Kinetically controlled optical resolution of racemic norbornene aldehyde derivatives

A new method for obtaining optically pure 5-norbornene 2-endo-aldehyde derivatives was developed. The reaction of a diastereomeric mixture of the ene acetals 2 and 2', derived from racemic norbornene aldehydes (+/-)-1 and chiral nonracemic (S,S)-hydrobenzoin 7, with NBS (0.5-0.6 eq.) in the presence of H(2)O proceeded in a kinetically controlled manner to give the optically pure hydroxy aldehydes 3 along with the intact ene acetals 2'. Both compounds 3 and 2' were converted into the optically pure norbornene aldehydes 1 and ent-1, respectively. This method opens the way to produce various types of 5-norbornene 2-endo-aldehydes with 3-exo- or 3-endo-substituents in optically pure forms.     

Synthesis of nucleic acid methylphosphonothioates.

The reagent obtained in situ by treating methylphosphonothioic dichloride with 1-hydroxy-6-trifluoromethylbenzotriazole could be used for the introduction of methylphosphonothioate linkages. The individual diastereomers of the protected dimer d-Tp(S,Me)A were applied in the synthesis of the chiral pure (R or S) hexamers d-[CpCpTp(S,Me)ApGpG]. The reagent showed also to be very effective for the preparation of the 3',5'-cyclic methylphosphonothioate of uridine.     

Synthesis and separation of diastereoisomers of 5'-O-(4,4'-dimethoxytrimethylphenyl)dithymidyl-(3,5')-4,4' -dimethoxytriphenylmethanephosphonate

"In solution" synthesis and separation of diastereoisomers of 5'-O-(4,4'-dimethoxytriphenylmethyl)dithymidyl (3',5') 4,4'-dimethoxytriphenylmethanephosphonate (DMTTDMTT) is described. One of diastereoisomers was successfully crystallized and characterized by means of HPLC.     

A stereospecifically 18O-labelled deoxydinucleoside phosphate block for incorporation into an oligonucleotide

Fully protected diastereoisomers of deoxyguanylyl (3' leads to 5') deoxyadenosine stereospecifically labelled on phosphorus with oxygen-18 have been synthesized by oxidation of phosphite triester intermediates in the presence of 18O-labelled water. The diastereoisomers have been chromatographically separated and their absolute configuration at phosphorus determined. (Rp)-[18O]deoxyguanylyl (3' leads to 5')deoxyadenosine has been prepared by complete deprotection of the parent diastereoisomer of the Sp configuration. Methylation of the former compound permits assignment of the absolute configurations of the methyl esters of N1-methyldeoxyguanylyl (3' leads to 5') N1-methyldeoxyadenosine.     

Synthesis and relative stereochemistry of the benzylic thioether diastereoisomers formed from glutathione and styrene oxide

The chemical reaction between (±) styrene oxide and glutathione produces both the benzylic and primary thioether positional isomers as a mixture of diastereoisomers (2, 5 and 3, 6), with a preference for the benzylic thioether isomers (66 : 34). Synthesis of the styrene oxide-glutathione conjugates from either (+)- or (?)- styrene oxide produces both positional isomers as single diastereoisomers. The benzylic thioether isomers (2 and 5) were prepared from protected 2-bromo-2-phenylethanol (8) and glutathione and were separated using hplc. The relative stereochemistry of the benzylic thioether isomers was assigned on the basis of the established chemical correlation between the optically pure styrene oxides and their precursors, the mandelic acids, as well as considerations of the mechanism of ring opening of epoxides by sulfur nucleophiles. The availability of the single diastereoisomers of the benzylic thioether isomers and the styrene oxideglutathione conjugates enables investigations concerned with the influence of chirality on the biotransformation and excretion of these conjugates.     

Photochemical synthesis of novel dideoxynucleosides

A series of 2',3'-dideoxynucleosides based on the apiose family was prepared from photochemical ring-expansion of a common cyclobutanone precursor. The starting ketone, (+/-) 3-[2'-(benzoyloxy)ethyl]-2,2-dimethylcyclobutanone (12) was prepared from commercially available (+/-)alpha-pinene. Since the optically pure antipodes of alpha-pinene are also commercially available, these nucleosides can be prepared optically pure using the identical procedure.     

Backbone modified oligodeoxynucleotides: stereoselective synthesis of methylphosphonates and HIV inhibitory capacity of phosphorothioates.

Oligonucleotides bearing phosphorothioate linkages of the HIV tatIII splice acceptor site were synthesized by automated solid phase synthesis. Especially 5'- and 3'-end capped thioates of the sequence 5'-ACACCCAATTCTGAAAATGG-3' show microM inhibition of HIV replication. ODN-methylphosphonates of defined stereochemistry were obtained with suitably modified proline phosphonamidite derivatives as monomeric building blocks. Asymmetric induction for nucleosidephosphonamidates up to 5:1 (Rp:Sp rsp. Sp:Rp depending on configuration of proline-moiety) could be reached. The intermediate phosphonamidite can be further reacted to dinucleoside methylphosphonates of enriched diastereomeric excess.     

Synthesis of enantiomerically pure D-FDOC, an anti-HIV agent

The beta-D-enantiomer of FDOC (2',3'-dideoxy-5-fluoro-oxacytidine) exhibits potent anti-HIV-1 activity. It was obtained in optically pure form by employing a tandem kinetic resolution/chiral salt crystallization protocol. In addition, conditions were developed that allowed the unwanted butyrate ester of the L-enantiomer of FDOC to be racemized. This material could then be recycled in future resolutions.     

Preparation of oligodeoxyribonucleoside methylphosphonates on a polystyrene support.

An efficient procedure is described for synthesizing deoxyribonucleoside methylphosphonates on polystyrene polymer supports which involves condensing 5'-dimethoxytrityldeoxynucleoside 3'-methylphosphonates. The oligomers are removed from the support and the base protecting groups hydrolyzed by treatment with ethylenediamine in ethanol, which avoids hydrolysis of the methylphosphonate linkages. Two types of oligomers were synthesized: those containing only methylphosphonate linkages, d-Np(Np)nN, and those which terminate with a 5' nucleotide residue, dNp (Np)nN. The latter oligomers can be phosphorylated by polynucleotide kinase, and are separated by polyacrylamide gel electrophoresis according to their chain length. Piperdine randomly cleaves the oligomer methylphosphonate linkages and generates a series of shorter oligomers whose number corresponds to the length of the original oligomer. Apurinic sites introduced by acid treatment spontaneously hydrolyze to give oligomers which terminate with free 3' and 5' OH groups. These reactions may be used to characterize the oligomers.     

Preparative method for isosilybin isolation based on enzymatic kinetic resolution of silymarin mixture

The flavonolignan isosilybin (1) is one of the silybin congeners contained in the silymarin complex, which is isolated from the seeds of the milk thistle (Silybum marianum). A number of recent studies have demonstrated that isosilybin is probably the most potent anticancer agent found in silymarin. It occurs as a mixture of two diastereoisomers, A and B. Lipase Novozym 435 was found to allow the preparative production of both optically pure isosilybin A and B in a diastereoisomeric purity of over 95%. A preparatory method based on the enzymatic resolution and other purification methods has been developed, enabling multigram amounts of both optically pure isosilybins A (1a) and B (1b) to be obtained.     

Assignment of the absolute configuration of P-chiral 5' mRNA cap analogues containing phosphorothioate moiety

Enzymatic cleavage of the P-chiral diastereoisomers of the 5' mRNA cap analogue bearing phosphorothioate moiety in alfa position of 5',5'-triphosphate bridge (m(7)Gppp(S)G D1 and D2) was performed by human Decapping Scavenger (DcpS) enzyme. Analysis of the degradation products allowed to estimate the absolute configuration at the asymmetric phosphorus atoms in examined compounds via correlation with the R(P) and S(P) diastereoisomers of guanosine 5'-O-(1-thiodiphosphate) (GDPalphaS).     

Use of methylphosphonic dichloride for the synthesis of oligonucleoside methylphosphonates.

Methylphosphonic dichloride was used to prepare protected deoxyribonucleoside 3'-methylphosphonate beta-cyanoethyl esters, d-[(MeO)2Tr]NpCNEt, and protected oligonucleoside methylphosphonates in solution. Reaction of d-[(MeO)2Tr]N with methylphosphonic dichloride gives d-[(MeO)2Tr]NpCl. The phosphonylation and subsequent esterification or condensation reactions are each complete within 60 min. The products are readily purified by "flash chromatography" on silica gel columns. d-[(MeO)2Tr]NpCl, or its tetrazole derivative, d-[(MeO)2Tr]Nptet, were tested as intermediates for the synthesis of oligothymidine methylphosphonates on a silica gel polymer support. The average yield per coupling step was 76% and did not increase with addition of more d-[(MeO)2Tr]TpCl. The formation of (5'-5') linked thymidine dimers indicated that the thymidine monomers are clustered closely together on the support. When N is ibuG, the yield for the coupling step on the support is very low. This may be due to steric hindrance of the 3'-phosphonate group by the N-2 isobutryl protecting group.     

Sequence specific hybridization properties of methylphosphonate oligodeoxynucleotides.

Methylphosphonate oligodeoxynucleotides (MPO's) with isomerically pure Rp-configurated methylphosphonates (MP's) were synthesized by block coupling of ApT and TpA dinucleoside methylphosphonates (DMP's, p indicating MP-linkage). Oligonucleotide duplexes (20 mers) with these Rp-MP's showed almost the same melting temperatures (Tm) as those with phosphorodiester bonds. Further a dependence of the duplex stability from the nucleosides (bases) adjacent to the MP moiety was observed. For the first time thermodynamic parameters for the duplex to coil transition of isomerically pure MP's were determined from the concentration dependence of the Tm. CD-spectra of the duplexes show structural changes which can be associated with the transition to a compact helix with higher helix winding angle.     

Structure of hybrid backbone methylphosphonate DNA heteroduplexes: effect of R and S stereochemistry.

Methyl phosphonate oligonucleotides have been used as antisense and antigene agents. Substitution of a methyl group for oxygen in the phosphate ester backbone introduces a new chiral center. Significant differences in physical properties and hybridization abilities are observed between the R(p) and S(p) diastereomers. Chirally pure methylphosphonate deoxyribooligonucleotides were synthesized, and the solution structures of duplexes formed between a single strand heptanucleotide methylphosphonate, d(Cp(Me)Cp(Me)Ap(Me)Ap(Me)Ap(Me)Cp(Me)A), hybridized to a complementary octanucleotide, d(TpGpTpTpTpGpGpC), were studied by NMR spectroscopy. Stereochemistry at the methylphosphonate center for the heptanucleotide was either RpRpRpRpRpRp (R(p) stereoisomer) or RpRpRpSpRpRp (S(p) stereoisomer, although only one of the six methylphosphonate centers has the S(p) stereochemistry). The results show that the methylphosphonate strands in the heteroduplexes exhibit increased dynamics relative to the DNA strand. Substitution of one chiral center from R(p) to S(p) has a profound effect on the hybridization ability of the methylphosphonate strand. Sugars in the phosphodiester strand exhibit C(2)(') endo sugar puckering while the sugars in the methyl phosphonate strand exhibit an intermediate C(4)(') endo puckering. Bases are well stacked on each other throughout the duplex. The hybridization of the methylphosphonate strand does not perturb the structure of the complementary DNA strand in the hetero duplexes. The sugar residue 5' to the S(p) chiral center shows A-form sugar puckering, with a C(3)(')-endo conformation. Minor groove width in the R(p) stereoisomer is considerably wider, particularly at the R(p) vs S(p) site and is attributed to either steric interactions across the minor groove or poorer metal ion coordination within the minor groove.     

Large-scale separation of silybin diastereoisomers using lipases

The flavonolignan silybin (1), isolated from the seeds of milk thistle (Silybum marianum), occurs in nature as an equimolar mixture of two diastereoisomers, silybin A and silybin B, that exhibit different biological activities. The preparative production of optically pure silybin A and B in a diastereoisomeric purity greater than 95% was accomplished using immobilized Candida antarctica lipase B (Novozym 435) in a combination of two reactions: regioselective acetylation of a natural silybin mixture (1) and subsequent stereoselective alcoholysis of the resulting 23-O-acetylsilybin (2). Several grams of the optically pure substances can be produced within one week using this new, robust and scalable process, which is selective, mild and high-yielding.     

C15 and C17 Aldehydes,from Japanese Domestic Tobacco,Nicotiana tabacum cv. Suifu

Optical isomers of methyl jasmonate were resolved from a commercial mixture via the formation of bornyl jasmonate diastereoisomers. The symmetry and magnitude of the optical rotation spectra, and the observation that only one isomer, (+)-methyl epijasmonate had the characteristic methyl jasmonate odor indicated that these preparations were optically pure.