OxymaPure Coupling Reagents: Beyond Solid-Phase Peptide Synthesis

OxymaPure [ethyl 2-cyano-2-(hydroxyimino)acetate] is an exceptional reagent with which to suppress racemization and enhance coupling efficiency during amide bond formation. The tremendous popularity of OxymaPure has led to the development of several Oxyma-based reagents. OxymaPure and its derived reagents are widely used in solid- and solution-phase peptide chemistry. This review summarizes the recent developments and applications of OxymaPure and Oxyma-based reagents in peptide chemistry, in particular in solution-phase chemistry. Moreover, the side reaction associated with OxymaPure is also discussed. 1 Introduction 2 Oxyma-Based Coupling Reagents 2.1 Aminium/Uronium Salts of OxymaPure 2.2 Phosphonium Salts of OxymaPure 2.3 Oxyma-Based Phosphates 2.4 Sulfonate Esters of OxymaPure 2.5 Benzoate Esters of OxymaPure 2.6 Carbonates of OxymaPure Derivatives 3 OxymaPure Derivatives 4 Other Oxime-Based Additives and Coupling Reagents 5 Side Reactions Using OxymaPure Derivatives 6 Conclusion 7 List of Abbreviations.We are grateful to the American Chemical Society (ACS) Green Chemistry Institute (GCI) (Pharmaceutical Roundtable Ignition Grant) for funding S.R.M. In addition, the work in laboratory of the authors was funded in part by the following: the South African Agency for Science and Technology Advancement, The National Research Foundation (NRF) (Blue Sky’s Research Programme, Grant No. 120386) and the University of KwaZulu-Natal (South Africa); the Ministerio de Ciencia e Innovación (Spanish Ministry of Science, Innovation) (RTI2018-093831-B-100), the Departament d’Innovació, Universitats i Empresa, Generalitat de Catalunya (2017 SGR 1439) and the Ministerio de Economía y Competitividad; and the International Scientific Partnership Program ISPP at King Saud University, Saudi Arabia, Riyadh (ISPP# 0061)

(<i>E</i>)‑Ethyl-2-cyano-2-(((2,4,6-trichlorobenzoyl)oxy)imino)acetate: A Modified Yamaguchi Reagent for Enantioselective Esterification, Thioesterification, Amidation, and Peptide Synthesis

Here, the synthesis and applications of (<i>E</i>)-ethyl-2-cyano-2-(((2,4,6-trichlorobenzoyl)­oxy)­imino)­acetate as a racemization suppressing and easily recyclable version of the Yamaguchi reagent that can be used for amide and peptide synthesis are reported. We demonstrated its application in racemization-free esterification, thioesterification, amidation, and peptide bond formation. We successfully synthesized oligopeptides on the solid support in dimethylformamide as well as in solution (dichloromethane) by applying this coupling reagent. It is important to note that a mixed-anhydride-based method provides peptide-forming reactions as good as the current methods using built-in coupling reagents. Mechanism investigation, racemization suppression, and recyclability are also discussed

Minimizing side reactions during amide formation using DIC and oxymapure in solid-phase peptide synthesis

N,N’-diisopropylcarbodiimide (DIC) and OxymaPure are the reagents of choice for the formation of peptide bonds in Solid-Phase Peptide Synthesis (SPPS). The combined use of DIC and OxymaPure in model cases can be accompanied by a serious side reaction with the ultimate formation of oxadiazole and HCN. Here we demonstrate, in a practical case, that the first step of the reaction (formation of an adduct) takes place with approximately 1% respect to the amount of OxymaPure. Furthermore, we found that the second part of the side-reaction (formation of oxadiazole and HCN) can be minimized and even suppressed with an appropriate protocol for the sequence of reagent addition. Thus, the best conditions are preactivation of the Fmoc-amino acid with only DIC for 2–5 min followed by addition of the resulting mixture to the peptide-resin. OxymaPure can be added to the resin before adding the preactivation mixture or after first adding the preactivation mixture, the latter approach giving the best results. Ultimately, in standard research conditions, the combined use of DIC and OxymaPure as coupling method in SPPS is safe.We thank Yoav Luxembourg (Luxembourg Bio Technologies) for encouraging this work and for the generous gift of the DIC and OxymaPure. The authors acknowledge the American Chemical Society (ACS) Green Chemistry Institute (GCI) Pharmaceutical Roundtable Ignition grant awarded to SRM. The work was partially funded by National Research Foundation (NRF) (Blue Sky’s Research Programme # 120386).Peer reviewe

Ethyl 2‑Cyano-2-(4-nitrophenylsulfonyloxyimino)acetate-Mediated Lossen Rearrangement: Single-Pot Racemization-Free Synthesis of Hydroxamic Acids and Ureas from Carboxylic Acids

Ethyl 2-cyano-2-(4-nitrophenylsulfonyloxyimino)­acetate (4-NBsOXY) mediated Lossen rearrangement and its application for the synthesis of ureas is demonstrated. Required hydroxamic acids for the Lossen rearrangements were synthesized from carboxylic acids using the same reagent. Finally, reaction of an amine with the produced isocyanate resulted in urea. Good yields without racemization were achieved under milder and simpler reaction conditions. Reactions are compatible with common <i>N</i>-protecting groups, such as Boc, Fmoc, Cbz, and benzyl, as well as various OH protecting groups, such as ^tBu and Bzl. Conversion from carboxylic acid to urea is achieved in one pot. Most importantly, byproducts Oxyma [ethyl 2-cyano-2-(hydroxyimino)­acetate] and 4-nitrobenzenesulfonic acid can be recovered easily and can be recycled to prepare the reagent. Thus, the method is environmentally friendly and cost-effective

A Practical Peptide Synthesis Workflow Using Amino-Li-Resin

Herein we report a practical approach for peptide synthesis using second-generation fibrous polyacrylamide resin (Li-resin, "Li" is coming from the name of its inventor, Yongfu Li). This resin with the corresponding handle was used for solid phase peptide synthesis (SPPS) using a fluorenylmethoxycarbonyl (Fmoc) approach. We reveal that the most appropriate mixing and filtration strategy when using amino-Li-resin in SPPS is via shaking and gravity filtration, instead of mechanical stirring and suction filtration used with other resins. The strategy was demonstrated with the SPPS of H-Tyr-Ile-Ile-Phe-Leu-NH2, which contains the difficult sequence Ile-Ile. The peptide was obtained with excellent purity and yield. We are confident that this strategy will be rapidly implemented by other peptide laboratories.The research was funded by the National Research Foundation (NRF) (Blue Sky’s Research Programme # 120386).Peer reviewe

Solid-Phase Synthesis of an "Inaccessible" hGH-Derived Peptide Using a Pseudoproline Monomer and SIT-Protection for Cysteine

The solid-phase peptide synthesis (SPPS) of the C-terminal sequence of hGH with one extra Tyr attached to its N-terminus (total of 16 residues with a disulfide bridge) has been accomplished for the first time by optimizing several synthetic parameters. First of all, the two Ser residues (positions 9 and 13 of the molecule) have been introduced as a single amino acid, Fmoc-Ser(ψMe,Mepro)-OH, demonstrating that the acylation of these hindered moieties is possible. This allows us to avoid the use of the corresponding dipeptides, Fmoc-AA-Ser(ψMe,Mepro)-OH, which are very often not commercially available or very costly. The second part of the sequence has been elongated via a double coupling approach using two of the most effective coupling methods (DIC-OxymaPure and HATU-DIEA). Finally, the disulfide bridging has been carried out very smoothly by a chemoselective thiol-disulfide interchange reaction between a SIT (sec-isoamyl mercaptan)-protected Cys residue and the free thiol of the second Cys. The synthesis of this short peptide has evidenced that SPPS is a multifactorial process which should be optimized in each case.The authors wish to thank Yoav Luxembourg (Luxembourg Biotechnology, Nes Ziona, Israel) for a generous gift of coupling reagents.Peer reviewe

Understanding OxymaPure as a Peptide Coupling Additive: A Guide to New Oxyma Derivatives

An in silico study, using the GALAS algorithm available in ACD/PhysChem Suite, was performed to calculate the pK a(s) of various oximes with potential application as peptide coupling additives. Among the known oximes and predicted structures, OxymaPure is superior based on the pK a values calculated, confirming the results described in the literature and validating this algorithm for further use in that field. Among the nondescribed oximes, based on pK a calculation, ethyl 2-(hydroxyimino)-2-nitroacetate seems to be a potential candidate to be used as an additive during peptide coupling.The authors wish to thank Sanji K. Bhal (Advanced Chemistry Development, Inc, ACD/Labs) for the help with the review of the article manuscript.Peer reviewe

tert-Butylethylcarbodiimide as an Efficient Substitute for Diisopropylcarbodiimide in Solid-Phase Peptide Synthesis: Understanding the Side Reaction of Carbodiimides with OxymaPure

The undesired reaction between carbodiimides (peptide coupling reagent) and OxymaPure (peptide coupling additive), which takes place in very low extension during peptide bond formation, is dependent on the steric hindrance around the carbodiimide backbone. Carbodiimides containing tertiary substituents on N such as di-tert-butylcarbodiimide do not activate the carboxylic group properly; the presence of secondary substituents such as in the case of diisopropylcarbodiimide (DIC) leads to the formation of oxadiazole and HCN; finally, primary substituents render an adduct of oxadiazine and no formation of HCN. tert-Butylethylcarbodiimide (TBEC), which is a hybrid of primary and tertiary substituents, leads to the formation of oxadiazine with no concomitant formation of HCN. Furthermore, TBEC outperforms DIC in terms of yield and minimization of racemization as it is demonstrated herein.Thanks go to Yoav Luxembourg and Gyorgy Orosz (Luxembourg Bio Technologies) for encouraging this work and for the generous gift of TBEC, DIC, and OxymaPure. This work was partially funded by the National Research Foundation (NRF) (Blue Sky’s Research Programme # 120386).Peer reviewe

Amide Formation: Choosing the Safer Carbodiimide in Combination with OxymaPure to Avoid HCN Release

It has been reported that DIC can react with OxymaPure to render an oxadiazole compound with the concomitant formation of HCN. Here we demonstrate that this reaction is not a feature of all carbodiimides but rather depends on the alkyl structure that flanks the two N atoms of the carbodiimide. Furthermore, we have identified two carbodiimides, TBEC and EDC·HCl, whose reaction with OxymaPure is exempt from HCN formation.We thank Yoav Luxembourg (Luxembourg Bio Technologies) for encouraging this work. This project received funding from the European Union’s Horizon 2020 Research and Innovation Programme under Marie Skłodowska-Curie Grant Agreement 861190 (PAVE). O.L.’s work was funded by PAVE. The authors acknowledge an American Chemical Society (ACS) Green Chemistry Institute (GCI) Pharmaceutical Roundtable Ignition Grant awarded to S.R.M. The work carried out in South Africa was partially funded by the National Research Foundation (NRF) (Blue Sky’s Research Programme 120386). Peptide preparation was performed at the NANBIOSIS–CIBER BBN Peptide Synthesis Unit (U3).Peer reviewe