Synthesis and photochemical properties of photo-cleavable crosslinkers

Article abstract of DOI:10.1016/j.tetlet.2009.03.009

We report herein the synthesis and the development of homo-bifunctional photo-cleavable sulfhydryl group cross-linkers that are able to react and then to photorelease two cysteines leading to the photoregulation of cross-linkers cleavage.

Full text of DOI:10.1016/j.tetlet.2009.03.009

Tetrahedron Letters 50 (2009) 2434–2436
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Tetrahedron Letters
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Synthesis and photochemical properties of photo-cleavable crosslinkers
*
Ziad Omran, Alexandre Specht
Laboratoire de Chimie Bioorganique, UMR 7199 CNRS/Université de Strasbourg, Faculté de Pharmacie, 74 route du Rhin, 67400 Illkirch, France
a r t i c l e i n f o
a b s t r a c t
Article history:
We report herein the synthesis and the development of homo-bifunctional photo-cleavable sulfhydryl
group cross-linkers that are able to react and then to photorelease two cysteines leading to the photore-
gulation of cross-linkers cleavage.
Received 4 February 2009
Revised 2 March 2009
Accepted 4 March 2009
Available online 9 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Photochemistry
Caged compounds
Cross-linkers
To follow physiological events in real time, photolabile protect-
ing groups offer the means to control the delivery of bioactive
materials, by rendering the molecule of interest biologically inert
(or caged) by means of a chemical modification with this photo-
removable group. The photochemical reaction allows a temporal
and spatial control of the release of active biomolecules, switching
on (or off) the targeted process within organized biological sys-
tems. Dynamics studies can therefore be undertaken using time-
resolved analytical methods.1–5
Photochemical triggering of biological events can be achieved
either from ‘caged’ biological effectors or from ‘caged’ proteins.1–5
Clearly, it is more advantageous to cage directly a protein,
because a stoichiometric irreversible modification leads to a stable
inactivated enzyme or receptor that requires a minimum amount
of photons for its reactivation. Only a few examples of protein
caging are found in the literature, due to the difficulty in targeting
a suitable caging group to the desired site of the protein.6,7 This has
been successfully achieved by selective modification of strategic
cysteine residues, either natural,8,9 or incorporated by site-directed
mutagenesis.10 In most cases of protein ‘caging’, 2-nitrobenzylbro-
mide or 4-hydroxy-phenacyl bromide derivatives were used as
photo-removable groups. Alternatively, unnatural amino acid
mutagenesis enabled direct incorporation of photolytic precursors
of various amino acids at a precise position in the sequence.11
The extension of protein caging to residues other than cysteines is
more difficult and requires a targeted site-directed modification.12
structure and function of proteins, or protein complexes.13
A
typical cross-linking reagent can contain either two identical
reactive sites (homo-bifunctional) or two different reactive sites
(hetero-bifunctional), which are connected with a carbon chain
spacer bridging a defined interval. Therefore, two functional
groups in the proteins (e.g., amine or sulfhydryl groups) can be
cross-linked.
We report here the synthesis and the development of homo-
bifunctional photo-cleavable sulfhydryl group crosslinkers. Their
reactivity towards cysteines and the subsequent photolytic reac-
tion have been studied, showing that these chemical probes were
able to react and then photorelease two cysteines leading to a
photo-controlled cleavage of these cross-linkers. Thus, our new
photo-cleavable crosslinkers should be very interesting tools for
the photoregulation of biological activities (Fig. 1). For example,
transport proteins, which need to undergo an important structural
rearrangement to catalyze the transport of specific molecules or
ions across membrane bilayer,14,15 could be locked in one confor-
mation by using both site-directed double cysteine mutants of
the transporter and intramolecular cross-linking. The UV photoly-
sis of such modified transporters should rapidly and efficiently
cleave the crosslinker, leading to the triggering of their activities.
The photocleavage of an intermolecular crosslinking combined
with mass spectrometry should overcome many of the challenges
associated with other crosslinking reagent for studying protein
structure and protein–protein interactions.16
Chemical cross-linking,
between different molecules (intermolecular) or parts of a mole-
cule (intramolecular), has allowed to gain further insight into
a
process forming covalent bonds
The synthesis of the photo-cleavable crosslinkers 1a–e is out-
lined in Scheme 1. Starting from veratrole, we prepared diketones
2a–e17 by Friedel–Crafts reaction in very good yields. The regiose-
lective nitration18 of the symmetric diketones 2a–e was properly
achieved by treatment of the latter with the nitronium cation
which was generated in situ by the action of the trifluoroacetic acid
* Corresponding author. Tel.: +33 390244166; fax: +33 390244306.
E-mail address: specht@bioorga.u-strasbg.fr (A. Specht).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.03.009

Z. Omran, A. Specht / Tetrahedron Letters 50 (2009) 2434–2436
2435
Figure 1. Proposed mechanism for the triggering of protein activities using photo-cleavable crosslinkers: spatio-temporal control of protein conformational changes or
protein–protein interactions.
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
HNO3, TFA
CH2Cl2, -60
67-84%
AlCl3
CH2Cl2
n
n
+
Cl
Cl
C
n
NO2 O2N
85-96%
2a-e
3a-e
n
compound
OH
OH
Br
Br
4
5
6
7
8
a
b
c
d
e
O
O
O
O
O
O
O
O
NaBH4
n
CH3COBr, HBr
65-90%
n
MeOH, CH2Cl2
94-100%
NO2 O2N
NO2 O2N
4a-e
1a-e
Scheme 1. Synthesis of the photo-cleavable crosslinkers 1a–e.
on nitric acid,19 leading to ortho-nitro diketones 3a–e. Diols 4a–e
were obtained by reduction of the diketones using sodium borohy-
dride. Final treatment of the obtained alcohols by acetyl bromide
and hydrobromic acid20 afforded the sulfhydryl group crosslinkers
1a–e in good yields.
These new photo-cleavable crosslinkers were designed to
modulate distances from 6 to 12 angstroms between the two sulf-
hydryl reactive groups, by the use of different carbon chain spacers
(Table 1).
ment of the decay of the probes. Our photo-cleavable cross-linkers
react with thiols within 2–2.5 h at the concentrations used.
The hydrolytic stability of our crosslinkers was checked by HPLC
analysis of 50 lM solution of 1a–e in a mixture 50:50 of EtOH and
phosphate buffer (50 mM, pH 7.3) at room temperature in the ab-
sence of light. A t1/2 for the hydrolysis of 9 h was measured, indi-
cating that the hydrolysis of our cross-linkers 1a–e is four to five
times slower than their sulfhydryl substitution reactions.
The photolytic reactions of 6a–e (50 lM in a mixture 50:50 of
To demonstrate the ability to photoregulate the cross-linking of
two sulfhydryl groups, we used the N-benzoylcysteine methyl es-
ter 521 to facilitate the UV detection of the released sulfhydryl
group by HPLC analysis.
Accordingly, crosslinkers 1a–e were reacted with excess
amount of N-benzoylcysteine methyl ester 5 in a slightly alkaline
medium to yield the ‘caged’ cysteines 6a–e (Scheme 2).
Their reactivities toward thiols and their half-times of reaction
in the presence of 20-fold excess of N-benzoylcysteine methyl es-
ter 5 in a mixture 50:50 of EtOH and phosphate buffer (50 mM,
pH 7.3) were checked by HPLC analysis. As the reaction rate varies
linearly with the concentration of the reagents (pseudo-first order
reaction), their reactivities were adjusted for an accurate measure-
EtOH and phosphate buffer (50 mM, pH 7.3), containing 0.5 mM
DTT at k = 365 nm) were analyzed by UV spectroscopy and HPLC
(Fig. 2). All six compounds showed an increase in absorbance at
375 and 280 nm and decrease at 320 nm. The isobestic points at
350 and 300 nm indicate that the photolytic reaction is homoge-
nous. The HPLC analysis of these reactions depicted the concomi-
tant disappearance of the starting compounds and an almost
quantitative liberation of 2 equiv of the unmasked thiol derivative
5 (Table 1). The latter was quantified by HPLC after an HPLC cali-
bration of 5. The expected nitroso side products 7a–e could not
be detected due to their chemical instability.
Quantum yields of the photolytic reactions for 6a–e were deter-
mined by competition with the 2-(nitrophenyl)ethyl-ATP reference
molecule.22 Quantum yields between 0.43 and 0.57 (Table 1) were
determined in a mixture 50:50 of EtOH and phosphate buffer
(50 mM, pH 7.2) leading to very high photochemical efficiencies
(up to 5260 MÀ1 cmÀ1 at 352 nm) for the fragmentation of our
photo-cleavable crosslinkers.
Table 1
Summary of the properties of 6a–e (e352 absorption coefficient at 352 nm, d the
distance between the two sulfhydryl groups, / quantum yield)
Compound
e352
d (Å)
Photo-released
Cysteine (%)
/
In conclusion, we have described here the synthesis and the
characterization of homo-bifunctional photo-cleavable sulfhydryl
group crosslinkers with carbon chain spacers ranging from 6 to
12 angstroms. These chemical probes are able to connect and then
to photorelease two cysteines leading to the photoregulation of
cross-linker cleavage. Therefore, they should be very interesting
6a
6b
6c
6d
6e
9350
9280
9230
8860
8980
6.4
7.8
9.3
10.4
11.7
200
191
200
195
198
0.44
0.43
0.57
0.51
0.51

2436
Z. Omran, A. Specht / Tetrahedron Letters 50 (2009) 2434–2436
Br Br
O
O
n
O
O
NO2 O2N
1a-e
O
COOMe
S
S
SH
O
O
O
O
k
N
H
n
h
NO2 O2N
5
6a-e
O
O
O
O
O
n
O
NO ON
7a-e
Scheme 2. Photoregulation of the cross-linking of two sulfhydryl groups.
Figure 2. Compound 6c (50 lM in a mixture 50:50 of EtOH and phosphate buffer 50 mM, pH 7.3, containing 0.5 mM DTT) was exposed to the 364 nm line of a Hg lamp. (A)
UV spectrum recording during the photolysis of 6c. (B) HPLC analysis at 250 nm before and after 30 min of irradiation of 6c. Compound 6c has a retention time of 28.4 min
and the appearing peak at 18.5 min corresponds to the N-benzoylcysteine methyl ester 5.
6. Loudwig, S.; Bayley, H. In Dynamic Studies in Biology, Phototriggers,
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This work was supported by the Université de Strasbourg, the
CNRS, and the HFSP grant RGY0069 (A.S.). The authors thank Pro-
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