164

POLYMER-SUPPORTED REAGENTS

Vol. 11

POLYPEPTIDE SYNTHESIS,
SOLID-PHASE METHOD

Introduction

Peptides play key structural and functional roles in biochemistry, pharmacology,
and neurobiology, and are important probes for research in enzymology, immunol-
ogy, and molecular biology. A peptide is a chain of 3–50 amino acid building blocks,
taken from among the 20 genetically encoded

L

-residues, or unusual ones. The se-

quences can be linear, cyclic, or branched. A rapid, efficient, and reliable method
for the chemical synthesis of peptides is of utmost importance. Synthesis can
confirm structures of naturally occurring substances and make them available in
greater quantities for further investigations, can delineate antigenic determinants

Encyclopedia of Polymer Science and Technology. Copyright John Wiley & Sons, Inc. All rights reserved.

Vol. 11

POLYPEPTIDE SYNTHESIS, SOLID-PHASE METHOD

165

to allow preparations of artificial vaccines, and can lead to potent new drugs by
judicious chemical substitutions that change functional groups and/or conforma-
tions of the parent peptide. These synthetic methods can also be extended to the
study of proteins, which are defined as polypeptide chains of 50 or more residues.

The last century has witnessed the development of an extraordinary array

of techniques for the chemical assembly of amino acids to form peptides. They can
be subdivided into solution and solid–phase methods. The former have evolved
since the beginning of the twentieth century (1–3); although quite powerful, they
also require considerable labor, time, and skill because of the unpredictable sol-
ubility characteristics of intermediates and because of the lack of a method for
automation. Recognizing the mechanical and physical limitations of the earlier
chemical precedents, but retaining their strengths, in 1959 R. B. Merrifield of
the Rockefeller University conceived the method of solid–phase peptide synthe-
sis (SPPS). The solid–phase method has since been expanded to include other
oligomeric systems, including DNA and polysaccharides, and to include combi-
natorial libraries. In recognition of the maturation and impact of this body of
work, Merrifield was honored with the 1984 Nobel Prize in Chemistry (4). Many
comprehensive reviews of SPPS have been written over the years (5–20).

Solid-phase synthesis is based on the covalent attachment (anchoring) of

the growing peptide chain to an insoluble polymeric support or resin carrier, so
that unreacted soluble reagents can be removed by filtration and washing without
loss of the peptide–resin. Subsequently, the insoluble peptide–resin is extended
by a series of addition cycles, which are required to proceed with high yields
and fidelities. Excess soluble reagents drive the reactions to completion. Because
of the speed and simplicity of the repetitive steps, the solid-phase procedure is
amenable to automation. After the chain has been established, it is necessary to
release (cleave) the crude peptide from the support under conditions that do not
affect sensitive residues in the sequence. Purification and characterization follow
to ensure and verify that the desired structure has been obtained.

Strategy and Tactics

A general strategy for stepwise solid-phase synthesis of linear polypeptides is
shown in Figure 1. An insoluble polymeric support (Resin) is functionalized with
a reactive group, typically an aminomethyl moiety. The first amino acid is attached
to this functional group through a linker, usually a handle. A handle is defined as a
bifunctional spacer that on one end typically contains a carboxyl group for coupling
to an aminomethyl group on the polymeric support. The other end attaches to the
first amino acid and acts as a permanent protecting group during chain assembly
(7). The first amino acid is then attached to the handle by the C

α

-carboxyl group

as either an ester or amide linkage depending on whether the desired peptide will
contain a C-terminal acid or amide group, respectively. Each amino acid contains
a temporary protecting group (T, Fig. 1) on the N

α

-amino group and a permanent

protecting group (P, Fig. 1) on a reactive side-chain functional group if necessary.
The peptide chain is then elaborated in the C

→ N direction by repeated cycles of

deprotection of the temporary N

α

-protecting group followed by coupling of the next

protected amino acid. This deprotection/coupling cycle is repeated until all amino

166

POLYPEPTIDE SYNTHESIS, SOLID-PHASE METHOD

Vol. 11

Fig. 1.

Generic stepwise solid-phase synthesis of linear polypeptides. T, temporary pro-

tection; P

x

, permanent side-chain protection; R

x

, amino acid side chain.

acids of the peptide have been introduced. The peptide is then cleaved from the
polymeric support, and the permanent protecting groups are removed; these last
two steps are often combined in a single procedure. This strategy with C

→ N chain

assembly offers the advantage that racemization is negligible. Variations on this
general scheme exist that allow other C-terminal modifications such as thioesters,
esters, or hydrazides to be synthesized. The chain may also be attached to the resin
via either a side-chain functional group or the peptide backbone to allow elongation
in the N

→ C direction as well as C → N (bidirectional synthesis) and to allow

on-resin cyclization between the termini and/or a side chain for the synthesis

Vol. 11

POLYPEPTIDE SYNTHESIS, SOLID-PHASE METHOD

167

Table 1. Statistics of Chain Assembly in SPPS

a

Total number of residues

Average % yield of repetitive

deprotection/coupling cycles

5

10

25

50

100

80.0

32.8

10.7

0.4

0.0

0.0

90.0

59.0

34.9

7.2

0.5

0.0

95.0

77.4

59.9

27.7

7.7

0.6

99.0

95.1

90.4

77.8

60.5

36.6

99.5

97.5

95.1

88.2

77.8

60.6

99.9

99.5

99.0

97.5

95.1

90.5

a

One can select a sequence length from the column headings and an average percent yield

over all deprotection/coupling cycles from the left-most column of numbers. The number at
the intersection of the selected column and row represents the theoretical percentage of pep-
tide chains on resin that will contain the correct sequence after linear assembly has been
completed. This theoretical percentage yield does not include side reactions and manipu-
lative losses that will result from the cleavage and the purification steps that follow chain
assembly in a synthesis.

of lactams, see References 3,5,7,8,10,12, and 16–20 for further information on
C-terminal modifications.

Because the growing peptide is not isolated and purified after each repet-

itive deprotection/coupling cycle (Fig. 1), the fidelity of each step must proceed
in near quantitative fashion. Table 1 contains the percent of correct peptide se-
quence that will be found on the resin at the end of a synthesis as a function of
the average percent yield over all deprotection/coupling cycles and as a function
of the total number of residues in the sequence. Yields of 95% per cycle, what
would be considered fantastic in a solution synthesis, are unacceptably low for
even peptides as short as 10 amino acids, as only 59.9% of the correct sequence
would be assembled on the resin. Yields must be greater than 99% for each repet-
itive deprotection/coupling cycle to have any hope of isolating the desired product.
The data of Table 1 also show that for sequences of greater than 25 amino acids,
increasing the yield of each cycle by a few tenths of a percent makes a substan-
tial difference in the overall yield. Thus, double coupling of each amino acid is
routinely done on longer syntheses. Fortunately, the art of peptide synthesis has
progressed to the level that yields greater than 99.5% for each repetitive cycle can
generally be achieved. The impurities are often deletion peptides that differ from
the desired sequence by only one or two amino acids and that may be hard if not
impossible to remove during purification. Thus, for longer sequences many pep-
tide chemists routinely include a capping step with acetic anhydride, after double
coupling and before deprotection, to terminate the chain before the next cycle is
begun. The termination peptides will then differ in length from the desired prod-
uct by many amino acids and will be easier to remove from the desired product
during purification.

In the repetitive deprotection/coupling cycle of Figure 1 are three classes

of protecting groups. The first is the temporary N

α

-protection. The remaining

two classes contain permanent groups, the side-chain-protection and the peptide-
linker bond, that remain intact during chain assembly. Implicit in this repetitive
cycle is that the two permanent classes are stable to the conditions used to remove

168

POLYPEPTIDE SYNTHESIS, SOLID-PHASE METHOD

Vol. 11

Fig. 2.

Commonly used protection scheme based on graduated acidolysis. Temporary N

α

-

amino protection is provided by the tert–butyloxycarbonyl (Boc) group, removed at each
step by the moderately strong trifluoroacetic acid (TFA). Permanent benyzl- and cyclohexyl-
based side-chain–protecting groups and the phenylacetamidomethyl (PAM) ester handle
linkage are cleaved simultaneously at the end of the synthesis by HF to yield the free
peptide acid.

the temporary N

α

-protection. If all classes are removed by the same chemical

mechanism, for example acidolysis, differential stability between classes can only
be achieved by designing protecting groups that are cleaved at different kinetic
rates. This often necessitates very harsh conditions for final removal of the two
classes of permanent protection. The alternative is to design protecting groups for
each class that are cleaved by different or orthogonal mechanisms (7,21). Ideally, a
fully orthogonal protection scheme would allow each class of protecting group to be
cleaved in any order without removing the other two classes (22). Orthogonal pro-
tection schemes allow milder overall conditions, because deprotection strategies
are based upon mechanism rather than kinetics.

Many combinations of temporary and permanent protecting groups have

been developed over the years. Only two have survived the test of time and are
routinely used today. The first is an improved version of the original Merrifield
system. This system is a non-orthogonal scheme based on graduated acid labil-
ity to differentiate temporary from permanent protecting groups (Fig. 2). The
N

α

-tert-butyloxycarbonyl (Boc) group (23–25) is used for temporary protection.

The Boc group is stable to alkali and nucleophiles but is rapidly removed by
organic and inorganic acids (7). The Boc group is usually removed by trifluoroacetic
acid (TFA) (20–50%) in dichloromethane (DCM) or HCl (4N) in dioxane. Perma-
nent side-chain–protecting groups are ether, ester, and urethane derivatives based
on benzyl alcohol “fine-tuned” with electron-donating methoxy or methyl groups
or electron-withdrawing halogens to achieve the proper level of stability to acid.
Alternatively, ether and ester derivatives based on cyclopentyl or cyclohexyl al-
cohol are sometimes used to mitigate certain side reactions. These permanent
side-chain–protecting groups and the peptide-handle linkage are cleaved by the
strong Lewis acid anhydrous HF. In neat anhydrous HF, cleaved protecting groups
exist as carbocations that may alkylate sensitive side chains of residues such as
tryptophan. Thus, acid cleavages are always carried out in the presence of carbo-
cation scavengers such as anisole. Scavengers such as dimethyl sulfide, p-cresol,

Vol. 11

POLYPEPTIDE SYNTHESIS, SOLID-PHASE METHOD

169

Fig. 3.

A mild two-dimensional orthogonal protection scheme for solid-phase synthesis.

Temporary N

α

-amino protection is provided by the 9-fluorenylmethyloxycarbonyl (Fmoc)

group, removed by a base-catalyzed elimination mechanism, typically with piperidine. Per-
manent tert-butyl-based side-chain-protecting groups and the p-alkoxybenzyl (PAB) ester
handle linkage are both cleaved by treatment with TFA to yield the free peptide acid at the
end of the synthesis. Details in text.

and p-thiocresol have been recommended in conjunction with a two-stage, low–
high HF cleavage method that is said to provide extra control and higher purities
(see section 3.4, Cleavage).

The Merrifield system is still popular today and up until the mid-1980s

was the most commonly used synthetic strategy. In the last 15 years a two-
dimensional orthogonal strategy based on the N

α

-9-fluorenylmethyloxycarbonyl

(Fmoc) group (26) has emerged as the method of choice for the synthesis of most re-
search peptides (Fig. 3). The temporary Fmoc group is removed by a

β-elimination

mechanism with piperidine (20–50%) in N,N-dimethylformamide (DMF) or N-
methylpyrollidinone (NMP) (27–30). Permanent protection is now provided by
ether, ester, and urethane derivatives based on t-butyl alcohol, triphenylmethanol,
or 9-hydroxyxanthene, which are cleaved by TFA at the same time the peptide is
released from the resin. Whereas the anhydrous HF used in the final deprotec-
tion step in Boc chemistry requires a special apparatus made of all fluorocarbon
(Kel F) lines and trained operators to safely cleave and isolate the peptide, the
milder conditions for final cleavage in Fmoc strategies, TFA (90%), have allowed
a wide variety of scientists of diverse training to now synthesize peptides by the
solid-phase method. Once again, scavengers are necessary to remove reactive car-
bocations during final TFA cleavage. The scavenger cocktails are covered in the
section on cleavage.

A three-dimensional orthogonal protecting scheme for the synthesis of lac-

tams is illustrated in Figure 4. The side chains of aspartic acid and lysine are
protected with ester and urethane derivatives, respectively, of allyl alcohol. The
allyl-based protecting groups may be cleaved under neutral conditions medi-
ated by Pd(0) while the fully protected peptide is still attached to the resin.
The lactam ring may then be formed taking advantage of the psuedo-dilution
effect of the peptide–resin. Fmoc removal followed by final TFA cleavage from the
5-(4-aminomethyl-3,5-dimethoxyphenoxy)valeric acid (PAL) handle results in the
side chain-to-side chain cyclic peptide with a C-terminal amide.

Side-chain–protecting groups keep reactive side chains from coupling with

the next incoming activated amino acid and thus forming a branch point in the

170

POLYPEPTIDE SYNTHESIS, SOLID-PHASE METHOD

Vol. 11

Fig. 4.

A mild three-dimensional orthogonal protection scheme for side chain-to-side

chain cyclization strategies. The allyl alcohol based side-chain protection of Asp and Lys
are removed with Pd(0) without affecting the temporary Fmoc protection or the permanent
peptide–PAL handle linkage. The lactam bond between Asp and Lys can now be formed
on resin, followed by sequential treatment with piperidine to remove the Fmoc group and
TFA to cleave the peptide from the PAL handle to yield the side chain-to-side chain cyclic
C-terminal amide peptide.

peptide, they protect sensitive residues from degradation during synthesis, and
they can greatly increase the solubility of certain residues in the common solvents
used in peptide synthesis. Because the occurrence of many of the degradation side
reactions are sequence-dependent, for the synthesis of research peptides in which
process development is often not an option, a maximal protecting group strategy
is recommended in which all trifunctional amino acids are protected. The recom-
mended protecting groups for each trifunctional amino acid that are commercially
available and compatible with either Boc or Fmoc chemistries are listed in Tables
2 and 3, respectively. These permanent side-chain–protecting groups are cleaved
at the same time as the peptide–resin linkage is cleaved.

For the synthesis of cyclic peptides, either lactams (58,59) (Fig. 4) or multiple

disulfide-containing (60) peptides, and for the synthesis of protected fragments,
permanent protecting groups must be selected whose cleavage mechanism is or-
thogonal to that used to remove the N

α

-protecting group and to that used to cleave

the peptide–resin linker. If on-resin cyclization is to be attempted, the orthogonal
protecting group does not have to be stable to the final cleavage/deprotection condi-
tions. All that is necessary is that the orthogonal side chain group can be removed
without affecting the N

α

-protection, the other permanent side-chain–protected

amino acids, and the peptide-linker bond. Table 4 summarizes the commercially
available amino acid derivatives for both Boc and Fmoc chemistries with side-
chain protection that is cleavable by mechanisms orthogonal to either acidolysis
and/or piperidine treatment.

The standard methodology of SPPS involves attachment of the first amino

acid to the resin via the C

α

-carboxyl. Synthesis then proceeds in the C

→ N direc-

tion. The majority of anchoring linkages have been designed to provide C-terminal
ester or amide linkages to provide upon cleavage, respectively, peptide acids or
amides (Table 5). The original linker used by Merrifield was a benzyl ester formed
by the reaction between the first protected amino acid and chloromethyl groups on

Table 2. Recommended Commercially Available Side-chain–Protected Amino Acids Compatible with Boc Chemistry
and Removed by HF During Final Deprotection/Cleavage

a

Amino acid and
protecting group

Abbreviation

Stability

Reference

Structure

Arginine

Mesitylene-2-sulfonyl

Arg(Mts)

TFA
Piperidine
Pd(0)
Hydrazine

31–33

4-Toluenesulfonyl

Arg(Tos)

TFA
Piperidine
Pd(0)
Hydrazine

31

Aspartic/glutamic

Cyclohexyl

Asp/Glu(OcHex)

TFA
Piperidine
Pd(0)
Hydrazine

31,32,34–36

Asparagine/glutamine

9-Xanthenyl

Asn/Gln(Xan)

b

Piperidine
Pd(0)
Hydrazine

8,31,37,38

171

Table 2. (Continued)

Amino acid and
protecting group

Abbreviation

Stability

Reference

Structure

Cysteine

4-Methylbenzyl

Cys(Meb)

TFA
Piperidine

7,31,39,46

Histidine

Benzyloxymethyl

His(Bom)

TFA
Piperidine
Pd(0)
Hydrazine

31,36,41

Lysine

2-Chlorobenzyloxycarbonyl

Lys(2-ClZ)

TFA
Piperidine
Pd(0)
Hydrazine

2,31,36,39

Methionine

Sulfoxide

Met(O)

TFA
Piperidine
Pd(0)
Hydrazine

2,31,40,42–
44

172

Serine/threonine

Benzyl

Ser/Thr(Bzl)

TFA
Piperidine
Pd(0)
Hydrazine

32,36

Tryptophan

Formyl

Trp(CHO)

TFA
Piperidine
Pd(0)
Hydrazine

2,31,32

Cyclohexyloxycarbonyl

Trp(Hoc)

TFA
Piperidine
Pd(0)
Hydrazine

45

Tyrosine

2,6-Dichlorobenzyl

Tyr(Cl

2

-Bzl)

TFA
Piperidine
Pd(0)
Hydrazine

31,32,36,39

a

The boldfaced atoms and structures to the right are the functional group being protected.

b

The Xan group is removed by TFA during subsequent deprotection steps. Xan prevents dehydration of Asn/Gln during coupling and

greatly increases the solubility of these residues.

173

Table 3. Recommended Commercially Available Side-chain–Protected Amino Acids Compatible with Fmoc Chemistry and
Removed by TFA During Final Deprotection/Cleavage

a

Amino acid and protecting group

Abbreviation

Stability

Reference

Structure

Arginine

2,2,4,6,7-Pentamethyldihydrobenzofuran-

5-sulfonyl

Arg(Pbf)

Piperidine
Pd(0)
Hydrazine

46,47

Aspartic/glutamic

tert-Butyl

Asp/Glu(O-t-Bu)

Piperidine
Pd(0)
Hydrazine

2,48

Asparagine/glutamine

Triphenylmethyl

Asn/Gln(Trt)

Piperidine
Pd(0)
Hydrazine

49

174

Cysteine

Triphenylmethyl

Cys(Trt)

Piperidine
Pd(0)
Hydrazine

2,50–52

9-Xanthenyl

Cys(Xan)

b

Piperidine
Pd(0)
Hydrazine

53

Histidine

Triphenylmethyl

His(Trt)

Piperidine
Pd(0)
Hydrazine

11

175

Table 3. (Continued)

Amino acid and protecting group

Abbreviation

Stability

Reference

Structure

Lysine

tert-Butyloxycarbonyl

Lys(Boc)

Piperidine
Pd(0)
Hydrazine

2,51

Serine/threonine

tert-Butyl

Ser/Thr(t-Bu)

Piperidine
Pd(0)
Hydrazine

51,54,55

Tryptophan

tert-Butyloxycarbonyl

Trp(Boc)

Piperidine
Pd(0)
Hydrazine

47,56,57

Tyrosine

tert-Butyl

Tyr(t-Bu)

Piperidine
Pd(0)
Hydrazine

51,54,55

a

The boldfaced atoms and structures to the right are the functional group being protected.

b

The Xan group can be removed from Cys with 1% TFA without affecting the peptide–resin linkage (53).

176

Table 4. Commercially Available Amino Acids with Side-chain Protection That Is Cleavable by Mechanisms Orthogonal to Either
Acidolysis and/or Piperidine Treatment

a

Amino acid and
protecting group

Abbreviation

Stability

Cleavage

Reference

Structure

Aspartic/glutamic

Allyl

Asp/Glu(OAl)

TFA
HF
Piperidine
Hydrazine

Pd(0)

52,62,63

4-

{N-[1-(4,4-Dimethyl-2,6-

dioxocyclohexylidene)-3-
methylbutyl]-amino

}-

benzyl

Asp/Glu(ODmab)

TFA
Piperidine
Pd(0)

Hydrazine

64

Fluorenylmethyl

Asp/Glu(OFm)

TFA
HF
Pd(0)
Hydrazine

Piperidine

65

Cysteine

Acetamidomethyl

Cys(Acm)

HF
TFA
Piperidine
Hydrazine

I

2

Hg(II)
Tl(Tfa)

3

2,60,66,67

177

Table 4. (Continued)

Amino acid and
protecting group

Abbreviation

Stability

Cleavage

Reference

Structure

tert-Butysulfenyl

Cys(S-t-Bu)

TFA
Piperidine
Pd(0)
Hydrazine

Thiols
Phosphines

68,69

Lysine

Allyloxycarbonyl

Lys(Aloc)

HF
TFA
Piperidine
Hydrazine

Pd(0)

63

4,4-Dimethyl-2,6-

dioxocyclohex-1-ylidene–
ethyl

Lys(Dde)

TFA
Piperidine
Pd(0)

Hydrazine

70–73

a

The boldfaced atoms and structures to the right are the functional group being protected. For a more comprehensive review of orthogonal protecting groups

see Reference 61.

178

Table 5. Resin Linkers and Handles

a

Cleavage

Resulting

Linker/handle

conditions

C-terminus

Reference

Structure

4-Chloromethyl resin

HF

Acid

8,74

4-Hydroxymethylphenylacetic acid (PAM)

HF

Acid

75,76

9-(Hydroxymethyl)-2-fluoreneacetic acid (HMFA) Piperidine

Acid

67,77,78

4-Nitrobenzophenone oxime resin

HOPip

AA

− +

N(n-

Bu)

4

AA-NH

2

Acid

b

Amide

80–82

α-Bromophenacyl

h

ν (350 nm)

Acid

83

179

Table 5. (Continued)

Cleavage

Resulting

Linker/handle

conditions

C-terminus

Reference

Structure

3-(4-Hydroxymethylphenoxy)propionic acid

(PAB) (Wang)

TFA

Acid

84–86

4-(2



,4



-Dimethoxyphenylhydroxymethyl)-

phenoxymethyl
resin

Dilute TFA

Acid

87

2-Chlorotrityl chloride resin

TFA, HOAc

Acid

88,89

5-(4-Hydroxymethyl-3,5-

dimethoxyphenoxy)valeric acid
(HAL)

Dilute TFA

Acid

90

Hydroxy–crotonyl–aminomethyl resin

(HYCRAM)

Pd(0)

Acid

91–93

180

3-Nitro-4-hydroxymethylbenzoic acid (ONb)

h

ν (350 nm)

Acid

22,94–96

4-(2



-Aminoethyl)-2-methoxy-5-nitro–

phenoxypropionic
acid

h

ν (350 nm)

Amide

97

4-Methylbenzhydrylamine resin (MBHA)

HF

Amide

98,99

4-(2



,4



-

Dimethoxyphenylaminomethyl)phenoxymethyl
resin (Rink)

Dilute TFA

Amide

87

5-(4-Aminomethyl-3,5-dimethoxyphenoxy)valeric

acid (PAL)

TFA

Amide

100,101

181

Table 5. (Continued)

Cleavage

Resulting

Linker/handle

conditions

C-terminus

Reference

Structure

5-(9-Aminoxanthen-3-oxy)valeric acid (XAL)

Dilute TFA

Amide

13,102,
103

3-Nitro-4-aminomethylbenzoic acid (Nonb)

h

ν (350 nm)

Amide

104

4-(4-Fmoc–aminomethyl-3,5-diethoxy–

phenoxy)butyric acid
(BAL)

TFA

Amide

105

4-(

α-Mercaptobenzyl)phenylacetic acid

HF

Thioester

106–108

a

Structural diagrams are oriented so that the resin or point of attachment to support is on the far right, and the site for anchoring the C-terminal amino acid

residue is on the far left. Benzyl ester linkages may also be cleaved by a range of nucleophiles to create acids, esters, or other derivatives. Consult References
7,9, and 20 for further examples.

b

Cleavage by aminolysis results in 0.6–2% racemization (79).

182

Vol. 11

POLYPEPTIDE SYNTHESIS, SOLID-PHASE METHOD

183

polystyrene resin (Table 5). The simple benzyl ester linkage was found to be slowly
cleaved by TFA and has now been replaced by the phenylacetamidomethyl (PAM)
ester handle (Fig. 2) which is stable to TFA but readily cleaved by HF. The preferred
loading technique for ester linkages is to attach the first amino-acid to the handle
in solution forming a “preformed handle.” The preformed handle may be purified in
solution before coupling to an amino-functionalized resin. The preformed handle
strategy gives one greater control over the loading in that the formation of the
amide bond between the handle and the resin can easily be driven to completion
(75,76). For synthesizing peptide acids by Fmoc chemistries, p-alkoxybenzyl esters
(PAB), “Wang resins” (Fig. 3, Table 5), are the preferred handles. Both PAM and
Wang resins preloaded with the first amino acid are now commercially available
at low cost and high purity from a variety of vendors (see Appendix). Finally, for a
detailed discussion of side reactions that can occur when forming the ester bond
between the first amino acid and either a handle- or a resin-bound hydroxyl group,
see Reference. 20 (pp. 129–132).

The recommended linker for synthesizing C-terminal amides compatible

with Boc chemistries is the MBHA linker (Table 5). For Fmoc chemistries either
the PAL (Fig. 4) or the Rink handles are recommended (Table 5).

Many other handles exist that are cleavable by orthogonal mechanisms

for the synthesis of protected peptide fragments; these are also summarized in
Table 5. A novel strategy for the synthesis of cyclic peptides on resin is to attach
the peptide to the resin by a backbone nitrogen rather that the C

α

-carboxyl. The N-

and C-termini then remain free for further functionalization such as cyclization,
esterification, or thioester formation. This backbone amide linker (BAL) is listed
in Table 5. Other strategies such as attaching the peptide to the resin via the side
chain of trifunctional amino acids and more comprehensive listings of linkers and
handles are covered in References 9,12, and 109–111.

Procedures

Deprotection/Coupling Cycle.

Tables 6 and 7 show representative de-

protection/coupling cycles for Boc- and Fmoc-based syntheses, respectively. When

Table 6. General Boc Chemistry SPPS

a

Step

Description

Solvents/chemicals

Time

b

, min

1.

Deprotection

20–50% TFA in DCM or NMP

1

+ 30

2.

Wash

DCM or NMP

4

× 2

3.

Neutralization

5% DIEA in DMF or NMP

2

× 2

4.

Wash

DMF or NMP

4

× 2

5.

Activation and coupling

c

,d,e

Selection from Table 8

6.

Wash

DMF or NMP

4

× 2

a

Boc–Asn and Boc–Gln are side-chain–protected.

b

Times are for batchwise synthesis. If a flow apparatus is used, the time is the combined

times of a step, eg, 2

× 2 min = 4 min of flow.

c

When double coupling, steps 1 through 4 are omitted between couplings.

d

When capping, the coupling reagents of Table 8 are replaced with acetic anhy-

dride/DIEA (10 equiv each), and steps 1 through 4 are omitted as when double coupling.

e

Steps 3 and 5, neutralization and coupling, may be combined into one step (112–116).

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POLYPEPTIDE SYNTHESIS, SOLID-PHASE METHOD

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Table 7. General Fmoc Chemistry SPPS

a

Step

Description

Solvents/chemicals

Time

b

, min

1.

Wash

DMF or NMP

3

× 1

2.

Deprotection

Piperidine/DMF or NMP (1:4)

1

+ 20

3.

Wash

DMF or NMP

3

× 1

4.

Activation and coupling

c

,d

Selection from Table 8

5.

Wash

DMF or NMP

3

× 1

a

Fmoc–Asn and Fmoc–Gln are side-chain–protected.

b

Times are for batchwise synthesis. If a flow apparatus is used, the time is the combined

times of a step, eg, 3

× 1 min = 3 min of flow.

c

When double coupling, steps 1 through 3 are omitted between couplings.

d

When capping, the coupling reagents of Table 8 are replaced with acetic anhy-

dride/DIEA (10 equiv each), and steps 1 through 3 are omitted as when double coupling.

double coupling or capping, the deprotection step is skipped and the protocol is
repeated, starting at the wash step before the activation and coupling steps.

The recommended coupling reagents and their respective protocols are listed

in Table 8. The structures of the coupling reagents are shown in Figure 5. Each
lab has a preference for coupling reagent, reaction time of each step, and solvents
(DCM, DMF, or NMP) used. Each coupling reagent has certain limitations. N,N



-

Diisopropylcarbodiimide (DIPCDI) activation in DMF or NMP is a slow process
that takes about 1 h to reach completion. In DCM the reaction is instantaneous
(117). Thus, preactivation in a separate vessel while deprotection is occurring
is often done with carbodiimide activation in DMF or NMP. Activated arginine
derivatives undergo a slow side reaction, where the guanido group attacks the ac-
tivated C

α

-carboxyl to form a six-membered ring. The amino acid is then no longer

able to couple. This side reaction does not affect the purity of the peptide, it only
nonproductively consumes the activated arginine residue. Thus, long preactiva-
tion of arginine by carbodiimides is not recommended to avoid the loss of the acti-
vated amino acid before it can couple to the free amino group of the peptide–resin.
The reagents benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexaflu-
orophosphate

(BOP),

2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium

hexafluorophosphate

(HBTU),

and

O-(7-azabenzotriazol-1-yl)-1,1,3,3-

tetramethyluronium hexafluorophosphate (HATU) must contain excess N-
methylmorpholine (NMM) or N,N-diisopropylethylamine (DIEA) for efficient
coupling (118–124). The excess tertiary amines present in these coupling cocktails
(Table 8, entries 2–4) will racemize cysteine (125). Either the DIPCDI or the
pentafluorophenyl active ester protocols are recommended for coupling this amino
acid. The BOP reagent releases the carcinogen hexamethylphosphoramide, so
the waste stream from this reagent must be handled with care. Excess unreacted
HBTU and HATU can cap unreacted amino groups (126,127); preactivation
should always be done in a separate vessel and the amino acid derivative should
always be present in slight excess to these two coupling reagents. Because of this
side reaction, HBTU and HATU are not recommended as reagents for on-resin
cyclizations. Pentafluorophenyl esters (OPfp) couple slowly as compared to the
other methods, addition of HOBt can increase the reaction rate (128,129).

The coupling times listed in Table 8 are only approximate, and for manual

syntheses the actual length of the coupling step is determined by monitoring for
unreacted amino groups by the sensitive ninhydrin test.

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POLYPEPTIDE SYNTHESIS, SOLID-PHASE METHOD

185

Fig. 5.

Structures of coupling reagents of Table 8.

Many other side reactions that can occur during coupling have been doc-

umented and are listed in the more comprehensive reviews (5–8, 10–20). How-
ever, if one uses the protected amino acids listed in Tables 2–4 and the deprotec-
tion/coupling protocols of Tables 6–8, these other side reactions generally are not
a problem during chain elongation.

Monitoring.

A key assumption in solid-phase procedures is that coupling

reactions can be made to go to completion (Table 1). A number of qualitative and
quantitative methods of monitoring these steps exist, including some with a possi-
bility of “real-time” feedback based on the kinetics of appearance or disappearance
of soluble chromophores measured in a flow-through system. Most accurate and
meaningful are qualitative and quantitative tests for the presence of unreacted
amines after a coupling step. Such tests should ideally be negative before proceed-
ing to the next deprotection/coupling cycle.

The most widely used qualitative test for monitoring unreacted amines is the

ninhydrin test (130). For monitoring imines (secondary amines) such as proline,

186

POLYPEPTIDE SYNTHESIS, SOLID-PHASE METHOD

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Table 8. Coupling Protocols for SPPS

Entry

Solvents/chemicals

Time

a

, min

1.

Protected amino acid (4 equiv) in DMF or NMP

5

DIPCDI (4 equiv)/HOBt (4 equiv) in DMF

b

,c

60

2.

Protected-amino acid (4 equiv) in DMF or NMP

5

BOP or PyBOP (3 equiv)/NMM (4.5 equiv)/HOBt (3 equiv)

20

in DMF or NMP

b

3.

Protected amino acid (4 equiv) in DMF or NMP

5

HBTU (3.6 equiv)/DIEA (7.2 equiv)/HOBt (4 equiv) in DMF

d

20

4.

Protected amino acid (4 equiv) in DMF or NMP

5

HATU (3.6 equiv)/DIEA (7.2 equiv)/HOAt (4 equiv) in DMF

d

20

e

5.

Protected amino acid pentafluorophenyl ester (4 equiv)

60

in DMF or NMP

a

The first line in the second column is the preactivation time, and the second line is the

coupling time. Entry 5 is the coupling time only.

b

The reagents can be added directly to the reaction vessel without preactivation, eg, during

on-resin cyclizations to form a lactam.

c

Earlier

references

on

the

carbodiimide

method

of

activation

use

N,N



-

dicyclohexylcarbodiimide (DCC) as the coupling reagent. DCC can cause severe
dermatitis when handled, and the urea by-product is prone to precipitate in the lines of
automated synthesizers. DCC is no longer recommended for use in solid-phase synthesis.

d

Preactivation in a separate vessel is required to avoid capping the N

α

-amino group.

e

Coupling times as short as 1 min have been recommended with the reagents of entry 4

(115,116).

the isatin test is used (131). These tests, which are carried out on an aliquot of
peptide–resin, are easy, reliable, and require only a few minutes to run, making it
possible to make a quick decision on how to proceed. A highly accurate quantitative
version of the ninhydrin test has been described (132). The ninhydrin test is not
amenable to automation, however. Other monitoring techniques exist that are
nondestructive and may be carried out on the whole batch of resin. These tests
are time consuming and not widely used.

As an alternative to monitoring unreacted N

α

-amino groups, soluble re-

actants and coproducts can be analyzed. Continuous measurement of electrical
conductivity can be used to evaluate Fmoc coupling and Fmoc deprotection effi-
ciencies (133–135). The progress of Fmoc chemistry can be evaluated by observing
the decrease of absorbance at 300 nm when Fmoc amino acids are taken up dur-
ing coupling, and by the increase in absorbance at 300 nm when the Fmoc group
is released with piperidine (136). Software on modern peptide synthesizers can
evaluate the kinetics of these deprotection/coupling steps and lengthen reaction
times if necessary.

Continuous flow monitoring is inherently insensitive for direct judgment of

reaction end points. In a typical coupling procedure with a fourfold excess of amino
acid, absorbance drops from 4.00 to 3.05 or 3.01 units with 95 or 99% coupling,
respectively. It is difficult to distinguish the approximate 1% difference between
3.05 and 3.01 accurately. In contrast, the 400% difference between 0.05 and 0.01
is easily measured when monitoring unreacted N

α

-amino groups.

An essential, although time-consuming, way to monitor the course of solid-

phase synthesis is total hydrolysis of aliquots of the peptide–resin removed after

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POLYPEPTIDE SYNTHESIS, SOLID-PHASE METHOD

187

Table 9. Preview in the Sequential Degradation of a
Hypothetical Peptide Made by Solid-Phase Synthesis

a

Edman cycle

%

1

2

3

4

Correct

A-B-C-D-E-F-resin 90

A

B

C

D

Des(A)

B-C-D-E-F-resin

0.4

B

C

D

E

Des(B)

A-C-D-E-F-resin

0.4

A

C

D

E

Des(C)

A-B-D-E-F-resin

0.2

A

B

D

E

Des(D)

A-B-C-E-F-resin

9.0

A

B

C

E

Preview

b

, %

0.4 0.8 1.0 10

a

Refs. 138–140.

b

With respect to expected residue.

certain synthetic steps. The technique of internal reference amino acids (IRAA) is
often used to measure yields and chain retention accurately (11,101,137).

Invasive monitoring of both synthetic efficiency and amino acid composi-

tion of peptide–resins can be achieved by a powerful quantitative variation of the
Edman sequential degradation, called “preview analysis” (138–140). To illustrate
preview analysis (Table 9), assume that the desired sequence A-B-C-D-E has been
assembled on 90% of the peptide–resins, that deletions of A, B, and C have occurred
at 0.4, 0.4, and 0.2%, respectively, and that due to a significant error at the step to
incorporate D, 9% of the chains lack this latter residue. After one cycle of Edman
degradation, the residue B is quantitatively determined by sensitive analytical
techniques at 0.4% of the expected A, even though B should only be viewed in the
second cycle. Thus the 0.4% preview of B reflects that amount of deletion of A.
The second cycle of Edman degradation in Table 9 shows the amplification effect
inherent in the method, because now 0.8% preview of C is noted. It is difficult to
ascribe a low degree of deletion to specific positions in the chain, but an average
deletion can be assumed because the precision increases with each Edman step.
In fact, the average level of deletions in a typical synthesis is

<0.3% per step.

The fourth degradation of the example (Table 9) illustrates the dramatic jump in
preview reflecting a failure in the corresponding stage of the synthesis. Because
this level is large, the specific location of the deletion can be identified.

Recently, on-resin mass spectrometry (MS) via the MALDI (matrix-assisted

laser desorption/ionization) technique has been developed. This method can be
used as a semi-on-line monitoring technique during SPPS (141). An especially
elegant approach for on-resin MS is to use the photolabile

α-methylphenacyl es-

ter linker (Table 5,

α-bromophenacyl entry), which is cleaved directly upon laser

photolysis and MALDI ionization (142,143).

Resins.

The solid-phase support (Resin, Fig. 1) is a well-solvated poly-

meric matrix in which the peptide chains are attached throughout a resin bead.
The polymeric backbone is a flexible superstructure through which reagents
and solvents can easily flow and once swollen has the physical properties of
a gel (144,145). This is in contrast to the common perception that solid-phase
chemistry only occurs on the surface of each polymer bead. Because of the perme-
able nature of the polymers, reaction rates approach but are slightly slower than
that found in solution. Commercially available polymers have functional group
loadings typically in the 0.2–1.0 mmol/g range. As an example of the distribution

188

POLYPEPTIDE SYNTHESIS, SOLID-PHASE METHOD

Vol. 11

of peptide chains within a polymer, polystyrene contains approximately 10 mmol/g
phenyl groups. Thus, a polystyrene resin with an aminomethyl substitution of 0.5
mmol/g contains a functional group on about every 20th phenyl ring. By the end
of the synthesis a resin may easily double in mass and volume but will remain a
well-solvated gel in the common solvents used in SPPS. This increase in size does
not appreciably affect reaction rates, and couplings generally continue to proceed
to completion (144).

Polymeric supports must be compatible with either of the two modes of SPPS.

The first is the batchwise method, where the reactions are agitated by mechan-
ical means such as shaking, vortexing, or bubbling with an inert gas such as
nitrogen. Reagents are removed by filtration either with suction or with nitrogen
pressure. The second mode is the continuous-flow method. In this mode reagents
and solvents are pumped through columns packed with resins. Resins for use in
continuous-flow SPPS must have sufficient mechanical stability to be stable to the
back pressures that develop during synthesis.

Two polymeric supports have classically been used in batchwise SPPS. The

first is polystyrene polymer cross–linked with 1% of 1,3-divinyl benzene. A typical
dry bead has a diameter of 50

µm that swells five- to sixfold in DCM or DMF (144).

The second was developed on the basis of the idea that the polymeric backbone
should be of similar chemical composition, a polyamide, as the peptide backbone
(11). Thus, copolymerized dimethylacrylamide, N,N’-bisacryloylethylenediamine,
and acryloylsarcosine methyl ester, or polyamide resin, was developed (146).
Polyamide supports are commercially known as Pepsyn resin. Many other sup-
ports have been developed that are compatible with batchwise SPPS and are
summarized in the more comprehensive reviews (7,10–20,109).

The original supports designed to withstand the high flow rates and re-

sulting back pressures of continuous-flow SPPS are low density, highly perme-
able inorganic matrices that have polyamide embedded within. These matrices
include polyamide–kieselguhr (Pepsyn K) (147) and polyamide–Polyhipe (148).
These polyamide-embedded resins have now been largely replaced by polyethy-
lene glycol–polystyrene graft supports that have superior swelling properties in
a range of solvents and have excellent physical and mechanical properties for
both batchwise and continuous-flow SPPS (13,149–154). These graft polymers are
commercially available as PEG-PS, TentaGel (Rapp Polymere GmbH, T ¨

ubingen,

Germany), and ArgoGel (Argonant, Foster City, Calif.)

Other resins compatible with both batchwise and continuous-flow SPPS are

poly(N-[2-(4-hydroxyphenyl)ethyl]acrylamide) (core Q) (155), polyethylene glycol
dimethylacrylamide (PEGA) resins (156,157), and cross–linked ethoxylate acry-
late resin (CLEAR). CLEAR has excellent swelling properties in a wide range of
polar and nonpolar solvents ranging from water to DCM (158).

Cleavage.

Upon completion of chain assembly the peptide must be cleaved

from the resin. In standard Boc and Fmoc chemistries cleavages simultaneously
remove side-chain–protecting groups as the peptide is liberated from the resin
by acidolytic conditions. A variety of unwanted side reactions can occur during
cleavage, so careful attention to detail is necessary to successfully complete this
step in a synthesis. Detailed discussions of the fine points of cleavage are covered
in the reviews (5–8,10–20).

Fmoc chemistries utilize the moderate-strength acid TFA to cleave the PAB

ester and either Rink or PAL amide linkages. TFA simultaneously cleaves the

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POLYPEPTIDE SYNTHESIS, SOLID-PHASE METHOD

189

Table 10. Recommended Cocktails for Final Cleavage of the Peptide–Resin
Linker and the Permanent Side-Chain–Protecting Groups Used in Fmoc-Based
Synthesis

a

Cocktail

Ratio of ingredients, v/v

Reference

Reagent K TFA/phenol/thioanisole/ethanedithiol/water, 82.5:5:5:2.5:5

165,166

Reagent R TFA/thioanisole/ethanedithiol/anisole, 90:5:3:2

101

Reagent B TFA/phenol/water/triisopropylsilane, 88:5:5:2

166,167

a

The peptide–resin is swollen in the cocktail, approximately 5 mL/g resin, for 1–2 h. The

cleavage solution is then added to 10 volumes of ice-cold diethyl ether to precipitate the
peptide. The crude peptide is then isolated by centrifugation to form a pellet that is washed
with cold ether. After washing, the pellet may be dissolved, and the peptide purified by
preparative reversed–phase HPLC.

side-chain–protecting groups listed in Table 3. In TFA the cleaved tert-butyl
groups exist either as cations or tert-butyl trifluoroacetate (159–162). The trityl,
9-xanthenyl, and Pbf groups also exist as cations. Alkylation of Trp, Met, and to
a lesser degree Cys and Tyr by these reactive species is prevented by including
scavengers in the reaction mixture (47,163,164). Table 10 contains three cocktails
that effectively scavenge reactive intermediates and also help prevent oxidation
of Met to Met(O) and oxidation of Trp by ozone generated in the reaction mixture
from dissolved oxygen (165,168). Once again the choice of cocktail is a preference
of each lab.

Final cleavage in Boc-based syntheses is usually achieved with anhydrous

HF. HF cleaves both the PAM and MBHA handles along with the side-chain–
protecting groups listed in Table 2 (31). HF cleavage procedures require an all-
fluorocarbon apparatus, since this strong acid will dissolve glassware. Just as in
the Fmoc TFA procedure, HF cleavages must contain carbonium ion scavengers.
The standard cleavage conditions are HF-anisole (9:1) at 0

◦

C for 1 h (8). To depro-

tect Trp(CHO) and Met(O), a two-stage “low–high HF” procedure is used in which
low HF concentrations of 20–25% in the presence of 4-thiocresol and dimethyl
sulfide first deprotect these two residues and others as well. Final cleavage from
the resin is then achieved by increasing the HF concentration to near that found
in the standard procedure. This low–high procedure is said to give better control
and better product purities than the standard procedure (31).

Purification.

Reversed phase HPLC is the standard method for purifi-

cation of synthetic peptides. This technique can generally remove the accumu-
lated low level systematic by-products present in the crude peptide isolated after
cleavage. The homogeneity of purified peptides should be checked by at least two
orthogonal techniques, either, analytical reversed-phase or ion-exchange HPLC,
or capillary zone electrophoresis (169). Mass spectrometry must also be used to
confirm that the molecular ion of the isolated product has the correct mass. Amino
acid analysis is used to confirm the correct amino acid composition. Sequencing
information either by automated Edman degradation or by mass spectrometry
techniques may also be done, although sequence analysis is not routinely required.

Side Reactions.

Solvents and Reagents.

To assure that each deprotection/coupling cycle

is completed with the necessary high fidelities illustrated in Table 1, all solvents
and reagents must be of the highest grade available. In Fmoc-based syntheses,

190

POLYPEPTIDE SYNTHESIS, SOLID-PHASE METHOD

Vol. 11

Fig. 6.

Formation of a diketopiperazine from a D-valyl-L-proline sequence anchored as

an o-nitrobenzyl ester. The example shown is designed to accentuate the potential problem,
in that susceptible amino acid residues and a very good leaving group toward intramolec-
ular aminolysis were both chosen. In this example, the level of diketopiperazine formation
depended significantly on the N

α

-amino-protecting group used, with

>75% side reaction

occurring in 2 min under Fmoc removal conditions and

∼40% shortly after neutralizataion

following Boc removal (22). See References 7,170, and 171 for additional information on
mechanistic details regarding diketopiperazine formation, and References 105,112, and
171–173 for coupling protocols that mitigate the problem.

low levels of contaminating secondary amines found either in the DMF (dimethy-
lamine) or in the DIEA used during an activation/coupling step can prematurely
deprotect the Fmoc group. The highest purity, “peptide synthesis grade,” of these
reagents should always be purchased; old reagents should not be used in synthe-
ses. Secondary amines can be detected with the ninhydrin test.

Diketopiperazine Formation during Attempted Incorporation of Third

Amino Acid.

The free N

α

-amino group of an anchored dipeptide is poised

for acid- or base-catalyzed intramolecular attack upon the C-terminal carbonyl
(Fig. 6). The formation and release of the six-membered-ring diketopiperazine
from the resin is kinetically favorable for residues that can form cis-peptide bonds,
for example, Gly, Pro, N-methylamino acids, and

D

-amino acids in either of the

dipeptide residues. A hydroxymethyl group remains on the resin. Diketopiper-
azine formation is also a problem when the third amino acid is added to a BAL-
anchored (Table 5) sequence (105) or to an activated leaving group such as the
O-nitrobenzyloxy handle (22). However, for most syntheses this problem can be
adequately handled.

Succinimide Formation and

α→β Rearrangement, Particularly at

Asn–Gly Sequences.

Asn- and to a lesser extent Asp-containing peptides can

cyclize to form a succinimide derivative (Fig. 7). The formation of the five-
membered succinimide ring is kinetically favorable. The ring can open to yield
either

α- or β-Asp peptides. The Asn–Gly sequence has the greatest tendency to

rearrange (174). This rearrangement can be a significant problem, especially if a
peptide is left to stand for too long in dilute acid solutions, such as the aqueous

Vol. 11

POLYPEPTIDE SYNTHESIS, SOLID-PHASE METHOD

191

Fig. 7.

Succinimide formation at the Asn–Gly sequence. The ultimate ratio of

α/β peptides

is determined by the thermodynamic stability of each sequence.

0.1% TFA commonly used for purification by preparative reversed-phase HPLC
(174–176), or even if stored with residual acid in the solid state (177).

Acid-Sensitive Side Chains and Bonds.

Trp is very sensitive to acidic con-

ditions and to a lesser extent so is Tyr. Both can easily react with carbocations and
activated molecular oxygen species. To avoid repeated exposure to acid, multiple-
Trp–containing peptides are best synthesized by Fmoc strategies. Use of one of
the cleavage cocktails listed in Table 10 during final cleavage is essential to avoid
degradation of Trp and Tyr residues. Finally, the Asp Pro bond is acid-labile, and
Fmoc chemistries are recommended for peptides that contain this sequence (178).

Problems with Methionine and Cysteine.

Sulfur-containing amino acids

are susceptible to oxidation and alkylation. The cocktails of Table 10 will pro-
tect these residues during cleavage. Met is very susceptible to oxidation to me-
thionine sulfoxide, Met(O), while standing in acidic solutions that contain dis-
solved oxygen. Low levels of this side reaction are very hard to avoid during
HPLC purification of peptides that contain this residue. C-terminal-esterified Cys
residues can be racemized by repeated piperidine deprotection treatments during
Fmoc SPPS (179,180). This side reaction is manageable if the protecting groups of
Table 3 are used. Alternatively, attachment of C-terminal Cys residues to the XAL
handle (Table 5) via the side chain followed by chain elongation has been shown
to prevent racemization (181). The use of either Trt or Xan side-chain protection
also diminishes the formation of 3-(1-piperidinyl)alanine by-products (Fig. 8) that
can be a serious side reaction with C-terminal Cys during Fmoc SPPS (181,182).

192

POLYPEPTIDE SYNTHESIS, SOLID-PHASE METHOD

Vol. 11

Fig. 8.

Base–catalyzed elimination of C-terminal Cys residues attached to the resin via an

ester linkage. 1,4-Addition of piperidine results in the formation of a 3-piperidinylalanine
derivative, which can undergo chain elongation in subsequent cycles.

Prognosis

Solid-phase synthesis has evolved to where even the nonspecialist can easily make
peptides of 50 amino acids or more in length. It is important to remember that
each synthetic procedure has limitations, and that even in the hands of highly
experienced workers, sequences will arise that defy facile preparation.

This article has touched on the key issues of chain assembly, cleavage, and

purification. More challenging issues such as modification by phosphorylation
(183–185), glycosylation, prenylation, and sulfation, are covered in References 20
and 186. The appendix contains strategies for finding synthesis apparatus, amino
acid derivatives, resins, handles, and chemical reagents on the Internet. Research
continues in other challenging areas such as disulfide bond formation, lactam cy-
clization, combinatorial methods, and improved reagents, handles, and resins.
Finally, there is the exciting area of chemical ligation, where peptide fragments
synthesized by the solid-phase method are chemoselectively condensed together
to form proteins, of both natural and designed sequences (187–190). With the in-
creasing number of peptide and protein targets identified by genomic sequencing,
improvements in synthesis, and especially purification and isolation are needed to
accelerate research in the field of proteomics (191–193). Thus, even now 45 years
after the introduction of SPPS by Merrifield, much challenging and exciting work
remains to be done.

Appendix

Solid-phase Peptide Synthesis and the Internet.

The Internet con-

tains many sites of interest for the peptide chemist. Since Internet addresses
tend to regularly change, we have chosen to list terms that one can use in a search

Vol. 11

POLYPEPTIDE SYNTHESIS, SOLID-PHASE METHOD

193

engine rather than addresses. One should search the term contained in the quo-
tation marks to find the specific site or topic of interest. Combining various parts
of the terms in quotations while searching can make a search more accurate.

Peptide Societies.

The first three societies have sites in English. The last

three are in the native language of the respective country.

(1) “American Peptide Society”
(2) “Australian Peptide Society”
(3) “European Peptide Society”—The European Peptide Society Web site con-

tains the best links to manufacturers of automated synthesizers and
reagents for SPPS at the time of publication of this review

(4) “Chinese Peptide Society”
(5) “Japanese Peptide Society”
(6) “Korean Peptide Society”

General SPPS Information.

(1) “ABRF Peptide Synthesis Research Group”—This Association of Biomolec-

ular Resource Facilities (ABRF) Web site contains summaries of difficult
peptide syntheses attempted by its members. This is a good resource to
see how various procedures actually work when used by practicing peptide
chemists.

(2) “NRC peptide synthesis database”—This database, run by the National Re-

search Council of Canada, contains a wealth of information about amino
acid derivatives.

Consumables and Automated/Manual Synthesizers.

Many manufac-

tures of protected amino acids, resins, coupling agents, and automated/manual
synthesizers exist.

(1) “Fmoc amino acids,” “Boc amino acids,” or “protected amino acids”—Trying

either of these phrases will lead to the majority of the commercial amino
acid vendors. Many of these vendors also sell resins and coupling reagents.

(2) “Solid-phase resins”—To find manufactures of resins for solid-phase syn-

thesis

(3) “Solid-phase coupling reagents” or “peptide coupling reagents”—To find

commercial sources for the coupling reagents of Figure 5

(4) “Solid-phase synthesis vessels,” “Solid-phase synthesis glassware,” “peptide

synthesis vessels,” or “combinatorial chemistry glassware”—To find reac-
tion vessels for manual SPPS

(5) “Peptide synthesizers” or “Solid-phase synthesizers”—To find automated

SPPS machines

(6) “HF apparatus”—This term will find the special all-fluoropolymer appara-

tus necessary for final cleavage with HF.

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POLYPEPTIDE SYNTHESIS, SOLID-PHASE METHOD

Vol. 11

BIBLIOGRAPHY

“Polypeptide Synthesis, Solid-Phase Method,” in EPST 1st ed., Suppl. Vol. 1, pp. 492–510,
by M. Manning, Medical College of Ohio at Toledo; “Polypeptide Synthesis, Solid-Phase
Method,” in EPSE 2nd ed., Vol. 12, pp. 811–858, by George Barany, Nancy Kneib-Cordonier,
Daniel G. Mullen, University of Minnesota.

1. F. M. Finn and K. Hofmann, in H. Neurath and R. L. Hill, eds. The Proteins, Vol. 2,

Academic Press, New York, 1976, pp. 105–253.

2. M. Bodanszky and A. Bodanszky, The Practice of Peptide Synthesis, Springer-Verlag,

Berlin, 1984.

3. M. Goodman, A. Felix, L. Moroder, and C. Toniolo, eds., Synthesis of Peptides and

Peptidomimetics (Houben-Wey E22: Methods of Organic Chemistry), Georg Thieme
Verlag, Stuttgart, 2002.

4. B. Merrifield, Science 232, 341–347 (1986).
5. B. W. Erickson and B. W. Merrifield, in H. Neurath and R. L. Hill, eds., The Proteins,

Vol. 2, Academic Press, New York, 1976, pp. 255–527.

6. C. Birr, Aspects of the Merrifield Peptide Synthesis, Springer-Verlag, Heidelberg, 1978.
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G

EORGE

B

ARANY

N

ANCY

K

NEIB

-C

ORDONIER

D

ANIEL

G. M

ULLEN

The University of Minnesota

200

POLYSACCHARIDES

Vol. 11

POLY(PHENYLENE ETHER).

See P

OLYETHERS

,

AROMATIC

.

POLY(p-PHENYLENEVINYLENE).

See Volume 7.

POLYPHOSPHAZENES.

See Volume 7.

POLYPROPYLENE.

See P

ROPYLENE

P

OLYMERS

.