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Occurrence and Minimization of Cysteine Racemization during Stepwise Solid-Phase Peptide Synthesis(1)(,)(2)
Yongxin Han1, Fernando Albericio, George Barany
1Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, and Department of Organic Chemistry, University of Barcelona, 08028-Barcelona, Spain.
The Journal of Organic Chemistry
|June 27, 1997
Summary
Cysteine racemization during peptide synthesis is a significant challenge. This study identifies conditions and protecting groups that minimize unwanted D-amino acid formation, enabling more accurate peptide synthesis.
Area of Science:
- Organic Chemistry
- Biochemistry
- Medicinal Chemistry
Background:
- Peptide synthesis conventionally assumes minimal racemization of N,S-protected cysteine derivatives.
- Widely used coupling reagents and protocols can lead to significant cysteine racemization during stepwise peptide incorporation.
Purpose of the Study:
- To systematically investigate cysteine racemization levels under various coupling conditions.
- To evaluate the impact of different beta-thiol protecting groups on racemization.
- To identify optimized protocols for minimizing cysteine racemization in peptide synthesis.
Main Methods:
- A quantitative model system using HPLC to resolve D- and L-cysteine containing peptides (H-Gly-Cys-Phe-NH(2)).
- Systematic variation of coupling reagents (e.g., BOP, HBTU, HATU), additives (HOBt, HOAt), bases (DIEA, NMM, TMP), solvents (DMF, CH(2)Cl(2)-DMF), and preactivation times.
- Evaluation of beta-thiol protecting groups: S-acetamidomethyl (Acm), S-triphenylmethyl (Trt), S-2,4,6-trimethoxybenzyl (Tmob), and S-9H-xanthen-9-yl (Xan).
Main Results:
- Standard protocols with phosphonium/aminium salts and preactivation resulted in 5-33% racemization.
- Avoiding preactivation reduced racemization by 6-7 fold.
- Using a weaker base (TMP), reducing base equivalents, and employing a mixed solvent system (CH(2)Cl(2)-DMF) further decreased racemization.
- Optimized conditions achieved <1% racemization per step in Fmoc solid-phase peptide synthesis.
Conclusions:
- Cysteine racemization is a critical issue in peptide synthesis, contrary to prior assumptions.
- Careful selection of coupling conditions, protecting groups, and reaction parameters is essential for minimizing racemization.
- Developed protocols enable efficient and accurate incorporation of cysteine residues in solid-phase peptide synthesis.