Novel base-labile protecting groups for 5'-hydroxy function in solid-phase oligonucleotide synthesis

The 6-(levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl (LMMoNBz) and 2-(levulinyloxymethyl)-5-methoxy-4-nitrobenzoyl (LMMpNBz) groups were developed as novel base-labile protection for the 5'-hydroxy function in solid-phase oligonucleotide synthesis. A comparative study of the LMMoNBz, LMMpNBz and 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) protecting groups for oligonucleotide synthesis proved strong feasibility for the LMMoNBz group.     

Synthesis of oligonucleotides using the 2-(levulinyloxymethyl)-5-nitrobenzoyl group for the 5'-position of nucleoside 3'-H-phosphonate and -H-phosphonothioate derivatives.

Synthetic studies on phosphodiester, phosphorothioate, and phosphorodithioate-linked oligonucleotides in terms of 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) group as the base-labile protecting group for the 5'-hydroxyl groups of nucleoside 3'-H-phosphonate and -H-phosphonothioate derivatives, are described.     

Oligonucleotide synthesis using the 2-(levulinyloxymethyl)-5-nitrobenzoyl group for the 5'-position of nucleoside 3'-phosphoramidite derivatives.

A comparative study on the utility of 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) and 2-(levulinyloxymethyl)benzoyl (LMBz) protecting groups for the 5'-positions of nucleoside 3'-phosphoramidite derivatives in the oligonucleotide synthesis is presented in terms of the syntheses of TpTpT, TpTpTpT, and UpCpApGpUpUpGpG. In addition we describe the synthesis, using the LMNBz protecting group, of the CpCpA terminus triplet of tRNAs bearing exocyclic amino groups with 15N-labeling, and the trimer Gp[A*]pG containing 2'-O-(beta-D-ribofuranosyl)adenosine ([A*]), the latter of which is found at position 64 in the yeast initiator tRNA(Met).     

Novel Base-Labile Protecting Groups for 5′-Hydroxy Function in Solid-Phase Oligonucleotide Synthesis

Abstract

The 6-(levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl (LMMoNBz) and 2-(levulinyloxymethyl)-5-methoxy-4-nitrobenzoyl (LMMpNBz) groups were developed as novel base-labile protection for the 5′-hydroxy function in solid-phase oligonucleotide synthesis. A comparative study of the LMMoNBz, LMMpNBz and 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) protecting groups for oligonucleotide synthesis proved strong feasibility for the LMMoNBz group.     

Synthesis and enzymatic transformations of 5-halo-6-methoxy-5,6-dihydro derivatives of 5-[1-methoxy-2-halo (or 2,2-dihalo)ethyl]-2'-deoxyuridines as potential herpes simplex virus inhibitors

The 5-halo-6-methoxy-5,6-dihydro derivatives of 5-[1-methoxy-2-halo(or 2,2-dihalo)ethyl]-2'-deoxyuridines (3-12) were synthesized and investigated as potential anti-herpes agents. These 5,6-dihydro derivatives were designed to act as potential prodrugs to 5-[1-methoxy-2-halo(or 2,2-dihalo)ethyl]-2'-deoxyuridines (2a-e), with enhanced metabolic stability, and ready conversion to the parent molecules. These 5,6-disubstituted-5,6-dihydro analogs are stable to E. coli thymidine phosphorylase, and undergo regeneration of the 5,6-olefinic bond to provide parent moieties (2a-e), upon incubation with glutathione at 37 degrees C. The compounds (3-12) themselves were found to be non-inhibitory against herpes simplex virus type-1 (HSV-1), likely due in part to their inability to undergo conversion to parent compounds in cell culture medium.     

2,2-Bis(ethoxycarbonyl)- and 2-(alkylaminocarbonyl)-2-cyano-substituted 3-(pivaloyloxy)propyl groups as biodegradable phosphate protections of oligonucleotides

Oligonucleotides bearing biodegradable phosphate protecting groups have been synthesized on a solid support. For this purpose, two dimeric building blocks, viz. 5'-O-(4,4'-dimethoxytrityl)-(R(P),S(P))-O(P)-[2,2-bis(ethoxycarbonyl)-3-(pivaloyloxy)propyl]-P-thiothymidylyl-(3',5')-thymidine 3'-[O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite] (1) and 5'-O-(4,4'-dimethoxytrityl)-(R(P),S(P))-O(P)-[2-cyano-2-(2-phenylethylaminocarbonyl)-3-(pivaloyloxy)propyl]thymidylyl-(3',5')-thymidine 3'-(H-phosphonate) (2), were prepared. Phosphoramidite 1 was incorporated into an phosphorothioate oligothymidylate sequence on a base-labile hydroquinone-O,O'-diacetic acid linker (Q-linker) and on a photolabile 4-alkoxy-5-methoxy-2-nitrobenzyl carbonate linker (11). H-Phosphonate 2 was, in turn, incorporated into an oligothymidylate sequence only on the photolabile linker. Kinetics of the removal of the protecting groups by porcine liver esterase and subsequent retro aldol condensation/phosphate elimination were then studied. While the pro-oligonucleotide that contained only one phosphate protection gave the deprotected phosphorothioate oligonucleotide in a quantitative yield, the enzymatic step was markedly decelerated upon increasing the number of protection groups, and hence chain cleavage started to compete.     

Isolation and characterization of 3-methoxy-2(5H)-furanone as the principal nephrotoxin from Narthecium ossifragum (L.) Huds

The principal substance in Narthecium ossifragum (L.) Huds, responsible for the nephrotoxic effects on cattle, moose, goats and other ruminants has been isolated and identified by X-ray crystallography as 3-methoxy-2(5H)-furanone. The Fourier-transform infra-red, 1H and 13C nuclear magnetic resonance, and mass spectra are also given. The concentration in four different batches of plant material varied from 113 to 344 microg g(-1) (wet weight). Extracts of N. ossifragum and fractions derived from them, including purified 3-methoxy-2(5H)-furanone, were each dosed intraruminally, to young goats. 3-Methoxy-2(5H)-furanone of 99.9% purity (15 mg kg(-1) live weight) caused increased concentration of creatinine in serum within 2-3 days, typical of kidney damage caused by N. ossifragum, while toxic effect was obtained down to 4 mg kg(-1) live weight with less purified material (> or = 95%). Toxic effect was also obtained with synthesized 3-methoxy-2(5H)-furanone (30 mg kg(-1) live weight). The isomer 4-methoxy-2(5H)-furanone, detected in some of the batches of the plant material, was not toxic when dosed at 60 mg kg(-1) live weight.     

The Effect of Linker Length on the Photomodification of Tyr Residue in N-(Tyrosyl)-N"-(5-azido-2-nitrobenzoyl)-diaminoalkanes

N-(Tyrosyl)-N"-(5-azido-2-nitrobenzoyl)-1,4-diaminobutane containing a Tyr residue connected with the photoreactive aryl azide group through a diaminobutylene linker was synthesized as a model for studying the photomodification of Tyr residues in proteins. This compound and the compound with a shorter, 1,2-diaminoethylene linker, obtained previously, were subjected to a computer modeling to find their minimal energy conformations. The aromatic rings of Tyr and 5-azido-2-nitrobenzoic acid residues in the latter compound were localized in parallel planes at a distance of approximately 0.3 nm between them and were shown to be implicated in stacking interaction. On the contrary, the planes of aromatic rings of the former compound with a longer diaminobutylene linker were found to be situated perpendicularly to each other, with the distance between the centers of the rings being approximately 0.6 nm. The computer analysis was confirmed by experimental results: when studying the photomodification of the compound with the diaminobutylene linker, neither stable products of the Tyr photomodification nor unstable products capable of transformation into stable products in the dark were found. On the contrary, such products were previously identified in the case of the compound with diaminoethylene linker. The formation of amino, nitro, azoxy and azo derivatives was common for the photomodification of both compounds.     

The possible involvement of 3-methoxy-2(5H)-furanone in the etiology of Narthecium asiaticum maxim. associated nephrotoxicity in cattle

Two samples of Narthecium asiaticum Maxim leaves collected in Japan were found to contain 103 microg(-1) and 160 microg g(-1) dry matter of 3-methoxy-2(5H)-furanone respectively. 3-Methoxy-2(5h)-furanone was suggested to be the toxic principle of N. asiaticum causing nephrotoxicity in cattle in Japan. Two other furanones, which are thought to be non-toxic, were also isolated from the two samples. These were 4-methoxy-2(5H)-furanone and 5-hydroxy-4-methoxy-2(5H)-furanone.     

Preparation of single-enantiomer biofunctional molecules with (S)-2-methoxy-2-(1-naphthyl)propanoic acid

(RS)-beta-Ionol and (RS)-2-methyl-4-octanol were resolved by using (S)-2-methoxy-2-(1-naphthyl)propanoic acid [(S)-MalphaNP acid]. The specific stereochemistry of each MalphaNP ester was elucidated by 2D NMR analyses, and shielding by the 1-naphthyl group was observed in both the 1H- and 13C-NMR spectra. Solvolysis of the individual (S)-MalphaNP esters gave four single-enantiomer alcohols. The normal-phase HPLC elution order of each MalphaNP ester is also discussed.     

Comparative evaluation of 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) rapid colorimetric assays for antimicrobial susceptibility testing of staphylococci and ESBL-producing clinical isolates

A new generation of water soluble tetrazolium salts have recently become available and in this study we compared a colorimetric assay developed using one of these salts, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8), with a previously developed 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) colorimetric assay to determine which agent is most suitable for use as a colorimetric indicator in susceptibility testing. The MICs of 6 antibiotics were determined for 33 staphylococci using both colorimetric assays and compared with those obtained using the British Society for Antimicrobial Chemotherapy reference broth microdilution method. Absolute categorical agreement between the reference and test methods ranged from 79% (cefuroxime) to 100% (vancomycin) for both assays. No minor or major errors occurred using either assay with very major errors ranging from zero (vancomycin) to seven (cefuroxime). Analysis of the distribution of differences in the log(2) dilution MIC results revealed overall agreement, within the accuracy limits of the standard test (+/-1 log(2) dilution), using the XTT and WST-8 assays of 98% and 88%, respectively. Further studies on 31 ESBL-producing isolates were performed using the XTT method with absolute categorical agreement ranging from 87% (nitrofurantoin) to 100% (ofloxacin and meropenem). No errors were noted for either ofloxacin or meropenem with overall agreement of 91%. The data suggests that XTT is more reliable and accurate than WST-8 for use in a rapid antimicrobial susceptibility test.     

Discovery of 5-substituted 2-amino-4-chloro-8-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-7,8-dihydropteridin-6(5H)-ones as potent and selective Hsp90 inhibitors

A new series of compounds, 5-substituted 2-amino-4-chloro-8-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-7,8-dihydropteridin-6(5H)-ones, have been designed and identified as potent and selective inhibitors of Hsp90. These compounds demonstrated nanomolar potency toward both Hsp90-regulated Her2 degradation and the growth of a panel of human tumor cell lines in cell-based assays. High selectivity of these compounds toward Hsp90 was evident given that they did not inhibit a panel of 34 kinases at 10 μM. The structure–activity relationship (SAR) of this series is reported here.     

Synthesis and antimycobacterial evaluation of newer 1-cyclopropyl-1,4-dihydro-6-fluoro-7-(substituted secondary amino)-8-methoxy-5-(sub)-4-oxoquinoline-3-carboxylic acids

Thirty-four newer 1-cyclopropyl-1,4-dihydro-6-fluoro-7-(substituted secondary amino)-8-methoxy-5-(sub)-4-oxoquinoline-3-carboxylic acids were synthesized from 1,2,3,4-tetrafluoro benzene and evaluated for in vitro and in vivo antimycobacterial activities against Mycobacterium tuberculosis H37Rv (MTB), multi-drug resistant M. tuberculosis (MDR-TB) and Mycobacterium smegmatis (MC(2)) and also tested for the ability to inhibit the supercoiling activity of DNA gyrase. Among the synthesized compounds, 7-(1-(4-methoxybenzyl)-3,4,5,6,7,8-hexahydroisoquinolin-2(1H)-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-5-nitro-4-oxoquinoline-3-carboxylic acid (13n) was found to be the most active compound in vitro with MIC of 0.16 and 0.33 microM against MTB and MDR-TB, respectively. In the in vivo animal model 13n decreased the bacterial load in lung and spleen tissues with 2.54 and 2.92-log10 protections, respectively, at the dose of 50mg/kg body weight. Compound 13n also inhibited the supercoiling activity of mycobacterial DNA gyrase with IC(50) of 30.0 microg/ml.     

Effect of linker length on the photomodification process of tyrosine residues in N-(tyrosyl)-N'-(5-azido-2-nitrobenzoyl)diaminoalkanes]

N-(Tyrosyl)-N'-(5-azido-2-nitrobenzoyl)-1,4-diaminobutane, containing a Tyr residue connected with the photoreactive aryl azide group through the diaminobutylene linker, was synthesized as a model for studying the photomodification of Tyr residues in proteins. This compound and the compound with a shorter, 1,2-diaminoethylene linker, obtained previously, were subjected to computer modeling to find their minimal-energy conformations. The aromatic rings of Tyr and 5-azido-2-nitrobenzoic acid residues in the latter compound were localized in parallel planes at a distance of approximately 0.3 nm between them and were shown to be implicated in stacking interactions. On the contrary, the planes of aromatic rings of the former compound with a longer, diaminobutylene linker were found to be situated perpendicularly to each other, with the distance between the centers of the rings being approximately 0.6 nm. The computer analysis was confirmed by experimental results: when studying the photomodification of the compound with the diaminobutylene linker, neither stable products of the Tyr photomodification nor unstable products capable of transformation into stable products in the dark were found. On the contrary, such products were previously identified in the case of the compound with diaminoethylene linker. The formation of amino, nitro, azoxy, and azo derivatives was common for the photomodification of both compounds.     

First total synthesis of (5Z,9Z)-(+/-)-2-methoxy-5,9-octadecadienoic acid, a marine derived methoxylated analog of taxoleic acid

The first total synthesis for the sponge derived (5Z,9Z)-(+/-)-2-methoxy-5,9-octadecadienoic acid, an analog of taxoleic acid, was accomplished in seven steps and in a 10% overall yield. It was again corroborated that the best strategy to prepare these cis,cis dimethylene interrupted double bonds is the double-alkyne bromide coupling reaction of 1,5-hexadiyne, which provides the advantage of achieving a 100% cis stereochemical purity for both double bonds after hydrogenation under Lindlar conditions. The alpha-methoxy functionality was best prepared via the Mukaiyama reaction of (4Z,8Z)-heptadecadienal with trimethylsilyl cyanide and triethylamine followed by acid hydrolysis. Selective methylation of the hydroxyl group of (5Z,9Z)-(+/-)-2-hydroxy-5,9-octadecadienoic acid was achieved with sodium hydride/methyl iodide when tetrahydrofuran was used as solvent. Complete spectral data is presented, for the first time, for this unusual marine alpha-methoxylated fatty acid.     

柯拉斯那沉香中2-(2-苯乙基)色酮类化学成分研究

为研究柯拉斯那(Aquilaria crassna Pierre ex Lecomte)沉香的化学成分。实验采用多种柱色谱方法从该沉香中分离得到9个2-(2-苯乙基)色酮类化合物,通过现代波谱学技术分别鉴定为6-甲氧基-2-[2-(3′-羟基-4′-甲氧基苯基)乙基]色酮(1)、5-羟基-6-甲氧基-2-[2-(3′-羟基-4′-甲氧基苯基)乙基]色酮(2)、tetrahydrochromone F(3)、6-甲氧基-2-[2-(3′-甲氧基-4′-羟基苯基)乙基]色酮(4)、6-甲氧基-7-羟基-2-[2-(4′-甲氧基苯基)乙基]色酮(5)、6,7-二甲氧基-2-[2-(3′-羟基-4′-甲氧基苯基)乙基]色酮(6)、6,7-二甲氧基-2-[2-(4′-甲氧基苯基)乙基]色酮(7)、6-羟基-2-[2-(4′-羟基苯基)乙基]色酮(8)、5-羟基-2-[2-(2′-羟基苯基)乙基]色酮(9)。化合物2、3和5～9均为首次从柯拉斯那所得沉香中分离得到。采用MTT法对单体化合物的细胞毒活性进行测试,测试结果表明,化合物1,2和4具有微弱的细胞毒活性。     

Synthesis of (+)-(R)- and (−)-(S)-5-hydroxy-2-methyl-2-dipropylaminotetralin: Effects on rat hippocampal output of 5-HT, 5-HIAA,and DOPAC as determined by in vivo microdialysis

Racemic 5-methoxy-2-methyl-2-dipropylaminotetralin ( 3 ) has been prepared by a short synthetic route, in which the N,N-dipropyliminium perchlorate of 5-methoxy-2-tetralone ( 4 ) is a key intermediate. Racemic 3 was resolved by crystallization of the corresponding diastereomeric di-p-toluoyltartrates. The enantiomeric excess (%ee) of the phenolic derivatives of (+)-(R)- and (?)-(S)-3 [(+)-(R)- and (?)-(S)-2] was determined by ¹HNMR spectroscopic analysis of the corresponding diastereomeric (?)-(R)-1,1′-binaphthyl-2,2′-diylphosphoric acid salts utilizing ¹³C satellites. X-ray crystallography established the absolute configuration of (?)-(S)-2 · HCl. The enantiomers of 2 were tested for hippocampal output of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, and dihydroxyphenylacetic acid in rats by use of in vivo microdialysis. The (?)-(S)-enantiomer appeared to affect 5-HT-turnover, whereas (+)-(R)- 2 was inactive. Results obtained provide support for the previously reported hypothesis that the inactivity of (?)-(S)- 2 at central DA receptors is caused by the steric bulk of the C(2)-methyl group. This makes it possible to define a “DA D2 receptor essential volume.” © 1993 Wiley-Liss, Inc.     

Laccase-mediated synthesis of 2-methoxy-3-methyl-5-(alkylamino)- and 3-methyl-2,5-bis(alkylamino)-[1,4]-benzoquinones

The synthesis of 5-alkylamino- and 2,5-bis(alkylamino)-[1,4]-benzoquinones, showing structural similarity to natural mitomycins, was performed through coupling of 2-methoxy-3-methylhydroquinone with primary amines such as n-octylamine, geranylamine and cyclooctylamine using laccases from Myceliophthora thermophila (MtL) and Pycnoporus cinnabarinus SBUG-M 1044 (PcL). Product spectra of laccase reactions differ due to reaction systems pH values (pH 7.0 for MtL and pH 5.0 for PcL) applied to assure enzymes optimal catalytic efficiency. The MtL- and PcL-mediated formation of monoaminated products was achieved at equimolar reactant concentrations with amine coupling at the meta-position to benzoquinones methyl group. Increased formation of diaminated products occurred in PcL-mediated reactions and generally when the amine was supplied in excess. Diamination entailed elimination of the benzoquinone methoxy group (amination in para-position to the first amine substituent). Six products were synthesised and characterised by NMR and HR-MS analysis. The laccase-mediated amine coupling to 2-methoxy-3-methylhydroquinone confers two of the essential pharmaceutical active motifs from mitomycins: (i) a stable 1,4-benzoquinoic parent structure and (ii) a biological active alkylation function (NH).     

Two crystalline pentofuranosyl bromides: tri-O-(p-nitrobenzoyl)-β-D-ribofuranosyl bromide and tri-O-(p-nitrobenzoyl)-α,β-D-xylofuranosyl bromide

Methyl α,β-D-ribofuranoside was p-nitrobenzoylated to give methyl tri-O-(p-nitrobenzoyl)-β-D-ribofuranoside (2),and this was treated with HBr in acetic acid to give tri- O-(p-nitrobenzoyl)-β-D-ribofuranosyl bromide (3). Bromide 3 could be converted into 2,5-anhydro-3,4,6-tri-O-(p-nitrobenzoyl)-D-allononitrile (4) with Hg(CN)₂, or hydrolyzed to 2,3,5-tri-O-(p-nitrobenzoyl)-D-ribose (5). On p-nitro- benzoylation, 5 gave tetra-O-(p-nitrobenzoyl)-β-D-ribofuranose (6). The synthesis of tri-O-(p-nitrobenzoyl)-α-β-D-xylofuranosyl bromide (11) started with methyl 3,5-O-isopropylidene-β-D-xyldfuranoside (7), which was p-nitrobenzoylated to give ester 8; this was then hydrolyzed, and the product p-nitrobenzoylated to give methyl tri-O-(p-nitrobenzoyl)-β-D-xylofuranoside (10) which, on treatment with HBr in CH₂Cl₂, afforded the desired bromide (11). Nucleophilic replacement with Hg(CN)₂ afforded 2,5-anhydro-3,4,6-tri-O-(p-nitrobenzoyl)-D-gulononitrile (12).     

Comparative evaluation of 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium,monosodium salt (WST-8) rapid colorimetric assays for antimicrobial susceptibility testing of staphylococci and ESBL-producing clinical isolates

A new generation of water soluble tetrazolium salts have recently become available and in this study we compared a colorimetric assay developed using one of these salts, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8), with a previously developed 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) colorimetric assay to determine which agent is most suitable for use as a colorimetric indicator in susceptibility testing. The MICs of 6 antibiotics were determined for 33 staphylococci using both colorimetric assays and compared with those obtained using the British Society for Antimicrobial Chemotherapy reference broth microdilution method. Absolute categorical agreement between the reference and test methods ranged from 79% (cefuroxime) to 100% (vancomycin) for both assays. No minor or major errors occurred using either assay with very major errors ranging from zero (vancomycin) to seven (cefuroxime). Analysis of the distribution of differences in the log₂ dilution MIC results revealed overall agreement, within the accuracy limits of the standard test (± 1 log₂ dilution), using the XTT and WST-8 assays of 98% and 88%, respectively. Further studies on 31 ESBL-producing isolates were performed using the XTT method with absolute categorical agreement ranging from 87% (nitrofurantoin) to 100% (ofloxacin and meropenem). No errors were noted for either ofloxacin or meropenem with overall agreement of 91%. The data suggests that XTT is more reliable and accurate than WST-8 for use in a rapid antimicrobial susceptibility test.