Lipase catalyzed inclusion of gastrodigenin for the evolution of blue light emitting peptide nanofibers

Article abstract of DOI:10.1039/c4cc02484b

We report lipase catalysed regioselective inclusion of gastrodigenin (p-hydroxybenzyl alcohol: HBA) to a peptide Nmoc-Leu-Trp-OH at physiological pH 7.4. The resultant Nmoc-Leu-Trp-HBA from a reaction cycle of dissipative self-assembly evolves into blue light emitting peptide nanofibers.

Full text of DOI:10.1039/c4cc02484b

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Lipase catalyzed inclusion of gastrodigenin for the
evolution of blue light emitting peptide
nanofibers†
Cite this: Chem. Commun., 2014,
50, 8685
Received 4th April 2014,
Accepted 13th June 2014
Dnyaneshwar B. Rasale, Indrajit Maity and Apurba K. Das*
DOI: 10.1039/c4cc02484b
www.rsc.org/chemcomm
We report lipase catalysed regioselective inclusion of gastrodigenin The enzyme lipase is known to hydrolyze carboxylic acid ester9 in
(p-hydroxybenzyl alcohol: HBA) to a peptide Nmoc-Leu-Trp-OH at aqueous medium but it also undergoes esterification10 or trans-
physiological pH 7.4. The resultant Nmoc-Leu-Trp-HBA from a esterification11 reactions in organic solvents. The simple visual
reaction cycle of dissipative self-assembly evolves into blue light colorimetric assay that responds to enzymatic reactions is the area
emitting peptide nanofibers.
of biosensing applications.12 Enzymatic conjugation of fluorophores
with enhanced emission properties in aqueous medium is more
Self-assembly1 is a spontaneous process in which a number of promising for bioassay and imaging applications.13
components form an organized structure. Inspired from natural
In this paper, we report lipase from Candida rugosa (CRL)
systems, several synthetic systems such as low molecular weight catalyzed incorporation of gastrodigenin p-hydroxybenzyl alco-
organic molecules have been adapted to develop nanoscale hol (HBA) in Nmoc-protected peptides (Nmoc = naphthalene-2-
architectures.2 In general, these self-assembling systems remain methoxy carbonyl) followed by evolution of a dissipative blue
in a local thermodynamic minimal state due to minimization of light emitting hydrogel (Fig. 1). A very recent study shows the
energy or continuous influx of chemical energy. To date, various use of HBA as a promising candidate for the establishment of
self-assembled architectures have been reported which are under anti-angiogenic treatment strategies in cancer therapy.14
thermodynamic equilibrium.3 Recently, there has been growing Herein, we exploit CRL for the inclusion of HBA regioselectively
interest to develop dissipative self-assembly (DSA) systems,
which have potential to adapt themselves. These DSA systems
consist of non-assembling entities, which can form different
nanostructures depending on external stimuli through activa-
tion of an energy source. Despite a constant source of energy,
the non-equilibrium counterpart of the DSA system leads to
dis-assembly due to deactivation of the building blocks caused
by energy dissipation. So far, few examples of dissipative self-
assembly4 have been reported. van Esch et al. reported an
excellent example of dissipative self-assembly of a molecular
gelator by using a chemical fuel.5 Herein, we have used the
enzyme lipase as an energy source to meet the requirement of
dissipative self-assembly.
Ajayaghosh et al. exploited controlled donor self-assembly and
energy transfer to form a white light emitting organogel.6 However,
biocatalytic evolution of blue light emitting biomaterials7 in
aqueous media is not yet reported in the literature. Xu and Ulijn
are the pioneers of enzyme-catalysed peptide self-assembly.8
Fig. 1 Schematic representation of lipase (CRL) catalysed regioselective
inclusion of HBA to Nmoc-peptides in aqueous medium. Lipase is used
as a source of energy for the reaction cycle of the dissipative system.
The corresponding ester evolves into a blue light emitting material upon
the self-assembly process at pH 7.4.
Department of Chemistry, Indian Institute of Technology Indore, Indore 452017,
India. E-mail: apurba.das@iiti.ac.in
† Electronic supplementary information (ESI) available: Synthesis and experi-
mental procedures. See DOI: 10.1039/c4cc02484b
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in the Nmoc based peptides via ester bond formation. Supra- were unable to form self-supporting hydrogels (Table S1, ESI†).
molecular chemistry presents the ability to drive the chemical Moreover, the HPLC analysis showed poor conversion of Nmoc-
processes reversibly through covalent and non-covalent inter- LY-HBA (12%) and Nmoc-YW-HBA (20%) (Fig. S2 and S3, ESI†). In
actions that result in more stable entities under the pressure of general, lipase favours ester hydrolysis reactions instead of ester-
self-organization.15 Keeping this idea in mind, we forced ification reactions in aqueous medium. This may be the reason
reverse ester hydrolysis of Nmoc-dipeptide with HBA using for the poor yield of esterification reactions of HBA with non-
CRL in aqueous medium at physiological pH 7.4.
assemblingpeptides Nmoc-LY 2 and Nmoc-YW 3. The exceptional
We have successfully synthesized three dipeptides bearing activity and higher yield by CRL towards regioselective incorpora-
an N-terminal hydrophobic aromatic naphthalene-2-methoxy tion of HBA into Nmoc-LW 1 in aqueous medium is driven by
carbonyl moiety. The peptide Nmoc-LW 1 (20 mmol Lꢀ1, 20.04 mg) supramolecular ordering of peptides, which is influenced by
(L: leucine, W = tryptophan) and p-hydroxybenzyl alcohol (HBA, hydrogen bonding, p–p stacking and hydrophobic interactions
80 mmol Lꢀ1, 19.84 mg) were dissolved in 2 mL of water at higher ofpeptides. The synthesizedNmoc-LW-HBA leads todis-assembly
pH by adding 0.5 M sodium hydroxide. The pH was adjusted to 7.4 upon addition of OHꢀ. The increased concentration of OHꢀ
by adding 0.1 M hydrochloric acid followed by 0.5 mg mLꢀ1 CRL. hydrolysed Nmoc-LW-HBA to its parent Nmoc-LW peptide. The
The resulting colorless solution was incubated at 37 1C for 24 h. dissipation of energy deactivated the building blocks to its
The HPLC analysis (Fig. S1, ESI†) showed 24% conversion of dis-assembled state despite a constant source of energy which is
Nmoc-LW-HBA ester at 24 h upon enzymatic activation. However, the enzyme lipase herein. We have synthesized Nmoc-LW-HBA by
the colorless solution turned light pink, which later turned into a a chemical method to study the self-assembly behaviour in
dark pink self-supporting hydrogel when the conversion reached aqueous medium. The chemical incorporation of HBA to Nmoc-
57% after 8 days (Fig. 2B). The self-assembly of small peptides peptides is quite difficult due to the presence of two hydroxyl
bearing N-terminal hydrophobic moieties has been reported by groups on HBA which leads to the mixture of two products
several groups.16 The HPLC kinetics showed that the initial rate of (Fig. S4, ESI†). Moreover, chemical incorporation of HBA with
esterification was faster which was relatively slow after 8 days of Nmoc-protected dipeptide having hydrophobic amino acids gives
enzyme reaction.
direct esterified products which are poorly soluble in aqueous
The 91% conversion was observed after 28 days of enzyme medium (Fig. S5, ESI†). The chemically synthesized Nmoc-LW-
reaction. This activated product self-assembles to form a self- HBA was unable to self-assemble in aqueous medium. Hence, the
supporting hydrogel at pH 7.4. Two other dipeptides Nmoc-LY 2 biocatalytic method is more promising and highly selective. The
(L:leucine, Y =tyrosine)andNmoc-YW3 (Y: tyrosine, W = tryptophan) CRL isan ideal biocatalystthatallows regioselectiveincorporation
of HBA to the Nmoc-LW 1 peptide in aqueous medium. The
benzylic hydroxyl group of HBA is linked to the carboxylic acid
of Nmoc-LW 1 via an esterification reaction upon treatment
with CRL. The CRL catalyzed regioselective ester product was
confirmed by 1H NMR, which is further supported by ESI-MS and
HPLC (Fig. 2C, Fig. S1 and S6, ESI†).
The flow behaviour and rigidity of the self-supporting hydro-
gel formed by Nmoc-LW-HBA ester was investigated by its
rheological properties (Fig. S7 and S8, ESI†). To evaluate the
storage modulus (G0) and loss modulus (G00), a typical frequency
sweep experiment was carried out. The frequency sweep data
show that G0 is higher than G00, which is an indication of the
viscoelastic nature of the hydrogel. The viscoelastic nature of
hydrogel 1 can be ascribed to the supramolecular ordering of
peptide molecules in aqueous medium.
The supramolecular ordering of peptides is driven by var-
ious non-covalent interactions including hydrogen bonding
and p–p stacking interactions.17 Circular dichroism spectra of
the solution of Nmoc-LW 1 and HBA (Fig. 4D) and the corre-
sponding gel Nmoc-LW-HBA help to evaluate the supramolecular
ordering of hydrogel 1. The CD spectrum of the Nmoc-LW-HBA
ester hydrogel shows a negative band near 193 nm and a positive
band near 216 nm which are attributed to p - p* and n - p*
transition of the CONH groups of the peptide backbone (Fig. 4D).
Fig. 2 (A) HPLC trace analysis of enzyme catalyzed esterification of
Nmoc-LW 1 to Nmoc-LW-HBA with its corresponding ESI-MS. (B) Real
time HPLC analysis for the formation of Nmoc-LW-HBA. (C) Partial
1H NMR of Nmoc-LW 1 and its corresponding dried Nmoc-LW-HBA
The intense negative peak at 193 nm indicates the existence of a
turn-type b-sheet conformation in the hydrogel state.18 The UV-vis
and fluorescence studies were performed to evaluate the supra-
molecular ordering of the self-assembled hydrogel. The UV-vis
hydrogel synthesized via
DMSO-d6.
a CRL catalyzed esterification reaction in
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Fig. 3 (A) Optical images of hydrogel Nmoc-LW-HBA 1 under daylight
and under UV light (365 nm). (B) Normalized emission spectra of solution
of Nmoc-LW 1 with HBA (lex = 280 nm) and the hydrogel formed by
Nmoc-LW-HBA (lex = 365 nm) and (C) the fluorescence microscopy
image of gel at 2 mmol Lꢀ1 concentration.
Fig. 4 (A) UV-vis absorption spectrum of hydrogel 1 at 2 mmol Lꢀ1
concentration. (B) Emission decay curves of solution of 1 and HBA and
the corresponding self-assembled hydrogel 1 monitored at 455 nm (IRF:
instrument response function). (C) AFM image of Nmoc-LW-HBA hydrogel
indicating dense nanofibrillar networks in the gel phase medium and
(D) CD spectra of a solution of Nmoc-LW 1 with HBA and the hydrogel
of Nmoc-LW-HBA.
spectra of HBA and Nmoc-LW 1 in aqueous solutions were
recorded at 2 mmol Lꢀ1 concentration. Both the solutions
exhibit UV-vis absorption spectra in the range of 250 to
300 nm (Fig. S9, ESI†). However, the UV-vis absorption spectrum Upon excitation at 365 nm, the hydrogel gives strong emission
of Nmoc-LW-HBA exhibits a broad peak at 372 nm along with at 470 nm (Fig. 3B). The solution of Nmoc-LW and HBA gives
two peaks near 220–300 nm (Fig. 4A). The absorption peak at weak emission in the range of 400–500 nm upon excitation at
372 nm for hydrogel Nmoc-LW-HBA was further confirmed by 365 nm (Fig. S13, ESI†). This happens due to strong p–p
fluorescence excitation spectra at two different emission wave- stacking interactions of the aromatic fluorophores in the gel
lengths (Fig. S10 and S11, ESI†). The higher red shift in the phase. The observed significant red shift of emission maxima is
UV-vis spectrum is due to the self-organization of gelator mole- attributed to the strong supramolecular ordering of peptide
cules in the hydrogel state. The self-supporting Nmoc-LW-HBA molecules in the gel phase. Moreover, the emission peak
hydrogel emits blue fluorescence upon exposure to UV light appearing at 455–470 nm corresponds to blue light in the
(illuminated at 365 nm) (Fig. 3A).
visible region of the electromagnetic spectrum.
The self-assembly of Nmoc-LW-HBA was observed upon
The time resolved fluorescence study was acquired using our
gradual conversion of Nmoc-LW 1 to Nmoc-LW-HBA via a time correlated single photon counting (TCSPC) setup (Fig. 4B).
CRL catalyzed esterification reaction. The self-assembled dark The decay curve of the Nmoc-LW-HBA hydrogel was recorded
pink hydrogel under day light showed bright blue fluorescence to investigate the higher order aggregates in the gel phase
under UV light which is further examined by steady state medium (Table S2, ESI†). The average lifetime of the peptide
fluorescence spectroscopy (Fig. 3B). The self-assembled hydrogel was exhibited as 1.39 ns with lifetime components of
Nmoc-LW-HBA peptide is composed of three fluorophore 0.84 ns (81%) and 3.77 ns (19%). However, the average lifetime
moieties. In general, the assembled naphthalene double ring of the solution of Nmoc-LW and HBA is 1.25 ns, which shows
emits at 320 nm to 350 nm upon excitation at 280 nm.19 fast biexponential decay with lifetime components of 0.90 ns
However, the tryptophan moiety emits in the range of 300 nm (90%) and 4.44 ns (10%). Such difference in decay lifetime is
to 350 nm, which depends upon the microenvironments. In evidence of non-radiative energy transfer20 in solution. How-
this case, emission spectra were recorded upon excitation at ever, such processes are hampered in the self-assembled hydro-
280 nm based on UV-vis absorption spectra. The emission gel state due to the rigid and close molecular packing through
maxima exhibit at 350 nm for the solution of Nmoc-LW 1 with hydrogen bonding and p–p stacking interactions, which results
HBA, a characteristic peak of tryptophan and naphthalene in a higher lifetime.
chromophores. To further confirm the individual emission of
Transmission electron microscopy (TEM), scanning electron
HBA and Nmoc-LW 1 solutions, emission spectra were recorded microscopy (SEM) and atomic force microscopy (AFM) were
upon excitation at 280 nm. HBA gives emission at 305 nm used to reveal the nanostructural morphology of the blue light
whereas Nmoc-LW 1 exhibits emission at 354 nm (Fig. S12, emitting self-assembled hydrogel. As shown in the AFM image
ESI†). However, upon excitation at 280 nm, the self-assembled (Fig. 4C), the hydrogel has an interwoven nanofibrous network
Nmoc-LW-HBA hydrogel shows a strong emission at 455 nm. with an average height of 7 nm. The transmission electron
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networks with an average diameter of 90 nm (Fig. S14, ESI†).
SEM images reveal morphological transformation of the Nmoc-
LW-HBA hydrogel at different time scales (Fig. S15, ESI†). SEM
images of the isolated products Nmoc-YW-HBA and Nmoc-LY-HBA
show entangled nanofibers and long fibrillar structures respec-
tively (Fig. S16, ESI†). The microscopic studies demonstrate the
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self-assembled nanofibers (Fig. 3C). The fluorescence microscopy
image reveals that the fluorescent hydrogel is composed of blue
light emitting entangled self-assembled nanofibers.21
In summary, a blue light emitting hydrogel upon bicatalytic
incorporation of gastrodigenin (p-hydroxybenzyl alcohol, HBA)
to an Nmoc-protected dipeptide has been discovered. Here, the
enzyme lipase is used as a source of energy to exploit dissipative
self-assembly. The resultant ester formation of a reaction cycle of
dissipative self-assembly is an ideal example to achieve complex
nanostructures in their self-assembled state. Besides, the lipase
propensity towards the hydrolysis of ester bonds in aqueous
medium, its catalytic activity is exploited in the development of
fluorescent peptide nanostructures via an esterification reaction.
The blue light emitting hydrogel could be an ideal biomaterial
for bioimaging and bioanalysis of various cell processes.
AKD sincerely acknowledges CSIR, New Delhi, India, for
financial support. DR and IM are indebted to CSIR, New Delhi,
India, for their fellowships. We thank SAIF, NEHU, Shillong, for
the assistance of EM facility.
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8688 | Chem. Commun., 2014, 50, 8685--8688
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