1. Studies on sensitivity to racemization of activated residues in couplings of N-benzyloxycarbonyldipeptides
N L Benoiton, Y C Lee, R Steinaur, F M Chen Int J Pept Protein Res. 1992 Dec;40(6):559-66. doi: 10.1111/j.1399-3011.1992.tb00441.x.
A series of 24 peptides Z-Gly-Xaa(R)-OH where Xaa = 15 different residues and R = H, NH2, tBu, Bzl, Trt, Mtr, and StBu were coupled with valine benzyl ester in dimethylformamide or dichloromethane at +5 degrees. The accompanying racemization was determined by analysis of the epimeric products by normal phase high-performance liquid chromatography (HPLC) for Xaa(R) = Met, Cys(StBu) and Lys(Z) and by reversed-phase HPLC after removal of benzyl-based protecting groups for Xaa(R) = Ser(tBu), Thr(tBu) and Arg(Mtr). The coupling methods examined included mixed anhydride (MxAn) at -5 degrees, and N,N'-dicyclohexylcarbodiimide (DCC), benzotriazol-1-yl-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluroniumhexafluorophosp hate (HBTU) in the presence of 1-hydroxybenzotriazole (HOBt). Very few couplings gave stereochemically pure products. The order of sensitivity to racemization of residues depended on the method of coupling and the solvent. It varied most when comparing MxAn to HOBt-assisted reactions; it varied moderately when comparing HOBt-assisted reactions. There was less variation in comparing BOP and HBTU reactions that are initiated by the same mechanism. Leu, Nle, Phe, Asn, Lys(Z) and Asp(OBzl) are identified as the residues least sensitive to racemization. DCC-HOBt generally led to less epimerization than the other methods.
2. An alternative solid phase peptide fragment condensation protocol with improved efficiency
N Mihala, J Bódi, A Gömöry, H Süli-Vargha J Pept Sci. 2001 Oct;7(10):565-8. doi: 10.1002/psc.352.
The success of solid phase peptide synthesis is often limited by the aggregation of the growing peptide chains on the resin. Working from the results of a study of model coupling reactions in solution between Z-Gly-Phe-OH and H-Phe-OBzl, we have achieved higher efficiency in the repetitive solid phase fragment condensation of VGVAPG, in a 3:1 chloroform-phenol solvent system, using diisopropylcarbodiimide (DIC) as coupling agent, and a combination of 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbt) and its tetrabutyl ammonium salt as additive, than in DMF with DIC and HODhbt alone.
3. Ligand bindings of bovine carboxypeptidase B. III. Hydrophobic activators in dipeptide hydrolysis
K Kuroda, H Akanuma, Y Sukenaga, H Sugihara, M Yamasaki J Biochem. 1980 Jun;87(6):1681-9. doi: 10.1093/oxfordjournals.jbchem.a132912.
Several hydrophobic compounds acted as activators in dipeptide (Bz-Gly-L-Arg-OH, Z-Gly-L-Phe-OH) hydrolysis by bovine carboxypeptidase B. These hydrophobic compounds include Bz-Gly-OH, Z-Gly-OH, Z-L-Phe-OH, and Z-L-Phe-GLy-OH. These compounds were indicated to bind to the secondary substrate binding sites which is proposed to be responsible for substrate activation kinetics in dipeptide hydrolysis. Of the compounds Z-L-Phe-OH alone acted also as a inhibitor at higher concentrations, indicating that it binds to both primary and secondary sites as the dipeptide substrates do. Comparison of the activation effects of the compounds employed indicated that hydrophobic interaction played an important role in binding to the secondary site. Substrate and modifier binding constants were also determined and the results indicated that modifier binding increased both affinity and catalytic rate constant of the primary site. On the other hand, Z-Gly-OH and Z-L-Phe-Gly-OH inhibited the hydrolyses of tri and tetrapeptide substrates. This observation suggests that the secondary site is contained in the extended active center which the enzyme possibly has.
