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The syntheses of novel rigid dendritic ligands and their ruthenium complexes, in which 1,3,5-phenylene-based hydrocarbon dendron units were directly connected to a phenanthroline ligand at the 3 and 8 positions, are described. Preliminary absorption and fluorescence results are indicative of highly efficient energy transfer within the rigid metallodendrimers.

The phosphoenolpyruvate carboxylase (PEPC) isozyme involved in C4 photosynthesis is known to undergo reversible regulatory phosphorylation under illuminated conditions, thereby decreasing the enzyme's sensitivity to its feedback inhibitor, L-malate. For the direct assay of this phosphorylation in intact maize leaves, phosphorylation state-specific antibodies to the C4-form PEPC were prepared. The antibodies were raised in rabbits against a synthetic phosphorylated 15-mer peptide with a sequence corresponding to that flanking the specific site of regulatory phosphorylation (Ser15) and subsequently purified by affinity-chromatography. Specificity of the resulting antibodies to the C4-form PEPC phosphorylated at Ser15 was established on the basis of several criteria. The antibodies did not react with the recombinant root-form of maize PEPC phosphorylated in vitro. By the use of these antibodies, the changes in PEPC phosphorylation state were semi-quantitatively monitored under several physiological conditions. When the changes in PEPC phosphorylation were monitored during the entire day with mature (13-week-old) maize plants grown in the field, phosphorylation started before dawn, reached a maximum by mid-morning, and then decreased before sunset. At midnight dephosphorylation was almost complete. The results suggest that the regulatory phosphorylation of C4-form PEPC in mature maize plants is controlled not only by a light signal but also by some other metabolic signal(s). Under nitrogen-limited conditions the phosphorylation was enhanced even though the level of PEPC protein was decreased. Thus there seems to be some compensatory regulatory mechanism for the phosphorylation.

The syntheses of aureobasidin A (AbA) derivatives with alkyl chains and their in vitro structure-biological activity relationships are discussed. The analogs replaced at positions 6, 7, or 8 of AbA with either L-glutamic acid, delta-hydroxy-L-norvaline, or delta-hydroxy-N-methyl-L-norvaline are prepared. The gamma-carboxyl or delta-hydroxyl group of these new amino acids was coupled with acids, alcohols, or amines with alkyl chains. While the analogs having L-glutamic acid residue at positions 6 or 8 showed weak activity, esterification of the gamma-carboxyl group with benzyl or shorter alkyl (C4 or C6) alcohols, significantly enhanced the activities. Introduction of longer alkyl (C14) chain to the same amino acids residues at positions 6, 7, or 8 resulted in total loss of antifungal activity. Among the lipophilic analogs in [L-Glu6] derivatives, the C6 alcohol ester showed the strongest antifungal activity against Candida spp. so far tested. None of the derivatives showed activity against Cryptococcus neoformans.

The first total synthesis of antifungal cyclic depsipeptide aureobasidin A is described. The synthesis was achieved mainly using bromotris(pyrrolidino)phosphonium hexafluorophosphate (PyBroP) as a coupling reagent. Peptide cyclization was carried out between L- allo -isoleucine (L- a lle 1 ) and L- Pro 9 residues in the linear nonapeptide at the final step of the synthesis. Synthesized aureobasidin A was completely identical with the natural antibiotic with respect to antifungal activity and physicochemical properties. Unusual reactions due to N -methylamino acid, an oxazoline-mediated reaction and an N , O -acyl migration, are also described.

A chemical synthesis was performed of an octapeptide with a phosphonic ester linkage, which corresponds to a variation of the partial sequence of a gene product of erb B-2. The phosphonic ester linkage was successfully prepared through a coupling reaction of magnesium salt of the hydroxypeptide with phosphonochloridate, without damage of the peptide bond. The free aminophosphonic peptide as the final product gave a single peak on the ODS column, and a reasonable ratio of the component amino acids. The reaction series exploited here might be useful as a general procedure for preparation of the phosphonic peptide.

A main component of ganglioside M5 from egg of the sea urchin, Anthocidaris crassispina, Neu5Gcα2 → 6Glcβ1 → 1Cer (1), which has N-glycolyl group in neuraminic acid part, as well as phytosphingosine and saturated nonhydroxy fatty acid in ceramide part was first synthesized starting from N-acetylneuraminic acid.

The isomers of sialic acid with 6-acetylamino group, 1a and 1b were synthesized from furanose 3a and 3b respectively obtained by aldol condensation of D -glucose with oxalacetic acid.

A main component of ganglioside M5 from egg of the sea urchin, Anthocidaris crassispina, Neu5Gcα2 → 6Glcβ1 → 1Cer (1), which has N-glycolyl group in neuraminic acid part, as well as phytosphingosine and saturated nonhydroxy fatty acid in ceramide part was first synthesized starting from N-acetylneuraminic acid.

Chemistry-based investigation is reviewed which led to identification of the active entities responsible for the immunostimulating potencies of peptidoglycan and lipopolysaccharide. Though these glycoconjugates which ubiquitously occur in wide range of bacteria as the essential components of their cell envelopes have long been known to enhance the immunological responses of higher animals, neither the precise chemical structures required nor the mechanism of their action had been [corrected] elucidated until early 1970s. Chemical synthesis of partial structures of peptidoglycan proved N-acetylmuramyl-L-alanyl-D-isoglutamine to be the minimum structure responsible for the activity and led to later identification of its receptor protein Nod2 present in animal cells. Another active partial structure of peptidoglycan, gamma-D-glutamyl-meso-diaminopimelic acid, and its receptor Nod1 were also identified as well. With regard to lipopolysaccharide, its glycolipid part named lipid A was purified and the structure studied. Chemically synthesized lipid A according to the newly elucidated structure exhibited full activity described for lipopolysaccharide known as endotoxin. Synthetic homogeneous lipid A and its structural analogues and labeled derivatives enabled precise studies of their interaction with receptor proteins and the mechanism of their action. Chemical synthesis of homogeneous partial structures of peptidoglycan and lipopolysaccharide gave unequivocal evidences for the concept that definite small molecular parts of these complex macromolecular bacterial glycoconjugates are specifically recognized by their respective receptors and trigger our defense system now widely recognized as innate immunity.

The isomers of sialic acid with 6-acetylamino group, 1a and 1b were synthesized from furanose 3a and 3b respectively obtained by aldol condensation of D -glucose with oxalacetic acid.

Total synthesis of a lanthionine peptide nisin was achieved by the successive condensations of four segments including cyclic lanthionine peptide parts and the C-terminal linear segment including dehydroalanine, followed by the deprotection procedure with anhydrous HF. Modified reaction conditions for the Hofmann degradation were applied to prepare the dehydroalanine residue from the 2,3-diaminopropionic acid residue. The final deprotection with anhydrous HF proceeded without decomposition of the dehydroamino acid residues which are generally labile in acidic media. The synthetic nisin was completely identical with the natural one in all respects, resulting in a confirmation of the proposed structure.

In this paper, we compared the chemical and biological activities of natural and synthetic clavamines, N-[2, 4-dihydroxyphenylacetyl-4-L-asparaginyl)-N'-[N-(L-arginyl-glycyl-L-alanyl)-8-amino-4-azaoctanoyl]-1, 5-pentane-diamine (DPA→Asn→Cad←Ptr←Ala←Gly←Arg)[DPA, 2, 4-dihydroxyphenylacetic acid; Cad, 1.5-pentanediamine (cadaverine); Ptr, 8-amino-4-azaoctanoic acid (putreanine, or carboxyethylputrescine)], and found that both were identical. Both compounds showed the same Rf value in thin-layer chromatography and the same retention time in high performance liquid chromatography (HPLC). Both hydrolyzates consisted of the same components, such as Gly, DPA, Ala, Asp, Arg and Ptr in gas chromatography. The Edman degradation of both compounds also gave exactly the same sequence. The 1H-NMR spectrum and fast atom bombardment (FAB) mass spectrum of the synthetic compound coincided with those of the natural one. Both were active on the insects used. Clavamine was the main component of the insecticidal activity in the crude venom. By this synthetic study, the structure of a new venom insecticide was thus established. The synthetic compound in abundant quantity will be advantageous for practical use as an insecticide over natural ones, because the latter is available only in small amounts. Also, it will be useful for the exploitation of a new field of biochemistry for polyamines.

Nephritogenoside (1), proposed as a nephritogenic triglycosylhenicosapeptide with asparagine N-α-glycoside linkage, was first synthesized by the coupling of triglycosyldipeptide active ester with the nonadecapeptide (3–21) using allyloxycarbonyl group as the final protecting group. A β-anomeric glycopeptide was also prepared.

The structure of ancovenin, a new peptide inhibitor of angiotensin I converting enzyme was determined chemically. Ancovenin is composed of 16 amino acid residues including three lanthionine residues which form a complex tricyclic structure bridged by sulfide bonds. Moreover, this peptide includes dehydroalanine as one of its unusual amino acids. In the present study, plausible mechanisms of the Edman degradation of dehydroalanine and lanthionine-containing peptides were also elucidated.

The ability of synthetic lipid A analogs to induce prostaglandin synthesis in macrophages was compared with that of native lipopolysaccharide. The synthetic preparations comprised monomeric or dimeric derivatives of D-glucosamine with different patterns of substitution by phosphate and tetradecanoic, (R)-3-hydroxytetradecanoic, and (R)-3-tetradecanoyloxytetradecanoic acid. All of these preparations are structurally distinct from native lipid A (principally regarding the position of fatty acid substitution) and hence have been previously shown to be endotoxically inactive in many biological tests. It was found that many of these synthetic samples exhibit strong activity in inducing prostaglandin E2 and prostaglandin F2 alpha, with some of them having activity comparable to that of lipopolysaccharide. Experiments with macrophages of C3H/HeJ mice enabled us to differentiate between endotoxin-specific (in the case of dimeric preparations) and endotoxin-nonspecific (for monomeric preparations) mechanisms for the induction of prostaglandins. These results indicate that there is a difference in the mechanism of induction of prostaglandin synthesis between monomeric lipid A's and dimeric or native lipid A structures.

Clavamine, a new insecticidal toxin isolated from the venom of Joro spider (Nephila clavata) was totally synthesized in order to confirm the proposed structure. In clavamine, a fragment of glycylalanine is inserted in the structure of NSTX-3, a New Guinean spider toxin. The synthesis determined the chemical structure of clavamine unambiguously.

GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha1--6fucosyltransferase (alpha1-6FucT; EC 2.4.1.68), which catalyzes the transfer of fucose from GDP-Fuc to N-linked type complex glycopeptides, was purified from a Triton X-100 extract of porcine brain microsomes. The purification procedures included sequential affinity chromatographies on GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-2Manalpha1- 2)Manbeta1-4GlcNAcbet a1-4GlcNAc-Asn-Sepharose 4B and synthetic GDP-hexanolamine-Sepharose 4B columns. The enzyme was recovered in a 12% final yield with a 440, 000-fold increase in specific activity. SDS-polyacrylamide gel electrophoresis of the purified enzyme gave a major band corresponding to an apparent molecular mass of 58 kDa. The alpha1-6FucT has 575 amino acids and no putative N-glycosylation sites. The cDNA was cloned in to pSVK3 and was then transiently transfected into COS-1 cells. alpha1-6FucT activity was found to be high in the transfected cells, as compared with non- or mock-transfected cells. Northern blotting analyses of rat adult tissues showed that alpha1-6FucT was highly expressed in brain. No sequence homology was found with other previously cloned fucosyltransferases, but the enzyme appears to be a type II transmembrane protein like the other glycosyltransferases.

A protein having a high-affinity binding site for [3H]mepyramine (MBP) was purified to homogeneity from rat liver membranes. The purified MBP has a single type of binding site for [3H]mepyramine with Kd value of 18.5 nM, and its molecular weight was determined to be 56,000 by SDS polyacrylamide gel electrophoresis. Amino acid sequences of twelve tryptic peptides derived from MBP are highly homologous with those of rat debrisoquine 4-hydroxylase (cytochrome P450 2D1) and other rat P450 2D subfamily members. In immunoblotting analysis, an antibody against rat P450 2D1 stained a band corresponding to MBP with Mr of 56,000; its migration position was clearly different from that of rat P450 2D1. Substrates and inhibitors of debrisoquine 4-hydroxylase potently displace [3H]-mepyramine binding to MBP. Quinine and quinidine showed 400 and 80 times, respectively, higher affinity for MBP than for debrisoquine 4-hydroxylase. These results suggest that MBP is a novel P450 2D family member.

A chromogenic substrate 1, 5-bromo-4-chloroindol-3-yl 5-acetamido-3,5-dideoxy-alpha-D-glycero-D-galacto-2-nonulopyranosidon ic acid (X-Neu5Ac), has been synthesized to facilitate the screening of bacterial colonies or plaques for the detection of either natural or mutant neuraminidase activity. Substrate 1 was hydrolyzed by neuraminidase isolated from Clostridium perfringens to release a halogenated indol-3-ol 2 that undergoes rapid aerobic oxidation to form the dark blue pigment, 5,5'-dibromo-4,-4'-dichloroindigo 3. Preliminary kinetic studies indicate that this compound is a good substrate (Km 0.89 x 10(-3) M) for neuraminidase and is quite stable under identical conditions in the absence of enzyme. These results suggest that X-Neu5Ac 1 can be useful to screen for bacterially-encoded enzyme production directly on agar plates.

A rice bran polysaccharide designated RON was subjected either to partial hydrolysis with formic acid or to partial degradation by ultrasonic irradiation. A significant change in the molecular size was also observed during simple chromatography of RON on a strongly acidic ion exchange resin, although the apparent molecular weight of RON had been assumed to be more than 1 x 10(6) daltons (Da). This fact indicates that RON exists as molecular aggregates, presumably mediated by metal cations. Degradation products with average molecular weights above ca. 1 x 10(4) Da which were obtained by any of the three methods still retained the following activities of RON: in vivo antitumor activity against Meth-A fibrosarcoma in mice by oral administration, and in vitro macrophage stimulatory effects to induce tumoricidal activity and interleukin 1 production. This molecular size was proven to be the minimum requisite for these activities because smaller fragments were scarcely active. The aggregation was characteristic of RON but not essential for its antitumor activity because definite, though slightly reduced, activity was exhibited even by the smaller fragments obtained after the ion exchange resin treatment.

Three novel lipid A analogs, which have an alpha- or beta-glycosidically bound phosphonooxyethyl group instead of the alpha-glycosyl phosphate group of natural lipid A, were synthesized. The first analog (2) had an alpha-phosphonooxyethyl group on the identical acylated disaccharide 4'-phosphate structure found in natural lipid A (from Escherichia coli) and hence differed from the latter only in the nature of the acidic group at position 1. The second one (3) had tetradecanoyl groups in place of the two (R)-3-hydroxytetradecanoyl groups bound to the 2- and 3-hydroxyl function of 2, retaining the alpha-phosphonooxyethyl group. The structure of the third analog (4) was the same as that of 3 except that the phosphonooxyethyl group of the former was beta-oriented. Compounds 2 and 3 exhibited potent activity against Meth A at the same level as natural lipid A, whereas 4 showed less activity. This fact revealed that the glycosidic phosphate is not a prerequisite for the antitumor activity of lipopolysaccharide. It can be replaced with a phosphonooxyethyl group without any loss of activity provided that the alpha-anomeric configuration at C-1 is retained. The replacement of the hydroxytetradecanoyl groups with tetradecanoyl groups does not change the activity either.

A site-specific ring opening reaction for the antifungal depsipeptide aureobasidin A occurred on treatment with HF at room temperature for 1 hour. The open-chain peptide thus obtained was recyclized to afford the original aureobasidin A without any modification.

The structure of lepidopteran was determined by sequential analysis using the Edman method for the whole molecule as well as several fragment peptides obtained by enzymatic and chemical cleavages. Consequently, lepidopteran was found to be a mixture of congeners composed of 35 amino acids possessing one replaceable amino acid residue of lysine with δ-hydroxylysine.

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.

Thirteen 6-O-acyl-N-acetylmuramyl-L-alanyl-D-isoglutamines (6-O-acyl-MDPs), including four inactive D-isoasparagine and L-isoglutamine analogs, were tested for their pyrogenicity and immunopotentiating activity to stimulate primary humoral and cellular immune responses in guinea pigs to a model protein antigen, ovalbumin, when administered in various vehicles. Among them, derivatives whose muramic acid residue was substituted by alpha-branched (and beta-hydroxylated) higher fatty acids at the carbon-6 position, especially 6-O-(2-tetradecylhexadecanoyl)-MDP (B3O-MDP) and, to a lesser extent, 6-O-(3-hydroxy-2-docosylhexacosanoyl)-MDP (BH48-MDP) and its L-serine analog [BH48-MDP(L-Ser)], were found to exert strong adjuvant activity in both the induction of delayed-type hypersensitivity and the stimulation of circulating precipitating antibody levels when combined with nonirritating vehicles (liposomes, squalene-in-water emulsion, and phosphate-buffered saline). These vehicles did not efficiently support the adjuvant activity of MDP, the parent molecule of the above lipophilic derivatives. Pyrogenicity tests showed that introduction of alpha-branched higher fatty acid groups but not of straight, long-chain fatty acids at the 6-position of the muramic acid residue resulted in marked decrease of the pyrogenicity inherent to MDP via intravenous administration.

On the N,O-acyl migration reaction of β-hydroxy-α-amino acids with concentrated sulfuric acid, a configuration of the carbon atom carrying hydroxyl group was found to be inverted through the reaction. Thus, threonine residue in peptide was converted to allothreonine residue and vice versa in the selective chemical cleavage of threonine peptide employing the N,O-migration with concentrated sulfuric acid. The reaction mechanism involving SNi elimination of O-sulfate formed as an intermediate was suggested. The inversion did not occur in the N,O-acyl migration reaction with other mineral acids than sulfuric acid.

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.

Silkworm, Bombyx mori , produces self-denfense substance lepidopteran in a haemolymph when it is vaccinated with killed Escherichia coli . Lepidopteran family contains at least three congeners A, B and C, all of which were isolated and determined in amino acid sequences. Furthermore, total synthesis of lepidopteran A was achieved. For elucidation of structure-activity relationship of lepidopteran, twenty one different kinds of peptide fragments were then synthesized. From results of antibacterial tests for those peptides, it is concluded that N-terminal region particularly the Arg -Trp2 sequence is very important for exhibition of the biological activity. The minimal sequence of lepidopteran providing significant bioactivity is the peptide fragment (1–18). α-Helix content in the peptide chain does not necessarily guarantee a manifestation of the activity. An important factor to strengthen the activity seems to be a hydrophobicity in the molecule in addition to the N-terminal region. The assumption was confirmed by preparation of long aliphatic amide of the fragment (1–18) which showed almost comparable activity to that of the original lepidopteran.

Title compounds ( 1a,b ) and an analogous disaccharide ( 2a ) were synthesized via a reaction of a ketosidic fluoride of 3-deoxy-D- manno -2-octulosonic acid (KDO) ( 3 ) with phosphorylated acyl glucosamine di- and monosaccharides.

Two new ureido amino acids, lividine ( 1 ) and grateloupine ( 2 ), were isolated from Grateloupia livida and Grateloupia filicina respectively. The structures of both amino acids were assumed mainly by NMR spectra and determined as N w -carbamoyl-L-citrulline for lividine and as N -carbamoyl- Y -aminobutyric acid for grateloupine by comparison with the synthetic compounds respectively.

The peptide N -acetylmuramyl- l -alanyl- d -isoglutamine (MDP), which has adjuvant activities, and 17 of its derivatives and analogs were synthesized and assayed to elucidate the structure necessary for adjuvant activity in induction of experimental allergic encephalomyelitis (EAE) in guinea pigs. The results revealed the importance of the d configuration and the α-carboxamide group of the isoglutaminyl residue of MDP for adjuvant activity. Replacement of the l -alanyl residue of MDP by d -alanine, but not by l -serine or glycine, resulted in a marked decrease in the activity. The β-methyl glycoside of MDP was found to be more active than the α-methyl derivative. 6- O -Stearoyl- N -acetylmuramyl- l -alanyl- d -isoglutamme showed activity.

Thirty two semisynthetic tuberactinomycins were prepared by introduction of various amino acids to amino group of tuberactinamine N, a cyclic peptide moiety of tuberactinomycin N and O, which was isolated from natural tuberactinomycin N by acid treatment with liberation of γ-hydroxy-β-lysine of the branched part. Among introduced amino acids, β-amino acids were synthesized from corresponding α-amino acids by modified Arndt-Eistert reaction. After couplings of N-protected amino acids with tuberactinamine N, deprotections were carried out to give semisynthetic tuberactinomycins. From their minimum inhibitory concentrations against many bacteria, it was suggested that a branched part effects significantly to the strength of antimicrobial activities though most important active site must locate in the cyclic peptide moiety, and basicity and/or hydrophobicity of the branched part seemed to strengthen the antibacterial activity especially.

Dityromycin is a peptide antibiotic isolated from the culture broth of the soil microorganism, Streptomyces sp. strain No. AM-2504, showing activities against Bacillus, Corynebacterium, and Clostridium. Dityromycin was clarified to be composed of N-MeVal, Pro, Val, Phenylglycine, CH3NH2, N,N-Me2Thr and di-α-amino acid containing diphenylether moiety. The peptide sequence except one labile amino acid residue was determined by partial hydrolysis, chemical cleavage, and the Edman degradation. The unknown amino acid was finally deduced to be the dehydroamino acid containing epoxide, resulting in an elucidation of whole structure of the antibiotic.

The structure of the oligosaccharide portion of an E.coli Re lipopolysaccharide was determined as α-KDO-(2→4)-α-KDO-(2→6)-β-GlcN-α(1→6)-α-GlcN, bisphosphorylated at positions 1 and 4′. Taking into account the previous determination of the acylation pattern of the GlcN disaccharide, the total structure of E.coli Re LPS was thus established.

Interferon-inducing, pyrogenic and proclotting enzyme of horseshoe crab activation activities of chemically synthesized lipid A analogues were investigated and compared with the same activities of a natural lipid A. These analogues are nonphosphorylated, C-1 or C-4′ monophosphorylated and C-1,4′ bisphosphorylated derivatives of β-1,6-linked d-glucosamine disaccharide possessing both ester-bound and amide-bound fatty acid substituents. Fatty acid substituents of the analogues are tetradecanoyl (C14), (R)-3-hydroxytetradecanoyl (C14-OH) or (R)-3-tetradecanoyloxytetradecanoyl [C14-O-(C14)] groups. The biological activities of the samples were assayed after solubilization with triethylamine and complexing with bovine serum albumin. Interferon-inducing activity was exhibited by both the C-1 monophosphorylated compounds examined. Ester-bound and amide-bound fatty acid substituents of these compounds are both C14 or C14 and C14-OH, respectively. Nonphosphorylated, C-4′ monophosphorylated and C-1,4′ bisphosphorylated compounds possessing the same fatty acid substituents as those of the C-1 monophosphorylated compounds showed no detectable interferon-inducing activity. C-4′ monophosphorylated compounds possessing C14-OH as ester-bound and C14-OH or C14-O-(C14) as amide-bound fatty acid substituents exhibited interferon-inducing activity, but nonphosphorylated compounds possessing the same fatty acid substituents did not. None of the analogues exhibited significant pyrogenicity nor proclotting enzyme of horseshoe crab activation activity under the conditions employed in this study.

Total syntheses have been achieved of capreomycin IA and IB according to our newly proposed structures. The β-lysine residue in the branch was introduced to the cyclic peptide moiety which was prepared by cyclization of the corresponding pentapeptide. Deprotection followed by conversion of β,β-diethoxyalanine residue to β-ureidodehydroalanine residue afforded the desired products, which were identical with natural capreomycins in all respects.

Various structural analogs of N-acetylmuramyl-L-alanyl-D-isoglutamine, which had been shown to be the minimum structure required for the immunoadjuvant activity of bacterial cell walls, were synthesized in order to investigate in more detail the relationship between the activity and chemical structures. Particularly, the compounds of the structures corresponding to the cell walls of special bacteria without immunoadjuvant activity were prepared in this study. Several novel procedures including the preparation of a branched glutamyl peptide were newly exploited to accomplish the syntheses of muramyl peptides with complicated structures. As a result of this synthetic study, new informations were obtained on the structural requirement for the immunoadjucant activity.

The structure/activity relationship of lipid A, a bioactive center of endotoxic lipopolysaccharides, in the activation of the clotting enzyme cascade of a horseshoe crab amoebocyte lysate (Limulus activity) and the complement system in human serum, was examined using synthetic lipids A and related compounds. Regarding Limulus activity, a newly developed colorimetric method, which utilizes a mixture of recombined clotting factors and a chromogenic substance, was much more sensitive for detecting changes in the chemical structure of test compounds than the conventional gelation method using the amoebocyte whole lysate. (beta 1-6)-D-Glucosamine disaccharide bisphosphates, which had neither 3-hydroxyacyl nor 3-acyloxyacyl groups, and acylglucosamine phosphates, which in structure correspond or are analogous to the non-reducing or reducing moieties of lipids A and biosynthetic disaccharide lipid A precursors showed only negligible activity in the colorimetric tests, but they exhibited a distinct though much weaker gelation activity than the parent disaccharide molecules. The assay results obtained by the colorimetric Limulus test correlate better with the pyrogenicity of the test synthetic compounds than those given by the gelation method, although the dependence of pyrogenicity on chemical structure is greater. The presence of 3-hydroxyacyl groups on the bisphosphorylated (beta 1-6)-D-glucosamine disaccharide backbone is the prerequisite for effective activation of the clotting enzyme cascade of horseshoe crab amoebocyte lysate, while the presence of an adequate number (one or two) of 3-acyloxyacyl groups on the disaccharide bisphosphate backbone is needed for full pyrogenicity. Complement activation, on the other hand, showed structural requirements quite different from those for the colorimetric Limulus activity and the pyrogenicity. The disaccharide compounds that had only non-hydroxylated acyl groups, acylated glucosamine phosphates that had the structure of the non-reducing portion of lipids A and biosynthetic disaccharide precursors, which were scarcely active in the colorimetric Limulus test, caused complement activation comparable to or sometimes stronger than that of the parent disaccharide molecules. Acylglucosamine phosphates, corresponding in structure to the reducing moiety of disaccharide compounds, however, showed little activity.

The experimental system utilized in investigating the correlation between the chemical structures of muramyl peptides and their protective activities in the sepsis type of systemic infections caused by Escherichia coli was applied in evaluating the enhancement of resistance to infection induced by 32 synthetic glycopeptide analogs, including 6-O-acyl derivatives and 1-alpha-O-benzyl derivatives of muramyl dipeptide (N-acetyl muramyl-L-alanyl-D-isoglutamine). In assessing the 6-O-acyl derivatives of muramyl dipeptide, we found that the degree of protective activity was attributable to the kinds of fatty acids introduced. Acylation of the 6-hydroxy group on the muramic acid moiety in muramyl dipeptide with natural mycolic acid or a synthetic fatty acid possessing either an alpha-branched or an alpha-branched, beta-hydroxylated group resulted in a decrease in or a disappearance of the protective activity of muramyl dipeptide. Acylation with a normal fatty acid or an iso fatty acid resulted in a retention or enhancement of muramyl dipeptide activity. The activity of acylated derivatives containing linear fatty acids was stimulated by increasing the chain length up to 18 carbon atoms. The highest degree of protective activity occurred with the derivatives acylated with straight-chain fatty acids, particularly with the derivatives acylated with palmitic acid and arachidic acid. Benzylation of the 1-hydroxy group of muramyl dipeptide resulted in a decrease in or a loss of protective activity.

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.

Streptolidine (roseonine, geamine) (1), a guanidino amino acid component of the streptothricin group antibiotics, was synthesized starting from D-ribose (8). The epimino compound (11b) was prepared from 8 in five synthetic steps. Azidolysis of 11b gave the arabino-type compound (12b), which in turn was converted to the key intermediate tribenzyloxycarbamido lactone (23b) by oxidation of C-1. The lactone 23b was rearranged to the free lactam (4) after deprotection, and then guanidinated with cyanogen bromide. The identity of the hydrolysis product of this guanidino lactam with natural streptolidine in all respects certified the proposed structure involving the steric configurations.

Conformational analyses of peptide antibiotics tuberactinomycins were performed by NMR spectroscopy of 100 MHz. Studies on temperature dependence and deuterium exchange in amide proton resonances revealed the presence of an intramolecular hydrogen bond participated with α-amide proton of the guanidino amino acid residue in the cyclic part of each molecule. The same result was obtained in tuberactinamine N, which is the cyclopeptide moiety without amino acid side chain in tuberactinomycins N and O. The common rigid structure fixed by hydrogen bonding in the cyclic part seems to be a requisite for the exhibition of microbial activity in tuberactinomycin family.

The activation of murine B cells and macrophages by synthetic lipid A analogues, solubilized with triethylamine and complexed with bovine serum albumin, were investigated in vitro. The analogues used are nonphosphorylated, C-1 or C-4′ monophos-phorylated and C-1,4′ diphosphorylated derivatives of β-1,6-linked D-glucosamine disaccharide possessing both ester- and amide-bound fatty acid substituents. They were divided into 4 groups, A, B, C and D in terms of the fatty acid substitution. Ester- and amine-bound fatty acids of the analogues are both tetradecanoic acids (C14) in group A, C14 and (R)-3-hydroxytetradecanoic acids (C14-OH) in group B, both C14-OH in group C and C14-OH and (R)-3-tetradecanoyloxytetradecanoic acids in group D. Mitogenic activity was exhibited in spleen cells from C3H/HeN mice by the C-1 monophosphorylated analogues in groups A and B, and by the C-4′ monophosphorylated analogues in groups B, C and D, but not in cells from C3H/HeJ mice. Nonphosphorylated analogues in groups A, B and C, and C-4′ monophosphorylated analogue in group A showed negative mitogenic activity. Only the nonphosphorylated analogue in group D exhibited mitogenic activity in spleen cells from C3H/HeJ mice as well as those from C3H/HeN mice. None of the other analogues exhibited the activity in C3H/HeJ spleen cells. Polyclonal B cell activation activity was exhibited by the C-1 monophosphorylated analogues in groups A and B, and by the C-4′ monophosphorylated analogues in groups C and D. Nonphosphorylated analogues in all groups and C-4′ monophosphorylated analogues in groups A and B showed negative PBA activity. None of the analogues tested could induce any cytostatic macrophages from thioglycollate-elicited peritoneal macrophages.