Design and synthesis of new immonium-type coupling reagents

Article abstract of DOI:10.1002/ejoc.200500630

A new family of immonium-type coupling reagents is de-scribed here. The differences in the carbocation skeletons of these reagents can be correlated with differences in stability and thus reactivity. The dihydroimidazole derivatives are highly unstable to air, whereas the salts derived from dimethylamine are the most stable and the pyrrolidino derivatives are of intermediate stability. These results should be taken into account mainly when coupling reagents are deposited in open vessels, such in some automatic synthesizers. As regards both coupling yield and retention of configuration, HOAt derivatives have been confirmed to be superior to those of HOBt in all cases. For peptides containing hindered residues, fluoroformamidinium salts are more convenient than the HOAt-based reagents. Wiley-VCH Verlag GmbH & Co. KGaA, 2006.

Full text of DOI:10.1002/ejoc.200500630

FULL PAPER
DOI: 10.1002/ejoc.200500630
Design and Synthesis of New Immonium-Type Coupling Reagents[‡]
[a]
[a]
[b]
Ayman El-Faham,* Sherine N. Khattab, Mohamed Abdul-Ghani, and
Fernando Albericio*[
c,d]
Keywords: Amides / Combinatorial chemistry / Peptides / Racemization / Solid-phase synthesis
A new family of immonium-type coupling reagents is de- in open vessels, such in some automatic synthesizers. As re-
scribed here. The differences in the carbocation skeletons of
these reagents can be correlated with differences in stability
and thus reactivity. The dihydroimidazole derivatives are
highly unstable to air, whereas the salts derived from dime-
thylamine are the most stable and the pyrrolidino derivatives
are of intermediate stability. These results should be taken
into account mainly when coupling reagents are deposited
gards both coupling yield and retention of configuration,
HOAt derivatives have been confirmed to be superior to
those of HOBt in all cases. For peptides containing hindered
residues, fluoroformamidinium salts are more convenient
than the HOAt-based reagents.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2006)
The activation of a carboxylic acid for the formation of phase methodologies.[2a,3] The formerly predominant car-
an amide bond is usually carried out using the so-called bodiimide and active-ester techniques have been replaced
peptide coupling reagents.[ During the last decade there with onium salts based upon 1-hydroxybenzotriazole
2]
[
4]
has been an evolution in the development of new activation (HOBt, 3a) and 7-aza-1-hydroxybenzotriazole (HOAt,
[
5]
methods and their application to both solution and solid- 3b). Among current methods of choice for peptide coup-
Scheme 1.
ling reagents are the aminium/uronium derivatives (also
known as Knorr reagents ), which have become popular
because of their high efficiency and low tendency towards
racemization of the amino acid or peptide residue. These
‡] For abbreviations see ref.[
1]
[6]
[
[
a] Department of Chemistry, Faculty of Science, Alexandria Uni-
versity,
Ibrahimia 21321, P. O. Box 246, Alexandria, Egypt
[
[
[
b] Department of Chemistry, Faculty of Science, Beirut Arab Uni- salts are prepared by reaction of a compound of the type
HOX (HOBt 3a, HOAt 3b, HODbht 3c, and HOSu 3d)
c] Barcelona Biomedical Research Institute, Barcelona Science with the chloroformamidinium salt 2 derived from an urea
versity,
P. O. Box 11-5020, Beirut, Lebanon
Park, University of Barcelona,
such as tetramethylurea (TMU, 1; Scheme 1). Examples of
0
8028 Barcelona, Spain
these common aminium/uronium salts are the 1-hydroxy-
d] Department of Organic Chemistry, University of Barcelona,
8028 Barcelona, Spain
[
6,7]
0
benzotriazole (HBTU) derivatives 4a,
the 7-aza-1-hy-
Eur. J. Org. Chem. 2006, 1563–1573
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1563

A. El-Faham, S. N. Khattab, M. Abdul-Ghani, F. Albericio
FULL PAPER
droxybenzotriazole (HATU) derivatives 4b,[5] the 3-hy-
droxy-3,4-dihydro-1,2,3-benzotriazin-4-one (HDTU) deriv-
[
6,8]
atives 4c,
the 1-hydroxy-1,2-dihydro-2-pyridone (HPTU)
[
9]
derivatives 4e, and the hydroxysuccinimide (HSTU) deriv-
[
10]
atives 4d. The majority of these compounds are commer-
cially available. Further investigation has led to other amin-
[
11]
[12]
ium-type reagents such as HBPyU (5),
HAPyU (6),
[
13]
[12]
[12]
HBMDU (7),
HAMDU (8),
HBPipU (9a),
and
[
12]
HAPipU (9b).
In 1998, Ramage et al. reported the synthesis of HOCt
[14]
(10),
which was designed for Fmoc solid-phase peptide
Onium salt mediated amide formation generally involves
synthesis. HOCt exhibits high coupling efficiency, but it two steps: activation, in which the coupling reagent reacts
should be used only as an additive in combination with car- with an N-protected amino acid to form an active carboxyl,
bodiimide reagents because its corresponding Knorr reagent and coupling, whereby the active carboxyl reacts with the
[
2,17]
decomposes during synthesis. This has also been observed amino component to form the peptide bond.
Thus, the
[9]
during the preparation of imidazolidonium-type reagents.
coupling efficiency depends on the nature of the leaving
Recently, Xu and Li[ have reported an alternative path- group of the onium salt, which is also the leaving group of
way to enhance the coupling efficiency by modifying the the active carboxyl. Therefore, the coupling efficiency can
carbon skeleton of uronium salts by replacing one of the be enhanced by modifying the leaving group of the onium
substituted amino groups with a hydrogen, alkyl, or aryl salts. This approach has proven successful for cases in
group (Scheme 2). The authors described that using these which HOAt-based onium salts were substituted for
types of immonium reagents to synthesize peptides not only HOBt-, HOPfp-, HOSu-, or HODhbt-derived onium
15]
[
2,6,7,8,13,18]
enhanced the coupling efficiency but also substantially sup- salts.
pressed the extent of racemization under relatively mild
conditions.
We present here a new approach to enhance the coupling
efficiency based on the modification of the structures of
[
15]
More recently, Carpino et al.[16] reported the new coup- the carbocation skeleton moiety of aminium/uronium-type
ling reagents HDATU (11), and HDAPyU (12), which they reagents, which feature relatively high reactivity and low ra-
prepared by a method analogous to that used for the prepa- cemization during peptide bond formation.
ration of HDTU (4c),[ which is the HODhbt uronium salt
8]
These uronium-type reagents can be readily prepared by
reagent. As expected, 11 and 12 are more reactive than 4c. treating N,N-dialkylcarbamoyl chloride (13) with secondary
Scheme 2.
1564
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 1563–1573

Immonium-Type Coupling Reagents
FULL PAPER
Scheme 3.
amines such as diethylamine, pyrrolidine, or piperidine to their stability in solution and in the solid state was exam-
1
give the corresponding urea derivatives 14 (Scheme 3, ined by HPLC and H NMR analysis. Furthermore, evalu-
Table 16). Then, the urea derivatives react with oxalyl chlo- ation of a given compound’s stability in solution can pro-
[
17]
ride to yield the corresponding chloro-salts 15 (Scheme 3, vide information about its reactivity.
HATU (4b),
Table 17), which are stabilized by the formation of a PF6 HAPyU (6), HAM PyU (16a), HAM PipU (16c), HAE -
2
2
2
salt. Subsequent reaction with HOXt (3a or 3b) in the pres- PyU (16e), HAE PipU (16g), HATeU (16i), TFFH (17a),
2
ence of a tertiary amine such as NEt affords the desired BTFFH (17b), DMFFH (17c), DEFFH (17d), and TEFFH
3
compounds 16 as crystalline, stable solids (Scheme 3, (17e) are stable solids at 25 °C. DMF solutions (0.6 ) of
Tables 18 and 19).
HATU (4b), HAPyU (6), HAM PyU (16a), HAM PipU
2 2
Furthermore, the chloride salts 15 can be converted into (16c), HAE PyU (16e), HAE PipU (16g), and HATeU (16i)
2
2
their corresponding fluorides 17 by treatment with KF are stable under nitrogen at 25 °C for 3–4 weeks. DMF
3 equiv.) in acetonitrile at 60 °C for 3–4 h (Scheme 3, solutions (0.6 ) of the reagents shown in Table 6 exposed
(
Table 20).
to the atmosphere are stable for 1–2 weeks. DMF solutions
0.6 ) of the fluoride salts TFFH (17a), BTFFH (17b),
DMFFH (17c), DEFFH (17d), and TEFFH (17e), are
(
Stability of HOXt Uronium-Type Reagents
In order to determine the compatibility of HOAt-based stable for 1–2 days, but in the presence of DIEA (0.6 )
1
coupling reagents with automated peptide synthesizers, they are only stable for 1 h, as determined by H NMR
Eur. J. Org. Chem. 2006, 1563–1573
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1565

A. El-Faham, S. N. Khattab, M. Abdul-Ghani, F. Albericio
FULL PAPER
spectroscopy. The data in Table 1 show that, as expected, Table 3. Hydrolytic stability of DMF solutions of uronium salt deriva-
tives in the presence of DIEA.
aza derivatives are less stable, and therefore more reactive,
than the benzotriazole analogs.[
17]
Coupling reagent
2 min
30 min
60 min
HATU (4b)
HBTU (4a)
HAPyU (6)
HAPipU (9b)
HATeU (16i)
HBTeU (16j)
93
95
59
65
95
97
80
90
84
83
62
76
37
65
67
79
53
77
59
66
36
62
9
43
43
66
28
62
38
36
Table 1. Hydrolytic stability of DMF solutions of uronium salt deriva-
tives in open vials.
[a]
Coupling reagent
5 h
24 h
48 h
HATU (4b)
HBTU (4a)
HAPyU (6)
HBPyU (5)
HAPipU (9b)
HAMDU (8)
HBMDU (7)
HATeU (16i)
HBTeU (16j)
99
100
96
97
98
0
0
100
100
98
100
100
95
98
79
84
95
0
76
86
20
40
81
0
HAM PyU (16a)
2
HBM PyU (16b)
2
HAE PyU (16e)
2
HAM PipU (16c)
2
0
0
or in convergent strategies during the fragment coupling
steps because the yields tend to be lower than for other
couplings.
92
95
85
90
88
85
88
50
83
60
HAM
HBM
HAE
2
PyU (16a)
PyU (16b)
PyU (16e)
2
2
Activation of Z-Aib-OH
[a] All reagents have a stability greater than 95% in a closed vial, but
HAMDU (8) and HBMU (7) are totally unstable.
As mentioned above, the formation of a peptide bond
between two amino acids involves two steps. The first step
Furthermore, the nature of the carbon skeleton of a com- is the activation of the carboxyl group of one residue, and
pound is of marked importance to the stability of the com- the second step is the nucleophilic attack of the amino
pound. Both dihydroimidazole [HAMDU (8), HBMDU group of the other amino acid derivative at the active car-
(7)] derivatives are very unstable, whereas their correspond- boxylic group. To study the activation step of the carboxylic
ing dimethylamine salts are the most stable and the pyrroli- acid, Z-Aib-OH was chosen because of its sterically hin-
dino derivatives are of intermediate stability. These results dered carboxyl group.
should be taken into account mainly for those syntheses
Reaction of Z-Aib-OH with p-chloroaniline (PCA), a
carried out in automatic synthesizers, where the coupling rather poor nucleophilic amine, in the presence of a coup-
reagents are dispensed in open vessels. As expected, all the ling reagent was used to compare the relative coupling rates
coupling reagents are more stable when their DMF solu- for various reagents. As the formation of intermediate Z-
tions are stored under N2,[ conditions which are em- Aib-OXt (active ester) is usually very fast (Table 4), half-
19]
1
ployed in some automatic synthesizers (Table 2).
lives were determined by H NMR spectroscopy by moni-
toring the disappearance of the benzylic CH residue (δ =
2
Table 2. Hydrolytic stabilty of uronium salt derivatives in DMF under 5.2 ppm) of the active ester and the appearance of the ben-
nitrogen.
zylic unit (δ = 5.02 ppm) of the Z-Aib-PCA formed
Coupling reagent
1 day
3 days
7 days
24 days
(Table 5).
HATU (4b)
HAPyU (6)
HAPipU (9b)
HATeU (16i)
100
100
100
100
100
100
100
98
96
98
100
98
100
100
92
84
88
94
88
94
95
87
68
72
90
75
89
90
Table 4. Rate of formation of active ester (Z-Aib-OAt) in DMF as sol-
vent with TMP as base.[
a]
Coupling rea-
gent
4b
6
16a
16e
16i
16c
16g
HAM
HAE
HAM
2
PyU (16a)
PyU (16e)
PipU (16c)
2
t1/2 [min]
3
Ϸ2
Ϸ2
Ϸ2
7
4
4
2
[a] t1/2 of formation of active ester by all of the coupling reagents is less
than 2 min (almost complete conversion within 2 min) when DIEA is
used, as opposed to 5–8 min when TMP is used, or around 16 min for
The stability of these compounds was also examined in
the presence of DIEA (Table 3) because peptide bond for- the case of tetraethyl derivatives.
mation is usually carried out in the presence of at least one
Table 5. Approximate half-lives for the disappearance of Z-Aib-OXt in
DMF in the presence of p-chloroaniline.
extra equivalent of base. Analysis of these results confirms
that the various coupling reagents rapidly degrade in the
absence of a carboxylic acid function.
t
1/2
DMF
This observation has practical consequences for both so- HATU (4b)
75–85 min
10–11 h
70–80 min
70–80 min
9–10 h
HBTU (4a)
HAPyU (6)
lid-phase and solution strategies. Thus, if activation of a
carboxylic acid is slow, then the coupling reagents will be
degraded and will no longer be able to activate the carboxyl
function. Under these conditions, aza derivatives are more
HAM
HBM
HAE
2
PyU (16a)
PyU (16b)
2
2
PyU (16e)
PipU (16c)
PipU (16g)
HATeU (16i)
90 min
labile than the benzotriazole derivatives and pyrrolidino de- HAM
2
100 min
100 min
100 min
HAE
2
rivatives are more labile than dimethylamino and dieth-
ylamino derivatives. This is important for cyclization steps
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 1563–1573

Immonium-Type Coupling Reagents
FULL PAPER
The results shown in Table 5 are in agreement with the
previously discussed observation (Table 1). Thus, aminium
salts derived from HOAt (4b) are more effective than those
derived from HOBt (4a). Examination of the activation at
2
min indicates that the aza derivatives are more reactive
than their corresponding benzotriazole analogs (the NMR
spectra show a 10–15% conversion for the aza derivatives
but almost no reaction for the those of HOBt). Further-
more, formation of the active-ester species competes with
hydrolysis of the coupling reagents. Thus, the most reactive
onium salt, HAMDU, gives the poorest activation as com-
pared to the less reactive HATU (4b), HATeU (16i), HAM2-
PyU (16a), and HAPyU (6), due to the fact that after a few
seconds no activation reagent is present in the medium.
These models involve stepwise coupling and [2+1] seg-
ment coupling as well. For the sensitive coupling of the Z-
Racemization
To investigate the configuration retention induced for the Phg-OH to H-Pro-NH to give 18, HATU (4b), HAPyU
2
new coupling reagents, several previously studied model (6), HAM PyU (16a), HAM PipU (16c), HAE PyU (16e),
2
2
2
peptide systems 18–23 were examined.[
12]
HAE PipU (16g), and HATeU (16i) give a greater conser-
2
Table 6. Yield and racemization during the formation of Z-Phg-Pro-NH
2
in DMF (solution-phase synthesis).[a]
Base
4b
4a
6
16[a]
16b
16e
16f
16i
16j
TMP
Yield [%]
77.9
2.1
81.2
6.4
78.9
2.3
80.1
2.9
80.4
3.7
78.1
4.9
78.1
6.2
85.2
3.5, 3.0
86.1
6.8, 6.7
 [%]
DIEA
Yield [%]
78.4
3.1
80.1
8.2
77.9
3.2
82.3
2.7
80.4
3.4
82.3
4.1
78.7
4.9
88.1
1.5, 1.7
84.6
6.2, 6.3
 [%]
[
a] The  and  forms of the test tripeptide have been described elsewhere.[12] The t
R
’s of  and  were determined by co-injection with authentic
and pure samples of .
Table 7. Yield and racemization during the formation of Z-Phe-Val-Pro-NH
2
(2 + 1) in DMF (solution-phase synthesis).[a]
Base
4b
4a
6
16a
16b
16e
16f
16i
16j
TMP
Yield [%]
83.2
5.3
81.2
14.2
85.5
3.5
82.1
4.1
83.4
14.2
83.2
4.9
84.5
15.7
86.3
9.0
84.5
18.3
 [%]
DIEA
Yield [%]
85.8
13.9
89.7
27.4
88.9
10.8
83.6
13.6
83.9
30.1
85.1
14.9
86.1
30.4
89.0
16.0
89.3
37.3
 [%]
[
a] The  and  forms of the test tripeptide have been described elsewhere.[8] The t
R
’s of  and  were determined by co-injection with
authentic and pure samples.
Table 8. Yield and racemization during formation of Z-Phe-Val-Pro-NH
2
19 (2 + 1) in DMF (solid-phase synthesis).[a]
Base
4b
4a
6
16a
16b
16e
16f
16i
16j
TMP
Yield [%]
93.9
9.4
95.2
13.6
93.4
8.8
93.2
16.9
93.4
22.9
92.1
17.2
92.3
29.8
92.1
22.2
91.2
39.5
 [%]
DIEA
Yield [%]
98.1
22.2
97.6
24.8
95.7
15.6
94.6
18.9
95.3
26.7
96.5
23.4
–
96.3
25.3
–
 [%]
[
a] The  and  forms of the test tripeptide have been described elsewhere.[8] The t
R
’s of  and  were determined by co-injection with
authentic and pure samples.
Eur. J. Org. Chem. 2006, 1563–1573
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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A. El-Faham, S. N. Khattab, M. Abdul-Ghani, F. Albericio
Finally, the test hexapeptide Z-Gly-Gly-Val-Ala-Gly-
FULL PAPER
vation of chirality than their benzotriazole analogs. Like-
wise, HAM PyU (16a) performs slightly better than Gly-NH (23) was assembled on a solid phase by coupling
2
2
HAPyU (6; Table 6).
Z-Gly-Gly-Val-OH to H-Ala-Gly-Gly-PAL-resin. This
For the well-studied segment coupling of Z-Phe-Val-OH coupling has previously been shown to be a sensitive test for
[
8,12,16]
to H-Pro-NH , which leads to tripeptide 19,
the best checking the performance of coupling reagents and bases
2
[
12]
results were obtained with HATU (4b), HAPyU (6), HAM2- (Table 12).
PyU (16a), and other HOAt derivatives in both solution
(Table 7) and on a solid phase (Table 8).
For the rather non-sensitive case of segment coupling of
Rate of Formation of the Protected Amino Acid Fluorides
Z-Gly-Phe-OH to H-Pro-NH , which leads to tripeptide 22,
2
HOAt-derived reagents again perform better than their
N-Protected amino acid (0.1 mmol), DIEA (0.1 mmol),
HOBt analogues (Table 9).
and fluoroformamidinium salt (0.1 mmol) were dissolved in
Studies with tripeptide models 20 and 21 also confirmed the corresponding solvent (DMF or DCM, 0.5 mL). The
the previous results (Tables 10 and 11).
reaction mixture was monitored by IR spectroscopy, which
Table 9. Yield and racemization during the formation of Z-Gly-Phe-Pro-NH
2
22 (2 + 1) in DMF (solution-phase synthesis).[a]
Base
4b
4a
6
16a
16b
16e
16f
16i
16j
TMP
Yield [%]
86.8
0.8
84.8
3.6
86.8
0.6
88.6
0.6
86.1
3.8
79.8
0.9
78.1
2.6
78.0
1.3
76.9
3.1
 [%]
DIEA
Yield [%]
90.1
1.6
88.9
5.9
89.8
1.3
88.9
1.0
86.1
4.7
83.3
1.2
79.3
3.9
80.1
1.8
78.9
3.9
 [%]
[
a] The  and  forms of the test tripeptide have been described elsewhere.[8] The t
authentic and pure samples.
R
’s of  and  were determined by co-injection with
Table 10. Yield and racemization during the formation of Z-Gly-Phe-Val-OMe 20 (2 + 1) in DMF (solution-phase synthesis).[a]
Base
4b
4a
6
16a
16b
16e
16f
16i
16j
TMP
Yield [%]
90.1
0.9
84.8
5.9
86.6
0.6
85.1
0.9
84.4
4.5
86.1
0.8
84.2
4.1
86.3
0.8
87.1
4.8
 [%]
DIEA
Yield [%]
86.2
1.6
88.9
3.6
84.1
1.3
85.0
1.6
86.4
3.4
82.3
1.8
82.7
10.1
87.1
1.5
86.3
11.8
 [%]
[
a] The  and  forms of the test tripeptide have been described elsewhere.[8] The t
authentic and pure samples.
R
’s of  and  were determined by co-injection with
Table 11. Yield and racemization during the formation of Z-Phe-Val-Ala-OMe 21 (2 + 1) in DMF (solution-phase synthesis).[a]
Base
4b
4a
6
16a
16b
16e
16f
16i
16j
TMP
Yield [%]
82.1
Ͻ0.1
83.3
2.9
83.2
Ͻ0.1
80.1
0.6
83.4
3.5
82.2
0.4
80.2
10.4
89.3
0.39
85.6
12.4
 [%]
DIEA
Yield [%]
83.2
2.1
88.9
5.6
84.1
2.3
85.0
2.6
86.4
6.4
82.3
2.8
84.7
13.1
86.2
2.8
90.1
16.1
 [%]
[
a] The  and  forms of the test tripeptide have been described elsewhere.[8] The t
authentic and pure samples.
R
’s of  and  were determined by co-injection with
Table 12. Yield and racemization during the formation of Z-Gly-Gly-Val-Ala-Gly-Gly-NH
2
-resin (3 + 3) in DMF (solid-phase synthesis).[a]
Base
4b
4a
6
16a
16b
16e
16f
16i
16j
TMP
Yield [%]
97.8
2.1
94.8
4.6
97.9
2.3
96.3
2.3
93.4
11.3
95.6
9.4
95.2
12.5
96.2
9.9
95.6
12.8
 [%]
DIEA
Yield [%]
92.1
29.9
95.9
36.8
96.7
41.4
–
–
–
–
–
–
 [%]
[
a] The test tripeptide has been described elsewhere.[8,12] The t
R
’s of  and  were determined by co-injection with authentic and pure samples.
1568
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 1563–1573

Immonium-Type Coupling Reagents
FULL PAPER
allows facile tracking of the formation of the corresponding ration was detected for Ile72 onto Asn, Ile69 onto Asp, and
–
1
acid fluoride (1844 cm ). For unhindered amino acids such Val onto Gln. Peptide purity was determined by reverse-
as Ala, Leu, Gly, His, Phe, Ser, and Met, three to seven phase HPLC analysis (Table 13) after cleavage of the pep-
minutes is sufficient for complete conversion to the acid tide from the resin by treatment with TFA/H O (9:1) for
2
fluoride in DMF or DCM. In the case of hindered amino 2 h at room temperature.
acids such as Val or Ile seven to ten minutes is necessary,
The results outlined in Table 13 reflect the superiority of
and in the case of the most hindered residue, Aib, the com- aza derivatives to HOBt analogs. Furthermore, long preac-
plete conversion takes about 25 min. However, even in these tivation times are shown to be detrimental. Finally, al-
latter cases (Val, Ile, Aib), more than 50–75% of acid fluo- though all aza derivatives gave good results, HATU (4b),
ride was formed after seven minutes of preactivation. HAPyU (6), HAM PyU (16a), and HAE PyU (16e) gave
2
2
Furthermore, the rate of formation can be increased by the best results (Ͼ80% purity).
using two equivalents of DIEA. Thus, for Val and Ile the
acid fluoride formation is almost complete within seven
minutes. Based on these experiments, the recommended
Hindered Amino Acids
maximum preactivation time for the salts is 7–10 min.
Based on IR studies, the order of reactivity is as follows:
TFFH (17a), BTFFH (17b) Ϸ DMFFH (17c), DEFFH
In a more demanding example, H-Tyr-Aib-Aib-Phe-Leu-
NH was manually assembled on PAL-PEG-PS-resin using
2
amino acid/activator (4 equiv.), base (8 equiv.), with a 30-
min coupling time, except for the case of Aib-Aib, which
required 1 h. Peptide purity was determined by reverse-
phase HPLC analysis after cleavage of the peptide from the
(17d), TEFFH (17e) Ͼ DFIH (17f). The poor results ob-
tained for DFIH are due to its decomposition during preac-
tivation.
resin by treatment with TFA/H O (9:1) for 2 h at room tem-
2
perature (Tables 14 and 15).
Comparison of Coupling Techniques
The results shown in Table 14 confirm that aza deriva-
In order to demonstrate the effectiveness of the new tives are more convenient for the synthesis of these de-
HOAt-based reagents and compare their performance to manding peptides, and that preactivation can be detrimen-
that of HOBt analogues, the common decapeptide model tal to the quality of the final product.
ACP(65–74) was assembled on a solid phase.[ The peptide
19]
The same synthesis was carried out with fluorofor-
was manually elongated on an Fmoc-Gly-Wang-PEG-PS- mamidinium salts. The results, which are shown in Table 15,
resin. Coupling times were shortened and excesses of rea- indicate that for this peptide these salts are more convenient
gents were reduced in order to highlight the differences be- than HOAt-based uronium salts.
tween the various coupling reagents studied, as described
In conclusion, a new family of immonium-type coupling
[
19]
previously.
Under these conditions, incomplete incorpo- reagents that differ in the structure of their carbocation
Table 13. Purity of ACP(65–74) using a 1.5-fold excess of amino acids and reagents and 1.5-min coupling time.[a]
Coupling Reagent
P.T.[b]
Yield [%]
ACP
Des-Asn
Des-Val
Des-Ala
Des-Ile69
Des-Ile72
Des-Ile, Ile
HATU (4b)
HATU (4b)
HBTU (4a)
HBTU (4a)
HAPyU (6)
HAPyU (6)
HBPyU (7)
HBPyU (7)
20–30 s
7 min
20–30 s
7 min
20–30 s
7 min
20–30 s
7 min
20–30 s
20–30 s
20–30 s
7 min
7 min
7 min
20–30 s
7 min
20–30 s
7 min
20–30 s
7 min
20–30 s
7 min
71.9
70.3
70.5
70.0
73.1
73.4
70.0
70.0
71.2
70.2
70.9
76.0
78.0
78.0
70.1
67.3
68.6
69.1
66.0
68.0
70.1
69.3
80.6
80.7
45.8
48.9
88.1
70.1
55.7
54.0
81.3
78.3
58.9
60.2
71.9
68.9
79.7
60.3
72.5
66.8
42.3
40.1
75.6
63.3
0.7
10.5
0.8
10.5
0.64
10.4
0.8
10.3
0.79
2.5
0.76
10.1
12.0
13.0
7.77
6.8
1.8
0.5
2.6
0.9
1.5
0.8
2.6
1.0
1.53
1.83
2.4
2.5
2.0
2.5
2.44
1.4
10.6
5.7
11.0
5.8
9.4
2.4
1.1
3.2
1.2
3.8
0.66
1.5
1.1
3.1
0.43
0.98
1.0
5.8
5.3
5.8
1.6
14.9
0.8
3.6
1.3
5.2
0.6
15.8
2.45
1.1
6.4
4.5
0.9
4.9
5.3
5.8
1.23
1.53
4.9
6.1
1.0
1.6
2.65
2.2
11.9
5.2
13.8
6.1
–
–
–
1.4
0.9
5.6
–
3.2
0.7
5.6
–
5.8
7.3
–
0.5
4.3
6.7
–
HAM
HAM
HB M
2
PyU (16a)
PipU (16c)
2
–
–
2
PyU (16b)
PyU (16b)
0.8
4.5
–
4.2
6.8
–
HBM
HAM
2
2
PyU (16a)
PipU (16c)
PyU (16e)
PyU (16e)
HAM
HAE
HAE
2
–
–
2
2.82
2.1
2.7
2.7
3.4
5.9
2.6
2.6
2.16
1.7
–
2.6
6.3
7.9
–
2
HATeU (16i)
HATeU (16i)
HBTeU (16j)
HBTeU (16j)
1.6
7.5
2.3
10.5
1.6
HAE
HAE
2
PipU (16g)
PipU (16g)
10.1
2.7
2
7.6
1.9
[
a] All peptides (ACP and des-amino acid structures) were confirmed by peak overlap in the presence of authentic samples prepared by standard
protocols and identified by mass spectral analysis on a Perseptive Biosystems Voyager DE type MALDI-TOF instrument using sinapinic acid as
matrix. Crude ACP(65–74) was analyzed by HPLC [Nova Pak C18 60-Å column (3.9×150 mm, 4 µm), linear gradient over 25 min of 5 to 35%
–1
3 2
CH CN in H O/0.1% TFA; flow rate: 1.0 mLmin ]. [b] P.T: preactivation time.
Eur. J. Org. Chem. 2006, 1563–1573
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1569

A. El-Faham, S. N. Khattab, M. Abdul-Ghani, F. Albericio
Table 14. Percentage of des-Aib (4-mer) obtained during solid-phase assembly of the pentapeptide.[a]
FULL PAPER
Coupling reagent
HATU (4b)
HBTU, 4a)
HAPyU (6)
(HBPyU, 5)
HAM
(HBM
2
PyU (16a)
PyU, 16b)
HAE
(HBE PyU, 16f)
2
PyU (16e)
2
HATeU (16i)
(HBTeU, 16j)
(
2
2
0–30 s P.T.[b]
7.9
43)
22
58)
23
(42)
24
18.9
(45)
23.6
(59)
21.2
(44)
25.1
(57)
22.3
(45)
24.2
(58)
(
7
min P.T.[b]
(
(57)
[
a] Tetrapeptide (des-Aib) was confirmed by peak overlap in the presence of an authentic sample. The crude H-Tyr-Aib-Aib-Phe-Leu-NH
2
was
analyzed by HPLC [Nova Pak C18 60-Å column (3.9×150 mm, 4 µm), linear gradient over 25 min of 10 to 90% CH CN in H O/0.1% TFA; flow
rate: 1.0 mLmin ]. [b] P.T.: preactivation time.
3
2
–1
Table 15. Pentapeptide purity and relative amount of des-Aib for solid-phase syntheses.
Coupling reagent
TFFH
17a)
BTFFH
(17b)
DFIH
(17f)
DMFFH
(17c)
DEFFH
(17d)
TEFFH
(17e)
(
Pentapep [%]
Des-Aib [%]
95.2
4.8
95.8
4.2
92.2
7.8
94.6
5.4
94.7
5.3
94.9
5.1
skeletons has been described. These differences have a UV Lamp Model CM-10 (254 nm). Melting points were obtained
in open capillary tubes using a Gallenkamp Sanyo melting point
marked influence on the stability of the reagent. The salts
apparatus and are uncorrected. IR spectra were recorded with a
derived from dimethylamine are the most stable to air,
Shimadzu 8300 series Fourier Transformer instrument as KBr pel-
whereas the pyrrolidino derivatives are the least stable and
–
1
lets. The absorption bands ( ν˜ max) are given in wavenumbers (cm ).
NMR spectra were recorded with a Bruker Avance 300 MHz spec-
trometer at room temperature. Tetramethylsilane (TMS) was used
as reference for all NMR spectra, with chemical shifts reported in
ppm relative to TMS. Elemental analyses were carried out at the
the remaining derivatives are of intermediate stability. These
results should be taken into account mainly when coupling
reagents are deposited in open vessels, such as in some
automatic synthesizers. HOAt derivatives are confirmed to
be superior to HOBt derivatives in terms of both coupling Microanalytical Laboratories of the Beirut Arab University, Leb-
yield and retention of configuration for all cases. Although anon. All solvents used for recrystallization, extraction, column
all aza derivatives gave good results, HATU (4b), HAPyU chromatography, and TLC were of commercial grade, distilled be-
fore use, and stored under dry conditions.
(
6), HAM PyU (16a), and HAE PyU (16e) gave the best
2 2
results. For peptides containing the rather hindered Aib res-
idue, the fluoroformamidinium salts proved to be more con-
venient than the HOAt-based reagents.
(18),[12] Z-Phe-Val-Pro-NH
The model peptides Z-Phg-Pro-NH
2 2
[8,12]
[12]
[12]
(
19),
Z-Gly-Phe-Val-OMe (20), Z-Phe-Val-Ala-OMe (21),
[8,12]
Z-Gly-Phe-Pro-NH
2
(22),
and Z-Gly-Gly-Val-Ala-Gly-Gly-
NH
methods.
2
(23)[
8,12]
were analyzed according to previously described
Experimental Section
General Procedure for the Preparation of Urea Derivatives: N,N-
Dialkylcarbamoyl chloride (0.6 mol) was added dropwise to a stir-
ring mixture of the secondary amine (0.5 mol) and triethylamine
General: TLC was performed on silica plates (8×4 cm) from Albet
using suitable solvent systems and visualization with a Spectroline
1
Table 16. Yield, b.p., H NMR spectroscopic, and elemental analytical data of urea derivatives.
Compd. Yield B.p.
1H NMR (CDCl
δ [ppm]
3
)
Elemental analysis: calcd. (found)
[%]
[°C]
C
H
N
1
4a
4b
4c
4d
4e
79
78
81
81
85
85
93
90
97
75
1.7–2.1 (m, 4 H), 2.83 (s, 6 H), 3.35 (m, 4 H)
1.6–2.1 (m, 6 H), 2.83 (s, 6 H), 3.35 (m, 4 H)
1.2 (t, 6 H), 1.8–2.1 (m, 4 H), 3.1–3.7 (m, 8 H)
1.2 (t, 6 H), 1.8–2.1 (m, 6 H), 3.1–3.7 (m, 8 H)
1.1 (t, 12 H), 3.18 (q, 8 H)
59.12 (59.21)
61.50 (61.32)
63.49 (63.42)
65.18 (65.43)
62.75 (62.85)
9.92 (9.76)
19.7 (19.51)
1
10.32 (10.33) 17.93 (18.11)
10.66 (10.43) 16.45 (16.51)
10.49 (11.05) 15.20 (15.41)
11.70 (11.69) 16.26 (16.35)
1
1
1
1
Table 17. Yield, m.p., H NMR spectroscopic, and elemental analytical data of chloroformamidinium salts.
Compd. Yield M.p. Elemental analysis: calcd. (found)
1H NMR (CDCl
3
)
[%]
[°C]
δ [ppm]
C
H
N
1
5a
5b
5c
5d
5e
89
88
90
90
88
93–95
99–101
2.0–2.13 (m, 4 H), 3.47 (s, 6 H), 3.9–4.0 (m, 4 H)
1.8–2.1 (m, 6 H), 3.27 (s, 6 H), 3.9–4.0 (m, 4 H)
27.41(27.37)
29.95 (29.73)
4.60 (4.52)
5.03(4.92)
5.42(5.29)
5.74(5.79)
5.99(5.90)
9.14 (9.01)
8.74(8.81)
8.37 (8.31)
8.03 (8.11)
8.32 (8.41)
1
1
141–1431 1.39 (t, 6 H), 2.0–2.5 (m, 4 H), 3.9 (q, 4 H), 4.14–4.29 (m, 4 H) 32.29 (32.14)
163–165
104–106
1
1.39 (t, 6 H), 1.8–2.3 (m, 6 H), 3.9 (q, 4 H), 4.14–4.29 (m, 4 H) 34.43(34.24)
1.38 (t, 12 H), 3.72 (q, 8 H) 32.11 (32.21)
1
1570
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Eur. J. Org. Chem. 2006, 1563–1573

Immonium-Type Coupling Reagents
FULL PAPER
1
Table 18. Yield, m.p., H NMR spectroscopic, and elemental analytical data of uronium salts derived from HOAt.
Compd. Yield M.p.
1H NMR (CDCl
δ [ppm]
3
)
Elemental analysis: calcd. (found)
[%]
[°C]
C
H
N
1
6a
6c
6e
6g
6i
86
83
86
88
89
181
1.9–2.18 (m, 4 H), 3.19 (s, 6 H), 3.8–3.95 (m, 4 H), 7.91 (dd, 1 H), 35.48 (35.38)
8.14 (dd, 1 H), 8.8 (dd, 1 H)
1.9–2.18 (m, 6 H), 3.19 (s, 6 H), 3.8–3.95 (m, 4 H), 7.91 (dd, 1 H), 37.15 (37.38)
8.14 (dd, 1 H), 8.8 (dd, 1 H)
4.22 (4.13)
20.69 (20.49)
(
dec)
188
dec)
176
dec)
1
4.56 (4.63)
4.87 (4.80)
5.17 (5.21)
5.31 (5.23)
20.00 (20.29)
19.35 (19.19)
18.75 (18.89)
19.26 (19.12)
(
1
1.3 (t, 6 H), 1.9–2.2 (m, 4 H), 3.4–3.89 (m, 8 H), 7.95 (dd, 1 H),
8.1 (dd, 1 H), 8.8 (dd, 1 H)
38.72 (38.60)
40.18 (40.31)
38.54 (38.46)
(
1
182–183 1.3 (t, 6 H), 1.9–2.2 (m, 6 H), 3.4–3.89 (m, 8 H), 7.95 (dd, 1 H),
.1 (dd, 1 H), 8.8 (dd, 1 H)
168–170 1.12 (t, 6 H), 1.46 (t, 6 H), 3.39 (q, 4 H), 3.7 (q, 4 H), 7.92 (dd,
H), 8.0 (dd, 1 H), 8.85 (dd, 1 H)
8
1
1
1
Table 19. Yield, m.p., H NMR spectroscopic, and elemental analytical data of uronium salts derived from HOBt.
Compd. Yield M.p. Elemental analysis: calcd. (found)
1H NMR (CDCl
3
)
[%] [°C] δ [ppm]
C
H
N
1
6b
6d
6f
6h
6j
83
84
85
82
87
153–155 1.9–2.2 (m, 4 H), 3.2 (s, 6 H), 3.8–4.0 (m, 4 H), 7.6 (d, 1 H), 7.92 38.53 (38.4)
d, 1 H), 7.96 (d, 1 H), 8.03 (d, 1 H)
167–168 1.7–2.2 (m, 6 H), 3.2 (s, 6 H), 3.8–4.0 (m, 4 H), 7.6 (d, 1 H), 7.92 40.10 (39.88)
d, 1 H), 7.96 (d, 1 H), 8.03 (d, 1 H)
118–120 1.3 (t, 6 H), 1.9–2.2 (m, 4 H), 3.6–4.1 (m, 8 H), 7.6 (d, 1 H), 7.92 41.58 (41.42)
d, 1 H), 7.96 (d, 1 H), 8.03 (d, 1 H)
132–135 1.3 (t, 6 H), 1.6–2.1 (m, 6 H), 3.4–3.9 (m, 8 H), 7.6 (d, 1 H), 7.92 42.95 (42.86)
d, 1 H), 7.96 (d, 1 H), 8.03 (d, 1 H)
154–156 1.18 (t, 12 H), 3.42 (q, 8 H), 7.6 (d, 1 H), 7.92 (d, 1 H), 7.96 (d,
H), 8.03 (d, 1 H)
4.48 (4.39)
17.28 (17.18)
(
1
4.81 (4.58)
5.18 (4.93)
4.92 (5.01)
5.56 (5.50)
16.70 (16.82)
16.16 (16.01)
15.66 (15.73)
16.09 (15.98)
(
1
(
1
(
1
41.38 (41.45)
1
1
Table 20. Yield, m.p., H NMR spectroscopic, and elemental analytical data of fluoroformamidinium salts.
Compd. Yield
%]
M.p.
[°C]
1H NMR (CDCl
δ [ppm]
3
)
Elemental analysis: calcd. (found)
[
C
H
N
1
7a
7b
7c
7d
92
91
93
92
110
153
115
108
3.35 (d)
1
1.98–2.03 (m, 8 H), 3.75–3.84 (m, 8 H)
1.97–2.04 (m, 4 H), 3.36(d, 6 H), 3.77–3.82 (m, 4 H)
1.41 (t, 6 H), 1.98–2.0 (m, 4 H), 3.78 (dq, 4 H), 3.94–4.05 (m,
1
1
28.98 (28.83)
33.97(33.85)
4.86 (4.79)
5.70 (5.61)
9.65 (9.70)
8.80 (8.78)
2
CH )
1
7e
83
89
118
168
1.29 (t, 12 H), 3.58 (dq, 8 H)
2.9 (s, 6 H), 3.88 (d, 4 H)
33.76 (33.81)
6.30 (6.24)
8.75 (8.82)
1
7f
(
0.5 mol) in dry DCM (400 mL) at 0 °C. When the addition was ether. The residue obtained was dissolved in DCM, and a saturated
complete, the mixture was stirred overnight at room temperature. aqueous potassium hexafluorophosphate (KPF ) solution was
The reaction mixture was then washed with H O (100 mL) in order
to dissolve the inorganic salt formed (Et N·HCl), and the mixture
was subsequently washed with 10% aqueous HCl, saturated aque-
ous Na CO , H O, and saturated aqueous NaCl solution (100 mL
each). Finally, the organic solvent was dried with anhydrous
Na SO , filtered, and the solvent removed under reduced pressure.
6
2
added at room temperature with vigorous stirring for 10–15 min.
The organic layer was collected, washed once with water (100 mL),
dried with anhydrous MgSO , filtered, and the solvent was re-
4
moved under reduced pressure. The crude product was recrys-
tallized from DCM/diethyl ether.
3
2
3
2
2
4
Synthesis of Fluoroformamidinium Salts:[20] Pre-dried KF (0.3 mol)
was added to a stirring solution of chloroformamidinium salt
The oily residue obtained was purified by vacuum distillation. Ana-
lytical data for the urea derivatives are given in Table 16, those of
the chloroformamidinium salts in Table 17, those of uronium salts
derived from HOAt in Table 18, those of uronium salts derived
from HOBt in Table 19, and those of fluoroformamidinium salts
in Table 20.
(
0.1 mol) in dry CH
heated at 60 °C for 3 h, then cooled down to room temp., filtered,
and washed with CH CN (2×50 mL). The CH CN solutions were
3
CN (200 mL). The reaction mixture was
3
3
combined and concentrated under vacuum, and the residue was
dissolved in hot DCM then filtered while hot. The filtrate was con-
centrated to approximately one-third of the original volume and
diethyl ether was added with vigorous stirring to promote the for-
mation of a white solid. No further crystallization was needed. All
of the fluoroformamidinium salts were stored in capped plastic
vials at room temperature for about six months then tested. Neither
General Procedure for the Preparation of Chloroformamidinium
[
20]
Salts: Oxalyl chloride (100 mmol) was added dropwise to a solu-
tion of the urea derivative (100 mmol) in dry DCM (300 mL) at
room temperature over a period of 5 min. The reaction mixture
was stirred under reflux for 3 h, and the solvent was removed under
reduced pressure. The residue was washed with anhydrous diethyl
1
hydrolysis nor urea formation was observed by H NMR spec-
ether (2×100 mL), then bubbled with N
2
to remove the diethyl
troscopy.
Eur. J. Org. Chem. 2006, 1563–1573
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1571

A. El-Faham, S. N. Khattab, M. Abdul-Ghani, F. Albericio
FULL PAPER
Synthesis of Uronium/Aminium Salts: The chloroformamidinium
salt (5 mmol) was added to a stirring solution of HOXt (5 mmol)
and triethylamine (5 mmol) in dry DCM (50 mL). The reaction
mixture was stirred at room temperature overnight, filtered, washed
with DCM (10 mL), and the residue was recrystallized from
[
1] Abbreviations not defined in text: Aib = α-aminoisobutyric acid;
ACP = acyl carrier protein decapeptide (65–74); DCC = dicyclo-
hexylcarbodiimide; DCM = dichloromethane; DIC = diisopro-
pylcarbodiimide; DIEA = diisopropylethylamine; DMF = di-
methyl formamide; HOBt = 1-hydroxybenzotriazole; HOAt = 7-
3
CH CN/diethyl ether.
aza-1-hydroxybenzotriazole; HOPfp
=
pentafluorophenol;
HAPyU = 1-(1-pyrrolidinyl-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-
methylene)-N-methylmethanaminium) hexafluorophosphate N-
oxide; HATU = N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyri-
din-1-yl-methylene]-N-methylmethanaminium hexafluorophos-
phate N-oxide; HBTU = N-[(1H-benzotriazol-1-yl)-(dimeth-
ylamino)methylene] N-methylmethanaminium hexafluorophos-
phate N-oxide; HBPyU = N-[(1H-benzotriazol-1-yl)-bis(pyrrolidi-
nyl)-methylene]-N-methylmethanaminium hexafluorophosphate
N-oxide; HDTU = O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-
Model Segment Coupling Reaction: Test couplings were carried out
as described previously for Z-Phg-Pro-NH
2
(18),[ Z-Phe-Val-Pro-
12]
[
8,12]
[12]
NH
(
2
(19),
21),[12] Z-Gly-Phe-Pro-NH
(22),
2
Z-Gly-Phe-Val-OMe (20),
Z-Phe-Val-Ala-OMe
[8,12]
and Z-Gly-Gly-Val-Ala-Gly-
[
8,12]
2
Gly-NH (23).
ACP(65–74)
(H-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-OH)
The peptide was manually assembled on an Fmoc-
[
19]
Synthesis:
Gly-Wang-PEG-PS-resin (0.17 mmolg ) with Fmoc-amino acids
–1
1,2,3,3-tetramethyluronium hexafluorophosphate; HAM PyU =
2
O-(1H-1,2,3-triazolo[4,5-b]pyridin-1-yl)-1,1-dimethyl-3,3-tetra-
(
(
1.5-fold excess), coupling reagent (1.5-fold excess), and DIEA
threefold excess). The preactivation times used are given in
methyleneuronium hexafluorophosphate; HBM
benzotriazol-1-yl)-1,1-dimethyl-3,3-tetramethyleneuronium hexa-
fluorophosphate; HAE PyU = O-(1H-1,2,3-triazolo[4,5-b]pyridin-
-yl)-1,1-diethyl-3,3-tetramethyleneuronium hexafluorophos-
phate; HBE PyU =O-(1H-benzotriazol-1-yl)-1,1-diethyl-3,3-tetra-
2
PyU = O-(1H-
Table 18, the coupling time was 1.5 min for each amino acid, and
the deprotection (piperidine/DMF, 2:8) time was 7 min. The pep-
2
1
tide was cleaved from the resin with TFA/H
temperature. The crude peptide was run on HPLC using the follow-
ing conditions: linear gradient of CH CN and H O containing
.1% TFA each, from 5% to 35% in 25 min, C-18 Nova Pak col-
umn (4 µm, 60 Å, 3.9×150 mm), detection at 220 nm.
2
O (9:1) for 2 h at room
2
methyleneuronium hexafluorophosphate; HATeU = O-(1H-1,2,3-
triazolo[4,5-b]pyridin-1-yl)-1,1,3,3-tetraethyluronium hexafluoro-
phosphate; HBTeU = O-(1H-benzotriazol-1-yl)-1,1,3,3-tetraeth-
yluronium hexafluorophosphate; TFFH = tetramethylfluorofor-
mamidinium hexafluorophosphate; DmFFH = 1,2-dimethyl-3,3-
3
2
0
Solid-Phase Synthesis of H-Tyr-Aib-Aib-Phe-Leu-NH
2
Using Uron-
tetramethylenefluoroformamidinium
hexafluorophosphate;
[
19]
ium Salts:
The pentapeptide was manually assembled on an
DeFFH 1,2-diethyl-3,3-tetramethylenefluoroformamidinium
=
–1
hexafluorophosphate; TeFFH = tetraethylfluoroformamidinium
hexafluorophosphate, TFA = trifluoroacetic acid; TMP = 2,4,6-tri-
methylpyridine (collidine); 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].
Fmoc-PAL-PEG-PS-resin (0.18 mmolg ) with the preactivation
times in Table 19, using Fmoc-amino acid (4 equiv.), coupling rea-
gent (4 equiv.), and DIEA (8 equiv.) in DMF at a total concentra-
tion of 0.3 . A coupling time of 30 min was used for all amino
acids except Aib-Aib, for which the time was 60 min. The peptide
2
was cleaved from the resin with TFA/H O (9:1) at room tempera-
[
2] a) F. Albericio, L. A. Carpino, Methods Enzymol., Solid-Phase Pep-
ture for 2 h. The solution was then filtered and the TFA removed
under vacuum. The crude peptide was precipitated by addition of
cold diethyl ether, and run on HPLC [C-18 Nova Pak column
tide Synthesis (Ed.: G. B. Fields), Academic Press, Orlando, FL,
1997; vol. 289, pp. 104–126; b) F. Albericio, S. A. Kates, in Solid-
Phase Synthesis A Practical Guide (Eds.: S. A. Kates, F. Albericio),
Marcel Dekker, New York, 2000, pp. 275–330; c) F. Albericio, R.
Chinchilla, D. J. Dodsworth, C. Najera, Org. Prep. Proced. Int.
3 2
(4 µm, 60 Å, 3.9×15 mm), linear gradient of CH CN and H O
containing 0.1% TFA each, from 10% to 90% in 25 min, detection
at 220 nm.] The retention times for the pentapeptide and for the
des-Aib tetrapeptide were 12.03 and 12.30 min, respectively.
2001, 33, 203; d) S. Y. Han, Y. A. Kim, Tetrahedron 2004, 60, 2447;
e) N. L. Benoiton, Chemistry of Peptide Synthesis, CRC, Boca Ra-
ton (FL), 2006.
[
[
[
[
3] J. M. Humphrey, A. R. Chaberlin, Chem. Rev. 1997, 97, 2243.
4] W. König, R. Geiger, Chem. Ber. 1970, 103, 788.
5] L. A. Carpino, J. Am. Chem. Soc. 1993, 115, 4397.
Solid-Phase Synthesis of H-Tyr-Aib-Aib-Phe-Leu-NH
2
Using Fluo-
roforamidinium Salts: The pentapeptide was manually assembled
–1
on an Fmoc-PAL-PEG-PS-resin (0.18 mmolg ) with 7 min preac-
tivation time using Fmoc-amino acid (5 equiv.), coupling reagent
6] K. Knorr, A. Trzeciak, W. Bannwarth, D. Gillesseu, Tetrahedron
Lett. 1989, 30, 1927.
(5 equiv.), and DIEA (10 equiv.) in DMF at a total concentration
[7] a) V. Dourtoglou, B. Gross, V. Lambropoul, C. Ziodrou, Synthesis
1984, 572; b) I. Abedmoty, F. Albericio, L. A. Carpino, B. M. Fox-
man, S. A. Kates, Lett. Pept. Sci. 1994, 1, 57.
of 0.3 . The coupling time used was 30 min for all amino acids,
except for Aib-Aib, which required 60 min. The pentapeptide was
cleaved from the resin with TFA/H O (9:1) at room temperature
2
for 2 h. The solution was then filtered and the TFA removed under
vacuum. The addition of cold diethyl ether precipitated the penta-
peptide. The crude peptide was run on HPLC using a C-18 Nova
[
8] L. A. Carpino, A. El-Faham, F. Albericio, J. Org. Chem. 1995, 60,
561.
9] S. S. M. Hassan, M. M. Ali, A. M. Attwiya, Talanta 2001, 54,1153.
10] H. J. Gruber, G. Kada, B. Pragl, C. Riener, C. D. Hahn, G. S.
3
[
[
Harms, W. Ahrer, T. G. Dax, K. Hohenthanner, H.-G. Knaus, Bi-
oconjugate Chem. 2000, 11, 161.
Pak column (4 µm, 60 Å, 3.9×15 mm), linear gradient of CH
3
CN
and H O containing 0.1% TFA each, from 10% to 90% in 25 min,
2
[
11] a) S. Q. Chen, J. Xu, Tetrahedron Lett. 1992, 33, 647; b) J. Coste, E.
detection at 220 nm. The retention times for the pentapeptide and
for the des-Aib tetrapeptide were 12.03 and 12.30 min, respectively.
Frerot, P. Jouin, Tetrahedron Lett. 1991, 32, 1967.
[12] L. A. Carpino, A. El-Faham, J. Org. Chem. 1994, 59, 695.
[
13] Y. Kiso, Y. Fujiwara, T. Kimura, A. Nishitani, K. Akaji, Int. Pept.
Protein Res. 1992, 40, 308.
14] a) L. Jiang, D. Davison, G. Tennant, R. Ramage, Tetrahedron 1998,
4, 14233; b) N. Robertson, L. Jiang, R. Ramage, Tetrahedron 1999,
5, 2713.
15] a) P. Li, C. Xu, Tetrahedron 2000, 56, 4437; b) P. Li, J. C. Xu, Tetra-
hedron Lett. 2000, 41, 721; c) P. Li, C. Xu, J. Pept. Res. 2000, 55,
110.
[16] L. A. Carpino, J. Xia, A. El-Faham, J. Org. Chem. 2004, 69, 54.
[
Acknowledgments
5
5
Prof. L. A. Carpino, University of Massachusetts, Amherst, USA,
is thanked for his support and advice. The authors are indebted to
the National Council for Scientific Research (CNRS-48-08-03) for
partial support of the work in Lebanon.
[
1572
www.eurjoc.org
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 1563–1573

Immonium-Type Coupling Reagents
FULL PAPER
[
17] F. Albericio, J. M. Bofill, A. El-Faham, S. A. Kates, J. Org. Chem.
998, 63, 9678.
18] L. A. Carpino, A. El-Faham, Tetrahedron 1999, 55, 6813.
19] a) L. A. Carpino, A. El-Faham, C. A. Minor, F. Albericio, J. Chem.
Soc., Chem. Commun. 1994, 201; b) F. Albericio, M. Cases, J. Al-
sina, S. A. Triolo, L. A. Carpino, S. A. Kates, Tetrahedron Lett.
1997, 38, 4853.
[20] A. El-Faham, Org. Prep. Proced. Int. 1998, 30, 477.
Received: August 18, 2005
1
[
[
Published Online: January 16, 2006
Eur. J. Org. Chem. 2006, 1563–1573
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1573