Photo-crosslinking of a self-assembled coumarin-dipeptide hydrogel

Article abstract of DOI:10.1039/c5nj00038f

The self-assembly and photo-crosslinking of a lysine dipeptide functionalized with 7-(diethylamino)-3-coumarin carboxylic acid (7-DAC) is described. Self-assembly of the dipeptide in water and PBS produces a hydrogel comprised of uniform, micrometer length nanofibers. Irradiation of the nanofibers at 365 nm induces dimerization of the coumarin groups, which crosslinks and stabilizes the noncovalent nanofibers and enhances the mechanical properties.

Full text of DOI:10.1039/c5nj00038f

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Photo-crosslinking of a self-assembled
coumarin-dipeptide hydrogel†
Cite this: New J. Chem., 2015,
39, 3225
Se Hye Kim,a Yuan Sun,a Jonah A. Kaplan,b Mark W. Grinstaff*b and
Jon R. Parquette*a
Received (in Montpellier, France)
5th January 2015,
Accepted 30th March 2015
DOI: 10.1039/c5nj00038f
www.rsc.org/njc
The self-assembly and photo-crosslinking of a lysine dipeptide with limited functionality to be jointly capable of undergoing
functionalized with 7-(diethylamino)-3-coumarin carboxylic acid controlled self-assembly and subsequent chemical crosslinking.13
(7-DAC) is described. Self-assembly of the dipeptide in water and Previously, we described the b-sheet assembly of dilysines,
PBS produces a hydrogel comprised of uniform, micrometer length functionalized with naphthalenedimide (NDI) appended to the
nanofibers. Irradiation of the nanofibers at 365 nm induces dimerization a-amino position, assembled into 1-D nanostructures in aqueous
of the coumarin groups, which crosslinks and stabilizes the noncovalent medium.14 The b-sheet assembly of these dipeptides in water
nanofibers and enhances the mechanical properties.
was driven by hydrophobic effects and p–p interactions of the
NDI group. Intermolecular electrostatic interactions among adjacent
The self-assembly of peptides is an efficient method to synthesize lysine ammonium groups attenuate the assembly process and
functional materials of interest for applications in in vivo imaging,1 reduce formation of insoluble aggregates. Furthermore, we found
drug delivery,2 and tissue engineering.3 This approach offers a that N-Fmoc derivatives of these dipeptides formed self-supporting
versatile strategy to create nanostructures of high complexity, hydrogels comprised of well-defined nanobelts.15
with the potential for self-healing and structural responsivity.4
Coumarin is ideally suited to serve as the crosslinking
Noncovalent structures are generally less robust than covalently functionality for dipeptide structures. The planar structure of
formed analogs, and thus self-asssembled nanostructures often the chromophore strongly promotes intermolecular p–p stacking
degrade or reorganize with changes in temperature and solvent. to drive self-assembly in water, similar to the NDI chromophore.
The utility of these materials in many applications requires 7-Diethylamino coumarin (7-DAC) also undergoes a reversible
greater physical robustness and mechanical stability. One strategy photodimerization upon irradiation at 365 nm, and dimer
to improve the stability of soft materials is to chemically crosslink cleavage at 265 nm.16 Photodimerization requires the coumarin
the nanostructure following the self-assembly process. This strategy groups to be positioned within 4.2–4.5 Å of each other.17 Thus,
is currently being used to stabilize hydrogels,5 micelles,6 liposomes7 the self-assembly process must proximally position the coumarin
supramolecular gels,8 dendrimers,9 polymeric nanoparticles10 and groups to permit dimerization, thereby circumventing the for-
amphiphilic nanotubes.11 Herein, we report the self-assembly and mation of nonspecific crosslinks between randomly positioned
photocrosslinking of a coumarin–dipeptide conjugate in aqueous chromophores.11a Coumarins also find broad utility as fluorescent
media as a method to increase the robustness of the assembled probes18 in biology and medicine due to their photostability, low
nanostructure.
cytotoxicity, and high fluorescence.19
The b-sheet self-assembly of short peptides12 provides a potential
Accordingly, 7-DAC chromophores were appended to both
strategy to create nanostructured materials from simple, accessible the N-terminal and N-e side chain positions of a dilysine
building blocks. The challenge in creating robust assemblies from peptide, as shown in Fig. 1. Two 7-DAC chromophores were
short dipeptides stems from the difficulty in designing structures conjugated to the dipeptide, resulting in compound A, in an
analogous synthetic scheme as that used in constructing Fmoc-
KK(NDI)-NH2 dipeptides. In this case, the Fmoc groups were
replaced with 7-DAC units, which should be similarly capable of
intermolecular p–p interactions. Indeed, self-assembly was
observed for these compounds in water, saline, and PBS. The
critical aggregation concentration (CAC) of freshly dissolved
solutions of A in PBS (w/o calcium and magnesium), prepared
a Department of Chemistry and Biochemistry, The Ohio State University,
100 W. 18th Ave., Columbus, Ohio 43210, USA.
E-mail: parquett@chemistry.ohio-state.edu; Fax: +1 614 292 1685
b Departments of Biomedical Engineering and Chemistry, Boston University,
590 Commonwealth Ave., Boston, MA 02215, USA. E-mail: mgrin@bu.edu
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nj00038f
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The UV-vis absorption spectrum of A in TFE (0.25 mM)
displayed absorption maxima at 264 and 428 nm (Fig. 2c).
Changing solvent from TFE to either water or PBS resulted in
reduced intensity and blue-shifted absorptions at 417 nm,
indicative of the presence of H-type face-to-face p-stacking
within the fibers. Similarly, the circular dichroism (CD) spectra
exhibited excitonic couplets with zero-crossings in the range of
400–425 nm in PBS and water (Fig. 2d). In contrast, a flat line
was recorded in TFE, consistent with the monomolecular state
of A in this solvent. Given the absence of an induced CD effect
in TFE, the couplets observed in PBS and water must arise from
the relative intermolecular orientation of the coumarin chromo-
phores within the aggregate. The electric transition moment of
the excitation band at 417 nm for 7-DAC runs approximately
along the axis connecting the 3- and 7-positions of the coumarin
structure.21 Accordingly, the opposite sign of the couplets in
water and PBS indicated the presence of an M- and P-type helical
packing orientation of the coumarin chromophores in water and
PBS, respectively (Fig. 3). Deconvolution of the Fourier-transform
infrared (FTIR) spectroscopy of A in PBS (10 mM, D2O) displayed
an amide I band at 1630 cmꢁ1, consistent with the presence
of b-sheet structure as a stabilizing feature of the nanofibers
(Fig. S1, ESI†).22
The photoinduced dimerization of the coumarin chromo-
phores was explored as a method to crosslink and stabilize the
self-assembled nanofibers. Accordingly, solutions of A in TFE
(250 mM), a solvent in which no aggregation takes place, did not
show significant changes in UV spectra after irradiation at
365 nm over 8 hours (Fig. 4c). In contrast, the absorption
bands at 260 and 417 nm progressively decreased by 50% and
30% in PBS and water, respectively, upon irradiation for 8 h
(Fig. 4a and b). Without further UV exposure, the bright yellow
hydrogel remained intact for 43 weeks; however, after irradiating
for 48 h, the hydrogel slowly darkened in shade (Fig. 4a). Prolonged
irradiation at 365 nm (47 d) resulted in an insoluble yellow
precipitate as dimerization further progressed (Fig. 4; inset,
Fig. S2, ESI†), resulting in flat-lines in both CD and UV-vis
spectra. Fully crosslinked water-insoluble nanostructures were
collected by centrifugation and washed with water to remove any
dipeptide monomers or oligomers. The crosslinked solid was
highly soluble in TFE, a solvent that could be expected to disrupt
the self-assembled nanofibers, based on the monomolecular nature
of A in this solvent. However, TEM imaging of the crosslinked
materials that were dissolved with TFE revealed the presence of
nanofibers with uniform diameters of 15 ꢀ 1 nm (Fig. 4d),
comparable to the nanofiber dimensions produced in water or PBS.
Fig. 1 Structural design of coumarin dipeptide hydrogel (10 mM in water,
wt% = 0.76%), DAC-KK(DAC)-NH2 (A).
without preincubation, was 380 mM, measured using the solvo-
chromatic dye Nile Red (Fig. S4 in the ESI†).
The self-assembly of A was investigated in both HPLC-grade
water (pH 7.0) and PBS (pH 7.4) by transmission electron
microscopy (TEM). Samples were prepared by diluting 10 mM
solutions after 24 h to 250 mM. TEM images of A in H2O and
PBS showed the formation of micron-length nanofibers with
uniform diameters of 18 ꢀ 1 nm (Fig. 2a and b). In contrast, no
observable aggregates were identified by TEM in 2,2,2-trifluoro-
ethanol (TFE) (0.25 mM), even after 1 week aging at 10 mM.
Although fibers formed in both PBS and water exhibited similar
dimensions, there was more apparent intertwining and bundling
of the fibers in PBS, compared with the mostly parallel arrays of
fibers formed in water. This increased bundling likely arises
from salt-induced electrostatic screening experienced by the
ammonium side-chain of A in PBS, compared with water. These
repulsive electrostatic interactions attenuate aggregation and would
be greater in pure water, leading to less interfiber associations and
entanglements.20
Fig. 2 TEM images of self-assembled dipeptide A in (a) PBS (pH 7.4) and
(b) water (pH 7.0). (Carbon-coated copper grid, 2 wt% uranyl acetate as
negative stain) (c) UV-vis spectra and (d) CD spectra of A in H2O, PBS and
TFE. All solutions were prepared at 10 mM and freshly diluted to 0.25 mM
after 1 day aging.
Fig. 3 Notional depiction of cross-linked, b-sheet nanostructure of A.
3226 | New J. Chem., 2015, 39, 3225--3228
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Fig. 4 Time dependent UV-vis spectra of A (a) in H2O (pH = 7.0), (b) in
PBS (pH = 7.4), and (c) in TFE upon UV irradiation. Inset pictures display the
hydrogel (10 mM) of A in water and the formation of insoluble precipitation
after UV irradiation at 365 nm. (d) TEM image of internally photocrosslinked
fibrils. All samples (250 mM) were irradiated at 365 nm with UV lamp
(6 W/0.16 Amps) for 80 hours. TEM samples were prepared from the
insoluble polymeric system (after UV irradiation) that was redissolved in TFE.
Fig. 5 Oscillatory shear testing of hydrogels prepared from A at 10 mM
showing stress sweeps of gels in pure water for both non-irradiated and
irradiated hydrogels (a); summarized elastic storage moduli (G0) for non-
irradiated hydrogels prepared in various aqueous solutions (i.e., water, PBS,
and saline) (b); non-irradiated hydrogels in saline (0.9% NaCl) (c); and
compressive properties of non-irradiated hydrogels prepared in saline (d).
Thus, although extensive photocrosslinking disrupts the hydrogel
structure, the self-assembled nanofibers remain intact in otherwise
denaturing solvents such as TFE.
used for spectroscopic analysis. Nonetheless, important variables
such as ionic strength and UV irradiation can tune the mechanical
properties of these gels, and further studies are warranted to
understand these triggers on self assembly and/or reorganization.
In summary, we describe a facile method to cross-link self-
assembled, b-sheet forming dipeptides. The structural design
follows that of Fmoc-KK(NDI)-NH2 dipeptide, whereby the
Fmoc and NDI chromophores are replaced with 7-DAC. The
resultant dipepetide A self-assembles into b-sheet nanofibers
that efficiently gel in aqueous media. The mechanical properties of
hydrogels prepared from A are strongly dependent on their aqueous
environment, suggesting an additional means to tune their physical
properties. Irradiation at 365 nm crosslinks the coumarin moieties
and retains the nanofiber structure. Although partial crosslinking
enhances the mechanical stability of the hydrogel, extensive irradia-
tion disrupts the hydrogel structure, resulting in a water-insoluble
material. However, the nanostructure remains intact in TFE, a
potentially denaturing solvent, which dissolves the non-crosslinked
material. This versatile strategy to stabilize nanostructures formed by
the b-sheet assembly of small dipeptides will facilitate the design of
additional soft materials for applications that require more robust or
responsive physical properties.
The mechanical properties of hydrogels prepared from A
were investigated by oscillatory shear testing (Fig. 5). In pure
water (10 mM, 23 1C), the hydrogels were weak and reversible with
an elastic storage modulus, G0, of B20 Pa (Fig. 5a). UV-irradiation
provided a modest increase in stiffness (G0 E 150 Pa), with a loss of
their reversibility characteristics. Reversibility was determined by
subjecting the same sample to a subsequent stress sweep and
comparing these two values (Fig. S6, ESI†). While more mechanical
and spectroscopic studies will help determine the supramolecular
effect of photodimerization within the hydrogel (i.e., intrafiber
versus interfiber linkages), the oscillatory shear data suggest a
majority of intrafiber crosslinks are due to the close association
and positioning required for individual molecules to dimerize. As
such, the majority of individual nanofibers stiffen, but these large
fibers are incapable of reassembling after being disrupted by
excessive shear strains. Gels (10 mM) prepared in ionic environ-
ments (e.g., saline/0.9% NaCl) exhibited 100-fold greater elastic
moduli (G0 E 20 kPa) (Fig. 5b) and possessed a compressive
modulus, E, of 11.4 kPa (Fig. 5c), whereas hydrogels prepared in
water were too weak for compression tests. The effect of increasing
peptide hydrogel stiffness by increasing ionic strength and/or
changing pH has been described by other researchers in detail,23
and is due to the increasing hydrophobicity of the di-lysine pep-
tides, which lowers their solubility and increases their solid-like
(precipitate) characteristics. Unlike those prepared in pure water,
Experimental
these hydrogels did not experience any appreciable changes in For the synthesis of 7-(diethylamino)-3-coumarin carboxylic acid
stiffness after UV-irradiation. This was due to the reduced solubility, (7-DAC), a solution of 4-diethylaminosalicylaldehyde (193.25 mg,
optical transparency, and molecular mobility within these gels, 1 mmol) and dimethyl malonate (158.5 mg, 1.2 mmol) in MeOH
which was not an issue with the lower concentrations (250 mM) (1 mL) was prepared. Piperidine (0.01 mM, 0.1 mmol) with
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