Preparation of large macrocyclic peptides using the oxime resin

Article abstract of DOI:10.1016/j.tetlet.2007.05.033

We exploited a peptide cyclization-cleavage reaction on oxime resin (PCOR) to obtain in one key step large macrocyclic peptides. Herein, we report on the different parameters affecting the cyclization-oligomerization reaction, whether to favor cyclic mono

Full text of DOI:10.1016/j.tetlet.2007.05.033

Tetrahedron Letters 48 (2007) 4929–4933
Preparation of large macrocyclic peptides using the oxime resin
Jean-Philippe Blanchette, Patrick Ferland and Normand Voyer*
Universite´ Laval, De´partement de chimie et Centre de recherche sur la fonction, la structure et l’inge´nierie des prote´ines,
Pavillon Vachon, Que´bec, Canada G1K 7P4
Received 21 October 2006; accepted 7 May 2007
Available online 16 May 2007
Abstract—We exploited a peptide cyclization–cleavage reaction on oxime resin (PCOR) to obtain in one key step large macrocyclic
peptides. Herein, we report on the different parameters affecting the cyclization–oligomerization reaction, whether to favor cyclic
monomer or cyclic oligomers formation.
Ó 2007 Elsevier Ltd. All rights reserved.
Cyclic peptides have been isolated from many natural
sources over the years and they constitute a useful class
of compounds for drug discovery.1–3 Indeed, the cycliza-
tion of the linear precursor brings conformational
constraints, which results in enhanced receptor affini-
ties.4–6 Additionally, macrocyclic peptides are useful
synthon in the preparation of unique tubular shape
nanoscale materials.7,8 However, the cyclization of
linear peptides is the key step in the synthesis and is
often quite difficult to achieve in high yield with all-L
amino acid residues.9,10
opment of artificial molecular channels,17,18 we sought
to prepare large macrocyclic peptides that could be cou-
pled onto helical peptide frameworks. Herein, we report
the synthesis of 9 novel large macrocyclic peptides
containing Pro-Gly sequences by exploiting oligo-
merization–cyclization–cleavage sequential reactions
on oxime resin (Scheme 1). Thus, we investigated that
methodology of oligomerization–cyclization with 3 lin-
ear sequences varying from 6 to 8 amino acids and con-
taining turn inducing tetrad Pro-Gly-Pro-Gly in order to
get a better understanding of the oligomerization–cycli-
zation phenomenon.
It has already been demonstrated that cyclization during
synthesis of peptides on oxime resin was an undesirable
side effect and that it is more prevalent at the coupling of
the third amino acid, as the formation of diketopipera-
zines is favored.11–14 In this case, cyclization becomes
less important with longer peptides as well as being
sequence dependent.14–16 Noteworthy of mention, it was
described that dimerization followed by the cyclization
of a pentapeptide lead to 23% of a cyclic 10-mer.15
The formation of this side product is interesting because
it suggests that it could be possible to obtain larger
macrocycles made from repeating units in one key
step.
Synthesis of the linear peptide sequences is shown in
Scheme 2. The first amino acid of the sequence, Boc-
Ala–OH was coupled overnight using diisopropylcarbo-
diimide as coupling reagent. Then the resin loading was
determined by the Kaiser’s nynhidrin test and quantita-
tive cleavage with n-propylamine. The Boc protecting
group was removed using 50% TFA/CH2Cl2, while the
second amino acid was being activated using classical
SPPS conditions with DIC and HOBt. Couplings were
allowed to proceed 2 h after addition of DIEA. Depro-
tection and coupling steps were repeated to obtain the
desired linear sequences, which were cleaved using
n-propylamine as nucleophile to determine the purity
and structure of the peptides n-propylamide using
Controlling the formation of those large cyclic com-
pounds with important surface area could lead to inter-
esting products that could find applications in material
science and supramolecular synthesis. Toward the devel-
1
HPLC, MS, and H NMR spectroscopy.
To further investigate the oligomerization–cyclization–
cleavage reaction, we verified the effect of different
parameters in order to develop an efficient method to
orient the reaction toward the formation of cyclic mono-
mers or cyclic oligomers. We investigated the effect of
*
Corresponding author. Tel.: +1 418 656 2131x3613; fax: +1 418 656
7916; e-mail: normand.voyer@chm.ulaval.ca
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.05.033

4930
J.-P. Blanchette et al. / Tetrahedron Letters 48 (2007) 4929–4933
intramolecular
intermolecular
O
O
H
H
O
N
N
N
N
N
H2N
N
H
N
H
O
O
H
O
O
O
O
O
O
O
O
O
H
H
N
N
O
O
N
HN
HN
O
O
NH
O
H
N
N
O
HN
O
O
O
N
O
NH
O
HN
O
HN
O
O
NH
O
N
N
O
N
NH
N
H
O
N
H
O
O
N
H
O
O
O
Intramolecular product
O
Cyclic dimer
+
Cyclic trimer
Cyclic tetramer
.......
Inter-Intramolecular products
Scheme 1. Synthesis of cyclic monomeric and oligomeric peptides on the oxime resin.
N
OH
BOC-Ala-OH
DIC, 24 h
BOC-Ala
O
OH
NO2
TFA/CH2Cl2 50%
+
H3N Ala
O
+
DIC, HOBt, 0 °C, 30 min
BOC-AAn-OH
BOC-AAn-OBt
DIEA, 2 h
Repeat steps n-1 times
BOC-AAn-Ala-O
-Ala-O
BOC-AAn... AA
2
Scheme 2. Synthesis of linear peptide sequences on the oxime resin.
the solvent and resin loading, acting as a pseudo-dilu-
tion factor, on the cyclization of 6, 7, and 8-amino acid
peptide sequences. All cyclizations were done with
2.0 equiv of DIEA and AcOH during 24 h after depro-
tection of the Boc protecting group. The results for the
cyclization of the hexapeptide sequence Glu(OBzl)-
(Pro-Gly)2-Ala are shown in Table 1.
The first observation that can be made from those
results is that the yields vary from 20% to 60% for
isolated cyclic peptides, which is quite high for the
cyclization of an all-L hexapeptide sequence. Also, as
expected, the solvent influences greatly the oligomeriza-
tion reaction. In all cases with the same resin loading,
the reaction produces a higher ratio of cyclic dimer

J.-P. Blanchette et al. / Tetrahedron Letters 48 (2007) 4929–4933
4931
Table 1. Cyclization of a hexapeptide linear sequence at different loadings and in different solventsa
O
O
H
H
O
N
O
N
N
N
N
1) 50% TFA/CH2Cl2
2) Cyclization
N
H
H
O
O
O
O
O
O
Solvent
Resin loading (mmol/g)
Yieldb (%)
Ratioc (%)
Dimer
Monomer
Trimer
CH2Cl2
DMF
CH2Cl2
DMF
CH2Cl2
DMF
0.3
0.3
0.8
0.8
1.2
1.2
31
29
42
36
60
23
36
65
23
37
14
30
49
25
55
41
51
46
15
10
22
22
35
24
a Cyclization were performed using 2.0 equiv of DIEA and AcOH for 24 h.
b Overall yield of isolated cyclic peptides.
c Relative ratio determined using reverse-phase HPLC.
and cyclic trimer in dichloromethane than in dimethyl-
formamide. The same conclusion can be made with
the increase of resin loading for the same solvent.
explained by both the different lengths of the chain
and the terminal amino acid, as the terminal glycine is
less hindered than glutamic acid. Results for the hepta-
peptide sequence are in agreement with previous obser-
vations. For identical resin loading, the cyclic monomer
is favored in dimethylformamide when compared to
dichloromethane. Also, lower resin loadings favor the
intramolecular cyclization, leading to higher proportion
of cyclic monomer. In the appropriate conditions, low
loading in dimethylformamide, it is possible to obtain
51% of isolated cyclic peptides containing 91% of the
cyclic monomer. Cyclization results for this methodo-
logy for the linear octapeptide sequence Ala-Gly-
Glu(OBzl)-(Pro-Gly)2-Ala are shown in Table 3.
This result can be explained by the fact that higher resin
loading leads to more intermolecular reactions. There-
fore, it is possible to exert a certain control over this type
of macrocyclization. For example, under appropriate
conditions, we can achieve the cyclization of a hexa-
peptide with a 60% isolated yield containing 51% of
the cyclic dimer (12 aa) and 35% of the cyclic trimer
(18 aa). It is interesting to point out that in this case,
instead of being a byproduct, the 12 amino acid ring
becomes the major product of the reaction. In the case
of the heptapeptide sequence Gly-Glu(OBzl)-(Pro-
Gly)2-Ala, the effect of the same parameters on cycliza-
tion were studied and the results are shown in Table 2.
Studies on the cyclization of the 8 amino acid sequence
demonstrated the same trends as with other sequences.
The use of dichloromethane with high resin loading
favor formation of large macrocycles, while a lower
resin loading in dimethylformamide enhances the
The yields of isolated cyclic compounds are a little high-
er in this case, varying from 37% to 70%. This can be
Table 2. Cyclization of an heptapeptide linear sequence at different loadings and in different solventsa
O
O
O
H
O
H
1) 50% TFA/CH2Cl2
2) Cyclization
N
N
N
N
N
N
O
N
H
H
O
O
H
O
O
O
O
Solvent
Resin loading (mmol/g)
Yieldb (%)
Ratioc (%)
Dimer
Monomer
Trimer
CH2Cl2
DMF
CH2Cl2
DMF
0.3
0.3
0.8
0.8
1.2
62
51
70
37
62
72
91
62
74
49
22
8
27
20
36
6
1
11
6
CH2Cl2
15
a Cyclization were performed using 2.0 equiv of DIEA and AcOH for 24 h.
b Overall yield of isolated cyclic peptides.
c Relative ratio determined using reverse-phase HPLC.

4932
J.-P. Blanchette et al. / Tetrahedron Letters 48 (2007) 4929–4933
Table 3. Cyclization of an octapeptide linear sequence at different loadings and in different solventsa
O
O
O
H
H
O
H
N
N
N
O
N
N
1) 50% TFA/CH2Cl2
2) Cyclization
N
N
N
H
H
H
O
O
O
O
O
O
O
Solvent
Resin loading (mmol/g)
Yieldb (%)
Ratioc (%)
Dimer
Monomer
Trimer
CH2Cl2
DMF
CH2Cl2
DMF
CH2Cl2
DMF
0.3
0.3
0.8
0.8
1.2
1.2
34
39
61
25
34
58
59
75
44
54
39
47
27
18
36
32
38
34
14
7
20
14
23
19
a Cyclization were performed using 2.0 equiv of DIEA and AcOH for 24 h.
b Overall yield of isolated cyclic peptides.
c Relative ratio determined using reverse-phase HPLC.
formation of cyclic monomer. If we compare the oppo-
site cases for this sequence, we can better highlight the
impact of those parameters on the cyclization–cleavage
reaction. With a loading of 0.3 mmol/g in dimethylform-
amide, it is possible to obtain 75% of cyclic monomer
from the isolated cyclic peptides obtained in 39% yield.
With a resin loading of 1.2 mmol/g in dichloromethane,
only 39% of the cyclic monomer was produced whereas
38% of the cyclic dimer (16 aa) and 23% of the cyclic
trimer (24 aa) were found in the crude macrocycle
mixture obtained with a 34% yield.
notable difference in the case of the hexapeptide is a
slight loss of yield of cyclization and a weak increase
in the dimer formation using Cs+. Results for the cycli-
zation of the heptapeptide and octapeptide sequences
were comparable with the ones obtained with the hexa-
peptides. These results suggest that it will be difficult to
orient the oligomerization–cyclization–cleavage reac-
tions by using different metal ions, but more investiga-
tions are needed to determine the effects of other
divalent metal ions on the outcome of the oligomeriza-
tion–cyclization–cleavage reactions.
Since it has been reported in the literature that the pres-
ence of ions can affect macrocyclization reactions, we
studied the reaction using the hexapeptide sequence in
the presence of different monovalent cations. Cycliza-
tion results are shown in Table 4.
In summary, we have demonstrated the possibility of
preparing in good yields very large peptide macrocycles
containing the turn inducing tetrad Pro-Gly-Pro-Gly
and containing up to 24 amino acids by exploiting the
nucleophilic cleavage susceptibility of the oxime resin.
By choosing the proper conditions, we have shown that
it is possible to influence in some ways the outcome of
the oligomerization–cyclization–cleavage reaction to
favor the formation of the cyclic monomers or the larger
The cyclization were performed at high loading with the
same procedure, this time adding 5.0 equiv of sodium,
potassium and cesium acetate, respectively. The only
Table 4. Cyclization of a hexapeptide sequence in the presence of different ionsa
O
O
O
H
H
N
O
N
N
N
N
1) 50% TFA/CH2Cl2
N
H
O
O
H
O
O
O
2) Cyclization
CH3COO-M+
O
Resin loading (mmol/g)
M+
Yieldb (%)
Ratioc (%)
Dimer
Monomer
Trimer
1.2
1.2
1.2
1.2
None
Na+
K+
Cs+
60
47
46
33
14
10
12
10
51
58
60
64
35
32
28
26
a Cyclization were performed using 2.0 equiv of DIEA and AcOH, with 5.0 equiv of corresponding acetate during 24 h.
b Overall yield of isolated cyclic peptides.
c Relative ratio determined using reverse-phase HPLC.

J.-P. Blanchette et al. / Tetrahedron Letters 48 (2007) 4929–4933
4933
3. Sarabia, F.; Chammaa, S.; Ruiz, A. S.; Ortiz, L. M.;
cyclic dimers and trimers. Although the amino acid
sequence surely influences the oligomerization–cycliza-
tion process investigated as previously demonstrated in
the literature, the results reported clearly illustrate the
usefulness of the methodology for the preparation of
very large macrocyclic peptides. We are currently study-
ing the generality of this useful method toward large
macrocyclic peptides using different peptidic sequences
as well as preparing large macrocyclic compounds that
could be very promising in supramolecular chemistry
and development of new nanomaterials.
Herrera, F. J. L. Curr. Med. Chem. 2004, 11, 1309–
1332.
4. Kessler, H. Angew. Chem., Int. Ed. Engl. 1982, 21, 512–
523.
5. Aumailley, M.; Gurrath, M.; Muller, G.; Calvete, J.;
¨
Timpl, R.; Kessler, H. FEBS Lett. 1991, 291, 50–54.
6. Gurrath, M.; Muller, G.; Kessler, H.; Aumailley, M.;
¨
Timpl, R. Eur. J. Biochem. 1992, 210, 911–921.
7. Ghadiri, M. R.; Granja, J. R.; Milligan, R. A.; McRee, D.
E.; Khazanovich, N. Nature. 1993, 366, 324–327.
8. Bong, D. T.; Clark, T. D.; Granja, J. R.; Ghadiri, M. R.
Angew. Chem., Int. Ed. 2001, 40, 988–1011.
9. Tang, Y. C.; Xie, H. B.; Tian, G. L.; Ye, Y. H. J. Pept.
Res. 2002, 60, 95–103.
10. Haddadi, M. E.; Cavelier, F.; Vives, E.; Azmani, A.;
Verducci, J.; Martinez, J. J. Pept. Sci. 2000, 6, 560–
570.
Acknowledgments
This work was supported by the NSERC of Canada, the
´
FQRNT, and the Universite Laval.
11. Nishino, N.; Xu, M.; Mihara, H.; Fujimoto, T. Bull.
Chem. Soc. Jpn. 1992, 65, 991–994.
¨
12. Osapay, G.; Bouvier, M.; Taylor, J. W. Techniques in
Supplementary data
Protein Chemistry II; Academic Press: New York, 1991,
pp 221–231.
13. Kaiser, E. T.; DeGrado, W. F. J. Org. Chem. 1980, 45,
1295–1300.
Contains experimental procedure, HPLC, and spectral
data for cleaved linear sequences and cyclic isolated
products. Supplementary data associated with this arti-
cle can be found, in the online version, at doi:10.1016/
j.tetlet.2007.05.033.
14. Davies, J. S. J. Pept. Sci. 2003, 9, 471–501.
15. Mihara, H.; Yamabe, S.; Niidome, T.; Aoyagi, H.;
Kumagai, H. Tetrahedron Lett. 1995, 36, 4837–4840.
16. Nishino, N.; Xu, M.; Mihara, H.; Fujimoto, T.;
Ueno, Y.; Kumagai, H. Tetrahedron Lett. 1992, 33,
1479–1482.
References and notes
17. Voyer, N.; Robitaille, M. J. Am. Chem. Soc. 1995, 117,
6599–6600.
18. Biron, E.; Otis, F.; Meillon, J.-C.; Robitaille, M.; Lamo-
the, J.; Van Hove, P.; Cormier, M.-E.; Voyer, N. Bioorg.
Med. Chem. 2004, 12, 1279–1290.
1. Wipf, P. Chem. Rev. 1995, 95, 2115–2134.
2. Lambert, J. N.; Mitchell, J. P.; Roberts, K. D. J. Chem.
Soc., Perkin Trans. 1 2001, 471–484.