Ronald Kluger


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)

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


Connecting and cross-linking two hemoglobins


upper route is fast and leads to destruction of thiamin

lower route is slow - accelerated by enzyme



·         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.


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.



·         Aminoacyl phosphate monoesters


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)


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

Site 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

·         Self-Assembly of a Functional Triple Protein: Hemoglobin-Avidin-Hemoglobin via Biotin-Avidin Interactions Serena Singh and Ronald Kluger, Biochemistry, 2016 55  2875-2882

·         Enhanced Nitrite Reductase Activity and Its Correlation with Oxygen Affinity in Hemoglobin bis-Tetramers Aizhou Wang and Ronald Kluger “” Biochemistry 2016 55, 6793–6799.

·         Cross-Linked Hemoglobin bis-Tetramers from Bioorthogonal Coupling Resist Extravasation and Its Consequences in the Circulation”, Aizhou Wang, Serena Singh, Binglan Yu, Donald B. Bloch, Warren M. Zapol, and Ronald Kluger, submitted to Transfusion, May 2018

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.

·          Increased Efficiency in Biomimetic Lewis Acid-Base Pair Catalyzed Monoacylation of Diols by Acyl Phosphate Monoesters” Yuyang Li and Ronald Kluger, FACETS 2017 2 682-689.

·         Lead-Catalyzed Aqueous Benzoylation of Carbohydrates with an Acyl Phosphate Ester Yuyang Li and Ronald Kluger, J Org Chem 2018 ASAP.

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.

·         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.

·         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.

·         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

·         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

·         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. 2016 138 7568-7573.

·         The reactivity of lactyl-oxythiamin implies the role of the amino-pyrimidine in thiamin catalyzed decarboxylation Yasaman Heidari, Graeme W. Howe and Ronald Kluger “” Bioorg. Chem. 2016 69, 153–158.

·         The Need for an Alternative to Radicals as the Cause of Fragmentation of a Thiamin-Derived Breslow Intermediate, Michael Bielecki and Ronald Kluger, Angew. Chem. Int. Ed. 2017 56  6321–6323

·         Carbon Kinetic Isotope Effects and the Mechanisms of Acid Catalyzed Decarboxylation of 2,4-Mimethoxybenzoic Acid and CO2 Incorporation into 1,2-Dimethoxybenzene Adelle A Vandersteen, Graeme A. Howe, Barbara Sherwood Lollar and Ronald Kluger J. Am Chem. Soc. 2017 139  15049-15053

·         Charge Dispersion and Its Effects on the Reactivity of Thiamin-Derived Breslow Intermediates Michael Bielecki, Graeme W. Howe, and Ronald Kluger “” Biochemistry (ASAP 2018).


Current Group members and their research areas:



Yuyang Li

Aminoacylation of RNA

Michael Bielecki

Thiamin enzyme intermediates

Aizhou Wang

Circulation of hemoglobin bis-tetramers and applications to lung perfusion

Jimmy Lee

Hemoglobin conjugates

Elijah Lai

Alternative reactions of thiamin adducts



Group Photo


Some Former Students and Postdoctorals

Current Position

Jik Chin

Professor, University of Toronto

Wing-Cheong Tsui

CEO, HK Biotech (Cambridge, UK)

Greg Thatcher

Professor, University of Illinois School of Medicine

David Zechel

Professor, Queen's University

Tim Smyth

Professor, Univesity of Limerick

Michael Brandl

Senior Research Scientist, Gilead Pharmaceuticals

John Paul Pezacki

Professor, University of Ottawa

Stephen Bearne

Professor, Dalhousie University

Scott Taylor

Professor, University of Waterloo

Neil Branda

Professor, Simon Fraser University

Andrew Grant

Professor. Mount Allison University

Qingyan Hu

Senior Scientist, Regeneron Pharmaceuticals

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 Cardiologist, BC Children’s Hospital

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.

Victoria Stergiopoulos

Physician, St. Michael’s Hospital, Toronto

Andrew Pelling

Professor, Dept of Physics, Univ of Ottawa

Ruxandra Serbanescu

Professor, Dept of Physics, Univ of Toronto

Gerald Gish

Scientist, Cancer Biology, Lunenfeld Institute

Last updated June 22, 2016

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