Synthesis and properties of a new conformational antigen which models an extracellular region of β1-adrenoreceptor

Article abstract of DOI:10.1134/S1068162009030029

Two fragments corresponding to the 125-133 and 206-218 sequences of a molecule of the β1-adrenoreceptor (autoantibodies to this protein are often found in patients with dilated cardiomyopathy) were synthesized by the solid phase method with the use of Fmoc technology. Two new conformational antigens were prepared by directed (regioselective) and undirected (spontaneous) formation of intramolecular and intermolecular disulfide bridges between the corresponding cysteine residues of the synthesized peptides. One of these antigens consisted of a mixture of disulfide isomers, and another antigen was an isomer with a natural arrangement of S-S bridges. Immunosorbents were obtained by immobilization of the synthesizes antigens on the bromocyanogen-activated sepharose and applied to the removal of autoantibodies in a β1- adrenoreceptor from the blood plasma of patients. We demonstrated that the sorbents on the basis of the conformational antigens were more effective in comparison with those containing linear peptide precursors.

Full text of DOI:10.1134/S1068162009030029

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2009, Vol. 35, No. 3, pp. 285–295. © Pleiades Publishing, Ltd., 2009.
Original Russian Text © M.V. Sidorova, M.E. Pal’keeva, A.S. Molokoedov, A.A. Az’muko, A.V. Sekridova, M.V. Ovchinnikov, P.A. Levashov, O.I. Afanasieva, Yu.V. Berestetskaya,
M.I. Afanasieva, O.A. Razova, Zh.D. Bespalova, S.N. Pokrovskii, 2009, published in Bioorganicheskaya Khimiya, 2009, Vol. 35, No. 3, pp. 311–322.
Synthesis and Properties of a New Conformational Antigen
which Models an Extracellular Region of b1-Adrenoreceptor
M. V. Sidorova1, M. E. Pal’keeva, A. S. Molokoedov, A. A. Az’muko, A. V. Sekridova,
M. V. Ovchinnikov, P. A. Levashov, O. I. Afanasieva, Yu. V. Berestetskaya, M. I. Afanasieva,
O. A. Razova, Zh. D. Bespalova, and S. N. Pokrovskii
Russian Cardiological Research and Production Complex, Russian Ministry of Public Health,
ul. 3-ya Cherepkovskaya 15a, 121552 Russia
Received November 7, 2008; in final form, December 2, 2008
Abstract—Two fragments corresponding to the 125–133 and 206–218 sequences of a molecule of the
β1-adrenoreceptor (autoantibodies to this protein are often found in patients with dilated cardiomyopathy) were
synthesized by the solid phase method with the use of Fmoc technology. Two new conformational antigens were
prepared by directed (regioselective) and undirected (spontaneous) formation of intramolecular and intermo-
lecular disulfide bridges between the corresponding cysteine residues of the synthesized peptides. One of these
antigens consisted of a mixture of disulfide isomers, and another antigen was an isomer with a natural arrange-
ment of S–S bridges. Immunosorbents were obtained by immobilization of the synthesizes antigens on the bro-
mocyanogen-activated sepharose and applied to the removal of autoantibodies in a β1-adrenoreceptor from the
blood plasma of patients. We demonstrated that the sorbents on the basis of the conformational antigens were
more effective in comparison with those containing linear peptide precursors.
Key words: peptide antigens, spontaneous and regioselective formation of disulfide bonds, β1-adrenoreceptor,
dilated cardiomyopathy.
DOI: 10.1134/S1068162009030029
INTRODUCTION
of a β1-adrenoreceptor are most often found in patients
with DCMP [7].
Dilated cardiomyopathy is one of the main reasons
of heavy cardiac decompensation and the most frequent
reason for heart transplantation.2 Pathogenesis of this
disease is not studied in detail. A hypotheses of chronic
virus infection and genetic determination are proposed,
but recent published data point to an autoimmune char-
acter of this heavy pathology [1, 2]. Clinical evidence
demonstrates that procedures of therapeutic apheresis
of immunoglobulins considerably improve the state of
patients with DCMP [3–5]. Autoantibodies that are
found in the serum of DCMP patients mainly belong to
the immunoglobulins of G class (IgG) and are antibod-
ies against self-antigens such as a β1-adrenoreceptor,
myosin, adenine nucleotide translocator, sarcolemma
laminin, protein of the M2 muscarinic receptor, and
others [6]. It was established that precisely autoanti-
bodies to the second extracellular loop of the molecule
The introduction of the peptide corresponding to the
sequence of the second loop of a β1-adrenoreceptor in
rabbits has been shown to result in DCMP development
[8]. Hence, the presence of autoantibodies of this spec-
ificity plays a key role in pathogenesis of this disease.
Moreover, the presence of antibodies in a β1-adrenore-
ceptor has been found to be one of the factors of the
development of tachyarrythmia and acute cardiac dec-
ompensation [9, 10].
β1-Adrenoreceptor has three extracellular and three
intracellular polypeptide sequences (loops) with seven
transmembrane α-helical domains. Two disulfide
bridges (Cys209 – Cys215 in the second loop and Cys131
–
Cys216 between the first and the second loop) are in the
molecule (Fig. 1) [11, 12].
Peptide fragments of the second extracellular loop
of a β1-adenoreceptor are known to be antigenic deter-
minants which can be bound to autoantibodies in this
protein [7, 13]. One of the approaches to DCMP treat-
ment is the removal of autoantibodies in a β1-adenore-
ceptor from blood plasma of patients by specific immu-
noabsorption on columns with sorbents which contain
antigenic peptide fragments of this protein. For exam-
ple, Coraffin® columns [3] are proposed for this pur-
pose. Their active component is a mechanical mixture
1
Corresponding author; phone: +7 (495) 414-6716; e-mail: pep-
tide@cardio.ru
2
Abbreviations: Acm, acetamidomethyl; DCMP, dilated cardi-
omyopathy; DIC, N,N'-diisopropylcarbodiimide; Fmoc, 9-fluore-
nylmethyloxycarbonyl; HOBt, 1-hydroxybenzotrazole; NMP,
N-methylpyrrolidone; TIBS, triisobutylsilane; Pmc, 2,2,5,7,8-
pentamethylchromane-6-sulfonyl; SPyr, 2-pyridinesulfenyl;
SPPS, solid phase peptide synthesis. Other abbreviations corre-
spond to IUPAC-IUB: Eur. J. Biochem, 1994, vol. 183, pp. 9–37.
285

286
SIDOROVA et al.
NH
2
Loop 2
N
Y
D
209
R
C
R
P
206
E
K
Loop 1
A
C
215
125
D
C
Y
E
216
G
Loop 3
218
V
S
F
F
C
E
L
S
D
W
R
F
L
V
E
P
D
R
E
A
T
C
R
131
133
N
R
H
121
200
(III)
221
(IV)
W
(I)
(II)
(V)
(VI)
(VII)
Cellular
membrane
Cytosol
COOH
Fig. 1. Structure of a β -adrenoreceptor [3]. Fragments of the polypeptide chain chosen for the synthesis of conformational antigens
1
125
131
133
206
209
are marked with gray color: Glu-Tyr-Gly-Ser-Phe-Phe- Cys-Glu- Leu (II) and Ala-Arg-Arg- Cys-Tyr-Asn-Asp-Pro-
215
216
218
Lys- Cys- Cys-Asp- Phe (IV).
of fragments of the first and the second extracellular Cys216 sequence, which is located in the second extra-
loops (the Glu-Tyr-Gly-Ser-Phe-Phe-Cys-Glu-Leu cellular loop of a β1-adrenoreceptor, participate in the
nonapeptide and Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro- formation of S–S bridges. This Cys–Cys sequence is an
Lys-Cys-Cys-Asp-Phe tridecapeptide corresponding to important structural element of a number of proteins
the 125–133 and 206–218 sequences of the β1-adenore- [14]. It creates the possibility of binding and approxi-
ceptor, respectively [3]) immobilized on the bromocy- mation of three segments of the amino acid chain.
anogen-activated agarose matrix. From our point of
According to the published data, the disulfide bond
between the Cys131 and Cys216 residues plays an impor-
tant role in supporting the conformation of the ligand-
binding site of the protein [15]. The presence of disul-
fide bridges in a molecule of a β1-adrenoreceptor most
probably contributes to the antigenic properties of this
protein (the existence of a conformational antigenic
view, the Coraffin® columns have a significant disad-
vantage. Each of the two sorbents in the mechanical
mixture contains one of the aforementioned peptides
and binds autoantibodies specific to linear antigenic
determinants which are located only in the first or only
in the second extracellular loop of a β1-adrenoreceptor.
As it has been mentioned above, two disulfide bonds determinant to which autoantibodies can form is quite
are present in the native molecule. Disulfide bonds are possible). Thus, the affinity of such a sorbent as Coraf-
known to have various functions in proteins. They sta- fin® will be inevitably insufficient for removal of all
bilize folded proteins. The thiol–disulfide exchange can autoantibodies to the β1-adrenoreceptor. This conclu-
be a basis for regulation of enzymatic activity, and so sion was experimentally proven [3] and could possibly
forth. Moreover, both cysteine residues of the Cys215- explain the fact that no new data on the application of
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 3 2009

SYNTHESIS AND PROPERTIES OF A NEW CONFORMATIONAL ANTIGEN
287
t
t
t
t
t
t
t
OBu Bu
Bu
AcmOBu
PmcPmc Trt
Trt
Boc Trt Acm
OBu
Bu
OBu
Fmoc-Glu-Tyr-Gly-Ser-Phe-Phe-Cys-Glu-Leu-
Fmoc-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-
P
P
125
131
133
206
209
215 216
218
1) 20% piperidine
2) TFA, TIBS
Acm
1) 20% piperidine
2) TFA, TIBS
SH Acm
SH
H-Glu-Tyr-Gly-Ser-Phe-Phe-Cys-Glu-Leu-OH
H-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-OH
125
131
133
206
209
215 216
218
(I)
(III)
Hg(OAc) , 30% AcOH
Hg(OAc) , 50% AcOH
2
2
SH
SH
133
216
H
OH
H
OH
125
131
(II)
SH
206
209
215
218
SH
(IV)
H O , pH 8
2
2
133
133
218
H
OH
H
OH
OH
125
131
125
131
+
216
206
216
H
OH
218
H
206
209
215
209
215
(V)
(VI)
Scheme A. Synthesis of the conformational antigen by simultaneous undirected (spontaneous) formation of the disulfide bridges.
Here and in the next schemes, numeration of the amino acid residues in peptides corresponds to the fragments of the amino acid
sequence of a β -adrenoreceptor.
1
SH
SH
215
216
H
OH
206 209
218
Acm
(III)
H O , pH 8
2
2
133
216
H
OH
H
OH
125
131
206 209
215
218
(I)
Acm
Acm
(VII)
l /AcOH
2
131
131
H
OH H
OH
125
133
125
133
131
216
SH
+
+
+
H
OH
H
OH
125
133
206 209
215
218
216
131
SH
(II)
(IX)
H
OHH
OH
206
209
215
218
125
133
(V)
(VIII)
209
215
Scheme B. Synthesis of the conformational antigen by the subsequent formation of the intramolecular Cys –Cys bridge by the
131
216
action of hydrogen peroxide and intermolecular Cys –Cys bridge by the action of iodine.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 3 2009

288
SIDOROVA et al.
Acm
Acm
131
216
H
OH
H
OH
125
133
206
209
215
218
(I)
(X)
Acm
SH
2,2'-dithiopyridine,
Hg(OAc) , 50% AcOH
2
pH 6.8
Acm
Acm
131
OH
133
216
H
H
OH
125
206
209
215
218
SH
SPyr
(II)
(XI)
NH OAc
4
pH 6.8
131
OH
H
133
125
209
215
H
OH
216 218
206
Acm
Acm
(XII)
l /AcOH
2
131
H
OH
125
133
209
215
H
OH
206
216 218
(V)
131
216
Scheme C. Synthesis of the conformational antigen by subsequent regioselective formation of the intramolecular Cys –Cys
209
215
bridge in the reaction of thiol–disulfide exchange and intramolecular Cys –Cys bridge by the action of iodine.
these columns in clinical practice has appeared for the fin® system. We made attempts to create new confor-
last five years.
mational synthetic antigens on the basis of theses pep-
tides in which the peptide chains were connected with
disulfide bonds.
On this basis, the problem of creation of a high-
affinity synthetic antigen whose structure and antigenic
properties will be close to those of the natural antigen,
a β1-adrenoreceptor, is urgent. To solve this problem,
we chose the already known peptides that corresponded
to the 125–133 and 206–218 sequences of both loops of
RESULTS AND DISCUSSION
The chosen nonapeptide and tridecapeptide were
a β1-adrenoreceptor and were earlier used for the Coraf- prepared by automatic solid phase peptide synthesis
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 3 2009

SYNTHESIS AND PROPERTIES OF A NEW CONFORMATIONAL ANTIGEN
289
using Fmoc technology. The syntheses were performed analytical HPLC and by determination of free SH
on a Wang polymer with a hydroxymethylphenyloxym- groups in the oxidation reaction mixture by the Ellman
ethyl anchoring group. The following protection groups reaction [19].
were used for blocking of the side functions of amino
Mass spectrometry of the reaction mixture demon-
acids: But for hydroxyl functions of serine and tyrosine;
strated that the product or products with å 2680 were
But for carboxyl functions of glutamic and aspartic
mainly formed after treatment with hydrogen peroxide.
acids; Boc for ε-amino function of lysine; Trt for car-
This molecular mass corresponded to the target asym-
boxylamide group of asparagine, and Pmc for guani-
metrical dimer (V). Minor peaks with molecular
dine function of arginine. A combination of Trt and
masses of 2184 and 3174 corresponded to dimers of
Acm groups was used for protection of sulfhydryl func-
nonapeptide and tridecapeptide, respectively. The
tions of cysteine residues in SPPS depending on the
minor products were separated from the main dimer by
chosen scheme of the subsequent formation of the dis-
HPLC. According to analytical HPLC, this dimer con-
ulfide bridges (see SchemesA–C). The acid-labile trityl
sisted of two substances with the same molecular mass
protections were removed by the treatment with triflu-
and close retention times. We proposed on the basis of
oroacetic acid simultaneously with the cleavage of the
mass spectrometry that these substances were disulfide
peptide from the polymeric support upon the comple-
isomers (V) and (VI) (Scheme A). Note that the forma-
tion of SPPS. Acm protections are stable under these
tion of a third isomer with a disulfide bond between
conditions [16]. These groups were cleaved by treat-
Cys215 and Cys216 is theoretically possible. However, it
ment with mercury acetate (for the preparation of pep-
is known from published data that the formation of a
tide thiols) or with iodine (for simultaneous cleavage of
disulfide bridge between neighboring cysteine residues
Acm groups and formation of S–S bridges) [16]. An
is usually impossible [20]. Dimers are the main reac-
amino acid chain was elongated stepwise starting from
tion products in this case. In addition, the geometry of
the ë-terminus by the carbodiimide method in the pres-
a peptide chain in natural peptides prevents the forma-
ence of 1-hydroxybenzotriazole. Upon the completion
tion of a disulfide bridge between neighboring cysteine
of SPPS, the peptides were cleaved from the carrier
residues [14]. We found that the product of spontaneous
with the simultaneous removal of the acid-labile pro-
cyclization contained 28.5% of isomer (V) with the nat-
tecting groups by treatment with trifluoroacetic acid
ural location of S–S bridges. Its retention time was
with the addition of H O and TIBS for binding of trityl
2
identical to that of product (V) of the regioselective
synthesis according to HPLC (Scheme B, see below).
The content of the second isomer in the reaction mix-
ture was 61.0%, and it can probably be identified as iso-
mer (VI) according to the aforementioned published
data (Scheme Ä).
carbcations. The synthesized peptides were purified by
preparative HPLC to a homogeneity of 95–97% and
used for the preparation of peptide antigens with intra-
molecular and intermolecular disulfide bridges accord-
ing to Schemes A–C.
The simplest method for preparation of peptide anti-
gens with disulfide bonds is the simultaneous undi-
rected (spontaneous) formation of all disulfide bridges
by the action of a mild oxidant (for example, air oxygen
or hydrogen peroxide). A mixture of disulfide isomers,
symmetrical and asymmetrical dimers, or oligomers is
usually obtained by this method. However, spontaneous
cyclization sometimes occurs with high regioselectiv-
ity, especially in the case of rather large molecules with
thermodynamically profitable physiologically active
conformation [17]. Linear precursors of the corre-
sponding peptides with completely protected cysteine
residues are necessary for spontaneous formation of
disulfide bridges. Therefore, Acm derivatives of non-
apeptide (I) and tridecapeptide (III) were synthesized
on the solid phase and treated with mercury acetate in
aqueous acetic acid for the removal of Acm protection
[16]. Peptide (I) was deprotected in 50% acetic acid
because it was poorly soluble in 30% acetic acid. Non-
apeptide (II) and tridecapeptide (IV) contained com-
pletely deprotected cysteine residues (see Scheme A).
The next stage was the preparation of a peptide anti-
gen with a natural arrangement of intramolecular and
intermolecular disulfide bonds (Schemes B and C).
According to Scheme B, the 206–218 sequence was
synthesized by SPPS and cleaved from the polymer
with the simultaneous removal of trityl protection
groups with the formation of peptide (III). The
intramolecular S–S bridge between Cys209 and Cys215
was formed by the treatment of tridecapeptide (III)
with hydrogen peroxide at pH 8 with a high yield. Then,
we tried to form an intramolecular disulfide bond
between Cys131 and Cys216 by the treatment of the cor-
responding Cys(Acm) derivatives of nonapeptide (I)
and tridecapeptide (VII) with iodine in acetic acid [16].
This reaction gave the target asymmetrical dimer (V)
with a yield of 14.5%, a symmetrical dimer of nonapep-
tide (VIII) with a yield of 17.7%, and nonapeptide (II)
and tridecapeptide (IX) with free Cys216 residues (the
yields were 15.0 and 52.2%, respectively).
All of these compounds were isolated from the reac-
The mixture of dimers was prepared by the undirected tion mixture by HPLC and identified by mass spec-
(spontaneous) formation of S–S bridges in an equimo- trometry. Thus, the main product of the synthesis
lar mixture of nonapeptide (125–133 (II) and tride- according to Scheme B was tridecapeptide (IX) instead
capeptide (206–218 (IV) by the action of hydrogen per- of the target dimer (V). We proposed that the presence
oxide [18] (Scheme A). The reaction was monitored by of the intramolecular S–S bridge in the tridecapeptide
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 3 2009

290
SIDOROVA et al.
A
220
(XII)
(XI)
(II)
ch2
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
min
Fig. 2.Analytical HPLC of the reaction mixture of the preparation of dimer (XII) by formation of the intermolecular disulfide bridge
131
216
between Cys and Cys via the reaction of thiol–disulfide exchange. Peaks were designated by figures of peptides in Scheme C.
The contents of target dimer (XII), thiol of nonapeptide (II), and the SPyr derivative of tridecapeptide (XI) in the reaction mixture
were 76, 7, and 5%, respectively.
made steric hindrance for formation of the intermolec- gram of the reaction mixture of the preparation of dimer
ular disulfide bond.
(XII) from the Cys216(SPyr) derivative of tridecapeptide
(XI) and nonapeptide thiol (II) by the method of thiol–
disulfide exchange at pH 6.8 in an aqueous–alcohol
medium is given in Fig. 2. Dimer (XII) was isolated
from the reaction mixture by HPLC on a column with a
Vydac sorbent. On the final stage of the synthesis, the
internal S–S bridge between Cys209 and Cys215 of the
[Cys(Acm)]2 derivative of dimer (XII) was formed by
the treatment with iodine in acetic acid. The yield of
target dimer (V) was 64.0%. Homogeneity of the prod-
uct was confirmed by analytical HPLC (Fig. 3). The
product was characterized by MALDI-TOF mass spec-
trometry. Note that peaks of molecular ions of mono-
mers (nonapeptide (II) and tridecapeptide (IX)) were
present in the mass spectra of the peptides with inter-
molecular disulfide bridges, along with peaks of molec-
ular ions of the dimer products. It is known from the
published data that several protective groups can be
cleaved [21] and S–S bonds can be reduced [22] during
mass spectrometry of peptides.
Therefore, we decided to form the intramolecular
disulfide bond between Cys131 and Cys216 at the first
and the internal disulfide bridge after that in the course
of the preparation of dimer (V) according to Scheme C.
In addition, we used a regioselective variation of the
formation of disulfide bridges in order to avoid side
symmetric dimers of the nonapeptide and tridecapep-
tide. For this reason, we prepared the linear precursor of
tridecapeptide (X) by SPPS. Cys209 and Cys215 of this
peptide were protected by Acm groups, and Cys216
remained unprotected. The crude SPPS product was
treated with 2,2’-dithiopyridine for the introduction of
temporary 2-pyridinesulfenyl protection (SPyr) into the
Cys216 residue. This protection was stable in acidic con-
ditions and the activated sulfhydryl group of cysteine
for reactions of thiol–disulfide exchange [16]. In partic-
ular, SPyr protection was removed by the action of pep-
tide thiols with the formation of disulfides. Earlier,
SPyr protection was successfully used for regioselec-
tive formation of disulfide bonds in syntheses of com-
Thus, two conformational peptide antigens in which
plex peptides with several S–S bridges [16]. We suc- linear antigenic determinants were connected with dis-
ceeded in the synthesis of the target peptide antigen (V) ulfide bonds were prepared on the basis of nonapeptide
according to Scheme C. All the reactions proceeded and tridecapeptide fragments of a β1-adrenoreceptor. A
with high yields of the target products. The chromato- comparison of the products of undirected and regiose-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 3 2009

SYNTHESIS AND PROPERTIES OF A NEW CONFORMATIONAL ANTIGEN
291
lective formation of S–S bridges by analytical HPLC
A
220
demonstrated that the undirected synthesis (Scheme A)
gave a mixture of isomer (V) (28.5%) with a natural
arrangement of S–S bridges and an isomer with the pro-
posed structure (VI) (61.4%), whereas the product of
the regioselective synthesis (Scheme C) is an individual
compound (V) with natural arrangements of S–S
bridges.
(V)
During the next stage, we prepared sorbents with
immobilized peptide antigens and studied the binding
of autoantibodies to a β1-adrenoreceptor by these sor-
bents.
Peptide antigens, including the mixture of dimers
(V) and (VI), dimer (V), nonapeptide (I), and tride-
capeptide (III), were immobilized on the bromocyano-
gen-activated sepharose according to the standard pro-
cedure [23] of immobilization of protein ligands on an
agarose matrix. The content of immobilized ligand was
evaluated by spectrophotometry (λ 280 nm) from the
difference of the amounts of the peptide used for the
reaction and the peptide remaining in the solution after
the completion of incubation with the matrix. The sorp-
tion properties of the prepared sorbents were examined
by the method of affinity chromatography with blood
plasma of the DCMP patients (patients 1, 2, 4, 5, and 6),
a patient with tachyarrythmia (patient 3), and healthy
donors (Fig. 4). As one can see from the figure, the con-
formational antigen prepared from the mixture of disul-
fide isomers according to Scheme A bound a signifi-
cantly larger amount of the autoantibodies to the β1-
adrenoreceptor from the blood plasma of the patients in
comparison with linear peptides (I) and (III). The sor-
bent with an immobilized ligand (V) more effectively
bound the antibodies than that with the same content of
ligand (V) + (VI).
ch2
15 16 17 18 19 20 21 22 23 24
min
Fig. 3. Analytical HPLC of the conformational peptide anti-
gen (V) with a natural arrangement of disulfide bonds.
The analytical HPLC was performed on a Gilson
chromatograph (France) equipped with a Vydac C18
column (4.6 × 250 mm, 5 µm, pore size of 300 Ang-
stroem, Sigma, United States). The column was eluted
with a gradient from 10 to 70% of buffer B (80% ace-
tonitrile in 0.1% trifluoroacetic acid) in buffer A (0.1%
trifluoroacetic acid) within 30 min at a flow rate of
1 ml/min at 226 nm. The preparative HPLC was per-
formed on a Beckman chromatograph (United States)
equipped with a Diasorb-C16 130T column (25 ×
250 mm, 10 µm) at 220 nm or a Vydac Protein & Pep-
tide C18 column (10 × 250 mm, 10 µm) at 226 nm.
Buffers A and B were used as eluents. Mass spectra
were recorded on a mass spectrometer with a time-off-
flight VISION 2000 base (Thermobioanalysis Corp.,
Finnigan, United States) and an impulse nitrogen laser
of class 3B with an irradiation length of 337 nm by the
MALDI method using 2,4,6-trihydroxyacetophenone
Thus, the content of the recognized structures in the
plasma of patients is considerably higher if antigen (V)
with the natural arrangement of disulfide bonds is used.
The efficacy of the sorbent with ligand (V) + (VI) is also
comparable with that of the sorbent with ligand (V) for
the diagnosis and treatment of DCMP.
EXPERIMENTAL
Derivatives of L-amino acids (Bachem, Switzer-
land), DIC, HOBt, TIBS, mercury acetate, iodine
(Fluka, Switzerland), L-ascorbic acid (Sigma, United
States), and 50% hydrogen peroxide (Solvay Interox,
Belgium) were used in this study. Hydrogen peroxide
was diluted with deionized water to the desired concen-
tration and titrated with a 1 M solution of KMnO4
before performing the reactions. N-Methylpyrrolidone,
dichloromethane, piperidine, methanol, and trifluoro-
acetic acid (Applied Biosystems GmbH, Germany)
were used for the peptide synthesis. Sepharose 4Fast
Flow (GE Healthcare Bio-Science AB, Sweden) was
used for preparation of the affinity sorbents. Acetoni-
trile (Technopharm, Russia) was used for HPLC.
1
matrix. H NMR spectra were recorded on a WH-500
Bruker spectrometer (500 MHz, Germany) in DMSO-
d6 at 300 K at a peptide concentration of 2–3 mg/ml.
Chemical shifts were measured relative to tetramethyl-
silane. Nitrogen in cylinders of the “os. ch.” (especially
pure) quality (Russia) was used in this study. The IgG
concentration was determined using a reagent kit from
Vektor-Best (Russia). Optical absorption of solutions
was evaluated on an Akvilon SF-103 spectrophotome-
ter and a Uniplan analyzer (Pikon, Russia).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 3 2009

292
SIDOROVA et al.
250
200
150
100
50
0
Healthy Patient1 Patient 2 Patient 3 Patient 4
Patient 5 Patient 6
(average
from four)
Mixture of peptides (I) and (III)
Mixture of isomers (V) and (VI)
Isomer (V)
Fig. 4. Amount of bound autoantibodies to the β -adrenoreceptor from the plasma of patients 1, 2, 4, 5, and 6 with dilated cardi-
1
omyopathy, patient 3 with tachyarrythmia, and healthy donors on the sorbents with the following immobilized synthetic antigens:
the mixture of linear peptides (I) and (III) (black columns) and the conformational antigens prepared by spontaneous (light gray
columns) and regioselective (dark gray columns) formation of the disulfide bridges.
Solid phase peptide synthesis was carried out on a approximately 1 ml and mixed with anhydrous ether.
The precipitate was filtered, washed with ether, and
dried.
431A automatic peptide synthesizer (Applied Biosys-
tems, United States) according to Fmoc technology
starting from 0.25 mmol of commercial Nα-Fmoc-ami-
noacylpolymers (Bachem, Switzerland). A copolymer
of styrene with 1% divinyl benzene with a hydroxyme-
thylphenyloxymethyl anchoring group (the Wang poly-
mer) was used as a carrier. The contents of starting
Fmoc-Leu and Fmoc-Phe were 0.73 and 0.67 mmol per
1 g of the protected polymer, respectively. The amino
acid chains were elongated according to the standard
programs for a one-step condensation of Fmoc-amino
acids. A synthetic cycle involved activation of the
attached Nα-Fmoc-amino acid (1 mmol, fourfold excess
in relation to amino groups of the resin) in the presence
of equivalent amounts of DIC and HOBt in NMP for 20
min, deprotection of α-amino groups with 20% solu-
tion of piperidine in NMP for 20 min, condensation of
the activated derivative of Nα–Fmoc–amino acid with
amino groups in NMP for 70 min, and all the necessary
intermediate washings of the peptidylpolymer. Upon
the completion of SPPS, Nα-deprotected peptidylpoly-
mers were suspended in the mixture of TFA (10 ml),
water (0.5 ml), and TIBS (0.5 ml) and stirred for 1 h at
25°ë. The polymer was filtered and washed with TFA
H-Glu-Tyr-Gly-Ser-Phe-Phe-Cys(Acm)-Glu-
Leu-OH (I) (the 125–133 sequence of b1-adrenore-
ceptor). Crude product (I) of SPPS was fractionated on
a column with Diasorb eluted with a concentration gra-
dient of buffer B in buffer A (from 100% of buffer A,
0.5%/min) at a flow rate of 10 ml/min. Fractions con-
taining the target products were evaporated for the
removal of acetonitrile and lyophilized. The yield of
peptide (I) was 0.148 g (51% relatively to the starting
amino acid attached to the polymer); Rt 21.65 min; 99%
purity; mass spectrum: M 1165.5 (Mcalculated for
ë54ç72N10O17S was 1164).
H-Glu-Tyr-Gly-Ser-Phe-Phe-Cys-Glu-Leu-OH (II).
The solution of mercury acetate (0.03 g, 0.090 mmol)
in 50% acetic acid (1.5 ml) was added to the solution of
nonapeptide (I) (0.050 g, 0.043 mmol) in 50% acetic
acid (5 ml). The reaction mixture was stirred for 1.5 h
at 20°ë, and hydrogen sulphide was bubbled through
the solution for 30 min. The precipitate of mercury sul-
phide was filtered and washed with 50% acetic acid (2 ×
5 ml). The filtrate was evaporated to a volume of
approximately 2 ml and fractionated on the column
(3 × 1 ml). The filtrate was evaporated to a volume of with Diasorb. The column was eluted with a concentra-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 3 2009

SYNTHESIS AND PROPERTIES OF A NEW CONFORMATIONAL ANTIGEN
293
tion gradient of buffer B in buffer A (from 20 to 80%,
0.5%/min) at a flow rate of 10 ml/min. Fractions con-
taining the target product were joined, evaporated for
the removal of acetonitrile, and lyophilized. The yield
of compound (II) was 0.038 g (80.0%); Rt 22.12 min;
purity of 97.2%; mass spectrum: M 1095.2 (Mcalculated
for ë51ç67N9O16S was 1094.2).
H-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-
Cys-Asp-Phe-OH H-Glu-Tyr-Gly-Ser-Phe-Phe-
Cys-Glu-Leu-OH(V) according to Scheme B. The
solution of compound (VII) (30 mg, 0.018 mmol) and
nonapeptide (I) (21 mg, 0.018 mmol) in 70% aqueous
acetic acid (18 ml) was treated with a solution of iodine
(45 mg, 0.18 mmol) in 70% acetic acid (60 ml) for 1 h
at 20°ë on stirring. The 5% solution of L-ascorbic acid
in deionized water was added to the reaction mixture to
complete decoloration. The reaction mixture was evap-
orated to a volume of approximately 2 ml, diluted with
water (20 ml), and fractionated on the column with Dia-
sorb. The elution was performed under the conditions
described for compound (I). Acetonitrile was evapo-
rated from the fractions containing the target product
and byproducts, and the residue was lyophilized. The yield
of dimer (V) was 7 mg (14.4%); Rt 19.60 min; mass spec-
trum: å 2680.3, 1587.6*, and 1093.0* (Mcalculated for
H-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-
Cys(Acm)-Asp-Phe-OH (III) (the 206-218 sequence
of b1-adrenoreceptor). Raw product (III) of SPPS was
purified as described for compound (I). The yield of
peptide (III) was 0.093 g (32.0% relatively to the start-
ing amino acid attached to the polymer); Rt 13.98 min;
purity of 97.2%; Mass spectrum: M 1161.3 (Mcalculated
for ë68ç104N22O21S3 was 1162).
H-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-
Cys-Asp-Phe-OH (IV) was prepared from compound
(III) (0.032 g, 0.019 mmol) by cleavage of the Acm
group according to the procedure described for peptide
(II) in 30% acetic acid. The yield of compound (IV)
was 0.027 g (90.0%); Rt 14.59 min; purity of 95.2%;
mass spectrum: M 1591.9 (Mcalculated for ë65ç99N21O20S3
was 1590.8).
ë116ç162N30O36S4 was 2680). The yield of dimer (VIII)
was 7 mg (17.7%); Rt 25.15 min; mass spectrum:
M 2185.2 (Mcalculated for ë102ç132N18O32S2 was 2184). The
yield of compound (II) was 3 mg (15.0%); Rt 22.12 min;
mass spectrum: å 1098.0 (Mcalculated for ë51ç67N9O16S1
was 1094.2. The yield of compound (IX) was 15 mg
(52.2%); Rt 14.20 min; mass spectrum: M 1589.0 Mcal-
culated for ë65ç99N21O20S3 was 1588.0).
Scheme A of the synthesis of the conformational
antigen. The solution of nonapeptide (II) (7 mg,
6 µmol) and tridecapeptide (IV) (9 mg, 6 µmol) in 10%
aqueous dioxane (20 ml) was brought to pH 8 by the
addition of 5% aqueous ammonium hydroxide (1 ml) in
portions of 0.1 ml and treated with 3% aqueous hydro-
gen peroxide for 10 min at 20°ë. The formation of the
S–S bond was monitored by the reaction with the Ell-
man reagent. Upon completion of the reaction, the mix-
ture was acidified to pH 4–5, the dioxane was evapo-
rated, and the residue was fractionated on the column
with Diasorb. The column was eluted as described for
compound (I). Fractions containing the main peak were
joined, evaporated for the removal of acetonitrile, and
lyophilized. The yield of the mixture of products (V)
and (VI) was 0.013 g (81%); Rt 19.60 min (28.5%) and
H-Ala-Arg-Arg-Cys(Acm)-Tyr-Asn-Asp-Pro-Lys-
Cys(Acm)-Cys(SPyr)-Asp-Phe-OH (XI). The solu-
tion of 2,2'-dithiopyridine (0.100 g, 0.454 mmol) in the
mixture of phosphate buffer (pH 6.8, 5 ml) and ethanol
(5 ml) was added to the solution of raw tridecapeptide
(X) (0.470 g, 0.271 mmol) cleaved from the polymer in
the mixture of the same buffer (20 ml) and ethanol
(10 ml). The reaction mixture was stirred for 1 h at
20°ë. The formation of the S–S bond was monitored by
HPLC and with the use of the Ellman reagent. Upon
completion of the reaction, acetic acid (3.5 ml) was
added to the reaction mixture to pH 4, the alcohol was
evaporated, and the residue was fractionated on the col-
umn with the Vydac sorbent. The column was eluted
with a concentration gradient of buffer B in buffer A
(from 0 to 10% within 5 min and from 10 to 60% within
100 min) at a flow rate of 3 ml/min. Fractions contain-
ing the target product were joined, and acetonitrile was
evaporated. The residue was diluted with water and lyo-
philized. The yield of compound (XI) was 0.361 g
(78.0%); Rt 16.52 min; purity of 95.0%; mass spectrum:
M 1839, 1732.9* (Mcalculated for ë75ç112N24O22S4 was
1842).
20.34 (61.4%); mass spectrum: å 2680.7, 1588.8*,
1094.1* (Mcalculated for C116H162N30O36S4 was 2680).
The H-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-
Lys-Cys-Cys(Acm)-Asp Phe-OH (VII) was prepard
from compound (III) (0.032 g, 0.019 mmol) by forma-
tion of the intramolecular S–S bond by the procedure
described for the preparation of the mixture of products
(V) and (VI). The yield of compound (VII) was 0.030 g
(94.7%); Rt 3.48 min; purity of 97.0%; mass spectrum:
M 1660.1 (Mcalculated for ë68ç102N22O21S3 was 1660).
H-Ala-Arg-Arg-Cys(Acm)-Tyr-Asn-Asp-Pro-
Lys-Cys(Acm)-Cys-Asp-Phe-OH H-Glu-Tyr-Gly-
Ser-Phe-Phe-Cys-Glu-Leu-OH (XII). The solution
*Hereafter, peaks corresponding to the products with the disulfide
bonds which are reduced in the course of mass spectrometry are
marked with asterisks.
of nonapeptide (II) (0.015 g, 0.014 mmol) in an ammo-
nium acetate buffer (pH 6.8, 5 ml) was added to the
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 3 2009

294
SIDOROVA et al.
solution of tridecapeptide (XI) (0.025 g, 0.014 mmol) added to the settled gel of the examined sorbent
in the same buffer (7 ml). The reaction mixture was (100 µl), and stirred for 30 min. Plasma after the chro-
stirred at 20°ë. The formation of the S–S bond was matography was separated by centrifugation at 12000 g
monitored by HPLC. Acetic acid (pH 4, 1.2 ml) was for 1 min. The supernatant was collected, and the sor-
added 30–40 min later, and the reaction mixture was bent was washed with 0.01 M KH2PO4–NaOH (pH 7.0)
fractionated on the column with the Vydac sorbent. The with 0.15 M NaCl (3 × 500 µl). Immunoglobulins were
column was eluted under the conditions described for removed from the sorbent by two subsequent incuba-
compound (XI). Fractions containing the target prod- tions with the mixture of 0.2 M glycine–HCl (pH 2.5)
uct were joined, evaporated for the removal of aceto- (250µl) for 5 min and pouring of the solution. The
nitrile, and lyophilized. The yield of compound (XII) obtained solutions were joined and brought to pH 4.5
was 0.023 g (57.1%); Rt 20.83 min; purity of 95.6%; by the addition of a 10% solution of NaOH, and the
immunoglobulin content was determined in the solu-
tions.
mass spectrum: M 2824.8, 1733.0* (Mcalculated for
ë122ç174N32O38S4 was 2826).
The concentration of the ligands in the solutions was
determined on a spectrophotometer according to the
optical absorption of the solutions at 280 nm, taking
into account the extinction coefficients which were
measured for the standard (1 mg/ml) solutions of every
peptide. The protein concentration was determined by
the Bradford method [24] at 595 nm.
Peptide (V) was prepared according to Scheme C
by cleavage of Acm groups from dimer (XII) (0.02 g,
0.007 mmol) with the simultaneous formation of the
intramolecular S–S bridge by the procedure described
above. The formation of the S–S bond was monitored
by HPLC. The purification was carried out on the col-
umn with the Vydac sorbent. Elution was performed
under the conditions described for compound (XI).
Fractions containing the target product were joined,
evaporated for the removal of acetonitrile, diluted with
water, and lyophilized. The yield of compound (V) was
0.012 g (64%); Rt 19.60 min; purity of 99.5%; mass
spectrum: M 2680.6, 1588.9*, 1093.0* (Mcalculated for
ë116ç162N30O36S4 was 2680).
ACKNOWLEDGEMENTS
This study was supported by State Contract
no. 02.512.11.2100 from 09.04.2007: Creation of Bio-
specific Sorbents on the Basis of Oligopeptides.
REFERENCES
Immobilization of the peptides. A settled
sepharose gel (5 ml) was washed with the solution of
KOH (4.48 g) and KH2PO4 (4.28 g) in water (20 ml),
and a suspension in the same solution (10 ml) was pre-
pared. The solution of BrCN (1 ml) in dioxane
(0.6 g/ml) was added to the suspension at 5°ë, and the
reaction mixture was shaken for 10 min at intervals of
25–30 s. The gel was washed with water (50 ml) at 5°ë
and with the buffer mixture of 0.2 M H3BO3 n-dash
NaOH with pH 8.0 (10 ml) and suspended in the same
buffer mixture (total volume was 10 ml). Solutions of
the peptides in the mixture of dimethylsulfoxide and
water (3 : 7) with concentration of 2.7 mg/ml were used
for the immobilization. In each case, 1.5 ml of the solu-
tion of the corresponding peptide was added to 2 ml of
the suspension of activated gel. The reaction mixture
was stirred for 10 h at 20°ë, mixed with a 1 M solution
of ethanolamine in water (0.5 ml, pH 8.0), and stirred
for 1 h. The gel was washed with water (50 ml) and a
buffer mixture (25 ml) of 0.1 M KH2PO4 n-dash NaOH
(pH 7.0). The amount of immobilized ligand was eval-
uated from the difference between its starting amount
and its amount in the first washings from the sorbent
after the covalent immobilization. The final content of
the immobilized ligand proved to be 1.1 and 1.0 µmol
per 1 ml of the settled gel for the mixture of (V) and
(VI) and for compound (V), respectively.
1. San Martín, M.A., García, Á., Rodríguez, F.J., and Terol, I.,
Rev. Esp. Cardiol., 2002, vol. 55, no. 5, pp. 514–524.
2. Fu, M. and Matsui, S., 2002, vol. 51, no. 4, pp. 208–212.
3. Rönspeck, W., Brinckmann R,, Egner R., Gebauer F.,
Winkler D., Jekow P., Wallukat G., Müller J., Kunze R,
Ther. Apher. Dial., 2003, vol. 7, no. 1, pp. 91–97.
4. Staudt, A., Schaper, F., Stangl, V., Plagemann, A., Bohm, M.,
Merkel, K., Wallukat, G., Wernecke, K.D., Stangl, K., Bau-
mann, G., and Felix, S.B., Circulation, 2001, vol. 103, no. 22,
pp. 2681–2686.
5. Konovalov, G.A., Belenkov, Yu.N., Zvezdkin, P.V., Che-
byshev, A.N., Semin, S.N., Kuznetsova, Yu.V., Ada-
mova, I.Yu., Kipor, S.G., and Pokrovskii, S.N., Kardio-
logiya, 2002, no. 6, pp. 123–127.
6. Matoba, Y., Okubo, H., and Nosé, Y., Artificial Organs,
2004, vol. 28, no. 2, pp. 171–181.
7. Magnusson, Y., Wallukat, G., Waagstein, F., Hjalmarson, A.,
and Hoebeke, J., Circulation, 1994, vol. 89, no. 6, pp. 2760–
2767.
8. Matsui, S., Fu, M.L., Hayase, M., Katsuda, S., Yamagu-
chi, N., Teraoka, K., Kurihara, T., and Takekoshi, N., J.
Card. Fail., 1999, vol. 5, no. (3), pp. 246–254.
9. Zhang, L., Hu, D., Li, J., Wu, Y., Liu, X., and Yang, X.,
Chin. Med. J. (Engl.), 2002, vol. 115, no. 8, pp. 1127–
1131.
10. Chiale, P.A., Ferrari, I., Mahler, E., Vallazza, M.A., and
Elizari, M.V., Circulation, 2001, vol. 103, no. 13,
pp. 1765–1771.
Binding of immunoglobulins to the sorbents was
examined by the method of volume sorption at 20°ë. A
sample of blood plasma of a patient (500 µl) was
diluted two times with the physiological solution,
11. Frielle, T., Collins, S., Daniel, K.W., Caron, M.G.,
Lefkowitz, R.J., and Kobilka, B.K., Proc. Natl. Acad.
Sci. USA, 1987, vol. 84, pp. 7920–7924.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 3 2009

SYNTHESIS AND PROPERTIES OF A NEW CONFORMATIONAL ANTIGEN
295
12. Tanea, K., Magnussonb, Y., Viguera, M., Lengagnea, R., 18. Sidorova, M.V., Molokoedov, A.S., Az’muko, A.A.,
Hjalmarsonb, A., Guilleta, J.B., and Hoebeeke, J., Bio-
chimie, 1994, vol. 76, pp. 159–164.
13. Iwata, M.,Yoshikawa, T., Baba,A.,Anzai, T., Nakamura, I.,
Wainai, Y., Takahashi, T., and Ogawa, S., Circulation Res.,
2001, vol. 88, pp. 578–586.
14. Shul’ts, G. and Shirmer, R., Printsipy strukturnoi orga-
nizatsii belkov (Principles of Structural Organization of
Proteins), Moscow: Mir, 1982 [Russian translation].
Kudryavtseva, E.V., Krause, E., Ovchinnikov, M.V., and
Besralova, Zh.D., Bioorg. Khim., 2004, vol. 30, pp. 115–
125 [Russ. J. Bioorg. Chem. (Engl. Transl.), vol. 30,
pp. 101–110].
19. Stewart, J.M. and Young, J.D., Solid Phase Peptide Syn-
thesis, 2nd ed., Rockford, Il.: Pierse Chemical Co., 1984.
20. Khimiya Polipeptidov (Polypeptide Chemistry), Mos-
cow: Mir, 1977 [Russian translation].
15. Fraser, C.M., J. Biol. Chem., 1989, vol. 264, pp. 9266–
21. Schmidt, M., Krause, E., Beyermann, M., and Bienert, M.,
9270.
Peptide Res., 1995, vol. 8, pp. 238–242.
16. Andreu, D, Albericio, F, Sole, N.A, Munson, M.C, Fer-
rer, M, and Barany, G, in Peptide Synthesis Protocol,
Pennington, M.W. and Dunn, B.M.., Eds., New Jersey,
Totowa: Humana Press Inc, 1994, pp. 91–169.
22. Nguyen, D.N., Becker, G.W., and Riggin, R.M., J. Chro-
matography A, 1995, vol. 705, pp. 21–45.
23. Porath, J., Biochimie, 1973, vol. 55, pp. 943–951.
17. Kudryavtseva, E.V., Sidorova, M.V., Molokoedov, A.S.,
Ovchinnikov, M.V., and Bespalova, Zh.D., Bioorg. 24. Bradford, M., Anal. Biochem., 1976, vol. 56, pp. 248–
Khim., 1999, vol. 25, pp. 107–116.
252.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 3 2009