COMU: A third generation of uronium-type coupling reagents

Article abstract of DOI:10.1002/psc.1204

COMU is a third generation of uronium-type coupling reagent based on ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma) as well as amorpholino carbon skeleton. The presence of the morpholino group has a marked influence on the solubility, stability and reactivity of the reagent. COMU performed extremely well in the presence of only 1 equiv. of base, thereby confirming the effect of the hydrogen bond acceptor in the reaction. The by-products of COMU are water soluble and easily removed, making it an excellent choice of coupling reagent for solution-phase peptide synthesis. Finally, COMU shows a less hazardous safety profile than benzotriazole-based reagents, such as HATU and HBTU, which in addition exhibit unpredictable autocatalytic decompositions and therefore a higher risk of explosion. Furthermore, in contrast to benzotriazole-based reagents, COMU is significantly less likely to cause allergic reaction. Copyright

Full text of DOI:10.1002/psc.1204

Protocol
Received: 28 September 2009
Revised: 18 October 2009
Accepted: 22 October 2009
Published online in Wiley Interscience: 30 November 2009
(
www.interscience.com) DOI 10.1002/psc.1204
COMU: A third generation of uronium-type
coupling reagents
a,b,c∗
a,d,e∗
Ayman El-Faham
and Fernando Albericio
COMU is a third generation of uronium-type coupling reagent based on ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma) as
well as a morpholino carbon skeleton. The presence of the morpholino group has a marked influence on the solubility, stability
and reactivity of the reagent. COMU performed extremely well in the presence of only 1 equiv. of base, thereby confirming the
effect of the hydrogen bond acceptor in the reaction. The by-products of COMU are water soluble and easily removed, making
it an excellent choice of coupling reagent for solution-phase peptide synthesis. Finally, COMU shows a less hazardous safety
profile than benzotriazole-based reagents, such as HATU and HBTU, which in addition exhibit unpredictable autocatalytic
decompositions and therefore a higher risk of explosion. Furthermore, in contrast to benzotriazole-based reagents, COMU is
significantly less likely to cause allergic reaction. Copyright ꢀc 2009 European Peptide Society and John Wiley & Sons, Ltd.
Keywords: coupling reagent; uronium salt; Oxyma; peptide synthesis; solid-phase methodology
REACTION SCHEME
GENERAL OPTIMISED PROCEDURE
Fmoc-Rink-Amide-AR (100 mg, 0.59 mmol/g) was deprotected by 20% piperidine (1mL) in DMF for 10 min., washed with DMF (2x1 mL), DCM
(
2x1 mL) and then DMF (2x1 mL). Fmoc-Leu-OH (3 equiv.), COMU (3 equiv.) and DIEA (6 equiv.) were mixed in DMF (0.5 mL) and activated for
1
-2 min and then added to the resin. The reaction mixture was then stirred slowly for 1 min and allowed to couple for 30min (1-h. double coupling only
for Aib-Aib). The resin was washed with DMF and then deprotected by 20 % piperidine in DMF for 7 min. It was then washed again with DMF, DCM,
and DMF, and then coupled with the next amino acid. Coupling and deprotection steps were repeated to obtain the pentapeptide. The crude product was
analyzed by HPLC, with the following conditions: linear gradient of 20 to 80 % CH CN/0.1 % TFA in H O/0.1 % TFA over 30 min, Symmetry Waters
3
2
C18 column (4.6 x 150 mm, 4 µm), detection at 220 nm, flow rate 1.0 ml/min, t penta = 10.87min (99.74%), t des-Aib = 11.15 min (0.26%).
R
R
Scope and Comments
∗
Correspondenceto:Ayman El-FahamandFernandoAlbericio,InstituteforResearchinBiomedicine,
Barcelona Science Park, Baldiri Reixac 10, 08028-Barcelona, Spain.
Peptidesynthesisisbasedonthepropercombinationofprotecting
groups and a suitable choice of coupling method [1–3]. Almost all
peptide bonds formed are currently carried out in the presence of
HOBt (1) or its derivatives (HOAt, 2; 6-Cl-HOBt, 3) [4–10].
E-mail: aymanel faham@hotmail.com; albericio@irbbarcelona.org
a
b
c
Institute for Research in Biomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028-Barcelona,
Spain
Alexandria University, Faculty of Science, Department of Chemistry, P.O. Box 246 Ibrahimia, 21321
Alexandria, Egypt
King Saud University, Department of Chemistry, College of Science, P.O. Box 2455, 1451-Riyadh,
Kingdom of Saudi Arabia
d
e
CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona
Science Park, Baldiri Reixac 10, 08028-Barcelona, Spain
DepartmentofOrganicChemistry,UniversityofBarcelona,MartíiFranqu e´ s1-11,08028-Barcelona,
Spain
Abbreviations used: Aib, α-aminoisobutyric acid; COMU, 1-[(1-(cyano-2-ethoxy-2-oxo-
ethylideneaminooxy)-dimethylamino-morpholinomethylene)]methanaminium hexafluorophos-
phate; DIEA, N,Ndiisopropylethylamine; HATU, N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]
pyridin-1-yl-methylene)-N-methylmethanaminium hexafluorophosphate N-oxide; NMP, N-
methylpyrrolidone; TMP, 2,4,6-trimethylpyridine or collidine. Source: Amino acids and peptides
are abbreviated and designated following the rules of the IUPAC-IUB Commission of Biochemical
Nomenclature [IUPAC-IUB Commission on Biochemical Nomenclature Symbols for Amino-Acid
Derivatives and Peptides. Recommendations (1971). J. Biol. Chem. 1972; 247: 977–983.
J. Pept. Sci. 2010; 16: 6–9
Copyright ꢀc 2009 European Peptide Society and John Wiley & Sons, Ltd.

COMU
Recent reports have confirmed the explosive properties of HOBt
derivatives [11]. In a previous study [12], we showed that Oxyma
4) is an excellent replacement for HOBt and its analogs. Here
form present during the coupling. This feature differs to that of
benzotriazole derivatives, in which the the N-form is predominant.
(
we devised a third generation of uronium salt, COMU (5), which
involves the combination of a morpholonium-based immonium
moiety as proton acceptor [13], and Oxyma (4) as leaving group to
provide a superior and safe coupling reagent for amide formation.
Experimental Procedure
General procedure for preparation of COMU [16,17]
Dimethylcarbamoyl chloride (0.6 mol) was added dropwise to a
stirring mixture of DCM (400 ml) and 4 N NaOH (250 ml) and
morpholine (0.5 mol) at 0 C. When the addition was completed,
The oxygen in the imminium structure increased the stability of
the coupling reagent compared with the tetramethyl derivatives.
Furthermore, Oxyma derivatives have higher stability than the
benzotriazole derivatives, HATU and HBTU. These observations are
of practical relevance for both solid-phase and solution strategies.
Althoughatypicalprotocolfortheuseofthesereagentsinvolves
◦
the mixture was stirred for 3 h at r.t. The organic layer was
collected and the aqueous layer was washed with DCM (100 ml).
The combined DCM solution was washed with a saturated solution
of NaCl (2 × 100 ml). Finally, the organic solvent was dried over
anhydrous MgSO4 and filtered. The solvent was then removed
under reduced pressure to give an oily residue. The product was
2
equiv. of base, usually DIEA, the presence of the morpholinium
moiety also allows good results with COMU when just 1 equiv. of
DIEA is used [14]. Alternatively, the less basic TMP (2 or even 1
equiv.) can be used instead of DIEA and provides good yields and
reduces racemization [14].
A further characteristic of COMU is that the course of reaction
can be followed as a result of change of color, depending on the
type of base used. Once the reaction is complete, the solution
becomes colorless to yellow, again depending on the type of base
used (Figure 1).
To check the effectiveness of COMU, the demanding leu-
enkephalinderivativeH-Tyr-Aib-Aib-Phe-Leu-NH2 [14]waschosen
as an example (see General Optimal Procedure). Synthesis with
COMU (5) led to only 0.26% of des-Aib when 2 equiv. of DIEA
was used, while HDMA and HDMB gave 1% and 10% of des-Aib,
respectively. In contrast, HATU and HBTU gave 17% and 53% of
des-Aib, respectively. These results are consistent with what is
discussed earlier.
◦
distilled and collected at bp 127–129 C as a colorless oil with a
9
3% yield.
N,N-Dimethyl-4-morpholinecarboxamide) H NMR (CDCl3):
δ 2.84 (s, 6H, 2 CH3), 3.22–3.20 (m, 4H, 2CH2), 3.68–3.70 (m, 4H,
1
(
13
2
CH2) ppm. C NMR (CDCl3): a¨ 38.6, 47.5, 66.9, 165.0 ppm. (The
NMR of the crude product was 99.8% pure, so we can use it directly
for the next step without further purification.)
Oxalylchloride(100 mmol)inDCM(100 ml)wasaddeddropwise
to a solution of urea (N,N-Dimethyl-4-morpholinecarboxamide)
A
B
Furthermore, COMU is compatible with microwave-assisted
peptide synthesizers [15]. Consistent with previous reports,
COMU displayed higher efficiency than HATU/HBTU in the
demanding synthesis of the Aib derivative of the leu-enkephalin
pentapeptide and produced no Oxyma-based by-products. Thus,
the combination of microwave irradiation and COMU resulted in
a similar performance to that observed by manual synthesis in
considerably shorter time.
C
In conclusion, we have introduced a third generation of O-form
uronium-type coupling reagent that differs in immonium moiety
and also in the leaving group. The presence of the morpholino
group has a marked influence on the polarity of the carbon
skeleton, which affects the solubility, stability and the reactivity of
the reagent. Remarkably, Oxyma derivatives often gave similar or
better results than the aza derivatives and performed extremely
well in the presence of only 1 equiv. of base. This observation
confirms the effect of the oxygen as a hydrogen bond acceptor in
the reaction. Although the acidity of Oxyma derivatives is similar
to that HOBt and higher than HOAt, the excellent performance of
the former can be explained by the fact that the O-form is the only
Figure 1. (a) Time 0; (b) after 2 min of the addition of COMU and TMP;
(c) after 1 h of the addition of COMU and TMP.
J. Pept. Sci. 2010; 16: 6–9
Copyright ꢀc 2009 European Peptide Society and John Wiley & Sons, Ltd.
www.interscience.com/journal/psc

EL-FAHAM AND ALBERICIO
(
100 mmol) in dry DCM (200 ml) at r.t. over 5 min. The reaction
•
After the coupling has taken place, the resin has a color, which
is due to the retention of some Oxyma. This is removed for the
piperidine treatment or by further washings with alcohol.
mixture was stirred under reflux for 3 h. and the solvent was
removed under vacuum. The residue was washed with anhydrous
ether (2×100 ml) and then bubbled with N2 to remove the excess
of ether. The residue was highly hygroscopic, and was therefore
dissolved directly in DCM, and a saturated aqueous potassium
hexafluorophosphate (100 mmol in 50 ml H2O) solution was then
added at r.t. with vigorous stirring for 10–15 min. The organic
layer was collected, washed once with water (100 ml), dried over
anhydrous MgSO4 and filtered. The solvent was removed under
reduced pressure to give a white solid which recrystallized from
DCM-ether or acetonitrile-ether to give white crystals in a yield of
Base
•
•
The use of immonium/onium salts requires careful attention to
the tertiary base used.
For the comparison experiments using distinct coupling
reagents and because of discrepancies in racemization levels
when using different samples of commercial tertiary amines,
DIEA (Aldrich, 99%) and NMM (Aldrich, 99%) are distilled first
◦
◦
fromninhydrinandthenfromCaH2 (bp126 Cand114–116 C,
respectively) and stored over molecular sieves. TMP (Eastman
◦
1
8
3
4
9.6%, m.p. 94–95 C; H NMR (CD3COCD3): δ 3.39 (s, 6H; 2CH3),
1
3
◦
.75 (t, 4H; 2CH2), 3.86 ppm (t, 4H; 2CH2); C NMR (CD3COCD3): δ
Kodak, 97%) is distilled from CaH2 (bp 170–172 C) and stored
4.36, 52.82, 65.99, 162.79 ppm).
over molecular sieves. Other bases should be treated similarly.
For regular synthesis, untreated DIEA, which may contain
various amounts of primary or secondary amines (positive
ninhydrin test), leads to enhanced racemization (2–3%). In
contrast, TMP (Aldrich, 99%), taken directly from the bottle,
gives racemization levels comparable to those obtained with
material distilled over CaH2.
The chlorine salt (20 mmol) was added to a solution of oxime
•
◦
potassiumsalt(20 mmol)inacetonitrile(50 ml)at0 C.Thereaction
mixture was stirred at this temperature for 30 min and allowed to
reach r.t. while stirring for 6 h. The crude product was filtered and
washed with acetonitrile. The solvent was concentrated to a small
volume (1/4) under reduced pressure. Dry ether was then added
to afford the product (COMU) as white crystals in a yield of 88.8%,
◦
Pre-activation time
m.p. 159–60 C.
1
H NMR (CD3COCD3): δ 1.38 (t, 3H; CH3), 3.41 (s, 6H; 2CH3), 3.82
•
The pre-activation time is crucial for the optimization of
the yield and the level of racemization. However, in some
synthesizers, pre-activation time is dictated by the instrument,
while in others and for manual syntheses, it can and should be
modulated.
(
(
t, 4H; 2CH2), 3.89 (t, 4H; 2CH2), 4.48 ppm (q, 2H; CH2); 13C NMR
CD3COCD3): δ 13.48, 40.70, 49.94, 64.59, 66.04, 106.76, 135.03,
1
56.14, 160.61 ppm.
•
•
In solution-phase coupling, pre-activation is not required so
the coupling reagent is added last at 0 C.
General procedure for coupling reaction using COMU in
solution-phase
◦
IncorporatingHOAt, HOBtorOxymainimmonium/oniumsalts,
the activation of ordinary amino acids gives the corresponding
OAt, OBt, Oxyma esters almost instantly. Thus, in such cases,
the pre-activation time should be kept to a minimum, since
the activated species can give rise to several side-reactions,
including the following:
COMU (0.25 mmol) was added to a mixture of the N-protected
amino acid (0.25 mmol), the amino component (0.25 mmol) and
base (0.50 mmol or 0.75 mmol in case of ester hydrochloride) in
◦
◦
DMF (2 ml) at 0 C and the reaction mixture was stirred at 0 C for
h and at r.t. for 2–3 h. The mixture was diluted with EtOAc (25 ml)
1
and extracted with 1 N HC1 (2 × 5 ml), 1 N NaHCO3 (2 × 5 ml) and
saturated NaCl (2 × 5 ml). The EtOAc was then dried with MgSO4,
the solvent was removed, and the crude peptide was directly
analyzed by HPLC and NMR.
-
Racemization, either by direct formation of the enolate or
by formation of oxazolone, which is prone to racemize.
Loss of reactivity by: (i) formation of the oxazolone, which
is also less reactive than the active esters; (ii) hydrolysis of
the active intermediates; (iii) by shift of the active ester from
the O-acyl to the N-acyl, which is less reactive. However, this
rearrangement has not been detected for COMU.
δ-Lactam formation during the activation of Arg, which is
not incorporated into the growing peptide chain, but results
in a lower yield.
-
General procedure for coupling reaction using COMU in solid-
phase
-
-
N-Protected amino acid (3 equiv.), base (6 equiv.) and COMU (3
equiv.) were pre-activated in DMF (0.3 M) for 1 min and then added
totheamino-resinwithmanualstirringfor2–5 minandallowedto
stand at r.t. for 10–30 min (1 h for hindered residues or 1-h double
coupling, also). The resin was filtered and washed with DMF.
Cyano derivatives or α-aminocrotonic acid formation for
Asn/Gln and Thr, respectively. These two side-reactions
are less frequent that that described above.
General Notes
Acknowledgements
•
COMUshouldbetreatedlikeotherrelatedstandalonecoupling
reagents, such as HATU or HBTU.
This work was partially supported by CICYT (CTQ2009-07758).
The Generalitat de Catalunya (2009SGR 1024), Luxembourg Bio
Technologies, Ltd., the Institute for Research in Biomedicine, and
the Barcelona Science Park.
Solvent
•
•
DMF (Fisher HPLC grade) is aspirated with a stream of N2 for
5 min and stored over molecular sieves.
In the microwave mode, NMP is more recommended than
DMF, which can provoke formylation of the amino group (this
is unrelated to the use of COMU or other coupling method).
1
References
1
Lloyd-Williams P, Albericio F, Giralt E. Chemical Approaches to the
Synthesis of Peptides and Proteins. CRC: Boca Raton (FL, USA), 1997.
www.interscience.com/journal/psc
Copyright ꢀc 2009 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2010; 16: 6–9

COMU
2
3
4
5
6
Goodman M, ed., Synthesis of Peptides and Peptidomimetics. Houben-
11 Wehrstedt KD, Wandrey PA, Heitkamp D. Explosive properties of
1-hydroxybenzotriazoles. J. Hazard. Mat. 2005; A126: 1–7.
12 Subir o´ s-Funosas R, Prohens R, Barbas R, El-Faham A, Albericio F.
Oxyma: an efficient additive for peptide synthesis to replace
benzotriazole-based HOBt and HOAt with a lower risk of explosion.
Chem. Eur. J. 2009; 15: 9394–9403.
13 El-Faham A, Albericio F. Immonium-type coupling reagents:
application in solution and solid phase peptide synthesis. Organic
Lett. 2007; 9: 4475–4477.
14 El-Faham A, Subir o´ s-Funosas R, Prohens R, Albericio F. COMU, a safer
and more effective replacement for benzotriazole based uronium
coupling reagents. Chem. Eur. J. 2009; 15: 9404–9416.
15 Subiros-Funosas R, Acosta GA, El-Faham A, Albericio F. Microwave
irradiationandCOMU:asuperiortoolforSPPS.TetrahedronLett.2009;
50: 6200–6202.
Weyl,MethodsofOrganicChemistry, Vol. E22a, Thieme: Stuggart·New
York, 2002.
Amblard M, Fehrentz J-A, Martinez J, Subra G. Methods and
protocols of modern solid phase peptide synthesis. Mol. Biotech.
2006; 33: 239–254.
Dourtoglou V, Ziegler JC, Gross B. O-Benzotriazolyl-N,N-tetr-
amethyluronium hexafluorophosphate: a new and effective reagent
for peptide coupling. Tetrahedron Lett. 1978; 19: 1269–1272.
Albericio F,Carpino LA.Couplingreagentsandactivation.InMethods
in Enzymology, Solid-Phase Peptide Synthesis, Vol. 289, Fields GB (ed.).
Academic Press: Orlando, 1997; 104–126.
Humphrey JM, Chamberlin AR. Chemical synthesis of natural
product peptides: coupling methods for the incorporation of
noncoded amino acids into peptides. Chem. Rev. 1997; 97:
2
243–2266.
Kaminski ZJ. Triazine-based condensingreagents. Biopolymers 2000;
5: 140–165.
16 El-Faham A, Khattab SN, Abdul-Ghani M, Albericio F. Design and
synthesis of a new immonium type coupling reagents. Eur. J. Org.
Chem. 2006; 6: 1563–1573.
7
8
9
5
Albericio F, Chinchilla R, Dodsworth DJ, Najera C. New Trends in
Peptide Coupling Reagents. Org. Prep. Proc. Int. 2001; 33: 203–303.
Han S-Y, Kim Y-A. Recent development of peptide coupling reagents
in organic synthesis. Tetrahedron 2004; 60: 2447–2467.
17 El-Faham A. New syntheses of bis(tetramethylene)fluoro-
formamidinium hexafluorophosphate (BTFFH) and 1,3-dimethyl-
2-fluoro-4,5-dihydro-1H-imidazolium hexafluorophosphate (DFIH).
Utility in peptide coupling reactions. Org. Prep. Procd. Int. 1998; 30:
477–481.
1
0 Montalbetti CAGN, Falque V. Amide bond formation and peptide
coupling. Tetrahedron 2005; 61: 10827–10852.
J. Pept. Sci. 2010; 16: 6–9
Copyright ꢀc 2009 European Peptide Society and John Wiley & Sons, Ltd.
www.interscience.com/journal/psc