Catalytic dephosphorylation of adenosine monophosphate (AMP) to form supramolecular nanofibers/hydrogels

Article abstract of DOI:10.1039/c2cc16723a

The use of enzyme to instruct the self-assembly of the nucleoside of adenosine in water provides a new class of molecular nanofibers/hydrogels as functional soft materials.

Full text of DOI:10.1039/c2cc16723a

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COMMUNICATION
Catalytic dephosphorylation of adenosine monophosphate (AMP) to form
supramolecular nanofibers/hydrogelsw
Xuewen Du, Junfeng Li, Yuan Gao, Yi Kuang and Bing Xu*
Received 30th October 2011, Accepted 16th December 2011
DOI: 10.1039/c2cc16723a
The use of enzyme to instruct the self-assembly of the nucleoside of
adenosine in water provides a new class of molecular nanofibers/
hydrogels as functional soft materials.
to connect benzyl groups to nucleoside derivatives for making
hydrogelators.8 Jamieson et al. demonstrated that the addition
of potassium ions into the mixture of guanosine gelator and a
non-gelator guanosine leads to a very stable hydrogel.9 Recently,
Barthelemy et al. reported the self-assembly of a glycosyl-
nucleoside-lipid to form nanofibers and nanotubes and suggested
that the carbohydrate moieties provide a suitable environment to
deliver nucleic acids into human cells.10 These developments
clearly highlight the promising opportunity presented by the
hydrogelators made of nucleosides.5
This communication reports the use of enzyme to instruct the
self-assembly of the nucleoside of adenosine in water for
developing supramolecular nanofibers/hydrogels as a novel type
of soft biomaterials. Hydrogels, composed of a solid network in
water, have been a type of fascinating materials that find many
useful applications. Although the network usually is made of
covalently crosslinked polymers, certain small molecules (referred
as hydrogelators) are able to self-assemble in water to form
nanofibers. When their density is sufficiently high, the nanofibers
entangle to form the network and result in hydrogels, which is
called supramolecular hydrogels.1 A variety of small molecules
can act as hydrogelators, including the derivatives of nucleosides.
Nucleosides, consisting of a nucleobase and a sugar (i.e., ribose or
deoxyribose), are the building blocks of nucleic acids (i.e., RNA
or DNA).2 Because of their biological relevance and importance,
they have attracted considerable research efforts in the area of
using nucleosides as the building blocks for generating hydrogels
as a new type of biomaterials. For example, Shimizu et al.
first reported the hydrogels made of nucleotide-appended
bolaamphiphiles. They found that nucleotides connected by
long spacers are capable of gelling water effectively through
spontaneous formation of a fibrous network. They also suggested
that hydrogen bonds involving the 50-hydroxyl group of the
deoxyribose moiety, hydrophobic interaction between the long
oligomethylene chains, and p–p stacking of the thymine residues
result in the effective hydrogelation.3 After this early report,
several other groups also have developed hydrogels built upon
nucleosides.4–10 Among them, Barthelemy et al. prepared a
family of uridine phosphocholine amphiphiles that self-assemble
to form supramolecular structures or materials including vesicles,
fibers, hydrogels, and organogels.4 Oda and co-workers have
developed cationic gemini surfactants that have nucleotides
as the counterions and form hydrogels upon the addition of
complementary nucleoside bases.6 Kim et al. used click chemistry
The above developments not only validate the suitability of
nucleosides to serve as a motif in hydrogelators, but also
illustrate several ways to trigger the formation of those
hydrogels, such as the change of temperatures,4 the addition
of ions,6 or the adjustment of pH.3 Despite the effectiveness of
these methods for identifying hydrogelators made of nucleosides,
a biocompatible method to generate the hydrogels would be
more beneficial for the applications of the hydrogelators of
nucleosides in a biological environment. Thus, we intend to use
an enzyme-catalysed reaction to form the hydrogels of nucleo-
sides because (i) enzyme-catalyzed hydrogelation is able to take
place in vitro and in vivo;11 (ii) the exceptional selectivity and high
efficiency offered by enzyme-catalysed reactions allow more
precise control of hydrogelation; (iii) enzymatic hydrogelation
of nucleosides is yet to be explored. Among the possible
candidates of nucleosides and enzymes, we choose adenosine2
and alkaline phosphatase (ALP)2 as the nucleoside and the
enzyme, respectively, for this investigation of the enzymatic
formation of supramolecular nanofibers/hydrogels of nucleo-
sides. Scheme 1A shows the process of catalytic dephosphory-
lation of adenosine monophosphate to form supramolecular
nanofibers and hydrogels. Adenosine is the nucleobase in
several important bioactive molecules,12 including adenosine-
50-triphosphate (ATP), a multifunctional nucleotide used in
cells as a coenzyme, and cyclic adenosine monophosphate
(cAMP), a second messenger molecule.13 ALP, a readily avail-
able hydrolase, is able to remove phosphate groups from a wide
range of substrates, including alkaloids, nucleotides, and proteins.
Thus, the use of ALP to trigger the formation of supramolecular
nanofibers/hydrogels consisting of an adenosine motif may offer
new opportunity to explore the promise of the hydrogelators of
nucleosides.
Department of Chemistry, Brandeis University, 415 South St.,
Waltham, MA 02454, USA. E-mail: bxu@brandeis.edu;
Fax: +1 781-736-2516; Tel: +1 781-736-5201
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c2cc16723a
Based on the above rationale, we made the hydrogelator (3)
and the corresponding precursor (4) by connecting adenosine
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Fig. 1 Optical images of (A) the solution of 4 (7.0 mg of 4 in 200 mL
of water at pH 7.4); (B) the gel formed by adding alkaline phosphatase
to the solution of 4; (C) the gel of 3 (4.0 mg of 3 in 200 mL of water at
pH 7.4); (D) the gel of 3 treated by adding 1 eq. poly(T)10 (2.0 wt%,
pH 7.4).
Scheme 1 (A) The illustration to show the concept of enzyme-
catalyzed formation of nanofibers and hydrogels. (B) The synthetic
route of the hydrogelator of adenosine (3) and the corresponding
precursor (4).
and adenosine monophosphate (AMP), respectively, with a small-
molecule hydrogelator naphthalene-Phe-Phe-Lys (NapFFK, 1), a
tripeptide derivative made by the conjugation of 2-(naphthalen-
2-yl)acetic acid with Phe-Phe-Lys, because 1 forms a hydrogel at a
low critical gelation concentration (0.8 wt%).14 ALP dephos-
phorylates 4 to generate 3, which has a minimum gelation
concentration of 0.8 wt%. Transmission electron microscopy
(TEM) reveals that the molecules of 3 self-assemble in water
to form long and uniform nanofibers; rheology study confirms
that the enzyme triggers the hydrogelation of 3. This preliminary
study not only opens a new way to synthesize supramolecular
nanostructures of nucleosides, but also has potential to provide
the assembly of nucleosides for a range of possible new appli-
cations.
Fig. 2 Transmission electron microscope (TEM) images, obtained by
negative staining,16 of (A) the solution of 4; (B) the gel formed by the
addition of the phosphatase; (C) the gel of 3 formed at pH 7.4; (D) the
gel of 3 treated by adding 1 eq. poly(T)10
.
Scheme 1 shows the synthetic route and structure of the
hydrogelator (3) and the precursor (4). According to our
previously reported procedure, we synthesized 1 using solid
phase peptide synthesis (SPPS).15 Nucleophilic substitution
of the chloride on 2 yields 3, which is phosphorylated at the
50-hydroxyl group of the ribose of adenosine to afford 4.
Dissolving well in water (Fig. 1A), 4 exhibits excellent stability
in water and hardly shows any dephosphorylation in the
absence of alkaline phosphatase (ALP).
strengthens the nanofiber networks that serve as the matrices of
the hydrogel.
As shown in the TEM images, the aqueous solution of the
precursor (4) hardly shows any well-formed nanofibers
(Fig. 2A). After the treatment of ALP, the formed hydrogel
contains long nanofibers with diameters of 11–17 nm and
lengths beyond several microns (Fig. 2B). Some of the nano-
fibers appear to form bundles that consist of 2–3 individual
nanofibers. Unlike the hydrogel formed by the addition of
ALP, the TEM of the hydrogel of 3 formed by the change of
pH shows low-density of nanofibers and relatively low degree
of crosslinking (Fig. 2C). Notably, the intermolecular inter-
action through the interbase binding between 3 and poly(T)10
results in a more effective cross-linked network that consists of
well-dispersed nanofibers (Fig. 2D), whose diameters range
from 12 to 24 nm. This result indicates that the use of the
single strand nucleic acids may be an effective approach for
modulating the mechanical properties of the hydrogels of
nucleosides.
After the successful synthesis of 4, we tested enzymatic
hydrogelation using 4. After dissolving 7.0 mg of 4 in
200 mL of buffer at pH = 7.4 to make a clear solution
(Fig. 1A), we added 10 units of alkaline phosphatase (ALP)
into the solution. As shown in Fig. 1B, a transparent gel forms
after the addition of ALP, indicating that the ALP converts
the precursor 4 to the desired hydrogelator 3, which self-
assembles in water to form the nanofibers and affords the
hydrogel. LC-MS also confirms the conversion of 4 to 3. We
also found that 3 can self-assemble in water and form a weak
hydrogel (Fig. 1C) at the concentration of 2.0 wt% and
pH 7.4. The addition of oligomeric nucleic acid poly(T)10 to
the hydrogel of 3 results in a new hydrogel (Fig. 1D) that
exhibits significantly increased elasticity, suggesting that the
Watson–Crick interaction between A (on 3) and T (on poly(T)10)
One characteristic property of gels is their viscoelasticity,
which reflects the mechanical properties of the hydrogels.
According to the rheological measurement, the strain sweep
and frequency sweep provide sufficient data for studying the
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Chem. Commun., 2012, 48, 2098–2100 2099

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new, biofunctional supramolecular assemblies that contribute
to the development of novel biomaterials, which certainly
deserve further exploration. In addition, this approach should
be applicable for other nucleosides, such as GMP, cAMP,
ADP or ATP, that are substrates of an array of enzymes and
carry important functions.
We are grateful for financial support from the NIH, and an
HFSP grant (RGP0056/2008). We thank the EM facility at
Brandeis University for assistance.
Notes and references
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In conclusion, we have successfully demonstrated that, with
proper molecular design, the integration of enzymatic reaction
and self-assembly provides a feasible approach to create the
molecular hydrogels of adenosine. Of all the nucleosides and
nucleotides, the recognition of adenosine 50-monophosphate
(AMP) is vital17 since nucleotide phosphates such as AMP are
important because of their role in bioenergetics, metabolism,
and transfer of genetic information. This work, thus, provides
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2100 Chem. Commun., 2012, 48, 2098–2100
This journal is The Royal Society of Chemistry 2012