Special Issue Review Received: 19 November 2013
Revised: 22 November 2013
Accepted: 22 November 2013
Published online in Wiley Online Library: 7 January 2014
(wileyonlinelibrary.com) DOI 10.1002/psc.2602
Templated native chemical ligation: peptide chemistry beyond protein synthesis‡ Olalla Vázquez and Oliver Seitz* Native chemical ligation (NCL) is a powerful method for the convergent synthesis of proteins and peptides. In its original format, NCL between a peptide containing a C-terminal thioester and another peptide offering an N-terminal cysteine has been used to enable protein synthesis of unprotected peptide fragments. However, the applications of NCL extend beyond the scope of protein synthesis. For instance, NCL can be put under the control of template molecules. In such a scenario, NCL enables the design of conditional reaction systems in which, peptide bond formation occurs only when a specific third party molecule is present. In this review, we will show how templates can be used to control the reactivity and chemoselectivity of NCL reactions. We highlight peptide and nucleic-acid-templated NCL reactions and discuss potential applications in nucleic acid diagnosis, origin-of-life studies and gene-expression-specific therapies. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. Keywords: native chemical ligation; templated chemistry; PNA; DNA; RNA
Introduction
78
Chemical synthesis provides fascinating opportunities for the preparation of well-defined biomolecules tailored to the needs of biological applications. For example, chemical synthesis has enabled studies of the role of protein posttranslational modifications such as prenylation [1,2], glycosylation [3–5], phosphorylation [6–9] and ubiquitination [10]. Biocompatible labelling chemistries have been used to install reporter groups at biomolecules or to induce oligomerization on or even within living cells [11–13]. In such efforts, one of the challenges of biocompatible chemistry lies within the required tolerance for water, which readily reacts with electrophiles. Furthermore, the ubiquitous presence of a multitude of functional groups occurring in bystander (bio)molecules calls for exceptionally high degrees of chemoselectivity. In addition, biocompatible transformations should proceed with high rate constants to drive reactions when the concentration of participating functional groups is low. The most recent efforts in the development of suitable biocompatible chemistries include various forms of electrocyclic reactions [14,15]. However, the challenge remains to combine water tolerance with high reactivity at high chemoselectivity. Templated reactions can meet this challenge. Templates bind some or all reactants and align the functional groups such that a reaction is facilitated (Figure 1). In other words, templates enhance the proximity of the reactive groups. The reaction can proceed in a quasi-intramolecular fashion at high effective molarity. As a result, templated reactions can provide product under dilution conditions where non-templated reactions cease to proceed. Furthermore, the template-induced alignment of functional groups helps increase the chemoselectivity of a reaction. In this account, we will focus on the native chemical ligation (NCL) reaction. NCL is the most widely used chemoselective ligation technique for the synthesis of native proteins, because it is one of the very few nonenzymatic reactions known that efficiently connects two unprotected peptide segments to generate a protein with an amide bond at the reaction site [16,17]. NCL involves a two-step process composed of (1) a rapid thiol exchange
J. Pept. Sci. 2014; 20: 78–86
step between a peptide thioester and the sulfhydryl group of the N-terminal cysteine residue in the other peptide segment, which prompts (2) an intramolecular nucleophilic attack by the α-amino group of the N-terminal cysteine (S → N acyl rearrangement) to form the final amide bond (Figure 2). This last step is the driving force of the reaction because the S → N acyl rearrangement is irreversible at physiological conditions and kinetically accessible because of the five-membered transition state. The NCL proceeds at mild reaction conditions in water, and the reaction preferably occurs at 1,2-aminothiol structures such as an N-terminal cysteine. However, the NCL has to be performed within a quite narrow reactivity range, because the thioesters involved are prone to hydrolysis, which usually precludes the use of basic conditions and/or high temperatures. NCL reactions usually proceed at peptide concentration in the millimolar range. The use of templates, which increase the effective molarity of the reactants by binding to the template, would enable reactions at lower concentrations. This was first demonstrated by Ghadiri [18], who showed that a leucine zipper domain (32-residue α-helical peptide) of the transcription factor GCN4 accelerates its own formation through NCL by templating the association of the two NCL peptide precursors of 15- and 17-residues fragments at micromolar concentrations (Figure 3). A single α-helix structure of this coiled coil domain acted as a template preorganizing the benzyl thioester and the cysteinyl reactive groups of the peptide fragments. The preorganization of the reactants on the template was mediated by interhelical * Correspondence to: Oliver Seitz, Institut für Chemie, Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2. D-12489 Berlin, Germany. E-mail: oliver. [email protected] ‡
This article is published in Journal of Peptide Science as part of the Special Issue devoted to contributions presented at the Chemical Protein Synthesis Meeting, April 3-6, 2013, Vienna, edited by Christian Becker (University of Vienna, Austria). Institut für Chemie, Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2, D-12489 Berlin, Germany
Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
TEMPLATED NATIVE CHEMICAL LIGATION Biography Olalla Vázquez graduated in Chemistry (Hons) in 2004 at the University of Santiago de Compostela, Spain. In 2005, she joined the laboratory of Prof. José Luis Mascareñas and Prof. Eugenio Vázquez and completed her PhD in 2010. She received the National Research Award for PhD Students and the European Young Chemists Award. Since 2011, she is a postdoctoral Marie Curie fellow in the group of Prof. Oliver Seitz at Humboldt-Universität zu Berlin, Germany, where she is studying nucleic-acid-templated peptidyl transfer reactions.
Biography Figure 2. Principle of native chemical ligation.
Oliver Seitz obtained his PhD from the University of Mainz in 1995. After postdoctoral research at the Scripps Research Institute in La Jolla, he moved to the University of Karlsruhe, Germany. In 2000, he became group leader at the Max-Planck Institute of Molecular Physiology in Dortmund, Germany, and in 2003, he was appointed Full Professor at the Humboldt-Universität zu Berlin. His research interests include peptide and nucleic acid chemistry, molecular diagnostics, DNA/RNA-directed chemistry and the development of tools for the biomolecular control of protein-protein and protein-nucleic acid interactions. interactions, which triggered the NCL and accelerated the initial rates of the product formation by 500-fold. The feasibility of an autocatalytic formation of peptide bonds pointed to a plausible mechanism for molecular evolution of polypeptides under prebiotic conditions.
Introduction of the Native Chemical Ligation Principle in Nucleic-acid-templated Chemistry Templated synthesis has frequently been studied in nucleic acid chemistry. The pioneering work of Shabarova, Orgel, von Kiedrowski and Joyce was focused on gene synthesis and molecular
J. Pept. Sci. 2014; 20: 78–86
evolution of DNA/RNA [19–24]. Therefore, this work was primarily concerned with the development of methods that lead to the formation of phosphodiester linkages. In the last two decades, it became apparent that nucleic-acid-templated (NA-templated) chemical reactions offer incredible opportunities for the programmable synthesis of functional molecules beyond the scope of canonical nucleic acid chemistry. NA-templated reactions proceed with high sequence fidelity. This has been used to detect specific DNA/RNA targets in complex environments. The advances and the state of the art in the field have been recently summarized in several reviews [25–29]. In this article, we review the current progress of NA-templated NCL, which provides interesting opportunities for connections between the nucleic acid and the protein worlds. In an early contribution (1996), Joyce extrapolated a concept of peptide and protein chemistry to NA-templated chemistry (Figure 4) [30]. It was demonstrated that a peptide acyl chain
Figure 4. Template-directed peptidyl transfer with an oligonucleotide.
Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jpepsci
79
Figure 1. Templates accelerate chemical reactions by conferring proximity enhancements.
Figure 3. Templated native chemical ligation via formation of coiled coils.
VÁZQUEZ AND SEITZ (
Revised: 22 November 2013
Accepted: 22 November 2013
Published online in Wiley Online Library: 7 January 2014
(wileyonlinelibrary.com) DOI 10.1002/psc.2602
Templated native chemical ligation: peptide chemistry beyond protein synthesis‡ Olalla Vázquez and Oliver Seitz* Native chemical ligation (NCL) is a powerful method for the convergent synthesis of proteins and peptides. In its original format, NCL between a peptide containing a C-terminal thioester and another peptide offering an N-terminal cysteine has been used to enable protein synthesis of unprotected peptide fragments. However, the applications of NCL extend beyond the scope of protein synthesis. For instance, NCL can be put under the control of template molecules. In such a scenario, NCL enables the design of conditional reaction systems in which, peptide bond formation occurs only when a specific third party molecule is present. In this review, we will show how templates can be used to control the reactivity and chemoselectivity of NCL reactions. We highlight peptide and nucleic-acid-templated NCL reactions and discuss potential applications in nucleic acid diagnosis, origin-of-life studies and gene-expression-specific therapies. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. Keywords: native chemical ligation; templated chemistry; PNA; DNA; RNA
Introduction
78
Chemical synthesis provides fascinating opportunities for the preparation of well-defined biomolecules tailored to the needs of biological applications. For example, chemical synthesis has enabled studies of the role of protein posttranslational modifications such as prenylation [1,2], glycosylation [3–5], phosphorylation [6–9] and ubiquitination [10]. Biocompatible labelling chemistries have been used to install reporter groups at biomolecules or to induce oligomerization on or even within living cells [11–13]. In such efforts, one of the challenges of biocompatible chemistry lies within the required tolerance for water, which readily reacts with electrophiles. Furthermore, the ubiquitous presence of a multitude of functional groups occurring in bystander (bio)molecules calls for exceptionally high degrees of chemoselectivity. In addition, biocompatible transformations should proceed with high rate constants to drive reactions when the concentration of participating functional groups is low. The most recent efforts in the development of suitable biocompatible chemistries include various forms of electrocyclic reactions [14,15]. However, the challenge remains to combine water tolerance with high reactivity at high chemoselectivity. Templated reactions can meet this challenge. Templates bind some or all reactants and align the functional groups such that a reaction is facilitated (Figure 1). In other words, templates enhance the proximity of the reactive groups. The reaction can proceed in a quasi-intramolecular fashion at high effective molarity. As a result, templated reactions can provide product under dilution conditions where non-templated reactions cease to proceed. Furthermore, the template-induced alignment of functional groups helps increase the chemoselectivity of a reaction. In this account, we will focus on the native chemical ligation (NCL) reaction. NCL is the most widely used chemoselective ligation technique for the synthesis of native proteins, because it is one of the very few nonenzymatic reactions known that efficiently connects two unprotected peptide segments to generate a protein with an amide bond at the reaction site [16,17]. NCL involves a two-step process composed of (1) a rapid thiol exchange
J. Pept. Sci. 2014; 20: 78–86
step between a peptide thioester and the sulfhydryl group of the N-terminal cysteine residue in the other peptide segment, which prompts (2) an intramolecular nucleophilic attack by the α-amino group of the N-terminal cysteine (S → N acyl rearrangement) to form the final amide bond (Figure 2). This last step is the driving force of the reaction because the S → N acyl rearrangement is irreversible at physiological conditions and kinetically accessible because of the five-membered transition state. The NCL proceeds at mild reaction conditions in water, and the reaction preferably occurs at 1,2-aminothiol structures such as an N-terminal cysteine. However, the NCL has to be performed within a quite narrow reactivity range, because the thioesters involved are prone to hydrolysis, which usually precludes the use of basic conditions and/or high temperatures. NCL reactions usually proceed at peptide concentration in the millimolar range. The use of templates, which increase the effective molarity of the reactants by binding to the template, would enable reactions at lower concentrations. This was first demonstrated by Ghadiri [18], who showed that a leucine zipper domain (32-residue α-helical peptide) of the transcription factor GCN4 accelerates its own formation through NCL by templating the association of the two NCL peptide precursors of 15- and 17-residues fragments at micromolar concentrations (Figure 3). A single α-helix structure of this coiled coil domain acted as a template preorganizing the benzyl thioester and the cysteinyl reactive groups of the peptide fragments. The preorganization of the reactants on the template was mediated by interhelical * Correspondence to: Oliver Seitz, Institut für Chemie, Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2. D-12489 Berlin, Germany. E-mail: oliver. [email protected] ‡
This article is published in Journal of Peptide Science as part of the Special Issue devoted to contributions presented at the Chemical Protein Synthesis Meeting, April 3-6, 2013, Vienna, edited by Christian Becker (University of Vienna, Austria). Institut für Chemie, Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2, D-12489 Berlin, Germany
Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
TEMPLATED NATIVE CHEMICAL LIGATION Biography Olalla Vázquez graduated in Chemistry (Hons) in 2004 at the University of Santiago de Compostela, Spain. In 2005, she joined the laboratory of Prof. José Luis Mascareñas and Prof. Eugenio Vázquez and completed her PhD in 2010. She received the National Research Award for PhD Students and the European Young Chemists Award. Since 2011, she is a postdoctoral Marie Curie fellow in the group of Prof. Oliver Seitz at Humboldt-Universität zu Berlin, Germany, where she is studying nucleic-acid-templated peptidyl transfer reactions.
Biography Figure 2. Principle of native chemical ligation.
Oliver Seitz obtained his PhD from the University of Mainz in 1995. After postdoctoral research at the Scripps Research Institute in La Jolla, he moved to the University of Karlsruhe, Germany. In 2000, he became group leader at the Max-Planck Institute of Molecular Physiology in Dortmund, Germany, and in 2003, he was appointed Full Professor at the Humboldt-Universität zu Berlin. His research interests include peptide and nucleic acid chemistry, molecular diagnostics, DNA/RNA-directed chemistry and the development of tools for the biomolecular control of protein-protein and protein-nucleic acid interactions. interactions, which triggered the NCL and accelerated the initial rates of the product formation by 500-fold. The feasibility of an autocatalytic formation of peptide bonds pointed to a plausible mechanism for molecular evolution of polypeptides under prebiotic conditions.
Introduction of the Native Chemical Ligation Principle in Nucleic-acid-templated Chemistry Templated synthesis has frequently been studied in nucleic acid chemistry. The pioneering work of Shabarova, Orgel, von Kiedrowski and Joyce was focused on gene synthesis and molecular
J. Pept. Sci. 2014; 20: 78–86
evolution of DNA/RNA [19–24]. Therefore, this work was primarily concerned with the development of methods that lead to the formation of phosphodiester linkages. In the last two decades, it became apparent that nucleic-acid-templated (NA-templated) chemical reactions offer incredible opportunities for the programmable synthesis of functional molecules beyond the scope of canonical nucleic acid chemistry. NA-templated reactions proceed with high sequence fidelity. This has been used to detect specific DNA/RNA targets in complex environments. The advances and the state of the art in the field have been recently summarized in several reviews [25–29]. In this article, we review the current progress of NA-templated NCL, which provides interesting opportunities for connections between the nucleic acid and the protein worlds. In an early contribution (1996), Joyce extrapolated a concept of peptide and protein chemistry to NA-templated chemistry (Figure 4) [30]. It was demonstrated that a peptide acyl chain
Figure 4. Template-directed peptidyl transfer with an oligonucleotide.
Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jpepsci
79
Figure 1. Templates accelerate chemical reactions by conferring proximity enhancements.
Figure 3. Templated native chemical ligation via formation of coiled coils.
VÁZQUEZ AND SEITZ (
