Peptide synthesis 2- -1,1,3,3-tetramethyluronium hexafluorophosphate

Peptide synthesis reagents such as diisopropylcarbodiimide (DIC), benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP), 2-(lH-benzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (FlOBt), piperidine, A-methylmorpholine (NMM), trifluoroacetic acid (TEA), triisopropylsilane (TIS), A -diisopropylethylamine (DIPEA, DIEA), l,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,2-ethanedithiol (EDT), and 4-dimethylaminopyridine (DMAP) may be obtained from Sigma-Aldrich, Chemlmpex, and Novabiochem. 

For both the preliminary and fined test peptides, it was necessary to prepare all possible isomers as standards for analysis. These reference peptides were synthesized using a PE-ABD 430A peptide synthesizer configured for FastMoc synthesis protocols. Piperidine (20%) in N-methylpyrrolidone (NMP) was used to deprotect the N-terminal amino acid. To activate each amino acid in the reaction vessel, the following was used 1.0 mM amino acid, 1.0 mM 2-(lH-benzotriazol-l-yl)-l-l-2,3, tetramethyluronium hexafluorophosphate (HBTU) in 1 M 1-hydroxybenzotriazole (HOBT) in NMP, 2.0 mM diisopropylethylamine (DIEA). Each His residue was double-coupled. HBTU, DIEA, piperidine, DMF and NMP were purchased from ABI. Methylene chloride was purchased from Burdick and Jackson. 

Figure 2.2 Modern solid phase peptide synthesis. Process begins with a-N terminal Fmoc deprotection of resin bound C-terminal amino acid residue with piperidine (mechanism illustrated). Peptide link formation follows (typical solvent Al-methylpyrrolidone [NMP]) by carboxyl group activation with dicyclohexylcarbodiimide (DCC) (mechanism illustrated) in presence of hydroxybenzotriazole (HOBt). HOBt probably replaces DCC as an activated leaving group helping to reduce a-racemization during peptide link formation. Other effective coupling agents used in place of DCC/HOBt are HBTU 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate Py-BOP benzotriazole-l-yl-oxy-tns-pyrrolidino-phosphonium hexafluorophosphate. The Process of a-N deprotection, and peptide link formation, continues for as many times as required (n-times), prior to global deprotection and resin removal.

Lansbuiy and coworkers have demonstrated diat the amount of aspartimide formed in convergent iV-glycopeptide synthesis correlates directly with the amount of base present in the reaction mixture 34, 36). For example, coupling of the aspartimide-prone peptide 1 and 7V-acelyl-p-glucosaminylamine with 2-(1 H-benzotriazol-1 -y 1)-1,1,3,3 -tetramethyluronium hexafluorophosphate... 

Another potential problem with DCC is that at the completion of the reaction some DCU remains in solution with the product, necessitating additional purification. Water-soluble carbodiimide derivatives such as l-Cyclohexyl-3-(2-morpholinoethyl)carbodiimide Metho-p-toluenesulffonate and l-Ethyl-3-(3 -dimethylaminopropyl)carbodiimide Hydrochloride (EDCI) obviate this problem, as they are removed by a simple extraction. Many newer coupling agents have been developed for peptide synthesis and other acylation reactions. These include Benzotriazol-l-yloxytris(dimethylamino)phosphonium Hexafluorophosphate (BOP)," 0-Benzotriazol-l-yl-N,N,N, N -tetramethyluronium Hexafluorophosphate (HBTU)," Bis(2-oxo-3-oxazolidinyl)phosphinic Chloride (BOP-Cl), and (1 //-1,2,3-benzotriazol-1 -yloxy)tris(pyrTolidino)phosphonium hexafluorophosphate (PyBOP). In addition to linear and polymeric amides, lactams of various ring sizes have been synthesized using these methods (eq 1)."... 

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