Investigation of Sequence Clipping and Structural Heterogeneity of an HIV Broadly Neutralizing Antibody by a Comprehensive LC-MS Analysis

CAP256 is one of the highly potent, broadly neutralizing monoclonal antibodies (bNAb) designed for HIV-1 therapy. During the process development of one of the constructs, an unexpected product-related impurity was observed via microfluidics gel electrophoresis. A panel of complementary LC-MS analyses was applied for the comprehensive characterization of CAP256 which included the analysis of the intact and reduced protein, the middle-up approach, and a set of complementary peptide mapping techniques and verification of the disulfide bonds. The designed workflow allowed to identify a clip within a protruding acidic loop in the CDR-H3 region of the heavy chain, which can lead to the decrease of bNAb potency. This characterization explained the origin of the additional species reflected by the reducing gel profile. An intra-loop disulfide bond linking the two fragments was identified, which explained why the non-reducing capillary electrophoresis (CE) profile was not affected. The extensive characterization of CAP256 post-translational modifications was performed to investigate a possible cause of CE profile complexity and to illustrate other structural details related to this molecule’s biological function. Two sites of the engineered Tyr sulfation were verified in the antigen-binding loop, and pyroglutamate formation was used as a tool for monitoring the extent of antibody clipping. Overall, the comprehensive LC-MS study was crucial to (1) identify the impurity as sequence clipping, (2) pinpoint the clipping location and justify its susceptibility relative to the molecular structure, (3) lead to an upstream process optimization to mitigate product quality risk, and (4) ultimately re-engineer the sequence to be clip-resistant.

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Greener approach toward the generation of dimedone derivatives

The use of dimedone in green chemistry has been described for the synthesis of selective heterocyclic motifs which are both pharmacologically and industrially important. The objective of this review is to summarize some of the selected recent advances of dimedone in the synthesis of organic compounds utilizing green chemistry procedures.     

Enhanced permeation, leaf retention, and plant protease inhibitor activity with bicontinuous microemulsions

Bicontinuous microemulsions (BCMEs) have excellent solubulizing properties along with low interfacial tension and aqueous content that can be controlled. In this work, water soluble plant protease inhibitor (PI), well characterized for its activity against insect pests, was incorporated into a BCME system and explored for permeation on hydrophobic leaf surfaces and protease inhibition activity. The bicontinuous nature of the microemulsion containing water:2-propanol:1-butanol (55:35:10 w/w) was characterized using conductivity and self-diffusion coefficient measurements. The PI was soluble in the water-rich bicontinuous domains, stable in the microemulsions, and protease inhibition activity was retained for a prolonged duration. The microemulsions ensured greater wettability and a wider spread of the PI on hydrophobic leaf surfaces as revealed by contact angle measurements. Significantly, trypsin inhibition activity assays of the PI recovered from the leaves after delivery from the microemulsion indicated a significant increase in the PI retention on the leaf. This BCME enabled greater leaf permeation and retention of the PI can be attributed to a temporary disruption of the waxy leaf surface followed by self-repair without causing any long term damage to the plant.     

Mechanical and electronic approaches to improve the sensitivity of microcantilever sensors

Advances in the field of micro electro mechanical systems and their uses now offer unique opportunities in the design of ultrasensitive analytical tools. The analytical community continues to search for cost-effective, reliable, and even portable analytical techniques that can give reliable and fast response results for a variety of chemicals and biomolecules. Microcantilevers （MCLs） have emerged as a unique platform for label-free chem-sensor or bioassay. Several electronic designs, including piezoresistive, piezoelectric, and capacitive approaches, have been applied to measure the bending or frequency change of the MCLs upon exposure to chemicals. This review summarizes mechanical, fabrication, and electronics approaches to increase the sensitivity of MCL sensors.     

Titania-silica nanoparticles ensemblies assisted heterogeneous catalytic strategy for the synthesis of pharmacologically significant 2,3-diaryl-3,4-dihydroimidazo[4,5-b]indole scaffolds

Present paper elicits the multicomponent reaction (MCR) strategy assisted by titania nanoparticles hosted on silica (TiO₂.SiO₂ NPs) as heterogeneous catalyst to synthesize a series of pharmacologically significant 2,3-diaryl-3,4-dihydroimidazo[4,5-b]indole derivatives. To the best of our information, the use of isatin as one of the precursors was hitherto unreported. The decrease in reaction time, low catalyst loading, high product yield (up to 92%), and excellent reusability of the catalyst (up to 7 cycles) put this protocol under the umbrella of green chemistry tenets. Characterization of catalysts was achieved through a number of techniques viz., energy-dispersive X-ray (EDX) spectroscopy, field emission scanning electron microscopy (FESEM), powder X-ray diffraction (XRD), fourier transform infrared (FTIR) spectra of adsorbed pyridine, temperature-programmed desorption of ammonia, and porosity measurements by nitrogen adsorption (Brunauer–Emmett–Teller [BET] method). Also, the structures of synthesized compounds were corroborated on the basis of FTIR, nuclear magnetic resonance (NMR), mass, and elemental analyses data.     

Tuning of Bistability,Thermal Stability of the Metastable States,and Application Prospects in the C3-Symmetric Designs of Multiple Azo(hetero)arenes Systems

Light-responsive molecular systems with multiple photoswitches in C₃-symmetric designs have enormous application potential. The design part of such molecular systems is critical due to its influence in several properties associated with the photoswitches. In order to tune, and in the evaluation of the design–property relationship, we synthesized 18 tripodal systems with variations in the core, linkers, connectivity, and azo(hetero)arene photoswitches. Through extensive spectroscopic and computational studies, we envisaged the factors controlling near-quantitative photoisomerization in both the directions (bistability) and the thermal stability of the metastable states. Furthermore, we also evaluated the impact of designs in obtaining reversible photo-responsive sol-gel phase transitions, solvatochromism, photo- and thermochromism.     

Ring-chain isomerism in conjugated guanylhydrazones: Experimental and theoretical study

Treatment of 1,3-diaryl-propene-2-one with aminoguanidine under acidic conditions for a short reaction period (1?h) delivers - in accordance with a literature report - the corresponding guanylhydrazones. However, when the reaction time was increased to 12?h, formation of the ring annulated product 4,5-dihydro-1H-pyrazole-1-carboximidamide was observed. This is the first case of ring-chain isomerism in conjugated guanylhydrazones. The acyclic conjugated guanylhydrazone and the corresponding annulated product (4,5-dihydro-1H-pyrazole-1-carboximidamide) could be clearly distinguished by means of UV and NMR spectroscopy. The formation of the ring isomer was further confirmed by single crystal XRD analysis. The time-dependent ¹H NMR study indicated the gradual transformation of the open-chain compound into the cyclic one. The mechanistic insights into the formation of these two products were explored using quantum chemical methods which revealed that the ring isomer is thermodynamically more stable than the open-chain isomer by 6–11?kcal/mol and the barrier for cyclization was found to be 31.37?kcal/mol.     

Dirhodium tetraprolinate-catalyzed asymmetric cyclopropanations with high turnover numbers

The bridged dirhodium tetraprolinate Rh(2)(S-biTISP)(2) (2) catalyzes the asymmetric cyclopropanation reaction between methyl phenyldiazoacetate and styrene at room temperature with high turnover number (92 000) and turnover frequency (4000 per h). [reaction: see text]