Four-dimensional orthogonal solid-phase synthesis of new scaffolds based on cyclic tetra-β-peptides

Article abstract of DOI:10.1016/S0040-4039(02)00203-4

A four-dimensional orthogonal protecting scheme that involves the acid-labile BAL linker in conjunction with Fmoc, Alloc and pNb protecting groups, which can be removed by β-elimination, allyl transfer, and reductive hydrolysis, respectively, allows the solid-phase preparation of scaffolds based on a cyclic tetra-β-peptide with free amino side chains ready for further elaboration.

Full text of DOI:10.1016/S0040-4039(02)00203-4

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 2029–2032
Four-dimensional orthogonal solid-phase synthesis of new
scaffolds based on cyclic tetra-b-peptides†
Miriam Royo,* Josep Farrera-Sinfreu, Laia Sole´ and Fernando Albericio*
Departament of Organic Chemistry, University of Barcelona, E-08028 Barcelona, Spain
Received 13 January 2002; revised 23 January 2002; accepted 25 January 2002
Abstract—A four-dimensional orthogonal protecting scheme that involves the acid-labile BAL linker in conjunction with Fmoc,
Alloc and pNb protecting groups, which can be removed by b-elimination, allyl transfer, and reductive hydrolysis, respectively,
allows the solid-phase preparation of scaffolds based on a cyclic tetra-b-peptide with free amino side chains ready for further
elaboration. © 2002 Published by Elsevier Science Ltd.
In medicinal chemistry, small cyclic peptides are excel-
lent scaffolds for the incorporation of functional groups
that can interact with the corresponding receptor.1
Although the vast majority of cyclic peptides described
in the literature are based on a-amino acids, there is
increasing interest in the design and synthesis of cyclic
peptides derived from b-amino acids. For example,
Seebach2 and Gellman3 have independently demon-
strated that peptides based on b-amino acids have a
well-defined secondary structure as well as excellent
stability against degradation by proteases. Further-
more, Seebach has also shown that cyclic b-peptides are
arranged in the solid state as tubular structures with a
tight net of pleated-sheet-type hydrogen bonds.4 That
supposes that those type of cyclic peptides present
secondary structure. Given the interest in these systems,
it would be advantageous to have at our disposal a
versatile method for the preparation of cyclic b-peptides
with protected functionalized side-chains that, after
deprotection, could be further manipulated for the
preparation of libraries based on the cyclic b-peptide
scaffold. In order to gain maximum advantage from the
synthetic effort, the ideal method would involve
anchoring the cyclic peptide on a solid support.5,6 We
describe here the preparation of a solid-phase anchored
cyclic b-peptide with protected side-chains and also
report some examples of the modified scaffold.
The only valid strategy for the solid-phase preparation
of cyclic peptides with protected side-chains is to
anchor the peptide through the backbone amide nitro-
gen. In order to obtain a completely flexible system it is
necessary to have at least a four-dimensional orthogo-
nal protection scheme (Fig. 1).7,8 More specifically, the
scheme requires the following: a permanent protecting
group to anchor the first amino acid to the support;
two semi-permanent protecting groups, one for the
protection of the C-terminal carboxylic acid group and
the other for the side-chains; and finally a temporary
protecting group for the protection of the b-amino
function.
Abbreviations: Alloc, allyloxycarbonyl; DIEA, N,N-diisopropylethyl-
amine; DIPCDI, N,N%-diisopropylcarbodiimide; DMF, N,N-
dimethylformamide; EtOAc, ethyl acetate; HOAc, acetic acid; HOBt,
hydroxybenzotriazole; IRAA, internal reference amino acid; PS,
polystyrene; PyAOP, 7-azabenzotriazol-1-yl-oxytris(pyrrolidino)-
phosphonium hexafluorophosphate; SPOS, solid-phase organic syn-
thesis; SPPS, solid-phase peptide synthesis; TBTU, N-[(1H-benzotria-
zol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium tetra-
fluoroborate N-oxide; TFA, trifluoroacetic acid. Amino acid
symbols denote L-configuration unless otherwise noted.
Keywords: b-amino acid; combinatorial chemistry; high-throughput
synthesis; libraries; protecting groups.
* Corresponding authors. Tel.: 34 93 402 90 58; fax: 34 93 339 78 78;
e-mail: royo@qo.ub.es; albericio@qo.ub.es
This paper is dedicated to Professor Joan A. Subirana on the
†
Figure 1. General protecting strategy for the solid-phase
preparation of scaffolds based on cyclic tetra-b-peptides.
occasion of his 65th birthday.
0040-4039/02/$ - see front matter © 2002 Published by Elsevier Science Ltd.
PII: S0040-4039(02)00203-4

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M. Royo et al. / Tetrahedron Letters 43 (2002) 2029–2032
was followed by incorporation of Fmoc-b-alanine and
Na-Alloc-Nb-Fmoc- -Dapa. After incorporation of the
For the anchoring of the backbone, a backbone amide
linker (BAL)9 resin (X1) was chosen, which at the end
of the process will liberate the peptide by treatment
with acid. The protection of the b-amino function was
performed with the fluorenylmethoxycarbonyl (Fmoc)10
group (X3), which is removed by a b-elimination reac-
tion. The side-chains of the amino acids were protected
with allyl-based groups11 (X4), which can be removed
by an allyl transfer reaction in the presence of Pd(0).
Finally, the p-nitrobenzyl group (pNb)12,13 (X2), which
is removed by reductive hydrolysis, was used for the
protection of the carboxylic acid group.
L
last residue, the removal of the pNb group was accom-
plished by treatment with SnCl2/HOAc/phenol/
DMF.12,13 Subsequent removal of the Fmoc group was
followed by cyclization with PyAOP/DIEA16 to give the
cyclic b-tetrapeptide in which the two a-amino func-
tions of the two Dapa residues were protected with the
Alloc group. Removal of the Alloc group with
17
Pd(PPh3)4 in the presence PhSiH3 left the two amino
functions free for further manipulation.18
After removal of the Alloc group, different aliquots of
the resin were derivatized separately. For example,
phenylacetic acid (PhAcOH) was incorporated using
DIPCDI/HOBt and the Arg-Gly-Asp (RGD)19 receptor
recognition motif was constructed using a stepwise
Fmoc/tBu strategy.
As a key trifunctional b-amino acid, Na-Alloc-Nb-
Fmoc-L-diaminopropionic acid (Dapa) was chosen and
this was elongated through the b-amino function.14 The
first cyclic peptide to be synthesized was c( -Dapa-
bAla- -Dapa-bAla), which has as side-chains the two
a-amino functions of the two Dapa residues.
L
L
The same scheme was used to prepare BAL-c[
Dapa(Boc)-bAla- -Dapa(Boc)-bAla], which after treat-
ment with TFA gave c( -Dapa-bAla- -Dapa-bAla)
L-
Our strategy started with the addition of 5-(4-formyl-
3,5-dimethoxyphenoxy)valeric acid (PALdehyde) to an
amino-functionalized PS solid support through TBTU/
DIEA coupling. This step was followed by on-resin
reductive amination using the appropriate amino acid
(usually b-alanine), pNb ester hydrochloride salt and
NaBH3CN in DMF (Fig. 2). In the case where the
b-amino group is attached to a BAL resin, the acylation
of the secondary amine was found to be faster than the
comparable reaction with a-amino acids anchored to
the same BAL resin.9 Quantitative yields were obtained
L
L
L
with the side-chain amino function of the Dapa
residues free for subsequent functionalization in solu-
tion. Finally, the simplest cyclic tetra-b-peptide,
c(bAla)4, was also prepared in good yield and with
excellent purity.20
In order to confirm any advantages inherent in our
cyclization on-resin method, the linear peptide was
prepared on a chloroꢀtrityl-(ClꢀTrt-)21 resin by follow-
ing the scheme shown in Fig. 3. Assembly of the
peptide chain was followed by removal of the Alloc
groups and PhAcOH was then incorporated as
in the acylation of Na-Alloc-Nb-Fmoc-L-Dapa by
using PyAOP/DIEA15 in DMF. Deprotection of the
b-amino function of the Dapa residue with piperidine
Figure 2. General strategy for the solid-phase preparation of scaffolds based on cyclic tetra-b-peptides containing Dapa residues.

M. Royo et al. / Tetrahedron Letters 43 (2002) 2029–2032
2031
Figure 3. General strategy for the preparation of scaffolds based on cyclic tetra-b-peptides using a ClꢀTrt-resin.
described above. Cleavage of the peptide with TFA and
subsequent cyclization in solution gave c[ -Dapa-
(PhAc)-bAla- -Dapa(PhAc)-bAla]. Comparison of the
The Fmoc group was removed using piperidine/DMF
L
(1:4) (2×15 min). Incorporation of the rest of the pro-
tected amino acids and acids (10 equiv.) was performed
with DIPCDI (10 equiv.) and HOBt (10 equiv.) in
DMF for 2 h.
L
two methods shows that cyclization on solid-phase
using a BAL resin, where the whole process was per-
formed on the solid-phase, gave better results than
cyclization in solution after synthesis of the linear pep-
tide on a ClꢀTrt-resin. Thus, while the linear peptide
was never detected by HPLC in the crude cyclic pep-
tides obtained in solid-phase after 2 h of reaction, in
solution large amounts of the linear peptide were
detected after 16 h of reaction (the ratio cyclic versus
linear was 1:4).22
Removal of the pNb group was carried out with 8 M
SnCl2 in DMF containing 1.6 mM HOAc and 0.2%
phenol (2×30 min, 60°C).
Solid-phase cyclization was carried out with PyAOP (5
equiv.) and DIEA (10 equiv.) in DMF for 2 h at 25°C.
Removal of the Alloc group was achieved with
Pd(PPh3)4 (0.1 equiv.) in the presence of PhSiH3 (24
equiv.) in CH2Cl2 under Ar (2×20 min, 25°C).
In conclusion, a solid-phase strategy has been devel-
oped that uses a tetra-orthogonal protecting scheme
and allows the preparation of cyclic tetra-b-peptides
with free amino side chains ready for further elabora-
tion. Cyclic tetra-b-peptide libraries prepared in this
way will be suitable for both on- and off-resin
screening.
Cleavage of the cyclic peptide from the resin was car-
ried out with TFA/CH2Cl2 (9:1) for 2 h at 25°C.
SPPS on ClTrt-resin. Fmoc-bAla-OH (0.5 equiv.)24 and
DIEA (2 equiv.) in CH2Cl2 were added to a ClꢀTrtCl-
resin (1.35 mmol/g) for 2 h at 25°C. The reaction was
terminated by addition of MeOH and stirring for 15
min.
1. Experimental
SPPS on BAL-resin. PALdehyde (5 equiv.) and TBTU
(5 equiv.) were dissolved in DMF and then DIEA (10
equiv.) was added. After 1 min preactivation, this solu-
tion was added to the amino-functionalized PS. Cou-
pling was allowed to proceed at 25°C for 2 h, after
which time the resin was negative to the Kaiser ninhy-
drin test.
The rest of the manipulations were carried out in a
similar way to those described above.
Acknowledgements
M.R. is a ‘Ramon y Cajal’ fellow (MCyT, Spain). The
excellent technical assistant of Mr. Miklos Winkler
(Eo¨tvo¨s University, Budapest, Hungary) is especially
acknowledged. This work was partially supported by
CICYT (BQU2000-0235), Generalitat de Catalunya
[Grup Consolidat, and Centre de Refere`ncia en
Biotecnologia].
Mixtures of b-alanine pNb ester hydrochloride23 (10
equiv.) and NaBH3CN (10 equiv.) in DMF were added
to the PALdehydeꢀIRAA-resins (1 equiv.). The reac-
tions were allowed to proceed for 1 h at 25°C. The
resins were then washed with CH2Cl2 and MeOH and
finally dried.
Na-Alloc-Nb-Fmoc-
L
-Dapa (5 equiv.), PyAOP (5
References
equiv.), and DIEA (10 equiv.) in DMF were added to
amino acyl ester BAL-resins and the mixtures were
allowed to react for 2 h at 25°C. After washing with
DMF and CH2Cl2, the couplings were repeated for a
further 2 h with fresh reagents.
1. Royo, M.; Van Den Nest, W.; del Fresno, M.; Frieden,
A.; Yahalom, D.; Rosenblatt, M.; Chorev, M.; Albericio,
F. Tetrahedron Lett. 2001, 42, 7387–7391 and references
cited therein.

2032
M. Royo et al. / Tetrahedron Letters 43 (2002) 2029–2032
2. Seebach, D.; Matthews, J. L. Chem. Commun. 1997,
2015–2022.
Carpino, L. A.; Kates, S. A. Tetrahedron Lett. 1997, 38,
4853–4856.
3. (a) Appella, D. H.; Barchi, J. J., Jr.; Durell, S. R.;
Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 2309–2310;
(b) Barchi, J. J., Jr.; Huang, X.; Appella, D. H.; Chris-
tianson, L. A.; Durell, S. R.; Gellman, S. H. J. Am.
Chem. Soc. 2000, 122, 2711–2718.
4. Seebach, D.; Matthews, J. L.; Meden, A.; Wessel, T.;
Baerlocher, C.; McCusker, L. B. Helv. Chim. Acta 1997,
80, 173–182.
5. For a review on cyclic peptides, see: Rovero, P. In
Practical Solid-Phase Synthesis: A Book Companion;
Kates, S. A.; Albericio, F., Eds.; Marcel Dekker: New
York, 2000; pp. 331–364.
6. For reviews on solid-phase chemistry, see: (a) Solid-Sup-
ported Combinatorial and Parallel Synthesis of Small-
Molecular-Weight Compound Libraries; Obrecht, D.;
Villalgordo, J. M., Eds.; Pergamon, 1998; (b) Combinato-
rial Chemistry: A Practical Approach; Bannwarth, W.;
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cal Solid-Phase Synthesis: A Book Companion; Kates, S.
A.; Albericio, F., Eds.; Marcel Dekker: New York, 2000;
(d) Seneci, P. Solid-Phase Synthesis and Combinatorial
Technologies; John Wiley & Sons: New York, 2001.
7. An orthogonal system is defined as a set of completely
independent classes of protecing groups, such that each
class of group can be removed in any order and in the
presence of all other classes (Barany, G.; Albericio, F. J.
Am. Chem. Soc. 1985, 107, 4936–4942, and references
cited therein).
17. Thieriet, N.; Alsina, J.; Giralt, E.; Guibe´, F.; Albericio,
F. Tetrahedron Lett. 1997, 38, 7275–7278.
18. If the Alloc groups are removed after the incorporation
of the Dapa derivative and before the removal of the
Fmoc group, different building blocks could be incorpo-
rated at each Dapa residue. Furthermore, the same
results can be achieved if the Dapa residues are incorpo-
rated with different Na protection, for instance Alloc and
methyltrityl (Mtt), which is also compatible with this
scheme.
19. (a) Rouslahti, E.; Pierschbacher, M. D. Science 1987,
238, 491–497; (b) Brooks, P. C.; Clark, C. F.; Cheresh, D.
A. Science 1994, 264, 569–571; (c) Cox, D.; Aoki, T.;
Seri, J.; Motoyama, Y.; Yoshida, K. Med. Res. Rev.
1994, 14, 194–195.
20. Solid-phase elongation of all linear peptides took place
with essentially quantitative yields. All cyclic peptides
were characterized by HPLC and MALDI-TOF-MS. c[
Dapa(Alloc)-bAla- -Dapa(Alloc)-bAla]: HPLC (78%
purity); MS calcd for C20H30N6O8 482.21, found 483.96
[M+H]+, 506.34 [M+Na]+, 522.32 [M+K]+; c[
-Dapa-
(PhAc)-bAla- -Dapa(PhAc)-bAla]: HPLC (72% purity);
MS calcd for C28H34N6O6 550.25, found 552.24 [M+H]+,
574.26 [M+Na]+, 590.24 [M+K]+; c[
-Dapa(RGD)-bAla-
-Dapa(RGD)-bAla]: HPLC (65% purity). MS calcd for
C36H62N18O14 970.47, found 973.06 [M+H]+, 995.06 [M+
Na]+, 1011.06 [M+K]+. c[
-Dapa-bAla- -Dapa-bAla]:
L-
L
L
L
L
L
L
L
HPLC (70% purity). MS calcd for C12H22N6O4 314.17,
found 315 [M+H]+, 338 [M+Na]+, 353 [M+K]+; c(bAla)4:
HPLC (73% purity). MS calcd for C12H20N4O4 284.15,
found 284 [M+H]+, 307 [M+Na]+, 323 [M+K]+.
8. For a review of orthogonal protecting groups, see:
Albericio, F. Biopolymers (Peptide Sci.) 2000, 55, 123–
139.
21. Barlos, K.; Gatos, D.; Scha¨fer, W. Angew. Chem., Int.
Ed. Engl. 1991, 30, 590–593.
22. Even worse results were obtained when cyclization of the
9. (a) Jensen, K. J.; Alsina, J.; Songster, M. F.; Va´gner, J.;
Albericio, F.; Barany, G. J. Am. Chem. Soc. 1998, 120,
5441–5452; (b) Alsina, J.; Jensen, K. J.; Albericio, F.;
Barany, G. Chem. Eur. J. 1999, 5, 2787–2795.
10. Carpino, L. A.; Han, G. Y. J. Org. Chem. 1972, 37,
3404–3405.
11. For reviews on allyl-based protecting groups, see: Guibe´,
F. Tetrahedron 1997, 53, 13509–13556 and 1998, 54,
2967–3042.
12. Hocker, M. D.; Caldwell, C. G.; Macsata, R. W.; Lyttle,
M. H. Peptide Res. 1995, 8, 310–315.
13. Romanovskis, P.; Spatola, A. F. J. Peptide Res. 1998, 52,
356–374.
14. Subirana and co-workers were the first to show that
b-amino acid polymers derived from aspartic acid adopt
helical structures. Ferna´ndez-Sant´ın, J. M.; Aymam´ı, J.;
Rodr´ıguez-Gala´n, A.; Mun˜oz-Guerra, S.; Subirana, J. A.
Nature 1984, 311, 53–54. Dapa is the aminated analogue
of Asp.
15. Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio,
F. J. Chem. Soc., Chem. Commun. 1994, 201–203.
16. Albericio, F.; Cases, M.; Alsina, J.; Triolo, S. A.;
precursor
H-L-Dapa(Alloc)-ßAla-L-Dapa(Alloc)-ßAla-
OH was attempted.
23. b-Alanine pNb ester hydrochloride was prepared by reac-
tion of Boc-bAla-OH (1 equiv.) with p-nitrobenzyl alco-
hol (1 equiv.) in the presence of DIEA (1 equiv.) in
CH2Cl2 for 16 h at 25°C. The residue was suspended in
EtOAc, washed with 10% aqueous Na2CO3, 0.1N
aqueous HCl, brine, dried (MgSO4), and concentrated in
vacuo. The Boc-bAla-OpNb intermediate, a white solid,
was dissolved in 4N HCl/dioxane and stirred at 25°C for
1 h. The homogeneous reaction mixture was concentrated
in vacuo and washed with Et2O to provide the product as
a white solid (81% overall yield).
24. When ClꢀTrt-resin of loading superior to 1 mmol/g is
used, a limited incorporation of the first amino acid is
advisable to avoid the formation of deletion peptides
arising from interchain aggregation. Chiva, C.; Vilaseca,
M.; Giralt, E.; Albericio, F. J. Peptide Sci. 1999, 5,
131–140.