Synthesis,Protonation Behavior,Conformational Analysis,and Regioselective Enzymatic Acylation of the Novel Diamino Analogue of (E)-5-(2-Bromovinyl)-2′-deoxyuridine (BVDU)

Abstract

(E)-3′,5′-Diamino-5-(2-bromovinyl)-2′,3′,5′-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. The protonation behavior of 5 has been studied by means of pH-metric measurements and NMR spectroscopy. This study allows the determination of the basicity constants and the stepwise protonation sites. Thus, the main species at physiological pH is the monoprotonated form. The conformational analysis of this nucleoside analogue was also carried out through ¹H NMR spectroscopy. In addition, a convenient synthesis of N-3′ and N-5′ acylated derivatives was developed by regioselective enzymatic acylation. Thus, Candida antarctica lipase B (CAL-B) selectively acylated the 5′-amino group, thus furnishing nucleosides 8. On the other hand, immobilized Pseudomonas cepacia lipase (PSL-C) exhibited the opposite selectivity, conferring acylation at the 3′-amino group, thus affording derivatives 9.     

Synthesis and antiviral activity assay of novel (E)-3',5'-diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine

(E)-3',5'-diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. In contrast with BVDU, compound 5 did not show activity against herpes simplex virus or varicella-zoster virus.     

Synthesis, resolution and anticonvulsant activity of chiral N-1'-ethyl,N-3'-(1-phenylethyl)-(R,S)-2'H,3H,5'H-spiro-(2-benzofuran-1,4'-imidazolidine)-2',3,5'-trione diastereomers

Four new N-1',N-3'-disubstituted-2'H,3H,5'H-spiro-(2-benzofuran-1,4'-imidazolidine)-2',3,5'-triones bearing a chiral N-3' substituent were synthesized, resolved and their anticonvulsant activity was obtained and determined that the activity was not stereoselective.     

Acylated flavonol glycosides from the forage legume, Onobrychis viciifolia (sainfoin)

Ten acylated flavonol glycosides were isolated from aqueous acetone extracts of the aerial parts of the forage legume, Onobrychis viciifolia, and their structures determined using spectroscopic methods. Among these were eight previously unreported examples which comprised either feruloylated or sinapoylated derivatives of 3-O-di- and 3-O-triglycosides of kaempferol (3,5,7,4'-tetrahydroxyflavone) or quercetin (3,5,7,3',4'-pentahydroxyflavone). The diglycosides were acylated at the primary Glc residue of O-α-Rhap(1→6)-β-Glcp (rutinose), whereas the triglycosides were acylated at the terminal Rha residues of the branched trisaccharides, O-α-Rhap(1→2)[α-Rhap(1→6)]-β-Galp or O-α-Rhap(1→2)[α-Rhap(1→6)]-β-Glcp. Identification of the primary 3-O-linked hexose residues as either Gal or Glc was carried out by negative ion electrospray and serial MS, and cryoprobe NMR spectroscopy. Analysis of UV and MS spectra of the acylated flavonol glycosides provided additional diagnostic features relevant to direct characterisation of these compounds in hyphenated analyses. Quantitative analysis of the acylated flavonol glycosides present in different aerial parts of sainfoin revealed that the highest concentrations were in mature leaflets.     

Biocatalytic synthesis,structural elucidation,antioxidant capacity and tyrosinase inhibition activity of long chain fatty acid acylated derivatives of phloridzin and isoquercitrin

Our present investigation describes the regioselective enzymatic acylation of two series of acylated derivatives of phloridzin and isoquercitrin with six different long chain saturated, mono- and poly-unsaturated fatty acids. The biocatalytic synthesis was optimized to achieve 81–98% yields, using immobilized lipase B, from Candida antarctica (Novozym 435®), in acetone at 45 °C. The synthesized esters have been analyzed by ¹H NMR, ¹³C NMR spectroscopy and evaluated for their antioxidant capacity and tyrosinase inhibition, using in vitro assays. Among all the phloridzin and isoquercitrin derivatives, the greatest potential for inhibition of tyrosinase activity (p ?0.05) was exhibited by the α-linolenic acid ester of isoquercitrin.     

N-(2-Carboxyethyl)chitosans: regioselective synthesis,characterisation and protolytic equilibria

N-(2-Carboxyethyl)chitosans were obtained by reaction of low molecular weight chitosan with a low degree of acetylation and 3-halopropionic acids under mild alkaline media (pH 8-9, NaHCO3) at 60 degrees C. The chemical structure of the derivatives obtained was determined by 1H and 13C NMR spectroscopies. It was found that alkylation of chitosan by 3-halopropionic acids proceeds exclusively at the amino groups. The products obtained are described in terms of their degrees of carboxyethylation and ratio of mono-, di-substitution and free amine content. The protonation constants of amino and carboxylate groups of a series of N-(2-carboxyethyl)chitosans were determined by pH-titration at ionic strength 0.1 M KNO3 and 25 degrees C.     

Complete lipopolysaccharide of Plesiomonas shigelloides O74:H5 (strain CNCTC 144/92). 2. Lipid A, its structural variability, the linkage to the core oligosaccharide, and the biological activity of the lipopolysaccharide

Plesiomonas shigelloides is a Gram-negative bacterium associated with waterborne infections, which is common in tropical and subtropical habitats. Contrary to the unified antigenic classification of P. shigelloides, data concerning the structure and activity of their lipopolysaccharides (LPS and endotoxin) are limited. This study completes the structural investigation of phenol- and water-soluble fractions of P. shigelloides O74 (strain CNCTC 144/92) LPS with the emphasis on lipid A heterogeneity, describing the entire molecule and some of its biological in vitro activities. Structures of the lipid A and the affinity-purified decasaccharide obtained by de-N,O-acylation of P. shigelloides O74 LPS were elucidated by chemical analysis combined with electrospray ionization multiple-stage mass spectrometry (ESI-MS(n)), MALDI-TOF MS, and NMR spectroscopy. Lipid A of P. shigelloides O74 is heterogeneous, and three major forms have been identified. They all were asymmetric, phosphorylated, and hexaacylated, showing different acylation patterns. The beta-GlcpN4P-(1-->6)-alpha-GlcpN1P disaccharide was substituted with the primary fatty acids: (R)-3-hydroxytetradecanoic acid [14:0(3-OH)] at N-2 and N-2' and (R)-3-hydroxydodecanoic acid [12:0(3-OH)] at O-3 and O-3'. The heterogeneity among the three forms (I-III) of P. shigelloides O74 lipid A was attributed to the substitution of the acyl residues at N-2' and O-3' with the secondary acyls: (I) cis-9-hexadecenoic acid (9c-16:1) at N-2' and 12:0 at O-3', (II) 14:0 at N-2' and 12:0 at O-3', and (III) 12:0 at N-2' and 12:0 at O-3'. The pro-inflammatory cytokine-inducing activities of P. shigelloides O74 LPS were similar to those of Escherichia coli O55 LPS.     

Controllable synthesis of polymerizable ester and amide prodrugs of acyclovir by enzyme in organic solvent

A facile control of the acylation position at the primary hydroxyl and amino of acyclovir, respectively, was achieved and five polymerizable acyclovir prodrugs were synthesized. Various reaction conditions were studied in detail. Thus, lipase acrylic resin from Candida antarctica (CAL-B) in pyridine or acetone showed high chemo-selectivity toward the primary hydroxyl of acyclovir. However, lipase PS 'Amano' (PS) in DMSO selectively acylated the amino group. The selectivity of PS could be adjusted by changing reaction solvents. The acyclovir vinyl derivatives obtained would be important monomers used for the preparation of macromolecular nucleoside drugs.     

Carbonylbisphosphonate and (diazomethylene)bisphosphonate analogues of AZT 5(')-diphosphate

A novel nucleotide analogue is described, in which the alpha,beta-phosphoric anhydride oxygen of a nucleoside 5(')-diphosphate is replaced by a carbonyl group: the carbonylbisphosphonate analogue 5 of 2('),3(')-dideoxy-3(')-azidothymidine 5(')-diphosphate (AZT 5(')-diphosphate). 5 was synthesized from tetramethyl (diazomethylene)bisphosphonate 1 via the trimethyl ester 4 of the corresponding AZT 5(')-(diazomethylene)bisphosphonate 6, which is also a new type of nucleotide analogue. The ultimate product 5 was isolated by reverse-phase HPLC, and characterized by 31P, 13C, and 1H NMR; and by high-resolution mass spectrometry. The ketone group of 5 is a visible chromophore (yellow) and reversibly forms a colorless hydrate. The ketone hydrate 'pK' is about 4.2 when excess of magnesium ion is present. The potential of such analogues as novel inhibitors of enzymes mediating nucleotide-dependent biochemical processes is discussed.     

7-deazainosine derivatives: synthesis and characterization of 7- and 7,8-substituted pyrrolo [2,3-d]pyrimidine ribonucleosides

The synthesis of model 7 deazapurine derivatives related to tubercidin and toyocamycin has been performed. Tubercidin derivatives were obtained by simple conversion of the amino group of the heterocyclic moiety of the starting 7-deazadenosine compounds, into a hydroxyl group. Preparation of toyocamycin derivatives was accomplished by treatment of the silylated 6-bromo-5-cyanopyrrolo[2,3-d]pyrimidin-4-one with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-d-ribofuranose. The glycosylation reaction afforded a mixture of 8-bromo 7-cyano 2',3',5' tri-O-benzoyl 7-deazainosine and 6-bromo-5-cyano-3-(2',3',5'-tri-O-benzoyl-beta-d-ribofuranosyl)pyrrolo[2,3-d]-pyrimidin-4-one isomers: The structures were assigned on the basis of NMR spectroscopy studies. Next deprotection treatment gave the novel 7-deazainosine ribonucleosides.     

Synthesis of 1-(5'-O-benzoyl-3'-deoxy-beta-D-glycero-pentofuran-2'-ulosyl)uracil and its alpha-anomer by [1,2]-hydride shift

In contrast with direct tosylation of 5'-O-benzoyl- (1d) or 5'-O-pivaloyl-1-beta-D-lyxofuranosyl-uracil (1e) with TsCl/pyridine, tosylation of the 2', 3'-O-dibutylstannylene derivatives (4d,e) of these compounds proved to give the 3'-O-tosyl derivatives 2d, e selectively. Coversion of 2d as a model to 1-(5'-O-benzoyl-3'-deoxy-beta-D-glycero-pentofuran-2'-ulosyl )uracil (5-beta) by base-induced [1,2]-hydride shift was examined under various reaction conditions, and the alpha/beta ratio of the product mixture (5-alpha, beta) determined by 1H NMR spectroscopy in each case. The BzOLi/DMF combination has proved to be most profitable for obtaining 5-beta.     

Coupling of drug protonation to the specific binding of aminoglycosides to the A site of 16 S rRNA: elucidation of the number of drug amino groups involved and their identities

2-Deoxystreptamine (2-DOS) aminoglycoside antibiotics bind specifically to the central region of the 16S rRNA A site and interfere with protein synthesis. Recently, we have shown that the binding of 2-DOS aminoglycosides to an A site model RNA oligonucleotide is linked to the protonation of drug amino groups. Here, we extend these studies to define the number of amino groups involved as well as their identities. Specifically, we use pH-dependent 15N NMR spectroscopy to determine the pK(a) values of the amino groups in neomycin B, paromomycin I, and lividomycin A sulfate, with the resulting pK(a) values ranging from 6.92 to 9.51. For each drug, the 3-amino group was associated with the lowest pK(a), with this value being 6.92 in neomycin B, 7.07 in paromomycin I, and 7.24 in lividomycin A. In addition, we use buffer-dependent isothermal titration calorimetry (ITC) to determine the number of protons linked to the complexation of the three drugs with the A site model RNA oligomer at pH 5.5, 8.8, or 9.0. At pH 5.5, the binding of the three drugs to the host RNA is independent of drug protonation effects. By contrast, at pH 9.0, the RNA binding of paromomycin I and neomycin B is coupled to the uptake of 3.25 and 3.80 protons, respectively, with the RNA binding of lividomycin A at pH 8.8 being coupled to the uptake of 3.25 protons. A comparison of these values with the protonation states of the drugs predicted by our NMR-derived pK(a) values allows us to identify the specific drug amino groups whose protonation is linked to complexation with the host RNA. These determinations reveal that the binding of lividomycin A to the host RNA is coupled to the protonation of all five of its amino groups, with the RNA binding of paromomycin I and neomycin B being linked to the protonation of four and at least five amino groups, respectively. For paromomycin I, the protonation reactions involve the 1-, 3-, 2'-, and 2"'-amino groups, while, for neomycin B, the binding-linked protonation reactions involve at least the 1-, 3-, 2', 6'-, and 2"'-amino groups. Our results clearly identify drug protonation reactions as important thermodynamic participants in the specific binding of 2-DOS aminoglycosides to the A site of 16S rRNA.     

Metabolism of the carbocyclic analogue of (E)-5-(2-iodovinyl)-2'-deoxyuridine in herpes simplex virus-infected cells. Incorporation of C-IVDU into DNA

The carbocyclic analogues of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU), in which the sugar moiety is replaced by a cyclopentane ring and which have been designated as C-BVDU and C-IVDU, respectively, are, like their parent compounds BVDU and IVDU, potent and selective inhibitors of herpes simplex virus type 1 (HSV-1) and, to a lesser extent, herpes simplex virus type 2 (HSV-2) replication. We have now synthesized the radiolabeled C-IVDU analogue, C-[125I]IVDU, and determined its metabolism by HSV-infected and mock-infected Vero cells. C-[125I]IVDU was effectively phosphorylated by HSV-1-infected cells and, to a lesser extent, HSV-2-infected cells. C-[125I]IVDU was not phosphorylated to an appreciable extent by either mock-infected cells or cells that had been infected with a thymidine kinase-deficient mutant of HSV-1. Furthermore, C-[125I]IVDU was incorporated into both viral and cellular DNA of HSV-1-infected Vero cells. This finding represents the first demonstration of the incorporation of a cyclopentylpyrimidine into DNA.     

The effect of chemical modification of 3-(3-amino-3-carboxypropyl)uridine on tRNA function.

The minor base 3-(3-amino-3-carboxypropyl)uridine (acp3U) in Escherichia coli tRNAPhe was acylated with the N-hydroxysuccinimide esters of acetic, phenoxy-acetic, and naphthoxyacetic acid, as well as the ester of 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-glycine. The derivatives of tRNAPhe formed were all capable of accepting phenylalanine. There were only minor effects on the kinetic parameters of these derivatives for E. coli phenylalanyl-tRNA synthetase. There was no effect on the ability of tRNAPhe to participate in poly(U)- or poly(ACU)-directed polypeptide synthesis or in the poly(U)-stimulated binding to E. coli ribosomes. The rate of photodynamic cross-linking of 4-Srd 8 to Cyd 13 was decreased in tRNAs containing the acetyl and dansyl-glycyl derivatives of acp3U, indicating that acylation of this base may perturb the tertiary structure of the tRNA. This base in tRNAPhe does not appear to play any role in the known biological functions of tRNAPhe.     

Synthesis and analgesic activity of N-(N-acetyl-L-amino-acyl)-5-methoxytryptamines

5-Methoxytryptamine was acylated with N-acetyl-L-amino acids to give rise the corresponding N-(N-acetyl-L-amino acyl)-5-methoxytryptamines. The analgesic activity was evaluated by the tail flick test. Among the 6 compounds, the analgesic potency of N-(N-acetyl-tryptophanyl)-5-methoxytryptamine (5e) and N-(N-acetyl-glycyl)-5-methoxytryptamine (5a) are much more potent than that of melatonin.     

Efficient chemoenzymatic synthesis of (S)- and (R)-5-(1-aminoethyl)-2-(cyclohexylmethoxy)benzamide: key intermediate for Src-SH2 inhibitor

A facile chemoenzymatic synthesis of both the S and R forms of 5-(1-aminoethyl)-2-(cyclohexylmethoxy)benzamide a key intermediate of non-peptidic Src SH2 inhibitors is described. Both the enantiomers were synthesized in high optical purity (>99% ee) by reduction followed by lipase-mediated acylation of the precursor 6 in one-pot. Immobilized Pseudomonas cepacia lipase offered high degree of enantioselectivity with spontaneity.     

2,5-oligoadenylate-peptide nucleic acids (2-5A-PNAs) activate RNase L

To potentiate the 2-5A (2',5'-oligoadenylate)-antisense and peptide nucleic acid (PNA) approaches to regulation of gene expression, composite molecules were generated containing both 2-5A and PNA moieties. 2-5A-PNA adducts were synthesized using solid-phase techniques. Highly cross-linked polystyrene beads were functionalized with glycine tethered through a p-hydroxymethylbenzoic acid linker and the PNA domain of the chimeric oligonucleotide analogue was added by sequential elongation of the amino terminus with the monomethoxytrityl protected N-(2-aminoethyl)-N-(adenin-1-ylacetyl)glycinate. Transition to the 2-5A domain was accomplished by coupling of the PNA chain to dimethoxytrityl protected N-(2-hydroxyethyl)-N-(adenin-1-ylacetyl)glycinate. Finally, (2-cyanoethyl)-N,N-diisopropyl-4-O-(4,4-dimethoxytrityl)butylphosphor amidite and the corresponding (2-cyanoethyl)-N,N-diisopropylphosphoramidite of 5-O-(4,4'-dimethoxytrityl)-3-O-(tert-butyldimethylsilyl)-N6-benzoyladeno sine were the synthons employed to add the 2 butanediol phosphate linkers and the four 2',5'-linked riboadenylates. The 5'-phosphate moiety was introduced with 2-[[2-(4,4'-dimethoxytrityloxy)ethyl]sulfonyl]ethyl-(2-cyanoethyl) -N,N-diisopropylphosphoramidite. Deprotection with methanolic NH3 and tetraethylammonium fluoride afforded the desired products, 2-SA-pnaA4, 2-5A-pnaA8 and 2-5A-pnaA12. When evaluated for their ability to cause the degradation of two different RNA substrates by the 2-5A-dependent RNase L, these new 2-5A-PNA conjugates were found to be potent RNase L activators. The union of 2-5A and PNA presents fresh opportunities to explore the biological and therapeutic implications of these unique approaches to antisense.     

The preparation of three fluorescence-labeled derivatives of testosterone

Three fluorescence-labelled derivatives of testosterone were prepared consisting of the steroid separated from the fluorochrome by a hydrocarbon "bridge". "Bridges" of different lengths (C2 to C7) were used as the length required to avoid steric hindrance effects by the fluorochrome in studies on steroid-protein binding was unknown. The three derivatives prepared were: 17 beta-hydroxy--4-androsten-3-one 3-(O-(N-(2'-mercapto)ethyl)carbamoylmethyl)oxime, 17 beta-hydroxy-4-androsten-3-one 3-(O-(N-(3'-amino)propyl)carbamoylmethyl)oxime and 17 beta-hydroxy-4-androsten-3-one 3-(O(N-(7'-amino)heptyl)carbamoylmethyl)oxime. These were then coupled with either a dansyl or a fluorescein molecule. Overall yields were sufficient and the products immunoreactive with anti-testosterone antiserum.     

5-[3-(E)-(4-azido-2,3,5,6-tetrafluorobenzamido)propenyl-1]-2'-deoxy- uridine-5'-triphosphate substitutes for thymidine-5'-triphosphate in the polymerase chain reaction.

The DNA targets may be labeled and simultaneously amplified in the polymerase chain reaction (PCR) using a pair of respective primers after elongation with nucleoside-5'-triphosphates carrying photoreactive groups. The amplified DNA may be subsequently photoactivated by irradiation above 300 nm, resulting in photo-cross-linking of the strands. For this goal 5-[3-(E)-(4-azido-2,3,5,6-tetrafluorobenzamido)propenyl-1]-, 5-{N-[N'-(4-azido-2,3,5, 6-tetrafluorobenzoyl)-3-aminopropionyl]aminomethyl}-, and 5-{N-[N'-(2-nitro-5-azidobenzoyl)-3-aminopropionyl]aminomethyl}-2'-de oxyuridine-5'-triphosphate (VII, VIa, and VIb) derivatives have been synthesized. It was found that VII is capable of efficiently elongating DNA primers with both Klenow fragment DNA polymerase I and Thermus aquaticus DNA polymerase. Thereto, it turned out to provide quantitative incorporation in DNA as revealed by the formation of the full-length amplificate by PCR in the presence of this photoreactive analogue without any dilution with natural dTTP. On the contrary, it was found, that incorporation of VIa and VIb do not permit further DNA replication.     

Stereospecific synthesis of (2S)-2-methyl-3-(2',6'-dimethyl-4'-hydroxyphenyl)-propionic acid (Mdp) and its incorporation into an opioid peptide

To examine the effect of replacing the N-terminal amino group in opioid peptides with a methyl group on biological activity, a stereospecific synthesis of the tyrosine analogue (2S)-2-methyl-3-(2',6'-dimethyl-4'-hydroxyphenyl)-propionic acid (Mdp) was performed. The enkephalin analogue (2S)-Mdp-D-Ala-Gly-Phe-Leu-NH2 turned out to be a quite potent delta opioid antagonist and a somewhat less potent mu antagonist, indicating that a positively charged N-terminal amino group is not a conditio sine qua non for the binding of opioid peptides to delta and mu receptors but may be required for signal transduction.