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For the purpose of a total synthesis of bacitracin A, a synthetic method of the thiazoline peptide was investigated. A benzyloxycarbonylaminoalkylimino ethyl ether derived from glycine, L-alanine, L-valine, L-leucine or L-isoleucine was coupled with ethyl L-cysteinate hydrochloride to afford the corresponding thiazoline peptide, that is, a dehydrated form of cysteine peptide. While L-cystine was obtained from acid hydrolyzates of those thiazoline peptides, hydrolysis of them after treatment with alkali or ammonia gave DL-cystine. Furthermore, alkaline treatment caused a racemization of the amino acid residue adjacent to the thiazoline ring too.

Endotoxin caused cytotoxic damages of mouse bone marrow cells and an increase in thromboplastin (Tp) activity of membrane fragments of the cells. Activities of synthetic lipid A analogs were compared with those of natural lipid A. Injection of 50μg of 406 increased cytotoxicity of the cells from 16.1±0.7 (control) to 26.4±1.7%, and the activity was comparable to that of natural lipid A (27.4±1.2%, dye exclusion test). Compounds 404 and 405 also induced cytotoxicity, however, compound 403 had no activity, compounds 503 to 506, which have double acyl groups at C2′ and C3′ positions, increased cytotoxicity. Among these analogs, compound 506 had the highest activity (from control, 16.1±1.9 to 32.6±1.6%). Compound 406 or 506 caused dose-dependent induction of Tp activity in the cells. Tp activity in the cells increased by injection of as little as 6μg of compound 406, 506 or by natural lipid A. Tp activity in the cells induced by compound 406 decreased after incubation with either Concanavalin A or Polymyxin B.These findings indicate that active principle of endotoxin, as to the induction of cytotoxicity and Tp activity in bone marrow cells, is the lipid A portion. Hydroxymyristic acid at C3′ position and two phosphate groups at Cl and C4′ positions were thought to be necessary for the induction of both activities. Present studies further supported our hypothesis that membrane perturbation due to endotoxin would result in the induction of Tp activity.

A new amino acid named galantinamic acid was found as one of the constituent amino acids in a peptide antibiotic galantin I. The primary structure of galantinamic acid was determined chemically and spectrometrically to be 6,10-diamino-2,3,5-trihydroxydecanoic acid.

The chemical structure of capreomycin, antituberculous peptide antibiotic, was revised from the results of NMR-analysis in comparison with tuberactinomycins. Capreomycin IA and IB were concluded to possess the similar amino acid sequences in their cyclic peptide moieties to those of tuberactionomycins.

4-Azidobutyryl esters formed from hydroxyl compounds and 4-azidobutyryl chloride were shown to be cleaved via reduction to 4-aminobutyrate followed by intramolecular O→N-acyl migrations to regenerate the free hydroxyl groups. High susceptibility of azide group to reduction enables the selective removal of this acyl group leaving many other functional groups unchanged.

Owl monkeys (Aotus trivirgatus griseimembra) were effectively immunized against a human malaria parasite, Plasmodium falciparum. Two injections of antigen, primarily mature segmenters with fully developed merozoites, mixed with adjuvant (6-O-stearoyl-N-acetylmuramyl-L-alanyl-D-isoglutamine and liposomes) were administered intramuscularly at a 4-week interval. Approximately 2 weeks after the second vaccination, the monkeys were challenged with the homologous strain of P. falciparum. All immunized monkeys survived the challenge. The substitution of Freund's complete adjuvant is an encouraging step toward the development of an effective and safe vaccine for human malaria.

A new amino acid, galantinic acid, was found as one of the constituent amino acids in a peptide antibiotic galantin I. The structure of galantinic acid was determined to be (2S,4S,5S)-5-amino-2-carboxymethyl-4-hydroxytetrahydropyran on the basis of NMR and CD studies.

Reduction of 7,8-didemethyl-8-hydroxy-[5-2H]-5-deazariboflavin by the selenium-containing hydrogenase from Methanococcus vannielii gave a C-5 chirally labeled 1,5-dihydro derivative. The absolute configuration of the chiral label was shown to be (R) by comparison of the chemically degraded product with authentic samples of known absolute configurations. Therefore, the steric course of the enzymic reactions involving the 8-hydroxy-5-deazaflavin cofactor can be defined as follows: (a) reduction occurs on the si face of the 5-deazaflavin molecule; (b) oxidation proceeds by the abstraction of the pro-S hydrogen at C-5 of the 1,5-dihydro-5-deazaflavin. Thus, the selenium-containing hydrogenase and 8-hydroxy-5-deazaflavin-dependent NADP+ reductase from M. vannielii are si face specific.

Tuberactinomycin O, one of the four congeners of the antituberculous peptide tuberactinomycin, was totally synthesized. The beta-ureidodehydroalanine moiety was constructed from beta,beta-diethoxyalanine with excess urea in acidic medium after a cyclization reaction of a pentapeptide was finished. Cyclization was carried out by means of the 1-succinimidyl ester method. To the cyclic pentapeptide, beta-lysine was introduced as the branched moiety and then deprotected to afford tuberactinomycin O which was completely identified with the natural form of the antibiotic.

Six analogs of an antituberculous antibiotic capreomycin were synthesized in order to clarify the structure-activity relationship, especially with regard to the significance of the β-amino group in α,β-diaminopropionic acid residue as well as the position of linkage of the branch residue, β-lysine, to the cyclic peptide moiety. All the synthetic products were found to have the same conformations in solution as those of the natural antibiotics in terms of NMR spectra. It was found from their antibacterial activities that an amino group located at β-position of the α,β-diaminopropionic acid residue adjacent to ureidodehydroalanine residue remarkably strengthens the biological activity, and that the position of a branch does not significantly influence the antibacterial potency.

Adjuvant and antitumor activities of synthetic 6-O-"mycoloyl"-N-acetylmuramyl-L-alanyl-D-isoglutamine were examined. All the synthetic 6-O-corynomycoloyl-, 6-O-mocardomycoloyl-, and 6-O-mycoloyl-N-acetylmuramyl-L-alanyl-D-isoglutamine were active as adjuvants for cell-mediated immune responses. However, 6-O-mycoloyl-N-acetylmuramyl-L-alanyl-D-isoglutamine was less active as an adjuvant on circulating antibody formation. It was shown that pyrogenic activity of N-acetylmuramyldipeptide was reduced by 6-O-acylation with mycolic acid, but not with nocardomycolic or corynomycolic acid. Tumor-suppression activity was observed by the synthetic 6-O-mycoloyl-N-acetylmuramyl-L-alanyl-D-isoglutamine by using transplantable tumor in syngenic mice.

Cyclic guanidino amino acid, capreomycidine, is a component of antituberculous peptides, capreomycin and tuberactinomycin N and O. Its epimer was also found in protease inhibitors, chymostatin and elastatinal. A new synthetic approach to DL-capreomycidine via β-hydroxyornithine was studied for the purpose of total synthesis of capreomycidine and epicapreomycidine of natural forms or its hydroxy derivative, tuberactidine.

Tuberactinamine N, the cyclic peptide moiety of tuberactinomycin N, was obtained in a crystalline state through liberation of gamma-hydroxy-beta-lysine from tuberactinomycin N by acid treatment. Tuberactinamine N possesses an intramolecular hydrogen bond in its molecule and showed antibacterial activities comparable to those of the original antibiotics. Conversion of tuberactinomycin N to O was achieved through coupling of diacyl-beta-lysine with tuberactinamine N followed by removal of the protecting groups.

Activation of peritoneal macrophages from guinea pigs by various bacterial cell walls, M-1 endo-N-acetylmuramidase enzymatically digested bacterial cell walls and synthetic muramyl dipeptides was studied in terms of stimulation of [14C] glucosamine incorporation. All test bacterial cell wall preparations significantly increased a [14C]glucosamine uptake by the macrophages. Some of the water-soluble M-1 enzyme digests also exerted stimulating effects on macrophages, although the activity of the digests was found to be weaker than those of original cell walls. Furthermore, an adjuvant-active synthetic MurNAc-L-Ala-D-isoGln (MDP) showed a weak but significant activity, whereas an adjuvant-inactive analog, MurNAc-L-Ala-L-iso-Gln, did not show a significant activity, at least with the dose of 100 microgram. Additional studies with 6-O-acyl derivatives of MDP revealed that 6-O-(2-tetradecylhexadecanoyl)-MDP and 6-O-(3-hydroxy-2-tetradecyl-octadecanoyl)-MDP exhibit stronger macrophage-stimulating effects than MDP. It can be concluded from the above findings that MDP is the essential structure responsible for stimulating the activity of cell walls on guinea pig peritoneal macrophages, but it requires a particle state, which results from an additive character of lipophilicity, to exert the activity fully and effectively.

Total syntheses of [Ala3, Ala4]-, [Ala3]-, and [Ala4]-tuberactinomycin O corresponding to deoxy analogs of natural antibiotic tuberactinomycin O were achieved for the purpose of elucidation of relationship between chemical structure and biological activity. Cyclization of the pentapeptide intermediates at two different positions was investigated. Minimum inhibitory concentrations of the synthetic analogs against Mycobacterium or Corynebacterium indicated that both hydroxy groups of two serine residues of tuberactinomycins are not required for antibacterial activity, nor participated to drug-resistancy of bacilli. These analogs were found to have the same conformations in solution as those of natural antibiotic from the results of NMR, ORD, and CD measurements.

The immunogenicity and antigenicity of synthetic Escherichia coli lipid A (compound 506) and its 1- and 4'-monophosphorylated derivatives (compounds 505 and 504, respectively) and nonphosphorylated derivative (compound 503) were compared with those of bis- and 4'-monophosphorylated natural free lipid A from E. coli. The synthetic compounds under study were either coated onto sheep erythrocytes (except for the water-insoluble preparation 503) or incorporated into liposomes and used for the immunization of rabbits. Both types of immunogens (the latter representing fully synthetic immunogens) resulted in high-titered polyclonal antisera which were characterized before or after absorption in a passive hemolysis assay as well as in a passive hemolysis inhibition assay with the synthetic compounds as test antigens. All antisera were found to react with their corresponding homologous antigens coated onto sheep erythrocytes, with titers of up to 2,048, and were comparable to those antisera obtained after immunization with natural lipid A exposed on the bacterial surface after acid hydrolysis. Antisera against bisphosphorylated compound 506 were highly specific for the homologous antigens, showing no interaction with compounds 504 and 505 in the passive hemolysis test. The same held true for the absorption experiments in which glutaraldehyde-fixed sheep erythrocytes were sensitized with the respective antigens. Antisera against monophosphorylated compounds 504 and 505 exhibited, besides their expected homologous reactivity, complete cross-reactivity with compound 506, but they did not cross-react with each other. Thus, anti-504 and anti-505 antibodies recognized distinct antigenic determinants, being related to the ester linked 4'-phosphate or the glycosidically linked 1-phosphate, respectively. Both antigenic determinants were also expressed by bisphosphorylated compound 506 used as an antigen; however, upon immunization, only antibodies against compound 506 were elicited.

A new amino acid, galantinic acid, was found as one of the constituent amino acids in a peptide antibiotic galantin I. The structure of galantinic acid was determined to be (2S,4S,5S)-5-amino-2-carboxymethyl-4-hydroxytetrahydropyran on the basis of NMR and CD studies.

Cell walls isolated from four bacterial species (Streptococcus pyogenes, Lactobacillus plantarum, Streptomyces gardneri, and Nocardia corynebacteriodes), which exhibited the adjuvant effect of stimulating cellular and humoral immune responses against ovalbumin in guinea pigs, caused the slow-starting and long-lasting contraction of guinea pig ileal strips suspended in Tyrode solution. In contrast to these cell walls active in immunoadjuvancy, those isolated from five bacterial species (Micrococcus lysodeikticus, Staphylococcus epidermidis, Arthrobacter atrocyaneus, Corynebacterium insidiosum, and Ampullariella regularis), which lacked immunoadjuvancy at least in intact walls, caused no or very weak contraction of the ileal strips. Further study demonstrated that both a monomer and a polymer of disaccharide-stem peptides, which were obtained by enzymatic degradation of S. epidermis cell wall peptidoglycans, displayed similar contractile effects. It was finally revealed that guinea pig ileum strips showed a definite contractile response to N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) and 6-O-stearoyl- and 6-O-(2-tetradecylhexadecanoyl)-MDPs, but not to their analogs, whose C-terminal amino acid was L-isoglutamine or D-isoasparagine in place of D-isoglutamine and which lacked adjuvancy. 6-O-(3-Hydroxy-2-docosylhexacosanoyl)-MDP, on the other hand, caused a slow and lasting relaxation of the ileum strips, but its L-isoglutamine and D-isoasparagine analogs did not.

threo-3-Methyl-D-cysteine as a moiety of β-methyllanthionine in the peptide antibiotic nisin was synthesized stereospecifically. The reaction of (2R,3R)-3-methyl-2-aziridinecarboxylic acid derivative prepared from D-threonine with thiobenzoic acid gave the β-mercapto α-amino acid derivative of the same configuration as the starting material. A stereochemistry of the product was retained as a result of double inversion mechanism through the reactions. Thus, we could prepare threo-3-methyl-D-cysteine, i.e., (2S,3S)-2-amino-3-mercaptobutanoic acid which is required for the synthetic study of nisin.

4-Azidobutyryl esters formed from hydroxyl compounds and 4-azidobutyryl chloride were shown to be cleaved via reduction to 4-aminobutyrate followed by intramolecular O→N-acyl migrations to regenerate the free hydroxyl groups. High susceptibility of azide group to reduction enables the selective removal of this acyl group leaving many other functional groups unchanged.

Chemical syntheses are described of polyacylated β(1→6) glucosamine disaccharide 1,4′-bis(phosphate), which corresponds to the proposed structure of E. coli lipid A, and of its dephospho derivatives. The synthetic bisphosphate proved to be identical with the corresponding natural specimen. The chemical structure of lipid A was thus established.

This paper is concerned with the adjuvant activity in stimulatory immunoglobulin E production against ovalbumin (OA) by bacterial cell walls, cell wall peptidoglycan (PG), and their PG fragments and synthetic N -acetylmuramyl (MurNAc) dipeptides in A/J mice. A PG isolated from Streptococcus pyogenes , PG subunit polymer and dimer obtained from Staphylococcus epidermidis , and water-soluble fragments of cell walls or PG prepared from Nocardia corynebacteriodes and Streptomyces gardneri were found to enhance both the primary and secondary responses of anti-OA immunoglobulin E antibody production. It was suggested that the PG portion, either intact or highly degraded, was capable of enhancing the immunoglobulin E antibody production, and there was no need for the non-PG moiety or intactness of PG structure for the adjuvant activity. This finding was confirmed and extended by the use of synthetic MurNAc dipeptides. Among eight MurNAc dipeptides tested, MurNAc- l -Ala- d -isoGln, MurNAc- l -Ala- d -Gln, MurNAc- l -Ala- d -Glu, and MurNAc- l -Ser- d -isoGln were found active as an adjuvant in the stimulation of the primary and secondary reaginic anti-OA antibody production in a similar way to the cell wall PG and their fragments. None of the synthetic MurNAc- l -Ala- l -isoGln, MurNAc- l -Ala- l -Gln, MurNAc- l -Ala- l -Glu, and MurNAc- l -Ala- d -isoAsn, on the other hand, stimulated the anti-OA immunoglobulin E antibody production in either primary or secondary response, indicating the importance for the adjuvancy in immunoglobulin E production of the configuration of the glutamic acid residues adjacent to the l -Ala (or l -Ser) in muramyl dipeptides.

Various modified substrates of lysine monooxygenase were examined to determine whether they were oxygenated or oxidized. Among various methyllysines tested, N ϵ - and δ-methyllysine underwent predominantly an oxygenative decarboxylation, producing the corresponding acid amides, while γ-methyllysine underwent predominantly an oxidative deamination with an α-keto acid as the reaction product. β-Methyllysine was inactive as substrate. All four methyllysines decreased the cooperativity of the enzyme with the normal substrate, lysine. Furthermore, lysine oxygenation was competitively inhibited by all of them except for β-methyllysine, which was much less inhibitory than the other methyllysines. Other analogs with a chloro or hydroxyl group at either the δ or the γ position were both oxygenated and oxidized. Analogs with a modified carboxyl or α-amino group were inactive as substrates.

The binding of [ 14 C]tuberactinomycin O, an antibiotic closely related to viomycin, to E. coli ribosomes has been examined by equilibrium dialysis method. The antibiotic has been observed to bind to the 70S ribosome, which possesses two binding sites: one on the 30S ribosomal subunit and another on the 50S subunit. The affinity for the large subunit is greater than that for the small subunit. The binding to both ribosomal subunits is reversed by viomycin, indicating that tuberactinomycin O and viomycin have the same binding sites on the ribosome. The results seem to be in accordance with the previous finding that viomycin exhibits dual actions on ribosomal function: the inhibition of fMet-tRNA F (initiation) and inhibition of translocation of peptidyl-tRNA.

Seven acyl derivatives of N-acetylmuramyl-L-alanyl-D-isoglutamine, which had previously been shown to be a minimum structure required for the immunoadjuvant activity, were synthesized in order to evaluate the significance of lipophilicity on the adjuvant activity and on the possible antitumor effect. Among them, are included 6-O-docosanoyl, stearoyl, lauroyl, octanoyl, butyryl, acetyl, and 4,6-di-O-acetyl derivatives. Some of these compounds were found to serve an improvement in the administration method to living body.

Synthesis of threo-3-methyl-D-cysteine, a moiety of β-methyllanthionine as a component of the peptide antibiotics nisin and subtilin, was achieved via (2R,3R)-1-t-butoxycarbonyl-3-methyl-2-aziridinecarboxamide derived from D-threonine. An addition of thioacetic acid or thiobenzoic acid to the aziridinecarboxamide gave S-acyl-β-mercapto-α-amino acid amide derivatives which were hydrolyzed directly or after the preformation of disulfide bond to afford the desired amino acid.

For the purpose of the verification of an expectation that the structural analogs of D-alanyl-D-alanine which participates in the biosynthesis of cell wall peptides might inhibit the growth of microorganisms, alanyl-1-aminoethanesulfonic acid was synthesized by the mixed anhydride method. Four diastereomers were separated and their absolute configurations were deduced on the basis of X-ray analysis. None of these dipeptide analogs and their benzyloxycarbonyl or t-butoxycarbonyl derivatives showed appreciable activity against E. coli.

The effect of synthetic lipid A analogs on murine spleen cells was studied. The preparations represented D-glucosamine and D-glucosaminyl-beta 1,6-D-glucosamine disaccharide derivatives substituted in different combinations by ester- and amide-bound fatty acids and by phosphate groups. Significant mitogenic activity was demonstrated with a number of synthetic disaccharide preparations; however, their potency was lower than that of lipid A. The synthetic preparations were not mitogenic for spleen cells from C3H/HeJ mice. Furthermore, the mitogenicity of the synthetic preparations was abolished after binding with polymyxin B. The results indicate that for expression of mitogenicity, a phosphate group at position 1 of the reducing glucosamine and amide-bound acyloxyacyl residues are important factors. Some of the synthetic preparations containing the diglucosamine backbone and expressing relatively low mitogenicity suppressed B-cell mitogenicity of lipid A. Although these preparations were lytic for erythrocytes, they did not affect the viability of the splenic lymphocytes. Suppression was seen when the synthetic preparations were added simultaneously with or after the lipid A mitogen, but optimal suppression was expressed when the preparations were added to the system 3 h before lipid A. Washing of the cells before the addition of lipid A did not affect the results. The suppression was not due to the induction of suppressor cells by the synthetic preparations. The disaccharide preparations did not inhibit T-cell mitogenicity of concanavalin A. In contrast to the disaccharide preparations, the monosaccharide preparations suppressed mitogenicity of both lipid A and concanavalin A, probably because of their direct toxicity for lymphocytes.

As a basic study in the synthetic approach to immunoactive cell wall peptidoglycan whose backbone is a β(1–4)-linked saccharide of alternating glucosamine and muramic acid, methods for the direct condensation of these sugar components were studied and O-(N-acetyl-β-muramyl)-(1→4)-N-acetyl-D-glucosamine and O-(N-acetyl-β-D-glucosaminyl)-(1→4)-N-acetylmuramic acid were synthesized. In the synthesis of the latter disaccharide, previous formation of an ester bond between the carboxyl group of muramic acid and the 6-hydroxyl group of glucosamine proved to facilitate the glycoside bond fromation between these sugar moieties.

6-Phosphate of N-acetylmuramyl-L-alanyl-D-isoglutamine and its lipophilic derivatives, i.e., 6-(octadecyl hydrogenphosphate) and 6-(2-docosyltetracosyl hydrogenphosphate) were synthesized after the structural feature of mycobacterial cell walls for the purpose to modify and possibly to enhance the immunostimulating activity of the muramyl dipeptide which is the minimum requisite for the activity. As expected, the latter more lipophilic phosphate showed a high activity in a tumor regression test.

The enantioface-differentiating hydrogenation of methyl 3-oxotetradecanoate over (R,R)-tartaric acid-NaBr-modified nickel gave methyl (R)-3-hydroxytetradecanoate(III) in 85% e.e.. After HI was converted to dicyclohexylammonium salt of 3-hydroxytetradecanoic acid (I), the salt was recrystallized three times from acetonitrile and was then treated with acid to give optically pure (R)-I in a good yield.

Mouse peritoneal macrophages, when treated with a lipophilic derivative of muramyl dipeptide either in vitro or in vivo by intraperitoneal injection, showed a more than fivefold increase in their ability to generate superoxide anion after stimulation of the macrophages with phorbol myristate acetate. This response was more than twice that observed with the parent molecule, muramyl dipeptide (MDP). Unlike MDP, which has a systemic effect, the lipophilic derivative, [B30]-MDP, did not alter the response of peritoneal macrophages when given subcutaneously in the flank, suggesting that [B30]-MDP remains localized at the site of injection. The enhanced effect of [B30]-MDP over MDP appeared to be due to the inherent lipophilicity of the molecule, and was probably not due to either stimulation of T lymphocytes or activation of the alternative pathway of complement.

The effects of 61 synthetic tuberactinomycin derivatives on polypeptide synthesis were tested in bacterial cell-free systems. Di-β-lys-capreomycin IIA was more effective than the natural product. Palmitoyl tuberactinamine N inhibited the growth of tuberactinomycin-resistant mutants and was not a ribosome inhibitor.

Viomycin inhibited polypeptide synthesis and translocation of peptidyl-tRNA on ribosomes derived from a sensitive strain of M. smegmatis (R-15), but not significantly on ribosomes from viomycin-resistant mutants (R-31 and R-43). The binding of [14C]tuberactinomycin O, a viomycin analog, to ribosomal particles was studied by Millipore filter method. The sensitive ribosome exhibited higher affinity for the antibiotic than the resistant ribosomes. The resistance was localized on the large ribosomal subunit in a mutant (R-31), and on the small subunit in another mutant (R-43). The binding of the drug to the sensitive ribosomal subunit was markedly reduced by combination with the resistant pair subunit, and the entire ribosome became resistant to the antibiotic.

Four stereoisomers of β-methyllanthionine were synthesized by coupling of N-enzyloxycarbonyl-3-methyl-D-cysteine with β-chloroalanine. 1H NMR analysis and retention time in amino acid analysis were studied on these compounds.