Synthesis of oligonucleotides containing Fapy.dG (N(6)-(2-deoxy-alpha,beta-D-erythropentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine) using a 5'-dimethoxytrityl dinucleotide phosphoramidite

Fapy.dG (N(6)()-(2-deoxy-alpha,beta-d-erythropentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine) is a modified purine lesion produced by a variety of DNA-damaging agents, which shows interesting biochemical properties. The previous method for synthesizing oligonucleotides containing Fapy.dG utilized a reverse dinucleotide phosphoramidite, which also required the synthesis of the appropriate reverse phosphoramidites. An improved method for synthesizing oligonucleotides containing Fapy.dG, which does not require reverse phosphoramidites, is described. Fapy.dG containing dinucleotide phosphoramidites containing 5'-thymidine (11a) or 5'-deoxycytidine (15) are prepared and employed in oligonucleotide synthesis. Oligonucleotide purity is assayed using the DNA repair enzyme formamidopyrimidine DNA glycosylase and by ESI-MS.     

(R)-2,4-Dihydroxybutyramide seco-pseudonucleosides: new versatile homochiral synthons for synthesis of modified oligonucleotides

[reaction: see text] Two series of seco-pseudonucleoside synthons were synthesized from (R)-(+)-alpha-hydroxy-gamma-butyrolactone and (R)-(-)-pantolactone by aminolysis, side-chain protection, dimethoxytritylation, and phosphitylation or solid-phase attachment. The phosphoramidites and solid supports were used in automated DNA synthesis to prepare oligonucleotides modified with one or more 2,4-dihydroxybutyramide units bearing side-chain reporter groups. These new oligonucleotide modification reagents allow the introduction of a label into any desired position within an oligonucleotide chain during solid-phase assembly.     

Synthesis of 2′-O-[2-[(N,N-dialkylamino)oxy]ethyl]-modified oligonucleotides: hybridization affinity, resistance to nuclease, and protein binding characteristics

Synthesis of a series of 2′-O-[2-[(N,N-dialkylamino)oxy]ethyl]-modified 5-methyluridine nucleoside phosphoramidites and solid supports are described. Using these monomers, modified oligonucleotides containing phosphodiester linkages were synthesized in high yields. These modified oligonucleotides showed enhanced binding affinity to the complementary RNA (and not to DNA) and excellent nuclease stability with t_1/2>24 h. The human serum albumin binding properties of modified oligonucleotides have been evaluated to assess their transport and toxicity properties.     

pH-Independent triplex formation: hairpin DNA containing isoguanine or 9-deaza-9-propynylguanine in place of protonated cytosine

Triplex-forming oligonucleotides (TFOs) containing 2'-deoxyisoguanosine (2), 7-bromo-7-deaza-2'-deoxyisoguanosine (2) as well as the propynylated 9-deazaguanine N7-(2'-deoxyribonucleoside) were prepared. For this the phosphoramidites 9a, b of the nucleoside 1 and, the phosphoramidites 19, 20 of compound 3b were synthesized. They were employed in solid-phase oligonucleotide synthesis to yield the protected 31-mer oligonucleotides. The deblocking of the allyl-protected oligonucleotides containing 1 was carried out by Pd(0)[PPh3]4-PPh3 followed by 25% aq. NH3. Formation of the 31-mer single-stranded intramolecular triplexes was studied by UV-melting curve analysis. In the single-stranded 31-mer oligonucleotides the protonated dC in the dCH(+)-dG-dC base triad was replaced by 2'-deoxyisoguanosine (1), 7-bromo-7-deaza-2'-deoxyisoguanosine (2) and, 9-deaza-9-propynylguanine N7-(2'-deoxyribonucleoside) (3b). The replacement of protonated dC by compounds 1 and 3b resulted in intramolecular triplexes which are formed pH-independently and are stable under neutral conditions. These triplexes contain "purine" nucleosides in the third pyrimidine rich strand of the oligonucleotide hairpin.     

The 3-(N-tert-butylcarboxamido)-1-propyl group as an attractive phosphate/thiophosphate protecting group for solid-phase oligodeoxyribonucleotide synthesis

Among the various phosphate/thiophosphate protecting groups suitable for solid-phase oligonucleotide synthesis, the 3-(N-tert-butylcarboxamido)-1-propyl group is one of the most convenient, as it can be readily removed, as needed, under thermolytic conditions at neutral pH. The deprotection reaction proceeds rapidly (t(1/2) approximately 100 s) through an intramolecular cyclodeesterification reaction involving the amide function and the release of the phosphate/thiophosphate group as a 2-(tert-butylimino)tetrahydrofuran salt. Incorporation of the 3-(N-tert-butylcarboxamido)-1-propyl group into the deoxyribonucleoside phosphoramidites 1a-d is achieved using inexpensive raw materials. The coupling efficiency of 1a-d in the solid-phase synthesis of d(ATCCGTAGCTAAGGTCATGC) and its phosphorothioate analogue is comparable to that of commercial 2-cyanoethyl deoxyribonucleoside phosphoramidites. These oligonucleotides were phosphate/thiophosphate-deprotected within 30 min upon heating at 90 degrees C in Phosphate-Buffered Saline (PBS buffer, pH 7.2). Since no detectable nucleobase modification or significant phosphorothioate desulfurization occurs, the 3-(N-tert-butylcarboxamido)-1-propyl group represents an attractive alternative to the 2-cyanoethyl group toward the large-scale preparation of therapeutic oligonucleotides.     

烷基修饰寡聚脱氧核苷酸磷酸残基的化学合成及稳定性研究

分别采用格氏试剂和三氯化磷三步取代法合成了4个新的烷基修饰磷酸残基的亚磷酸酰胺单体, 其结构经1H NMR和31P NMR表征. 利用这些单体合成模型序列5'-dTTTx TT-3', 考察了单体及寡聚核苷酸序列在DNA/RNA合成条件下的稳定性, 提出了固相合成含有烷基修饰磷酸残基的寡聚核苷酸序列裂解及脱保护条件.     

Phosphorylating reagent-free synthesis of 5'-phosphate oligonucleotides by controlled oxidative degradation of their 5'-end

The 5'-phosphorylated oligonucleotides (5'-pONs) are currently synthesized using expensive and sensitive modified phosphoramidite reagents. In this work, a simple, cost-effective, efficient, and automatable method is presented, based on the controlled oxidation of the 5'-terminal alcohol followed by a β-elimination reaction. The latter reaction leads to the removal of the terminal 5'-nucleoside and subsequent formation of the 5'-phosphate moiety. Thus, chemical phosphorylation of oligonucleotides (DNA or RNA) is achieved without using modified phosphoramidites.     

2'-Ribose-ferrocene oligonucleotides for electronic detection of nucleic acids

We have synthesized two novel phosphoramidites with a ferrocenyl moiety at the 2'-ribose position linked through a butoxy linker. Using automated DNA/RNA synthesis techniques, oligonucleotides containing ferrocene at various positions were prepared and characterized by HPLC, MALDI-TOF mass spectrometry, and electrochemistry. Thermal stability studies of the ferrocene-modified DNA duplexes revealed that introduction of one or two ferrocenyl complexes does not result in an observed change of the T(m) values of the corresponding DNA duplexes when compared to the nonmodified hybrids. These data indicate that the introduction of a ferrocenyl group at the 2'-position of the ribose ring containing either a purine or pyrimidine base has no effect on the stability of the modified DNA. The electrochemical behavior of the ferrocene-containing DNA was examined by cyclic voltammetry. The modified 2'-ferrocene-oligonucleotides are electrochemically active and can be used as signaling probes for the electronic detection of nucleic acids on bioelectronic sensors.     

Thermolytic properties of 3-(2-pyridyl)-1-propyl and 2-[N-methyl-N-(2-pyridyl)]aminoethyl phosphate/thiophosphate protecting groups in solid-phase synthesis of oligodeoxyribonucleotides

Thermolytic groups may serve as alternatives to the conventional 2-cyanoethyl group for phosphate/thiophosphate protection in solid-phase oligonucleotide synthesis to prevent DNA alkylation by acrylonitrile generated under the basic conditions used for oligonucleotide deprotection. Additionally, thermolytic groups are attractive in the context of engineering a "heat-driven" process for the synthesis of oligonucleotides on diagnostic microarrays. In these regards, the potential application of pyridine derivatives as thermolytic phosphate/thiophosphate protecting groups has been investigated. Specifically, 2-pyridinepropanol and 2-[N-methyl-N-(2-pyridyl)]aminoethanol were incorporated into deoxyribonucleoside phosphoramidites 7a-d and 9, which were found as efficient as 2-cyanoethyl deoxyribonucleoside phosphoramidites in solid-phase oligonucleotide synthesis. Whereas the removal of 3-(2-pyridyl)-1-propyl phosphate/thiophosphate protecting groups from oligonucleotides is effected within 30 min upon heating at 55 degrees C in concentrated NH4OH or in an aqueous buffer at pH 7.0, cleavage of 2-[N-methyl-N-(2-pyridyl)]aminoethyl groups occurs spontaneously when their phosphate or phosphorothioate esters are formed during oligonucleotide synthesis. The deprotection of these groups follows a cyclodeesterification process generating the bicyclic salts 13 and 14 as side products. These salts do not alkylate or otherwise modify any DNA nucleobases and do not desulfurize a phosphorothioate diester model under conditions mimicking large-scale oligonucleotide deprotection.     

Phosphoramidites and solid supports based on N-substituted 2,4-dihydroxybutyramides: universal reagents for synthesis of modified oligonucleotides

A general and convenient method for synthesis of modified oligonucleotides by use of new non-nucleoside phosphoramidites is reported. A chiral 1,3-diol backbone of the modifying reagents is generated either from (R)-(+)-α-hydroxy-γ-butyrolactone or (R)-(−)-pantolactone. Aliphatic amines were acylated with the lactones to give the corresponding N-substituted 2,4-dihydroxybutyramides. After protection of a side chain, if necessary, the diols were converted into phosphoramidites or solid supports suitable for use in oligonucleotide synthesis. The reagents allow single, multiple or combined introduction of various functions (e.g., alkylamine, imidazole and pyrene residues) into synthetic oligonucleotides. The structures of the conjugates were confirmed by MALDI-TOF mass spectrometry.     

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.     

Synthesis of stable azide and alkyne functionalized phosphoramidite nucleosides

The use of CuAAC chemistry to crosslink and stabilize oligonucleotides has been limited by the incompatibility of azides with the phosphoramidites used in automated oligonucleotide synthesis. Herein we report optimized reaction conditions to synthesize azide derivatives of thymidine and cytidine phosphoramidites. Investigation of the stability of the novel phosphoramidites using ³¹P NMR at room temperature showed less than 10% degradation after 6?h. The azide modified thymidine was successfully utilized as an internal modifier in the standard phosphoramidite synthesis of a DNA sequence. The synthesized azide and alkyne derivatives of pyrimidines will allow efficient incorporation of azide and alkyne click pairs into nucleic acids, thus widening the applicability of click chemistry in investigating the chemistry of nucleic acids.     

Synthesis of the First Tellurium‐Derivatized Oligonucleotides for Structural and Functional Studies

We report here the first synthesis of Te‐nucleoside phosphoramidites and Te‐modified oligonucleotides. We protected the 2′‐tellurium functionality by alkylation and found that the Te functionality is compatible with solid‐phase synthesis and that the Te oligonucleotides are stable during deprotection and purification. In addition, the redox properties of the Te functionalities have been explored. We found that the telluride and telluoxide DNAs are interchangeable by redox reactions. At elevated temperature, the Te‐DNA can also be site‐specifically fragmented oxidatively or reductively when 2′‐TePh functionality is present, whereas elimination of the nucleobase is observed in the presence of 2′‐TeMe. Moreover, the stability of the DNA duplexes derivatized with the Te functionalities has been investigated. Our Te derivatization of nucleic acids provides a novel approach for investigating DNA damage as well as for structure and function studies of nucleic acids and their protein complexes.     

Photocaging strategy for functionalisation of oligonucleotides and its applications for oligonucleotide labelling and cyclisation

We have used a photocaging strategy to develop novel phosphoramidites and expand the repertoire of protecting groups for modification of oligonucleotides by solid-phase synthesis. We synthesised five photolabile phosphoramidites and four new photolabile controlled pore glasses (CPGs). By using these photolabile phosphoramidites and CPGs, modified oligodeoxynucleotides (ODNs) with phosphate, amine, acid, thiol and carbonyl moieties at 5' and/or 3' ends were readily synthesised. To the best of our knowledge, this is the first report of introducing a carbonyl at the 5' end and thiol groups at both ends of ODNs with photolabile modifiers. Terminal labelling was also easily realised in solution or by on-column solid-phase synthesis. By using the photolabile amine modifier and the photolabile acid CPG, cyclisation of an oligodeoxynucleotide was achieved with good yields. This study provides an alternative way to introduce functional groups into oligonucleotides and expand the scope of oligonucleotide bio-orthogonal labelling.     

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.     

Thermolytic 4-methylthio-1-butyl group for phosphate/thiophosphate protection in solid-phase synthesis of DNA oligonucleotides

The thermolabile 4-methylthio-1-butyl phosphate/thiophosphate protecting group for DNA oligonucleotides has been investigated for its potential application to a "heat-driven" process for either oligonucleotide synthesis on diagnostic microarrays or, oppositely, to the large-scale preparation of therapeutic oligonucleotides. The preparation of phosphoramidites 10a-d is straightforward, and the incorporation of these amidites into oligonucleotides via solid-phase techniques proceeds as efficiently as that achieved with 2-cyanoethyl deoxyribonucleoside phosphoramidites. The versatility of the 4-methylthio-1-butyl phosphate/thiophosphate protecting group is exemplified by its facile removal from oligonucleotides upon heating for 30 min at 55 degrees C in an aqueous buffer under neutral conditions or within 2 h at 55 degrees C in concentrated NH(4)OH. The deprotection reaction occurs through an intramolecular cyclodeesterification mechanism leading to the formation of sulfonium salt 18. When mixed with deoxyribonucleosides and N-protected 2'-deoxyribonucleosides or with a model phosphorothioate diester under conditions approximating those of large-scale (>50 mmol) oligonucleotide deprotection reactions, the salt 18 did not significantly alter DNA nucleobases or desulfurize the phosphorothioate diester model to an appreciable extent.     

Improved syntheses of bis(beta-cyclodextrin) derivatives, new carriers for gadolinium complexes

In the last decade a number of reports have been published on the synthesis and characterization of bridged cyclodextrin dimers (bis-CDs) connected with linkers of different lengths and structures. These dimers, having two hydrophobic cavities in close proximity, display much higher binding affinities and molecular selectivities than parent CDs, forming stable supramolecular adducts. We describe new synthetic protocols for the preparation of bis(beta-CDs) bearing 2-2', 3-3' and 6-6' bridges. Some of the critical steps were carried out either under high-intensity ultrasound (US) or microwave (MW) irradiation. Bis(beta-CDs) containing 6-6' ureido- and thioureido-bridges were prepared in high yields by a MW-promoted aza-Wittig reaction using polymer-bound triphenylphosphine, while those containing 2,2' and 3,3' bridges were prepared from mono-alkenyl beta-CDs by the cross-metathesis reaction (homodimerization) in the presence of 2(nd)-generation Grubbs catalyst under sonochemical conditions. By these improved protocols CD dimers could be obtained in gram amounts to prepare stable adducts of bis-CDs with contrast agents (CAs) containing gadolinium(iii) chelates. In the case of Gd(iii) chleate "G-1" the inclusion complexes were found to be 2 to 3 orders of magnitude more stable than that formed by beta-CD (K(ass) = 4.3 x 10(4) M(-1)vs 8.0 x 10(2) M(-1)). Relaxivity increased as well by factors of 3 and 4, viz. from 9.1 mM(-1) s(-1) (beta-CD) to 27.7 and 35 mM(-1) s(-1).     

Diamondoid-modified DNA

We prepared novel C5-modified triphosphates and phosphoramidites with a diamondoid functionally linked to the nucleobase. Using primer extension experiments with different length templates we investigated whether the modified triphosphates were enzymatically incorporated into DNA and whether they were further extended. We found that all three modified nucleotides can be incorporated into DNA using a single-nucleotide incorporation experiment, but only partially using two templates that demand for multiple incorporation of the modified nucleotides. The modified phosphoramidites were introduced into oligonucleotides utilizing DNA synthesizer technology. The occurring oligonucleotide structures were examined by circular dichroism (CD) and melting temperature (T(m)) measurements and were found to adapt similar helix conformations as their unmodified counterparts.     

Synthesis and properties of novel l-isonucleoside modified oligonucleotides and siRNAs

Antisense oligonucleotides and siRNAs are potential therapeutic agents and their chemical modifications play an important role to improve the properties and activities of oligonucleotides. Isonucleoside is a type of nucleoside analogue, in which the nucleobase is moved from C-1 to other positions of ribose. In this report, a novel isonucleoside containing a 5'-CH(2)-extended chain at the sugar moiety was synthesized, thus isoadenosine and isothymidine were incorporated into a DNA single strand and siRNA. It was found that isonucleoside modified oligonucleotides can form stable double helical structures with their complementary DNA and RNA and the stability towards nuclease and ability to activate RNase H are more promising compared with the unmodified, natural analogues. In siRNA, passenger strand modified with isonucleoside () at 3' or 5' terminal can retain the silencing activity and minimize the passenger strand specific off-target effect.     

Molybdenum carbonyl complexes with pyridylimino acidato ligands

Reactions of [Mo(CO)4(pip)2] with pyridine-2-carbaldehyde and the appropriate amino ester or amino acid produce complexes with chelated pyridylimino ligands bearing, respectively, an ester (1) or carboxylate (2a,b, 4, 5a,b) pendant arm, the structures of which have been determined by X-ray crystallography. In the case of alpha-amino acids, the resulting imino carboxylate complexes are unstable towards decarboxylation, this being complete for (R)-2-phenylglycine. The products of decarboxylation 3a-c were isolated and characterized, including X-ray structure determinations for and . In contrast, the derivatives of beta-alanine (4) and 3- and 4-aminobenzoic acids (5a,b) are stable towards decarboxylation. The structure determinations show that the pyridyliminocarboxylate complexes crystallize as salts with piperidinium cations, forming hydrogen-bonded ion-pair dimers featuring twelve- or eight-membered rings. Protonation of carboxylate complexes with 2 M HCl in CH2Cl2/water yields the corresponding neutral complexes 6a,b containing a free carboxylic acid functionality.