From partial to full agonism: identification of a novel 2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole as a full agonist of the human GPR119 receptor.

A novel GPR119 agonist based on the 2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole scaffold was designed through lead optimization starting from pyrazole-based GPR119 agonist 1. The design is centered on the conformational restriction of the core scaffold, while minimizing the change in spatial relationships of two key pharmacophoric elements (piperidine-carbamate and aryl sulfone).

Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase.

Vaccinia virus DNA polymerase catalyzes duplex-by-duplex DNA joining reactions in vitro and many features of these recombination reactions are reprised in vivo. This can explain the intimate linkage between virus replication and genetic recombination. However, it is unclear why these apparently ordinary polymerases exhibit this unusual catalytic capacity. In this study, we have used different substrates to perform a detailed investigation of the mechanism of duplex-by-duplex recombination catalyzed by vaccinia DNA polymerase. When homologous, blunt-ended linear duplex substrates are incubated with vaccinia polymerase, in the presence of Mg2+ and dNTPs, the appearance of joint molecules is preceded by the exposure of complementary single-stranded sequences by the proofreading exonuclease. These intermediates anneal to form a population of joint molecules containing hybrid regions flanked by nicks, 1-5 nt gaps, and/or short overhangs. The products are relatively resistant to exonuclease (and polymerase) activity and thus accumulate in joining reactions. Surface plasmon resonance (SPR) measurements showed the enzyme has a relative binding affinity favoring blunt-ended duplexes over molecules bearing 3'-recessed gaps. Recombinant duplexes are the least favored ligands. These data suggest that a particular combination of otherwise ordinary enzymatic and DNA-binding properties, enable poxvirus DNA polymerases to promote duplex joining reactions.

A novel mutator of Escherichia coli carrying a defect in the dgt gene, encoding a dGTP triphosphohydrolase.

A novel mutator locus in Escherichia coli was identified from a collection of random transposon insertion mutants. Several mutators in this collection were found to have an insertion in the dgt gene, encoding a previously characterized dGTP triphosphohydrolase. The mutator activity of the dgt mutants displays an unusual specificity. Among the six possible base pair substitutions in a lacZ reversion system, the G.C-->C.G transversion and A.T-->G.C transition are strongly enhanced (10- to 50-fold), while a modest effect (two- to threefold) is also observed for the G.C-->A.T transition. Interestingly, a two- to threefold reduction in mutant frequency (antimutator effect) is observed for the G.C-->T.A transversion. In the absence of DNA mismatch repair (mutL) some of these effects are reduced or abolished, while other effects remain unchanged. Analysis of these effects, combined with the DNA sequence contexts in which the reversions take place, suggests that alterations of the dGTP pools as well as alterations in the level of some modified dNTP derivatives could affect the fidelity of in vivo DNA replication and, hence, account for the overall mutator effects.

Enzymatic processing of replication and recombination intermediates by the vaccinia virus DNA polymerase.

Poxvirus DNA polymerases play a critical role in promoting virus recombination. To test if vaccinia polymerase (E9L) could mediate this effect by catalyzing the post-synaptic processing of recombinant joint molecules, we prepared substrates bearing a nick, a 3'-unpaired overhang, a 5' overhang, or both 3' and 5' overhangs. The sequence of the 5' overhang was also modified to permit or preclude branch migration across the joint site. These substrates were incubated with E9L, and the fate of the primer strand characterized under steady-state reaction conditions. E9L rapidly excises a mispaired 3' strand from a DNA duplex, producing a meta-stable nicked molecule that is a substrate for ligase. The reaction was not greatly affected by adding an unpaired 5' strand, but since such molecules cannot be processed into nicked intermediates, the 3'-ended strand continued to be subjected to exonucleolytic attack. Incorporating homology into the 5' overhang prevented this and permitted some strand assimilation, but such substrates also promoted strand-displacement DNA synthesis of a type predicted by the 1981 Moyer and Graves model for poxvirus replication. Single-strand annealing reactions are used by poxviruses to produce recombinant viruses and these data show that virus DNA polymerases can process DNA in such a manner as to both generate single-stranded substrates for such reactions and to facilitate the final processing of the reaction products.

Total Synthesis of (-)-lasubine II by the conjugate addition and intramolecular acylation of an amino ester with an acetylenic sulfone.

[reaction: see text] The conjugate addition of methyl (S)-(2-piperidyl)acetate (3) to 2-(3,4-dimethoxyphenyl)-1-(p-toluenesulfonyl)ethyne (4), followed by LDA-promoted intramolecular acylation, stereoselective reduction, and desulfonylation, afforded (-)-lasubine II.

Synthesis of the quinolizidine alkaloids (-)-lasubine II and (+/-)-myrtine by conjugate addition and intramolecular acylation of amino esters with acetylenic sulfones.

The conjugate additions of (2-piperidyl)acetate esters to acetylenic sulfones, followed by LDA-promoted intramolecular acylations, afforded cyclic enaminones that were readily converted into the corresponding 4-substituted 2-keto- or 2-hydroxyquinolizidines by stereoselective reduction and desulfonylation. This procedure was applied to the total synthesis of (-)-lasubine II from methyl (S)-(2-piperidyl)acetate and 2-(3,4-dimethoxyphenyl)-1-(p-toluenesulfonyl)ethyne. Similarly, methyl (+/-)-(2-piperidyl)acetate and 1-(p-toluenesulfonyl)propyne provided (+/-)-myrtine.