Through the looking glass – a new world of
proteins enabled by chemical synthesis{
Stephen Kent,* Youhei Sohma, Suhuai Liu, Duhee Bang, Brad Pentelute and
Kalyaneswar Mandal
‘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.
Keywords: chemical protein synthesis; chemical ligation; ester insulin; erythropoietin; glycoprotein mimetics; mirror-image proteins;
racemic protein crystallography
Background
“In the field of protein synthesis, it is my confident hope that
tomorrow’s deeds will catch up with today’s titles, and that
we shall truly be able to obtain enzymatically active proteins
as synthetic substances: as materials composed of a single
molecular species.”
J. Rudinger [1]
The total chemical synthesis of proteins – particularly enzymes –
was one of the grand challenges of 20th century organic chemistry.
The great chemist Emil Fischer had, in early pursuit of that goal,
articulated the ‘peptide theory’ of protein (and enzyme) structure
and had gone on to pioneer the field of chemical peptide synthesis.
In the three decades after the Second World War, organic chemists
addressed with renewed enthusiasm this goal of total synthesis of
protein molecules that have enzymatic activity. Novel methods of
peptide synthesis were developed, including a wide range of protecting
group and activation chemistries. Initially, these tools were
applied to the chemical synthesis of biologically active peptides
found in nature. Then, as the covalent and X-ray structures of
enzyme protein molecules became available from the work of
Anfinsen, Moore and Stein, Phillips, Harker, and others, these
methods for the chemical synthesis of peptide chains were applied
to the total chemical synthesis of enzymes. Classical solution methods
of peptide synthesis culminated in the attempted total synthesis of
‘consensus lysozyme’ by Kenner and co-workers [2]. Also in the
1970s, Gutte and Merrifield reported the use of stepwise solid phase
peptide synthesis to make RNase A with high enzymatic activity [3].
Nonetheless, despite the powerful methods that had been
developed for the chemical synthesis of peptide chains, through
the early 1990s reproducible total synthesis was limited to only
the smallest proteins containing ~50 amino acids [4–7]. The main
problems limiting the size of synthetically accessible peptide
chains were the insolubility of protected peptide segments and
racemization in solution synthesis, and the statistical accumulation
of error peptide byproducts in stepwise solid phase synthesis.
In 2007, Durek reported the total chemical synthesis of human
lysozyme (130 amino acid residues; eight Cys residues in four
disulfides) and the determination of the high-resolution X-ray
structure of the synthetic enzyme, which had full catalytic activity
(Figure 1) [8]. Contemporaneously, Torbeev reported the total
chemical synthesis of a 203 amino acid residue covalent dimer of
the HIV-1 protease protein molecule, also characterized by
high-resolution X-ray crystallography and with full enzymatic
activity (Figure 2) [9]. Each of these total syntheses was the work
of just one talented young scientist using manual synthetic
methods. Clearly, a transformation of our ability to make proteins
by chemical synthesis had taken place in the preceding ~15 years.
* Correspondence to: Stephen Kent, University of Chicago, Chicago, IL 60637, USA.
E-mail: skent@uchicago.edu
{ The Josef Rudinger Lecture Award of the European Peptide Society was
assigned to Professor Stephen Kent on occasion of the 31st European Peptide
Symposium, Copenhagen, September 5–9, 2010. The present review is based
on this lecture.
University of Chicago, Chicago, IL 60637, USA
J. Pept. Sci. 2012; 18: 428–436 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
Review
Received: 27 April 2012 Accepted: 30 April 2012 Published online in Wiley Online Library: 4 June 2012
(wileyonlinelibrary.com) DOI 10.1002/psc.2421
428
Chemical Ligation
“. . . the synthesis of slightly “generalized” enzymes, that is
of simplified structures formally derived from the natural
enzymes, . . . in such a way as to eliminate as many synthetic
complications as possible while maintaining the structures
which appear essential. If this should enable us to obtain a homogeneous
substance with the appropriate enzymic properties,
the loss of “naturalness” will have been worth it.”
J. Rudinger [1]
The concept that enabled individual researchers to carry out
the robust, reproducible total synthesis of enzyme protein molecules
was ‘chemical ligation’, the chemoselective covalent condensation
of unprotected peptides to give long polypeptide
chain products [10]. The chemical ligation principle is shown in
Figure 3. The key underlying concept is the formation of a nonnative
(analogue) structure at the site of covalent joining of the
peptide segments; formation of this unnatural structure, not
found in a protein’s polypeptide chain, allowed the use of a pair
of unique, mutually reactive functional groups – one on each of
the peptide segments to be condensed – and thus did away with
the need for protecting groups to direct the outcome of the
condensation reaction. The result was a substantial simplification
of the synthetic process and enabled the direct preparation of
the long polypeptide chains found in protein molecules. Unprotected
peptide segments are usually soluble in aqueous organic
solvent mixtures and can be readily prepared by highly optimized
SPPS, purified by reverse-phase HPLC, and characterized by LCMS.
Chemoselective reaction with the formation of an unnatural structure
at the ligation site also obviated racemization. Additionally,
the full-length target polypeptide chain resulting from the chemical
ligation of unprotected peptide segments is obtained in final
form without the need for further chemical manipulations.
Thioester-forming chemical ligation, from the reaction of a
peptide-thiocarboxylate and a bromoacetyl-peptide, was used in
the initial demonstrations of chemical ligation for the total
synthesis of proteins. Mirror-image forms of the HIV-1 protease
enzyme molecule were prepared by thioester-forming chemical
ligation and served to illustrate the reciprocal chiral specificity of
mirror-image proteins acting on chiral substrates [11,12]. More
complex protein constructs were also prepared using thioesterforming
and oxime-forming ligation chemistries in combination
[13]. Subsequently, thioester-forming chemical ligation was
extended to the thioester-mediated formation of a native amide
bond linking the two unprotected peptide segments (‘native
chemical ligation’) [14].
Ester Insulin
“In the 50 years since the determination of the insulin structure
by Sanger, there have been sizable accomplishments in
the chemical synthesis of insulin, but we still lack a high-yield
synthesis of this molecule. This is an obstacle to the development
of improved insulin analogs. . .”
J.P. Mayer, F. Zhang, and R.D. DiMarchi [15]
The total chemical synthesis of human insulin has been one of
the principal synthetic objectives of organic chemistry. Reproducible
total syntheses of human insulin and analogues were
reported in the 1970s [4,5]. Nevertheless, because of the challenge
of accurate recombination of the two-chain insulin molecule
from individual synthetic A and B chains, an efficient total
chemical synthesis of insulin has continued to elude the research
community [15]. In the course of research aimed at a synthetic
route to a chemical analogue of proinsulin, we noted that the
side chain carboxyl of GluA4 was in contact with the side chain
hydroxyl of ThrB30. We envisioned an ester bond linking those
two side chains and set out to examine the feasibility of a total
chemical synthesis of the insulin protein molecule via an ‘ester
insulin’ intermediate (Figure 4). The ester-linked polypeptide
precursor was prepared by a combination of SPPS and native
chemical ligation; this depsipeptide chain folded efficiently at
physiological pH in the presence of a redox couple to give ‘ester
insulin’. Multidimensional NMR and LCMS data were consistent
with the formation of three native disulfide bonds in ester insulin.
Simple saponification at 4
C gave fully active human insulin in
greater than 90% yield [16].
Ester insulin is the key to the efficient total chemical synthesis
of insulin analogues and will enable the application of the principles
of medicinal chemistry to the further optimization of the
properties of the insulin molecule for human therapeutic use [17].
Erythropoietin
“. . . access to homogeneous and structurally definable
glycoproteins could be of significant value for the systematic
study of the effects of protein glycosylation on biology-level
performance. . . In particular, de novo chemical synthesis might
provide a powerful path to designed glycoproteins as it offers,
in principle, precise structural control while providing opportunities
for systematic variation of both the structures of the
glycodomains and their location within the peptide sequence.”
C. Kan and S.J. Danishefsky [18]
The glycoprotein erythropoietin (EPO) stimulates the production
of erythrocytes and is widely used as a treatment for anemia; the
recombinant EPO used as a human therapeutic is produced in
genetically engineered mammalian cell cultures and consists of a
number of different glycoforms with variable sialic acid content.
A total synthesis of the EPO glycoprotein is a current objective
for organic chemistry groups throughout the world in much the
same way that insulin was in the 1970s. The goal is the production
Biography
Stephen Kent took his BSc in
Chemistry and Biochemistry
and his MSc (Thesis: ‘Peptide
sequences by mass spectrometry’)
in his native New Zealand.
He completed a PhD in Organic
Chemistry at the University of
California, Berkeley (Thesis:
‘Specific chemical modification
and 13
C-NMR properties of the lysine at the active site of
alcohol dehydrogenase’). From 1974 to 1981, he worked with
R. Bruce Merrifield at the Rockefeller University in New York.
Subsequently, Kent held academic positions at the California
Institute of Technology, Bond University, and The Scripps
Research Institute before joining the University of Chicago
where he is now Professor of Chemistry and Professor of Biochemistry
and Molecular Biology.
THROUGH THE LOOKING GLASS
J. Pept. Sci. 2012; 18: 428–436 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci
429
of defined glycoforms in order to correlate the chemical structure
of the EPO molecule with its biological activities [18].
In 2003, we reported the design and total chemical synthesis of
‘synthetic erythropoiesis protein’ (SEP), a glycoprotein mimetic of
50,825 Da with full biological activity and extended half-life in vivo
(Figure 5) [19]. The target 166 amino acid polypeptide chain was assembled
by the native chemical ligation of four synthetic peptides.
Two of these peptide segments had identical covalently attached
‘glycan mimetic’ moieties; these branched oligo{ethyleneoxideamide}
molecules were made by conventional synthetic chemistry,
were monodispersed, and had defined covalent structure. Each glycan
mimetic was designed to increase the hydrodynamic volume
of SEP, and each had four carboxylates to provide negative charges
in the same way as a natural sialic acid. This synthetic tour de force
involved more than 25 researchers and was carried out in a commercial
research laboratory.
More recently, in our university laboratory, Suhuai Liu undertook
the fully convergent synthesis of [Lys24,38,83
]EPO from five
Figure 1. Total chemical synthesis of human lysozyme. (A) Scheme for the convergent chemical synthesis of the 130 amino acid polypeptide chain
(sequence inset) and its folding to give human lysozyme containing four disulfide bonds; (B) LCMS characterization of the synthetic enzyme molecule;
(C) a portion of the 2Fo-Fc electron density map with fitted structural model, used to determine the high-resolution (1.04 Å) X-ray structure of the
synthetic enzyme [adapted from Ref. [8]].
KENT ET AL.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2012; 18: 428–436
430
synthetic peptide segments by convergent chemical ligation
(Figure 6) [20]. By contrast with the synthesis of synthetic erythropoiesis
protein a decade earlier, synthesis of [Lys24,38,83
]EPO was
accomplished by a single individual using manual methods. A
key feature of the approach was the use of kinetically controlled
ligation [21] in a convergent synthetic scheme; another key
aspect of the synthesis was selective desulfurization of Cys residues
in the presence of protected Cys(Acm) residues, enabling
native chemical ligation at Xaa-Ala sites [22,23]. Final removal
of the Acm groups and folding with concomitant formation of
two native disulfides gave synthetic [Lys24,38,83
]EPO that had full
biological activity in a factor-dependent cell line proliferation
assay. In collaboration with Danny Lee in the Margaret Brimble
Lab at the University of Auckland, we are extending this work
to the use of triazole-forming ‘click’ chemistry for the total
chemical synthesis of a neoglycan form of EPO. This work is
based on our recent report of the compatibility of native chemical
ligation and triazole-forming click chemistry in the preparation
of synthetic neoglycopeptides [24].
Racemic Protein Crystallography
“. . . more importantly, since we expect P1- to be significantly
more probable than other symmetries, we predict that
racemic protein mixtures will crystallize more readily than
samples consisting only of the biological enantiomer.”
S.W. Wukovitz and T.O. Yeates [25]
In 2008, we reported the use of racemic protein crystallography
to determine the X-ray structure of the snow flea antifreeze
protein (sfAFP). This novel protein was found to have an unprecedented
structure made up entirely of polyproline type II helices
connected by reverse turns [26]. In the course of that work, we
observed that a racemic mixture made up of D-sfAFP and L-sfAFP
crystallized much more readily than the L-sfAFP protein alone.
This facile crystallization was also observed for a quasi-racemic
mixture of D-sfAFP with a L-[SeCH2]sfAFP.1
In a prescient theoretical
paper describing the frequency of occurrence of the ‘biological’
space groups in protein crystallography, Wukovitz and
Yeates had predicted the more facile crystallization of racemic
Figure 2. Total chemical synthesis of a covalent dimer of HIV-1 protease. (A) Scheme for the convergent synthesis of the 203 amino residue polypeptide
chain from four synthetic peptide segments; (B) Fourier transform ion cyclotron resonance electrospray mass spectrometric characterization of the synthetic
enzyme molecule; (C) X-ray structure of the synthetic enzyme; (D) catalytic activity of the synthetic enzyme (adapted from Ref. [9]).
Figure 3. The chemical ligation principle [10].
1
The protein molecule L-[SeCH2]sfAFP contains a ‘pseudo-Gln’ residue made
by alkylating selenocysteine with bromoacetamide, where the D-protein contains
D-Gln, to produce a heavy atom analogue to solve the phase problem
in X-ray analysis [26].
THROUGH THE LOOKING GLASS
J. Pept. Sci. 2012; 18: 428–436 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci
431
Figure 4. Ester insulin (adapted from Ref. [16]).
Figure 5. Synthetic erythropoiesis protein (reprinted from [32], with permission from Elsevier).
KENT ET AL.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2012; 18: 428–436
432
protein mixtures in centrosymmetric space groups [25]. In multiple
instances, encompassing more than a dozen proteins, we have
verified this prediction.
Recently, we designed an analogue of the model protein crambin
in which an ion pair between the side chain guanidinium of Arg10
and the a-carboxylate of Asn46 was replaced by a covalent (amide)
bond [27]. This protein was designed to contain a novel linear-loop
polypeptide chain covalent topology not yet observed in nature.
We set out to chemically synthesize the linear-loop polypeptide
chain, to fold it to form a protein molecule, and to determine the
X-ray structure of this novel protein analogue. In order to effect
the synthesis, we developed the ‘kinetically controlled ligation’
method for the condensation of a peptide-thioarylester with a
Cys-peptide-thioalkylester to give a product peptide-thioalkyl-ester
Figure 6. Total chemical synthesis of [Lys24,38,83
]EPO (adapted from Ref. [20]).
Figure 7. X-ray structure of a novel topological analogue of crambin
(adapted from Ref. [27]).
THROUGH THE LOOKING GLASS
J. Pept. Sci. 2012; 18: 428–436 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci
433
Figure 8. Total chemical synthesis of VEGF-A. The observed mass of the unfolded monomer was 11,932.2 Æ 0.7 Da; the observed mass of the folded
synthetic VEGF-A protein molecule was 23,848.7 Æ 1.2 Da (adapted from Ref. [29]).
KENT ET AL.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2012; 18: 428–436
434
[21]; then, we applied kinetically controlled ligation to the synthesis
of the key branched thioester intermediate, followed by intramolecular
native chemical ligation and folding with concomitant formation
of three disulfide bonds as shown by LCMS [27].
Our challenge was to determine the folded structure of this
novel protein molecule. Attempts to crystallize the protein analogue
on its own were not successful. The topological analogue
(‘topologue’) protein molecule was readily crystallized from a
quasi-racemic mixture with D-crambin, and the structure was
solved by molecular replacement using inverted coordinates
for L-crambin. The high-resolution X-ray data showed that the
topological analogue had a unique folded structure in which
the N-terminal of the polypeptide chain penetrated the macrolactam
ring and is pinned in that position by two disulfide
bonds (Figure 7) [27]. Facile crystallization of the quasi-racemic
protein mixture was key to determination of the structure of
this novel topological protein analogue.
Mirror-Image Protein Therapeutics
“A method is described that uses a biologically encoded library
for the identification of D-peptide ligands, which should be resistant
to proteolytic degradation. In this approach, a protein
is synthesized in the D-amino acid configuration and used to
select peptides from a phage display library expressing random
L-amino acid peptides. For reasons of symmetry, the
mirror images of these phage-displayed peptides interact
with the target protein of the natural handedness.”
Peter S. Kim and associates [28]
In 1996, Peter Kim and colleagues at the Whitehead Institute at the
Massachusetts Institute of Technology published a novel ‘mirror image
phage display’ technique for the systematic development of D-peptide
ligands for a natural protein target molecule [28]. This ingenious method
uses themirror-image D-protein formof target proteinmoleculeto screen
phage-displayed peptide libraries and select high-affinity peptide ligands.
The selected peptide ligands are chemically synthesized in mirror--
image D-peptide form; because of the reciprocal nature of chiral interac-
tionsatthemolecularlevel,theseD-peptideswillhavethesamespecificity
and affinity for the natural L-protein form of the target molecule. The
limiting feature of this approach to the development of D-peptide ligands
is the ability to synthesize the natural target as a D-protein molecule.
In collaboration with the Sachdev Sidhu Lab at the University of
Toronto, we have set out to extend Kim’s approach to screen phagedisplayed
protein libraries against the mirror-image D-protein form
of vascular endothelial growth factor (D-VEGF-A). First, we set out to
establish efficient chemical synthesis of L-VEGF-A (Figure 8) [29].
The VEGF-A protein molecule is a covalent homodimer: each 102
amino acid residue monomer polypeptide chain has three intrachain
disulfides, and there are two disulfide bonds connecting the monomers
in the VEGF-A molecule. The target 102 residue polypeptide
chain was made from three synthetic peptide segments by sequential
‘one pot’ native chemical ligation reactions. A single purification step
gave high purity product with the correct mass. Folding was carried
out at pH8.4 in aqueous 0.15M guanidineÁHCl in the presence of a redox
couple and, over a period of several days, gave a near quantitative
yield of folded VEGF-A with the correct mass by direct infusion electrospray
mass spectrometry [29]. Synthetic L-VEGF-A was fully active in a
human umbilical vein endothelial cell proliferation assay; the chemically
synthesized protein was crystallized, and its X-ray structure was
determined to 1.8Å resolution and was identical, within experimental
error, to the reported structure of recombinant VEGF-A [29].
The mirror-image protein D-VEGF-A has been prepared using a
similar synthetic strategy and screened against phage-displayed
libraries of variants of a small protein scaffold. These results will be
reported elsewhere [30].
Conclusion
“The chemical ligation approach. . . breaks the conceptual
shackles imposed by the peptide bond, frees us from the linear
paradigm of the genetic code, and opens the world of proteins
to the entire repertoire of chemistry.”
S. Kent and associates [31]
Total protein synthesis based on modern chemical ligation
methods enables the reproducible preparation of protein molecules
of a size that less than 20years ago was considered to be inaccessible
to chemistry. Key to this ability is the covalent chemical
condensation of unprotected peptide segments by means of an
unnatural link between the two reacting segments. In the native
chemical ligation refinement of this approach, the initial thioester-linked
intermediate rearranges to give a native amide bond
at the ligation site. Enzymes can be prepared in crystalline form
and with full catalytic activity. An efficient route to the total chemical
synthesis of human insulin and analogues has been devised on
the basis of the use of an ester-linked synthetic intermediate that
folds in quantitative yield and that can be simply converted in high
yield to fully active human insulin. Thus, two of the grand challenges
of synthetic protein chemistry have been successfully
realized.
Modern chemical ligation methods have also enabled the design
and total chemical synthesis of ‘synthetic erythropoiesis protein’, a
glycoprotein mimetic of defined chemical structure with a molecular
weight of 50,825g/mol that has full biological activity and an extended
half-life in vivo.
Most significantly, chemical protein synthesis enables the routine
preparation of ‘mirror-image’ D-protein molecules. Mirror-image enzyme
molecules display reciprocal chiral specificity – a D-enzyme will
act only on the mirror image of the natural substrate. The highly ordered
crystals necessary for determination of high-resolution X-ray
structures form with great ease from racemic protein mixtures. This facile
crystallization of racemic protein mixtures overcomes the principal
obstacle to determination of protein structures by X-ray diffraction
and has proven useful for the determination of the structure of novel
proteins and novel protein analogues. Mirror-image protein molecules
can also be used to develop D-peptide (and D-protein) ligands specific
for the natural form of a target protein molecule. Such D-peptide molecules
are invisible to biochemical metabolism and may have advantages
as human therapeutics.
In the future, we can anticipate the elucidation of important biological
questions enabled by chemical protein synthesis and the broader
application of mirror-image proteins to the development of novel human
therapeutics.
References
1 Rudinger J. In Chemistry and Biology of Peptides – Proceedings of the
3rd American Peptide Symposium, Meienhofer J (ed.) Ann Arbor
Science Publishers, 1972; 729–735.
2 Kenner GW. Towards synthesis of proteins. Proc. R. Soc. Lond. A 1977;
353: 441–457.
3 Gutte B, Merrifield RB. The synthesis of ribonuclease A. J. Biol. Chem.
1971; 246: 1922–1941.
THROUGH THE LOOKING GLASS
J. Pept. Sci. 2012; 18: 428–436 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci
435
4 Sieber P, Kamber B, Hartmann A, Jöhl A, Riniker B, Rittel W. Totalsynthese
von Humaninsulin unter gezielter Bildung der Disulfidbindungen. Helv.
Chim. Acta 1974; 57: 2617–2621.
5 Eisler K, Kamber B, Riniker B, Rittel W, Sieber P, De Gasparo M, Marki F.
Synthesis and biological activity of five D-Cys analogs of human insulin.
Bioorg. Chem. 1979; 8: 443–450.
6 Heath WF, Merrifield RB. A synthetic approach to structure–function
relationships in the murine epidermal growth factor molecule. Proc.
Natl. Acad. Sci. U.S.A. 1986; 83: 6367–6371.
7 Woo DD-L, Clark-Lewis I, Chait BT, Kent SBH. Chemical synthesis in
protein engineering: total synthesis, purification, and covalent structural
characterization of a mitogenic protein, human transforming growth
factor-a. Protein Eng. 1989; 3: 29–37.
8 Durek T, Torbeev VY, Kent SBH. Convergent chemical synthesis and
high resolution X-ray structure of human lysozyme. Proc. Natl. Acad.
Sci. U.S.A. 2007: 104: 4846–4851.
9 Torbeev VY, Kent SBH. Convergent chemical synthesis and crystal
structure of a 203 amino acid ‘covalent dimer’ HIV-1 protease enzyme
molecule. Angew. Chem. Int. Ed. 2007; 46: 1667–1670.
10 Schnölzer M, Kent SBH. Constructing proteins by dovetailing unprotected
synthetic peptides: backbone engineered HIV protease. Science
1992; 256: 221–225.
11 deLisle Milton RC, Milton SCF, Kent SBH. Total chemical synthesis of a
D-enzyme: the enantiomers of HIV-1 protease demonstrate reciprocal
chiral substrate specificity. Science 1992; 256: 1445–1448.
12 deLisle Milton RC, Milton SCF, Schnölzer M, Kent SBH. Synthesis of
proteins by chemical ligation of unprotected peptide segments:
mirror-image enzyme molecules, D- & L-HIV protease analogues. In
Techniques in Protein Chemistry IV, Angeletti R (ed.). Academic Press:
New York, 1993; 257–267.
13 Canne LE, Ferré-D’Amaré AR, Burley SK, Kent SBH. Total chemical synthesis
of a unique transcription factor-related protein: cMyc-Max. J.
Am. Chem. Soc. 1995; 117: 2998–3007.
14 Dawson PE, Muir TW, Clark-Lewis I, Kent SBH Synthesis of proteins by
native chemical ligation. Science 1994; 266: 776–779
15 Mayer JP, Zhang F, DiMarchi RD. Insulin structure and function. Biopolymers
2007; 88: 687–713.
16 Sohma Y, Hua Q-X, Whittaker J, Weiss MA, Kent SBH. Design and total
synthesis of [GluA4(ObThrB30)]insulin (‘ester insulin’): a minimal
proinsulin surrogate that can be chemically converted into human
insulin. Angew. Chem. Int. Ed. 2010; 49: 5489–5493.
17 Musiol H-J, Moroder L. Two-chain insulin from a single-chain
branched depsipeptide precursor: the end of a long journey. Angew.
Chem. Int. Ed. 2010; 49: 7624–7626.
18 Kan C, Danishefsky SJ. Recent departures in the synthesis of peptides
and glycopeptides. Tetrahedron 2009; 65: 9047–9065.
19 Kochendoerfer GG, Chen S-Y, Mao F, Cressman S, Traviglia S, Shao H,
Hunter C, Low D, Cagle N, Carnevali M, Gueriguian V, Keogh P, Porter
H, Stratton SM, Wiedeke MC, Wilken J, Tang J, Levy JJ, Miranda LP,
Crnogorac M, Kalbag S, Botti P, Schindler-Horvath J, Savatski L, Adamson
JW, Kung A, Kent SBH, Bradburne JA. Design and chemical synthesis of a
homogeneous polymer-modified erythropoiesis protein. Science 2003;
299: 884–887.
20 Liu S, Pentelute BL, Kent SBH. Convergent chemical synthesis of
[Lysine24,38,83
]human erythropoietin. Angew. Chem. Int. Ed. 2012; 51: 993–999.
21 Bang D, Pentelute BL, Kent SBH. Kinetically-controlled ligation for the
convergent chemical synthesis of proteins. Angew. Chem. Int. Ed.
2006; 45: 3985–3988.
22 Pentelute BL, Kent SBH. Selective desulfurization of cysteine in the
presence of Cys(Acm) in polypeptides obtained by native chemical
ligation. Org. Lett. 2007; 9: 687–690.
23 Wan Q, Danishefsky SJ. Free-radical-based, specific desulfurization of
cysteine: a powerful advance in the synthesis of polypeptides
and glycopolypeptides. Angew. Chem. Int. Ed. 2007; 46: 9248–9252.
24 Lee DJ, Mandal K, Harris PWR, Brimble MA, Kent SBH. A one-pot
approach to neoglycopeptides using orthogonal native chemical
ligation and click chemistry. Org. Lett. 2009; 11: 5270–5273.
25 Wukovitz SW, Yeates TO. Why protein crystals favour some space-groups
over others. Nat. Struct. Biol. 1995; 2: 1062–1067.
26 Pentelute BL, Gates ZP, Tereshko V, Dashnau J, Vanderkooi JM,
Kossiakoff AA, Kent SBH. X-ray structure of snow flea antifreeze
protein determined by racemic crystallization of synthetic protein
enantiomers. J. Am. Chem. Soc. 2008; 130: 9695–9701.
27 Mandal K, Pentelute BL, Bang D, Gates ZP, Torbeev VY, Kent SBH Design,
total chemical synthesis, and X-ray structure of a protein having a novel
polypeptide chain topology. Angew. Chem. Int. Ed. 2012; 51: 1481–1486.
28 Schumacher TNM, Mayr LM, Minor Jr. DL, Milhollen MA, Burgess MW,
Kim PS. Identification of D-peptide ligands through mirror-image
phage display. Science 1996; 271: 1854–1857.
29 Mandal K, Kent SBH. Total chemical synthesis of biologically active
vascular endothelial growth factor. Angew. Chem. Int. Ed. 2011; 50:
8029–8033.
30 Mandal K, Uppalapati M, Ault-Riché D, Kenney J, Lowitz J, Sidhu S,
Kent SBH. X-Ray structure of a diastereochiral complex of VEGF-A
and a D-protein antagonist designed by phage display. Proc. Natl.
Acad. Sci. U.S.A. 2012; submitted.
31 Baca M, Canne L, Chai R, Dawson P, Muir T, Smith R, Walker S, Kent
SBH. A general total synthesis of proteins: chemical ligation of
unprotected peptides. 23rd European Peptide Symposium, Braga,
1994 September 4–10, Portugal, Abstract. L07.
32 Casi G, Hilvert D. Convergent protein synthesis. Curr. Opin. Struct. Biol.
2003; 13: 589–594.
KENT ET AL.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2012; 18: 428–436
436