Morpholine-based immonium and halogenoamidinium salts as coupling reagents in peptide synthesis

Article abstract of DOI:10.1021/jo702622c

(Chemical Equation Presented) Here we describe a new family of N-form immonium-type coupling reagents that differ in their carbocation skeleton structure. The N-methylpiperazine derivative failed to form immonium salts, while the thiomorpholine derivative did not give better results than the coupling reagents currently used. The presence of the morpholine had a marked influence on the solubility and stability as well as the reactivity of the reagent. Finally, the fluoroamidinium salt performed extremely well in the presence of only 1 equiv of base, thereby confirming the effect of the proton acceptor in the reaction.

Full text of DOI:10.1021/jo702622c

Morpholine-Based Immonium and Halogenoamidinium Salts as
Coupling Reagents in Peptide Synthesis1
Ayman El-Faham*,†,‡ and Fernando Albericio*,†,§,|
Institute for Research in Biomedicine, Barcelona Science Park, Josep Samitier 1, 08028-Barcelona, Spain,
Department of Chemistry, Faculty of Science, Alexandria UniVersity, Ibrahimia 21321, Alexandria, Egypt,
Department of Organic Chemistry, UniVersity of Barcelona, Mart´ı i Franque´s 1-11, 08028-Barcelona,
Spain, and CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine,
Barcelona Science Park, Josep Samitier 1-5, E-08028 Barcelona, Spain
albericio@pcb.ub.cat; aymanel_faham@hotmail.com
ReceiVed December 11, 2007
Here we describe a new family of N-form immonium-type coupling reagents that differ in their carbocation
skeleton structure. The N-methylpiperazine derivative failed to form immonium salts, while the
thiomorpholine derivative did not give better results than the coupling reagents currently used. The presence
of the morpholine had a marked influence on the solubility and stability as well as the reactivity of the
reagent. Finally, the fluoroamidinium salt performed extremely well in the presence of only 1 equiv of
base, thereby confirming the effect of the proton acceptor in the reaction.
Introduction
Although a considerable amount of work has been done to
identify the best leaving groups,3,6 less attention has been paid
Peptide synthesis depends on the proper combination of
protecting groups and the right choice of the coupling
method.2 The two main classes of coupling techniques are
(i) those that require in situ activation of the carboxylic acid
and (ii) those that require an activated species that has previ-
ously been prepared (usually from an in situ activation
step), isolated, purified, and characterized.3 Among the first,
the predominance of carbodiimides is being replaced with
the stand-alone reagents, such as immonium and phosphon-
ium salts, phosphates, and phosphinates.3 Immonium salts,4
probably the most widely used and most powerful salts,5 are
formed by a leaving group and an immonium skeleton
(Figure 1). The mechanism involves an attack of the car-
boxylate on the electron-deficient carbon of the immonium
salt to give the corresponding acyloxyamidinium salt, which
is unstable and reacts with the leaving group originally pre-
sented in the immonium coupling reagent to give the active
species, which undergoes aminolysis to give the target amide
bond (Scheme 1). The driving force of this process is the
generation of urea. Couplings involving immonium salts are
carried out with an excess of base, which can promote
racemization.
(1) Abbreviations not defined in text: Aib, R-aminoisobutyric acid; DCM,
dicloromethane; DIEA, N,N-diisopropylethylamine; DMF, N,N-dime-
thylformamide; HOBt, 1-hydroxybenzotriazole; HOAt, 7-aza-1-hydroxy-
benzotriazole; HOPfp, pentafluorophenol; HATU, N-[(dimethylamino)-
1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene)-N-methylmethanaminium
hexafluorophosphate N-oxide; HBTU, N-[(1H-benzotriazol-1-yl)(dimethy-
lamino)methylene]N-methylmethanaminium hexafluorophosphate N-oxide;
HAM2PipU, O-(1H-1,2,3-triazolo[4,5-b]pyridin-1-yl)-1,1-dimethyl-3,3-pen-
tamethyleneuronium hexafluorophosphate; HBM2PipU, O-(1H-benzotriazol-
1-yl)-1,1-dimethyl-3,3-pentamethyleneuronium hexafluorophosphate; TFFH,
tetramethylfluoroformamidinium hexafluorophosphate, TFA ) trifluoro-
acetic acid; TMP, 2,4,6-trimethylpyridine (collidine); Z, benzyloxycarbonyl;
Boc, tert-butyloxycarbonyl. DMCH, N-(Chloro(morpholino)methylene)-N-
methylmethanaminium hexafluorophosphate; HDMA, 1-((dimethylamino)-
(morpholino)methylene)-1H-[1,2,3]triazolo[4,5-b]pyridinium hexafluoro-
phosphate 3-oxide; HDMB, 1-((dimethylamino)(morpholino)methylene)-
1H-benzotriazoliumhexafluorophosphate3-oxide;4-HDMA,3-((dimethylamino)-
(morpholino)methylene)-1H-[1,2,3]triazolo[4,5-b]pyridinium hexafluoro-
phosphate 1-oxide; 6-HDMCB, 6-chloro-1-((dimethylamino)(morpholino)-
methylene)-1H-benzotriazolium hexafluorophosphate 3-oxide; 6-HDMFB,
6-trifluoromethyl-1-((dimethylamino)(morpholino)methylene)-1H-benzot-
riazolium hexafluorophosphate 3-oxide; HDMPfp, 1-((dimethyamino)-
(morpholino))oxypentafluorophenyl metheniminium hexafluorophosphate;
HDMS, 1-((dimethyamino)(morpholino))oxypyrrolidine-2,5-dione metha-
naminium hexafluorophosphate; HDTMA, 1-((dimethylamino)(thiomor-
pholino)methylene)-1H-[1,2,3]triazolo[4,5-b]pyridinium hexafluorophos-
phate 3-oxide; HDTMB, 1-((dimethylamino)(thiomorpholino)methylene)-
1H-benzotriazolium hexafluorophosphate 3-oxide; DMFH, N-(fluoro(morpho-
lino)methylene)-N-methylmethanaminium hexafluorophosphate. Amino ac-
ids and peptides are abbreviated and designated following the rules of the
IUPAC-IUB Commission of Biochemical Nomenclature (J. Biol. Chem.
1972, 247, 977).
† Institute for Research in Biomedicine.
‡ Alexandria University.
§ University of Barcelona.
| CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and
Nanomedicine.
10.1021/jo702622c CCC: $40.75 © 2008 American Chemical Society
Published on Web 03/06/2008
J. Org. Chem. 2008, 73, 2731-2737
2731

El-Faham and Albericio
FIGURE 1. X-ray crystallography of HDMA (A) and HDMB (B).
SCHEME 1. General Structure of Immonium Coupling Reagents and Mechanism of the Amide Formation Using These
Reagents
to the immonium part. Furthermore, in these cases, no additional
heteroatoms have been included.3,6a,i,l,o,7 Recently, we have
shown that the incorporation of a proton acceptor in the immon-
ium part showed superiority to those described previously.8
Herein, this concept has been extended to other leaving groups,
paying special attention to the use of halogens such as F and
Cl.
corresponding chloro salts, which were stabilized by the
formation of PF6 salts (4i,ii). Subsequent reaction with the
N-hydroxylamines (8a-f) and the pentafluorophenol (8g) in the
form of the potassium salt or in the presence of Et3N rendered
the target compounds (5, 6).
Furthermore, reaction of the urea derived from the N-
methylpiperazine (3iii) with oxalyl chloride did not give the
(6) (a) Henklein, P.; Beyermann, M.; Bienert, M.; Knorr, R. In Proceed-
ings of the 21st European Peptide Symposium; Giralt, E., Andreu, D., Eds.;
ESCOM, Science: Leiden, 1991; pp 67-68, (b) Knorr, R.; Trzeciak, A.;
Bannwarth, W.; Gillessen, D. In Peptide, Proc. Eur. Pept. Symp.; Jung,
G., Bayer, E., Eds.; de Gruyter: Berlin, 1989; pp 37-39. (c) Carpino, L.
A. J. Am. Chem. Soc. 1993, 115, 4397-4398.(d) Carpino, L A.; El-Faham,
A.; Minor, C. A.; Albericio, F. J. Chem. Soc., Chem. Commun. 1993, pp.
201-203. (e) Wijkmans, J. C. H. M.; Kruijtzer, J. A. W.; van der Marel,
G. A.; van Boom, J. H.; Bloemhoff, W. Recl. TraV. Chim. Pays-Bas 1994,
113, 394-397.(f) Carpino, L. A.; El-Faham, A.; Albericio, F. J. Org. Chem.
1995, 60, 3561-3564. (g) Carpino, L. A.; El-Faham, A. J. Am. Chem. Soc.
1995, 117, 5401-5402., (h) El-Faham, A. Chem. Lett. 1998, pp. 671-
672. (i) Habermann, J.; Kunz, H. J. Prakt. Chem. 1998, 340, 233-239. (j)
Garner, P.; Anderson, J. T.; Dey, S.; Youngs, W. J.; Galat, K. J. Org. Chem.
1998, 63, 5732-5733. (k) Bailen, M. A.; Chinchilla, R.; Dodsworth, D. J.;
Najera, C. J. Org. Chem. 1999, 64, 8936-8939. (l) El-Faham, A. Lett. Pept.
Sci. 2000, 7, 113-121. (m) Albericio, F.; Bailen, M. A.; Chinchilla, R.;
Dodsworth, D. J.; Najera, C. Tetrahedron 2001, 57, 9607-9613. (n) Marder,
O.; Shvo, Y.; Albericio, F. Chim. Oggi 2002, 20 (7/8), 37-41. (o) Vendrell,
M.; Ventura, R.; Ewenson, A.; Royo, M.; Albericio, F. Tetrahedron Lett.
2005, 46, 5383-5386.
Results and Discussion
Taking a N-containing 6-membered ring structure as a base,
the corresponding nonsymmetrical immonium salts containing
O, S, and NCH3 were prepared by treating N,N-dimethylcar-
bamoyl chloride with the morpholine (2i), thiomorpholine (2ii),
and N-methylpiperazine (2iii) to give the urea derivatives 3
(Scheme 2). Urea derivatives (3i,ii) from morpholine and
thiomorpholine were reacted with oxalyl chloride to yield the
(2) (a) Lloyd-Williams, P.; Albericio, F.; Giralt, E. Chemical Approaches
to the Synthesis of Peptides and Proteins; CRC: Boca Raton, 1997. (b)
Goodman, M., Ed. Synthesis of Peptides and Peptidomimetics; Houben-
Weyl, Methods of Organic Chemistry; Thieme: Stuggart, New York, 2002;
Vol. E22a. (c) Amblard, M.; Fehrentz, J-A.; Martinez, J.; Subra, G. Mol.
Biotech. 2006, 33, 239-254.
(3) (a) Albericio, F.; Carpino, L. A. In Methods of Enzymology, Solid-
Phase Peptide Synthesis; Fields, G. B., Ed.; Academic Press: Orlando, 1997;
Vol. 289, pp 104-126. (b) Humphrey, J. M.; Chamberlin, A. R. Chem.
ReV. 1997, 97, 2243-2266. (c) Kaminski, Z. J. Biopolymers 2000, 55, 140-
165. (d) Albericio, F.; Chinchilla, R.; Dodsworth, D. J.; Najera, C. Org.
Prep. Proc. Int. 2001, 33, 203-303. (e) Han, S-Y.; Kim, Y-A. Tetrahedron
2004, 60, 2447-2467. (f) Montalbetti, C. A. G. N.; Falque, V. Tetrahedron
2005, 61, 10827-10852.
(7) (a) Kiso, Y.; Kimura, T.; Fujiwara, Y.; Sakikawa, H.; Akaji, K. Chem.
Pharm. Bull. 1990, 38, 270-272. (b) Coste, J.; Fre´rot, E.; Jouin, P.; Castro,
B. Tetrahedron Lett., 1991, 32, 1967-1970. (c) Chen, S.; Xu, J. Tetrahedron
Lett., 1992, 33, 647-650. (d) Akaji, K.; Kuriyama, N.; Kimura, T.; Fujiwara,
Y.; Kiso, Y. Tetrahedron Lett. 1992, 33, 3177-3180.(e) El-Faham, A. Org.
Prep. Proc. Int. 1998, 30, 477-481. (f) Klose, J.; El-Faham, A.; Henklein,
P.; Carpino, L. A.; Bienert, M. Tetrahedron Lett. 1999, 40, 2045-2048.(g)
El-Faham, A.; Khattab, S.N.; Abdul-Ghani, M.; Albericio, F. Eur. J. Org.
Chem. 2006, pp. 1563-1573.
(4) Dourtoglou, V.; Ziegler, J. C.; Gross, B. Tetrahedron Lett. 1978,
1269-1272.
(5) Albericio, F.; Bofill, J. M.; El-Faham, A.; Kates, S. A. J. Org. Chem.
1998, 63, 9678-9683.
(8) El-Faham, A.; Albericio, F. Org. Lett. 2007, 9, 4475-4477.
2732 J. Org. Chem., Vol. 73, No. 7, 2008

Coupling Reagents in Peptide Synthesis
TABLE 1. Hydrolytic Stability of Immonium-Type Coupling
Reagents in DMF (Open Vials)
SCHEME 2. Procedure Followed for the Preparation of the
Novel Proton Acceptor Immonium-Type Coupling Reagents
entry
coupling reagent
5 h (%)
24 h (%)
48 h (%)
1
2
3
4
HATU (9)
99
100
100
100
95
98
96
76
86
83
90
HBTU (10)
HDMA (5a)
HDMB (5b)
100
TABLE 2. Solubility in DMF of the Immonium-Type Coupling
Reagents
entry
coupling reagent
wt/1 mL
molarity
1
2
3
4
5
6
7
HATU (9)
HBTU (10)
HCTU (11)
HDMA (5a)
HDMB (5b)
6-HDMCB (5d)
6-HDMFB (5e)
0.165
0.175
0.207
0.285
0.350
0.456
0.475
0.43
0.46
0.50
0.68
0.83
1.00
1.02
To determine the compatibility of the new coupling reagents
(5) with peptide synthesis in both manual and automatic mode,
their solubility and stability in solution and in the solid state
was examined via 1H NMR analysis. Table 1 indicates that the
oxygen in the structure increases the stability of the coupling
reagent when compared with the tetramethyl derivatives (entries
1 vs 3 and 2 vs 4). Reagents 5a,b showed stability greater than
95% in a closed vial. The presence of the oxygen in the skeleton
showed the most remarkable effects on the stability of the most
hygroscopic and less stable chloro derivatives (4i). The NMR
spectra showed only 18% hydrolysis (2% after 1 week and 10%
after 1 month) after 2 months of storage at room temperature
in a closed plastic vial.12 These observations have a practical
implication for both solid-phase and solution strategies. Thus,
if the activation of a carboxylic acid is slow, the coupling
reagents will be degraded and no longer be able to activate the
carboxyl function. This is crucial for cyclization steps or in
convergent strategies during the fragment coupling steps because
yields tend to be lower than for other couplings.
Table 2 indicates that the presence of the oxygen atom in
the carbon skeleton is of marked importance for the solubility
of the compound. Thus, all morpholine derivatives (5) were
more soluble than the dimethylamine ones (9-11) (entries 1
vs 4, 2 vs 5, 3 vs 6). Furthermore, 6-HDMCB (5d) and
6-HDMFB (5e) were the most soluble and could be used to
prepare up to 1 M solution. This enhanced solubility should
be used to prepare more concentrated solution to enhance
coupling rates and to remove the excess and the urea
side products during the workup after the coupling in a solution
mode approach.
expected product, but only an oily residue that could not be
purified or identified by NMR spectra.
Finally, the chloro salts (4i) were converted into their
corresponding fluoride (7) by reaction with KF (Scheme 2).
X-ray crystallography showed that both HDMA (5a) and
HDMB (5b) were in the N-form, as expected when compared
with similar derivatives.9 Furthermore, the 13C NMR for these
two compounds indicated displacements of the imino carbon
[CdN+(Me2)] 149.65 ppm for HDMA (5a) and 150.65 ppm
for HDMB (5b), which are consistent with those reported
[151.63 and 151.42 ppm respectively for the counterparts HATU
(9) and HBTU (10)10] for this kind of compound.11
(9) Abdelmoty, I.; Albericio, F.; Carpino, L. A.; Foxman, B. M.; Kates,
S. A. Lett. Pept. Sci. 1994, 1, 57-67.
(10) Representative immonium salt coupling reagents:
The efficacy of the new morpholine derivatives to reduce
racemization was examined using two models systems (Z-Phg-
OH + H-Pro-NH2 and Z-Phe-Val-OH + H-Pro-NH2) in
solution.13 Table 3 [(1 + 1) model] indicates that in all cases
the morpholine derivatives resulted in improved control of
racemization when compared with their tetramethylamino
counterparts (entries 2, 5, 14, 16 vs 1, 4, 13, 15, respectively).
A good example is the case of the chloro salts (entries 16 vs
15) where racemization decreased from 29.9% to 6.9%. For the
morpholine derivatives (entries 2, 5, 14), the use of only 1 equiv
(11) Carpino, L. A.; Imazumi, H.; El-Faham, A.; Ferrer, F. J.; Zhang,
C.; Lee, Y.; Foxman, B. M.; Henklein, P.; Hanay, C.; Mugge, C.; Wenschuh,
H.; Klose, J.; Beyermann, M.; Bienert, M. Angew. Chem., Int. Ed., 2002,
41, 442-445.
(12) In contrast, the tetramethyl (TCFH) derivatives have been described
as very hygroscopic compounds (El-Faham, A. Chem. Lett. 1998, 7, 671-
673.
(13) Carpino, L. A.; El-Faham, A. J. Org. Chem. 1994, 59, 695-698.
J. Org. Chem, Vol. 73, No. 7, 2008 2733

El-Faham and Albericio
TABLE 3. Yield and Racemization during the Formation of
TABLE 4. Yield and Racemization during the Formation of
Z-Phe-Val-Pro-NH2 (2 + 1) in DMFa
Z-Phg-Pro-NH2 in DMFa
entry
1
coupling reagent
base (equiv)
yield (%)
DL (%)
ref
8
entry
1
coupling reagent
base (equiv)
yield (%)
LDL (%)
ref
8
HATU (9)
DIEA (2)
DIEA (1)
DIEA (2)
DIEA (1)
DIEA (2)
DIEA (2)
DIEA (1)
DIEA (2)
DIEA (1)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (1)
DIEA (2)
DIEA (1)
DIEA (2)
DIEA (2)
DIEA (1)
DIEA (2)
DIEA (1)
78.4
74.8
81.2
82.3
83.0
80.2
75.0
80.8
82.3
81.3
84.5
84.1
80.1
79.6
80.0
83.7
80.1
77.4
82.6
83.7
80.0
81.9
80.3
80.3
78.4
3.1
2.4
1.6
1.6
2.1
8.2
5.3
3.8
3.1
3.5
1.5
0.6
15.7
14.9
18.9
3.7
7.1
6.5
4.9
1.2
29.9
6.9
5.2
3.3
2.4
HATU (9)
DIEA (2)
DIEA (1)
TMP (2)
TMP (1)
DIEA (2)
DIEA (1)
TMP (2)
TMP (1)
DIEA (2)
DIEA (2)
DIEA (1)
TMP (2)
DIEA (2)
DIEA (1)
TMP (2)
TMP (1)
DIEA (2)
DIEA (1)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (1)
TMP (2)
85.8
83.2
78.0
76.1
89.3
87.4
86.2
84.1
81.3
89.7
78.6
81.2
88.7
86.3
87.1
80.1
82.2
79.9
81.3
75.8
78.3
89.1
80.0
88.9
76.5
85.5
13.9
11.0
5.3
4.9
10.5
5.1
2
HDMA (5a)
8
3
4
HDTMA (6a)b
HBTU (10)
2
HDMA (5a)
8
8
8
3.7
3.8
5
HDMB (5b)
3
4
HDTMA (6a)
HBTU (10)
22.6
27.7
16.3
14.2
20.3
11.5
13.3
10.5
29.8
13.9
31.4
37.1
35.2
37.8
28.9
11.3
10.6
4.1
6
7
8
HDTMB (6b)b
6-HDMCB (5d)
6-HDMFB (5e)
HDMPfp (5g)
HDMS (5f)
8
8
8
9
5
HDMB (5b)
10
11
12
13
HSTU (15)b
4-HDMA (5c)
TFFH (14)
6
7
8
HDTMB (6b)
6-HDMCB (5d)
6-HDMFB (5e)
HDMPfp (5g)
HDMS (5f)
14
DMFH (7)
9
15
16
TCFH (13)
DMCH (4i)
10
11
12
13
HSTU(15)b
4-HDMA (5c)
HAM2PipU (12)
17
HAM2PipU (12)
a Couplings were carried out without preactivation, as described in the
Experimental Section. LL and DL forms of the test dipeptide have been
described elsewhere.13 The tR of LL and DL were identified by coinjection
with pure samples of LL. b The HPLC showed a peak at 5.6 min for the
urea, which indicates that the thiomorpholine urea derivatives do not wash
out completely during the normal work up.
a
LLL and DL forms of the test tripeptide have been described elsewhere13
and coinjected with authentic and pure samples. b Extra peaks were observed
at 17.3 min (25.0%) and at 17.6 min (6.1%).
TABLE 5. Yield and Racemization during the Formation of
Z-Gly-Phe-Pro-NH2, Z-Gly-Phe-Ala-OMe, and Z-Gly-Phe-Val-OMe
(2 + 1) in DMF (Solution-Phase Synthesis)a
of base caused a slight increase in yield and a decrease in
racemization, while for the tetramethylamine derivatives (entries
1, 4, 13) the decrease in racemization was also accompanied
by a decrease in yield.14 In this model, the thiomorpholine
derivatives showed a similar performance to the morpholine ones
(entries 3, 6 vs 2, 4). Interestingly, the chloro (5d, entry 7) and
the trifluoromethyl (5e, entry 8), which contains the less reactive
and expensive benzotriazoles when compared with the aza
derivatives, performed well and are good alternatives to the aza
derivatives. The compounds that contained less reactive leaving
groups rendered more racemization (entries 9-12). Finally,
HDMA (5a) compared favorably with HAM2PipU (12), which
contains a piperidine ring instead of the morpholine to discharge
the possible six-membered ring effect.
A similar tendency was observed when the most demanding
[2 + 1], Z-Phe-Val-OH + H-Pro-NH2, model was used. The
largest discrepancy with the previous experiments is the poorer
behavior of the thiomorphonilo derivatives (entries 3, 6) as
indicated in Table 4. As expected, the use of the less basic TMP
considerably reduced the level of racemization when compared
with DIEA.
For the rather nonsensitive case of segment coupling, such
as Z-Gly-Phe-OH to H-Val-OMe, Z-Phe-Val-OH to H-Ala-
OMe, and Z-Gly-Phe-OH to H-Pro-NH2 leading to correspond-
ing tripeptides,13 the immonium salts derived from morpholine
again performed better than diemthylamino derivatives (Table 5).
In order to demonstrate the effectiveness of the new mor-
pholine coupling reagents in solid-phase mode, the nonapeptide
H-Met-Pro-Pro-Glu-Val-Lys-Phe-Leu-NH2 was assembled on
the solid-phase. The peptide was manually elongated on an
Fmoc-Rink-Amide-resin. Coupling times were reduced (15 min)
as well as the excess of reagents [3 equiv for the coupling
entry
1
peptide
coupling reagent base (equiv) yield (%) l (%)
Z-Gly-Phe-Pro-NH2 HATU (9)
DIEA (2)
DIEA (1)
TMP (2)
TMP (1)
DIEA (2)
DIEA (1)
TMP (2)
DIEA (2)
DIEA (2)
DIEA (1)
TMP (2)
DIEA (2)
DIEA (1)
TMP (2)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
TMP (2)
DIEA (2)
TMP (2)
DIEA (2)
TMP (2)
DIEA (2)
TMP (2)
DIEA (2)
TMP (2)
DIEA (2)
TMP (2)
90.1
80.1
86.8
76.9
89.9
88.9
86.9
78.8
88.9
78.9
84.8
89.6
88.4
87.3
79.1
90.3
78.9
84.5
91.2
83.4
84.1
85.6
90.1
89.3
89.8
88.9
90.1
86.8
89.9
88.6
88.9
84.8
89.7
88.3
1.6
0.9
0.9
0.8
1.0
0.9
0.9
1.6
5.9
4.1
3.6
2.8
2.1
2.9
3.9
3.6
1.5
2.8
12.5
11.3
32.0
33.7
2
HDMA (5a)
3
4
HDTMA (6a)
HBTU (10)
5
HDMB (5b)
6
7
8
HDTMB (6b)
4-HDMA (5c)
6-HDMCB (5d)
6-HDMFB (5e)
HSTU (15)
HDMS (5f)
HDMPfp (5g)
HPyOPfp
9
10
11
12
13
14 Z-Gly-Phe-Ala-OMe HDMA (5a)
15 HDMB (5b)
16 Z-Gly-Phe-Val-OMe HATU (9)
0.06
0.12
0.03
0.11
1.56
0.82
0.65
0.65
5.90
3.57
2.90
3.39
18
17
19
HDMA (5a)
HBTU (10)
HDMB (5b)
a
LL and DL forms of the test dipeptide have been described elsewhere.
The tR of LL and DL were identified by coinjection with authentic and pure
samples of LL.
reagent and 6 equiv for DIEA (3 equiv for the fluoro derivative
DMFH)] with preactivation time (5 min). Peptide purity as
determined by reversed-phase HPLC analysis: HDMA (5a),
88%; HATU (9), 84%; HDMB (5b), 91%; HBTU (10), 81%;
(14) The most unstable chloro derivative (4i, entry 16) does not follow
this trend.
2734 J. Org. Chem., Vol. 73, No. 7, 2008

Coupling Reagents in Peptide Synthesis
TABLE 6. Yield for the Coupling of Fmoc-Aib-OH onto the
Aib-Phe-Leu-NH-resin Calculated by Tyr-Aib-Aib-Phe-Leu-NH2 vs
a
Tyr-Aib-Phe-Leu-NH2
base
(equiv)
penta
(%)
Des-Aib
(%)
entry
1
coupling reagent
HATU (9)
ref
8
DIEA (2)
DIEA (1)
DIEA (2)
DIEA (1)
DIEA (2)
DIEA (2)
DIEA (1)
DIEA (2)
DIEA (1)
DIEA (2)
DIEA (2)
DIEA (1)
DIEA (2)
DIEA (1)
DIEA (2)
DIEA (2)
DIEA (1)
DIEA (2)
DIEA (1)
DIEA (2)
DIEA (1)
DIEA (2)
DIEA (1)
DIEA (2)
83
68
98
90
80
47
33
89
64
75
99
39
78
30
98
95
94
96
99
9
17
32
2
2
HDMA (5a)
8
10
20
53
67
10
36
25
<1
62
22
70
2
5
6
4
<1
91
90
56
51
<1
3
4
HDTMA (6a)
HBTU (10)
8
8
5
HDMB (5b)
6
7
HDTMB (6b)
6-HDMCB (5d)
FIGURE 2. Extent of coupling with 1-2 min of preactivation in the
presence of 6 equiv of DIEA, Fmoc-amino acids, and coupling reagents
(3 equiv), except for DMFH (7) (5 equiv), 15 min coupling.
8
8
6-HDMFB (5e)
9
10
4-HDMA (5c)
TFFH (13)
11
12
13
14
DMFH (7)
TCFH (13)
10
43
49
99
DMCH (4i)
DMCH (9)/6-Cl-HOBt
a Tetrapeptide (des-Aib) was confirmed by peak overlap in the presence
of an authentic sample. The crude H-Tyr-Aib-Aib-Phe-Leu-NH2 was
analyzed by HPLC. HPLC-MS showed the right mass for the penta at
612.0.
group has a marked influence on the polarity of the carbon
skeleton, which affects the solubility and stability as well as
the reactivity of the reagent (yield and control of the racem-
ization). The salt derived from morpholine derivatives are the
most stable to air. These results should be taken into account
when coupling reagents are placed in open vessels, such as in
some automatic synthesizers. HOAt derivatives were confirmed
to be superior to HOBt ones in terms of both coupling yield
and retention of configuration for all cases. Remarkably, the
6-ClHOBt derivative gave equally good results as the aza
derivatives. Finally, the fluoride salt performed extremely well
in the presence of only 1 equiv of base, thereby confirming the
effect of the O proton acceptor in the reaction.
FIGURE 3. Extent of coupling with 7 min preactivation in the presence
of 6 equiv of DIEA, Fmoc-amino acidsv and coupling reagents (3 equiv)
except for DMFH (7) (5 equiv), 15 min coupling time.
6-HDMCB (5d), 91%; DMFH (7), 89%. Furthermore, the extent
of each coupling was calculated using the UV spectroscopy for
the dibenzofulvene (DBF) adduct at λmax of 300.5 (Figures 2
and 3). Again, the morpholine derivatives gave purity in all cases
around 90%, while the purity of the tetramethylamine derivatives
(9, 10) was around 81-84%. Again, HDMB (5b) and 6-HD-
MCB (5d) performed similarly to the aza derivatives HDMA
(5a) and HATU (9), which contain the most expensive HOAt.
Remarkably interesting is the result with DMFH (7), which
performed extremely well with only 1 equiv of base.
In a more demanding example, H-Tyr-Aib-Aib-Phe-Leu-
NH26d was manually assembled on Fmoc-Rink Amide.AM-resin
using amino acid/activator (3 equiv), DIEA (6 equiv) or (3
equiv), using 30 min coupling time except for the case of Aib-
Aib, for which 1 h was used. Yields for the coupling of Fmoc-
Aib-OH onto the Aib-containing resin were determined by
reversed-phase HPLC analysis, after cleavage of the peptide
from the resin (Table 6). The best results were obtained with
HDMA (5a) and 6-HDMCB (5d) with 2 equiv of base, while
the fluoro salt DMFH (7) gave better results with 1 equiv of
base. In addition, DMCH (4) in the presence of 6-ClHOBt gave
equally good results as 5a and 7. Again, the thiomorpholine
derivatives (6a,b) gave slightly worse results when compared
with the morpholine ones.
Experimental Section
General Procedure for the Preparation of Urea Derivatives.6g
N,N-Dialkylcarbamoyl chloride (0.6 mol) was added dropwise to
a stirring mixture of secondary amine (0.5 mol) and triethylamine
(TEA) (0.5 mol)15 in DCM (400 mL) at 0 °C. When the addition
was completed, the mixture was stirred for 3-4 h at room
temperature. The reaction mixture was basified with 10% NaOH,
and then the organic layer was collected and the aqueous layer
washed with 100 mL of DCM. The combined DCM solution was
washed with H2O (2 × 100 mL) and saturated solution of NaCl (2
× 100 mL). Finally, the organic solvent was dried over anhydrous
MgSO4 and filtered, and the solvent was removed under reduced
pressure. The oily residue obtained was purified by vacuum
distillation.
N,N-Dimethyl-4-morpholinecarboxamide, 3i.16 The urea de-
rivative was distilled and collected at 127-129 °C as a colorless
Conclusions
In conclusion, herein a new family of N-form immonium-
type coupling reagents that differ in their carbocation skeleton
structure have been described. The presence of the morpholino
(15) Alternatively, NaOH can be used instead of TEA (ref 8).
(16) Solomo, T. (Ciba-Geigy A.-G.). Ger. Offen. 2206366 19720831,
1972, Patent written in German. Application: DE 72-2206366 19720210.
J. Org. Chem, Vol. 73, No. 7, 2008 2735

El-Faham and Albericio
oil with a 92.4% yield. 1H NMR (CDCl3): δ 2.84 (s, 6H, 2 CH3),
3.22-3.20 (m, 4H, 2CH2), 3.68-3.70 (m, 4H, 2CH2) ppm. 13C
NMR (CDCl3): δ 38.6, 47.5, 66.9, 165.0 ppm.
3-((Dimethylamino)(morpholino)methylene)-1H-[1,3]triazolo-
[4,5-b]pyridinium Hexafluorophosphate 1-Oxide (4-HDMA, 5c).
The product was obtained as a pale yellow solid (88.9% yield).
1
N,N-Dimethyl-4-thiomorpholinecarboxamide, 3ii.16 The pure
urea was obtained as light brown crystals with an 89.5% yield.
Mp: 61-62 °C. 1H NMR (CDCl3): δ 2.62-2.65 (m, 4H, 2CH2),
2.81 (s, 6H, 2CH3), 3.48-3.50 (m, 4H, 2CH2) ppm. 13C NMR
(CDCl3): δ 27.4, 38.77, 5.38, 165.2 ppm. Anal. Calcd for C7H14N2-
OS: C, 48.25; H, 8.10; N, 16.08. Found: C, 48.35; H, 8.15, N,
16.18.
Mp: 208-210 °C dec. H NMR (CD3COCD3): δ 3.30 (s, 3H,
CH3), 3.71 (s, 3H, CH3), 3.75-4.02 (m, 4H, 2CH2), 4.11-4.16
(m, 4H, 2CH2),7.86-7.89 (dd, 1H,ar), 8.58-8.61 (dd,1H,ar), 9.09-
9.11 (dd,1H,ar) ppm. 13C NMR (CD3COCD3): δ 42.1, 42.5, 50.7,
51.8, 66.1, 123.3, 126.3, 155.4 ppm. Anal. Calcd for C12H17F6N6O2P
(422): C, 34.13; H, 4.06; N, 19.90. Found: C, 34.22; H, 4.11; N,
19.78.
N,N-4-Trimethyl-1-piperazinecarboxamide, 3iii.17,18 The pure
urea derivative was collected at 120-122 °C as a pale yellow oil
(1-2 mmHg) following method A, in 87.4% yield (74.7 g). IR
6-Chloro-1-((dimethylamino)(morpholino)methylene)-1H-
benzotriazolium hexafluorophosphate 3-Oxide (6-HDMCB, 5d).8
The product was obtained as a white solid (93.5% yield). Mp: 193-
(BaF2): 2936-2791 (Sp,3 H), 1650 (CO) cm-1 1H NMR
.
1
194 °C dec. H NMR (CD3COCD3): δ 3.31(s, 3H, CH3), 3.69(s,
(CDCl3): δ 2.20 (s, 3H, CH3), 2.31-2.34 (m, 4H, 2CH2), 2.75 (s,
6H, 2CH3), 3.18-3.20 (m, 4H, 2CH2) ppm. 13C NMR (CDCl3): δ
38.7, 46.4, 46.9, 55.1, 165.0 ppm.
3H, CH3), 3.94-3.951(m, 4H, 2CH2), 4.12-4.14 (m, 4H, 2CH2),
7.96-8.03 (qd, 2H, ar), 8.12-8.13 (dd,1H, ar) ppm. 13C NMR
(CD3COCD3): δ 41.8, 42.3, 50.8, 51.8, 66.2, 66.4, 115.8, 116.2,
132.8, 133.9, 150.5 ppm. Anal. Calcd for C13H17ClF6N5O2P (455):
C, 34.26; H, 3.76; N, 15.37. Found: C, 34.39; H, 3.83; N, 15.54.
6-Trifluoromethyl-1-((dimethylamino)(morpholino)methylene)-
1H-benzotriazolium Hexafluorophosphate 3-Oxide (6-HDMFB,
5e). The product was obtained as a white solid (81.5% yield). Mp:
N-(Chloro(morpholino)methylene)-N-methylmethanamini-
um Hexafluorophosphate (DMCH, 4i).8 Oxalyl chloride (100
mmol) in dichloromethane (DCM) (100 mL) was added dropwise
to a solution of urea derivative 3i (100 mmol) in dry DCM (200
mL) at room temperature over 5 min. The reaction mixture was
stirred under reflux for 3 h, the solvent was removed under vacuum,
and the residue was washed with anhydrous ether (2 × 100 mL)
and then bubbled with N2 to remove excess of the ether. The white
solid obtained was dissolved in DCM (500 mL), and a saturated
aqueous KPF6 solution (18.4 g in 50 mL H2O) was added at room
temperature with vigorous stirring for 10-15 min. The organic layer
was collected, washed once with water (50 mL), dried over
anhydrous MgSO4, and filtered, and the solvent was then removed
under reduced pressure to give a white solid which recrystallized
from DCM ether to give white crystals (28.9 g, 89.6% yield). Mp:
1
194-195 °C. H NMR (CD3COCD3): δ 3.34 (s, 3H, CH3), 3.72
(s, 3H, CH3), 3.95-3.961 (m, 4H, 2CH2), 4.08-4.15 (m, 4H,
2CH2), 8.24-8.27 (qd, 2H, ar), 8.43 (t,1H, ar) ppm. 13C NMR (CD3-
COCD3): δ 41.8, 42.4, 50.9, 51.7, 66.2, 66.4, 114.5, 116.4, 129.9,
150.5 ppm. Anal. Calcd for C14H17F9N5O2P (489): C, 34.37; H,
3.50; N, 14.31. Found: C, 34.49; H, 3.63; N, 14.50.
1-((Dimethyamino)(morpholino))oxypentafluorophenyl Meth-
anaminium Hexafluorophosphate (HDMPfp, 5f). The product
1
was obtained as a white solid (91% yield). Mp: 202-203 °C. H
NMR (CD3COCD3): δ 3.38 (s, 6H, CH3), 3.80-3.83 (m, 4H, CH2),
3.86-3.89 (m, 4H, 2CH2) ppm. 13C NMR (CD3COCD3): δ 40.6,
49.3, 65.7, 159.7 ppm. Anal. Calcd for C13H15F11N2O2P (471): C,
33.13; H, 3.21; N, 5.94. Found: C, 33.33; H, 3.13; N, 6.12.
1-((Dimethyamino)(morpholino))oxypyrrolidine-2,5-dione
Methanaminium Hexafluorophosphate (HDMS, 5g). The product
was obtained as a white solid (78.3% yield). Mp: 192-194 °C.
1H NMR (CD3COCD3): δ 3.03 (s, 4H, 2CH2), 3.35 (s, 6H, 2CH3),
3.82-3.85 (m, 8H, 4CH2) ppm. 13C NMR (CD3COCD3): δ 25.9,
49.4, 65.8, 161.5, 170.2 ppm. Anal. Calcd for C11H19F6N3O4P
(402): C, 32.84; H, 4.76; N, 10.45. Found: C, 32.69; H, 4.70; N,
10.58.
1
94-95 °C. H NMR (CD3COCD3): δ 3.39 (s, 6H, 2CH3), 3.75(t,
4H, 2CH2), 3.86 (t, 4H, 2CH2) ppm. 13C NMR (CD3COCD3): δ
44.4, 52.8, 66.0 ppm. Anal. Calcd for C7H14ClF6N2OP (322): C,
26.06; H, 4.37; N, 8.68. Found: C, 25.94; H, 4.43; N, 8.79.
General Method for the Synthesis of Immonium-Type
Coupling Reagents, 5a-g, 6a,b. The chloro salt 4 (6.45 g) was
added to a solution of HOXt (8) (20 mmol) and TEA (20 mmol,
2.8 g) in DCM (50 mL) or KOXt (20 mmol) in CH3CN (40 mL)
at 0 °C. The reaction mixture was stirred at this temperature and
then left at room temprature overnight. The solid was filtered and
washed with cooled DCM. The white solid was recrystallized from
CH3CN-ether.
1-((Dimethylamino)(thiomorpholino)methylene)-1H-[1,3]tria-
zolo[4,5-b]pyridinium Hexafluorophosphate 3-Oxide (HDTMA,
6a). The crude chloro salt (4ii)19 (20 mmol) at 0 °C was added to
a solution of HOAt (2.72 g, 20 mmol) and TEA (2.8 g, 20 mmol)
in DCM (50 mL). The reaction mixture was stirred at this
temperature under N2 and left at room temperature overnight. The
solid was filtered and washed with cooled DCM. The pale yellow
solid was recrystallized from CH3CN ether to give white crystals
(75.6%). Mp: 197-199 °C dec. 1H NMR (CD3COCD3): δ 2.76-
2.77 (m, 1H, CH), 2.97-3.01 (dt, 1H, CH), 3.14-3.23 (m, 2H,
CH2), 3.56 (s, 3H, CH3), 3.71 (s, 3H, CH3), 3.72-3.73 (dt, 1H,
CH), 3.96-4.00 (dt, 1H, CH), 4.11-4.18 (m, 1H, CH), 4.39-4.45
(dt, 1H, CH), 8.00-8.03 (dd, 1H, ar), 8.47-8.50 (dd,1H, ar), 8.84-
8.85 (dd, 1H, ar) ppm. 13C NMR (CD3COCD3): δ 14.7, 27.0, 38.8,
45.5, 59.7, 120.5, 128.7, 135.2, 140.2, 150.2 ppm. Anal. Calcd for
C12H17F6N6OPS (438): C, 32.88; H, 3.91; N, 19.17. Found: C,
32.67; H, 4.03; N, 19.31.
1-((Dimethylamino)(morpholino)methylene)-1H-[1,3]triazolo-
[4,5-b]pyridinium Hexafluorophosphate 3-Oxide (HDMA, 5a).8
The product was obtained as a white solid (91.2% yield). Mp: 194-
195 °C dec. 1H NMR (CD3COCD3): δ 3.27 (s, 3H, CH3), 3.64 (s,
3H, CH3), 3.85-3.89 (m, 4H, 2CH2), 4.00-4.07 (m, 4H, 2CH2),
7.93-7.96 (dd, 1H, ar), 8.44-8.47 (dd,1H, ar), 8.76-8.77 (dd, 1H,
ar) ppm.13 C NMR (CD3COCD3): δ 41.7, 42.3, 50.8, 51.6, 66.2,
66.4, 124.4, 127.84, 149.65 ppm. MS m/z using MALDI with ACH
matrix 422.2.Anal. Calcd for C12H17F6N6O2P (422): C, 34.13; H,
4.06; N, 19.90. Found: C, 34.31; H, 4.17; N, 20.13.
1-((Dimethylamino)(morpholino)methylene)-1H-benzotriazo-
lium hexafluorophosphate 3-Oxide (HDMB, 5b).8 The product
was obtained as a white solid (7.54 g, 88.27% yield). Mp: 196-
197 °C dec. 1H NMR (CD3COCD3): δ 3.22 (s, 3H, CH3), 3.51 (s,
3H, CH3), 3.66-3.88 (m, 4H, 2CH2), 4.03-4.06 (m, 4H, 2CH2),
7.65-7.96 (dt, 1H, ar), 7.86-7.92 (dm,2H, ar), 7.98.7.99 (dd, 1H,
ar) ppm. 13C NMR (CD3COCD3): δ 41.8, 42.2, 50.8, 51.5, 66.2,
66.4, 110.0, 114.6, 127.5, 133.5 ppm. Anal. Calcd for C13H18F6N5O2P
(421): C, 37.06; H; 4.31; N, 16.62. Found: C, 36.98; H, 4.26; N,
16.73. MS m/z using MALDI with ACH matrix 421.23.
1-((Dimethylamino)(thiomorpholino)methylene)-1H-benzot-
riazolium Hexafluorophosphate 3-Oxide (HDTMB, 6b). The
white solid obtained was recrystallized from CH3CN ether to give
white crystals (80.1% yield). Mp: 190-191 °C dec. 1H NMR (CD3-
COCD3): δ 2.67-2.70 (m, 1H, CH), 2.91-2.96 (m, 1H, CH), 2.97
(s, 3H, CH3), 3.33-3.48 (m, 5H, CH2, CH3), 3.65-3.69 (m, 1H,
(17) Kushner, S.; Brancone, L. M.; Hewitt, R. I.; McEwen, W. L.;
Subbarow, Y.; Stewart, H. W.; Turner, R. J.; Denton, J. J. J. Org. Chem.
1948, 13, 144-153.
(18) Angier, R. B.; Murdock, K. C.; Curran, W. V.; Sollenberger, P. Y.;
Casey, J. P. J. Org. Chem., 1968, 11, 720-729.
(19) 4ii was used directly without storage because it is very sensitive to
the moisture. The reaction should be under nitrogen atmosphere.
2736 J. Org. Chem., Vol. 73, No. 7, 2008

Coupling Reagents in Peptide Synthesis
CH), 3.88-3.95 m, 1H, CH), 4.09-4.12 (m, 1H, CH), 8.00-8.03
(dd, 1H, ar), 7.68-7.72 (td,1H, ar), 7.86-7.88 (d, 1H, ar), 7.93-
7.97 (d, 1H, ar), 8.08 (d, 1H, ar) ppm. 13C NMR (CD3COCD3): δ
14.7, 27.3, 40.6, 40.8, 42.8, 43.1, 52.9, 53.2, 115.4, 116.5, 127.9,
133.6, 150.8 ppm. Anal. Calcd for C13H18F6N5OPS (437): C, 35.70;
H, 4.15; N, 16.01. Found: C, 35.87; H, 4.06; N, 16.22.
N-(Fluoro(morpholino)methylene)-N-methylmethanamini-
um Hexafluorophosphate (DMFH, 7). Predried KF (0.3 mol) was
added to a stirring solution of chloroformamidinium salt 4i (0.1
mol) in dry CH3CN (200 mL). The reaction mixture was stirred at
room temperature for 6 h, filtered, and washed with CH3CN (2 ×
50 mL). The CH3CN solutions were combined and concentrated
under vacuum, and the residue was dissolved in hot DCM and then
filtered. The filtrate was concentrated to approximately 1/3 of the
original volume, and ether was added with vigorous stirring to
promote the formation of a white solid (89.5 yield). Mp: 92-93 °C.
1H NMR (CH3CN-d6): δ 3.31 (d, 6H, 2CH3), 3.70-3.81 (m, 4H,
2CH2) 3.84-3.86 (m, 4H, 2CH2) ppm.13 C NMR (acetone-d6): 39.3,
43.6, 159.6 ppm. Anal. Calcd for C7H14F7N2OP (306): C, 27.46;
H, 4.61; N, 9.15. Found: C, 27.55; H, 4.64; N, 9.33.
Analysis of the Extent of Coupling Using UV during the
Solid-Phase Synthesis of H-Met-Pro-Pro-Glu-Val-Lys-Phe-Leu-
NH2. After each coupling, 4-6 mg of the resin was taken, washed
with DMF, DCM, and ether, and then dried under vacuum to obtain
the exact weight. The dried resin was treated with 20% piperidine
in DMF (2 mL) for 10 min and then diluted with ethanol up to 25
mL. The extent of coupling was calculated according to the λmax at
300.5 for the dibenzofulvene adduct.
Solid-Phase Synthesis of H-Tyr-Aib-Aib-Phe-Leu-NH2.8 The
pentapeptide was manually assembled on a Fmoc-PAL-PEG-PS-
resin (0.18 mmol/g) with the preactivation time as indicated in Table
6, using Fmoc-amino acid (4 equiv), coupling reagent (4 equiv),
and DIEA (8 equiv) in DMF at a total concentration of 0.3 M. A
coupling time of 30 min was used for all amino acids except for
Aib-Aib, for which the time was 60 min. The peptide was cleaved
from the resin using TFA-H2O (9:1) at room temperature for 2 h.
The solution was filtered, and the TFA was removed under vacuum.
The crude peptide was precipitated by addition of cold ether and
analyzed on HPLC [Waters Symmetry Column C18, 5 µm, 4.6 ×
150 mm, linear gradient of CH3CN and H2O containing 0.1% TFA
each one, from 10% to 90% in 25 min, detection at 220 nm.] The
tR for the pentapeptide and for the des-Aib tetrapeptide was 8.80
and 9.10 min, respectively.
Model Segment Coupling Reaction. Test couplings were carried
out as previously described for Z-Phg-Pro-NH2,20 Z-Phe-Val-Pro-
NH2,6f,20 Z-Gly-Phe-Val-OMe,20 Z-Phe-Val-Ala-OMe,20 and Z-Gly-
Phe-Pro-NH2.6f,20
Solid-Phase Synthesis of H-Met-Pro-Pro-Glu-Val-Lys-Phe-
Leu-NH2. The peptide was manually elongated on an Fmoc-Rink
Amide-resin (0.7 mmol/g). Coupling times were 15 min; excesses
and reagents were 3 equiv for the Fmoc-amino acids and the
coupling reagent and 6 equiv for DIEA; pre-activation time 1-2
min. Peptide purity was determined after cleavage of the peptide
from the resin by treatment with TFA-H2O (9:1) for 2 h at room
temperature by reverse-phase HPLC analysis (using Symmetry
Waters C18 4 µ, 4.6 × 150 mm.), linear gradient over 30 min of 10
to 90% CH3CN in H2O/0.1% TFA, flow rate 1.0 mL/min, tR
nonapeptide ) 10.95 min).
Acknowledgment. This study was partially supported by
CICYT (CTQ2006-03794/BQU, PETRI), ISCIII (CIBER-BBN
0074), the Generalitat de Catalunya (2005SGR 00662), the
Institute for Research in Biomedicine, and the Barcelona Science
Park.
Supporting Information Available: Characterization material.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(20) Carpino, L. A.; El-Faham, A. J. Org. Chem. 1994, 59, 695-698.
JO702622C
J. Org. Chem, Vol. 73, No. 7, 2008 2737