1. Inhibition of growth of HeLa cells by new synthetic protease inhibitors
S Mori, Y Kozaki, M Kato, A Tendo, Y Kikawa, H Sekine, M Muramatu J Biochem. 1984 Jun;95(6):1617-23. doi: 10.1093/oxfordjournals.jbchem.a134774.
Tryptic hydrolysis of benzoyl-DL-arginine p-nitroanilide was competitively inhibited by phenyl and substituted phenyl esters of trans-4-guanidinomethylcyclohexanecarboxylic acid (GMCHA), amidinopiperidine-4-carboxylic acid (APCA), amidinopiperidine-3-carboxylic acid (AP3CA), amidinopiperidine-4-acetic acid (APAA), amidinopiperidine-4-propionic acid (APPA), amidinopiperidine-3-propionic acid (AP3PA), amidinopiperidine-4-butyric acid (APBA), and amidinopiperidine-3-butyric acid (AP3BA). The 4-tert-butylphenyl (tBP) ester of APPA was the most effective inhibitor, its K1 value being 5.0 X 10(-7) M. The free acids and phenols had no inhibitory effect at 10(-3) M. The tBP esters of GMCHA, APPA, and APBA caused 50-60% inhibition of growth of HeLa cells, their effects being dose-dependent, while same esters of APCA, AP3PA, APPA, AP3PA, and AP3BA inhibited the growth by 30-40%. The phenyl esters of these were less inhibitory than the tBP esters. A protease preparation obtained from HeLa cells by sonication and ultrafiltration through a molecular sieve membrane strongly hydrolyzed the fluorescent peptides Boc-Val-Pro-Arg-MCA and Bz-Arg-MCA. This proteolytic activity was not affected by soybean trypsin inhibitor but was strongly inhibited by the tBP esters of GMCHA, APAA, and APBA, their effects roughly paralleling their inhibitions of the growth of HeLa cells.
2. Solid-Phase Total Synthesis of Bacitracin A
Jinho Lee, John H. Griffin, Thalia I. Nicas J Org Chem. 1996 Jun 14;61(12):3983-3986. doi: 10.1021/jo960580b.
An efficient solid-phase method for the total synthesis of bacitracin A is reported. This work was undertaken in order to provide a general means of probing the intriguing mode of action of the bacitracins and exploring their potential for use against emerging drug-resistant pathogens. The synthetic approach to bacitracin A involves three key features: (1) linkage to the solid support through the side chain of the L-asparaginyl residue at position 12 (L-Asn(12)), (2) cyclization through amide bond formation between the alpha-carboxyl of L-Asn(12) and the side chain amino group of L-Lys(8), and (3) postcyclization addition of the N-terminal thiazoline dipeptide as a single unit. To initiate the synthesis, Fmoc L-Asp(OH)-OAllyl was attached to a PAL resin. The chain of bacitracin A was elaborated in the C-to-N direction by sequential piperidine deprotection/HBTU-mediated coupling cycles with Fmoc D-Asp(OtBu)-OH, Fmoc L-His(Trt)-OH, Fmoc D-Phe-OH, Fmoc L-Ile-OH, Fmoc D-Orn(Boc)-OH, Fmoc L-Lys(Aloc)-OH, Fmoc L-Ile-OH, Fmoc D-Glu(OtBu)-OH, and Fmoc L-Leu-OH. The allyl ester and allyl carbamate protecting groups of L-Asn(12) and L-Lys(8), respectively, were simultaneously and selectively removed by treating the peptide-resin with palladium tetrakis(triphenylphosphine), acetic acid, and triethylamine. Cyclization was effected by PyBOP/HOBT under the pseudo high-dilution conditions afforded by attachment to the solid support. After removal of the N-terminal Fmoc group, the cyclized peptide was coupled with 2-[1'(S)-(tert-butyloxycarbonylamino)-2'(R)-methylbutyl]-4(R)-carboxy-Delta(2)-thiazoline (1). The synthetic peptide was deprotected and cleaved from the solid support under acidic conditions and then purified by reverse-phase HPLC. The synthetic material exhibited an ion in the FAB-MS at m/z 1422.7, consistent with the molecular weight calculated for the parent ion of bacitracin A (MH(+) = C(73)H(84)N(10)O(23)Cl(2), 1422.7 g/mol). It was also indistinguishable from authentic bacitracin A by high-field (1)H NMR and displayed antibacterial activity equal to that of the natural product, thus confirming its identity as bacitracin A. The overall yield for the solid-phase synthesis was 24%.
3. Quinone cross-linked polysaccharide hybrid fiber
Yoshiko Kuboe, Hitomi Tonegawa, Kousaku Ohkawa, Hiroyuki Yamamoto Biomacromolecules. 2004 Mar-Apr;5(2):348-57. doi: 10.1021/bm034363d.
The present article describes the synthesis of the N-(Lys-Gly-Tyr-Gly)-chitosan using the water-soluble active ester method, the preparation of the N-(Lys-Gly-Tyr-Gly)-chitosan-gellan hybrid fibers, and the reinforcement of the hybrid fibers by enzymatic cross-linking between the N-grafted peptides chains of chitosan. The cationic polysaccharide chitosan was treated with Boc-Lys(Z)-Gly-Tyr(Bzl)-Gly (4-hydroxyphenyl)dimethylsulfonium methyl sulfate ester in DMF-0.15 M acetic acid to incorporate the peptides into the side chain amino groups of chitosan followed by the acidic removals of the Z and Bzl groups. The degrees of N substitution were estimated to be 2.0 and 10 molar % by changing the molar ratios of the amino groups of the parent chitosan and the active ester. The resulting cationic N-(Lys-Gly-Tyr-Gly)-chitosan was spun into the hybrid fibers with the anionic polysaccharide gellan in water. The tensile strengths of the N-(Lys-Gly-Tyr-Gly)-chitosan hybrid fibers were superior to those of the original chitosan-gellan fibers. The mechanical strengths of the hybrid fibers further increased upon enzymatic oxidation using tyrosinase. Based on these results, we concluded that the covalent cross-linking due to the enzyme oxidation between the grafted peptides significantly contributed to reinforcement of the polysaccharide hybrid fibers. The present results afford a new methodology for the reinforcement achieved by the polymer modification inspired by a biological process.
