Divergent Supramolecular Gelation of Backbone Modified Short Hybrid δ-Peptides

Article abstract of DOI:10.1021/acs.biomac.8b01684

The ordered supramolecular assemblies of short peptides have been recently gaining momentum due to their widespread applications in biology and materials sciences. In contrast to the α-peptides, limited success has been achieved from the backbone modified

Full text of DOI:10.1021/acs.biomac.8b01684

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Article
Divergent Supramolecular Gelation of
Backbone Modified Short Hybrid #-Peptides
Rahi M. Reja, Rajat Patel, Vivek Kumar, Anjali Jha, and Hosahudya N Gopi
Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 12 Feb 2019
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Divergent Supramolecular Gelation of Backbone
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Modified Short Hybrid δ-Peptides
Rahi M. Reja†, Rajat Patel†, Vivek Kumar†, Anjali Jha§, Hosahudya N. Gopi*†
§Nanobioscience Group, Agharkar Research Institute, G. G. Agarkar Road, Pune-411004, India
†Department of Chemistry, Indian Institute of Science Education and Research, Dr.HomiBhabha
Road, Pune-411008, India E-mail: hn.gopi@iiserpune.ac.in
ABSTRACT
The ordered supramolecular assemblies of short peptides have been recently gaining momentum
due to their wide spread applications in biology and materials sciences. In contrast to the α-
peptides, limited success has been achieved from the backbone modified peptides. The proteolytic
stability and conformational flexibility of the backbone modified peptides composed of -,- and
δ-amino acids can be explored to design ordered supramolecular gels and self-assembled materials.
In this article, we are reporting the divergent supramolecular gels from a new class of short hybrid
dipeptides composed of conformationally flexible new β(O)-δ5-amino acids. The hybrid dipeptide
composed of β3- and β(O)-δ5-Phe showed the formation of transparent gels from the aromatic
solvents, while the dipeptide composed of β(O)-δ5-Phe showed the thixotropic gel in phosphate
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buffered saline (PBS). In contrast, no organic or hydrogels were observed from the dipeptides
composed of alternating α- and β(O)-δ5-Phe as well as 4 and β(O)-δ5-Phe. The organogelation
property displayed by the β3,(O)-δ5-Phe dipeptide was further explored to recover the oil spills
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from the oil-water mixture. The thixotropic hydrogels displayed by the (O)-δ5,(O)-δ5-Phe
dipeptide was further utilized as matrix along with cell culture medium to grow the cells in 2D-
cell culture. Replacing the backbone -CH2- with “O” in the δ-Phe leads to the drastic change in
the supramolecular behavior of δ-peptides. Overall, the short dipeptides from different backbone
modified amino acids showed the divergent gelation properties and these properties can be further
explored to design new functional biomaterials.
KEYWORDS: δ-Peptides, organo and hydrogels, stimuli responsive, thixotropic, oil-spill
recovery, 2D-cell culture, proteolytic stability.
INTRODUCTION
The central theme in the fields of nanoscience and nanotechnology is the formation of highly
ordered self-assembled materials in nanoscale.1 The fascinating properties displayed by the
nanostructures have attracted considerable attention in recent years due to their widespread
applications in various fields including energytechnologies, catalysis, biomedicaland chemical
biology.2-5 In addition to the numerous types of nano-assemblies derived from the small organic
molecules, the biomacromolecules such as lipids, proteins, carbohydrates and nucleic acids, often
self-assembled into highly ordered supramolecular architectures.6-9 The hierarchical
supramolecular structures of biomacromolecules are stabilized by various types of non-covalent
interactions such as H-bonding, salt bridge interactions, van der Waals interactions and aromatic
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pi-stacking.10 Along with the biomacromolecules, shortpeptides have displayed a rich
supramolecular diversity and have been serving as excellent tools to design various types of self-
assembled biomaterials.11-13 In fact, the finding of self-assembled peptide nanotube from a Phe-
Phe dipeptide by the Gazit and colleagues has opened new avenues in the generation of soft
materials from the short peptides.14 The dipeptide nanotubes have been explored as templates to
cast silver nanowires, as drug delivery agents, light harvesting systems, energy storage materials
and antimicrobial candidates etc.15-18 In addition, the self-assembling properties of peptides have
been finding applications in the fields of tissue engineering as well as nanoelectronics.19, 20Along
with the -peptides, efforts have also been made in the literature to understand the molecular self-
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assemblies of β- and -peptides.21,22 A variety of supramolecular architectures such as tapes, fibers,
ribbons, polyhedrons, nanotubes and vesicles have also been derived from the supramolecular
assemblies of β- and -peptides.23-25Recently, Malhotra et al. demonstrated the antimicrobial
properties of self-assembled α,-hybrid peptides.26 The advantage of β- and –peptides over the α-
peptides is that they are proteolytically stable and required short sequences to attain folded
structures.27
In addition to the biomaterials, the low molecular weight gelators (LMWG) have also attracted
considerable attention due to their applications in oil spill recovery,28,29 toxicity remediation
devices,30,31 electro-optical displays,32 drug delivery systems,33,34 tissue engineering,35 wound
healing36 etc. The groups of Gazit,19 Uljin,38 Xu,39Adams40 and others41,42 have examined the
potential of aromatic-aromatic interactions in the design of different organo and hydrogels from
the short peptide sequences. Further, Banerjee and colleagues showed the influence of fatty acids
on the supramolecular hydrogelation of Phe-Phe dipeptides.28,33 We have been interested in
understanding the conformational and self-assembling properties of peptides composed of various
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types of non-natural β- and -amino acids.43,44 In comparison to β- and -amino acids, structural
and self-assembling properties of peptides composed of higher homologous amino acids such as
δ-amino acids are scarcely examined in the literature. Nevertheless, Hoffman and colleagues
investigated the possible conformational space available to the peptides composed of δ-amino
acids using theoretical calculations.45 Apart from the theoretical calculations, the groups of Huc,46
Sharma47 and Chakraborty48 have examined the structural properties of peptides and foldamers
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Scheme1: Sequences of the dipeptides under investigations.
containing various types of δ-amino acids. Recently, we have shown the superior and spontaneous
supramolecular gelation properties from γ4Phe-γ4Phe dipeptide over the β3Phe-β3Phe and Phe-Phe
dipeptides.49As γ4Phe-γ4Phe showed excellent gelation over the β- and -dipeptide counterparts,
we have hypothesized that δ5Phe-δ5Phe dipeptide motifs can be served as better hydrogelators than
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the -dipeptide counterpart. More importantly, as the length of the δ5-amino acids is equal to the
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length of an -dipeptide, they can be used as surrogates of α-dipeptides to overcome the proteolytic
cleavage. In this context, we have synthesized peptide P1 and examined its hydrogelation
properties in phosphate saline buffer. In sharp contrast to 4Phe-4Phe, the new peptide δ5Phe-
δ5Phe failed to give hydrogels in PBS and aqueous buffers. In order to increase the solubility and
conformational flexibility of the peptides composed of δ5-Phe we considered to introduce
heteroatom oxygen (O) into the backbone of δ5-Phe. We sought to systematically investigate the
gelation properties of the hybrid peptides composed of Xxx-β(O)-δ5-Phe, where Xxx is α, β3, 4 or
β(O)-δ5-Phe (Scheme 1). Here, we report the formation of the stimuli responsive, injectable,
biocompatible hydrogels from the Boc-β(O)-δ5-Phe-β(O)-δ5-Phe dipeptide, its utility in 2D-cell
culture and organogels from Boc-β3Phe-β(O)-δ5-Phe dipeptide and its potential application in oil
spill recovery. Under identical conditions, both Boc-αPhe-β(O)-δ5-Phe and Boc-4Phe-β(O)-δ5-
Phe did not give either organic or hydrogels.
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EXPERIMENTAL SECTION
Materials
All chemical and reagents including amino acids were purchased from commercial sources.
Solvents ethyl acetate and petroleum ether (60−80 °C) were distilled before to use. Solvent
tetrahydrofuran (THF) was dried by reflexing on sodium metal wire and distilled before to use.
Silica gel 120−200 mesh was used to purify the protected amino acids and peptides in column
1
chromatography. The H NMR spectra of protected amino acids and peptides were recorded on
400 MHz (or 13C on 100 MHz) in the solvent deuterated chloroform (CDCl3). The acid labile Boc
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and alkyl esters (ethyl or methyl esters) were used for the N- and C-terminus protections,
respectively. The Boc-(S)-β3-Phe was synthesized starting from Boc-Phe by the reported
protocol.50 The Boc-(R)-γ4-Phe was synthesized starting from Boc-(S)-Phe aldehyde through
Witting reaction followed by the reduction of double bonds using H2, Pd/C (10%). Finally, ethyl
ester of Boc-4-Phe was subjected to saponification as reported earlier.43 The β(O)-δ5-Phe was
synthesized starting from Boc-protected amino alcohol using reported protocol.51 The carbon
analogous of β(O)-δ5-Phe was synthesized from Boc-(S)-β3-Phe. All peptide couplings reactions
were achieved using the coupling reagent N-ethyl-N′-(3-(dimethylaminopropyl)carbodiimide
hydrochloride (EDC.HCl) and 1-hydroxybenzotriazole (HOBt) and N,N-diisopropylethylamine
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(DIEA). Finally, the pure peptides were characterized by H and C NMR spectroscopy and
authenticated by high resolution mass spectroscopy (HRMS).
General Procedure for the Solution Phase Peptides (P1-P5) Synthesis:
All peptide (P1-P5) were synthesized through solution phase peptide chemistry as reported
earlier.49 In brief, 1 mmol of N-Boc-protected-amino acid was dissolved in 5 mL of DMF and the
solution was to cooled to 0 °C. Subsequently, 1mmol of 1EDC·HCl and 1 mmol of HOBt and 2
mmol of DIEA were added reaction at 0 °C. Immediately, 1.1 mmol of methyl ester of amino
acid to the reaction mixture and reaction was allowed to come to the room temperature. The
reaction mixture was stirred for about 12 h. The reaction progress was monitored using TLC. After
completion, 50 mL of ethyl acetate was added to the reaction mixture followed by 50 mL of
saturated sodium chloride solution. The ethyl acetate layer was separated and the aqueous layer
was extracted again with ethyl acetate (50 mL  3). The combined ethyl acetate layer was washed
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with 10% Na2CO3 (20 mL  3), 5% HCl (20 mL  3 ), water (20 mL  3), finally with saturated
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sodium chloride solution (30 mL  2). The solution was dried over anhydrous sodium sulfate and
evaporated under vacuum to obtain crude peptides. These crude peptides were purified using
column chromatography with EtOAc/hexane solvent system. Finally, peptides were subjected to
saponification in methanol solution using 1N NaOH. The peptide acid obtained after saponification
were directly used gelation studies.
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Scheme 2: Synthesis of N-Boc-δ5-Phe-OEt form N-Boc-3Phe-OH
Scheme3: Synthesis of N-Boc-(O)-δ5-Phe-OtBu form N-Boc amino alcohol.
Synthesis of N-Boc-δ5-Phe-OH
N-Boc-δ5-Phe-OH was synthesized starting from N-Boc-(S)-β3-Phe-OH. Briefly, N-Boc-(S)-β3-
Phe-Weinreb amide (1.6 g, 5 mmol) was converted to aldehyde through the reduction of Weinreb
amide using LAH (228 mg, 6 mmol) and subjected Wittig reaction to get N-Boc-α,-unsaturated-
δ5-Phe-OEt. The Wittig product was purified using column chromatography by EtOAc/hexane
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solvent system. After that, the N-Boc-α,-unsaturated-δ5-Phe-OEt was reduced using H2/Pd-C to
get N-Boc-δ5-Phe-OEt. Further, the N-Boc-δ5-Phe-OEt was subjected saponification using 1N
NaOH in EtOH to obtain N-Boc-δ5Phe-OH and directly used for the peptides synthesis without
purification.
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Synthesis of N-Boc-(O)-δ5-Phe-OtBu:
The N-Boc-β(O)-δ-Phe was synthesized as reported earlier.51 Briefly, tetra- butylammonium
hydrogensulfate (340 mg, 1 mmol) was dissolved in solution of sodium hydroxide (10 g, 250 mmol
in 10 mL of water and 15 mL of toluene). To this biphasic reaction mixture, tert-butyl
bromoacetate (2.2 mL, 15 mmol) was added slowly at room temperature. The reaction mixture
was cooled to 0 °C, followed by slow addition of N-Boc-phenyl alanine alcohol (2.5 g, 10 mmol)
in toluene (15 mL). The reaction was allowed to come to room temperature and the stirring was
continued for about 3 h. The organic phase was separated from the aqueous phase. The aqueous
solution was again extracted with diethyl ether (30 mL 2). The combined organic layer was
washed with saturated sodium chloride solution (30 mL  2), dried over anhydrous sodium sulfate
and evaporated under reduced pressure. The crude product of N-Boc-(O)-δ5-Phe-OtBu was
purified by column chromatography using EtOAc/hexane solvent system.
Gelation Study of Peptides P1, P2 and P4
Peptides P1 and P4 (10 mg each) were placed in the separate sample vials. To each vial of peptides,
1 mL of toluene was added. The peptide solution in toluene was heated on a hot air gun to dissolve
the peptides. The clear solution of peptides in toluene was cooled to room temperature and
sonicated for about 10 min. The organo gelation property of peptide P1 and P4 was confirmed by
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the inverted sample vial experiment. For the hydrogelation, 10 mg of peptide P2 was dissolved in
1 mL of 1× PBS buffer at pH 7.4 and gently heated on a hot air gun to make it a clear solution and
cooled to room temperature. Upon standing for about 12 h, the solution slowly transforms to gel
state and the gelation property was further confirmed by the inverted sample vial experiment.
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Morphology Study of Peptide P1, P2 and P4
FE-SEM experiment was carried out by drop casting 5L of gel on SiO2/Si substrate. The samples
were dried at room temperature under vacuum and the samples on SiO2/Si substrate were again
coated with gold. The images were taken using scanning electron microscope using tungsten
filament as electron source operated at 10 kV.
Rheology Experiment
The rheology experiments were carried out on preformed gel samples of organogel (in toluene)
and hydrogel (in 1× PBS) at a concentration of 10 mg/mL using rheometer. All experiments were
carried out single time.
Oil Spill Recovery Experiment
Peptide P4 shows phase selective gelation in hydrocarbon solvent. In a typical experiment the
solution of peptide P4 (30 mg in 3 mL) in toluene was added to the oil-water mixture composed
of 10 mL of oil and 30 mL of saturated NaCl solution. To mimic the sea water saturated NaCl
solution was used. Instructively, upon treatment of the peptide solution the oil transformed into
gels in less than 5 min. The peptide-oil gel was scooped out from the oil/water mixture and
subjected to vacuum distillation and the oil was recovered from the oil water mixture.
Proteolytic Stability of Peptide P2
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Proteolytic stability of peptide P2 was carried out using reported protocol.37 The peptide P2 (1
mM) was dissolved in 1 mL of HEPES buffer (pH 7.4) containing 5% DMSO. Then, 10L of 5M
chymotrypsin enzyme was added to the peptide solution and incubated at 37 ˚C for about 24 h.
After that, 50 L from the above mentioned stock solution was injected in RP-HPLC. The HPLC
was carried out using ACN/H2O at flow rate of 1 mL/min. Same procedure was followed to
understand the proteolytic stability of peptide Boc-Phe-Phe-OH.
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Effect of Hydrogel from Peptide P2 on Cell Viability
Cells were cultured in complete growth media (DMEM/F12) with 10% (v/v) (fetal bovine serum)
FBS and 100 U/mL penicillin in humidified 5% (v/v) CO2 at 37 °C. Gel was prepared by diluting
peptideP2 dissolved in PBS to 10-fold using DMEM cell culture medium. This was coated on the
96-well cell culture plate and incubated further for about 3 h. Cells suspension in complete growth
medium was pipetted into each well (pre-coated with gel) at a count of 10,000 cells/well. The plate
was further incubated for 18 h at 37 °C under 5% of CO2. After 18 h of incubation complete growth
media was removed and cells were washed with PBS. Cell viability assay was carried out after 18
h using calcein staining. Calcein AM stain at 2 µM final concentration was added to each cells and
incubated for 45 min and then images were taken at 10 with fluorescence microscope.
Experiments were performed as four independent replicates. The data represented in bar graph are
further analyzed. Significance values indicated by * for p < 0.05, ns ~ non-significant for p > 0.05;
in comparison to the respective control cell samples.
RESULT AND DISCUSSION
Gelation Study
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The sequences of δ- and hybrid δ-peptides are given in the Scheme 1. The β3- and 4-Phe were
synthesized as reported earlier.50,43 The δ5-Phe was synthesized starting from the Boc-β3-Phe as
shown in the Scheme 2. The new β(O)-δ5-Phe was synthesized starting from β-amino alcohol as
shown in the Scheme 3. To understand their ordered self-aggregation properties, the purified
peptide P1 was subjected to the gelation studies in 1 phosphate buffer saline (1PBS) at pH=7.4
as well as in aromatic organic solvents. Peptide P1 failed to give hydrogels in aqueous buffer
however it gelates various aromatic organic solvents. The organogels of P1 in various aromatic
solvents are shown in the Figure 1. In contrast, peptide P2 gave stable gels in 1 PBS at pH 7.4 at
the concentration of 10 mg/mL dissolved upon gently heating and standing for about 12 h. Inspired
by the spontaneous hydrogelation of P2, we have systematically probed the gelation properties of
hybrid dipeptides consisting Xxx-β(O)-δ5-Phe sequences (P3-P5, Scheme 1). Among the all
hybrid peptides, P4 has shown excellent gelation property in a variety of aromatic solvents such
as toluene, benzene, xylene and mesitylene at the concentration of 10 mg/mL. Other peptides (P3
and P5) were found to be either insoluble or not inducing the gelation in aromatic organic solvents.
All hybrid peptides (P3, P4 and P5) were found to be insoluble in 1 PBS and failed to give
hydrogels. The gels derived from the peptides P1, P2 and P4 were found to be external stimuli
responsive. The transition of gel to sol can be achieved by either mechanical forces or by increasing
the temperature. In contrast to P3 and P5, the hybrid peptide P4 consisting of β- and β(O)-δ5-Phe
may be reached the threshold of hydrophobicity and hydrophilicity which probably responsible for
the gelation of aromatic organic solvents.28 Being side-chains were constant among all the
peptides, the backbone conformational flexibility is probably responsible for the gelation
properties of the peptides.28 Further, FT-IR spectroscopy was used to study the conformation of
peptide P1, P2 and P4 in the xerogel state. The C=O stretching (amide I) and NH bending (amide
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II) frequencies around ~1650 cm-1 and ~1700 cm-1 respectively suggest the β-sheet character of
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peptides in the gel state (See ESI).33
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Morphology Study
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To understand supramolecular assembly formed by the peptides P1, P2 and P4, the FE-SEM was
carried out. As both P3 and P5 did not show any gelation properties, we have not subjected them
for the morphology studies. Though the peptides P1, P2 and P4 displayed the fibrillar morphology
however they are different from one another. Peptide P1 and P4 displayed densely packed fibrillar
network compared to peptide P2.
Figure 1: Examination for the formation of gel in (A) Peptide P1 (conc. 10 mg/mL) in toluene (B)
Peptide P2 (conc. 10 mg/mL) in 1 PBS at pH 7.4. (C) Peptide P3 (conc. 10 mg/mL) in toluene.
(D) Peptide P4 (conc. 10 mg/mL) in toluene. (E) Peptide P5 (conc. 10 mg/mL) in toluene. (F) FE-
SEM images of the organogel of peptide P4 in toluene. (G) FE-SEM images of the hydrogel of
peptide P2 in 1 PBS.
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To gain insight into the viscoelasticity and self-healing properties of organogels of P1 and P4 and
hydrogels of P2, we carried out the rheological measurements. These results are shown in Figure
2. The larger value of the storage modulus (G') compared to the loss modulus (G'') in the strain
sweep experiment at constant angular frequency, signifying the elastic nature of both organogels
(P1 and P4) and hydrogels (P2). The constant values of G' and G'' in a frequency sweep
experiments further support the stable organic and hydrogels from these peptides.
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Figure 2: (A) Strain sweep rheological analysis of P1 organogel in toluene (10 mg/mL) at
constant angular frequency (1 rad/sec). (B) Strain sweep rheological analysis of the P2 hydrogel
in 1  PBS (10 mg/mL) at constant angular frequency (1 rad/Sec). (C) Strain sweep rheological
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analysis of P4 organogel in toluene (10 mg/mL) at constant angular frequency (1 rad/Sec). (D)
Step-strain rheology experiment of peptide P2 hydrogel (10 mg/mL) at constant frequency (1
rad/Sec).
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Stimuli Responsive Properties of Organo and Hydrogels
We further noticed that the organogels of the peptides P1 and P4 and hydrogel of peptide P2 were
found to be external stimuli responsive. The transition from gel to sol can be achieved using either
Figure 3: Stimuli responsive nature of the (A) organogel of peptide P4 and (B) hydrogel of peptide
P2. (C) Injectable nature of the hydrogel from peptide P2. (D) Slow release of the dye from
hydrogel matrix to the 1× PBS over about 18 h.
heat or mechanical shaking. Upon cooling and resting, the sol state slowly transformed into the
gel state. By applying the mechanical strain, the hydrogel was transformed to sol and after
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removing the strain the sol slowly transformed again into the gel state. This thixotropic nature of
the gels was further supported by the rheology experiments (Figure 2D). As these types of
injectable hydrogels have been finding applications in the drug delivery, we have examined the
potential of P2 hydrogel as a drug delivery system. We encapsulated 500µL of 1mM proflavine
solution in the gel matrix and 500µL of 1 PBS was added on the top of the hydrogel matrix. The
slow release of the drug was monitored using UV-Vis spectroscopy. The release kinetics suggested
that ~35% of the drug was released from gel to the buffer in 18 h (See ESI).
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Oil Spill Recovery by Organogel from Peptide P4
Inspired by the gelation of peptide P4 in various aromatic solvents, we sought to investigate
whether it can be used to selectively gelate the crude oil from the oil-water mixture. The large
scale separation of oil from oil-water mixture has been one of the big challenges in worldwide due
Figure 4: Phase selective gelation in oil/water mixture and oil spill recovery.
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to the increasing release of oil contaminated waste water and marine oil spills to the seas which
severely damage the marine eco-system.52-53 There are numerous methods available for the oil-
spill recovery, however peptide based amphiphiles attracted considerable attention in recent years
in oil spill recovery.54 Recently Basak et al. have demonstrated the oil spill recovery using
ethanolic solution of small peptides composed of aromatic amino acids.28 The schematic process
of oil spill recovery by the peptide P4 is shown in the Figure 4. To understand the selective gelation
of crude oil, the solution of peptide P4 (30 mg in 3 mL) in toluene was added to the oil-water
mixture composed of 10 mL of oil and 30 mL of saturated NaCl solution. Instructively, upon
treatment of the peptide solution, the oil transformed into gel in less than 5 min. The peptide-oil
gel was scooped out from the oil/water mixture and subjected for vacuum distillation. Through this
process we have successfully recovered the oil from the oil-salt water mixture. Overall, the self-
assembling nature of the dipeptide P4 was found to be an excellent alternative for recovering the
oil in oil spills.
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Biocompatibility Studies of the Peptide P2 Hydrogel:
As many thixotropic peptides based hydrogels have been using as a cell culture matrix, we sought
to examine the biocompatibility of the peptide P2 hydrogel and its utility as matrix in the 2D cell
culture. To understand the biocompatibility, peptide gel was mixed with DMEM media at a ratio
of 1:10 and the cells were grown over the gel matrix. We have examined biocompatibility of the
P2 hydrogel using both cancer cell line LN229 and normal cell line HEK293T. After 18h, the live
cells were stained with calcein AM and imaged using fluorescence microscope. The difference in
the growth of the cells over the gel matrix compared to the control experiment without peptide gel
are shown in the Figure 5. These results suggest that hydrogel from δ-peptideis biocompatible and
this type of peptides can be used as matrix along with the culture media. As the δ-amino acid can
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serve as a dipeptide mimetic, we have examined the proteolytic stability of peptide P2 along with
the control -dipeptide Boc-Phe-Phe-OH using the protease chymotrypsin. These results are
shown in Figure 6. In contrast to -dipeptide, the HPLC analysis suggests that peptide P2 was
found to be stable to chymotrypsin.
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Figure 5: Growth of the cells over gel matrix after 18 h. (A) and (C) are the growth of the cells
over gel matrix. (B) and (D) are the control experiment without any hydrogel. The viable cells
are stained with calcein AM stain. (Scale bar for all images are 400 m)
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Figure 6: HPLC analysis depicting the stability of peptides (A) P2 and (B) control peptide Boc-
Phe-Phe-OH against the protease chymotrypsin.
CONCLUSION
In conclusion, we have demonstrated the formation of self-assembled divergent gels from a new
class of short peptides consisting of δ-Phe. These δ-amino acids may serve as surrogates of -
dipeptide. The dipeptide (P1) composed of backbone homologated δ5-Phe was found to be
insoluble in aqueous buffers, however gave stable gels in aromatic organic solvents. In contrast,
the dipeptide composed (O)-δ5-Phe showed remarkable, transparent, thixotropic hydrogels from
the phosphate saline. Replacing backbone –CH2- with “O” atom showed phenomenal change in
their supramolecular gelation properties. The subtle change in the backbone leads to a drastic
change in the molecular behavior of these peptides. Among the hybrid dipeptidesP3, P4 and P5,
surprisingly only peptide P4 (β3/(O)-δ5) gave organogels in aromatic solvents. Further, we have
demonstrated the utility of β3/(O)-δ5-hybrid peptide P4 in the oil spill recovery through a phase
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selective gelation process. In addition, we have shown the potential applications of the dipeptide
P2 hydrogel as drug delivery system as well as matrix to grow the cells. Both P2 and P4 were
found to be sensitive to the external heat as well as mechanical shaking. The biocompatibility,
proteolytic stability and thixotropic nature of the new δ-peptide may serve as potential alternatives
to the existing peptide gels and it can be used as injectable and drug delivery agents. Overall, the
study reported here can open new avenues for the design of biomaterials, injectable gels and drug
delivery agents.
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ASSOCIATED CONTENT
Supporting Information
Details of the peptides and amino acids characterization, gelation property, 1H, 13C NMR and
HRMS spectra are available in the supporting information.
AUTHOR INFORMATION
Corresponding Author
*Dr. Hosahudya N. Gopi, Department of Chemistry, Indian Institute of Science Education and
Research, Dr. Homi Bhabha Road, Pune-411008. Email: hn.gopi@iiserpune.ac.in
Funding Sources
Science and Engineering Research Board (SERB), Department of Science and Technology, Govt.
of India (EMR/2014/ 000606)
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ACKNOWLEDGMENT
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R. M. R is thankful to IISER Pune for research fellowship. R. P and V. K are thankful to DST-
India for the Inspire Fellowship. A.J thanks Inspire Faculty award (DST-India). H. N. G thanks
SERB (EMR/2014/ 000606), Department of Science and Technology, Govt. of India for the
financial support.
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ABBREVIATIONS
TLC, thin layer chromatography; PBS, phosphate-buffered saline; FE-SEM, field emission
scanning electron microscope; NMR, nuclear magnetic resonance.
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Divergent Supramolecular Gelation of Backbone
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Modified Short Hybrid δ-Peptides
Rahi M. Reja†, Rajat Patel†, Vivek Kumar†, Anjali Jha§, Hosahudya N. Gopi*†
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§Nanobioscience Group, Agharkar Research Institute, G. G. Agarkar Road, Pune-411004, India
†Department of Chemistry, Indian Institute of Science Education and Research, Dr. Homi
Bhabha Road, Pune-411008, India E-mail: hn.gopi@iiserpune.ac.in
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