Novel protein science enabled by total chemical synthesis

Chemical synthesis is a well‐established method for the preparation in the research laboratory of multiple‐tens‐of‐milligram amounts of correctly folded, high purity protein molecules. Chemically synthesized proteins enable a broad spectrum of novel protein science. Racemic mixtures consisting of d‐protein and l‐protein enantiomers facilitate crystallization and determination of protein structures by X‐ray diffraction. d‐Proteins enable the systematic development of unnatural mirror image protein molecules that bind with high affinity to natural protein targets. The d‐protein form of a therapeutic target can also be used to screen natural product libraries to identify novel small molecule leads for drug development. Proteins with novel polypeptide chain topologies including branched, circular, linear‐loop, and interpenetrating polypeptide chains can be constructed by chemical synthesis. Medicinal chemistry can be applied to optimize the properties of therapeutic protein molecules. Chemical synthesis has been used to redesign glycoproteins and for the a priori design and construction of covalently constrained novel protein scaffolds not found in nature. Versatile and precise labeling of protein molecules by chemical synthesis facilitates effective application of advanced physical methods including multidimensional nuclear magnetic resonance and time‐resolved FTIR for the elucidation of protein structure–activity relationships. The chemistries used for total synthesis of proteins have been adapted to making artificial molecular devices and protein‐inspired nanomolecular constructs. Research to develop mirror image life in the laboratory is in its very earliest stages, based on the total chemical synthesis of d‐protein forms of polymerase enzymes.

[1]  S. Sidhu,et al.  A Potent d-Protein Antagonist of VEGF-A is Nonimmunogenic, Metabolically Stable, and Longer-Circulating in Vivo. , 2016, ACS chemical biology.

[2]  J. Vanderkooi,et al.  Mirror image forms of snow flea antifreeze protein prepared by total chemical synthesis have identical antifreeze activities. , 2008, Journal of the American Chemical Society.

[3]  C. Hackenberger,et al.  A supramolecular peptide synthesizer. , 2013, Angewandte Chemie.

[4]  Ramkrishna Adhikary,et al.  Transparent Window Vibrational Probes for the Characterization of Proteins With High Structural and Temporal Resolution. , 2017, Chemical reviews.

[5]  Inversion of the Side-Chain Stereochemistry of Indvidual Thr or Ile Residues in a Protein Molecule: Impact on the Folding, Stability, and Structure of the ShK Toxin. , 2017, Angewandte Chemie.

[6]  S. Kent.  Bringing the science of proteins into the realm of organic chemistry: total chemical synthesis of SEP (synthetic erythropoiesis protein). , 2013, Angewandte Chemie.

[7]  W. Lindner,et al.  Chirality in Drug Research: FRANCOTTE:CHIRALITY O-BK , 2006 .

[8]  Oliver Hartley,et al.  Medicinal chemistry applied to a synthetic protein: development of highly potent HIV entry inhibitors. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[9]  O. Melnyk,et al.  One-pot chemical synthesis of small ubiquitin-like modifier protein–peptide conjugates using bis(2-sulfanylethyl)amido peptide latent thioester surrogates , 2015, Nature Protocols.

[10]  E. Baker,et al.  Radiation Damage and Racemic Protein Crystallography Reveal the Unique Structure of the GASA/Snakin Protein Superfamily. , 2016, Angewandte Chemie.

[11]  G. Makhatadze,et al.  Total chemical synthesis and X-ray crystal structure of a protein diastereomer: [D-Gln 35]ubiquitin. , 2005, Angewandte Chemie.

[12]  Lei Liu,et al.  Practical Chemical Synthesis of Atypical Ubiquitin Chains by Using an Isopeptide-Linked Ub Isomer. , 2017, Angewandte Chemie.

[13]  W. M. Westler,et al.  Protein conformational dynamics in the mechanism of HIV-1 protease catalysis , 2011, Proceedings of the National Academy of Sciences.

[14]  Anthony A Kossiakoff,et al.  X-ray structure of snow flea antifreeze protein determined by racemic crystallization of synthetic protein enantiomers. , 2008, Journal of the American Chemical Society.

[15]  A. Brik,et al.  Convergent versus sequential protein synthesis: the case of ubiquitinated and glycosylated H2B. , 2015, Angewandte Chemie.

[16]  P. Dawson,et al.  Post-Translational Backbone Engineering through Selenomethionine-Mediated Incorporation of Freidinger Lactams. , 2018, Angewandte Chemie.

[17]  Todd O. Yeates,et al.  Why protein crystals favour some space-groups over others , 1995, Nature Structural Biology.

[18]  P. Dawson,et al.  Thermodynamics of a designed protein catenane. , 2003, Journal of molecular biology.

[19]  A. Delmas,et al.  Synthesis of a biologically active triazole-containing analogue of cystatin A through successive peptidomimetic alkyne-azide ligations. , 2012, Angewandte Chemie.

[20]  S. Kent,et al.  Total chemical synthesis of a D-enzyme: the enantiomers of HIV-1 protease show reciprocal chiral substrate specificity [corrected]. , 1992, Science.

[21]  R. Payne,et al.  Rapid and efficient protein synthesis through expansion of the native chemical ligation concept , 2018 .

[22]  S. Kent,et al.  Protein Backbone Engineering through Total Chemical Synthesis: New Insight into the Mechanism of HIV-1 Protease Catalysis ☆ , 2000 .

[23]  K. Tezuka,et al.  Chemical synthesis of erythropoietin glycoforms for insights into the relationship between glycosylation pattern and bioactivity , 2016, Science Advances.

[24]  Brian J. Smith,et al.  Extending Halogen-based Medicinal Chemistry to Proteins , 2016, The Journal of Biological Chemistry.

[25]  S. Kent,et al.  Catalytic contribution of flap-substrate hydrogen bonds in "HIV-1 protease" explored by chemical synthesis. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[26]  J. Bode.  Chemical Protein Synthesis with the α-Ketoacid-Hydroxylamine Ligation. , 2017, Accounts of chemical research.

[27]  T. Muir,et al.  Expressed Protein Ligation, a Novel Method for Studying Protein-Protein Interactions in Transcription* , 1998, The Journal of Biological Chemistry.

[28]  S. Kent,et al.  Structural engineering of the HIV‐1 protease molecule with a β‐turn mimic of fixed geometry , 1993, Protein science : a publication of the Protein Society.

[29]  Chong Li,et al.  D-peptide inhibitors of the p53–MDM2 interaction for targeted molecular therapy of malignant neoplasms , 2010, Proceedings of the National Academy of Sciences.

[30]  S. Kent.  Chemical protein synthesis: Inventing synthetic methods to decipher how proteins work. , 2017, Bioorganic & medicinal chemistry.

[31]  John P. Overington,et al.  Protein engineering and design. , 1989, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[32]  G. Petsko,et al.  Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site , 2013, Proceedings of the National Academy of Sciences.

[33]  T. Wucherpfennig,et al.  Formation and rearrangement of homoserine depsipeptides and depsiproteins in the α-ketoacid-hydroxylamine ligation with 5-oxaproline. , 2014, Angewandte Chemie.

[34]  D. Baker,et al.  The coming of age of de novo protein design , 2016, Nature.

[35]  Stephen B H Kent,et al.  Total chemical synthesis of biologically active vascular endothelial growth factor. , 2011, Angewandte Chemie.

[36]  Dieter Willbold,et al.  Mirror image phage display--a method to generate D-peptide ligands for use in diagnostic or therapeutical applications. , 2009, Molecular bioSystems.

[37]  A. Brzozowski,et al.  Insulin-like Growth Factor 1 Analogs Clicked in the C Domain: Chemical Synthesis and Biological Activities. , 2017, Journal of medicinal chemistry.

[38]  Lei Liu,et al.  A synthetic molecular system capable of mirror-image genetic replication and transcription. , 2016, Nature chemistry.

[39]  O. Seitz,et al.  Features of Auxiliaries That Enable Native Chemical Ligation beyond Glycine and Cleavage via Radical Fragmentation. , 2018, Chemistry.

[40]  M. Weiss,et al.  Scope and Limitations of Fmoc Chemistry SPPS-Based Approaches to the Total Synthesis of Insulin Lispro via Ester Insulin. , 2017, Chemistry.

[41]  C. Weller,et al.  Chemically Sumoylated Histone H4 Stimulates Intranucleosomal Demethylation by the LSD1-CoREST Complex. , 2017, ACS chemical biology.

[42]  Chuan-fa Liu,et al.  Peptide Weinreb amide derivatives as thioester precursors for native chemical ligation. , 2017, Organic & biomolecular chemistry.

[43]  M. Weiss,et al.  Fully convergent chemical synthesis of ester insulin: determination of the high resolution X-ray structure by racemic protein crystallography. , 2013, Journal of the American Chemical Society.

[44]  T. Muir,et al.  Synthesis of proteins by native chemical ligation. , 1994, Science.

[45]  E. Baker,et al.  A functional role of Rv1738 in Mycobacterium tuberculosis persistence suggested by racemic protein crystallography , 2015, Proceedings of the National Academy of Sciences.

[46]  F. Perler,et al.  The mechanism of intein-mediated protein splicing: variations on a theme. , 2005, Protein and peptide letters.

[47]  S. Kent.  Racemic & quasi-racemic protein crystallography enabled by chemical protein synthesis. , 2018, Current opinion in chemical biology.

[48]  J. Bode,et al.  A radical approach to posttranslational mutagenesis , 2016, Science.

[49]  T. Wucherpfennig,et al.  Product Selectivity in KAHA Ligations: Ester vs. Amide Formation with Cyclic Hydroxylamines , 2017 .

[50]  S. Kent,et al.  Ionization state of the catalytic dyad Asp25/25' in the HIV-1 protease: NMR studies of site-specifically 13C labelled HIV-1 protease prepared by total chemical synthesis. , 2012, Organic & biomolecular chemistry.

[51]  F. Bezanilla,et al.  Total chemical synthesis of biologically active fluorescent dye-labeled Ts1 toxin. , 2014, Angewandte Chemie.

[52]  M. Hong,et al.  Chemical ligation of the influenza M2 protein for solid‐state NMR characterization of the cytoplasmic domain , 2015, Protein science : a publication of the Protein Society.

[53]  S. Kent,et al.  Engineering an unnatural Nα‐anchored disulfide into BPTI by total chemical synthesis: structural and functional consequences , 1999 .

[54]  M. Sánchez-Hidalgo,et al.  Discovering the Bacterial Circular Proteins: Bacteriocins, Cyanobactins, and Pilins* , 2012, The Journal of Biological Chemistry.

[55]  Lei Liu.  Protein Ligation and Total Synthesis II , 2015 .

[56]  R. Rudolph,et al.  Routes to active proteins from transformed microorganisms. , 1991, Current opinion in biotechnology.

[57]  A. Wlodawer,et al.  Total chemical synthesis and high-resolution crystal structure of the potent anti-HIV protein AOP-RANTES. , 1999, Chemistry & biology.

[58]  S. Kent,et al.  Total chemical synthesis and biological activities of glycosylated and non-glycosylated forms of the chemokines CCL1 and Ser-CCL1. , 2014, Angewandte Chemie.

[59]  G. Verdine,et al.  Total Chemical Synthesis and Folding of All-l and All-d Variants of Oncogenic KRas(G12V). , 2017, Journal of the American Chemical Society.

[60]  Stephen Z. D. Cheng,et al.  Giant molecules: where chemistry, physics, and bio-science meet , 2017, Science China Chemistry.

[61]  Wolfgang Lindner,et al.  Chirality in drug research , 2006 .

[62]  C. Birck,et al.  Dissecting mechanism of coupled folding and binding of an intrinsically disordered protein by chemical synthesis of conformationally constrained analogues. , 2017, Chemical communications.

[63]  S. Kent,et al.  Design, total chemical synthesis, and X-ray structure of a protein having a novel linear-loop polypeptide chain topology. , 2012, Angewandte Chemie.

[64]  S. Sidhu,et al.  Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography , 2012, Proceedings of the National Academy of Sciences.

[65]  P. R. Elliott,et al.  Synthesis of Poly-Ubiquitin Chains Using a Bifunctional Ubiquitin Monomer , 2017, Organic letters.

[66]  Michael S. Kay,et al.  Potent D-peptide inhibitors of HIV-1 entry , 2007, Proceedings of the National Academy of Sciences.

[67]  A. Goldberg,et al.  Protein degradation and protection against misfolded or damaged proteins , 2003, Nature.

[68]  C. Hill,et al.  Design of a Potent d-Peptide HIV-1 Entry Inhibitor with a Strong Barrier to Resistance , 2010, Journal of Virology.

[69]  S. Kent,et al.  A one-pot total synthesis of crambin. , 2004, Angewandte Chemie.

[70]  J. Berg,et al.  The structure of a centrosymmetric protein crystal , 1993, Proteins.

[71]  A. Conibear,et al.  Advancing the Frontiers of Chemical Protein Synthesis-The 7th CPS Meeting, Haifa, Israel. , 2018, Cell chemical biology.

[72]  A. Ferré-D’Amaré,et al.  TOTAL CHEMICAL SYNTHESIS OF A UNIQUE TRANSCRIPTION FACTOR-RELATED PROTEIN : CMYC-MAX , 1995 .

[73]  S. Kent,et al.  Total chemical synthesis of enzymatically active human type II secretory phospholipase A2. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[74]  David J Craik,et al.  Thematic Minireview Series on Circular Proteins , 2012, The Journal of Biological Chemistry.

[75]  K. Mislow.  Introduction to stereochemistry , 1966 .

[76]  T. Ha,et al.  Single-Molecule Studies of HIV-1 Protease Catalysis Enabled by Chemical Protein Synthesis , 2011 .

[77]  Ting F. Zhu,et al.  Total chemical synthesis of a thermostable enzyme capable of polymerase chain reaction , 2017, Cell Discovery.

[78]  Han Liu,et al.  P-B Desulfurization: An Enabling Method for Protein Chemical Synthesis and Site-Specific Deuteration. , 2017, Angewandte Chemie.

[79]  S. Kent,et al.  Editorial overview: synthetic biomolecules. , 2014, Current opinion in chemical biology.

[80]  A. Warshel.  Electrostatic Origin of the Catalytic Power of Enzymes and the Role of Preorganized Active Sites* , 1998, The Journal of Biological Chemistry.

[81]  A. Gautier.  Structure determination of α-helical membrane proteins by solution-state NMR: emphasis on retinal proteins. , 2014, Biochimica et biophysica acta.

[82]  J. Bode,et al.  Chemical synthesis of a homoserine-mutant of the antibacterial, head-to-tail cyclized protein AS-48 by α-ketoacid–hydroxylamine (KAHA) ligation† †Electronic supplementary information (ESI) available: Experimental procedures, supplementary results, and spectroscopic data for new compounds. See DOI: 1 , 2017, Chemical science.

[83]  S. Klußmann,et al.  A thermostable d-polymerase for mirror-image PCR , 2017, Nucleic acids research.

[84]  A. Deniz,et al.  Site-Specific Three-Color Labeling of α-Synuclein via Conjugation to Uniquely Reactive Cysteines during Assembly by Native Chemical Ligation. , 2018, Cell chemical biology.

[85]  Dirk Schnappinger,et al.  Inhibition of Respiration by Nitric Oxide Induces a Mycobacterium tuberculosis Dormancy Program , 2003, The Journal of experimental medicine.

[86]  D. Woolfson,et al.  Controlling the Assembly of Coiled–Coil Peptide Nanotubes , 2015, Angewandte Chemie.

[87]  W. Li,et al.  Polyubiquitin chains: functions, structures, and mechanisms , 2008, Cellular and Molecular Life Sciences.

[88]  S. Kent,et al.  Constructing proteins by dovetailing unprotected synthetic peptides: backbone-engineered HIV protease. , 1992, Science.

[89]  S. Kent,et al.  Convergent chemical synthesis and crystal structure of a 203 amino acid "covalent dimer" HIV-1 protease enzyme molecule. , 2007, Angewandte Chemie.

[90]  Carlos R. Baiz,et al.  An Introduction to Protein 2D IR Spectroscopy , 2013 .

[91]  P. Dawson,et al.  Synthesis of native proteins by chemical ligation. , 2000, Annual review of biochemistry.

[92]  S. Kent,et al.  Through the looking glass – a new world of proteins enabled by chemical synthesis , 2012, Journal of Peptide Science.

[93]  B. Meier,et al.  Segmental isotope labelling and solid-state NMR of a 12 × 59 kDa motor protein: identification of structural variability , 2018, Journal of biomolecular NMR.

[94]  S. Boxer,et al.  Electric Fields and Enzyme Catalysis. , 2017, Annual review of biochemistry.

[95]  Yun-Kun Qi,et al.  Total Chemical Synthesis of Modified Histones , 2018, Front. Chem..

[96]  Shan Liu,et al.  Highly Selective Targeting of Hepatic Stellate Cells for Liver Fibrosis Treatment Using a d-Enantiomeric Peptide Ligand of Fn14 Identified by Mirror-Image mRNA Display. , 2017, Molecular pharmaceutics.

[97]  C. Anfinsen.  Principles that govern the folding of protein chains. , 1973, Science.

[98]  R. Zuckermann,et al.  Protein side-chain translocation mutagenesis via incorporation of peptoid residues. , 2011, ACS chemical biology.

[99]  R. Delden,et al.  Absolute Asymmetric Synthesis: The Origin, Control, and Amplification of Chirality. , 1999, Angewandte Chemie.

[100]  T. Yeates,et al.  (Quasi-)racemic X-ray structures of glycosylated and non-glycosylated forms of the chemokine Ser-CCL1 prepared by total chemical synthesis. , 2014, Angewandte Chemie.

[101]  S. Kent.  Total chemical synthesis of proteins. , 2009, Chemical Society reviews.

[102]  J. Adamson,et al.  Design and Chemical Synthesis of a Homogeneous Polymer-Modified Erythropoiesis Protein , 2003, Science.

[103]  Lei Liu,et al.  Blocking of the PD-1/PD-L1 Interaction by a D-Peptide Antagonist for Cancer Immunotherapy. , 2015, Angewandte Chemie.

[104]  S. Kent.  The critical role of peptide chemistry in the life sciences , 2015, Journal of peptide science : an official publication of the European Peptide Society.

[105]  Lorenz M. Mayr,et al.  Identification of d-Peptide Ligands Through Mirror-Image Phage Display , 1996, Science.

[106]  Stephen K. Burley,et al.  X-Ray Structures of Myc-Max and Mad-Max Recognizing DNA Molecular Bases of Regulation by Proto-Oncogenic Transcription Factors , 2003, Cell.

[107]  Klavs F Jensen,et al.  A fully automated flow-based approach for accelerated peptide synthesis. , 2017, Nature chemical biology.

[108]  R. Norton,et al.  Reinvestigation of the biological activity of d-allo-ShK protein , 2017, The Journal of Biological Chemistry.

[109]  J. W. Ward,et al.  Sequence-Specific Peptide Synthesis by an Artificial Small-Molecule Machine , 2013, Science.

[110]  E. Johansson,et al.  Divergent Protein Synthesis of Bowman–Birk Protease Inhibitors, their Hydrodynamic Behavior and Co-crystallization with α-Chymotrypsin , 2017, Synlett.

[111]  D Cowburn,et al.  Chemical ligation of folded recombinant proteins: segmental isotopic labeling of domains for NMR studies. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[112]  Lianjie Xu,et al.  Topology: a unique dimension in protein engineering , 2017, Science China Chemistry.

[113]  G Goldstein,et al.  The complete amino acid sequence of ubiquitin, an adenylate cyclase stimulating polypeptide probably universal in living cells. , 1975, Biochemistry.

[114]  T. Narumi,et al.  Direct synthesis of N-terminal thiazolidine-containing peptide thioesters from peptide hydrazides. , 2018, Chemical communications.

[115]  F. Bezanilla,et al.  Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET , 2017, Proceedings of the National Academy of Sciences.

[116]  Andrew E. Bruno,et al.  Statistical Analysis of Crystallization Database Links Protein Physico-Chemical Features with Crystallization Mechanisms , 2013, PloS one.

[117]  Convergent Total Synthesis of Histone H2B Protein with Site-Specific Ubiquitination at Lys120 , 2017 .

[118]  Brian W Matthews.  Racemic crystallography—Easy crystals and easy structures: What's not to like? , 2009, Protein science : a publication of the Protein Society.

[119]  G. Debelouchina,et al.  Ubiquitin Utilizes an Acidic Surface Patch to Alter Chromatin Structure , 2016, Nature chemical biology.

[120]  A. Mackay.  Crystal Enigma , 1989, Nature.

[121]  Lei Liu,et al.  Monomer/Oligomer Quasi-Racemic Protein Crystallography. , 2016, Journal of the American Chemical Society.

[122]  Kenneth A. Miller,et al.  Asymmetric total syntheses of (+)- and (-)-versicolamide B and biosynthetic implications. , 2009, Nature chemistry.

[123]  David Baker,et al.  De novo design of covalently constrained mesosize protein scaffolds with unique tertiary structures , 2017, Proceedings of the National Academy of Sciences.

[124]  D. Craik,et al.  Plant cyclotides: A unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif. , 1999, Journal of molecular biology.

[125]  B. Kieffer,et al.  Total chemical synthesis and biophysical properties of a designed soluble 24 kDa amyloid analogue† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc01790e , 2018, Chemical science.

[126]  S. Boxer,et al.  Vibrational Stark effects calibrate the sensitivity of vibrational probes for electric fields in proteins. , 2003, Biochemistry.

[127]  Duhee Bang,et al.  Dissecting the energetics of protein α-helix C-cap termination through chemical protein synthesis , 2006, Nature chemical biology.

[128]  G. Lippens,et al.  Synthesis, folding, and structure of the β‐turn mimic modified B1 domain of streptococcal protein G , 1999, Protein science : a publication of the Protein Society.

[129]  S. Gellman,et al.  Effects of Single α-to-β Residue Replacements on Structure and Stability in a Small Protein: Insights from Quasiracemic Crystallization. , 2016, Journal of the American Chemical Society.

[130]  A. Tokmakoff,et al.  Efficient Total Chemical Synthesis of 13C=18O Isotopomers of Human Insulin for Isotope‐Edited FTIR , 2016, Chembiochem : a European journal of chemical biology.

[131]  M. Weiss,et al.  Design and folding of [GluA4(ObetaThrB30)]insulin ("ester insulin"): a minimal proinsulin surrogate that can be chemically converted into human insulin. , 2010, Angewandte Chemie.

[132]  H. Neurath.  The Golden Years of Protein Science: Banquet Address at the First European Symposium of The Protein Society, Davos, Switzerland, May 30, 1995 , 1995, Protein science : a publication of the Protein Society.

[133]  N. Fujii,et al.  Screening of a virtual mirror-image library of natural products. , 2016, Chemical communications.