Adjuvant activity of some nonpyrogenic hydrophobic analogues of muramyl dipeptide in enhancing the primary humoral and cell mediated immune responses in guinea pig model

Five muramyl dipeptide analogues synthesized by derivatization of gamma-carboxyl of D-isoglutamine residue of MDP into alkyl amides or incorporation of lysine residue at the site via epsilon-NH2 function were evaluated for immuno-adjuvant activity. Derivatization of gamma-carboxyl of D-isoglutamine into butyl, octyl and dibutyl residues stimulated delayed type of hypersensitivity (DTH) response, the maximum stimulation being observed with octyl amide. Introduction of lauryl amide residue abolished DTH response. The antibody response was impaired with all the alkyl amide analogues except for the lysyl amide derivative with which the response was higher than MDP. Correlation was observed between DTH response and macrophage migration.     

Biological activity of synthetic subunits of streptococcus peptidoglycan. III. Relationship of subunit and analogue structure to adjuvant activity in cell-mediated immunity

Each of a series of synthetic peptidoglycan subunits and subunit analogues was injected in combination with streptococcus type M24 antigen extract. The substances tested were: (8a) N-acetylmuramyldipeptide (MDP) and the following derivatives thereof: MDP modified in positions C3 and C4, or with L-alanine substituted by L-2-aminobutyric acid or with the peptide chain prolonged (by three lysines or a polylysine); (b) some synthetically prepared peptides: a hexapeptide, a tridecapeptide and an octadecapeptide. Configurations in positions C3 and C4 were found essential for the adjuvant effect. Adjuvant activity, though somewhat lower than in MDP, was pronounced in the analogue containing the L-2-aminobutyryl residue. Surprisingly, potent adjuvant effect was displayed by the hexapeptide; prolongation of the peptidic chain was not effective. The use of a polymeric carrier for MDP increased the adjuvant effect. Contrary to expectation, streptococcal antigens used with immunoadjuvant materials showed that induced delayed hypersensitivity was type related.     

Structural specificity of synthetic peptide adjuvant for induction of experimental allergic encephalomyelitis

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.     

Mammalian folylpoly-gamma-glutamate synthetase. 3. Specificity for folate analogues

A variety of folate analogues were synthesized to explore the specificity of the folate binding site of hog liver folylpolyglutamate synthetase and the requirements for catalysis. Modifications of the internal and terminal glutamate moieties of folate cause large drops in on rates and/or affinity for the protein. The only exceptions are glutamine, homocysteate, and ornithine analogues, indicating a less stringent specificity around the delta-carbon of glutamate. It is proposed that initial folate binding to the enzyme involves low-affinity interactions at a pterin and a glutamate site and that the first glutamate bound is the internal residue adjacent to the benzoyl group. Processive movement of the polyglutamate chain through the glutamate site and a possible conformational change in the protein when the terminal residue is bound would result in tight binding and would position the gamma-carboxyl of the terminal glutamate in the correct position for catalysis. Steric limitations imposed on the internal glutamate residues that loop out and additional steric constraints imposed by binding of different pterin moieties would be expected to effect slight conformational changes in the protein and/or the terminal glutamate and would explain the decrease in on rate and catalytic rate with increased polyglutamate chain length, and the differential effect of one-carbon substitution on the catalytic rate with polyglutamate derivatives. The 4-amino substitution of folate increases the on rate for monoglutamate derivatives but severely impairs catalysis with diglutamate derivatives. Pteroylornithine derivatives are the first potent and specific inhibitors of folylpolyglutamate synthetase to be identified and may act as analogues of reaction intermediates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific degradation of pectins via a carbodiimide-mediated Lossen rearrangement of methyl esterified galacturonic acid residues.

A specific, chemical degradation of the methyl esterified galacturonic acid residues of pectins is described. These residues are converted, with hydroxylamine, to hydroxamic acids, and then, with a carbodiimide, to isoureas; the latter undergo a Lossen rearrangement on alkaline hydrolysis. The isocyanates formed are hydrolysed to 5-aminoarabinopyranose derivatives, which spontaneously ring open to give 1,5-dialdehydes. The latter are reduced, in situ, to avoid peeling reactions, with sodium borohydride to give substituted arabitol residues. Thus, overall, partially esterified pectins are specifically cleaved to generate a series of oligogalacturonic acids bearing an arabitol residue as aglycone. Analysis of oligomers so generated discloses the pattern of contiguous nonesterification in a variety of pectins of differing degrees of esterification. Other potential applications are described.     

Structure of the major glycolipid from Rothia dentocariosa

Structural studies of the major glycolipid isolated from Rothia dentocariosa were carried out by specific chemical degradation and nuclear magnetic resonance spectroscopy. The glycolipid was found to be a dimannosylacylmonoglyceride in which the carbohydrate part was the glycerol-linked dimannoside alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-sn-Gro, and the internal mannose was esterified at C-6 by fatty acid residue. The other fatty acyl chain substituted the primary methylene position of glycerol. The occurrence of this glycolipid is limited to the related microorganisms. The structural characteristics can facilitate the differentiation of some genera.     

Biosynthesis of unnatural bacteriochlorophyll c derivatives esterified with α,ω-diols in the green sulfur photosynthetic bacterium Chlorobaculum tepidum

Unnatural bacteriochlorophyll (BChl) c derivatives possessing a hydroxy group at the terminus of a hydrocarbon chain at the 17-propionate were biosynthesized in the green sulfur photosynthetic bacterium Chlorobaculum tepidum. Addition of exogenous 1,8-octanediol, 1,12-dodecanediol, and 1,16-hexadecanediol in acetone to liquid cultures resulted in accumulation of BChl c monoesterified with the corresponding diols. The relative ratios of the novel BChl c derivatives esterified with 1,8-, 1,12-, and 1,16-diols to totally producing BChl c were 8.2, 50.2, and 57.6% in the cells grown with additive α,ω-diols at concentrations of 1.5, 0.06, and 0.06 mM, respectively, at the final concentration. The homologue composition of BChl c derivatives esterified with these α,ω-diols was similar to that of original, coexisting BChl c esterified with farnesol (BChl c(F)), suggesting that esterification of α,ω-diols occurred at the last step of the BChl c biosynthetic pathway by BChl c synthase, BchK, in the same manner as in BChl c(F). Chlorosomes, which were isolated from cells grown in the presence of exogenous α,ω-diols, contained a ratio and a composition of BChl c derivatives esterified with the diols similar to those in the whole cells, indicating that these BChl c derivatives were actually present in chlorosomes. Q(y) absorption bands of C. tepidum cells containing the novel BChl c derivatives were shifted to a shorter wavelength, although their bandwidths were analogous to those of cells obtained by normal cultivation. Circular dichroism spectra of cells that had BChl c derivatives esterified with α,ω-diols exhibited S-shaped signals in the Q(y) region, whose polarities were the reverse of those of cells grown in the normal medium and by supplementation with neat acetone as a control experiment. These spectral features of C. tepidum possessing BChl c derivatives esterified with α,ω-diols imply that the novel BChl c derivatives possessing a hydroxy group at the terminus of a hydrocarbon chain affect their self-assembly in chlorosomes.     

The adjuvant activity of synthetic N-acetylmuramyl-dipeptide: evidence of initial target cells for the adjuvant activity.

MurNAc-l-Ala-d-isoGln (N-acetylmuramyl-l-alanyl-d-isoglutamine, MDP), a synthetic compound, acts as an adjuvant on the humoral immune response and on the T cell-mediated immune response. In this report, we attempted to directly demonstrate the initial target cells of MDP for its adjuvant activity in vitro by using cell separation procedures.It was demonstrated that MDP enhanced the immune response following direct interaction with antigen-stimulated T and B lymphocytes, but nonstimulated lymphocytes, shortly after triggering by antigen, and that there was no macrophage requirement for MDP to elicite the adjuvant action in the primary anti-SRBC PFC response in vitro. It has also been demonstrated that the adjuvant activity of MDP is due to an enhancing effect which is different from the possible mitogenic activity to spleen cells and MDP replaces neither a function of macrophages, which is substituted by 2-mercaptoethanol nor a helper function of T cells.     

Characterization of a renal medium chain acyl-CoA synthetase responsible for glycine conjugation in mouse kidney mitochondria.

Glycine conjugation of a series of benzoic acid derivatives was investigated in mouse kidney mitochondria. The chlorine and methyl substitutions in the para- and meta-positions of the benzene ring yielded an increase in glycine conjugation. The acids with a methoxy group showed a low degree of glycine conjugation. In addition, the acids with nitro or amino groups were conjugated to a slight extent with glycine. The in vitro conjugation of salicylic acid with glycine occurred not in liver but in kidney. The specificity of the renal medium chain acyl-CoA synthetase catalyzing the first reaction of glycine conjugation was also examined. The enzyme accepted not only medium chain fatty acids but also aromatic and arylacetic acids. The highest activity was shown with hexanoic acid. High activities were observed for benzoic acid derivatives with alkyl and alkoxyl groups in the para- and meta-positions of the benzene ring. An ortho-substituted acid exhibited no activity. In addition, the enzyme was less active with valproic acid, tranexamic acid, indomethacin and ketoprofen. The enzyme was inhibited by diflunisal, 2-hydroxydodecanoic acid and salicylic acid, which did not act as substrates. There was a poor correlation between the activity of the medium chain acyl-CoA synthetase and glycine conjugation of eleven substituted benzoic acids. These findings suggest that the present medium chain acyl-CoA synthetase is involved in glycine conjugation of the substituted acids in mouse kidney mitochondria, but there may be a larger contribution of another isoenzyme.     

Muramyl dipeptide induces acute joint inflammation in the mouse

Acute joint inflammation was produced in BALB/c mice by a single intravenous injection of synthetic muramyl dipeptide (MDP), its stereoisomers and 6-O-acyl derivatives of MDP. Four adjuvant-active, but not five adjuvant-inactive MDP analogs induced acute swelling and erythema of the ankles and wrists which were detected around 6-10 hr, reached the maximum severity by 18-24 hr and subsided by days 3 to 4 after injection. Introduction of the stearoyl group, but not the alpha-branched long chain fatty acid group into the C-9 hydroxyl group of MDP enhanced and prolonged the joint lesions compared with MDP.     

Solid phase synthesis of peptides containing novel amino acids, substituted 3-benzimidazolealanines

A direct solid-phase synthesis of a series of substituted benzimidazole-containing peptides is described. The method involves on-resin formation of new amino acids containing benzimidazole derivatives in the side chain. The heterocycle conjugates were obtained by reaction between aldehydes and peptides containing β-(3,4-diaminophenyl)alanine residue, immobilized on a polymeric solid support.     

Structural and serological characterization of the major glycolipid from Rothia mucilaginosa

Structural studies on the major glycolipid isolated from Rothia mucilaginosa were carried out utilising specific chemical degradation, NMR spectroscopy and matrix-assisted laser-desorption/ionization time of flight mass spectrometry (MALDI TOF-MS). The glycolipid was found to be a dimannosylacylmonoglyceride in which the carbohydrate part was the glycerol-linked dimannoside alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-sn-Gro (Man A-Man B-Gro), of which Man B was esterified at O-6 by a fatty acid residue. A second fatty acid substituted the secondary methylene position of the glycerol residue, in contrast to the glycolipid previously found in R. dentocariosa and Saccharopolyspora strains, in which the second fatty acid esterified the primary methylene position of glycerol. Results of the ELISA experiment with rabbit specific antibacterial sera indicate that these two major glycolipids are antigenic, and the patterns of serological reactivity are similar but not identical.     

Dendritic cell maturation induced by muramyl dipeptide (MDP) derivatives: monoacylated MDP confers TLR2/TLR4 activation

6-O-acyl-muramyldipeptides (MDP) with various lengths of fatty acid chains were examined for their dendritic cell (DC) maturation activity expressed through TLRs. Judging from anti-TLR mAb/inhibitor-blocking analysis, MDP derivatives with a single octanoyl or stearoyl fatty acid chain were found to activate TLR2 and TLR4 on human DCs, although intact and diacylated MDP expressed no ability to activate TLRs. Human DC activation profiles by the monoacylated MDP were essentially similar to those by Calmette-Guerin (BCG)-cell wall skeleton (CWS) and BCG-peptidoglycan (PGN) based on their ability to up-regulate costimulators, HLA-DR, beta(2)-microglobulin, and allostimulatory MLR. Monoacylated MDP induced cytokines with similar profiles to BCG-CWS or -PGN, although their potency for induction of TNF-alpha, IL-12p40, and IL-6 was less than that of BCG-CWS or -PGN. The MDP derivatives initiated similar activation in normal mouse macrophages, but exhibited no effect on TLR2/4-deficient or MyD88-deficient mouse macrophages. Mutation of d-isoGln to l-isoGln in monoacylated MDP did not result in loss of the DC maturation activity, suggesting marginal participation of nucleotide-binding oligomerization domain 2, if any, in monoacyl MDP-dependent DC maturation. These results define the adjuvant activity of 6-O-acyl MDP compounds at the molecular level. They target TLR2/TLR4 and act through the MyD88-dependent pathway in DCs and macrophages. Hence, the unusual combined activation of TLR2 and TLR4 observed with Mycobacterium tuberculosis is in part reflected in the functional properties of monoacylated MDP compounds. These findings infer that the essential minimal requirement for TLR2/4-mediated adjuvancy of BCG lies within a modified MDP.     

NMR Studies of the MDP Conjugates with Amino-Acridine/Acridone Derivatives

Summary ¹H and¹³C NMR studies allowed us to determine the structure of anticancer active MDP analogs modified at the C-terminus with amino-acridine/acridone derivatives. The products contain an isoglutamine residue without contamination by their isomers containing a glutamine residue as could be expected based on the literature data.     

Pulsefluorimetry of tyrosyl peptides

The fluorescence quantum yield and the fluorescence decay of aqueous solutions of derivatives containina a single tyrosine residue have been measured at different pH. In these derivatives tyrosine was substituted on its amino end (series I) or/and, on its carboxyl end (series II), by acyl, amino or amino acyl groups. The fluorescence decays of series I derivatives are monoexponential regardless to the ionization state of their amino group. Upon deprotonation of the α-amino group, the quantum yields and the lifetimes increase in the case of dipeptides, and slightly decrease, for the tripeptides. The quantum yield and the lifetime increase with the side chain length of the aliphatic residue adjacent to the tyrosine residue, (the fluorescence of Val Tyr anion being identical to that of free Tyrosine). Quite different is the behavior of series II derivatives: their decays at pH 5.5 must be described by two exponential terms, one of them decaying with a short time constant (about 0.5 ns) and little side chain effect is observed. The fluorescence intensity increases upon deprolonalion of the α-amino proup (though to a lesser extent than for series I derivatives); a nearly monoexponential decay is observed at basic pH for dipeptides. but not for tyrosine amide, amide or dipeptides, or tripeptides. The following interpretation of our results is proposed: fluorescence quenching occurs in molecular conformations in which a peptide carbonyl can come in contact with the phenolic chromophore. This condition depends mainly on the value of the angle x₁ which determines the conformation of the tyrosyl residue around its C_α-C_β bond. It appears that the rotamer in which quenching occurs are not the same for series I and series II derivatives, which can explain the different behavior of these two kinds of compounds. The interpretation of the fluorescence properties is developed taking into account on one side the relative population of the rotamers in the ground state, which is given by studies of crystals and of solutions, and on the other side the possibility of an exchange between these rotamers during the excited state time. In this scheme the protonated α-amino groups would act to reinforce the quenching efficiency of the carbonyl. At last it is found that the radiative lifetime of the phenolic chromophore is the same for all the compounds studies.     

NMR Studies of the MDP Conjugates with Amino-Acridine/Acridone Derivatives

¹H and ¹³C NMR studies allowed us to determine the structure of anticancer active MDP analogs modified at the C-terminus with amino-acridine/acridone derivatives. The products contain an isoglutamine residue without contamination by their isomers containing a glutamine residue as could be expected based on the literature data.     

Polyphenol fatty acid esters as serine protease inhibitors: a quantum-chemical QSAR analysis

We investigated the ability of polyphenol fatty acid esters to inhibit the activity of serine proteases trypsin, thrombin, elastase and urokinase. Potent protease inhibition in micromolar range was displayed by rutin and rutin derivatives esterified with medium and long chain, mono- and polyunsaturated fatty acids (1e–m), followed by phloridzin and esculin esters with medium and long fatty acid chain length (2a–d, 3a–d), while unmodified compounds showed only little or no effect. QSAR study of the compounds tested provided the most significant parameters for individual inhibition activities, i.e. number of hydrogen bond donors for urokinase, molecular volume for thrombin, and solvation energy for elastase. According to the statistical analysis, the action of elastase inhibitors is opposed to those of urokinase and thrombin. Cluster analysis showed two groups of compounds: original polyphenols together with rutin esters with short fatty acid chain length and rutin esters with long fatty acid chain length.     

Critical molecular regions for elicitation of the sweetness of the sweet-tasting protein, thaumatin I

Thaumatin is an intensely sweet-tasting protein. To identify the critical amino acid residue(s) responsible for elicitation of the sweetness of thaumatin, we prepared mutant thaumatin proteins, using Pichia pastoris, in which alanine residues were substituted for lysine or arginine residues, and the sweetness of each mutant protein was evaluated by sensory analysis in humans. Four lysine residues (K49, K67, K106 and K163) and three arginine residues (R76, R79 and R82) played significant roles in thaumatin sweetness. Of these residues, K67 and R82 were particularly important for eliciting the sweetness. We also prepared two further mutant thaumatin I proteins: one in which an arginine residue was substituted for a lysine residue, R82K, and one in which a lysine residue was substituted for an arginine residue, K67R. The threshold value for sweetness was higher for R82K than for thaumatin I, indicating that not only the positive charge but also the structure of the side chain of the arginine residue at position 82 influences the sweetness of thaumatin, whereas only the positive charge of the K67 side chain affects sweetness.     

Alcohol Induced Reversal of Enantioselectivity in a Lipase Catalyzed Resolution of 2-Chloropropionic Acid

Alcohol induced reversal of enantioselectivity in the esterification of 2-chloropropionic acid using lipase from Candida cylindracea has been investigated. It was found that an alcohol having substituents both at the α- and the β-carbon preferentially esterified the S-acid, while a straight chain alcohol preferentially esterified the R-acid. Intermediate enantioselectivities were obtained with alcohols having substituents either at the α- or the β-carbon, but still in favor of the R-acid. An acyl binding domain composed of three subsites is proposed for this lipase; one for the hydrocarbon chain, a second for a methyl substituent and a third for an electronegative substituent.