Ronald Kluger

ron_08

Professor - Organic and Biological Chemistry

Department of Chemistry, University of Toronto, 80 St. George Street Toronto, Ontario, Canada M5S 3H6

E-mail: r.kluger(at)utoronto.ca

Phone: (416)-978-3582

Office: Davenport Building 444

Laboratories: Davenport Building 450 and 451

 

Research Interests

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

kluger1

Connecting and cross-linking two hemoglobins

 

upper route is fast and leads to destruction of thiamin

lower route is slow - accelerated by enzyme

thiamin_scheme

·          

·         Decarboxylation mechanisms.

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 commitment", 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.

decarboxylation_catalysis

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.

hydrolytic_decarboxylation

 

·         Aminoacyl phosphate monoesters

 aminoacylation

Reactions are selectively catalyzed by lanthanides (recognition of diol in water)

Aminoacylation of RNA

 

Learn more about this with a slide presentation: click here

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.

Selected Publications (by Area)

Overview

The article summarizes the origins of current research as well as earlier activities.

Selective Alteration of Proteins

Chemical alterations permit systematic studies of proteins that would dissociate - these have many applications.

·         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. A useful overview of the status of dealing with hypertension caused by red cell subsitutes

·         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. Testing if bis-tetramers do indeed avoid hypertension . the results are positive. The work was done by Francine Lui from our group in the NO-focussed lab of Dr. Zapol at Harvard Medical School.

·         Increasing Efficiency in Protein.Protein Coupling: Subunit-Directed Acetylation and Phase-Directed CuAAC (.Click Coupling.) in the Formation of Hemoglobin Bis-Tetramers. Aizhou Wang and Ronald Kluger. Biochemistry 2014 53 6793.6799. The novel use of an acetyl protecting group to direct coupling to the beta sub units.

·         Subunit-Specific Serial Cross-Linking Permits Highly Efficient CuAAC Formation of Functional Hemoglobin bis-Tetrameric Oxygen Carriers Serena Singh, Ying Yang, Ina Dubsinsky, and Ronald Kluger .Org. Biomol. Chem. 2015 13 11118-11128

Acyl phosphate monoesters as biomimetic reagents

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 Salts
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 Kluger Org. Biomol. Chem.2011 9 , 5645-5647.

·         Solid phase lanthanum catalysis of monoacylation of diols in water by acyl phosphate monoesters. Raj S. Dhiman, Aizhou Wang, and Ronald Kluger .. Can. J. Chem. 2015 93, 445-450.

Catalyzed decarboxylation: thiamin intermediates, addition of water, and reversibility

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.

·         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 Brød 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 rather than steric interference in the reaction 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 Brød bases catalyzed decarboxylation by promoting the departure of bicarbonate from an addition intermediate.

·         Carbon Kinetic Isotope Effects Reveal Variations in Reactivity of Intermediates in the Formation of Protonated Carbonic Acid Adelle A. Vandersteen, Scott O. C. Mundle, Georges Lacrampe-Couloume, Barbara Sherwood Lollar, and Ronald Kluger J. Org. Chem. 2013 78  12176.12181.

Variation of isotope effects reveals the reactivity of otherwise inaccessible intermediates.

·         Avoiding CO2 in Catalysis of Decarboxylation Ronald Kluger, Graeme W. Howe, and Scott O.C. Mundle . Adv. Phys. Org. Chem. 2013 47 85-128.

·         Decarboxylation without CO2: Why Bicarbonate Forms Directly as Trichloroacetate Is Converted to Chloroform. Graeme Howe and Ronald Kluger J. Org. Chem. 2014 79 10972.10980. Why is base necessary for this reaction? Does it prevent reversion?

·         Catalyzing Decarboxylation by Taming Carbon Dioxide Ronald Kluger Pure Appl. Chem. 2015 87 353-360.
A condensed overview of recent work

·         Decarboxylation, CO2 and the Reversion Problem, Ronald Kluger Acc. Chem. Res. 2015 48 2843.2849.
A perspective on how decarboxylation can be facilitated and how carboxylation could be catalyzed

·         Lithium-stabilized nucleophilic addition of thiamin to a ketone provides an efficient route to mandelylthiamin, a critical pre-decarboxylation intermediate Michael Bielecki, Graeme W. Howe, and Ronald Kluger Bioorg. Chem. 2015 64 124-129.
Lithium ions stabiliz
e addition of a nucleophilic carbene to a ketone

·         Decarboxylation, CO2 and the Reversion Problem Ronald Kluger Acc. Chem. Res 2015 48 2843.2849.

·         How Acid-Catalyzed Decarboxylation of 2,4-Dimethoxybenzoic Acid Avoids Formation of Protonated CO2 Graeme W Howe, Adelle A. Vandersteen, and Ronald Kluger, J. Am. Chem. Soc. ASAP. (June 2016).

Group members and their research areas:

Name

Area

Chung-Woo Fung (Technical assistant)

instrumentation, HPLC, mass spectrometry

Yuyang Li

Aminoacylation of RNA

Alan Amin

Aminoacylation of RNA

Michael Bielecki

Thiamin enzyme intermediates

Graeme Howe

Decarboxylation catalysis

Ina Dubinsky (postdoctoral)

Hemoglobin coupling

Serena Singh

Efficient formation of bis-tetramers

Aizhou Wang

Subunit reaction specificity

Nathan Wu

Elimination reactions of addition products in enzyme reactions

Wendy He

New derivatives of thiamin intermediates

Yasaman Heidari

Reaction patterns of addition products

 

GROUP SAFETY NOTICE


group_photo_2014

 

Not So Recent Group Photo

 

Some Former Students and Postdoctorals

Current Position

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Wing-Cheong Tsui

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Greg Thatcher

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David Zechel

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Tim Smyth

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Michael Brandl

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John Paul Pezacki

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Stephen Bearne

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Scott Taylor

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Neil Branda

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Andrew Grant

Professor. Mount Allison University

Qingyan Hu

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Scott Mundle

Professor, University of Windsor and Great Lakes Inst. for Environmental Research

John Lam

Lead Program Manager, Microsoft, Seattle

Francine Lui

Scientist, Northern Biologicals, Toronto

Jolanta Wodzinska

Manager, Non-Clinical Discovery, ApoPharma Inc., Toronto

Ian Moore

Application Scientist, SCIEX

Steve Brookes

Manager, Drug Development, Therapure Biopharma Inc

Amer Alagic

Research Director, Microbix Biosystems

Dongxin Hu

Senior Project Manger, A.U.G. Signals, Toronto

Elizabeth Wilson

Analytical Scientist, Amgen, Cambridge, USA

Lisa Cameron

‎Director, Commercial QC Laboratories, Teva Canada

Glenn Ikeda

Patent Manager, Teva Canada

Vittorio De Stefano

Managing Director, VAR-X Holding Corporation (Investments)

Vince Mazza

Managing Partner, Traverse Capital Partners

Philip Wasserstein

Neurologist, Palo Alto Medical Foundation

Steven Rathgeber

Pediatric Resident, University of British Columbia

Khashayar Karimian

CEO, Arasto Pharmaceutical Chemicals, Tehran

Xianfeng Li

Research Leader, Anacor Pharmaceuticals, San Francisco

Yonghong Song

Staff Scientist, Myokardia Pharmaceuticals, San Francisco

Ian Gray

Deputy Director, Pharmaceutical & Scientific Services, Sanofi Pasteur, London UK

Belinda Tsao Nivaggioli

CEO, Avencia Pharmaceuticals, California

Marcel Trachsel

CEO, INT/EXT Communications AG, Basel

Elizabeth Wilson

Staff Scientist, Amgen, Cambridge, Mass.

Last updated June 22, 2016

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