Synthesis of new pyrazolo[1,2,3]triazines by cyclative cleavage of pyrazolyltriazenes

We describe the synthesis of so far synthetically not accessible 3,6-substituted-4,6-dihydro-3H-pyrazolo[3,4-d][1,2,3]triazines as nitrogen-rich heterocycles. The target compounds were obtained in five steps, including an amidation and a cyclative cleavage reaction as key reaction steps. The introduction of two side chains allowed a variation of the pyrazolo[3,4-d][1,2,3]triazine core with commercially available building blocks, enabling the extension of the protocol to gain other derivatives straightforwardly. Attempts to synthesize 3,7-substituted-4,7-dihydro-3H-pyrazolo[3,4-d][1,2,3]triazines, the regioisomers of the successfully gained 3,6-substituted 4,6-dihydro-3H-pyrazolo[3,4-d][1,2,3]triazines, were not successful under similar conditions due to the higher stability of the triazene functionality in the regioisomeric precursors and thus, the failure of the removal of the protective group.

Preliminary Study on the Simulation of a Radiation Damage Analysis of Biodegradable Polymers

In this study, biodegradable poly(L-lactide-co-ε-caprolactone) (PLCL) and poly(L-co-d,l lactide) (PLDLA) were evaluated using Geant4 (G4EmStandardPhysics_option4) for damage simulation, in order to predict the safety of these biodegradable polymers against gamma ray sterilization. In the PLCL damage model, both chain scission and crosslinking reactions appear to occur at a radiation dose in the range 0–200 kGy, but the chain cleavage reaction is expected to be relatively dominant at high irradiation doses above 500 kGy. On the other hand, the PLDLA damage model predicted that the chain cleavage reaction would prevail at the total irradiation dose (25–500 kGy). To verify the simulation results, the physicochemical changes in the irradiated PLCL and PLDLA films were characterized by GPC (gel permeation chromatography), ATR-FTIR (attenuated total reflection Fourier transform infrared), and DSC (difference scanning calorimetry) analyses. The Geant4 simulation curve for the radiation-induced damage to the molecular weight was consistent with the experimentally obtained results. These results imply that the pre-simulation study can be useful for predicting the optimal irradiation dose and ensuring material safety, particularly for implanted biodegradable materials in radiation processing.

Development of A Recombinant Murine Luteinizing Hormone Binding Protein As A Selective Hormone Inhibitor

Physiological levels of luteinizing hormone (LH), in concert with follicle stimulating hormone (FSH), promote ovarian follicular development and ovulation. However, high LH levels associated with ovarian dysfunction have been shown to inhibit these processes. Thus, developing a selective LH inhibitor could be potentially useful for treating ovarian dysfunction. Here, we developed a mouse LH-binding protein (mLBP) composed of the extracellular domain of LH receptors as a selective inhibitor of mouse LH. After transient introduction of mLBP expressing vectors into Expi293F cells, mLBP was obtained as a soluble protein via a cleavage reaction with thrombin. The binding ability of mLBP for mouse LH was confirmed using sera containing high LH and FSH collected from ovariectomized (OVX) mice. The bioactivity of mLBP was demonstrated by inhibition of cAMP and testosterone productions induced by OVXmouse serum in mouse Leydig MLTC-1 cells expressing LH receptors. In contrast, mLBP did not bind mouse FSH and inhibit cAMP production induced by OVX-mouse serum in 293 cells expressing mouse FSH receptors. The mLBP also showed binding affinity to human LH (hLH), and inhibited hLH-induced cAMP production in MLTC-1 cells. Thus, the mLBP selectively suppresses the action of LH and is a potential therapeutic agent for ovarian dysfunction.

U7 deciphered: the mechanism that forms the unusual 3′ end of metazoan replication-dependent histone mRNAs

In animal cells, replication-dependent histone mRNAs end with a highly conserved stem–loop structure followed by a 4- to 5-nucleotide single-stranded tail. This unique 3′ end distinguishes replication-dependent histone mRNAs from all other eukaryotic mRNAs, which end with a poly(A) tail produced by the canonical 3′-end processing mechanism of cleavage and polyadenylation. The pioneering studies of Max Birnstiel's group demonstrated nearly 40 years ago that the unique 3′ end of animal replication-dependent histone mRNAs is generated by a distinct processing mechanism, whereby histone mRNA precursors are cleaved downstream of the stem–loop, but this cleavage is not followed by polyadenylation. The key role is played by the U7 snRNP, a complex of a ∼60 nucleotide U7 snRNA and many proteins. Some of these proteins, including the enzymatic component CPSF73, are shared with the canonical cleavage and polyadenylation machinery, justifying the view that the two metazoan pre-mRNA 3′-end processing mechanisms have a common evolutionary origin. The studies on U7 snRNP culminated in the recent breakthrough of reconstituting an entirely recombinant human machinery that is capable of accurately cleaving histone pre-mRNAs, and determining its structure in complex with a pre-mRNA substrate (with 13 proteins and two RNAs) that is poised for the cleavage reaction. The structure uncovered an unanticipated network of interactions within the U7 snRNP and a remarkable mechanism of activating catalytically dormant CPSF73 for the cleavage. This work provides a conceptual framework for understanding other eukaryotic 3′-end processing machineries.

Kinetic Study of the Avocado Sunblotch Viroid Self-Cleavage Reaction Reveals Compensatory Effects between High-Pressure and High-Temperature: Implications for Origins of Life on Earth

A high pressure apparatus allowing one to study enzyme kinetics under pressure was used to study the self-cleavage activity of the avocado sunblotch viroid. The kinetics of this reaction were determined under pressure over a range up to 300 MPa (1–3000 bar). It appears that the initial rate of this reaction decreases when pressure increases, revealing a positive ΔV≠ of activation, which correlates with the domain closure accompanying the reaction and the decrease of the surface of the viroid exposed to the solvent. Although, as expected, temperature increases the rate of the reaction whose energy of activation was determined, it appeared that it does not significantly influence the ΔV≠ of activation and that pressure does not influence the energy of activation. These results provide information about the structural aspects or this self-cleavage reaction, which is involved in the process of maturation of this viroid. The behavior of ASBVd results from the involvement of the hammerhead ribozyme present at its catalytic domain, indeed a structural motif is very widespread in the ancient and current RNA world.

Synthesis of new pyrazolo[1,2,3]triazines by cyclative cleavage of pyrazolyltriazenes

We describe the synthesis of so far synthetically not accessible 3,6-substituted-4,6-dihydro-3H-pyrazolo[3,4-d][1,2,3]triazines as nitrogen-rich heterocycles. The target compounds were obtained in five steps, including an amidation and a cyclative cleavage reaction as key reaction steps. The introduction of two side-chains allowed a variation of the pyrazolo[3,4-d][1,2,3]triazine-core with commercially available building blocks, enabling the extension of the protocol to gain other derivatives straightforwardly. Attempts to synthesize 3,7-substituted-4,7-dihydro-3H-pyrazolo[3,4-d][1,2,3]triazines, the regio-isomers of the successfully gained 3,6-substituted-4,6-dihydro-3H-pyrazolo[3,4-d][1,2,3]-3H-triazines, were not successful under similar conditions due to the higher stability of the triazene functionality in the regio-isomeric precursors and thus the failure of the removal of the protective group.