Photochemistry and thermal decarboxylation of alpha-phosphoryloxy-p-nitrophenylacetates.

Alpha-carboxy-4-nitrobenzyl phosphate 4 and its derived monomethyl phosphate ester 5 were synthesized and purified by anion-exchange chromatography. A gradient of LiCl was necessary for elution of the anion-exchange column to avoid unexpected thermal decarboxylation that occurred during vacuum evaporation when the volatile triethylammonium bicarbonate buffer was used. Photolysis of each compound was accompanied by decarboxylation, and 4 released inorganic phosphate with near-100% stoichiometry. Time-resolved infrared spectroscopy of the photolysis reaction, coupled with density functional theory calculations of vibrational frequencies, enabled us to infer a mechanism for the photolytic pathway, although there was some evidence for a second pathway also being operative. In contrast to the results for 4, photolysis of 5 appeared to release little or no monomethyl phosphate.

Studies of decarboxylation in photolysis of alpha-carboxy-2-nitrobenzyl (CNB) caged compounds.

Photolysis of alpha-carboxy-2-nitrobenzyl (CNB) caged compounds, studied here by time-resolved IR and UV spectroscopy, involves at least two pathways. In one, a conventional 2-nitrobenzyl type rearrangement takes place to release the photoprotected species via rapid decay of an aci-nitro intermediate. The alpha-carboxylate moiety of the CNB group is retained and the final by-product from this pathway is 2-nitrosophenylglyoxylate. Direct measurements of product formation confirmed that release via this pathway is faster for CNB-caged compounds than for related caged compounds without an alpha-carboxylate substituent and a rationale for the faster release rate is proposed. In a second pathway, photodecarboxylation of the starting material occurs: this pathway leads only to a slow, minor release of the photoprotected species. The extent to which the latter pathway contributes is affected by the nature of buffer salts in the irradiated solution. It was more prominent in an amine-based buffer (MOPS) than in phosphate buffer.

Fluorescent ligands for the hemagglutinin of influenza A: synthesis and ligand binding assays.

Two fluorescent conjugates of sialic acid have been prepared, with a convenient synthetic route that involves preparation of an unsaturated benzyl ester by cross-metathesis, followed by combined hydrogenation/ hydrogenolysis to provide a sialoside bearing a delta-carboxybutyl group, suitable for coupling with the chosen fluorophores. The fluorescent conjugates bound to bromelain-cleaved hemagglutinin (BHA) with affinities in the low microM range. Binding was accompanied by approximately 4.5-fold fluorescence enhancement for the dansyl conjugate 1 and approximately 3-fold fluorescence quenching for the pyrene conjugate 3.

A series of related nucleotide analogues that aids optimization of fluorescence signals in probing the mechanism of P-loop ATPases, such as actomyosin.

We have synthesized a set of ATP and ADP analogues that have a fluorophore linked to the nucleotide via the 3'-position of the ribose moiety. Combinations of three different coumarins are each attached via different length linkers. A linker based on propylenediamine increases the separation between the nucleotide and fluorophore relative to that of the previously reported ethylenediamine-linked coumarin nucleotides [Webb, M. R., and Corrie, J. E. T. (2001) Biophys. J. 81, 1562-1569]. A synthesis of 3'-amino-3'-deoxyATP is described using a combination of chemical and enzymatic procedures, mostly from published methods for synthesis of this compound but with some modifications that improved the convenience of the experimental procedures. This compound is used as a basis of a series of analogues with effectively a zero-length linker. Fluorescence properties of all these analogues are described, together with the kinetics of their interaction with rabbit skeletal myosin subfragment 1 in the presence and absence of actin. One particular analogue, deac-aminoATP [3'-(7-diethylaminocoumarin-3-carbonylamino)-3'-deoxyadenosine 5'-triphosphate], shows a 17-fold enhancement of fluorescence upon binding to this (skeletal) myosin II. As the diphosphate, it exhibits a large signal change upon dissociation from the actomyosin, with kinetics similar to those of natural ADP. The ability of this set of analogues to produce large signals indicated potential uses when scarce proteins are studied in small amounts.

Photolytic cleavage of 1-(2-nitrophenyl)ethyl ethers involves two parallel pathways and product release is rate-limited by decomposition of a common hemiacetal intermediate.

Time-resolved FTIR spectroscopic studies of the flash photolysis of several 1-(2-nitrophenyl)ethyl ethers derived from aliphatic alcohols showed that a long-lived hemiacetal intermediate was formed during the reaction. Breakdown of this intermediate was rate-limiting for product release. One of these compounds (methyl 2-[1-(2-nitrophenyl)ethoxy]ethyl phosphate, 9) was studied in detail by a combination of time-resolved FTIR and UV-vis spectroscopy. In addition, product studies confirmed clean photolytic decomposition to the expected alcohol, 2-hydroxyethyl methyl phosphate, and the 2-nitrosoacetophenone byproduct. At pH 7.0, 1 degrees C, the rate constant for product release was 0.11 s(-1), very much slower than the 5020 s(-1) rate constant for decay of the photochemically generated aci-nitro intermediate (pH 7.0, 2 degrees C). Time-resolved UV-vis measurements showed that the hemiacetal intermediate is formed by two competing pathways, with fast (approximately 80% of the reaction flux) and slow (approximately 20% of the flux) components. Only the minor, slower path is responsible for the observed aci-nitro decay process. These competing reactions are interpreted with the aid of semiempirical PM3 calculations of reaction barriers. Furthermore, AMSOL calculations indicate that the pK(a) of the nitronic acid isomer formed by photolysis is likely to determine partition into the alternate paths. These unusual results appear to be general for 1-(2-nitrophenyl)ethyl ethers and contrast with a related 2-nitrobenzyl ether that photolyzed without involvement of a long-lived hemiacetal.