WO2005021150A2

WO2005021150A2 - Labelled polymeric materials

Info

Publication number: WO2005021150A2
Application number: PCT/GB2004/003689
Authority: WO
Inventors: Paul O'brien; Nigel L. Pickett; Andrew J. Sutherland
Original assignee: The University Of Manchester
Priority date: 2003-09-01
Filing date: 2004-08-27
Publication date: 2005-03-10
Also published as: WO2005021150A3

Abstract

A polymeric bead labelled with at least one nanoparticle associated with a moiety which is covalently incorporated in the polymer of the bead. In a preferred embodiment the moiety is a ligand for the nanoparticle and is most preferably a phosphine oxide ligand.

Description

Labelled Polymeric Materials

The present invention relates to labelled polymeric materials and more paticularly to polymeric beads labelled with at least one nanoparticle.

Background of the invention

Approaches to synthetic organic chemistry have developed rapidly over recent years with the increased demand for high speed synthesis and screening techniques. Combinatorial techniques, such as 'split and mix' and high-throughput parallel synthesis are now used routinely. We are concerned in particular with approaches that involve the on-support synthesis and in situ screening of large numbers of diverse molecules prepared using split-and-mix techniques. Whilst there has been much success in synthesising libraries of this type, a major problem with this approach is being able to identify exactly which molecule is attached to a particular bead. To this end, a number of innovative deconvolution approaches have been developed to encode individual beads. These include: chemical encoding with molecular tags¹; organic fluorophores ; fluorescent colloids ; Raman fingerprints ; and radio frequency transponders . More recently, Nie et al have utilised fluorescent inorganic semiconductor quantum dots. This last approach is most attractive since quantum dots offer significant advantages over conventional fluorescent dyes since they are brighter, more photostable materials with narrow emission bands that can be excited by any wavelength greater than the energy of their lowest transition. These properties also allow optical barcoding of polymer supports by combining different colour quantum dots with different intensity levels. However, the approach described to generate the quantum dot encoded materials simply involved embedding the quantum dots into the outer layers of 1- 2 Dm resin beads then sealing with a final silica layer. Whilst encoded materials of this type appear to be ideal for the optical encoding of biomolecules⁶ the method of quantum dots immobilisation is non-covalent in nature and thus materials generated in this manner would not be suitable for widespread application in solid phase organic synthesis. We thus elected to establish the feasibility of incorporating quantum dots covalently into the polymer matrices of supports of the type used routinely in the solid phase synthesis of combinatorial libraries. A recent publication by Emrick et al⁸ described an elegant approach to incorporate quantum dots covalently into spin and solution cast polymer films by pre-coating quantum dots with polymerisable ligand 1. We were intrigued to investigate whether such an approach could be extended to incorporate quantum dots into polymer beads utilising well established suspension polymerisation techniques. We believed that if successful, this new procedure would enable the facile production of many of the bead-types that are available commercially for combinatorial chemistry applications but with the added benefit of quantum dot encoding.

Summary of the Invention

The present invention provides a polymeric bead labelled with at least one nanoparticle associated with a moiety which is covalently incorporated in the polymer of the bead.

"Beads" in accordance with the invention may for example have a maximum cross- sectional size of 30 to 70 microns (e.g. about 50 microns) and may or may not be spherical.

Beads in accordance with the invention may incorporate a single nanoparticle or a plurality of nanoparticles.

The moiety (which is covalently incorporated in the polymer of the bead) is preferably a ligand for the nanoparticle and is most preferably a phosphine oxide ligand.

The polymer of the bead may be an addition polymer.

It is particularly preferred that the polymer of the bead is an addition polymer and that the moiety (e.g. a ligand as aforesaid), prior to its covalent incorporation in the polymer of the bead, included at least one ethylenically unsaturated double bond. The addition polymer may for example be comprised of styrene and/or divinylbenzene residues.

The preferred ligand employed in the invention is a phosphine oxide of the formula P(0)R₁ R₂ R₃ where R_1} R₂ and R₃ are the same or different hydrocarbyl groups, at least one of which, prior to covalent incorporation of the ligand into the polymer of the bead, included at least one ethylenically unsaturated double bond.

Ri and R₂ are preferably n-octyl groups and R₃ is preferably selected from groups of the formula -(CH₂)₅CH₂CH=CH₂ and -CH₂-Ar-CH-CH₂ where Ar is a para- phenylene group.

The bead may incorporate reactive groups (e.g. functional groups) and may be used in solid phase organic synthesis, e.g. in combinatorial synthesis. Polymeric beads as contemplated by the invention may be used as optically encoded solid supports in solid phase organic chemistry.

The beads may be produced by a suspension polymerisation technique. A preferred such technique employs a monomer mixture comprised of the nanoparticle associated with a ligand of the formula P(0) R_ls R and R₃ are as defined above and at least one of styrene and/or divinyl benzene.

The nanoparticles may be nanocrystals, e.g. quantum dots. Nanoparticles for use in the invention may for example be produced as disclosed in US-B-6 379 635. The nanoparticle may be a CdSe quantum dot.

The invention also provides nanoparticles associated with a moiety having a polymerisable group (e.g. an ethylenically unsaturated group). The moiety may be a ligand as described above.

Description of Drawing

In the drawings:

Fig. 1 schematically illustrates preparation of resin beads from a mixture of styrene, divinylbenzene and a quantum dot (QD) material associated with a ligand incorporating ethylenically unsaturated groups; Fig. 2 illustrates fluorescent emission finge rinting of quantum dot-containing polymer beads; and

Fig. 3 shows photoluminescence spectra of quantum dot-containing polymer beads (excitation wavelength = 380nm).

Description of Preferred Embodiment

Quantum dots were synthesised from (Li )[Cd₁₀Se₄(SPh)₁₆] and hexadecylamine as described previously⁹ to give CdSe core particles capped with hexadecylamine. Ligand exchange was then performed to displace the hexadecylamine with one of two polymerisable ligands 1 or 5 (Scheme 1) ϊ'⁸'¹⁰. The structure of each of the ligands is based on the commonly used quantum dot capping agent trioctylphosphine oxide (TOPO) 2, since TOPO has a good affinity for CdSe quantum dots. Success of ligand exchange was confirmed by 1H NMR and by observing the increase in solubility of the coated quantum dots in styrene. Polymerisable ligand 1 was synthesised using the two-step protocol described by

Emrick et al ' and a variation of this route was used to construct ligand 5 (Scheme 1). Specifically, dibutylphosphite 3 was converted into dioctylphosphine oxide 4 by treatment with octylmagnesium bromide followed by an aqueous acid quench. A subsequent sodium hydride mediated alkylation step

Scheme 1 Synthesis of polymerisable ligand 5 with 8-bromo-l-octene furnished polymerisable ligand 5 in good yield.* To facilitate the generation of a relatively large number of beaded materials, a scaled-down suspension polymerisation procedure (20ml reaction volume), utilising a Carousel™ reaction station, was used to generate a series of quantum dot-containing polymer beads (Table 1).^§'¹² For each of the polymer samples described in Table 1, simple visual inspection indicated that quantum dots had indeed been incorporated successfully. However, in the case of beads produced using ligand 5 the extent of quantum dot incorporation, as evidenced by microanalysis, was very low (Table 1). Thus all subsequent investigations utilised beads constructed using ligand 1. Each sample was further analysed by i) fluorescent emission fingerprinting (Figure

Table 1 Compositions of quantum dot-containing beads generated using the small- scale suspension polymerisation procedure.

Entry Mole % Mass QD QD Ligand Microanalysis % DVB / nig C : H Cd

1 2 6.8 1 90.57 7.70 0.72 ^a 87.96 : 7.41 0.67 ^b 89.69 8.01 0.75 ^c

2 20 6.8 1 87.60 7.50 0.19 ^a 90.31 8.22 0.17 ^c

3 2 2.7 5 90.84 8.06 < 0.01 ^a

4 20 2.7 5 92.06 7.95 <0.01 ^a

^a Before Soxhlet extraction. After Soxhlet extraction with dichloromethane for 4 h. After Soxhlet extraction with dichloromethane for 12 h.

2), ii) photoluminescence spectroscopy (Figure 3) and iii) elemental microanalysis after extended periods of Soxhlet extraction (Tablel). Most importantly, both fluorescent emission fingerprinting and photoluminescence spectroscopy of the polymer beads exhibited strong emissions at approximately 522nm corresponding directly with the emission observed for the hexdecylamine coated quantum dots prior to ligand exchange (522 nm) (Figures 2 and 3). Moreover, as Figure 2 shows, two different regions of the same bead give essentially identical emission spectra indicating that quantum dot incorporation is uniform. In addition, the data in Table 1 shows clearly that the amount of cadmium within the beads remains fairly constant, within experimental error, even after prolonged periods of Soxhlet extraction, with dichloromethane (entries 1 and 2). This finding indicates that the dots are incorporated into the beads irreversibly, since the hexadecylamine, ligand 1 coated dots are highly soluble in cold dichloromethane.

In conclusion, we have shown that it is possible to incorporate quantum dots into polystyrene beads by using suspension polymerisation protocols. We are currently extending the protocols described herein to prepare a range of beads with various functional groups and colour/intensities of quantum dots for utilisation in solid phase synthesis. These materials will then be tested for their ability to withstand a wide range of reagents and reaction conditions commonly employed in synthetic organic chemistry.

Notes and references f Quantum dots, 20mg, prepared according to a previously published procedure⁹, were dissolved in 1,4-dioxane, 4ml, containing a large excess of the polymerisable ligand, 1 mmol. The solution was then stirred for 20 hours at room temperature. Centrifugation and drying resulted in ligand exchanged quantum dots (14mg for the p- vinylbenzylDOPO and lOmg for p-octenylDOPO batches).

X Product structures were confirmed by 1H and ¹³C NMR and APCI mass spectrometry.

§ Small-scale suspension polymerisation procedure. Aqueous polvinylalcohol (PVA) solution (20ml, 1% wt/wt, 87-89% hydrolyzed PVA, average MW 85,000-146,000) was prepared and transferred to the reaction vessel (screw cap boiling tube attached to a N₂/vacuum manifold system) and degassed with N for 20 minutes. A monomer mixture - styrene (10 mmol), divinylbenzene and quantum dots (Table 1) - was prepared and stirred for 5 minutes. ALBN (76 μmol) was added to the monomer mixture then the resultant mixture was stirred for a further 15 minutes with N₂ degassing. The dark orange monomer solution (colour due to the quantum dots) was added to the PVA solution while rapidly stirring with a cross-shaped magnetic stirrer. A two phase system resulted with orange droplets being suspended in the uncoloured aqueous PVA solution. The suspension was stirred at room temperature for 30 minutes before the temperature was increased to 70°C and stirring was continued for a further 4 hours. The resin beads were then collected over a 38 μτa sieve and washed in situ with copious amounts of water. The damp beads were then transferred to a sintered funnel and washed with methanol (100 cm³), MeOH/THF, 1/1 (100 cm³), THF (100 cm³), CH₂C1₂ (100 cm³) and acetone (100 cm³).

1 Z.-J. Ni, D. Maclean, C. P. Holmes, M. N. Murphy, B. Ruhland, J. W. Jacobs, E. M. Gordon, M. A. Gallop, J. Med Chem., 1996, 39, 1601; M. H. J. Ohlmeyer, R. N. Swanson, L. W. Dillard, J. C. Reader, G. Asouline, R. Kobayashi, W.C. Still, Proc. Natl. Acad. Sci., 1993, 90, 10922. B.J. Egner, S. Rana, H. Smith, N. Bouloc, J.C. Frey, W.S. Brocklesby and M. Bradley, Chem. Commun., 1997, 735. B.J. Battersby, G.A. Lawrie, A.P.R. Johnston and M. Trau, Chem. Commun., 2002, 1435. H. Fenniri, L. Ding, A.E. Ribbe and Y. Zyianov, J. Am. Chem. Soc, 2001, 123, 8151. TranSort ™, www.irori.com M. Han, X. Gao, J.Z. Su & S. Nie, Nature Biotechnology, 2001, 19, 631. C.B. Murray, CR. Kagan, M.G. Bawendi, Annu. Rev. Mater. Sci., 2000, 30, 545; T. Trindade, P. O'Brien, NX. Pickett, Chem. Mater., 2001, 13, 3843; A.J. Sutherland, Current Opinion in Solid State and Materials Science, 2002, 6, 365. H. Skaff, M.F. Ilker, E.B. Coughlin and T. Emrick, J. Am. Chem. Soc, 2002, 124, 5729. S.L. Cumberland, K.M. Hanif A. Javier, G.A. Khitrov, G.F. Strouse, S.M. Woessner and C.S. Yun, Chem. Mater., 2002, 14, 1576. P Reiss, J. Bleuse and A Pron, Nano Letters, 2002, 2 781. R.H. Williams and L.A. Hamilton, J. Am. Chem. Soc, 1955, 77, 3411; N. Platzer, F. Dardoise, W. Bergeret, J.C. Gautier and S. Raynal, Phosphorus, Sulfur and Silicon and Related Elements, 1986, 27, 275.M.C. McCairn, S.R. Tonge and A.J. Sutherland, J. Org. Chem., 2002, 67, 4847

Claims

1. A polymeric bead labelled with at least one nanoparticle associated with a moiety which is covalently incorporated in the polymer of the bead.

2. A bead as claimed in claim 1 incorporating a single said nanoparticle.

3. A bead as claimed in claim lmincorporating a plurality of said nanoparticle.

4. A bead as claimed in any one of claims 1 to 3 wherein the moiety is a ligand for the nanoparticle.

5. A bead as claimed in claim 4 wherein the ligand is a phosphine oxide.

6. A bead as claimed in any one of claims 1 to 5 wherein the polymer of the bead is an addition polymer.

7. A bead as claimed in any one of claims 1 to 3 wherein the polymer of the bead is an addition polymer and said moiety, prior to its covalent incorporation in the polymer of the bead, included at least one ethylenically in saturated double bond.

8. A bead as claimed in claim 8 wherein the moiety is ligand for the nanoparticle.

9. A bead as claimed in claim 8 wherein the ligand is a phosphine oxide of the formula P(0)Ri R₂ R₃ where R_1; R₂ and R₃ are the same or different hydrocarbyl groups, at least one of which, prior to covalent incorporation of the ligand into the polymer of the bead, included at least one ethylenically unsaturated double bond.

10. A bead as claimed in claim 9 wherein Rγ and R₂ are n-octyl groups and R₃ is selected from groups of the formula -(CH₂)₅CH₂CH=CH₂ and -CH₂-Ar-CH=CH₂ where Ar is a para-phenylene group.

11. A bead as claimed in any one of claims 7 to 10 wherein the polymer of the bead comprises polystyrene and/or polydivinylbenzene.

12. A bead as claimed in any one of claims 7 to 10 having reactive groups.

13. A bead as claimed in any one of claims 1 to 12 wherein the nanoparticle comprises CdSe.

14. The use of beads as claimed in any one of claims 1 to 13 in solid phase organic synthesis, e.g. combinational chemistry.

15. A method of producing a bead as claimed in any one of claims 1 to 14 wherein polymerisation to form the bead and covalenty incorporate said moiety is effected by means of suspension polymerisation.

16. A method as claimed in claim 15 wherein the mixture to be polymerised comprised the nanoparticle associated with said moiety and at least one monomer for forming the polymer.

17. A method as claimed in claim 16 wherein the polymerisation is a free radical polymerisation.

18. A method as claimed in claim 16 or 17 wherein the monomer mixture comprises the nanoparticle associated with a ligand of the formula P(0) R_ls R₂ and R₃ as defined in claim 9 or 10 and at least one of styrene and/or divinyl benzene.

19. Nanoparticles associated with a moiety having a polymerisable group (e.g. an ethylenically unsaturated group).

20. Nanoparticles as claimed in claim 19 wherein the moiety is a ligand.

21. Nanoparticles as claimed in claim 20 wherein the ligand is of the formula P(0) R_\, R₂ and R₃ as defined in claim 9 or 10.

22. Molecular structures that contain one or more polymerisable moieties, none or one or more other chemically reactive moieties and one or more moieties capable of associating in some way to one or more nanoparticles, such as a quantum dot, and a process whereby polymerisation of the molecule, by one of many well known polymerisation procedures, either in the presence or absence of other polymerisable molecules results in the encapsulation of the nanoparticles, either singly or en masse into polymers of any desired shape or size for example a spherical, 50 micrometer, chemically reactive polymer bead.