Peptide Notes

Overview of Peptide Synthesis

Introduction

Proteins are present in every living cell and possess a variety of biochemical activities. They
appear as enzymes, hormones, antibiotics, and receptors. They compose a major portion of
muscle, hair, and skin. Consequently, scientists have been very interested in synthesizing them in
the laboratory. This interest has developed into a major synthetic field known as Peptide
Synthesis. The major objectives in this field are four-fold:

1. To verify the structure of naturally occurring peptides as determined by degradation

techniques.
2. To study the relationship between structure and activity of biologically active protein and
peptides and establish their molecular mechanisms.
3. To synthesize peptides that are of medical importance such as hormones and vaccines.
4. To develop new peptide-based immunogens.

Solid Phase Peptide Synthesis (SPPS)

The fundamental premise of this technique involves the incorporation of N-α-amino acids into a
peptide of any desired sequence with one end of the sequence remaining attached to a solid
support matrix. While the peptide is being synthesized usually by stepwise methods, all soluble
reagents can be removed from the peptide-solid support matrix by filtration and washed away at
the end of each coupling step. After the desired sequence of amino acids has been obtained, the
peptide can be removed from the polymeric support.

The general scheme for solid phase peptide synthesis is outlined in Figure 1. The solid support is
a synthetic polymer that bears reactive groups such as -OH. These groups are made so that they
can react easily with the carboxyl group of an N-α-protected amino acid, thereby covalently
binding it to the polymer. The amino protecting group (X) can then be removed and a second N-α-
protected amino acid can be coupled to the attached amino acid. These steps are repeated until
the desired sequence is obtained. At the end of the synthesis, a different reagent is applied to
cleave the bond between the C-terminal amino acid and the polymer support; the peptide then
goes into solution and can be obtained from the solution.

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Fmoc Strategy in SPPS

The crucial link in any polypeptide chain is the amide bond, which is formed by the condensation
of an amine group of one amino acid and a carboxyl group of another. Generally, an amino acid
consists of a central carbon atom (called the α-carbon) that is attached to four other groups: a
hydrogen, an amino group, a carboxyl group, and a side chain group. The side chain group,
designated R, defines the different structures of amino acids. Certain side chains contain
functional groups that can interfere with the formation of the amide bond. Therefore, it is
important to mask the functional groups of the amino acid side chain.

The general scheme which outlines the strategy of Fmoc synthesis is shown in Figure 2. Initially,
the first Fmoc amino acid is attached to an insoluble support resin via an acid labile linker.
Deprotection of Fmoc, is accomplished by treatment of the amino acid with a base, usually

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piperidine. The second Fmoc amino acid is coupled utilizing a pre-activated species or in situ
activation. After the desired peptide is synthesized, the resin bound peptide is deprotected and
detached from the solid support via TFA cleavage.

Fmoc Cleavage

The removal of peptides in solid phase peptide synthesis is primarily done by acidolysis. The Fmoc
chemistry employs the use of weak acids such as TFA or TMSBr. Various scavengers are included
to protect the peptide from carbocations generated during cleavage which can lead to side
reactions. These additives usually include thiol compounds, phenol, and water.

The following protecting groups are compatible with TFA and TMSBr cleavage:

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 Peptide Notes

Arg(Boc)2 Cys(Acm) Lys(Boc)

Arg(Mtr) Cys(Trt) Lys(Fmoc)
Arg(Pbf) Gln(Tmob) Lys(Mtt)
Arg(Pmc) Gln(Trt) Ser(tBu)
Asn(Tmob) Glu(OtBu) Thr(tBu)
Asn(Trt) His(Boc) Tyr(tBu)
Asp(OtBu) His(Trt)

Depending on the type of protecting groups present, certain combinations of scavengers must be
used. For instance, when either Boc and t-Butyl groups are present, their carbocation
counterparts (t-butyl cations and t-butyltrifluoroacetate) can react with Trp, Tyr, and Met to form
their t-butyl derivatives. While EDT is a very efficient scavenger for t-butyl trifluoroacetate, it
does not protect Trp from t-butylation. Therefore, water must be added in order to suppress
alkylation. The indole ring of Trp and the hydroxyl group of Tyr are especially susceptible to the
reactivity of the cleaved Pmc group. Again, water has been shown to be effective in suppressing
this reaction. Similar occurrences can happen with the Trt and Mtr groups. Therefore, scavengers
in the appropriate combination will greatly reduce the amount of side reactions.

Boc Strategy in SPPS

The general scheme which outlines the strategy of Boc synthesis is shown in Figure 3. Initially,
the first Boc amino acid is attached to an insoluble support resin via a HF cleavable linker.
Deprotection of Boc, is accomplished by treatment of the amino acid with TFA. The second Boc
amino acid is coupled utilizing a pre-activated species or in situ activation. After the desired
peptide is synthesized, the resin bound peptide is deprotected and detached from the solid
support via HF cleavage.

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Boc Cleavage

The Boc chemistry employs the use of strong acids such as HF, TFMSOTf, or TMSOTf. Various
additives, usually thiol compounds are added to protect the peptide from the carbocations
generated during cleavage.

The following protecting groups are compatible with HF cleavage:

Arg(Mts) Cys(4-MeOBzl) His(Z)

Arg(Tos) Glu(OBzl) Lys(Cl-Z)
Asp(OBzl) Glu(OcHex) Ser(Bzl)
Asp(OcHex) His(Bom) Thr(Bzl)

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Cys(Acm) His(Dnp) Trp(CHO)

Cys(4-MeBzl) His(Tos) Tyr(Br-Z)
Asp(OtBu) His(Trt)

The following protecting groups are compatible with TFMSOTf cleavage:

Arg(Mts) His(Bom) Met(O)

Asp(OBzl) His(Dnp)) Ser(Bzl)
Cys(Acm) His(Tos) Thr(Bzl)
Cys(4-MeBzl) His(Z) Trp(CHO)
Glu(OBzl) Lys(Cl-Z) Tyr(Br-Z)

The following protecting groups are compatible with TMSOTf cleavage:

Arg(Mts) Glu(OcHex) Trp(CHO)

Arg(Mbs) His(Bom) Trp(Mts)
Asp(OBzl) Lys(Cl-Z) Tyr(Br-Z)
Asp(OcHex) Met(O) Tyr(Bzl)
Cys(Acm) Ser(Bzl) Tyr(Cl-Bzl)
His(Bom) Thr(Bzl)

General Coupling Methods in SPPS

Coupling reactions in SPPS require the acylation reactions to be highly efficient to yield high-
purity peptides.

Coupling Methods in Fmoc SPPS

The most widely used coupling method in Fmoc SPPS is the activated ester method either pre-
formed (pre-activated species) or in situ (without pre-activation). Initially, the p-nitrophenyl and
N-hydroxysuccinimide (ONSu) activated esters were the predominantly used forms (1-2).
However, even in the presence of HOBt, the coupling reactions tended to be slow. In addition,
ONSu esters of Fmoc amino acids were prone to the formation of the side product succinimido-
carbonyl-β-alanine-N-hydroxysuccinimide ester (3-4). The most commonly used activated esters
presently are the pentafluorophenyl (OPfp) ester and the 3-hydroxy-2,3-dihydro-4-oxo-benzo-
triazone (ODhbt) ester (5-7). In the presence of HOBt, the rate of reaction is very rapid and the
reaction is efficient with minimal side product formation. On the other hand, many coupling
reactions can be done in situ using activating reagents such as DCC, HBTU, TBTU, BOP, or BOP-
Cl. The direct addition of carbodiimide is considered to be the best choice (8-13). HBTU and TBTU

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would rank second, followed by BOP and finally BOP-Cl. With regards to ester coupling, the
following order was found: BOP/HOBt > carbodiimide/HOBt ~ carbodiimide/ODhbT > DCC/OPfp
(14-15).

More recently, 1-hydroxy-7-azabenzotriazole (HOAt) and its corresponding uronium salt analog O-
(7-azabenzotrizol-1-yl)-1,1,3,3, tetra-methyluronium hexafluorophosphate (HATU) have been
developed and found to have a greater catalytic activity than their HOBt and HBTU counterparts.
The use of HOAt and HATU enhances coupling yields, shortens coupling times, and reduces
racemization. Consequently, these reagents are suitable for the coupling of sterically hindered
amino acids, thereby ensuring greater success in the synthesis of difficult peptides (16-17).

Coupling Methods in Boc SPPS

The carbodiimides, primarily DCC, were the coupling reagents of choice for many years (18). The
major drawbacks encountered were the precipitation of dicyclohexylurea during the activation and
acylation processes and the numerous side reactions associated with its usage. Several
carbodiimides which produced soluble ureas were developed, such as diisopropylcarbodiimide
(DIC), t-butyl methyl- and t-butylethyl-carbodiimides (19-22), but these did not resolve the
problem of side reactions. Consequently, new types of activating agents were developed. The first
of these was BOP (23), PyBroP (24-25) PyBOP (26), HBTU (27), TBTU (28), and HATU (29). All of
these reagents require bases for activation.

All of the DCC and DCC-related derivatives discussed previously work by the formation of the
symmetrical anhydride. The symmetrical anhydrides are usually very reactive and have been
used extensively in SPPS, especially in Boc synthesis (30-33). Attempts at incorporating
symmetrical anhydrides to Fmoc amino acids were met with some difficulties (34-36). For
instance, symmetrical anhydrides prepared from N-(3-dimethylamino propyl)-N'-ethyl-
carbodiimide•HCl, upon formation of the 2-alkoxy-5(4H)-oxazolone intermediate, rearranged in
the presence of carbodiimides and tertiary amines (37). Also, not all of the Fmoc symmetrical
anhydrides are soluble in DCM or remain insoluble regardless of the solvent used (38).

An alternative to the symmetrical anhydride is the mixed anhydride which is a carboxylic-

carbonate or carboxylic-phosphinic mixed anhydride. Typically, these anhydrides are prepared by
reacting either isobutyl- or isopropyl-chloroformate and substituted phosphinic chlorides with the
N-α-protected amino acid (39-42). The reaction is typically rapid with little or no side reactions
(43-46).

A type of mixed anhydride, N-carboxyanhydrides (NCA's), also known as Leuchs' anhydride have
been widely used for the preparation of polyamino acids (47). This class of compounds combines
N-α-protection with carboxyl group activation. Once reacted with another amino acid or peptide
residue, the NCA releases carbon dioxide as its only by-product. NCA derivatives are easily
prepared by treating α-amino acids with phosgene (48-51). The resulting NCA derivatives usually
crystallize out and are ready for use under strictly defined conditions. These conditions require
the pH to be carefully controlled during synthesis. At pH < 10, the peptide-carbamate (produced
by the reaction between the NCA and the peptide or amino acid residue) tends to lose carbon

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dioxide with the generation of a free α-amino end group with resulting polymerization. At pH
10.5, hydrolytic decomposition of the NCA occurs. Therefore, the reaction is performed at pH
10.2. Another required condition is that the reaction proceeds for 2 minutes at 0°C with vigorous
stirring. The resulting product is free of racemization and bears a free α-amino group that can be
extended by addition of another anhydride.

Solution Phase Synthesis

Stepwise condensation is based on the repetitive addition of single N-α-protected amino acids to
a growing amino component, generally starting from the C-terminal amino acid of the chain to be
synthesized. The process of coupling individual amino acids can be accomplished through
employment of the carbodiimide (52-53), the mixed carbonic anhydride (54-55), or the N-
carboxyanhydride methods (56-57). The carbodiimide method involves coupling N- and C-
protected amino acids by using DCC as the coupling reagent. Essentially, this coupling reagent
promotes dehydration between the free carboxyl group of an N-protected amino acid and the free
amino group of the C-protected amino acid, resulting in the formation of an amide bond with
precipitation of the by- product, N,N'-dicylcohexylurea. This method, however, is hampered by
side reactions which can result in racemization (58-59) or in the presence of a strong base, the
formation of 5(4H)-oxazolones (60) and N-acylureas (61). Fortunately, these side reactions can
be minimized, if not altogether eliminated, by adding a coupling catalyst such as N-
hydroxysuccinimide (HOSu) or 1-hydroxybenzotriazole (HOBt). In addition, this method can be
employed to synthesize the active ester derivatives of N-protected amino acids (62). In turn, the
resulting activated ester will react spontaneously with any other C-protected amino acid or
peptide to form a new peptide.

In cases where separation of the activated ester from the by-product dicyclohexylurea proves to
be difficult, the mixed carbonic anhydride method can be employed. This method consists of two
stages: the first stage involves activating the carboxyl group of an N-α-protected amino acid with
an appropriate alkyl chlorocarbonate, such as ethylchlorocarbonate (63), or preferably
isobutylchlorocarbonate (64). Activation occurs in an organic solvent in the presence of a tertiary
base. The second stage involves reacting the carbonic anhydride with a free amine component of
an amino acid or a peptide unit. The carbonic anhydride is usually added at a 14-fold excess over
the amine component.

The mixed carbonic anhydride method is noted for being highly effective at low temperatures,
resulting in high yields and pure products. However, it does have its short-comings. For instance,
there is a tendency for the anhydride derivative to undergo racemization as a result of the strong
activation of the carbonyl group. This problem does not occur when N-α-urethane protecting
groups, such as Cbz or t-Boc, are employed (65-66). Furthermore, as a result of their high
reactivity, mixed carbonic anhydrides are prone to the formation of 5(4H)-oxazolones (67),
urethanes (68-69), diacyimides (70-71), esters (72), and are subject to disproportion (73-74).
Conditions which prompt such side reactions to occur are high temperatures, prolonged activation
times (the time interval between the addition to the alkylchlorocarbonate and the amine
component after the mixed anhydride is formed), steric bulk of the amine component, and
incomplete formation of the mixed anhydride. Fortunately, most of these side reactions, except

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 Peptide Notes

for oxazalone and urethane formation, can be substantially reduced by performing the reaction at
low temperature (~ -15°C) and allowing for shorter activation times (~ 1-2 min). To minimize the
formation of oxazolone and urethane derivatives, the following conditions must be implemented:
1) dried organic solvents such as ethyl acetate, tetrahydrofuran, t-butanol, or acetonitrile must
be used (75); 2) the tertiary base, N-methylmorpholine, should be used (76); and 3) Cbz- or Boc-
N-α-protected amino acids must be utilized (77).

Although isobutyl- and ethylchlorocarbonate are typically used to form carbonic anhydrides, other
coupling reagents do exist. For example, N-ethyloxycarbonyl-2-ethyloxy-1,2-dihydroquinoline
(EEDQ) (78) and N-isobutyloxy-carbonyl-2-isobutyloxy-1,2-dihydroquinoline (IIDQ) (79) were
developed to react with the carboxyl component to form the ethyl- or isobutylcarbonate
derivative. Unlike the classical anhydride procedure, EEDQ and IIDQ do not require base nor low
reaction temperatures. Typically, the procedure involves reacting equimolar amounts of the
carboxyl and amine components in an organic solvent (a wide variety of solvents can be used)
(80) at 0.1 M to 0.4 M concentrations. Then EEDQ or IIDQ is added in 5-10% excess and the
mixture is allowed to stir for 15-24 hours at room temperature. After removal of the solvent, in
vacuo, the residue is dissolved in ethyl acetate and washed with 1N NaHCO3, 10% citric acid, and
salt water, then dried with Na2SO4 (anhydrous), and evaporated. The product can then be
recrystallized or purified by chromatography. While this method circumvents the use of base, it is
still subject to racemization and urethane side product formation at levels comparable to those
found in the classical anhydride approach. Consequently, its only advantage may be that it is
easy and convenient to use. It should be noted that a detailed comparison of the two methods
has not been carried out to this date.

HPLC Analysis and Purification (81-84)

Analytical HPLC utilizes columns and pumping systems that can withstand and deliver very high
pressures enabling the use of very fine particles (3-10 microns) as packing material.
Consequently, peptides can be resolved with a high degree of resolution in a short time interval (i.
e., minutes).

Two common HPLC purification methods are, ion exchange and reverse phase. Ion exchange
HPLC is based on direct charge interactions between the peptide and the stationary phase. The
column support is derivatized with an ionic species that maintains a particular charge over a
certain pH range, while the peptide or peptide mixture exhibits an opposite charge which is
dependent on its amino acid composition. Separation is dependent on charge interactions. The
peptide is eluted by changing the pH, the ionic strength, or both. Typically, a solution of low ionic
strength is used; the ionic strength of the solution is then gradually or step-wise increased until
the peptide is eluted from the column. One example of ion exchange separation incorporates the
use of strong cation exchange columns such as sulfoethylaspartimide which separates on the
basis of positive charge at an acidic pH.

Reverse phase HPLC conditions are essentially the reverse of normal phase chromotography. The
peptide binds on the column through hydrophobic interactions and is eluted by decreasing the

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ionic strength (i.e., increasing the hydrophobicity of the eluent). Generally, the column supports
are composed of hydrocarbon alkane chains which are covalently attached to silica. These chains
range from C4 to C18 carbon atoms in length. Since elution from the column is a function of the
hydrophobicity, the longer chain hydrocarbon columns are better for small, highly charged
peptides. On the other hand, large hydrophobic peptides elute better using short chain
hydrocarbon supports. However, in general practice, these two types of columns can be used
interchangeably with little significant differences. Other types of supports consist of aromatic
hydrocarbons such as phenyl groups.

A typical run usually consist of two buffers, 0.1% TFA-H2O and 80% acetonitrile/0.1% TFA-H2O,
which are mixed using a linear gradient with a flow rate which will give a 0.5% to 1.0% change
per minute. Typical columns for analytical and purification runs are 4.6 x 250 mm (3-10 microns)
and 22 x 250 mm (10 microns), respectively. If radial packed columns are used, then column
sizes are 8 x 100 mm (3-10 microns) and 25 x 100 mm (10 microns), respectively. A variety of
other buffers can contain many different types of reagents such as 0.1% heptafluorobutyric acid,
0.1% phosphoric acid, dilute HCl, formic acid (5-60%, pH 2-4), 10-100 mM NH4HCO3, sodium/
ammonium acetate, TFA/TEA, sodium or potassium phosphate, or triethylammonium phosphate
(pH 4-8). In addition, water miscible eluents can also be added such as methanol, propanol, and
isopropanol. Therefore, many combinations of solvents and additives for a buffer are possible. It
should be noted that silica-based reverse phase column packing must not be exposed to high pH's
or even slightly basic pH's for extended periods of time because the column can be destroyed at
those pH levels.

The crude peptide obtained from SPPS will contain many by-products which are a result of
deletion or truncated peptides as well as side products stemming from cleaved side chains or
oxidation during the cleavage and deprotection process. Earlier purification methods included ion
exchange, partition, and counter current chromatography. Recent purification methods include
reverse phase HPLC which is generally successful with peptides containing 60 residues or less. In
conjunction, ion exchange HPLC can be used in cases where reverse phase HPLC does not work.

Typically, analytical HPLC results are used to determine the purification conditions. For example,
if a peptide elutes out at 30% (0.1% TFA) aqueous acetonitrile (determined by analytical HPLC
analysis), a buffer containing a lower concentration of acetonitrile is chosen such that the peptide
peak will come out 4-5 minutes after the solvent peak under isocratic conditions (e.g., 28%
(0.1% TFA) aqueous acetonitrile). The purification conditions will entail using a linear gradient of
16-35% (0.1% TFA) aqueous acetonitrile over one or two hours depending on the type of column
chosen. The collected fractions will then be checked by analytical HPLC, using the buffer chosen
for isocractic conditions.

Handling and Storage of Peptides

Peptides have widely varying solubility properties. The main problem associated with the
dissolution of a peptide is secondary structure formation. This formation is likely to occur with all
but the shortest of peptides and is even more pronounced in peptides containing multiple

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hydrophobic amino acid residues. Secondary structure formation can be promoted by salts. It is
recommended first to dissolve the peptide in sterile distilled or deionized water. Sonication can be
applied if necessary to increase the rate of dissolution. If the peptide is still insoluble, addition of
a small amount of dilute (approximately 10%) acetic acid (for basic peptides) or aqueous
ammonia (for acidic peptides) can facilitate dissolution of the peptide.

For long-term storage of peptides, lyophilization is highly recommended. Lyophilized peptides can
be stored for years at temperatures of -20°C or lower with little or no degradation. Peptides in
solution are much less stable. Peptides are susceptible to degradation by bacteria so they should
be dissolved in sterile, purified water.

Peptides containing methionine, cysteine, or tryptophan residues can have limited storage time in
solution due to oxidation. These peptides should be dissolved in oxygen-free solvents. To prevent
the damage caused by repeated freezing and thawing of peptides, dissolving the amount needed
for the immediate experiment and storing the remaining peptide in solid form is recommended.

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Peptide Notes

55. Denkewalter, R. G. et al. J. Amer. Chem. Soc. 88, 3163 (1966).

56. Hirschmann, R. et al. J. Org. Chem. 32, 3412 (1967).
57. Anderson, G. W. and Callahan, F. M., J. Amer. Chem. Soc. 80, 2902-3000 (1958).
58. Hofman, K et al. Amide Resin, J. Amer. Chem. Soc. 80, 1486-1489 (1958).
59. Konig, W. and Geiger, R. Chem. Ber. 80, 103 (1970).
60. Wunsch, E. and Dress, F. Chem, Ber. 9, 110-120 (1966).
61. Bodanszky, M., Acta Chim. Acad. Sci. Hung 10, 335-346 (1956).
62. Wieland, T. and Bernhard, H. Justus Liebigs Ann. Chem. 572, 190-194 (1951).
63. Anderson, G. W. et al. J. Amer. Chem. Soc. 89, 5012-5017 (1967).
64. Determann, H. Peptides, Proceedings of the 8th European Peptide Symposium,
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65. Weygand, F. et al. Angew. Chem, Int. Ed. Engl. 2, 183-188 (1963).
66. Vaughan, J. R., Jr. J. Amer. Chem. Soc. 74, 6137-6139 (1952).
67. Anderson, G. W. et al. J. Amer. Chem. Soc. 89, 5012-5017 (1967).
68. Battersby, A. R. and Robinson, J. C. J. Chem. Soc. 259-269 (1955).
69. Bodanszky, M. and Tolle, J. C. Int. J. Peptide Protein Res. 10, 380-384 (1977).
70. Merrifield, R. B. et al. J. Org. Chem. 39, 660-668 (1977).
71. Goodman, M. and Stueben, K. C. J. Org. Chem. 27, 3409-03416 (1962).
72. Koppel, K. D. and Renick, R. J. J. Org. Chem. 23, 1565-1567 (1958).
73. Tarbell, D. S. Acc. Chem. Res. 2, 296-300 (1969).
74. Albertson, N. F., Org. React. 12, 157-355 (1962).
75. Longosz, E. J. and Tarbell, D. S. J. Org. Chem. 26, 2161-2169 (1961).
76. Anderson, G. W. Peptides: Chemistry and Biochemistry, Weinstein, B. and Lande, S.,
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77. Anderson, G. W. et al. J. Amer. Chem. Soc. 89, 5012-5017 (1967).
78. Belleau, D. and Malek, G. J. Amer. Chem. Soc. 90, 1651-1652 (1968).
79. Kiso, Y. et al. Chem. Pharm. Bull. 21, 2507-2510 (1973).
80. Belleau, D. and Malek, G. J. Amer. Chem. Soc. 90, 1651-1652 (1968).
81. Reviews in High Performance Liquid Chromatography of Peptides and Proteins:
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82. River, J. et al. J. Chromatography 288, 303 (1984).
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