A novel synthetic peptide polymer with cyclic RGD motifs supports serum-free attachment of anchorage-dependent cells.

Cell adhesivity is a basic biological principle, which provides mechanisms for construction of multicellular organisms, tissue genesis, migration and individual cell survival. In vivo, the cell adhesive environment is provided by extracellular matrix molecules, neighboring cell surfaces and soluble factors delivered either by tissue cells or by blood circulation. The exact molecular composition of the microenvironment of a cell is not properly understood. The nondefined molecular composition of "native" adhesive components hinders their application when defined culture conditions are necessary, as, for an example, growing human cells for further clinical application. Applying large, substrate-coating molecules as backbones for carrying specific adhesive peptide motifs provides a relatively cheap, reproducible, and chemically defined group of synthetic adhesion molecules. Here, we report on the design, synthesis, and testing of a novel cyclic RGD-containing coating material, which promotes initial attachment, spreading, survival, and proliferation of a number of different cell types. The potent adhesive polypeptide-brush, composed of poly[Lys(DL-Ala(m))] branched chain polypeptide (AK) and multiple copies of cyclic(arginyl-glycyl-aspartyl-D-phenylalanyl-cysteine) pentapeptide prevents anoikis and supports cell attachment in the absence of serum or other biological additives. The defined conditions for cell maintenance make this material a promising candidate for coating artificial cell substrates even for therapeutic applications.

Isotype distribution and fine specificity of the antibody response of inbred mouse strains to four compounds belonging to a new group of synthetic branched polypeptides.

A new group of synthetic branched polypeptides was developed to initiate a systematic study of the relationships between the chemical structure (charge, size, primary structure, configuration and conformation), the carrier potential and the antigenic properties of these biodegradable and biocompatible macromolecules. This model system has two main advantages over the previously used ones: (i) the side chains grafted to the poly(L-lysine) backbone are composed of about three DL-Ala and a single chain-terminating amino acid with different absolute configuration and/or identity, and (ii) the conformation of these polypeptides is characterized in solution. The size, charge and inside area of the four molecules selected for this study were identical; however, the identity, the absolute configuration of the chain-terminating amino acids (D-Leu, Leu, Phe or D-Phe) and, in consequence, the conformation of the macromolecules were different. The qualitative and quantitative features of the antibody response induced by the four polypeptides were characterized in inbred mouse strains by IgM and IgG type antibody levels, as well as by isotype distribution and fine specificity of antibodies produced during the primary and memory response. The intensity of the memory response and the characteristics of subclass distribution were dependent on the conformation of the branched polypeptides. These molecules carry at least two types of antigenic determinants. One is ordered to the tetrapeptide side chain, the expression of which proved to be inversely correlated with the backbone-originated helix content of the molecules. The other antigenic determinant corresponds to the common inside area of the polypeptides which is less conformation-dependent and therefore common to all four polypeptides.

Defensins purified from human granulocytes bind C1q and activate the classical complement pathway like the transmembrane glycoprotein gp41 of HIV-1.

The transmembrane glycoprotein gp41 of HIV-1 contains a C1q binding domain (HIVenv 583-610) and activates the human complement system through the classical pathway. Based on structural and functional similarities between human defensins (human neutrophil peptide, HNP 1-3) and synthetic peptides representing the env 583-610 region of HIV-1, we found it interesting to investigate the C1q binding and complement activating ability of human defensins. Human defensins were purified and characterized by size exclusion chromatography, ultrafiltration, gel electrophoresis and HPLC. The complement activating ability of the purified peptides was assessed in a solid-phase immunoassay. Defensins, fixed to an ELISA plate, were able to bind the C1q subcomponent of the first complement component (C1), triggering the classical pathway of complement activation which led to C4b binding to the plate. Reduction and subsequent alkylation of disulfide bridges of defensins greatly decreased the C1q binding ability but complement activation (C4b binding) remained high. Further acetylation of the reduced defensin peptide resulted in a molecule which bound very little or no C1q but still activated the complement cascade. These phenomena indicate that defensins interact with the complement system via C1q-dependent and C1q-independent mechanisms, and extend the number of functional similarities between defensins and gp41 of HIV-1 to include C1q binding and complement activation.

Antitumour effect of a gonadotropin-releasing-hormone antagonist (MI-1544) and its conjugate on human breast cancer cells and their xenografts.

Our gonadotropin-releasing hormone (GnRH) antagonist analogue MI-1544 ([Ac-D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10]GnRH) was developed as a potential contraceptive material, because it decreased the luteinizing hormone level without unfavourable side-effects. The antagonist was covalently bound to poly[Lys-(Ac-Glu0.96-DL-Ala3.1)] (AcEAK)-a branched polypeptide having a polylysine backbone--resulting in a MI-1544-AcEAK conjugate. According to our in vitro experiments the MI-1544 induced a 33%-35% decrease in cell numbers of MCF-7 and MDA-MB-231 human breast cancer cell lines at a dose of 30 microM. The biodegradable polymeric carrier, AcEAK, alone inhibited cell proliferation by only 13%-15%, while the MI-1544-AcEAK conjugate, applied at the same dose, was capable of producing 45%-50% inhibition of cell proliferation. Our in vivo experiments using immunosuppressed mice showed that MI-1544, applied twice daily s.c., inhibited the growth of oestrogensensitive and -insensitive xenografts by 65% and 30% respectively. This effect was potentiated (70%) in both types of xenografts by the presence of the polymeric carrier in the conjugate; however, the carrier by itself did not cause tumour growth inhibition. The polymeric polypeptide carrier is supposed to increase the stability of the GnRH antagonist and to prevent the rapid excretion of the covalently bound peptide molecule. The antagonist and its conjugate may have various direct and indirect effects on breast cancer cells and, as a consequence, the new GnRH antagonist conjugates are suitable for treating an extended range of breast cancers.

Synthesis and functional studies of tuftsin analogs containing isopeptide bond.

In the present paper a new approach is reported, to increase the resistance of tuftsin toward enzymatic cleavage by the introduction of an isopeptide bond into the molecule. The tetrapeptides H-Lys(Thr)-Pro-Arg-OH and H-Lys(Ala)-Pro-Arg-OH, the pentapeptides H-Thr-Lys(Ala)-Pro-Arg-OH, H-Thr-Lys(Thr)-Pro-Arg-OH and H-Ala-Lys(Ala)-Pro-Arg-OH and their For- and Boc-protected derivatives were built up by stepwise elongation of the chain, using conventional solution-phase methods. Preliminary experiments confirmed that from the Lys residue in position 2 of tuftsin the alpha-peptide bond between the Thr and Lys is cleaved with a significantly higher rate by leucine aminopeptidase than the epsilon-peptide bond. Several of the isopeptide derivatives increased to a higher extent the interleukin (IL-1) secretion by monocytes than tuftsin or [Ala1]-tuftsin.

Synthesis and conformational studies of poly(L-lysine) based branched polypeptides with Ser and Glu/Leu in the side chains.

In a new group of polypeptides, the branches were composed of DL-Ala oligopeptide, L-serine and L-leucine or L-glutamic acid residues. The synthesis of eight different side-chain combinations is described. In the first group, Ser was attached directly to the epsilon-amino groups of polylysine, and Leu or Glu was situated at the side chain end (poly[Lys(X(i)-DL-Ala(m)-Ser(j))]). Alternatively, Leu or Glu was positioned next to the polylysine backbone (poly[Lys(Ser(j)-DL-Ala(m)-X(i))], where X=L-Leu or L-Glu and m approximately 3-6, i

Peripheral versus central potencies of N-type voltage-sensitive calcium channel blockers.

The ability of a series of synthetic analogues of omega-conopeptides MVIIA (SNX-111) and TVIA (SNX-185) to prevent electrically-evoked norepinephrine release from rat tail artery and hippocampal slice preparations was determined in an effort to identify voltage-sensitive calcium channel (VSCC) blockers that selectively target N-type VSCCs in central nervous system tissue. Electrical field stimulation (3 Hz, 1 ms in duration. 80 V for 1 min) caused a high and consistent tritium outflow from rat tail artery and hippocampal slice preparations preloaded with [3H]-norepinephrine. All conopeptides, chosen for their selective affinities for high-affinity SNX-111 binding sites (i.e., N-type VSCCs) over high-affinity omega-conopeptides MVIIC (SNX-230) binding sites (i.e., P/Q-type VSCCs), produced a concentration-dependent inhibition of calcium dependent electrically-evoked tritium outflow from both tail arteries and hippocampal slices: IC50s ranged from 1.2 nM to 1.2 microM. Blocking potencies (IC50s) in the tail artery assay were significantly correlated with those measured in the hippocampal slice preparation (r = 0.91, P = 0.00000012). There was a significant correlation between IC50s for blockade of hippocampal norepinephrine release and the inhibition of high-affinity [125I]-SNX-I11 binding in rat brain synaptosomes (r = 0.76, P = 0.00028). Blockade of hippocampal norepinephrine release was not significantly correlated with the inhibition of high-affinity SNX-230 binding (r = 0.46, P = 0.056). Maximum inhibition of tritium outflow in the tail artery assay was 22+/-1.4% of control, approximating the value (20.9+/-16.0% of control) obtained in the absence of extracellular Ca2+. In contrast, the maximum inhibition of tritium release from hippocampal slices was 36.8+/-2.5% of control (P < 0.05, compared to that of the tail artery assay). These results suggest that (1) N-type VSCCs alone mediate low frequency electrical stimulation-evoked neurotransmitter release from peripheral sympathetic efferents (tail artery) while both N-type and non-N type(s) mediate neurotransmitter release from CNS neurons (hippocampus); and (2) analogues of omega-conopeptides MVIIA and TVIA do not differentiate between N-type VSCCs mediating norepinephrine release from central and peripheral neural tissues.

Conformational study of linear and cyclic peptides corresponding to the 276-284 epitope region of HSV gD-1.

The results of conformational analysis of linear and cyclic peptides from the 276SALLEDPVG(284) sequence of glycoprotein D of Herpes simplex virus are presented. The epitope peptides were synthesized by SPPS and on resin cyclization was applied for preparation of cyclic compounds. Circular dichroism spectroscopy, Fourier-transform infrared spectroscopy and nuclear magnetic resonance (NMR) were used to determine of the solution structure of both linear and cyclic peptides. The results indicated that the cyclopeptides containing the core of the epitope (DPVG) as a part of the cycle have more stable beta-turn structure than the linear peptides or the cyclic analogues, where the core motif is not a part of the cycle. NMR study of H-SALLc(EDPVGK)-NH(2) confirm presence of a type I beta-turn structure which includes the DPVG epitope core.

Gamma scintigraphy of 111In-labelled branched chain polypeptides (BCP) with a poly(L-lysine) backbone in mice with mammary carcinoma: effect of charge on biodistribution and tumour imaging potential.

Radiolabelled synthetic branched chain polypeptides (BCP) represent a new and novel range of materials with potential as radiopharmaceuticals. Preliminary imaging studies have been undertaken with 111In-labelled BCP in mice with subcutaneously transplanted mammary carcinoma. Four polypeptides each with a poly(L-lysine) backbone and side chains of DL-alanine residues were studied. These were AK, which is polycationic, EAK which is amphoteric, having additional glutamic acid residues at the end of the side chains, and AcEAK (anionic) and SucEAK (highly polyanionic) where the terminal glutamic acid amino groups were acetylated or succinylated respectively. Radiolabelling was achieved by previous conjugation with DTPA. Serial images up to 48 hours showed marked retention of 111In-labelled polycationic AK and polyanionic SucEAK in the liver and spleen, with renal uptake also being visible in the case of AK. 111In-labelled EAK and AcEAK showed longer blood survival with some liver uptake, but tumour uptake was also visualized by 24 hours with both of these polypeptides. These studies demonstrate the feasibility of using 111In-labelled synthetic branched chain polypeptides as radiopharmaceuticals for gamma scintigraphy and the visualization of tumours by modification of the side chain structure. These materials warrant further study.

Comparison of two continuous fungal bioreactors for posttreatment of anaerobically pretreated weak black liquor from kraft pulp mills.

The purpose of this work was to evaluate and compare two continuous systems of posttreatment of anaerobically pretreated weak black liquor (WBL). The first system consisted of a packed bed reactor (PBR) with Trametes versicolor (Tv) immobilized on wood cubes of holm oak (biocubes). The second system was a fluidized bed reactor (FBR) with Lentinus edodes (Le) immobilized on wood cubes of holm oak. The reactors operated for 65 days at a hydraulic retention time (HRT) of 5 days, at 28 degrees C, with continuous aeration. Response variables monitored were conventional and specific, unit, net removal efficiency (eta and eta(sun), respectively) of chemical oxygen demand (COD), color, and ligninoids, and enzymatic activities of manganese peroxidase (MnP), lignin peroxidase (LiP), laccase (Lac) and proteases. The PBR showed an average color eta superior to that of the FBR (52.42 +/- 21.78% and 25.34 +/- 14.38% for PBR and FBR, respectively); removals of COD and ligninoids presented a similar pattern to that of color. Lac activity was significantly larger in PBR than in FBR. Activity of MnP in PBR was higher than that of the FBR (0.004 and 0.002 U MnP/mL, respectively). This difference could be ascribed to the different fungi present in each bioreactor. LiP activity was very low in both reactors. Average value of proteases was almost double in the FBR as compared with PBR (0.472 and 0.209 U Proteases/mL, respectively). During the last 2 weeks of operation, biocubes in the FBR experienced a significant loss of the attached Le biomass, probably by attrition. This and higher protease activity in the FBR could explain the lower pollutant removals achieved in the FBR. Overall, PBR with immobilized Tv showed a better performance than the FBR with Le for the posttreatment of the recalcitrant anaerobic effluent. Extended and sustained pollutant removal (65 days) was achieved in the PBR, although more research is needed to evaluate bioreactor performance at shorter hydraulic retention times.

Effect of tuftsin and oligotuftsins on chemotaxis and chemotactic selection in Tetrahymena pyriformis.

The chemotactic properties of tuftsin (H-TKPR-OH), tuftsin derivatives (H-KPR-OH, H-TKPKG-NH(2), Ac-TKPKG-NH(2)) and TKPKG-based oligotuftsins (T20, T30, T40) were investigated in Tetrahymena pyriformis GL. In contrast to its effects on Mammalia, tuftsin elicited chemorepellent or neutral responses; truncation of the N-terminal part (KPR) led to similar results, though with more neutral effects. The significance of the C-terminal part of the molecule was revealed by the chemoattractant properties of TKPKG, which are nevertheless abolished by acylation. Among the oligotuftsins, T20 and T40 were chemoattractants at higher concentrations (10(-9)-10(-6) M), while T30 had a wide-ranging chemorepellent effect, indicating that chemotaxis is elicited in Tetrahymena only by ligands with optimal physicochemical characters (mass, conformation, etc.). The chemotactic selection data indicated that tuftsin-induced chemotaxis results from fairly short-term signalling in Tetrahymena.

Efficacy of the phosphorylation of synthetic peptides by purified catalytic subunit of PKA (PKAcat) from bovine lens depends on the amino acid sequence of the peptides.

Protein kinase (PK) A catalytic (PKAcat) subunit was purified to homogeneity from bovine lens using a 100-kDa cut-off membrane filtration followed by different chromatographic procedures. The molecular weight of PKAcat was found to be 41 kDa. The kinase phosphorylates histone IIIs and other synthetic modified peptides of VRKRTLRRL with different amino acid environment. The extent of phosphorylation depends not only on the presence of Ser or Thr (phosphorylating residues) but also on other surrounding amino acid residues. Although some peptides compete in phosphorylating histone, they are not very significant. The result suggests that the extent of phosphorylation depends on the amino acid residue(s) surrounding phosphorylable residue(s) on the peptide.

Application of Marfey's reagent in racemization studies of amino acids and peptides.

Reversed-phase high-performance liquid chromatography and pre-column derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey's reagent) were used for monitoring racemization in peptides, amino acids and their derivatives by separation of optical isomers of amino acids. The technique was applied to the analysis of biologically active peptides, amino acids, their N- and C-protected derivatives, branched polypeptides based on polylysine, and endothiopeptides, and to the detection of stereochemical consequences of side reactions and hydrolysis. The chromatographic samples were mixtures of L- and D-amino acids obtained by hydrolysis of different peptides and derivatives. Baseline separations could be achieved on an ODS-Hypersil column with methanol-acetonitrile-acetate buffer (pH 4) mixtures as the eluents. The rates of racemization were calculated.

Carrier design: conformational studies of amino acid (X) and oligopeptide (X-DL-Alam) substituted poly (L-lysine).

The present study was undertaken to examine the influence of the reversal of the side-chain sequential order on the conformation of branched polypeptides. At the same time, the influence of the optically active amino acid joined directly to the poly (L-Lys) backbone and the DL-Ala oligomer grafted as chain-terminating fragment were separately analyzed. Therefore two sets of polypeptides were synthesized corresponding to the general formula poly[Lys-(Xi)] (XK) and poly[Lys-(DL-Alam-Xi)] (AXK) when X = Ala, D-Ala, Leu, D-Leu, Phe, D-Phe, Ile, Pro, Glu, D-Glu, or His. For coupling amino acid X to polylysine, three types of active ester methods were compared: the use of pentafluorophenyl or pentachlorophenyl ester, and the effect of the addition of an equimolar amount of 1-hydroxy-benzotriazole. After cleavage of protecting groups, AXK polypeptides were synthesized by grafting short oligo(DL-Ala) chains to XK by using N-carboxy-DL-Ala anhydride. The CD measurements performed in water solutions of various pH values and ionic strengths were used for classification of the polypeptide conformations as either ordered (helical) or unordered. Different from what was observed with the unsubstituted poly (L-Lys), poly[Lys-(Xi)] type polypeptides can adopt ordered structure even under nearly physiological conditions (pH 7.3, 0.2M NaCl). These data suggest that the introduction of amino acid residue with either (ar) alkyl side chain (Ala, Leu, Phe) or negatively charged side chain (Glu) promotes markedly the formation of ordered structure. Comparison of chiroptical properties of poly[Lys-(DL-Alam-Xi)] and of poly[Lys-(Xi)] reveals that side-chain interactions play an important role in the stabilization of ordered solution conformation of AXK type branched polypeptides. The results give rather conclusive evidence that not only hydrophobic interactions, but also ionic attraction, can be involved in the formation and stabilization of helical conformation of branched polypeptides.

Conjugation of epitope peptides with SH group to branched chain polymeric polypeptides via Cys(Npys).

Since bioconjugates may play an important role as therapeutics in the future, the development of new and effective conjugation strategies is necessary. For the attachment of peptide-like molecules to carriers, there are two main coupling methods involving amide or disulfide bonds. Conjugation through an amide bond can be achieved in several well-defined ways known from peptide chemistry. However, the formation of disulfide bridges between cysteine-containing peptides and carrier molecules still has some problems. In this paper, we describe a novel approach in which the carrier polypeptide is modified by 3-nitro-2-pyridinesulfenyl (Npys)-protected cysteine and this derivative has been applied for conjugation of Cys-containing epitope peptides with poly(L-lysine)-based branched polypeptides. Considering the stability of Npys group in the presence of pentafluorophenol, Boc-Cys(Npys)-OPfp dervivative was selected for introduction to the N-terminal of branches of polypeptides backbone. The branches of the polymers were built up from oligo(DL-alanine) (poly[Lys(DL-Ala(m))], AK) and elongated by an optically active amino acid [poly[Lys(X(i)-DL-Ala(m))], XAK]. We found that the nature of X (Glu, Ser, Thr) has great influence on the incorporation of the protected cysteine residue. Herpes simplex virus and adenovirus epitope peptides were conjugated to Boc-Cys(Npys)-modified polypeptides. Results indicate that the incorporation of epitope peptides depends on the number of Npys group on the polymers as well as on the presence/absence of Boc-protecting group on the Cys residue. This new class of Cys(Npys)-derivatized branched polypeptides is stable for a couple of months and suitable for effective preparation of epitope peptide conjugates possessing increased water solubility.

Stability of Asp-Pro bond under high and low energy collision induced dissociation conditions in the immunodominant epitope region of herpes simplex virion glycoprotein D.

A series of truncated Herpes simplex virion peptides studied by fast atom bombardment mass spectrometry under high and low energy collision induced dissociation conditions showed preferential fragmentation of the aspartyl-proline amide bond, compared to other peptide bonds. Electrospray ionization investigation proved that this favoured fragmentation can not be attributed to only the known proline effect, as a change from Asp to Asn in the peptide yielded an Asn-Pro bond which was found to be stable under the same ionization conditions. This mass spectrometric behaviour is in good agreement with the observation that DP bonds are sensitive to acidic conditions.

Biodegradability of synthetic branched polypeptide with poly(L-lysine) backbone.

A detailed investigation is reported about the biodegradation of poly[Lys(DL-Alam)], m approximately 3, (AK) the common inside area of a branched polypeptide model system developed by our group over the last decade. Enzymatic hydrolysis was carried out by the exopeptidase aminopeptidase M, or the endopeptidase trypsin, or their mixture. Ion-exchange column chromatography, paper electrophoresis and thin-layer chromatography were utilised to achieve separation of metabolites. Breakdown products were identified by the aid of synthetic oligopeptides representing the potential fragments (DL-Ala2, DL-Ala3, Lys(DL-Alam), m = 1-3). The kinetics and the degree of enzymatic degradation were determined. The ratio of peptide/amino acid amounts in the hydrolysate was found to be 1.07 after 24 h treatment with aminopeptidase M, 3.0 with trypsin and 1.3 with aminopeptidase - trypsin mixture. The overall results indicated that the proteolysis of AK by an aminopeptidase M and trypsin mixture proceeds stepwise at multiple sites on the polypeptide chain. The degradation is significantly retarded as compared to that of alpha- or epsilon-polylysine. A mechanism of degradation is suggested based on the experimental results.

The influence of the side-chain sequence on the structure-activity correlations of immunomodulatory branched polypeptides. Synthesis and conformational analysis of new model polypeptides.

New branched polypeptides were synthesized for a detailed study of the influence of the side-chain structure on the conformation and biological properties. The first subset of polypeptides were prepared by coupling of tetrapeptides to poly[L-Lys]. These polymers contain either DL-Ala3-X [poly[Lys-(X-DL-Ala3)n]] or X-DL-Ala3 [poly[Lys-(DL-Ala3-X)n] (n less than or equal to 1)] tetrapeptide side chains. Another group of branched polymers comprise a mixture of DL-Alam and of DL-Alam-X oligomeric branches in a random distribution [poly[Lys-(DL-Alam-Xi)] (i less than 1, m approximately 3)]. In each subset the X = Leu or Phe derivatives were prepared. The N-protected tetrapeptides were synthesized by conventional liquid phase methods and were coupled as active esters. The degree of racemization was found relatively high both for active esters and coupled derivatives, when optically active amino acids were in the C-terminal position of the tetrapeptides. In the case of the poly[Lys-(Leu-DL-Ala3)n] derivative, comparative experiments were carried out using various methodical alterations. The highest stereochemical homogeniety could be achieved when the tetrapeptide active ester was synthesized by the "backing off" method. CD spectra of poly[Lys-(Xi-DL-Alam)] (i less than 1, m approximately 3) and of poly[Lys-(X-DL-Ala3)n] were analyzed and compared to those of poly[Lys-(DL-Alam-Xi)] and of poly[Lys-(DL-Ala3-X)n]. All measurements were performed in water solutions of varying pH values and ionic strengths. The data obtained suggest that branched polypeptides containing a mixture of two different types of oligomeric side chains (DL-Alam and DL-Alam-Xi or Xi-DL-Alam) distributed randomly adopt an almost identical conformation to those that comprise only the respective tetrapeptide (DL-Ala3-X or X-DL-Ala3) branches. The results also indicate that the tendency to form an ordered structure is determined by the identity and the position of the chiral amino acid X (Phe or Leu) in the side chain.

Synthesis and immunological studies of alpha-conotoxin chimera containing an immunodominant epitope from the 268-284 region of HSV gD protein.

We have synthesized and characterized new chimeric peptides by inserting an epitope of the glycoprotein D (gD) of herpes simplex virus (HSV) serotype 1 as 'guest' sequence in the 'host' structure of alpha-conotoxin GI, a 13-residue peptide (ECCNPACGRHYSC) isolated from the venom of Conus geographus. The 276-284 region of HSV gD-1 selected for these studies is highly hydrophilic and adopts a beta-turn. The alpha-conotoxin GI also contains a beta-turn in the 8-12 region, stabilized by two disulfide bridges at positions 2-7 and 3-13. Thus, the tetramer sequence of alpha-conotoxin, 8Arg-His-Tyr-Ser12 has been replaced by Asp-Pro-Val-Gly (DPVG), identified previously as the epitope core. The syntheses were performed by Fmoc strategy on Rink resin and DTNB or air oxidation were applied for the formation of the first 3-13 disulfide bond in the presence of guanidinium hydrochloride. For the formation of the second disulfide Cys2-Cys7 three different oxidation procedures [iodine in 95% acetic acid, air oxidation in dimethyl sulfoxide/1 M HCl or Tl(tfa)3 in trifluoroacetic acid (TFE)] were compared. The high-performance liquid chromatography purified peptides were characterized by electrospray mass spectrometry and amino acid analysis. The bicyclic HSV-alpha-[Tyr1]-conotoxin chimeric peptide and native alpha-conotoxin GI showed similar circular dichroism spectra in phosphate-buffered saline (PBS) and in a PBS-TFE 1:1 (v/v) mixture, which might suggest that these compounds also share similar secondary structures. In immunologic studies the characteristics of the primary and of the memory immunoglobulin (Ig) M- and IgG-type antibody responses showed that the bicyclic HSV-alpha-[Tyr1]-conotoxin chimera is capable to induce strong antibody responses in C57/Bl/6 mice but was poorly immunogenic in CBA and BALB/c mice. Data obtained with the C57/Bl/6 serum indicate that the polyclonal antibodies recognize the DPVG motif presented in the bicyclic HSV-alpha-[Tyr1]-conotoxin and some reactivity was also found with the monocyclic but not with the linear form of the chimera. Results with two IgM type monoclonal antibodies from a bicyclic HSV-alpha-[Tyr1]-conotoxin immunized C57/Bl/6 mouse also point to the specific interaction with the DPVG sequence. Taken together these studies suggest, that the relative intensity of DPVG-specific responses was found to be dependent on the mouse strain and on the conformation of the chimeric molecules. We found that the IgM monoclonal antibodies are able to recognize the linear DPVG sequence, while the majority of IgG antibodies is directed to the same motif in a conformation stabilized by double cyclization.