The use of N-urethane-protected N-carboxyanhydrides (UNCAs) in amino acid and peptide synthesis.

N-Urethane-protected N-carboxyanhydrides (UNCAs) are very reactive amino acid derivatives. They have been successfully used in peptide synthesis, in both solution and solid phase. We have demonstrated that UNCAs are interesting starting materials for the synthesis of various amino acid derivatives. Chemoselective reduction of UNCAs with sodium borohydride led the corresponding N-protected beta amino alcohols. Reaction of UNCAs with Meldrum's acid, followed by cyclisation, yielded enantiomerically pure tetramic acid derivatives. Diastereoselective reduction of tetramic acid derivatives produced [4S,5S)-N-alkoxycarbonyl-4-hydroxy-5-alkylpyrrolidin-2-ones derived from amino acids, which after hydrolysis yielded statine and statine analogues. Tetramic acid derivatives could also be obtained by reaction of UNCAs with benzyl ethyl malonate in the presence of sodium hydride to yield gamma-N-benzyloxycarbonylamino-beta-oxodicarboxyl esters followed by hydrogenolytic deprotection and decarboxylation. UNCAs also reacted with phosphoranes to produce the ketophosphorane in excellent yields. Subsequent oxidation with oxone or with [bis(acetoxy)-iodo]-benzene produced vicinal tricarbonyl derivatives. These reactions usually proceeded smoothly and with high yields.

Synthesis of an IgG fragment decapeptide exhibiting phagocytosis stimulating activity of polymorphonuclear leucocytes.

Fragment 335-344 of human IgG was synthesized stepwise, using active esters of N-protected amino acids and benzotriazolyloxytris (dimethylamino)phosphonium hexafluorophosphate for coupling. This fragment could be liberated from the carrier molecule by the two specific enzymes responsible for the release of tuftsin from the same carrier molecule: tuftsin endocarboxy-peptidase cleaves at the Arg-Glu bond and leukokininase at the Lys-Thr bond. This decapeptide fragment, Thr-Ile-Ser-Lys-Ala-Lys-Gly-Gln-Pro-Arg showed significant activity in stimulating phagocytosis of human polymorphonuclear leucocytes.

New synthetic and natural tuftsin-related compounds and evaluation of their phagocytosis-stimulating activity.

The preparation of large amounts of tuftsin (50 g) as well as purification by preparative HPLC are presented. Synthesis of several analogues exhibiting significant phagocytosis-stimulating activity of human PMN are described and discussed. On the other hand, the synthesis of a natural human IgG decapeptide fragment 335-344 that could be liberated from the same carrier molecule is presented. This decapeptide fragment, Thr-Ile-Ser-Lys-Ala-Lys-Gly-Gln-Pro-Arg, showed interesting activity in stimulating phagocytosis of human polymorphonuclear leukocytes.

Synthesis and some pharmacological properties of Z-Tyr(SO3H)-Met-Gly-Trp-Met-Asp(Phe-NH2)-OH, a 32-beta-aspartyl analogue of cholecystokinin (pancreozymin) 27-33.

The heptapeptide carbobenzoxy-L-tyrosyl(O-sulfate)-L-methionylglycyl-L-tryptophyl-L-methionyl-L- aspartyl-beta-L-phenylalanine amine (Z-32-beta-Asp-CCK-27-33) was synthesized and tested for its ability to stimulate secretion from dispersed pancreatic acini in vitro, to increase protein secretion from cat pancreas in vivo, and to cause contraction of guinea pig gallbladder in situ. In increasing amylase secretion in vitro, the Z-32-beta-Asp-CCK-27-33 was equal in efficacy with but approximatively one-third as potent as the Boc-CCK-27-33, and when tested in vivo its activity is approximately 10 Ivy dog units (Idu)/microgram. In stimulation of the contraction of the gallbladder, it showed an activity lower than 1 Idu/microgram. This analogue has more pancreozyminic activity than cholecystokin-like activity. This seems to indicate different affinities for the two receptors.

Activated N-nitrosocarbamates for regioselective synthesis of N-nitrosoureas.

A practical and convenient method for synthesizing antitumor compounds, N-alkyl-N-nitrosoureas, regioselectively nitrosated on the nitrogen atom bearing the alkyl group is proposed. N-Alkyl-N-nitrosocarbamates are interesting intermediates in these syntheses and yield, by reaction with amino compounds, the regioselectively nitrosated N-alkyl-N-nitrosoureas. As an interesting example, N,N'-bis[(2-chloroethyl)nitrosocarbamoyl]cystamine, a new attractive oncostatic derivative, has been prepared. The cytotoxic activity of these various compounds were tested on L1210 leukemia.

Bactericidal activity of tuftsin.

The biological activities of the phagocytosis stimulating tetrapeptide, Thr-Lys-Pro-Arg are discussed. A brief account on the stimulation by tuftsin of phagocytosis of various particles, including bacteria was reported. Stimulation of bactericidal activity by this tetrapeptide was investigated in vitro as well as in vivo. The potency of tuftsin to enhance blood clearing of Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Serratia marcescens by mouse peritoneal macrophages was demonstrated. Bactericidal activity and effects of tuftsin on this phenomenon were studied in liver and spleen of mice. Tuftsin stimulates these activities. Same experiments were performed in infected leukemic mice by Serratia marcescens or Escherichia coli. Results on blood clearing and bactericidal activities in liver and spleen were reported and compared to those of healthy and leukemic untreated animals. Tuftsin was found to present interesting stimulatory effects on the bactericidal activity of phagocytes.

Microbiological transformation of 1,2-diphenyl-3,5 dioxo-4-n-butyl pyrazolidine (phenylbutazone).

Microbiological conversion of phenylbutazone with Rhizopus arrhizus Fischer gave 4-hydroxy phenylbutazone in excellent yield. Spectral studies, mass spectrometry, in particular, were made of the obtained metabolite; other experiments performed with the intact microorganism or with the cell-free system, as well as studies with the well-known metabolic inhibitor, SKF 525 A, indicated that the obtained 4-hydroxy phenylbutazone was not an artefact. Similar experiments were performed on oxyphenbutazone.

[Lengthening of the peptide chain at the C-terminal end of a glycosylamino acid]. / Allongement de la chaîne peptidique du côté C-terminal d'un glycosylaminoacide

An O-glycodipeptide was synthesized by lengthening the peptide chain on the C-terminal side of a glycosylamino acid unit. N-(Benzyloxycarbonyl)-3-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-L-threonine o-nitrophenyl ester and pentachlorophenyl ester were condensed with glycine benzyl ester to give both the same glycodipeptide, [N-(benzyloxycarbonyl)-3-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-L-threonyl]glycine benzyl ester (9). The two anomers, N-(benzyloxycarbonyl)-3-O-(2,3,4,6-tetra-O-acetyl-alpha- and beta-D-glucopyranosyl)-L-threonine pentachlorophenyl ester were also prepared. 1H-N.m.r. studies ascertained the structure and anomeric configuration expected.