New Photoreactive Cleavable Reagents with Trifluoromethyldiazirine Group

Article abstract of DOI:10.1023/A:1023268617727

Photoreactive crosslinking reagents that simultaneously contain a trifluoromethyldiazirine and an o-nitrobenzyl groups were synthesized for the first time. Photochemical properties of the reagents were studied, and the possibility of separate activation o

Full text of DOI:10.1023/A:1023268617727

Russian Journal of Bioorganic Chemistry, Vol. 29, No. 2, 2003, pp. 177–184. Translated from Bioorganicheskaya Khimiya, Vol. 29, No. 2, 2003, pp. 200–207.
Original Russian Text Copyright © 2003 by Mchedlidze, Sumbatyan, Bondar’, Taranenko, Korshunova.
New Photoreactive Cleavable Reagents
with Trifluoromethyldiazirine Group
1
M. T. Mchedlidze*, N. V. Sumbatyan*, D. A. Bondar’*,
M. V. Taranenko*, and G. A. Korshunova**
*Faculty of Chemistry, Moscow State University, Vorob’evy gory, Moscow, 119899 Russia
**Belozerskii Institute of Physicochemical Biology, Moscow State University, Vorob’evy gory, Moscow, 119899 Russia
Received December 21, 2001; in final form, March 29, 2002
Abstract—Photoreactive crosslinking reagents that simultaneously contain a trifluoromethyldiazirine and an
o-nitrobenzyl groups were synthesized for the first time. Photochemical properties of the reagents were studied,
and the possibility of separate activation of the diazirine group and o-nitrobenzyl linker was shown.
Key words: aryl(trifluoromethyl)diazirines, cleavable photoreactive reagents, photolysis
1
INTRODUCTION
In this work, we suggest new photoreactive cleav-
able reagents (I)–(IV). They have two photolabile
groups that are activated upon UV irradiation: p-triflu-
oromethyldiazirine group, which allows the covalent
binding of the contacting molecules at the contact
point; and Ó-nitrobenzyl group, which enables the split-
ting off of the useless part of the ligand. The photo-
chemical properties of these compounds were studied,
and a possibility of separate activation of diazirine and
nitrobenzyl groups was shown.
Photoaffinity modification is an effective approach
for studying interactions between biological molecules
[1]. Under irradiation, the photolabile group of one of
2
the interacting molecules generates a highly reactive
species that attacks neighboring groups with the forma-
tion of crosslinks. The study of the resulting covalent
complexes furnishes one with the information on the
structural organization of a system and on the nature of
interacting molecules [2–4]. However, the difficulties
in isolation and analysis of the cross-linked products
hinder their successful identification. The use of photo-
reactive reagents that can be selectively cleaved after
the covalent linking to a biological molecule may help
approach to the solution of this problem (Fig. 1). Such
reagents allow one to remove the useless part of the
ligand molecule and, in this way, to facilitate the detec-
tion and isolation of the resulting products.
RESULTS AND DISCUSSION
The choice of diazirine group as a photolabile func-
tion was not accidental. The photolysis of this group
proceeds with the formation of the highly reactive car-
bene under mild conditions (irradiation at 350–360 nm)
that are harmless for biological molecules. Moreover,
this group is stable within a wide range of conditions,
e.g., in thiol buffers with pH values varying from 0 to
14. The cleavable site, Ó-nitrobenzyl group, decom-
poses under the action of UV light of the same wave-
lengths; it is widely used for the synthesis of photo-
labile protecting groups, resins with photolabile linkers
for the solid phase synthesis, and photocleavable conju-
gates of peptides with oligonucleotides [14–18].
Scheme 1 shows the synthesis of reagents (I) and
(II). Starting 4-[3-(trifluoromethyl)-3H-diazirin-3-
yl]toluene (V) was obtained by a modified Nassal pro-
cedure [19–21]. 2-Nitro-4-[3-(trifluoromethyl)-3H-
diazirin-3-yl]toluene (VI) was synthesized by nitrating
(V) with a HNO3/H2SO4 mixture at 0°ë. Carrying out
the reaction at a low temperature allows one to avoid
the formation of dinitroderivative and to obtain the tar-
get product (VI) with a good yield.
The photoreagents that, in addition to the trifluo-
romethyldiazirine group, contain some chemically
cleavable groups (cis-diol, disulfide, or ester) are
known [5–12]. Their cleavage proceeds under the
action of chemical reagents, which imposes consider-
able limitations on their use for studying molecular
interactions in biological systems. An alternative
approach is based on the application of compounds that
contain a photocleavable site, i.e., a group cleavable
upon the UV irradiation. Fang et al. [13] were the first
who synthesized a bifunctional reagent that contained
two groups (m-nitrophenoxy and trifluoromethyldiazir-
ine) capable of a selective cleavage at different pH on
irradiation at 350 nm.
1
Please send correspondence to phone, +7 (095) 939-5520 or e-
mail, manana_m@imail.ru.
Abbreviations: NBS, N-bromosuccinimide.
2-Nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]ben-
zoic acid (I) was obtained by oxidation of (VI) by anal-
2
1068-1620/03/2902-0177$25.00 © 2003 MAIK “Nauka /Interperiodica”

178
MCHEDLIDZE et al.
N
N
N
N
N
N
N
N
CF3
CF3
CF3
F3C
NO2
NO2
NO2
NO2
CH2Br
CH2NH2 · HCl
CH2NCS
COOH
(I)
(II)
(III)
(IV)
ogy with the method that was used for oxidation of the silica gel in yield of 33%. Its structure was proven by
diazirine-containing reagents [19]. Potassium perman- mass spectrometry and UV and IR spectroscopy.
ganate in aqueous pyridine was used as an oxidizing
2-Nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]ben-
agent. A relatively low yield of the reaction product
zylamine hydrochloride (III) (Scheme 2) was synthe-
(31%) was apparently caused by a partial destruction of
sized using the modified Delepine reaction [22]. The
the diazirine cycle upon a long heating and by heavy
classical synthesis implies the interaction of a bromide
losses upon purification.
with methenamine (hexamethylenetetramine) in etha-
All the attempts to obtain 2-nitro-4-[3-(trifluorome-
thyl)-3H-diazirin-3-yl]benzyl bromide (II) by the bro-
mination of (VI) were unsuccessful. The conditions of
rigorous radical bromination were tested, but the
desired product was not detected; instead, only the
starting compound was isolated from the reaction mix-
ture. We suggest an alternative approach based on the
synthesis of 4-[3-(trifluoromethyl)-3H-diazirin-3-
yl]benzyl bromide (VII) by brominating the modified
toluene (V) with subsequent nitration leading to the tar-
get (II). NBS was used to brominate (V) according to
the procedure in [20].
Reagent (II) was prepared by nitrating substituted
benzyl bromide (VII) with nitric acid. We have found
that use of nitric acid containing nitrogen oxides favors
the formation of large amounts of by-products. The
same tendency was also observed when the reaction
was carried out at the temperatures from 0 to +5°ë. At
the same time, the reaction was not completed at a sub-
stantially lower temperature (–20°ë). On the basis of
the model experiments, we determined the optimal con-
ditions for the synthesis: the use of 90% nitric acid
freed from nitrogen oxides and the reaction tempera-
tures from –5°C to –10°C. Nevertheless, TLC showed
the formation of three products with Rf 0.57, 0.4, and
0.3. The target product (II) (Rf 0.4) was isolated from
nol followed by the decomposition of the resulting
methenaminium salt by gaseous hydrogen chloride.
This procedure gives the target amine with 40% yield.
The use of chloroform as a solvent [23] instead of eth-
anol greatly increases the solubility of the reacting sub-
stances, which results in increase in yield of the reac-
tion product: we obtained the methenaminium salt
(VIII) a white crystalline substance in 90% yield. The
salt was decomposed by two methods. The first of them
consisted in the treatment with a 8 : 1 methanol–con-
centrate HCl mixture [24] and resulted in the target
amine hydrochloride (III). The second method
involved the decomposition of the methenaminium salt
with sulfur dioxide to give 2-nitro-4-[3-(trifluorome-
thyl)-3H-diazirin-3-yl]benzylaminomethyl hydrogen
sulfite (IX), which was stable on storage. It can be
transformed into the amine by the treatment with 25%
hydrochloric acid and water vapor [25].
2-Nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]ben-
zyl isothiocyanate (IV) was obtained by the reaction of
amine (III) with carbon disulfide in pyridine with DCC
as a coupling reagent (Scheme 2). The reaction was car-
ried out for 12 h at room temperature. The coupling
reactions with the use of DCC are accompanied by the
formation of dicyclohexylurea, which usually precipi-
tates and can be easily removed from the reaction mix-
the reaction mixture by a column chromatography on ture. However, in our case, any precipitate was not
Receptor Ligand
hν
(a)
(b)
(c)
Photolabile
group
Cleavable
group
Fig. 1. The principle of the photoaffinity modification by the use of a cleavable reagent: (a) the formation of a ligand–receptor non-
covalent complex, (b) the formation of a covalent complex upon the irradiation, (c) the cleavage (upon the action of light or chemical
reagents) and the removal of the ballast part of the ligand from the ligand–receptor complex.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 2 2003

NEW PHOTOREACTIVE CLEAVABLE REAGENTS
179
N
N
N
N
F3C
N
N
F3C
F3C
HNO3/H2SO4
KMnO4
NO2
NO2
CH3
CH3
(V)
COOH
(VI)
(I)
NBS
N
N
N
N
F3C
F3C
HNO3
NO2
CH2Br
(VII)
CH2Br
(II)
Scheme 1. The synthesis of 2-nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]toluene (VI), 2-nitro-4-[3-(trifluoromethyl)-3H-diazi-
rin-3-yl]benzoic acid (I), and 2-nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl bromide (II).
N
N
N
N
N
N
N
N
N
CF3
CF3
CF3
CF3
N
N
N
HCl
CS2, DCC
NO2
NO2
NO2
NO2
N
CH2Br
CH2NH2 · HCl
CH2NCS
+
N
N
H2C
Br–
(VIII)
(II)
(III)
(IV)
N
BocAlaOH, DCC
N
F3C
SO2
HCl
N
N
N
CF3
NO2
O
BocNH
NH
CH3
NO2
(X)
H2C NH CH2 OSO2H
(IX)
Scheme 2. The synthesis of 2-nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzylamine hydrochloride (III), 2-nitro-4-[3-(trifluo-
α
romethyl)-3H-diazirin-3-yl]benzyl isothiocyanate (IV), and 2-nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzylamide of N -
Boc-alanine (X).
formed, because dicyclohexylthiourea is easily soluble presence of DCC to give (X). After the elimination of
in organic solvents. We solve this problem by the dicy-
clohexylthiourea removal by a column chromatography
on silica gel (see the Experimental section).
Boc group, ninhydrin test of the reaction product
showed the presence of amino group in the resulting
compound, and an absorption band corresponding to
2-Nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]ben- the diazirine ring (363 nm, ε 170 å–1 cm–1) was
zylamine (III) was coupled with Nα-Boc-alanine in the observed in its UV spectrum.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 2 2003

180
MCHEDLIDZE et al.
H
O
X R
OH
hν
+ R–XH
CH2 X R
O
+
N
O
O
N
N
O–
where X = O, NH, or S.
Scheme 3. The cleavage of o-nitrobenzyl derivative under the UV light action.
A characteristic band in the region of 342–353 nm ness of the absorbing layer (cm), and t is the irradiation
(ε 160–640 å–1 cm–1) is observed in the electronic
absorption spectra of ethanol solutions of diazirines
(I)–(IV) (Table 1), which is in a good agreement with
literature data [19, 26, 27]. The UV irradiation with a
wavelength ~360 nm results in the photolytic decompo-
sition of the diazirine group to give a carbene. In this
case, the intensity of the light absorption at the maxi-
mum corresponding to the diazirine ring decreases
along with an increase in the irradiation time. The half-
decay time is determined as the time necessary for half
of the molecules to undergo a photolytic decomposi-
tion. If the intensity of the light irradiation is known, the
quantum yield (ϕ) for the decomposition of a photo-
labile group may be determined as:
time (s);
∆m = ∆cNAV = ∆A/εNAV, where (∆c is a change in a
substance concentration (mol/l), ∆A is the change in the
optical absorption, NA is Avogadro’s number (mol–1),
and V is the volume of solution (ml).
The photolysis of compounds containing
Ó-nitrobenzyl group proceeds according to the mecha-
nism of intramolecular oxidation–reduction as shown
in Scheme 3. In this case, Ó-nitrosobenzaldehyde is
formed, which follows from the appearance of a peak in
the UV-range of 300–400 nm corresponding to the
absorption of nitroso group [28].
We studied the kinetics of the selective photode-
composition of diazirine group and nitrobenzyl linker
by the example of 2-nitro-4-[3-(trifluoromethyl)-3H-
diazirin-3-yl]benzylamide of Nα-ÇÓÒ-alanine (X),
which is a simplified model of the photoreactive protein
molecule that can be used for studying the nucleic acid–
protein and protein–protein interactions.
ϕ = ∆m/∆n,
where ∆n is the number of light quanta absorbed in a
definite time, and ∆m is the number of the mols of diaz-
irine reacted for the time. These values can be specified
as follows:
The kinetic study was carried out by recording the
UV spectra of an ethanolic solution of (X) after its irra-
diation for a definite time. A mercury lamp emitting
monochromatic light at 365 nm was used as a light
source for irradiation. An intensity of the absorption at
363 nm corresponding to diazirine group was observed
to decrease during the first 25 min of photolysis
(Fig. 2a). Clear isobestic points observed in the spectra
indicate a photochemical reaction of the first order. As
known [29], the products of carbene insertion into O–H
bond are formed upon the photolysis of diazirines in
alcohols. Therefore, 2-nitro-4-(2,2,2-trifluoromethyl-1-
ethoxyethyl)benzylamide of Nα-ÇÓÒ-alanine (XI)
seems to be the major product in the initial stage of the
photolysis of (X) (Scheme 4). The absorption maxi-
mum at 358 nm corresponding to nitroso group was
observed to appear and to grow at longer irradiation
times (Fig. 2b). This indicated the decomposition of
nitrobenzyl group, which presumably resulted in the
formation of 2-nitroso-4-(2,2,2-trifluoro-1-ethoxy-
ethyl)benzaldehyde (XII) and another product, amide
of Nα-ÇÓÒ-alanine (XIII), which was detected by TLC
in the irradiated solution.
∆n = Iabsorbedt = I0εlct, where Iabsorbed is the intensity
of light absorbed by a substance (quanta/s), I0 is the
intensity of the light irradiating the reaction vessel
(quanta/s), ε is the molar absorption coefficient (M–1 cm–
1), c is concentration of a substance (mol/l), l is a thick-
Table 1. The UV spectra of the synthesized photoreagents
(I)–(IV)
Compound
λmax, nm
ε, M–1 cm–1
(I)
256
291
343
251
295
342
220
260
353
220
246
353
19900
1300
200
(II)
12600
1300
160
(III)
(IV)
5500
1800
330
8400
11600
640
The data that characterize the photochemical prop-
erties of the diazirine and nitrobenzyl groups in (X) are
presented in Table 2. One can see that these groups sig-
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NEW PHOTOREACTIVE CLEAVABLE REAGENTS
181
CH3
CH3
N
N
C2H5O
C2H5OH, hν
CH2 NH
CH NHBoc
CH2 NH
CH NHBoc
F3C
F3C
O
O
NO2
NO2
(X)
(XI)
hν
CH3
O
H
C2H5O
F3C
+
H2N
CH NHBoc
O
NO
(XII)
(XIII)
α
Scheme 4. Scheme 4. The scheme of the UV photolysis of 2-nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzylamide of N -Boc-
alanine (X).
nificantly differ in their half-decay times (τ1/2; 7.4 and may be useful to study their intermolecular contacts by
the method of photoaffinity modification.
67 min, respectively) and quantum yields (ϕ) of the
photochemical decomposition. This means that the
photolysis of (X) is selective: diazirine group first
forms a short-lived carbene and then the nitrobenzyl
linker is degraded.
EXPERIMENTAL
TLC was carried out on the precoated Silica gel
60 F254 plates (Merck, Germany) using the following
solvent systems: (A) 65 : 10 petroleum ether–chloro-
form, (B) 4 : 1 hexane–ethyl acetate, (C) 2 : 1 chloro-
form–hexane, (D) 50 : 25 : 2 benzene–acetone–acetic
acid; and (E) hexane. Substances were detected by their
UV absorption. Column chromatography was per-
formed on Silica gel 60 (70–230 mesh; Merck, Ger-
Thus, new photoreactive cleavable reagents that
include a trifluoromethyldiazirine group are here
described. The presence of chemically active groups
[carboxyl in (I), bromomethyl in (II), amino in (III),
and isothiocyanate in (IV)] helps use these reagents for
the modification of amino, carboxyl, and sulfhydryl
groups as the ligands within biological molecules that many).
A
1
(a)
(b)
0.2
85 min
35 min
75 min
55 min
0 min
1 min
2 min
4 min
8 min
15 min
0
0
300
350
400
340
350
360
370
λ, nm
α
Fig. 2. The kinetics of the 2-nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzylamide of N -Boc-alanine (X) photolysis. The
absorption spectra of the solution of (X) in ethanol (c 1.35 mM) are shown at various irradiation times: (a) a decrease in the intensity
of the peak at 363 nm, corresponding to diazirine group for 0–15 min; (b) an increase in the absorption at 358 nm (this peak corre-
sponds to nitroso group) for 35–85 min.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 2 2003

182
MCHEDLIDZE et al.
Table 2. The kinetic characteristics of the photolysis of 2-nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzylamide of
Nα-Boc-alanine (X)
Photolabile group
τ1/2, min
k, min–1
ϕ
CF3
7.4 ± 1.4
(9.3 ± 1.7) × 10–2
0.98 ± 0.33
N
N
CH2
NO2
66.9 ± 15.8
(1.03 ± 0.23) × 10–2
0.109 ± 0.032
IR spectra were recorded on a Specord 40 instru- sodium sulfate and evaporated. The residue was dis-
ment (Germany). Mass spectra were determined using solved in ethanol (8 ml) and precipitated with water
a Finnigan MAT INCOS 50 instrument using the elec- (25 ml) to give (I) as a white crystalline substance;
tron impact ionization. UV spectra were measured in a yield 120 mg (31%); Rf 0.27 (B); UV (hexane, λmax, nm
single beam mode on an Aminco DW 2000 spectropho-
tometer at the wavelength range of 200–500 nm and
scanning rate of 3 nm/s in quartz cells with an optical
path length of 1 cm.
Syntheses of substances containing trifluoromethyl-
diazirine group were carried out under the conditions of
moderate illumination.
4-[3-(Trifluoromethyl)-3H-diazirin-3-yl]toluene (V)
was synthesized as described in [19]. 4-[3-(Trifluoro-
methyl)-3H-diazirin-3-yl]benzyl bromide (VII) was
obtained according to the method [20] in 74% yield.
2-Nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]-
toluene (VI). A 95% H2SO4 (1.4 ml) was carefully
added dropwise to a stirred cooled (0°ë) 4-[3-(trifluo-
romethyl)-3H-diazirin-3-yl]toluene (V) (400 mg,
2 mmol). Then a nitrating mixture [97% HNO3–fuming
HNO3–concentrate H2SO4 (0.29 + 0.1 + 0.55 ml,
respectively)] was dropwise added. The reaction mix-
ture was stirred for 1 h, poured on ice and extracted
with diethyl ether (2 × 10 ml). The extract was washed
with 10% sodium bicarbonate (2 × 10 ml) and water
(logε ): 256 (4.3), 291 (3.1), and 343 (2.3); MS, m/z
247 [M –N2]+, 167 [M + H – N2–CCF3]+.
2-Nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]ben-
zyl bromide (II). 4-[3-(Trifluoromethyl)-3H-diazirin-
3-yl]benzyl bromide (VII) (1.36 g, 4.87 mmol) was
added dropwise to a cooled (–10°ë) 90% nitric acid
(9 ml, 0.17 mmol) depleted of nitrogen oxides. The
reaction mixture was extracted with diethyl ether
(2 × 20 ml); the extract was washed with a saturated
sodium bicarbonate solution and water, dried over
sodium sulfate, filtered, and evaporated on a rotary
evaporator. The resulting yellow oil was dissolved in a
solvent system A (1 ml) and chromatographed on silica
gel using system A as an eluent. Three fractions were
collected with Rf 0.57, 0.4, and 0.3. Target product (II)
was in fraction with Rf 0.4; yield 511 mg (33%); Rf 0.4
(A) and 0.13 (E); UV (ethanol, λmax, nm (logε ): 251
(4.1), 295 (3.1), and 342 (2.2); IR (ν, cm–1): 1704
(NO2), 1608 (N=N); MS, m/z: 297 [M + H – N2]+, 295
[M – H – N2]+, 251 [M + H – N2 – NO2]+, 249 [M – H –
(2 × 10 ml), dried over anhydrous sodium sulfate, and N2 – NO2]+.
evaporated to give (VI) as an yellowish oil; yield of
350 mg (70%); Rf 0.75 (A) and 0.22 (E); UV (diethyl
ether), λmax, nm ( logε ): 250 (4.0), 288 (3.0), and 358
(2.3); MS, m/z: 245 [M]+.
2-Nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]ben-
zoic acid (I). Potassium permanganate (1 g, 5.6 mmol)
was added to a stirred solution of (VI) (350 mg,
1.4 mmol) in a 10 : 7 pyridine–water mixture (17 ml).
The reaction mixture was stirred at 50°ë for 15 h,
cooled to room temperature, diluted with water (50 ml),
acidified to pH 2 with 1 N sulfuric acid, discolored by
the addition of saturated Na2S2O5 solution, and then
extracted with diethyl ether (2 × 100 ml). The organic
2-Nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]ben-
zylamine hydrochloride (III). Method 1. Compound
(II) (372 mg, 1.15 mmol) was added to a solution of
methenamine (170 mg, 1.2 mmol) in chloroform
(2 ml). The reaction mixture was stirred for two days,
and the precipitated methenaminium salt (483 mg,
90%) was filtered and decomposed by the treatment
with a 1 : 8 conc. HCl–methanol mixture (10 ml) for
three days at room temperature. The precipitated
ammonium chloride was filtered off, and the filtrate
was evaporated to dryness to yield 306 mg (90%) of
(III).
Method 2. Compound (II) (100 mg, 0.31 mmol) was
phase was alkalized with 1 N NaOH to pH 10 and added to a solution of methenamine (43 mg,
extracted with water. The water phase was acidified 0.31 mmol) in chloroform (1 ml). The reaction mixture
with 1 N sulfuric acid to pH 2 and extracted with was stirred for two days, and the precipitated methen-
diethyl ether. The extract was dried over anhydrous aminium salt (143 mg, 90%) was filtered and then dis-
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NEW PHOTOREACTIVE CLEAVABLE REAGENTS
183
The measured intensity (I0) was 1.5 × 1016 quanta/s.
solved in water (2 ml). Gaseous SO2 was passed
through the solution for 1.5 h, which was accompanied
with warming-up of the reaction mixture and the for-
mation of a white precipitate 2-nitro-4-[3-(trifluorome-
thyl)-3H-diazirin-3-yl]benzylaminomethyl hydrogen
sulfite (IX). The filtered precipitate was suspended in
25% HCl (5 ml) and aqueous vapor was passed though
the suspension for 15 min. The resultant clear solution
was cooled, and a white precipitate was formed. It was
filtered and washed with an ice water (2 × 2 ml) to yield
68 mg (75%) of (III); Rf 0.4 (B); UV (ethanol, λmax, nm
After irradiation for 75 min, Nα-ÇÓÒ-alanine amide was
detected in the solution by TLC, Rf 0.38 in 9 : 1 chloro-
form–methanol; its spot coincided with that for the ref-
erence compound. On spraying the plate with 0.2% nin-
hydrin in acetone containing 10% acetic acid with sub-
sequent heating to 100°ë, a purple staining
characteristic of amino acids was observed.
ACKNOWLEDGMENTS
(logε ): 220 (3.74), 260 (3.24), and 353 (2.51).
We are grateful to Cand. Sci. (Chem.) V.L. Drutse
for the use of his instruments.
This work was supported by the Russian Foundation
for Basic Research.
2-Nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]ben-
zyl isothiocyanate (IV). Compound (III) (50 mg,
0.17 mmol) was dissolved in pyridine (2.5 ml), and car-
bon disulfide (100 µl, 1.7 mmol) was added to the
stirred solution. The reaction mixture was cooled to
0°ë, treated with DCC (55 mg, 0.27 mmol), stirred for
4 h, and then evaporated. The residue was chromato-
graphed on a silica gel column eluted with the system
C to yield 30 mg (58%) of (IV); Rf 0.7 (B) and 0.9 (C);
UV (ethanol, λmax, nm (logε ): 220 (3.92), 246 (4.06),
and 353 (2.8); IR (cm–1): 2080 (NCS), 1704 (NO2),
1612 (N=N); MS, m/z: 269 [M – H – S]+, 244 [M –
NCS]+, 229 [M – NCS – N]+.
2-Nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]ben-
zylamide of Na-Boc-alanine (X). Nα-Boc-alanine
(8.7 mg, 46 µmol) was dissolved in dichloromethane
(1.5 ml) and treated with 1-hydroxybenzotriazole
(9 mg, 67 µmol). The reaction mixture was cooled on
ice, treated with (11 mg, 54 µmol) DCC, and stirred for
1 h. A solution of (III) (16 mg, 50 µmol) and diisopro-
pylethylamine (20 µl, 54 µmol) in dichloromethane
(1 ml) was added to the reaction mixture, and stirring
was continued for one day. The precipitate of dicyclo-
hexylurea was filtered off, the filtrate was evaporated,
the residue was dissolved in ethyl acetate (10 ml), and
the solution was washed with 10% sodium bicarbonate
(2 × 5 ml), 0.1 N sulfuric acid (2 × 5 ml), water (2 ×
5 ml), and a saturated solution of sodium chloride (2 ×
5 ml). The ethyl acetate extract was dried over anhy-
drous sodium sulfate and evaporated to yield 10 mg
(50%) of (X); Rf 0.73 (D); UV (ethanol, λmax, nm
REFERENCES
1. Singh, A., Thornton, E.R., and Westheimer, F.H., J. Biol.
Chem., 1962, vol. 23, pp. 3006–3008.
2. Bayley, H., Photogenerated Reagents in Biochemistry
and Molecular Biology, Work, T. and Burdon, R., Eds.,
Amsterdam: Elsevier, 1983, vol. 12, p. 187.
3. Brunner, J., Annu. Rev. Biochem., 1993, vol. 62, pp. 483–
514.
4. Kotzyba-Hilbert, F., Kapfer, I., and Goedner, M., Angew.
Chem., Int. Ed., 1995, vol. 34, pp. 1296–1312.
5. Brunner, J. and Richards, F., J. Biol. Chem., 1980,
vol. 225, pp. 3319–3329.
6. Kogon, A.A., New Photoreactive Reagents for Studying
Biological Macromoleculs by the Method of Fast Photo-
affinity Crosslinking, Cand. Sc. (Chem.) Dissertation,
Moscow: Moscow State Univ., 1990.
7. Savage, M.D., Mattson, G., Desai, S., Nielander, G.W.,
Morgensen, S., and Conklin, E.J., Avidin–Biotin Chem-
istry: a Handbook, Illinois: Pierce Chemical Co., 1992.
8. Kogon, A., Peletskaya, E., Quinn, T., and Folk, W.,
Abstracts of Papers, 37 Annual West Central States Bio-
chemistry and Molecular Biology Conf., Research and
Education, Univ. of Missouri, Columbia, 1994, p. 60.
9. Brunner, J., Trends in Cell Biol., 1996, vol. 6, pp. 154–
157.
10. Bochkarev, D. and Kogon, A., Anal. Biochem., 1992,
vol. 204, pp. 90–95.
11. Kogon, A., Bochkarev, D., Baskunov, B., and Cherpa-
(logε ): 319 (3.9), 333 (2.52), and 363 (2.2). The pres-
ence of a free amino group was proven by the ninhydrin
test after the removal of Boc group.
kov, A., Liebigs Ann. Chem., 1992, pp. 879–881.
12. Kempin, U., Kanoaka, Y., and Hatanaka, Y., Heterocy-
cles, 1998, vol. 49, pp. 465–468.
The photolysis of 2-nitro-4-[3-(trifluoromethyl)-
3H-diazirin-3-yl]benzylamide of Na-Boc-alanine
(X). A 1.35 mM solution of (X) in ethanol was irradi-
ated in a quartz cell with an optical path length of 1 cm.
A Mineralight UVGL-58 mercury lamp (with an inte-
grated filter), which emits a monochromatic light with
a wavelength of 365 nm with intensity of 700 µW/cm2,
was used as a light source for the irradiation. The inten-
sity of the incident light (in quanta/s) required to deter-
mine the quantum yield was measured by actinometry
using the ferrioxalate–phenanthroline technique [30].
13. Fang, K., Hashimoto, M., Jokush, S., Turro, N.J., and
Nakanishi, K., J. Am. Chem. Soc., 1998, vol. 120,
pp. 8543–8544.
14. Pillai, K., Synthesis, 1980, pp. 1–26.
15. Lloyd-Williamsetal, F., Tetrahedron, 1993, vol. 49,
pp. 11065–11133.
16. Olejnik, J., Krzymanska-Olejnik, E., and Roths-
child, K.J., Nucleic Acids Res., 1998, vol. 26, pp. 3572–
3576.
17. Wei, Y., Yan, Y., Pei, D., and Gong, B., Bioorg. Med.
Chem. Letters, 1998, vol. 8, pp. 2419–2422.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 2 2003

184
MCHEDLIDZE et al.
18. Olejnik, J., Ludemann, H.-C., Krzymanska-Olejnik, E.,
Berkenkamp, S., Hillenkamp, F., and Rothschild, K.J.,
Nucleic Acids Res., 1999, vol. 27, pp. 4626–4631.
nicheskie reaktsii, Moscow: Inostrannaya Literatura,
1965, Sbornik 8, p. 263, London, Chapman&Hall.
25. Houben-Weyl, vol. XI, 1957, p. 106.
19. Nassal, M., Liebigs Ann. Chem., 1983, pp. 1510–1523.
26. Brunner, J. and Semenza, G., Biochemistry, 1981,
20. Nassal, M., J. Am. Chem. Soc., 1984, vol. 106, pp. 7540–
vol. 20, pp. 7174–7182.
7545.
27. Dolder, M., Michel, H., and Sigrist, H., J. Protein Chem.,
21. Topin, A.N., Vestn. Mosk. Gos. Univ., Ser. 2: Khim.,
1990, vol. 9, pp. 407–415.
1995, vol. 36, pp. 583–587.
28. Wieboldt, R., Ramesh, D., Carpenter, B.K., and
22. Delepine, M., Compt. Rend., 1895, vol. 120, p. 501.
Hess, G.P., Biochemistry, 1994, vol. 33, pp. 1526–1533.
23. Tietze, L.F. and Eicher, T., Reaktionen und Synthesen im
organisch-chemischen Praktikum und Forschungslabo-
ratorium, Stuttgart: Georg Tieme, 1991. Translated
under the title Preparativnaya organicheskaya khimiya,
Moscow: Mir, 1999.
24. Angial, S., Organic Reactions, vol. 8, New York: John
Wiley & Sons, 1954. Translated under the title Orga-
29. Brunner, J., Senn, H., and Richards, F.M., J. Biol. Chem.,
1980, vol. 255, pp. 3313–3318.
30. Eksperimental’nye metody khimicheskoi kinetiki (Exper-
imental Methods of Chemical Kinetics), Ema-
nuel’, N.M. and Sergeev, G.B., Eds., Moscow: Vysshaya
Shkola, 1980, pp. 132–135.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 2 2003