ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2017, Vol. 43, No. 4, pp. 351–358. © Pleiades Publishing, Ltd., 2017. Original Russian Text © M.V. Sidorova, M.E. Palkeeva, A.A. Az’muko, M.V. Ovchinnikov, A.S. Molokoedov, T.V. Sharf, E.E. Efremov, S.P. Golitsyn, 2017, published in Bioorganicheskaya Khimiya, 2017, Vol. 43, No. 4, pp. 339–346.
Solid-Phase Fragment Condensation for Synthesis of Peptides from the Immunodominant Sequence of β1-Adrenoreceptor M. V. Sidorova1, M. E. Palkeeva, A. A. Az’muko, M. V. Ovchinnikov, A. S. Molokoedov, T. V. Sharf, E. E. Efremov, and S. P. Golitsyn Russian Cardiology Research and Production Complex of the Russian Health Ministry, Moscow, 121552 Russia Received October 5, 2016; in final form, October 31, 2016
Abstract⎯The P26 peptide corresponding to the 197–222 sequence of the second extracellular loop of the β1-adrenoreceptor (β1-AR) was synthesized by solid-phase fragment condensation on the Wang polymer. Pentapeptide fragments were prepared on the 2-chlorotrityl resin. The racemization degree of the C-terminal alanine residue of the pentapeptide was experimentally evaluated for the synthetic H-Glu-Ser-Asp-Glu-AlaArg-OH hexapeptide β1-АR-(202–207) which was prepared by the 5 + 1 fragment condensation with the use of various condensing agents. A content of the diastereoisomeric peptide in the products of the fragment condensation was determined by HPLC on a reversed phase. The D-alanine-containing hexapeptide was specially synthesized and used for a comparison. The minimum racemization degree of the C-terminal alanine residue was observed if complex F was applied to the synthesis of the hexapeptide. Keywords: the second extracellular loop of the β1-adrenoreceptor, solid-phase peptide synthesis, convergent peptide synthesis, fragment condensation, racemization of a C-terminal amino acid residue DOI: 10.1134/S1068162017040112
INTRODUCTION The β1-adrenoreceptor (β1-AR) is located in the cardiomyocytes and plays an important role in the regulation of a cardiac functioning. The IgG autoantibodies to β1-AR are an important marker of a number of autoimmune pathologies. The autoantibodies participate in the development of the myocardial ischemia, arterial hypertension, dilated cardiomyopathy, “idiopathic” ventricular arrhythmia, and other cardiac pathologies [1]. The antibodies to the protein are known to be identified in 50% of patients with such types of arrhythmia and in 35% of patients with disorders of myocardial conduction [2]. In addition, the antibodies to β1-AR are found in 10% of healthy per1 Corresponding
author: phone: +7 (495) 414-67-16; fax: +7 (495) 414-67-86; e-mail:
[email protected]. Abbreviations: Ahx, 6-aminohexanoyl; Acm, acetamidomethyl; β1-АR, β1-adrenoreceptor; Boc, tert-butyloxycarbonyl; BOP, benzotriazole-1-yloxy-tris(trimethylamino)phosphonium hexafluorophosphate; Bt, biotinyl; But, tert-butyl; DCC, N,N'-dicyclohexylcarbodiimide; DCM, dichloromethane; DIC, N,N'diisopropylcarbodiimide; DIEA, diisopropylethylamine; DMA, dimethylacetamide; DMSO, dimethylsulfoxide; DTT, dithiothreitol; Fmoc, 9-fluorenylmethyloxycarbonyl; HOBt, 1-hydroxybenzotriazole; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; 4-MePip, 4-methylpiperidine; NMP, N-methylpyrrolidone; Pbf, 2,2,4,6,7 –pentamethyldihydrobenzofuran-5-sulfonyl; TBTU, N,N,N',N'-tetramethyl-О(benzotriazole-1-yl)uronium tetrafluoroborate; TIBS, triisobutylsilane; EIA, enzyme immunoassay; SPS, solid-phase synthesis; SPFC, solid-phase fragment сondensation.
sons [3]. Peptide fragments of the second extracellular loop of β1-AR, in particular the HWWRAESDEARRCYNDPKCCDFVTNR (197–222) sequence, have been found to be the β1-AR antigenic determinants and to interact with the antibodies to β1-AR in EIA [1, 4]. Different groups of studies demonstrated that long-time (6 and 14 months) immunization of animals with the β1-АR-(197–222) and β1-АR-(197–223) peptides resulted in morphological changes in cardiomyocytes, suggesting a development of cardiomyopathy [5, 6]. In this connection, studies of the β1-AR immunodominant epitopes are very important for creation of novel diagnostic and prognostic methods when dealing with certain categories of patients. This study continues investigations in the field of synthesis of the peptides which correspond to the β1AR antigenic determinants [7, 8] and can react with the autoantibodies to this protein in EIA in the course of blood analysis of patients with cardiovascular pathologies [9]. The goal of this study is an optimization of the synthesis of the 26-member peptide (P26) corresponding to the 197–222 sequence of the second extracellular loop of β1-AR and a preparation of the peptide conjugate with biotin. These peptides are promising for the creation of antigenic diagnostic drugs.
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RESULTS AND DISCUSSION The H-His-Trp-Trp-Arg-Ala-Glu-Ser-Asp-GluAla-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-AspPhe-Val-Thr-Asn-Arg-OH sequence of the target peptide (P26) contains many trifunctional amino acids, suggesting potential complications in the course of a solid-phase synthesis of this peptide and postsynthetic procedures [10]. The residues of Trp and Cys are especially subject to an oxidation, and the -Asp-Prosequence is sensitive to an acidic medium [11]. This peptide was synthesized according to the modern Fmoc-scheme that was the best for SPS of the Asp-Pro-containing peptides [10]. The base-labile protection of the α-amino group was combined with the acid-labile protecting groups of the side chains of the amino acids [12]: Boc for Lys and Trp; But for Thr, Ser, and Tyr; OBut for Asp and Glu; and Trt for His, Cys, and Asn. First, the P26 polypeptide was test-synthesized on the Wang polymer by a stepwise elongation of the peptide chain using the uronium salts for the formation of the amide bonds (TBTU/DIEA). We chose the feedback deprotection program for α-amino group of the peptidylpolymer among the standard program set for a Tribute-UV automatic synthesizer, i.e., the deprotection procedure was automatically repeated if the Fmoc-protecting group was not completely cleaved according to the UV-monitoring. We observed a progressive decrease in the yields at the stage of the peptide bond formation during the last 7– 8 cycles of SPS based on the UV-monitoring. We thought that this process was mainly associated with the decrease in the yields of the peptide bond formation. As a result, “false peptides” with missing individual amino acid residues, which were typical side products in SPS, were formed. A crude product of the stepwise SPS that was performed with the use of trityl-protecting groups on the sulfhydryl function of the cysteine residues was a rather complex mixture which contained no more than 25–28% of the target product. The two-step purification by gel-chromatography on Sephadex G-25 in 2% acetic acid and HPLC on a reversed phase gave the peptide with a low yield (8% in relation to the first amino acid). Mass spectrometry of the crude SPS product and impurities which were obtained during the purification of the P26 peptide demonstrated that products with molecular masses that corresponded to peptides with a cleavage of peptide bonds were present along with the side products with misses of separate amino acid residues (their molecular masses were
close to that of the target peptide). The peptide with molecular mass that corresponded to the H-Ser-AspGlu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-OH sequence was isolated. The N→O-acyl migration in the Ser203 residue probably occurred in the course of the final deprotection of the P26 peptide with trifluoroacetic acid, and two side-reactions (a hydrolysis of the ester that was formed as a result of the migration and a cleavage of the Asp-Pro bond) took place during the gel-chromatography on Sephadex G-25 in 2% acetic acid. Thus, these two side-processes resulted in this impurity. An inverse O→N acyl migration at pH 8.5–9 was impossible for the peptide with three cysteine residues with free sulfhydryl groups due to a high probability of thiol-disulfide conversions with the formation of peptide oligomers. The difficulties during the test-synthesis of the P26 peptide by the stepwise SPS made us to change this initial synthetic scheme. First, we decided to use Acm-protecting group for the sulfhydryl functions of the cysteine residues. Second, we proposed a solid-phase fragment condensation (SPFC) for the synthesis of this peptide on the same polymer by subsequent attachment of two pentapeptide fragments to the stepwise-synthesized hexadecapeptidylpolymer 1 (see Scheme 1). The Acm-protecting group is widely used for the synthesis of cysteine-containing peptides, because it is stable under the conditions of the amide bond formation and deprotection of α-amino groups and side functions of amino acids. The Acm-protection is removed by the treatment with salts of mercury and silver [13] for preparation of free thiols or by the treatment with iodine for the preparation of cyclic disulfides [13]. The use of this protection prevents the N → O acyl migration in the Ser and Thr residues of a crude SPS product owing to a possibility of the twostep deprotection of a peptide. On the first step, all the functional groups of the peptide except the cysteine sulfhydryl group are deprotected, and the inverse O → N acyl migration at pH 8.5 and the subsequent purification of the Cys(Acm)-protected peptide are possible. On the second step, the cysteine residues are deprotected, and the target product is finally purified.
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t
H-Arg(Pbf)-Arg(Pbf)-Cys(Acm)-Tyr(But)-Asn(Trt)Asp(OBut)-Pro-Lys(Boc)-Cys(Acm)-Cys(Acm)-Asp(OBt)Phe-Val-Thr(But)-Asn(Trt)-Arg(Pbf)- P Peptidylpolymer 1
t
Fmoc-Glu(OBu )-Ser(Bu )Asp(OBut)-Glu(OBut)-Ala-OH (FI)
353
+
Complex F
Boc-His(Boc)-Trp(Boc)Trp(Boc)-Arg(Pbf)-Ala-OH (FII)
+
Fmoc-Glu(OBut)-Ser(But)-Asp(OBut)-Glu(OBut)-Ala-Arg(Pbf)Arg(Pbf)-Cys(Acm)-Tyr(But)-Asn(Trt)-Asp(OBut)-Pro-Lys(Boc)Cys(Acm)-Cys(Acm)-Asp(OBut)-Phe-Val-Thr(But)-Asn(Trt)Peptidylpolymer 2 Arg(Pbf)- P Complex F
Boc-His(Boc)-Trp(Boc)-Trp(Boc)-Arg(Pbf)-Ala-Glu(OBut)-Ser(But)-Asp(OBut)-Glu(OBut)-Ala-Arg(Pbf)Arg(Pbf)-Cys(Acm)-Tyr(But)-Asn(Trt)-Asp(OBut)-Pro-Lys(Boc)-Cys(Acm)-Cys(Acm)-Asp(OBut)-Phe-ValPeptidylpolymer 3 Thr(But)-Asn(Trt)-Arg(Pbf)- P TFA H-His-Trp-Trp-Arg-Ala-Glu-Ser-Asp-Glu-Ala-Arg-Arg-Cys(Acm)-Tyr-Asn-Asp-Pro-LysCys(Acm)-Cys(Acm)-Asp-Phe-Val-Thr-Asn-Arg-OH[Cys(Acm)3]P26 1) Hg(OAc)2 2) H2S H-His-Trp-Trp-Arg-Ala-Glu-Ser-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-AspPhe-Val-Thr-Asn-Arg-OH(P26) Scheme 1. The solid-phase fragment condensation of the P26 polypeptide.
As a rule, the fragment condensation both by the conventional and solid-phase methods is known to give a product of higher quality in comparison with the stepwise synthesis [14–16]. In addition, purification of peptides that are prepared by the fragment condensation is considerably easier. Therefore, we decided to use the fragment condensation in the last steps of the synthesis even though “convenient” (optically inactive or slightly racemizing) amino acid residues for dividing into peptide blocks were absent in the sequence of the target product. Racemization of optically active amino acid residues on C-termini of peptide fragments are known to be possible during their fragment condensation. However, previously, we have successfully performed SPFC of peptide blocks with optically active C-terminal amino acid residues. The fragment with the C-terminal free arginine was used in the synthesis of the 1–42 fragment of β-amyloid [15]. This fragment was attached by the DCC/HOBT method in the presence of one equivalent of pyridine hydrobromide for a protonation of the guanidine function. The RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
subsequent enantiomeric analysis of a hydrolysate of this peptide on a column with a chiral phase demonstrated that this peptide did not contain significant amounts of D-arginine, and its enatiomeric composition corresponded to that of the product of the stepwise synthesis. It was reported [16] that the DCC condensation of the peptide block with the C-terminal glutamic acid residue in the presence of HOBt in DMSO gave almost no racemization. The F1 and F2 protected fragments for the fragment condensation of the synthesis of the target peptide were prepared by the solid-phase method on 2chlorotritylchloride resin (see Experimental). We evaluated the degree of the possible racemization of the C-terminal alanine residue after the attachment of the F1 pentapeptide fragment (Scheme 1) to the Nterminal H-Arg(Pbf)-residue of aminopeptidylpolymer 1 with the use of different reagents under the conditions of a model experiment. We synthesized the HGlu-Ser-Asp-Glu-DAla-Arg-OH hexapeptide with D-alanine residue as a comparison substance. This Vol. 43
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(а)
reomers with LAla and DAla in the crude product of the fragment condensation was determined by HPLC after the deprotection (Fig. 1).
1 2
HPLC of the reaction mixtures of the fragment condensation of peptide FI with H-Arg(Pbf)-polymer revealed the racemization of the C-terminal residue in the peptide block. However, the racemization degree considerably depended on the chosen method for the amide bond formation. The content of the diastereomeric peptide in the reaction mixture was lower than 8% if the condensation was performed for 16 h with the use of complex F (one of variations of the carbodiimide method) (Fig. 1b, Table 1). At the same time, the application of BOP/HOBt/ DIEA (2 h) resulted in the 35% content of the diastereomeric peptide (Fig. 1a, Table 1). The unexpectedly high racemization (approximately 20%) of the C-terminal alanine residue was also observed when the carbodiimide method (DIC/HOBt) in DMSO was used. Thus, complex F proved to be the most convenient for the condensation of fragment FI, and this method was used for the synthesis of the P26 peptide. In addition, the model experiment demonstrated that the diastereoisomeric impurities could be highly probably separated from the target product, because the pair of the model diastereomers was fractionated by HPLC as well-defined peaks (Fig. 1).
1 (b)
2
2 (c)
8 9 10 11 12 13 14 15 16 17 18 19 20 min Fig. 1. Profiles of the analytical HPLC of the crude products of the synthesis of (peak 1) the H-Glu-Ser-Asp-GluAla-Arg-OH model peptide corresponding to the 202–207 sequence of β1-АR by the (5 + 1) fragment condensation on the polymer with the use of (a) BOP/HOBt/DIEA (2 h) and (b) complex F in NMP (16 h) in comparison with (c) the H-Glu-Ser-Asp-Glu-DAla-Arg-OH model peptide (peak 2) on the Kromasil column (4.6 × 250 mm, 5μm). The peptides were eluted with the gradient of buffer B (80% acetonitrile in 0.1% TFA) in buffer A (0.1% TFA) from 0 to 40% within 40 min at a flow rate of 1 mL/min at 220 nm (conditions 3).
peptide could be formed if the condensation of the fragment with the peptidylpolymer or aminoacylpolymer was accompanied by the racemization of the Cterminal amino acid residue. Complex F (an adduct of N,N'-dicyclohexylcarbodiimide and pentafluorophenol in a molar ratio of 1 : 3) [17], DIC/HOBt in DMSO [16], or the Castro’s Reagent (BOP/HOBt/DIEA) [18] were used for the condensation. The content of diaste-
Based on these results, the fragment condensation during the synthesis of the target polypeptide was performed with the use of complex F in NMP [17]. The reaction completeness was controlled by the qualitative ninhydrin test [19]. The condensation was finished within 4 h. The prepared Cys-protected crude product of SPS contained ≈70% of the target peptide and was purified by the preparative HPLC to 96% homogeneity. The peptide had a correct molecular mass according to the MALDI mass spectrometry. The Acm-protecting groups were cleaved by the treatment with mercury acetate in 30% acetic acid. The deprotection reaction was performed without complications, and the substance after the removal of the mercury sulfide was purified by HPLC. The TPFS methodology was also applied to the conjugation of the P26 peptide with biotin. The corresponding N-terminal fragment (FII) was synthesized by the solid-phase method on the chlorotritylchloride resin to which biotin was attached via the 6-aminohexane-acid spacer. The obtained fragment FIII (Table 2) was condensed with the 21-member peptidylpolymer 2 using complex F. Yields of the TPFS-prepared peptides were significantly higher than those after the stepwise chain elongation (see Table 3). The impurity (3% relative to the first amino acid) with the molecular mass (3597.7) that corresponded to that calculated for the target peptide (Мcalcd = 3598.1) was isolated during the preparation of the biotinylated peptide (Bt-Ahx-P26) along with the target product.
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Table 1. Evaluation of the racemization level of LAla in the model experiment of preparation of the H-Glu-Ser-Asp-GluAla-Arg-OH hexapeptide by the fragment condensation HPLC data Method of the synthesis/ condensing agent
Peptide
H-Glu-Ser-Asp-Glu-LAla-Arg-OH H-Glu-Ser-Asp-Glu-LAla-Arg-OH H-Glu-Ser-Asp-Glu-DAla-Arg-OH H-Glu-Ser-Asp-Glu-LAla-Arg-OH
the diastereomer content*, %
TPFS (5+1), BOP/HOBt/DIEA, 2 h, NMP TPFS (5+1), complex F, 16 h, NMP Stepwise SPS/ TBTU/NMM TPFS (5+1), DIC/HOBt, 4 h, DMSO
LAla
DAla
54 82 0 67
35 8.0 90 21
retention time of the diastereomers, Rt, min LAla 14.48 14.48 14.48
DAla 15.60 15.60 15.60 15.60
* The content of the diastereomeric hexapeptides (%) in the crude products of SPS is given.
Table 2. Characteristics of the peptide fragments*
Peptide
Fmoc-Glu(OBut)-Ser(But)-Asp(OBut)-Glu(OBut)-Ala-OH (FI) Boc-His(Boc)-Trp(Boc)-Trp(Boc)-Arg(Pbf)-Ala-OH (FII) Bt-Ahx-His-Trp(Boc)-Trp(Boc)-Arg(Pbf)-Ala-OH (FIII)
Yield, %
Rf in TLC (system)
Homogeneity (%) according to HPLC after the cleavage of all the protecting groups
82.0
0.48 (А)
88.5 (conditions 3)
74.2 75.8
0.31(B) 0.24(B)
91.2 (conditions 2) 90.1 (conditions 2)
* The HPLC conditions and the compositions of the chromatographic systems for TLC are given in the Experimental.
This substance was very probably the diastereomer of the target peptide. Thus, we proposed the combined scheme for the solid-phase synthesis of the P26 peptide that corresponded to the 197–222 immunodominant sequence of the second extracellular loop of β1-АR. This scheme combined the stepwise elongation of the peptide chain on the first 16 steps of the synthesis and the fragment condensation on the finishing steps of the synthesis and provided the preparation of the P26 peptide with the yield that was high enough for EIA. EXPERIMENTAL Derivatives of L-amino acids (Fluka, NovaBiochem and Bachem, Switzerland), DIEA, HOBt, TBTU, (Fluka, Switzerland), and TIBS (Aldrich, United States) were used in this study. DMF, N-methylpyrrolidone (NMP), dichloromethane, 4-methylpiperidine, and TFA (Fluka, Switzerland) were used for the synthesis. Acetonitrile and isopropanol (Panreac, Spain) were applied to the synthesis and purification of the peptides. The TLC of the peptide fragments was performed on Kieselgel 60 F 254 plates (Merck, Germany) in the following chromatographic systems: (А), chloroform–methanol–acetic acid (90 : 8 : 2) and RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
(B), chloroform–methanol–acetic acid (9 : 1 : 0.5). The peptides on the plates were detected in UV-light and by the Сl2–benzidine treatment. The analytical HPLC of the peptides was carried out on a Knauer chromatograph (Germany) on a YMC-Pack column (4.6 × 250 mm, 10 μm, Japan) (conditions 1) and a VydacC18 300Е column (4.6 × 250 mm, 5 μm, United States) (conditions 2). The peptides were eluted with a gradient of buffer B (80% acetonitrile in 0.1% TFA) in buffer A (0.1% TFA) (from 10 to 70% within 30 min) at a flow rate of 1 mL/min at 220 nm in both cases. The analytical HPLC in the model experiments was performed on a Gilson chromatograph (France) on a Kromasil column (4.6 × 250 mm, 5 μm, Sweden). The peptides were eluted from the column with a gradient of buffer B in buffer A (from 0 to 40% within 40 min) at a flow rate of 1 mL/min at 220 nm (conditions 3). The preparative HPLC of the peptides was carried out on a Knauer chromatograph (Germany) on a column with the Eurosphere ODS sorbent (20 × 250 mm). The peptides were eluted with the 0.5%/min-gradient of buffer B (80% acetonitrile in 0.1% TFA) in buffer A (0.1% TFA) at a flow rate of 10 mL/min at 226 nm (conditions 4). Vol. 43
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Table 3
Peptide
Designation
Molecular Yield, mass %
HPLC (conditions1) Rt, min
homogeneity, %
MALDI-MS, m/z
HWWRAESDEARRC(Acm)YNDPKC(Acm)C(Acm)DFVTNR
[Cys(Acm)3]-P26
3471.6 48.0(а) 15.59
96.2
3471.18
Bt-Ahx-HWWRAESDEARRC(Acm)YNDPKC(Acm)C(Acm)-DFVTNR
Bt-Ahx[Cys(Acm)3]-P26
3811.0 32.5(а) 17.96
94.8
3811.04
HWWRAESDEARRCYNDPKCCDFVTNR* Р26
3258.6
8.4(а) 16.51
90.2
3258.1
HWWRAESDEARRCYNDPKCCDFVTNR
3258.6 51.7(b) 16.51
97.7
3258.1; 3296.1 [M + K]+
3598.1 50.7(b) 18.59
95.0
3597.5; 3619.6 [M + Na]+ 3635.6 [M + K]+
Р26
Bt-Ahx-HWWRAESDEARRCYNDPKCCD- Bt-Ahx-Р26 FVTNR
* The peptide that was prepared by the stepwise SPS is marked by an asterisk. (а) The yields are given relatively to the first amino acid which is attached to the polymer. (b) The yields are given relatively to the starting [Cys(Acm)3]-derivative.
Solid-Phase Peptide Synthesis of the Peptide Fragments We used the following protecting groups in SPS: Fmoc for α-amino functions; Acm for sulfhydryl group of Cys; But for hydroxyl groups of Ser, Thr, and Tyr; OBut for carboxyl groups of Asp and Glu; Boc for ε-amino group of Lys, imidazole group of the N-terminal His residue (fragment II), and indole ring of Trp; Pbf for guanidine group of Arg; and Trt for caboxamide group of Asn and imidazole group of His. The peptide fragments were synthesized on 2-chlorotritylchloride resin (Iris Biotech, Germany) with 1.56 equivalents of Cl/g. For the attachment of the first amino acid, the solution of Fmoc-Ala-OH (2.3 g, 7.25 mmol) in DCM (40 mL) was added to the resin (4.0 g, 6.24 mmol). DIEA (3.2 mL, 18.8 mmol) was added, and the suspension was stirred for 30 min at 25°С. The polymer was filtered and washed with DCM (2 × 40 mL), DMF (2 × 40 mL), and DCM (2 × 40 mL). The residual chlorine was cleaved by the treatment with the mixture of DCM, MeOH, and DIEA (32 : 6 : 2), and the polymer was washed with DCM (2 × 40 mL) and DMF (2 × 40 mL). The content of the first amino acid was determined on a spectrophotometer and proved to be 0.70 mmol/g. Fragments (FI, FII, and FIII) were synthesized in an automatic regime on a Tribute-UV synthesizer (Protein Technologies Inc., United States) according to the standard programs starting from the C-terminus from 0.9 g (0.63 mmol) of the Fmoc-aminoacylpolymer. The SPS cycle involved the following basic steps:
(1) Deprotection of α-amino groups by the treatment with 25% 4-МеPip/DMF for 10 min; (2) Washing with DMF; (3) Condensation with the fourfold excess of Fmoc-Xaa/TBTU/HOBt in the presence of 2 equivalents of DIEA in DMF for 1 h; (4) Washing with DMF. After the synthesis was completed, the protected peptides were cleaved from the polymer by the treatment with the mixture (15 mL) of AcOH–trifluoroethanol–DCM (1 : 2 : 7) for 40 min. The polymer was filtered off and washed with the deprotecting mixture (2 × 10 mL). The filtrate was evaporated, and the product was precipitated with anhydrous ether, dried, crystallized from the appropriate solvent, and dried again. The structure of the synthesized fragments and their homogeneity was confirmed by 1H NMR spectroscopy and TLC. In addition, the samples of the synthesized peptides were treated with trifluoroacetic acid and analyzed by the analytical HPLC. The content of the main substance in the samples proved to be ≈90%. Synthesis of the [Cys(Acm)3]-P26 Polypeptide H-His-Trp-Trp-Arg-Ala-Glu-Ser-Asp-Glu-Ala-ArgArg-Cys(Acm)-Tyr-Asn-Asp-Pro-Lys-Cys(Acm)Cys(Acm)-Asp-Phe-Val-Thr-Asn-Arg-OH. The Wang polymer (co-polymer of styrene and 1% divinylbenzene with 4-hydroxymethylphenoxymethyl anchoring group) with the attached Fmoc-Arg(Pbf)-OH (0.67 mmol/g) (NovaBiochem, Switzerland) was used for SPS. The peptide was synthesized on an automatic Tribute-UV peptide synthesizer (Protein Technolo-
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gies Inc.) according to the standard programs starting from the C-terminus from 0.55 g (0.37 mmol) of the Fmoc-aminoacylpolymer. The synthetic cycle was described above for the syntheses of the fragments. The resin sample (50 mg) was taken on the stage of hexadecapeptidylpolymer 1, treated with the deprotecting mixture (5 mL of TFA, 0.25 mL of TIBS, and 0.25 mL of deionized water) for 2 h. The polymer was filtered off, and the filtrate was evaporated. The product was precipitated with anhydrous ether. The quality of the 16-member intermediate peptide was evaluated by HPLC on the Vydac C18 300Е column in conditions 2. The content of the basic substance in the sample was 80.3% (Rt = 13.03 min). The Nα-Fmoc-protection was removed from hexadecapeptidylpolymer 1, and the solution of the FmocGlu(OBut)-Ser(But)-Asp(OBut)-Glu(OBut)-Ala-OH fragment (FI) (0.75 g, 0.75 mmol) and complex F (0.56 g, 1.0 mmol) in NMP (7 mL) was added to the polymer with a free amino group. The peptidylpolymer was filtered 4 h later (the negative ninhydrin test) and washed according to the synthetic protocol. Peptidylpolymer 2 that contained 21-member peptide was divided into two parts (0.9 g in each). One part of the polymer was condensed with the Boc-His(Boc)Trp(Boc)-Trp(Boc)-Arg(Pbf)-Ala-OH (FII) fragment after the removal of the Fmoc-group and the BtAhx-His-Trp(Boc)-Trp(Boc)-Arg(Pbf)-Ala-OH (FIII) fragment was attached to another part of the Nαdeprotected polymer using half the amounts of all the reagents. The final deprotection with the simultaneous cleavage of the [Cys(Acm)3]-P26 peptide from peptidylpolymer 3 was performed by the treatment with the mixture of TFA (10 mL), deionized water (0.25 mL), TIBS (0.25 mL), phenol (0.3 g), and DTT (0.25 g) for 2 h. The polymer was filtered off and washed with the deprotecting mixture (2 × 2 mL). The filtrate was evaporated and mixed with anhydrous ether. The precipitate was filtered, washed with DCM (3 × 3 mL) and ether (3 × 5 mL), and dried in a vacuum desiccator. The resulting substance was dissolved in water, alkalized to pH 8.5 with 25% aqueous NH4OH, and lyophilized. The crude [Cys(Acm)3]-P26 peptide after SPS (0.64 g), which contained 72% of the main substance according to HPLC, was divided into portions (150 mg in each) and purified on the column with the Eurosphere ODS sorbent in conditions 4. The fractions that contained the target product were joined and lyophilized. The yield was 308 mg (48% in relation to the polymer-attached first amino acid); Rt 15.59 min; the homogeneity was 96.2% (HPLC in conditions 1 and 2). MALDI-MS: found m/z 3471.18; calcd. М 3471.6. The biotinylated Bt-Ahx-His-Trp-Trp-Arg-AlaGlu-Ser-Asp-Glu-Ala-Arg-Arg-Cys(Acm)-Tyr-AsnAsp-Pro-Lys-Cys(Acm)-Cys(Acm)-Asp-Phe-Val-ThrAsn-Arg-OH peptide (Bt-Ahx-[Cys(Acm)3]-P26) was RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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synthesized and purified as described for [Cys(Acm)3]P26. Its characteristics are given in Table 3. H-His-Trp-Trp-Arg-Ala-Glu-Ser-Asp-Glu-Ala-ArgArg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-ValThr-Asn-Arg-OH (Р26). The solution of Hg(OAc)2 (83.4 mg, 0.268 mmol) in 30% AcOH (4 mL) was added to the solution of the [Cys(Acm)3]-P26 peptide (156 mg, 0.044 mmol) in 30% AcOH (14 mL). The reaction mixture was stirred for 2 h at 25°С and subsequently purged with hydrogen sulfide for 20 min and nitrogen for 10 min. The precipitate of mercury sulfide was separated. The filtrate was diluted with distilled water to the volume of 50 mL and fractionated on the column with the Eurosphere ODS sorbent in conditions 4. The yield of the P26 peptide was 85.4 mg (51.7%). Its characteristics are given in Table 3. The biotinylated peptide was prepared as described for the peptides with free α-amino group. Its characteristics are given in Table 3. ACKNOWLEDGMENTS This study was performed within the framework of the project “Creation of a diagnostic kit that is based on the competitive enzyme immunoassay for a determination of the level of autoantibodies to the β1adrenoreceptor in patients with idiopathic rhythm disorders, myocardial conduction, and cardiovascular pathologies” and the agreement no. 14.604.21.0068 (27.06.2014) of the Federal Targeted Program “Investigations in the area of foreground directions of development of the scientific and technical complex of Russia” (2014–2020). The unique identifier of the project was RFMEFI60414X0068. REFERENCES 1. Magnusson, Y., Wallukat, G., Waagstein, F., Hjalmarson, A., and Hoebeke, J., Circulation, 1994, vol. 89, no. 6, pp. 2760–2767. 2. Deubner, N. and Berliner, D., Eur. J. Heart Fail., 2010, vol. 12, pp. 753–762. 3. Liu, H.R., Zhao, R.R., Zhi, J.M., Wu, B.W., and Fu, M.L., Autoimmunity, 1999, vol. 29, pp. 43–51. 4. Magnusson, Y., Hoeyer, S., Lengagne, R., Chapot, M.-P., Guillet, J.-G., Strosberg, A.A.D., and Hoebeke, J., Clin. Exp. Immunol., 1989, vol. 78, pp. 42–48. 5. Matsui, S., Fu, M., Hayase, M., Katsuda, C., Yamaguchi, N., Teraoka, K., Kurihara, T., and Takekoshi, N., J. Cardiovasc. Pharm., 2003, vol. 42, pp. 99–103. 6. Lin, Z., Haijun, B., Jue, T., Xiaoliang, W., Suli, Z., Zhongmei, H., Li, Y., Rongrui, Z., Xin, M., and Huirong, L., Int. J. Cardiol., 2011, vol. 149, pp. 89–94. 7. Sidorova, M.V., Pal’keeva, M.E., Molokoedov, A.S., Az’muko, A.A., Sekridova, A.V., Ovchinnikov, M.V., Levashov, P.A., Afanasieva, O.I., Berestetskaya, Yu.V., Afanasieva, M.I., Rasova, O.A., Bespalova, Zh.D., and Pokrovskii, S.N., Russ. J. Bioorg. Chem., 2009, vol. 35, no. 3, pp. 285–295. Vol. 43
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Translated by L. Onoprienko
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