Synthesis of native proteins by chemical ligation.

TL;DR: The facile access to novel analogs provided by chemical protein synthesis has led to original insights into the molecular basis of protein function in a number of systems.

Abstract: ▪ Abstract In just a few short years, the chemical ligation of unprotected peptide segments in aqueous solution has established itself as the most practical method for the total synthesis of native proteins. A wide range of proteins has been prepared. These synthetic molecules have led to the elucidation of gene function, to the discovery of novel biology, and to the determination of new three-dimensional protein structures by both NMR and X-ray crystallography. The facile access to novel analogs provided by chemical protein synthesis has led to original insights into the molecular basis of protein function in a number of systems. Chemical protein synthesis has also enabled the systematic development of proteins with enhanced potency and specificity as candidate therapeutic agents.

Summary

INTRODUCTION: Protein Science in the Postgenome Era

• An important current objective in biomedical research is to understand the molecular basis of the numerous and intricate biological activities of proteins and therefore to be able to predict and control these activities.
• These attempts to incorporate other amino acids have had very limited successobtaining adequate amounts of pure protein from the cell-free translation systems can be a significant challenge (8) , and many unnatural amino acids are simply not compatible with ribosomal polypeptide synthesis (9) .
• The use of synthetic chemistry promises the unlimited variation of the covalent structure of a polypeptide chain with the objective of understanding the molecular basis of protein function.

DOMAINS: Building Blocks of the Protein World

• Because proteins are large molecules, applying chemical synthesis to them is a considerable challenge.
• Furthermore, the biological functions of proteins originate in the tertiary structure of the protein molecule-that is, in the precise three-dimensional folded structure of the polypeptide chain.
• Protein domains are defined as autonomous units of folding and, frequently, of function (17, 18) .
• The challenge confronting the chemist is, first, the total synthesis of folded domains and then the ability to stitch these domains together to build complex protein molecules.

CHEMICAL PROTEIN SYNTHESIS: The State of the Art in 1990

• Since last reviewed in this journal (19) , total chemical synthesis of native proteins has made a number of important contributions to biomedical research.
• The existence of this virally encoded aspartyl proteinase had been postulated based on an analysis of viral nucleic acid sequence data, and molecular genetic studies had indicated that its action in processing the gag-pol polyprotein was essential to the viral life cycle (21) .
• The first preparations of the enzyme of defined molecular composition were produced by chemical synthesis (22) , using a highly optimized version of stepwise solid-phase peptide synthesis (19) .
• In a strikingly important contribution, total chemical synthesis was also used to prepare large amounts of homogeneous enzyme for the determination of the original crystal structures of the HIV-1 protease molecule .
• These structural data were made freely available to the research community and formed the foundation for the successful worldwide programs of structure-based drug design (28) that led to the development of the highly effective protease inhibitor class of acquired immune deficiency syndrome therapeutic agents (29) .

SYNTHETIC-PEPTIDE CHEMISTRY: Useful but Bounded

• At the start of the decade of the 1990s total chemical synthesis, by the standard methods of peptide chemistry of even a small protein molecule remained a daunting task, often requiring large teams and taking years to complete, with no guarantee of success.
• Classical solution synthetic chemistry involves the preparation of fully protected peptide segments and their subsequent condensation in organic solvents for the convergent synthesis of large polypeptides (36) .
• This poor solubility made such protected peptide segments difficult to work with, and the low concentrations attainable for reacting segments often led to slow and incomplete reactions (37, 38) .
• This ingenious chemical synthesis method, the progenitor of all polymersupported organic chemistry, was introduced in 1963 by Merrifield (13) .
• Such methods have not found widespread use.

CHEMICAL LIGATION OF UNPROTECTED PEPTIDE SEGMENTS

• As recently as 1991 (48) , the challenge remained: namely, to develop methods that enable the general application of the tools of chemistry to the world of the protein molecule.
• Based on this premise, in the early 1990s the principle of chemoselective reaction (49) was adapted to enable the use of unprotected peptide segments in chemical protein synthesis (50) .
• The product polypeptide is obtained directly in final form.
• A variety of ligation chemistries has been used (Table 1 ), and the chemical ligation of unprotected peptide segments has provided access to a range of protein targets.

NATIVE CHEMICAL LIGATION

• Only a single reaction product is obtained, even in the presence of additional Cys residues in either segment.
• Simply mixing together two peptide segments that contain correctly designed, mutually reactive functionalities led to the formation of a single polypeptide product containing a native peptide bond at the ligation site.
• This highly chemoselective reaction is performed in aqueous solution at neutral pH under denaturing conditions.
• Detailed studies of mechanistic aspects of the native chemical ligation reaction have been published (63, 65) .

Conformationally Assisted Ligation

• In some cases, folding conditions can be used to accelerate the rate of native chemical ligation (76) .
• In these cases, the revealed N and C terminals of the peptide fragments are located in close proximity at the site of chain scission.
• This greatly increases the collision frequency, and a weakly activated C-terminal group such as a thioester can be used to religate the fragments.
• Total synthesis of proteins using this approach has been demonstrated with the chymotrypsin inhibitor CI2 .
• When two synthetic peptide segments spanning the CI2 molecule, one incorporating a C-terminal thioester and the other an N-terminal cysteine, are mixed together under folding conditions, conformationally-assisted ligation proceeds in <2 min, compared with several hours for chemical ligation under denaturing conditions (76) .

SCOPE OF NATIVE CHEMICAL LIGATION FOR THE SYNTHESIS OF PROTEINS

• The first applications of native chemical ligation were to small, Cys-rich proteins such as disulfide-cross-linked secretory proteins or the zinc-finger proteins.
• In all, >300 biologically active proteins from >20 different families have been successfully prepared by total chemical synthesis with this method.
• These are still early results in what will surely be more widespread application of the method, but they demonstrate routine synthetic access to single-domain proteins and suggest that native chemical ligation will provide the basis of a general synthetic access to the world of proteins.

FOLDING SYNTHETIC PROTEINS

• The activity of a protein molecule originates in the precise tertiary structure of the folded polypeptide chain.
• (Top) Ribbon structure of the crystalline dimer.
• The successful syntheses of such proteins suggests that this ability to accurately fold synthetic polypeptide chains may hold true both for single domains and for more complex proteins.
• Control of the folding process can be particularly important in the production of proteins that are toxic to the cell, such as proteolytic enzymes.
• This control over enzymatic activity was one of the key features of the success of chemical protein synthesis in the early work on the HIV-1 protease (20) .

Noncoded Amino Acids

• Demonstrating the power of the chemical protein synthesis method, large amounts of each protein analog were made, purified, and fully characterized.
• Noncoded amino acids are frequently found in native proteins in vivo.
• One common modification of this type is γ -carboxy-glutamic acid (Gla), found for example in the eponymous Gla domains in plasma proteins.
• This multidomain protein consists of an N-terminal Gla domain that contains 11 Gla residues, which is followed by a thrombin-sensitive region, three epidermal growth factor domains, and a sex hormone-binding globulinlike region.
• Folding of this polypeptide chain produced a three-domain protein, microProtein S, that displayed anticoagulant cofactor activity.

Precise Covalent Modification

• The ability to prepare native proteins by total synthesis, using chemical ligation of unprotected peptide segments, provides a convenient and general route to sitespecific modification of the protein molecule.
• The full range of synthetic peptide and peptidomimetic chemistry (86) is at the command of the researcher who wants to make precise and controlled changes in a protein's covalent structure.
• Such changes are not limited by the genetic code or by the strictures of the ribosomal machinery.
• An early example of the utility of this approach was the total chemical synthesis of (BTD) HIV-1 protease (87), a protein in which the Gly-Gly sequence found in a β-turn in the native protein (20) was replaced by the sterically constrained bicyclic compound BTD, a rigid mimetic of type II β-turn geometry (88) .
• The resulting enzyme showed full activity and a significantly enhanced thermostability (87) .

Site-Specific Tagged Proteins

• Chemical synthesis enables the specific labeling of a protein molecule at unique site(s).
• Such specific modification is less likely to perturb the structure or activity of the protein than uncontrolled reaction with labeling reagents that stochastically target all amino or other particular functional groups in the protein.
• Fluorescently tagged proteins are extremely useful tools for biology and drug discovery, and synthesis of native proteins by chemical ligation allows the facile incorporation of fluorescent dye molecules at any desired position in a protein molecule.
• Dye chelator-labeled proteins have been made for time-resolved fluorescence studies, in which it is possible to largely eliminate background emission by use of suitably "time-gated" detection (C Hunter, G Kochendoerfer, 89a).
• Finally, total chemical synthesis allows the ready introduction of affinity tags, such as biotin, at precise sites in the protein molecule, while preserving biological activity, again something that is straightforward with chemistry.

Backbone Engineering

• Another intriguing example of site-specific modification of the protein molecule, enabled by chemical ligation, is the covalent modification of the polypeptide backbone itself.
• A functionally important peptide bond (i.e. backbone amide) in the HIV-1 protease molecule was site-specifically replaced by a thioester moiety in each monomer of the homodimeric protein molecule, to investigate the direct involvement of that specific peptide bond in the mechanism of action of the enzyme, as suggested by the X-ray crystallographic data (20, 56) .
• The results of these studies showed that the two flap regions of the homodimeric native HIV-1 protease molecule work analogously to the single flap moiety in the two-domain, single-polypeptide chain, cell-encoded aspartyl proteinases (90) .
• More recently, an engineered backbone structure was introduced into bovine pancreatic trypsin inhibitor, by replacing one Cys residue involved in forming a disulfide bond with an NGly, to investigate the effects of such a substitution on the folding, activity, structure, and stability of the resulting protein molecule (92) .
• The side chain of the Cys residue has effectively been moved to the backbone amide N atom.

Protein Medicinal Chemistry

• Synthetic access enables the systematic application of the principles of medicinal chemistry to the protein molecule itself.
• An example is the total chemical synthesis of the potent anti-HIV molecule AOP-RANTES (79) .
• The chemical protein analog NNY-RANTES, which resulted from the first phase of this program, is >30-fold more effective as an anti-HIV compound and has been shown to prevent HIV infection at low nanomolar concentrations in the huPBL-SCID mouse model for acuired immune deficiency syndrome (93) .
• NNY-RANTES is the most potent known anti-HIV compound.
• It is believed to work by inhibiting receptor recycling (94) , thus clearing CCR5 from the surface of peripheral blood cells, a mechanism distinct from current clinical therapies for acquired immune deficiency syndrome.

Rapid Access to Functional Gene Products

• In the past few years, an important new application has emerged for chemical protein synthesis-to enable rapid access to functional wild-type protein molecules directly from gene sequence data .
• Success of the genome projects has resulted in the discovery of >100,000 new proteins (1).
• The probable roles of many of these predicted proteins can be tentatively assigned by analogy to proteins of known function, using bioinformatics.
• Nevertheless, the precise biochemical properties of a mature gene product can only be assessed at the level of the protein molecule itself.
• Synthesis of native proteins by chemical ligation of unprotected peptides can provide access in a matter of days to large (10 + mg) amounts of functional protein molecules of exquisite homogeneity, based directly on gene sequence data.

Structural Biology

• Facile access to the large (i.e. multiple tens of milligram) amounts of high-purity preparations produced by chemical protein synthesis can be of great utility for studies of protein structure by NMR spectroscopy and by X-ray crystallography.
• New methods for NMR spectroscopy have considerably enhanced the speed with which the structure of small (i.e. <200-aa-residue) proteins can be determined.
• Examples include, the chemokine SDF-1α (81), the chemical protein analog AOP-RANTES , and the mirror-image enzyme molecule D-HIV-1 protease .
• Such high-throughput structure determination will require access to great numbers of proteins in high purity and large amount.
• Chemical protein synthesis by the methods described here is well suited to provide the proteins needed for genomic structural biology.

Expressed Protein Ligation

• From its inception, the native chemical ligation method was also envisioned for use with peptides that are produced by recombinant means (62) .
• There are now multiple examples of the chemical ligation of recombinant peptides.
• These recombinant products can be reacted with synthetic peptide-thioesters to generate native polypeptides of hybrid biological and chemical origin.
• More recently, intein-based protein expression vectors have been adapted to generate polypeptide thioesters by recombinant means for use in native chemical ligation (104, 105) .
• With the approaches described above, both the peptidethioester and the N-terminal Cys peptide can be of recombinant origin.

Solid-Phase Protein Synthesis

• The principles of polymer-supported organic synthesis (13, 19, 111) have been applied to the chemical ligation of unprotected peptide segments in aqueous solution [(112); Figure 17 ].
• In solid-phase chemical ligation, unprotected peptide segments of 35-50 amino acids (i.e. ∼5 kDa each) are used as building blocks to assemble the target polymer-bound polypeptide by consecutive ligation on a water-compatible polymer support.
• Strategies for segment condensation in both the N-to-C and C-to-N directions have been used successfully for solid-phase protein synthesis (112) and alternative linker chemistries developed (112a).
• Target molecules have been constructed from as many as eight peptide segments by solid-phase chemical ligation [e.g. the polypeptide of the tissue plasminagen activator catalytic domain; M w 25,000 (W Lu, unpublished data), and the polypeptide chain of the enzyme secretory PLA2 GV has beenassembled in a single day Figure 16 Expressed protein ligation (104) .
• This process uses intein-mediated (73) preparation of a recombinant peptide-α thioester, which is then reacted with a Cys-peptide segment by native chemical ligation to prepare the desired product.

Membrane Proteins

• An important aspect of the study of proteins which have been predicted from gene sequence data is the integral membrane class of proteins.
• Computer-aided analysis of the predicted open reading frames from a number of completely sequenced (13, 19, 111) are applied to solid-phase protein synthesis.
• After removal of the Cys-protecting group (PG), successive rounds of ligation can be carried out to give the polymer-bound target polypeptide.
• Yet integral membrane proteins are difficult to express at high levels by recombinant-DNA-based methods and have proven hard to isolate in homogeneous form in chemically defined media (114) .
• The M2 protein had previously proven refractory to multiple attempts at expression by recombinant-DNA-based methods (W Degrado, personal communication), but was readily obtained by chemical ligation of unprotected synthetic peptides.

Glycoprotein Synthesis

• Recently, the Bertozzi laboratory (116) reported the first total synthesis of a glycoprotein, using native chemical ligation in conjunction with innovative methods for the synthesis of glycopeptide-α thioesters.
• One of the most important applications of chemical protein synthesis will be the systematic preparation of glycoforms of gylcosylated proteins as homogeneous molecular species of defined covalent structure, to establish the role of the carbohydrate moiety in the biological function of the glycoprotein.

FUTURE DEVELOPMENTS Ligation Sites

• In its current form, native ligation chemistry uses a Cys residue at the site of formation of the new peptide bond joining two unprotected peptide segments.
• The work of Muir and coworkers is illustrative of this expedient but effective approach (66, 104, 110) .
• Also, biological researchers frequently insert Cys residues into a polypeptide chain to investigate the structurefunction relationships in a protein molecule (119) or as a site for the introduction of a spectroscopic probe, such as an electron spin resonance label (120) .
• This proven utility of arbitrarily introduced Cys residues provides considerable flexibility in synthetic design for the preparation of functional protein molecules by native chemical ligation at Cys.
• Additionally, it would be desirable to have the option to use thioester-mediated chemical ligation at residues other than Cys.

Size of Protein Targets

• To date, it has proved possible to make every protein that has been attempted by the chemical ligation of unprotected peptide segments in aqueous solution, even integral membrane proteins.
• Some targets are significantly more work than others-especially if there are multiple intermediate ligation products to handle.
• The recently developed solid-phase protein synthesis method (see above), using polymer-supported chemical ligation (112), provides a very effective means for the ready isolation of these intermediate products and will significantly simplify syntheses requiring ligation of multiple segments.
• The work of their own and others' laboratories, including the laboratories of Offord (University of Geneva, Switzerland) and Muir (Rockefeller University, New York, NY), has failed to show any inherent size limitations for application of the chemical ligation method, up to several-hundred kilodaltons in the latter case (122) .
• Folding of chemically synthesized polypeptide chains to form native proteins, in which significant problems might have been anticipated, is usually straightforward for the domain size proteins made to date.

Chemical Synthesis of Peptide Segments

• Virtually any target protein can be prepared by total chemical synthesis, provided that a suitable set of high-purity peptide-thioester segments is available.
• Ironically, for many researchers the most challenging aspect of applying the chemical ligation method to proteins is making the peptide segments.
• To date, the principal constraint on widespread application of the native ligation method has been the lack of methods for the facile chemical synthesis of unprotected peptide-α thioester segments.
• The need to make large numbers of analogs of thousands of native proteins by chemical ligation, and hence to prepare many tens-of-thousands of peptide segments, provides an unprecedented impetus for the development of efficient methods of peptide synthesis.
• The authors can look forward with confidence to the development of radically improved methods for the rapid, costeffective preparation of large numbers of unprotected peptide-thioester segments for use in chemical protein synthesis (124) .

SUMMARY AND CONCLUSIONS

• Total synthesis by the chemical ligation of unprotected peptide segments can now provide general access to native proteins of ≤30 kDa in size.
• This size range encompasses the structural and functional domains that are the modular building blocks of function in the protein world, from enzymes to receptors, from signal transduction adaptor molecules to large multisubunit protein assemblies.
• This will enable the dissection at the level of the protein molecule of important biochemistry, such as the intracellular signal transduction pathways.
• The stage is now set for the application of the tools of chemistry to the entire universe of proteins.
• "Where nucleic acids are the codes, proteins are the substance of life" (125) .

Figures

Figure 6 Total synthesis of a covalent heterodimeric transcription factor, cMyc-Max, by convergent chemical ligation (57). (Upper left) Molecular model of the covalent construct, bound to cognate duplex DNA. (Upper right) Synthetic scheme—each B/HLH/Z domain was assembled by thioester-forming chemical ligation of two peptide segments; these polypeptide products were then covalently linked by oxime-forming chemical ligation to yield a synthetic protein construct with two N terminals and no C terminal. (Lower right) Circular dichorism measurements showed that the covalent cMyc-Max construct folded correctly and was preordered even in the absence of cognate DNA. (Lower left) The covalent cMyc-Max heterodimer was active in a gel shift assay for DNA binding. Adapted from Reference 57 and Ferr´-D’Amare AR (1995. PhD thesis).

Figure 15 From gene sequence direct to functional protein, using chemical protein synthesis.

Figure 13 Three-dimensional crystal structure of the mirror image protein molecule, D-HIV-1 protease (20). The protein was prepared by thioester-forming chemical ligation of peptide segments synthesized with D-amino acids (52). (Left) Molscript representation of the ligated chemically synthesized D-protein molecule, displayed as the L-form for comparison purposes (20). (Right) Molscript representation of recombinant L-HIV-1 protease, prepared inE. coli (81a). The close similarity of the folded structures of the synthetic ligated D-protein and the recombinant L-protein is clearly evident.

Figure 17 Solid-phase chemical ligation (112). Native chemical ligation of unprotected peptide segments and the principles of polymer-supported synthetic organic chemistry (13, 19, 111) are applied to solid-phase protein synthesis. In the example shown, the C-terminal segment of the target polypeptide is attached by a cleavable linker to a water-compatible support. The next segment as a peptide-αthioester is reacted by native chemical ligation, to give the polymer-bound ligation product. After removal of the Cys-protecting group (PG), successive rounds of ligation can be carried out to give the polymer-bound target polypeptide. After cleavage from the polymer support, the product is purified and folded to give the target protein molecule.

Figure 3 Principles of chemical ligation (48, 50). Uniquely reactive functionalities are incorporated into each peptide by chemical synthesis. Mutual chemoselective reaction of these moieties allows the use of completely unprotected peptide segments, which are prepared by standard means and can be readily purified and characterized by sensitive, high-resolution methods. Reaction is carried out in aqueous solution in the presence of chaotropes, such as 6 M guanidine-HCl, to increase the concentration of reacting segments and speed up the reaction. The product polypeptide is obtained directly in final form.

Figure 16 Expressed protein ligation (104). This process uses intein-mediated (73) preparation of a recombinant peptide-αthioester, which is then reacted with a Cys-peptide segment by native chemical ligation to prepare the desired product.

Figure 14 Total chemical synthesis of a multidomain protein: a tethered-dimer form of the HIV-1 protease. (Left) Convergent ligation synthetic strategy (85). Thioester-forming ligation was used to make each of the 101-residue monomers, and then directed disulfide formation was used to make the 202-residue synthetic dimer. TheNH moiety of Ile50, in one monomer only, was substituted for by an O by incorporation of an ester at that position in chemical synthesis of the peptide segment (20). (Right) Analytical data. (Top right). HPLC, showing high-purity product and autolysis fragments consistent with the full enzymatic activity of the backbone-engineered 22-kDa protein. (Bottom right) Electrospray mass spectrometry data showing the high purity and correct observed mass of the synthetic construct.

Figure 5 Backbone-engineered HIV-1 protease by chemical ligation (56). (Top) Design of the variant enzyme. (Top left) H bonding of “water 301” by the amideNH of Ile50 at the tip of each flap structure. (Top right) Sulfur atoms replacing theseNH moieties, thus deleting the H-bonding potential. (Bottom) Synthetic scheme. Nucleophilic thioesterforming ligation, with inversion of configuration at the ‘D-Ile50’ chiral center, to give the desired 99-residue polypeptide, which is folded to form the homodimeric enzyme molecule.

Figure 8 Raw analytical HPLC data from the synthesis by native chemical ligation of the 110-residue polypeptide chain of the enzyme barnase (63). The two unprotected peptide segments, (1-48)αCOSbenzyl and Cys49-110, were reacted in aqueous solution at pH 7 in the presence of a thiol catalyst. Data after 7 h show a nearly quantitative reaction to form a single product. (Insert) Electrospray mass spectrometric data for the ligated 110-residue polypeptide,Mw 12,343.

Figure 11 Applications of native chemical ligation.

TABLE 1 Chemistries used for the synthesis of native proteins by chemical ligation of unprotected peptide segments

TABLE 2 Selected proteins prepared by total synthesis using native chemical ligationa

Figure 10 Conformationally assisted chemical ligation, exemplified for the chymotrypsin inhibitor C12. (A, left) Thioester-mediated chemical ligation at Cys under standard denaturing conditions occurs over several hours. (B, right) The same ligation reaction under folding conditions, in which the two segments associate to increase the collision frequency between the reacting functionalities, is complete within 3 min (76).

Figure 19 Chemical synthesis of an integral membrane protein (115). The 97-residue polypeptide chain of the influenza M2 protein was prepared by native chemical ligation and folded to form the active tetrameric form. (Insert) Electrospray mass spectrometric data showing the desired product, mass 11,170 Da.

Figure 7 Native chemical ligation (62). Unprotected peptide segments are reacted by means of reversible thiol/thioester exchange to give thioester-linked initial reaction products. Uniquely, the thioester-linked intermediate involving an N-terminal Cys residue (boxed) is able to undergo nucleophilic rearrangement by a highly favored intramolecular mechanism; this step is irreversible (under the conditions used) and gives a polypeptide product that is linked by a native amide (i.e. peptide) bond. Only a single reaction product is obtained, even in the presence of additional Cys residues in either segment. The product polypeptide is subsequently folded to give the desired synthetic protein molecule.

Figure 18 Size of synthetic polypeptides accessible by chemical ligation.

Figure 12 Crystal structure of AOPRANTES (79). This chemically modified chemokine protein was prepared by total synthesis, using native chemical ligation. X-ray diffraction was used to determine the structure to 1.6-Å resolution. (Top) Ribbon structure of the crystalline dimer. (Bottom) 2Fo-Fc electron

TABLE 3 Some structural motifs successfully folded as synthetic proteinsa

Figure 1 Crystal structures of chemically synthesized HIV-1 protease. These were the original high-resolution structures (23, 25–27) of this protein and guided the subsequent drug design programs. The synthetic protein preparation used for X-ray crystallography containedL-α-amino-n-butyric acid residues in place of the two Cys residues in each subunit. (Left) Molscript representation of the synthetic enzyme in complex with the substratederived inhibitor MVT101 (25). (Right upper panel) 2Fo-Fc electron density map for the side chains of the unnatural amino acids used to replace the two Cys residues in each subunit of the synthetic enzyme (23). (Lower panel) Side chains of theL-α-amino-n-butyric acid residues superimposed on the mercury atoms from Cys-containing enzyme (24) that has been crystallized in the same space group. This shows that the side chains of the unnatural amino acid have the same conformation as the natural Cys side chains. (Adapted from References 23 and 25).

Figure 2 Historical progress in the size of synthetically accessible polypeptides.

Full text

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Annu. Rev. Biochem. 2000. 69:923–60
Copyright
c
 2000 by Annual Reviews. All rights reserved
SYNTHESIS OF NATIVE PROTEINS
BY
CHEMICAL LIGATION
∗
Philip E. Dawson
1
and Stephen B. H. Kent
2
1
The Scripps Research Institute, La Jolla, California 92037; e-mail: dawson@scripps.edu;
2
Gryphon Sciences, South San Francisco, California 94080; e-mail: skent@gryphonsci.
com
Key Words chemical protein synthesis, thioester, protein, peptide, solid phase
synthesis, polymer-supported synthesis, protein engineering
■ Abstract In just a few short years, the chemical ligation of unprotected peptide
segments in aqueous solution has established itself as the most practical method for the
total synthesis of native proteins. A wide range of proteins has been prepared. These
synthetic molecules have led to the elucidation of gene function, to the discovery of
novel biology, and to the determination of new three-dimensional protein structures by
both NMR and X-ray crystallography. The facile access to novel analogs provided by
chemicalproteinsynthesishasledtooriginalinsightsintothemolecularbasisofprotein
function in a number of systems. Chemical protein synthesis has also enabled the
systematic developmentof proteins with enhanced potency and specificity as candidate
therapeutic agents.
CONTENTS
INTRODUCTION: Protein Science in the Postgenome Era ................... 924
DOMAINS: Building Blocks of the Protein World
......................... 925
CHEMICAL PROTEIN SYNTHESIS: The State of the Art in 1990
.............926
SYNTHETIC-PEPTIDE CHEMISTRY: Useful but Bounded
.................926
CHEMICAL LIGATION OF UNPROTECTED PEPTIDE SEGMENTS
.........929
NATIVE CHEMICAL LIGATION
.................................... 933
BIOCHEMICAL PEPTIDE LIGATION
................................ 935
Protein Splicing
................................................ 935
Conformationally Assisted Ligation
.................................. 938
SCOPE OF NATIVE CHEMICAL LIGATION FOR THE SYNTHESIS
OF PROTEINS
................................................. 939
∗
At the time of the invitation, as now, Stephen Kent is President and Chief Scientist at
Gryphon Sciences. Gryphon Sciences is focused on the development and sale of enhanced
protein therapeutics using chemical protein synthesis. The core technology of the company
is largely the subject matter of the chapter we have submitted.
0066-4154/00/0707-0923/$14.00
923

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924 DAWSON
¥
KENT
FOLDING SYNTHETIC PROTEINS ..................................940
CASE STUDIES IN THE APPLICATION OF CHEMICAL
PROTEIN SYNTHESIS
........................................... 944
Noncoded Amino Acids
.......................................... 944
Precise Covalent Modification
...................................... 945
Site-Specific Tagged Proteins
...................................... 945
Backbone Engineering
........................................... 946
Protein Medicinal Chemistry
....................................... 946
Rapid Access to Functional Gene Products
............................. 947
Structural Biology
.............................................. 947
CURRENT DEVELOPMENTS
...................................... 949
Expressed Protein Ligation
........................................ 949
Solid-Phase Protein Synthesis
...................................... 950
Membrane Proteins
............................................. 951
Glycoprotein Synthesis
........................................... 953
FUTURE DEVELOPMENTS
........................................ 954
Ligation Sites
.................................................. 954
Size of Protein Targets
........................................... 955
Chemical Synthesis of Peptide Segments
.............................. 956
SUMMARY AND CONCLUSIONS
................................... 956
INTRODUCTION: Protein Science in the Postgenome Era
An important current objective in biomedical research is to understand the molecu-
larbasis ofthe numerousand intricatebiological activitiesof proteins and therefore
to be able to predict and control these activities. The importance of this goal is
dramatically increased today because of the explosive success of the genome-
sequencing projects, which have revealed hundreds of thousands of new proteins,
but only as predicted sequence data (1). For the biologist, elucidation of the bi-
ological function of a predicted protein molecule is thus a challenge of great
significance. In the words of Freeman Dyson, “[In the post-genome era], proteins
will emerge as the big problem and the big opportunity. When this revolution oc-
curs, it will have a more profound effect than the Human Genome Project on the
future of science and medicine” (2).
For the past 20 years, most studies of the molecular basis of protein action have
been carried out by recombinant DNA-based expression of proteins in genetically
engineered cells (3). From its introduction, this powerful method revolutionized
the study of proteins by enabling the production of large amounts of proteins of
defined molecular composition and by allowing the systematic variation of the
amino acid sequence of proteins (4). Expression of proteins in engineered cells
is now a mature technology, and its scope and limitations are well understood:
(a) Small proteins (i.e. <30 kDa) are easier to express than large, multidomain
proteins; (b) folding of large-protein molecules can also be a challenge; (c) product

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CHEMICAL PROTEIN SYNTHESIS 925
heterogeneity is frequently a problem, caused by uncontrolled processing of the
nascent polypeptide in the cell; and (d) the overexpression of proteins that are toxic
to the cell, such as proteases, can be difficult (5).
Additionally, because the cell is used as a protein factory, such molecular bi-
ology studies are inherently limited to the 20 genetically encoded amino acids.
Efforts have been made to use cell-free synthesis to expand the repertoire of ri-
bosomal synthesis to include noncoded amino acids as building blocks (6, 7).
These attempts to incorporate other amino acids have had very limited success—
obtaining adequate amounts of pure protein from the cell-free translation systems
can be a significant challenge (8), and many unnatural amino acids are simply not
compatible with ribosomal polypeptide synthesis (9).
Chemical synthesis is an attractive alternative to biological methods of protein
production. The use of synthetic chemistry promises the unlimited variation of
the covalent structure of a polypeptide chain with the objective of understanding
the molecular basis of protein function. Chemistry also promises the ability to
systematically tune the properties of a protein molecule in a completely general
fashion.
This vision was one of the prime imperatives of organic chemistry in the time of
Emil Fischer at the beginning of the 20th century. In a 1905 letter to Adolf Baeyer,
Fischer wrote, “My entire yearning is directed toward the first synthetic enzyme.
If its preparation falls into my lap with the synthesis of a natural protein material,
I will consider my mission fulfilled” (10). In the decades since then, the challenge
of applying the methods of chemistry to the study of protein action has stimulated
numerous advances in synthetic methods. Historically, these advances included
the use of novel reversible protecting groups (11), novel activation methods for the
formation of covalent bonds (12), and even polymer-supported synthesis (13), all
of which sprang from the drive to apply the science of chemistry to the study of
proteins.
DOMAINS: Building Blocks of the Protein World
Because proteins are large molecules, applying chemical synthesis to them is a
considerable challenge. Furthermore, the biological functions of proteins orig-
inate in the tertiary structure of the protein molecule—that is, in the precise
three-dimensional folded structure of the polypeptide chain. The typical protein
molecule is ∼30 kDa in size and consists of two ∼15-kDa domains (14–16);
each domain has a polypeptide chain length of ∼130 (±40) amino acids (14–16).
Protein domains are defined as autonomous units of folding and, frequently, of
function (17, 18). As such, domains are the building blocks of the protein world.
The challenge confronting the chemist is, first, the total synthesis of folded do-
mains and then the ability to stitch these domains together to build complexprotein
molecules.

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July 12, 2000 11:26 AR102 CHAP29
926 DAWSON
¥
KENT
CHEMICAL PROTEIN SYNTHESIS: The State
of the Art in 1990
Since last reviewed in this journal (19), total chemical synthesis of native pro-
teins has made a number of important contributions to biomedical research. It is
notable that the Kent laboratory at the California Institute of Technology used total
chemical synthesis based on predicted gene sequence data to carry out pioneering
studies of human immunodeficiency virus 1 (HIV-1) protease enzyme (20). The
existence of this virally encoded aspartyl proteinase had been postulated based on
an analysis of viral nucleic acid sequence data, and molecular genetic studies had
indicated that its action in processing the gag-pol polyprotein was essential to the
viral life cycle (21). For this reason, the HIV-1 protease was, early on, proposed as
an important target for drug development. The first preparations of the enzyme of
defined molecular composition were produced by chemical synthesis (22), using a
highly optimized version of stepwise solid-phase peptide synthesis (19). This work
proved that the active form of the HIV-1 protease was a homodimer consisting of
two identical 99-residue polypeptide chains, and it showed that the chemically
synthesized enzyme accurately processed the putative cleavage sites in the viral
gag-pol translation product (22).
In a strikingly important contribution, total chemical synthesis was also used to
prepare large amounts of homogeneous enzyme for the determination of the orig-
inal crystal structures of the HIV-1 protease molecule (Figure 1). The structure of
the unliganded synthetic enzyme (23) corrected a seriously flawed low-resolution
structure (24) that had been obtained by using protein derived from recombinant
expression in Escherichia coli. Even more significantly, use of chemically synthe-
sized enzyme provided the first high-resolution cocrystal structures of the HIV-1
protease molecule complexed with substrate-derived inhibitors (25–27). These
structural data were made freely available to the research community and formed
the foundation for the successful worldwide programs of structure-based drug de-
sign (28) that led to the development of the highly effective protease inhibitor class
of acquired immune deficiency syndrome therapeutic agents (29).
SYNTHETIC-PEPTIDE CHEMISTRY: Useful but Bounded
Despite successful syntheses of the HIV-1 protease (22) and of a limited number
of other proteins (30–35), at the start of the decade of the 1990s total chem-
ical synthesis, by the standard methods of peptide chemistry of even a small
protein molecule remained a daunting task, often requiring large teams and tak-
ing years to complete, with no guarantee of success. The routine, reproducible
preparation of synthetic polypeptides of defined chemical structure was limited
to products of ∼50 amino acid residues (19; Figure 2). This size limitation ap-
plied equally to synthesis by solution or by solid-phase methods, but for differing
reasons.

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July 12, 2000 11:26 AR102 CHAP29
CHEMICAL PROTEIN SYNTHESIS 927
Figure 1 Crystal structures of chemically synthesized HIV-1 protease. These were the
original high-resolution structures (23, 25–27) of this protein and guided the subsequent
drug design programs. The synthetic protein preparation used for X-ray crystallography
contained
L-α-amino-n-butyricacidresiduesinplaceofthetwoCysresiduesineachsubunit.
(Left) Molscript representation of the synthetic enzyme in complex with the substrate-
derived inhibitor MVT101 (25). (Right upper panel) 2Fo-Fc electron density map for the
side chains of the unnatural amino acids used to replace the two Cys residues in each subunit
of the synthetic enzyme (23). (Lower panel) Side chains of the
L-α-amino-n-butyric acid
residues superimposed on the mercury atoms from Cys-containing enzyme (24) that has
been crystallized in the same space group. This shows that the side chains of the unnatural
amino acid have the same conformation as the natural Cys side chains. (Adapted from
References 23 and 25).
Figure 2 Historical progress in the size of synthetically accessible polypeptides.

Frequently asked questions

Q1. What are the contributions in this paper?

The facile access to novel analogs provided by chemical protein synthesis has led to original insights into the molecular basis of protein function in a number of systems.