Abstract:The deprotection step is crucial in order to secure a good quality product in Fmoc solid phase peptide synthesis. 9-Fluorenylmethoxycarbonyl (Fmoc) removal is achieved by a two-step mechanism reaction favored by the use of cyclic secondary amines; however, the efficiency of the reaction could be affected by side reactions and by-product formation. Several aspects have to be taken into consideration when selecting a deprotection reagent: its physicochemical behavior, basicity (pKa) and polarity, concentration, and time of reaction, toxicity and disposability of residues and, finally, availability of reagents. This report presents a comparison of the performance of three strategies for deprotection using microwave-assisted Fmoc peptide synthesis. Four peptide sequences were synthesized using Rink amide resin with a Liberty Blue™ automated synthesizer and 4-methylpiperidine (4MP), piperidine (PP), and piperazine (PZ) as Fmoc removal reagents. In the first instance all three reagents behaved similarly. A detailed analysis showed a correlation between the hydrophobicity and size of the peptide with the yield and purity of the obtained product. The three reagents are interchangeable, and replacement of piperidine could be advantageous regarding toxicity and reagent handling.
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.
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.
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