Preparation of ceramide conjugates and derivatives of sphingolipid analogues
11760779 · 2023-09-19
Assignee
Inventors
- Nicolai Vladimirovich Bovin (Moscow, RU)
- Stephen Micheal Henry (Auckland, NZ)
- Alexander Borisovich Tuzikov (Moscow, RU)
Cpc classification
C07K7/02
CHEMISTRY; METALLURGY
C07K19/00
CHEMISTRY; METALLURGY
A61K47/549
HUMAN NECESSITIES
A61K47/64
HUMAN NECESSITIES
A61K47/542
HUMAN NECESSITIES
C07K1/1077
CHEMISTRY; METALLURGY
International classification
C07K9/00
CHEMISTRY; METALLURGY
A61K47/64
HUMAN NECESSITIES
C07K1/107
CHEMISTRY; METALLURGY
Abstract
The preparation of water dispersible ceramide conjugates and derivatives of sphingolipid analogues is described. The conjugates and analogues are prepared by reacting a succinimidyl carbonate of a β-Ala derivative with the primary amine of a functionalised spacer. Despite their dispersibility in water, the ceramide conjugates and derivatives of sphingolipid analogues spontaneously incorporate into the plasma membranes of cells.
Claims
1. A ceramide conjugate of the structure: ##STR00026## where F is H or comprises a functional moiety, M is a monovalent cation, n is the integer 1, 2, 3 or 4, p is the integer 1, 2 or 3 and R is selected from the group consisting of C.sub.14-18 alkyl or alkenyl.
2. The conjugate of claim 1 where F is selected from the group consisting of: ##STR00027## where q is the integer 2, 3 or 4, r is the integer 3, 4 or 5 and Glyc is a mono-, di-, tri- or oligosaccharide linked via a glycosidic bond.
3. The conjugate of claim 2 of the structure: ##STR00028##
4. The conjugate of claim 2 of the structure: ##STR00029## where Glyc is an aminoalkyltrisaccharide of the structure: ##STR00030##
5. A method of preparing the ceramide conjugate of claim 1 comprising the step of N-acylation of a 1-carbamate derivative of 2-amino-4-octadecen-3-ol with an activated fatty acid.
6. The method of claim 5 where the activated fatty acid is the N-oxysuccinimide ester of the fatty acid.
7. The method of claim 5 where the fatty acid is stearic acid.
8. The method of claim 5 where F is selected from the group consisting of: ##STR00031## where q is the integer 2, 3 or 4, r is the integer 3, 4 or 5 and Glyc is a mono-, di-, tri- or oligosaccharide linked via a glycosidic bond.
9. The method of claim 5 where n and p are each the integer 2.
Description
BRIEF DESCRIPTION OF DRAWINGS
(1)
(2)
(3)
(4)
(5)
(6)
DESCRIPTION OF EMBODIMENTS
(7) As stated in the publications of Anderson et al (2014a, 2014b and 2015) and Compton et al (2014), although α-galactosyl ceramide has considerable biological activity, it does have limitations such as poor solubility. The disclosures of these publications are directed to providing compounds with improved in vivo efficacy as mediated by improved targeting of the CD1 protein of NKT cells. These studies are to be distinguished from the present invention where it has been sought to provide ceramide conjugates and sphingolipid analogues that have improved solubility but are functionally equivalent to their naturally occurring counterparts with regard to their ability to incorporate and become distributed in the lipid bilayer of cell membranes. The invention resides at least in part in the use of a β-Ala derivative (IV or XIV) as an intermediate in the preparation of both the ceramide conjugates and the derivatives of sphingolipid analogues. The use of such intermediates was found to be necessary to provide a succinimidyl carbonate that would react with the amine (V or XV). For example, the intermediate (XII) was found to be reactive towards β-Ala, but not the amine (XV)
(8) Chemistry
(9) Ceramide Conjugates
(10) Initial attempts to activate natural ceramide with DSC for the purposes of preparing ceramide conjugates were unsuccessful. The mixture of products obtained did not react with the amine (V). This was attributed to the formation of a cyclic carbonate of ceramide as opposed to the desired succinimidyl carbonate. It was concluded that natural ceramide was not suitable for direct conjugation via a cabamoyl linkage. The ceramide conjugates were therefore prepared via a 1-carbamate derivative of 2-amino-4-octadecen-3-ol with the ceramide moiety being formed by amidation of this intermediate. The preparation of a ceramide conjugate where the functional moiety is biotin has been prepared according to Scheme 1A-C. The amine (V) used in this scheme was prepared according to an adaptation of the method described in the publication of Bovin et al (2009). The ceramide conjugates prepared according to the described method have the advantageous property of being readily dispersible in water.
(11) Preparation of a Ceramide Conjugate (X)
(12) Scheme 1A
(13) To a stirred solution of 2-N.sub.3,3-Bzl-sphingosine (1)(102.7 mg, 0.239 mmol) in a mixture of dichloromethane (3 mL) and dimethylformamide (2 mL) DSC (122.5 mg, 0.478 mmol) and triethylamine (33.2 μL, 0.239 mmol) were added. The mixture was stirred for 20 hours at ambient temperature before being evaporated in vacuum (oil pump). The residue was dissolved in chloroform and extracted three times with water (3×4 mL). The chloroform extract was evaporated and the residue was thoroughly dried in vacuum. The yield of 2-N.sub.3,3-Bzl-sphingosine-ONSu (II) as a white solid was 130 mg (95%). TLC: 2-N.sub.3,3-Bzl-sphingosine (I) R.sub.f 0.42, 2-N.sub.3,3-Bzl-sphingosine-ONSu (II) R.sub.f 0.32 (15:5:1 (v/v/v) hexane/chloroform/2-propanol).
(14) To a stirred solution of 2-N.sub.3,3-Bzl-sphingosine-ONSu (II) (130 mg, 0.227 mmol) in a mixture of dichloromethane (2 mL) and dimethylformamide (2 mL) a solution of β-Ala (40.5 mg, 0.405 mmol: 405 μL of solution 100 mg/mL β-Ala and 86 μL/mL trifluoroacetic acid in DMSO) and triethylamine (253 μL, 1.816 mmol) were added. The mixture was stirred for 19 hours at ambient temperature before evaporating in vacuum (oil pump) and drying. The residue was extracted in a mixture of chloroform (4 mL), water (4 mL) and 2M hydrochloric acid (0.12 mL). The chloroform layer was washed twice with water (2×4 mL), evaporated and the residue was dried in vacuum. The crude material was purified on a silica gel column (volume circa 80 ml) in 15:5:1 (v/v/v) hexane/chloroform/2-propanol eluted with 15:5:1 (v/v/v) hexane/chloroform/2-propanol including 0.5% (v/v) acetic acid. Collected fractions were evaporated and the residue dissolved in chloroform. The solution of the residue was washed twice with water (2×3 mL), diluted with acetonitrile (3 mL) and evaporated. Thorough drying of the residue yielded 105.5 mg (85%) of pure 2-N.sub.3,3-Bzl-sphingosine-β-Ala (III) as a colorless syrupy glass. TLC: R.sub.f 0.12 (15:5:1 (v/v/v) hexane/chloroform/2-propanol); R.sub.f 0.49 (4:1 (v/v) chloroform/2-propanol).
(15) .sup.1H NMR of 2-N.sub.3, 3-Bzl-sphingosine-β-Ala (III) (700 MHz, [D]CHCl.sub.3/[D.sub.4]CH.sub.3OH 1:1, 30° C.): δ 8.210 (m, 2H; orto-H of Bzl), 7.766 (m, 1H; para-H of Bzl), 7.636 (m, 2H; meta-H of Bzl), 6.114 (m, 1H; ═CH), 5.754 (m, 2H; CH═ and ═C—CH—O), 4.383 (dd, J=11.4, 4.6 Hz, 1H; OCH), 4.274 (dd, J=11.4, 7.7 Hz, 1H; OCH′), 4.172 (m, 1H; CH—N.sub.3), 3.565 (t, J=6.6 Hz, 2H; NCH.sub.2 of β-Ala), 2.693 (t, J=6.6 Hz, 2H; CH.sub.2CO of β-Ala), 2.260 (q, J=7.1, 7.1, 6.8 Hz, 2H; ═C—CH.sub.2), 1.565 (m, 2H; ═C—C—CH.sub.2), 1.440 (m, 20H; 10 CH.sub.2), 1.043, (t, J=7.1 Hz, 3H; CH.sub.3) ppm.
(16) ##STR00020##
Scheme 1B
(17) To a stirred solution of 2-N.sub.3,3-Bzl-sphingosine-β-Ala (111)(40 mg, 73.4 μmol) in 1,2-dichloroethane (1 mL) a solution of DSC (37.6 mg, 147 μmol: 470 μL of 80 mg/mL solution in dimethylformamide) and triethylamine (15.3 μL, 110 μmol) were added, and the mixture was stirred for 1.5 hours at ambient temperature. The reaction mixture was acidified with acetic acid (100 μL) and placed on a Sephadex LH-20 column (volume 90 mL) and eluted with 2:1 (v/v) chloroform/2-propanol including 0.5% (v/v) acetic acid. Fractions containing 2-N.sub.3,3-Bzl-Sphingosine-β-Ala-ONSu (IV) were combined, evaporated and the residue dried in vacuum. The yield of 2-N.sub.3,3-Bzl-sphingosine-β-Ala-ONSu (IV) as a white solid was 42.4 mg (90%). TLC: R.sup.f 0.56 (15:5:1 (v/v/v) hexane/chloroform/2-propanol).
(18) To a stirred suspension of the biot-CMG(2) amine (V) (44 mg, 34.7 μmol) in dimethyl sulfoxide (2 mL) a solution of 2-N.sub.3,3-Bzl-sphingosine-β-Ala-ONSu (IV) (24.5 mg, 38.1 micromole) in 1,2-dichloroethane (0.49 mL), water (0.7 mL) and 1M aqueous sodium bicarbonate (34.7 μL) were added. The mixture was stirred for 2 hours at ambient temperature before acidifying the reaction mixture with acetic acid (6 μL) and placing on a Sephadex LH-20 column (volume 130 mL) and eluting with 40:14:10:1 (v/v/v/v) water/methanol/2-propanol/chloroform. Fractions containing 2-N.sub.3,3-Bzl-sphingosine-β-Ala-CMG(2)-biot (VI) were combined, evaporated, the residue dissolved in water (3 mL) and then freeze-dried. The yield of 2-N.sub.3,3-Bzl-sphingosine-β-Ala-CMG(2)-biot (VI) as a white solid was 56.7 mg (90% based on biot-CMG(2) amine (V)). TLC: R.sub.f 0.64 (1:3:1 (v/v/v) dichloromethane/ethanol/water); R.sub.f 0.40 (1:3:1 (v/v/v) dichoromethane/ethanol/water including 2% (v/v) acetic acid volume).
(19) .sup.1H NMR of 2-N.sub.3,3-Bzl-sphingosine-β-Ala-CMG(2)-biot (VI) (700 MHz, [D.sub.2]H.sub.2O/[D.sub.4]CH.sub.3OH 1:1, 30° C.): δ 8.035 (m, 2H; orto-H of Bzl), 7.653 (m, 1H; para-H of Bzl), 7.507 (m, 2H; meta-H of Bzl), 5.951 (m, 1H; ═CH), 5.602 (m, 2H; CH═ and ═C—CH—O), 4.581 (dd, J=7.9, 5.0 Hz, 1H; NHCH of biotin), 4.396 (dd, J=7.9, 4.5 Hz, 1H; NHCH of biotin), 4.333-3.899 (total 35H; 4 CH.sub.2COO, 12 NCH.sub.2CO, CH.sub.2O, CH—N.sub.3), 3.469-3.351 (m, 6H; NCH.sub.2CH.sub.2N and CH.sub.2N of β-Ala), 3.289 (m, 1H; NHCHCH of biotin), 2.985 (dd, J=12.9, 4.8 Hz, 1H; NHCHCH of biotin), 2.761 (d, J=12.9 Hz, 1H; NHCHCH of biotin), 2.554 (broad t, 2H; CH.sub.2CO of β-Ala), 2.354 (m, 2H; COCH.sub.2 of biotin), 2.086 (broad, 2H; ═C—CH.sub.2), 1.756 (m, 1H; COCH.sub.2CH.sub.2CH.sub.2CH of biotin), 1.677 (m, 2H; COCH.sub.2CH.sub.2CH.sub.2CH.sub.2 of biotin), 1.593 (m, 1H; COCH.sub.2CH.sub.2CH.sub.2CH of biotin), 1.462 (m, 2H; COCH.sub.2CH.sub.2CH.sub.2CH.sub.2 of biotin), 1.353 (m, 2H; ═C—C—CH.sub.2), 1.224 (m, 20H; 10 CH.sub.2), 0.865 (t, J=7.0 Hz, 3H; CH.sub.3) ppm.
(20) MALDI TOF mass-spectrum of 2-N.sub.3,3-Bzl-sphingosine-β-Ala-CMG(2)-biot (VI) (C.sub.73H.sub.108N.sub.20O.sub.27S, MW isotopic=1729). M/z 1704: M(−N.sub.2+2H)+H; 1726: M(−N.sub.2+2H)+Na; 1730: M+H; 1752; MNa+H; 1768: MK+H; 1774: MNa.sub.2+H; 1790: MNaK+H. Instrument: FLEX-PC, DHB matrix.
(21) To a stirred solution 2-N.sub.3,3-Bzl-sphingosine-β-Ala-CMG(2)-biot (VI) (49.1 mg, 27.01 μmol) in water (4.91 mL), methanol (9.82 mL) and triethylamine (0.737 mL) were added. The mixture was kept for 77 hours at ambient temperature before evaporating the reaction mixture and thoroughly drying the residue in vacuum and freeze drying. The 2-N.sub.3-sphingosine-β-Ala-CMG(2)-biot (VII) was used without purification. TLC: R.sub.f 0.34 (1:3:1 (v/v/v) dichloromethane/ethanol/water including 2% (v/v) acetic acid).
(22) .sup.1H NMR of 2-N.sub.3-sphingosine-β-Ala-CMG(2)-biot (VII) (700 MHz, [D.sub.2]H.sub.2O/[D.sub.4]CH.sub.3OH 1:1, 30° C.): δ 5.812 (m, 1H; CH═), 5.518 (m, 1H; ═CH), 4.593 (dd, J=7.9, 5.0 Hz, 1H; NHCH of biotin), 4.409 (dd, J=7.9, 4.5 Hz, 1H; NHCH of biotin), 4.272-3.934 (total 35H; 4 CH.sub.2COO, 12 NCH.sub.2CO, CH.sub.2O, CH—N.sub.3), 3.720 (m, 1H; ═C—CH—O), 3.459-3.352 (m, 6H; NCH.sub.2CH.sub.2N and CH.sub.2N of β-Ala), 3.304 (m, 1H; NHCHCH of biotin), 2.998 (dd, J=12.9, 4.9 Hz, 1H; NHCHCH of biotin), 2.769 (d, J=12.9 Hz, 1H; NHCHCH of biotin), 2.557 (t, J=6.5 Hz, 2H; CH.sub.2CO of β-Ala), 2.360 (m, 2H; COCH.sub.2 of biotin), 2.095 (m, 2H; ═C—CH.sub.2), 1.771 (m, 1H; COCH.sub.2CH.sub.2CH.sub.2CH of biotin), 1.696 (m, 2H; COCH.sub.2CH.sub.2CH.sub.2CH.sub.2 of biotin), 1.610 (m, 1H; COCH.sub.2CH.sub.2CH.sub.2CH of biotin), 1.469 (m, 2H; COCH.sub.2CH.sub.2CH.sub.2CH.sub.2 of biotin), 1.418 (m, 2H; ═C—C—CH.sub.2), 1.281 (m, 20H; 10 CH.sub.2), 0.898 (t, J=7.1 Hz, 3H; C.sub.3) ppm.
(23) Scheme 1C
(24) To a stirred solution of the unpurified 2-N.sub.3-sphingosine-β-Ala-CMG(2)-biot (VII) (27.01 μmol) in water (1.5 mL), methanol (4.5 mL), dithiothreitol (150 mg) and triethylamine (30 μL) were added. The mixture was stirred for 48 hours at ambient temperature and the reaction mixture evaporated to dryness. The residue vas dissolved in 2 ml of 2:1 (v/v) water/2-propanol, placed on a Sephadex LH-20 column (volume 90 mL) and eluted with 2:1 (v/v) water/2-propanol including 0.05 M Py-HOAc. Fractions containing sphingosine-β-Ala-CMG(2)-biot (VIII) were combined, evaporated and the residue dried in vacuum. The yield of sphingosine-β-Ala-CMG(2)-biot (VIII) as a white solid was 44.3 mg (89% based on 2-N.sub.3,3-Bzl-Sphingosine-β-Ala-CMG(2)-biot (VII) if calculated as tripyridunium salt). TLC: R.sub.f 0.29 (1:3:1 (v/v/v) dichloromethane/ethanol/water including 2% (v/v) acetic acid), ninhydrin-positive.
(25) To a stirred solution of sphingosine-β-Ala-CMG(2)-biot (VIII) (44.3 mg, 24.1 μmol) in a mixture of water (2 mL) and 2-propanol (3 mL) 1 M aqueous sodium bicarbonate (160 μL) and a solution of N-oxysuccinimide ester of stearic acid (20.3 mg, 53 micromole) in 1,2-dichloroethane (0.27 mL) were added, and the mixture stirred for 8 hours at ambient temperature. Additional portions of N-oxysuccinimide ester of stearic acid (20.3 mg, 53 μmol) in 1,2-dichloroethane (0.27 mL) and 1 M aqueous sodium bicarbonate (160 μL) were added and the mixture was stirred for 15 hours at ambient temperature. The reaction mixture was acidified with acetic acid (18 μL), evaporated and the residue dried in vacuum. The reaction products were separated on a silica gel column (volume 75 mL) prepared in 4:1 (v/v) chloroform/methanol and eluted with 2:6:1 (v/v/v) chloroform/methanol/water. Chromatography was accompanied by self-oxidation
(26) ##STR00021##
oxidation of the biotin group into biotin (S-oxide). Repeated (twice) separation of ceramide-β-Ala-CMG(2)-biot (X), ceramide-β-Ala-CMG(2)-biot(S-oxide) (IX) and minor sphingosine-β-Ala-CMG(2)-biot(S-oxide) (oxidation of unreacted sphingosine-β-Ala-CMG(2)-biot (VIII)) on a silica gel column (volume 75 mL) prepared in 4:1 (v/v) chloroform/methanol and eluted with 1:2:1 (v/v/v) dichloromethane/ethanol/water including 1% Py provided 20.5 mg (yield 45%) of pure freeze-dried ceramide-β-Ala-CMG(2)-biot(S-oxide) (IX). TLC: ceramide-β-Ala-CMG(2)-biot (X) R.sub.f 0.62; ceramide-β-Ala-CMG(2)-biot(S-oxide)(IX) R.sub.f 0.54; sphingosine-β-Ala-CMG(2)-biot(S-oxide) R.sub.f 0.46 (1:3:1 (v/v/v) dichloromethane/ethanol/water including 1% (v/v) Py).
(27) .sup.1H NMR of ceramide-β-Ala-CMG(2)-biot(S-oxide) (IX) (700 MHz, [D.sub.2]H.sub.2O/[D.sub.4]CH.sub.3OH 2:1, 30° C.): δ 5.769 (m, 1H; CH═), 5.454 (m, 1H; ═CH), under water (NHCH of biotin-S-oxide), 4.721 (dd, J=8.9, 5.5 Hz, 1H; NHCH of biotin-S-oxide), 4.331-3.897 (total 36H; 4 CH.sub.2COO, 12 NCH.sub.2CO, CH.sub.2O, CHN, ═C—CH—O), 3.639 (d, J=13.5, 1.9 Hz, 1H; NHCHCH of biotin-S-oxide), 3.446-3.344 (m, 7H; NCH.sub.2CH.sub.2N, CH.sub.2N of β-Ala and NHCHCH of biotin-S-oxide), 3.219 (dd, J=13.5, 6.7 Hz, 1H; NHCHCH of biotin-S-oxide), 2.536 (broad t, 2H; CH.sub.2CO of β-Ala), 2.394 (t, J=7.3 Hz, 2H; COCH.sub.2 of biotin-S-oxide), 2.208 (broad t, 2H; COCH.sub.2 of stearoyl), 2.030 (m, 2H; ═C—CH.sub.2), 1.901 (m, 2H; COCH.sub.2CH.sub.2 of stearoyl), 1.734 and 1.593 (m, 6H; 3CH.sub.2 of biotin-S-oxide), 1.304 (broad s, 50H; 25CH.sub.2), 0.902 (t, J=6.9 Hz, 3H; CH.sub.3), 0.894 (broad t, J=7.1 Hz, 3H; CH.sub.3) ppm.
(28) MALDI TOF mass-spectrum of ceramide-β-Ala-CMG(2)-biot(S-oxide) (IX) (C.sub.84H1.sub.40N.sub.18O.sub.28S, MW=1881). M/z 1882: M+H; 1904: MNa+H; 1920: MK+H; 1926: MNa.sub.2+H; 1942: MNaK+H; 1948: MNa.sub.3+H; 1964: MKNa.sub.2+H; 1970: MNa.sub.4+H; 1986: MKNa.sub.3+H; 1992: MNa.sub.4+Na; 2008: MNa.sub.4+K. Instrument: FLEX-PC, DHB matrix.
(29) To a stirred solution of ceramide-β-Ala-CMG(2)-biot(S-oxide) (IX) (20.5 mg, 10.89 μmol) in a water (1.314 mL) N-methyl-mercaptoacetamide (373 μL) was added and the mixture kept for 69 hours at 40° C. The reaction mixture was placed on a Sephadex LH-20 column (volume 90 mL) and eluted with 2:1 (v/v) water/2-propanol including 0.03 M acetic acid and 0.06 M Py). Fractions containing pure ceramide-β-Ala-CMG(2)-biot (X) were combined, evaporated and the residue thoroughly dried in vacuum. The residue was dissolved in water (1 mL), titrated to pH 6.5 with 0.1 M sodium bicarbonate and freeze-dried. The yield of ceramide-β-Ala-CMG(2)-biot (X) as a white solid was 15.8 mg (74%). TLC: R.sub.f 0.64 (1:3:1 (v/v/v) dichloromethane/ethanol/water).
(30) .sup.1H NMR of ceramide-β-Ala-CMG(2)-biot (X) (700 MHz, [D.sub.2]H.sub.2O/[D.sub.4]CH.sub.3OH 2:1, 30° C.): δ 5.764 (m, 1H; CH═), 5.443 (m, 1H; ═CH), 4.603 (dd, J=7.9, 4.9 Hz, 1H;
(31) ##STR00022##
NHCH of biotin), 4.423 (dd, J=7.9, 4.6 Hz, 1H; NHCH of biotin), 4.312-3.964 (total 36H; 4 CH.sub.2COO, 12 NCH.sub.2CO, CH.sub.2O, CHN and ═C—CH—O), 3.444-3.352 (m, 6H; NCH.sub.2CH.sub.2N and CH.sub.2N of β-Ala), 3.313 (m, 1H; NHCHCH of biotin), 2.997 (dd, J=12.9, 4.8 Hz, 1H; NHCHCH of biotin), 2.778 (d, J=12.9 Hz, 1H; NHCHCH of biotin), 2.552 (t, J=6.5 Hz, 2H; CH.sub.2CO of β-Ala), 2.365 (m, 2H; COCH.sub.2 of biotin), 2.206 (broad t, 2H; COCH.sub.2 of stearoyl), 2.039 (m, 2H; ═C—CH.sub.2), 1.759, 1.691, 1.601, 1.558 and 1.469 (m, total 8H; 6H of biot and COCH.sub.2CH.sub.2 of stearoyl), 1.390 (m, 2H; ═C—C—CH.sub.2), 1.299 (m, 48H; 24 CH.sub.2), 0.905 (t, J=6.6 Hz, 3H; CH.sub.3), 0.895 (broad t, J=7.0 Hz, 3H; CH.sub.3) ppm.
(32) MALDI TOF mass-spectrum of ceramide-β-Ala-CMG(2)-biot (X) (C.sub.84H.sub.140N.sub.18O.sub.27S, MW=1865). M/z 1866: M+H; 1888: M+Na; 1904: M+K; 1910: MNa+Na; 1926: MNa+K; 1932: MNa.sub.2+Na; 1948: MNa.sub.2+K; 1954: MNa.sub.3+Na; 1970: MNa.sub.3+K; 1976: MNa.sub.4+Na. Instrument: FLEX-PC, DHB matrix.
(33) Derivatives of Sphingolipid Analogues
(34) Sphingolipid analogues comprising a 2-(alkyl)alkyl membrane anchor, e.g. 2-(teradecyl)hexadecyl, are mimetics of the ceramide conjugates that retain the advantageous property of being dispersible in water. In preferred embodiments of these structural mimetics, the fully saturated dialkyl alcohol 2-(tetradecyl)hexadecanol is substituted for the acylated sphingosine of ceramide. A sphingolipid analogue where the functional moiety is biotin has been prepared according to Scheme 2.
(35) The amine (XV) is prepared according to the method described in the publication of Bovin et al (2009). The preparation of the β-Ala derivative (XIV) was found to be necessary to provide a succinimidyl carbonate that would react with the amine (XV). The intermediate (XII) was found to be reactive towards β-Ala, but not the amine (XV).
(36) The preparation of glycosphingolipid analogues according to Scheme 3 is also anticipated. In both schemes it will be recognised that a range of sphingolipid analogues may be prepared using homologues of 2-(tetradecyl)hexadecanol. Homologues comprising alkyl chains of a length comparable with that of ceramide are preferred.
(37) ##STR00023##
Preparation of a Sphingolipid Analogue (XVI)
(38) To a stirred solution of 2-(tetradecyl)hexadecanol (XI) (Katayama Chemical Industries Co., Limited) (15.3 mg, 34.87 μmol) in a mixture of dichloromethane (1 mL) and dimethylformamide (0.6 mL) a solution of DSC (71.5 mg, 279 μmol) in dimethylformamide (0.893 mL) and triethylamine (19.4 μL, 139 μmol) were added. The mixture was stirred for 24 hours at ambient temperature and then the reaction mixture acidified with acetic acid (96 μL) before being placed on a Sephadex LH-20 column (volume 90 mL) and eluted with 2:1 (v/v) trichloromethane/2-propanol plus 0.5% (v/v) acetic acid. Fractions containing the intermediate (XII) were combined, evaporated and the residue dried in vacuum. The yield of the intermediate (XII) as a white solid was 19.1 mg (94%). TLC: R.sub.f 0.35 (15:5:1 (v/v/v) hexane/trichloromethane/2-propanol).
(39) To a stirred solution of the intermediate (XII) (19.1 mg, 32.94 μmol) in a mixture of 1,2-dichloroethane (1 mL) and dimethylformamide (2 mL) a volume of 106 μL of a 100 mg/mL solution of β-Ala (10.6 mg, 119 μmol) with 86 μL/mL trifluoroacetic acid in DMSO and triethylamine (66 μL, 475 μmol) were added. The mixture was stirred for 17 hours at ambient temperature before being acidified with acetic acid (60 μL) and evaporated with 5 mL of 2-propanol to a minimum volume. The sample was placed on a Sephadex LH-20 column (volume 90 mL) and eluted with 2:1 (v/v) trichloromethane/2-propanol plus 0.5 M Py.AcOH. Fractions containing the intermediate (XIII) were combined, evaporated and the residue dried in vacuum. The yield of the intermediate (XIII) as a white solid was 17.4 mg (95%). TLC: R.sub.f 0.38 (2:4:1 (v/v/v) hexane/trichloromethane/2-propanol). .sup.1H NMR of intermediate (XIII) (700 MHz, [D]CHCl.sub.3/[D.sub.4]CH.sub.3OH 1:1, 30° C.): δ 4.098 (d, J=5.4 Hz, 2H; CH.sub.2O), 3.549 (t, J=6.5 Hz, 2H; CH.sub.2N of β-Ala), 2.684 (t, J=6.5 Hz, 2H; CH.sub.2CO of β-Ala), 1.766 (m, 1H; OCH.sub.2CH), 1.433 (m, 52H; 26 CH.sub.2), 1.046, (t, J=7.1 Hz, 6H; 2 CH.sub.3) ppm.
(40) To a stirred solution of the intermediate (XIII) (11.3 mg, 20.4 μmol) in a mixture of 1,2-dichloroethane (1 mL) and dimethylformamide (0.35 mL) a 131 μL volume of a 80 mg/mL solution of DSC (10.5 mg, 41 μmol) in dimethylformamide and triethylamine (4.3 μL, 31 μmol) were added. The mixture was stirred for 2 hours at ambient temperature and the reaction mixture acidified with acetic acid (100 μL). The acidified mixture was placed on Sephadex LH-20 column (volume 90 mL) and eluted with 2:1 (v/v) trichloromethane/2-propanol plus 0.5% (v/v) acetic acid. Fractions containing the activated intermediate (XIV) were combined, evaporated and the residue dried in vacuum. The yield of the activated intermediate (XIV) as a white solid was 12.7 mg (96%). TLC: R.sub.f=0.72 (2:4:1 (v/v/v) hexane/trichloromethane/2-propanol).
(41) To a stirred suspension of the amine (XV) (22.3 mg, 17.56 μmol) in dimethyl sulfoxide (1.5 mL) a solution of the activated intermediate (XIV) (12.7 mg, 19.51 μmol) in 1,2-dichloroethane (0.25 mL), water (0.35 mL) and 1 M aqueous sodium bicarbonate (35.2 μL) were added, and the mixture stirred for 5 hours at ambient temperature. The reaction mixture was acidified with acetic acid (3 μL), evaporated with 3 mL of 1:1 (v/v) 2-propanol/water to a minimum volume and placed on a Sephadex LH-20 column (volume 90 mL). The column was eluted with 1:2 (v/v) 2-propanol/water plus 3% (v/v) dichloromethane and 0.3% (v/v) Py by volume). Fractions containing the sphingolipid analogue (XVI) were combined, evaporated and the residue thoroughly dried in vacuum. The residue was dissolved in water (1 mL), titrated to pH 6.5 with 0.1 M sodium bicarbonate and freeze-dried. The yield of the sphingolipid analogue (XVI) as a white solid was 29.2 mg (91% based on the amine (XV)). TLC: R.sub.f 0.61 (2:6:1 (v/v/v) trichloromethane/methanol/water). .sup.1H NMR of the sphingolipid analogue (XVI) (see
(42) Avidinylated functional moieties may be conjugated to the ceramide conjugate (X) or the sphingolipid analogue (XVI) under biocompatible conditions exploiting non-covalent avidin-biotin binding. Alternatively, it is anticipated that ceramide conjugates and sphingolipid analogues may be prepared by covalent attachment of the functional moiety. For example, it is anticipated that glycosphingolipid analogues may be prepared according to Scheme 3. In accordance with this scheme equimolar amounts of the activated β-Ala derivative (XIV) and the diamine (XVII) are reacted to provide the membrane anchor (XVIII). The membrane anchor (XVIII) is then reacted with an activated saccharide, such as the N-succimidyl carbamate of a 3-aminopropylglycoside (XIX).
(43) ##STR00024##
(44) A non-limiting example of a 3-aminopropylglycoside (XIX) is 3-aminopropyl-α-D-galactopyranosyl-(1,3)-β-D-galactopyranosyl-(1.fwdarw.4)-2-acetamido-2-deoxy-β-D-glucopyranoside (Galα3Galβ4GlcNAcβ-). This 3-aminopropylglycoside may be prepared according to Scheme 4.
(45) The glycosyl acceptor (3-trifluoroacetamidopropyl)-2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-4-O-(2,4-di-O-acetyl-6-O-benzyl-(3-D-galactopyranosyl)-β-D-glucopyranoside (XXII) was prepared according to the method disclosed in the publication of Pazynina et al (2008). A mixture of the glycosyl acceptor (XXII) (500 mg, 0.59 mmol), thiogalactopyranoside (XXI) (576 mg, 1.18 mmol), NIS (267 mg, 1.18 mmol), anhydrous CH.sub.2Cl.sub.2 (25 ml) and molecular sieves 4 Å (500 mg) was stirred at −45° C. for 30 min under an atmosphere of Ar. A solution of TfOH (21 μl, 0.236 mmol) in anhydrous CH.sub.2Cl.sub.2 (0.5 ml) was then added. The reaction mixture was stirred for 2 h at −45° C. and the temperature was then increased to −20° C. over 4 h. The mixture was kept at −20° C. overnight. Then extra amounts of thiogalactopyranoside (XXI)(144 mg, 0.295 mmol), NIS (66 mg, 0.295 mmol) and TfOH (5 μl, 0.06 mmol) were added and the stirring maintained at −20° C. for 2 h before being allowed to slowly warm up to r.t. (1 h). A saturated aqueous solution of Na.sub.2S.sub.2O.sub.3 was then added and the mixture filtered. The filtrate was diluted with CHCl.sub.3 (300 ml), washed with H.sub.2O (2×100 ml), dried by filtration through cotton wool, and concentrated. Gel filtration on LH-20 (CHCl.sub.3-MeOH) afforded the product (XXIII) (600 mg, 80%), as a white foam.
(46) .sup.1H NMR (700 MHz, CDCl.sub.3, characteristic signals), δ, ppm: 1.78-1.82 (m, 4H, CHCHC, OC(O)CH.sub.3), 1.84-1.90 (m, 1H, CHCHC), 1.91, 1.94, 1.97, 1.98, 2.06 (5 s, 5×3H, 4 OC(O)CH.sub.3, NH(O)CH.sub.3), 3.23-3.30 (m, 1H, NCHH), 3.59-3.65 (m, 1H, NCHH), 4.05 (m, 1H, H-2.sup.1), 4.33 (d, 1H, J.sub.1,2 7.55, H-1.sup.I), 4.40 (d, 1H, J 12.04, PhCHH), 4.42 (d, 1H, J.sub.1,2 8.07, H-1.sup.II), 4.45 (d, 1H, J 11.92, PhCHH), 4.48 (d, 1H, J 12.00, PhCHH), 4.50 (d, 1H, J 12.00, PhCHH), 4.52 (d, 1H, J 12.04, PhCHH), 4.54 (d, 1H, J 12.00, PhCHH), 4.57 (d, 1H, J 12.00, PhCHH), 4.64 (d, 1H, J 11.92, PhCHH), 4.99 (dd≈t, 1H, J 8.24, H-2.sup.II), 5.08-5.13 (m, 2H, H-3.sup.I, H-3.sup.III), 5.23 (d, 1H, J.sub.1,2 3.31, H-1.sup.III), 5.46 (d, 1H, J.sub.3,4 2.25, H-4.sup.II), 5.54 (d, 1H, J.sub.3,4 3.11, H-4.sup.III), 7.20-7.40 (m, 20H, ArH); 7.49-7.54 (m, 1H, NHC(O)CF.sub.3). R.sub.f 0.4 (PhCH.sub.3—AcOEt, 1:2).
(47) The product (XXIII) (252 mg, 0.198 mmol) was deacetylated according to Zemplen (8 h, 40° C.), neutralized with AcOH and concentrated. The TLC (CH.sub.3Cl-MeOH, 10:1) analysis of the obtained product showed two spots: the main spot with R.sub.f 0.45, and another one on the start line (ninhydrin positive spot) that was an indication of partial loss of trifluoroacetyl. Therefore, the product was N-trifluoroacetylated by treatment with CF.sub.3COOMe (0.1 ml) and Et.sub.3N (0.01 ml) in MeOH (10 ml) for 1 h, concentrated and subjected to column
(48) ##STR00025##
chromatography on silica gel (CHCl.sub.3-MeOH, 15:1) to afford the product (XXIV) as a white foam (163 mg, 77%), R.sub.f 0.45 (CH.sub.3Cl-MeOH, 10:1). The product (XXIV) was subjected to hydrogenolysis (200 mg Pd/C, 10 ml MeOH, 2 h), filtered, N-defluoroacetylated (5% Et.sub.3N/H.sub.2O, 3 h) and concentrated. Cation-exchange chromatography on Dowex 50X4-400 (H.sup.+) (elution with 5% aqueous ammonia) gave the product (XXV) (90 mg, 98%) as a white foam.
(49) .sup.1H NMR (D.sub.2O, characteristic signals), δ, ppm: 1.94-1.98 (m, 2H, CCH.sub.2C), 2.07 (s, 3H, NHC(O) CH.sub.3), 3.11 (m, J 6.92, 2H, NCH.sub.2), 4.54 and 4.56 (2d, 2H, J.sub.1,28.06, J.sub.1,2 7.87, H-1.sup.I and H-1.sup.II), 5.16 (d, 1H, J.sub.1,2 3.87, H-1.sup.III). R.sub.f 0.3 (EtOH-BuOH-Py-H.sub.2O—AcOH; 100:10:10:10:3). Although the foregoing schemes are illustrated referencing the use of DSC the use of other homo-bifunctional cross-linkers such as disuccinimidylglutarate, disuccinimidyladipate and disuccinimidylpimelate is also contemplated.
(50) Biology
(51) Ceramide Conjugates
(52) Incorporation of a Ceramide Conjugates into Cell Membranes
(53) A 50 μL volume of packed red blood cells was re-suspended in an equal volume of a dispersion in phosphate buffered saline (PBS) (pH 7.2) of the ceramide conjugate (X). The re-suspended cells were incubated at 37° C. for two hours and then washed once with PBS. The modified cells were diluted with PBS to provide a suspension at a concentration equivalent to 5% of the packed cell volume. A 30 μL volume of the diluted suspension was then mixed with an equal volume of a 0.1 mg/mL solution of Avidin Alexa Flour™ 488 in PBS. The mixture was incubated at room temperature for 30 minutes before washing and re-suspending the modified cells in PBS. The fluorescence of cells in a 5 μL volume was observed using a microscope (Olympus BX51) at 400× magnification. Photomicrographs (1.903 seconds exposure time) are presented in
(54) The modified cells prepared using dispersions of the ceramide conjugate (X) at different concentrations (0 (control), 0.5, 5 and 50 μM) were scored. The results are presented in Table 1.
(55) The observations form fluorescent labelling of the modified cells confirmed that both the ceramide conjugate (X) and the construct designated biotin-CMG(2)-Ad-DOPE spontaneously incorporated into cell membranes.
(56) TABLE-US-00001 TABLE 1 Comparison of the fluorescence scores observed for red blood cells modified to incorporate either the ceramide conjugate (X) or the construct designated biotin-CMG(2)-Ad-DOPE from the publication of Bovin et al (2009). Fluorescence score ceramide Concentration (μM) conjugate biotin-CMG(2)-Ad-DOPE 0 0 — 0.5 0 to 1+ 0 5 1+ 2+ 50 2 to 3+ 3+
(57) Derivatives of Sphingolipid Analogues
(58) Incorporation of a Sphingolipid Analogue into Cell Membranes
(59) A 50 μL volume of packed red blood cells was re-suspended in an equal volume of a dispersion in phosphate buffered saline (PBS) (pH 7.2) of the sphingolipid analogue (XVI). The re-suspended cells were incubated at 37° C. for two hours and then washed once with PBS. The modified cells were diluted with PBS to provide a suspension at a concentration equivalent to 5% of the packed cell volume. A 30 μL volume of the diluted suspension was then mixed with an equal volume of a 0.1 mg/mL solution of Avidin Alexa Flour™ 488 in PBS. The mixture was incubated at room temperature for 30 minutes before washing and re-suspending the modified cells in PBS. The fluorescence of cells in a 5 μL volume was observed using a microscope (Olympus BX51) at 400× magnification. Photomicrographs (1.903 seconds exposure time) are presented in
(60) The modified cells prepared using dispersions of the sphingolipid analogue (XVI) at different concentrations (0 (control), 0.5, 5 and 50 μM) were scored. The results are presented in Table 2.
(61) Modified cells prepared using either the sphingolipid analogue (XVI) or the construct designated biotin-CMG(2)-Ad-DOPE from the publication of Bovin et al (2009) were indistinguishable.
(62) Although the invention has been described with reference to embodiments or examples it should be appreciated that variations and modifications may be made to these embodiments or examples without departing from the scope of the invention. Where known equivalents exist to specific elements, features or integers, such equivalents are incorporated as if specifically referred to in this specification. Variations and modifications to the embodiments or
(63) TABLE-US-00002 TABLE 2 Comparison of the fluorescence scores observed for red blood cells modified to incorporate either the sphingolipid analogue (XVI) or the construct designated biotin-CMG(2)-Ad-DOPE from the publication of Bovin et al (2009). Fluorescence score sphingolipid Concentration (μM) analogue biotin-CMG(2)-Ad-DOPE 0 — — 0.5 0 0 5 2+ 2+ 50 3+ 3+
examples that include elements, features or integers disclosed in and selected from the referenced publications are within the scope of the invention unless specifically disclaimed. The advantages provided by the invention and discussed in the description may be provided in the alternative or in combination in these different embodiments of the invention.
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