycarbonyl derivative. For example, the
release of thymidine from 65b is reported to be twice as fast as that for the α-substituted derivative;
however, the nitrostyrenes (i.e., 67) are photolabile and thus can compete with the starting material for
incident light.
The quantum efficiencies for the release of thymidine varied depending on the substituents on 65.
Substitutions at the benzylic carbon appeared to give the highest release efficiencies, whereas the chloro
derivatives gave slightly better conversions, as seen in Table 69.18.
The high quantum efficiency and respectable protection and deprotection yields obtained for the
methyl-substituted 2-(o-nitrophenyl)ethoxycarbonyl photoprotecting group prompted Pirrung et al.66 to
use this protecting group for the solid-phase synthesis of oligodeoxynucleotides.
Thiopixyl and Coumaryl
Coleman and Boyd67 introduced the 9-phenylthioxanthyl or S-pixyl photoprotecting group for the four
principal nucleosides, thymidine and three other benzoyl protected nucleoside bases. The chromophore
was synthesized in three steps, starting with thioxanthone, through Grignard addition of phenylmagnesium bromide, followed by dehydration with trimethylsilyl chloride and dimethylsulfoxide to give the 9chloro-9-phenylthioxanthene. Treatment with the corresponding alcohol in a dry solution of pyridine
and dimethylaminopyridine (DMAP) afforded the photoremovable protected hydroxy derivatives 68 in
good yields (Table 69.19). Irradiation of 68 in aqueous acetonitrile resulted in the release of the nucleoside
or alcohol along with 69 (Eq. (69.34)).
Ph
OR
Ph
OH
hν, 300 nm
S
+
CH3CN(aq)
ROH
S
68
69
X
CH2
R=
CH2
O
CH2
OH
X = Thymine, N-Bz-Adenine,
N-Bz-Cytosine, N-iBu-Guanine
(69.34)
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TABLE 69.19 Synthesis and Deprotection Yields
for Various Derivatives of 68
R
Protectiona
(% Yield)
Deprotectionb
(% Yield)
80
94
79
86
82
92
93
95
97
96
75
89
CH2-benzyl
CH2-(E)styryl
Thymidine
N-Bz-adenosine
N-Bz-cytidine
N-iBu-guanosine
a
b
Based on isolated yields after column chromatography.
Substrate concentrations were approximately
0.1 mM; yields were determined by HPLC.
Mechanistically, this photorelease reaction occurs via photosolvolysis of the aryl ether carbon–oxygen
bond. The resulting resonance stabilized S-pixyl carbocation reacts with water to form 69. Control
experiments showed that the photoprotected alcohols are stable under thermal conditions; that is, refluxing in aqueous acetonitrile resulted in no detectable decomposition. Each of the caged products was
obtained as a solid, a convenience for laboratory purification and manipulation. The best deprotection
yields were obtained with solvent mixtures containing the maximum concentration of water permissible,
limited only by the solubility of the protected alcohol. Concentrations of water ranged from 40 to 60%,
depending on the type of alcohol used. In most cases, excellent deprotection yields were obtained.
A factor that limits the versatility of the S-pixyl group is the wavelength range required for photolysis.
The range of excitation wavelengths from 200 to 300 nm overlaps with a number of functional groups
that could compete with the incident light. For example, the pyrimidine base cytosine has substantial
absorptivity at 300 nm. As a result, extended irradiation times were required to effect deprotection of
the N-Bz-cytidine analog and a lower yield was obtained, as seen from Table 69.19.
Coumaryl photoprotecting groups have also been used for the protection of alcohols. A recent study
by Lin and Lawrence68 described the synthesis and photorelease of caged diols using a coumaryl acetal
derivative 70 (Eq. (69.35)).
H R
O
O
H
Br
O
hν, 348 nm
H
OH
Br
+
HO
O
O
1:1 CH3OH/buffer
HO
pH 7.4
70
OPh
SPh
Φ = 0.0041
70b
Φ = 0.0053
OH
Me
O
Me
70a
R
O
71
Me
R=
O
70c
Φ = 0.0051
N
Me
Ph
70d
Φ = 0.0278
(69.35)
Acetal 70 was prepared in a two-step sequence starting with oxidation of 6-bromo-7-hydroxy-4hydroxymethylcoumarin with manganese dioxide, followed by addition of the corresponding diol in
1348_C69.fm Page 31 Monday, October 13, 2003 3:22 PM
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Photoremovable Protecting Groups
anhydrous toluene. Protection yields ranged from 94% for 70a to 15% for 70d. Photolysis of 70 at 348
nm in a methanol/aqueous buffer solution afforded the free diol accompanied by the aldehyde photoproduct 71. The specific deprotection yields were not provided but in all cases were reported to exceed
75%.
The photorelease mechanism is not well understood but is speculated to proceed through an intramolecular ion pair69 generated from photoheterolysis of the carbon oxygen bond, as shown in Scheme 11.
Attack of a water molecule at the electrophilic carbon generates a hemiacetal that eliminates the alkoxy
group to afford the diol and byproduct 71.
1
O
70
hν
O
R
*
R
O
O
O
HO
O
HO
O
HO
R
HO
O
O
HO
O
HO
Br
O
Br
Br
Br
R
H2O
71
O
O
R
+
OH
HO
O
O
SCHEME 11 Mechanism proposed for the photorelease of caged diols from 70.
This particular coumaryl variant offers a unique advantage in essentially being able to protect both
hydroxyl groups of a diol using only one equivalent of the protecting group. In addition, the derivatives
in this study were shown to be stable to hydrolysis in aqueous solvents at neutral or basic pH. For example,
incubation of compounds 70a–d in a 1:1 methanol/buffer solution (pH 7.4) resulted in no detectable
degradation after a period of two weeks. Despite these advantages, the range of applications of the coumarin
group is limited thus far to 1,2- and 1,4-diols (1,3-diols were found to be inert to photolysis). Like the onitrobenzyl group, the coumaryl group also has the disadvantage of producing a highly absorbing photoproduct. In addition, further studies need to be carried out in order to elucidate the mechanism of the
photocleavage.
Benzoin
In 1995, Pirrung and Bradley70 reported the use of dimethoxybenzoin (DMB) carbonate to protect various
alcohols, including the 5′-hydroxyl group of nucleosides. The DMB carbonate was synthesized in three
steps, starting with methylation of carbonyldiimidazole with methyl triflate followed by addition of 2(3,5-dimethoxyphenyl)-2-hydroxy-1-phenylethanone to form a relatively stable activated acylating agent.
Treatment with an alcohol under basic conditions in nitromethane furnished the protected alcohol 72
in yields that ranged from 42 to 95%.
Irradiation of 72 at 350 nm resulted in release of the alcohol and formation of dimethoxybenzofuran
11 (Eq. (69.36)). A wide variety of alcohols, including a thiol, were explored in this protection/deprotection scheme. Excellent deprotection yields were obtained, as high as 98% for the protected thymidine
derivative. The mechanism as discussed earlier is postulated to proceed through intramolecular cyclization followed by demotion to form a zwitterionic intermediate. Expulsion of the alcohol occurs either
concomitantly with the release of carbon dioxide or by a stepwise decarboxylation of the initially released
carbonate 73.
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RO
O
O
MeO
Ph
O
hν, 350 nm
O
O
RO
THF
OMe
-CO2
(36)
ROH
73
-11
72
PhCH2OH
88%
PhCH2SH
N-Bz-adenosine
N-iBu-guanosine
thymidine
95%
97%
96%
98%
C8H17
OH
HO
96%
94%
(69.36)
Attractive features of the DMB photoprotecting group are that the benzofuran photoproduct 11 is
inert and exhibits strong fluorescence at 396 nm, allowing the deprotection conversions to be monitored
spectroscopically. In the same study, the DMB group was successfully used to synthesize two trinucleotides
bearing adjacent thymidine residues, demonstrating its potential in solid-phase DNA synthesis. Compared with nitrobenzyl photoprotecting groups, the rate of release of substrate is much faster for the
DMB group70 (krelease ~ 108–109 s–1). The main disadvantages of the DMB group are the competition for
incident light by the photoproducts and poor solubility in aqueous media.
Other
The anthraquinon-2-ylmethoxycarbonyl (Aqmoc) photoprotecting group is a relatively recent addition
to the chromophores used as photoremovable groups. In a recent application for alcohols, Furuta et al.71
reported that the caged derivative could be synthesized in two steps from anthraquinonylmethanol by
treatment with 4-nitrophenylchloroformate and DMAP followed by coupling to the desired alcohol with
DMAP to provide 74 in good yields (Table 69.20). Photolysis of 74 in 50% aqueous tetrahydrofuran
(THF) at 350 nm resulted in the release of the alcohol (Eq. (69.37)).
TABLE 69.20 Yields and Quantum Efficienciesa for Aqmoc Derivatives
Aqmoc Derivative
74a
74b
a
b
Protection
(% Yield)
Φ
Deprotection
(% Yield)
76
86
0.1
Not determined
68
91b
For the disappearance of starting material.
Determined by HPLC.
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Photoremovable Protecting Groups
O
O
O
OR
hν, 350 nm
ROH
photoproducts
+
THF/H2O
O
NH2
74
N
O
R=
CH2
O
O
N
CH2
N
N
O
O
O
OH OH
74a
74b
(69.37)
The photoproducts from the anthraquinone moiety were not fully characterized; however, in the case of
74a, an anthraquinon-2-ylmethanol tetrahydrofuranyl ether was isolated, probably the result of hydrogen
atom abstraction from the solvent. Also, a small amount of bis(1,2,3,4-di-O-isopropylidene-D-galactopyranosyl) carbonate was obtained after photolysis of 74a, likely the result of attack of the released alcohol
on the starting material.
Little is known about the photorelease mechanism of the Aqmoc group. Stern–Volmer analysis showed
that the triplet excited state is the reactive excited state that leads to release of the alcohol. The rate
constant determined for 74a (4.6 × 106 s–1) is consistent with rapid release of the carbonate followed by
a slower, rate-limiting loss of carbon dioxide to give the free alcohol. Additional studies are needed to
confirm this mechanism.
Recently, it was shown that nitro-substituted aryl carbamates could be used as photolabile protecting
groups for alcohols.72 N-Methyl-N-(o-nitrophenyl)carbamate 75 (Eq. (69.38)) was synthesized in two
steps, beginning with acylation of N-methyl-2-nitroaniline with phosgene followed by nucleophilic
addition of the alcohol, either as an alkoxide or in the presence of DMAP and triethylamine. An alternative
synthetic route involved the generation of the corresponding chloroformate from the alcohol and phosgene followed by nucleophilic addition of the nitroaniline. The carbamate derivatives 75a-d were synthesized in yields ranging from 58 to 94%.
Photolysis of carbamate 75 at various wavelengths led to deprotection of the alcohol, which was
accompanied by formation of nitrosoaniline 76 as a byproduct. Deprotection yields for all derivatives
were not provided; however, the reported yields for 75c and 75d were 100% and 91%, respectively. Such
high yields are a definite advantage in terms of both synthetic and biological applications. Another
attractive feature is the solubility in ethanol and water, solvents suitable for biological studies. Despite
these advantages, two main limitations are worthy of note. First, the carbamate cages are susceptible to
hydrolysis, particularly in basic media,73 thus limiting their use to aqueous solvents with relatively neutral
pH. Second, longer irradiation wavelengths were found to result in decreased deprotection yields; for
example, when 75c was irradiated at 312 nm, a quantitative deprotection yield was obtained. The yield
dropped to 76% when the photolysis was carried out at 365 nm.
NO2
NO
OR hν, 254-365 nm
N
O
N
ROH
H +
EtOH/H2O
76
75
O
R=
CH3
75a
CH2Ph
75b
I
Ph
N
H
75c
75d
N
O
CO2Me
(69.38)
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TABLE 69.21 Caged Silyl Derivatives
and Their Corresponding Deprotection
Yields in Methanol
Silyl Ether
Protection
(% Yield)
Deprotection
(% Yield)
77a
77b
77c
77d
77e
77f
82
80
77
79
81
85
94a
89
90
92
86a
90
a
Based on the yield of the byproduct 78.
Silyl photoprotecting groups have recently been developed for primary and secondary alcohols.74 The
silyl cage, (2-hydroxy-3-naphthylvinyl)-diisopropylsilyl ether 77, was synthesized in nine steps starting
with the commercially available naphthalene-2,3-diol. Irradiation at 350 nm in methanol triggered the
release of the alcohol, accompanied by the formation of a cyclic silyl byproduct 78 (Eq. (69.39)). This
byproduct is likely formed via intramolecular attack of the naphthol oxygen at silicon following a
trans,cis-isomerization of the starting material. Synthetic and photochemical yields are listed in
Table 69.21.
Si
OR
OH
hν, 350 nm
O
CH3OH
77
R=
i-Pr
Si
+
78
O
t-Bu
T
DMT-O
O
T
Me
77b
77c
H3C
CH3
OTBS
77a
ROH
77d
77e
CH3
77f
(69.39)
Byproduct 78 exhibits its strongest absorption in the region below 310 nm and, therefore, does not
significantly compete with 77 for incident light. The yields from Table 69.21 are sufficiently high to enable
the practical use of the silyl photoprotecting group in synthetic applications; however, the silyl cages lack
the water solubility necessary for application in aqueous media. Like triisopropylsilyl ethers, the cages
are also susceptible to cleavage in the presence of acidic media or solutions containing fluoride such as
1-N HCl or TBAF.75
The importance of nitric oxide (NO) in bioregulatory processes and other physiological functions
prompted the development of photoprotecting groups specifically designed for its release. A most recent
example is the synthesis and photochemistry of a series of naphthylmethyl and naphthylallyl diazeniumdiolates.76 These derivatives, represented by 79 in Eq. (69.40), were prepared from reaction of 1-(N,Ndiethylamino)-diazen-1-ium-1,3-diolate (81) with the corresponding alkyl bromide. Photolysis produced
a mixture of products that resulted from two different reaction pathways. Path a is a nonproductive
pathway that leads to the formation of nitrosamine 80 and oxime byproducts. Path b leads to diazeniumdiolate 81, which collapses to give free NO, along with diethylamine and other photoproducts. The
extent to which the reaction follows one pathway over another is dependent on the substituents present
on the naphthyl ring. As Table 69.22 shows, the best deprotection yields were obtained with a methoxy
group at the 5 and 8 positions of the ring.
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Photoremovable Protecting Groups
TABLE 69.22 NO Cages and Their Corresponding
Deprotection Yields and Quantum Efficiencies
NO cage
Protection
(% Yield)
Φ
Deprotectiona,b
(% Yield)
79a
79b
79c
79d
79e
90
25
30
36
5
0.007
Not determined
0.12
0.12
0.66
1
1
25
40
95
a
b
O
X
N
O
N
NEt2
Based on the disappearance of starting material (using HPLC)
and the amount of NO measured; thermal decomposition of
81 was found to produce 1.5 equivalents of NO.
For 79a–c, a wavelength of 300 nm was used; for 79d,e, the
wavelength was 350 nm.
a
O
Et2N N
n
hν
n
N
O
oximes
80
CH3CN(aq)
X
Ar
+
Y
79
b
Et2N
O
N
N
O
+
Ar
2 NO
CH2
n
+
n = 0,1
81
+
other
photoproducts
n = 0, X = Y = H
n = 0, X = H, Y = Me
n = 1, X = Y = H
n = 0, X = OMe, Y = H
n = 1, X = OMe, Y = H
79a
79b
79c
79d
79e
Et2NH
(69.40)
The likely mechanism proceeds through photosolvolysis of the carbon–oxygen bond, resulting in a
resonance-stabilized carbocation. Subsequent release of NO occurs from the diazeniumdiolate 81. Acidic
conditions that protonated the amine greatly enhanced the rate of NO release. Derivative 79e undergoes
clean deprotection and exhibits an excellent quantum efficiency, making it the most attractive of the NO
cages studied thus far. Some additional advantages are its absorption beyond 300 nm (λmax = 336 nm)
and its stability in acidic and basic solutions at room temperature for up to 24 hr. Unfortunately, it suffers
from a low protection yield of only 5%, and its solubility is limited to 20 µM in 95% aqueous acetonitrile.
Despite these shortcomings, its development may pave the way for similar methoxy-substituted naphthylallyl derivatives with increased solubility in aqueous media and higher protection yields.
A unique photoprotecting group for alcohols and thiols was reported in the mid 1990s.77 Benzoylbenzoate ester 82 (Eq. (69.41)) was synthesized in one step from DCC coupling of the corresponding alcohol
or thiol to 2-benzoylbenzoic acid. Photolysis at ~300 nm in the presence of cyclohexylamine, an electron
donor, afforded 3-phenylphthalide 84 along with the free alcohol or thiol.
O
Ph
CO2R
OH
hν
cyclohexylamine
CO2R
O
+
Ph
Ph
O
ROH
or RSH
1:1 PhH:CH3CN
82
83
84
R = n-C12H25-, c-C12H23-, cholesteryl-, geranyl-, 2',3'-isopropylidene uridinyl-, n-C12H25 (thioester) (69.41)
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*3
O
Ph
O
hν
Ar
Ph
ISC
NH2
NH2
O
Ar
Ph
Ar
Ar = o-benzoate ester
NH2
Ph
OH
OH
Ar
85
NH
NH2
OH
Ph
Ph
Ar
Ar
+
83
86
SCHEME 12 Proposed photoreduction mechanism of benzoylbenzoate esters.78
TABLE 69.23 Data for Benzoylbenzoate Esters
R
n-C12H25c-C12H23CholesterylGeranyl2′,3′-isopropylidene uridinyl
n-C12H25 (thioester)
a
b
c
Protection
(% Yield)
Deprotectiona
(% Yield)
76
50
67
63
79
76
95
85
100
90b
90c
60
Yield of the recovered alcohol (thiol) was determined with NMR.
sec-Butylamine was used as the electron donor; the solvent was
1:1 benzene/isopropanol.
sec-Butylamine was the electron donor; photolysis was carried
out with a uranium filter.
The mechanism outlined in Scheme 1278 involves electron transfer from the amine to the ketone in
the excited state followed by intermolecular proton transfer to generate radical pair 85–86; a second
electron transfer and proton exchange lead to the reduced alcohol 83, which lactonizes to form 84
concurrently with release of the alcohol. In general, the benzoylbenzoate photoprotecting group worked
well for the particular substrates studied. Synthesis yields for the benzoylbenzoate cages were respectable,
and the deprotection occurred in most cases with good recoveries of the alcohol (Table 69.23). Problems
were encountered in the photolysis of the caged thiol that led to the formation of side products and thus
a lower overall deprotection yield.
While the benzoylbenzoate cages exhibit good deprotection yields for alcohols, their application is
limited to organic solvents. The presence of an electron donor (i.e., aliphatic amine) is also required, a
necessity that may complicate the reaction mixture in the presence of other sensitive functional groups.
Finally, the release process must be inherently a slow one due to the ground state lactonization process
involved.
69.7 Phenols and Other Weak Acids
o-Nitrobenzyl
The photolabile o-nitrobenzyl derivative was utilized to protect the phenolic OH group of serotonin.79
The serotonin type-3 receptor is the only ligand-gated ion channel in the 5-HT receptor family.80,81 The
protection of the phenolic hydroxy group of serotonin required four steps, as shown in Scheme 13. The
substrate was released upon excitation with 337-nm laser pulses. The signal decay from pulsed laser
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Photoremovable Protecting Groups
NH2
HO
NO2 COOCH3
O
1) di-t-BOC anhydride (97%)
N
H
2) K+ t-butylate
N
H
N
H
NO2 COOCH3
Serotonin
BOC
N-Boc-OMeCNB-5HT
Br
1) 3.5% K2CO3
MeOH/H2O 1:1
(85%)
Methyl-2-bromo-2(2-nitrophenyl) acetate
2) FCH2COOH
CH2Cl2/Acetone/H2O
10:10:1 (90%)
(15%)
NO
NO2 COOH
O
COOH
hν, 337 nm
O
+
Serotonin
NH2
-H+
N
H
O-CNB-5HT
SCHEME 13 Protection and photochemical deprotection of serotonin.
studies gave a time constant of 16 µs, and the quantum yield was determined to be 0.03. The rate of
decay of the intermediate was observed to be pH dependent. The caged serotonin showed good solubility
in buffered aqueous media (in excess of 2 mM); however, the authors suggested that the caged compound
was subject to hydrolysis in the dark on standing.
This photoremovable protecting group has been employed as a photocleavable linker to reagents bound
to Au surfaces.82 4-Hydroxy-stilbene was linked to 6-bromohexyl-3-nitro-4-bromomethylbenzoate in
51% yield, then thiolated by trimethylsilylthioxy dehalogenation in THF, followed by desilylation in situ.
The self-assembled monolayers of long-chain alkyl thiolate on bulk polycrystalline gold were constructed.
Upon irradiation at 350 nm, the Z,E-photoisomerization attained a photostationary state within 25 min,
while the dissociation took about 60 min; however, sensitization with 1,4-dibromonaphthalene (ET =
58.1 kcal/mol) produced a cleaner photoisomerization. The unidirectional isomerization, from cis to
trans, by both direct irradiation and sensitization was followed by the release of a bound chain from the
metal surface.
O
O
hν, 350 nm
ON
HO
+
O2N
O
O
O(CH2)6SH
O(CH2)6SH
hν, 350 nm
hν, 350 nm
O
O
HO
hν, 350 nm
O2N
ON
+
O
O
O(CH2)6SH
SCHEME 14
O(CH2)6SH
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Benzoin
The ubiquinol oxidizing enzyme is a redox active enzyme that requires a fast two-electron reduction of
ubiquinone. 3′,5′-Dimethoxybenzoin (DMB) caged ubiquinol83 was synthesized to study the detailed
enzymatic mechanism of the fast electron-transfer process in redox active enzymes. The monosilylated
ubiquinol was coupled to the protecting group to form the o-nitrophenylcarbonate ester of 3′,5′dimethoxyphenyl(phenyldithiane) in 55% yield. Upon photolysis, DMB caged ubiquinol generated
ubiquinone with a rate greater than 106 s–1(Eq. (69.42)).
O
OH
H3CO
H3CO
CH3
CH3
H3CO
H3CO
O
O
hn, 355 nm
O
O
-CO2
OCH3
+
H3CO
O
O
OCH3
OCH3
11
(69.42)
Pirrung and Bradley70 also applied DMB (3′,5′-dimethoxybenzoin) to protect the phenolic group, demonstrating that 4-methoxyphenol was released upon irradiation at 350 nm in 90% yield (Eq. (69.43)).
H3CO
O
C
O
O
hn, 350 nm
H3CO
H3CO
H3CO
Ph
+
T HF
O
OH
OCH3
-CO2
11
90%
O
OCH3
(69.43)
69.8 Amines
o-Nitrobenzyl
There are few variations for effective amine photoremovable protecting groups. The o-nitrobenzyl group
remains the most popular group and among the many examples the studies by Cameron and Frechet
are noteworthy.84 In general, o-nitrobenzylcarbamates of aliphatic amines upon photolysis release the
amine in good yield. For example, cyclohexylamine is released from its o-nitrobenzyl carbamate 87 as
the corresponding free carbamate upon irradiation in THF at 254 nm (Eq. (69.44)).
NO2 R2
O
O
hν, 254 nm
N
H
NO R2
O
THF
+
CO2 +
H2N
R1
R1
87
(69.44)
Subsequent loss of carbon dioxide frees the amine. Quantum efficiencies varied depending on the
substituents present at the ortho and benzylic positions (Table 69.24). The best efficiencies were obtained
with two o-nitro groups on the aryl ring, likely increasing the probability for the hydrogen atom abstraction by one of the nitro groups.
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Photoremovable Protecting Groups
TABLE 69.24 Protection Yieldsa and Quantum
Efficiencies for Various Substituted
o-Nitrobenzyl Carbamates
R1
R2
H
NO2
H
NO2
H
NO2
H
H
Me
Me
o-Nitrophenyl
2,6-Dinitrophenyl
a
Protection
(% Yield)
Φ
71
78
71
71
79
52
0.13
0.62
0.11
0.35
0.26
0.28
Deprotection yields were not provided.
A problem that is not entirely avoidable is the formation of imine byproducts via reaction of the
released amine with the aldehydic group in the photoproduct. This occurrence could be suppressed with
alkyl or aryl substitution at the benzylic position, leading to the formation of a less reactive ketone in
comparison with the nitroso aldehyde formed with no substitution at the benzylic position. Imine
byproduct formation is also less likely to occur in relatively nonpolar solvent systems, such as THF, which
ultimately limits the application of the o-nitrobenzyl carbamate photoprotecting group to nonaqueous
systems in this regard.
Benzoin Derivatives
In the mid-1990s, Pirrung and Huang85 extended the use of the benzoin photoprotecting group to the
release of amines by synthesizing m,m′-dimethoxybenzoin (DMB) carbamates. The DMB derivatives
were synthesized by coupling the corresponding amine with benzoin carbonyl chloride that had been
elaborated by reaction of carbonyldiimidazole with the methyl triflate of 88 followed by nucleophilic
addition of DMB. Irradiation in benzene or THF at 350 nm produced the free amine, carbon dioxide,
and the benzofuran byproduct (Eq. (69.45)).
R2
N
O
R1
hν, 350 nm
O
R1NHR2
MeO
benzene
+
11
+
CO2
O
OMe
88
(69.45)
Five different amines were protected and recovered in moderate to good yields as shown in Table 69.25.
The DMB carbamates appeared to work well for a variety of amines in the presence of other functional
groups, such as alcohols or esters; however, the reaction is limited to secondary amines, as primary amines
were found to undergo intramolecular cyclization leading to byproducts that are inert to photolysis.
Arylsulfonamides
Corrie and Papageorgiou86 have reported the synthesis and photochemistry of various methoxysubstituted arylsulfonamides. Similar derivatives had been previously found to undergo single electron
transfer reactions in the excited state, leading to cleavage of the sulfur–nitrogen bond.87,88 It was
reasoned that such a process could be used for the rapid release of neurologically active amines.
The arylsulfonamide derivatives were synthesized in several steps, starting from 1,5-dimethoxynaphthalene. Photolysis of 89 in phosphate buffer (pH 7.0) in the presence of ascorbate resulted in release of
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TABLE 69.25 Protection and Deprotection Yields for DMB Carbamates
Amine
Protection (% Yield)
Deprotection (% Yield)
85
89a
76
79
N
H
90
56
CO2t-Bu
90
73
88
97a
NH
N
H
N
H
HO
Ph
N
H
a
The amine was recovered as the corresponding hydrochloride salt.
TABLE 69.26 Synthesis and Photochemistry Yields of Arylsulfonamides
R1
(CH2)3OPO3–2
(CH2)3OPO3–2
Me
a
b
R2
Protection
(% Yield)
Deprotectiona
(% Yield)
71
61
59
35
22b
53
CO2–
CH2CO2–
CO2Me
Yields were determined using quantitative amino acid analysis; irradiations
were only carried out to approximately 50% conversion of the starting
material in the presence of 10-mM ascorbate.
Photolyzed in buffer solution only.
the free amine in low to moderate yields, accompanied by the reduced arylsulfonyl byproduct 90 (Eq.
(69.46) and Table 69.26).
OMe
OMe
hν
1:2 buffer/MeOH
ascorbate
OR1
SO2NH
89
+
H2N
R2
OR1
SO2-
R2
90
(69.46)
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Photoremovable Protecting Groups
OMe
OMe
89
hν
H2O
SET
-R2CH2NH2
back electron
transfer
90
OR1
OR1
O
S
O
O
NHCH2R2
S
O
SCHEME 15 Proposed mechanism for amino acid release from arylsulfonamides.
The mechanism is thought to involve an excited state intramolecular single eletron trnasfer from the
electron-rich naphthalene to the sulfonamide group (Scheme 15); release of the amine then occurs via
assistance from the neighboring oxygen, leading to a radical cation that is subsequently reduced to 90
by ascorbate.
It was speculated that, in the case of the glycine and β-alanine substrates, a competitive electron transfer
was occurring from the carboxylate group to the radical cation of 90. Such a process would yield a
carboxyl radical that would undergo subsequent decarboxylation, leading to a mixture of side products
and thus a lower yield of the free amino acid. Spectroscopic evidence using LFP combined with FTIR
spectroscopy supported this hypothesis. Low yields were still encountered even in the presence of
increased amounts of ascorbate, suggesting that the electron transfer was taking place within a solvent
cage.
Despite this shortcoming, the arylsulfonamide group may still hold promise as a photoremovable
protecting group for amines lacking a carboxylate moiety. Further studies would need to be carried out
to fully establish its capabilities in this regard.
69.9 Conclusion
A wide variety of photoremovable protecting groups have been added to the veteran o-nitrobenzyl series.
Each new group has been developed to address the shortcomings of the o-nitrobenzyl group or to add
features such as faster rates for release, extension of the absorption range into the near UV-visible region,
improved solubility, higher efficiencies, improved conversions and yields, and more benign photoproducts from the protecting group. Extensions and applications of this chemistry to two photon excitation
processes, to traceless reagents in combinatorial chemistry and photolithography, as orthogonal reagents
in synthesis, and to time-resolved spectroscopic techniques wil make even more demands on the design,
synthesis, and development of new photoremovable protecting groups.
Even now, however, no single photoremovable protecting group fulfills all nine criteria Sheehan and
Lester had suggested (outlined at the beginning of this chapter). Nevertheless, important progress has
been achieved as evidenced by the growing number of applications reported for many of these photoremovable groups, especially in biological studies. Applications in synthesis, combinatorial chemistry, micro
arrays, and photolithography are also forthcoming. These fields have benefited and will continue to draw
the interest of the science community as improvements of existing systems and discovery of new photoactive protecting groups are developed. This field of photoremovable protecting groups is still in its
infancy.
Acknowledgments
We acknowledge the support of the Department of Energy, University of Kansas and the National Science
Foundation.
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References and Notes
1. (a) Kaplan, J. H., Forbush, B. I., and Hoffman, J. F., Rapid photolytic release of adenosine 5′triphosphate from a protected analogue: utilization by the Na:K pump of human red blood cell
ghosts, Biochemistry, 17, 1929–1935, 1978. (b) Engels, J. and Schlaeger, E.-J., Synthesis, structure,
and reactivity of adenosine cyclic 3′,5′-phosphate benzyl triesters, J. Med. Chem., 20, 907-911, 1977.
2. Givens, R. S. and Kueper III, L. W., Photochemistry of phosphate esters, Chem. Rev., 93, 55–66,
1993.
3. Adams, S. R. and Tsien, R. Y., Controlling cell chemistry with caged compounds, Ann. Rev. Physiol.,
18, 755–784, 2000.
4. Corrie, J. E. T. and Trentham, D. R., Synthetic, mechanistic and photochemical studies of phosphate
esters of substituted benzoins, J. Chem. Soc., Perkin Trans. 1, 2409–2417, 1992.
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