University of Birmingham Fluorescent and chemico-fluorescent responsive polymers from dithiomaleimide and dibromomaleimide functional monomers

A new class of brightly ﬂ uorescent and pro ﬂ uorescent methacrylate and acrylate monomers is reported. The ﬂ uorescent monomers contain the dithiomaleimide (DTM) ﬂ uorophore, which imparts a large Stokes shift (up to 250 nm) and bright emission. Furthermore, the simple and e ﬃ cient chemistry of the DTM group, as well as its excellent processability (highly soluble, neutral functional group) makes monomer preparation straightforward. Copolymerisation at 10 mol% loading with a range of hydrophobic and hydrophilic monomers is demonstrated by RAFT polymerisation. Reactions proceed to high monomer conversion with excellent control over molecular weight ( Đ M < 1.3) under standard polymerisation conditions. Incorporation of these ﬂ uorescent DTM-functional monomers has little e ﬀ ect on polymer properties, with PEG (meth)acrylate copolymers retaining their water solubility and thermoresponsive behaviour. A thiol-exchange reaction is also possible, whereby the thiol ligands of the pendent DTM groups can be exchanged by conjugate addition – elimination with an alternative thiol. Monomers containing the dibromomaleimide (DBM) group gave pro ﬂ uorescent copolymers. Reaction of the DBM group with thiols (to form the DTM group) corresponds to a chemico- ﬂ uorescent response, leading to an OFF-to-ON switching of ﬂ uorescence. This post-polymerisation functionalisation is shown to be fast and highly e ﬃ cient (>95% conversion in 3 h), and by using thiols of di ﬀ erent polarities can be used to progressively tune the LCST cloud point of a thermoresponsive polymer over a range of 11 (cid:1) C. Therefore, both DTM and DBM functional monomers provide a simple and e ﬀ ective tool for ﬂ uorescent labelling of (meth)acrylate polymers.


Introduction
The polymerisation of dye molecules that are functionalised with a vinyl group (uorescent vinyl monomers) allows incorporation of uorophore units along a polymer backbone. These uorescent vinyl monomers are highly versatile, as they are compatible with reversible-deactivation radical polymerisation (RDRP) processes, 1 such as nitroxide mediated polymerisation (NMP), 2 atom transfer radical polymerisation (ATRP), 3 and reversible addition-fragmentation chain transfer (RAFT) polymerisation. 4 Copolymers, block copolymers and homopolymers of uorescent vinyl monomers have found a myriad of applications in organic electronic devices, sensor materials, studying the physical properties of polymers, and for labelling polymer materials (nanoparticles, hydrogels, membranes etc.) for uorescence detection/imaging in biomedical applications. 5 Polymers can also be uorescently labelled using an end-group modication approach, 6 but there are several advantages of using uorescent vinyl monomers. For example when using these monomers the degree of uorophore incorporation (and hence its concentration in the nal polymer) can be simply varied by altering the monomer feed, as opposed to an endgroup labelling approach which is limited to one or two uorophores per chain. Another advantage is that it doesn't require any modication to either the initiator or the nal polymer. Furthermore if a uorescent vinyl monomer is used, the resultant polymer end-groups remain 'available', allowing for further modication of the polymer by end-group functionalisation or conjugation. The importance and utility of uorescent vinyl monomers is illustrated by the wealth of variations that have been investigated, with a recent review of the literature nding over 200 different examples. 5 Popular amongst these include vinyl monomers based on polyaromatic hydrocarbons (such as naphthalene, 7 pyrene, 8 perylene, 9 and anthracene 10 ), uorescein, 11 rhodamine, 12 coumarin, 13 naphthalimide, 14 BODIPY, 15 and oxadiazole 16 uorophores. However, the molecular weight and relative dimensions of many of these frequently used uorophores are signicant, with the result that they can signicantly alter polymer properties. For example the incorporation of highly hydrophobic aromatic uorophores can dramatically affect the solubility of hydrophilic polymers in aqueous solution, which has been shown to result in polymer aggregation and increased surface adsorption from solution. 17 As well as permanently uorescent labels, it is highly desirable to be able to generate an emissive uorophore from a non-uorescent labelling agent (i.e. a latent uorophore or pro-uorophore) upon completion of a targeted reaction. As opposed to the attachment of an already emissive species, use of a prouorophore gives a clear indication that uorescent labelling has been achieved at the desired location, as an OFF-to-ON change in emission will occur. For polymeric systems this has been achieved by using a quenched uorophore where the target reaction results in the loss of the quenching group. For example, copper-catalysed azide-alkyne cycloaddition (CuAAC) of 'quenched' 3-azidocoumarin with an alkyne functional polymer leads to the formation of an emissive triazole-coumarin functional polymer. 18 Cleavage of a quenching 'trimethyl lock' from a rhodamine functional polymer by intracellular esterases has also been used to generate an OFF-to-ON change in emission, 19 as has the nitroxide exchange reaction of an NMP synthesised polymer with an isoindoline conjugated to (and therefore quenched by) a nitroxide. 20 Fluorescence emission can also be triggered where the labelling reaction is also the uorophore forming reaction, i.e. two non-uorescent groups react to form a uorophore. These are much rarer, as they require the reaction that generates the uorophore to be highly efficient, if it is to have utility as a labelling reaction for macromolecules. The tetrazole-alkene/azirine-alkene cycloaddition results in emissive products as demonstrated by Lin and colleagues, 21 and this reaction has been used for protein-polymer conjugation (PEGylation), 22 and for polymer-polymer conjugation both in solution and on a silicon or cellulose surface. 23 We have recently demonstrated that the conjugate-addition of dibromomaleimide (DBM) with thiols results in the formation of a uorescent dithiomaleimide (DTM) product. 24 This fast and highly efficient reaction has been utilised for PEGylation, 24,25 disulde bridging of proteins for bioconjugation, 26 glycoprotein synthesis, 26a,27 polymer end-group functionalisation, 28 polymer-polymer conjugation, 29 and the synthesis of cyclic 30 and sequence-ordered polymers. 31 We have also shown that the DTM uorophore can be incorporated into a block copolymer micelle at the junction between the core forming poly(lactide) block, and the corona forming poly(PEG acrylate) block. Due to the DTM group's small size and intermediate polarity, it had no detrimental effect on block copolymer selfassembly. In the micellar state the DTM does not self-quench leading to a signicant increase in emission, and a concentration-independent emission and anisotropy prole over 3 orders of magnitude concentration range. 32 Furthermore, timedomain uorescence-lifetime imaging (FLIM) was shown to be able to resolve differences in the supramolecular state in vitro, differentiating assembled and dis-assembled micelles. However, one drawback to this approach is the use of a DTM functional dual ROP initiator/RAFT agent for the block copolymer synthesis, limiting the versatility of this approach.
Herein we report the synthesis and RAFT copolymerisation of novel methacrylate and acrylate uorescent DTM monomers, and chemico-uorescent responsive DBM monomers. We demonstrate that the uorescent DTM monomers result in the formation of highly emissive polymers, while the DBM monomers give access to chemico-uorescent responsive polymers (Fig. 1). We show that these DBM functional polymers undergo a fast and highly efficient conjugation-induced uorescent labelling reaction with thiols, to form uorescent products. We believe that these new DTM and DBM monomers allow a much more versatile and efficient route to polymers labelled with the highly desirable DTM uorophore.

Monomer synthesis
Novel dithiomaleimide (DTM) and dibromomaleimide (DBM) monomers were prepared according to Scheme 1. The methacrylate derivatives dithiomaleimide methylmethacrylate (DTMMA) and dibromomaleimide methylmethacrylate (DBMMA), were synthesised by alkylation of butanethiol-DTM 24 (1) or commercially available 2,3-DBM with bromoacetyl methacrylate 33 (the latter being prepared in a single step according to literature). The acrylate derivatives dithiomaleimide acrylate (DTMA) and dibromomaleimide acrylate (DBMA) were prepared likewise by alkylation with bromoacetyl acrylate 34 (which was also prepared in a single step according to literature). Monomers were puried by ash column chromatography and characterised by 1 H and 13 C NMR spectroscopy (Fig. S1-S4 †) and high resolution mass spectroscopy (see ESI †). HPLC of the Fig. 1 Strategy for the preparation of fluorescent polymers using DTM monomers, and profluorescent polymers that undergo a chemicofluorescent response using DBM monomers.
uorescent monomers DTMMA and DTMA demonstrated the presence of a single uorescent species (Fig. S5 †), whose excitation and emission spectra (recorded in CHCl 3 ) were very similar to the butanethiol-DTM precursor (1). 24 They had a broad excitation spectra with maxima at $260 and $420 nm, with the corresponding emission maximum at $520 nm (Fig. 2), indicating that the emissive properties of the DTM group had not been compromised by incorporation into (meth)acrylate monomers. In this study a simple n-butyl thiol ligand was chosen for the DTM, however it should be noted that a range of alternative functionality could be incorporated into these DTM monomers by varying the choice of thiol ligand, as demonstrated previously. 24

Polymerisation of uorescent DTM monomers
Polymers containing the DTM uorophore could be accessed directly, simply by copolymerisation of the uorescent DTM monomers with (meth)acrylates. We investigated the RAFT polymerisation of DTMMA and DTMA using commercially available chain transfer agents (CTAs). For methacrylate polymerisations 2-cyano-2-propyl benzodithioate was chosen, and for acrylate polymerisations cyanomethyl dodecyl trithiocarbonate was used, to give optimum control over molecular weight for these monomer classes. 4 All polymerisations were performed using typical conditions, namely as a solution in 1,4-dioxane, heating at 65 C, with the radical initiator AIBN and 10 mol% loading of the DTM monomer;  Table 1).
Copolymerisation of DTMMA with the hydrophobic monomer methyl methacrylate (MMA) displayed linear rst order consumption of both monomers, with a linear increase of molecular weight with conversion (as measured by SEC), and low dispersities throughout (Đ M < 1.2), indicating a good control over molecular weight during the polymerisation (Fig. S6 †). Both MMA and DTMMA were consumed at an approximately equivalent rate, to a nal conversion at 9 h of 84% for MMA and 87% for DTMMA. The polymer (P1) was isolated by precipitation into methanol, with 1 H NMR spectroscopy of the puried product revealing incorporation of DTMMA at the expected 10 mol% loading (Fig. S7 †). SEC analysis of P1 (THF eluent) indicated a narrow molecular weight distribution (Đ M ¼ 1.13), with incorporation of the DTM functional group and retention of the dithiobenzoate RAFT end-group indicated by absorption maxima at 413 nm and 307 nm respectively for the polymer peak in the 3D chromatogram collected using a PDA detector (Fig. S8 †). The fact that both MMA and DTMMA were consumed at an approximately equivalent rate, and that the nal polymer had both monomers incorporated at their initial feed ratio, as well as having a narrow molecular weight distribution, suggests a random copolymerisation.
Copolymerisation of DTMMA with the hydrophilic monomer oligoethylene glycol methacrylate (OEGMA, M n ¼ 300 Da) at a 10 mol% loading of DTMMA also proceeded with linear kinetics, and good control over molecular weight (Fig. S9 †). Again both monomers were consumed at an equivalent rate, with the puried polymer (P2) showing the expected 10 mol% loading of DTMMA by 1 H NMR spectroscopy (Fig. S10 †). A narrow molecular weight distribution (Đ M ¼ 1.23), and incorporation of DTM and dithiobenzoate groups was again shown by SEC with a PDA detector (Fig. S11 †). Despite the choice of a hydrophobic n-butyl thiol ligand in the DTMMA monomer, the OEGMA copolymer P2 retained its water solubility and thermoresponsive behaviour. 35 The LCST cloud point of P2 in water (18.2 MU cm) was measured at 10 g l À1 as T c ¼ 50.2 AE 0.0 C  during the heating cycle, and 50.0 AE 0.1 C for the cooling cycle (Fig. S12 †). An analogous homopolymer of POEGMA prepared by RAFT (P9, M n ¼ 10.1 kDa) was found to have T c ¼ 65.8 AE 0.0 C and 65.7 AE 0.1 C for heating and cooling respectively, indicating that copolymerisation with DTMMA had caused an increase in hydrophobicity. Similar success was observed for copolymerisation of DTMA with hydrophobic tert-butyl acrylate (tBA) and hydrophilic triethyleneglycol monomethylether acrylate (TEGA) 36 monomers at a 10 mol% loading of DTMA using the same reaction conditions as in the methacrylate polymerisations (Scheme 2). Again, linear rst order polymerisation kinetics, a linear increase of molecular weight with conversion, and low dispersities were observed (Fig. S13 †). 1 H NMR revealed incorporation of DTMA at 9 mol% and 10 mol% for tBA and TEGA copolymerisations respectively (Fig. S14 and S15 †), while SEC analysis showed narrow molecular weight distributions and incorporation of the DTM and trithiocarbonate chromophores (Fig. S16 †). The water solubility and thermoresponsive behaviour of the P(TEGA-co-DTMA) copolymer was also retained, with an LCST cloud point at 10 g l À1 in water (18.2 MU cm) of T c ¼ 37.3 AE 0.3 C during the heating cycle, and 36.9 AE 0.2 C for the cooling cycle (Fig. S17 †). The analogous PTEGA homopolymer (P10, M n ¼ 10.1 kDa) prepared by RAFT had T c ¼ 65.5 AE 0.0 C (heating cycle) and 65.2 AE 0.0 C (cooling cycle) suggesting that the introduction of the hydrophobic DTMA monomer had caused an increase in hydrophobicity, as was observed for DTMMA (P2).
The uorescence spectra (in CHCl 3 ) of the PDTMMA and PDTMA copolymers (P1-4) are very similar to that of the monomers, retaining the excitation maxima at $260 nm and $420 nm and emission maximum at $520 nm (Fig. 2). The dependence of emission on polymer concentration was studied, using P3 as an example. 2D excitation-emission spectra were collected at concentrations of 5, 1, 0.5 and 0.1 mM (Fig. S18 †). At the higher concentrations aggregate/multimer or dimer emission was observed, while at 0.1 mM emission corresponded to that of uorophore unimers, in accord with the spectrum of the small molecule DTM 1 at the same concentration. This indicates that once the polymer is sufficiently diluted to avoid interchain quenching, there is no signicant intra-chain quenching caused by neighbouring DTM containing repeat units. Molar emission (integrated emission intensity divided by molar concentration) for P3 in CHCl 3 also demonstrates this lack of self-quenching, with a region of concentration independent emission between 0.1 mM and 1 mM (Fig. S19 †).
The results presented in this section demonstrate that the C]C double bond of the DTM motif was unreactive toward radical polymerisation for DTMMA and DTMA monomers, and that no adverse effects on DTM uorescence are caused by incorporation into a polymeric structure, in line with previous ndings. 24,32 Polymerisation of chemico-uorescent responsive DBM monomers again demonstrated linear rst order kinetics, a linear increase of molecular weight with conversion and low dispersity, indicating good control over the polymerisation (Fig. S20 †). 1 H NMR spectroscopy of the puried polymers (P5 and P6) revealed incorporation of DBMMA at 11 mol% and 13 mol% loading, while 13 C NMR of P5 provided additional proof of the incorporation of the DBM monomer as evidenced by the characteristic resonance of the DBM C]C at 129.9 ppm, and C]O at 166.7 ppm (Fig. S21 and S22 †). SEC of P5 and P6 showed narrow molecular weight distributions with Đ M ¼ 1.12 and 1.24 respectively (Fig. S23 †).
Copolymerisations initially proceeded with linear rst order consumption of monomer, however once a certain total monomer conversion had been reached ($60% and $35% for tBA and TEGA respectively) a complete retardation of polymerisation was observed (Fig. S24 †). No loss of control over the evolution of molecular weight was observed (Đ M # 1.2 throughout the polymerisations), indicating that chain transfer to DBM or branching via DBM C]C double bond polymerisation were not the cause of the retardation. To demonstrate that this retardation is caused by the DBM group we conducted a series of RAFT polymerisations of tBA in the presence of 2,3-dibromo-N-methyl-maleimide (DBMM) at a range of DBMM loadings. By measuring initial rates of monomer consumption the order of reaction was found to be À0.68 for DBMM (Fig. S25 †), using the method of Bell et al. 37 In comparison, RAFT polymerisations of MMA in the presence of DBMM gave an order of reaction ¼ À0.14 for DBMM, explaining why no signicant retardation of polymerisation was observed for copolymerisations of DBMMA. This suggests that interaction of the propagating radical with the DBM group is the cause of the retardation, with either the greater stability of the methacrylate radical over the acrylate radical, or its greater steric bulk decreasing this effect. The external order of butanethiol-dithio-N-methyl-maleimide (DTMM 32 ) in tBA and MMA RAFT polymerisations was found to be 0.08 and À0.01 respectively, indicating that the DTM group doesn't interfere in the polymerisations. This is in line with copolymerisations of DTMMA and DTMA (above), and with previous reports. 24,32, 38 1 H NMR spectra of P7 and P8 showed incorporation of DBMA at 11 mol% in both cases ( Fig. S26 and 27 †), with no obvious deviation from the expected product. The 13 C NMR spectrum of P7 clearly showed peaks attributed to the DBM group's C]C (129.8 ppm) and C]O (166.6 ppm) resonances. SEC again revealed good control over molecular weight, with Đ M ¼ 1.14 and 1.24 for P7 and P8 respectively (Fig. S28 †). In line with previous results for polymerisations with a DBM functional RAFT agent, 28 this data suggests that the polymerisation retardation does not lead to loss of the DBM group.
Post polymerisation functionalisation of chemico-uorescent responsive P(OEGMA-co-DBMMA) Post-polymerisation functionalisation of polymers derived from the DBM monomers gives an alternative route to uorescent DTM containing polymers (Fig. 1). The pendent DBM units undergo a highly efficient conjugation reaction with two equivalents of a thiol, allowing introduction of further functionality along the polymer backbone, while simultaneously inducing a chemico-uorescent response resulting in OFF-to-ON switching of uorescence emission. The efficient conjugation of pendent reactive groups along a thermoresponsive polymer backbone has been shown to allow subtle tuning of poly(N-isopropylacrylamide) and POEGMA LCST cloud points. 39 We therefore anticipated that the reaction of P(OEGMA-co-DBMMA) with thiols would also allow for LCST cloud point modication.
To demonstrate this approach we performed the reaction of P(OEGMA 27.6 -co-DBMMA 4.7 ) (P11) with a range of thiols (HS-R) bearing different functional groups (R) according to Scheme 3 and Table 2. P11 was rst dissolved in pH 6 buffer (100 mM sodium phosphate, 150 mM NaCl) at 10 g l À1 , before addition of a small excess of thiol (12 eq. relative to the polymer which corresponds to 2.6 eq. per DBM group) as a 1 M solution in DMF.
The reaction was found to be very fast, with the immediate formation of the yellow/green DTM uorophore observed. Aer purication by dialysis 1 H NMR spectroscopy analysis revealed new resonances attributed to the successfully added thiols. For those products where the new resonances didn't overlap with major POEGMA resonances, conversion was calculated as $95% for double thiol substitution (Fig. S29 †). SEC revealed slight changes in polymer M n as a result of the substitution of -Br with -SR. There was some low molecular weight tailing for P13 due to interactions between the column and the pendent diol groups, while the acid functional P12 failed to elute entirely due to column interactions. The use of a UV-vis SEC detector conrmed transformation of the pendent groups to DTMs, as the polymers absorbed at the DTM absorption maximum (l max ¼ 420 nm in the CHCl 3 SEC solvent) as shown in Fig. S30. † Fluorescence spectra recorded in CHCl 3 solution showed the presence of the DTM uorophore, with excitation maxima at $420 nm and corresponding emission maxima of $520 nm, therefore indicating that the P11 had successfully undergone an OFF-to-ON switching of uorescence emission (Fig. S31 †). LCST cloud point measurements (at 10 g l À1 in 18.2 MU cm water) of the thiol substituted polymers P12-15 revealed that the transition temperature could be subtly tuned either above or below that of the initial polymer through R group choice, with a 11 C range in cloud points obtained ( Fig. 3 and Table 2). The trend in LCST cloud points was found to follow the relative polarities of the thiols used, suggesting that water solubility of the pendent DTM groups was the determining factor in cloud point temperature.
In order to monitor the rate of site-group modication the absorbance due to the DTM uorophore was measured in situ during a post-polymerisation functionalisation reaction. The UVvis spectrum of a solution of P12 in pH 6 buffer was measured. A concentration of 1 g l À1 was required to obtain absorbance <1 at the l max of 409 nm, and this absorbance was taken to correspond to 95% conversion for the post-polymerisation functionalisation reaction (Table 2). Then, to a 1 g l À1 solution of P11 in buffer was added 12 eq. of HS(CH 2 ) 2 CO 2 H as a 1 M solution in DMF, and the absorbance at 409 nm monitored as a function of time. Even at this 10Â dilution from the optimum conditions, the reaction was found to be very fast, reaching 50% conversion within 10 min, and >95% conversion aer 3 h (Fig. 4).

Reversible chemico-uorescent response by thiol-exchange
The work of Baker and Caddick has shown that it is possible to perform a thiol-exchange reaction on DTM, resulting in the elimination of the original thiol ligands and addition of two new thiol ligands. 26a This is due to retention of the maleimide C]C double bond in the DTM group, which therefore allows further conjugate addition-elimination reactions to occur. Furthermore, we have previously shown that the DTM which contains thiophenol (-SPh) ligands has a drastically decreased emission, due to conjugation of the phenyl groups to the DTM ring. 24 Therefore, by performing a thiol-exchange reaction on an emissive DTM-functional polymer with thiophenol, it should be possible to achieve an ON-to-OFF switching of uorescence.
To illustrate this possibility a thiol-exchange reaction was performed using the emissive P(TEGA 31.6 -co-DTMA 3.4 ) copolymer (P4). In order to achieve complete conversion an excess of thiophenol was used, corresponding to 10 eq. per -SBu ligand in P4 (Scheme 4). P4 was dissolved in pH 6 buffer at 10 g l À1 , before addition of thiophenol as a 2.5 M solution in DMF. Emission (l ex ¼ 435 nm) was monitored during the reaction, with a drastic reduction in emission observed within 1 hour (Fig. S32 †). Aer purication by dialysis, 1 H NMR spectroscopy analysis of the product (P16) revealed new resonances attributed to the successfully added -SPh groups, with loss of resonances corresponding to the -SBu groups (Fig. S33 †).

Conclusions
Methacrylate and acrylate monomers bearing dithiomaleimide (DTM) or dibromomaleimide (DBM) functional groups have been synthesised, and successfully polymerised by RAFT polymerisation. The uorescent DTM monomers were found to give uorescent copolymers with MMA, OEGMA, tBA, and TEGA with good control over molecular weight distribution. Copolymers with OEGMA and TEGA retained their thermoresponsive properties, with the 10 mol% loading of DTM monomers causing a decrease in LCST cloud point.
The DBM monomers gave chemico-uorescent responsive copolymers with MMA, OEGMA, tBA and TEGA, again with good control over molecular weight distribution. Polymerisations with DBM acrylate were found to reach a limiting conversion, due to retardation by the DBM group, however no loss of DBM functionality or molecular weight control was observed. The DBM copolymer with OEGMA was shown to undergo a highly efficient chemico-uorescent responsive conjugation with thiols in aqueous media, leading to an OFF-to-ON switching of uorescence emission. These substituted OEGMA copolymers retained an LCST cloud point, which could be progressively tuned by judicious choice of thiol in the conjugation-induced uorescent labelling reaction.
It was also possible to exchange the thiol ligands on the pendent DTM groups of a DTM-functional copolymer. This thiol-exchange reaction was demonstrated using thiophenol, as the resultant dithiophenol maleimide group has drastically reduced emission. Therefore this thiol-exchange reaction results in an ON-to-OFF switching of uorescence emission, demonstrating that the chemico-uorescent response of these polymers can be considered to be reversible.
These new monomers present a straightforward and versatile approach for the labelling of polymeric materials with the DTM uorophore. The effectiveness of DTM labelled polymer nanoparticles for in vitro uorescence-lifetime imaging microscopy (FLIM) has previously been demonstrated, 32 and we anticipate that these new (meth)acrylate monomers will present a convenient route to a wide range of new DTM labelled uorescent materials.