N,O-Bis(trimethylsilyl)acetamide/N-hydroxysuccinimide ester (BSA/NHS) as coupling agents for dipeptide synthesis

Article abstract of DOI:10.1016/j.cclet.2015.11.012

A method using N,O-bis(trimethylsilyl)acetamide/N-hydroxysuccinimide ester (BSA/NHS) as coupling agents for dipeptide synthesis is descried. The coupling reaction between N-hydroxysuccinimide (NHS) esters and amines could be performed under mild conditions with N,O-bis(trimethylsilyl)acetamide (BSA) as coupling reagent and no additional acid/base is required. All byproducts and excessive reactants are water soluble or hydrolysable and easy to eliminate through water-washing at the purification stage. Moreover, all the reactants are inexpensive and widely used in conventional drug production.

Full text of DOI:10.1016/j.cclet.2015.11.012

G Model
CCLET 3489 1–4
Chinese Chemical Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
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Original article
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N,O-bis(trimethylsilyl)acetamide/N-hydroxysuccinimide ester
(BSA/NHS) as coupling agents for dipeptide synthesis
*
Q1 Ye Huang, Wen-Hua Feng
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Department of New Drug Research and Development, Institute of Materia Medica & Peking Union Medical College, Chinese Academy of Medical Sciences,
Beijing 100050, China
A R T I C L E I N F O
A B S T R A C T
Article history:
A method using N,O-bis(trimethylsilyl)acetamide/N-hydroxysuccinimide ester (BSA/NHS) as coupling
agents for dipeptide synthesis is descried. The coupling reaction between N-hydroxysuccinimide (NHS)
esters and amines could be performed under mild conditions with N,O-bis(trimethylsilyl)acetamide
(BSA) as coupling reagent and no additional acid/base is required. All byproducts and excessive reactants
are water soluble or hydrolysable and easy to eliminate through water-washing at the purification stage.
Moreover, all the reactants are inexpensive and widely used in conventional drug production.
ß 2015 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Received 29 September 2015
Received in revised form 26 October 2015
Accepted 5 November 2015
Available online xxx
Keywords:
Dipeptide synthesis
Solution-phase
N,O-bis(trimethylsilyl)acetamide
N-hydroxysuccinimide ester
Water washing
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1. Introduction
decades of optimization, shortcomings of solid-phase synthesis 31
still limited its applications in commercial-scale manufacture. 32
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Since du Vigneaud published the first solution-phase synthesis
of oxytocin in 1953 [1], it has been found that peptides participate
in various biocatalytic processes [2]. With assistance of recent
advances in synthetic [3] and drug delivery technologies [4],
peptide-based anti-cancer [5], anti-diabetic [6], anti-microbial [7],
anti-fungal [8] drugs and intrinsic hormonal analogues have been
discovered [9]. Therefore, it is imperative to develop large scale
synthetic approaches for the production of complex peptides.
Traditional solution-phase methodologies are the most
widely used approaches for industrial production of approved
peptide-based pharmaceuticals. In general, they have no scale
limitations, and each step can be monitored precisely. However,
significant shortcomings of solution-phase methodologies, such
as consumption of a large amount of organic solvents, tedious
protection/de-protection steps and difficult purification process-
es, still hamper the development of scale-up routes of peptide
syntheses. Solid-phase methodologies, in which the peptides of
relatively complex sequences could be synthesized using
automated and rapid synthesis/workup procedures, were pio-
neered by Bruce Merrifield to overcome the problems in
traditional solution-phase methodologies [10]. However, after
For example, lack of scalability, inadequate in-process controls 33
and low purity of the final products still need improvement 34
[11]. Alternative approaches consisting of soluble polymer- 35
tagged liquid-phase reactions have also been developed to 36
ensure both high reaction rate and real time reaction monitoring 37
[12]. In this method, peptide syntheses could be performed in the 38
solution phase but work-up is accomplished in a manner similar 39
to solid-phase synthesis where most of the excess organic 40
reagents and activated amino acids are removed by simple 41
solidification and filtration procedures. However, the high-cost 42
of soluble polymers, the time-consuming process of solidifica- 43
tion/crystallization in each coupling cycle and the inherent scale 44
limitation as observed in solid-phase methodologies represent 45
serious challenges for large scale synthesis of peptides.
46
Here we describe a simplified dipeptide synthesis strategy 47
using the N-hydroxysuccinimide (NHS) ester of amino acids and a 48
silylation agent N,O-bis(trimethylsilyl)acetamide (BSA) as
a
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coupling agent (Fig. 1) The coupling reactions between NHS esters 50
and amines occur efficiently under mild conditions with BSA only 51
and no additional acid/base is required. Besides, excessive BSA and 52
other byproducts are water soluble or hydrolysable [13] and easy 53
to remove by simple water-washing at the purification stage. 54
Consequently, less racemization, fewer units of operation, and 55
simpler purification processes can be achieved compared to other 56
*
Corresponding author.
solution-phase methodologies.
57
E-mail address: fwh@imm.ac.cn (W.-H. Feng).
http://dx.doi.org/10.1016/j.cclet.2015.11.012
1001-8417/ß 2015 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Y. Huang, W.-H. Feng, N,O-bis(trimethylsilyl)acetamide/N-hydroxysuccinimide ester (BSA/NHS) as
coupling agents for dipeptide synthesis, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.11.012

G Model
CCLET 3489 1–4
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Y. Huang, W.-H. Feng / Chinese Chemical Letters xxx (2015) xxx–xxx
Table 1
Synthesis of N-Boc protected dipeptides via the BSA/NHS methoda.
Entry Product
Time (h) Solvent BSA/AA/NHS esterb Yield (%)c
1
Boc-Phe-Pro-OH 48
Boc-Phe-Pro-OH
DCM
DCM
DCM
DCM
THF
ꢀ/1.1/1
Trace
94.3
64.4
58.7
44.5
78.8
63.2
83.2
82.1
81.5
91.1
85.4
2
8
2.2/1.1/1
4.4/1.1/1
1.1/1.1/1
2.2/1.1/1
2.2/1.1/1
2.2/1.1/1
2.2/1.1/1
2.2/1.1/1
2.2/1.1/1
2.2/1.1/1
2.2/1.1/1
3
Boc-Phe-Pro-OH 12
Boc-Phe-Pro-OH 24
4
5
Boc-Phe-Pro-OH
Boc-Phe-Pro-OH 24
Boc-Phe-Pro-OH
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Fig. 1. Synthesis of t-butyloxycarbonyl (Boc)-protected dipeptide using BSA and
6
THF
NHS ester as coupling agents.
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8
DMF
DMF
DCM
DCM
DCM
DCM
8
Boc-Phe-Pro-OH 24
Boc-Leu-Phe-OH 10
Boc-Ala-Phe-OH 10
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2. Experimental
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Boc-Ala-Pro-OH
Boc-Ile-Val-OH
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All reactions were performed under a nitrogen atmosphere
using anhydrous techniques unless otherwise noted. 1H NMR
(300 MHz) on a Varian Mercury 300 spectrometer was recorded in
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a
Reaction conditions: unprotected amino acid (AA) reacted with BSA at room
temperature, followed by the addition of N-Boc protected NHS ester (NHS ester).
b
DMSO-d6 or CDCl3. Chemical shifts are reported in
d (ppm) units
The molar ratio of BSA to amino acid and NHS ester.
c
relative to the internal standard tetramethylsilane (TMS). All the
reactions were monitored by thin-layer chromatography (TLC)
analysis on pre-coated silica gel G plates at 254 nm under UV lamp
or HPLC analysis.
Isolated yield.
by Boc group. Most N-Boc protected NHS ester could be purchased,
those commercially unavailable N-Boc protected NHS esters were
readily obtained by coupling the corresponding N-Boc protected
amino acids with N-hydroxysuccinimide (NHS-OH) in the presence
of N,N-dicyclohexylcarbodiimide (DCC) [14]. The resulting bypro-
ducts containing the dicyclohexyl urea could be removed by
filtration though a short pad of silica gel. After concentration of the
filtrate, pure active esters could be recrystallized from various
solvent systems. The solid N-Boc protected NHS esters were stable
at ꢀ4 8C for a long period of time.
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2.1. General procedure for the preparation of N-Boc protected
dipeptide
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Under argon protection, BSA (2.2 equiv.) was added to a
solution of amino acid (1.1 equiv.) in anhydrous dichloromethane.
After the mixture was stirred for 1–8 h at 23 8C, a solution of N-Boc
protected NHS ester (1 equiv.) in dichloromethane was added. The
reaction mixture was stirred at 23 8C under argon until all active
ester was consumed as judged by TLC analysis. The reaction
mixture was washed with brine, dried over Na2SO4 and
concentrated in vacuo to provide a white solid. The isolated
product was recrystallized from diethyl ether/n-hexane to yield
the targeted dipeptide as a white solid.
The coupling conditions and purification processes were
optimized first through the synthesis of various dipeptides
(Table 1). When no BSA was added, the coupling product was
hardly detected (entry 1). Through screening various reaction
conditions for the synthesis of Boc-Phe-Pro-OH, we found that the
coupling efficiency and yield were optimal when 1.1 equiv. of
unprotected proline reacted with 2.2 equiv. of BSA first in
dichloromethane (DCM) at room temperature, followed by the
addition of 1 equiv. of N-Boc protected Phe NHS ester (enrty 2). The
ratio of BSA was important that either excessive (entry 3) or
insufficient (entry 4) BSA would reduce the coupling yield
significantly. Meanwhile, the ratio of each reagent was important
not only for coupling efficiency, but also for purification process.
NHS esters were insoluble in water, but soluble in organic solvents.
In contrary, unprotected amino acid and BSA were either soluble in
water or easily hydrolyzed in water. Therefore, the molar quantity
of the NHS ester should be slightly lower than the unprotected
amino acid and BSA to guarantee the NHS ester to be exhausted
completely. Then the excessive unprotected amino acids and BSA
could be easily removed by simply washing with water.
All the unprotected amino acids are insoluble in organic
solvents. So the addition order of reagents is quite important and
unprotected amino acids should react with BSA first to increase its
solubility and nucleophilicity. According to our data, the solubility
of most amino acids improved significantly after being silylated
with BSA. Among them, the silylation of proline was the fastest and
it became soluble in dichloromethane after just 1 h reaction with
BSA. But it took hours for other unprotected amino acids to be
silylated and dissolve in dichloromethane. Therefore, the slightly
lower yield when C-terminal was unprotected amino acids other
than proline may be owing to their relatively poorer solubility. For
the same reason, when C-terminal was several unprotected
hydrophilic amino acids, such as aspartic acid, glutamic acid
and cysteine, nearly no dipeptide products were obtained. Besides,
unprotected basic amino acids that have two amino groups, such as
arginine, lysine and histidine, are also unsuitable for the BSA/NHS
method. The solubility of silylated amino acid in THF and DMF was
N-Boc-
(m/z): 363.2 [M + H]+; 307.1 (M-(CH3)2C = CH2). 1H NMR
(400 MHz, CDCl3): 7.26 (m, 5H), 5.39 (d, 1H, J = 8.6 Hz), 4.69–
L-phenylalanine-L-proline (Boc-Phe-Pro-OH): ESI-MS
d
4.49 (m, 2H), 3.63–3.52 (m, 1H), 3.01 (m, 3H), 2.28–2.16 (m, 1H),
2.10–1.98 (m, 1H), 1.86 (m, 2H), 1.39 (s, 9H).
N-Boc-
287.2 [M + H]+; 231.1 (M-(CH3)2C = CH2). 1H NMR (400 MHz,
CDCl3): 4.60 (dd, 1H, J = 8.1, 3.9 Hz), 4.49 (s, 1H), 3.73 (q, 1H,
L-alanine-L-proline (Boc-Ala-Pro-OH): ESI-MS (m/z):
d
J = 8.0 Hz), 3.59 (m, 1H), 2.31–2.00 (m, 4H), 1.43 (s, 9H), 1.34 (d,
J = 6.9 Hz, 3H).
N-Boc-
(m/z): 337.2 [M + H]+; 281.1 (M-(CH3)2C = CH2). 1H NMR
(400 MHz, CDCl3): 7.32–7.10 (m, 5H), 6.85 (d, 1H, J = 7.5 Hz),
L-alanine-L-phenylalanine (Boc-Ala-Phe-OH): ESI-MS
d
5.36–5.06 (m, 1H), 4.82 (q, 1H, J = 6.5 Hz), 4.21 (s, 1H), 3.20 (dd, 1H,
J = 14.0, 5.5 Hz), 3.03 (dd, 1H, J = 14.3, 6.4 Hz), 1.43 (s, 9H), 1.26 (s,
3H).
N-Boc-
(m/z): 379.2 [M + H]+; 323.2 (M-(CH3)2C = CH2). 1H NMR
(400 MHz, CDCl3): 7.29–7.11 (m, 5H), 6.97–6.78 (m, 1H), 5.13
L-leucine-L-phenylalanine (Boc-Leu-Phe-OH): ESI-MS
d
(d, 1H, J = 8.8 Hz), 4.92–4.73 (m, 1H), 4.21 (d, 1H, J = 8.2 Hz), 3.26–
3.10 (m, 1H), 2.98 (dd, 1H, J = 13.9, 6.4 Hz), 1.59 (m, 2H), 1.44 (m,
10H), 0.89 (t, 6H, J = 7.0 Hz).
N-Boc-
331.2 [M + H]+; 275.2 (M-(CH3)2C = CH2). 1H NMR (400 MHz,
CDCl3): 6.87 (d, 1H, J = 8.7 Hz), 5.34 (d, 1H, J = 9.0 Hz), 4.62 (dd,
L-isoleucine-L-valine (Boc-Ile-Val-OH): ESI-MS (m/z):
d
1H, J = 8.6, 4.8 Hz), 3.97 (t, 1H, J = 8.2 Hz), 2.00 (m, 2H), 1.43 (s, 9H),
1.22 (m, 2H), 0.93 (m, 12H).
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3. Results and discussion
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In order to avoid racemization under alkaline conditions for the
deprotection step, all the N-terminus of NHS ester were protected
Please cite this article in press as: Y. Huang, W.-H. Feng, N,O-bis(trimethylsilyl)acetamide/N-hydroxysuccinimide ester (BSA/NHS) as
coupling agents for dipeptide synthesis, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.11.012

G Model
CCLET 3489 1–4
Y. Huang, W.-H. Feng / Chinese Chemical Letters xxx (2015) xxx–xxx
3
Fig. 2. HPLC analysis of Boc-Phe-Pro-OH synthesized using the BSA/NHS strategy. Chromatographic conditions: Instrument: Waters Acquity UPLC; Chromatographic column:
Waters UPLC BEH C18 reversed-phase column (1.7
m
m, 50 ꢁ 2.1 mm); Flow rate: 0.2 mL/min; Column temperature: 40 8C; Mobile phase: phase A: 0.1% formic acid aqueous
solution; phase B: CH3CN; gradient conditions: 0 min ! 6 min: 95% phase A ! 5% phase A; 6 min ! 8 min: 5% phase A; 8 min ! 8.1 min: 5% phase A ! 95% phase A;
8.1 min ! 10 min: 95% phase A ! 5% phase A.
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clearly worse and even proline could not totally dissolve after
reacting with BSA for 24 h. Consequently, the coupling efficiency
was lower when the solvent was changed to THF (entry 5) or DMF
(entry 7), and the time for achieving an acceptable yield in this two
solvents was longer too (entries 6 and 8).
Excessive addition of BSA would silylated the carboxyl group
and amino group simultaneously. When the carboxyl group and
amino group were both silylated, it has been proved that acylating
agents reacted with N-trimethylsilyl group exclusively [15]. The
selectivity may be owing to the stability of silicon–oxygen bond is
higher than that of silcon–nitrogen bond and silylation could
increase the nucleophilicity of amines. Therefore, NHS esters react
with amines selectively under these mild conditions (Scheme 1).
After all active ester being consumed as judged by TLC analysis,
processes. When the dipeptides were produced in the form of 191
hydrochloride salts, they would be more hygroscopic. Above all, we 192
could synthesize the dipeptide products in good yields and high 193
purity in significantly shorter reaction time with simpler 194
purification processes.
195
4. Conclusion
196
In summary, the BSA/NHS strategy has been successfully 197
utilized in the rapid, large scale solution-phase synthesis of various 198
dipeptides. Through the BSA/NHS strategy, coupling reaction was 199
completed under neutral and mild conditions that involve no extra 200
coupling reagents or acid/base. Excessive reagents and byproducts 201
were removed using water or saturated sodium chloride solution 202
rather than several rounds of acidic and basic aqueous extractions. 203
Moreover, all the reactants are inexpensive and widely used in 204
conventional drug production. Above all, the BSA/NHS strategy has 205
the potential to be applied in further commercial-scale manufac- 206
the dipeptide product was isolated through
a convenient
purification process. The silicon–oxygen bond could be easily
hydrolyzed by water to produce the targeted N-Boc protected
dipeptide [15d,e]. Besides, excessive BSA and amino acids are
either hydrolysable or water soluble and the water-insoluble NHS
esters are exhausted, all the excessive reagents and byproducts
could be removed simply by water or saturated sodium chloride
solution wash. The coupling reaction and purification process were
both performed under mild conditions and no additional coupling
reagents or acid/base were involved. Consequently, racemization
was minimal. As expected, none of excessive reactants, byproducts
or the racemized products was detected by HPLC analysis (Fig. 2).
Then the coupling and purification method was verified through
the synthesis of another four dipeptides, all of which were
obtained in high isolated yield of above 80 percent (entries 9–12).
The N-Boc protecting group was subsequently cleaved using
trifluoroacetic acid/dichloromethane (1:2) and the pure depro-
tected dipeptide was obtained after additional recrystallization
from diethyl ether. More impurities could be observed when pure
trifluoroacetic acid was utilized as the deprotection reagent,
leading to lower yields and more complicated purification
ture of other peptide drugs.
207
Acknowledgment
208
This research was financially supported by the National Science 209
and Technology Major Project of China (No. 521042).
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References
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coupling agents for dipeptide synthesis, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.11.012

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Please cite this article in press as: Y. Huang, W.-H. Feng, N,O-bis(trimethylsilyl)acetamide/N-hydroxysuccinimide ester (BSA/NHS) as
coupling agents for dipeptide synthesis, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.11.012