Thermally reversible and irreversible interstrand photocrosslinking of 5-chloro-2'-deoxy-4-thiouridine modified DNA oligonucleotides.

We describe highly efficient interstrand photocrosslinking of a DNA duplex containing 5-chloro-2'-deoxy-4-thiouridine (ClSdU) in one strand, proceeding via a two-step photochemical cascade, involving the formation of a thermally reversible crosslink between ClSdU and thymidine in the target strand and its subsequent conversion to a thermally stable fluorescent crosslink. These results show that ClSdU has great potential to be a valuable DNA photo-crosslinking reagent for chemical biology applications.

Photochemical behavior of 2-azidopurine tri-O-acetylribonucleoside in aqueous solution: unprecedented transformation into 1-(5'-O-acetyl-ß-D-ribofuranosyl)-5-[(2-oxo-1,3,5-oxadiazocan-4-ylidene)amino]-1H-imidazole-4-carbaldehyde.

The photochemical behavior of 2-azidopurine 2',3',5'-tri-O-acetylribonucleoside has been investigated in aqueous solution under aerobic and anaerobic conditions. The two major processes under anaerobic irradiation of 2-azidopurine 2',3',5'-tri-O-acetylribonucleoside involve unprecedented transformation into 1-(5'-O-acetyl-ß-D-ribofuranosyl)-5-[(2-oxo-1,3,5-oxadiazocan-4-ylidene)amino]-1H-imidazole-4-carbaldehyde and photoreduction to respective 2-aminopurine derivative, whereas under aerobic conditions these two processes occur to a much lesser extent and photooxidation to respective 2-nitropurine derivative dominates. The structures of photoproducts formed were confirmed by NMR and high-resolution electrospray ionization mass spectral data.

Highly efficient fluorescent interstrand photo-crosslinking of DNA duplexes labeled with 5-fluoro-4-thio-2'-o-methyluridine.

The formation of a fluorescent photoadduct between 5-fluoro-4-thiouridine ((FS) U), in the sequence context 5'-A(FS) UA-3' and incorporated into a synthetic oligonucleotide either at its 3'- or 5'-end, and one of the thymines of the TAT motif in a complementary target DNA strand led to photo-crosslinking of the two strands for several oligonucleotide constructs. Enzymatic digestion, MS, UV, and fluorescence spectral analyses of the interstrand crosslinked oligonucleotides revealed the identity of the thymidine that participates in the photo-crosslinking reaction as well as the diastereomeric structures of the crosslinks. The proposed pathways of interstrand photo-crosslinking are supported by experiments with isotopically labeled oligonucleotide constructs and visualized by means of molecular dynamics simulations.

Photochemistry of 6-amino-2-azido, 2-amino-6-azido and 2,6-diazido analogues of purine ribonucleosides in aqueous solutions.

The photochemistry of 6-amino-2-azidopurine, 2-amino-6-azidopurine and 2,6-diazidopurine ribonucleosides has been investigated in aqueous solutions under aerobic and anaerobic conditions. Near UV irradiation of 6-amino-2-azido-9-(2',3',5'-tri-O-acetyl-ß-D-ribofuranosyl)purine and 2-amino-6-azido-9-(2',3',5'-tri-O-acetyl-ß-D-ribofuranosyl)purine in the presence of oxygen leads to efficient formation of 6-amino-2-nitro-9-(2',3',5'-tri-O-acetyl-ß-D-ribofuranosyl)purine and 2-amino-6-nitro-9-(2',3',5'-tri-O-acetyl-ß-D-ribofuranosyl)purine. Under anaerobic conditions, both azidopurine ribonucleosides preferentially undergo photoreduction to 2,6-diamino-9-(2',3',5'-tri-O-acetyl-ß-D-ribofuranosyl)purine. The structures of the photoproducts formed were confirmed by UV, NMR and HR ESI-TOF MS spectral data. The photoproducts observed in this study for the aminoazidopurines are distinctly different from those observed previously for 6-azidopurine. When no amino group is present, the photochemistry of 6-azidopurine leads to the formation of a 1,3,5-triazepinone nucleoside. The energetics of the 6-nitreno moiety along both oxidation and ring expansion pathways was calculated using the nudged elastic band (NEB) method based on density functional theory (DFT) using DMol3. The role of the 2-amino group in regulating the competition between these pathways was elucidated in order to explain how the striking difference in reactivity under irradiation arises from the greater spin density on the 6-nitreno-9-methyl-9H-purin-2-amine, which essentially eliminates the barrier to oxidation observed in 6-nitreno-9-methyl-9H-purine. Finally, the importance of tetrazolyl intermediates for the photochemical activation of azide bond cleavage to release N2 and form the 6-nitreno group was also corroborated using the DFT methods.

Photoaddition of 5-bromouracil to uracil in oligonucleotides leading to 5,5'-bipyrimidine-type adducts: mechanism of the photoreaction.

5-Bromouracil ((Br)U) modified di- and hexanucleotides having (Br)U flanked on the 5' or the 3' side by uracil (U) have been synthesized, and their photochemical reactivity was examined under the conditions of irradiation with near UV light. The results indicate that the primary photochemical process in all of these compounds involves the formation of an intermediate cyclobutane phodoadduct composed of (Br)U and U, which undergoes further photochemically and thermally induced transformations to 5,5'-bipyrimidine type adducts.

5-Fluoro-4-thiouridine phosphoramidite: new synthon for introducing photoaffinity label into oligodeoxynucleotides.

The synthesis of phosphoramidite of 5-fluoro-4-thio-2'-O-methyluridine is described. An appropriate set of protecting groups was optimized including the 4-thio function introduced via 4-triazolyl as the 4-(2-cyanoethyl)thio derivative, and the t-butyldimethyl silyl for 2' and 3' hydroxyl protection, enabling efficient synthesis of the phosphoramidite. These protecting groups prevented unwanted side reactions during oligonucleotide synthesis. The utility of the proposed synthetic route was proven by the preparation of several oligonucleotides via automated synthesis. Photochemical experiments confirmed the utility of the synthon.

Photoinduced fluorescent cross-linking of 5-chloro- and 5-fluoro-4-thiouridines with thymidine.

Two highly fluorescent, thermally stable diastereomeric photoadducts, 3a,b, are formed when either 5-chloro-4-thiouridine, 1, or 5-fluoro-4-thiouridine, 2, are photoexcited with 366 nm UV light in the presence of thymidine (T). 5-Fluoro-4-thiouridine, 2, exhibits photoreactivity much higher than that of the 5-chloro derivative 1. In both cases the photoreaction is very clean, leading to highly eficient conversion of the 5-halogeno-4-thiouridine (1, 2) and T to photoadducts 3a,b. The identity and structure of 3a was confirmed using mass spectrometry and 2-D NMR. The epimeric relationship of 3a,b was established by UV circular dichroism spectroscopy. 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.

Generation of thiyl radicals by the photolysis of 5-iodo-4-thiouridine.

The photochemistry of 2',3',5'-tri-O-acetyl-5-iodo-4-thiouridine (3) in deoxygenated 1:1 CH(3)CN-H(2)O pH 5.8 (phosphate buffer) solution has been studied by means of steady-state and nanosecond laser flash photolysis methods. Under steady-state irradiation (lambda > or = 334 nm), the stable photoproducts were iodide ion, 2',3',5'-tri-O-acetyl-4-thiouridine (4), and two disulfides. The disulfides were the symmetrical bis-(2',3',5'-tri-O-acetyl-5-iodo-4-thiouridine) (5) and unsymmetrical 6, which contains both 4-thiouridine and 5-iodo-4-thiouridine residues. The formation of the dehalogenated photoproduct suggests that C(5)-I bond cleavage is a primary photochemical step. Attempts to scavenge the resulting C(5)-centered radical by suitable addends, bis-(N-alpha-acetyl)cystine-bis-N-ethylamide or benzene, were unsuccessful. Analysis of the photoproducts formed under these conditions showed that the S-atom is the reactive center. The photoproduct 4, obtained by irradiation of 3 in CD(3)CN-H(2)O, followed by reversed-phase HPLC isolation using nonlabeled eluents, did not contain deuterium. An analogous experiment performed in CH(3)CN-D(2)O gave deuterated product 4-d with 88% of the deuterium incorporated at C(5). Transient absorption observed upon laser excitation (lambda= 308 nm) of 3 was assigned to the 4-uridinylthiyl radical on the basis of the similarity of this spectrum with that obtained upon laser photolysis of the disulfide: bis-(2',3',5'-tri-O-acetyl-4-thiouridine) 14. On the basis of the results of steady-state and laser photolysis studies, a mechanism of the photochemical reaction of 3 is proposed. The key mechanistic step is a transformation of the C(5)-centered radical formed initially by C(5)-I bond cleavage into a long-lived S-centered radical via a 1,3-hydrogen shift. Theoretical calculations confirmed that the long-lived S-centered radical is the most stable radical derived from the 4-thiouracil residue.

Solvatochromism of a novel betaine dye derived from purine.

A novel solvatochromic betaine dye has been synthesized from xanthosine and characterized spectroscopically by UV-vis in a broad range of solvents. The dye 9-(2',3',5'-tri-O-acetyl-beta-d-ribofuranosyl)-2-(pyridinium-1-yl)-9H-purin-6-olate, 1a, exhibits solvent-induced spectral band shifts that are (2)/(3) as large as that of the betaine known as Reichardt's dye, which forms the basis of the E(T)(30) solvent polarity scale. Moreover, the dye 1a is a ribonucleoside and hence has the potential application as a polarity probe for application in RNA oligonucleotides. The isomeric dye 6-(pyridinium-1)-yl-9H-purin-2-olate, 2a, has also been synthesized and exhibits slightly smaller solvatochromic band shifts. The new betaine dyes have also been studied by comparing the experimental and calculated solvatochromic shifts based on the calculation of the UV/vis absorption spectra, using a combination of methods with density functional theory (DFT). The COSMO continuum dielectric method, an applied electric field term in the Hamiltonian, and time-dependent density functional theory (TD-DFT) methods were used to obtain absorption energies, ground-state dipole moments, and the difference dipole moment between the ground and excited states. The calculations predict a lower energy absorption band of charge-transfer character that is highly solvatochromic, and a higher energy absorption band that has pi-pi character which is not solvatochromic, in agreement with the experimental data. For Reichardt's dye the difference dipole moment between the ground and excited state (Deltamu = mu(e) - mu(g)) was also calculated and compared to experiment: Deltamu(calcd) = -6 D and Deltamu(exptl) = -9 +/- 1 D.(1) The ground-state dipole moment was found to be mu(g)(calcd) = 18 D and mu(g)(exptl) = 14.8 +/- 1.2 D.(1).

Intra- and intermolecular electronic relaxation of the second excited singlet and the lowest excited triplet states of 1,3-dimethyl-4-thiouracil in solution.

Intramolecular processes of deactivation of 1,3-dimethyl-4-thiouracil (DMTU) from the second excited singlet (S2) (pi, pi*) and the lowest excited triplet (T1) (pi, pi*) states have been studied using perfluoro-1,3-dimethylcyclohexane (PFDMCH) as a solvent. The spectral and photophysical (PP) properties of DMTU in CCl4, hexane and water have also been described. For the first time, the fluorescence from S2 state DMTU has been observed. The picosecond lifetime of DMTU in the S2 state (tau(S2)) in PFDMCH has been proposed to be determined by a very fast intramolecular reversible process of hydrogen abstraction from the ortho methyl group by the thiocarbonyl group. The shortening of tau(S2) in CCl4 is interpreted to be caused by the intermolecular interactions between DMTU (S2) and the solvent. Results of the phosphorescence decay as a function of DMTU concentration were analyzed using the Stern-Volmer formalism, which enabled determination of the intrinsic lifetime of the T1 state (tau0(T1)) and rate constants of self-quenching (k(sq)). The lifetimes, tau0(T1), of DMTU in PFDMCH and CCl4 are much longer than the values hitherto obtained in more reactive solvents. The PP properties of DMTU both in the S2 and T1 states have been shown to be determined by the thiocarbonyl group.