Scalable synthesis of substituted 2,7-dimethyl-9-phenylxanthen-9-ol (DMPx-OH): useful for the preparation of crystalline 5′-O-DMPx-protected nucleosides

A convenient single-step synthesis of several 2,7-dimethyl-9-phenylxanthen-9-ol (DMPx-OH) analogs has been accomplished using a Friedel-Crafts reaction. Treatment of various DMPx-OH with unprotected 2′-O-methoxyethyl-ribonucleosides (MOE) in the presence of B(C₆F₅)_3, as a Lewis Acid catalyst, furnished 5′-O-protected derivatives of 2′-MOE-ribonucleosides in good yields. The deprotection of the DMPx groups was accomplished by acid hydrolysis under very mild conditions. Among the five DMPx analogs synthesized, the 2,7-dimethyl-9-(4-nitrophenyl)xanthene-9-yl group furnished crystalline products enabling non-chromatographic isolation of 5′-O-protetced nucleosides.     

Probing of PSE acetal protection for nucleoside chemistry

The use of phenylsulfonylethylidene (PSE) acetal as a new 3′,5′-bridged protecting group in nucleoside chemistry is reported. The PSE acetal demonstrates to be compatible with Lewis acids used in standard glycosylation reactions. In addition, a selective 2′-O-deacylation from a 3′,5′-O-(phenylsulfonyl)-2′-O-acetyl nucleoside can be achieved, giving access to subsequent chemical modifications in 2′ position. However, the PSE acetal cleavage surprisingly appeared to be purine/pyrimidine base dependent.     

Facile synthesis of 2′-O-cyanoethyluridine by ring-opening reaction of 2,2′-anhydrouridine with cyanoethyl trimethylsilyl ether in the presence of BF3·Et2O

In this Letter, a facile method for the synthesis of 2′-O-cyanoethyluridine, which is a key intermediate in the synthesis of fully and partially 2′-O-cyanoethylated oligoribonucleotides as well as unmodified oligoribonucleotides, was developed by the ring-opening reaction of 2,2′-anhydrouridine with 2-cyanoethyl trimethylsilyl ether in the presence of BF₃·Et₂O in dimethylacetamide. The 2′-O-cyanoethyluridine 3′-phosphoramidite derivative was converted into the 2′-O-cyanoethyl-4-N-acetylcytidine 3′-phosphoramidite derivative by a series of reactions involving displacement of the 4-(1H-1,2,4-triazol-1-yl)uridine derivative with ammonia followed by acetylation.     

Synthesis of DNA conjugates by solid-phase fragment condensation via aldehyde-nucleophile coupling

Oligodeoxyribonucleotides were synthesized that contain a novel nucleoside, 2′-O-(2,3-dihydroxypropyl)cytidine. Its 2′-diol group was blocked by an allyloxycarbonyl protecting group. Selective deprotection of diol group(s) of the support-immobilized blocked oligodeoxyribonucleotide by Pd(0) followed by periodate oxidation resulted in generation of the 2′-aldehyde group(s) on solid-phase. The modified oligonucleotides were used to prepare a number of conjugates with acridine, biotin and N-modified laminin peptides by oxime, hydrazone and hydrazine formation. The method may be applicable to the synthesis of oligonucleotide-peptide conjugates.     

A theoretical study on the elimination reaction of acrylonitrile from 2′-O-cyanoethylated nucleosides by Bu4NF

The elimination reaction of acrylonitrile from 2′-O-cyanoethylated nucleosides by Bu₄NF was studied computationally. The transition structures for the reaction of 3-methoxypropionitrile with Me₄NF were located at the MP2, B3LYP, and B3LYP-SCRF(Dipole) levels with 6-31 + G* basis set. The α-hydrogen of the cyano group was removed via a proton transfer from the α-carbon to the oxygen with a syn-periplanar arrangement rather than the deprotonation reaction with F^?. In the presence of an excess amount of Me₄NF, the activation energy decreased by the coordination of Me₄N⁺ to the cyano group. The reaction of 3-methoxypropionitrile with Bu₄NF and 2′-O-cyanoethylated uridine with Me₄NF also occurred via a proton transfer mechanism with lower activation energies. The reactivity tendency corresponds to the amount of the negative charge on the oxygen atom and part of the reason for the faster elimination with F^? rather than with DBU concerns entropy. The experimental results are well explained by these calculations.     

Linear free energy relationships and kinetic isotope effects reveal the chemistry of the Ado 2′-OH group

Using kinetic isotope effects (KIE) and Hammett correlations, we show that the main role of the adenosine 2′-OH group on deprotonation by the non nucleophilic base DBU during external acyl group transfer is to generate enhanced electron density on the attacking nucleophile through ionization. The small primary KIEs (1.2 and 1.6) and the large Hammett reaction constants (+2.25 and +3.19) obtained for the ethanolysis of 2′/3′-O-p-substituted benzoyl 5′-O-trityl adenosines and 2′-deoxyadenosines are consistent with an A_N + D_N reaction mechanism. The implications of our results are discussed in terms of chemical contributions of the 2′-OH group in the ribosome catalysis of peptide bond formation.     

PhBCl2 promoted reductive opening of 2,4-O-p-methoxybenzylidene: new regioselective differentiation of position 2 and 4 of α-l-iduronyl moieties in disaccharide building blocks

We describe a new protocol for the challenging differentiation of the position 2^′ and 4^′ of l-iduronyl moieties located at the nonreducing end of various disaccharide building blocks. This methodology is based on the introduction of a 2^′,4^′-O-p-methoxybenzylidene group, followed by a totally regioselective reductive opening of this acetal by the PhBCl₂/Et₃SiH reagent system. l-Iduronyl moieties protected by a 4^′-O-p-methoxybenzyl group were thus obtained regioselectively and efficiently.     

Carbohydrate-derived spiroketals: stereoselective synthesis of di-d-fructose dianhydrides

A one-pot synthesis of di-d-fructose dianhydrides (DFAs) having the 1,6,9,13-tetraoxadispiro[4.2.4.2]tetradecane and 1,7,10,15-tetraoxadispiro[5.2.5.2]hexadecane skeleton has been accomplished. The methodology relies on the ability of per-O-protected 1,2-O-isopropylidene β-d-fructofuranose and β-d-fructopyranose derivatives to undergo a tandem acetal cleavage-intermolecular glycosylation-intramolecular spiroketalization process by reaction with suitable acid promoters, such as boron trifluoride etherate or trifluoromethanesulfonic acid, in apolar organic solvents. Spirocyclization proceeds then under irreversible reaction conditions to give binary mixtures of di-d-fructofuranose (α,α and α,β diastereomers) or di-d-fructopyranose 1,2′:2,1′ dianhydrides (β,β and α,β), respectively, the stereochemical outcome being dependent on the non-participating or participating character of the protecting groups. Thus, benzylated and allylated derivatives afford, preferentially, the non-symmetric DFAs (α,β), with diastereomeric excess up to 92%. In contrast, the use of participating benzoyl groups favours the C₂-symmetric diastereomer in both series.     

Synthesis and anti-HCV activity of 1-(1′,3′-O-anhydro-3′-C-methyl-β-d-psicofuranosyl)uracil

Synthesis of a novel 1′,2′-oxetane-uridine bearing a 2′-C-methyl substituent, [1-(1′,3′-O-anhydro-3′-C-methyl-β-d-psicofuranosyl)uracil], is described. Key to its construction was the use of 6-O-(p-toluoyl)-1,2:3,4-di-O-isopropylidene-3-C-methyl-d-psicofuranose as a nucleosidation substrate, which itself was derived from d-fructose. Anti-HCV activity was examined for the corresponding triphosphate which was not found to be an inhibitor of HCV NS5B 1b wild type polymerase in vitro. The 1′,2′-oxetane uridine triphosphate without 2′-C-methyl substitution was similarly inactive, however, the guanosine analog displayed modest inhibition (IC₅₀ = 10 μM).     

Novel and efficient regioselective enzymatic approach to 3′-, 5′- and 3′,5′-di-O-crotonyl 2′-deoxynucleoside derivatives

Regioselective syntheses of several O-crotonyl 2′-deoxynucleoside derivatives have been efficiently achieved using a biocatalytic methodology. While Candida antarctica lipase B (CAL-B) afforded the 5′-O-acylated compounds, immobilized lipase from Pseudomonas cepacia (PSL-C) provided the 3′-O-crotonylated analogs. Since classical chemical approaches did not work appropriately due to side isomerization reactions, a mixture of both lipases was used to achieve a useful synthetic route toward diacylated nucleosides.     

An efficient and versatile synthesis of GlcNAcstatins—potent and selective O-GlcNAcase inhibitors built on the tetrahydroimidazo[1,2-a]pyridine scaffold

We report a novel approach to the synthesis of GlcNAcstatins—members of an emerging family of potent and selective inhibitors of peptidyl O-GlcNAc hydrolase build upon tetrahydroimidazo[1,2-a]pyridine scaffold. Making use of a streamlined synthetic sequence featuring de novo synthesis of imidazoles from glyoxal, ammonia and aldehydes, a properly functionalised linear GlcNAcstatin precursor has been efficiently prepared starting from methyl 3,4-O-(2′,3′-dimethoxybutane-2′,3′-diyl)-α-d-mannopyranoside. Subsequent ring closure of the linear precursor in an intramolecular S_N2 process furnished the key fused d-mannose-imidazole GlcNAcstatin precursor in excellent yield. Finally, a sequence of transformations of this key intermediate granted expeditious access to a variety of the target compounds bearing a C(2)-phenethyl group and a range of N(8) acyl substituents. The versatility of the new approach stems from an appropriate choice of a set of acid labile permanent protecting groups on the monosaccharide starting material. Application was demonstrated by the synthesis of GlcNAcstatins containing polyunsaturated and thiol-containing amido substituents.     

Synthesis of 2′-hydrazine oligonucleotides and their efficient conjugation with aldehydes and 1,3-diketones

Oligodeoxyribonucleotides that contain a novel nucleoside, 2′-O-(2-hydrazinoethyl)uridine, were synthesised by NaBH₃CN reduction of hydrazones formed from 2′-O-(2-oxoethyl)oligonucleotides with FmocNHNH₂, followed by concd aq NH₃ deprotection. The 2′-hydrazine oligonucleotides obtained were then used to synthesise a number of conjugates with aldehydes via hydrazone formation and with1,3-diketones via pyrazole formation. The method was shown to be applicable for the preparation of oligonucleotide-peptide conjugates.     

Efficient conjugation and preferential DNA binding of oligonucleotides containing 2′-O-(2-oxoethyl)arabinouridine

Oligodeoxyribonucleotides were synthesized that contain a novel nucleoside, 2′-O-(2-oxoethyl)arabinouridine. Whereas such oligonucleotides showed only a slight reduction in the T_M values of their complexes with complementary DNA, a significant destabilization was observed in the case of duplexes formed with RNA. This may be explained by the C_2′-endo conformation of 2′-O-(2,3-dihydroxypropyl)arabinouridine as demonstrated by NMR experiments in D₂O. The modified oligonucleotides were used to synthesize a number of conjugates with dyes, biotin and a N-modified laminin peptide, by hydrazone and oxime formation. We suggest that the 2′-arabinoaldehyde-containing DNA duplexes may be valuable tools for affinity modification of DNA-binding proteins.     

Cyclization versus oligomerization of SP- and RP-5′-OH-N-benzoyl-2′-deoxycytidine-3′-O-(2-thio-4,4-pentamethylene-1,3,2-oxathiaphospholane)s

The S_P-isomer of 5′-OH-N⁴-benzoyl-2′-deoxycytidine-3′-O-(2-thio-4,4-pentamethylene-1,3,2-oxathiaphospholane) undergoes DBU-promoted intramolecular cyclization providing as a sole product S_P-deoxycytidine cyclic 3′,5′-O,O-phosphorothioate. Unexpectedly, the R_P-counterpart yields a mixture of products consisting of R_P-deoxycytidine cyclic 3′,5′-O,O-phosphorothioate and macrocyclic oligo(deoxycytidine phosphorothioate)s. The results of molecular modeling indicate that the dychotomy observed for the R_P substrate may result from remarkably higher energy of the corresponding transition states, caused by the presence of bulky ‘spiro’ pentamethylene substituent at the position C4 in the oxathiaphospholane ring.     

An improved method for synthesizing antennary β-d-mannopyranosyl disaccharide units

The merits of an indirect protecting method for hydroxyl groups using allyl groups via allyloxycarbonyl groups in the synthesis of antennary β-d-mannopyranosyl disaccharides from β-d-galactopyranosyl disaccharides were studied. Regioselective allyloxycarbonylation and conversion reactions involving simultaneous double S_N2 nucleophilic substitution at C-2′ and C-4′ of benzyl O-[β-d-galactopyranosyl]-(1-4)-3,6-di-O-benzyl-2-deoxy-2-N-phthalimido-β-d-glucopyranoside were examined for comparison with the direct allylation method. The required β-d-mannopyranosyl disaccharide having proper protecting groups was obtained using this indirect method in 52% yield. In contrast, the reported direct allylation method using methyl O-(β-d-galactopyranosyl) disaccharide gave the corresponding β-d-mannopyranosyl disaccharide in only 7.5% yield.     

Synthesis and hybridization properties of 2′-O-(tetrazol-5-yl)ethyl-modified oligonucleotides

2′-O-(1H-Tetrazol-5-yl)ethyladenosine was synthesized using 2′-O-cyanoethyladenosine derivative as a key intermediate. The 2′-O-(1H-tetrazol-5-yl)ethyl modifications exhibited intriguing properties such as the change in the structure of the tetrazole residue between a protonated and a deprotonated form. The T_m experiments of various oligodeoxynucleotides having a 2′-O-(1H-tetrazol-5-yl)ethyl-modified adenosine showed reduced hybridization affinity in comparison to the unmodified oligonucleotides toward their complementary oligodeoxynucleotides. The mechanism of the reduced hybridization affinity was discussed on the basis of the structure and the physicochemical properties of the tetrazole moiety.     

Synthesis and biological evaluation of 5′-O-dicarboxylic fatty acyl monoester derivatives of anti-HIV nucleoside reverse transcriptase inhibitors

A number of 5′-O-dicarboxylic fatty acyl monoester derivatives of 3′-azido-3′-deoxythymidine (zidovudine, AZT), 2′,3′-didehydro-2′,3′-dideoxythymidine (stavudine, d4T), and 3′-fluoro-3′-deoxythymidine (alovudine, FLT) were synthesized to improve the lipophilicity and potentially the cellular delivery of parent polar 2′,3′-dideoxynucleoside (ddN) analogs. The compounds were evaluated for their anti-HIV activity. Three different fatty acids with varying chain length of suberic acid (octanedioic acid), sebacic acid (decanedioic acid), and dodecanedioic acid were used for the conjugation with the nucleosides. The compounds were evaluated for anti-HIV activity and cytotoxicity. All dicarboxylic ester conjugates of nucleosides exhibited significantly higher anti-HIV activity than that of the corresponding parent nucleoside analogs. Among all the tested conjugates, 5′-O-suberate derivative of AZT (EC₅₀ = 0.10 nM) was found to be the most potent compound and showed 80-fold higher anti-HIV activity than AZT without any significant toxicity (TC₅₀ >500 nM).     

Synthesis of 2′-O,3′-O bicyclic adenosine analogues using ring closing metathesis

The synthesis of 2′-O,3′-O bicyclic adenosine derivatives is presented as the first examples of a new family of 13-membered ring bicyclic nucleoside analogues. Cyclisation was achieved through ring closing metathesis (RCM) on a diene intermediate using Grubbs’ catalyst. The Z and E isomers were purified and characterised.     

Regioselective synthesis of O- and O-cyclopyrimidine nucleoside analogues

Regioselective synthesis of two new series of cyclonucleoside analogues from the 1,2-carbonucleoside of uracil 1a: O²,7′-cyclonucleosides (3a-c) and O⁶,7′-cyclonucleosides (4a-c), analogues of pyrimidine (cyclohexane derivatives) is reported. Synthesis of O²-cyclonucleoside analogues was performed by activation of the hydroxymethyl group of carbocyclic moiety and using the carbonyl group at position 2 of the heterocyclic base as a nucleophile. Synthesis of O⁶-cyclonucleoside analogues was achieved by nucleophilic attack of the 7′-hydroxyl group on the electron-deficient 6-position and subsequently dehydrohalogenation in basic conditions.     

Concise and efficient synthesis of 3′-O-triphosphates of 2′-deoxyadenosine and 2′-deoxycytidine

We describe concise and efficient synthesis of 2′-deoxyadenosine-3′-O-triphosphate (2′-d-3′-ATP) and 2′-deoxycytidine-3′-O-triphosphate (2′-d-3′-CTP) which are well known for their various biological applications. One-pot synthetic methodology was used to convert N⁶-Benzoyl-5′-O-levulinoyl-2′-deoxyadenosine into N⁶-Benzoyl-5′-O-levulinoyl-2′-deoxyadenosine-3′-O-triphosphate in 72% yield. One-step concurrent deprotection of N⁶-Benzoyl and 5′-O-levulinoyl groups using concentrated aqueous ammonia resulted in 2′-d-3′-ATP in 75% yield. The same synthetic strategy was successfully employed to convert N⁴-Benzoyl-5′-O-levulinoyl-2′-deoxycytidine into 2′-d-3′-CTP in 66% yield.