Fluorescent red emitting functionalizable calcium indicators

Abstract

Compounds of formula I ##STR00001##
and a process for manufacturing the compounds. A method of using the compounds for the detection of calcium ions and a method of detecting intracellular calcium are also described.

Claims

1. A compound of formula I ##STR00053## or a salt thereof, wherein Z represents H, alkyl, CH.sub.2—OAc, Na.sup.+ or K.sup.+; R.sup.1, R.sup.2, R.sup.3 and R.sup.4 represent each independently H, halo, alkyl, COR.sup.11, OR.sup.11, SR.sup.11 or NR.sup.11R.sup.12, wherein R.sup.11 and R.sup.12 represent each independently H, alkyl or aryl; m represents 0, 3 or 4; W represents O, NR.sup.9, S or CR.sup.9R.sup.10, wherein R.sup.9 and R.sup.10 represent each independently H or alkyl; L represents a single bond or a linker selected from the group comprising alkyl, aryl, alkylaryl, arylalkyl, polyethylene glycol (PEG), polypropylene glycol (PPG), peptide, aminocarbonyl, alkylaminocarbonyl, aminothiocarbonyl or a combination thereof; optionally additionally comprising a residue of a reactive group through which L is bounded to Y selected from carbonyl group or triazolo group; Y represents a reactive function selected from the group comprising N.sub.3, amino, alkylamino, COOH, amide, maleimide, alkyne, SH, OH, ester, N-hydroxysuccinimide ester, N-hydroxyglutarimide ester, maleimide ester, acid anhydride, acid halide, halo, nitro, nitrile, isonitriles, acrylamide, aldehyde, ketone, acetals, ketals, anhydride, glutaric anhydride, succinic anhydride, maleic anhydride, thiocyanate, isothiocyanate, isocyanate, hydrazide, hydrazines, hydrazones, ethers, oxides, cyanates, diazo, diazonium, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids, sulfates, sulfenic acids, amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamic acids thiohydroxamic acids, allenes, ortho esters, sulfites, enamines, ynamines, ureas, pseudoureas, semicarbazides, carbodiimides, carbamates and imines; or a bioactive group selected from amino acid, peptide, protein, antibody, enzyme, polysaccharide, dextran, benzylguanine, lipid, lipid assembly, fatty acid, nucleoside, nucleotide, oligonucleotide, hapten, aptamer, biotin, avidin, synthetic polymer, polymeric microparticle, nanoparticle, fluorophore, chromophore, radioisotope, macrocyclic complexes of radioisotope, and combinations thereof; R.sup.5 and R.sup.6 each independently represent H, alkyl or halo; R.sup.7 and R.sup.8 each independently represent H, alkyl; or R.sup.5 and R.sup.7 are linked together in a single alkyl moiety to form a ring with adjacent carbon and nitrogen atoms; or R.sup.6 and R.sup.8 are linked together in a single alkyl moiety to form a ring with adjacent carbon and nitrogen atoms; and X represents O, NR.sup.9, S, CR.sup.9R.sup.10, Se or Si, wherein R.sup.9 and R.sup.10 represent each independently H or alkyl.

2. The compounds according to claim 1, wherein, when R.sup.5 and R.sup.7, or R.sup.6 and R.sup.8, are linked together in a single alkyl moiety, the single alkyl moiety is propyl.

3. The compound according to claim 1, of formula Ia ##STR00054## or a salt thereof, wherein W, L, Y, R.sup.5, R.sup.6, R.sup.7, R.sup.8 and X are as previously defined.

4. The compound according to claim 1, of formula Ib ##STR00055## or a salt thereof, wherein W, L, Y, R.sup.5, R.sup.6, R.sup.7, R.sup.8 and X are as previously defined.

5. The compound according to claim 1, selected from the group comprising compounds of formula Ia-1, Ia-2 and Ib-1: ##STR00056## ##STR00057##

6. Process for manufacturing a compound of formula I according to claim 1, comprising performing a Vilsmeier-Haack reaction on a compound of formula III ##STR00058## wherein Z, R.sup.1, R.sup.2, R.sup.3, R.sup.4, m, W, L and Y are as previously defined; leading to compound of formula II ##STR00059## wherein Z, R.sup.1, R.sup.2, R.sup.3, R.sup.4, m, W, L and Y are as previously defined; and further comprising a step of rhodamine formation on the aldehyde function of compound of formula II, to afford compound of formula I.

7. The compound according to claim 2, of formula Ia ##STR00060## or a salt thereof, wherein W, L, Y, R.sup.5, R.sup.6, R.sup.7, R.sup.8 and X are as previously defined.

8. Method of detecting intracellular calcium comprising: adding a compound according to claim 1 to a sample containing at least one cell; incubating the sample for a time sufficient for the compound to be loaded into the cell; illuminating the sample at an exciting wavelength that generates a fluorescent response from the indicator; detecting the fluorescent response.

9. Method according to claim 8, further comprising: stimulating the cell; monitoring changes in the intensity of the fluorescent response from the indicator; and correlating the changes in fluorescence intensity with changes in intracellular calcium levels.

10. Kit for performing a calcium assay, comprising a compound according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a drawing schematically representing the red-emitting calcium indicator of the invention.

(2) FIG. 2: Normalized absorption and emission spectra of Ia-1 (5 μM in water, 30 mM MOPS, 100 mM KCl, pH 7.2).

(3) FIG. 3: Absorption spectra of Ia-1 (5 μM) in presence of different concentration of calcium (30 mM MOPS, 100 mM KCl, pH 7.2).

(4) FIG. 4: Emission spectra of Ia-1 (5 μM) in presence of different concentration of calcium (30 mM MOPS, 100 mM KCl, pH 7.2).

(5) FIG. 5: Fluorimetric titration of Ia-1 (5 μM) against Ca.sup.2+ in a buffer containing (in mM) 100 KCl and 30 MOPS (pH 7.2).

(6) FIG. 6: Fluorimetric titration of Ia-1-Dextran-6000 conjugate against Ca.sup.2+ in a buffer containing (in mM) 100 KCl and 30 MOPS (pH 7.2).

(7) FIG. 7: Normalised fluorimetric titrations against Ca.sup.2+ of Ia-1 (diamond points) and its dextran-6000 conjugate Ia-1-Dextran-6000 (triangular points).

(8) FIG. 8: Two-photon excitation of Ia-1. a) Two-photon excitation spectrum, average of five independent measurements. The individual measurements are indicated by grey circles. b) Plot of fluorescence intensity vs. excitation power. The individual measurements are indicated by grey circles; power function fit to the data (black line).

(9) FIG. 9: Imaging Ca.sup.2+ in layer 2/3 neurons in vivo. a) A layer 2/3 pyramidal neuron filled with Alexa Fluor 488 and Ia-1-Dextran-6000. b) Action potential (AP) c) Ca.sup.2+ transients.

(10) FIG. 10: Imaging Purkinje cells in vivo using bulk loading of Ia-1. a) Configuration of AM-ester (Ib-1) injection and imaging. b) Resulting staining of tissue 60 minutes after injection of indicator. c) Active Purkinje cell dendrites identified using a spatial PCA/ICA algorithm. d) Fluorescence traces from the identified dendrites. e) Stimulus triggered averages of the complete traces in d (20 repetitions).

(11) FIG. 11: [Ca.sup.2+] transients in cerebellar slices imaged with Ia-1. a-c) Parallel fiber stimulation. a) Purkinje cell filled with 100 μM Alexa Fluor 488 and 200 μM Ia-1-Dextran via patch-pipette (scale bar: 25 μm). The white arrow head marks the stimulation site. b) Segment of dendrite showing the region imaged in c using a line scan. c) Voltage trace (top) showing spontaneous spiking of the Purkinje cell with a parallel fiber stimulus evoked increase in spiking frequency. Stimulus evoked Ca.sup.2+ transients (bottom) at s1, s2 and d, the two spines and the underlying dendritic shaft, respectively (average of three trials). Stimulus timing is indicated at the bottom of the traces. d-f) Climbing fiber stimulation. d) Purkinje cell filled with 300 μM Ia-1, with region of interest indicated by yellow rectangle. e) Region of interest with measurement points indicated. Note that points 1-3 and 4-6 are on different spiny branchlets while points 7 and 8 are in the background. f) Ca.sup.2+ transients following climbing fiber activation recorded at 2.8 kHz (traces averaged over 26 stimulations).

(12) FIG. 12: Dual color functional imaging in vitro and in vivo. a-c) Combined imaging of [Glutamate] and [Ca.sup.2+] a) A Purkinje cell expressing iGluSnFR was filled with 200 μM Ia-1-Dextran. The image shows the basal fluorescence of Ia-1. b) Double pulse stimulation of the climbing fiber triggers spatially different patterns of glutamate release and Ca.sup.2+ influx (maximum dF/F.sub.0 images; the inset at the top shows the two evoked complex spikes). Note the breaks between regions showing iGluSnFR activation (indicated by white arrows) c) Fluorescence traces for Ia-1 and iGluSnFR following single pulse climbing fiber stimulation (top inset). Note the absence of a fluorescent transient for iGluSnFR in the “Ca.sup.2+ only” region. d-f) Odor-evoked calcium responses in olfactory bulb glomeruli. d) Juxtaglomerular neurons and mitral cell dendritic tufts expressing YFP demarcate glomeruli in a Kv3.1-eYFP mouse. e) Olfactory sensory neuron glutamatergic terminals, labeled with Ia-1-Dextran, clearly filled the inner boundaries of most glomeruli (Red channel). f) A 3 s application of 30% isoamyl acetate reliably triggered presynaptic calcium responses in several glomeruli.

(13) FIG. 13: Normalised fluorimetric titrations against Ca.sup.2+ of Ia-1, Ic-1 and Ic-2.

EXAMPLES

(14) The present invention is further illustrated by the following examples.

(15) I. Synthesis

(16) I.1. Materials and General Methods

(17) All the solvents were of analytical grade. Chemicals were purchased from commercial sources. .sup.1H-NMR and .sup.13C-NMR were measured on a Bruker avance 111-300 MHz spectrometer with chemical shifts reported in ppm (TMS as internal standard). Mass spectra were measured on a Focus GC/DSQ II spectrometer (ThermoScientific) for IC and an API 3000 spectrometer (Applied Biosystems, PE Sciex) for ES. All pH measurements were made with a Mettler Toledo pH-Meter. Fluorescence spectra were recorded on a JASCO FP-8300 spectrofluorometer. Absorption spectra were determined on a VARIAN CARY 300 Bio UV-Visible spectrophotometer. All measurements were done at a set temperature of 25° C. The purity of the dyes were checked by RP-HPLC C-18, eluant: ACN 0.1% TFA/Water 0.1% TFA, method: 20/80 to 100/0 within 20 min then 100/0 for 10 min. detection at λ.sub.Abs=254 nm. The apparent dissociation constant for calcium (Kd Ca.sup.2+) was measured with a calcium calibration buffer kit from Invitrogen.

(18) I.2. Synthesis of Compound Ia-1 and Ib-1

(19) ##STR00021## ##STR00022##

(20) ##STR00023##

(21) To a solution of 5-fluoro-2-nitrophenol (14.90 g, 94.84 mmol) in DMF (75 mL) were added dibromoethane (40.90 mL, 472.2 mmol, 5 eq) and K.sub.2CO.sub.3 (26.30 g, 189.7 mmol, 2 eq), the mixture was allowed to stir at 70° C. for 2 h. The solvents were evaporated and the product was extracted with EtOAc, washed with water (3 times) and brine (2 times). The organic phase was dried over MgSO.sub.4, filtered and evaporated to reach a volume of 200 mL. The symmetric dinitro compound crystallizes first and was filtered off. The filtrate was then allowed to crystallize to obtain 20.12 g of 1 (80%) as a yellow powder. .sup.1H-NMR (300 MHz, DMSO-d6): δ 8.04 (dd, J.sub.a-b=9.1 Hz, J.sub.a-F=6.1 Hz, 1H, H.sub.a), 7.37 (dd, L.sub.c-F=11.0 Hz, J.sub.c-b=2.6 Hz, 1H, H.sub.c), 7.02 (ddd, J.sub.b-a=9.1, J.sub.b-F=7.8 Hz, J.sub.b-c=2.6 Hz, 1H, H.sub.b), 4.56-4.53 (m, 2H, CH.sub.2O), 3.84-3.81 (m, 2H, CH.sub.2Br). .sup.13C-NMR (75 MHz, DMSO-d6): δ 164.82 (d, .sup.1J.sub.F-C=251 Hz, CF), 152.81 (d, .sup.3J.sub.C-F=12 Hz, CO), 136.17 (d, .sup.4J.sub.F-C=3 Hz, CNO.sub.2), 127.62 (d, .sup.3J.sub.F-C=11 Hz, C.sub.a), 108.01 (d, .sup.2J.sub.F-C=23 Hz, C.sub.b), 103.45 (d, .sup.2J.sub.F-C=27 Hz, C.sub.c), 69.78 (CH.sub.2O), 30.39 (CH.sub.2Br). MS (CI), calcd for C.sub.8H.sub.11BrFN.sub.2O.sub.3 [M+NH.sub.4].sup.+280.9, found 281.0.

(22) ##STR00024##

(23) To a solution of 1 (19.79 g, 74.96 mmol) in DMF (75 mL) were added 2-nitrophenol (11.46 g, 82.45 mmol, 1.1 eq) and K.sub.2CO.sub.3 (15.63 g, 112.4 mmol, 1.5 eq), the mixture was allowed to stir overnight at 70° C. The solvent was evaporated and the product was extracted with DCM, washed with HCl (1M) and brine (2 times). The organic phase was dried over MgSO.sub.4, filtered and evaporated to reach a volume of 200 mL. The product crystallized and was filtered to obtain 12.00 g of 2 (50%) as a yellow powder. .sup.1H-NMR (300 MHz, DMSO-d6): δ 8.01 (dd, J.sub.a-b=9.1 Hz, J.sub.a-F=6.1 Hz, 1H, H.sub.a), 7.86 (dd, J.sub.g-f=8.1 Hz, J.sub.g-e=1.6 Hz, 1H, H.sub.g), 7.67 (ddd, .sup.3J=8.5, 7.4, .sup.4J.sub.e-g=1.7 Hz, 1H, H.sub.e), 7.45-7.39 (m, 2H, H.sub.c, H.sub.d), 7.15 (ddd, .sup.3J=8.1 Hz, 7.4 Hz, .sup.4J=1.1 Hz, 1H, H.sub.f), 7.01 (ddd J.sub.b-a=9.1, J.sub.b-F=7.8 Hz, J.sub.b-c=2.6 Hz, 1H, H.sub.b), 4.59-4.54 (m, 4H, 2CH.sub.2O). .sup.13C-NMR (75 MHz, DMSO-d6): δ 164.86 (d, .sup.1J.sub.F-C=251 Hz, CF), 153.34 (d, .sup.3J.sub.C-F=11.9 Hz, CO), 150.82 (Cq Ar), 139.74 (Cq Ar), 136.15 (d, .sup.4J.sub.F-C=3.7 Hz, CNO.sub.2), 134.33 (C.sub.e), 127.58 (d, .sup.3J.sub.F-C=11 Hz, C.sub.a), 124.85 (C.sub.g), 121.05 (C.sub.f), 115.55 (C.sub.d), 107.90 (d, .sup.2J.sub.F-C=24 Hz, C.sub.b), 103.57 (d, .sup.2J.sub.F-C=27.7 Hz, C.sub.e), 68.57 (CH.sub.2O), 67.93 (CH.sub.2O). MS (ES+), calcd for C.sub.14H.sub.11FN.sub.2O.sub.6Na [M+Na].sup.+ 345.0, found 345.3. HRMS (ES+), calcd for C.sub.14H.sub.11FN.sub.2O.sub.6Na [M+Na].sup.+ 345.0493, found 345.0501.

(24) ##STR00025##

(25) To a stirred solution of 2 (5.91 g, 18.34 mmol) in DMSO (53 mL) was added NaOH 20% (11.5 mL) the solution turned yellow and was allowed to stir at room temperature overnight. 50 mL of water and 10 mL HCl (1M) were then added and the product was extracted 3 times with EtOAc. The organic phase was washed 3 times with water before being dried over MgSO.sub.4, the solution was filtered and evaporated and crystallized in EtOAc to obtain 4.46 g of 3 (76%) as a yellow powder. .sup.1H-NMR (300 MHz, DMSO-d6): δ 7.90-7.85 (m, 2H, H.sub.a, H.sub.g), 7.67-7.64 (dd, .sup.3J=8.7 Hz, .sup.4J=1.5 Hz, 1H, H.sub.e), 7.48 (d, .sup.3J=8.4 Hz, 1H, H.sub.d), 7.16 (t, .sup.3J=7.7 Hz, 1H, H.sub.f), 6.66 (d, .sup.4J=2.2 Hz, 1H, H.sub.e), 6.51 (dd, .sup.3J=9.0, .sup.4J=2.2 Hz, 1H, H.sub.b), 4.55-4.54 (m, 2H, CH.sub.2O), 4.45 (t, J=3.8 Hz, 2H, CH.sub.2O). .sup.13C-NMR (75 MHz, DMSO-d6): δ 163.87 (Cq Ar), 154.51 (Cq Ar), 150.98 (Cq Ar), 139.79 (Cq Ar), 134.36 (C.sub.e), 131.12 (Cq Ar), 128.18 (C.sub.a or C.sub.g), 124.86 (C.sub.a or C.sub.g), 121.03 (C.sub.f), 115.75 (C.sub.d), 108.01 (C.sub.b), 101.56 (C.sub.c), 68.06 (CH.sub.2O), 67.87 (CH.sub.2O). MS (CI), calcd for C.sub.14H.sub.16N.sub.3O.sub.7 [M+NH.sub.4].sup.+338.0, found 337.7. HRMS (ES+), calcd for C.sub.14H.sub.13N.sub.2O.sub.7[M+H].sup.+ 321.0717, found 321.0722.

(26) ##STR00026##

(27) To a solution of 3 (4.86 g, 15.19 mmol) in DMF (50 mL) were added dibromohexane (11.12 mL, 45.56 mmol, 3 eq) and K.sub.2CO.sub.3 (3.16 g, 22.78 mmol, 1.5 eq). The mixture was allowed to stir at 70° C. for 12 h. The solvents were evaporated and the product was extracted with EtOAc washed with water (3 times) and brine (2 times). The organic phase was dried over MgSO.sub.4, filtered and evaporated. The crude was purified by column chromatography on silica gel (Cyclohexane/EtOAc: 7/3) to obtain the crude 4 which was crystallized in a mixture of EtOAc and cyclohexane (3/7) to obtain 2.97 g of pure 4 (40%) as a off white powder. Rf=0.22 (Cyclohexane/EtOAc, 7/3). .sup.1H-NMR (300 MHz, CDCl.sub.3): δ 8.00 (d, J=9.1 Hz, 1H, H.sub.a), 7.86 (dd, J=8.1, 1.6 Hz, 1H, H.sub.g), 7.64-7.58 (m, 1H, H.sub.e), 7.33 (dd (in solvent peak), 1H, Hd), 7.14-7.09 (m, 1H, H.sub.f), 6.66 (d, J=2.4 Hz, 1H, H.sub.c), 6.57 (dd, J=9.1, 2.4 Hz, 1H, H.sub.b), 4.60-4.51 (m, 4H, 2CH.sub.2O), 4.08 (t, J=6.4 Hz, 2H, CH.sub.2O), 3.47 (t, J=6.7 Hz, 2H, CH.sub.2Br), 1.96-1.85 (m, 4H, 2CH.sub.2), 1.56 (dt, J=7.1, 3.5 Hz, 4H, 2CH.sub.2). .sup.13C-NMR (75 MHz, CDCl.sub.3): δ 164.35 (Cq), 154.63 (Cq), 151.96 (Cq), 140.43 (Cq), 134.37 (C.sub.e), 133.34 (Cq), 128.32 (C.sub.a), 125.56 (C.sub.g), 121.43 (C.sub.f), 116.14 (C.sub.d), 106.89 (C.sub.b), 102.02 (C.sub.c), 68.83 (CH.sub.2O), 68.70 (CH.sub.2O), 68.65 (CH.sub.2O), 33.81 (CH.sub.2Br), 32.63 (CH.sub.2), 28.85 (CH.sub.2), 27.86 (CH.sub.2), 25.19 (CH.sub.2). MS (ES+), calcd for C.sub.20H.sub.23BrN.sub.2O.sub.7Na [M+Na].sup.+ 505.0, found 505.5. HRMS (ES+), calcd for C.sub.20H.sub.24BrN.sub.2O.sub.7 [M+H].sup.+ 483.0767, found 483.0772.

(28) ##STR00027##

(29) To a solution of 4 (5.00 g, 10.35 mmol) in EtOAc (100 mL) and methanol (30 mL) was added Pd/C (1.10 g). The solution was stirred and degassed before H.sub.2 was allowed to bubble in the solution for 5 h. The solution was then filtered off celite and rinsed with EtOAc under an atmosphere of argon. The solvents were evaporated and the residue was dissolved in acetonitrile (50 mL), to this solution were added, methyl bromoacetate (12.0 mL, 124.2 mmol, 12 eq) and DIEA (23.0 mL, 124.2 mmol, 12 eq) before being warmed up to 80° C. The solution was allowed to stir overnight at 80° C. The solvents were evaporated, the product was extracted with dichloromethane (DCM) and the organic layer washed with water, was dried over MgSO.sub.4, filtered and evaporated. The crude was purified by column chromatography on silica gel (Cyclohexane/EtOAc: 7/3) to give 3.71 g of 5 (50%) as a yellowish syrup containing some impurities (visible between 2 and 3 ppm in .sup.1H NMR) that could not be removed at this stage. Rf=0.51 (Cyclohexane/EtOAc, 6/4). .sup.1H-NMR (300 MHz, CDCl.sub.3): δ 6.85-6.74 (m, 5H), 6.39 (d, J=2.7 Hz, 1H), 6.31 (dd, J=8.7, 2.7 Hz, 1H), 4.20 (m, 4H, CH.sub.2O), 4.08 (s, 4H, 2CH.sub.2N), 4.02 (s, 4H, 2CH.sub.2N), 3.81 (t, J=6.4 Hz, 2H, CH.sub.2O), 3.50 (d, J=7.4 Hz, 12H, 4 OMe), 3.36 (t, J=6.8 Hz, 2H, CH.sub.2Br), 1.85-1.80 (m, 2H, CH.sub.2), 1.71-1.67 (m, 2H, CH.sub.2), 1.42 (t, J=3.6 Hz, 4H, 2 CH.sub.2). MS (ES+), calcd for C.sub.32H.sub.43BrN.sub.2O.sub.11Na [M+Na].sup.+735.2, found 735.8.

(30) ##STR00028##

(31) To a solution of 5 (3.71 g, 5.218 mmol) in DMF (10 mL) was added NaN.sub.3 (1.02 g, 15.65 mmol, 3 eq). The solution was stirred at 80° C. overnight. The product was extracted with EtOAc and washed with water (3 times) and brine (2 times), the organic phase was dried over MgSO.sub.4, filtered and concentrated to give 3.52 g of 6 (quant) as a yellowish syrup. .sup.1H-NMR (300 MHz, CDCl.sub.3): δ 6.84-6.73 (m, 5H), 6.39 (d, J=2.5 Hz, 1H), 6.31 (dd, J=8.7, 2.5 Hz, 1H), 4.19 (d, 4H, CH.sub.2O), 4.08 (s, 4H, 2CH.sub.2N), 4.01 (s, 4H, 2CH.sub.2N), 3.81 (t, J=6.4 Hz, 2H, CH.sub.2O), 3.48 (d, J=7.3 Hz, 12H, 4 OMe), 3.20 (t, J=6.8 Hz, 2H, CH.sub.2N.sub.3), 1.70-1.64 (m, 2H, CH.sub.2), 1.60-1.51 (m, 2H, CH.sub.2), 1.38 (m, 4H, 2 CH.sub.2). Impurities between 2 and 3 ppm could not be removed. MS (ES+), calcd for C.sub.32H.sub.44N.sub.5O.sub.11 [M+H].sup.+ 674.3, found 674.3. HRMS (ES+), calcd for C.sub.32H.sub.44N.sub.5O.sub.11 [M+H].sup.+ 674.3032, found 674.3054.

(32) ##STR00029##

(33) To a solution of 6 (1.22 g, 1.81 mmol) in DMF (5 mL) was added POCl.sub.3 (1.35 mL, 14.48 mmol, 8 eq) dropwise without cooling. After addition the solution was allowed to stir for 40 min and then water (50 mL) was added followed by slow addition of a saturated solution of NaHCO.sub.3 to reach a pH of 8. The product was extracted with DCM and washed twice with brine before being dried over MgSO.sub.4 filtrated and evaporated. The crude was purified by column chromatography on silica gel (Cyclohexane/EtOAc: 6/4) to give 505 mg of 7 (40%) as a yellow syrup. Rf=0.25 (Cyclohexane/EtOAc, 5/5). .sup.1H-NMR (300 MHz, CDCl.sub.3): δ 10.23 (s, 1H, CHO), 7.27 (s, 1H, Ha), 6.86-6.75 (m, 4H, Hd, He, Hf, Hg), 6.39 (s, 1H, Hc), 4.26 (d, J=2.4 Hz, 4H, 2CH.sub.2O), 4.06 (d, J=3.3 Hz, 4H, 2CH.sub.2N), 4.02 (d, J=5.8 Hz, 4H, 2CH.sub.2N), 3.96 (t, J=6.3 Hz, 2H, CH.sub.2O), 3.49 (2s, 12H, 2 OMe), 3.22 (t, J=6.8 Hz, 2H, CH.sub.2N.sub.3), 1.77 (t, J=7.1 Hz, 2H, CH.sub.2), 1.57 (t, J=7.0 Hz, 2H, CH.sub.2), 1.45-1.35 (m, 4H, CH.sub.2). .sup.13C-NMR (75 MHz, CDCl.sub.3): δ 187.99 (CHO), 171.94 (COOMe), 171.64 (COOMe), 158.88 (Cq Ar), 157.35 (Cq Ar), 150.21 (Cq Ar), 139.40 (Cq Ar), 133.40 (Cq Ar), 122.44 (CH Ar), 121.86 (CH Ar), 119.19 (CH Ar), 118.28 (Cq Ar), 118.07 (Ca), 113.45 (CH Ar), 97.79 (Cc), 68.93 (CH.sub.2O), 67.53 (CH.sub.2O), 66.81 (CH.sub.2O), 53.36 (2CH.sub.2N), 53.32 (2CH.sub.2N), 51.69 (OMe), 51.65 (OMe), 51.35 (CH.sub.2N.sub.3), 30.19 (CH.sub.2), 29.07 (CH.sub.2), 28.82 (CH.sub.2), 26.92 (CH.sub.2), 26.50 (CH.sub.2), 25.70 (CH.sub.2). MS (ES+), calcd for C.sub.33H.sub.44N.sub.5O.sub.12 [M+H].sup.+ 702.3, found 702.2. HRMS (ES+), calcd for C.sub.33H.sub.44N.sub.5O.sub.12 [M+H].sup.+ 702.2981, found 702.3008.

(34) The position of the carbonyl was confirmed by further NMR investigations using a HMBC (Heteronuclear Multiple Bond Correlation) experiment.

(35) Numbering for X-Rhodamines

(36) ##STR00030##

(37) To a solution of aldehyde 7 (300 mg, 0.428 mmol) in propionic acid (5 mL) was added 8-hydroxyjulolidine (161 mg, 0.856 mmol, 2 eq) and PTSA (8 mg, 0.042 mmol, 0.1 eq). The solution was protected from light and stirred at room temperature overnight. To the brown mixture was added a solution of chloranil (103 mg, 0.428 mmol, 1 eq) in DCM (10 mL), the reaction turned dark and was allowed to stir overnight at room temperature. The dark purple solution was evaporated to dryness. The residue was purified by column chromatography on silica gel (gradient of 100% DCM to 9/1 DCM/Methanol) to obtain 130 mg of 8 (30%) as a purple solid after lyophilisation (dioxane/water: 1/1). Rf=0.32 (DCM/MeOH, 9/1). .sup.1H-NMR (300 MHz, CDCl.sub.3): δ 7.84 (d, J=8.1 Hz, 1H, H Ar), 7.06 (d, J=7.9, 1H, H Ar), 6.97-6.86 (m, 5H, H Ar, H.sub.7), 6.71 (d, J=2.9 Hz, 1H, H Ar), 4.47-4.40 (m, 4H, CH.sub.2O), 4.21 (s, 4H, NCH.sub.2COOMe), 4.11 (s, 4H, NCH.sub.2COOMe), 3.87 (t, J=6.1 Hz, 2H, CH.sub.2O), 3.67 (s, 6H, 2 OMe), 3.56 (m, 14H, 2 OMe, H.sub.1, H.sub.4), 3.11 (d, J=7.0 Hz, 2H, CH.sub.2N.sub.3), 3.04 (t, J=6.3 Hz, 4H, H.sub.6), 2.75 (q, J=6.2 Hz, 4H, H.sub.3), 2.13-2.10 (m, 4H, H.sub.5), 2.00 (t, J=5.5 Hz, 4H, H.sub.2), 1.49-1.34 (m, 4H, CH.sub.2), 1.19-1.03 (m, 4H, CH.sub.2). .sup.13C-NMR (75 MHz, CDCl.sub.3): δ 171.97 (CO ester), 171.56 (CO ester), 153.04 (C Ar), 152.74 (C Ar), 152.31 (C Ar), 152.09 (C Ar), 151.02 (C Ar), 150.43 (C Ar), 144.79 (C Ar), 139.41 (C Ar), 138.16 (C Ar), 132.61 (C Ar), 128.20 (CH Ar), 127.15 (CH Ar), 126.33 (CH Ar), 123.34 (C Ar), 122.64 (CH Ar), 122.61 (CH Ar), 121.91 (CH Ar), 119.54 (CH Ar), 113.89 (C Ar) (CH Ar), 113.43 (C Ar), 113.35 (C Ar), 105.16 (C Ar), 69.10 (CH.sub.2O), 67.70 (CH.sub.2O), 67.19 (CH.sub.2O), 53.66 (NCH.sub.2COOMe), 53.52 (NCH.sub.2COOMe), 51.73 (4 OMe), 51.16 (CH.sub.2N.sub.3), 50.97 (C.sub.1 or C.sub.4), 50.52 (C.sub.1 or C.sub.4), 28.82 (CH.sub.2), 28.73 (CH.sub.2), 27.72 (C.sub.3), 26.26 (CH.sub.2), 25.52 (CH.sub.2), 20.83 (C.sub.2), 20.00 (C.sub.6), 19.85 (C.sub.5). MS (ES+), calcd for C.sub.57H.sub.68N.sub.7O.sub.12 [M].sup.+1042.5, found 1042.9. HRMS (ES+), calcd for C.sub.57H.sub.68N.sub.7O.sub.12 [M].sup.+1042.4920, found 1042.4949.

(38) ##STR00031##

(39) To a solution of 8 (100 mg, 0.090 mmol) in methanol (6 mL) were added, KOH (504 mg, 9.00 mmol, 100 eq) followed by 2 mL of water, the mixture was stirred overnight. The solution was diluted with aq HCl (1M) and extracted with CHCl.sub.3 until the aqueous phase became slightly pink. The organic phase was then dried over MgSO.sub.4, filtered and concentrated. The residue was purified on a reverse phase column C-18 using acetonitrile (0.1% TFA) and water (0.1% TFA) mixture as eluant (20% acetonitrile to 60%). The solvents were evaporated and 80 mg of Ia-1 (˜90%) were obtained as a purple solid after lyophilisation (dioxane/water, 1/1). MS (ES+), calcd for C.sub.53H.sub.60N.sub.7O.sub.12 [M].sup.+986.4, found 986.4. HRMS (ES+), calcd for C.sub.53H.sub.60N.sub.7O.sub.12 [M].sup.+986.4294, found 1042.4329.

(40) ##STR00032##

(41) To a solution of Ia-1 (50 mg, ˜50 μmol) in chloroform were added bromomethyl acetate (80 μL, 500 μmol, 1 eq) and NEt.sub.3 (60 μL, 400 μmol, 8 eq). The solution was protected from light and allowed to stir at room temperature overnight. The reaction was monitored by TLC (DCM/MeOH, 9/1). The solvents were evaporated and the crude was purified by column chromatography on silica gel (gradient of 100% DCM to 9/1 DCM/Methanol) to obtain 30 mg of Ib-1 (˜45%) as a purple solid after lyophilisation (dioxane/water, 1/1). Rf=0.45 (DCM/MeOH, 9/1). MS (ES+), calcd for C.sub.65H.sub.76N.sub.7O.sub.20 [M].sup.+1274.5, found 1274.5. HRMS (ES+), calcd for C.sub.65H.sub.76N.sub.7O.sub.20 [M].sup.+1274.5140, found 1274.5128.

(42) I.3. Synthesis of Dextran Conjugates Ia-1-Dextran

(43) Dextran 6,000 MW (Sigma-Aldrich, ref: 31388) and dextran 1,500 MW (Sigma-Aldrich, ref: 31394) were propargylated as described by Nielsen et al. (Nielsen et al., Biomacromolecules, 2010, 11, 1710-1715). The .sup.1H-NMR showed that the functionalized dextrans were propargylated once evry glucose unit.

(44) Final MW Dextran 6,000: ˜9,800 g.Math.mol.sup.−1

(45) Final MW Dextran 1,500: ˜2,400 g.Math.mol.sup.−1

(46) Conjugation of Dextran 6,000.

(47) To a solution of propargylated dextran 6,000 (30 mg, ˜3 μmol) in water (3 mL) was added Ia-1 (8 mg, 8 μmol, 2.6 eq) in methanol (1 mL) and an heterogeneous solution of CuSO.sub.4.5H.sub.2O (4 mg, 16 μmol, 5.3 eq) and sodium ascorbate (4 mg, 20 μmol, 6.6 eq) in water (500 μL). The solution was allowed to stir in the dark at room temperature overnight. The solvents were evaporated and the residue was dissolved in 1 mL of EDTA solution (0.1 M) and eluted through a G-25 column (eluant water) to give 24 mg of Ia-1-Dextran 6,000 conjugate (˜60% yield).

(48) Conjugation of Dextran 1,500.

(49) To a solution of propargylated dextran 1500 (30 mg, ˜12.5 μmol) in DMF (1 mL) was added Ia-1 (4.5 mg, 4.5 μmol, 0.3 eq) in DMF (200 μL) and a heterogeneous solution of CuSO.sub.4.5H.sub.2O (4 mg, 16 μmol, 1.3 eq) and sodium ascorbate (4 mg, 20 μmol, 1.6 eq) in water (100 μL). The solution was allowed to stir in the dark at 50° C. overnight. The solvents were evaporated and the residue was dissolved in 1 mL of EDTA solution (0.1 M) and eluted through a G-25 column (eluant water to give 20 mg of Ia-1-Dextran 1,500 conjugate (˜58% yield).

(50) I.4. Synthesis of compound Ic-1 (n° 17 below) and Ic-2 (n° 18 below)

(51) ##STR00033## ##STR00034##
HCl, THF, rt; (e) 1,6-dibromohexane (3 equiv), K.sub.2CO.sub.3 (3 equiv), DMF, 70° C., 59% for 7, 83% for 8 over two steps; (f) With substrate 7: SnCl.sub.2.2H.sub.2O (8 equiv), conc. HCl, EtOH, 80° C.; (g) With substrate 8: H.sub.2, Pd/C, AcOEt/MeOH (4:1), rt; (h) BrCH.sub.2CO.sub.2Me (12-15 equiv), DIEA (12-15 equiv), acetonitrile, 80° C., 48% for 9, 42% for 10 over two steps; (i) NaN.sub.3 (3 equiv), DMF, 80° C., 92% for 11, 97% for 12; (j) Vilsmeier reagent (3 equiv), DMF, 60° C., 55% for 13, 56% for 14; (k) 8-hydroxyjulolidine (2 equiv), TfOH (0.15-0.3 equiv), DCM, rt, then p-chloranil (1 equiv), rt, 43% for 15, 39% for 16; (l) 10 M KOH, MeOH, rt, 40% for 17, 77% for 18.

(52) ##STR00035##

(53) To a solution of potassium hydroxide (42.9 g, 764 mmol) in water (150 mL) was added portionwise 5-fluoro-2-nitrophenol (24.0 g, 153 mmol). The mixture was heated at 90° C. for 24 h then the temperature was raised up to 100° C. After refluxing for 19 h, the orange solution was cooled to room temperature then diluted in water and washed with aq. 1M HCl. The aqueous layer was extracted with ethyl acetate then the combined organic layers were washed with brine then dried over MgSO.sub.4, filtered and concentrated to afford 1 (22 g, 93%) as a pale orange solid.

(54) ##STR00036##

(55) A solution of 1 (4.75 g, 30.64 mmol) and 3,4-dihydropyran (7 mL, 76.61 mmol) in CH.sub.2Cl.sub.2 (150 mL) was cooled to 0° C. then camphorsulfonic acid (0.355 g, 1.53 mmol) was added. The yellow solution was stirred at 0° C. for 20 min then triethylamine (0.300 mL) was added and the mixture was concentrated. The residue was taken up in CH.sub.2Cl.sub.2 (50 mL) then hexanes (400 mL) was added in order to precipitate the product. After 2 h at room temperature then 2 days at −18° C., the solid was filtered then purified by flash chromatography (cyclohexane/ethyl acetate 95:5 to 92:8) to afford 2 (6.29 g, 86%) as a yellow solid.

(56) ##STR00037##

(57) To a solution of 4-chloro-2-nitrophenol (20 g, 0.115 mmol) in N,N-dimethylformamide (100 mL) was added 1,2-dibromoethane (50 mL, 576 mmol) then potassium carbonate (32 g, 230 mmol). The mixture was heated at 70° C. for 2 h 30, cooled to room temperature then diluted with ethyl acetate and filtered through a celite pad. The filtrate was concentrated to dryness then taken up in ethyl acetate, washed with brine and dried over MgSO.sub.4, filtered and concentrated. The residue was purified by flash chromatography (cyclohexane/ethyl acetate 9:1 to 85:15) to afford 3 (21.6 g, 67%) as a yellowish solid.

(58) ##STR00038##

(59) 5-fluoro-2-nitrophenol (10.3 g, 65.56 mmol) was treated following the procedure which gave 3 to afford 4 (9.91 g, 57%) as a yellowish solid after flash chromatography (cyclohexane/ethyl acetate 9:1 to 85:15).

(60) ##STR00039##

(61) To a solution of 2 (3.86 g, 16.14 mmol) and 3 (4.98 g, 17.75 mmol) in N,N-dimethylformamide (20 mL) was added potassium carbonate (3.34 g, 24.2 mmol). The mixture was heated overnight at 70° C. then cooled to room temperature, diluted in ethyl acetate and filtered through a celite pad. The filtrate was concentrated to dryness then the residue was taken up in ethyl acetate and washed with aq. 1M HCl. The aqueous layer was extracted with ethyl acetate then the combined organic layers were washed with brine then dried over MgSO.sub.4, filtered and concentrated. The residue was recrystallized from ethyl acetate/petroleum ether then the solid was filtered off and washed with cold petroleum ether to afford 5 (6.04 g, 85%) as a yellow solid.

(62) ##STR00040##

(63) Compounds 2 (5.38 g, 22.49 mmol) and 4 (6.53 g, 24.74 mmol) were treated following the procedure which gave 5 to afford 6 (9.50 g, 81%) as a yellow solid after flash chromatography (cyclohexane/ethyl acetate 9:1 to 4:1).

(64) ##STR00041##

(65) To a solution of 5 (6.04 g, 13.76 mmol) in a 2:1 mixture of THF/water (150 mL) was added conc. HCl (15 mL). The solution was stirred at room temperature for 1 h 30 then diluted in ethyl acetate and washed with brine. The aqueous layer was extracted with ethyl acetate then the combined organic layers were dried over MgSO.sub.4, filtered and concentrated to dryness.

(66) The residue was dissolved in N,N-dimethylformamide (45 mL) then 1,6-dibromohexane (6.30 mL, 41.28 mmol) and potassium carbonate (2.85 g, 20.64 mmol) were added. The mixture was stirred at 70° C. for 2 h 30 then diluted with ethyl acetate and filtered through a celite pad. The filtrate was concentrated then taken up in CH.sub.2Cl.sub.2 and washed with aq. 1M HCl. The aqueous layer was extracted with CH.sub.2Cl.sub.2 then the combined organic layers were washed with brine then dried over MgSO.sub.4, filtered and concentrated. The residue was purified by flash chromatography (cyclohexane/ethyl acetate 95:5 to 85:15) then the residue was taken up in ethyl acetate and hexanes (200 mL) was added. After triturating for 10 min then cooling at −25° C. for 1 h, the precipitate was filtered off to afford 7 (4.21 g, 59%) as an off white solid.

(67) ##STR00042##

(68) Compound 6 (7.52 g, 17.80 mmol) was treated following the procedure which gave 7 to afford 8 (7.36 g, 83%) as a yellowish solid after flash chromatography (cyclohexane/ethyl acetate 9:1 to 4:1) followed by a precipitation from hexanes.

(69) ##STR00043##

(70) To a suspension of 7 (2.59 g, 5.0 mmol) in absolute ethanol (40 mL) was added SnCl.sub.2.2H.sub.2O (9.0 g, 40 mmol) and conc. HCl (6.5 mL). The mixture was stirred in the dark at 80° C. for 2 h then cooled to room temperature and brought to pH>11 with dropwise addition of aq. 3M NaOH. A grey precipitate started forming and the solution turned gradually from yellow to reddish. The resulting suspension was diluted with water then extracted with diethyl ether (5×100 mL). The combined organic layers were dried over MgSO.sub.4, filtered and concentrated to dryness to afford the crude amino derivative as a dark brown oil.

(71) The residue was dissolved in acetonitrile (10 mL) then methyl bromoacetate (7.1 mL, 75 mmol) and N,N-diisopropylethylamine (13.1 mL, 75 mmol) were added. The mixture was stirred in the dark and under argon at 80° C. for 38 h then cooled to room temperature. The mixture was diluted with CH.sub.2Cl.sub.2 then washed with satd. aq. NaHCO.sub.3 and the aqueous layer was extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over MgSO.sub.4, filtered and concentrated to dryness. The crude residue was purified by flash chromatography (cyclohexane/ethyl acetate 9:1 to 7:3) to afford 9 (1.78 g, 48%) as a brownish syrup.

(72) ##STR00044##

(73) To a solution of 8 (0.490 g, 0.977 mmol) in a 4:1 mixture of ethyl acetate/methanol (10 mL) was added 10% w/w palladium on carbon (0.100 g). The suspension was stirred at room temperature under hydrogen atmosphere for 3 h then filtered through a celite pad. The filtrate was concentrated to dryness to afford the crude amino derivative as a dark brown oil.

(74) The residue was dissolved in acetonitrile (2 mL) then methyl bromoacetate (1.1 mL, 11.7 mmol) and N,N-diisopropylethylamine (2 mL, 11.7 mmol) were added. The mixture was stirred in the dark and under argon at 80° C. for 20 h then cooled to room temperature. The mixture was diluted with CH.sub.2Cl.sub.2 then washed with satd. aq. NaHCO.sub.3 and the aqueous layer was extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over MgSO.sub.4, filtered and concentrated to dryness. The crude residue was purified by flash chromatography (cyclohexane/ethyl acetate 9:1 to 7:3) to afford 9 (0.301 g, 42%) as a brownish syrup.

(75) ##STR00045##

(76) To a solution of 9 (1.02 g, 1.37 mmol) in N,N-dimethylformamide (6 mL) was added sodium azide (0.270 g, 3.06 mmol). The solution was strirred in the dark and under argon at 80° C. for 21 h then cooled to room temperature and diluted with ethyl acetate. After washing twice with water, the combined aqueous layers were extracted with ethyl acetate. The combined organic layers were washed with brine then dried over MgSO.sub.4, filtered and concentrated to dryness to afford 11 (0.897 g, 92%) as a brownish syrup.

(77) ##STR00046##

(78) Compound 10 (0.287 g, 0.393 mmol) was treated following the procedure which gave 11 to afford 12 (0.264 g, 97%) as a brownish syrup.

(79) ##STR00047##

(80) A solution of phosphoryl chloride (0.350 mL, 3.73 mmol) in N,N-dimethylformamide (0.700 mL) was stirred at 0° C. for 1 h then added dropwise to a solution of 11 (0.880 g, 1.24 mmol) in N,N-dimethylformamide (4 mL). The mixture was stirred in the dark at 60° C. for 1 h 30 then cooled to room temperature before diluting with ethyl acetate and adding satd. aq. NaHCO.sub.3. The aqueous layer was extracted with ethyl acetate then the combined organic layers were washed with brine, dried over MgSO.sub.4, filtered and concentrated. The crude residue was purified by flash chromatography (cyclohexane/ethyl acetate 9:1 to 3:2) to afford 13 (0.506 g, 55%) as an orange syrup.

(81) ##STR00048##

(82) Compound 12 (0.234 g, 0.338 mmol) was treated following the procedure which gave 13 to afford 14 (0.135 g, 56%) as an orange oil after flash chromatography (cyclohexane/ethyl acetate 3:2 to 1:1).

(83) ##STR00049##

(84) To a solution of 13 (0.110 g, 0.149 mmol) in CH.sub.2Cl.sub.2 (1 mL) was added 8-hydroxyjulolidine (0.056 g, 0.299 mmol) then trifluoromethanesulfonic acid (4 μL, 0.045 mmol). The solution was stirred overnight in the dark at room temperature then p-chloranil (0.037 g, 0.149 mmol) was added and the brown solution turned dark. After stirring in the dark at room temperature for 4 h, the purple mixture was concentrated. The crude residue was purified by flash chromatography (CH.sub.2Cl.sub.2/methanol 100:0 to 95:5) to afford 15 (0.075 g, 43%) as a dark purple solid. .sup.1H NMR (CD.sub.3OD, 300 MHz) δ 6.94 (s, 2H), 6.91-6.80 (m, 4H), 6.60 (td, J=8.2 Hz, J=2.7 Hz, 1H), 4.48-4.36 (m, 4H), 4.12 (s, 4H), 4.10 (s, 4H), 3.92 (t, J=5.7 Hz, 2H), 3.61 (s, 6H), 3.57-3.50 (m, 12H), 3.09-2.99 (m, 6H), 2.84-2.67 (m, 4H), 2.14-2.06 (m, 4H), 2.02-1.94 (m, 4H), 1.47-1.39 (m, 2H), 1.34-1.24 (m, 2H), 1.14-1.04 (m, 2H), 1.02-0.94 (m, 2H); .sup.13C NMR (CD.sub.3OD, 75 MHz) δ 173.5, 173.3 (4C, 4C═O), 154.6, 154.4, 153.7, 153.6, 152.2, 153.0, 152.4, 137.0, 133.8, 128.3, 124.9, 123.7, 121.4, 121.3, 114.6, 114.5, 106.4, 101.3, 70.1, 68.9 (2C), 54.7 (2C), 52.2, 52.1, 51.8, 51.4, 29.9, 29.8, 28.7, 27.2, 26.7, 21.9, 21.0, 20.9.

(85) ##STR00050##

(86) Compound 14 (0.117 g, 0.162 mmol) was treated following the procedure which gave 15 to afford 16 (0.075 g, 39%) as a deep purple solid after flash chromatography (CH.sub.2Cl.sub.2/methanol 100:0 to 94:6). .sup.1H NMR (CD.sub.3OD, 300 MHz) δ 6.94 (s, 2H), 6.91-6.80 (m, 4H), 6.60 (td, J=8.2 Hz, J=2.7 Hz, 1H), 4.48-4.36 (m, 4H), 4.12 (s, 4H), 4.10 (s, 4H), 3.92 (t, J=5.7 Hz, 2H), 3.61 (s, 6H), 3.57-3.50 (m, 12H), 3.09-2.99 (m, 6H), 2.84-2.67 (m, 4H), 2.14-2.06 (m, 4H), 2.02-1.94 (m, 4H), 1.47-1.39 (m, 2H), 1.34-1.24 (m, 2H), 1.14-1.04 (m, 2H), 1.02-0.94 (m, 2H); .sup.13C NMR (CD.sub.3OD, 75 MHz) δ 173.5, 173.3 (4C, 4C═O), 154.6, 154.4, 153.7, 153.6, 152.2, 153.0, 152.4, 137.0, 133.8, 128.3, 124.9, 123.7, 121.4, 121.3, 114.6, 114.5, 106.4, 101.3, 70.1, 68.9 (2C), 54.7 (2C), 52.2, 52.1, 51.8, 51.4, 29.9, 29.8, 28.7, 27.2, 26.7, 21.9, 21.0, 20.9.

(87) ##STR00051##

(88) To a solution of 15 (0.114 g, 0.105 mmol) in methanol (7 mL) was added aq. 10 M KOH (1 mL). The mixture was stirred in the dark at room temperature for 20 h then diluted with chloroform and washed with aq. 1 M HCl. The aqueous layer was extracted with chloroform then the combined organic layers were dried over MgSO.sub.4, filtered and concentrated. The crude residue was purified on a reverse phase column C-18 using acetonitrile (0.1% TFA) and water (0.1% TFA) as eluant (20% ACN to 60%) to afford 17 (i.e. compound or formula Ic-1) (0.043 g, 40%) as a deep purple solid after lyophilization (water/dioxane 1:1).

(89) MS (ES+): C.sub.53H.sub.59ClN.sub.7O.sub.12.sup.+: cald 1020.39. Found: 1020.4

(90) ##STR00052##

(91) To a solution of 16 (0.055 g, 0.052 mmol) in methanol (4 mL) was added aq. 10 M KOH (0.5 mL). The mixture was stirred in the dark at room temperature for 2 h then diluted with chloroform and washed with aq. 1 M HCl. The aqueous layer was extracted with chloroform then the combined organic layers were dried over MgSO.sub.4, filtered and concentrated. The residue was taken up in a 1:1 mixture of water/dioxane then freeze-dried to afford 18 (i.e. compound or formula Ic-2) (0.040 g, 77%) as a deep purple solid.

(92) MS (ES+): C.sub.53H.sub.59FN.sub.7O.sub.12.sup.+ cald: 1004.42. Found: 1004.3

(93) II. Optical Properties of Compound Ia-1 and Derivatives Thereof

(94) II.1. Absorption and Emission Spectra

(95) Normalised absorption and emission spectra of Ia-1 (5 μM in water, 30 mM MOPS, 100 mM KCl, pH 7.2) were determined and are reported in FIG. 2. Absorption and emission spectra of Ia-1 (5 μM) in presence of different concentration of calcium (30 mM MOPS, 100 mM KCl, pH 7.2) were also determined and are represented in FIGS. 3 and 4 respectively.

(96) II.2. Determination of Dissociation Constants

(97) A fluorimetric titration of Ia-1 (5 μM) against Ca.sup.2+ in a buffer containing (in mM) 100 KCl and 30 MOPS (pH 7.2) was performed. The resulting curve of titration is reported in FIG. 5. The line fits a Hill profile from the average of three independent titrations and gave an apparent dissociation constant of 258 nM.

(98) A fluorimetric titration of Ia-1-Dextran-6000 conjugate against Ca.sup.2+ in a buffer containing (in mM) 100 KCl and 30 MOPS (pH 7.2) was also conducted. The resulting curve of titration is reported in FIG. 6. The line hit Hill profile and gave an apparent dissociation constant of 295 nM.

(99) The normalised fluorimetric titrations against Ca.sup.2+ of Ia-1 and its dextran-6000 conjugate Ia-1-Dextran-6000 are represented in FIG. 7.

(100) A fluorimetric titration of Ic-1 and Ic-2 against Ca.sup.2+ in a buffer containing (in mM) 100 KCl and 30 MOPS (pH 7.2) was also conducted. The line hit Hill profile and gave an apparent dissociation constant of 1.70 μM for Ic-1 and 0.32 μM for Ic-2. The normalised fluorimetric titrations against Ca.sup.2+ of Ia-1, Ic-1 and Ic-2 are represented in FIG. 13.

(101) II.3. Determination of the Quantum Yield

(102) Determination of Ia-1 Fluorescence Quantum Yields.

(103) The quantum yields φ of Ia-1 were calculated from the slope of the integrated spectral emission (545 to 700 nm) of Ia-1 in the presence (2 mM) or absence (0 mM, 10 mM EGTA) of Ca.sup.2+ vs. absorbance at 535 nm using rhodamine 101 (φ=1.0 in absolute ethanol) as a reference standard. A solvent correction was applied for the comparison of the fluorescence quantum yields of Ia-1 and rhodamine 101. The quantum yields φ were calculated using the following equation where φ is the quantum yield, s is the value of the observed slope and η is the refractive index of the solvent used.

(104) $\phi ={\phi }_{\mathrm{ref}}\frac{s}{s_{\mathrm{ref}}}.Math.\frac{{\eta }^2}{{\eta }_{\mathrm{ref}}^2}$ The calculations gave: φ.sub.Calcium free=0.0089 φ.sub.Ca2+=0.4541
II.4. Two-Photon Excitation of Compound Ia-1

(105) Experiments of two-photon excitation of Ia-1 were conducted. Results are represented on FIG. 8. The power function fit to the data gives a power of 1.92±0.10—within one SD of 2.0, as expected for two-photon excitation evidencing that the measured fluorescence was indeed two-photon excited.

(106) III. Ex Vivo and In Vivo Evaluation

(107) III.1. Material and Methods

(108) Animals

(109) All procedures were approved by the local ethical review committee and performed under license from the UK Home Office in accordance with the Animal (Scientific Procedures) Act 1986. For in vivo preparations, analgesics (Carprofen) were provided as needed.

(110) Slicing

(111) Parasagittal cerebellar slices (200 μm) were made using standard techniques from C57BL6/J mice (Harlan) at postnatal days 25-29. Artificial CSF (ACSF) for both slicing and recording contained the following (in mM): 125 NaCl, 2.5 KCl, 26 NaHCO.sub.3, 1.25 NaH.sub.2PO.sub.4, 25 glucose, 1 MgCl.sub.2, and 2 CaCl.sub.2, and was bubbled with 5% carbon dioxide, 95% oxygen. Slices were continuously superfused with ACSF during the experiment. Slice experiments were performed at room temperature.

(112) For high speed imaging experiments, acute 260 μm thick slices were obtained from the cerebellar vermis of P60 mice and superfused with extracellular saline medium.

(113) Electrophysiology and Imaging in Cerebellum

(114) Full frame and linescan two-photon imaging was performed using microscopes optimized for in vitro (Prairie Technologies) or in vivo (MOM, Sutter) experiments. Two photon excitation was provided by a pulsed Ti:Sa laser (MaiTai HP, Newport), tuned to a central wavelength of 890 to 920 nm. The microscopes were controlled by ScanImage 3.5 and 3.7.1. Patch-clamp pipettes were filled with an internal solution containing (in mM): K-methanesulfonate 133, KCl 7, HEPES 10, Mg-ATP 2, Na.sub.2ATP 2, Na.sub.2GTP 0.5, EGTA 0.05, 0.1 Alexa Fluor 488 and Ia-1-Dextran as indicated; pH 7.2. Recordings from visually identified Purkinje cells were made using a Multiclamp 700B amplifier (Molecular Devices). Data were lowpass filtered at 4 kHz and acquired at 20 kHz using an ITC-18 digitizer (Instrutech) controlled by AxoGraph X (http://www.axographx.com/). Electrical stimuli were delivered via a theta-glass bipolar electrode filled with ACSF using a constant current stimulus isolator (DS-3, Digitimer). When using electrical stimulation, 10 μM SR-95531 (Sigma or Tocris) was added to the perfusion medium.

(115) Climbing fiber stimulation-evoked transient [Ca.sup.2+] changes in Purkinje cell spines were recorded at high acquisition rate (>2 kHz) by two-photon random-access microscopy, a technique is based on the use of acousto-optic deflectors (AODs), which enable selective scanning of defined points. Purkinje cells were recorded in current-clamp mode, using 2-3MΩ patch pipettes containing 300 μM Ia-1. Recordings were obtained by use of a Multiclamp 700B (Molecular Devices). Following the dialysis of Ia-1, Purkinje cells in slices were imaged under a 25× Leica water immersion objective (HCX IRAPO L 25×/0.95). Two-photon excitation was produced by a pulsed Ti:Sa laser (Chameleon Vision Plus, Coherent) coupled into the transmitted light pathway of the microscope by a dichroic filter (740dcsx, Chroma Technology Corporation) and tuned to a central wavelength of 890 nm. A custom-made user interface based on National Instrument cards programmed under Labview was used to operate the AODs and coordinate the scanning protocols and signal acquisition. A multifunction card (NI-PCI-MIO 16 E-4) was used to pass all the triggers necessary to synchronize the imaging and the electrophysiology and to control the piezo-electric device that moves the objective in Z. Fluorescence photons were detected by cooled AsGaP photomultipliers (H7421-40, Hamamatsu) discriminated and counted on a fast digital card.

(116) Virus Injection

(117) Young (P19) C57BL6/J mice were anesthetized using isoflurane, an incision was made into the scalp and a small (˜0.5 mm) craniotomy was performed over lobule V of the cerebellar vermis. A widebore (˜50 μm) micropipette containing viral suspension (AAV1.hSyn.iGluSnFr.WPRE.SV40, University of Pennsylvania Vector Core) was inserted through the craniotomy and carefully lowered 1.0 mm into the brain. Using application of low pressure 400-800 nL viral suspension were slowly injected (10-20 minutes). After the injection further 5-10 minutes were waited before retraction of the injection pipette. The scalp was glued and sutured and the mouse left to recover. At least 7 days incubation time were allowed prior to further experiments.

(118) In Vivo Imaging of Olfactory Sensory Neuron Terminals

(119) Kv3.1-eYFP mice (8-10 week-old) were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). Ia-1-Dextran-6000 was dissolved 2.5% w/v in a solution of aCSF (in mM: 125 NaCl, 2.5 KCl, 1.25 NaH.sub.2PO.sub.4, 25 NaHCO.sub.3, 1 MgCl.sub.2, 2 CaCl.sub.2 and 25 glucose) with 0.2% Triton X-100 (Sigma-Aldrich). 8 μl of this solution was injected in the mouse naris, and mice were left on their backs to recover from anesthesia. 7 days later, an acute craniotomy was performed over the dorsal olfactory bulb and the brain stabilized with 3.5% agar for imaging. To activate olfactory sensory neurons (OSNs), odors were applied in a 1 ml/min flux of filtered, humidified air supplemented with 30% oxygen. eYFP and Ia-1 fluorescence was collected in two separate channels (“green” and “red”, respectively) of a custom-built two-photon laser scanning microscope, with the femtosecond pulsed excitation beam set to 910 nm.

(120) In Vivo Bulk Loading and Imaging

(121) Adult C57BL6 mice (6-9 weeks; Harlan) were anesthetized with isoflurane, supplemented with 1 mg/kg chlorprothixene. A 1.5-2 mm craniotomy was performed over cerebellar lobule V. Care was taken to leave the dura mater intact. Ib-1 was prepared and injected using standard methods. A 50 μg aliquot was dissolved in 20% Pluronic-127 in DMSO (Invitrogen) and then diluted 1:10 in saline (150 mM NaCl, 2.5 mM KCl, 10 mM HEPES, pH 7.4). This solution was filtered and injected into the cerebellum under visual guidance using a patch-pipette and 500-750 mbar pressure for 1-3 minutes. After injection the preparation was left to incubate for up to 1.5 hours prior to imaging. This helped improve labeling and lower background fluorescence.

(122) Data Analysis and Statistics

(123) Imaging data were analyzed using ImageJ (http://rsbweb.nih.gov/ij/). Extracted fluroescence traces, linescans and electrophysiological data were analyzed using in house routines programmed in IgorPro versions 5 or 6.2 (Wavemetrics) and in pClamp 10 (Molecular Device Inc).

(124) III.2. In Vivo Ca.sup.2+ Imaging in Layer 2/3 Pyramidal Neurons

(125) Ca.sup.2+ was imaged in layer 2/3 neurons in vivo as reported in FIG. 9.

(126) FIG. 9a represents a layer 2/3 pyramidal neuron (˜200 μm below the brain surface), filled with 100 μM Alexa Fluor 488 and 200 μM Ia-1-Dextran-6000.

(127) FIG. 9b reports action potential (AP) trains evoked by current injection of increasing duration.

(128) The corresponding Ca.sup.2+ transients were recorded by line-scanning the proximal dendrite (red line in a) at 500 Hz and displayed as percentage change in “red-over-green” ratio from baseline (FIG. 9c). Fluorescence traces are aligned to AP onset and color-coded to match the number of APs (top). The peak amplitudes (circle) and the area under the curve (triangle) of the fluorescence trace were plotted against the number of action potentials (bottom). While the area increases nearly linearly, the peak amplitude saturates, as expected for a high affinity indicator.

(129) III.3. Population Imaging of Purkinje Cells In Vivo Using AM Bulk Loading

(130) Purkinje cells were imaged in vivo using bulk loading of Ia-1. Results are reported in FIG. 10.

(131) FIG. 10a shows the configuration of AM-ester (Ib-1) injection and imaging. FIG. 10b presents the resulting staining of tissue 60 minutes after injection of indicator. Purkinje cells can be seen as vertical stripes with occasional brighter spots (presumably corresponding to dendrites). Active Purkinje cell dendrites identified using a spatial PCA/ICA algorithm are depicted in FIG. 10c.

(132) Fluorescence traces from the identified dendrites are recorded in FIG. 10d. Stimulus timing is indicated by the underlying grey bars. Stimulus triggered averages of the complete traces in d (20 repetitions) is represented in FIG. 10e. Note that all cells except for the third (red) show a stimulus-locked response.

(133) In the past decade, population imaging of neurons using bulk loading of acetoxymethyl ester (AM) derivatives of [Ca.sup.2+] indicators has become one of the most common methods to monitoring neuronal activity. This has opened a wide new field of applications for these AM esters. Yet, nearly all recent bulk loading studies rely on either Oregon Green-488 BAPTA-1-AM (OGB-1) or Fluo-4-AM, again limiting the possibility to multiplex indicators for different signalling species. Here the Applicant demonstrates that the AM derivative of Ia-1 (i.e. Ib-1) is a suitable red-emitting alternative to these indicators. A series of three experiments was performed in which Ib-1 was bolus injected in the cerebellar vermis (FIG. 10a). In all experiments, the resulting labelling was comparable to that obtained with OGB-1 in similar conditions. The main difference was the need for a longer incubation period prior to onset of imaging (60-75 minutes compared to 45-60 minutes for OGB-1 and 15-30 minutes for Fluo-4). In all experiments fluorescence traces extracted for identified dendrites (FIG. 10c) showed clear complex spike activity with a good signal-to-noise ratio (FIG. 10d) as expected for this preparation. Using electrocutaneous stimulation of the hind limb it was possible to evoke responses time-locked to the stimulus in a high fraction of dendrites (FIG. 10e). Taken together, these data show that Ib-1 is similarly suited for population imaging experiments as OGB-1 AM or Fluo-4 AM, with the advantage of leaving the green detection channel free for additional indicators.

(134) IV. Discussion

(135) In cuvette calibration experiments (paragraph II.2), Ia-1 was found to have a K.sub.D of 258±8 nM, with a 50-fold (±2) increase of fluorescence on binding [Ca.sup.2+] and a maximum quantum yield of 0.45 (paragraph II.3). Besides suitability for single photon excitation, Ia-1 is also effectively two-photon excited (paragraph II.4).

(136) To minimize the subcellular compartmentalization typical for red emitting fluorescent probes, 1.5 and 6 kD dextran conjugates were obtained by click chemistry and used in the further experiments.

(137) Using two-photon microscopy and simultaneous patch-clamp recording, it was verified that Ia-1 gives signals comparable to commonly used green emitting [Ca.sup.2+] probes. For this purpose, Purkinje cells were filled in cerebellar slices with Ia-1-Dextran-6000 and Alexa Fluor-488 via patch-clamp pipettes (FIG. 11a). Electrical stimulation of parallel fiber inputs to Purkinje cells resulted in an increase in spike frequency and [Ca.sup.2+] transients that were recorded using line-scan imaging (FIG. 11b). Even mild stimulation (2 pulses at 100 Hz) yielded large fluorescence transients (>100% dF/F.sub.0) with a high signal-to-noise ratio in single spines (FIG. 11c). As reported in previous studies, transients were larger and faster in dendritic spines than in the dendritic shaft.

(138) Next, high-speed random access microscopy (FIG. 11d) was used to verify that Ia-1 reports [Ca.sup.2+] transients with kinetics comparable to commonly used indicators. During stimulation of the climbing fiber, fluorescence traces were acquired from multiple spines (range: 4 to 14) at rates over 1 kHz (range 2.2 to 4.8 kHz) (FIGS. 11e and 11f). The rise time τ=1.30±0.26 ms (exponential fit; n=59 spines from 7 cells), was not significantly different from/significantly faster than the kinetics found for Fluo-4.

(139) Having verified the suitability of Ia-1 for in vitro experiments, in vivo patch-clamp recordings were then performed from neocortical layer 2/3 pyramidal neurons in anesthetized mice with concomitant [Ca.sup.2+] imaging (FIG. 9), showing that spiking activity in these neurons resulted in large fluorescence transients, that showed a linear amplitude vs. spike number relation for low spike numbers, saturating for higher spike numbers, as expected for a high affinity calcium indicator. Taken together these experiments demonstrate that Ia-1 is a calcium indicator well suited for a wide range of neuroscience experiments, with a signal quality comparable to previously used high-affinity green emitting probes.

(140) To complete the functional imaging toolbox, a means of imaging cell populations, rather than single cells is needed. In the past decade this has commonly been achieved using bulk loading of calcium indicators in the AM-ester form (Ib-1). An AM-ester of Ia-1 (i.e. Ib-1) was thus synthesized and used to bulk load cerebellar neurons in vivo (FIG. 10). It was found a labeling identical to that commonly found in experiments using Oregon Green-488 BAPTA-1 AM (OGB-1 AM) as well as comparable spontaneous and sensory evoked responses. This indicates that Ib-1 is a powerful addition to the optophysiological toolbox.

(141) Making use of the strongly overlapping two-photon excitation spectra of eGFP and Ia-1, a set of experiments was conducted, which were previously not possible: Simultaneous imaging of glutamate release onto Purkinje cells (using iGluSnFR) and the resulting post-synaptic [Ca.sup.2+] increase (using Ia-1-Dextran-6000). In these experiments acute cerebellar slices of P27-P29 mice were prepared 7 to 9 days after viral transfection of the cerebellar vermis with iGluSnFR. Visually identified Purkinje cells showing green fluorescence were whole-cell recorded and filled with Ia-1-Dextran (FIG. 12a). Electrical stimulation of the glutamatergic climbing fiber input evoked clear fluorescence transients in both color channels (FIG. 12b). We found, glutamate signals to be confined to a distinct subsections of the dendritic (i.e. limited to sites of synaptic release), whereas the resulting [Ca.sup.2+] transients were global, with comparable amplitudes over different regions in the dendritic tree (FIG. 12c). These experiments demonstrate the potential of two-channel functional imaging, with the red emission and high sensitivity of Ia-1 being an ideal match for numerous other indicators emitting in the green-yellow spectral band.

(142) To verify that dual color imaging is also possible in vivo we used Ia-1-Dextran-6000 to report presynaptic activity in anesthetized Kv3.1-eYFP adult mice. In the olfactory bulb of these mice, mitral and tufted cells, as well as a population of periglomerular neurons, strongly express eYFP and their somata and processes clearly demarcate the external glomerular boundaries (FIG. 12d). Olfactory sensory neuron (OSN) terminals, labeled with Ia-1, filled the inner glomerular boundaries (FIG. 12e). In single glomeruli (n=8 animals) we could record presynaptic calcium responses with an excellent signal to noise ratio. FIG. 12f shows a typical example in which presynaptic calcium responses were selectively evoked by odor presentation in a subset of glomeruli. These responses adapted strongly at this high odorant concentration, as reported previously. These data clearly indicate that Ia-1 can be used in vivo as an efficient red calcium-sensitive dye in the presence of eYFP.