Fluorescent polymers from non-fluorescent photoreactive monomers

Article abstract of DOI:10.1039/c4cc07792j

A facile, fast and ambient-temperature avenue towards highly fluorescent polymers is introduced via polymerizing non-fluorescent photoreactive monomers based on light-induced NITEC chemistry, providing a platform technology for fluorescent polymers. The resulting polypyrazolines were analyzed in depth and the photo-triggered step-growth process was monitored in a detailed kinetic study.

Full text of DOI:10.1039/c4cc07792j

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Communication
Fluorescent Polymers from Non-Fluorescent Photoreactive Monomers
Jan O. Mueller,a,b Dominik Voll,a,b Friedrich G. Schmidt,c Guillaume Delaittre*a,d and Christopher
Barner-Kowollik*a,b
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
The concept of step-growth polymerization – first investigated
50 by Carothers17 – has recently been revived by introducing novel
A facile, fast and ambient-temperature avenue towards highly
fluorescent polymers is introduced via polymerizing non-
fluorescent photoreactive monomers based on light-induced
NITEC chemistry, providing a platform technology for
10 fluorescent polymers. The resulting polypyrazolines were
analyzed in depth and the photo-triggered step-growth
process was monitored in a detailed kinetic study.
efficient conjugation tools referred to as click chemistry.18
A
popular example is the copper-catalyzed azide alkyne
cycloaddition, which was applied for instance to produce
conjugated polymers,19 poly(ferrocene)s,20 or palladium
55 containing polymers.21 To the best of our knowledge, there are
only few examples where light-triggered cycloaddition reactions
have been applied in the context of step-growth polymerization.
Although polyimides were generated by polymerization of bis-
maleimides with photoenol dilinkers22 or benzene23 and the
60 dimerization reactions of coumarin or cinnamate groups were
employed for polymerizing oligoethyleneglycol monomers,24 the
NITEC approach has not yet been employed and neither of the
former examples produces a fluorescent product.
During the last decades the importance of photo-induced
processes has rapidly increased not only in organic or
15 biochemistry, yet also in materials science and polymer
chemistry. The need for efficient light-triggered ligation
techniques is particularly present in the field of polymer science,
where post-polymerization transformations and purification
procedures can be extremely challenging. Due to their appealing
20 properties (e.g. high yields, facile conduction, limited – if any –
side reactions, spatiotemporal control), a variety of photo-induced
ligation techniques has been established in polymer-focused
applications including (hetero) Diels–Alder systems (photoenol1
and o-naphthoquinone2), [2+2]-cycloadditions,3 thioaldehyde
25 based processes,4 and [3+2]-cycloaddition systems.5 Among the
later reactions, the most widely employed system is the nitrile
imine-mediated tetrazole-ene cycloaddition (NITEC), which has
been used in numerous examples for e.g., protein-polymer
conjugation reactions,6 spatially resolved surface modification on
30 various substrates,7 and the generation of fluorescent single-chain
nano particles.8 Especially the biocompatibility of the NITEC
process, during which a nitrogen molecule is released and a
pyrazoline species is formed, widens the scope of this
Scheme 1 General reaction scheme for the photo-induced polyaddition of
65
M1 and M2 forming Ppy1 and Ppy2 respectively.
Firstly, new photoreactive monomers had to be designed to
enable a step-growth polymerization process (Scheme 1). In order
to avoid the common problems with inaccurate molarities of the
two reactive groups – typical for "A-A + B-B"-type of
methodology allowing
for
instance
in-vivo
protein
35 modifications.9 Herein, we provide a novel application for light-
70 polymerizations – our novel monomers were designed to be of
the type A-B, expressing both functional species combined in one
molecule. The synthetic pathway is simple (Scheme S1,
Supporting Information): A carboxylic acid-functionalized diaryl
tetrazole is transformed into the corresponding acyl chloride and
75 conjugated with a hydroxy terminal spacer molecule attached to
the activated double bond species, either an acrylate (M1) or a
fumerate (M2)(see Scheme 1). The molar masses of the resulting
monomers are 394.4 g mol-1 (M1) and 480.5 g mol-1 (M2).
Subsequent to the monomer preparation, the reaction conditions
80 for the novel polycycloaddition had to be explored since a variety
of parameters were expected to influence the polymerization
product: concentration, total batch size, irradiation time, and
triggered ligation techniques, presenting
strategy based on NITEC chemistry.
a polymerization
The NITEC concept enables addressing another highly topical
field of research by generating a cycloadduct with fluorescent
40 properties.10 Fluorescent polymeric materials have recently been
employed for imaging applications,11 bio-sensing,12 fluorescence
resonance energy transfer (FRET) analysis,13 and polymeric light-
emitting diodes.14 They typically either consist of conjugated
polymer backbones15 or fluorescence markers which are attached
45 to non-fluorescent polymer backbones.16 In our current approach,
the fluorescent species is incorporated into the polymer backbone
forming the connection between the monomer units in a step-
growth polymerization process.
This journal is © The Royal Society of Chemistry [year]
[journal], [year], [vol], 00–00 | 1

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Fig. 2
a) Image of a vial containing fluorescent Ppy2 solution excited
40
under UV-irradiation (365 nm) of a hand-held TLC lamp. b) UV-Vis
(dashed) and fluorescence (plain) spectra of M2 (black) and
poly(pyrazoline) (Ppy2, red).
common analytical techniques (NMR, SEC, MS) was employed
for each polymerization sample. Fortunately, the NITEC system
45 is invariant to oxygen or water, as evidenced in earlier studies,25
facilitating the sample preparation to only dissolving the
crystalline monomer and sealing the crimp-top vials to avoid
evaporation of the solvent. Subsequent to the irradiation of M2,
the solvent (THF) was removed under reduced pressure and the
50 resulting crude polymer mixture was analyzed by size-exclusion
chromatography (SEC), nuclear magnetic resonance (NMR)
spectroscopy, electrospray-ionization mass spectrometry (ESI-
MS), UV-Visible spectroscopy (UV-Vis), and fluorescence
spectroscopy. NMR analysis demonstrated that only the desired
55 cycloaddition occurs, excluding the existence of any side
reactions (Fig. 1a). Comparing the spectra of monomer and
polymer, a shift of the aromatic protons (a, b, c, d) to higher
fields (a', b', c', d') can be observed. Furthermore, the resonances
assigned to the fumerate double bond disappear (e), along with
60 the appearance of the pyrazoline signals (e', e'').
SEC analysis of the crude polymer produced within 8 h using
3 UV lamps reveals a mixture of low molar mass termination
product (34 – 36 min retention time) and a polymer distribution
(34 – 26 min retention time) with overall molecular weight values
65 of Mn = 2,500 g mol-1 and Mw = 12,000 g mol-1 (green line
Fig. 1b). In order to carefully examine both types of product, the
polymer was separated from the lower molar mass moieties by
precipitation in cold methanol yielding 60 wt.% polymer and
40 wt.% cyclic oligomers. SEC analysis of both fractions
70 (Fig. 1b) displays the success of this facile separation method, as
the precipitating product (red line) is almost quantitatively free of
oligomeric species present in the non-precipitating fraction
(dashed gray line). Thus, a polypyrazoline of Mn = 7,400 g mol-1
and Mw = 12,900 g mol-1 could be isolated. The low molar mass
75 material was additionally analyzed by ESI-MS in order to verify
its cyclic structure (Fig. S3). The mass spectrum confirms the
NMR results which suggest that open-chain oligomers containing
residual tetrazole units have been converted into pyrazoline
species. The m / z values perfectly match the predicted masses for
80 cycles composed of 2, 3, and 4 monomer units.
Fig. 1
a) NMR spectra of monomer M2 (top) and the crude
polypyrazoline Ppy2 (bottom). b) SEC chromatograms of monomer M2
(black), its crude polymerization product (green), its non-precipitating
termination product (dashed gray), and its precipitated polymer fraction
(red). Polymerization conditions: M2 (2 mg) was dissolved in THF
(100 mg mL-1) and irradiated for 8 h (108 W).
5
irradiation intensity. Therefore, the polymerization reactions were
conducted in a custom built photo-reactor, which can be equipped
10 with 1, 3, or 5 lamps (for lamp specification and schematic
picture of the photoreactor, refer to Fig. S10-S11). Upon UV light
exposure, the excited tetrazole moieties each release a nitrogen
molecule, forming a highly reactive nitrile imine species, which
subsequently reacts with the dipolarophile units present in an
15 equimolar amount in a [3+2]-cycloaddition. This cycloadduct can
potentially either connect two monomers, oligomers, or
polymeric species in a step-growth polymerization process or in
an intramolecular ,-cycloaddition generating cyclic species
and terminating the polymerization (Scheme 1).
In order to explore this novel photopolymerization concept, the
reaction conditions had to be established. M1 was employed for
the initial light-triggered reactions in order to determine the
influence of the monomer concentration and of the total amount
of monomer per batch (Fig. S1 and S2). Since the solubility of
25 Ppy1 is reduced in organic solvents suitable for analytic methods,
in-depth characterization was conducted with Ppy2 and the
kinetic study refers to the polymerization process of M2. The
concentration was chosen to be as high as possible since the
probability for intermolecular reaction between two different
30 polymer chains is less likely in diluted systems, where
intramolecular cycle formation is favored. This assumption was
verified in an initial concentration study with M1, depicted in
Fig. S1. Consequently, a concentration of 100 mg mL-1 was
employed for all further polymerization reactions. In addition, the
35 total batch size needed to be established. Since the reaction time
to achieve full conversion scales with the total amount of
monomer utilized (Fig. S2), the minimum amount required for
20
The most appealing property of the polypyrazolines along with
the novel polymerization technique presented here lies in the
fluorescence of the pyrazoline moieties (Fig. 2a). The monomer

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Fig. 3
a) 1H NMR spectra of the raw product obtained by polymerization of M2 produced with 5 UV-lamps after different irradiation times. The
displayed region contains the signals a, b, c, a' and b' (refer to Fig. 1a). b) Kinetic plot of conversion vs. irradiation time calculated by integrating the
respective NMR spectra. The kinetic plot parameters (Table S2) and additional kinetic data are available in the Supporting Information (Fig. S9). c)
5
Corresponding Mw vs. conversion plot. Mw values were obtain via SEC analysis of the crude polymerization mixtures. The Carothers equation is provided
in the Supporting Information (Eq. S2).
themselves do not show any fluorescence, which is demonstrated
for M2 in Fig. 2b. When the fluorescence spectrum is recorded
data sets correlate well with Carothers theory except for very high
conversions where the molar masses of the experimental data still
after irradiation with UV-light, the product exhibits strong 50 fit the shape of the curve, yet the experimental values are lower.
10 fluorescence in a broad emission spectrum ranging from 450 nm
to 750 nm (Fig. 2a). further appealing feature of the
pyrazoline-based material is that, besides its biocompatibility as
demonstrated in previous studies,26 it absorbs light at longer
This observation can be explained by the effect of the termination
reaction producing low molar mass material, which lowers the
overall molar mass of the polymer mixture. A termination
reaction – here a ring closure – was not taken into account in the
A
wavelengths than 400 nm (dashed red line in Fig. 2a) and thus 55 original theoretical work of Carothers.
15 fluorescence can be induced at wavelengths suitable for imaging
applications in living organisms.
In order to investigate the polymerization process in detail, its
Conclusions
In summary, we introduce an efficient light-induced
progress was monitored by NMR and SEC analysis, determining
the conversion and the molar mass of the produced material
20 (Fig. 3) simultaneously. In the following, the results of the photo-
polymerization of M2 are being discussed exemplarily. Proton
NMR spectroscopy is the ideal tool to monitor the reaction
process of the current system as the signals of monomer and
product can be integrated without significant overlap. The
25 overview spectrum (Fig. 1a) which compares the signals of M2
and the polymer reveals that the pyrazoline protons (e', e'' in
Fig. 1a, top) and the spacer resonances (k, k') can both serve as
indicators for the conversion, as well as the aromatic protons (a,
b, a', b') which are depicted in Fig. 3a (Fig. S7 and S8). Either
30 integration approach results in conversion values within a NMR
typical 5 % error. The conversion was calculated according to
equation S1 (Supporting Information). Consequently, the
resulting conversions were plotted against the reaction time under
variation of the irradiation intensity (1, 3, or 5 lamps, each 36 W).
35 As expected, the reaction time necessary to achieve full
conversion is reduced from 24 h to 4 h when the irradiation
power is increased from 36 W to 180 W. Noteworthy is the
exceptional agreement of the experimental data points and an
exponential fit attests a reaction of pseudo first-order kinetics for
40 all employed irradiation intensities (Eq. S2 Fig. S9, and
Table S2).
polycondensation as a facile avenue to fluorescent polymers. The
step-growth polymerization of newly designed photoreactive
60 monomers – each containing a photosensitive tetrazole and a
dipolarophile unit joint by a short linker – is demonstrated. In a
facile-to-conduct polymerization process polypyrazolines were
successfully generated at ambient temperature and atmosphere,
without any catalyst. The polymerization kinetics were followed
65 by NMR and SEC and satisfy classical Carothers conditions,
despite the generation of cyclic low molar mass termination
product accumulating during the polymerization process. The
intense fluorescence of the final product provides a technology
platform for future in-vivo imaging applications of the generated
70 polymers.
Notes and references
a Preparative Macromolecular Chemistry, Institut für Technische Chemie
und Polymerchemie, Karlsruhe Institute of Technology (KIT),
Engesserstr. 18, 76128 Karlsruhe, Germany. Email: christopher.barner-
75 kowollik@kit.edu
b Institut für Biologische Grenzflächen, Karlsruhe Institute of Technology
(KIT), Hermann-von-Helmholtz Platz 1, 76344 Eggenstein-
Leopoldshafen, Germany
c Evonik Industries AG, Paul-Baumann-Strasse 1, 45764 Marl, Germany
80 dInstitute of Toxicology and Genetics, Karlsruhe Institute of Technology
(KIT),
Hermann-von-Helmholtz-Platz
1,
76344
Eggenstein-
In order to obtain a molar mass vs. conversion plot (Fig. 3c)
the SEC data of the crude polypyrazoline samples were evaluated
(Fig. S6-S8). Again, all three different irradiation intensities were
45 followed and the weight-average molar masses (Mw) were
compared to the respective theoretical molar mass evolution
given by Carothers equation (Eq. S3).27 The values of all three
Leopoldshafen, Germany, Email: guillaume.delaittre@kit.edu
† Electronic Supplementary Information (ESI) available: 1H NMR
spectra of M1 and the kinetic study of M2, 13C NMR and ESI-MS spectra
85 of M1 and M2, SEC chromatograms of the kinetic study, the total mass
study, and the concentration study, utilized materials, synthetic
procedures. See DOI: 10.1039/b000000x/

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Table of Contents
Fluorescent Polymers from Non-Fluorescent Photoreactive Monomers
Jan O. Mueller,a,b Dominik Voll,a,b Friedrich G. Schmidt,c Guillaume Delaittre*a,d and Christopher
5 Barner-Kowollik*a,b
a Preparative Macromolecular Chemistry, Institut für Technische Chemie und Polymerchemie, Karlsruhe Institute of Technology (KIT), Engesserstr. 18,
76128 Karlsruhe, Germany. Email: christopher.barner-kowollik@kit.edu, guillaume.delaittre@kit.edu.
b
Institut für Biologische Grenzflächen, Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz Platz 1, 76344 Eggenstein-Leopoldshafen,
10 Germany
c Evonik Industries AG, Paul-Baumann-Strasse 1, 45764 Marl, Germany
dInstitute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen,
Germany
15
20
25
For table of contents use only!
A facile, fast and ambient-temperature avenue towards highly fluorescent polymers is introduced via polymerizing non-
fluorescent photoreactive monomers based on light-induced NITEC chemistry, providing a platform technology for fluorescent
30 polymers. The resulting polypyrazolines were analyzed in depth and the photo-triggered step-growth process was monitored in a
detailed kinetic study.