Synthesis of N-Aminopyrazinium Analogs of Cytidine and 2′-Deoxycytidine

Abstract

N-Aminopyrazine analogues of cytidine and 2′-deoxycytidine were prepared from 1-(β-D-ribofuranosyl)-1,2-dihydro-2-oxopyrazine and 1-(2-deoxy-β-D-ribofuranosyl)-1,2-dihydro-2-oxopyrazine, respectively, by amination with O-mesitylenesulfonylhydroxylamine.     

Synthesis of Oligonucleotides Containing 2′-Deoxyguanosine Adducts of Nitropyrenes

Two different approaches to synthesize oligonucleotides containing the 2 ′-deoxyguanosine adducts formed by nitropyrenes are described. A direct reaction of an unmodified oligonucleotide with an activated nitropyrene derivative is a convenient biomimetic approach for generating the major adducts in DNA. A total synthetic approach, by contrast, involves several synthetic steps, including Buchwald-Hartwig Pd-catalyzed coupling, but can be used for incorporating both the major and minor adducts in DNA in high yield.     

The Three-Dimensional Structure of 3′-Deoxycytidine

Abstract

Structural analysis of 3′-deoxycytidine and comparison with 2′-deoxynucleosides reveals no noticeable effect on the conformation of the molecule due to the lack of 3′-oxygen atom. There are two crystallographically independent molecules and both adopt the anti conformation with C3′-endo sugar puckering. A ‘head-to-tail’ packing of the molecules along the b axis results in a virtual ‘2′-5′ polycytidylic acid chain.     

Synthesis of Triple Helix Forming Oligonucleotides Containing 2′-Deoxyformycin A

Abstract

N⁷-Benzoyl-2′-deoxyformycin A (5) was prepared from formycin A and incorporated into the triple helix forming oligonucleotide PRE2ap at CG inversion sites. The modified oligonucleotide containing three substitutions of 2′-deoxyformycin A displayed a 10-fold increase in binding affinity as compared to its unmodified counterpart. This provided a method to accommodate CG inversion sites within target sites for antiparallel triple helix formation.     

Synthesis of 2′-Modified Oligonucleotides Containing Aldehyde or Ethylenediamine Groups

Abstract

Oligonucleotides carrying 2′-aldehyde groups were synthesized and coupled to peptides containing an N-terminal cysteine, aminooxy or hydrazide group to give peptide-oligonucleotide conjugates in good yield. The synthesis of a novel phosphoramidite reagent for the incorporation of 2′-O-(2,3-diaminopropyl)uridine into oligonucleotides was also described. Resultant 2′-diaminooligonucleotides may be useful intermediates in further peptide conjugation studies.     

Synthesis and Properties of 2′-O-Methylribonucleotide Methylphosphonate Containing Chimeric Oligonucleotides

Abstract

2′-O-methylribonucleoside methylphosphonamidites are synthesized and incorporated into oligonucleotides to obtain chimeric antisense oligonucleotides. The resulting oligonucleotide binds to their target RNA/DNA sequences efficiently and stable in a medium containing bovine serum.     

Synthesis and Properties of Oligonucleotides Containing 5-Aza-2′-deoxycytidine

Abstract

The preparation of a protected derivative of 5-aza-2′-deoxycytidine carrying the 2-(p-nitrophenyl)ethyl group is described. The new derivative is useful for the preparation of oligonucleotides containing 5-aza-2′-deoxycytidine using a special methodology that avoids the use of ammonia.     

Synthesis and Photochemical Conversion of Oligonucleotides Containing 2-Chloro-2′-Deoxyadenosine

Abstract

The rate and velocity of the photoconversion of 2-chloro-2′-deoxyadenosine into 2′-deoxyisoguanosine within oligonucleotides was found to be sequence-specific and depends on the nearest neighbor.     

The Parallel and Antiparallel Triplex Formation and Stability of Self Complementary Oligonucleotides Containing 2′-Fluoro-arabinosyl Thymine and 5-Methyl-2′-Deoxycytidine

Abstract

We examined the effects of 1–(2-deoxy -2-fluoro-β-D-arabinofuranosyl)-thymine (or FMAU, a potent antiviral nucleoside) on the stability of duplex and triplexes. When compared the stability of the self-complementary 5′-A₅T₅ duplex with 5′-A₅X₅ (X = FMAU), duplex containing FMAU has much higher melting temperature (T_m). 5′-A₆T₅T₃X₃T₅F₃X₃ and T₃X₃T₅A₆T₅F₃X₃ form the parallel and antiparallel triplexes T₃X₃: A₆:X₃X₃, respectively. The former exhibited the typical T:A:T triplex behavior with only one melting temperature at 70 °C and 45 °c in 1.0 M and 0.2 M NaCl solution, respectively, whereas the latter has two T_m values at 56 °C and 28 °C in 1.0 M solution. FMAU clearly stabilize the triplex structure as A₆T₂₂ which forms the parallel triplex T₆:A₆:T₆ has also only one T_m at 54 °C and 37 °C in high and iow salt concentration solutions, respectively. A 31mer 5′-TCCTCCTTTTTTAGGAGGATTTTTTGGTGGT and 5′-TCCTCCTTTTTTAGGAGGATTTTTTX'X'TX'X'T (X' = 2′-deoxy-5-methylcytidine) were prepared to study their triplex forming potential. The former was found to have a week interaction of the Watson-Crick duplex with the mismatched third-strand at all pH. The latter formed a stable triplex at lower pH consistent with required protonation on the 5-methylcytosine base. For these studies we developed a simple PC desktop spreadsheet program to calculate the first derivative profile of the melting curve data.

This paper is dedicated to Prof. Jacques H. van Boom on the occasion of his 60th birthday.     

Novel Method for the Synthesis of 2′ -Phosphorylated Oligonucleotides

We have developed a new method for the preparation of oligodeoxyribonucleotides and oligo(2′-O-methylribonucleotides) that contain a 2′-phosphorylated ribonucleoside residue, and optimized it to avoid 2′ -3′ -isomerization and chain cleavage. Structures of the 2′ -phosphorylated oligonucleotides were confirmed by MALDI-TOF MS and enzymatic digestion, and the stability of their duplexes with DNA and RNA was investigated. 2′-Phosphorylated oligonucleotides may be useful intermediates for the introduction of various chemical groups for a wide range of applications.     

Synthesis and Some Properties of Modified Oligonucleotides. II. Oligonucleotides Containing 2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl Pyrimidine Nucleosides

Abstract

In order to find the effects of unnatural nucleosides on the stability of duplex, several oligonucleotides containing 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-uracil(FAU),-cytosine (FAC) and -thymine (FMAU) were synthesized by two alternative approaches: phosphoramidite method on an ABI 392 synthesizer and H-phosphonate procedure on our GeneSyn I universal module synthesizer. It was shown from the melting profiles that the presence of FMAU has a large stabilizing effect on the duplex. Replacement of thymidine with FAU, or deoxycytidine with FAC resulted in the formation of less stable duplexes. Temperature-dependent CD spectroscopy demonstrated that the structures of the fluorine containing oligomers are very similar to those of unmodified oligomers.     

A Rapid Solid Phase Method for the Synthesis of 3′-Thiol Group Containing Oligonucleotides

Abstract

A rapid solid phase method for the synthesis of 3′-thiol group containing oligonucleotides is described.     

Stabilization of RNA Bulges by Oligonucleotides Containing 2′-Naphthylmethyl-2′-deoxytubercidine

Abstract

A novel nucleoside analogue, 2′-naphthylmethyl-2′-deoxytubercidine, is synthesized and incorporated in oligonucleotides that stabilize bulges in partially complementary RNA.     

Synthesis and Incorporation of 2′-O-Methyl-Pseudouridine into Oligonucleotides

Abstract

A short multigram synthesis of 2′-O-methylpseudouridine and its phosphoramidite derivative is described which avoids the use of protecting groups on the nitrogens. A binding study of oligonucleotides containing this modification suggest an increased binding affinity to RNA when compared to oligonucleotides incorporating 2′-O-methyluridine.     

Synthesis and Activity of Oligonucleotides Containing a Biologically Active Nucleoside at the 2′End

Abstract

The antiviral activity of (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVdUrd), acyclovir and other antiherpetic nucleosides depends on a selective phosphorylation by the herpesvirus-induced thymidine kinase in the infected cells. Viruses not encoding a specific thymidine kinase (TK) activity are resistant to the action of these nucleoside analogues. The nucleoside monophosphates are as such poorly taken up by the cells. In order to circumvent the necessity of intracellular phosphorylation, we synthesized four core oligonucleotides bearing a biological active nucleoside at the 2′end. It was hypothesized that these core oligonucleotides, like core 2–5A itself, would be taken up within the cell and that, following intracellular 2′-5′ phosphodiesterase cleavage, the 5′-monophosphate of the active product would be formed. In these circumstances, activity against TK- strains could be expected.     

Synthesis and Evaluation of 2′-Deoxy-2′-Spirodiflurocyclopropyl Nucleoside Analogs

The preparation of 2′-deoxy-2′-siprodifluorocyclopropany-lnucleoside analogs has been achieved from α-d-glucose in several steps. The key step in the synthesis was the introduction of the difluorocyclopropane through a difluorocarbene type reaction at the 2′-position. Then, a series of novel 2′-deoxy-2′-spirodifluorocyclopropanyl nucleoside analogs were synthesized using the Vorbrüggen method. All the synthesized nucleosides were characterized and subsequently evaluated against hepatitis C and influenza A virus strains in vitro.     

Commercial-Scale Synthesis of Protected 2′-Deoxycytidine and Cytidine Nucleosides

Abstract

Transformation of 2′-deoxyuridine and uridine analogs to protected 2′-deoxycytidine and cytidine analogs has been investigated by two different methods. First, traditional triazolation protocol and second p-nitrophenoxylation method. Our studies conclude that the triazolation method is better and suitable for commercial scale--up.     

Concise and Efficient Synthesis of 3′-O-Tetraphosphates of 2′-Deoxyadenosine and 2′-Deoxycytidine

We describe concise and efficient synthesis of biologically very important 3′-O-tetraphosphates namely 2′-deoxyadenosine-3′-O-tetraphosphate (2′-d-3′-A4P) and 2′-deoxycytidine-3′-O-tetra-phosphate (2′-d-3′-C4P). N⁶-benzoyl-5′-O-levulinoyl-2′-deoxyadenosine was converted into N⁶-benzoyl-5′-O-levulinoyl-2′-deoxyadenosine-3′-O-tetraphosphate in 87% yield using a one-pot synthetic methodology. One-step concurrent deprotection of N⁶-benzoyl and 5′-O-levulinoyl groups using concentrated aqueous ammonia resulted 2′-d-3′-A4P in 74% yield. The same synthetic strategy was successfully employed to convert N⁴-benzoyl-5′-O-levulinoyl-2′-deoxycytidine into 2′-d-3′-C4P in 68% yield.     

Oligonucleotides Containing N7-Cyanoborane-2′-deoxyguanosine

Abstract

For synthesis of N⁷-cyanoborane-containing oligonucleotides, the 5′-DMT protecting group is not a suitable precursor because the boronated nucleoside is incompatible with DMT cations released during deprotection of the oligonucleotide. As an alternative to DMT, we have investigated use of the 5′-Fmoc protecting group. We found that the cyanoborane group is stable during synthesis and deprotection conditions used with Fmoc derivatives.