Identification and suppression of decomposition during carbodiimide-mediated reactions of Boc-amino acids with phenols, hydroxylamines and amino acid ester hydrochlorides.
N-tert-Butoxycarbonylamino acids (Boc-Xaa-OH) were coupled with p-nitrophenol (HONp) in dichloromethane using N,N'-dicyclohexylcarbodiimide (DCC) and N-ethyl-N'(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and the products were identified and quantitated by high-performance liquid chromatography. Boc-Xaa-OH with Xaa = Val was coupled also with pentafluorophenol (HOPf) and hydroxy-containing additives (HOR), and the products were similarly determined as the methylamides. EDC-mediated reactions of Boc-Xaa-OH gave 8-25% of Boc-Xaa-Xaa-OR as well as Boc-Xaa-OR for R = Ph, Np, Pf, benzotriazole (Bt) and 5-norbornene-endo-2,3-dicarboxamide; DCC-mediated couplings, 5-7% for R = Np and Bt. No dimer was formed in couplings with N-hydroxysuccinimide or 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine. Dimerization was eliminated from DCC-mediated reactions by the addition of 1 equiv. of N-methylmorpholine, from the EDC-mediated reactions by carrying them out in pyridine. Dimerization is attributed to formation of the intermediate 2-alkoxy-5(4H)-oxazolone that undergoes fragmentation to the N-carboxyanhydride, which reacts with the alcohol giving amino acid ester. Ester produces dimer by aminolysis of the O-acylisourea. Decomposition (1.4%) was also detected by analysis for H-Val-Phe-OMe in DCC-mediated reactions of Boc-valine with H-Phe-OMe, and was demonstrated to be caused by the hydrochloride of the ester salt that had been neutralized with N-methylmorpholine. Decomposition was eliminated by the addition of 5% of pyridine, which also had the beneficial effect of suppressing N-acylurea formation.
Benoiton
N L
NL
Department of Biochemistry, University of Ottawa, Ontario, Canada.
Lee
Y C
YC
Chen
F M
FM
D000596:Amino Acids / Q000737:chemistry*; D004024:Dicyclohexylcarbodiimide / Q000737:chemistry*; D004952:Esters; D005563:Formic Acid Esters / Q000737:chemistry*; D006851:Hydrochloric Acid / Q000737:chemistry; D006898:Hydroxylamines / Q000737:chemistry; D008752:Methylene Chloride / Q000737:chemistry; D008956:Models, Chemical; D009596:Nitrophenols / Q000737:chemistry; D010455:Peptides / Q000138:chemical synthesis; D011725:Pyridines / Q000737:chemistry
D000596:Amino Acids; D004952:Esters; D005563:Formic Acid Esters; D006898:Hydroxylamines; D009596:Nitrophenols; D010455:Peptides; D011725:Pyridines; C000345:t-butyloxycarbonyl group; D004024:Dicyclohexylcarbodiimide; D008752:Methylene Chloride; C023666:pyridine; D006851:Hydrochloric Acid; C024836:4-nitrophenol