Overview of Custom 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:
-
To verify the structure of naturally occurring peptides as determined by degradation techniques.
-
To study the relationship between structure and activity of
biologically active protein and peptides and establish their molecular
mechanisms.
-
To synthesize peptides that are of medical importance such as hormones and vaccines.
-
To develop new peptide-based immunogens.
Solid Phase Peptide Synthesis (SPPS)
The fundamental premise of this technique involves the incorporation of N-a-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-a-protected
amino acid, thereby covalently binding it to the polymer. The amino
protecting group (X) can then be removed and a second N-a-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.
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 a-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 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:
|
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.
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)
|
|
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-b-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
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-a-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-a-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 a-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 dioxide with the generation of a free a-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 a-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-a-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-a-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-a-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 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-a-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 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 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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