Cross-Electrophile C-PIII Coupling of Chlorophosphines with Organic Halides: Photoinduced PIII and Aminoalkyl Radical Generation Enabled by Pnictogen Bonding.

Pnictogen bonding (PnB) has gained recognition as an appealing strategy for constructing novel architectures and unlocking new properties. Within the synthetic community, the development of a straightforward and much simpler protocol for cross-electrophile C-PIII coupling remains an ongoing challenge with organic halides. In this study, we present a simple strategy for photoinduced PnB-enabled cross-electrophile C-PIII couplings using readily available chlorophosphines and organic halides via merging single electron transfer (SET) and halogen atom transfer (XAT) processes. In this photomediated transformation, the PnB formed between chlorophosphines and alkyl amines facilitates the photogeneration of PIII radicals and α-aminoalkyl radicals through SET. Subsequently, the resulting α-aminoalkyl radicals activate C-X bonds via XAT, leading to the formation of carbon radicals. This methodology offers operational simplicity and compatibility with both aliphatic and aromatic chlorophosphines and organic halides.

Expression of glycoproteins bearing complex human-like glycans with galactose terminal in Hansenula polymorpha.

Glycoproteins derived from Hansenula polymorpha can not be used for therapeutic purposes due to their high-mannose type asparagine-linked (N-linked) glycans, which result in immune reactions and poor pharmacokinetic behaviors in human body. Previously, we reported that the trimannosyl core N-linked glycans (Man(3)GlcNAc(2)) intermediate can be generated in endoplasmic reticulum in HpALG3 and HpALG11 double-mutant H. polymorpha. Here, we describe the further modification of the glycosylation pathway in this double-defect strain to express glycoproteins with complex human-like glycans. After eliminating the impact of HpOCH1, three glycosyltransferases were introduced into this triple-mutant strain. When human ß-1,2-N-acetylglucosaminyltransferase I (hGnTI) was efficiently targeted in early Golgi, more than 95 % glycans attached to the glycoproteins were added one N-acetylglucosamine (GlcNAc). With subsequently introduction of rat ß-1,2-N-acetylglucosaminyltransferase II (rGnTII) and human ß-1,4-galactosyltransferase I (hGalTI), several glycoengineered strains can produce glycoproteins bearing glycans with terminal N-acetylglucosamine or galactose. The expression of glycoproteins with glycan Gal(2)GlcNAc(2)Man(3)GlcNAc(2) represents a significant step toward the ability to express fully humanized glycoproteins in H. polymorpha. Furthermore, several shake-flask and bioreactor fermentation experiments indicated that, although the cells do display a reduction in growth rate, the glycoengineered strains are still suitable for high-density fermentation.

Development of an α-amylase reporter system for efficient screening of clones with highly expressed heterologous protein in Hansenula polymorpha.

To check feasibility and effectiveness of the α-amylase reporter system, two vectors were designed and tested using hepatitis B virus surface antigen (HBsAg) and Homo sapiens granulocyte-macrophage colony stimulating factor 2 (hGM-CSF2) as a model. By integrating the vector containing two independent cassettes into the same genome locus, high-producing clones of HBsAg (or hGM-CSF2) were screened using the α-amylase as a reporter. Results show there was a positive correlation (Correlation coefficient, R (2) > 0.95) between the yield of recombinant proteins and the α-amylase activity of corresponding transformants, which was independent of the gene dosage.

Nonsense mutations of replicase and movement protein genes contribute to the attenuation of an avirulent tomato mosaic virus.

Three recovery mutants of an avirulent Tomato mosaic virus genus: (Tobamovirus) (ToMV-K) with back mutations of the replicase and/or movement protein (MP) genes, have been constructed by site-directed mutagenesis, and infectious plasmids (pToMV-K) were obtained. The rescued phenotypes of the progeny viruses showed that the replicase and MP recovery mutant (ToMV-K(rase-mp)) induced severe symptoms on both systemic and necrotic plants similar to those induced by the virulent strain. The replicase back mutant (ToMV-K(rase)) produced chlorosis and mosaic symptoms on N. tabacum cv. Huangmiaoyu (systemic host), while the MP recovery mutant (ToMV-K(mp)) produced no systemic symptoms on Huangmiaoyu tobacco. Sequencing of the cDNA of progeny viruses revealed that the "back mutants" maintained these mutation sites during infection. Protein immunoblots indicated that the 98 and 126 kDa proteins were expressed in the plants systemically infected by ToMV-K and pToMV-K, whereas no 98 kDa protein was detected in the plants infected by ToMV. The MPs (27 kDa) of ToMV-K and pToMV-K in the plants were smaller in size than those (30 kDa) of ToMV and pToMVK(rase-mp). These data suggest that ToMV-K replicates and spreads by expressing the truncated 98 and 126 kDa replicases and 27 kDa MP in plants. The opal mutation at nucleotides (nt) 2670-2672 of the replicase gene mainly contributes to the attenuation of ToMV-K, whereas the mutations at nt 5632-5664 of the MP gene attenuate the induced symptoms.