Abdalla E. A. Hassan

High Fidelity of Base Pairing by 2-Selenothymidine in DNA

The base pairs are the contributors to the sequence-dependent recognition of nucleic acids, genetic information storage, and high fidelity of DNA polymerase replication. However, the wobble base pairing, where T pairs with G instead of A, reduces specific base-pairing recognition and compromises the high fidelity of the enzymatic polymerization. Via the selenium atomic probing at the 2-position of thymidine, we have investigated the wobble discrimination by manipulating the steric and electronic effects at the 2-exo position, providing a unique chemical strategy to enhance the base pair specificity. We report here the first synthesis of the novel 2-Se-thymidine ((Se)T) derivative, its phosphoramidite, and the Se-DNAs. Our biophysical and structural studies of the 2-Se-T DNAs reveal that the bulky 2-Se atom with a weak hydrogen-bonding ability can largely increase mismatch discriminations (including T/G wobble and T/C mismatched base pairs) while maintaining the (Se)T/A virtually identical to the native T/A base pair. The 2-Se atom bulkiness and the electronic effect are probably the main factors responsible for the discrimination against the formation of the wobble (Se)T/G base pair. Our investigations provide a potential novel tool to investigate the specific recognition of base pairs, which is the basis of high fidelity during replication, transcription, and translation. Furthermore, this Se-atom-specific substitution and probing are useful for X-ray crystal structure and function studies of nucleic acids.

Novel RNA base pair with higher specificity using single selenium atom

Specificity of nucleobase pairing provides essential foundation for genetic information storage, replication, transcription and translation in all living organisms. However, the wobble base pairs, where U in RNA (or T in DNA) pairs with G instead of A, might compromise the high specificity of the base pairing. The U/G wobble pairing is ubiquitous in RNA, especially in non-coding RNA. In order to increase U/A pairing specificity, we have hypothesized to discriminate against U/G wobble pair by tailoring the steric and electronic effects at the 2-exo position of uridine and replacing the 2-exo oxygen with a selenium atom. We report here the first synthesis of the 2-Se-U-RNAs as well as the 2-Se-uridine (SeU) phosphoramidite. Our biophysical and structural studies of the SeU-RNAs indicate that this single atom replacement can indeed create a novel U/A base pair with higher specificity than the natural one. We reveal that the SeU/A pair maintains a structure virtually identical to the native U/A base pair, while discriminating against U/G wobble pair. This oxygen replacement with selenium offers a unique chemical strategy to enhance the base pairing specificity at the atomic level.

Hydrogen bond formation between the naturally modified nucleobase and phosphate backbone

Natural RNAs, especially tRNAs, are extensively modified to tailor structure and function diversities. Uracil is the most modified nucleobase among all natural nucleobases. Interestingly, >76% of uracil modifications are located on its 5-position. We have investigated the natural 5-methoxy (5-O-CH3) modification of uracil in the context of A-form oligonucleotide duplex. Our X-ray crystal structure indicates first a H-bond formation between the uracil 5-O-CH3 and its 5′-phosphate. This novel H-bond is not observed when the oxygen of 5-O-CH3 is replaced with a larger atom (selenium or sulfur). The 5-O-CH3 modification does not cause significant structure and stability alterations. Moreover, our computational study is consistent with the experimental observation. The investigation on the uracil 5-position demonstrates the importance of this RNA modification at the atomic level. Our finding suggests a general interaction between the nucleobase and backbone and reveals a plausible function of the tRNA 5-O-CH3 modification, which might potentially rigidify the local conformation and facilitates translation.

Synthesis, structure and imaging of oligodeoxyribonucleotides with tellurium-nucleobase derivatization

We report here the first synthesis of 5-phenyl–telluride–thymidine derivatives and the Te-phosphoramidite. We also report here the synthesis, structure and STM current-imaging studies of DNA oligonucleotides containing the nucleobases (thymine) derivatized with 5-phenyl-telluride functionality (5-Te). Our results show that the 5-Te-DNA is stable, and that the Te-DNA duplex has the thermo-stability similar to the corresponding native duplex. The crystal structure indicates that the 5-Te-DNA duplex structure is virtually identical to the native one, and that the Te-modified T and native A interact similarly to the native T and A pair. Furthermore, while the corresponding native showed weak signals, the DNA duplex modified with electron-rich tellurium functionality showed strong topographic and current peaks by STM imaging, suggesting a potential strategy to directly image DNA without structural perturbation.

Synthesis and antimicrobial evaluation of novel pyrazolones and pyrazolone nucleosides.

The synthesis of a novel series of 4-arylhydrazono-5-methyl-1,2-dihydropyrazol-3-ones 4a–h, and their N 2-alkyl and acyclo, glucopyranosyl, and ribofuranosyl derivatives is described. K2CO3 catalyzed alkylation of 4a–h with allyl bromide, propargyl bromide, 4-bromobutyl acetate, 2-acetoxyethoxymethyl bromide, and 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide proceeded selectively at the N 2-position of the pyrazolinone ring. Glycosylation of 4a with 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose under Vorbruggen glycosylation conditions gave the corresponding N 2-4-arylhydrazonopyrazolone ribofuranoside 9a in good yield. Conventional deprotection of the acetyl protected nucleosides furnished the corresponding 4-arylhydrazonopyrazolone nucleosides in good yields. Selected numbers of the newly synthesized compounds were screened for antimicrobial activity. Compounds 4b, 12a, and 14d showed moderate activities against Aspergillus flavus, Penicillium sp., and Escherichia coli.

Lithium Hexamethyldisilazane Transformation of Transiently Protected 4-Aza/Benzimidazole Nitriles to Amidines and their Dimethyl Sulfoxide Mediated Imidazole Ring Formation.

Trimethylsilyl-transient protection successfully allowed the use of lithium hexamethyldisilazane to prepare benzimidazole (BI) and 4-azabenzimidazole (azaBI) amidines from nitriles in 58-88% yields. This strategy offers a much better choice to prepare BI/azaBI amidines than the lengthy, low-yielding Pinner reaction. Synthesis of aza/benzimidazole rings from aromatic diamines and aldehydes was affected in dimethyl sulfoxide in 10-15 min, while known procedures require long time and purification. These methods are important for the BI/azaBI-based drug industry and for developing specific DNA binders for expanded therapeutic applications.

Construction and structure studies of DNA-bipyridine complexes as versatile scaffolds for site-specific incorporation of metal ions into DNA

The facile construction of metal–DNA complexes using ‘Click’ reactions is reported here. A series of 2′-propargyl-modified DNA oligonucleotides were initially synthesized as structure scaffolds and were then modified through ‘Click’ reaction to incorporate a bipyridine ligand equipped with an azido group. These metal chelating ligands can be placed in the DNA context in site-specific fashion to provide versatile templates for binding various metal ions, which are exchangeable using a simple EDTA washing-and-filtration step. The constructed metal–DNA complexes were found to be thermally stable. Their structures were explored by solving a crystal structure of a propargyl-modified DNA duplex and installing the bipyridine ligands by molecular modeling and simulation. These metal–DNA complexes could have wide applications as novel organometallic catalysts, artificial ribonucleases, and potential metal delivery systems.

Cyano-modification on Uridine Decrease the Base Pairing Stability and Specificity through Neighbouring Disruption in RNA Duplex

5‐Cyanomethyluridine (cnm5U) and 5‐cyanouridine (cn5U), the two uridine analogues, were synthesized and incorporated into RNA oligonucleotides. Base‐pairing stability and specificity studies in RNA duplexes indicated that cnm5U slightly decreased the stability of the duplex but retained the base‐pairing preference. In contrast, cn5U dramatically decreased both base‐pairing stability and specificity between U:A and other noncanonical U:G, U:U, and U:C pairs. In addition, the cn5U:G pair was found to be stronger than the cn5U:A pair and the other mismatched pairs in the context of a RNA duplex; this implied that cn5U might slightly prefer to recognize G over A. Our mechanistic studies by molecular simulations showed that the cn5U modification did not directly affect the base pairing of the parent nucleotide; instead, it weakened the neighboring base pair in the 5′ side of the modification in the RNA duplexes. Consistent with the simulation data, replacing the Watson–Crick A:U pair to a mismatched C:U pair in the 5′‐neighboring site did not affect the overall stability of the duplex. Our work reveals the significance of the electron‐withdrawing cyano group in natural tRNA systems and provides two novel building blocks for constructing RNA‐based therapeutics.

CHARACTERIZATION OF GLUTATHIONE-HOMOCYSTINE TRANSHYDROGENASE AS A NOVEL ISOFORM OF GLUTATHIONE S-TRANSFERASE FROM ASPERGILLUS FLAVIPES

Glutathione-homocystine transhydrogenase/oxidoreductase (GHTHase) is an enzyme that catalyzes the reversible oxidation/reduction of reduced glutathione (GSH) and homocystine (Hcy2) to oxidized glutathione and homocysteine (Hcy). GHTHase is implicated in regulation of the metabolism of homocysteine and glutathione. Aspergillus flavipes JF831014 exhibits the highest productivity of GHTHase, using GSH as electron donor and Hcy2 as acceptor. GHTHase yield from A. flavipes has been nutritionally optimized to reach the maximum activity (21.14 U/mg) using GSH (0.4%) combined with Hcy2 (0.01%) and glucose (0.4%), NADH + H (30 mM) at medium initial pH 7.8. The yield of GHTHase was increased about 1.2 times upon starvation of the culture of A. flavipes for two days, as compared to the non-sulfur starved culture. The GHTHase activity was increased by 13.7 fold with total yield of 8.8%. According to denaturing PAGE, GHTHase had 35 kDa, and 75 kDa by non-denaturing PAGE, gel-filtration and DLS analysis, ensuring its homodimeric identity. The enzyme displayed a highest activity at pH 6.5 – 7.6, 40°C, and pH stability within 6.0 – 8.0. GHTHase has higher affinity for GSH (Km = 14.3 mM) and cysteine (Km = 15.1 mM) as hydrogen donor for Hcy2 reduction, other than CDNB for glutathione S-transferase. The variant catalytic response to standard glutathione S-transferase substrates, esteem the unique catalytic properties of GHTHase. The enzyme was significantly inhibited by iodoacetate, hydroxylamine, and propargylglycine, revealing their sulfur active site dependence. Thus, a new isoform of glutathione S-transferase, GHTHase, with unique potency to reduce Hcy2 to soluble Hcy using GSH as hydrogen donor, was discovered. The specificity of GHTHase to oxidize toxic insoluble Hcy2 (cardiovascular disorder risk factor) can be a novel route to attack cardiovascular diseases.