Modular functionalization and hydrogel formation: via red-shifted and self-reporting [2+2] cycloadditions

Article abstract of DOI:10.1039/d0cc07429b

We present a modularly applicable, red-shifted and self-reporting photodynamic covalent crosslinker, abbreviated qStyPy, that performs [2+2] cycloadditions upon irradiation with 470 nm in water. The rational design of qStyPy increases its hydrophilicity due to a permanent charge and features a broad emission in the far-red/near-infrared regime as a readout for the cycloadduct formation, rendering qStyPy suitable for biomedical applications. This journal is

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DOI: 10.1039/D0CC07429B
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Modular functionalization and hydrogel formation via red-shifted
and self-reporting [2+2] cycloadditions
Simon Ludwanowski,*b-e Daniel Hoenders,a-e Kubra Kalayci,f,g Hendrik Frisch,f,g Christopher Barner-
Received 00th January 20xx,
Accepted 00th January 20xx
Kowollik,f,g Andreas Walther*a-e
DOI: 10.1039/x0xx00000x
We present a modularly applicable, red-shifted and self-reporting with light of the wavelengths above 430 nm (Fig. 1). These red-
photodynamic covalent crosslinker, abbreviated qStyPy, that shifted photodynamic covalent crosslinkers (PCCs) are
performs [2+2] cycloadditions upon irradiation with 470 nm in bioorthogonal and may be used for instance to prepare
water. The rational design of qStyPy increases its hydrophilicity due extracellular matrices in cell culturing.11,12 Although such PCCs
to a permanent charge and features a broad emission in the far- show a beneficial red-shifting, they are strongly hydrophobic,
red/near-infrared regime as readout for the cycloadduct formation, which limits their applications in aqueous biological systems.
rendering qStyPy suitable for biomedical applications.
To overcome this drawback, we rationally designed a
multifunctional quaternized PCC, named qStyPy (1-trimethyl-
Photoresponsive interactions have been widely exploited in ammonium-6-pentafluorstyryl-pyrene), which is based on the
(bio)material science,1 because they enable spatiotemporal core structure of styrylpyrene and features (i) the ability to
control over bond formation and/or cleavage.2 In addition to perform [2+2] cycloadditions upon light excitation, (ii) a
beneficial non-covalent interactions,3 photo-induced [2+2] and permanent charge to increase its solubility in water, and (iii) a
[4+4] cycloadditions are particularly promising because they modular and quick functionalization with thiolated
may form covalent bonds in the absence of a catalyst, while (bio)macromolecules (e.g., DNA, peptides and polyethylene
they are still reversible in nature using light or temperature.4 glycol (PEG)) by the metal-free para-fluoro-thiol reaction (PFTR;
The most common molecules, which undergo
a
[2+2] Fig. 1).13 The PFTR proceeds nearly quantitatively in presence of
cycloaddition upon light irradiation, are thymine,5 stilbene,6 a base via nucleophilic aromatic substitution (SNAr), orthogonal
cinnamic acid,7 coumarin,8 and styrylpyrene.9 Considering at RT to other nucleophiles, such as primary amines and hydroxy
biological applications, however, these photo-induced ligations groups, and under comparably mild conditions due to its self-
generally rely on (strong) UV light which can be harmful to propagating nature.14 The functionalized qStyPy-thioether (R–
biological specimens, such as cells. More recently, we and qStyPy) enables to perform the [2+2] cycloaddition with light of
others exploited red-shifted [2+2] cycloadditions on the the wavelength of λ = 470 nm.
material scale using derivatives of styrylpyrene,10
acrylamidylpyrene11 and styrylquinoxaline,12 which function
a. A3BMS Lab, Department of Chemistry, University of Mainz, Duesbergweg 10-14,
55128 Mainz, Germany. E-Mail: andreas.walther@uni-mainz.de
b. Institute for Macromolecular Chemistry, University of Freiburg, Stefan-Meier-
Straße 31, 79104 Freiburg, Germany. E-Mail: simon.ludwanowski@makro.uni-
freiburg.de
c. Freiburg Materials Research Center (FMF), University of Freiburg, Stefan-Meier-
Straße 21, 79104 Freiburg, Germany.
d. Freiburg Center for Interactive Materials and Bioinspired Technologies (FIT),
University of Freiburg, Georges-Köhler-Allee 105, 79110 Freiburg, Germany.
e. Cluster of Excellence livMatS @ FIT – Freiburg Center for Interactive Materials and
Fig. 1 Thiol-reactive PCC undergoing self-reporting [2+2] cycloadditions by blue light.
Previous studies have shown that the [2+2] cycloadditions of styrylpyrene,10
acrylamidylpyrene11 and styrylquinoxaline12 function best using light of the wavelength
of λ1 = 435 nm, λ2 = 455 nm, and λ3 = 470 nm, respectively. Here, we introduce a
quaternized styrylpyrene (qStyPy) derivative with an increased water-solubility that can
be readily and modularly functionalized with (bio)macromolecules (R-qStyPy) via SNAr
reactions and enables to perform the [2+2] cycloaddition with blue light (λ = 470 nm).
Bioinspired Technologies, University of Freiburg, Georges-Köhler-Allee 105, D-
79110 Freiburg, Germany.
f. Centre for Materials Science, Queensland University of Technology (QUT), 2
George Street, Brisbane, QLD 4000, Australia.
g. School of Chemistry and Physics, Queensland University of Technology (QUT), 2
George Street, Brisbane, QLD 4000, Australia.
†
Electronic Supplementary Information (ESI) available. See DOI: 10.1039/
x0xx00000x
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The rational design of the modularly applicable, thiol- (highlighted by arrows; Fig. 3b,c). In agreement with previous
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reactive and red-shifted qStyPy includes specific functional studies,17 this
DOI: 10.1039/D0CC07429B
moieties. Firstly, the quaternized ammonium group increases
the water-solubility as a result of the permanent charge and at
the same time enables triplet state (T1) formation upon
excitation. Previous studies on the photodimerization of
coumarin suggest that the [2+2] cycloaddition proceeds via the
triplet state T1,15 which has been proposed to be the case for
styrylpyrene as well.10 This is in agreement with our previous
study on the non-quaternized analogue StyPy (bottom right, Fig.
2a).16 Upon excitation, StyPy undergoes a twisted intra-
molecular charge transfer process, which concurrently
diminishes the singlet-to-triplet state conversion via inter-
system crossing. Consequently, StyPy does not form a [2+2]
cycloadduct due to the absence of the T1 state. Secondly, qStyPy
features a pentafluorophenyl ring, which enables the facile and
modular functionalization of (bio)macromolecules via the PFTR
in presence of DBU (diazabicycloundecene; Fig. 2b).
Fig. 2 Synthesis and functionalization of qStypy. (a) Reaction conditions: (i) 1.) NaN3, CuI,
N,N’-dimethyl-ethylenediamine, sodium ascorbate, N2; 2.) NaSH · x H2O, 38 %; (ii) MeI,
K2CO3, N2, 100 %; (iii) pentafluorostyrene, Pd(OAc)2, NEt3, K2CO3, N2, 62 %, (iv) MeOTf,
92 %. (b) Facile and modular tagging via PFTR. Reaction conditions: (v) R-SH, DBU, > 90 %.
(c) 1H-NMR spectrum of qStyPy. (d) Red-shifted UV/Vis spectrum of R-qStyPy after
thioether formation in comparison to qStyPy (blue).
qStyPy can be readily prepared in a four-step synthesis with
an overall yield of 21 %, starting from commercial 1,6-dibromo-
pyrene (Fig. 2a). The reaction conditions are outlined in the
caption and detailed in the Supplementary Information (Fig. S1).
The desired product was obtained in high purity (> 97 %)
according to 1H- and 19F-NMR spectroscopy (Fig. 2c; Fig. S2). The
combination of the strongly electron-withdrawing pentafluoro-
phenyl ring and the electron-deficient quaternized ammonium
group of qStyPy gives rise to an acceptor-π-acceptor (A-π-A)
architecture with an absorption maximum at λabs = 374 nm (Fig.
2d). The subsequent functionalization of qStyPy via PFTR lowers
the energy gap between the highest occupied and the lowest
unoccupied molecular orbital, because the thioether acts as an
electron donor with respect to the A-π-A system. Thus, the
absorption of R–qStyPy is red-shifted compared to qStyPy (Fig.
2d) with a maximum at λabs = 388 nm and a broad absorbance
above 450 nm. In summary, the PFTR enables the facile
conjugation of (macro)molecules of interest with qStyPy and at
the same time red-shifts the UV/Vis spectrum of the product,
which allows to perform the [2+2] cycloaddition with blue light.
Initially, we characterized the activity of the [2+2]
cycloaddition in acetonitrile as function of the incident
wavelength (410 – 480 nm). Specifically, we employed a PEG-
modified-qStyPy (mPEG44-qStyPy), obtained by coupling qStyPy
to polyethylene glycol methyl ether thiol (mPEG44-SH,
Mn = 2,000 g·mol-1; Fig. S3) via PFTR, and irradiated the samples
(1.0 mg·mL-1, oxygen-free; Fig. 3a) with an equal number of
photons (22.1 mmol) using a tunable laser. Subsequently, we
acquired UV/Vis spectra of a diluted solution, normalized to the
isosbestic point at 363 nm (red circle; Fig. 3b), and calculated
the conversion based on the absorbance at 400 nm (dashed
line). Fig. 3c indicates that the highest activity is obtained using
an irradiation wavelength of 430 nm yielding a conversion of
approximately 85 %. Shorter and longer wavelengths lead to a
decreased activity (dashed curve, guide to the eye), whereas
light above 470 nm yields a conversion of less than 13 %. In
contrast, the irradiation with 470 nm of an aqueous solution
is due to enforced hydrophobic interactions of the extended π–
systems in aqueous solution, which bring the PCCs transiently
into proximity (Fig. S4). Hence, this beneficial association of
qStyPy in water leads to the acceleration of the [2+2] ligation.
Subsequently, we analyzed the kinetics of the [2+2]
cycloaddition of mPEG44-qStyPy. To this end, we dissolved
mPEG44-qStyPy (1.0 mg·mL-1) in PB (phosphate buffer, 1.0 mM,
pH 7.4) and irradiated the solution with a 470 nm LED. The high
molecular weight of mPEG44-qStyPy facilitates the analysis of
the [2+2] cycloaddition via size exclusion chromatography (SEC;
Fig. 3d). The detection wavelength was set to 363 nm (isosbestic
point). Fig. 3d displays the chromatograms as a function of the
irradiation time. The peak at an elution time of approximately
32 min (starting material) decreases, while the peak at ca. 30
min increases (cycloadduct) with an increasing irradiation time.
The normalization of the area under the curve in presence of
the isosbestic point enables a directly calculation of the
conversion of the cycloaddition by relating the deconvoluted
Gaussian curves (Fig. 3e). The reaction rate illustrates pseudo-
first-order kinetics with
a
maximum conversion of
approximately 76 % in less than 1.5 min, which can be fitted by
a mono-exponential decay (colored graph). The limit regarding
the maximum conversion is likely related to the development of
a
photostationary state or to reaching a subcritical
concentration for still allowing efficient reactions of the short-
living triplet state. The adduct is stable in water (pH 7.4) and any
degradation is ≪ 1 % within 7 days.
After analyzing the increased activity of qStyPy in water, we
exploited the [2+2] cycloaddition on the material scale to form
light-responsive hydrogels. Thus, we functionalized star-shaped
4arm-PEG-SH (sPEG-SH, Mn, MALDI = 20,780 Da) with qStyPy via
PFTR to prepare sPEG-qStyPy (Mn = 23,110 Da; Fig. 4a). We
obtained the desired product with a degree of functionalization
of df > 98 %, underscoring the efficiency of the thiol-reactive
moiety (Fig. S5). Subsequently, we dissolved sPEG-qStyPy in PB
leads to
a significantly higher conversion of ca. 78 %
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to obtain a mass concentration of 7.0 wt%. The concentration is properties in oscillation mode over time (f = 1 HzV,ieγw=Ar1tic%le ;OnFliinge.
DOI: 10.1039/D0CC07429B
close to the overlap concentration c* of sPEG-OH (Mn = 20 kDa, 4b). The storage (G’) and the loss modulus (G’’) present the
elastic and the viscous properties, respectively. Before
irradiation, sPEG-qStyPy behaves as a viscoelastic liquid (G’’ >
G’; Fig. 4c, black curves) over the entire frequency range (0.01 –
100 rad·s-1) at 25 °C. Although some interactions between the
associative sPEG-qStyPy polymers are macroscopically visible by
the increased viscosity, the dynamics of the supramolecular
interactions are too high at 25 °C to form a percolating network.
Upon irradiation with 470 nm, G’ steeply increases within the
first 1.5 h to approximately 5.5 kPa and a sol-gel transition takes
place (G’ > G’’, highlighted by an arrow; Fig. 4b) after ca. 20 min.
Consequently, the [2+2] cycloaddition of qStyPy leads to a
percolating network with covalent crosslinks. As the triplet state
of qStyPy only persists with a short half-life, the probability of
the [2+2] cycloaddition decreases with a proceeding network
formation as a result of a hampered diffusion of qStyPy units.
Nevertheless, the stiffness of the hydrogel steadily increases,
and G’ reaches approximately 7.3 kPa upon irradiation for 20 h
(Fig. 4b). The corresponding frequency sweep (blue curves; Fig.
4c) indicates that G’ and G’’ slightly depend on the angular
frequency. This phenomenon suggests that the network is
Fig. 3 Photoreactivity of mPEG44-qStypy. (a) Scheme of the [2+2] cycloaddition of
mPEG44-qStyPy. (b) UV/Vis spectra normalized to the isosbestic point at 363 nm
composed of covalent crosslinks and sacrificial supramolecular
crosslinks (sticky π–π interactions; Fig. 4a), since a purely
(highlighted by a red circle) and acquired as a function of the irradiation wavelength. (c)
covalent network would not show a frequency-dependent
Action plot calculated from the absorbance at 400 nm (dashed line in b). The highest
behavior within the frequency range. The amplitude sweep
activity is obtained using 430 nm, whereas shorter and longer wavelengths lead to a
decreased activity (dashed curve, guide to the eye). The activity at 470 nm significantly shows an elastically crosslinked network with a yield point
increases in water as opposed to acetonitrile due to enforced hydrophobic interactions
of qStyPy in aqueous solution (highlighted by an arrow). (d) Irradiation time-dependent
SEC using 470 nm. The chromatograms were acquired at a detection wavelength of
363 nm, at which the starting material and the cycloadduct absorb equally (isosbestic
(ΔG’ > 5 %; Fig. 4d) at a strain amplitude of γy ≈ 250 % and a flow
point (G’ = G’’) at γf ≈ 590 %, which leads to the failure of the
network. The mechanical properties of the hydrogel, especially
the stiffness and the elasticity, underscore the efficiency of the
[2+2] cycloaddition using visible light.
point). (e) Kinetic profile of the [2+2] cycloaddition. The reaction rate shows pseudo-first-
order kinetics with a turnover of approximately 76 %.
In addition to the red-shifted cycloaddition and the
increased water-solubility, qStyPy self-reports on the formation
of its [2+2] cycloadduct as seen by in-situ UV/Vis and
fluorescence spectroscopy of sPEG-qStyPy (50 µM) dissolved in
PB (Fig. 4e,f; see Fig. S10 for experimental setup). The UV/Vis
spectra in Fig. 4e show that reaction proceeds continuously
from the molecularly dissolved sPEG-qStyPy (purple spectrum)
to the [2+2] cycloadduct (red spectrum) with a clear isosbestic
point at 363 nm (highlighted by an arrow). The isosbestic point
indicates the clean transformation from the starting material to
c* = 6.9 wt%; Fig. S6), which allows to derive distinct structure-
property relationships, as it minimizes chain entanglements but
guarantees close proximity of the end groups. The dissolution
of sPEG-qStyPy in water yields a highly viscous and transparent
liquid with a zero-shear viscosity of η0 > 5.5 · 104 mPa·s (Fig. S7).
The zero-shear viscosity is higher than that of pristine sPEG-OH
at the overlap concentration (η0 = 4.3 mPa·s; Fig. S6), which
indicates the association of the sPEG-qStyPy based on reversible
supramolecular interactions. However, the terminal charge of
qStyPy, as designed by us, is of critical importance for increased
solubility of sPEG-qStyPy in contrast to other hydrophobic
molecules undergoing [2+2] cycloadditions. For instance, a
related sPEG-styrylpyrene (Mn = 22 kDa, 4arm) without a
quaternized ammonium group leads to macroscopic phase
segregation at 7.0 wt% as shown in Fig. S8. Clearly, the strongly
hydrophobic terminal moieties of the non-quaternized
analogue result in an inhomogeneous dissolution of sPEG-
styrylpyrene in water. Thus, the terminal charge of qStyPy
enables the homogeneous distribution of sPEG-qStyPy in
aqueous solution, which is a requirement for the formation of a
model network in contrast to the hydrophobic styrylpyrene.
To induce crosslinking, we irradiated sPEG-qStyPy in-situ
through a glass bottom plate inside a rheometer with a fiber-
coupled 470 nm LED (Fig. S9) and measured the rheological
the cycloadduct in
corresponding fluorescence spectra show
a
chemically selective fashion. The
continuous
a
alteration with a new emission maximum at λem = 655 nm (Fig.
4f). Hence, the progress of the [2+2] cycloaddition can easily be
tracked either by the absorbance at 400 nm (dashed line; Fig.
4e) or by the ratio of the two emission maxima (Fl655 nm / Fl550 nm
;
Fig. 4g). While UV/Vis measurements are only an option in
highly diluted and transparent samples, the fluorescence
measurements are also feasible in turbid samples and on the
material scale. Thus, the strongly red- shifted fluorescence with
a broad emission in the far-red/near- infrared regime is a
particularly interesting feature for biological applications. Fig.
4g illustrates the kinetic profile of the [2+2] cycloaddition using
UV/Vis and fluorescence spectroscopy. The former indicates a
pseudo-first-order reaction at the beginning that slows down
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over time because of the hampered diffusion of qStyPy units. stemming from cell and tissue autofluorescence. We anticipate
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Based on the absorbance at 400 nm, approximately 71 % of
that these design principles are of direct DimOpI:o10rt.1a0n3c9e/Di0nCtCh0e7f4i2e9ldB
of photoresponsive ligations to further increase the
hydrophilicity of common hydrophobic molecules undergoing
[2+2] photocycloadditions and to further red-shift the
underlying excitation wavelengths. Despite the beneficial
design of qStyPy, one needs to investigate its potential toxicity
in biomedical applications, as it may intercalate into DNA due to
its positive charge and its extended π–system.
Conflicts of interest
There are no conflicts to declare.
Notes and references
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Fig. 4 Crosslinking of sPEG-qStyPy to form model networks by blue light. (a) Scheme of
the [2+2] cycloaddition of sPEG-qStyPy to form a network consisting of covalent and
sacrificial supramolecular crosslinks. (b) In-situ irradiation of sPEG-qStyPy with 470 nm.
The steep increase of G’ leads to a sol-gel transition after ca. 20 min and an overall
stiffness of 7.3 kPa upon irradiation for 20 h. (c) Frequency sweeps before (black curves)
3
4
and after irradiation (blue curves). Before irradiation, sPEG-qStyPy behaves as
viscoelastic liquid over the entire frequency range. After irradiation, G’ and G’’ show a
slight frequency dependency, which indicates network based on covalent and
a
a
supramolecular crosslinks. (d) The amplitude sweep after irradiation reveals a highly
elastic hydrogel with a yield point at γy ≈ 250 % and a flow point at γf ≈ 590 %. Time-
dependent (e) UV/Vis and (f) fluorescence measurements. The spectra stepwise evolve
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qStyPy is converted into the cycloadduct within 20 h of
irradiation. In contrast, the kinetic profile of mPEG44-qStyPy on
a molecular level (Fig. 3c) shows a faster cycloaddition reaching
a steady state already within 1.5 min. Hence, it becomes
evident that the [2+2] cycloaddition is highly dependent on the
mobility of the qStyPy units due to the short-living triplet state.
In conclusion, the rational design of the thiol-reactive
qStyPy enables to functionalize (bio)macromolecules of interest
and to perform [2+2] cycloadditions outside the harmful UV-
regime in aqueous solution as well as on the material scale to
form hydrogel model networks. Due to the quaternized
ammonium moiety, the water solubility of qStyPy is critically
enhanced compared to the state-of-the-art PCCs, which leads
to the homogeneous dissolution of macromolecular qStyPy-
conjugates. Considering that the mechanical properties of
hydrogels are dependent on the overall polymer content, the
enhanced water solubility will extend the tuning range of the
resulting materials in terms of their stiffness and elasticity,
which is of great importance in the field of tissue engineering
and 3D bioprinting. Moreover, qStyPy self-reports on the
formation of its [2+2] cycloadduct via a broad emission within
the far-red/near-infrared regime as opposed to common PCCs.
This feature is particularly interesting considering complex
biological samples and materials, as it enables to readily track
the progress of the cycloaddition with minimal perturbation
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