Synthesis of peptide dendrimers based on a β-cyclodextrin core with guest binding ability

Article abstract of DOI:10.1016/S0040-4039(03)01432-1

The synthesis of three first-order dendrimers based on a β-cyclodextrin core containing fourteen Val, Phe and Val-Phe residues is described. The guest binding ability of the tetradecavalent peptidyl β-cyclodextrin derivative has been tested by calorimetric titration and the thermodynamic parameters for the complex formation with adamantanecarboxylic acid were obtained.

Full text of DOI:10.1016/S0040-4039(03)01432-1

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 6125–6128
Synthesis of peptide dendrimers based on a b-cyclodextrin core
with guest binding abilityꢀ
Abdullah M. A. Muhanna,a Emilia Ortiz-Salmero´n,b Luis Garc´ıa-Fuentes,b
Juan J. Gime´nez-Mart´ıneza and Antonio Vargas-Berenguela,*
a
´
Area de Qu´ımica Orga´nica, Universidad de Almer´ıa, 04120 Almer´ıa, Spain
bDepartamento de Qu´ımica-F´ısica, Bioqu´ımica y Qu´ımica Inorga´nica, Universidad de Almer´ıa, 04120 Almer´ıa, Spain
Received 17 March 2003; revised 6 June 2003; accepted 7 June 2003
Abstract—The synthesis of three first-order dendrimers based on a b-cyclodextrin core containing fourteen Val, Phe and Val-Phe
residues is described. The guest binding ability of the tetradecavalent peptidyl b-cyclodextrin derivative has been tested by
calorimetric titration and the thermodynamic parameters for the complex formation with adamantanecarboxylic acid were
obtained.
© 2003 Elsevier Ltd. All rights reserved.
Peptide dendrimers are highly branched structures usu-
ally consisting of (1) a core or template bearing several
branching points responsible for the spatial architecture
and the dendrimeric character and (2) multiple copies
of bioactive peptides attached onto the core.1 Examples
of those kind of compounds are the so called Multiple
Antigen Peptides (MAP) developed in order to amplify
synthetic peptide immunogens.1,2 Multivalent peptide
systems have been used as well in protein de novo
design based on the template-assembled synthetic
proteins (TASP) concept.1,3
We have been working on a similar concept but using
carbohydrates as biological markers. We reported the
synthesis of a variety of per-substituted b-CD deriva-
tives branched and/or hyperbranched with O-, S-gly-
cosides and glycopyranosylamines having enhanced
affinity with carbohydrate-specific receptor proteins
and with inclusion complexation abilities.7
The majority of the the reported CDs with grafted
amino acids are monosubstituted derivatives in which
the amino acid8–10 or the peptide6,8,11–14 have been
bound either directly or via a spacer. Peptidyl-cyclodex-
trins consisting in a neuropeptide derivative bound to
two CD rings have been also reported.6 The synthesis
of per-substituted CDs with phenylalanine and cysteine
has been described15 and also symmetrically-substituted
CDs with the C-terminal peptide amide of gastrin have
been also reported.14,16
Cyclodextrins4 (CD) derivatives bearing peptides may
be useful as carriers for transporting drugs to biological
targets containing specific peptide receptors.5,6 Thus,
the peptide bio-recognizability together with the CD
host–guest complexation properties would provide a
potential site-specific drug carrier system. In addition,
the well-defined torus-shaped structure of CDs4 pro-
vides a versatile scaffold for the construction of
branched structures of bioactive molecules such as pep-
tides. Therefore, CDs are suitable templates for the
application of the MAP concept as a means to increase
the peptide-receptor binding and hence improve the site
specificity of the drug delivery system.
Here we describe the synthesis of four first-order amino
acid/peptide dendrimers based on a b-CD core follow-
ing a convergent approach. The synthesis was carried
out by first constructing the amino acid or peptide
dendrons, followed by their coupling to the key tem-
plate heptakis 6-deoxi-6-iodo-b-CD derivative17 (1).
The branching unit 3,3%-iminobispropylamine was con-
verted into the bis(carbobenzyloxy) derivative 2 by
regioselective reaction with benzylic alcohol, carbonyl
diimidazole and KOH (Scheme 1). Compound 2 was
then transformed into the N-chloroacetylated derivative
3 by a three-steps synthesis as reported elsewhere.7c We
used the amino acid derivatives of H-Val-OMe (10) and
ꢀ Supplementary data associated with this article can be found at
doi:10.1016/S0040-4039(03)01432-1
* Corresponding author. Tel.: +34-950-015315; fax: +34-950-015481;
e-mail: avargas@ual.es
0040-4039/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)01432-1

6126
A. M. A. Muhanna et al. / Tetrahedron Letters 44 (2003) 6125–6128
Scheme 1. Reagents and conditions: (i) DCI, BnOH, KOH, toluene, reflux, 64.7%. (ii) Ref. 7b. (iii) (ClCH2CO)2O, NaHCO3,
CH3CN, rt, 76% (for 7), 74% (for 8), 84% (for 9). (iv) (a) thiourea, acetone, rt, (b) Na2SO3, H2O, 62% (for 10), 59% (for 11), 61%
(for 12), 61% (for 21), 55% (for 22), 53% (for 23), 58% (for 24). (v) Cs2CO3, DMF, rt, 81% (for 13), 80% (for 14), 78% (for 15),
86.8% (for 16). (vi) (a) TFA, dry CH2Cl2, rt, 2–6 h; (b) (ClCH2CO)2O, DIPEA, CH3CN, rt, 92% (for 17), 90% (for 18), 86% (for
19), 91% (for 20).
H-Phe-OMe (11) and the peptide derivative of H-Phe-
Val-OMe (12) as the N-mercaptoacetyl derivatives for
their coupling at their N-terminus with 3. Compounds
10–12 were synthesized from the methyl esters 4–6. The
amino acid methyl esters 4 and 5 were treated with
chloracetic acid and sodium bicarbonate to obtain the
N-chloroacetylated amino acids 7 and 8 in 76 and 74%
yield, respectively. The peptide methyl ester 6 was
subsequently subjected to hydrogenolysis and
chloroacetylation to isolate N-chloracetyl peptide
derivative 9 in 84% yield. Reaction of compounds 7–9
with thiourea followed by treatment with sodium sul-
phite afforded the thiol derivatives 10–12 in 58–62%
yield. The bis-branched amino acid and peptide deriva-
tives were readily obtained by the reaction of thiols
10–12 with 3 in the presence of Cs2CO3 in DMF at
room temperature. Under these conditions the bis-
branched derivatives 13–15 were obtained in 81, 80 and
79% yield, respectively. In order to prepare a multiva-
lent diepitopic2b model system we also carried out the
sequential nucleophilic displacement of the two chloride
groups of 3 by the in situ formed cesium thiolate
derivatives of 10 and 11, respectively, obtaining the
difunctional bis-branched amino acid 16 in 78% overall
yield. Acidolytic removal of the Boc-N protecting
group in 13–16 using TFA in CH2Cl2 followed by
treatment with chloracetic anhydride in MeOH
afforded 17–20 in 86–92% yield. Then, the N-chlo-
racetyl derivatives 17–20 were treated with thiourea and
sodium sulfite to give the thiols 21–24 (61, 55, 58 and
53%, respectively). The coupling of the dendrons 21–24
to the CD core was accomplished by reaction of thiols
21–24 with CD template 1 in the presence Cs2CO3 at
60°C in dry DMF.18a The products were isolated as the
corresponding peracetylated derivatives 25–28 in 62–
72% yield18b (Scheme 2). De-O-acetylation of 25–27
afforded the valine, phenylalanine and Phe-Val-con-
taining 14-mer 29–31 in 82, 78–82% yield. Similarly,
de-O-acetylation of 28 yielded the diepitopic-like tetra-
deca branched CD 32, containing Phe and Val residues,
in 77% yield.
The amino acid and peptidyl CDs 25–32 were charac-
terised by NMR spectroscopic techniques with COSY,
HMQC, and HMBC experiments and MALDI-TOF
mass spectrometry. The room-temperature 1H NMR
spectra showed a considerable broadening of the sig-
nals. This indicates a restricted mobility on the NMR
time scale due to the presence of amide bonds in the
CD derivatives. However, it is also possible that some
conformational isomerism19a or ring distortion19b may
occur as a consecuence of the persubstitution on the
CD primary face. When measurements of the NMR
data were performed at 80°C, the resolution of the

A. M. A. Muhanna et al. / Tetrahedron Letters 44 (2003) 6125–6128
6127
torus. 13C NMR spectra of 25–32 display only one
anomeric carbon signal at 96.3–102.7 ppm. In addition,
they show signals at 57.5 and 57.3 ppm for compounds
25 and 29, at 53.3 ppm for compounds 26 and 30 and
at 57.2–58.14 and 53.3–54.0 for compounds 27, 28, 31
and 32, corresponding to the Val and Phe CH a-car-
bons, respectively.
The inclusion complexation behaviour of the tetra-
decapeptidyl CD 31 with the guest molecule adaman-
tanecarboxylic acid (AC) was tested at pH 7.4 by using
isothermal titration calorimetry (ITC) (Table 1). ITC
measurements provide direct determination of n, the
stoichiometry, DH°, the binding enthalpy change, and
K, the affinity constant. From measurements of K, the
binding free energy, DG°, can be calculated and hence
the binding entropy change, DS°, determined.22 In pre-
vious studies we have not detected by ITC the forma-
tion of inclusion complexes between some naphtalene
derivative guests and CDs tetradecabranched with gly-
coside residues.7b,c Thus, the steric congestion on the
primary face of CD due to the attachment of fourteen
branches at the CD lower rim could lead to a confor-
mational distortion of the CD torus.19 The distortion,
due to the rotation of one or more glucose units around
the glycosidic linkages, would alterate the cavity shape,
thus preventing guest penetration. In order to investi-
gate whether or not the hyperbranched b-CDs could be
used as guest receptors we chose AC that, unlike the
naphtalene derivative guests used before,7c,d provides a
structure with a quasispheric symmetry. In addition,
AC forms a very strong 1:1 inclusion complex with
b-CD.20 Stepwise addition of aliquots of AC-containing
solution to a solution of 31 led to a decrease in the
extent of released heat indicating binding interaction
between AC and 31. From the performed ITC experi-
ments we found that the best fit for the three variables,
n, DH°, and K was for a 1:2 (AC/31) stoichiometry.
The complexation of AC with 31 is exothermic and
entropy-driven and affords a less stable complex with
AC than the native b-CD complex, with a binding
constant value, K, of 3.78 M−1.
Scheme 2. Reagents and conditions: (i) (a) Cs2CO3, DMF,
60°C, 7 days, (b) Ac2O, Py, DMAP, 40°C, 48 h, 72% (for 25),
69% (for 26), 65% (for 27), 62% (for 28). (ii) NaOMe, MeOH,
82% (for 29), 78% (for 30), 76% (for 31), 77% (for 32).
spectra was improved considerably, allowing the assign-
ment of the spectroscopic signals. The 1H NMR spectra
show that the ratios of the integrals for the signals of
the appended residue protons and for the signals of
those belonging to the CD core are in accordance with
the structures of the products. The 13C NMR spectra of
25–32 show carbon signals at 51.0–51.9 ppm revealing
the presence of the methoxycarbonyl groups on the CD
In summary, we report the synthesis of tetradecavalent
amino acid and peptide dendrimers based on a b-CD
core. The persubstitution of the primary face of the CD
with fourteen peptide residues modifies the CD cavity
shape but has the ability to form a complex with AC.
Table 1. Thermodynamics of binding of guest AC with peptidyl CD 31 in H2O at 25°C and pH 7.4 (10 mM sodium
phosphate)
Host
n (AC:host)
K×10−4 (M−1
)
−DG° (kcal mol−1
)
−DH° (kcal mol−1
)
TDS° (kcal mol−1
)
b-CDa
b-CDb
31
1:1
1:1
1:2
4.00
2.89 0.86
0.38 0.01
–
5.40 0.1
4.50 0.03
1.99 0.03
–
6.10 0.02
4.89 0.02
1.59 0.03
2.90 0.03
a Ref. 20.
b Ref. 21.

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A. M. A. Muhanna et al. / Tetrahedron Letters 44 (2003) 6125–6128
Stoddart, J. F.; Alker, D.; Harding, V. D. J. Org. Chem.
Acknowledgements
1996, 61, 903–908.
18. (a) Procedure for the synthesis of 27: A mixture of 1 (200
mg, 0.08 mmol), 23 (1.11 g, 1.12 mmol), Cs2CO3 (549 mg,
1.68 mmol) in dry DMF (10 mL) was stirred at 60°C under
Ar for 7 days. After this time the reaction mixture was
filtered and, then was treated with Ac2O (12 mL), pyridine
(8 mL), DMAP (cat.) and stirred for 48 h at 40°C. The
reaction crude was poured into ice/H2O, then aqueous HCl
(5%, 100 mL) was added, and the mixture was extracted
with EtOAc (2×200 mL). The combined organic phases
were washed with aqueous HCl (5%, 100 mL), saturated
NaHCO3 (2×100 mL), and then with water (2×100 mL).
The organic layer was dried (Na2SO4), filtered, and the
solvent was evaporated to give a crude product that was
purified by flash column chromatography to give 27 (443
mg, 65%) as a solid.
We thank the Spanish Ministry of Science and Technol-
ogy for financial support (Grants BQU2000-1159) and
the University of Almer´ıa for
a
scholarship
(A.M.A.M.).
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(b) NMR data for peptidyl CD 27: H NMR (300 MHz,
(CD3)2SO, 353 K): l=8.14–7.94 (m, 28H, NH), 7.82 (bs,
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