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The reactions involving the 5-hexenyl or the 6-heptenyl radical cyclization of a distant double bond at C4' and the radical center at C2' of the ribofuranose ring of thymidine are used for synthesizing North-type conformationally constrained cis-fused bicyclic five-membered and six-membered carbocyclic analogues of LNA and ENA. Enhanced lifetimes have made the carbocyclic-LNA and carbocyclic-ENA modifications very good candidates as potential antisense therapeutic agents.

The molecular structures and dynamics of 1',2'-azetidine- and 1',2'-oxetane-fused thymidines, as well as their 2',4'-fused thymine (T) counterparts are determined to understand how the chemical nature of the conformational constraints of the sugar moiety in ON/RNA(DNA) dictates the duplex structure and reactivity. The results have shown that the nature of the sugar conformational constraints steer the physicochemical property of the constraint 1-thyminyl moiety, which plays a vital part in tuning the strength of hydrogen bonding in the basepairing.

The study describes the design, synthesis and phosphorylation pattern of the new bicyclo[3.1.0]hexane template that exhibits highly strong and increased chemical and enzymatic stability due to the absence of a true glycosyl bond. The template is found to be extremely rigid that makes it useful for sculpting other carbocyclic nucleosides, which also makes it easier to interact with the herpes simplex thymidine kinase.

The X-ray crystal structure determination technique is employed to study the structure of the independently generated C5'-nucleosidyl radicals in thymidine and 2'-deoxyguanosine, which are found to be highly unstable. The results reveal that the process of cyclization of the 2'-deoxyguanosin-5'-yl radical has an extremely high rate constant, as it is highly stereoselective in nature.

The synthesis of 2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T), physicochemical, nuclear magnetic resonance (NMR) and computational structural studies of aza-ENA-T and biochemical studies of aza-ENA-T containing oligonucleotides are reported. The aza-ENA-T modification in the antisense oligonucleotide (AON) has improved RNA target affinity, comparable RNase H cleavage rate and higher blood serum stability.

The practical synthesis of the 2'-deoxy-4'-thioribonucleosides derivatives which would be easily converted into the corresponding phosphoramidite units for 4'-thioDNA synthesis is investigated. The desired compounds are synthesized from 4'-thioribonucleoside derivatives through bromination, followed by radical reduction at room temperature.

Reporting the synthesis of a 1'-aminomethylthymidine, together with that of oligonucleotides containing this nucleoside, prepared via the 3'-phosphoramidite of the aminomethylnucleoside. The synthesis of the oligonucleotides included the conversion of residues in the interior of protected oligonucleotides on solid supports in microwave-assisted conversions.

Recent work has focused on developing Raman spectroscopy as a noninvasive probe of DNA interactions with solvents, intercatants, proteins, and other ligands. Here, we report the Raman spectra of thymine in eight solvents and thymidine in nine solvents obtained with visible excitation. Raman spectra under acidic, neutral, and basic conditions were also obtained of both thymine and thymidine. Changes in both the frequencies and intensities of several of the vibrational bands in the 800-1800 [cm.sup.-1] region are observed. No evidence of deprotonation in the different solvents is observed for either thymine or thymidine. Correlations of the observed frequency shifts of specific vibrational modes with characteristic properties of the solvent for both thymine and thymidine show a significant correlation with acceptor and donor numbers, measures of the hydrogen-bonding ability of the solvent, in both thymine and thymidine. These results are interpreted in terms of hydrogen-bonding interactions between the N-H protons of the thymine base and lone pairs of electrons on the solvent molecules and between the solvent hydrogens and lone pairs on C=O sites. The solvent-dependent intensity in vibrational bands of thymine between 1500 and 1800 [cm.sup.-1] indicates a strong interaction between thymine and solvent at the C=O and N-H sites that leads to separation of the C=O stretches from the C=C stretch. The intensity variations with solvent were much smaller for thymidine than for thymine, perhaps as a result of replacing the [N.sub.1] proton by the sugar. These results suggest that Raman spectroscopy is uniquely sensitive to specific interactions of thymine and thymidine with their environment. Key words: Raman spectroscopy, thymine, thymidine, solvent effects, hydrogen bonding.

Two isomers of methanocarba (MC) thymidine are used to determine whether kinases and polymerases discriminate between their substartes on the basis of sugar conformation. A combined solid-state and solution conformational analysis of both compounds, coupled with the direct measurement of mono suggests that kinases prefer substrates that adopt the S sugar conformation.

The synthesis and spectroscopic properties of a novel Ru(bpy(sub 2))(sub 2)(4-m-4'-cam-bpy)(super 2+) are reported. Incorporation of this ruthenium-thymidine in an oligodeoxynucleotide does not significantly change the duplex structure and the favorable photophysical properties of Ru(bpy(sub 2))(sub 2)(4-m-4'-cam-bpy)(super 2+) complex are retained when the complex is covalently attached to DNA.

2',4'-Propylene-bridged thymidine (carba-ENA-T) and five 8'-Me/N[H.sub.2]/OH modified carba-ENA-T analogues are prepared through intramolecular radical addition to C[double bond]N of the tethered oxime-ether. The RNA strand in all of the carba-ENA derivatives modified oligodeoxynucleotides (AON):RNA hybrid duplexes is digested by RNase H1 with high efficiency, even at twice the rate of those of the native and ENA modified counterpart.

A five-enzyme in vitro pathway is used for verifying that ORF36 has catalyzed oxidation of biogenic thymidine diphosphate (TDP)-L-epi-vancosamine and for determining whether ORF36 has displayed catalytic competence for any of its biosynthetic progenitors, which are candidate substrates for nitrososynthases in vivo. The studies have mapped substrate determinants and have described a minimal monooxygenase mechanism for amino sugar oxidation by ORF36.

Multiple kinetic isotope effect (KIE) measurements were used to characterize the transition state of human thymidine phosphorylase (hTP) which is responsible for thymidine (dT) homeostasis and angiogenesis activity. Transition state analysis revealed a stepwise mechanism with a 2-deoxyribocation formed early and a higher energetic barrier for nucleophilic attack of a water molecule on the high energy intermediate and predicted that deoxyribose adopted a mild 3'-endo conformation during nucleophilic capture.

A new class of conformationally constrained nucleosides, [alpha]-L-ribo-carbocyclic LNA thymidine ([alpha]-L-carba-LNA-T (LNA = locked nuclei acid) analogues and a novel ' double-locked' [alpha]-L-ribo-configured tetracyclic thymidine in which both the sugar puckering and glycosidic torsion are simultaneously constrained, are prepared by a key step involving 5-exo free-radical intramolecular cyclization. The improved nuclease resistances and efficient RNase H recruitment capabilities have elevated the [alpha]-L-carba-LNA-modified nucleotides into a new class of locked nucleic acids for RNA targeting therapeutics.

The crystal structure of human deoxycytidine kinase (dCK) in complex with the cytidine analogue 5-methyldeoxycytidine (5-Me-dC) is solved in order to understand the determinants preventing thymidine phosphorylation by wild-type (WT) dCK. The studies have shown that it is in fact residue Asp133 of dCK that is most responsible for discriminating against the thymine base and dCK variants have improved activity with 5-substituted deoxycytidine and thymidine analogoues.

Photochromic nucleosides are designed that have combined the structural features and molecular recognition properties of nucleic acids with the light-sensitivity of diarylethenes, where the target compounds have a 7-deazaadenosine unit linked to a thiophene as the second aryl functionality via a 1,2-cyclopentenyl linker. The molecular switch is developed that has displayed both photochromism and acidichromism, whereby showing the possibility to gate the spectral properties to multiple stimuli.

The inactive conformation of the loop of residues 181-197 which prevents substrate binding was stabilized to demonstrate the replacement of V3L in human thymidylate synthase which affects its kinetic properties and intracellular stability. The mechanism leading to the high [K.sub.m,app] values in the crystal structure of a V3F*FdUMP binary complex could be applied to facilitate the design of compounds acting as allosteric inhibitors of hTS.

The [super]BrdU-bearing G-quadruplex DNA was exposed to UVA light to gain insight into the efficient formation of the G[8-5]U intrastrand cross-link, where the C8 of guanine in the external G-tetrad is covalently linked with the C5 of its adjacent 3' uracil in the loop region. The formation of intrastrand cross-link in G-quadruplex motifs might account for the photocytotoxic effect induced by [super]BrdU incorporation, and the [super]BrdU-mediated photo-cross-linking might constitute a useful method for monitoring the different conformations of G-quadruplex folding.

The synthesis of the novel 2-Se-thymidine ([super Se]) derivative, its phosphoramidite, and the Se-DNAs is described. The studies have provided a novel tool to examine the specific recognition of base pairs, which is the basis of high fidelity during replication, transcription and translation.

Two highly fluorescent, thermally stable diastereomeric photoadducts are formed when either 5-chloro-4-thiouridine (1), or 5-fluoro-4-thiouridine (2) are photoexcited with UV light in the presence of thymidine (T). The studies have shown that the geometry of the fluorescent photoadduct is consistent with formation of an interstrand cross-link in a DNA duplex if 1 or 2 is flanked by T in an opposite strand.

A novel substrate for cyclobutane pyrimidine dimer (CPD) photolyase that absorbs only weakly around 265 nm: a modified thymidine 10-mer with a central CPD and all bases, except the one at the 3' end, replaced by 5,6-dihydrothymine which virtually does not absorb around 265 nm is reported. The novel substrate attributed with low background absorption around 265 nm and the defined structure could be used as a promising tool for fast and ultrafast transient absorption studies on pyrimidine dimer splitting by CPD photolyase.

The first chemically characterized DNA radical-mediated cross-link resulting from formal abstraction of a hydrogen atom from the thymidine methyl group, 5-(2'-deoxyuridinyl)methyl radical with the opposing 2'-deoxyadenosine is reported. This is the first example of double-strand cleavage during nucleotide excision repair of cross-linked DNA that does not already contain a strand break in the vicinity of the cross-link.

The photoelectron spectroscopy measurements in aqueous microjets were used to obtain vertical ionization energies of the nucleosides cytidine and deoxythymidine in water. Accurate aqueous-phase vertical ionization potentials provide a significant improvement to the corrected gas-phase values and could be applied to assess the threshold energies for photooxidation and oxidation free energies of solvent-exposed DNA components as well as delocalization and charge-hopping mechanisms in DNA ionized by radiation.

Nonpolar nucleoside analogues of systematically varied size and shape are used for probing the mechanisms by which the two human thymidine kinases (TK1 and TK2) recognize and phosphorylate their substrate, thymidine. The results have shown that nucleoside recognition mechanisms for the two classes of enzymes are very different and nonpolar nucleosides are likely to be active in the nucleotide salvage pathway in human cells.

The bacteriophage RB69 DNA polymerase (RB69 pol) and the metal ion exchange-inert nucleotide analogue rhodium(III) deoxythymidine triphosphate (Rh*dTTP) are used to explore the requirements of metal binding to the 'A' site and to the 'B' site, independently. The implications of data obtained for the reverse rate of the enzyme isomerization, closed to open, of a DNA polymerase complex are also discussed for the fidelity of DNA polymerization by RB69 pol.

The kinetic analysis of human deoxycytidine kinase (dCK) variants is performed to mutate and select the optimal residues at position 104 and an alanine at position 133. The unique catalytic properties observed for several of the mutants suggested them to be good candidates for suicide-gene/protein-therapy applications.

The new mechanisms are described by which Q509L in HIV-1 reverse transcriptase (RT) has increased zidovudine (AZT) resistance when combined with the thymidine analogue mutations D67N, K70R and T215F. The studies have shown how a mutation in the ribonuclease H (RNase H) domain of RT increases AZT resistance and have highlighted how the polymerase and RNase H domains of RT function in concert to confer drug resistance.

The studies of the interstrand cross-links produced by phenylselenyl substituted derivatives of thymidine and 5-methyl-2'-deoxycytidine in duplex DNA when oxidized by Nal[O.sub.4] is reported. The versatile cross-linking reactions of thymidine and 5-methyl-2'-deoxycytidine molecules could be used as mechanistic probes and tools for biotechnology.

The time-dependent density functional theory (TDDFT) is used for studying the lowest excited states of the 5'-thymidine monophosphate (5'-dTMPH) radical anion as a model of DNA. A [pi]* and [sigma]* coupling has led to a rapid dissociative process that has resulted in DNA strand cleavage.

A study was conducted to investigate the excited state dynamics of 4-thiothymidine ([S.sub.4]-TdR) and its photosensitization to molecular oxygen in solution with UVA irradiation. The photosensitization from triplet ([S.sub.4]-TdR) has given rise to singlet oxygen [O.sub.2] with a quantum yield of 0.50 in the presence of molecular oxygen.

The density functional theory treatment of a simple model for DNA, the nucleotide, 5'-thymidine monophosphate (5'-dTMPH) was employed to investigate the mechanisms of low-energy electron (LEE) attachment and subsequent single-strand break (SSB) formation. The results supports the hypothesis that transiently bound electrons (shape resonances) to the virtual molecular orbitals of the neutral molecule likely play a major role in the cleavage of the sugar-phosphate [C.sub.5']-[O.sub.5'] bond in DNA, resulting in the direct formation of single strand breaks without significant molecular relaxation.

Function and structure analysis investigates the Se derivatization on the nucleobases. The first synthesis of the 4-Se-thymidine phosphoramidite, its incorporation, and the thermostability and crystal structure studies of the DNAs containing 4-Se-thymidine changes, is described.

The photochemical behavior of the 3'-N-sulfamate analogue of thymidylyl(3'-5')thymidine (TnsoT) using a major C'3'-endo sugar pucker and an increased base stacking ability when compared to its natural counterpart thymidylyl(3'-5')thymidine (TpT) is investigated. The results have shown that the cyclobutane pyrimidine dimer (CPD) and the oligonucleotides containing (6-4) photoproduct surrogates arise from distinct minor stacked conformations.

A sensitive probe of the nature of the H-transfer step is applied in search of the effect of W80 substitution to M in exocyclic thymidylate synthase (ecTS). The findings are in accordance with an alteration of the pre- and reorganization of the system toward and after the H-transfer step, but no significant in the H-transfer step per se.

An accessible and simple fluorescent dT analogue is demonstrated to effectively probe the DNA microenvironment and display different emission in a duplex versus single-stranded oligonucleotides. The synthesis and photophysical characteristics of a series of simple and responsive thymidine analogues, where a 2'-deoxy-U core is conjugated to aromatic five-membered heterocycles, including furan, thiophene, oxazole, thiazle is described.

A series of tridentate ligands derived from the nucleosides thymidine and uridine is developed starting from the amine analogues. The complexes provide novel potential inhibitors for nucleoside kinases as well as thymidine analogues, endowing them with suitable properties for therapeutic and diagnostic applications.

A new method of inhibiting thymidylate synthase in cells instead of substrate analogue inhibitors is presented. The experiments suggest that a similar dominant-negative response to the DM of TS could be affected in tumor cells, for which preliminary evidence is described.

L-thymidine (L-TdR) a substrate for herpes simplex virus type 1 thymidine kinase (HSV-1 TK), reduces HSV-1 multiplication in HeLa cells. HSV-1 TK phosphorylates the L and D enantiomers of TdR to their corresponding monophosphates (MPs) with identical efficacy.

Several experiments are conducted to reveal a new two-stage one-pot method for the enzymatic biosynthesis of TDP-L-mycarose from the readily available precursors, glucose-1-phosphate and thymidine. The process described demonstrates the use of enzymes in the synthesis of several unstable TDP-sugar derivatives and hence can be widely used for the identification of more such enzymes in addition to the synthesis of other unusual sugars.

The enhanced base-pairing and the effect of the thio groups at the 2- and 4-positions on the hybridization properties of DNAs containing them and on DNA replication as well are evaluated. Increased stability and hybridization selectivity of 2-thiothymidine deoxyribosides (2S) and the high efficiency of polymerase replication of both isomers might prove useful in a number of applications.

The enzymatic synthesis of two Pse DNA isomers by using the two alpha-Se-TTP diastereomers(Sp and Rp) is studied. The quantitative enzymatic approach is useful for synthesis of longer DNAs with multiple Pse groups in large scale for X-ray crystal structure determination of DNAs and DNA-protein complexes by the MAD phasing technique.

A conceptually novel dinucleotide cleavage strategy that in principle could be applicable to all nonrepeating dinucleotide sequences is developed. Sequence-specific, synergistic interactions between 5'-amino-5'-deoxythymidine and ribonucleotides are used to demonstrated that specific dinucleotide cleavage may be achieved through the use of appropriately modified nucleotides.

The mechanism of hydroxide catalysis for phosphodiester hydrolysis by examining (super D2O)k, ionic strength effects, and (super 18)k(sub nuc) to probe the hydroxide-catalyzed hydrolysis of thymidine-5'-p-nitrophenyl phosphate (T5-PNPP), is investigated. The results are found to be most consistent with a direct nucleophilic attack by hydroxide, making phosphodiester hydrolysis distinct from both ester and amide hydrolysis.

Researchers have reported 5'-acylamido-substituted templates that are capable of accelerating the incorporation of deoxyadenosine , deoxyguanosine and thymidine residues in single nucleotide extensions. The reaction rates measured are far from the diffusion limit and cholic acid residue is devoid of cationic groups providing electrostatic attraction or electrostatic catalysis and indicates that the catalytic microenvironment set-up by this residue can be improved.

The adaptation of the strategy of caged thymidine nucleobases to aptamer technology is described. Light induced aptamers will provide an opportunity for the spatial and temporal control of bioactive molecules and will enhance the application of aptamers in the context of cellular and biological environments.