Available online at www.sciencedirect.com
ScienceDirect Editorial overview: Synthetic Biomolecules Stephen BH Kent and Paul F Alewood Current Opinion in Chemical Biology 2014, 22:viii–xi For a complete overview see the Issue Available online 16th October 2014 http://dx.doi.org/10.1016/j.cbpa.2014.09.037 S1367-5931/# 2014 Elsevier Ltd. All right reserved.
Stephen BH Kent Department of Chemistry and Department of Biochemistry & Molecular Biology, University of Chicago, USA e-mail: [email protected] Stephen Kent is professor of Chemistry, and Professor of Biochemistry & Molecular Biology, at The University of Chicago. He received his B.Sc. from Victoria University of Wellington, his M.Sc., from Massey University, New Zealand, and his Ph.D.[Chemistry], from the University of California, Berkeley. Kent and his colleagues invent new synthetic chemistries and use them to elucidate the molecular basis of protein function.
Paul F Alewood Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Australia e-mail: [email protected] Paul Alewood is professor of Chemical and Structural Biology in the Institute for Molecular Bioscience at the University of Queensland. He has an international reputation in the field of bioactive peptides and synthetic proteins and is the author of over 300 publications in high quality journals. He is also an inventor on 12 patents. His principal research areas are in the fields of peptide, protein and medicinal chemistry.
Synthetic chemistry applied to peptides and proteins, an idea whose time has come This issue of Current Opinion in Chemical Biology focuses on synthetic peptides and synthetic proteins and their applications in research. Notably, ten of the articles in this issue comprise a representative sample of an important and rapidly growing field of chemical biology, the application of synthetic chemistries to the study of biologically active protein molecules. A majority of the articles deal with aspects of chemical ligation for the total or semi-synthesis of protein molecules. All of this current work is based on the original chemical ligation principle: the chemoselective covalent condensation of unprotected peptides enabled by formation of a non-native bond at the ligation site. [1] It is now generally understood that introduction of an unnatural structure in the act of ligating two peptides can both preserve biological function and provide a uniquely chemical analog of the parent protein molecule, useful for biochemical and biological research. Several of the chemistries discussed (native chemical ligation; Thr/Ser ligation; KAHA ligation) are extensions of this fundamental principle, in that the initial unnatural ligation product rearranges to form a native amide (peptide) bond in the final product. These ligation chemistries enable the ready preparation of a wide range of analog proteins not readily accessible by molecular biology. A number of ingenious applications of the different ligation chemistries are described. Other topics that are discussed in the articles found in this issue include the site-specific chemical modification — a different kind of ligation chemistry — of proteins, protein semisynthesis by enzyme-catalyzed transamidation, peptide catalysts for stereoselective organic transformations, and approaches to the synthesis of peptide libraries by facile transamidation between peptide molecules. Several articles describe the application of ligation chemistries or more conventional chemistries to studies of distinct families of protein molecules: relaxins, chemokines, glycoproteins, mirror image proteins, and selenoproteins.
Peptides A major focus of synthetic organic chemistry in recent years is the development of chiral catalysts for stereoselective synthesis. Lewandowski and Wennemers describe a body of highly innovative work on the design and application of effective peptide catalysts for stereoselective organic transformations. These are short chain peptides of 2–10 amino acids and are often Pro-containing. Polymer-supported forms of these peptides can be reused as catalysts, with applications both at lab scale and for industrial scale syntheses Current Opinion in Chemical Biology 2014, 22:viii–xi
www.sciencedirect.com
Editorial overview: Synthetic Biomolecules Kent and Alewood ix
of chirally-pure complex molecules. The properties of these synthetic peptide catalysts are reminiscent of enzymes, and possible implications of this similarity for the evolution of protein enzymes are considered.
Native chemical ligation of unprotected peptides A different kind of peptide chemistry is discussed by Malins and Payne, namely the native chemical ligation of unprotected peptides followed by desulfurization of the Cys or other b- (or g-) thiol amino acid at the ligation site. This converts the thiol-containing amino acid needed for native chemical ligation into one of the natural amino acids found in protein molecules. Based on the original work of Yan and Dawson [2], novel Cys surrogates for 13 of the 20 proteinogenic amino acids have been developed in recent years and applied to chemical protein synthesis. Similarly, selenocysteine has been used in native chemical ligation followed by chemoselective deselenization. Payne also discusses recent work on novel routes to peptide-thioesters and their reagent equivalents, by the more widely used Fmoc chemistry solid phase peptide synthesis, and improved thiol catalysts for native chemical ligation. Peptide libraries have been widely used in research in the past two decades. Melnyk and Agouridas describe recent approaches to the development of dynamic covalent native peptide libraries by selective peptide transamidation/ amide metathesis reactions. Some N-(2-sulfanylethyl) amides are able to rearrange into transient thioesters by spontaneous acyl migration from nitrogen to sulfur, and are thus able to act as latent thioesters. This section of the current issue has a very insightful discussion of mechanistic aspects of S-to-N acyl migration and implications for the native chemical ligation reaction. N,S-acyl shift systems are a basis for reversible amide bond chemistries that can operate in aqueous media. Selenopeptides can also participate in transamidation and amide metathesis reactions. Further studies are needed to accelerate the amide metathesis reactions and thus enable transamidation chemistry to be applied to the generation of native peptide combinatorial libraries.
New ligation chemistries for protein synthesis With the upsurge of interest in chemical synthesis and semi-synthesis of protein molecules, there has emerged a veritable cottage industry of proposed novel ligation chemistries. Most of these new chemistries have been found wanting in terms of practical utility. Harmand, Murar, and Bode review several of the most promising new ligation chemistries that are being developed and used as alternatives to native chemical ligation. The KAHA ligation from the Bode laboratory involves the reaction of a peptide-aketoacid with a 5-oxaproline-peptide. Originally this reaction was thought to directly give an amide-linked ligation product. Recently, the KAHA www.sciencedirect.com
ligation has been shown to give ester linkages (‘depsipeptides’) as the initial products; these rearrange at alkaline pH to give the desired native amide link [3]. This discovery extends the utility of this novel chemistry to the preparation of ester-containing ‘backbone engineered’ protein molecules. The alkyne-azide ‘click’ reaction has also been applied to chemical ligation of suitably functionalized unprotected peptides to give triazolelinked ligation products. This represents the application to protein science of this very useful chemistry and exemplifies the original chemical ligation concept, i.e. chemoselective reaction enabled by formation of an unnatural structure at the ligation site [1]. In nature, there are numerous families of proteins containing Cys residues (e.g. zinc-finger proteins) or disulfide bonds (e.g. chemokines and other secretory proteins), thus rendering them synthetically accessible in a straightforward fashion using the original native chemical ligation reaction. Nonetheless, it is often the case that Cys residues are not suitably located for disconnection into conveniently sized peptide segment building blocks, or that a target protein molecule contains few or no Cys residues. For that reason, amide-forming chemical ligation at nonCys sites would increase synthetic versatility. Chi Lung Lee and Xuechen Li describe the chemoselective reaction between an N-terminal serine or threonine of an unprotected peptide segment and a C-terminal salicylaldehyde ester of another unprotected peptide segment. This reaction initially gives an N,O-benzylidene acetal linked product, which upon acidolysis produces a native peptide bond at the site of ligation. The scope and limitations of Ser/Thr ligation are discussed, along with methods for the synthesis of the requisite peptide-salicylaldehyde esters. Serine/threonine ligation has been applied to the synthesis of peptide natural products, cyclic peptides, and proteins.
Site-specific protein modification In elucidating the roles of protein post-translational modifications, a particular challenge is the preparation of homogenous protein preparations with defined site-specific post-translational modifications. Schumacher and Hackenberger discuss novel chemistries for the site-specific functionalization of protein molecules, and their applications. The work discussed includes innovative linkerdesign and novel conjugation chemistries that enable the preparation of protein constructs containing ‘turn-on’ fluorescent labels and the chemical installation of posttranslational modifications. They also discuss the use of specific chemical reactions to give protein–PEG conjugates, and the application of these types of chemistry to the preparation of antibody–drug conjugates, an emerging area of great activity in the pharmaceutical industry. In addition to post-translational side chain modifications, the polypeptide chain of protein molecules can be Current Opinion in Chemical Biology 2014, 22:viii–xi
x Synthetic biomolecules
post-translationally modified, either in nature or in the laboratory. Schmohl and Schwarzer describe and discuss the use of the enzyme sortase A, a bacterial transpeptidase, to effect this different kind of site-specific protein modification. The substrate specificity of this enzyme is well understood, and it can be used as a peptide ligase for the sortase-mediated labeling of proteins using appropriately labeled synthetic peptide substrates. The synthetic peptide substrates can be engineered to facilitate the enzyme-catalyzed ligation reaction. The sortase A enzyme molecule itself can also be engineered to improve the catalytic properties for these and other applications.
Protein targets Chemokines are a subclass of cytokines with important functions in the inflammatory response. Panitz and BeckSickinger briefly review the total synthesis of chemokines by native chemical ligation, and then focus on the use of semi-synthesis by expressed protein ligation to study and expand the properties of chemokine protein molecules. Semi-synthesis has been applied to site-specific labeling and the preparation of unique chemical analogues for the elucidation of structure–activity relationships in selected chemokines. They describe the design and preparation of a light-activated chemokine construct, and the covalent attachment of chemokines to surfaces for more-controlled study of their chemotactic properties. Members of the family of insulin-related small protein molecules play diverse roles in human biology. Hossain and Wade described the total chemical synthesis of the relaxins by random combination of synthetic A-chain and B-chain peptides. A more sophisticated approach to the total chemical synthesis of relaxins involves the use of unique combinations of Cys side chain protecting groups to enable regioselective disulfide bond formation. The relaxins can also be produced by recombinant DNAbased expression. In combination, these various approaches to the preparation of relaxins enable the systematic exploration of structure–function relationships in these important protein molecules. A particularly widespread and important post-translational modification of proteins is the site-specific covalent attachment of carbohydrates. The resulting glycoproteins have altered biological properties in vivo. Okamoto, Izumi, and Kajihara point out the importance of being able to prepare homogeneous glycoproteins of known chemical structure. They describe synthetic strategies for the total chemical synthesis of glycoproteins, and the application of these methods to the preparation of two distinct types of glycoprotein, an EPO analog having an intact N-linked sialyloligosaccharide; and, a highly glycosylated antifreeze protein/polypeptide. Chemoenzymatic synthesis of a glycosylated IgG protein is also described. Most notably, the chemical synthesis of a deliberately ‘misfolded’ glycoprotein provided a unique Current Opinion in Chemical Biology 2014, 22:viii–xi
unnatural glycoprotein as a novel probe for glycobiology. A chemically synthesized glycosylated chemokine of defined covalent structure was used to explore the use of quasi-racemic crystallography for the determination of glycoprotein X-ray structures. All protein molecules found in nature are made up of Lamino acids and the achiral amino acid glycine. Mirror image proteins are made up of D-amino acids and Gly, and are enantiomers of the corresponding natural protein molecules. Such D-proteins can only be made by chemistry. Le Zhao and Wuyuan Lu describe the use of Dproteins in racemic protein crystallography to facilitate the crystallization of recalcitrant proteins and the determination of their X-ray structures. D-proteins are also essential for mirror image phage display, the use of genetically encoded libraries for the development of D-peptide ligands and candidate D-protein therapeutics. The use of D-enantiomeric defensins casts new lights on their biochemical mechanism of action. Selenocysteine is often called the twenty-first natural amino acid and is found in ribosomally-translated protein molecules. Norman Metanis and Don Hilvert give a broad overview of selenoproteins, which are being discovered in increasing numbers. They discuss the biosynthesis of selenoproteins and their unique redox and thermodynamic properties. Selenoproteins can be made by total chemical synthesis using native chemical ligation at selenocysteine, and can also be prepared by semi-synthesis using expressed protein ligation. The utility of artificial diselenide bonds in disulfide-containing proteins is briefly touched on, a topic that will be of great importance in the coming years.
Characterization of synthetic proteins With the increasing number of publications reporting total chemical synthesis of proteins, it is important to re-emphasize the need for meticulous characterization of the synthetic products as defined chemical compounds. Proteins are large molecules with complex structures. In addition to conventional chemical characterizations including purity, mass, and covalent structure, there is a unique attribute that must also be documented for a synthetic protein molecule: namely, that the synthetic product has the correct folded ‘tertiary’ structure of a protein molecule that gives rise to its properties including its biological functions. That folded structure is encoded by the linear amino acid sequence of the protein’s polypeptide chain. A disturbing feature of a number of papers in the current literature, even in prestigious journals [4], is that putative synthetic protein products are not fully characterized. In many cases, the data that is provided — often only reported in the on-line supporting information — do not support either the homogeneity of the synthetic www.sciencedirect.com
Editorial overview: Synthetic Biomolecules Kent and Alewood xi
product or its claimed structure. This is not acceptable practice for scientific research.
Determination of the connectivities for disulfide bondcontaining proteins: for example, by enzymatic digestion, LCMS peptide mapping.
The complete characterization of a synthetic protein would be as follows:
Tertiary structure
Homogeneity Show that the purified synthetic protein is a pure, single molecular species by at least two analytical techniques based on distinct separation principles: for example, analytical HPLC (hydrophobicity); isoelectric focusing (charge). If mass spectrometry (MS) is used to evaluate the purity of the synthetic protein, the MS data should be from direct infusion electrospray MS or its equivalent, so that the data show all molecular species present in the synthetic product. It is most emphatically not acceptable to report MS data at a single time point from an LCMS run, especially if that point does not correspond to the high point of the UV absorbance chromatogram!
Show that the protein has a defined folded structure: for example, by multidimensional NMR fingerprinting. Determine the folded structure of the synthetic protein; for example, by X-ray crystallography. Only then, when it has been shown that the synthetic product is a pure, homogeneous protein molecule with correct covalent structure and defined tertiary structure, can the properties of the synthetic protein be studied and correlated with its chemical structure in a valid fashion. Few reported total syntheses of protein molecules meet that standard of characterization. An example that does can be found here [5].
References 1.
Schno¨lzer M, Kent SBH: Constructing proteins by dovetailing unprotected synthetic peptides: backbone engineered HIV protease. Science 1992, 256:221-225.
2.
Yan LZ, Dawson PE: Synthesis of peptides and proteins without cysteine residues by native chemical ligation combined with desulfurization. J Am Chem Soc 2001, 123:526-533.
3.
Thomas G. Wucherpfennig, Florian Rohrbacher, Dr. Vijaya R. Pattabiraman and Prof. Dr. Jeffrey W. Bode, Formation and Rearrangement of Homoserine Depsipeptides and Depsiproteins in the a-Ketoacid–Hydroxylamine Ligation with 5-Oxaproline. Angew Chem Int Ed, Article first published online: 22 SEP 2014, DOI: 10.1002/anie.201406097].
4.
Wang P, Dong S, Shieh JH, Peguero E, Hendrickson R, Moore MA, Danishefsky SJ: Erythropoietin derived by chemical synthesis. Science 2013, 342:1357-1360.
5.
Durek T, Vetter I, Wang CI, Motin L, Knapp O, Adams DJ, Lewis RJ, Alewood PF: Chemical engineering and structural and pharmacological characterization of the a-scorpion toxin OD1. ACS Chem Biol 2013, 8:1215-1222.
Covalent structure Report the measured monoisotopic mass of the synthetic protein; alternatively, for lower resolution measurements, report the measured mass with its experimental uncertainty, and compare that data with the high point on the isotope envelope. Direct experimental verification of the amino acid sequence of the full-length synthetic polypeptide chain: for example, by enzymatic digestion, followed by LC-MS/MS sequencing.
www.sciencedirect.com
Current Opinion in Chemical Biology 2014, 22:viii–xi
ScienceDirect Editorial overview: Synthetic Biomolecules Stephen BH Kent and Paul F Alewood Current Opinion in Chemical Biology 2014, 22:viii–xi For a complete overview see the Issue Available online 16th October 2014 http://dx.doi.org/10.1016/j.cbpa.2014.09.037 S1367-5931/# 2014 Elsevier Ltd. All right reserved.
Stephen BH Kent Department of Chemistry and Department of Biochemistry & Molecular Biology, University of Chicago, USA e-mail: [email protected] Stephen Kent is professor of Chemistry, and Professor of Biochemistry & Molecular Biology, at The University of Chicago. He received his B.Sc. from Victoria University of Wellington, his M.Sc., from Massey University, New Zealand, and his Ph.D.[Chemistry], from the University of California, Berkeley. Kent and his colleagues invent new synthetic chemistries and use them to elucidate the molecular basis of protein function.
Paul F Alewood Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Australia e-mail: [email protected] Paul Alewood is professor of Chemical and Structural Biology in the Institute for Molecular Bioscience at the University of Queensland. He has an international reputation in the field of bioactive peptides and synthetic proteins and is the author of over 300 publications in high quality journals. He is also an inventor on 12 patents. His principal research areas are in the fields of peptide, protein and medicinal chemistry.
Synthetic chemistry applied to peptides and proteins, an idea whose time has come This issue of Current Opinion in Chemical Biology focuses on synthetic peptides and synthetic proteins and their applications in research. Notably, ten of the articles in this issue comprise a representative sample of an important and rapidly growing field of chemical biology, the application of synthetic chemistries to the study of biologically active protein molecules. A majority of the articles deal with aspects of chemical ligation for the total or semi-synthesis of protein molecules. All of this current work is based on the original chemical ligation principle: the chemoselective covalent condensation of unprotected peptides enabled by formation of a non-native bond at the ligation site. [1] It is now generally understood that introduction of an unnatural structure in the act of ligating two peptides can both preserve biological function and provide a uniquely chemical analog of the parent protein molecule, useful for biochemical and biological research. Several of the chemistries discussed (native chemical ligation; Thr/Ser ligation; KAHA ligation) are extensions of this fundamental principle, in that the initial unnatural ligation product rearranges to form a native amide (peptide) bond in the final product. These ligation chemistries enable the ready preparation of a wide range of analog proteins not readily accessible by molecular biology. A number of ingenious applications of the different ligation chemistries are described. Other topics that are discussed in the articles found in this issue include the site-specific chemical modification — a different kind of ligation chemistry — of proteins, protein semisynthesis by enzyme-catalyzed transamidation, peptide catalysts for stereoselective organic transformations, and approaches to the synthesis of peptide libraries by facile transamidation between peptide molecules. Several articles describe the application of ligation chemistries or more conventional chemistries to studies of distinct families of protein molecules: relaxins, chemokines, glycoproteins, mirror image proteins, and selenoproteins.
Peptides A major focus of synthetic organic chemistry in recent years is the development of chiral catalysts for stereoselective synthesis. Lewandowski and Wennemers describe a body of highly innovative work on the design and application of effective peptide catalysts for stereoselective organic transformations. These are short chain peptides of 2–10 amino acids and are often Pro-containing. Polymer-supported forms of these peptides can be reused as catalysts, with applications both at lab scale and for industrial scale syntheses Current Opinion in Chemical Biology 2014, 22:viii–xi
www.sciencedirect.com
Editorial overview: Synthetic Biomolecules Kent and Alewood ix
of chirally-pure complex molecules. The properties of these synthetic peptide catalysts are reminiscent of enzymes, and possible implications of this similarity for the evolution of protein enzymes are considered.
Native chemical ligation of unprotected peptides A different kind of peptide chemistry is discussed by Malins and Payne, namely the native chemical ligation of unprotected peptides followed by desulfurization of the Cys or other b- (or g-) thiol amino acid at the ligation site. This converts the thiol-containing amino acid needed for native chemical ligation into one of the natural amino acids found in protein molecules. Based on the original work of Yan and Dawson [2], novel Cys surrogates for 13 of the 20 proteinogenic amino acids have been developed in recent years and applied to chemical protein synthesis. Similarly, selenocysteine has been used in native chemical ligation followed by chemoselective deselenization. Payne also discusses recent work on novel routes to peptide-thioesters and their reagent equivalents, by the more widely used Fmoc chemistry solid phase peptide synthesis, and improved thiol catalysts for native chemical ligation. Peptide libraries have been widely used in research in the past two decades. Melnyk and Agouridas describe recent approaches to the development of dynamic covalent native peptide libraries by selective peptide transamidation/ amide metathesis reactions. Some N-(2-sulfanylethyl) amides are able to rearrange into transient thioesters by spontaneous acyl migration from nitrogen to sulfur, and are thus able to act as latent thioesters. This section of the current issue has a very insightful discussion of mechanistic aspects of S-to-N acyl migration and implications for the native chemical ligation reaction. N,S-acyl shift systems are a basis for reversible amide bond chemistries that can operate in aqueous media. Selenopeptides can also participate in transamidation and amide metathesis reactions. Further studies are needed to accelerate the amide metathesis reactions and thus enable transamidation chemistry to be applied to the generation of native peptide combinatorial libraries.
New ligation chemistries for protein synthesis With the upsurge of interest in chemical synthesis and semi-synthesis of protein molecules, there has emerged a veritable cottage industry of proposed novel ligation chemistries. Most of these new chemistries have been found wanting in terms of practical utility. Harmand, Murar, and Bode review several of the most promising new ligation chemistries that are being developed and used as alternatives to native chemical ligation. The KAHA ligation from the Bode laboratory involves the reaction of a peptide-aketoacid with a 5-oxaproline-peptide. Originally this reaction was thought to directly give an amide-linked ligation product. Recently, the KAHA www.sciencedirect.com
ligation has been shown to give ester linkages (‘depsipeptides’) as the initial products; these rearrange at alkaline pH to give the desired native amide link [3]. This discovery extends the utility of this novel chemistry to the preparation of ester-containing ‘backbone engineered’ protein molecules. The alkyne-azide ‘click’ reaction has also been applied to chemical ligation of suitably functionalized unprotected peptides to give triazolelinked ligation products. This represents the application to protein science of this very useful chemistry and exemplifies the original chemical ligation concept, i.e. chemoselective reaction enabled by formation of an unnatural structure at the ligation site [1]. In nature, there are numerous families of proteins containing Cys residues (e.g. zinc-finger proteins) or disulfide bonds (e.g. chemokines and other secretory proteins), thus rendering them synthetically accessible in a straightforward fashion using the original native chemical ligation reaction. Nonetheless, it is often the case that Cys residues are not suitably located for disconnection into conveniently sized peptide segment building blocks, or that a target protein molecule contains few or no Cys residues. For that reason, amide-forming chemical ligation at nonCys sites would increase synthetic versatility. Chi Lung Lee and Xuechen Li describe the chemoselective reaction between an N-terminal serine or threonine of an unprotected peptide segment and a C-terminal salicylaldehyde ester of another unprotected peptide segment. This reaction initially gives an N,O-benzylidene acetal linked product, which upon acidolysis produces a native peptide bond at the site of ligation. The scope and limitations of Ser/Thr ligation are discussed, along with methods for the synthesis of the requisite peptide-salicylaldehyde esters. Serine/threonine ligation has been applied to the synthesis of peptide natural products, cyclic peptides, and proteins.
Site-specific protein modification In elucidating the roles of protein post-translational modifications, a particular challenge is the preparation of homogenous protein preparations with defined site-specific post-translational modifications. Schumacher and Hackenberger discuss novel chemistries for the site-specific functionalization of protein molecules, and their applications. The work discussed includes innovative linkerdesign and novel conjugation chemistries that enable the preparation of protein constructs containing ‘turn-on’ fluorescent labels and the chemical installation of posttranslational modifications. They also discuss the use of specific chemical reactions to give protein–PEG conjugates, and the application of these types of chemistry to the preparation of antibody–drug conjugates, an emerging area of great activity in the pharmaceutical industry. In addition to post-translational side chain modifications, the polypeptide chain of protein molecules can be Current Opinion in Chemical Biology 2014, 22:viii–xi
x Synthetic biomolecules
post-translationally modified, either in nature or in the laboratory. Schmohl and Schwarzer describe and discuss the use of the enzyme sortase A, a bacterial transpeptidase, to effect this different kind of site-specific protein modification. The substrate specificity of this enzyme is well understood, and it can be used as a peptide ligase for the sortase-mediated labeling of proteins using appropriately labeled synthetic peptide substrates. The synthetic peptide substrates can be engineered to facilitate the enzyme-catalyzed ligation reaction. The sortase A enzyme molecule itself can also be engineered to improve the catalytic properties for these and other applications.
Protein targets Chemokines are a subclass of cytokines with important functions in the inflammatory response. Panitz and BeckSickinger briefly review the total synthesis of chemokines by native chemical ligation, and then focus on the use of semi-synthesis by expressed protein ligation to study and expand the properties of chemokine protein molecules. Semi-synthesis has been applied to site-specific labeling and the preparation of unique chemical analogues for the elucidation of structure–activity relationships in selected chemokines. They describe the design and preparation of a light-activated chemokine construct, and the covalent attachment of chemokines to surfaces for more-controlled study of their chemotactic properties. Members of the family of insulin-related small protein molecules play diverse roles in human biology. Hossain and Wade described the total chemical synthesis of the relaxins by random combination of synthetic A-chain and B-chain peptides. A more sophisticated approach to the total chemical synthesis of relaxins involves the use of unique combinations of Cys side chain protecting groups to enable regioselective disulfide bond formation. The relaxins can also be produced by recombinant DNAbased expression. In combination, these various approaches to the preparation of relaxins enable the systematic exploration of structure–function relationships in these important protein molecules. A particularly widespread and important post-translational modification of proteins is the site-specific covalent attachment of carbohydrates. The resulting glycoproteins have altered biological properties in vivo. Okamoto, Izumi, and Kajihara point out the importance of being able to prepare homogeneous glycoproteins of known chemical structure. They describe synthetic strategies for the total chemical synthesis of glycoproteins, and the application of these methods to the preparation of two distinct types of glycoprotein, an EPO analog having an intact N-linked sialyloligosaccharide; and, a highly glycosylated antifreeze protein/polypeptide. Chemoenzymatic synthesis of a glycosylated IgG protein is also described. Most notably, the chemical synthesis of a deliberately ‘misfolded’ glycoprotein provided a unique Current Opinion in Chemical Biology 2014, 22:viii–xi
unnatural glycoprotein as a novel probe for glycobiology. A chemically synthesized glycosylated chemokine of defined covalent structure was used to explore the use of quasi-racemic crystallography for the determination of glycoprotein X-ray structures. All protein molecules found in nature are made up of Lamino acids and the achiral amino acid glycine. Mirror image proteins are made up of D-amino acids and Gly, and are enantiomers of the corresponding natural protein molecules. Such D-proteins can only be made by chemistry. Le Zhao and Wuyuan Lu describe the use of Dproteins in racemic protein crystallography to facilitate the crystallization of recalcitrant proteins and the determination of their X-ray structures. D-proteins are also essential for mirror image phage display, the use of genetically encoded libraries for the development of D-peptide ligands and candidate D-protein therapeutics. The use of D-enantiomeric defensins casts new lights on their biochemical mechanism of action. Selenocysteine is often called the twenty-first natural amino acid and is found in ribosomally-translated protein molecules. Norman Metanis and Don Hilvert give a broad overview of selenoproteins, which are being discovered in increasing numbers. They discuss the biosynthesis of selenoproteins and their unique redox and thermodynamic properties. Selenoproteins can be made by total chemical synthesis using native chemical ligation at selenocysteine, and can also be prepared by semi-synthesis using expressed protein ligation. The utility of artificial diselenide bonds in disulfide-containing proteins is briefly touched on, a topic that will be of great importance in the coming years.
Characterization of synthetic proteins With the increasing number of publications reporting total chemical synthesis of proteins, it is important to re-emphasize the need for meticulous characterization of the synthetic products as defined chemical compounds. Proteins are large molecules with complex structures. In addition to conventional chemical characterizations including purity, mass, and covalent structure, there is a unique attribute that must also be documented for a synthetic protein molecule: namely, that the synthetic product has the correct folded ‘tertiary’ structure of a protein molecule that gives rise to its properties including its biological functions. That folded structure is encoded by the linear amino acid sequence of the protein’s polypeptide chain. A disturbing feature of a number of papers in the current literature, even in prestigious journals [4], is that putative synthetic protein products are not fully characterized. In many cases, the data that is provided — often only reported in the on-line supporting information — do not support either the homogeneity of the synthetic www.sciencedirect.com
Editorial overview: Synthetic Biomolecules Kent and Alewood xi
product or its claimed structure. This is not acceptable practice for scientific research.
Determination of the connectivities for disulfide bondcontaining proteins: for example, by enzymatic digestion, LCMS peptide mapping.
The complete characterization of a synthetic protein would be as follows:
Tertiary structure
Homogeneity Show that the purified synthetic protein is a pure, single molecular species by at least two analytical techniques based on distinct separation principles: for example, analytical HPLC (hydrophobicity); isoelectric focusing (charge). If mass spectrometry (MS) is used to evaluate the purity of the synthetic protein, the MS data should be from direct infusion electrospray MS or its equivalent, so that the data show all molecular species present in the synthetic product. It is most emphatically not acceptable to report MS data at a single time point from an LCMS run, especially if that point does not correspond to the high point of the UV absorbance chromatogram!
Show that the protein has a defined folded structure: for example, by multidimensional NMR fingerprinting. Determine the folded structure of the synthetic protein; for example, by X-ray crystallography. Only then, when it has been shown that the synthetic product is a pure, homogeneous protein molecule with correct covalent structure and defined tertiary structure, can the properties of the synthetic protein be studied and correlated with its chemical structure in a valid fashion. Few reported total syntheses of protein molecules meet that standard of characterization. An example that does can be found here [5].
References 1.
Schno¨lzer M, Kent SBH: Constructing proteins by dovetailing unprotected synthetic peptides: backbone engineered HIV protease. Science 1992, 256:221-225.
2.
Yan LZ, Dawson PE: Synthesis of peptides and proteins without cysteine residues by native chemical ligation combined with desulfurization. J Am Chem Soc 2001, 123:526-533.
3.
Thomas G. Wucherpfennig, Florian Rohrbacher, Dr. Vijaya R. Pattabiraman and Prof. Dr. Jeffrey W. Bode, Formation and Rearrangement of Homoserine Depsipeptides and Depsiproteins in the a-Ketoacid–Hydroxylamine Ligation with 5-Oxaproline. Angew Chem Int Ed, Article first published online: 22 SEP 2014, DOI: 10.1002/anie.201406097].
4.
Wang P, Dong S, Shieh JH, Peguero E, Hendrickson R, Moore MA, Danishefsky SJ: Erythropoietin derived by chemical synthesis. Science 2013, 342:1357-1360.
5.
Durek T, Vetter I, Wang CI, Motin L, Knapp O, Adams DJ, Lewis RJ, Alewood PF: Chemical engineering and structural and pharmacological characterization of the a-scorpion toxin OD1. ACS Chem Biol 2013, 8:1215-1222.
Covalent structure Report the measured monoisotopic mass of the synthetic protein; alternatively, for lower resolution measurements, report the measured mass with its experimental uncertainty, and compare that data with the high point on the isotope envelope. Direct experimental verification of the amino acid sequence of the full-length synthetic polypeptide chain: for example, by enzymatic digestion, followed by LC-MS/MS sequencing.
www.sciencedirect.com
Current Opinion in Chemical Biology 2014, 22:viii–xi
