A faster solid phase peptide synthesis method using ultrasonic agitation

Article abstract of DOI:10.1016/j.tetlet.2019.05.069

Solid phase-supported synthesis is a widely used strategy in peptide chemistry. A factor which limits the product purity is the individual stages yields. Here, we reported that the use of ultrasonic agitation allows to reduce tenfold the time of synthesis in the Fmoc strategy and improve the purity of the final product. Our method is a promising alternative to traditional synthetic methods and microwave synthesizers.

Full text of DOI:10.1016/j.tetlet.2019.05.069

Accepted Manuscript
The faster peptide synthesis on the solid phase using ultrasonic agitation
Grzegorz Wołczański, Halina Pł óciennik, Marek Lisowski, Piotr Stefanowicz
PII:
S0040-4039(19)30530-1
https://doi.org/10.1016/j.tetlet.2019.05.069
TETL 50841
DOI:
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
3 April 2019
30 May 2019
31 May 2019
Please cite this article as: Wołczański, G., Pł óciennik, H., Lisowski, M., Stefanowicz, P., The faster peptide synthesis
on the solid phase using ultrasonic agitation, Tetrahedron Letters (2019), doi: https://doi.org/10.1016/j.tetlet.
2
019.05.069
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The faster peptide synthesis on the solid phase using ultrasonic agitation.
Grzegorz Wołczański, Halina Płóciennik, Marek Lisowski, Piotr Stefanowicz

1
Tetrahedron Letters
The faster peptide synthesis on the solid phase using ultrasonic agitation
a
a
a
a
Grzegorz Wołczański *, Halina Płóciennik Marek Lisowski and Piotr Stefanowicz
,
,
a
Faculty of Chemistry, University of Wrocław, F. Joliot-Curie 14, 50-383 Wrocław, Poland, e-mail: Grzegorz.wolczanski@chem.uni.wroc.pl
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
S
OLID PHASE
CHEMISTRY. THE FACTOR WHICH LIMITS THE PURITY OF THE PRODUCT IS THE
INDIVIDUAL STAGES YIELDS. HERE WE REPORTED THAT THE USE OF ULTRASONIC
AGITATION ALLOWS TO REDUCE TENFOLD THE TIME OF THE SYNTHESIS IN THE MOC
STRATEGY AND IMPROVE THE PURITY OF THE FINAL PRODUCT. OUR METHOD IS A
PROMISING ALTERNATIVE TO TRADITIONAL SYNTHETIC METHODS AND MICROWAVE
SYNTHESIZERS
-SUPPORTED SYNTHESIS IS A WIDELY USED STRATEGY IN PEPTIDE
,
F
Available online
,
.
Keywords:
peptide synthesis; SPPS; ultrasonication; coupling
reaction conditions
2
009 Elsevier Ltd. All rights reserved.
Introduction
available in most laboratories all over the world, what makes our
method readily accessible for everyone interested.
Similarly to microwaves, ultrasound is regarded as a green
activation technique. However, in contrast to microwaves,
influence of ultrasounds on chemical systems is still poorly
understood. Ultrasonic irradiation (consisting of pressure waves
that are not quantized) is not absorbed by single molecules,
though it is partially converted into heat due to the nonlinear
phenomenon of cavitation [1]. In recent years, interest in the use
of ultrasound in organic synthesis has increased [2]. The use of
ultrasound allows in some cases to reduce drastically the reaction
time and implement of processes not available in other ways, and
may give a better selectivity. The early peak of interest in the use
of ultrasound in chemistry took place in the late 70s and 80s, and
at this time Shimonishi et al. reported the first solid phase peptide
synthesis using ultrasonic waves [3]. Later, Shaw et al.
demonstrated a new SPPS protocol for sporopollenin (the outer,
highly resistant exine of Lycopodium clavatum spores) using a
sonical agitation [4]. Other applications of sonical agitation in
broadly understood peptide chemistry have been also reported,
e.g. attaching of the first residue to the Merrifield resin [5],
cleavage from the resin [6] or synthesis of various kinds of
peptidomimetics in solution [7]. Despite a drastic reduction of the
reaction times, to the best of our knowledge Shimonishi’s and
Shaw’s works still remain the only readily available data on the
direct use of ultrasound in the solid phase peptide synthesis. The
reasons of the lack of interest in the sonical solid phase peptide
synthesis can be explained by focusing of the scientific
Results and discussion
The temperature of water in the ultrasonic bath was maintained
between 25 and 30 °C, and the room temperature was at similar
level. The cavitation phenomenon causes a lot of local
temperature increasing, which provides to heating the whole
solution. Therefore, the temperature of the water bath was
monitored after each cycle of sonication. If the temperature was
higher than 30 °C, we exchanged water in the bath but you can
use any type of a circulator with a cooling instead of doing that.
All of the sonical peptide syntheses experiments were performed
in the sonical cleaning bath obtained from Bandelin (Germany) -
SONOREX DIGITEC DT 103 H with ultrasonic peak output:
5
60 W (it corresponds to 4 times ultrasonic nominal output); and
ultrasonic frequency: 35 kHz.
At the first stage, we optimized the times of both coupling
reaction and Fmoc deprotection at sonical conditions. As we
found
by
spectrophotometric
determination
of
piperidine-dibenzofulvene adduct, the Fmoc group was fully
removed from the Fmoc-Val-Wang polystyrene resin after 1 min
of sonication in 25% piperidine in DMF. The coupling reaction
was checked by coupling of Fmoc-Val-OH (3 eq.) using TBTU
(3 eq.) and DIEA (6 eq.) to the H-Val-Wang resin. The
conditions were optimized for 20 mg of resin with loading of 0.7
mmol/g. A portion of the Fmoc-Val-Wang resin was placed in 2
mL syringe reactor and swelled for 5 min with sonication before
Fmoc deprotection. The syringe reactors were equipped with a
ring made of polyurethane foam enables to float like a bobber
fishing. Concentration of Fmoc-Val-OH in the coupling mixture
was 0.1M. Yield of the reaction was determined
spectrophotometrically by measuring the amount of the
dibenzofulvene adduct released from the Fmoc-Val-Val-Wang
t
community on the chemical aspects of the Fmoc/ Bu strategy
when the two works were published and the later development of
microwave techniques. The results obtained more than 30 years
ago require a reminder and re-validation today. Here, we
t
demonstrate the solid phase peptide synthesis with the Fmoc/ Bu
strategy on commercially available solid supports, accelerated by
the ultrasonic agitation. We used a common ultrasonic bath

2
Tetrahedron Letters
resin. The spectra measured for samples with the coupling time
As difficult sequences, we chose three peptides described
earlier as models during validation of the microwave-assisted
solid phased peptide synthesis [9]. One of them is the TAT
longer than 5 min have the same intensities (after normalization)
in the measured range between 270 and 320 nm, what means that
5
minutes of sonication is enough for a successful coupling.
peptide (Fmoc-GRKKRRQRRRPPQ-NH ), a cationic cell-
2
To determine the influence of sonical agitation on the efficiency
of solid phase peptide synthesis, we carried out a comparative
studies on a series of model peptides, including so called difficult
sequences. During synthesis of longer peptides we increased the
coupling time using sonical agitation to 10 minutes, and the time
of sonication in 25% piperidine in DMF to 2 minutes. Between
subsequent synthetic steps (after coupling or Fmoc deprotection),
the resin was washed successively with DMF. As a standard
protocol we used a procedure, commonly used in our research
team, with the coupling time of 2 hours and Fmoc deprotection
performed by incubation of the resin in 25% piperidine for 25
minutes at room temperature in a syringe reactor placed on the
rotator. In all the experiments, the concentration of Fmoc amino
acid derivatives in the coupling mixture was 0.15-0.20 M.
penetrating 48-60 fragment of the HIV-1 TAT protein [10],
which is difficult to synthesize due to the multiple arginine
residues. The peptide was cleaved from the resin with the
N-terminal Fmoc group as a chromophore for HPLC analysis
with UV detection at 210 nm. During optimization of the TAT
peptide synthesis we found that longer time of the coupling
reaction at sonical conditions is needed – 20 minutes of
agitation. This sequence was the most difficult of our models.
Using both an H-Rink amide ChemMatrix® resin and a poly-
styrene based Fmoc-Rink-MBHA resin we obtained a much
better purity of the TAT peptide synthesized under sonical
conditions than using the classical approach (Figure 1).
0
,5
,45
,4
One of the simplest model peptides chosen for comparative
studies was Met-enkephalin (H-YGGFM-OH). This choice was
made to determine if the sonication conditions can cause
oxidation of Met to Met(O), as it is known that during a
sonication free radicals may be formed (especially at higher
frequencies) [1,2,8]. Both syntheses were performed on a
Fmoc-Met-Wang polystyrene resin with loading of 0.7 mmol/g.
Met-enkephalin synthesized ultrasonically does not differ from
that obtained in the classical way. In both cases we found traces
of the oxidized form in the ESI-MS spectra, but the oxidized
compound peak intensities in the cases of sonical and classical
synthesis are only 2.95 and 2.86%, respectively, of those of the
desired product which was derived from the absolute intensities
of the monoisotopic peaks. Thus, the sonication does not
influence the Met residue oxidation in the peptide. Additionally,
at the sonical conditions we obtained the product with a better
purity (64%) than in the case of classical synthesis (51%).
0
0
0
0
0
0
,35
0,3
,25
0,2
,15
0,1
,05
0
0
5
10
15
20
t [min.]
25
30
35
40
1
0,9
0,8
0,7
0,6
0,5
0,4
,3
0,2
0
0,1
0
0
5
10
15
20
t [min.]
25
30
35
40
Figure 1. HPLC chromatograms (detection at 210 nm) of crude
Fmoc-TAT-NH peptide synthesized classically (top) and
sonically (down) on H-Rink amide ChemMatrix® resin.
2
.
Table 1. Comparison between the sonical and the classical solid phase peptide synthesis.
Peptide
Method*
Classical:
Sonical
Solid support
Time*[min.]
600
Purity**[%]
51,1
Met-enkephalin
Met-enkephalin
Fmoc-UB33-43-OCam
Fmoc-UB33-43-OCam
Fmoc-Met-Wang, polystyrene, 0.72 mmol/g
Fmoc-Met-Wang, polystyrene, 0.72 mmol/g
H-Rink ChemMatrix®, 0.4-0.6 mmol/g
H-Rink ChemMatrix®, 0.4-0.6 mmol/g
H-Rink ChemMatrix®, 0.4-0.6 mmol/g
H-Rink ChemMatrix®, 0.4-0.6 mmol/g
Fmoc-Rink MBHA, polystyrene, 0.68 mmol/g
Fmoc-Rink MBHA, polystyrene, 0.68 mmol/g
H-Rink ChemMatrix®, 0.4-0.6 mmol/g
H-Rink ChemMatrix®, 0.4-0.6 mmol/g
Fmoc-Rink MBHA, polystyrene, 0.68 mmol/g
Fmoc-Rink MBHA, polystyrene, 0.68 mmol/g
H-Rink ChemMatrix®, 0.4-0.6 mmol/g
H-Rink ChemMatrix®, 0.4-0.6 mmol/g
Fmoc-Rink MBHA, polystyrene, 0.68 mmol/g
Fmoc-Rink MBHA, polystyrene, 0.68 mmol/g
48
64,4
71,3
Classical
Sonical
1860
237
89,9
Fmoc-TAT48-60-NH
Fmoc-TAT48-60-NH
Fmoc-TAT48-60-NH
Fmoc-TAT48-60-NH
2
2
2
2
Classical
Sonical
1920
284
24,2
79,4
Classical
Sonical
1920
284
21,8
60,6
Ac-B2m58-69-NH
Ac-B2m58-69-NH
Ac-B2m58-69-NH
Ac-B2m58-69-NH
2
2
2
2
Classical
Sonical
1860
204
69,7
82,1
Classical
Sonical
1860
204
39,1
51,2
H-ACP65-74-NH
H-ACP65-74-NH
H-ACP65-74-NH
H-ACP65-74-NH
2
2
2
2
Classical
Sonical
1500
120
64,4
74,6
50,7
Classical
Sonical
1500
120
56,8
t
β-endorphin (31 amino acids)
Sonical
Fmoc-Glu( Bu)-Wang, polystyrene, 0.64 mmol/g
360
47,8
a
In all cases the TBTU/DIEA coupling method was used. Classical and sonical method are described in the supplementary materials. Under sonical conditions
the syringe with the resin was sonicated in a ultrasonic cleaning bath; in classical conditions the syringe was shaked using rotary shaker.
b
The total time of synthesis including coupling reactions and Fmoc-deprotections steps, without washing and peptides cleavage from the resin
.

3
The acetylated B2m peptide derived from amyloidogenic
human β-2-microglubulin (fragment 58-69) [11] was chosen
as a second model. This peptide was synthesized with the
containing 30 amino acid residues may be synthesized
manually in one workday. Nowadays, we do not have
automatic peptide synthesizers based on the ultrasound
technology. However, if one reviews carefully the
development of microwave peptide synthesis in recent years,
it can be expected that the automation of the sonical
synthesis of peptides will lead in the future to equally drastic
reduction of synthesis times and increase of the purity of
raw products.
8
2.1% (ChemMatrix) and 51.2% (MBHA) purity using a
sonical agitation in comparison to 69.7% and 39.1%,
respectively, using classical SPPS. We also synthesized the
acyl
carrier
protein
(ACP)
fragment
65-74
(
H-VQAAIDYING-NH ), which is widely used as a
2
standard to demonstrate the solid phase peptide efficiency
9,12]. The ACP peptide was synthesized with a better
[
purity and about ten times faster using a sonical agitation
than with the classical approach using both types of resins,
H-Rink ChemMatrix® and Fmoc-Rink MBHA. Results of
the syntheses are summarized in Table 1.
To demonstrate general applicability of the sonical agitation
in solid phase peptide synthesis, we synthesized
caboxamidomethyl (Cam) ester of the ubiquitin fragment
UB(33-43) cleaved from the resin with N-terminal Fmoc
group. The peptide Cam esters may be used potentially in
the synthesis of larger peptides through peptide fragments
enzymatic ligation reactions and head-to-tail cyclizations
[13]. The Cam ester was introduced by coupling of
bromoacetic acid to the H-Rink ChemMatrix resin using a
known method [14]. Then, 3 equiv. Fmoc-Leu-OH and 3
equiv. DIEA were dissolved in DMF and incubated with the
resin overnight or sonicated for 15 minutes. However, the
synthesis times given in Table 1 include only the time of a
peptide synthesis using the TBTU/DIEA method (in this
case 9 residues instead of 10). The UB(33-43) sequence
contains two Pro residues, which may induce turns
hindering attachment of contiguous residues. It makes this
synthesis difficult. Although both syntheses were performed
on a ChemMatrix® resin dedicated for difficult sequences,
the purity of the peptide synthesized sonically was almost
twice better than of that obtained with the classical
approach.
Figure 3. Direct comparison of the coupling reaction kinetics
using a sonical agitation (orange) and a classical approach
(blue).
Direct comparison of the coupling efficiency using standard
mixing and ultrasound agitation was carried out on example
of one of the TAT peptide synthesis steps. The choice was
dictated by difficulties during synthesis of this peptide. The
coupling of Fmoc-Arg(Pbf)-OH to the H-Arg(Pbf)-Pro-Pro-
Gln(Trt)-Rink MBHA resin was chosen as a model coupling
reaction. 300 mg of the H-Arg(Pbf)-Pro-Pro-Gln(Trt)-Rink
MBHA resin with loading of 0.68 mmol/g were prepared
using a sonical agitation. After drying, the resin was splitted
into 20 mg portions. Then, the samples were incubated with
a coupling mixture (3 equiv. Fmoc-Xaa-OH – 0.08M, 3
equiv. TBTU, 6 equiv. DIEA in 500L DMF) for various
time periods using the rotator as a shaker or sonical
agitation. After a coupling step, the resin was treated
immediately with 25% piperidine in DMF to quench the
reaction and remove the Fmoc group. Then, the
peptidyl-resin
was
derivatized
using
§
2
,4-dinitrofluorobenzen . After cleavage from the resin with
a subsequent evaporation under nitrogen and lyophilization,
the samples were dissolved in 10mL of acetonitrile/water
(50:50) and analysed using HPLC with the UV detection at
Figure 2. HPLC chromatogram and ESI-MS spectrum of crude
β-endorphin synthesized using the sonical solid phase peptide
synthesis method.
360 nm. The time dependence of coupling yields shows
directly that the sonical agitation accelerates the reaction
(Figure 3). However, the coupling was not quantitative even
after 24 hours of coupling at room temperature and plateau
of conversion obtained using sonical agitation is lower than
for the classical approach. It may be explained by two
factors. First, the competition between the coupling reaction
and formation of lactam from the activated arginine
derivative surely have a significant contribution to a lower
“sonical” yield. Second, sonication may increase both
reactions rates, causing a faster reduction of the active
arginine derivative concentration. This disadvantage may be
omitted by coupling repetitions for difficult amino acid
β-Endorphin was synthesized sonically as an example of
longer peptides. The synthesis was performed on a
t
Fmoc-Glu( But)-Wang polystyrene resin with loading of
0
.65 mmol/g, using 3- fold excess of reagents during each
coupling step. The purity of a crude peptide (synthesized
using 10 minutes of sonical agitation per each residue
coupling reaction) is around 50% (Figure 2). This peptide
was obtained using our sonical manual solid phase peptide
synthesis method in about 8 hours, showing that peptides

4
Tetrahedron
derivatives. Nonetheless, we obtained good purities of the
of the reaction time and improvement of the product purity
in comparison to the standard procedure. Our method is
easily accessible to all those interested without the need to
purchase an additional equipment. We showed also that a
sonication even at the elevated temperature (70°C) is free of
racemization. The sonical solid phase peptide synthesis
began to be used in our team as a routine method of
synthesis of peptides and peptide conjugates (examples are
available in Supplementary Material, section 6. Additional
experimental data). Its main advantage in relation to the
microwave synthesis is the ability to run many parallel
syntheses using a simpler source of energy (ultrasonic bath).
TAT peptides synthesized sonically. However, during the
synthesis of model peptides we used twice more
concentrated solutions of coupling reagents which is easier
to do with a larger portion of a resin.
Conflicts of interest
There are no conflicts to declare.
References and notes
§
2,4-dinitrophenyl (DNP) derivatives of peptides are commonly
used in HPLC analysis with UV detection.
Figure 4. The investigation of racemization. Comparison of
1
R. B. Nasir Baig, R. S. Varma, Chem. Soc. Rev. 2012, 41, 1559;
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1
significant H NMR spectra of models peptides synthesized
2
manually using the classical approach at room temperature vs
sonical method at elevated temperature. Chemical shifts of His
and Cys α-protons and methylene group of Acm (left panels),
and γ-methyl protons of Val (right panels) show that sonication
at 70°C does not cause racemization.
Cravotto, P. Cintas, Chem. Eur. J., 2007, 13, 1902; C.
Stavarache, A.-M. Poscan, M. Vinatour, T.J. Mason,
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2
D.H. Bremner, Ultrasonics Sonochemistry, 1994, 1, 119; T.J.
Mason, Chem. Soc. Rev., 1997, 26, 443; G.J. Price, Ultrasonics
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One of the most important factors describing the efficiency
of solid phase peptide synthesis is lack of racemization
during coupling reactions, leading to only one desired
diastereoisomer of the peptide. We investigated the
influence of sonical agitation at room and higher
temperatures on racemization. The racemization of His and
Cys, the most susceptible residues [15], was tested during
dipeptides H-Cys(Acm)-Val-OH and H-His(Trt)-Val-OH
Ultrasonics Sonochemistry, 2017, 35
, 1; B. Banerjee,
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3
S. Takahashi, Y. Shimonishi, Chemistry Letters, 1974, 51; S.
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syntheses. The peptides were synthesized on
a
4
5
6
7
R. Adamson, S. Gregson, G. Shaw, Int. J. Peptide Protein Res.,
1
983, 22, 560
Fmoc-Val-Wang polystyrene resin. Both diasteroisomers
were synthesized using the classical approach as reference
samples. The temperature of water in the ultrasonic bath was
stabilized at the desired level (30, 50 or 70°C). Then, a
freshly prepared coupling mixture was mixed with the
portion of the H-Val-Wang resin in a syringe reactor and
placed immediately in the ultrasonic bath. Peptides were
partially purified using Sep-Pak columns. The racemization
M.V. Anuradha, B. Ravindranath, Tetrahedron, 1995, 51
671
,
,
,
5
M.V. Anuradha, B. Ravindranath, Tetrahedron, 1995, 51
5675
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1
123; R.M. Srivastava, R.A.W. Neves Filho, C.A. da Silva, A.J.
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Vasconcelos, A.C. Beltran Rodrigues, E. L. Bastos, H.A.
1
was examined using H NMR spectroscopy, and the
assignment of signals was based on information from the
COSY spectra. We did not find any evidence of
racemization even at 70°C, for both Cys and His residues
Stefani, ChemistrySelect, 2016, 6, 1287
(Figure 4). As in the microwave assisted solid phase peptide
8
9
P. Riesz, D. Berdahl, C.L. Christman, Environmental Health
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during coupling reactions using various activation methods
needs more detailed studies.
2
001, 29, 167
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0 Ch. Bechara, S. Sagan, FEBS Letters, 2013, 587, 1693
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2
003, 325, 249

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6
Tetrahedron
Sonication accelerates couplings in solid phase

peptide synthesis


Sonical peptide synthesis gives products with
higher purity than classical approach
Sonication do not cause the racemization of
sensible residues (Cys, His)