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o-Nitrobenzyloxycarbonyl and a number of related groups have been tested for the photolabile protection of nucleoside 5'-hydroxyls. The rates of photodeprotection were found to vary by approximately 17-fold in a series of 5'-O-protected thymidine derivatives irradiated at 365 nm under identical conditions. The homologous 2-(o-nitrophenyl)ethoxycarbonyl group and its derivatives were found to be removed approximately 2-fold faster than the corresponding o-nitrobenzyloxycarbonyl group, possibly due to an increased rate of o~-hydrogen abstraction by the photo-excited nitro group. Photolysis rates were affected by substitutions on both the phenyl ring and m-carbon, with the strongest rate enhancements caused by the presence of a methyl or second o-nitrophenyl group in the tx-position. Among the ring-substituted derivatives studied, o-nitro and o-iodo had the strongest enhancement effects on photodeprotection, while an o-fluoro group reduced the rate of photodeprotection. In general, substitutions at other positions on the phenyl ring had less effect on photolysis rates. © 1997 Elsevier Science Ltd.The use of photolabile protecting groups in nucleic acid 1-8, carbohydrate 9, and peptide 10-12 chemistry has been well established. More recently, photolabile protection of the 5'-hydroxyl of 2'-deoxyribonucleoside 3'-phosphoramidites has been employed in the solid-phase synthesis of DNA probe arrays. 13-16 Useful photolabile protecting groups must be stable to mild chemical treatments, but photolytically cleaved in high yield by irradiation at wavelengths which do not damage the protected molecule.The o-nitrobenzyl group or groups containing this photosensitive moiety have been used for photolabile protection of hydroxyl, carboxyl, amino, thiol, and carbonyl functions. 17 The removal of photolabile groups from a protected hydroxyl oxygen by irradiation at wavelengths >320 nm involves abstraction of a hydrogen from the m-methylene carbon by the excited nitro group followed by rearrangement to o-nitrosobenzaldehyde and the deprotected alcohol. In the case of the 2-nitrobenzyloxycarbonyl group, carbon dioxide is also released (Fig. 1). The quantum yield of photodeprotection may be strongly influenced by substitution on the phenyl ring or methylene carbon of the o-nitrobenzyl group. Reichmanis et al. 18 reported a 5-fold increase in quantum yield for photocleavage of the o-nitrobenzyl esters of trimethylacetic acid when the parent ester is substituted with an s-methyl group. 4247

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Dedicated with best wishes to Prof. Edgar Heilbronner on the occasion of his 80th birthdayThe photochemistry of several 2-(2-nitrophenyl)ethyl-caged compounds including caged thymidine nucleosides was studied by nanosecond laser flash photolysis and stationary illumination experiments with quantitative HPLC analysis for quantum yields and product distribution. Effects of solvent basicity and acidity were investigated by varying the H 2 O content and HCl concentration, respectively, in MeCN/H 2 O mixtures. For all compounds 1 ± 7 investigated, intramolecular H abstraction by the nitro group from the exocyclic a-position with respect to the aryl moiety was found to be the primary process. The protolytic dissociation equilibrium of the resulting aci-nitro compound was kinetically characterized in the 0.1 ± 10 ms time region. In general, two reaction channels compete for the aci-nitro compound and its anion: b-elimination of the caged compound occurs from the anion, while from the undissociated aci-nitro compound, a nitrosobenzene derivative is formed with no release of the caged compound. The yield ratio of these two reaction channels can be controlled through shifts in the protolytic dissociation equilibrium of the aci-nitro compound. In solutions with either low basicity (H 2 O-free MeCN) or high acidity (higher concentration of HCl in H 2 O/MeCN), two as yet unidentified products are formed, each one specifically for one of the mentioned conditions. Introduction. ± Photolabile protecting groups play an important role in synthetic organic chemistry [1], for caging of biologically active molecules [2], and for lightdirected, combinatorial solid-phase syntheses of biopolymers [3]. A particularly attractive application is the generation of so-called high-density DNA chips [4] [5] which are needed in various types to perform the sequencing by hybridisation (SBH) method for oligonucleotide and gene sequencing [6]. The o-nitrobenzyl (oNB) type of protecting groups has been commonly used since 1901 when the photoreaction was discovered [7], and its reaction mechanism has been investigated in detail [8]. So far, the [(a-methyl-2-nitropiperonyl)oxy]carbonyl ([1-(6-nitro-1,3-benzodioxol-5-yl)-ethoxy]carbonyl; MeNPOC) group has been the preferred choice in DNA-chip production [4].The photochemical cleavage mechanism of the (o-nitrobenzyl)oxy function is triggered by the abstraction of a benzylic H-atom by the excited nitro group [9]. Recently, a new type of photolabile protecting group was developed on the basis of the [2-(2-nitrophenyl)ethoxy]carbonyl functionality [10] [11] which is cleaved by a lightinduced b-elimination process with formation of o-nitrostyrene, CO 2 , and the corresponding alcohol. It was suggested [10] that, in analogy to the o-nitrobenzyl groups, the cleavage of the [2-(2-nitrophenyl)ethoxy]carbonyl moiety, too, should start with the formation of an aci-nitro intermediate, through H abstraction by the nitro group at the exocyclic a-position with respect to the aryl moiety. Based on results from laser f...

We have synthesized a highly fluorescent (quantum yield 0.88) guanosine analog, (3-methyl-8-(2-deoxy-beta-D-ribofuranosyl) isoxanthopterin (3-Mi) in a dimethoxytrityl, phosphoramidite protected form, which can be site-specifically inserted into oligonucleotides through a 3',5'-phosphodiester linkage using an automated DNA synthesizer. Fluorescence is partially quenched within an oligonucleotide and the degree of quench is a function of the fluorophore's proximity to purines and its position in the oligonucleotide. As an example of the potential utility of this class of fluorophores, we developed a continuous assay for HIV-1 integrase 3'-processing reaction by incorporating 3-MI at the cleavage site in a double-stranded oligonucleotide identical to the U5 terminal sequence of the HIV genome. Integrase cleaves the 3'-terminal dinucleotide containing the fluorophore, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. Substitution of the fluorophore for guanosine at the cleavage site does not inhibit integrase activity. This assay is specific for the 3'-processing reaction. The change in fluorescence intensity is linear over time and proportional to the rate of the reaction. This assay demonstrates the potential utility of this new class of fluorophore for continuous monitoring of protein/DNA interactions.

Water-soluble UV-A/B-absorbing pigments are secreted by cells of the cosmopolitan terrestrial cyanobacterium Nostoc commune. The pigments constitute a complex mixture of monomers with molecular masses of up to 1801 Da. Two different chromophores with absorption maxima at 312 and 335 nm are linked to different amino acids and to oligosaccharides consisting of galactose, glucose, xylose, glucuronic acid, and glucosamine. The 335 nm chromophore is a 1,3-diaminocyclohexen derivative, while the chromophore with an absorption maximum at 312 nm is most likely a 3-aminocyclohexen-1-one derivative. These UV-inducible substances are the first mycosporines to be described covalently linked to oligosaccharides. The pigments are located in the extracellular glycan sheath of Nostoc colonies, where they form complexes of extremely high molecular mass that are attached noncovalently to the glycan sheath. Pigments occur in concentrations that permit the cells to attenuate a significant part of incident UV-B radiation.

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Nitric oxide synthases (NOS) are homodimeric enzymes that NADPH-dependently convert L-arginine to nitric oxide and L-citrulline. Interestingly, all NOS also require (6R)-5,6,7,8-tetrahydro-L-biopterin (H 4 Bip) for maximal activity although the mechanism is not fully understood. Basal NOS activity, i.e. that in the absence of exogenous H 4 Bip, has been attributed to enzyme-associated H 4 Bip. To elucidate further H 4 Bip function in purified NOS, we developed two types of pterin-based NOS inhibitors, termed anti-pterins. In contrast to type II anti-pterins, type I anti-pterins specifically displaced enzyme-associated H 4 Bip and inhibited H 4 Bip-stimulated NOS activity in a fully competitive manner but, surprisingly, had no effect on basal NOS activity. Moreover, for a number of different NOS preparations basal activity (percent of V max ) was frequently higher than the percentage of pterin saturation and was not affected by preincubation of enzyme with H 4 Bip. Thus, basal NOS activity appeared to be independent of enzyme-associated H 4 Bip. The lack of intrinsic 4a-pterincarbinolamine dehydratase activity argued against classical H 4 Bip redox cycling in NOS. Rather, H 4 Bip was required for both maximal activity and stability of NOS by binding to the oxygenase/dimerization domain and preventing monomerization and inactivation during L-arginine turnover. Since anti-pterins were also effective in intact cells, they may become useful in modulating states of pathologically high nitric oxide formation.