Enzymic synthesis of polynucleotides containing 5.6-methylene- and 5,6-dihydropyrimidines

Abstract

1 -(/3-D-Ribofuranosyl>(a+j3)5,6-methyleneuracil 5 '-diphosphate (lb), a bicyclic isomer of 5-methyluridine, was synthesized and investigated as a substrate for polynucleotide phosphorylase from Micrococcus luteus. In the same way as dihydrouridine 5'-diphosphate (Mb), lb could not be converted directly into a homopolymer by the enzyme. However, both lb and Mb could be copolymerized with ADP, IDP, CDP, or UDP into polymers which contained 5-40 residues of lb or Mb per 100 [of the normal] nucleotide units and were shown to be distributed internally in the polynucleotide chain. The ratios obtained depended on the rel-In electronic and spectral terms the cyclopropane ring may be regarded as an intermediate between a carbon-carbon single and double bond; e.g., the bond length of cyclopropane (Bastiansen et al., 1964) bond angles of vicinal hydrogens (Bastiansen et al., 1964) and CH hybridization (Wiberg, 1964) lie between the corresponding values of ethane and ethylene. Electronically, such an intermediacy is demonstrated by its ability to stabilize a photoexcited state (Yoshida and Ogoski, 1970) or an incipient carbonium ion (Deno et Brown and Cleveland, 1966). In a purely steric sense this comparison fails, since the methylene group adds considerable bulk to the corresponding ethane or ethylene. Thus, investigation of the relative activities of substrates with ethane, ethylene, and cyclopropane unit variations in a dynamic enzymic system might permit an assessment of the relative importance of steric vs. electronic factors.The recent synthesis (Kunieda and Witkop, 1971) of 1 -(0-D-ribofuranosyl>a(or /3)-5,6-methyleneuracil ("cyclo-5-methyluridine," I) permits a test of this approach in the field of nucleic acids, since I can be considered an intermediate between uridine (II), 5-methyluridine, and their dihydro derivatives. The cyclopropane ring contributes to the aromaticity which shows up in the ultraviolet spectrum of I (Xmax 245 nm, shoulder), intermediate between II(Xmax 260 nm)and III (Xmax 206 nm). If electronic factors, i.e., aromaticity, were overriding in determining the ability of I, II, and III to serve as substrates, the order should be II>I>III. If steric factors involving the 5,6 bond of uridine were important, then the order should be II>III>I. Initial results with compound I in biological systems have been discouraging. I does not arrest the growth of HeLa and L5178Y cells nor production of vaccinia virus in HeLa cells when tested at 10-4 m (C. Heidelberger, personal communication), is inactive against Vaccinia, Polio III, Herpes, Coe, Adeno, Rhino (two strains

Development of Reactions of 6- and 5-Substituted 1,3-Dimethyluracils with Dimethylsulfoxonium Methylide

“…Chromatography of the residue (silica gel using petroleum ether−EtOAc 1:1 as eluent) afforded, first, 1,3-dimethylcyclothymine ( 46a ) as a colorless oil (25 mg, 16%). The TLC mobility and 1 H and 13 C NMR spectra of 46a matched that of an authentic sample . The second product, collected as a colorless oil (65 mg, 42%), was identified as 16 by comparison of its 1 H and 13 C NMR spectra with an authentic sample …”

“…Uracils such as 44 and 45 are unknown and are of biochemical and medical interest as thymine mimics and for the great reactivities expected for their cyclopropene double bonds. Reaction of 1,3-dimethyluracil ( 2 , Z = H) with 3 has been previously reported to give cyclothymine 46a , 1,3- dibenzyluracil, and phenylmercuric bromodichloromethane yield cyclothymine 46b , and ( E )- and ( Z )-cyclothymines 46c are obtained by addition−displacements of 5-bromo-1,3-dimethyluracil by sodium ethyl phenylacetate …”