Determination by HPLC fluorescence analysis of the natural enantiomers of sex pheromones in the New World screwworm fly, Cochliomyia hominivorax.

Bioassays of six racemic synthesized candidate sex pheromone compounds against male New World screwworm Cochliomyia hominivorax (Coquerel) flies showed that the most potent bioactivity was found with 6-acetoxy-19-methylnonacosane and 7-acetoxy-15-methylnonacosane compared with four other isomeric acetoxy nonacosanes and a larger aliphatic ketone. As all these methyl-branched compounds have two asymmetric carbons and four possible enantiomers, characterization of the natural enantiomers was essential. All four enantiomers for the two most bioactive isomers of the natural sex pheromone were synthesized for bioassay. Hydrolysis and derivatization of these enantiomers with different fluorescent reagents was followed by column-switched high-performance liquid chromatography. The use of two linked, reversed-phase columns of different polarity held at sub-ambient temperatures allowed good separation of each enantiomer. This analysis applied to natural material was successful, as (6R,19R)-6-acetoxy-19-methylnonanocosane, and (7R,15R)- and (7R,15S)-7-acetoxy-15-methylnonanocosane were detected in extracts of recently colonized female flies.

Synthesis of the L-enantiomer of 4'-C-ethynyl-2'-deoxycytidine.

The L-enantiomer of 4'-C-ethynyl-2'-deoxycytidine (2) was synthesized, but did not show any activity against HIV-1 up to 100 microM.

Some applications of a chiral fluorometric reagent, (S)-TBMB carboxylic acid.

Molecular design and applications of a fluorometric chiral agent, (S)-TBMB carboxylic acid, are briefly reviewed. The agent, possessing an asymmetric 1,3-benzodioxole skeleton, was designed as a novel class of chiral agent that functions also as a benzoate chromophore for exciton chirality CD methods. The utility of this agent has been demonstrated in an application to determine enantiomeric amino acids, acyl-sn-glycerols, glycosyl-sn-glycerols, and other chiral alcohols and amines.

4'-Ethynyl nucleoside analogs: potent inhibitors of multidrug-resistant human immunodeficiency virus variants in vitro.

A series of 4'-ethynyl (4'-E) nucleoside analogs were designed, synthesized, and identified as being active against a wide spectrum of human immunodeficiency viruses (HIV), including a variety of laboratory strains of HIV-1, HIV-2, and primary clinical HIV-1 isolates. Among such analogs examined, 4'-E-2'-deoxycytidine (4'-E-dC), 4'-E-2'-deoxyadenosine (4'-E-dA), 4'-E-2'-deoxyribofuranosyl-2,6-diaminopurine, and 4'-E-2'-deoxyguanosine were the most potent and blocked HIV-1 replication with 50% effective concentrations ranging from 0.0003 to 0.01 microM in vitro with favorable cellular toxicity profiles (selectivity indices ranging 458 to 2,600). These 4'-E analogs also suppressed replication of various drug-resistant HIV-1 clones, including HIV-1(M41L/T215Y), HIV-1(K65R), HIV-1(L74V), HIV-1(M41L/T69S-S-G/T215Y), and HIV-1(A62V/V75I/F77L/F116Y/Q151M). Moreover, these analogs inhibited the replication of multidrug-resistant clinical HIV-1 strains carrying a variety of drug resistance-related amino acid substitutions isolated from HIV-1-infected individuals for whom 10 or 11 different anti-HIV-1 agents had failed. The 4'-E analogs also blocked the replication of a non-nucleoside reverse transcriptase inhibitor-resistant clone, HIV-1(Y181C), and showed an HIV-1 inhibition profile similar to that of zidovudine in time-of-drug-addition assays. The antiviral activity of 4'-E-thymidine and 4'-E-dC was blocked by the addition of thymidine and 2'-deoxycytidine, respectively, while that of 4'-E-dA was not affected by 2'-deoxyadenosine, similar to the antiviral activity reversion feature of 2',3'-dideoxynucleosides, strongly suggesting that 4'-E analogs belong to the family of nucleoside reverse transcriptase inhibitors. Further development of 4'-E analogs as potential therapeutics for infection with multidrug-resistant HIV-1 is warranted.

4'-C-substituted-2'-deoxynucleosides: a family of antiretroviral agents which are potent against drug-resistant HIV variants.

In the past years, a variety of 4'-C-substituted-2'-deoxynucleosides (4'SdNs) were designed, synthesized, and examined as potential therapeutics against human immunodeficiency virus (HIV) infection and certain such analogues proved to exert potent activity against HIV-1 in vitro. Unlike currently available nucleoside reverse transcriptase (RT) inhibitors such as 3'-azido-3'-deoxythymidine (AZT), which have the 2',3'-dideoxy configuration and thereby cause DNA chain termination in the elongating proviral DNA, 4'SdNs do retain the 3'-alpha-OH moiety but also appear to work against retrovirus as proviral DNA chain terminators. Several 4'SdNs have been shown to be active against various laboratory and clinical HIV-1 strains including known drug-resistant HIV-1 variants. Among such 4'SdNs is 4'-azido-2'-deoxythymidine (4'-AZT) which exerts potent antiviral activity against wild type and AZT-resistant clinical HIV strains. More anti-HIV 4'SdNs have recently been reported including 4'-ethynylthymidine, 4'-ethynyl-2'-deoxy-D-ribofuranosyl-2,6-diaminopurine (4'-E-dDAP), 4'-ethynyl-2'-deoxyguanosine (4'-E-dG) and 4'-ethynyl-2'-deoxyadenosine (4'-E-dA). The latter three analogues are highly potent against HIV-1 and HIV-2 with EC50 values ranging from 0.0003 to 0.01 microM and have favorable cytotoxicity profiles with selective index (SI) values ranging from 975 to 2600. These 4'-ethynyl-2'-deoxynucleosides also exert potent activity against all known drug-resistant HIV-1 variants including the multi-dideoxynucleoside-resistant HIV and the variants with the 6-base pair inserts. Some of these compounds have favorable pharmacological properties and further development as potential therapeutics against HIV-1 infection is warranted.

Syntheses of 4'-C-ethynyl-beta-D-arabino- and 4'-C-ethynyl-2'-deoxy-beta-D-ribo-pentofuranosylpyrimidines and -purines and evaluation of their anti-HIV activity.

4'-C-Ethynyl-beta-D-arabino- and 4'-C-ethynyl-2'-deoxy-beta-D-ribo-pentofuranosylpyrimidine and -purine nucleosides were synthesized and evaluated for their in vitro anti-HIV activity. The key intermediate, 4-C-ethynyl- or 4-C-triethylsilylethynyl-D-ribo-pentofuranose, was prepared from D-glucose and glycosidated with various pyrimidine or purine bases. The arabinopyrimidine derivatives were prepared from the corresponding ribo derivatives via O(2),2'-anhydro nucleosides. The 2'-deoxy-ribo derivatives were synthesized by radical reduction of 2'-bromo or 2'- phenoxythiocarbonyloxy nucleosides. Among these 4'-C-ethynyl nucleosides, seven analogues proved to be potent against HIV-1 in vitro with EC(50) values ranging from 0.0003 to 0. 03 microM. These compounds also exerted activity against clinical and multi-dideoxy-nucleoside-resistant HIV-1 strains with comparable EC(50) values. Three such 4'-C-ethynyl-2'-deoxypurine analogues including 4'-C-ethynyl-2'-deoxyadenosine and 4'-C-ethynyl-2, 6-diamino-2'-deoxypurine were less cytotoxic [selectivity indices (SIs): 975-2733] than three 4'-C-ethynyl-2'-deoxycytidine analogues (SIs: 63-363). 4'-C-Ethynyl-5-fluoro-2'-deoxycytidine was least toxic (SI: >3333) and potent against all HIV strains tested.

Absolute configuration of a ceramide with a novel branched-chain fatty acid isolated from the epiphytic dinoflagellate, Coolia monotis.

The absolute configuration of the chiral center at the C15 position of a novel branched-chain fatty acid derived from a new ceramide isolated from the epiphytic dinoflagellate Coolia monotis was determined to be of R from by reversed-phase HPLC after cleavage to 12-methylpentadecanoic acid and subsequent conversion with the chiral fluorescent reagent, (1R,2R)-2-(2,3-anthracenedicarboximido)cyclohexanol.

Synthesis of 2-C-methyl-D-erythritol and 2-C-methyl-L-threitol; determination of the absolute configuration of 2-C-methyl-1,2,3,4-butanetetrol isolated.

2-C-Methyl-D-erythritol (A) and 2-C-methyl-L-threitol (B) were respectively synthesized from D-glucose and D-galactose. The 2-C-methyl-1,2,3,4-butanetetrol compound (C) recently isolated from Phlox sublata L was confirmed to be A by comparing the CD and 1H-NMR spectra of its tri-O-benzoate with those of A and B.

Practical synthesis of the disaccharide epitope, D-galactopyranosyl-alpha-1,3-D-galactopyranose, by using 1,2;5,6-di-O-cyclohexylidene-alpha-D-galactofuranose as the glycosyl acceptor.

D-Galactosyl-alpha-1,3-D-galactopyranose (1) was chemically prepared in a good yield by coupling phenyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-galactopyranoside (5) or 2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl bromide (8) with 1,2:5,6-di-O-cyclohexylidene-alpha-D-galactofuranose (3) with subsequent de-O-benzylation and de-O-cyclohexylidenation of the resulting protected alpha1,3-disaccharide.

Development of phosphine reagents for the high-performance liquid chromatographic-fluorometric determination of lipid hydroperoxides.

Phosphine reagents were designed and synthesized as a new type of fluorescent reagents for the determination of lipid hydroperoxides in foodstuff and biological materials. All phosphine reagents prepared had no fluorescence but their oxides, which were produced by the reaction of the phosphines with hydroperoxides, had strong fluorescence. Among the phosphine reagents prepared, diphenyl-1-pyrenylphosphine had the most suitable properties as a fluorescent reagent and was successfully applied to the determination of hydroperoxides by batch, flow injection and HPLC post-column methods.

Synthesis of 4'-ethynyl-purine nucleosides possessing anti-HIV activity.

Searching for more effective anti-HIV agents, we have prepared 4'-ethynyl-purine nucleosides. They were derived in several steps from 4-C-triethylsilylethynyl ribose, which was used as an intermediate in the synthesis of pyrimidine nucleosides. The adenine derivative exhibited significant anti-HIV activity and favorable cytotoxicity profile in vitro.

Synthesis of 4'-C-ethynyl-beta-D-arabino- and 4'-C-ethynyl-2'-deoxy-beta- D-ribo-pentofuranosyl pyrimidines, and their biological evaluation.

4'-C-Ethynyl-beta-D-arabino-pentofuranosyl thymine (14) and cytosine (16), and 4'-C-ethynyl-2'-deoxy-beta-D-ribo-pentofuranosyl thymine (25) and cytosine (27) were synthesized by properly protected 4'-C-hydroxy-methyl-3,5-di-O-benzyl-alpha-D-ribo-pentofuranose (1) from D-glucose. Among them, 2'-deoxy derivatives 25 and 27 exhibited antiviral activity, while cytidine derivatives 16 and 27 inhibited the growth of neoplastic cells.

Synthesis of novel 4'-C-methyl-pyrimidine nucleosides and their biological activities.

Two novel 4'-C-methylnucleosides, 4'-methylBVDU 9 and 4'-methylBVaraU 10, were synthesized. The former was derived from 3',5'-di-O-acetyl-2'-deoxy-4'-C-methyluridine 12, and the latter was produced via glycosylation between 4-C-methyl-D-ribose derivative 11 and a silylated bromovinyl uracil. 4'-MethylBVDU 9 exhibited particularly potent anti-varicella-zoster virus (VZV) activity in vitro.

Synthesis of 4'-substituted nucleosides and their biological evaluation.

4'-C-Ethynyl-beta-D-arabino- and 4'-C-ethynyl-beta-D-2'-deoxy-ribo-pentofuranosyl pyrimidines were synthesized. Most of these 4'-ethynyl nucleosides showed interesting biological activities.

Lipase-catalyzed Kinetic Resolution of (±)-trans- and cis-2-Azidocycloalkanols.

The lipase-catalyzed kinetic resolution of trans- and cis-2-azidocycloalkanols and the preparation of enantiomerically pure trans- and cis-2-aminocycloalkanols are described. Four kinds of lipases were screened for the acetylation of trans- and cis-2-azidocycloalkanols. Among them, Pseudomonas sp. lipases (lipase PS and lipase AK, Amamo Pharmaceutical Co.) showed the highest enantioselectivity. These products were converted to the corresponding 2-aminocycloalkanols to determine their enantiomeric excess (ee) and absolute configurations by HPLC and CD analyses, using (S)-TBMB carboxylic acid [(S)-2-tert-butyl-2-methyl-1,3-benzodioxole-4-carboxylic acid] as the chiral conversion reagent. The results of the CD analysis proved N,O-bis-(S)-TBMB carboxylated cis-2-aminocycloalkanols to adopt a predominantly N-equatorial conformation. The partially resolved trans- and cis-2-aminocycloalkanols, except for trans-2-aminocyclopentanol, were recrystallized from ethyl acetate to give enantiomerically pure forms.

Enatiomeric Separation of Branched Fatty Acids after Conversion with trans-2-(2,3-Anthracenedicarboximido)cyclohexanol, a Highly Sensitive Chiral Fluorescent Conversion Reagent.

(1R,2R)-2-(2,3-Anthracenedicarboximido)cyclohexanol was synthesized as a highly sensitive chiral fluorescent conversion reagent. The diastereomeric derivatives of chiral branched fatty acids that had methyl ethyl chirality from the 2 to 12 position were separated into 2 peaks by reversed-phase HPLC and detected at the 10(-15) mole level by fluorometry.

Synthesis of 4'-C-Ethynyl-ß-D-ribo-pentofuranosyl Pyrimidines.

The 4'-C-ethynyl-ß-D-ribo-pentofuranosylpyrimidines were prepared from D-glucose through properly protected 4'-C-formyl-D-ribo-ribofuranose as the key intermediate, and preliminary biological tests against some viruses and tumor cells showed that the compounds were not active.

Simultaneous Determination of Lipid Hydroperoxides by HPLC-Post Column Systems.

Two HPLC-post column systems, a conventional-size system and a semi-micro system, for the simultaneous determination of lipid hydroperoxides were developed. The hydroperoxides of free fatty acids, phosphatidylcholines, triacylglycerols and cholesterol esters were individually determined at the pmol level with good reproducibility by using gradient elution and post-column detection with diphenyl-1-pyrenylphosphine.