Triflic acid catalyzed reductive coupling reactions of carbonyl compounds with O-, S-, and N-nucleophiles.

Highly efficient metal-free reductive coupling reactions of aldehydes and ketones with a range of nucleophiles in the presence of triflic acid (1-5 mol%) as the catalyst are presented. The reactions can be performed at ambient temperature without exclusion of moisture or air. A range of symmetrical and unsymmetrical ethers were obtained by this method in high yields and short reaction times. For the first time, the influence of additional functionalization has been studied. Furthermore, the formation of thioethers from ketones (by addition of unmodified thiols) and of sulfonamides from either aldehydes or ketones has been achieved under catalytic conditions.

A new catalytic oxidative cleavage reaction to furnish lactones.

A highly efficient oxidative cleavage reaction of THF and THP alcohols to gamma- and delta-lactones using catalytic PCC (1 mol%) and periodic acid as terminal oxidant is presented.

Characterization of neutralizing epitopes within the major capsid protein of human papillomavirus type 33.

BACKGROUND: Infections with papillomaviruses induce type-specific immune responses, mainly directed against the major capsid protein, L1. Based on the propensity of the L1 protein to self-assemble into virus-like particles (VLPs), type-specific vaccines have already been developed. In order to generate vaccines that target a broader spectrum of HPV types, extended knowledge of neutralizing epitopes is required. Despite the association of human papillomavirus type 33 (HPV33) with cervical carcinomas, fine mapping of neutralizing conformational epitopes on HPV33 has not been reported yet. By loop swapping between HPV33 and HPV16 capsid proteins, we have identified amino acid sequences critical for the binding of conformation-dependent type-specific neutralizing antibodies to surface-exposed hyper variable loops of HPV33 capsid protein L1. RESULTS: Reactivities of monoclonal antibodies (mAbs) H33.B6, H33.E12, H33.J3 and H16.56E with HPV16:33 and HPV33:16 hybrid L1 VLPs revealed the complex structures of their conformational epitopes as well as the major residues contributing to their binding sites. Whereas the epitope of mAb H33.J3 was determined by amino acids (aa) 51-58 in the BC loop of HPV33 L1, sequences of at least two hyper variable loops, DE (aa 132-140) and FGb (aa 282-291), were found to be essential for binding of H33.B6. The epitope of H33.E12 was even more complex, requiring sequences of the FGa loop (aa 260-270), in addition to loops DE and FGb. CONCLUSION: These data demonstrate that neutralizing epitopes in HPV33 L1 are mainly located on the tip of the capsomere and that several hyper variable loops contribute to form these conformational epitopes. Knowledge of the antigenic structure of HPV is crucial for designing hybrid particles as a basis for intertypic HPV vaccines.

The German R&D Program for CO2 Utilization-Innovations for a Green Economy.

Carbon capture and utilization (CCU) is a field of key emerging technologies. CCU can support the economy to decrease the dependency on fossil carbon raw materials, to stabilize electricity grids and markets with respect to a growing share of fluctuating renewable energy. Furthermore, it can contribute to mitigate anthropogenic CO2 emissions. The German Federal Ministry of Education and Research has provided substantial financial support for research and development projects, stimulating research, development, and innovations in the field of CO2 utilization. This review provides an overview over the most relevant funding measures in this field. Examples of successful projects demonstrate that CCU technologies are already economically viable or technologically ready for industrial application. CCU technologies as elements of a future "green economy" can contribute to reach the ambitious German sustainability targets with regard to climate protection as well as raw material productivity.

Development, validation, and standardization of polymerase chain reaction-based detection of E. coli O157.

A diagnostic polymerase chain reaction assay was developed for the detection of E. coli O157 as the first part of a multicenter validation and standardization project. The assay is based on amplification of sequences of the rfbE O157 gene and includes an internal amplification control. The selectivity of the assay was evaluated against 155 strains, including 32 E. coli O157, 38 E. coli non-O157, and 85 non-E. coli. It was shown to be highly inclusive (100%) and exclusive (100%). The assay has a 100% detection probability of approximately 2 x 10(3) cells per reaction.

Toward an international standard for PCR-based detection of foodborne Escherichia coli O157: validation of the PCR-based method in a multicenter interlaboratory trial.

The performance of a polymerase chain reaction (PCR) method for detection of Escherichia coli O157, previously validated on DNA extracted from pure cultures, was evaluated on spiked cattle swabs through an interlaboratory trial, including 12 participating laboratories from 11 European countries. Twelve cattle swab samples, spiked at 4 levels (0, 1-10, 10-100, and 100-1000 colony-forming units, in triplicate) with E. coli O157 were prepared centrally in the originating laboratory; the receiving laboratories performed pre-PCR treatment followed by PCR. The results were reported as positive when the correct amplicons were present after gel electrophoresis. The statistical analysis, performed on 10 sets of reported results, determined the diagnostic sensitivity to be 92.2%. The diagnostic specificity was 100%. The accordance (repeatability) was 90.0%, calculated from all positive inoculation levels. The concordance (reproducibility) was 85.0%, calculated from all positive inoculation levels. The concordance odds ratio (degree of interlaboratory variation calculated from all positive inoculation levels) was 1.58, indicating the robustness of the PCR method. Thus, the interlaboratory variation due to personnel, reagents, minor temperature or pH fluctuations and, not least, thermal cyclers, did not affect the performance of the method, which is currently being considered as part of an intenational PCR standard.

Chemical chaperones improve protein secretion and rescue mutant factor VIII in mice with hemophilia A.

Inefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking.

Transgene loss and changes in the promoter methylation status as determinants for expression duration in nonviral gene transfer for factor IX.

Advances in delivery techniques and in expression construct design have renewed interest in nonviral gene transfer. Here, we test plasmid or bacterial backbone free minicircle vectors for factor IX (FIX) expression by hydrodynamic liver-directed delivery. Both constructs are driven by a hepatic control region, the human α(1)-antitrypsin promoter, which results in long-term expression in FIX knockout mice. However, levels of expression were higher and expression loss over time was reduced when using minicircles. Even at the highest expression levels (>700% of normal) FIX was fully functional. Transgene loss was the main determinant for expression loss over time for both vector types. A significant effect of gene silencing was observed only for the plasmid, not for the minicircle vector. To determine the influence of promoter methylation, we performed bisulfite-mediated conversion and sequencing of vector DNA on days 14 and 100 after gene transfer. We determined a higher frequency of methyl-protected cytosines in CpGs and a lower degree of demethylation at bacterial Dcm methylation sequences near transcription factor-binding sites in the α(1)-antitrypsin promoter in plasmid compared with minicircle mice on day 100. Therefore, the methylation status might reflect differences in the levels and durability of expression. Judging from the high levels of functional FIX obtained, small fractions of liver or single liver segments should be sufficient to reach therapeutic efficacy in translating hydrodynamic delivery to humans. However, transgene loss remains to be addressed to further guarantee sustained expression over time.

A concise and scalable route to L-azidohomoalanine.

A concise and highly efficient synthetic route to L-azidohomoalanine (L-Aha) and its homologues is presented here. These chemically modified amino acids are used for the introduction of bioorthogonal handles into proteins. The described route avoids major problems of previously reported methods including expensive starting materials, low efficiency, and lack of scalability. Starting from inexpensive N-Boc-O-Bn-L-aspartic acid, gram quantities of L-Aha hydrochloride can be prepared with high purity. The reactions can be completed within 1 week and the products can be incorporated into proteins using L-methionine auxotrophs.

New S-adenosyl-L-methionine analogues: synthesis and reactivity studies.

Two new and complementary synthetic strategies for 5'-N-chloroethylamino-5'-deoxyadenosines are presented. Additionally, the reaction kinetics of their conversion into aziridines under typical enzyme assay conditions is reported using time-resolved NMR spectroscopy. A stable photocaged derivative of 5'-N-chloroethylamino-5'-deoxyadenosine has also been synthesized, and its stability and activation in aqueous solution at physiological pH have been examined.

Factor VIII-eGFP fusion proteins with preserved functional activity for the analysis of the early secretory pathway of factor VIII.

Considering the difficulty in detecting factor (F)VIII in vivo, fluorescently labelled FVIII protein provides a tool to analyse the intracellular localisation, bio distribution, and pharmacokinetics of the protein in living organisms. Here, we report the use of FVIII full length and B-domain deleted proteins, fused to enhanced green fluorescent protein (eGFP) at the C-terminus of the coagulation protein via a nine amino acid spanning linker. Comparison of the FVIII-eGFP fusion proteins to their unlabelled counterparts showed no impairment with respect to recombinant expression levels, intracellular processing, specific coagulant activity and decay at physiological temperature. Confocal live cell imaging demonstrated ER-Golgi-transport of B-domain deleted FVIII-eGFP in vesicular tubular carriers. Using temperature blocks and release experiments, imaging of FVIII-eGFP fusion proteins enabled for the first time the visualisation of the early secretory pathway of B-domain deleted FVIII in living cells and in particular highlighted the apparent deficit of active transport carriers, an observation consistent with the low rates of FVIII secretion seen in recombinant expression systems.

Protein scaffold and expression level determine antiviral activity of membrane-anchored antiviral peptides.

Cell membrane-anchored (ma) antiviral peptides derived from the C-terminal heptad repeat of the HIV-1 transmembrane glycoprotein gp41 (C-peptides) and expressed from retroviral vectors were shown to efficiently inhibit HIV-1 entry into target cells. Here, we analyzed the influence of the vector backbone, the scaffold modules that anchor the peptide to the membrane and the length of the C-peptide on expression level and antiviral activity. In general, antiviral activity was determined primarily by the density of the C-peptide on the cell surface. By influencing expression levels, the scaffold elements indirectly also determined antiviral activity. Additional direct effects of the scaffold on antiviral activity were minor. At comparable expression levels, the elongated C-peptide (maC46) was found to be more potent than the shorter maC36. On the basis of these findings, a dose-response assay was established that quantifies antiviral activity relative to the expression level of the antiviral gene product. Taken together, these data demonstrate the importance of analyzing the efficacy of therapeutic genes relative to the dose of the gene product.