Chirospecific syntheses of 2H- and 13C-labeled 1-O-alkyl-2-O-alkyl'-sn-glycero-3-phosphoethanolamines and 1-O-alkyl-2-O-alkyl'-sn-glycero-3-phosphocholines

A convenient sequence for the synthesis of 1-O-alkyl-2-O-alkyl'-sn-glycero-3-phospholipids was demonstrated starting from 2,3-O-isopropylidene-sn-glycerol, which was first alkylated with 1-bromohexadecane, then converted to the corresponding benzylidene analog. Other less convenient methods to prepare 2,3-O-benzylidene-1-O-hexadecyl-sn-glycerol were also investigated. The key step in the synthesis was the reduction of 2,3-O-benzylidene-1-O-hexadecyl-sn-glycerol with lithium aluminum hydride-aluminum chloride to give 3-O-benzyl-1-O-hexadecyl-sn-glycerol as the major product in 79% yield. The syntheses of 1-O-hexadecyl-2-O-hexadecyl-(1',1'-d2,-sn-glycero-3-phosphoethanolamine and 1-O-hexadecyl-2-O-hexadecyl-(1'-13C)-sn-glycero-3-phosphoethanolamine as well as the correspondingly labeled sn-glycero-3-phosphocholine analogs were then performed. The optical purities of the synthetic intermediates and the ether lipids were established by a novel 1H-NMR method.     

Improved synthesis of 5-hydroxylysine (Hyl) derivatives.

The synthesis of 5-hydroxylysine (Hyl) derivatives for incorporation by solid-phase methodologies presents numerous challenges. Hyl readily undergoes intramolecular lactone formation, and protected intermediates often have poor solubilities. The goals of this work were twofold: first, develop a convenient method for the synthesis of O-protected Fmoc-Hyl; secondly, evaluate the efficiency of methods for the synthesis of O-glycosylated Fmoc-Hyl. The 5-O-tert-butyldimethylsilyl (TBDMS) fluoren-9-ylmethoxycarbonyl-Hyl (Fmoc-Hyl) derivative was conveniently prepared by the addition of tert-butyldimethylsilyl trifluoromethanesulfonate to copper-complexed Hyl[epsilon-tert-butyloxycarbonyl (Boc)]. The complex was decomposed with Na+ Chelex resin and the Fmoc group added to the alpha-amino group. Fmoc-Hyl(epsilon-Boc, O-TBDMS) was obtained in 67% overall yield and successfully used for the solid-phase syntheses of 3 Hyl-containing peptides. The preparation of Fmoc-Hyl[epsilon-Boc, O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)] was compared for the thioglycoside, trichloroacetimidate and Koenigs-Knorr methods. The most efficient approach was found to be Koenigs-Knorr under inverse conditions, where Fmoc-Hyl(epsilon-Boc)-OBzl and peracetylated galactosyl bromide were added to silver trifluoromethanesulfonate in 1,2-dichloroethane, resulting in a 45% isolated yield. Side-reactions that occurred during previously described preparations of glycosylated Hyl derivatives, such as lactone formation, loss of side-chain protecting groups, orthoester formation, or production of anomeric mixtures, were avoided here. Research on the enzymology of Lys hydroxylation and subsequent glycosylation, as well as the role of glycosylated Hyl in receptor recognition, will be greatly aided by the convenient and efficient synthetic methods developed here.     

Synthesis and biological activity of 4'-C-hydroxymethyl-2'-fluro-D-arabinofuranosylpurine nucleosides

A series of 4'-C-hydroxymethyl-2'-fluoro-D-arabinofuranosylpurine nucleosides was prepared and evaluated for cytotoxicity. The details of a convenient synthesis of the carbohydrate precursor 4-C-hydroxymethyl-3,5-di-O-benzoyl-2-fluoro-alpha-D-arabinofuranosyl bromide (13) are presented. Proof of the structure and configuration at all chiral centers of the sugars and the nucleosides were obtained by proton NMR. All five target nucleosides were evaluated for cytotoxicity in human tumor cell lines. The 4'-C-hydroxymethyl clofarabine analogue (16beta) showed slight cytotoxicity in CCRF-CEM leukemia cells.     

Solid-phase route to Fmoc-protected cationic amino acid building blocks

Diamino acids are commonly found in bioactive compounds, yet only few are commercially available as building blocks for solid-phase peptide synthesis. In the present work a convenient, inexpensive route to multiple-charged amino acid building blocks with varying degree of hydrophobicity was developed. A versatile solid-phase protocol leading to selectively protected amino alcohol intermediates was followed by oxidation to yield the desired di- or polycationic amino acid building blocks in gram-scale amounts. The synthetic sequence comprises loading of (S)-1-(p-nosyl)aziridine-2-methanol onto a freshly prepared trityl bromide resin, followed by ring opening with an appropriate primary amine, on-resin N(β)-Boc protection of the resulting secondary amine, exchange of the N(α)-protecting group, cleavage from the resin, and finally oxidation in solution to yield the target γ-aza substituted building blocks having an Fmoc/Boc protection scheme. This strategy facilitates incorporation of multiple positive charges into the building blocks provided that the corresponding partially protected di- or polyamines are available. An array of compounds covering a wide variety of γ-aza substituted analogs of simple neutral amino acids as well as analogs displaying high bulkiness or polycationic side chains was prepared. Two building blocks were incorporated into peptide sequences using microwave-assisted solid-phase peptide synthesis confirming their general utility.     

Acceptor activity of 4-N-acetylcytidine in the synthesis of (3'-5')-internucleotide bond catalyzed by pancreatic nuclease]

Cytidine and 4-N-acetylcytidine were compared as phosphate acceptors in dinucleoside monophosphate synthesis catalyzed by pancreatic ribonuclease with uridine-2',3'-cyclophosphate and cytidine-2',3'-cyclo phosphate as phosphate donors. Because of low solubility of 4-N-acetylcytidine in water, the synthesis was carried out in aqueus-organic media. The results obtained indicate that acetylation of the exoaminogroup of cytidine decreases its acceptor activity. For the first time uridilyl-(3'-5')-4-N-acetylcytidine and cytidilyl-(3'-5')-4-N-acetylcytidine are prepared enzymatically by pancreatic ribonuclease.     

Lipid derivatives of (2'-5')oligo A

The synthesis of adenylyl-(2'-5')-adenylyl-(2'-5')-2', 3'-O-(1-methoxyhexadecylidene)-adenosine (III) and its 5'-phosphorylated analogue (V) is described. Phosphorylation was achieved by (2-cyanoethyl)-phosphodichloridite agent followed by iodine oxidation.     

The first total synthesis of the marine fatty acid (+/-)-9-methoxypentadecanoic acid: a synthetic route towards mid-chain methoxylated fatty acids

The marine fatty acid (+/-)-9-methoxypentadecanoic acid was synthesized for the first time in seven steps (7.8% overall yield) starting from commercially available 9-decen-1-ol. The key step in the synthesis was the coupling of pentylmagnesium bromide with 1-benzyloxy-9,10-epoxydecane under 1,5-cyclooctadiene copper (I) chloride catalysis. Nuclear magnetic resonance data are provided for the first time for this type of methoxylated fatty acids and the synthetic approach utilized is of general applicability since it can be used in the synthesis of other mid-chain methoxylated fatty acids. This synthetic methodology should afford sufficient quantities of these fatty acids for biological evaluation. The spectral data obtained for the title compound will also be helpful in subsequent characterizations of other mid-chain methoxylated fatty acids using nuclear magnetic resonance spectroscopy.     

A chromogenic substrate for phosphatidylinositol-specific phospholipase C: 4-nitrophenyl myo-inositol-1-phosphate.

A chromogenic water-soluble substrate for phosphatidylinositol-specific phospholipase C was synthesized starting from myo-inositol employing isopropylidene and 4-methoxytetrahydropyranyl protecting groups. In this analogue of phosphatidylinositol, 4-nitrophenol replaces the diacylglycerol moiety, resulting in synthetic, racemic 4-nitrophenyl myo-inositol-1-phosphate. Using this synthetic substrate a rapid, convenient and sensitive spectrophotometric assay for the phosphatidylinositol-specific phospholipase C from Bacillus cereus was developed. Initial rates of the cleavage of the nitrophenol substrate were linear with time and the amount of enzyme used. At pH 7.0, specific activities for the B. cereus enzyme were 77 and 150 mumol substrate cleaved min-1 (mg protein)-1 at substrate concentrations of 1 and 2 mM, respectively. Under these conditions, less than 50 ng quantities of enzyme were easily detected. The chromogenic substrate was stable during long term storage (6 months) as a solid at -20 degrees C.     

Effects of 8-chlorodeoxyadenosine on DNA synthesis by the Klenow fragment of DNA polymerase I

8-chloro-2'-deoxyadenosine (8-Cl-dAdo) was incorporated into synthetic DNA oligonucleotides to determine its effects on DNA synthesis by the 3'-5' exonuclease-free Klenow fragment of Escherichia coli DNA Polymerase I (KF-). Single nucleotide insertion experiments were used to determine the coding potential of 8-Cl-dAdo in a DNA template. KF- inserted TTP opposite 8-Cl-dAdo in the template, but with decreased efficiency relative to natural deoxyadenosine. Running-start primer extensions with KF- resulted in polymerase pausing at 8-Cl-dAdo template sites during DNA synthesis. The 2'-deoxyribonucleoside triphosphate analogue, 8-Cl-dATP, was incorporated opposite thymidine (T) approximately two-fold less efficiently than dATP.     

Novel heterobifunctionalized polystyrene-polyethylene glycol resin for simultaneous preparation of free and immobilized peptides and biological activity detected by confocal microscopy

Summary Fast and convenient binding assays using synthetic peptides are of utmost and increasing importance, especially in the search for lead structures or in the field of diagnostics. A polymeric support suitable for solid-phase peptide synthesis was functionalized with two different anchor groups. The interior part of the aminomethylated polystyrene-1%-divinylbenzene resin beads, comprising about 98% of the total loading capacity, was modified by the acid-labile ADPV anchor whereas the 2% outer surface of the polymer was covalently coated with a PEG 10 000 derivative which renders the resin surface hydrophilic and biocompatible. The novel resin was characterized by introducing marker amino acids and by infrared spectroscopy. Employing this bifunctionalized resin for peptide synthesis, free as well as polymer-bound peptides were obtained which were tested for recognition by antibody. The resin-bound peptides proved to be suitable for ELISA and fluorescence assays, as shown by confocal laser microscopic investigations. Peptides from the interior part were obtained in high yield and purity as analyzed by HPLC, electrospray mass spectrometry and Edman degradation.     

Development of recombinant, immobilised beta-1,4-mannosyltransferase for use as an efficient tool in the chemoenzymatic synthesis of N-linked oligosaccharides.

The preparation of the conserved core structure of asparagine-linked oligosaccharides found in eukaryotic glycoproteins is an important step towards the synthesis of homogeneous neoglycoproteins. So far, however, the convenient generation of the Manbeta4GlcNAcbeta4GlcNAc (Gn2M) core trisaccharide has proved to be a major obstacle because of the inherent difficulties associated with the synthesis of beta-mannosides. Here we report the overproduction in Escherichia coli of full-length and transmembrane-deleted yeast beta-1, 4-mannosyltransferases as novel N-terminal fusions bearing a decahistidinyl sequence and the minimal human Myc epitope. The recombinant enzymes were highly active and were amenable to immobilisation by nickel(II) chelation and to immunodetection with an anti-Myc monoclonal antibody. The immobilised, transmembrane-deleted enzyme exhibited an apparent Km of 14 microM for the synthetic acceptor substrate analogue, phytanyl-pyrophosphoryl-alpha-N,N'-diacetylchitobioside (PPGn2), under saturating donor conditions. This figure is comparable to those previously reported for native and recombinant yeast beta-1, 4-mannosyltransferases with, respectively, the natural dolichyl-linked acceptor and PPGn2. The validity of the reaction product was confirmed by chromatographic and spectroscopic analysis.     

5'-modified agonist and antagonist (2'-5')(A)n analogues: synthesis and biological activity

Two 5'-modified (2'-5')(A)4 oligomers with an increased resistance to phosphatase degradation were synthesized and evaluated for their ability to develop an antiviral response when introduced into intact cells by microinjection or by chemical conjugation to poly(L-lysine). The enzymatic synthesis of 5'-gamma-phosphorothioate and beta,gamma-difluoromethylene (2'-5')(A)4 from adenosine 5'-O-(3-thiotriphosphate) and adenosine beta,gamma-difluoromethylenetriphosphate by (2'-5')-oligoadenylate synthetase is described. The isolation and characterization of these (2'-5')(A)4 analogues were achieved by high-performance liquid chromatography. The structures of 5'-modified tetramers were corroborated by enzyme digestion. These two 5'-modified tetramers compete as efficiently as natural (2'-5')(A)4 for the binding of a radiolabeled (2'-5')(A)4 probe to ribonuclease (RNase) L. Nevertheless, at the opposite to 5'-gamma-phosphorothioate (2'-5')(A)4, beta,gamma-difluoromethylene (2'-5')(A)4 failed to induce an antiviral response after microinjection in HeLa cells. In addition, it behaves as an antagonist of RNase L as demonstrated by its ability to inhibit the antiviral properties of 5'-gamma-phosphorothioate (2'-5')(A)4 when both are microinjected in HeLa cells. The increased metabolic stability of 5'-gamma-phosphorothioate (2'-5')(A)4 as compared to that of (2'-5')(A)4 was first demonstrated in cell-free extracts and then confirmed in intact cells after introduction in the form of a conjugate to poly(L-lysine). Indeed, 5'-gamma-phosphorothioate (2'-5')(A)4-poly(L-lysine) conjugate induces protein synthesis inhibition and characteristic ribosomal RNA cleavages for longer times than unmodified (2'-5')(A)4-poly(L-lysine) in the same cell system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solid-phase total synthesis and antimicrobial activities of loloatins A-D

The first total synthesis of the decapeptide antibiotics loloatins A-D (1-4), originally isolated from the marine bacterial isolate MK-PNG-276A, possibly in the genus Bacillus, was accomplished by solid-phase peptide synthesis (SPPS), followed by 'head-to-tail' cyclization of the activated linear precursors, without protection of nucleophilic side-chain functions, on a safety-catch resin. The synthetic peptides were equally active as the natural products isolated from the bacterial source and found to possess similar bacterial selectivity as other members in the family of amphipathic antimicrobial cyclic decapeptides.     

SYNTHESIS AND BIOLOGICAL ACTIVITY OF 4′-C-HYDROXYMETHYL-2′-FLUORO- D-ARABINOFURANOSYLPURINE NUCLEOSIDES

A series of 4′-C-hydroxymethyl-2′-fluoro-D-arabinofuranosylpurine nucleosides was prepared and evaluated for cytotoxicity. The details of a convenient synthesis of the carbohydrate precursor 4-C-hydroxymethyl-3,5-di-O-benzoyl-2-fluoro-α-D-arabinofuranosyl bromide (13) are presented. Proof of the structure and configuration at all chiral centers of the sugars and the nucleosides were obtained by proton NMR. All five target nucleosides were evaluated for cytotoxicity in human tumor cell lines. The 4′-C-hydroxymethyl clofarabine analogue (16β) showed slight cytotoxicity in CCRF-CEM leukemia cells.     

Thermodynamics of 7-methylguanosine cation stacking with tryptophan upon mRNA 5' cap binding to translation factor eIF4E

All eukaryotic nuclear transcribed mRNAs possess the cap structure, consisting of 7-methylguanosine linked by the 5'-5' triphosphate bridge to the first nucleoside. The goal of the present study is to dissect the enthalpy and entropy changes of association of the mRNA 5' cap with eIF4E into contributions originating from the interaction of 7-methylguanosine with tryptophan. The model results are discussed in the context of the thermodynamic parameters for the association of eIF4E with synthetic cap analogues.     

Nucleoside 5'-(beta, gamma-peroxytriphosphates)

Procedures for the synthesis, purification, and characterization of beta, gamma-peroxy analogues of the eight common ribo- and deoxyribonucleoside triphosphates have been developed. Although adenosine 5'-(beta, gamma-peroxytriphosphate) was stable to conditions in most biochemical systems, incubation of a solution of the analogue at 100 degrees C led to formation of AMP and ATP, as well as ADP. NAD+ pyrophosphorylase was the only enzyme among 13 tested for which adenosine 5'-(beta, gamma-peroxytriphosphate) was a good substrate, but the analogue was an effective inhibitor for a number of kinases. The peroxy compounds tested inactive with Escherichia coli RNA polymerase and DNA polymerase I, as well as with wheat germ RNA polymerase II.     

Preparation of protected peptide amides using the Fmoc chemical protocol. Comparison of resins for solid phase synthesis.

Different resins were examined for their potential use in the solid phase synthesis of protected peptide amides using the 9-fluorenylmethoxycarbonyl (Fmoc) chemical protocol. The model protected peptide amide BocTyr-Gly-Gly-Phe-Leu-Arg(Pmc)NH2 (1) was synthesized on both the acid-labile 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin (Rink amide resin) (2) and on resins containing the base-labile linker 4-hydroxymethylbenzoic acid. Of the resins examined only the methylbenzhydrylamine resin containing the 4-hydroxymethylbenzoic acid linkage, which was cleaved by ammonolysis in isopropanol, gave the model peptide 1 in good overall yield (53% including functionalization). Thus the synthesis of protected peptide amides by solid phase synthesis using Fmoc-protected amino acids with t-butyl-type side chain protecting groups is feasible. The choice of peptide-resin linkage and its cleavage conditions, however, are critical to the success of such syntheses. The potential application of this synthetic strategy to the preparation of novel peptide amides is discussed.     

An improved synthesis of the C-linked glucuronide of N-(4-hydroxyphenyl)retinamide

Retinoic acid analogues such as N-(4-hydroxyphenyl)retinamide (4-HPR) are effective chemopreventatives and chemotherapeutics for numerous types of cancer. The C-linked analogue of the O-glucuronide of 4-HPR (4-HPRCG) has been shown to be a more effective agent. The synthetic route to this molecule has been significantly improved by access to a key C-benzyl-glucuronide intermediate through employment of a Suzuki coupling reaction between an exoanomeric methylene sugar and an aryl bromide. Preliminary evidence shows 4-HPRCG has chemotherapeutic activity.     

Diastereoselective synthesis of aminobacteriohopanetetrol, a biomarker for methanotrophic bacteria: confirmation of the absolute configuration

A short and convenient strategy was developed for the first stereoselective chemical synthesis of aminobacteriohopanetetrol (= (1R,2R,3S,4S)-5-amino-1-[(22R)-hopan-30-yl]pentane-1,2,3,4-tetrol; 1), a typical biomarker for methanotrophic bacteria. Comparison of the NMR spectra of the synthetic and natural (peracetylated) product enabled us to unambiguously corroborate the absolute configuration of the functionalized pentyl side chain of 1.