V. Haridas

Self-assembling tryptophan-based designer peptides as intracellular delivery vehicles

A series of tryptophan-based peptides W1a, b-W4a, b, with diverse architectures were designed and synthesized. These tryptophan containing peptides can self-assemble to spherical particle. This self-assembled system was demonstrated to encapsulate rhodamine B and penetrate the cell membrane.

Sortase-Based Bio-organic Strategies for Macromolecular Synthesis

Protein ligation sorted: SrtA is one of the molecules nature uses to perform chemoselective ligation on amazingly complex protein molecules. Sortase-mediated ligation (SML) with chemoselective reactions will find a variety of applications in chemistry, biology, and medicine in the near future.

Dendrons and dendrimers as pseudochaperonins for refolding of proteins

Peptide dendrimers are screened for “artificial chaperone” (protein refolding) activity by a sensitive fluorescence based assay. The refolding with largest dendrimer is found to help in recovering biological activity of >90% in the case of unfolded lipases and amylases. The refolding yields decrease down to 14% with a decrease in the complexity and hydrophobicity of the dendron/dendrimer. CD spectroscopy confirms the correct refolding in terms of secondary structure contents of the proteins. The DLS data indicates that presence of the dendrons/dendrimers facilitates protein refolding by preventing the aggregation of proteins.

A supramolecular approach to metal ion sensing: cystine-based designer systems for Cu2+, Hg2+, Cd2+ and Pb2+ sensing

We have synthesized two cystine containing molecules S1 (pyrene-labelled) and S2 (tryptophan-labelled) and demonstrated that the former can detect Cu2+, and the latter can detect Hg2+ in acetonitrile. The 1:1 mixture of S1 and S2 forms a heterodimeric system S1:S2, which was confirmed by mass spectrometric, UV-Visible and fluorescence spectroscopic studies. Additional proof for the formation of S1:S2 came from 1H NMR, CD, ITC, ultramicroscopic and computational studies. The supramolecularly assembled S1:S2 detects Pb2+ in nanomolar levels. A control compound S3 containing tryptophan and pyrene units showed totally different binding properties compared to S1, S2 and S1:S2. DFT studies on S1:S2 establish that S1 in S1:S2 adopts an extended conformation, thereby keeping the pyrene units proximal to the indole moiety of S2 for energy transfer. The binding of Pb2+ with S1:S2 brings the two pyrene units into proximity resulting in a folded structure and the formation of the excimer. These results clearly demonstrate a hitherto unknown and unexpected organization of two fluorescent molecules leading to a new supramolecular system capable of Pb2+ detection.

Natural macromolecular antifreeze agents to synthetic antifreeze agents

Many living systems native to the Arctic and Antarctic regions express antifreeze proteins (AFPs) or antifreeze glycoproteins (AFGPs) that recognize and bind to specific faces of ice crystals, thereby inhibiting ice growth. The non-colligative freezing point depression induced by these proteins results primarily from their unique chemical structures. This review describes the various classes of AF(G)Ps, their structural hierarchy, their mechanisms of action, and novel synthetic antifreeze compounds. The mechanism of action of AF(G)Ps displays a high degree of precision present in natural systems. The varied chemical structures of AFPs with similar antifreeze activities suggest convergent evolution. Structural studies of AFPs from insects, plants and bacteria have revealed unusual beta helical structures. A variety of AF(G)P analogs have been synthesized and have revealed the mechanisms underlying the action of AF(G)Ps. The utility of AF(G)Ps and their analogs in cryopreservation, cryosurgery, and the food industry motivates the development of new artificial antifreeze agents.

Modulation of prion polymerization and toxicity by rationally designed peptidomimetics

Misfolding and aggregation of cellular prion protein is associated with a large array of neurological disorders commonly called the transmissible spongiform encephalopathies. Designing inhibitors against prions has remained a daunting task owing to limited information about mechanism(s) of their pathogenic self-assembly. Here, we explore the anti-prion properties of a combinatorial library of bispidine-based peptidomimetics (BPMs) that conjugate amino acids with hydrophobic and aromatic side chains. Keeping the bispidine unit unaltered, a series of structurally diverse BPMs were synthesized and tested for their prion-modulating properties. Administration of Leu- and Trp-BPMs delayed and completely inhibited the amyloidogenic conversion of human prion protein (HuPrP), respectively. We found that each BPM induced the HuPrP to form unique oligomeric nanostructures differing in their biophysical properties, cellular toxicities and response to conformation-specific antibodies. While Leu-BPMs were found to stabilize the oligomers, Trp-BPMs effected transient oligomerization, resulting in the formation of non-toxic, non-fibrillar aggregates. Yet another aromatic residue, Phe, however, accelerated the aggregation process in HuPrP. Molecular insights obtained through MD (molecular dynamics) simulations suggested that each BPM differently engages a conserved Tyr 169 residue at the α2-β2 loop of HuPrP and affects the stability of α2 and α3 helices. Our results demonstrate that this new class of molecules having chemical scaffolds conjugating hydrophobic/aromatic residues could effectively modulate prion aggregation and toxicity.

Cysteine-based fluorescence “turn-on” sensors for Cu2+ and Ag+

We designed and synthesized two metal ion binding molecules 3a and 3b based on cysteine. In 3a, pyrene is used as a fluorescent probe, while 3b contains tryptophan, which acts as a fluorescent probe as well as facilitates metal ion binding. Detailed spectroscopic, calorimetric, microscopic and computational studies revealed the binding mode and the plausible structures of the complexes.

Novel peptidylated surfaces for interference-free electrochemical detection of cardiac troponin I

Novel peptidylated surfaces were designed to minimise interferences when electrochemically detecting cardiac troponin I in complex biological samples. Disulfide-cored peptide dendrons featuring carbomethoxy groups were self-assembled on gold electrodes. The carbomethoxy groups were deprotected to obtain carboxylic groups used to immobilise antibodies for cardiac troponin I marker. The chemisorption of two types of peptides, one containing triazole and the other with native peptide bonds, on a gold substrate was studied by quartz crystal microbalance (QCM), surface plasmon resonance (SPR) and X-ray photoelectron spectroscopy (XPS). Peptides formed ordered self-assembled monolayers, contributing to a more efficient display of the subsequently immobilised antibodies towards their binding to the antigen. As a result, electrochemical immunosensors prepared by self-assembly of peptides afforded higher sensitivities for cardiac troponin I than those prepared by the chemisorption of alkane thiolated compounds. Triazolic peptide-modified immunosensors showed extraordinary sensitivity towards cardiac troponin I [1.7µA/(ng/mL) in phosphate buffer], but suffered from surface fouling in 10% serum. Modification with non-triazolic peptides gave rise to anti-fouling properties and still enabled the detection of cardiac troponin I at pg/mL concentrations in 10% serum without significant matrix effects.

Vesicles: self-assembly beyond biological lipids

Self-assembly is a powerful strategy for the development of various intricate supramolecular architectures through non-covalent interactions. Non-covalent interactions can be utilized to produce tubular, vesicular, spherical, fibril, toroidal and helical morphologies. Amongst different types of morphologies, vesicles are of great interest due to their potential use in the drug encapsulation, drug delivery, and as nanoscale reaction vessels. These applications inspired chemists to develop various synthetic molecules that display vesicular self-assembly. This review presents recent examples of synthetic systems that show vesicular self-assembly.

Peptide dendrons as thermal stability amplifiers for IgG1 monoclonal antibody biotherapeutics

Biotherapeutics such as monoclonal antibodies (mAbs) have a major share of the pharmaceutical industry for treatment of life-threatening chronic diseases such as cancer, skin ailments, and immune disorders. Instabilities such as aggregation, fragmentation, oxidation, and reduction have resulted in the practice of storing these products at low temperatures (-80 to -20 °C). However, reliable storage at these temperatures can be a challenge, particularly in developing and underdeveloped countries; hence, lately, there has been a renewed interest in creating formulations that would offer stability at higher temperatures (25 to 55 °C). Most therapeutic formulations contain excipients such as salts, sugars, amino acids, surfactants, and polymers to provide stability to the biotherapeutic, but their efficacy at high temperatures is limited. The current work proposes the use of peptide dendrons of different generations to create formulations that would be stable at high temperature. Among these dendrons, third-generation lysine dendron L6 has been identified to provide the highest stability to mAbs, as demonstrated by a host of analytical techniques such as size-exclusion chromatography (SEC), dynamic light scattering (DLS), Nanoparticle tracking Analysis (NTA), and circular dichroism (CD). The biocompatibility of these dendrons was confirmed by hemolytic activity tests. Non-interference of the dendrons with the activity of the mAb was confirmed using a surface plasmon resonance (SPR) based activity assay. We hope that this study will stimulate utilization of such higher-generation dendrons for enhancing the thermal stability of mAbs.