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

Antibodies are of importance for the field of proteomics, both as reagents for imaging cells, tissues, and organs and as capturing agents for affinity enrichment in massspectrometry-based techniques. It is important to gain basic insights regarding the binding sites (epitopes) of antibodies and potential cross-reactivity to nontarget proteins. Knowledge about an antibody's linear epitopes is also useful in, for instance, developing assays involving the capture of peptides obtained from trypsin cleavage of samples prior to mass spectrometry analysis. Here, we describe, for the first time, the design and use of peptide arrays covering all human proteins for the analysis of antibody specificity, based on parallel in situ photolithic synthesis of a total of 2.1 million overlapping peptides. This has allowed analysis of on-and off-target binding of both monoclonal and polyclonal antibodies, complemented with precise mapping of epitopes based on full amino acid substitution scans. The analysis suggests that linear epitopes are relatively short, confined to five to seven residues, resulting in apparent off-target binding to peptides corresponding to a large number of unrelated human proteins. However, subsequent analysis using recombinant proteins suggests that these linear epitopes have a strict conformational component, thus giving us new insights regarding how antibodies bind to their antigens. Molecular & Cellular

Based upon the photolabile [2-(2-nitrophenyl)propoxy]carbonyl group (NPPOC), a large number of modified 2-(2-nitrophenyl)propanol derivatives substituted at the phenyl ring (see 23 ± 34 and 57 ± 76) as well as at the side-chain (see 85 ± 92 and 95 ± 98) were synthesized to improve the photoreactivity of this new type of photolabile entity. The phenyl moiety was also exchanged by the naphthalenyl group (see 102, 103, 105, 108, 110, 113, and 114), the thienyl substituent (see 115, 117, 118, and 120), and the benzothienyl substituent (see 121). The 2-(2-nitroaryl-and heteroaryl)propanols were converted with diphosgene into the corresponding carbonochloridates, which reacted subsequently with thymidine to the thymidine 5'-(protected carbonates) 123 ± 178 as the main reaction products. In several cases, the corresponding 3'-carbonates and 3',5'-dicarbonates 179 ± 212 were also isolated and characterized. Photolysis studies under standardized conditions (see Table) indicated that the rate of photocleavage varies in a broad range depending on the substituents. So far, thepropyl carbonates] 141 and 142, and 5'-[2-(2-nitro-5-thienylphenyl)propyl carbonates] 143 and 144 showed the best properties regarding fast and uniform deprotection. Since the nucleobases of 213 ± 215 do not influence the photocleavage features, in general, the new type of photolabile building blocks allows in form of their 3'-phosphoramidites the photolithographic formation of high-quality biochips.

Focal molography is a next-generation biosensor that visualizes specific biomolecular interactions in real time. It transduces affinity modulation on the sensor surface into refractive index modulation caused by target molecules that are bound to a precisely assembled nanopattern of molecular recognition sites, termed the 'mologram'. The mologram is designed so that laser light is scattered at specifically bound molecules, generating a strong signal in the focus of the mologram via constructive interference, while scattering at nonspecifically bound molecules does not contribute to the effect. We present the realization of molograms on a chip by submicrometre near-field reactive immersion lithography on a light-sensitive monolithic graft copolymer layer. We demonstrate the selective and sensitive detection of biomolecules, which bind to the recognition sites of the mologram in various complex biological samples. This allows the label-free analysis of non-covalent interactions in complex biological samples, without a need for extensive sample preparation, and enables novel time- and cost-saving ways of performing and developing immunoassays for diagnostic tests.

Conditions and kinetics of triplet sensitization as a method for increasing the light sensitivity of photolabile protecting groups used for the photolithographic synthesis of oligonucleotide microarrays were quantitatively studied with the photolabile 2-(2-nitrophenyl)propyl protecting group in homogeneous solutions and on glass substrates by using laser flash photolysis, continuous illumination with HPLC analysis, fluorescence dye labelling, and hybridization. In terms of efficiency and avoidance of chemical side reactions, 9H-thioxanthen-9-one was the most-suitable sensitizer. Both in solution and on a glass substrate, the photostationary kinetics were quantitatively modelled and the relevant kinetic parameters determined. While the sensitization kinetics was diffusion-controlled both in solution and on the chip, the photostationary kinetics was essentially of zero order only on the chip because here the triplet-quenching effect of the released photoproduct 2-(2-nitrophenyl)propene was suppressed as a consequence of the inhomogeneous reaction that took place in a narrow diffusion zone above the surface from where the photoproducts could quickly escape. The kinetic simulation allowed quantitative estimate of the density of reactive groups on the surface. It was further demonstrated that, with 9H-thioxanthen-9-one as a sensitizer, high-density oligonucleotide microarrays of high quality can be produced with one-third of the normal exposure time.

Light as an external trigger is a valuable and easily controllable tool for directing chemical reactions with high spatial and temporal accuracy. Two o-nitrobenzyl derivatives, benzoyl- and thiophenyl-NPPOC, undergo photo-deprotection with significantly improved efficiency over that of the commonly used NPPOC group. The two- and twelvefold increase in photo-deprotection efficiency was proven using photolithograph synthesis of microarrays.

BackgroundDNA microarrays are a core element of modern genomics research and medical diagnostics, allowing the simple and simultaneous determination of the relative abundances of hundreds of thousands to millions of genomic DNA or RNA sequences in a sample. Photolithographic in situ synthesis, using light projection from a digitally-controlled array of micromirrors, has been successful at both commercial and laboratory scales. The advantages of this synthesis method are its ability to reliably produce high-quality custom microarrays with a very high spatial density of DNA features using a compact device with few moving parts. The phosphoramidite chemistry used in photolithographic synthesis is similar to that used in conventional solid-phase synthesis of oligonucleotides, but some unique differences require an independent optimization of the synthesis chemistry to achieve fast and low-cost synthesis without compromising microarray quality.ResultsHigh microarray quality could be maintained while reducing coupling time to a few seconds using DCI activator. Five coupling activators were compared, which resulted in microarray hybridization signals following the order ETT > Activator 42 > DCI ≫ BTT ≫ pyridinium chloride, but only the use of DCI led to both high signal and highly uniform feature intensities. The photodeprotection time was also reduced to a few seconds by replacing the NPPOC photolabile group with the new thiophenyl-NPPOC group. Other chemical parameters, such as oxidation and washing steps were also optimized.ConclusionsHighly optimized and microarray-specific phosphoramidite chemistry, along with the use of the very photosensitive thiophenyl-NPPOC protecting group allow for the synthesis of high-complexity DNA arrays using coupling times of 15 s and deprotection times of 9 s. The resulting overall cycle time (coupling to coupling) of about 50 s, results in a three-fold reduction in synthesis time.

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