Oxidative transformations of nucleoside fluorenemethyl H-phosphonoselenoate diesters

9-Fluorenemethyl H-phosphonoselenoate monoester has been used to produce thymidine 3'-O-phosphoroselenoate monoester from which various P(V) derivatives containing multiple modifications at phosphorus were obtained; e.g., thymidine 3'-O-phosphoroselenofluoridate, 3'-O-phosphoroselenothioate, or 3'-O-phosphorodiselenoate monoesters.     

A new carotenoid glucoside ester from Chondromyces apiculatus

The carotenoid composition of the myxobacterium Chondromyces apiculatus is reported. A new acyclic carotenoid glucoside ester was isolated and its structure determined as 1′-glucosyloxy-3′,4′-didehydro-1′,2′-dihydro-ψ,ψ-carotene monoester.     

Optical purity of (3S,3'S)-astaxanthin from Haematococcus pluvialis

Haematococcus pluvialis cultivated in a N-deficient medium produced astaxanthin (1 % of total carotenoids), the monoester (76 %) and diester (7 %) of astaxanthin, β,β-carotene (1 %), an adonirubin ester (3 %), (3R,3′R,6′R)-lutein (7 %), violaxanthin (2 %) and neoxanthin (1 %). The CD values of the mono- and diesters of astaxanthin, the HPLC properties of astaxanthin monoester further esterified with (?)-camphanic acid and the optical purity of astaxanthin [determined by HPLC analysis of the diester of(?)-camphanic acid] produced by saponification of the natural mono- and diesters of astaxanthin in the absence of oxygen showed that this green alga synthesizes pure (3S,3′S)-astaxanthin esters.     

Synthesis of Thymidine Derivatives 3′-Modified with a Polar Three-Atom Group as Potential Anti-HIV-1 Agents

Abstract

3′-Modified thymidine analogs, such as 3′-O-[(methylthio)thio-carbonyl]thymidine (4), 3′-O-thiocarbamoylthymidine (7), and N-(3′-deoxy-thymidin-3′-y1)phosphoramidates (9a, b), were synthesized from thymidine derivatives (1), (5), and (8), respectively, as potential anti-human immunodeficiency virus type 1 (HIV-1) agents. No significant activity against HIV-1 was, however, observed with any of these compounds.     

Synthesis of High Quality Phosphorothioate Oligonucleotides as Antisense Drugs. Use of I-Linker in the Elimination of 3′-Terminal Phosphorothioate Monoesters

Abstract

Detritylation of a 5′-O-DMT-2′-deoxyadenosine moiety attached to solid support under acidic condition leads to depurination during oligonucleotide synthesis. Deprotection followed by reversed phase HPLC purification leads to desired oligonucleotide contaminated with significant levels of 3′-terminal phosphorothiaote (3′-TPT) monoester (n?1)-mer. However, it is demonstrated that attachment of dA nucleoside through its exocyclic amino group to solid support leads to substantial reduction of 3′-TPT formation thereby improving the quality of oligonucleotide synthesized.     

New Dinucleoside Analogues via Cross-Coupling Metathesis

Synthesis of 3′-3′, 5′-5′, and 3′-5′ dimeric thymidine, linked by an olefinic chain between glycosidic moieties is described. Cross metathesis reaction of 3′ or 5′ O-allyl analogues of thymidine led to the expected 3′-3′ and 5′-5′ dimeric compounds, respectively. In order to obtain the 3′-5′ dimer, 5′-O-allyl and 3′-O-allyl monomers were first linked by their free 3′ OH and 5′ OH groups through a glutaryl spacer; ring closing metathesis was then operated upon this temporary dimer, followed by glutaryl removal.     

Partial purification and characterization of thymidylate kinase from embryonic chick liver.

Thymidylate kinase from the livers of 18-day-old chick embryos was concentrated 423-fold. The purification procedure included acid precipitation, ammonium sulfate fractionation, gel filtration on Sephadex G-100 and G-75 Super Fine, and ion-exchange chromatography on Diethylaminoethyl Sephadex A-50. This enzyme was found to be very labile but could be stabilized for long periods of time by its substrate (thymidine 5′-monophosphate) in the presence of 2-mercaptoethanol. Enzymes responsible for the formation of thymidine 5′-diphosphate and thymidine 5′-triphosphate, respectively, were separated during fractionation procedures. Thymidylate kinase from chick embryo liver was found to be a single protein having a molecular weight of approximately 46,000, Michaelis constant approximately 8 × 10^?5m, and a broad pH optimum between 6.6 and 8.6. A 2–3 mm requirement of Mg²⁺ above the adenosine 5′-triphosphate concentration was shown to be necessary for maximum enzyme activity. The enzyme appears to be competitively inhibited by thymidine, thymidine 5′-diphosphate, and thymidine 5′-triphosphate and noncompetitively inhibited by adenosine 5′-diphosphate.Thymidylate kinase enzymes isolated from two stages of developing embryonic liver and adult chick liver were shown to be identical.     

Iodine Monochloride Facilitated Deglycosylation,Anomerization, and Isomerization of 3-Substituted Thymidine Analogues

The reaction of thymidine, 3-mono-, and 3,3′,5′-trialkylsubstitued thymidine analogues with iodine monochloride (ICl) was investigated. Treatment with ICl resulted in rapid deglycosylation, anomerization, and isomerization of thymidine and 3-substituted thymidine analogues under various reaction conditions leading to the formation of the nucleobases as the major products accompanied by minor formation of α-furanosidic-, α-pyranosidic-, and β-pyranosidic nucleosides. On the other hand, 3,3′,5′-trisubstitued thymidine analogues were only deglycosylated and anomerized. These results are similar to those observed for the acidic hydrolysis of the glycoside bond in nucleosides, but were presumably caused by the Lewis acid character of an iodine electrophile.     

Efficient Transformation of Thymidine into 2′,3′-Didehydro-2′,3′-Dideoxy-Thymidine (D4T) Involving Opening of a 2,3′-Anhydro Derivative by Phenylselenol

Abstract

A new, high-yielding method for introduction of the selenophenyl residue at the 3′-position of thymidine is reported. This reaction avoided any strongly basic or reductive reagent, thus allowing the use of benzoate ester as a protective group at O-5′. Further oxidation-elimination sequence followed by basic deprotection afforded 2′,3′-didehydro-2′,3′-dideoxythymidine (D4T) in 67.5% overall yield from thymidine.     

Synthesis and Antiviral Evaluation of 3′-Substituted Thymidine Analogues Derived from 3′-Amino-3′-deoxythymidine

Abstract

To assess the structure-activity relationship for antiviral activity, a series of 3′-deoxy-3′-N-functionalized thymidine analogues were synthesized. Several of these thymidine analogues show moderate in vitro activity against HIV-1 and HIV-2.     

A NEW APPROACH TO THE SYNTHESIS OF 4′-CARBON-SUBSTITUTED NUCLEOSIDES: DEVELOPMENT OF A HIGHLY ACTIVE ANTI-HIV AGENT 2′, 3′-DIDEHYDRO-3′-DEOXY-4′-ETHYNYLTHYMIDINE

Oxidation of 3′-O-TBDMS-4′,5′-unsaturated thymidine 3 with dimethyldioxirane (DMDO) allowed the isolation of the epoxide 4. Upon reacting with organosilicon reagents in the presence of SnCl₄, 4 underwent stereoselective ring opening to give 4′-α-allyl (6), 4′-α-(2-bromoallyl) (7), 4′-α-(cyclopenten-3-yl) (8), and 4′-α-cyano (9) derivatives of thymidine. Reactions of the 3′-epimer 12 with organoaluminum reagents gave 4′-α-methyl (13), 4′-α-vinyl (14), and 4′-α-ethynyl (15) analogues. Compounds 13–15 were transformed into corresponding 2′,3′-didehydro-3′-deoxy derivatives. Evaluation of their ability to inhibit the replication of HIV in cell culture showed that 4′-ethynyl-d4T (19) is more potent and less toxic than the parent compound d4T.     

New Efficient Synthesis of Thymidine Cyclic 3′, 5′- Phosphorofluoridate and Its Sulfur Analogue via the Phosphoroamidite Route

Abstract

We describe the convenient synthesis of thymidine cyclic 3′, 5′-phosphorofluoridate 6, which is superior to that previously reported. Our procedure is based on a sequence of reactions utilizing 3 as the key substrate. Similar sequence of reaction leads to the sulfur analogues of 6 the thymidine cyclic 3′, 5′-phosphorofluoridothioate 7.     

Conformationally Locked Nucleosides. Synthesis of Oligodeoxynucleotides Containing 3′-Amino-3′-deoxy-3′-N,5′(R)-C-ethylenethymidine

Abstract

3′-Amino-3′-deoxy-5′-O-(4,4′-dimethoxytrityl)-3′-N,5′(R)-C-ethylenethymidine (6) was synthesized starting from 3′-azido-3′-deoxythymidine. Condensation of 6 with 5′-O-(H-phosphonyl)thymidine and 5′-O-(p-nitrophenoxycarbonyl)thymidine derivatives gave dinucleotide and dinucleoside derivatives, respectively, which were incorporated into oligodeoxynucleotides (ODNs). Tm data of the modified ODNs are also presented.     

Methodology for the Synthesis of Dinucleoside Monophosphates Containing A 2′-Deoxy-3-Isoadenosine Unit: 3-iso-dApT and Tp(3-iso-dA)

Abstract

2′ -Deoxy-3-isoadenylyl(3′-5′)thymidine and thymidylyl-(3′-5′)-2′-deoxy-3-isoadenosine have been synthesized by mild protection/deprotection methodology that circumvents facile N3-Cl′ hydrolytic cleavage of the 2′-deoxy-3-isoadenosine moiety.

     

3′-3′,3′-5′ and 5′-5′ TpT-Amides: Synthesis,Characterization and Alkaline Hydrolysis

Abstract

A simple procedure is described for the preparation of the title compounds 1, 8 and 9. 3′-3′ or 3′-5′ or 5′-5′ TpT was reacted with a twofold molar excess of TPS in anhydrous DMF, at room temperature, for 5 min, followed by a 1 min in situ treatment of the reaction mixture with excess 7.0 N NH₄OH, at 0°C. The alkaline hydrolysis of 1, 8 and 9 proceeds without the assistance of 3′- and 5′-hydroxyl groups resulting in equimolar mixtures of thymidine (4) and thymidine 3′-phosphoramidate (6) (for the 3′-3′ isomer) or thymidine 5′-phosphoramidate (7) (for the 5′-5′ isomer) or 6 and 7 in equal quantities (for the 3′-5′ isomer).     

Synthesis,In Vitro Anti-HIV Activity,and Biological Stability of 5′-O-Myristoyl Analogue Derivatives of 3′-Fluoro-2′,3′-Dideoxythymidine (FLT) as Potential Bifunctional Prodrugs of FLT

Abstract

A group of 5′-O-myristoyl analogue derivatives of FLT (2) were evaluated as potential anti-HIV agents that were designed to serve as prodrugs to FLT. 3′-Fluoro-2′,3′-dideoxy-5′-O-(12-methoxydodecanoyl)thymidine (4) (EC₅₀ = 3.8 nM) and 3′-fluoro-2′,3′-dideoxy-5′-O-(12-azidododecanoyl)thymidine (8) (EC₅₀ = 2.8 nM) were the most effective anti-HIV-1 agents. There was a linear correlation between Log P and HPLC Log retention time for the 5 ′-O-FLT esters. The in vitro enzymatic hydrolysis half-life (t½), among the group of esters (3–8) in porcine liver esterase, rat plasma and rat brain homogenate was longer for 3′-fluoro-2′,3′-dideoxy-5 ′-O-(myristoyl)thymidine (7), with t½ values of 20.3, 4.6 and 17.5 min, respectively.     

Oligo(2′-<Emphasis Type="Italic">O</Emphasis>-methylribonucleotides) and their derivatives: III. 5′-Mono- and 5′-bispyrenyl derivatives of oligo(2′-<Emphasis Type="Italic">O</Emphasis>-methylribonucleotides) and their 3′-modified analogues: Synthesis and properties

5′-Pyrenylmethylphosphamide and 5′-bispyrenylmethylphosphordiamide derivatives of oligo(2′-O-methylribonucleotides) and their analogues with thymidine attached at their 3′-termini by a 3′-3′-phosphodiester internucleotide bond (“inverted” thymidine) were synthesized. The effect of the pyrene residue(s) on the thermal stability of duplexes of the modified oligonucleotides with RNA and DNA was studied. A possibility of detection of hybridization of 5′-mono- and 5′-bispyrenyl derivatives with RNA and DNA targets in solution was demonstrated according to the changes in fluorescence. 5′-Pyrenylphosphamide derivatives of oligo(2′-O-methylribonucleotides) and their inverted analogues were shown to be used as sensitive probes for the detection of single nucleotide polymorphisms in RNA and DNA by the method of thermal duplex denaturation with fluorescence change registration.     

DNA synthesis in cultured human fibroblasts: Regulation by 3′ : 5′-cyclic amp

The addition of serum to density-inhibited human fibroblast cultures induced a wave of DNA synthesis, measured as [³H] thymidine incorporation into acid-precipitable material, beginning after 8–12 hr and reaching maximum levels at 16–24 hr. Addition of dibutyryl-3′ : 5′-cyclic AMP (DBcAMP) together with serum inhibited [³H] thymidine incorporation by 75–95%. When DBcAMP was added for the first 4 hr of serum stimulation and then removed, the wave of DNA synthesis was not delayed. This suggested that serum could induce DNA synthesis even though cyclic AMP concentrations were maintained at high levels by DBcAMP during this initial period. These results are inconsistent with the hypothesis that it is the immediate transient reduction in 3′ : 5′-cyclic AMP concentration following the addition of serum that triggers DNA synthesis. By contrast, DBcAMP added 8 hr after serum inhibited [³H] thymidine incorporation to the same extent as DBcAMP added at the same time as serum. This indicated that a step essential for DNA synthesis and occurring late in G₁ was inhibited by high concentrations of 3′ : 5′-cyclic AMP.     

Isolation of 3-(2-carboxyethyl)thymine following in vitro reaction of β-propiolactone with calf thymus DNA

3-(2-Carboxyethyl)thymine (3-CET) was synthesized from β-propiolactone (BPL) and dThd5′P at pH 9.0–9.5 via the intermediate 3-(2-carboxyethyl)thymidine-5′-monophosphoric acid (3-CEdThd5′P). 3-CEdThd5′P was converted to 3-CET by hydrolysis in 1.5 N HCl at 100°C for 2 h. The structure of 3-CET was assigned on the basis of UV spectra, electron impact (EI) and isobutane chemical ionization mass spectra and the EI mass spectrum of a trimethylsilyl derivative of 3-CET. BPL was reacted in vitro with calf thymus DNA at pH 7.5. 100 A units of BPL-reacted DNA yielded, following perchloric acid hydrolysis and preparative paper chromatography, 3 A units of 3-CET. Reaction of BPL with the phosphodiester thymidylyl-(3′-5′)thymidine gave 3-(2-carboxyethyl)thymidylyl-(3′-5′)-3-(2-carboxyethyl)thymidine (～3%). Phosphotriester formation was not detected.