Ramamurthy Charubala

Synthesis and properties of 3′-deoxyadenylate trimer dA2′p5′A2′p5′A

Abstract The trimeric 3′-deoxyadenylyl-(2′→5′)-3′-deoxyadenylyl-(2′→5′)-3′-deoxyadenosine ( 1 2 ) was synthesized via the phosphotriester approach starting from cordycepine ( 1 ). Various physical data have been determined and compared with those of the ribo-A2′p5′A2′p5′A analog.

Synthesis of O6-p-nitrophenylethyl guanosine and 2′-deoxyguanosine derivatives

Abstract The p-nitrophenylethyl group is introduced into the O 6 -position of 2′-deoxyguanosine and guanosine via the Mitsunobu-reaction to yield valuable building blocks for oligonucleotide syntheses.

Specific interaction of the diastereomers 7(R)- and 7(S)-tetrahydrobiopterin with phenylalanine hydroxylase: implications for understanding primapterinuria and vitiligo

Pterin‐4a‐carbinolamine dehydratase (PCD) is an essential component of the phenylalanine hydroxylase (PAH) system, catalyzing the regeneration of the essential cofactor 6(R)‐L‐erythro‐5,6,7,8‐tetrahydrobiopterin [6(R)BH4]. Mutations in PCD or its deactivation by hydrogen peroxide result in the generation of 7(R,S)BH4, which is a potent inhibitor of PAH that has been implicated in primapterinuria, a variant form of phenylketonuria, and in the skin depigmentation disorder vitiligo. We have synthesized and separated the 7(R) and 7(S) diastereomers confirming their structure by NMR. Both 7(R)‐ and 7(S)BH4 function as poor cofactors for PAH, whereas only 7(S)BH4 acts as a potent competitive inhibitor vs. 6(R)BH4 (Ki2.3–4.9 µM). Kinetic and binding studies, as well as characterization of the pterin‐enzyme complexes by fluorescence spectroscopy, revealed that the inhibitory effects of 7(R,S)BH4 on PAH are in fact specifically based on 7(S)BH4 binding. The molecular dynamics simulated structures of the pterin‐PAH complexes indicate that 7(S)BH4 inhibition is due to its interaction with the polar region at the pterin binding site close to Ser‐251, whereas its low efficiency as cofactor is related to a suboptimal positioning toward the catalytic iron. 7(S)BH4 is not an inhibitor for tyrosine hydroxylase (TH) in the physiological range, presumably due to the replacement of Ser‐251 by the corresponding Ala297. Taken together, our results identified structural determinants for the specific regulation of PAH and TH by 7(S)BH4, which in turn aid in the understanding of primapterinuria and acute vitiligo. —Pey, A. L., Martinez, A., Charubala, R., Maitland, D. J., Teigen, K., Calvo, A., Pfleiderer, W., Wood, J. M., Schallreuter, K. U. Specific interaction of the diastereomers 7(R)‐ and 7(S)‐tetrahydrobiopterin with phenylalanine hydroxylase: implications for understanding primapterinuria and vitiligo FASEB J. 20, E1451–E1464 (2006)

Inhibition of Epstein-Barr virus-associated nuclear antigen (EBNA) induction by (2′,5′)oligoadenylate and the cordycepin analog: Mechanism of action for inhibition of EBV-induced transformation

Abstract We have previously shown that interferon inhibits Epstein-Barr virus-induced host cell DNA synthesis and morphologic transformation of human adult peripheral blood leukocytes and that core (2′,5′)oligoadenylate and the cordycepin analog can replace interferon with respect to the blockage of EBV-induced functions. The present communication describes the inhibition of Epstein-Barr virus-associated nuclear antigen (EBNA) in EBV-infected human adult peripheral blood leukocytes and the inhibition of EBV-induced morphologic transformation of human umbilical cord leukocytes by interferon, core (2′,5′)oligoadenylate and the core cordycepin analog. These data suggest that the antiviral mechanism of interferon, core (2′,5′)oligoadenylate and the core cordycepin analog is through the inhibition of EBV early functions, possibly transcription or translation of EBNA.

The p-Nitrophenylethyl Group - An Universal Blocking Group in Nucleoside and Nucleotide Chemistry

Abstract The development of the p-nitrophenylethyl (NPE) group for phosphate, phosphite and amide protection as well as the p-nitrophenyleth-oxycarbonyl (NPEOC) group for amino and hydroxy protection offers the first universally applicable blocking groups in nucleoside, nucleotide and oligonucleotide chemistry.

Synthesis and properties of adenylate trimers A2′p5′A2′p5′A, A2′p5′A3′p5′A and A3′p5′A2′p5′A

Abstract Triadenylates with (2′-5′)(2′-5′) and mixed (2′-5′)(3′-5′) and (3′-5′)(2′-5′) linkages respectively were synthesized via the phosphotriester approach followed by deblocking of the fully protected intermediates. The isomeric trimers were characterized by NMR- and CD-spectra and show very similar hypochromicity effects.

A new solid phase approach for rapid syntheses of oligonucleotides bearing a 3′-terminal phosphate group

Abstract Synthesis of pentanucleotide ApTpCpTpTp containing a 3′-terminal phosphomonoester group was accomplished with the help of the new phosphate-solid phase link 2-(4-carboxyphenyl-mercapto)ethanol; a similar molecule, 2-benzylsulfonylethanol, revealed interesting features as a temporary 3′-phosphate protecting group.

2′,5′-Adenylate and Cordycepin Trimer Cores: Metabolic Stability and Evidence for Antimitogenesis without 5′-Rephosphorylation

Abstract The effect of the 2′,5′-adenylate and cordycepin trimer cores on DNA and protein synthesis in human umbilical cord lymphocytes, lymphoblasts, peripheral blood lymphocytes and Epstein-Barr virus infected lymphocytes and their metabolism in tissue culture medium have been studied. [32P]Adenylate and [32P]- and [3H]cordycepin trimer cores were synthesized either enzymatically or chemically and added to cells in culture. The half-lives of the 2′,5′-A3 core and 2′,5′-3′dA3 core in tissue culture were 3 and 17 hr, respectively. Chromatographic analysis of the TCA-soluble extracts of the lymphocytes and lymphoblasts treated with 2′,5′-[3H]A3 showed that 0.25% of the 32P was identified as AMP, ADP, ATP and inorganic phosphate. By the more sensitive 2′,5′-p3A4[32P]pCp radiobinding assay, 2′,5′-A3 was detected in the TCA supernatants; however, there was no 5′-rephosphorylation. With the [3H]- and [32P]cordycepin trimer core, 0.55% and 1.3% of the radioactivity was in the TCA soluble extracts. Although ther...

Pteridine Nucleosides—New Versatile Building Blocks in Oligonucleotide Synthesis

Abstract Chemical syntheses of 1-(2-deoxy-β-D-ribofuranosyl)lumazines and isopterins as well as 8-(2-deoxy-β-D-ribofuranosyl)-4-amino-7(8H)pteridones and -isoxanthopterins have been developed to make the structural analogs of the naturally occurring 2′-deoxyribonucleosides in the pteridine series available. The corresponding phosphoramidites have been used in machine-aided solid-support syntheses leading to new types of fluorescence labeled oligonucleotides. The effects of the various fluorophors on duplex formation and as labels for enzyme reactions is demonstrated.

Synthesis of a modified 2′, 5′-Adenylate trimer with a 2′,3′-Di-O-(2-Carboxyethyl)-ethylidene terminal group

Abstract The trimer of 2′, 5′-oligoadenylic acid with a (2-carboxyethyl)ethylidene group ({=1}{=6}) at the 2′-terminal adenosine moiety and its 3′-deoxyadenosine analog ({=1}{=7}) have been synthesized by the phosphotriester method.