Linker phosphoramidite reagents for the attachment of the first nucleoside to underivatized solid-phase supports

New linker phosphoramidite reagents containing a cleavable 3′-ester linkage are used for attaching the first nucleoside to the surface of a solid- phase support. Inexpensive, underivatized amino supports, such as long chain alkylamine controlled-pore glass, can serve as universal supports. No modifications to phosphoramidite coupling conditions are required and, after synthesis, treatment with NH₄OH releases the products with 3′-OH ends. No 3′-dephosphorylation is required. Phosphoramidite reagents containing a succinate and sulfonyl diethanol linkage between the nucleoside and phosphoramidite group are particularly advantageous and can be used to create both 3′-OH and 5′-phosphate ends on oligonucleotides. Reproducibility and quality of oligonucleotide synthesis is demonstrated for either column and 96-well plate formats on low-, medium- or high-loading CPG supports.     

SYNTHESIS of 2′-DEOXY-2′-FLUOROGUANYL-(3′,5′)-GUANOSINE

ABSTRACT

The protected analogue of 2-amnio-6-chloropurine arabinoside (3b) was subjected to reaction with diethylaminosulfur trifluoride (DAST) and subsequently treated with NaOAc in Ac₂O/AcOH to give N ²,O ^3′,O ^5′-triacetyl-2′-deoxy-2′-fluoroguanosine (5a). After deacetylation of the sugar moiety and protection of 5′-OH by a 4,4′-dimethoxytrityl group, this nucleoside component was converted to 2′-deoxy-2′-fluoroguanyl-(3′,5′)-guanosine (6c, GfpG).     

Lipase-catalyzed acylation of quercetin with cinnamic acid

Acylation of quercetin with cinnamic acid catalyzed by Candida antarctica lipase B (CAL-B) or Pseudomonas cepacia lipase C (PCL-C) was investigated. Specifically, the effects of reaction duration, incubation temperature, and molar ratio of substrates on bioconversion yield, initial rate of reaction, and regioselectivity were investigated. Three new acylated quercetin analogues were produced: quercetin 4′-cinnamate (C₂₄H₁₆O₈), quercetin 3′,4′-dicinnamate (C₃₃H₂₂O₉), and quercetin 7,3′,4′-tricinnamate (C₄₂H₂₈O₁₀). The effects of the lipase-catalyzed acylation conditions on the bioconversion yields varied across the conditions. The initial rate of reaction of acylation of quercetin with cinnamic acid catalyzed by CAL-B and PCL-C was similar. In the presence of CAL-B, acylation mainly took place at the C-4′-OH, generating mostly quercetin 4′-cinnamate; whereas with PCL-C, acylation mainly took place at both the 4′- and 3′-hydroxyls, generating quercetin 3′,4′-dicinnamate. Thin-layer-chromatography analysis showed that the three acylated quercetin analogues had higher lipophilicity when compared with quercetin. In silico investigation revealed that quercetin 4’-cinnamate and quercetin 3′,4′-dicinnamate are likely to be orally active pharmacological drugs.     

Aminoacyl-tRNA Analogues; Synthesis,Purification and Properties of 3′-Anthraniloyl Oligoribonucleotides

Abstract

Reaction of isatoic anhydride with adenosine, adenosine 5′-phosphate, oligoribonucleotides or with the E. coli tRNA^Val led to attachment of an anthraniloyl residue at 2′-or 3′-OH groups of 3′-terminal ribose residue. No protection of the S'-hydroxyl group or internal 2′-hydroxyl groups is required for this specific reaction. Anthraniloyl-tRNA which is an analogue of aminoacyl-tRNA forms a ternary complex with EF-Tu*GTP. The anthraniloyl-residue is used as a fluorescent reporter group to monitor interactions with proteins.

     

Secondary structure in polyuridylic acid: Non-classical hydrogen bonding and the function of the ribose 2′-hydroxyl group

The role of non-classical hydrogen bonding in RNA structure has been investigated using polyuridylic acid, which has a labile ordered structure at temperatures near 0 °C, as a model system. By comparing the proton nuclear magnetic resonance spectrum of poly(U) in the transition region with that of uridine and the dimer UpU we find evidence that both the imino N(₃)-H and the ribosyl 2′-OH protons are hydrogen bonded. The characteristics of the former are consistent with participation in N(₃)-HOC bonding primarily between residues in the same strand. As yet we cannot unambiguously assign the acceptor for the 2′-OH in ordered poly(U): because of its apparent stability and the acceptable stereochemistry, we presently favor a bond between ribose 2′-OH and O(1′) connecting adjacent nucleotides of the same strand. This arrangement could contribute to the co-operativity of the poly(U) helix formation. The recently proposed 2′-OHO(1′) interactions in crystalline yeast transfer RNA^Phe suggest similar interactions might play a role in the conformational stability of natural RNAs. A second conformational transition below the major transition in the ultraviolet can be detected in poly(U) by monitoring the H(₆) proton of uracil.     

Spin Labeled Nucleoside Analogues: 4′-Hydroxymorpholin-2′-Ylpurines and Pyrimidines

Abstract

Upon borane-pyridine reduction, a series of nucleoside dialdehyde dioximes 2 underwent cyclization to the corresponding 4′-hydroxymorpholin-2′-ylpurines or pyrimidines 3 from which the peracetyl derivatives 4 were prepared. At room temperature, compounds 3 and 4 exist as a mixture of invertomers in which the 4′S (equatorial 4′-OH or 4′-OAc) predominates. A 14 kcal/mol, nitrogen inversion barrier was estimated from variable temperature experiments. N.O.E. and ³J_CH measurements established the anti conformation of the base-“sugar” bond. Compounds 3 spontaneously oxidized to the corresponding aminoxyl free radicals, EPR spectra of which showed that they existed in a chair conformation.     

Configurational effects on conformational properties of cyclic nucleotides. I. Theoretical calculations of conformer preferences in α-nucleoside 3′,5′ cyclic monophosphates

A comparative study has been made of the configurational effects on the conformational properties of α- and β-anomers of purine and pyrimidine nucleoside 3′,5′,-cyclic monophosphates and their 2′-arabino epimers. Correlation between orientation of the base and the 2′-hydroxyl group have been studied theoretically using the PCILO (Perturbative Configuration Interaction using Localized Orbitals) method. The effect of change in ribose puckering on the base-hydroxyl interaction has also been studied. The result show that steric repulsions and stabilizing effects of intramolecular hydrogen bonding between the base and the 2′-hydroxyl (OH) group are of major importance in determining configurations of α-anomers and 2′-arabino-β-epimers. For example, hydrogen bonding between the 2′-hydroxyl group and polar centers on the base ring is clearly implicated as a determinant of syn-anti preferences of the purine (adenine) or pyrimidine (uracil) bases in α-nucleoside 3′,5′-cyclic monophosphates. Moreover, barrier heights for interconversion between conformers are sensitive to ribose pucker and 2′-OH orientations. The result clearly show that a change in ribose-ring pucker plays an essential role in relieving repulsive interaction between the base and the 2′-hydroxyl group. Thus a C2′-exo-C3′-endo (₂T³) pucker is favored for α-anomers in contrast with the C4′-exo-C3′-endo (₄T³) from found in β-compounds.     

SYNTHESIS OF NEW CLASSES OF BORON-CONTAINING NUCLEOTIDES

Four different types of boron-modified nucleotides are reported: P-boranophos-phorothioates, P-cyanoboranophosphates, P-boranomethylphosphonates, and P3′-N5′-boranophosphoramidates. Synthesis of dinucleoside borano-phosphorothioates and nucleoside P-borano-P-thiomonophosphates via a lithium sulfide method is described. The Li₂S method also provides an alternative way to synthesize phosphorothioates through a dinitrophenyl P(V) phosphotriester precursor. The mechanism of Li₂S substitution was investigated.     

Variation in isomeric products of a phosphodiesterase from the chloroplasts of Phaseolus vulgaris in response to cations

ABSTRACT

Fast-atom bombardment mass spectrometry (FABMS), and collisionally-induced dissociation and mass-analyzed ion kinetic energy spectrum scanning (CID/MIKES) have been used to examine cation effects on a Phaseolus chloroplast complex phosphodiesterase activity. The kinetic parameters of the activity, and the effects of Li⁺, Na⁺, K⁺, Mg²⁺, Mn²⁺ and Fe³⁺ upon them, were determined with 3′,5′-cyclic AMP, -GMP and -CMP, and 2′,3′-cyclic AMP, -GMP and -CMP as substrates. Irrespective of the presence of cations and of the complex nucleotidase, the preferred substrate is a 3′,5′-cyclic nucleotide, not a 2′,3′-cyclic nucleotide. In the presence of the nucleotidase 3′,5′-cyclic AMP and 3′,5′-cyclic GMP are the best substrates, unless Fe³⁺ ions are present. Mg²⁺ and Mn²⁺ stimulate hydrolysis of 3′,5′-cyclic AMP and 3′,5′-cyclic GMP by the complex. However, Fe³⁺ inhibits these activities but stimulates the hydrolysis of 3′,5′-cyclic CMP. Kinetic data indicate that each of these six substrates is hydrolyzed at a single, common, catalytic site. Differentiation of the phosphodiesterase isomeric mononucleotide products by FABMS CID/MIKES analysis indicates that in the absence of ions and after removal of the nucleotidase, the 3′-ester linkage of the 3′,5′-cyclic substrates was hydrolyzed exclusively. Addition of monovalent and divalent ions results in hydrolysis of both the 5′- and 3′-ester linkages.     

Substrate/Inhibitor Properties of Human Deoxycytidine Kinase (dCK) and Thymidine Kinases (Tk1 And Tk2) Towards the Sugar Moiety of Nucleosides,Including O′-Alkyl Analogues

Abstract

Nucleoside analogues with modified sugar moieties have been examined for their substrate/inhibitor specificities towards highly purified deoxycytidine kinase (dCK) and thymidine kinases (tetrameric high-affinity form of TK1, and TK2) from human leukemic spleen. In particular, the analogues included the mono-and di-O′-methyl derivatives of dC, dU and dA, syntheses of which are described. In general, purine nucleosides with modified sugar rings were feebler substrates than the corresponding cytosine analogues. Sugar-modified analogues of dU were also relatively poor substrates of TK1 and TK2, but were reasonably good inhibitors, with generally lower K_i values vs TK2 than TK1. An excellent discriminator between TK1 and TK2 was 3′-hexanoylamino-2′,3′-dideoxythymidine, with a K_i of ～600 μM for TK1 and ～0.1 μM for TK2. 3′-OMe-dC was a superior inhibitor of dCK to its 5′-O-methyl congener, consistent with possible participation of the oxygen of the (3′)-OH or (3′)-OMe as proton acceptor in hydrogen bonding with the enzyme. Surprisingly α-dT was a good substrate of both TK1 and TK2, with K_i values of 120 and 30 μM for TK1 and TK2, respectively; and a 3′-branched α-L-deoxycytidine analogue proved to be as good a substrate as its α-D-counterpart. Several 5 ′-substituted analogues of dC were     

CycloSaligenyl Pronucleotides of 5-Iodo and 5-Trifluoromethyl-1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluorobenzene Mimics of Thymidine: Synthesis and Evaluation of this Pronucleotide Monophosphate Delivery System for Compounds with Potential Anticancer Activity

Abstract

A group of unnatural 1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluorobenzenes possessing a 5-I or 5-CF₃ substituent, that were originally designed as thymidine mimics, were coupled via their 5′-OH group to a cyclosaligenyl (cycloSal) ring system having a variety of C-3 substituents (Me, OMe, H). The 5′-O-cycloSal-pronucleotide concept was designed to effect a thymidine kinase-bypass, thereby providing a method for the intracellular delivery and generation of the 5′-O-monophosphate for nucleosides that are poorly phosphorylated. The 5′-O-cycloSal pronucleotide phosphotriesters synthesized in this study were obtained as a 1:1 mixture of two diastereomers that differ in configuration (S _P or R _P) at the asymmetric phosphorous center. The (S _P)- and (R _P)-diastereomers for the 5′-O-3-methylcycloSal- and 5′-O-3-methoxycycloSal derivatives of 1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-iodobenzene were separated by silica gel flash column chromatography. This class of cycloSal pronucleotide compounds generally exhibited weak cytotoxic activities in a MTT assay (CC₅₀ values in the 10^?3 to 10^?4 M range), against a number of cancer cell lines (143B, 143B-LTK, EMT-6, Hela, 293), except for cyclosaligenyl-5′-O-[1′-(2,4-difluoro-5-iodophenyl)-2′-deoxy-β-D-ribofuranosyl]phosphate that was more potent (CC₅₀ values in the 10^?5 to 10^?6 M range), than the reference drug 5-iodo-2′-deoxyuridine (IUDR) which showed CC₅₀ values in the 10^?3 to 10^?5 M range.     

A New Method for the Synthesis of 2′-O-Benzyladenosine Using Mitsunobu Reaction

Abstract

A new method to introduce a benzyl group onto the 2′-OH of purine ribonucleoside is described. Thus, 6-chloropurine 3′-O-benzoylriboside and its 5′-O-trityl congener were condensed with benzyl alcohol using the Mitsunobu reaction to give the 2′-O-benzyl derivative. The yields were varied from 4.6 to 62.9% depending on the solvent used. The product was converted to adenosine, indicating that the stereochemistry at C-2′ is retained.     

Synthesis of 5a,5a′-dicarba-d-glucobioses from conformationally restricted carbaglucosyl triflates using SN2-type inversion with carbaglucosyl nucleophiles

Novel carbohydrate mimics were designed which contain two 5a-carba-d-glucose residues, one each at reducing and nonreducing end, and thus these mimics are 5a,5a′-dicarba-d-glucobioses. Dicarbadisaccharides have attractive features such as stability against endogenous degradative enzymes and being resistant to glycation reactions such as the Maillard reaction. For the synthesis of dicarba-β-d-isomaltose derivatives, the carbaglucosyl triflate locked in ⁴C₁ conformation was synthesized by protecting with butane-2,3-diacetal group or benzylidene group. Then, 5a,5a′-dicarba-β-d-maltose and 5a,5a′-dicarba-α,β-d-trehalose were synthesized by the S_N2-type inversion reaction using 4,6-O-benzylidene carbaglucosyl triflate with 4-OH and 1-OH carba-β-d-glucose derivatives, respectively, and similarly 5a,5a′-dicarba-α-d-isomaltose with 6-OH carba-α-d-glucose derivative.     

PNA-DNA Chimeras Containing 5-Alkynyl-pyrimidine PNA Units. Synthesis,Binding Properties,and Enzymatic Stability

Abstract

Three chimeric dimer synthons (oeg_t^NHT, oeg_up^NHT and oeg_uh^NHT) containing thymine (t), 5-(l-propynyl)-uracil (up) and 5-(1-hexyn-1-yl)-uracil (uh) PNA units with N-(2-hydroxyethyl)glycine (oeg) backbone were synthesized in solution and incorporated into T₂₀ oligonucleotide analogues, using standard P-amidite chemistry. Insertion of dimer blocks led to destabilization of duplexes with dA₂₀ target. The smallest T _m drops were found for chimeras containing oeg_up^NHT dimers. Incorporation of the chimeric synthons into the 3′-end of T₂₀ brought about growing resistance to 3′-exonucleolytic (SV PDE) cleavage in the order of oeg_t^NHT < oeg_up^NHT < oeg_uh^NHT. Due to different endonuclease activities of 3′- and 5′-exonucleases applied, placing of five consecutive dimers at the 5′-terminus resulted in a relatively smaller, but also side-chain dependent, stabilization towards the hydrolysis by 5′-exonuclease (BS PDE). Neither exonucleases (SV and BS PDE) nor an endonuclease (Nuclease P₁) could hydrolyse the unnatural phosphodiester bond linking the 3′-OH of thymidine to the terminal OH of N-(2-hydroxyethyl)glycine PNA backbone.     

Investigation of a Facile Synthetic Method for Phosphorothioate Dimer Synthons In Oligonucleotide Phosphorothioates Synthesis

Abstract

A facile synthetic method of a phosphorothioate dimer block was investigated. Dinucleoside phosphite triester intermediates were obtained in one-pot synthesis by the coupling of a protected nucleoside bearing free 5′-OH and a protected nucleoside bearing free 3′-OH in the presence of phosphorous trichloride (PCl₃) and 1,2,4-triazole. The intermediates were easily sulfurized to afford the desired phosphorothioate dimer blocks in 33-64% overall yields.     

Selective Biocatalytic Acylation Studies on 5′-O-(4,4′-Dimethoxytrityl)-2′,3′-Secouridine: An Efficient Synthesis of UNA Monomer

Lipozyme® TL IM (Theremomyces lanuginosus lipase immobilized on silica) in toluene catalyzes the acylation of the 2 ′-OH over the 3 ′-OH group in 5 ′-O-(4,4 ′-dimethoxytrityl)-2 ′,3 ′-secouridine (5 ′-O-DMT-2 ′,3 ′-secouridine) in a highly selective fashion in moderate to almost quantitative yields. The turn over during benzoyl transfer reactions mediated by vinyl benzoate or benzoic anhydride was faster than in acyl transfer reactions with vinyl acetate or C₁ to C₅ acid anhydrides; except in the case of butanoic anhydride. The 2 ′-O-benzoyl-5 ′-O-DMT-2 ′,3 ′-secouridine obtained by Lipozyme® TL IM catalyzed benzoylation of 5 ′-O-DMT-2 ′,3 ′-secouridine was successfully converted into its 3 ′-O-phosphoramidite derivative in satisfactory yield, which is a building block for the preparation of oligonucleotides containing the uracil monomer of UNA (unlocked nucleic acid).     

CHIMERIC RNA WITH MODIFIED BACKBONES: ALTERNATING METHYLENE(METHYLIMINO) LINKED PHOSPHODIESTER BACKBONE OLIGONUCLEOTIDES WITH 2′-OH AND 2′-OMe GROUPS

Synthesis of a novel ribo-MMI dimer with 2′-OH and 2′-OMe in 5′- and 3′-nucleosides, respectively is presented. The synthesis was accomplished by reductive coupling of 3′-deoxy-3′-C-formyluridine and 2′-O-methyl-5′-O-methylaminouridine via a thioacetal as the key intermediate for the top part of the dimer. Incorporation of ribo- MMI dimers into oligonucleotides increased binding affinity for target RNA.     

Role of aspartic acid residues D87 and D89 in APS kinase domain of human 3′-phosphoadenosine 5′-phosphosulfate synthase 1 and 2b: A commonality with phosphatases/kinases

3′-phosphoadenosine 5′-phosphosulfate (PAPS) is synthesized in two steps by PAPS synthase (PAPSS). PAPSS is comprised of ATP sulfurylase (ATPS) and APS kinase (APSK) domain activities. ATPS combines inorganic sulfate with α-phosphoryl of ATP to form adenosine 5′-phosphosulfate (APS) and PPi. In the second step APS is phosphorylated at 3′-OH using another mole of ATP to form PAPS and ADP catalyzed by APSK. The transfer of gamma-phosphoryl from ATP onto 3′-OH requires Mg₂⁺ and purported to involve residues D₈₇GD₈₉N. We report that mutation of either aspartic residue to alanine completely abolishes APSK activity in PAPS formation. PAPSS is an, unique enzyme that binds to four different nucleotides: ATP and APS on both ATPS and APSK domains and ADP and PAPS exclusively on the APSK domain. The thermodynamic binding and the catalytic interplay must be very tightly controlled to form the end-product PAPS in the forward direction. Though APS binds to ATPS and APSK, in ATPS domain, the APS is a product and for APSK it is a substrate. DGDN motif is absent in ATPS and present in APSK. Mutation of D₈₇ and D₈₉ did not hamper ATPS activity however abolished APSK activity severely. Thus, D₈₇GD₈₉N region is required for stabilization of Mg²⁺-ATP, in the process of splitting the γ-phosphoryl from ATP and transfer of γ-phosphoryl onto 3′-OH of APS to form PAPS a process that cannot be achieved by ATPS domain. In addition, gamma³²P-ATP, trapped phosphoryl enzyme intermediate more with PAPSS2 than with PAPSS1. This suggests inherent active site residues could control novel catalytic differences. Molecular docking studies of hPAPSS1with ATP + Mg²⁺ and APS of wild type and mutants supports the experimental results.     

THE SYNTHESIS OF DITHYMIDINE BORANOPHOSPHATE BY THE OXATHIAPHOSPHOLANE APPROACH

The application of the oxathiaphospholane approach for the synthesis of dithymidine boranphospate was evaluated. It was shown, that although the nucleoside-3′-O-oxathiaphospholane-borane complexes 2 or 6 could not be chromatographically separated into diastereomerically pure species due to their apparent instability to moisture, they can be successfully applied to the non-stereocontrolled formation of internucleotide boranophosphate bond by reaction with 5′-OH-nucleoside in the presence of DBU. Attempts to apply the related dithiaphospholane approach for the preparation of dithymidine boranophosphorothioate were unsuccessful.     

Anti-complement activity of polynucleotides.

A biologic assay system, based on complement (C′) inhibition, is described to unravel structural differences among polynucleotides. The C′ system appears particularly suitable to distinguish (1) homo- from copoly-ribonucleotides, (2) deoxyribo- from 2′-OH and other 2′-modified polynucleotides, and (3) single homopolynucleotides from double- or triple-stranded complexes.From these studies a number of polynucleotides emerged with potent anti-C′ activities, worthy of further investigation. The most active polymers were (G)_n (polyguanylic acid), (dCc1)_n [poly(2′-chloro-2′deoxycytidylic acid)] and (dUz)_n [poly(2′-azido-2′-deoxyuridylic acid)].