Volume 26, Number 7
by Christina Hood, German Fuentes, Hirendra Patel, Karen Page,
Mahendra Menakuru, and Jae H. Park
TECHNICAL ARTICLE
Low-Cost, Fast, Conventional Peptide Synthesis
With HCTU and Automated Peptide Synthesizers
Out of a concern for purity and yield, many peptide
chemists today choose to extend their reaction times
excessively in order to obtain as high a purity and
yield as possible. Typical reaction times for conventional Fmoc solid-phase peptide synthesis (SPPS) are
deprotection times of 10–30 min, and coupling times
of 20 min to over an hour, which can result in cycle
times of up to 2 hr. As a result, conventional peptide
synthesis chemistry is perceived as slow, but this is
not necessarily the case.
Historically, the biggest contribution to increasing the speed of peptide synthesis was undoubtedly the invention of solid-phase peptide synthesis by Bruce Merrifield in 1963. 1 By attaching
the growing peptide chain to a solid support,
his method eliminated time-consuming purification steps and paved the way for automating the
process. Since then, advances in automation and
chemistry have made it possible to increase the
speed at which peptides can be made. Most chemistry methods have focused on the development
of more efficient activators for the coupling step.
O-benzotriazole-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HBTU) was the first of
these activators, and was introduced in 1990
for performing FastMoc™ chemistry (Applied
Biosystems, Foster City, CA) with coupling
times of 10–30 minutes.2
In 1993, Carpino introduced the activator O-(7azabenzotriazole-1-yl)-N,N,N′N′-tetramethyluronium hexafluorophosphate (HATU), which was
used four years later by Alewood and Miranda to
perform 1–2 min couplings with Boc chemistry.3,4
Similar fast methods have not been developed for
Fmoc chemistry, and for many laboratories, HATU
is too expensive to use for all but the most difficult
couplings. In 2002, the activator 1H-benzotriazolium 1-[bis(dimethylamino)methylene]-5-chlorohexafluorophosphate (1-),3-oxide (HCTU) was
introduced by Luxembourg Laboratories (Rehovat,
Israel), and is available at a significantly lower price
than HATU.5
The authors tested the efficiency of HCTU
by synthesizing a phosphorylated peptide
(H- CRRKGpSQKVS-NH 2 ) using HBTU and
HCTU, and found that HCTU produced the higherpurity peptide (data not shown). They then synthesized the 65–74 fragment of the acyl carrier protein
(65–74ACP) (H-VQAAIDYING-OH) using HCTU;
HATU; HBTU; benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluoro-phosphate (PyBOP);
and O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU). They found that
HCTU and HATU produced peptides of extremely
similar purity, while the remaining activators had
additional impurities (data not shown). From this,
it was concluded that HCTU was a highly efficient
coupling reagent.
The goal was to synthesize peptides as quickly and
inexpensively as possible. The authors were able
to achieve extremely rapid coupling times using
the activator HCTU. This paper demonstrates
this on seven peptides with a variety of properties:
long, short, hydrophobic, hydrophilic, cyclic, and
peptides containing D-amino acids and pseudoproline dipeptides.
Table 1
Summary of reaction and synthesis times for linear peptides; peptide lengths are also given
Peptide
GHRP-6
Oxytocin
65–74
ACP
G-LHRH
C-peptide
hAmylin1–37
β-Amyloid1–42
a
b
Length
6-mer
9-mer
10-mer
10-mer
31-mer
37-mer
42-mer
Dep. time
2 × 30 sec
2 × 30 sec
2 × 30 sec
2 × 30 sec
2 × 1.5 min
2 × 1 min
2 × 1 min
Coup. time
2 × 1 min
2 × 1 min
2 × 1 min
2 × 1 min
2 × 2 min
2 × 2.5 min
1 × 5 min
Cycle timea
14 min
14 min
14 min
14 min
18 min
19 min
17 min
Synth. timeb
1.4 hr
2.1 hr
2.1 hr
2.3 hr
9 hr
10.8 hr
11.6 hr
Determined from Prelude with 0.5 min × 6 DMF top washes.
Cycle time × number of cycles.
Experimental
Peptides were synthesized as described previously
on either a Symphony® or Prelude™ peptide synthesizer from Protein Technologies, Inc. (Tucson,
AZ).6 The Prelude automated peptide synthesizer
was used to save money on expensive monomer
additions, because its Single-Shot™ delivery feature delivers the entire contents of an amino acid
bottle to a specified reaction vessel without priming or waste. HPLC and mass spectrometry analysis were also carried out as described previously.6
Results and
discussion
Figure 1
HPLC of crude GHRP-6. This 6-mer peptide was synthesized
in 1.4 hr with 2 × 30 sec deprotection times and 2 × 1 min coupling times.
Using HCTU as the coupling reagent, the
authors were able to synthesize the shorter peptides (10 residues or less) using 1-min deprotection times and 2-min coupling times. The
reaction times for the longer peptides (over 30
residues) were reduced to 2–3 min for deprotection and 5 min for coupling, resulting in cycle
times of 14–19 min (Table 1). Specific results for
each peptide are detailed below.
a
Peptide containing
D-amino acids
GHRP-6 (H-HwAWfK-NH2) is a growth hormone releasing peptide that contains two amino
acids with D-configuration (D-Trp and D-Phe).7
GHRP-6 was synthesized with 2 × 30 sec deprotection times and 2 × 1 min coupling times,
resulting in a total synthesis time of 1.4 hr (Figure
1). D-amino acids were added using the SingleShot delivery feature on the Prelude.
b
Oxytocin
Oxytocin (H-CYIQNCPLG-NH 2) is a component of the prohormone. It is located at the
N-terminal end of the sequence and contains a
disulfide bridge between Cys-1 and Cys-6. Linear oxytocin was synthesized with deprotection
times of 2 × 30 sec and coupling times of 2 × 1
min, resulting in a total synthesis time of 2.1
hr (Figure 2a). It was cyclized on the resin for 2
× 40 min using thallium (III) trifluoroacetate
delivered by the Prelude’s Single-Shot delivery
feature prior to cleavage (Figure 2b). The cyclization time was not optimized.
Figure 2
HPLCs of crude a) linear and b) cyclized oxytocin. The
9-mer linear peptide was synthesized in 2.1 hr with 2 × 30 sec deprotection times and 2 × 1 min coupling times. It was then cyclized for 2 × 40
min for a total synthesis time of 3.4 hr.
continued
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Volume 26, Number 7
TECHNICAL ARTICLE
control of the Symphony and Prelude peptide
synthesizers. G-LHRH was synthesized in 2.3
hr using deprotection times of 2 × 30 sec and
coupling times of 2 × 1 min (Figure 4).
a
C-peptide
Chain A of the human proinsulin C-peptide9
(H-EAEDLQVGQVELGGGPGAGSLQPLALE GLG-OH) (Q is replaced with G)
was synthesized using reaction times of 2 ×
1.5 min and 2 × 2 min for deprotection and
coupling, respectively, in a total synthesis
time of 9 hr (Figure 5).
Figure 3
HPLC of crude 65–74ACP. This 10-mer peptide was synthesized in 2.1 hr with 2 × 30 sec deprotection times and 2 × 1 min coupling times.
Human amylin1–37
Human amylin1–37 (H-KCNTATCATQRLA
NFLVHSSNNFGAILSSTNVGSNTYNH2) is a major component of the amyloid
deposits found in the pancreases of type-II
diabetes patients and contains a disulfide bridge between Cys-2 and Cys-7. 10
Linear hAmylin 1–37 was synthesized with
deprotection times of 2 × 1 min, and acylation times of 2 × 2.5 min, resulting in a
total synthesis time of 10.8 hr (Figure 6a).
Pseudoproline dipeptides were incorporated
into the sequence using the Prelude’s Single-Shot delivery feature. Fmoc-Ala-ThrΨMe,Mepro-OH was coupled at position A8T,
Fmoc-Ser-Ser-Ψ Me,Mepro-OH was coupled
at S 19S, and Fmoc-Leu-Ser-Ψ Me,Mepro-OH
was coupled at position L 27S. The peptide
was then cyclized on the resin in 10 min by
treatment with thallium (III) trifluoroacetate
delivered by the Prelude’s Single-Shot delivery feature, producing the cyclized peptide in
a total synthesis time of 11 hr (Figure 6b).
Figure 4
HPLC of crude G-LHRH. This 10-mer peptide was synthesized in 2.3 hr with 2 × 30 sec deprotection times and 2 × 1 min coupling times.
b
Figure 6
HPLCs of crude a) linear and b) cyclized hAmylin1-37.
The 37-mer linear peptide was synthesized in 10.8 hr with 2 × 1 min
deprotection times and 2 × 2.5 min coupling times. It was then cyclized
in 10 min for a total synthesis time of 11 hr.
a
β-Amyloid1–42
Synthesis of the human β-amyloid1-42 peptide
(H-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA-OH) by conventional SPPS has been reported to be difficult due to on-resin aggregation and the high
hydrophobicity of the C-terminal segment.11
The authors synthesized β-amyloid1–42 with
deprotection times of 2 × 1 min and acylation
times of 1 × 5 min for a total synthesis time of
11.6 hr (Table 1, Figure 7a). The peptide was
then purified by analytical HPLC (Figure 7b).
The HPLC product peaks were slightly broadened, as seen before with this peptide.12
Figure 5
HPLC of crude C-peptide. This 31-mer peptide was
synthesized in 9 hr with 2 × 1.5 min deprotection times and 2 × 2 min
coupling times.
The 65–74 fragment of the
acyl carrier protein
65–74
ACP is a well-known diffi cult sequence used
to test new synthesis protocols and used at Protein
Technologies, Inc. for quality control purposes.
ACP was synthesized with 2 × 30 sec deprotection
times and 2 × 1 min couplings for a total synthesis
time of 2.1 hr. HPLC analysis of the crude peptide
showed a significant prepeak due to incomplete
coupling of the valine (data not shown). However, it was found that this prepeak could be eliminated by coupling the valine in 1:1 dimethylformamide:dimethylsulfoxide (DMF:DMSO) for 2 × 5
min (Figure 3).8
G-LHRH
G-LHRH (H-GHWSYGLRPG-NH2) is a modified
version of the luteinizing hormone releasing hormone, and is also used as a test peptide for quality
b
Conclusion
The authors demonstrated that HCTU is a
highly efficient coupling agent by using it to synthesize seven peptides with deprotection times of
3 min or less and coupling times of 5 min or less.
Combining fast chemistry with peptide synthesizers like the Prelude or Symphony, which have
been optimized for fast fluid deliveries, resulted
in cycle times as short as 14 min. Cost-savings
were realized using HCTU as a less expensive
activator, and minimizing reagent loss with the
Prelude’s Single-Shot delivery feature.
Figure 7
HPLCs of a) crude and b) purified human β-amyloid1-42.
This 42-mer peptide was synthesized in 11.6 hr with 2 × 1 min deprotection times and 1 × 5 min coupling times.
References
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Merrifield, R.B. Solid phase peptide synthesis 1. Synthesis of a tetrapeptide. J. Am. Chem. Soc.1963, 85,
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TECHNICAL ARTICLE
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a) Quibell, M.; Turnell, W.G.; Johnson, T. Preparation and purification of β-amyloid (1–43) via soluble, amide backbone protected intermediates. J.
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Burdik, D.; Soreghan, B.; Kwon, M.; Kosmoksi, J.;
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The authors are with Protein Technologies, Inc., 4675 S.
Coach Dr., Tucson, AZ 85714, U.S.A. ; tel.: 520-629-9626;
fax: 520-629-9806; e-mail: [email protected]. All
tables and figures were adapted from Ref. 6.
6/11/08 10:33:16 AM