Visible Light Activation of Nucleophilic Thiol-X Addition via Thioether Bimane Photocleavage for Polymer Cross-Linking

Article abstract of DOI:10.1021/acs.biomac.8b01153

On-demand photo-uncaging of reactive thiols have been employed in engineering biomaterial scaffolds for regulation of cellular activities. A drawback of the current photo-uncaging chemistry is the utilization of high energy UV light or 2-photon laser light, which may be harmful to cells and cause undesired side reactions within the biological environment. We introduce an effective approach for the caging of thiol using monobromobimane, which can be removed under irradiation of light at = 420 nm and in the presence of electrophiles, such as acrylate, propiolate and maleimide, for trapping of the newly release thiol. This chemical approach can be used in visible light-induced polymer coupling and cross-linking for the preparation of cell-laden hydrogels.

Full text of DOI:10.1021/acs.biomac.8b01153

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Article
Visible Light Activation of Nucleophilic Thiol-X Addition via
Thioether Bimane Photocleavage for Polymer Crosslinking
Vinh X. Truong, Fanyi Li, and John S Forsythe
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01153 • Publication Date (Web): 18 Sep 2018
Downloaded from http://pubs.acs.org on September 19, 2018
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Visible Light Activation of Nucleophilic ThiolꢀX
Addition via Thioether Bimane Photocleavage for
Polymer Crosslinking
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Vinh X. Truong,* † Fanyi Li, †‡ and John S. Forsythe †.
AUTHOR ADDRESS.
†Department of Materials Science and Engineering, Monash Institute of Medical Engineering,
Monash University, Clayton, 3800 VIC, Australia.).
‡CSIRO Manufacturing, Bayview Avenue, Clayton, VIC 3168, Australia
KEYWORDS. Bioorthogonal, Michael addition, photoꢀcrosslinking, visible light, nucleophilic
thiolꢀX, hydrogel, polymer coupling.
ABSTRACT. Onꢀdemand photoꢀuncaging of reactive thiols have been employed in engineering
biomaterial scaffolds for regulation of cellular activities. A drawback of the current photoꢀ
uncaging chemistry is the utilization of high energy UV light or 2ꢀphoton laser light, which may
be harmful to cells and cause undesired side reactions within the biological environment. We
introduce an effective approach for the caging of thiol using monobromobimane, which can be
removed under irradiation of light at λ = 420 nm and in the presence of electrophiles, such as
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acrylate, propiolate and maleimide, for trapping of the newly release thiol. This chemical
approach can be used in visible lightꢀinduced polymer coupling and crosslinking for the
preparation of cellꢀladen hydrogels.
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Introduction
Photochemistry has emerged as a powerful tool for polymer modifications which are
indispensable in advanced functional materials and biomedicine.1ꢀ4 Light can endow spatial and
temporal control in applications such as surface patterning, engineering 3D biomaterials
structures, and onꢀdemand drug release.1 Photoꢀinduced free radical polymerisation is perhaps
the most popular chemical approach towards construction of biomaterial scaffolds with
spatiotemporal regulation over the chemical and physical properties.4 However, the generated
free radicals may react with biological components such as cells and tissues,5 causing irreversible
damage. Although employment of photoꢀinduced thiolꢀene reaction has allowed for
encapsulation of cells6ꢀ8 and proteins9 without affecting their bioactivity, photoꢀmodulation of
gel properties still requires the generation of reactive free radicals, which can have significant
effect on radicalꢀsensitive cells.10
To circumvent the generation of free radicals, photoremovable protecting groups (PPG) have
been recently employed for photoꢀrelease of clickable functionalities in lightꢀtriggered polymer
crosslinking.2 For example, the photocleavage of oꢀnitrobenzyl (oꢀNB) derivatives was used to
generate aldehyde,11ꢀ13 ketone14 or alkoxyamine14,
for oxime or imine ligation in the
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construction of bioorthogonal hydrogels. The oꢀNB group has also been used in photoꢀuncaging
of reactive thiol which can participate in subsequent Michael addition for modification of
hydrogel microstructures.16ꢀ18 Caging of the thiol group prevents its oxidation to disulfide under
air exposure, thus increasing the storage of the thiolꢀcontaining polymers as well as the
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efficiency of polymer ligation, via thiolꢀnucleophilic addition, prior to uncaging.1 However,
deprotection of oꢀNB thioether produces nitrosobenzaldehyde which can also react with the free
thiols or amines within biological environments.
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Figure 1. Comparison of PPGs used in photoꢀuncaging of thiols for engineering hydrogel
structures.
The group of Shoichet utilised 6ꢀbromoꢀ7ꢀhydroxycoumarin (BrꢀCmr, Figure 1) to mask thiol
groups within an agarose gel which, upon irradiation of either UV light or twoꢀphoton near
infrared light, exposed free thiol.19ꢀ21 Hence immobilisation of different growth factors in a
spatial and temporal manner could be achieved, via thiolꢀmaleimide addition, for guiding the
differentiation of encapsulated stem cells. Similar to oꢀNB photoꢀuncaging, UV light irradiation
of BrꢀCmr group leads to photoisomerization sideꢀproduct that can react with the newly released
thiol, thus reducing the efficiency of the subsequent thiolꢀnucleophilic addition. To circumvent
the photoisomerization of BrꢀCmr, Shoichet and coꢀworkers recently employed
nitrodibenzofuran (NDBF) for concealment of active thiols within hyaluronic hydrogels.22
Nevertheless, removal of the aforementioned PPGs can only by activated by UV light or twoꢀ
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photon nearꢀinfrared light,23 which may be damaging to cells and tissues at high dosage of
light.24ꢀ26
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For a PPG to be expedient in biomaterials engineering, it should fulfil several requirements2
including: (i) negligible toxicity of the photoprotecting group and released byꢀproducts; (ii) high
uncaging efficiency with minimal side reactions of the photoꢀreleased species under irradiation
of wavelength ≥ 365 nm; and (iii) adequate solubility and stability in biological media. Ideally
the photolabile precursor should also (iv) be easily synthesised and conjugated to the polymer
structure. Taking all these prerequisites into consideration, we selected bimane chromophore as
the PPG to mask the thiol group on polymer chains. Herein we report an efficient method for
caging the thiol group using bromobimane. The photoꢀactivated thiol can then be utilised in
visible lightꢀcontrolled polymer coupling, via nucleophilic thiol addition to various electrophiles
including acrylate, propiolate and maleimide. In turn, we demonstrate the catalystꢀfree visible
lightꢀtriggered crosslinking of poly(ethylene glycol) suitable for the fabrication of cellꢀladen
hydrogels.
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Experimental Section
General Considerations
Materials. Solvents (CH2Cl2, diethyl ether, petroleum ether, acetone, tetrahydrofurane, and
methanol) were purchased from VWR in HPLC grade. 4ꢀarm PEG10kꢀOH was purchased from
Jenkem Tech, USA. 4ꢀarm PEG10kꢀNH2 was synthesised from 4ꢀarm PEG10kꢀOH according to
a previously published procedure with the degree of conversion from ꢀOH to ꢀNH2 group of
96%.27 Tetramethylrhodamine (TRITC) conjugated streptavidin was purchased from Thermo
Fisher Scientific. All other chemicals were purchased from SigmaꢀAldrich and used as received.
All synthesized compounds were stored in a freezer at ꢀ20 °C and covered with aluminium foil.
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Glassware for synthesis was first treated in a base bath (KOH in isopropanol 2 M) overnight,
rinsed with water, submerged in an acid bath (HCl 1 M) for 2ꢀ4 h, rinsed with reverse osmosis
water and dried in an oven at 120 °C for 6ꢀ12 h.
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Chromatography. Thinꢀlayer chromatography (TLC) was performed on silica gel 60 F254
alumina sheets (Merck) and visualized by UV light or potassium permanganate solution. Column
chromatography was run on silica gel 60 (0.04ꢀ0.06 mm, 230ꢀ400 mesh ASTM, Merck).
NMR spectra were recorded on a Bruker Avance III 400 MHz or 600 MHz with a 5 mm
broadband autoꢀtunable probe with Zꢀgradients at 293 K. Chemical shifts are reported as δ in
parts per million (ppm) and referenced to the chemical shift of the residual solvent resonances
(CDCl3 δ = 7.26 ppm), couplings are shown as s: singlet, d: doublet, t: triplet, m: multiplet.
Polymer samples were prepared at a concentration of 20 mg mLꢀ1. In most spectra traces of water
appears as a broad singlet at around 1.5ꢀ2.5 ppm. NMR spectra were processed using
MestReNova software.
Synthesis Procedures
Synthesis
of
3ꢀ(bromomethyl)ꢀ2,5,6ꢀtrimethylꢀ1H,7Hꢀpyrazolo[1,2ꢀa]pyrazoleꢀ1,7ꢀdione
(monobromo bimane). A solution of hydrazine hydrate (6 mL, 0.16 mol) in ethanol (60 mL) was
added dropwise to a solution of ethyl methylacetoacetate (17 mL, 0.12 mol) under stirring at
ambient temperature. The solution was stirred for 2 h and concentrated in vacuo to give product
as white solid (yield: 10.5 g, 78%).
The above product was suspended in chloroform (500 mL) and Cl2 was bubbled through the
solution via a glass pipette at a rate at which the solution was gently refluxed at ambient
temperature. The white solid quickly dissolved into the solution and the mixture turned yellow
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after 2 h. The solution was concentrated in vacuo to give yellow oil product which was used
directly in the next step.
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The above product was dissolved in CH2Cl2 (100 mL) and K2CO3 (17 g, 0.12 mol) was added.
The solution was cooled on an ice bath and water (2 mL) was added slowly. The solution was
then stirred at ambient temperature for 24 h, filter, and washed with saturated NH4Cl solution.
The organic phase was dried (MgSO4) and concentrated in vacuo to give product as pale yellow
solid which is mostly synꢀbimane as indicated by 1H NMR (total yield: 12 g, ca. 52%).
The above product (2 g, ca. 10.4 mmol) was dissolved in CH2Cl2 (20 mL) and cooled to 0 °C,
a solution of Br2 (0.5 mL, 9.72 mmol) in CH2Cl2 (40 mL) was added dropwise over 1 h. The
solution was stirred at ambient temperature for 2 h and washed with water (100 mL), dried
(MgSO4) and concentrated in vacuo. The residue was purified by column chromatography
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running on ethyl acetate to give product as yellow solid (yield: 1.21 g, 45.9%). H NMR (δ,
ppm): 4.25 (s), 2.38 (s), 1.84 (s), 1.79 (s); 13C NMR (δ, ppm): 6.8, 11.4, 17.5, 113, 115, 142, 144,
157, 158.
Synthesis of 4ꢀarm PEGꢀSH (1). 4ꢀarm PEGꢀOH (5 g, 0.5 mmol), mercaptopropionic acid
(2.16 g, 20 mmol) and paraꢀtoluenesulfonic acid (10 mg, catalytic amount) were added to a
solution of cyclohexane (100 mL) in a 250 mL round bottom flask. The solution was heated at
100 °C under DeanꢀStark conditions for 16 h. The solution was cooled to ambient temperature
and concentrated in vacuo. The residue was dissolved in dichloromethane (50 mL), washed with
saturated NaHCO3 solution (50 mL), water (50 mL), brine (50 mL), dried (MgSO4), concentrated
to ca. 5 mL and precipitated by dropwise addition in diethyl ether (200 mL). The polymer
product was collected as white powder (yield: 4.6 g, 88.5%). The degree of endꢀgroup
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functionalization, calculated based on integration of NMR chemical shifts, was 99%. H NMR
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(δ, ppm): 4.24ꢀ4.26 (t, J = 4.8 Hz), 3.61 (broad s), 3.57 (s), 2.75ꢀ2.77 (q, J = 8.4, 7.3 Hz), 2.69 (t,
= 7 Hz), Mn = 14300 g molꢀ1, Ð = 1.13.
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Synthesis of 4ꢀarm PEGꢀSꢀbimane (2). 4ꢀarm PEGꢀSH (1 g, 0.1 mmol) and monobromobimane
(120 mg, 0.48 mmol) were dissolved in CH2Cl2, NEt3 (48 mg, 0.48 mmol) was added and the
solution was stirred at ambient temperature for 5 h. It was then precipitated into Et2O to give
product as pale yellow solid (yield: 0.96 g, ca. 95%). The degree of functionalization was 100%.
1H NMR (δ, ppm): 4.25ꢀ4.27 (t, J = 4.8 Hz), 3.63 (broad s), 3.4 (s), 2.83ꢀ2.86 (t, J = 7.1 Hz),
2.67ꢀ2.68 (t, J = 7.1 Hz), 2.39 (s), 1.9 (s), 1.83 (s); Mn = 15100 g molꢀ1, Ð = 1.21.
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Synthesis of 4ꢀarm PEGꢀacrylate (4a). 4ꢀarm PEGꢀOH (1 g, 0.1 mmol) and NEt3 (80 mg, 0.8
mmol) were dissolved in CH2Cl2 (20 mL) and the solution was cooled on an ice bath. Acryloyl
chloride (72.4 mg, 0.8 mmol) in CH2Cl2 (5 mL) was added dropwise. The solution was allowed
to warm to ambient temperature and stirred for 6 h after that it was washed with water (50 mL
x2), brine (50 mL), dried (MgSO4), and concentrated in vacuo to ca. 2 mL and precipitated by
dropwise addition in diethyl ether (100 mL) to give product as white solid (yield: 0.88 g, ca.
88%). The degree of endꢀgroup functionalization was 99%. 1H NMR (δ, ppm): 6.40ꢀ6.44 (dd, J =
1.4, 15.9), 6.11ꢀ6.18 (dd, J = 10.5 Hz, 6.6 Hz), 5.82ꢀ5.85 (dd, J = 1.4 Hz, 8.8 Hz), 4.30ꢀ4.31 (t, J
= 4.8 Hz), 3.72 (broad s), 3.66 (s). Mn = 13300 g molꢀ1, Ð = 1.07.
Synthesis of 4ꢀarm PEGꢀpropiolate. 4ꢀarm PEGꢀOH (2 g, 0.2 mmol), propiolic acid (0.14 g, 2
mmol), pꢀtoluene sulfonic acid (10 mg, catalytic amount) were added to a solution of
cyclohexane (100 mL) in a 250 mL round bottom flask. The solution was heated at 100 °C under
DeanꢀStark conditions for 16 h. The solution was cooled to ambient temperature and
concentrated in vacuo. The residue was dissolved in dichloromethane (50 mL), washed with
saturated NaHCO3 solution (50 mL), water (50 mL), brine (50 mL), dried (MgSO4), concentrated
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to ca. 2 mL and precipitated by dropwise addition in diethyl ether (100 mL). The polymer
product was collected as white powder (yield: 1.7 g, ca. 85%). The degree of endꢀgroup
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functionalization was 99%. H NMR (δ, ppm): 4.34ꢀ4.36 (t, J = 4.8 Hz), 3.66 (bs), 3.65 (s), 3.0
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(s). Mn = 13900 g molꢀ1, Ð = 1.09.
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Synthesis of 2,5ꢀdioxopyrrolidinꢀ1ꢀyl 3ꢀ(2,5ꢀdioxoꢀ2,5ꢀdihydroꢀ1Hꢀpyrrolꢀ1ꢀyl)propanoate (Nꢀ
hydroxysuccinimide maleimide). Glycine (1.5 g, 20 mmol) and maleic anhydride (2.35 g, 2
mmol) were dissolved in acetic acid (50 mL) and the solution was stirred at 130 °C for 5 h. The
solution was then allowed to cool to ambient temperature and poured into water (200 mL),
extracted with EtOAC (100 mL x 3), dried (MgSO4), and concentrated in vacuo to give product
as white crystal.
The above product was redissolved in CH2Cl2 (50 mL) and to the solution was added
EDC.HCl (3.82 g, 20 mmol) and Nꢀhydroxysuccinimide (2.26 g, 20 mmol). The solution was
stirred at ambient temperature for 2 h, washed with water (50 mL), brine (50 mL), dried
(MgSO4), concentrated and purified by column chromatography eluting with EtOAC/CH2Cl2
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(v/v = 1/4) to give product as white crystal (total yield: 4.2 g, 79%). H NMR (δ, ppm): 6.73 (s),
3.93 (t, J = 7.0 Hz), 3.03 (t, J = 7.0 Hz), 2.82 (bs).
Synthesis of 4ꢀarm PEGꢀmaleimide. Nꢀhydroxysuccinimide maleimide (109 mg, 0.41 mmol)
and 4ꢀarm PEG10kꢀNH2 (1 g, 0.1 mmol) were dissolved in CH2Cl2 (10 mL) and the solution was
stirred at ambient temperature for 4 h. The mixture was then precipitated into Et2O (200 mL) to
give product as white powder (yield: 1 g, ca. 92%). The degree of endꢀgroup functionalization
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was 97%. H NMR (δ, ppm): 6.84 (bs), 6.71 (s), 3.84ꢀ3.87 (t, J = 7.2 Hz), 3.66 (bs), 3.42 (bs),
2.52ꢀ2.55 (t, J = 7.2 Hz). Mn = 14400 g molꢀ1, Ð = 1.13.
Characterization Methods
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Light irradiation is undertaken using a Mightex's WheeLED wavelengthꢀswitchable LED for λ
= 420 nm (builtꢀin filter). The intensity of light irradiance on the gel sample was tuned to the
desired intensity using a RMꢀ12 radiometer (Opsytec) with a sensor VISBG 400ꢀ570 nm.
Ellman’s test: The amount of free thiol released under visible light irradiation was determined
using the Ellman’s test. In a typical procedure, an aqueous solution of 4ꢀarm PEGꢀSbimane (1.5
mL, 0.05 mM, ca. 0.2 mM of the –Sbimane endgroup, Tris buffered pH 7.4) in a quartz cuvette
equipped with a stirring bar was first corrected as background on the Cary60. To this solution
was added a solution of 5,5'ꢀdithiobisꢀ(2ꢀnitrobenzoic acid) (0.2 mM in Tris buffered pH 7.4)
and the mixture was irradiated with blue light (420 nm, 20 mW cmꢀ2) under stirring for
predetermined periods of time. The solution was allowed to react with stirring in the dark for 10
min and the absorbance at 412 nm was recorded. Lꢀcysteine solutions (0.025ꢀ0.2 mM) were used
as standards for calculation of the amount of thiol release. Each measurement was done in
triplicate.
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Quantum efficiency. Data of the progress of Ellman’s assay were plotted in Sigmaplot 12.5 and
fitted via nonlinear regression analysis to a firstꢀorder process. The quantum efficiency (Φu) was
calculated using the equation Φu = (Iσt70%), where I is the irradiation intensity in Einstein cmꢀ2 sꢀ
1, σ is the decadic extinction coefficient (103 × ε, molar extinction coefficient at 420 nm) in cm2
mol−1, and t70% is the irradiation time in seconds for 70% conversion to the product.23 The light
intensity was reꢀmeasured using potassium ferrioxalate actinometry.
Size exclusion chromatography (SEC) was performed using a Shimadzu modular system
comprised of a SILꢀ20AD automatic injector, an RIDꢀ10A differential refractiveꢀindex detector
and a 50 × 7.8 mm guard column followed by three KFꢀ805L columns (300 × 8 mm, bead size:
10 ꢁm, pore size maximum: 5000 Å). N,NꢀDimethylacetamide (HPLC grade, 0.03% w/v LiBr)
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at 50 °C was used for the analysis with a flow rate of 1 mL minꢀ1. Reaction time course samples
dissolved in DMAc were filtered through 0.45 ꢁm PTFE filters before injection. The SEC
calibration was performed with narrowꢀpolydispersity polystyrene standards ranging from 500 to
2 × 106 g molꢀ1.
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Fluorescence spectrum was obtained using a Shimadzu RFꢀ5301 PC fluorescence
spectrophotometer. 2.5 mg of (reaction time course) sample was diluted in 3 mL of 1:2
acetonitrile/H2O and then further diluted 1 in 10 (200 µL added to 1.8 mL of H2O). Fluorescence
spectra were run using excitation wavelength 390 nm and emission scanned between 400 and
800 nm.
Rheological data were recorded using an Anton Paar Physica rheometer with a plateꢀplate
configuration. The lower plate is made of quartz and the upper plate is made of stainless steel
with a diameter of 15 mm. A liquid light guild, which was connected to the light source was
equipped below the quartz plate. In a typical experiment, a polymer solution (50 µL, 10 wt%) of
4ꢀarm PEGꢀSH/PEGꢀSbimane and a solution of 4ꢀarm PEGꢀAcr/Pro/Mal (50 µL, 10 wt%) were
mixed on a vortex mixer for 5 seconds. This solution was placed on the lower plate and the upper
plate was lowered to a measurement gap of 0.2 mm. A layer of paraffin oil was applied on the
edge of the stainless steel plate to prevent dehydration of hydrogel and the test was started by
applying a 1% strain with the frequency of 0.1 Hz on the sample. In all rheological tests, an
induction period of 20ꢀ200 s was observed after light illumination and before a sharp increase in
the storage modulus.
Hydrogel patterning. A solution (300 µL) of 4ꢀarm PEGꢀAcr (c = 8 mM) and 4ꢀarm PEGꢀ
Sbimane (c = 10 mM) was casted into a circular Teflon mould with a diameter of 1 cm. The
solution was then subjected to light irradiation (λ = 420 nm, I = 20 mW cmꢀ2) for 10 min, after
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which a yellow solid gel was formed. The gel was then immersed in an aqueous solution of
biotinꢀmaleimide (c = 0.1 mM, Sigma Aldrich) for 1 h. A photomask was then placed on the
surface of the gel and the light irradiation was applied on the materials for 20 min. The hydrogel
was then placed in an aqueous solution of TRITCꢀstreptavidin (c = 0.1 mg mLꢀ1) for 1 h. The
material was then washed with water/ethanol solution (v/v = 8/2) was then subjected to imaging
under a Nikon fluorescent microscope.
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Human mesenchymal stem cell culture. hMSCs (Bone marrowꢀderived) were cultured in low
glucoseꢀDMEM (1 g Lꢀ1 Dꢀglucose and 110 mg Lꢀ1 sodium pyruvate) with 100 µg mLꢀ1
penicillinꢀstreptomycin and 10% (v/v) fetal bovine serum (FBS) supplements and maintained at
37 °C and 5% CO2. Passage 6 cells were used in this work.
hBMSC encapsulation and viability test. hMSCs were harvested by TrypLETM Express
treatment, spun down and resuspended in three different electrophileꢀcontaining crosslinkers
stock solutions (4ꢀarm PEGꢀArc, 4ꢀarm PEGꢀPro and 4ꢀarm PEGꢀMal). The cell/crosslinker
mixtures were then mixed thoroughly with 4ꢀarm PEGꢀSbimane to achieve the final cell density
of 2.5 million cells mLꢀ1. Triplicate samples (25 µL/each) per condition were prepared in a
culture dish and subsequently exposed to the 420 nm light (I = 20 mW cmꢀ2) for 10 minutes.
The crosslinked cellꢀladen hydrogel samples were rinsed twice with culture media and
maintained at 37 °C and 5% CO2. To test cell viability, live/dead staining was performed after 24
hrs encapsulation. Gels were washed with phosphate buffer saline (PBS) solution and incubated
with Live/Dead staining kit (Life Technologies, USA) for 30 minutes at room temperature. The
samples were then rinsed thrice with PBS and imaged using a Nikon fluorescent microscope.
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Results and Discussion
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Figure 2. a) Scheme of the caging thiol endꢀgroups using monobromobimane, and H NMR
spectra of the polymers (CDCl3, 600 MHz); b) UVꢀvis spectra of the 4ꢀarm PEGꢀSH in aqueous
solution (0.05 mM) before and after caging with bimane, and after irradiation at λ = 420 nm for
30 min; and c) graph showing the trapping of bimaneꢀprotected thiol, determined by Ellman’s
test, followed by irradiation at different time periods (error bars represent standard deviations of
3 repeats).
The bimane structure is highly compact and this group has good solubility in aqueous solvent
which is useful for preparation of water soluble conjugated polymers for biological studies.28
Bromobimane has high reactivity towards free thiol and has been used for fluorescent tagging of
proteins.28ꢀ30 The thioꢀbimane ether molecules have been shown to be highly stable and nonꢀtoxic
to cells.30 Here we employ this reagent to protect thiol endꢀgroups of a 4ꢀarm PEGꢀSH (1) via
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thiolꢀbromo reaction (Figure 2a). Although bromobimane is commercially available, this reagent
is expensive which prompts us to prepare bromobimane from reported procedures.31, 32 In our
hands, the synthesis of bromobimane can be streamlined to a continuous 3ꢀstep process with only
one column chromatography purification, and an overall yield of 17.9% in gram scale (See
Supporting Information for details). This yield is lower than the yield previously reported for
monobromobimane synthesis, which was ca. 24%, and the lower yield may be due to some
intermediates not purified in our synthesis procedure.28,29 The 4ꢀarm PEGꢀSH can be easily
prepared by Fischer’s esterification of 4ꢀarm PEGꢀOH using paraꢀtoluenesulfonic acid as the
catalyst, and complete conversion of the –OH to –SH group was obtained.
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Treatment of the 4ꢀarm PEGꢀSH with bromobimane in dichloromethane and in the presence of
1
trimethylamine led to complete caging of the –SH group as indicated by H NMR analysis
(Figure 2a). The resultant polymer (2) has absorption and emission maxima at 388 nm (molar
extinction coefficient ε388 = 6.8 x 103 L molꢀ1 cmꢀ1) and 472 nm respectively (Stoke’s shift of 84
nm) in aqueous solution (Figure 2b and Figure S10). Based on the UVꢀvis spectrum we selected
a 420 nm LED light source, with an irradiance tuned at 20 mW cmꢀ2, to investigate the
photocleavage of the thioꢀbimane group. Under stirring in a Trisꢀbuffered (pH 7.4) solution
containing Ellman’s reagent, 5,5'ꢀdithiobisꢀ(2ꢀnitrobenzoic acid), we observed circa 70% of the
Sbimane was photoꢀreacted with Ellman’s reagent (Figure 2c) after 20 minutes of light
irradiation. The quantum yield of this photoreaction was calculated to be 0.02. The incomplete
release of the thiol may be due to the competing absorbance of the bimaneꢀhydroxyl product and
competing recombination reaction as shown below.
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Figure 3. a) Structures of the PEG with electrophiles endꢀgroups for thiolꢀaddition; b) 1H NMR
spectra (CDCl3, 400 MHz) of the polymers after light treatment showing chemical shifts from
the thiol addition reactions; and c) SEC traces of 2 before and after irradiation at 420 nm with
and without the electrophiles, indicating polymer ligation via thiolꢀnucleophilic addition.
We next analyzed 2 after photoꢀuncaging by extracting the polymer from aqueous solution
using dichloromethane followed by precipitation in diethyl ether. To our surprise, we did not
observe free thiol, as indicated by the Ellman’s test, when examining 1H NMR spectrum of the 4ꢀ
arm PEGꢀSbimane (2) after light irradiation in the absence of the Ellman’s reagent (Figure 3).
This indicates that no reaction occurred when irradiating aqueous solution of PEGꢀSbiman. On
the other hand, when solution (Trisꢀbuffered, pH 7.4) of 2 was irradiated in the presence of linear
PEGs having electrophilic endꢀgroups (Figure 3a) such as acrylate (Acr), propiolate (Pro) and
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maleimide (Mal), we observed formation of polymerꢀpolymer coupling product as indicated by
the peaks at higher molecular weight in SEC traces (Figure 3c). The presence of the thiolꢀ
nucleophilic addition adducts is also evident in the 1H NMR spectra (Figure 3b) of the polymers
extracted from the reacting solution. In the reaction of photoꢀreactive thiol with propiolate, the
chemical shifts of the major alkenyl protons display a vicinal coupling constant (3JHH) of 10 Hz,
which is characteristic of the cisꢀisomer. This is in agreement with previous reports33, 34 on
nucleophilic thiolꢀpropiolate addition in PBS, which typically produces cisꢀalkenyl thioether.
Integration of the corresponding chemical shifts allows us to calculate the efficiency of the free
thiol reacting with the electrophilic counterparts. Specifically, the acrylate group gave the highest
trapping efficiency of the photoꢀreleased thiol with 72% conversion observed, whereas only 63%
and 51% conversions were calculated for propiolate and maleimide respectively. The lower
conversion of Sbimane in the reaction with maleimide may be due to photoꢀcycloaddition side
reactions of the maleimide group.35 The undefined nature of the Sbimaneꢀmaleimide
photoreaction is further shown by the bimodal characteristic of the SEC trace of the polymer
product.
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Scheme 1. Suggested mechanism for the photoꢀuncaging of thiol bimane in the presence of an
electrophile.
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Based on our observation of 1H NMR data and previous work on photolysis quenching of ester
bimane by Sigh and coꢀworkers,36 we propose a mechanism for the photoꢀuncaging of thioether
bimane as outlined in Scheme 1. After photon absorption, the bimane thioether is converted to
the singlet excited state S1, which then can fluoresce or undergo heterolytic CꢀS bond cleavage to
form a zwitterion intermediate. The zwitterion intermediate can recombine back to the ground
state thioether bimane or react with electrophiles such as electronꢀdeficient alkene/alkyne in
water to form the respective thioether and hydroxyl bimane byꢀproduct. The competing
recombination reaction, as opposed to solvent trapping and hydrolysis in aqueous solvent, may
contribute to the stability of 2 in solution under light irradiation and in the absence of
electrophiles. Furthermore, the incomplete photocleavage in the presence of electrophiles, as
seen in the Ellman’s test and nucleophilic addition with acrylate, propiolate and maleimide, is
probably due to the recombination of the zwitterion intermediate. Since the hydroxyl bimane byꢀ
product has a maximum absorbance of 325 nm in water, a blue shift from the absorbance of the
Sbimane group,37 it would be expected the competing absorbance of this molecule is minimal.
Meanwhile, we observed excellent stability of 2 in phosphate buffer saline solution under
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1
ambient storage condition and under light exposure for 2 weeks, i.e. no changes observed in H
NMR spectrum. This high stability of the protected thiol under light and in the absence of the
electrophiles can be highly advantageous for storage of materials and retaining the
photoreactivity of protected thiols before photoꢀinduced conjugation.
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Figure 4. a) Scheme of polymer crosslinking via spontaneous and lightꢀtriggered thiolꢀ
nucleophilic additions; b) Gelation profiles of spontaneous thiolꢀnucleophilic addition followed
by the increase in storage moduli of the polymer solutions (c = 10 mM) at different pH; Gelation
profiles of thiolꢀnucleophilic addition via lightꢀtriggered release of thiol under irradiation of λ =
420 nm and I = 20 mW cmꢀ2 and at c) pH 6.9 and d) pH 7.4, dashed green lines indicate when
the light was switched on; and e) temporal control of the gelation process for Sbimane/Acr gel in
PBS pH 7.4 by blue light (420 nm), highlighted areas indicate when the light was turned on.
Having proof that the photoꢀuncaging of thioether bimane could occur effectively in the
presence of a nucleophile trap, we next prepared 4ꢀarm PEG linkers containing similar
electrophiles (4a-c) and assessed the polymer crosslinking under visible light irradiation using
rheological tests. For comparison, we first studied the gelation of 4ꢀarm PEGꢀSH with 4ꢀarm
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PEG linkers in phosphate buffer saline (Figure 4a). As seen in Figure 4b, the gelation kinetics,
followed by the increase in the storage modulus of the mixture of the investigated crosslinkers, is
highly dependent on the nature of the electrophile and pH. For acrylate endꢀgroup, a pH ≥ 7.4 is
required for efficient crosslinking with complete gelation occurring after 40ꢀ60 min of mixing
the crosslinker and 4ꢀarm PEGꢀSH. Gelation was faster for the propiolate endꢀgroup at similar
pH, with complete gelation in less than 10 min at pH 7.4. The SH/Pro crosslinking could also
proceed effectively at slightly basic pH 7.1 (Figure S11). The maleimide group is highly reactive
towards free thiol, as gels were observed to form almost instantaneously upon mixing PBS
solutions (pH > 7.0) of 4ꢀarm PEGꢀSH and 4ꢀarm PEGꢀMal. We were only able to obtain part of
the gelation profile for SH/Mal crosslinking on the rheometer using distilled water (pH 6.9) as
the medium.
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Replacing 4ꢀarm PEGꢀSH (1) with 4ꢀarm PEGꢀSbimane (2) allows for gelation to occur only
when the polymer solutions were subjected to irradiation at λ = 420 nm and I = 20 mW cmꢀ2
(Figure 4c). Interestingly, crosslinking could occur for 4ꢀarm PEGꢀAcr in solution with pH 6.9,
confirming that the released thiol is in anionic form. The gelation, featured by the crossꢀover
between G’ and G”, generally occurred after 1ꢀ2 min of irradiation for both Sbimane/Acr and
Sbimane/Pro solutions, whereas gelation happened more rapidly for Sbimane/Mal solution due to
the higher reactivity of maleimide towards nucleophiles. In our [4+4] hydrogel system with
equivalent molar ratio of the reacting groups, a 33% conversion of the functional groups is
required for gelation to occur according to Flory Stockmayer’s theory.38, 39 In the absence of
electrophileꢀcontaining linkers, no gelation was observed for 4ꢀarm PEGꢀSbimane under light
irradiation (Figure S12a). Addition of hydrogen peroxide, which is known to induce thiol
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oxidation, to solution of 2 resulted in a slight increase in the storage modulus after ca. 30 min of
irradiation (Figure S12b). This shows that oxidation during light irradiation is not significant.
The gelation onset for Sbimane/Acr and SBimane/Pro occurred more rapidly when pH of the
solution was raised to 7.4 (Figure 4d) and the effect of basic pH on the gelation kinetics is more
profound for the Sbimane/Acr mixture, with complete gelation, featured by the G’ value
reaching a plateau, happened after ca. 20 min of blue light irradiation. This fast gelation indicates
a faster trapping of the photoꢀreactive thiol compared to the observed results from Ellman’s test,
and this may be due to the higher reactivity of the acrylate compared to 5,5'ꢀdithiobisꢀ(2ꢀ
nitrobenzoic acid) used in Ellma’s assay. In comparison, slower gelation kinetics was observer
for Sbimane/Pro and Sbimane/Mal mixtures, which is consistent to the lower trapping efficiency
of both the Pro and Mal groups compared to the Acr group in reactions of 4ꢀarm PEGꢀSH with
linear MeOꢀPEGs. To account for the lower conversion of Pro and Mal in the reaction with
photoꢀreactive thiols, we irradiated 4a, 4b, and 4c separately in PBS pH 7.4 and in the absence of
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2 for 1 h. H NMR analysis of the polymers after light treatment shows that the Acr group
remained intact whilst some side reactions occurred in both 4b and 4c solutions, resulting in 38%
and 15% reduction of the Pro and Mal groups respectively (Figure S6 and S9). The loss of the
alkyne in 4b structure is accompanied by the loss of the ester group, indicating hydrolysis of the
propiolate, whereas the loss of the maleimide in 4c may be due to both and photolysis and freeꢀ
radical dimerization.35
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Figure 5. Photoꢀpatterning of hydrogel containing Sbimane group with biotinꢀmaleimide
under irradiation of visible light, and subsequent reaction with TRITCꢀstreptavidin. Image
showing red fluorescently labelled streptavidin in the patterned region.
A distinct advantage of the photocrosslinking over spontaneous mixingꢀinduced crosslinking
in hydrogel preparation is the ability to fineꢀtune the gel mechanical properties in a temporal and
spatial fashion. To demonstrate the temporal control over gel properties, we undertook a
rheological experiment in which the storage and loss moduli of the Sbimane/Acr solution pH 6.9
were monitored while blue light was switched on and off continuously. As seen in Figure 4e, the
polymerization was totally halted when the light was turned off, and resumed only when the light
was on, demonstrating the defined controlled over photoꢀuncaging and trapping process. To
demonstrate the spatial control over conjugation process, we first prepared a hydrogel containing
unreated Sbimane groups within the gel network. Upon photolysis under visible light irradiation
and in the presence of biotinꢀmaleimide the Sbimane group reacted with maleimide in the
irradiated regions (Figure 5). The immobilization of the biotin in the patterned area was
confirmed by the specific reaction of biotin with TRITCꢀstreptavidin, which can then be
visualized under a fluorescence microscope.
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Table 1. Physical properties of hydrogels formed by spontaneous and photoꢀtriggered thiolꢀ
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nucleophilic addition.
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Hydrogels a
G’asꢀprepared
(kPa)
G’swollen
(kPa)
EWCb
Gel
fractionc
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(%)
SH/Acrd
22.9 ± 1.7
18.5 ± 1.2
19.8 ± 1.9
20.8 ± 2.1
13.6 ± 2.7
14.1 ± 2.6
13.1 ± 1.1
8.7 ± 0.8
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88 ± 3
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72 ± 4
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Sbimane/Male
a: hydrogels were prepared from 100 µL solution of polymer with a total polymer
concentration of 10 mM (each reacting polymer solution had the concentration of 5 mM) and a
resultant thickness of 1 mm, reported values are the averages of 3 samples and errors represent
standard deviations; : Equilibrium water content (EWC) was calculated from the following
equation: (wswollenꢀwdry)x100%/wswollen in which wswollen is the weight of fully swollen gel and wdry
b
c
is the weight of dried gel; : gel fraction was calculated from the ratio of dry gel weight over
weight of polymer before gelation; d: pH 7.6; e: pH 7.1; f: pH 6.9; e: pH 7.4.
We further characterised physical properties of the hydrogels prepared by both spontaneous
polymerization and photoꢀpolymerization, and the data are presented in Table 1. All photoꢀ
polymerized gels were prepared from solutions with a thickness of 1 mm to minimise light
attenuation in photocrosslinking, and the irradiation time for photocrosslinking was 30 min at I
= 20 mW cmꢀ2. The molar absorptivity of the polymer solutions were found to be 1.9 x 103 L
molꢀ1 cmꢀ1 and the resultant gels are transparent. The expected bimane hydroxyl molecule In
general, the asꢀprepared hydrogels, with an initial water content of 90.9%, has robust mechanical
strength with the storage moduli in the range of 14ꢀ23 kPa. These hydrogels were highly water
absorbent with the equilibrium water contents varying from 94ꢀ96%. The G’ values were also
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observed to decrease as the gels were swollen in water.40, 41 Compared to spontaneouslyꢀformed
hydrogels, photocrosslinked gels have lower crosslinking efficiency, as shown by the lower
storage moduli and gel fraction, indicating the photoꢀinduced polymerized network is less
defined than the spontaneously formed network. This may be due to several factors including:
light attenuation, competing recombination of the ionꢀpair intermediate (Scheme 1) and light
absorption of the hydroxyl bimane byꢀproduct. Nevertheless, lightꢀinduced crosslinked gels
displayed good mechanical strength, even in their fully swollen state with the storage modulus
values in the range of 5ꢀ12 kPa. The physical properties of photocrosslinked gels may be tuned
within this range by altering the water content of the preꢀgel solutions, molar stoichiometry of
the reacting groups, or time of irradiation.15, 41ꢀ44
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Figure 6. Liveꢀdead staining of hMSCs 24ꢀh postꢀencapsulation (live cell = green, dead cell =
red, scale bar = 100 µm).
To assess the potential utility of our photoꢀuncaging/thiolꢀnucleophilic conjugation in
biological studies, we carried out polymer crosslinking using all three electrophileꢀcontaining
crosslinkers in the presence of hMSCs. The gelation condition was set at c = 10 mM and
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irradiation at λ = 420 nm, I = 20 mW cmꢀ2 for 10 min, which produced gels having thickness of
2ꢀ3 mm and moduli in the range of 2ꢀ4 kPa. Rheological analysis of the gelation process using
cell culture media as the solvent showed similar kinetics as PBS pH 7.4, i.e. rapid gelation upon
light irradiation (Figure S13). Live/dead staining of the encapsulated cells after 24 h postꢀ
gelation shows high percentage of live cells (>90% cell viability, Figure 6), which suggests the
photocrosslinking process and hydroxyl bimane byꢀproduct are both nonꢀtoxic to living cells. In
our cell encapsulation study, PEGꢀonly hydrogel was used which limits the culture duration
because PEG does not contain cell binding sequence. Therefore the viability of the cells
decreased with increasing culture time.43 For tissue engineering applications, hydrogels prepared
from naturally derived polymers such as hyaluronic acid1 and gelatin may be used to support
cells growth.7, 12, 43 These polymers can also be modified to attach the Sbimane group for spatialꢀ
tuning of gel stiffness and/or temporalꢀcontrolled activation of biomolecules.
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Conclusions
In conclusion, we present an efficient approach for caging of the thiol group using
bromobimane. Photoꢀuncaging of the thioether bimane under irradiation of blue light, coupled
with trapping of the anionic thiols by electrophiles can proceed rapidly and efficiently in water
and without the requirement of catalyst, providing a means for construction of photoꢀcrosslinked
gels with good mechanical properties. Importantly, the bimane group fulfils all requirements for
a photocleavable caging group suitable for biology related studies. These requirements, as
demonstrated in our work, include good water solubility, high stability in biological media and
minimal toxicity to cells. The nucleophiles, including propiolate and maleimide, employed in
this work were found to partially degrade under the light treatment conditions, nevertheless we
expect that more stable electrophiles without the ester group such as vinylsulfone will
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circumvent the stability issues encountered in the propiolate and maleimide groups. We believe
the caging and photouncagingꢀcoupling process introduced in this work can be effectively
employed in a range of applications including molecular biology and spatiotemporal control of
biomaterials properties to direct cellꢀmaterials interaction in regenerative medicine.
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ASSOCIATED CONTENT
Supporting Information. The following files are available free of charge.
NMR spectra of the synthesized compounds, additional UVꢀvis and fluorescent spectra, and
additional rheological data (file type, PDF).
AUTHOR INFORMATION
Corresponding Author
Vinh X. Truong. Email: vinh.truong@monash.edu
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Funding Sources
The research was supported under the Australian Research Council's Discovery Projects funding
scheme (DP160101591). F.L. acknowledges support from the New Horizons MonashꢀCSIRO
Council Joint PhD Scholarship Program.
ACKNOWLEDGMENT
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Dr Francessca Ercole is acknowledged for helping with running SEC on the polymer samples.
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ABBREVIATIONS
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PEG, poly(ethylene glycol); oꢀNB, orthoꢀnitrobenzyl; NDBF, nitrodibenzofuran ; PPG,
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photoremovable protecting group; Cmr, coumarin; TLC, thinꢀlayer chromatography; SEC, size
exclusion chromatography; Acr, acrylate; Pro, propiolate; Mal, maleimide; PBS, phosphate
buffer saline.
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