High-efficiency solid phase peptide synthesis (he -Spps)

Article abstract of DOI:10.1021/ol4036825

A series of improvements to the standard solid phase peptide synthesis (SPPS) process allowing for significant gains in product purity along with only a 4 min standard cycle time and a 90% reduction in total waste produced is reported. For example, syntheses of the well-known ^65-74acyl carrier protein (ACP) and ^1-42β-amyloid peptides were accomplished with 93 and 72% purity (UPLC-MS) in only 44 and 229 min, respectively.

Full text of DOI:10.1021/ol4036825

Letter
pubs.acs.org/OrgLett
High-Efficiency Solid Phase Peptide Synthesis (HE-SPPS)
Jonathan M. Collins,* Keith A. Porter, Sandeep K. Singh, and Grace S. Vanier
CEM Corporation, 3100 Smith Farm Road, Matthews, North Carolina 28104, United States
*
S Supporting Information
ABSTRACT: A series of improvements to the standard solid
phase peptide synthesis (SPPS) process allowing for significant
gains in product purity along with only a 4 min standard cycle
time and a 90% reduction in total waste produced is reported.
6
5−74
For example, syntheses of the well-known
protein (ACP) and
acyl carrier
β-amyloid peptides were accomplished
with 93 and 72% purity (UPLC−MS) in only 44 and 229 min, respectively.
1
−42
olid phase peptide synthesis (SPPS) has proven invaluable
performance liquid chromatography (UPLC) with extended
gradient times coupled to a mass spectrometer.
The selected peptides were first synthesized using conven-
S
for the successful construction of a diverse array of natural
1
and modified peptide sequences. Fmoc chemistry is the
dominant method in use, which features the base labile α-
amino Fmoc protecting group along with acid labile side chain
tional room temperature methods at a 0.1 mmol scale in order
9
to establish a baseline purity level. Common polystyrene resins
2
10
1−42
protection and peptide-resin linkage.
were used for each synthesis. The one exception was
β-
Although SPPS is instrumental for peptide production, it is
not without challenges. The process is complicated by the well
documented occurrence of aggregation thought to originate
amyloid for which a 0.16 mmol/g loading PAL-PEG-PS resin
was used because of the longer peptide length. The use of
microwave for the TFA cleavage step in Fmoc SPPS
demonstrated the ability to shorten this step to 30 min at 38
°C as confirmed by our results. Therefore, for all subsequent
experiments the MW cleavage method was used in order to
save time.
3
primarily from intermolecular hydrogen bonding. This creates
inaccessibility of the reactive end of the chain thereby leading to
more difficult deblocking or acylation steps. Additionally, steric
effects can lower reaction rates when the terminal amine is
secondary (i.e., proline, N-substituted amino acids) or when
bulky side chain protecting groups are present. An incomplete
reaction at any step in the synthesis leads to deletion sequences,
which can be extremely hard to separate from the desired target
sequence. At the same time incomplete removal of previously
used peptide reagents can lead to impurities such as
substitutions and addition sequences by undesirably participat-
ing in a subsequent step. As a result SPPS is typically performed
with long reaction times, large excesses of reagents, and many
Even with long synthesis times, relatively low purity was
1−42
obtained for the JR 10-mer, ABRF 1992, thymosin, and
β-
amyloid peptides (Table 1, entry 3−5, 9−10, 14). The ABC 20-
mer was prepared in good purity using the standard
conventional times (Table 1, entry 6, 7) but failed to give
any target using shorter reaction times (Table 1, entry 8). For
the thymosin peptide the purity increased from 37 to 47%
(
Table 1, entry 11) with the combined use of the more
11
aggressive HCTU activation and switching to a hydrophilic
resin (0.52 mmol/g loading Rink Amide ChemMatrix) under
standard long reaction times. Attempts to reduce the
deprotection and coupling times under the same conditions,
even with the added benefit of 10 equiv of the coupling
reagents failed to improve the crude purity from 36% (Table 1,
entry 12).
4
repetitive washing steps between each step. For example, a 100
mg scale production of a peptide 20 amino acids in length can
take 24 h to complete and produce several liters of chemical
waste. Efforts to make SPPS maximally efficient therefore must
address the efficiency of both reaction steps in each cycle and
the overall washing process.
Microwave irradiation was then applied to both the
deprotection and coupling steps using previously reported
Our goal was to develop a high-efficiency (HE)-SPPS
method that could be applied to any sequence with the goal of
dramatically increasing the efficiency of the SPPS process.
Microwave (MW) irradiation has been previously applied to
SPPS and we explored this tool for the deprotection, coupling,
12−14
conditions.
For activation we chose to use the well-known
carbodiimide (DIC) based techniques with additives such as
HOBt, HOAt, and Oxyma. For the present study we used
15
5
−8
Oxyma as our additive of choice, as hydroxybenzotriazole
and resin cleavage steps.
We identified a group of 6 known
derivatives such as HOBt and HOAt have been classified as
peptides from 10 to 42 amino acid length (Table 1) that
together contain a range of synthesis challenges and common
side reactions. Therefore, any method improvements that
would hold constant across all 6 of these peptides would have
strong support for general use in SPPS. In order to fully resolve
any impurities that may be present, we utilized ultra-high-
16
Class 1c explosives. Often, the use of DIC/(HOBt or
Oxyma) activation is associated with an initial preactivation
Received: December 19, 2013
©
XXXX American Chemical Society
A
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Organic Letters
Letter
Table 1. Baseline Conventional Synthesis Results
a
f
entry
peptide
deprotection time (min)
activation
coupling time (min)
UPLC purity (%)
crude yield (%)
e
e
1
2
3
4
5
6
7
8
9
65−74ACP
65−74ACP
JR 10-mer
JR 10-mer
5 + 10
5 + 10
5 + 10
0.5 + 3
5 + 10
5 + 10
5 + 10
0.5 + 3
5 + 10
5 + 10
5 + 10
0.5 + 3
0.5 + 3
5 + 10
0.5 + 3
DIC/Oxyma
HBTU/DIEA
DIC/Oxyma
HCTU/DIEA
DIC/Oxyma
DIC/Oxyma
HBTU/DIEA
DIC/Oxyma
DIC/Oxyma
HCTU/DIEA
HCTU/DIEA
HCTU/DIEA
HCTU/DIEA
DIC/Oxyma
HCTU/DIEA
60
30
60
5
38 (39 )
94 (96 )
b
90
24
e
e
42 (42 )
80 (80 )
c
35
56
58
82
ABRF 1992
ABC 20-mer
ABC 20-mer
ABC 20-mer
thymosin
60
60
30
5
e
e
70 (70 )
94 (96 )
e
e
68 (70 )
98 (94 )
<5
−
e
e
60
30
30
5
35 (37 )
79 (81 )
10
11
12
13
14
15
thymosin
34
47
36
25
56
14
70
65
56
68
85
72
c
thymosin
c
d
thymosin
thymosin
5
1
−42β-amyloid
60
1−42
β-amyloid
5
a
Peptide Sequences: 65−74ACP: VQAAIDYING, JR 10-mer: WFTTLISTIM-NH , ABRF 1992: GVRGDKGNPGWPGAPY, ABC 20-mer:
2
1−42
VYWTSPFMKLIHEQCNRADG-NH , thymosin: SDAAVDTSSEITTKDLKEKKEVVEEAEN-NH , β-Amyloid: DAEFRHDSGYEVHHQKLV-
2
2
b
c
FFAEDVGSNKGAIIGLMVGGVVIA-NH . Resin = 0.44 mmol/g of Gly-Wang ChemMatrix. Resin = 0.52 mmol/g of Rink Amide ChemMatrix.
2
d
e
f
1
0 equiv used. MW resin cleavage method. Crude yield corresponds to lyophilized product without any purification.
step because of formation of the initial O-acylisourea
proceeding faster in a nonpolar solvent such as DCM, while
the subsequent acylation step proceeds faster in a more polar
medium such as DMF or NMP. We felt that microwave
irradiation would allow both processes to occur rapidly in a
single solvent, thereby simplifying the overall process.
feedback, as aggressive temperature ramping could lead to
17
undesirable temperature overshooting. We used an internal
fiber optic probe (Figure S4, Supporting Information) during
MW irradiation and found that we could reproducibly reach a
control temperature of 90 °C with maximum temperature of 92
°C in only 20 s during both the deprotection and coupling
steps. Through optimization we showed that equal or higher
purity could be obtained for each peptide in the set using a
single 1 min deprotection and 2 min coupling.
Initial attempts to use microwave irradiation proved
encouraging, as shown in Table 2. Each of the six peptides
Table 2. Baseline MW Synthesis Results
Next we focused on optimization of the washing that is
required during SPPS. Since both DMF and NMP are used
routinely for washing, we investigated both. It was noted that
the resin maintained a residual temperature of 50 °C or higher
immediately after draining each reaction. We reasoned that this
could provide a benefit to the washing process as a higher
diffusion was more likely to occur at elevated temperature. To
our satisfaction we found that 3 washes of only 1, 2, and 3 mL
with NMP and 2, 2, and 3 mL with DMF for standard
polystyrene resins were required as confirmed by UPLC−MS
analysis. The one exception to this was the low loaded PAL-
PEG-PS resin used for 1 β-amyloid, which required 4 × 4 mL
washing with DMF after the deprotection step because of its
higher mass from significantly lower substitution.
b
deprotection
UPLC purity
(%)
crude yield
(%)
a
entry
peptide
reagent
65−74ACP
1
2
3
4
5
6
A
C
B
A
A
A
91
60
79
71
58
67
99
74
96
91
87
87
JR 10-mer
ABRF 1992
ABC 20-mer
thymosin
1
−42β-Amyloid
a
A = 20% piperidine w/0.1 M Oxyma; B = 10% piperazine; C = 20%
b
piperidine. Crude yield corresponds to lyophilized product without
−42
any purification.
Postcoupling washes are typically done to prevent residual
activated amino acid from undesirably coupling during the
subsequent deblocking step. A closer analysis reveals inherent
protection in the SPPS process against this occurring. First, the
residual activated amino acid remaining after vessel draining
will be very small compared to the large excess of deblocking
base added. The deblocking base, as a secondary amine, should
react rapidly with the residual activated amino acid present in a
solution phase process providing complete destruction of the
investigated were synthesized in the same or higher purity than
the optimized conventional conditions with an average cycle
time of around 30 min. This is in agreement with previous
reports that show microwave irradiation can allow for not only
fast deprotection and coupling reactions, but in many cases also
higher purity synthesis results. We then began a series of
sequential optimization steps to achieve our goal of increasing
the efficiency of SPPS. First, we focused on optimizing the MW
conditions used for the deprotection and coupling steps with
the goal of performing both steps as rapidly as possible. We
recognized that significant time could be saved by performing
the deprotection step in a single stage as opposed to the 2-stage
deprotection used as standard practice. Additionally, we also
reasoned that both the deprotection and coupling steps could
be performed significantly faster by more aggressive application
of microwave power while also maintaining a higher temper-
ature. This approach requires precise and rapid temperature
14
1
8,19
ester.
Initially almost all peptide chains should be blocked
with Fmoc from the previous coupling reaction further limiting
the ability of the activated ester to couple in a kinetically slower
solid phase reaction. We therefore reasoned that washing could
possibly be eliminated after the coupling step. This was indeed
confirmed with every peptide tested in the data set and
eliminated a major portion of the synthesis process and
chemical waste.
B
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Organic Letters
Letter
Piperidine can be difficult to obtain because of its
classification as a controlled substance. As an alternative,
piperazine has been previously investigated both convention-
ally and in MW SPPS. However, piperazine has an
approximate maximum concentration in DMF or NMP of
the three Asp residues in thymosin under optimized conditions
was only 0.91%. However, the ABC 20-mer containing a C-
terminal Asp-Gly segment had a % D Asp content of 4.94%.
Adding 0.1 M Oxyma to the 10% piperazine cocktail reduced
this to 3.57%, while use of the Fmoc-Asp(OtBu)-(Dmb)Gly-
OH derivative led to only 0.36% D Asp (Table S1 (SI)). This
demonstrates that aspartic acid is in general protected during
the optimized deblocking conditions, but less aggressive
deblocking conditions should be considered for the Asp-Gly
case.
2
0
12
6%. We reasoned that a new solvent system that increases
piperazine concentration without negatively affecting solubility
of the peptide-resin complex would be useful. We found that a
mixture of EtOH and NMP (10:90) allowed dissolution of
piperazine at 10%. Pleasingly this new mixture increased the
effectiveness of Fmoc deblocking to levels that matched 20%
piperidine.
Finally, in the synthesis of the ABRF 1992 peptide, we found
that a single arginine coupling at 25 min room temperature
followed by 2 min with MW irradiation at 75 °C was as efficient
We next focused on improving the coupling methods of
cysteine and histidine because of their epimerization sensitivity
13
as the previously described double coupling method. This
allows for both a 50% reduction in the expensive Fmoc-
Arg(Pbf)-OH derivative and a slight time savings. Taken
together these optimizations along with fast automation
allowed for significant improvements as shown in Table 5.
The purity increased for all peptides with a standard cycle time
of slightly longer than 4 min and a major waste reduction.
21
and longer coupling time. We investigated whether lack of a
hindered amine present with the in situ carbodiimide approach
would allow cysteine and histidine to be coupled under more
aggressive temperature conditions using the ABC 20-mer
12
peptide with known sensitivity to these side reactions. Using
the more aggressive two minute coupling method at 90 °C for
cysteine led to only 1.04% D-Cys as shown in Table 3.
Table 5. Fully Optimized SPPS Methods
Table 3. Epimerization of Fmoc-Cys(Trt)-OH
crude
a
total
waste
(mL)
entry ramp (min) hold (min) max temp (°C)
activation
% D
UPLC
purity (%)
yield
synthesis b
time (min)
entry
peptide
(%)
1
2
3
4
5
6
7
NA
NA
2
30
60
4
rt
HBTU/DIEA
DIC/Oxyma
DIC/Oxyma
HBTU/DIEA
DIC/Oxyma
DIC/HOBt
DIC/HOAt
1.38
0.34
1.14
16.7
1.04
3.73
2.01
1
2
3
4
5
6
65−74ACP
93
67
82
73
61
72
98
72
97
95
95
87
44
154
170
272
340
468
1019
rt
JR 10-mer
ABRF 1992
ABC 20-mer
49
50
90
90
90
90
97
0.5
0.5
0.5
0.5
1.5
1.5
1.5
1.5
127
131
229
thymosin
1
−42β-Amyloid
a
Crude yield corresponds to lyophilized product without any
b
purification. Does not include a 5 min resin swell time.
Importantly, this shows that the in situ carbodiimide activation
method is uniquely suitable for cysteine under elevated
temperatures, as lack of a hindered amine limits epimerization
from direct enolization. Since histidine displayed significant
epimerization, we then attempted to utilize a recently
introduced side chain protecting group, Mbom, that protects
the π-nitrogen on the imidazole side chain. An optimized 3
min coupling step at 80 °C led to only 1.43% D-His (Table 4,
entry 5).
Aspartimide formation can accumulate during each depro-
tection step in Fmoc chemistry leading to α,β-aspartyl peptides
and α- and β-piperidides (Scheme S1 (SI)). This base catalyzed
side reaction typically occurs in Asp-X sequences [X = Gly, Asn,
Ser, Thr] with Asp-Gly being the worst. The % D content of
An optimized process for SPPS is presented that allows for
complete cycle times of approximately 4 min along with a
significant reduction (approximately 90%) in total chemical
waste compared to traditional methods. Key features of HE-
SPPS are newly developed microwave enhanced reaction
conditions, a novel low-cost deblocking cocktail, low-cost in
situ carbodiimide based activation, optimized washing, and
ultrafast automation. This new method should have a
significant impact on the assembly of difficult peptides, high
throughput production, and larger scale peptide production as
it offers improvements in synthesis efficiency, speed, and
chemical usage.
22
Table 4. Epimerization of Fmoc-His(Trt)-OH and Fmoc-
His(π-Mbom)-H
ASSOCIATED CONTENT
Supporting Information
■
*
S
entry ramp (min) hold (min) max temp (°C)
activation
% D
Experimental procedure, UPLC−MS chromatograms and data,
and C.A.T. epimerization analysis. This material is available free
of charge via the Internet at http://pubs.acs.org.
1
2
3
4
5
6
7
8
9
NA
NA
2
30
60
4
rt
HBTU/DIEA
DIC/Oxyma
DIC/Oxyma
HBTU/DIEA
DIC/Oxyma
DIC/Oxyma
DIC/Oxyma
DIC/HOBt
DIC/HOAt
1.79
1.42
3.24
25.6
1.43
23.2
2.96
15.3
18.9
rt
50
90
80
90
90
90
90
0.5
0.5
0.5
0.5
0.5
0.5
1.5
2.5
1.5
1.5
1.5
1.5
AUTHOR INFORMATION
Corresponding Author
■
*
a
a
E-mail: jon.collins@cem.com.
Notes
The authors declare the following competing financial
interest(s): Authors are employees of CEM Corporation,
which manufactures the peptide synthesizer used in this study.
a
Fmoc-His(π-Mbom) used for entries 5 and 7.
C
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Organic Letters
Letter
ACKNOWLEDGMENTS
■
Joseph Lambert, David Herman, Eric Williamson, Tony
Baranski, Joshua Foster, and Alicia Stell of CEM Corporation
are acknowledged for support with the rapid automation
development. Dedicated to the memory of Stacey Palasek, a
gifted scientist and respected colleague at CEM Corporation,
who would have undoubtedly contributed to this work.
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■
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(
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(
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a
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7
5 °C, first coupling; 5 min at 75 °C, second coupling) for the results
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dx.doi.org/10.1021/ol4036825 | Org. Lett. XXXX, XXX, XXX−XXX