Fluorosurfactants

Abstract

The present invention provides a surfactant having a formula selected from the group consisting of: B—((X).sub.x—(CH.sub.2).sub.a-A).sub.n(VI), (A-(CH.sub.2).sub.a—(X).sub.x—B—(X).sub.x—(CH.sub.2).sub.a-A).sub.n (IV), (A-(CH.sub.2).sub.a—(X).sub.x—B).sub.n (V), and (B).sub.n—(X).sub.x—(CH.sub.2).sub.a-A (VII), wherein A is a perfluoropolyether; a is a positive integer; X is either a covalent bond or a linking group; x is a positive integer; B is a polyalkylene oxide unit; n is a positive integer greater than 1 and, in compounds comprising more than one A, B, X, a and x, each may be the same or different. The present invention also relates to methods of making such surfactants, uses of such surfactants and emulsions comprising such surfactants.

Claims

1. A surfactant of formula (II),
A-(CH.sub.2).sub.a—(X).sub.x—B—(X).sub.x—(CH.sub.2).sub.a-A  (II), wherein, each A is a perfluoropolyether and comprises a repeat unit of the formula:
—[CF(CF.sub.3)CF.sub.2O].sub.b—, wherein h is a positive integer; a is a positive integer; X is either a covalent bond or a linking group; x is a positive integer; B is a polyalkylene oxide unit; and each A, X, a, and x may be the same or different.

2. A surfactant as claimed in claim 1, wherein each A comprises a repeat unit of the formula:
—[CF.sub.2CF.sub.2O].sub.c—[CF(CF.sub.3)CF.sub.2O].sub.b—, wherein b is a positive integer and c is 0 or a positive integer; or wherein each A consists of the formula:
CF.sub.3CF.sub.2CF.sub.2O—[CF(CF.sub.3)CF.sub.2O].sub.b—CF(CF.sub.3)—, wherein b is a positive integer.

3. A surfactant as claimed in claim 1, wherein b is an integer from 1 to 100.

4. A surfactant as claimed in claim 1, wherein each a is an integer from 1 to 5.

5. A surfactant as claimed in claim 1, wherein at least one X is a covalent bond.

6. A surfactant as claimed in claim 1, wherein at least one X is a linking group selected from the group consisting of —C(O)NH—, —C(O)NMe-, —NHC(O)—, —NMeC(O)—, —C(O)S—, —SC(O)—, —C(O)O—, —OC(O)—, —OC(O)O—, —OC(O)NH—, —OC(O)NMe-, —O—, —S—, —NHC(O)NH—, —NMeC(O)NH—, —NHC(O)NMe-, —NHC(O)O—, —NMeC(O)O—, —SO.sub.2NH—, —NHSO.sub.2—, —NHSO.sub.2—C.sub.6H.sub.4—O—, —O—C.sub.6H.sub.4—SO.sub.2NH—, and linking groups of the formula -D-(E).sub.h-(G).sub.d- and -(G).sub.d-(E).sub.h-D-, wherein D is selected from NH, NMe, C(O), CO.sub.2, O and SO.sub.g wherein g is 0, 1 or 2, E is selected from alkylene, optionally substituted arylene and optionally substituted heteroarylene, h is 0 or 1, G is selected from C(O)NH, CO.sub.2, NH, NMe, O, C(O), S and SO.sub.2NH, and d is 0 or 1.

7. A surfactant as claimed in claim 1, wherein each x is 1.

8. A surfactant as claimed in claim 1, wherein each B comprises a group selected from a group consisting of a polyethylene oxide unit, a polypropylene oxide unit, and a unit of the formula
—[CH.sub.2CH.sub.2O].sub.e—, wherein e is a positive integer.

9. A surfactant as claimed in claim 1, wherein each B consists of a unit of the formula:
—[CH.sub.2].sub.r—[CH.sub.2CH.sub.2O].sub.e—[CH.sub.2].sub.r, wherein e is a positive integer and r and r′ are each independent 0, 1, 2, 3, 4 or 5.

10. A surfactant as claimed in claim 1, wherein each B consists of the formula:
—[CH(CH.sub.3)CH.sub.2O].sub.f—[CH.sub.2CH.sub.2O].sub.e—[CH.sub.2CH(CH.sub.3)O].sub.f′—CH.sub.2CH(CH.sub.3)—, wherein e, f and f′ are each independently a positive integer.

11. A surfactant as claimed in claim 10, wherein e is an integer from 1 to 100.

12. A surfactant as claimed in claim 10, wherein f and f′ are each independently an integer from 1 to 50.

13. A surfactant as claimed in claim 1 selected from the group consisting of: ##STR00018## ##STR00019## wherein each b, e, f and f′ are each independently a positive integer.

14. A surfactant as claimed in claim 1 selected from the group consisting of: ##STR00020## wherein each b, e, f and f′ are each independently a positive integer.

15. A method for making a surfactant as claimed in claim 1, the method comprising: reacting a compound of the formula (VIII)
A-(CH.sub.2).sub.a—Y  (VIII), wherein A is a perfluoropolyether and comprises a repeat unit of the formula:
—[CF(CF.sub.3)CF.sub.2O].sub.b—, wherein b is a positive integer; a is a positive integer; and Y comprises a nucleophilic group, a leaving group, or an isocyanate group, with a compound of the formula (XI)
Z—B—Z  (XI), wherein B is a polyalkylene oxide, and each Z comprises a nucleophilic group, a leaving group or an isocyanate group.

16. A method as claimed in claim 15, wherein each A comprises a repeat unit of the formula:
—[CF.sub.2CF.sub.2O].sub.c—[CF(CF.sub.3)CF.sub.2O].sub.b—, wherein h is a positive integer and c is 0 or a positive integer; or wherein each A consists of the formula:
CF.sub.3CF.sub.2CF.sub.2O—[CF(CF.sub.3)CF.sub.2O].sub.b—CF(CF.sub.3)—, wherein b is a positive integer.

17. A method as claimed in claim 15, wherein a is an integer from 1 to 5.

18. A method as claimed in claim 15, wherein each B comprises a group selected from a group consisting of a polyethylene oxide unit, a polypropylene oxide unit, and a unit of the formula:
—[CH.sub.2CH.sub.2O].sub.e, wherein e is a positive integer.

19. A method as claimed in claim 15, wherein B consists of the formula —[CH.sub.2].sub.r—[CH.sub.2CH.sub.2O].sub.e—[CH.sub.2].sub.r— or —[CH(CH.sub.3)CH.sub.2O].sub.f—[CH.sub.2CH.sub.2O].sub.e[CH.sub.2CH(CH.sub.3)].sub.f—CH.sub.2CH(CH.sub.3)—, wherein e, f and f′ are each independently a positive integer and r and r′ are each independently 0, 1, 2, 3, 4 or 5.

20. A method as claimed in claim 15, wherein Y is selected from NH2, NHMe, OH, SH, NCO, Cl, Br, I, OMe, OEt, OTs, OMs, OTf, OC.sub.6H.sub.4NO.sub.2, NHC(O)L, C(O)L, OC(O)L, SO.sub.2L and OC.sub.6H.sub.4SO.sub.2L, wherein L is selected from Cl, Br, I, OMe, OEt, OFT, OTs, OMs, OTf and OC.sub.6H.sub.4NO.sub.2 and/or wherein Z is selected from NH.sub.2, OH, SH, NCO, Cl, Br, I, OMe, OEt, OH, OTs, OMs, OTf, OC.sub.6H.sub.4NO.sub.2, NHC(O)L, C(O)L, OC(O)L, SO.sub.2L and OC.sub.6H.sub.4SO.sub.2L, wherein L is selected from Cl, Br, I, OMe, OEt, OH, OTs, OMs, OTf and OC.sub.6H.sub.4NO.sub.2.

21. A method as claimed in claim 15, wherein the compound of the formula (VIII) is selected from the group consisting of CF.sub.3CF.sub.2CF.sub.2O—[CF(CF.sub.3)CF.sub.2O].sub.b—CF(CF.sub.3)—CH.sub.2OC.sub.6H.sub.4SO.sub.2Cl, CF.sub.3CF.sub.2CF.sub.2O—[CF (CF.sub.3)CF.sub.2O].sub.b—CF(CF.sub.3)—CH.sub.2SO.sub.2Cl, CF.sub.3CF.sub.2CF.sub.2O—[CF(CF.sub.3)CF.sub.2O].sub.b—CF(CF.sub.3)—CH.sub.2OC, (O)OC.sub.6H.sub.4NO.sub.2, CF.sub.3CF.sub.2CF.sub.2O—[CF(CF.sub.3)CF.sub.2O].sub.b—CF(CF.sub.3)—CH.sub.2OH, CF.sub.3CF.sub.2CF.sub.2O—[CF(CF.sub.3)CF.sub.2O].sub.b—CF(CF.sub.3)—CH.sub.2NCO, CF.sub.3CF.sub.2CF.sub.2O—[CF(CF.sub.3)CF.sub.2O].sub.b—CF(CF.sub.3)—CH.sub.2NH.sub.2 and CF.sub.3CF.sub.2CF.sub.2O—[CF(CF.sub.3)CF.sub.2O].sub.b—CF(CF.sub.3)—CH.sub.2NHMe, wherein b is an integer from 1 to 50 and/or wherein the compound of formula (XI) is selected from TsO—CH.sub.2CH.sub.2—[OCH.sub.2CH.sub.2].sub.c—OTs, MsO—CH.sub.2CH.sub.2—[OCH.sub.2CH.sub.2].sub.e—OMs, NO.sub.2C.sub.6H.sub.4OC(O)O—CH.sub.2CH.sub.2—[OCH.sub.2CH.sub.2].sub.e—OC(O)OC.sub.6H.sub.4NO.sub.2, OCN—CH.sub.2CH.sub.2—[OCH.sub.2CH.sub.2].sub.e—NCO, H.sub.2N—[CH.sub.2].sub.3—[OCH.sub.2CH.sub.2].sub.e—CH.sub.2—NH.sub.2 and N.sub.2N—[CH(CH.sub.3)CH.sub.2O].sub.f—[CH.sub.2CH.sub.2O].sub.e—[CH.sub.2CH(CH.sub.3)O].sub.f—CH.sub.2CH(CH.sub.3)—NH.sub.2 wherein e is an integer from 1 to 100 and f and f are each independently an integer from 1 to 50.

22. A composition comprising a surfactant, wherein the surfactant has a formula selected from the group consisting of
A-(CH.sub.2).sub.a—(X).sub.x—B—(X).sub.x—(CH.sub.2).sub.a-A  (II), wherein, each A is a perfluoropolyether and comprises a repeat unit of the formula:
—[CF(CF.sub.3)CF.sub.2O].sub.b—, wherein h is a positive integer; a is a positive integer; X is either a covalent bond or a linking group; x is a positive integer; B is a polyalkylene oxide unit; and each A, X, a and x may be the same or different.

23. The composition as claimed in claim 22, in the form of an emulsion comprising: a discontinuous aqueous phase; a continuous oil phase; and said surfactant.

24. A method of preparing an emulsion comprising: (i) preparing an aqueous phase; (ii) preparing an oil phase; and (iii) mixing said aqueous phase, said oil phase and a surfactant having a formula selected from the group consisting of:
A-(CH.sub.2).sub.a—(X).sub.x—B—(X).sub.x—(CH.sub.2).sub.a-A  (II), wherein, each A is a perfluom olyether and comprises a repeat unit of the formula:
—[CF(CF.sub.3)CF.sub.2O].sub.b— wherein b is a positive integer; a is a positive integer; X is either a covalent bond or a linking group; x is a positive integer; B is a polyalkylene oxide unit; and A, X, a and x may be the same or different, to form said emulsion.

25. A method as claimed in claim 24, wherein said mixing is by a flow focus junction of a microfluidic device.

26. A method comprising performing one or more chemical and/or biological reactions, and/or biological processes in the discontinuous aqueous phase of an emulsion as claimed in claim 23.

Description

BRIEF DESCRIPTION OF FIGURES

(1) These and other aspects of the invention will now be further described, by way of example only, with reference to the accompanying figures in which:

(2) FIG. 1 is an IR spectrum of the acyl chloride product derived from the reaction of Krytox 157 FSL with oxalyl chloride;

(3) FIG. 2 is an IR spectrum of methyl ester (XII);

(4) FIG. 3 is a .sup.1H NMR spectrum of methyl ester (XII);

(5) FIG. 4 is an IR spectrum of alcohol (XIII);

(6) FIG. 5 is a .sup.1H NMR spectrum of alcohol (XIII);

(7) FIG. 6 is a .sup.1H NMR spectrum of tosylated polyethylene glycol (XIV);

(8) FIG. 7 is a .sup.1H NMR spectrum of surfactant (IIa);

(9) FIG. 8 is an IR spectrum of activated carbonate ester product (XV);

(10) FIG. 9 is a .sup.1H NMR spectrum of activated carbonate ester product (XV);

(11) FIG. 10 is an IR spectrum of surfactant (IIb);

(12) FIG. 11 is a .sup.1H NMR spectrum of surfactant (IIb);

(13) FIG. 12 is an IR spectrum of amide (XVI);

(14) FIG. 13 is a .sup.1H NMR spectrum of amide (XVI);

(15) FIG. 14 is a .sup.1H NMR spectrum of amine (XVII);

(16) FIG. 15 is an IR spectrum of surfactant (IIj);

(17) FIG. 16 is a .sup.1H NMR spectrum of surfactant (IIj);

(18) FIG. 17 is a diagrammatic illustration of a polydimethylsiloxane (PDMS) biochip with a flow focusing cross junction nozzle of 40 μm×40 μm;

(19) FIG. 18 is a microscope image of emulsion droplets generated on a polydimethylsiloxane (PDMS) biochip with a flow focusing cross junction nozzle of 40 μm×40 μm;

(20) FIG. 19 shows microscope images of a droplet emulsion sample comprising surfactant (Ha) taken before (left hand image) and after (right hand image) PCR thermal cycles;

(21) FIG. 20 shows microscope images of a droplet emulsion sample comprising surfactant (IIb) taken before (left hand image) and after (right hand image) PCR thermal cycles;

(22) FIG. 21 shows an electrophoresis analysis of emulsion PCR product resulting from the droplet emulsion sample comprising surfactant (IIa);

(23) FIG. 22 shows an electrophoresis analysis of emulsion PCR product resulting from the droplet emulsion sample comprising surfactant (IIb);

(24) FIG. 23a is a microscope image of surfactant (IIa) stabilized droplets containing cells after incubation for 2 hours at 37° C.;

(25) FIG. 23b is a bar graph showing the percentage of viable cells in surfactant (IIa) stabilized droplets containing cells after incubation for 2 hours at 37° C.;

(26) FIG. 24a is a microscope image of surfactant (IIb) stabilized droplets containing cells after incubation for 2 hours at 37° C.;

(27) FIG. 24b is a bar graph showing the percentage of viable cells in surfactant (IIb) stabilized droplets containing cells after incubation for 2 hours at 37° C.;

EXAMPLES

(28) Materials

(29) All starting materials employed are commercially available. Krytox™ 157 FSL (MW=2103 Dalton) was obtained from DuPont. Jeffamine® 900 and polyethylene glycol (MW=950-1050 Dalton) were obtained from Sigma Aldrich.

(30) FC72, Anhydrous Novec 7100™ and Novec 7500™ were obtained from 3M. Oxalyl chloride, polymer-supported 4-dimethylaminopyridine, DBU (1,8-diazabicyclo(5.4.0)undec-7-ene), anhydrous methanol, sodium borohydride, diglyme, trimethylamine, methylamine, pyridine, methylmorpholine, poly(ethylene glycol) bis(3-aminopropyl) terminated, p-nitrophenylorthochloroformate, anhydrous tetrahydrofuran, anhydrous toluene, phenylsilane, polymer-supported piperidine, dichloromethane, 4-toluene sulfonylchloride, ammonium hydroxide and Fe.sub.3(CO).sub.12 were obtained from Sigma Aldrich.

(31) Anhydrous dimethylformamide (DMF), anhydrous sodium sulphate, ammonium carbonate, hydrochloric acid, anhydrous magnesium sulfate, anhydrous diethyl ether, toluene, hexane, 3-aminopropyl silica gel, sodium hydride and methanol, were obtained from Sigma Aldrich.

(32) Analysis Methods

(33) Infra-Red (IR) spectroscopy analysis was performed using a Perkin Elmer Spectrum One IR machine with diamond ATR accessory.

(34) Nuclear Magnetic Resonance (NMR) spectroscopy analysis was performed using a 500 MHz Bruker AVANCE III HD NMR spectrometer with DCH-Cryoprobe.

Example 1: Synthesis of Surfactant (IIa)

(35) ##STR00008##

(36) Step 1

(37) 90 g of Krytox™ 157 FSL (Mw=2103 Daltons) were placed in a 250 mL round bottom flask, equipped with a magnetic stirrer bar and sealed with a rubber seal. The flask was evacuated and refilled with nitrogen three times to de-gas the Krytox™ polymer. 75 mL of anhydrous Novec™ 7100 was added by syringe to dissolve the Krytox™. Then 105 mL of oxalyl chloride was added by syringe at room temperature followed by catalytic amounts of anhydrous DMF (one drop from a syringe needle). The reaction was stirred at room temperature overnight, decanted into a clean 250 mL round bottom flask and evaporated to dryness. Yield of acyl chloride (off-white opaque oil): quantitative. IR carbonyl stretch at 1807 cm.sup.−1. The IR spectrum for the acyl chloride product is shown in FIG. 1.

(38) Step 2

(39) 17 g of the acyl chloride product from step 1 were dissolved in anhydrous Novec™ 7100 and 2 g of polymer supported 4-dimethylaminopyridine (1.5 eq.) was added. The mixture was protected with nitrogen and 5 mL of anhydrous methanol was syringed in at room temperature. The reaction was stirred at room temperature overnight. The polymer supported 4-dimethylaminopyridine was filtered off and the filtrate evaporated to dryness. Yield of methyl ester (XI) (clear oil): 16.9 g (99.4%). IR carbonyl stretch at 1792 cm.sup.−1, .sup.1H NMR peak at 4.0 (s). The IR spectrum for the methyl ester product (XII) is shown in FIG. 2. The .sup.1H NMR spectrum for the methyl ester product (XII) is shown in FIG. 3.

(40) Step 3

(41) 3 g of sodium borohydride was placed in a 100 mL round bottom flask and 5 mL of anhydrous Novec™ 7100 and 4 mL of diglyme were added. The mixture was placed under nitrogen in an ice bath. 16.9 g of the methyl ester product (XII) from step 2 was dissolved in 15 mL of anhydrous Novec™ 7100 and slowly added to the suspended sodium borohydride by syringe. Then the flask was fitted with a reflux condenser, protected with nitrogen and heated to 75° C. for one hour. The reaction was allowed to cool to room temperature, was diluted to 150 mL with Novec™ 7100 and quenched by pouring into 100 mL aqueous ammonium chloride/hydrochloric acid buffer (pH=7 after quench). The phases were separated and the fluorophilic phase extracted with 100 mL of water and dried over anhydrous sodium sulphate. The drying agent was removed by filtration and the filtrate evaporated to dryness. Yield of alcohol (XIII) (clear oil): 14.4 g (86%), .sup.1H NMR peaks at 3.8 (s) and 4.25 (dd). The IR spectrum for the alcohol product (XIII) is shown in FIG. 4. The .sup.1H NMR spectrum for the alcohol product (XIII) is shown in FIG. 5.

(42) Step 4

(43) 50 g of polyethylene glycol (Mw=950-1050 Dalton) was dissolved in 300 mL of dichloromethane and protected with nitrogen. 16.2 mL of pyridine was added by syringe followed by 25.7 g of 4-toluene sulfonylchloride in 100 mL of dichloromethane. The reaction was stirred at room temperature overnight. The reaction was extracted twice with 100 mL of 1 M hydrochloric acid, dried with 200 mL of saturated brine and dried over anhydrous magnesium sulfate. The drying agent was filtered off and the clear filtrate was evaporated to dryness. The oily residue was extracted three times with 50 mL of anhydrous diethyl ether and then dried in vacuo. The oily residue was refluxed in a Dean-Stark apparatus in 100 mL of toluene where a further 1 mL of water was removed. The solution was evaporated to dryness. Yield of tosylated polyethylene glycol (XIV) (off white clear oil): 47 g (72%). The .sup.1H NMR spectrum for the tosylated polyethylene glycol product (XIV) is shown in FIG. 6.

(44) Step 5

(45) 0.86 g of sodium hydride was suspended in 40 mL of anhydrous Novec™ 7100 and protected with nitrogen. 75.2 g of the alcohol product (XIII) from step 3 was dissolved in 35 mL of anhydrous Novec™ 7100 and added to the sodium hydride suspension. The suspension was warmed to 40° C. for three hours until no more gas evolved. The reaction was allowed to cool to room temperature. 22.6 g of the tosylated polyethylene glycol product (XIV) from step 4 was dissolved in 100 mL of anhydrous tetrahydrofuran and added to the suspension in 25 mL aliquots by syringe. The reaction was stirred at room temperature overnight and then heated to 65° C. for one day. The reaction was cooled to room temperature and extracted twice with 75 mL of methanol. The fluorous layer was concentrated to near dryness and the oily residue was purified by column chromatography eluting with 10% methanol in Novec™ 7100. Yield of surfactant (IIa): 12.9 g. The .sup.1H NMR spectrum of the surfactant product (IIa) is shown in FIG. 7.

Example 2: Synthesis of Surfactant (IIb)

(46) ##STR00009##

(47) Steps 1 to 3 were carried out in the same manner as for Example 1.

(48) Step 4

(49) 10.5 g of the alcohol product (XIII) from step 3 was placed in a 100 mL round bottom flask, equipped with a magnetic stirrer bar, sealed with a rubber seal and evacuated and refilled with nitrogen three times. Then 20 mL of anhydrous Novec™ 7100 were added by syringe followed by 2.8 mL of trimethylamine and 0.12 mL of pyridine. The solution was cooled in a water-ice bath and p-nitrophenylorthochloroformate in 10 mL of anhydrous tetrahydrofuran was slowly added by syringe. An off-white precipitate formed. The reaction was allowed to warm to room temperature and stirred for four days. The reaction was quenched with 50 mL of aqueous ammonium carbonate solution, the layers separated and the organic layer was extracted with a further 50 mL of aqueous ammonium carbonate. The fluorous layer was evaporated to dryness and purified by column chromatography. Yield of activated carbonate ester (XV) (clear oil): 6.15 g (54%). .sup.1H NMR peaks at 4.95 (m), 7.4 (d) and 8.85 (d). The IR spectrum for the activated carbonate ester product (XV) is shown in FIG. 8. The .sup.1H NMR spectrum for the activated carbonate ester product (XV) is shown in FIG. 9.

(50) Step 5

(51) 1.08 g of Jeffamine 900 was placed in a 100 mL round bottom flask. 6 g of the activated carbonate ester product (XV) from step 4 was dissolved in 15 mL of Novec™ 7100 and added to the Jeffamine. 1 g of polymer supported piperidine was added and the reaction was stirred at room temperature protected under nitrogen for three days. The polymer supported piperidine had coagulated/clumped and turned bright yellow. The reaction was filtered off and the clear filtrate was purified by column chromatography with 10% methanol in Novec™ 7100 as the eluent. Yield of surfactant (IIb) (yellow waxy solid): 2.84 g (42%). IR carbonyl stretch at 1743 cm.sup.−1. The IR spectrum for the surfactant product (IIb) is shown in FIG. 10. The .sup.1H NMR spectrum for the surfactant product (Mb) is shown in FIG. 11.

Example 3: Synthesis of Surfactant (IIc)

(52) ##STR00010##

(53) Step 1 was carried out in the same manner as for Example 1.

(54) Step 2

(55) To a stirred solution of the acyl chloride product from step 1 (45.14 g; Mw=2329.5; 19.37 mmol) in Novec 7500 (50 mL), that was cooled to ˜4° C. in an ice bath under N.sub.2, was added ammonium hydroxide (48.5 mL, 7.989 M, 20 mol equivalents) via a syringe and the reaction mixture was stirred rapidly overnight. 50 mL methanol and 20 mL THF were added to the reaction solution with high speed stirring, and then the mixture was allowed to settle. The top layer (mainly a mixture of water/methanol/THF) was separated off and discarded. To the remaining bottom layer, was added 50 mL methanol and 20 mL THF again with high speed stirring, followed by evaporation to remove most of the volatile component. To the residue, was added 55 mL Novec 7100 and 20 mL methanol. The resultant solution was stirred at 700 rpm for 10 minutes. Then, the solution was allowed to settle in a separation funnel, and the top layer was separated and discarded. The collected fluorous phase was washed once more with 20 mL water and then dissolved in 50 mL methanol and 50 mL THF. The resultant clear solution was dried with anhydrous magnesium sulphate. After filtration, the filtrate solution was concentrated to dryness in vacuo giving amide product (XVI) as a colourless oil (yield: 47.537 grams, 98%). IR: 1740 cm.sup.−1. The IR spectrum for the amide product (XVI) is shown in FIG. 12. The .sup.1H NMR spectrum for the amide product (XVI) is shown in FIG. 13.

(56) Step 3

(57) To solid Fe.sub.3(CO).sub.12 (1.05 g) under nitrogen, was added anhydrous toluene (52 mL) at room temperature with stirring. To the resultant dark green solution (˜0.04 M), was added a solution of the amide product (XVI) from step 2 (47.18 grams, Mw=2310, 20.42 mmol) in anhydrous Novec 7500 (92 mL) via a syringe, followed by addition of phenylsilane (10.08 mL, 81.7 mmol) using a separate syringe. The mixture was stirred at 132° C. (aluminium block temperature) for 2 days. On cooling to room temperature, a mixture of 15 mL methanol, 7 mL 5N HCl aqueous solution and 8 mL water were added portionwise into the reaction using a Pasteur pipette with stirring, followed by a mixture of 25 mL methanol and 25 mL 2N NaOH aq. solution, and finally 10 mL 2N NaOH. After stirring for a further 10 minutes, the resultant mixture was filtered over celite. The red aqueous top layer and fluorous bottom layer were separated in a separating funnel and the aqueous phase was back extracted with 50 mL Novec 7500. The combined organic phase was dried with anhydrous sodium sulphate. After filtration, it was concentrated in vacuo giving a yellow-brown oil, 43.4 grams. The concentrated residue was dissolved again in 80% Novec 7100/hexane (65 mL), and purified on an Interchim SiHC cartridge (220 g, 50 μm diameter spherical silica gel) and was eluted as follows: (1) 80% Novec 7100/Hexane (700 mL), (2) Novec 7100 (2000 mL), (3) 0.5% MeOH/Novec 7100 (800 mL) and finally (4) pure Novec7100 (1000 mL) consecutively.

(58) The product containing fractions were combined and concentrated in vacuo to give amine product (XVII) as a white hazy oil (yield: 26.908 grams, 57.37%). The .sup.1H NMR spectrum for the amine product (XVII) is shown in FIG. 14.

(59) Step 4

(60) To a stirred solution of the amine product (XVII) from step 3 (4.313 grams, Mw=2231, 1.933 mmol) in anhydrous Novec 7100 (8.0 mL) and THF (1.4 mL), was added Hunig's base (0.35 mL, 2.03 mmol) via syringe, followed by a warm solution of p-NO.sub.2Ph-OCOO—PEO-OCOO-p-NO.sub.2Ph (1 gram, Mn˜1098, 0.91 mmol) in THF (7 mL) together with DBU (0.306 mL, 2.03 mmol). The mixture was then stirred at 40° C. for two days, and then evaporated to dryness. The residue was dissolved in in Novec7100 (50 mL), and the solution was treated with 4 g of 3-aminopropyl silica gel with gentle stirring for 10 minutes. After filtration of the solid, the filtrate was treated with another 2 g of 3-aminopropyl silica gel with gentle stirring for 10 minutes. The same procedure was repeated once more with 0.7 g of 3-aminopropyl silica gel. The final filtrate was concentrated in vacuo to dryness to give a pale yellow oil, which was purified on an lnterchim SiHC cartridge (25 g, 50 μm diameter spherical silica gel). The cartridge was eluted as follows: (1) Novec 7100 (100 mL), (2) 3% MeOH/Novec 7100 (100 mL), (3) 6% MeOH/Novec 7100 (100 mL) and finally (4) 10% MeOH/Novec 7100 (300 mL) consecutively. The product containing fractions were combined and concentrated in vacuo to give carbamate product (IIc) as a white hazy oil (yield: 377 mg).

Example 4: Synthesis of Surfactant (IIg)

(61) ##STR00011##

(62) Steps 1 to 3 were carried out in the same manner as for Example 3.

(63) Step 4

(64) To a stirred solution of PEG-bis(isocyanate) (0.988 g, Mn˜2000, 0.49 mmol) in anhydrous THF (10 mL) at 35° C., was added the amine product (XVII) from step 3 (4.536 grams, Mw=2231, 1.976 mmol) in Novec 7500 (7.5 mL) and followed by DBU (0.225 mL, 1.482 mmol). The resultant mixture was stirred at 35° C. overnight. Then to it, was added 3-aminopropyl silica gel (2.604 g) and Novec 7100 (5 mL). The suspension was gently stirred at room temperature for 20 minutes. After filtration of the solid, the filtrate was treated with another 1.06 g 3-aminopropyl silica gel with gentle stirring for further 10 minutes. The final filtrate was concentrated in vacuo to dryness to give a yellow oil, which was purified on an Interchim SiHC cartridge (25 g, 50 μm diameter spherical silica gel). The cartridge was eluted as follows: (1) Novec 7100 (100 mL), (2) 3% MeOH/Novec 7100 (100 mL), (3) 6% MeOH/Novec 7100 (100 mL) and finally (4) 10% MeOH/Novec 7100 (300 mL) consecutively. The product containing fractions were combined and concentrated in vacuo to give urea product (IIg) as a light yellow oil (yield: 477 mg).

Example 5: Synthesis of Surfactant (IIj)

(65) ##STR00012##

(66) Steps 1 to 4 were carried out in the same manner as for Example 1 and Example 2.

(67) Step 5

(68) The activated Krytox carbonate ester product (XV), 38.09 grams, 15.47 mmol) from step 4 was placed in a 250 mL round bottom flask fitted with magnetic stirrer bar and 50 mL dropping funnel with septum. The apparatus was degassed by applying vacuum and refilled with nitrogen 3 times. Dry Novec 7100 (stored over anhydrous Na.sub.2SO.sub.4, 30 mL) was added by syringe to dissolve the activated carbonate ester, followed by anhydrous tetrahydrofuran (50 mL) by syringe. Poly(ethylene glycol) bis(3-aminopropyl) terminated (Mn˜1500, 10.44 grams, 6.96 mmol) was dissolved in anhydrous tetrahydrofuran (25 mL) and dry Novec 7100 (stored over anhydrous Na.sub.2SO.sub.4, 15 mL) under nitrogen protection in a 100 mL round bottom flask fitted with septum. 4-Methylmorpholine (2.8 mL=2.58 grams, 23.21 mmol) was added by syringe to the poly(ethylene glycol) bis(3-aminopropyl) terminated solution. The solution with 4-methylmorpholine was then added to the dropping funnel and added slowly to the stirring activated Krytox carbonate ester over 30 minutes. The reaction was stirred at 35° C. heating block temperature for 17 hours. The reaction was then evaporated to dryness. The residue was re-dissolved in 200 mL of Novec 7100. 3-Aminopropyl functionalised silica gel (15 grams, 15 mmol) was added to the solution, stirred for 10 minutes and removed by filtration over a filter frit. Another 15 grams (15 mmol) of 3-aminopropyl functionalised silica gel was added four more times and removed by filtration over a frit. After the fifth aliquot of 3-aminopropyl functionalised silica gel, the mixture was filtered over Celite and filter paper under suction. The removal of 4-nitrophenol related impurities was complete when a small amount of 3-aminopropyl functionalised silica gel (20 mg) showed no traces of yellow when a few drops of the filtrate were added. The clear filtrate was evaporated to dryness on a rotary evaporator (40° C., 270 mbar, then 50° C., 0-5 mbar) to an opaque, colourless oily wax (15 g). The waxy residue was dissolved in 50 mL of Novec 7100.

(69) A flash chromatography cartridge (Puriflash HC Spherical Silica, 50 um, 25 g) was primed with 100 mL Novec 7100 (p=8 psi). The solution of crude surfactant was applied by syringe. The column was eluted using 100 ml of neat Novec 7100, then 100 mL of 5% methanol in Novec 7100 and finally 300 mL of 10% methanol in Novec 7100. Ten fractions of 50 mL each were collected. Fractions 6-8 were combined and evaporated to dryness on a rotary evaporator (40° C., 270 mbar, then 70° C., 0-5 mbar) to yield a clear waxy solid IIj (total yield: 3.863 grams, 9%). The IR spectrum for the surfactant product (IIj) is shown in FIG. 15. The .sup.1H NMR spectrum for the surfactant product (IIj) is shown in FIG. 16.

Example 6: Proposed Synthesis of Surfactant (IIh)

(70) ##STR00013##

(71) Step 1 is carried out in the same manner as for Example 1.

(72) Step 2

(73) To a stirred solution of the acyl chloride product from step 1 in Novec 7100 that is cooled in an ice bath, is added a cool aqueous solution of methylamine. The reaction mixture is then allowed to warm to room temperature, and stirring continued overnight. The reaction solution is mixed with saturated brine, and the mixture filtered. The water layer is separated off and the organic fraction dried with anhydrous sodium sulphate, filtered and concentrated in vacuo giving an oil, which was dissolved in FC72. The mixture is filtered through celite and concentrated in vacuo to give amide product (XVIII).

(74) Step 3

(75) To solid Fe.sub.3(CO).sub.12 under nitrogen, is added anhydrous toluene at room temperature with stirring. To the resultant dark green solution is added a solution of the amide product (XVIII) from step 2 in anhydrous Novec 7500, followed by addition of phenylsilane. The mixture is stirred at 130° C. (aluminium block temperature) for 2 days. On cooling to room temperature, methanol and HCl aq. solution are added into the reaction. The resultant solid is filtered off, and washed with Novec 7100. The aqueous phase and fluorous layer are then separated and the aqueous phase back extracted with Novec 7100. The combined organic phase is dried with anhydrous sodium sulphate, filtered and concentrated in vacuo giving an oil, which is dissolved in amixture of hexane and Novec 7100. The mixture is purified on an Interchim SiHC cartridge (25 g, 15 μm diameter spherical silica gel) to give amine product (XIX).

(76) Step 4

(77) To a stirred solution of the amine product (XIX) from step 3 in anhydrous Novec 7100 and THF, is added Hunig's base followed by a warm solution of NO.sub.2PhOCOO—(CH.sub.2CH.sub.2O).sub.eCO.sub.2PhNO.sub.2 in THF together with DBU. The mixture is then stirred at 40° C. for two days, and evaporated to dryness. The residue is dissolved in in Novec7100 and the solution is treated with 3-aminopropyl silica gel with gentle stirring. The mixture is filtered and the filtrate concentrated in vacuo. The resultant residue is purified on an Interchim SiHC cartridge to give carbamate product (IIh).

Example 7: Proposed Synthesis of Surfactant (IIi)

(78) ##STR00014##

(79) Steps 1 to 3 are carried out in the same manner as for Example 6.

(80) Step 4

(81) To a stirred solution of PEG-bis(isocyanate) in anhydrous THF at 35° C., is added the amine product (XIX) from step 3 in Novec 7500, followed by DBU. The resultant mixture is stirred at 35° C. overnight then 3-aminopropyl silica gel and Novec 7100 is added. The mixture is stirred at room temperature and then filtered. The filtrate is concentrated in vacuo and the residue purified on an Interchim SiHC cartridge to give urea product (IIi).

Example 8: Proposed Synthesis of Surfactant (IId)

(82) ##STR00015##

(83) Steps 1 to 3 are carried out in the same manner as for Example 3.

(84) Step 4

(85) The amine product from step 3 is converted to the corresponding isocyanate.

(86) Step 5

(87) The isocyanate product from step 4 is reacted with Jeffamine 900 to provide the surfactant product (IId).

Example 9: Proposed Synthesis of Surfactant (IIe)

(88) ##STR00016##

(89) Steps 1 to 3 are carried out in the same manner as for Example 1.

(90) Step 4

(91) The alcohol product from step 3 is converted to the sulfonyl chloride.

(92) Step 5

(93) The sulfonyl chloride product from step 4 is reacted with Jeffamine 900 to provide the surfactant product (IIe).

Example 10: Proposed Synthesis of Surfactant (IIf)

(94) ##STR00017##

(95) Steps 1 to 3 are carried out in the same manner as for Example 1.

(96) Step 4

(97) The alcohol product from step 3 is converted to the corresponding phenyl ether.

(98) Step 5

(99) The phenyl ether product from step 4 is converted to the sulfonyl chloride.

(100) Step 6

(101) The sulfonyl chloride product from step 5 is reacted with Jeffamine 900 to provide the surfactant product (IIf).

Example 11: Emulsion Generation Using Surfactants

(102) Emulsion droplets were generated on a polydimethylsiloxane (PDMS) Pico-Gen™ biochip (Sphere Fluidics Limited) with a flow focusing cross junction nozzle of 40 μm×40 μm. This biochip is illustrated diagrammatically in FIG. 17 which shows the emulsion outlet (1) (at the top left of the diagram), aqueous inlet (2) (indicated by the left hand side arrow in the main diagram), oil inlet (3) (indicated by the right hand side arrow in the main diagram) and flow focusing cross junction (4) (indicated by the arrows in the magnified inset).

(103) Novec™ 7500 was used as the continuous oil phase and polymerase chain reaction (PCR) mix solution (see below table) was used as the aqueous phase. 5% (w/w) of purified surfactant (IIa) from Example 1 or surfactant (IIb) from Example 2 was dissolved in the continuous oil phase prior to mixing of the oil and aqueous phases in the microfluidic device. Table 1 shows the composition of the PCR mix solution.

(104) PCR Mix Solution

(105) Platinum® Taq DNA Polymerase kit (Life Technologies, #10966)

(106) Jurkat genomic DNA sample (Thermo Fisher Scientific, #SD1111)

(107) ACTB primer set (Jena Bioscience GmbH, #PCR-253)

(108) dNTP Mix, 10 mM each (Thermo Fisher Scientific, #R0191)

(109) Nuclease-free Water, 50 mL (Life Technologies, #AM9937)

(110) TABLE-US-00001 TABLE 1 Reagent Volume (μL) Final Concentration Nuclease-free Water 435.6 n/a Platinum Taq buffer 60 n/a MgCl.sub.2 18 1.5 mM dNTP (10 mM) 12 0.2 mM Primers 12 0.3 μM DNA sample 60  3.65 ng/μL Platinum Taq enzyme 2.4 0.4 unit/50 μL Master mix volume 600 n/a

(111) The oil flow rate was 300 μL/hr and the aqueous flow rate was 300 μL/hr. Droplet generation frequency was about 1,000 Hz, and droplet volume was around 80-87 μL (53.5-55 μm in diameter). FIG. 18 shows droplets (5) generated at the flow focusing junction (4) where an oil phase (6) (indicated by the vertical arrows) and an aqueous phase (7) (indicated by the horizontal arrow) meet. The droplets are stable within the microfluidic channel.

(112) This demonstrates the successful formation of an emulsion comprising surfactant (IIa) or (IIb) within a microfluidic device. The droplets formed are stable within the microfluidic channel, showing that the surfactants successfully stabilised the droplets, preventing coalescence.

Example 12: Emulsion PCR

(113) The droplet emulsion samples generated in Example 11 using surfactant (IIa) and surfactant (IIb) were each placed in a G-Strom Thermal Cycler System (Labtech.com), and the thermal cycle program shown in Table 2 was run.

(114) TABLE-US-00002 TABLE 2 Temperature Time # Cycles 95° C. 2 min 1 95° C. 30 sec 35 59° C. 30 sec 72° C. 30 sec 72° C. 2 min 1  4° C. ∞ ∞

(115) Droplet images were taken under a Zeiss microscope with a Mikrotron Hi-Speed camera before and after the PCR thermal cycles. FIG. 19 shows microscope images of the droplet emulsion sample comprising surfactant (IIa) before (left hand side image) and after (right hand side image) the PCR thermal cycles were run. FIG. 20 shows microscope images of the droplet emulsion sample comprising surfactant (IIb) before (left hand side image) and after (right hand side image) the PCR thermal cycles were run. The images show that the surfactant (IIa) and surfactant (IIb) were each functionally active by stabilising the droplets and stopping coalescence even during thermal cycles.

(116) The PCR product was then analysed with standard agarose gel DNA electrophoresis. Emulsion PCR was also run analogously using the commercially available fluorous surfactant Pico-Surf™ 1 (Sphere Fluidics Limited) in place of surfactant (IIa) or (IIb) in the droplet emulsion, and the product used as a positive control in the electrophoresis analysis.

(117) FIG. 21 shows the electrophoresis result for the emulsion PCR product resulting from the droplet emulsion sample comprising surfactant (IIa). In FIG. 21, Lane 1: PCR product in bulk; Lane 2: PCR product in emulsion stabilized with Pico-Surf™ 1; and Lane 3: PCR product in emulsion stabilized with surfactant (IIa). This shows that the PCR product in the surfactant (IIa) stabilized droplet emulsion gives a clear product band as bright as that of the PCR product from the Pico-Surf™ 1 stabilized droplet emulsion.

(118) FIG. 22 shows the electrophoresis result for the emulsion PCR product resulting from the droplet emulsion sample comprising surfactant (IIb). In FIG. 22, Lane 1: molecule ladder; Lane 2: PCR product in bulk; Lane 3: PCR product in emulsion stabilized with Pico-Surf™ 1; and Lane 4: PCR product in emulsion stabilized with surfactant (IIb). This shows that the PCR product in the surfactant (IIb) stabilized droplet emulsion gives a clear product band as bright as that of the PCR product from the Pico-Surf™ 1 stabilized droplet emulsion.

Example 13: Cell Viability in Surfactant Stabilized Emulsions

(119) 1.2×10.sup.6 Chinese hamster ovary (CHO) cells from a suspension culture were pelleted (300×g, 5 min), re-suspended in 1 mL encapsulation media (CHO cell growth media, 16% OptiPrep™, 1% Pluronic® F-68) and passed through a 30 μm CeliTrics® cell strainer. Cells were encapsulated in 300 μL droplets using 5% (w/w) surfactant (IIa), surfactant (IIb) or Pico-Surf™ 1 in Novec™ 7500. 200 μL of emulsion was collected for each sample and after collection placed in a 37° C. CO.sub.2 incubator for 2 hr before being processed. Non-encapsulated CHO cells were kept in parallel as a viability control.

(120) In order to assess viability, samples were de-emulsified by mixing with an equal volume of Pico-Break™ (Sphere Fluidics Limited), followed by transferring 100 μL of the aqueous phase (containing CHO cells) into a fresh 1.5 mL reaction tube. 5 μL of Solution 18 (AO•DAPI, Chemometec, #910-3018) were added to the cells, mixed, and 10 μL of each sample was loaded in a chamber of an NC-Slide A8™ (Chemometec, #942-0003). Non-encapsulated CHO cells (100 μL) were directly mixed with 5 μL of Solution 18 prior to loading on an NC-Slide A8™. Cell viability was determined using the Viability and Cell Count Assay program on a NucleoCounter® NC-250™ instrument.

(121) FIG. 23 shows the results of the cell viability study for cells encapsulated in droplets using surfactant (IIa). FIG. 23a shows a microscope image of surfactant (IIa) stabilized droplets containing cells after incubation for 2 hours at 37° C. The scale bar represents 100 μm. The arrows indicate cells (which appear as small white circles) in the droplets. FIG. 23b shows the percentage of viable cells in the samples from non-encapsulated CHO cells (Bulk-left), cells encapsulated in droplets using Pico-Surf™ 1 (middle), and cells encapsulated in droplets using surfactant (IIa) (right) after incubation for 2 hours at 37° C. This shows that the viability of cells encapsulated in droplets using surfactant (IIa) is comparable to the viability of cells encapsulated in droplets using Pico-Surf™ 1 and to the viability of non-encapsulated cells.

(122) FIG. 24 shows the results of the cell viability study for cells encapsulated in droplets using surfactant (lib). FIG. 24a shows a microscope image of surfactant (IIb) stabilized droplets containing cells after incubation for 2 hours at 37° C. The scale bar represents 100 μm. The arrows indicate cells (which appear as small white circles) in the droplets. FIG. 24b shows the percentage of viable cells in the samples from non-encapsulated CHO cells (Bulk-left), cells encapsulated in droplets using Pico-Surf™ 1 (middle), and cells encapsulated in droplets using surfactant (IIb) (right) after incubation for 2 hours at 37° C. This shows that the viability of cells encapsulated in droplets using surfactant (IIb) is comparable to the viability of cells encapsulated in droplets using Pico-Surf™ 1 and to the viability of non-encapsulated cells. The inset image in FIG. 24b shows a typical fluorescent image for the cell viability measurement. When viewed in colour, viable cells appear green and dead cells appear red and the image confirms the afore-described result.

(123) No doubt many other effective alternatives will occur to the skilled person. It will be understood that the invention is not limited to the described embodiments and encompasses modifications apparent to those skilled in the art lying within the spirit and scope of the claims appended hereto.