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

Direct PCR sequencing with boronated nucleotides provides an alternative to current PCR sequencing methods. The positions of boranophosphate-modified nucleotides incorporated randomly into DNA during PCR can be revealed directly by exonuclease digestion to give sequencing ladders. Cytosine nucleotides, however, are especially sensitive to exonuclease digestion and provide suboptimal sequencing ladders. Therefore, a series of 5-substituted analogs of 2'-deoxycytidine 5'-(alpha-P-borano)triphosphates (dCTPalphaB) were synthesized with the hope of increasing the nuclease resistance of deoxycytosine residues and thereby enhancing the deoxycytosine band intensities. These dCTP analogs contain a boranophosphate modification at the alpha-phosphate group in 2'-deoxycytidine 5'-triphosphate (dCTP) as well as a 5-methyl, 5-ethyl, 5-bromo or 5-iodo substitution for the 5-hydrogen of cytosine. The two diastereomers of each new dCTP derivative were separated by reverse phase HPLC. The first eluted diastereomer (putatively Rp) of each dCTP analog was a substrate for T7 DNA polymerase (Sequenase) and had an incorporation efficiency similar to normal dCTP and dCTPalphaB, with the 5-iodo-dCTPalphaB analog being the least efficient. Substitution at the C-5 position of cytosine by alkyl groups (ethyl and methyl) markedly enhanced the dCTPalphaB resistance towards exonuclease III (5-Et-dCTPalphaB >5-Me-dCTPalphaB >dCTPalphaB approximately 5-Br-dCTPalphaB >5-I-dCTPalphaB), thereby generating DNA sequences that better define the deoxycytosine positions. The introduction of modified dCTPalphaB should increase the utility of direct DNA sequencing with boronated nucleoside 5'-triphosphates.

Two novel hydrolytic dehalogenases, thermostable L-2-haloacid dehalogenase (L-DEX) inducibly synthesized by 2-chloropropionate (2-CPA) and nonthermostable DL-2-haloacid dehalogenase (DL-DEX) induced by 2-chloroacrylate, were purified to homogeneity from Pseudomonas sp. strain YL. DL-DEX consisted of a monomer with a molecular weight of about 36,000 and catalyzed the dehalogenation of L and D isomers of 2-CPA to produce D-and L-lactates, respectively. It acted on 2-haloalkanoic acids with a carbon chain length of 2 to 4. The maximum activity on DL-2-CPA was found at pH 10.5 and 45°C. L-DEX, composed of two subunits with identical molecular weights of 27,000, catalyzes the dehalogenation of L-2-haloalkanoic acids to produce the corresponding D-2-hydroxyalkanoic acids. The enzyme acts not only on short-carbon-chain 2-haloacids such as monochloroacetate and monoiodoacetate in aqueous solution but also on long-carbon-chain 2-haloacids such as 2-bromohexadecanoate in n-heptane. L-DEX is thermostable: it retained its full activity upon heating at 60°C for 30 min. The pH and temperature optima for dehalogenation of L-2-CPA were 9.5 and 65°C, respectively. L-DEX was strongly inhibited by modification of carboxyl groups with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and Woodward reagent K, but DL-DEX was not.

Nucleoside boranophosphates, in which one of the phosphate oxygens is replaced by a borane group, are isoionic and isoelectronic analogues of naturally occurring nucleotides. Boranophosphates also are biochemically important congeners of phosphorothioates and methylphosphonates. We have developed a convenient one-pot method to synthesize the set of ribonucleoside (A, U, G, and C) 5'-(alpha-P-borano)triphosphates. Phosphitylation of the 2',3'-protected ribonucleoside with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one gives the 5'-phosphite intermediate 2 which undergoes in situ substitution in the presence of pyrophosphate to give the cyclic intermediate, P(2),P(3)-dioxo-P(1)-ribonucleosidylcyclotriphosphate 3. Immediate oxidation of compound 3 with amine.borane complex results in ribonucleoside 5'-(alpha-P-borano)cyclotriphosphate 4. Subsequent reaction of compound 4 with water followed by ammonium hydroxide yields the crude product as a diastereomeric mixture of ribonucleoside 5'-(alpha-P-borano)triphosphate 6. Pure compound 6 is isolated in 30-45% overall yield using ion-exchange chromatography. The separation of two diastereomers of ribonucleoside 5'-(alpha-P-borano)triphosphate 6 is achieved by reverse phase HPLC.

Many organic compounds were found to be substrates for halogenation reactions catalyzed by the non-hemetype bromoperoxidase found in the red alga Corallina pilulifera. Anisole, 1 -methoxynaphthalene and thiophene were converted to o and p-bromoanisoles, 1 -methoxy-4-bromonaphthalene and 2-bromothiophene respectively. Regiospecificity of the enzymatic bromination of anisole was tested and found to be the same as in the chemical reaction with NaOBr. The enzyme also acted on substituted alkenes such as styrene, cyclohexene, trans-cinnamic acid, trans-cinnamyl alcohol and cis-propenylphosphonic acid, to give the respective bromohydrin compounds or decarboxylated bromo compound. These bromohydrin compounds were always mixtures of stereoisomers. In the light of the above findings together with the previous studies concerning the halogenation mechanism, the bromoperoxidase of C. pilulifera was considered to have no specific restriction site for these substrates. Although the reaction mechanism of the chloroperoxidase appears to be still a matter of controversy [18-221, the lack of substrate specificity and stereospecificity of the chloroperoxidase-catalyzed halogenation can be explained in terms of the molecular halogen or hypophalous acid addition chemistry in water, the halogen being released from the enzyme into the reaction mixture in the presence of halide anions and hydrogen peroxide [ l l , 121. It has also been found that chloroperoxidase was unstable under the reaction conditions because the released active molecular halogen denatured the enzyme [12]. As a result this enzyme can not be used as a halogenating agent in a continuous halogenation processes.In previous studies Itoh et al. examined the distribution [23, 241, enzymatic properties [23, 25, 261 and reaction mechanisms [28] of bromoperoxidases derived from coralline algae (Corallinaceae, Rhodophyta). The bromoperoxidase of Corallina pilulifera is clearly distinguishable from the usual haloperoxidases because it has non-heme iron as a prosthetic group [26]. In addition the enzyme was found to be stable under normal reaction conditions [12], thus, making it suitable for use in halogenation processes [27]. It has already been reported that cyclic P-diketones, phenol, o-hydroxybenzyl alcohol [23] and nitrogen-containing heterocycles [I21 serve Correspondence to N. Itoh, Research Laboratory, Amano Pharmaceutical Co. Ltd., Nishiharu, Nishikasugai-gun, Aichi, Japan-481Abbreviations. GC, gas chromatography; EI-MS, electron impact mass spectrometry.as substrates for this enzyme, and the halogenation occurs at its active site [27]. In this paper we describe new substrates and clarify the regiospecificity and stereospecificity of this enzyme's halogenation reaction, which contribute not only to its use as a synthetic tool in organic chemistry but also to the further clarification of its halogenation mechanism. The enzyme's biological roles in algae are also discussed on the basis of the results. EXPERIMENTAL PROCEDURE Preparation of bromoperoxidaseBromoperoxid...