Supramolecular hydrogels based on β-amino acid derivatives

Article abstract of DOI:10.1039/b516133a

Here we report on a new class of supramolecular hydrogels based on dipeptides that consist of β-amino acids, which may confer proteolytic resistance to the hydrogels for biomedical applications. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b516133a

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Supramolecular hydrogels based on b-amino acid derivatives
Zhimou Yang,a Gaolin Lianga and Bing Xu*ab
Received (in Cambridge, UK) 14th November 2005, Accepted 6th December 2005
First published as an Advance Article on the web 6th January 2006
DOI: 10.1039/b516133a
calculated with relative accuracy, the confirmation of b-amino
Here we report on a new class of supramolecular hydrogels
based on dipeptides that consist of b-amino acids, which may
confer proteolytic resistance to the hydrogels for biomedical
applications.
acids based hydrogelators should provide a new way to tailor the
stability of hydrogels in biological environment and ultimately
expand the ranges of applications of the hydrogels as biomaterials.
Scheme 1 illustrates the chemical structures of the two
hydrogelators, which are dipeptidic mimics linked to naphthalene
groups via amide bonds. The syntheses of both compounds are
simple and straightforward: The N-hydroxyl succinimide (NHS)
activated ester of 2-(naphthalen-2-yloxy)acetic acid reacts with
glycine to afford 2-(2-(naphthalen-2-yloxy)acetamido)acetic acid
(5), and 2-(naphthalen-2-yl)acetic acid reacts with b3-phenylalanine
to afford 3-(2-(naphthalen-2-yl)acetamido)-3-phenylpropanoic
acid (4), respectively. The NHS assisted coupling between 5
and b3-alanine gives 1 in 67% yield, and the coupling between 4
and b3-phenylalanine affords 2 in 72% yield.
Small molecules with appropriate hydrophobicity and hydrophi-
licity can self-assemble into nanofibers in water as the matrices of
supramolecular hydrogels. Because these 3-D, fibrillar matrices
may act as a versatile scaffold for biosensing, cell culturing, and
incorporating therapeutic agents,1–3 supramolecular hydrogels are
attracting considerable amount of research interest. Notably, the
demonstration of self-assembled oligopeptide nanofibers or
nanotubes, which gel water to produce hydrogels for biomedical
applications (e.g., as scaffolds to promote the growth of neurons,1
to induce biomineralization,3 or to assist cell adhesion4), has
stimulated the research efforts on low-molecular-weight hydro-
gelators.5,6 Being used in vivo, these oligopeptide-based scaffolds
are biodegradable through proteolytic enzymes catalyzed hydro-
lysis.7,8 However, such an inherent susceptibility towards enzymes
shorten the in vivo lifetime of these peptide-based hydrogels, thus
reducing their efficacy and limiting their scope of applications
when long-term bioavailability is required.
Both 1 and 2 act as hydrogelators under proper conditions (e.g.,
concentration, temperature, and pH). After 5 mg of 1 is suspended
in 1.0 mL of water, the adjustment of the pH value of the
suspension to 4.8 results in a clear solution. To adjust the pH value
of the solution back to 4.3 gives a transparent hydrogel (Gel I,
Fig. 1A). A gel–sol phase transition happens at 45 uC, and cooling
the solution back to room temperatures regenerates Gel I. These
two procedures can be repeated for many times without affecting
the quality of the hydrogel. Similarly, 5 mg of 2 in 1.0 mL of water
also forms a slightly opaque hydrogel (Gel II, Fig. 1B) by carefully
adjusting pH or temperature (the pH range and temperature of
gel–sol phase transition are 6.2–6.5 and y48 uC, respectively).
Both Gels I and II are stable at room temperature for several
months.
Proteolysis is a common disadvantage for peptide-based
therapeutic agents. Therefore, many efforts have focused on
designing and synthesizing non-peptide molecules that mimic the
structures and functions of peptides or proteins to achieve
prolonged or controlled stability and bioavailability of those
molecules.7 Among the peptidomimics,9 b-peptides, which contain
b-amino acids, have received the most intensive attention due to
their improved biostability.7,10 Despite the rapid progress in the
design and synthesis of b-peptides, the application of b-amino
acids for controlling the bioavailability of supramolecular hydro-
gels remains unexplored because whether a b-amino acid derivative
would act as a hydrogelator remains unknown (although
Hanabusa et al. have shown that some b-amino acid containing
alkylamides form organogels11). In this paper, we presented the
first synthesis and characterization of hydrogelators that are
b-amino acid derived peptidomimics.
Fig. 2 shows the rheological data of Gel I and Gel II with
dynamic frequency sweep. The values of their storage moduli (G9)
Compound 1 (C10H7OCH2CONH-a-Gly-b3-HAla-COOH) is
composed of a a-amino acid (glycine) and a b-amino acid (b3-
alanine), and compound 2 (C10H7CH2CONH-b3-HPhe-b3-HPhe-
COOH) of two b-amino acids (b3-phenylalanine). Because large
amounts of other b-amino acids are readily available and the
conformation of b-amino acid containing peptidomimics can be
Scheme 1 The chemical structures of the b-amino acid derivatives
synthesized in this work. Among them, 1 and 2 are hydrogelators.
aDepartment of Chemistry, The Hong Kong University of Science &
Technology, Clear Water Bay, Hong Kong, China.
E-mail: chbingxu@ust.hk; Fax: 852 2358 1594; Tel: 852 2358 7351
bBioengineering Program, The Hong Kong University of Science &
Technology, Clear Water Bay, Hong Kong, China
Fig. 1 Optical images of the hydrogels formed by (A) 1 and (B) 2.
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Fig. 2 Frequency dependence of the dynamic storage moduli (G9) and
the loss moduli (G0) of Gel I and Gel II.
exceed that of their loss moduli (G0) by a factor of 5–6, indicating
that these two samples are hydrogels.12 Both the values of G9 of
the hydrogels exhibit weak dependence on frequency (from 0.1 to
100 rad s21) at the stress above 20 Pa, suggesting that the matrices
of these two gels have good tolerance to external force. The
comparison of the G9 values of the hydrogels indicates that the
storage modulus of Gel II is slightly larger than that of Gel I,
suggesting that Gel II is elastically stronger than Gel I, which
agrees with the stronger p–p interactions offered by b3-phenyla-
lanine. In addition, Gels I and II display relatively weaker elasticity
than that of the hydrogels formed by Fmoc-amino acid
derivatives.6 This observation indicates the weaker p–p interaction
of naphthalene groups than that of Fmoc groups and weaker
hydrogen bond networks due to the conformational flexibility
associated with b-amino acids. To further study the microstructure
of Gels I and II, we obtained the transmission electron micrograph
(TEM) images of the hydrogels. As shown in Fig. 3A, uniform
sized and well-dispersed small fibrils (about 25 nm) constitute the
matrices for Gel I. The sizes of fibrils in Gel II, unlike in Gel I, are
less uniform (ranging from 20 to 80 nm), and the small fibrils
exhibit tendency to tangle with each other and form large
bundles, which further confirms the stronger p–p stacking ability
of b3-phenylalanine and agrees with higher elasticity of Gel II.
We obtained the emission spectra of 1 and 2 in solution phase
and gel phase for investigating the molecular arrangements in the
hydrogels. Both compounds exhibit broad emission peaks in
solution phase with the maxima at 357 nm for 1, and 330 nm for 2.
The asymmetric shapes of the emission peaks of 1 and 2 in solution
phase and the extensions of their tails in the longer wavelength
region indicate that both 1 and 2 have strong tendency to form
dimers or oligomers even in diluted solution phase (,1025 M),
which should contribute to the hydrogelation of 1 or 2. In the gel
phases, the centers of the emission peaks show considerable red-
shifts (from 357 to 382 nm for 1; from 330 to 350 nm for 2),
suggesting that 1 and 2 form efficient p–p stacking in their
respective gel phases. The pronounced emission above 400 nm for
Gel I suggests that the p–p interaction of naphthalene groups in
Gel I is stronger than that of Gel II (because Ueno et al. recently
showed that caged excimer of naphthalene group to give a broad
emission peak over 400–550 nm13). This result also agrees well with
Fig. 3 TEM images of (A) Gel I and (B) Gel II and (C) emission spectra
of compound 1 or 2 in solution and gel phase (lex. = 272 nm).
the aggregation behavior of 1 and 2 in solution phases, as indicated
by the emission spectra of 1 and 2.
To explore the structural parameters of the b-amino acid
derivatives for hydrogelation, we also synthesized compounds 3
and 4 and tested if 3 or 4 would form a hydrogel. Neither 3 nor 4
acts as a hydrogelator. The structural difference between 1 and 3 is
that b3-HAla replaces the glycine, which obviously renders 3 more
hydrophobic because of the introduction of CH2 moiety. As the
consequence of increased hydrophobicity, 3 fails to act as a
hydrogelator. Under the same condition for 1 forming a hydrogel,
3 forms metastable vesicles to give a milky solution, which turn
into a precipitate overnight at room temperature. On the other
hand, 4 is one b3-HPhe less than 3. In addition to the decrease of
the p–p interactions, the structural removal of phenylalanine
reduces the topological polar surface area (tPSA)14 up to 30%.
Hence, 4 fails to form hydrogels but gives a precipitate. The above
results further confirm that the balanced hydrophobic interactions
and hydrogen bonding play key roles in the supramolecular
hydrogels of 1 or 2.
Thus, we proposed a possible hydrogen bond network and p–p
interactions that sustain the 3-D fibril network (Fig. 4). For Gel I,
the hydrogen bonds are likely to form between two layers of
molecules of 1. One molecule of 1 connects with at least two other
molecules of 1 through hydrogen bonds from amide groups
(Fig. 4A, B). This supramolecular arrangement should allow the
partial overlap of two naphthalene groups to explain the broad
emission peak centered at 400 nm of Gel I. It also permits the
formation of nanofibers of 1. For Gel II, the hydrogen bonds
between the carboxylic acid group and the amide group next to the
naphthalene may provide the necessary interaction to form a
superhelical structure (Fig. 4C, D). In this superstructure, the
overlap between naphthalenes is less dominant, which also explains
why the emission spectra of Gel II only display relatively weak
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K. L. Niece, E. Beniash, D. A. Harrington, J. A. Kessler and S. I. Stupp,
Science, 2004, 303, 1352.
2 X. J. Chen, J. C. Stendahl, M. S. Baker, X. M. Zhang, K. L. Niece,
S. I. Stupp and D. B. Kaufman, Cell Transplant., 2003, 12, 160;
S. Kiyonaka, K. Sada, I. Yoshimura, S. Shinkai, N. Kato and
I. Hamachi, Nat. Mater., 2004, 3, 58; B. G. Xing, C. W. Yu,
K. H. Chow, P. L. Ho, D. G. Fu and B. Xu, J. Am. Chem. Soc., 2002,
124, 14846; Z. M. Yang and B. Xu, Chem. Commun., 2004, 2424.
3 J. D. Hartgerink, E. Beniash and S. I. Stupp, Science, 2001, 294, 1684.
4 S. G. Zhang, T. C. Holmes, C. M. Dipersio, R. O. Hynes, X. Su and
A. Rich, Biomaterials, 1995, 16, 1385.
5 P. Terech and R. G. Weiss, Chem. Rev., 1997, 97, 3133; I. Yoshimura,
Y. Miyahara, N. Kasagi, H. Yamane, A. Ojida and I. Hamachi, J. Am.
Chem. Soc., 2004, 126, 12204; L. A. Estroff and A. D. Hamilton,
Angew. Chem., Int. Ed., 2000, 39, 3447; L. A. Estroff and
A. D. Hamilton, Chem. Rev., 2004, 104, 1201; J. H. Jung, Y. Ono,
K. Hanabusa and S. Shinkai, J. Am. Chem. Soc., 2000, 122, 5008;
F. M. Menger and K. L. Caran, J. Am. Chem. Soc., 2000, 122, 11679;
S. Kiyonaka, K. Sugiyasu, S. Shinkai and I. Hamachi, J. Am. Chem.
Soc., 2002, 124, 10954; H. Kobayashi, A. Friggeri, K. Koumoto,
M. Amaike, S. Shinkai and D. N. Reinhoudt, Org. Lett., 2002, 4, 1423;
Z. M. Yang, H. W. Gu, D. G. Fu, P. Gao, J. K. Lam and B. Xu, Adv.
Mater., 2004, 16, 1440; Z. M. Yang, H. W. Gu, Y. Zhang, L. Wang and
B. Xu, Chem. Commun., 2004, 208; Z. M. Yang, K. M. Xu, L. Wang,
H. W. Gu, H. Wei, M. J. Zhang and B. Xu, Chem. Commun., 2005,
4414; Y. Zhang, Z. M. Yang, F. Yuan, H. W. Gu, P. Gao and B. Xu,
J. Am. Chem. Soc., 2004, 126, 15028; S. Yamaguchi, I. Yoshimura,
T. Kohira, S. Tamaru and I. Hamachi, J. Am. Chem. Soc., 2005, 127,
11835; A. Heeres, C. Van der Pol, M. Stuart, A. Friggeri, B. L. Feringa
and J. Van Esch, J. Am. Chem. Soc., 2003, 125, 14252; K. J. C. van
Bommel, C. van der Pol, I. Muizebelt, A. Friggeri, A. Heeres,
A. Meetsma, B. L. Feringa and J. van Esch, Angew. Chem., Int. Ed.,
2004, 43, 1663; K. J. C. Van Bommel, M. C. A. Stuart, B. L. Feringa
and J. Van Esch, Org. Biomol. Chem., 2005, 3, 2917; S. Bhuniya,
S. M. Park and B. H. Kim, Org. Lett., 2005, 7, 1741; D. K. Kumar,
D. A. Jose, A. Das and P. Dastidar, Chem. Commun., 2005, 4059;
N. M. Sangeetha and U. Maitra, Chem. Soc. Rev., 2005, 34, 821;
A. M. Bieser and J. C. Tiller, Chem. Commun., 2005, 3942;
S. S. Mahajan, R. Paranji, R. Mehta, R. P. Lyon and W. M. Atkins,
Bioconjugate Chem., 2005, 16, 1019; A. R. Hirst and D. K. Smith,
Chem.–Eur. J., 2005, 11, 5496; M. Suzuki, S. Owa, M. Yumoto,
M. Kimura, H. Shirai and K. Hanabusa, Tetrahedron Lett., 2004, 45,
5399; M. Suzuki, S. Owa, M. Kimura, A. Kurose, H. Shirai and
K. Hanabusa, Tetrahedron Lett., 2005, 46, 303; M. Suzuki, M. Yumoto,
H. Shirai and K. Hanabusa, Org. Biomol. Chem., 2005, 3, 3073.
6 Y. Zhang, H. W. Gu, Z. M. Yang and B. Xu, J. Am. Chem. Soc., 2003,
125, 13680.
Fig. 4 Possible molecular arrangements in Gel I and Gel II. The possible
hydrogen bonds would allow supramolecular chains of (A) 1 and (C) 2;
CPK model of the supramolecular chains of (B) 1 and (D) 2.
intensity above 400 nm. The chain of 2 showed in Fig. 4C, D
should also be hydrophobic and tends to aggregate into nanofibers
with a range of widths. These supramolecular chains in Gel I or
Gel II further grow into a 3-D fibrillar matrix via self-assembly,
which traps the water molecules and leave spaces for the
incorporation of drug molecules within it and stopped the flow
of the liquid.
7 D. Seebach and J. L. Matthews, Chem. Commun., 1997, 2015.
8 H.-W. Jun, V. Yuwono, S. E. Paramonov and J. D. Hartgerink, Adv.
Mater., 2005, 17, 2612.
In summary, we have developed two novel hydrogels which
formed by self-assembly of the b-amino acid derivatives. In a
biological environment, this type of hydrogels should have
prolonged bioavailability in comparison with the hydrogels formed
by a-amino acid derivatives. Further work will focus on the use of
these hydrogels to incorporate and deliver therapeutic agents and
the examination of their stabilities in vitro and in vivo.
B. X. acknowledges the financial support from RGC
(Hong Kong) and EHIA (HKUST).
9 A. Giannis, Angew. Chem., Int. Ed. Engl., 1993, 32, 1244.
10 D. H. Appella, L. A. Christianson, I. L. Karle, D. R. Powell and
S. H. Gellman, J. Am. Chem. Soc., 1996, 118, 13071; D. Seebach,
S. Abele, K. Gademann, G. Guichard, T. Hintermann, B. Jaun,
J. L. Matthews and J. V. Schreiber, Helv. Chim. Acta, 1998, 81, 932;
D. F. Hook, P. Bindschadler, Y. R. Mahajan, R. Sebesta, P. Kast and
D. Seebach, Chem. Biodiversity, 2005, 2, 591; T. A. Martinek and
F. Fulop, Eur. J. Biochem., 2003, 270, 3657; E. A. Porter, X. F. Wang,
H. S. Lee, B. Weisblum and S. H. Gellman, Nature, 2000, 404, 565.
11 K. Hanabusa, J. Tange, Y. Taguchi, T. Koyama and H. Shirai, J. Chem.
Soc., Chem. Commun., 1993, 390.
12 S. Yao, U. Beginn, T. Gress, M. Lysetska and F. Wu¨rthner, J. Am.
Chem. Soc., 2004, 126, 8336.
Notes and references
1 T. C. Holmes, S. de Lacalle, X. Su, G. S. Liu, A. Rich and S. G. Zhang,
Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 6728; G. A. Silva, C. Czeisler,
13 H. Ikeda, Y. Iidaka and A. Ueno, Org. Lett., 2003, 5, 1625.
14 P. Ertl, B. Rohde and P. Selzer, J. Med. Chem., 2000, 43, 3714.
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