Nagendra K. Sharma

Type II Isopentenyl Diphosphate Isomerase: Probing the Mechanism with Alkyne/Allene Diphosphate Substrate Analogues

Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the basic five-carbon building blocks of isoprenoid molecules. Two structurally unrelated classes of IDIs are known. Type I IPP isomerase (IDI-1) utilizes a divalent metal in a protonation-deprotonation reaction. In contrast, the type II enzyme (IDI-2) requires reduced flavin, raising the possibility that the reaction catalyzed by IDI-2 involves the net addition or abstraction of a hydrogen atom. As part of our studies of the mechanism of isomerization for IDI-2, we synthesized allene and alkyne substrate analogues for the enzyme. These molecules are predicted to be substantially less reactive toward proton addition than IPP and DMAPP but have similar reactivities toward hydrogen atom addition. This prediction was verified by calculations of gas-phase heats of reaction for addition of a proton and of a hydrogen atom to 1-butyne (3) and 1,2-butadiene (4) to form the 1-buten-2-yl carbocation and radical, respectively, and related affinities for 2-methyl-1-butene (5) and 2-methyl-2-butene (6) using G3MP2B3 and CBS-QB3 protocols. Alkyne 1-OPP and allene 2-OPP were not substrates for Thermus thermophilus IDI-2 or Escherichia coli IDI-1 but instead were competitive inhibitors. The experimental and computational results are consistent with a protonation-deprotonation mechanism for the enzyme-catalyzed isomerization of IPP and DMAPP.

Expanding the repertoire of pyrrolidyl PNA analogues for DNA/RNA hybridization selectivity: aminoethylpyrrolidinone PNA (aepone-PNA)

New PNA analogues derived from aminoethylpyrrolidin-5-one backbone show stabilization of aepone-PNA:DNA hybrids and destabilization of the corresponding RNA hybrids compared to unmodified PNA.

PNA C–C+i-motif: superior stability of PNA TC8 tetraplexes compared to DNA TC8 tetraplexes at low pH

Study of self-assembly of PNA TC8 monitored by UV thermal transition at 295 nm indicates formation of a C-C+ tetraplex (i-motif) in acidic pH, with higher stability than the analogous dTC8.

Instability of Amide Bond Comprising the 2-Aminotropone Moiety: Cleavable under Mild Acidic Conditions

An unusual hydrolysis/solvolysis of the classical acyclic amide bond, derived from N-troponylaminoethylglycine (Traeg) and α-amino acids, is described under mild acidic conditions. The reactivity of this amide bond is possibly owed to the protonation of the troponyl carbonyl functional group. The results suggest that the Traeg amino acid is a potential candidate for protecting and caging of the amine functional group of bioactive molecules via a cleavable amide bond.

Synthesis and spectroscopic studies of berberine immobilized modified cellulose material

This report describes the synthesis, characterization and spectroscopic studies of berberine immobilized modified cellulose materials, which could be a promising new biocompatible fluorescence material because berberine is a natural fluorescent molecule having important pharmacological aspects including selective binding with DNA G-quadruplex. Thus, berberine immobilized cellulose materials could be applicable in the screening of G-quadruplexing DNA sequence by bio-imaging techniques.

Helical supramolecular self-assembly by prolamide thymidine/uridine analogues

ABSTRACT This report describes the syntheses of rationally designed non-sugar nucleoside as prolamide nucleosides which contain prolyl ring and pyrimidine nucleobases (uracil/thymine) via acetamide bonds. These nucleosides have propensity to form distinctive self-assembly supramolecular helical structures ubiquitously through Watson-Crick/reverse type of hydrogen bonding with nucleobases. Moreover, the prolyl acetamide backbone groups- carbonyl (-C = O) and hydroxyl (-OH) group, are also involved in strengthening of self-assembled helical structures. Importantly, both prolamide thymidine and prolamide uridine have shown two distinctive helical structural patterns, in spite of containing the same backbone. Hence thymine and uracil moieties of prolamide nucleosides are responsible for unique supramolecular helical structural architectures. Graphical Abstract

Hybrid DNA i-motif: Aminoethylprolyl-PNA (pC5) enhance the stability of DNA (dC5) i-motif structure

This report describes the synthesis of C-rich sequence, cytosine pentamer, of aep-PNA and its biophysical studies for the formation of hybrid DNA:aep-PNAi-motif structure with DNA cytosine pentamer (dC5) under acidic pH conditions. Herein, the CD/UV/NMR/ESI-Mass studies strongly support the formation of stable hybrid DNA i-motif structure with aep-PNA even near acidic conditions. Hence aep-PNA C-rich sequence cytosine could be considered as potential DNA i-motif stabilizing agents in vivo conditions.

Dideoxy nucleoside triphosphate (ddNTP) analogues: Synthesis and polymerase substrate activities of pyrrolidinyl nucleoside triphosphates (prNTPs)

The dideoxynucleoside triphosphates (ddNTPs) terminate the bio-polymerization of DNA and become essential chemical component of DNA sequencing technology which is now basic tool for molecular biology research. In this method the radiolabeled or fluorescent dye labeled ddNTP analogues are being used for DNA sequencing by detection of the terminated DNA fragment after single labeled ddNTP incorporation into DNA under PCR conditions. This report describes the syntheses of rationally designed novel amino-functionalized ddNTP analogue such as Pyrrolidine nucleoside triphosphates (prNTPs), and their polymerase activities with DNA polymerase by LC-MS and Gel-electrophoretic techniques. The Mass and PAGE analyses strongly support the incorporation of prNTPs into DNA oligonucleotide with Therminator DNA polymerase as like control substrate ddNTP. As resultant the DNA oligonucleotide are functionalized as amine group by prNTP incorporation with polymerase. Hence prNTPs provide opportunities to prepare demandable conjugated DNA with other biomolecules/dyes/fluorescence molecule without modifying nucleobase structure.

Methylene Salicylicacidyl Hexamer (MSH) Has DNAse Activity

AbstractSalicylic acid and formaldehyde form heterogenous methyl/methylene salicylicacidyl oligomers and polymers in presence of sulfuric acid (H2SO4) and sodium nitrite (NaNO2). One of the oligomers as aurintricarboxylic acid (ATA), methelene bridged salicylic acid trimer, has been identified and explored in biochemical research, which is a potent inhibitor of many biological processes. A very few reports are also available on dimer, trimer, and tetramer of methelene bridged salicylic acids from same reaction mixture. Herein, we report the isolation and biochemical screening of partial purified low-molecular component as methylene salicylicacidyl hexamer (MSH) from the above reaction mixture. The interaction of methylene salicylicacidyl oligomer with DNA was studied by agarose and polyacrylamide gel electrophoresis, which suggest that methylene salicylicacidyl oligomer has DNAse activity. So far, no such significant reports are available on low-molecular oligomer of methelene bridged salicylic acids. In further, we also attempted to investigate the nature of nuclease activity, which clearly indicates DNA exonuclease type of activity. Further studies are needed to establish the mechanism of actions. Graphical AbstractSynthesis of methylene salicylic acic (MSH) and DNAase studies