Department of Chemistry, University of Toronto, 80 St. George Street Toronto, Ontario, Canada M5S 3H6
Office: Davenport Building 444
Laboratories: Davenport Building 450 and 451
o The site-specific modification of proteins and enzymes by designed organic compounds is an area of growing importance. We have developed new methods to modify the oxygen-carrier protein, hemoglobin, so it can be used for a number of applications. For example, we have produced multifunctional reagents that cross-link hemoglobin at specific sites, introducing properties that make the material suitable to be used as an alternative for red cells in blood transfusions. We extended the method to include ways to combine cross-linking within a protein and connecting with a second (or additional) protein. This permits the interactions of assembled proteins to be studied in a defined system. We recently have improved the process by applying the CuAAC click procedure that we adapted specifically for coupling of two cross-linked hemoglobin-azides with bis-alkynes
Connecting and cross-linking two hemoglobins
lower route is slow - accelerated by enzyme
Reversibility. The replacement of a carboxyl group by a proton appears to be a simple matter of forming forming CO2 by breaking a C-C bond. However, catalytic and isotope effect patterns remind us that the CO2 is a powerful electrophile, making the process easily reversible prior to separation of the CO2 molecule from the residual carbanion. We are examining the modes by which this process can be accelerated despite the reversibility. We find that enzymes may function by enhancing the "forward committment", which can be diagnosed by an increased 12C/13C kinetic isotope effect. We also have found that in some cases decarboxylation is accelerated in acidic solutions. We are examining related reactions involving proton transfers and the resulting steric effects that are associated with reorganization.
Hydrolytic Route. We have also found that there is a distinct alternative to the loss of CO2: acid-catalyzed addition of water to the carboxyl group and formation of carbonic acid. Protonated CO2 is an impossible intermediate yet in some cases reactions proceed in highly acidic solutions. This route is analogous to ester hydrolysis and appears to be accessible in systems that provide adequate leaving groups. We are examining the extent to which this process occurs in the many reactions that are reported to undergo acid catalyzed decarboxylation.
We have found that lanthanide ions are particularly effective catalysts for the addition of oxygen nucleophiles to acyl phosphate monoesters in general. Our research is aimed at extending and improving the approach.
Reactions are selectively catalyzed by lanthanides (recognition of diol in water)
Aminoacylation of RNA
Learn more about this with a slide presentation: click here
Biomimetic peptide coupling
The aminoacyl phosphates are also efficient reagents for producing peptide bonds in water by reaction with amino acid esters. This mimics the non-ribosomal formation of peptides. We are expanding this as a method to produce peptides under conditions that are amenable to biological catalysts.
Chemical alterations permit systematic studies of proteins that would dissociate - these have many applications.
generation of dendritic arrays of cross-linked hemoglobin: symmetry and
Dongxin Hu and Ronald Kluger Org. Biomol. Chem. 2008 6 151 – 156. By producing a three-fold symmetrical six-sited reagent, we can efficiently connect hemoglobin tetramers to one another. These larger species are expected to be useful in applications related to blood substitutes and drug delivery but have been difficult to produce efficiently.
Functional cross-linked hemoglobin bis-tetramers: Geometry and cooperativity Dongxin Hu and Ronald Kluger “”, Biochemistry 2008 47 12551-12561 () DOI: 10.1021/bi801452b
Polyethylene Glycol Conjugation Enhances the Nitrite Reductase Activity of Native and Cross-Linked Hemoglobin. Francine E. Lui, Pengcheng Dong, and Ronald Kluger Biochemistry; 2008; 47; 10773-10780. DOI: 10.1021/bi801116k The beneficial effect of PEG on hemoglobin as a red cell substitute correlates with its ability to convert nitrite to nitric oxide.
Hemoglobin bis-tetramers via cooperative azide-alkyne coupling. Jonathan S. Foot, Francine E. Lui and Ronald Kluger, Chem. Commun., 2009 7315-7317. Using "click chemistry" to couple proteins avoids competing hydrolysis of the reagents. The reaction is auto-catalytic because the first product is more soluble than one of the reactants.
Enhancing Nitrite Reductase Activity of Modified Hemoglobin: Bis-tetramers and Their PEGylated Derivatives Francine E. Lui and Ronald Kluger Biochemistry 2009 48 11912–11919. Increasing size and adding PEG chains overcomes problems with oxygen delivery and increases the rate of NO formation.
Protein-Protein Coupling And Its Application To Functional Red Blood Cell Substitutes” Ronald Kluger, Jonathan S. Foot, and Adelle A. Vandersteen Chem. Commun.(Feature Article), 2009 45 1194-1202. An overview of chemical approaches to defined materials produced from hemoglobin as red cell alternatives.
Efficient CuAAC click formation of functional hemoglobin bis-tetramers Ying Yang and Ronald Kuger Chem. Commun., 2010 46 7557-7559. Used of the CuAAC reaction to produce coupled proteins effectively.
Red Cell Substitutes from Hemoglobin – Do We Start All Over Again? Curr. Opin. Chem. Biol. 2010 14 538-543. A critical examination of the state of the field.
Reviving artificial blood: meeting the challenge of NO scavenging by hemoglobin. Francine E. Lui and Ronald Kluger ChemBiochem 2010 11 1816-1824.
Hemodynamic Responses to a Hemoglobin Bis-Tetramer and its Polyethylene Glycol Conjugate Francine E. Lui, Binglan Yu, David M. Baron, Chong Lei, Warren M. Zapol and Ronald Kluger Transfusion 2012 52 974-982
Acyl phosphate esters occur in nature but their use as reagents has been developed in our lab. They have very useful properties, especially as electrophiles in water.
Biomimetic Aminoacylation of
Ribonucleotides and RNA with Aminoacyl Phosphate Esters and Lanthanum
Svetlana Tzvetkova and Ronald Kluger J. Am. Chem. Soc. 2007 129 15848– 15854
This is the first example of a direct selective reaction that adds an aminoacyl group to the 3'-terminus of tRNA, in direct analogy to the biochemical process but without the restricions imposed by the genetic code.
pKa-Dependent Formation of Amides in Water from an Acyl Phosphate Monoester and Amines Jolanta Wodzinska and Ronald Kluger J. Org. Chem. 2008 73 4753-4754. Acyl phosphates can distinguish amines based on their basicity and thus acylate selectively.
Magnesium ion enhances lanthanum-promoted monobenzoylation of a monosaccharide in water Raj S. Dhiman and Ronald Kluger “, Org. Biomol. Chem. 2010 8 2006 –2008. Since La coordinates to the by-product, adding magnesium allows the La to be catalytic by replacing it in the by-product.
Aminoacylation of nucleosides and nucleotides Direct catalytic aminoacylation of the end of an RNA-like molecule. Sohyoung Her and Ronald Kluger Org. Biomol. Chem. 2011 9, 676–678.
Biomimetic peptide bond formation in water with aminoacyl phosphate esters Activation by an analogue of the AMP derivative effectively couples amino acids. Raj S. Dhiman, Liliana Guevara Opinska, and Ronald KlugerOrg. Biomol. Chem.2011 9 , 5645-5647.
Thiamin promotes reactions in patterns that reflect those of related enzymes. The role of the protein is clear if they are compared side by side. There are remarkable differences. Our results have led us to reconsider the role of thiamin in enzyme-catalyzed decarboxylation as well as the general process with other catalysts. We have found previously unknown routes for decarboxylation that are also amenable to consideration of how CO2 could be utilized for synthesis.
Making thiamin work faster: acid promoted separation of carbon dioxide. Qingyan Hu and Ronald Kluger, J. Am. Chem. Soc. 2005 127 12242-12243.
Protein-enhanced decarboxylation of the covalent intermediate in benzoylformate decarboxylase – desolvation or acid catalysis?, Ronald Kluger and Daria Yu Bioorg. Chem. 2006 34 337-344.
Accelerating Unimolecular Decarboxylation by Pre-Associated Acid Catalysis in Thiamin-Derived Intermediates: Implicating Brønsted Acids as Carbanion Traps in Enzymes. Ronald Kluger, Glenn Ikeda, Qingyan Hu, Pengpeng Cao, and Joel Drewry. J. Am. Chem. Soc. 2006 128 15856-15864. The discovery that an acid derived from pyridine can accelerate the decarboxylation of the conjugate of thiamin and benzoylformate resolves fundamental issues on how decarboxylation can be accelerated on an enzyme by preventing the return of carbon dioxide to the carbanion. The implications are very general and will lead to new areas of research.
Thiamin Diphosphate Catalysis: Enzymic and Nonenzymic Covalent Intermediates Ronald Kluger and Kai Tittmann, Chem. Rev. 2008 108 1797-1833. The importance of intermediates is illustrated by the interaction of knowledge from analysis of parallel enzymic and nonenzymic reactions.
Catalyzing separation of Carbon Dioxide in Thiamin diphosphate Promoted Decarboxylation” Ronald Kluger and Steven Rathgeber FEBS J. 2008 275 6089-6100. A review of previously unknown mechanisms for enhancing the rate of decarboxylation in enzymes.
Internal Return of Carbon Dioxide in Decarboxylation: catalysis of Separation and 12C/13C Kinetic Isotope Effects Scott O. C. Mundle, Steven Rathgeber, Georges Lacrampe-Couloume, Barbara Sherwood Lollar, and Ronald Kluger. J. Am. Chem. Soc., 2009, 131, 11638–11639 DOI: 10.1021/ja902686h. The formation of carbon dioxide is surprisingly reversible and this can be seen from the change in carbon isotope effect with addition of catalysts that block the reverse reaction.
Decarboxylation via Addition of Water to a Carboxyl Group: Acid-Catalysis of Pyrrole-2-Carboxylic Acid Scott O. C. Mundle and Ronald Kluger, J. Am. Chem. Soc., 2009, 131, 11674–11675 DOI: 10.1021/ja905196n. Protonation of pyrrole-2-carboxylic acid occurs on carbon, creating a delocalized structure that activates the carboxyl group for addition of water and cleavage of the C-C bond, resulting in the release of protonated carbonic acid. This mechanism of decarboxylation presents an alternative to the normally seen direct formation of carbon dioxide and can be subject to catalysis as well as serve as a source of a carboxylating agent.
Hydrolytic Decarboxylation of Carboxylic Acids and the Formation of Protonated Carbonic Acid Scott O. C. Mundle, Georges Lacrampe-Couloume, Barbara Sherwood Lollar, and Ronald Kluger J. Am. Chem. Soc.2010 132 , 24530-2436. In-depth examination of the addition mechanism for decarboxylation.
The role of pre-association in Brønsted acid-catalyzed decarboxylation and related processes” Ronald Kluger and Scott O. C. Mundle Adv. Phys. Org. Chem. 2010 44 357-375. Can decarboxylation be promoted by preventing CO2 from adding back as soon as it forms?
Investigating the mechanism of heteroaromatic decarboxylation using solvent kinetic isotope effects and Eyring transition state theory, Scott O.C. Mundle, Liliana Guevara Opinska, Ronald Kluger and Andrew Dicks J. Chem. Educ.2011 88 1004–1006. An experiment that lets undergraduates discover a mechanism with a simple kinetic study in our area of research.
Protonated Carbonic Acid and Reactive Intermediates in the Acidic Decarboxylation of Indole-Carboxylic Acids Adelle A. Vandersteen, Scott O.C. Mundle and Ronald Kluger J. Org. Chem.2012 77 6505–6509. This class of reactions follows a pattern where elimination of protonated carbonic acid is the key step.
Origins of steric effects in general base catalyzed enolization: Solvation and electrostatic attraction Scott O. C. Mundle, Graeme W. Howe and Ronald Kluger J. Am. Chem. Soc.2012 134 1066-1070. The source of steric effects in small molecule enolization is the result of disruption of solvation ratther steric interference in the reaciton itself.
Base-Catalyzed Decarboxylation of Mandelylthiamin: Direct Formation of Bicarbonate as an Alternative to Formation of CO2 Graeme W. Howe, Michael Bielecki, and Ronald Kluger J. Am. Chem. Soc.2012 134 20621–20623. We discovered that Brønsted bases catalyzed decarboxylation by promoting the departure of bicarbonate from an addition intermediate.
Chung-Woo Fung (Technical assistant)
instrumentation, HPLC, mass spectrometry
|Yuyang Li||Aminoacylation of RNA|
|Michael Bielecki||Thiamin enzyme intermediates|
|Dr. Scott Mundle||13-C kinetic isotope effects (in Professor Barbara Sherwood Lollar's group) - applications to contaminant hydrology|
|Graeme Howe||Decarboxylation catalysis|
|Liliana Guevara Opinska||Thiamin enzyme mechanisms|
|Erika Siren||Protein-protein conjugation|
|Serena Singh||Efficient formation of bis-tetramers|
|Aizhou Wang||Subunit reaction specificity|
Last updated October 18, 2013