Structural requirement for the agonist activity of the TLR2 ligand Pam2Cys

Synthetic lipopeptides have demonstrated great potential as a vaccine strategy for eliciting cellular and humoral immunity. One of the most potent lipid moieties used is S-[2,3-bis(palmitoyloxy)propyl]cysteine (Pam2Cys). Pam2Cys binds to and activates dendritic cells by engagement of Toll like receptor 2 (TLR 2). In this study, we have investigated the structural requirement of the agonist activity of Pam2Cys by varying the three structural elements of the core structure S-(2,3-dihydroxypropyl)-cysteine namely (1) the α-amino group of the cysteine residue (2) the sulphur atom of the cysteine residue and (3) the 2,3-dihydroxypropyl moiety. Four novel analogues of Pam2Cys were made and each of these analogues were incorporated into vaccine constructs and examined for immunogenicity. Our results demonstrate that (1) the potency of the peptide vaccine is least affected by removal of the amino group (2) substitution of the sulphur atom with an amide bond leads to significant reduction of biological activity (3) removal of the amino group and at the same time substitution of the sulphur with an amide bond significantly decreases the biological activity (4) in the two analogues in which the sulphur atom is replaced with an amide bond the analogue containing the 1,3-dihydroxypropyl moiety demonstrates higher activity than the one which contains 2,3-dihydroxypropyl. In conclusion, the results demonstrate strict structural requirements for agonist activity of the TLR2 ligand Pam2Cys.     

The N-hydroxysuccinimide ester of Boc-[S-(3-nitro-2-pyridinesulfenyl)]-cysteine: a heterobifunctional cross-linking agent

Synthetic cysteine-containing peptides were unidirectionally conjugated to albumin via disulfide bonds using the S-(3-nitro-2-pyridinesulfenyl) derivative of cysteine. This method employs the N-hydroxysuccinimide ester of Boc-[S-(3-nitro-2-pyridinesulfenyl)]-cysteine, a protected amino acid derivative used in peptide synthesis, as a heterobifunctional cross-linking agent. The disulfide bonds in the conjugates are formed by the reaction of free thiols with S-(3-nitro-2-pyridinesulfenyl) groups. Bovine albumin was conjugated in this manner to several synthetic peptides derived from human fibrin. Amino acid analysis of these conjugates demonstrated incorporations of from 6 to 11 peptide molecules per molecule of protein.     

Genotoxicity of carcinogenic N-nitrosopropylamine derivatives in the hepatocyte primary culture/DNA-repair test

The genotoxicity of N-nitrosodipropylamine, 8 of its oxidized derivatives and N-nitroso-2,6-dimethylmorpholine was examined in the hepatocyte primary culture (HPC)/DNA repair test. Nine N-nitrosamines which are known to be carcinogenic and mutagenic were clearly positive in the HPC/DNA-repair test. N-Nitroso(2,3-dihydroxypropyl) (2-hydroxypropyl)amine did not elicit DNA repair, but showed a borderline mutagenic response in the Salmonella/microsome test. Thus, the HPC/DNA-repair test displays a comparable capacity to the bacterial mutagenesis test for detecting the genotoxic effects of this class of carcinogens.     

Synthesis and characteristics of modified oligodeoxyribonucleotides containing 2'-O-(2,3-dihydroxypropyl)uridine and 2'-O-(2-exoethyl)uridine

A new uridine derivative, 2'-O-(2,3-dihydroxypropyl)uridine, and its 3'-phosphoramidite were obtained for direct introduction into oligonucleotides during automated chemical synthesis. Oligonucleotides 10 to 15 nt long harboring 2'-O-(2,3-dihydroxypropyl)uridine residues were synthesized; periodate oxidation of these oligomers gave oligonucleotides containing 2'-O-(2-oxoethyl)uridine residues. The presence of a reactive aldehyde group in 2' position of the carbohydrate moiety was confirmed by the interaction with p-nitrophenylhydrazine and methionine methyl ester. The thermostability of DNA duplexes containing modified units is practically indistinguishable from that of the natural analogues.     

Synthesis of the mitogenic S-[2,3-bis(palmitoyloxy)propyl]-N-palmitoylpentapeptide from Escherichia coli lipoprotein

The N-terminal pentapeptide of the lipoprotein from the outer membrane of Escherichia coli was obtained by coupling S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteine to O-tert-butylseryl-O-tert-butyl-seryl-asparaginyl-alanine tert-butyl ester followed by deprotection with trifluoroacetic acid. The tetrapeptide was built up from alanine tert-butyl ester with N-9-fluorenylmethyloxycarbonyl protected amino acids. S-[2,3-Bis(palmitoyloxy)propyl]-N-palmitoylcysteine was obtained from N,N'-dipalmitoylcystine di-tert-butyl ester via reduction to the thiol, and S-alkylation with racemic 3-bromo-1,2-propanediol followed by esterification with palmitic acid in the presence of dicyclohexylcarbodiimide/dimethylaminopyridine and deprotection with trifluoroacetic acid. The compounds were characterized unequivocally by 13C-NMR and mass spectra. The diastereomers of S-[2,3-bis(palmitoyloxy)propyl]-N-palmitoylcysteine tert-butyl ester with opposite configuration at the propyl-C-2 atom could be separated on a silica-gel column.     

Aliphatic analogues of nucleotides: synthesis and affinity towards nucleases

DL-1-(2,3-Dihydroxypropyl)thymine was prepared by Hilbert-Johnson reaction of 2,4-dinethoxy-5-methylpyrimidine with allyl bromide followed by the osmium tetroxide catalyzed hydroxylation of the l-allyl-4-methoxy-5-methylpyrimidin-2-one obtained as an intermediate. The D-glycero enantiomer, R-1-(2,3-dihydroxypropyl)thymine and the corresponding 1-substituted uracil derivative were prepared from 3-O-p-toluenesulfonyl-1, 2-O-isopropylidene-D-glycerine and sodium salt of 4-methoxy-5-methylpyrimidin-2-one or 4-methoxypyrimidin-2-one followed by treatment with hydrogen chloride in ethanol. The phosphorylation of the above 2,3-dihydroxypropyl derivatives with phosphoryl chloride in triethyl phosphate afforded the corresponding 3-phosphates which were transformed into the 2′,3′-cyclic phosphates by the condensation with N,N′-dicyclohexylcarbodiimide. The latter compounds of the D-glycero configuration are split by some microbial RNases to the 3-phosphates.     

Immunostimulants and Toll-like receptor ligands obtained by screening combinatorial lipopeptide collections.

Synthetic lipopeptides carrying the head group of bacterial lipoproteins are specific ligands of Toll-like receptors (TLR). The three fatty acids containing lipopeptides with the tripalmitoyl-S-glyceryl-cysteinyl N-terminus (Pam(3)Cys) are agonists of TLR2. The structurally related lipopeptides with a head group lacking the fatty acyl residue at the amino-terminus (Pam(2)Cys) stimulate TLR2 and 6. To investigate the influence of the peptide chain of lipohexapeptides with a free N-terminus with regard to their ability to enhance B-cell proliferation, a randomized S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-pentapeptide amide collection Pam(2)CysXXXXX and 5 x 19 subcollections (Pam(2)CysOXXXX, Pam(2)CysXOXXX, Pam(2)CysXXOXX, Pam(2)CysXXXOX, Pam(2)CysXXXXO, O: all protein amino acids except Cys) were prepared by parallel solid-phase synthesis. The collection represents synthetic lipopeptide analogues of the numerous bacterial lipoproteins and of mycoplasma lipoprotein. Each of the 95 subcollections is characterized by one defined and four degenerated amino acid positions thus comprising 19(4) individual lipopeptides with free N-terminal amino groups. High-performance liquid chromatography electrospray mass spectrometry (HPLC-ESI-MS) was applied for the analytical characterization of the lipohexapeptide amide subcollections and for the individual lipohexapeptide amides. The subcollections were tested for polyclonal activation of murine spleen cells, deconvolution led to highly active single S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-pentapeptide amides.     

Synthesis of novel immunologically active tripalmitoyl-S-glycerylcysteinyl lipopeptides as useful intermediates for immunogen preparations

The synthesis and characterization of lipopeptides consisting of the lipoamino acid N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteine (Pam3Cys-OH) and different peptide segments and/or spacer molecules is described. Pam3Cys-peptides, which are derived from the immunologically active N-terminus of bacterial lipoprotein, were obtained either by solution or solid phase peptide synthesis. In particular, the amphiphilic and water-soluble lipohexapeptides Pam3Cys-Ser-(Lys)4 and Pam3Cys-Ser-(Glu)4 proved to be potent macrophage and B-cell activators and non-toxic, non-pyrogenic immune adjuvants in combination with or covalently linked to antigens and haptens.     

"Single Addition" substrates for the synthesis of specific oligoribonucleotides with polynucleotide phosphorylase. Synthesis of 2'-(alpha-methoxyethy) nucleoside 5'-diphosphates.

A number of synthetic methods for the preparation of the 2-O-(alpha-methoxyethyl) derivatives of the 5-diphosphates of adenosine, cytidine, guanosine, and uridine have been studied in order to provide nucleotide substrates that can be applied to the synthesis of specific oligoribonucleotides using polynucleotide phosphorylase. The reaction of nucleoside 5-diphosphates with methyl vinyl ether for a limited time produces low yields of the corresponding 2-O-(alpha-methoxyethyl) derivatives because the rate of methoxyethylation of the 3-hydroxyl groups. A study of the rates of acidic hydrolysis of alpha-methoxyethyl groups in the 2 and 3 positions of nucleosides and nucleotides has been made, and the results obtained form the basis of a more efficient method for the synthesis of the blocked nucleoside diphosphates. The method involves the reaction of nucleoside 5-diphosphates with methyl vinyl ether to give the corresponding 2,3-di-O-(alpha-methoxyethyl)nucleoside 5-diphosphates, and exploits the fact that, in the acidic hydrolysis of these derivatives, the rate of removal of the 3-methoxyethyl group is about twice that of the group in the 2 position. Alternative syntheses were based on the phosphorylation of methoxyethylated nucleosides and nucleotides. The derivatives, 2-O- and 2,3-di-O-(alpha-methoxyethyl)uridine, were prepared by the methoxyethylation of 3,5-di-O-acetyluridine and 5-O-acetyluridine followed by removal of the acetyl groups. The corresponding guanosine derivatives were made by the synthetic routes: (i) guanosine leads to O-2,O-3,O-5,N-2-tetrabenzoylguanosine leads to 2-N-benzoylguanosine leads to O3-acetyl-N-2,O5-dibenzoylguanosine leads to 2-O-(alpha-methoxyethyl)guanosine, and (ii) 2,3-O-isopropylideneguanosine leads to N-2,O5-diacetyl-2,3-O-isopropylideneguanosine leads to N-2,O-5-diacetylguanosine leads to 2,3-di-O-(alpha-methoxyethyl)guanosine. These methoxyethylated nucleosides were converted to the corresponding 5-phosphates by reaction with cyanoethyl phosphate and dicyclohexylcarbodiimide, and then to the corresponding 5-diphosphates by subsequent reaction with 1,1-carbonyldiimidazole and inorganic phosphate.     

Synthesis and Properties of Modified Oligodeoxyribonucleotides Containing 2"-O-(2,3-Dihydroxypropyl)uridine and 2"-O-(2-Oxoethyl)uridine

A new uridine derivative, 2"-O-(2,3-dihydroxypropyl)uridine, and its 3"-phosphoramidite were obtained for direct introduction into oligonucleotides during automated chemical synthesis. Oligonucleotides 10 to 15 nt long harboring 2"-O-(2,3-dihydroxypropyl)uridine residues were synthesized; periodate oxidation of these oligomers gave oligonucleotides containing 2"-O-(2-oxoethyl)uridine residues. The presence of a reactive aldehyde group in 2" position of the carbohydrate moiety was confirmed by the interaction withp-nitrophenylhydrazine and methionine methyl ester. The thermostability of DNA duplexes containing modified units does not practically differ from that of the natural analogues.     

Activation of superoxide formation and lysozyme release in human neutrophils by the synthetic lipopeptide Pam3Cys-Ser-(Lys)4. Involvement of guanine-nucleotide-binding proteins and synergism with chemotactic peptides.

Upon exposure to the bacterial chemotactic peptide fMet-Leu-Phe, human neutrophils release lysozyme and generate superoxide anions (O2.-). The synthetic lipoamino acid N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam3Cys), which is derived from the N-terminus of bacterial lipoprotein, when attached to Ser-(Lys)4 [giving Pam3Cys-Ser-(Lys)4], activated O2.- formation and lysozyme release in human neutrophils with an effectiveness amounting to about 15% of that of fMet-Leu-Phe. Palmitic acid, muramyl dipeptide, lipopolysaccharide and the lipopeptides Pam3Cys-Ala-Gly, Pam3Cys-Ser-Gly, Pam3Cys-Ser, Pam3Cys-OMe and Pam3Cys-OH did not activate O2.- formation. Pertussis toxin, which ADP-ribosylates guanine-nucleotide-binding proteins (G-proteins) and functionally uncouples formyl peptide receptors from G-proteins, prevented activation of O2.- formation by fMet-Leu-Phe and inhibited Pam3Cys-Ser-(Lys)4-induced O2.- formation by 85%. Lipopeptide-induced exocytosis was pertussis-toxin-insensitive. O2.- formation induced by Pam3Cys-Ser-(Lys)4 and fMet-Leu-Phe was enhanced by cytochalasin B, by a phorbol ester and by a diacylglycerol kinase inhibitor. Addition of activators of adenylate cyclase and removal of extracellular Ca2+ inhibited O2.- formation by fMet-Leu-Phe and Pam3Cys-Ser-(Lys)4 to different extents. Pam3Cys-Ser-(Lys)4 synergistically enhanced fMet-Leu-Phe-induced O2.- formation and primed neutrophils to respond to the chemotactic peptide at non-stimulatory concentrations. Our data suggest the following. (1) Pam3Cys-Ser-(Lys)4 activates neutrophils through G-proteins, involving pertussis-toxin-sensitive and -insensitive processes. (2) The signal transduction pathways activated by fMet-Leu-Phe and Pam3Cys-Ser-(Lys)4 are similar but not identical. (3) In inflammatory processes, bacterial lipoproteins and chemotactic peptides may interact synergistically to activate O2.- formation, leading to enhanced bactericidal activity.     

Preparation and characterization of N epsilon-(2,3-dihydroxypropyl)-L-lysine and its phenylthiohydantoin derivative

Convenient methods for preparative synthesis of N epsilon-(2,3-dihydroxypropyl)-L-lysine and its phenylthiohydantoin derivative are described. The former compound was characterized by elemental analysis, melting point, and ion-exchange chromatography and the latter by elemental analysis, melting point, UV-spectrum, HPLC and thin-layer chromatography. This study was performed for investigations of lysine residues in proteins.     

A simple method for monitoring the cysteine content in synthetic peptides.

A new method for the determination of the cysteine content of synthetic peptides is described. For this purpose the classical amino acid analysis is not suitable, as cysteine may undergo oxidative dimerisation to cystine during the hydrolysis or evaporation. The intact peptide was reacted with N-ethylmaleimide (NEM) yielding a stable S-(N-ethylsuccinimido)-cysteine derivative, which could be separated by RP-HPLC and characterised by mass spectrometry. For the quantitative determination less than 0.1 microM peptide was sufficient.     

攀援孔药花化学成分研究

从攀援孔药花全草95%乙醇提取物中首次分离得到19个化合物,通过波谱数据或与已知物对照,它们分别鉴定为:(2S,3S,4R)-2-[(2R)-2-羟基-二十一酰胺基]-二十一烷-1,3,4-三醇(1)、(2S,3S,4R)-2-二十四酰胺基-十八烷-1,3,4-三醇(2)、胡萝卜甙(3)、β-谷甾醇(4)、(20S,22E,24R)-5α,8α-表二氧-麦角甾-6,22-二烯-3β-醇(5)、6β-羟基-豆甾-4-烯-3-酮(6)、十六烷酸-1-甘油酯(7)、桦木酸(8)、大黄素(9)、二十二烷酸-1-甘油酯(10)、对羟基苯甲醛(11)、十七烷酸-1-甘油酯(12)、金色酰胺醇乙酸酯(13)、十九烷酸-1-甘油酯(14)、棕榈酸(15)(、E)-p-香豆酸(16)、(22E,24S)-24-甲基-5α-胆甾-7,22-二烯-3β,5α,6β-三醇(17)、2-去氧-β-蜕皮激素(18)和auranamide(19)。     

UV-Photolysis of S-(cis-l-Propenyl)-L-cysteine in Oxygen-free Aqueous Solution

From the comparisons of mass spectral fragmentations and gas chromatographic retention times with reference compounds, volatile flavor products from UV-photolysis of S-(cis-l-propenyl)-L-cysteine in oxygen-free aqueous solutions were identical with propanal, 2-methyl-2-pentenal, n-propyl mercaptan, allyl mercaptan, 1-propenyl mercaptan, 2-methylthiophene, 3-methylthiophene, 2,3-dimethylthiophene, 2,4-dimethylthiophene, 2,5-dimethylthiophene, 3,4-dimethylthiophene, n-propyl 1-propenyl sulfide and di- 1-propenyl sulfide. Moreover, from the comparison of two-dimentional thin-layer chromatograms with reference compounds, ninhydrin-positive products with interest in terms of the degradation mechanism were identical with alanine and cystine. It seems that above-mentioned thiophene derivatives are produced via 1-propenyl thiyl radicals.     

Pseudoarginine: synthesis and properties of derivatives of delta-(1-imidazolyl)norvaline.

An analogue of arginine has been synthesized in which an imidazole ring occupies the position of the guanidino group of the natural amino acid. It was expected that peptides containing this amino acid when protonated might bind at enzymic sites specific for arginine, but that the pK of the imidazole ring, near 7, would facilitate entry of such peptides into cells, in contrast to peptides containing arginine. Other analogues of arginine can be visualized with a low side-chain pK, including isomers of the imidazole derivative which is the subject of this paper. These are viewed as 'pseudoarginines'. Our initial observations concern the properties of delta-(1-imidazolyl)norvaline in which a ring nitrogen atom is attached to norvaline, which thus becomes comparable to the guanidino delta-nitrogen. Its synthesis is described along with several derivatives examined as substrates or inhibitors. Potential ligands containing delta-(1-imidazolyl)norvaline (ImNva) did not give evidence of interaction with trypsin or plasma kallikrein, serine proteinases which bind arginine derivatives. However, clostripain, a bacterial cysteine proteinase specific for arginine, was readily inactivated by Cbz-Phe-ImNva-CH2F and the rate of inactivation showed an acid pH-dependence not observed, for example, in the inactivation of clostripain by Bz-Phe-LysCH2F.     

Synthetic lipopeptide analogs of bacterial lipoprotein are potent polyclonal activators for murine B lymphocytes

The lipoprotein from the outer membrane of Escherichia coli and other Enterobacteriaceae is a potent polyclonal activator for B lymphocytes. To determine the molecular structure responsible for the biologic activity of lipoprotein, a well-defined series of analogs of its N-terminal part was synthesized: S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-cysteine, -cysteine methyl ester, -cysteinyl-serine, -cysteinyl-seryl-serine, -cysteinyl-seryl-seryl-asparagine, and -cysteinyl-seryl-seryl-asparaginyl-alanine. All compounds were tested for mitogenic activity toward spleen cells from BALB/c, LPS-non-responder C3H/HeJ, and congenitally athymic C3H/Tif/Bom/nu/nu mice, measuring the incorporation of [3H]thymidine into DNA. Lymphocyte activation was confirmed by determination of the incorporation of [3H]uridine into RNA and [3H]leucine into protein. The synthetic lipopeptides were also investigated for their ability to stimulate B lymphocytes into immunoglobulin secretion, as shown by a hemolytic plaque assay. Throughout our studies, the compounds carrying two to five amino acids exhibited strong stimulation activity toward B lymphocytes comparable to native lipoprotein. In contrast, products containing only one amino acid, cysteine or cysteine methyl ester, were only marginally active, indicating that to obtain full biologic activity the presence of the hydrophilic dipeptide structure is necessary. All compounds exhibited only a marginal effect on thymocytes. Thus, a series of defined synthetic fragments of a bacterial outer membrane component exhibits a pronounced mitogenic and polyclonally stimulating activity towards B lymphocytes. The substances will be valuable tools for more detailed investigations on the molecular mechanisms of B cell activation.     

A reinvestigation of the synthesis of arsonolipids (2,3-diacyloxypropylarsonic acids)

A reinvestigation of the reactions leading to arsonolipids (2,3-diacyloxypropylarsonic acids) has been carried out in order to understand why the yields of their preparation were only moderate, although they are better than those reported for 2,3-diacyloxypropylphosphonic acid (phosphotidic acid). Thus, the reaction of glycidol and of 3-chloro-1,2-propanediol with alkaline sodium arsenite, "Na3AsO3", gives the desired product, 2,3-dihydroxypropylarsonic acid, and approximately 10% of an arsenic-containing glycerol dimer which is removed during the preparation of these arsonolipids. The step which is mainly responsible for the diminished yields is due to the reaction of the -As(SPh)2 or -AsO3H- precursor with the activated acid chlorides or carboxylic acid anhydrides to give an intermediate which cyclizes with the primary hydroxy group of the 2,3-dihydroxypropyl moiety. This cyclization does not allow the primary hydroxy group to be acylated. Such cyclization could not be avoided with RCOCl/py, (RCO)2O/DMAP, or RCOOH/DCC/DMAP acylating systems.     

The influence of various adjuvants on antibody synthesis following immunization with an hapten.

For the production of specific antibodies to the hapten MATP (4-Amino-1,2,2-trimethyl-phenylphosphonate) in Balb/c mice various non-toxic adjuvants were compared to Freund's complete adjuvant (FCA). For immunization the hapten MATP was coupled to the carrier human serum albumin (HSA). The immunostimulating effect of the synthetic lipopeptides Pam3Cys-OH, Pam3Cys-Ser-Ser-Asn-Ala and different concentrations of the lipohexapeptide Pam3Cys-Ser-(Lys)4 (Pam3Cys = S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N- palmitoyl-(R)-cysteine as well as of aluminium hydroxide were tested. IgG antibody titers in serum were determined in ELISA. In dose-response studies 50 micrograms Pam3Cys-Ser-(Lys)4 per mouse was the most effective dose with a long period of high antibody levels after the second booster. Pam3Cys-Ser-Ser-Asn-Ala provoked only low antibody titers. Immunostimulation with Pam3Cys--OH did not result in an increased production of specific antibodies. Compared to the control group an enhanced antibody synthesis could be provoked with aluminium hydroxide. However, the increase was much smaller than by using FCA. The lipopeptide Pam3Cys-Ser-(Lys)4 turned out to be a very potent adjuvant. One week after booster injection into mice 50 micrograms of this substance helped to elicit a higher antibody titer than FCA. Hence, as far as the degree of antibody production is concerned, Pam3Cys-Ser--(Lys)4 represents an alternative adjuvant to FCA.     

Chemical synthesis of cell-permeable apoptotic peptides from in vivo produced proteins

In vivo synthesis of peptides by bacterial expression has developed into a reliable alternative to solid-phase peptide synthesis. A significant drawback of in vivo methods is the difficulty with which gene products can be modified post-translationally. Here, we present a method for the facile modification of peptides generated in bacterial hosts after cyanogen bromide cleavage at C-terminal methionines. Reaction of the resulting homoserine lactones with propargylamine allows efficient and selective modification with a wide variety of chemicals such as fluorescent dyes, biotin derivatives, polyprenyls, lipids, polysaccharides, or peptides. Attachment of the cell penetrating peptide octa-arginine (R(8)) to peptides derived from the proapoptotic tumor suppressor Bak BH3 led to efficient cellular uptake and subsequent cytochrome c release from mitochondria, culminating in induction of apoptosis similar to that observed with peptides linked to R(8) via the peptide backbone. These results highlight the significant potential for use of such tools in live cells.