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E Darzynkiewicz

Publications and source records attributed to E Darzynkiewicz.

47 records · Page 3Linked to original sources
PubMed1976

Conformation in aqueous medium of the neutral, protonated and anionic forms of 9-beta-D-arabinofuranosyladenine.

Proton magnetic resonance spectroscopy was employed to study the solution conformations of the neutral, protonated and dissociated forms of the therapeutically active 9-beta-D-arabinofuranosyladenine (araA). In particular, in strongly basic medium, increasing alkalinity led to pronounced changes in chemical shifts and coupling constants of some pentose protons, due to ionization of the pentose hydroxyls, especially the 2'-OH. The neutral form of araA may be characterized as approx. 25% C(2')endo and approx. 60% gauche-gauche, hence somewhat different from that of the therapeutically active 1-beta-D-arabinofuranosylcytosine (araC). By contrast, the conformations of the anionic forms of both of these are identical, predominantly (greater than 80%) C(2')endo and gauche-gauche. With the aid of the 3'-O-methyl derivatives of araA and araC, where only the 2'-OH ionizes, and the accompanying conformational changes are similar, it follows that the conformation C(2')endo and gauche-gauche for all the foregoing is constrained to this form via a strong intramolecular hydrogen bond, viz. O(5')H...O(2')(-). The influence of the foregoing hydrogen bond on the chemical shifts of the adenine H(8) in the araA anion points to the existence of the latter in the form anti. A similar effect of the doubly ionized phosphate group on H(8) in 5'-araAMP shows the nucleotide to also prefer the form anti, as previously demonstrated for 5'-AMP. The conformations of the sugar rings of the neutral forms of araA and adenosine in aqueous medium differ appreciably, whereas in the solid state they are very similar. PMR spectroscopy is shown to be an effective method for following sugar hydroxyl dissociation. The extent of ionization of a given hydroxyl is provided by the resulting chemical shifts of neighbouring (geminal and vicinal) protons. When ionization is accompanied by a change in conformation, the process may be followed also by changes in proton-proton vicinal coupling constants.

Adenine Nucleotides↗
PubMed1976

Methylation of adenosine in strongly alkaline medium: Preparation and Properties of o'-methyl derivatives of adenosine and N6-methyladenosine.

In strongly alkaline aqueous medium, 9-substituted adenines, including adenine nucleosides, are relatively resistant to alkylation of the ring nitrogens and the exocyclic amino group. This fact was utilized to obtain the various possible Omicron'-methyl derivatives of adenosine by dimethyl sulfate treatment of the latter in alkaline medium, followed by separation of the products on a Dowex OH-column. In strongly alkaline aqueous dimethyl sulfoxide, several derivatives additionally methylated on the amino group were obtained, including N6,O2'-dimethyladenosine, a component located recently at the 5' terminus of animal cell and viral mRNAs. The latter was also prepared by diazomethane methylation of N6-methyladenosine in the presence of SnCl2. Alkylation in alkaline medium possesses the advantage that the products are not limited to those involving etherification of cis hydroxyls and is applicable to adenine nucleosides with sugar components other than ribose. The properties of the various O'-methylderivatives, including proton magnetic resonance data, are presented in detail.

Adenosine↗
PubMed1976

Preparation and properties of the O-methyl derivatives of 9-beta-D-arabinofuranosyladenine.

The previously reported resistance of 9-substituted adenines to ring alkylation in alkaline medium was profited from to prepare all seven possible O' methyl derivatives of the therapeutically important 9-beta-D-arabinofuranosyladenine (araA) by mild methylation of the latter with dimethylsulfate in aqueous alkaline medium. All the products were fractionated and isolated in a single step on a Dowex OH- column. The sequence of elution of the various derivatives was strikingly similar to that for the O' methyl derivatives of 1-beta-D-arabinofuranosylcytosine, previously reported, suggestive of a similar sequence of acidities of the sugar hydroxyls. The products were identified by various criteria, including PMR spectroscopy, extensive data for which are supplied. The 2'-O-methyl and 3'-O-methyl derivatives of araA exhibited appreciably lower susceptibility to calf intestinal adenosine deaminase than the parent araA. The 5'-O-methyl analogue was fully resistant to enzymatic deamination.

Chromatography, Ion Exchange↗
PubMedDate not supplied

Hydrolysis of some mRNA 5'-cap analogs catalyzed by the human Fhit protein--and lupin ApppA hydrolases.

Hydrolysis of the following four cap analogs: m7G(5')ppp(5')A, m7G(5')ppp(5')m6A, m7G(5')ppp(5')m2'OG and m7G(5')ppp(5')2'dG catalyzed by homogeneous human Fhit protein and yellow lupin Ap3A hydrolase has been investigated. The hydrolysis products were identified by HPLC analysis and the K(m) and Vmax values calculated based on the data obtained by the fluorimetric method.

Acid Anhydride Hydrolases↗
PubMedDate not supplied

Spectrophotometric determination of the pK values for dissociation of the sugar hydroxyls in pyrimidine arabinonucleosides.

Spectrophotometric titration in the ultraviolet has been employed to determine the pK values for dissociation of the sugar hydroxyls in pyrimidine arabinonucleosides and some of their O'-methyl and O'-ethyl derivatives. The order of dissociation of the sugar hydroxyls in the arabinofuranose ring was 2'-OH greater than 3'-OH greater than 5'-OH. The higher acidity (lower pK) of the 2'-OH was interpreted in terms of formation of an intramolecular hydrogen bond of the form 5'-OH....2'-O(--) and the accompanying changes in conformation of the arabinose ring. The various factors affecting the dissociation of specific hydroxyls in some of the O'-alkyl derivatives are discussed in relation to steric, conformational and other effects.

Arabinofuranosyluracil↗
PubMedDate not supplied

Fluorescence studies on association of human translation initiation factor eIF4E with mRNA cap-analogues.

Binding of a long series of mono- and dinucleotide analogues of the 7-methylguanosine containing 5'-mRNA-cap to human protein translation initiation factor eIF4E has been investigated by means of fluorescence. A new methodological approach in gathering and analysis of the fluorescence data provided us with very accurate values of the association equilibrium constant K and normalized, maximal quenching of the protein fluorescence delta Fmax, during titration of eIF4E by various cap-analogues. The results confirm participation of at least two conserved tryptophan residues of eIF4E in interaction with 7-methylguanine, as has been described recently for murine eIF4E, complexed with 7-methyl-GDP in crystal (Marcotrigiano et al., 1997, Cell 89, 951), and for yeast eIF4E, complexed with the same ligand in solution (Matsuo et al., 1997, Nature Struct. Biol. 4, 717). On the other hand binding by eIF4E of unmethylated guanine nucleotides and N2,N2,7-trimethylguanine containing nucleotides differ substantially from the way of binding of the regular mRNA-cap. Influence of the structural features of the cap-analogues, especially the type of the second nucleoside in the dinucleotide caps, on their association with eIF4E and biological activities in in vitro protein translation systems has been discussed in light of the known structures of the eIF4E-7-methyl-GDP complexes in crystal and solution.

Animals↗