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5 Solid-Phase Synthesis
5.1 THE IDEA OF SOLID-PHASE SYNTHESIS
Solid-phase synthesis means synthesis on a solid support. It is a technology that
dates back to the early 1960s — an era when ion exchange on functionalized
polystyrene beads was the prominent method for purification and analysis of small
charged molecules. R. B. Merrifield, an immunologist working in the laboratory of
D. W. Woolley at the Rockefeller Institute in New York, was required to synthesize
analogues of biologically active peptides. In the process, Merrifield perceived that
the approach of the day, made up of successive coupling and deprotection reactions
in solution, followed by extractions at each step to eliminate unconsumed reactants
and secondary products, was very labor intensive and repetitive, and he concluded
that there was a need for a rapid and automatic method for the synthesis of peptides.
He suggested that the synthesis be carried out with the first residue attached to an
insoluble support (Figure 5.1), so that purification could be achieved by simple
filtration instead of extraction. According to his proposal, a protected amino acid is
anchored to an insoluble functionalized support by a bond that resists all chemistries
employed during assembly of the peptide. The amino group is deprotected, and
additional residues are introduced successively. Each reaction is followed by filtra-
tion, which removes unconsumed reactants and secondary products that are dissolved
in the solvent. Final deprotection detaches the peptide from the support. Three
advantages were envisioned by Merrifield: high yields achieved by forcing the
reactions to completion, less manipulation and consequently less time, and mini-
mized losses of material because reactions and purification would take place without
removing the peptide support from the reaction vessel. Successful implementation
of the method was announced in 1962, with the first publication appearing the
following year. For the first 10 years or so, the method faced considerable opposition

Temporarily protected amino acid Pg1-Xaa-OH

Functionalized insoluble support Y-Polymer
First residue attachment Pg1-Xaa-Polymer
Amino deprotection H-Xaa-Polymer
Coupling Pg1-Xbb(Pg2)-OH Pg1-Xbb(Pg2)-Xaa-Polymer
Repeat amino deprotection Pg1-Xcc-Xbb(Pg2)-Xaa-Polymer
and coupling
Final deprotection and
release of peptide H-Xcc-Xbb-Xaa-OH

FIGURE 5.1 The idea of solid-phase synthesis as conceived by Merrifield in 1959. Protecting
group Pg1 is selectively removed after the addition of each residue.

125

© 2006 by Taylor & Francis Group, LLC

 126 Chemistry of Peptide Synthesis

by the traditionalists, but this resistance gradually dissipated. Twenty years of devel-
opment and refinement by Merrifield and colleagues and others culminated in the
award in 1984 of the Nobel Prize in Chemistry to Merrifield for development of a
methodology for chemical synthesis on a solid matrix. The method has since found
no end of applications. It is interesting to note that an analogous approach was
employed by others in 1963 to synthesize a dipeptide, the amino-terminal residue
being fixed to a polystyrene support through an amide bond.1,2

1. RB Merrifield. Federation Proc 21, 412, 1962.

2. RB Merrifield. Solid phase synthesis. I. The synthesis of a tetrapeptide. J Am Chem
Soc 85, 2149, 1963.

5.2 SOLID-PHASE SYNTHESIS AS DEVELOPED BY

MERRIFIELD
Initial attempts to develop the solid-phase method (see Section 5.01) involved attach-
ment of the first residue to a functionalized copolymer of styrene and divinylbenzene,
with the trade name Dowex 50, as the benzyl ester with benzyloxycarbonyl for
protection of α-amino groups and selective deprotection by acidolysis with hydrogen
bromide in acetic acid (see Section 3.5). The method was inefficient, however,
because of the significant loss of the chain from the support at each step. The
anchoring linkage was stabilized to acidolysis by nitration of the phenyl ring of the
benzyl moiety (see Section 3.19), and this allowed the first successful synthesis of
a peptide; namely, L-leucyl-L-alanylglycyl-L-valine. Unreacted amino groups were
capped by acetylation with triethylammonium acetate after each coupling. The chain
was detached from the resin by saponification. The structure was established by
comparison with an authentic sample prepared in solution using 4-nitrophenyl esters
(see Section 2.9) for peptide-bond formation. It was obvious, however, that the
combination of two benzyl-based protectors was not going to be satisfactory. For-
tunately, the tert-butoxycarbonyl protector (see Section 3.6) had just become avail-
able, and an improved methodology was developed. Figure 5.2 shows the scheme
employed in 1964 by Merrifield to prepare bradykinin, which contains nine residues
— the first biologically active peptide synthesized by his method. The first residue
was attached to a polystyrene-divinylbenzene copolymer as the benzyl ester by
reaction of Boc-Nω-nitroarginine with the chloromethylated polymer (see Section
5.7) in the presence of triethylamine. The Boc-protector was removed by acidolysis
with hydrogen chloride in acetic acid, and the amine hydrochloride that was gener-
ated was neutralized with triethylamine. Liberated tert-butoxycarbonyl produces
isobutene and carbon dioxide.
The next residues were attached successively by dicyclohexylcarbodiimide-
mediated coupling of Boc–amino acids with the free amino groups. The use of excess
Boc–amino acid eliminated the need for capping after coupling. The last Boc-group
and the benzyl-based side chain and carboxy-terminal protectors were removed at
the end of the synthesis by acidolysis with hydrogen bromide in trifluoroacetic acid;
the latter was used instead of acetic acid to avoid acetylation of hydroxymethyl side
chains (see Section 6.6). Catalytic hydrogenolysis of the peptide removed the nitro

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 Solid-Phase Synthesis 127

Chloromethyl polymer
O R1
(CH3)3COC NHCHCO2H ClCH2 PS
Anchoring of first residue Benzyl ester
O R1 O
1. Deprotection (CH3)3 COC NHCHC OCH2 PS
HCl/CH3CO2H Boc-amino-
2. Neutralization CO2 acyl polymer
R1 O
(C 2H5)3N (CH3)2C=CH2 NH2CHC OCH2 PS
Coupling boc-amino acid Aminoacyl polymer
C6H11N=C=NC6H11 O R2 O R1 O
(CH3)3COC NHCHC NHCHC OCH2 PS
Complete deprotection, Boc-dipeptidyl polymer
release of chain R2 O R1
HBr/CF3CO2H NH3CHC NHCHCO 2H BrCH2 PS
Dipeptide

FIGURE 5.2 Synthesis of a peptide on a solid support according to Merrifield in 1964.

PS = polystyrene. Initially, the Nαprotector was benzyloxycarbonyl, removed by HBr in
CH3CO2H, followed by final deprotection with the same reagent at 78˚C. The current protocol
employs CF3CO2H and HF, respectively.

groups. A 68% yield of bradykinin was obtained after purification of the crude
product on a weakly acidic cation-exchange resin. Four days were required for the
synthesis, and 4 more days were required to purify the product. All reactions except
deprotection had been carried out in dimethylformamide. However, dichloromethane
proved superior for couplings because less N-acyl-N,N′-dicyclohexylurea (see Sec-
tion 2.2) is formed, and hence a smaller excess of Boc–amino acid is required.
Peptide-bond formation employing activated esters had been examined, but the tactic
was rejected on the basis that the reactions did not go to completion. It was later
shown that activated esters are suitable for solid-phase synthesis if a solvent more
polar than dichloromethane (see Section 7.6) is employed. Present-day protocol
involves use of 50% trifluoroacetic acid in dichloromethane for deprotection of α-
amino groups, and hydrogen fluoride for final deprotection and release of the chain
from the support.2–6

2. RB Merrifield. Solid phase synthesis. I. The synthesis of a tetrapeptide. J Am Chem

Soc 85, 2149, 1963.
3. RB Merrifield. Solid phase synthesis. II. The synthesis of bradykinin. J Am Chem
Soc 86, 304, 1964.
4. RB Merrifield. Solid-phase synthesis. III. An improved synthesis of bradykinin.
Biochemistry 3, 1385, 1964.
5. RB Merrifield. Solid phase synthesis. IV. The synthesis of methionyl-lysyl-bradyki-
nin. J Org Chem 29, 3100, 1964.
6. RB Merrifield. Solid-phase peptide synthesis. Endeavour 3, 1965.

5.3 VESSELS AND EQUIPMENT FOR SOLID-PHASE SYNTHESIS

The unique feature of solid-phase synthesis is the elimination of unconsumed reac-
tants and secondary products by filtration. The latter is possible because chain

© 2006 by Taylor & Francis Group, LLC

 128 Chemistry of Peptide Synthesis

Stirrer Teflon-threaded
Stopper
closures
Drying tube

Paddle 180⬚
Solvent
Resin beads
Sintered glass
Stopcock

B Suction A C

FIGURE 5.3 Reaction vessels for solid-phase synthesis. (A) 10–300-mL vessel (0.5–10 g of
resin) affixed to a rotating-arc shaker; (B) 0.5–8-L (20–200 mm in diameter) cylindrical
container with stirrer for up to 500 g of resin; (C) Ananth vessel with two 80-mL chambers.10

assembly is carried out on an insoluble support that is in the form of small beads,
so the essential is a reaction vessel fitted at the bottom with a fritted glass disk and
an outlet through which suction can be applied to remove the solvent containing the
undesired components (Figure 5.3, A). The flow of liquid is controlled by a stopcock.
Reactants and solvent are introduced through an upper entrance port that is closed
by a stopper or screw cap. Agitation is achieved by fixing the vessel to a rotating-
arc shaker. Simple movement of the support and not vigorous shaking is desired, so
that the beads (see Section 5.7) are not damaged. Vessels of a variety of shapes and
designs are available, including a two-chamber vessel (Figure 5.3, C) that allows
synthesis of two different peptides at the same time. Some vessels have an inlet at
the bottom for introducing a stream of nitrogen for agitation, with an appropriate
outlet at the top. For synthesis on a larger scale, a vessel of increased diameter can
be selected, into which is inserted a paddle connected to a mechanical stirrer for
mixing the components (Figure 5.3, B). Monitoring of reactions can be achieved by
withdrawing an aliquot of resin through the entrance port. Various arrangements of
connected containers and three-way valves permit the delivery and removal of
solvents and reagents without opening the vessels. An alternative involves synthesis
under continuous-flow conditions (Figure 5.4), in which case the support is station-
ary, the solvent flows in a cycle through the support, and the reagents are injected
into the moving solvent. Chain assembly employing the above systems is referred

Pump
Wash solvents, Reactants
deprotector,
neutralizer
Waste or Detector Column
collector

FIGURE 5.4 Schematic representation of a continous-flow system for the solid-phase syn-
thesis of peptides. Solvent is forced through the system by a pump. The support is in the
form of a column that is stationary. A reaction is monitored by measuring the change in
absorbance of the solvent stream.

© 2006 by Taylor & Francis Group, LLC

 Solid-Phase Synthesis 129

to as “manual” solid-phase synthesis. Instruments controlled by a programmer or

computer produce peptides by “automated” synthesis.2,7–10

2. RB Merrifield. Solid phase synthesis. I. The synthesis of a tetrapeptide. J Am Chem

Soc 85, 2149, 1963.
7. RB Merrifield, JM Stewart, N Jernberg. Instrument for automated synthesis of pep-
tides. Anal Chem 38, 1905, 1966.
8. V Gut, J Rudinger. Rate measurement in solid phase peptide synthesis, in E. Bricas,
ed. Peptides 1968. Proceedings of the 9th European Peptide Symposium, North-
Holland, Amsterdam, 1968, pp 185-188.
9. TJ Lukas, MB Prystowsky, BW Erickson. Solid-phase peptide synthesis under con-
tinous-flow conditions. Proc Natl Acad Sci USA 78, 2791, 1981.
10. M Anantharamaiah, A Gawish, M Iqbal, SA Khan, CG Brouilette, JP Segrest. In CA
Peeters, ed. Peptides of Biological Fluids, New York, Permagon, 1986, p 34.

5.4 A TYPICAL PROTOCOL FOR SOLID-PHASE

SYNTHESIS
Solid-phase synthesis involves the combination of reagents with functional groups
that are located on the surface and on the inside of beaded polymers. The beads are
immersed in solvent containing the reagents, which approach the solvated sites by
diffusion. Success is contingent on the sites being accessible to the reagents. Com-
plete reaction is encouraged by use of a large excess of reagent; complete removal
of soluble components is achieved by repeated washing and filtration. A typical
protocol for the synthesis of a 14-mer on polystyrene resin using Boc/Bzl chemistry
appears in Figure 5.5. The peptide resin is suspended in dichloromethane, then
methanol, and then dichloromethane. The latter swells the resin beads, and the more
polar methanol shrinks the beads (see Section 5.7). Alternating swelling and shrink-
ing serves to expose reacting sites. Methanol is also used to remove reagents that are
presented in dimethylformamide because it is miscible with the two other solvents.

Min Min
1. CH2Cl2 wash, 80 mL 3 X2 8. CH2Cl2 wash, 80 mL 3 X3
2. CH3OH wash, 30 mL 3 X2 9. Boc-Xaa-OH (10 mmol)
3. CH2Cl2 wash, 80 mL 3 X2 in 30 mL DMF + DCC
4. CF3CO2H-CH2Cl2 (10 mmol) in DMF, 30 X1
(1:1), 70 mL 10 X2 10 . CH3OH wash, 40 mL 3 X2
5. CH2Cl2 wash, 80 mL 3 X2 11 . Et2N-DMF (1:8), 70 mL 5 X2
6. Et2N-DMF (1:8), 70 mL 5 X2 12 . CH3OH wash, 30 mL 3 X2
7. CH3OH wash, 40 mL 3 X2 13 . CH2Cl2 wash, 80 mL 3 X2

FIGURE 5.5 Schedule for the solid-phase synthesis of somatostatin, a 14-mer, on 10 g of

resin reacted with 5 mequiv of the first amino acid, adapted from J. Rivier, J. Am. Chem. Soc.
96:2986, 1974. Min = time of mixing; X2 = two times; DCC = dicyclohexylcarbodiimide.
Step 4 included 5% of (CH2SH)2 to prevent the oxidation of tryptophan. When the ninhydrin
test on an aliquot after step 13 was negative, step 1 followed; when positive, steps 9–13 were
repeated.

© 2006 by Taylor & Francis Group, LLC

 130 Chemistry of Peptide Synthesis

O O
R1 O R1 O
OH A
+ NH2CC N CC R1 O
OH H H
O 2H2O O H2O O CC
O O O N O
HO
N
B NH2 +
HO
O O 2H2O O O
Ruhemann’s purple Amine Ninhydrin

FIGURE 5.6 Reaction of ninhydrin (trioxohydrindene hydrate) with the amino group of a
bound residue (A) generates the Schiff’s base. Hydrolysis after shift of the double bond
generates the aldehyde and another amine which reacts (B) with a second molecule of
ninhydrin to give an equilibrium mixture of the anion depicted and its tetraoxo form with a
maximum of absorbance at 570 nm.

Each wash is for a selected period of time and is carried out twice to completely
effect the change in solvent. Step 4, followed by a dichloromethane wash, removes
the Boc-group. Step 6 neutralizes the trifluoroacetate anion that is bound by ionic
interaction. Step 9 is the coupling reaction. Step 11 serves to remove any Boc–amino
acid that might be bound to the resin by adsorption. Step 13 is followed by a test
for unreacted amino groups to verify that the coupling has gone to completion. An
aliquot of beads is heated with a solution containing ninhydrin and other components.
Two molecules of ninhydrin combine with an amino group with the liberation of
three molecules of water (Figure 5.6) to produce a purple color, the intensity of
which depends on the nature of the amino-terminal residue. The resin beads remain
colorless if they do not contain cationic sites that combine with the anionic form of
the chromophore (see Section 5.16). If the test is positive, a second coupling, steps
9–13, is effected, or the amino groups are permanently blocked or “capped” by
acetylation or some other amide-bond forming reaction to prevent chain extension
at these sites. The reagent for capping should not contain alcohol together with base
so that fission by transesterification at the anchored residue is avoided. Final depro-
tection, which also releases the chain from the resin, is effected by strong acid. The
synthesis described in Figure 5.5 was carried out manually on a scale up to 100 g
of peptide-resin at the time when the solid-phase approach was still considered
controversial. A multitude of protocols have emerged, a significant detail being the
use of dichloromethane as the solvent for couplings.11–13

11. E Kaiser, RL Colescott, CD Bossinger, PI Cook. Color test for detection of free
terminal amino groups in the solid-phase synthesis of peptides. Anal Biochem 34,
595, 1970.
12. JEF Rivier. Somatostatin. Total solid phase synthesis. J Am Chem Soc 96, 2986, 1974.
13. VK Sarin, SBH Kent, JP Tam, RB Merrifield. Quantitative monitoring of solid-phase
peptide synthesis by the ninhydrin reaction. Anal Biochem 117, 147, 1981.

© 2006 by Taylor & Francis Group, LLC

 Solid-Phase Synthesis 131

5.5 FEATURES AND REQUIREMENTS FOR

SOLID-PHASE SYNTHESIS
There are several features that are characteristic of solid-phase synthesis. There is
no loss of material accompanying the synthetic steps because the peptide is never
taken out of the vessel. Secondary products issuing from the reagents and protectors
do not affect the purity of the peptide because they are quantitatively removed after
each reaction. Reactions can be encouraged to go to completion by the use of an
excess of reagents. The question of the solubility of the peptide does not arise as it
does for syntheses carried out in solution. No purification of intermediates is effected
during the synthesis, and operations are repetitive and have been automated. How-
ever, monitoring the course of reactions is not straightforward, though some auto-
mated systems incorporate measurement devices, and synthetic impurities resemble
the target molecule (see following), which makes them difficult to remove at the
end of the synthesis.
The requirements for solid-phase synthesis are diverse. The support must be
insoluble, in the form of beads of sufficient size to allow quick removal of solvent
by filtration, and stable to agitation and inert to all the chemistry and solvents
employed. For continuous-flow systems, the beads also must be noncompressible.
Reactions with functional groups on beads imply reaction on the inside of the beads
as well as on the surface. Thus, it is imperative that there be easy diffusion of reagents
inside the swollen beads and that the reaction sites be accessible. Accessibility is
facilitated by a polymer matrix that is not dense and not highly functionalized. A
matrix of defined constitution allows for better control of the chemistry. Easier
reaction is favored by a spacer that separates the matrix from the reaction sites.
Coupling requires an environment of intermediate polarity such as that provided by
dichloromethane or dimethylformamide; benzene is unsuitable as solvent.
Reactions in solid-phase synthesis should be complete to avoid the formation
of mixtures of failure sequences, which creates a formidable purification challenge.
As an example, a 99% yield at each of 30 steps of the synthesis of a 15-mer gives
as product a 74% yield of target peptide and a 26% yield of peptides that lack one
residue. This is assuming that all unreacted groups participate in the next step. When
an amino group becomes unavailable for chain growth, the substance produced is
referred to as a truncated peptide. Truncated peptides that resume growth give rise
to peptides containing deletion sequences. Production of failure sequences resulting
from incomplete coupling may be avoided by effecting a second acylation or by
capping (see Section 5.4); however, there is no guarantee that this action will achieve
the objective. Removal of reagents should be complete, achieved by repetitive
washing with appropriate solvents. Successful solid-phase synthesis requires that
there be no reactions on the side chains and main chain during assembly, including
premature removal of protecting groups, which includes the carboxy-terminal pro-
tector. In view of the use of liberal amounts of solvents and amino acid derivatives
in large excess, all materials should be pure, and the chiral integrity of the residues
must be preserved throughout the synthesis.6,14

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 132 Chemistry of Peptide Synthesis

6. RB Merrifield. Solid-phase peptide synthesis. Endeavour 3, 1965.

14. S Hancock, DJ Prescott, PR Vagelos, GR Marshall. Solvation of the polymer matrix.
Source of truncated and deletion sequences in solid phase synthesis. J Org Chem 38,
774, 1973.

5.6 OPTIONS AND CONSIDERATIONS FOR

SOLID-PHASE SYNTHESIS
Solid-phase synthesis is a technology by which the synthesis of a peptide is simpli-
fied. The chemistry for synthesis on a solid support is the same as that for synthesis
in solution, except that the protector of the carboxy terminus is linked to an insoluble
support, either directly or indirectly. Peptides that are to be cyclized may be anchored
through the functional group of a side chain (see Section 5.24). Chain assembly is
by addition of single residues, which is the approach also employed for synthesis
in solution. The nature of the appendage on the carboxy terminus provides the unique
difference. The appendage consists of a polymer matrix attached to a protector. The
nature of the linkage between the protector and the first residue, as well as the
method employed to anchor the first residue, is dictated by the nature of the target
peptide. The stability that is required of the anchoring bond depends on the condi-
tions that are employed to remove the protectors on the amino terminus of the
growing peptide chain, or vice versa. The consequence of the above is that the choice
of one option limits the choices for the other options because most are interdepen-
dent. The variables include the type of support, the nature of the anchoring bond
and how it is created, the method of coupling, the nature of the amino-terminus
protector and the deprotecting reagent, and the method of final deprotection.
There are three major types of target molecules: peptides, which implies a free
carboxy terminus; protected peptides, which usually implies a free carboxy terminus;
and peptide amides. Resins are either microporous gels or composites. The more
common gel is a polystyrene–divinylbenzene copolymer (see Section 5.7), an alter-
native is the polar polydimethylacrylamide (see Section 5.8), and recently made
popular are the polyethyleneglycol–polystyrene adducts (see Section 5.9). The link-
ers (see Section 5.10) to which the peptides are bound are substituted benzyl alcohols
for the synthesis of peptides and substituted benzyl amines for the synthesis of
amides. Protectors are either tert-butoxycarbonyl for the amino termini with benzyl
or variants for the side-chain functional groups or 9-fluorenylmethoxycarbonyl for
the amino termini with tert-butyl for the side-chain functional groups (see Section
3.20). Bond formation is by use of carbodiimides or variations thereof, occasionally
symmetrical anhydrides or activated esters, and onium salt-based reagents (see
Section 5.15). Each coupling method has its attractive features and limiting impli-
cations. Some residues such as asparaginyl and glutaminyl may require special
consideration. Selection among the options is often influenced by the experience of
the operator and the traditions of the laboratory.

© 2006 by Taylor & Francis Group, LLC