Download Chemical synthesis of proteins

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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
existed for over 30 years. However, the construction 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 reactions1. The creation of protein-like molecular architectures 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 constructing 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 stepwise and are then purified and covalently linked to
create the desired protein. Fragment assembly can be
further subdivided into two distinct methods: (1) convergent 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 constructed stepwise and purified, and then non-covalently
driven to associate into protein-like structures. Combinations 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 aminoacid–linker–support complex is thoroughly washed
with solvent. The next amino acid (which is also Naprotected) is then coupled to the amino-acid–linker–
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–
linker–support 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–
linker–support complex is cleaved to obtain the peptide as a free acid or an amide, depending on the chemical nature of the linker. Ideally, the cleavage reagent
C
J.A. Borgia and G.B. Fields ( [email protected]) 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)
should also remove the amino acid side-chain-protecting 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 typically removed by trifluoroacetic acid (TFA) and the free
N terminus is neutralized by a tertiary amine. The peptide 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 electron-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-fluorenylmethyloxycarbonyl (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 NaFmoc protection is provided primarily by ether, ester
and urethane derivatives based on t-butanol. These
derivatives are removed at the same time as the appropriate 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 synthesis using both the Boc and the Fmoc solid-phase
chemical methods.
Stepwise synthesis of proteins
Many impressive protein syntheses have been performed 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 protease6–9 (99 residues per chain); monomeric
peptide chains were initially synthesized by Boc chemistry. After purification, individual peptides associate
into an active homodimer. This synthetic HIV-1
protease was used in X-ray crystallography to determine its structure, both with and without known
0167-7799/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(00)01445-1
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A
NH
X
O
CH
C
OH +
Linker
Resin
Anchoring
Repetitive cycle
A
NH
X
O
CH
C
Linker
Resin
Nα-deprotection
A
NH
Y
O
CH
C
OH + H2N
X
O
CH
C
Linker
Resin
Coupling
A
NH
Y
O
CH
C
NH
X
O
CH
C
Linker
Resin
(1) Nα-deprotection
(2) Cleavage
(3) Side-chain
deprotection
H2N
Z
O
CH
C
NH
R
O
CH
C
NH
n
Y
O
CH
C
NH
X
O
CH
C
NH2
OH
trends in Biotechnology
Figure 1
Solid-phase peptide synthesis. An N a-derivatized amino acid is attached to an insoluble 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 aminoacid–linker–support complex. The deprotection–coupling cycle is repeated until the
desired sequence of amino acids has been generated. Finally, the peptide–linker–support
complex is cleaved to obtain the peptide as a free acid or an amide, depending on
the chemical nature of the linker. Ideally, the cleavage reagent should also remove
the amino acid side-chain-protecting groups, which are stable to the N a-deprotection
conditions.
HIV-1 protease inhibitors bound8,10,11. Another recent
application of stepwise synthesis is the construction of
bovine pancreatic-trypsin inhibitor and its analogs (58
residues) by Fmoc chemistry12–14. The analogs were
used for the biophysical characterization of folding
intermediates and to dissect folding pathways13,14.
Stepwise assembly can proceed either in a linear
fashion or on a template. Mutter et al. developed
the template-assembled synthetic protein (TASP)
approach, based on the concept that templates would
promote secondary-structure formation and minimize
the aggregation of larger complexes15,16. TASP molecules containing a four-a-helix bundle17 and a
combination four a-helix-bundle-and-b-barrel-like
structure18 were constructed by stepwise SPPS on a
template; the templates were typically small cyclic
peptides. ‘Minicollagens’ (76–121 residues) have also
been assembled by stepwise Fmoc chemistry on a
branched template19,20. These minicollagens have been
used in biochemical studies in order to identify cellular recognition sites within interstitial collagens, which
might be important in wound healing and platelet
aggregation19,21.
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The ability to assemble proteins in a stepwise fashion
is limited by the synthetic efficiency of each step. As
discussed by Kent22, the synthesis of a 100-residue protein with 99.9% efficiency at each step provides a 90%
overall yield of the desired product; 97% efficiency
provides a 5% overall yield. The effective solvation of
the peptide–resin complex is perhaps the most crucial
condition for efficient chain assembly; ‘difficult couplings’ during SPPS have been attributed to the poor
solvation of the growing chain. Infrared and nuclear
magnetic resonance spectroscopy have shown that
intermolecular b-sheet aggregates can lower the coupling efficiency23–25. Both the Boc and the Fmoc
chemistries appear to be susceptible to the same difficult couplings26–28, and side-by-side syntheses for moderate-length chains (~30 residues) are comparable29,30.
Coupling efficiencies are enhanced by the addition of
polar solvents31–34 and the use of in situ neutralization
protocols35. It has also been suggested that adding
chaotropic salts to organic solvents can disrupt b-sheet
aggregates and thus improve overall yields36,37. Furthermore, the use of a lower substitution level of resin
in order to avoid interchain crowding can also improve
the overall synthetic efficiency38.
A different approach to circumventing potential
aggregation is reversible protection of the amino acid
amide nitrogen using Na-Fmoc-Na(2-Fmoc-hydroxy4-methoxybenzyl) derivatives39. The formation of
b-sheet structure can be minimized by using amide
nitrogen protection every sixth to eighth amino
acid39,40. Alternatively, incorporating ‘pseudoproline’
residues can be used to disrupt intermolecular b-sheet
formation41.
Assembling proteins from fragments
Convergent synthesis of protected peptide fragments
In contrast to the stepwise approach, convergent
synthesis uses protected peptide fragments to make proteins. The ‘convergence’ of the protein fragments can
be performed either in solution or on the solid phase
(Fig. 2). The advantage of convergent protein synthesis is that fragments of the desired protein are synthesized, purified and characterized (which ensures that
each fragment is of high integrity), and only then
assembled into the complete protein. Thus, the cumulative effects of stepwise synthetic errors are minimized.
Convergent synthesis requires ready access to pure, partially protected peptide segments, which are needed as
building blocks. Preparing these intermediates by SPPS
depends on several levels of selectively cleavable protecting groups and linkers. Some of these protection
schemes are orthogonal; that is, they involve two or
more classes of group, which are removed by different
chemical mechanisms and so can be removed in any
order and in the presence of other classes. Examples of
orthogonality for convergent synthesis include the combination of the Boc–benzyl strategy with nucleophilelabile, base-labile, palladium-labile or photolabile
linkers, and the combination of Fmoc–tertiary-butyl
strategy with dilute-acid-labile, palladium-labile or
photolabile linkers. Such combinations have been
successful at generating Na-amino- and side-chainprotected segments with free Ca-carboxyl groups42,43.
There are many examples of protein synthesis by the
convergent approach in solution or in the solid phase.
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+
+
Repetitive
cycle
Repetitive
cycle
+ H2N
+ H2N
(1) Cleavage
(2) Purification
Nα-deprotection
+ H2N
n′
n
Coupling
n′
n
Nα-deprotection
+ H2N
n′′
n′
n
Coupling
n′′
n′
n
(1) Nα-deprotection
(2) Cleavage and
side-chain
deprotection
(3) Purification
H2N
n′′
n′
n
OH
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 resinbound 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 convergentsynthesis approach in solution44, and green fluorescent
protein (238 residues) was assembled in solution using
26 protected fragments prepared by solid-phase methods45. 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),
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Androctonus australis Hector toxin II (64 residues; Ref. 52),
l-Cro DNA binding protein53 (66 residues), prothymosin 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-chromatographic purification and the organic solvents traditionally used for coupling reactions43,56; mixed-solvent
systems can be used to improve some solubility problems56. Second, the individual rates for coupling fragments are substantially lower than those for activated
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N terminal
(a)
C terminal
Product
O
O
SH
S
Br
O
(b)
O
O
SH
O
Br
S
O
SH
O
S
N
N
O
O
(c)
O
O
H
H2 N
O
H
O
N
O
R
R
H
H2N
N
H
H
H2N
O
O
O
O
H
N
N
H
O
N
H
O
O
H
H2N
N
N
H
N
trends in Biotechnology
Figure 3
N- and C-terminal groups used for non-native chemoselective ligation.
The product structures shown are produced when the groups react
under aqueous conditions. Ligation by the groups in (a) results in
thioester bond formation; groups in (b) form a thioether bond. The
ligation strategies depicted in (c) all use an aldehyde for the Nterminal ligation group.
amino acid species in traditional stepwise synthesis, and
the reaction times are often increased to enhance the
fragment-coupling efficiency. Third, the amino acid
can racemize at the C terminus of a segment during
coupling, but this can be avoided by constructing
fragments that have either a Gly or a Pro C-terminus.
Chemoselective ligation of unprotected peptide
fragments
As an alternative to the convergent approach, methods have been developed that can link unprotected peptide fragments. Known as ‘chemoselective ligation’, the
original strategies resulted in the formation of thioester
or oxime bonds between peptide fragments. For example, the reaction of a peptide containing a C-terminal
thioacid with a peptide containing an N-terminal
bromoacetyl group results in a synthetic protein product
containing a thioester bond57,58 (Fig. 3a). This approach
has been used to construct HIV-1 protease57, a four-ahelix TASP molecule59, a folded b-sandwich fibronectin domain model60 and a tethered dimer of HIV-1
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protease61. A convenient linker has been described that
produces C-terminal thioacids after Boc chemistry62.
A variation of the thioester approach, in which a peptide C-terminal thiol (such as Cys) is reacted with an
N-terminal bromoacetyl or maleimido peptide to form
a thioether bond (Fig. 3b), has been used to construct
a triple-helical domain from the macrophage scavenger
receptor63,64, linked cytoplasmic domains from the
aIIbb3 integrin65, a b-meander TASP molecule66 and a
129-residue tripod protein67. Linkers that produce Cterminal thiols have been described for both Boc68 and
Fmoc69 chemistries. To prevent the decomposition of
thioester linkers during Fmoc syntheses, an Fmoc
deprotection solution containing 1-methylpyrrolidine,
hexamethyleneimine and 1-hydroxybenzotriazole in
N-methylpyrrolidone-dimethylsulfoxide (1:1) should
be used70. As an alternative to thioester and thioether
bonds, peptides containing either aldehyde or Nterminal aminooxyacetyl groups can be ligated to form
oxime bonds71,72 (Fig. 3c). An aldehyde-containing
peptide can also be ligated to a peptide containing a
weak nucleophilic base, such as hydrazide, N-terminal
Cys or N-terminal Thr, to form hydrazone, thiazolidine
and oxazolidine linkages, respectively73–76 (Fig. 3c).
Chemoselective ligation was made more attractive by
‘native chemical ligation’, in which an amide bond
(rather than a thioester, thioether or oxime bond) is
generated between fragments58,77 (Fig. 4). A peptide
with a C-terminal thioacid is converted to a 5-thio-2nitrobenzoic acid ester and then reacted with a peptide
with an N-terminal Cys residue. The initial thioester
ligation product spontaneously rearranges, producing
an amide bond and regenerating the free sulfhydryl on
the Cys. The ligation strategy has been extended further to make use of other thiol additives and their
respective reactivities78. The trityl-associated mercaptoproprionic-acid leucine (TAMPAL) resin allows the
convenient generation of any amino acid as a C-terminal thioester79. Native chemical ligation can proceed
intramolecularly to create cyclized proteins80,81.
Conformationally assisted protein ligation, in which
the C- and N-termini to be ligated are brought into
close proximity by peptide folding, has been shown to
eliminate the absolute need for an N-terminal Cys
residue82. Amide bonds can also be generated by
chemoselective ligation methods that result in thiazolidine linkages via an O→N acyl shift to form hydroxymethyl thiazolidine83. Chemoselective ligation can be
performed for multiple fragments84,85 and in either the
N→C or the C→N direction in the solid phase86. A
modular chemoselective-ligation strategy was used to
synthesize a covalently linked dimer of the b–HLH–Z
domains of the cMyc and Max transcription factors87.
The individual cMyc and Max domains were assembled by native chemical ligation and then linked via
oxime bond formation.
Chemoselective ligation has recently been extended
to allow ‘expressed protein ligation’. The gene for a
desired protein is cloned into an intein vector and
expressed. The splicing process that occurs at the
recombinant-protein–intein junction is intercepted by
a thiol, generating the a-thioester recombinant protein. The recombinant protein is then ligated to a peptide or protein that has an N-terminal Cys residue88,89.
This approach also allows a synthetic peptide to be
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(a)
(b)
+ HS
Repetitive
cycle
+
Repetitive
cycle
+ H2N
+ H2N
(1) Nα-deprotection
(2) Cleavage and side-chain
deprotection
(3) React with R-Br
(4) Purification
H2N
+
SR
H 2N
HS
n′
R1
N
(1) Nα-deprotection
(2) Cleavage and side-chain
deprotection
(3) Purification
n
NH2
OH
O
N
H
SR′
O R2
H2N
S
H
O R3
N
H
O
H
R1
N
N
H
H
O R3
O N
N
H
O
S
O R2
H2N
n′
HS
n
NH2
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 converted to a thioester, allowing the two fragments to react in aqueous solution to form a thioester bond between them. Spontaneous rearrangement 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 distinct structures. For example, Sakakibara et al. showed
that synthetic peptides of the sequence (Pro–Pro–Gly)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
TIBTECH JUNE 2000 (Vol. 18)
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
G
P
P*
+ H2N
G
P
P*
n′
n
Nα-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
O
O
G
P
P*
G
P
P*
G
P
P*
G
P
P*
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-ahelix-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 2,29bipyridine-4,49-dicarboxylic acid into a 15-residue
peptide and complexing with Fe21 (Ref. 93). The
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individual strands, designated ‘pepy’, were 35% a-helical; 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-ioninduced 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
methods
Cumulative effects of synthetic inefficiencies
Convergent synthesis
Fragments are of high integrity; cumulative effects
of stepwise synthetic errors are minimized
Solubility of fragments in aqueous environment
Racemization during fragment coupling
Efficiency of fragment coupling
Chemoselective ligation Fragments are of high integrity; cumulative effects
of stepwise synthetic errors are minimized
Fragments are typically soluble in aqueous environments
Limited residue possibilities at the ligation site
Hydrophobic (transmembrane) fragments may
have limited solubility in aqueous environment
Directed assembly
Redundancy of protein structure
Control of assembly size
Fragments are of high integrity; cumulative effects
of stepwise synthetic errors are minimized
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 complexation, 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 tails97–102. Examining peptideamphiphile structure using CD and one- and two dimensional NMR spectroscopy has shown that this approach
can nucleate or stabilize a-helical and collagen-like
triple-helix structures98,100–103. 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 incorporation 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.
TIBTECH JUNE 2000 (Vol. 18)
There are several feasible approaches for the chemical
synthesis of proteins, each with benefits and limitations
(Table 1). Stepwise solid-phase synthesis is straightforward but will always be limited by the cumulative
effects of synthetic inefficiencies. Convergent protein
synthesis uses fragments of high integrity, but solubilization 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 traditional 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 racemization. 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.
References
1 Fields, G.B., ed. (1997) Solid-phase peptide synthesis. Methods in
Enzymology 289
2 Merrifield, B. (1986) Solid phase synthesis. Science 232, 341–347
3 Angeletti, R.H. et al. (1997) Six-year study of peptide synthesis.
Methods Enzymol. 289, 697–717
4 Remmer, H.A. and Fields, G.B. (2000) Chemical synthesis of
peptides. In Peptide and Protein Drug Analysis (Reid, R.E., ed.),
pp. 133–169, Marcel Dekker
5 Gutte, B. and Merrifield, R.B. (1971) The synthesis of ribonuclease
A. J. Biol. Chem. 246, 1722–1741
6 Schneider, J. and Kent, S.B.H. (1988) Enzymatic activity of a
synthetic 99 residue protein corresponding to the putative HIV-1
protease. Cell 54, 363–368
7 Nutt, R.F. et al. (1988) Chemical synthesis and enzymatic activity
of a 99-residue peptide with a sequence proposed for the human
immunodeficiency virus protease. Proc. Natl. Acad. Sci. U. S. A.
85, 7129–7133
249
TTEC June 2000
4/5/00
11:13 am
Page 250
REVIEWS
8 Wlodawer, A. et al. (1989) Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-1 protease. Science 245,
616–621
9 deL. Milton, R.C. et al. (1992) Total chemical synthesis of a
D-enzyme: the enantiomers of HIV-1 protease show demonstration
of reciprocal chiral substrate specificity. Science 256, 1445–1448
10 Miller, M. et al. (1989) Structure of complex of synthetic HIV-1
protease with a substrate-based inhibitor at 2.3 Å resolution.
Science 246, 1149–1152
11 Swain, A.L. et al. (1990) X-ray crystallographic structure of a
complex between a synthetic protease of human immunodeficiency
virus 1 and a substrate-based hydroxyethylamine inhibitor. Proc.
Natl. Acad. Sci. U. S. A. 87, 8805–8809
12 Ferrer, M. et al. (1992) Solid-phase synthesis of bovine pancreatic
trypsin inhibitor (BPTI) and two analogues. Int. J. Pept. Protein Res.
40, 194–207
13 Ferrer, M. et al. (1995) Partially folded, molten globule and molten
coil states of bovine pancreatic trypsin inhibitor. Nat. Struct. Biol.
2, 211–217
14 Pan, H. et al. (1995) Extensive nonrandom structure in reduced
and unfolded bovine pancreatic trypsin inhibitor. Biochemistry 34,
13974–13981
15 Mutter, M. (1988) Nature’s rules and chemist’s tools: a way for
creating novel proteins. Trends Biochem. Sci. 13, 260–265
16 Mutter, M. and Vuilleumier, S. (1989) A chemical approach to
protein design: template-assembled synthetic proteins (TASP).
Angew. Chem., Int. Ed. Engl. 28, 535–554
17 Mutter, M. et al. (1992) Template-assembled synthetic proteins
with four-helix-bundle topology. J. Am. Chem. Soc. 114, 1463–1470
18 Mutter, M. et al. (1989) The construction of new proteins: a
template-assembled synthetic protein (TASP) containing both a
4-helix bundle and b-barrel-like structure. Proteins 5, 13–21
19 Grab, B. et al. (1996) Promotion of fibroblast adhesion by triplehelical peptide models of type I collagen-derived sequences. J. Biol.
Chem. 271, 12234–12240
20 Barnes, M.J. et al. (1996) The use of collagen-based model peptides
to investigate platelet-reactive sequences in collagen. Biopolymers
40, 383–397
21 Morton, L.F. et al. (1997) The platelet reactivity of synthetic peptides based on the collagen III fragment a1(III)CB4. J. Biol. Chem.
272, 11044–11048
22 Kent, S.B.H. (1980) New aspects of solid phase peptide synthesis.
In Biomedical Polymers (Goldberg, E.P. and Nakajima, A., eds),
pp. 213–242, Academic Press
23 Live, D.H. and Kent, S.B.H. (1983) Correlation of coupling rates
with physicochemical properties of resin-bound peptides in solid
phase synthesis. In Peptides: Structure and Function (Hruby, V.J. and
Rich, D.H., eds), pp. 65–68, Pierce Chemical
24 Mutter, M. et al. (1985) The impact of secondary structure
formation in peptide synthesis. In Peptides: Structure and Function
(Deber, C.M. et al., eds), pp. 397–405, Pierce Chemical
25 Ludwick, A.G. et al. (1986) Association of peptide chains during
Merrifield solid-phase peptide synthesis. J. Am. Chem. Soc. 108,
6493–6496
26 Meister, S.M. and Kent, S.B.H. (1983) Sequence-dependent
coupling problems in stepwise solid phase peptide synthesis: occurrence, mechanism, and correction. In Peptides: Structure and Function
(Hruby, V.J. and Rich, D.H., eds), pp. 103–106, Pierce Chemical
27 Young, J.D. et al. (1990) Coupling efficiencies of amino acids in
solid phase synthesis of peptides. Peptide Res. 3, 194–200
28 van Woerkom, W.J. and van Nispen, J.W. (1991) Difficult couplings
in stepwise solid phase peptide synthesis: predictable or just a
guess? Int. J. Pept. Protein Res. 38, 103–113
29 Atherton, E. et al. (1983) Peptide synthesis, part 3: comparative
solid-phase syntheses of human b-endorphin on polyamide
supports using t-butoxycarbonyl and fluorenylmethoxycarbonyl
protecting groups. J. Chem. Soc., Perkin Trans. 1, 65–73
30 Wade, J.D. et al. (1986) Solid-phase synthesis of a-human atrial
natriuretic factor: comparison of the Boc–polystyrene and Fmoc–
polyamide methods. Biopolymers 25, S21–S37
31 Fields, G.B. and Fields, C.G. (1991) Solvation effects in solid-phase
peptide synthesis. J. Am. Chem. Soc. 113, 4202–4207
250
32 Fields, G.B. et al. (1992) Principles and practice of solid-phase
peptide synthesis. In Synthetic Peptides: A User’s Guide (Grant, G.A.,
ed.), pp. 77–183, W.H. Freeman
33 Hyde, C. et al. (1992) Internal aggregation during solid phase
peptide synthesis: dimethyl sulfoxide as a powerful dissociating
solvent. J. Chem. Soc., Chem. Commun. 1573–1575
34 Miranda, L.P. and Alewood, P.F. (1999) Accelerated chemical
synthesis of peptides and small proteins. Proc. Natl. Acad. Sci. U.S.A.
96, 1181–1186
35 Alewood, P. et al. (1997) Rapid in situ neutralization protocols for
Boc and Fmoc solid-phase chemistries. Methods Enzymol. 289,
14–29
36 Stewart, J.M. and Klis, W.A. (1990) Polystyrene-based solid phase
peptide synthesis: the state of the art. In Innovation and Perspectives
in Solid Phase Synthesis (Epton, R., ed.), pp. 1–9, Solid Phase
Conference Coordination, Birmingham, UK
37 Thaler, A. et al. (1991) Lithium-salt effects in peptide synthesis,
part II: improvement of degree of resin swelling and of efficiency
of coupling in solid-phase synthesis. Helv. Chim. Acta 74, 628–643
38 Tam, J.P. and Lu, Y-A. (1995) Coupling difficulty associated with
interchain clustering and phase transition in solid phase peptide
synthesis. J. Am. Chem. Soc. 117, 12058–12063
39 Johnson, T. et al. (1993) A reversible protecting group for the
amide bond in peptides: use in the synthesis of ‘difficult sequences’.
J. Chem. Soc., Chem. Commun. 369–372
40 Hyde, C. et al. (1994) Some ‘difficult sequences’ made easy: a study
of interchain association in solid-phase peptide synthesis. Int. J.
Pept. Protein Res. 43, 431–440
41 Wöhr, T. et al. (1996) Pseudo-prolines as a solubilizing, structuredisrupting protection technique in peptide synthesis. J. Am. Chem.
Soc. 118, 9218–9227
42 Lloyd-Williams, P. et al. (1993) Convergent solid-phase peptide
synthesis. Tetrahedron 49, 11065–11133
43 Albericio, F. et al. (1997) Convergent solid-phase peptide synthesis.
Methods Enzymol. 289, 313–336
44 Yajima, H. and Fujii, N. (1981) Totally synthetic crystalline
ribonuclease A. Biopolymers 20, 1859–1867
45 Nishiuchi, Y. et al. (1998) Chemical synthesis of the precursor
molecule of the Aequorea green fluorescent protein, subsequent
folding, and development of fluorescence. Proc. Natl. Acad. Sci.
U. S. A. 95, 13549–13554
46 Vuilleumier, S. and Mutter, M. (1993) Synthetic peptide and
template-assembled synthetic protein models of the hen egg white
lysozyme 87–97 helix. Biopolymers 33, 389–400
47 Feng, Y. et al. (1996) Collagen-based structures containing the
peptoid residue N-isobutylglycine (Nleu): synthesis and biophysical studies of Gly–Pro–Nleu sequences by circular dichroism,
ultraviolet absorbance and optical rotation. Biopolymers 39, 859–872
48 Feng, Y. et al. (1997) Collagen-based structures containing the
peptoid residue N-isobutylglycine (Nleu): synthesis and biophysical
studies of Gly–Nleu–Pro sequences by circular dichroism and
optical rotation. Biochemistry 36, 8716–8724
49 Hendrix, J.C. et al. (1992) Studies related to a convergent fragment-coupling approach to peptide synthesis using the Kaiser
oxime resin. J. Org. Chem. 57, 3414–3420
50 Hendrix, J.C. and Lansbury, P.T. (1992) Synthesis of a protected
peptide corresponding to residues 1–25 of the b-amyloid protein
of Alzheimer’s disease. J. Org. Chem. 57, 3421–3426
51 Dalcol, I. et al. (1995) Convergent solid phase peptide synthesis:
an efficient approach to the synthesis of highly repetitive protein
domains. J. Org. Chem. 60, 7575–7581
52 Grandas, A. et al. (1989) Convergent solid phase peptide synthesis
VII: good yields in the coupling of protected segments on a solid
support. Tetrahedron 45, 4637–4648
53 Atherton, E. et al. (1990) Solid phase fragment condensation – the
problems. In Innovation and Perspectives in Solid Phase Synthesis
(Epton, R., ed.), pp. 11–25, Solid Phase Conference Coordination,
Birmingham, UK
54 Barlos, K. et al. (1991) Synthesis of prothymosin a (ProTa) – a
protein consisting of 109 amino acid residues. Angew. Chem., Int.
Ed. Engl. 30, 590–593
55 Peluso, S. et al. (1997) Protein mimetics (TASP) by sequential
TIBTECH JUNE 2000 (Vol. 18)
TTEC June 2000
4/5/00
11:13 am
Page 251
REVIEWS
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
condensation of peptide loops to an immobilised topological
template. Tetrahedron 53, 7231–7236
Sakakibara, S. (1999) Chemical synthesis of proteins in solution.
Biopolymers (Peptide Sci.) 51, 279–296
Schnölzer, M. and Kent, S.B.H. (1992) Constructing proteins by
dovetailing unprotected synthetic peptides. Science 256, 221–225
Muir, T.W. et al. (1997) Protein synthesis by chemical ligation of
unprotected peptides in aqueous solution. Methods Enzymol. 289,
266–298
Dawson, P.E. and Kent, S.B.H. (1993) Convenient total synthesis
of a 4-helix TASP molecule by chemoselective ligation. J. Am.
Chem. Soc. 115, 7263–7266
Williams, M.J. et al. (1994) Total chemical synthesis of a folded
b-sandwich protein domain: an analog of the tenth fibronectin
type 3 module. J. Am. Chem. Soc. 116, 10797–10798
Baca, M. et al. (1995) Chemical ligation of cysteine-containing
peptides: synthesis of a 22 kDa tethered dimer of HIV-1 protease.
J. Am. Chem. Soc. 117, 1881–1887
Canne, L.E. et al. (1995) A general method for the synthesis of
thioester resin linkers for use in the solid phase synthesis of
peptide-a-thioacids. Tetrahedron Lett. 36, 1217–1220
Tanaka, T. et al. (1993) A synthetic model of collagen structure taken
from bovine macrophage scavenger receptor. FEBS Lett. 334, 272–276
Tanaka, T. et al. (1996) Synthetic collagen-like domain derived
from the macrophage scavenger receptor binds acetylated lowdensity lipoprotein in vitro. Protein Eng. 9, 307–313
Muir, T.W. et al. (1994) Design and chemical synthesis of a neoprotein structural model for the cytoplasmic domain of a multisubunit cell-surface receptor: integrin aIIbb3 (platelet GPIIb–IIIa).
Biochemistry 33, 7701–7708
Nefzi, A. et al. (1995) Chemoselective ligation of multifunctional
peptides to topological templates via thioether formation for TASP
synthesis. Tetrahedron Lett. 36, 229–230
McCafferty, D.G. et al. (1995) Engineering of a 129-residue
tripod protein by chemoselective ligation of proline-II helices.
Tetrahedron 51, 9859–9872
Englebretsen, D.R. et al. (1995) A novel thioether linker: chemical synthesis of a HIV-1 protease analogue by thioether ligation.
Tetrahedron Lett. 36, 8871–8874
Ramage, R. et al. (1996) Methodology for chemical synthesis of
proteins. In Innovation and Perspectives in Solid Phase Synthesis and
Combinatorial Libraries 1996 (Epton, R., ed.), pp. 1–10, Mayflower
Scientific, Kingswinford, UK
Li, X. et al. (1998) Direct preparation of peptide thioesters using
an Fmoc solid-phase method. Tetrahedron Lett. 39, 8669–8672
Tuchscherer, G. (1993) Template assembled synthetic proteins:
condensation of a multifunctional peptide to a topological template via chemoselective ligation. Tetrahedron Lett. 34, 8419–8422
Rose, K. (1994) Facile synthesis of homogeneous artificial proteins.
J. Am. Chem. Soc. 116, 30–33
Rao, G. and Tam, J.P. (1994) Synthesis of peptide dendrimer.
J. Am. Chem. Soc. 116, 6975–6976
Spetzler, J.C. and Tam, J.P. (1995) Unprotected peptides as building
blocks for branched peptides and peptide dendrimers. Int. J. Pept.
Protein Res. 45, 78–85
Shao, J. and Tam, J.P. (1995) Unprotected peptides as building
blocks for the synthesis of peptide dendrimers with oxime, hydrazone and thiazolidine linkages. J. Am. Chem. Soc. 117, 3893–3899
Tam, J.P. and Spetzler, J.C. (1997) Multiple antigen peptide
system. Methods Enzymol. 289, 612–637
Dawson, P.E. et al. (1994) Synthesis of proteins by native chemical ligation. Science 266, 776–779
Dawson, P.E. et al. (1997) Modulation of reactivity in native
chemical ligation through the use of thiol additives. J. Am. Chem.
Soc. 119, 4325–4329
Hackeng, T.M. et al. (1999) Protein synthesis by native chemical
ligation: expanded scope by using straightforward methodology.
Proc. Natl. Acad. Sci. U. S. A. 96, 10068–10073
Camarero, J.A. et al. (1998) Chemical synthesis of a circular
protein domain: evidence for folding-assisted cyclization. Angew.
Chem., Int. Ed. Engl. 37, 347–349
Camarero, J.A. and Muir, T.W. (1999) Biosynthesis of a head-to-tail
TIBTECH JUNE 2000 (Vol. 18)
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
cyclized protein with improved biological activity. J. Am. Chem.
Soc. 121, 5597–5598
Beligere, G.S. and Dawson, P.E. (1999) Conformationally assisted
protein ligation using C-terminal thioester peptides. J. Am. Chem.
Soc. 121, 6332–6333
Liu, C.F. and Tam, J.P. (1994) Peptide segment ligation strategy
without use of protecting groups. Proc. Natl. Acad. Sci. U. S. A.
91, 6574–6588
Tam, J.P. et al. (1999) Orthogonal ligation strategies for peptide
and protein. Biopolymers (Peptide Sci.) 51, 311–332
Beligere, G.S. and Dawson, P.E. (1999) Synthesis of a three zinc
finger protein, Zif 268, by native chemical ligation. Biopolymers
(Peptide Sci.) 51, 363–369
Canne, L.E. et al. (1999) Chemical protein synthesis by solid phase
ligation of unprotected peptide segments. J. Am. Chem. Soc. 121,
8720–8727
Canne, L.E. et al. (1995) Total chemical synthesis of a unique transcription factor-related protein: cMyc–Max. J. Am. Chem. Soc.
117, 2998–3007
Cotton, G.J. et al. (1999) Insertion of a synthetic peptide into a
recombinant protein framework: a protein biosensor. J. Am. Chem.
Soc. 121, 1100–1101
Ayers, B. et al. (1999) Introduction of unnatural amino acids into
proteins using expressed protein ligation. Biopolymers (Peptide Sci.)
51, 343–354
Sakakibara, S. et al. (1968) Synthesis of poly-(L-prolyl-L-prolylglycyl) of defined molecular weights. Bull. Chem. Soc. Jpn. 41, 1273
Mayo, K.H. and Fields, G.B. (1997) Peptides as models for understanding protein folding. In Protein Structural Biology in Bio-Medical
Research (Allewell, N. and Woodward, C., eds), pp. 567–612,
JAI Press, Greenwich, CT, USA
Ghadiri, M.R. et al. (1992) A convergent approach to protein
design: metal ion-assisted spontaneous self-assembly of a polypeptide into a triple-helix bundle protein. J. Am. Chem. Soc. 114,
825–831
Lieberman, M. and Sasaki, T. (1991) Iron(II) organizes a synthetic
peptide into three-helix bundles. J. Am. Chem. Soc. 113, 1470–1471
Sasaki, T. and Lieberman, M. (1993) Between the secondary structure and the tertiary structure falls the globule: a problem in de novo
protein design. Tetrahedron 49, 3677–3689
Suzuki, K. et al. (1998) Metal ion induced self-assembly of a
designed peptide into a triple-stranded a-helical bundle: a novel
metal binding site in the hydrophobic core. J. Am. Chem. Soc. 120,
13008–13015
Ghadiri, M.R. et al. (1992) Design of an artificial four-helix bundle
metalloprotein via a novel ruthenium(II)-assisted self-assembly
process. J. Am. Chem. Soc. 114, 4000–4002
Berndt, P. et al. (1995) Synthetic lipidation of peptides and amino
acids: monolayer structure and properties. J. Am. Chem. Soc. 117,
9515–9522
Yu, Y-C. et al. (1996) Self-assembling amphiphiles for construction
of protein molecular architecture. J. Am. Chem. Soc. 118,
12515–12520
Yu, Y-C. et al. (1997) Construction of biologically active protein
molecular architecture using self-assembling peptide-amphiphiles.
Methods Enzymol. 289, 571–587
Fields, G.B. et al. (1998) Protein-like molecular architecture:
biomaterial applications for inducing cellular receptor binding and
signal transduction. Biopolymers 47, 143–151
Yu, Y-C. et al. (1998) Minimal lipidation stabilizes protein-like
molecular architecture. J. Am. Chem. Soc. 120, 9979–9987
Yu, Y-C. et al. (1999) Structure and dynamics of peptideamphiphiles incorporating triple-helical protein-like molecular
architecture. Biochemistry 38, 1659–1668
Fields, G.B. et al. Peptide-amphiphile induction of a-helical and
triple-helical structures. In Synthetic Macromolecules with Higher
Structural Order (Khan, I.M., ed.), American Chemical Society,
Washington, DC, USA (in press)
Kochendoerfer, G.G. et al. (1999) Total chemical synthesis of
the integral membrane protein influenza A virus M2: role of
its C-terminal domain in tetramer assembly. Biochemistry 38,
11905–11913
251