Ultrafast Infra-red Spectroscopy as a Probe of Chemical Reaction Mechanisms
The timescales for reactions in solution are very fast, with lifetimes of reactive or energized intermediates that can be as short as a few picoseconds. Nevertheless, these intermediates can be observed, and the flow of energy released by exothermic reactions can be followed using techniques based on ultrafast infra-red spectroscopy. Two-dimensional infra-red spectroscopy (2DIR) provides further insights into structural changes and energy flow in molecules on these very short timescales. This talk will introduce the techniques of ultrafast time resolved infra-red and 2DIR spectroscopy and illustrate their application to the study of the mechanisms of chemical reactions. Examples will be drawn from our work on radical reactions in solution, photochemistry of hydrogen-bonded DNA base pairs, and vibrational dynamics in transition metal complexes.
Synthetic Molecules and Materials as Models of the Oxygen Evolving Complex of Photosystem II
Efficient catalytic water oxidation is an important reaction for the development of solar hydrogen as a source of green energy. Extraction of high energy electrons from water for the preparation of fuel leaves O2 as a byproduct. Nature boasts the only truly effective catalyst for this reaction, the oxygen evolving complex (OEC), a tetramanganese-calcium-oxo cluster. Though decades of research has gone to the synthesis of biologically-inspired Mn-O clusters, these systems fall short in biomimetic reactivity in comparison to other metal systems (Ru, Co, Ni, and Cu for example), which begs the question: Why have we been unable to mimic the biological chemistry with manganese?
Geometric rearrangements, and changes in coordination number are known to occur in the OEC, but current model chemistry relies heavily on chelation to stabilize synthetic clusters, rendering them inert, and without open coordination sites. Our group at Temple is exploring a new approach to manganese cluster synthesis, using sterics of bridging and terminal ligands to direct sterics and coordination number. Through our approach, we are able to access biologically relevant geometries, and prepare unchelated clusters with significant reactivity seen rarely (or never) in decades of chelate clusters. These reactivities include N-N and C-N bond cleavage, hydrogen atom transfer, ligand exchange, reductive elimination, and cluster rearrangement.
A second topic is the exploration of solid-state layered manganese oxides (termed birnessite) in the chemical and electrochemical oxidation of water. The birnessite phase of MnO2 is typically viewed as a poor water oxidation catalyst, but modification of the material by the introduction of highly active “defect” sites turns this material from a poor catalyst into an active catalyst for water oxidation. The studies being undertaken may shed light on the identity and structure of these sites and aid the development of rubust and affordable catalysts for this important reaction.
Ultrafast Vibrational Sum-Frequency Spectroscopy and Dynamics at Mineral/Aqueous Interfaces
The properties of water at interfaces are important in many disciplines. However, it is not clear what effects the presence of the surface, the charge that can develop on the surface, the solution ionic strength, and the interfacial electric field, have on how interfacial water molecules communicate with each other, e.g., how thermal (vibrational) energy flows. To address these issues we investigated the ultrafast vibrational population and dephasing dynamics of the O-H stretch using IR pump-vibrational Sum Frequency Generation (vSFG) probe at the water/mineral interfaces. Contrary to previous reports, the vibrational lifetime of the O-H stretch at the silica/water interface is ~ 600 fs, a factor 2-3 slower than bulk water, when the surface is neutral. Charging the SiO 2 interface appears to lead to a dramatic acceleration of vibrational relaxation. Experiments on the effect of ionic strength, suggest that the primary reason for accelerated dynamics at pH=6
is the sampling of water within the Debye length that has bulk-like solvation. The pH dependent structuring of interfacial water and the influence of electrolyte also impact interfacial reactivity.
A newly developed SFG spectrometer, based on a novel ultrabroadband optical parametric amplifier generating IR pulses in the ~2800-6000 cm -1 range bandwidths >2000 cm -1 in the near-IR range, allows vSFG spectroscopy, including low-intensity features such as non-hydrogen bonded OH vibrations and combination [stretch+bend] and overtone bands of interfacial water. Access to these modes opens up opportunities for investigations of a broad range of interfaces.
- The Effect of Electric Fields on the Ultrafast Vibrational Relaxation of Water at a Charged Solid-LiquidInterface as Probed by Vibrational Sum Frequency Generation, A. Eftekhari-Bafrooei and E. Borguet, J.Phys. Chem. Lett.,, 2, 1353-1358 (2011)
- Experimental Correlation Between Interfacial Water Structure and Mineral Reactivity. Dewan, S.;Yeganeh, M. S.; Borguet, E., J. Phys. Chem. Lett., 2013, 4 (11), 1977-1982.
- Observation of the Bending Mode of Interfacial Water at Silica Surfaces by Near Infrared VibrationalSum-Frequency Generation Spectroscopy of the [stretch+bend] Combination Bands. Oleksandr Isaienko, Satoshi Nihonyanagi, Devika Sil, and Eric Borguet, J. Phys. Chem. Lett., 4, 531-535, (2013)
Playing dice withzeolite secondary building blocks
Our laboratory has been using an organic soluble phosphate monoester (ArO)P(O)(OH) (Ar = 2,6-diisopropylphenyl) as the primary building unit (PBU) to assemble a large number of polyhedral molecules that resemble one or more of zeolite secondary building units and display various functions.1-4 While the reaction of this phosphate with a divalent metal ion (e.g. Zn2+) in a donor solvent predominantly leads to the isolation of stable tetranuclear metal phosphates [(ArO)PO3Zn(L)]4 which possess a Zn4O12P4 D4R SBU inorganic core. In recent times, however, we have unraveled that it is possible to also isolate other SBUs, starting from the same set of reactants, but by making small variations in the reaction conditions. Now it is possible to isolate hitherto unknown discrete D6R and D8R SBUs (which possessZn6O18P6 and Zn8O24P8cores, respectively) by switching the solvent from methanol to acetonitrile and the co-ligand from DMSO to either 4-formylpyridine5 or 4-cyanopyridine.6From a series of experimental observations it has now become apparent that, irrespective of the conditions employed, S4R SBUs are formed as the initial products. It is quite intuitive to conclude that a face-to-face fusion of two S4R blocks will lead to the formation of a D4R SBU. The explanation for the formation of larger SBUs such as D6R and D8R from a S4R however would need a different two-stage mechanism involving (a) side-by-side fusion of two or more S4Rsand (b) a constructive folding to close up the double-n-ring (n = 4, 6, or 8) SBUs. One cannot discount the possibility of misfolding in step (b), which will lead to the isolation of polymeric chains with a staircase conformation. Similarly, formation of larger S6R and S8R SBUs in the initial phase of the reaction cannot also be ignored. A rationalization of these building principles will be presented in this lecture.
1. Kalita, A.C.; Roch-Marchal, C.;Murugavel, R.Dalton Trans., 2013, 26, 9755.
2. Kalita, A.C.; Murugavel, R.Inorg. Chem., 2014, 53, 3345.
3. Kalita, A.C.; Gogoi, N.;Jangir, R.; Kupuswamy, S.; Walawalkar, M. G.; Murugavel, R.Inorg. Chem.,2014, 53, 8959.
4. Kalita, A.C.; Sharma, K.; Murugavel, R. Cryst. Eng. Comm.2014, 16, 51.
5. Gupta, S.K.; Dar, A. A.; Rajeshkumar, T.; Kuppuswamy, S.; Langley, S. K.; Murray, K. S.; Rajaraman, G.; Murugavel, R. Dalton Trans. 2015, 44, 5587.
6. Gupta, S.K.; Kuppuswamy, S.; Walsh, J. P. S.; McInnes, E. J. L.; Murugavel, R. Dalton Trans. 2015, 44, 5961.
7. Gupta, S.K.; Kalita, A.C.; Murugavel, R. unpublished
8. Dar, A.; Murugavel, R. unpublished
9. Dar, A.; Sharma, S. K.; Murugavel, R. Inorg. Chem. 2015,54, 7953.
10. Dar, A.; Gupta, S. K.; Sen. S.; Patwari, G. N.; Murugavel, R. Inorg. Chem. 2015,54, 0000.
EPR and the Binding of Molecules to Cytochrome P450 Enzymes
The cytochromes P450 are a large enzymes superfamily found in every branch of life. Some isoforms help synthesize essential compounds and are attractive targets for antibiotics. Other isoforms metabolize many pharmaceutical drugs and are one source of drug interactions. Many drugs directly bind heme in the active site but our pulsed EPR studies show a new binding mode with a water as a bridge between heme and drug, sometimes retaining enzymatic activity
Seeing enzymes in action
Single-molecule studies of electron-transfer between copper centers of small laccase (SLAC) from S. coelicolor
Single molecule enzymology provides an unprecedented level of detail about aspects of enzyme mechanisms which have been difficult to probe in bulk. One such aspect is intramolecular electron transfer (ET) which is a recurring theme in the research on oxidoreductases. Recently, we introduced a technique to study ET in enzymes at single molecule level by means of confocal fluorescence microscopy (PNAS. 2008, 105, 3250).
I will present recent results on an enzyme, small laccase (SLAC), from S. coelicolor which converts O2 to H2O with concomitant oxidation of organic substrate(s). SLAC is unique, among commonly studied multicopper oxidases (MCO), in its structure (being a homo-trimer of two-domain monomers) and function where it employs a so-called type 1 (T1) Cu site, a trinuclear Cu centre (TNC) and a tyrosine residue (Y108) to catalyze this process which has a high activation barrier. I have measured, for the first time, intramolecular ET rates between the T1 and TNC of SLAC during turnover, one molecule at a time. The distribution across many molecules shows an average ET rate ~450 s-1 independent of substrate concentration, consistent with the proposed enzyme mechanism and with the results of transient kinetics experiments. The activation energy for ET amounts to 350 meV and varies from molecule to molecule with a spread of ±25 meV. Experiments are underway to measure other microscopic rate constants in the enzymatic cycle which have never been measured in bulk. The method is suitable to study ET in a wide range of redox active enzymes in-vitro as well as in-vivo.
Cells’ Armour to Prevent Oxidative Stress
How redox non-innocent residues influence enzyme catalysis and protect cells from oxidative damage?
For a long time, amino acid residues have been thought to provide backbone which holds the protein active site in place and directs substrates/products in specific orientations to catalyze chemical reactions. However, ample evidence has appeared in recent literature which signifies the role of redox-active amino acid residues like tyrosine or tryptophan in enzyme catalysis (Chem. Rev. 1998, 98, 705). Such new information has greatly improved our understanding of reaction rates and enzyme catalysis in modern biochemistry.
I will present recent results on two different classes of enzymes: heme containing Rice-α-oxygenase (RαO) from Oryza sativa and Cu-containing small laccase (SLAC) from Streptomyces coelicolor which form tyrosyl radicals during turnover. RαO catalyzes the α-oxidation of long chain fatty acids whereas laccases catalyzes the oxidation of phenols, aromatic amines, etc. I have made use of a variety of spectroscopic methods and enzyme kinetics to study the mechanism of operation of these two enzymes. For RαO, I have demonstrated reversible H• abstraction of the substrate by a Y379• that is formed during enzyme turnover. A very large, weekly temperature dependent kinetic isotope effect (~50) has been observed which is consistent with nuclear tunneling. For SLAC, I have shown that Y108 residue, which resides close to the trinuclear Cu cluster, gets oxidized when there is a shortage of reducing equivalents in the milieu. It is proposed that such reversible oxidation of key protein residues is (one of) the defense mechanisms of cells to prevent oxidative damage of critical machinery by the reactive oxygen species generated during O2 metabolism by the enzymes.
Molecular Probes and Templates for DNA Structures
DNA exists in variety of structural forms supported by canonical (A, B and Z-DNA) and non-canonical [H-DNA, G-quadruplex, i-motif, A-motif etc] hydrogen bonding interactions. Small molecules play key role in the study of DNA structures, biological significance, and to treat diseases related to their structure and function. On the other hand, the unique molecular recognition, persistence length and size of DNA inspire researchers to create novel molecular architectures for numerous applications. In this context, we have been developing mutually templated novel small molecule-ss DNA architectures using canonical and non-canonical hydrogen bonding interactions of nucleobases. In this talk, I shall present our recent results on fluorescence probes and nucleobase conjugates to study canonical and non-canonical hydrogen bonded DNA structures as well as to create hybrid DNA ensembles for applications ranging from biology to materials science.
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3. N. Narayanaswamy, S. Das, P. K. Samanta, K. Banu, S. K. Dhar, S. K. Pati and T. Govindaraju, Nucleic Acids Res. (2015)DOI: 10.1093/nar/gkv875.
4. N. Narayanaswamy, M. Unnikrishnan, M. Gupta and T. Govinadaraju. Bioorg. Med. Chem. Lett. 2015, 25, 2395.
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Molecular Tools for the Manipulation of Size, Surface Chemistry and Assemblies of Metal Nanoparticles
Nanoscale particles have been envisioned to be the building blocks of a wide variety of future technologies including catalysis, electronic, optical and information technologies.As the nanoparticles have enhanced surface activity a layer of organic molecules are often used as passivating or capping agents. The surface functioanlization assumes significance not just for their stability in diverse solvent media but defines the way nanoparticles interact either with themselves or with the environment/biological systems. For example, water dispersibility is an essential criterion to realize bio-applications of nanoparticles.On the other hand, dispersions in organic media can be utilized to obtain interesting assemblies. In this connection, we have been working on a procedure called “digestive ripening” process in which a colloidal metal suspension in a solvent is refluxed at or above the solvent boiling temperature in the presence of the surface active agent like thiols resulting in the conversion of a highly polydisperse colloid into a monodispersed one (s< 5%). It is hypothesized that the thiols bind and remove reactive surface atoms/clusters from big nanoparticles and redeposit them on smaller nanoparticles. In this way, large particles become smaller, while small particles become larger and eventually, an equilibrium size is obtained that is specific to each of the digestive ripening agent used. While the original work on digestive ripening has been largely carried out with gold nanocrystals, it has recently been extended to several other nanoparticle systems. Once again, the mechanism of this process is not understood, which is extremely important to generate nanocrystals with controlled and desired size distributions. In this presentation we will review the state of the art in digestive ripening and some of our recent experimental results that we hope will help in developing a mechanistic model for digestive ripening.
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P. Sahu and B. L. V. Prasad, Nanoscale, 2013, 5, 1768 – 1771
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D. S. Sidhaye and B. L. V. Prasad, New J. Chem. 2011, 35, 755–763
Nano-bioconjugation of Mutant Cytochrome P450cam (CYP101) for Biocatalysis
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Design and Construction of Protein Molecules with Novel Properties
One of the most important challenges in chemical biology is to understand the molecular basis of protein function. Developing new methodologies using chemistry and the ability to apply those methodologies to proteins plays a crucial role in addressing this challenge. I propose to direct my future research program towards applying chemistry to design and engineer novel protein molecules, and to systematically develop a new class of therapeutics with controlled biochemical properties. Part of my research will be focused on the design and total chemical synthesis of medicinally relevant protein molecules with complex polypeptide backbone topologies that are rare or do not occur in natural proteins. I will develop a novel technology, using virtual structure-based screening of peptide fragment libraries from protein data bank, to identify small protein molecules of opposite handedness that will be used as candidate therapeutics. A significant portion of my research will be dedicated to develop and apply chemistry tools to enhance the conformational rigidity, introduced by the incorporation of fixed elements of secondary structures that will improve the stability and receptorbinding affinity of the small protein drug candidates. I will also explore and extend racemic and quasi-racemic crystallography to unravel complex biological questions. All these research projects will be pursued with the goal of addressing a wide range of questions having implications in fundamental as well as in