Convenient synthesis of a propargylated cyclic (3'-5') diguanylic acid and its "click" conjugation to a biotinylated azide

The ribonucleoside building block, N2-isobutyryl-2'-O-propargyl-3'-O-levulinyl guanosine, was prepared from commercial N2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-propargyl guanosine in a yield of 91%. The propargylated guanylyl(3'-5')guanosine phosphotriester was synthesized from the reaction of N2-isobutyryl-2'-O-propargyl-3'-O-levulinyl guanosine with N2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-3'-O-[(2-cyanoethyl)-N,N-diisopropylaminophosphinyl] guanosine and isolated in a yield of 88% after P(III) oxidation, 3'-/5'-deprotection, and purification. The propargylated guanylyl(3'-5')guanosine phosphotriester was phosphitylated using 2-cyanoethyl tetraisopropylphosphordiamidite and 1H-tetrazole and was followed by an in situ intramolecular cyclization to give a propargylated c-di-GMP triester, which was isolated in a yield of 40% after P(III) oxidation and purification. Complete N-deacylation of the guanine bases and removal of the 2-cyanoethyl phosphate protecting groups from the propargylated c-di-GMP triester were performed by treatment with aqueous ammonia at ambient temperature. The final 2'-desilylation reaction was effected by exposure to triethylammonium trihydrofluoride affording the desired propargylated c-di-GMP diester, the purity of which exceeded 95%. Biotinylation of the propargylated c-di-GMP diester was easily accomplished through its cycloaddition reaction with a biotinylated azide derivative under click conditions to produce the biotinylated c-di-GMP conjugate of interest in an isolated yield of 62%.     

Synthesis of 3'-thioamido-modified 3'-deoxythymidine-5'-triphosphates and their use as chain terminators in Sanger-DNA sequencing

The thioamide derivatives 3'-deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-[(2-methyl-1-thioxo- propyl)amino]thymidine 1 and 3'-deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-((6-([(9H-(fluo-ren-9- ylmethoxy)carbonyl]-amino)-1-thioxohexyl)amino) thymidine 2 were synthesized by regioselective thionation of their corresponding amides 7 and 8 with 2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide (Lawesson's reagent). The thioamides were converted into the corresponding 5'-triphosphates 3 and 4. Compound 3 was chosen for DNA sequencing experiments and 4 was further labelled with fluorescein.     

Synthesis of two 8-[(anthraquinone-2-yl)-linked]-2'-deoxyadenosine 3'-benzyl hydrogen phosphates for studies of photoinduced hole transport in DNA

The challenge in working with anthraquinone-2'-deoxyadenosine (AQ-dA) conjugates is that they are insoluble in water and only sparingly soluble in most organic solvents. However, water-soluble AQ-dA conjugates with short linkers are required for study of their electrochemical and intramolecular electron transfer properties in this solvent prior to their use in laser kinetics investigations of photoinduced hole (cation) transport in DNA. This article first describes the synthesis of a water-soluble, ethynyl-linked AQ-dA conjugate, 8-[(anthraquinone-2-yl)ethynyl]-2'-deoxyadenosine 3'-benzyl hydrogen phosphate, based on initial formation of a 5'-O-(4,4'-dimethoxytrityl) (5'-O-DMTr) intermediate. Because intended H2 over Pd/C reduction of the ethynyl linker in 5'-O-DMTr-protected 2'-deoxyadenosines cleaves the DMTr protecting group and precipitates multiple side products, this work also describes the synthesis of an ethylenyl-linked AQ-dA conjugate, 8-[2-(anthraquinone-2-yl)ethyl]-2'-deoxyadenosine 3'-benzyl hydrogen phosphate, starting with a 5'-O-tert-butyldiphenylsilyl protecting group.     

New reagents and methods for the synthesis of internal and 3'-labeled DNA

The syntheses of two new nucleoside phosphoramidites containing a hydroxyl functionality masked by a levulinate protecting group are presented; N(4)-(2-(ethylene glycol-2-levulinate)ethyl)-5-methyl-5'-(4,4'-dimethoxytrityl)-3'-O-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxycytidine 1 and 5-(N-(6-O-levulinoyl-1-aminohexyl)-3(E)-acrylamido)-5'-(4,4'-dimethoxytrityl)-3'-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxyuridine 3. Optimization of solid-phase-supported synthetic parameters for incorporation of these into DNA, removal of the levulinate group by exposure to dilute hydrazine, and subsequent attachment of dye labels is described. Synthesis of the known compound 5-(N-(6-trifluoroacetylaminohexyl)-3(E)-acrylamido)-5'-(4,4'-dimethoxytrityl)-3'-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxyuridine 2 (1), containing a masked amine at the end of an alkyl chain attached at the 5 position, was also revisited using new techniques developed for 3.     

Mung bean (Phaseolus aureus) nuclease. A mechanistic investigation of the DNA-cleavage reaction using a dinucleoside phosphorothioate.

Mung-bean (Phaseolus aureus) nuclease has been found to cleave the Sp diastereoisomer of 5'-O-thymidyl 3'-O-(2'-deoxyadenosyl)phosphorothioate, (Sp)-d[Ap(S)T], in 18O-labelled water with inversion of configuration at phosphorus to give (Sp)-thymidine 5'-[16O, 18O]phosphorothioate, the stereochemistry of which was deduced by methylation to (Rp,Sp)-thymidine 5'-S-methyl-O-methyl-[16O,18O]phosphorothioate and 31P-n.m.r. analysis. This result is consistent with a mechanism involving a direct 'in-line' attack of water on DNA for the nuclease-catalysed reaction without the involvement of a covalent nucleotidylated-enzyme intermediate.     

Endowing RNase H-inactive antisense with catalytic activity: 2-5A-morphants

A convergent synthetic approach was used to conjugate 2',5'-oligoadenylate (2-5A, p5'A2' [p5'A2'](n)()p5'A) to phosphorodiamidate morpholino oligomers (morphants). To provide requisite quantities of 2-5A starting material, commercially and readily available synthons for solid-phase synthesis were adapted for larger scale solution synthesis. Thus, the tetranucleotide 5'-phosphoryladenylyl(2'-->5')adenylyl(2'-->5')adenylyl(2'-->5')adenosine (p5'A2'p5'A2'](2)p5'A2', tetramer 2-5A, 9) was synthesized starting with 2',3'-O-dibenzoyl-N(6),N(6)-dibenzoyl adenosine prepared from commercially available 5'-O-(4-monomethoxytrityl) adenosine. Coupling with N(6)-benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-(tert-butyldimethylsilyl) adenosine-2'-(N,N-diisopropyl-2-cyanoethyl)phosphoramidite, followed by oxidization and deprotection, generated 5'-deprotected dimer 2-5A. Similar procedures lengthened the chain to form protected tetramer 2-5 A. The title product 9 p5'A(2'p5'A)(3) (tetramer 2-5A) was obtained through phosphorylation of the terminal 5'-hydroxy of the protected tetramer and removal of remaining protecting groups using concentrated ammonium hydroxide-ethanol (3:1, v/v) at 55 degrees C and tetrabutylammonium fluoride (TBAF) in THF at room temperature, respectively. The 2-5A-phosphorodiamidate morpholino antisense chimera 11 (2-5A-morphant) was synthesized by covalently linking an aminolinker-functionalized phosphorodiamidate morpholino oligomer with periodate oxidized 2-5A tetramer (p5'A2'[p5'A2'](2)p5'A). The resulting Schiff base was reduced with cyanoborohydride thereby transforming the ribose of the 2'-terminal nucleotide of 2-5A N-substituted morpholine. RNase L assays demonstrated that this novel 2-5A-antisense chimera had significant biological activity, thereby providing another potential tool for RNA ablation.     

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.     

Synthesis of 8-(2"-hydroxyethoxy)adenosine-5',2"-phosphate derivative

Reaction of 8-bromo-2',3'-O-isopropylidene-5'-O-(tetrahydropyran-2-yl) adenosine (Ib) with lithium 2-(tetrahydropyran-2-yloxy) ethoxide, followed by removal of the tetrahydropyran-2-yl groups, afforded 8-(2'-hydroxyethoxy)-2',3'-O-isopropylideneadenosine (II). Successive treatment of II with n-butyllithium and with methyl dichlorophosphate provided the 5',2'-(methyl phosphate) derivative (IIIa and IIIb).     

An approach to the stereoselective synthesis of Sp-dinucleoside phosphorothioates using phosphotriester chemistry.

An approach to the stereoselective synthesis of Sp- dinucleoside phosphorothioates has been investigated which utilizes phosphotriester chemistry. The stereoselectivity of internucleotide bond formation between N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine-3'-O-(S2-cyano-e thyl) phosphorothioate (3) and 3'-O-acetylthymidine has been studied using either mesitylenesulphonyl-5-(pyridin-2-yl)tetrazole (MSPy) or 1-mesitylenesulphonyl-3-nitro-1,2,4-triazole (MSNT) as the activating agent. The removal of the cyanoethyl group from the protected dinucleoside phosphorothioate has been studied, and conditions are reported which provide rapid deprotection without concomittant desulphurisation.     

Conformationally locked nucleosides. Synthesis of oligodeoxynucleotides containing 3'-amino-3'-deoxy-3'-N,5'(R)-C-ethylenethymidine

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.     

Synthesis and electrochemistry of anthraquinone-oligodeoxynucleotide conjugates

Electroactive oligodeoxynucleotides (ODNs) with specific base sequences have a potential application as electrical sensors for DNA molecules. To this end, a phosphoramidite that bears a 9, 10-anthraquinone (AQ) group tethered to the 2'-O of the uridine via a hexylamino linker, 2'-O-[6-[2-oxo(9, 10-anthraquinon-2-yl)amino]hexyl]-5'-O-(4,4'-dimethoxytrityl)uridi ne 3'-[2-(cyanoethyl)bis(1-methylethyl)phosphoramidite] (3), has been synthesized and used to prepare three ODNs with tethered AQs using standard phosphoramidite chemistry. The synthetic methodology thus allows the synthesis of ODNs with electroactive tags attached to given locations in the base sequence. Cyclic voltammetric behavior of these AQ-ODN conjugates was examined in aqueous buffer solutions at a hanging mercury drop electrode. At slow sweep rates, nearly reversible two-electron waves characteristic of an adsorbed anthraquinone/hydroquinone redox couple was observed for all of the AQ-ODN conjugates. Approximate Langmuirian isotherms were found for the AQ-ODNs with molecular footprints, calculated from the saturation coverages, that scaled with molecular size. The cyclic voltammetric response of the duplexes formed from the AQ-ODNs and their complementary ODN was complicated by the competitive adsorption of the individual ODNs and possibly the duplex species as well.     

Synthesis and enzymatic deprotection of fully protected 2'-5' oligoadenylates (2-5A): towards a prodrug strategy for short 2-5A

Fully protected pA2'p5'A2'p5'A trimers 1a and 1b have been prepared as prodrug candidates for a short 2'-5' oligoadenylate, 2-5A, and its 3'-O-Me analog, respectively. The kinetics of hog liver carboxyesterase (HLE)-triggered deprotection in HEPES buffer (pH?7.5) at 37° has been studied. The deprotection of 1a turned out to be very slow, and 2-5A never appeared in a fully deprotected form. By contrast, a considerable proportion of 1b was converted to the desired 2-5A trimer, although partial removal of the 3'-O-[(acetyloxy)methyl] group prior to exposure of the adjacent phosphodiester linkage resulted in 2',5'→3',5' phosphate migration and release of adenosine as side reactions.     

2',5'-Oligoadenylates chiral at phosphorus: enzymatic synthesis, properties, and biological activities of 2',5'-phosphorothioate trimer and tetramer analogues synthesized from (SP)-ATP alpha S

The enzymatic synthesis and characterization of (RP)-2',5'-AMPS trimer and tetramer (SP)-5'-O-(1-thiotriphosphates) from chirally substituted (SP)-[alpha-35S]ATP alpha S by 2',5'-oligoadenylate synthetase from interferon-treated L cell extracts are described. The (RP)-ATP alpha S isomer is not a substrate for the synthetase. The identification of the trimer and tetramer analogues (molar ratio 70:30) was accomplished by high-performance liquid chromatography and subsequent separation by charge using DEAE-cellulose thin-layer chromatography. The digestion of the analogue by snake venom phosphodiesterase I (SVPD) to [alpha-35S]ATP alpha S and [35S]AMPS but not by T2 RNase demonstrated the presence of the 2',5' linkage. The assignment of RP configuration of the 2',5'-phosphorothiodiester linkage was based on the highly specific stereoselectivity of SVPD for RP diastereomers [Burgers, P. M. J., & Eckstein, F. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4978-4800; Bryant, F. R., & Benkovic, S. J. (1979) Biochemistry 18, 2825-2828; Nelson, P. S., Bach, C. T., & Verheyden, J. P. H. (1984) J. Org. Chem. 49, 2314-2317]. This suggests that the synthesis of the phosphorothioate analogues proceeded via inversion of configuration at the chiral phosphorus of (SP)-ATP alpha S. The putative (RP)-2',5'-AMPS tetramer (SP)-5'-O-(1-thiotriphosphate) displaced the 2',5'-p3A4[32P]pCp analogue from 2',5'-oligoadenylate-dependent endonuclease 5 times more efficiently than did equimolar concentrations of authentic 2',5'-adenylate tetramer triphosphate. Furthermore, in studies using the calcium phosphate coprecipitation technique, the 2',5'-phosphorothioate trimer and tetramer analogues inhibited protein synthesis better than did 2',5'-adenylate trimer and tetramer triphosphates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Separation of [Sp]-3'-O-(5'-dimethoxytritylthymidyl)-5'-O-(3'camphanoyl thymidyl)-O-methyl phosphite.

First isolation of diastereoisomerically enriched (d.p. 95%) [Sp]-3'-O-(5'-dimethoxytrityl tjymidyl)-5'-O-(3'-camphanoyl thymidyl)-O-methyl phosphite (1) allowed to assign, via sulfuration and deprotection, its absolute configuration as Sp. Its reactions with methyl iodide, dimethoxytrityl chloride, or water, respectively, allowed to confirm correctness of assignment of absolute configuration in diastereisomers of TPMeT, and to assign absolute configuration in corresponding diastereoisomers of TPDMTT and TPHT.     

Synthesis and properties of 5'-triphosphoryl 2'-5' oligoadenylates (2-5A), and a general method for synthesis of 3', 5'-bisphosphorylated oligonucleotides.

2'-5'-Linked oligoadenylic acid 5'-triphosphates (2-5A) having chain lengths of 2-4 have been synthesized by polymerization of 3'-O-(o-nitrobenzyl)-N-benzoyladenosine 5'-phosphate followed by 5'-triphosphorylation via the imidazolidates. A large scale preparation of 5'-O-phosphoryladenylyl-(2'-5')-adenylyl-(2'-5')-adenosine was performed by the phosphotriester method using 5'-O-monomethoxytrityl-3'-O-(o-nitrobenzyl)-N-benzoyladenosine 2'-O-p-chlorophenylphosphate and 5'-O-phosphorodianilido-3'-O-(o-nitrobenzyl)-N-benzoyladenosine 2'-O-p-chlorophenylphosphate as intermediates. The trimer was also triphosphorylated by the imidazolide method. CD spectra for 5'-mono and triphosphorylated 2'-5' adenylates were measured as well as their UV hypochromicities. This triester method was also applied to the synthesis of 3',5'-bisphosphorylated protected oligoadenylic acids with natural 3'-5' linkages which could be used for further condensations to yield 5'-phosphorylated polynucleotides.     

Process development for the synthesis of 5'-O-(4,4'-dimethoxytrityl)-N2-isobutyryl-2'-O-(2-methoxyethyl)-guanosine--a potential precursor for the second generation antisense oligonucleotides: an improved process for the preparation of 2'-O-alkyl-2,6-diaminopurine riboside

An efficient four step process for the preparation of 5'-O-(4,4'-dimethoxytrityl)-N2-isobutyryl-2'-O-(2-methoxyethyl)-guanosine 1 was developed. Direct 2'-O-alkylation of 2,6-diaminopurine riboside 2 was accomplished via inexpensive and commercially available reagents such as KOH, DMSO and alkyl halides at room temperature in 4-6 hrs. Pure 2'-O-(2-methoxyethyl)-DAPR 3 was isolated by crystallization from methanol. Enzymatic deamination of 3 followed by selective N2-isobutyrylation and 5'-O-dimethoxytritylation furnished desired 1 in high yield and purity. Fully optimized four step synthetic process has been scaled up to the pilot plant level.     

Supported synthesis of ferrocene modified oligonucleotides as new electroactive DNA probes

The use of 1-[3-O-(2-cyanoethyl-N,N-diisopropylphosphor amidityl)propyl]ferrocene and 1-[3-O-dimethoxytrityl propyl]-1'-[3'-O-(2-cyanoethyl-N,N-diisopropylphosphoramidityl) propyl] ferrocene as reactive synthons for DNA/RNA synthesizer allows to generate ferrocene-labelled oligonucleotides with remarkable DNA detection properties.     

Dependence of properties of polyclonal antibodies to (2'-5')-oligoadenylates on the method of hapten binding to an immunogen

To elucidate the antibody-(2'-5')oligoadenylate relation to the mode of the hapten-immunogen conjugation, a new (2'-5')oligoadenylic acid trimer derivative containing a 2'-terminal N6-(5-carboxypentyl)adenosine and its 125I-labeled immunogenic conjugate were synthesized. The immunization with this conjugate and with a conjugate based on the 2',3'-O-[1-(2-carboxyethyl)]ethylidene derivative of the (2'-5')triadenylic acid gave antisera with different affinities toward modified (2'-5')oligonucleotides. Epitopes involved in the (2'-5')oligomer-binding to different antisera were found.     

Lipid derivatives of (2'-5')oligo A

The synthesis of adenylyl-(2'-5')-adenylyl-(2'-5')-2', 3'-O-(1-methoxyhexadecylidene)-adenosine (III) and its 5'-phosphorylated analogue (V) is described. Phosphorylation was achieved by (2-cyanoethyl)-phosphodichloridite agent followed by iodine oxidation.     

Reagent for introducing pyrene residues in oligonucleotides.

A novel pyrenyl-containing phosphoramidite reagent, N-[4-(1-pyrenyl)butyryl]-O1-(4,4'-dimethoxytrityl)-O2- [(diisopropylamino)(2-cyanoethoxy)phosphino]-3-amino-1 ,2-propanediol (5), has been synthesized from 4-(1-pyrenyl)butanoic acid in four steps with the 52% overall yield and used to incorporate pyrene residue(s) into oligonucleotides. Oligonucleotides 6 and 7, bearing one or two pyrenes at the 5'-terminus, have been prepared by means of that reagent, characterized with fluorescence spectra, and successfully used as primers in a polymerase chain reaction.