One-pot synthesis of flexible poly-p-(phenyleneethynylene)s with controlled conjugated length

Abstract

A novel monomer design for the synthesis of PPE-type polymers containing conjugated segments of well-defined length connected by flexible linkers under Sonogashira reaction conditions is presented. The resulting polymers retain the photophysical properties of a fully conjugated PPE. The extent of incorporation of the flexible units along the backbone is governed by the comonomer feed ratio and can be varied in a statistically predictable fashion.

Claims

1. A conjugated polymer, comprising a poly(p-phenylene-ethynylene) (PPE) having a flexible linker between a portion of phenylene units, comprising the structure: ##STR00002## wherein R.sub.1 and R.sub.2 independently are substituents that provide solubility and/or a functionality for bonding or associating with a molecule, wherein X is a flexible unit of at least two atoms that do no conjugate with the adjacent phenylenes, and wherein x+y is 2 to 100,000 and x is 0.01y to 100y.

2. The conjugated polymer according to claim 1, wherein the R.sub.1 and R.sub.2 are independently a polyethyleneoxide oligomer or an alkyl group, optionally terminated with a functionality comprising at least one amine, carboxylic acid, thiol, hydroxy, or any combination thereof.

3. The conjugated polymer according to claim 1, wherein the (PPE) has the structure: ##STR00003##

4. The conjugated polymer according to claim 3, wherein R.sub.1 is: ##STR00004## and R.sub.2 is: ##STR00005##

5. The conjugated polymer according to claim 1, wherein x is 0.1y to 10y.

6. The conjugated polymer according to claim 1, further comprising at least a fourth repeating unit.

7. A complex comprising, a conjugated polymer according to claim 1 and a molecule having a complementary functionality for complexation to a functionality of at least one of R.sub.1 and R.sub.2.

8. The complex according to claim 7, wherein the molecule is a biopolymer.

9. The complex according to claim 7, wherein the molecule is hyaluronic acid.

10. The complex according to claim 7, wherein the complex is in the form of a nanoparticle.

11. A method of preparing a conjugated polymer according to claim 1 comprising: providing at least one diacetylene substituted phenylene monomer and/or at least one dihalo substituted phenylene monomer; providing at least one monomer comprising a flexible linker at least two phenylenes substituted with acetylene functionality or halo phenylene functionality; providing a Sonogashira catalyst; and combining the at least one diacetylene substituted phenylene monomer and/or at least one dihalo substituted phenylene monomer with the at least one monomer comprising a flexible linker with the Sonogashira catalyst, wherein condensation occurs to form the conjugated polymer according to claim 1.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a reaction scheme for the preparation of terpolymer PPEs having a flexible linker according to an embodiment of the invention.

(2) FIG. 2 is a .sup.1HNMR spectrum of a terpolymer PPE having a flexible linker according to an embodiment of the invention.

(3) FIG. 3A is a composite of normalized absorption spectra, and FIG. 3B is a composite of normalized emission spectra of polymers P1, P2, P4 and P6 in DMF for various co- and terpolymer PPEs according to an embodiment of the invention.

(4) FIG. 4 is a composite of normalized emission spectra of polymers P4, P5, and P6 in DMF according to an embodiment of the invention.

(5) FIG. 5 shows a reaction scheme for an alternate preparation of co- or terpolymer PPEs, according to an embodiment of the invention.

(6) FIG. 6 shows a reaction scheme for the synthesis of a di(haloaryl) flexible spacer, according to an embodiment of the invention.

(7) FIG. 7 is a .sup.1HNMR spectrum of a terpolymer PPE having a flexible linker according to an embodiment of the invention.

(8) FIG. 8A is a composite of normalized FTIR spectra, FIG. 8B is a composite of normalized absorption spectra, and FIG. 8C is a composite of normalized emission spectra of polymers PPB, PPE, and PE-dPPB for various co- and terpolymer PPEs according to an embodiment of the invention.

(9) FIG. 9 shows structures for the components and a scheme for the formation of a complex between hyaluronic acid and an amine substituted PPE, according to an embodiment of the invention.

(10) FIG. 10 shows composite absorption and emission spectra for uncomplexed PPEs and their complex with hyaluronic acid, according to an embodiment of the invention.

(11) FIG. 11 shows micrographs of PPE-hyaluronic acid complexed nanoparticles, according to an embodiment of the invention.

(12) FIG. 12A shows structures of PPEs and FIG. 12B shows plots of Golgi localization affinities for the structures of FIG. 12A.

(13) FIG. 13 shows a plot of cell viability for their exposure to the PPEs of FIG. 12A, according to an embodiment of the invention.

DETAILED DISCLOSURE

(14) A novel monomer design for the synthesis of PPE-type polymers containing conjugated segments of well-defined length connected by flexible linkers under Sonogashira reaction conditions has been developed. The resulting polymers retain the photophysical properties of a fully conjugated PPE. The extent of incorporation of the flexible units along the backbone is governed by the comonomer feed ratio and can be varied in a statistically predictable fashion. Increased flexible content translates to the formation of segments of shorter conjugation length, and the precise control of the amount of flexibility therefore provides a means to control the length of conjugated segments within a polymer chain.

(15) Specifically, the subject invention provides a novel monomer design under modified Sonogashira conditions for the formation of PPE-type polymers with precisely controlled amount of the flexible component and well-defined length of the corresponding conjugated segments. Increasing the amount of flexible units at the expense of the conjugated ones leads to the formation of shorter conjugated segments, and the conjugated length can thus be shortened in a statistically predictable fashion. Optical properties of the fully conjugated backbone are preserved in polymers with flexible content less than 25%.

(16) The incorporation of a variable proportion of a particular monomer into a polymer formed under Sonogashira conditions requires the use of a three-monomer system with one A-type monomer and a variable feed ratio of two different B-type monomers. This system was utilized by Swager et al..sup.12 who introduced a variable amount of anthracene units into the PPE polymers using a three-monomer one-pot Sonogashira-type polymerization with a fixed amount of the diacetylene monomer and a variable ratio of the aryl halide and di-bromoanthracene monomers. Stoichiometric incorporation was unfortunately not observed, presumably due to the difference in aryl halide reactivity. However, other coupling chemistries (e.g. Wittig reaction.sup.13, Suzuki coupling.sup.14) have successfully utilized such a ternary system to control the statistical, random percent incorporation of a particular functional unit into a polymer backbone.

(17) The success of the stoichiometric incorporation of the flexible unit into the CP backbone under Sonogashira conditions rests heavily on minimizing the likelihood of Glaser-type homocoupling through careful monomer design and optimized reaction conditions. Since the monomer reactivity towards the Sonogashira reaction is heavily governed by the choice of aryl halide and the electronic substitution of its aromatic ring, the stoichiometry of the aryl iodide monomer A was kept constant throughout the polymer series. The feed ratio of acetylene comonomers B1 (conjugated) and B2 (flexible) was varied to achieve the controlled conjugation length modulation, as indicated in the reaction scheme shown in FIG. 1.

(18) The factors that determine the reactivity of the acetylene monomer towards Sonogashira coupling are widely unexplored, although electron-donating groups have been recently shown to enhance the nucleophilicity of the copper acetylide, which increases the rate of the transmetalation step..sup.15 The predicted pK.sub.as for the B1 and B2 acetylene protons are, however, very similar (23.4 and 23.7 respectively),.sup.16 and electronic effects can therefore be ruled out as a contributing factor towards any reactivity difference.

(19) Monomer A with a fixed amount of incorporation was decorated with ethylene oxide side-chains containing pendant guanidinium groups. The guanidinium group is an amine-rich functionality naturally occurring in the amino acid guanidine, consisting of two delocalized primary amines and one secondary amine with a high pK.sub.a of around 13..sup.17 This functional group has been employed in a wide variety of synthetic materials to improve solubility and cellular uptake,.sup.18 and it can be included in the polymers for future use in cellular applications.

(20) The design of the flexible diacetylene monomer B2 is based on modified cystine, a naturally occurring biomolecule. In addition to its non-conjugated, flexible nature, it provides a biodegradable moiety for applications in intracellular gene delivery..sup.19 The fully conjugated complementary diacetylene monomer B1 has been decorated with long ethylene oxide chains to preserve polymer solubility.

(21) The optimization of the polymerization conditions was carried out in order to find a suitable system for the incorporation of the biodegradable, flexible, disulfide-containing monomer B2. The initial polymerization of B2 with aryl iodide A (e.g., attempt at P6, a polymer with the highest possible flexible content) under typical Sonogashira conditions (Pd[Cl.sub.2(PPh.sub.3).sub.2], CuI, DMF/morpholine) did not yield any polymers with considerable molecular weight, an observation contrary to polymerizations containing a structurally similar flexible monomer lacking the disulfide bond. Due to the lack of use of disulfide-containing reactants in Sonogashira coupling reactions in the literature, together with reported complexation of thiols, and disulfides with the palladium metal,.sup.20 the presence of monomer B2 in the Sonogashira coupling cycle may inhibit the reaction through complexation with the palladium species, where the monomer acts as a bidentate ligand coordinating through the sulfur and nitrogen atoms.

(22) Sonogashira reactions often use ligands to accompany the palladium source for improved catalytic cycle efficiency..sup.3 Therefore the introduction of a ligand with stronger affinity than B2 towards palladium should lead to efficient polymerization despite the presence of the disulfide-containing monomer.

(23) A screening of reaction conditions using monomers A and B2 under systematically varied combinations of Pd[Cl.sub.2(PPh.sub.3).sub.2], Pd[(PPh.sub.3).sub.4] and Pd(OAc).sub.2 with bidentate ligands 1,4-bis(diphenylphosphino)-butane (dppb), 1,1-bis(diphenylphosphino)ferrocene (dppf), and 2,2-bipyridyl (bpy) showed that the presence of dppf in the coupling reaction greatly improves the resulting polymer molecular weights. The optimized reaction conditions (Pd(OAc).sub.2, dppf, CuI, DMF/THF/DIPA) were therefore used to prepare all polymers containing disulfide monomer B2 (FIG. 1).

(24) The resulting series of polymers with variable proportion of flexible, non-conjugated biodegradable linker B2 was prepared by varying the relative stoichiometric ratio of monomers B1 and B2 from 1:0 (no linker B2, fully conjugated PPE, P1) to 0:1 (only linker B2, conjugated trimer units, P6). The physical and photophysical properties are summarized in Table 1, below. The flexible content is denoted as x and represents the percentage of B2 out of total amount of diacetylene monomer used. The conjugation length is represented by n which denotes the number of arylene units within the conjugated segment. For example, P6 is synthesized exclusively from the reaction between monomers A and B2, resulting in a polymer with three consecutive arylene conjugated units connected via the cystine linker. For P6 x is therefore 1, and n is equal to 3. The total flexible content in P3 would be 50%a half of the amount of the B2 proportion x, owing to the fact that the iodo monomer A and the combined acetylenes are always reacted at a 1:1 ratio.

(25) TABLE-US-00001 TABLE 1 Comparison of the Physical and Photophysical Properties of polymers P1-P6 with varying conjugation length. A B1 B2 M.sub.n .sub.max,abs .sub.max,em QY Polymer equiv. equiv. (=x) n.sup.a (g/mol).sup.b PDI.sup.c (nm).sup.d (nm).sup.d,e (%).sup.f P1 1 1 0 n/a 18,800 1.28 441 476 25 P2 1 0.90 0.10 21 10,700 1.50 425 473 17 P3 1 0.75 0.25 9 11,800 1.47 417 472 20 P4 1 0.50 0.50 5 12,200 1.50 399 469 13 P5 1 0.75 0.25 3/5 5,300 1.54 361 453 6 P6 1 0 1 3 13,400 1.70 358 406 5 .sup.aStatistically predominant number of phenylene rings within a conjugated segment. .sup.bDetermined by gel permeation chromatography in THF. .sup.cPDI (polydispersity index) = M.sub.w/M.sub.n. .sup.dMeasured in DMF. .sup.eExcitation wavelength 440, 420, 415, 395, 360 and 355 nm in DMF. .sup.fQuantum yield in DMF measured relative to diphenylanthracene standard.

(26) The incorporation of the flexible unit was evaluated by .sup.1H NMR spectroscopy, as shown in FIG. 2. The analysis of predicted copolymer structures examined the alkoxy protons on the side-chain of monomers A and B1 (H.sub.EO, 4.2 ppm), the guanidinium NH protons characteristic to monomer A (H.sub.A, combination of 11.4 and 12.4 ppm), the methoxy protons characteristic to monomer B1 (3.2 ppm), and the aromatic protons (H.sub.B, 7.45 and 7.65 ppm) and amine NH (H.sub.B, 10.4 ppm) characteristic to monomer B2. All proton peaks were integrated relative to the ethylene oxide proton peak H.sub.EO, and were in good agreement with the integration values predicted by theoretical copolymer analysis. An example spectrum of polymer P3 is presented in FIG. 2. Based on the B1:B2 feeding ratio (0.75:0.25), the predicted polymer structure calls for H.sub.EO proton integration of 3.5 (0.75*4H+0.25*2H). The observed integrations of the remaining peaks correspond well to the predicted values: for H.sub.A, predicted 1H (0.75*1H+0.25*1H), observed 0.98 (0.29+0.69), for H.sub.B predicted 0.5H (0.25*2H), observed 0.48 (average of 0.43 and 0.53), and for H.sub.B predicted 0.25, observed 0.16 (due to deuterium exchange). The observed integration ratios for all polymers are reported in the Supporting Information together with percent deviations from predicted values. While .sup.1H NMR data point towards stoichiometrically controlled B2 incorporation, the technique only provides information about the average sample, and it is therefore not a reliable tool for the prediction of the length of the individual conjugated segments.

(27) A more direct evidence of conjugation length modulation is demonstrated by absorption and emission spectra of the polymers P1-P6, which clearly show a blue shift in absorbance maxima with increasing flexible, non-conjugated linker content (i.e. higher x, lower n) (FIG. 3A). This shift indicates that the predominant conjugated units within the polymers indeed structurally correspond to the average units characterized by NMR. A similar trend was expected with the fluorescence maxima of the six polymers (FIG. 3B).

(28) The experimental observations, however, only show this trend with the last three polymers in the series, P4-P6 (FIG. 4). The polymer P6 with the shortest conjugated segments (x=1, n=3) exhibits blue fluorescence at 406 nm as expected, while in polymer P4, in which the B1/B2 feed ratio statistically favors the predominant formation of pentamers (n=5), the emission maximum is significantly red-shifted at 469 nm (>60 nm Stokes shift). Polymer P5, which contains both trimers and pentamers based on the monomer feed ratio (B1:B2 0.25:0.75), shows two emission maxima, one at the wavelength of the trimer (n=3, 408 nm) and one significantly red shifted (453 nm) corresponding to pentamer (n=5). The overlap between the trimer emission and the pentamer absorption maxima (408 nm and 399 nm respectively) leads to resonance energy transfer (RET) from the shorter, higher-energy trimer units to the longer, lower energy pentamers, resulting in the pentamer units being the major emitting species. This explanation is consistent with the observed P5 emission spectrum which features the predominant red-shifted emission at 453 nm. Past studies of a structurally related conjugated poly-p-(phenylynevinylene)s (PPVs) observed a similar trend..sup.13 Furthermore, the emission peaks of the shorter polymers undergoing RET are very broad, while polymers with increased conjugation length and consequent decrease of the amount of RET (P1 and P2), vibrational structure can be observed. No further significant red-shifting of emission maxima occurs with additional increase in conjugation length past pentamer (P4.fwdarw.P1), suggesting that the effective conjugation length for this series of PPEs is reached with approximately 5 aromatic segments. Theoretical calculations on a series of oligo p-phenylene-ethynylenes.sup.21 agree that while in perfectly planar systems the effective conjugation length is saturated at around 10 aromatic units, this number decreases to around 5 when the planarity of the conjugated segment is disturbed. Thus by keeping the amount of the flexible linker to below 25% of the total polymer content (P4 and longer), it is possible to fabricate flexible polymers with good solubility and photophysical properties exhibiting those of a fully-conjugated PPE polymer backbone.

(29) Advantageously, this step-growth polymerization, which is expected to be completely random, leads to a series of polymers with relatively well-defined, predictable pattern of monomer incorporation as evidenced by the UV absorbance trend. One explanation is that a preferential formation of the PPE-conjugated oligomeric segments happens between monomers A and B1, which are subsequently linked up into a polymer in a reaction with B2. To test this hypothesis, Synthesis of polymer P3 was carried out by the sequential addition of monomers. A (1 eq) and B1 (0.75 eq) were allowed to react for 2 hours in the absence of linker B2. The resulting oligomers P3a exhibited photophysical properties similar to the one-pot polymer P3. When the remaining monomer B2 (0.25 eq) was added and the polymerization allowed to proceed overnight, polymer P3b exhibited an increase in polymer molecular weight relative to oligomers P3a and conserved photophysical properties analogous to those of P3. The number average molecular weight of the polymer P3b more than doubled compared to oligomers P3a, and .sup.1H NMR clearly demonstrates the incorporation of linker B2 into the polymer (see SI).

(30) TABLE-US-00002 TABLE 2 Sequential addition experiment. M.sub.n .sub.max,abs .sub.max,em Polymer A B1 B2 (g/mol) PDI (nm) (nm) QY (%) P1 1 1 0 18,800 1.28 441 476 25 P3 1 0.75 0.25 11,800 1.47 417 472 20 P3a 1 0.75 2,000 2.15 425 476 12 P3b 1 0.75 0.25 5,100 1.90 420 472 10

(31) This hypothesis assumes that B1 will undergo coupling in the Sonogashira cycle more readily than B2. The acetylene proton pK.sub.a values are comparable between B1 and B2, so the formation of the copper acetylide is not considered a contributing factor in the monomer reactivity. In the Sonogashira catalytic cycle the transmetalation step is typically the rate-determining step,.sup.15 during which the copper acetylide is transferred to the palladium-arene-iodide complex. Preferential incorporation of B1 into the polymer appears to be due to the compact structure of the monomer which allows for a more straightforward transmetalation event compared to the large, flexible, long and freely rotating monomer B2. However, the copper cycle which facilitates the addition of the alkyne species to the palladium catalytic cycle is not well understood, and other factors such as monomer solubility can play a role in determining its reactivity in this complex system.

(32) In one embodiment, the subject invention provides a three-monomer system with a variable feed ratio of a flexible, biodegradable, non-conjugated unit under Sonogashira coupling conditions that leads to the stoichiometric incorporation of the monomers into the polymer backbone, allowing for the tuning of the length of the conjugated segments within the polymer. In accordance with the subject invention, polymers with 25% or less flexible content exhibit photophysical properties analogous to that of a fully-conjugated PPE backbone. The subject invention facilitates the controlled incorporation of additional features such as flexibility and biodegradability into the conventional PPE polymers without the loss of their excellent photophysical properties.

(33) In embodiments of the subject invention, the flexible linker can be provided by a diacetylide monomer or the dihaloaryl monomer. The use of a flexible linker between two aryl halides is shown in FIG. 5 that is prepared as shown in FIG. 6. In this embodiment of the invention, the conjugation length depends upon the coupling conditions, where a Glaser coupling promotes long conjugation sections from the coupling of diacetylene monomers and short (trimer) conjugation lengths from Sonogashira coupling. A proton NMR spectrum of a polymer made according to the scheme of FIG. 5 is shown in FIG. 7. Composite FTIR, absorbance, and emission spectra, for the different PPEs formed by Glaser coupling, Sonogashira coupling, and competitive coupling is shown in FIG. 8A, FIG. 8B and FIG. 8C.

(34) The PPEs with flexible linkers can form complexes between the amines and carboxylic acid groups of polymers, including biopolymers, for example, a complex with hyaluronic acid, as illustrated in FIG. 9. The complexation can be observed by absorption and emission spectroscopy, as shown in FIG. 10. The complexation from solution results in the formation of nanoparticles, as shown in FIG. 11.

(35) The PPEs with flexible linkers can be modified by the side groups attached to the aromatic units of the polymers. For example as can be seen in FIG. 12A, the structure can be varied, and depending of the polymers microstructure, the structural variation leads to selectivity of the polymers for specific cells, as indicated the Golgo localization selectivity illustrated in FIG. 12B. The structures and photophysical properties for the polymers of FIG. 12A are tabulated in Table 3, below. The functional groups effected toxicity to cells depending on the side groups, as shown in FIG. 13.

(36) TABLE-US-00003 TABLE 3 Structures and properties of polymers prepared from competitive Glaser and Sonogashira coupling. Abs Emission Hydrodynamic Zeta M.sub.n .sub.max.sup.c .sub.max.sup.d Quantum radius Potential CPN Type (kDa).sup.a PDI.sup.b (nm) (nm) Yield.sup.e (d .Math. nm).sup.f (mV).sup.f P1 PPE 11.8 1.43 427 492 2% 71.22 +42.3 P2 PPE 16.4 1.49 433 496 3% 60.55 +20.2 P3 PPE 10.7 1.64 420 496 2% 58.34 +44.3 P4 PE-d-PPB 22.3 2.28 444 500 1% 87.41 +46.2

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