Dynamic and static quenching of 1,N6-ethenoadenine fluorescence in nicotinamide 1,N6-ethenoadenine dinucleotide and in 1,N6-etheno-9-(3-(indol-3-yl) propyl) adenine.

Abstract

For nicotinamide 1,N6-ethenoadenine dinucleotide (ENAD+), the fluorescent analog of NAD+, in neutral aqueous solution the quantum yield has been determined to be 0.028 and the fluorescent lifetime, 2.1 nsec. Simultaneous determination of quantum yields and lifetimes of eNAD+ and of the "half molecule" EAMP allows the calculation of the percentage of stacked and open conformations of the dinucleotide. At 250 in neutral aqueous solution there is 45 I 5% of stacked forms. The value of the fluorescent lifetime observed for ENAD+ is sensitive to fluorescent impurities, especially those containing the E-adenosine moiety, and a purification procedure using high performance liquid chromatography was devised to obtain fluorescently homogeneous preparations.In order to study the effect on e-adenosine fluorescence caused by the possible close proximity of a tryptophan in a polypeptide chain or protein, we have prepared 1,N6-etheno- These studies relate to intramolecular stacking interactions within the ENAD+ molecule and set a probable upper limit for the interactions within the NAD+ molecule itself. It is also important to delineate possible interactions between the adenosine moiety of various coenzymes and the individual amino acids, e.g., tryptophan, of various proteins. To do so on a simplified level we synthesized 1,N6-etheno-9-[3- (indol-3-yl)propyl]adenine (EAde9-Cs-Ind3) by reaction of 9-[3-(indol-3-yl)propyl]adenine (Ade9-C3-Ind3) (25, 26) with chloroacetaldehyde, thus providing a model in which indole is used as a neutral substitute for tryptophan. From simultaneous measurement of the fluorescence lifetime and the quantum yield of EAde9-C3-Ind3 relative to those of the "half molecules" 1,N6-etheno-9-propyladenine (EAde9-C3) and 3-propylindole (Ind3-Cs), we are able to predict the fluorescence quenching effect of bringing the E-adenine moiety into close proximity with the indole of tryptophan. (27.6 MPa). The eluate was monitored by absorbance at 254 nm, recorded on a Hewlett-Packard recorder modified as described previously (27). As a criterion of purity we used identical lifetimes determined by both phase shift and modulation (28,29). This technique is very sensitive to the presence of more than a single fluorescent decay, and the presence of EAMP in our preparation would be expected to 3966

The 1.8 Å Cholix Toxin Crystal Structure in Complex with NAD+ and Evidence for a New Kinetic Model

“…lision of the nicotinamide and ⑀-adenine moieties (45). It appears that the nicotinamide and ⑀-adenine moiety continue interacting even in the bound state, which is consistent with our idea that nicotinamide forms dynamic interactions (Fig.…”

Design, Synthesis, and Characterization of Sulfamide and Sulfamate Nucleotidomimetic Inhibitors of hHint1

“…24 Therefore, one might predict that compounds 4 and 5 are likely to encounter a higher entropic penalty upon binding to hHint1. Consequently, one might propose that removal of the indole group in compound 5 would likely increase the binding affinity by decreasing the entropic cost of binding to hHint1.…”

Synthesis and Characterization of Stacked and Quenched Uridine Nucleotide Fluorophores

“…It had a high quantum yield and a long fluorescence lifetime and could be used to follow ATP interactions primarily by polarization studies (6 -8). In contrast, as in case of NAD, significant population of etheno-NAD was in a folded conformation in aqueous solution as a result of aromatic interactions between the pyridine and the modified adenine moiety, leading to dynamic collisional quenching of fluorescence and short fluorescence life-time (9). This stacked and quenched fluorophore was brilliantly used to establish negative co-operativity for glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle for the binding of the tetrameric apoenzyme to the coenzyme.…”