- Improved Fmoc Solid-Phase Peptide Synthesis of Oxytocin with High Bioactivity
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We described here the synthesis of oxytocin by an improved Fmoc solid-phase peptide synthesis (SPPS) method with a Rink-Amide resin as the solid support, HBTU as the coupling reagent, Fmoc-protected amino acids as the building blocks, and piperazine for Fmoc removal as a substitute for the standard reagent piperidine. Unlike previously reported syntheses, the removal of the S -Acm protecting group of Cys and cyclization forming the disulfide bond were carried out by using iodine on the resin with the fully protected peptide chains. Finally, a crude oxytocin with a purity of 92% was obtained by simultaneous cleavage of the peptide chains from the resin and removal of all side-chain protecting groups with trifluoroacetic acid containing the scavengers (yield 85%). The crude peptide was purified by using preparative RP-HPLC to obtain oxytocin (high purity 99.3%) with a bioactivity of 588 IU/mg, the highest reported so far in the literature. This investigation provides a contribution in efforts for the large-scale synthesis of oxytocin in high purity under mild conditions with iodine for on-resin disulfide bond formation and a substitute for the standard Fmoc-deprotecting reagent piperidine, a controlled substance.
- Sun, Pengcheng,Tang, Wenli,Huang, Yu,Hu, Bi-Huang
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Read Online
- Disulfide bond formation in S-Acetamidomethyl cysteine-containing peptides by the combination of silver trifluoromethanesulfonate and dimethylsulfoxide / aqueous HCI
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S-Acetamidomethyl (Acm) cysteine was found to be converted quantitatively to cystine by deprotection of the Acm group with silver trifluoromethanesulfonate (AgOTf) followed by dimethylsulfoxide (DMSO) / aqueous hydrochloric acid (HCl) treatment. No significant side reactions were observed with oxidation-sensitive amino acids such as Met, Tyr, and Trp under these reaction conditions. This method has been applied successfully to the syntheses of oxytocin and a Trp-containing peptide, urotensin II.
- Tamamura, Hirokazu,Otaka, Akira,Nakamura, Junko,Okubo, Kaori,Koide, Takaki,Ikeda, Kyoko,Fujii, Nobutaka
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- APPLICATION OF DIMETHYLSULFOXIDE(DMSO)/TRIFLUOROACETIC ACID(TFA) OXIDATION TO THE SYNTHESIS OF CYSTINE-CONTAINING PEPTIDE
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S-protected cysteine derivatives as well as cysteine were converted to cystine by action of DMSO/TFA; as examples, two model peptides, oxytocin and an α-human calcitonin gene-related peptide (α-hCGRP), were prepared by this reaction.
- Otaka, Akira,Koide, Takaki,Shide, Atsuko,Fujii, Nobutaka
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Read Online
- (S)-9-Fluorenylmethyl-L-cysteine, a Useful HF-stable Derivative for Peptide Synthesis
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The reliability of the (S)-9-fluorenylmethyl-L-cysteine, a completely HF-stable cysteine derivative, has been tested on a solid phase synthesis of oxytocin.
- Ruiz-Gayo, Mariano,Albericio, Fernando,Pedroso, Enrique,Giralt, Ernest
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Read Online
- “On-Resin” Disulfide Peptide Synthesis with Methyl 3-Nitro-2-pyridinesulfenate
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New methodologies for the construction of full peptide structures with all disulfide bonds on the resin are attractive for the development of solid phase peptide synthesis. Detailed reaction conditions for the on-resin disulfide bond formation have been investigated using a mild and chemically stable oxidizing reagent, methyl 3-nitro-2-pyridinesulfenate (Npys-OMe). Monocyclic oxytocin, MCH and bicyclic α-conotoxin ImI were synthesized in both semi-automated and full-automated protocols. It was found that on-resin intramolecular disulfide bond formation with Npys-OMe proceeds with the minimal formation of peptide oligomers by adopting a solvent system with 0.4 M LiCl/DMF. Crude peptides with complete disulfide bond patterns can be obtained in high purity using both protocols. This minimized the RP-HPLC purification step and the desired peptides were obtained with better yields. To our knowledge, this is the first fully automated construction of a bicyclic disulfide peptide on resin with more than 50 % purity in Fmoc-based SPPS. These results suggest that Npys-OMe is a useful reagent for the disulfide bond formation in automated protocols.
- Kobayashi, Kiyotaka,Taguchi, Akihiro,Cui, Yan,Shida, Hayate,Muguruma, Kyohei,Takayama, Kentaro,Taniguchi, Atsuhiko,Hayashi, Yoshio
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supporting information
p. 956 - 963
(2021/02/01)
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- Detection of Thiol Functionality and Disulfide Bond Formation by Polyoxometalate
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The detection of thiol functionality and intramolecular disulfide bond formation of peptides using the α-Keggin type polyoxometalate molybdenum-oxygen cluster (H3PMo12O40·nH2O) is described. Our method entails the addition of this polyoxometalate to solutions of thiol, whereupon the color of the solution changes from colorless to deep blue. Reduction of the polyoxometalate from Mo(VI) to Mo(V) occurs with concomitant oxidation of the thiol functionality, to form disulfide bonds. To exemplify the utility this phenomenon, we accomplished the oxidation of glutathione, reduced linear oxytocin, bactenecin, and α-conotoxin SI; all of which proceeded smoothly and in good conversion in 24 h to less and were accomplished by a change in the color of the reaction solutions.
- Konno, Hiroyuki,Yasumiishi, Haruto,Aoki, Reika,Nitanai, Ikumi,Yano, Shigekazu
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supporting information
p. 745 - 749
(2020/11/10)
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- Sustainable Peptide Synthesis Enabled by a Transient Protecting Group
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The growing interest in synthetic peptides has prompted the development of viable methods for their sustainable production. Currently, large amounts of toxic solvents are required for peptide assembly from protected building blocks, and switching to water as a reaction medium remains a major hurdle in peptide chemistry. We report an aqueous solid-phase peptide synthesis strategy that is based on a water-compatible 2,7-disulfo-9-fluorenylmethoxycarbonyl (Smoc) protecting group. This approach enables peptide assembly under aqueous conditions, real-time monitoring of building block coupling, and efficient postsynthetic purification. The procedure for the synthesis of all natural and several non-natural Smoc-protected amino acids is described, as well as the assembly of 22 peptide sequences and the fundamental issues of SPPS, including the protecting group strategy, coupling and cleavage efficiency, stability under aqueous conditions, and crucial side reactions.
- Avrutina, Olga,Knauer, Sascha,Koch, Niklas,Kolmar, Harald,Meusinger, Reinhard,Uth, Christina
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supporting information
p. 12984 - 12990
(2020/06/01)
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- Selective disulfidation reagent using nitrogen-containing compound and method for producing disulfide-containing compound
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The present invention provides a means capable of selectively introducing a disulfide bond with respect to two free thiol groups located in a molecule of an organic compound such as a peptide, or the like, in a short time by a simple treatment and also by a chemically stable method. A nitrogen-containing compound represented by Chemical Formula 1 below or a salt thereof: The symbols shown in Chemical Formula 1 are the same as defined in the specification.
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Page/Page column 26-28; 34-35; 39
(2020/12/02)
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- Palladium-Mediated Direct Disulfide Bond Formation in Proteins Containing S-Acetamidomethyl-cysteine under Aqueous Conditions
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One of the applied synthetic strategies for correct disulfide bond formation relies on the use of orthogonal Cys protecting groups. This approach requires purification before and after the deprotection steps, which prolongs the entire synthetic process and lowers the yield of the reaction. A major challenge in using this approach is to be able to apply one-pot synthesis under mild conditions and aqueous media. In this study, we report the development of an approach for rapid disulfide bond formation by employing palladium chemistry and S-acetamidomethyl-cysteine [Cys(Acm)]. Oxidation of Cys(Acm) to the corresponding disulfide bond is achieved within minutes in a one-pot operation by applying palladium and diethyldithiocarbamate. The utility of this reaction was demonstrated by the synthesis of the peptide oxytocin and the first total chemical synthesis of the protein thioredoxin-1. Our investigation revealed a critical role of the Acm protecting group in the disulfide bond formation, apparently due to the generation of a disulfiram in the reaction pathway, which significantly assists the oxidation step.
- Laps, Shay,Sun, Hao,Kamnesky, Guy,Brik, Ashraf
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supporting information
p. 5729 - 5733
(2019/04/14)
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- Rapid Photolysis-Mediated Folding of Disulfide-Rich Peptides
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Structure–activity relationship studies are a highly time-consuming aspect of peptide-based drug development, particularly in the assembly of disulfide-rich peptides, which often requires multiple synthetic steps and purifications. Therefore, it is vital to develop rapid and efficient chemical methods to readily access the desired peptides. We have developed a photolysis-mediated “one-pot” strategy for regioselective disulfide bond formation. The new pairing system utilises two ortho-nitroveratryl protected cysteines to generate two disulfide bridges in less than one hour in good yield. This strategy was applied to the synthesis of complex disulfide-rich peptides such as Rho-conotoxin ρ-TIA and native human insulin.
- Patil, Nitin A.,Karas, John A.,Wade, John D.,Hossain, Mohammed Akhter,Tailhades, Julien
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p. 8599 - 8603
(2019/06/04)
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- Building bridges for highly selective, potent and stable oxytocin and vasopressin analogs
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Oxytocin (OT) is an exciting potential therapeutic agent, but it is highly sensitive to modification and suffers extensive degradation at elevated temperature and in vivo. Here we report studies towards OT analogs with favorable selectivity, affinity and potency towards the oxytocin receptor (OTR), in addition to improving stability of the peptide by bridging the disulfide region with substituted dibromo-xylene analogs. We found a sensitive structure-activity relationship in which meta-cyclized analogs (dOTmeta) gave highest affinity (50 nM Ki), selectivity (34-fold), and agonist potency (34 nM EC50, 87-fold selectivity) towards OTR. Surprisingly, ortho-cyclized analogs demonstrated OTR and vasopressin V1a receptor subtype affinity (220 nM and 69 nM, respectively) and pharmacological activity (294 nM and 35 nM, respectively). V1a binding and selectivity for ortho-cyclized peptides could be improved 6-fold by substituting a neutral residue at position 8 with a basic amino acid, providing potent antagonists (14 nM IC50) that displayed no activation of the OTR. Furthermore, xylene-bridged analogs demonstrated increased stability compared to OT at elevated temperature, demonstrating promising therapeutic potential for these analogs which warrants further study.
- Beard, Rhiannon,Stucki, Andy,Schmitt, Muriel,Py, Gabrielle,Grundschober, Christophe,Gee, Antony D.,Tate, Edward W.
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p. 3039 - 3045
(2018/03/29)
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- Synthesis of Peptide Disulfide-Bond Mimics by Using Fully Orthogonally Protected Diaminodiacids
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A new strategy was developed for the synthesis of peptide disulfide-bond mimics using fully orthogonally protected diaminodiacids. This method overcomes the previous problems of heavy-metal contamination and poor compatibility with Fmoc chemistry and provides a practical avenue for the efficient preparation of peptide disulfide-bond mimics.
- Wang, Tao,Fan, Jian,Chen, Xiao-Xu,Zhao, Rui,Xu, Yang,Bierer, Donald,Liu, Lei,Li, Yi-Ming,Shi, Jing,Fang, Ge-Min
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supporting information
p. 6074 - 6078
(2018/09/27)
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- 3-Nitro-2-pyridinesulfenates as Efficient Solution- and Solid-Phase Disulfide Bond Forming Agents
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In this paper, a new disulfide-forming agent based on the finding that alkoxy 3-nitro-2-pyridinesulfenates (Npys-OR) can oxidize thiol groups is reported. Methyl 3-nitro-2-pyridinesulfenate (Npys-OMe), which is easily prepared from 3-nitro-2-pyridinesulfenyl chloride in a one-step reaction and has a reduction peak potential (Epc) of ?0.541 V versus Ag/AgCl, produces the cyclic nonapeptide oxytocin from its linear form in good yield (92 %) with minimal oligomer formation. Npys-OMe in the solid phase also demonstrated excellent results in oxytocin synthesis. Other disulfide-containing peptides, such as α-human atrial natriuretic peptide and α-conotoxin ImI, were also successfully synthesized. During these syntheses, no side reactions of methionine (Met) and tryptophan (Trp) residues or the S-acetamidomethyl (Acm) protecting group were detected. These results suggested that Npys-OMe or its solid-phase analog provides a new strategy for regioselective disulfide bond formation to assist the synthesis of complex disulfide-rich peptides.
- Taguchi, Akihiro,Kobayashi, Kiyotaka,Kotani, Akira,Muguruma, Kyohei,Kobayashi, Misaki,Fukumoto, Kentarou,Takayama, Kentaro,Hakamata, Hideki,Hayashi, Yoshio
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supporting information
p. 8262 - 8267
(2017/06/23)
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- NOVEL COMPOUND, PRODUCTION METHOD THEREFOR, AND APPLICATION THEREFOR
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[Problems] To provide a novel peptide synthesis technique that is completely different than heretofore, and to provide a novel compound that enables the synthesis/creation of a novel artificial functional protein and the synthesis/creation of a novel functional peptide, as well as a method for producing the same. [Solution] A compound represented by formula (I) or a salt thereof.
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- Systematic N-methylation of oxytocin: Impact on pharmacology and intramolecular hydrogen bonding network
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Oxytocin (OT) is a peptide hormone agonist of the OT receptor (OTR) that plays an important role in social behaviors such as pair bonding, maternal bonding and trust. The pharmaceutical development of OT as an oral peptide therapeutic has been hindered historically by its unfavorable physicochemical properties, including molecular weight, polarity and number of hydrogen bond donors, which determines poor cell permeability. Here we describe the first systematic study of single and multiple N-methylations of OT and their effect on physicochemical properties as well as potency at the OT receptor. The agonist EC50and percent effect for OTR are reported and show that most N-methylations are tolerated but with some loss in potency compared to OT. The effect of N-methylation on exposed polarity is assessed through the EPSA chromatographic method and the results validated against NMR temperature coefficient experiments and the determination of NMR solution structures. We found that backbone methylation of residues not involved in IMHB and removal of the N-terminal amine can significantly reduce the exposed polarity of peptides, and yet retain a significant OTR agonist activity. The results of this study also expose the potential challenge of using the N-methylation strategy for the OT system; while exposed polarity is reduced, in some cases backbone methylation produces a significant conformational change that compromises agonist activity. The data presented provides useful insights on the SAR of OT and suggests future design strategies that can be used to develop more permeable OTR agonists based on the OT framework.
- Sciabola, Simone,Goetz, Gilles H.,Bai, Guoyun,Rogers, Bruce N.,Gray, David L.,Duplantier, Allen,Fonseca, Kari R.,Vanase-Frawley, Michelle A.,Kablaoui, Natasha M.
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supporting information
p. 3513 - 3520
(2016/07/20)
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- Metal-Free Photocatalytic Aerobic Oxidation of Thiols to Disulfides in Batch and Continuous-Flow
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Disulfides represent significant molecular and structural features in various biologically active compounds and fine chemicals. Therefore, the development of mild, efficient and sustainable methods to access disulfides is of great importance. Here, we describe the development of a mild metal-free photocatalytic aerobic oxidation of thiols to disulfides using Eosin Y and visible-light irradiation. A continuous flow procedure was developed to accelerate the photocatalytic process, enabling the preparation of disulfides with high purity in a timeframe of minutes. The mildness and applicability of our method was exemplified by the flow synthesis of the cyclic peptide hormone, oxytocin, requiring only a 200s reaction time and an efficient one-pot batch protocol for the preparation of the therapeutic thiuram disulfide, disulfiram.
- Talla, Ali,Driessen, Brian,Straathof, Natan J. W.,Milroy, Lech-Gustav,Brunsveld, Luc,Hessel, Volker,No?l, Timothy
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supporting information
p. 2180 - 2186
(2015/07/27)
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- N-chlorosuccinimide, an efficient peptide disulfide bond-forming reagent in aqueous solution
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A novel method has been developed for the efficient formation of peptide disulfide bonds under aqueous conditions using N-chlorosuccinimide. Complete disulfide bond formation is achieved in 15 min with solvent mixtures containing water and acetonitrile. The Royal Society of Chemistry 2013.
- Postma, Tobias M.,Albericio, Fernando
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p. 14277 - 14280
(2013/09/02)
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- N-Chlorosuccinimide, an efficient reagent for on-resin disulfide formation in solid-phase peptide synthesis
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N-Chlorosuccinimide is described as a widely applicable on-resin disulfide-forming reagent. Disulfide bond formation was completed within 15 min in DMF. This strategy was successfully used in the synthesis of oxytocin and a regioselective synthesis of an α-conotoxin. Moreover, disulfide formation with N-chlorosuccinimide was found to be compatible with oxidation-prone methionine and tryptophan.
- Postma, Tobias M.,Albericio, Fernando
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supporting information
p. 616 - 619
(2013/04/10)
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- Revisiting oxytocin through the medium of isonitriles
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The reaction of thioamino acids and N-terminal peptides, mediated by hindered isonitriles and hydroxybenzotriazole, gives rise to peptide bonds. In one pathway, oxytocin was synthesized by eight such reiterative amidations. In another stereospecific track, oxytocin was constructed by native chemical ligation, wherein the two building blocks were assembled by thioacid amine amidation. The NMR spectra of oxytocin and dihydrooxytocin suggest a high level of preorganization in the latter, perhaps favoring oxidative folding.
- Wang, Ting,Danishefsky, Samuel J.
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supporting information; experimental part
p. 13244 - 13247
(2012/09/25)
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- Trimethoxyphenylthio as a highly labile replacement for tert-butylthio cysteine protection in fmoc solid phase synthesis
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Trimethoxyphenylthio (S-Tmp) is described as a novel cysteine protecting group in Fmoc solid phase peptide synthesis replacing the difficult to remove tert-butylthio. S-Tmp and dimethoxyphenylthio (S-Dmp) were successfully used for cysteine protection in a variety of peptides. Moreover, both groups can be removed in 5 min with mild reducing agents. S-Tmp is recommended for cysteine protection, as it yields crude peptides of high purity.
- Postma, Tobias M.,Albericio, Fernando,Giraud, Matthieu
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supporting information
p. 5468 - 5471,4
(2012/12/13)
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- Trimethoxyphenylthio as a highly labile replacement for tert-butylthio cysteine protection in fmoc solid phase synthesis
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Trimethoxyphenylthio (S-Tmp) is described as a novel cysteine protecting group in Fmoc solid phase peptide synthesis replacing the difficult to remove tert-butylthio. S-Tmp and dimethoxyphenylthio (S-Dmp) were successfully used for cysteine protection in a variety of peptides. Moreover, both groups can be removed in 5 min with mild reducing agents. S-Tmp is recommended for cysteine protection, as it yields crude peptides of high purity.
- Postma, Tobias M.,Giraud, Matthieu,Albericio, Fernando
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supporting information
p. 5468 - 5471
(2013/01/15)
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- Modulating oxytocin activity and plasma stability by disulfide bond engineering
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Disulfide bond engineering is an important approach to improve the metabolic half-life of cysteine-containing peptides. Eleven analogues of oxytocin were synthesized including disulfide bond replacements by thioether, selenylsulfide, diselenide, and ditelluride bridges, and their stabilities in human plasma and activity at the human oxytocin receptor were assessed. The cystathionine (Ki = 1.5 nM, and EC50 = 32 nM), selenylsulfide (Ki = 0.29/0.72 nM, and EC50 = 2.6/154 nM), diselenide (Ki = 11.8 nM, and EC50 = 18 nM), and ditelluride analogues (Ki = 7.6 nM, and EC50 = 27.3 nM) retained considerable affinity and functional potency as compared to oxytocin (Ki = 0.79 nM, and EC50 = 15 nM), while shortening the disulfide bridge abolished binding and functional activity. The mimetics showed a 1.5-3-fold enhancement of plasma stability as compared to oxytocin (t 1/2 = 12 h). By contrast, the all-d-oxytocin and head to tail cyclic oxytocin analogues, while significantly more stable with half-lives greater than 48 h, had little or no detectable binding or functional activity.
- Muttenthaler, Markus,Andersson, Asa,De Araujo, Aline D.,Dekan, Zoltan,Lewis, Richard J.,Alewood, Paul F.
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experimental part
p. 8585 - 8596
(2011/02/28)
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- Counterion exchange process for peptides
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The invention encompasses a process for purifying a peptide comprising loading a peptide onto a RP-HPLC column; washing the column with an aqueous solution of a pharmaceutically acceptable counterion salt; and eluting the peptide from the column with a solvent mixture of a organic solvent and an acid of the pharmaceutically acceptable counterion, wherein the aqueous solution has a pH of at least about 6.
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Page/Page column 11
(2008/06/13)
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- The use of hydrogen peroxide for closing disulfide bridges in peptides
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The use of hydrogen peroxide for the formation of disulfide bridges was studied in 15 peptides of various lengths and structures. The oxidation of peptide thiols by hydrogen peroxide was shown to proceed under mild conditions without noticeable side reactions of Trp, Tyr, and Met residues. Yields of the corresponding cyclic disulfides were high and mostly exceeded those obtained with other oxidative agents, in particular, iodine. It was established that the use of hydrogen peroxide in organic medium also provided sufficiently high yields when large-scale syntheses of oxytocin and octreotide (up to 10 g) were carried out.
- Sidorova,Molokoedov,Az'muko,Kudryavtseva,Krause,Ovchinnikov,Bespalova
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p. 101 - 110
(2007/10/03)
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- SUBSTANCES IN ORDER TO ACHIEVE PREFERENCE AND CREATE ACCEPTANCE
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The present invention relates to the use of substances with oxytocin activity in order to achieve preference and create acceptance. It also relates to a composition comprising at least one substance with oxytocin activity in order to achieve preference and/or create acceptance. It also relates to a composition containing at least one substance with oxytocin activity and at least one flavouring or fragrance agent in order to create acceptance.
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- Discovery of a highly selective and efficient reagent for formation of intramolecular disulfide bonds in peptides
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We have discovered that trans-[Pt(en)2Cl2]2+ (en = ethylenediamine) is a highly selective and efficient reagent for the quantitative formation of intramolecular disulfide bonds in peptides. A series of 14 dithiol peptides which form disulfide-containing rings ranging in size from 14 to 53 atoms were used to characterize the reagent. The dithiol peptides are cleanly and rapidly converted to their disulfide forms by a slight excess of the platinum complex under mild reaction conditions (slightly acidic and neutral media). For all the dithiol peptides studied, including a penicillamine-derived peptide, the oxidation yields range from 97% to 100%. No side reactions were observed, including no oxidation of the methionine side chain. The reaction kinetics for oxidation of reduced pressinoic acid were found to be second order overall: rate = k'[Pt(IV)][dithiol peptide], where k' is a pH-dependent second-order rate constant. Values of 0.60 ± 0.01, 3.5 ± 0.2, and 22 ± 1 M-1 s-1 were determined for k' at pH 3.0, 4.0, and 5.0, respectively (25 °C and 0.45 M ionic strength). A reaction mechanism for oxidation of dithiol peptides by [Pt(en)2Cl2]2+ is proposed. [Pt(en)2Cl2]2+ and its reduction product [Pt(en)2]2+ are essentially substitution inert under the conditions used for disulfide formation, they are nontoxic, and they are readily separated from peptides by HPLC. The characteristics of [Pt(en)2Cl2]2+ and its reaction properties with dithiol peptides suggest that [Pt(en)2Cl2]2+ is a universal reagent for the rapid and quantitative formation of intramolecular disulfide bonds in peptides.
- Shi, Tiesheng,Rabenstein, Dallas L.
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p. 6809 - 6815
(2007/10/03)
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- Trans-Dichlorotetracyanoplatinate(IV) as a Reagent for the Rapid and Quantitative Formation of Intramolecular Disulfide Bonds in Peptides
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Oxidation of cysteine thiol groups by trans-dichlorotetracyanoplatinate(IV) to form intramolecular peptide disulfide bonds has been studied for a series of dithiol peptides ranging from 4 to 15 amino acid residues in length. The dithiol peptides are rapidly and quantitatively transformed to their intramolecular disulfide forms by a slight excess of [Pt(CN)4Cl2]2-, as shown by HPLC. Quantitative analyses by HPLC and by spectrophotometric titration confirm a [Pt(IV)]:[dithiol peptide] stoichiometry of 1:1. Under the low pH conditions used, oxidation to form a 38-membered ring in the case of reduced somatostatin is as rapid as that to form much smaller rings, suggesting that ring closure is not the rate-determining step. The oxidation rates increase as the pH is increased. Time-resolved spectra show two isosbestic points, indicating that no peptide-platinum intermediates accumulate to a significant amount. A reaction mechanism similar to that for reduction of [Pt(CN)4Cl2]2- by monothiols is proposed. [Pt(CN)4Cl2]2- is a mild oxidant and essentially substitution inert; its reduction product, [Pt(CN)4Cl2]2-, is stable, has no redox chemistry with peptides, and does not form complexes with peptides. Moreover, [Pt(CN)4Cl2]2- and [Pt(CN)4Cl2]2- are nontoxic and readily separable from peptides by HPLC, and the cost of the Pt(IV) complex is negligible compared with that of peptides. The only unwanted side reaction observed with [Pt(CN)4Cl2]2- is oxidation of the sulfur of methionine to the sulfoxide form. These characteristics and the results of this study suggest that [Pt(CN)4Cl2]2- is an excellent reagent for the formation of intramolecular peptide disulfide bonds.
- Shi, Tiesheng,Rabenstein, Dallas L.
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p. 4590 - 4595
(2007/10/03)
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- Novel solid-phase reagents for facile formation of intramolecular disulfide bridges in peptides under mild conditions
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The controlled formation of intramolecular disulfide bridges in peptides, while avoiding unwanted oligomerization, is a significant challenge. Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid), was developed originally in the context of an assay for measuring free thiol concentration under physiological conditions. The present studies demonstrate that this reagent, when bound through two sites to a suitable solid support (PEG-PS, modified Sephadex, or controlled-pore glass), is an effective mild oxidizing reagent that promotes the formation of disulfide bridges. Rates and yields of the reactions were determined as a function of pH, excess of oxidizing reagent, resin loading, and parent support, for the preparation of oxytocin and deaminooxytocin (9 residues, disulfide bridge between residues 1 and 6), somatostatin (14 residues, disulfide bridge between residues 3 and 14), α-conotoxin SI (13 residues, disulfide bridges between residues 2 and 7; 3 and 13), and apamin (18 residues, disulfide bridges between residues 1 and 11; 3 and 15). Cystine dimers of these peptide models formed, if at all, in relatively low amounts. Use of solid-phase Ellman's reagents to oxidize the linear precursors of conotoxin or apamin (tetrathiols) gave as the main products the correctly paired regioisomers. Particular advantages of the overall approach include fast reaction rates over a wide range of pH, from 2.7 to 6.6; easy purification of disulfide-containing products; and the specificity and reusability of the reagents.
- Annis, Ioana,Chen, Lin,Barany, George
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p. 7226 - 7238
(2007/10/03)
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- Thiol/disulfide exchange reactions of captopril and penicillamine with arginine vasopressin and oxytocin
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The kinetics and equilibria of the reaction of the thiol-containing drugs captopril (D-3-mercapto-2-methylpropanoyl-L-proline, CpSH) and penicillamine (β, β-dimethylcysteine, PSH) with the disulfide bonds of the neurohypophyseal peptide hormones arginine vasopressin (AVP) and oxytocin (OT) have been characterized. CpSH reacts with AVP and OT by thiol/disulfide interchange to form two peptide-CpSH mixed disulfides, which in turn react with another molecule of CpSH to form the reduced peptide and CpSSCp. Forward and reverse rate constants and the equilibrium constant are reported for both steps in the reaction of CpSH with AVP and OT at pH 7.00. The rate constant for the first step (k1) is much larger than that for the second step (k2). Also, once formed, the peptide-CpSH mixed disulfides rapidly undergo intramolecular thiol/disulfide interchange with reformation of the cyclic peptide and CpSH. PSH reacts with AVP and OT by the same two-step reaction sequence; however, the rate of the second step is very slow due to steric hindrance from the methyl groups of PSH and the PSH moiety of the peptide-PSH mixed disulfides. Using rate constants determined in this study and PSH levels in the plasma of patients on PSH therapy, it is predicted that in vivo reduction of the disulfide bonds of AVP and OT by PSH and CpSH has little effect on the plasma half-lives of AVP or OT.
- Rabenstein, Dallas L.,Yeo, Pauline L.
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p. 109 - 118
(2007/10/02)
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- Investigation of the dimethylsulfoxide-trifluoroacetic acid oxidation system for the synthesis of cystine-containing peptides
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Disulfide bonds of peptides were effectively established between S- protected cysteine residues as well as free cysteine residues by the action of dimethylsulfoxide in trifluoroacetic acid. Oxytocin and α-human calcitonin gene-related peptide were synthesized using this oxidation system. The feasibility of this method for the formation of two disulfide bridges of apamin was also examined.
- Koide,Otaka,Fujii
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p. 1030 - 1034
(2007/10/02)
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- Disulfide bond formation using the silyl chloride-sulfoxide system for the synthesis of a cystine peptide
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An efficient method for disulfide bond formation in peptides by the silyl chloride-sulfoxide system is described. Methyltrichlorosilane in trifluoroacetic acid, in the presence of diphenyl sulfoxide, is found to cleave various S-protecting groups of cysteine to form cystine directly within 10-30 min. No side reactions were observed with nucleophilic amino acids such as Met, His, or Tyr, except for Trp, under the reaction conditions of the silyl chloride-sulfoxide treatment. A chlorination of the indole moiety of unprotected Trp, rather than sulfur-sulfur bond formation, is a dominant reaction when the peptide containing unprotected Trp is treated with the chlorosilane-sulfoxide. However, the disulfide bond can be formed efficiently with no modification at the indole ring by treatment of the peptide having a formyl-protected Trp residue with the silyl chloride-sulfoxide system. The formyl group is removed by a brief treatment at basic pH without affecting the disulfide bond formed by the silyl chloride-sulfoxide treatment. This new disulfide bond forming reaction in trifluoroacetic acid is successfully applied to the syntheses of oxytocin, human brain natriuretic peptide, and somatostatin without any solubility problem.
- Akaji, Kenichi,Tatsumi, Tadashi,Yoshida, Makoto,Kimura, Tooru,Fujiwara, Yoichi,Kiso, Voshiaki
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p. 4137 - 4143
(2007/10/02)
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- USE OF SEPHADEX LH-20 FOR SOLID-PHASE SYNTHESIS OF OXYTOCIN
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The solid-phase synthesis of oxytocin on commercial Sephadex LH-20 is described.The biological activity of the product obtained was 132 AU/mg, and its oxytocin content, according to HPLC, was 28.2percent.It was shown that the amount of oxytocin and the biological activity do not depend on the degree of addition of Gly9 to the polymer matrix within the range of 0.6-1.6 mmoles of Gly9/g of resin; after the regeneration, the Sephadex LH-20 can be re-used for solid-phase synthesis of oxytocin.
- Zel'tser, I. E.,Antonov, A. A.,Karel'skii, V. N.,Prikhodkina, E. A.,Ovsepyan, A. M.,Zhuravleva, E. E.
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- PREPARATION AND APPLICATION OF NEW ACID LABILE ANCHOR GROUPS FOR THE SYNTHESIS OF PEPTIDE AMIDES BY FMOC SOLID PHASE SYNTHESIS
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The synthesis and application of new linkage agents for the preparation of peptide amides using a modified Fmoc strategy is described.
- Breipohl, G.,Knolle, J.,Stueber, W.
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p. 5651 - 5654
(2007/10/02)
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- Analogs of neurohypophysial hormones with inhibition effects
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The invention pertains to analogs of neurohypophysial hormones evidencing inhibition effects of the chemical formula I STR1 where A is CH3 CO or H, V is a residue of penicilamine or cysteine, B is CH3 or H, X is a residue of isoleucine or phenylalanine, Y is a residue of penicilamine or cysteine, and Z is a residue of leucine or lysine. These analogues evidence inhibition effects towards certain biological activities of neurohypophysial hormones.
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- Fluorenylmethoxycarbonylamino Acid Active Esters in Solid Phase Peptide Synthesis Using p-Alkoxybenzyl Alcohol Resin: Synthesis of Oxytocin
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A superior solid phase synthesis of the pituitary hormone, oxytocin (I) has been accomblished by the condensation of fluorenylmethoxycarbonyl(Fmoc)-amino acid active esters in the presence of 1-hydroxybenzotriazole (HOBt)
- Sivanandaiah, K. M.,Gurusiddappa, S.
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p. 139 - 140
(2007/10/02)
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