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.     

The 4-(N-dichloroacetyl-N-methylamino)benzyloxymethyl group for 2'-hydroxyl protection of ribonucleosides in the solid-phase synthesis of oligoribonucleotides

Emerging RNA-based technologies for controlling gene expression have triggered a high demand for synthetic oligoribonucleotides and have motivated the development of ribonucleoside phosphoramidites that would exhibit coupling kinetics and coupling efficiencies comparable to those of deoxyribonucleoside phosphoramidites. To fulfill these needs, the novel 4-(N-dichloroacetyl-N-methylamino)benzyloxymethyl group for 2'-hydroxyl protection of ribonucleoside phosphoramidites 9a-d has been implemented (Schemes 1 and 2). The solid-phase synthesis of AUCCGUAGCUAACGUCAUGG was then carried out employing 9a-d as 0.2 M solutions in dry MeCN and 5-benzylthio-1H-tetrazole as an activator. The coupling efficiency of 9a-d averaged 99% within a coupling time of 180 s. Following removal of all base-sensitive protecting groups, cleavage of the remaining 2'-[4-(N-methylamino)benzyl] acetals from the RNA oligonucleotide was effected in buffered 0.1 M AcOH (pH 3.8) within 30 min at 90 degrees C. RP-HPLC and PAGE analyses of the fully deprotected AUCCGUAGCUAACGUCAUGG were comparable to those of a commercial RNA oligonucleotide sharing an identical sequence. Enzymatic digestion of the RNA oligomer catalyzed by bovine spleen phosphodiesterase and bacterial alkaline phosphatase revealed no significant amounts of RNA fragments containing (2'-->5')-internucleotidic phosphodiester linkages or noteworthy nucleobase modifications.     

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.     

Enhanced stereoselectivity in internucleotidic bond formation by the use of the chiral ribose moiety of thymidine

This paper deals with the synthesis of new cyclic thymidine 3'-phosphoramidite building blocks having a covalent linker between the trityl type 5'-hydroxyl protecting group and the phosphorus atom attached to the 3'-hydroxyl group of thymidine. The ring structures were designed to reduce the conformational freedom around the phosphorus center so that the stereoselectivity in the internucleotide linkage formation would be improved. The linkers were also designed to be removed readily by treatment with aqueous ammonia. These building blocks were synthesized in good yield by one-pot cyclization of the diol precursors with dichloro(N,N-diisopropylamino)phosphine, despite their large-membered ring. Various activators having 1H-tetrazole, imidazole, and triazole structures were investigated to find the best selectivity in the synthesis of thymidylyl(5'-3')thymidine phosphorothioate. It turned out that our cyclic phosphoramidites gave preferentially the R(p) diastereoisomer in high coupling yield applicable to the solid-phase synthesis of oligodeoxynucleotides. It should be noted that high stereoselectivity was achieved without any chiral sources other than the 2'-deoxyribose moiety itself. The mechanistic studies revealed the importance of the steric bulk and the acidity of the activators. It was also found that the steric bulk of the alcoholic nucleophile was an important factor that determined the stereoselectivity in our systems.     

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.     

DNA microarray synthesis by using PDMS molecular stamp (Ⅱ)——Oligonucleotide on-chip synthesis using PDMS stamp

Based on the standard phosphoramidites chemistry protocol, two oligonucleotides synthetic routes were studied by contact stamping reactants to a modified glass slide. Route A was a contact coupling reaction, in which a nucleoside monomer was transferred and coupled to reactive groups (OH) on a substrate by spreading the nucleoside activated with tetrazole on a polydimethylsiloxane (PDMS) stamp. Route B was a contact detritylation, in which one nucleoside was fixed on the desired synthesis regions where dimethoxytrityl (DMT) protecting groups on the 5'-hydroxyl of the support-bound nucleoside were removed by stamping trichloroacetic acid (TCA) distributed on features on a PDMS stamp. Experiments showed that the synthetic yield and the reaction speed of route A were higher than those of route B. It was shown that 20 mer oligonucleo-tide arrays immobilized on the glass slide were successfully synthesized using the PDMS stamps, and the coupling efficiency showed no difference between the PDMS stamping and the co     

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.     

3'-nitrophenylpropyloxycarbonyl (NPPOC) protecting groups for high-fidelity automated 5' --> 3' photochemical DNA synthesis

[structure: see text]. The most powerful DNA microarrays would be prepared by photolithography with free 3'-ends that could be processed enzymatically. A photoremovable group that could be removed in quantitative yield would ensure high purity of the synthesized probes. We have developed new pyrimidine building blocks for 5' --> 3' DNA synthesis with high cycle yields using the NPPOC (3'-nitrophenylpropyloxycarbonyl) protecting group. These phosphoramidites were proved in automated photochemical DNA synthesis on a modified synthesizer.     

Synthesis of 2'-N-methylamino-2'-deoxyguanosine and 2'-N,N-dimethylamino-2'-deoxyguanosine and their incorporation into RNA by phosphoramidite chemistry

The 2'-hydroxyl groups within RNA contribute in essential ways to RNA structure and function. Previously, we designed an atomic mutation cycle (AMC) that uses ribonucleoside analogues bearing different C-2'-substituents, including -OCH(3), -NH(2), -NHMe, and -NMe(2), to identify hydroxyl groups within RNA that donate functionally significant hydrogen bonds. To enable AMC analysis of the nucleophilic guanosine cofactor in the Tetrahymena ribozyme reaction and at other guanosines whose 2'-hydroxyl groups impart critical functional contributions, we describe here the syntheses of 2'-methylamino-2'-deoxyguanosine (G(NHMe)) and 2'-N,N-dimethylamino-2'-deoxyguanosine (G(NMe(2))) and their corresponding phosphoramidites. The key step in obtaining the nucleosides involved S(N)2 displacement of 2'-β-triflate from an appropriate guanosine derivative by methylamine or dimethylamine. We readily obtained the G(NMe(2)) phosphoramidite and incorporated it into RNA. However, the G(NHMe) phosphoramidite posed a significantly greater challenge due to lack of a suitable -2'-NHMe protecting group. After testing several strategies, we established that allyloxycarbonyl (Alloc) provided suitable protection for 2'-N-methylamino group during the phosphoramidite synthesis and the subsequent RNA synthesis. This work enables AMC analysis of guanosine's 2'-hydroxyl group within RNA.     

Thermolytic carbonates for potential 5'-hydroxyl protection of deoxyribonucleosides

Thermolytic groups structurally related to well-studied heat-sensitive phosphate/thiophosphate protecting groups have been evaluated for 5'-hydroxyl protection of deoxyribonucleosides as carbonates and for potential use in solid-phase oligonucleotide synthesis. The spatial arrangement of selected functional groups forming an asymmetric nucleosidic 5'-O-carbonic acid ester has been designed to enable heat-induced cyclodecarbonation reactions, which would result in the release of carbon dioxide and the generation of a nucleosidic 5'-hydroxyl group. The nucleosidic 5'-O-carbonates 3-8, 10-15, and 19-21 were prepared and were isolated in yields ranging from 45 to 83%. Thermolytic deprotection of these carbonates is preferably performed in aqueous organic solvent at 90 degrees C under near neutral conditions. The rates of carbonate deprotection are dependent on the nucleophilicity of the functional group involved in the postulated cyclodecarbonation reaction and on solvent polarity. Deprotection kinetics increase according to the following order: 4 < 5 < 10 < 6 < 12 < 7 < 13 < 8 < 14 congruent with 19-21 and CCl4 < dioxane < MeCN < t-BuOH < MeCN:phosphate buffer (3:1 v/v, pH 7.0) < EtOH:phosphate buffer (1:1 v/v, pH 7.0). Complete thermolytic deprotection of carbonates 7, 8, 13, and 14 is achieved within 20 min to 2 h under optimal conditions in phosphate buffer-MeCN. The 2-(2-pyridyl)amino-1-phenylethyl and 2-[N-methyl-N-(2-pyridyl)]aminoethyl groups are particularly promising for 5'-hydroxyl protection of deoxyribonucleosides as thermolytic carbonates.     

DNA microarray synthesis by using PDMS molecular stamp (II)

Based on the standard phosphoramidites chemistry protocol, two oligonucleotides synthetic routes were studied by contact stamping reactants to a modified glass slide. Route A was a contact coupling reaction, in which a nucleoside monomer was transferred and coupled to reactive groups (OH) on a substrate by spreading the nucleoside activated with tetrazole on a polydimethylsiloxane (PDMS) stamp. Route B was a contact detritylation, in which one nucleoside was fixed on the desired synthesis regions where dimethoxytrityl (DMT) protecting groups on the 5’-hydroxyl of the support-bound nucleoside were removed by stamping trichloroacetic acid (TCA) distributed on features on a PDMS stamp. Experiments showed that the synthetic yield and the reaction speed of route A were higher than those of route B. It was shown that 20 mer oligonucleotide arrays immobilized on the glass slide were successfully synthesized using the PDMS stamps, and the coupling efficiency showed no difference between the PDMS stamping and the conventional synthesis methods.     

DNA microarray synthesis by using PDMS molecular stamp (II) Oligonucleotide on-chip synthesis using PDMS stamp

Based on the standard phosphoramidites chemistry protocol, two oligonucleotides synthetic routes were studied by contact stamping reactants to a modified glass slide. Route A was a contact coupling reaction, in which a nucleoside monomer was transferred and coupled to reactive groups (OH) on a substrate by spreading the nucleoside activated with tetrazole on a polydimethylsiloxane (PDMS) stamp. Route B was a contact detritylation, in which one nucleoside was fixed on the desired synthesis regions where dimethoxytrityl (DMT) protecting groups on the 5’-hydroxyl of the support-bound nucleoside were removed by stamping trichloroacetic acid (TCA) distributed on features on a PDMS stamp. Experiments showed that the synthetic yield and the reaction speed of route A were higher than those of route B. It was shown that 20 mer oligonucleotide arrays immobilized on the glass slide were successfully synthesized using the PDMS stamps, and the coupling efficiency showed no difference between the PDMS stamping and the conventional synthesis methods.     

Synthesis of Photolabile 5′‐O‐Phosphoramidites for the Photolithographic Production of Microarrays of Inversely Oriented Oligonucleotides

The photolabile 3′‐O‐{[2‐(2‐nitrophenyl)propoxy]carbonyl}‐protected 5′‐phosphoramidites ( 16 – 18 ) were synthesized (see Scheme) for an alternative mode of light‐directed production of oligonucleotide arrays. Because of the characteristics of these monomeric building blocks, photolithographic in situ DNA synthesis occurred in 5′→3′ direction, in agreement with the orientation of enzymatic synthesis. Synthesis yields were as good as those of conventional reactions. The resulting oligonucleotides are attached to the surface via their 5′‐termini, while the 3′‐hydroxy groups are available as substrates for enzymatic reactions such as primer extension upon hybridization of a DNA template (see Fig. 2). The production of such oligonucleotide chips adds new procedural avenues to the growing number of applications of DNA microarrays.     

Triplet‐Sensitized Photodeprotection of Oligonucleotides in Solution and on Microarray Chips

Conditions and kinetics of triplet sensitization as a method for increasing the light sensitivity of photolabile protecting groups used for the photolithographic synthesis of oligonucleotide microarrays were quantitatively studied with the photolabile 2‐(2‐nitrophenyl)propyl protecting group in homogeneous solutions and on glass substrates by using laser flash photolysis, continuous illumination with HPLC analysis, fluorescence dye labelling, and hybridization. In terms of efficiency and avoidance of chemical side reactions, 9H‐thioxanthen‐9‐one was the most‐suitable sensitizer. Both in solution and on a glass substrate, the photostationary kinetics were quantitatively modelled and the relevant kinetic parameters determined. While the sensitization kinetics was diffusion‐controlled both in solution and on the chip, the photostationary kinetics was essentially of zero order only on the chip because here the triplet‐quenching effect of the released photoproduct 2‐(2‐nitrophenyl)propene was suppressed as a consequence of the inhomogeneous reaction that took place in a narrow diffusion zone above the surface from where the photoproducts could quickly escape. The kinetic simulation allowed quantitative estimate of the density of reactive groups on the surface. It was further demonstrated that, with 9H‐thioxanthen‐9‐one as a sensitizer, high‐density oligonucleotide microarrays of high quality can be produced with one‐third of the normal exposure time.     

Synthesis of 2'-O-methoxyethylguanosine using a novel silicon-based protecting group

A short and efficient synthesis of 2'-O-methoxyethylguanosine (8) is described. Central to this strategy is the development of a novel silicon-based protecting group (MDPSCl(2), 2) used to protect the 3',5'-hydroxyl groups of the ribose. Silylation of guanosine with 2 proceeded with excellent regioselectivity and in 79% yield. Alkylation of the 2'-hydroxyl group of 6 proceeded with methoxyethyl bromide and NaHMDS and afforded compound 7 in 85% yield, without any noticeable cleavage of the silyl protecting group and without the need to protect the guanine base moiety. Finally, deprotection of 7 was achieved using TBAF and produced 8 in 97% yield.     

Reaction of amides of 3-imidazoline and 3-imidazoline-3-oxide derivatives with sodium hypobromite

Conclusions The reaction of 2,2,5,5-tetramethyl-3-imidazoline-1-oxyl-4-carboxamide with sodium hypobromite leads to 4-amino-2,2,5,5-tetramethyl-3-imidazoline-1-oxyl. 2,2,5,5-Tetramethyl-3-imidazoline-3-oxide-1-oxyl-4-carboxamide under the same conditions gives 3-hydroxy-2,2,5,5-tetramethylimidazolidin-4-one-1-oxyl.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 1, pp. 228–231, January, 1979.     

Assessment of 4-nitrogenated benzyloxymethyl groups for 2'-hydroxyl protection in solid-phase RNA synthesis

The search for a 2'-OH protecting group that would impart ribonucleoside phosphoramidites with coupling kinetics and coupling efficiencies comparable to those of deoxyribonucleoside phosphoramidites led to an assessment of 2'-O-(4-nitrogenated benzyloxy)methyl groups through solid-phase RNA synthesis using phosphoramidites 2a-d, 12a, and 14a. These phosphoramidites exhibited rapid and efficient coupling properties. Particularly noteworthy is the cleavage of the 2'-O-[4-(N-methylamino)benzyloxy]methyl groups in 0.1 M AcOH, which led to U19dT within 15 min at 90 degrees C. [reaction: see text]     

Syntheses of two 5-hydroxymethyl-2'-deoxycytidine phosphoramidites with TBDMS as the 5-hydroxymethyl protecting group and their incorporation into DNA

5-Hydroxymethylcytosine (5-hmC) is a newly discovered DNA base modification in mammalian genomic DNA that is proposed to be a major epigenetic mark. We report here the syntheses of two new versions of phosphoramidites III and IV from 5-iodo-2'-deoxyuridine in 18% and 32% overall yields, respectively, with TBDMS as the 5-hydroxyl protecting group. Phosphoramidites III and IV allow efficient incorporation of 5-hmC into DNA and a "one-step" deprotection procedure to cleanly remove all the protecting groups. A "two-step" deprotection strategy is compatible with ultramild DNA synthesis, which enables the synthesis of 5hmC-containing DNA with additional modifications.     

cis-Tetrahydrofuran-3,4-diol structure as a key skeleton of new protecting groups removable by self-cyclization under oxidative conditions

A variety of carbonate-type acyl groups having a cis-tetrahydrofuran-3,4-diol (1,4-anhydroerythritol) backbone structure and a TrS or MMTrS group have been examined as new "protected" protecting groups of the 5'-hydroxyl group of nucleosides. These acyl groups were designed in a manner where they could be deprotected by I(2)-promoted removal of the TrS or MMTrS group followed by self-cyclization involving an intramolecular attack of the once-generated neighboring hydroxyl group on the acyl carbon. It turned out that these acyl groups could be introduced into the 5'-hydroxyl group of a 3'-O-protected thymidine derivative by use of the corresponding acyl imidazolides or 4-nitrophenyl esters as well as by reaction with carbonyldiimidazole or 4-nitrophenyl chloroformate. Among the acyl groups tested, it was found that the CTFOC group could be easily introduced into the 5'-hydroxyl group of 3'-masked deoxyribonucleoside derivatives and rapidly removed under mild conditions using iodine.     

Synthesis of adenylyl-(3'----5')-guanosine and some analogues as probes to explore the molecular mechanism of stimulation of influenza virus RNA polymerase

Influenza virus mRNA synthesis is primed by a capped oligonucleotide which is cleaved off from a cellular mRNA by a viral protein. The dinucleotide A3'p5'G can be used as a primer for the viral RNA polymerase mediated RNA synthesis in a cell-free system. Analogues of A3'p5'G have therefore been synthesized using the phosphotriester approach, and their priming ability for the influenza virus mRNA synthesis has been determined. An absence of the 2'-hydroxyl function in the guanosine residue in the dinucleotide, as in A3'p5'dG, drastically decreased its priming ability. Similarly, an alteration of the 3'----5' phosphate linkage to a 2'----5' phosphodiester linkage affected the priming ability quite severely. However a dinucleotide, with the 2'-hydroxyl function omitted in the adenosine moiety, as in dA3'p5'G, could still stimulate the mRNA synthesis. None of the modified dinucleotides inhibited A3'p5'G or globin mRNA primed influenza mRNA synthesis.