Modern Peptide and Protein Chemistry: Reaching New Heights
- Christian P. R. Hackenberger*Christian P. R. Hackenberger*Email: [email protected]Leibniz-Institut fur Molekulare Pharmakologie, Robert-Roessle-Strasse 10, Berlin 13125, GermanyScripps Research Institute, Chemistry Department, 10550 North Torrey Pines Road, BCC123, La Jolla, California 92037, United StatesTsinghua University, Department of Chemistry, Beijing 100084, ChinaOsaka University, Institute for Protein Research, 3-2 Yamadaoka, Osaka 565-0871, JapanMore by Christian P. R. Hackenberger
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- Philip E. Dawson*Philip E. Dawson*Email: [email protected]Leibniz-Institut fur Molekulare Pharmakologie, Robert-Roessle-Strasse 10, Berlin 13125, GermanyScripps Research Institute, Chemistry Department, 10550 North Torrey Pines Road, BCC123, La Jolla, California 92037, United StatesTsinghua University, Department of Chemistry, Beijing 100084, ChinaOsaka University, Institute for Protein Research, 3-2 Yamadaoka, Osaka 565-0871, JapanMore by Philip E. Dawson
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- Yong-Xiang Chen*Yong-Xiang Chen*Email: [email protected]Leibniz-Institut fur Molekulare Pharmakologie, Robert-Roessle-Strasse 10, Berlin 13125, GermanyScripps Research Institute, Chemistry Department, 10550 North Torrey Pines Road, BCC123, La Jolla, California 92037, United StatesTsinghua University, Department of Chemistry, Beijing 100084, ChinaOsaka University, Institute for Protein Research, 3-2 Yamadaoka, Osaka 565-0871, JapanMore by Yong-Xiang Chen
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- Hironobu Hojo*Hironobu Hojo*Email: [email protected]Leibniz-Institut fur Molekulare Pharmakologie, Robert-Roessle-Strasse 10, Berlin 13125, GermanyScripps Research Institute, Chemistry Department, 10550 North Torrey Pines Road, BCC123, La Jolla, California 92037, United StatesTsinghua University, Department of Chemistry, Beijing 100084, ChinaOsaka University, Institute for Protein Research, 3-2 Yamadaoka, Osaka 565-0871, JapanMore by Hironobu Hojo
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SPECIAL ISSUE
This article is part of the
Peptides, proteins, and their derivatives have attracted growing interest from both academics and the pharmaceutical industry over the past decades, creating a renaissance in the field of peptide/protein therapeutics. This resurgence not only benefits from but also indeed has stimulated the rapid development of new protein chemistry to access and study these biomolecules. Thus, we are honored to serve as guest editors of this Special Issue of The Journal of Organic Chemistry on Modern Peptide and Protein Chemistry, which includes a variety of cutting-edge contributions from leading experts in the field. This high-quality work will help readers to experience the extensive and in-depth exploration of peptide and protein chemistry and synthesis with an emphasis on the most current progress and functional applications.
Milestones in Peptide/Protein Chemistry
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The synthesis of peptides and proteins has been gradually advanced during the past century along with the prosperous development of organic chemistry. Several milestone accomplishments originate from the first description and synthesis of a peptide bond in the glycyl-glycine dipeptide by Emil Fischer, (1) who is considered the founder of peptide synthesis. Max Bergmann, one of Fischer’s students, and his doctoral student Leonidas Zervas, introduced the carbobenzoxy (Cbz, Z) group as the first reversible amine protection group in organic synthesis. (2) Early peptide syntheses were performed in solution and were successfully applied for the first assembly of a polypeptide hormone, oxytocin, by Vincent du Vigneaud, (3) who was awarded the Nobel Prize in Chemistry in 1955.
A leap in practical preparation of more complex peptides was the advent of solid-phase peptide synthesis (SPPS) strategies, pioneered by Bruce Merrifield, (4−7) who also received a Nobel Prize in Chemistry, in 1984, for “his development of methodology for chemical synthesis on a solid matrix”. The refinement of SPPS strategies has enabled the routine and efficient synthesis of numerous peptides, small proteins, and their synthetic analogues. The subsequent development of chemoselective ligation, in particular native chemical ligation (NCL) by Stephen Kent in 1994 (8) has opened the field of proteins to organic synthesis by facilitating the assembly of multiple unprotected peptide fragments, obtained from SPPS. These reactions, together with the development of semisynthetic strategies, (9) have pushed the size limitation of synthetic proteins to a few hundreds of amino acids.
Besides the approaches to synthesize native peptides and proteins, various synthetic methodologies for accessing derivatives with diversified structures have also been developed, enabling a host of high-precision hypotheses about structure–function relationships to be explored. These include backbone and side-chain modifications, in which the latter can be introduced by biorthogonal modification reactions, including for example cycloaddition (10,11) or Staudinger reactions. (12) Importantly, the site-specific introduction of posttranslational modifications into peptides and proteins has been critical for the understanding of their essential role in biological processes. These developments have laid a solid foundation for the current renaissance of peptide and protein therapeutics and simultaneously pushed basic biological and chemical research to new heights.
Current progresses in this Special Issue
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This Special Issue of The Journal of Organic Chemistry on Modern Peptide and Protein Chemistry contains 48 research articles from 16 countries. These research articles cover various aspects of modern synthesis and modification of peptides and proteins.
For example, novel peptide ligation methods are featured in this issue. Aspartic acid forming α-ketoacid–hydroxylamine (KAHA) ligations were realized by using (S)-4,4-difluoro-5-oxaproline. Diselenide-selenoester ligation (DSL) was successfully expanded to phenylalanine and leucine at the ligation site via utility of β-selenophenylalanine and β-selenoleucine, followed by application in constructing a glycopeptide and protein UL22A. The utilization of N-terminal Cys/Sec-containing peptides for NCL was facilitated by discovering copper-mediated deprotection of thiazolidine and selenazolidine derivatives. Chemical synthesis of functional proteins, such as homogeneous granulocyte-macrophage colony-stimulating factor and human chemokine CXCL14 containing methionine sulfoxide, were achieved by using Se-auxiliary-mediated or S-mediated NCL. Alternative chemoselective reactions, such as metal-mediated Suzuki–Miyaura cross-coupling and azide-yne cycloaddition are refined and applied to peptides. Finally, enzyme-mediated peptide synthesis using an immobilized peptide ligase is demonstrated.
Furthermore, new synthetic approaches to construct native peptides and peptide mimetics were described in many contributions to this issue. For example, epimerization-free preparation of C-terminal Cys peptide acid by Fmoc SPPS was realized by using pseudoproline-type protecting group. A new two-photon cleavable thiol protection group, based on a methoxy-substituted nitrodibenzofuran structure, was developed for SPPS. Along those lines, synthetic schemes for peptide and amino acid mimetics are reported here, such as thioamide peptides, indolizidinone dipeptide mimetics and β2-homologous amino acids.
Another challenge in the field is the requirement to improve the stability, cell permeability, and pharmacologic potency of peptides and proteins. Site-specific modification of these biomolecules by additional chemical moieties provides a general and efficient solution. For example, lipidation at the N-terminus of peptides increases the cytotoxicity of lipovelutibols. PEGylation near a patch of nonpolar surface residues increases the conformational stability of the WW domain. A trimethylammonium cation modification at the C-terminus of Vancomycin improves the pharmacological properties of this antimicrobial agent. A few nonproteinogenic residues, like dehydroamino acids, chiral alkenyl cyclopropane amino acids and aza-glycine, were incorporated within peptides. Meanwhile, late-stage modification provides a simple way to prepare site-specifically modified polypeptides, for instance, by developing sulfonium triggered thiol–yne reaction for cysteine modification, or by using sophisticated disulfide-bond formation reaction and cyanobenzothiazole-mediated cysteine dual labeling. In addition, conformationally strained cyclic peptides are the focus of several publications due to their great potential in therapeutic development. Different peptide cyclization strategies, for example, by using NCL followed copper-mediated deprotection of selenazolidine and employing optimized ring closing metathesis reaction with desallyl side products suppressed, are also reported.
A few publications in this Special Issue are relevant to construct biologically active peptide/protein derivatives for interfering with biomolecule recognition, reflecting the advantages of their specificity and efficacy at their targets. For example, a MUC1 glycopeptide library for positional scanning was constructed and applied to reveal the importance of PDTR epitope glycosylation for lectin binding. A peptide-based multiepitopic vaccine platform was built via click reactions. A cell-permeable cyclic peptidyl inhibitor is presented that modulates the Keap1-Nrf2 interaction.
At last, we would like to highlight an important publication, which concerns the safety of researchers working in the field of peptide synthesis. This study reports the anaphylaxis induced by peptide coupling agents including HATU, HBTU, and HCTU and reminds everyone to employ proper protection against potential health hazard of these reagents.
Outlook
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Highly efficient synthetic methodologies to access peptides and proteins and their derivatives with diversified structures have accelerated their translational biomedical research and development. Although a wealth of exciting advances in various aspects of peptide and protein chemistry have been reported in this Special Issue and elsewhere, many unsolved problems remain, and complicated challenges still need to be met. We are expecting even more innovative contributions to this field, which can benefit from multidisciplinary thinking and technology that is evolving at high speed. For example, it is still demanding to push the size and complexity of synthetic proteins to greater heights with the help of new chemical strategies and even enzyme-catalyzed reactions. Developments in biosynthesis will help to realize the construction of some unique peptide structures inside the biological systems. Data-mining and machine-learning technologies might facilitate the structural design as well as the synthetic planning of bioactive proteins and their derivatives. As the guest editors of this Special Issue to celebrate the progression of Modern Peptide and Protein Chemistry, we are looking forward to witnessing and participating in the inevitably prosperous future of this field.
Figure 1
Figure 1. The guest editors of this issue (from left to right Christian Hackenberger, Philip Dawson, Yong-Xiang Chen and Hironobu Hojo) at the eighth Chemical Protein Synthesis Meeting in Berlin.
Author Information
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- Christian P. R. Hackenberger, Leibniz-Institut fur Molekulare Pharmakologie, Robert-Roessle-Strasse 10, Berlin 13125, Germany; Scripps Research Institute, Chemistry Department, 10550 North Torrey Pines Road, BCC123, La Jolla, California 92037, United States; Tsinghua University, Department of Chemistry, Beijing 100084, China; Osaka University, Institute for Protein Research, 3-2 Yamadaoka, Osaka 565-0871, Japan,
http://orcid.org/0000-0001-7457-4742,
- Philip E. Dawson, Leibniz-Institut fur Molekulare Pharmakologie, Robert-Roessle-Strasse 10, Berlin 13125, Germany; Scripps Research Institute, Chemistry Department, 10550 North Torrey Pines Road, BCC123, La Jolla, California 92037, United States; Tsinghua University, Department of Chemistry, Beijing 100084, China; Osaka University, Institute for Protein Research, 3-2 Yamadaoka, Osaka 565-0871, Japan,http://orcid.org/0000-0002-2538-603X,
- Yong-Xiang Chen, Leibniz-Institut fur Molekulare Pharmakologie, Robert-Roessle-Strasse 10, Berlin 13125, Germany; Scripps Research Institute, Chemistry Department, 10550 North Torrey Pines Road, BCC123, La Jolla, California 92037, United States; Tsinghua University, Department of Chemistry, Beijing 100084, China; Osaka University, Institute for Protein Research, 3-2 Yamadaoka, Osaka 565-0871, Japan,http://orcid.org/0000-0003-3518-0139,
- Hironobu Hojo, Leibniz-Institut fur Molekulare Pharmakologie, Robert-Roessle-Strasse 10, Berlin 13125, Germany; Scripps Research Institute, Chemistry Department, 10550 North Torrey Pines Road, BCC123, La Jolla, California 92037, United States; Tsinghua University, Department of Chemistry, Beijing 100084, China; Osaka University, Institute for Protein Research, 3-2 Yamadaoka, Osaka 565-0871, Japan,http://orcid.org/0000-0003-0916-1233,
Biographies
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Christian P. R. Hackenberger
Christian P. R. Hackenberger completed his graduate studies in chemistry at the University of Freiburg and the University of Wisconsin—Madison and his doctoral studies in 2003 at the RWTH Aachen. After a postdoctoral position at MIT, he started his own group at the Freie Universität Berlin in 2005. In 2012, he was appointed Leibniz-Humboldt Professor for Chemical Biology at the Leibniz-Research Institute for Molecular Pharmacology and the Humboldt Universität zu Berlin. His group works on the development of new chemoselective and bioorthogonal reactions, the identification and analysis of novel PTMs, the engineering of protein-based pharmaceuticals, and novel approaches to functional protein synthesis and delivery, in particular for the labeling and modification of different antibody formats. He is cofounder of the start-up “Tubulis Technologies”, which ventures into engineering better tolerable cancer drugs based on protein– and antibody–drug conjugates.
Philip E. Dawson
Phil Dawson is a Professor in the Department of Chemistry, Scripps Research in La Jolla, CA, and Dean of the Skaggs Graduate School of Chemical and Biological Sciences. He received an A.B. (1992) in Chemistry from Washington University and Ph.D. (1996) from Scripps Research under the guidance of Steve Kent. After pursuing postdoctoral work at Caltech with Harry Gray and Tom Meade, he returned to Scripps as an Assistant Professor. He has served as President of the American Peptide Society, the Board of Directors for FASEB, and cochaired the 22nd American Peptide Symposium and the GRC on Biology and Chemistry of Peptides. He has published over 180 papers and has been honored with an Alfred P. Sloan Foundation fellowship, the Vincent du Vigneaud Award, the Max Bergmann Kreis Gold Medal, the Zervas Award and the RSC MedImmune Protein and Peptide Science Award. Professor Dawson is a pioneer of chemoselective ligation methods for macromolecule synthesis and modification and has applied these tools broadly to better understand biological systems.
Yong-Xiang Chen
Yong-Xiang Chen graduated from Hunan University with a B.S. degree in 2002. She then received a Ph.D. degree under the guidance of Prof. Yan-Mei Li from Tsinghua University in 2007 after which she had worked in the group of Prof. Herbert Waldmann at Max-Planck Institution of Molecular Physiology in Dortmund as an Alexander von Humboldt postdoctoral fellow. Since 2011, she has joined Tsinghua University as an associate professor in the Department of Chemistry. Her current research interests include synthesis of peptides and proteins with posttranslational modifications; application of them in elucidating or interfering the molecular processes of related biological events.
Hironobu Hojo
Hironobu Hojo obtained his Ph.D. in organic chemistry from Osaka University in 1994 under the guidance of Prof. Saburo Aimoto on the development of the chemical method for protein synthesis. He then moved to the Osaka City University as lecturer and worked on the development of novel biomaterials. In 1998, he moved to Tokai University as an associate professor. There, he started to develop a facile method for glycoprotein synthesis by collaboration with Prof. Yoshiaki Nakahara. He was promoted to professor of Tokai University in 2007. He moved to the present position, Professor of the Institute for Protein Research, Osaka University, in 2013 and is developing a chemical approach toward the understanding of protein and glycoprotein function.
References
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This article references 12 other publications.
- 1Fischer, E.; Fourneau, E. Ueber einige Derivate des Glykocolls. Ber. Dtsch. Chem. Ges. 1901, 34, 2868– 2877, DOI: 10.1002/cber.190103402249Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaD28XitFyitbc%253D&md5=47ac4c6496102fee68b1f555a323213eOver some derivatives of the glycine. [machine translation]Fischer, Emil; Fourneau, ErnestBerichte der Deutschen Chemischen Gesellschaft (1901), 34 (), 2868-77CODEN: BDCGAS; ISSN:0365-9496.[Machine Translation of Descriptors]. Experiments to combine the amino acids developing from the protein materials by hydrolysis by anhydride formation again to larger complexes were implemented by SCHAAL, GRIMAUX, SCHIFF, SCHUeTZENBERGER, LILIEINFELD, BALBIANO among others, however always led to amorphous, with difficulty characterizable products. As known, α-amino acids, as well as their ester, (E. FISCHER, Ber. Dtsch. Chem. Ges. 34. 435; C. 1901. I. 678) can be transported into dimolecular anhydrides, their simplest representative the glycine anhydride (2.5-Diacipiperazin) of CURTIUS and GOEBEL (J. pr. Chem. [2] 37. 173) is. After the observations of the authors this compound splits up as follows when short boiling with concentrated hydrochloric acid: (see original document for graphics) the new substance, which is the simplest anhydrous product of the glycine (glycine), became glycine mentioned under use of the described glycyl for the remainder of NH2.CH2.CO. From the glycine anhydride, alcoholic and hydrochloric acid results the analogous glycineethylester from boiling with; NH2.CH2.CO.NH.CH2.COOC2H5, which, as also, easily again into glycine anhydride changes the acid itself and is distinguished by the reactivity of its NH2-groups. Representation of the glycine anhydride (2.5-Diacipiperazins). 100 g glycine ester (previously cited) are shifted under cool ones with 60 g water; at room temperature then the glycine anhydride separates to 67% of the theory crystallized formation within 1-2 days. The same is only a weak base, whose salts are divided by water or alcohol. The steady chlorine hydrate obtained of CURTIUS and GOEBEL by boiling with strong hydrochloric acid to water belongs to not this base, but the methylene glycol; it forms also, if one lets stand the glycine anhydride with cold concentrated hydrochloric acid several days. From 95 percent alcohol, it crystallized with 1H2O; from concentrated hydrochloric acid it separates in large crystals, with rapid cool one as mash of fine needles formation. With the decomposition with silver oxide, it supplies the free glycine, C4H8O3N2. Pearl-grey-glossy lamellas from water + alcohol; decomposing rapidly heats up, after preceding darkening with 215-220° without the one which can be melted; easily soluble into hot water; very few soluble in alcohol; insoluble in ethers; freshly precipitated copper oxide dissolves to a low-blue liquid, from which with the vaporization itself the copper salt as light-blue, already into cold water easily soluble consisting of small prisms. Crystal masses separate. Difference of the glycine anhydride, which supplies a hardly colored liquid when short boiling with copper oxide only. Glycineethylester, C6H12O3N2, forms by saturate glycine anhydride in 270 ccm for a cooled suspension of 10 g absolute alcohol with HCl and rapid heating up to the boiling. When cooling off, then the chlorine hydrate, C6H13O3N2. separates HCl, in glossy needles off, which heats up, rapidly, against 182° (corrected) under decomposition melting and into cold water easily soluble, into boiling alcohol are rather easily soluble. For the production of the free ester, the aqueous solution of the salt with soda lye, laminated over with chloroform, is shifted. Needles from chloroform petroleum-ethene; melting point 88-89° (corrected); in water with strongly alkaline reaction very easily soluble; very easily soluble also in chloroform and alcohol, more heavily in acetone, rather little soluble in ethers; alcoholic goes with boiling up for itself, in aqueous solution, as well as its lower effect. Ammoniac or Na-ethylate again in glycine anhydride over. With heating up, for instance, on 190° beside much glycine anhydride, it supplies small quantities of a body, which gives the biuret coloring with alkali and cuprous salts and probably with the "Biuret base" from CURTIUS and GOEBEL is identical. Through treatment into cold solution with phenylisocyanate, aqueous with normal caustic soda solution transferred, the ester goes into the sodium salt of the phenylcarbaminoglycines, C6H5.NH.CO.NH.CH2.CO.NH.COOH, over, as more closely crystal mash separates and diluted with. Acetic acid one divides. Silk-glossy needles from little water, melting against 175° (corrected) under decomposition, rather easily soluble in hot alcohol, very little soluble in ethers; gives no biuret reaction. By 1/2 hour boiling with alcohol of concentrated sulfuric acid into the ethyl ester, C13H17O4N3 is transferred; crystals from the twenty-way quantity hot water; melting point 165-166° easily soluble into hot alcohol; few soluble in toluene, chloroform; very few soluble in ethers. The caboxylethylglycineester, C2H5O2C.NH.CH2.CO.NH.CH2.CO2C2H5, is obtained from the glycineester and chlorine coal NSA your ester into cold soda solution; frequently to bunches mirror-image-eats from little water, flat combined prisms or mirror-image-eats from acetic ester, melting point 87° (corrects); easily soluble in hot water, alcohol, acetone, benzene, more heavily in acetic esters, ethers, petroleum-ethene; no biuret reaction gives. By 24-hour effect of liquid ammonia, as well as by 4-bis 5-hour heating up with the twenty-way quantity with 0° saturated alcoholic. Ammonia on 100° the ester goes into the carbaminoglycineethylester, NH2.CO.NH.CH2.CO.NH.CH2.CO2C2H5 or C2H5O2C.NH.CH2.CO.NH.CH2.CO.NH2, over; fine, usually hexagonal lamellas from water or alcohol; melting, rapidly heats up, against 183° (corrected) under decomposition; very easily soluble in hot water, alcohol; few soluble in chloroform, benzene, ether; becomes of cold aqueous alkalis lower formation crystallized acid of a saponified, which against 180° under decomposition melting and gives no biuret reaction. The esters of the amino acids affect heavier than ammonia the caboxylethylglycineester; but C4H9. developed with leucineester; CH(NH2).COOC2H5, lower exit of alcohol with 130° a compound C15H27O6N3, which with 109-110° melting and the biuret reaction shows.
- 2Bergmann, M.; Zervas, L. Max Bergmann und Leonidas Zervas: Über ein allgemeines Verfahren der Peptid-Synthese. Ber. Dtsch. Chem. Ges. B 1932, 65, 1192– 1201, DOI: 10.1002/cber.19320650722Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaA38XkvFWmsA%253D%253D&md5=33b633a0b5c7479127b16f948ed6916eA general process for the synthesis of peptidesBergmann, Max; Zervas, LeonidasBerichte der Deutschen Chemischen Gesellschaft [Abteilung] B: Abhandlungen (1932), 65B (), 1192-1201CODEN: BDCBAD; ISSN:0365-9488.In the method of synthesizing peptides which consists in stabilizing the amino group of 1 acid with a protective group R, then so altering the CO2H group as to enable it to couple with a 2nd amino acid and removing the group R after the coupling has been effected, the difficulty lies in finding a group R which can be so readily split off again that the peptide union is not at the same time appreciably attacked. B. and Z. have found that the group PhCH2OCO (called carbobenzoxy for short) is capable of much wider application for this purpose than Ac or MeC6H4SO2; by means of the readily available chloride, PhCH2OCOCl, it can easily be introduced into amino acids of the most varied types and split off again as PhMe and CO2 by catalytic hydrogenation in open vessels. By this means there have been prepd. a no. of peptides which it had hitherto not been possible to obtain. Thus, carbobenzoxy-d-glutamic acid (I), heated with Ac2O, gives an optically active anhydride (II) which readily adds NH3 to form the amide HO2CCH2CH2CH(NHR)CONH2 (III), and III on hydrogenation yields isoglutamine (IV), different from natural glutamine, showing that in IV it is the α-CO2H group to the NH2 which is changed to CONH2. The compd. (V) obtained from II with di-Et d-glutamate is therefore assigned the Structure HO2CCH2CH2CH(NHR) CONHCH(CO2Et)CH2CH2CO2Et; on hydrogenation it gives a d-glutamyl-d-glutamic acid (VI), m. 205°, entirely different from the compd., m. 167-8°, obtained by Blanchetiere by thermal condensation of glutamic: acid and subsequent alkali hydrolysis of the resulting anhydride (C. A. 19, 638). VI is rapidly split by pancreatin, according to preliminary expts. by H. Schleich. l-Isoasparagine (VII) and l-aspartyl-l-tyrosine (VIII) were synthesized in the same way from carbobenzoxy-l-aspartic acid (IX). VIII is not split by either com. pancreatin or fresh exts. of the mucosa of the pig's intestine. It remains to be detd. whether in this case a β-peptide is formed or special ferment specificities obtain for-α-aspartyl peptides. The synthesis of glueopeptides of d-glucosamine is described in the following abstr; that of glycylproline, m. 185°, [α]D20 -112.6° (water), and of d-lysyl-d-glutamic acid, m. 197°, [α]D19 22.9° (water), will be described elsewhere, together with the behavior of the peptides toward ferments. It is worthy of note that these carbobenzoxy derivs. of amino acids completely retain their optical activity both when treated with Ac2O and when converted into the chlorides. Benzyl chloroformate (X) is obtained in sufficiently pure form in about 60-g. yield from 240 g. of cold 20% phosgene soln. in PhMe and 45 cc. PhCH2OH allowed to stand 0.5 hr. in ice and 2 hrs. at room temp., and freed from the excess of phosgene with dry CO2 or N and from the PhMe by evapn. in vacuo, below 60°. Attempts to purify it by distn. result in considerable decompn. N-Carbobenzoxyglycocoll (72% from glycocoll in cold 4 N NaOH slowly treated alternately with X and 4 N NaOH), m. 120° (all m. ps. cor.); 6.3 g. gives with PCl5 in ether 5.3 g. of the chloride, m. 43°, decompg. into the anhydride CO.NH.CH2.CO.O and PhCH2Cl. N-Caybobenzoxy-dl-alanine, m. 114-5°. d-Isomer, m. 84° [α]D17 -14.3° (AcOH); chloride, oil regenerating an acid with [α]D18 -14.2°. N-Carbobenzoxy-di-serine (42% yield), m. 125°. N,N-Dicarbobenzoxy-l-cystine, m. 123°, [α]D20 -91.7° (AcOH); the chloride, m. 67-8°, readily couples with H2NCH2CO2Et to dicarbobenzoxy-l-cystyldiglycine Et ester, m. 166°, also obtained through the hydrazide and azide instead of the chloride. I (15 g. from 8.8 g. d-glutamic acid), m. 120°, [αD19 -7.1° (AcOH); II, m. 94°, [α]D19 -44.1° (AcOH). N-Carbobenzoxy-d-isoglutamine (III) (5.3 g. from 6 g. II), m. 175° (N-carbobenzoxy-d-glutamine, m. 137°). IV, obtained with H and Pd sponge in aq. MeOH contg. a little AcOH, [α]D22 21.1° (water). IX, m. 116°, [α]D18 9.6° (AcOH); anhydride (4.1 g. from 5.4 g. of the acid), m. 84°, [α]D19 -39.8° (AcOH); N-Carbobenzoxy-l-isoasparagine (2 g. from 2.7 g. of the anhydride), m. 164°, [α]D18 6.9° (AcOH) (Me ester, m. 121°, [α]D20 9.0° (AcOH)); VII, needles with 1 H2O which is not lost in a high vacuum at 78°, [α]D18 15.5° (0.1 N HCl), evolves NH3 with NaOH only on heating. Carbobenzoxy-l-asparagine, m. 165° (mixed m. p. with the iso-compd., 153°), [α]D18 7.6° (AcOH); Me ester, m. 150°, [α]D20 -2.0° (AcOH). Carbobenzoxy-dl-phenylalanine (with Oskar Fritz Leinert) , m. 103°. N-Carbobenzoxy-l-tyrosine Et ester (80% yield), m. 78°, [α]D25 -4.7° (alc.); free acid, needles with 2 H2O, m. 101°, [α]20 11.1° (AcOH). N-Monocarbobenzoxy-d-arginine (5.5 g. from 5.3 g. arginine), m. 175°, [α]D20 -9.2° (0.2 N HCl). N-Carbobenzoxy-l-histidine (yield poor), m. 209°. Carbobenzoxyglycylglycine (70% yield), m. 178°. Di-Et N-carbobenzoxy-d-glutamyl-d-glutamate (V) (2.8 g. from 3 g. II), m. 137°; free acid, from the ester and somewhat more than 3 mols. of N NaOH at 18° (80% yield), sinters about 145°, m. around 176°; VI, [α]D18 19.9° in water contg. 1 mol. HCl. Carbobenzoxy-l-aspartyl-l-tyrosine Et ester (2.2 g. from 4 g. of the anhydride), m. 203°; free acid, needles with 1 H2O, m. 110°; VIII, darkens around 230°, [α]D18 60.1° in water contg. 1 mol. HCl.
- 3Vigneaud, V. d.; Ressler, C.; Swan, C. J. M.; Roberts, C. W.; Katsoyannis, P. G.; Gordon, S. The Synthesis of an Octapeptide Amide with the Hormonal Activity of Oxytocin. J. Am. Chem. Soc. 1953, 75 (19), 4879– 4880, DOI: 10.1021/ja01115a553Google ScholarThere is no corresponding record for this reference.
- 4Merrifield, R. B. Solid phase peptide synthesis I: Synthesis of a tetrapeptide. J. Am. Chem. Soc. 1963, 85, 2149– 2154, DOI: 10.1021/ja00897a025Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF3sXksVajsLg%253D&md5=4b5fa1ad0e76fc3e48b037b39138fbf5Solid phase peptide synthesis. I. The synthesis of a tetrapeptideMerrifield, R. B.Journal of the American Chemical Society (1963), 85 (14), 2149-54CODEN: JACSAT; ISSN:0002-7863.A new approach to the chem. synthesis of polypeptides was investigated. It involved the stepwise addition of protected amino acids to a growing peptide chain which was bound by a covalent bond to a solid resin particle. This provided a procedure whereby reagents anti by-products were removed by filtration, and the recrystn. of intermediates was eliminated. The advantages of the new method were speed and simplicity of operation. The feasibility of the idea was demonstrated by the synthesis of the model tetrapeptide L-leucyl-L-alanylglycyl-L-valine. The peptide was identical with a sample prepd. by the standard p-nitrophenyl ester procedure.
- 5Merrifield, B Solid phase synthesis. Science 1986, 232, 341– 347, DOI: 10.1126/science.3961484Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL28XltFGguro%253D&md5=b10960ef70b5506cae35a326c1906fd6Solid phase synthesisMerrifield, BruceScience (Washington, DC, United States) (1986), 232 (4748), 341-7CODEN: SCIEAS; ISSN:0036-8075.A review with 74 refs.
- 6Merrifield, R. B.; Stewart, J. M.; Jernberg, N. Instrument for automated synthesis of peptides. Anal. Chem. 1966, 38, 1905– 1914, DOI: 10.1021/ac50155a057Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF2sXhvVyntw%253D%253D&md5=d9a1168c832477266dd05ba11ccf6188Instrument for automated synthesis of peptidesMerrifield, Robert B.; Stewart, John Morrow; Jernberg, NilsAnalytical Chemistry (1966), 38 (13), 1905-14CODEN: ANCHAM; ISSN:0003-2700.cf. CA 64, 809h. An instrument is described for the automated synthesis of peptides, in which a peptide chain is synthesized in a stepwise manner while one end of the chain is covalently attached to an insol. solid support, a chloromethylated styrene-divinylbenzene copolymer bead. The C-terminal amino acid is coupled as a benzyl ester to the resin and the peptide chain grows by condensation at the amino end with N-acylated amino acids. The protecting group is tert-BuO2C and activation is usually by the carbodiimide or active ester routes. All of the reactions, including the intermediate purification procedures, are conducted within a single reaction vessel and a stepping drum programmer controls and sequences the operation of the various components. A saving of time and effort and larger overall yields were observed by using the instruments for peptide synthesis.
- 7Merrifield, R. B. New approaches to the chemical synthesis of peptides. Recent Prog. Horm Res. 1967, 23, 451– 482, DOI: 10.1016/B978-1-4831-9826-2.50013-1Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF1cXltVSjug%253D%253D&md5=4ccb38a1479c5a7cdec2755b940ee1aeNew Approaches to the chemical synthesis of peptidesMerrifield, Robert B.Recent Progress in Hormone Research (1967), 23 (), 451-76, discussion 476-82CODEN: RPHRA6; ISSN:0079-9963.Rapid, simple and efficient approaches to the problem of peptide synthesis are reviewed which are based on the principle of synthesis without isolation of intermediates or on the use of polymeric support. 43 references.
- 8Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Synthesis of proteins by native chemical ligation. Science 1994, 266 (5186), 776– 9, DOI: 10.1126/science.7973629Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXitVGgtrw%253D&md5=1d352d817ab48851500610dcd32d1136Synthesis of proteins by native chemical ligationDawson, Philip E.; Muir, Tom W.; Clark-Lewis, Ian; Kent, Stephen B. H.Science (Washington, D. C.) (1994), 266 (5186), 776-9CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A simple technique has been devised that allows the direct synthesis of native backbone proteins of moderate size. Chemoselective reaction of two unprotected peptide segments gives an initial thioester-linked species. Spontaneous rearrangement of this transient intermediate yields a full-length product with a native peptide bond at the ligation site. The utility of native chem. ligation was demonstrated by the one-step prepn. of a cytokine contg. multiple disulfides. The polypeptide ligation product was folded and oxidized to form the native disulfide-contg. protein mol. Native chem. ligation is an important step toward the general application of chem. to proteins.
- 9Muir, T. W.; Sondhi, D.; Cole, P. A. Expressed protein ligation: a general method for protein engineering. Proc. Natl. Acad. Sci. U. S. A. 1998, 95 (12), 6705– 10, DOI: 10.1073/pnas.95.12.6705Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXjslyntLg%253D&md5=bc924251576f3475f02b00c0a484c1fdExpressed protein ligation: A general method for protein engineeringMuir, Tom W.; Sondhi, Dolan; Cole, Philip A.Proceedings of the National Academy of Sciences of the United States of America (1998), 95 (12), 6705-6710CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A protein semisynthesis method-expressed protein ligation-is described that involves the chemoselective addn. of a peptide to a recombinant protein. This method was used to ligate a phosphotyrosine peptide to the C terminus of the protein tyrosine kinase C-terminal Src kinase (Csk). By intercepting a thioester generated in the recombinant protein with an N-terminal cysteine contg. synthetic peptide, near quant. chem. ligation of the peptide to the protein was achieved. The semisynthetic tail-phosphorylated Csk showed evidence of an intramol. phosphotyrosine-Src homol. 2 interaction and an unexpected increase in catalytic phosphoryl transfer efficiency toward a physiol. relevant substrate compared with the non-tail-phosphorylated control. This work illustrates that expressed protein ligation is a simple and powerful new method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and biophys. probes into proteins of any size.
- 10Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem., Int. Ed. 2002, 41 (14), 2596– 9, DOI: 10.1002/1521-3773(20020715)41:14<2596::AID-ANIE2596>3.0.CO;2-4Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xls1Ohsr4%253D&md5=4603664be6639353b5e70f19b9f8d59fA stepwise Huisgen cycloaddition process: copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynesRostovtsev, Vsevolod V.; Green, Luke G.; Fokin, Valery V.; Sharpless, K. BarryAngewandte Chemie, International Edition (2002), 41 (14), 2596-2599CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)1,4-Disubstituted 1,2,3-triazoles I (R1 = PhCH2, PhCH2OCH2, 1-adamantyl, etc.; R2 = HO2C, Ph, PhOCH2, Et2NCH2, etc.) were readily and cleanly prepd. via highly efficient and regioselective copper(I)-catalyzed cycloaddn. of azides R1N3 with terminal alkynes R2C≡CH in 82-93% yields.
- 11Tornøe, C. W.; Christensen, C.; Meldal, M. Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 2002, 67 (9), 3057– 64, DOI: 10.1021/jo011148jGoogle Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XisVeks7w%253D&md5=6b3b805572783873952871f1b69f46ddPeptidotriazoles on Solid Phase: [1,2,3]-Triazoles by Regiospecific Copper(I)-Catalyzed 1,3-Dipolar Cycloadditions of Terminal Alkynes to AzidesTornoe, Christian W.; Christensen, Caspar; Meldal, MortenJournal of Organic Chemistry (2002), 67 (9), 3057-3064CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)The cycloaddn. of azides to alkynes is one of the most important synthetic routes to 1H-[1,2,3]-triazoles. This work reports a novel regiospecific copper(I)-catalyzed 1,3-dipolar cycloaddn. of terminal alkynes to azides on solid-phase. Primary, secondary, and tertiary alkyl azides, aryl azides, and an azido sugar were used successfully in the copper(I)-catalyzed cycloaddn. producing diversely 1,4-substituted [1,2,3]-triazoles in peptide backbones or side chains. The reaction conditions were fully compatible with solid-phase peptide synthesis on polar supports. The copper(I) catalysis is mild and efficient (>95% conversion and purity in most cases) and furthermore, the x-ray structure of 2-azido-2-methylpropanoic acid has been solved, to yield structural information on the 1,3-dipoles entering the reaction. Novel Fmoc-protected amino azides were prepd. from Fmoc-amino alcs. by Mitsunobu reaction.
- 12Saxon, E.; Bertozzi, C. R. Cell surface engineering by a modified Staudinger reaction. Science 2000, 287 (5460), 2007– 10, DOI: 10.1126/science.287.5460.2007Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXhvF2ru7g%253D&md5=91685aaba684aa4dfee86f44fef09886Cell surface engineering by a modified Staudinger reactionSaxon, Eliana; Bertozzi, Carolyn R.Science (Washington, D. C.) (2000), 287 (5460), 2007-2010CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Selective chem. reactions enacted within a cellular environment can be powerful tools for elucidating biol. processes or engineering novel interactions. A chem. transformation that permits the selective formation of covalent adducts among richly functionalized biopolymers within a cellular context is presented. A ligation modeled after the Staudinger reaction forms an amide bond by coupling of an azide and a specifically engineered triarylphosphine. Both reactive partners are abiotic and chem. orthogonal to native cellular components. Azides installed within cell surface glycoconjugates by metab. of a synthetic azidosugar were reacted with a biotinylated triarylphosphine to produce stable cell-surface adducts. The tremendous selectivity of the transformation should permit its execution within a cell's interior, offering new possibilities for probing intracellular interactions.
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Figure 1
Figure 1. The guest editors of this issue (from left to right Christian Hackenberger, Philip Dawson, Yong-Xiang Chen and Hironobu Hojo) at the eighth Chemical Protein Synthesis Meeting in Berlin.
References
ARTICLE SECTIONSThis article references 12 other publications.
- 1Fischer, E.; Fourneau, E. Ueber einige Derivate des Glykocolls. Ber. Dtsch. Chem. Ges. 1901, 34, 2868– 2877, DOI: 10.1002/cber.190103402249Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaD28XitFyitbc%253D&md5=47ac4c6496102fee68b1f555a323213eOver some derivatives of the glycine. [machine translation]Fischer, Emil; Fourneau, ErnestBerichte der Deutschen Chemischen Gesellschaft (1901), 34 (), 2868-77CODEN: BDCGAS; ISSN:0365-9496.[Machine Translation of Descriptors]. Experiments to combine the amino acids developing from the protein materials by hydrolysis by anhydride formation again to larger complexes were implemented by SCHAAL, GRIMAUX, SCHIFF, SCHUeTZENBERGER, LILIEINFELD, BALBIANO among others, however always led to amorphous, with difficulty characterizable products. As known, α-amino acids, as well as their ester, (E. FISCHER, Ber. Dtsch. Chem. Ges. 34. 435; C. 1901. I. 678) can be transported into dimolecular anhydrides, their simplest representative the glycine anhydride (2.5-Diacipiperazin) of CURTIUS and GOEBEL (J. pr. Chem. [2] 37. 173) is. After the observations of the authors this compound splits up as follows when short boiling with concentrated hydrochloric acid: (see original document for graphics) the new substance, which is the simplest anhydrous product of the glycine (glycine), became glycine mentioned under use of the described glycyl for the remainder of NH2.CH2.CO. From the glycine anhydride, alcoholic and hydrochloric acid results the analogous glycineethylester from boiling with; NH2.CH2.CO.NH.CH2.COOC2H5, which, as also, easily again into glycine anhydride changes the acid itself and is distinguished by the reactivity of its NH2-groups. Representation of the glycine anhydride (2.5-Diacipiperazins). 100 g glycine ester (previously cited) are shifted under cool ones with 60 g water; at room temperature then the glycine anhydride separates to 67% of the theory crystallized formation within 1-2 days. The same is only a weak base, whose salts are divided by water or alcohol. The steady chlorine hydrate obtained of CURTIUS and GOEBEL by boiling with strong hydrochloric acid to water belongs to not this base, but the methylene glycol; it forms also, if one lets stand the glycine anhydride with cold concentrated hydrochloric acid several days. From 95 percent alcohol, it crystallized with 1H2O; from concentrated hydrochloric acid it separates in large crystals, with rapid cool one as mash of fine needles formation. With the decomposition with silver oxide, it supplies the free glycine, C4H8O3N2. Pearl-grey-glossy lamellas from water + alcohol; decomposing rapidly heats up, after preceding darkening with 215-220° without the one which can be melted; easily soluble into hot water; very few soluble in alcohol; insoluble in ethers; freshly precipitated copper oxide dissolves to a low-blue liquid, from which with the vaporization itself the copper salt as light-blue, already into cold water easily soluble consisting of small prisms. Crystal masses separate. Difference of the glycine anhydride, which supplies a hardly colored liquid when short boiling with copper oxide only. Glycineethylester, C6H12O3N2, forms by saturate glycine anhydride in 270 ccm for a cooled suspension of 10 g absolute alcohol with HCl and rapid heating up to the boiling. When cooling off, then the chlorine hydrate, C6H13O3N2. separates HCl, in glossy needles off, which heats up, rapidly, against 182° (corrected) under decomposition melting and into cold water easily soluble, into boiling alcohol are rather easily soluble. For the production of the free ester, the aqueous solution of the salt with soda lye, laminated over with chloroform, is shifted. Needles from chloroform petroleum-ethene; melting point 88-89° (corrected); in water with strongly alkaline reaction very easily soluble; very easily soluble also in chloroform and alcohol, more heavily in acetone, rather little soluble in ethers; alcoholic goes with boiling up for itself, in aqueous solution, as well as its lower effect. Ammoniac or Na-ethylate again in glycine anhydride over. With heating up, for instance, on 190° beside much glycine anhydride, it supplies small quantities of a body, which gives the biuret coloring with alkali and cuprous salts and probably with the "Biuret base" from CURTIUS and GOEBEL is identical. Through treatment into cold solution with phenylisocyanate, aqueous with normal caustic soda solution transferred, the ester goes into the sodium salt of the phenylcarbaminoglycines, C6H5.NH.CO.NH.CH2.CO.NH.COOH, over, as more closely crystal mash separates and diluted with. Acetic acid one divides. Silk-glossy needles from little water, melting against 175° (corrected) under decomposition, rather easily soluble in hot alcohol, very little soluble in ethers; gives no biuret reaction. By 1/2 hour boiling with alcohol of concentrated sulfuric acid into the ethyl ester, C13H17O4N3 is transferred; crystals from the twenty-way quantity hot water; melting point 165-166° easily soluble into hot alcohol; few soluble in toluene, chloroform; very few soluble in ethers. The caboxylethylglycineester, C2H5O2C.NH.CH2.CO.NH.CH2.CO2C2H5, is obtained from the glycineester and chlorine coal NSA your ester into cold soda solution; frequently to bunches mirror-image-eats from little water, flat combined prisms or mirror-image-eats from acetic ester, melting point 87° (corrects); easily soluble in hot water, alcohol, acetone, benzene, more heavily in acetic esters, ethers, petroleum-ethene; no biuret reaction gives. By 24-hour effect of liquid ammonia, as well as by 4-bis 5-hour heating up with the twenty-way quantity with 0° saturated alcoholic. Ammonia on 100° the ester goes into the carbaminoglycineethylester, NH2.CO.NH.CH2.CO.NH.CH2.CO2C2H5 or C2H5O2C.NH.CH2.CO.NH.CH2.CO.NH2, over; fine, usually hexagonal lamellas from water or alcohol; melting, rapidly heats up, against 183° (corrected) under decomposition; very easily soluble in hot water, alcohol; few soluble in chloroform, benzene, ether; becomes of cold aqueous alkalis lower formation crystallized acid of a saponified, which against 180° under decomposition melting and gives no biuret reaction. The esters of the amino acids affect heavier than ammonia the caboxylethylglycineester; but C4H9. developed with leucineester; CH(NH2).COOC2H5, lower exit of alcohol with 130° a compound C15H27O6N3, which with 109-110° melting and the biuret reaction shows.
- 2Bergmann, M.; Zervas, L. Max Bergmann und Leonidas Zervas: Über ein allgemeines Verfahren der Peptid-Synthese. Ber. Dtsch. Chem. Ges. B 1932, 65, 1192– 1201, DOI: 10.1002/cber.19320650722Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaA38XkvFWmsA%253D%253D&md5=33b633a0b5c7479127b16f948ed6916eA general process for the synthesis of peptidesBergmann, Max; Zervas, LeonidasBerichte der Deutschen Chemischen Gesellschaft [Abteilung] B: Abhandlungen (1932), 65B (), 1192-1201CODEN: BDCBAD; ISSN:0365-9488.In the method of synthesizing peptides which consists in stabilizing the amino group of 1 acid with a protective group R, then so altering the CO2H group as to enable it to couple with a 2nd amino acid and removing the group R after the coupling has been effected, the difficulty lies in finding a group R which can be so readily split off again that the peptide union is not at the same time appreciably attacked. B. and Z. have found that the group PhCH2OCO (called carbobenzoxy for short) is capable of much wider application for this purpose than Ac or MeC6H4SO2; by means of the readily available chloride, PhCH2OCOCl, it can easily be introduced into amino acids of the most varied types and split off again as PhMe and CO2 by catalytic hydrogenation in open vessels. By this means there have been prepd. a no. of peptides which it had hitherto not been possible to obtain. Thus, carbobenzoxy-d-glutamic acid (I), heated with Ac2O, gives an optically active anhydride (II) which readily adds NH3 to form the amide HO2CCH2CH2CH(NHR)CONH2 (III), and III on hydrogenation yields isoglutamine (IV), different from natural glutamine, showing that in IV it is the α-CO2H group to the NH2 which is changed to CONH2. The compd. (V) obtained from II with di-Et d-glutamate is therefore assigned the Structure HO2CCH2CH2CH(NHR) CONHCH(CO2Et)CH2CH2CO2Et; on hydrogenation it gives a d-glutamyl-d-glutamic acid (VI), m. 205°, entirely different from the compd., m. 167-8°, obtained by Blanchetiere by thermal condensation of glutamic: acid and subsequent alkali hydrolysis of the resulting anhydride (C. A. 19, 638). VI is rapidly split by pancreatin, according to preliminary expts. by H. Schleich. l-Isoasparagine (VII) and l-aspartyl-l-tyrosine (VIII) were synthesized in the same way from carbobenzoxy-l-aspartic acid (IX). VIII is not split by either com. pancreatin or fresh exts. of the mucosa of the pig's intestine. It remains to be detd. whether in this case a β-peptide is formed or special ferment specificities obtain for-α-aspartyl peptides. The synthesis of glueopeptides of d-glucosamine is described in the following abstr; that of glycylproline, m. 185°, [α]D20 -112.6° (water), and of d-lysyl-d-glutamic acid, m. 197°, [α]D19 22.9° (water), will be described elsewhere, together with the behavior of the peptides toward ferments. It is worthy of note that these carbobenzoxy derivs. of amino acids completely retain their optical activity both when treated with Ac2O and when converted into the chlorides. Benzyl chloroformate (X) is obtained in sufficiently pure form in about 60-g. yield from 240 g. of cold 20% phosgene soln. in PhMe and 45 cc. PhCH2OH allowed to stand 0.5 hr. in ice and 2 hrs. at room temp., and freed from the excess of phosgene with dry CO2 or N and from the PhMe by evapn. in vacuo, below 60°. Attempts to purify it by distn. result in considerable decompn. N-Carbobenzoxyglycocoll (72% from glycocoll in cold 4 N NaOH slowly treated alternately with X and 4 N NaOH), m. 120° (all m. ps. cor.); 6.3 g. gives with PCl5 in ether 5.3 g. of the chloride, m. 43°, decompg. into the anhydride CO.NH.CH2.CO.O and PhCH2Cl. N-Caybobenzoxy-dl-alanine, m. 114-5°. d-Isomer, m. 84° [α]D17 -14.3° (AcOH); chloride, oil regenerating an acid with [α]D18 -14.2°. N-Carbobenzoxy-di-serine (42% yield), m. 125°. N,N-Dicarbobenzoxy-l-cystine, m. 123°, [α]D20 -91.7° (AcOH); the chloride, m. 67-8°, readily couples with H2NCH2CO2Et to dicarbobenzoxy-l-cystyldiglycine Et ester, m. 166°, also obtained through the hydrazide and azide instead of the chloride. I (15 g. from 8.8 g. d-glutamic acid), m. 120°, [αD19 -7.1° (AcOH); II, m. 94°, [α]D19 -44.1° (AcOH). N-Carbobenzoxy-d-isoglutamine (III) (5.3 g. from 6 g. II), m. 175° (N-carbobenzoxy-d-glutamine, m. 137°). IV, obtained with H and Pd sponge in aq. MeOH contg. a little AcOH, [α]D22 21.1° (water). IX, m. 116°, [α]D18 9.6° (AcOH); anhydride (4.1 g. from 5.4 g. of the acid), m. 84°, [α]D19 -39.8° (AcOH); N-Carbobenzoxy-l-isoasparagine (2 g. from 2.7 g. of the anhydride), m. 164°, [α]D18 6.9° (AcOH) (Me ester, m. 121°, [α]D20 9.0° (AcOH)); VII, needles with 1 H2O which is not lost in a high vacuum at 78°, [α]D18 15.5° (0.1 N HCl), evolves NH3 with NaOH only on heating. Carbobenzoxy-l-asparagine, m. 165° (mixed m. p. with the iso-compd., 153°), [α]D18 7.6° (AcOH); Me ester, m. 150°, [α]D20 -2.0° (AcOH). Carbobenzoxy-dl-phenylalanine (with Oskar Fritz Leinert) , m. 103°. N-Carbobenzoxy-l-tyrosine Et ester (80% yield), m. 78°, [α]D25 -4.7° (alc.); free acid, needles with 2 H2O, m. 101°, [α]20 11.1° (AcOH). N-Monocarbobenzoxy-d-arginine (5.5 g. from 5.3 g. arginine), m. 175°, [α]D20 -9.2° (0.2 N HCl). N-Carbobenzoxy-l-histidine (yield poor), m. 209°. Carbobenzoxyglycylglycine (70% yield), m. 178°. Di-Et N-carbobenzoxy-d-glutamyl-d-glutamate (V) (2.8 g. from 3 g. II), m. 137°; free acid, from the ester and somewhat more than 3 mols. of N NaOH at 18° (80% yield), sinters about 145°, m. around 176°; VI, [α]D18 19.9° in water contg. 1 mol. HCl. Carbobenzoxy-l-aspartyl-l-tyrosine Et ester (2.2 g. from 4 g. of the anhydride), m. 203°; free acid, needles with 1 H2O, m. 110°; VIII, darkens around 230°, [α]D18 60.1° in water contg. 1 mol. HCl.
- 3Vigneaud, V. d.; Ressler, C.; Swan, C. J. M.; Roberts, C. W.; Katsoyannis, P. G.; Gordon, S. The Synthesis of an Octapeptide Amide with the Hormonal Activity of Oxytocin. J. Am. Chem. Soc. 1953, 75 (19), 4879– 4880, DOI: 10.1021/ja01115a553Google ScholarThere is no corresponding record for this reference.
- 4Merrifield, R. B. Solid phase peptide synthesis I: Synthesis of a tetrapeptide. J. Am. Chem. Soc. 1963, 85, 2149– 2154, DOI: 10.1021/ja00897a025Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF3sXksVajsLg%253D&md5=4b5fa1ad0e76fc3e48b037b39138fbf5Solid phase peptide synthesis. I. The synthesis of a tetrapeptideMerrifield, R. B.Journal of the American Chemical Society (1963), 85 (14), 2149-54CODEN: JACSAT; ISSN:0002-7863.A new approach to the chem. synthesis of polypeptides was investigated. It involved the stepwise addition of protected amino acids to a growing peptide chain which was bound by a covalent bond to a solid resin particle. This provided a procedure whereby reagents anti by-products were removed by filtration, and the recrystn. of intermediates was eliminated. The advantages of the new method were speed and simplicity of operation. The feasibility of the idea was demonstrated by the synthesis of the model tetrapeptide L-leucyl-L-alanylglycyl-L-valine. The peptide was identical with a sample prepd. by the standard p-nitrophenyl ester procedure.
- 5Merrifield, B Solid phase synthesis. Science 1986, 232, 341– 347, DOI: 10.1126/science.3961484Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL28XltFGguro%253D&md5=b10960ef70b5506cae35a326c1906fd6Solid phase synthesisMerrifield, BruceScience (Washington, DC, United States) (1986), 232 (4748), 341-7CODEN: SCIEAS; ISSN:0036-8075.A review with 74 refs.
- 6Merrifield, R. B.; Stewart, J. M.; Jernberg, N. Instrument for automated synthesis of peptides. Anal. Chem. 1966, 38, 1905– 1914, DOI: 10.1021/ac50155a057Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF2sXhvVyntw%253D%253D&md5=d9a1168c832477266dd05ba11ccf6188Instrument for automated synthesis of peptidesMerrifield, Robert B.; Stewart, John Morrow; Jernberg, NilsAnalytical Chemistry (1966), 38 (13), 1905-14CODEN: ANCHAM; ISSN:0003-2700.cf. CA 64, 809h. An instrument is described for the automated synthesis of peptides, in which a peptide chain is synthesized in a stepwise manner while one end of the chain is covalently attached to an insol. solid support, a chloromethylated styrene-divinylbenzene copolymer bead. The C-terminal amino acid is coupled as a benzyl ester to the resin and the peptide chain grows by condensation at the amino end with N-acylated amino acids. The protecting group is tert-BuO2C and activation is usually by the carbodiimide or active ester routes. All of the reactions, including the intermediate purification procedures, are conducted within a single reaction vessel and a stepping drum programmer controls and sequences the operation of the various components. A saving of time and effort and larger overall yields were observed by using the instruments for peptide synthesis.
- 7Merrifield, R. B. New approaches to the chemical synthesis of peptides. Recent Prog. Horm Res. 1967, 23, 451– 482, DOI: 10.1016/B978-1-4831-9826-2.50013-1Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF1cXltVSjug%253D%253D&md5=4ccb38a1479c5a7cdec2755b940ee1aeNew Approaches to the chemical synthesis of peptidesMerrifield, Robert B.Recent Progress in Hormone Research (1967), 23 (), 451-76, discussion 476-82CODEN: RPHRA6; ISSN:0079-9963.Rapid, simple and efficient approaches to the problem of peptide synthesis are reviewed which are based on the principle of synthesis without isolation of intermediates or on the use of polymeric support. 43 references.
- 8Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Synthesis of proteins by native chemical ligation. Science 1994, 266 (5186), 776– 9, DOI: 10.1126/science.7973629Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXitVGgtrw%253D&md5=1d352d817ab48851500610dcd32d1136Synthesis of proteins by native chemical ligationDawson, Philip E.; Muir, Tom W.; Clark-Lewis, Ian; Kent, Stephen B. H.Science (Washington, D. C.) (1994), 266 (5186), 776-9CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A simple technique has been devised that allows the direct synthesis of native backbone proteins of moderate size. Chemoselective reaction of two unprotected peptide segments gives an initial thioester-linked species. Spontaneous rearrangement of this transient intermediate yields a full-length product with a native peptide bond at the ligation site. The utility of native chem. ligation was demonstrated by the one-step prepn. of a cytokine contg. multiple disulfides. The polypeptide ligation product was folded and oxidized to form the native disulfide-contg. protein mol. Native chem. ligation is an important step toward the general application of chem. to proteins.
- 9Muir, T. W.; Sondhi, D.; Cole, P. A. Expressed protein ligation: a general method for protein engineering. Proc. Natl. Acad. Sci. U. S. A. 1998, 95 (12), 6705– 10, DOI: 10.1073/pnas.95.12.6705Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXjslyntLg%253D&md5=bc924251576f3475f02b00c0a484c1fdExpressed protein ligation: A general method for protein engineeringMuir, Tom W.; Sondhi, Dolan; Cole, Philip A.Proceedings of the National Academy of Sciences of the United States of America (1998), 95 (12), 6705-6710CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A protein semisynthesis method-expressed protein ligation-is described that involves the chemoselective addn. of a peptide to a recombinant protein. This method was used to ligate a phosphotyrosine peptide to the C terminus of the protein tyrosine kinase C-terminal Src kinase (Csk). By intercepting a thioester generated in the recombinant protein with an N-terminal cysteine contg. synthetic peptide, near quant. chem. ligation of the peptide to the protein was achieved. The semisynthetic tail-phosphorylated Csk showed evidence of an intramol. phosphotyrosine-Src homol. 2 interaction and an unexpected increase in catalytic phosphoryl transfer efficiency toward a physiol. relevant substrate compared with the non-tail-phosphorylated control. This work illustrates that expressed protein ligation is a simple and powerful new method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and biophys. probes into proteins of any size.
- 10Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem., Int. Ed. 2002, 41 (14), 2596– 9, DOI: 10.1002/1521-3773(20020715)41:14<2596::AID-ANIE2596>3.0.CO;2-4Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xls1Ohsr4%253D&md5=4603664be6639353b5e70f19b9f8d59fA stepwise Huisgen cycloaddition process: copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynesRostovtsev, Vsevolod V.; Green, Luke G.; Fokin, Valery V.; Sharpless, K. BarryAngewandte Chemie, International Edition (2002), 41 (14), 2596-2599CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)1,4-Disubstituted 1,2,3-triazoles I (R1 = PhCH2, PhCH2OCH2, 1-adamantyl, etc.; R2 = HO2C, Ph, PhOCH2, Et2NCH2, etc.) were readily and cleanly prepd. via highly efficient and regioselective copper(I)-catalyzed cycloaddn. of azides R1N3 with terminal alkynes R2C≡CH in 82-93% yields.
- 11Tornøe, C. W.; Christensen, C.; Meldal, M. Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 2002, 67 (9), 3057– 64, DOI: 10.1021/jo011148jGoogle Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XisVeks7w%253D&md5=6b3b805572783873952871f1b69f46ddPeptidotriazoles on Solid Phase: [1,2,3]-Triazoles by Regiospecific Copper(I)-Catalyzed 1,3-Dipolar Cycloadditions of Terminal Alkynes to AzidesTornoe, Christian W.; Christensen, Caspar; Meldal, MortenJournal of Organic Chemistry (2002), 67 (9), 3057-3064CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)The cycloaddn. of azides to alkynes is one of the most important synthetic routes to 1H-[1,2,3]-triazoles. This work reports a novel regiospecific copper(I)-catalyzed 1,3-dipolar cycloaddn. of terminal alkynes to azides on solid-phase. Primary, secondary, and tertiary alkyl azides, aryl azides, and an azido sugar were used successfully in the copper(I)-catalyzed cycloaddn. producing diversely 1,4-substituted [1,2,3]-triazoles in peptide backbones or side chains. The reaction conditions were fully compatible with solid-phase peptide synthesis on polar supports. The copper(I) catalysis is mild and efficient (>95% conversion and purity in most cases) and furthermore, the x-ray structure of 2-azido-2-methylpropanoic acid has been solved, to yield structural information on the 1,3-dipoles entering the reaction. Novel Fmoc-protected amino azides were prepd. from Fmoc-amino alcs. by Mitsunobu reaction.
- 12Saxon, E.; Bertozzi, C. R. Cell surface engineering by a modified Staudinger reaction. Science 2000, 287 (5460), 2007– 10, DOI: 10.1126/science.287.5460.2007Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXhvF2ru7g%253D&md5=91685aaba684aa4dfee86f44fef09886Cell surface engineering by a modified Staudinger reactionSaxon, Eliana; Bertozzi, Carolyn R.Science (Washington, D. C.) (2000), 287 (5460), 2007-2010CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Selective chem. reactions enacted within a cellular environment can be powerful tools for elucidating biol. processes or engineering novel interactions. A chem. transformation that permits the selective formation of covalent adducts among richly functionalized biopolymers within a cellular context is presented. A ligation modeled after the Staudinger reaction forms an amide bond by coupling of an azide and a specifically engineered triarylphosphine. Both reactive partners are abiotic and chem. orthogonal to native cellular components. Azides installed within cell surface glycoconjugates by metab. of a synthetic azidosugar were reacted with a biotinylated triarylphosphine to produce stable cell-surface adducts. The tremendous selectivity of the transformation should permit its execution within a cell's interior, offering new possibilities for probing intracellular interactions.