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5 Solid phase peptide synthesis 5.1 Bruce Merrifield and the Rockefeller University 5.2 New York life in the 1970s 5.3 Emil Fischer and the early years of peptide chemistry 5.4 A Golden Age of peptide synthesis 5.5 Total chemical synthesis of enzymes: claims and counterclaims 5.6 Fixing solid phase peptide synthesis 5.7 Optimized SPPS

7 Mirror image proteins 7.1 Mirror image enzymes 7.2 Mirror image drug discovery 7.3 Racemic protein crystallography 7.4 Correcting “D-allo-ShK” protein

3 Novel peptide and protein science 3.1 Hepatitis B virus immunology 3.2 Synthetic erythropoiesis protein 3.3 Ester insulin

Appendix: Vita, Chemistree, Holographs, Publications, Glossary, Links and literature, Image sources, Index

4 Formative influences 4.1 Childhood and early education 4.2 Undergraduate education 4.3 Scientific apprenticeships 4.4 University of California Berkeley 4.5 PhD research 4.6 Berkeley life

1 Total chemical synthesis of proteins 1.1 Chemical ligation — the true breakthrough 1.2 Native chemical ligation 1.3 Convergent synthesis of proteins 1.4 Total protein syntheses enabled by chemical ligation 1.5 Impact on total chemical protein synthesis

2 Chemistry of enzyme catalysis 2.1 Role of hydrogen bonds in catalysis 2.2 Structure of the tetrahedral intermediate 2.3 Site-specific labeling 2.4 An artificial catalytic apparatus

6 An efflorescence of scientific creativity 6.1 A computer-controlled peptide synthesizer 6.2 Instrumentation development at Caltech 6.3 Crystal structure of the HIV-1 protease 6.4 Life at Caltech and Pasadena 6.5 The Scripps Research Institute 6.6 Life in the world of biotech 6.7 University of Chicago

At the conclusion of 2019 I was contacted by the Division on the History of Chemistry of the German Chemical Society and asked to consider writing an autobiography, as part of a series of books on the life and work of eminent scientists. I accepted the invitation. The resulting written account is intended to provide insight into what motivated me to embark on a career of scientific research, the formative influences that shaped the way I approached research, how I came to focus my research on understanding the chemistry of enzyme catalysis, what I achieved in the course of a lifelong pursuit of that knowledge, what factors stimulated my original scientific insights, and how those ideas were received by the scientific community. [...]

8 Afterword 8.1 Acceptance and rejection of ideas 8.2 Creativity in research 8.3 An idiosyncratic approach to science 8.4 Some thoughts on academic science 8.5 A few last words of thanks

HIV-1 protease is indispensable for virus propagation and an important therapeutic target for antiviral inhibitors to treat AIDS. As such inhibitors are transition-state mimics, a detailed understanding of the enzyme mechanism is crucial for the development of better anti-HIV drugs. Here, we used room-temperature joint X-ray/neutron crystallography to directly visualize hydrogen atoms and map hydrogen bonding interactions in a protease complex with peptidomimetic inhibitor KVS-1 containing a reactive nonhydrolyzable ketomethylene isostere, which, upon reacting with the catalytic water molecule, is converted into a tetrahedral intermediate state, KVS-1TI. We unambiguously determined that the resulting tetrahedral intermediate is an oxyanion, rather than the gem-diol, and both catalytic aspartic acid residues are protonated. The oxyanion tetrahedral intermediate appears to be unstable, even though the negative charge on the oxyanion is delocalized through a strong n → π* hyperconjugative interaction into the nearby peptidic carbonyl group of the inhibitor. To better understand the influence of the ketomethylene isostere as a protease inhibitor, we have also examined the protease structure and binding affinity with keto-darunavir (keto-DRV), which similar to KVS-1 includes the ketomethylene isostere. We show that keto-DRV is a significantly less potent protease inhibitor than DRV. These findings shed light on the reaction mechanism of peptide hydrolysis catalyzed by HIV-1 protease and provide valuable insights into further improvements in the design of protease inhibitors.

We report a general approach to engineering multivalent d-proteins with antibody-like activities in vivo. Mirror-image phage display and structure-guided design were utilized to create a d-protein that uses receptor mimicry to antagonize vascular endothelial growth factor A (VEGF-A). Selections against the d-protein form of VEGF-A using phage-displayed libraries of two different domain scaffolds yielded two proteins that bound distinct receptor interaction sites on VEGF-A. X-ray crystal structures of the d-protein/VEGF-A complexes were used to guide affinity maturation and to construct a heterodimeric d-protein VEGF-A antagonist with picomolar activity. The d-protein VEGF-A antagonist prevented vascular leakage in a rabbit eye model of wet age-related macular degeneration and slowed tumor growth in the MC38 syngeneic mouse tumor model with efficacies comparable to those of approved antibody drugs, and in contrast with antibodies, the d-protein was non-immunogenic during treatment and following subcutaneous immunizations.

A racemic protein mixture can be used to form centrosymmetric crystals for structure determination by X-ray diffraction. Both the unnatural d-protein and the corresponding natural l-protein are made by total chemical synthesis based on native chemical ligation-chemoselective condensation of unprotected synthetic peptide segments. Racemic protein crystallography is important for structure determination of the many natural protein molecules that are refractory to crystallization. Racemic mixtures facilitate the crystallization of recalcitrant proteins, and give diffraction-quality crystals. Quasi-racemic crystallization, using a single d-protein molecule, can facilitate the determination of the structures of a series of l-protein analog molecules.

Total chemical synthesis of proteins has been rendered practical by the chemical ligation principle: chemoselective condensation of unprotected peptide segments equipped with unique, mutually reactive functional groups, enabled by formation of a non-native replacement for the peptide bond. Ligation chemistries are briefly described, including native chemical ligation – thioester-mediated, amide-forming reaction at Xaa-Cys sites – and its extensions. Case studies from the author’s own works are used to illustrate the utility and applications of chemical protein synthesis. Selected recent developments in the field are briefly discussed.

ShK toxin is a cysteine-rich 35-residue protein ion-channel ligand isolated from the sea anemone Stichodactyla helianthus. In this work, we studied the effect of inverting the side chain stereochemistry of individual Thr or Ile residues on the properties of the ShK protein. Molecular dynamics simulations were used to calculate the free energy cost of inverting the side-chain stereochemistry of individual Thr or Ile residues. Guided by the computational results, we used chemical protein synthesis to prepare three ShK polypeptide chain analogues, each containing either an allo-Thr or an allo-Ile residue. The three allo-Thr or allo-Ile-containing ShK polypeptides were able to fold into defined protein products, but with different folding propensities. Their relative thermal stabilities were measured and were consistent with the MD simulation data. Structures of the three ShK analogue proteins were determined by quasi-racemic X-ray crystallography and were similar to wild-type ShK. All three ShK analogues retained ion-channel blocking activity.

Ts3 is an alpha scorpion toxin from the venom of the Brazilian scorpion Tityus serrulatus. Ts3 binds to the domain IV voltage sensor of voltage-gated sodium channels (Nav ) and slows down their fast inactivation. The covalent structure of the Ts3 toxin is uncertain, and the structure of the folded protein molecule is unknown. Herein, we report the total chemical synthesis of four candidate Ts3 toxin protein molecules and the results of structure-activity studies that enabled us to establish the covalent structure of biologically active Ts3 toxin. We also report the synthesis of the mirror image form of the Ts3 protein molecule, and the use of racemic protein crystallography to determine the folded (tertiary) structure of biologically active Ts3 toxin by X-ray diffraction.

Isotope-edited two-dimensional Fourier transform infrared spectroscopy (2 D FTIR) can potentially provide a unique probe of protein structure and dynamics. However, general methods for the site-specific incorporation of stable (13) C=(18) O labels into the polypeptide backbone of the protein molecule have not yet been established. Here we describe, as a prototype for the incorporation of specific arrays of isotope labels, the total chemical synthesis-via a key ester insulin intermediate-of 97 % enriched [(1-(13) C=(18) O)Phe(B24) ] human insulin: stable-isotope labeled at a single backbone amide carbonyl. The amino acid sequence as well as the positions of the disulfide bonds and the correctly folded structure were unambiguously confirmed by the X-ray crystal structure of the synthetic protein molecule. In vitro assays of the isotope labeled [(1-(13) C=(18) O)Phe(B24) ] human insulin showed that it had full insulin receptor binding activity. Linear and 2 D IR spectra revealed a distinct red-shifted amide I carbonyl band peak at 1595 cm(-1) resulting from the (1-(13) C=(18) O)Phe(B24) backbone label. This work illustrates the utility of chemical synthesis to enable the application of advanced physical methods for the elucidation of the molecular basis of protein function.

Under suitable conditions, trifluoromethanesulfonic acid performs comparably to hydrogen fluoride for the on-resin global deprotection of peptides prepared by Boc chemistry solid phase peptide synthesis (SPPS). Obviation of hydrogen fluoride in Boc chemistry SPPS enables the straightforward synthesis of peptide-αthioesters for use in native chemical ligation.

With the goal of investigating electronic aspects of the catalysis of peptide bond hydrolysis, an analogue of HIV-1 protease was designed in which a non-peptide hydroxy-isoquinolinone artificial catalytic apparatus replaced the conserved Asp25–Thr26–Gly27 sequence in each 99-residue polypeptide chain of the homodimeric enzyme molecule. The enzyme analogue was prepared by total chemical synthesis and had detectable catalytic activity on known HIV-1 protease peptide substrates. Compared with uncatalyzed hydrolysis, the analogue enzyme increased the rate of peptide bond hydrolysis by ∼108-fold. Extensions of this unique approach to the study of enzyme catalysis in HIV-1 protease are discussed.

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.

The Dark Energy Spectroscopic Instrument (DESI) is under construction to measure the expansion history of the Universe using the Baryon Acoustic Oscillation technique. The spectra of 35 million galaxies and quasars over 14000 square degrees will be measured during the life of the experiment. A new prime focus corrector for the KPNO Mayall telescope will deliver light to 5000 fiber optic positioners. The fibers in turn feed ten broad-band spectrographs. We describe the DESI corrector optics, a series of six fused silica and borosilicate lenses. The lens diameters range from 0.8 to 1.1 meters, and their weights 84 to 237 kg. Most lens surfaces are spherical, and two are challenging 10th-order polynomial aspheres. The lenses have been successfully polished and treated with an antireflection coating at multiple subcontractors, and are now being integrated into the DESI corrector barrel assembly at University College London. We describe the final performance of the lenses in terms of their various parameters, including surface figure, homogeneity, and others, and compare their final performance against the demanding DESI corrector requirements. Also we describe the reoptimization of the lens spacing in their corrector barrel after their final measurements are known. Finally we assess the performance of the corrector as a whole, compared to early budgeted estimates.

The Dark Energy Spectroscopic Instrument (DESI) is under construction to measure the expansion history of the Universe using the Baryon Acoustic Oscillation technique. The spectra of 35 million galaxies and quasars over 14000 square degrees will be measured during the life of the experiment. A new prime focus corrector for the KPNO Mayall telescope will deliver light to 5000 fiber optic positioners. The fibers in turn feed ten broad-band spectrographs. We present an overview of the instrumentation, the main technical requirements and challenges, and the current status of the project. © 2018 SPIE., This research is supported by the Director, Office of Science, Office of High Energy Physics of the U.S. Department of Energy under Contract No. DEAC0205CH1123, and by the National Energy Research Scientific Computing Center, a DOE Office of Science User Facility under the same contract; additional support for DESI is provided by the U.S. National Science Foundation, Division of Astronomical Sciences under Contract No. AST-0950945 to the National Optical Astronomy Observatory; the Science and Technologies Facilities Council of the United Kingdom; the Gordon and Betty Moore Foundation; the Heising-Simons Foundation; the National Council of Science and Technology of Mexico, and by the DESI Member Institutions. The authors are honored to be permitted to conduct astronomical research on Iolkam Du'ag (Kitt Peak), a mountain with particular significance to the Tohono O'odham Nation.

ShK toxin from the sea anemone Stichodactyla helianthus is a 35-residue protein that binds to the Kv1.3 ion channel with high affinity. Recently we determined the X-ray structure of ShK toxin by racemic crystallography, in the course of which we discovered that d-ShK has a near-background IC50 value ∼50,000 times lower than that of the l-ShK toxin. This lack of activity was at odds with previously reported results for an ShK diastereomer designated d-allo-ShK, for which significant biological activity had been observed in a similar receptor-blocking assay. As reported, d-allo-ShK was made up of d-amino acids, but with retention of the natural stereochemistry of the chiral side chains of the Ile and Thr residues, i.e. containing d-allo-Ile and d-allo-Thr along with d-amino acids and glycine. To understand its apparent biological activity, we set out to chemically synthesize d-allo-ShK and determine its X-ray structure by racemic crystallography. Using validated allo-Thr and allo-Ile, both l-allo-ShK and d-allo-ShK polypeptide chains were prepared by total chemical synthesis. Neither the l-allo-ShK nor the d-allo-ShK polypeptides folded, whereas both l-ShK and d-ShK folded smoothly under the same conditions. Re-examination of NMR spectra of the previously reported d-allo-ShK protein revealed that diagnostic Thr and Ile signals were the same as for authentic d-ShK. On the basis of these results, we conclude that the previously reported d-allo-ShK was in fact d-ShK, the true enantiomer of natural l-ShK toxin, and that the apparent biological activity may have arisen from inadvertent contamination with trace amounts of l-ShK toxin.

The burial of hydrophobic side chains in a protein core generally is thought to be the major ingredient for stable, cooperative folding. Here, we show that, for the snow flea antifreeze protein (sfAFP), stability and cooperativity can occur without a hydrophobic core, and without α-helices or β-sheets. sfAFP has low sequence complexity with 46% glycine and an interior filled only with backbone H-bonds between six polyproline 2 (PP2) helices. However, the protein folds in a kinetically two-state manner and is moderately stable at room temperature. We believe that a major part of the stability arises from the unusual match between residue-level PP2 dihedral angle bias in the unfolded state and PP2 helical structure in the native state. Additional stabilizing factors that compensate for the dearth of hydrophobic burial include shorter and stronger H-bonds, and increased entropy in the folded state. These results extend our understanding of the origins of cooperativity and stability in protein folding, including the balance between solvent and polypeptide chain entropies.

Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: β-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.

Misfolding of proinsulin variants in the pancreatic β-cell, a monogenic cause of permanent neonatal-onset diabetes mellitus, provides a model for a disease of protein toxicity. A hot spot for such clinical mutations is found at position B8, conserved as glycine within the vertebrate insulin superfamily. We set out to investigate the molecular basis of the aberrant properties of a proinsulin clinical mutant in which residue Gly(B8) is replaced by Ser(B8). Modular total chemical synthesis was used to prepare the wild-type [Gly(B8)]proinsulin molecule and three analogs: [D-Ala(B8)]proinsulin, [L-Ala(B8)]proinsulin, and the clinical mutant [L-Ser(B8)]proinsulin. The protein diastereomer [D-Ala(B8)]proinsulin produced higher folding yields at all pH values compared with the wild-type proinsulin and the other two analogs, but showed only very weak binding to the insulin receptor. The clinical mutant [L-Ser(B8)]proinsulin impaired folding at pH 7.5 even in the presence of protein-disulfide isomerase. Surprisingly, although [L-Ser(B8)]proinsulin did not fold well under the physiological conditions investigated, once folded the [L-Ser(B8)]proinsulin protein molecule bound to the insulin receptor more effectively than wild-type proinsulin. Such paradoxical gain of function (not pertinent in vivo due to impaired secretion of the mutant insulin) presumably reflects induced fit in the native mechanism of hormone-receptor engagement. This work provides insight into the molecular mechanism of a clinical mutation in the insulin gene associated with diabetes mellitus. These results dramatically illustrate the power of total protein synthesis, as enabled by modern chemical ligation methods, for the investigation of protein folding and misfolding.

Ts1 toxin is a protein found in the venom of the Brazilian scorpion Tityus serrulatus. Ts1 binds to the domain II voltage sensor in the voltage-gated sodium channel Nav and modifies its voltage dependence. In the work reported here, we established an efficient total chemical synthesis of the Ts1 protein using modern chemical ligation methods and demonstrated that it was fully active in modifying the voltage dependence of the rat skeletal muscle voltage-gated sodium channel rNav1.4 expressed in oocytes. Total synthesis combined with click chemistry was used to label the Ts1 protein molecule with the fluorescent dyes Alexa-Fluor 488 and Bodipy. Dye-labeled Ts1 proteins retained their optical properties and bound to and modified the voltage dependence of the sodium channel Nav. Because of the highly specific binding of Ts1 toxin to Nav, successful chemical synthesis and labeling of Ts1 toxin provides an important tool for biophysical studies, histochemical studies, and opto-pharmacological studies of the Nav protein.

The enzyme sortase A is a ligase which catalyzes transpeptidation reactions.1, 2 Surface proteins, including virulence factors, that have a C terminal recognition sequence are attached to Gly5 on the peptidoglycan of bacterial cell walls by sortase A.1 The enzyme is an important anti-virulence and anti-infective drug target for resistant strains of Gram-positive bacteria.2 In addition, because sortase A enables the splicing of polypeptide chains, the transpeptidation reaction catalyzed by sortase A is a potentially valuable tool for protein science.3 Here we describe the total chemical synthesis of enzymatically active sortase A. The target 148 residue polypeptide chain of sortase AΔN59 was synthesized by the convergent chemical ligation of four unprotected synthetic peptide segments. The folded protein molecule was isolated by size-exclusion chromatography and had full enzymatic activity in a transpeptidation assay. Total synthesis of sortase A will enable more sophisticated engineering of this important enzyme molecule.

CCL1 is a naturally glycosylated chemokine protein that is secreted by activated T-cells and acts as a chemoattractant for monocytes. Originally, CCL1 was identified as a 73 amino acid protein having one N-glycosylation site, and a variant 74 residue non-glycosylated form, Ser-CCL1, has also been described. There are no systematic studies of the effect of glycosylation on the biological activities of either CCL1 or Ser-CCL1. Here we report the total chemical syntheses of both N-glycosylated and non-glycosylated forms of (Ser-)CCL1, by convergent native chemical ligation. We used an N-glycan isolated from hen egg yolk together with the Nbz linker for Fmoc chemistry solid phase synthesis of the glycopeptide-(α) thioester building block. Chemotaxis assays of these glycoproteins and the corresponding non-glycosylated proteins were carried out. The results were correlated with the chemical structures of the (glyco)protein molecules. To the best of our knowledge, these are the first investigations of the effect of glycosylation on the chemotactic activity of the chemokine (Ser-)CCL1 using homogeneous N-glycosylated protein molecules of defined covalent structure.

Peptide chemistry plays a key role in the synthesis and study of protein molecules and their functions. Modern ligation methods enable the total synthesis of enzymes and the systematic dissection of the chemical basis of enzyme catalysis. Predicted developments in peptide science are described. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

Erythropoietin, commonly known as EPO, is a glycoprotein hormone that stimulates the production of red blood cells. Recombinant EPO has been described as "arguably the most successful drug spawned by the revolution in recombinant DNA technology". Recently, the EPO glycoprotein molecule has re-emerged as a major target of synthetic organic chemistry. In this article I will give an account of an important body of earlier work on the chemical synthesis of a designed EPO analogue that had full biological activity and improved pharmacokinetic properties. The design and synthesis of this "synthetic erythropoiesis protein" was ahead of its time, but has gained new relevance in recent months. Here I will document the story of one of the major accomplishments of synthetic chemistry in a more complete way than is possible in the primary literature, and put the work in its contemporaneous context.

Erythropoietin, bekannt als EPO, ist ein hormonelles Glycoprotein, das die Produktion von roten Blutkorperchen stimuliert. Rekombinantes EPO wurde als “die wohl erfolgreichste Droge, die durch die Revolution der rekombinanten DNA-Technologie hervorgebracht wurde”, beschrieben. In jungerer Zeit hat das EPO-Glycoproteinmolekul eine Renaissance als bedeutendes Ziel der organischen Synthesechemie erfahren. In diesem Kurzaufsatz mochte ich auf fruhere wichtige Arbeiten zur chemischen Synthese eines masgeschneiderten EPO-Analogons eingehen, das volle biologische Aktivitat und verbesserte pharmakokinetische Eigenschaften aufwies. Das Design und die Synthese dieses “synthetischen Erythropoeseproteins” waren damals ihrer Zeit voraus und haben in den letzten Monaten wieder neue Bedeutung gewonnen. Ich mochte hier die Geschichte einer der wesentlichen Leistungen der Synthesechemie etwas detaillierter dokumentieren, als es in der Primarliteratur moglich ist, wobei auch auf den heutigen Kontext eingegangen werden soll.

The Photoactive Yellow Protein (PYP) is a structural prototype for the PAS superfamily of proteins, which includes hundreds of receptor and regulatory proteins from all three kingdoms of life. PYP itself is a small globular protein that undergoes a photocycle involving a series of conformational changes in response to light excitation of its p-coumaric acid chromophore, making it an excellent model system to study the molecular basis of signaling in the PAS super family. To enable novel chemical approaches to elucidating the structural changes that accompany signaling in PYP, we have chemically synthesized the 125 amino acid residue protein molecule using a combination of Boc chemistry solid phase peptide synthesis and native chemical ligation. Synthetic PYP exhibits the wildtype photocycle, as determined in photobleaching studies. Planned future studies include incorporation of site-specific isotopic labels into specific secondary structural elements to determine which structural elements are involved in signaling state formation using difference FTIR spectroscopy.

Efficient total synthesis of insulin is important to enable the application of medicinal chemistry to the optimization of the properties of this important protein molecule. Recently we described "ester insulin"--a novel form of insulin in which the function of the 35 residue C-peptide of proinsulin is replaced by a single covalent bond--as a key intermediate for the efficient total synthesis of insulin. Here we describe a fully convergent synthetic route to the ester insulin molecule from three unprotected peptide segments of approximately equal size. The synthetic ester insulin polypeptide chain folded much more rapidly than proinsulin, and at physiological pH. Both the D-protein and L-protein enantiomers of monomeric DKP ester insulin (i.e., [Asp(B10), Lys(B28), Pro(B29)]ester insulin) were prepared by total chemical synthesis. The atomic structure of the synthetic ester insulin molecule was determined by racemic protein X-ray crystallography to a resolution of 1.6 Å. Diffraction quality crystals were readily obtained from the racemic mixture of {D-DKP ester insulin + L-DKP ester insulin}, whereas crystals were not obtained from the L-ester insulin alone even after extensive trials. Both the D-protein and L-protein enantiomers of monomeric DKP ester insulin were assayed for receptor binding and in diabetic rats, before and after conversion by saponification to the corresponding DKP insulin enantiomers. L-DKP ester insulin bound weakly to the insulin receptor, while synthetic L-DKP insulin derived from the L-DKP ester insulin intermediate was fully active in binding to the insulin receptor. The D- and L-DKP ester insulins and D-DKP insulin were inactive in lowering blood glucose in diabetic rats, while synthetic L-DKP insulin was fully active in this biological assay. The structural basis of the lack of biological activity of ester insulin is discussed.

We have systematically explored three approaches based on 9-fluorenylmethoxycarbonyl (Fmoc) chemistry solid phase peptide synthesis (SPPS) for the total chemical synthesis of the key depsipeptide intermediate for the efficient total chemical synthesis of insulin. The approaches used were: stepwise Fmoc chemistry SPPS; the "hybrid method", in which maximally protected peptide segments made by Fmoc chemistry SPPS are condensed in solution; and, native chemical ligation using peptide-thioester segments generated by Fmoc chemistry SPPS. A key building block in all three approaches was a Glu[O-β-(Thr)] ester-linked dipeptide equipped with a set of orthogonal protecting groups compatible with Fmoc chemistry SPPS. The most effective method for the preparation of the 51 residue ester-linked polypeptide chain of ester insulin was the use of unprotected peptide-thioester segments, prepared from peptide-hydrazides synthesized by Fmoc chemistry SPPS, and condensed by native chemical ligation. High-resolution X-ray crystallography confirmed the disulfide pairings and three-dimensional structure of synthetic insulin lispro prepared from ester insulin lispro by this route. Further optimization of these pilot studies could yield an efficient total chemical synthesis of insulin lispro (Humalog) based on peptide synthesis by Fmoc chemistry SPPS.

Under suitable conditions, trifluoromethanesulfonic acid performs comparably to hydrogen fluoride for the on-resin global deprotection of peptides prepared by Boc chemistry solid phase peptide synthesis (SPPS). Obviation of hydrogen fluoride in Boc chemistry SPPS enables the straightforward synthesis of peptide

In this paper, we have used total chemical synthesis of RNase A analogues in order to probe the molecular basis of enzyme catalysis. Our goal was to obligately fill the adenine-binding pocket on the enzyme molecule, and to thus pre-orient the imidazole side chain of His119 in its catalytically productive orientation. Two designed analogues of the RNase A protein molecule that contained an adenine moiety covalently bound to distinct amino acid side chains adjacent to the adenine binding pocket were prepared. A crystal structure of one analogue was determined at 2.3 A resolution. Kinetic data for RNA transphosporylation and 2',3' cyclic mononucleotide hydrolysis were acquired for the adenine-containing RNase A analogue proteins. As anticipated, the presence of a covalently attached adenine on the enzyme molecule decreased the rate of transphosphorylation and increased the rate of hydrolysis, although the magnitude of the effects was small. This work illustrates the use of total protein synthesis to investigate the chemistry of enzyme catalysis in ways not possible through traditional biochemistry or molecular biology.

Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are involved in a large variety of physiological processes. Regulatory β-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or β1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) β1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the β1-subunit within the α/β1-subunit complex.

Polypeptides composed entirely of d-amino acids and the achiral amino acid glycine (d-proteins) inherently have in vivo properties that are proposed to be near-optimal for a large molecule therapeutic agent. Specifically, d-proteins are resistant to degradation by proteases and are anticipated to be nonimmunogenic. Furthermore, d-proteins are manufactured chemically and can be engineered to have other desirable properties, such as improved stability, affinity, and pharmacokinetics. Thus, a well-designed d-protein therapeutic would likely have significant advantages over l-protein drugs. Toward the goal of developing d-protein therapeutics, we previously generated RFX001.D, a d-protein antagonist of natural vascular endothelial growth factor A (VEGF-A) that inhibited binding to its receptor. However, RFX001.D is unstable at physiological temperatures (Tm = 33 °C). Here, we describe RFX037.D, a variant of RFX001.D with extreme thermal stability (Tm95 °C), high affinity for VEGF-A (Kd = 6 nM), and improved receptor blocking. Comparison of the two enantiomeric forms of RFX037 revealed that the d-protein is more stable in mouse, monkey, and human plasma and has a longer half-life in vivo in mice. Significantly, RFX037.D was nonimmunogenic in mice, whereas the l-enantiomer generated a strong immune response. These results confirm the potential utility of synthetic d-proteins as alternatives to therapeutic antibodies.

‘Chemical ligation’ – the regioselective and chemoselective covalent condensation of unprotected peptide segments – has enabled the synthesis of polypeptide chains of more than 200 amino acids. An efficient total chemical synthesis of the insulin molecule has been devised on the basis of a key ester-linked intermediate that is chemically converted to fully active human insulin. Enzyme molecules of defined covalent structure and with full enzymatic activity have been prepared and characterized by high-resolution X-ray crystallography. A ‘glycoprotein mimetic’ of defined chemical structure and with a mass of 50,825 Da, has been prepared and shown to have full biological activity and improved pharmacokinetic properties. d-Protein molecules that are the mirror images of proteins found in the natural world have been prepared by total chemical synthesis. Racemic protein mixtures, consisting of the d-enantiomers and l-enantiomers of a protein molecule, form highly ordered centrosymmetric crystals with great ease; this has enabled the determination of the crystal structures of recalcitrant protein molecules. A protein with a novel linear-loop covalent topology of the peptide chain has been designed and synthesized and its structure determined by facile crystallization as the quasi-racemate with the d-form of the native protein molecule. We have developed an optimized total chemical synthesis of biologically active vascular endothelial growth factor-A; total synthesis of the mirror-image protein will be used to systematically develop d-protein antagonists of this important growth factor. The total chemical synthesis of proteins is now a practical reality and enables access to a new world of protein molecules. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

Original synthetic and structure determination methods were used to make a protein molecule with an unprecedented linear-loop polypeptide chain topology, and to characterize its X-ray structure.

Erythropoietin (EPO) is a glycoprotein hormone that plays important roles in regulating the production of red blood cells (erythrocytes).[1] Since human EPO was first isolated and purified from urine in 1977,[2] the structure and physiological properties of EPO have been thoroughly studied.[3] Mature human EPO found in nature consists of a polypeptide chain of 165 amino acids with four covalently attached oligosaccharides,[4] one of which is an O-linked oligosaccharide at Ser126, and the other three are N-linked oligosaccharides at residues Asn24, 38, 83.[ 5 ] EPO is used as a therapeutic agent to treat anemia caused by chronic kidney disease.[6] Commercial EPO is prepared using recombinant DNA technology. The carbohydrate moieties of both native and recombinant EPO[7] are heterogenerous and composed of multiple glycans with different lengths and composition at each glycosylation site.[5a,8] This heterogeneity makes it difficult to evaluate the effects of carbohydrate on EPO’s pharmacokinetic properties and, more importantly, complicates the understanding of mechanism of EPO’s action at the molecular level. For these reasons, it is important to develop an alternative strategy to prepare homogeneous EPO.

In the image: The design and synthesis of peptidomimetics that maintain the configuration of the triad Asp-Thr-Gly found in the catalytic site of the HIV-1 protease (see scheme) are described. By using regioselective nitration and reductive lactamisation, the bicyclic mimetics were constructed in a simple fashion and further equipped with appropriate protecting groups suitable for application in solid-phase peptide synthesis.

Understanding the chemistry of enzyme catalysis has been an important objective of chemical research since the time of Emil Fischer. The goal is a detailed understanding of the molecular basis of the chemistry occurring in the enzyme–substrate complex. The physical organic chemistry of enzyme catalysis is potentially more accessible than the study of the corresponding reaction(s) in solution because, in principle, in the enzyme–substrate complex every aspect of the reaction environment (proximity, angles of attack, electronics of reactants/catalytic functionalities, polarity/dielectric constant of the reaction medium) can be controlled. Realizing such control in experimental practice will require the application of new methods. Total chemical synthesis has an important contribution to make, in that it can provide the researcher with atom-by-atom control of the structure (electronic, covalent, and geometric) of both the substrate and the