COMU: A safer and more effective replacement for benzotriazole-based uronium coupling reagents

Article abstract of DOI:10.1002/chem.200900615

We describe a new family of uronium-type coupling reagents that differ in their iminium moieties and leaving groups. The presence of the morpholino group in conjunction with an oxime derivative-especially ethyl 2-cy ano-2- (hydroxyimino) acet ate (Oxyma)-had a marked influence on the solubilities, stabilities, and reactivities of the reagents. Finally, the new uronium salt derived from Oxyma (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. COMU also showed a less hazardous safety profile than the benzotriazolebased HDMA and HDMB, which exhibited unpredictable autocatalytic decompositions. Furthermore, the Oxyma moiety contained in COMU suggests a lower risk of explosion than in the case of the benzotriazole derivatives.

Full text of DOI:10.1002/chem.200900615

DOI: 10.1002/chem.200900615
COMU: A Safer and More Effective Replacement for Benzotriazole-Based
Uronium Coupling Reagents**
[
a, b, c]
[a, d]
[e]
Ayman El-Faham,*
Ramon Subirꢀs Funosas,
Rafel Prohens, and
[
a, d, f]
Fernando Albericio*
Abstract: We describe a new family of
uronium-type coupling reagents that
differ in their iminium moieties and
leaving groups. The presence of the
morpholino group in conjunction with
an oxime derivative—especially ethyl
uronium salt derived from Oxyma
(COMU) performed extremely well in
the presence of only 1 equiv of base,
thereby confirming the effect of the hy-
drogen bond acceptor in the reaction.
COMU also showed a less hazardous
safety profile than the benzotriazole-
based HDMA and HDMB, which ex-
hibited unpredictable autocatalytic de-
compositions. Furthermore, the Oxyma
moiety contained in COMU suggests a
lower risk of explosion than in the case
of the benzotriazole derivatives.
2-cyano-2-(hydroxyimino)acetate
Keywords: coupling
Oxyma peptides
synthesis · uronium salts
reagents
·
(
Oxyma)—had a marked influence on
·
· solid-phase
the solubilities, stabilities, and reactivi-
ties of the reagents. Finally, the new
[
1]
Introduction
method. Nowadays, almost all peptide bonds are formed
in the presence of 1-hydroxybenzotriazole (HOBt, 1,
Figure 1, left) or its derivatives (HOAt, 2; 6-Cl-HOBt,
[2]
Peptide synthesis is based on an appropriate combination of
protecting groups and
a suitable choice of coupling
[
a] Prof. A. El-Faham, R. S. Funosas, Prof. F. Albericio
Institute for Research in Biomedicine, Barcelona Science Park
Baldiri Reixac 10, 08028 Barcelona (Spain)
Fax : (+34)93-403-71-26
E-mail: aymanel_faham@hotmail.com
albericio@irbbarcelona.org
[
[
b] Prof. A. El-Faham
Department of Chemistry, College of Science
King Saud University, P.O. Box 2455
Riyadh 11451 (Saudi Arabia)
[
c] Prof. A. El-Faham
Figure 1. General structures of HOBt derivatives (left), immonium/uroni-
um salts (center), and the most commonly used immonium salts (right).
Department of Chemistry, Faculty of Science
Alexandria University, Ibrahimia 21321, Alexandria (Egypt)
d] R. S. Funosas, Prof. F. Albericio
CIBER-BBN, Networking Centre on Bioengineering
Biomaterials and Nanomedicine, Barcelona Science Park
Baldiri Reixac 10, 08028 Barcelona (Spain)
[
3,4]
3
).
HOBt derivatives are therefore either used in combi-
nation with a carbodiimide or another coupling agent or are
built into a stand-alone reagent such as an immonium
[
e] R. Prohens
Plataforma de Polimorfisme i Calorimetria
Serveis Cientificotꢀcnics, University of Barcelona
Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona (Spain)
[HATU (4), HBTU (5), HCTU, (6), Figure 1, right] or phos-
[5,6]
phonium (PyAOP, PyBOP, PyClock) salt.
An onium salt
consists of two parts: a leaving group (YL) and the iminium
moiety (Figure 1, center).
[
f] Prof. F. Albericio
Department of Organic Chemistry, University of Barcelona
Martꢁ i Franquꢂs 1–11, 08028 Barcelona (Spain)
Recently we showed that the incorporation of a hydrogen
bond acceptor in the iminium part resulted in performances
[
**] All abbreviations used in the text are given in reference [1].
[7]
superior to those described previously. As reported in our
previous work, the presence of an oxygen in the iminium
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200900615.
9404
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Chem. Eur. J. 2009, 15, 9404 – 9416

FULL PAPER
moiety confers more solubility on the reagent, enhances
coupling yields, and decreases racemization, thereby allow-
ing the use of just 1 equiv of base. HDMA (7), HDMB (8),
and 6-HDMCB (9) are thus more efficient in terms of cou-
pling efficiency and reduction of racemization than their
counterparts HATU (4), HBTU (5), and HCTU (6). Impor-
tantly, 6-HDMCB (9), which is consistently superior to
HDMB (8), often performs in a similar manner to HATU
Figure 3. Structures of Oxyma and the new uronium salt.
(
4), which is one of the most powerful commercially avail-
[7]
able immonium coupling reagents known to date. It is im-
chlorides 12a or 12b with morpholine (13a) or pyrrol ACHTUNGTRENNUNGi dine
portant to note that all these reagents exist in their N-
(13b) to give the urea derivatives 14a–c (Scheme 1). Urea
derivatives (14a–c) were treated with phosgene or oxalyl
chloride to yield the corresponding chloro salts, which were
stabilized by the formation of hexafluorophosphate salts
[8]
forms,
which are less reactive than the O-forms
[9]
(
Figure 2).
(
15a–c). Subsequent treatment with oxime derivatives (17a–
d), obtained by nitrosation from the active methylene com-
pounds 16a–d, in the form of their potassium salts or in the
presence of Et N provided the target compounds (18a–l,
3
Scheme 1).
1
3
Interestingly, the C NMR spectra of these compounds
indicated displacements of the carbocationic carbon of
1
56.11 ppm for HOTU (18a, the hexafluorophosphate
[12]
count ACHUTNGRNEUNGe rpart of TOTU, already described in the literature)
Figure 2. General structures of the dimethyl-morpholino immonium salts,
which are superior to their tetramethyl counterparts.
and 156.14 ppm for COMU (18c). These displacements are
consistent with those reported for this kind of compound in
[13,9]
the O-form.
X-ray crystallography confirmed this hy-
Recent reports have confirmed the explosive properties of
HOBt derivatives. In our preceding paper, we showed
pothesis (Figure 4).
[10]
[11]
that Oxyma (10, Figure 3) is an
excellent
replacement
for
HOBt and its analogues. Here
we report a new uronium salt,
COMU (11, Figure 3), which
represents the combination of a
morpholonium-based immoni-
um moiety, introduced in our
previous work, and Oxyma (10)
as leaving group, as a superior
and safe coupling reagent for
amide formation.
Results and Discussion
Scheme 1 shows the uronium
salts prepared for the first
screening. We tested four dis-
tinct oximes (17a–d) and sever-
al iminium moieties, including
dimethyl-morpholino, pyrrolidi-
no-morpholino, and dimethyl-
pyrrolidino systems, the last of
these as a reference for the role
of the pyrrolidino moiety. The
corresponding unsymmetrical
uronium salts were prepared by
treating N,N-dialkyl car ACHTUNGRENNUbG am ACHTUNGTRENNGNUo yl Scheme 1. Procedure followed for the preparation of the oxime-based uronium-type coupling reagents.
Chem. Eur. J. 2009, 15, 9404 – 9416
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9405

F. Albericio, A. El-Faham et al.
Table 2. Effect of oxygen on the solubility of the immonium-/uronium-
type coupling reagents in DMF.
Entry
Coupling reagent
Wt/1 mL
Molarity
1
2
3
4
5
6
7
8
HATU (4)
HBTU (5)
0.165
0.175
0.420
0.620
0.410
0.520
0.320
0.430
0.43
0.46
1.09
1.44
1.20
1.36
0.79
0.98
HOTU (18a)
COMU (18c)
HTODC (18e)
HDMODC (18g)
HTOPC (18k)
HDMOPC (18l)
COMU (18c) and HDMODC (18g) were the most soluble.
They were used to prepare solutions of up to 1.5m and
showed clearly higher solubilities than the benzotriazole de-
rivatives 4 and 5 (Table 2, entries 4, 6 vs. 1, 2). This in-
creased solubility can be used to prepare more concentrated
solutions in order to enhance coupling yields and to facili-
tate the removal of the excess of coupling reagent and the
urea side-products during the workup in a solution-mode
approach.
A further characteristic of the Oxyma derivative 18c is
that the course of reaction can be followed due to a change
in color, which depends on the type of base used. Thus,
2 min after the addition of the coupling reagent, a solution
of 18c has turned orange-red when DIEA is used as a base,
and pink in the case of TMP. Once the reaction is complete,
the solutions become colorless and yellow, respectively
Figure 4. X-ray crystallographic structure of COMU.
To determine the compatibilities of the new coupling re-
agents with peptide synthesis in both manual and automatic
modes, their solubilities and stabilities in solution and in the
1
solid state were examined by H NMR analysis. The pres-
ence of the oxygen in the iminium structure increased the
stabilities of the coupling reagent relative to their tetrameth-
yl derivatives (entries 3 vs. 4, Table 1). Furthermore, Oxyma
Table 1. Hydrolytic stabilities [%] of immonium/uronium-type coupling
reagents in DMF (open vials).
(
Figure 5).
Entry
Coupling reagent
5 h [%]
24 h [%]
48 h [%]
1
2
3
4
HATU (4)
HBTU (5)
HOTU (18a)
COMU (18c)
99
95
98
95
100
76
86
84
93
100
100
100
derivatives have greater stabilities than the benzotriazole
derivatives HATU (4) and HBTU (5) (Figure 1). All these
reagents showed stabilities greater than 95% in a closed
vial. These observations are of practical relevance for both
solid-phase and solution strategies: when the activation of a
carboxylic acid is slow and the coupling reagent is not
stable, it is degraded and no longer able to activate the car-
boxylic function. This feature is crucial in cyclization steps
or for segment coupling steps in convergent strategies, in
which the excess of the carboxylic function is either absent
Figure 5. A) 2 min after the addition of COMU (pink with TMP and
orange-red with DIEA). B) 1 h after the addition of COMU.
(
cyclization) or low (segment coupling) and couplings are
therefore very slow.
Table 2 indicates that the presence of the oxygen atom in
the carbon skeleton and the leaving group are of marked
relevance for the solubilities of the compounds. Thus, all di-
methyl-morpholino derivatives (18c, 18g, and 18l) were
more soluble than their tetramethyl counterparts (18a, 18e,
and 18k) (Table 2, entries 3 vs. 4, 5 vs. 6, and 7 vs. 8). Fur-
thermore, ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma,
A preliminary screening on the efficiency of Oxyma-
based coupling reagents 18a and 18c, in the coupling of hin-
dered amino acids, was examined with two model systems
(Fmoc-Val-OH + H-Val-NH2 and Z-Aib-OH + H-Val-
OMe) in solution. The reaction mixtures were followed by
HPLC; the tR values for the starting Fmoc-Val-OH and Z-
Aib-OH were 20.89 min and 18.25 min, respectively, and
10) derivatives were more soluble than dicyano and cyano-
pyridinyl ones (Table 2, entries 3, 4 vs. 5, 6 and 7, 8). Thus,
those for the products (Fmoc-Val-Val- NH and Z-Aib-Val-
2
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Benzotriazole-Based Uronium Coupling Reagents
FULL PAPER
OMe) were 19.88 min and 20.90 min, respectively. The
Oxyma-based coupling reagents were more reactive than
the benzotriazole derivatives (Tables 3 and 4, 18c, 18a vs. 7,
The novel uronium coupling reagents were tested and
compared with classical immonium salts (including the ben-
zotrizole derivatives 19, 20, and 21, containing pyrrolidino-
morpholino systems, Figure 6) with the aid of these models,
which involve stepwise and also
Table 3. Levels of coupling of Fmoc-Val-Val-NH
2
with use of several dif-
[
2+1]
segment
coupling
ferent coupling reagents and a range of equivalents of DIEA in DMF as
[
a]
(Tables 5 and 6). For the step-
wise coupling of Z-Phg-OH to
H-Pro-NH2 to produce Z-Phg-
a solvent.
HATU (4)
yield [%]
HDMA (7)
yield [%]
COMU (18c) HOTU (18a)
yield [%] yield [%]
time
min]
[
Pro-NH2,
the
oxime-based
2
equiv 1 equiv 2 equiv 1 equiv 2 equiv 1 equiv 2 equiv 1 equiv
COMU (18c), HOTU (18a),
HTODC (18e), and HDMODC
(18g) gave better conservation
of chirality than the benzotria-
zole-based HATU (4), HBTU
5
1
2
3
6
1
83.0
87.6
90.5
92.5
93.0
94.0
70.0
76.0
80.0
82.0
82.0
83.0
94.8
95.0
96.4
98.0
99.0
99.0
80.0
85.0
90.0
93.5
95.5
96.0
95.1
96.0
98.0
98.5
100.0 96.0
100.0 98.0
82.0
86.0
90.1
94.5
85.0
89.0
91.0
93.0
94.0
96.0
71.0
78.0
83.0
86.0
87.0
88.0
0
0
0
0
Figure 6. General structures of
benzotriazole-based immoni-
um salts containing pyrrolidi-
no-morpholino systems.
20
(
5), HDMA (7), HDMB (8),
[
a] HPLC conditions: linear gradient of 10 to 90% CH
3
CN/0.1% TFA in
[7]
À1
and 6-HDMCB (9) (Table 5).
2
H O/0.1%TFA over 30 min, detection at 200 nm. Flow rate=1 mL min
The dimethyl-morpholino de-
rivative COMU (18c) induced
less racemization than other Oxyma derivatives containing
different iminium moieties (18a, 18b, 18d) and than other
uronium salts containing different oxime substituents (18g,
Column: Waters C18, 5 mm, 4.6ꢄ150 mm (Waters Dual Wavelength De-
tector and Waters 717 Plus auto sampler). t (Fmoc-Val-OH)=20.89 min,
(Fmoc-Val-Val-NH )=19.88 min.
R
t
R
2
Table 4. Levels of coupling of Z-Aib-Val-OMe with use of several differ-
ent coupling reagents and a range of equivalents of DIEA in DMF as a
solvent.
1
8l). In the oxime series, the worst results were obtained
[
a]
HATU HBTU HDMA (7)
COMU (18c) HOTU (18a)
yield [%] yield [%]
2
Table 5. Yields and racemization for the formation of Z-Phg-Pro-NH in
DMF (solution-phase synthesis).
[
a]
(
4)
(5)
yield [%]
time
min]
yield
yield
[%]
[
Coupling reagent
Base (equiv)
Yield [%]
d,l [%]
[%]
2
equiv 2 equiv 2 equiv 1 equiv 2 equiv 1 equiv 2 equiv 1 equiv
HATU (4)
DIEA (2)
TMP (2)
DIEA (1)
DIEA (2)
TMP (2)
DIEA (1)
DIEA (2)
TMP (2)
DIEA (1)
DIEA (2)
TMP (2)
DIEA (1)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
TMP (2)
TMP (1)
DIEA (2)
TMP (2)
78.4
77.9
74.8
80.2
81.2
75.0
81.2
80.3
82.3
80.8
79.9
82.3
84.5
89.9
88.9
90.1
78.9
90.0
80.3
88.2
91.0
93.0
89.3
89.8
89.3
90.1
88.7
90.2
85.3
86.0
3.1
2.1
2.4
8.2
6.4
5.3
1.6
3.9
1.6
3.8
7.8
3.1
1.5
2.6
4.9
2.1
0.17
1.20
0.70
0.12
0.90
0.40
0.31
0.32
0.39
0.40
0.44
0.35
28.9
13.6
2
5
1
2
3
6
1
80.0
85.0
87.0
89.0
90.0
91.0
92.0
70.0
76.0
78.0
80.0
81.0
83.0
83.0
89.0
91.0
93.0
94.0
96.0
97.0
98.0
80.5
86.5
88.0
89.5
90.0
91.0
91.0
89.0
92.0
93.0
95.0
97.0
98.0
99.0
85.0
89.0
90.0
91.0
93.0
96.0
97.0
85.0
88.0
89.0
90.0
91.0
91.3
92.0
69.0
74.0
77.0
82.0
86.0
86.0
86.0
HBTU (5)
HDMA (7)
HDMB (8)
0
0
0
0
20
[
H
a] HPLC conditions: linear gradient of 10 to 90% CH
O/0.1%TFA over 30 min, detection at 200 nm, flow rate=1 mL min
Column: Waters C18, 5 mm, 4.6ꢄ150 mm (Waters Dual Wavelength De-
tector and Waters 717 Plus auto sampler). t (Z-Aib-OH)=18.25 min, t
Z-Aib-Val-OMe)=20.90 min. t (Z-Aib-OAt)=22.30 min, t (Z-Aib-
OBt)=22.80 min, t (Z-Aib-Oxyma)=23.90 min.
3
CN/0.1% TFA in
À1
2
,
R
R
6-HDMCB (9)
HMPyA (19)
HMPyB (20)
HMPyC (21)
HOTU (18a)
(
R
R
R
4
). Again, the morpholine derivatives were superior to their
COMU (18c)
tetramethyl counterparts (Tables 3 and 4, 18c, 7 vs. 18a, 4).
In both cases, COMU (18c) was superior to HATU (4), the
most potent of the currently commercially available cou-
pling reagents. This superiority was more remarkable when
only 1 equiv of base was used (Table 3 and Table 4), thereby
reaffirming the hydrogen bond acceptor role of the oxygen
in the morpholino moiety.
Once these encouraging results with the Oxyma-based
COMU (18c) had been obtained, a deeper study using sev-
eral oxime derivatives were carried out. Two model pep-
tides, Z-Phg-Pro-NH and Z-Phe-Val-Pro-NH , were used to
TMP (1)
HDmPyOC (18b)
HMPyOC (18d)
HTODC (18e)
HDMODC (18g)
HDmPyODC (18 f)
HMPyODC (18h)
HTOPC (18k)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
HDMOPC (18l)
[
7]
[
a] ll and dl forms of the test dipeptide are described elsewhere. The
values for ll and dl were identified by co-injection with authentic and
pure samples of ll. HPLC system: linear gradient of 20 to 50% CH CN/
.1% TFA in H O/0.1%TFA over 30 min, detection at 200 nm Water
Symmetry C18 5 mm 4.6ꢄ150 mm, tR ll =26.01 min., tR dl =27.40 min.
t
R
2
2
3
study the retention of configuration achieved with the new
coupling reagents.
0
2
[7]
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9407

F. Albericio, A. El-Faham et al.
Table 6. Yields and racemization for the formation of Z-Phe-Val-Pro-
Table 7. Yields and racemization for the formation of Z-Phe-Val-Pro-
NH (2+1) in DMF (solid-phase synthesis).
2
[
a]
[a]
NH
2
(2+1) in DMF (solution-phase synthesis).
Coupling reagent
Base (equiv)
Yield [%]
ldl [%]
Coupling reagent
Base (equiv)
Yield [%]
ldl [%]
HATU (4)
DIEA (2)
DIEA (1)
TMP (2)
TMP (1)
DIEA (2)
DIEA (1)
TMP (2)
DIEA (2)
DIEA (1)
TMP (2)
TMP (1)
DIEA (2)
DIEA (1)
TMP (2)
TMP (1)
DIEA (2)
TMP (2)
TMP (2)
TMP (2)
TMP (2)
DIEA (2)
TMP (2)
TMP (1)
DIEA (2)
TMP (2)
TMP (1)
DIEA (2)
TMP (2)
DIEA (2)
TMP (2)
TMP (2)
85.8
83.2
78.0
76.1
89.7
78.6
81.2
89.3
87.4
86.2
84.1
88.7
86.3
87.1
80.1
79.9
80.8
90.1
86.8
89.9
91.2
88.7
80.3
91.3
89.8
90.3
92.0
88.0
92.3
91.0
89.1
82.3
88.0
90.0
87.2
89.4
88.3
79.8
90.2
87.2
13.9
11.0
5.3
HATU (4)
HBTU (5)
HDMA (7)
HDMB (8)
6-HDMCB (9)
HOTU (18a)
TMP (2)
TMP (2)
TMP (2)
TMP (2)
TMP (2)
TMP (2)
TMP (1)
TMP (2)
TMP (1)
TMP (2)
TMP (1)
TMP (2)
TMP (1)
90.0
89.0
90.0
92.0
92.0
91.0
85.0
94.0
92.0
92.0
86.0
94.0
88.0
13.0
27.0
12.0
23.0
20.0
27.1
25.0
23.0
21.0
38.0
28.9
25.7
23.3
4.9
HBTU (5)
HDMA (7)
27.7
16.3
14.2
10.5
5.1
COMU (18c)
3.7
3.8
HTODC (18e)
HDMODC (18g)
HDMB (8)
20.3
11.5
13.3
10.5
13.9
6.5
4.1
15.3
8.7
23.6
7.4
7.5
19.3
7.0
3.5
[
a] The coupling was performed with Fmoc-Pro-Rink amide-PS-resin and
3
equiv of Z-Phe-Val-OH, 3 equiv of coupling reagent, 6 equiv of base
6
-HDMCB (9)
(
TMP), and preactivation for 10–30 s in DMF at RT. The peptide was re-
covered after deblocking with water in TFA (10%) for 1 h at RT. The
solvent was removed under vacuum and then washed with hexane. The
crude peptide was injected into the HPLC system by using a previously
reported method. Extra peaks related to the starting material (Z-Phe-
Val-OH) were observed in 3–5% percentages. lll and ldl forms of the
HMPyA (19)
HMPyB (20)
HMPyC (21)
HOTU (18a)
[7]
[7]
test tripeptide are described elsewhere. Samples were co-injected with
authentic and pure samples of lll.
COMU (18c)
HDmPyOC (18b)
HMPyOC (18d)
HTODC (18e)
20.7
7.9
Fmoc-Aib-OH onto the Aib-containing resin were deter-
mined by reversed-phase HPLC analysis, after cleavage of
the peptide from the resin (Table 8). The best results were
obtained with the Oxyma-based COMU (18c) and HOTU
26.3
10.2
17.7
16.3
17.9
13.2
22.1
23.2
43.1
40.7
43.6
40.2
[
b]
TMP (1)
DIEA (2)
(
18a), with higher per ACHTUNGTREUNNNGc ent ACTHUNGTRENNUNGa ges of target pentapeptide being
HDMODC (18g)
[
b]
TMP (1)
TMP (2)
TMP (2)
TMP (2)
TMP (1)
TMP (2)
TMP (1)
HDmPyODC (18 f)
HMPyODC (18h)
HTOPC (18k)
Table 8. The percentages of des-Aib (H-Tyr-Aib-Phe-Leu-NH
2
) obtained
during solid-phase assembly of the pentapeptide (H-Tyr-Aib-Aib-Phe-
[
a]
2
Leu-NH ).
HDMOPC (18l)
Coupling reagent
Base (equiv)
Pentapeptide [%]
des-Aib [%]
[
7]
[
a] lll and ldl forms of the test tripeptide are described elsewhere.
Samples were co-injected with authentic and pure samples of lll. HPLC
system: linear gradient of 20% to 80% CH CN/0.1% TFA in H O/
.1%TFA over 30 min, detection at 200 nm, Waters C18 5 mm 4.6ꢄ
50 mm column, tR lll =19.98 min., tR ldl =21.05 min. [b] Extra peak relat-
HATU (4)
DIEA (2)
DIEA (1)
DIEA (2)
DIEA (1)
DIEA (2)
DIEA (1)
DIEA (2)
DIEA (1)
DIEA (2)
DIEA (1)
DIEA (2)
83.0
68.0
47.0
33.0
98.0
90.0
89.0
64.0
98.7
39.0
99.0
0.3
87.5
99.7
29.3
99.86
85.5
95.3
27.4
41.3
17
32
53
67
<1
10
10
36
1.3
62.0
1.0
99.7
12.5
0.26
70.7
0.14
13.5
4.7
HBTU (5)
HDMA (7)
HDMB (8)
3
2
0
1
ed to the starting material (Z-Phe-Val-OH) was observed in 3–5% ratio.
6
-HDMCB (9)
with the cyano-pyridinyl system (18k, 18l) because of the
higher acidity of the corresponding oxime.
For the same model but with the reaction carried out on
solid-phase, COMU (18c) gave the best coupling yield, to-
gether with levels of racemization similar to those seen with
the HOBt derivatives (Table 7).
HOTU (18a)
COMU (18c)
[
b]
c]
DIEA (1)
DIEA (2)
DIEA (2)
DIEA (1)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
[
[
b]
[c]
HTODC (18e)
HDMODC (18g)
HTOPC (18k)
To check the effectiveness of the new reagents, the de-
manding Leu-enkephalin derivative H-Tyr-Aib-Aib-Phe-
72.9
58.3
[6f]
HDMOPC (18 L)
Leu-NH2 was manually assembled on Fmoc-RinkAmide-
AM-resin with the use of amino acid/activator (3 equiv),
DIEA (6 or 3 equiv) and 30 min coupling times, except in
the case of Aib-Aib, for which 1 h double coupling was
used. Percentages of incorporation for the coupling of
[
a] Pentapeptide and deletion tetrapeptide des-Aib were confirmed by
peak overlap in the presence of authentic samples. HPLC-MS showed
the correct mass for the pentapeptide at 612.0. [b] 1 h standard single
coupling was performed for Aib-Aib with only 1 equiv of base. [c] 30 min
standard single coupling was performed for Aib-Aib with 2 equiv of base.
9408
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ꢃ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 9404 – 9416

Benzotriazole-Based Uronium Coupling Reagents
FULL PAPER
obtained than with HDMA (7) or HATU (4). Synthesis with
COMU (18c) led to only a 0.26% yield of des-Aib when
remove Et NH) DMF, as in the rest of experiments, in order
2
to examine the effect of the solventꢅs purity (entries 1–6 vs.
7–12). The assay with standard DMF reflected the huge dif-
ference in reactivity between HOAt- and HOBt-derived
uronium salts, with an impressive—for such demanding con-
ditions—91% yield of pentapeptide being obtained with
HDMA (7), whereas HDMB (8) only afforded a poor 7%
(entries 1, 2). The Oxyma-based COMU (18c) gave a much
higher purity than HDMB but, unlike in the previous syn-
2
equiv of DIEA were used, whereas its tetramethyl deriva-
tive 18a gave a 1% yield under the same conditions. For a
30 min coupling, 18c gave a 0.14% yield of des-Aib whereas
18a gave a 12.5% yield. This observation indicates that the
morpholino moiety increases the reactivities of uronium
salts relative to tetramethyl derivatives. These results are
consistent with what is discussed above.
In view of the superiority showed by the morpholino-con-
taining uronium salts over their tetramethyl counterparts,
the effect of the leaving group was further tested by compar-
ing the benzotriazole-based HDMA (7) and HDMB (8) and
the Oxyma-based COMU (18c) in the manual solid-phase
thesis of H-Tyr-Aib-Aib-Phe-Leu-NH , it was far from that
2
afforded by HDMA (43%, entry 3). The experiment was re-
peated, but with preactivation with only 1 equiv of base, a
second equiv being added once the coupling mixture had
been added onto the resin, in order to examine whether the
base had any effect on the stability of the active ester during
preactivation. The results showed no significant variation,
except in the case of COMU (18c), for which the yield rose
from 43% to 55% (due to the higher rate of coupling for
the MeLeu residue), suggesting a high reactivity of the
Oxyma-derived active ester (Table 9, entries 4, 5, and 6).
The percentage of pentapeptide also increased to the same
extent with COMU (18c) when the experiment with 2 equiv
DIEA was carried out with treated, instead of standard,
DMF, confirming the positive effect of the higher purity of
the solvent, although this was not noticeable in the experi-
ments with HDMA and HDMB (Table 9, entries 7, 8, and
9). Finally, an experiment was conducted with only 1 equiv
DIEA, displaying the same tendency as observed previous-
ly: the performance of COMU (18c) was superior to that of
the HOBt derivative but not as potent as that of the HOAt
one (Table 9, entries 10, 11, and 12).
assembly of H-Tyr-MeLeu-MeLeu-Phe-Leu-NH on Fmoc-
2
RinkAmide-AM-PS-resin. The strategy followed for the
assay began with the elongation of resin-bound tripeptide
H-MeLeu-Phe-Leu-resin by use of DIC/Oxyma (10) in
30 min couplings. Quantitative yields were verified by
means of the Kaiser test for primary amines. After this pre-
liminary step, comparisons were made for the stepwise in-
corporation of the two last residues, with use of the corre-
sponding immonium/uronium salt and Fmoc-amino acid.
Samples were preactivated for 20–30 s with DIEA (2 or
1
equiv relative to uronium salt/Fmoc-amino acid) in order
to avoid guanidylation of the growing peptide chain. The
coupling times were shortened to 5 min, so that significant
differences in the reactivities of the coupling reagents could
arise. After cleavage from the resin with 90% TFA/10%
H O and lyophilization, relative performances were checked
2
in terms of percentages of pentapeptide and deletion pep-
tides, as determined by reversed-phase HPLC analysis
The effectiveness of COMU (18c) was compared with
that of HOTU (18a) in the synthesis of the common deca-
(
Table 9).
[6f]
The experiments were carried out either in standard
99.8% purity, as determined by GC) or in treated (anhy-
peptide model ACP (65–74) on solid-phase (Table 10).
(
drous, dried over molecular sieves and bubbled with N to
2
Table 10. Preparation of ACP (65–74) (H-Val-Gln-Ala-Ala-Ile-Asp-Tyr-
Ile-Asn-Gly-NH ) with use of the different coupling reagents (2 equiv)
and 2 min coupling times.
2
[
a]
Table 9. Percentages of H-Tyr-MeLeu-MeLeu-Phe-Leu-NH
deletion peptides obtained in solid-phase assembly in 5 min with use of
2
and related
69
72
Coupling re-
agent
ACP
[%]
Des-Asn
[%]
Des-Val
[%]
Des-Ile
[%]
Des-Ile
[%]
[
a]
various dimethyl-morpholino immonium/uronium salts.
Entry Coupling conditions Coup. Penta des-
reagent [%] MeLeu
%]
HOTU (18a) 66.0
COMU (1cc) 79.4
0.27
0.74
2.72
–
21.7
18.0
7.56
0.98
des-Tyr Trip
[%]
[%]
[
a] The HPLC-MS showed the correct mass for the ACP at 1065.0.
[
1
2
3
4
5
6
7
8
9
1
1
1
HDMA 91.4
HDMB 7.0
COMU 42.9
HDMA 91.5
4.5
42.2
48.5
5.0
42.4
39.5
6.7
43.0
38.5
18.5
37.8
51.9
3.8
9.7
4.4
3.1
9.4
3.0
3.6
9.2
3.3
7.4
8.9
6.3
0.3
41.1
4.2
0.4
41.3
2.0
0.3
41.0
2.2
0.4
47.6
8.2
2
equiv
DIEA
standard
DMF
The peptide was manually elongated on a Fmoc-Rink
Amide-AM-resin (0.7 mmolg ). Coupling times of 2 min
À1
2
equiv
HDMB
6.9
[
b]
DIEA
were used and excesses of reagents were 2 equiv for cou-
pling reagents and amino acids and 4 equiv for DIEA. In-
COMU 55.5
HDMA 89.4
2
equiv
72
69
HDMB
6.8
corporation was detected for Ile onto Asn and for Ile
DIEA
treated
DMF
COMU 56.0
HDMA 73.7
onto Asp. Peptide purity was determined by reversed-phase
HPLC analysis, after cleavage of the peptide from the resin
0
1
2
1
equiv
HDMB
5.7
DIEA
by treatment with TFA/H O (9:1) for 2 h at RT. HPLC anal-
2
COMU 33.6
ysis again showed a better performance of the morpholino-
containing derivative than for the tetramethyl analogue:
some deletion peptides, such as des-Val, were not observed
with COMU (18c) and the percentage of pentapeptide ob-
[
a] HPLC-MS showed the correct mass for the pentapeptide at 695.5.
Fmoc-amino acids were preactivated for 20–30 s. [b] Fmoc-amino acids
were preactivated with only 1 equiv DIEA, with addition of another
1
equiv onto the resin after the first addition.
Chem. Eur. J. 2009, 15, 9404 – 9416
ꢃ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9409

F. Albericio, A. El-Faham et al.
tained was higher than that obtained with HOTU (18a)
ing from the starting induction period with no thermal
signal and their unexpected steep initiation. Therefore, a
temperature alarm in an industrial process is not effective
with compounds that show this kinetic behavior. Neverthe-
less, a dynamic DSC experiment can provide only indica-
tions of the autocatalytic nature of decomposition.
(
Table 10, entry 1 vs 2).
The safety profile of COMU (18c), the most efficient of
the uronium salts tested, was also considered and compared
to those of HDMA (7) and HDMB (8). This issue was
highly relevant in view of the explosive properties of benzo-
triazole-based additives and derived stand-alone coupling
reagents, which limit their transportation. Although explo-
sivity has never been reported for HDMA (7) or HDMB
In contrast, COMU decomposed in a more constant
manner that did not resemble this autocatalytic pattern. The
normalized exothermic DH values should also be noted:
À1
À1
À1
(
8), the fact that they contain HOBt/HOAt and that other
209 kJmol
for HDMB, 245 kJmol
for HDMA, and
immonium salts, such as TBTU, have also shown explosive
183 kJmol for COMU. These observations indicate that in
the event of an explosion, COMU would have less thermal
severity. The DTad value (adiabatic temperature rise), calcu-
lated from experimentally ascertained exothermic DH
values, also shows this relative severity: 2488C for HDMB
(8), 2908C for HDMA (7), and 2148C for COMU (18c). To
conclude with the information that can be extracted from
the DSC assay, the onset temperature at which decomposi-
tion began was lower in the case of the experiment with
COMU (18c) (1608C) than with HDMA (7) or HDMB (8)
(1778C and 1808C, respectively).
[10]
properties
means that a certain safety risk is assumed.
Our main interest was focused on checking whether the
novel coupling reagent COMU (18c) would display the
common pattern observed in explosive compounds: high re-
[14]
lease of pressure associated with rapid decomposition.
The thermal risk was assessed through a combination of
two calorimetry assays: Differential Scanning Calorimetry
(
DSC) and Accelerating Rate Calorimetry (ARC). A pre-
liminary assessment of the risk associated with a given de-
composition can be determined by means of a dynamic DSC
experiment. The heat released during this assay is measured
by comparing it with a reference that has undergone the
same thermal process. In addition, the relative decomposi-
tion kinetics (which are linked to explosivity) are highlight-
A further evaluation of the risks associated with these
compounds was carried out under adiabatic conditions by
[17]
the ARC technique.
By this approach, the pressure re-
leased and the DTad can be directly determined. The assay
begins with the application of the “heat-wait-seek” method,
and when self-heating of the sample at a rate higher than
[
15]
ed. In this assay, samples are heated in a closed crucible
under a flow of N from 30 to 3008C at a constant heating
2
À1
À1
rate of 108Cmin . Diagrams displaying the heat flow as a
0.028Cmin is detected, the experiment is changed to adia-
function of time and temperature showed a distinct differ-
ence in decomposition behavior between benzotriazole-
based HDMA (7) and HDMB (8) and the novel COMU
batic mode. When decomposition occurs, the temperature
and the pressure rise, and once the temperature reaches
values above 3008C the assay is stopped manually. In all
cases the pressure rises detected were relatively low, in com-
parison with those of related additives. In particular, the
released pressure measured in the COMU (18c) experiment
was similar to that seen with HDMA (7) (53 vs. 55 bar) and
slightly higher than that seen with HDMB (8) (24 bar)
(
Figure 7). During the decomposition of HDMA (7) and
[11]
(
Figure 8).
Additionally, the increase in temperature during decom-
position (DT ) was considerably lower with COMU (18c)
ad
(
1
648C) than with HDMA (7) or HDMB (8) (1648C and
218C, respectively), thereby confirming the lower thermal
Figure 7. Thermograms showing heat flow versus temperature and time
in the DSC experiments with HDMA (7), HDMB (8), and COMU (18c).
HDMB (8) the release of heat was slow at the beginning
and increased very sharply, reaching a maximum and finally
decreasing. This decomposition profile as observed for
HDMA (7) and HDMB (8) resembles that of an autocata-
lytic reaction, in which the product also acts as a catalyst,
the rate of the reaction increasing at the same time as the
Figure 8. Decomposition profiles observed during ARC experiments with
HDMB (8, &), HDMA (7, ^), and COMU (18c, ~) showing released
pressure (bar) as a function of time (min).
[16]
conversion. These self-accelerating reactions warrant spe-
cial attention because of their great unpredictability, result-
9410
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ꢃ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 9404 – 9416

Benzotriazole-Based Uronium Coupling Reagents
FULL PAPER
severity of COMU observed in the DSC assay (Figure 9).
Also consistent with the previous assay were the distinct ki-
netic profiles of COMU (18c) and the benzotriazole-based
Table 11. Experimentally determined and calculated values obtained
from dynamic DSC and ARC assays with the different stand-alone cou-
pling reagents.
DSC
ARC
calcd DTad Onset Dp
Coupling
reagent
Onset DH
8C] [kJmol
exptl DTad
À1
[a]
[
A
H
U
G
R
N
U
G
]
[8C]
[8C]
ACHTUNGTRENUNNG[ bar] [8C]
HDMA (7)
HDMB (8)
COMU (18c) 160
177
180
245
209
183
290
248
214
119
122
91
55
24
53
164
121
64
[
(
2
a] Calculated DTad (8C) was obtained from the general formula DTad
À1
À1
À1
À1
8C)=DH (Jmol )/
A
H
U
G
R
N
U
G
A
H
U
G
R
N
N
(gmol )·c
p
(kJkg 8C )], with estimated Cp=
À1
À1
kJkg 8C
.
follow autocatalytic or non-autocatalytic kinetics. Tempera-
tures were set at 108C below the onset observed in the cor-
responding dynamic DSC assay, and remained constant for
4
80 min. As a result, we obtained thermograms showing
Figure 9. Decomposition profiles observed during ARC experiments with
HDMB (8, &), HDMA (7, ^), and COMU (18c, ~) showing temperature
heat flow versus time (Figure 10). As would be expected for
(
8C) as a function of time (min).
immonium salts. Whereas the temperatures in the HDMA
7) and HDMB (8) experiments increased slowly at the be-
(
ginning and rose suddenly to their maxima (similarly to the
DSC results, suggesting autocatylic kinetics), in the COMU
(
18c) experiment the temperature reached approximately
one third of the total DTad over a period of time longer than
had been required for the whole decomposition of HDMA
(
7) and HDMB (8). These slower kinetics could enable pres-
sure originating from decomposition to be released into the
environment.
Figure 10. Thermograms showing heat flow versus time for isothermal
DSC experiments set at 108C below the onset temperatures of HDMA
(7, 1678C), HDMB (8, 1708C), and COMU (18c, 1508C).
With regard to the onset temperatures, ARC allows more
accurate determination than DSC, which is known to suffer
from uncertainty due to the smaller scale. For COMU (18c),
decomposition began at a lower temperature than for
HDMA (7) and HDMB(8) (918C vs. 1198C and 1228C). For
safe working, it is recommended that the temperature of a
given compound be maintained at values at which the time
to maximum rate under adiabatic conditions is longer than
an autocatalytic reaction, HDMA (7) and HDMB (8) each
defined a bell-shaped heat release curve, reported for this
type of kinetics, in which the reaction accelerated, passing
through a maximum of heat release and then decreasing. In
contrast, COMU (18c) displayed a typical non-autocatalytic,
nth-order kinetic profile, in which the rate of heat release
[18]
2
4 h. This temperature can commonly be estimated after
running an ARC assay, by subtraction of 50 K from the ob-
[19]
[16]
served onset. This safety value is at a lower temperature
decreased uniformly with time. The former distinct kinetic
with COMU (18c) than with HDMA (7) and HDMB (8)
profile strongly suggests the risk of a thermal runaway,
which may lead to a sudden explosion, and consequently the
safety measures that need to be taken.
(
418C vs. 698C and 728C). Although peptide chemistry is
usually performed at room temperature, and therefore
below this safety threshold value, these results show that
COMU has less thermal stability than benzotriazole-based
immonium salts that also contain the morpholino moiety.
The experimentally determined and calculated values deter-
mined from calorimetric studies and discussed above are
shown in Table 11.
To study further the autocatalytic natures of the decom-
positions of HDMA (7) and HDMB (8), as suggested by the
results obtained from the dynamic DSC and ARC experi-
ments, in contrast with the results obtained for COMU
Conclusions
In conclusion, we describe a new class of O-form uronium-
type coupling reagents that differ in their immonium moiet-
ies and also in the leaving groups. The presence of the mor-
pholino group has a marked influence on the polarity of the
carbon skeleton, which affects the solubility, stability, and
the reactivity of the reagent. As would be expected, HOAt
derivatives were in all cases confirmed to be superior to
HOBt ones in terms both of coupling yields and of retention
(
18c), we performed isothermal DSC assays. This technique
is the most reliable way to detect whether decompositions
Chem. Eur. J. 2009, 15, 9404 – 9416
ꢃ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9411

F. Albericio, A. El-Faham et al.
of configuration. Remarkably, Oxyma derivatives often gave
results similar to or even better than those obtained with
the aza derivatives, and also performed extremely well in
the presence of only 1 equiv of base, thereby confirming the
effect of the oxygen as a hydrogen bond acceptor in the re-
action. The excellent performances of Oxyma derivatives,
despite their acidities being similar to that of HOBt and
higher than that of HOAt, can be explained by the fact that
the O-form is the only form present during the coupling. In
the benzotriazole derivatives, the N-form is the predominant
one.
scopic, so it was dissolved directly in CH
tassium hexafluorophosphate (100 mmol in 50 mL water, KPF
added at room temperature with vigorous stirring for 10–15 min. The or-
ganic layer was collected, washed once with water (100 mL), dried over
anhydrous MgSO , and filtered. The solvent was removed under reduced
4
pressure to give a white solid that was recrystallized from CH
or acetonitrile/ether to give white crystals.
2
Cl
2
, and saturated aqueous po-
) was
6
2 2
Cl /ether
4
-[(Dimethylamino)chloromethylene]morpholin-4-iminium hexafluoro-
[7]
phosphate (DMCH, 15a): The salt was obtained as white crystals in a
1
yield of 89.6% (28.9 g). M.p. 94– 958C; H NMR (CD
s, 6H; 2CH ), 3.75 (t, 4H; 2CH ), 3.86 ppm (t, 4H; 2CH
(CD COCD ): d=44.36, 52.82, 65.99, 162.79 ppm.
3 3
COCD ): d=3.39
13
(
3
2
2
); C NMR
3
3
N-[Chloro(pyrrolidin-1-yl)methylene]-N-methylmethanaminium
hexa-
[23]
By means of calorimetry techniques, the safety profile of
COMU (18c) was compared with those of benzotriazole-
based HDMA (7) and HDMB (8). Although the pressure
rises with the three coupling reagents were similar, COMU
fluorophosphate (DmPyCH, 15b):
The product was obtained as a
1
white solid in a yield of 89.0%. M.p. 93–958C; H NMR (CD
3
COCD
3
):
d=2.00–2.13 (m, 4H; 2CH
2
), 3.49 (s, 6H; 2CH
3
), 3.90–4.02 ppm (m, 4H;
2
CH
2
).
1
-[Chloro(morpholino)methylene]pyrrolidinium
hexafluorophosphate
(
18c) decomposed in a much slower and more controlled
(
MPyCH, 15c): The chloro salt was obtained by the method described
manner, with less thermal severity but greater predictabili-
above. The product was obtained as a white solid in a yield of 83.9%.
[
20]
1
M.p. 99–1008C; H NMR ([D
.87 (t, 4H; 2CH ), 4.00 (t, 4H; 2CH
): d=25.80, 51.75, 55.97, 65.97, 154.85 ppm; elemental
analysis (%) calculated for C OP (348.65): C 31.00, H 32.69, N
8.03; found: C 31.00, H 32.69, N 8.31.
6
]acetone): d=2.10–2.14(m, 4H; 2CH
2
),
ty.
Moreover, the decomposition of HDMA (7) and
3
2
2
), 4.04–4.06 ppm (m, 4H; 2CH
2
);
HDMB (8), unlike that of COMU (18c), displayed autocata-
lytic kinetics, being highly unpredictable and difficult to
detect before ending up in thermal runaway.
1
3
C NMR (CDCl
3
9
6 2
H16ClF N
General Procedure for preparation of uranium-type coupling reagents:
The chloro salt (20 mmol) was added at 08C to a solution of an oxime
potassium salt or a benzotriazole derivative (20 mmol) in acetonitrile
Experimental Section
(
50 mL). The reaction mixture was stirred at this temperature for 30 min
and was then allowed to come to room temperature with stirring over
h. The crude product was filtered and washed with acetonitrile. The sol-
General: TLC was performed on silica plates (8ꢄ4 cm, Albet) in suitable
6
solvent systems with visualization with
a Spectroline Model CM-10
vent was concentrated to a small volume (1/4) under reduced pressure,
and dry ether was then added to afford the product as a white solid in a
pure state.
UV lamp (254 nm). Melting points were measured in open capillary
tubes with a Buchi B-540 melting point apparatus and were uncorrected.
Infrared (IR) spectra were recorded on a Thermo Nicolet series Fourier
Transformer instrument as KBr pellets. The absorption bands (nmax) are
Synthesis of the potassium salt of hydroxycarbonimidoyl dicyanide
[21]
(
17a): Sodium nitrite (14.2 g, 206 mmol) was slowly added at 08C (20–
À1
given in wavenumbers (cm ). A Shimadzu UV-250/PC instrument was
3
0 min addition) to a solution of malononitrile (9.06 g, 138 mmol) in
used as a UV/Vis spectrophotometer. NMR spectra were recorded on a
Varian mercury 400 MHz spectrometer at room temperature. Tetrame-
thylsilane (TMS) was used as reference for all NMR spectra, with chemi-
cal shifts reported as ppm relative to TMS. HPLC analyses were carried
out with a Waters Symmetry Column C18, 5 mm, 4.6ꢄ150 mm with dual l
absorbance detector. HPLC-MS analyses were carried out with a Waters
Symmetry Column C18, 5 mm, 4.6ꢄ150 mm with dual detector. All sol-
vents used for recrystallization, extraction, column chromatography, and
TLC were of commercial grade, distilled before use, and stored under
dry conditions.
acetic acid (20 mL) and water (50 mL). This mixture was then stirred at
the same temperature for 45 min. After quenching of the reaction with
HCl (2n, 100 mL), the compound was extracted three times with ether
(
2 4
3ꢄ100 mL). The extracts were dried over anhydrous Na SO , and the
ether was removed under vacuum to give an oily residue. The oily prod-
uct was added slowly to a cold solution of KOH (8.0g) in MeOH
(
100 mL), and then the reaction mixture was stirred for 20 min. Excess
ether was added to afford the potassium salt as yellow crystals in a yield
13
of 14.1g (77.5%). C NMR (DMSO): d=107,40, 113.57, 119.78 ppm.
Synthesis of the potassium salt of diethyl 2-(hydroxyimino)malonate
[
7]
N,N-Dimethylmorpholine-4-carboxamide (DMU, 14a): The urea deriv-
[22]
(
17c): A solution of diethyl malonate (16 g, 0.1 mol) in glacial acetic
ative was distilled and collected at 127–1298C as a colorless oil in a yield
acid (17.5 mL, 0.3 mol) was stirred vigorously at 0–58C while a solution
of NaNO (20.7 g, 0.3 mol) in water (250 mL) was added dropwise over
–4 h. The ice bath was removed and the mixture was stirred vigorously
1
of 92.4% (73g from 0.5 mol reaction). H NMR (CDCl
3
): d=2.84 (s, 6H;
);
2
2
CH
3
), 3.22–3.2 (m, 4H; 2CH
C NMR (CDCl ): d=38.62, 47.51, 66.89, 164.96 ppm.
N,N-Dimethylpiperidine-1-carboxamide (DmPyU, 14b):
urea was obtained as a colorless oil at 98–1008C in a yield of 85.5%.
2
), 3.68–3.70 ppm (m, 4H; 2CH
2
3
1
3
3
for a further 20 h. The nitrosation was carried out in three-necked flasks
with appropriate fittings and a small vent to allow nitric oxide to escape.
[
24,25]
The pure
The reaction mixture was extracted with CH
00 mL portions). The combined CH Cl extracts were dried over anhy-
drous Na SO . The CH Cl was removed under vacuum and the resulting
oily product was dissolved in CH Cl (400 mL) and then stirred with an-
hydrous K CO (32g) for 15 min. After filtration, the CH Cl was re-
2 2
Cl (400 mL and then 3ꢄ
1
H NMR (CDCl
3
): d=1.81–2.10 (m, 4H; 2CH
).
2 3
), 2.81(s, 6H; 2CH ),
1
2
2
3
.15–3.18 ppm (m, 4H; 2CH
2
2
4
2
2
Morpholino(pyrrolidin-1-yl)methanone (MPyU, 14c): The urea derivative
was distilled and collected at 127–1298C as a colorless oil in a yield of
2
2
2
3
2
2
1
9
2
2.4% (73g from 0.5 mol reaction). H NMR (CDCl
3
): d=2.84 (s, 6H;
moved until 200 mL was reached, ether was added until the solution
became cloudy, and the mixture was then kept in the refrigerator over-
1
3
CH
3
), 3.22–3.2(m, 4H; 2CH
): d=38.62, 47.51, 66.89, 164.96 ppm.
General Procedure for the synthesis of chlorouronium salts:
chloride (100 mmol) in CH Cl (100 mL) was added dropwise at room
temperature over 5 min to a solution of urea derivative (100 mmol) in
dry CH Cl (200 mL). The reaction mixture was stirred under reflux for
h, and the solvent was removed under vacuum. The residue was washed
with anhydrous ether (2ꢄ100 mL) and was then bubbled with N to
remove the excess of the ether. The obtained residue was very hygro-
2
), 3.68–3.70 ppm (m, 4H; 2CH
2
);
C NMR (CDCl
3
night to afford off-white crystals in 63.4% yield. M.p. 116–1188C;
1
[
26]
3 3
H NMR (CDCl ): d=1.24–1.29 (q, 6H; 2CH ), 4.20–4.29 ppm (m, 4H;
Oxalyl
2
CH
2
).
2
2
[
23]
Synthesis of 2-pyridylhydroxyiminoacetonitrile (17d):
A solution of
2
2
sodium nitrite (4.5 g, 0.065 mol in 5 mL water) was added slowly, with
cooling, to a solution of 2-pyridylacetonitrile (2.2 g, 0.019 mol) in glacial
acetic acid (4.5 mL). After 12 h standing, the precipitate was filtered off,
washed with water, dried, and then recrystallized from ethanol to afford
3
2
9412
www.chemeurj.org
ꢃ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 9404 – 9416

Benzotriazole-Based Uronium Coupling Reagents
FULL PAPER
the product in 65% yield. M.p. 220–2228C (lit. mp. 219–2228C, 68%);
1-{[1-(Dicyanomethyleneaminooxy)-dimethylamino-morpholinomethyle-
ne]}methanaminium hexafluorophosphate (HDMODC, 18g): The prod-
1
H NMR ([D
6
]DMSO: d=7.45–7.52 (1H), 7.87–7.90 (2H), 8.67 (d, 1H),
1
4.12 ppm (OH).
uct was obtained as white solid in a yield of 5.7 g (74.8%). M.p. 118–
1
[
24]
1198C; H NMR ([D ]acetone): d=3.42 (s, 6H; 2CH ), 3.80–3.88 ppm
General Procedure for the preparation of urea derivatives: The N,N-
dialkylcarbamoyl chloride (0.6 mol) was added dropwise at 08C to a stir-
ring mixture of secondary amine (0.5 mol) and triethylamine (TEA, 0.5
mol) in CH Cl (400 mL). When the addition was complete, the mixture
2 2
was stirred for 3–4 h at room temperature. The reaction mixture was basi-
6
3
1
3
(
m, 8H; 4CH
2
); C NMR ([D
6
]acetone): d=40.97, 49.93, 65.92, 105.13,
1
08.15, 119.84, 159.77 ppm; elemental analysis (%) calcd for
P (381.21): C 31.51, H 3.70, N 18.37; found: C 31.68, H 3.84,
10 14 6 5 2
C H F N O
N 18.61.
fied with NaOH (10%), and the organic layer was then collected and the
1-[(Dicyanomethyleneaminooxy)(morpholino)methylene]pyrrolidinium
aqueous layer was washed with CH
solution was washed with H O (2ꢄ100 mL) and saturated solution of
NaCl (2ꢄ100 mL). Finally, the organic solvent was dried over anhydrous
MgSO and filtered, and the solvent was removed under reduced pres-
2
Cl
2
(100 mL). The combined CH
2
Cl
2
hexafluorophosphate (HMPyODC, 18h): The product was obtained as
light yellow solid in a yield of 6.4 g (78.6%). M.p. 158–1598C; H NMR
1
2
([D
3.86–3.88 (m, 4H; 2CH
([D
elemental analysis (%) calcd for C12
.96, N 17.20; found: C 35.60, H 4.06, N 17.56.
6
]acetone): d=2.11–2.14 (m, 4H; 2CH
2
), 3.79–3.85 (m, 4H; 2CH
2
),
1
3
4
2
), 3.98–4.01 ppm (m, 4H; 2CH
2
); C NMR
sure. The oily residue obtained was purified by vacuum distillation.
6
]acetone): d=25.28, 49.15, 65.95, 105.07, 108.17, 119.48, 157.00 ppm;
P (407.25): C 35.39, H
16 6 5 2
H F N O
O-[Cyano(ethoxycarbonyl)methylidene)amino]-1,1,3,3-tetramethyluroni-
um hexafluorophosphate (HOTU, 18a): The product was obtained as a
white solid in a yield of 6.32g (82.1%). M.p. 135–1378C (dec). The trie-
thylamine/HOXt approach gave a lower yield (68.7%) than the potassi-
3
O-[(Diethoxycarbonylmethylidene)amino]-1,1,3,3-tetramethyluronium
hexafluorophosphate (HTODeC, 18i): The product was obtained as a
1
1
um salt strategy, maybe due to the washing with water. H NMR
pale yellow oil in a yield of 84.5%. H NMR (CDCl
3
): d=1.35 (t, 6H;
); elemental
P (433.28): C 33.26, H 5.12, N 9.70;
(
2
1
[D
H; CH
6
]acetone): d=1.37 (1, 3H; CH
3
), 3.37 (s, 12H; 4CH
]acetone): d=13.46, 40.71, 64.56, 106.78, 135.09,
17 6 4 3
56.11, 161.43 ppm; elemental analysis (%) calculated for C10H F N O P
3
), 4.82 ppm (q,
2CH
analysis (%) calcd for C12
found: C 33.44, H 5.25, N 9.98.
-[(1,3-Diethoxy-1,3-dioxoprop-2-ylideneaminooxy)-dimethylamino-mor-
3 3 2
), 3.18 (s, 12H; 4CH ), 4.39–4.48 ppm (q, 4H; 2CH
1
3
2
); C NMR ([D
6
H
22
F
6
N
3
O
5
(
386.23): C 31.10, H 4.44, N 14.51; found: C 31.34, H 4.35, N 14.75.
1
1
-[(1-(Cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-pyrroli-
pholinomethylene)]methanaminium hexafluorophosphate (HDMODeC,
dinomethylene)]methanaminium hexafluorophosphate (HDmPyOC,
18j): The product was obtained as pale yellow oil in a yield of 84.3%.
1
1
8b): The product was obtained as a white solid, in a yield of 6.8g
H NMR (CDCl
), 3.61 (t, 2H; CH
4.50 ppm (q, 4H; 2CH
P (475.32): C 35.38, H 5.09, N 8.84; found: C 35.53, H 5.28,
3
): d=1.35 (t, 6H; 2CH
), 3.78 (t, 2H; CH
); elemental analysis (%) calcd for
3
), 3.21(s, 6H; 2CH
3
), 3.35 (t,
1
(
82.7%). M.p. 126–1278C (dec); HNMR ([D
6
]acetone): d=1.37 (t, 3H;
), 3.95–3.99(m, 4H;
]acetone): d=13.47, 25.09,
0.40, 51.58, 64.52, 106.74, 134.82, 156.12, 158.66 ppm; elemental analysis
P (412.27): C 34.96, H 4.65, N 13.59;
2H; CH
2
2
2
), 3.87 (t, 2H; CH
2
), 4.37–
CH
2
4
3
), 2.10, 2.13 (m, 4H; 2CH
2
), 3.36 (s, 6H; 2CH
3
2
1
3
CH
2
), 4.48 ppm (q, 2H; CH
2
); C NMR ([D
6
C
14
H
24
F
6
N
3
O
6
N 9.11.
(
%) calculated for C12
found: C 35.07, H 4.79, N 13.72.
-[(1-(Cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-mor-
19 6 4 3
H F N O
N-{[Cyano(pyridin-2-yl)methyleneaminooxy](dimethylamino)methylene}-
N-methylmethanaminium hexafluorophosphate (HTOPC, 18k): The
1
product was obtained as a light reddish brown solid in a yield of 6.2 g
1
pholinomethylene)]methanaminium hexafluorophosphate (COMU, 18c):
(82.7%). M.p. 169–1718C; H NMR ([D
6
]acetone): d=3.30 (s, 12H;
The product was obtained as white crystals in a yield of 7.6 g (88.8%),
and decomposes without melting at 159.908C according to dynamic DSC
3
4CH ), 7.57–7.61 (m, 1H), 7.94 (td, 1H), 8.10 (dd, 1H), 8.70 ppm (dd,
1
3
1H); C NMR ([D
142.46, 146.67, 150.68, 162.03 ppm; elemental analysis (%) calcd for
OP (391.25): C 36.84, H 4.12, N 17.90; found: C 37.00, H 4.21,
6
]acetone): d=40.48, 107.56, 122.52, 127.87, 138.18,
1
(
Figure 3). H NMR ([D
6
]acetone): d=1.38 (t, 3H; CH
), 3.89 (t, 4H; 2CH ), 4.48 ppm (q, 2H; CH
]acetone): d=13.48, 40.70, 49.94, 64.59, 66.04, 106.76,
35.03, 156.14, 160.61 ppm; elemental analysis (%) calcd for
P (428.27): C 33.65, H 4.47, N 13.08; found: C 33.79, H
3
), 3.41 (s, 6H;
2
CH
3
), 3.82 (t, 4H; 2CH
2
2
2
);
12 16 6 5
C H F N
N 18.11.
1
3
C NMR ([D
6
1
N-{[Cyano(pyridin-2-yl)methyleneaminooxy](dimethylamino)methylene}-
N-morpholinomethanaminium hexafluorophosphate (HDMOPC, 18l):
12 19 6 4 4
C H F N O
4
.59, N 13.30.
The product was obtained as a light brown solid in a yield of 7.74 g
1
1
-[(1-Cyano-2-ethoxy-2-oxoethylideneaminooxy)(morpholino)methyle-
(89.4%). M.p. 154–1558C; H NMR ([D
6
]acetone): d=3.34 (s, 12H;
ne]pyrrolidinium hexafluorophosphate (HMPyOC, 18d): The product
was obtained as white solid in a yield of 8.2 g (90.3%). M.p. 171–1728C;
4CH
3
), 3.79–3.83 (m, 8H; 4CH
2
), 7.58–7.62 (m, 1H), 7.96 (td, 1H), 8.12
1
3
(dd, 1H), 8.71 ppm (dd, 1H); C NMR ([D
6
]acetone): d=40.76, 49.79,
1
H NMR ([D
6
]acetone): d=1.26 (t, 3H; CH
.65–3.68 (m, 4H; 2CH ), 3.74–3.76 (m, 4H; 2CH
CH ), 4.37–4.40 ppm (q, 2H; CH
5.09, 49.20, 51.57, 55.98, 51.76, 64.54, 66.06, 106.69, 134.63, 156.11,
57.88 ppm; elemental analysis (%) calcd for C14 P (454.31): C
7.01, H 4.66, N 12.33; found: C 37.25, H 4.78, N 12.50.
3
), 1.98–2.02 (m, 4H; 2CH
), 3.84–3.87 (m, 4H;
2 6
); C NMR ([D ]acetone): d=13.49,
2
),
66.13, 107.60, 122.53, 127.92, 138.23, 142.74, 146.63, 150.69, 161.06 ppm;
elemental analysis (%) calcd for C14 P (433.29): C 38.81, H
4.19, N 16.16; found: C 39.04, H 4.33, N 16.47.
-[Morpholino(pyrrolidinium-1-ylidene)methyl]-1H-[1.2.3]triazolo
3
2
2
1
3
2
2
18 6 5 2
H F N O
1
3
2
1
ACHTUNGTRENNUNG[ 4,5-
21 6 4 4
H F N O
I]pyridine 3-oxide hexafluorophosphate (HMPyA, 19): The product was
obtained as a white solid in a yield of 7.3 g (81.3%). M.p. 206–2088C
1
O-[(Dicyanomethylidene)amino]-1,1,3,3-tetramethyluronium hexafluoro-
(dec); H NMR ([D
6
]acetone): d=2.11–2.15 (m, 2H; CH
), 3.48–3.63 (m, 2H; CH ), 3.79–4.16 (m, 10H; 5CH
1H), 8.53 (dd, 1H), 8.85 ppm (dd, 1H); C NMR ([D
25.09, 41.74, 42.35, 50.82, 51.58, 66.19, 66.42, 124.37, 127.84, 149.65 ppm;
elemental analysis (%) calcd for C14 P (448.30): C 37.51, H
4.27, N 18.75; found: C 37.75, H 4.42, N 19.02.
-[Morpholino(pyrrolidinium-1-ylidene)methyl]-1H-benzo[d]-
[1.2.3]triazole 3-oxide hexafluorophosphate (HMPyB, 20): The product
2
), 2.18–2.30 (m,
), 8.02 (dd,
]acetone): d=
phosphate (HTODC, 18e): The product was obtained as a white solid in
2H; CH
2
2
2
1
13
a yield of 5.0 g (74.0%). M.p. 180–1818C (dec); H NMR ([D
6
]acetone):
6
1
3
d=3.27 (s, 12H; 4CH
08.21, 119.65, 160.67 ppm; elemental analysis (%) calcd for
OP (339.18): C 28.33, H 3.57, N 20.65; found: C 28.52, H 3.65,
3 6
) ppm; C NMR ([D ]acetone): d=40.80, 105.10,
1
19 6 6 2
H F N O
8 12 6 5
C H F N
N 20.88.
1
ACHTUNGTRENNUNG
1
-{[1-(Dicyanomethylideneaminooxy)-dimethylamino-pyrrolodinomethy-
lene]}methanaminium hexafluorophosphate (HDmPyODC, 18 f): The
product was obtained as a light yellow solid, in a yield of 5.4 g (74%).
was obtained as a white solid in a yield of 7.4 g (82.8%). M.p. 203–2048C
1
(dec); H NMR ([D
6
]acetone): d=2.12–2.15 (m, 2H; CH
), 3.42–3.58 (m, 2H; CH ), 3.80–4.26 (m, 10H; 5CH
1H), 7.97–8.02 (m, 2H), 8.07 ppm (d, 1H); C NMR ([D
25.09, 41.76, 42.23, 50.81, 51.51, 66.19, 66.41, 109.99, 114.56, 127.52,
2
), 2.16–2.29 (m,
), 7.75 (td,
]acetone): d=
1
M.p. 146–1478C (dec). H NMR ([D
6
]acetone): d=1.97–2.00 (m, 4H;
2H; CH
2
2
2
1
3
13
2
CH
[D ]acetone): d=25.07, 40.41, 51.77, 105.06, 108.23, 119.30, 157.86 ppm;
elemental analysis (%) calculated for C10 OP (365.22): C 32.89, H
2
), 3.28 (s, 6H; 2CH
3
), 3.96–4.04 (m, 4H; 2CH
2
) ppm. C NMR
6
(
6
H
14
F
6
N
5
143.54 ppm. elemental analysis (%) calcd for C15
H
20
F
6
N
5
O
2
P (447.32): C
3
.86, N 19.18; found: C 33.12, H 3.99, N 19.51.
40.28, H 4.51, N 15.66; found: C 40.45, H 4.66, N 15.89.
Chem. Eur. J. 2009, 15, 9404 – 9416
ꢃ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9413

F. Albericio, A. El-Faham et al.
5
ACHTUNGTRENNUNG
-Chloro-1-[morpholino(pyrrolidinium-1-ylidene)methyl]-1H-benzo[d]-
[1.2.3]triazole 3-oxide hexafluorophosphate (HMPyC, 21): The product
was obtained as a white solid in a yield of 7.96 g (82.7%). M.p. 217–
the pentapeptide. The crude product was analyzed by HPLC with a
linear gradient of 20 to 80% CH CN/0.1% TFA in H O/0.1% TFA over
3
2
30 min on a Symmetry Waters C18 column (4.6ꢄ150mm, 4 mm), with de-
1
À1
2
2
7
188C (dec); H NMR ([D
.29 (m, 2H; CH ), 3.45–3.65 (m, 2H; CH
.99 (d, 1H), 8.02 (d, 1H), 8.12 ppm (dd, 1H); C NMR ([D
6
]acetone): d=2.10–2.15 (m, 2H; CH
2
), 2.19–
),
]acetone):
tection at 220 nm, flow rate 1.0 mLmin , tR (penta)=8.82 min, tR (des-
2
2
), 3.81–4.09 (m, 10H; 5CH
2
Aib)=9.10 min.
1
3
6
2
Synthesis of H-Tyr-MeLeu-MeLeu-Phe-Leu-NH on solid phase: Tripep-
d=25.93, 52.89, 53.36, 66.09, 66.42, 115.79, 115.84, 132.57, 132.62, 134.06,
34.12, 147.38 ppm; elemental analysis (%) calculated for
P (481.76): C 37.40, H 3.98, N 14.54; found: C 37.65, H
tide H-MeLeu-Phe-Leu-NH
mide-Aminomethyl-PS-resin (0.63 mmolg ), after Fmoc removal with
piperidine in DMF (20%, 2ꢄ5 min). The resin was washed with DMF
2
was manually assembled on Fmoc-RinkA-
1
À1
15 6 5 2
C H19ClF N O
4
.13, N 14.79.
Fmoc-Val-Val-NH
as follows: Tetramethylfluoroformamidiniun hexafluorophosphate
(
2 2
ꢄ10), CH Cl (ꢄ10), and DMF (ꢄ10). Residues were introduced after
2
in solution phase: An authentic sample was prepared
30 min coupling, with preactivation of Fmoc-amino acids (3 equiv) with
Oxyma (3 equiv) and DIC (3 equiv) in DMF for 1.5 min. Quantitative in-
corporation was checked at each step by use of the Kaiser test for pri-
(
(
TFFH, 1 mmol) was added at 08C to a mixture of Fmoc-Val-OH
1 mmol), and DIEA (1 mmol) in DMF (5 mL) and the reaction mixture
2
mary amines. Sample cleavage (10 mg) with TFA/H O (9:1) confirmed
2
was activated for 5 min, followed by the addition of H-Val-NH (1 mmol)
and DIEA (1 mmol). The reaction mixture was stirred at 08C for 1 h and
at RT for 3h. Water (50 mL) was added and the precipitate was collected
and washed with sat. Na
the tripeptide in >99.5% purity, as analyzed by reversed-phase HPLC
+
and ESI-MS ([M+H] =405.32). The two last residues, Tyr and MeLeu,
were introduced by preparation of 0.3m solutions of coupling reagent
2 3
CO and HCl (1n), and was then dried in air
(
3 equiv) and Fmoc-amino acid (3 equiv) in standard or treated DMF,
and preactivation of the mixture with DIEA (3 or 6 equiv) for 20–30 s.
The peptide chain was cleaved from the resin with TFA/H O (9:1) over
h at room temperature. The solution was filtered and the resin was
washed with CH Cl (1 mLꢄ2), which was removed together with TFA
under nitrogen flow. The crude peptide was purified with cold Et
and recrystallized from ethyl acetate/hexane to give a white solid in a
1
yield of 87.0%. M.p. 226–2288C. H NMR ([D
2H; 4CH ), 1.80–2.00 (m, 2H; 2CH), 3.87 (t, 1H; CH), 4.10 (t, 1H;
CH), 4.18–4.20 (m, 3H; CH, CH ), 7.02 (t, 2H; NH), 7.28–7.85 ppm (m,
2, ar, NH).
This sample was used as an authentic sample and injected into the HPLC
Waters 2487) under the following conditions: linear gradient of 10 to
6
]DMSO): d=0.95 (t,
2
1
3
2
2
2
2
1
2
O
(2 mLꢄ3) and after lyophilization, purity was checked on reversed-phase
HPLC, with use of a Waters SunFire C18 Column (3.5 mm, 4.6ꢄ100mm),
(
9
2
0% CH
3 2
CN/0.1% TFA in H O/0.1% TFA over 30 min, detection at
with a 20% to 50% linear gradient of 0.036% TFA in CH
TFA in H O over 8 min, with detection at 220 nm. The t of the penta-
peptide was 6.5 min, whereas the t values of des-MeLeu, des-Tyr, and
were 5.0, 5.7, and 3.0 min respective-
3
CN/0.045%
À1
00 nm. Flow rate=1 mL min . Column: Waters C18, 5 mm, 4.6ꢄ150mm
2
R
(
Waters Dual Wavelength Detector and Waters 717 Plus auto sampler).
=20.89 min (Fmoc-Val-OH), t =19.88 min (Fmoc-Val-Val-NH ). This
R
t
R
R
2
tripeptide H-MeLeu-Phe-Leu-NH
ly.
2
reaction was repeated under several sets of coupling conditions and sam-
ples (10 mL) were taken at different intervals. In addition, the reaction
was quenched by addition of acetonitrile/water (1:1, 2 mL), and a sample
of the solution (10 mL) was then injected into the HPLC system under
the same conditions as described above.
Synthesis of ACP (65–74) (H-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-
[6d]
NH
2
): The peptide was elongated manually on an Fmoc-Rink Amide-
À1
AM-resin (0.7 mmolg ). Coupling times of 2 min were used, and excess-
es of reagents were 2 equiv for Fmoc-amino acids and coupling reagents
Z-Aib-Val-OMe in solution phase: An authentic sample was prepared as
follows: TFFH (0.264 g, 1 mmol) was added at 08C to a mixture of Z-
Aib-OH (0.237 g, 1 mmol), and DIEA (0.174 mL, 1 mmol) in DMF
72
and 4 equiv for DIEA. Incorporation was detected for Ile onto Asn and
69
for Ile onto Asp. Peptide purity was determined by reversed-phase
HPLC analysis (Symmetry Waters C18 (4.6ꢄ150mm, 4 mm), linear gradi-
(
5 mL) and the reaction mixture was activated for 8 min, followed by ad-
ent over 30 min of 10 to 90% CH
3
CN in H
des-Asn=10.8 min, t
des-Val=8.43 min), after cleavage
2
O/0.1% TFA, flow rate
dition of H-ValO-Me·HCl (0.169 g, 1 mmol) and DIEA (2 mmol). The re-
action mixture was stirred at 08C for 1 h and at RT for 3 h. Water
À1
1
.0 mL min , t
R
decapeptide=10.43 min, t
R
R
des-
72
69
Ile =7.5 min, t
R
des-Ile =9.1 min, t
R
(
50 mL) was added, and the precipitate was collected and washed with
of the peptide from the resin by treatment with TFA/H
room temperature.
2
O (9:1) for 2 h at
sat. Na CO and HCl (1n), and was then dried in air and recrystallized
from dichloromethane/hexane (white needles after two days at room
2
3
General Procedure for dynamic differential scanning calorimetry assays:
The thermal behavior of HDMA (7b), HDMB (8), and COMU (18c)
temperature) to give a white solid in 87.0% yield. M.p. 76–778C;
1
H NMR ([D
6
]DMSO): d=0.85 (t, 6H; 2CH
.18 (m, 1H; CH), 3.72 (s, 3H; OCH ), 4.49–4.53 (m, 1H; CH), 5.09 (s,
H; CH ), 5.36 (brs, 1H; NH), 7.28–7.37 (m, 6H; ar, NH) ppm. This
3 3
), 1.56 (d, 6H; 2CH ), 2.13–
was tested. Samples (1 mg) were heated from 308C to 3008C at a rate of
2
2
3
À1
1
08Cmin in a closed high-pressure crucible with N
2
flow in a Mettler–
2
Toledo DSC-30 differential scanning calorimeter. Diagrams showing heat
flow as a function of temperature and time were obtained.
sample was used as an authentic sample and injected into the HPLC
Waters 2487) under the following conditions: linear gradient of 10 to
(
9
2
0% CH
3
CN/0.1% TFA in H
2
O/0.1%TFA over 30 min, detection at
General Procedure for isothermal differential scanning calorimetry
assays: The autocatalytic natures of HDMA (7b), HDMB (8), and
COMU (18c) was tested. Samples (1 mg) were heated to 108C below the
onset temperatures detected in the dynamic DSC [HDMA (7): 1678C,
HDMB (8): 1708C, and COMU (17c): 1508C] for 480 min in a closed
high-pressure crucible with N2 flow in a Mettler–Toledo DSC-30 differen-
tial scanning calorimeter. Diagrams showing heat flow as a function of
time were obtained.
À1
00 nm. Flow rate=1 mL min . Column: Waters C18, 5 mm, 4.6ꢄ150mm.
(
Waters Dual Wavelength Detector and Waters 717 Plus auto sampler).
(Z-Aib-OH)=18.25 min, t (Z-Aib-Val-OMe)=20.90 min, t (Z-Aib-
OAt)=22.30 min, (Z-Aib-OBt)=22.80 min, (Z-Aib-Oxyma)=
3.90 min.
Model segment coupling reaction: Test couplings were carried out as pre-
t
R
R
R
t
R
t
R
2
[
2
7]
[7]
viously described for Z-Phg-Pro-NH
2
and Z-Phe-Val-Pro-NH .
[
6d]
Synthesis of Tyr-Aib-Aib-Phe-Leu-NH
2
on solid phase:
Fmoc-Rink
General Procedure for ARC experiments: Assays were carried out on an
Accelerating Rate Calorimeter (ARC) from Thermal Hazard Technolo-
gy, with use of ARCTC-HC-MCQ (Hastelloy) test cells. Samples [1.837g
of HDMB (8), 1.605 of HDMA (7), and 2.350g of COMU (18c)] were
introduced into the calorimetric test cell at room temperature, without
stirring. The cell was heated at the initial temperature (308C) and the
“heat-wait-seek” method was applied. This procedure consisted of heat-
ing the sample by 58C and, after 15 min of equilibrium, measuring
À1
Amide-PS (100 mg, 0.7 mmolg ) was deblocked with piperidine in DMF
20%, 10 mL) for 10 min and washed with DMF (2ꢄ10 mL), CH
0 mL), and then DMF (2ꢄ10 mL). Fmoc-Leu-OH (3 equiv), coupling
reagent (3 equiv), and DIEA (6 equiv) were mixed in DMF (0.5 mL) for
activation (1–2 min) and then added to the resin. The reaction mixture
was then stirred slowly for 1 min and allowed to couple for 30 min (1 h,
double coupling only for the Aib-Aib). The resin was washed with DMF,
and then deblocked with piperidine in DMF (20%) for 7 min. It was
(
1
2 2
Cl (2ꢄ
À1
whether self-heating was occurring at a rate higher than 0.028Cmin
then again washed with DMF, CH
next amino acid. Coupling and deblocking steps were repeated to provide
2
Cl
2
, and DMF and coupled with the
(default sensitivity threshold). When self-heating was detected, the
system was changed to adiabatic mode. After decomposition, the assay
9414
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ꢃ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 9404 – 9416

Benzotriazole-Based Uronium Coupling Reagents
FULL PAPER
[
27]
was stopped when temperature rose above 3008C. The phi factors
were: 2.02371 (HDMA), 2.76093 (HDMB), and 2.40644 (COMU).
ods of Organic Chemistry, Vol. E22a (Ed.: M. Goodman), Thieme,
Stuttgart, 2002; c) M. Amblard, J.-A. Fehrentz, J. Martinez, G.
Subra, Mol. Biotechnol. 2006, 33, 239–254.
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3] a) F. Albericio, L. A. Carpino in Methods in Enzymology, Solid-
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mers 2000, 55, 140–165; d) F. Albericio, R. Chinchilla, D. J. Dods-
worth, C. Najera, Org. Prep. Proced. Int. 2001, 33, 203–303; e) S-Y.
Han, Y-A. Kim, Tetrahedron 2004, 60, 2447–2467; f) C. A. G. N.
Montalbetti, V. Falque, Tetrahedron 2005, 61, 10827–10852.
Acknowledgements
This work was partially supported by CICYT (CTQ2006–03794/BQU),
Luxembourg Bio Technologies, Ltd., the Generalitat de Catalunya
(
2005SGR 00662), the Institute for Research in Biomedicine, and the
Barcelona Science Park. RS-F thanks the Ministerio de Educaciꢆn y
Ciencia for a FPU PhD fellowship. We also thank the Calorimetry Plat-
form at the Barcelona Science Park for their support in the DSC and
ARC experiments.
[
[
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[
1] Abbreviations not defined in text: Aib, a-aminoisobutyric acid;
DIEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide;
HOBt, 1-hydroxybenzotriazole; HOAt, 7-aza-1-hydroxybenzotria-
zole; N-HATU, N-[(dimethylamino)-1H-1,2,3-triazolo
-yl-methylene)-N-methylmethanaminium hexafluorophosphate N-
oxide; N-HBTU, N-[(1H-benzotriazol-1-yl)-(dimethylamino)methy-
lene]N-methylmethanaminium hexafluorophosphate N-oxide;
DMCH,4-((dimethyamino)chloromethylene)morpholin-4-Iminium
hexaflurophosphate HDMA, 1-((dimethylimino)morpholino>
methyl)3-H-[1,2,3]triazolo[4,5-b]pyridine-1–3-olate hexafluorophos-
phate; HDMB, 1-((dimethylimino)morpholino>methyl)3-H-benzo-
[1,2,3]triazolo-1-ium-3-olate hexafluorophosphate; 6-HDMCB, 1-
(dimethylimino)morpholino>methyl)3-H-6-chlorobenzo[1,2,3] tria-
ACHTUNGTRENNU[GN 4,5-b]pyridin-
1
1
15, 4397–4398; d) L. A. Carpino, A. El-Faham, C. A. Minor, F. Al-
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AHCTUNGTRENNUNG
3
1
561–3564; g) L. A. Carpino, A. El-Faham, J. Am. Chem. Soc. 1995,
17, 5401–5402; h) A. El-Faham, Chem. Lett. 1998, 671–672; i) J.
ACHTUNGTRENNUNG
Habermann, H. Kunz, J. Prakt. Chem. 1998, 340, 233–239; j) P.
Garner, J. T. Anderson, S. Dey, W. J. Youngs, K. Galat, J. Org.
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Faham, Lett. Pept. Sci. 2000, 7, 113–121; m) F. Albericio, M. A.
Bailen, R. Chinchilla, J. D. Dodsworth, C. Najera, Tetrahedron 2001,
(
ACHTUNGTRENNUNG
zolo-1-ium-3-olate hexafluorophosphate; DMU, N,N-dimethylmor-
pholine-4-carboxamide; DmPyU, N,N-Dimethylpiperidine-1-carbox-
amide;
DmPyCH, (Chloro(pyrrolidin-1-yl)methylene)-N-methylmethanami-
nium hexafluorophosphate; MPyCH, 1-(chloro(morpholino)methyl-
ene)pyrrolidinium Hexafluorophosphate; HOTU, O-[Cyano(ethoxy-
carbonyl)methylidene)amino]-1,1,3,3-tetramethyluronium Hexa-
MPyU,
Morpholino(pyrrolidin-1-yl)methanone;
N-
AHCTUNGTRENNUNG
5
2
7, 9607–9613; n) O. Marder, Y. Shvo, F. Albericio, Chim. Oggi
002, 20, 37–41; o) M. Vendrell, R. Ventura, A. Ewenson, M. Royo,
ACHTUNGTRENNUNG
F. Albericio, Tetrahedron Lett. 2005, 46, 5383–5386.
fluorophosphate; HdmPyOC, 1-[(1-(Cyano-2-ethoxy-2-oxoethylide-
neaminooxy)-dimethylamino-pyrrolodinomethylene)] methanamini-
[
[
[
7] a) A. El-Faham, F. Albericio, Org. Lett. 2007, 9, 4475–4477; b) A.
El-Faham, F. Albericio, J. Org. Chem. 2008, 73, 2731–2737.
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schuh, J. Klose, M. Beyermann, M. Bienert, Angew. Chem. 2002,
um
Hexafluorophosphate; COMU,
1-[(1-(Cyano-2-ethoxy-2-
oxoethylideneaminooxy)-dimethylamino-morpholinomethylene)]
methanaminium Hexafluorophosphate; HMPyOC, 1-((1-cyano-2-
ethoxy-2-oxoethylideneaminooxy)(morpholino)methylene) pyrro
inium Hexafluorophosphate; HTODC, O-[(Dicyanomethylidene)-
amino]-1,1,3,3-tetramethyluronium Hexafluorophosphate;
HDmPyODC, 1-[(1-(dicyanomethylideneaminoxy)-dimethylamino-
pyrrolodinomethylene)] methanaminium Hexafluorophosphate;
HDMODC, 1-[(1-(dicyanomethyleneaminooxy)-dimethylamino-
morpholinomethylene)] methanaminium Hexafluorophosphate
HMPyODC, 1-((dicyanomethyleneaminooxy)morpholinomethyl-
ene)pyrrolidinium Hexafluorophosphoate; HTODeC, O-[(diethoxy-
ACHTUNGTNERNUNGl id-
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
1
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[
[
[
A
H
U
G
R
N
U
G
ACHTUNGTRENNUNG
2
AHCTUNGTRENNUNG
12] G. Breipohl, W. Koenig, Ger. Offen. DE 90-4016596, 1991.
ACHTUNGTRENNUNG
carbonylmethylidene)amino]-1,1,3,3-tetramethyluronium Hexafluor-
ophosphate; HDMODeC, 1-[(1,3-diethyoxy-1,3-dioxopropan-2-yli-
deneaminooxy)-dimethylamino-morpholinomethylene)] methanami-
nium Hexafluorophosphate; HTOPC, N-[(cyano(pyridine-2-yl)me-
thyleneaminooxy)(dimethylamino)methylene)-N-Methylmethanami-
nium Hexafluorophosphate; HDMOPC, N-[(cyano(pyridine-2-
yl)methyleneaminooxy)(dimethylamino)methylene)-N-morpholino-
methanaminium Hexafluorophosphate; HMPyA, 1-(morpholino-
[13] L. A. Carpino, P. Henklein, B. M. Foxman, I. Abdelmoty, B. Costi-
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A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(pyrrolidinium-1-ylidene)methyl)-1H-[1,2,3]triazolo
oxide Hexafluorophospahte; HMPyB, 1-(morpholino(pyrrolidinium-
-ylidene)methyl)-1H-benzo[d][1,2,3]triazole 3-oxide Hexafluoro-
phosphate; HMPyC, 5-chloro-1-(morpholino(pyrrolidinium-1-ylide-
ne)methyl)-1H-benzo[d][1,2,3]triazole 3-oxide hexafluorophosphate;
A CHTUNGTRENNUN[G 4,5-I]pyridine 3-
1
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
Amino acids and peptides are abbreviated and designated following
the rules of the IUPAC-IUB Commission of Biochemical Nomencla-
ture [J. Biol. Chem. 247, 977 (1972)].
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to the Synthesis of Peptides and Proteins, CRC, Boca Raton, 1997;
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[
Chem. Eur. J. 2009, 15, 9404 – 9416
ꢃ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9415

F. Albericio, A. El-Faham et al.
[
[
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Received: March 8, 2009
2
229–2230.
Published online: July 20, 2009
9416
www.chemeurj.org
ꢃ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 9404 – 9416