Supramolecular nanofibers and hydrogels of nucleopeptides

Article abstract of DOI:10.1002/anie.201103641

Watson-Crick interactions: The conjugation of nucleobases (B=thymine, adenine, cytosine, and guanine; see picture) with small peptides affords a novel kind of supramolecular nanofibers and hydrogelators, which exhibit a high biocompatibility and biostability and are regarded as promising new biomaterials.

Full text of DOI:10.1002/anie.201103641

DOI: 10.1002/anie.201103641
Supramolecular Hydrogels
Supramolecular Nanofibers and Hydrogels of Nucleopeptides**
Xinming Li, Yi Kuang, Hsin-Chieh Lin, Yuan Gao, Junfeng Shi, and Bing Xu*
Integration of nucleobases with small peptides generates a
novel kind of nucleopeptides as biocompatible and biostable
supramolecular hydrogelators. As a class of molecules that
contain both nucleobases and amino acids, nucleopeptides
bear considerable biological and biomedical importance.[1]
Naturally occurring nucleopeptides, such as willardiine-con-
taining nucleopeptides and peptidyl nucleosides, are anti-
biotics against microorganisms.[2] A number of unnatural
nucleobase containing peptides, such as peptide nucleic acids
(PNA), have found successful applications in biology and
biomedicine (as an analog of DNA).[3,4] Such biological
significances render nucleopeptides as attractive targets for
heterocyclic chemistry and useful molecules for studying
biology, which has achieved considerable success.[5] There is,
however, little work to use nucleopeptides for developing
novel class of materials.[6] Thus, we decide to explore the
potential of nucleopeptides to serve as building blocks for
biomaterials. Among many possible choices of the types of
materials, we chose to generate hydrogels[7] of nucleopeptides
for two simple reasons: 1) supramolecular hydrogels, result-
ing from molecular self-assembly in water that form entan-
gled nanofibers, have exhibited considerable promises for
applications in biomedicine because of the inherent biocom-
patibility and biodegradability associated with the supra-
molecular nanofibers;[8] 2) despite their versatility and impor-
tance, small nucleopeptides have been hardly explored for
hydrogels. Thus, the primary goal of this work is to design,
synthesize, and evaluate molecular hydrogelators[7a,b,9] made
of nucleopeptides.
Despite the existence of several well-characterized forms
of nucleopeptides (chiral nucleopeptides, achiral nucleo-
pseudopeptides, or peptidyl and amino nucleosides),[1b] it is
unknown which types of nucleopeptides would be optimal for
generating molecular hydrogelators that form nanofibers and
hydrogels. Based on the fact that the dipeptide, l-Phe-l-Phe
(FF; l-Phe =l-phenylalanine), forms nanotube structures[10]
and aromatic rings interact with neighboring nucleobases to
stabilize designed DNA structures,[4a] we hypothesize that the
conjugation of FF with a nucleobase leads to a molecular
hydrogelator. Such a rationale turns out to be valid. As shown
in Scheme 1, the connection of a nucleobase (adenine,
Scheme 1. Molecular structures and shapes of the hydrogelators and
corresponding precursors based on nucleopeptides.
guanine, thymine, or cytosine) to the dipeptides segment
(FF), affords a novel series of nucleopeptides (1) as hydro-
gelators that self-assemble in water to form nanofibers and
produce hydrogels at a concentration of 2.0 wt% and a pH
value around 5. Molecular mechanics (MM) calculations
indicate that the Hoogsteen interactions among nucleobases
promote the formation of the nanofibers. The conjugation of
tyrosine phosphate to 1 yields another group of nucleopep-
tides, precursor 2, which undergoes catalytic dephosphoryla-
tion to generate hydrogelator 3 that forms supramolecular
nanofibers and hydrogels at low concentrations (2.0 wt%)
and physiological pH value. Surprisingly, both 2 and 3 exhibit
significant resistance to proteinase K, a powerful digestive
enzyme. This result unambiguously confirms the unique
advantage of the nucleobase. Moreover, circular dichroism
(CD) experiments and rheological measurements indicate
that the nucleobases of the nucleopeptidic hydrogelators,
after self-assembly, are able to interact with the nucleic acids
through Watson–Crick H-bonding. Because nucleobases are
an important class of biofunctional motifs, this work not only
illustrates the first example of nucleopeptides as hydrogela-
tors made by an enzymatic reaction, but also provides a facile
way to explore the potential applications of nucleopeptides as
biomaterials, which may lead to a new and general platform to
examine specific biological functions (e.g. binding to DNA
and RNA) of a dynamic supramolecular system that is able to
interact with both proteins and nucleic acids.
[*] Dr. X. M. Li, Y. Kuang, Dr. H.-C. Lin, Y. Gao, J. F. Shi, Prof. B. Xu
Department of Chemistry, Brandeis University
415 South Street, Waltham, MA 02454 (USA)
E-mail: bxu@brandeis.edu
[**] This work was partially supported by NIH (R01CA142746), NSF
(DMR 0820492), a HFSP grant (RGP0056/2008), and start-up grant
from Brandeis University. The images were taken at Brandeis EM
facilities.
Figure 1a shows the typical synthetic route exemplified by
the process for making the hydrogelators based on adenine.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201103641.
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9365

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to form nanofibers with the width of 15, 9,
and 10 nm, respectively, and the nanofibers
entangle to trap water and result in hydro-
gels (Table 1) at a concentration of 2.0 wt%
and under slight acid condition (pH 5.0).
Like 2A, precursors 2G and 2T, at a
concentration of 2.0 wt% and pH 7.4, upon
addition of ALP (10 U) turn into hydro-
gelators 3G and 3T, respectively. This
enzymatic conversion leads to the forma-
tion of nanofibers 3G and 3T and results in
the corresponding hydrogels shown in
Table 1. TEM reveals that the diameters of
the nanofibers of 3G (14 nm) and 3T
(9 nm) are similar to those of the nanofibers
of 1G and 1T, respectively. At a concen-
tration of 2.0 wt% and pH 7.4, 3C self-
assembles to afford both nanoparticles
(11 nm) and short, thin nanofibers (4 nm
in diameter and about 200 nm long), but
fails to form well-defined nanofiber net-
Figure 1. a) Synthetic route of a hydrogelator (1A) and a precursor (2A) based on adenine.
1) Dicyclohexylcarbodiimid (DCC) and N-hydroxysuccinimid (NHS), 2) l-phenylalanine, 3)
l-tyrosine phosphate, and 4) trifluoroacetic acid (TFA). b) Dephosphorylation process
catalyzed by alkaline phosphatase (ALP) that converts 2A to 3A and results in nanofibers
and a hydrogel.
Following the procedures reported by Nieddu and co-work-
ers[11] for making nucleobase acetic acids, we first synthesized
bis(tert-butyloxycarbonyl) (bis-Boc) protected adenine, (N6-
bis-Boc-adenine-9-yl)-acetic acid (4). After being activated
by N-hydroxysuccinimide (NHS), 4 reacts with l-Phe to
afford 5, which undergoes the same NHS activation and
phenylalanine coupling to give the key intermediate 6.
Subsequent removal of the Boc-protecting groups with
trifluoroacetic acid (TFA) yields the nucleopeptides (1A) in
47% total yield. Encouraged by the observation that 1A,
acting as a hydrogelator, self-assembles to form nanofibers
with the diameter of 16 nm (Figure 2a) and results in a
hydrogel at the concentration of 2.0 wt% and pH of 5.0, we
used the NHS-activated intermediate 6 to react with l-Tyr
phosphate to obtain 7, which forms the precursor 2A after
deprotection of the Boc groups. The dephosphorylation
process of precursor 2A catalyzed by an enzyme (Figure 1b)
leads to a translucent hydrogel of nucleopeptide 3A (Table 1)
at physiological pH value. A 31P NMR study confirms that the
precursor (2A) completely transforms into the hydrogelator
(3A) in 12 h after the addition of alkaline phosphatase (ALP,
see Figure S2 in the Supporting Information), and the TEM
images (Figure 2) of the negative stained hydrogel of 3A
reveals nanofibers with a width of 20 nm, confirming that
nanofibers of 3A act as the matrices to sustain the hydrogel
(with a storage modulus around 2082 Pa at 2.0 wt%).
The formation of the nanofibers and the hydrogel of 1A
or 3A indicates that the direct attachment of a purine or
pyrimidine base to a small peptide is a valid approach to
design hydrogelators of nucleopeptides. To examine the
generality of this approach, we used the synthetic procedures
similar to Figure 1a to produce the nucleopeptides consisting
of other nucleobases (G, T, or C) and examined their
capability to form nanofibers and hydrogels. As revealed by
TEM (Figure 2), hydrogelators 1G, 1T, and 1C self-assemble
Figure 2. Transmission electron micrographs of the hydrogels formed
by 1A, 1G, 1T, 1C, 3A, 3G, 3T and the solution of 3C (scale
bar=100 nm).
works to provide effective matrices that warrant a hydrogel of
3C.
We measured the rheological properties of the hydrogels
to gain further insight on their characteristics. As shown in
Table 1, the hydrogel of 1G exhibits the highest storage
modulus (12.6 KPa), the hydrogels of 1A and 1T possess
relatively high storage moduli of 8.1 and 6.3 KPa, respectively,
and the hydrogel of 1C has the lowest storage modulus
(26 Pa). The storage moduli of the hydrogels of 3G and 3T
are 682 and 2.9 Pa, respectively, indicating that the hydrogel
of 3T possesses much weaker mechanical strength than those
of the hydrogels 3A and 3G (Table 1). The relatively high
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Table 1: The conditions and properties of the nucleopeptidic hydrogelators and corresponding supramolecular nanofibers and hydrogels.
Sample
1A
1G
1T
1C
3A
3G
3T
3C
concentration [wt%]
pH
2.0
5.0
2.0
5.0
2.0
5.0
2.0
5.0
2.0
7.4
2.0
7.4
2.0
7.4
2.0
7.4
optical images
width of nanofibers [nm]
critical strain [%]
16
1.0
8090
>500
15
0.8
12613
>500
9
1.2
6346
>500
10
0.6
26
>500
20
0.4
2082
>500
14
2.0
682
>500
9
5[a]
–
8.0
2.9
>500
storage modulus G’ [Pa]
–
IC50 [mm][b]
>500
[a] These thin nanofibers have low quantity and coexist with nanoparticles, thus fail to result in a hydrogel. [b] Concentration required for 50 %
inhibition of cell viability.
storage moduli of hydrogels of 1A, 1G, 3A, and 3G may stem
from purine bases that favor the formation of Hoogsteen base
pair,[12] in addition to strong p–p interactions of purine
nucleobases that contain two fused five- and six-membered
heterocyclic rings. Moreover, the lower storage moduli of the
hydrogels of 3 relative to those of the corresponding hydro-
gels of 1 suggest that the presence of tyrosine may reduce the
efficiency of the noncovalent interactions required for the
stabilization of self-assembled nanostructures, thus resulting
in a relatively weak viscoelastic property of those hydrogels.
Furthermore, the addition of an oligomeric deoxyadeno-
sine (A10) to the hydrogel of 1Tor 3Tresults in a more stable
hydrogel (see Figures S3 and S6 in the Supporting Informa-
tion), as demonstrated by the increase of storage modulus
(G’) from 6.3 KPa (of hydrogel 1T) to 14.3 KPa (of the
hydrogel of 1T and A10), or from 2.9 Pa (of hydrogel 3T) to
12.0 Pa (of the hydrogel of 3T and A10; see Figure S6 in the
Supporting Information). This result suggests that Watson–
Crick interactions between the self-assembly of 1T (or 3T)
and A10 favor molecular aggregation and enhance the
mechanical strength of the hydrogels. To further examine
Watson–Crick H-bonding between complementary nucleo-
bases among the hydrogelators, we use hydrogelators of 1T
and 1A (or 3Tand 3A) to prepare a mixed hydrogel and find
that the storage modulus (G’) increases from 6.3 KPa (of
hydrogel 1T) to 18 KPa (of the hydrogel of 1T and 1A), or
from 2.9 Pa (of hydrogel 3T) to 150 Pa (of the hydrogel of 3T
and 3A). The mixed hydrogel of the mismatched nucleobases
(i.e. 1T and 1G or 1T and 1C) exhibits, however, little
increase of the storage moduli (see Figure S8 in the Support-
ing Information) in comparison to that of hydrogel 1T. These
results indicate that these nucleopeptidic hydrogelators
preserve Watson–Crick interactions of the nucleobases.
We used circular dichroism (CD) to study the super-
structures of these nanofibers of self-assembled nucleopep-
tides in the gel phase. The hydrogels of 1 have the common
feature of b-sheet structure according to the CD spectra with
a positive peak near 195 nm and a negative peak around
210 nm (see Figure S3 in the Supporting Information),
suggesting that these nucleopeptides arrange into b-sheet-
like configurations. The hydrogels of 3A, 3G, and 3T display
the common feature of CD spectra with a positive peak near
195 nm and a negative peak around 210 nm, which also
suggests that the nucleopeptides adopt b-sheet-like config-
urations. The CD spectrum of the 3C solution exhibits a
positive peak near 203 nm and a negative peak around
215 nm, which red-shifts relative to the signals of typical b-
sheet configuration. The red-shifted signal related to b-sheet-
like configurations is likely associated with a structure, which
is twisted relative to the standard planar b-sheet structure,
agreeing with the fact that the increase in b-sheet twisting
causes disorder and results in short nanofibers and nano-
particles, which leads to weak mechanical strength.[13] Overall,
the signals of b-sheet structures (i.e. transitions at 195–
225 nm) of 1 are stronger than those of 3, following the trend
that the storage modulus of 1 is larger than that of 3. The CD
signals with broad bands around 300 nm among the hydrogels
1 and 3 likely originate from the formation of mesophases of
hydrogelators because they locate far from the chromophoric
absorption region (ca. 270 nm) of the hydrogelators (see
Figure S4 in the Supporting Information).
Similar to other nucleobase-containing small molecules
that bind with nucleic acids through Watson–Crick interac-
tion,[14] hydrogelators 1T or 3T also bind to oligomeric
deoxyadenosine (e.g. A10), which results in distinctive
changes in the CD spectra. For example, comparing to the
CD of hydrogel 1T, the CD of the 1T–A10 mixed gel (see
Figure S5 in the Supporting Information) exhibits the
decreased ellipticity of positive bands at 192 and 228 nm
and negative bands around 205, 247, and 287 nm. The CD
spectrum of the 3T–A10 mixed gel (see Figure S5 in the
Supporting Information) shows that the addition of A10 both
changes the intensity of the bands at 195 and 205 nm and
creates a new band at 303 nm that possibly is a result of the
conformational change of the self-assembled structures of 3T
induced by A10.[14a–c] In addition, compared to the solution of
1T or 2T, the CD spectra of the mixed solution of A10 with
hydrogelator 1T (or precursor 2T) shows slight changes in the
band shape, indicating the relatively weak interaction in the
solution state (see Figure S5 in the Supporting Information).
We also used molecular mechanical (MM) calculations to
evaluate noncovalent interactions[15] and simulate the width
of the nanofibers of 1. As shown in Figure 3a, the simulated
widths of the nanofibers are 15, 16, 9, and 11 nm for
nucleopeptides 1A, 1G, 1T, and 1C, respectively, which
correlate well with experimental observations (Figure 2).
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Communications
proteinase K; more than 40% of 2G or 3G remain
after 4 h of the incubation with proteinase K.
Although less than 10% of 1T or Nap-FFY[19]
remain after 4 h of incubation with proteinase K
(see Figure S13 in the Supporting Information), the
excellent or fair resistance to enzymatic digestion,
exhibited by the nucleopeptides 2 and 3, confirms the
unique advantage of nucleobases. Because of their
high resistance to proteases, the hydrogel formed by
hydrogelator 3T, 3A, or 3C promises to serve as new
biomaterial for applications that require long-term
biostability. In addition, this result suggests that the
incorporation of nucleobase may be an effective
approach for improving the biostability of other
small peptidic hydrogelators.
In conclusion, this work demonstrates the gen-
eration of a new type of hydrogelators based on
conjugates of nucleobases and short peptides that
self-assemble in water to afford supramolecular
hydrogels upon a pH- or enzymatic trigger, and
introduces a new, simple, and general approach for
developing soft, biocompatible materials from nucle-
opeptides. This work provides a facile way to explore
new applications of nucleopeptides as functional
Figure 3. a) Width of fibers of hydrogels 1A, 1C, 1G and 1T based on trans-
mission electron micrographs (in gray) and molecular mechanical calculations (in
black). Cell viability test for 72 h of b) 1A, 1C, 1T, and 1G and c) 2A, 2C, 2T, and
2G. d) Optical images of HeLa cells on the surface 0 and 20 h after creation of
scratchs in the presence of hydrogel 3T (by adding 27.7 mm 3T to the media).
According to the simulations, the widths of the nano-
fibers in hydrogels of 1A and 1G are likely a result of
Hoogsteen base pair formation[12,16] by adenine or
guanine nucleobases (see Figures S10 and S11 in the
Supporting Information). In addition, MM calcula-
tions support the formation of b-sheet-like structures.
To verify the biocompatibility of the hydrogelators,
we added hydrogelator 1 or precursor 2 to the culture
of HeLa cells and measured the proliferation of the
cells. According to the MTT assay shown in Figure 3,
Figure 4. Time-dependent course of the digestions of hydrogelators a) 2T, 2C,
2G, and 2A, and b) 3T, 3A, 3G, and compound 3C by proteinase K.
after being incubated with a hydrogelator (1A, 1T, or
1C) at a concentration of 500 mm or a precursor (2A,
2T, or 2C) for 72 h, the cell viability remains at 100%.
Although the cell viability decreases slightly when they
are incubated with 500 mm 1G or 2G for 72 h, the concen-
tration required for 50 % inhibition (IC50) is still > 500 mm.
These results prove that nucleopeptides 1, 2, and, 3 are
biocompatible. We also used a simple wound-healing assay[17]
to examine the capability of the nanofibers and hydrogels of 3
to serve as a material in which cell–matrix interactions are
maintained. As shown in Figure 3d, the presence of the
hydrogel of 3T in the cell culture has little inhibitory effect on
the migration of the cells, which further supports the
biocompatibility of 3.
Besides biocompatibility, biostability is also an essential
requisite for a biomaterial. Thus, we examine the stability of
hydrogelators by incubating them with proteinase K, a
powerful protease that hydrolyzes a wide range of peptidic
substrates and cleaves the peptide bond adjacent to the
carboxyl group of aliphatic and aromatic amino acids with
blocked alpha amino groups.[18] As shown in Figure 4, more
than 85% of 2T, 3T, or 3A, more than 70% of 2A or 3C, and
above 50% of 2C remain after 24 h of the incubation with
biomaterials because of the facile incorporation of bioactive
peptides or molecular recognition motifs[20] into nucleobases.
Received: May 27, 2011
Revised: July 19, 2011
Published online: August 26, 2011
Keywords: biocompatibility · hydrogels · nanofibers ·
.
nucleopeptides · supramolecular chemistry
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