1. Use of thiol acids in peptide segment coupling in non-aqueous solvents
D Yamashiro, J Blake Int J Pept Protein Res. 1981 Oct;18(4):383-92. doi: 10.1111/j.1399-3011.1981.tb02996.x.
Methods for preparing protected peptide thiol acids by solution and solid-phase procedures have been developed. The coupling of boc-Gly-Ala-SH to polymer-bound Leu was effected with dicyclohexylcarbodiimide (DCC), 2,4-dinitrofluorobenzene (FDNB), N-bromosuccinimide (NBS), and heat. In each case, the resulting H-Gly-Ala-leu-OH was analyzed for the content of H-Gly-D-Ala-Leu-OH (Izumiya test). The sensitivity of the test (0.1% epimer) was increased to 0.003% by developing a double chromatographic procedure involving partition chromatography prior to ion-exchange chromatography. The amounts of DL epimer found in the H-Gly-Ala-Leu-OH for the various tests were (solvent and temperature in parentheses): DCC (CH2Cl2, 0 degrees), 0.012%; DCC (DMF, 24 degrees),0.04%; FDNB (DMF, 24 degrees), 0.53%; NBS (DMF, 24 degrees), 0.46%, heat (DMF, 60 degrees), 0.86%. Under the same conditions conventional couplings of Boc-Gly-Ala-OH gave: DCC (DMF, 24 degrees), 16.5%; DCC/1-hydroxybenzotriazole (DMF, 24 degrees), 0.13%. In a competitive coupling of equimolar amounts of Boc-Gly-Ala-SH and Boc-Gly-Ala-OH with DCC in DMF the level of DL epimer was 0.21%, indicating that the thiol acid can be selectively coupled in the presence of carboxyl groups. In a variation of the test Boc-Gly-Leu-SH coupled with DCC (CH2Cl2, 0 degrees) to resin bound Ala with an epimer level of 0.05%.
2. Peptide processing in the central nervous system
N Marks, A Suhar, M Benuck Adv Biochem Psychopharmacol. 1981;28:49-60.
1. A thiol proteinase from human pituitaries was purified approximately 400 fold and shown to have different chromatographic properties from that of calf brain. Among substrates cleaved were myelin basic protein, histones, beta-lipotropin, neurophysin, and Substance P. 2. The enzyme showed properties associated with a cathepsin-B like enzyme: dependence on -SH groups, pH optimum of 6.5, inhibition by leupeptin and a synthetic analog, Boc-D-Phe-Pro-arginal, and cleavage of dipeptidyl arylamides with basic residues adjacent to or penultimate to the chromatographic grouping. 3. Membranes present in the P2 fraction of rat brain contained three or more enkephalinases when submitted to DEAE-cellulose chromatography. Further purification on an IgG-Sepharose affinity column prepared with antibody to lung angiotensin converting enzyme indicated the presence of dipeptidyl carboxypeptidase(s) with properties distinct from those of ACE. In addition, the DEAE-cellulose fractions contained various aminopeptidase activities when tested with Leu-Gly-Gly, Leu-Nap, and Ala-Ala-Nap as substrates.
3. Protection of asparagine and glutamine during N alpha-Bpoc-based solid-phase peptide synthesis
R I Carey, H Huang, J L Wadsworth, C S Burrell Int J Pept Protein Res. 1996 Mar;47(3):209-13. doi: 10.1111/j.1399-3011.1996.tb01346.x.
In this paper we describe the synthesis and properties of Bpoc-Asn(Trt)-OH, Bpoc-Asn(Trt)-OPfp, Bpoc-Gln(Trt)-OH and Bpoc-Gln(Trt)-OPfp. These derivatives are highly soluble in CH2Cl2 and can be coupled efficiently in solid-phase peptide synthesis. The peptides, acetyl-Ala-Phe-Asn(Trt)-Gly-Leu-Ala-O-Dbf-SH and Boc-Cys(Acm)-Ala-Phe-Gln(Trt)-Gly-Leu-Ala-O-Dbf-SH (where O-Dbf-SH is the peptide ester of 4-mercapto-6-hydroxydibenzofuran) were synthesized by stepwise solid-phase peptide synthesis using N alpha-Bpoc amino acids. We have observed that less than 0.1% of the trityl group is removed from the carboxamide of Gln and Asn during a standard 15 min N alpha-Bpoc deprotection in 0.5% TFA in CH2Cl2.
