In this study we report the in vitro activities of four cyclic opioid peptides with various seque... more In this study we report the in vitro activities of four cyclic opioid peptides with various sequence length/macrocycle size and N-methylamino acid residue content. N-Methylated amino acids were incorporated and cyclization was employed to enhance conformational rigidity to various extent. The effect of such modifications on ligand structure and binding properties were studied. The pentapeptide containing one endocyclic and one exocyclic N-methylated amino acid displayed the highest affinity to the mu-opioid receptor. This peptide was also shown to be a full agonist, while the other analogs failed to activate the mu opioid receptor. Results of molecular docking studies provided rationale for the explanation of binding properties on a structural basis.
Journal of The American Society for Mass Spectrometry, 2012
Derivatization of peptides as quaternary ammonium salts (QAS) is a known method for sensitive det... more Derivatization of peptides as quaternary ammonium salts (QAS) is a known method for sensitive detection by electrospray ionization tandem mass spectrometry. Hydrogens at α-carbon atom in N,N,N-trialkylglycine residue can be easily exchanged by deuterons. The exchange reaction is base-catalyzed and is dramatically slow at lower pH. Introduced deuterons are stable in acidic aqueous solution and are not back-exchanged during LC-MS analysis. Increased ionization efficiency, provided by the fixed positive charge on QAS group, as well as the deuterium labeling, enables the analysis of trace amounts of peptides.
Results Dimeric peptides were synthesized using standard Fmoc solid-phase strategy (Fig. 1). The ... more Results Dimeric peptides were synthesized using standard Fmoc solid-phase strategy (Fig. 1). The crosslinking of N-terminal amino groups was achieved on solid support through the reaction with pentafluorophenyl esters of poly (ethyleneglycol) biscarboxylic acid PEG ( ...
We investigated the formation of imidazole, benzimidazole and quinoxaline moieties through conden... more We investigated the formation of imidazole, benzimidazole and quinoxaline moieties through condensation with various aldehydes and α-dicarbonyl compounds. The imidazole synthesis utilizes the N-terminal or side chain amino group of amino acids, whereas a derivate of ...
Argireline, a peptide with the sequence: Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2, also known as Acetyl Hex... more Argireline, a peptide with the sequence: Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2, also known as Acetyl Hexapeptide-8, reduces facial lines and wrinkles by destabilization of the formation of the SNARE complex (SNAP Receptor, soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor), thus preventing muscle contraction. It is a biosafe cosmetic alternative to the botulinum toxin. The method of choice in bioactive peptide analysis is reversed-phase high performance liquid chromatography coupled with mass spectrometry (LC-MS). The aim of this work as to present the properties of Argireline and the analysis of cosmetic products containing this peptide. Previous reports on possible Argireline transformations in cosmetic formulations have not confirmed deacetylation, whereas the oxidation of methionine residue was detected by our team. As the biological activity of the oxidized Argireline is not known, further biological studies, as well as efficient analytical procedures for tra...
As part of our continuing studies on the structure-activity relationships of cyclic pentapeptides... more As part of our continuing studies on the structure-activity relationships of cyclic pentapeptides based on the structure of endomorphin-2 (EM-2), we report here the synthesis and biological activities of a new series of analogues of a general sequence Tyr/Dmt-c[d-Lys-Phe-Phe-Asp]NH2 (where Dmt = 2',6'-dimethyltyrosine), incorporating fluorinated amino acids: 4-fluorophenylalanine (4-F-Phe), 2,4-difluorophenylalanine (2,4-F-Phe), or 4-trifluoromethylphenylalanine (4-CF3-Phe) instead of the Phe residue in position 3 or 4. Depending on the fluorinated amino acid residue and its position in the sequence, analogues were mixed, high affinity MOP/KOP receptor agonists, MOP/DOP/KOP agonists, or selective KOP agonists. The in vitro potencies and efficacies of all novel analogues were assessed in calcium mobilization assay. The most potent analogues, Dmt-c[d-Lys-Phe-4-F-Phe-Asp]NH2 and Dmt-c[d-Lys-Phe-2,4-F-Phe-Asp]NH2, were tested in vivo in the mouse hot-plate test. They produced strong antinociceptive effect not only after intracerebroventricular but also after intraperitoneal injection, indicating that they were able to cross the blood-brain barrier.
A new and straightforward solid-phase synthesis of a series of site-specific Amadori-modified pep... more A new and straightforward solid-phase synthesis of a series of site-specific Amadori-modified peptides is described. The method involves reductive alkylation of the e-amino groups of lysine with 2,3:4,5-di-O-isopropylidene-b-D D-arabino-hexos-2-ulo-2,6pyranose in the presence of sodium cyanoborohydride on a solid support.
After a short description of the results of Victor Najjar's research on tuftsin and of the discov... more After a short description of the results of Victor Najjar's research on tuftsin and of the discoveries done by other authors in the early stage of tuftsin investigation, the current state of work on tuftsin is presented, based mainly on the literature published in the years 1984 -1997. The presentation follows this order: the occurrence of tuftsin and retro-tuftsin sequences in proteins, their synthesis and biology, the antigenic properties of tuftsin, its influence on phagocytic cells, and other biologic activities of tuftsin, including antimicrobial, antiviral, antitumor and central effects, and the search for tuftsin superactive analogs.
As part of our continuing studies on the structure-activity relationships of cyclic pentapeptides... more As part of our continuing studies on the structure-activity relationships of cyclic pentapeptides based on the structure of endomorphin-2, we report here the synthesis and biological activities of a new series of analogs incorporating 2', 3' or 4'-methylphenylalanine (MePhe) residues into positions 3 or 4 of the parent cyclopeptide, Dmt-c[d-Lys-Phe-Phe-Asp]NH2 (Dmt=2',6'-dimethyltyrosine). Analogs with MePhe in position 4 showed a row of magnitude increased μ-opioid receptor (MOP receptor) affinity as compared with a parent compound. The in vitro potencies of the new analogs were determined in calcium mobilization assay performed in Chinese Hamster Ovary (CHO) cells expressing human recombinant opioid receptors and chimeric G proteins. All analogs were strong μ/κ (MOP/KOP) receptor agonists and weak δ (DOP) receptor agonists. In the in vivo hot-plate test in mice, the MePhe(4)-modified peptides showed remarkable antinociceptive activity after intracerebroventricular (i.c.v.) administration which was most likely due to the concomitant activation of more than one opioid receptor type.
The cyclization of linear analogs based on endomorphin-2 structure, Tyr/Dmt-d-Lys-Phe-Phe-Asp-NH2... more The cyclization of linear analogs based on endomorphin-2 structure, Tyr/Dmt-d-Lys-Phe-Phe-Asp-NH2 and Tyr/Dmt-d-Cys-Phe-Phe-Cys-NH2 (where Dmt=2',6'-dimethyltyrosine), resulting in obtaining lactam or disulfide derivatives, was studied using liquid chromatography combined with on-line mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS). In case of cyclization via an amide bond, the formation of the cyclic monomers, cyclic but not linear dimers and even traces of cyclic trimers was observed. Disulfide bridge containing peptides was obtained by the solid-phase synthesis of the linear sequences, followed by either in-solution or on-resin cyclization. In case of the in-solution cyclization, the expected cyclic monomers were the only products. When oxidation of the cysteine residues was performed when the peptides were still on the resin, cyclic monomer and two cyclodimers, parallel and antiparallel, were found. Digestion of the isolated cyclodimers with α-chymotrypsin allowed for their unambiguous identification. The comparison of the cyclic monomer/dimer ratios for analogs with Tyr versus Dmt in position 1 revealed that the presence of the exocyclic Dmt favored formation of the cyclic monomer, most likely due to the increased steric bulk of this amino acid side-chain as compared with Tyr.
Journal of The American Society for Mass Spectrometry, 2013
The fragmentation of peptides containing quaternary ammonium group, but lacking easily mobilizabl... more The fragmentation of peptides containing quaternary ammonium group, but lacking easily mobilizable protons, was examined with the aid of deuteriumlabeled analogs and quantum-chemical modeling. The fragmentation of oligoproline containing quaternary ammonium group involves the mobilization of hydrogens localized at αand γor δ-carbon atoms in the pyrrolidine ring of proline. The study of the dissociation pattern highlights the unusual proline residue behavior during MS/MS experiments of peptides.
Microwave-assisted (MW) reactions are of special interest to the chemical community due to faster... more Microwave-assisted (MW) reactions are of special interest to the chemical community due to faster reaction times, cleaner reactions and higher product yields. The adaptation of MW to solid phase peptide synthesis resulted in spectacular syntheses of difficult peptides. In the case of Merrifield support, used frequently in synthesis of special peptides, the conditions used in product cleavage are not compatible with off-resin monitoring of the reaction progress. The application of MW irradiation in product removal from Merrifield resin using trifluoroacetic acid (TFA) was investigated using model tetrapeptides and the effects were compared with standard trifluoromethanesulphonic acid (TFMSA) cleavage using elemental analysis as well as chromatographic (HPLC) and spectroscopic (IR) methods. The deprotection of benzyloxycarbonyl and benzyl groups in synthetic bioactive peptides was analyzed using LC-MS and MS/MS experiments. In a 5 min microwave-assisted TFA reaction at low temperature, the majority of product is released from the resin, making the analytical scale MW-assisted procedure a method of choice in monitoring the reactions carried out on Merrifield resin due to the short reaction time and compatibility with HPLC and ESI-MS conditions.
The review presents three hypotheses concerning the amino acid complementarity: 1) the Mekler-Bla... more The review presents three hypotheses concerning the amino acid complementarity: 1) the Mekler-Blalock antisense hypothesis; 2) the Root-Bernstein approach based on stereochemical complementarity of amino acids and anti-amino acids coded by anticodons read in parallel with the coding DNA strand; 3) Siemion hypothesis resulting from the periodicity of the genetic code. The current state of knowledge as well as the results of the implementations of these hypotheses are compared. A special attention is given to Root-Bernstein and Siemion hypotheses, which differ in only few points of the complementarity prediction. We describe methods of investigation of peptide-antipeptide pairing, including circular dichroism, mass spectrometry, affinity chromatography and other techniques. The biological applications of complementarity principle are considered, such as search for bioeffector-bioreceptor interaction systems, the influence of peptide-antipeptide pairing on the activity of peptide hormones, and the application of antipeptides in immunochemistry. The possible role of amino acid-anti-amino acid interactions in the formation of the spatial structures of peptides, proteins and protein complexes is discussed. Such problems as the pairing preferences of protein-protein interfaces, the role of the pairing in the creation of disulfide bonds and the possible appearance of such interactions in beta-structure are also examined. The main intention of the paper is to bring the complementarity problem to the attention of the scientific community, as a possible tool in proteomics, molecular design and molecular recognition.
The central nervous system (CNS) and the immune system were for many years considered as two auto... more The central nervous system (CNS) and the immune system were for many years considered as two autonomous systems. Now, the reciprocal connections between them are generally recognized and very well documented. The links are realized mainly by various immuno- and neuropeptides. In the review the influence of the following immunopeptides on CNS is presented: tuftsin, thymulin, thymopoietin and thymopentin, thymosins, and thymic humoral factor. On the other side, the activity in the immune system of such neuropeptides as substance P, neurotensin, some neurokinins, enkephalins, and endorphins is discussed.
In this study we report the in vitro activities of four cyclic opioid peptides with various seque... more In this study we report the in vitro activities of four cyclic opioid peptides with various sequence length/macrocycle size and N-methylamino acid residue content. N-Methylated amino acids were incorporated and cyclization was employed to enhance conformational rigidity to various extent. The effect of such modifications on ligand structure and binding properties were studied. The pentapeptide containing one endocyclic and one exocyclic N-methylated amino acid displayed the highest affinity to the mu-opioid receptor. This peptide was also shown to be a full agonist, while the other analogs failed to activate the mu opioid receptor. Results of molecular docking studies provided rationale for the explanation of binding properties on a structural basis.
Journal of The American Society for Mass Spectrometry, 2012
Derivatization of peptides as quaternary ammonium salts (QAS) is a known method for sensitive det... more Derivatization of peptides as quaternary ammonium salts (QAS) is a known method for sensitive detection by electrospray ionization tandem mass spectrometry. Hydrogens at α-carbon atom in N,N,N-trialkylglycine residue can be easily exchanged by deuterons. The exchange reaction is base-catalyzed and is dramatically slow at lower pH. Introduced deuterons are stable in acidic aqueous solution and are not back-exchanged during LC-MS analysis. Increased ionization efficiency, provided by the fixed positive charge on QAS group, as well as the deuterium labeling, enables the analysis of trace amounts of peptides.
Results Dimeric peptides were synthesized using standard Fmoc solid-phase strategy (Fig. 1). The ... more Results Dimeric peptides were synthesized using standard Fmoc solid-phase strategy (Fig. 1). The crosslinking of N-terminal amino groups was achieved on solid support through the reaction with pentafluorophenyl esters of poly (ethyleneglycol) biscarboxylic acid PEG ( ...
We investigated the formation of imidazole, benzimidazole and quinoxaline moieties through conden... more We investigated the formation of imidazole, benzimidazole and quinoxaline moieties through condensation with various aldehydes and α-dicarbonyl compounds. The imidazole synthesis utilizes the N-terminal or side chain amino group of amino acids, whereas a derivate of ...
Argireline, a peptide with the sequence: Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2, also known as Acetyl Hex... more Argireline, a peptide with the sequence: Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2, also known as Acetyl Hexapeptide-8, reduces facial lines and wrinkles by destabilization of the formation of the SNARE complex (SNAP Receptor, soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor), thus preventing muscle contraction. It is a biosafe cosmetic alternative to the botulinum toxin. The method of choice in bioactive peptide analysis is reversed-phase high performance liquid chromatography coupled with mass spectrometry (LC-MS). The aim of this work as to present the properties of Argireline and the analysis of cosmetic products containing this peptide. Previous reports on possible Argireline transformations in cosmetic formulations have not confirmed deacetylation, whereas the oxidation of methionine residue was detected by our team. As the biological activity of the oxidized Argireline is not known, further biological studies, as well as efficient analytical procedures for tra...
As part of our continuing studies on the structure-activity relationships of cyclic pentapeptides... more As part of our continuing studies on the structure-activity relationships of cyclic pentapeptides based on the structure of endomorphin-2 (EM-2), we report here the synthesis and biological activities of a new series of analogues of a general sequence Tyr/Dmt-c[d-Lys-Phe-Phe-Asp]NH2 (where Dmt = 2',6'-dimethyltyrosine), incorporating fluorinated amino acids: 4-fluorophenylalanine (4-F-Phe), 2,4-difluorophenylalanine (2,4-F-Phe), or 4-trifluoromethylphenylalanine (4-CF3-Phe) instead of the Phe residue in position 3 or 4. Depending on the fluorinated amino acid residue and its position in the sequence, analogues were mixed, high affinity MOP/KOP receptor agonists, MOP/DOP/KOP agonists, or selective KOP agonists. The in vitro potencies and efficacies of all novel analogues were assessed in calcium mobilization assay. The most potent analogues, Dmt-c[d-Lys-Phe-4-F-Phe-Asp]NH2 and Dmt-c[d-Lys-Phe-2,4-F-Phe-Asp]NH2, were tested in vivo in the mouse hot-plate test. They produced strong antinociceptive effect not only after intracerebroventricular but also after intraperitoneal injection, indicating that they were able to cross the blood-brain barrier.
A new and straightforward solid-phase synthesis of a series of site-specific Amadori-modified pep... more A new and straightforward solid-phase synthesis of a series of site-specific Amadori-modified peptides is described. The method involves reductive alkylation of the e-amino groups of lysine with 2,3:4,5-di-O-isopropylidene-b-D D-arabino-hexos-2-ulo-2,6pyranose in the presence of sodium cyanoborohydride on a solid support.
After a short description of the results of Victor Najjar's research on tuftsin and of the discov... more After a short description of the results of Victor Najjar's research on tuftsin and of the discoveries done by other authors in the early stage of tuftsin investigation, the current state of work on tuftsin is presented, based mainly on the literature published in the years 1984 -1997. The presentation follows this order: the occurrence of tuftsin and retro-tuftsin sequences in proteins, their synthesis and biology, the antigenic properties of tuftsin, its influence on phagocytic cells, and other biologic activities of tuftsin, including antimicrobial, antiviral, antitumor and central effects, and the search for tuftsin superactive analogs.
As part of our continuing studies on the structure-activity relationships of cyclic pentapeptides... more As part of our continuing studies on the structure-activity relationships of cyclic pentapeptides based on the structure of endomorphin-2, we report here the synthesis and biological activities of a new series of analogs incorporating 2', 3' or 4'-methylphenylalanine (MePhe) residues into positions 3 or 4 of the parent cyclopeptide, Dmt-c[d-Lys-Phe-Phe-Asp]NH2 (Dmt=2',6'-dimethyltyrosine). Analogs with MePhe in position 4 showed a row of magnitude increased μ-opioid receptor (MOP receptor) affinity as compared with a parent compound. The in vitro potencies of the new analogs were determined in calcium mobilization assay performed in Chinese Hamster Ovary (CHO) cells expressing human recombinant opioid receptors and chimeric G proteins. All analogs were strong μ/κ (MOP/KOP) receptor agonists and weak δ (DOP) receptor agonists. In the in vivo hot-plate test in mice, the MePhe(4)-modified peptides showed remarkable antinociceptive activity after intracerebroventricular (i.c.v.) administration which was most likely due to the concomitant activation of more than one opioid receptor type.
The cyclization of linear analogs based on endomorphin-2 structure, Tyr/Dmt-d-Lys-Phe-Phe-Asp-NH2... more The cyclization of linear analogs based on endomorphin-2 structure, Tyr/Dmt-d-Lys-Phe-Phe-Asp-NH2 and Tyr/Dmt-d-Cys-Phe-Phe-Cys-NH2 (where Dmt=2',6'-dimethyltyrosine), resulting in obtaining lactam or disulfide derivatives, was studied using liquid chromatography combined with on-line mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS). In case of cyclization via an amide bond, the formation of the cyclic monomers, cyclic but not linear dimers and even traces of cyclic trimers was observed. Disulfide bridge containing peptides was obtained by the solid-phase synthesis of the linear sequences, followed by either in-solution or on-resin cyclization. In case of the in-solution cyclization, the expected cyclic monomers were the only products. When oxidation of the cysteine residues was performed when the peptides were still on the resin, cyclic monomer and two cyclodimers, parallel and antiparallel, were found. Digestion of the isolated cyclodimers with α-chymotrypsin allowed for their unambiguous identification. The comparison of the cyclic monomer/dimer ratios for analogs with Tyr versus Dmt in position 1 revealed that the presence of the exocyclic Dmt favored formation of the cyclic monomer, most likely due to the increased steric bulk of this amino acid side-chain as compared with Tyr.
Journal of The American Society for Mass Spectrometry, 2013
The fragmentation of peptides containing quaternary ammonium group, but lacking easily mobilizabl... more The fragmentation of peptides containing quaternary ammonium group, but lacking easily mobilizable protons, was examined with the aid of deuteriumlabeled analogs and quantum-chemical modeling. The fragmentation of oligoproline containing quaternary ammonium group involves the mobilization of hydrogens localized at αand γor δ-carbon atoms in the pyrrolidine ring of proline. The study of the dissociation pattern highlights the unusual proline residue behavior during MS/MS experiments of peptides.
Microwave-assisted (MW) reactions are of special interest to the chemical community due to faster... more Microwave-assisted (MW) reactions are of special interest to the chemical community due to faster reaction times, cleaner reactions and higher product yields. The adaptation of MW to solid phase peptide synthesis resulted in spectacular syntheses of difficult peptides. In the case of Merrifield support, used frequently in synthesis of special peptides, the conditions used in product cleavage are not compatible with off-resin monitoring of the reaction progress. The application of MW irradiation in product removal from Merrifield resin using trifluoroacetic acid (TFA) was investigated using model tetrapeptides and the effects were compared with standard trifluoromethanesulphonic acid (TFMSA) cleavage using elemental analysis as well as chromatographic (HPLC) and spectroscopic (IR) methods. The deprotection of benzyloxycarbonyl and benzyl groups in synthetic bioactive peptides was analyzed using LC-MS and MS/MS experiments. In a 5 min microwave-assisted TFA reaction at low temperature, the majority of product is released from the resin, making the analytical scale MW-assisted procedure a method of choice in monitoring the reactions carried out on Merrifield resin due to the short reaction time and compatibility with HPLC and ESI-MS conditions.
The review presents three hypotheses concerning the amino acid complementarity: 1) the Mekler-Bla... more The review presents three hypotheses concerning the amino acid complementarity: 1) the Mekler-Blalock antisense hypothesis; 2) the Root-Bernstein approach based on stereochemical complementarity of amino acids and anti-amino acids coded by anticodons read in parallel with the coding DNA strand; 3) Siemion hypothesis resulting from the periodicity of the genetic code. The current state of knowledge as well as the results of the implementations of these hypotheses are compared. A special attention is given to Root-Bernstein and Siemion hypotheses, which differ in only few points of the complementarity prediction. We describe methods of investigation of peptide-antipeptide pairing, including circular dichroism, mass spectrometry, affinity chromatography and other techniques. The biological applications of complementarity principle are considered, such as search for bioeffector-bioreceptor interaction systems, the influence of peptide-antipeptide pairing on the activity of peptide hormones, and the application of antipeptides in immunochemistry. The possible role of amino acid-anti-amino acid interactions in the formation of the spatial structures of peptides, proteins and protein complexes is discussed. Such problems as the pairing preferences of protein-protein interfaces, the role of the pairing in the creation of disulfide bonds and the possible appearance of such interactions in beta-structure are also examined. The main intention of the paper is to bring the complementarity problem to the attention of the scientific community, as a possible tool in proteomics, molecular design and molecular recognition.
The central nervous system (CNS) and the immune system were for many years considered as two auto... more The central nervous system (CNS) and the immune system were for many years considered as two autonomous systems. Now, the reciprocal connections between them are generally recognized and very well documented. The links are realized mainly by various immuno- and neuropeptides. In the review the influence of the following immunopeptides on CNS is presented: tuftsin, thymulin, thymopoietin and thymopentin, thymosins, and thymic humoral factor. On the other side, the activity in the immune system of such neuropeptides as substance P, neurotensin, some neurokinins, enkephalins, and endorphins is discussed.
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Papers by Alicja Kluczyk