The Effect of Linker Length on the Photomodification of Tyr Residue in N-(Tyrosyl)-N′-(5-azido-2-nitrobenzoyl)-diaminoalkanes

Article abstract of DOI:10.1023/A:1012980517479

N-(Tyrosyl)-N′-(5-azido-2-nitrobenzoyl)-1,4-diaminobutane containing a Tyr residue connected with the photoreactive aryl azide group through a diaminobutylene linker was synthesized as a model for studying the photomodification of Tyr residues in proteins

Full text of DOI:10.1023/A:1012980517479

Russian Journal of Bioorganic Chemistry, Vol. 27, No. 6, 2001, pp. 362–369. Translated from Bioorganicheskaya Khimiya, Vol. 27, No. 6, 2001, pp. 408–416.
Original Russian Text Copyright © 2001 by Mishchenko, Kozhanova, Denisov, Markushin, Alekseev, Cherepanova, Ivanova.
The Effect of Linker Length on the Photomodification
of Tyr Residue in N-(Tyrosyl)-N'-(5-azido-2-nitrobenzoyl)-
diaminoalkanes
1
E. L. Mishchenko*, L. A. Kozhanova**, A. Yu. Denisov*, Yu. Ya. Markushin*,
P. V. Alekseev*, N. I. Cherepanova***, and T. M. Ivanova*
*Novosibirsk Institute of Bioorganic Chemistry, Siberian Division, Russian Academy of Sciences,
prosp. Akademika Lavrent’eva 8, Novosibirsk, 630090 Russia
**Limnological Institute, Siberian Division, Russian Academy of Sciences, Ulan-Batorskaya ul. 3, Irkutsk, 664033 Russia
***Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. Miklukho-Maklaya 16/10, GSP Moscow, 117997 Russia
Received January 16, 2001; in final form, May 11, 2001
Abstract—N-(Tyrosyl)-N'-(5-azido-2-nitrobenzoyl)-1,4-diaminobutane containing a Tyr residue connected
with the photoreactive aryl azide group through a diaminobutylene linker was synthesized as a model for study-
ing the photomodification of Tyr residues in proteins. This compound and the compound with a shorter,
1,2-diaminoethylene linker, obtained previously, were subjected to a computer modeling to find their minimal
energy conformations. The aromatic rings of Tyr and 5-azido-2-nitrobenzoic acid residues in the latter com-
pound were localized in parallel planes at a distance of approximately 0.3 nm between them and were shown
to be implicated in stacking interaction. On the contrary, the planes of aromatic rings of the former compound
with a longer diaminobutylene linker were found to be situated perpendicularly to each other, with the distance
between the centers of the rings being approximately 0.6 nm. The computer analysis was confirmed by exper-
imental results: when studying the photomodification of the compound with the diaminobutylene linker, neither
stable products of the Tyr photomodification nor unstable products capable of transformation into stable prod-
ucts in the dark were found. On the contrary, such products were previously identified in the case of the com-
pound with diaminoethylene linker. The formation of amino, nitro, azoxy and azo derivatives was common for
the photomodification of both compounds.
Key words: amino acid photoderivatives; tyrosine, 5-azido-2-nitrobenzoyl derivatives, photoderivatives
1
INTRODUCTION
Previously, we synthesized a model compound, N-
(tyrosyl)-N'-(5-azido-2-nitrobenzoyl)-1,2-diaminoet-
hane (Ia), in which the tyrosine and 5-amino-2-
nitrobenzoic acid residues are connected by a diamino-
ethylene linker [4]. We demonstrated that the triplet
The method of photoaffinity modification is widely
used for studies of enzyme active sites as well as pro-
tein–nucleic acid and protein–protein interactions in
supramolecular structures. The amino acid residues 4-nitrobenzoylnitrene formed upon the irradiation of an
(Ia) aqueous solution with the light with λ 313–365 nm
can efficiently modify the Tyr residue in the same
model molecule to give the stable product, cyclo-[1-(4'-
nitro-3'-benzoyl)-2-(aminotyrosyl)-N,N'-ethylenedi-
amine] (IIa). In addition, an unstable product IIb was
subjected to photomodification were determined upon
the studies of protein binding sites by means of sub-
strate analogs with aryl azide groups [1]. The substrate
analogs containing a 5-azido-2-nitrobenzoic acid resi-
due were shown to modify the Tyr residue in the major-
ity of cases [1–3]. However, the modification products
have not yet been identified, because it is hard to accu-
mulate sufficient amounts of homogeneous photomod-
ified peptides for the NMR study. Only electron absorp-
tion spectra were recorded for a few peptides contain-
ing Tyr residue modified by a derivative of 5-azido-2-
nitrobenzoic acid. These spectra remain the only phys-
icochemical characteristic of such products [2, 3].
NO2
O
+
O
H3N
C
CH
NH(CH2)nNHC
CH2
n = 2 (Ia)
n = 4 (Ib)
N3
1
To whom correspondence should be addressed; e-mail:
smlen@niboch.nsc.ru.
OH
1068-1620/01/2706-0362$25.00 © 2001 MAIK “Nauka /Interperiodica”

THE EFFECT OF LINKER LENGTH ON THE PHOTOMODIFICATION
363
also found, which was almost completely converted
O
into (IIa) in the dark. The electronic absorption spectra
of (II‡) (λmax 413 nm) and a peptide fragment of the
Klenow fragment whose Tyr766 was modified by a
5-azido-2-nitrobenzoic acid residue introduced into the
U residue of the primer of the primer–matrix duplex
(λmax 410 nm) [3] were similar in the area of 300–
450 nm. Therefore, the structures of this stable product
with a modified Tyr residue and (IIa) should be similar.
+
C
NH(CH2)2NHCOCHNH3
NO2
CH2
H
N
HO
(IIa)
Since the Tyr and 5-azido-2-nitrobenzoic acid resi-
dues in (Ia) are connected by a short artificial linker, it
was important to reveal the effect of an increase in the
linker length on the intramolecular photomodification
of the Tyr residue. In this study, we synthesized the
model compound, N-(tyrosyl)-N'-(5-azido-2-nitroben-
zoyl)-1,4-diaminobutane (Ib), whose linker is by two
methylene groups longer than that of compound (Ia),
and studied its photolysis.
formations of (Ia) corresponding to the minimum
energy, the Tyr and 5-azido-2-nitrobenzoic acid resi-
dues are situated in parallel planes at a distance of
approximately 0.3 nm and participate in stacking inter-
action. On the other hand, according to the same com-
puter modeling, the aromatic rings of the reactive resi-
dues in (Ib) are disposed perpendicularly to each other
at a distance of approximately 0.6 nm between the ring
centers (Fig. 1). The distance between the nitrogen
atom of aryl azide nearest to the aromatic ring and the
hydrogen atom of Tyr in the ortho-position to its
hydroxyl group is 0.48 nm in (Ia) and 0.70 nm in (Ib).
These computation data suggest that a longer linker in
the model (Ib) removes the reactive residue from the
target to be modified and, therefore, decreases the prob-
ability of contact between them.
RESULTS AND DISCUSSION
We presumed that the distance between the Tyr and
5-azido-2-nitrobenzoic acid residues in the new model
(Ib) would be as short as in the previous model (Ia) [4],
and this distance would be comparable with that
between the reacting sites during the protein photoaf-
finity modification. At the same time, the elongation of
the linker by two methylene groups in (Ib) could pro-
vide for a less dense spatial structure of the two inter-
acting aromatic rings and allow the modification of not
only ortho-position of Tyr residue [to give the product
similar to (IIa)], but also other atoms in (Ib) (e.g., ben-
zyl carbon atom in this residue). For example, it is
known that the toluene photomodification by perfluoro-
aryl azides proceeds through the introduction of singlet
arylnitrenes into ortho- and para-positions of the tolu-
ene aromatic ring and through the reaction of triplet
arylnitrenes with benzyl carbon atom of toluene [5].
These results were experimentally verified by study-
ing the photolysis of (Ib) in 0.15% aqueous methanol at
concentrations of 0.031 and 0.062 mM by irradiation
for 40 s. At 0.062 mM concentration, (Ib) was irradi-
ated under aerobic (solutions both saturated and unsat-
urated with oxygen) and anaerobic conditions. Only
aerobic conditions without saturation with oxygen were
used at 0.031 mM concentration of (Ib). The resulting
mixtures of products were analyzed by microcolumn
reversed-phase HPLC immediately after irradiation and
after storage in the dark at 20°C for 1–24 h.
Eight peaks were revealed in the mixture of photol-
ysis products (0.062 mM, aerobic conditions without
saturation with oxygen) immediately after the irradia-
tion of (Ib) (Fig. 2a). Compounds corresponding to
these peaks were identified as the photoconversion
products (IIIb)–(VIIb) according to their spectral char-
acteristics and MALDI MS (Scheme 1). Note that no
stable products of the Tyr photomodification were
found in the mixture of photolysis products.
Aryl azide derivative of tyrosine (Ib) was prepared
by acylation of N-(5-azido-2-nitrobenzoyl)-1,4-diami-
nobutane with N-succinimide ester of Boc-DL-
Tyr(Boc) followed by the removal of Boc-protection.
The structure of (Ib) was confirmed by 1H NMR, UV,
and IR spectroscopies and MALDI MS (the data are
1
given in the Experimental section). H NMR parame-
ters of the Tyr and 5-azido-2-nitrobenzoic acid residues
in (Ib) corresponded to the published data (cf. [6] and
[4], respectively). Product (Ib) was stable at storage for
six months at –20°ë as a pellet and in methanol solu-
tion (50 mM) according to TLC and reversed-phase
HPLC.
The quantitative composition of the products
changed after the reaction mixture storage in the dark at
20°ë for 1–24 h (the dark stage of reaction) (Fig. 2b).
It was found that the content of (IIIb), (Vb), and (VIb)
(peaks 4, 7, and 8, respectively) and of the compounds
corresponding to peaks 2 and 3 increased due to the
decomposition of the unstable compound (VIIb) (peak
6), which practically disappeared 20 h after the begin-
ning of the dark stage. The quantitative data are given
in the table.
Before studying the photomodification of (Ib), the
preferred conformations of models (Ia) and (Ib) were
analyzed using computer modeling, in order to check
the possibility of the intramolecular modification of Tyr
residue by a highly reactive nitrene. The method of lim-
ited molecular mechanics and dynamics with regard to
A comparison of the chromatographic profiles of the
water environment [7] demonstrated that, in one of con- photolysis products of model compounds (Ia) (Fig. 2c)
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 27 No. 6 2001

364
MISHCHENKO et al.
(Ia)
(Ib)
Fig. 1. Spatial structures of the model compounds (Ia) and (Ib) corresponding to the minimal-energy conformations under aqueous
conditions (water molecules are not shown).
and (Ib) (Fig. 2a) demonstrated that the photolysis pro- tion of Tyr (IIa) (peak 6). No stable products of the Tyr
cesses of these compounds differ. The main difference photomodification were found among the compounds
was found in the group of peaks 1–6 (Fig. 2c), which corresponding to peaks 1–4 (Fig. 2a). It should be noted
contained the product of intramolecular photomodifica- that the compounds corresponding to peaks 2 and 3
11
12
13
+
hν
+
+
R'
R'
N3
R'
NH2
N N
R'
R'
NO2
O
R''
R''
R''
R''
R''
(Ib)
(IIIb)
N N
(VIb)
(IVb)
(Vb)
R'
R'
+
+
+ other photolysis
R'
NO
products
R''
R''
R''
(VIIb)
(unstable)
+
R' = NO2
NH3
10
9
8
7
6
R'' = C(O)NHCH2CH2CH2CH2NHC(O)CH
5 CH2
4
3
1
2
OH
Scheme 1. The photolysis products (Ib). Here and further in the Experimental section, atoms are numerated for con-
1
venience of the interpretation of H NMR spectra.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 27 No. 6 2001

THE EFFECT OF LINKER LENGTH ON THE PHOTOMODIFICATION
365
A220
0.15
(IVb)
(VIIb)
(‡)
(Vb)
(VIb)
(IIIb)
5
6
4
3
7
2
8
1
0.15
(b)
4
5
7
2 3
8
1
6
(VIIa)
1
(c)
8
0.15
(unstable)
(IVa)
3
(IIa)
(Va)
7
5 6
9
(VIa)
10
4
2
0.17
(d)
(IIIb)
(Vb)
5
10
15
20
25
30
min
Fig. 2. Analysis of the products of 40-s photolysis of (c) (Ia) and (a, b, d) (Ib) (in concentration of 0.062 mM in 0.15% aqueous
methanol) (a) just after the irradiation, (b) after the storage in the dark for 20 h at 20°C, and (d) after the concentration of the com-
bined fractions 2 and 3 (Fig. 2a) by the microcolumn reversed-phase HPLC in a linear gradient of acetonitrile (5 to 20%) in 0.1%
TFA for 32 min.
were unstable upon the concentration and their struc- (Ia) at various concentrations, structure (VIIa) of
tures were not determined. The retention times of peaks nitroso derivative of (Ia) was presumably ascribed to
7–10 in Fig. 2c and peaks 5–8 in Fig. 2a were close to the unstable product from peak 8 in Fig. 2c, which
each other, which suggests that the corresponding com- almost completely disappears at storage in the dark for
pounds probably have similar chemical structures. Pre- 20 h. Accordingly, compounds eluted as peaks 5–8
viously, we identified substances corresponding to (Fig. 2a) probably are nitro, nitroso, azoxy, and azo
peaks 7, 9, and 10 in Fig. 2c as nitro (IVa), azoxy (Va), derivatives of (Ib). Therefore, the same numbers [e.g.,
and azo (VIa) derivatives of (Ia), respectively [4]. On (IVa) to the substance from peak 7 in Fig. 2c and (IVb)
the basis of electronic spectroscopy and quantitative to that from peak 5 in Fig. 2a] were attributed to the
analysis of stable products formed after irradiation of photolysis products of (Ia) and (Ib), which can be con-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 27 No. 6 2001

366
MISHCHENKO et al.
Relative content of the products (A) immediately after photolysis of (Ib) and (B) after 20-h storage in the dark (according to
reversed-phase HPLC*)
Products and their relative content, %
(VIIb)
(unstable)
peak 6
unidentified compounds
peak 2 peak 3
Concentration
of (Ib), mM
(IIIb)
peak 4
(IVb)
peak 5
(Vb)
peak 7
(VIb)
peak 8
A
B
A
B
A
B
A
B
A
B
A
B
A
B
0.062
14.8 23.0 20.1 20.2 14.0 15.8
7.6 9.0 13.1
1.5 4.6 28.5
–
12.2 14.8 8.5 12.0
0.062 saturation with
O2
9.65 17.3 45.7 45.5
–
–
7.0
3.9 12.6 2.2
8.3
0.062 saturation with 19.2 19.2
Ar
–
–
–
–
62.4 62.4
–
–
–
–
–
–
0.031
14.5 18.8 20.6 20.6
7.1
7.0
4.0 3.9 35.8
–
–
28.7
–
7.46
* For HPLC conditions, see the Experimental section. The amounts of photoproducts were estimated by integration of the corresponding
chromatographic peaks obtained at 220 nm. A, the chromatographic profiles were obtained just after the irradiation of (Ib) (see Fig. 2a);
B, the chromatographic profiles were obtained after the storage of the photolysis mixture for 20 h in the dark at 20°C (see Fig. 2b).
sidered to be the corresponding derivatives of the same among the products of the anaerobic irradiation of (Ib)
chemical nature.
(see table).
The unstable substances eluted in fractions 2 and 3
The stable products of the photolysis of (Ib) were
(Fig. 2a) were formed from (VIIb) at the dark stage
processes. These products were absent just after the
irradiation of the low-concentration (0.031 mM) solu-
tion of (Ib), but they appeared after the dark stage of the
decomposition reaction of (VIIb) (table). These prod-
ucts also formed immediately after the irradiation of the
concentrated (0.062 mM) solution of (Ib), and their
content also increased after the dark stage due to the
decomposition of (VIIb) (Figs. 2a, 2b, table).
The mixture formed after the irradiation of
0.062 mM solution of (Ib) under aerobic conditions
without saturation with oxygen was kept in the dark at
20°ë for 4 days and analyzed by the reversed-phase
HPLC. In this case, products corresponding to peaks 2
and 3 were absent from the mixture, while the amounts
of amino derivative (IIIb) (peak 4) and azoxy deriva-
tive (Vb) (peak 7) increased by 15.3 and 8.3%, respec-
tively, in comparison with their amounts just after the
irradiation of (Ib). At the same time, a new peak with
the retention time of 22.49 min was observed in the
chromatogram (data not shown). Practically complete
conversion of these compounds into the amine (IIIb)
(λmax 385 nm) and azoxy derivative (Vb) (λmax 342 nm
(Fig. 2d) also proceeded upon the concentration of the
combined fractions 2 and 3 according to reversed-phase
HPLC, UV-spectroscopy, and MALDI MS.
obtained in the amounts sufficient for their identifica-
1
tion by H NMR, UV-spectroscopy, and MALDI MS
and by the comparison with the analogous products of
the photolysis of (Ia) (see [4] and the Experimental
section).
The structure of aromatic amine (IIIb) was ascribed
to the product in peak 4 of Fig. 2a, because it contained
a Tyr residue similar to that in (Ib) and changes in the
chemical shifts of H11, H12, and H13 in (IIIb) were
characteristic of an amino substituted derivative [8].
The electronic absorption spectra of (IIIa) (λmax
384 nm [4]) and (IIIb) (λmax 385 nm) are similar.
Products eluted in fractions 2 and 3 (Fig. 2a) and
compound (VIIb) from peak 6 could not be identified
because of their instability. It was established that all
the unstable compounds appeared only in the course of
photolysis under aerobic conditions (see table). It is
known that the irradiation of 4-nitrophenyl azide results
in the triplet nitrene, which can intensively interact with
oxygen to give (4-nitrophenyl)nitroso oxide in the form
of a biradical or a zwitterion. Rapid dimerization of this
nitroso oxide proceeds through an intermediate unsta-
ble cyclic diperoxide and results in 1,4-dinitrobenzene
and 1-nitroso-4-nitrobenzene [9, 10]. We ascribed to
(VIIb) a probable nitroso structure, since the electron
absorption spectra of (VIIa) (λmax 278 nm, [4]) and
(VIIb) (λmax 278 nm) practically coincided and were
similar to that of 1-nitroso-4-nitrobenzene (λmax 282 nm,
Nitrosobenzene is known to be easily reduced to
form phenylhydroxylamine. The interaction of
nitrosobenzene with phenylhydroxylamine leads to
[11]). It was found by the quantitative analysis of the azoxybenzene, and the disproportionation of phenylhy-
photolysis products that the saturation of the solutions droxylamine gives aniline and azoxybenzene [12]. We
(0.062 mM) of (Ib) with oxygen and subsequent irradi- can presume that (VIIb) would also undergo similar
ation under aerobic conditions results in a more than conversions in the presence of methanol as a reducing
twofold increase in the content of nitro derivatives agent (Scheme 2). It is probable that the compound
(IVb) and (VIIb). The two compounds were absent eluted in fraction 3 corresponds to N-(tyrosyl)-
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THE EFFECT OF LINKER LENGTH ON THE PHOTOMODIFICATION
367
R'
NH2
2H+, 2e
2H+, 2e
R''
R''
R'
NO
R'
NHOH
(IIIb)
–2H+, –2e
R''
R''
+
(VIIb)
R'
N N
R'
O
R''
(Vb)
+
R'
N N
R'
+
O
R''
R''
R' = NO2
NH3
(Vb)
R'' = C(O)NHCH2CH2CH2CH2NHC(O)CH
CH2
OH
Scheme 2. Conversions of the hypothetic nitroso compound (VIIb) in the presence of reducing agent (methanol).
N'-[N-(2-nitro-5-hydroxylaminobenzoyl)]-1,4-diamino- logs [4], capable of converting into stable analogs dur-
butane, because its electronic absorption spectrum is
similar to that of N-(4-nitophenyl)hydroxylamine (λmax
356 nm) [13]. Data about the products of interaction of
phenols and their analogs with aromatic nitroso com-
pounds lack in the literature. At the same time, the
nitroso group is known to be analogous to the carbonyl
group and enters similar reactions. Since the reaction
with alcohols with the formation of unstable hemiace-
tals is characteristic of carbonyl-containing com-
pounds, we can presume that the compound eluted in
fraction 2 results from a similar interaction of the
nitroso group of (VIIb) with the Tyr hydroxyl group.
ing the dark stage.
The importance of an optimal linker length for the
effective intramolecular modification of the tyrosine
phenyl group in various compounds was demonstrated
in a number of publications [15–18]. For example, the
intramolecular alkylation was found to proceed in the
compounds of the general formula Ph(CH2)nCH2Cl,
where n = 2 or 3; in the case of a longer linker it gave a
high yield of the cyclization product, whereas only its
trace amounts formed in the case of a shorter linker
[15]. For compounds of the general formula
PhCO(CH2)nCH2Cl (n = 4–8), the short linker (n = 4)
ensured a high yield of the cyclization product, whereas
the successive elongation of the linker to n = 5–8
resulted only in the intermolecular alkylation products
[16].
It is well known that the irradiation of aromatic
azides results in the splitting off of a nitrogen molecule
and the formation of a singlet arylnitrene [10, 14].
However, the irradiation of 4-nitrophenyl azide easily
gives a triplet 4-nirtophenylnitrene due to the extremely
rapid transition of the singlet 4-nitrophenylnitrene into
its basic triplet state [9]. As follows from the studies of
photolysis of the model compounds (Ia) [4] and (Ib),
their irradiation results in the reactions characteristic of
the triplet 4-nitrophenylnitrene [9]: dimerization with
formation of substituted azobenzenes (VIa) and (VIb),
reduction to the substituted 4-nitroanilines (IIIa) and
(IIIb), and interaction with oxygen giving rise to nitro
products (IVa) and (IVb) and substituted azoxyben-
zenes (Va) and (Vb) (see Scheme 1). However, the
analysis of the photolysis products of (Ib) did not
reveal products of the photomodification of the Tyr res-
In our case, the linker elongation by two methylene
groups inhibited the modification of the ortho-C–H
bond in the Tyr residue; the corresponding product
(IIa) arose only under the irradiation of the model com-
pound (Ia) with the shorter linker. No other modifica-
tion variants were found either. On the basis of calcula-
tions and experimental data, we can conclude that the
linker elongation by two methylene groups prevents the
realization of the molecule conformation in which the
Tyr and 5-azido-2-nitrobenzoic acid residues are in an
optimal position for the formation of a stable modifica-
idue: neither stable (analogs of IIa) nor unstable ana- tion product.
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MISHCHENKO et al.
EXPERIMENTAL
nitrobenzoyl)-1,4-diaminobutane (36 mg, 0.11 mmol)
in anhydrous dioxane (2 ml) was added to a solution of
Boc-DL-Tyr(Boc) N-succinimide ester (57.5 mg,
0.12 mmol) in anhydrous dioxane (2 ml). The reaction
mixture was stirred for 10 min in the dark, evaporated
to dryness, and treated with water (5 ml). The product
was extracted with ethyl acetate (2 × 2 ml). The extract
was dried over anhydrous Na2SO4 for 24 h, filtered, and
evaporated. The oily residue was dissolved in dioxane
(1 ml), and the solution was dropwise added to water
(5 ml). The water layer was decanted, and the oily res-
idue was dried in a desiccator over P2O5 for several
days, dissolved in methanol (1 ml), and treated with
methanol (0.4 ml) saturated with HCl. The solution was
kept for 48 h at room temperature and poured into cool
ether (14 ml). The oily residue was several times
washed with ether, dissolved in methanol (1 ml) and
precipitated with ether (10 ml). The precipitate was fil-
tered, washed with ether (2 ml), and dried in a vacuum
to give (Ib); yield 30 mg (47%); Rf 0.82 (A), 0.1 (B); RT
21.91 min (for HPLC conditions, see below); UV (10%
aqueous methanol), λmax, nm (ε, å–1 cm–1): 318 (8800);
Trifluoroacetic acid, 5-azido-2-nitrobenzoic acid
(Sigma, United States), and 2,5-dihydroxybenzoic acid
(Aldrich, United States) were used in this study. N-(5-
Azido-2-benzoyl)-1,4-diaminobutane and N-succinim-
ide ester of Boc-DL-Tyr(Boc) were synthesized in the
technological laboratory of NIBKh, Siberian Division,
Russian Academy of Sciences by the methods
described in [4] and [19], respectively. Acetonitrile,
1,4-dioxane, N,N-dimethylformamide, benzene, pyri-
dine, acetone, methanol, ethanol, methylene chloride,
diethyl ether, ethyl acetate, and n-hexane were purified
according to the standard procedures. All the reagents
used were of the reagent grade.
The electronic absorption spectra were recorded on
a Shimadzu UV-2100 spectrophotometer (Japan) in
quartz cells with a 0.1-cm optical pathway. IR spectra
were recorded on a Bruker IFS 66 spectrometer (Ger-
many) in KBr pellets. 1H NMR spectra (δ, ppm, J, Hz)
were recorded on a Bruker AM 400 spectrometer (Ger-
many) at the working frequency of 400.13 MHz in D2O
at 25°ë. The chemical shifts were calculated relative to
the residual solvent signal (water, δ 4.80 ppm). The
NMR spectra were interpreted with the use of a Sadtler
catalog of NMR spectra (Sadtler Research Laborato-
ries) and other widely used sources of the NMR spec-
tral information.
Mass spectra were registered on a VISION 2000
MALDI time-of-flight mass spectrometer (Thermo Bio
Analysis, UK) using a VSL-337ND nitrogen laser
(337 nm, impulse of 3 ns, energy of 150–200 µJ/imp)
for ionization. 2,5-Dihydroxybenzoic acid was used as
a matrix. The samples for the MALDI TOF MS were
prepared according to the following procedures: aque-
ous solutions of the tested compounds (0.1–0.5 mM,
0.3 µl) were mixed with a solution (0.3 µl) of the matrix
(20 mg/ml) in 20% aqueous acetonitrile, dried, and
analyzed. The spectra were obtained by registering pos-
itive ions in the reflection regime.
IR (KBr, νmax, cm–1): 1342, 1517 (NO2), 1653
1
(C=Oamide), 2123 (N3); H NMR: 8.21 (1 H, d, J 9.0,
H11), 7.38 (1 H, dd, J 9.0 and 2.5, H12), 7.22 (1 H, d,
J 2.5, H13), 7.14 (1 H, d, J 8.5, H1 and H4), 6.82 (1 H,
d, J 8.5, H2 and H3), 3.3–3.2 (4 H, m, H7 and H10),
3.18 (1 H, d, J 7.0, H6), 3.1–3.0 (2 H, m, H5), 1.6–1.5
2
(4 H, m, H8 and H9); MALDI, m/z: 386.6 [M + H –
NO]+, and 400.5 [M + H – O]+, 416.5 [M + H]+, and 438
[M + Na]+ for (IIIb).
Irradiation of the samples was performed using a
DRSh-1000 Hg high pressure lamp (1000 W). Two
spectral lines (313 and 365 nm) were selected by a
combination of glass filters. The intensity of the inci-
dent light (J = 1.35 ± 0.01) × 1015 photons s–1 cm–2) was
measured by a ferrioxalate Hatchard–Parker actinome-
ter [21].
A 0.031 or 0.062 mM solution of (Ib) (accuracy was
±5%) in 0.15% aqueous methanol was irradiated. The
molar extinction coefficient of (Ib) was determined as a
sum of absorption coefficients of Tyr (ε275 1230 M–1 cm–1
TLC was performed on DC-Alufolen Kieselgel 60
F254 precoated plates (Merck, Germany) in the follow-
ing chromatographic systems: (A) 4 : 1 methanol–
ammonia and (B) 9 : 1 methylene chloride–methanol.
[22]) and aryl azide (ε318 8800 M–1 cm–1 [2]).
Analytical microcolumn reversed-phase HPLC was
performed on a MiliChrom A-02 microcolumn liquid
chromatograph (EcoNova, Russia) [20]. The photolysis
products were isolated on a MiliChrom-4 microcolumn
liquid chromatograph (Orel, Russia).
The spatial structures of (Ia) and (Ib) were calcu-
lated by the method of limited molecular mechanics
and dynamics using the Amber 4.0 program [7]. Visu-
alization of the structures and rotation around some
chemical bonds were performed with the use of the
HyperChem program (HyperCube). All the calcula-
tions were performed on personal Pentium computers.
Aerobic conditions of irradiation were provided
for by simply using 0.062 mM solution of (Ib) in 0.15%
aqueous methanol or blowing oxygen through the solu-
tion for 20 min at 0°ë.
Anaerobic conditions were provided for by bub-
bling argon through 0.062 mM solution of (Ib) for
20 min at 0°ë.
The photolysis products were analyzed by
reversed-phase HPLC on a Nucleosil 100-5C18 micro-
column (2 × 75 mm, 5 µm (Macherey-Nagel, Ger-
2
A photolysis of (Ib) with the formation of amine (IIIb) proceeds
N-(DL-Tyrosyl)-N'-(5-azido-2-nitrobenzoyl)-1,4-
diaminobutane (Ib). A solution of N-(5-azido-2-
simultaneously with the laser ionization (λ 337 nm) of (Ib) in the
MALDI method.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 27 No. 6 2001

THE EFFECT OF LINKER LENGTH ON THE PHOTOMODIFICATION
369
many)) in a linear gradient of acetonitrile (5 to 20%) in
We thank Dr. N.P. Gritsan for her interest in this
0.1% TFA for 32 min at a flow rate of 100 µl/min just study and valuable criticism during the preparation of
after the irradiation of (Ib) or after the storage of reac- the manuscript.
tion solution in the dark at room temperature for 1–24 h.
The photolysis products were characterized by their
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ACKNOWLEDGMENTS
399.
This study was supported by the Program of Scien-
tific Cooperation between Eastern Europe and Switzer-
land (SCOPES 2000–2003) and, partially, by grant
7SUPJ062336.
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RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 27 No. 6 2001