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N-Acetylmuramyl peptides of successive length corresponding to the partial structures of cell wall in Staphylococcus aureus were synthesized in order to elucidate the minimum effective structure responsible for immunoadjuvant activity of bacterial cell walls. In view of the finding that N-acetylmuramyl-L-alanyl-D-isoglutamine was the least structure moiety for exhibition of the activity, nine analogs of either N-acetylmuramyl amino acid or N-acetylmuramyl dipeptide were also synthesized.

Cell walls isolated from 29 strains of 24 gram-positive bacterial species, whose peptidoglycans belong to the group A type of Schleifer and Kandler's classification, with one exception (Arthrobacter sp.), were shown to activate the complement cascade in pooled fresh human serum mainly through the alternative pathway and partly through the classical one. The complement-activating effect of cell walls (5 species) possessing group B type peptidoglycan, except those of Corynebacterium insidiosum, was weaker than that of the walls with group A type peptidoglycan. Preparations of peptidoglycan isolated from cell walls of Staphylococcus aureus, Streptococcus pyogenes, and Lactobacillus plantarum also activated the alternative pathway of the complement cascade, but less effectively than the respective parent cell walls. A water-soluble "polymer" of peptidoglycan subunits (SEPS), which was prepared from Staphylococcus epidermidis peptidoglycans by treatment with a cross-bridge degrading endopeptidase, retained most of the complement-activating ability of the parent cell walls. A peptidoglycan "monomer," SEPS-M, which was obtained by hydrolysis of the glycan chain of SEPS with endo-N-acetylmuramidase to disaccharide units did not activate complement. In conformity with this finding, neither synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) nor MDP-L-Lys-D-Ala activated the complement cascade. Among several lipophilic derivatives of MDP, 6-O-(3-hydroxy-3-docosylhexacosanoyl)-MDP-L-Lys-D-Ala (BH48-MDP-L-Lys-D-Ala) and 6-O-(2-tetradecylhexadecanoyl)-MDP (B30-MDP) were shown to activate complement through the alternative as well as the classical pathway and exclusively through the classical pathway, respectively. The finding that a D-isoasparagine analog of B30-MDP caused the same effect as the parent molecule strongly suggests that the activation of complement by B30-MDP is different from that caused by cell wall peptidoglycans and a water-soluble "polymer" of peptidoglycan subunits.

Biological activities of two groups of synthesized lipid A analogs, the counterpart of biosynthetic precursor, Lehmann's Ia type, 406, and E. coli lipid A type, 506, as well as their non-phosphorylated, and mono-phosphorylated analogs were investigated. The activities employed included four bone marrow cell reactions in mice, mice skin reaction, leukocytes migration in rabbits' cornea, and hemagglutination. Compound 406 and 506 elicited bone marrow reactions in mice and hemagglutination of mouse RBC, although 406 failed to elicit hemorrhage and necrosis also in mice skin. Compound 406 did not elicit corneal reaction in rabbits. The results suggest that for elicitation of this reaction and mice skin reaction, acyloxyacyl structure is required. Cytotoxicity and thromboplastin production of four bone marrow reactions had been reported by us to be endotoxic reactions, since these had not been elicited by peptidoglycan of Lactobacillus and Staphylococcus (1981) and 300 series synthesized analogs (1984) which did not have endotoxic structures. From these results, it seems that these two marrow reactions and hemagglutination require, as does the limulus test, the lipid A part structure as is present in 406.

Lipopolysaccharides (LPS) are well known inducers of interleukin 1 (IL 1). Here we show that synthetic heptaacyl Salmonella minnesota (compound 516) and synthetic E. coli type (compound 506) lipid A, as well as monodephospho part structures thereof, are able to induce IL 1 production in human mononuclear cells. The 4'-monodephospho part structure (compound 505) was found to be the most active IL 1 inducer as compared to compound 506 and the 1-monodephospho part structure (compound 504). Synthetic lipid A precursor Ia, lacking nonhydroxylated fatty acids (compound 406), and its 1- or 4'-monodephospho part structures (compounds 404 and 405) did not induce IL 1 production. IL 2 is not produced during stimulation with the synthetic compounds. IL 1 activity was partially heat labile and could be inhibited by a rabbit antiserum against human leukocytic pyrogen. These results show that the lipid A component of LPS is active in inducing IL 1 and that the presence of 3-acyloxyacyl residues is necessary for IL 1 induction.

Ausgehend von dem aus der Vorstufe (I) dargestellten Alkohol (IIa) werden durch verschiedene Folgereaktionen bzw. deren Wiederholung die Analogen (IIb) und (IIc) dargestellt; die aus (IIa)-(IIc) erhaltlichen Bromderivate (IId)-(IIf) werden in die Ester (IV) und (V) und weiter in die Sauren (VIa)-(VIc) bzw. (VIIa)-(VIIc) ubergefuhrt.

α-Glycosyl phosphates and 4-phosphates of N-acylglucosamine derivatives were prepared in high yields via the reaction of corresponding O-lithium salts with dibenzyl or diphenyl phosphorochloridate at low temperature.

Muramyl dipeptide, which does not induce epithelioid granuloma when injected alone dissolved in phosphate-buffered saline, could induce extensive granulomas in guinea pigs when chemically conjugated with branched, but not linear, fatty acids. Peptidoglycan fragments of Staphylococcus epidermidis could evoke epithelioid granulomas when incorporated in a water-in-oil emulsion. These findings suggest the importance of a lipid bound to muramyl dipeptide for granuloma formation. In view of the fact that mycobacteria uniquely contain large amounts of branched fatty acids, it was proposed that the complex of muramyl dipeptide and branched fatty acids, mostly mycolic acids, is a structure in tubercle bacilli responsible for tubercle formation.

N -Acetylmuramyl- l -alanyl- d -isoglutamine (MDP) induced the chemotaxis of human blood monocytes without mediation of serum complement. Specificity (dependency on chemical structure) of the chemotactic activity of MDP was evidenced by a negligible activity of analogs lacking most of the immunomodulating activities of MDP.

A strain of Streptoverticillium cinnamoneum produced a peptide antibiotic named lanthiopeptin, which contained four unusual amino acids, erythro-beta-hydroxyaspartic acid, mesolanthionine, threo-beta-methyllanthionine and lysinoalanine. Lanthiopeptin showed antiviral activity against herpes simplex virus type 1 KOS strain infection in Vero cells by cytopathic effect reduction assay. The structure of lanthiopeptin is similar to that of ancovenin.

The peptide antibiotic longicatenamycin produced by a Streptomyces strain S-520 is a complex mixture of several congeners. The determination of the total structure of longicatenamycin was performed on the mixture using the procedure of N-bromosuccinimide oxidation followed by EDMAN degradation. The structure of the most abundant congener of longicatenamycin can be represented as cyclo (glycyl-L-2-amino-6-methylheptanoyl-D-valyl-D-ornithyl-threo-beta-hydroxy-L-glutamyl-5-chloro-D-tryptophyl) (Fig. 1: 1=4, m=0, n=3). There are three amino acid positions in which variations are found. The antibiotic complex contains compounds in which L-2-amino-6-methyl-heptanoic acid is replaced with either L-2-amino-5-methylhexanoic acid of L-2-amino-7-methyloctanoic acid; further D-valine with D-isoleucine as well as D-ornithine with D-lysine.

Intracutaneous injection of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) in guinea pigs caused an extensive necrotic reaction in footpads prepared by injection of heat-killed Mycobacterium tuberculosis in water-in-mineral-oil emulsion. We examined a variety of analogs and derivatives of muramylpeptides for their ability to provoke this reaction. A maximum and a minimum structure responsible for the necrotic reaction were found to be N-acetylglycosaminyl-beta(1-4)-N-acetylmuramyl-tripeptide (GlcNAc-MurNAc-L-Ala-D-isoGln-meso-A2pm) and MDP, respectively. An unexpected finding was that GlcNAc-MurNAc-tetrapeptides having L-amino acids at their C termini, unlike comparable compounds having C-terminal D-amino acids, exhibited definite necrosis-inducing activity, probably due to their tendency to undergo in vivo degradation to GlcNAc-MurNAc-tripeptide. Introduction of some acyl groups, especially the stearoyl group, to the 6-O position of the muramic acid or the peptide moiety of muramylpeptides increased the necrosis-inducing activity of the parent molecules. However, this was not observed with 1-thio-muramic acid analogs of MDP. Modification of the alpha- or gamma-carboxyl groups of the glutamic acid residues of muramylpeptides tended to decrease their necrosis-inducing ability. Analogs and derivatives of muramylpeptides which are capable of inducing necrosis at a primed site, with few exceptions, exhibited powerful adjuvanticity against ovalbumin in guinea pigs. However, the reverse was not necessarily true.

2,2′-N:3,3′-O-Tetrakis[(R)-3-hydroxytetradecanoyl]-β(1-6)-D-glucosamine disaccharide 1,4′-bis(phosphate) and its dephospho derivatives were synthesized. The bisphosphate prepared was shown to be identical with a natural biosynthetic precursor of lipid A which corresponds to the lipophilic part of lipopolysaccharide (LPS) in bacterial cell wall. The synthetic bis- and monophosphates exhibited many of typical endotoxic activities of LPS. Consequently, this work established the chemical structure of the biosynthetic precursor of lipid A and elucidated the fundamental structure required for the expression of these activities.

Hypusine, a new basic amino acid occurring in the homogenate of bovine brain tissue, was synthesized to determine the absolute structure. Nα-Benzyloxycarbonyl-L-lysine benzyl ester was coupled with (S)- or (R)-4-benzyloxycarbonylamino-1-bromo-2-butanol derived from L- or D-malic acid, respectively. The products were deprotected by catalytic hydrogenation. One of the synthetic compounds, i.e., (2S,9R)-2,11-diamino-9-hydroxy-7-azaundecanoic acid, was completely identical with natural hypusine in all respects.

Cross-reactivities of synthetic lipid A analogues with monoclonal and conventional antibodies against Salmonella lipid A were studied. It was shown that the in vitro antigenicity of a synthetic compound 506, β-(1→6) D-glucosamine disaccharide 1,4′-bisphosphate, which is acylated at 2′-amino and 3′-hydroxyl groups with ( R )-3-dodecanoyloxytetradecanoyl and ( R )-3-tetradecanoyloxytetradecanoyl groups, respectively, and has ( R )-3-hydroxytetradecanoyl groups at 2-amino and 3-hydroxyl groups, was practically indistinguishable from that of the natural E . coli lipid A preparation, and that both phosphates in positions 1 and 4′ as well as ester- and amide-linked fatty acyl residues, particularly 3-acyloxyacyl group, of the glucosamine disaccharide are involved in the cross-reactivity of lipid A as important antigenic determinants.

A cyclic guanidino amino acid, L-epicapreomycidine, isolated from hydrolyzates of protease inhibitors such as chymostatin and elastatinal was synthesized. This amino acid is a diastereomer of L-capreomycidine which is a component amino acid of tuberactinomycin N,O and capreomycin. A new synthetic route from β-hydroxy-DL-orni thine via an aziridine compound to DL-capreomycidine was applied to the synthesis of L-epicapreomycidine starting from erythro-β-hydroxy-L-ornithine. A convenient and one-step technique to obtain L-erythro form from a mixture of four diastereomers of β-hydroxyornithine by means of enzymatic reaction was newly exploited. The synthetic L-epicapreomycidine obtained was identical with natural one in all respects, thus confirming the proposed structure.

For the investigation on the action mechanism of immunoadjuvant active N-acetylmuramyl-L-alanyl-D-isoglutamine, its labeled compound, i.e., N-acetylmuramyl-L-[U-14C]alanyl-D-isoglutamine was synthesized. For this purpose, an entirely novel synthetic route for the muramyl dipeptide was exploited using cold materials and applied to the preparation of the labeled compound. A convenient synthetic procedure of isoglutamine derivatives is also described.

The recently chemically synthesized Escherichia coli lipid A and the natural free lipid A of E. coli were compared with respect to their endotoxic activities in the following test systems: lethal toxicity, pyrogenicity, local Shwartzman reactivity, Limulus amoebocyte lysate gelation capacity, tumour necrotizing activity, B cell mitogenicity, induction of prostaglandin synthesis in macrophages, and antigenic specificity. It was found that synthetic and natural free lipid A exhibit identical activities and are indistinguishable in all tests.

threo-γ-Hydroxy-L-β-lysine which is a constituent amino acid of antibiotics tuberactinomycin A and N, was synthesized from threo-β-hydroxy-L-ornithine derivative by the Arndt-Eistert reaction. The synthetic amino acid was identical with the natural sample in all respects. Furthermore, a route was established to prepare a protected γ-hydroxy-β-lysine derivative useful for the peptide synthesis.

Two disaccharide dipeptides, i.e., O-(N-acetyl-β-D-glucosaminyl)-(1→4)-N-acetylmuramyl-L-alanyl-D-isoglutamine (2) and O-(N-acetyl-β-muramyl-L-alanyl-D-isoglutamine)-(1→4)-N-acetyl-D-glucosamine (3), which correspond to partial structures of cell wall peptidoglycan were synthesized via the same glucosamine disaccharide as a common intermediate. Thus, selective introduction of lactic acid moiety on either one of the sugar moieties of the intermediate gave two disaccharides respectively which contain muramic acid on either reducing or nonreducing side. Coupling of the peptide moiety followed by deprotection afforded 2 and 3. The results of biological tests showed that both 2 and 3 have the same magnitude of immunostimulating activities as those of N-acetylmuramyl-L-alanyl-D-isoglutamine which is the minimum structure required for expression of the activities.

6-O-(2-Tetradecylhexadecanoyl)-N-acetylmuramyl-L-alanyl-D-isoglutamine (B3O-MDP) and 6-O-(3-hydroxy-2-docosylhexacosanoyl)-MDP (BH48-MDP) were examined for immunopotentiating activity in stimulating the primary immune responses of guinea pigs to ovalbumin and for adverse reactions at the injection site and in the regional lymph nodes when administered in combination with several nonirritating vehicles. B30-MDP was found to potently stimulate both humoral and cellular immune responses, the latter by induction of delayed-type hypersensitivity, irrespective of the administration vehicle examined (liposomes, a squalene in water emulsion, Intralipid, phosphate-buffered saline, and Nikkol HCO-60 glucose solution), although the minimum effective dose was dependent on the vehicle used. Reactions in the footpad receiving the injection were negligible. Noticeable local reaction consisted of swelling of the lymph nodes in the region of the injection, but the swelling was only noted with higher doses and subsided 3 to 4 weeks after immunization, unlike the persistent swelling caused by the administration of water-in-mineral oil emulsions with or without B30-MDP. BH48-MDP and its L-serine analog, BH48-MDP(L-Ser), which has L-serine in place of L-alanine in MDP, in combination with the squalene-in-water emulsion, also intensely stimulated both cellular and humoral antiovalbumin immune responses, but the effects of these compounds seemed to be influenced to a greater degree by the vehicles than by B30-MDP. Thus, B30-MDP was chosen from a series of a 6-O-acyl derivatives of MDP as the most promising candidate for possible application in practical vaccines.

For the purpose of study on racemization of exo methine carbon atom of thiazoline, oxazoline or imidazoline ring, the following four compounds were synthesized from amino acid imino ether: 2-(1-benzyloxycarbonylaminoethyl)-2-thiazoline (I), methyl 2-(1-benzyloxycarbonylaminoethyl)-2-thiazoline-4-carboxylate (II), methyl 2-(1-benzyloxycarbonylaminoethyl)-2-oxazoline-4-carboxylate (III), and 2-(1-benzyloxycarbonylaminoethyl)-2-imidazoline (IV). A racemization rate of the alanine residue was measured from an optical rotation of 2,4-dinitrophenyl derivative obtained from the acid hydrolyzate and from hydrogen-deuterium exchange reaction at α-carbon atom. All these compounds were found to be racemized very easily particularly in the presence of proton source. From the experimental results, a plausible mechanism for racemization peculiar to such heterocyclic compounds through protonation-deprotonation process was presented.

Aus dem nach bekannten Methoden dargestellten Tetrapeptidester (Ia) bzw. -hydrazid (Ib) wird schrittweise nach der Azid-Methode das Hexapeptidderivat (IIa) erhalten.

A nonenzymic model for the biosynthesis of thyroxine from diiodotyrosine is described. In this reaction, l -thyroxine is formed from 3,5-diiodo- l -tyrosine at neutral pH and at room temperature in the presence of sodium glyoxylate, cupric acetate, and oxygen. The reaction proceeds rapidly through transamination via a metal chelate of the Schiff base of diiodotyrosine and glyoxylic acid, followed by oxidative coupling of the 4-hydroxy-3,5-diiodophenylpyruvic acid thus formed, with diiodotyrosine.

For the purpose of making a total synthesis of bacitracin A, a protected heptapeptide with a ring structure, Nα- (formyl-L-isoleucyl)-cyclo-(L-lysyl-δ-tosyl-D-ornithyl-L-isoleucyl-D-phenyl-alanyl-im-benzyl-L-histidyl-α-methyl-β-L-aspartyl) was first prepared by a high dilution method. After deformylation, it was coupled with a tetrapeptide corresponding to the N-terminal part of bacitracin A, the benzyloxycarbonyl-L-isoleucyl-S-benzyl-L-cysteinyl-L-leucyl-D-glutamic acid (α-azide)-γ-t-butyl ester, to afford an undecapeptide derivative. This was converted to a hydrazide, which was then coupled with β-benzyl-D-isoasparaginate to give the Nα-(benzyloxycarbonyl-L-isoleucyl-S-benzyl-L-cysteinyl-L-leucyl-γ-t-butyl-D-glutamyl-D-isoleucyl)-cyclo-(L-lysyl-δ-tosyl-D-ornithyl-L-isoleucyl-D-phenylalanyl-im-benzyl-L-histidyl-β-L-aspartyl)-D-isoasparagine β-benzyl ester. The removal of all protecting groups from it gave a free dodecapeptide, which has the same amino acid sequence, including optical properties, as that ...

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.

1-Succinimidyl esters of various aliphatic acids and amino acids were reduced with sodium borohydride in tetrahydrofuran to the corresponding alcohols under mild conditions in fairly good yields. The facile reaction offers a convenient method for the preparation of alcohols from carboxylic acids, and can be effectively applied to a selective conversion of an appropriate carboxyl group to a hydroxymethyl group in the presence of other functional groups.

A new cyclopropyl amino acid, carnosadine, was isolated from red alga Grateloupia carnosa and the structure was determined to be 1-amino-2-guanidino-methylcylopropane-1-carboxylic acid from the results of NMR, mass spectrometry and coloring tests.