The α,5-dicarboxy-2-nitrobenzyl caging group, a tool for biophysical applications with improved hydrophilicity: Synthesis, photochemical properties and biological characterization

Article abstract of DOI:10.1111/j.1751-1097.2010.00803.x

Earlier we reported on the synthesis of α,4-dicarboxy-2-nitrobenyzl caged compounds (Schaper, K. et al. [2002] Eur. J. Org. Chem., 1037-1046). These compounds have the advantage of an increased hydrophilicity compared with the well-established α-carboxy-2

Full text of DOI:10.1111/j.1751-1097.2010.00803.x

Photochemistry and Photobiology, 2010, 86: 1247–1254
The a,5-Dicarboxy-2-nitrobenzyl Caging Group, a Tool for Biophysical
Applications with Improved Hydrophilicity: Synthesis, Photochemical
Properties and Biological Characterization
Klaus Schaper*, Sayed Abdollah Madani Mobarekeh, Peter Doro and Daniela Maydt
Group for Organic Photochemistry, Institute for Organic Chemistry and Macromolecular Chemistry,
Heinrich-Heine-University Du¨sseldorf, Du¨sseldorf, Germany
Received 11 May 2010, accepted 13 August 2010, DOI: 10.1111/j.1751-1097.2010.00803.x
bathochromic absorption compared with biological material
(e.g. DNA basis and amino acids), (f) the release of the active
ABSTRACT
Earlier we reported on the synthesis of a,4-dicarboxy-2-nitro-
benyzl caged compounds (Schaper, K. et al. [2002] Eur. J. Org.
Chem., 1037–1046). These compounds have the advantage of an
increased hydrophilicity compared with the well-established a-
carboxy-2-nitrobenzyl caged compounds; however, the release of
the active compound becomes slower due to the introduction of
the additional carboxy group. Based upon theoretical calcula-
tions we predicted that the release would become faster when the
additional carboxy group is moved to the 5-position. Here we
describe the synthesis and the photochemical and biological
characterization of an a,5-dicarboxy-2-nitrobenyzl caged com-
pound. The high hydrophilicity of the new caging group is
maintained due to the fact that the additional carboxy moiety is
preserved, while the release of the active species from the new
derivative is even faster than for the reference, an a-CNB caged
compound.
compounds has to be fast and the (g) photoreaction should
have a high quantum yield. In order to fulfill these demands
Hagen introduced in recent years the coumarin-type caged
compounds which photolyze extremely fast at rather long
wavelengths (40–45).
Bathochromic absorbing caged compounds of the o-nitro-
benzyl type can be achieved by the synthesis of bichromo-
phoric systems consisting of one caged compound and one
sensitizer (46,47), or as R. Y. Tsien showed by introducing
donor groups into the chromophore to give the 4,5-dimethoxy-
2-nitrobenzyl group (3) (see Scheme 2) (48). Compounds of
type 3 and 4 exhibit some unusual photophysical properties
(49–51). The question of an increased hydrophilicity, in order
to enhance water solubility and biological inertness, was
NO2
X
NO
ν
h
+
HX
O
INTRODUCTION
1
2
In 1901, Silber and Ciamician discovered the photochemistry
of o-nitrobenzyl (NB) compounds (1). In the 1960s, Barltop
and Woodward used this discovery in order to create photo-
labile protecting groups (2–4). A prototype reaction is shown
in Scheme 1. In 1977, Engels used this type of photochemistry
for the first time in order to release bioactive compounds (5,6)
and, 1 year later, Kaplan took this idea one step further by
using laser photolysis and he coined the expression ‘‘caged
compound’’ for photolabile protected compounds (7). In
contrast to conventional mixing techniques the use of caged
compounds allows kinetic investigations on a microsecond
time scale on cell surfaces (8–14) or within cells (15–17). Since
then various kinds of caging groups have been introduced (18–
39). However, the o-nitrobenzyl protecting group and its
numerous derivatives are still the most widely used ones.
A caged compound has to meet a number of characteristics
in order to be a useful tool in biophysical studies, namely it has
to be (a) water-soluble, (b) hydrolytically stable and (c) the side
products of the photoreaction and (d) the caged compound
itself has to be biologically inert, (e) it should exhibit a
Scheme 1. Photoreaction of the prototype of a caged compounds of
the o-nitrobenzyl type. The nitro moiety in 1 is reduced to a nitroso
group while the a-position is oxidized. The molecule HX is released
and o-nitrosobenzaldehyde 2 is formed.
O
O
NO2
X
NO2
X
O
O
3
4
6
NO2
NO2
O
X
X
O
COOH
COOH
5
HOOC
NO2
NO2
X
X
HOOC
COOH
COOH
7
8
Scheme 2. Overview over some specially designed caged compounds
of the o-nitrobenzyl type.
*Corresponding author email: schaper@klaus-schaper.de (Klaus Schaper)
ꢀ 2010 The Authors. JournalCompilation. The AmericanSocietyofPhotobiology 0031-8655/10
1247

1248 Klaus Schaper et al.
temperature and then stored over night at 5ꢁC and the residue was
filtered off. The aqueous solution was extracted with ethyl acetate
(3 · 50 mL). The organic layer was dried over sodium sulfate and
evaporated at room temperature to give a combined yield of 3.00 g
(13.3 mmol, 64%) 12 as a yellow solid. mp. 170–171ꢁC. 1H-NMR
(DMSO-d6, 200 MHz, Fig. S1): d = 13,5 and 12.8 (2 * s, 1H each,
COOH), 8.16 (d, 3JHH = 8.4 Hz, 1H, Ar-3-H), 8.11 (d,
successfully addressed by Hess, who introduced an a-carboxy
group in order to give 5 (a-carboxy-2-nitrobenyzl [a-CNB]).
The additional a-carboxy group has additionally a positive
influence on the rate and quantum yield of the release of the
caged compound (25,52–54). We took up that idea and tried to
further improve the properties of these compounds by intro-
ducing additional carboxy groups into compound 4 to give the
a-carboxy-4,5-methylendioxy-2-nitrobenyl group 6 (51) and
into 5 to give 7 the a,4-dicarboxy-2-nitrobenzyl group (a,4-
DCNB) (55), which indeed exhibits an increased hydrophilic-
ity. However, the release of the active compound is slower for
a,4-DCNB compared with a-CNB 5 (see Table 1 for more
details). Based on our earlier theoretical predictions we
assumed that we could increase the rate of release for the
active compound by moving the carboxy group from the 4- to
the 5-position to give a,5-dicarboxy-2-nitrobenzyl group (a,5-
DCNB 8) (55). Therefore we set out to synthesize a a,5-DCNB
caged aspartate 17.
4JHH = 1.5 Hz,
1H,
Ar-6-H),
8.05
(dd,
3JHH = 8.4 Hz,
4JHH = 1.8 Hz, 1H, Ar-4-H), 4.07 (s, 2H, Ar-CH2) ppm. IR (film):
v~ = 3500–2500 (m-OH), 1678 (m C=O), 1615, 1587, (m and d ring), 1521
(mas NO2), 1400 (d CH2), 1348 (ms NO2), 1295, 1124 (m C–O) cm)1
.
tert-Butyl a-[5-(tert-butoxycarbonyl)-2-nitrophenyl]acetate (13). A
solution of 3.40 g (15.1 mmol) dicarboxylic acid 12 and 0.30 mL
perchloric acid (70%) in 80.0 mL (69.0 g, 0.594 mol) tert-butyl acetate
was stirred at room temperature for 48 h. The clear orange solution
was neutralized with saturated sodium bicarbonate solution and
extracted with ether (2 · 50 mL). The combined organic layers were
washed with water (2 · 40 mL), dried over sodium sulfate and
concentrated under reduced pressure. The remaining oil was dissolved
in n-hexane ⁄ ether (4 ⁄ 1) and filtered through a silica gel column to give
2.95 g (8.74 mmol, 58%) compound 13 as a pale yellow solid. mp.
92–94ꢁC. Anal. for C17H23NO6 Calc. C: 60.52, H: 6.87, N: 4.15, Found
C: 60.31, H: 7.03, N: 4.10. 1H-NMR (CDCl3, 500 MHz, Figure S2
3
and S3): d = 8.10 (d, JHH = 8.5 Hz, 1H, Ar-3-H), 8.03 (dd,
=
4
3
3JHH = 8.5 Hz, JHH = 1.8 Hz, 1H, Ar-4-H), 7.94 (d, JHH
MATERIALS AND METHODS
1.8 Hz, 1H, Ar-6-H), 3.99 (s, 2H, Ar-CH2), 1.61 and 1.44 (2 s, 9 H
each, tert-butyl) ppm. 13C-NMR (CDCl3, 125 MHz, Fig. S4):
d = 168.8 (b-C), 163.62 (Ar-COOR), 151.2 (Ar-2-C), 136.1 (Ar-5-C),
134.3 (Ar-1-C), 130.5 (Ar-3-C), 129.3 (Ar-4-C), 125.1 (Ar-6-C), 82.6,
82.1 (C(CH3)3), 40.9 (a-C), 28.1, 28.0 (C(CH3)3) ppm. IR (film):
v~ = 2979 (mas CH alkyl), 2930 (ms CH alkyl), 1729, 1725 (m C=O),
1614, 1587 (m and d ring), 1526 (mas NO2), 1393, 1343 (d C(CH3)3), 1298
Starting materials. N-(tert-butoxycarbonyl)-O1-(tert-butyl)-D-aspartic
acid was obtained from Bachem Biochemica GmbH. The synthesis of
thiophenoxyacetonitrile 10, (5-carboxy-2-nitrophenyl)acetonitrile 11
and O-[a,5-bis-(tert-butoxycarbonyl)-2-nitrobenzyl] acetic acid S1 is
described in the Supporting Information. NMR-spectra for com-
pounds 12, 13, 14, 15 and 17 are shown in the Supporting Information.
Chromatography. Sephadex LH20 (BioChemica) was used for
column chromatography of the final product 17. All other chromato-
graphic purifications were carried out on silica gel 60 (0.040–
0.063 mm; Merck).
(ms NO2), 1224, 1153 (m C–O) cm)1
.
tert-Butyl a-bromo-a-[5-(tert-butoxycarbonyl)-o-nitrophenyl]acetate
(14). A solution of 2.50 g (7.41 mmol) diester 13, 1.40 g (7.89 mmol)
N-bromosuccinimide and catalytic amounts of dibenzoylperoxide
(DBPO) in 50 mL tetrachloromethane was heated to reflux for 16 h.
The mixture was then filtered and the clear solution was concentrated
2-(5-Carboxy-2-nitrophenyl)acetic acid (12). A mixture of 4.50 g
(20.8 mmol) nitrile 11 in 100 mL 20% hydrochloric acid was stirred
under reflux for 6 h. The mixture was allowed to cool to room
Table 1. Calculated total energies according to Klopmans theory (70) and observed life times for the decay of the aci-nitro-intermediates for
a-CNB D-aspartate (23), a,5-DCNB D-aspartate (17) and a,4-DCNB D-aspartate (24) (55).
Z
E
DE
DE
Compound
*
s1 (ls)†
*
s2 (ls)†
F
kJ molꢀ1
kJ molꢀ1
NO2
COO
)94.2
26
4 (64%)
)90.3
416
62 (36%)
0.19
0.01
O
COO
NH3
23
O
NO2
COO
OOC
)91.8
)84.5
56
4 (89%)
)82.8
)79.6
340
55 (11%)
0.10
0.01
O
COO
NH3
17
O
OOC
NO2
COO
111
1 (73%)
508
172 (27%)
0.14
0.01
O
COO
NH3
24
O
*For these calculations the acetates were used instead of the D-aspartates; †The numbers in brackets refer to the contribution of this exponent to the
decay of the aci-nitro intermediate.

Photochemistry and Photobiology, 2010, 86 1249
3
7.99 (2 d, JHH = 8.5 Hz and JHH = 8.5 Hz, 1H, Ar-3-H), 6.64 and
3
in vacuum. The product and starting material were separated through
column chromatography (silica gel, hexane ⁄ ethyl acetate 4:1 (to yield
1.64 g (3.93 mmol, 53%) 14 as a pale yellow solid. mp. 53–55ꢁC. Anal.
for C17H22BrNO6 and ½ C4H8O2 and ¼ C6H14 (The amount of ethyl
acetate and hexane was determined by 1H-NMR.) Calc. C: 51.10, H:
6.17, N: 2.91 Found: C: 52.19, H: 6.31, N: 3.09. 1H-NMR (CDCl3,
6.63 (2s (Integration: 1:1.1), 1H, a-H), 5.52 and 5.45 (2 d,
3
3JHH = 7.0 Hz and JHH = 7.0 Hz, 1H, N-H), 4.60—4.40 (m, 1H,
2¢-CH), 3.18-2.74 (m, 2H, 3¢-H), 1.60 and 1.59 (2 s, 9 H, BOC) 1.44,
1.43, 1.42, 1,393, 1.386 and 1.38, (6 s, 27 H, 3 x tert-butyl) ppm (for
the numbering of atoms see Scheme 3). The signals were assigned using
the data of acetate derivate S1 as reference. (see Supporting Informa-
4
500 MHz, Fig. S5): d = 8.28 (d, JHH = 1.8 Hz, 1H, Ar-6-H), 8.08
3
4
~
(dd, JHH = 8.5 Hz, JHH = 1.8 Hz, 1H, Ar-4-H), 8.02 (d,
3JHH = 8.5 Hz, 1H, Ar-3-H), 5.97 (s, 1H, Ar-CH), 1.65 and 1.43 (2
s, 9 H each, tert-butyl) ppm. 13C-NMR (CDCl3, 125 MHz, Fig. S6):
d = 164.6 (b-C), 163.1 (Ar-COOR), 149.9 (Ar-2-C), 136.6 (Ar-5-C),
133.2 (Ar-1-C), 132.9 (Ar-3-C), 130.7 (Ar-4-C), 125.2 (Ar-6-C), 82.8
and 82.1 (2 * C(CH3)3), 65.9 (a-C), 28.4 and 28.1 (C(CH3)3) ppm. IR
tion), IR (film): v = 3352 (m NH), 2977 (mas CH2), 2933 (ms CH2), 1715,
1694 (m C=O), 1613, 1590 (m and d ring), 1537 (mas NO2), 1456, 1394
(d C(CH3)3), 1456 (m C–N), 1368 (d C(CH3)3), 1304 (ms NO2), 1159
(m C–O) cm)1
.
O4-(a,5-dicarboxy-2-nitrobenzyl)-D-aspartic acid (17). A solution of
210 mg (0.336 mmol) 15 in a mixture of 15 mL dichloromethane and
5 mL trifluoro acetic acid was stirred at room temperature under
nitrogen for 16 h. The mixture was concentrated under reduced
pressure, 10 mL toluene was added and the solvent was evaporated
again. The procedure was repeated once and the residual solid was
purified on Sephadex LH20 with water as eluent giving 98 mg
(0.28 mmol, 82%) as a roughly 1.4:1 mixture of diastereomers of 17
(estimated from the 1H-NMR integrals of the benzylic proton) as a
white and very hygroscopic powder. mp. 136–138ꢁC. Anal. for
C13H12N2O10*H2O: Calc. C: 41.72, H: 3.77, N: 7.48, Found C:
41.74, H: 3.87, N: 7.57. 1H-NMR (D2O, 500 MHz, Fig. S8): d = 8.14
~
(film): v = 2978, (mas CH alkyl), 2934 (ms CH alkyl), 1721, 1677 (m
C=O), 1608, 1587 (m and d ring), 1531 (mas NO2), 1456, 1394 (d
C(CH3)3), 1306 (ms NO2), 1161, ([m C–O) cm)1
.
O4-[a,5-bis-(tert-butoxycarbonyl)-2-nitrobenzyl]-N-(tert-butoxycarbonyl)-
O1-tert-butyl-D-aspartic acid (15). A mixture of 200 mg (0.691 mmol)
N-(tert-butoxycarbonyl)-O1-(tert-butyl)-D-aspartic acid (16), 330 mg
(0.793 mmol) t-butyl a-bromo-a-(4-tert-butoxycarbonyl-2-nitrophe-
nyl) acetate (14) and 140 mg (0.920 mmol) 1,8-diazabicy-
clo[5.4.0]undec-7-ene (DBU) in 30 mL absolute benzene were heated
under reflux for 5 h. The mixture was allowed to cool to room
temperature and then 50 mL water and 50 mL ethyl acetate were
added. The phases were separated and the aqueous layer was extracted
with ethyl acetate (2 · 30 mL). The combined organic layers were
dried over sodium sulfate and concentrated under reduced pressure.
The product 15 was purified by column chromatography (silica gel,
n-hexane ⁄ ethyl acetate 4:1) yielding 237 mg (0.380 mmol, 55%) of a
roughly 1.1:1 mixture of diastereomers (estimated from the 1H-NMR
integrals of the benzylic proton) as a pale brown viscous oil. 1H-NMR
3
(s (broad), 1H, Ar-6-H), 8.14 (dd (broad), JHH = 8.6 Hz,
3
4JHH = 1.5 Hz, 1H, Ar-4-H), 8.09 (d (broad), JHH = 8.6 Hz, 1H,
Ar-3-H), 6.61 and 6.59 (2 s, (Integration 1:1.4), 1H, a-H), 4.12 (t,
3JHH = 6.01 Hz, 1H, 2¢-H), 3.21- 3.08 (m, 2H, 3¢-H) ppm. 13C-NMR
(D2O, 125 MHz, Fig. S9): 172.1 and 172.0 (a-C-COOH), 170.6 and
170.5 (Ar-COOH), 168.78 and 168.75 and 167.82 and 167.81 (1¢-C,
4¢-C), 150.4 (Ar-2-C), 136.2 (Ar-5-C), 131.9 (Ar-6-C), 130.7 (Ar-1-C),
130.0 (Ar-3-C), 126.1 (Ar-4-C), 72.6 (a-C), 52.6 (2¢-C), 25.2 (3¢-C) ppm.
IR (KBr): v~ = 3600-2500 (m OH and NH), 2979 CH, 1730, 1671
(m C=O), 1534 (mas NO2), 1458 (ms CH2, CH), 1367 (ms NO2), 1190
4
(CDCl3 200 MHz, Fig. S7): d = 8.24 and 8.20 (2 d, JHH = 1.8 Hz,
3
4JHH = 1.7 Hz, 1H, Ar-6-H), 8.10 and 8.09 (2 dd, JHH = 8.5 Hz,
3JHH = 8.5 Hz, JHH = 1.8 Hz and 1.7 Hz, 1H, Ar-4-H), 8.02 and
(m C–O) cm)1
.
4
11% NaOH
NO2
NO2
CN
DMSO
S
CN
HOOC
HOOC
9
10
11
20% HCl
Δ
-BuOAc
t
NO2
COO -Bu
NO2
HClO4
t
COOH
-BuOOC
t
t
HOOC
13
12
NBS
DBPO
CCl4
DBU
benzene
Δ
NO2
NO2
COO -Bu
t
COO -Bu
t
-BuOOC
t
-BuOOC
O
COO -Bu
t
Br
14
15
O
NHBoc
HO
COO -Bu
t
TFA
CH2Cl2
O
NHBoc
16
3
2
NO2
4
1
COOH
α
5
HOOC
1'
6
3'
4'
O
2' COOH
17
O
NH2
Scheme 3. Synthetic procedure leading to an a,5-dicarboxy-2-nitrobenzyl protected D-aspartate.

1250 Klaus Schaper et al.
General characterization. The 200 MHz 1H-NMR spectra were
measured on a Bruker DXR-200, 500 MHz 1H-NMR and 125 MHz
13C-NMR were measured on a Bruker DXR-500. IR-spectra were
recorded on a Bruker Vector 22, UV spectra were recorded on a Perkin
Elmer Lambda 19, melting points were determined using a Reichardt
Thermovar, Buchi 510.
undergo inter system crossing (ISC) 173. The triplet excited
state 173 leads to the formation of the aci-nitro intermediate 18
as well, unless the aromatic ring is donor substituted (51). The
aci-nitro intermediate 18 exists in two diasteromeric forms,
which usually decay with two distinct rate constants within
microseconds to milliseconds in order to form the isoxazol 19.
This step is thought to be the rate limiting step in the reaction
if the leaving group is a carboxylic acid moiety. In the
following steps the D-aspartate 21 and the nitroso byproduct of
the photoreaction 20 is released. A side reaction discovered by
Corrie, the decarboxylation of the caged compound in the
a-position is omitted from this scheme (62).
The 13C-NMR spectra were assigned by increment systems (56) and
by comparison with references (57). 1H-NMR spectra were assigned by
increments (56). IR spectra were assigned by using references (58).
Transient spectroscopy. The setup for the transient absorption
experiments used in order to determine the rates of the reaction was
described earlier (55,59). The quantum yields were extracted from the
transient spectra (55). For all measurements 0.1 M phosphate buffer at
pH = 7 was used. A sample trace is shown in the Supporting
Information.
Earlier we reported on the synthesis of a,4-DCNB caged
compounds (7). These compounds are due to their increased
hydrophilicity advantageous compared with the well-
established a-CNB caged compounds (5). However, for a,4-
DCNB caged compounds (7) the aci-nitro intermediate decays
significantly slower than the respective aci-nitro intermediate
for a-CNB caged compounds (5) (55). Thus the release of the
active compound is slower. We were able to explain this
behavior using the Klopman equation (Eq. [1]). The term ETot
is a measure for the reactivity for a reaction between a
nucleophile and an electrophile, such as the ring-closure
reaction from 18 to 19 (see Scheme 4). The first term in the
Klopman equation describes the electrostatic interaction
RESULTS
Synthesis
Title compound 17 was synthesized as shown in Scheme 3.
Photochemical properties
The photochemistry of o-nitrobenzyl caged compounds has
been investigated thoroughly (15,21,52,60–68). The general
accepted mechanism is shown in Scheme 4. Upon irradiation
the excited state of the caged compound 171 is formed which
can either react to form the aci-nitro intermediate 18 or
NO2
COO
OOC
O
COO
NH3
17
O
ν
h
1
3
NO2
COO
NO2
ISC
COO
OOC
OOC
O
COO
NH3
O
COO
NH3
O
O
171
173
OH
NO2H
COO
N
O
O
OOC
OOC
OOC
O
COO
NH3
COO
NH3
O
O
19
18
NO
COO
O
COO
NH3
+
OOC
O
O
20
21
Scheme 4. Mechanism of the photoreaction of caged compounds of the o-nitrobenzyl-type.

Photochemistry and Photobiology, 2010, 86 1251
between the electrophile and the nucleophile, where qN is the
charge of the nucleophilic atom, qE is the charge of the
electrophilic atom and RNE is the distance between those two
atoms. The second term describes the orbital interaction,
where cHOMO is the orbital coefficient of the HOMO at the
nucleophilic atom, cLUMO is the orbital coefficient of the
LUMO at the electrophilic atom, EHOMO is the energy of the
HOMO, ELUMO is the energy of the LUMO and bHL is the
resonance integral.
20
0
-20
-40
-60
2
1
qNqE 2ðcHOMOcLUMO
b
HLÞ
DETot
¼
þ
ð1Þ
4p 20 RNE
EHOMO ꢀ ELUMO
All the necessary data to compute DETot, that is the
distance of the nucleophilic center to the electrophilic center,
the Mulliken charges, the coefficients for the HOMO and the
LUMO at the respective centers and the energy of HOMO
and LUMO were taken from ab initio calculations
(HF ⁄ STO6-31G**). The resonance integral bHL was estimated
using an approximation developed by Pariser and Parr (69)
(Eq. [2]). The results of these calculations are shown in
Table 1. More details regarding these calculations were
published earlier (55).
-10
0
10
20
30
40
50
60
Time (ms)
Figure 2. a,5-DCNB D-aspartate (17) was tested for it utility in
biophysical experiments. As test system the excitatory amino acid
carrier 1 (EAAC1) expressed in human embryonic kidney cells was
used. A sample trace is shown.
The quantum yield for a,5-DCNB D-aspartate (17) is 0.10
and somewhat lower than for the reference compounds 22 and
23 (see Table 1). However, they are sufficiently high for most
applications in biophysical studies.
ꢀ
ꢁ
ꢀ5:6864 ꢁ RNE
bHLðRNEÞ ¼ ꢀ6442eV ꢁ exp
ð2Þ
A
Biological characterization
Additionally we calculated the DETot-values for a,5-DCNB
compounds and predicted that a,5-DCNB D-aspartate (17)
should photolyze faster than the a,5-DCNB derivative (23),
but slower than the a-CNB derivative (22). For the fast and
major component of the decay of the aci-nitro intermediate
this prediction is confirmed by transient absorption spectros-
copy (for the respective life times see Table 1). A graphical
representation of the measured life times versus the calculated
Klopman energies yields a linear plot (see Fig. 1, left panel,
lower trace and right panel). For the slow and minor
component the error bars for the life times are much larger
and the values do not change significantly for the three
compounds.
The utility of a,5-DCNB D-aspartate (17) for biophysical
experiments was tested. The excitatory amino acid carrier 1
(EAAC1) expressed in human embryonic kidney cells was used
as test system (55). No response of the cells was observed upon
exposure to 17 and the response of the cells to D-aspartate was
not inhibited. Upon photolysis of 17, D-aspartate is released, a
chloride channel is opened and the resulting current can be
recorded (see Fig. 2). Hydrolysis of 17 in a dark reaction
would lead to free D-aspartate in the solution and thus to
activation of the receptor prior to photolysis. This is not
observed and therefore we can conclude that 17 is hydro-
lytically stable for at least several hours in aqueous solution.
DISCUSSION AND CONCLUSION
125
700
E-isomers
The motivation of our work was the design and synthesis of an
o-nitrobenzyl caging group with improved properties. For this
purpose we wanted to combine the high hydrophilicity of a,4-
DCNB type compounds 7 with the faster release of the active
species in a-CNB type compounds 5. This goal was achieved
by the synthesis of a,5-DCNB type compounds 8. By moving
the carboxy group from position 4 (7) to 5 (8), the increased
hydrophilicity (for more details see Supporting Information)
was kept. An increased hydrophilicity leads in principle to a
better solubility of the compound in aqueous buffer at pH = 7
and the caged compound and the side products of the
photoreaction are more likely to be biologically inert.
Z-isomers
4-DCNB
600
100
500
4-DCNB
75
400
α-CNB
5-DCNB
300
5-DCNB
50
25
0
200
100
0
α-CNB
4-DCNB
5-DCNB
α-CNB
-95
-90
-85
-80
-75
-70
-95
-90
-85
-80
The new a,5-DCNB caged compound proved to be hydro-
lytically stable. This had to be expected, because it is well
known that the respective a-CNB derivative is hydrolytically
stable and there is no reason to suspect that the carboxy
moiety in 5-position should influence that behavior.
ΔETot/(kJ/mol)
ΔETot/(kJ/mol)
Figure 1. Time constants for the decay of the aci-nitro-intermediates
of a-CNB D-aspartate (23), a,5-DCNB D-aspartate (17) and a,4-DCNB
D-aspartate (24) plotted versus the calculated Klopman energies. The
right panel shows an enlarged view of the lower trace in the left panel.

1252 Klaus Schaper et al.
Figure S4. 13C-NMR (DMSO, 125 MHz) spectrum of 13.
1
Figure S5. H-NMR (DMSO, 500 MHz) spectrum of 14.
1,0
0,8
0,6
0,4
0,2
0,0
23
17
Figure S6. 13C-NMR (DMSO, 125 MHz) spectrum of 14.
1
Figure S7. H-NMR (DMSO, 200 MHz) spectrum of 15.
1
Figure S8. H-NMR (DMSO, 200 MHz) spectrum of 17.
Figure S9. 13C-NMR (DMSO, 125 MHz) spectrum of 17.
1
Figure S10. H-NMR (DMSO, 500 MHz) spectrum of S1.
Figure S11. 13C-NMR (DMSO, 125 MHz) spectrum of S1.
Figure S12. HMQC (DMSO, 500 MHz) spectrum of S1,
cutout.
Figure S13. Transient absorbance change produced by
1 mM 17 after photolysis with a 10 ns, 308 nm laser flash in
black. The wavelength of the analysis light was 430 nm with a
path length of 10 mm (T = 22ꢁC and pH = 7.0 [100 mM
phosphate buffer]). The red line represents the best fit to the
data with a double exponential decay function. The lower
panel shows the fitting residuals.
0
200
400
600
t/μs
800
1000
Figure 3. Comparison of the relative decay of the aci-nitro-interme-
diate of a-CNB D-aspartate (23) and a,5-DCNB D-aspartate (17).
Table S1. In order to get an estimate of the hydrophilicity of
the caging groups, we determined Rf values for the respective
caged toluic acids, namely a-CNB caged toluic acid S2, a,4-
DCNB caged toluic acid S3 and a,5-DCNB caged toluic acid
S4 for different acetone ⁄ water mixtures on silica gel.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting information sup-
plied by the authors. Any queries (other than missing material)
should be directed to the corresponding author for the article.
The absorption properties of the new a,5-DCNB caged
compound are very comparable to the absorption properties of
a-CNB and a,4-DCNB caged compounds. No distinct absorp-
tion band can be detected above 260 nm. The molar absorp-
tivity at 308 nm is e = 1900 L mol)1 cm)1 and at 350 nm it is
e = 470 L mol)1 cm)1 (data obtained in acetonitrile). Com-
pared with the a-CNB D-aspartate (22) and the a,4-DCNB
D-aspartate (23) the a,5-DCNB D-aspartate (17) only has the
disadvantage of a somewhat lower quantum yield (0.1 for 17
compared with 0.14 for 23 and 0.19 for 22). However, the
product e · F which in dilute solution (e · c · d < 0.1) can
be used to compare the efficiency of uncaging is still sufficiently
high for 17 in order to be a useful tool.
The rate of decay of the aci-nitro intermediate and thus the
rate of release of the active compound was increased as
predicted. Indeed, the situation is even better than expected.
The time constant for the fast component is for a,5-DCNB
D-aspartate (17) about 120% higher compared with a-CNB D-
aspartate (22) and the slow components are the same within
the error margins; however, the fast component is for a,5-
DCNB D-aspartate (17) responsible for 89% of the overall
reaction, while it is only responsible for 64% of the overall
reaction for a-CNB D-aspartate (22). Due to this, the fast
phase is for our new derivative much more dominating and the
release of the active compound is much faster for a,5-DCNB
D-aspartate (17) (see Fig. 3). For example in order to get a
95% release of the active compound it will take more than
800 ls for a-CNB D-aspartate (22), while it will take 300 ls for
a,5-DCNB D-aspartate (17).
REFERENCES
1. Silber, P. and G. Ciamician (1901) Chemische Lichtwirkungen.
Chem. Ber. 34, 2040–2046.
2. Patchornik, A., B. Amit and R. B. Woodward (1970) Photosen-
sitive protecting groups. J. Am. Chem. Soc. 92, 6333–6335.
3. Barltrop, J. A., P. J. Plant and P. Schofield (1966) Photosensitive
protective groups. Chem. Commun. 822–823.
4. Barltrop, J. A. and P. Schofield (1962) Photosensitive protecting
groups. Tetrahedron Lett. 3, 697–699.
5. Engels, J. and R. Reidys (1978) Synthesis and application of
photolabile guanosine 3¢,5¢-phosphoric-o-nitrobenzylester. Exper-
ientia 34, 14–15.
6. Engels, J. and E. J. Schlaeger (1977) Synthesis, structure, and
reactivity of adenosine cyclic 3¢,5¢-phosphate benzyl triesters. J.
Med. Chem. 20, 907–911.
7. Kaplan, J. H., B. Forbush and J. F. Hoffman (1978) Rapid
photolytic release of adenosine 5¢-triphosphate from a protected
analog—Utilization by Na–K pump of human red blood-cell
ghosts. Biochemistry 17, 1929–1935.
8. Hess, G. P., D. J. Cash and H. Aoshima (1979) Acetylcholine
receptor-controlled ion fluxes in membrane-vesicles investigated
by fast reaction techniques. Nature 282, 329–331.
9. Udgaonkar, J. B. and G. P. Hess (1987) Chemical kinetic mea-
surements of a mammalian acetylcholine-receptor by a fast-reac-
tion technique. Proc. Natl Acad. Sci. USA 84, 8758–8762.
10. Muralidharan, S., G. M. Maher, W. A. Boyle and J. M. Nerbonne
(1993) Caged phenylephidrine—Development and application to
probe the mechanism of a-receptor mediated vasoconstriction.
Proc. Natl Acad. Sci. USA 90, 5199–5203.
Acknowledgements—We thank C. Grewer (Binghamton University)
for providing the biophysical data. This work was supported by the
Deutsche Forschungsgemeinschaft (SFB 663, project B6).
11. Walker, J. W., H. Martin, F. R. Schmitt and R. J. Barsotti (1993)
Rapid release of an a-adrenergic receptor ligand from photolabile
analogs. Biochemistry 32, 1338–1345.
12. Wieboldt, R., K. R. Gee, L. Niu, D. Ramesh, B. K. Carpenter and
G. P. Hess (1994) Photolabile precursors of glutamate—Synthesis,
photochemical properties, and activation of glutamate receptors
on a microsecond time-scale. Proc. Natl Acad. Sci. USA 91, 8752–
8756.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
1
Figure S1. H-NMR (DMSO, 200 MHz) spectrum of 12.
1
Figure S2. H-NMR (DMSO, 500 MHz) spectrum of 13.
13. Wieboldt, R., D. Ramesh, B. K. Carpenter and G. P. Hess (1994)
Synthesis and photochemistry of photolabile derivatives of gam-
ma-aminobutyric-acid for chemical kinetic investigations of the
Figure S3. 1H-NMR (DMSO, 500 MHz) spectrum of 13,
cutout.

Photochemistry and Photobiology, 2010, 86 1253
gamma-aminobutyric-acid receptor in the millisecond time region.
Biochemistry 33, 1526–1533.
14. Grewer, C., J. Jager, B. K. Carpenter and G. P. Hess (2000) A new
photolabile precursor of glycine with improved properties: A tool
for chemical kinetic investigations of the glycine receptor. Bio-
chemistry 39, 2063–2070.
33. Park, C. H. and R. S. Givens (1997) New photoactivated pro-
tecting groups .6. p-hydroxyphenacyl: A phototrigger for chemical
and biochemical probes. J. Am. Chem. Soc. 119, 2453–2463.
34. Walbert, S., W. Pfleiderer and U. E. Steiner (2001) Photolabile
protecting groups for nucleosides: Mechanistic studies of the 2-(2-
nitrophenyl)ethyl group. Helv. Chim. Acta 84, 1601–1611.
15. McCray, J. A. and D. R. Trentham (1989) Properties and uses of
photoreactive caged compounds. Annu. Rev. Biophys. Biophys.
Chem. 18, 239–270.
16. Theriot, J. and T. Mitchison (1991) Actin tracks—Reply. Nature
354, 363–363.
17. Scheidig, A. J., C. Burmester and R. S. Goody (1998) Use of caged
nucleotides to characterize unstable intermediates by X-ray crys-
tallography. In Caged Compounds (Edited by G. Mariott).
Academic Press, Orlando, Florida. Methods Enzymol. 291, 251–
264.
35. Woll, D., S. Walbert, K. P. Stengele, T. J. Albert, T. Richmond, J.
Norton, M. Singer, R. D. Green, W. Pfleiderer and U. E. Steiner
(2004) Triplet-sensitized photodeprotection of oligonucleotides in
solution and on microarray chips. Helv. Chim. Acta 87, 28–45.
36. Woll, D., S. Walbert, K. P. Stengele, R. Green, T. Albert, W.
Pfleiderer and U. E. Steiner (2003) More efficient photolitho-
graphic synthesis of DNA-chips by photosensitization. Nucleo-
sides Nucleotides Nucleic Acids 22, 1395–1398.
37. Huang, Y. H. H., S. R. Sinha, O. D. Fedoryak, G. C. R.
Ellis-Davies and D. E. Bergles (2005) Synthesis and character-
18. Bochet, C. G. (2002) Photolabile protecting groups and linkers. J.
Chem. Soc., Perkin Trans. 1, 125–142.
ization of 4-methoxy-7-nitroindolinyl-D-aspartate,
a
caged
compound for selective activation of glutamate transporters and
N-methyl-D-aspartate receptors in brain tissue. Biochemistry 44,
3316–3326.
19. Pirrung, M. C. and V. S. Rana (2005) Photoremovable protecting
groups in DNA synthesis and microarray fabrication. In Dynamic
Studies in Biology: Phototriggers, Photoswitches and Caged Com-
pounds (Edited by M. Goldner and R. S. Givens), pp. 341–368.
Wiley, New York.
20. Morrison, H. (1993) Biological Applications of Photochemical
Switches. John Wiley & Sons, Inc., New York, Chichester,
Bribane, Toronto, Sigapore.
21. Marriott, G. (1998) Caged Compounds. Academic Press, San
Diego, London, Boston, New York, Sydney, Tokyo, Toronto.
22. Mayer, G. and A. Heckel (2006) Biologically active molecules with
a ‘‘light switch.’’ Angew. Chem. Int. Ed. 45, 4900–4921.
23. Hagen, V., C. Dzeja, S. Frings, J. Bendig, E. Krause and U. B.
Kaupp (1996) Caged compounds of hydrolysis-resistant analogues
of cAMP and cGMP: Synthesis and application to cyclic nucleo-
tide-gated channels. Biochemistry 35, 7762–7771.
24. Dzeja, C., V. Hagen, S. Frings, J. Bendig, E. Krause and U. B.
Kaupp (1996) Studies of Ca2 + permeation in CNG channels
using caged compounds. Prog. Biophys. Mol. Biol. 65, PG103–
PG103.
25. Niu, L., K. R. Gee, K. Schaper and G. P. Hess (1996) Synthesis
and photochemical properties of a kainate precursor and activa-
tion of kainate and AMPA receptor channels on a microsecond
time scale. Biochemistry 35, 2030–2036.
26. Graciani, N. R., D. S. Swanson and J. W. Kelly (1995) Phos-
phomonoesters and phosphodiesters derived from the photohy-
drolysis of 2-methoxy-5-nitrophenyl substituted phosphotriesters.
Tetrahedron 51, 1077–1086.
27. Geibel, S., A. Barth, S. Amslinger, A. H. Jung, C. Burzik, R. J.
Clarke, R. S. Givens and K. Fendler (2000) P3-2-(4-hydroxyphe-
nyl)-2-oxo ethyl ATP for the rapid activation of the Na+,K+-
ATPase. Biophys. J. 79, 1346–1357.
38. Canepari, M., L. Nelson, G. Papageorgiou, J. E. T. Corrie and D.
Ogden (2001) Photochemical and pharmacological evaluation of
7-nitroindolinyl-and 4-methoxy-7-nitroindolinyl-amino acids as
novel, fast caged neurotransmitters. J. Neurosci. Methods 112,
29–42.
39. Canepari, M., L. Nelson, G. Papageorgiou, J. E. T. Corrie and D.
Ogden (2001) Pharmacological evaluation of 7-nitroindoline-
caged neurotransmitters and 4-methoxy-7-nitroindoline-caged
L-glutamate. Biophys. J. 80, 484.
40. Hagen, V., B. Dekowski, N. Kotzur, R. Lechler, B. Wiesner, B.
Briand and M. Beyermann (2008) {7-[bis(carboxymethyl)
amino]coumarin-4-yl}methoxycarbonyl derivatives for photore-
lease of carboxylic acids, alcohols ⁄ phenols, thioalcohols ⁄
thiophenols, and amines. Chem. Eur. J. 14, 1621–1627.
41. Schmidt, R., D. Geissler, V. Hagen and J. Bendig (2007) Mecha-
nism of photocleavage of (coumarin-4-yl)methyl esters. J. Phys.
Chem. A 111, 5768–5774.
42. Hagen, V., B. Dekowski, V. Nache, R. Schmidt, D. Geissler, D.
Lorenz, J. Eichhorst, S. Keller, H. Kaneko, K. Benndorf and B.
Wiesner (2005) Coumarinylmethyl esters for ultrafast release of
high concentrations of cyclic nucleotides upon one- and two-
photon photolysis. Angew. Chem. Int. Ed. 44, 7887–7891.
43. Schmidt, R., D. Geissler, V. Hagen and J. Bendig (2005) Kinetics
study of the photocleavage of (coumarin-4-yl)methyl esters.
J. Phys. Chem. A 109, 5000–5004.
44. Geissler, D., Y. N. Antonenko, R. Schmidt, S. Keller, O. O.
Krylova, B. Wiesner, J. Bendig, P. Pohl and V. Hagen (2005)
(Coumarin-4-yl)methyl esters as highly efficient, ultrafast photot-
riggers for protons and their application to acidifying membrane
surfaces. Angew. Chem. Int. Ed. 44, 1195–1198.
28. Allen, D. G., J. Lannergren and H. Westerblad (1999) The use
of caged adenine nucleotides and caged phosphate in intact
skeletal muscle fibres of the mouse. Acta Physiol. Scand. 166,
341–347.
29. Gee, K. R., L. W. Kueper, J. Barnes, G. Dudley and R. S. Givens
(1996) Desyl esters of amino acid neurotransmitters. Phototriggers
for biologically active neurotransmitters. J. Org. Chem. 61, 1228–
1233.
45. Hagen, V., S. Frings, B. Wiesner, S. Helm, U. B. Kaupp and J.
Bendig (2003) [7-(dialkylamino)coumarin-4-yl]methyl-caged com-
pounds as ultrafast and effective long-wavelength phototriggers of
8-bromo-substituted cyclic nucleotides. Chembiochem 4, 434–442.
46. Smirnova, J., D. Woll, W. Pfleiderer and U. E. Steiner (2005)
Synthesis of caged nucleosides with photoremovable protecting
groups linked to intramolecular antennae. Helv. Chim. Acta 88,
891–904.
30. Givens, R. S., D. Heger, B. Hellrung, Y. Kamdzhilov, M. Mac,
P. G. Conrad, E. Cope, J. I. Lee, J. F. Mata-Segreda, R. L.
Schowen and J. Wirz (2008) The photo-Favorskii reaction of
p-hydroxyphenacyl compounds is initiated by water-assisted,
adiabatic extrusion of a triplet biradical. J. Am. Chem. Soc. 130,
3307–3309.
31. Givens, R. S., P. S. Athey, B. Matuszewski, L. W. Kueper, J. Y.
Xue and T. Fister (1993) Photochemistry of phosphate-esters—
a-Keto phosphates as a photoprotecting group for caged phos-
phate. J. Am. Chem. Soc. 115, 6001–6012.
32. Givens, R. S., J. F. W. Weber, A. H. Jung and C.-H. Park. (1998)
New photoprotecting groups: desyl- and p-hydroxyphenacyl
phosphate and carboxylate esters. In Caged Compounds (Edited
by G. Marriott). Academic Press, San Diego, London, Boston,
New York, Sydney, Tokyo, Toronto, Methods Enzymol. 291,
1–29.
47. Woll, D., S. Laimgruber, M. Galetskaya, J. Smirnova, W.
Pfleiderer, B. Heinz, P. Gilch and U. E. Steiner (2007) On the
mechanism of intramolecular sensitization of photocleavage of the
2-(2-nitrophenyl)propoxycarbonyl (NPPOC) protecting group.
J. Am. Chem. Soc. 129, 12148–12158.
48. Adams, S. R., J. P. Y. Kao and R. Y. Tsien (1989) Biologically
useful chelators that take up Ca2 + upon illumination. J. Am.
Chem. Soc. 111, 7957–7968.
49. Gorner, H. (2005) Effects of 4,5-dimethoxy groups on the time-
resolved photoconversion of 2-nitrobenzyl alcohols and 2-nitro-
benzaldehyde into nitroso derivatives. Photochem. Photobiol. Sci.
4, 822–828.
50. Schaper, K., M. Etinski and T. Fleig (2009) Theoretical
investigation of the excited states of 2-nitrobenzyl and 4,5-methyl-
endioxy-2-nitrobenzyl caging groups. Photochem. Photobiol. 85,
1075–1081.

1254 Klaus Schaper et al.
51. Bley, F., K. Schaper and H. Gorner (2008) Photoprocesses of
molecules with 2-nitrobenzyl protecting groups and caged organic
acids. Photochem. Photobiol. 84, 162–171.
52. Milburn, T., N. Matsubara, A. P. Billington, J. B. Udgaonkar, J.
W. Walker, B. K. Carpenter, W. W. Webb, J. Marque, W. Denk,
J. A. McCray and G. P. Hess (1989) Synthesis, photochemistry,
61. Schworer, M. and J. Wirz (2001) Photochemical reaction mecha-
nisms of 2-nitrobenzyl compounds in solution I. 2-nitrotoluene:
thermodynamic and kinetic parameters of the aci-nitro tautomer.
Helv. Chim. Acta 84, 1441–1458.
62. Corrie, J. E. T., V. R. N. Munasinghe, D. R. Trenthama and A.
Barth (2008) Studies of decarboxylation in photolysis of alpha-
carboxy-2-nitrobenzyl (CNB) caged compounds. Photochem.
Photobiol. Sci. 7, 84–97.
and biological-activity of a caged photolabile acetylcholine-
receptor ligand. Biochemistry 28, 49–55.
53. Gee, K. R., L. Niu, K. Schaper, V. Jayaraman and G. P. Hess
(1999) Synthesis and photochemistry of a photolabile precursor of
N-methyl-D-aspartate (NMDA) that is photolyzed in the micro-
second time region and is suitable for chemical kinetic investiga-
tions of the NMDA receptor. Biochemistry 38, 3140–3147.
54. Gee, K. R., L. Niu, K. Schaper and G. P. Hess (1995) Caged
bioactive carboxylates—Synthesis, photolysis studies, and bio-
logical characterization of a new caged N-methyl-D-aspartic acid.
J. Org. Chem. 60, 4260–4263.
55. Schaper, K., S. A. Madani Mobarekeh and C. Grewer (2002)
Synthesis and photophysical characterization of a new, highly
hydrophilic caging group. Eur. J. Org. Chem. 1037–1046.
56. Hesse, M., H. Meier and B. Zeeh (1984) Spektroskopische
Methoden in der organischen Chemie. Georg Thieme Verlag,
Stuttgart, New York.
63. Kaplan, J. H. (1990) Photochemical manipulation of divalent-
cation levels. Annu. Rev. Physiol. 52, 897–914.
64. Gee, K. R., B. K. Carpenter and G. P. Hess (1998) Synthesis,
photochemistry, and biological characterization of photolabile
protecting groups for carboxylic acids and neutrotranmitters. In
Caged Compounds (Edited by G. Marriott). Academic Press, San
Diego, London, Boston, New York, Sydney, Tokyo, Toronto,
Methods Enzymol. 291, 30–50.
65. Adams, S. R. and R. Y. Tsien (1993) Controlling cell chemistry
with caged compounds. Annu. Rev. Physiol. 55, 755–784.
66. Corrie, J. E. T. and D. R. Trentham (1993) Caged nucleotides and
neurotransmitters. In Biological Applications of Photochemical
Switches (Edited by H. Morrison). John Wiley & Sons, Inc., New
York, Chichester, Brisbane, Toronto, Singapore. Bioorganic
Photochemistry Series, 2, 243–305.
57. Kalinowski, H. O., S. Berger and S. Braun (1984) 13C-NMR
Spektroskopie. Georg Thieme Verlag, Stuttgart, New York.
58. Gunzler, H. and H. Bock (1990) IR-Spektroskopie. VCH
Verlagsgesellschaft, Weinheim.
59. Grewer, C., S. A. Madani Mobarekeh, N. Watzke, T. Rauen and
K. Schaper (2001) Substrate translocation kinetics of excitatory
amino acid carrier 1 probed with laser-pulse photolysis of a new
photolabile precursor of D-aspartic acid. Biochemistry 40, 232–
240.
67. Hess, G. P. (1993) Determination of the chemical mechanism of
neurotransmitter receptor-mediated reactions by rapid chemical
kinetic techniques. Biochemistry 32, 989–1000.
68. Cohen, B. E., B. L. Stoddard and D. E. Koshland (1997) Caged
NADP and NAD. Synthesis and characterization of functionally
distinct caged compounds. Biochemistry 36, 9035–9044.
69. Pariser, R. and R. G. Parr (1953) A semi-empirical theory of the
electronic spectra and electronic structure of complex molecules. 2.
J. Chem. Phys. 21, 767–776.
60. Peng, L., J. Wirz and M. Goeldner (1997) Synthesis and charac-
terization of photolabile compounds releasing noracetylcholine in
the microsecond time range. Angew. Chem. Int. Ed. 36, 398–400.
70. Klopman, G. (1968) Chemical reactivity and concepts of charge-
and frontier controlled reactions. J. Am. Chem. Soc. 90, 223–
234.