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By using the X-ray and infrared spectroscopic characteristics of various synthetic analogues and partial structures of lipid A in the dried state, a comparison of these compounds with their natural counterparts was undertaken. As judged by their X-ray diffraction and infrared spectroscopic features, the compounds tested could be divided into two main groups. The first group covered those samples synthesized in accordance with a previously assumed structure, while those synthesized in accordance with present knowledge on the lipid-A primary structure formed the second group. Members of the first group were characterized by a liquid-like, alpha-type arrangement of their fatty acyl chains in a non-lamellar supramolecular structure, while all members of the second group formed bilayered phases with a much more ordered, beta-type conformation of their fatty acyl chains. Synthetic Escherichia coli-type lipid A (compound 506), proved to be essentially identical to its natural counterpart with respect to those conformational properties accessible by our methods. The synthetic hepta-acyl species of Salmonella minnesota lipid A (compound 516) revealed an unexpected conformational behaviour, whereby a fatty-acyl-chain packing could be detected which was different from the hexagonal arrangement found for all other compounds of the second group.

In the series of synthetic study on peptide antibiotic nisin, a bicyclic sulfide part ring D-E in this molecule was successfully synthesized. Conjunctive sulfide rings were constructed either by one step or stepwise desulfurization reaction from corresponding disulfides by use of hexaethylphosphorus triamide. Thus, all of the ring moieties in nisin were prepared for total synthesis of this antibiotic

The structure of ancovenin, a new peptide inhibitor of angiotensin I converting enzyme, was determined to be a unique tricyclic peptide which comprises sixteen amino acid residues including dehydroalanine and three sulfide amino acids as unusual components.

6-O-Acyl derivatives of N-acetylmuramyl- l -alanyl- d -isoglutamine (MDP) and synthetic, low toxicity lipid-A analogues were examined for their ability to enhance the potency of current viral vaccines. 6-O-(2-Tetradecylhexadecanoyl)-MDP (B30-MDP) in non-irritative vehicles such as physiological saline, phosphate-buffered saline (PBS), squalene-PBS emulsion, Intralipid or liposomes, significantly stimulated the primary and secondary antibody production of guinea-pigs against influenza split or subunit vaccine and inactivated the hepatitis B virus surface (HBs) antigen. Mice seemed less responsive to the adjuvanticity of B30-MDP than guinea-pigs. Two low toxicity lipid A analogues, acylated β(1–6)- d -glucosamine disaccharide bisphosphates (which do not have amide-bound or ester-bound 3-acyloxyacyl groups unlike fully toxic Escherichia coli-type lipid A), caused significantly enhanced antibody responses, primary or secondary, when administered to mice by incorporation into liposomes with inactivated HBs antigen.

Two new phospholipids, 2-N; 3-O-bis (R)-3-hydroxytetradecanoyl-glucosamine 1-phosphate and 2-N-(R) -3-hexadecanoyloxytetradecanoyl-3-O-(R)-3-hydroxytetradecanoylglucosamine 1-phosphate, were synthesized and identified with natural lipid X and lipid Y respectively.

Several synthetic acylated glucosamine monophosphates, with structures corresponding to the nonreducing or reducing moiety of the lipid A of the Escherichia coli or Salmonella minnesota type, and a synthetic compound corresponding to a biosynthetic disaccharide lipid A precursor (designated Ia or IVA) were examined for their endotoxic and related bioactivities in comparison with those of the synthetic and bacterial parent molecules, i.e., acylated beta(1-6)-D-glucosamine disaccharide bisphosphates. Some of the test monosaccharide compounds were definitely active in most of the in vitro assays. Their activities, except for complement activation, however, were weaker than those of the reference compounds, synthetic and bacterial acylated disaccharide bisphosphates. The differences between the test monosaccharide and disaccharide compounds were much more apparent in in vivo assays, in which the test acylated glucosamine monophosphates were scarcely active, though some test compounds exhibited weak lethal toxicity in galactosamine-loaded mice and were weakly active in pyrogenicity, immunoadjuvant activity, and possible tumor necrosis factor and alpha and beta interferon-inducing ability in Mycobacterium bovis BCG- and Propionibacterium acnes-primed mice, respectively. Mixture at an equimolar ratio of acyl glucosamine monophosphates, each of which has the structure of the reducing or nonreducing moiety of the reference disaccharide compound, did not restore the endotoxic or associated bioactivities of the corresponding parent molecules. No essential differences in bioactivity were noted between synthetic and bacterial monosaccharide compounds, i.e., lipid X, whose structure corresponds to the reducing moiety of E. coli-type lipid A.

Binding studies were performed with a [14C]-labelled derivative of viomycin, tuberactinomycin 0 (TUM O). TUM O bound to 30S and 50S subunits. The binding component was the RNA, since ribosomal proteins did not bind the drug. Other RNAs such as tRNA, phage RNA (MS2), and homopolynucleotides also bound the drug. Striking differences in the binding capacity of the various homopolynucleotides were found. Poly(U) bound strongly, poly(G) and poly(C) bound intermediately, whereas poly(A) showed a very low binding. DNA also bound TUM O, although with native DNA the binding was only weak. Finally the effects of viomycin on the assembly in vitro of the 50S subunit from E. coli were tested. A very strong inhibition was found: when the reconstitution was performed at 0.5 x 10(-6) M viomycin the particles formed sedimented at about 50S, but showed a residual activity of less than 10%. The inhibitory power of viomycin with respect to the in vitro assembly is more pronounced than that found in in vitro systems for protein synthesis.

An amino acid composition of the pep tide antibiotic longicatenamycin was investigated. Four new amino acids, i.e., thero-β-hydroxy-L-glutamic acid, L-2-amino-5-rnethylhexanoic acid, L-2-amino-6-methylheptanoic acid, and L-2-amino-7-methyloctanoic acid, which were reported by Shoji et al., were reconfirmed by comparison with the synthetic specimens. Racemic forms of the latter three amino acids were synthesized through acetaminomalonate method. Furthermore, 5-chloro-D-tryptophan was newly isolated from the hydrolyzate of the antibiotic. This structure was assigned by NMR, MS and ORD. A steric configuration of this amino acid was certified by degradation to D-aspartic acid through ozonization. 5-Chloro-DL-tryptophan was synthesized from 3-cyanopropionaldehyde and p-chlorophenylhydrazine by Fischer’s indole synthesis.

To elucidate the stereospecificity of the selenium-containing hydrogenase from Methanocuccus vannielii , ( R ) - and ( S ) - [4- 2 H 1 -3,4-dihydro-7-hydroxy-1-hydroxyethylquinolinone were synthesized as authentic samples for comparison with the compound which was derived from the enzymic reaction product.

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.

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.

Structural principles of the three regions of the lipopolysaccharide molecule are briefly discussed. One of the recently available, chemically synthesized lipid A analogues has been tested for biologic activities. The preparation was found to be toxic, pyrogenic, and mitogenic. The amounts, however, needed for expression of effects similar to those of lipid A were about 500 times higher. These investigations are aimed at verifying the proposed structure of lipid A and at evaluating structure-activity relationships.

A synthetic compound (compound 516), beta(1-6)-linked D-glucosamine disaccharide 1,4'-bisphosphate, which is acylated by (R)-3-hexadecanoyloxytetradecanoyl, (R)-3-hydroxytetradecanoyl, (R)-3-dodecanoyloxytetradecanoyl, and (R)-3-tetradecanoyloxytetradecanoyl groups at positions 2,3,2', and 3', respectively, exhibited in vitro antigenic reactivity of high specificity comparable to that of free lipid A from Salmonella minnesota R595. This was confirmed by an enzyme-linked immunosorbent assay and an enzyme-linked immunosorbent assay inhibition test with monoclonal and conventional antibodies. The results of comparative analysis performed with several synthetic lipid A analogs as well as three monosaccharide derivatives suggested that the complete structure involving both phosphate groups at the C-1 and C-4' positions and the 3-acyloxyacyl groups at the C-2, C-2', and C-3' positions of the glucosamine disaccharide are required for the expression of the serological specificity of Salmonella-type lipid A. This was deduced from the observations that compound 506, a synthetic Escherichia coli-type lipid A which has the same structure as that of compound 516, except that 3-hydroxytetradecanoyl group is substituted for an acyloxyacyl residue at the C-2 position, exhibited significantly reduced antigenic reactivity as compared with compound 516 and that the replacement by the hydrogen atom of the phosphoryl group at the C-1 position or by 3-hydroxytetradecanoyl or tetradecanoyl groups of acyl residues at the 2, 3, 2', and 3' positions of compound 516 results in a marked reduction of reactivity with monoclonal antibodies 5G and 36G. Similar results were obtained by assays with conventional rabbit antibodies, but the structural difference between compounds 516 and 506 could not be distinguished by these polyclonal antibodies. The results of cross-reactions among synthetic analogs with monoclonal antibodies 161M and 1-9M, which have been confirmed to exhibit different serological specificities from the 5G or 36G antibody, also suggested that the nature and linkage of fatty acyl residues as well as the backbone structure of lipid A play an important role in determining serological specificity of the lipid A molecule.

The applicability and the present limitations of the laser microprobe mass analyser LAMMA -500 as an instrument for the structural analysis of higher molecular weight, non-volatile, bio-organic compounds (less than or equal to 2000 u) were investigated. For this purpose mass spectra of various synthetic and natural compounds representing cell wall components of Gram-negative bacteria, e.g. phospholipids and lipid A-like molecules were studied. In several cases these spectra exhibited relatively simple and interpretable patterns with a prominent quasi-molecular ion originating from alkali attachment. For one group of the compounds studied--synthetic lipid A-like molecules containing a phosphate moiety--the spectra were rather complicated and lacked pronounced quasi-molecular peaks. Possible reasons for this observation are discussed.

Summary N-acetylmuramyl- l -alanyl- d -isoglutamine, a synthetic substance of a minimal structure required for the adjuvant activity of bacterial cell walls was found to activate macrophages in mice, whereas its diastereomer, N-acetylmuramyl- l -alanyl- l -isoglutamine, which is inactive as adjuvant did not activate them.

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.

Long-chain acyl derivatives of the disaccharide dipeptide, which is a common building unit of bacterial cell walls, were synthesized in order to test their immunostimulating activity.

A novel and very accurate method was established for the determination of the optical purity of a peptide by use of the following procedure: (1) hydrolysis of the peptide in deuterium chloride, (2) gas chromatographic separation of each amino acid enantiomer on a chiral phase, and (3) determination of the D/L ratio by mass fragmentography. In this manner, one can estimate the true chiral purity of each amino acid residue with an accuracy of ∼0.2%. The recemization effected during hydrolysis could be eliminated in principle, since the artificially formed DL-amino acids are necessarily labeled at the α-position with deuterium and can thus be distinguished mass spectrometrically from the D- and L-isomer originally present in the peptide. The versatility of the method was proven by analysis of model peptides, as well as by a racemization test in fragment condensation.

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.

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.

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.

Mycobacteria in the adjuvant used for induction of experimental autoimmune encephalomyelitis (EAE) in guinea pigs can be replaced by synthetic N -acetylmuramyl- l -alanyl- d -isoglutamine. A combination of synthetic encephalitogenic peptides and muramyl dipeptides induces EAE effectively at a dose on the microgram level. In this system, the synthetic heptapeptide, H-Trp-Gly-Ala-Glu-Gly-Gln-Arg-OH, with a sequence identical to those of residues 116 to 122 of the basic protein of human myelin, was the shortest peptide causing EAE. These compounds form a simple system which should be useful in studies on the mechanism of the cell-mediated autoimmune reaction.

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 title compound was synthesized and identified with a partial hydrolysis product from natural streptothricin. This work confirmed the presence of the proposed glycosyl guanidine structure in the antibiotic and established a method for its preparation.

Several linear and branched all-trans-multiprenylacetic acids were synthesised, and introduced to the 6 position of the sugar moiety of muramyl dipeptide and its analog, via an amino acid as a linking unit. Compared with the saturated stearoyl derivative, all the compounds having a multiprenylacetyl group exhibited more potent adjuvant activity on the induction of delayed-type hypersensitivity to N-acetyl-3-(4-arsonophenylazo)-L-tyrosine. The derivatives with larger branched side chains tended to have increased activity.

γ-Hydroxy-β-lysine is a new basic amino acid isolated from the hydrolyzates of antitubercular peptides, tuberactinomycin A and N. In order to confirm the structure and establish the stereochemistry of the amino acid, two diastereoisomers of γ-hydroxy-DL-β-lysine were synthesized by Arndt-Eistert synthesis via β-hiydroxyornithine. Synthetic erythro and threo isomers were found to be identical spectroscopically and chromatographically with the corresponding forms obtained from the antibiotics respectively.

α-Ketopyranosyl fluorides, 2 and 3, of 4,5:7,8-di-O-isopropylidene KDO methyl and benzyl esters were prepared and shown to act as effective glycosyl donors. X-Ray structure analysis of 2 and 1H NMR study established the boat (B3,6) conformation of the di-O-isopropylidene derivatives of KDO and enabled the assignment of their anomeric configurations.

Ethyl 2-(1-benzyloxycarbonylaminoethyl)-R-Δ2-thiazoline-4-carboxylate (III), ethyl 2-(1-benzyloxycarbonylamino-2-methyl-n-butyl)-R-Δ2-thiazoline-4-carboxylate (VI) and their acid amide derivatives (V, VII and VIII) were prepared by either the iminoether coupling method or the dehydration method. As a general character of thiazoline peptide, it was found that the amino acid residue connected to thiazoline ring at N-terminal side is completely racemized at the stage of thiazoline ring formation, and further epimerized, in the case of isoleucine, by treatment with the basic reagent.

A simple synthesis of various forms of radioactive L-thyroxine, carrying the label (I/sup 131/ or C/sup 14/) either in the phenolic ring or in the nonphenolic ring and in the side chain, is described, It is based on the coupling of 4hydroxy-3,5-diiodophenylpyruvic acid and 3,5-diiodotyrosine. Labeled 4- hydroxy-3,5-diiodophenylpyruvic acid was prepared by iodination of p- hydroxybenzaidehyde foilowed by condensation with acetylglycine and hydrolysis of the azlactone formed, or by enzymic oxidative deamination of diiodotyrosine. (auth)

Synthesis of thiazoline peptide from cysteine peptide by dehydration with hydrochloric acid in non-aqueous solvent was investigated. By this method, ethyl 2-benzyloxycarbonylaminomethyl-R-Δ2-thiazoline-4-carboxylate and 2 - benzyloxycarbonylaminomethyl-R-Δ2-thiazoline-4-carbonyl-L-leucine ethyl ester were prepared and isolated in pure states. This synthetic method could give a promising route to a total synthesis of bacitracin A. However, the cysteine residue in the latter thiazoline peptide ester was found to be easily racemized under the basic condition.

Tuberactidine (III) is a guanidino amino acid component of an ti tubercular pep tides, tuberactinomycin A and B, while the known guanidino amino acid capreomycidine (V) is involved in tuberactinomycin N and O. The structure of tuberactidine was assigned to be α-(4-hydroxy-2-iminohexahydro-6-pyrimidinyl)glycine and its stereochemistry was also established. During isolation of tuberactidine from an eluate in the column chromatography of tuberactinomycin hydrolyzate, it was converted to Nα-formyltuberactidine dimer (I). In this dimer, structural alternation between a carbinolamine form of cyclol type and an amide lactone form was noticed depending to a pH of the solution. I was hydrolyzed to tuberactidine and viomycidine (IV) with hydrobromic acid. In addition, a plausible pathway for the formation of viocidic acid (VII), one of the artifacts formed during hydrolysis of tuberactinomycin, was proposed.

For the purpose of synthesis of antibiotic bacitracin of six amino acids-membered ring formula, the following intermediate peptides, i.e., cyclo-(Nα-benzy]oxycarbonyl-L-lysyl-Nδ-cyclopentyloxycarbonyl-D-ornithyl-L-isojeucyl-D-phenylalanyl-L-histidyl-α-methyl-L-aspartyl) (III) and Nα-benzyloxycarbonyl-Ne-t-butyloxycarbonyl-L-lysyl-Nδ-cyclopentyloxycarbonyl-D-ornithyl-L-isoleucyl-D-phenylalanyl-Nim-benzyl-L-histidyl-L-aspartyl-D-isoasparagine benzyl ester (IV) were prepared. In this synthesis, cyclopentyloxycarbonyl group was introduced to protect δ-amino group of ornithine residue and cleavability of this protecting group for hydrogen fluoride was investigated.

A protected heptapeptide, the formyl-L-isoleucyl-e-benzyloxycarbonyl-L-lysyl-δ-5-tosyl-D-ornithyl-L-isoleucyl-D-phenylalanyl-im-benzyl-L-histidyl-L-aspartic acid α-methyl-β-benzyl ester, was synthesized by the azide method and the carbodiimide method. It is an intermediate peptide for the total synthesis of the antibiotic bacitracin A, the structure of which is still ambiguous at a few points. β-Benzyl benzyloxycarbonyl-L-aspartate was prepared by a novel method similar to that used in the synthesis of γ-benzyl glutamate. The α-methyl-β-benzyl L-aspartate prepared could be useful in the synthesis of branched aspartic acid peptide. Phenomena of dimorphism were observed in crystals of δ-tosyl-D-ornithine and δ-tosyl-D-ornithyl-L-isoleucyl-D-phenylalanine ethyl ester hydrobromide, and two crystal forms of each compound were isolated in pure states.

Threonine peptide was converted to an oxazolidone derivative via an O-chlorocarbonyl derivative by treatment with phosgene and dimethylaniline followed by refluxing in xylene, wherever the threonin...

In the synthetic study of bacitracins, peptide fragments corresponding to a branched part of bacitracin F, i.e., (S)-2-(2-methylbutyryl)thiazole-4-carboxylic acid 1-succinimidyl ester (III) and L-leucyl-γ-t-butyl-D-glutamyl-L-isoleucine (IV), were prepared. As a macrocyclic part of bacitracin A and F of cycloheptapeptide formula, cyclo- (Nα-benzyloxycarbonyl-L-lysyl-Nδ-cyclopentyloxycarbonyl-D-ornithyl-L-isoleucyl-D-phenylalanyl-L-histidyl-β-benzyl-D-aspartyl-L-asparaginyl) (V) was synthesized through cyanomethyl ester method.

A new basic amino acid, hypusine, was isolated from the homogenate of bovine brain tissue by ion-exchange column chromatography. The structure of this amino acid was determined to be N 6 -4-amino-2-hydroxybutyl)-2,6-diaminohexanoic acid on the basis of its physical properties involving NMR and mass spectra, as well as chemical degradation including periodate oxidation and reduction with HI and P.

Asparagine peptide was selectively cleaved by the Hofmann rearrangement followed by an alkaline treatment at the peptide linkage in which an amino group of the asparagine residue participated. Upon reaction of N-benzyloxycarbonyl-L-alanyl-L-asparagine with one equivalent of bromine and three equivalents of aqueous sodium hydroxide solution at 60°C, 1-(N-benzyloxycarbonyl-L-alanyl)-2-oxoimidazolidine-5-carboxylic acid was obtained in quantitative yield. When this was treated with an aqueous methanolic sodium hydroxide solution at room temperature, a cleavage reaction occurred to give N-benzyloxycarbonyl-L-alanine and 2-oxo-imidazolidine-5-carboxylic acid. Under the same condition of the rearrangement, N-benzyl-oxycarbonyl-L-asparaginyl-L-phenylalanine was converted to 1-benzyloxycarbonyl-2-oxoimidazo-lidine-5-carbonylphenylalanine. Since glutamine peptide could not give the corresponding ring compound, this cleavage reaction was found to be specific to asparagine peptide.

The structure of putreanine, a new amino acid isolated from mammalian brain, was studied by its dissociation constants, mass spectrum, NMR spectrum and ORD. The structure N-(4-aminobutyl)-3-aminopropionic acid was assigned to it from the results obtained. This amino acid was synthesized by condensation of 4-phthalimino-1-bromobutane and ethyl β-alaninate followed by add hydrolysis. The identity of the synthetic compound with the natural amino acid confirmed the structural assignment.

For a total synthesis of bacitracian A, a synthetic method to secure unprotected thiazoline peptides must be established. 2-Aminomethyl-R-Δ2-thiazoline-4-carboxamide, 2-aminomethyl-R-Δ2-thiazoline-4-carboxylic acid, 2-aminomethyl-R-Δ2-thiazoline-4-carbonyl-L-leucine were synthesized by either the iminoether coupling method or by the dehydration method using N-benzyloxycarbonyl group and benzyl ester as protecting groups.

An optical active 2-(2-methylbutyryl)thiazole-4-carboxylic acid obtained from bacitracin F by an acid hydrolysis was synthesized starting from L-isoleucine. Applying the usual methods for the peptide synthesis, 2-(2-methylbutyryl)thiazole-4-carbonyl-L-leucyl-D-glutamic acid α-methyl-γ-t-butyl ester corresponding to the N-terminal tetrapeptide of bacitracin F, was then synthesized from the thiazole carboxylic acid. UV spectrum of the synthetic tetrapeptide was very similar to that of bacitracin F.