Ethyl cyanohydroxyiminoacetate is obtained in the reaction of ethyl cyanoacetate and nitrous acid in 87% yield. Because of the rapid hydrolysis of the ester, the reaction should be carried out at pH 4.5, in buffered phosphoric acid the product can even be obtained in virtually quantitative yield. The compound can be purified by recrystallization from ethanol or ethyl acetate. Compared with the benzotriazole derivatives 1-hydroxybenzotriazole and 1-hydroxy-7-azabenzotriazole , ethyl cyanohydroxyiminoacetate exhibits a markedly slowed thermal decomposition on heating.
Owing to the simple preparative accessibility, the uncritical behavior at temperatures below 80 °C and in particular because of the high yields and the low racemization of the peptides obtained, ethyl cyanohydroxyiminoacetate has now become widely used as an additive in peptide syntheses. Ethyl cyanohydroxyiminoacetate can be used as a coupling additive in the conventional peptide linking in solution, as in automated Merrifield synthesis on a solid-phase peptide synthesis, together with coupling reagents such as carbodiimides, diisopropylcarbodiimide or the water-soluble 1-ethyl-3-carbodiimide ). For example, the stepwise liquid-phase synthesis of the dipeptide Z-L-Phg-L-Val-OMe yields the LL-product with 81-84% which is free from racemic DL dipeptide, using From N-protected Z-L-α-phenylglycine and L-valine methyl ester with the coupling reagent DIC and the additive ethyl cyanohydroxyiminoacetate. More recently, a variety of derivatives of ethyl cyanohydroxyiminoacetate have been developed as acylation reagents, such as Fmoc-oxyma for the transfer of the fluorenylmethoxycarbonyl protective group or the coupling reagent COMU which is readily soluble as a dimethylmorpholine-uronium salt and which, like Oxyma, is superior to the standard additive HOBt for the suppression of racemization and acylation efficiency and is comparable to HOAt without presenting an explosion risk such as the benzotriazoles. With water-soluble derivatives of ethyl cyanohydroxyiminoacetate as additive and DIC as coupling reagent even in weakly basic aqueous solutions the linking of protected amino acids to oligopeptides is possible with a yield of 95% and a diastereomeric excess of> 99% using the model substances Z-L-Phg-OH and L-H-Pro-NH2. In the coupling of amino acids, frequently occurring secondary reactions largely suppressed, which would be the formation of symmetricalacid anhydrides, racemization and epimerization and the cyclization to oxazolinones or - especially for dipeptides - to 2,5-diketopiperazines.
