Biomedical subjects
Publications and source records attributed to A Kanavarioti.
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We have undertaken a complete kinetic analysis of the template-directed oligoguanylate synthesis originated in Orgel's laboratory (Inoue and Orgel, 1982). The reaction of guanosine 5'-phospho-2-methylimidazolide, 2-MelmpG, with ribooligoguanylates all 3'-5' linked, designated n3 with n = 7-12, was studied in the presence/absence of the complementary template polycytidylic acid, poly(C). Conditions were chosen where poly(C) and 2-MelmpG are in large excess over the oligoguanylate. In the absence of the template at 37 degrees C the reaction leads to three isomeric oligomers that are elongated by one monomer unit. They are the 3'-5' linked, (n + 1)3, the 2'-5' linked, (n + 1)2, and the pyrophosphate product, (n + 1)p, formed in an approximate ratio 1:2:5. In the presence of the template the reaction is 20-fold faster and yields products n + 1, n + 2, n + 3 etc. as long as 2-MelmpG is available. Most importantly the formation of the natural, 3'-5' linked isomer, is enhanced selectively by 140-fold at 37 degrees C. Qualitative observations allow the conclusion that this enhancement is temperature dependent and increases with decreasing temperature. For example, at 1 degree C only the 3'-5' linked isomers were detected. Initial rates for the disappearance of the n3 oligoguanylate were determined at 1, 23, and 37 degrees C. It was found that the pseudo-first order rate constant for oligoguanylate elongation was linearly proportional to the 2-MelmpG concentration. This implies that the reaction complex poly(C).n3.2-MelmpG does not accumulate under the reaction conditions, a conclusion which is also supported by infrared data (Miles and Frazier, 1982). The implication of the above results with respect to chemical evolution is that lower temperatures, i.e., close to freezing, enhance the regioselectivity of these template-directed reactions and that one way to improve replication models may be sought in finding conditions that favor stable reaction complexes.
We have studied the hydrolysis of guanosine 5'-phospho-2-methylimidazolide, 2-MeImpG, in aqueous buffered solutions of various pH's at 75 degrees C and 37 degrees C. At 75 degrees C and pH < or = 1.0, two kinetic processes were observed spectrophotometrically: the first and more rapid one is attributed to the hydrolysis of the phosphoimidazolide P-N bond; the second and much slower one, to the cleavage of the glycosidic bond. At 37 degrees C, pH 2.0, the spectrophotometrically determined rate constant of P-N bond hydrolysis was confirmed by using high pressure liquid chromatography, HPLC. With the latter technique it was possible to separate reactants and products and also to extend the pH-rate profile into the neutral region where rates are slower and, therefore, difficult to measure spectrophotometrically. The pH-rate profiles at both temperatures exhibit similar behavior. At pH < 2 the pseudo-first-order rate constant increases with decreasing pH; in the region 2 < pH < 7 there is a plateau followed by a decrease for pH > 7. These data are consistent with a reactivity order zwitterion > anion for P-N bond hydrolysis. It is noteworthy that P-N bond hydrolysis in phosphoimidazolides is very slow compared to other phosphoramidates. This may be one of the reasons why this compound showed extraordinary ability in forming long oligomers under template-directed conditions.
An earlier study of the reaction of phosphoimidazolide activated nucleosides (ImpN) in aqueous phosphate buffers indicated two modes of reaction of the phosphate monoanion and dianion. The first mode is catalysis of the hydrolysis of the P-N bond in ImpN's which leads to imidazole and nucleoside 5'-monophosphate. The second represents a nucleophilic substitution of the imidazole to yield the nucleoside 5'-diphosphate. This earlier study thus served as a model for the reaction of ImpN with nucleoside monophosphates (pN) because the latter can be regarded as phosphate derivatives. In the present study we investigated the reaction of guanosine 5'-phosphate-2-methylimidazolide, 2-MeImpG, in the presence of pN (N = guanosine, adenosine and uridine) in the range 6.9 less than or equal to pH less than or equal to 7.7. We observed that pN's do act as nucleophiles to form NppG, and as general base to enhance the hydrolysis of the P-N bond in 2-MeImpG, i.e. pN show the same behavior as inorganic phosphate. The kinetic analysis yields the following rate constants for the dianion pN2-: knpN = 0.17 +/- 0.02 M-1 h-1 for nucleophilic attack and khpN = 0.11 +/- 0.07 M-1 h-1 for general base catalysis of the hydrolysis. These rate constants which are independent of the nucleobase compare with kp.2 = 0.415 M-1 h-1 and khp2. = 0.217 M-1 h-1 for the reactions of HPO4(2-). In addition, this study shows that under conditions where pN presumably form stacks, the reaction mechanism remains unchanged although in quantitative terms stacked pN are somewhat less reactive. Attack by the 2'-OH and 3'-OH groups of the ribose moiety in amounts greater than or equal to 1% is not observed; this is attributed to the large difference in nucleophilicity in the neutral pH range between the phosphate group and the ribose hydroxyls. This nucleophilicity rank is not altered by stacking.