New azobenzene derivatives for directed modification of proteins

Article abstract of DOI:10.1134/S1068162009050033

Derivatives of azobenzene which contained a maleimide group in one of the benzene rings (for binding to a protein cysteine residue) and maleimide, hydroxyl, or carboxyl substitutes in another benzene ring were synthesized. The reactivity of these compound

Full text of DOI:10.1134/S1068162009050033

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2009, Vol. 35, No. 5, pp. 549–555. © Pleiades Publishing, Ltd., 2009.
Original Russian Text © Le Thi Hien, B. Schierling, A.Yu. Ryazanova, T.S. Zatsepin, E.M. Volkov, E.A. Kubareva, T.I. Velichko, A. Pingoud, T.S. Oretskaya, 2009, published in
Bioorganicheskaya Khimiya, 2009, Vol. 35, No. 5, pp. 610–617.
New Azobenzene Derivatives
for Directed Modification of Proteins
Le Thi Hiena, B. Schierlingb, A. Yu. Ryazanovaa, T. S. Zatsepina, E. M. Volkova, E. A. Kubarevaa,
T. I. Velichkoa, A. Pingoudb, and T. S. Oretskayaa
,1
a Chemistry Department, Faculty of Bioengineering and Bioinformatics,
A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Moscow, 119991 Russia
b Institute of Biochemistry, Justus-Liebig University, Giessen, Germany
Received March 31, 2009; in final form, April 28, 2009
Abstract—Derivatives of azobenzene which contained a maleimide group in one of the benzene rings (for
binding to a protein cysteine residue) and maleimide, hydroxyl, or carboxyl substitutes in another benzene ring
were synthesized. The reactivity of these compounds towards a cysteine residue of a protein and their optical
properties in a free form and after their attachment to the mutant forms of the SsoII restriction endonuclease
were studied.
Key words: maleimidoazobenzene; the SsoII restriction endonuclease; proteins, modification of cysteine residues
DOI: 10.1134/S1068162009050033
INTRODUCTION
azobenzene derivatives [8, 9]. As a rule, cysteine resi-
dues of a protein are modified by both the symmetrical
azobenzene derivatives bis(chloroacetamidosulfo)azoben-
zene [4] and bis(iodoacetamido)azobenzene [10] and its
asymmetrical derivatives (4-maleimidobenzophenone
[11] and maleimidoazobenzeneglutamic acid [12]). It is
important to note that the compounds with a maleimide
group are more reactive towards sulfhydryl groups than
the chloroacetamide and iodoacetamide derivatives.
The chemical modification of proteins plays an
important role both for the investigation of their func-
tioning and for the creation of protein constructions
with novel properties.2 Such constructions are often
used as instruments in molecular biology, in particular,
for the induction of protein activity in a desired
moment. For example, DNA polymerase from Thermus
aquaticus can be reversibly inactivated by treatment
with formaldehyde, resulting in the blocking of the
lysine residue in the binding site. The regeneration of
the native enzyme proceeds through hydrolysis of the
Schiff base upon heating to 95°C [1].
Azobenzene undergoes cis-trans-(syn-anti)-isomer-
ization induced by illumination with light of appropri-
ate wavelengths (Fig. 1a). The isomerization proceeds
quickly and effectively and is practically independent
of the environment and polarity of the solvent. Azoben-
zene derivatives have been frequently introduced into
proteins for modulation of their activity [2–5]. Such
reagents are of interest because azobenzene is isomer-
ized by illumination with light with a wavelength of
more than 300 nm and, thus, allows for protein activa-
tion/deactivation in vivo changes of the protein activity
either due to the direct alteration of the conformation of
the azobenzene derivatives with subsequent steric
hindrances [6, 7] or due to a change in the protein
conformation because of cis-trans-isomerization of the
We proposed to use only the asymmetrical deriva-
tives of azobenzene for the regulation of the accessibil-
ity of the active site of enzyme and synthesized confor-
mationally flexible compounds with hydroxyl and car-
boxyl substitutes. Further, in case the desirable effect
would be achieved, the other derivatives will be synthe-
sized.
In this study, we prepared previously unknown
derivatives of azobenzene with a maleimide group (for the
binding to a protein cysteine) in one of the benzene rings
and with a maleimide, hydroxyl, or carboxyl substitute in
another ring. The reactivity of the obtained derivatives
was confirmed with the model proteins, mutant forms
of the SsoII restriction endonuclease (R.SsoII). SH
groups of cysteine residues of the protein were modi-
fied, and the photoisomerization of the protein-bound
azobenzene derivatives was also demonstrated (Fig.
1b). Such derivatives were proposed for modulation of
the DNA binding to the following proteins: restriction
endonucleases, DNA-methyltransferases, and tran-
scription factors. The possibility of the intracellular
activation/deactivation of these enzymes will give
novel information on their functioning and allow regu-
lating their activity in cells.
1
Corresponding author; phone: (495) 939-5411; fax: (495) 939-
3181; e-mail: oretskaya@belozersky.msu.ru.
2
Abbreviations: NBS, N-bromosuccinimide; PEG-Mal, (methyl-
polyethylenglycol ) -polyethylenglycol -maleimide; R.SsoII,
12 3
4
the SsoII restriction endonuclease; SDS, sodium dodecylsulfate.
549

550
LE THI HIEN et al.
N
365 nm
470 nm
N
N
N
trans-
(anti)
cis-
(syn)
(‡)
365 nm
470 nm
(b)
Fig. 1. The cis-trans-isomerization of azobenzene (a) in its free form and (b) its derivative attached to the mutant form of the
R.SsoII(C33S/C60S/R174C) induced by illumination with light with certain wavelengths.
RESULTS AND DISCUSSION
nitrobenzenes by tin (II) chloride [16]. 4-Aminotolu-
ene-3-sulfonic acid (I) was oxidized by sodium perbo-
rate in the presence of boric acid in glacial acetic acid.
As a rule, radical bromination is performed in nonpolar
haloid-containing solvents, and the choice of solvents
was rather small. Compound (II) was practically insol-
uble in dichloromethane and tetrachloromethane and,
thus, the attempts of bromination with N-bromosuccin-
imide during illumination or in the presence of 3-chlo-
roperoxybenzoic acid in the next stage were unsuccess-
ful. Compound (II) was converted into its dioctyl ester
[17] in order to increase its solubility in low-polar sol-
vents, but diester (III) was also insoluble in the afore-
mentioned solvents. As a result, we gave up the prepa-
Synthesis of Azobenzene Derivatives
Azobenzene derivatives have often been used for pro-
tein modification [7, 10–13]. The goal of this study was
the synthesis of new derivatives of azobenzene which
contained the maleimide group for binding to a protein
cysteine residue in one of the benzene rings and substi-
tutes with different functional groups in the other ring.
We also planned to prepare azobenzene derivatives with
sulfogroups in the aromatic ring, which would increase
the solubility of the modifying agents in water.
In the first stage, we attempted the synthesis of a
water-soluble derivative of azobenzene (Scheme 1).
The most frequently used methods of the preparation of
azobenzenes are the oxidation of anilines by hypochlo-
ride [14] or sodium perborate [15] or the reduction of ration of water-soluble sulfoderivatives of azobenzene.
SO3H
SO3H
SO3H
NaBO3/H3BO3/AcOH
H3C
NH2
H3C
N N
(II)
CH3
(I)
1) SOCl2
2) n-C8H17OH
NBS/hν/RCO3H
C8H17O3S
SO3C8H17
SO3H
N N
SO3H
H3C
N N
CH3
BrH2C
CH2Br
CH2Br
(III)
NBS/hν
SO3C8H17
N N
SO3C8H17
BrCH2
Scheme 1.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 5 2009

NEW AZOBENZENE DERIVATIVES
551
4,4'-Bis(maleimido)azobenzene (IV) was prepared was synthesized according to the procedure [19], but in
from N,N'-azobenzenedimaleic acid (IVa) similarly to another combination of solvents. The target product
the previous synthesis of bisarylmaleimides from (IV) was isolated from the reaction mixture by absorp-
bisarylmaleamine acids [18] (Scheme 2a). Acid (IVa) tion chromatography on a column with silica gel.
O
O
O
O
H2N
N
O
CH3COONa
(CH3CO)2O
HN
OH
N
N
NH2
N
NH
HO
O
(IVa)
O
O
N
O
N
N
N
O
(a)
(IV)
O
O
N
O
N
O
N
O
OH
N
N
HS
N
N
N
N
N
OH
O
S
O
O
(V)
O
O
N
O
O
N
COOH
O
N
HS
N
N
N
COOH
O
S
O
O
(VI)
(b)
O
Scheme 2.
The preparation of monosubstituted bismaleimides tic for trans-isomers of azobenzene derivatives. The
in organic solvents has not been described in literature, maximum is shifted to 325–330 nm in the case of the
although the interaction of maleimides with thiols has cis-isomers. All of the synthesized derivatives of
been carefully studied, especially in an aqueous azobenzene were quickly and reversibly isomerized
medium [20]. We studied the attachment of thiols to under light illumination at 365 and 470 nm, indepen-
azobenzene (IV) varying solvent, temperature, and dently of the structure of the substitutes in the benzene
ratio of the initial compounds and chose the optimal rings. The absorption spectra of compound (IV) in
conditions for this reaction. The solution of the thiols DMSO are given as an example (Fig. 2).
was slowly dropwise added to the solution of com-
pound (IV) in dioxane. Using of other solvents (tet-
Modification of Proteins
with Azobenzene Derivatives
rahydrofurane, dimethylformamide, or methylene chlo-
ride) instead of dioxane was possible. The reaction
products were maleimidoazobenzenes with one or two
thiol molecules incorporated, which were separated by
column chromatography. Derivatives of maleimidoa-
zobenzene with 2-mercaptoethanol and 3-mercapto-
propionic acid, in particular, compound (V) with a
hydroxyl group and compound (VI) with a carboxyl
group, were prepared by this procedure with yields of
25–30% (Scheme 2b).
Mutant forms of the SsoII restriction endonuclease
(R.SsoII) were used as model proteins for the attach-
ment of the azobenzene derivatives. These mutant pro-
teins contained one or two cysteine residues:
R.SsoII(C33S/C60S/R174C) or R.SsoII(C33S/C60S/
S171C/R174C), respectively. Results of the X-ray anal-
ysis of the complex of the Ecl18kI (R.Ecl18kI) restric-
tion endonuclease with a substrate [21] served as the
We have studied the efficacy of the reversible basis for the construction of the mutant forms of
cis-trans-isomerization of the prepared compounds. R.SsoII. R.Ecl18kI is an isoschizomer and the nearest
A peak with a maximum at 340–350 nm is characteris- homologue of R.SsoII. These enzymes are distin-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 5 2009

552
1.4
LE THI HIEN et al.
The tenfold excess of the solution of azobenzene
(‡)
trans-
derivative (IV), (V), or (VI) in DMSO was added to the
protein solution at room temperature. The efficacy of
the reaction with azobenzenes was determined via
adding an excess of PEG-Mal to an aliquot of the reac-
tion mixture. The yield of the conjugate of the protein
with PEG-Mal was determined according to the gel
analysis by the ImageQuant program and was no higher
than 5% (Fig. 3, line 4). Hence, the yield of the protein
modified by reagents (IV), (V), and (VI) was approxi-
mately 95%. Further, the proteins modified by the
azobenzene derivatives were separated from the nonre-
acted reagent by gel filtration. The efficacy of the reac-
tion of the mutant forms of the restriction endonuclease
with reagents (IV), (V), and (VI) was evaluated by spec-
trophotometry as a ratio of the intensity of the peak at
340 nm, which was characteristic of the azobenzene
derivatives to the intensity of the protein peak at 280 nm.
The extinction coefficients of the R.SsoII mutant form
ε280 and the azobenzene derivatives were taken to be
42000 M–1 cm–1 [22] and 22900 M–1 cm–1 [23], respec-
tively. The yield of the protein modified with deriva-
tives (IV), (V), or (VI) was about 85%. Thus, com-
pounds (IV), (V), and (VI) were shown to be useable
for the effective and selective modification of proteins.
The substitutes have practically no influence on the
reactivity of the maleimide group. A comparison of the
optical properties of the azobenzene derivatives with
those attached to the cysteine residues of protein
revealed no significant differences (Fig. 4).
0 min
1 min
2 min
5 min
10 min
1.2
1.0
0.8
0.6
0.4
0.2
cis-
0
1.4
(b)
trans-
2 min
1.2
1.0
0.8
0.6
0.4
0.2
0
1 min
0 min
cis-
250
300
350
400
450
500
Wavelength, nm
Further, we plan to perform an intramolecular
crosslinking of proteins by the azobenzene derivative (IV).
Such a crosslinking changes the protein structure by the
isomerization of the azobenzene derivative induced by
illumination. Compounds (V) and (VI) will be used for
regulation of the activity of R.SsoII during the forma-
tion of the enzyme–substrate complex. We propose to
modify the enzyme by these derivatives in the immedi-
ate vicinity of the DNA-binding site. Changes in the
conformation of the protein-attached azobenzene deriv-
atives will allow closing or opening the DNA-binding
site of R.SsoII. The proposed scheme of control of the
R.SsoII reactivity through the isomerization of the pro-
tein-attached compound (V) is illustrated in Fig. 1b.
Fig. 2. Absorption spectra of 4,4'-bismaleimidoazobenzene
in DMSO in the concentration of 100 μM: (a) trans cis
isomerization at 365 nm and (b) cis trans-isomeriza-
tion during the illumination with blue light with a wave-
length of 470 nm.
guished by one amino acid residue (Val232 in
R.Ecl18kI corresponds to Ile in R.SsoII). R.SsoII has a
molecular mass of 37.4 kDa.
The accessibility of cysteine residues of R.SsoII
mutant forms for modification with synthesized
reagents (IV), (V), and (VI) was assessed via polyeth-
ylene glycol, modified by maleimide group (PEG-Mal
with a molecular mass of 2.3 kDa) and the availability
of the cysteine residues of the mutant forms of R.SsoII
for the modification by the prepared reagents (IV), (V),
and (VI) was determined. The use of PEG-Mal had the
following advantages:
EXPERIMENTAL
The following reagents were used in this study: azo-
dianiline (Acros, Belgium), maleic anhydride, acetic
anhydride, thionyl chloride, boric acid (Reakhim, Rus-
sia), 2-mercaptoethanol (Amresco, United States),
(1) PEG-Mal contained a maleimide group and was
a chemical analogue of the compounds synthesized by us.
(2) PEG-Mal had a rather large molecular mass, and 3-mercaptopropionic acid (Merck, Germany), sodium
SDS-polyacrylamide gel electrophoresis could be used perborate (NaBO3 · 4H2O), N-bromosuccinimide
as a standard method of the analysis of such reaction (Fluka, Germany), 4-aminotoluene-3-sulfonic acid
mixtures. PEG-Mal practically quantitatively interacts (Aldrich, United States), 3-chloroperoxybenzoic acid
with proteins, resulting in the appearance of the corre- (Lancaster,
Great
Britain),
and
(methyl-
sponding band of the conjugate in polyacrylamide gel polyethyleneglycol12)3-polyethyleneglycol4-maleimide
(Fig. 3, line 2).
of 2.3-kDa molecular mass (PEG-Mal, Pierce, United
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 5 2009

NEW AZOBENZENE DERIVATIVES
553
M
1
2
3
4
1.0
(‡)
kDa
0 min
3 min
7 min
10 min
60
0.8
50
40
Conjugate of the
0.6
0.4
0.2
protein with PEG-Mal
Protein
30
Fig. 3. Analysis of the interaction of the mutant form of
R.SsoII(C33S/C60S/R174C) (5 μM) with PEG-Mal
(500 μM) and (or) (V) (50 μM) by Laemmli gel electro-
phoresis. The gel was stained with the Coomassie G250
solution. The protein was incubated with (line 2) PEG-Mal,
(line 3) compound (V), and (line 4) initially with compound
(V) and, then, with PEG-Mal. Line 1: protein control. M are
the marker proteins. The molecular masses are indicated on
the left.
0
1.0
(b)
2 min
1 min
0 min
0.8
0.6
0.4
0.2
0
States). The absolute solvents were prepared by the
standard methods.
Mass spectra were recorded on a system that con-
sisted of an ACQUITY UPLC liquid chromatograph
and a TQD tandem quadrupole mass-spectrometric
detector (Waters, United States).
NMR spectra were recorded on the Avance 400
spectrometer (Bruker, Germany) at 25°C. CHCl3
served as an internal standard.
TLC was performed by an ascending method on
Kieselgel 60 F254 plates (Merck, Germany). The sub-
stances with absorption in the UV area were detected at
254 nm.
250
300
350
400
450
500
Wavelength, nm
Fig. 4. Absorption spectrum of the conjugate of
R.SsoII(C33S/C60S/S171C/R174C) with compound (VI):
(a) trans
UV light with
(b) cis
cis-isomerization during illumination with
The derivatives of azobenzene were isolated by
chromatography on a column with silica gel (Kieselgel
60, 0.040–0.063, Merck, Germany).
a
wavelength of 365 nm and
trans-isomerization during illumination with
blue light with a wavelength of 470 nm.
Synthesis of sulfoderivatives of azobenzene.
4,4'-Azotoluene-3,3'-disulfonic acid (II). A mixture
of 4-aminotoluene-3-sulfonic acid (I) (4 g, 0.02 mol),
sodium perborate (NaBO3 · 4H2O) (2.9 g, 0.028 mol),
and boric acid (1.05 g, 0.017 mol) in glacial acetic acid
(100 ml) was stirred at 60°C for 6 h. The mixture
became dark orange in one hour. White precipitate and
dark resins (different oxidation products) were filtered
off, and the dark orange filtrate was evaporated to dry-
ness and dissolved in water. The crystals of target prod-
uct (II) were precipitated via cooling the solution to
4°C. The yield of compound (II) was 1.4 g (35%); MS:
ë14H14N2O6S2 (M–); calcd/found 369.03/369.11.
pound was insoluble in most of the organic solvents.
Our attempts to isolate it and to confirm its structure
failed.
4,4'-Bis(maleimido)azobenzene (IV). Maleic
anhydride (1.8 g, 17.7 mmol) was added to the solution
of diazoaniline (1.5 g, 7.1 mmol) in DMF (20 ml). The
reaction mixture was stirred for 1 h and diluted with
dioxane (20 ml). The precipitate was filtered and dried.
Sodium acetate (1.8 g, 21.3 mmol) and acetic anhydride
(13.4 ml, 142 mmol) were added to the precipitate. The
reaction mixture was heated to boiling and kept for
45 min. The precipitate was completely dissolved. The
reaction was monitored by TLC in a mixture of chloro-
form and ethanol (95 : 5). Rf of the product was 0.83.
The excess of acetic anhydride was hydrolyzed by the
addition of cool water. The precipitate was filtered,
washed with a 10% solution of Na2CO3 and with water
to pH 7, and dried. Product (IV) was isolated by chro-
Dioctyl ester of 4,4'-azotoluene-3,3'-disulfonic
acid (III). Thionyl chloride (SOCl2) (4 ml) was drop-
wise added to compound (II) (1 g). Another 4 ml of
SOCl2 and anhydrous dioxane (4 ml) were added one
hour later, and the reaction mixture was kept for 1 h
until stopping of the liberation of hydrogen chloride
and sulfur dioxide. The mixture was evaporated to dry-
ness, reevaporated with dioxane (3 × 5 ml), and stirred
for 2 h at 40°C in octanol (7 ml). The obtained com- matography on silica gel in chloroform with a yield of
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 5 2009

554
LE THI HIEN et al.
1.69 g (64%). 1H NMR (CDCl3, δ, ppm): 8.0 (4 H, d), 10–20 μl of a buffer containing 62 mM Tris-HCl (pH
7.7 (4 H, d), 7.3 (4 H, d).
6.8), 2% SDS (m/v), 0.7 M 2-mercaptoethanol, 10%
glycerol (v/v), and 0.1% Bromophenol Blue (m/v). Pro-
tein markers of molecular mass (10 μl, Fermentas,
Lithuania) were applied to one of the lines. The gel was
stained with a Coomassie R250 solution (Fermentas,
Lithuania) or Coomassie G250 (Serva, Germany).
4-Maleimido-4'-[2-(2-hydroxyethylthio)succin-
imido]azobenzene (V). The solution of 2-mercapto-
ethanol (0.4 mmol) in dioxane (2 ml) was added to the
solution of compound (IV) (150 mg, 0.4 mmol) in
dioxane (20 ml) within 30 min during stirring. The
reaction was monitored by TLC in the mixture of chlo-
roform and ethanol (95 : 5). Product (V) (Rf 0.23) was
isolated by chromatography on silica gel in the ethanol
gradient in chloroform (0–5%) with a yield of 45 mg
The protein modification by derivatives (IV), (V),
or (VI). 1 μl of 50 mM solution of azobenzene deriva-
tive (IV), (V), or (VI) in DMSO was added to a 1 ml of
5 μM solution of a protein in a 20 mM potassium phos-
phate buffer pH 7.4, 150 mM KCl. The reaction mix-
ture was kept for 15 min at room temperature and cen-
trifuged for 5 min at 12000 g. The proteins modified
with the azobenzene derivatives were separated from
the nonreacted reagent by gel filtration on an Akta Puri-
fier chromatograph on a HiTrap column with a volume
of 5 ml with Sephadex G-25 Superfine (GE Healthcare,
United States). The proteins were eluted with a 20 mM
Tris-HCl buffer (pH 7.5) containing 20 mM KCl.
1
(25%). H NMR (CDCl3, δ, ppm): 8.0 (4 H, d), 7.7
(4 H, d), 7.3 (2 H, s), 4.2 (1 H, s), 3.7 (2 H, m), 3.0
(2 H, m), 2.7 (2 H, m); MS: C22H18N4O5S (MH)+;
calcd/found: 451.10/451.24.
4-Maleimido-4'-[2-(2-carboxyethylthio)succin-
imido]azobenzene (VI). The solution of 3-mercapto-
propionic acid (35 μl, 0.4 mmol) in dioxane (2 ml) was
added to the solution of compound (IV) (150 mg,
0.4 mmol) in dioxane (20 ml) within 30 min during stir-
ring. The reaction was monitored by TLC in the mix-
ture of chloroform and ethanol (95 : 5). Product (VI)
with Rf 0.11 was isolated by chromatography on silica
gel in the gradient of ethanol in chloroform (from 0 to
10%) with a yield of 55.5 mg (29%); 1H NMR (CDCl3,
δ, ppm): 12.4 (1 H, s), 8.0 (4 H, d), 7.7 (4 H, m),
7.3 (2 H, s), 4.2 (1 H, s), 3.7 (2 H, m), 3.0 (2 H, m),
2.7 (2 H, m); MS: C23H18N4O6S (MH)+; calcd/found:
479.09/478.94.
Modification of the proteins with PEG-Mal.
2 μl of 5 mM solution of PEG-Mal (2.3 kDa) in DMSO
was added to 20 μl of 5 μM solution of the protein in a
20 mM potassium phosphate buffer (pH 7.4) contain-
ing 150 mM KCl. The reaction mixture was kept for
15 min at room temperature.
The photoisomerization of the free azobenzene
derivatives was performed on a Cary Eclipse spectrof-
luorimeter (Varian, United States) in a quartz cuvette
with an optical path of 1 cm. The absorption spectra of
these solutions were recorded on a Cary 50 UV-Vis
Preparation of the mutant forms of R.SsoII.
We performed site-specific mutagenesis of the R.SsoII
gene by the Kirsch method [24]. The Arg(AGA)174 codon
in the R.SsoII(C33S/C60S/R174C) gene was exchanged
for Cys(TGT), and the Ser(TCT)171 and Arg(AGA)174
codons in the R.SsoII(C33S/C60S/S171C/R174C) gene
were exchanged for Cys(TGC) and Cys(TGT) codons,
respectively. The pACMS7/pQESso9 C33S/C60S plas-
mids were used as starting plasmids, where pQESso9
C33S/C60S contained the R.SsoII gene without cys-
teine residues. These plasmids were kindly presented
by Doctor V. Pingoud (Germany). DNA sequencing
over the entire coding region confirmed the presence of
the corresponding codon substitutions in them. The
spectrophotometer
(Varian,
United
States).
The solution volume was 200 μl. The isomerization
degree was determined according to the height of the
peak at 325–350 nm. The solution of the azobenzene
derivatives before the illumination contained a mixture
of cis- and trans-isomers, and the trans-isomer pre-
dominated. The solution was illuminated with light
with a wavelength of 470 nm for 1 min in order to con-
vert the compound into the trans-conformation. The
mutant forms of the R.SsoII recombinant proteins with height of the peak maximum was not changed with fur-
the HiS6-tag on their N-termini were produced in the
JM109 Escherichia coli cells and isolated by affinity
chromatography of Ni-NTA-agarose as described in
[22].
ther illumination. The solution was subsequently illu-
minated at 365 nm for 1, 2, 5, and 10 min for the prep-
aration of the cis-isomer. After that, reverse isomeriza-
tion was performed by the subsequent illumination of
the solution at 470 nm for 1 and 2 min, and the height
of the maximum achieved the starting value.
Laemmli gel electrophoresis [25] was performed
in flat PAAG (10 × 8 × 0.1 cm) in a buffer containing
25 mM Tris, 250 mM glycine, 0.1% SDS (m/v) at
pH 8.3, and a field strength of 14 V/cm. The separating
gel consisted of 0.375 M Tris-HCl (pH 8.8), 14.25%
acrylamide, 0.75% N,N'-methylenebisacrylamide, and
0.1% SDS. The concentrating gel contained 0.125 M
Tris-HCl (pH 6.8), 3.9% acrylamide, 0.1%
N,N'-methylenebisacrylamide, and 0.1% SDS. Samples
Photoisomerization of the proteins modified with
derivatives of azobenzene was carried out using a UV
lamp with a wavelength of 365 nm and a lamp of blue
light with a wavelength of 470 nm (Bachofer, Ger-
many). The absorption spectra of these solutions were
recorded on a NanoDrop ND-1000 spectrophotometer
were heated for 5 min at 95°C and applied to the gel in (Peqlab Biotechnologie GmbH, Germany).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 5 2009

NEW AZOBENZENE DERIVATIVES
555
11. Luo, Y., Wu, J., Li, B., Langsetmo, K., Gergely, J., and
ACKNOWLEDGMENTS
Tao, T., J. Mol. Biol., 2000, vol. 296, pp. 899–910.
We are grateful to V.N. Tashlitsky (Chemistry
Department, M.V. Lomonosov Moscow State
University) for recording of the mass spectra and
V. Pingoud (Institute of Biochemistry, Justus-Liebig
12. Volgraf, M., Gorostiza, P., Numano, R., Kramer, R., Isa-
coff, E., and Trauner, D., Nat. Chem. Biol, 2006, vol. 2,
pp. 47–52.
13. Pozhidaeva, N., Cormier, M., Chaudhari, A., and Wool-
ley, A., Bioconjugate Chem., 2004, vol. 15, pp. 1297–
1303.
University,
Giessen,
Germany)
for
the
pACMS7/pQESso9 C33S/C60S plasmids.
This study was supported by DFG-RFBR Program
International Research Training Groups (grants
nos. 08-04-91974 and GRK 1384), and RFBR grant
07-04-00545.
14. Zhang, Z., Burns, D.C., Kumita, J.R., Smart, O.S., and
Woolley, G.A., Bioconjugate Chem., 2003, vol. 14,
pp. 824–829.
15. Menta, S.M. and Vakilwala, M.V., J. Am. Chem. Soc.,
1952, vol. 74, pp. 563–564.
16. Cook, A.H., J. Chem. Soc., 1938, pp. 876–881.
REFERENCES
17. Sintezy organicheskikh preparatov (Syntheses of Organic
Preparations), Johnson, W.S., Ed., Moscow: Inostr.
Liter., 1956, vol. 6, pp. 81–83.
18. Lifits, A.L., Veitsman, A.A., and Distanova, L.Ya.,
Metody polucheniya khimicheskikh reaktivov i prepara-
tov (Methods of Obtaining of Chemical Reagents and
Preparations), Lastovskii, R.P., Ed., Moscow:
NIITEKhIM, 1970, issue 22, pp. 118–122.
19. Lifits, A.L., Veitsman, A.A., and Distanova, L.Ya.,
Metody polucheniya khimicheskikh reaktivov i prepara-
tov (Methods of Obtaining of Chemical Reagents and
Preparations), Lastovskii, R.P., Ed., Moscow:
NIITEKhIM, 1970, issue 22, pp. 114–117.
1. Ivanov, I., Loffert, D., Kang, J., Ribbe, J., and Stein-
ert, K., Method for Reversible Modification of Thermo-
stable Enzymes, 2001,US Patent 6,183,998.
2. Renner, C. and Moroder, L., ChemBioChem, 2006,
vol. 7, pp. 868–878.
3. Dong, S., Loweneck, M., Schrader, T., Schreier, W.,
Zinth, W., Moroder, L., and Renner, C.A., Chem. Eur. J.,
2006, vol. 12, pp. 1114–1120.
4. Burns, D.C., Zhang, F., and Woolley, G.A., Nature Pro-
tocols, 2007, vol. 2, pp. 251–258.
5. Burns, D.C., Flint, D.G., Kumita, J.R., Feldman, H.J.,
Serrano, L., Zhang, Z., Smart, O.S., and Woolley, G.A.,
Biochemistry, 2004, vol. 43, pp. 15329–15338.
20. Derek, G.S., Blumenfeld, O.O., and Konigsberg, W.,
Biochem. J., 1964, vol. 91, p. 589.
6. Gorostiza, P. and Isacoff, E., Mol. Biosyst., 2007, vol. 3,
21. Bochtler, M., Szczepanowski, R.H., Tamulaitis, G., Gra-
zulis, S., Czapinska, H., Manakova, E., and Siksnys, V.,
EMBO J., 2006, vol. 25, pp. 2219–2229.
22. Sud’ina, A.E., Restriction Endonucleases SsoII, RsrGI,
and MboI: Study of Properties and Determination of
DNA-Binding Motifs, Cand. Sci. (Chem.) Dissertation,
Moscow: Lomonosov Moscow State University, 2004.
23. Umeki, N.,Yoshizawa, T., Sugimoto,Y., Mitsui, T., Wak-
abayashi, K., and Maruta, S., J. Biochem., 2004,
vol. 136, pp. 839–846.
pp. 686–704.
7. Fortin, D.L., Banghart, M.R., Dunn, T.W., Borges, K.,
Wagenaar, D.A., Gaudry, Q., Karakossian, M.H.,
Otis, T.S., Kristan, W.B., Trauner, D., and Kramer, R.H.,
Nature Methods, 2008, vol. 5, pp. 331–338.
8. Woolley, A., Jaikaran, A., Berezovski, M., Calarco, J.,
Krylov, S., Smart, O., and Kumita, J., Biochemistry,
2006, vol. 45, pp. 6075–6084.
9. Nakayama, K., Endo, M., and Majima, T., Bioconjugate
Chem., 2005, vol. 16, pp. 1360–1366.
10. Kumita, J., Smart, O., and Woolley, A., Proc. Natl. Acad.
Sci. USA, 2000, vol. 97, pp. 3803–3808.
24. Kirsch, R.D. and Joly, E., Nucleic Acid Res., 1998,
vol. 26, pp. 1848–1850.
25. Laemmli, U.K., Nature, 1970, vol. 227, pp. 680–685.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 5 2009