- Reaction medium engineering in enzymatic peptide fragment condensation: Synthesis of eledoisin and LH-RH
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The influence of different reaction systems on α-chymotrypsin-catalyzed synthesis of eledoisin and LH-RH peptides from (7+4) and (5+5) fragments was investigated. The peptide yield was determined in the following systems: buffered aqueous media, frozen solutions, organic media, and cosolvent mixtures. The experimental set up was tailored to allow the screening of an array of conditions with minimum consumption of peptide fragments (2.1 and 2.5mM). The best yields (22% yield for eledoisin and 68% yield for LH-RH) were obtained in buffered aqueous solutions. It was found that the choice of buffer had a strong influence on the peptide yield; boric-borate and ammonium acetate buffers at pH 9, gave the best results. In buffered aqueous systems, both syntheses were scaled up by using a 10-fold increase in fragment concentration (21 and 25mM). Under these conditions the yields rose to 57% and 80% of eledoisin and LH-RH, respectively. Moreover, during the synthesis of eledoisin and in the presence of boric-borate buffer pH 9, the peptide precipitated from the reaction medium preventing a secondary hydrolysis and facilitating the in situ product purification. Copyright (C) 1998 Elsevier Science Ltd.
- Bjoerup, Peter,Torres, Josep Lluis,Adlercreutz, Patrick,Clapes, Pere
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- α-Chymotrypsin-catalyzed (3 + 7) segment synthesis of the luteinizing hormone releasing hormone
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The formation of the luteinizing hormone releasing hormone (LHRH) Glp-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (1), from the segments Glp-His-Trp-OEt (2) and H-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (3) is catalyzed by α-chymotrypsin. A yield of 71% was achieved when the reaction was performed at -15°C in a solution which contained 38 vol% DMSO and was saturated with KCl.
- Schuster,Aaviksaar,Jakubke
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- IMPROVEMENTS IN SOLID PHASE PEPTIDE SYNTHESIS
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An improved method of deprotection in solid phase peptide synthesis is disclosed. In particular the deprotecting composition is added in high concentration and small volume to the mixture of the coupling solution, the growing peptide chain, and any excess activated acid from the preceding coupling cycle, and without any draining step between the coupling step of the previous cycle and the addition of the deprotection composition for the successive cycle. Thereafter, the ambient pressure in the vessel is reduced with a vacuum pull to remove the deprotecting composition without any draining step and without otherwise adversely affecting the remaining materials in the vessel or causing problems in subsequent steps in the SPPS cycle.
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Paragraph 0071-0076
(2017/08/01)
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- Solid-phase peptide synthesis in highly loaded conditions
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The use of very highly substituted resins has been avoided for peptide synthesis due to the aggravation of chain-chain interactions within beads. To better evaluate this problem, a combined solvation-peptide synthesis approach was herein developed taking as models, several peptide-resins and with peptide contents values increasing up to near 85%. Influence of peptide sequence and loading to solvation characteristics of these compounds was observed. Moreover, chain-chain distance and chain concentration within the bead were also calculated in different loaded conditions. Of note, a severe shrinking of beads occurred during the α-amine deprotonation step only when in heavily loaded resins, thus suggesting the need for the modification of the solvent system at this step. Finally, the yields of different syntheses in low and heavily loaded conditions were comparable, thus indicating the feasibility of applying this latter "prohibitive" chemical synthesis protocol. We thought these results might be basically credited to the possibility, without the need of increasing molar excess of reactants, of carrying out the coupling reaction in higher concentration of reactants - near three to seven folds - favored by the use of smaller amount of resin. Additionally, the alteration in the solvent system at the α-amine deprotonation step might be also improving the peptide synthesis when in heavily loaded experimental protocol.
- Nakaie, Clovis R.,Oliveira, Eliandre,Vicente, Eduardo F.,Jubilut, Guita N.,Souza, Sinval E.G.,Marchetto, Reinaldo,Cilli, Eduardo M.
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experimental part
p. 101 - 109
(2011/06/25)
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- Solid-Phase Synthesis of Peptide Amides on a Polystyrene Support Using Fluorenylmethoxycarbonyl Protecting Groups
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We have investigated new routes for the synthesis of acid-sensitive amino acids using N-fluorenylmethoxycarbonyl amino acids and mild acid labile side chain protection in order to avoid HF cleavage as final step.The 4-(benzyloxy)benzylamine resin, applied successfully by Pietta and Brenna, proved to be unsuitable for the synthesis of peptide amides since we have found that trifluoroacetic acid treatment of peptide-resins yield the peptide still bearing a p-hydroxybenzyl group on the carboxamide nitrogen.We have successfully used the 2,4-dimethoxybenzhydrylamine support for the synthesis of peptide amides.Cholecystokinin octapeptide and GnRH were synthesized on the 2,4-dimethoxybenzhydrylamine resin and cleaved from the polymer with trifluoroacetic acid-thioanisole (4:1), and 95percent trifluroacetic acid containing phenol (2.5percent) and dimethyl sulfide (2.5percent), respectively.The final cleavage conditions had to be optimized in order to obtain good yields, suggesting that side chain protections of certain amino acids, possibly arginine and tryptophan, may have to be further refined.
- Penke, Botond,Rivier, Jean
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p. 1197 - 1200
(2007/10/02)
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- Synthesis of Gonadoliberin, a Gonadotropin Releasing Hormone
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A simple laboratory synthesis of gonadoliberin was elaborated basing on classical solution methods and using minimal protection of the side-chain functions.In the final step a hexapeptide segment was condensed with the corresponding tetrapeptide and the obtained product was purified by a single step silica gel chromatography.The total efficiency of the whole synthesis was 15percent.
- Rzeszotarska, Barbara,Masiukiewicz, Elzbieta,Kmiecik-Chmura, Halina
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p. 791 - 798
(2007/10/02)
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- Synthetic decapeptide having the activity of the luteinizing hormone releasing hormone and method for manufacturing the same
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A synthetic decapeptide, L-pglutamyl-L-histidyl-L-tryptophanyl-L-seryl-L-tyrosyl-glycyl-L-leucyl-L-arginyl-L-prolyl-glycinamide which has the hormonal activities of the luteinizing hormone releasing hormone (LRH) of the hypothalamus gland of mammals is produced by utilizing as the key starting materials, the amino acids, glutamic acid or pyroglutamic acid, histidine, tryptophan, serine, tyrosine, glycine, leucine, arginine, and proline. Synthesis of the decapeptide is accomplished by coupling, in appropriate combinations of appropriate protected forms of the amino acids, and finally deprotecting to yield the amide, L-pglutamyl-L-histidyl-L-tryptophanyl-L-seryl-L-tyrosyl-glycyl-L-leucyl-L-arginyl-L-prolyl-glycinamide.
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- Synthesis and biological activity of some analogs of the gonadotropin releasing hormone
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Twenty one analogs of gonadotropin releasing hormone 2 (GnRH), were synthesized by the solid phase method. The derivatives were 3 (GnRH methyl ester), GnRH N methyl amide, GnRH free acid, 2 (Gn RH N terminal hexapeptide), and also [Ac Ala1], [Ac gly1], [D 1], [Pro1], [Arg2], [Tyr(Me)3], [Ser5], [des Gly6], [Sar6], [des Pro9], [des Gly10, Pro NH29], [des Gly10, Pro NHC2H59], [Tyr11], [Tyr(Me)35], [des His2, des Pro9], [des His2, Sar6], and [Sar6 des Tyr5] GnRH. All analogs were purified at both the protected and the deblocked stage. The final products were characterized by chemical and physical methods and assayed in vitro for both LH and FSH release using rat pituitaries. Those derivatives which showed less than 0.2% of the activity of GnRH itself were further tested for inhibition of gonadotropin release.
- Arnold,Flouret,Morgan,Rippel,White
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p. 314 - 319,315, 316
(2007/10/05)
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