WO2014188605A1 - Synthesis of pdms-based polyrotaxane, purified pdms-based polyrotaxane and pdms-based polyrotaxane derivatives - Google Patents

Abstract

The present invention provides a new synthetic process of PDMS (polydimethylsiloxane)-based polyrotaxane, which comprises a step of reacting pseudopolyrotaxane consisting of PDMS and CDs (cyclodextrins) with an end-capping agent, by (a) a radical coupling reaction, or (b) a microwave coupling reaction. By the method of the present invention, PDMS-based polyrotaxane can be obtained with high filling ratio of CDs on the PDMS chain.

Description

Description

Title of Invention

Synthesis of PDMS-Based Polyrotaxane, Purified PDMS-Based

Polyrotaxane and PDMS-Based Polyrotaxane derivatives

Technical Field

[ 0 0 0 1 ]

The present invention relates to a synthesis of PDMS (polydimethylsiloxane) -based polyrotaxane, the characterization of a purified polyrotaxane and synthesis of polyrotaxane derivatives.

Background Art

[ 0 0 0 2]

Pseudopolyrotaxanes (PPRs) are physical complexes of macro -cyclic molecules such as cyclodextrins (CDs) threaded on a polymer chain. PPRs are unstable because the macro-cyclic molecules and the polymer chain are not bonded covalently. An end-capping agent with bulky groups should be added at both ends of polymer chain to prevent dethreading of the macro-cyclic molecules from polymer chain. The end-capped pseudopolyrotaxane is called "polyrotaxane" (PR). The nature of the polymer, its molecular weight, the choice of the macrocycle and filling ratio of the macrocyclic molecules on the polymer chain, determine the properties of the polyrotaxane and define its applications. Polyrotaxanes are used as/in materials for various fields such as hydrogels, drug carriers, tissue scaffolds, coatings, photoelectric devices, porous silica materials, insulated molecular wire of conducting materials and the like.

[0 0 0 3 ]

Cyclodextrins (CDs) are the macrocycles of choice for the majority of polyrotaxanes and several excellent reviews have reported CDs-based polyrotaxane synthesis and applications (Wenz, G.; Han, B.H.,' Muller, A. Chem. Rev. 2006, 106, 782-817. Nakashima, N.; Kawabuchi, A. Journal of inclusion Phenomena and Molecular Recognition in Chemistry 1998, 32, 363-373. Nepogodiev, S.A.; Stoddart, F. J. Chem. Rev. 2006, 98, 782-817. Harada, A.,^' Hashidzume, A.; Takashima, Y. Advances in Polymer Science 2006, 201, 1-43. Huang, F.,^' Gibson, H. W. Prog. Polym. Sci. 2005, 30, 982-1018.)

[ 0 0 0 4 ]

Since Harada et al. first introduced the one based on a- CD and Poly-ethyleneglycol (PEG) in 1990 (Harada, A.; Kamachi, M. J. Chem. Soc. Commun. 1990, 1322 -1323), many different linear polymers have been used to construct novel polyrotaxanes with α-, β-, or γ-CDs. Among the polymers used, Poly-dimethylsiloxane (PDMS) offers very specific properties. Since Silicon-based organic polymers are particularly known for their unique rheological properties, PDMS is widely used for its intrinsic flexibility and thermostability. Fields of application include solf lithography, contact lenses, lubricants, and shampoos (Smith, A. L. Chemical Analysis 1974, 41, Analysis of silicones.).

In particular, in the field of contact lens such as soft contact lens (SCL) and hard contact lens, PDMS is used as a base material for high oxygen permeability contact lens materials. However, PDMS based material is generally hydrophobic and therefore, surface treatment is often required so as to provide a hydrophilic surface. But hydrophilic PDMS based material could be obtained by including PDMS polymer inside the CDs like in polyrotaxane.

In addition, PDMS is unstable in acidic conditions. Therefore, use of PDMS based materials for medical implants can be problematic. But in the case of polyrotaxane, the PDMS is covered by the CDs and thus, the stability of PDMS could be increased.

[ 0 0 0 5 ]

Surprisingly, despite the obvious advantages of developing PDMS based polyrotaxane as a new class of Organic -Inorganic material (Kato, K .; Komatsu, H. ; Ito, K. Macromolecules 2010, 43, 8799-8804.) very few groups have published their synthesis. The formation of Poly-dimethylsiloxane inclusion complexes with β-CD and γ-CD were first reported by Harada et al. in 2000 (a) Okumura, H.; Okada, M. >' Kawaguchi, Y. ; Harada, A. Macromolecules 2000, 33, 4297-4298, b) Okumura, H. ,^' Kawaguchi, Y. ; Harada, A. Macromolecules 2001, 34, 6338-6343). But it was not until 2007 that the synthesis of the PDMS-polyrotaxane began to be published.

[ 0 0 0 6 ]

The inventors of the present invention know only two groups that have reported the successful coupling reaction of α,ω-telechelic polydimethylsiloxane with specific blocking groups preventing the dethreading of the larger cyclodextrins (Kato, K.J Inoue, K.J Kidowaki, M.»^' Ito, K. Macromolecules 2009, 42, 7129-7136; a) Sukhanova, T. E.; Perminova, M. P.; Bronnikov, S. V.; Grigor'ev, A. I.; Volkov, A. Ya.J Gubanova, G. N.J Kutin, A. A.; Marangoci, N.; Pinteala, M.; Harabagiu, V.; Simionescu, B. Russian Journal of Applied Chemistry 2008, 81, 2145-2150; b) Marangoci, N.J Farcas, A. ; Pinteala, M.; Harabagiu, V.,^* Simionescu, B. Sukhanova, T. E.; Perminova, M. P.; Grigor'ev, A. L,^" Gubanova, G. N.J Bronnikov, S. V. J. Inch Phenom. Macrocycl. Chem. 2009, 63, 34, 355-364.)

[ 0 0 0 7 ]

Ito et al. demonstrated the synthesis of PDMS-polyrotaxane sparsely populated with γ-CD (WO2008/155953). Modification of the hydroxyl groups of the γ-CD improved their solubility in various solvents which allowed characterization. Rotaxanation and purity of the macromolecule was finally proven by SEC (GPC). The first Organic-Inorganic hybrid slide-ring gel was reported in the same publication.

[ 0 0 0 8 ]

The filling ratio referred in the present specification is a ratio of the total number of [CDs threaded on one PMDS polymer chain] to the maximum number of [CDs which can be threaded on the same PDMS polymer chain, the "ideally full-packed PR"]. More specific definitions are described later.

With the nature of the polymer, its molecular weight and the nature of the macrocyclic compound, the filling ratio determines the properties of the polyrotaxane and defines its applications.

To the best of the inventor's knowledge, a method to control the synthesis of PDMS-based polyrotaxane with different filhng ratio (low to high filling ratio) has not been clearly established yet. One reason is that an appropriate methodology to evaluate the filling ratio had not been established if the purity of the product is not proven (no free macrocycles).

[ 0 0 0 9 ]

Changing the polymer backbone of the polyrotaxane with PDMS is not straightforward and specific end-capping synthesis methods have to be developed.

Obviously dethreading of the macrocycles can become an issue when making densely packed PDMS-polyrotaxane. [Summary of Invention]

[ 0 0 1 0 ]

In this study, a new method for synthesis of PDMS-based polyrotaxanes is provided which allows the controlling of the filling ratio of polyrotaxane and the obtainment of densely packed polyrotaxane (High filling ratio PR). Also a new method to provide pure PR is provided. As a result, an exact filling ratio can be determined.

The inventors of the present invention assumed that the low conversion yield [PPR into PR] and low filling ratio of the corresponding PDMS-based PR are due to: (i) intrinsic hydrophobic nature of the PDMS polymer chain, (ii) extremely poor accessibility of the PDMS telechelic functions to the capping agents (difference of solubility of PDMS, PDMS-based PPR and the capping agents), (iii) PDMS's "thicker" main chain polymers which requires larger CDs (β-CD or γ-CDs). Conditions (i), (ii) and (iii) result in the de-threading of the CDs from PDMS before coupling the PDMS with an end-capping agent.

The present invention relates to, but is not limited to, a method of synthesis of a polyrotaxane consisting of PDMS and CDs with end-capping agent.

The present invention provides a method of synthesizing

PDMS-based polyrotaxane with controlled filling ratio via radical end-coupling or microwave coupling to prevent the CDs from dethreading. As the result of the investigation and experiments by the inventors of the present invention, it is recognized that PDMS-based polyrotaxane with high filling ratio of CDs can be obtained. The pure polyrotaxane was isolated by preparative column chromatography and characterized by NMR spectroscopy and mass spectrometry. The detailed structural analysis demonstrates without ambiguity the obtainment of the polyrotaxane molecular necklace.

[ 0 0 1 1 ]

Herein we disclose two methods to synthesize polyrotaxane with a high filling ratio: (i) a radical coupling reaction and (ii) a microwave coupling reaction.

1. Radical coupling reaction:

As already reported, the radical coupling reaction allowed the obtainment of full packed Polyethylene oxide (PEO) based polyrotaxanes in very high yield (95% conversion yield) (N. JARROUX, P. GUEGAN, H. CHERADAME, L. AUVRAY. The Journal of Physical Chemistry B, 109, 23816-23822 (2005)). This almost quantitative conversion yield into the polyrotaxane was actually achieved by reaction of 1-pyrene butyric acid N-hydroxy succinimide ester (PBS) on the methacrylate telechelic functions of the POE polymer chain previously threaded in crCDs (Model Reaction). Depending on the nature of the polymer backbone, its molecular weight and the number of CDs on the macromolecule, eliminating the free macrocycles can be quite challenging, and characterization is quite complex.

To the inventor's best knowledge, this is the first complete characterization of a pure PDMS-based polyrotaxane. The detailed structural analysis demonstrates without ambiguity the obtainment of the polyrotaxane molecular necklace with a high filling ratio. 2. Microwave coupling reaction:

Taking advantages of advances in microwave chemistry, the goal of this study is to work in conditions with no solvent to prevent polyrotaxane dethreading, and still get the polyrotaxane in a good yield. The reaction studied herein is the alcoholysis or an aminolysis of isocyanate or thioisocyanate functions assisted by microwaves to block the pseudopolyrotaxane. The synthesis of high molecular weight compounds attributed to the PR was proved. To the inventor's best knowledge this is the first time the PDMS-based polyrotaxane has been synthesized (a) in conditions with no solvent, (b) under microwave irradiation.

In addition to the above, the microwave coupling reaction is not limited to use of PDMS in the preparation of polyrotaxane. In other words, linear polymer chains which are known to be used for the polyrotaxane can be used for the microwave coupling reaction.

[ 0 0 1 2 ]

Hereinafter, we explain in more detail two methods to synthesize polyrotaxane with high filling ratio: (i) radical coupling reaction and (ii) microwave coupling reaction.

[ 0 0 1 3 ]

(i) Radical coupling reaction

The radical coupling reaction is a fast and quantitative method to obtain the full packed POE -based polyrotaxane in a very high yield (JARROUX, P. GUEGAN, H. CHERADAME, L. AUVRAY, The Journal of Physical Chemistry B, 109, 23816-23822 (2005)). However, the same method did not allow obtaining the PDMS'based polyrotaxane in high yield due to^: (i) the intrinsic hydrophobic nature of the PDMS polymer chain, (ii) extremely poor accessibility of the PDMS telechelic functions to the capping agents (difference of solubility of PDMS, PDMS'based PPR and the capping agents), (iii) PDMS's "thicker" main chain polymers which requires larger CDs (β-CD or γ-CDs). Conditions (i), (ii) and (iii) result in the de-threading of the CDs from PDMS before coupling. Thus, the inventors of the present invention investigated to dense a method to obtain the PDMS-based PR in relatively good yield with high filling ratio.

In the conditions used for the synthesis of POE-based polyrotaxane, the reaction mixture is too compact (like paste). But working in more dilute conditions by adding solvents leads to dethreading. Then, the inventors found that adding a solution, preferably a saturated solution of CDs or of [CD/compound A] complexes to the reaction mixture is efficient to increase the conversion yield [PPR into PR] and the PR filling ratio. The reaction mixture is then fluid which certainly allows for increasing the accessibility of the PDMS telechelic functions to the end-capping agent. Because the reaction mixture is saturated with CDs, the CDs already threaded on the PDMS will not dethread and even so the CDs added to saturate the reaction mixture should replace the CDs that would eventually dethread.

Different filling ratios shall be obtained by modifying the concentration of the solution of CDs or of [CD/compound A] complexes. This shall allow for making PDMS-based polyrotaxane with different filhng ratios, enlarging the range of material application investigations.

The solution of [CDs^compound A] complexes is prepared by stirring

CDs with Compound A, which has a good affinity to the cavity of the CDs or has a chemical structure similar to the end-capping agent or has a similar chemical structure of the bulky group of the end-capping agent. By adding the solution of complexes in the reaction mixture, the end-capping agents will not be trapped in the cavity of the CDs, which could reduce the efficacy of the end-coupling reaction. The end-capping agent should be free to react on the PDMS extremities (first hypothesis). Actually, the reaction with only free CDs also gives good results.

[ 0 0 1 4 ]

A first aspect of the present invention is a method of preparing a PDMS-based polyrotaxane comprising a step of reacting pseudopolyrotaxane consisting of PDMS and CDs with an end-capping agent, by (i) a radical coupling reaction.

Another aspect of the present invention is a method of preparing a PDMS-based polyrotaxane comprising steps of

(a) preparing a solution, preferably a saturated solution of CDs or [CDs^compound A] complexes, compound A having a good affinity to the cavity of the CDs or having a chemical structure similar to the end-capping agent,

(b) adding the mixture solution prepared in step (a) to a mixture of pseudopolyrotaxane, radical initiator and end-capping agent, and stirring the resulted mixture, and

(c) working up the reaction mixture in step (b) to thus obtain a PDMS-based polyrotaxane. [ 0 0 1 5 ]

(ii) Microwave coupling reaction

The present invention provides another method of synthesis of polyrotaxane with high filling ratio via microwave coupling reaction. The nature of the linear polymer chain used to construct the polyrotaxane is not limited and includes linear polymer chains which are known to be used for polyrotaxane such as polydimethylsiloxane (PDMS), polyoxyethylene (POE) polymer, polylysine polymer, polycarbonate polymer, and the like (including copolymer). The synthesis of polyrotaxane via microwave coupling reaction is preferable for hydrophobic polymer chains such as polydimethylsiloxane (PDMS) as the method allows working with no solvent restraining dethreading, which is one of the limiting factor with this type of polymer chains.

Previous experiments made on the polyrotaxane synthesis permitted to determine that the de -threading is the limiting factor of the coupling reaction.

Taking advantage of advances in microwave chemistry, the goal of this study is to work in conditions with no solvent to prevent polyrotaxane dethreading, and still obtain the polyrotaxane in a good yield.

The reaction studied is the alcoholysis or aminolysis of isocyanate or thioisocyanate functions assisted by microwaves to block the pseudopolyrotaxane with end-capping agent. A variety of end-capping agents were used to study the effect of the nature of the bulky groups on the reaction. Also, reactions with solvent and with no solvent were studied for comparison.

The studies show the feasibility of the polyrotaxane synthesis assisted by microwaves is easier and faster than conventional methods. In fact, the use of reactants in mass without solvent presents many advantages for industrialization. This point is also advantageous in controlling the number of CDs borne by the polyrotaxane. Without solvent, no dethreading should be observed and densely packed PR should be obtained. Adding known amount of solvent should allow the control of the de -threading and obtainment of the PR with different filling ratio.

[ 0 0 1 6 ]

One aspect of the present invention is a method of preparing a PDMS-based polyrotaxane comprising a step of reacting pseudopolyrotaxane consisting of PDMS and CDs with end-capping agent, (ii) under irradiation of microwave.

[ 0 0 1 7 ]

Another aspect of the present invention is a method of preparing a PDMS-based polyrotaxane comprising steps o£

(a) when necessary, the PPRs is prepared with modified cyclodextrins to further prevent the reaction of the end-capping agent on the hydroxyl groups of the cyclodextrins,

(b) mixing the pseudopolyrotaxane and end-capping agent,

(c) irradiating the mixture obtained in step (b) with a microwave, and

(d) working up the reaction mixture in step (c) to thus obtain a

PDMS -based polyrotaxane.

[ 0 0 1 8 ]

The advantages and characteristics of microwave coupling reactions are as follows.

1. Protection of the hydroxyl groups on the CD may be necessary in the microwave reaction to avoid possible side reaction on the OH of the CDs (e.g. NCO on the OH).

2. The use of a polymer or copolymer as an end-capping agent is possible even if the characterization of the obtained polyrotaxane is more difficult.

3. The use of a solid end-capping agent such as CDs, without dissolving in a solvent, is possible.

4. To avoid the heterogeneity of the media and to increase the reaction yield, the use of end-capping agents liquid at the experimental conditions is preferable.

5. The use of solvent may lead to dethreading. [Brief Description of Drawings]

[ 0 0 1 9 ]

Figure 1 shows steps of the preparation of pseudopolyrotaxane.

Figure 2 shows steps of the preparation of complex of γ-CD and pyrene. Figure 3 shows steps of radical synthesis of polyrotaxane.

Figure 4 shows SEC characterization in DMF of the polyrotaxane (run 4) before and after purification.

Figure 5 shows ID O-NMR spectra of pseudopolyrotaxane (A) and ID

Ή-NMR spectra of pure polyrotaxane extracted from run 4 product of reaction (B).

Figure 6 shows 2D ¹H-NMR spectra of pure polyrotaxane.

Figure 7 shows Maldi-Tof MS spectrum of pure polyrotaxane.

Figure 8 shows Ή NMR of FB78f in DMSO"d₆.

Figure 9 shows SEC chromatogram of FB78f in DMF.

Figure 10 shows Maldi-Tof MS spectrum of FB78f in linear mode.

Figure 11 shows Maldi-Tof MS spectrum of FB78f in reflector mode.

Figure 12 shows attribution of peaks on Maldi-Tof spectrum of FB78f in reflector mode.

Figure 13 shows Ή NMR Spectrum of FB81pf in DMSO-d₆.

Figure 14 shows SEC chromatogram of FB81pf in DMSO"d₆.

Figure 15 shows Ή NMR Spectrum of FB86pf in DMSO-d₆.

Figure 16 shows SEC chromatogram of FB86pf in DMSO"d₆. Figure 17 shows Maldi-Tof MS Spectrum of FB87pf in linear mode in DMF.

Figure 18 shows attribution of peaks on Maldi-Tof spectrum of FB87f in reflector mode (figure 20) corresponding to the functionalization of PDMS by copolymer PDPSPDMS-1.

Figure 19 shows attribution of peaks on Maldi-Tof spectrum of FB87f in reflector mode, (figure 20) corresponding to the functionalization of PDMS by copolymer PDPSPDMS-2.

Figure 20 shows Maldi-Tof MS spectrum of FB87pf in reflector mode (zoom).

Figure 21 shows SEC Chromatogram of FB95pf in DMF.

Figure 22 shows SEC Chromatogram of FBlOlpf in DMF.

Figure 23 shows a conceptural scheme of diiscocyanate PDMS and silylated CDs PseudoPR (above) / diamine PDMS and native CDs PseudoPR (under).

Figure 24 shows SEC Chromatograms of reactants used at control.

Figure 25 shows FB132 analysis. The PR quantity is deduced once the calculation of free CDs and FITC is done in the raw product.

Figure 26 shows steps for modification of polyrotaxane.

Figure 27 shows ID Ή-NMR spectrum and SEC chromatogram of modified polyrotaxane FP92orgp.

Figure 28 shows SEC chromatogram of the Polyrotaxane prepared via radical synthesis from [PDMS/Si-γ CD] based PPR.

[Description of Embodiments]

[ 0 0 2 0 ]

1. Polvdimethylsiloxane (PDMS)

In the present specification, polydimethylsiloxane (PDMS) polymer having the following repeating unit-^'

[ 0 0 2 1 ]

In the above formula, n means a number of repeating unit and preferably ranges from 10 to 2000.

The number average molecular weight of polydimethylsiloxane is preferably from 700 to 150000 and more preferably, from 6000 to 75000, and most preferably from 5000 to 11000.

Preferable terminal groups of polydimethylsiloxane used in the methods of the present invention depend on the type of reactions. Therefore, the terminal groups of PDMS are described in each section of "Radical coupling" and "Microwave coupling reaction", below.

The above described PDMS chain can be prepared in accordance with known methods in the art from a commercially available PDMS derivative such as PDMS-diOH available from Shin-Etsu Chemical, co. Ltd.

[ 0 0 2 2 ]

2. Cvclodextrins (CDs)

Cyclodextrins are a group of structurally related natural products formed during enzymatic degradation of starch. These cyclic oligosaccharides consist of (a-l,4)-linked a-D-glucopyranose units and contain a somewhat lipophilic central cavity and a hydrophilic outer surface. Due to the chair conformation of the glucopyranose units, the cyclodextrins are shaped like truncated cones rather than perfect cylinders. The hydroxyl functions are orientated to the cone exterior with the primary hydroxyl groups of the sugar residues at the narrow edge of the cone and the secondary hydroxyl groups at the wider edge. The central cavity is lined by the skeletal carbons and ethereal oxygens of the glucose residues, which gives it a lipophilic character. The polarity of the cavity has been estimated to be similar to that of an aqueous ethanolic solution. α-, β-, and γ-Cyclodextrins consist respectively of six, seven, and eight alpha-pyranoses connecting with alpha 1→4 glycoside bond.

Preferably β-cyclodextrin (β-CD) and γ-cyclodextrin (γ-CD) are used in the present invention as cyclodextrins (CDs). Most preferably, γ-cyclodextrin (γ-CD) is used. In the present invention, hydroxy group(s) on the cyclodextrins (CDs) can be modified with a variety of groups, including trialkylsilyl group such as trimethylsilyl group, methacryloyl group or acryloyl group, and the like.

Silylation reaction of cyclodextrins is reported by Harabagiu, V. et al.

{Carbohydrate Polymers. 2004, 56, 301-311.). The silylation of cyclodextrins can be performed, for example, in anhydrous conditions, under an inert atmosphere.

Methacrylation or acrylation of cyclodextrins also can be conducted by a known method for methacrylation or acrylation of a hydroxy group.

[ 0 0 2 3 ]

3. Pseudopolyrotaxane (PPR)

In the present specification, pseudopolyrotaxane (PPR) is a complex of a linear polymer such as PDMS, and a macrocyclic compound such as CD, wherein the macrocyclic compounds threaded onto the linear polymer chain.

PPR can be prepared by mixing, for example, PDMS and an excess molar amount of CD (e.g. n Y"CD>2/3n Siloxane Units) in water for approximately 1 hour to 48 hours at an ambient temperature (using magnetic stirrer, ultrasounds and the like). Pseudopolyrotaxane can be obtained as white crystalline precipitate by collecting by filtration or lyophilization, centrifugation and the like.

Modified CD such as SiCD (silylated cyclodextrin) can be used to prepare the pseudopolyrotaxane by a similar method as those described above for the preparation of PPR with non-modified CD. However, the method may be modified and an organic solvent such as CH2CI2 can be used for complexation of PDMS and SiCD.

[ 0 0 2 4 ]

4. End-capping agent for the radical coupling reaction and for microwave coupling reaction

In the present invention, an end-capping agent is a compound having a bulky group to prevent dethreading of CD from PPR and having a functional or an active group that is reactive with the terminal group of linear polymer chain such as polydimethylsiloxane.

Any compound can be used as an end-capping agent if the compound is bulky enough to prevent dethreading of CD from the PDMS chain. More preferably, the end-capping agent is a tricyclic, tetracyclic or pentacyclic aliphatic/aromatic compound, or a large sugar, dendrimer, cyclic silicone. Most preferably, the end-capping agent contains as a bulky group triphenyl, phenalene, phenanthrene, anthracene, fluoranthene, acephenanthrylene, aceanthrylene, triphenylene, pyrene, chrysene, naphthacene, pleiadene, picene, perilene, pentaphene, pentacene, phthalocyanins, cyclodextrins, di or tri-adamantane, cucurbiturils, or crownethers. However, the end-capping agent is not limited to the exemplified compounds.

The end-capping agent is functional or has active groups which shall be reactive with the terminal groups of linear polymer chain such as PDMS chain. For example, the functional or active groups of the end-capping agent can be a halogen atom, carboxyl group, hydroxy group, amino group, isocyanate, thioisocyanate group, unsaturated double bond, aldehyde group, amido group, diazo group, or the like. These groups may be further activated to increase the reactivity. For example, a carboxyl group can be activated by a known method, for example, by N- hydroxy succinimide (NHS), and the like.

[ 0 0 2 5 ]

5. Compound A

In the radical synthesis of polyrotaxane of the present invention, a complex of compound A with CD, [CD -Compound A], may be used.

The use of the [CD/Compound A] complex is to avoid complexation of the end-capping agent with the CDs added to saturate the reaction medium. The use of the [CD/Compound A] complex may not be needed if the blocking group of the end-capping agent has no affinity with the CDs.

Preferably, Compound A has-^'

(i) a good affinity to the cavity of the CDs, (ii) a similar structure to that of a end-capping agent or a part of the end-capping agent that has good affinity to the cavity of the CDs, and/or

(iii) has a similar chemical structure to the bulky part of the end-capping agent used in the synthesis of the polyrotaxane.

[ 0 0 2 6 ]

More preferably, compound A is a tricyclic, tetracyclic or pentacyclic aliphatic/aromatic compound.

Most preferably, compound A is aromatic cycle compound selected from the group consisting of phenalene, pnenanthrene, anthracene, fluoranthene, acephenanthrylene, aceanthrylene, triphenylene, pyrene, chrysene, naphthacene, pleiadene, picene, perilene, pentaphene and pentacene. Preferable aliphatic cycle compounds include adamantane.

[ 0 0 2 7 ]

6. Radical coupling reaction

The one characteristic of the method of the present invention is to bind pseudopolyrotaxane consisting of PDMS and CDs with end-capping agent by a radical coupling reaction.

Preferably, the radical coupling reaction is conducted in the presence of a radical initiator. Any type of radical initiator can be used. Examples of radical initiators include persulfate such as Na2S20s (sodium persulfate), peroxides and the like. Preferably, the concentration of pseudopolyrotaxane in the radical coupling reaction mixture ranges from 2xlO^'3mol.L^'1 to lOxlO^mol.L^"1.

Preferably, the concentration of end-capping agent in the radical coupling reaction mixture ranges from 20xlO^"3mol.L^'1 to lOOxlO^mol.L^"1.

Preferable molar ratio of pseudopolyrotaxane to end-capping agent ranges from 1/2 to 1/20, and more preferably from 1/5 to 1/15.

[ 0 0 2 8 ]

Examples of preferred solvent used in the radical coupling reaction include DMSO, DMF, Et20, H2O, pentane, acetonitrile, acetone, and a mixture thereof. Use of at least water is preferred to form radicals. Preferred examples include a mixture of at least two solvents. More preferably, examples of solvent include a mixture of at least two solvents, which at least comprises DMSO (or DMF) and H2O. Examples of a mixture of solvents include DMSO/H₂O or DMSO/Et₂O/H₂O, DMSO/pentane/H₂O, and the like. DMSO/Et₂O/H₂O is preferred.

The radical coupling reaction is preferably conducted at a temperature of from 18 degrees C to 30 degrees C, more preferably at 25 degrees C, for 1 minute to 48 hours. But a person skilled in the art can select the temperature suitably depending on the other conditions such as a kind of solvent(s) to be used.

[ 0 0 2 9 ]

Examples of the terminal groups of polydimethylsiloxane used in the radical coupling reaction include those described for the examples of terminal groups for the end-capping agent.

Preferable examples of terminal groups of polydimethylsiloxane in the radical coupling reaction include moieties with unsaturated double bond such as methacryloyl or acryloyl groups, or carboxy group or NHS activated ester.

[0 0 3 0 ]

The radical synthesis proposed is a coupling reaction of a methacrylate (or acrylate) functional group of PDMS with an activated end-capping agent such as tricyclic, tetracyclic or pentacyclic aliphatic/aromatic compound with an activated ester of a carboxylic acid.

Examples PDMS having a methacrylate (or acrylate) functional group include PDMS-dimethacrylate (PDMS-DiMA), and PDMS-diacrylate. For example, PDMS-dimethacrylate is reacted with PBS (pyrene butyric acid N -hydroxysuccinimide ester).

[ 0 0 3 1 ]

7. Microwave coupling reaction

The one characteristic of the method of the present invention is to bind pseudopolyrotaxane, consisting of linear polymer chain (such as PDMS, POE and the like) and CDs, with an end-capping agent by irradiating it with microwaves. The microwave coupling reaction in the present invention uses microwave irradiation to enhance the bonding reaction between the end-capping agent and the functional groups on the linear polymer chain

Any bonding type between the linear polymer chain and end-capping agent can be used as long as the reaction proceeds under microwave irradiation. For example, ester, ether, urethane, ureido, amide and carbamate bond can be used.

Therefore, the terminal groups of linear polymer chain and of end-capping agent can be selected depending on the bonding type to be used in the reaction.

[ 0 0 3 2 ]

In the present specification, carbamate, urea, thiocarbamate or thiorea and the like can be used as a bonding between the linear polymer chain and end-capping agent. Therefore, alcoholysis or aminolysis of isocyanate or thioisocyanate functions can be used as the reaction between the functional groups on linear polymer chain and on end-capping agent.

This coupling involves the reaction of -OH or -N¾ onto -NCO (isocyanate)/-NCS (thioisocyante). Accordingly examples of linear polymer chain include those having -OH, -NH2, -NCO or NCS end functional groups, such as PDMS-DiNCO, PDMS-DiNCS, PDMS-DiOH, and PDMSdiNH₂.

In the case of PDMS-DiNCO, the end capping agent can be any bulky group having an Η, -NH2 functional group. In the case of PDMS-DiNCS, the end capping can be any bulky group having an -NH2. In the case of PDMS-DiOH, the end capping can be any bulky group having an -NCO. In the case of PDMS-D1NH2 , the end capping can be any bulky group having an -NCO or -NCS.

[ 0 0 3 3 ]

The method to prepare a linear polymer chain such as PDMS having isocyanate or thioisocyanate group or hydroxyl or amino group as the terminal group is well known in the art and reported in, for example, WO2004/063795 (Macromonomer Al).

Also, the end-capping agent with isocyanate or thioisocyanate group or hydroxyl or amino group as the terminal group can be easily prepared by well known methods in the art. Also, compounds commercially available can be used. For example triphenyl aminosilane (Sigma Aldrich), trityl isothiocyanate (Sigma Aldrich) and the like can be used.

[ 0 0 3 4 ]

The microwave coupling reaction of the present invention can be conducted without solvent. It is very unique and surprising that polyrotaxane can be prepared without solvent.

However, a solvent may be used in the microwave reaction.

Examples of solvent include DMF, DMSO and the like. The amount of solvent can be appropriately determined. For example, it may range from 0 to 30 mL per 1 g of pseudopolyrotaxane.

Preferable molar ratio of pseudopolyrotaxane to end-capping agent ranges from 1/2 to 1/20, and more preferably 1/5 to 1/15.

The microwave coupling reaction is preferably conducted at a temperature of from 100 degrees C to 250 degrees C, more preferably at 150 degrees C, for 0.01 minute to 15 minutes. But a person skilled in the art can select the temperature and time suitably depending on the other conditions such as a kind of solvent(s) to be used. The power of the microwave can be 800W to 1200W.

[ 0 0 3 5 ]

8. Purification Methods

Depending on the nature of the polymer backbone, its molecular weight or the number of CDs on the macromolecule, eliminating the free macrocycles can be quite challenging. Numerous methods commonly used to purify polymers such as precipitation, filtration, ultrafiltration, dialyses, etc., have been investigated to isolate the pure polyrotaxane. Although the purity was improved, no method appeared to be efficient at isolating the macromolecule.

SEC analysis samples always showed the presence of a large number of free CDs. These results suggested the formation of strong aggregates between the polyrotaxane and the free cyclodextrins! this even in the presence of lithium salts or Urea, which allows the breaking of hydrogen bonds.

The inventors of the present invention found that free CDs can be removed efficiently by a preparative SEC. The chromatogram of the polyrotaxane obtained by extraction evidenced the absence of free CDs (Figure 4).

Size exclusion chromatography (SEC) analysis were carried out in dimethylformamide (DMF) using a Waters Chromatography columns coupled with U.V (λ = 345 nm) and refractive -index detection. For example, two preparative columns (WAT025861) were used in series to isolate the pure polyrotaxane (Eluent: DMF, Flow rate: 2.0 ml/min, Injection Volume: 400 μΐ (24mg/injection)). After two injections the columns are regenerated by washing with blend of solvent to limit the diffusion phenomena and adsorption of CD.

[ 0 0 3 6 ]

Purification of the PR by Modification of the CDs: The inventors of the present invention also found it possible to purify the Polyrotaxane samples by modification of the CDs in the polyrotaxane samples. Examples of modification include silylation, methylation, acetylation, and the like of the crude polyrotaxane. Modification changes the solution behavior of the polyrotaxane and therefore, the purification can be successfully conducted. In particular, modified CDs such as silylated, methacrylated or acetylated CDs could easily be removed from the mixture.

[ 0 0 3 7 ]

9 . Filling ratio (FR)

In the present specification, filling ratio is defined as Na Nf, wherein Na is the total number of CDs, e.g., γ-CDs, per one polydimethylsiloxane polymer of the synthesized polyrotaxane and Nf is the total number of CDs, e.g., γ-CD per one polydimethylsiloxane polymer, of the corresponding polyrotaxane ideally full packed with γ-CDs on polydimetylsiloxane. The phrase "ideally full packed" means that PDMS is fully covered by CDs.

[100% filling ratio = PDMS is fully covered by CDs.]

According to Harada's findings, one γ-CD cavity accommodates 1.5 siloxane units (2 nsi(CH3)2 = 3 n_rco). At this stoechiometry, the γ-CDs should be almost closed packed from end to end of the α,ω-dimethacrylate PDMS chain. 100% filling ratio of polyrotaxane made from a 4600 PDMS Mw is equivalent to 40 γ-CDs per PDMS chain. The filling ratio can be estimated from the O-NMR spectrum at the condition that Polyrotaxane samples is pure (Purity controlled by SEC Chromatography). The filling ratio can then be estimated directly from the end-capping groups/CD peaks integration ratio on Ή-ΝΜΈ, spectrum.

The polyrotaxane filling ratio can also be estimated by use of Maldi-Tof MS spectrum. The macromolecule is generally characterized on Maldi-Tof MS spectrum by a wide range of molar masses shaped into broad "humps" spaced by the approximate value of the macrocycle threaded on the polymer backbone. Number of humps indicates the number of CDs on the chain.

Examples

[ 0 0 3 8 ]

Common materials for Radical and Microwave synthesis

Cyclodextrins (γ-CD) were supplied by Nippon Food company Ltd.

They were used after being dried under vacuum for 16 hours at room temperature.

PDMS were kindly provided by Shin-Etsu Chemical Co. Ltd. The number average molecular weight of polymer samples and the rate of functionalization 2 were verified by Ή NMR.

Materials for Radical synthesis :

,ω-dimethacrylate polydimethylsiloxane (α,ω-dimethacrylate PDMS, Mn (number average molecular weight) = 4600 g.mol^'1) was kindly provided by Shin-Etsu Chemical Co. Ltd. The number average molecular weight of polymer samples and the rate of functionalization 2 were verified by Ή NMR. The radical initiator, i.e., sodium persulfate, 1-pyrene butyric acid N- hydroxy succinimide ester (PBS), pyrene and Sinapinic acid (SA), were purchased from Sigma-Aldrich Co. and were used as received. Ultra pure water was prepared by passing distilled water through a Quantum Ultra pure Organex Cartridge (QTUM000EX, Millipore), dimethylsulfoxide (DMSO), dimethylformamide (DMF), diethyl ether (ether) and pentane were purchased from SDS Carlo Erba and were used as received.

Materials for Microwave synthesis- Branched polyethyleneimine (BPEI) (From Aldrich)

Poly (dimethylsiloxane - ca dip he ny lsiloxane) , dihy droxy

(cPDPS-PDMS) (form Aldrich)

Triphenyl silanol (TPOH) (from Aldrich)

Triphenyl aminosilane (TPNH2) (From Aldrich)

Trityl isothiocyanate (TITC (From Aldrich)

Fluorescein isothiocyanate (FITC) (From Aldrich)

Materials for Modification of CDs- l-(trimethylsilyl)imidazole (TMSI) and Isocyanatoethyl methacrylate (IEM) were purchased from Sigma-Aldrich Co. and were used as received.

Dimethylformamide (DMF), Chloroform (CHCL3) were purchased from SDS Carlo Erba and were dried over CaHfo and distilled under nitrogen just before use.

[0 0 3 9 ]

Analysis

Size exclusion chromatography (SEC) analysis was carried out in dimethylformamide (DMF) using a Waters Chromatography column (WAT045810) coupled with U.V (λ = 345 nm) and refractive -index detection. The analytical experiments were performed at a flow rate of 0.3 ml/min and the injection volume was 50 pL (l mg/mL). The calibration method was described previously (Jarroux Ν. Guegan P.,^" Cheradame H.; Auvray L.; The Journal of Physical Chemistry B 2005, 109, 23816-23822.). Two preparative columns (WAT025861) were used in series to extract the pure polyrotaxane (Eluent: DMF, Flow rate: 2.0 ml/min, Injection Volume: 400 pL (24mg/injection)).

[ 0 0 4 0 ]

Ή NMR spectra were recorded on a Bruker Avance 600 MHz NMR spectrometer equipped with a cryoprobe in dimethylsulfoxide (DMSO-de) at 298K. One-dimensional spectra were acquired with 64 scans and 16 000 data points. The NOESY experiment was carried out with a mixing time of 200 ms, 2048 data points x 256 increments x 128 scans and a spectral width of 9000 Hz in both dimensions. The data were zero-filled to give 4096 x 512 data matrix prior to Fourier transformation. Excitation sculpting sequence (Hwang T.-K, and Shaka 1995) was used for suppression of the residual water signal at 3.6 ppm for all spectra.

[ 0 0 4 1 ]

Maldi-Tof MS (matrix- associated laser desorption ionization mass spectrometry): Characterizations of polyrotaxanes by Maldi-Tof MS were performed using a Perseptive Biosystems Voyager-DE Pro STR Time of Flight mass spectrometer (Applied Biosystems/MDS Sciex, Foster City, CA), equipped with a nitrogen UV laser (λ=337 nm) pulsed at a 20 Hz frequency. The mass spectrometer was operated both in the positive and negative ion reflector mode with an accelerating potential of +/-20 kV and a grid percentage equal to 70%. Mass spectra were recorded with the laser intensity set just above the ionization threshold (2800 in arbitrary units, on our instrument) to avoid fragmentation and maximize the resolution (pulse width 3 ns). Time delay between laser pulse and ion extraction was set to 450 ns. A set of parameters in the linear mode was also tested consisting of an accelerating potential of +/- 25 kV, a grid percentage of 93% and an extraction delay of 800 ns and the laser power was adjusted to 3200. Typically, mass spectra were obtained by accumulation of 200-1000 laser shots according to detection mode for each analysis and processed using Data Explorer 4.0 software (Applied Biosystems).

Samples containing polyrotaxanes were prepared at 76 mg/mL in water and Sinapinic acid (SA) at 20 mg/mL in methanol water 1/1 (v/v) were used as the matrix. Samples for Maldi-Tof MS analysis concentration were prepared by mixing 0.5 to 1 μΐ of sample and one volume of matrix. Then, one microlitter of the mixture was deposited via "dried droplet" method on a mirror polished stainless steel MALDI target and allowed to dry at room temperature under atmospheric pressure for periods of five and twenty minutes. External calibration was performed using proteins mixture provided by manufacturer.

[ 0 0 4 2 ]

Example 1

I. Preliminary study for radical reaction of polyrotaxane

The influence of several co-solvents and co-agents in the radical reaction has been examined to improve the accessibility of the PDMS extremities by the end-capping groups. Finally the insolubility of the PDMS in the solvents used in the model reaction (DMSO/H₂0 33/66 v/v) was solved by adding Et20 as another co-solvent [(DMSO/Et20 50/50 v/v) /

H₂0 33/66 v/v] (run l). Still important dethreading of the yCDs was evidenced by SEC analysis which suggests that only 14wt% of the pseudopolyrotaxane were transformed into the polyrotaxane. This conversion yield was improved by saturating the reaction medium with additional γ-CDs to limit dethreading of the molecular macrocycles already threaded on the polymer backbone (run 2).

Unfortunately, at those concentrations the medium was too heterogeneous and compact to observe any significant change and the yield hardly increased up to 27wt%. The reaction mixture was then diluted 5 times maintaining the saturation in yCDs and adjusting the amount of Na2S20e to keep the initiator concentration constant (run 3). The yield increased up to 78wt%.

In all runs, the end-capping agent (PBS) was used in excess as compared with the number of PDMS extremities (PBS/ PDMS =10). In run 3, however, the end-capping agents (pyrene derivative) could have been trapped in the cavities of the γ-CDs added in a large amount, thus limiting the reaction efficacy. This eventuality was investigated in the last run where the cavities of the γ-CDs used to saturate the reaction medium were previously filled with pyrene molecules (run 4). The conversion yield increased moderately up to 81wt%. The result was not significant to validate our assumption.

[ 0 0 4 3 ] Effect of reaction medium composition on the polyrotaxane synthesis conversion yield.

a The Mw of the^"Pseudo-PR was estimated to be equivalent to the full packed Pseudo-PR [CD:(Si(C¾)₂) is equivalent to 2:3] (Harada and al. Macromolecules, 2000, 33,

4297-4298)

b The percentage of PR was estimated from the crude product SEC chromatograph directly from the ratio of the PR peak area over the overall summed peak area (including the signal of PR, free CDs, DMSO and pyrene derivatives (PBS, pyrene, etc)). This value is just qualitative for comparison. c The percentage of PR was estimated from SEC chromatograph of the crude product after calibration and quantification of all residues present in the sample such as free CDs, trace of DMSO and pyrene derivatives (PBS, pyrene, etc). It is worth noticing that the values of %PR calculated with^(c) or without calibration* are very similar.

dThe polyrotaxane conversion yield was estimated according to the equation: [Wt% Yield = %PRx(mCrudeProduct/mPseudo-PE)].

e For information: Without blocking the PDMS extremities the PDMS/yCD inclusion complex breaks in SEC experimental conditions. The Pseudo-PR SEC chromatograph in DMF clearly shows one peak corresponding to the free CDs (Sensitivity for PDMS is too low to observe the polymer). Undoubtedly the high molecular peak observed in the different runs can by attributed to the PR.

[ 0 0 4 4 ]

II. Radical synthesis of polyrotaxane

1. Preparation of pseudopolyrotaxane:

Preparation steps are indicated in Figure 1.

A saturated aqueous solution of γ-cyclodextrins (lOg in 43mL) was added into a 100 mL round flask containing α,ω-dimethacrylate poly-dimethylsiloxane (PDMS) (0.921g). According to Harada's findings, one γ-CD cavity accommodates 1.5 siloxane units (2 nsi(cH3)2 = 3 n_rco)>^" at this stochiometry the γ-CDs should be almost closed packed from end to end of the ,ω-dimethacrylate PDMS chain. The mixture was stirred at room temperature for 24 h. The pseudopolyrotaxane PDMS/y-CD complexes were obtained as white crystalline precipitate collected by filtration, and then dried under vacuum (mass = 9.3912 g.). The mass yield was 86%.

PDMS' peaks: 0.0 ppm ((CH₃)₂Sr), 0.45-0.55 ppm (-CH₂-Si), 1.6-1.7ppm (-CH₂-CH₂-Si), 1.8-1.9 ppm (CH₃-C=), 4.0-4.1ppm (-CH₂-O), 5.5 and 6ppm (CH₂=), and cyclodextrin's peaks: 3.5-3.6 ppm (m, 1H, H₄), 3.6-3.7 ppm (m, 1H, ¾), 3.8-3.85 ppm (m, 1H, H₅), 3.85-3.95 ppm (m, 1H, H₆), 3.9-4.0 ppm (m, 1H, H₃) 4.3-4.5 ppm (t, 1H, OH₆), 4.6-4.8 ppm (s, 1H, Hi=H anomeric), 5.3-5.6 ppm (2 x d, 1H, OH₃ and OH₂).

[ 0 0 4 5 ]

2. Preparation of a complex of γ-CD and Pyrene^

Preparation steps are indicated in Figure 2.

γ-CD (3.48g) and pyrene (0.543g) were added in 10 mL of pure water and then mixed for 24 h. The solution became turbid because of the formation of a light yellow precipitate. The solution was kept apart for the polyrotaxane synthesis. [ 0 0 4 6 ]

3. Radical synthesis of polyrotaxane (run 4):

Steps of radical synthesis are indicated in Figure 3.

A powder mixture of pseudopolyrotaxane PDMS/yCD (l.69g), 1-pyrenebutyric acid N-hydroxysuccimide ester (0.116g) and Sodium persulfate (0.411mg) was prepared into a 100 mL round flask. The reaction started by adding lOmL of the aqueous 1^1 pyrene/yCD complexes solution and 5mL of DMSO/Et₂0 (50^50 v/v). The mixture was stirred for 18 hours at room temperature. The reaction was then quenched by plunging the reaction flask into liquid nitrogen followed by freeze drying.

The coupling efficacy was estimated by size exclusion chromatography (SEC) analysis of the crude polyrotaxane products. The conversion yield of pseudopolyrotaxane into polyrotaxane was estimated at 81wt%.

[ 0 0 4 7 ]

II. Purification of polyrotaxane

1. Isolation by Preparative SEC (Size Excluding Chromatography) The crude polyrotaxane was purified by preparative SEC. 80 mg of pure polyrotaxane was isolated.

Figure 4 shows SEC characterization of the polyrotaxane (run 4) before and after extraction by preparative SEC in DMF. The chromatogram of the polyrotaxane obtained by extraction evidenced the absence of free yCDs.

[ 0 0 4 8 ]

2. Pure polyrotaxane Characterisation

ID Ή-NMR Figures 5(A) and 5(B) show ID ^!H-NMR spectra of pseudopolyrotaxane (A) and pure polyrotaxane extracted from run 4 product of reaction (B), recorded at 298K in DMSO-de. The inset in (B) shows a zoom of ID ¹H-NMR spectrum of polyrotaxane between 0.6 and ^•1.5 ppm. PDMS methyl protons and γ-CDs protons are labeled. Disappearance of PDMS peaks demonstrates that the PDMS is in a confined environment with low mobility.

[0 0 4 9 ]

2D ^!H-NMR

Figure 6 shows 2D ^!H-NMR spectra of pure polyrotaxane, demonstrating interaction of PDMS methyl protons with CDs' protons inside the cavity. The presence of PDMS inside the CDs' cavity is proven.

[ 0 0 5 0 ]

MALDI TOF MS

Figure 7 shows Maldi-Tof MS spectra of pure polyrotaxane.

a. Linear Mode: The presence of compounds with masses of 28000 g.mol ¹ was clearly evidenced in the pure polyrotaxane with a number of yCD threaded on the PDMS polymer chain superior to 18.

b. Reflector Mode: Demonstrates the possible recombinations of the α,ω-dimethacrylate polydimethylsiloxane bearing one yCD, with the capping agent 1-pyrene butyric acid N-hydroxy succinimide ester (PBS). The analysis clearly demonstrated that one pyrenyl group is bulky enough to block PDMS polymer chain extremities and to prevent the γ-CDs from dethreading.

[ 0 0 5 1 ]

3. Filling ratio (FR)

Both Maldi-Tof MS experiment in the linear mode and ID ^!H-NMR showed the possibility to cover the PDMS polymer chain up to 20-40% with γ-CD.

Since the ID O-NMR peaks characteristic of PDMS Methyl protons are not visible, the polyrotaxane filling ratio was estimated directly from the pyrene end-capping groups/CD peak integration ratio. The calculation gave between 7 and 14 γ-CDs per polyrotaxane macromolecule depending on the number of pyrene end-capping groups attached to the PDMS chain (2 to 4 Py / PDMS chain,^' see discussion bellow). The average filling ratio is most likely to be in the range between 20 and 40 % (Close packing is defined to be 100%, i.e. 41 γ-CDs on a 4600 Mw PDMS chain).

The macromolecule is generally characterized on Maldi-Tof MS spectrum by a wide range of molar masses shaped into broad "humps" spaced by the approximate value of the macrocycle threaded on the polymer backbone. Number of humps indicates the number of CDs on the chain. Using those optimized conditions, compounds with masses of 28000 g.mol ¹ were clearly evidenced in the pure polyrotaxane with a number of y-CD threaded on the PDMS polymer chain superior to 18.

[ 0 0 5 2 ]

Example 2

Microwave coupling reaction of polyrotaxane

(A) Synthesis with [PDMS-DiNCO/Si- y CDl based PPR

1. Synthesis of PDMS-PiNCO

Diisocyanate poly(dimethylsiloxane), PDMS-DiNCO (sample 14), was synthesized in accordance with a known method in the art (ref. WO2004/063795 (Macromonomer AD).

The pseudopolyrotaxane was prepared with persilylated γ-CDs (Si-yCDs) to prevent the reaction of the isocyanate functions on the CDs' hydroxyl groups. The synthesis of the Si-yCDs and the pseudopolyrotaxane (PDMS-DiNCO / Si-yCDs inclusion complex) are described below.

[ 0 0 5 3 ]

2. Persilylation of cvclodextrins

Persilylation reaction of cyclodextrins was reported previously

(Harabagiu, V.; Simionescu; B. C,^" Pinteala, M.>^' Merrienne, C^' Mahuteau, J.; Guegan, P.; Cheradame, H., Carbohydrate Polymers. 2004, 56, 301-311.).

Persilylation of γ-CD was conducted as described below.

The persilylation was performed in anhydrous conditions, under an inert atmosphere. One gram of yCD was introduced in a flask where an N2 flow was maintained forl5 minutes before adding 20 mL of anhydrous DMF. After 15 minutes under N2 flow, N-trimethylsilylimidazole TMSI (Aldrich) were dropped at 20°C under magnetic stirring. One hour later, the mixture was diluted with 20 mL chloroform to avoid an eventual precipitation. The reaction was stirred for 1 hour under dynamic N₂ and under static N2 for at least 24 hours. The reaction was stopped by slowly adding 80 mL of water. CHCI3 (around 40mL) was added into the reaction flask. The organic phase was washed 3 times with pure water (300 mL). The product of reaction was extracted in the organic phase and dried under vacuum. Persilylated y CDs (2.29g) were then obtained.

The degree of silylation was estimated by calculating the ratio of the relative peak integrations of the unmodified yCD hydroxyl group (OH2 OH3 at 5,5 and 5,4 ppm et OH6 at 4,4 ppm) and the yCD anomeric proton (HI at 4.8 ppm). NMR Characterisation of FB70 (persilylated yCDs) allows calculating the % of silylation to 100%.

[ 0 0 5 4 ] 3. Synthesis of Pseudopolyrotaxane from PDMS-DiNCO and persilylated γ-CDs

6.65g of persilylated γ-CDs (Si-yCD) and 250 mg of diisocyanate poly(dimethylsiloxane) were solubilized in 6.1 mL of dry dichloromethane under an inert gas. The quantity of dichloromethane is adjusted in order to have the same concentration of Si- CDs versus solvent as with native CDs in water.

The reaction mixture was stirred at room temperature for at least 12 hours. The reaction mixture became turbid but no precipitate was obtained. The product of reaction was collected by evaporation of the solvent and dried under vacuum. 6.899 g of pseudopolyrotaxane PDMS/Si-yCD was so obtained.

The pseudopolyrotaxane was then stored under inert gas at low temperature.

[ 0 0 5 5 ]

4. Study of the microwave coupling reaction of [PDMS-DiNCO/Si-YCDlbased Pseudopolyrotaxane

In all examples, reactants are just mixed into the microwave reaction vessels and set under microwave irradiation. Example (i): γ-CD was used as end-capping group (FB76-78, 82). The following table shows the conditions of the reactions.

[ 0 0 5 6 ]

Characterisation of the run 3 (FB78) is given thereafter (200°C without solvent).

The product of reaction was washed first with water to remove free

CDs. Unexpectedly the product of reaction was soluble in water and high molecular weight compounds attributed to the PR were found in the water phase (FB78f).

[ 0 0 5 7 ]

NMR Spectrum (Figure 8): Characteristic peaks of CDs and

PDMS are observed in FB78f. Detailed analysis evidenced the de-silylation of the CDs certainly due to the heat generated in the microwave. In this case, desilylation rate was estimated at 59% (comprising 90% of desilylation of the OH2-OH3 functions).

[ 0 0 5 8 ]

SEC Chromatogram (Figure 9) shows the presence of high molecular weights compounds and free CDs. Because of the partial desilylation of the CDs, the quantification of free CDs couldn't be done.

[ 0 0 5 9 ]

The MALDI-TOF mass spectrum in the linear mode. (Figure 10) shaped into broad "humps" is characteristic of the polyrotaxane. Detectable peaks were seen in the range of m/z 2300 to 40000 with an averaged inter-peak spacing of about 2000 m/z corresponding to the molecular weight of partially persilylated γ-CD. The analysis clearly evidenced the production of the polyrotaxane with a various numbers of CDs threaded on the PDMS polymer.

[ 0 0 6 0 ]

MALDI Tof MS in reflector mode (Figure 11) permitted to precisely attribute the peaks of low molecular weight compounds in the sample and to confirm the coupling reaction leading to the formation of the polyrotaxane structure.

Other populations corresponded to the functionalization of the diisocyanate PDMS by γ-CDs, as shown in Figure 12.

[ 0 0 6 1 ] Experiment Conclusions^ high molecular weights have been evidenced in MALDI TOF MS and confirmed by SEC.

Other experiments with γ-CD as end-capping agent were explored. In particular, reaction in the presence of a solvent (in this case DMF) allowed a better blending of the reactants and reactivity seemed better. However, the presence of solvent increased the dethreading of the CDs, which was not the goal of the study. In addition, decreasing the temperature of reaction allowed reducing the rate of desilylation, but the reaction yield was lower.

[ 0 0 6 2 ]

Example (ii) BPEI was used as end-capping group (FB79-81, 83).

As it is known that the reactivity of the isocyanate functions is higher with an amine function than with an alcohol function, an end-capping agent with an amine function was used. The branched polyethyleneimine (BPEI) is a polymer of high molecular weight (about 25000 g.moM) and good "steric congestion".

[ 0 0 6 3 ]

Characterisation of the run 7 (FB81) is described thereafter (200°C without solvent). The product of reaction was first washed with water to remove BPEI in excess. High molecular weights compounds attributed to the PR were extracted in acetone (FB81pf).

Figure 13 shows Ή-NMR Spectrum of FB81pf in DMSO-DG.

Figure 14 shows SEC chromatogram of FB81pf in DMF.

SEC chromatogram of FB81pf didn't show signals generally attributed to free CDs. But the NMR spectrum showed the presence of CDs (silylated or not) in the sample. The results suggested that partially desilylated CDs in the sample were borne by the polyrotaxane. The amount of BPEI seemed to be very low according to NMR and the product might be quite pure.

Maldi Tof MS: The product was not analysed because of the size of the BPEI, the product would not be able to desorb from the Maldi Tof MS matrix. Experiment conclusions · high molecular weights attributed to the polyrotaxane have been evidenced. Additional experiments with BPEI were performed in the presence of a solvent. The addition of solvent favoured the accessibility of BPEI which was limited by BPEI high viscosity. As observed previously, in the presence of solvent the coupling reaction was more efficient but dethreading was observed.

[0 0 6 4 ]

Example (hi) cPDPSPDMS was used as an end-capping group (FB84-87).

The use of an end-capping agent with high viscosity limits the reactivity. A derivative of PDMS was chosen as the end-capping agent in order to keep the same type of composition as the polymer included in the polyrotaxane. We used Polydiphenylsiloxane-Polydimethylsiloxane co-polymer (cPDPSPDMS) as the end-capping agent.

[ 0 0 6 5 ] Characterisations of the run 11 (FB86) are described thereafter (200°C without solvent).

The product of reaction was first washed with pentane to eliminate excess of cPDPSPDMS. High molecular weight compounds attributed to the PR were then extracted in the acetone phase (FB86pf).

[ 0 0 6 6 ]

Figure 15 and Figure 16 show respectively Ή NMR Spectrum in DMF and SEC Chromotogram of FB86 in different extraction phases.

1. The Pentane phase (FB86f - 63% in mass): NMR showed the presence of a large amount of PDPSPDMS. This analysis permitted the estimation of the rate of CDs desilylation to 64%. The product was analyzed by SEC in DMF, in which PDPSPDMS is not soluble. Although presence of high molecular weights was evidenced, mostly free CDs were present in the sample.

2. The acetone phase (FB86pf ^■ 4% in mass): NMR showed a lower rate of CDs desilylation (41%) and showed the presence of PDPSPDMS. SEC showed a larger proportion of high molecular weights versus free CDs in this phase.

3. The insoluble part (FB86pp - 3% in mass): mostly free desilylated CDs (90%).

[ 0 0 6 7 ] Figure 17 shows MALDI Tof MS of FB87pf in the linear mode. Desorption of FB86pf was difficult and MALDI Tof characterization of the product was impossible. However MALDI Tof characterization of the product of run 12 conducted in DMF was possible (FB87). Indeed de-threading occurred in the presence of solvent and run 12 permitted to obtain the polyrotaxane with very few CDs (or no CDs at all) which permit a better desorption on MALDI TOF matrices.

The analysis of MALDI TOF MS in linear mode of FB87pf (run at 200°C with DMF) evidenced the presence of different populations separated by around 2000 uma, which corresponds to the molecular weight of a partially desilylated CD (around 50%).

[ 0 0 6 8 ]

Figure 20 shows the MALDI Tof MS spectrum of FB87pf in the reflector mode.

Figures 18 and 19 show the attribution some peaks seen on the

MALDI Tof MS of FB87pf in the reflector mode (normal and zoom).

In reflector mode, the populations evidenced the PDMS diisocyanate functionalized by different cPDPSPDMS copolymers (cPDPSPDMS 1 and 2) having different molecular weights.

[ 0 0 6 9 ]

The use of a liquid end-capping agent with a low viscosity permitted to work in media with better homogeneity. The accessibility to PDMS extremities was certainly increased and the reactivity was better. So we targeted end-capping agents that are liquids at the temperature of the reaction. Triphenyl end-capping agents with an amine or an alcohol functional group were examined.

[ 0 0 7 0 ]

Example (iv) TPOH was used as end-capping group (FB93-98) Triphenyl silanol (TPOH) is liquid at 150°C.

[ 0 0 7 1 ]

Characterisation of the run 15 (FB95) is described thereafter (150°C without solvent).

The product of reaction was first washed with acetonitrile to eliminate excess of TPOH. High molecular weight compounds attributed to the PR were then extracted in the acetone phase (FB95pf). 6% in mass of the product of reaction were extracted in the acetone phase and SEC showed 32.3% of high molecular weight compounds.

Figure 21 shows SEC Chromatogram of FB97pf in DMF. [ 0 0 7 2 ]

Other experiments-^' Better results are obtained in the presence of solvent. The yield was also increased when increasing the molar ratio of end-capping agent over the number of PDMS extremities from 5 to 50 times. This result correlated with the difficulties to blend the reactants in the absence of solvent.

[ 0 0 7 3 ]

Example (v) TPNH2 was used as end-capping group (FB99-104) Triphenyl aminosilane (TPNH₂) is liquid at 60°C.

[ 0 0 7 4 ]

Characterizations of the run 21 (FB101) are described thereafter (l50°Cwithout solvent ).

The product of reaction was first washed with acetonitrile to eliminate excess of TPOH. High molecular weight compounds attributed to the PR were then extracted in the acetone phase (FBlOlpf).

6% in mass of the product of reaction were extracted in the acetone phase and SEC showed 16.5% of high molecular weight compounds.

Figure 22 shows SEC Chromatogram of FBlOlpf in DMF.

Contrary to expectation the reaction was less effective with TPNH2 than with TPOH. This result could be explained by the higher volatility of TPNH₂ versus TPOH. Indeed, the TPNH2 is in gaseous form at the temperature of the reaction under microwaves and could not react with the PDMS isocyanates.

In the presence of DMF, the reaction was more effective with TPNH2 than TPOH (65.5% of high molecular mass by SEC in pf phase versus 29.7% for TPOH). The addition of DMF permitted the "trapping" of the TPNH2 in the liquid phase. [ 0 0 7 5 ]

(B) Synthesis with [PDMS-DiNH₂ /Native vCDsl based Pseudopolyrotaxane Previous experiments showed the feasibility of the condensation under microwave irradiation to synthesize the polyrotaxane. However, the protection of the hydroxyl groups on the CDs is necessary to prevent their reaction on the PDMS isocyanate telechelic functions. Modification of the CDs is time consuming and so is the preparation of PDMS-DiNCO.

To dodge those difficulties, a [PDMS"diNH₂/Natives yCDs] based pseudopolyrotaxane was prepared to react with end-capping agents having isocyanate (-NCO) or isothiocyanate (-NCS) reactive function (see Figure 23). Indeed, the reactivity of isocyanate and the isothiocyanate is known to be better with -NH2 reactional groups than with—OH reactional groups. And the end-capping agents should react in priority with the -NH2 at the extremities of the PDMS chains. This way the protection of the hydroxyl groups on the CDs is not necessary and the polyrotaxane can be synthesized under microwave irradiation directly with PPR prepared with native cyclodextrins.

[ 0 0 7 6 ]

Example (vi) TITC and FITC were used as end capping group

(FB130-134)

Trityl isothiocyanate (TITC - liquid at 138°C) and fluorescein isothiocyanate (FITC - liquid at 315°C) were then used as bulky group. In addition, the isothiocyanate function is known to be more selective than the isocyanate function to react on -NH2 groups. This allows working with PPR bearing native CDs and not silylated CDs like in previous examples.

The different experiments with TITC and FITC were carried out using the same protocol (150°C for 5min) with different ratios (bulky group = molar amount of extremity x5 and xl5) , and with and without the presence of solvents.

[ 0 0 7 7 ]

*Yield is obtained by quantification in SEC of the free CDs and bulky group still present in the crude product of reaction.

[ 0 0 7 8 ]

The followings are observed in the above-described study.

(i) The reactions made with 5 times more blocking agent than NH₂ functions (molar) showed a lower yield than the ones made with 15 times the amount of blocking agent. The proportion of bulky group used is very important when the reaction takes place without solvent because of the lack of accessibility between reactants.

(ii) The reactions without solvent showed higher quantity of PR than the runs conducted with solvent. Reactions without solvent have the advantage of limiting dethreading evidenced in runs conducted with solvent which also showed a lower yield.

(iii) The results obtained permits to demonstrate a higher efficiency of the FITC versus TITC as blocking agent, even if the FITC is a molecule more complex than TITC and solid at the temperature of reaction. The reactivity of FITC with amine functions seems to be higher than TITC.

[ 0 0 7 9 ]

MALDI TOF characterization was conducted on several runs.

In the FB131 and FB132, the characterization showed the presence of cyclodextrin and glucopyranose units.

The presence of FITC or TITC limits desorption of the polyrotaxane entities. Indeed, the FITC and the TITC have a higher absorbance than the matrices usually used to desorb the polyrotaxane. This phenomenon implies that the laser sends the energy to the molecule instead of the matrix, and leads to the degradation of the molecule.

In the FB133, the spectrum shows different populations of CDs modified by FITC groups (up to 6 FITC attached to the cyclodextrin). The evidence of the secondary reaction (isothiocyanate function reaction with alcohol functions) brings the proof of the feasibility of the principal reaction (isothiocyanate function with amine function). Unfortunately, the PRs cannot be desorbed from the MALDI Tof matrix and could not be characterised.

[ 0 0 8 0 ]

SEC Chromatograms of FITC and TITC showed peaks in the same high molecular range as the polyrotaxane (Figure 24). SEC analyses showed the same profiles whatever the temperature or the presence of LiBr salts (0.1 M) which showed that the presence of peaks in the high molecular weight range were not due to aggregates. SEC Chromatograms of polyrotaxane samples: SEC analysis was conducted on crude product in order to quantify the yield of reaction. Quantification of polyrotaxane was done after subtraction of the signal corresponding to the exact amount of end-capping agent used in the reaction. SEC Chromatograms of FB132 clearly shows the presence of polyrotaxane (Figure 25).

[ 0 0 8 1 ] Example 3

Modification of polyrotaxane

1. Silylation and Methacrylation of polyrotaxane:

Polyrotaxane was modified by silylation and methacrylation (Figure 26).

The silylation reaction of cyclodextrins was reported previously (Harabagiu, V.; Simionescu,^' B. C>^' Pinteala, M.; Merrienne, C.,^' Mahuteau, J.; Guegan, P.; Cheradame, H., Carbohydrate Polymers. 2004, 56, 301-311.). The silylation of polyrotaxane was performed in anhydrous conditions, under an inert atmosphere ((Jarroux N.5 Guegan P.; Cheradame H.J Auvray L.; The Journal of Physical Chemistry B '2005, 109, 23816-23822.).).

A crude polyrotaxane sample (FB91) was prepared via radical synthesis using conditions described in the paragraphs under the title of "II. Radical synthesis of polyrotaxane, 3. Radical synthesis of polyrotaxane (run 4)".

1.54 g of crude polyrotaxane (FB91) was introduced in a flask where an N2 flow was maintained during 15 minutes before adding 20 mL of anhydrous DMF. When the polyrotaxane was totally dissolved, 0.260 mL of Isocyanatoethyl methacrylate (IEM) and, 15 minutes later, 8.14 mL of N-trimethylsilylimidazole (TMSI) (Aldrich) were added dropwise at 20°C under magnetic stirring. 1 hour later, the mixture was diluted with 20 mL chloroform to avoid an eventual precipitation. The reaction was stirred for 1 hour under dynamic N2 and under static N2 during 8 days. The reaction was stopped by slowly adding 80 mL of water. [ 0 0 8 2 ]

2. Purification Protocol:

A large amount of CH2CL2 (around 300mL) was added into the reaction flask. The organic phase was washed 3 times with pure water (300 mL). The product of reaction was extracted in the organic phase and dried under vacuum. 1.8 g was then obtained (FB92org). Emulsion proves the good silylation yield and makes the extraction of the organic phase difficult.

The product of reaction was then stirred in an excess of DMSO for 24 hours (around 50 mL per gram of dried product). And the product remaining in the flask was collected by centrifugation and dried under vacuum. 514 mg of pure polyrotaxane was isolated as confirm by SEC analysis (FB92orgp).

The amount of purified polyrotaxane, 514 mg, corresponds to 70 wt% of the polyrotaxane product contained in the crude product used to start this reaction (FB91).

[ 0 0 8 3 ] 3. Characterizations^

ID Ή-NMR

Figure 27 (Top) shows ID Ή ΝΜΒ, spectrum of polyrotaxane (FB92orgp). The degree of methacrylation was estimated by calculating the ratio of the relative peak integrations of the methacrylate protons (at 6 ppm) and the γ-CD anomeric proton (HI at 4.8 ppm). The degree of silylation was estimated by calculating the ratio of the relative peak integrations of the unmodified γ-CD hydroxyl group (OH2 OH3 at 5.5 and 5.4 ppm et OH6 at 4.4 ppm) and the γ-CD anomeric proton (HI at 4.8 ppm).

The degree of methacrylation and silylation were calculated respectively around 1.2% and 70%.

[ 0 0 8 4 ]

SEC Chromatography

The chromatogram of the polyrotaxane modified polyrotaxane FB92orgp obtained evidences the absence of free CDs. (see Figure 28 (bottom)). Pure polyrotaxane was obtained. [ 0 0 8 5 ]

Example 4

Polyrotaxane synthesis with modified γ-CDs 1. Preparation of Pseudopolyrotaxane with persilylated γ-CDs^ Synthesis of the pseudopolyrotaxane with persilylated γ-CDs and α,ω-dimethacrylate poly(dimethylsiloxane) in

CH₂Cl₂/Pseudo-polyrotaxane PDMS/ySiCD (Silylation reaction of cyclodextrins was reported previously (Harabagiu, V.,^* Simionescu! B. C;

Pinteala, M.; Merrienne, C.^" Mahuteau, J.; Guegan, P.; Cheradame, H.,

Carbohydrate Polymers. 2004, 56, 301-311.))

Ill mg of persilylated γ-CDs (γ-SiCD) were solubilized in 5 mL of

CH2CL2 in order to be at the limit of solubility. 23 mg ,ω-dimethacrylate poly(dimethylsiloxane) (number average molecular weight: M> = 4600 g.mol ¹) Was added to this solution of γ-SiCD. According to Harada's findings, one γ-CD cavity accommodates 1.5 siloxane units (2 nSi(CHs)2 = 3 ηγ-CD); at this stochiometry the γ-CDs should be almost closed packed from end to end of the α,ω-dimethacrylate PDMS chain. The reaction was stirred at room temperature for 24 hours. The reaction mixture became turbid but no precipitate was obtained. The product of reaction was collected by evaporation of the solvent and dried under vacuum. 134 mg of pseudopolyrotaxane PDMS/ySiCD was so obtained. [ 0 0 8 6 ]

2. Preparation of a complex of γ-SiCD and Pyrene^

γ-SiCD (1355 mg) and pyrene (90 mg) were added in 1.7 mL of pure water and then mixed for 24 hours. The solution became turbid because of the formation of a precipitate. The solution was kept apart for the polyrotaxane synthesis. [ 0 0 8 7 ]

3. Synthesis of Polyrotaxane with the Pseudo-PR bearing persilylated γ-CDs

A powder mixture of pseudopolyrotaxane PDMS/ySiCD (134 mg), 1-pyrenebutyric acid N-hydroxysuccimide ester (14.3 mg) and Sodium persulfate (79 mg) was prepared in a 50 mL round flask. The reaction started by adding 1.7 mL of the aqueous 1^1 pyrene/ γ-SiCD complexes solution and 0.9 mL of DMSO/Et₂O (50-50 v/v). The mixture was stirred for 12 hours, at room temperature. The reaction was then quenched by plunging the reaction flask into liquid nitrogen followed by freeze drying (mass 1,672 g).

[ 0 0 8 8 ]

SEC chromatography (Figure 28) proved the feasibility to get the polyrotaxane from modified γ-SiCD based pseudopolyrotaxane. Consequently the possibility to form the pseudopolyrotaxane with modified yCD and the method were proven. The presence of high molecular weight compounds was confirmed by SEC.

Claims

1. A method of preparing a PDMS(polydimethylsiloxane) -based polyrotaxane, comprising a step of reacting pseudopolyrotaxane consisting of PDMS and CDs (cyclodextrins) with an end-capping agent, by (a) a radical coupling reaction, or (b) a microwave coupling reaction.

2. The method of claim 1, wherein the (a) radical coupling reaction comprises the following steps^

(i) preparing a solution, preferably a saturated solution of CDs or [CDs^compound A] complexes, compound A having a good affinity to the cavity of the CDs or having a chemical structure similar to the end-capping agent,

(ii) adding the mixture solution prepared in step (i) to a mixture of pseudopolyrotaxane, radical initiator and end-capping agent, and stirring the resulted mixture, and

(iii) working up the reaction mixture obtained in step (ii) to thus obtain a PDMS-based polyrotaxane.

3. A method of preparing a polyrotaxane, comprising a step of reacting pseudopolyrotaxane consisting of a linear polymer chain and CDs (cyclodextrins) with end-capping agent, by (b) a microwave coupling reaction, wherein the (b) microwave coupling reaction comprises the following steps:

(i) mixing pseudopolyrotaxane and end-capping agent,

(ii) irradiating the mixture obtained in step (i) with a microwave, and (iii) working up the reaction mixture obtained in step (ii) to thus obtain the corresponding linear polymer chain based polyrotaxane.

4. The method of any one of claims 1 to 3, further comprising a step of isolating the polyrotaxane by a preparative SEC (size extrusion chromatography) technique.

5. A PDMS (polydimethylsiloxane) -based polyrotaxane having a filling ratio (%) ranging from 20% to 40%, wherein the filling ratio (%) is defined as "Na/Nf x 100", wherein Na is the number of CDs(cyclodextrins) per one PDMS polymer chain of a polyrotaxane and Nf is the number of CDs per one PDMS polymer chain wherein the polydimethylsiloxane is highly packed with CDs.

6. A method of purification of polyrotaxane, comprising a step of isolating polyrotaxane by a preparative SEC (size extrusion chromatography) technique.

7. A method of purification of polyrotaxane, comprising a step of modification of the CDs of the polyrotaxane and a step of isolation of the modified polyrotaxane.