High-throughput five minute microwave accelerated glycosylation approach to the synthesis of nucleoside libraries

The Vorbrüggen glycosylation reaction was adapted into a one-step 5 min/130 degrees C microwave assisted reaction. Triethanolamine in acetontrile containing 2% water was determined to be optimal for the neutralization of trimethylsilyl triflate allowing for direct MPLC purification of the reaction mixture. When coupled with a NH3/methanol deprotection reaction, a high-throughput method of nucleoside library synthesis was enabled. The method was demonstrated by examining the ribosylation of 48 nitrogen containing heteroaromatic bases that included 25 purines, four pyrazolopyrimidines, two 8-azapurines, one 2-azapurine, two imidazopyridines, two benzimidazoles, three imidazoles, three 1,2,4-triazoles, two pyrimidines, two 3-deazapyrimidines, one quinazolinedione, and one alloxazine. Of these, 32 yielded single regioisomer products, and six resulted in separable mixtures. Seven examples provided inseparable regioisomer mixtures of -two to three compounds (16 nucleosides), and three examples failed to yield isolable products. For the 45 single isomers isolated, the average two-step overall yield +/- SD was 26 +/- 16%, and the average purity +/- SD was 95 +/- 6%. A total of 58 different nucleosides were prepared of which 15 had not previously been accessed directly from glycosylation/deprotection of a readily available base.     

Pyrrolo-dC oligonucleotides bearing alkynyl side chains with terminal triple bonds: synthesis, base pairing and fluorescent dye conjugates prepared by the azide-alkyne "click" reaction

5-(Octa-1,7-diynyl)-2'-deoxyuridine was converted into the furano-dU derivative 7 by copper-catalyzed cyclization; the pyrolodC-derivative 3 was formed upon ammonolysis. The bicyclic nucleosides 3 and 7 as well as the corresponding non-cyclic precursors 4 and 6 all containing terminal C[triple bond]C bonds were conjugated with the non-fluorescent 3-azido-7-hydroxycoumarin 5 employing the copper(I)-catalyzed Huisgen-Sharpless-Meldal cycloaddition "click reaction". Strongly fluorescent 1H-1,2,3-triazole conjugates (30-33) are formed incorporating two fluorescent reporters-the pyrdC nucleoside and the coumarin moiety. Oligonucleotides incorporating 6-alkynyl and 6-alkyl 7H-pyrrolo[2,3-d]pyrimidin-2(3H)-one nucleosides (3 and 2f) have been prepared by solid-phase synthesis using the phosphoramidite building blocks 10 and 13 ; the pyrrolo-dC oligonucleotides are formed during ammonia treatment. The duplex stability of oligonucleotides containing 3 and related derivatives was studied. Oligonucleotides with terminal triple bonded nucleosides such as 3 are more stabilizing than those lacking a side chain with terminal unsaturation; open-chain derivatives (4) are even more efficient. The click reaction was also performed on oligonucleotides containing the pyrdC-derivative and the fluorescence properties of nucleosides, oligonucleotides and their coumarin conjugates were studied.     

Synthesis of Novel Nucleic Acid Mimics via the Stereoselective Intermolecular Radical Coupling of 3'-Iodo Nucleosides and Formaldoximes(1)

A highly convergent free radical coupling of alkyl iodides and oximes, mediated by bis(trimethylstannyl) benzopinacolate (8), has been utilized to prepare a series of dimeric nucleosides as mimics of natural nucleic acids. The systematic optimization of the reaction conditions allowed for the single-step conversion of the appropriate iodides and oximes into the 2'-deoxy dimers 9 in moderate to excellent yields. For example, the reaction of 3'-deoxy-3'-iodo-5'-(triphenylmethyl)thymidine (6a) with 3'-O-(tert-butyldiphenylsilyl)-5'-O-(methyleneimino)thymidine (7a) in the presence of 8 in degassed benzene gave an 81% yield of 3'-de(oxyphosphinico)-3'-(methyleneimino)-5'-O-(triphenylmethyl)thymidylyl-(3'-->5')-3'-O-(tert-butyldiphenylsilyl)thymidine (9a). Similarly prepared were dimers containing both pyrimidine (thymine, 5-methylcytosine) and purine (adenine, guanine) bases. The reaction was highly stereoselective, giving only a single dimeric species having the ribo-configuration of the newly introduced C-3'-branched methylene moiety. Also prepared were dimers 16, incorporating 2'-O-methyl ribonucleosides in both halves of the dimer. This required the synthesis of 3'-deoxy-3'-iodo-2'-O-methyl nucleosides 12 as well as 2'-O-methyl-5'-O-methyleneimino nucleosides 15. For example, 5'-O-(tert-butyldiphenylsilyl)-3'-deoxy-3'-iodo-2'-O-methyl-5-methyluridine (12e) was prepared in 80% yield by displacement of the corresponding triflate with Bu(4)NI. Also prepared were the suitably protected 3'-deoxy-3'-iodo adenosine and guanosine derivatives. Compounds 15 were prepared in high yield by a regioselective Mitsunobu reaction to give the corresponding 5'-O-phthalimido nucleosides 13, which were subsequently converted to the requisite oximes 15. In the 2'-O-methyl series, the pinacolate coupling reaction proceeded with efficiency equal to that observed for the 2'-deoxy series 9, but with slightly less stereoselectivity, giving predominantly the C-3'ribo products 16, contaminated with 5-25% of the epimeric material. Mixed base dimers containing both pyrimidine and purine bases at all possible positions, including purine-purine dimers were prepared. The hydroxylamine or methyleneimino (MI) backbone of several representative dimers so prepared was converted via methylation to give the corresponding methylenemethylimino (MMI)-linked compounds, which are novel phosphate surrogates for use in antisense oligonucleotides.     

Synthesis of pCpCpA-3'-NH-phenylalanine as a ribosomal substrate

[reaction: see text] The trinucleotide cytidylyl(3'-->5'phosphoryl)cytidylyl(3'-->5'phosphoryl)-3'-deoxy-3'-(L-phenylalanyl) amido adenosine (CpCpA-NH-Phe) was synthesized by phosphoramidite chemistry from 3'-amino-3'-deoxyadenosine as the ribosomal substrate. The 3'-amino-3'-deoxyadenosine was first converted to 3'-(N-tert-butyloxycarbonyl-L-phenylalanine)amido-3'-deoxy-6-N,6-N,2'-O-tribenzoyl-adenosine and then coupled with cytidine phosphoramidite to produce the fully protected CpCpA-NH-Phe-Boc. The title product was obtained after removing all protection groups and then radiolabeled with (32)P to yield pCpCpA-NH-Phe, which demonstrated high activity for the peptidyl transferase reaction in the ribosome.     

Synthesis of the ribosomal P-site substrate CCA-pcb

[reaction: see text] CCA-pcb (cytidylyl-(3'5')-cytidylyl-(3'5')-3'(2')-O-(N-(6-D-(+)-biotinoylaminohexanoyl)-L-phenylalanyl)adenosine), a ribosomal P-site substrate, was synthesized by phosphoramidite chemistry in 26 steps with an overall yield of 18%, starting from biotin. The synthesis relies on the judicious selection of orthogonal silyl protecting groups for the 5'-hydroxyls and acid-labile protecting groups (DMTr, AcE, and MeE) at other reactive sites to ensure the intactness of the labile ester. Both 3'-esterification and nucleotide coupling were accomplished by in situ activation with imidazolium ions.     

An improved phosphoramidite approach for the chemical synthesis of 3′,5′-cyclic diguanylic acid

3′,5′-Cyclic diguanylic acid (c-di-GMP) plays important roles as a signaling and effector molecule in prokaryotes as well as inducing innate and adaptive immune responses in mammalian cells through activation of cell death pathways. An improved phosphoramidite method for the synthesis of c-di-GMP is reported herein. The method is based on the use of an unprecedented 5′-O-formyl ester, which can efficiently and chemoselectively be cleaved from a dinucleotide phosphoramidite intermediate to permit a 1H-tetrazole-mediated cyclocondensation reaction leading to a fully protected c-di-GMP product in a yield of 78%. The native c-di-GMP is isolated in an overall yield of 36% based on the commercial ribonucleoside used as starting material.     

铱催化的MBH乙酸酯与吲哚酮类化合物的烯丙基化反应

研究了一种新的铱催化的Morita-Baylis-Hillman(MBH)乙酸酯与3-苯基取代的吲哚酮的烯丙基烷基化反应, 发现铱催化的区域选择明显不同于钯催化的反应, 直接、 高效地合成了一类未见报道的具有新结构的 3,3-二取代的吲哚酮类化合物. 通过对亚磷酰胺、 双膦及单膦等配体、 金属源、 溶剂、 碱以及反应温度的筛选, 获得了适合该反应的催化体系及最优条件: 以[Ir(COD)Cl]₂(摩尔分数5%)和亚磷酰胺配体(L6, 摩尔分数10%)为催化剂, CH₃CN为溶剂, Cs₂CO₃为碱, 于?30 ℃反应25 h. 在最优条件下, 对不同类型取代基的底物进行了考察, 发现底物普适性良好, 产率最低为84%, 最高可达98%. 同时还发现, 底物取代基的电性对反应产率影响不大, 一些其它类型的双膦和单膦配体对催化反应也有较好的催化效果.     

Determination of aliphatic amines in air by on-line solid-phase derivatization with HPLC-UV/FL

An easy, rapid, and efficient method using on-line solid-phase derivatization in HPLC is developed for the trace determination of aliphatic amines in air. Some fundamental studies on stop-flow, on-line, solid-phase derivatizations in HPLC are also investigated, such as optimization of the reaction detection HPLC system and band broadening. Air is sampled with silica gel tubes from different sites, including sewage areas, fish cleaning and processing rooms, and an organoleptic lab of the U.S. Food & Drug Administration (FDA). The trapped amines are desorbed with an acidic aqueous-organic solution, followed by pH adjustment of the eluates to pH 10. The resulting solution is directly injected into an on-line, precolumn, solid-phase derivatization and reversed-phase HPLC-UV/FL system, not requiring any further sample workup steps. The percent derivatizations are as high as 88 +/- 5% (n = 3) for primary amines, and 75 +/- 4% (n = 3) for diethylamine under optimized conditions (60 degrees C for 10 min). The recoveries for all amines are above 90%. The method is validated by a single-blind, spiked experiment with 1.1-4.4% relative standard deviation (RSD) in the range of 15-47 ppm. These results are confirmed by a GC-FID method performed in another lab. Amines are quantitated via calibration plots, with final concentrations from 0.02 to 0.38 mg/m3 air. It is suggested that this newer approach for the determination of amines and polyamines, using polymeric solid-phase reagents on-line, precolumn in HPLC, should prove generally successful for other amines and other sample types in the future.     

Syntheses and base-pairing properties of locked nucleic acid nucleotides containing hypoxanthine, 2,6-diaminopurine, and 2-aminopurine nucleobases

Second generation 2'-O,4'-C-methylene-linked nucleotides 1-3 containing hypoxanthine, 2,6-diaminopurine, and 2-aminopurine nucleobases were synthesized and incorporated into locked nucleic acid (LNA) oligonucleotides by means of the automated phosphoramidite method. The required phosphoramidite monomeric units were efficiently prepared via convergent synthesis. The glycosyl donor 4 was stereoselectively coupled with hypoxanthine and 6-chloro-2-aminopurine to give the 4'-C-branched nucleosides 5 and 17. The methods for conversion of 5 and 17 into phosphoramidites 11, 25, and 29 were developed and described in full details for the first time. Hybridization properties of LNA octamers containing the new LNA nucleotides were assessed against perfect and singly mismatched DNA. The binding studies revealed that all LNA octamers hybridize very efficiently to DNA following Watson-Crick base-pairing rules with increased binding affinity compared to the DNA analogues. The unique properties of the nucleotides 1-3 make them very useful for further strengthening of the LNA technology.     

Highly efficient syntheses of [methyl-11C]thymidine and its analogue 4'-[methyl-11C]thiothymidine as nucleoside PET probes for cancer cell proliferation by Pd(0)-mediated rapid C-[11C]methylation

Pd(0)-mediated rapid couplings of CH(3)I (and then [(11)C]CH(3)I) with excess 5-tributylstannyl-2'-deoxyuridine and -4'-thio-2'-deoxyuridine were investigated for the syntheses of [methyl-(11)C]thymidine and its stable analogue, 4'-[methyl-(11)C]thiothymidine as PET probes for cancer diagnosis. The previously reported conditions were attempted using Pd(2)(dba)(3)/P(o-CH(3)C(6)H(4))(3) (1?:?4 in molar ratio) at 130 °C for 5 min in DMF, giving desired products only in 32 and 30% yields. Therefore, we adapted the current reaction conditions developed in our laboratory for heteroaromatic compounds. The reaction using CH(3)I/stannane/Pd(2)(dba)(3)/P(o-CH(3)C(6)H(4))(3)/CuCl/K(2)CO(3) (1?:?25?:?1?:?32?:?2?:?5) at 80 °C gave thymidine in 85% yield. Whereas, CH(3)I/stannane/Pd(2)(dba)(3)/P(o-CH(3)C(6)H(4))(3)/CuBr/CsF (1?:?25?:?1?:?32?:?2?:?5) including another CuBr/CsF system promoted the reaction at a milder temperature (60 °C), giving thymidine in 100% yield. Chemo-response of thiothymidine-precursor was different from thymidine system. Thus, the above optimized conditions including CuBr/CsF system gave 4'-thiothymidine only in 40% yield. The reaction using 5-fold amount of CuBr/CsF at 80 °C gave much higher yield (83%), but unexpectedly, the reaction was accompanied by a considerable amount of undesired destannylated product. Such destannylation was greatly suppressed by changing to a CuCl/K(2)CO(3) system using CH(3)I/stannane/Pd(2)(dba)(3)/P(o-CH(3)C(6)H(4))(3)/CuCl/K(2)CO(3) (1?:?25?:?1?:?32?:?2?:?5) at 80 °C, giving the 4'-thiothymidine in 98% yield. The each optimized conditions were successfully applied to the syntheses of the corresponding PET probes in 87 and 93% HPLC analytical yields. [(11)C]Compounds were isolated by preparative HPLC after the reaction conducted under slightly improved conditions, exhibiting sufficient radioactivity of 3.7-3.8 GBq and specific radioactivity of 89-200 GBq μmol(-1) with radiochemical purity of ≥99.5% for animal and human PET studies.     

Nucleotides. Part XLVI. The synthesis of phospholipid conjugates of antivirally active nucleosides by the improved phosphoramidite methodology

The application of the improved phosphoramidite strategy for the synthese of oligonucleotides using β-eliminating protecting groups to phospholipid chemistry offers the possibility to synthesize phospholipid conjugates of AZT ( 6 ) and cordycepin. The synthesis of 3′-azido-3′-deoxythymidine ( 6 ) was achieved by a new isolation procedure without chromatographic purification steps in an overall yield of 50%. Protected cordycepin ( = 3′-de-oxyadenosine) derivatives, the N⁶,2′-bis[2-(4-nitrophenyl)ethoxycarbonyl]cordycepin ( 12 ) and the N⁶,5′-bis[2-(4-nitrophenyl)ethoxycarbonyl]cordycepin ( 13 ) wre prepared by known methods and direct acylation of N⁶-[2-(4-nitrophenyl)ethoxycarbonyl]cordycepin ( 9 ), respectively. These protected nucleosides and the 3′-azido-3′-de-oxythymidine ( 6 ) reacted with newly synthesized and properly characterized lipid-phosphoramidites 21–25 , catalyzed by 1H-tetrazole, to the corresponding nucleoside-phospholipid conjugates 26–38 in high yield. The deprotection was accomplished via β-elimination with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in aprotic solvents to give analytically pure nucleoside-phospholipid diesters 39–51 as triethylammonium or sodium salts. The newly synthesized compounds were characterized by elemental analyses and UV and ¹H-NMR spectra.     

Preparation, identification and analysis of stereoisomeric anti-benzo[a]pyrene diol epoxide-deoxyguanosine adducts using phenyl liquid chromatography with diode array, fluorescence and tandem mass spectrometry detection

Benzo[a]pyrene, a common environmental pollutant, can be metabolized into reactive anti-benzo[a]pyrene diol epoxide (anti-BPDE), which predominantly binds to deoxyguanine in DNA and forms four stereoisomeric adducts. To characterize the stereochemistry of these adduct isomers, preparation of single adducted deoxyguanosine (dG) is required for efficient enantiomeric analysis. Here, we demonstrate an improved method for preparation, identification, and analysis of four BPDE-adducted dGs, including (+)-trans-, (-)-trans-, (+)-cis-, and (-)-cis-anti-BPDE-N(2)-dG. These stereoisomerically adducted nucleosides were first synthesized by a direct reaction of (+/-)-anti-BPDE with dG, followed by optimized solid-phase extraction (SPE) and HPLC purification. The reaction of (+/-)-anti-BPDE and dG displayed a yield as high as 45%. The developed preparation method does not require any enzymatic digestion. Based on highly efficient separation achieved by optimization of stationary phase and mobile phase, LC-UV-MS/MS and LC-diode array detection (DAD)-fluorescence detection (FL) methods were established for characterization and analysis of the four stereoisomeric anti-BPDE-dGs. The established LC-DAD-FL method may provide characterization and analysis of four stereoisomeric anti-BPDE-dGs and two interfering anti-BPDE tetrols by taking advantage of their distinct fluorescence quenching.     

Determination of aromatic and branched-chain amino acids in plasma by HPLC with electrogenerated Ru(bpy)3(3+) chemiluminescence detection

An HPLC method is described for the electrochemiluminescence (ECL) detection of amino acids, following cycloaddition reaction of their amino groups with divinyl sulfone (DVS), using electrogenerated tris(bipyridine)ruthenium(III). The derivatization reaction conditions were examined, with the optimum conditions found to be 40 mM DVS (pH 8.0) at 50 degrees C for 15 min. Detection limits for the 15 amino acids examined varied greatly (0.04-8.0 pmol) using a standard solution by flow injection analysis (FIA). These optimized conditions were used for HPLC determination of the amino acids in human plasma. A linear relationship was obtained up to 100 pmol on a column for aromatic and branched-chain amino acids. Recoveries of Tyr, Met, Val, Leu, Ile, Phe and Trp when added to human plasma (1 micromol/10 ml plasma, n=5) were 101.5+/-1.1, 99.0+/-1.2, 98.0+/-1.4, 101.1+/-1.6, 95.1+/-1.6, 99.2+/-1.5 and 97.7+/-1.3 % (mean+/-S.D.) respectively. The concentrations of the amino acids in the plasma are in good agreement with other published data.     

Oligonucleotide duplexes containing N8-glycosylated 8-aza-7-deazaguanine and self-assembly of 8-aza-7-deazapurines on the nucleoside and the oligomeric level

The 8-aza-7-deazaguanine N8-(2'-deoxy-beta-D-ribofuranoside) (1) was synthesized, converted into the phosphoramidite 4 and incorporated into oligonucleotides. Nucleoside 1 forms stable base pairs with 2'-deoxy-5-methylisocytidine in DNA with antiparallel chain orientation (aps) and with 2'-deoxycytidine in duplexes with parallel chains (ps). According to the CD spectra self-complementary oligonucleotides d(1-m5isoC)3 and d(1-C), form autonomous DNA-structures. Neither the nucleoside 1 nor the regularly linked 8-aza-7-deaza-2'-deoxyguanosine form G-like tetrads while the regularly linked 8-aza-7-deaza-2'-deoxyisoguanosine gives higher molecular assemblies which are destroyed by bulky 7-bromo substituents. This was verified on monomeric nucleosides by ESI-MS spectrometry and on oligonucleotides by HPLC analysis.     

Study by means of high-performance liquid chromatography of solutes that decrease theophylline/protein binding in the serum of uremic patients.

Substantial changes in protein binding of drugs occur during the progression of renal insufficiency. Protein-bound uremic solutes play a role in the inhibition of drug protein binding. We previously demonstrated that hippuric acid in uremic ultrafiltrate was an inhibitor of the theophylline protein binding. The present study was undertaken to extend the yield of protein-bound uremic solutes by displacing ligands in uremic serum from their binding sites by five deproteinization methods. The inhibitory effect on theophylline protein binding of the deproteinized uremic serum was higher than with ultrafiltrate (p < 0.05). The influence of 30 semi-preparative HPLC fractions from deproteinized uremic serum on the theophylline protein binding was evaluated to identify the responsible compounds and to compare their relative individual impact. The theophylline protein binding was calculated as a percentage (bound versus total). The most important decrease of the protein binding was observed in HPLC fractions 6, 10 to 13, 15 and 28 with protein binding of: 61.5 +/- 10.8, 64.5 +/- 7.6, 60.9 +/- 10.1, 47.5 +/- 3.3, 60.0 +/- 6.7, 60.7 +/- 6.3 and 61.3 +/- 6.9%, respectively versus 69.1 +/- 2.4% for control serum (p < 0.05). The responsible compounds were characterized in the fractions by co-elution: 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), indole-3-acetic acid, indoxyl sulfate, hippuric acid, p-hydroxyhippuric acid and tryptophan. Their concentration was determined by analytical HPLC and a solution containing these compounds at the same concentration as in deproteinized uremic serum was composed. This solution was added to control serum and decreased the theophylline protein binding from 69.0 +/- 4.4% to 61.3 +/- 1.3%, which was less important than in genuine uremic serum (44.4 +/- 3.8%, p < 0.05). Dose-response curves with the characterized compounds revealed that the most important role in binding inhibition could be attributed to hippuric acid and CMPF. Our data suggests that the yield of protein binding inhibiting compounds is more important with deproteinized uremic serum than with uremic ultrafiltrate. The identified uremic compounds are not entirely representative for the decreased protein binding of theophylline, indicating that additional factors than those identified in this study affect the protein binding as well.     

O6-(benzotriazol-1-yl)inosine derivatives: easily synthesized, reactive nucleosides

A novel class of O6-(benzotriazol-1-yl)inosine as well as the corresponding 2'-deoxy derivatives can be conveniently prepared by a reaction between sugar-protected or -unprotected inosine or 2'-deoxyinosine nucleosides and 1H-benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP). The reaction appears to proceed via a nucleoside phosphonium salt, and in the absence of any additional nucleophile, the released 1-hydroxybenzotriazole undergoes reaction with the formed phosphonium salt leading to the requisite O6-(benzotriazol-1-yl)inosine or 2'-deoxyinosine derivatives. Isolation and characterization of the phosphonium salt as well as analysis by 31P{1H} NMR appear to be consistent with this reaction pathway. The resulting O6-(benzotriazol-1-yl)inosine derivatives are effective as electrophilic nucleosides, undergoing facile reactions with a variety of nucleophiles such as alcohols, phenols, amines, and a thiol. Unusual and challenging nucleoside derivatives such as an aryl-bridged dimer, a nucleoside-amino acid conjugate, and a nucleoside-nucleoside dimer have also been synthesized from the O6-(benzotriazol-1-yl)-2'-deoxyinosine derivative. Finally, a fully protected DNA building block, the O6-(benzotriazol-1-yl)-2'-deoxyinosine 5'-O-DMT 3'-O-phosphoramidite, has been prepared and a preliminary evaluation of its use for DNA modification has been performed. Results from these studies indicate several important facts: A single, simple methodological approach provides a class of stable, isolable ribo and 2'-deoxyribonucleoside derivatives that possess excellent reactivity for SNAr chemistry with a wide range of nucleophiles. Also, a benzotriazolyl nucleoside phosphoramidite appears to be a suitable reagent for incorporation into DNA for purposes of site-specific DNA modification.     

Determination of the oxidation products of the reaction between alpha-pinene and hydroxyl radicals by high-performance liquid chromatography

In this paper a method is described for determining and quantifying the degradation products of the reaction of alpha-pinene with hydroxyl radicals. The study is carried out in a fast-flow reactor equipped with a specially designed microwave cavity (type Surfatron) allowing to operate at pressures up to 100 Torr (1 Torr=133.322 Pa). The semi-volatile products are collected on a liquid nitrogen trap (LN2 trap) coated with a 2,4-dinitrophenylhydrazine (2,4-DNPH) solution and the batch samples are subsequently analyzed by HPLC. In order to perform quantitative measurements the batch samples contained two internal standards: benzaldehyde-2,4-DNPH and tolualdehyde-2,4-DNPH. In the experiments carried out at 50 Torr and 100 Torr, HPLC measurements showed that the semi-volatile products formaldehyde, acetaldehyde, acetone, campholenealdehyde and pinonaldehyde could be quantified as oxidation products for the alpha-pinene/OH reaction, with pinonaldehyde being the main product. Assuming that all these five oxidation products have the same collection efficiency on the LN2 trap, one arrives at the following relative product yields (expressed in mol %) at 50 and 100 Torr, respectively: 9.7+/-0.7 and 6+/-5 for formaldehyde; 1.1+/-0.1 and 0.9+/-0.5 for acetaldehyde; 16+/-1 and 6+/-2 for acetone; 11+/-2 and 5.5+/-0.7 for campholenealdehyde; 63+/-3 and 82+/-7 for pinonaldehyde.     

Synthesis of 2'-O-[2-[(N,N-dimethylamino)oxy]ethyl] modified nucleosides and oligonucleotides.

A versatile synthetic route has been developed for the synthesis of 2'-O-[2-[(N,N-dimethylamino)oxy]ethyl] (abbreviated as 2'-O-DMAOE) modified purine and pyrimidine nucleosides and their corresponding nucleoside phosphoramidites and solid supports. To synthesize 2'-O-DMAOE purine nucleosides, the key intermediate B (Scheme 1) was obtained from the 2'-O-allyl purine nucleosides (13a and 15) via oxidative cleavage of the carbon-carbon bond to the corresponding aldehydes followed by reduction. To synthesize pyrimidine nucleosides, opening the 2,2'-anhydro-5-methyluridine 5 with the borate ester of ethylene glycol gave the key intermediate B. The 2'-O-(2-hydroxyethyl) nucleosides were converted, in excellent yield, by a regioselective Mitsunobu reaction, to the corresponding 2'-O-[2-[(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)oxy]ethyl] nucleosides (18, 19, and 20). These compounds were subsequently deprotected and converted into the 2'-O-[2-[(methyleneamino)oxy]ethyl] derivatives (22, 23, and 24). Reduction and a second reductive amination with formaldehyde yielded the corresponding 2'-O-[2-[(N,N-dimethylamino)oxy]ethyl] nucleosides (25, 26, and 27). These nucleosides were converted to their 3'-O-phosphoramidites and controlled-pore glass solid supports in excellent overall yield. Using these monomers, modified oligonucleotides containing pyrimidine and purine bases were synthesized with phosphodiester, phosphorothioate, and both linkages (phosphorothioate and phosphodiester) present in the same oligonucleotide as a chimera in high yields. The oligonucleotides were characterized by HPLC, capillary gel electrophoresis, and ESMS. The effect of this modification on the affinity of the oligonucleotides for complementary RNA and on nuclease stability was evaluated. The 2'-O-DMAOE modification enhanced the binding affinity of the oligonucleotides for the complementary RNA (and not for DNA). The modified oligonucleotides that possessed the phosphodiester backbone demonstrated excellent resistance to nuclease with t(1/2) > 24 h.     

Stereospecific syntheses of 3'-deuterated pyrimidine nucleosides and their site-specific incorporation into DNA

[reaction: see text] 2'-Deoxy-3'-deutero pyrimidines have been synthesized in high yields and incorporated into deoxyoligonucleotides using standard phosphoramidite chemistry. A key synthetic step is a stereospecific reduction of 3'-keto nucleosides using sodium triacetoxyborodeuteride to give 3'-deuterated thymidine and 2'-deoxy uridine nucleosides. Conversion of the corresponding phorphoramidites 7a and 7b to 4-triazolo derivatives has, for the first time, enabled incorporation of 2'-deoxy-3'-deutero cytidine and 2'-deoxy-3'-deutero-5-methyl cytidine into oligonucleotides.