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FLUORESCENT CELL MARKERS

20170082642 ยท 2017-03-23

    Inventors

    Cpc classification

    International classification

    Abstract

    The preparation and use of fluorescent cell markers of the structure F-S.sub.1-S.sub.2-L is described where F is a fluorophore, S.sub.1-S.sub.2 is a spacer linking F to L, and L is a diacyl lipid.

    Claims

    1) A fluorescent cell marker of the structure:
    FS.sub.1-S.sub.2L including the substructure: ##STR00027## where: F is a fluorophore; S.sub.1-S.sub.2 is a spacer linking F to L; L is a lipid selected from the group consisting of diacyl- and dialkyl-glycerolipids, including glycerophospholipids; m and n are independently 3 to 6; R.sub.1 is O or S; and * is other than H.

    2) The fluorescent cell marker of claim 1 where F is selected from the group consisting of: fluorophores of fluorescein, Oregon Green, Pennsylvania Green, Tokyo Green, eosin, BODIPY, BODIPY TR, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 594, Texas Red, Lucifer Yellow, tetramethylrhodamine and their derivatives.

    3) The fluorescent cell marker of claim 1 where F is selected from the group consisting of: fluorophores of fluorescein, BODIPY and their derivatives.

    4) The fluorescent cell marker of claim 1 where the sum of in and n is 6 to 9 and * is C or N.

    5) The fluorescent cell marker of claim 1 where F is the fluorophore of fluorescein or one of its derivatives and S.sub.1 is a C.sub.3-5-diaminoalkyl derivative selected from the group consisting of: 1,3-diaminopropyl, 1,4-diaminobutyl, or 1,5-aminopentyl derivatives.

    6) The fluorescent cell marker of claim 1 where F is the fluorophore of fluorescein or one of its derivatives and S.sub.1 is a C.sub.3-5-aminoalkylthioureidyl.

    7) The fluorescent cell marker of claim 1 where F is the fluorophore of fluorescein or one of its derivatives and S.sub.1 is 5-((5-aminopentyl) thioureidyl.

    8) The fluorescent cell marker of claim 1 where F is the fluorophore of fluorescein or one of its derivatives and S.sub.2 is selected. from the group including: CO(CH.sub.2).sub.3CO, CO(CH.sub.2).sub.4CO (adipate), CO(CH.sub.2).sub.5CO and CO(CH.sub.2).sub.5)NHCO(CH.sub.2).sub.5CO.

    9) The fluorescent cell marker of claim 1 where F is the fluorophore of fluorescein or one of its derivatives and S.sub.2 as CO(CH.sub.2).sub.4CO (adipate).

    10) The fluorescent cell marker of claim 1 where F is the fluorophore of fluorescein or one of its derivatives and the structure includes the substructure: ##STR00028## where m and n are independently 3 to 5 and * is other than H.

    11) The fluorescent cell marker of claim 1 where F is the fluorophore BODIPY or one of its derivatives and S.sub.1 is a C.sub.2-5-alkionyldiamine.

    12) The fluorescent cell marker of claim 1 where F is the fluorophore BODIPY or one of its derivatives and S.sub.1 is propionyl ethyldiamine.

    13) The fluorescent cell marker of claim 1 where F is the fluorophore BODIPY or one of its derivatives and S.sub.2 is selected from the group consisting of: CO(CH.sub.2).sub.3CO, CO(CH.sub.2).sub.4CO (adipate) and CO(CH.sub.2).sub.5CO.

    14) The fluorescent cell marker of claim 1 where F is the fluorophore of BODIPY or one of its derivatives and S.sub.2 is CO(CH.sub.2).sub.4CO (adipate).

    15) The fluorescent cell marker of claim 1 where F is the fluorophore of BODIPY or one of its derivatives and the structure includes the substructure: ##STR00029## where p, q and r are independently 3 to 5 and * is other than H.

    16) The fluorescent cell marker of claim 15 where the sum of p, q and r is 8.

    17) The fluorescent cell marker of claim 16 where p is 2, q is 2 and r is 4.

    18) The fluorescent cell marker of claim 1 where L is selected from the group consisting of diacylglycerolipids, phosphatidate, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol, and diphosphatidyl glycerol derived from one or more of trans-3-hexadecenoic acid, cis-5-hexadecenoic acid, cis-7-hexadecenoic acid, cis-9-hexadecenoic acid, cis-6-octadecenoic acid, cis-9-octadecenoic acid, trans-9-octadecenoic acid, trans-11-octadecenoic acid, cis-11-octadecenoic acid, cis-11-eicosenoic acid or cis-13-docsenoic acid.

    19) The fluorescent cell marker of claim 18 where the lipid is derived from one or more cis-desaturated fatty acids.

    20) The fluorescent cell masker of claim 19 where L is selected from the group consisting of: 1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE) and rac-1,2-dioleoylglycerol (DOG).

    21) A fluorescent cell marker with the structure: ##STR00030## designated KODE-fluorescein (I) and where M is typically H, but may be replaced by another monovalent cation such as Na.sup.+, K.sup.+ or NH.sub.4.sup.+.

    22) A fluorescent cell marker with the structure: ##STR00031## designated KODE-Oregon Green (II) and where M is typically H, but may be replaced by another monovalent cation such as Na.sup.+, K.sup.+ or NH.sub.4.sup.+.

    23) A fluorescent cell marker with the structure: ##STR00032## designated KODE-Tokyo Green (III) and where M is typically H, but may be replaced by another monovalent cation such as Na.sup.+, K.sup.+ or NH.sub.4.sup.+.

    24) A fluorescent cell marker with the structure: ##STR00033## designated KODE-Pennsylvania Green (IV) and where K is typically H, but may be replaced by another monovalent cation such as Na.sup.+, K.sup.+ or NH.sub.4.sup.+.

    25) A fluorescent cell marker with the structure: ##STR00034## designated KODE-BODIPY (V) and where M is typically H, but may be replaced by another monovalent cation such as Na.sup.+, K.sup.+ or NH.sub.4.sup.+.

    26) A method of marking cells or multi-cellular structures including the step of: Contacting a suspension of cells or multi-cellular structures with a cell marker of claim 1 for a time and at a temperature sufficient to allow incorporation of the marker into the membrane of the cell or multi-cellular structure.

    27) A cell or multi-cellular structure incorporating a cell marker of claim 1.

    28) A cell or multi-cellular structure produced by the method of claim 26.

    Description

    BRIEF DESCRIPTION OF FIGURES

    [0043] FIG. 1. Red blood cells following contact with cell marker (I) viewed with a fluorescence microscope at 470 nm under 250 magnification.

    [0044] FIG. 2. Structure of cell marker designated KODE-fluorescein (I).

    [0045] FIG. 3. Structure of cell marker designated KODE-Oregon Green (II).

    [0046] FIG. 4. Structure of cell marker designated KODE-Tokyo Green (III).

    [0047] FIG. 5. Structure of cell marker designated KODE-Pennsylvania Green (IV).

    [0048] FIG. 6. Structure of cell marker designated KODE-BODIPY (V).

    [0049] FIG. 7. .sup.1H-NMR spectrum of the cell marker designated KODE-BODIPY (V).

    DETAILED DESCRIPTION

    [0050] The specification accompanying international application no. PCT/NZ2005/000052 (publication no. WO 2005/090368) describes water soluble synthetic molecules that are constructs of the structure F-S.sub.1-S.sub.2-L.

    [0051] In these constructs F is a carbohydrate and the constructs spontaneously and stably incorporate into lipid bi-layers, including cell membranes.

    [0052] The preferred constructs described in the specification accompanying the international application comprise the substructure:

    ##STR00009##

    where n 3 to 5, X is H or C, and * is other than H.

    [0053] M is typically H, but may be replaced. by another monovalent cation such as Na.sup.+, K.sup.+ or NH.sub.4.sup.+.

    [0054] F is a fluorophore in the constructs of the present invention with different physicochemical properties to those of carbohydrate. The spacer (S.sub.1-S.sub.2) is selected to provide a construct that can be readily dispersed in aqueous vehicles such as saline.

    [0055] Whilst not wishing to be bound by theory it is believed the cell markers of the present invention spontaneously incorporate into the lipid bi-layer of the cell membrane via their diacyl lipid tail. The fluorophore moiety is therefore expressed at the cell surface. The cell markers of the present invention can be used to mark cells without modification of the proteins expressed at the surface of the cell.

    [0056] The likelihood of cell functions mediated by proteins expressed at the cell surface is reduced. Furthermore, the likelihood of the cell marker becoming uniformly distributed in the two dimensions of the lipid bilayer is increased. The mobility of the fluorophore is not dependent on the mobility of the cell surface expressed proteins to which the fluorophore might otherwise be conjugated.

    [0057] Additional advantages are anticipated to accrue as the cell markers may allow studies on cell membrane dynamics independent of protein function and cycling. Cells labeled using the cell markers of the present invention may still be identified by conventional means and used in established biological methods such as fluorescence activated cell sorting (FACS) systems.

    [0058] For the preparation of KODE-fluorescein (I), FITC is first conjugated with a diamine such as 1,5-diaminopentyl (cadaverine). The conjugated FITC is then reacted with an activated lipid (I-A) prepared as described in international application number PCT/NZ2005/000052.

    [0059] A number of fluorescent compounds are available commercially as cadaverine derivatives. The cell markers where F is one of the fluorophores designated in Table 1 may be prepared.

    TABLE-US-00001 TABLE 1 Fluorophores (represented as neutrally charged protonated species). Fluorophore Designation [00010]embedded image Fluorescein (6-isomer) [00011]embedded image Fluorescein (5-isomer) [00012]embedded image Fluorescein (4-isomer) [00013]embedded image Oregon Green (5-isomer) [00014]embedded image Pennsylvania Green (5-isomer) [00015]embedded image Tokoyo Green (5-isomer) [00016]embedded image Eosin (5-isomer) [00017]embedded image BODIPY [00018]embedded image BODIPY TR [00019]embedded image Alexa Fluor 350 [00020]embedded image Alexa Fluor 405 [00021]embedded image Alexa Fluor 488 (5-isomer) [00022]embedded image Alexa Fluor 568 (5-isomer) [00023]embedded image Alexa Fluor 594 (5-isomer) [00024]embedded image Texas Red (5-isomer) [00025]embedded image Lucifer Yellow [00026]embedded image Tetramethylrhodamine (5-isomer)

    [0060] For the preparation of KODE-BODIPY (V), BODIPY may alternatively be conjugated with an alkionyl diamine such propionly ethylenediamine (BODIPY FL FDA). The conjugated BODIPY is then reacted with an activated lipid (L-A) prepared as described in the specification accompanying international application no. PCT/NE2005/000052.

    EXAMPLE 1

    Preparation of Activated 1,2-O-distereoyl-sn-glycero-3-phosphatidylethanolamine (DSPE) and Activated 1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (L-A)

    [0061] To a solution of bis(N-hydroxysuccinimidyl) adipate (A) (70 mg, 205 mol) in dry N,N-dimethylformamide (1.5 ml) were added DOPE or DSPE (L) (40 mol) in chloroform (1.5 ml) followed by triethylamine (7 l). The mixture was kept for 2 h at room temperature, then neutralized with acetic acid and partially concentrated in vacuo.

    [0062] Column chromatography (Sephadex LH-20, 1:1 chloroform-methanol, 0.2% acetic acid) of the residue yielded the activated lipid (L-A) (37 mg, 95%) as a colorless syrup; TLC (chloroform-methanol-water, 6:3:0.5): R.sub.f=0.5 (DOPE-A), R.sub.f=0.55 (DSPE-A).

    [0063] .sup.1H NMR (CDCl.sub.3/CD.sub.3OD, 2:1), : DSPE-A-5.39 (mm, 1H, OCH.sub.2CHOCH.sub.2O), 4.53 (dd, 1H, J=3.42, J=11.98, CCOOHCHCHOCH.sub.2O), 4.33 (dd, 1H, J=6.87, J=11.98, CCOOHCHCHOCH.sub.2O), 4.23 (m, 2H, POCH.sub.2CH.sub.2NH.sub.2), 4.15 (m, 2H, CH.sub.2OP), 61 (m, 2H, POCH.sub.2CH.sub.2-NH.sub.2), 3.00 (s, 4H, ONSuc), 2.81 (m, 2H, CH.sub.2CO (Ad), 2.48 (m, 4H, 2(CH.sub.2CO), 2.42 (m, 2H, CH.sub.2CO (Ad), 1.93 (m, 4H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO), 1.78 (m, 4H, 2(COCH.sub.2CH.sub.2) 1,43, 1.47 (2 bs, 40H, 20CH.sub.2), 1.04 (m, 6H, 2CH.sub.3).

    [0064] DOPE-A-5.5 (m, 4H, 2(CHCH), 5.39 H, (m, 1H, OCH.sub.2-CHOCH.sub.2O), 4.58 (dd, 1H, J=3.67, J=11.98, CCOOHCHCHO CH2O), 4.34 (dd, 1H, J=6.61, J=11.98, CCOOHCHCHOCH.sub.2O), 4.26 (m, 2H, POCH.sub.2CH.sub.2NH.sub.2), 4.18 (m, 2H, CH.sub.2OP), 3.62 (m, 2H, POCH.sub.2CH.sub.2NH.sub.2), 3.00 (s, 4H, ONSuc), 2.8 (m, 2H, CH.sub.2CO (Ad), 2.50 (m, 4H, 2(CH.sub.2CO), 2.42 (m, 2H, CH.sub.2CO (Ad), 2.17 (m, 8H, 2(CH.sub.2CHCHCH.sub.2), 1.93 (m, 4H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO), 1.78 (m, 4H, 2(COCH.sub.2CH.sub.2), 1,43, 1.47 (2 bs, 40H, 20CH.sub.2), 1.04 (m, 6H, 2CH.sub.3).

    [0065] Condensation of DOPE-A with ((5-aminopentyl)thioureidyl) fluorescein (fluorescein cadaverine)

    [0066] To a solution of activated DOPE (L-A) (5 mg, 5.2 mol) in N,N-dimethylformamide (0.5 ml) 3 mg (4.6 mol) of fluorescein cadaverine dihydrobromide salt and 5 l of triethylamine were added. The mixture was kept for 2 h at room temperature, then 10 l of 3% aq. NH.sub.3 were added and the mixture was kept at room temperature for 1 h.

    [0067] Column chromatography (Sephadex LH-20, 1:1 chloroform-methanol, followed by silica gel, ethyl acetate-isopropanol-water, 6:3:1) of the mixture yielded 4.2 mg (67%) KODE-fluorescein (I), R.sub.f 0.5 (ethyl acetate-isopropanol-water, 6:3:1).

    [0068] .sup.1H NMR (CDCl.sub.3/CD.sub.3OD, 1:1), : KODE-fluorescein (I)-8.38 (bs, 1H, aromatic proton of fluorescein), 8.15 (dd, 1H, J=1.7, J=8.3, aromatic proton of fluorescein) 7.30(d, 1H, J=8.3, aromatic proton of fluorescein), 6.87 (m, 4H, aromatic protons of fluorescein), 6.72 (dd, 2H, J=2.4, J=8.8, aromatic protons of fluorescein), 5.50 (m, 4H, 2(CHCH), 5,38 (m, 1H, OCH.sub.2CHOCH.sub.2O), 4.58 (dd, 1H, J=6.6, J.sub.gem=11.8, HHCOC(O)), 4.34 (dd, 1H, J=3.2, J.sub.gem=11.8, HHCOC(O)), 4.14 (m, 2H, OCHCH.sub.2OP) (4.1 (m, 2H, POCH.sub.2CH.sub.2NH) 3.80 (m, 2H, NCH.sub.2(CH.sub.2).sub.3CH.sub.2NHCS) 3.39 and 3.58 (2m, 22H, NCH.sub.2CH.sub.2OP and NCH.sub.2(CH.sub.2).sub.3CH.sub.2NHCS) 2.48 (m, 4H, 2(CH.sub.2CO), 2.39 (m, 4H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO), 2.19 (m, 8H, 2(CH.sub.2CHCHCH.sub.2), 1.84 (m, 2H, CH.sub.2 fluorescein cadaverine), 1.8 (m, 10H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO, 2(COCH.sub.2CH.sub.2, and CH.sub.2 fluorescein cadaverine), 1.62 (m, 2H, CH.sub.2 fluorescein cadaverine) 1,42, 1.46 (2 bs, 40H, 20 CH.sub.2), 1.05 (m, 6H, 2 CH.sub.3).

    [0069] Association of RODE-fluorescein (I) With Cell Membranes

    [0070] KODE-fluorescein (I) readily associates with the membrane of red blood cells. Insertion of the molecule is observed when dispersions of the molecule at concentrations greater than 0.1 mg/ml are contacted with suspensions of the red blood cells.

    [0071] A medium to strongly fluorescing cell was considered to indicate a uniform distribution of the molecule across the cell membrane (FIG. 1). The incorporation and distribution appears to be stable for a period of at least 40 days when cells are stored in the dark.

    EXAMPLE 2

    [0072] Activated 1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) (L-A) was prepared as described in Example 1.

    [0073] Condensation of DOPE-A with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylenediamine, hydrochloride (BODIPY FL EDA)

    [0074] To a solution of 15 mg (15.5 mol) activated DOPE in CH.sub.2Cl.sub.2 (0.5 ml), 5 mg (13.5 mol) of BODIPY FL EDA in N,N-dimethylformamide (0.3 ml) and 5 l of triethylamine were added. The mixture was kept for 2 h at room temperature.

    [0075] Column chromatography (Sephadex LH-20, 1:1 chloroform-methanol) of the mixture yielded 14.2 mg (75%) KODE-BODIPY (I), Et.sub.3N-salt; MW 1289.6, R.sub.f 0.3 (ethyl acetate-isopropanol-water, 6:3:1).

    [0076] .sup.1H NMR (CDCl.sub.3/CD.sub.3OD, 1:1): 7.40 (s, 1H, aromatic proton of BODIPY), 7.12 (d, 1H, J==3.8 aromatic proton of BODIPY), 6.47 (d, 1H, J=3.8 aromatic proton of BODIPY), 6.32 (s, 1H, aromatic protons of BODIPY), 5.50 (m, 4H, 2(CHCH), 5.38 (m, 1H, OCH.sub.2CHOCH.sub.2O), 4.58 (dd, 1H, J=3.2, J.sub.gem=11.8, HHCO(O)), 4.33 (dd, 1H, J=6.6, J=11.8, HHCOC(O)), 4.16 (t, 2H, J=5.6, POCH.sub.2CH.sub.2NH), 4.1 (m, 2H, OCHCH.sub.2OP), 3.60 (t, 2H, POCH.sub.2CH.sub.2NH), 3.46, 3.42 and 2.8 (3m, 4H, 2H, 2H, CH.sub.2CH.sub.2C(O)NH(CH.sub.2).sub.2NH of BODIPY), 2.70 (s, 3H, CH.sub.3 of BODIPY), 2.48 (m, 4H, 2(CH.sub.2CO), 2.45 (s, 3H, CH.sub.3 of BODIPY), 2.37 (m, 4H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO), 2.19 (m, 8H, 2(CH.sub.2CHCHCH.sub.2), 1.8 (m, 8H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO, 2(COCH.sub.2CH.sub.2)), 1.46, 1.43 (2 bs, 40H, 20 CH.sub.2), 1.05 (m, 6H, 2 CH.sub.3); 3.31 (q, 6H, J=7.4, 3CH.sub.2 of Et.sub.3N), 1.50 (t, 9H, J=7.4, 3CH.sub.3 of Et.sub.3N).

    [0077] Although the invention has been described by way of exemplary embodiments it should be appreciated that variations and modifications may be made with out departing from the scope of the invention. Furthermore where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in this specification.