Gordon P. Reid

[16] Synthesis and properties of caged nucleotides

Publisher Summary Photosensitive precursors have been termed “caged compounds.” This chapter focuses on caged compounds that yield nucleotides or nucleotide analogs on photolysis. In this approach, a biological preparation is equilibrated with a caged nucleotide, and it is then illuminated using a near-UV light pulse to liberate the nucleotide that initiates a biological response. Such a strategy has been used to make time-resolved measurements of the actions of ATP and ATPγS on muscle fibers, the Na+/K+ ion pump ATPase, and sarcoplasmic reticulum vesicles and of GTP analogs on sensory neurons. These nucleotides are common in that they contain weakly acidic phosphate groups and the photolabile precursors used are 1-(2-nitrophenyl)ethyl phosphate esters. The esters have advantageous photochemical properties. This chapter describes the synthesis and characterization of 1-(2-nitrophenyl)ethyl phosphate esters of nucleotides using as representative examples caged ATP, ATPβ, γNH, and GTPγS.

Synthesis and absolute stereochemistry of the two diastereoisomers of P3-1-(2-nitrophenyl)ethyl adenosine triphosphate (‘caged’ ATP)

1 -(2-Nitrophenyl)ethanol was resolved by fractional crystallisation of its diastereoisomeric (1S)-cam-phanates. The absolute stereochemistry of the (S)-alcohol was determined using the Horeau kinetic resolution procedure, and subsequently confirmed by X-ray crystallography of its (1S)-camphanate ester. The resolved alcohols were converted to (R)- and (S)-1-(2-nitrophenyl)ethyl phosphates, each of which was condensed with adenosine diphosphate to give the (R)- and (S)-1-(2-nitro-phenyl)ethyl P3-esters of adenosine triphosphate.

Time-resolved crystallography on H-ras p21

We describe here the results obtained to date on a project aimed at characterizing the changes occurring in the protein product (p21) of the H-ras proto-oncogene during and as a result of hydrolysis of GTP at its active site. The approach used involves crystallization of p21 with a photosensitive precursor of GTP (caged GTP) at the active site followed by generation of GTP by photolysis and collection of X-ray diffraction data using the Laue method at a synchrotron source. The structure of p21 complexed with a single diastereomer of caged GTP is presented here. In contrast to crystals obtained with mixed diastereomers, the nucleotide appears to bind in a manner which is very similar to that of other guanine nucleotides (GDP, GTP, GppNHp). The current state of time resolved structural experiments using these crystals is presented.