Synthesis of bifunctional peptide derivatives based on a β-cyclodextrin core with drug delivery potential

Article abstract of DOI:10.1016/j.tetlet.2009.11.118

A selective, versatile, robust methodology for bifunctionalization of β-cyclodextrin is achieved allowing the attachment of peptides in varying C- and/or N-terminal combinations on resin using Fmoc SPPS. Two linkers are attached to cyclodextrin enabling selective binding to the resin (or a peptide attached to the resin). Continuation of peptide growth and/or cleavage from the resin follows, thus various combinations of peptide-cyclodextrin species are achieved. A model peptide (Gly-Ala) is used in this study to illustrate the potential of this system for attaching one or more bioactive peptides for drug transport and release purposes.

Full text of DOI:10.1016/j.tetlet.2009.11.118

Tetrahedron Letters 51 (2010) 800–803
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of bifunctional peptide derivatives based on a b-cyclodextrin core
with drug delivery potential
Rachel J. White, Paul G. Plieger, David R. K. Harding *
Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
A selective, versatile, robust methodology for bifunctionalization of b-cyclodextrin is achieved allowing
the attachment of peptides in varying C- and/or N-terminal combinations on resin using Fmoc SPPS.
Two linkers are attached to cyclodextrin enabling selective binding to the resin (or a peptide attached
to the resin). Continuation of peptide growth and/or cleavage from the resin follows, thus various com-
binations of peptide–cyclodextrin species are achieved. A model peptide (Gly-Ala) is used in this study to
illustrate the potential of this system for attaching one or more bioactive peptides for drug transport and
release purposes.
Received 3 September 2009
Revised 25 November 2009
Accepted 27 November 2009
Available online 1 December 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Comprised of six, seven or eight
nose units, the cyclodextrins (a, b, and c, respectively) are cyclic
oligosaccharides with an overall shape reminiscent of a truncated
cone. The narrow rim of this cone bears seven primary hydroxy
groups and the wider rim bears 14 secondary hydroxy groups.
Selective modification of one or more hydroxy groups has, in the
past, allowed for the grafting of various molecules with new recog-
nition functions, yet bifunctionalization still remains a challenge.
a
-1,4-linked
D
-(+)-glucopyra-
one or two peptides in various combinations; for example, the
attachment of b-CD to the C- and/or N-terminus of peptides as well
as the functionalization of b-CD with differing peptidyl chains
(Scheme 1).
1
,2
Scheme 2 outlines a six-step process to bifunctionalize cyclo-
dextrin 1 (an example of step (i) in Scheme 1) to allow for its use
in Fmoc SPPS for the attachment of one or more peptides. The
end product 7, containing both amino and carboxylic functional
groups, is set up for peptide synthesis. All compounds were charac-
terized by high- or low-resolution mass spectrometry and/or NMR
studies. Selective mono-derivatization of the primary hydroxy
3
,4
Despite the difficulties associated with bifunctionalization, a num-
ber of groups have recently made substantial progress in selective
bifunctionalization via the utilization of benzylation for protec-
3
–5
11
tion.
group of b-CD (1) was reported by Byun et al.
Examples of peptide-functionalized cyclodextrins are numerous,
so it is somewhat surprising that bifunctionalized cyclodextrins
bearingtwodifferentpeptidylunitshaveyet tobe reported. Ourpur-
suit of new drug carriers incorporating the recognition signalling
capabilities of bioactive molecules, for example, peptides, has led
us to develop a versatile route to cyclodextrin functionalization.
There are a large number of cellular receptors for which peptides ap-
pear to be the most versatile targeting agents. There are many pub-
lications on the grafting of single amino acids onto cyclodextrins, but
little has been reported on multi-peptidyl-cyclodextrin com-
This reaction allows the selective replacement of one primary
hydroxy group by a tosyl group. Thus, treatment of b-CD (1) with
tosyl chloride and sodium hydroxide in water at 0 °C afforded 2
A
11
(mono-6 -(4-methylbenzenesulfonyl)-b-cyclodextrin).
Next, 2
was treated with 35% aqueous ammonia, refreshed daily, for seven
A
days. Recrystallization from water gave a 90% yield of 3 (mono-6 -
amino-b-cyclodextrin) which was found to be a stable, easy to han-
2
A
dle compound. Protecting the mono-6 -amino-b-cyclodextrin (3)
with a Cbz group using Cbz-OSu and sodium hydrogen carbonate
A
in dioxane/water at room temperatureafforded mono-6 -carboben-
6
–8
12
pounds. Of thefew knownexamples, only a small numberof these
employ solid-phase peptide synthesis techniques for grafting
peptides onto cyclodextrin off resin, and these result in an all or no
zyloxyamino-b-cyclodextrin, 4. Scale-up to 5 g of purified material
was readily achieved by recrystallization from water. RP-HPLC puri-
fication using a stepwise methanol/water/TFA gradient allowed for
confirmation via complete NMR characterization (see Supplemen-
7
,9,10
functionalization of the b-cyclodextrin (b-CD) hydroxy groups.
1
3
We now report, for the first time, bifunctionalization of b-CD with
a series of model peptides using solid-phase peptide synthesis. This
synthetic approach allows the functionalization of cyclodextrin to
tary data). Succinylation of 4
resulted in the isolation of
A
mono-6 -carbobenzyloxyamino-succinyl-b-cyclodextrin (5) in a
reasonable yield (40%). Dissolution in water followed by filtration
of any insolubles gave the product in the filtrate suitable for use in
the next step. A mixture of regioisomers of 5 was used for the
remainder of the experiment. The ester linkage is stable under all
*
Corresponding author. Tel.: +64 63569099; fax: +64 63505682.
E-mail address: D.R.Harding@massey.ac.nz (D.R.K. Harding).
0
040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.11.118

R. J. White et al. / Tetrahedron Letters 51 (2010) 800–803
801
X
X
OH
O
X
= Functional group
O
O
OH
OH
OH
O
O
O
O
O
O
=
=
N- or C-terminal peptide
Protecting group
OH
OH
OH
5
5
X
X
OH
X
OH
O
O
O
O
O
O
OH
OH
OH
OH
OH
OH
RESIN
X
+
O
O
O
OH
OH
OH
5
OH
OH
OH
(ii)
OH
HO
OH
OH
HO
(
i)
O
O
O
HO
O
O
O
HO
5
X
X
OH
O
OH
O
X
X
OH
OH
7
O
O
O
OH
OH
OH
O
O
O
β
-Cyclodextrin
OH
OH
OH
5
Scheme 1. Derivatization of b-cyclodextrin using solid-phase peptide synthesis. (i) Synthesis of bifunctional b-cyclodextrin to enable selective peptidyl attachment in SPPS.
(
ii) Attachment of peptides to b-cyclodextrin using SPPS to synthesise a number of C- and/or N-terminus-derivatized products.
Ts
firmed by ESI-MS studies as per Sforza et al.14 Currently, it is not
possible to differentiate between the (AB/AG), (AC/AF) and (AD/AE)
pairs using mass spectrometry.
OH
O
OH
a
b
O
O
O
OH
OH
OH
O
O
O
OH
OH
OH
In the presence of a twofold excess of sodium chloride, un- and
disubstituted b-CDs randomly break at the acetal junctions, giving
rise to different polyglucose sodium-containing fragments. There-
fore, the relative abundances of the di-, mono- and unsubstituted
fragments generated from the three different regioisomeric pairs
(AB/AG), (AC/AF) and (AD/AE) by ESI-MS can be compared to those
determined by statistical analysis (see Supplementary data).14
Figure 1 shows the identification of the three regioisomers achieved
by ESI-MS studies showing three glucose units and one or no dehy-
drated groups (m/z 642 and 742, respectively). The ratio values for
regioisomer peaks were found to be in agreement with literature
7
6
1
2
Cbz
NH
NH
2
O
OH
OH
O
c
O
O
OH
OH
OH
O
OH
O
O
O
OH
OH
OH
OH
6
6
3
4
O
OH
Cbz
NH
O
O
OH
O
O
d
O
e
OH
OH
OH
O
O
O
OH
OH
OH
5
14
values and consistent with the expected pattern of fragmentation
5
O
(inset, Fig. 1). The 642 peak is higher for the AD isomer, the 742 peak
is higher for the AB isomer, and the two peaks are of similar intensity
OH
O
NH
2
OH
O
O
O
f
for the AC isomer (left, Fig. 1). Hydrogenolysis of the Cbz-protecting
O
O
OH
OH
OH
A
O
O
O
group gave mono-6 -amino-succinyl-b-cyclodextrin,
6
as
a
OH
OH
OH
12
A
5
regioisomeric mixture. Fmoc protection of 6 produced mono-6 -
fluorenylmethyloxycarbonylamino-succinyl-b-cyclodextrin 7, as a
regioisomeric mixture which could be purified via recrystallization
from water. For complete NMR analysis, RP-HPLC was first
performedusing a stepwise methanol/water/TFA gradient. This gave
the elution pattern of (AC/AF):(AD/AE):(AB/AG) isomers as
confirmed by ESI-MS studies (see Supplementary data). Compound
6
O
Fmoc
OH
O
NH
O
OH
O
O
OH
OH
OH
O
O
O
OH
OH
OH
5
7
has been shown to be stable under all standard Fmoc SPPS reaction
7
conditions (see Supplementary data) with the expected loss of the
Fmoc group using 20% piperidine in DMF (2 ꢀ 10 min). This robust
method enables the attachment of one peptide to 7 using SPPS by
the selective removal of the Fmoc-protecting group (Scheme 3).
Scheme 2. Bifunctional synthesis of b-CD. Reagents and conditions: (a) tosyl
1
1
2
chloride, NaOH, H
NaHCO , dioxane/water, RT, overnight, 51%; (d) succinic anhydride, py, rt overnight,
0%; (e) H , Pd/C, DMF, rt, 90%; (f) Fmoc-OSu, NaHCO , dioxane/water, rt, overnight,
0%.
2 3
O, 0–5 °C, 5 h, 38%; (b) 35% aq NH , 7 days, 90%; (c) Cbz-OSu,
3
4
4
2
3
15
Although this work does not report new chemistry, the syn-
thetic approach and versatility allows for the addition and growth
of C- and/or N-terminal peptides with the same and/or different
functional properties on a solid-phase support. Peptide attachment
off resin in solution using stepwise or ligation synthetic protocols
is also possible (data not shown). Direct coupling to unfunctional-
ized b-CD was unsuccessful under all Fmoc procedures used in this
study. In addition, the succinyl linker is not removed at the end of
Fmoc SPPS reaction conditions (see Supplementary data). Isomeric
purification was achieved by RP-HPLC using a stepwise methanol/
water/TFA gradient followed by an acetonitrile/water/TFA gradient.
This afforded a 1:2:2 ratio of the (AB/AG):(AC/AF):(AD/AE) regioi-
somers with the elution pattern of (AD/AE):(AC/AF):(AB/AG) as con-

8
02
R. J. White et al. / Tetrahedron Letters 51 (2010) 800–803
A
Figure 1. Experimental ESI-isomer spectra (left) with the expected pattern of fragmentation (inset); structures of mono-6 -carbobenzyloxyamino-succinyl-b-cyclodextrin, 5
isomers. AB/AG isomer (top), AC/AF isomer (middle), AD/AE isomer (bottom).
this multi-step synthesis. Potentially, by using a resin that
generates a peptide with acid functionality, the succinyl group is
available for further functionalization.
were studied under various amino acid coupling conditions using
the Fmoc UV–vis coupling method for the detection of the loss
9
of the Fmoc group (see Supplementary data). It was found that
the best method of coupling was with EDC/DIEA in excess in a
50% pyridine/DMF solvent system overnight followed by one
recoupling. This gave a coupling percentage for 7 of 25% after a sec-
ond recoupling. Even with a third consecutive recoupling, the cou-
pling percentage did not improve. It is thought that the length of
the spacer arm (the succinyl group) between 7 and the resin might
not be long enough to enable efficient coupling. Standard Fmoc-
coupling conditions were used for the addition of Fmoc amino
This system has great developmental potential for the transpor-
tation of drugs and/or other molecules. Owing to the large variety
of cellular receptors, peptides appear to be amongst the most ver-
satile compounds for such targeting purposes. The grafting of pep-
7
tides onto cyclodextrin also adds future therapeutic dimensions.
To establish proof-of-concept, the attachment of a short model
peptide to 7 at both the N- and C-terminus was achieved using
Fmoc SPPS to give 8 and 9, respectively (Scheme 3). The coupling
conditions for the attachment of 7 to resin and/or amino acids
0
0
acids [TBTU (O-(benzotriazol-1-yl)-N,N,N ,N -tetramethyluronium

R. J. White et al. / Tetrahedron Letters 51 (2010) 800–803
803
O
Peptide
NH2
O
NH
O
OH
O
OH
O
OH
O
OH
O
O
OH
O
HO
O
HO
O
HO
OH
OH
O
O
O
a, b, c, d, e
O
OH
OH
OH
5
O
HO
H2N
O
a, b, c, d, e
5
NH
8
7
O
O
Peptide
Peptide
O
NH2
NH
O
NH
O
OH
O
NH2
O
OH
a, b, c, d, e
O
O
O
O
O
O
OH
OH
OH
O
O
O
OH
OH
OH
O
O
O
OH
OH
OH
5
OH
OH
OH
5
1
0
9
Scheme 3. Peptide synthesis using bifunctionalized b-cyclodextrin, 7. Reagents and conditions: (a) Rink resin (0.073 mmol/g), DMF, 3 h; (b) 20% piperidine in DMF,
ꢀ 10 min; (c) amino acid (4 equiv), TBTU (4 equiv), HOBt (4 equiv), DIEA (8 equiv) in DMF, rt, overnight or 7 (4 equiv), EDC (4 equiv), DIEA (8 equiv), py/DMF, rt overnight;
d) DMF; (e) TFA (30%), CH Cl , rt, 30–60 min, 21% (for 8), 15% (for 9), 3% (for 10) where the peptide is Ala-Gly.
2
(
2
2
tetrafluoroborate) and DIEA (diisopropylethylamine) in DMF in
excess], resulting in pure 21% (10.4 mg) and 15% (7.4 mg) yields
for 8 and 9, respectively (Scheme 3). The pure products were char-
acterized by HR-MS. The attachment of 7 at both the C- and N-ter-
mini of a peptide was also achieved (Scheme 3) using the same
reaction conditions producing 10 in a pure 3% (2.4 mg) yield as
confirmed by LR-MS (MALDI-TOF).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2009.11.118.
References and notes
1.
Challa, R.; Ahuja, A.; Javed, A.; Khar, R. K. A. A. P. S. PharmSciTech 2005, 6, E329–E357.
We have demonstrated a unique, versatile and robust method
for the bifunctionalization of b-CD. It was interesting to note that
a 1:2:2 ratio for the (AB/AG):(AC/AF):(AD/AE) isomers for the
bifunctional cyclodextrin complex 5 was obtained indicating a ste-
ric preference in the overall complex. Isomer separation was
achieved using RP-HPLC with characterization by ESI-MS studies.
Furthermore, the attachment of peptides selectively to either the
C- and/or N-terminus using Fmoc SPPS has been demonstrated.
This system has great potential for the transportation of drugs
and/or other molecules, especially as CD is reported as being bio-
2
. Khan, A. R.; Forgo, P.; Stine, K. J.; D’Souza, V. T. Chem. Rev. 1998, 98, 1977–1996.
3. Tabushi, I.; Nabeshima, T.; Kitaguchi, H.; Yamamura, K. J. Am. Chem. Soc. 1982,
104, 2017–2019.
4.
5.
6.
Sollogoub, M. Eur. J. Org. Chem. 2009, 1295–1303.
Guieu, S.; Sollogoub, M. Angew. Chem., Int. Ed. 2008, 120, 7168–7171.
Muhanna, A. M. A.; Ortiz-Salmeron, E.; Garcia-Fuentes, L.; Gimenez-Martinez, J.
J.; Vargas-Berenguel, A. Tetrahedron Lett. 2003, 44, 6125–6128.
Pean, C.; Creminon, C.; Wijkhuisen, A.; Grassi, J.; Guenot, P.; Jehan, P.; Dalbiez,
J.-P.; Perly, B.; Djedaini-Pilard, F. J. Chem. Soc., Perkin Trans. 2 2000, 853–863.
Yu, H.; Makino, Y.; Fukudome, M.; Xie, R.-G.; Yuan, D.-Q.; Fujita, K. Tetrahedron
Lett. 2007, 48, 3267–3271.
7.
8.
9.
Dodziuk, H.; Demchuk, O. M.; Schilf, W.; Dolgonos, G. J. Mol. Struct. 2004, 693,
145–151.
1
,2,6,7
compatible.
Studies are continuing into the attachment and
10. Tsutsumi, H.; Ikeda, H.; Mihara, H.; Ueno, A. Bioorg. Med. Chem. Lett. 2004, 14,
analysis of potential bioactive peptidyl-cyclodextrin complexes
for their potential use in drug transportation and delivery.
723–726.
1
1
1. Byun, H.-S.; Zhong, N.; Bittman, R. Org. Synth. 2000, 77, 225–230.
2. Chan, W. C.; White, P. D. Fmoc Solid Phase Peptide Synthesis: A Practical
Approach; Oxford University Press: New York, 2002.
Acknowledgements
13. Schaschke, N.; Musiol, H.-J.; Assfalg-Machleidt, I.; Machleidt, W.; Moroder, L.
Bioorg. Med. Chem. Lett. 1997, 7, 2507–2512.
1
4. Sforza, S.; Galaverna, G.; Corradini, R.; Dossena, A.; Marchelli, R. J. Am. Chem.
This work was supported by grants from Massey University and
the Marsden Fund (RSNZ). R.J.W. thanks Massey University for a
Vice Chancellor’s scholarship.
Soc. 2003, 14, 124–135.
15. Ashton, P. R.; Koeniger, R.; Stoddart, J. F.; Alker, D.; Harding, V. D. J. Org. Chem.
1996, 61, 903–908.