Re-evaluating the stability of COMU in different solvents

Article abstract of DOI:10.1002/psc.3024

COMU is uronium-type coupling reagent based on OxymaPure. It showed several advantages over classical benzotriazole-based coupling reagents such as higher solubility, water-soluble byproduct, and monitoring the reaction by changing of color. Although COMU is well known to perform excellent in solution, but its hydrolytic stability in DMF limits its use in automatic peptide synthesizer. Herein, we evaluated the hydrolytic stability of COMU in γ-valerolactone (GVL), acetonitrile (ACN) and N-formylmorpholine (NFM) and compared its stability against DMF. The stability of COMU after 24?h was found to be 88 and 89% in GVL and ACN, respectively, when compared in DMF (14%). Further, the demanding Aib-ACP decapeptide and JR decapeptide were successfully synthesized using COMU dissolved in GVL or ACN while Fmoc amino acids were dissolved in DMF. Copyright

Full text of DOI:10.1002/psc.3024

Research Article
Received: 11 April 2017
Revised: 5 June 2017
Accepted: 14 June 2017
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/psc.3024
Re-evaluating the stability of COMU in
different solvents
a,b
b
b,c
d,e
Ashish Kumar, Yahya E. Jad, Beatriz G. de la Torre, Ayman El-Faham *
a,d,f,g
and Fernando Albericio
*
COMU is uronium-type coupling reagent based on OxymaPure. It showed several advantages over classical benzotriazole-based
coupling reagents such as higher solubility, water-soluble byproduct, and monitoring the reaction by changing of color. Although
COMU is well known to perform excellent in solution, but its hydrolytic stability in DMF limits its use in automatic peptide synthe-
sizer. Herein, we evaluated the hydrolytic stability of COMU in γ-valerolactone (GVL), acetonitrile (ACN) and N-formylmorpholine
(NFM) and compared its stability against DMF. The stability of COMU after 24 h was found to be 88 and 89% in GVL and ACN, re-
spectively, when compared in DMF (14%). Further, the demanding Aib-ACP decapeptide and JR decapeptide were successfully
synthesized using COMU dissolved in GVL or ACN while Fmoc amino acids were dissolved in DMF. Copyright © 2017 European
Peptide Society and John Wiley & Sons, Ltd.
Data S1. HPLC chromatograms
Keywords: COMU; peptide-coupling reagent; solid-phase peptide synthesis; green solvent; γ-valerolactone
explained based on the fact that COMU contains C―O bond
which is comparatively more reactive than C―N bond present
Introduction
in HMDB [14,18]. However, this higher reactivity of C―O bond
Peptide synthesis involves appropriate combination of protecting
present in COMU is responsible for its low hydrolytic stability
groups and a suitable choice of coupling method [1,2]. The use
in DMF where it degrades into its precursors: OxymaPure and
of carbodiimides as coupling reagents [3] was a major event in
dimethylmorpholineurea. This instability property limits its uses
the history of peptide synthesis. The addition of phenols and
as coupling reagent in automated peptide synthesis as pointed
N-hydroxy derivatives [4] to carbodiimides during the coupling
reaction leads to the suppression of racemization and an
enhancement of yield because of the inhibition of side-reactions
such as formations of oxazolone or N-acylurea. Later, stand-alone
out by our group and others [19,20].
Our group was involved for many years in finding a suitable
solvent, which assures the stability of COMU for more than a few
coupling reagents such as immonium [4] and phosphonium
salts, phosphates and phosphinates [5–10] were reported as
*
Correspondence to: Fernando Albericio, School of Chemistry and Physics,
University of KwaZulu-Natal, Durban 4001, South Africa. E-mail: albericio@ukzn.
ac.zaAyman El-Faham, Department of Chemistry, College of Science, King Saud
University, PO Box 2455, Riyadh 11451, Saudi Arabia. E-mail: aelfaham@ksu.
edu.sa
efficient reagents for peptide synthesis. Benzotriazole-based
coupling reagents were most commonly employed since its first
report in 1970. However, the explosive nature of benzotriazoles
led to its classification as Class 1 explosive materials and
therefore requires great care during transportation and storage,
which can restrict their use. Later, in 2009, OxymaPure and its
dimethylmorpholine uronium salt COMU [11] were reported as
an efficient additive and coupling reagent, respectively, for
peptide synthesis (Figure 2) [12]. OxymaPure and COMU
displayed a remarkable efficiency to inhibit racemization and
an impressive coupling capability, superior to the performance
shown by HOBt derivatives and comparable to those of HOAt
derivatives. In addition, they had a lower risk of explosion than
that of benzotriazole derivatives [13–17], which reflected on
the GSK’s reagent guides because COMU was classified as one
of the greenest coupling reagent. Furthermore, COMU showed
a better solubility than the classical benzotriazole derivatives in
DMF, water-soluble byproducts, and enabling of color-
monitoring of the reaction [15]. The superiority of OxymaPure-
based uronium salts such COMU when compared with HOBt
counterparts such as HMDB and HDMA (Figure 1) can be
a School of Chemistry and Physics, University of KwaZulu-Natal, Durban 4001,
South Africa
b School of Health Sciences, University of KwaZulu-Natal, Durban 4001, South
Africa
c School of Laboratory of Medicine and Medical Sciences, University of KwaZulu-
Natal, Durban 4001, South Africa
d Department of Chemistry, College of Science, King Saud University, PO Box 2455,
Riyadh 11451, Saudi Arabia
e Chemistry Department, Faculty of Science, Alexandria University, PO Box 426,
Ibrahimia, 12321, Alexandria, Egypt
f
Department of Organic Chemistry, University of Barcelona, 08028, Barcelona,
Spain
g CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and
Nanomedicine, Barcelona Science Park, Barcelona 08028, Spain
J. Pept. Sci. 2017
Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

A. KUMAR ET AL.
steric hindrance. It was found in our earlier reports that along with
desired decapeptide, des-Aib is the most common side-product
observed because of the highly steric hindered nature of the Aib
residue [21].
Stepwise Fmoc-SPPS was utilized to synthesize the peptide
model. 0.5 M stock solution of COMU in GVL and ACN was prepared
and stored for 24 h to mimic the automatic SPPS. The common
Fmoc-RinkAmide-AM-PS resin and fast coupling process [28] for
Figure 1. COMU, HDMA and HDMB.
10 min were utilized, except for the challenging coupling of
Aib-Aib where 30-min double coupling was performed. At the
end, the coupling was carried out in a mixture DMF-GVL/ACN
(2:1) because Fmoc amino acids were dissolved in DMF. The HPLC
chromatogram of the obtained product matched what was
obtained from other standard protocols [21]. In addition,
H-RinkAmide-ChemMatrix resin was also used to synthesize the
model in similar fashion using COMU stored in ACN. HPLC
chromatogram of Aib-ACP decapeptide is shown in Figure 2.
Furthermore, the more challenging and difficult sequence
Jung and Redemann decapeptide (JR decapeptide; H-Trp-Phe-
hours. Unfortunately, most of the solvents such as tetrahydrofuran
(THF), 2-methyltetrahydrofuran (2-MeTHF) or cyclopentyl methyl
ether (CPME) which are known to give an excellent performance
in peptide synthesis using DIC and OxymaPure were found to be
incompatible with COMU due to its insolubility in those solvents
[
21]. In our earlier reports, it was found that solvents like
γ-valerolactone (GVL), acetonitrile (ACN) and N-formylmorpholine
NFM) can dissolve COMU satisfactorily [22–24]. Hence, in this
(
report, we evaluated the solubility as well as stability of COMU in
above mentioned solvents.
2
Thr-Thr-Leu-Ile-Ser-Thr-Ile-Met-NH ) [19,29,30] was also chosen
as a model. This peptide is considered to be more challenging
than the Aib-ACP decapeptide and is currently used as peptide
model for analyzing synthetic strategies. Again, non-optimized
standard conditions (single coupling, 30 min) were used to
evidence if there are differences among solvents. Thus, manual
stepwise Fmoc-SPPS was utilized in combination with
H-RinkAmide-ChemMatrix resin in a mixture of DMF with GVL
or ACN (2:1). Again, the HPLC chromatograms of the obtained
products when DMF-GVL (2:1) (Figure 3(B)) and DMF-ACN (2:1)
(Figure 3(C)) were used matched what was obtained from the
standard protocol (Figure 3(A)). All the relavent HPLC chromato-
grams can be found with the supporting information.
Result and Discussion
To study the solubility of COMU in GVL, ACN and NFM, 0.5 M
solution of COMU in different mentioned solvents was prepared
similar to that of DMF which is used under automatic peptide
synthesizer [21,25].
The stability of COMU in GVL, ACN and NFM was studied
along with DMF as solvent. Solution of COMU in above
mentioned solvents (0.2 M) was prepared and stored in closed
vials, without any special care such as bubbling N . Table 1
2
clearly indicates the instability of COMU in DMF which is found
in agreement with those described in literature [26], confirming
the incompatibility of COMU and DMF in automatic peptide Experimental
synthesizer. However, it is observed from Table 2 that COMU is
quite stable in GVL and ACN even after 48 h clearly indicating
Materials
its application as coupling reagent using GVL and ACN as solvent
in automatic peptide synthesizer during SPPS. Unfortunately,
COMU in NFM, which has a close structure to DMF, unlike GVL
and ACN was found to be unstable.
In order to determine the compatibility of COMU as coupling
reagent in peptide synthesizer for SPPS, the previously studied
sequence Aib-ACP decapeptide (H-Val-Gln-Aib-Aib-Ile-Asp-Tyr-Ile-
The solvents used were of HPLC reagent grade and purchased from
commercial suppliers. Amino acid derivatives purchased from GL
Biochem (Shanghai) Ltd, and Iris Biotech GMBH. Fmoc amino acids
contained the following side-chain protecting groups: Fmoc-Asn(-
t
t
Trt)-OH, Fmoc-Asp (O Bu), Fmoc-Glu (O Bu), Fmoc-Gln (Trt)-OH,
t
Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser( Bu)-OH, Fmoc-
t
t
Thr( Bu)-OH, and Fmoc-Tyr( Bu)-OH. GVL and NFM purchased from
Sigma-Aldrich. Analytical HPLC was performed on Agilent 1100 sys-
tem using Phenomenex C18 column (3 μm, 4.6 × 50 mm) and
Chemstation software used for data processing. Buffer A: 0.1%
TFA in H O, buffer B: 0.1% TFA in CH CN in HPLC.
Asn-Gly-NH ) was employed as a challenging model. This is the
2
modified version of the model peptide, ACP (64–74), which is
probably the most used model for determining the efficiency of
new SPPS protocols [22,27]. This peptide is prone to aggregation
2
3
[21] hence makes it optimum for evaluation of compatibility of
COMU as coupling reagent in SPPS. In addition, two alanine
residues in positions 66 and 67 in the parent sequence were
substituted with two Aib residues with the intent of introducing
Hydrolytic Stability Test
Ten milliliters of 0.2 M solution of COMU in GVL, DMF, ACN and
NFM was stored in a closed HPLC vial at room temperature. At
Table 1. Hydrolytic stability test of 0.2 M COMU in GVL, NFM and DMF
up to 48 h
Table 2. Hydrolytic stability test of 0.2 M COMU in GVL and ACN up to
11 days
Solvent
2 min
30 min
1 h
4 h
6 h
24 h
48 h
Solvent 1st 2nd 3rd 4th 5th 8th 9th 10th 11th
DMF
GVL
94
90
92
91
92
90
92
89
81
90
91
88
67
90
90
73
58
90
89
51
14
88
89
45
0
day day day day day day day day day
84
89
3
ACN
NFM
GVL 90
ACN 92
87
89
84
89
81
89
78
89
64
85
60
85
55
82
53
79
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J. Pept. Sci. 2017

COMU STABILITY
Figure 2. HPLC chromatograms of Aib-ACP decapeptide: (A) COMU in GVL assembled on RinkAmide-AM-PS resin. (B) COMU in ACN assembled on
RinkAmide-AM-PS resin. (C) COMU in ACN assembled on RinkAmide-AM-ChemMatrix resin.
each interval time period, 10.0 μl of aliquot was taken and diluted
to 0.5 ml with CH CN and immediately 1.0 μl was injected into
RP-HPLC with gradient 5–30% of CH CN (0.1% TFA)/H O (0.1%
Amount of COMU was calculated according to the integration of
the peak area at 220 nm of the signal associated with the COMU
with respect to the corresponding leaving group. All HPLC
chromatograms are available as the Supporting Information.
3
3
2
TFA) over 15 min, flow rate: 1.0 ml/min, detection at 220 nm.
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A. KUMAR ET AL.
Figure 3. HPLC chromatograms of JR decapeptide assembled on H-RinkAmide ChemMatrix resin: (A) freshly prepared COMU in DMF, (B) COMU in GVL for
24 h and (C) COMU in ACN (24 h).
SPPS of Aib-ACP decapeptide (H-Val-Gln-Aib-Aib-Ile-Asp-Tyr-
Ile-Asn-Gly-NH2)
of solvent and soluble reagent. The Fmoc-RinkAmide-AM-PS
(0.74 mmol/g, 100 mg) was washed with DMF, DCM and DMF
(2 × 7 ml each). Fmoc removal was carried out with 20%
piperidine in DMF (6 ml) for 1 min and 7 min. Parallel to this,
The SPPS was carried out manually in plastic syringe fitted with a
polyethylene porous disk attached to vacuum for quick removal
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J. Pept. Sci. 2017

COMU STABILITY
H-RinkAmide-ChemMatrix resin (0.46 mmol/g, 122 mg) was washed
with DMF, MeOH, DCM, 1% TFA in DCM and 5% DIEA in DCM. After
this, resins were washed with DMF, DCM and DMF (2 × 7 ml each)
followed by coupling with Fmoc-Gly-OH (5 equiv in 1.25 ml of
DMF)/COMU [5 equiv in 0.74 ml of COMU in GVL/ACN (stock solu-
tion kept for 24 h)]/DIEA (10 equiv) for 10 min. After coupling, the
resin was again washed as mentioned before and then deblocked
using 20% piperidine in DMF for 1 min and 7 min followed by
washing using DMF, DCM and DMF. This protocol of coupling
and Fmoc-deprotection was repeated until decapeptide was
synthesized. However, double coupling was performed for Aib at
position 3 for 30 min during SPPS. Peptide was cleaved from
gave another example that green solvents can open new chances
to overcome some limitations that were reported by using the
traditional synthetic strategies.
Acknowledgments
This work was funded in part by the following: National Research
Foundation (NRF) and the University of KwaZulu-Natal (South
Africa); the International Scientific Partnership Program ISPP at King
Saud University (ISPP# 0061) (Saudi Arabia); MEC (CTQ2015-67870-
P), the Generalitat de Catalunya (2014 SGR 137) (Spain). We thank
Luxembourg Biotech. for the generous gift of coupling reagents
and for supporting this research.
2
the resin by treatment with cocktail TFA-TIS-H O (95:2.5:2.5) for
2
h at room temperature. The TFA was removed under stream of
nitrogen, and the crude peptide was purified by washing with cold
diethyl ether (3 × 10 ml). The ratio of decapeptide and des-Aib
peptide was determined by HPLC analysis using a Phenomenex References
C
18 column (3 μm, 4.6 × 50 mm) with a linear gradient of 10–50%
of CH CN (0.1% TFA)/H O (0.1% TFA) over 15 min, flow rate:
.0 ml/min, detection at 220 nm. All HPLC chromatograms are
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available as the Supporting Information.
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Met-NH )
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The SPPS was carried out manually in plastic syringe fitted with a
polyethylene porous disk attached to vacuum for quick removal
of solvent and soluble reagent. The H-RinkAmide ChemMatrix
6
(
1
0.45 mmol/g, 164.44 mg) was washed with DMF, MeOH, DCM,
% TFA in DCM, 5% DIEA in DCM, DMF, DCM and DMF (2 × 7 ml
each). Fmoc removal was carried out with 20% piperidine in DMF
6 ml) for 1 and 7 min. After this, resin was washed with DMF,
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DCM and DMF (2 × 7 ml each) followed by coupling with Fmoc-
Met-OH (5 equiv in 1.25 ml of DMF)/COMU [5 equiv in 0.74 ml of
COMU in GVL/ACN (stock solution kept for 24 h) and fresh DMF]/
DIEA (10 equiv) for 30 min. After coupling, the resin was again
washed as mentioned before and then deblocked using 20%
piperidine in DMF for 1 and 7 min followed by washing using
DMF, DCM and DMF. This protocol of coupling and
Fmoc-deprotection was repeated until decapeptide was
synthesized. Peptide was cleaved from the resin by treatment with
2
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1
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1
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TFA was removed under stream of nitrogen, and the crude peptide
was purified by washing with cold diethyl ether (3 × 10 ml). The
percentage of target decapeptide was determined by HPLC
analysis using a Phenomenex C18 column (3 μm, 4.6 × 50 mm) with
a linear gradient of 15–60% of CH CN (0.1% TFA)/H O (0.1% TFA)
3 2
over 15 min, flow rate: 1.0 ml/min, detection at 220 nm. All HPLC
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1
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decapeptide and J R decapeptide by using a 24 h stored solution
of COMU which is not possible in case of DMF because of its high
hydrolytic hydrolysis. Thus, the main role of GVL and ACN is to
overcome the instability issue of COMU when dissolved in DMF.
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and ACN is classified as a polar aprotic solvent [23], this work also
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Data S1. HPLC chromatograms
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