CELL-PENETRATING FLUORESCENT DYES WITH SECONDARY ALCOHOL FUNCTIONALITIES
20190367737 ยท 2019-12-05
Inventors
- Alexey BUTKEVICH (Goettingen, DE)
- Vladimir N. Belov (Goettingen, DE)
- Stefan W. Hell (Goettingen, DE)
- Dirk KAMIN (Goettingen, DE)
- Sven SIDENSTEIN (Goettingen, DE)
- Heydar SHOJAEI (Goettingen, DE)
- Kirill KOLMAKOV (Magdeburg, DE)
- Viktor V. SOKOLOV (St. Petersburg, RU)
Cpc classification
G01N33/94
PHYSICS
C09B69/008
CHEMISTRY; METALLURGY
C09B11/28
CHEMISTRY; METALLURGY
C09B11/24
CHEMISTRY; METALLURGY
International classification
C09B11/28
CHEMISTRY; METALLURGY
C09B11/24
CHEMISTRY; METALLURGY
G01N33/94
PHYSICS
C09B69/00
CHEMISTRY; METALLURGY
Abstract
The invention relates to novel cell-penetrating fluorescent dyes with secondary alcohol functionalities having one of the following general formulae I-III and 4: The invention also relates to the use of these compounds for optical microscopy and imaging techniques.
##STR00001##
Claims
1-20. (canceled)
21. A fluorescent dye of compounds of the general formulae selected from the group consisting of Ia, Ib, IIa, IIb, IIIa, IIIb, 4a and 4b below ##STR00031## which exist in equilibrium between an open zwitterionic form (a) and a closed form (b), wherein in formulae Ia, Ib, IIa, IIb, IIIa, and IIIb: XO, C(Alkyl).sub.2, Si(Alkyl).sub.2 or Ge(Alkyl).sub.2 with alkyl being a C.sub.1-C.sub.4 alkyl group, selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl and sec-butyl; R.sup.1 and R.sup.2 in structures Ia, Ib, IIa and IIb are independently CF.sub.3CH.sub.2, a C.sub.1-C.sub.6 alkyl group, or a substituted C.sub.1-C.sub.6 alkyl group; R.sup.3 and R.sup.4 in structures Ia, Ib, IIa, IIb, IIIa, and IIIb are independently hydrogen; a C.sub.1-C.sub.6 alkyl group or substituted C.sub.1-C.sub.6 alkyl group; a carboxyl group or a derivative of the carboxyl group selected from the group consisting of a lower alkyl, aryl and hetaryl ester, in which the alkyl, aryl or hetaryl ester is optionally substituted; a primary or secondary alkyl, aryl or hetaryl carboxamide (CONHR, CONRR), in which the alkyl, aryl or hetaryl part(s) (R, R) is/are optionally substituted; a N-hydroxysuccinimidyl ester or another ester of formula CO.sub.2X, where X is a leaving group, selected from the group consisting of F, N.sub.3, SR, and OR with R=aryl or hetaryl; CONHNH.sub.2 or CONRNRR, with R, R, and R being independently H, lower alkyl, in which the alkyl, aryl or hetaryl part is optionally substituted; CONHOH or CONROR, with R, and R being independently H, lower alkyl, aryl or hetaryl, in which the alkyl, aryl or hetaryl part is optionally substituted; COCHN.sub.2 or COCRN.sub.2 with R=lower alkyl, aryl or hetaryl, in which the alkyl, aryl or hetaryl part is optionally substituted; an amino group, lower alkylamino group or dialkylamino group, the alkyl part(s) of which may be optionally substituted with a carboxyl group or a derivative of the carboxyl group selected from the group consisting of a lower alkyl, aryl and hetaryl ester, in which the alkyl, aryl or hetaryl part is optionally substituted; azido group; N-maleimidoalkyl group, iodoacetamide or any other reactive group suitable for conjugation to biomolecules; an arylthio or alkylthio group, the aryl or alkyl part of which may be optionally substituted with an uncharged group selected from the group consisting of an azide group N.sub.3, carboxyl group, and a derivative of the carboxyl group selected from the group consisting of a lower alkyl, aryl and hetaryl ester, in which the alkyl, aryl or hetaryl part is optionally substituted; an alkyl, aryl or hetaryl amide (NHCOR, NHCORR), in which the alkyl, aryl or hetaryl part(s) (R, R) is(are) optionally substituted; optionally, the carbon chains in R.sup.1, R.sup.2, R.sup.3 and R.sup.4 contain double or triple bonds and/or cycles, and/or uncharged groups, selected from the group consisting of hydroxyl, and one, two, three and four heteroatoms; R.sup.5H, F or Cl; R.sup.6, R.sup.9, R.sup.1, R.sup.11H or OH and wavy bonds () in structures Ia, Ib, IIa, IIb, IIIa, IIIb above denote all possible isomers if one or more substituents from the group consisting of R.sup.6, R.sup.9, R.sup.10, and R.sup.11 is/are OH; R.sup.7, R.sup.8 are independently H, a C.sub.1-C.sub.4 alkyl group, F, Cl, Br or I; provided (1) that all dyes represented by the formulae Ia, Ib, IIa, IIb, IIIa, IIIb contain at least one substituted 3-hydroxy-1,2,3,4-tetrahydroquinoline fragment (with an element CH.sub.2CH (OH) CH.sub.2) incorporated into the structures of the formulae Ia, Ib, IIa, IIb, IIIa, IIIb and in the structural formula below, ##STR00032## with an additional provision (2) for structures IIIa and IIIb that at least one of R.sup.6, R.sup.9, R.sup.10 or R.sup.11 is a hydroxyl group; ##STR00033## wherein Alkyl in structure 4 is a C.sub.1-C.sub.6 alkyl group, selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, pentyl, isopentyl, 2-methylbutyl, 2,2-dimethylpropyl, hexyl, isohexyl, 2-methylpentyl, 3-methylpentyl, and 2,2-dimethylbutyl; R in structures 4a and 4b may be hydrogen, a C.sub.1-C.sub.6 alkyl group or substituted C.sub.1-C.sub.6 alkyl group, a carboxyl group or a derivative of the carboxyl group selected from the group consisting of a lower alkyl, aryl or hetaryl ester, in which the alkyl, aryl or hetaryl part is optionally substituted; a primary or secondary alkyl, aryl or hetaryl carboxamide (CONHR, CONRR), in which the alkyl, aryl or hetaryl part(s) (R, R) is (are) optionally substituted; a N-hydroxysuccinimidyl ester or another reactive ester of formula CO.sub.2X, where X is a leaving group, selected from the group consisting of F, N.sub.3, SR, and OR with R=aryl or hetaryl; CONHNH.sub.2 or CONRNRR with R, R, R being independently H, C.sub.1-C.sub.6 alkyl, aryl or hetaryl, in which the alkyl, aryl or hetaryl part(s) is (are) optionally substituted; CONHOH or CONROR with R, R being independently H, lower alkyl, aryl or hetaryl, in which the alkyl, aryl or hetaryl part(s) is (are) optionally substituted; COCHN.sub.2 or COCRN.sub.2 with R=lower alkyl, aryl or hetaryl, in which the alkyl, aryl or hetaryl part is optionally substituted; an amino group, lower alkylamino group or dialkylamino group, the alkyl part(s) of which may be optionally substituted with a carboxyl group or a derivative of the carboxyl group selected from the group consisting of a lower alkyl, aryl or hetaryl ester, in which the alkyl, aryl or hetaryl part is optionally substituted; a primary or secondary alkyl, aryl or hetaryl amide (NHCOR, NHCORR), in which the alkyl, aryl or hetaryl part(s) (R, R) is (are) optionally substituted; azido group; N-maleimidoalkyl group, iodoacetamide or any other reactive group suitable for conjugation to biomolecules; optionally, the carbon chains in the group R may contain double or triple bonds and/or cycles, and/or uncharged groups as well as one, two, three or four heteroatoms.
22. A fluorescent dye according to claim 21 wherein R.sup.3 and R.sup.4 in structures Ia, Ib, IIa, IIb, IIIa, IIIb are selected from the group consisting of hydrogen; a C.sub.1-C.sub.6 alkyl group or substituted C.sub.1-C.sub.6 alkyl group; a carboxyl group or a derivative of the carboxyl group selected from the group consisting of a lower alkyl, aryl and hetaryl ester, in which the alkyl, aryl or hetaryl part is optionally substituted with a functional group as defined below; a primary or secondary alkyl, aryl or hetaryl carboxamide (CONHR, CONRR), in which the alkyl, aryl or hetaryl part(s) (R, R) is (are) optionally substituted with a functional group as defined below; a N-hydroxysuccinimidyl ester or another reactive ester of formula CO.sub.2X, where X is a leaving group selected from the group consisting of F, N.sub.3, SR, and OR with R=aryl or hetaryl; CONHNH.sub.2 or CONRNRR, with R, R, R being independently H, lower alkyl, aryl or hetaryl, in which the alkyl, aryl or hetaryl part is optionally substituted with a functional group as defined below; CONHOH or CONROR, with R, R being independently H, lower alkyl, aryl or hetaryl, in which the alkyl, aryl or hetaryl part is optionally substituted; COCHN.sub.2 or COCRN.sub.2 with R=lower alkyl, aryl or hetaryl, in which the alkyl, aryl or hetaryl part is optionally substituted with a functional group as defined below; an amino group, lower alkylamino group or dialkylamino group, the alkyl part(s) of which may be optionally substituted with a functional group of a carboxyl group or a derivative of the carboxyl group selected from the group consisting of a lower alkyl, aryl or hetaryl ester, in which the alkyl, aryl or hetaryl part is optionally substituted with a functional group as defined below; azido group; N-maleimidoalkyl group, iodoacetamide or any other reactive group suitable for conjugation to biomolecules; an arylthio or alkylthio group, the aryl or alkyl part of which may be optionally substituted with an uncharged group selected from the group consisting of an azide group N.sub.3, carboxyl group, and a derivative of the carboxyl group selected from the group consisting of a lower alkyl, aryl or hetaryl ester, in which the alkyl, aryl or hetaryl part is optionally substituted with a functional group as defined below; an alkyl, aryl or hetaryl amide (NHCOR, NHCORR), in which the alkyl, aryl or hetaryl part(s) (R, R) is (are) optionally substituted with a functional group as defined below; optionally, the carbon chains in R.sup.1, R.sup.2, R.sup.3 and R.sup.4 may contain double or triple bonds and/or cycles, and/or uncharged groups, selected from the group consisting of hydroxyl, one, two, three and four heteroatoms; R.sup.5H, F or Cl; R.sup.6, R.sup.9, R.sup.10, R.sup.11H or OH and the wavy bonds () in structures Ia, Ib, IIa, IIb, IIIa, IIIb above denote all possible isomers if one or more substituents from the set R.sup.6, R.sup.9, R.sup.10, R.sup.11 is/are OH; R.sup.7, R.sup.8 are independently H, a C.sub.1-C.sub.4 alkyl group, F, Cl, Br or I; provided (1) that all dyes represented by the formulae Ia, Ib, IIa, IIb, IIIa, IIIb contain at least one substituted 3-hydroxy-1,2,3,4-tetrahydroquinoline fragment (with an element CH.sub.2CH(OH)CH.sub.2) incorporated into the structures as shown in formulae Ia, Ib, IIa, IIb, IIIa, and IIIb and in the structural formula below, ##STR00034## with an additional provision (2) for structures IIIa and IIIb, that at least one of R.sup.6, R.sup.9, R.sup.10 or R.sup.11 is a hydroxyl group; ##STR00035## wherein alkyl in structures 4a and 4b is a C.sub.1-C.sub.6 alkyl group selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, pentyl, isopentyl, 2-methylbutyl, 2,2-dimethylpropyl, hexyl, isohexyl, 2-methylpentyl, 3-methylpentyl, and 2,2-dimethylbutyl; R in structures 4a and 4b may be hydrogen, a C.sub.1-C.sub.6 alkyl group or substituted C.sub.1-C.sub.6 alkyl group, a carboxyl group or a derivative of the carboxyl group selected from the group consisting of a lower alkyl, aryl or hetaryl ester, in which the alkyl, aryl or hetaryl part is optionally substituted with a functional group as defined below; a primary or secondary alkyl, aryl or hetaryl carboxamide (CONHR, CONRR), in which the alkyl, aryl or hetaryl part(s) (R, R) is (are) optionally substituted with a functional group as defined below; a N-hydroxysuccinimidyl ester or another reactive ester of formula CO.sub.2X, where X is a leaving group selected from the group consisting of F, N.sub.3, SR, OR with R=aryl or hetaryl; CONHNH.sub.2 or CONRNRR with R, R, R being independently H, C.sub.1-C.sub.6 alkyl, aryl or hetaryl, in which the alkyl, aryl or hetaryl part(s) is (are) optionally substituted with a functional group as defined below; CONHOH or CONROR with R, R being independently H, lower alkyl, aryl or hetaryl, in which the alkyl, aryl or hetaryl part(s) is (are) optionally substituted; COCHN.sub.2 or COCRN.sub.2 with R=lower alkyl, aryl or hetaryl, in which the alkyl, aryl or hetaryl part is optionally substituted with a functional group as defined below; an amino group, lower alkylamino group or dialkylamino group, the alkyl part(s) of which may be optionally substituted with a functional group selected from the group consisting of a carboxyl group and a derivative of the carboxyl group selected from the group consisting of lower alkyl, aryl and hetaryl ester, in which the alkyl, aryl or hetaryl part is optionally substituted; a primary or secondary alkyl, aryl or hetaryl amide (NHCOR, NHCORR), in which the alkyl, aryl or hetaryl part(s) (R, R) is (are) optionally substituted with a functional group as defined below; azido group; N-maleimidoalkyl group, iodoacetamide or any other reactive group suitable for conjugation to biomolecules; wherein the term a functional group as defined below refers to a group selected from the following groups: cycloalkenyl, alkenyl selected from the group consisting of vinyl, allyl, propen-1-yl, propen-2-yl, 1,3-butadien-1-yl, 1,3-budadien-2-yl, and 1,2-propadien-1-yl, and alkynyl selected from the group consisting of ethynyl, propynyl, propargyl, and aryl selected from the group consisting of phenyl, 1-naphthyl, 2-naphthyl, 9-anthracenyl-, pyren-1-yl, pyren-2-yl; halogen atoms selected from the group consisting of F, Cl, Br, and I, azido groups, nitroso groups, diazo groups, diazocarbonyl groups, hydroxy groups, protected hydroxy groups OR, with R=acyl selected from the group consisting of formyl, acetyl, tetrahydropyranyl, t-butyl, allyl, propargyl, phenyl, p-chlorophenyl, p-methoxy-phenyl, p-nitrophenyl, perfluorophenyl, perchlorophenyl, benzyl, 2-picolyl, 4-picolyl, diphenylmethyl, triphenylmethyl, trialkylsilyl, triphenylsilyl, and diphenylalkylsilyl, thiol groups, protected thiol groups SR with R as defined above for OR, primary and secondary amino groups, hydrazo groups, hydroxylamino groups, carbonyl groups in aldehydes and ketones, protected carbonyl groups, hydrazones, oximes, thioketones, sulfoxides, sulfones, sulfonic acid residues and their salts, carboxylic acid groups COOH and ester groups COOR, with R=alkyl selected from the group consisting of CH.sub.3, C.sub.2H.sub.5, and t-butyl; allyl, propargyl, phenyl, p-chlorophenyl, p-methoxyphenyl, p-nitrophenyl, perfluorophenyl, perchlorophenyl, benzyl, 2-picolyl, 4-picolyl, diphenylmethyl, triphenylmethyl, trialkylsilyl, triphenylsilyl, and diphenylalkylsilyl; primary, secondary and tertiary amido groups, a phosphorodiamidite group, C.sub.2H.sub.5, isopropyl, allyl, tert-butyl, phenyl, a phosphoroamidite group [OP (NR.sub.2)(OR)], with R CH.sub.3, C.sub.2H.sub.5, isopropyl, allyl, tert-butyl, phenyl, RH, alkyl, aryl, hetaryl, a phosphorodiamidite group [OP(NR.sub.2)], with RCH.sub.3, C.sub.2H.sub.5, isopropyl, allyl, tert-butyl, phenyl], phosphoric acid residues OP(O)(OR)(OR), with R (R)H, CH.sub.3, C.sub.2H.sub.5, allyl, tert-butyl, phenyl, NH.sub.2; phosphonic acid residues P(O)(OR)(OR), with R(R)H, CH.sub.3, C.sub.2H.sub.5, allyl, tert-butyl, phenyl, NH.sub.2; a N-phthalimido group, and heterocyclic residues; optionally, the carbon chains in the group R may contain double or triple bonds and/or cycles, and/or uncharged groups as well as one, two, three or four heteroatoms.
23. A fluorescent dye according to claim 22, wherein the term a functional group as defined below on each occurrence refers to a hydroxy group.
24. A compound of the structures IIIa and IIIb according to claim 21 which contains two hydroxyl groups and wherein either R.sup.6=R.sup.9OH and R.sup.10=R.sup.11H, or R.sup.6=R.sup.9H and R.sup.10=R.sup.11OH.
25. A compound of the structures IIIa and IIIb according to claim 22 which contains one hydroxyl group and wherein either R.sup.6=R.sup.9OH and R.sup.10=R.sup.11H, or R.sup.6=R.sup.9H and R.sup.10=R.sup.11OH.
26. A compound of the structures IIIa and IIIb according to claim 21 which contains one hydroxyl group and wherein either R.sup.6OH and R.sup.9=R.sup.10=R.sup.11H, or R.sup.10OH and R.sup.6=R.sup.9=R.sup.11H.
27. A compound of the structures IIIa and IIIb according to claim 22 which contains one hydroxyl group and wherein either R.sup.6OH and R.sup.9=R.sup.10=R.sup.11H, or R.sup.10OH and R.sup.6=R.sup.9=R.sup.11H.
28. A fluorescent dye according to claim 21 representing one of the compounds 5-R.sup.3, wherein R.sup.1=R.sup.2CH.sub.2CF.sub.3, R.sup.6OH and R.sup.3CO.sub.2H, its reactive ester, or amide with a linker bearing one terminal amino or carboxylic acid group as indicated in the structural formulae below ##STR00036##
29. A fluorescent dye according to claim 22 representing one of the compounds 5-R.sup.3, wherein R.sup.1=R.sup.2CH.sub.2CF.sub.3, R.sup.6OH and R.sup.3=CO.sub.2H, its reactive ester, or amide with a linker bearing one terminal amino or carboxylic acid group as indicated in the structural formulae below ##STR00037##
30. A fluorescent dye according to claim 21 representing one of the compounds 14a,b-R.sup.3, wherein either R.sup.1=R.sup.2OH, or R.sup.1 OH and R.sup.2H, and wherein R.sup.3=CO.sub.2H, its reactive ester, or amide with a linker bearing one terminal amino or carboxylic acid group. ##STR00038##
31. A fluorescent dye according to claim 22 representing one of the compounds 14a,b-R.sup.3, wherein either R.sup.1=R.sup.2OH, or ROH and R.sup.2H, and wherein R.sup.3=CO.sub.2H, its reactive ester, or amide with a linker bearing one terminal amino or carboxylic acid group ##STR00039##
32. A fluorescent dye of structures 4a and 4b according to claim 21 where alkyl is a straight or branched C.sub.1-C.sub.6 alkyl chain; R is CO.sub.2H or a reactive ester, or a functional group capable of participating in a ligation reaction selected from the group consisting of an azide, alkyne, strained alkene and tetrazine reactive group, which functional group is connected with the phenyl group through a linker, or R is any ligand forming a covalent bond with a biological target of interest, or a noncovalent ligand binding to a biological target of interest selected from the group consisting of a docetaxel and jasplakinolide derivative.
33. A fluorescent dye of structures 4a and 4b according to claim 22 where alkyl is a straight or branched C.sub.1-C.sub.6 alkyl chain; R is CO.sub.2H or a reactive ester, or a functional group capable of participating in a ligation reaction selected from the group consisting of an azide, alkyne, strained alkene or tetrazine reactive group, which functional group is connected with the phenyl group through a linker, or R is any ligand forming a covalent bond with a biological target of interest, or a noncovalent ligand binding to a biological target of interest selected from the group consisting of a docetaxel or jasplakinolide derivative.
34. A fluorescent dye according to claim 32 representing the compounds 4-R, wherein R=CO.sub.2H or its reactive ester, as indicated in the structural formulae below ##STR00040##
35. A fluorescent dye according to claim 32 representing one of the compounds 33, 33-NHS, 33-Halo, 33-tubulin, 33-lysosome and 34 as indicated in the structural formulae below ##STR00041## ##STR00042##
36. A method for preparing germa- and silaxanthones comprising the following steps: a) a regioselective bromination of di-O-TIPS-protected bis(3-hydroxyphenyl)silanes or -germanes to the corresponding di-O-TIPS-protected bis(2-bromo-5-hydroxyphenyl)silanes or -germanes using an electrophilic brominating reagent; b) double metal-halogen exchange on the dibromide resulting from the step a) followed by reaction with a carbamoyl chloride; c) optionally further deprotection/reprotection steps.
37. The method according to claim 36 for preparing the compounds 24, 37-OH below or their O-protected analogs, such as compounds 25 in Scheme 4 and 37 in Scheme 5: ##STR00043##
38. Use of the compounds according to claim 21 as reagents for conjugation or bioconjugation.
39. The use according to claim 36, wherein the conjugation or bioconjugation comprises formation of at least one covalent chemical bond or at least one molecular complex with a chemical entity or substance selected from the group consisting of an amine, thiol, carboxylic acid, aldehyde, alcohol, aromatic compound, heterocycle selected from the group consisting of tetrazine, alkyne, alkene including strained and bicyclic alkenes selected from the group consisting of trans-cyclooctene, cyclopropene and norbornene derivatives, organic azide, dye, amino acid, amino acid residue coupled to any chemical entity, peptide, protein, antibody, single-domain antibody, carbohydrate including a carbohydrate residue attached to a protein, nucleic acid, toxin, lipid, virus, and virus-like particle.
40. A conjugate or bioconjugate comprising a dye according to claim 21 coupled via at least one covalent chemical bond or at least one molecular complex to a chemical entity or substance selected from the group consisting of amine, thiol, carboxylic acid, aldehyde, alcohol, aromatic compound, heterocycle, selected from the group consisting of tetrazine, alkyne, and alkene including strained and bicyclic alkenes selected from the group consisting of trans-cyclooctene, cyclopropene and norbornene derivatives, organic azide, dye, amino acid, an amino acid residue coupled to any chemical entity, peptide, protein selected from the group consisting of enzymes, immunoglobulins, antibodies, single-domain antibodies, and carbohydrates including a carbohydrate residue attached to a protein, nucleic acid, toxin, lipid, virus, and a virus-like particle.
41. A conjugate or bioconjugate comprising a dye according to claim 35 coupled via a covalent bond to Pepstatin A, Jasplakinolide derivatives, Docetaxel derivative, Cabazitaxel derivative and Aminophalloidin depicted below ##STR00044##
42. Use of the compounds according to claim 21 as cell permeant substances penetrating through membranes of living and fixed cells.
43. Use of the conjugates of the compounds according to claim 40 as cell permeant substances penetrating through membranes of living and fixed cells.
44. Use of the compounds according to claim 21 for tracking and monitoring dynamic processes in a sample or in an object.
45. Use of the conjugates of the compounds according to claim 40 for tracking and monitoring dynamic processes in a sample or in an object.
46. Use of the compounds according to claim 21 or of their conjugates according to claim 40 as fluorescent tags, analytical reagents and labels in optical microscopy, imaging techniques, protein tracking, nucleic acid labelling and flow cytometry.
47. Use of the compounds according the conjugates of the compounds according to claim 40 as fluorescent tags, analytical reagents and labels in optical microscopy, imaging techniques, protein tracking, nucleic acid labelling and flow cytometry.
48. The use according to claim 46 wherein the optical microscopy and imaging techniques comprise confocal microscopy, structured illumination microscopy, stimulated emission depletion microscopy [STED], fluorescence correlation spectroscopy [FCS], a combination of STED and FCS (STED-FCS), fluorescence recovery after photobleaching [FRAP], fluorescence lifetime imaging [FLIM], ground state depletion with individual molecular return [GSD or GSDIM], RESOLFT microscopy, fluorescence resonant energy transfer [FRET], single molecule switching techniques selected from the group consisting of single molecule localization microscopy [SMLM], photoactivation localization microscopy [PALM, PALMIRA, fPALM], and stochastic optical reconstruction microscopy [STORM].
Description
FIGURES
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GENERAL MATERIALS AND METHODS
[0104] Thin Layer Chromatography
[0105] Normal phase TLC was performed on silica gel 60 F.sub.254 (Merck Millipore, Germany). For TLC on reversed phase silica gel 60 RP-18 F.sub.254s (Merck Millipore) was used. Preparative TLC was performed on HPTLC Silica gel 60 F.sub.254 with concentrating zone 102.5 cm (Merck Millipore). Compounds were detected by exposing TLC plates to UV-light (254 or 366 nm) or heating with vanillin stain (6 g vanillin and 1.5 mL conc. H.sub.2SO.sub.4 in 100 mL ethanol).
[0106] Column Chromatography
[0107] Silica gel 60 with a particle size of 40-63 m was purchased from Merck Millipore. Reversed phase column chromatography was performed on POLYGOPREP 60-50 C.sub.18 (Macherey Nagel GmbH & Co. KG, Germany). Deactivated silica gel 60 was purchased from MP Biomedical. Routine separation was performed with an automated Isoleram One system (Biotage AG, Sweden) with commercially available cartridges.
[0108] Absorption Spectroscopy
[0109] Absorption spectra were recorded with a Varian Cary 4000 UV-Vis double-beam spectrophotometer (Agilent Technologies, USA). For the determination of the absorption spectra, quartz cells with a 1 cm path length were used. Emission spectra and fluorescence quantum yield were obtained on a Quantaurus-QY Absolute PL quantum yield spectrometer C11347 (Hamamatsu Photonics, Japan) or on a Varian Cary Eclipse fluorescence spectrometer (Agilent Technologies, USA).
[0110] Nuclear Magnetic Resonance (NMR)
[0111] NMR Spectra were recorded on an Agilent 400MR DD2 spectrometer at room temperature (RT; 25 C.). All spectra are referenced to tetramethylsilane as an internal standard (=0.00 ppm) using the signals of the residual protons of CHCl.sub.3 (7.26 ppm) in CDCl.sub.3, CHD.sub.2OD (3.31 ppm) in CD.sub.3OD, CHD.sub.2COCD.sub.3 (2.05 ppm) in (CD.sub.3).sub.2CO or DMSO-d.sub.5 in DMSO-d.sub.6. Multiplicities of the signals are described as follows: s=singlet, br. s=broad singlet, d=doublet, t=triplet, q=quartet, m=multiplet. Coupling constants .sup.nJ.sub.x,y are given in Hz, where n is the number of bonds between the coupled nuclei x and y. For .sup.13C-signals, which were revealed by indirect detection by HSQC, only resonances of the carbon atoms linked to H-atoms were recorded.
[0112] Mass-Spectrometry (MS)
[0113] Low resolution mass spectra (50-3500 m/z) with electro-spray ionization (ESI) were obtained on a Varian 500-MS spectrometer (Agilent Technologies, USA). High resolution mass spectra (ESI-HRMS) were obtained on a Bruker micro TOF (ESI-TOF-MS) spectrometer (Bruker Corporation, USA).
[0114] High-Performance Liquid Chromatography (HPLC)
[0115] HPLC system (Knauer, Germany): Smartline pump 1000 (2) with 10 mL pump-head, UV detector 2500, column thermostat 4000, mixing chamber, injection valve with a 20 or 50 L loop for the analytical and 500 L loop for preparative columns; 6-port-3-channel switching valve; analytical column: Eurospher-100 C18 5 m (if not stated otherwise), or Kinetex C18 100, 5 m, 2504 mm, 1.2 mL/min; preparative column: Kinetex C18 100, 5 m, 25020 mm, 10 mL min/mL, solvent A: water+0.1% v/v trifluoroacetic acid (TFA); solvent B: MeCN+0.1% v/v TFA (if not stated otherwise.) For isolation and purification of the acid sensitive dyes or their derivatives, acetonitrileaqueous systems containing 0.05-0.1 M of Et.sub.3N*H.sub.2CO.sub.3 buffer (pH=8; Sigma, or self-prepared from 1 M aq. Et.sub.3N and CO.sub.2 gas obtained by evaporation of solid CO.sub.2).
[0116] Cell Culture and Live-Labeling
[0117] Either HeLa or U2OS cells were used. Cells were grown on #1.5 coverslips and cultured at 37 C. with 5% CO2 in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS (fetal bovine serum), 1% sodium pyruvate and 1% penicillin/streptomycin. The U2OS cell line is a stable cell line which has a stable expression of the vimentin-HaloTag fusion protein (M. Ratz, I. Testa, S. W. Hell, S. Jakobs, Sci. Rep. 2015, 5, 9592). For the expression of vimentin-HaloTag fusion protein in HeLa cells, cells were transfected with 3 g DNA/well using TurboFect Transfection Reagent (Fermentas/Thermo Scientific, Life Technologies brand) in a 6 well plate. 24 hrs after transfection cells were incubated with the respective dye (typically 1 M) for 20 minutes at growth conditions, washed for 10-30 minutes in HDMEM at 37 C. and placed in an imaging chamber with pre-warmed HDMEM (37 C., DMEM lacking Phenol Red, 10 mM HEPES, pH7.4 1% penicillin/streptomycin). Live STED imaging was performed at room temperature.
[0118] Live STED Imaging
[0119] STED and confocal counterpart images were acquired using the commercially available two-color STED 775 quad scanning microscope from Abberior Instruments (Gbttingen, Germany) equipped with an Olympus IX83 microscope stand and an Olympus UplanSApo 100/1.4 OIL objective.
[0120] Dyes were excited respective their excitation maxima with either a pulsed 561 nm or a pulsed 640 nm laser. The STED laser was pulsed at 775 nm.
[0121] Fluorescence detection was done in two spectral detection channels, 605-625 nm and 650-720 nm.
[0122] For compound 5-Halo the excitation laser was pulsed at 532 nm and the STED laser at 631 nm (
[0123] Imaging and image processing was done with ImSpector software. Images are displayed as raw data unless stated in the figure legend. Pixel size was usually 20-30 nm.
Example 1
Synthesis of Fluorescent Rhodamine Dyes and their Precursors
N,N-Bis-(2,2,2-trifluoroethyl)-6-carboxy-Q-rhodamine-,-diol (5)
[0124] Compound 1-TBS.
[0125] A mixture of 3-(tert-butyldimethyl-silyloxy)aniline (prepared according to M. S. Hossain et al., Org. Biomol. Chem. 2015, 13, 5082-5085)(2.24 g, 10.0 mmol) and epichlorohydrin (0.94 g, 10.0 mmol) in acetic acid (20 mL) was stirred overnight at RT. The mixture was poured into aq. NaHCO.sub.3 (30 g in 300 mL water), extracted with ethyl acetate (350 mL), and the combined organic layers were washed with brine and dried over MgSO.sub.4. The product 1-TBS was isolated by flash column chromatography (Biotage SNAP Ultra 100 g; gradient 0% to 5% ethyl acetateCH.sub.2Cl.sub.2) as light yellow oil, yield 0.84 g (27%). .sup.1H NMR (400 MHz, CDCl.sub.3): 7.02 (t, J=8.0 Hz, 1H), 6.29-6.23 (m, 2H), 6.16 (t, J=2.2 Hz, 1H), 4.09-4.03 (m, 1H), 3.68 (dd, J=11.3, 4.5 Hz, 1H), 3.63 (dd, J=11.2, 6.1 Hz, 1H), 3.35 (dd, J=13.3, 4.4 Hz, 1H), 3.21 (dd, J=13.3, 7.1 Hz, 1H), 0.98 (s, 9H), 0.19 (s, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3): 156.8, 149.1, 129.9, 110.1, 106.8, 105.2, 69.9, 47.7, 47.1, 25.7, 18.2, 4.4. ESI-MS, positive mode: m/z (rel. int., %)=316.3 (100) [M+H].sup.+.
[0126] Compound 1-Me.
[0127] A mixture of 3-methoxyaniline (m-anisidine; 2.46 g, 20.0 mmol) and epichlorohydrin (2.03 g, 22.0 mmol, 1.1 eq) in acetic acid (7 mL) was stirred overnight at RT. The mixture was poured into aq. NaHCO.sub.3 (15 g in 200 mL water), extracted with ethyl acetate (250 mL), the combined organic layers were washed with brine and dried over MgSO.sub.4. The product 1-Me was isolated by flash column chromatography (Teledyne Isco RediSep Rf 120 g; gradient 0% to 5% ethyl acetateCH.sub.2Cl.sub.2) as light yellow oil, yield 1.66 g (38%). .sup.1H NMR (400 MHz, CDCl.sub.3): 7.10 (t, J=8.1 Hz, 1H), 6.32 (ddt, J=8.2, 2.5, 0.7 Hz, 1H), 6.27 (ddt, J=8.1, 2.3, 0.6 Hz, 1H), 6.22 (t, J=2.3 Hz, 1H), 4.06 (ddtd, J=7.2, 6.1, 4.5, 0.5 Hz, 1H), 3.77 (s, 3H), 3.69-3.58 (m, 2H), 3.36 (dd, J=13.3, 4.4 Hz, 1H), 3.21 (dd, J=13.3, 7.2 Hz, 1H). .sup.13C NMR (101 MHz, CDCl.sub.3): 160.9, 149.2, 130.2, 106.3, 103.3, 99.4, 69.8, 55.2, 47.7, 47.1.
[0128] Compounds 2a-TBS and 2b-TBS.
[0129] A solution of 1-TBS (343 mg, 1.09 mmol) and N,N-diethylaniline (810 mg, 5.43 mmol, 5 eq) in 1,2-dichlorobenzene (15 mL) was heated at 140 C. (bath temperature) in a sealed vessel for 5 days. Upon cooling, 1 M NaOH (10 mL) was added, the organic layer was separated, and the aqueous layer containing viscous residue was extracted with CH.sub.2Cl.sub.2 (20 mL). The combined organic layers were washed with 1 M NaOH, water and dried over MgSO.sub.4. TLC control (SiO.sub.2/20% ethyl acetate in CH.sub.2Cl.sub.2): R.sub.f (product)=0.3. The product was isolated by flash column chromatography (Teledyne Isco RediSep Rf 24 g; gradient 0% to 20% ethyl acetateCH.sub.2Cl.sub.2) as light yellow oil, yield 0.84 g (27%), as a 2:1 mixture of regioisomers 2a-TBS and 2b-TBS, used in the next step without separation. Major 7-isomer (2a-TBS): .sup.1H NMR (400 MHz, CDCl.sub.3): 6.81 (d, J=8.3 Hz, 1H), 6.23-6.17 (m, 1H), 6.04 (d, J=2.3 Hz, 1H), 4.23-4.18 (m, 1H), 3.32-3.28 (m, 1H), 3.23-3.20 (m, 1H), 2.97 (ddt, J=16.1, 4.3, 1.3 Hz, 1H), 2.75-2.67 (m, 1H), 0.97 (s, 9H), 0.18 (s, 6H). Minor 5-isomer (2b-TBS): .sup.1H NMR (400 MHz, CDCl.sub.3): 6.87 (t, J=8.0 Hz, 1H), 6.23-6.17 (m, 2H), 4.27-4.22 (m, 1H), 3.28-3.24 (m, 1H), 3.20-3.17 (m, 1H), 2.85 (dd, J=17.4, 4.7 Hz, 1H), 2.80-2.73 (m, 1H), 1.00 (s, 9H), 0.23 (s, 3H), 0.22 (s, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3; 2a-TBS/2b-TBS): 155.0, 154.8, 145.1, 144.3, 131.1, 126.9, 111.5, 110.3, 108.1, 107.3, 105.5, 63.6, 63.4, 47.6, 47.3, 34.9, 30.7, 25.8, 25.7, 18.3, 18.2, 4.2, 4.4. ESI-MS, positive mode: m/z (rel. int., %)=250.2 (100) [M+H].sup.+.
[0130] Compound 2a-Me (7-Isomer).
[0131] A solution of 1-Me (1.61 g, 7.46 mmol) and N,N-diethylaniline (5.56 g, 37.3 mmol, 5 eq) in 1,2-dichlorobenzene (50 mL) was heated at 160 C. (bath temperature) in a sealed vessel for 5 days. Upon cooling, the volatiles were removed on a rotavapor (bath temperature 75 OC), the residue was dissolved in CH.sub.2Cl.sub.2 (100 mL), washed with 1 M NaOH and water (50 mL each). The organic layer was dried over MgSO.sub.4, filtered and dried in vacuo at 85 C. to remove most of N,N-diethylaniline. TLC control (SiO.sub.2/20% ethyl acetate in CH.sub.2Cl.sub.2): R.sub.f (product)=0.3. The product was isolated by flash column chromatography (Teledyne Isco RediSep Rf 80 g; gradient 0% to 30% ethyl acetateCH.sub.2Cl.sub.2) as light yellow oil, yield 468 mg (35%) of 2a-Me (7-isomer, nearly free from 5-isomer). .sup.1H NMR (400 MHz, CDCl.sub.3): 6.88 (d, J=8.3 Hz, 1H), 6.28 (dd, J=8.3, 2.5 Hz, 1H), 6.10 (d, J=2.5 Hz, 1H), 4.22 (qd, J=4.6, 2.3 Hz, 1H), 3.74 (s, 3H), 3.31 (dtd, J=11.3, 1.6, 0.8 Hz, 1H), 3.22 (ddd, J=11.3, 5.1, 2.0 Hz, 1H), 3.02-2.92 (m, 1H), 2.78-2.67 (m, 1H), 2.32 (br.s, 1H). .sup.13C NMR (101 MHz, CDCl.sub.3): 159.2, 144.6, 131.4, 111.1, 104.2, 99.6, 63.7, 55.3, 47.7, 34.9. ESI-MS, positive mode: m/z (rel. int., %)=180.2 (100) [M+H].sup.+.
[0132] Compound 3-Me (Method A).
[0133] A solution of 2a-Me (205 mg, 1.14 mmol) and triethylamine (289 mg, 2.86 mmol, 2.5 eq) in CH.sub.2Cl.sub.2 (10 mL) was cooled in dry ice-acetone bath, and trifluoroacetic anhydride (TFAA; 365 L, 551 mg, 2.62 mmol, 2.3 eq) was added. The resulting mixture was stirred for 10 min, allowed to warm up to RT and stirred for 1 h. The mixture was diluted with CH.sub.2Cl.sub.2 (10 mL), washed sequentially with water, 10% aq. KHSO.sub.4, sat. aq. NaHCO.sub.3, brine (15 mL of each) and dried over MgSO.sub.4. The filtrate was evaporated, the residue was dissolved in THF (10 mL) and BH.sub.3.THF (1 M in THF, 8 mL, 8 mmol) was added. The reaction mixture was refluxed under argon overnight, cooled in ice-water bath and quenched by careful addition of methanol. The solution was evaporated, 1 N NaOH (15 mL) was added to the residue and the mixture was stirred for 30 min at RT. The reaction mixture was extracted with diethyl ether (320 mL), the organic layer was washed with sat. aq. NaHCO.sub.3, brine (20 mL of each) and dried over MgSO.sub.4. The product was isolated by flash column chromatography (Bchi Sepacore Silica HP 12 g; gradient 0% to 10% ethyl acetate CH.sub.2Cl.sub.2), yielding 230 mg (77% over 2 steps) of 3-Me as yellowish oil.
[0134] Method B:
[0135] Solid 2,2,2-trifluoroethyl(mesityl)iodonium trifluoromethanesulfonate (T. Umemoto, Y. Gotoh, J. Fluorine Chem. 1986, 31, 231-236; G. L. Tolnai, A. Szkely, Z. Mak, T. Gti, J. Daru, T. Bihari, A. Stirling, Z. Novk, Chem. Commun. 2015, 51, 4488-4491) was added portionwise over 2-3 min to a solution of 2a-Me (90 mg, 0.5 mmol) and 2,6-lutidine (87 L, 80 mg, 0.75 mmol, 1.5 eq) in dry CH.sub.2Cl.sub.2 (3 mL), and the resulting clear solution was stirred at RT for 1 h. The mixture was diluted with sat. aq. NaHCO.sub.3 (10 mL), extracted with CH.sub.2Cl.sub.2 (320 mL), the combined extracts were washed with brine and dried over Na.sub.2SO.sub.4. The product was isolated by flash column chromatography (Bchi Sepacore Silica HP 12 g; gradient 20% to 100% ethyl acetatehexane), fractions containing the product were evaporated and dried in vacuo to remove any residual 2,6-lutidine. Yield 118 mg (90%) of 3-Me as viscous yellowish oil. .sup.1H NMR (400 MHz, CDCl.sub.3): 6.92 (dt, J=8.2, 1.0 Hz, 1H), 6.33 (dd, J=8.2, 2.4 Hz, 1H), 6.28 (d, J=2.3 Hz, 1H), 4.21 (qd, J=5.4, 2.7 Hz, 1H), 3.96-3.83 (m, 1H), 3.82-3.72 (m, 1H), 3.77 (s, 3H), 3.50 (dt, J=11.7, 2.1 Hz, 1H), 3.29 (ddd, J=11.7, 5.7, 1.8 Hz, 1H), 3.05-2.95 (m, 1H), 2.74 (ddt, J=15.9, 5.6, 1.2 Hz, 1H), 2.14 (br.s, 1H). .sup.13C NMR (101 MHz, CDCl.sub.3): 159.5, 144.3, 131.4, 125.6 (q, J=283.3 Hz), 112.0, 103.4, 98.6 (q, J=1.8 Hz), 63.3, 56.5, 55.4, 53.5 (q, J=32.8 Hz), 35.6. .sup.19F NMR (376 MHz, CDCl.sub.3): 69.8. ESI-MS, positive mode: m/z (rel. int., %)=262.1 (100) [M+H].sup.+.
[0136] Compound 3-H (from 2a,b-TBS).
[0137] A solution of mixed isomers 2a,b-TBS (169 mg, 0.605 mmol) and triethylamine (153 mg, 1.51 mmol, 2.5 eq) in CH.sub.2Cl.sub.2 (10 mL) was cooled in dry ice-acetone bath, and trifluoroacetic anhydride (TFAA; 193 L, 292 mg, 1.40 mmol, 2.3 eq) was added. The resulting mixture was stirred at 78 OC for 10 min, allowed to warm up to RT and stirred for 1 h. The mixture was diluted with CH.sub.2Cl.sub.2 (10 mL), washed sequentially with water, 10% aq. KHSO.sub.4, sat. aq. NaHCO.sub.3, brine (15 mL of each) and dried over MgSO.sub.4. The filtrate was evaporated, the residue was dissolved in THF (10 mL) and BH.sub.3.THF (1 M in THF, 4.5 mL, 4.5 mmol) was added. The reaction mixture was refluxed under argon overnight, cooled in ice-water bath and quenched by careful addition of methanol. The solution was evaporated, the residue was treated with 1 M NaOH (20 mL), extracted with diethyl ether (10 mL), the organic layer was discarded, and the aqueous layer was carefully neutralized with 1 M HCl and then with phosphate buffer to pH 7.5. The resulting aqueous solution was saturated with NaCl and extracted with diethyl ether (320 mL), the combined extracts were dried over MgSO.sub.4. The products were isolated by flash column chromatography (Interchim Puriflash 15 m 25 g; gradient 0% to 40% ethyl acetateCH.sub.2Cl.sub.2), yielding 45 mg (30%) of 3-H (less polar 7-isomer, eluted first) as yellowish oil, along with 12 mg (8%) of impure 5-regioisomer.
[0138] Compound 3-H (from 3-Me).
[0139] A solution of 3-Me (650 mg, 3.0 mmol), thiophenol (0.40 g, 3.6 mmol) and K.sub.2CO.sub.3 (20 mg) was heated in N-methylpyrrolidone at 180 C. (bath temperature) for 24 h. The reaction mixture was cooled down, diluted with diethyl ether (100 mL) and washed with 1 M NaOH (20 mL). The organic layer was discarded, and the aqueous layer was neutralized to pH8 with 5% aq. citric acid. The title product (and, partially, thiophenol) was extracted with ether (320 mL). Combined organic solutions were dried (Na.sub.2SO.sub.4) and evaporated. The residue was subjected to chromatography on 25 cm.sup.3 SiO.sub.2 (Isolera), and the title compound (0.4 g, 65%) isolated by elution with a CH.sub.2Cl.sub.2-EtOAc mixture (10-50% EtOAc). .sup.1H NMR (400 MHz, CD.sub.3CN): 6.78 (dt, J=8.0, 1.0 Hz, 1H), 6.63 (s, 1H), 6.19 (br.d, J=2.6 Hz, 1H), 6.14 (dd, J=8.0, 2.3 Hz, 1H), 4.05 (dddd, J=11.9, 7.1, 4.7, 3.1 Hz, 1H), 4.00-3.81 (m, 2H), 3.42 (ddd, J=11.6, 3.3, 1.9 Hz, 1H), 3.21-3.14 (m, 1H), 3.05 (d, J=5.1 Hz, 1H), 2.87 (ddt, J=15.5, 4.7, 1.3 Hz, 1H), 2.58 (ddt, J=15.5, 7.1, 1.2 Hz, 1H). .sup.19F NMR (376 MHz, CD.sub.3CN): 70.7. .sup.13C NMR (101 MHz, CD.sub.3CN): 157.2, 145.7, 131.9, 127.2 (q, J=282.7 Hz), 113.1, 105.9, 99.6 (q, J=1.6 Hz), 63.7, 57.3, 53.7 (q, J=32.2 Hz), 36.3. ESI-MS, positive mode: m/z (rel. int., %)=248.1 (100) [M+H].sup.+.
[0140] Compound 4 (Scheme 2) was prepared according to a published procedure (G. Mudd, I. Prez Pi, N. Fethers, P. G. Dodd, O. R. Barbeau, M. Auer, Methods Appl. Fluoresc. 2015, 3, 045002).
N,N-Bis-(2,2,2-trifluoroethyl)-6-carboxy-Q-rhodamine-,-diol (5-OH)
[0141] Compound 3 (25 mg, 0.1 mmol), compound 4 (15 mg, 0.072 mmol) and p-toluenesulfonic acid monohydrate (5 mg) were heated in propionic acid (1 mL) at 80 C. overnight. DDQ (16 mg, 0.07 mmol) was added to the reaction mixture at room temperature (RT), it was heated to 40 C., and stirred at this temperature for 1 h. All volatile materials have been removed in vacuo, and the residue dissolved in CH.sub.2Cl.sub.2 (100 mL). The organic solution was washed with water and sat. aq. NaHCO.sub.3. Combined aqueous solutions were extracted with CH.sub.2Cl.sub.2 (20 mL). Combined organic solutions were dried (Na.sub.2SO.sub.4), filtered, and dichloromethane was evaporated in vacuo. The residue was dissolved in MeOH (3 mL), and 2 M aq. NaOH (0.5 mL) was added to the methanolic solution. The reaction mixture was kept overnight at room temperature, and all volatile materials were evaporated in vacuo. The residue was dissolved in aqueous MeCN and acidified with aq. TFA. TLC on reversed phase (VWR International, TLC Siliga gel 60 RP-18 F.sub.254s) in the solvent system MeCN/H.sub.2O=1:2 (+0.3% HCOOH) revealed a brightly fluorescent orange spot. The title dye was isolated by RP chromatography on Polygoprep 60-50 C.sub.18 (Macherey Nagel). Elution with aqueous acetonitrile (MeCN/H.sub.2O=1:2+0.1% TFA) followed by direct lyophilization of the pooled fractions containing diastereomers of dye 5 afforded 8 mg of red voluminous solid (mixture of 3 diastereomers). HPLC: analytical column: Kinetex C18 100, 5 m, 2504.6 mm, 1.2 mL/min; solvent A: water+0.1% v/v trifluoroacetic acid (TFA); solvent B: MeCN+0.1% v/v TFA. Analytical method: A/B: 70/300/100 in 20 min, UV-VIS detection with dioden array. t.sub.R=5.4 min (isomers 1+2 are inseparable under these conditions), 5.6 min (isomer 3) in ratio ca. 2.5:1. Substances with t.sub.R=5.4 min and t.sub.R=5.4 min were isolated by preparative HPLC; preparative column: Kinetex C18 100, 5 m, 25020 mm, 10 mL min/mL, solvent A: water+0.05% v/v trifluoroacetic acid (TFA); solvent B: MeCN. By analytical HPLC using another buffer [solvent A: water+0.05 M Et.sub.3N*H.sub.2CO.sub.3 buffer with pH=8; solvent B: MeCN; method: A/B: 80/20.fwdarw.0/100 in 20 min, UV-VIS detection with dioden array], it turned out that the substance with t.sub.R=5.4 min (see above) was a mixture of two isomers (1+2) with t.sub.R=5.0 min and 5.2 min (2:1). Isomers 1+2 were used in the preparation of an NHS ester and an amide from Halo-Tag-amine (see below). Isomers differ in mutual orientations of hydroxyl groups (syn and anti). For the syn-isomer, an additional pair of diastereomers is possible, due to the hindered rotation of the phenyl ring with 2 carboxylic acid groups (see
N-Hydroxysuccinimidyl eater of N,N-bis-(2,2,2-trifluoro-ethyl)-6-carboxy-Q-rhodamine-,-diol (5-NHS) and its conjugate with Halo-Tag amine (5-Halo)
[0142] Three stock solutions (A, B and C) were prepared. Solution A: 22 mg HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate) in dry DMF (0.10 mL); solution B: Et.sub.3N (10 mg) in dry DMF (0.10 mL); solution C: N-hydroxysuccinimide (19 mg) in dry DMF (0.10 mL). To a solution of dye 5 (1.7 mg, 2.6 mol) in dry DMF (0.10 mL), solutions A, B and C were added successively (14 L of each solutions) under argon, and the mixture was stirred for 30 min at room temperature. HPLC analysis indicated the conversion degree of 94-95%. (Eurospher-100 C18 5 m 2504 mm, 1.2 mL/min; solvent A: water+0.1% v/v trifluoroacetic acid (TFA); solvent B: MeCN+0.1% v/v TFA. A/B: 30/70-100/0, 25 min, t.sub.R=6.7 min (compound 5), 8.3/8.6 min (mono-NHS esters, mixture of diastereomers). The product (6-NHS ester of dye 5, a stable compound 5-NHS) can be isolated by preparative HPLC (after removing DMF in vacuo), or used in situ. In the latter case, Halo-Tag-amine (H.sub.2N(CH.sub.2).sub.2O(CH.sub.2).sub.2O(CH.sub.2).sub.6Cl=C.sub.10H.sub.22ClNO.sub.2, 1.5 mg, 6.7 M) was added to the reaction mixture as a solution in DMF (20-40 L), and the reaction mixture was stirred at 40 C. After several hours, HPLC control indicated that the conversion to new products with t.sub.R=11.9/12.2 min (5-Halo, mixture of dye diastereomers) was nearly complete (93%; HPLC area). After removing DMF in vacuo, amide 5-Halo was isolated by preparative HPLC (1 mg, 46%), dissolved in DMSO (400 L), and this stock-solution was used (after dilution with aqueous PBS to 1-5 M) in labeling experiments with living cells (see section with imaging results). ESI-MS (C.sub.41H.sub.44C.sub.1F.sub.6N30.sub.8, 855.27), positive mode: m/z (rel. int., %)=856 (100) [M+H].sup.+. HR-MS, ESI (positive mode): m/z=878.2591 (found [M+Na].sup.+), 878.2613 (calc.).
Hydroxylated ROX Dyes (13a,b)
7-(tert-Butyl-dimethyl-silanyloxy)-1,2,3,4-tetrahydroquinoline (6)
[0143] in Scheme 2 was prepared according to the published method of S. M. Pauff and S. C. Miller (Org. Lett. 2011, 13, 6196-6199) with modifications (Kolmakov et al., Chem. Eur. J. 2015, 21, 13344-13356).
[0144] Compound 7.
[0145] Compound 6 (90 mg) was dissolved in neat epichlorohydrin (1 mL), and the solution was heated at 80 C. for 30 h. The reaction mixture was applied onto column with SiO.sub.2 (10 g) packed in hexane-dichloromethane mixture (3:1). Elution with hexane-dichloromethane (3:1.fwdarw.1:1) afforded 60 mg of compound 7 (oil). .sup.1H NMR (400 MHz, CDCl.sub.3): =6.80 (dd, J=8.1, 0.9 Hz, 1H), 6.16 (d, J=2.3 Hz, 1H), 6.13 (dd, J=8.1, 2.3 Hz, 1H), 4.15 (m, 1H), 3.70 (dd, J=11.2, 4.3 Hz, 1H), 3.62 (dd, J=11.3, 5.4 Hz, 1H), 3.35 (dd, J=6.4, 4.0 Hz, 1H), 3.30 (dd, J=6.4, 4.0 Hz, 1H), 3.30 (td, J=5.3, 1.3 Hz, 2H), 2.70 (t, J=6.4 Hz, 2H), 2.49 (d, J=4.6 Hz, 1H), 1.92 (m, 2H), 0.98 (s, 9H), 0.20 (s, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3): =156.8, 149.0, 129.9, 110.1, 106.8, 105.2, 69.9, 47.7, 47.1, 25.70, 18.2, 4.4. MS (ESI): m/z (positive mode, %)=356 (100) [M+H].sup.+.
Alcohol 8a and oxirane 8b (tert-butyl-dimethyl-[[1-(oxiran-2-ylmethyl)-3,4-dihydro-2H-quinolin-7-yl]-oxy]silane
[0146] Amine 6 (1.60 g, 6.08 mmol) was dissolved in a mixture of epichlorohydrine (7.28 g, 79.1 mmol, 6.15 mL) and 1,2-dichlorobenzene (15 mL). The mixture was heated at 140 C. (oil bath temperature) for 18 h in a thick-walled screw-cup test tube. Then all volatile materials were distilled-off under reduced pressure (0.5-1 mbar, bath temperature 50-100 C.) leaving 2.64 g of colorless oil. TLC (hexane-ethyl acetate, 3:1, or dichloromethane-ethyl acetate, 10:1) indicated complete consumption of the starting material (compound 6) and formation of two new compounds. The residual oil was subjected to chromatography over regular silica gel (Isolera, RediSep Rf cartridge with 40 g SiO.sub.2, hexane-ethyl acetate, from 95:5 to 70:30). Alcohol 8a was isolated as a red-brown viscous oil (1.02 g, 52%); oxirane 8b (0.52 g, 27%)as a light yellow viscous oil.
[0147] Compound 8a. .sup.1H NMR (400 MHz, CDCl.sub.3): =6.70 (dt, J=8.1, 0.9 Hz, 1H), 6.16 (d, J=8.1 Hz, 1H), 4.36 (dddd, J=14.4, 7.7, 6.8, 4.9 Hz, 1H), 3.16-2.99 (m, 3H), 2.90-2.61 (m, 4H), 2.37 (d, J=9.3 Hz, 1H), 2.10-1.92 (m, 2H), 0.99 (s, 9H), 0.20/0.21 (2s, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3): =152.7, 143.3, 126.8, 115.1, 110.4, 107.4, 63.3, 55.4, 50.4, 31.50, 27.1, 25.8, 25.6, 22.3, 18.2, 4.1. MS (ESI): m/z (positive mode, %)=320.2 (100) [M+H].sup.+. HR-MS (C.sub.18H.sub.29NO.sub.2Si): 320.2048 (found M+H), 320.2040 (calc.); 342.1864 (found for M+Na), 342.1860 (calc.).
[0148] Compound 8b. .sup.1H NMR (400 MHz, CDCl.sub.3): =6.77 (ddt, J=7.8, 1.0, 0.5 Hz, 1H), 6.16-6.03 (m, 2H), 3.52 (dd, J=12 and 3.2 Hz, 1H), 3.39-3.25 (m, 3H), 3.15 (dddd, J=4.6, 4.0, 3.2, 2.7 Hz, 1H), 2.81-2.75 (m, 1H), 2.68 (t, J=6.4 Hz, 2H), 2.58 (dd, J=5.0, 2.7 Hz, 1H), 2.00-1.80 (m, 2H), 0.97 (s, 9H), 0.19 (s, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3): =154.9, 146.1, 129.5, 115.6, 107.7, 103.0, 52.9, 50.4, 50.3, 45.4, 27.3, 25.8, 22.4, 18.2, 4.4. MS (ESI): m/z (positive mode, %)=320 (100) [M+H].sup.+. HR-MS (C.sub.18H.sub.29NO.sub.2Si): 320.2041 (found M+H), 320.2040 (calc.).
[0149] 8a-Acetate.
[0150] In an argon atmosphere, alcohol 8a (0.66 g, 2.1 mmol) was dissolved in pyridine (2.22 g, 28.0 mmol, 2.26 mL) in a screw-cup tube containing a stirring bar. Acetic anhydride (1.19 g, 11.6 mmol, 1.10 mL) was added at room temperature, and the reaction mixture was stirred for 18 h. The course of the reaction was monitored by TLC using dichloromethane-hexane mixture (3:1) with 10% v/v of ethyl acetate. The reaction mixture was concentrated in vacuo to ca. 3/10 of its initial volume and diluted with ethyl acetate (100 mL). The organic solution was shaken with saturated aq. KHSO.sub.4 (20 mL); an aqueous phase was separated and extracted with EtOAc (100 mL). The combined organic solutions were washed with water, saturated aqueous NaHCO.sub.3 and brine. The organic phase was dried over Na.sub.2SO.sub.4, and the solvent was evaporated in vacuo. The residuea reddish-brown viscous oilwas subjected to chromatography on regular SiO.sub.2. Elution with dichloromethane-hexane (1:1 to 1:0) gave acetate 9 (0.564 g, 75%) as a red-orange viscous oil. .sup.1H NMR (400 MHz, CDCl.sub.3): =6.68 (dt, J=8.1, 0.9 Hz, 1H), 6.11 (d, J=8.1 Hz, 1H), 5.26 (me, 1H), 3.26 (ddd, J=11.3, 3.5, 1.7 Hz, 1H), 3.17-2.92 (m, 4H), 2.76-2.62 (m, 3H), 2.07 (s, 3H), 2.02-1.91 (m, 2H), 0.99 (s, 9H), 0.21 (s, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3): =170.8, 152.1, 143.0, 126.8, 114.6, 109.5, 106.8, 66.9, 52.3, 50.1, 27.9, 27.1, 25.8, 22.2, 21.4, 18.3, 4.1. MS (ESI): m/z (positive mode, %)=362.2 (100) [M+H].sup.+, HR-MS (C.sub.20H.sub.31NO.sub.3Si): 384.1964 (found for M+Na), 384.1965 (calc. for M+Na).
[0151] Phenol 9.
[0152] A solution of 8a-acetate (0.560 g, 1.55 mmol) in acetonitrile (14 mL) was prepared in a polyethylene screw-cap bottle and cooled with ice. An aqueous HF solution (0.2 mL of 50-55% solution) was added; the ice bath was removed, and the reaction mixture was stirred at room temperature for 20 h. TLC (dichloromethane/ethyl acetate, 10:1) indicated the full conversion of the staring material. The reddish reaction solution was diluted with H.sub.2O (50 mL) and extracted four times with ethyl acetate (50 mL and 325 mL). The combined organic solutions were washed with brine and dried over Na.sub.2SO.sub.4. The solvents were removed under reduced pressure, and the residue (0.46 g of a green viscous semi-solid) was subjected to chromatography (PuriFlash HC, 25 g SiO.sub.2, 15 m, dichloromethane/ethyl acetate 95:5.fwdarw.80:20). Yield0.314 g (82%) of phenol 9 as a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3): =7.26 (s, 1H), 6.69 (d, J=8.0, 1H), 6.07 (d, J=8.0 Hz, 1H), 5.26 (tdd, J=6.8, 5.8, 3.5 Hz, 1H), 4.48 (s, 1H), 3.28 (ddd, J=11.4, 3.5, 1.7 Hz, 1H), 3.19-2.98 (m, 4H), 2.78-2.66 (m, 3H), 2.07 (s, 3H), 2.11-1.89 (m, 2H). .sup.13C NMR (101 MHz, CDCl.sub.3): =170.7, 152.0, 142.9, 127.1, 114.3, 105.2, 103.3, 66.6, 52.2, 50.1, 27.1, 22.2, 21.3. MS (ESI): m/z (positive mode, %)=248.1 (100) [M+H].sup.+. HR-MS (C.sub.14H.sub.17NO.sub.3): 270.1105 (found for M+Na), 270.1101 (calc.).
[0153] Benzophenone 12a.
[0154] In a screw-cap bottle, 8-hydroxyjulolidine 10 (0.60 g, 3.17 mmol) and trimellitic anhydride 11 (1.83 g, 9.51 mmol) were suspended in acetic acid (15 mL) and flushed with argon. Conc. H.sub.2SO.sub.4 (1 drop) was added, and the reaction mixture was gradually heated in an oil bath (up to 100 C.) and stirred for 2.5 h. The precipitate dissolved at about 80-90 C., and a red solution formed. After about 0.5 h, a new precipitate was formed. After cooling to room temperature, the purple suspension was transferred into centrifuge test-tubes, sonicated for several seconds in an ultrasonic bath, centrifuged, and the purple supernatant solution was discarded. The precipitate was washed with acetic acid (5 mL); the same sonicationcentrifugation cycles were repeated 5-6 times, until the supernatant solution became colorless. The solid residue was dried in vacuo to obtain 0.48 g (40%) of benzophenone 12a with traces of 12b (>95:<5). Beige solid; TLC: ACN/H.sub.2O/DCM, 9:1:1. HPLC (Eurospher-100 C18 5 m 2504 mm, 1.2 mL/min; solvent A: water+0.1% v/v trifluoroacetic acid (TFA); solvent B: MeCN+0.1% v/v TFA). A/B: 30/70-100/0, 25 min, t.sub.R=9.7 min (12a), 10.7 min (12b). .sup.1H NMR (400 MHz, DMSO-d.sub.6) 12.82 (s, 1H), 8.09 (dd, J=8.1, 1.7 Hz, 1H), 8.02 (d, J=8.1 Hz, 1H), 7.76 (d, J=1.7 Hz, 1H), 6.39 (s, 1H), 3.24 (td, J=7.4, 4.8 Hz, 4H), 2.58 (t, J=6.4 Hz, 2H), 2.40 (t, J=6.2 Hz, 2H), 1.91-1.78 (m, 2H), 1.80-1.69 (m, 2H). C.sub.21H.sub.19NO.sub.6 (381.12124). MS (ESI): m/z (negative mode, %)=380 (100) [MH].sup..
[0155] Compounds 13a-c.
[0156] Benzophenone 12a (23 mg, 0.060 mmol) and acetate 9 (15 mg, 0.061 mmol) were dissolved in 2.3 mL DMF, trimethylsilyl polyphosphate (170 mg) in DMF (0.4 mL) were added, and the reaction mixture was heated under argon for 3 h at 100 C. (or 2 h at 120 C.). After cooling down to room temperature, the reaction mixture was diluted with 40 mL of the half-saturated brine and transferred into a separation funnel with CH.sub.2Cl.sub.2 (40 mL) and 5 mL acetonitrile. After shaking and separation of the organic layer, the aqueous phase was extracted with CH.sub.2Cl.sub.2-acetonitrile mixture (40 mL+5 mL) and with CH.sub.2Cl.sub.2 (20 mL). Combined organic solutions were washed with water (20 mL), and the aqueous extract was washed with CH.sub.2Cl.sub.2 (40 mL). HPLC analysis of the reaction mixture: columnEurospher-100 C18 5 m, 2504 mm, 1.2 mL/min; solvent A: water+0.1% v/v trifluoroacetic acid (TFA); solvent B: MeCN+0.1% v/v TFA A/B=70/30-0/100 in 20 min, detection at 580 nm. Retention times (t.sub.R), min (peak areas, %, compounds): 10.2 (30, 13b), 11 (46, 13a), 12.3 (24, 13c). Combined organic solutions were evaporated in vacuo. and the residue was applied onto a column with regular SiO.sub.2 (55 g). Elution with acetonitrile-water mixture (10:1.fwdarw.5:1+5% v/v CH.sub.2Cl.sub.2) afforded magenta-colored fractions; first these with compound 13c, and theninseparable mixture of mono- and diacetates of dyes 13a and 13b (displaying a single spot on TLC). These fractions were combined, evaporated in vacuo, and the residue was dissolved in 10 mL of 0.1 M aq. NaOH. After keeping overnight at room temperature, the reaction mixture was acidified with TFA (0.2 mL). Then the reaction mixture saturated with NH.sub.4Cl, and extracted twice with CH.sub.2Cl.sub.2 acetonitrile mixtures (5:1 and 5:2, 60 mL and 70 mL, respectively). Combined organic solutions were evaporated in vacuo, and the residue was subjected to chromatography on regular SiO.sub.2 using the solvent system CH.sub.2Cl.sub.2MeOHH.sub.2O (75/30/2). Dyes 13a and 13b were isolated in approximately equal amounts and additionally purified by RP chromatography on RP C18 (Macherey Nagel, Polygoprep 60-50) using acetonitrile-water mixture (1/5.fwdarw.1/1; +0.2% v/v HCOOH). Homogeneous fractions were pooled, evaporated and afforded diastereomers 13a and and 7.7 min (in ratio ca. 2:3:1; HPLC conditions as above). .sup.1H NMR (400 MHz, CD.sub.3OD, 13a, mixture of 3 diastereomers): =8.17 (dd, J=8.1 and 1.7 Hz, 1H), 8.06 (d, J=7.9 Hz, 1H), 7.78-7.71 (m, 1H), 6.85-6.79 (m, 1H), 4.57 (m, 1H), 4.36 (m, 2H), 3.64 (br. d, J=13.0 Hz, 2H), 3.50 (dq, J=17.5, 6.1 Hz, 4H), 3.38 (dd, J=13.4, 5.2 Hz, 1H), 3.21-3.13 (m, 1H), 3.08 (t, J=6.5 Hz, 1H), 2.70 (s, 4H), 2.12 (m, 2H), 1.99-1.89 (m, 4H). .sup.1H NMR (400 MHz, CD.sub.3OD, compound 13b; main diastereomer): =8.16 (dd, J=8.1 and 1.7 Hz, 1H), 8.04 (d, J=8.0 Hz, 1H), 7.73 (m, 1H), 6.85 (s, 2H), 4.36 (m, 2H, CHOH), 3.65 (br. d, J=13.5 Hz, 2H), 3.48 (m, J=5.6 Hz, 4H), 3.38 (dd, J=13.3 and 5.2 Hz, 2H), 3.18 (m, 4H), 2.71 (m, 4H), 1.96 (m, 4H) ppm. The following spectral data mere obtained for aqueous solutions containing PBS buffer (pH 7.4). 13a: absorption .sub.max, nm (, M.sup.1 cm.sup.1)=574 (55000), emission .sub.max, nm (.sub.fl-fluorescence quantum yield)=597 (0.74absolute value; excitation at 540-550 nm). 13b: absorption .sub.max, nm (, M.sup.1 cm.sup.1)=564 (60000), emission .sub.max, nm (.sub.fl-fluorescence quantum yield)=588 (0.63absolute value; excitation at 550 nm). 13a (C.sub.33H.sub.30N.sub.2O.sub.6, M=550). HR-MS (ESI): m/z (positive mode)=551.2181 (found [M+H].sup.+), 551.2177 (calculated). MS (ESI): m/z (negative mode, %)=549 (100) [MH].sup.. 13b (C.sub.33H.sub.30N.sub.2O.sub.7, M=566). HR-MS (ESI): m/z (negative mode)=565.1976 (found [MH].sup.), 565.1980 (calculated).
##STR00026##
[0157] General Method for Obtaining Mono-NHS Eaters 14a-c-NHS.
[0158] A dye (13a, 13b or 13c) in an amount of ca. 3.5 mg (ca. 6.3 M) is dissolved in the mixture of DMF (1.5 mL) and CH.sub.2Cl.sub.2 (0.5 mL) and treated with Et.sub.3N (portions of 2.5 L) and disuccinimidyl carbonate (portions of 5 mg). After addition of each portion, the course of the reaction is monitored by HPLC [columnEurospher-100 C18 5 m, 2504 mm, 1.2 mL/min; solvent A: water+0.1% v/v trifluoroacetic acid (TFA); solvent B: MeCN+0.1% v/v TFA A/B=70/30-0/100 in 20 min, detection at 580 nm] and/or TLC (regular SiO.sub.2, ACN/H.sub.2O, 5:1). The reaction was complete, when 20 mg of DSC and 15 L of Et.sub.3N were added. The spot of the double NHS ester was also detected (with highest R.sub.f; blue color), and (sometimes) the spot of the mono-NHS ester of the sterically more hindered carboxylic acid group. The latter had the lowest R.sub.f-value (and gave the bluish spot). The reaction mixture was diluted with the equal volume of dry CAN and applied onto a column with regular SiO.sub.2 (10 g). Elution with ACN/CH.sub.2Cl.sub.2/H.sub.2O (10:1:1) followed by ACN/H.sub.2O (5:1) afforded the required mono-NHS esters 14a-c-NHS as magenta-colored solutions. They were concentrated, filtered through the Rotilabo membrane filters (attached to syringes; in order to remove SiO.sub.2) and lyophilized. 14a-NHS (C.sub.37H.sub.33N.sub.3O.sub.8, M=647). MS (ESI): m/z (positive mode, %)=648 (100) [M+H].sup.+; 14b-NHS (C.sub.37H.sub.33N.sub.3O.sub.9, M=663.2217). HR-MS (ESI): m/z (positive mode, %)=664.2252 (found [M+H].sup.+), 664.2290 (calculated).
[0159] General method for obtaining mono-amides 14a-c-NH(CH.sub.2).sub.2O(CH.sub.2).sub.2O(CH.sub.2).sub.6Cl.
[0160] Mono-NHS esters 14a-c-NHS in amounts of ca. 1 mg (ca. 2 M) were dissolved in ca. 200 L of the solution of Cl(CH.sub.2).sub.6O(CH.sub.2).sub.2O(CH.sub.2).sub.2NH.sub.2 in DMF (20 mg/mL), and Et.sub.3N (10 L) was added with stirring. The course of the reaction was monitored by TLC. When the reaction was complete (ca. 2 h), the reaction mixture was diluted with CH.sub.2Cl.sub.2 acetonitrile mixture (1:3) and extracted with equal volume of the saturated aqueous NaHCO.sub.3 solution (lower phase). The upper organic layer was separated and extracted with half-saturated aqueous NH.sub.4Cl acidified with trifluoroacetic acid (TFA; in order to remove excess of the amine). The organic solution was evaporated in vacuo, and the residue applied onto a column with regular SiO.sub.2 (5 g). Elution with CH2Cl2methanol (10:1.fwdarw.8:1) afforded the title amides which were additionally purified by chromatography on regular SiO.sub.2 (5 g) using MeCNH.sub.2O mixture (10:1.fwdarw.5:1). Pure fractions were pooled, concentrated, filtered through the Rotilabo syringe filters (0.22 m), and lyophilized. 14a-NH(CH.sub.2).sub.2O(CH.sub.2).sub.2O(CH.sub.2).sub.6Cl (C.sub.43H.sub.50N.sub.3O.sub.7Cl): HR-MS (ESI): m/z (positive mode, %)=756.3384 (found [M+H].sup.+), 756.3410 (calculated). HPLC [columnKinetex 5 m C18 100, 2504 mm, 1.2 mL/min; solvent A: 0.05 M Et.sub.3N*H.sub.2CO.sub.3 buffer (TEAB); solvent B: MeCN, A/B=70/30-0/100 in 20 min, dioden-array detection at 600 nm]; t.sub.R=9.7 and 10.5 min (in ratio 1:1)2 diastereomers differing in the mutual position of OH and COOH groups; see main text. The following spectral data were obtained for aqueous solutions containing PBS buffer (pH 7.4). Absorption .sub.max, nm (, M.sup.1 cm.sup.1)=578 (55000), emission .sub.max, nm (.sub.fl)=601 (0.74relative value obtained with Rhodamine 101 as a reference dye with .sub.fl=0.96 in ethanol; excitation at 540 nm). 14b-NH(CH.sub.2).sub.2O(CH.sub.2).sub.2O(CH.sub.2).sub.6Cl (C.sub.43H.sub.50N.sub.3O.sub.8Cl): HR-MS (ESI): m/z (positive mode, %)=772.3329 (found [M+H].sup.+), 772.3359 (calculated). HPLC [columnKinetex 5 m C18 100, 2504 mm, 1.2 mL/min; solvent A: 0.05 M Et.sub.3N*H.sub.2CO.sub.3 buffer (TEAB); solvent B: MeCN, A/B=70/30-0/100 in 20 min, diode array detection in the range 200-700 nm]; t.sub.R=8.0 and 8.0 min (in ratio 1:1)2 diastereomers differing in the mutual position of OH and COOH groups; see main text. The following spectral data were obtained for aqueous solutions containing PBS buffer (pH 7.4). Absorption .sub.max, nm (, M.sup.1 cm.sup.1)=575 (63000), emission .sub.max, nm (.sub.fl)=598 (0.55relative value obtained with Rhodamine 101 as a reference dye with .sub.fl=0.96 in ethanol; excitation at 540 nm). 14c-NH(CH.sub.2).sub.2O(CH.sub.2).sub.2O(CH.sub.2).sub.6Cl. The following spectral data were obtained for aqueous solutions containing PBS buffer (pH 7.4). Absorption .sub.max, nm (, M.sup.1 cm.sup.1)=582 (55000), emission .sub.max, nm (.sub.fl)=605 (0.64relative value obtained with Rhodamine 101 as a reference dye with .sub.fl=0.96 in ethanol; excitation at 540 nm).
Example 2
Synthesis of Ge-Rhodamine Dyes
Synthesis of Tetramethyl-Ge-Rhodamine 19
[0161] Compound 15.
[0162] A solution of 3-bromo-N,N-dimethylaniline (2.32 g, 11.6 mmol, 2 eq) in anhydrous THF (40 mL) was degassed on a Schlenk line and cooled to 78 C. under argon. n-butyllithium (5.1 mL of 2.5 M solution in hexanes, 12.76 mmol, 2.2 eq) was injected with a syringe quickly dropwise, and the mixture was stirred at 78 OC for 1 h. Dimethylgermanium dichloride (1.01 g, 5.8 mmol), dissolved in anhydrous THF (3 mL), was injected dropwise with a syringe. The mixture was allowed to warm up to RT and stirred for 2.5 h. Brine (50 mL) was then added, the mixture was extracted with ethyl acetate (340 mL), and the combined organic layers were dried over Na.sub.2SO.sub.4. TLC control (SiO.sub.2/10% ethyl acetate in hexane): R.sub.f (product)=0.37. The product 15 was isolated by flash column chromatography (Biotage SNAP Ultra 50 g, gradient 1% to 10% ethyl acetate-hexane over 15 column volumes) as colorless oil, yield 1.56 g (78%). .sup.1H NMR (400 MHz, CDCl.sub.3): 7.27 (ddd, J=8.3, 7.1, 0.5 Hz, 2H), 6.93 (br.d, J=2.9 Hz, 2H), 6.90 (dt, J=7.1, 1.0 Hz, 2H), 2.96 (s, 8H), 0.66 (s, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3): 150.3, 141.1, 128.7, 122.2, 117.9, 113.1, 40.8, 2.7. ESI-MS, positive mode: m/z (rel. int., %)=345.2 (100) [M+H].sup.+. HR-MS (ESI, positive mode): 345.1387 [M+H].sup.+ (found), 345.1384 (calculated for C.sub.18H.sub.27N.sub.2Ge, [M+H].sup.+).
[0163] Compound 16.
[0164] N-Bromosuccinimide (NBS; 1.69 g, 9.51 mmol, 2.1 eq) was added portionwise to a solution of 15 (2.61 g, 4.34 mmol) in acetonitrile (25 mL), cooled in ice-water bath. Yellow color appeared first, followed by precipitation. The addition of NBS was terminated when yellow color of the suspension abruptly turned brownish, and the mixture was left stirring in ice-water bath for 1 h. TLC control (SiO.sub.2/CH.sub.2Cl.sub.2): R.sub.f (product)=0.65, R.sub.f (starting material)=0.55. Sat. aqueous NaHCO.sub.3 (40 mL) was added, the mixture was extracted with CH.sub.2Cl.sub.2 (340 mL), the combined extracts were washed with brine and dried over Na.sub.2SO.sub.4. The product 16 was isolated by flash column chromatography (Biotage SNAP Ultra 50 g, gradient 10% to 100% CH.sub.2Cl.sub.2 hexane over 15 CV) as yellowish solid, yield 2.00 g (88%). .sup.1H NMR (400 MHz, CDCl.sub.3): 7.36 (d, J=8.7 Hz, 2H), 6.75 (d, J=3.2 Hz, 2H), 6.58 (dd, J=8.8, 3.2 Hz, 2H), 2.87 (s, 12H), 0.87 (s, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3): 149.1, 141.5, 132.8, 120.9, 116.8, 115.0, 40.7, 0.1. ESI-MS, positive mode: m/z (rel. int., %)=501.0 (100) [M+H].sup.+. HR-MS (ESI, positive mode): 500.9590 [M+H].sup.+ (found), 500.9581 (calculated for C.sub.18H.sub.25N.sub.2GeBr.sub.2, [M+H].sup.+).
[0165] Compound 17.
[0166] A solution of 16 (500 mg, 0.994 mmol) in anhydrous THF (20 mL) was degassed on a Schlenk line and cooled to 78 C. under argon. tert-Butyllithium (2.4 mL of 1.7 M solution in pentane, 3.98 mmol, 4 eq) was added dropwise, and the resulting bright yellow solution was stirred at 78 C. for 1.5 h. Dimethylcarbamyl chloride (100 L, 1.09 mmol) was then injected dropwise with a Hamilton syringe. The mixture was stirred at 78 C. for 30 min, allowed to warm up to RT and quenched with sat. aq. NH.sub.4Cl (10 mL). TLC control (SiO.sub.2/ethyl acetate: R.sub.f (product)=0.50 (bright yellow), R.sub.f (impurity)=0.71 (colorless). The mixture was extracted with ethyl acetate (320 mL) and the combined organic layers were dried over Na.sub.2SO.sub.4. The product 17 was isolated by flash column chromatography (Sepacore Silica HP 12 g; gradient 10% to 100% ethyl acetatehexane) as bright yellow solid, quickly turning greenish, yield 264 g (72%). .sup.1H NMR (400 MHz, CDCl.sub.3): 8.42 (d, J=9.0 Hz, 2H), 6.80 (dd, J=9.1, 2.8 Hz, 2H), 6.72 (d, J=2.8 Hz, 2H), 3.08 (s, 12H), 0.60 (s, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3): 185.2, 151.5, 143.3, 132.1, 129.7, 114.3, 112.7, 40.1, 1.3. ESI-MS, positive mode: m/z (rel. int., %)=371.1 (100) [M+H].sup.+. HR-MS (ESI, positive mode): 371.1175 [M+H].sup.+ (found), 371.1177 (calculated for C.sub.19H.sub.25N.sub.2OGe, [M+H].sup.+).
[0167] Compound 19.
[0168] In a 100 mL round-bottom flask, a degassed solution of compound 18 (Scheme 7; (223 mg, 0.54 mmol, 2 eq) in anhydrous THF (10 mL) was cooled to 78 C. tert-Butyllithium (0.32 mL of 1.7 M solution in pentane, 0.54 mmol, 2 eq) was added dropwise, and the resulting yellow-orange solution was stirred at 78 C. for 1 h. A solution of ketone 17 (100 mg, 0.27 mmol) in THF (8 mL) was added quickly dropwise, the light orange mixture was allowed to warm up to room temperature (RT) and stirred for 3.5 h. The mixture was then cooled in ice-water bath and acetic acid (1.4 mL) was added. The resulting blue solution was evaporated to a viscous residue, 6 N HCl (16 mL) was added, and the mixture was stirred at 80 C. (bath temperature) overnight. The mixture was adjusted to pH 1-2 with NaHCO.sub.3, extracted with CH.sub.2Cl.sub.2 (450 mL), washed with 0.1 HCl (250 mL), brine and dried over Na.sub.2SO.sub.4. The combined organic layers were washed with brine and dried over Na.sub.2SO.sub.4. The product was isolated by flash column chromatography (Sepacore Silica HP 12 g; gradient 0% to 10% methanolCH.sub.2Cl.sub.2) and freeze-dried from dioxane to give 19 as blue fluffy solid (47 mg, 34% yield). UV-Vis (PBS 7.4): .sub.max ()=634 nm (97000 M.sup.1cm.sup.1); fluorescence (PBS 7.4): .sub.excit=590 nm, .sub.em=655 nm; Of, (relative to Oxazine 1, O.sub.fi=0.14 in ethanol)=0.43. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.29 (dd, J=7.9, 1.3 Hz, 1H), 8.23 (dd, J=1.3, 0.8 Hz, 1H), 8.05 (dd, J=8.0, 0.7 Hz, 1H), 6.98 (d, J=2.9 Hz, 2H), 6.86 (d, J=8.9 Hz, 2H), 6.52 (dd, J=8.9, 2.9 Hz, 2H), 2.96 (s, 12H), 0.81 (s, 3H), 0.80 (s, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3): 170.4, 169.5, 153.4, 149.6, 140.4, 134.1, 131.6, 131.2, 130.7, 127.5, 127.1, 126.3, 117.5, 112.7, 40.5, 0.8, 2.2. ESI-MS, positive mode: m/z (rel. int., %)=519.1 (100) [M+H].sup.+. HR-MS (ESI, positive mode): 519.1330 [M+H].sup.+ (found), 519.1339 (calculated for C.sub.27H.sub.28N.sub.2O.sub.4Ge, [M+H].sup.+).
##STR00027##
[0169] Compound 19-Halo.
[0170] PyBOP (30 L of 20 mg/100 L DMF stock solution, 11.6 mol, 1.5 eq) was added to a solution of 19 (4 mg, 7.74 mol), HaloTag Amine (02)(2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine; 3.5 mg, 15.63 mol, 2 eq) and trimethylamine (12 L) in DMF (100 L). After 1 h, the solvent was evaporated in vacuo at RT, and the product was isolated by preparative TLC (silica, 3% methanolCH.sub.2Cl.sub.2, then CH.sub.2Cl.sub.2) giving an impure material, which was repurified by preparative HPLC (Kinetex 5 m C18 100, 21 mm25 cm, 11 mL/min, isocratic 65/35 A/B, Aacetonitrile, Bwater+0.05% TFA). Yield 3.8 mg (68%), purity (HPLC) 95%. HPLC (30/70-100/0 A/B over 20 min, Kinetex 5 m C18 100 4.6250 mm, 1.2 mL/min, detection at 254 nm or 636 nm): =12.50 min. .sup.1H NMR (400 MHz, CDCl.sub.3): 7.99 (dd, J=7.9, 0.7 Hz, 1H), 7.93 (dd, J=7.9, 1.4 Hz, 1H), 7.91 (dd, J=1.4, 0.8 Hz, 1H), 6.96 (d, J=2.9 Hz, 2H), 6.84 (d, J=8.9 Hz, 2H), 6.78 (br.s, 1H), 6.50 (dd, J=8.9, 2.9 Hz, 2H), 3.71-3.63 (m, 6H), 3.59-3.55 (m, 2H), 3.49 (t, J=6.7 Hz, 2H), 3.41 (t, J=6.7 Hz, 2H), 2.96 (s, 12H), 1.71 (m, 2H+H.sub.2O), 1.57-1.25 (m, 6H), 0.79 (s, 3H), 0.79 (s, 3H). ESI-MS, positive mode: m/z (rel. int., %)=724.3 [M+H].sup.+. HR-MS (ESI, positive mode): 724.2572 (found), 724.2571 (calculated for C.sub.37H.sub.49ClGeN.sub.3O.sub.5, [M+H].sup.+).
[0171] Compound 20.
[0172] TSTU (N,N,N,N-tetramethyl-O(N-succinimidyl)-uronium tetrafluoroborate; 20 L of 14 mg/100 L DMSO stock solution, 9.1 mol, 1.2 eq) was added to a solution of 19 (4 mg, 7.74 mol) and DIEA (N-ethyldiisopropylamine; 12 L) in DMSO (200 L). After stirring for 5 min, 8-aminooctanoic acid (20 L of 15 mg/100 L DMSO stock suspension, prepared by sonication; 18.2 mol, 2.3 eq) was added, the resulting suspension was sonicated at RT for 10 min followed by vigorous stirring for 15 min. Water (50 L) was then added, and the mixture was stirred for further 30 min. Acetic acid (50 L) was added, and the solvents were evaporated in vacuo at RT. The product 20 was isolated by preparative HPLC (Kinetex 5 m C18 100, 21 mm25 cm, 11 mL/min, isocratic 50/50 A/B, Aacetonitrile, Bwater+0.05% TFA). Yield 2.5 mg (49%). HPLC (30/70-100/0 A/B over 20 min, Kinetex 5 m C18 100 4.6250 mm, 1.2 mL/min, detection at 254 nm or 636 nm): =8.60 min. .sup.1H NMR (400 MHz, DMSO-d.sub.6): 8.75 (t, J=5.6 Hz, 1H), 8.11 (dd, J=8.0, 1.4 Hz, 1H), 8.03 (br.d, J=8.2 Hz, 1H), 7.86 (br.s, 1H), 7.05 (br.s, 2H), 6.71 (br.d, J=9.0 Hz, 2H), 6.61 (br.d, J=8.1 Hz, 2H), 3.24 (q, J=6.7 Hz, 2H), 2.94 (br.s, 12H), 2.17 (t, J=7.4 Hz, 2H), 1.49 (m, 4H), 1.27 (m, 6H), 0.77 (s, 3H), 0.68 (s, 3H). ESI-MS, positive mode: m/z (rel. int., %)=660.4 (100) [M+H].sup.+.
[0173] Compound 19-Tubulin.
[0174] TSTU (N,N,N,N-tetramethyl-O(N-succinimidyl)uronium tetrafluoroborate; 10 L of 17 mg/100 L DMF stock solution, 5.7 mol, 1.5 eq) was added to a solution of 20 (2.5 mg, 3.8 mol) and DIEA (N-ethyldiisopropylamine; 10 L) in DMSO (200 L), and the reaction mixture was stirred for 1 h. DIEA (20 L) followed by 3-H.sub.2NXT.HCO.sub.2R (3-aminodocetaxel formate (G. Lukinaviius et al., Nat. Methods 2014, 11, 731-733); 4.3 mg, 5.7 mol, 1.5 eq, dissolved in 100 L DMF+100 L DMSO) were added, and the reaction mixture was left stirring at RT overnight. The solvents were evaporated in vacuo at RT, and the product was isolated by preparative HPLC (Kinetex 5 m C18 100, 10 mm25 cm, gradient 30/70-80/20 A/B over 20 min; Aacetonitrile, Bwater+0.05% TFA). Yield 0.73 mg (14%), determined spectrophotometrically (G. Lukinaviius et al., Nat. Methods 2014, 11, 731-733). HPLC (20/80-80/20 A/B over 20 min, Kinetex 5 m C18 100 4.6250 mm, 1.2 mL/min, detection at 650 nm): =14.90 min. ESI-MS, positive mode: m/z (rel. int., %)=1371.5 [M+Na].sup.+. HR-MS (ESI, positive mode): 1371.5111 (found), 1371.5165 (calculated for C.sub.73H.sub.86N.sub.4O.sub.16GeNa, [M+Na].sup.+).
[0175] Hydroxylated Ge-Rhodamine (33)
[0176] Compound 21.
[0177] A solution of 3-bromophenol (8.1 g, 46.8 mmol) and imidazole (3.5 g, 51.5 mmol, 1.1 eq) in CH.sub.2Cl.sub.2 (55 mL) was cooled in ice-water bath. To the cold mixture, a solution of triisopropylsilyl chloride (TIPSCl; 10.4 mL, 49.2 mmol, 1.05 eq) in CH.sub.2Cl.sub.2 (40 mL) was added dropwise over 20 min. The resulting suspension was removed from ice-water bath and stirred at RT overnight. TLC control (SiO.sub.2/10% EtOAc in hexane): R.sub.f (product)=0.70, R.sub.f (3-bromophenol)=0.18. The mixture was diluted with water (150 mL), extracted with CH.sub.2Cl.sub.2 (350 mL), the combined organic layers were washed with brine and dried over Na.sub.2SO.sub.4. The product 21 was isolated by flash chromatography on a 1 L glass Bchner funnel with 200 g silica (3.5 cm layer), eluting with 0%.fwdarw.5%.fwdarw.10% CH.sub.2Cl.sub.2 hexane in 400 mL portions. Colorless oil, yield 15.07 g (98%). .sup.1H NMR (400 MHz, CDCl.sub.3): 7.08-7.06 (m, 2H), 7.05-7.03 (m, 1H), 6.84-6.77 (m, 1H), 1.32-1.18 (m, 3H), 1.10 (d, J=7.2 Hz, 18H).
[0178] Compound 22.
[0179] A solution of TIPS-protected bromophenol 21 (3.54 g, 10.76 mmol, 2 eq) in anhydrous THF (40 mL) was degassed on a Schlenk line and cooled to 78 C. under argon. n-Butyllithium (4.7 mL of 2.5 M solution in hexanes, 11.84 mmol, 2.2 eq) was injected with a syringe quickly dropwise, and the mixture was stirred at 78 C. for 1 h, turning into a thin suspension. Dimethylgermanium dichloride (0.62 mL, 5.38 mmol), dissolved in anhydrous THF (2.5 mL), was injected dropwise with a syringe. The mixture was allowed to warm up to RT (the solids dissolved), and the resulting solution was stirred at RT for 4.5 h. Brine (40 mL) was then added, the mixture was extracted with ethyl acetate (340 mL), and the combined organic layers were dried over Na.sub.2SO.sub.4. TLC control (SiO.sub.2/10% CH.sub.2Cl.sub.2 in hexane): R.sub.f (product)=0.34, R.sub.f (impurity)=0.50. The product 22 was isolated by flash column chromatography (Teledyne Isco RediSep Rf 80 g, isocratic in pure hexane for 5 CV, then gradient 0% to 20% CH.sub.2Cl.sub.2 hexane over 8 CV) as colorless oil, yield 2.79 g (86%). .sup.1H NMR (400 MHz, CDCl.sub.3): 7.23-7.17 (m, 2H), 7.01 (dt, J=7.1, 1.1 Hz, 2H), 6.98-6.96 (m, 2H), 6.85 (ddd, J=8.1, 2.6, 1.1 Hz, 2H), 1.27-1.16 (m, 6H), 1.08 (d, J=7.2 Hz, 36H), 0.60 (s, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3): 155.9, 141.6, 129.1, 126.2, 125.0, 120.2, 18.1, 12.8, 3.0. ESI-MS, positive mode: m/z (rel. int., %)=625.3 (100) [M+Na].sup.+. HR-MS (ESI, positive mode): 25.2897 [M+Na].sup.+ (found), 625.2930 (calculated for C.sub.32H.sub.56O.sub.6Si.sub.2GeNa, [M+Na].sup.+).
[0180] Compound 23.
[0181] N-Bromosuccinimide (1.62 g, 9.11 mmol, 2.1 eq) was added portionwise over 5 min to a solution of 22 (2.61 g, 4.34 mmol) in the mixture of acetonitrile (60 mL) and pyridine (8.5 mL). The resulting mixture was stirred at 60 C. (bath temperature) overnight (22 h), turning into a brown solution. TLC control (SiO.sub.2/5% CH.sub.2Cl.sub.2 in hexane): R.sub.f (product)=0.27, R.sub.f (starting material)=0.19. The solvents were removed on a rotary evaporator; the residue was redissolved in CH.sub.2Cl.sub.2 (120 mL) and the solution was washed with sat. aqueous NaHCO.sub.3, water, brine (100 mL each) and dried over Na.sub.2SO.sub.4. The product 23 was isolated by flash column chromatography (Teledyne Isco RediSep Rf 80 g, gradient 0% to 25% CH.sub.2Cl.sub.2 hexane over 15 CV) as viscous colorless oil with purity 85%, yield 2.64 g (70% considering purity). The material was used in the next step without further purification. .sup.1H NMR (400 MHz, CDCl.sub.3): 7.35 (d, J=8.6 Hz, 2H), 6.85 (d, J=3.0 Hz, 2H), 6.74 (dd, J=8.6, 3.0 Hz, 2H), 1.23-1.12 (m, 6H), 1.05 (d, J=7.0 Hz, 36H), 0.84 (s, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3): 155.1, 142.4, 133.5, 127.5, 122.5, 120.9, 18.0, 12.8, 0.3. ESI-MS, positive mode: m/z (rel. int., %)=783.1 (100) [M+Na].sup.+. HR-MS (ESI, positive mode): 783.1103 [M+Na].sup.+ (found), 783.1119 (calculated for C.sub.32H.sub.54O.sub.2Si.sub.2GeBr.sub.2Na, [M+Na].sup.+).
[0182] Compound 24.
[0183] A solution of 23 (2.9 g, 3.82 mmol) in anhydrous THF (60 mL) was degassed on a Schlenk line and cooled to 78 C. under argon. n-Butyllithium (3.2 mL of 2.5 M solution in hexanes, 8.02 mmol, 2.1 eq) was added dropwise, and the resulting light yellow solution was stirred at 78 C. for 5.5 h. Dimethylcarbamyl chloride (0.36 mL, 3.82 mmol) was then injected dropwise with a syringe. The mixture was stirred at 78 C. for 30 min, turning nearly colorless, and was then allowed to warm up to RT and stirred for further 1.5 h. TLC control (SiO.sub.2/10% ethyl acetate in hexane): R.sub.f (product)=0.62 (yellow upon heating with NaOH), R.sub.f (starting material)=0.72. The reaction mixture was quenched with sat. aq. NH.sub.4Cl (30 mL), extracted with ethyl acetate (330 mL), and the combined organic layers were dried over Na.sub.2SO.sub.4 The product was isolated by flash column chromatography (Teledyne Isco RediSep Rf 80 g, gradient 0% to 100% A:B, A=10% ethyl acetate-hexane, B=hexane) as light yellow viscous oil with purity 70%, yield 2.13 g (62% considering purity). The impure material was used directly for deprotection. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.37 (d, J=8.7 Hz, 2H), 7.02 (d, J=2.6 Hz, 2H), 6.98 (dd, J=8.7, 2.6 Hz, 2H), 1.36-1.21 (m, 6H), 1.13 (d, J=7.3 Hz, 36H), 0.59 (s, 6H). ESI-MS, positive mode: m/z (rel. int., %)=629.3 (100) [M+H].sup.+. HR-MS (ESI, positive mode): 629.2898 [M+H].sup.+ (found), 629.2903 (calculated for C.sub.33H.sub.55GeO.sub.3Si.sub.2, [M+H].sup.+).
[0184] Tetrabutylammonium fluoride trihydrate (2.68 g, 8.5 mmol, 2.5 equiv), dissolved in THF (20 mL), was added a solution of crude di-TIPS ether from the previous step (2.13 g, 70% purity) in THF (25 mL), cooled in ice-water bath. The resulting bright yellow solution was stirred at 0 C. for 1 h. TLC control (SiO.sub.2/50% ethyl acetate in hexane): R.sub.f (product)=0.26 (yellow with NaOH), R.sub.f (impurity)=0.48, R.sub.f (starting material)=0.76. Sat. aq. NH.sub.4Cl (50 mL) was added followed by minimal amount of water necessary to dissolve the solids, the mixture was extracted with ethyl acetate (340 mL), the combined organic layers were washed with brine and dried over Na.sub.2SO.sub.4. The product was isolated by flash column chromatography (Teledyne Isco RediSep Rf 80 g; gradient 20% to 80% ethyl acetate-hexane) to give pure 24 as white solid. Yield 475 mg (39% over 2 steps). .sup.1H NMR (400 MHz, acetone-d.sub.6): 9.09 (s, 2H), 8.31 (d, J=8.7 Hz, 2H), 7.12 (d, J=2.6 Hz, 2H), 7.00 (dd, J=8.8, 2.6 Hz, 2H), 0.60 (s, 6H). .sup.13C NMR (101 MHz, acetone-d.sub.6): 161.1, 144.7, 134.1, 133.3, 119.9, 117.6, 1.9. ESI-MS, positive mode: m/z (rel. int., %)=339.0 (100) [M+Na].sup.+. HR-MS (ESI, positive mode): 339.0052 [M+Na]+(found), 339.0050 (calculated for C.sub.15H.sub.14GeO.sub.3Na, [M+Na].sup.+).
[0185] Compound 25.
[0186] A solution of 24 (475 mg, 1.51 mmol) and imidazole (308 mg, 4.53 mmol, 3 eq) in CH.sub.2Cl.sub.2 (30 mL) and DMF (3 mL) was cooled in ice-water bath. To the cold mixture, a solution of tert-butyldimethylsilyl chloride (TBSCl; 684 mg, 4.53 mmol, 3 eq) in CH.sub.2Cl.sub.2 (5 mL) was added quickly dropwise. The resulting white suspension was removed from ice-water bath and stirred at RT for 4 h. TLC control (SiO.sub.2/10% EtOAc in hexane): R.sub.f (product)=0.56, R.sub.f (starting material)=0. The mixture was diluted with water (300 mL), extracted with CH.sub.2Cl.sub.2 (350 mL), the combined organic layers were washed with water (200 mL), sat. aq. NaHCO.sub.3, brine (100 mL each) and dried over Na.sub.2SO.sub.4. The product 25 was isolated by flash column chromatography (Teledyne Isco RediSep Rf 40 g; gradient 0% to 100% A:B, A=10% ethyl acetate-hexane, B=hexane). White solid, yield 817 g (100%). .sup.1H NMR (400 MHz, CDCl.sub.3): 8.37 (d, J=8.7 Hz, 2H), 6.98 (d, J=2.6 Hz, 2H), 6.95 (dd, J=8.7, 2.6 Hz, 2H), 1.01 (s, 18H), 0.60 (s, 6H), 0.26 (s, 12H). .sup.13C NMR (101 MHz, CDCl.sub.3): 186.2, 158.8, 143.8, 134.8, 132.7, 123.9, 121.3, 25.8, 18.5, 1.7, 4.2. ESI-MS, positive mode: m/z (rel. int., %)=545.2 (100) [M+H].sup.+. HR-MS (ESI, positive mode): 545.1958 [M+H].sup.+ (found), 545.1962 (calculated for C.sub.27H.sub.43O.sub.3Si.sub.2Ge, [M+H].sup.+).
[0187] Compound 27.
[0188] In a 100 mL round-bottom flask, a degassed solution of compound 26 (Scheme 9; 1.04 g, 2.9 mmol, 2 eq) in anhydrous THF (10 mL) and pentane (5 mL) was cooled to 100 C. (bath temperature, diethyl etherliquid N.sub.2). n-Butyllithium (1.2 mL of 2.5 M solution in hexanes, 2.9 mmol, 2 eq) was carefully introduced through a needle along the wall of the flask. Clear solution quickly turned orange and then brown-orange; it was stirred at 100 C. for 10 min, and the solution of ketone 25 (787 mg, 1.45 mmol) in THF (8 mL) was injected over 1-2 min along the wall of the flask. The flask was then placed into a 78 bath (dry iceacetone) and the brown solution was stirred for 10 min. The cooling bath was removed, the mixture was allowed to warm up to RT and stirred for further 30 min. TLC control (SiO.sub.2/10% ethyl acetate in hexane): R.sub.f (product)=0.35 (purple upon heating with NaOH), R.sub.f (starting material)=0.47. The reaction mixture was quenched with water (50 mL), adjusted to pH4 with acetic acid, extracted with ethyl acetate (330 mL), the combined organic layers were washed with brine and dried over Na.sub.2SO.sub.4. The product was isolated by flash column chromatography (Biotage SNAP Ultra 100 g; gradient 0% to 100% A:B, A=15% ethyl acetatehexane, B=hexane) and freeze-dried from dioxane to give 27 as white solid (501 mg, 46% yield). .sup.1H NMR (400 MHz, CDCl.sub.3): 8.18 (dd, J=8.0, 1.3 Hz, 1H), 8.08 (dd, J=1.3, 0.7 Hz, 1H), 7.99 (dd, J=8.0, 0.7 Hz, 1H), 7.10 (d, J=2.7 Hz, 2H), 6.96 (d, J=8.7 Hz, 2H), 6.65 (dd, J=8.7, 2.7 Hz, 2H), 1.60 (s, 9H), 0.98 (s, 18H), 0.795 (s, 3H), 0.790 (s, 3H), 0.19 (s, 12H). .sup.13C NMR (101 MHz, CDCl.sub.3): 169.4, 164.5, 155.5, 153.0, 140.6, 137.0, 136.2, 130.4, 129.7, 127.8, 126.2, 125.9, 125.5, 120.2, 91.2, 82.6, 28.3, 25.8, 18.4, 0.5, 1.9, 4.21, 4.23. ESI-MS, positive mode: m/z (rel. int., %)=749.3 (100) [M+H].sup.+. HR-MS (ESI, positive mode): 749.2732 [M+H].sup.+ (found), 749.2753 (calculated for C.sub.39H.sub.55O.sub.6GeSi.sub.2, [M+H].sup.+).
[0189] Compound 28.
[0190] Tetrabutylammonium fluoride trihydrate (850 mg, 2.68 mmol, 4 eq), dissolved in THF (5 mL), was added a solution of 27 (500 mg, 0.67 mmol) in THF (7 mL), cooled in ice-water bath. The resulting blue solution with intense red fluorescence was stirred at 0 C. for 30 min. TLC control (SiO.sub.2/10% ethyl acetate in CH.sub.2Cl.sub.2): R.sub.f (product)=0.31, R.sub.f (starting material)=0.83. Sat. aq. NH.sub.4Cl (20 mL) was added followed by the minimal amount of water necessary to dissolve the solids, the mixture was extracted with ethyl acetate (325 mL), the combined organic layers were washed with brine and dried over Na.sub.2SO.sub.4. The product was isolated by flash column chromatography (Teledyne Isco RediSep Rf 24 g; gradient 0% to 30% ethyl acetateCH.sub.2Cl.sub.2), evaporated to viscous colorless oil and freeze-dried from dioxane to give 28 as white solid (393 mg, contains 0.65 mol dioxane per mol of product, 100% yield). .sup.1H NMR (400 MHz, CDCl.sub.3): 8.18 (dd, J=8.0, 1.3 Hz, 1H), 8.03 (dd, J=1.3, 0.8 Hz, 1H), 7.99 (dd, J=8.0, 0.8 Hz, 1H), 7.08 (d, J=2.8 Hz, 2H), 6.86 (d, J=8.7 Hz, 2H), 6.60 (dd, J=8.7, 2.8 Hz, 2H), 6.40 (d, J=4.0 Hz, 2H), 1.59 (s, 9H), 0.67 (s, 3H), 0.63 (s, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3): 170.6, 164.7, 155.7, 153.4, 140.8, 137.2, 134.9, 130.5, 129.4, 127.9, 126.3, 125.8, 120.9, 115.9, 92.1, 83.1, 28.3, 0.3, 1.9. ESI-MS, positive mode: m/z (rel. int., %)=543.1 (100) [M+Na].sup.+. HR-MS (ESI, positive mode): 543.0838 [M+Na].sup.+ (found), 543.0839 (calculated for C.sub.27H.sub.26GeO.sub.6Na, [M+Na].sup.+).
[0191] Compound 29.
[0192] Trifluoromethanesulfonic anhydride (Tf.sub.2O; 155 L, 0.92 mmol, 4 eq) was added dropwise to a solution of 28 (133 mg of material containing 0.65 mol dioxane per mol of 28, 0.23 mmol) and pyridine (150 L, 1.84 mmol, 8 eq) in dry CH.sub.2Cl.sub.2 (5 mL), cooled in ice-water bath. Pink color appeared upon addition of each drop and vanished immediately. The flask was then removed from the cooling bath, and the mixture was stirred at RT for 1 h. TLC control (SiO.sub.2/10% ethyl acetate in hexane): R.sub.f (product)=0.18 (pink with NaOH). The mixture was diluted with cold water (20 mL), extracted with CH.sub.2Cl.sub.2 (320 mL), the combined extracts were washed with brine and dried over Na.sub.2SO.sub.4. The product was isolated by flash column chromatography (Sepacore Silica HP 12 g; gradient 0% to 40% ethyl acetate-hexane) and freeze-dried from dioxane to give 29 as white fluffy solid, yield 173 mg (96%). .sup.1H NMR (400 MHz, CDCl.sub.3): 8.26 (dd, J=8.0, 1.2 Hz, 1H), 8.16 (dd, J=1.3, 0.7 Hz, 1H), 8.05 (dd, J=8.0, 0.8 Hz, 1H), 7.54 (d, J=2.7 Hz, 2H), 7.31 (d, J=8.8 Hz, 2H), 7.16 (dd, J=8.8, 2.7 Hz, 2H), 1.62 (d, J=0.8 Hz, 9H), 0.93 (s, 3H), 0.92 (s, 3H). .sup.19F NMR (376 MHz, CDCl.sub.3): 72.78. .sup.13C NMR (101 MHz, CDCl.sub.3): 168.1, 164.0, 150.8, 149.5, 143.3, 142.5, 137.7, 131.4, 129.2, 128.4, 127.0, 126.7, 125.7, 122.1, 118.8 (q, J=320.8 Hz), 89.2, 83.2, 28.3, 0.9, 1.7. ESI-MS, positive mode: m/z (rel. int., %)=807.0 (100) [M+Na].sup.+. HR-MS (ESI, positive mode): 806.9805 [M+Na].sup.+ (found), 806.9824 (calculated for C.sub.29H.sub.24O.sub.10GeS.sub.2F.sub.6Na, [M+Na].sup.+).
[0193] Compound 31.
[0194] A mixture of 29 (170 mg, 0.22 mmol), 3-(tert-butyldimethylsilyloxy)azetidine 30 (123 mg, 0.66 mmol, 3 eq), Pd.sub.2(dba).sub.3 (20 mg, 0.022 mmol, 10 mol %), XPhos (31 mg, 0.066 mmol, 30 mol %) and K.sub.2CO.sub.3(91 mg, 0.66 mmol, 3 eq) in dry dioxane (2 mL) was degassed on a Schlenk line and stirred at 100 C. under argon (bath temperature) in a sealed flask for 2 h. TLC control showed incomplete conversion (SiO.sub.2/hexaneCH.sub.2Cl.sub.2 ethyl acetate 70:25:5, stained by heating with 1 M NaOH): R.sub.f (product)=0.2 (blue), R.sub.f (monosubstitution product)=0.33 (violet), R.sub.f (starting material)=0.38 (pink). Additional portions of Pd.sub.2(dba).sub.3 (20 mg, 0.022 mmol, 10 mol %) and XPhos (31 mg, 0.066 mmol, 30 mol %) were added, the mixture was degassed stirred at 100 C. for further 2 h (4 h total time). The consumption of both the starting material and the monotriflate intermediate was verified by TLC, and upon cooling the brown mixture was diluted with water (20 mL), extracted with CH.sub.2Cl.sub.2 (320 mL), the combined organic layers were washed with brine and dried over Na.sub.2SO.sub.4. The filtrate was evaporated on Celite and the product was isolated by flash column chromatography (Interchim Puriflash 15 m 25 g; isocratic in pure CH.sub.2Cl.sub.2 until the first peak is eluted (6 CV), then gradient 0% to 100% A:B, A=10% ethyl acetate in CH.sub.2Cl.sub.2, BCH.sub.2Cl.sub.2) and freeze-dried from 1,4-dioxane to yield 84 mg (45%) of 31 as light yellow fluffy solid. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.16 (dd, J=8.0, 1.3 Hz, 1H), 8.03 (t, J=1.0 Hz, 1H), 7.97 (dd, J=8.0, 0.7 Hz, 1H), 6.88 (d, J=8.6 Hz, 2H), 6.67 (d, J=2.6 Hz, 2H), 6.26 (dd, J=8.7, 2.6 Hz, 2H), 4.74 (tt, J=6.3, 5.0 Hz, 2H), 4.15 (td, J=6.5, 1.3 Hz, 4H), 3.65 (dd, J=7.6, 5.0 Hz, 4H), 1.59 (s, 9H), 0.89 (s, 18H), 0.78 (s, 3H), 0.78 (s, 3H), 0.07 (s, 12H). .sup.13C NMR (101 MHz, CDCl.sub.3): 169.7, 164.6, 153.6, 150.9, 139.8, 136.8, 132.3, 130.1, 130.0, 127.2, 125.94, 125.92, 116.6, 112.0, 92.2, 82.4, 62.5, 62.2, 62.1, 28.3, 25.9, 18.1, 0.6, 1.8, 4.8. ESI-MS, positive mode: m/z (rel. int., %)=859.4 (100) [M+H].sup.+. HR-MS (ESI, positive mode): 859.3586 [M+H].sup.+ (found), 859.3599 (calculated for C.sub.45H.sub.65N.sub.2O.sub.6Si.sub.2Ge, [M+H].sup.+).
[0195] Compound 32.
[0196] Tetrabutylammonium fluoride trihydrate (93 mg, 294 mol, 3 equiv), dissolved in THF (5 mL), was added a solution of 31 (84 mg, 98 mol) in THF (10 mL), cooled in ice-water bath. The reaction mixture was stirred at 0 C. for 1 h. Water (20 mL), acetic acid (0.5 mL) and brine (20 mL) were then added, and the mixture was extracted with ethyl acetate (320 mL), the combined organic layers were dried over Na.sub.2SO.sub.4, filtered, evaporated and re-evaporated twice with ethyl acetate (220 mL). The product was isolated by flash column chromatography (Sepacore Silica HP 12 g; gradient 20% to 100% ethyl acetateCH.sub.2Cl.sub.2) and freeze-dried from 1,4-dioxane to give 32 as light green fluffy solid. Yield 60 mg (97%). .sup.1H NMR (400 MHz, CD.sub.3OD): 8.16 (dd, J=8.1, 1.3 Hz, 1H), 7.96 (dd, J=8.0, 0.7 Hz, 1H), 7.90 (t, J=1.0 Hz, 1H), 6.79 (d, J=8.7 Hz, 2H), 6.73 (d, J=2.6 Hz, 2H), 6.28 (ddd, J=8.7, 2.7, 0.9 Hz, 2H), 4.85 (s, 2H+H.sub.2O), 4.63 (tt, J=6.4, 4.8 Hz, 2H), 4.16-4.07 (m, 4H), 3.67-3.56 (m, 4H), 1.55 (s, 9H), 0.76 (s, 3H), 0.70 (s, 3H). .sup.13C NMR (101 MHz, CD.sub.3OD): 171.5, 165.6, 155.7, 152.5, 140.3, 138.3, 133.0, 131.1, 130.5, 128.1, 126.9, 126.5, 117.6, 113.4, 83.6, 63.0, 62.5, 28.3, 0.1, 1.7. ESI-MS, positive mode: m/z (rel. int., %)=631.2 (100) [M+H].sup.+. HR-MS (ESI, positive mode): 31.1851 [M+H].sup.+ (found), 631.1865 (calculated for C.sub.33H.sub.37N.sub.2O.sub.6Ge, [M+H].sup.+).
[0197] Compound 33.
[0198] Trifluoroacetic acid (1.6 mL) was added to a solution of 32 (50 mg, 79.5 mol) in CH.sub.2Cl.sub.2 (8 mL), cooled in ice-water bath. The cooling bath was removed, and the reaction mixture was stirred at RT for 3 h. The solvents were evaporated, and the residue was re-evaporated twice with toluene (220 mL). The product was isolated by flash column chromatography (20 g silica, acetonitrileCH.sub.2Cl.sub.2 water 10:1:0.5, then 10:1:1), followed by chromatography on a reversed phase (RP-C.sub.18 10 g; gradient 67% to 33% water-acetonitrile). Fractions containing the product were evaporated, dissolved in 1,4-dioxane with addition of minimal amount of water, a small amount of viscous insoluble material was centrifuged off, the supernatant was microfiltered and freeze-dried to give 51 mg (79%) blue fluffy solid (trifluoroacetate salt, containing 1.4 mol of 1,4-dioxane per mol of the dye). HPLC (10/90-100/0 over 25 min, column 4250 mm, 1.2 mL/min, detection at 254 nm): =10.32 min. UV-Vis (PBS 7.4): .sub.max (e)=631 nm (61000 M.sup.1 cm.sup.1); fluorescence (PBS 7.4): .sub.excit=590 nm, .sub.em=651 nm; .sub.fl(abs.)=0.60. .sup.1H NMR (400 MHz, CD.sub.3OD): 8.22 (br.d, J=7.0 Hz, 1H), 8.07 (br.s, 1H), 7.92 (d, J=7.0 Hz, 1H), 6.78 (d, J=8.6 Hz, 2H), 6.73 (d, J=2.5 Hz, 2H), 6.25 (dd, J=8.6, 2.6 Hz, 2H), 4.63 (tt, J=6.2, 4.8 Hz, 2H), 4.16-4.08 (m, 4H), 3.62-3.57 (m, 4H), 0.74 (s, 3H), 0.71 (s, 3H). .sup.19F NMR (376 MHz, CD.sub.3OD): 76.95. .sup.13C NMR (101 MHz, CD.sub.3OD): 171.7, 154.1, 152.5, 141.4, 133.4, 130.1, 128.3, 126.4, 117.7, 112.9, 94.8, 63.0, 62.5, 0.5, 2.6. ESI-MS, positive mode: m/z (rel. int., %)=575.2 (100) [M+H].sup.+. HR-MS (ESI, positive mode): 575.1225 (found), 575.1238 (calculated for C.sub.29H.sub.29N.sub.2O.sub.6Ge, [M+H].sup.+).
##STR00028## ##STR00029##
[0199] Compound 33-Halo.
[0200] PyBOP (20 L of 20 mg/100 L stock solution, 7.41 mol, 1.5 equiv) was added to a solution of 33 (4 mg, 5.82 mol), HaloTag Amine (02)(2-(2-((6-chlorohexyl)-oxy)ethoxy)ethanamine; 2.0 mg, 8.73 mol, 1.5 equiv) and DIEA (N-ethyldiisopropylamine; 10 L) in DMF (100 L). After 1 h, the solvent was evaporated in vacuo at RT, and the product 33-Halo was isolated by preparative TLC (silica, 5% methanolCH.sub.2Cl.sub.2, then 10% methanolCH.sub.2Cl.sub.2). Yield 4.3 mg (95%), purity (HPLC) 95%. HPLC (10/90-100/0 A/B over 25 min, column 4250 mm, 1.2 mL/min, detection at 254 nm or 640 nm): =14.60 min. UV-Vis (PBS 7.4): .sub.max=639 nm; fluorescence (PBS 7.4): .sub.excit=610 nm, .sub.em=660 nm. ESI-MS, positive mode: m/z (rel. int., %)=780.3 [M+H].sup.+. HR-MS (ESI, positive mode): 780.2471 (found), 780.2470 (calculated for C.sub.39H.sub.49ClGeN.sub.3O.sub.7, [M+H].sup.+).
[0201] Compound 33-Lysosome.
[0202] To a solution of 33 (2 mg, 3.5 mol) in dry DMSO (400 L) under argon, DIEA (N-ethyldiisopropylamine; 16 L, 91 mol) and TSTU (1.5 mg, 4.6 mol) were added. The reaction mixture was stirred for 5 min, and 1,6-diaminohexane (1.6 mg, 13.3 mol) was then added. The reaction mixture was sonicated for 15 min, followed by addition of water (20 L) and stirring for 30 min (HPLC control). The mixture was quenched by addition of acetic acid (6.3 L, 105 mol) and freeze-dried. The product was isolated by prep. HPLC (column Kinetex 5 m C18 100, 10 mm25 cm, gradient 20/80 to 50/50 A/B over 20 min, Aacetonitrile, Bwater+0.05% TFA) to give 1.5 mg (64%) of 33a as blue solid, which was used in the next step as follows. HPLC (20/80-100/0 A/B over 10 min, column Kinetex 2.6 m C18 100 4.675 mm, 1 mL/min, detection at 254 nm or 650 nm): =2.9 min (33a), 3.6 min (33).
[0203] Pepstatin A (1.9 mg, 2.7 mol) was dissolved in dry DMSO (200 L), DIEA (3.6 L, 21.4 mol) was added, followed by TSTU (3.2 mol; 10 L of 4.3 mg/50 L stock solution in DMSO), and the solution was stirred for 80 min at rt. To the resulting solution of Pepstatin A NHS ester, DIEA (3.6 L, 21.4 mol) and a solution of 33a (1.5 mg, 2.2 mol) in DMSO (200 L) were added and the mixture was stirred for 60 min at rt. The solvent was removed by freeze-drying, and the product was isolated by prep. HPLC (column Kinetex 5 m C18 100, 10 mm25 cm, gradient 30/70 to 100/0 A/B over 20 min, Aacetonitrile, Bwater+0.05% TFA). HPLC (20/80-80/20 A/B over 10 min, column Kinetex 2.6 m C18 100 4.675 mm, 1 mL/min, detection at 254 nm or 650 nm): =6.3 min (33-lysosome).
[0204] Compound 34.
[0205] TSTU (N,N,N,N-tetramethyl-O(N-succinimidyl)-uronium tetrafluoroborate; 20 L of 18 mg/100 L DMSO stock solution, 12 mol, 1.2 eq) was added to a solution of 33 (6 mg, 10 mol) and DIEA (N-ethyldiisopropylamine; 12 L) in DMSO (250 L). After stirring for 5 min, 8-aminooctanoic acid (20 L of 18 mg/100 L DMSO stock suspension, prepared by sonication; 23 mol, 2.3 eq) was added, the resulting suspension was sonicated at RT for 10 min followed by vigorous stirring for 15 min. Water (50 L) was then added, and the mixture was stirred for further 30 min. Acetic acid (50 L) was added, and the solvents were evaporated in vacuo at RT. The product was isolated by preparative HPLC (Kinetex 5 m C18 100, 21 mm25 cm, 11 mL/min, gradient 20/75-70/30 A/B over 20 min, Aacetonitrile, Bwater+0.05% TFA). Yield 2.5 mg (49%). HPLC (20/80-70/30 A/B over 20 min, Kinetex 5 m C18 100 4.6250 mm, 1.2 mL/min, detection at 254 nm): =10.90 min. .sup.1H NMR (400 MHz, DMSO-d.sub.6): 8.75 (t, J=5.6 Hz, 1H), 8.11 (dd, J=8.0, 1.4 Hz, 1H), 8.03 (br.d, J=8.2 Hz, 1H), 7.86 (br.s, 1H), 7.05 (br.s, 2H), 6.71 (br.d, J=9.0 Hz, 2H), 6.61 (br.d, J=8.1 Hz, 2H), 3.24 (q, J=6.7 Hz, 2H), 2.94 (br.s, 12H), 2.17 (t, J=7.4 Hz, 2H), 1.49 (m, 4H), 1.27 (m, 6H), 0.77 (s, 3H), 0.68 (s, 3H). ESI-MS, positive mode: m/z (rel. int., %)=716.2 [M+H].sup.+. HR-MS (ESI, positive mode): 716.2374 (found), 716.2394 (calculated for C.sub.37H.sub.44N.sub.3O.sub.7Ge, [M+H].sup.+).
[0206] Compound 33-Tubulin.
[0207] TSTU (N,N,N,N-tetramethyl-O(N-succinimidyl)uronium tetrafluoroborate; 13 L of 20 mg/100 L DMF stock solution, 8.67 mol, 1.5 eq) was added to a solution of 34 (5.78 mol in 500 L DMSO) and DIEA (N-ethyldiisopropylamine; 16 L), and the reaction mixture was stirred for 1 h. DIEA (30 L) followed by 3-H.sub.2NXT.HCO.sub.2H (3-aminodocetaxel formate; 6.5 mg, 8.7 mol, 1.5 eq, dissolved in 150 L DMF) were added, and the reaction mixture was left stirring at RT overnight. The solvents were evaporated in vacuo at RT, and the product was isolated by preparative HPLC (Kinetex 5 m C18 100, 10 mm25 cm, isocratic 50/50 A/B, Aacetonitrile, Bwater+0.05% TFA). Yield 1.59 mg (20%), determined spectrophotometrically. HPLC (10/90-80/20 A/B over 2 min, column 4250 mm, 1.2 mL/min, detection at 254 nm): =15.83 min. ESI-MS, positive mode: m/z (rel. int., %)=1405.5 (100) [M+H].sup.+. HR-MS (ESI, positive mode): 1405.5237 (found), 1405.5244 (calculated for C.sub.75H.sub.87N.sub.4O.sub.18Ge, [M+H].sup.+).
[0208] Hydroxylated Si-Rhodamine JF.sub.646 (42)
[0209] Compound 35.
[0210] A solution of TIPS-protected bromophenol (21; 5.27 g, 16.02 mmol, 2 eq) in anhydrous THF (60 mL) was degassed on a Schlenk line and cooled to 78 C. under argon. n-Butyllithium (7.05 mL of 2.5 M solution in hexanes, 17.62 mmol, 2.2 eq) was injected with a syringe quickly dropwise, and the mixture was stirred at 78 C. for 1 h, turning into a thick suspension. Dichlorodimethylsilane (0.97 mL, 8.01 mmol) was added dropwise with a syringe. The mixture was allowed to warm up to RT (the solids dissolved), and the resulting solution was stirred at RT for 2 h. Brine (50 mL) and water (20 mL) were then added, the mixture was extracted with ethyl acetate (350 mL), and the combined organic layers were dried over Na.sub.2SO.sub.4. TLC control (SiO.sub.2/10% CH.sub.2Cl.sub.2 in hexane): R.sub.f (product)=0.36, R.sub.f (starting material)=0.55. The product 35 was isolated by flash column chromatography (90 g silica, gradient 0% to 30% CH.sub.2Cl.sub.2 hexane) as colorless oil, yield 4.12 g (93%). .sup.1H NMR (400 MHz, CDCl.sub.3): 7.23-7.18 (m, 2H), 7.06 (dt, J=7.2, 1.1 Hz, 2H), 7.02-7.00 (m, 2H), 6.88 (ddd, J=8.1, 2.6, 1.1 Hz, 2H), 1.29-1.16 (m, 6H), 1.08 (d, J=7.1 Hz, 36H), 0.51 (s, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3): 155.7, 139.8, 129.0, 126.8, 125.6, 120.8, 18.1, 12.8, 2.3. ESI-MS, positive mode: m/z (rel. int., %)=595.3 (100) [M+K].sup.+. HR-MS (ESI, positive mode): 595.3215 [M+K].sup.+ (found), 595.3220 (calculated for C.sub.32H.sub.56O.sub.2Si.sub.3K, [M+K].sup.+).
[0211] Compound 36.
[0212] N-Bromosuccinimide (2.33 g, 13.07 mmol, 2.1 eq) was added portionwise over 5 min to a solution of 35 (3.46 g, 6.22 mmol) in the mixture of acetonitrile (85 mL) and chloroform (25 mL). The resulting solution was stirred at 60 C. (bath temperature) overnight (16 h), turning light yellow. TLC control (SiO.sub.2/5% CH.sub.2Cl.sub.2 in hexane): R.sub.f (product)=0.44, R.sub.f (starting material)=0.36. The solvents were removed on a rotary evaporator; the residue was redissolved in CH.sub.2Cl.sub.2 (150 mL) and the solution was washed with sat. aqueous NaHCO.sub.3, water, brine (100 mL each) and dried over Na.sub.2SO.sub.4. The product 36 was isolated by flash column chromatography (100 g silica, gradient 0% to 15% CH.sub.2Cl.sub.2 hexane) as colorless oil, yield 3.27 g (74%). .sup.1H NMR (400 MHz, CDCl.sub.3): 7.34 (d, J=8.6 Hz, 2H), 6.97 (d, J=3.0 Hz, 2H), 6.76 (dd, J=8.6, 3.0 Hz, 2H), 1.25-1.15 (m, 6H), 1.07 (d, J=7.3 Hz, 36H), 0.72 (s, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3): 155.0, 140.0, 133.8, 128.5, 123.0, 121.0, 18.0, 12.8, 1.0. ESI-MS, positive mode: m/z (rel. int., %)=737.2 (100) [M+Na].sup.+. HR-MS (ESI, positive mode): 737.1664 [M+Na] (found), 737.1673 (calculated for C.sub.32H.sub.54Br.sub.2O.sub.2Si.sub.3Na, [M+Na].sup.+).
[0213] Compound 37.
[0214] A solution of 36 (4.06 g, 5.69 mmol) in anhydrous THF (80 mL) was degassed on a Schlenk line and cooled to 78 C. under argon. n-Butyllithium (5 mL of 2.5 M solution in hexanes, 12.5 mmol, 2.2 eq) was added dropwise, and the mixture was stirred at 78 C. for 3.5 h, turning into a clear light yellow solution. Dimethylcarbamyl chloride (0.53 mL, 5.69 mmol, 1 eq) was then injected dropwise with a syringe. The mixture was stirred at 78 C. for 30 min, allowed to warm up to RT and stirred for further 30 min. TLC control (SiO.sub.2/10% ethyl acetate in hexane): R.sub.f (product)=0.59 (yellow upon heating with NaOH), R.sub.f (starting material)=0.67. The reaction mixture was quenched with sat. aq. NH.sub.4Cl (30 mL), extracted with ethyl acetate (340 mL), and the combined organic layers were dried over Na.sub.2SO.sub.4. The product 37 was isolated by flash column chromatography (Teledyne Isco RediSep Rf 80 g, gradient 0% to 100% A:B, A=10% ethyl acetate-hexane, B=hexane) as light yellow viscous oil, yield 2.46 g (74%). .sup.1H NMR (400 MHz, CDCl.sub.3): 8.36 (d, J=8.7 Hz, 2H), 7.08 (d, J=2.5 Hz, 2H), 7.02 (dd, J=8.7, 2.6 Hz, 2H), 1.36-1.25 (m, 6H), 1.13 (d, J=7.3 Hz, 36H), 0.45 (s, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3): 186.1, 159.3, 141.2, 134.5, 132.4, 123.6, 121.8, 18.1, 12.9, 1.4. ESI-MS, positive mode: m/z (rel. int., %)=583.3 [M+H].sup.+. HR-MS (ESI, positive mode): 583.3428 [M+H].sup.+ (found), 583.3454 (calculated for C.sub.33H.sub.55O.sub.3Si.sub.3, [M+H].sup.+).
[0215] Compound 38.
[0216] In a 50 mL round-bottom flask, a degassed solution of 26 (Scheme 9; 427 mg, 1.2 mmol, 2 eq) in anhydrous THF (4 mL) and pentane (2 mL) was cooled to 100 C. (bath temperature, diethyl etherliquid N.sub.2). n-Butyllithium (0.48 mL of 2.5 M solution in hexanes, 1.2 mmol, 2 eq) was carefully introduced through a needle along the wall of the flask. Clear solution quickly turned orange and then purple; it was stirred at 100 C. for 10 min, and the solution of ketone 37 (349 mg, 0.6 mmol) in THF (2 mL) was injected over 1-2 min along the wall of the flask. The flask was then placed into a 78 bath (dry iceacetone) and the stirring was continued for 10 min. The cooling bath was removed, the mixture was allowed to warm up to RT and stirred for further 30 min. TLC control (SiO.sub.2/10% ethyl acetate in hexane): Rf (product)=0.45 (purple upon heating with NaOH), Rf (starting material)=0.62. The reaction mixture was quenched with water (20 mL), adjusted to pH4 with acetic acid, extracted with ethyl acetate (320 mL), the combined organic layers were washed with brine and dried over Na.sub.2SO.sub.4. The product was isolated by flash column chromatography (Interchim Puriflash HP 15 m 25 g; gradient 0% to 20% ethyl acetate-hexane); fractions containing the product were repurified by flash column chromatography (Grace Reveleris HP 12 g; gradient 30% to 100% CH.sub.2Cl.sub.2 pentane over 20 CV), and the product was freeze-dried from 1,4-dioxane to give 38 as white solid. Yield 186 mg (39%). .sup.1H NMR (400 MHz, acetone-d.sub.6): 8.17 (dd, J=8.0, 1.3 Hz, 1H), 8.04 (d, J=8.0 Hz, 1H), 7.81 (dd, J=1.3, 0.7 Hz, 1H), 7.35 (d, J=2.7 Hz, 2H), 7.05 (d, J=8.7 Hz, 2H), 6.93 (dd, J=8.8, 2.7 Hz, 2H), 1.54 (s, 9H), 1.36-1.24 (m, 6H), 1.11 (d, J=7.4 Hz, 36H), 0.74 (s, 3H), 0.61 (s, 3H). .sup.13C NMR (101 MHz, acetone-d.sub.6): 170.0, 164.5, 156.53, 156.52, 138.4, 137.7, 137.0, 130.9, 130.0, 129.0, 128.7, 126.7, 125.7, 125.0, 122.6, 90.3, 82.9, 28.2, 18.3, 13.4, 0.3, 0.4. ESI-MS, positive mode: m/z (rel. int., %)=787.5 (100) [M+H].sup.+. HR-MS (ESI, positive mode): 787.4224 [M+H].sup.+ (found), 787.4240 (calculated for C.sub.45H.sub.67O.sub.6Si.sub.3, [M+H].sup.+).
[0217] Compound 39.
[0218] Tetrabutylammonium fluoride trihydrate (290 mg, 0.92 mmol, 4 eq), dissolved in THF (3.5 mL), was added a solution of 38 (180 mg, 0.23 mmol) in THF (3 mL), cooled in ice-water bath. An intense blue solution with red fluorescence formed. The solution was stirred at 0 C. for 30 min. TLC control (SiO.sub.2/10% ethyl acetate in CH.sub.2Cl.sub.2): Rf (product)=0.47 (orange; purple with NaOH). Sat. aq. NH.sub.4Cl (20 mL) was added, the mixture was extracted with ethyl acetate (320 mL), the combined organic layers were washed with brine and dried over Na.sub.2SO.sub.4. The filtrate was evaporated to light orange oil, which was used directly in the next step.
[0219] Trifluoromethanesulfonic anhydride (Tf.sub.2O; 155 L, 0.92 mmol, 4 eq) was added dropwise to a solution of the crude material from the deprotection step and pyridine (150 L, 1.84 mmol, 8 eq) in dry CH.sub.2Cl.sub.2 (5 mL), cooled in ice-water bath. Pink color appeared upon addition of each drop and vanished immediately. The flask was then removed from the cooling bath, and the yellow mixture was stirred at RT for 1 h. TLC control (SiO.sub.2/10% ethyl acetate in hexane): R.sub.f (product)=0.22 (purple with NaOH). The mixture was diluted with cold water (30 mL), extracted with CH.sub.2Cl.sub.2 (320 mL), the combined extracts were washed with brine and dried over Na.sub.2SO.sub.4. The product was isolated by flash column chromatography (Sepacore Silica HP 12 g; gradient 0% to 20% ethyl acetatepentane) and pure fraction were evaporated to give 39 as white foam, yield 153 mg (90%). .sup.1H NMR (400 MHz, CDCl.sub.3): 8.21 (dd, J=8.0, 1.3 Hz, 1H), 8.04 (dd, J=8.0, 0.8 Hz, 1H), 7.91 (t, J=1.0 Hz, 1H), 7.57 (d, J=2.6 Hz, 2H), 7.28 (d, J=8.9 Hz, 2H), 7.21 (dd, J=8.9, 2.7 Hz, 2H), 1.58 (s, 9H), 0.81 (s, 3H), 0.71 (s, 3H). .sup.19F NMR (376 MHz, CDCl.sub.3): 72.77. ESI-MS, positive mode: m/z (rel. int., %)=739.3 (100) [M+H].sup.+.
[0220] Compound 40.
[0221] A mixture of 39 (150 mg, 0.2 mmol), 3-(tert-butyldimethylsilyloxy)azetidine 30 (112 mg, 0.6 mmol, 3 eq), Pd.sub.2(dba).sub.3 (18 mg, 0.02 mmol, 10 mol %), XPhos (29 mg, 0.06 mmol, 30 mol %) and K.sub.2CO.sub.3 (83 mg, 0.6 mmol, 3 eq) in dry dioxane (2 mL) was degassed on a Schlenk line and stirred at 100 C. under argon (bath temperature) in a sealed flask for 3 h. TLC control (SiO.sub.2/pentaneCH.sub.2Cl.sub.2 ethyl acetate 70:25:5, stained by heating): R.sub.f (product)=0.50 (blue), R.sub.f (starting material)=0.61 (colorless). Upon cooling, the reaction mixture was diluted with water (20 mL), extracted with CH.sub.2Cl.sub.2 (320 mL), the combined organic layers were washed with brine and dried over Na.sub.2SO.sub.4. The filtrate was evaporated on Celite, and the product was isolated by flash column chromatography (Interchim Puriflash 15 m 25 g; gradient 0% to 100% A:B, A=hexaneCH.sub.2Cl.sub.2 ethyl acetate 70:25:10, B=hexaneCH.sub.2Cl.sub.2 70:25) and freeze-dried from 1,4-dioxane to yield 145 mg (89%) of 40 as light yellow fluffy solid. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.11 (dd, J=8.0, 1.3 Hz, 1H), 7.96 (dd, J=8.0, 0.7 Hz, 1H), 7.81 (s, 1H), 6.85 (d, J=8.7 Hz, 2H), 6.69 (d, J=2.6 Hz, 2H), 6.33 (dd, J=8.8, 2.6 Hz, 2H), 4.78-4.69 (m, 2H), 4.19-4.12 (m, 4H), 3.69-3.62 (m, 4H), 1.55 (s, 9H), 0.89 (s, 18H), 0.66 (s, 3H), 0.58 (s, 3H), 0.07 (s, 12H). .sup.13C NMR (101 MHz, CDCl.sub.3): 170.3, 164.4, 155.4, 150.7, 137.3, 136.1, 132.7, 129.9, 129.0, 127.7, 125.7, 125.1, 116.3, 113.3, 82.3, 62.5, 62.1, 28.2, 25.9, 18.1, 0.2, 0.6, 4.8. ESI-MS, positive mode: m/z (rel. int., %)=813.4 (100) [M+H].sup.+. HR-MS (ESI, positive mode): 813.4120 [M+H].sup.+ (found), 813.4145 (calculated for C.sub.45H.sub.65N.sub.2O.sub.6Si.sub.3, [M+H].sup.+).
[0222] Compound 41.
[0223] Tetrabutylammonium fluoride trihydrate (142 mg, 0.45 mmol, 3 eq), dissolved in THF (10 mL), was added a solution of 40 (122 mg, 0.15 mmol) in THF (15 mL), cooled in ice-water bath. The reaction mixture was stirred at 0 C. for 1 h. Water (20 mL), acetic acid (1.5 mL) and brine (20 mL) were then added, and the mixture was extracted with ethyl acetate (320 mL), the combined organic layers were dried over Na.sub.2SO.sub.4, filtered, evaporated and re-evaporated twice with ethyl acetate (220 mL). The product was isolated by flash column chromatography (13 g silica; gradient 10% to 80% ethyl acetateCH.sub.2Cl.sub.2) and freeze-dried from 1,4-dioxane to give 41 as light green fluffy solid. Yield 84 mg (96%). .sup.1H NMR (400 MHz, CD.sub.3OD): 8.12 (dd, J=8.0, 1.3 Hz, 1H), 7.97 (dd, J=8.1, 0.7 Hz, 1H), 7.69 (t, J=1.0 Hz, 1H), 6.77 (d, J=2.8 Hz, 2H), 6.76 (d, J=8.9 Hz, 2H), 6.35 (dd, J=8.9, 2.8, 2H), 4.63 (tt, J=6.5, 4.8 Hz, 2H), 4.17-4.09 (m, 4H), 3.64-3.57 (m, 4H), 1.51 (d, J=0.8 Hz, 9H), 0.64 (s, 3H), 0.54 (s, 3H). .sup.13C NMR (101 MHz, CD.sub.3OD): 172.0, 165.4, 157.4, 152.4, 138.7, 136.9, 133.4, 130.9, 129.7, 128.5, 126.7, 125.6, 117.3, 114.6, 93.2, 83.5, 68.1, 63.0, 62.5, 28.3, 0.1, 0.5. ESI-MS, positive mode: m/z (rel. int., %)=585.3 (100) [M+H].sup.+. HR-MS (ESI, positive mode): 585.2429 [M+H].sup.+ (found), 585.2415 (calculated for C.sub.33H.sub.37N.sub.2O.sub.6Si, [M+H].sup.+).
[0224] Compound 42.
[0225] Trifluoroacetic acid (2.8 mL) was added to a solution of 41 (84 mg, 144 mol) in CH.sub.2Cl.sub.2 (14 mL), cooled in ice-water bath. The cooling bath was removed, and the reaction mixture was stirred at RT for 6 h. The solvents were evaporated, and the residue was re-evaporated twice with toluene (220 mL) and freeze-dried from 1,4-dioxane, giving 101 mg of impure material. The product was isolated by chromatography on a reversed phase (RP-C.sub.18 10 g; gradient 67% to 33% wateracetonitrile). Fractions containing the product were evaporated and freeze-dried from 1,4-dioxane to give 70 mg (92%) of 42 as blue fluffy solid (free base). HPLC (10/90-100/0 over 25 min, column 4250 mm, 1.2 mL/min, detection at 254 nm): =9.98 min. UV-Vis (PBS 7.4): .sub.max ()=641 nm (51000 M.sup.1 cm.sup.1); fluorescence (PBS 7.4): .sub.excit=610 nm, .sub.em=662 nm; .sub.fl (relative to Oxazine 1, .sub.fl=0.14 in ethanol)=0.42. .sup.1H NMR (400 MHz, CD.sub.3OD): 8.22 (dd, J=7.9, 1.3 Hz, 1H), 8.00 (d, J=8.0 Hz, 1H), 7.83 (d, J=1.0 Hz, 1H), 6.78 (d, J=2.6 Hz, 2H), 6.74 (d, J=8.7 Hz, 2H), 6.39 (dd, J=8.7, 2.7 Hz, 2H), 4.66 (tt, J=6.4, 4.8 Hz, 2H), 4.16 (ddd, J=7.4, 6.4, 1.0 Hz, 4H), 3.66-3.60 (m, 4H), 0.64 (s, 3H), 0.54 (s, 3H). .sup.13C NMR (101 MHz, CD.sub.3OD): 171.7, 156.3, 152.5, 137.8, 133.5, 131.3, 130.5, 128.9, 126.8, 126.6, 117.5, 114.3, 63.0, 62.5, 0.1, 1.2. ESI-MS, positive mode: m/z (rel. int., %)=529.2 (100) [M+H].sup.+. HR-MS (ESI, positive mode): 529.1787 (found), 529.1789 (calculated for C.sub.29H.sub.29N.sub.2O.sub.6Si, [M+H].sup.+).
##STR00030##
[0226] Compound 42-Halo.
[0227] PyBOP (20 L of 22 mg/100 L stock solution, 8.52 mol, 1.5 equiv) was added to a solution of 42 (3 mg, 5.68 mol), HaloTag Amine (O2) (2-(2-((6-chlorohexyl)-oxy)ethoxy)ethanamine; 1.9 mg, 8.52 mol, 1.5 equiv) and DIEA (N-ethyldiisopropylamine; 10 L) in DMF (100 L). After 1 h, the solvent was evaporated in vacuo at RT, and the product 42-Halo was isolated by preparative TLC (silica, 10% methanolCH.sub.2Cl.sub.2, then 100% CH.sub.2Cl.sub.2). Yield 3.0 mg (72%), purity (HPLC) 96%. HPLC (10/90-100/0 over 25 min, column 4250 mm, 1.2 mL/min, detection at 254 nm): i=14.98 min. ESI-MS, positive mode: m/z (rel. int., %)=734.3 [M+H].sup.+. HR-MS (ESI, positive mode): 734.3011 (found), 734.3023 (calculated for C.sub.39H.sub.49ClSiN.sub.3O.sub.7, [M+H].sup.+).
Example III
STED Optical Microscopy of Cells Using Exemplary Novel Dyes of the Invention
[0228] Dye 5: In order to provide a suitable STED wavelength (STED=stimulated emission depletion, method of the super-resolution optical microscopy providing the optical resolution beyond the diffraction limit), the 660 nm STED laser of the commercial Leica STED microscope, such as Leica TCS SP8 STED 3X, can be advantageously used. Indeed, the difference between the emission maximum and the STED wavelength is 100-170 nm (for the dyes with small Stokes shifts).
[0229]
[0230] Dye 13a: ROX dyes belong to the so-called big four group of dyes (FAM, JOE [6-JOE has absorption and emission maxima at 520 nm and 548 nm, respectively, =75 000 M.sup.1 cm.sup.1], TAMRA and ROX) dominating in the DNA sequencing, and may be efficiently excited with an argon ion laser emitting at 488 nm (and 514 nm). As demonstrated here, the fluorescence of 6-carboxy ROX dyes (13a-c) may be efficiently switched off with 775 nm STED laser.
[0231]
[0232] Comparison of the mono-hydroxylated dye 13a and the commercially available 6-ROX dye (13c;
[0233]
[0234] In the case of germanium-containing dyes19 and 33hydroxylation was combined with the incorporation of the nitrogen atoms into azetidine rings. The spectral properties of dye 19 (with dimethylamino auxofluoric groups) and dye 33 (with 3-hydroxyazetidine groups) are very similar. However, the fluorescence quantum yield of dye 33 in aqueous buffers (0.60) is higher than the fluorescence quantum yield of dye 19 (0.43). This finding illustrates the positive influence of hydroxyl groups on the fluorescence quantum yield of GeR dyes. The conjugates of both dyes (with docetaxel and HaloTag amine) enable a clear visualization of tubulin filaments and HaloTag-fusion proteins in living cells (see
[0235]
[0236]
[0237]
[0238]
[0239] Combination of hydroxylated dyes 13a and 33 enabled to realize two-color STED microscopy in living cells (
[0240]
[0241]
[0242] 33-lysosome probe showed a high fluorescence increase upon pepsin binding and only a moderate fluorescence intensity change upon nonspecific binding with BSA.
[0243] This demonstrates that 33-lysosome is a suitable probe for selectively staining the acidic compartments of lysosomes in the presence of native protein background.