Optical Detection of Photorelease Kinetics on Gold and Glass Surfaces using Streptavidin-Coupled Biotinylated Photolabile Protecting Groups for Nucleosides.

Five biotinylated photolabile compounds of the general structure Bt-L1 -NPPOC-X-L2 were synthesized, in which Bt represents a biotin unit, L1 is a 3,6-dioxa-n-octane or an n-hexane spacer, NPPOC is the photolabile protecting group 2-(2-nitrophenyl)propoxycarbonyl, and X is a thymidine unit as a representative nucleoside or a direct linkage to L2 , an ω-mercapto- or ω-aminohexoyl linker, for coupling to a substrate surface. These compounds served for testing the photocleavage kinetics in self-assembled monolayers on gold or glass by using surface plasmon resonance (SPR) on gold or reflectometric interference spectroscopy (RIfS) on glass, whereby the biotin moiety offered the possibility to increase the bulkiness of the leaving group by binding to streptavidin, which thereby largely enhanced the SPR or RIfS signals. The photokinetics, found to consist in a dominating fast stage and a less contributing slow stage, were quantitatively analyzed, and the quantum yield of the fast part reached values up to almost 1 in favorable cases. A direct comparison of the results from SPR and RIfS yielded almost identical results. The present investigations pave the way to in situ monitoring of the photolithographic synthesis of DNA chips.

X-ray photoelectron spectroscopy- and surface plasmon resonance-detected photo release of photolabile protecting groups from nucleoside self-assembled monolayers on gold surfaces.

The formation of self-assembled monolayers (SAMs) on gold by 2-(5-iodo-2-nitrophenyl) propoxycarbonyl (I-NPPOC)-protected thymidine with an attached mercaptohexyl succinate linker and the kinetics of photochemical release of the I-NPPOC group were monitored using X-ray photoelectron spectroscopy (XPS) and surface plasmon resonance (SPR) detection. In the XPS spectra, the iodine peaks allowed for specific and accurate monitoring of the presence and loss of I-NPPOC groups on the surface. In the SPR experiment, the overall signal change on photoillumination is in accord with a theoretical estimation of the density of I-NPPOC groups in a dense monolayer. The kinetics roughly follow a biexponential time dependence with two very different time constants, corresponding to photochemical quantum yields of 0.22 and 0.0032, respectively.

Intramolecular sensitization of photocleavage of the photolabile 2-(2-nitrophenyl)propoxycarbonyl (NPPOC) protecting group: photoproducts and photokinetics of the release of nucleosides.

Novel photolabile protecting groups based on the 2-(2-nitrophenyl)propoxycarbonyl (NPPOC) group with a covalently linked thioxanthone as an intramolecular triplet sensitizer exhibit significantly enhanced light sensitivity under continuous illumination. Herein we present a detailed study of the photokinetics and photoproducts of nucleosides caged with these new protecting groups. Relative to the parent NPPOC group, the light sensitivity of the new photolabile protecting groups is enhanced by up to a factor of 21 at 366 nm and is still quite high at 405 nm, the wavelength at which the sensitivity of the parent compound is practically zero. A new pathway for deprotection of the NPPOC group proceeding through a nitroso benzylalcohol intermediate has been discovered to complement the main mechanism, which involves beta elimination. Under standard conditions of lithographic DNA-chip synthesis, some of the new compounds, while maintaining the same chip quality, react ten times faster than the unmodified NPPOC-protected nucleosides.

On the mechanism of intramolecular sensitization of photocleavage of the 2-(2-nitrophenyl)propoxycarbonyl (NPPOC) protecting group.

A spectroscopic study of a variety of covalently linked thioxanthone(TX)-linker-2-(2-nitrophenyl)propoxycarbonyl(NPPOC)-substrate conjugates is presented. Herein, the TX chromophore functions as an intramolecular sensitizer to the NPPOC moiety, a photolabile protecting group used in photolithographic DNA chip synthesis. The rate of electronic energy transfer between TX and NPPOC was quantified by means of stationary fluorescence as well as nanosecond and femtosecond time-resolved laser spectroscopy. A dual mechanism of triplet-triplet energy transfer has been observed comprising a slower mechanism involving the T1(pipi*) state of TX with linker-length-dependent time constants longer than 20 ns and a fast mechanism with linker-length-dependent time constants shorter than 3 ns. Evidence is provided that the latter mechanism is due to energy transfer from the T2(npi*) state which is in fast equilibrium with the fluorescent S1(pipi*) state. In the case of direct linkage between the aromatic rings of TX and NPPOC, the spectroscopic properties are indicative of one united chromophore which, however, still shows the typical NPPOC cleavage reaction triggered by intramolecular hydrogen atom transfer to the nitro group.