Phenylalanine-containing cyclic dipeptides-the lowest molecular weight hydrogelators based on unmodified proteinogenic amino acids

Article abstract of DOI:10.1039/c3cc44110e

Cyclic dipeptides (diketopiperazines-DKPs) that are based on the proteinogenic amino acid phenylalanine in combination with serine, cysteine, glutamate, histidine and lysine are described as simple and remarkable low molecular weight hydrogelators. Blends

Full text of DOI:10.1039/c3cc44110e

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
Phenylalanine-containing cyclic dipeptides – the lowest
molecular weight hydrogelators based on unmodified
proteinogenic amino acids†
Cite this: Chem. Commun., 2013,
49, 7818
Received 31st May 2013,
Accepted 3rd July 2013
Alexander J. Kleinsmann and Boris J. Nachtsheim*
DOI: 10.1039/c3cc44110e
www.rsc.org/chemcomm
Cyclic dipeptides (diketopiperazines – DKPs) that are based on the
proteinogenic amino acid phenylalanine in combination with
serine, cysteine, glutamate, histidine and lysine are described as
simple and remarkable low molecular weight hydrogelators.
Blends of selected DKPs show remarkable pH-dependent properties
and can be applied as easy to tune materials in drug delivery.
Fig. 1 Investigated cyclic dipeptides.
Physical molecular hydrogels based on low molecular weight open-chained di- or oligopeptides. Therefore, we synthesized a small
gelators (LMWGs) have become an important class of materials set of DKPs containing the lipophilic amino acid phenylalanine and
due to their versatile applications as innovative soft materials a variety of hydrophilic amino acids, in particular glycine (1), serine
in injection based drug delivery and tissue engineering.1 The (2), cysteine (3), glutamate (4), histidine (5) and lysine (6) (Fig. 1).
gel-resulting self-assembly process of LMWGs is initiated by In an initial experiment we were intended to verify the principle
non-covalent forces such as hydrogen bonding, p-stacking or hydrogelation ability of cyclic dipeptides 1–6. Thus, we heated an
van der Waals interactions. Above all, amino acids and small aqueous suspension containing 4 wt% of each DKP until the solid
peptides are popular LMWGs since they are easy to vary and completely dissolved. Subsequently, the saturated peptide-containing
often show promising biocompatibility.2,3 However, intermolec- solution was cooled to rt or even lower. To our great delight, each
ular hydrogen bonding networks between amide bonds alone, in DKP 1–6 formed stable hydrogels under these conditions (Fig. 2).
particular in water, are in general not sufficient to make a small
DKPs 1–3 and 5 form opaque hydrogels. Glutamate-containing
peptide or amino acid an efficient hydrogelator. Thus, function- DKP 4 yields a transparent hydrogel (at pH 6.0 in phosphate buffer).
alization of the peptide N- or C-terminus with protecting groups The lysine-containing DKP 6 also yields a transparent hydrogel. To
capable of excessive p-stacking interactions is necessary. Low further elucidate their thermal stability, we examined the sol–gel
molecular weight peptide based hydrogelators which only contain transition temperature of these hydrogelators systematically as a
unmodified proteinogenic amino acids are rare.4 In our search for function of DKP-concentration (ESI,† Fig. S1A). Cyclo(L-Phe-L-Cys) 3
the smallest amino acid based units that can form higher ordered was the most effective hydrogelator since it formed a stable hydrogel
structures in an aqueous environment,5 we herein want to present down to a remarkably low concentration of only 0.25 wt%. It shows a
cyclic dipeptides (diketopiperazines – DKPs) as representative notable high sol–gel transition temperature of up to 100 1C at 1 wt%.
examples for remarkable small LMWGs that self-assemble into In contrast, the corresponding serine-containing DKP 2 turned out to
nanofibers and form stable, self-healing hydrogels.6,7
be the least efficient hydrogelator since high peptide loadings (at least
Due to the rigidity of the 6-membered ring, we predicted that 4 wt%) are needed for a hydrogelation with a low sol–gel transition
the self-assembly of DKPs into higher ordered structures via
cooperative intermolecular hydrogen bonding should be entro-
pically much more favourable than it would be for comparable
University of Tuebingen, Institute of Organic Chemistry, Auf der Morgenstelle 18,
D-72076 Tuebingen, Germany. E-mail: boris.nachtsheim@uni-tuebingen.de;
Fax: +49 7071 295897; Tel: +49 7071 29 72035
† Electronic supplementary information (ESI) available: Synthetic procedures,
spectroscopic data as well as 1H and 13C spectra of DKPs 1–6, rheological
Fig. 2 Macroscopic appearance of DKP-based hydrogels; (A) cyclo(L-Phe-Gly) 1.
(B) Cyclo(L-Phe-L-Ser) 2. (C) Cyclo(L-Phe-L-Cys) 3. (D) Cyclo(L-Phe-L-Glu) in phosphate
measurements and concentration dependent sol–gel transition temperatures of
buffer at pH 6.0. (E) Cyclo(L-Phe-L-His). (F) Cyclo(L-Phe-L-Lys).
1–6 and blends of 4 and 6. See DOI: 10.1039/c3cc44110e
c
7818 Chem. Commun., 2013, 49, 7818--7820
This journal is The Royal Society of Chemistry 2013

View Article Online
Communication
ChemComm
temperature of 12 1C. In general, all DKPs, except 2, form stable of 1 showed porous, but highly ordered planes (Fig. 3A and B). Each
hydrogels at rt with a low concentration of 2 wt%. Cyclo(L-Phe-Gly) 1 plane is woven by knotted nanofibers. DKP 2 also consists of porous
which bears no hydrophilic functional group forms stable hydrogels planes (Fig. 3C and D) which contain entangled nanofibers and
down to a concentration of 1 wt%. To the best of our knowledge, 1 is partly microcrystalline domains. DKP 3 is constructed by undefined
the lowest molecular weight peptide-based hydrogelator containing nanosheets (Fig. 3E and F) while the glutamate-containing DKP 4
only proteinogenic amino acids that has been described so far.8,9
Next, we were interested in the shape and morphology of the phosphate buffer).
DKPs 5 and 6 form dense lamellar sheets with bundled nano-
(Fig. 3E and F) forms a defined porous network at pH 6.0 (in
hydrogels on a nanoscale. Therefore, we prepared xerogels of the
corresponding hydrogels through freeze drying and performed fiber connections between the sheets (Fig. 3I–L). The viscoelastic
scanning electron microscopy (SEM) (Fig. 3). Xerogel morphology properties of the hydrogels were investigated by rheological strain
sweep experiments (ESI,† Fig. S2–S7A). The values of the storage
modulus G0 are in the remarkable range of 103–106 Pa. A strong age-
dependent storage modulus was observed for the lysine containing
hydrogel 6 which increases from 697 Pa for a freshly prepared gel
sample to 146 kPa for a gel sample aged for 20 h prior to the
rheological measurement. On the other hand, the freshly prepared
sample showed the highest mechanical stability with a high critical
strain level of 10% deformation. Further time sweep experiments
showed a remarkable increase of G0 upon aging for all DKPs (ESI,†
Fig. S2–S7D). In agreement with comparable hydrogels, the loss
moduli (G00) were found to be at least one order of magnitude lower
than G0 indicating that the hydrogels described herein are true
physical hydrogels and not viscoelastic fluids.10 Frequency sweep
experiments of all hydrogels showed a very weak dependency on the
angular frequency suggesting that the gel matrices have excellent
tolerance towards external forces (ESI,† Fig. S2–S7B). Furthermore,
most of the hydrogels (except of 2) showed a significant thixotropic
behaviour and thus have self-healing properties (ESI,† Fig. S2–S7C).
We further asked ourselves whether we can tune the mechanical
and morphological properties of the hydrogel by mixing two or more
DKPs. Due to recent reports regarding tuneable two-component
hydrogels based on the interaction between diamines and carboxylic
acids,11,12 we studied the gelation behaviour of a 1 : 1 mixture
between the glutamate containing DKP 4 and the lysine containing
DKP 6.
We initially investigated the sol–gel transition temperature of the
blended hydrogel and found remarkable pH-dependency. While pure
4 only formed stable hydrogels at pH 6.0 and pure 6 only formed
stable hydrogels in basic solution (pH 8–11), the blend formed a clear
hydrogel within a remarkably widespread pH-range between pH 2 and
pH 11. The sol–gel temperature reaches a maximum at pH 2 (79 1C)
and a minimum at neutral pH-values (27 1C, Fig. 4B). The viscoelastic
properties showed significant pH-dependency as well, which
was inverse to the sol–gel temperatures with a maximum G0 value
Fig. 3 SEM images of xerogels. Unless otherwise stated, gelation was performed
in an unbuffered aqueous solution (A and B) cyclo(L-Phe-Gly) 1 at pH = 7. (C and D)
Cyclo(L-Phe-L-Ser) 2 at pH = 7. (E and F) Cyclo(L-Phe-L-Cys) 3 at pH = 7. (G and H)
Cyclo(L-Phe-L-Glu) 4 at pH = 6.0 (phosphate buffer). (I and J) Cyclo(L-Phe-L-His) 5 at
pH = 8. (K and L) Cyclo(L-Phe-L-Lys) 6 at pH = 10–10.5.
Fig. 4 (A) SEM image of a freeze-dried xerogel (2 wt% 4 and 2 wt% 6 at pH 7).
(B) pH-dependent sol–gel temperature of a 1 : 1 blend of 4 and 6 (2 wt% each)
compared to pure DKPs (2 wt%).
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 7818--7820 7819

View Article Online
ChemComm
Communication
ˇ
759–773; ( f ) J. Kopecek and J. Yang, Angew. Chem., Int. Ed., 2012, 51,
7396–7417; (g) C. Tomasini and N. Castellucci, Chem. Soc. Rev.,
2013, 42, 156–172; (h) A. Dasgupta, J. H. Mondal and D. Das, RSC
Adv., 2013, 3, 9117–9149.
3 For selected recent examples for peptide-based LMW hydrogelators see:
(a) V. J. Nebot, J. Armengol, J. Smets, S. F. Prieto, B. Escuder and
J. F. Miravet, Chem.–Eur. J., 2012, 18, 4063–4072; (b) S. Marchesan,
C. D. Easton, F. Kushkaki, L. Waddington and P. G. Hartley, Chem.
Commun., 2012, 48, 2195–2197; (c) R. Orbach, I. Mironi-Harpaz, L. Adler-
Abramovich, E. Mossou, E. P. Mitchell, V. T. Forsyth, E. Gazit and
D. Seliktar, Langmuir, 2012, 28, 2015–2022; (d) K. A. Houton,
K. L. Morris, L. Chen, M. Schmidtmann, J. T. A. Jones, L. C. Serpell,
G. O. Lloyd and D. J. Adams, Langmuir, 2012, 28, 9797–9806;
(e) S. Grigoriou, E. K. Johnson, L. Chen, D. J. Adams, T. D. James
and P. J. Cameron, Soft Matter, 2012, 8, 6788–6791; ( f ) M. Hughes, P.
W. J. M. Frederix, J. Raeburn, L. S. Birchall, J. Sadownik, F. C. Coomer,
I. H. Lin, E. J. Cussen, N. T. Hunt, T. Tuttle, S. J. Webb, D. J. Adams
and R. V. Ulijn, Soft Matter, 2012, 8, 5595–5602; (g) G. Pont, L. Chen,
D. G. Spiller and D. J. Adams, Soft Matter, 2012, 8, 7797–7802;
(h) X. Zhang, X. Chu, L. Wang, H. Wang, G. Liang, J. Zhang, J. Long
and Z. Yang, Angew. Chem., Int. Ed., 2012, 51, 4388–4392; (i) C. Ou,
J. Zhang, X. Zhang, Z. Yang and M. Chen, Chem. Commun., 2013, 49,
1853–1855; ( j) J. Nanda, A. Biswas and A. Banerjee, Soft Matter, 2013,
9, 4198–4208; (k) R. Wieduwild, M. Tsurkan, K. Chwalek,
P. Murawala, M. Nowak, U. Freudenberg, C. Neinhuis, C. Werner
and Y. Zhang, J. Am. Chem. Soc., 2013, 135, 2919–2922.
4 For examples of low molecular weight peptide self-assemblies/hydro-
gels see: (a) M. Reches and E. Gazit, Science, 2003, 300, 625–627;
(b) N. S. de Groot, T. Parella, F. X. Aviles, J. Vendrell and S. Ventura,
Biophys. J., 2007, 92, 1732–1741; (c) M. J. Krysmann, V. Castelletto,
A. Kelarakis, I. W. Hamley, R. A. Hule and D. J. Pochan, Biochemistry,
2008, 47, 4597–4605; (d) J. Naskar, G. Palui and A. Banerjee, J. Phys.
Chem. B, 2009, 113, 11787–11792; (e) A. Saiani, A. Mohammed,
H. Frielinghaus, R. Collins, N. Hodson, C. M. Kielty, M. J. Sherratt
and A. F. Miller, Soft Matter, 2009, 5, 193–202; ( f ) S. Marchesan,
C. D. Easton, F. Kushkaki, L. Waddington and P. G. Hartley, Chem.
Commun., 2012, 48, 2195–2197.
Fig. 5 Drug release experiment with BSA (dotted lines) and tetracycline
(continuous lines) in different blend hydrogels of DKPs 4 and 6. (DKP 4 : DKP 6).
at pH 7 (ESI,† Fig. S8A). However, the fastest regeneration is
observed at pH 10 (ESI,† Fig. S8B). A SEM image of a freeze dried
sample at pH 7 showed a so far unseen morphological shape
with a highly ordered lamellar structure (Fig. 4A). Xerogel
morphologies for other pH values are shown in the ESI† (Fig. S9).
Finally, we were interested in an application of our newly found
hydrogel blends in a drug release experiment. We particular asked
ourselves whether we can modulate the drug releasing properties of
the hydrogel by simply varying the DKP ratio. Thus 1 : 1, 1 : 2 and 2 : 1
mixtures of 4 and 6 were investigated in the release of (1) tetracycline
(TC) as a lipophilic small molecule and (2) bovine serum albumin
(BSA) as a highly charged polypeptide as model substrates (Fig. 5).13
For both substrates a significant difference in drug release was
observed. While nearly all BSA was released after 24 h for a 2 : 1
mixture of 4 and 6, only 55% was released with the 1 : 2 mixture. The
hydrogel containing a 1 : 1 mixture of 4 and 6 lies in-between (75%).
For TC the fastest release was observed for the 1 : 1 and the 2 : 1
mixtures (82% and 80%) while the 1:2 mixture showed a signifi-
cantly delayed release (62%).
In summary, we herein present the remarkable hydrogelation
properties of phenylalanine-containing cyclic dipeptides (diketo-
piperazines – DKPs) which only contain proteinogenic amino acids
as building blocks. To the best of our knowledge these structures are
the smallest peptide-based hydrogelators that have been described
so far. Despite their simplicity and the low molecular weight of their
monomeric building blocks, the so formed hydrogels are remarkably
stable and self-healing. We could further demonstrate that a blend
of two well-chosen DKPs shows novel pH-dependent mechanical
properties and can be used as soft materials for delayed drug release.
We gratefully acknowledge the Baden-Wu¨rttemberg Stiftung
(Juniorprofessorenprogramm) for financial support.
5 S. Zhang, Acc. Chem. Res., 2012, 45, 2142–2150.
6 A. D. Borthwick, Chem. Rev., 2012, 112, 3641–3716.
7 For DKP-derived organo- and hydrogelators see: (a) Z. Xie, A. Zhang,
L. Ye, X. Wang and Z.-G. Feng, J. Mater. Chem., 2009, 19, 6100–6102;
(b) Z. Xie, A. Zhang, L. Ye and Z.-G. Feng, Soft Matter, 2009, 5,
1474–1482; (c) S. Manchineella and T. Govindaraju, RSC Adv., 2012,
2, 5539–5542; (d) K. Hanabusa, Y. Matsumoto, T. Miki, T. Koyama
and H. Shirai, J. Chem. Soc., Chem. Commun., 1994, 1401–1402.
8 An exceptional low molecular weight squalen-based hydrogelator
was recently described by Ohsedo and co-workers: Y. Ohsedo,
M. Miyamoto, M. Oono, K. Shikii and A. Tanaka, RSC Adv., 2013,
3, 3844–3847.
9 The so far lowest molecular weight hydrogleators with amide bonds
were described by Xu and Das: (a) J. Shi, Y. Gao, Z. Yang and B. Xu,
Beilstein J. Org. Chem., 2011, 7, 167–172; (b) D. K. Kumar, D. A. Jose,
P. Dastidar and A. Das, Langmuir, 2004, 20, 10413–10418.
10 (a) B. Adhikari, J. Nanda and A. Banerjee, Soft Matter, 2011, 7, 8913–8922;
(b) J. Nanda and A. Banerjee, Soft Matter, 2012, 8, 3380–3386;
(c) A. M. Smith, R. J. Williams, C. Tang, P. Coppo, R. F. Collins,
M. L. Turner, A. Saiani and R. V. Ulijn, Adv. Mater., 2008, 20, 37–41.
11 For a review see: (a) A. R. Hirst and D. K. Smith, Chem.–Eur. J., 2005,
11, 5496–5508; (b) L. E. Buerkle and S. J. Rowan, Chem. Soc. Rev.,
2012, 41, 6089–6102.
12 (a) A. R. Hirst, D. K. Smith, M. C. Feiters and H. P. M. Geurts, Langmuir,
2004, 20, 7070–7077; (b) A. R. Hirst, D. K. Smith, M. C. Feiters and
H. P. M. Geurts, Chem.–Eur. J., 2004, 10, 5901–5910; (c) Q. Wang, Z. Yang,
L. Wang, M. Ma and B. Xu, Chem. Commun., 2007, 1032–1034;
(d) Q. Wang, Z. Yang, X. Zhang, X. Xiao, C. K. Chang and B. Xu, Angew.
Chem., Int. Ed., 2007, 46, 4285–4289; (e) Q. Wang, Z. Yang, M. Ma,
C. K. Chang and B. Xu, Chem.–Eur. J., 2008, 14, 5073–5078; ( f ) X. Zhu,
P. Duan, L. Zhang and M. Liu, Chem.–Eur. J., 2011, 17, 3429–3437;
(g) A. R. Hirst, J. E. Miravet, B. Escuder, L. Noirez, V. Castelletto,
I. W. Hamley and D. K. Smith, Chem.–Eur. J., 2009, 15, 372–379;
(h) J. G. Hardy, A. R. Hirst and D. K. Smith, Soft Matter, 2012, 8, 3399–3406.
Notes and references
1 For reviews see: (a) A. R. Hirst, B. Escuder, J. F. Miravet and
D. K. Smith, Angew. Chem., Int. Ed., 2008, 47, 8002–8018;
(b) C. Yan and D. J. Pochan, Chem. Soc. Rev., 2010, 39, 3528–3540;
(c) D. M. Ryan and B. L. Nilsson, Polym. Chem., 2012, 3, 18–33;
(d) H. Wang and Z. Yang, Nanoscale, 2012, 4, 5259–5267; (e) Y. Li,
J. Rodrigues and H. Tomas, Chem. Soc. Rev., 2012, 41, 2193–2221.
2 For reviews see: (a) I. W. Hamley, Angew. Chem., Int. Ed., 2007, 46,
8128–8147; (b) J. Raeburn, A. Zamith Cardoso and D. J. Adams,
Chem. Soc. Rev., 2013, 42, 5143–5156; (c) E. K. Johnson, D. J. Adams
and P. J. Cameron, J. Mater. Chem., 2011, 21, 2024–2027; (d) J. W.
Steed, Chem. Commun., 2011, 47, 1379–1383; (e) A. M. Jonker, 13 T. Vermonden, R. Censi and W. E. Hennink, Chem. Rev., 2012, 112,
¨
D. W. P. M. Lowik and J. C. M. van Hest, Chem. Mater., 2011, 24,
2853–2888.
c
This journal is The Royal Society of Chemistry 2013
7820 Chem. Commun., 2013, 49, 7818--7820