Microwave irradiation and COMU: a potent combination for solid-phase peptide synthesis

Article abstract of DOI:10.1016/j.tetlet.2009.08.117

Here we demonstrate the compatibility of Oxyma-based uronium-type coupling reagent COMU with microwave-assisted peptide synthesizers. Consistent with previous reports, COMU displayed higher efficiency than benzotriazole classical immonium salts HATU and H

Full text of DOI:10.1016/j.tetlet.2009.08.117

Tetrahedron Letters 50 (2009) 6200–6202
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Microwave irradiation and COMU: a potent combination for solid-phase
peptide synthesis
Ramon Subiros-Funosas a,b, Gerardo A. Acosta a,b, Ayman El-Faham a,c,d, Fernando Albericio a,b,e,
*
a Institute for Research in Biomedicine (IRB Barcelona), Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain
b CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Baldiri Reixac 10-12, 08028 Barcelona, Spain
c Department of Chemistry, College of Science, King Saud University, PO Box 2455, Riyadh, Saudi Arabia
d Department of Chemistry, Faculty of Science, Alexandria University, Ibrahimia 21321, Alexandria, Egypt
e Department of Organic Chemistry, University of Barcelona, Martí i Franqués 1-11, 08028-Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2009
Revised 7 August 2009
Accepted 28 August 2009
Available online 2 September 2009
Here we demonstrate the compatibility of Oxyma-based uronium-type coupling reagent COMU with
microwave-assisted peptide synthesizers. Consistent with previous reports, COMU displayed higher effi-
ciency than benzotriazole classical immonium salts HATU and HBTU in the demanding synthesis of the
Aib derivative of Leu-Enkephalin pentapeptide and did not yield Oxyma-based byproducts. Thus, the
combination of microwave irradiation and COMU resulted in a similar performance in considerably
shorter time to that achieved by manual synthesis.
Keywords:
Coupling reagents
Microwave
Ó 2009 Elsevier Ltd. All rights reserved.
Peptide
Solid-phase synthesis
Oxyma
Uronium-type coupling reagent 1-[(1-(cyano-2-ethoxy-2-oxoe-
thylideneaminooxy)-dimethylamino-morpholinomethylene)] meth-
anaminium hexafluorophosphate, 1 (COMU, Fig. 1) is a more efficient
alternative to classical benzotriazole immonium salts in terms of
racemization suppression, coupling effectiveness, stability, and solu-
bility.1 In addition to the morpholino moiety, this superiority is
attributed to the introduction of ethyl 2-cyano-2-(hydroxyimi-
no)acetate, 2 (Oxyma, Fig. 1) in its structure. Oxyma displays high
control of optical purity and extension of coupling in demanding
sequences.2
Both Oxyma and its related uronium salt can be handled with a
considerably lower risk than its explosive benzotriazole counter-
parts, as determined by calorimetry techniques. However, the ther-
mal stability of Oxyma is relatively low.1,2 Nonetheless, no incident
has been reported during extreme coupling stability assays, carried
out using microwave irradiation at 80 °C.2
contrast, a similar experiment with microwave irradiation gave
rise to some of these undesired byproducts ( Fig. 2). In that
demanding experiment, the main byproduct resulted from the
formylation of the amino function (3, 47.9%), attack of the amino
group on the carboxylate (4) and the carbonyl of the oxime (5),
which was also hydrolyzed (6). In all the cases, impurities were de-
tected in the range 2–4%.2
In order to unequivocally determine the compatibility of the
Oxyma-derived coupling reagents with microwave-assisted solid-
phase peptide synthesis (SPPS), we compared the performance of
COMU with that of classical immonium salts HBTU 7 and HATU 8
in the synthesis of a true peptide model conducted in an auto-
mated peptide synthesizer with microwave irradiation (Fig. 3).3
Since its first appearance in 1986, microwave irradiation has
been established as a highly useful tool for solid-phase organic
Previous studies examined the stability of Oxyma toward the
nucleophilic N-terminus of the growing peptide chain in extreme
experiments. To enhance the likelihood of this side reaction, these
experiments used a high excess of the additive in the absence of
carbodiimide and Fmoc-AA-OH.2 In the assay conducted at room
temperature overnight, no Oxyma-based byproduct was found. In
CN
COOEt
N
CN
COOEt
OH
PF6
O
O
N
N
N
2
1
* Corresponding author. Tel.: +11 34 93 402 9058; fax: +11 34 93 339 7878.
E-mail address: albericio@pcb.ub.es (F. Albericio).
Figure 1. Structure of COMU and Oxyma.
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.08.117

R. Subiros-Funosas et al. / Tetrahedron Letters 50 (2009) 6200–6202
6201
based solid support that shows good swelling properties in both
polar and non-polar solvents.18–21 A conditioning protocol was fol-
lowed to remove any remaining base from the commercial resin
before its transfer with DMF into a suitable-sized polypropylene
vial. Solutions of reagents for coupling and deprotection steps were
placed in appropriate vessels (Table 1).
Short single couplings were applied to introduce the five resi-
dues (6 min at 80 °C), using 1 mmol of Fmoc-aminoacid (0.6 mmol
for Aib, in order to enhance relative differences), 1 equiv of cou-
pling reagent, and 2 equiv of base, relative to amino acid.22 Depro-
tection steps were conducted with a solution of piperidine in DMF
(Table 1) at 75 °C (3 min  2). Once automated synthesis of the
pentapeptide was completed (proceeding safely with COMU 1 un-
der the abovementioned conditions), the final cleavage from the
resin was accomplished with a 2-h treatment with 90% TFA/10%
H2O at room temperature. After precipitation with cold Et2O and
lyophilization, the samples were analyzed by reverse-phase
HPLC-PDA and HPLC–MS. An example of the crude mixtures ob-
tained is shown for the synthesis with COMU 1 (Fig. 4).
Two main compounds were observed in the crude mixtures: the
desired pentapeptides (H-Tyr-Aib-Aib-Phe-Leu-NH2) and des-Aib
(H-Tyr-Aib-Phe-Leu-NH2). Only traces of other possible deletion
peptides such as des-Tyr (H-Aib-Aib-Phe-Leu-NH2) and des-Aib,Tyr
(H-Aib-Phe-Leu-NH2) were detected. Some minor peaks were ob-
served and these were not related to the coupling reagents or dele-
tion peptides. The relative percentages of pentapeptide/des-Aib for
each coupling reagent are shown in Table 2. The highest percent-
age of pentapeptide was obtained with COMU 1 (92%). This perfor-
mance was better than that observed for HATU 8 (79%) or HBTU 7,
(23%). The manual synthesis with COMU at room temperature
afforded a higher percentage of the Aib-enkephalin pentapeptide
(99.7%);1 however, coupling times were much longer (60 min, dou-
ble coupling) than those in the abovementioned microwave syn-
thesis (6 min). These results therefore highlight the enhanced
efficiency of the method presented.
O
O
H
NH peptide
NH peptide
NC
4
3
N OH
OH
OH
HN
NC
HN
NH peptide
COOH
NH peptide
COOEt
NC
6
5
Figure 2. Side products obtained by reaction of Oxyma with a peptide in the
absence of carbodiimide and Fmoc-AA-OH.
synthesis, both in academy and in industry, by increasing product
yield and purity and reducing reaction times, in combination with
thermal effects.4–6 However, few studies have reported the appli-
cation of this approach in the field of peptide synthesis since the
early report of Wang and co-workers in 1991.7 This low general
acceptance could be partly attributed to the widespread belief that
the acceleration rate of coupling and deprotection steps also leads
to instability of coupling reagents and an increase in typical side
reactions, such as racemization and aspartimide formation. Never-
theless, recent studies have confirmed that these drawbacks can be
controlled with an adequate choice of reaction conditions, and also
that resin loading is critical for optimal microwave-assisted pep-
tide synthesis.8,9 The availability of peptide synthesizers equipped
with microwave irradiation has undoubtedly contributed to the
sudden increasing popularity of this tool in SPPS, which has proved
compatible with both Fmoc and Boc approaches in the assembly of
difficult sequences including phospho and glycopeptides, cymant-
rene-peptide bioconjugates, and cyclotides.10–16
One of the key advantages offered by microwave irradiation
(which cannot be explained by thermal effects) derives from the
dipolar polarization mechanism, which provides extra energy for
the rotation of the molecules with a dipolar moment, such as pep-
tides.8,12 This effect is highly relevant during the elongation of long
and hydrophobic peptides, since the polar peptide backbone con-
stantly aligns with the electric field, thereby disrupting chain
aggregation. Thus, peptides of up to 30-mer can be prepared easily
overnight.8
In our experiment with COMU, carried out in more realistic con-
ditions than those abovementioned, no byproducts (4–6) related to
Table 1
Reagents and solvents used for microwave-assisted Fmoc-SPPS
To further demonstrate the strong performance of microwave-
assisted SPPS in challenging sequences, here we report the assem-
bly of the highly demanding Aib-analog of Leu-enkephalin penta-
peptide (H-Tyr-Aib-Aib-Phe-Leu-NH2) by means of a Liberty CEM
Microwave Peptide Synthesizer. The Aib-Aib sterically hindered
coupling is an excellent scenario to compare the performance of
COMU 1 and benzotriazole-based HBTU 7 and HATU 8,17 and also
to study the formation of the side reactions associated with the
presence of Oxyma during the coupling.
Reagent
Concentration
Solvent
Amino acid
Activator
Base
Fmoc-AA-OH
HBTU/HATU/COMU
DIEA
0.2 M
0.5 M
2 M
DMF
DMF
DMF
DMF
Deprotection
Piperidine
20% v/v
The synthesis was carried out in a 0.1 mmol scale of H-RinkA-
mide-ChemMatrix resin (loading = 0.42 mmol/g), a totally PEG-
N
N
N
N
PF6
N
X
O
Figure 4. HPLC analysis of the crude obtained from the experiment using COMU.
HPLC conditions: Waters SunFire C18 column (3.5
gradient 20–30% of 0.036% TFA in CH3CN/0.045% TFA in H2O over 8 min;
flow = 1.0 ml/min; detection at 220 nm; tR (penta) = 6.780 min, [M+H]+ = 611.35;
tR (des-Aib) = 7.119 min, [M+H]+ = 526.30.
HBTU, X=CH (7)
HATU, X=N (8)
l
m, 4.6 Â 100 mm); linear
Figure 3. Structure of benzotriazole-based immonium salts HBTU and HATU.

6202
R. Subiros-Funosas et al. / Tetrahedron Letters 50 (2009) 6200–6202
Table 2
References and notes
Relative percentages of pentapeptide/des-Aib with various coupling reagents
1. El-Faham, A.; Subirós-Funosas, R.; Prohens, R.; Albericio, F. Chem. Eur. J. in
press. doi:10.1002/chem.200900615.
2. Subirós-Funosas, R.; Prohens, R.; Barbas, R.; El-Faham, A.; Albericio, F. Chem.
Eur.J. in press. doi:10.1002/chem.200900614.
3. Carpino, L. A.; Imazumi, H.; El-Faham, A.; Ferrer, F. J.; Zhang, C.; Lee, Y.;
Foxman, B. M.; Henklein, P.; Hanay, C.; Mügge, C.; Wenschuh, H.; Klose, J.;
Beyermann, M.; Bienert, M. Angew. Chem., Int. Ed. 2002, 41, 441–445.
4. Giguere, R. J.; Bray, T. L.; Duncan, S. M.; Majetich, G. Tetrahedron Lett. 1986, 27,
4945–4948.
Coupling reagent
Pentapeptide (%)
Des-Aib (%)
Yield (%)
COMU (1)
HBTU (7)
HATU (8)
92.1
23.1
79.5
7.9
76.9
20.5
88.5
20.5
76.0
the Oxyma moiety contained in the coupling reagent were de-
tected. However, traces of guanidylation were found at the dipep-
tide stage23 with each coupling reagent, during the introduction of
the first Aib residue.
5. Lidstrom, P.; Tierney, J.; Wathey, B.; Westman, J. Tetrahedron 2001, 57, 9225–
9283.
6. de la Hoz, A.; Diaz-Ortiz, A.; Moreno, A. Chem. Soc. Rev. 2005, 34, 164–178.
7. Chen, S. T.; Chiou, S. H.; Wang, K. T. J. Chin. Chem. Soc. 1991, 38, 85–91.
8. Palasek, S. A.; Cox, Z. J.; Collins, J. M. J. Pept. Sci. 2007, 13, 143–148.
9. Coantic, S.; Subra, G.; Martinez, J. Int. J. Pept. Res. Ther. 2008, 14, 143–147.
10. Bacsa, B.; Desai, B.; Dibo, G.; Kappe, C. O. J. Pept. Sci. 2006, 12, 633–638.
11. Katrizky, A. R.; Khashab, N. M.; Yoshioka, M.; Haase, D. N.; Wilson, K. R.;
Johnson, J. V.; Chung, A.; Haskell-Luevano, C. Chem. Biol. Drug Des. 2007, 70,
465–468.
12. Cemazar, M.; Craik, D. J. J. Pept. Sci. 2008, 14, 683–689.
13. Brandt, M.; Gammeltoft, S.; Jensen, K. J. Int. J. Pept. Res. Ther. 2006, 12, 349–357.
14. Nagaike, F.; Onuma, Y.; Kanazawa, C.; Hojo, H.; Ueki, A.; Nakahara, Y.;
Nakahara, Y. Org. Lett. 2006, 8, 4465–4468.
15. Peindy N’dongo, H. W.; Ott, I.; Gust, R.; Schatzschneider, U. J. Organomet. Chem.
2009, 694, 823–827.
16. Leta Aboye, T.; Clark, R. J.; Craik, D. J.; Goransson, U. ChemBioChem 2008, 9,
103–113.
17. El-Faham, A.; Albericio, J. Org. Chem. 2008, 73, 2731–2737.
18. Garcia-Martin, F.; Quintanar-Audelo, M.; Garcia-Ramos, Y.; Cruz, L. J.; Gravel,
C.; Furic, R.; Côté, S.; Tulla-Puche, J.; Albericio, F. J. Comb. Chem. 2006, 8, 213–
220.
In summary, here we demonstrate the compatibility of Oxyma-
based COMU 1 with microwave-assisted SPPS, during the auto-
mated synthesis of Aib-enkephalin pentapeptide. The Oxyma moi-
ety contained in COMU also displayed high nucleophilic stability to
the N-terminus of the growing peptide chain, as shown by the ab-
sence of Oxyma-based byproducts. Consistent with the previous
reports, COMU showed better performance than classical immoni-
um salts HATU and HBTU, which yielded lower percentages of the
desired pentapeptide. On the basis of our findings, we conclude
that the combination of COMU and microwave irradiation is an
efficient approach for SPPS, as it yielded more than 90% of Aib-
enkephalin, a highly demanding pentapeptide, in considerably less
time than the conventional manual synthesis.
19. García-Martín, F.; White, P.; Steinauer, R.; Côté, S.; Tulla- Puche, J.; Albericio, F.
Biopolymers (Pept. Sci.) 2006, 84, 566–575.
20. de la Torre, B. G.; Jakab, A.; Andreu, D. Int. J. Pept. Res. Ther. 2007, 13,
265–270.
Acknowledgments
This work was partially supported by CICYT (CTQ2006-03794/
BQU), Luxembourg Bio Technologies, Ltd., the Generalitat de Catalu-
nya (2005SGR 00662), the Institute for Research in Biomedicine,
and the Barcelona Science Park. R.S.-F. thanks the Ministerio de Edu-
cación y Ciencia for a FPU Ph.D. fellowship.
21. Frutos, S.; Tulla-Puche, J.; Albericio, F.; Giralt, E. Int. J. Pept. Res. Ther. 2007, 13,
221–227.
22. Common microwave protocols were used, showing the compatibility of COMU,
HATU, and HBTU with those protocols.
23. Albericio, F.; Bofill, J. M.; El-Faham, A.; Kates, S. A. J. Org. Chem. 1998, 63, 9678–
9683.