Vellore Institute of Technology (VIT) is a private university located in&nbsp;Tamil Nadu, India. Founded in 1984, as Vellore Engineering College, the institution offers 20 undergraduate, 34 postgraduate, four integrated and four research programs. It has campuses in Vellore, Amravati, Bhopal and Chennai.

VIT is one of the top ranked private universities in India according to NIRF, THE and QS Rankings.&nbsp;Govt. of India has recognized&nbsp;VIT, Vellore as an&nbsp;Institution of Eminence. This has allowed VIT to take independent quality initiatives and move up in world ranking.

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VIT University

Conjugate Sn(iv)(pyropheophorbide a)dichloride-(peptide nucleic acid) catalyzes reduction of azobenzene derivatives in the presence of complementary nucleic acid (NA) upon irridiation with red light (660 nm). This is the first red light-induced NA-templated photoreduction. It is highly sensitive to single mismatches in the NA-template and can detect down to 5 nM NAs. This journal is © The Royal Society of Chemistry.

Department of Chemistry

School of Advanced Sciences

Vellore Campus

World University root node

Chemical Communications

Red light-triggered photoreduction on a nucleic acid template

Racemic mixtures frequently display properties that are different from those associated with their enantiopure counterparts, and are often characterized by higher propensity to form aggregates. Our previous research established that mixing of the enantiomers of Alzheimer amyloid β (Aβ) 42 peptides is an effective strategy to induce an oligomer-to-fibril conversion, which puts Aβ42 into a substantially less toxic state. Here, new insights into this chiral inactivation effect are presented. In addition to the commonly used Thioflavin T fibril formation assays, the use of the less aggregation-prone Aβ40 system allowed us to monitor peptide aggregation by NMR. Whereas enantiopure peptide was well soluble under the chosen experimental conditions and showed no sign of precipitation, addition of one equivalent of the mirror-image peptide triggered an instant and rapid aggregation, observable through the attenuation of the NMR signal. The racemic Aβ40 fibrils were found by transmission electron microscopy to be distinct in morphology, exhibiting a ~2-fold narrowing as compared with their enantiopure counterparts. © 2019 Wiley Periodicals, Inc.

Peptide Science

New insights into differential aggregation of enantiomerically pure and racemic Aβ40 systems

Amyloid β is an inherently disordered peptide that can form diverse neurotoxic aggregates, and its 42-amino-acid isoform is believed to be the agent responsible for Alzheimer's disease (AD). Cellular uptake of the peptide is a pivotal step for it to be able to exert many of its toxic actions. The cellular uptake process is complex, and numerous competing internalization pathways have been proposed. To date, it remains unclear which of the uptake mechanisms are particularly important for the overall process, and improvement of this understanding is needed, so that better molecular AD therapeutics can be designed. Chirality can be used as a unique tool to study this process, because some of the proposed mechanisms are expected to proceed in stereoselective fashion, whereas others are not. To shed light on this important issue, we synthesized fluorescently labeled enantiomers of amyloid β and quantified their cellular uptake, finding that uptake occurs in stereoselective fashion, with a typical preference for the l stereoisomer of ≈5:1. This suggests that the process is predominantly receptor-mediated, with likely minor contributions of non-stereoselective mechanisms. © 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Fulltext

ChemBioChem

Chirality Dependence of Amyloid β Cellular Uptake and a New Mechanistic Perspective

Racemates often have lower solubility than enantiopure compounds, and the mixing of enantiomers can enhance the aggregation propensity of peptides. Amyloid beta (Aβ) 42 is an aggregation-prone peptide that is believed to play a key role in Alzheimer's disease. Soluble Aβ42 aggregation intermediates (oligomers) have emerged as being particularly neurotoxic. We hypothesized that the addition of mirror-image d-Aβ42 should reduce the concentration of toxic oligomers formed from natural l-Aβ42. We synthesized l- and D-Aβ42 and found their equimolar mixing to lead to accelerated fibril formation. Confocal microscopy with fluorescently labeled analogues of the enantiomers showed their colocalization in racemic fibrils. Owing to the enhanced fibril formation propensity, racemic Aβ42 was less prone to form soluble oligomers. This resulted in the protection of cells from the toxicity of l-Aβ42 at concentrations up to 50 μm. The mixing of Aβ42 enantiomers thus accelerates the formation of non-toxic fibrils. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Angewandte Chemie - International Edition

Suppression of Oligomer Formation and Formation of Non-Toxic Fibrils upon Addition of Mirror-Image Aβ42 to the Natural l-Enantiomer

Amyloid beta-protein 42 plays an important role in the onset and progression of Alzheimer's disease. Familial mutations have identified the glutamate residue 22 as a hotspot with regard to peptide neurotoxicity. We introduce an approach to study the influence of systematic sidechain modification at this residue, employing chirality as a structural probe. Circular dichroism experiments reveal that charge-preserving alterations of the amino acid sidechain attenuate the characteristic random coil to β-sheet transition associated with the wildtype peptide. Removal of the negative charge from residue 22, a trait observed with all known familial mutations at this residue, gives rise to a peptide with limited random coil propensity and high β-sheet characteristics. Our approach can be extended to other residues of Aβ, as well as further amyloidogenic peptides. © 2016 Wiley Periodicals, Inc.

Chirality

Using chiral peptide substitutions to probe the structure function relationship of a key residue of Aβ42

Amyloidogenic peptides such as the Alzheimer’s disease-implicated Amyloid beta (Aβ), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (Aβ42) and amyloid beta 40 (Aβ40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (>95%) Aβ42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the Aβ40 peptide with identical success and without the need to alter the method. © 2017 Journal of Visualized Experiments.

Journal of Visualized Experiments

A tailored HPLC purification protocol that yields high-purity amyloid beta 42 and amyloid beta 40 peptides, capable of oligomer formation

Chemistry - A European Journal

Inside Cover: Introduction of d-Glutamate at a Critical Residue of Aβ42 Stabilizes a Prefibrillary Aggregate with Enhanced Toxicity (Chem. Eur. J. 34/2016)

The amyloid beta peptide 42 (Aβ42) is an aggregation-prone peptide that plays a pivotal role in Alzheimer′s disease. We report that a subtle perturbation to the peptide through a single chirality change at glutamate 22 leads to a pronounced delay in the β-sheet adoption of the peptide. This was accompanied by an attenuated propensity of the peptide to form fibrils, which was correlated with changes at the level of the fibrillary architecture. Strikingly, the incorporation of d-glutamate was found to stabilize a soluble, ordered macromolecular assembly with enhanced cytotoxicity to PC12 cells, highlighting the importance of advanced prefibrillary Aβ aggregates in neurotoxicity. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Introduction of d-Glutamate at a Critical Residue of Aβ42 Stabilizes a Prefibrillary Aggregate with Enhanced Toxicity

We developed a simple protocol for high-yielding synthesis of conjugates of a deuterated dihydro-N,N,N′,N′-tetramethylrhodamine (F) with oligodeoxyribonucleotides and a 2′-OMe RNA (a representative nuclease-resistant, chemically modified oligonucleotide) using easily accessible starting materials including NaBD4 and conjugates of oligonucleotides with N,N,N′,N′-tetramethylrhodamine (F). These compounds were found to be stable in air and insensitive to light at 525, 635 and 650 nm, whereas slow activation occurs upon their exposure to 470 nm light. However, at the conditions of the templated reaction, in the presence of a target nucleic acid and a photocatalyst based on the eosin structure, the Fis oxidized forming fluorescent F. This reaction is &gt;30-fold faster than the background reaction in the absence of the template. Moreover, the presence of a single mismatch in the target nucleic acid slows down the templated reaction by eightfold. These activatable dyes can potentially find applications as nucleic acid-specific probes for super-resolution imaging in live cells. © 2014 Springer-Verlag Berlin Heidelberg.

Histochemistry and Cell Biology

Nucleic acid-specific photoactivation of oligodeoxyribonucleotides labeled with deuterated dihydro-N,N,N′,N′-tetramethylrhodamine using green light

Photochemical, nucleic acid-induced reactions, which are controlled by nontoxic red light, are well-suited for detection of nucleic acids in live cells, since they do not require any additives and can be spatially and temporally regulated. We have recently described the first reaction of this type, in which a phenylselenyl derivative of thymidine (5′-PhSeT-ODNa) is cleaved in the presence of singlet oxygen (Fülöp, A., Peng, X., Greenberg, M. M., Mokhir, A. (2010) A nucleic acid directed, red light-induced chemical reaction. Chem. Commun. 46, 5659-5661). The latter reagent is produced upon exposure of a photosensitizer 3′-PS-ODNb (PS = Indium(III)- pyropheophorbide-a-chloride: InPPa) to >630 nm light. In 2012 we reported on a fluorogenic version of this reaction (Dutta, S., Flottmann, B., Heilemann, M., Mokhir, A. (2012) Hybridization and reaction-based, fluorogenic nucleic acid probes. Chem. Commun. 47, 9664-9666), which is potentially applicable for the detection of nucleic acids in cells. Unfortunately, its yield does not exceed 25% and no catalytic turnover could be observed in the presence of substrate excess. This problem occurs due to the efficient, competing oxidation of the substrate containing an electron rich carbon-carbon double bonds (SCH=CHS) in the presence of singlet oxygen with formation of a noncleavable product (SCH=CHSO). Herein we describe a related, but substantially improved photochemical, catalytic transformation of a fluorogenic, organic substrate, which consists of 9,10-dialkoxyanthracene linked to fluorescein, with formation of a bright fluorescent dye. In highly dilute solution this reaction occurs only in the presence of a nucleic acid template. We developed three types of such a reaction and demonstrated that they are high yielding and generate over 7.7 catalytic turnovers, are sensitive to single mismatches in nucleic acid targets, and can be applied for determination of both the amount of nucleic acids and potentially their localization. © 2013 American Chemical Society.

Bioconjugate Chemistry

Fluorogenic, catalytic, photochemical reaction for amplified detection of nucleic acids

We developed fluorogenic probes, which are photoactivated in the presence of specific nucleic acid templates with the release of fluorescent dyes. This templated reaction can be used to target specific nucleic acids in complex mixtures. We further demonstrate that this reaction can be monitored by single-molecule fluorescence imaging. © 2012 The Royal Society of Chemistry.

Hybridization and reaction-based fluorogenic nucleic acid probes

The autocatalytic photochemical reaction, which is potentially controlled by any selected nucleic acid, is highly sequence specific and not inhibited by its products, was developed. This reaction generates colored and fluorescent products, which can be monitored by the naked eye. © 2011 The Royal Society of Chemistry.

An autocatalytic chromogenic and fluorogenic photochemical reaction controlled by nucleic acids

chemical equation presented A helping strand: A method to selectively form copper nanoparticles in solution using double-stranded DNA has been developed. The size of the nanoparticles is controlled by the length of the dsDNA template, and single-stranded DNA did not act as a template (see scheme). Single-stranded overhangs in dsDNA were used to prepare a nanostructure in which two metallized dsDNA segments were linked together by a nonmetallized rigid linker. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.