BITS Faculty Publications

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1,3,5-Trisubstituted benzenes as fluorescent photoaffinity probes for human carbonic anhydrase II capture
(RSC, 2013) Addy, Partha Sarathi
The ‘capture’ of proteins by small molecules via irreversible cross-linking mediated by photo-irradiation is of interest in the field of proteomics (for reviews see ref. 1). The technique has the potential for profiling protein-binding by small molecules, an objective of importance both for basic cell biology and in pharmaceutical science. Capture compounds, or photoaffinity probes, are typically endowed with three functions comprising (i) a selectivity function, such as an enzyme inhibitor, (ii) a photo-cross linking group (capture function) and (iii) a sorting group to enable separation of the captured protein from biological mixtures, such as biotin or an alkyne for subsequent modification. The captured protein(s) can be isolated using streptavidin beads and identified by mass spectrometry or Western blotting (for examples see ref. 2)
Aggregation induced emission based luminogenic (aiegenic) probes for the biomarker detection
(Wiley, 2025-01) Addy, Partha Sarathi
Various biomarkers such as proteins play key roles in controlling crucial biochemical processes. The critical concentration of the biomarkers is important to maintain a healthy life. In fact, imbalance in concentration or irregular activity of these can lead to various diseases like Cancer, Alzheimer's etc. Therefore, the disease related biomarkers and their timely detection are key to control the illness. In the literature, a few activity-based probes for the detection of such biomarkers are available. As per the requirement an ideal probe should be very specific to recognize the target analyte and that could be achieved by virtue of having a robust structure and stimuli responsive nature. In this regard, several fluorescent probes are of great choice. Although these fluorescent probes face certain challenges such as aggregation caused quenching, which heavily affects the sensitivity and photostability is another major concern for many fluorescent probes. To overcome these challenges aggregation-induced emissive fluorescent probes found to be an excellent alternative. Aggregation induced emissive luminogens (AIEgens) offer higher signal to noise ratios and found to possess better photostability during sensing and imaging. In the present review we have summarized the development of AIEgenic probes for sensing and imaging of disease related biomarkers. We believe this review could be a guide to design efficient AIEgenic probes for the diagnostics development.
Aggregation-induced emission: a curious case of molecular luminescence
(Springer, 2024-11) Addy, Partha Sarathi
Aggregation-induced emission (AIE) is an interesting luminescence phenomenon. This process describes how unity in the molecular world can produce bright emissions (luminescence). Chemists have adopted this technique to design various luminescent materials to develop efficient diagnostics and therapeutics. In this article, a concise description of AIE and its development is documented. The application of AIE is significant in the field of materials and biology. In the last part of the article, a brief description of various uses is presented.
ChemInform Abstract: Design and Synthesis of Azobenzene Template Based Sulfonamide for Capture of HCAII: Dependence of Efficiency on E—Z Geometry
(Wiley, 2014-08-28) Addy, Partha Sarathi
A Chemoselective Rapid Azo-Coupling Reaction (CRACR) for Unclickable Bioconjugation
(ACS, 2017) Addy, Partha Sarathi
Chemoselective modification of complex biomolecules has become a cornerstone of chemical biology. Despite the exciting developments of the past two decades, the demand for new chemoselective reactions with unique abilities, and those compatible with existing chemistries for concurrent multisite-directed labeling, remains high. Here we show that 5-hydroxyindoles exhibit remarkably high reactivity toward aromatic diazonium ions and this reaction can be used to chemoselectively label proteins. We have previously genetically encoded the noncanonical amino acid 5-hydroxytryptophan in both E. coli and eukaryotes, enabling efficient site-specific incorporation of 5-hydroxyindole into virtually any protein. The 5-hydroxytryptophan residue was shown to allow rapid, chemoselective protein modification using the azo-coupling reaction, and the utility of this bioconjugation strategy was further illustrated by generating a functional antibody–fluorophore conjugate. Although the resulting azo-linkage is otherwise stable, we show that it can be efficiently cleaved upon treatment with dithionite. Our work establishes a unique chemoselective “unclickable” bioconjugation strategy to site-specifically modify proteins expressed in both bacteria and eukaryotes.
Defining the current scope and limitations of dual noncanonical amino acid mutagenesis in mammalian cells
(RSC, 2017) Addy, Partha Sarathi
The ability to site-specifically incorporate two distinct noncanonical amino acids (ncAAs) into the proteome of a mammalian cell with high fidelity and efficiency will have many enabling applications. It would require the use of two different engineered aminoacyl-tRNA synthetase (aaRS)/tRNA pairs, each suppressing a distinct nonsense codon, and which cross-react neither with each other, nor with their counterparts from the host cell. Three different aaRS/tRNA pairs have been developed so far to expand the genetic code of mammalian cells, which can be potentially combined in three unique ways to drive site-specific incorporation of two distinct ncAAs. To explore the suitability of using these combinations for suppressing two distinct nonsense codons with high fidelity and efficiency, here we systematically investigate: (1) how efficiently the three available aaRS/tRNA pairs suppress the three different nonsense codons, (2) preexisting cross-reactivities among these pairs that would compromise their simultaneous use, and (3) whether different nonsense-suppressor tRNAs exhibit unwanted suppression of non-cognate stop codons in mammalian cells. From these comprehensive analyses, two unique combinations of aaRS/tRNA pairs emerged as being suitable for high-fidelity dual nonsense suppression. We developed expression systems to validate the use of both combinations for the site-specific incorporation of two different ncAAs into proteins expressed in mammalian cells. Our work lays the foundation for developing powerful applications of dual-ncAA incorporation technology in mammalian cells, and highlights aspects of this nascent technology that need to be addressed to realize its full potential.
Expanding the genetic code of mammalian cells
(Portlandpress, 2017-04-13) Addy, Partha Sarathi
In the last two decades, unnatural amino acid (UAA) mutagenesis has emerged as a powerful new method to probe and engineer protein structure and function. This technology enables precise incorporation of a rapidly expanding repertoire of UAAs into predefined sites of a target protein expressed in living cells. Owing to the small footprint of these genetically encoded UAAs and the large variety of enabling functionalities they offer, this technology has tremendous potential for deciphering the delicate and complex biology of the mammalian cells. Over the last few years, exciting progress has been made toward expanding the toolbox of genetically encoded UAAs in mammalian cells, improving the efficiency of their incorporation and developing innovative applications. Here, we provide our perspective on these recent developments and highlight the current challenges that must be overcome to realize the full potential of this technology.
A facile Garratt–Braverman cyclization route to intercalative DNA-binding bis-quinones
(Elsiever, 2012-01-04) Addy, Partha Sarathi
Bispropargyl ethers (both symmetrical and non-symmetrical) equipped with 1,4-dimethoxyaryl groups were synthesized. Under strongly basic conditions (KOBut/toluene/reflux), these ethers underwent Garratt–Braverman type cyclization to the tetramethoxy bi-aryl systems in high yields presumably via the bisallenes. The products could be successfully converted to the bis-quinones via CAN-mediated demethylation cum oxidation. This two-step protocol offers a simple route to bis-quinones, connected by C1–C2′ bonds, in good yields. Fluorescence based EB-displacement assay, CD spectroscopy and viscosity measurements confirmed the DNA-binding ability of the synthesized quinones via intercalation.
Garratt-Braverman Cyclization: a Powerful Tool for C-C Bond Format
(Thieme, 2012-08) Addy, Partha Sarathi
Development of new strategies for C–C bond formation remains in the forefront of organic synthesis. The base-mediated rearrangement of bis-propargyl sulfones via bis-allenes generated in situ, now known as the Garratt–Braverman cyclization (GBC), leads to the formation of two new C–C bonds. The reaction has recently drawn attention from organic chemists due to the wide scope as well as interesting mechanism. This report aims to give an account of the developments in this area with particular emphasis on synthetic applications.
Glucose Directly Promotes Antifungal Resistance in the Fungal Pathogen, Candida spp
(Elsiever, 2014-09-12) Addy, Partha Sarathi
Effects of glucose on the susceptibility of antifungal agents were investigated against Candida spp. Increasing the concentration of glucose decreased the activity of antifungal agents; voriconazole was the most affected drugs followed by amphotericin B. No significant change has been observed for anidulafungin. Biophysical interactions between antifungal agents with glucose molecules were investigated using isothermal titration calorimetry, Fourier transform infrared, and 1H NMR. Glucose has a higher affinity to bind with voriconazole by hydrogen bonding and decrease the susceptibility of antifungal agents during chemotherapy. In addition to confirming the results observed in vitro, theoretical docking studies demonstrated that voriconazole presented three important hydrogen bonds and amphotericin B presented two hydrogen bonds that stabilized the glucose. In vivo results also suggest that the physiologically relevant higher glucose level in the bloodstream of diabetes mellitus mice might interact with the available selective agents during antifungal therapy, thus decreasing glucose activity by complex formation. Thus, proper selection of drugs for diabetes mellitus patients is important to control infectious diseases.
Label-assisted laser desorption/ionization mass spectrometry (LA-LDI-MS): an emerging technique for rapid detection of ubiquitous cis-1,2-diol functionality
(RSC, 2014) Addy, Partha Sarathi
The Label-Assisted Laser Desorption/Ionization (LA-LDI) technique has recently been applied to the detection of small molecules through a Time of Flight (TOF) mass spectrometric measurement. By excluding the external matrix, the mass spectrum becomes a lot cleaner being free of matrix-related peaks and noise. In this paper, we report a Label-Assisted Laser Desorption/Ionization Mass Spectrometry (LA-LDI-MS) based method for detection of various cis-1,2-diols, including the ones ubiquitous in biological samples.
Labeling Proteins at Site-Specifically Incorporated 5-Hydroxytryptophan Residues Using a Chemoselective Rapid Azo-Coupling Reaction
(Springer, 2019-07-23) Addy, Partha Sarathi
Chemoselective protein labeling is a valuable tool in the arsenal of modern chemical biology. The unnatural amino acid mutagenesis technology provides a powerful way to site-specifically introduce nonnatural chemical functionalities into recombinant proteins, which can be subsequently functionalized in a chemoselective manner. Even though several strategies currently exist to selectively label recombinant proteins in this manner, there is considerable interest for the development of additional chemoselective reactions that are fast, catalyst-free, use readily available reagents, and are compatible with existing conjugation chemistries. Here we describe a method to express recombinant proteins in E. coli site-specifically incorporating 5-hydroxytryptophan, followed by the chemoselective labeling of this residue using a chemoselective rapid azo-coupling reaction.
Laser desorption ionization mass spectrometry: Recent progress in matrix-free and label-assisted techniques
(Wiley, 2017-10-13) Addy, Partha Sarathi
The MALDI-based mass spectrometry, over the last three decades, has become an important analytical tool. It is a gentle ionization technique, usually applicable to detect and characterize analytes with high molecular weights like proteins and other macromolecules. The earlier difficulty of detection of analytes with low molecular weights like small organic molecules and metal ion complexes with this technique arose due to the cluster of peaks in the low molecular weight region generated from the matrix. To detect such molecules and metal ion complexes, a four-prong strategy has been developed. These include use of alternate matrix materials, employment of new surface materials that require no matrix, use of metabolites that directly absorb the laser light, and the laser-absorbing label-assisted LDI-MS (popularly known as LALDI-MS). This review will highlight the developments with all these strategies with a special emphasis on LALDI-MS.
Mutually Orthogonal Nonsense-Suppression Systems and Conjugation Chemistries for Precise Protein Labeling at up to Three Distinct Sites
(ACS, 2019-03-25) Addy, Partha Sarathi
Site-specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into a protein is an emerging technology with tremendous potential. It relies on mutually orthogonal engineered aminoacyl-tRNA synthetase/tRNA pairs that suppress different nonsense/frameshift codons. So far, up to two distinct ncAAs have been incorporated into proteins expressed in E. coli, using archaea-derived tyrosyl and pyrrolysyl pairs. Here we report that the E. coli derived tryptophanyl pair can be combined with the archaeal tyrosyl or the pyrrolysyl pair in ATMW1 E. coli to incorporate two different ncAAs into one protein with high fidelity and efficiency. By combining all three orthogonal pairs, we further demonstrate simultaneous site-specific incorporation of three different ncAAs into one protein. To use this technology for chemoselectively labeling proteins with multiple distinct entities at predefined sites, we also sought to identify different bioconjugation handles that can be coincorporated into proteins as ncAA-side chains and subsequently functionalized through mutually compatible labeling chemistries. To this end, we show that the recently developed chemoselective rapid azo-coupling reaction (CRACR) directed to 5-hydroxytryptophan (5HTP) is compatible with strain-promoted azide–alkyne cycloaddition (SPAAC) targeted to p-azidophenylalanine (pAzF) and strain-promoted inverse electron-demand Diels–Alder cycloaddition (SPIEDAC) targeted to cyclopropene-lysine (CpK) for rapid, catalyst-free protein labeling at multiple sites. Combining these mutually orthogonal nonsense suppression systems and the mutually compatible bioconjugation handles they incorporate, we demonstrate site-specific labeling of recombinantly expressed proteins at up to three distinct sites.
Nontoxic Aggregation-Induced Emissive Luminogen for the Detection of Amyloid Fibrils and Cellular Protein Aggregates
(ACS, 2023-10) Chowdhury, Rajdeep; Addy, Partha Sarathi
Protein misfolding and aggregation resulting in amyloid formation is directly linked to various diseases. Hence, there is keen interest in developing probes for the selective detection of such misfolded aggregated proteins. In this paper, we have shown the use of a nontoxic aggregation-induced emissive luminogen (AIEgen), BIDCPV, for the selective detection of insulin amyloid fibrils and their various stages of formation. We further verified the selective response of BIDCPV toward amyloid fibrils by testing the probe against Aβ 42 peptides, which is well known to form the fibrils. Additionally, the low toxicity, efficient cellular internalization capability, and photostability make BIDCPV a unique candidate for sensing protein aggregates inside mammalian cells.
One-Pot Synthesis of Vinylogous Cyano Aminoaryls (VinCAs) as Benzenic Fluorophores: Tailoring Diverse Emission Colors for White Light Emitting Materials and Cell Imaging
(ACS, 2024-05) Ghosh, Sarbani; Chowdhury, Rajdeep; Addy, Partha Sarathi
Donor–acceptor-based organic small molecules with an electronic push–pull effect can demonstrate intramolecular charge transfer to show interesting photoluminescence properties. This is an essential criterion for designing fluorogenic probes for cell imaging studies and the development of organic light-emitting diodes. Now, to design such optical materials sometimes it is necessary to tune the band gap by controlling the energies of the highest occupied molecular orbital and lowest unoccupied molecular orbital. Typically, the band gaps could be modulated by installing unsaturated handles between electron-rich donors and electron-deficient acceptors. However, these methods are often synthetically and economically challenging due to the involvement of expensive catalysts and difficult reaction setups. In our present study, we show a straightforward, cost-effective method for obtaining a series of donor–acceptor-type Vinylogous Cyano Aminoaryls (VinCAs) with diverse emission colors. Further studies reveal that these VinCAs can serve as effective cell imaging agents, showcasing potential use in chemical biology. Additionally, these molecules could be further used to generate white light emission (WLE), showing their potential utility in advanced lighting technologies
An orthogonalized platform for genetic code expansion in both bacteria and eukaryotes
(Nature, 2017-02-13) Addy, Partha Sarathi
In this study, we demonstrate the feasibility of expanding the genetic code of Escherichia coli using its own tryptophanyl–tRNA synthetase and tRNA (TrpRS–tRNATrp) pair. This was made possible by first functionally replacing this endogenous pair with an E. coli–optimized counterpart from Saccharomyces cerevisiae, and then reintroducing the liberated E. coli TrpRS–tRNATrp pair into the resulting strain as a nonsense suppressor, which was then followed by its directed evolution to genetically encode several new unnatural amino acids (UAAs). These engineered TrpRS–tRNATrp variants were also able to drive efficient UAA mutagenesis in mammalian cells. Since bacteria-derived aminoacyl–tRNA synthetase (aaRS)–tRNA pairs are typically orthogonal in eukaryotes, our work provides a general strategy to develop additional aaRS–tRNA pairs that can be used for UAA mutagenesis of proteins expressed in both E. coli and eukaryotes.
An Oxidative Bioconjugation Strategy Targeted to a Genetically Encoded 5-Hydroxytryptophan
(Wiley, 2018-04-12) Addy, Partha Sarathi
Approaches that enable the chemoselective, covalent modification of proteins in a site-specific manner have emerged as a powerful technology for a wide range of applications. The electron-rich unnatural amino acid 5-hydroxytryptophan was recently genetically encoded in both Escherichia coli and eukaryotes, thereby allowing its site-specific incorporation into virtually any recombinant protein. Herein, we report the chemoselective conjugation of various aromatic amines to full-length proteins under mild, oxidative conditions that target this site-specifically incorporated 5-hydroxytryptophan residue.
Polyaromatic label-assisted laser desorption ionization mass spectrometry (LA-LDI MS): a new analytical technique for selective detection of zinc ion
(RSC, 2014) Addy, Partha Sarathi
An external matrix-free LDI technique for selective detection zinc ion is described. An internal polyaromatic label helped desorption cum ionization processes. The method is free of cross-interference which as often faced in fluorescence based methods.
Polymer with Competing Depolymerization Pathways: Chain Unzipping versus Chain Scission
(ACS, 2020) Addy, Partha Sarathi
Interest in triggered depolymerization is growing, driven by needs in sustainable plastics, self-healing materials, controlled release, and sensory amplification. For many triggered depolymerization reactions, the rate-limiting step does not directly involve the stimulus, and therefore, depolymerization kinetics exhibit only weak or no correlation to the concentration and reactivity of the stimulus. However, for many applications, a direct relationship between the stimulus and the depolymerization kinetics is desired. Here we designed, synthesized, and studied a polymer in which a nucleophile-induced chain scission (NICS) mechanism competes with the chain unzipping pathway. We find that the choice of the chain end functionality and the character of the nucleophile determines which of these is the predominant pathway. The NICS pathway was found to be dependent on the stimulus concentration, in contrast to the chain unzipping mechanism. We demonstrate transferability of these molecular-scale, structure–property relationships to nanoscale materials by formulating the polymers into host nanoparticles.