Method for determining the presence or absence of a biomarker
09945852 ยท 2018-04-17
Assignee
Inventors
- Christopher David Gwenin (Gwynedd, GB)
- Mark Stephen Baird (Gwynedd, GB)
- Vanessa Valerie Gwenin (Gwynedd, GB)
- Mark Pitts (Gwynedd, GB)
Cpc classification
C07C321/06
CHEMISTRY; METALLURGY
C07H13/04
CHEMISTRY; METALLURGY
International classification
C07C321/06
CHEMISTRY; METALLURGY
Abstract
A method of determining the presence or absence in a sample of a biomarker, the method comprising: (a) linking an antigen to colloidal gold to provide a gold-antigen species; (b) contacting the gold-antigen species with the sample; (c) adding a diagnosis agent to the sample; and (d) observing the color of the sample.
Claims
1. A method of determining the presence or absence in a sample of an antibody indicative of exposure to mycobacteria, the method comprising: (a) linking an antigen to colloidal gold to provide a gold-antigen species; (b) contacting the gold-antigen species with the sample to produce a suspension comprising the gold-antigen species and the sample; (c) adding to the suspension a diagnosis agent selected from the group consisting of: sodium chloride, magnesium chloride, and potassium chloride, wherein the addition of the diagnosis agent to the suspension produces a different coloured suspension depending on whether the sample contains or does not contain the antibody; and (d) observing the colour of the sample, wherein the antigen includes at least one mycolic acid derived antigen selected from the group consisting of: (i) mycolic acids obtained from natural sources; (ii) synthetically prepared mycolic acids; (iii) salts of mycolic acids; (iv) esters of mycolic acids (i) and/or (ii); and (v) sulfur-containing mycolic acids and/or salts or esters thereof.
2. The method according to claim 1, wherein the mycobacteria is Mycobacterium tuberculosis.
3. The method according to claim 1, wherein the antigen is linked to the colloidal gold by a gold-sulfur bond.
4. The method according to claim 3, wherein the gold-sulfur bond is formed between the colloidal gold and a sulfur atom contained within the antigen molecule.
5. The method according to claim 3, wherein the gold-sulfur bond is formed between the colloidal gold and a sulfur-containing linker compound linked to the antigen.
6. The method according to claim 1, in which step (d) involves visually observing the presence or absence of a colour change to provide a qualitative assessment.
7. The method according to claim 1, wherein step (d) involves quantitative measurement of an intensity of light absorbance at one or more wavelengths and at one or more points in time.
8. The method of claim 1, wherein the mycolic acid derived antigen is selected from the group consisting of: (i) mycolic acids obtained from natural sources; (ii) synthetically prepared mycolic acids; and (v) sulphur-containing mycolic acids and/or salts or esters thereof.
Description
EXAMPLE 1
(1) Compound A was prepared as follows:
(2) ##STR00006##
(3) 2,2-dimethylpropinic acid 12-(phenyl-1-H-tetrazole-5-ylsulfonyl)-dodecyl ester was prepared using a method analogous to that described by Al Dulayymi, Baird, Roberts, Verschoor and Deysel in supplementary information to Tetrahedron 2007, 63, 2571-2592.
(4) Sodium bis(trimethylsilyl) amide (70 ml, 74.6 mmol) was added dropwise to a stirred solution of 10-bromodecanal (9 g, 38.2 mmol) and 2,2-dimethylpropinic acid 12-(phenyl-1-H-tetrazole-5-ylsulfonyl)-dodecyl ester (21.98 g, 45.9 mmol) in dry THF (200 ml) under nitrogen at 0 C. and then allowed to warm to room temperature. When TLC showed no starting material remaining the reaction mixture was quenched with a saturated aqueous solution of NH.sub.4Cl at 0 C. followed by petrol/ethyl acetate (10:1, 50 ml). The organic phase was separated and the aqueous layer was extracted with petrol/ethyl acetate (10:1, 3100 ml). The combined organic extracts were dried and evaporated. Column chromatography (petrol/ethyl acetate, 20:1) gave 2,2-dimethylpropanoic acid (22-bromo)-12-enyl-docos ester (12.60 g, 68%) as a colourless oil.
(5) This oil was dissolved in IMS/THF (1:1, 70 ml), palladium on carbon (1 g, 10%) was added and the solution stirred under hydrogen for 1 hour. Filtration through Celite, concentration in vacuo and purification by column chromatography (petrol/ethyl acetate, 10:1) gave 22-bromodocosyl pivalate (11 g, 87%) as a white solid.
(6) This white solid was mixed with 1-Phenyl-1H-tetrazole-5-thiol (4.40 g, 24.6 mmol) and anhydrous potassium carbotonate (6.81 g, 49.3 mmol) in acetone (250 ml) and stirred vigorously for 18 hours at room temperature. When TLC analysis indicated that the reaction was complete, water (500 ml) was added and the mixture was extracted with dichloromethane (1200 ml, 2100 ml). The combined organic phases were washed with brine (2200 ml), dried and the solvent was evaporated to give a white solid, 2,2-Dimethylpropanoic acid 22-(1-phenyl-1H-tetrazole-5-ylsulfanyl)docosyl ester (13 g, 84%).
(7) A solution of ammonium molybdate (VI) tetrahydrate (13.70 g, 11.09 mmol) in 35% H.sub.2O.sub.2 (50 ml) prepared and cooled in an ice bath was added to a stirred solution of the white solid (13 g, 22.18 mol) in THF/IMS (2:3) (150 ml) at 10 C. and stirred at room temperature for 2 hours. A further solution of ammonium molybdate (VI) tetrahydrate (6.85 g, 5.554 mmol) in 35% H.sub.2O.sub.2 (25 ml) was added and the mixture was stirred at room temperature for a further 18 hours. The mixture was poured into water (1 L) and extracted with dichloromethane (1250 ml, 3150 ml). The combined organic phases were washed with water (500 ml), dried and the solvent evaporated. Column chromatography (petrol/ethyl acetate, 5:1) gave compound A (2,2-dimethylpropanoic acid 22-(1-phenyl-1H-tetrazole-5-ylsulfonyl)docosyl ester, 12.4 g, 90%) as a white solid .sub.H: 7.61-7.60 (2H, m), 7.59-7.58 (3H, m), 4.04 (2H, t, J 6.65), 3.73 (2H, t, J 7.4), 1.95-1.92 (2H, m), 1.61 (2H, p, J 6.95), 1.50 (2H, p, J 6.65), 1.37-1.22 (34H, m), 1.19 (9H, s); c: 178.60, 171.08, 153.47, 133.03, 131.40, 129.66, 125.04, 64.42, 60.34, 55.98, 38.68, 29.66, 29.60, 29.53, 29.47, 29.42, 29.18, 29.15, 28.85, 28.57, 28.10, 27.16, 25.87, 21.90, 20.99; v.sub.max/cm.sup.1: 2917, 2854, 1725, 1594, 1500, 1472, 1344, 1285, 1157
EXAMPLE 2
(8) Compound B was prepared by the following method:
(9) ##STR00007##
(10) (2R,3R)-5-benzyloxy-3-(tert-butyl-dimethyl-silanyloxy)-2-(oxo-ethyl)pentanoic acid methyl ester (3.7 g, 9.38 mmol) was prepared according to the method published in Koza, G.; Theunissen, C.; Al-Dulayymi, J. R.; Baird, M. S. Tetrahedron, 2009, 65, 10099. This was dissolved in dry THF (100 ml) along with compound A (6.39 g, 10.32 mmol) and stirred at 10 C. before the addition of lithium bis(trimethylsilyl)amide (14.6 ml, 15.48 mmol). The reaction mixture turned bright yellow and was left to reach r.t. and stirred for one hour. When TLC indicated the reaction was complete, it was quenched by the addition of saturated aqueous NH.sub.4Cl (50 ml). The product was extracted with petrol/ethyl acetate (20:1, 3150 ml), dried over MgSO.sub.4, filtered and evaporated. Column chromotography (petrol/ethyl acetate, 20:1) gave (R)-2-[(E/Z)(R)-1-(tert-butyl-dimethyl-silanyloxy)-3-benzyloxy-propyl]-26-(2,2-dimthyl-propionyloxy)-hexacos-3-enoic acid methyl ester (5 g, 67%) as a colourless oil [Found [M+Na].sup.+: 809.5982; C.sub.48H.sub.86O.sub.6SiNa requires 809.6086; .sub.H: 7.33-7.28 (2H, m), 7.25-7.22 (3H, m), 5.41-5.34 (1H, m), 5.28-5.19 (1H, m), 4.44 (2H, s), 4.02-3.99 (1H, m), 3.59 (3H, s), 3.55-3.48 (2H, m), 2.58-2.54 (1H, m), 2.25-2.20 (2H, m), 1.78 (2H, p, J 5.35), 1.57 (2H, p, J 6.6), 1.29-1.19 (49H, m), 1.15 (9H, s), 0.85-0.79 (6H, m); .sub.C: 178.64, 174.00, 173.98, 138.48, 132.79, 128.31, 127.55, 127.46, 126.83, 72.89, 70.42, 66.24, 64.45, 52.24, 51.34, 51.24, 38.71, 33.74, 33.68, 32.53, 30.39, 29.70, 29.63, 29.60, 29.55, 29.51, 29.48, 29.36, 29.22, 29.10, 28.61, 27.27, 25.90, 17.95, 4.58, 4.87; v.sub.max/cm.sup.1: 2925, 2853, 1731, 1461, 1283, 1252, 1159, 1101.
EXAMPLE 3
(11) Compound C was prepared by the following method:
(12) ##STR00008##
(13) Palladium on carbon (1 g, 10%) was added to a solution of compound B (5 g, 6.35 mmol) in a mixture of THF (50 ml) and IMS (50 ml) and stirred under hydrogen for 2 days. Filtration through Celite and evaporation of the solvent was followed by column chromatography (petrol/ethyl acetate, 5:1) to give (R)-2-[((R)-1-(tert-butyl-dimethyl-silanyloxy)-3-hydroxy-propyl]-26-(2,2-dimthyl-propionyloxy)-hexacosanoic acid methyl ester (3 g, 67%) as a white solid.
(14) This white solid in dichloromethane (20 ml) was added to a stirred suspension of PCC (2.40 g, 11.15 mmol) in dichloromethane (130 ml) at room temperature and the mixture stirred vigorously for 2 hrs. When TLC showed no starting material remained the mixture was poured petrol/ethyl acetate (10:1, 300 ml), filtered through a pad of silica and Celite, washed with petrol/ethyl acetate (10:1) and evaporated. Column chromatography (petrol/ethyl acetate, 10:1) gave (R)-2-[(R)-1-(tert-butyl-dimethyl-silanyloxy)-3-oxo-propy]-26-(2,2-dimethyl-propionyloxy)-hexacosanoic acid methyl ester (2.46 g, 82%) as a colourless oil.
(15) Lithium bis(trimethylsilyl)amide (5.85 ml, 4.77 mmol) was added to a stirred solution of the colourless oil (2.46 g, 3.67 mmol) and 7-bromo (1-phenyl-1H-tetrazole-5-sulfonyl)heptyl (1.84 g, 4.77 mmol) in dry THF (100 ml) at 10 C. The reaction turned bright yellow and was left to reach r.t. and stirred for one hour under N.sub.2. When TLC showed that the reaction was complete the reaction mixture was quenched with a saturated aqueous solution of NH.sub.4Cl (50 ml). The product was extracted with petrol/ethyl acetate (20:1, 3150 ml), dried over MgSO.sub.4, filtered and evaporated. Column chromotography (petrol/ethyl acetate, 20:1) gave (R)-2-[(E/Z)(R)-10-bromo-1-(tert-butyl-dimethyl-silanyloxy)-dec-3-enyl]-26-(2,2-dimethyl-propionyloxy)-hexacosanoic acid methyl ester (2.3 g, 76%) as a colourless oil.
(16) Palladium on carbon (0.3 g, 10%) was added to a stirred solution of this colourless oil (2.30 g, 2.68 mmol) in IMS/THF (1:1, 100 ml) and hydrogenated for 1 hour. The solution was filtered over a bed of Celite and the solvent was evaporated. Column chromatography (petrol/ethyl acetate, 10:1) gave (R)-2-[(R)-10-bromo-1-(tert-butyl-dimethyl-silanyloxy)-decyl]-26-(2,2-dimethylpropionyloxy)-hexacosanoic acid methyl ester (2.0 g, 81%) as a colourless oil.
(17) 1.8 g (2.093 mmol) of this oil was stirred with 1-phenyl-1H-tetrazole-5-thiol (0.41 g, 2.29 mmol), and anhydrous potassium carbotonate (0.63 g, 4.60 mmol) acetone/THF (30/15 ml) under reflux for 2 hours. When TLC indicated the reaction was complete it was quenched with water (50 ml) extracted with dichloromethane (1100 ml, 275 ml). The combined organic layers were washed with brine (2100 ml), dried and the solvent was evaporated. Column chromatography (petrol/ethyl acetate, 5:1) gave (R)-methyl 2-((R)-1-(tert-butyldimethylsilyloxy)-10-(1-phenyl-1H-tetrazol-5-ylsulfanyl)decyl)-26-(pivaloyloxy)hexacosanoate (1.70 g, 85%) as a colourless oil.
(18) A solution of ammonium molybdate (VI) tetrahydrate (1.05 g, 0.85 mmol) in 35% H.sub.2O.sub.2 (15 ml), prepared and cooled in an ice bath was added to a stirred solution of this colourless oil (1.63 g, 1.70 mmol) in THF/IMS (15/20 ml) at 10 C. and stirred at room temperature for 2 hours. A further solution of ammonium molybdate (VI) tetrahydrate (0.52 g, 0.42 mmol) in 35% H.sub.2O.sub.2 (10 ml) was added and the mixture was stirred at room temperature for 18 hours. The mixture was poured into water (250 ml) and extracted with petrol/ethyl acetate (5:2, 2200 ml). The combined organic layers were washed with water (100 ml), dried and the solvent was evaporated. Column chromatography (petrol/ethyl acetate, 5:1 and then 1:1) gave (R)-methyl 2-((R)-1-(tert-butyldimethylsilyloxy)-10-(1-phenyl-1H-tetrazol-5-ylsulfonyl)decyl)-26-(pivaloyloxy)hexacosanoate (compound C, 1.28 g, 76%) as a colourless oil. Found [M+Na].sup.+: 1011.6958; C.sub.55H.sub.100O.sub.7SiN.sub.4Na requires 1011.6974]; [].sup.20.sub.D 7.64 (c 0.89, CHCl.sub.3); O.sub.H: 7.71-7.69 (2H, m), 7.62-7.60 (3H, m), 4.04 (2H, t, J 6.6), 3.92-3.88 (1H, m), 3.73 (2H, t, J 7.85), 3.65 (3H, s), 2.54-2.50 (1H, m), 1.95 (2H, p, J 7.6), 1.61 (2H, p, J 6.9), 1.53-1.15 (67H, m, including a singlet at 1.19), 0.86 (9H, s), 0.04 (3H, s), 0.02 (3H, s); .sub.C: 178.66, 175.08, 153.48, 133.03, 131.43, 129.70, 125.04, 73.15, 64.46, 55.99, 51.58, 51.23, 38.71, 33.61, 29.55, 29.44, 29.39, 29.21, 28.87, 28.59, 28.13, 27.84, 27.43, 27.19, 25.89, 25.74, 23.73, 21.94, 17.96, 4.37, 4.93; v.sub.max/cm.sup.1: 2925, 2853, 1731, 1463, 1344, 1284, 1254, 1154, 1099, 1074, 836, 775.
EXAMPLE 4
(19) Compound D was prepared by the following method:
(20) ##STR00009##
(21) (8S,9S)-8-methoxy-9-methylheptacosanal and 2,2-dimethylpropionic acid-7-(1-phenyl-1H-tetrazol-5-ylsulfonyl)-heptyl ester were prepared using a method analogous to that described by Al Dulayymi, Baird, Roberts, Verschoor and Deysel in supplementary information to Tetrahedron 2007, 63, 2571-2592.
(22) (8S,9S)-8-methoxy-9-methylheptacosanal (1.56 g, 3.56 mmol) and 2,2-dimethylpropionic acid-7-(1-phenyl-1H-tetrazol-5-ylsulfonyl)-heptyl ester (1.74 g, 4.27 mmol) were stirred in dry THF (50 ml) nitrogen at 10 C. Lithium bis(trimethylsilyl)amide (5.24 ml, 5.56 mmol, 1.06 M) was added dropwise between 12 C. and 5 C., the solution was stirred for 18 hrs. When TLC showed no starting material was left, dichloromethane (50 ml) and sat. aq. ammonium chloride (50 ml) were added. The aqueous layer was re-extracted with dichloromethane (2100 ml) and the combined organic layers were dried and evaporated to give a crude product. This was purified via column chromatography eluting with petrol/ether (10:1) to give a colourless oil, (E/Z)-2,2-dimethyl-propionic acid 15-methoxy-16-methyl-tetratriacont-7-enyl ester (1.78 g, 81%). Palladium on charcoal (0.2 g, 10%) was added to a stirred solution of the above product (1.78 g, 2.87 mmol) in THF (5 ml) and IMS (40 ml). The mixture was stirred under hydrogen at atmospheric pressure. When no more hydrogen was being absorbed the catalyst was removed via suction filtration through a pad of celite and was washed with THF (50 ml). The filtrate was evaporated to give a colourless oil, (15S,16S)-15-methoxy-16-methyltetratriacontyl pivalate (1.57 g, 88%).
(23) This colourless oil was added in THF (10 ml) was added slowly to a solution of lithium aluminium hydride (0.14 g, 3.79 mmol) in THF (20 ml) at 20 C. The reaction was allowed to reach RT then heated under reflux for 1 hr. When TLC showed no starting material was left the reaction was cooled to 20 C. and was quenched with sat. aq. sodium sulfate until a white precipitate formed. THF (30 ml) was added and the mixture was stirred for 30 mins, then filtered through a bed of silica and the solvent evaporated. The resulting solution was taken up in dichloromethane (50 ml) and washed with water (10 ml) and then dried. The solvent was evaporated and the crude product was purified via column chromatography eluting with petrol/ether (20:1, then 1:1) to give a white solid.
(24) N-Bromosuccinimide (0.44 g, 2.46 mmol, 1.3 mol. equiv.) was added in portions over 15 mins to a stirred solution of the white solid (1.04 g, 1.89 mmol) and triphenylphosphine (0.56 g, 2.14 mmol, 1.13 equiv) in dichloromethane (20 ml) at 0 C. The mixture was stirred at RT for 1 hr, under TLC indicated completion of the reaction. It was quenched with sat. aq. sodium meta-bisulfite (25 ml) then the aqueous layer was re-extracted with dichloromethane (220 ml) and the combined organic extracts washed with water (50 ml), dried and evaporated. The residue was treated with petrol/ether (1:1, 50 ml) heated at reflux for 30 mins and then filtered and washed with petrol/ether (1:1, 25 ml). The filtrate was evaporated and the resultant residue purified via column chromatography eluting with petrol/ether (10:1) to give a white solid, (15S,16S)-1-bromo-15-methoxy-16-methyl-tetratriacontane (0.76 g, 83%).
(25) This white solid (0.70 g, 1.14 mmol) in THF (3 ml) and acetone (3 ml) was added to a stirred solution of 1-phenyl-1H-tetrazole-5-thiol (0.22 g, 1.26 mmol, 1.1 mol. equiv.) and anhydrous potassium carbonate (0.55 g, 4.00 mmol, 3.5 mol. equiv.) in acetone (15 ml) at RT. The mixture was stirred at RT for 18 hrs, then the solvent was evaporated and the residue was diluted with petrol/ether (1:1, 20 ml) and water (20 ml). The aqueous layer was re-extracted with petrol/ether (1:1, 210 ml). The combined organic extracts were dried and evaporated to give a crude oil which was purified via column chromatography eluting with petrol/ether (10:1) to give a colourless oil, 5-((15S,16S)-15-methoxy-16-methyltetratriacontyl-1-sulfanyl)-1-phenyl-1H-tetrazole (0.76 g, 93%).
(26) m-Chloroperbenzoic acid (0.52 g, 3.04 mmol, 3 mol. equiv.) in dichloromethane (5 ml) was added slowly to this colourless oil (0.72 g, 1.01 mmol) and sodium hydrogen carbonate (0.38 g, 4.56 mmol, 4.5 mol. equiv.) in dichloromethane (5 ml) at 5 C. The mixture was stirred for 18 hrs at RT, when TLC analysis indicated completion of the reaction. The solvent was evaporated and the resultant residue was diluted with ethyl acetate (5 ml) and slowly quenched with sat. aq. sodium metabisulfite (2 ml). The aqueous layer was re-extracted with ethyl acetate (210 ml) and the combined organic extracts were washed with sat. aq. sodium hydrogen carbonate (10 ml) and then water (20 ml). The organic extract was then dried and evaporated and the resultant yellow oil purified via column chromatography eluting with petrol/ether (1:1) to give a white solid, 5-((15S,16S)-15-methoxy-16-methyltetratriacontyl-1-sulfonyl)-1-phenyl-1H-tetrazole (0.67 g, 89%), which showed .sub.H (500 MHz, CDCl.sub.3): 7.71-7.70 (2H, m), 7.69-7.61 (3H, m), 3.74 (2H, t, J 7.9 Hz), 3.34 (3H, s), 2.97-2.95 (1H, m), 1.96 (2H, pent, J 7.9 Hz), 1.63-1.58 (1H, m), 1.50 (2H, pent, J 7.6 Hz), 1.45-1.22 (56H, m), 0.89 (3H, t, J 6.6 Hz), 0.85 (3H, d, J 6.7 Hz); .sub.C (125 MHz, CDCl.sub.3): 153.5, 133.1, 131.5, 130.3, 125.1, 85.5, 57.7, 56.0, 35.3, 32.4, 31.9, 30.5, 30.0, 29.9, 29.7, 29.7, 29.62, 29.60, 29.5, 29.4, 29.2, 28.9, 26.5, 22.8, 22.0, 15.1, 14.4; v.sub.max: 2947, 2852, 1321, 1164, 1097 cm.sup.1; [].sup.23.sub.D=6.28 (CHCl.sub.3, 1.024 mol); [Found M+Na.sup.+: 753.50; C.sub.43H.sub.78N.sub.4NaO.sub.3S requires: 753.57].
EXAMPLE 5
(27) Compound E was prepared as follows:
(28) ##STR00010##
(29) 2,2-dimethyl-propionic acid 8-(1-phenyl-1H-tetrazole-5-sulfonyl)-octyl ester was prepared by a method analogous to that described by Al Dulayymi, Baird, Roberts, Verschoor and Deysel in supplementary information to Tetrahedron 2007, 63, 2571-2592.
(30) Lithium hexamethyldisilazide (7.76 ml, 8.22 mmol, 1.06M) was added dropwise to a stirred solution of 2,2-dimethyl-propionic acid 8-(1-phenyl-1H-tetrazole-5-sulfonyl)-octyl ester (2.67 g, 6.32 mmol) and (1S,2R)-2-((S)-4-(tert-butyldiphenylsilyloxy)butan-2-yl)cyclopropanecarbaldehyde (1.93 g, 5.27 mmol) in dry THF (50 ml) under nitrogen at 20 C. The temperature rose to 10 C. during the addition of the base, and a yellow solution resulted. The mixture was allowed to reach RT and was stirred for 2 hrs, when TLC showed no starting material was left and then cooled to 0 C. and quenched with sat. aq. ammonium chloride (100 ml). The product was extracted with petrol/ether (1:1, 350 ml). The combined organic layers were washed with brine (100 ml), dried and evaporated to give an oil, which was purified via column chromatography eluting with petrol/ether (7:1) to give (E/Z)-9-((1R,2S)-2-((R)-1-(tert-butyldiphenylsilyloxy)propan-2-yl)cyclopropyl)non-8-enyl pivalate (2.28 g, 75%). Dipotassium azo-dicarboxylate (45.20 g, 232.70 mmol) was added to a stirred solution of this pivalate (4.47 g, 7.76 mmol) in THF (200 ml) and methanol (100 ml) at 10 C. under nitrogen, giving a yellow precipitate. A solution of glacial acetic acid (10 ml) and THF (20 ml) was added dropwise over 48 hrs, after which a white precipitate had formed. The mixture was cooled to 0 C. and poured slowly into sat. aq. sodium hydrogen carbonate (50 ml) and then extracted with petrol/ether (1:1, 3100 ml). The combined organic layers were washed with water (50 ml), dried and evaporated to give a thick oil which slowly solidified. The residue was purified by column chromatography eluting in petrol/ether (10:1) to give a colourless oil, 2,2-dimethyl-propionic acid 9-((1S,2R)-2-(S)-4-(tert-butyldiphenylsilyloxy)butan-2-yl)cyclopropyl)nonyl pivalate (4.14 g, 93%).
(31) This colourless oil (4.14 g, 7.16 mmol) was dissolved in dry THF (20 ml) in a polyethylene vial under nitrogen at RT. Pyridine (2 ml) and HF.Pyridine (10.23 ml, 7.16 mmol) were added and the mixture stirred for 17 hrs at 45 C., when TLC showed no starting material was left. The mixture was diluted with petrol/ether (1:1, 20 ml) and neutralised with by adding to sat. aq. sodium hydrogen carbonate (25 ml) until no more carbon dioxide was liberated. The compound was extracted with petrol/ether (1:1, 250 ml) and washed with brine (100 ml), dried and evaporated. The resultant oil was purified via column chromatography eluting with petrol/ether (4:1) to give a colourless oil, 9-((1S,2R)-2-((S)-4-hydroxybutan-2-yl)cyclopropyl)nonyl pivalate (1.92 g, 79%).
(32) This colourless oil (0.42 g, 1.23 mmol) was added to a stirred suspension of PCC (0.67 g, 3.09 mmol, 2.5 mol. equiv.) in dichloromethane (10 ml). The reaction mixture was stirred for 2 hrs at RT, when TLC analysis confirmed completion of the reaction, and diluted with ether (50 ml). The mixture was filtered through a bed of silica and washed with ether (210 ml), the solvent evaporated and the product was purified via column chromatography eluting with petrol/ether (5:2) to give a colourless oil, 9-((1S,2R)-2-((S)-4-oxobutan-2-yl)cyclopropyl)nonyl pivalate (0.39 g, 93%), which showed .sub.H (500 MHz, CDCl.sub.3): 9.78 (1H, s), 2.50 (1H, ddd, J 15.75, 6.3, 1.9 Hz), 2.35 (1H, ddd, J 15.75, 7.9, 2.5 Hz), 1.61 (2H, pent, J 6.6 Hz), 1.32-1.13 (26H, m), 1.00 (3H, d, J 6.65 Hz), 0.49 (1H, m), 0.34-0.21 (3H, m); .sub.C (125 MHz, CDCl.sub.3): 202.9, 178.6, 64.4, 51.4, 38.7, 34.1, 33.9, 29.6, 29.51, 29.48, 29.2, 28.6, 27.2, 25.9, 25.6, 20.0, 18.8, 11.4; v.sub.max: 2924, 2878, 1727 cm.sup.1; [].sup.22.sub.D=+20.47 (CHCl.sub.3, 1.076 mol); [Found M+Na.sup.+: 361.24; C.sub.21H.sub.38NaO.sub.3 requires: 361.27].
EXAMPLE 6
(33) Compound F was prepared as follows:
(34) ##STR00011##
(35) Lithium hexamethyldisilazide (0.923 ml, 0.978 mmol, 1.06M) was added dropwise to a stirred solution of compound D (670 mg, 0.903 mmol) and compound E (255 mg, 0.752 mmol) in dry THF (10 ml) under nitrogen at 20 C. The reaction mixture rose to 10 C. during the addition of the base, and a yellow solution resulted. The mixture was allowed to reach RT and was stirred for 1 hr, when TLC showed no starting material was left. The reaction mixture was cooled to 0 C. and quenched with sat. aq. ammonium chloride (10 ml). The product was extracted with petrol/ether (1:1, 310 ml). The combined organic layers were washed with brine (20 ml), dried and evaporated to give an oil, which was purified via column chromatography eluting with petrol/ether (20:1) to give 9-((1S,2R)-2-[(E/Z)-(2S,19S,20S)-19-methoxy-20-methyloctatriacont-4-en-2-yl]cyclopropyl)nonyl pivalate (410 mg, 54%). Dipotassium azodicarboxylate (2.49 g, 12.83 mmol, 30 mol. equiv.) was added to a stirred solution of this compound (410 mg, 0.487 mmol) in THF (20 ml) and methanol (10 ml) at 10 C. under nitrogen, giving a yellow precipitate. A solution of glacial acetic acid (1 ml) and THF (2 ml) was added dropwise over 48 hrs, after which a white precipitate had formed. The mixture was cooled to 0 C. and poured slowly into sat. aq. sodium hydrogen carbonate (5 ml) and then extracted with petrol/ether (1:1, 325 ml). The combined organic layers were washed with water (10 ml), dried and evaporated to give a thick oil which slowly solidified. The residue was purified via column chromatography eluting in petrol/ether (10:1) to give a colourless oil, 9-((1S,2R)-2-((2S,19S,20S)-19-methoxy-20-methyloctatriacontan-2-yl)cyclopropyl)nonyl pivalate (400 mg, 97%).
(36) A solution of this colourless oil (400 mg, 0.4737 mmol) in THF (5 ml) was added slowly Lithium aluminium hydride (36.0 mg, 0.9479 mmol, 2 mol. equiv.) in THF (5 ml, HPLC grade) at 20 C. under nitrogen. The reaction was allowed to reach RT, then heated under reflux for 1 hr. When TLC showed no starting material was left the reaction mixture was cooled to 20 C. and was quenched with sat. aq. sodium sulfate until a white precipitate formed. The resultant mixture was stirred for 30 mins and then filtered through a bed of silica and the solvent evaporated. The product was purified via column chromatography eluting with petrol/ether (1:1) to give a colourless oil, 9-((1S,2R)-2-((2S,19S,20S)-19-methoxy-20-methyloctatriacontan-2-yl)cyclopropyl)nonan-1-ol (260 mg, 72%).
(37) This colourless oil (0.26 g, 0.343 mmol) was added to a stirred suspension of PCC (0.22 g, 1.03 mmol, 3 mol. equiv.) in dichloromethane (10 ml). The reaction mixture was stirred for 1 hr at RT, when TLC analysis confirmed completion of the reaction, then diluted with ether (10 ml). The mixture was filtered through a bed of silica and washed with ether (25 ml), the solvent evaporated and the product was purified via column chromatography eluting with petrol/ether (10:1) to give a colourless oil, 9-((1R,2R)-2-((2S,19S,20S)-19-methoxy-20-methyloctatriacontan-2-yl)cyclopropyl)-nonanal (0.24 g, 96%), which showed .sub.H (500 MHz, CDCl.sub.3): 9.77 (1H, br. t, J 1.85 Hz), 3.38 (3H, s), 2.97-2.95 (1H, m), 2.43 (2H, dt, J 1.85, 7.55 Hz), 1.99-1.97 (1H, m), 1.65-1.61 (1H, m), 1.56 (2H, m), 1.40-1.09 (78H, br. m including br. s at 1.27), 0.89 (6H, dt, J 2.85, 6.6 Hz), 0.85 (3H, d, J 6.6 Hz), 0.48-0.43 (1H, m), 0.22-0.18 (1H, m), 0.17-0.14 (1H, m), 0.13-0.09 (1H, m); .sub.C (125 MHz, CDCl.sub.3): 205.1, 85.5, 65.6, 57.7, 43.9, 38.1, 37.4, 35.4, 34.4, 32.8, 32.4, 31.9, 30.5, 30.0, 29.9, 29.7, 29.6, 29.5, 29.4, 29.3, 27.6, 27.3, 26.2, 25.8, 22.7, 19.7, 18.6, 16.5, 14.3, 10.5; v.sub.max: 2984, 2875, 1724 cm.sup.1; [].sup.19.sub.D=3.45 (CHCl.sub.3, 1.247 mol); [Found M+Na.sup.+: 781.72; C.sub.52H.sub.102NaO.sub.2 requires: 781.78].
EXAMPLE 7
(38) Compound G was prepared as follows:
(39) ##STR00012##
(40) Lithium bis(trimethylsilyl)amide (0.96 ml, 1.02 mmol, 1.06 M) was added to a stirred solution of compound F (0.494 g, 0.651 mmol) and compound C (0.773 g, 0.781 mmol) in dry THF (15 ml) at 0-5 C. The solution turned bright yellow/orange and was left to reach room temperature and stirred for 1 hour under N.sub.2 (g). When TLC showed no starting material remaining the reaction was quenched by addition of a saturated aqueous solution of NH.sub.4Cl (10 ml) at 20 C. The mixture was extracted with petrol/ethyl acetate (1:1, 315 ml) and the combined organic layers were dried, filtered and evaporated. Column chromatography (petrol/ethyl acetate, 20:1) gave (2R)-methyl 2-((1R)-1-(tert-butyldimethylsilyloxy)-19-((1R)-2-((2S,19S,20S)-19-methoxy-20-methyloctatriacontan-2-yl)cyclopropyl)nonadec-10-enyl)-26-(pivaloyloxy)hexacosanoate (0.849 g, 0.558 mmol, 86%) as a colourless oil; [].sub.D.sup.2 8.54 (c 1.19, CHCl.sub.3). This showed v.sub.max(film)/cm.sup.1: 2923 (CH), 2853 (CH), 1732 (CO) and 1463; .sub.H: 0.02 (3H, s, SiCH.sub.3), 0.05 (3H, s, SiCH.sub.3), 0.09-0.22 (3H, m, 3cyclopropane CH), 0.41-0.48 (1H, m, cyclopropane CH), 0.68 (1H, m, CH), 0.83-0.90 (23H, m, including a singlet at 0.87, SiC(CH.sub.3).sub.3), 1.26 (151H, m, including a singlet at 1.20), 1.62 (5H, m), 1.96 (2H, m, CH.sub.2), 2.02 (1H, m, CH), 2.53 (1H, ddd, J 3.6, 7.2 and 11.0, CHCH(CH.sub.2)CO), 2.96 (1H, m, CH.sub.2CH(OCH.sub.3)CH.sub.2), 3.35 (3H, s, OCH.sub.3), 3.66 (3H, s, OCH.sub.3), 3.91 (1H, m, CH.sub.2CH(O)CH), 4.05 (2H, t, J 6.6, CH.sub.2CH.sub.2O) and 5.37 (2H, m, CH.sub.2CHCHCH.sub.2); .sub.C: 4.9, 4.4, 10.5, 14.1, 14.9, 18.0, 18.6, 19.7, 22.6, 22.7, 23.7, 25.8, 25.9, 26.1, 26.2, 27.2, 27.3, 27.5, 27.6, 27.8, 28.6, 29.1, 29.2, 29.2, 29.3, 29.4, 29.5, 29.5, 29.6, 29.6, 29.7, 29.7, 29.8, 30.0, 30.0, 30.1, 30.5, 31.9, 32.4, 32.6, 33.7, 34.5, 35.3, 37.4, 38.1, 51.2, 51.6, 57.7, 64.5, 73.2, 85.5, 129.8, 129.9, 130.3, 130.4, 143.2, 175.2 and 178.7.
EXAMPLE 8
(41) Compound H, a sulfur-containing mycolic acid derived antigen of the present invention was prepared as follows:
(42) ##STR00013##
(43) Dipotassium azodicarboxylate was added in excess to a stirred solution of compound G (0.840 g, 0.552 mmol) in dry THF (10 ml) and methanol (5 ml) at 0 C. under N.sub.2 (g). Acetic acid (2 ml) in dry THF (4 ml) was added dropwise in small portions throughout the day at 0 C. The following morning further dipotassiumazodicarboxylate followed by more of the acetic acid in THF was added. Again, after stirring overnight, more dipotassiumazodicarboxylate was added, followed by more of the acetic acid in THF. After stirring for a 3.sup.rd night the reaction was quenched by adding the reaction mixture in small portions to a saturated solution of aqueous NaHCO.sub.3 (15 ml). The mixture was extracted with petrol/ethyl acetate (5:2, 325 ml) and the combined organic layers were dried, filtered and evaporated. Column chromatography (petrol, ethyl acetate, 20:1) gave (2R)-methyl 2-((1R)-1-(tert-butyldimethylsilyloxy)-19-((1R)-2-((2S,19S, 20S)-19-methoxy-20-methyloctatriacontan-2-yl)cyclopropyl)nonadecyl)-26-(pivaloyloxy)hexacosanoate (0.727 g, 0.477 mmol, 86%) as a colourless oil.
(44) This colourless oil (0.71 g, 0.47 mmol) was added to a stirred solution of potassium hydroxide (0.39 g, 6.99 mmol) in a mixture of THF (10 ml), methanol (10 ml) and water (1 ml). The mixture was heated under reflux at 70 C. and monitored by TLC. After 3 hours, when TLC showed no starting material remaining the reaction was quenched with water (10 ml) and extracted with ethyl acetate (315 ml). The combined organic extracts were dried, filtered and evaporated. Column chromatography (petrol/ethyl acetate, 10:1) gave (2R)-methyl 2-((1R)-1-(tert-butyldimethylsilyloxy)-19-((1R)-2-((2S,19S,20S)-19-methoxy-20-methyloctatriacontan-2-yl)cyclopropyl)nonadecyl)-26-hydroxyhexacosanoate (0.583 g, 0.41 mmol, 86%) as a white solid.
(45) A solution of this white solid (0.474 g, 0.327 mmol) and triethylamine (2 ml) in dry dichloromethane (25 ml) was cooled to 20 C. under N.sub.2 (g) and stirred for 30 minutes, followed by the addition of toluene sulfonyl chloride (0.081 g, 0.425 mmol) in one portion. The solution was kept in the refrigerator overnight. When TLC showed no starting material remaining the solvent was evaporated. Column chromatography (petrol/ethyl acetate, 10:1) gave (2R)-methyl 2-((1R)-1-(tert-butyldimethylsilyl)oxy)-19-((1R)-2-((2S,19S,20S)-19-methoxy-20-methyloctatriacontan-2-yl)cyclopropyl)nonadecyl)-26-(tosyloxy) hexacosanoate (0.333 g, 0.211 mmol, 65%) as a colourless oil.
(46) A solution of this colourless oil (0.399 g, 0.251 mmol) and potassium thioacetate (0.115 g, 1.003 mmol) in acetone (15 ml) was stirred at room temperature overnight. When TLC showed that no starting material remained the solvent was evaporated. Column chromatography (petrol/ethyl acetate, 20:1) gave (2R)-methyl 26-(acetylthio)-2-((1R)-1-(tert-butyldimethylsilyloxy)-19-((1R)-2-((2S,19S,20S)-19-methoxy-20-methyloctatriacontan-2-yl)cyclopropyl)nonadecyl)hexanoate (0.227 g, 0.152 mmol, 61%) as a colourless oil.
(47) The colourless oil (50 mg, 0.0333 mmol) was dissolved in dry THF (4 ml) in a dry polyethylene vial under N.sub.2 (g) at 0 C. Pyridine (98.2 mg, 7.77 mmol, 0.1 ml) and HF.Pyridine (88 mg, 0.8 ml) were added and the mixture stirred at 45 C. overnight. When TLC showed no starting material remaining, the mixture was added slowly to a saturated aqueous solution of NaHCO.sub.3 (10 ml). The solution was extracted with petrol/ethyl acetate (1:1, 315 ml) and the combined organic extracts were dried, filtered and evaporated. Column chromatography (petrol/ethyl acetate, 10:1) gave (2R)-methyl 26-(acetylthio)-2-((1R)-1-hydroxy-19-((1R)-2-((2S,19S,20S)-19-methoxy-20-methyloctatriacontan-2-yl)cyclopropyl)nonadecyl)hexacosanoate (41.1 mg, 0.0299 mmol, 90%) as a white solid.
(48) This white solid (14 mg, 0.010 mmol) was suspended in a 5% aqueous solution of TBAH (2 ml) and the solution was heated to 100 C. overnight. After this time TLC showed that the reaction was complete. The solution was cooled to room temperature and acidified to pH 1 with 1 M HCl and then extracted with diethyl ether (315 ml). The combined organic layers were dried, filtered and the solvent evaporated. Column chromatography (chloroform/methanol, 10:1) gave (2R)-2-((1R)-1-hydroxy-19-((1R)-2-((2S,19S,20S)-19-methoxy-20-methyloctatriacontan-2-yl)cyclopropyl)nonadecyl)-26-mercaptohexacosanoic acid (7.7 mg, 0.0058 mmol, 58%) as a white solid; [].sub.D.sup.2z 2.78 (c 0.77, CHCl.sub.3). This showed .sub.H: 0.08-0.20 (3H, m, 3cyclopropane CH), 0.44 (1H, m, cyclopropane CH), 0.66 (1H, m, CH), 0.86 (3H, d, J 6.9, CH.sub.3CH), 0.89 (3H, t, J 7.0, CH.sub.3CH.sub.2), 0.90 (3H, d, J 6.6, CH.sub.3CH), 1.26 (144H, m), 1.67 (8H, m), 2.47 (1H, dt, J 5.4 and 9.1 CHCH(CH.sub.2)CO), 2.69 (2H, t, J 7.4, CH2CH2S), 2.97 (1H, m, CH.sub.2CH(OCH.sub.3)CH.sub.2), 3.35 (3H, s, OCH.sub.3) and 3.91 (1H, m, CH2CH(OH)CH.sub.2).
EXAMPLE 9
(49) The following sulfur-containing linker compound I was prepared using the steps described below:
(50) ##STR00014##
(51) Magnesium turnings (13.50 g, 0.56 mol) were stirred in dry THF (100 ml) under N.sub.2 (g). 2-(6-Bromohexyloxy)tetrahydro-2H-pyran (60.95 g, 0.23 mol) in dry THF (100 ml) was added slowly whilst heating gently with a heat gun. After all the solution was added the reaction mixture was heated under reflux for 30 min.
(52) A stirred solution of S citronellyl bromide (10.00 g, 45.6 mmol) in dry THF (100 ml) was cooled to 78 C. under N.sub.2 (g). The THF solution of (6-(tetrahydro-2H-pyran-2-yloxy)hexyl)magnesium bromide was transferred into the same vessel and the resultant mixture was cooled to 78 C. LiCuCl.sub.4 (0.1 M in THF, 8 ml, 0.8 mmol) was added slowly in one portion and the temperature was seen to rise. The solution was left in the cooling bath for 2 days during which it slowly warmed to ambient temperature. The reaction was quenched with a saturated aqueous solution of NH.sub.4Cl (100 ml) and the resultant bright blue solution was extracted with ether (3100 ml). The combined organic phases were washed with brine (100 ml), dried, filtered and evaporated. Flash distillation gave 2-((R)-9,13-dimethyltetradec-12-enyloxy)tetrahydro-2H-pyran (10.28 g, 31.7 mmol, 70%) as a colourless oil.
(53) Following a known procedure, a stirred solution of this colourless oil (8.00 g, 24.7 mmol) in dichloromethane (200 ml) was cooled to 78 C. prior to treatment with O.sub.3 until a blue colour (liquid O.sub.3) was seen to persist in the cooled solution. N.sub.2 (g) was subsequently bubbled through the solution to remove excess O.sub.3 and avoid danger on warming. Triphenylphosphine (6.47 g, 24.7 mmol) was added to the cold solution, which was warmed to ambient temperature and stirred overnight. The solvent was removed by rotary evaporation. Column chromatography (petrol/ether, 2:1) gave (4R)-4-methyl-12-(tetrahydro-2H-pyran-2-yloxy)dodecanal (5.81 g, 19.5 mmol, 80%) as a colourless oil.
(54) (5-Carboxypentyl)triphenylphosphonium bromide (18.3 g, 40.2 mmol) was dissolved in 3:1 dry toluene/dry DMSO (200 ml). The solution was cooled to 0 C. and lithium bis(trimethylsilyl)amide (1.06 M in THF, 79.4 ml, 84.2 mmol) was slowly added maintaining a temperature of less than 0.3 C. The resultant bright red orange solution was warmed to ambient temperature over 3 hours and stirred at ambient temperature for a further 1 hour. The solution was cooled to 15 C. and (4R)-4-methyl-12-(tetrahydro-2H-pyran-2-yloxy)dodecanal (5.7 g, 19.1 mmol) was added as a solution in dry toluene (15 ml); the temperature was seen to rise on this addition. The solution was allowed to slowly return to ambient temperature and stirred overnight. A saturated aqueous solution of NH.sub.4Cl (200 ml) was added and the mixture extracted with ethyl acetate (4100 ml). The combined organic phases were washed with brine (100 ml), dried, filtered and evaporated. Column chromatography (petrol/ethyl acetate, 5:2) gave (10R)-10-methyl-18-(tetrahydro-2H-pyran-2-yloxy)octadec-6-enoic acid (3.85 g, 9.72 mmol, 51%) as a colourless oil.
(55) Palladium on carbon (10%, 2.0 g) was slowly added under a stream of N.sub.2 (g) to a stirred solution of this colourless oil (3.84 g, 9.69 mmol) in methanol (50 ml). The flask was connected to a hydrogenation apparatus which was purged of any air by repeated application of vacuum followed by refilling the system with H.sub.2 (g). The reaction was monitored by observing the amount of H.sub.2 (g) absorbed by a burette that is part of the apparatus. When the burette reading was steady the reaction was complete. The reaction mixture was then filtered through a pad of Celite, which was washed with copious methanol. The solvent was removed by rotary evaporation. The residue was suspended in dichloromethane (50 ml), washed with brine (20 ml), dried, filtered and evaporated. Column chromatography (petrol/ether, 1:1) gave (10S)-10-methyl-18-(tetrahydro-2H-pyran-2-yloxy)octadecanoic acid (3.14 g, 7.88 mmol, 81%) as a colourless oil.
(56) p-Toluene sulfonic acid monohydrate (372 mg, 1.95 mmol) was added to a stirred solution of this colourless oil (3.05 g, 7.82 mmol) in THF (25 ml), methanol (100 ml) and water (5 ml) at room temperature. The mixture was heated under reflux for 30 min. When TLC showed no starting material remaining a saturated aqueous solution of NaHCO.sub.3 (100 ml) and petrol/ethyl acetate (1:1, 100 ml) were added. The layers were separated and the aqueous layer was extracted with petrol/ethyl acetate (1:1, 3100 ml). The combined organic phases were washed with brine (100 ml), dried, filtered and evaporated. Column chromatography (petrol/ether, 2:1) gave (S)-methyl 18-hydroxy-10-methyloctadecanoate (1.87 g, 5.70 mmol, 74%) as a colourless oil.
(57) N-Bromosuccinimide (1.22 g, 6.86 mmol) was added in portions to a stirred solution of this colourless oil (1.50 g, 4.57 mmol) and triphenylphosphine (1.80 g, 6.86 mmol) in dichloromethane (50 ml) at 0 C. The reaction was stirred at room temperature for 1 hour or until there was no starting material remaining. The reaction was quenched with a saturated aqueous solution of Na.sub.2S.sub.2O.sub.5 (50 ml). The reaction mixture was separated and the aqueous layer extracted with dichloromethane (250 ml). The combined organic layers were washed with water, dried, filtered and evaporated to give a residue. This was treated with a mixture of petrol/ethyl acetate (1:1) (50 ml) and heated under reflux for 30 min. The solution was filtered, washed with petrol/ethyl acetate (1:1) and the solvent evaporated. Column chromatography (petrol/ethyl acetate, 5:1) gave (S)-methyl 18-bromo-10-methyloctadecanoate (1.56 g, 3.98 mmol, 87%) as a colourless oil.
(58) A solution of this colourless oil (500 mg, 1.28 mmol) and thiourea (148 mg, 1.94 mmol) in ethanol (10 ml) was heated under reflux for 2.5 hours. The solvent was evaporated and 5 M NaOH (4 ml) was added slowly with stirring and the solution was heated under reflux for another 2 hours. The aqueous solution was cooled in an ice bath and acidified with dilute HCl. The solution was then extracted with ether (315 ml), dried, filtered and evaporated. Column chromatography (petrol/ether, 1:1) gave (S)-18-mercapto-10-methyloctadecanoic acid (217 mg, 0.66 mmol, 51%) as a colourless oil; {Found (M+Na).sup.+: 353.2471, C.sub.19H.sub.38O.sub.2SNa requires: 353.2485}. This showed v.sub.max(film)/cm.sup.1: 3027 (broad OH), 2924 (CH saturated), 2853 (CH saturated), 2674 (SH), 1708 (CO) and 1463; .sub.H: 0.85 (3H, d, J 6.3, CH.sub.3CH), 1.08 (2H, m, CH.sub.2), 1.29 (23H, m, 11CH.sub.2 and CH), 1.63 (4H, m, 2CH.sub.2), 2.36 (2H, t, J 7.4, CH.sub.2CH.sub.2CO) and 2.54 (2H, q, J 7.4, CH.sub.2CH.sub.2SH); .sub.C: 19.7, 24.6, 24.7, 27.0, 28.4, 29.0, 29.1, 29.3, 29.5, 29.6, 29.9, 32.7, 34.0, 34.1, 37.1 and 180.3.
EXAMPLE 10
(59) An sulfur-containing linker compound J was prepared as follows:
(60) ##STR00015##
(61) Stearic acid (8.53 g, 30.0 mmol) was added to a solution of N-hydroxy succinimide (3.45 g, 30.0 mmol) in dry ethyl acetate (130 ml). A solution of DCC (6.18 g, 30 mmol) in dry ethyl acetate (10 ml) was added and the reaction mixture was stirred overnight at room temperature. DCU was removed by filtration and the filtrate was concentrated under reduced pressure. Recrystallisation (ethanol) gave 2,5-dioxopyrrolidin-1-yl stearate (8.51 g, 22.3 mmol, 74%) as a white solid.
(62) This white solid (5.29 g, 13.9 mmol) and 2-mercaptoethylamine hydrochloride (2.36 g, 20.8 mmol) were dissolved in dry dichloromethane (140 ml). Triethylamine (4.20 g, 5.8 ml, 41.6 mmol) that was dissolved in dry dichloromethane (5 ml) was added to the solution resulting in precipitation of a white solid. The reaction was allowed to stir overnight at room temperature. The following morning ethyl acetate (20 ml) and ethanol (10 ml) were added to form a clear solution. The organic solution was washed with dilute acid (315 ml) and water (215 ml) and the combined organic layers were dried, filtered and evaporated. Recrystallisation (chloroform) gave N-(2-mercaptoethyl)stearamide (2.66 g, 7.76 mmol, 56%) as a white solid; m.p: 63-65 C.; {Found (M+Na).sup.+: 366.2818, C.sub.20H.sub.41NOSNa requires: 366.2806}. This showed v.sub.max(nujol)/cm.sup.1: 3300 (NH), 2920 (CH saturated), 2853 (CH saturated), 1640 (CO), 1550 and 1464; .sub.H: 0.89 (3H, t, J 6.9, CH.sub.3CH.sub.2), 1.26 (28H, m), 1.64 (2H, m), 2.20 (2H, t, J 7.6, CH.sub.2CH.sub.2CO), 2.68 (2H, dt, J 6.4 and 8.4, NHCH.sub.2CH.sub.2SH), 3.45 (2H, q, J 6.2, NHCH.sub.2CH.sub.2SH) and 5.8 (1H, br s, NH); .sub.C: 14.1, 22.7, 24.8, 25.7, 29.3, 29.4, 29.5, 29.6, 29.7, 29.7, 31.9, 36.8, 42.2 and 173.3.
EXAMPLE 11
(63) Compound K was prepared as follows:
(64) ##STR00016##
(65) Lithium bis(trimethylsilyl)amide (4.14 ml, 4.39 mmol, 1.06 M) was added dropwise to a stirred solution of (R)-2-[(R)-1-(tert-butyldimethylsilanyloxy)-3-oxo-propyl]-hexacosanoic acid methyl ester (130) (1.30 g, 2.25 mmol) and 7-(1-phenyl-1H-tetrazol-5-ylsulfonyl)heptyl pivalate (160) (1.20 g, 2.93 mmol, 1.2 mol. equiv.) in dry THF (50 ml) at 15 C. The mixture was then stirred for 18 hrs at RT, when TLC analysis indicated completion of the reaction. Sat. aq. ammonium chloride (20 ml) and petrol/ether (1:1, 50 ml) were added. The aqueous layer was re-extracted with petrol/ether (1:1, 350 ml) and the combined organic extracts washed with brine (50 ml), dried and evaporated to give a yellow oil. The crude product was purified by column chromatography eluting with petrol/ether (20:1) to give a colourless oil, methyl 2-((R-(E,Z)-1-(tert-butyldimethylsilyloxy)-10-(pivaloyloxy)dec-3-nyl)hexacosanoate (1.23 g, 72%). Palladium on charcoal (10%, 0.5 g) was added to a stirred solution of methyl 2-((R-(E,Z)-1-(tert-butyldimethylsilyloxy)-10-(pivaloyloxy)dec-3-enyl)hexacosanoate (1.23 g, 1.54 mmol) in ethanol (20 ml) and THF (20 ml). The mixture was stirred while being hydrogenated at atmospheric pressure, and when hydrogen absorption was complete was filtered through a pad of celite and washed with ethyl acetate (100 ml). The filtrate was evaporated to give a colourless oil, methyl 2-((1R,2R)-1-(tert-butyldimethylsilyloxy)-10-(pivaloyloxy)decyl)hexacosanoate (1.12, 93%).
(66) This colourless oil (1.10 g, 1.14 mmol) in THF (10 ml) was added to a stirred solution of potassium hydroxide (1.19 g, 21.18 mmol, 15 mol. equiv.) in THF (20 ml), methanol (20 ml) and water (2 ml). The mixture was heated to 70 C. and reflux was maintained for 2 hrs. When TLC analysis indicated completion of the reaction the mixture was quenched with water (10 ml) and the aqueous layer extracted with ethyl acetate (350 ml). The combined organic extracts were dried and evaporated and the crude product purified via column chromatography eluting with petrol/ether (5:2) to give a colourless oil, methyl 2-((1R,2R)-1-(tert-butyldimethylsilyloxy)-10-hydroxydecyl)hexacosanoate (0.66 g, 67%).
(67) Triphenyl phosphine (0.29 g, 1.12 mmol, 1.2 mol. equiv.) was added to a stirred solution of the colourless oil (0.65 g, 0.935 mmol) in dry dichloromethane (20 ml) and then sodium hydrogen carbonate (0.10 g) was added. The mixture was cooled to 0 C. and N-bromosuccinimide (0.22 g, 1.22 mmol, 1.3 mol. equiv.) was added portion wise over 10 mins at 0-4 C. The reaction was stirred for 1 hr at 0-3 C., when TLC analysis showed completion of the reaction, sat. aq. sodium bisulfate (10 ml). The aqueous layer was re-extracted with dichloromethane (220 ml) and the combined organic layers were washed with water (20 ml), dried and the solvent evaporated. The resultant crude product was taken up in petrol/ether (1:1, 40 ml) and the mixture stirred for 30 mins, then the triphenylphosphonium oxide was filtered was washed with petrol/ether (1:1, 20 ml). The solvent was evaporated and the crude product purified via column chromatography eluting with petrol/ether (20:1) to give a white solid, methyl 2-((1R,2R)-10-bromo-1-(tert-butyldimethylsilyloxy)decyl)hexacosanoate (0.54 g, 76%).
(68) The white solid (0.54 g, 0.71 mmol) was dissolved in THF (1.5 ml) and acetone (1.5 ml) and added to a stirred solution of 1-phenyl-1H-tetrazole-5-thiol (0.15 g, 0.857 mmol, 1.2 mol. equiv.) and anhydrous potassium carbonate (0.29 g, 2.14 mmol, 3 mol. equiv.) in acetone (10 ml, HPLC grade) at RT. The mixture was stirred at RT for 18 hrs, then the solvent was evaporated and the residue was diluted with petrol/ether (1:1, 20 ml) and water (20 ml). The aqueous layer was re-extracted with petrol/ether (1:1, 310 ml). The combined organic extracts were dried and evaporated to give a crude oil which was purified via column chromatography eluting with petrol/ether (10:1) to give a colourless oil, methyl 2-((1R,2R)-1-(tert-butyldimethylsilyloxy)-10-(1-phenyl-1H-tetrazol-5-ylthio)decyl)hexacosanoate (0.45 g, 73%).
(69) m-Chloroperbenzoic acid (0.39 g, 1.58 mmol, 3 mol. equiv) in dichloromethane (2 ml) was added slowly to a stirred solution of the colourless oil (0.45 g, 0.53 mmol) and sodium hydrogen carbonate (0.20 g, 2.37 mmol, 4.5 mol. equiv.) in dichloromethane (5 ml) at 5 C. The mixture was stirred for 18 hrs at RT, when TLC analysis indicated completion of the reaction, and the solvent was evaporated. The resultant residue was diluted with ethyl acetate (5 ml) and slowly quenched with sat. aq. sodium metabisulfite (2 ml). The aqueous layer was re-extracted with ethyl acetate (210 ml) and the combined organic extracts were washed with sat. aq. sodium hydrogen carbonate (10 ml) and then water (20 ml). The organic extract was then dried and evaporated and the resultant yellow oil purified via column chromatography eluting with petrol/ether (1:1) to give a white solid, methyl 2-((1R,2R)-1-(tert-butyldimethylsilyloxy)-10-(1-phenyl-1H-tetrazol-5-ylsulfonyl)decyl)hexacosanoate (0.35 g, 75%), which showed .sub.H (500 MHz, CDCl.sub.3): 7.71-7.70 (2H, m), 7.64-7.60 (3H, m), 3.92-3.89 (1H, m), 3.74 (2H, t, J 8.2 Hz), 3.66 (3H, s), 2.53 (1H, ddd, J 3.8, 6.95, 11.05 Hz), 1.96 (2H, dist. pent, J 7.9 Hz), 1.51-1.20 (60H, br. m including br. s at 1.26), 0.89 (3H, t J 6.6 Hz), 0.87 (9H, s), 0.05 (3H, s), 0.02 (3H, s); .sub.C (125 MHz, CDCl.sub.3): 175.1, 154.5, 133.8, 130.0, 129.8, 123.9, 73.2, 61.8, 51.6, 51.2, 33.7, 33.4, 31.9, 29.8, 29.7, 29.6, 29.6, 29.4, 29.4, 29.1, 29.0, 28.7, 27.9, 27.7, 27.1, 25.8, 23.8, 22.7, 18.0, 15.2, 14.1, 4.4, 4.9; v.sub.max: 2919, 2848, 1721 1464 cm.sup.1; [].sup.21.sub.D=4.85 (CHCl.sub.3, 1.201 mol) [Found M+Na.sup.+: 911.52; C.sub.50H.sub.92NaO.sub.5SiN.sub.4S requires: 911.65].
EXAMPLE 12
(70) Compound L was prepared as follows:
(71) ##STR00017##
(72) Lithium hexamethyldisilazide (0.2920 ml, 0.309 mmol, 1.06M) was added dropwise to a stirred solution of compound K (183 mg, 0.206 mmol) and compound F (172 mg, 0.227 mmol) in dry THF (10 ml) under nitrogen at 20 C. The temperature rose to 10 C. during the addition of the base, and a yellow solution resulted. The mixture was allowed to reach RT and was stirred for 1 hr, when TLC showed no starting material was left, then cooled to 0 C. and quenched with sat. aq. ammonium chloride (10 ml). The product was extracted with petrol/ether (1:1, 310 ml). The combined organic layers were washed with brine (20 ml), dried and evaporated to give an oil, which was purified by column chromatography eluting with petrol/ether (20:1) to give methyl 2-((R)-(E/Z)-1-(tert-butyldimethylsilyloxy)-19-((1S,2R)-2-((2S,18S,19S)-18-methoxy-19-methyl heptatriacontan-2-yl)cyclopropyl)nonadec-10-enyl)hexacosanoate (80.6 mg, 28%). Dipotassium azodicarboxylate (0.33 g, 1.708 mmol, 30 mol. equiv.) was added to a stirred solution of the above compound (80.6 mg, 0.057 mmol) in THF (5 ml) and methanol (5 ml) at 10 C. under nitrogen, giving a yellow precipitate. A solution of glacial acetic acid (1 ml) and THF (2 ml) was added dropwise over 48 hrs, after which a white precipitate had formed. The mixture was cooled to 0 C. and poured slowly into sat. aq. sodium hydrogen carbonate (5 ml) and then extracted with petrol/ether (1:1, 310 ml). The combined organic layers were washed with water (10 ml), dried and evaporated to give a thick oil which slowly solidified. The residue was purified via column chromatography eluting in petrol/ether (10:1) to give a white solid, methyl 2-((R)-1-(tert-butyldimethylsilyloxy)-19-((1S,2R)-2-(2S,18S,19S)-18-methoxy-19-methylheptatriacontan-2-yl)cyclopropyl)nonadecyl) hexacosanoate (75.7 mg, 94%), which showed .sub.H (500 MHz, CDCl.sub.3): 3.92-3.90 (1H, m), 3.66 (3H, s), 3.35 (3H, s), 2.97-2.95 (1H, m), 2.54 (1H, ddd, J 3.75, 7.25, 11 Hz), 1.58-1.18 (150H, br. m including br. s at 1.27), 0.91-0.85 (21H, m), 0.48-0.44 (1H, m), 0.22-0.18 (1H, m), 0.17-0.14 (1H, m), 0.13-0.09 (1H, m), 0.05 (3H, s), 0.03 (3H, s); .sub.C (125 MHz, CDCl.sub.3): 175.1, 125.5, 85.5, 73.2, 65.9, 57.7, 51.6, 38.1, 37.4, 37.1, 35.8, 35.4, 34.5, 33.7, 32.8, 32.4, 31.9, 31.1, 30.5, 30.3, 30.1, 30.0, 29.9, 29.8, 29.7, 29.64, 29.60, 29.52, 29.49, 29.4, 27.8, 27.6, 27.5, 27.3, 26.2, 26.1, 25.8, 23.7, 22.7, 19.7, 18.6, 18.0, 14.9, 14.1, 10.5, 4.4, 4.9; v.sub.max: 2923, 2852, 1741, 1465 cm.sup.1; [].sup.24.sub.D=1.45 (CHCl.sub.3, 0.856 mol); [Found M+Na.sup.+: 1446.21; C.sub.95H.sub.190NaO.sub.4Si requires: 1446.43].
EXAMPLE 13
(73) Compound M, a sulfur-containing mycolic acid derivative was prepared as follows:
(74) ##STR00018##
(75) A dry polyethylene vial equipped with a rubber septum was charged with compound L (70 mg, 0.0494 mmol) in dry THF (4 ml) under nitrogen at 0 C. Pyridine (0.2 ml) and hydrogen fluoride-pyridine complex (0.2 ml, 0.140 mmol, 208 mol. equiv.) were added and the mixture stirred for 32 hrs at 43 C. When TLC analysis indicated completion of the reaction the mixture was neutralised by slowly pouring the mixture into sat. aq. sodium hydrogen carbonate (10 ml) until no more carbon dioxide was liberated. The product was extract with petrol/ether (1:1, 350 ml), dried and evaporated to give a white solid. This was purified via column chromatography eluting with petrol/ether (4:1) to give a white solid, methyl 2-((R)-1-hydroxy-19-((1S,2R)-2-((2S,18S,19S)-18-methoxy-19-methylheptatriacontan-2-yl)cyclopropyl)nonadecyl) hexacosanoate (35 mg, 54%).
(76) Lithium hydroxide monohydrate (20 mg, 0.835 mmol, 30 mol. equiv.) was added to a stirred solution of the white solid (35 mg, 0.0269 mmol) in THF (2.5 ml), methanol (0.3 ml) and water (0.3 ml) at RT. The mixture was stirred at 43 C. for 18 hrs, when TLC analysis indicated completion of the reaction, then cooled to RT and acidified with hydrochloric acid (5%, 1 ml) and the aqueous layer extracted with warm petrol/ether (1:1, 310 ml). The combined organic extracts were dried and evaporated, and then purified by column chromatography eluting with petrol/ethyl acetate (5:1) to give a white solid, (R)-2-((R)-1-hydroxy-19-((1S,2R)-2-((2S,18S,19S)-18-methoxy-19-methylheptatriacontan-2-yl)cyclopropyl)nonadecyl)hexacosanoic acid (24.0 mg, 69%).
(77) This white solid (25 mg, 0.0193 mmol)) was added to a solution of N hydroxy succinimide (2.2 mg, 0.0193 mmol) in dry ethyl acetate (2 ml). A solution of DCC (4.0 mg, 0.0193 mmol) in dry ethyl acetate (1 ml) was added and the reaction mixture was stirred overnight at room temperature. DCU was removed by filtration and the filtrate was concentrated under reduced pressure. Column chromatography (petrol/ethyl acetate, 5:1) gave (2R)-2,5-dioxopyrrolidin-1-yl 2-((1R)-1-hydroxy-19-((1R)-2-((2S,19S,20S)-19-methoxy-20-methyloctatriacontan-2-yl)cyclopropyl)nonadecyl)hexacosanoate (10.3 mg, 0.0074 mmol, 38%) as a white solid. The white solid (8.3 mg, 0.00597 mmol) and 2-mercaptoethylamine hydrochloride (1.0 mg, 0.00895 mmol) were suspended in dry dichloromethane (2 ml). Triethylamine (1.8 mg, 0.0179 mmol) that was dissolved in dry dichloromethane (1 ml) was added to the solution. The reaction was allowed to heat under reflux for 3 nights and more 2-mercaptoethylamine hydrochloride (1.0 mg, 0.00895 mmol) was added. The solution was again heated under reflux for a further 3 nights. Ethyl acetate (10 ml) and ethanol (5 ml) were added to the cooled solution to form a clear solution. The organic solution was then washed with dilute acid (310 ml) and water (210 ml). The organic solution was dried, filtered and evaporated. Column chromatography (petrol/ethyl acetate, 5:1) gave (2R)-2-((1R)-1-Hydroxy-19-((1R)-2-((2S,19S,20S)-19-methoxy-20-methyloctatriacontan-2-yl)cyclopropyl)nonadecyl)-N-(2-mercaptoethyl)hexacosanamide (3.7 mg, 0.00273 mmol, 46%) as a white solid.
EXAMPLE 14
(78) The N-hydroxysuccinimide ester of natural mycolic acid was prepared according to the following method:
(79) Natural mycolic acid (18 mg, 0.0138 mmol) was added to a solution of N hydroxy succinimide (2 mg, 0.0138 mmol) in dry ethyl acetate (2 ml). A solution of DCC (3 mg, 0.0138 mmol) in dry ethyl acetate (1 ml) was added and the reaction mixture was stirred overnight at room temperature. DCU was removed by filtration and the filtrate was concentrated under reduced pressure. Column chromatography (petrol/ethyl acetate, 5:2) gave the N-hydroxysuccinimide ester of natural mycolic acid (11.7 mg, 0.0084 mmol, 61%) as a white solid.
EXAMPLE 15
(80) The N-(2-mercaptoethyl)amide of natural mycolic acid was prepared according to the following method:
(81) The N-hydroxysuccinimide ester of natural mycolic acid (11.3 mg, 0.0081 mmol) and 2-mercaptoethylamine hydrochloride (1.5 mg, 0.0121 mmol) were suspended in dry dichloromethane (2 ml). Triethylamine (2.5 mg, 3.4 l, 0.0242 mmol) that was dissolved in dry dichloromethane (1 ml) was added to the solution. The reaction was allowed to heat under reflux for 3 nights and more 2-mercaptoethylamine hydrochloride (1.5 mg, 0.0121 mmol) was added. The solution was again heated under reflux for a further 3 nights. Ethyl acetate (15 ml) and ethanol (10 ml) were added to the cooled solution to form a clear solution. The organic solution was then washed with dilute acid (320 ml) and water (220 ml). The organic solution was dried, filtered and evaporated. Column chromatography (petrol/ethyl acetate, 5:2) gave the N-(2-mercaptoethyl)amide of natural mycolic acid (5.5 mg, 0.0040 mmol, 50%) as a white solid.
EXAMPLE 16
(82) The method of the first aspect of the present invention in embodiments in which the antigen includes a sulfur atom in the molecule may be carried out as follows:
(83) 10 mM phosphate buffer (pH 7.4) (10 ml), colloidal gold (10 ml, 0.01% Au) and 5 M thiolated antigen solution (2.5 ml) were put in a glass vial, and placed in a shaker overnight. 1 ml aliqouts from the aqueous layer of this solution was taken and centrifuged at 13,200 rpm for 25 minutes. The supernatant was removed and the remaining coated gold nanoparticles were re-suspended in 10 mM phosphate buffer (pH 7.4) (1 ml) before being re-combined and then transferred into the ELISA wells (180 l/well). Sera (20 l) (diluted to a 1 in 5,000 dilution in 10 mM phosphate buffer (pH 7.4)) was added to each well and left for 30 minutes. A saturated aqueous solution of NaCl (20 l) was added to each well and left for 15 minutes before reading the absorbance at 540 nm, 570 nm and 630 nm.
(84) The antigen solution is initially pink/red in colour. Upon addition of the saturated NaCl solution if the sera is positive for TB the antigen-gold complex remains in solution and no colour change is observed. If the sera is negative for TB the antigen-gold complex aggregates/precipitates from the solution and the mixture turns blue. Measuring the absorption of UV-visible light allows a quantitative assessment of the interaction to be made, i.e. the ratio of blue (negative) to red (positive) light can be measured.
EXAMPLE 17
(85) The method of the first aspect of the present invention in embodiments in which the antigen does not include a sulfur atom in the molecule but is linked via a sulfur-containing linker compound may be carried out as follows:
(86) 10 mM phosphate buffer (pH 7.4) (10 ml), colloidal gold (10 ml, 0.01% Au) and 5 M linker compound solution (2.5 ml) were put in a glass vial, and placed in a shaker for 16 hours. A 5 M antigen solution (antigen dissolved in hexane) (2.5 ml) was added to this vial, and left on a shaker for a further 16 hours. 1 ml aliqouts were taken from the aqueous layer of this solution and centrifuged at 7000 RCF for 12 minutes. The supernatant was removed and the remaining coated gold nanoparticles were re-suspended in 10 mM phosphate buffer (pH 7.4) (1 ml) before being re-combined and then transferred into the ELISA wells (180 l/well). Sera (20 l) (diluted to a 1 in 5,000 dilution in 10 mM phosphate buffer (pH 7.4)) was added to each well and left for 30 minutes. A saturated aqueous solution of NaCl (20 l) was added to each well and left for 15 minutes before reading the absorbance at 540 nm and 630 nm.
(87) The antigen solution is initially red in colour. Upon addition of the saturated NaCl solution if the sera is positive for TB the antigen-gold complex remains in solution and no colour change is observed. If the sera is negative for TB the antigen-gold complex aggregates/precipitates from the solution and the mixture turns blue. Measuring the absorption of UV-visible light allows a quantitative assessment of the interaction to be made, i.e. the ratio of blue (negative) to red (positive) light can be measured.
EXAMPLE 18
(88) Quantitative analysis assessment of the interaction when testing with a number of antigens was carried out. This was used to determine the level of aggregation/precipitation of the gold nanoparticles carrying the antigen. As described herein and with particular reference to the methods of example 16 and example 17, in the absence of an antibody to bind to the antigen, upon addition of saturated sodium chloride the antigen-coated gold nanoparticles aggregate and/or precipitate from solution. In an idealised situation in which no antibody is present there would be complete (i.e. 100%) aggregation. In an idealised situation in which all of the antigen is bonded to antibody there would be no aggregation (i.e. 0%) upon addition of the saturated sodium chloride solution.
(89) The percentage aggregation may be calculated as follows:
(90)
EXAMPLE 19
(91) Compound H was used as an antigen in the method of example 16. Three sera samples known to be positive and three sera samples known to be negative were used in the method.
(92) In each case the absorbance of the sample at 540 nm and the absorbance at 630 nm was measured. The results were used to calculate the percentage aggregation for each sample as described in example 18. The colour of the sample was also observed. The results are in table 1:
(93) TABLE-US-00001 Percentage Sample Positive/negative aggregation Colour 1 positive 16 pink 2 positive 30 pink 3 positive 17 pink 4 negative 60 blue 5 negative 64 blue 6 negative 60 blue
EXAMPLE 20
(94) Sulfur-containing linker compound J was coated onto gold nanoparticles according to the method of example 17, followed by the antigen below:
(95) ##STR00019##
(96) Three sera samples known to be positive and three sera samples known to be negative were used in the method.
(97) In each case the absorbance of the sample at 540 nm and the absorbance at 630 nm was measured. The results were used to calculate the percentage aggregation for each sample as described in example 18. The colour of the sample was also observed. The results are in table 2:
(98) TABLE-US-00002 Percentage Sample Positive/negative aggregation Colour 1 positive 0 pink 2 positive 0 pink 3 positive 0.16 pink 4 negative 77 blue 5 negative 71 blue 6 negative 54 purple
EXAMPLE 21
(99) Sulfur-containing linker compound J was coated onto gold nanoparticles according to the method of example 17, followed by a commercially available antigen from Sigma comprising the trehalose dimycolate of a mixture of natural mycolic acids.
(100) Three sera samples known to be positive and three sera samples known to be negative were used in the method.
(101) In each case the absorbance of the sample at 540 nm and the absorbance at 630 nm was measured. The results were used to calculate the percentage aggregation for each sample as described in example 18. The colour of the sample was also observed. The results are in table 3:
(102) TABLE-US-00003 Percentage Sample Positive/negative aggregation Colour 1 positive 35 pink 2 positive 12 pink 3 positive 18 pink 4 negative 94 blue 5 negative 80 blue 6 negative 66 purple
EXAMPLE 22
(103) An alternative method in which centrifugation of the gold nanoparticles before use is not carried out is described below. Similar results were obtained by this method.
(104) 10 mM phosphate buffer (pH 7.4) (10 ml), colloidal gold (10 ml, 0.01% Au) and 5 M thiolated stearic acid solution (2.5 ml) were put in a glass vial, and placed in a shaker for 16 hours. A 5 M antigen solution (antigen dissolved in hexane) (2.5 ml) was added to this vial, and left on a shaker for a further 16 hours. Aliquots from the aqueous layer are then transferred into the ELISA wells (180 l/well). Sera (20 l) (diluted to a 1 in 2,500 dilution in 10 mM phosphate buffer (pH 7.4)) was added to each well and left for 30 minutes. A saturated aqueous solution of NaCl (20 l) was added to each well and left for 15 minutes before reading the absorbance at 540 nm and 630 nm.
EXAMPLE 23
(105) Analysis has been carried out to determine whether the gold nanoparticleantigen complexes retain their stability and activity over time. The complexes were tested for stability and activity after periods of 1, 2, 3 and 6 months following formation, with storage at 4 C. UV-visible spectroscopy showed the same patterns for two serum samples after storing the complexes for the time periods mentioned above as were observed immediately following formation. Thus the complexes remain stable and active and are still able to distinguish positive and negative serum samples at least 6 months after their formation.