A photocleavable linker for the chemoselective functionalization of biomaterials

Article abstract of DOI:10.1039/c2jm35173k

The functionalization of matrices with "caged" functional groups and their subsequent selective uncaging is a promising approach for generating patterns of bioactive molecules to guide cell growth or recreate in vivo microarchitectures. To date, this has been limited to caged carboxylic acids, amines and thiols, functional groups found within biological systems. We present a bifunctional caged carbonyl linker as an alternative approach for the chemoselective functionalization of biomaterials. This linker was readily coupled to collagen, employed as a model biomaterial, and underwent rapid uncaging in aqueous media upon irradiation with ultraviolet light to yield free carbonyl groups. Modified surfaces proved to be non-adhesive to cells until the chemoselective reintroduction of adhesion following incubation of uncaged carbonyls with gelatin hydrazide, with native gelatin failing to elicit a cellular response.

Full text of DOI:10.1039/c2jm35173k

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PAPER
A photocleavable linker for the chemoselective functionalization of
biomaterials†
*
Liz O’Donovan and Paul A. De Bank
Received 2nd August 2012, Accepted 7th September 2012
DOI: 10.1039/c2jm35173k
The functionalization of matrices with ‘‘caged’’ functional groups and their subsequent selective uncaging
is a promising approach for generating patterns of bioactive molecules to guide cell growth or recreate
in vivo microarchitectures. To date, this has been limited to caged carboxylic acids, amines and thiols,
functional groups found within biological systems. We present a bifunctional caged carbonyl linker as an
alternative approach for the chemoselective functionalization of biomaterials. This linker was readily
coupled to collagen, employed as a model biomaterial, and underwent rapid uncaging in aqueous media
upon irradiation with ultraviolet light to yield free carbonyl groups. Modified surfaces proved to be non-
adhesive to cells until the chemoselective reintroduction of adhesion following incubation of uncaged
carbonyls with gelatin hydrazide, with native gelatin failing to elicit a cellular response.
potentially complex three-dimensional arrangements. To date,
Introduction
this approach has been pursued with caged amine,16–18 carboxylic
acid19,20 and thiol linkers21,22 which, following irradiation, are
coupled with biomolecules possessing complementary function-
ality (e.g. succinimide esters, amines and maleimides, respec-
tively). However, these functional groups are commonly found in
biological systems so, for the sequential functionalization of
matrices with multiple bioactive molecules or biopatterning in
the presence of live cells, specificity could be lost due to cross-
reactivity with existing functional groups.
Natural tissues consist of multiple cell types arranged in specific
three-dimensional (3D) configurations. It is now widely accepted
that, in order to promote optimal cell proliferation, migration,
differentiation and, ultimately, survival in vitro, this microenvi-
ronment should be replicated as closely as possible.1–4 Such
biomimetic culture systems are of great importance in a number
of biomedical applications such as tissue engineering, biosensors
and the development of cellular models of disease. Numerous
techniques for the spatial deposition of proteins and other
bioactive molecules for the patterning and guidance of cells in
both two and three dimensions have been described, including
microcontact printing,5–7 photo- and nanolithography,8–10
microfluidic patterning,11 inkjet printing,12,13 and photo-
immobilization.14,15 However, these techniques, as a whole, suffer
from a number of drawbacks, including a lack of cytocompati-
bility (precluding subsequent introduction of biomolecules in the
presence of living cells) and poor spatial resolution. Perhaps the
most promising approach for patterning biological cues, partic-
ularly in 3D, is the use of matrices where reactive functional
groups are caged by a photolabile protecting group (PPG). Upon
irradiation, photolysis of the PPG uncages the chemical moiety,
thus enabling the subsequent coupling of biomolecules to the
exposed functional group. With the use of photomasks or a
focused laser, patterned bioactive matrices can be generated,
while uncaging using multiphoton excitation extends this to
Aldehydes and ketones are not normally found within bio-
logical systems and can react chemoselectively with suitably
functionalized molecules such as hydrazides, thiosemicarbazides
or aminooxy compounds in the presence of biologically prevalent
groups.23–25 Hence, in order to pattern biomaterial matrices with
multiple bioactive molecules while eliminating the potential of
cross-reactivity, a caged aldehyde or ketone linker would be
highly desirable. This paper describes the development and
evaluation of such a molecule. The strategy for the design and
application of this linker is depicted in Fig. 1. This molecule
consists of three key components (Fig. 1a): a carbonyl group
caged with PPG 2,26 a poly(ethylene glycol) (PEG)-based linker,
and a terminal primary amine. PEG incorporation is desirable
for two reasons; firstly to provide a physical spacer between the
caged carbonyl and the material surface and, secondly, to render
the modified biomaterial cell-repulsive until chemoselective
ligation of an adhesive biomolecule, preventing non-specific
adhesion.27–29 A primary amine was chosen as the terminal
functionality in order to confer the ability to couple the linker to
the wide range of biomaterials that possess carboxylic acid
groups, either naturally (e.g. proteins, alginate) or following
minor chemical modification (e.g. hydrolysis of polyesters). The
strategy for the application of this linker is shown in Fig. 1b. The
Department of Pharmacy and Pharmacology, Centre for Regenerative
Medicine, University of Bath, Bath, BA2 7AY, UK. E-mail: p.debank@
bath.ac.uk; Fax: +44 (0)1225 386114; Tel: +44 (0)1225 384017
† Electronic supplementary information (ESI) available: Detailed
synthetic procedures and NMR spectra for all novel compounds. See
DOI: 10.1039/c2jm35173k
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Synthesis
The synthetic route to caged aldehyde 10 is shown in Scheme 1.
Detailed synthetic procedures and NMR spectra for 10 and
compounds 3 to 7 are provided in the ESI.†
Photolysis and UV/Vis spectroscopy of caged aldehyde 10 in
solution
Photolysis was performed on 10 (20 mM) in 50% v/v aqueous
methanol for various times by exposure to light with lmax ¼ 365
nm (26 mW cmꢀ2) using a 400 W UVA lamp (UV Light Tech-
nology, Ltd.). Following photolysis, solutions were analysed by
RP-HPLC using a Jasco HPLC system equipped with a Phe-
nomenex Gemini 5 mm C-18 column (150 ꢁ 4.4 mm) with a flow
rate of 1 mL per minute and a mobile phase of 1 : 1 acetonitrile–
water. The peak area of 10 at each time point was determined to
assess the extent of photolysis. UV/Vis spectra of 10 (20 mM) in
methanol were obtained using a Fluostar Omega multi-mode
microplate reader (BMG Labtech).
Functionalization and analysis of collagen-coated surfaces with
caged aldehyde 10
Depending on the experiment, either 24 well cell culture plates or
circular glass coverslips (12 mm diameter) were incubated with
collagen (0.01% w/v in 0.02 M acetic acid) for 1 hour, after which
the solution was aspirated and surfaces allowed to air dry.
Following washing with phosphate-buffered saline (PBS),
surfaces were incubated with a solution of EDC and NHS in PBS
(36.5 and 17.4 mM, respectively) for 1 hour. After rinsing with
PBS, activated surfaces were incubated with 10 in 1 : 1 methanol–
water for 1 hour. Surfaces were then rinsed with PBS prior to use.
For UV/Vis analysis, collagen-coated 24 well plates were func-
tionalized with various concentrations of 10 and each well
immersed in ultrapure water. UV/Vis spectra were obtained
Fig. 1 Strategy for the chemoselective functionalization of materials with
a caged aldehyde or ketone. (a) Structure of the proposed caged carbonyl
and its photolysis upon UV irradiation under aqueous conditions. (b)
Schematic of site- and chemoselective decoration of biomaterials with cell-
adhesive molecules following coupling of linker 1. Carboxylic acids on the
surface of a material (I) are activated and covalently coupled to the caged
carbonyl linker via an amide bond (II). Photolysis of the photolabile
protecting groups by irradiation with ultraviolet light yields free carbonyls
on the biomaterial (III) and this functionality is subsequently used to
chemoselectivelyligate a cell-adhesive hydrazide, or other suitablyreactive
species, to the surface (IV). R ¼ H or alkyl.
biomaterial surface, in this case with accessible carboxylic acids
(I), is activated with N-(3-dimethylaminopropyl)-N0-ethyl-
carbodiimide (EDC) and N-hydroxysuccinimide (NHS), then
coupled with linker 1 to decorate the surface with caged
carbonyls (II). Exposure of the surface to ultraviolet light under
aqueous conditions results in photolysis of the PPG, uncaging
the protected carbonyl (III). The biomaterial surface in both II
and III is cell-repulsive, and will only promote cell adhesion
following chemoselective ligation of a cell-adhesive molecule
with a hydrazide group, or other suitable functionality, to the
exposed carbonyl (IV), in this case via a hydrazone bond.
Experimental
Materials
Scheme 1 (a) 4-Bromobutanal (3), Bi(OTf)3, toluene, 80 ꢂC; (b)
NEt4CN, MeCN, reflux; (c) KOH, 2-methoxyethanol, reflux; (d)
N-hydroxysuccinimide, N,N0-diisopropylcarbodiimide, CH2Cl2, rt; and
(e) H2N(CH2CH2O)3(CH2)2NH2 (8), CH2Cl2 rt.
All reagents were purchased from Sigma-Aldrich (Dorset, UK)
unless stated otherwise and were used without further
purification.
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using a Fluostar Omega multi-mode microplate reader. Surfaces
functionalized with 10 (30 mM) were then irradiated with light at
lmax ¼ 365 nm (26 mW cmꢀ2) for 20 minutes to photolyze the
PPG, washed three times with ultrapure water and the UV/Vis
spectroscopy repeated.
Cell adhesion to functionalized surfaces
Murine C2C12 myoblasts (ECACC, #91031101) were cultured
in Dulbecco’s modified Eagle’s medium supplemented with
foetal bovine serum (10% v/v), penicillin (100 U mLꢀ1), strep-
tomycin (100 mg mLꢀ1) and amphotericin B (250 ng mLꢀ1). Cells
were maintained at 37 ꢂC in a humidified atmosphere containing
5% CO2.
Static water contact angle measurements
Collagen-coated circular glass coverslips functionalized with 10
(30 mM) were either left untreated or subjected to photolysis.
Photolyzed surfaces were incubated in PBS adjusted to pH 5.5
in the absence or presence of gelatin or gelatin hydrazide
(3 mg mLꢀ1) for 20 minutes at 37 ꢂC. Coverslips were then
washed with PBS and placed in the wells of a 6 well plate. Each
well was seeded with 1 ꢁ 105 C2C12 cells and their growth
monitored by phase contrast microscopy for 72 hours. Cells were
Collagen-coated circular glass coverslips functionalized with 10
(30 mM) were placed on microscope slides and 5 mL drops of
ultrapure water applied to the surface. Images of the drops were
obtained using a Veho VMS-004 USB microscope and a similar
experimental setup as that described by Lamour et al.30 Drop
analysis was performed using the ‘‘drop_analysis’’ plugin for
ImageJ (NIH, Bethesda, Maryland, USA) and contact angles
reported as the mean ꢃ standard deviation of three independent
experiments.
then stained with Hoechst 33342 in PBS (Invitrogen; 1 mg mLꢀ1
)
for 20 minutes at 37 ꢂC, and fixed in methanol at ꢀ20 ꢂC for 10
minutes prior to imaging using fluorescence microscopy.
Ligation of fluorescein-5-thiosemicarbazide to uncaged
aldehydes
Results and discussion
Synthesis of a caged aldehyde linker
Collagen-coated glass coverslips were functionalized with
various concentrations of 10 and the photolabile protecting group
photolyzed by irradiation for 20 minutes as described above to
uncage the aldehydes. As a positive, aldehyde-rich control,
collagen coated coverslips were incubated with sodium periodate
(2 mM) in ultrapure water for one hour at room temperature to
oxidize carbohydrate moieties. Coverslips were washed three
times with ultrapure water and then incubated with fluorescein-
5-thiosemicarbazide (1 mg mLꢀ1) in PBS for 20 minutes at room
temperature. Following washing with ultrapure water to remove
unbound thiosemicarbazide, the coverslips were imaged using
fluorescence microscopy (Leica DMI400B) to determine the
relative extent of fluorescein ligation to the surfaces. Images were
converted to greyscale and analysed using ImageJ to determine
the image area, mean grey value and integrated density. The
corrected fluorescence intensity (CFI) of each image was calcu-
lated using the equation CFI ¼ integrated density ꢀ (area ꢁ
mean grey value of control images). Surfaces functionalized with
10 (30 mM) and treated with fluorescein-5-thiosemicarbazide
without photolysis were used as controls. Data were collected
from three coverslips per condition and are presented as the
mean ꢃ standard deviation of three independent experiments.
The synthetic route to caged aldehyde linker 10 is depicted in
Scheme 1. Following the synthesis of the photolabile protecting
group,
4-(dimethylamino)-2-(hydroxydiphenylmethyl)phenol
2,26 a carbonyl with a suitable terminal functionality for subse-
quent installation of the PEG spacer was required. After
numerous unsuccessful attempts to protect a variety of aldehydes
and ketones with 2, the key step in the synthesis of 10 proved to be
the formation of novel acetal 4, derived from 2 and 4-bromo-
butanal 3, synthesized from 4-bromobutanol using Swern
conditions.
Unexpectedly, conversion of the bromo substituent to the
corresponding carboxylic acid proved initially challenging.
Original attempts to form a Grignard reagent through the
reaction of 4 with activated magnesium followed by the addition
of solid carbon dioxide were unsuccessful. However, an alter-
native route via conversion of bromide 4 to the corresponding
nitrile
5 using tetraethylammonium cyanide, followed by
hydrolysis to the acid 6 was successful, although severe hydro-
lysis conditions were required. Subsequent activation of acid 6
with N-hydroxysuccinimide and N,N0-diisopropylcarbodiimide
gave compound 7 in high yield. Reaction of activated ester 7 with
carbamate 8, prepared from tetraethylene glycol (TEG) using
three literature synthesis steps,32 led to the isolation of caged
aldehyde 10 in 40% yield following purification.
Functionalization of gelatin with pendant hydrazides
Gelatin from porcine skin (type B, 0.05 g) was dissolved in
ultrapure water (10 mL, pH 4) and stirred for 48 hours at room
temperature with the trifluoroacetate salt of 2,5-dioxopyrrolidin-
1-yl-3-(4-hydrazinyl-4-oxobutanamido)propanoate (0.50 g),
previously synthesized as described by Scott and Cwi.31 The
solution was then dialyzed for 72 hours against ultrapure water
and lyophilized to yield gelatin hydrazide. To confirm the pres-
ence of the hydrazide functionality, the modified gelatin (10 mg)
was treated with 4-hydroxybenzaldehyde (100 mg) in ultrapure
water (5 mL; pH 5.5) for 18 hours at room temperature. The
resultant modified protein was dialyzed and lyophilized as
described above and analysed by UV/Vis spectroscopy.
Functionalization of biomaterial surfaces with caged aldehydes
For its application in the chemoselective modification of mate-
rials with bioactive molecules, it was essential to demonstrate
that caged aldehyde 10 undergoes successful uncaging in an
aqueous environment. This was initially demonstrated in solu-
tion by irradiation of 10 at lmax ¼ 365 nm (26 mW cmꢀ2) and
subsequent HPLC determination of the remaining linker with
respect to exposure time. Uncaging under these conditions was
rapid, with complete photolysis of the PPG within 15 minutes
(Fig. 2). We then proceeded to demonstrate the utility of this
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Fig. 2 Progress of uncaging of 10 (20 mM) in 1 : 1 methanol–water
determined by monitoring HPLC peak area of the remaining compound.
approach in material functionalization by employing collagen
films as a model biomaterial substrate. Activation of exposed
carboxylic acids with EDC and NHS followed by incubation
with linker 10 resulted in its successful coupling to the films. This
was confirmed by UV/Vis spectroscopy of the modified surfaces,
where the presence of the surface-bound PPG chromophore was
clearly visible and concentration-dependent (Fig. 3), matching
the UV/Vis spectrum observed in solution (Fig. 3, dotted line).
Successful photolysis of the PPG from the functionalized
collagen in an aqueous environment was demonstrated following
irradiation for 20 minutes at lmax ¼ 365 nm (26 mW cmꢀ2), as
evidenced by the dashed line in Fig. 3, indicating the disap-
pearance of the PPG chromophore. The successful coupling of 10
to collagen films and the subsequent uncaging of the aldehyde
was further demonstrated by examining the static contact angle
of water droplets on the functionalized surfaces (Fig. 4). The
contact angle for unmodified collagen films was 37.55 ꢃ 3.61ꢂ
(Fig. 4a), in agreement with literature values and reflecting its
hydrophilic nature.33 Upon coupling linker 10 to collagen, the
water contact angle increased to 78.17 ꢃ 0.54ꢂ, indicating a
significantly more hydrophobic surface as might be expected
from the presence of the three phenyl groups in the PPG
Fig. 4 Successful modification and subsequent photolysis of collagen
films demonstrated by static water contact angle. Untreated collagen (a),
collagen modified with caged aldehyde 10 (b) and uncaged aldehydes on
modified collagen following photolysis (c).
(Fig. 4b). Successful photolysis of the PPG was demonstrated by
a further shift in the water contact angle to 28.56 ꢃ 0.97ꢂ,
resulting from the presence of the hydrophilic aldehyde and TEG
spacer on the surface of the collagen (Fig. 4c).
Following the successful demonstration of collagen function-
alization and subsequent uncaging of the protected aldehydes,
we then investigated the utilization of the exposed aldehydes for
chemoselective ligation to the collagen surface. Collagen films
were activated with EDC and NHS and then incubated with
various concentrations of linker 10. Modified films were irradi-
ated under the conditions described above and fluorescein-5-
thiosemicarbazide ligated to the resultant free aldehydes. To
generate a control, aldehyde-rich surface, collagen films were
Fig. 3 UV/Vis spectra of collagen films covalently modified with
different concentrations of caged aldehyde 10. The dotted line represents
the solution spectrum of 10 in methanol (20 mM), while the dashed line
represents the spectrum of a film modified with 10 (30 mM) and then
irradiated at lmax ¼ 365 nm (26 mW cmꢀ2).
Fig. 5 Effect of linker concentration on the fluorescence of collagen
films following coupling with linker 10, photolysis for 20 minutes and
chemoselective ligation of fluorescein-5-thiosemicarbazide. Collagen
films oxidized with sodium periodate (NaIO4) to generate aldehydes were
used as a comparison.
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treated with sodium periodate in order to oxidize available sialic
acids within the glycoprotein.34 The fluorescence of the films, a
measure of the number of available aldehydes that had under-
gone chemoselective ligation, was then determined and shown to
be dependent on linker concentration (Fig. 5), demonstrating the
successful uncaging of the surface-bound linker and the avail-
ability of the aldehydes for further functionalization.
A characteristic peak at ꢄ340 nm, corresponding to the
resultant aromatic hydrazone,35 was observed following its
reaction with gelatin hydrazide, confirming the successful
Chemoselective reintroduction of cell adhesion using caged
aldehydes
For linker 10 to have future applications in cell patterning and
guidance, it was essential to demonstrate that cell adhesivity and
growth on modified biomaterials could be selectively introduced
with no non-specific binding to regions that had not been irra-
diated (Fig. 1b(II)) or chemoselectively functionalized with an
adhesive species (Fig. 1b(III)). To achieve this, we chose gelatin
as a suitable cell-adhesive biomolecule. To incorporate hydrazide
functionalities within gelatin, i.e. generating ‘‘gelatin hydrazide’’,
we synthesized a short bifunctional linker containing a hydrazide
trifluoroacetate salt at one end and an N-hydroxysuccinimide
ester at the other.31 Upon incubation with gelatin, free amine
groups within the protein reacted with the NHS ester to form
amide bonds, thus decorating the gelatin with pendant hydra-
zides (Fig. 6a). To confirm the incorporation of hydrazide
moieties, the modified protein was incubated with 4-hydroxy-
benzaldehyde and, following purification, subsequently analysed
using UV/Vis spectroscopy.
Fig. 7 Fluorescence microscopy of C2C12 myoblasts 72 hours after
seeding on collagen-coated 12 mm diameter coverslips with different
surface modifications within 6 well culture dishes. (a) Collagen modified
with linker 10 (30 mM) and (b) collagen modified with linker 10 (30 mM)
and then UV irradiated to uncage protected aldehydes. (c) Uncaged
aldehydes incubated with gelatin (3 mg mLꢀ1) for 20 minutes at 37 ꢂC,
pH 5.5 and (d) uncaged aldehydes incubated with gelatin hydrazide (3 mg
mLꢀ1) under the same conditions as (c). Cell nuclei stained with Hoechst
33342 are shown in blue. Scale bars ¼ 200 mm.
Fig. 6 (a) Decoration of gelatin with hydrazide groups via the reaction
of free amines with the TFA salt of 2,5-dioxopyrrolidin-1-yl-3-(4-
hydrazinyl-4-oxobutanamido)propanoate. (b) UV/Vis spectra of
unmodified gelatin, gelatin hydrazide and gelatin hydrazide following
incubation with 4-hydroxybenzaldehyde (4-HB) in PBS.
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chemoselective reaction of the aldehyde with the pendant
hydrazides. Similar peaks were absent in native gelatin and
untreated gelatin hydrazide (Fig. 6b) and no evidence of
aromatic imine formation was observed following the reaction of
unmodified gelatin with 4-hydroxybenzaldehyde under the same
conditions (not shown), confirming the chemoselectivity of this
reaction.
aminooxy compounds to uncaged aldehyde groups offers the
possibility of decorating functional materials with multiple bio-
logically active molecules without unwanted cross-reactivity
towards existing functional groups of biomolecules already
present on the material. By making small changes to the design of
the linker’s terminal functionality, this general approach is
applicable to a large number of biomaterials other than those
with accessible carboxylic acids and can potentially be utilized
with both two- and three-dimensional substrates, either alone or
in combination with other caged functional groups. Amongst the
possible applications of this approach is the recapitulation of
tissue microarchitectures and cell niches for the advancement of
areas such as biosensing, tissue modelling, tissue engineering and
regenerative medicine.
Cell adhesion and growth was studied using C2C12 myoblasts
as a model cell line. Collagen films were generated on circular
glass coverslips, activated with EDC/NHS and functionalized
with caged aldehyde 10. These coverslips were then placed within
standard multiwell cell culture plates and seeded with C2C12s
following exposure of the films to various conditions. After 72
hours of culture, cell growth on the different coverslip surfaces
was compared with that on the surrounding tissue culture plastic.
As might be expected for a substrate rich in hydrophobic phenyl
groups, surfaces functionalized with 10 did not support cell
adhesion and growth (Fig. 7a), thus fulfilling the criterion of non-
irradiated surfaces being non-adhesive. Cells on the surrounding
plastic became confluent and grew up to the edge of the coverslip
but did not cross the boundary. Photolysis of the PPGs, resulting
in exposure of aldehydes on the coverslip surfaces, elicited similar
results, with no adhesion or growth of cells on these surfaces
(Fig. 7b). Again, this was expected due to the design of the linker,
where the TEG spacer generates a cell- and protein-repulsive
layer on the biomaterial surface. The final two surface treatments
demonstrated the chemoselective ligation of gelatin hydrazide to
the uncaged aldehydes. The reaction of a hydrazide with a
carbonyl to generate a hydrazone bond is pH-dependent and
favoured under acidic conditions.24 Hence, we incubated
collagen films, following functionalization with 10 and subse-
quent irradiation, with either gelatin or gelatin hydrazide for 20
minutes at pH 5.5 prior to cell seeding. These surfaces did not
promote cell adhesion and growth following incubation with
gelatin, suggesting that the native protein did not react with the
aldehydes under these conditions (Fig. 7c), resulting in a surface
similar to that in Fig. 7b. While gelatin possesses free amine
groups, which may be expected to react with aldehydes, the
susceptibility of any resultant imines to hydrolysis results in a
shift in the equilibrium of this reaction away from imine
formation.36 Conversely, however, aldehyde rich surfaces that
were incubated with gelatin hydrazide promoted cell adhesion
and growth in excess of the surrounding tissue culture plastic,
clearly demonstrating the successful chemoselective ligation of
this protein to uncaged aldehydes under these reaction condi-
tions (Fig. 7d). This is a versatile approach, which could readily
be employed for the hydrazide functionalization of other
bioactive proteins and peptides, and their subsequent chemo-
selective ligation to biomaterial surfaces and matrices.
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Conclusions
A bifunctional caged aldehyde linker that can be coupled to a
number of different biomaterials and readily undergoes photol-
ysis in aqueous media has been synthesized, and chemoselective
ligation of biomolecules to the resulting uncaged aldehydes has
been demonstrated. To our knowledge, this is the first reported
use of caged aldehydes to functionalize material surfaces. Che-
moselective ligation of hydrazides, thiosemicarbazides or
26 P. Wang, Y. Wang, H. Hu, C. Spencer, X. Liang and L. Pan, J. Org.
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21884 | J. Mater. Chem., 2012, 22, 21878–21884
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