Letters in Peptide Science, 7: 113–121, 2000. KLUWER/ESCOM © 2000 Kluwer Academic Publishers. Printed in the Netherlands. 113 Addition of HOXt (X = A or B) improves the efficiency of phenol-based coupling reagents during peptide synthesis Ayman El-Faham University of Alexandria, Faculty of Science, Department of Chemistry, PO Box 426, Ibrahimia, 21321 Alexandria, Egypt Received 15 January 2000; Accepted 22 February 2000 Key words: epimerization, phenol-based coupling reagent, solid phase peptide assembly Summary In the course of comparing the effectiveness of HATU, HBTU, and phenol-based coupling reagents, such as the pentafluorophenyl, 2-nitrophenyl, and 2,4,5-trichlorophenyluronium salts by (a) formation of Fmoc-Ala-Val-OtBu, (b) (2+1) segment coupling and (c) stepwise solid phase peptide assembly of typical model peptides such as the pentapeptide H-Tyr-Aib-Aib-Phe-Leu-NH 2 and ACP decapeptide (65–74), we found a striking improvement of the less effective phenol-based coupling reagents (HPyOPfp, HPyONp, and HPyOTcp), both with regard to reaction rate and extent of epimerization, when HOAt was added and a clear superiority of HAPyU (in the presence and absence of HOAt) relative to the compounds derived from HOBt, HOPfp, HONp, and HOTcp. Abbreviations: Aib, α-aminoisobutyric acid; DIEA, diisopropylethylamine; TMP, collidine, 2,4,6-tri- methyl- pyridine; DMF, N, N-dimethylformamide; HOBt, 1-hydroxybenzotriazole; HOAt, 7-aza-1-hydroxybenzotriazole; HAPyU, 1-(1-pyrrolidinyl-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene)-N-methylmethanaminium hexafluo- rophosphate N-oxide; HOPfp, pentafluorophenol; HONp = 2-nitrophenol; HOTcp, 2,4,5-trichlorophenol; HPyOPfp, bis(tetramethylene)pentafluorophenoxyformamidinium hexafluorophosphate; HpyONp bis(tetra- methylene)-2-nitrophenoxyformamidinium hexafluorophosphate HPyOTcp, bis(tetramethylene)-2,4,5-trichlo- rophenoxyformamidinium hexafluorophosphate; BTCFH, bis(tetramethylene)chloroformamidinium hexafluoro- phosphate. Amino acids and peptides are abbreviated and designated following the rules of the IUPAC-IUB Commission of Biochemical Nomenclature [J. Biol. Chem., 247 (1972) 977]. Introduction In the field of peptide synthesis, pentafluorophenyl, 2-nitrophenyl, and 2,4,5-trichlorophenyl esters of amino acids have been reported to be highly effi- cient in amide bond formation [1–3]. Habermann et al. [4] reported the synthesis and application of sev- eral pentafluorophenol-based in situ activation agents. These reagents were first investigated for coupling re- actions carried out in solution. In these studies, the uronium salt-based reagent HPyOPfp 3 proved to be highly efficient in certain cases. In a preliminary publication [5], we reported a striking improvement of the less effective HOPfp- based coupling reagent HPyOPfp 3, both with regard to reaction rate and extent of epimerization when HOAt was added to the reaction mixture. Due to the importance of these coupling reagents, we have exten- ded our studies to determine whether similar effects are possible for several new phenol-based in situ activ- ation reagents derived from the very inexpensive 2-ni- tro- and 2,4,5-trichlorophenol. Among these are: bis- (tetramethylene)-2-nitrophenoxyformamidinium hex- afluorophosphate (HPyONp) 4, and bis(tetramethy- lene)-2,4,5-trichlorophenoxyformamidinium hexaflu- orophosphate (HPyOTcp) 5.