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If not fertilized, synchronous apoptosis is induced in starfish eggs at approximately 11h after stimulation with the hormone, 1-methyladenine. In this study, a membrane-impermeant substrate of caspase-3, acetyl-Asp-Glu-Val-Asp-coumarylamido-4-methanesulfonic acid (Ac-DEVD-CAMS), was synthesized and microinjected into a starfish egg. Caspase-3 activity in unfertilized egg was detected approximately 30min before blebbing by quantifying the accumulation rate of a membrane-impermeant, fluorogenic product, 7-aminocoumarin-4-methanesulfonic acid (ACMS), using a photomultiplier mounted on a fluorescence microscope. When active recombinant human caspase-3 was microinjected into an egg at 3h after 1-methyladenine treatment, the injected caspase-3 activity decreased and disappeared within 2h. This decrease is probably due to proteasome-dependent degradation of the enzyme, since the injected caspase-3 was degraded and a proteasome inhibitor blocked its degradation. In contrast, in aged eggs at approximately 10h after 1-methyladenine treatment, no degradation of the injected caspase-3 was observed, suggesting that endogenous caspase-3 may stabilize at this point, therefore, inducing apoptosis.

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 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 monooleoyl glycerophospho heptapeptide was synthesized as a candidate of the endogenous pathogen of essential hypertension. Synthesis of the target compound was achieved by the fragment condensation of the phosphorylated tetrapeptide with a His-containing tripeptide.

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.

N-Acetylneuramimic acid (sialic acid) 1 was synthesized through relatively short steps from d -glucose via a key intermediate 1,5-lactone derivative obtained from the pyranose isomer of the aldol condensation products of d -glucose with oxalacetic acid.

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.

A spider toxin NSTX-3 obtained from the venom of Papua New Guinean spider, Nephila maculata was synthesized in order to confirm its proposed structure and to supply the sample for the biological tests. The structure of NSTX-3 is now established as 2,4-dihydroxyphenylacetyl-L-Asn→Cad←Pua←L-Arg, where Cad stands for cadaverine and Pua for putreanine.

A lysophosphatidylserine with 19:4 acyl group proposed as a candidate of DLIS-2, an endogenous digoxin-like substance was chemically synthesized. The synthetic compound showed a significant activity of Na + ,K + -ATPase inhibition, which has been accepted as a major indicator activity for the essential hypertension.

In a study on structure-activity relationship of spider toxin NSTX-3 obtained from venom of Nephila maculata, nine analogs or fragments of natural toxin were synthesized. Among them, des-Arg- NSTX-3 was found to be the minimum structure for blocking the excitatory transmission in the crustacean neuromuscular synapse. A positive charge of the arginine part was concluded to play the most important role for exhibition of the activity.

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.

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.

The structure of the lipid A component of E. coli lippopolysaccharide was determined by means of chemical and 2D-NMR methods unequivocally to be a glucosamine β(1′-6)-disaccharide 1,4′-diphosphate acylated at the two hydroxyl (positions C-3 and −3′) and the two amino groups.

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.

Total Synthesis of antibiotic althiomycin has been achieved starting from D-cysteine. The imide bond between thiazoline and the pyrrolinone part was constructed by coupling reaction of sodium salt of pyrrolinone with cysteine active ester or by photoreaction with diketene. The hydroxymethyl group attached on the carbon adjacent to C-2 of the thiazoline ring, was introduced by aldol condensation posterior to the thiazoline ring formation. The thiazole part was introduced in a final step in whole process of the total synthesis of the antibiotic. The synthetic althiomycin was identical with the natural antibiotic in all respects.

A synthetic route was established to construct the proposed structure of lipid A which consists of β (1–6) glucosamine disaccharide 1,4′-diphosphate O,N-acylated with (R)-3-hydroxy- or (R)-3-acyloxytetradecanoic acid.

Two antigens, cholera toxin (CT) and a synthetic albumin-conjugated 16-residue peptide derived from the circumsporozoite (CS) protein of Plasmodium falciparum sporozoites, were tested as immunogens in rabbits. The malaria peptide-albumin conjugate by itself was completely nonimmunogenic, and although cholera toxin was immunogenic it also expressed considerable native toxicity. After attachment of CT to liposomes containing ganglioside GM1, toxicity of CT was completely eliminated and antigenicity was enhanced. Therefore liposomes may be capable of reducing toxicity of certain potentially dangerous antigens such as toxins. After incorporation of the malaria peptide-albumin conjugate into liposomes a high titre of specific antibodies was induced against the malaria peptide but not against albumin. These antibodies also reacted with native CS protein. Three adjuvants, including lipid A and two types of lipophilic muramyl dipeptide, were compared and found to be effective in liposomes. Based on the conversion of synthetic P. falciparum CS peptide from a nonimmunogenic to an immunogenic form and on the 'toxoiding' effect of liposomes for CT, it is concluded that liposomes should be considered as being a useful carrier for antigens and adjuvants for vaccines for poorly antigenic or toxic substances.

The biological activities of synthetic glycolipids, which were chemically synthesized and based on the structure of lipid A of the lipopolysaccharide (LPS) from Escherichia coli, were examined with special reference to their adjuvant activity on the induction of delayed-type hypersensitivity, activity on the induction of tumour necrotic factor (TNF) and tumour regressive activity on line 10 hepatoma in strain 2 guinea pigs. Among them, a compound structurally corresponding to free E. coli lipid A (compound 506) as well as LPS exhibited potent adjuvant activity in the induction of delayed-type hypersensitivity in guinea pigs and TNF inducing activity in the sera of mice which were presensitized with Propionibacterium acnes. Compound 506 showed potent lethal toxicity in the intravenous administration of BALB/c mice presensitized with P. acnes. The regressive activity on line 10 hepatoma was observed by the multiple intralesional injection of squalane-treated compounds 504 and 505 in strain 2 guinea pigs.

The first total synthesis of streptothricin F was achieved and its structure was unequivocally confirmed.

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.

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.

A synthetic muramyl dipeptide (MDP), which has the minimal adjuvant-active structure that can substitute for Mycobacterium in complete Freund's adjuvant, could stimulate peritoneal macrophages of the guinea pig to produce T-cell activating soluble factors. The culture supernatant of MDP-stimulated macrophages could help the proliferative response of the T-lymphocyte-enriched fraction of lymph nodes to PHA. It also helped the antigenic activation of immune T lymphocytes deprived of macrophages to produce macrophage migration inhibitory factor (MIF). Gel filtration of the culture supernatant showed that both of the helping activities were found mainly in the fraction emerging at the 53,000- to 85,000-MW range. A series of MDP analogs which are known to be adjuvant active or inactive was tested for their ability to stimulate macrophages to produce these factors. The adjuvant activity of these synthetic compounds was found to be parallel to the ability to stimulate the production of these T-cell activating monokines. Since both of these activities are highly dependent on the precise stereochemical structures of the MDP analogs, the parallelism of these activities would suggest the possibility that the immunopotentiating effect of the MDP and its analogs is related at least partially to their activity to stimulate macrophages to produce T-cell activating monokines.

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.

6- O -(2-Deoxy-2-myristoylamino-6- O -myristoyl-β-D-glucopyranosyl)-2-deoxy-2-myristoylamino-3,4-di- O -myristoyl-D-glucopyranose ( 2 ) was prepared in a synthetic approach to lipid A which is the active center of bacterial endotoxin.

1-α-Monophosphate ( 2 ), 4′-monophosphate ( 3 ), and 1-α, 4′-diphosphate ( 4 ) of 6-0-(2-deoxy-2-tetradecanoylamino-6-0-tetradecanoyl-β-D-glucopyranosyl) -2-deoxy-2-tetradecanoylamino-3, 4-di -0-tetradecanoyl-D-glucopyranose were prepared in order to elucidate the role of phosphate moieties in endotoxic activity of lipid A.

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.

The first total synthesis of E. coli lipid A ( 1 ) is described. The synthetic compound was identical with a natural specimen and exhibited the full endotoxic activity. It was thus conclusively proved by this chemical synthesis that lipid A is the active principle of bacterial endotoxin.

A total synthesis of 2,2′-N; 3,3′-O-tetraquis[(R)-3-hydroxytetradecanoyl]-β(1–6)-D-glucosamine disaccharide 1,4′-diphosphate is described. This is the first confirmation of the fundamental structure of lipid A since the synthetic compound exhibited most of the characteristic biological activities of natural endotoxin.

1-Dephospho E. coli lipid A ( 3 ) was synthesized and shown to be identical with the corresponding derivative isolated from bacterial cells. This provides the unequivocal evidence supporting the structure of E. coli lipid A ( 2 ) recently proposed by us.

Total synthesis of a lanthionine peptide nisin was successfully achieved for the first time by application of new methods for preparations of dehydroalanine and lanthionine moieties, resulting in a confirmation of the proposed structure.

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.

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.

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.

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.