Tetrafluoroaryl azide as an N-Terminal capping group for click-To-dissolve diphenylalanine hydrogels

Article abstract of DOI:10.1039/d0ra01013h

The synthesis of a bioorthogonal-responsive low molecular weight diphenylalanine (PhePhe)-based hydrogel that is capped with a 4-Azido-2,3,5,6-Tetrafluorobenzyl carbamate self-immolative linker is reported. The hydrogelator (AzF₄-PhePhe) generates a stable hydrogel at 0.1 wt%, and rapidly reacts with the bioorthogonal reagent trans-cyclooctene (TCO), inducing a gel-To-solution transition. The critical gel concentration is five-fold lower than our previously synthesized non-fluorinated hydrogelator (Az-PhePhe), and the minimum concentration of TCO required for visible gel-To-solution transition in 24 hours is 1 mM. Doxorubicin can be encapsulated in the hydrogel and TCO-Triggered dissolution results in 76% and 89% release after 10 and 24 hours, respectively. Compared with our non-substituted aryl azide capping group used for Az-PhePhe, the tetrafluorinated aryl azide group improves the stability of the hydrogel in unbuffered water at a lower critical gel concentration, while improving sensitivity towards the bioorthogonal reagent TCO.

Full text of DOI:10.1039/d0ra01013h

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Tetrafluoroaryl azide as an N-terminal capping
group for click-to-dissolve diphenylalanine
hydrogels†
Cite this: RSC Adv., 2020, 10, 9234
*
Sumit Dadhwal, Jessica M. Fairhall, Sarah Hook and Allan B. Gamble
The synthesis of a bioorthogonal-responsive low molecular weight diphenylalanine (PhePhe)-based
hydrogel that is capped with a 4-azido-2,3,5,6-tetrafluorobenzyl carbamate self-immolative linker is
reported. The hydrogelator (AzF4-PhePhe) generates a stable hydrogel at 0.1 wt%, and rapidly reacts with
the bioorthogonal reagent trans-cyclooctene (TCO), inducing a gel-to-solution transition. The critical gel
concentration is five-fold lower than our previously synthesized non-fluorinated hydrogelator (Az-
PhePhe), and the minimum concentration of TCO required for visible gel-to-solution transition in 24
hours is 1 mM. Doxorubicin can be encapsulated in the hydrogel and TCO-triggered dissolution results
in 76% and 89% release after 10 and 24 hours, respectively. Compared with our non-substituted aryl
azide capping group used for Az-PhePhe, the tetrafluorinated aryl azide group improves the stability of
the hydrogel in unbuffered water at a lower critical gel concentration, while improving sensitivity
towards the bioorthogonal reagent TCO.
Received 24th January 2020
Accepted 20th February 2020
DOI: 10.1039/d0ra01013h
rsc.li/rsc-advances
proposed that this could be due to lower electrostatic repulsion
between aromatic p-systems.7 Hsu et al.6 reported the improved
1 Introduction
mechanical properties of hydrogels via formation of thinner
entangled nanobers using the pentauorophenyl moiety as
a hydrophobic capping group on the N-terminus of the dipep-
tide. They also reported the gel-to-sol transition temperature of
the pentauorophenyl-capped hydrogel was higher (45 ꢀC) than
body temperature, making it a potential scaffold for tissue
engineering.6 In another report from the same group,10 an N-4-
uorobenzyl-capped diphenylalanine hydrogelator exhibited
a critical gel concentration at 1.5 wt%, whereas the control
hydrogelator, N-benzyl-diphenylalanine formed a viscous solu-
tion at 5 wt% with a white precipitate, indicating that the
presence of one uorine atom on the benzylic capping group
was sufficient to form electrostatic p–p interactions between
uorinated and non-uorinated aromatic rings.
Short peptidic hydrogelators have been identied for their
potential use in biomedical applications. In particular, the use
of and scope of hydrophobic capping moieties on the N-
terminus or C-terminus of the short peptides has attracted
signicant attention in the last few decades.1,2 These amphi-
philic hydrogelators can partake in non-covalent interactions3
such as electrostatic, hydrogen bonding, p–p stacking, and
hydrophobic van der Waals interactions, to form stable cross-
linked brous gel networks.1,2,4,5 The use of halogen substitu-
ents, such as uorine, on the aromatic rings of the hydrogelator
backbone and hydrophobic N-terminal capping groups have
been reported to improve the relative strengths of the hydro-
gel.6–11 Ryan et al.9 reported an instant gelation and the forma-
tion of
a rigid hydrogel using Fmoc-protected (capped)
We previously reported the synthesis and characterisation of
an N-4-azidobenzylcarbamate-diphenylalanine (Az-PhePhe) 1
hydrogel that could be triggered to undergo a gel-to-solution
(gel–sol) transition upon addition of a strained alkene; trans-
cyclooct-4-enol/TCO (Scheme 1a).12 The critical gel concentra-
tion was found to be 0.5 wt% and hydrogel encapsulated with
model drug (doxorubicin) showed controlled drug release in
response to TCO addition, demonstrating that TCO-triggered
hydrogels have potential for further development as stimuli-
responsive drug delivery systems. However, in solution, the
soluble hydrogelator 1 was found to be toxic at high mM
concentrations in tumour and normal kidney cells, indicating
that a lower critical gel concentration would be ideal for bio-
logical applications. The Az-PhePhe 1 hydrogel also displayed
pentauorophenylalanine. The improved properties of the
hydrogel were due to intermolecular p–p interactions between
the peruorinated phenylalanine ring and an Fmoc-group of
adjacent molecules.9 In another example, Liyanage and Nilsson
explored the effect of electron-donating (e.g. OH, CH3, NH2) and
electron-withdrawing (e.g. NO2, CN, F) substituents on the
phenylalanine group of Fmoc-phenylalanine on the gel self-
assembly process.7 The authors reported rapid hydrogel
formation in the presence of electron withdrawing groups, and
School of Pharmacy, University of Otago, Dunedin, 9054, New Zealand. E-mail: allan.
gamble@otago.ac.nz
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra01013h
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Scheme 1 TCO-triggered gel–sol transition of; (a) dipeptide hydrogel using hydrogelator 4-azidobenzyl carbamate-PhePhe 1 (Az-PhePhe;
previous work),12 and (b) dipeptide hydrogel using hydrogelator 4-azidotetrafluorobenzyl carbamate-PhePhe 3 (AzF4-PhePhe; this work).
a relatively slow gel-to-sol transition in the presence of 5 mM the hydrogel (albeit slowly). However, mass spectrometry
concentrations of the trigger (TCO).
experiments show that aniline 4 is relatively stable under the
Our lab and others, have demonstrated that the rates of acidic conditions of the hydrogel, and suggests that the strength
bioorthogonal reactions, such as the 1,3-dipolar cycloaddition13 of the hydrogel decreases (i.e. dissolution begins) upon forma-
and Staudinger reduction,14,15 can be increased in the presence tion of aniline 4.
of uorine substituents on an aryl azide group. In particular, the
1,3-dipolar cycloaddition between TCO and an azide is almost
2 Experimental
2.1 Materials and general experimental
an order-of-magnitude faster when uorine-substituents are
present on the aryl azide ring.13 Therefore, we have been
investigating the effect of a uorine-substituted aryl azide N-
Materials and general experimental techniques were purchased
capping group on the critical gel concentration of the dipep-
and conducted, respectively, as per our previous reports.12,13,16
tide diphenylalanine (PhePhe) and sensitivity towards the bio-
All chemicals were purchased from commercial suppliers and
orthogonal trigger (TCO).
used without purication. 5-Hydroxy-1-cyclooctene (cis-cyclooct-
Herein we report on the use of the 4-azido-2,3,5,6-
4-enol) was purchased from Carbosynth Limited, UK. Diphe-
tetrauorobenzyl carbamate as
a removable hydrophobic
nylalanine and 4-amino-2,3,4,6-tetrauoro benzoic acid were
purchased from AK Scientic, USA. Doxorubicin hydrochloride
(DOX) was purchased from Lancrix Chemicals, Shanghai,
China. All other reagents were purchased from Sigma-Aldrich or
capping group for the N-terminus of PhePhe, giving hydro-
gelator 3 (AzF4-PhePhe; Scheme 1b). The uorinated N-capping
group improves the strength of the hydrogel and its stimuli-
responsiveness towards TCO. Dissolution of the AzF4-PhePhe
hydrogel is the result of a 1,3-dipolar cycloaddition, generating
aniline 4; via intermediates 3a–3c and an aldehyde by-product
(Fig. S1†), which can undergo 1,6-self-immolation to provide
dipeptide 2 and an azaquinone methide. Evidence suggests that
self-immolation of 4 to give 2, does occur during dissolution of
1
AK Scientic. H and 13C NMR spectra were recorded on a 400
MHz Varian MR spectrometer. Chemical shis are reported as
d in parts per million (ppm) and coupling constants are re-
ported as J values in Hz. High resolution electrospray ionization
mass spectra (ESI-MS) were recorded on a microTOFQ mass
spectrometer. HPLC was performed using an Agilent 1200
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system, equipped with a Phenomenex Synergi 4 mm Fusion-RP were recorded using a MegaView 3 camera (So Imaging System
¨
80A (150 ꢁ 4.6 mm) column, and a photodiode array detector. GmbH, Munster, Germany).
The applied mobile phases used for kinetic studies were: A, aq.
H2O + 0.1% formic acid; and B, MeCN + 0.1% formic acid. Flow
speed was 1 mL minꢂ1 and injection volumes were 50 mL.
Gradient mobile phase, 80% A/20% B to 100% B in 8 minutes, 2
minutes at 100% MeCN with 0.1% formic acid and returning to
starting conditions by 15 minutes.
2.6 Scanning electron microscopy (SEM)
SEM experiments were conducted using our previously reported
standard conditions.12 The gel (0.1 wt%) sample was dried onto
aluminum stubs overnight. Aer drying, the samples were
sputter coated in an Emitech K575X Peltier-cooled high reso-
lution sputter coater (EM Technologies Ltd, Kent, England).
They were coated with 5 nm of gold palladium. Samples were
2.2 Synthesis
The synthesis of trans-cyclooct-4-enol (TCO)16,17 was conducted viewed in a JEOL JSM-6700F eld emission scanning electron
according to literature procedures. The synthesis of (4-azido- microscope (JEOL Ltd, Tokyo, Japan) at an accelerating voltage
2,3,5,6-tetrauorobenzyloxycarbonyl)-L-phenylalanyl-L-phenylal- of 5 kV.
anine 3, and intermediates 6/7/8 from benzoic acid 5 are
described in the ESI.†
2.7 Infrared spectroscopy
Attenuated Total Reectance-Fourier Transform Infrared Spec-
troscopy (ATR-FTIR) was carried out using a Varian 3100 FTIR
2.3 Hydrogel preparation
Hydrogel preparation was conducted using a similar procedure (Excalibur Series) instrument equipped with an attenuated total
to our previous report.12 The critical gel concentration (CGC) reectance accessory (GladiATR, Piketech, USA). The experi-
was tested at 0.02 wt%, 0.03 wt%, 0.04 wt% 0.05 wt% and ments were conducted under the same conditions we previously
0.1 wt%. For 0.1 wt% hydrogel, AzF4-PhePhe 3 (1 mg) was dis- reported.12 Following formation of the hydrogel from
3
solved in 50 mL DMSO followed by addition of 950 mL aqueous (0.1 wt%), the sample was lyophilised and the FTIR spectrum
phase. Aer addition of aqueous phase, the solution turned was measured (sample prior to trigger). In parallel, TCO (5 mM)
opaque, followed formation of the transparent gel. Gel forma- was added to a 0.1 wt% hydrogel from 3 (trigger experiment).
tion, aer addition of the aqueous phase, took approx. 1 min at Following an incubation period of 12 hours (at 37 ꢀC), the
ꢀ
ꢀ
37 C and approx. 5 min at 25 C.
sample was lyophilized and re-analysed by FTIR. For the time-
dependent ATR-FTIR study, hydrogel from 3 was prepared in
ꢀ
different vials using water and incubated at 37 C with 0.5 mL
2.4 Rheology
TCO (5 mM) in water. At the stated time points, the samples
were lyophilised and analysed by ATR-FTIR. For all measure-
ments, the lyophilised gel sample was clamped directly on the
ATR diamond crystal and spectra was recorded over a range of
400–4000 cmꢂ1. The spectrum was the mean of 64 scans with
a resolution of 4 cmꢂ1. Data collected was analysed using Varian
Resolution Pro, v4.1.0.101 soware.
Rheology measurements were performed as per our previous
report.12 Measurements were performed using 40 mm parallel
plate on an Oscillatory HR-3 Rheometer (TA Instruments) at
25 ꢀC. For 0.1 wt% hydrogels, AzF4-PhePhe 3 (1 mg) was dis-
solved in DMSO (50 mL) and aer adding water (950 mL) or PBS
(pH 7.4, 950 mL) the sample was loaded on the rheometer and
le undisturbed for 10 minutes to form a brous gel network.
The LVER measurements (Fig. S2†) were done with 0.1 to 100%
oscillation strain. The storage (G0) and loss (G00) moduli were
2.8 HRMS analysis of TCO-triggered hydrogel
measured using dynamic frequency sweep at a xed strain of HRMS analysis was conducted using similar assay conditions to
1.0% in the frequency range 0.1 to 100 rad sꢂ1. Rheology our previous report.12 Hydrogel generated from 3 (0.1 wt%) was
measurements were also performed at different temperatures prepared in water (5% DMSO). For the pH experiments, nal pH
(25 and 37 ꢀC). For the time dependent rheology experiment, (7.4, 6.5) was adjusted using dilute HCl and/or NaOH (measured
hydrogel (0.1 wt%) was prepared on the rheometer followed by by a pH meter S220 Seven Compact, Mettler Toledo). This was
addition of 0.5 mL water containing TCO (5 mM). Rheology was followed by addition of 0.5 mL TCO dissolved in water, and
measured at various time points in the dark.
incubation at 37 ꢀC for 12 hours (or the time indicated). The gel/
solution was lyophilised and analysed by HRMS (ESI+ and/or
ESIꢂ).
2.5 Transmission electron microscopy (TEM)
TEM experiments were conducted using our previously reported
standard conditions.12 TEM was used to investigate the
2.9 NMR mechanistic studies
morphology of 0.1 wt% gels. The sample grids were prepared by A solution of AzF4-PhePhe 3 (4.3 mg) and internal standard (1-
depositing 10 mL of gel onto plasma-glowed TEM grids (300 uoro-2,4-dinitrobenzene; 1.4 mg) were dissolved in acetoni-
mesh, carbon-coated copper grids). Excess sample was carefully trile-d3 (0.75 mL; ampoule) under N2 (nal concentration of 3
removed aer one minute using blotting paper. The grids were was 10 mM). To initiate the 1,3-dipolar cycloaddition, TCO (4.7
dried completely before viewing. They were viewed on a Philips mg) was added to the above solution (nal TCO concentration
CM100 TEM (Philips Electron Optics, Eindhoven, The Nether- of 50 mM) and the NMR spectra (1H and 19F) were recorded at
lands) operated at an accelerating voltage of 100 keV. Images various time points. Aer 48 hours, 100 mL NMR sample was
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removed for the HRMS analysis and replaced with 100 mL D2O. percentage of DOX released was calculated from a standard
Aer addition of D2O to the NMR tube, the sample was mixed curve of DOX (Fig. S3†) in water measured at 485 nm.
(shaken) and spectra was recorded.
2.12 Cell viability study
Using the assay conditions from our previous studies,12 a stock
2.10 HPLC kinetic study
solution of AzF4-PhePhe 3 (1 mg mLꢂ1) was made using DMSO
A 6 mM stock of trans-cyclooctenol in water and a 1 mM stock of
(2.5%) in 1 mL of cell media (Eagle's Minimum Essential
AzF4-PhePhe 3 in acetonitrile were prepared. The kinetics assay
Medium; EMEM), followed by sonication to dissolve
3
was conducted using the conditions of our previous report.12 To
begin the 1,3-dipolar cycloaddition, 500 mL of trans-cyclooctenol
stock was added to 500 mL of compound 3 stock. The sample
was incubated at 37 ꢀC, and at 20 min intervals, an aliquot of 50
mL was injected onto the HPLC. The absorbance for compound
3 was measured at HPLC-UV: 254 nm tR ¼ 8.1 min. For control
experiments (run in triplicate): 500 mL of AzF4-PhePhe 3 stock
was added to 500 mL of water and incubated at 37 ꢀC, an aliquot
of 50 mL was injected onto the HPLC at time intervals 0 min and
24 hours. The second order rate constant, from triplicate runs,
was calculated as 0.0947 ꢃ 0.0098 Mꢂ1 sꢂ1 (n ¼ 3).
completely. Different concentrations of 3 were prepared using
the stock solution. Madin-Darby Canine Kidney [MDCK (NBL-
2)] cells [American Type Culture Collection (ATCC®), Mana-
ssas, VA, USA] were plated at a density of 7.5 ꢁ 103 cells per well.
Cells were incubated with 3 at different concentrations for 24
and 48 hours, followed by addition of 10 mL of resazurin. Aer
addition of resazurin, MDCK cells were incubated for 4 hours at
ꢀ
37 C. Fluorescence was measured at 544 nm (excitation) and
590 nm (emission) in a PolarStar Omega plate reader. Cell
viability was normalized against cell only (100%) and cell-free
controls (0%). Data were analysed in GraphPad Prism.
2.11 Doxorubicin (DOX) encapsulation and release studies
3 Results and discussion
3.1 Synthesis
Drug release studies from the hydrogel were conducted using
slightly modied conditions to our previous report.12 Speci-
cally, a 25 mL solution of AzF4-PhePhe 3 (0.5 mg) in DMSO was
prepared. In parallel, a 1 mg mLꢂ1 stock solution of DOX in
water was prepared, and from this stock a 100 mL aliquot was
diluted in 375 mL water. The water solution containing 100 mg
mLꢂ1 (0.17 mM) of DOX was added to the DMSO solution of 3.
The gel (0.1 wt% of 3, pH 6.12) was le undisturbed for 10 min.
The gel was then washed with water (3 ꢁ 0.5 mL) to remove any
trace amounts of untrapped DOX. Following this, 1 mL of TCO
(1 mM) in water was placed over the gel, and incubated at 37 ꢀC.
The synthesis of AzF4-PhePhe 3 is outlined in Scheme 2. 4-
Azido-2,3,5,6-tetrauorobenzyl alcohol
7 was prepared as
previously reported using the commercially available acid 5 (via
6),13 followed by conversion to the activated succinate ester 8.
Activated ester 8 was reacted with diphenylalanine 2 (PhePhe) in
the presence of N,N-diisopropylethylamine (DIPEA) to provide
AzF4-PhePhe 3.
3.2 Hydrogel formulation and characterisation
For the control experiment, water-only was added (without Dissolving AzF4-PhePhe 3 in DMSO followed by addition of
TCO). At the indicated time-points 0.1 mL water was removed water (nal DMSO conc. 5%); i.e. using the solvent switch
and replaced with the same amount of water containing TCO (or method, resulted in hydrogel formation. Aer addition of water
water-only). Care was taken so as to not disturb the gel. The into the DMSO solution of 3 (nal pH of gel ¼ 3.7) an opaque
absorbance of released DOX was measured with a plate reader solution was observed, that subsequently turned trans-
(PolarStar Omega, BMG Labtech) at 485 nm. The cumulative parent,18–20 indicating self-assembled network formation
Scheme 2 Synthesis of hydrogelator 3 (AzF4-PhePhe).
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Fig. 1 (a) Hydrogel formation using AzF4-PhePhe 3 at 0.05 and 0.1 wt%, and rheology studies conducted on the hydrogel formed at (b) 0.1 wt%,
(c) 0.3 wt%, and (d) 0.5 wt%.
(Fig. 1a).18 To determine the critical hydrogel concentration inuence of the C-terminal carboxylate on the hydrogel pH. At
with 3, tube inversion at varying concentrations of 3 was con- higher temperatures, aromatic p-stacking interactions are ex-
ducted (0.01–0.5 wt%), and gel formation was identied at pected to dominate self-assembly of short hydrophobic
0.05 wt%, however, a visibly superior gel at 0.1 wt% was iden- peptides,21 providing a plausible explanation for more rapid
tied (Fig. 1a). Rheology of the gel at 0.1 wt%, 0.3 wt%, and assembly of our hydrophobic hydrogelator 3 at 37 ꢀC compared
0.5 wt% indicated that at all three concentrations, a stable to lower temperatures. The same effect by temperature on time
hydrogel was formed (Fig. 1b–d). The linear viscoelastic region for gel formation was reported on a series of Fmoc-peptide-
(LVER) of the hydrogel at 0.1 wt% indicated that up to 10% based hydrogels.22
oscillation strain could be applied without destroying the
Hydrogel formation was also attempted in PBS at 25 ꢀC.
structure of the gel (Fig. S2†). Notably, the viscoelasticity of the However, there was no opaque suspension (as in DMSO : H2O)
gel at 0.1 wt% (Fig. 1b; G0 ꢄ 100 Pa and G00 ꢄ 10 Pa) was aer addition of PBS into a DMSO solution of 3, and subsequent
equivalent to the viscoelasticity we measured for the hydrogel formation of hydrogel was not observed. Instead the addition of
formed by Az-PhePhe 1 at ve-times the concentration (0.5 wt%: PBS (pH 7.4) to the DMSO solution of 3 resulted in a transparent
G0 ꢄ 100 Pa and G00 ꢄ 10 Pa).12 Gel strength increased at 0.3 and viscous solution that contained oating bres/precipitates
0.5 wt% (G0 ꢄ 1000 Pa and G00 ꢄ 100 Pa), to ten-times the (Fig. S5;† nal hydrogel pH ¼ 3.9), supporting the rheology
viscoelasticity reported by our original hydrogel using Az- data that showed no gel formation (Fig. S6†). Attempts to form
PhePhe 1. Overall, the visual critical gel concentration and the gel in PBS at 37 ꢀC failed, providing the same viscous
rheology studies on gel formation indicate that AzF4-PhePhe 3 solution. Notably, the use of PBS during the solvent switch
can form similar strength hydrogels to the non-uorinated Az- method still resulted in an acidic hydrogel (pH ¼ 3.9), indi-
PhePhe 1 at ve-times lower concentrations (0.1 wt% cf. cating that a change in pH was not responsible for the inhibi-
0.5 wt%).12 This supports the hypothesis that the presence of tion of bril formation. The salts present in the buffer appear to
uorine-substituents in the peptide structure improves and be inhibiting bril formation to some extent in the more
strengthens hydrogel formation. For stimuli-responsive studies hydrophobic uorinated hydrogelator 3, a phenomenon not
reported herein, 0.1 wt% hydrogel was selected as this; (1) had observed for the non-uorinated hydrogelator 1.12 In support of
a comparable gel viscoelasticity to Az-PhePhe 1 (providing our results, Nilsson and co-workers described the synthesis of
a direct comparison to our previous studies), and (2) enabled us uorine-containing hydrogelators and noticed a transparent
to work at lower concentrations of hydrogelator (preferred as we viscous solution and weak hydrogel with PBS.18 The authors
move towards biocompatible hydrogels).
hypothesised that solvent plays a key role in hydrogel forma-
The rate of hydrogel self-assembly from 3 was investigated at tion, and that salts such as phosphate can shield hydrogen
different temperatures (4, 25 and 37 ꢀC). Hydrogels did not form bonding and other non-covalent interactions between brils.18
at 4 ꢀC, whereas the self-assembly process took around 5
The morphology of the hydrogel formed with 3 (0.1 wt% at
minutes at 25 ꢀC and 1 minute at 37 ꢀC. Frequency sweep pH 3.7) was investigated by transmission electron microscopy
measurements examining the viscoelastic properties of the (TEM) and scanning electron microscopy (SEM) which showed
ꢀ
ꢀ
hydrogel from 3 at 25 C and 37 C indicated that stable gels the formation of highly dense and thin bres in the hydrogel
form in both cases, with slightly higher viscoelasticity observed (Fig. S7 and S8†). Morphology of AzF4-PhePhe 3 in PBS (nal pH
for the gel at 37 ꢀC (Fig. S4†). Upon addition of the hydrogelator 3.9) was also investigated using TEM. The image showed the
3, the pH of the gel formed was acidic (pH 3.7), indicating the formation of non-entangled bres which do not form a network
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as seen with water (Fig. S9 cf. Fig. S7†), supporting the visual of the hydrogel aer addition of TCO (5 mM) as seen by the
observations outlined above. Incubating 3 at 37 ꢀC in PBS for 24 decrease in storage (G0) and loss (G00) moduli over time (Fig. 2a
hours, and subjecting the sample to electron microscopy and b). Notably, aer only 0.5 hours, the storage and loss
(Fig. S9b†), revealed the same low density bres, with no gel moduli (and thus viscoelasticity), had decreased by ꢄ10-fold.
formation over time. Therefore, as previously reported, the data Over the next 3.5 hours the G0 and G00 continued to decrease, but
suggests that phosphate salts play some role in preventing only by ꢄ5-fold. The disappearance of the azide stretch in the
hydrogel formation from 3.18
time-dependent IR studies of the 1,3-dipolar cycloaddition
supported the rheology data, with only a trace amount of azide
remaining at 0.5 hours, and no remaining azide aer 1 hour
(Fig. 2c). Taken together, the results suggest that the 1,3-dipolar
cycloaddition is fast, but that hydrolysis of the triazoline and
imine (at least in part), is required for dissolution of the
hydrogel. Based on our previous studies of the 4-azido-2,3,5,6-
tertrauorobenzyl linker,13 there is also a possibility that the
self-immolative step for the AzF4-PhePhe 3 gel will be slower
than the non-substituted Az-PhePhe 1 gel. However, the
rheology and IR data did not provide evidence into whether or
not the nal 1,6-self-immolation was required for dissolution to
begin in the AzF4-PhePhe hydrogel, thus further studies (NMR
and mass spectrometry) were conducted (see below).
High resolution mass spectral (HRMS) analysis of the gel-to-
sol transition aer 4 hours incubation at 37 ꢀC with TCO (5
mM), followed by lyophilisation, supported the IR data above,
with the absence of a molecular ion (M + H and M + Na) indi-
cating complete consumption of the AzF4-PhePhe 3 (Fig. S13†).
The major peak in the spectrum (ESI-positive) was found at m/z
556.1460 (Fig. S13†), and could be assigned to the M + Na ion of
3.3 Triggered gel-to-solution transition
To investigate the stimuli-responsive nature of the hydrogel
formed by AzF4-PhePhe 3 in water, varying concentrations of
TCO (1, 3 and 5 mM) were added to the hydrogel. Visual analysis
indicated that the most rapid and complete gel-to-sol transition
(Fig. S10†) was observed within 4 hours at 37 ꢀC when the AzF4-
PhePhe hydrogel (0.1 wt%, pH 3.7) was incubated with 5 mM of
TCO. At 3 mM and 1 mM of TCO, the gel-to-sol transition was
visually evident, but the time required for complete dissolution
increased from 4 hours to ꢄ8 and 24 hours, respectively. The
gel-to-sol transition of the AzF4-PhePhe hydrogel was faster with
TCO (5 mM) than our previously reported non-uorinated azide
hydrogel (Az-PhePhe 1),12 which was 10 hours for complete gel-
to-sol phase transition. Overall, this result indicated that a tet-
rauoro-substitution pattern on the aryl azide linker has
improved the sensitivity of the hydrogel towards TCO. TEM
analysis also supported the gel-to-sol transition upon addition
of, and incubation with TCO (5 mM), with the entangled brous
network being degraded (Fig. S11†). Infrared (IR) spectroscopy
conrmed that a 1,3-dipolar cycloaddition had transpired via
the absence of the strong asymmetric N^N stretch of the azide
group at ꢄ2100 cmꢂ1 when 5 mM TCO was added (Fig. S12†).
To relate the visual gel–sol observations to mechanism, time-
dependent rheology and IR studies on AzF4-PhePhe 3 hydrogel
during the TCO-induced gel-to-sol transition were carried out
(Fig. 2). The rheology data demonstrated the gradual weakening
aniline,
4-amino-2,3,5,6-tetrauorobenzylcarbamate-PhePhe
dipeptide 4 (Scheme 1). Mass spectral analysis (ESI-negative)
of AzF4-PhePhe hydrogel 3 incubated with TCO (5 mM) for 12
hours (lyophilised), demonstrated that self-immolation of the 4-
amino-2,3,5,6-tetrauoro linker and release of dipeptide Phe-
Phe 2 does occur over prolonged incubation; as evidenced by
the peak at m/z 311.1403 that can be assigned to PhePhe 2 [M–
Fig. 2 Time-dependent rheology (a and b) and IR studies (c) for the incubation of AzF4-PhePhe 3 hydrogel (0.1 wt%) and TCO (5 mM).
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H], and m/z 435 that can be assigned to the imine 3e (Fig. S14†) hours at 37 ꢀC, and lyophilised. The dried sample was analysed
formed by condensation of PhePhe 2 and the carbocyclic alde- by HRMS (ESI-negative) which showed the formation of various
hyde (Fig. S1†). However, the imine (aldimine and ketamine) side products (Fig. S15 and S16†). Intermediates 3b–3d were
and/or aziridine reaction intermediates 3b–3d (Fig. S1†) were identied, along with imine 3e, the product of condensation
also identied by their [M–H] molecular ion at m/z 656.254 between PhePhe 2 and the aldehyde by-product (Fig. S1;†
(Fig. S14;† all identical in mass). As expected, the tetrauoro- generated by hydrolysis of imine 3b). When compared to the
substituent pattern led to an unstable triazoline intermediate HRMS experiment conducted for 12 hours at the lower pH of 3.7
3a (Fig. S1†); not identied in the mass spectra (Fig. S13 and (Fig. S14†), the notable difference was that the aniline 4 (Mw
S14†). Therefore, the HRMS data for the AzF4-PhePhe 3 dipep- 533 g molꢂ1) was virtually absent at pH 7.4 and 6.5, suggesting
tide hydrogel formed under relatively acidic conditions (pH 3.7) that the rate of 1,6-self-immolation was faster at higher pH.
suggests that initiation of the gel–sol transition (within 4 hours There was also a peak identied at m/z 311.1424 (Fig. S15;† pH
when 5 mM TCO used) appears to depend on the conversion of 7.4) and 311.1442 (Fig. S16;† pH 6.5), assigned as the [M–H] ion
the azide functionality to an aniline, and does not require of the dipeptide (PhePhe 2). This suggests that over time, the
complete 1,6-self-immolation and subsequent release of the aniline-capping group is removed from the dipeptide, with the
free dipeptide.
most plausible route via a 1,6-self-immolation. We do however
In our previous work we demonstrated that the pKa of the note that some of the self-immolation from aniline 4 may occur
imine generated aer cycloaddition of TCO with 4-azido-2,3,5,6- during the mass spectral analysis (see NMR and HRMS study
tetrauorobenzyl linkers was lower than the non-substituted 4- below).
azidobenzyl linker,13 and the HRMS evidence (Fig. S14†) in this
As nal conrmation for the stability of aniline 4, and
time-
study supports the formation of a relatively stable imine inter- possible reduced levels of 1,6-self-immolation,
a
mediate, even at acidic pH (3.7). While acidic pH should monitored solution phase 1H (Fig. 3) and 19F NMR (Fig. 4)
promote imine hydrolysis, protonation of the subsequent study was conducted.
product aniline 4, prior to a 1,6-self-immolation, would be ex-
As AzF4-PhePhe 3 was converted to intermediates 3b–3d and
pected to slow the self-immolation process. Therefore, the aniline 4, changes around the dipeptide backbone were difficult
hydrogel of 3 was prepared at higher pH (7.4 and 6.5), to see if to analyse in the 1H NMR spectrum (Fig. 3). However, the
1,6-self-immolation could be promoted. Once the acidity/ presence of an imine proton at d 7.96 (due to aldimine 3b;
basicity of the hydrogel was adjusted to the desired pH (using structure in Fig. S1†) and the appearance of an aldehyde peak (d
HCl and NaOH), TCO (5 mM) was added and incubated for 12 9.58) aer 4 hours of reaction, were indicative of aldimine
Fig. 3 1H NMR spectra of AzF4-PhePhe 3 and TCO cycloaddition in acetonitrile-d3. Int. Std. is 1-fluoro-2,4-dinitrobenzene.
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Fig. 4 19F NMR spectra of AzF4-PhePhe 3 and TCO cycloaddition in acetonitrile-d3.
formation and subsequent hydrolysis to aniline 4. The bridging produce aniline 4, and was identied in the 19F NMR spectrum
triazoline peaks (expected around d 3.5–3.7 and 4.2–4.5)13,16 aer 48 hours incubation (d ꢂ146.6, ꢂ156.2). Also evident was
were not signicant (below signal-to-noise of the spectrum), a peak at m/z 532.1520, which could be assigned to the major
supporting the HRMS data, where the triazoline 3a was not product of the NMR reaction, aniline 4. Although not observed
identied. Analysis of the 19F NMR spectrum (Fig. 4) further in the NMR reaction mixture (Fig. 3 and 4), PhePhe 2 was
conrmed complete reaction of AzF4-PhePhe 3 with TCO aer present in the HRMS (m/z 311.1414), indicating that under the
only 1 h of incubation (loss of uorine signals at d ꢂ144.6, conditions of the mass spectrum, some of aniline 4 must be
ꢂ154.0). Spectral evidence for the triazoline 3a was missing eliminating to provide the dipeptide 2.
(expected around d ꢂ144.5 and ꢂ149 ppm).13 Instead, direct
Based on previous work in our group, the 1,6-self-
evidence for formation of aldimine 3b could be observed aer immolation was faster in an aqueous environment.13,16 There-
immediate addition of TCO (Fig. 4), lending further support to fore 100 mL of the acetonitrile-d6 solution was replaced with D2O
the instability of the triazoline intermediate 3a when a tetra- (16% nal volume). However, in this instance, no further
uoroaryl group is used as the linker. Within 1 hour, aniline 4 changes to aniline 4 were observed over 24 hours (Fig. S17†).
had started to form (d ꢂ147.9, ꢂ163.9), along with other minor Overall, the NMR studies suggest that 1,6-self-immolation of
products that include the ketamine 3c, aziridine 3d, and the aniline 4 does not occur under the partial aqueous conditions of
enamine tautomer of aldimine 3b, and by 48 hours aniline 4 the NMR experiment. While this does not directly compare to
was the major product. Conrmation of the major product 100% aqueous conditions, the results are in contrast to the non-
being aniline 4, was provided by comparison of the 19F NMR at substituted azide/aniline linker, where the 1,6-self-immolation
48 hours to the 19F NMR spectra of the expected 1,6-self- can be promoted by the addition of only a small amount of
immolation product 4-amino-2,3,5,6-tetrauorobenzyl alcohol D2O.13,16
(Fig. S17†). The 19F NMR spectral comparison (Fig. S17†) clearly
To summarise the experiments above, TCO addition (1–5
indicated that the immolation had not transpired under the mM) results in relatively fast dissolution of AzF4-PhePhe derived
NMR solvent conditions, with no indication of peaks belonging hydrogels. The mechanistic studies suggest that self-
to the 4-amino-2,3,5,6-tetrauorobenzyl alcohol.
immolation does occur in 3, but that some of the initial visual
HRMS analysis of the NMR reaction aer 48 hours in dissolution for the hydrogel of 3 could be the result of a polarity
acetonitrile-d6 (Fig. S18†) indicated a product/intermediate with change; i.e. conversion of azide 3 to aniline 4 (Scheme 1b),
a molecular weight of 657 g molꢂ1 (m/z 656.2418), that could be especially under the acidic conditions of the hydrogel in this
assigned to aziridine 3d. Aziridine 3d was not expected to study. While this study provides signicant mechanistic insight
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into the TCO-driven dissolution process, future work using the (DOX) as a model drug cargo in the brous network of Az-
4-azido-2,3,5,6-tetrauorobenzyl linker and new variations of PhePhe 1, whereby TCO was added at a 5 mM concentration
linkers, will investigate self-immolative-driven dissolution of and resulted in approximately 87% release of DOX over 10
short peptide hydrogels to polarity-driven dissolution. The use hours.12 As discussed above, our early stimuli-responsive
of amide-linked N-capping groups6,11 that are TCO-triggerable, studies with a 0.1 wt% hydrogel of AzF4-PhePhe 3 and 5 mM
but not self-immolative, will be employed.
TCO, resulted in a visual gel–sol transition within 4 hours, while
1 mM TCO resulted in visual gel–sol transition of ꢄ24 hours. As
the aim of this work was; (1) to enable formation of hydrogel at
a lower critical gel concentration, and (2) exhibit increased
sensitivity towards the bioorthogonal trigger (TCO), a hydrogel
at 0.1 wt% AzF4-PhePhe 3 encapsulating DOX and the addition
of 1 mM TCO were used to study cargo encapsulation and
release. DOX (100 mg) was encapsulated in 0.5 mL of hydrogel 3
and le undisturbed for 10 minutes. Aer addition of water
3.4 Rate of 1,3-dipolar cycloaddition between AzF4-PhePhe 3
and TCO
The product distribution and rate of the 1,3-dipolar cycloaddi-
tion and triazoline/imine hydrolysis following reaction of TCO
and 3 in an organic/aqueous solution (MeCN : H2O, 1 : 1) was
analysed using HPLC (Fig. S19†). The rate was measured by the
disappearance of AzF4-PhePhe 3 (RT ¼ 8.1 min) over time, and
was measured under pseudo rst-order conditions (Fig. S20;† 6-
fold excess of TCO). The second-order rate constant was calcu-
lated as 0.095 ꢃ 0.010 Mꢂ1 sꢂ1, a rate comparable to our
previously reported bioorthogonal prodrug activation for
tetrauoro-substituted aryl azide prodrugs in MeCN : H2O,13
and approx. ve-times faster than the rate of our previously
reported non-substituted azido-hydrogel, Az-PhePhe 1.12
ꢀ
containing TCO (1 mM), the hydrogel was incubated at 37 C
and at the indicated time points, 0.1 mL of sample was taken
and replaced with fresh 0.1 mL water aliquot containing 1 mM
of TCO (0.1 mL water without TCO for controls) and incubation
continued. The cumulative release of DOX from the hydrogel
was analysed by UV absorbance (Fig. 5) and quantied using
a DOX-standard curve (Fig. S3†). The gel incubated with 1 mM
TCO in water released 89% of DOX in 24 hours whereas control
hydrogel (without TCO) released 24% of DOX in 24 hours.
When compared to the cargo release studies from the Az-
PhePhe 1 hydrogel aer addition of TCO (5 mM), the
amount of DOX released from the AzF4-PhePhe 3 hydrogel with
ve-fold less TCO (1 mM) aer 10 hours of incubation was
76%; comparable to the 87% DOX release for 1. This suggests
that a ve-fold lower critical gel concentration and ve-fold
lower bioorthogonal trigger can be used to release a high
level of drug cargo over 10 hours, with almost complete release
at 24 hours.
3.5 Cargo release from AzF4-PhePhe 3
Next, the AzF4-PhePhe 3 hydrogel was investigated for its drug
delivery potential. We have previously reported doxorubicin
3.6 Cytotoxicity of AzF4-PhePhe 3
Thordarson and co-workers have previously shown that the
cytotoxicity of N-Fmoc-PhePhe hydrogels is inuenced by the
concentration of the dissolved (soluble) peptide in the cell
culture media.23 In a separate publication using a 3D cell culture
they also demonstrated that the capping group on the low
molecular weight hydrogel also inuences cytotoxicity,24
Fig. 5 TCO-triggered (1 mM) drug cargo release from a 0.1 wt%
hydrogel of AzF4-PhePhe 3. Absorbance of doxorubicin diffusing into
the water on top of the hydrogel was measured at 485 nm. Error bars
represent the SD for triplicate experiments (n ¼ 3).
Fig. 6 Cell cytotoxicity of AzF4-PhePhe 3 against Madin-Darby Canine Kidney (MDCK) cells. Incubation times were (a) 24 hours and (b) 48 hours,
and ꢃSD is from triplicate runs (n ¼ 3).
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indicating the importance of determining the cytotoxicity of the
new AzF4-PhePhe 3 dipeptide and comparing it to our previ-
ously reported dipeptide Az-PhePhe 1.12 In a typical experiment,
Madin-Darby Canine Kidney (MDCK) cells were exposed AzF4-
PhePhe 3 for 24 or 48 hours, and cell viability determined at
concentrations up to 1.7 mM (Fig. 6). The experiments were
conducted under standard solution phase (see experimental
section for details), therefore mimicking dipeptide that would
leach from the hydrogel in a biological setting. The cell viability
was compared to control cells (without 3). Substantial cytotox-
icity of AzF4-PhePhe 3 (cell viability # 50%) was observed at
concentrations of 1.7 mM and 0.43–1.7 mM in the 24 and 48
hour incubation experiments, respectively. As a comparison,
our reported hydrogel Az-PhePhe 1, showed substantial cyto-
toxicity to MDCK cells at a concentration of 1 mM aer 24 hours
of incubation.12 The data suggests that while the differences in
cytotoxicity between are relatively small, the uorinated dipep-
tide 3 exhibits lower cytotoxicity than the non-uorinated
dipeptide 1.
Conflicts of interest
There are no conicts of interest to declare.
Acknowledgements
This research was supported in part by a contract from the
Health Research Council of New Zealand (A. B. G.). We thank
the Department of Chemistry at the University of Otago for use
of the NMR and HRMS instruments, and the Otago Micro and
Nanoscale Imaging for use of their facilities.
Notes and references
1 S. Fleming and R. V. Ulijn, Chem. Soc. Rev., 2014, 43, 8150–
8177.
2 X. Du, J. Zhou, J. Shi and B. Xu, Chem. Rev., 2015, 115, 13165–
13307.
3 M. J. Webber, E. A. Appel, E. W. Meijer and R. Langer, Nat.
Mater., 2016, 15, 13–26.
4 J. Raeburn, A. Zamith Cardoso and D. J. Adams, Chem. Soc.
Rev., 2013, 42, 5143–5156.
4 Conclusion
In summary, the use of a self-immolative 4-azido-2,3,5,6-
tetrauorobenzyl carbamate linker to protect (cap) the N-
terminus of a diphenylalanine dipeptide 2 and generate
a stimuli-responsive hydrogel is reported. The capped dipeptide
(AzF4-PhePhe 3) resulted in a hydrogel that formed a stable
brous network in water, but not in phosphate buffered saline
(PBS). In water (nal pH of 3.7), 3 formed a weak hydrogel at
0.05 wt%, and a strong hydrogel, as evidenced by its viscoelastic
properties, at 0.1 wt% of 3 (considered the critical gel concen-
5 J. Hoque, N. Sangaj and S. Varghese, Macromol. Biosci., 2019,
19, 1800259, DOI: 10.1002/mabi.201800259.
6 S.-M. Hsu, Y.-C. Lin, J.-W. Chang, Y.-H. Liu and H.-C. Lin,
Angew. Chem., Int. Ed., 2014, 53, 1921–1927.
7 W. Liyanage and B. L. Nilsson, Langmuir, 2016, 32, 787–799.
8 D. M. Ryan, S. B. Anderson and B. L. Nilsson, So Matter,
2010, 6, 3220–3231.
9 D. M. Ryan, S. B. Anderson, F. T. Senguen, R. E. Youngman
and B. L. Nilsson, So Matter, 2010, 6, 475–479.
tration). The absence of a hydrogel in PBS (nal pH 3.9) was 10 F.-Y. Wu, S.-M. Hsu, H. Cheng, L.-H. Hsu and H.-C. Lin, New
likely due to disruption of H-bonding and other interactions by J. Chem., 2015, 39, 4240–4243.
the phosphate salts, as reported by others.18 Reaction of the 11 S.-M. Hsu, J.-W. Cheng, F.-Y. Wu, Y.-C. Lin, T.-S. Lai,
AzF4-PhePhe 3 hydrogel with TCO resulted in dissolution of the H. Cheng and H.-C. Lin, RSC Adv., 2015, 5, 32431–32434.
hydrogel in 4–24 hours. At the lowest concentration tested 12 S. Dadhwal, J. M. Fairhall, S. K. Goswami, S. Hook and
(1 mM TCO), the complete visual dissolution took 24 hours. A. B. Gamble, Chem.–Asian J., 2019, 14, 1143–1150.
Encapsulation of doxorubicin (DOX) as a model drug cargo was 13 S. S. Matikonda, J. M. Fairhall, F. Fiedler, S. Sanhajariya,
conducted at 0.1 wt% of 3, and upon addition of TCO (1 mM)
dissolution resulted in 76% and 89% release of DOX aer 10
R. A. J. Tucker, S. Hook, A. L. Garden and A. B. Gamble,
Bioconjugate Chem., 2018, 29, 324–334.
¨
and 24 hours, respectively. The rate of the 1,3-dipolar cycload- 14 M. Sundhoro, S. Jeon, J. Park, O. Ramstrom and M. Yan,
dition between an AzF4-PhePhe 3 and TCO was determined Angew. Chem., Int. Ed., 2017, 56, 12117–12121.
under pseudo rst-order conditions in solution phase, and the 15 Y. Xie, L. Cheng, Y. Gao, X. Cai, X. Yang, L. Yi and Z. Xi,
second-order rate constant calculated as 0.095 ꢃ 0.010 Mꢂ1 sꢂ1
,
Chem.–Asian J., 2018, 13, 1791–1796.
16 S. S. Matikonda, D. L. Orsi, V. Staudacher, I. A. Jenkins,
F. Fiedler, J. Chen and A. B. Gamble, Chem. Sci., 2015, 6,
1212–1218.
ve-times faster than the reaction of TCO with Az-PhePhe 1.12
The ability to formulate AzF4-PhePhe 3 as a stable hydrogel at
0.1 wt%, and its improved sensitivity and rate of reaction with
the bioorthogonal trigger (TCO), indicates that the use of uo- 17 M. Royzen, G. P. A. Yap and J. M. Fox, J. Am. Chem. Soc., 2008,
rine in related peptide-based hydrogels could lead to drug 130, 3760–3761.
delivery systems with in vivo potential. Other endogenous trig- 18 D. M. Ryan, T. M. Doran, S. B. Anderson and B. L. Nilsson,
gers of azides, for example hydrogen sulde, explored by others Langmuir, 2011, 27, 4029–4039.
on short peptidic hydrogels,25 would also provide an alternate 19 D. M. Ryan, T. M. Doran and B. L. Nilsson, Langmuir, 2011,
strategy for dissolution of hydrogels with uorinated aryl azide- 27, 11145–11156.
capping groups. Peptidic and polymeric hydrogels incorpo- 20 D. M. Ryan, T. M. Doran and B. L. Nilsson, Chem. Commun.,
rating the tetrauoroaryl azide and other azide-functionalised 2011, 47, 475–477.
self-immolative linkers, and their activation mechanisms are 21 S. Debnath, S. Roy, Y. M. Abul-Haija, P. W. J. M. Frederix,
of current interest to our research group.
S. M. Ramalhete, A. R. Hirst, N. Javid, N. T. Hunt,
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Paper
S. M. Kelly, J. Angulo, Y. Z. Khimyak and R. V. Ulijn, Chem.– 24 J. P. Wojciechowski, A. D. Martin, A. F. Mason, C. M. Fife,
Eur. J., 2019, 25, 7881–7887.
S. M. Sagnella, M. Kavallaris and P. Thordarson,
22 R. Orbach, I. Mironi-Harpaz, L. Adler-Abramovich,
ChemPlusChem, 2017, 82, 383–389.
E. Mossou, E. P. Mitchell, V. T. Forsyth, E. Gazit and 25 R. Peltier, G. Chen, H. Lei, M. Zhang, L. Gao, S. S. Lee,
D. Seliktar, Langmuir, 2012, 28, 2015–2022.
23 W. T. Truong, Y. Su, D. Gloria, F. Braet and P. Thordarson,
Biomater. Sci., 2015, 3, 298–307.
Z. Wang and H. Sun, Chem. Commun., 2015, 51, 17273–
17276.
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