Genotyping the hepatitis B virus with a fragment of the HBV DNA polymerase gene in Shenyang, China

The hepatitis B virus (HBV) has been classified into eight genotypes (A-H) based on intergenotypic divergence of at least 8% in the complete nucleotide sequence or more than 4% in the S gene. To facilitate the investigation of the relationship between the efficacy of drug treatment and the mutation with specific genotype of HBV, we have established a new genotyping strategy based on a fragment of the HBV DNA polymerase gene. Pairwise sequence and phylogenetic analyses were performed using CLUSTAL V (DNASTAR) on the eight (A-H) standard full-length nucleotide sequences of HBV DNA from GenBank (NCBI) and the corresponding semi-nested PCR products from the HBV DNA polymerase gene. The differences in the semi-nested PCR fragments of the polymerase genes among genotypes A through F were greater than 4%, which is consistent with the intergenotypic divergence of at least 4% in HBV DNA S gene sequences. Genotyping using the semi-nested PCR products of the DNA polymerase genes revealed that only genotypes B, C, and D were present in the 50 cases, from Shenyang, China, with a distribution of 11 cases (22%), 25 cases (50%), and 14 cases (28%) respectively. These results demonstrate that our new genotyping method utilizing a fragment of the HBV DNA polymerase gene is valid and can be employed as a general genotyping strategy in areas with prevalent HBV genotypes A through F. In Shenyang, China, genotypes C, B, and D were identified with this new genotyping method, and genotype C was demonstrated to be the dominant genotype

The Hepatitis B Virus Ribonuclease H Is Sensitive to Inhibitors of the Human Immunodeficiency Virus Ribonuclease H and Integrase Enzymes

Nucleos(t)ide analog therapy blocks DNA synthesis by the hepatitis B virus (HBV) reverse transcriptase and can control the infection, but treatment is life-long and has high costs and unpredictable long-term side effects. The profound suppression of HBV by the nucleos(t)ide analogs and their ability to cure some patients indicates that they can push HBV to the brink of extinction. Consequently, more patients could be cured by suppressing HBV replication further using a new drug in combination with the nucleos(t)ide analogs. The HBV ribonuclease H (RNAseH) is a logical drug target because it is the second of only two viral enzymes that are essential for viral replication, but it has not been exploited, primarily because it is very difficult to produce active enzyme. To address this difficulty, we expressed HBV genotype D and H RNAseHs in E. coli and enriched the enzymes by nickel-affinity chromatography. HBV RNAseH activity in the enriched lysates was characterized in preparation for drug screening. Twenty-one candidate HBV RNAseH inhibitors were identified using chemical structure-activity analyses based on inhibitors of the HIV RNAseH and integrase. Twelve anti-RNAseH and anti-integrase compounds inhibited the HBV RNAseH at 10 μM, the best compounds had low micromolar IC50 values against the RNAseH, and one compound inhibited HBV replication in tissue culture at 10 μM. Recombinant HBV genotype D RNAseH was more sensitive to inhibition than genotype H. This study demonstrates that recombinant HBV RNAseH suitable for low-throughput antiviral drug screening has been produced. The high percentage of compounds developed against the HIV RNAseH and integrase that were active against the HBV RNAseH indicates that the extensive drug design efforts against these HIV enzymes can guide anti-HBV RNAseH drug discovery. Finally, differential inhibition of HBV genotype D and H RNAseHs indicates that viral genetic variability will be a factor during drug development. © 2013 Tavis et al

Photoinduced electron transfer reactions of alpha-cyclopropyl- and alpha-epoxy ketones. Tandem fragmentation-cyclization to bi-, tri-, and spirocyclic ketones

Kirschberg T, Mattay J. Photoinduced electron transfer reactions of alpha-cyclopropyl- and alpha-epoxy ketones. Tandem fragmentation-cyclization to bi-, tri-, and spirocyclic ketones. The Journal of Organic Chemistry. 1996;61(25):8885-8896.Reductive photoinduced electron transfer (PET) reactions have been performed with various bicyclic alpha-cyclopropyl-substituted ketones and tertiary amines. The reaction resulted in a regioselective cleavage of one cyclopropyl bond under formation of an exocyclic radical with an endocyclic enolate unit. In the case of bicyclic ketones with an unsaturated side chain, various bicyclic, spirocyclic, and tricyclic products are accessible via radical cyclization, depending on the position of the alkenyl substituent. In addition to triethylamine, N-silylated amines have also been used as electron donors, leading to a variety of compounds, among them are silylated fragmentation products, indicating that a proton is transferred from not only the amine radical cation but also the cationic silyl group. The intramolecular Paterno-Buchi reaction has also been studied for cyclopropane derivatives of the jasmone type leading to tetracyclic oxetanes. Finally, alpha-epoxy-substituted ketones have been investigated under PET conditions, yielding ring-opened products

FRAGMENTATION-CYCLIZATION REACTIONS BY PHOTOINDUCED ELECTRON-TRANSFER

KIRSCHBERG T, Mattay J. FRAGMENTATION-CYCLIZATION REACTIONS BY PHOTOINDUCED ELECTRON-TRANSFER. Tetrahedron Letters. 1994;35(39):7217-7220.Irradiation of unsaturated bicyclo[4.1.0]heptanones and bicyclo[3.1.0]hexanones under reductive PET conditions leads to a regioselective cleavage of the cyclopropane moiety followed by cyclization. By this method various types of ring anellated and spirocyclic compounds are accessible

Reductive cyclization of alpha-cyclopropylketones with alkynyl- and aryl-tethered substituents

Fagnoni M, Schmoldt P, Kirschberg T, Mattay J. Reductive cyclization of alpha-cyclopropylketones with alkynyl- and aryl-tethered substituents. Tetrahedron. 1998;54(23):6427-6444.Photoinduced electron transfer (PET) reactions of alpha-cyclopropyl-substituted ketones and triethylamine (TEA) were used to initiate the cyclopropylcarbinyl-homoallyl rearrangement. The intramolecular cyclization reaction onto triple bonds was performed yielding bicyclic and spirocyclic compounds. Furthermore, in some preliminary studies it was shown that even intramolecular aromatic substitutions are feasible. (C) 1998 Elsevier Science Ltd. All rights reserved

Photochemical reactions of cyclopropyl ketones by electron transfer

Schmoldt P, Kirschberg T, Fagnoni M, Mattay J. Photochemical reactions of cyclopropyl ketones by electron transfer. JOURNAL OF INFORMATION RECORDING. 1998;24(3-4):249-252.Photoinduced electron transfer (PET) from triethylamine (TEA) to cyclopropyl ketones initiated a cyclopropylcarbinyl-homoallyl rearrangement. Attack of the so formed homoallyl radicals on a terminally unsaturated side chain gave various bicyclic products the structure of which depends on the position and length of that side chain

PHOTOINDUCED ELECTRON-TRANSFER (PET) IN ORGANIC-SYNTHESIS - [3+2)-TYPE CYCLOADDITION, CYCLIZATION AND C-C BOND-CLEAVAGE REACTIONS

ALBRECHT E, AVERDUNG J, BISCHOF EW, et al. PHOTOINDUCED ELECTRON-TRANSFER (PET) IN ORGANIC-SYNTHESIS - [3+2)-TYPE CYCLOADDITION, CYCLIZATION AND C-C BOND-CLEAVAGE REACTIONS. Journal of Photochemistry and Photobiology A Chemistry. 1994;82(1-3):219-232.An area of growing interest, in organic photochemistry in particular, involves the use of light to induce an electron transfer from a donor (D) to an acceptor (A) molecule, designated photoinduced electron transfer (PET). Excitation of A or D leads to well-defined changes in their redox properties, i.e. A (D) becomes a stronger acceptor (donor). In general, the feasibility of producing radical ions can be predicted using the well-known Weller equation and, moreover, polar solvents and salts effects support their formation. Following a brief introduction, we focus on the synthetic applications of PET, and discuss [3+2] cycloadditions, reductive and oxidative cyclizations and C-C bond cleavage reactions. The PET ring cleavage of azirines results in the formation of 2-azaallenyl radical cations which may be trapped by dipolarophiles to give heterocyclic compounds. Using this method, we have developed a new route to heterophanes. Porphyrins can also be prepared by this procedure. Further studies aim ot reveal the scope and limitations of these new reactions. Radical cations and radical anions can attack pi bonds within the same molecule. Depending on the reaction conditions chosen to generate the radical ions, five- or six-membered ring systems can be obtained. Under PET reductive conditions, radical anions arise which preferentially form five-membered ring molecules (Baldwin rule). However, using PET oxidative conditions, radical cations are generated which preferentially form six-membered ring molecules due to their electrophilic character. In addition, we have developed a new reductive C-C bond cleavage reaction with annular cyclopropanes and cyclobutanes and have applied this method to a new short synthesis of the hirsutene ring system