The initial rates of incorporation of dTTP and thymidine 5'-O-(3-thiotriphosphate) (dTTP alpha S) into poly(dA) X oligo(dT) during template-directed synthesis by the large fragment of DNA polymerase I have been measured by using a rapid-quench technique. The rates were initially equal, indicating a nonrate-limiting chemical step. However, the rate of thionucleotide incorporation steadily diminished to 10% of its initial value as the number of consecutive dTMP alpha S residues in the primer strand increased. This anomalous behavior can be attributed to the helix instability inherent in phosphorothioate-containing duplexes. Positional isotope exchange experiments employing the labeled substrate [alpha-18O2]dATP have revealed negligible alpha, beta-bridging----beta-nonbridging isotope exchange in template-directed reactions of Escherichia coli DNA polymerase I (Pol I) both in the presence and in the absence of added inorganic pyrophosphate (PPi), suggesting rapid PPi release following the chemical step. These observations are consistent with a rate-limiting step that is tentatively assigned to a conformational change of the E X DNA X dNTP complex immediately preceding the chemical step. In addition, the substrate analogue (Sp)-dATP alpha S has been employed to examine the mechanism of the PPi exchange reaction catalyzed by Pol I. The net retention of configuration at the alpha-P is interpreted in terms of two consecutive inversion reactions, namely, 3'-hydroxyl attack, followed by PPi attack on the newly formed primer terminus. Kinetic analysis has revealed that while alpha-phosphorothioate substitution has no effect upon the initial rate of polymerization, it does attenuate the PPi exchange reaction by a factor of 15-18 fold.(ABSTRACT TRUNCATED AT 250 WORDS)
Ausgehend von den Komponenten (I) und (II) werden die Pterin‐Derivate (VI), (VI I) [== Zf‐Oxidationsprodukt von (VI)] bzw. (IX) synthetisiert.
l,l-Dialkyl-1H-diazirino[l,2-b]phthalazine-3,S-d~ones isomerize in boiling toluene into 2-(l-alken-l-yl)-4-hydroxy-l(2H)-phthalazinones and react with enamines and ynamines to yield derivatives of pyrazolo[l,2-b]-and indazolo[l,2-b]phthalazinediones.The thermal conversions of 1-(2,4-dinitropheny1)-3,3-dialkyl-and 1-(2,4-dinitropheny1)-2,3,3-trialkyldiaziridines into 2,4-dinitrophenylhydrazones and 2-alkyl-6-nitrobenzotriazole 1-oxides, respectively, and the addition of 3,3-dialkyl-and 1,3,3-trialkyldiaziridines to electrophilic acetylenes have been the subjects of earlier reports in this series.1$2 In the present paper the synthesis and reactions of 1-alkyl-and 1,l-dialkyl-1H-diazirino[l,2-b lphthalazine-3,8-diones (1-7) and the related systems 8 and 9 are described. These versatile compounds are thermally isomerized into 2-(1 -alken-1-yl) -4-hydroxy-1 -( 2H)phthalazinones and react with ynamines and enamines to give pyrazolo[l,2-b]-and indazolo[l,2-b]phthalazinediones. Reactions with I nitrones are presented in the following paper.Resultswere prepared by the addition of phthaloyl chloride to ethereal solutions of 3,3-dialkyldiaziridines containing triethylamine (Scheme I, Table I). The l-alkyllH-diazirino[l,2-b]phthalazine-3,8-diones 6 and 7 were synthesized by generating the 3-alkyldiaziridines in The Scheme I EtBN o-C,H,(COCl), + HN-NHvTable I 1-Alkyl-and 1,l-Dialkyl-1H-diazirino [ 1,2-b]phthalazine-3,8-dionesa Compd b R1 R2 Yield,c % Mp, OC 1 Me Me 58 98-99 2 Et Et 35 70-71 3 n -P r n -P r 47 108-109 4 -(CH2)5-75 110-111 5 Me Et 71 76-77 6 H n -Pr 61 71-72.5 110-111 7 H t -Bu 51 a Satisfactory analytical data for C, H, and N were obtained for all compounds listed in the table. Ed. Compounds 1-7 were recrystallized from cyclohexane. c Yields are reported on the basis of recrystallized products.situ from the corresponding chloral adducts, R b m C H ( O H ) C H C C l 3 , in the presence of o-phthaloyl chloride. Compounds 8 and 9 were made in much the same / \ manner as 1-5 by employing the appropriate diacid chlo-R' R2 ride and 3,3-~entamethylenediaziridine.The structures of 1-9 were assigned on the basis of elemental analyses, nmr spectroscopy, and by the hydrolysis H+* $r + ~1~2~0 of 2,4, and 7 into 4-hydroxy-l(2H)-phthalazinone (10) and the corresponding carbonyl compounds 3-pentanone, cyclohexanone, and pivalaldehyde, respectively (Scheme I). The nmr spectrum of l,l-dimethyl-lH-diazirino[1,2-0 1-7 10 blphthalazine-3,B-dione (1) taken in C6D6 shows two peaks a t 6 1.45 and 1.25 for the two methyl groups on the diaziridinyl carbon. On heating to about 60" the two peaks coalesce to a broad singlet which on further heating to 75O becomes a sharp singlet a t 6 1.55. If the temperature is lowered to 40° the two signals for the methyl groups reappear. The temperature variance of the nmr spectrum of 1 indicates inversions of the nitrogen atoms (Scheme 11) and is of some interest, since the N-aroyl moieties are perforce cis to 0 R1 0 CH,OH-H,O OH 0 8 9 3187
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