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N-Acetylmuramyl-L-alanyl-D-isoglutamine α-phenylglycoside was synthesized from α-phenyl-D-glucosaminide peracetate, which was obtained by fusing β-D-glucosamine pentaacetate with phenol and zinc chloride followed by deacetylation. α-Phenyl-N-acetylglucosaminide was converted in two steps into 4,6-O-isopropylidene-N-acetyl-D-muramic acid α-phenylglycoside, which was condensed with L-Ala-D-iGln benzyl ether using the activated ester method. Sequential removal of glycopeptide protecting groups via acid hydrolysis and catalytic hydrogenolysis gave the target product.

N-Acetylmuramyl- l -alanyl- d -isoglutamine or muramyl dipeptide is a cleavage product of peptidoglycan by lysozyme. This study explored the use of the temperature-programmed desorption mass spectrometry (TPDMS) in analysis of glycoside of muramyl dipeptide: O-{(4-tert-butylcyclohexyl)-2-acetamido-2,3-dideoxy-β- d -glucopyranoside-3-yl}- d -lactoyl- l -alanyl- d -isoglutamine (MDP) on the surface of fumed silica. Stages of pyrolysis of MDP in condensed state and on the silica surface have been determined. Three stages have been clear identified under pyrolysis of MDP on the silica surface. Kinetic parameters of thermal reactions on the fumed silica surface and in the condensed state have been calculated.

A relatively easy synthetic method is reported for the production of the immunoadjuvant glycopeptide, N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP). Most of the details in this method were also applied to the preparation of some analogs, that were synthesized in order to study structure-activity relationships.

A series of MDP (muramyldipeptide) or nor-MDP (normuramyldipeptide) analogues modified at the C-terminus post of the molecule by a formation of an ester bond between the carboxylic group of isoglutamine and the hydroxyl function of the respective derivatives of 4-carboxamide-acridine/9-acridone or 1-nitro-9-hydroxyalkylaminoacridines were synthesized as potential anticancer agents. The compounds O-(1-O-benzyl-N-acetyl-muramyl-l-alanyl-d-gamma-isoglutaminyl)-9-(ethylamino)-1-nitroacridine ester 3j and O-(1-O-benzyl-N-acetyl-muramyl-l-alanyl-d-gamma-isoglutaminyl)-9-propylamino-1-nitroacridine ester 3k exhibited high in vitro cytotoxic activity against a panel of human cell lines, prostate cancer and AIDS-related lymphoma (ARL). Analogue 3j was also active in vivo in the hollow fiber assay. Antitumor activity of both compounds were tested in vivo against difference human tumor xenograft, but only analogue 3k showed in vivo activity against sc UACC-62 melanoma in mice.

We have reported [unpublished data] the synthesis and QSAR of 5-substituted-2-(substituted benzenesulphonyl) glutamines which have shown the importance of steric factor on the aliphatic chain. N-Phthalyl isoglutamine, having the substitution at position 1 of the glutamic acid moiety, is the metabolite of recently approved thalidomide for different types of tumors by US FDA. Based on these, 36 new 1,5-N,N'-disubstituted-2-(substituted benzenesulphonyl) glutamamides were synthesized, as tools for further elucidation of the structural requirements for antitumor activity. All the synthesized compounds were tested for antitumor activity against Ehrlich Ascites Carcinoma (EAC) in Swiss albino mice using tumor weight as inhibitory parameter. Quantitative structure-activity relationship (QSAR) studies of these analogues revealed that the electron donating groups on the phenyl ring are found to be mandatory for the activity which was also proved by the negative coefficient of indicator parameter I(3,) for NO(2) group on the phenyl ring. Molecular volume (MV) and steric factor at R(5) position also plays a role in ligand-receptor interactions.

Partial structures of peptidoglycan, a potent immunostimulating glycoconjugate of bacteria, were synthesized for precise biological studies. A key disaccharide glucosaminyl-β(1–4)-muramic acid was prepared by stereoselective glycosylation of a N -Troc (Troc=2,2,2-trichloroethoxycarbonyl) muramic acid acceptor with a N -Troc-glucosaminyl trichloroacetimidate. The disaccharide was converted to either a disaccharide acceptor or a donor. They were then coupled together by the same glycosylation method to give a tetrasaccharide in a good yield. Octasaccharide was also obtained in a good yield in a similar manner. N -Acetylation and coupling with the dipeptide moiety of l -alanyl- d -isoglutamine followed by deprotection afforded the repeating peptidoglycan tetrasaccharide and octasaccahride peptide conjugates for the first time.

The surface of the melanoma BRO cells was shown to contain binding sites for N-acetylglucosaminyl-(β1-4)-N-acetylmuramyl-alanyl-D-isoglutamine (GMDP). Their number (1500 ± 200 per cell) and affinity (Kd= 10 ± 1.2 nM) were determined. The occurrence of these sites was found to correlate with the ability of the melanoma cells to react invitrowith GMDP by increasing the expression of melanoma-associated antigens (MAA). An increased number of the GMDP binding sites (5200 ± 500 per cell) was observed upon treating the melanoma BRO cells with tumor necrosis factor α (TNF-α). The mechanism of the TNF-α action most likely involves the unmasking of GMDP binding sites, initially expressed on the cell surface, by activating the endogenous protease that hydrolyzes surface proteins, in particular, highly glycosylated LAMP-2 protein exposed on the melanoma cell surface.

Ethyl 3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-D-glucopyranoside (5), prepared by benzylation of ethyl 2-deoxy-2-phthalimido-1-thio-β-D-glucopyranoside (4), was transformed by reaction with bromine into 3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl bromide (6). Thioglycoside 5 in the presence of methyl triflate and glycosylbromide 6 in the presence of silver triflate were used as glycosyl donors for condensation with benzyl 2-acetamido-3-O-allyl-6-O-benzyl-2-deoxy-α-D-glucopyranoside (7), to give benzyl 2-acetamido-3-O-allyl-6-O-benzyl-4-O-(3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-2-deoxy-α-D-glucopyranoside (8). Its reductive dephthaloylation with NaBH4/AcOH afforded benzyl 2-acetamido-3-O-allyl-4-O-(2-amino-3,4,6-tri-O-benzyl-2-deoxy-β-D-glucopyranosyl)- 6-O-benzyl-2-deoxy-α-D-glucopyranoside (11). Compound 11 was N-acylated to give benzyl 2-acetamido-4-O-(2-acylamino-3,4,6-tri-O-benzyl-2-deoxy-β-D-glucopyranosyl)-3-O-allyl-6-O-benzyl-2-deoxy-α-D-glucopyranosides (12a) or (12b). These compounds were converted into corresponding benzyl 2-acetamido-4-O-(2-acylamino-3,4,6-tri-O-benzyl-2-deoxy-β-D-glucopyranosyl)-6-O-benzyl-3-O-carboxymethyl-2-deoxy-α-D-glucopyranosides which, by condensation with H-L-Abu-D-isoGln(OBzl) followed by hydrogenolysis of protective benzyl groups, furnished glycopeptides 16a and 16b. Intramolecular O→N migration of the allyl protecting group followed by its reduction to the propyl residue by reaction of compound 8 with hydrazine or hydrazinium acetate, to give benzyl 2-acetamido-4-O-(3,4,6-tri-O-benzyl-2-deoxy-2-propylamino-β-D-glucopyranosyl)-6-O-benzyl-2-deoxy-α-D-glucopyranoside (9), is also described.

N-Acetylnormuramoyl-L-2-aminobutanoyl-D-isoglutamine (7) and its lipophilic 6-O-octadecanoyl (8) and 6-O-(2-tetradecylhexadecanoyl) (9) derivatives were prepared and their immunoadjuvant activity and pyrogenicity were tested. Compounds 8 and 9 are less pyrogenic than muramoyl-dipeptide (MDP) and norMDP analog 7. Both lipophilic derivatives 8 and 9 are better adjuvants than MDP in cell mediated immunity.

In an attempt to clarify ambiguities in earlier reports on the preclinical chemistry of Antineoplaston A10 (3-[(phenylacetyl)amino]-2, 6-piperidinedione; PAP), we detail herein hydrolysis studies with improved synthesis and characterization of PAP hydrolysis products, (phenylacetyl)-glutamine (PAG), (phenylacetyl)isoglutamine (PAIG), and (phenylacetyl)-glutamic acid (PAGA). Flash chromatography proved superior to extraction in the isolation of synthetic standards and hydrolysates. Synthesis of PAIG directly from commercial isoglutamine showed consistently better yields than the previously reported method. The 1H and 13C NMR and HPLC-MS data from the synthesized standards matched the data from the isolated PAP hydrolysates formed under acid and alkaline degradation conditions. Multiple quantum coherence NMR methods (HMQC and HMBC) and HPLC-MS-MS methods were applied to provide unambiguous structural assignments for key isomers PAG and PAIG. Previous PAP hydrolysis studies are shown to be reproducible, and the structures of hydrolysis products are elucidated in detail.

Summary In search for immunologically active dipeptides, a series of new N -(7-oxoacyl)- l -alanyl- d -isoglutamines has been synthesized where lipophilicity was varied by changing the 7-oxoacyl residue from 7-oxooctanoyl to 7-oxotetradecanoyl. The immunological properties of the compounds were examined in an in vivo immunorestoration test. It was found that lipophilicity had a significant influence on the potency of the compounds tested. Compounds between N -(7-oxodecanoyl)- l -alanyl- d -isoglutamine and N -(7-oxotetradecanoyl)- l -alanyl- d -isoglutamine showed biphasic influence on efficacy. The most active compound found was 7-oxododecanoyl- l -alanyl- d -isoglutamine, which also has an activity comparable to that of azimexone.

The syntheses of eight conformationally restricted analogues of N -acetylmuramyl- l -alanyl d -isoglutamine (MDP) based on the d -glucopyrano[2,3- d ]oxazolidine ring system are described.

The present experiments demonstrate that, similar to immunomodulatory muramyl dipeptide, its desmuramyl analog adamantylamide dipeptide is able to induce mild and fully reversible paw edema in mice. This effect is an immune-related phenomenon depending on the activation of T-cell/macrophage interactions and on production of prostaglandins. Possible involvement of certain immunoregulatory/inflammatory cytokines (e.g. IL-1, IL-2) has been suggested. The most probable intrinsic moiety of the adamantylamide dipeptide molecule responsible for triggering the edema formation is obviously D-isoglutamine.

The synthesis has been effected of derivatives of MDP having a spacer with an amino or carboxy group. The final stage of the synthesis was the condensation of Boc-L-Ala-D-iGln with 6-aminohexanol (followed by the two-stage replacement of the hydroxy by an azido group) or with benzyl 6-aminohexanoate.

α-Cyclopentyl D-glutamate was obtained from dicyclopentyl D-glutamate utilizing lipase-catalyzed hydrolysis. The monoester was then treated with NH 3 in methanol to produce D-isoglutamine.

Partial structures of peptidoglycan, a potent immunostimulating glycoconjugate of bacteria, were synthesized for precise biological studies. A key disaccharide glucosaminyl-β(1–4)-muramic acid was prepared by stereoselective glycosylation of a N -Troc (Troc=2,2,2-trichloroethoxycarbonyl) muramic acid acceptor with a N -Troc-glucosaminyl trichloroacetimidate. The disaccharide was converted to either a disaccharide acceptor or a donor. They were then coupled together by the same glycosylation method to give a tetrasaccharide in a good yield. Octasaccharide was also obtained in a good yield in a similar manner. N -Acetylation and coupling with the dipeptide moiety of l -alanyl- d -isoglutamine followed by deprotection afforded the repeating peptidoglycan tetrasaccharide and octasaccahride peptide conjugates for the first time.

In order to confirm that tert-butyl ester protection at the isoglutaminyl residue prevents the base-catalyzed iso-glutamine = glutamine rearrangement in title compounds, 6-0-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-N-acetylmura-moyl-L-alanyl-D-glutamine (7) was synthesized from α-benzyl glycoside of 6-0-(2-acetamido-3,4,6-tri-0-acetyl-2-deoxy-β-D-glucopyranosyl)-N-acetylmuramic acid (2) and Z-L-Ala-D-Glu(NH2)OBut via its α-tert-butyl ester 5. 1H and 13C NMR measurements were carried out with regioisomeric [β-GlcNAc-(1-→6)-MurNAc]-L-Ala-D-glutamine and -isoglutamine derivatives; the presented data show differences that enable distinction between the sugar-peptide regioisomers.

Four novel analogs of hepatitis B virus surface antigen (14-32)Pre-S2 region fragment attached covalently to N-acetylmuramic acid, N-acetylmuramyl-L-alanyl-D-isoglutamine, N-acetylnormuramic acid, and N-acetylnormuramyl-L-alanyl-D-isoglutamine were synthesized by the solid phase method. The ability of analogs to induce cellular and humoral immunity to native HBsAg was tested on rabbits. Cellular immune response occurred in vitro, and HBs antibodies were detected in all immunized animals. No additional adjuvants were used in the tests.

The 1-β-S-acyl-6-O-phosphono, 6-O-acyl-1-deoxy-4-O-phosphono, and 1-deoxy-6-O-(16-phosphorylhexadecanoyl) derivatives of N-acetylmuramoyl-l-alanyl-d-isoglutamine methyl ester were synthesized from corresponding intermediates with bis(2,2,2-trichIoroethyl) phosphorochloridate and diphenyl phosphorochloridate, respectively. The immunoadjuvant activity of each product was examined.

Two exocyclic olefins 10 and 22 readily elaborated from D-glucosamine have been transformed by Ferrier rearrangement reaction into amino-cyclohexanones 11 12 and 23 24. Reduction of ketone 11 with tri-t-butoxy-aluminium hydride followed by sequential acetylation and hydrogenolysis gave the acid 14 which was coupled with dipeptide 15 to provide 16. Alkaline hydrolysis of the latter afforded 1 a carbocyclic nor-analogue of MDP. The second target molecule 2 was prepared from an oxazolidine-pseudo-D-glucosamine 27 obtained by treatment of amino-cyclohexanone 23 with methoxymethylenetriphenylphosphorane and mercuric acetate-sodium borohydride reagents as key steps in the synthesis. On the other hand hydroboration of the olefin 31 yielded pseudo-L-idosamine derivative 28. Birch reduction of 27 followed by sequential benzylidenation etherification with (S)-α-chloropropionic acid and condensation of the resulted 36 with dipeptide 15 gave 36. Hydrogenolysis of the latter afforded the carbocyclic analogue 2 of MDP.

[reaction: see text] A synthesis of the glutarimide-derived metabolite of thalidomide, 5'-hydroxythalidomide (2), is described. The synthesis employed the lactone derivative of N-benzyloxycarbonyl (CBZ)-protected 4-hydroxyglutamic acid 12, which is prepared by a de novo route from diethyl acetamidomalonate. The reaction of 12 with 4-methoxybenzylamine gave the corresponding isoglutamine, which then provided the key CBZ-protected N-PMB-glutarimide 14 after dehydration. Deprotection of both the CBZ and PMB groups followed by phthalimidation and deacetylation of the 3-amino-5-acetoxyglutarimide 16 afforded 2.

The unusual amino acid diaminopimelic acid (DAP) was prepared by cross metathesis of appropriately protected vinyl glycine and allyl glycine derivatives. Catalytic hydrogenation of the cross-coupling product resulted in reduction of the double bond and the removal of protecting groups. The resulting compounds were appropriately protected for the polymer-supported and solution-phase synthesis of muramyl tripeptides 2 and 3, which differ in the amidation of the alpha-carboxylic acids of the isoglutamine and DAP moieties. Muramyl dipeptide (1, MDP), the DAP-containing muramyl tripeptide 3, and the lysine-containing muramyl tripeptides 4 and 5 induced TNF-alpha gene expression without TNF-alpha protein production in a human monocytic cell line. The observed block in translation could be removed by co-incubation with LPS, resulting in an apparent synergistic effect. Compound 2 did not induce TNF-alpha gene expression, neither did it exhibit a synergistic effect with LPS; this indicates that amidation of the alpha-carboxylic acids of the isoglutamine and DAP moieties results in a loss of biological activity. It is proposed that amidation of alpha-carboxylic acids is a strategy that may be used by pathogens to avoid detection by the innate immune system. Furthermore, the pattern recognition receptors Nod1 and Nod2 have been implicated in the possible induction of a synergistic effect of muropeptides with LPS.

Several conjugates of muramyl dipeptide (MDP) or nor-muramyl dipeptide (nor-MDP) with tuftsin were synthesized. Conjugates 8a–f were prepared by acylation of protected tuftsin with the isoglutamine carboxyl group of MDP or nor-MDP 2a–f. Also tuftsin analogue 6 (H-Thr-Lys-Pro-Arg(NO2)-OH) was obtained. All synthesized compounds were investigated at the Medical University of Gdansk. The biological activity of the examined compounds was estimated using in vitro cultures of human monocytes and lymphocytes. The substances displayed cytotoxic effects, as was revealed in the viability tests performed. The effects were most probably mediated by the induction of an oxidative burst in monocytes and the stimulation of redox enzymes in lymphocytes. In addition, the analogues turned out to be efficient stimulators of TNFα and IL6 secretion by monocytes and lymphocytes. Nevertheless, the secretion of cytokines did not affect the viability of the leukocyte population used in the experiments. The beneficial properties of the compounds examined (mainly 6, 3, 8a and 8c), which implies their usefulness as potential therapeutic agents, are connected with their rapid start of action and more efficient effects compared with tuftsin alone. An in vivo assay on animal models will be performed. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.

The 1-α-O- and 1-β-S-[(3R)-3-tetradecanoyloxytetradecanoyl] derivatives of N-acetylmuramoyl-L-alanyl-D-isoglutamine methyl ester (MDP methyl ester) were synthesized by coupling of the corresponding intermediate, the C-1-hydroxyl or C-1-β-thiol derivative of MDP methyl ester, with (3R)-3-tetradecanoyloxytetradecanoic acid. The immunoadjuvant activities of each product and their related compounds were examined.

The synthesis of two groups (Chart 1, types A and B) of conjugates of MDP (muramyldipeptide) and nor-MDP (normuramyldipeptide) with acridine/acridone derivatives and the synthesis of analogues of desmuramylpeptides (Chart 1, types C and D) containing acridine/ acridone derivatives have been described. In type A conjugates, the hydroxyl group at C6 of the sugar moiety was acylated with acridine/acridone N-substituted omega-aminoalkanocarboxylic acids (Scheme 1), whereas the conjugates of type B (Table 2) and three analogues of type C or D (Scheme 2) have an amide bond formed between the carboxylic group of isoglutamine and the amine function of the respective acridine/acridone derivatives. The preliminary screening data indicate that the analogues of groups A, C, and D exhibit small cytotoxic activity, whereas several analogues of type B, 4b, 4c, 4e, 4g, 4h, 4i, and 4l, exhibiting potent in vitro cytotoxic activity against a panel of human cell lines (Table 4), have been selected by the National Cancer Institute (NCI) Evaluation Committee for further testing. Analogues 4b and 4h were active in the in vivo hollow fiber assay (Table 5). Analogue 3a shows an immunostimulating effect on the cytotoxic activity of the NK cells obtained from the spleen of healthy and Ab melanoma bearing animals.

In the search for more potent and less toxic immunomodulators, adamantylamide dipeptide (AdDP) was synthesized by the covalent union of amantadine with the l -alanyl- d -isoglutamine residue of muramyldipeptide (MDP). The present experiments demonstrate the ability of AdDP, co-administered with a protein immunogen, to raise or enhance a humoral response in immunized animals. BALB/c mice were immunized either by the intraperitoneal (ip) or oral route with ovalbumin (Ova) alone or combined with either AdDP or CpG oligonucleotide (ODN-CpG), a proved adjuvant. A clear adjuvant dose–response relationship was observed on the increment of Ova-specific serum antibody titers when AdDP was used as adjuvant, irrespectively of the administration route. The IgG isotype analysis showed that AdDP promotes a consistent increment in IgG1 antibodies associated with a dominant Th2 response pattern. When administered by the oral route, AdDP was at least as efficient as ODN-CpG as adjuvant. Similar results were obtained in rabbits immunized by the oral route, suggesting that the adjuvanticity of AdDP is not restricted to the murine system. In conclusion, AdDP was shown to be a powerful and non-toxic adjuvant at both systemic and mucosal levels, which makes it a promising tool for vaccine development.

N-Glycopeptide mimetic with N-glycosylated isoasparagine and isoglutamine conjugates were synthesized by regioselective opening of unsymmetrical cyclic anhydride derivatives of l-aspartic acid and l-glutamic acid, using per-O-acetylated ?-d-glycopyranosyl amine. The ?-chloro derivative gave a mixture of asparagine and isoasparagine linked glycoconjugates, whereas the trifluoroacetamide derivatives gave predominantly the isoasparagine and isoglutamine linked glycoconjugates as the product. The X-ray crystal structure of the ?-chloro isoasparagine linked glycoconjugate showed unique pattern of hydrogen bonding. This journal is � the Partner Organisations 2014.

This study investigates the effect of some components of the Staphylococcus aureus cell wall [lipoteichoic acid (LTA), N-acetyl-muramyl-alanyl-d-isoglutamine (MD), muramic acid (MA) and protein A (PA)] in modulating expression of cell-surface adhesion molecules CD11a/CD18, CD11b/CD18 on monocytes qualitatively and quantitatively. Monocytes incubated with bacterial components presented different CD11b/CD18 expressions which were dose-dependent in contrast to controls. The results obtained demonstrated that lymphocytes incubated with bacterial components also increased the expression of CD11a/CD18. The modifications in activation of CD11a/CD18 and CD11b/CD18 expression are probably correlated with modifications of membrane fluidity measured as polarisation fluorescence (P).

The formulation consists of N -acetylglucosaminyl- N -acetylmuramyl- l -alanyl- d -isoglutamine, dimethyldioctadecylammonium chloride and the trace element zinc as an l -proline complex. This ternary synergistic composition shows promise as a new adjuvant.

A number of compounds, in their structure similar to the immunomodulatory agent adamantylamide-L-alanyl-D-isoglutamine (AdDP), a desmuramyl analog of muramyl dipeptide (MDP), were compared with respect to their in vivo immunobiological activity. The agents were represented by adamantylamide-peptides or -amino acids differing principally in the nature of glutaminyl moieties. They were all able to induce inflammatory reaction which was prevented by anti-T-cell (TH) mAb, silica, indomethacin and dexamethasone. Responsible for this effect proved to be glutaminyl moieties (glutamine, isoglutamine, or glutamic acid), irrespective of their stereospecificity. Unlike the immunomodulatory molecule of AdDP, neither of the compounds and amino acids were able to stimulate production of antibodies in immunized animals, however.

Silver triflate-promoted condensation of 3,4,6-tri-O-acetyl-2-deoxy-2- phthalimido-beta-D-glucopyranosyl bromide (1) with benzyl 2-acetamido-6-O-benzyl-2-deoxy-3-O- (methoxycarbonyl)methyl-alpha-D-glucopyranoside (4) afforded the key compound, benzyl 2-acetamido-6-O-benzyl-2-deoxy-3-O- (methoxy-carbonyl)methyl-4-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b eta-D- glucopyranosyl)-alpha-D-glucopyranoside (5), which after deprotection was transformed into acid 10. Condensation of 10 with the benzyl ester of L-alpha-aminobutanoyl-D-isoglutamine and deisopropylidenation of the product 11 afforded the benzyl ester of N-(2-O-[benzyl 2-acetamido-4-O-(2-acetamido-3-O-benzyloxymethyl-2- deoxy-beta-D-glucopyranosyl)-6-O-benzyl-2,3-dideoxy-alpha-D-glucopyra nosid-3- yl]glycoloyl)-L-alpha-aminobutanoyl-D-isoglutamine (12). Partial O-acylation of 12 and hydrogenolysis of protecting groups gave the 6-O-stearoyl- and 6-O-(2-tetradecylhexadecanoyl)-disaccharide-dipeptides 17 and 18, respectively. Pyrogenicity and adjuvant activity in cell-mediated immunity are reported.

The effect of methyl esterification of the isoglutamine residue in 6- O -acylated muramyl dipeptide (MDP) on some biological activities was investigated. Methyl esterification influenced more or less the expression of all activities tested. The adjuvant and colony stimulating factor (CSF)-inducing activities of 6- O -acylated MDP analogs carrying a 3-hexadecanoyloxytetradecanoyl [C 14 O(C 17 )] group were stronger than those of the corresponding analogs carrying a 2-tetradecylhexadecanoyl (B30) group. Macrophage activation, i.e. induction of tumor necrosis factor (TNF) and promotion of phagocytosis, by 6- O -C 14 O(C 16 )-MDP methyl esters was weaker.

2-Azido-2-deoxy-4,6- O -isopropylidene-3- O -[(1 R )-(methoxycarbonyl)ethyl]- a - d -glucopyranosyl trichloroacetimidate ( 3 a ) has been used as the glycosyl donor in the synthesis of glycosphingolipids 14 and 27 . Reaction of 3 a with (2 S , 3 R , 4 E )-2-azido-3-benzoyloxy-4-octadecen-1-ol ( 6 ) gave (2 S , 3 R , 4 E )-2-azido-1-{2-azido-2-deoxy-4,6- O -isopropylidene-3- O -[(1 R )-1-(methoxycarbonyl)ethyl]-β- d - glucopyranosyloxy}-3-benzoyloxy-4-octadecene ( 7 ), which was converted into (2 S , 3 R , 4 E )-1-{2-deoxy-2-hexadecanoylamino-3-O-[(2 R )-propanoyl-( l -alanyl- d -isoglutamine benzyl ester)-2-yl]-β- d -glucopyranosyloxy}-2-hexadecanoyl-amino-4-octadecen-3-ol ( 14 ). Reaction of 3 a with tert -butyldimethylsilyl 2-azido-3,6-di- O -benzyl-2-deoxy-β- d -glucopyranoside ( 15 ) gave tert -butyldimethylsilyl 2-azido-4- O -{2-azido-2-deoxy-4,6- O -isopropylidene-3- O -[(1 R )-1-(methoxycarbonyl)ethyl]- β- d -glucopyranosyl}-3,6-di- O -benzyl-2-deoxy-β- d -glucopyranoside ( 16 β), which was converted into 1,3,6-tri- O -acetyl-2-deoxy-4- O -{4,6-di- O -acetyl-2-deoxy-2-hexadecanoylamino-3- O -[(2 R )-propanoyl-( l -alanyl- d -isoglutamine methyl ester)-2-yl]-β- d -glucopyranosyl}-2-hexadecanoylamino- d -glucopyranose ( 27 ).

The conformation in solution in methyl sulfoxide of the immunoadjuvant peptidoglycan monomer (PGM), obtained by digestion with lysozyme of the linear peptidoglycan polymer isolated from Brevibacterium divaricatum , was studied by 1 H-n.m.r. spectroscopy. The temperature dependence of the chemical shift of the resonances of the amide protons suggested that the amino group of alanine-5 is involved in hydrogen bonding, most probably to the α-carbonyl of the isoglutamine which showed restricted rotation, as indicated by the large chemical shift non-equivalence for the resonances of the βCH 2 group. A cyclic structure is proposed for the C -terminal pentapeptide of PGM, which is further supported by various n.O.e. interactions involving the meso -diaminopimelic residue and the N -acetylmuramoyl group.

The medical indications for positive immunomodulation are becoming ever more widespread. In some situations, it is possible to use endogenous immunomodulators (IMs) or synthetics, but most of the experimentally tested IMs as well as those applied in clinical studies are of microbial origin. In the laboratories of the Institute of Pharmacology a new substance has been developed linking L-alanine-D isoglutamine residua of muramyl dipeptide with the antiviral l-amidoadamantan. This substance (adamantylamide dipeptide — AdDP) retains marked antiviral effects (Masihi et al. 1987) and shows positive IM properties as well (Masek et al. 1984). In our experiments in mice we tested the immunomodulatory properties of AdDP using several in vivo tests of cell mediated immunity. Selected were the regional graft-versus-host reaction (GVHR), regional host-versus-graft reaction (HVGR) and delayed type hypersensitivity (DTH) to SRBC (Sůla and Nouza 1984, Sůla et al 1988).

α-Cyclopentyl D-glutamate was obtained from dicyclopentyl D-glutamate utilizing lipase-catalyzed hydrolysis. The monoester was then treated with NH 3 in methanol to produce D-isoglutamine.

N-Acetylmuramyl-L-alanyl-D-isoglutamine corresponding to the minimum structure responsible for the immunoadjuvant activity of bacterial cell walls was acylated at the 6-hydroxyl group with various mycolic acids of long carbon chains in order to test their possible antitumor effects. Mycolic acids used were extracted from cell walls of Mycobacterium, Nocardia, and Corynebacterium. All the mycoloyl derivatives synthesized show antitumor activity based on a principle of immunotherapy.

Carboxypeptidase Y catalyzed reactions between a common acyl component (Bz-Ala-OMe) and a variety of amine compounds are described. The α-amino acid amides tested were—with the exception of isoglutamine— incorporated in high yields (70–95%). For the free α-amino acids, the yields were below 60% and fluctuated widely. For the cases examined, the enzyme only reacted with thel-enatiomers of these compounds. Dipeptides were not accepted as amine components. Glycinonitril, glycine-n-methyl amide and threonine-n-methyl amide reacted in low yields (

Bacterial cell walls and their building blocks have been investigated using thermally programmed pyrolysis at 50–500°C, followed by field ionization mass spectrometry of their pyrolysates. Spectra of selected model compounds, namely N-acetylglucosamine, N-acetylmuramic acid, N-acetylmuramyl- l -alanyl- d -isoglutamine and murein (peptidoglycan), isolated from the cell walls of Bacillus subtilis var. nig . WM were compared with the spectrum of the cell walls of this microorganism. The cell wall building blocks N-acetylmuramic acid and N-acetylmuramyl- l -alanyl- d -isoglutamine show pyrolysis products identical with N-acetylglucosamine, as the O-glycosidic bonds of the amino sugar derivatives are thermally very labile. In general, products at m/z 203, 185 and 167, generated by water elimination, as well as the characteristic peak at m/z 125 are observed. In addition, some peaks that originate from the O-glycosidic side-chain are registered. The murein, however, does not show any large, significant pyrolysis fragments from the peptide side-chains, owing to their strong cross-linkages. The pyrolysis of this biopolymer yields, in principle, only peaks characteristic of aminopolysaccharides: m/z 83, 85, 95, 97, 109, 111, 123, 125, 137, 139, 151, 167, 185 and 203. The peaks in the lower mass region are generally difficult to assign to a particular structure, such as an amino sugar. For a first analytical evaluation of the method, for instance for taxonomy problems, spectra obtained from the well described cell walls of Bacillus subtilis were also checked with the spectrum of cell walls of a yet unknown composition ( Propioni shermanii ). The pyrolysis spectrum of cell walls of Bacillus subtilis is very similar to that of murein and shows that it consists mainly of amino sugars. In contrast, the spectrum of cell walls from Propioni shermanii shows strong signals indicating neutral sugars: m/z 98, 110, 112, 126, 144 and 162.