- AJIPHASE: A Highly Efficient Synthetic Method for One-Pot Peptide Elongation in the Solution Phase by an Fmoc Strategy
-
We previously reported an efficient peptide synthesis method, AJIPHASE, that comprises repeated reactions and isolations by precipitation. This method utilizes an anchor molecule with long-chain alkyl groups as a protecting group for the C-terminus. To further improve this method, we developed a one-pot synthesis of a peptide sequence wherein the synthetic intermediates were isolated by solvent extraction instead of precipitation. A branched-chain anchor molecule was used in the new process, significantly enhancing the solubility of long peptides and the operational efficiency compared with the previous method, which employed precipitation for isolation and a straight-chain aliphatic group. Another prerequisite for this solvent-extraction-based strategy was the use of thiomalic acid and DBU for Fmoc deprotection, which facilitates the removal of byproducts, such as the fulvene adduct.
- Takahashi, Daisuke,Inomata, Tatsuji,Fukui, Tatsuya
-
-
Read Online
- Harnessing polarity and viscosity to identify green binary solvent mixtures as viable alternatives to DMF in solid-phase peptide synthesis
-
Solid-phase peptide synthesis (SPPS) enables routine synthesis of virtually any type of peptide sequence and is the preferred method for peptide synthesis in academia and the pharmaceutical industry alike. Still, SPPS typically requires significant amounts of hazardous solvents and thus suffers from a negative environmental footprint. Such drawbacks have spurred numerous initiatives for solvent substitution, reduction and recycling, and a handful solvents have recently been proposed as potential green alternatives toN,N-dimethylformamide (DMF). In this report, we recognise solvent viscosity and polarity in combination as key physicochemical parameters for SPPS and identify green binary solvent mixtures of dimethyl sulfoxide (DMSO) and 1,3-dioxolane or 2-methyl tetrahydrofuran that closely resemble DMF. In a series of reagent dissolution, resin swelling, peptide coupling and Fmoc-removal experiments we show that combining solvents offers unprecedented opportunities to predict and fine-tune the overall solvent properties for different aspects of SPPS. Lastly, the identified green binary solvent mixtures were employed for the synthesis of a range of challenging model peptides and peptide therapeutics on meaningful scale, demonstrating that binary solvent mixtures are viable green alternatives to DMF in SPPS.
- Albericio, Fernando,Dettner, Frank,Egelund, Peter H. G.,Haselmann, Kim F.,Jadhav, Sandip,Johansson Castro, Henrik,Krüger, Tobias,Lechner, Carolin,Liffert, Raphael,Martin, Vincent,Pedersen, Daniel Sejer,Sch?nleber, Ralph,Thordal Le Quement, Sebastian
-
-
Read Online
- Protein Modification at Tyrosine with Iminoxyl Radicals
-
Post-translational modifications (PTMs) of proteins are a biological mechanism for reversibly controlling protein function. Synthetic protein modifications (SPMs) at specific canonical amino acids can mimic PTMs. However, reversible SPMs at hydrophobic amino acid residues in proteins are especially limited. Here, we report a tyrosine (Tyr)-selective SPM utilizing persistent iminoxyl radicals, which are readily generated from sterically hindered oximes via single-electron oxidation. The reactivity of iminoxyl radicals with Tyr was dependent on the steric and electronic demands of oximes; isopropyl methyl piperidinium oxime 1f formed stable adducts, whereas the reaction of tert-butyl methyl piperidinium oxime 1o was reversible. The difference in reversibility between 1f and 1o, differentiated only by one methyl group, is due to the stability of iminoxyl radicals, which is partly dictated by the bond dissociation energy of oxime O-H groups. The Tyr-selective modifications with 1f and 1o proceeded under physiologically relevant, mild conditions. Specifically, the stable Tyr-modification with 1f introduced functional small molecules, including an azobenzene photoswitch, to proteins. Moreover, masking critical Tyr residues by SPM with 1o, and subsequent deconjugation triggered by the treatment with a thiol, enabled on-demand control of protein functions. We applied this reversible Tyr modification with 1o to alter an enzymatic activity and the binding affinity of a monoclonal antibody with an antigen upon modification/deconjugation. The on-demand ON/OFF switch of protein functions through Tyr-selective and reversible covalent-bond formation will provide unique opportunities in biological research and therapeutics.
- Ishiyama, Takashi,Kanai, Motomu,Maruyama, Katsuya,Oisaki, Kounosuke,Sakai, Kentaro,Seki, Yohei,Togo, Takaya
-
supporting information
p. 19844 - 19855
(2021/11/30)
-
- SYNTHETIC METHOD OF BIVALIRUNDIN
-
The Active Pharmaceutical Ingredient (API), Bivalirudin, is a 20 amino acid peptide containing one basic and 5 amino acid residues. With a chemical formula of C98H138N24O33 and a molecular weight of 2180.3 g/mol. Bivalirudin is also known as Angiomax. It is a direct thrombin inhibitor indicated for use as an anticoagulant. It provides a method for synthesizing bivalirudin and in particular to the synthetic method of bivalirudin by solid phase peptide synthesis using Fmoc-Leu-Wang resin as a carrier. The method has the advantages of high yield, less by-product and simple separation and purification, and it's time gaining than the prior art, and is suitable for pilot and industrial production.
- -
-
Page/Page column 12; 13
(2020/01/24)
-
- PEPTIDE SYNTHESIS METHOD
-
The present invention has an object of providing a peptide synthesis method using a carrier capable of reversibly repeating the dissolved state and the insolubilized state, wherein the problem of an amino acid active species existing in the reaction system in de-protection reaction can be easily solved. The present invention provides a peptide synthesis method comprising the following steps: a step of condensing an N-Fmoc protected amino acid with a peptide having a C-terminal protected with a carrier which is crystallized according to a change of a composition of a dissolving solvent, in the presence of a condensing agent, to obtain an N-Fmoc-C-carrier protected peptide, a step of adding an alkylamine having 1 to 14 carbon atoms or hydroxyl amine to the reaction system, a step of de-protecting the N-terminal, and a step of changing the composition of the solvent dissolving the C-carrier protected peptide, to crystallize and separate the peptide.
- -
-
Paragraph 0379-0380
(2018/08/20)
-
- IMPROVEMENTS IN SOLID PHASE PEPTIDE SYNTHESIS
-
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.
- -
-
Paragraph 0071-0076
(2017/08/01)
-
- Solid phase peptide synthesis via side chain attachment
-
The present application discloses peptides and peptaibols of high purity may be obtained by solid phase peptide synthesis using as the starting resin hydroxy amino acids, hydroxy amino acid amides, hydroxy amino alcohols or small peptides containing hydroxy amino acids attached to polymers through their side chain.
- -
-
Page/Page column 19; 26
(2016/08/10)
-
- PROCESS FOR PRODUCTION OF BIVALIRUDIN
-
The invention relates to methods for the preparation of high purity Bivalirudin. The polypeptide is prepared in a high purity of at least 98.5% (by HPLC), wherein the total impurities amount to less than 1.5%, comprising not more than 0.5% [Asp9-Bivalirudin] and each is impurity less than 1.0%, and preferably having a purity of at least about 99.0% by HPLC, wherein the total impurities amount to less than 1.0%, comprising not more than 0.5% [Asp9-Bivalirudin] and each impurity is less than 0.5%.
- -
-
Paragraph 0129; 0130
(2016/11/24)
-
- Buffer-based method for preparing bivalirudin drug product
-
A method for preventing the formation of a bivalirudin precipitate during preparation of a pharmaceutical drug product comprising about 250 mg of bivalirudin, a dried bivalirudin drug product, and a concentrated liquid bivalirudin drug product. The method for preventing the formation of a bivalirudin precipitate comprises (i) preparing an aqueous solution comprising a buffer and a pH greater than the isoelectric point of bivalirudin; (ii) adding bivalirudin salt to the aqueous solution to form a bulk solution; (iii) transferring the bulk solution to one or more vessels; and (iv) drying the bulk solution. The buffer may have a pKa of about 4 to less than 7, and a pH greater than the isoelectric point of bivalirudin. The pH of the bulk solution may be maintained at a level greater than the isoelectric point of bivalirudin. Further, the bulk solution may have a final pH of about 4 to about 7.
- -
-
-
- METHOD FOR PRODUCING BIVALIRUDIN
-
A method for producing bivalirudin using solid phase peptide synthesis by the following steps: a) mixing a Fmoc-amino acid resin or a Fmoc-peptide resin with a de-protective agent so as to remove Fmoc-; b) in the presence of a condensing agent, condensing a Fmoc- or Boc-amino acid with the amino acid or the peptide bound to the resin; c) repeating the steps a) and b) to yield a peptide resin represented by Formula I, (SEQ?ID?NO.?1) Boc-D-Phe1-Pro2-Arg(Pbf)3-Pro4-Gly5-Gly6-Gly7- Gly8-Asn(Trt)9-Gly10-Asp(OtBu)11-Phe12-Glu (OtBu)13-Glu(OtBu)14-Ile15-Pro16-Glu(OtBu)17-Glu (OtBu)18-Tyr(tBu)19-Leu20-Resin?(I) and d) in the presence of a cleavage agent, separating the peptide from the resin to yield bivalirudin represented by Formula II (SEQ ID NO. 2). D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe- Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu?(II) Based on its total volume, the de-protective agent is composed of between 3 and 20% of piperidine and between 0.5 and 10% of bicyclic amidine. The method is low in cost and the resultant bivalirudin has high purity.
- -
-
-
- Method of preparing bivalirudin
-
The present invention relates to a novel solid phase peptide synthesis method for Bivalirudin. This method contains following steps: serving Trityl Chloride Resin, 4-Methyltrityl Chloride Resin, 4-Methoxytrityl Chloride Resin, or 2-Cl Trityl Chloride Resin, or attaching of Wang Resin as a start raw material); according to general solid phase peptide synthesis rules, coupling protected amino acids after deprotection of Fmoc-protection group and then deprotecting side chain protection group; cleaving peptides from resin; and then obtaining crude Bivalirudin product. C18 high pressure liquid chromatography (HPLC) column is applied to purify the product of Bivalirudin. This method is suitable and effective for mass production, in addition to its features of high quality, low production cost, high synthetic yield, avoidance of usage of fatal toxic chemical such as HF, and less environmental pollution. The high yield rate of 99% is achieved for each synthetic step and total yield rate is 14%.
- -
-
-
