Novel proton acceptor immonium-type coupling reagents: Application in solution and solid-phase peptide synthesis

Article abstract of DOI:10.1021/ol701817u

(Chemical Equation Presented) A novel proton acceptor coupling reagent shows superiority to those described previously. The oxygen in the carbocation moiety confers more solubility to the reagent. Furthermore, it enhances coupling yields and decreases racemization, allowing the use of 1 equiv of base.

Full text of DOI:10.1021/ol701817u

ORGANIC
LETTERS
2007
Vol. 9, No. 22
4475-4477
Novel Proton Acceptor Immonium-Type
Coupling Reagents: Application in
Solution and Solid-Phase Peptide
Synthesis
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, and Department of Organic
Chemistry, UniVersity of Barcelona, Mart´ı i Franque´s 1-11, 08028-Barcelona, Spain
albericio@pcb.ub.es; aymanel_faham@hotmail.com
Received July 28, 2007
ABSTRACT
A novel proton acceptor coupling reagent shows superiority to those described previously. The oxygen in the carbocation moiety confers
more solubility to the reagent. Furthermore, it enhances coupling yields and decreases racemization, allowing the use of 1 equiv of base.
The most widely used coupling reagents are carbodiimides,
immonium and phosphonium salts, and N-triazinylammo-
nium salts.1 Immonium salts, such as HATU, HAPyU,
HBTU, HCTU, or TFFH,2 which possibly are the most
powerful,3 are formed by a leaving group and a carbocation
skeleton. Even though a substantial amount of work has been
done in the past few years to identify the best leaving
group,1,4 less attention has been paid to the nature of the
carbocation part.3,5 The first study on the design, preparation,
and application of novel immonium salts, which incorporate
a proton acceptor in their carbocation skeleton, is described
herein.
Couplings involving immonium salts (Figure 1) are carried
out with an excess of base, at least 2 equiv, and in the
presence of 1 equiv of the hydroxylamine derivative.1c In
this regard, we thought that the incorporation of a proton
(2) Abbreviations not defined in the text: Boc, tert-butyloxycarbonyl;
Cl-HOBt, 6-chloro-1-hydroxybenzotriazole; DCM, dichloromethane; DIEA,
diisopropylethylamine; DMF, N,N-dimethylformamide; Et3N, N,N,N-tri-
ethylamine; Fmoc, fluorenylmethoxycarbonyl; 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)-(dimethyl-
amino)methylene] N-methylmethanaminium hexafluorophosphate N-oxide;
HCTU, N-[(1H-6-chloro-benzotriazol-1-yl)-(dimethylamino)methylene] N-
methylmethanaminium hexafluorophosphate N-oxide; 6-HDMCB, 1-((di-
methylimino)(morpholino)methyl)3-H-6-chlorobenzo[1,2,3]triazolo-1-ium-3-olate
hexafluorophosphate; HMDA, 1-((dimethylimino)(morpholino)methyl)3-
H-[1,2,3]triazolo[4,5-b]pyridine-1-3-olate hexafluorophosphate; HMDB,
1-((dimethylimino)(morpholino)methyl)3-H-benzo[1,2,3]triazolo-1-ium-3-
olate hexafluorophosphate; HOAt, 7-aza-1-hydroxybenzotriazole; HOBt,
1-hydroxybenzotriazole; TFFH, tetramethylfluoroformamidinium hexafluo-
rophosphate; TFA, trifluoroacetic acid; TMP, 2,4,6-trimethylpyridine (col-
lidine); Z, benzyloxycarbonyl. Amino acids and peptides are abbreviated
and designated following the rules of the IUPAC-IUB Commission of
Biochemical Nomenclature [J. Biol. Chem. 1972, 247, 977].
† Barcelona Science Park.
‡ Alexandria University.
§ University of Barcelona.
(1) (a) Albericio, F.; Carpino, L. A. In Methods in 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.
(3) Albericio, F.; Bofill, J. M.; El-Faham, A.; Kates, S. A. J. Org. Chem.
1998, 63, 9678-9683.
10.1021/ol701817u CCC: $37.00
© 2007 American Chemical Society
Published on Web 09/28/2007

Scheme 1. Pathway Used for the Preparation of Novel Proton
Acceptor Immonium-Type Coupling Reagents
Figure 1. Representative immonium salt coupling reagents.
acceptor could have influenced the coupling reagent perfor-
mance in terms of reactivity and racemization control, as
well as its solubility and stability.
The unsymmetrical morpholine-based immonium salts can
be readily prepared by treating N,N-dimethyl carbamoyl
chloride with morpholine to give the corresponding urea
derivatives 5.6 Then, the urea derivative reacts with oxalyl
chloride to yield the corresponding chloro salts (Scheme 1),
which are stabilized by the formation of a PF6 salt 6.
Subsequent reaction with KOXt or HOXt in the presence of
a tertiary amine such as Et3N affords the desired compound
7 as crystalline and shelf-stable solids.7 X-ray crystallography
showed that both HDMA and HDMB were in the N form,
and this agreed with the 13C NMR spectral published data.8
The first property to be taken into account is the solubility
of the coupling reagent, which has a great influence on the
reaction yield and its performance in the automatic synthe-
sizers. Thus, the presence of the oxygen atom in the carbon
skeleton is of marked importance to the solubility of the
compound, increasing its solubility. Accordingly, morpholino
derivatives 7 are more soluble than dimethylamino deriva-
tives 1 and 2, where the salt 6-HDMCB (7c) is more soluble
and can be used to prepare up to a 1 M solution, while
morpholino salts derived from HOAt 7a or HOBt 7b can be
used to prepare up to a 0.75 M solution, as compared to
other dimethylamino derivatives 1-3, which can be used to
prepare up to a 0.5 M solution in DMF. This enhanced
solubility should help the removal during the workup after
the coupling of the urea formed in a solution mode approach.
Table 1 shows examples illustrating the effectiveness of
morpholino derivatives 7 in reducing racemization. All
Table 1. Extent of Conversion and Racemization during Coupling of Z-Phg-OH or Z-Phe-Val-OH and H-Pro-NH2 Using Immonium
Salt-Based Reagentsa
no.
1
AA1
AA2
coupling reagent
HATU
base (equiv)
yield
D-L isomer (%)
Z-Phg-OH
H-Pro-NH2
DIEA (2)
DIEA (1)
TMP (2)
DIEA (2)
DIEA (1)
TMP (2)
DIEA (2)
DIEA (1)
TMP (2)
DIEA (2)
DIEA (1)
TMP (2)
DIEA (2)
DIEA (2)
DIEA (1)
TMP (2)
DIEA (2)
DIEA (1)
TMP (2)
DIEA (2)
DIEA (1)
TMP (2)
DIEA (2)
DIEA (1)
TMP (2)
78.4
74.8
77.9
81.2
82.3
80.3
80.2
75.0
81.2
80.8
82.3
79.9
84.5
85.8
83.2
78.0
89.3
87.4
86.2
89.7
78.6
81.2
88.7
86.3
87.1
3.1
2.4
2.1
1.6
1.6
3.9
8.2
5.3
6.4
3.8
3.1
7.8
1.5
13.9
11.0
5.3
10.5
5.1
2
3
4
Z-Phg-OH
Z-Phg-OH
Z-Phg-OH
H-Pro-NH2
H-Pro-NH2
H-Pro-NH2
HMDA
HBTU
HMDB
5
6
Z-Phg-OH
Z-Phe-Val-OH
H-Pro-NH2
H-Pro-NH2
6-HDMCB
HATU
7
8
9
Z-Phe-Val-OH
Z-Phe-Val-OH
Z-Phe-Val-OH
H-Pro-NH2
H-Pro-NH2
H-Pro-NH2
HMDA
HBTU
HMDB
3.7
27.7
16.3
14.2
20.3
11.5
13.3
a
conc ) 0.1 mM, calculated by HPLC.
4476
Org. Lett., Vol. 9, No. 22, 2007

morpholino derivatives (nos. 2, 4, 5, 7, and 9) result in
improved control of racemization when compared to their
tetramethylamino counterparts (nos. 1, 3, 6, and 8). Interest-
ingly, morpholino derivatives perform especially well with
just 1 equiv of base. Thus, yields are similar or even slightly
better than those obtained when 2 equiv of base and/or
tetramethylamino derivatives are used.
To compare the relative coupling rates for various reagents
and the effect of the oxygen as a proton acceptor, reaction
of Fmoc-Val-OH with H-Val-NH2 in the presence of 1 or 2
equiv of DIEA was carried out. Table 2 indicates that the
Table 2. Extent of Conversion in the Preparation of
Fmoc-Val-Val-NH2 Using HATU/HDMA and DIEA (1 Equiv/2
Equiv) as a Base in DMF (Calculated by HPLC)
Figure 2. HPLC for the crude pentapeptide prepared in solution.
HATU
yield (%)
HDMA
yield (%)
In a more demanding example, H-Tyr-Aib-Aib-Phe-Leu-
NH2 was manually solid-phase assembled on Fmoc-Rink
Amide-AM-resin using amino acid/activator (3 equiv) and
DIEA (6 equiv) for a 30 min coupling, except for Aib-Aib,
which required 1 h. Yields for coupling of Fmoc-Aib-OH
onto H-Aib-Phe-Leu-NH-Rink Amide-AM-resin were de-
termined by reverse-phase HPLC analysis (integration of
pentapeptide vs tetrapeptide) after cleavage of the pep-
tide from the resin. The best results were obtained with
6-HDMCB (7c) (99% coupling) and HDMA (7a) (98%
coupling) and HDMB (7b) (89% coupling) over HATU (1)
(83% coupling) and HBTU (2) (47% coupling).
In conclusion, the novel proton acceptor coupling reagents
showed superiority to those described previously.9 The
oxygen in the carbocation moiety confers more solubility,
to the reagent, allowing us to carry out the reaction at
higher concentration and facilitating the removal of side
products when chemistry is carried out in solution. Further-
more, it enhances coupling yields and decreases racemiza-
tion, allowing the use of just 1 equiv of base. Results obtained
with HMDB and 6-HDMCB, which contain the less reactive
HOBt and Cl-HOBt, match those obtained with HATU,
which contains the most effective and expensive HOAt.
time (min)
2 equiv
1 equiv
2 equiv
1 equiv
5
10
20
30
60
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
95.2
96.4
98.0
99.0
99.0
80.0
85.0
90.0
93.5
95.5
96.0
120
morpholino derivative (HDMA) showed a better coupling
rate in both cases (1 and 2 equiv of DIEA). This indicates
that the oxygen atom increases reactivity and acts as a proton
acceptor during the coupling step.
To demonstrate the effectiveness of the oxygen on the
carbon skeleton of the new reagents in the solution phase,
the pentapeptide H-Tyr-Gly-Gly-Phe-Leu-NH2 was prepared
in solution without isolation and purification of the inter-
mediates using Boc chemistry and HDMB (7b) as a coupling
reagent. The purity of the pentapeptide tested by HPLC
analysis and HPLC-MS was excellent (Figure 2). The HPLC-
MS showed the correct mass for the pentapeptide. No traces
of the urea were detected confirming the high solubility of
these derivatives.
Acknowledgment. This study was partially supported
by CICYT (CTQ2006-03794/BQU), the Generalitat de
Catalunya (2005SGR 00662), Luxembourg Industries Ltd.,
the Institute of Research in Biomedicine, and the Barcelona
Science Park.
(4) Immonium salt derivatives of HOAt, such as HATU and HAPyU,
have been shown to be the most efficient in terms of both reactivity and
controlling racemization. This has been interpreted by the electron-
withdrawing influence of the nitrogen atom that effects stabilization of the
leaving group leading to greater reactivity. Second, the nitrogen makes a
classic neighboring group effect feasible which can both speed up the
reactivity and reduce loss of configuration. (a) Carpino, L. A. J. Am. Chem.
Soc. 1993, 115, 4397-4398. (b) Carpino, L. A.; El-Faham, A.; Minor, C.
A.; Albericio, F. J. Chem. Soc., Chem. Commun. 1993, 201-203.
(5) El-Faham, A.; Khattab, S. N.; Abdul-Ghani, M.; Albericio, F. Eur.
J. Org. Chem. 2006, 1563-1573.
Supporting Information Available: Experimental pro-
cedures and characterization material. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL701817U
(6) Reactions of urea 6 with other secondary amines did not led to
positive results [thiomorpholine, poor coupling reagent; N-methylpiperazine,
the target compound was not obtained].
(9) Interestingly, Kaminski, Papini, and co-workers have described the
N-methylmorpholinium derivative as the reagent of choice of its family of
reagents for their independent triazinylammonium system. Kaminski, Z.
J.; Kolesinska, B.; Kolesinska, J.; Sabatino, G.; Chelli, M.; Rovero, P.;
Blaszczyk, M.; Glowka, M. L.; Papini, A. M. J. Am. Chem. Soc. 2005,
127, 16912-16920.
(7) Patent pending.
(8) 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.
Org. Lett., Vol. 9, No. 22, 2007
4477