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Chemical synthesis of proteins
Jeffrey A. Borgia and Gregg B. Fields
The manipulation of protein structure enables a better understanding of the principles of protein folding, as well as the development
of novel therapeutics and drug-delivery vehicles. Chemical synthesis is the most powerful approach for constructing proteins
of novel design and structure, allowing for variation of covalent structure without limitations. Here we describe the various
chemical methods that are currently used for creating proteins of unique architecture and function.
hemical methods for assembling proteins have
C existed for over 30 years. However, the con-
struction of proteins has become more accessible
recently because of improvements in peptide-synthesis
efficiency, including the development of rapid coupling
reagents and the minimization of deleterious side reac-
tions1. The creation of protein-like molecular archi-
tectures is desirable for the studies of protein folding
and stability, as well as for the design of proteins geared
for specific functions.
There are three general chemical approaches to con-
structing proteins. The first is stepwise synthesis, in
which the entire protein is synthesized one amino acid
at a time. The second is fragment assembly, in which
individual peptide strands are initially constructed step-
wise and are then purified and covalently linked to
create the desired protein. Fragment assembly can be
further subdivided into two distinct methods: (1) con-
vergent synthesis of fully protected fragments; and (2)
chemoselective ligation of unprotected fragments. The
third approach to protein construction is directed
assembly, in which individual peptide strands are con-
structed stepwise and purified, and then non-covalently
driven to associate into protein-like structures. Com-
binations of these three general chemical approaches
can also be used for protein construction.
Solid-phase peptide synthesis (SPPS), developed by
Merrifield2, has proved to be the method of choice for
efficiently producing peptides and small proteins
(Fig. 1). Essentially, an Na-derivatized amino acid is
attached to a commercially available, insoluble (i.e.
solid) support via a linker moiety. The Na-protecting
group is then removed (deprotection) and the amino-
acidlinkersupport complex is thoroughly washed
with solvent. The next amino acid (which is also Na-
protected) is then coupled to the amino-acidlinker
support complex as either a preactivated species or
directly (in situ) in the presence of an activator. After
this reaction is complete, the nascent oligopeptide
linkersupport complex is washed with solvent, thus
removing unreacted material. The deprotection
coupling cycle is repeated until the desired sequence
of amino acids is generated. Finally, the peptide
linkersupport complex is cleaved to obtain the pep-
tide as a free acid or an amide, depending on the chemi-
cal nature of the linker. Ideally, the cleavage reagent
J.A. Borgia and G.B. Fields ( fieldsg@fau.edu) are at the Department
of Chemistry and Biochemistry and the Center for Molecular Biology and
Biotechnology, Florida Atlantic University, Boca Raton, FL 33431, USA.
TIBTECH JUNE 2000 (Vol. 18) 0167-7799/00/$ see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(00)01445-1
should also remove the amino acid side-chain-protect-
ing groups, which are stable to the Na-deprotection
conditions.
There are two main protocols that have been used
for the solid-phase chemical synthesis of proteins. The
first protocol uses the tertiary-butyloxycarbonyl (Boc)
group for Na-amino protection. The Boc group is typi-
cally removed by trifluoroacetic acid (TFA) and the free
N terminus is neutralized by a tertiary amine. The pep-
tide is removed from the resin with a relatively strong
acid, usually hydrogen fluoride. Side-chain-protecting
groups include ether, ester and urethane derivatives
based on benzyl alcohol, fine-tuned either with elec-
tron-donating methoxy or methyl groups, or with
electron-withdrawing halogens to give the proper level
of acid stability or lability. Alternatively, ether and ester
derivatives based on cyclopentyl or cyclohexyl alcohol
are sometimes used, because they moderate certain side
reactions. Side-chain-protecting groups are specifically
designed to be stable to repeated cycles of Boc removal,
yet to be cleanly cleaved by hydrogen fluoride.
The second protocol uses the 9-fluorenylmethyloxy-
carbonyl (Fmoc) group for Na-amino protection. The
Fmoc group is usually removed with piperidine
in N,N-dimethylformamide or N-methylpyrrolidone.
Side-chain protection that is compatible with Na-
Fmoc protection is provided primarily by ether, ester
and urethane derivatives based on t-butanol. These
derivatives are removed at the same time as the appro-
priate anchoring linkages by the use of TFA. The
milder conditions of the Fmoc protocol have led to its
being preferred by peptide laboratories3 but certain
deleterious side reactions are more prevalent in the
Fmoc protocol4. This article summarizes protein syn-
thesis using both the Boc and the Fmoc solid-phase
chemical methods.
Stepwise synthesis of proteins
Many impressive protein syntheses have been per-
formed using both the Boc and the Fmoc chemistries.
The earliest example was the construction of active
ribonuclease A5 (124 residues), which validated the use
of SPPS. More recently, stepwise solid-phase synthesis
was used to assemble another active enzyme, HIV-1
aspartyl protease69 (99 residues per chain); monomeric
peptide chains were initially synthesized by Boc chem-
istry. After purification, individual peptides associate
into an active homodimer. This synthetic HIV-1
protease was used in X-ray crystallography to deter-
mine its structure, both with and without known
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The ability to assemble proteins in a stepwise fashion

is limited by the synthetic efficiency of each step. As
X O discussed by Kent22, the synthesis of a 100-residue pro-
NH CH C OH + Linker Resin
tein with 99.9% efficiency at each step provides a 90%
A
overall yield of the desired product; 97% efficiency
provides a 5% overall yield. The effective solvation of
Anchoring
the peptideresin complex is perhaps the most crucial
X O condition for efficient chain assembly; difficult cou-
Repetitive cycle
A NH CH C Linker Resin plings during SPPS have been attributed to the poor
solvation of the growing chain. Infrared and nuclear
magnetic resonance spectroscopy have shown that
N-deprotection intermolecular b-sheet aggregates can lower the cou-
Y O X O pling efficiency2325. Both the Boc and the Fmoc
A NH CH C OH + H2N CH C Linker Resin chemistries appear to be susceptible to the same diffi-
cult couplings2628, and side-by-side syntheses for mod-
erate-length chains (~30 residues) are comparable29,30.
Coupling Coupling efficiencies are enhanced by the addition of
polar solvents3134 and the use of in situ neutralization
Y O X O protocols35. It has also been suggested that adding
A NH CH C NH CH C Linker Resin chaotropic salts to organic solvents can disrupt b-sheet
aggregates and thus improve overall yields36,37. Fur-
(1) N-deprotection thermore, the use of a lower substitution level of resin
(2) Cleavage in order to avoid interchain crowding can also improve
(3) Side-chain the overall synthetic efficiency38.
deprotection
A different approach to circumventing potential
aggregation is reversible protection of the amino acid
Z O R O Y O X O
amide nitrogen using Na-Fmoc-Na(2-Fmoc-hydroxy-
H2N CH C NH CH C NH CH C NH CH C NH2 4-methoxybenzyl) derivatives39. The formation of
n OH b-sheet structure can be minimized by using amide
trends in Biotechnology nitrogen protection every sixth to eighth amino
acid39,40. Alternatively, incorporating pseudoproline
Figure 1 residues can be used to disrupt intermolecular b-sheet
Solid-phase peptide synthesis. An N a-derivatized amino acid is attached to an insol- formation41.
uble support (resin) via a linker moiety. The N a-protecting group (A) is then removed
(deprotection). The next amino acid (which is N a-protected) is coupled to the amino- Assembling proteins from fragments
acidlinkersupport complex. The deprotectioncoupling cycle is repeated until the Convergent synthesis of protected peptide fragments
desired sequence of amino acids has been generated. Finally, the peptidelinkersupport In contrast to the stepwise approach, convergent
complex is cleaved to obtain the peptide as a free acid or an amide, depending on synthesis uses protected peptide fragments to make pro-
the chemical nature of the linker. Ideally, the cleavage reagent should also remove teins. The convergence of the protein fragments can
the amino acid side-chain-protecting groups, which are stable to the N a-deprotection be performed either in solution or on the solid phase
conditions. (Fig. 2). The advantage of convergent protein synthe-
sis is that fragments of the desired protein are synthe-
HIV-1 protease inhibitors bound8,10,11. Another recent sized, purified and characterized (which ensures that
application of stepwise synthesis is the construction of each fragment is of high integrity), and only then
bovine pancreatic-trypsin inhibitor and its analogs (58 assembled into the complete protein. Thus, the cumu-
residues) by Fmoc chemistry1214. The analogs were lative effects of stepwise synthetic errors are minimized.
used for the biophysical characterization of folding Convergent synthesis requires ready access to pure, par-
intermediates and to dissect folding pathways13,14. tially protected peptide segments, which are needed as
Stepwise assembly can proceed either in a linear building blocks. Preparing these intermediates by SPPS
fashion or on a template. Mutter et al. developed depends on several levels of selectively cleavable pro-
the template-assembled synthetic protein (TASP) tecting groups and linkers. Some of these protection
approach, based on the concept that templates would schemes are orthogonal; that is, they involve two or
promote secondary-structure formation and minimize more classes of group, which are removed by different
the aggregation of larger complexes15,16. TASP mol- chemical mechanisms and so can be removed in any
ecules containing a four-a-helix bundle17 and a order and in the presence of other classes. Examples of
combination four a-helix-bundle-and-b-barrel-like orthogonality for convergent synthesis include the com-
structure18 were constructed by stepwise SPPS on a bination of the Bocbenzyl strategy with nucleophile-
template; the templates were typically small cyclic labile, base-labile, palladium-labile or photolabile
peptides. Minicollagens (76121 residues) have also linkers, and the combination of Fmoctertiary-butyl
been assembled by stepwise Fmoc chemistry on a strategy with dilute-acid-labile, palladium-labile or
branched template19,20. These minicollagens have been photolabile linkers. Such combinations have been
used in biochemical studies in order to identify cellu- successful at generating Na-amino- and side-chain-
lar recognition sites within interstitial collagens, which protected segments with free Ca-carboxyl groups42,43.
might be important in wound healing and platelet There are many examples of protein synthesis by the
aggregation19,21. convergent approach in solution or in the solid phase.

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+ +

Repetitive Repetitive
cycle + H2N cycle + H2N

(1) Cleavage
(2) Purification N-deprotection

+ H2N
n n
Coupling

n n
N-deprotection

+ H2N
n n n
Coupling

n n n
(1) N-deprotection
(2) Cleavage and
side-chain
deprotection
(3) Purification

H2N OH
n n n
NH2
trends in Biotechnology

Figure 2
Convergent solid-phase peptide synthesis. Two fragments are assembled initially on the solid phase; one fragment is cleaved and purified
with the N a-amino and side-chain-protecting groups intact. The purified fragment is then coupled to the free N a-amino terminus of the resin-
bound fragment. After coupling is complete, the N a-amino protecting group is removed. A third fragment, which has been previously purified,
is coupled to the free N a-amino terminus of the resin-bound fragments. This process is repeated until the desired sequence is obtained; the
protein is then cleaved from the resin, simultaneously removing the side-chain-protecting groups, and purified.

Ribonuclease A was assembled using a convergent-
synthesis approach in solution44, and green fluorescent
protein (238 residues) was assembled in solution using
26 protected fragments prepared by solid-phase meth-
ods45. A TASP containing a four-a-helix bundle was
constructed by convergent synthesis in solution46.
Triple-helical minicollagens containing N-alkyl amino
acids have been assembled by incorporating a Kemp
triacid template after convergent synthesis47,48. The
solid-phase assembly of protected segments has proved
to be successful for b-amyloid protein49,50 (42 residues),
the N-terminal domain of g-zein protein51 (48 residues),
Androctonus australis Hector toxin II (64 residues; Ref. 52),
l-Cro DNA binding protein53 (66 residues), prothy-
mosin a (109 residues; Ref. 54) and a TASP containing
three peptide loops55.
Convergent protein synthesis faces several difficulties.
First, the protected fragments are often poorly soluble
in the aqueous solvents used for liquid-chromato-
graphic purification and the organic solvents tradition-
ally used for coupling reactions43,56; mixed-solvent
systems can be used to improve some solubility prob-
lems56. Second, the individual rates for coupling frag-
ments are substantially lower than those for activated

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protease61. A convenient linker has been described that

N terminal C terminal Product produces C-terminal thioacids after Boc chemistry62.
(a) O O A variation of the thioester approach, in which a pep-
tide C-terminal thiol (such as Cys) is reacted with an
SH Br S N-terminal bromoacetyl or maleimido peptide to form
O O a thioether bond (Fig. 3b), has been used to construct
a triple-helical domain from the macrophage scavenger
(b)
O O receptor63,64, linked cytoplasmic domains from the
SH Br S aIIbb3 integrin65, a b-meander TASP molecule66 and a
129-residue tripod protein67. Linkers that produce C-
terminal thiols have been described for both Boc68 and
O O
SH S Fmoc69 chemistries. To prevent the decomposition of
N N thioester linkers during Fmoc syntheses, an Fmoc
deprotection solution containing 1-methylpyrrolidine,
O O hexamethyleneimine and 1-hydroxybenzotriazole in
N-methylpyrrolidone-dimethylsulfoxide (1:1) should
(c) O O O
be used70. As an alternative to thioester and thioether
O H O
H H2 N N bonds, peptides containing either aldehyde or N-
terminal aminooxyacetyl groups can be ligated to form
R R
oxime bonds71,72 (Fig. 3c). An aldehyde-containing
O peptide can also be ligated to a peptide containing a
H H2N N weak nucleophilic base, such as hydrazide, N-terminal
H Cys or N-terminal Thr, to form hydrazone, thiazolidine
and oxazolidine linkages, respectively7376 (Fig. 3c).
Chemoselective ligation was made more attractive by
O O H O
native chemical ligation, in which an amide bond
H2N N
H N N (rather than a thioester, thioether or oxime bond) is
H H generated between fragments58,77 (Fig. 4). A peptide
with a C-terminal thioacid is converted to a 5-thio-2-
O O nitrobenzoic acid ester and then reacted with a peptide
O with an N-terminal Cys residue. The initial thioester
H2N N
ligation product spontaneously rearranges, producing
H an amide bond and regenerating the free sulfhydryl on
N N
H the Cys. The ligation strategy has been extended fur-
trends in Biotechnology ther to make use of other thiol additives and their
respective reactivities78. The trityl-associated mercap-
Figure 3 toproprionic-acid leucine (TAMPAL) resin allows the
N- and C-terminal groups used for non-native chemoselective ligation. convenient generation of any amino acid as a C-ter-
The product structures shown are produced when the groups react minal thioester79. Native chemical ligation can proceed
under aqueous conditions. Ligation by the groups in (a) results in intramolecularly to create cyclized proteins80,81.
thioester bond formation; groups in (b) form a thioether bond. The Conformationally assisted protein ligation, in which
ligation strategies depicted in (c) all use an aldehyde for the N- the C- and N-termini to be ligated are brought into
terminal ligation group. close proximity by peptide folding, has been shown to
eliminate the absolute need for an N-terminal Cys
amino acid species in traditional stepwise synthesis, and residue82. Amide bonds can also be generated by
the reaction times are often increased to enhance the chemoselective ligation methods that result in thiazo-
fragment-coupling efficiency. Third, the amino acid lidine linkages via an ON acyl shift to form hydroxy-
can racemize at the C terminus of a segment during methyl thiazolidine83. Chemoselective ligation can be
coupling, but this can be avoided by constructing performed for multiple fragments84,85 and in either the
fragments that have either a Gly or a Pro C-terminus. NC or the CN direction in the solid phase86. A
modular chemoselective-ligation strategy was used to
Chemoselective ligation of unprotected peptide synthesize a covalently linked dimer of the bHLHZ
fragments domains of the cMyc and Max transcription factors87.
As an alternative to the convergent approach, meth- The individual cMyc and Max domains were assem-
ods have been developed that can link unprotected pep- bled by native chemical ligation and then linked via
tide fragments. Known as chemoselective ligation, the oxime bond formation.
original strategies resulted in the formation of thioester Chemoselective ligation has recently been extended
or oxime bonds between peptide fragments. For exam- to allow expressed protein ligation. The gene for a
ple, the reaction of a peptide containing a C-terminal desired protein is cloned into an intein vector and
thioacid with a peptide containing an N-terminal expressed. The splicing process that occurs at the
bromoacetyl group results in a synthetic protein product recombinant-proteinintein junction is intercepted by
containing a thioester bond57,58 (Fig. 3a). This approach a thiol, generating the a-thioester recombinant pro-
has been used to construct HIV-1 protease57, a four-a- tein. The recombinant protein is then ligated to a pep-
helix TASP molecule59, a folded b-sandwich fibro- tide or protein that has an N-terminal Cys residue88,89.
nectin domain model60 and a tethered dimer of HIV-1 This approach also allows a synthetic peptide to be

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(a) (b)
+ HS +

Repetitive Repetitive
cycle + H2N cycle + H2N

(1) N-deprotection (1) N-deprotection

(2) Cleavage and side-chain (2) Cleavage and side-chain
deprotection deprotection
(3) React with R-Br (3) Purification
(4) Purification

H2N SR + H 2N NH2
n HS n OH

R1 O
N
N H SR O R3
O R2 H2N N
H
S O
H

H H
O R3
R1 O N N
N H
N H S O
O R2

H2N NH2
n HS n OH trends in Biotechnology

Figure 4
Native chemoselective ligation. Two fragments are assembled initially on the solid phase; in this specific example, one has a C-terminal
thioacid and the other has an N-terminal Cys residue. Both fragments are cleaved, side-chain deprotected and purified. The thioacid is con-
verted to a thioester, allowing the two fragments to react in aqueous solution to form a thioester bond between them. Spontaneous rearrange-
ment of the thioester bond results in a native amide bond between the fragments. The process can then be repeated to link additional
peptide fragments.

inserted between two recombinant proteins and has
been used to construct recombinant Src-homology-2
and -3 domains with a fluorescent probe between
them88.
Non-covalent directed assembly of proteins
Many initial studies on protein folding focused on
the ability of peptides to self-assemble and to form dis-
tinct structures. For example, Sakakibara et al. showed
that synthetic peptides of the sequence (ProProGly)n,
where n 5 10 or 20, could self-assemble to form
collagen-like triple helices90. Peptide self-assembly has
since been used to create a-helix-bundle and b-sheet
proteins91. Unfortunately, peptide self-assembly is not
a particularly easy process to regulate. Undesirable
molecular assemblies occur (such as the formation of a
mixture of a-helix dimers, trimers, tetramers, etc.) and
peptide chains form large aggregates of an unknown
order; controlling these peptide interactions could
minimize these difficulties. One approach to controlled
peptide assembly is to incorporate moieties on the
end of the peptide chains and to use the properties of
these moieties to drive peptides to interact in a specific
fashion (Fig. 5). The advantages of this approach
include: (1) the proper number of peptides will associate,
eliminating undesired aggregates; and (2) the moieties
themselves might act as templates, which could initiate
and stabilize structure formation.

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Repetitive
cycle + H2N

G P P* G P P*
n n

N-deprotection

O
OH + H2N G P P* G P P*
n n

Coupling

O
G P P* G P P*
n n

(1) Cleavage and

side-chain deprotection
(2) Purification
(3) Self-association
O
G P P* G P P*
O
G P P* G P P*
O
G P P* G P P*

Secondary structure
trends in Biotechnology

Figure 5
Directed self-assembly approach. The desired peptide sequence is assembled by standard solid-phase stepwise-synthesis methods. After
synthesis is complete, the N a-amino protecting group is removed from the resin-bound peptide and the moiety to be used for assembly is
incorporated. In this particular example, an alkyl chain has been coupled to a collagen-like sequence to create a peptide amphiphile. The
peptide amphiphile is then cleaved from the resin (which simultaneously removes the side-chain-protecting groups), purified and allowed to
self-associate in an aqueous environment.

One well-documented, directed self-assembly approach
uses chelators attached to peptides and appropriate
metals to initiate interstrand association. A triple-a-
helix-bundle protein has been created by incorporating
5-carboxy-2,29-bipyridine onto a 15-residue peptide
and using Co(II), Ni(II) or Ru(II) complexation92. The
circular dichroism (CD) spectrum of the Ru(II)-
complexed-peptide indicated that it had a left-handed,
supercoiled a-helical structure. A triple-a-helix-bundle
protein has also been created by incorporating 2,29-
bipyridine-4,49-dicarboxylic acid into a 15-residue
peptide and complexing with Fe21 (Ref. 93). The
individual strands, designated pepy, were 35% a-heli-
cal; the resultant protein [FeII(pepy)3] was 85% a-helical.
FeII(pepy)3 was found to exist in four stereoisomeric
states94. As an alternative to using chelators, a metal-ion-
induced triple-a-helix-bundle protein has been created
by incorporating selective His residues within a 31-residue
peptide sequence and using Ni(II) complexation95.
A Ru(II)-complexed four-a-helix bundle has been
constructed by incorporating a pyridyl group into the
N terminus of a 15-residue helix-forming peptide96.
The complex was .90% a-helical by CD spectroscopy.
Guanidinium-HCl-induced denaturation of the bundle

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Table 1. Benefits and limitations of chemical approaches for protein synthesis

Chemical approach Benefits Limitations

Stepwise synthesis Straightforward synthesis based on well-documented Cumulative effects of synthetic inefficiencies
methods

Convergent synthesis Fragments are of high integrity; cumulative effects Solubility of fragments in aqueous environment
of stepwise synthetic errors are minimized Racemization during fragment coupling
Efficiency of fragment coupling

Chemoselective ligation Fragments are of high integrity; cumulative effects Limited residue possibilities at the ligation site
of stepwise synthetic errors are minimized Hydrophobic (transmembrane) fragments may
Fragments are typically soluble in aqueous environments have limited solubility in aqueous environment

Directed assembly Fragments are of high integrity; cumulative effects Redundancy of protein structure
of stepwise synthetic errors are minimized Control of assembly size
Fragments are typically soluble in aqueous environments
Fragments are linked by noncovalent forces

was highly cooperative, with a change of free energy
in water (DGH2O) of 25.6 kcal mol21. This value is
comparable to that seen for many small, folded proteins,
for which DGH2O 5 25 to 215 kcal mol21.
In addition to the use of chelators and metal com-
plexation, the construction of peptide amphiphiles has
been used for directed self-assembly. A peptide head
group can form a distinct structural element; a
hydrophobic tail aligns the peptide strands and induces
secondary and tertiary structure formation as well as
providing a hydrophobic surface for self-association.
Peptide amphiphiles have been constructed using dialkyl
ester and monoalkyl tails97102. Examining peptide-
amphiphile structure using CD and one- and two dimen-
sional NMR spectroscopy has shown that this approach
can nucleate or stabilize a-helical and collagen-like
triple-helix structures98,100103. Peptide amphiphiles
thus provide a simple way of building stable protein
structural motifs using peptide head groups. The tight
alignment of the amino acids achieved through the
association of the lipid part of the molecule98,99 could
be a general tool for initiating protein folding.
Native chemical ligation and directed self-assembly
were used together to synthesize the influenza-A-virus
M2 protein104. This 97-residue monomeric protein
was constructed by ligating two fragments; the final
tetrameric protein channel self-assembled in micelles
or liposomes.
Directed assembly is limited by two factors. Current
approaches have described only homo-oligomeric
assemblies, which result in redundancy in the structural
elements within the protein. The assembly process can
also lead to the formation of extremely large peptide
aggregates, which may complicate studies of the desired
biomolecule.
Overview
Chemical synthesis is the most powerful approach for
constructing proteins of novel design and structure
because it allows the covalent structure to be varied
limitlessly. Chemical approaches allow the incorpora-
tion of non-native elements, such as N-substituted
and D-amino acids, and the replacement of backbone
amide bonds. Chemical methods might also be used to
generate proteins that cannot be produced by expression
systems because they are toxic.
There are several feasible approaches for the chemical
synthesis of proteins, each with benefits and limitations
(Table 1). Stepwise solid-phase synthesis is straight-
forward but will always be limited by the cumulative
effects of synthetic inefficiencies. Convergent protein
synthesis uses fragments of high integrity, but solu-
bilization of the protected fragments for purification
and subsequent coupling can be difficult. In addition,
individual rates for coupling fragments are substantially
lower than those for activated amino acid species in tra-
ditional stepwise synthesis, and there is always the risk
of racemization at the C terminus of each segment.
Careful attention to synthetic design and execution can
minimize these problems. Chemoselective ligation has
the same advantages as convergent protein synthesis,
but it is not subject to solubility problems or racemiz-
ation. At present, chemoselective ligation is only limited
by the choice of residues at the ligation site. Directed
assembly requires the fewest chemical steps of all of the
approaches, linking unprotected peptide fragments by
noncovalent methods, but it is limited by a redundancy
in the number of overall structures that can be created
and by the size of potential aggregates. The choice
of approach is very much dependent upon the desired
target protein.
Acknowledgments
The authors work was supported by National Institute
of Health grants CA77402, HL62427 and AR01929.
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