Dyes with phosphinic acid, phosphinate, phosphonate and phosphonamidate substituents as auxochromic groups and methods for preparing the same
10174202 · 2019-01-08
Assignee
Inventors
- Maksim Sednev (Wuerzburg, DE)
- Alexey BUTKEVICH (Goettingen, DE)
- Heydar SHOJAEI (Goettingen, DE)
- Vladimir Belov (Goettingen, DE)
- Stefan Hell (Goettingen, DE)
- Christian Wurm (Goettingen, DE)
- Dirk KAMIN (Goettingen, DE)
Cpc classification
C09B11/28
CHEMISTRY; METALLURGY
C07F9/655372
CHEMISTRY; METALLURGY
C07F9/6561
CHEMISTRY; METALLURGY
C09B23/04
CHEMISTRY; METALLURGY
C09B11/24
CHEMISTRY; METALLURGY
C07F9/65586
CHEMISTRY; METALLURGY
C07F9/65522
CHEMISTRY; METALLURGY
C07F9/4021
CHEMISTRY; METALLURGY
C07F9/4419
CHEMISTRY; METALLURGY
International classification
C09B5/24
CHEMISTRY; METALLURGY
C07F9/6561
CHEMISTRY; METALLURGY
C07F9/6558
CHEMISTRY; METALLURGY
C07F9/6553
CHEMISTRY; METALLURGY
C07F9/655
CHEMISTRY; METALLURGY
Abstract
Compounds of formula I are disclosed: ##STR00001##
wherein X.sup.1, X.sup.2, X.sup.3, X.sup.4 are independently H, F, Cl, Br, I, CN, NO.sub.2, OR.sup.1, SR.sup.1, NR.sup.1R.sup.2, COR.sup.1, COOR.sup.1, CONR.sup.1R.sup.2, PO.sub.3R.sup.1R.sup.2, SO.sub.2R.sup.1, SO.sub.3R.sup.1 or R.sup.3; R.sup.1 and R.sup.2 are, e.g., H, alkyl or aryl or optionally a ring; R.sup.3 is, e.g., alkyl, alkenyl, alkynyl, aryl or cycloalkyl; Y is OR.sup.1, NR.sup.1R.sup.2, or NR.sup.1R.sup.3; Q is O, S, SO.sub.2, NR, C(R.sup.3).sub.2, Si(R.sup.3).sub.2, Ge(R.sup.3).sub.2, P(O)R.sup.3 or P(O)OR.sup.3; Q and X.sup.1 can optionally form part of a ring; L and M are independently OR.sup.1, SR.sup.1, NR.sup.1R.sup.2 and R.sup.3; L and M can optionally form part of a ring; Z is O, S, NR.sup.1, CR.sup.1R.sup.3 or aryl; and Z and X.sup.4 can optionally form part of a ring.
Claims
1. A compound which is a fluorescent dye and has the structural formula: ##STR00139## wherein: each of X.sup.1, X.sup.2, X.sup.3 and X.sup.4 is a member independently selected from the group consisting of H, F, Cl, Br, I, CN, NO.sub.2, OR.sup.1, SR.sup.1, NR.sup.1R.sup.2, COR.sup.1, COOR.sup.1, CONR.sup.1R.sup.2, PO.sub.3R.sup.1R.sup.2, SO.sub.2R.sup.1, SO.sub.3R.sup.1 and R.sup.3, where: R.sup.1 and R.sup.2 are independently selected from H, alkyl, aryl or heteroaryl, and R.sup.1 and R.sup.2 can optionally form together a substituted or unsubstituted 4-7 membered ring; R.sup.3 is alkyl, alkenyl, alkynyl, aryl or cycloalkyl, optionally substituted with one or more heteroatoms independently selected from N, O, S, F, Cl, Br, I, N.sub.3, amine, OH, OR.sup.1, OCOR.sup.1, aryl, COOR.sup.1, CONR.sup.1R.sup.2, PO.sub.3H.sub.2 and SO.sub.3H, where R.sup.1 and R.sup.2 are defined as above; Y is selected from OR.sup.1, NR.sup.1R.sup.2, or NR.sup.1R.sup.3, where R.sup.1, R.sup.2 and R.sup.3 are defined as above; Q is selected from O, S, SO.sub.2, NR.sup.3, C(R.sup.3).sub.2, Si(R.sup.3).sub.2, Ge(R.sup.3).sub.2, P(O)R.sup.3, or P(O)OR.sup.3, where R.sup.3 is defined as above, and wherein Q and X.sup.1, taken together with the atoms to which they are bonded, can optionally form a substituted or unsubstituted 5-7 membered ring; L and M are independently selected from OR.sup.1, SR.sup.1, NR.sup.1R.sup.2 or R.sup.3, where R.sup.1, R.sup.2 and R.sup.3 are defined as above, and wherein L and M, taken together with the atoms to which they are bonded, can optionally form a substituted or unsubstituted 5-7 membered ring; and Z is selected from O, S, NR.sup.1, CR.sup.1R.sup.3 or aryl, where R.sup.1 and R.sup.3 are defined as above, and wherein Z and X.sup.4, taken with the atoms to which they are bonded, can optionally form a substituted or unsubstituted 5-7 membered ring.
2. The compound according to claim 1, wherein the amine is a member selected from the group consisting of NH.sub.2, NH(alkyl), NH(aryl), N(alkyl)(aryl) and N(alkyl).sub.2.
3. The compound according to claim 1, where Z and X.sup.4, taken with the atoms to which they are bonded, form a substituted or unsubstituted 5-7 membered ring, substituted with at least one additional heteroatom selected from the group consisting of N, O and S and/or at least one substituent selected from the group consisting of F, Cl, Br, I, CN, N.sub.3, B(OR.sup.1)(OR.sup.2), OR.sup.1, SR.sup.1, NR.sup.1R.sup.2, COR.sup.1, COOR.sup.1, CONR.sup.1R.sup.2, PO.sub.3R.sup.1R.sup.2, SO.sub.2R.sup.1, SO.sub.3R.sup.1 and R.sup.3, where R.sup.1, R.sup.2, R.sup.3 are defined as in claim 1.
4. The compound according to claim 1, having one of the following formulae Ia-Is: ##STR00140## wherein: each substituent X is a member independently selected from the group consisting of: H, F, Cl, Br, I, CN, NO.sub.2, OR.sup.1, SR.sup.1, NR.sup.1R.sup.2, COR.sup.1, COOR.sup.1, CONR.sup.1R.sup.2, PO.sub.3R.sup.1R.sup.2, SO.sub.2R.sup.1, SO.sub.3R.sup.1 and R.sup.3, where R.sup.1, R.sup.2, R.sup.3 are defined as in claim 1; the substituent R is selected from H and R.sup.3, where R.sup.3 is defined as in claim 1; Q is selected from O, S, SO.sub.2, NR.sup.3, C(R.sup.3).sub.2, Si(R.sup.3).sub.2, Ge(R.sup.3).sub.2, P(O)R.sup.3, and P(O)OR.sup.3, where R.sup.3 is defined as in claim 1, L, M, R.sup.1, R.sup.2 are defined as in claim 1; ##STR00141## wherein the substituents L, M, and R.sup.1 are defined as in claim 1, the substituents Q, R and X are defined as above; ##STR00142## wherein the substituents L, M, R.sup.1 and R.sup.2 are defined as in claim 1, the substituents Q, R and X are defined as above; ##STR00143## wherein: R.sup.1a and R.sup.2a are independently selected from H, alkyl, aryl or heteroaryl, and wherein R.sup.1a and R.sup.2a can optionally form together a substituted or unsubstituted 4-7 membered ring; R.sup.1b and R.sup.2b are independently selected from H, alkyl, aryl or heteroaryl, and wherein R.sup.1b and R.sup.2b can optionally form together a substituted or unsubstituted 4-7 membered ring; L and M are defined as in claim 1, Q and X are defined as above, and a positive charge is delocalized among atoms of the conjugated system in alternating positions such that structure Id above represents only one possible mesomeric structure; ##STR00144## wherein: R.sup.1a and R.sup.2a are independently selected from H, alkyl, aryl or heteroaryl, and wherein R.sup.1a and R.sup.2a can optionally form together a substituted or unsubstituted 4-7 membered ring; R.sup.1b and R.sup.2b are independently selected from H, alkyl, aryl or heteroaryl, and wherein R.sup.1b and R.sup.2b can optionally form together a substituted or unsubstituted 4-7 membered ring; L and M are defined as in claim 1, Q and X are defined as above, and a positive charge is delocalized among atoms of the conjugated system in alternating positions such that structure Ie above represents only one possible mesomeric structure; ##STR00145## wherein the substituents L, M, R.sup.1 and R.sup.2 are defined as in claim 1, Q, R and X are defined as above, and a positive charge is delocalized among atoms of the conjugated system in alternating positions such that structure If above represents only one possible mesomeric structure; ##STR00146## wherein the substituents L and M are defined as in claim 1, Q and X are defined as above, R.sup.a, R.sup.b and R.sup.c are independently selected from H and R.sup.3, where R.sup.3 is defined as in claim 1; ##STR00147## wherein the substituents L and M are defined as in claim 1, Q and X are defined as above, R.sup.a, R.sup.b and R.sup.c are independently selected from H and R.sup.3, where R.sup.3 is defined as in claim 1; ##STR00148## wherein the substituents L and M are defined as in claim 1, Q and X are defined as above, R.sup.a, R.sup.b and R.sup.c are independently selected from H and R.sup.3, where R.sup.3 is defined as in claim 1; ##STR00149## wherein the substituents L and M are defined as in claim 1, Q and X are defined as above, R.sup.a, R.sup.b, R.sup.c and R.sup.d are independently selected from H and R.sup.3, where R.sup.3 is defined as in claim 1, and a positive charge is delocalized among atoms of the conjugated system in alternating positions such that structure Ij above represents only one possible mesomeric structure; ##STR00150## wherein the substituents L and M are defined as in claim 1, Q and X are defined as above, R.sup.a, R.sup.b, R.sup.c and R.sup.d are independently selected from H and R.sup.3, where R.sup.3 is defined as in claim 1, and a positive charge is delocalized among atoms of the conjugated system in alternating positions such that structure Ik above represents only one possible mesomeric structure; ##STR00151## wherein the substituents L, M, R.sup.1 and R.sup.2 are defined as in claim 1, Q and X are defined as above, R.sup.a and R.sup.b are independently selected from H and R.sup.3, where R.sup.3 is defined as in claim 1, and a positive charge is delocalized among atoms of the conjugated system in alternating positions such that structure Il above represents only one possible mesomeric structure; ##STR00152## wherein the substituents L, M, R.sup.1 and R.sup.2 are defined as in claim 1, Q and X are defined as above, R.sup.a and R.sup.b are independently selected from H and R.sup.3, where R.sup.3 is defined as in claim 1, and a positive charge is delocalized among atoms of the conjugated system in alternating positions such that structure Im above represents only one possible mesomeric structure; ##STR00153## wherein the substituents L and M are defined as in claim 1, Q and X are defined as above, R.sup.a, R.sup.b and R.sup.c are independently selected from H and R.sup.3, where R.sup.3 is defined as in claim 1, and a positive charge is delocalized among atoms of the conjugated system in alternating positions such that structure In above represents only one possible mesomeric structure; ##STR00154## wherein the substituents L and M are defined as in claim 1, Q and X are defined as above, R.sup.a and R.sup.b are independently selected from H and R.sup.3, where R.sup.3 is defined as in claim 1; ##STR00155## wherein the substituents L and M are defined as in claim 1, Q and X are defined as above, R.sup.a and R.sup.b are independently selected from H and R.sup.3, where R.sup.3 is defined as in claim 1, and a positive charge is delocalized among atoms of the conjugated system in alternating positions such that structure Ip above represents only one possible mesomeric structure; ##STR00156## wherein the substituents L and M are defined as in claim 1, Q and X are defined as above, R.sup.a, R.sup.b and R.sup.c are independently selected from H and R.sup.3, where R.sup.3 is defined as in claim 1, and a positive charge is delocalized among atoms of the conjugated system in alternating positions such that structure Iq above represents only one possible mesomeric structure; ##STR00157## wherein the substituents L and M are defined as in claim 1, X is defined as above, R.sup.1a and R.sup.2a are independently selected from H, alkyl, aryl or heteroaryl, and wherein R.sup.1a and R.sup.2a can optionally form together a substituted or unsubstituted 4-7 membered ring, R.sup.1b and R.sup.2b are independently selected from H, alkyl, aryl or heteroaryl, and wherein R.sup.1b and R.sup.2b can optionally form together a substituted or unsubstituted 4-7 membered ring, and the positive charge is delocalized among the atoms of the conjugated system in alternating positions (only one mesomeric structure Ir is shown); and ##STR00158## wherein the substituents L and M are defined as in claim 1, X is defined as above, R.sup.a and R.sup.b are independently selected from H and R.sup.3, where R.sup.3 is defined as in claim 1, R.sup.1 and R.sup.2 are independently selected from H, alkyl, aryl or heteroaryl, and wherein R.sup.1 and R.sup.2 can optionally form together a substituted or unsubstituted 4-7 membered ring, and the positive charge is delocalized among the atoms of the conjugated system in alternating positions (only one mesomeric structure Is is shown).
5. The compound according to claim 1, wherein R.sup.1 of COOR.sup.1 is N-succinimidyl, N-phthalimidyl, N-tetrachlorphthalimidyl, pentachlorophenyl, pentafluorophenyl, 2,3,5,6-tetrafluorophenyl, 4-(hydroxysulfonyl)-2,3,5,6-tetrafluorophenyl [p-(HOSO.sub.2)C.sub.6F.sub.4], 1-benzotriazolyl or cyanomethyl.
6. The compound according to claim 1, wherein R.sup.1 of OR.sup.1 is N-succinimidyl, N-phthalimidyl, N-tetrachlorphthalimidyl, pentachlorophenyl, pentafluorophenyl, 2,3,5,6-tetrafluorophenyl, 4-(hydroxysulfonyl)-2,3,5,6-tetrafluorophenyl [p-(HOSO.sub.2)C.sub.6F.sub.4], 1-benzotriazolyl or cyanomethyl.
7. The compound according to claim 4 having a structural formula selected from: ##STR00159## ##STR00160## ##STR00161## ##STR00162## ##STR00163## ##STR00164## wherein the substituents R.sup.1, R.sup.2, R.sup.1a, R.sup.2a, R.sup.1b, R.sup.2b, R are defined as in claim 4.
8. The compound according to claim 1, having a structural formula selected from: ##STR00165## ##STR00166## ##STR00167## ##STR00168## ##STR00169## ##STR00170## ##STR00171##
9. The compound according to claim 1 in a form of a salt with organic or inorganic counterion(s), its cocrystal with another organic or inorganic compound(s), or a composition containing any of the dyes of claim 1.
10. A conjugate or bioconjugate comprising a compound according to claim 1 coupled via at least one covalent chemical bond or at least one molecular complex to a chemical entity or substance.
11. A method for preparing the compound according to claim 1, comprising the following steps: a) Reacting of a precursor compound having the following structure III: ##STR00172## wherein Q, X.sup.1, X.sup.2, X.sup.3, X.sup.4, Y and Z are defined as in claim 1, and A is either a hydrogen atom or a reacting group selected from the group consisting of CN, Cl, Br, I, triflate (OTf), and nonaflate (ONf), with phosphinic (hypophosphorous) acid H.sub.3PO.sub.2, phosphinites [R.sup.aOPR.sup.bR.sup.c], phosphonites [(R.sup.aO)(R.sup.bO)PR.sup.c], phosphites R.sup.cOP(OR.sup.a)(OR.sup.b), phosphoramidites R.sup.cR.sup.dNP(OR.sup.a)(OR.sup.b), where R.sup.a-d are H, alkyl or aryl, or salts of the corresponding esters, where at least one of R.sup.a-d is a metal; the reacting providing either directly a compound of claim 1, or an intermediate compound, which can be isolated or used as crude material for the following step; b) Optional alkylation of an intermediate phosphinic ester leuco derivative with an alkyl halide, alkyl sulfonate or a Michael acceptor, if step a) was performed with phosphinic (hypophosphorous) acid or its esters; c) Oxidizing of the leuco intermediate into a dye of the formula of claim 1 by oxidation with an organic or inorganic oxidant, or with an inorganic oxidant in the presence of catalytic amounts of an organic oxidant(s), or electrochemically, with or without subsequent acidic or basic hydrolysis; and d) Optionally performing post-synthetic modifications.
12. The method of claim 11, wherein step a) is catalyzed by a nucleophilic catalyst.
13. The method according to claim 11, wherein the organic oxidant is selected from 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), 2,3,5,6-tetrachloro-1,4-benzoquinone (p-chloranil), 3,4,5,6-tetrachloro-1,2-benzoquinone (o-chloranil), or other quinones, and/or the inorganic oxidant is selected from oxygen, hydrogen peroxide, iodine, periodate salts, Mn(OAc).sub.3, Pb(OAc).sub.4, PbO.sub.2, or K.sub.3[Fe(CN).sub.6].
14. The method according to claim 11, wherein step c) is performed in vitro or in vivo resulting in a detectable response.
15. The compound according to claim 1, which is a fluorescent label, probe, tracer or marker, as well as quencher in fluorescence energy transfer (FRET) experiments, imaging and optical microscopy.
16. The compound according to claim 15, which is effective for tracking and monitoring dynamic processes in a sample or in an object.
17. The compound according to claim 1, which is a fluorescent tag, analytical reagent or label suitable for use in optical microscopy, imaging techniques, protein tracking, nucleic acid labeling and flow cytometry.
18. The compound according to claim 17, wherein the optical microscopy and imaging methods comprise stimulated emission depiction microscopy, minimal photon fluxes techniques, single molecule switching techniques, single molecule localization microscopy, photoactivation localization microscopy, stochastic optical reconstruction microscopy, direct STORM; fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, fluorescence lifetime imaging, ground state depletion with individual molecular return and fluorescence resonant energy transfer.
19. The compound according to claim 4, having formula Ifa and/or Ifb: ##STR00173## wherein a positive charge is delocalized among atoms of the conjugated system in alternating positions such that structure Ifa above represents only one possible mesomeric structure.
20. The compound according to claim 4, having the following formula ##STR00174## wherein R.sup.a and R.sup.b are independently selected from H and R.sup.3.
21. A compound which is a fluorescent dye and has a structural formula selected from the group consisting of: ##STR00175##
Description
BRIEF DESCRIPTION OF THE FIGURES
(1)
(2)
(3)
(4) The present invention is further illustrated by the following specific but non-limiting examples.
EXAMPLE 1
Synthesis of Fluorescent Phosphorylated Coumarin Dyes and their Precursors
Dimethyl [3-(benzo[d]thiazol-2-yl)-7-(diethylamino)-2-oxo-2H-chromen-4-yl]phosphonate (1)
(5) ##STR00058##
(6) Compound 1. To a stirred suspension of NaH (17 mg of 60 wt. % in mineral oil, 0.429 mmol) in dry DMF (0.5 mL), cooled in ice-water bath, dimethyl phosphite (40 L, 0.429 mmol) was added in one portion. The resulting suspension was warmed up to rt and stirred for 30 min, turning into a clear solution, which was added to a stirred suspension of Coumarin 6 (50 mg, 0.143 mmol) in DMF (0.5 mL). The orange solid dissolved immediately and clear red-orange solution formed. The mixture was stirred at rt for 1 h, and the resulting pale orange solution was poured into water (30 mL) and brine (10 mL), extracted with EtOAc (415 mL), the combined extracts were dried over Na.sub.2SO.sub.4, filtered and evaporated. The residue was taken up in EtOAc (20 mL) and MeOH (5 mL), heated up to 70 C., and a solution of DDQ (32 mg, 0.143 mmol) in EtOAc (2 mL) was added quickly dropwise. The mixture was stirred at 70 C. for 5 min, cooled down to rt and evaporated. The crude product was purified by column chromatography twice (16 g SiO.sub.2, gradient 50% to 100% EtOAc/hexane, and 17 g SiO.sub.2, gradient 50% to 80% EtOAc/hexane) and lyophilized from 1,4-dioxane. Bright yellow solid, yield 34 mg (52%). .sup.1H NMR (400 MHz, CDCl.sub.3): 8.18 (d, J=9.3 Hz, 1H), 8.08 (ddd, J=8.1, 1.3, 0.7 Hz, 1H), 7.92 (ddd, J=8.0, 1.3, 0.7 Hz, 1H), 7.48 (ddd, J=8.2, 7.2, 1.3 Hz, 1H), 7.41 (ddd, J=8.3, 7.3, 1.2 Hz, 1H), 6.65 (dd, J=9.4, 2.7 Hz, 1H), 6.52 (dd, J=2.7, 1.7 Hz, 1H), 3.67 (s, 3H), 3.64 (s, 3H), 3.44 (q, J=7.1 Hz, 4H), 1.23 (t, J=7.1 Hz, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3): 5 162.2 (d, J=7.1 Hz), 160.0 (d, J=19.9 Hz), 156.6 (d, J=14.3 Hz), 152.8, 151.5, 142.3 (d, J=172.0 Hz), 137.1, 130.5 (d, J=2.7 Hz), 126.1, 125.6, 123.6, 121.7, 119.5 (d, J=6.4 Hz), 109.7, 107.2 (d, J=9.9 Hz), 97.4 (d, J=2.5 Hz), 67.2, 53.6, 53.5, 45.1, 12.6. .sup.31P NMR (162 MHz, CDCl.sub.3): 13.37. MS (ESI): m/z (positive mode, rel. int., %)=459.1 (100) [M+H].sup.+, 497.1 (45) [M+K].sup.+. HRMS (C.sub.22H.sub.23N.sub.2O.sub.5PS): m/z (positive mode)=459.1138 (found [M+H].sup.+), 459.1138 (calc.). UV/Vis (MeCN): .sub.max ()=439 nm (24000 M.sup.1cm.sup.1); fluorescence (MeCN): .sub.excit=470 nm, .sub.em=651 nm, .sub.fl=0.20. Standard: Abberior Star520SX tert-butyl ester, .sub.fl=0.27 (MeCN).
3-(Benzo[d]thiazol-2-yl)-2-oxo-2H-chromen-7-yl trifluoromethanesulfonate (2a)
(7) ##STR00059##
(8) Compound 2a. Pyridine (0.55 mL, 6.8 mmol, 4 eq) was added to a suspension of 3-(2-benzothiazolyl)umbelliferone (500 mg, 1.69 mmol) in CH.sub.2Cl.sub.2 (25 mL); a voluminous yellow precipitate formed. The suspension was cooled in ice-water bath, and triflic anhydride (572 L, 3.4 mmol, 2 eq) was added dropwise; the precipitate dissolved. The mixture was warmed up to rt and the resulting thin suspension was stirred at rt (room temperature) for 3 h. It was then cooled in ice-water bath, diluted with water (30 mL), re-extracted with CH.sub.2Cl.sub.2 (220 mL); the combined organic layers were washed with water and brine, dried over Na.sub.2SO.sub.4. The product was isolated by flash chromatography on Biotage Isolera system (12 g Sepacore Silica HP cartridge, gradient 20% to 100% CH.sub.2Cl.sub.2/hexane); the fractions containing the product were evaporated to lemon-yellow solid, which was triturated with hexane, filtered off, washed with hexane and dried in vacuo. Yield 630 mg (87%). .sup.1H NMR (400 MHz, CDCl.sub.3): 9.03 (s, 1H), 8.11-8.05 (m, 1H), 8.00-7.94 (m, 1H), 7.80 (d, J=8.6 Hz, 1H), 7.54 (ddd, J=8.3, 7.1, 1.3 Hz, 1H), 7.43 (ddd, J=8.2, 7.1, 1.1 Hz, 1H), 7.39-7.35 (m, 1H), 7.31 (dd, J=8.6, 2.4 Hz, 1H). .sup.19F NMR (376 MHz, CDCl.sub.3): 72.51. .sup.13C NMR (101 MHz, CDCl.sub.3): 158.9, 158.8, 154.2, 152.6, 151.6, 139.7, 137.1, 131.0, 126.9, 125.9, 123.3, 121.9, 121.5, 119.0, 118.8 (q, J=321.0 Hz), 118.7, 110.6. MS (ESI): m/z (positive mode, rel. int., %)=428.2 (100) [M+H].sup.+. HRMS (C.sub.17H.sub.8NO.sub.5S.sub.2F.sub.3): m/z (positive mode)=427.9867 (found [M+H].sup.+), 427.9869 (calc.).
3-(Benzo[d]thiazol-2-yl)-7-[3-(tert-butyldimethylsilyloxy)azetidin-1-yl]-2H-chromen-2-one (2b)
(9) ##STR00060##
(10) Compound 2b. A mixture of 2a (150 mg, 0.35 mmol), 3-(tert-butyldimethylsilyloxy)azetidine [prepared according to WO 2010015849 A2] (196 mg, 1.05 mmol, 3 eq), Pd.sub.2(dba).sub.3 (8 mg, 8.75 mol, 2.5 mol %), ()-BINAP (18 mg, 28 mol, 8 mol %) and potassium carbonate (97 mg, 0.7 mmol, 2 eq) in toluene (2 mL) was sealed in a 10 mL vial capped with a septum, degassed on a Schlenk line and stirred under argon at 100 C. (bath temperature) for 2 h. Yellow solution gradually turned into an orange suspension. Upon cooling down to rt, acetic acid (1 mL) was added to the reaction mixture, the mixture was diluted with CH.sub.2Cl.sub.2 (30 mL) and evaporated on Celite. The product was isolated by flash chromatography on Biotage Isolera system (12 g Sepacore Silica HP cartridge, gradient 50% to 100% CH.sub.2Cl.sub.2/hexane); the fractions containing the product were evaporated to bright orange solid, yield 98 mg (60%). .sup.1H NMR (400 MHz, CD.sub.3CN+1% TFA): 8.16 (ddd, J=8.2, 1.2, 0.7 Hz, 1H), 8.09 (dt, J=8.3, 0.9 Hz, 1H), 7.78 (ddd, J=8.4, 7.3, 1.2 Hz, 1H), 7.72-7.62 (m, 2H), 7.67 (s, 1H), 6.58 (dd, J=8.9, 2.1 Hz, 1H), 6.36 (dd, J=2.1, 0.7 Hz, 1H), 4.89 (tt, J=6.5, 4.2 Hz, 1H), 4.51-4.44 (m, 2H), 4.06-3.99 (m, 2H), 0.95 (s, 9H), 0.14 (s, 6H). .sup.13C NMR (101 MHz, nitrobenzene-d.sub.5): 162.1, 161.1, 157.2, 154.7, 153.6, 142.9, 137.2, 131.6, 126.8, 125.3, 123.2, 122.3, 113.3, 110.0, 109.9, 96.5, 62.8, 61.7, 26.0, 18.4, 4.9. MS (ESI): m/z (positive mode, rel. int., %)=465.3 (100) [M+H].sup.+. HRMS (C.sub.25H.sub.28N.sub.2O.sub.3SSi): m/z (positive mode) =465.1653 (found [M+H].sup.+), 465.1663 (calc.).
Dimethyl [3-(benzo[d]thiazol-2-yl)-7-(3-hydroxyazetidin-1-yl)-2-oxo-2H-chromen-4-yl]phosphonate (2)
(11) ##STR00061##
(12) Compound 2. To a stirred suspension of NaH (23 mg of 60 wt. % in mineral oil, 0.58 mmol, 3 eq) in dry DMF (0.5 mL), cooled in ice-water bath, dimethyl phosphite (53 L, 0.58 mmol, 3 eq) was added in one portion. The resulting suspension was warmed up to rt and stirred for 30 min, turning into a clear solution, which was added to a stirred suspension of 2b (90 mg, 0.194 mmol) in DMF (3 mL). The orange solid quickly dissolved and clear reddish-brown solution formed. The mixture was stirred at rt for 1 h, DMF was evaporated in vacuo at rt, and the residue was mixed with water (20 mL) and brine (20 mL). Acetic acid was added to pH 3, and the mixture was extracted with EtOAc (320 mL), the combined extracts were dried over Na.sub.2SO.sub.4, filtered and evaporated. The residue was taken up in EtOAc (20 mL) and MeOH (5 mL), heated up to 70 C., and a solution of DDQ (44 mg, 0.194 mmol, 1 eq) in EtOAc (3 mL) was added quickly dropwise. The resulting red-orange mixture was stirred at 70 C. for 5 min, cooled down to rt and evaporated on Celite. The product was isolated by flash chromatography on Biotage Isolera system (10 g Biotage SNAP Ultra cartridge, gradient 0% to 10% methanol/CH.sub.2Cl.sub.2); two fractions were collected, containing the product and the TBS-protected product. Both fractions were pooled together, evaporated and the mixture was used for deprotection. The material was dissolved in THF (7 mL), cooled in ice-water bath, and tetrabutylammonium fluoride trihydrate (92 mg, 0.291 mmol) was added. The resulting brown-yellow solution was allowed to warm up to rt and stirred for 1 h. The mixture was diluted with brine (15 mL), extracted with EtOAc (320 mL), the combined extracts were dried over Na.sub.2SO.sub.4, filtered and evaporated on Celite. The product was isolated by flash chromatography on Biotage Isolera system (12 g Sepacore Silica HP cartridge, gradient 0% to 50% methanol/ethyl acetate); the fractions containing the product were evaporated to brown-red solid, which was freeze-dried from aqueous dioxane to fluffy red solid, yield 20 mg (23% over 3 steps). .sup.1H NMR (400 MHz, acetic acid-d.sub.4): 11.57 (s, 1H), 8.16 (ddd, J=8.2, 1.2, 0.7 Hz, 1H), 8.10 (d, J=9.1 Hz, 1H), 8.03 (ddd, J=8.0, 1.3, 0.7 Hz, 1H), 7.57 (ddd, J=8.3, 7.2, 1.3 Hz, 1H), 7.50 (ddd, J=8.3, 7.2, 1.2 Hz, 1H), 6.48 (dd, J=9.1, 2.4 Hz, 1H), 6.33 (dd, J=2.4, 1.6 Hz, 1H), 4.86 (tt, J=6.6, 4.3 Hz, 1H), 4.34 (ddd, J=9.2, 6.6, 1.3 Hz, 2H), 3.97 (ddd, J=9.2, 4.4, 1.3 Hz, 2H), 3.72 (s, 3H), 3.69 (s, 3H). MS (ESI): m/z (positive mode, rel. int., %)=459.2 (100) [M+H].sup.+. HRMS (C.sub.21H.sub.19N.sub.2O.sub.6PS): m/z (positive mode)=459.0764 (found [M+H].sup.+), 459.0774 (calc.). UV/Vis (MeCN): .sub.max ()=424 nm (19000 M.sup.1cm.sup.1); fluorescence (MeCN): .sub.excit=470 nm, .sub.em=651 nm, .sub.fl=0.16. Standard: Abberior Star520SX tert-butyl ester, .sub.fl=0.27 (MeCN).
Di-tert-butyl [3-(benzo[d]thiazol-2-yl)-7-(diethylamino)-2-oxo-2H-chromen-4-yl]phosphonate (3)
(13) ##STR00062##
(14) Compound 3. To a stirred suspension of NaH (34 mg of 60 wt. % in mineral oil, 0.858 mmol) in dry DMF (1.5 mL), cooled in ice-water bath, di(tert-butyl) phosphite (173 L, 0.858 mmol) was added in one portion. The resulting suspension was stirred for 2 h at rt and for 30 min at 55 C. The resulting thin white suspension was added to a stirred suspension of Coumarin 6 (100 mg, 0.286 mmol) in DMF (0.8 mL). The solids dissolved immediately and clear light-orange solution formed. The mixture was stirred at rt for 1 h and then poured into sat. aq. NaHCO.sub.3 (50 mL), extracted with EtOAc (415 mL), the combined extracts were dried over Na.sub.2SO.sub.4, filtered and evaporated. The residue was redissolved in EtOAc (20 mL), heated up to 75 C., and a solution of DDQ (65 mg, 0.286 mmol) in EtOAc (3 mL) was added quickly dropwise. The mixture was stirred at 75 C. for 5 min, cooled down to rt and evaporated. The product was isolated by column chromatography (30 g SiO.sub.2, gradient 20% to 50% EtOAc/hexane) and lyophilized from 1,4-dioxane. Bright yellow-orange solid, yield 130 mg (84%). .sup.1H NMR (400 MHz, acetone-d.sub.6): 8.25 (br.s, 1H), 8.08-8.03 (m, 1H), 7.99 (ddd, J=8.1, 1.2, 0.6 Hz, 1H), 7.55-7.47 (m, 1H), 7.47-7.41 (m, 1H), 6.84 (dd, J=9.4, 2.7 Hz, 1H), 6.58 (dd, J=2.7, 1.5 Hz, 1H), 3.57 (q, J=7.2 Hz, 4H), 1.45 (s, 18H), 1.26 (t, J=7.1 Hz, 6H). .sup.31P NMR (162 MHz, acetone-d.sub.6): 5 0.60. .sup.13C NMR (101 MHz, acetone-d.sub.6): 209.9, 163.7 (d, J=7.0 Hz), 160.4, 157.5 (d, J=13.4 Hz), 154.1, 152.1, 138.2, 132.1 (d, J=2.0 Hz), 126.3, 125.7, 123.9, 122.3, 110.9, 109.8, 107.2 (d, J=7.9 Hz), 97.6 (d, J=2.6 Hz), 85.2 (d, J=7.3 Hz), 67.6, 45.2, 12.8. MS (ESI): m/z (positive mode, rel. int., %)=543.2 (55) [M+H].sup.+, 565.2 (35) [M+Na].sup.+, 581.2 (100) [M+K].sup.+. HRMS (C.sub.28H.sub.36N.sub.2O.sub.5PS): m/z (positive mode)=543.2077 (found [M+H].sup.+), 543.2077 (calc.). UV/Vis (MeCN): .sub.max ()=434 nm (39000 M.sup.1cm.sup.1); fluorescence (MeCN): .sub.excit=470 nm, .sub.em=658 nm, .sub.fl=0.29. Standard: Abberior Star520SX tert-butyl ester, .sub.fl=0.27 (MeCN).
[3-(Benzo[d]thiazol-2-yl)-7-(diethylamino)-2-oxo-2H-chromen-4-yl]phosphonic acid (4)
(15) ##STR00063##
(16) Compound 4. Trifluoroacetic acid (150 L) was added to a stirred solution of the dye 3 (82 mg, 0.15 mmol) in CH.sub.2Cl.sub.2 (5 mL). The orange color of the solution turned violet upon addition of the acid and eventually deep purple. The mixture was stirred at rt for 30 min, evaporated to dryness, the residue was dissolved in acetic acid and lyophilized. Yield 64 mg (99%), red-brown solid. .sup.1H NMR (400 MHz, acetic acid-d.sub.4): 9.06 (d, J=9.6 Hz, 1H), 8.14 (t, J=7.3 Hz, 2H), 7.76 (t, J=7.6 Hz, 1H), 7.66 (t, J=7.7 Hz, 1H), 6.91 (d, J=9.2 Hz, 1H), 6.61 (s, 1H), 3.61 (q, J=7.1 Hz, 4H), 1.30 (t, J=7.0 Hz, 6H). .sup.31P NMR (162 MHz, acetic acid-d.sub.4): 5.41. MS (ESI): m/z (negative mode, rel. int., %)=429.1 (100) [MH].sup.. HRMS (C.sub.20H.sub.19N.sub.2O.sub.5PS): m/z (negative mode)=429.0684 (found [MH].sup.), 429.0680 (calc.). UV/Vis (PBS 7.4): .sub.max ()=419 nm (22000 M.sup.1cm.sup.1); fluorescence (MeCN): .sub.excit=460 nm, .sub.em=613 nm, .sub.fl=0.04. Standard: Abberior Star520SX tert-butyl ester, .sub.fl=0.27 (MeCN).
EXAMPLE 2
Synthesis of Fluorescent Phosphorylated Xanthene Dyes and their Precursors
9-(Dimethoxyphosphoryl)-3,6-bis(dimethylamino)-9H-xanthenylium trifluoroacetate (5)
(17) ##STR00064##
(18) Compound 5. In a screw-cap test tube to a suspension of Pyronin Y (50 mg; 0.165 mmol) in CH.sub.2Cl.sub.2 (1 mL) P(OMe).sub.3 (20 mg; 0.165 mmol) was added at r.t. under argon. The resulting reaction mixture was warmed up to 40 C. and stirred for 2 h at this temperature. After cooling down to 0 C., DDQ (112 mg; 0.495 mmol) was added. The reaction mixture was stirred additionally for 15 min at 0 C. After dilution with MeCN (.sup.3 mL), the reaction mixture was directly subjected to column chromatography on SiO.sub.2 (30 g; MeCN.fwdarw.MeCN/H.sub.2O 10:1+0.1 v/v % of TFA) to afford 20 mg (29%) of a dark violet powder. .sup.1H NMR (400 MHz, CD.sub.3CN): =3.30 (s, 12H, 2NMe.sub.2), 3.85 (d, J.sub.HP=11.6 Hz, 6H, 2OMe), 6.74 (m, J.sub.HH=2.5 Hz, 2H.sub.ar), 7.13 (dd, J.sub.HH=9.9 and 2.5 Hz, 2H.sub.ar), 8.69 (d, J.sub.HH=9.9 Hz, 2H.sub.ar) ppm. .sup.13C NMR (100 MHz, CD.sub.3CN, APT): =40.4 (+), 53.3 (+, d, J.sub.CP=5 Hz), 96.4 (+), 110.0 (), 115.0 (+), 115.8 (, d, J.sub.CP=10.4 Hz), 132.0 (+, d, J.sub.CP=3.3 Hz), 157.0 (), 157.2 (, d, J.sub.CP=13.3 Hz) ppm. .sup.31P NMR (162 MHz, CD.sub.3CN): (=13.7 ppm. MS (ESI): m/z (positive mode, rel. int., %)=375.2 (100) [MCl].sup.+. HPLC: t.sub.R=8.5 min (96.5%), B/A=30/70-100/0 in 25 min, column 4250 mm, 1.2 mL/min, detection at 254 nm.
9-(Diisopropoxyphosphoryl)-3,6-bis(dimethylamino)-9H-xanthenylium trifluoroacetate (6)
(19) ##STR00065##
(20) Compound 6. In a screw-cap test tube to a suspension of Pyronin Y (50 mg; 0.165 mmol) in MeCN (1 mL) P(OiPr).sub.3 (34 mg; 0.165 mmol) was added at r.t. under argon. The resulting reaction mixture was warmed up to 60 C. and stirred for 1 h at this temperature. After cooling down to 0 C., DDQ (37 mg; 0.165 mmol) was added, and the reaction mixture was stirred for additional 10 min at 0 C. After warming up to r.t., the reaction mixture was directly subjected to column chromatography on SiO.sub.2 (30 g; MeCN.fwdarw.MeCN/H.sub.2O 10:1+0.1 v/v % of TFA) to afford 23 mg (30%) of a dark violet powder. .sup.1H NMR (400 MHz, CD.sub.3CN): =1.17 (d, J.sub.HH=6.2 Hz, 6H, OiPr), 1.43 (d, J.sub.HH=6.2 Hz, 6H, OiPr), 3.30 (s, 12H, 2NMe.sub.2), 4.85 (m, J.sub.HP=12.3 Hz, J.sub.HH=6.2 Hz, 2H, 2OiPr), 6.78 (m, J.sub.HH=2.4 Hz, 2H.sub.ar), 7.16 (dd, J.sub.HH=9.9, 2.6 Hz, 2H.sub.ar), 8.81 (d, J.sub.HH=9.9 Hz, 2H.sub.ar) ppm. .sup.13C NMR (100 MHz, CD.sub.3CN, APT): =22.9 (+, d, J.sub.CP=5.0 Hz), 23.2 (+, d, J.sub.CP=4.0 Hz), 40.4(+), 73.6 (+, d, J.sub.CP=5.7 Hz), 96.3 (+, J.sub.CP=1.6 Hz), 110.0 (), 114.8 (+), 115.4 (, d, J.sub.CP=10.5 Hz), 132.4 (+, J.sub.CP=3.3 Hz), 157.0 (), 157.3 (, J.sub.CP=13.0 Hz) ppm. .sup.31P NMR (162 MHz, CD.sub.3CN): =8.1 ppm. MS (ESI): m/z (positive mode, rel. int., %)=431.3 (100) [MCl].sup.+. HPLC: t.sub.R=13.5 min (97%), B/A=30/70-100/0 in 25 min, column 4250 mm, 1.2 mL/min, detection at 254 nm. UV/Vis (MeCN): .sub.max ()=617 nm (57638 M.sup.1cm.sup.1); fluorescence (MeCN): .sub.excit=585 nm, .sub.em=649 nm, .sub.fl=0.38. Standard: Oxazine 4, .sub.fl=0.63 (MeOH). UV/Vis (PBS 7.4): .sub.max ()=629 nm (52254 M.sup.1cm.sup.1); fluorescence (PBS 7.4): .sub.excit=585 nm, .sub.em=668 nm, .sub.fl=0.12. Standard: Oxazine 4, .sub.fl=0.63 (MeOH).
9-[(Diisopropylamino)(methoxy)phosphoryl]-3,6-bis(dimethylamino)-9H-xanthenylium trifluoroacetate (7)
(21) ##STR00066##
(22) Compound 7. In a screw-cap test tube to a suspension of Pyronin Y (50 mg; 0.165 mmol) in MeCN (1 mL) dimethyl N,N-diisopropylphosphoramidite (32 mg; 0.165 mmol) was added at r.t. under Ar. The reaction mixture was warmed up to 60 C. and stirred for 30 min at this temperature. After cooling down to 0 C., DDQ (37 mg; 0.165 mmol) was added, and the reaction mixture was stirred for additional 10 min at 0 C. After warming up to r.t., the reaction mixture was directly subjected to column chromatography on SiO.sub.2 (30 g; MeCN.fwdarw.MeCN/H.sub.2O 20:1+0.1 v/v % of TFA). Fractions containing the title compound were evaporated to dryness, dissolved in water and extracted with CH.sub.2Cl.sub.2 (3). The combined extracts were dried with Na.sub.2SO.sub.4 and evaporated to yield 32 mg (40%) of a dark violet solid. .sup.1H NMR (400 MHz, CD.sub.3CN): =1.23 (d, J.sub.HH=6.8 Hz, 6H, NiPr.sub.2), 1.26 (d, J.sub.HH=6.8 Hz, 6H, NiPr.sub.2), 3.29 (s, 12H, 2NMe.sub.2), 3.44-3.60 (m, 2H, NiPr.sub.2), 3.72 (d, J.sub.HP=11.5 Hz, 3H, OMe), 6.74-6.77 (m, 2H.sub.ar), 7.15 (dd, J.sub.HH=9.9 and 2.7 Hz, 2H.sub.ar), 8.89 (d, J.sub.HH=9.9 Hz, 2H.sub.ar) ppm. .sup.13C NMR (100 MHz, CD.sub.3CN, APT): =18.1 (+), 21.8 (+, d, J.sub.CP=2.7 Hz), 21.9 (+, d, J.sub.CP=2.7 Hz), 47.0 (+, m), 52.0 (+, d, J.sub.CP=5.8 Hz), 96.2 (+), 114.7 (+), 116.0 (, d, J.sub.CP=9.2 Hz), 132.2 (+, d, J.sub.CP=3.3 Hz), 156.9 (), 157.4 (, d, J.sub.CP=11.7 Hz) ppm. .sup.31P NMR (162 MHz, CD.sub.3CN): =20.1 ppm. MS (ESI): m/z (positive mode, rel. int., %)=444.4 (100) [MCl].sup.+. HPLC: t.sub.R=13.1 min (91%), B/A=30/70-100/0 in 25 min, column 4250 mm, 1.2 mL/min, detection at 254 nm. UV/Vis (MeCN): .sub.max ()=616 nm (52455 M.sup.1cm.sup.1); fluorescence (MeCN): .sub.excit=590 nm, .sub.em=649 nm, .sub.fl=0.39. Standard: Oxazine 4, .sub.fl=0.63 (MeOH). UV/Vis (PBS 7.4): .sub.max ()=628 nm (46051 M.sup.1cm.sup.1); fluorescence (PBS 7.4): .sub.excit=590 nm, .sub.em=664 nm, .sub.fl=0.11. Standard: Oxazine 4, .sub.fl=0.63 (MeOH).
3,6-Bis(dimethylamino)-9-(diphenylphosphoryl)-9H-xanthenylium trifluoroacetate (8)
(23) ##STR00067##
(24) Compound 8. In a screw-cap test tube to a solution of Pyronin Y (50 mg; 0.165 mmol) in acetonitrile (5 mL) methoxydiphenylphosphine (56 mg; 0.330 mmol) was added at r.t. under argon. The resulting reaction mixture was warmed up to 60 C. and stirred for 1 h at this temperature. After cooling down to 0 C., DDQ (75 mg; 0.330 mmol) was added. The reaction mixture was stirred additionally for 30 min at 0 C. and directly subjected to column chromatography on SiO.sub.2 (30 g; MeCN.fwdarw.5% to 50% H.sub.2O/MeCN+0.1 v/v % of TFA) to afford 18 mg (22%) of the product as violet solid. HPLC: t.sub.R=10.7 min (86%), B/A=30/70-100/0 in 25 min, column 4250 mm, 1.2 mL/min, detection at 254 nm.
2-[[3,6-Bis(dimethylamino)-9H-xanthen-9-yl](dimethylamino)phosphoryloxy]benzoic acid (9a)
(25) ##STR00068##
(26) Compound 9a. A suspension of Pyronin Y (151 mg; 0.5 mmol) and tetrabutylammonium iodide (185 mg; 0.5 mmol) in CH.sub.2Cl.sub.2 was sonicated briefly, and 2-dimethylamino-4H-1,3,2-benzodioxaphosphorin-4-one [prepared according to Gast, R.; Kaukorat, T.; Neda, I.; Schmutzler, R. Z. Naturforsch. 1993, 48b, 867-874] (211 mg, 1 mmol) was added. The resulting mixture was sonicated for 2 min and stirred vigorously for 1 h, during which time a bright pink solution turned into a deep purple thin suspension. Sodium hydroxide (1 mL of 10% in MeOH/H.sub.2O 1:1) was added followed by just enough MeOH to homogenize the mixture. After stirring for 10 min, AcOH (2 mL) was added, and the mixture was evaporated to dryness (re-evaporated several times with acetone). The residue was subjected to column chromatography (45 g of SiO.sub.2, gradient 10% to 40% MeOH/CH.sub.2Cl.sub.2). Fractions containing the product were pooled, evaporated to dryness, redissolved in 1,4-dioxane (20 mL), filtered through a 0.45 m PTFE membrane filter and freeze-dried, yielding the intermediate xanthene 9a (106 mg, 43% yield) as a fluffy violet solid. MS (ESI): m/z (negative mode, rel. int., %)=494.3 (100) [MH].sup.+. HRMS (C.sub.27H.sub.30N.sub.3O.sub.5P): m/z (negative mode)=494.1850 (found [MH].sup.), 494.1850 (calc.).
2-[[3,6-Bis(dimethylamino)-9H-xanthenylium-9yl](dimethylamino)phosphoryloxy]-benzoate (9)
(27) ##STR00069##
(28) Compound 9. The compound 9a (52 mg; 0.105 mmol) was dissolved in CH.sub.2Cl.sub.2 (5 mL), the solution was cooled in dry ice-acetone bath, and DDQ (24 mg; 0.105 mmol) in CH.sub.2Cl.sub.2 (3 mL) was added quickly dropwise. The resulting dark violet solution was allowed to warm up to rt and evaporated to dryness. The residue was subjected to column chromatography (30 g of SiO.sub.2, gradient 5% to 50% MeOH/CH.sub.2Cl.sub.2); the fractions containing the product were evaporated and re-purified by reversed-phase chromatography (15 g of RPC.sub.18, gradient 10% to 30% H.sub.2O/MeCN). The pure fractions were evaporated to yield the product as a bronze solid (40 mg, 77%). .sup.1H NMR (400 MHz, CD.sub.3OD): 9.14 (d, J=9.8 Hz, 2H), 7.43 (d, J=8.4 Hz, 1H), 7.32 (ddd, J=8.5, 6.4, 2.8 Hz, 1H), 7.14-7.04 (m, 4H), 6.72-6.68 (m, 2H), 3.28 (s, 12H), 2.75 (s, 3H), 2.64 (s, 3H) ppm. .sup.13C NMR (126 MHz, CD.sub.3OD): 170.3, 158.6 (d, J=11.7 Hz), 158.1, 153.8 (d, J=155.5 Hz), 149.2 (d, J=7.4 Hz), 135.3 (d, J=2.9 Hz), 131.3, 129.6 (d, J=6.2 Hz), 128.8, 124.8, 122.3 (d, J=3.0 Hz), 116.8, 116.7 (d, J=9.4 Hz), 97.2 (d, J=1.4 Hz), 40.9, 39.0, 34.9. .sup.31P NMR (162 MHz, CD.sub.3OD): 1.56 ppm. MS (ESI): m/z (positive mode, rel. int., %)=494.2 (100) [M+H].sup.+, 516.2 (91) [M+Na].sup.+. HRMS (C.sub.26H.sub.28N.sub.3O.sub.5P): m/z (positive mode)=494.1841 (found [M+H].sup.+), 494.1839 (calc.). UV/Vis (MeOH): .sub.max ()=589 nm (49000 M.sup.1cm.sup.1); fluorescence (MeOH): .sub.excit=540 nm, .sub.em=622 nm, .sub.fl=0.36. Standard: Atto 594, .sub.fl=0.85 (H.sub.2O). UV/Vis (PBS 7.4): .sub.max ()=603 nm (69000 M.sup.1cm.sup.1); fluorescence (PBS 7.4): .sub.excit=540 nm, .sub.em=638 nm, .sub.fl=0.09. Standard: Atto 594, .sub.fl=0.85 (H.sub.2O).
tert-Butyl 3-[(diethoxyphosphino)(methyl)amino]propanoate (10a)
(29) ##STR00070##
(30) Compound 10a. To a solution of tert-butyl 3-(methylamino)propionate [prepared according to WO2009/083614 A1] (500 mg; 3.14 mmol) and NEt.sub.3 (380 mg; 3.77 mmol) in a mixture of benzene and CHCl.sub.3 (20 mL; 3:1), a solution of diethyl chlorophosphite (490 mg; 3.14 mmol) in benzene (2 mL) was added dropwise at 0 C. The reaction mixture was refluxed for 2 h. After cooling down to r.t., the reaction mixture was diluted with n-hexane (.sup.20 mL) and filtered through a glass filter. The filtrate was evaporated and subjected to column chromatography (30 g of SiO.sub.2, Hex/EtOAc 1:3+0.1 v/v % of NEt.sub.3) to afford 392 mg (50%) of a colorless oil. .sup.1H NMR (400 MHz, CD.sub.3CN): =1.18 (t, J.sub.HH=7.0 Hz, 6H, 2OEt), 1.43 (s, 9H, tBu), 2.37 (t, J.sub.HH=7.0 Hz, 2H, CH.sub.2), 2.52 (d, J.sub.HP=6.6 Hz, 3H, NMe), 3.21 (dt, J.sub.HP=9.7 Hz, J.sub.HH=7.0 Hz, 2H, NCH.sub.2), 3.57-3.72 (m, 4H, 2OEt) ppm. .sup.31P NMR (162 MHz, CD.sub.3CN): =144.9 ppm.
tert-Butyl 3-[[[3,6-bis(dimethylamino)-9H-xanthen-9-yl](ethoxy)phosphoryl](methyl)-amino]propanoate (10b)
(31) ##STR00071##
(32) Compound 10b. In a screw-cap test tube to a suspension of Pyronin Y (50 mg; 0.165 mmol) in MeCN (1 mL) compound 10a (62 mg; 0.247 mmol) was added at r.t. under argon. The resulted mixture was stirred for 2 h at 60 C. After cooling down to r.t., the reaction mixture was diluted with CH.sub.2Cl.sub.2 (.sup.3 mL) and subjected to column chromatography (30 g of SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 30:1) to yield 37 mg (43%) of a brown oil. .sup.1H NMR (400 MHz, CD.sub.3CN): (=1.21 (t, J.sub.HH=7.0 Hz, 3H, OEt), 1.38 (s, 9H, tBu), 2.05-2.25 (m, 2H, CH.sub.2), 2.39 (d, J.sub.HP=8.2 Hz, 3H, NMe), 2.78-2.94 (m, 2H, NCH.sub.2), 2.93 (s, 12H, 2NMe.sub.2), 3.74-3.98 (m, 2H, OEt), 4.25 (d, J.sub.HP=20.9 Hz, 1H), 6.37 (m, J.sub.HH=2.2 Hz, 2H.sub.ar), 6.50 (dd, J.sub.HH=8.7 Hz and 2.6 Hz, 2H.sub.ar), 7.05 (dd, J.sub.HH=8.6 Hz and 2.4 Hz, 1H.sub.ar), 7.15 (dd, J.sub.HH=8.5 Hz and 2.5 Hz, 1H.sub.ar) ppm. .sup.31P NMR (162 MHz, CD.sub.3CN): =26.9 ppm. HPLC: t.sub.R=7.8 min (84%), B/A=30/70-100/0 in 25 min, column 4250 mm, 1.2 mL/min, detection at 254 nm.
3-[[[3,6-Bis(dimethylamino)-9H-xanthen-9-yl](ethoxy)phosphoryl]methyl)amino]-propanoic acid (10c)
(33) ##STR00072##
(34) Compound 10c. To a solution of compound 10b (33 mg; 0.064 mmol) in CH.sub.2Cl.sub.2 (1 mL) Et.sub.3SiH (37 mg; 0.319 mmol) and TFA (1 mL) were added dropwise. The resulting reaction mixture stirred for 1.5 h at r.t., and all volatiles were removed in vacuo. The residue was subjected to column chromatography (25 g of SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 10:1) to afford 27 mg (92%) of a bluish solid. .sup.1H NMR (400 MHz, CD.sub.3CN): =1.20 (t, J.sub.HH=7.0 Hz, 3H, OEt), 2.09-2.24 (m, 2H, CH.sub.2), 2.39 (d, J.sub.HP=8.1 Hz, 3H, NMe), 2.75-3.07 (m, 2H, NCH.sub.2), 2.93 (s, 12H, NMe.sub.2), 3.74-3.97 (m, 2H, OEt), 4.31 (d, J.sub.HP=21.1 Hz), 6.38-6.43 (m, 2H.sub.ar), 6.49-6.55 (m, 2H.sub.ar), 7.02-7.09 (m, 1H.sub.ar), 7.12-7.18 (m, 1H.sub.ar) ppm. .sup.31P NMR (162 MHz, CD.sub.3CN): =27.3 ppm. MS (ESI): m/z (negative mode, rel. int., %)=460.4 (100) [MH].sup..
9-[[(2-Carboxyethyl)(methyl)amino]ethoxy)phosphoryl]-3,6-bis(dimethylamino)-9H-xanthenylium trifluoroacetate (10)
(35) ##STR00073##
(36) Compound 10. To a solution of compound 10c (25 mg; 0.054 mmol) in MeCN (1 mL) DDQ (12 mg; 0.054 mmol) was added at 0 C. The resulted mixture was stirred for 30 min at r.t. and was then directly subjected to column chromatography (15 g of SiO.sub.2, MeCN.fwdarw.MeCN/H.sub.2O 1:1+0.1 v/v % of TFA to yield 22 mg (80%) of a dark violet solid. .sup.1H NMR (400 MHz, CDCl.sub.3): =1.41 (t, J.sub.HH=7.3 Hz, 3H, OEt), 2.54-2.73 (m, 2H, CH.sub.2), 2.80 (d, J.sub.HP=10.8 Hz, 3H, NMe), 3.27-3.50 (m, 2H, NCH.sub.2), 3.37 (s, 12H, 2NMe.sub.2), 4.18-4.37 (m, 2H, OEt), 6.74 (s, broad, 2H.sub.ar), 7.18 (dd, J.sub.HH=9.9 Hz, J.sub.HP=2.3 Hz, 2H.sub.ar), 8.86 (d, J.sub.HH=9.9 Hz, 2H.sub.ar) ppm. .sup.31P NMR (162 MHz, CDCl.sub.3): =17.7 ppm. MS (ESI): m/z (positive mode, rel. int., %)=460.2 (100) [MCl].sup.+. HPLC: t.sub.R=16.0 min (95%), B/A=20/80-100/0 in 25 min, column 4250 mm, 1.2 mL/min, detection at 636 nm. UV/Vis (MeCN): .sub.max ()=615 nm (22224 M.sup.1cm.sup.1); fluorescence (MeCN): .sub.excit=600 nm, .sub.em=652 nm, .sub.fl=0.46. Standard: Nile Blue, .sub.fl=0.27 (MeOH). UV/Vis (PBS 7.4): .sub.max ()=625 nm (13816 M.sup.1cm.sup.1); fluorescence (PBS 7.4): .sub.excit=610 nm, .sub.em=670 nm, .sub.fl=0.17. Standard: Nile Blue, .sub.fl=0.27 (MeOH).
9-[[[(2-Carboxyethyl)(methyl)amino]ethoxy)phosphoryl]-3,6-bis(dimethylamino)-9H-xanthenylium trifluoroacetate NHS ester (10-NHS)
(37) ##STR00074##
(38) Compound 10-NHS. To a solution of compound 10 (10 mg; 0.020 mmol) in MeCN (1 mL), N-hydroxysuccinimide (35 mg; 0.30 mmol), HATU (30 mg; 0.08 mmol) and Et.sub.3N (36 mg; 0.36 mmol) were added at r.t. under Ar. After stirring for 30 min, the reaction mixture was quenched with AcOH (21 L), diluted with CH.sub.2Cl.sub.2 and washed with water (2). The organic layer was dried with Na.sub.2SO.sub.4 and evaporated to give 10 mg (85%) of crude blue material. HPLC analysis (B/A=30/70-100/0 in 25 min, column 4250 mm, 1.2 mL/min, detection at 636 nm) showed the presence of two substances with t.sub.R=6.6 min (10%; the starting material) and t.sub.R=8.6 min (90%; the title compound). After purification by preparative HPLC, 3 mg (25%) of a violet solid were isolated. MS (ESI): m/z (positive mode, rel. int., %)=557.2 (100) [MCl].sup.+. HPLC: t.sub.R=8.6 min (94%), B/A=30/70-100/0 in 25 min, column 4250 mm, 1.2 mL/min, detection at 254 nm.
Ethyl [3,6-bis(dimethylamino)-9H-xanthenylium-9-yl]phosphonate (11)
(39) ##STR00075##
(40) Compound 11. Solutions of compound 10 in protic solvents (MeOH, H.sub.2O) spontaneously decompose (particularly in the presence of acids) with formation of compound 11. .sup.1H NMR (400 MHz, CD.sub.3CN): =1.04 (t, J.sub.HH=7.0 Hz, 3H, OEt), 3.27 (s, 12H, 2NMe.sub.2), 3.79 (dt, J.sub.HP=14.2 Hz, J.sub.HH=7.2 Hz, 2H, OEt), 6.77-6.80 (m, 2H.sub.ar), 7.18 (dd, J.sub.HH=9.8 Hz, J.sub.HH=2.6 Hz, 2H.sub.ar), 9.04 (d, J.sub.HH=9.8 Hz, 2H.sub.ar) ppm. .sup.31P NMR (162 MHz, CD.sub.3CN): =3.7 ppm. MS (ESI): m/z (positive mode, rel. int., %)=375.1 (100) [M+H].sup.+, 397.2 (47) [M+Na].sup.+. UV/Vis (MeCN): .sub.max ()=565 nm (63519 M.sup.1cm.sup.1); fluorescence (MeCN): .sub.excit=550 nm, .sub.em=595 nm, .sub.fl=0.44. Standard: Atto Rho11, .sub.fl=0.80 (H.sub.2O). UV/Vis (PBS 7.4): .sub.max ()=597 nm (66564 M.sup.1cm.sup.1); fluorescence (PBS 7.4): .sub.excit=580 nm, .sub.em=633 nm, .sub.fl=0.22. Standard: Atto 594, .sub.fl=0.85 (H.sub.2O).
Allyl 4-[(dimethoxyphosphino)oxy]butanoate (12a)
(41) ##STR00076##
(42) Compound 12a. To a solution of allyl 4-hydroxybutyrate (100 mg; 0.69 mmol) in CH.sub.2Cl.sub.2 (7 mL) a solution of tetrazole (0.45 M; 6.4 mL; 2.89 mmol) in MeCN and dimethyl N,N-diisopropylphosphorimidate (306 mg; 1.59 mmol) were added under Ar. The reaction mixture was stirred for 1 h at r.t., and then sat. aq. NaHCO.sub.3 was added (.sup.10 mL). The organic layer was separated, and the aq. layer was extracted with CH.sub.2Cl.sub.2 (2). Combined organic solutions were dried with Na.sub.2SO.sub.4 and evaporated. The residue was subjected to column chromatography (10 g of SiO.sub.2, hexane/EtOAc 6:1) to yield 103 mg (63%) of colorless oil. .sup.1H NMR (400 MHz, CD.sub.3CN): =1.84-1.92 (m, 2H, CH.sub.2), 2.42 (t, J.sub.HH=7.3 Hz, 2H, CH.sub.2), 3.47 (d, J.sub.HP=10.6 Hz, 6H, 2OMe), 3.81 (dt, J.sub.HP=7.5 Hz, J.sub.HH=6.3 Hz, 2H, OCH.sub.2), 4.56 (dt, J.sub.HH=5.5 and 1.5 Hz, 2H, OAll), 5.22 (m, J.sub.HH=10.5 and 1.4 Hz, 1H, OAll), 5.31 (m, J.sub.HH=17.3 and 1.6 Hz, 1H, OAll), 5.95 (m, J.sub.HH=17.3, 10.5 and 5.5 Hz, 1H, OAll) ppm. .sup.31P NMR (162 MHz, CD.sub.3CN): =140.1 ppm.
9-[[4-(Allyloxy)-4-oxobutoxy](methoxy)phosphoryl]-3,6-bis(dimethylamino)-9H-xanthen-9-ylium chloride (12)
(43) ##STR00077##
(44) Compound 12. In a screw-cap test tube to a suspension of Pyronin Y (50 mg; 0.165 mmol) in MeCN (1 mL) the compound 12a (58 mg; 0.247 mmol) was added at r.t. under argon. The resulting mixture was stirred overnight at 50 C. After cooling down to 0 C., DDQ (56 mg; 0.165 mmol) was added, and the reaction mixture was stirred for additional 10 min at 0 C. After warming up to r.t., the reaction mixture was directly subjected to column chromatography on SiO.sub.2 (30 g; MeCN.fwdarw.MeCN/H.sub.2O 20:1) to afford 20 mg (23%) of a violet solid. .sup.1H NMR (400 MHz, CD.sub.3CN): =1.92-1.97 (m, 2H, CH.sub.2, overlapped with solvent signal), 2.35-2.41 (m, J.sub.HH=7.3, 7.1 and 3.8 Hz, 2H, CH.sub.2), 3.31 (s, 12H, 2NMe.sub.2), 3.84 (d, J.sub.HP=11.6 Hz, 3H, OMe), 4.18 (ddt, J.sub.HP=10.3 Hz, J.sub.HH=7.8 and 6.3 Hz, 1H, OCH.sub.2), 4.28 (ddt, J.sub.HP=10.3 Hz, J.sub.HH=7.5 and 6.2 Hz, 1H, OCH.sub.2), 4.47-4.50 (m, J.sub.HH=5.4 and 1.5 Hz, 2H, OAll), 5.16-5.21 (m, J.sub.HH=10.5 and 1.4 Hz, 1H, OAll), 5.22-5.28 (m, J.sub.HH=17.3 and 1.6 Hz, 1H, OAll), 5.83-5.93 (m, J.sub.HH=17.2, 10.8 and 5.5 Hz, 1H, OAll), 6.77 (m, J.sub.HH=2.4 Hz, 2H.sub.ar), 7.15 (dd, J.sub.HH=9.9 and 2.6 Hz, 2H.sub.ar), 8.72 (d, J.sub.HH=9.9 Hz, 2H.sub.ar) ppm. .sup.31P NMR (162 MHz, CD.sub.3CN): =12.5 ppm. MS (ESI): m/z (positive mode, rel. int., %)=487.2 (100) [MCl].sup.+. HPLC: t.sub.R=12.2 min (95%), B/A=30/70-100/0 in 25 min, column 4250 mm, 1.2 mL/min, detection at 600 nm. UV/Vis (MeCN): .sub.max ()=621 nm (42690 M.sup.1cm.sup.1); fluorescence (MeCN): .sub.excit=610 nm, .sub.em=653 nm, .sub.fl=0.32. Standard: Atto 633, .sub.fl=0.64 (H.sub.2O). UV/Vis (PBS 7.4): .sub.max ()=632 nm (29540 M.sup.1cm.sup.1); fluorescence (PBS 7.4): .sub.excit=620 nm, .sub.em=669 nm, .sub.fl=0.17. Standard: Atto 633, CD.sub.fl=0.64 (H.sub.2O).
[3,6-Bis(dimethylamino)-9H-xanthen-9-yl]phosphinic acid (13a)
(45) ##STR00078##
(46) Compound 13a. A solution of Pyronin Y (1 g; 3.3 mmol) in aq. H.sub.3PO.sub.2 (50%, 4.4 g; 33.3 mmol) was stirred overnight at 100 C. After cooling down to r.t., the reaction mixture was diluted with H.sub.2O (.sup.25 mL) and subjected to reverse-phase column chromatography (100 g of RPSiO.sub.2, H.sub.2O.fwdarw.H.sub.2O/MeCN 5:1+0.1 v/v % of TFA to yield 1031 mg (94%) of the title product as a red solid. .sup.1H NMR (400 MHz, CD.sub.3OD): =3.06 (s, 12H, 2NMe.sub.2), 4.30 (d, J.sub.HP=15.5 Hz, 1H), 7.03-7.11 (m, 4H.sub.ar), 7.37-7.43 (m, 2H.sub.ar) ppm. With addition of NEt.sub.3 (1 eq.): .sup.1H NMR (400 MHz, CD.sub.3OD): =1.15 (t, J.sub.HH=7.2 Hz, 9H, NEt.sub.3), 2.88 (s, 12H, 2NMe.sub.2), 2.90-2.99 (m, 6H, NEt.sub.3), 3.82 (d, J.sub.HP=15.5 Hz, 1H), 6.39 (d, J.sub.HH=2.5 Hz, 2H.sub.ar), 6.49 (dd, J.sub.HH=8.5 and 2.6 Hz, 2H.sub.ar), 6.76 (d, J.sub.HP=504 Hz, 1H, PH) 7.11 (dd, J.sub.HH=8.5 Hz, J.sub.HP=2.0 Hz, 2H.sub.ar) ppm. .sup.31P NMR (162 MHz, CD.sub.3OD): =26.4 ppm. MS (ESI): m/z (positive mode, rel. int., %)=333.1 (100) [M+H].sup.+. MS (ESI): m/z (negative mode, rel. int., %)=331.2 (100) [MH].sup..
[3,6-Bis(dimethylamino)-9H-xanthenylium-9-yl]phosphinate (13)
(47) ##STR00079##
(48) Compound 13. To a solution of compound 13a (50 mg; 0.15 mmol) in MeOH (2 mL) DDQ (36 mg; 0.16 mmol) was added at 0 C. After stirring for 15 min, all volatiles were removed in vacuo, and the residue was dissolved in aq. NaHCO.sub.3 (.sup.50 mL). This solution was extracted with CH.sub.2Cl.sub.2 (450 mL), the aq. layer was acidified with conc. HCl to pH 0, saturated with NaCl and again extracted with CH.sub.2Cl.sub.2 (450 mL). The combined organic extracts from the second extraction were evaporated. The residue was subjected to reverse-phase column chromatography (25 g of RPSiO.sub.2, MeCN/H.sub.2O 1:1) to give 30 mg (61%) of a violet powder. Et.sub.3N (1 eq.) was added to the NMR samples. .sup.1H NMR (400 MHz, CD.sub.3OD): =1.28 (t, J.sub.HH=7.3 Hz, 9H, NEt.sub.3), 3.17 (q, J.sub.HH=7.3 Hz, 6H, NEt.sub.3), 3.29 (s, 12H, 2NMe.sub.2), 6.79-6.83 (m, 2H.sub.ar), 7.13 (dd, J.sub.HH=9.7 Hz and 2.6 Hz, 2H.sub.ar), 8.32 (d, J.sub.HP=547 Hz, 1H, PH) 8.71 (d, J.sub.HH=9.7 Hz, 2H.sub.ar) ppm. .sup.31P NMR (162 MHz, CD.sub.3OD): =2.1 ppm. MS (ESI): m/z (positive mode, rel. int., %)=331.1 (85) [M+H].sup.+, 353.1 (100) [M+Na].sup.+. UV/Vis (PBS 7.4): .sub.max ()=590 nm (59543 M.sup.1cm.sup.1); fluorescence (PBS 7.4): .sub.excit=560 nm, .sub.em=627 nm, .sub.fl=0.33. Standard: Oxazine 4, .sub.fl=0.63 (MeOH).
tert-Butyl 2-[[3,6-bis(dimethylamino)-9H-xanthen-9yl](ethoxy)phosphoryl]acetate (14a)
(49) ##STR00080##
(50) Compound 14a. In a screw-cap test tube to a suspension of Pyronin Y (50 mg; 0.165 mmol) in DMF (1 mL) tert-butyl 2-diethoxyphosphinoacetate (117 mg; 0.49 mmol) was added at r.t. under argon. The resulted mixture stirred overnight at 60 C. After cooling down to r.t., the reaction mixture was diluted with water (.sup.50 mL) and extracted with Et.sub.2O (350 mL). The combined ethereal solutions were dried with Na.sub.2SO.sub.4 and evaporated. The residue was subjected to column chromatography (20 g of SiO.sub.2, n-hexane/EtOAc 1:3) to give brown oil which was dried in vacuo overnight. Yield 35 mg (45%). .sup.1H NMR (400 MHz, CD.sub.3CN): =1.15 (t, =7.0 Hz, 3H, OEt), 1.46 (s, 9H, tBu), 2.65 (dd, J.sub.HP=14.7 Hz, J.sub.HH=13.9 Hz, 1H), 2.69 (dd, J.sub.HH=14.9 Hz, J.sub.HH=13.9 Hz, 1H), 2.94 (s, 12H, 2NMe.sub.2), 3.78-3.95 (m, 2H, OEt), 4.39 (d, J.sub.HP=15.8 Hz, 1H), 6.40 (s, broad, 1H.sub.ar), 6.41 (s, broad, 1H.sub.ar), 6.51-6.56 (m, 2H.sub.ar), 7.15 (dd, J.sub.HH=8.6 Hz, J.sub.HP=2.3 Hz, 1H.sub.ar), 7.22 (dd, J.sub.HH=8.6 Hz, J.sub.HP=2.4 Hz, 1H.sub.ar) ppm. .sup.31P NMR (162 MHz, CD.sub.3CN): S=41.5 ppm. MS (ESI): m/z (positive mode, rel. int., %)=497.2 (100) [M+Na].sup.+.
2-[[3,6-Bis(dimethylamino)-9H-xanthenylium-9-yl](ethoxy)phosphoryl]acetate (14)
(51) ##STR00081##
(52) Compound 14. To a solition of compound 14a (18 mg; 0.038 mmol) in CH.sub.2Cl.sub.2 (1 mL) Et.sub.3SiH (26 mg; 0.22 mmol) and TFA (1 mL) were added dropwise. The resulting reaction mixture was stirred for 1.5 h at r.t., and all volatiles were removed in vacuo. The residue was dissolved in MeCN (.sup.3 mL), the resulting solution was cooled down in an ice bath, and DDQ (10 mg; 0.046 mmol) was added. After 30 min stirring at 0 C., the reaction mixture was directly subjected to column chromatography (20 g of SiO.sub.2, MeCN.fwdarw.MeCN/H.sub.2O+0.1 v/v % of TFA). Fractions containing the blue substance were combined and freeze-dried. In order to get rid of residual SiO.sub.2, the product was dissolved in water, the resulted solution was saturated with NH.sub.4Cl and extracted with CH.sub.2Cl.sub.2 (5). Combined organic solutions were dried with Na.sub.2SO.sub.4 and evaporated to afford 10 mg (63%) of a blue solid. .sup.1H NMR (400 MHz, CD.sub.3CN): =1.33 (t, J.sub.HH=7.0 Hz, 3H, OEt), 3.29 (s, 12H, 2NMe.sub.2), 3.43-3.57 (m, 2H, CH.sub.2), 4.09 (ddt, J.sub.HP=15.1 Hz, J.sub.HH=10.2 and 7.0 Hz, 1H, OEt), 4.30 (ddt, J.sub.HP=15.2 Hz, J.sub.HH=10.3 and 7.0 Hz, 1H, OEt), 6.74-6.77 (m, 2H.sub.ar), 7.15 (dd, J.sub.HH=9.9 and 2.7 Hz, 2H.sub.ar), 8.85 (d, J.sub.HH=9.9 Hz, 2H.sub.ar) ppm. .sup.31P NMR (162 MHz, CD.sub.3CN): =31.4 ppm. MS (ESI): m/z (positive mode, rel. int., %)=417.1 (100) [M+H].sup.+. UV/Vis (MeCN): .sub.max ()=625 nm (22599 M.sup.1cm.sup.1); fluorescence (MeCN): .sub.excit=620 nm, .sub.em=656 nm, .sub.fl=0.32. Standard: Atto 633, .sub.fl=0.64 (H.sub.2O). UV/Vis (PBS 7.4): .sub.max ()=631 nm (47622 M.sup.1cm.sup.1); fluorescence (PBS 7.4): .sub.excit=620 nm, .sub.em=667 nm, .sub.fl=0.22. Standard: Nile Blue, .sub.fl=0.27 (MeOH).
Bis(triethylammonium) 3-[[3,6-bis(dimethylamino)-9H-xanthen-9-yl]oxidophosphoryl]-propanoate (15a)
(53) ##STR00082##
(54) Compound 15a. To a suspension of compound 13a (73 mg; 0.22 mmol) in CH.sub.2Cl.sub.2 (1 mL) acrylic acid (60 mg; 0.82 mmol) and N,O-bis(trimethylsilyl)acetimidate (BSA; 380 mg, 1.87 mmol) were added dropwise at 0 C. under Ar. A clear red solution formed. After stirring at r.t. overnight, the reaction mixture was quenched with MeOH (.sup.1 mL) at 0 C. and evaporated. The oily residue was subjected to reverse-phase column chromatography (30 g of RPSiO.sub.2, MeCN/H.sub.2O 1:10+0.1 v/v % of NEt.sub.3). Fractions containing the title product were evaporated, and the residue was redissolved in MeOH. The methanolic solution was filtered through a fine sintered glass filter and evaporated to afford 67 mg (50%) of a blue amorphous solid. .sup.1H NMR (400 MHz, CD.sub.3OD): =1.13-1.19 (m, 18H, 2NEt.sub.3), 1.58-1.67 (m, 2H, CH.sub.2), 2.25-2.33 (m, 2H, CH.sub.2), 2.88 (s, 12H, 2NMe.sub.2), 2.93-3.01 (m, 12H, 2NEt.sub.3), 3.90 (d, J.sub.HP=19.5 Hz), 6.39 (m, 2H.sub.ar), 6.49 (d, J.sub.HH=8.5 Hz, 1H.sub.ar), 6.50 (d, J.sub.HH=8.5 Hz, 1H.sub.ar), 7.16-7.23 (m, 2H.sub.ar) ppm. .sup.31P NMR (162 MHz, CD.sub.3OD): =37.3 ppm. MS (ESI): m/z (negative mode, rel. int., %)=403.4 (100) [M2NEt.sub.3H].sup..
[3,6-Bis(dimethylamino)-9H-xanthenyhum-9-yl](2-carboxyethyl)phosphinate (15)
(55) ##STR00083##
(56) Compound 15. To a solution of compound 15a (67 mg; 0.11 mmol) in MeCN (3 mL) DDQ (25 mg; 0.11 mmol) was added at 0 C. The resulting mixture was stirred at r.t. for 15 min and was then directly subjected to column chromatography (30 g of SiO.sub.2, MeCN.fwdarw.MeCN/H.sub.2O 10:1+0.1 v/v % of TFA.fwdarw.2:1+0.1 v/v % of TFA). Fractions containing the title compound were evaporated, and the residue was dissolved in H.sub.2O (.sup.20 mL). The solution was saturated with NaCl, acidified with TFA (.sup.1 mL) and extracted with CH.sub.2Cl.sub.2 (350 mL). The combined organic extracts were evaporated to give 34 mg (77%) of a violet solid. .sup.1H NMR (400 MHz, CD.sub.3OD): =2.09-2.22 (m, 2H, CH.sub.2), 2.45-2.58 (m, 2H, CH.sub.2), 3.28 (s, 12H, 2NMe.sub.2), 6.77 (s, 2H.sub.ar), 7.11 (d, J.sub.HH=9.0 Hz, 2H.sub.ar), 9.19 (d, J.sub.HH=9.0 Hz, 2H.sub.ar) ppm. MS (ESI): m/z (positive mode, rel. int., %)=403.1 (100) [M+H].sup.+. UV/Vis (PBS 7.4): .sub.max ()=598 nm (52094 M.sup.1cm.sup.1); fluorescence (PBS 7.4): .sub.excit=560 nm, .sub.em=636 nm, .sub.fl=0.29. Standard: Oxazine 4, .sub.fl=0.63 (MeOH).
tert-Butyl 6-(N-methylacrylamido)hexanoate (16a)
(57) ##STR00084##
(58) Compound 16a. To a solution of tert-butyl 6-(methylamino)hexanoate [prepared according to Epstein, M. G.; Reeves, B. D.; Maaty, W. S.; Fouchard, D.; Dratz, E. A.; Bothner, B.; Grieco, P. A. Bioconjugate Chem. 2013, 24, 1552-1561] (600 mg; 2.98 mmol) and Et.sub.3N (391 mg; 3.87 mmol) in CH.sub.2Cl.sub.2 (5 mL) a solution of acryloyl chloride (324 mg; 3.58 mmol) in CH.sub.2Cl.sub.2 (5 mL) was added dropwise at 0 C. under vigorous stirring. The resulting mixture was stirred overnight at r.t., diluted with Et.sub.2O, filtered through a glass filter and evaporated. The residue was subjected to column chromatography (30 g of SiO.sub.2, n-hexane/EtOAc 1:2) to afford 657 mg (86%) as colorless oil. .sup.1H NMR (400 MHz, CDCl.sub.3): =1.28-1.36 (m, 2H, CH.sub.2), 1.43 (s, 9H, tBu), 1.54-1.65 (m, 4H, 2CH.sub.2), 2.21 (t, J.sub.HH =7.4 Hz, 2H, CH.sub.2), 3.00 (br.s, 3H, NMe), 3.29-3.48 (m, 2H, NCH.sub.2), 5.66 (dd, J.sub.HH=10.4 and 2.0 Hz, 1H), 6.31 (dd, J.sub.HH=16.7 and 1.9 Hz, 1H), 6.55 (dd, J.sub.HH=16.7 and 10.4 Hz, 1H) ppm. MS (ESI): m/z (positive mode, rel. int., %)=278.2 (100) [M+Na].sup.+. HRMS (C.sub.14H.sub.25NO.sub.3P): m/z (positive mode)=278.1721 (found [M+Na].sup.+), 278.1727 (calc.).
Triethylammonium [3,6-bis(dimethylamino)-9H-xanthen-9-yl][3-[(6-tert-butoxy-6-oxohexyl)(methypamino]-3-oxopropyl]phosphinate (16b)
(59) ##STR00085##
(60) Compound 16b. To a suspension of compound 13a (100 mg; 0.30 mmol) and compound 16a (288 mg; 1.13 mmol) in CH.sub.2Cl.sub.2 (2 mL), N,O-bis(trimethylsilyl)acetimidate (BSA; 457 mg, 2.25 mmol) was added dropwise at 0 C. under argon. A clear red solution formed. After stirring at r.t. overnight and at 40 C. for 3 h, the reaction mixture was quenched with MeOH (.sup.5 mL) at 0 C. and evaporated. The oily residue was subjected to reverse-phase column chromatography (30 g of RPSiO.sub.2, MeCN/H.sub.2O 3:1+0.1 v/v % of NEt.sub.3). Fractions containing the title product were evaporated, and the residue was redissolved in MeOH. The methanolic solution was filtered through a fine sintered glass filter and evaporated to afford 30 mg (14%) of a red amorphous solid. .sup.1H NMR (400 MHz, CD.sub.3OD), mixture of 2 rotameric forms: =1.23 (t, J.sub.HH=7.3 Hz, 9H, NEt.sub.3), 1.25-1.34 (m, 2H, CH.sub.2), 1.42 (s, 9H, tBu), 1.49-1.64 (m, 6H, 3CH.sub.2), 2.15-2.27 (m, 4H, 2CH.sub.2), 2.79 (s, 3H, NMe), 2.89 (m, 12H, NMe.sub.2), 3.09 (q, J.sub.HH=7.3 Hz, 6H, NEt.sub.3), 3.31-3.44 (m, 2H, CH.sub.2), 3.93 (d, J.sub.HP=23.1 Hz, 1H), 6.37-6.40 (m, 2H.sub.ar), 6.48-6.52 (m, 2H.sub.ar), 7.23-7.28 (m, .sup.2H.sub.ar) ppm .sup.31P NMR (162 MHz, CD.sub.3OD), mixture of 2 rotameric forms: =39.0 (minor), 39.1 (major) ppm. MS (ESI): m/z (negative mode, rel. int., %)=586.3 (100) [MNEt.sub.3H].sup.. HRMS (C.sub.31H.sub.46N.sub.3O.sub.6P): m/z (negative mode)=586.3044 (found [MNEt.sub.3H].sup.+), 586.3051 (calc.).
Triethylammonium 6-[3-[[3,6-bis(dimethylamino)-9H-xanthenylium-9-yl]oxidophosphoryl]-N-methylpropanamido]hexanoate (16)
(61) ##STR00086##
(62) Compound 16. To a solution of compound 16b (30 mg; 0.051 mmol) in MeOH (2 mL) DDQ (12 mg; 0.051 mmol) was added at 0 C. The resulting mixture was stirred for 15 min at r.t. and was then directly subjected to reverse-phase column chromatography (25 g of RPSiO.sub.2, MeCN/H.sub.2O 1:2+0.1 v/v % of NEt.sub.3.fwdarw.10:1+0.1 v/v % of NEt.sub.3). Fractions containing the title product were evaporated, and the residue was redissolved in MeOH. The methanolic solution was filtered through a fine sintered glass filter and evaporated to afford 12 mg of a violet solid. This substance was dissolved in TFA (1 mL) and stirred for 30 min at r.t. The volatiles were evaporated and the residue was subjected to reverse-phase column chromatography (25 g of RPSiO.sub.2, MeCN/H.sub.2O 3:1+0.1 v/v % of HCOOH) to yield 8 mg (25%) of a violet solid. MS (ESI): m/z (negative mode, rel. int., %)=528.2 (100) [MNEt.sub.3H].sup.. HRMS (C.sub.27H.sub.36N.sub.3O.sub.6P): m/z (negative mode)=528.2256 (found [MNEt.sub.3H].sup.+), 528.2269 (calc.). HPLC: t.sub.R=16.1 min (100%), B/A=20/80-50/50 in 25 min, column 4.0250 mm, 1.2 mL/min, detection at 592 nm. UV/Vis (PBS 7.4): .sub.max ()=603 nm (10750 M.sup.1cm.sup.1); fluorescence (PBS 7.4): .sub.excit=565 nm, .sub.em=636 nm, .sub.fl=0.30. Standard: Oxazine 4, .sub.fl=0.63 (MeOH).
6-[3-[[3,6-Bis(dimethylamino)-9H-xanthenylium-9-yl]oxidophosphory]-N-methylpropanamido]hexanoic acid NHS ester (16-NHS)
(63) ##STR00087##
(64) Compound 16-NHS. To a solution of compound 16 (3 mg; 0.006 mmol) in MeCN (1 mL), N-hydroxysuccinimide (10 mg; 0.085 mmol), HATU (9 mg; 0.027 mmol) and NEt.sub.3 (10 mg; 0.10 mmol) were added at r.t. under Ar. After stirring for 1 h, the reaction mixture was evaporated to dryness, and the residue was subjected to column chromatography (20 g of SiO.sub.2, MeCN/H.sub.2O 4:1). Fractions containing the NHS-ester were combined and freeze-dried to give 1 mg (28%) of a dark blue solid. MS (ESI): m/z (positive mode, rel. int., %)=627.1 (45) [M+H].sup.+, 649.1 (100) [M+Na].sup.+. HPLC: t.sub.R=21.8 min (70%), B/A=20/80-50/50 in 25 min, column 4.0250 mm, 1.2 mL/min, detection at 254 nm.
3-[[2[2-(6-chlorohexyloxy)ethoxy]ethyl]amino]-3-oxopropyl[6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl]phosphinate (16-Halo)
(65) ##STR00088##
(66) HaloTag(O2)-acrylamide. To a solution of HaloTag Amine (O2) Ligand [Singh, V.; Wang, S.; Kool, E. T. J. Am. Chem. Soc. 2013, 135, 6184-6191] (300 mg, 1.34 mmol) and N-ethyldiisopropylamine (350 L, 2 mmol) in dry CH.sub.2Cl.sub.2 (5 mL), cooled in ice-water bath, acryloyl chloride (131 L, 1.61 mmol) dissolved in dry CH.sub.2Cl.sub.2 (1 mL) was added dropwise. The reaction mixture was stirred at 0 C. for 30 min and at rt for 2 h. The mixture was then diluted with CH.sub.2Cl.sub.2 (40 mL), washed with sat. aq. NaHCO.sub.3, brine and dried over Na.sub.2SO.sub.4. The product was isolated by column chromatography (20 g SiO.sub.2, gradient 0% to 5% methanol/EtOAc) and dried in vacuo to yield 325 mg (87%) of the product as colorless oil. The material contained .sup.30% of 3-hydroxypropionamide impurity and was used without further purification. .sup.1H NMR (400 MHz, DMSO-d.sub.6): 8.15 (t, J=5.8 Hz, 1H), 8.05 (t, J=5.2 Hz, impurity), 6.24 (dd, J=17.1, 10.1 Hz, 1H), 6.07 (dd, J=17.1, 2.3 Hz, 1H), 5.56 (dd, J=10.1, 2.3 Hz, 1H), 3.77 (t, J=6.4 Hz, impurity), 3.61 (t, J=6.6 Hz, 2H), 3.53-3.25 (m, 10H), 3.21 (q, J=5.8 Hz, impurity), 2.56 (t, J=6.4 Hz, impurity), 1.75-1.66 (m, 2H), 1.48 (tt, J=8.0, 6.4 Hz, 2H), 1.43-1.24 (m, 4H). .sup.13C NMR (101 MHz, DMSO-d.sub.6): 168.9 (impurity), 164.6, 131.7, 125.0, 70.2, 69.6, 69.41 (impurity), 69.40, 69.05 (impurity), 69.02, 45.3, 41.0 (impurity), 38.64 (impurity), 38.61, 38.2 (impurity), 32.0, 29.1, 26.1, 24.9. MS (ESI): m/z (positive mode, rel. int., %)=278.2 (29) [M+H].sup.+, 300.1 (100) [M+Na].sup.+, 316.1 (78) [M+K].sup.+. HRMS (C.sub.13H.sub.24NO.sub.3Cl): m/z (positive mode)=278.1518 (found [M+H].sup.+), 278.1517 (calc.).
(67) ##STR00089##
(68) Compound 16-Halo.
(69) To a suspension of 13a (60 mg, 0.18 mmol) in 1,2-dichloroethane (2 mL), cooled in ice-water bath, N,O-bis(trimethylsilyl)acetimidate (BSA; 350 L, 1.44 mmol) was added quickly dropwise. The resulting clear solution was stirred at 0 C. under N.sub.2 atmosphere for 10 min, followed by addition of HaloTag(O2)-acrylamide (278 mg, purity .sup.70%, .sup.0.7 mmol) in 1,2-dichloroethane (1.5 mL). The mixture was stirred at 70 C. under N.sub.2 atmosphere overnight, the solvent was evaporated, the residue was redissolved in CH.sub.2Cl.sub.2 (3 mL), cooled in dry ice-acetone bath followed by addition of DDQ (41 mg, 0.18 mmol) in CH.sub.2Cl.sub.2 (3 mL) quickly dropwise. The dark violet mixture was allowed to warm up to rt and stirred for 15 min. Trifluoroacetic acid (50 L) was added, the mixture was evaporated to dryness and the product was isolated by column chromatography (30 g SiO.sub.2, gradient 10% to 30% methanol/CH.sub.2Cl.sub.2) and lyophilized from aqueous 1,4-dioxane. Dark violet crystalline solid, yield 53 mg (48%). .sup.1H NMR (400 MHz, CD.sub.3OD): 9.43 (d, J=9.9 Hz, 2H), 7.99 (t, J=5.6 Hz, 1H), 7.13 (dd, J=9.9, 2.7 Hz, 2H), 6.81 (dd, J=2.7, 1.3 Hz, 2H), 3.56-3.51 (m, 6H), 3.43 (t, J=6.5 Hz, 2H), 3.42 (t, J=5.6 Hz, 2H), 3.31 (s, 12H), 3.20 (td, J=5.6, 4.0 Hz, 2H), 2.43-2.34 (m, 2H), 2.16-2.06 (m, 2H), 1.78-1.68 (m, 2H), 1.54 (dq, J=7.6, 6.6 Hz, 2H), 1.47-1.28 (m, 4H). MS (ESI): m/z (positive mode, rel. int., %)=608.3 (48) [M+H].sup.+, 630.3 (53) [M+Na].sup.+, 646.2 (100) [M+K].sup.+. HRMS (C.sub.13H.sub.24NO.sub.3Cl): m/z (positive mode)=608.2653 (found [M+H].sup.+), 608.2651 (calc.).
9-[(Diisopropylamino)(methoxy)phosphoryl]-1H,2H,3H5H,6H,7H,11H,12H,13H,15H,16H,17H-pyrido[3,2,1-ij]quinolizino[1,9:6,7,8]chromeno[2,3-f]quinolinium trifluoroacetate (17) and Methyl (1H,2H,3H,5H,6H,7H,11H,12H,13H,15H,16H,17H-pyrido[3,2,1-ij]quinolizino[1,9:6,7,8]chromeno[2,3-f]quinolinium-9-yl)phosphonate (18)
(70) ##STR00090##
(71) Compounds 17 and 18. In a screw-cap test tube to a suspension of the corresponding pyronine (50 mg; 0.13 mmol) in MeCN (1 mL) dimethyl N,N-diisopropylphosphoramidite (100 mg; 0.52 mmol) was added at r.t. under Ar. The resulting reaction mixture was warmed up to 60 C. and stirred for 2.5 h at this temperature. After cooling down to 0 C., DDQ (116 mg; 0.52 mmol) was added, and the reaction mixture was stirred for additional 10 min at 0 C. After warming up to r.t., the reaction mixture was diluted with MeCN (10 ml) and directly subjected to column chromatography on SiO.sub.2 (100 g; MeCN.fwdarw.MeCN/H.sub.2O 20:1+0.1 v/v % of TFA). Fractions containing the title compound were evaporated to dryness, dissolved in water and extracted with CH.sub.2Cl.sub.2 (3). Combined organic solutions were dried with Na.sub.2SO.sub.4 and evaporated to yield 40 mg of a dark blue solid. HPLC analysis (B/A=50/50-100/0 in 25 min, column 4250 mm, 1.2 mL/min, detection at 254 nm) showed the presence of two colored substances: 17 with t.sub.R=10.7 min (58%) and 18 with t.sub.R=18.3 min (42%). This mixture was subjected to reverse-phase column chromatography (30 g of RPSiO.sub.2, MeCN/H.sub.2O 2:1+0.1 v/v % of TFA.fwdarw.MeCN+0.1 v/v % of TFA.fwdarw.MeOH) to yield 8 mg (10%) of 17 and 7 mg (12%) of 18.
(72) Compound 17: MS (ESI): m/z (positive mode, rel. int., %)=548.3 (100) [MCl].sup.+. HRMS (C.sub.32H.sub.43N.sub.3O.sub.3P): m/z (positive mode)=548.3046 (found [MCl].sup.+), 548.3037 (calc.). UV/Vis (MeCN): .sub.max ()=649 nm (52455 M.sup.1cm.sup.1); fluorescence (MeCN): .sub.excit=610 nm, .sub.em=679 nm, .sub.fl=0.25. Standard: Oxazine 1, .sub.fl=0.11 (EtOH). UV/Vis (PBS 7.4): .sub.max ()=661 nm (46976 M.sup.1cm.sup.1); fluorescence (PBS 7.4): .sub.excit=650 nm, .sub.em=696 nm, .sub.fl=0.11. Standard: Atto 655, .sub.fl=0.30 (H.sub.2O).
(73) Compound 18: .sup.1H NMR (400 MHz, CDCl.sub.3): =1.94-2.02 (m, 4H, 2CH.sub.2), 2.03-2.11 (m, 4H, 2CH.sub.2), 2.84-2.90 (m, 4H, 2CH.sub.2), 2.91-2.98 (m, 4H, 2CH.sub.2), 3.43-3.52 (m, 8H, 4CH.sub.2), 3.59 (d, J.sub.HP=11.5 Hz, 3H, OMe), 8.95 (s, 2H.sub.ar) ppm. .sup.31P NMR (162 MHz, CD.sub.3CN): =5.0 ppm. UV/Vis (MeCN): .sub.max ()=596 nm (49565 M.sup.1cm.sup.1); fluorescence (MeCN): .sub.excit=580 nm, .sub.em=623 nm, .sub.fl=0.85. Standard: Atto 590, .sub.fl=0.80 (H.sub.2O). UV/Vis (PBS 7.4): .sub.max ()=630 nm (52286 M.sup.1cm.sup.1); fluorescence (PBS 7.4): .sub.excit=620 nm, .sub.em=666 nm, .sub.fl=0.17. Standard: Atto 637, CD.sub.fl=0.25 (H.sub.2O).
9-[[(2-Carboxyethyl)(methyl)amino](ethoxy)phosphoryl]-1H,2H,3H,5H,6H,7H,11H,12H,13H,15H,16H,17H-pyrido[3,2,1-ij]quinolizino[1,9:6,7,8]chromeno[2,3-f]quinolinium trifluoroacetate (19)
(74) ##STR00091##
(75) Compound 19. In a screw-cap test tube to a solution of the corresponding pyronine (100 mg; 0.26 mmol) in MeCN (2 mL) compound 10a (97 mg; 0.39 mmol) was added at r.t. under argon. The resulted mixture was stirred overnight at 60 C. After cooling down to r.t., the reaction mixture was diluted with CH.sub.2Cl.sub.2 (.sup.3 mL) and subjected to column chromatography (30 g of SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 30:1) to give 50 mg (31%) of a violet oil. The product from previous step was dissolved in a mixture of CH.sub.2Cl.sub.2 and TFA (1:1, 2 mL), and the resulted solution was stirred for 2 h at r.t. Afterwards, all volatiles were removed in vacuo, and the residue was subjected to column chromatography (25 g of SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 10:1). The crude product was dissolved in MeCN (3 mL), the resulted solution was cooled down with an ice bath, and DDQ (18 mg; 0.079 mmol) was added. After 10 min stirring at r.t, the reaction mixture was directly subjected to column chromatography (25 g of SiO.sub.2, MeCN.fwdarw.MeCN/H.sub.2O 10:1+0.1 v/v % of TFA to yield 27 mg of crude product which was further purified by additional column chromatography (20 g of SiO.sub.2, MeCN/H.sub.2O 10:1+0.1 v/v % of TFA) to afford 11 mg (7% based on the starting pyronine) of a dark blue solid. .sup.1H NMR (400 MHz, CDCl.sub.3): =1.35 (t, J.sub.HH=7.0 Hz, 3H, OEt), 1.99-2.14 (m, 8H, 4CH.sub.2), 2.60-2.67 (m, J.sub.HH=6.7 Hz, 2H, CH.sub.2), 2.77 (d, =10.5 Hz, 3H, NMe), 2.84-3.00 (m, 8H, 4CH.sub.2), 3.51-3.58 (m, 8H, 4NCH.sub.2), 3.58-3.66 (m, 2H, NCH.sub.2), 4.09-4.35 (m, 2H, OEt), 8.33 (s, 2H.sub.ar). .sup.31P NMR (162 MHz, CDCl.sub.3): =18.9 ppm. MS (ESI): m/z (positive mode, rel. int., %)=564.2 (100) [MCl].sup.+. HPLC: t.sub.R=12.0 min (96%), B/A=30/70-100/0 in 25 min, column 4250 mm, 1.2 mL/min, detection at 254 nm. UV/Vis (MeCN): .sub.max ()=648 nm (29373 M.sup.1cm.sup.1); fluorescence (MeCN): .sub.excit=630 nm, .sub.em=682 nm, .sub.fl=0.19. Standard: Atto 655, .sub.fl=0.30 (H.sub.2O). UV/Vis (PBS 7.4): .sub.max ()=659 nm (37214 M.sup.1cm.sup.1); fluorescence (PBS 7.4): .sub.excit=640 nm, .sub.em=705 nm, .sub.fl=0.11. Standard: Atto 655, .sub.fl=0.30 (H.sub.2O).
9-[[(2-Carboxyethyl)(methyl)amino](ethoxy)phosphoryl]-1H,2H,3H,5H,6H,7H,11H,12H,13H,15H,16H,17H-pyrido[3,2,1-ij]quinolizino[1,9:6,7,8]chromeno[2,3-f]quinolinium NHS ester trifluoroacetate (19-NHS)
(76) ##STR00092##
(77) Compound 19-NHS. To a solution of compound 19 (5 mg; 0.009 mmol) in MeCN (1 mL), N-hydroxysuccinimide (15 mg; 0.133 mmol), HATU (13 mg; 0.035 mmol) and NEt.sub.3 (16 mg; 0.16 mmol) were added at r.t. under Ar. After 1 h stirring, the reaction mixture was evaporated to dryness, the residue was dissolved in CH.sub.2Cl.sub.2 (.sup.20 mL) and washed with water (220 mL). The organic solution was dried with Na.sub.2SO.sub.4 and evaporated. The residue was subjected to column chromatography (20 g of SiO.sub.2, MeCN/H.sub.2O 15:1+0.1 v/v % of TFA). Fractions containing the NHS-ester were combined and freeze-dried to give 3 mg (50%) of a dark blue solid. MS (ESI): m/z (positive mode, rel. int., %)=661.3 (100) [MCl].sup.+. HPLC: t.sub.R=14.3 min (97%), B/A=30/70-100/0 in 25 min, column 4250 mm, 1.2 mL/min, detection at 254 nm.
10-(Diethylamino)-7-(dimethoxyphosphoryl)-3-(dimethylamino)-6,7-dihydro-5H-benzo[c]xanthenylium trifluoroacetate (20)
(78) ##STR00093##
(79) Compound 20. Trimethyl phosphite (88 L; 0.75 mmol, 3 eq) was added to a stirred solution of the dye H-hNR [Niu, G.; Liu, W.; Zhou, B.; Xiao, H.; Zhang, H.; Wu, J.; Ge, J.; Wang, P. J. Org. Chem. 2016, 81(17), 7393-7399.] (112 mg; 0.25 mmol) and tetrabutylammonium iodide (92 mg; 0.25 mmol) in dry CH.sub.2Cl.sub.2 (6 mL). The reaction mixture was stirred at rt for 3 h, eventually turning light blue. The mixture was evaporated to dryness on Celite, and the leuco dye was isolated by flash column chromatography (Biichi Sepacore Silica HP 12 g, gradient 50% to 100% EtOAc/hexane) and used directly in the next step.
(80) The material was dissolved in CH.sub.2Cl.sub.2 (5 mL), the solution was cooled in dry ice-acetone bath, and DDQ (57 mg; 0.25 mmol) in CH.sub.2Cl.sub.2 (5 mL) was added quickly dropwise. The resulting dark green solution was allowed to warm up to rt and stirred for 15 min. The mixture was evaporated to dryness Celite, and the product was isolated by flash column chromatography (Biichi Sepacore Silica HP 12 g, gradient 0% to 100% A:B, A5% H.sub.2O/MeCN+0.1% v/v TFA, BMeCN); the fractions containing the product were pooled and evaporated. The residue was dissolved in 1,4-dioxane, filtered through a 0.2 M PTFE membrane filter and lyophilized. Black solid, yield 130 mg (92%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): 8.57 (d, J=9.6 Hz, 1H), 8.14 (d, J=9.3 Hz, 1H), 7.22 (dd, J=9.7, 2.7 Hz, 1H), 7.16 (t, J=2.4 Hz, 1H), 6.91 (dd, J=9.3, 2.4 Hz, 1H), 6.75 (d, J=2.3 Hz, 1H), 3.82 (s, 2H), 3.79 (s, 2H), 3.61 (q, J=6.8 Hz, 4H), 3.30-3.24 (m, 2H), 3.22 (s, 7H), 2.95 (dd, J=8.5, 6.1 Hz, 2H), 1.21 (t, J=7.0 Hz, 7H). .sup.31P NMR (162 MHz, DMSO-d.sub.6): 14.64. .sup.19F NMR (376 MHz, DMSO-d.sub.6): 74.60. .sup.13C NMR (101 MHz, DMSO-d.sub.6): 164.1 (d, J=15.3 Hz), 158.3 (q, J=35.2 Hz), 155.7, 155.5 (d, J=12.9 Hz), 152.5, 146.7, 138.4 (d, J=170.3 Hz), 130.3, 129.6, 129.5, 125.6 (d, J=10.6 Hz), 115.0, 114.8 (d, J=12.1 Hz), 113.5 (d, J=1.5 Hz), 113.0, 110.5, 96.3, 53.31, 53.26, 44.8, 40.2, 27.0, 25.1, 12.5. MS (ESI): m/z (positive mode, rel. int., %)=455.2 (100) [M].sup.+. HRMS (C.sub.24H.sub.35H.sub.3O.sub.2PS): m/z (positive mode)=455.2099 (found [M].sup.+), 455.2094 (calc.). UV/Vis (MeCN): .sub.max () 681 nm (40000 M.sup.1cm.sup.1); fluorescence (MeCN): .sub.excit=630 nm, .sub.em=737 nm, .sub.fl=0.17 (abs.). UV/Vis (PBS 7.4): .sub.max ()=693 nm (29000 M.sup.1cm.sup.1); fluorescence (MeCN): .sub.excit=630 nm, .sub.em=754 nm, .sub.fl=0.02 (abs.).
EXAMPLE 3
Synthesis of Fluorescent Phosphorylated Thioxanthene Dyes and their Precursors
3,6-Bis(dimethylamino)-9H-thioxanthenylium perchlorate (21a)
(81) ##STR00094##
(82) Compound 21a. Powdered sulfur (10 g) was added in portions over 15 min to 30% SO.sub.3H.sub.2SO.sub.4 (25 mL), the resulting brown yellow suspension was cooled in ice-water bath and 4,4-bis(dimethylamino)diphenylmethane (9.5 g, 37.4 mmol) was added in portions at such a rate that the temperature of the reaction mixture remained below 20 C. (over .sup.10 min). The yellow suspension was stirred at rt for 1.5 h. The mixture was then poured on ice (.sup.250 mL), the dark purple mixture was allowed to warm up to rt, transferred into a 500 mL round-bottom flask and refluxed for 1 h. The resulting suspension was cooled down to rt, filtered through a layer of Celite, a solution of ZnCl.sub.2 (80 g in 150 mL water) was added and the mixture was left at 4 C. overnight. A dark red oil, containing the crystals of 3,6-bis(dimethylamino)thioxanthylium trichlorozincate [prepared according to WO 2010067078 A2 and Heterocyclic Chemistry, 1966, 3, p. 228], separated. The colorless supernatant was decanted off, the residue was dissolved in boiling water (150 mL) and NaClO.sub.4 solution (5 g in 10 mL water) was added. The resulting suspension was allowed to cool down to rt and then left in ice-water bath to complete crystallization. The crystals were filtered off, washed with water, Et.sub.2O/hexane (1:1) and Et.sub.2O, dried in vacuo. Small brown crystals, yield 606 mg (4%). MS (ESI): m/z (positive mode, rel. int., %)=283.1 (100) [M].sup.+.
9-[(Diisopropylamino)(methoxy)phosphoryl]-3,6-bis(dimethylamino)-9H-thioxanthen-9-ylium trifluoroacetate (21)
(83) ##STR00095##
(84) Compound 21. Dimethyl N,N-diisopropylphosphoramidite (181 L; 0.786 mmol) was added to a stirred suspension of 21a (100 mg; 0.262 mmol) and tetrabutylammonium iodide (97 mg; 0.262 mmol) in dry CH.sub.2Cl.sub.2 (4 mL). The reaction mixture, which quickly turned into a light-brown clear solution, was stirred at rt for 30 min. The mixture was evaporated to dryness, and the leuco dye was isolated by column chromatography (18 g SiO.sub.2, gradient 50% to 100% EtOAc/hexane) and used directly in the next step.
(85) The material was dissolved in CH.sub.2Cl.sub.2 (3 mL), the solution was cooled in dry ice-acetone bath, and DDQ (59 mg; 0.26 mmol) in CH.sub.2Cl.sub.2 (3 mL) was added quickly dropwise. The resulting turquoise-blue solution was allowed to warm up to rt, stirred for 15 min. The mixture was evaporated to dryness, and the residue was subjected to column chromatography (20 g of SiO.sub.2, gradient 0% to 5% H.sub.2O/MeCN, then 5% H.sub.2O/MeCN+0.5 v/v % TFA); the fractions containing the product were pooled and evaporated. The residue was dissolved in 1,4-dioxane (with addition of minimal amount of water to dissolve the solids), centrifuged, the supernatant was filtered through 0.2 M PTFE membrane filter and lyophilized. Blue solid, yield 145 mg (97%). MS (ESI): m/z (positive mode, rel. int., %)=460.2 (100) [M].sup.+. HRMS (C.sub.24H.sub.35N.sub.3O.sub.2PS): m/z (positive mode)=460.2184 (found [M].sup.+), 460.2182 (calc.). UV/Vis (MeCN): .sub.max ()=654 nm (64000 M.sup.1cm.sup.1); fluorescence (MeCN): .sub.excit=610 nm, .sub.em=728 nm, .sub.fl=0.10. Standard: Oxazine 1, .sub.fl=0.11 (EtOH).
EXAMPLE 4
Synthesis of Fluorescent Phosphorylated Acridine Dyes and their Precursors
9-(Dimethoxyphosphoryl)-3,6-bis(dimethylamino)-10-methylacridinium trifluoroacetate (22)
(86) ##STR00096##
(87) Compound 22. To a stirred suspension of NaH (33 mg of 60 wt. % in mineral oil, 0.825 mmol) in dry DMF (1 mL), cooled in ice-water bath, dimethyl phosphite (78 L, 0.825 mmol) was added in one portion. The resulting suspension was warmed up to rt and stirred for 30 min, turning into a clear solution. A suspension of 3,6-bis(dimethylamino)-10-methylacridinium iodide [prepared according to Rodriguez, M. E.; Azizuddin, K.; Zhang, P.; Chiu, S.; Lam, M.; Kenney, M. E.; Burda, C.; Oleinick, N. L. Mitochondrion 2008, 8, 237-246] (100 mg, 0.25 mmol) in DMF (1 mL) was added, and the resulting clear orange-brown solution was stirred at rt for 1 h and at 100 C. for 1 h. The reaction mixture was evaporated to dryness (bath temperature 60 C.) and re-evaporated with acetone. The intermediate leuco dye was isolated by column chromatography (15 g of SiO.sub.2, gradient 0% to 5% MeOH/EtOAc) and used directly in the next step.
(88) The material was dissolved in CH.sub.2Cl.sub.2 (3 mL), the solution was cooled in dry ice-acetone bath, and DDQ (30 mg; 0.13 mmol) in CH.sub.2Cl.sub.2 (2 mL) was added quickly dropwise. The resulting bright red-purple mixture was allowed to warm up to rt and stirred for 15 min. The mixture was evaporated to dryness, and the residue was subjected to column chromatography (15 g of SiO.sub.2, gradient 0% to 5% H.sub.2O/MeCN, then 5% to 10% H.sub.2O/MeCN+0.5 v/v % TFA); the fractions containing the product were pooled, evaporated and re-purified by column chromatography (18 g of SiO.sub.2, 5% H.sub.2O/MeCN+0.2 v/v % TFA). The residue after evaporation was dissolved in 1,4-dioxane (with addition of minimal amount of water to dissolve the solids), centrifuged, the supernatant was filtered through 0.2 M PTFE membrane filter and lyophilized. Purple solid, yield 65 mg (52%). .sup.13C NMR (126 MHz, CD.sub.3OD): 156.2 (d, J=1.8 Hz), 145.0 (dd, J=14.0, 2.9 Hz), 137.3 (d, J=172.6 Hz), 132.4 (d, J=4.1 Hz), 120.2 (dd, J=11.2, 2.2 Hz), 116.5, 94.3, 68.1, 54.1 (d, J=5.9 Hz), 40.7, 38.3. MS (ESI): m/z (positive mode, rel. int., %)=388.2 (100) [M].sup.+. HRMS (C.sub.20H.sub.22N.sub.3O.sub.3P): m/z (positive mode)=388.1785 (found [M].sup.+), 388.1785 (calc.). UV/Vis (MeOH): .sub.max ()=560 nm (43000 M.sup.1cm.sup.1); fluorescence (MeOH): .sub.excit=510 nm, .sub.em=620 nm, .sub.fl=0.95. Standard: Atto 495, .sub.fl=0.5 (EtOH).
10-(4-tert-Butoxy-4-oxobutyl)-3,6-bis(dimethylamino)acridinium iodide (23a)
(89) ##STR00097##
(90) Compound 23a (Atto 495 tert-butyl ester). A suspension of Acridine Orange base (265 mg, 1 mmol) and tert-butyl 4-iodobutyrate [prepared according to Hardouin, C.; Kelso, M. J.; Romero, F. A.; Rayl, T. J.; Leung, D.; Hwang, I.; Cravatt, B. F.; Boger, D. L. J. Med. Chem. 2007, 50(14), 3359-3368] in toluene (15 mL) was refluxed for 18 h. The reaction mixture was evaporated to dryness, and the residue was subjected to column chromatography (40 g of SiO.sub.2, gradient 5% to 10% EtOH/CH.sub.2Cl.sub.2), eluting the fluorescent band. The eluate was evaporated and the product was isolated by reversed-phase column chromatography (30 g RP-C.sub.18, gradient 50% to 20% H.sub.2O/MeCN+1 v/v % TFA), the fractions containing the product were pooled and the residue was lyophilized from H.sub.2O/MeCN (2:1). Orange solid, yield 165 mg (31%). .sup.1H NMR (400 MHz, CD.sub.3OD): 8.46 (d, J=3.3 Hz, 1H), 7.76 (dd, J=9.3, 2.3 Hz, 2H), 7.15 (dt, J=9.3, 2.2 Hz, 2H), 6.79 (br.s, 2H), 4.59-4.51 (m, 2H), 3.29 (s, 12H), 2.64 (dd, J=6.9, 4.9 Hz, 2H), 2.05 (dq, J=11.8, 6.4 Hz, 2H), 1.49 (s, 9H) ppm. .sup.13C NMR (101 MHz, CD.sub.3OD): 174.4, 157.5, 144.1, 144.0, 134.2, 118.3, 115.4, 93.7, 82.2, 48.1, 41.0, 31.9, 28.5, 21.4 ppm. MS (ESI): m/z (positive mode, rel. int., %)=408.3 (100) [M].sup.+. HRMS (C.sub.25H.sub.34N.sub.3O.sub.2): m/z (positive mode)=408.2643 (found [M].sup.+), 408.2646 (calc.).
10-(3-Carboxypropyl)-3,6-bis(dimethylamino)-9-(hydroxyhydrophosphoryl)acridinium trifluoroacetate (23)
(91) ##STR00098##
(92) Compound 23. A suspension of 23a (165 mg, 0.31 mmol) in 50% aq. H.sub.3PO.sub.2 (2 mL) was stirred at 100 C. for 4 days. The resulting solution was cooled down to rt and transferred directly on top of a reversed-phase column (15 g RP-C.sub.18), excess H.sub.3PO.sub.2 was washed off first with water, followed by the 50% to 30% H.sub.2O/MeCN gradient. Fractions containing the leuco dye were pooled, MeCN was evaporated and the residue was lyophilized, giving 100 mg of the leuco dye as a red solid. MS (ESI): m/z (negative mode, rel. int., %)=416.2 (100) [MH].sup..
(93) The leuco acid was dissolved in the mixture of CH.sub.2Cl.sub.2 (3 mL) and MeOH (3 mL), the solution was cooled in dry ice-acetone bath, and DDQ (54 mg; 0.24 mmol) in CH.sub.2Cl.sub.2 (4 mL) was added quickly dropwise. The resulting bright-pink suspension was allowed to warm up to rt and stirred for 15 min. The mixture was evaporated to dryness, and the residue was subjected to column chromatography (30 g of SiO.sub.2, gradient 20% to 50% H.sub.2O/MeCN, then 50% H.sub.2O/MeCN+1 v/v % TFA); the fractions containing the product were pooled and evaporated. Further purification was done by reversed-phase column chromatography (15 g RP-C.sub.18, gradient 10% to 40% H.sub.2O/MeCN+5 v/v % 0.1 M Et.sub.3NH.sup.+ HCO.sub.3.sup. in H.sub.2O). The pure fractions containing the product were evaporated to dryness, the residue was dissolved in acetic acid (.sup.50 mL), centrifuged, the supernatant was filtered through 0.2 M PTFE membrane filter and lyophilized; the impure fractions were re-chromatographed and treated again as described, giving the combined yield of 100 mg (61% over 2 steps) as a red solid. .sup.1H NMR (400 MHz, acetic acid-d.sub.4): 9.02 (d, J=9.8 Hz, 2H), 8.57 (d, J=567.5 Hz, 1H), 7.23 (dd, J=9.5, 1.9 Hz, 2H), 6.73 (s, 2H), 4.72-4.54 (m, 2H), 3.30 (s, 12H), 2.80 (t, J=6.1 Hz, 2H), 2.27 (td, J=11.9, 5.6 Hz, 1H). .sup.31P NMR (162 MHz, acetic acid-d.sub.4): 5.81. .sup.19F NMR (376 MHz, acetic acid-d.sub.4): 76.67. MS (ESI): m/z (positive mode, rel. int., %)=416.2 (100) [M].sup.+, 438.2 (48) [MH+Na].sup.+, 454.1 (34) [MH+K].sup.+. HRMS (C.sub.21H.sub.27N.sub.3O.sub.4P): m/z (positive mode)=416.1732 (found [M].sup.+), 416.1734 (calc.). UV/Vis (MeOH): .sub.max ()=527 nm (67000 M.sup.1cm.sup.1); fluorescence (MeOH): .sub.excit=490 nm, .sub.em=579 nm, .sub.fl=0.75. Standard: Atto 495, .sub.fl=0.5 (EtOH). UV/Vis (PBS 7.4): .sub.max ()=535 nm (38000 M.sup.1cm.sup.1); fluorescence (PBS 7.4): .sub.excit=490 nm, .sub.em=595 nm, .sub.fl=0.14. Standard: Rhodamine 6G, .sub.fl=0.95 (EtOH).
[10-(3-Carboxypropyl)-3,6-bis(dimethylamino)acridinium-9-yl]phosphinate NHS ester (23-NHS)
(94) ##STR00099##
(95) Compound 23-NHS. Triethylamine (20 L, .sup.140 mol) N-hydroxysuccinimide (50 L of a 1.13 M solution in DMF, 56.5 mol) and HATU (50 L of a 0.76 M solution in DMF, 38 mol) were added to a suspension of 23 (2 mg, 3.8 mol) in DMF (0.1 mL). The resulting clear bright-pink solution was stirred at rt for 1 h. The solvent was evaporated to dryness at rt, and the product was isolated from the residue by column chromatography (15 g of SiO.sub.2, gradient 10% to 25% H.sub.2O/MeCN in 5% increments). The fractions containing the product were pooled, evaporated at rt, the residue was dissolved in dioxane (with minimal amount of water added to dissolve the solids), centrifuged off the silica dust, filtered the supernatant through 0.2 M PTFE membrane filter and lyophilized the filtrate. Yield 1.2 mg (62%), purity 87% (HPLC), red solid. MS (ESI): m/z (positive mode, rel. int., %)=513.2 (100) [M+H].sup.+, 535.2 (51) [M+Na].sup.+. HPLC: t.sub.R=9.0 min (87%), B/A=30/70-100/0 in 25 min, column 4250 mm, flow 1.2 mL/min, detection at 254 nm.
EXAMPLE 5
Synthesis of Fluorescent Phosphorylated Carbopyronine Dyes and their Precursors
9-(Diisopropoxyphosphoryl)-3,6-bis(dimethylamino)-10,10-dimethyl-9,10-dihydro-anthracenylium trifluoromethanesulfonate (24)
(96) ##STR00100##
(97) Compound 24. In a Schlenk flask to a solution of 3,6-bis(dimethylamino)-10,10-dimethylanthrone [prepared according to WO 2012052435 A1] (20 mg; 0.065 mmol) in CH.sub.2Cl.sub.2 (1 ml) triflic anhydride (18 mg; 0.065 mmol) was injected under argon. The resulted blue colored reaction mixture stirred for 10 min at r.t., and P(OiPr).sub.3 (13 mg; 0.065 mmol) was injected thereto. After overnight stirring at r.t., all volatiles were evaporated in the flow of argon. The residue was subjected to reverse-phase chromatography (30 g of RPSiO.sub.2, MeCN/H.sub.2O 1:1+0.1 v/v % of TFA). Green-colored fractions were collected, combined and evaporated. After additional normal phase column chromatography (30 g of SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 20:1.fwdarw.10:1) 10 mg (25%) of a dark green solid were obtained. .sup.1H NMR (400 MHz, CD.sub.3CN): =1.18 (d, J.sub.HH=6.2 Hz, 6H, OiPr), 1.41 (d, J.sub.HH=6.2 Hz, 6H, OiPr), 1.66 (s, 6H, 2Me), 3.35 (s, 2NMe.sub.2), 4.92 (m, 2H, 2OiPr), 6.92 (dd, J.sub.HH=9.8 and 2.7 Hz, 2H.sub.ar), 7.13 (m, J.sub.HH=2.7 Hz), 8.80 (d, J.sub.HH=9.8 Hz) ppm. MS (ESI): m/z (positive mode, rel. int., %)=457.3 (100) [MOTf].sup.+. UV/Vis (MeCN): .sub.max ()=694 nm (37946 M.sup.1cm.sup.1); fluorescence (MeCN): .sub.excit=660 nm, .sub.em=779 nm, .sub.fl=0.03. Standard: Rhodamine 800, .sub.fl=0.25 (EtOH).
9-[(Allyloxy)(diisopropylamino)phosphoryl]-3,6-bis(dimethylamino)-10,10-dimethyl-9,10-dihydroanthracenylium trifluoromethanesulfonate (25)
(98) ##STR00101##
(99) Compound 25. In a Schlenk flask to a solution of 3,6-bis(dimethylamino)-10,10-dimethylanthrone (50 mg; 0.16 mmol) in CH.sub.2Cl.sub.2 (2 mL) triflic anhydride (46 mg; 0.16 mmol) was injected under argon. The resulted blue colored reaction mixture stirred for 10 min at r.t., and diallyl N,N-diisopropylphosphoramidite (40 mg; 0.16 mmol) was injected thereto. After overnight stirring at r.t., the reaction mixture was diluted with MeCN (.sup.5 mL) and directly subjected to column chromatography (30 g of SiO.sub.2, MeCN.fwdarw.MeCN/H.sub.2O 20:1.fwdarw.10:1.fwdarw.5:1.fwdarw.2:1). Green-colored fractions were collected, combined and evaporated. After additional reverse-phase column chromatography (20 g of RPSiO.sub.2, MeCN/H.sub.2O 1:1.fwdarw.5:1+0.1 v/v % of TFA) 18 mg (21%) of a dark green solid were obtained. .sup.1H NMR (400 MHz, CDCl.sub.3): =1.28 (d, J.sub.HH=6.7 Hz, 6H, NiPr), 1.33 (d, J.sub.HH=6.8 Hz, 6H, NiPr), 1.69 (s, 6H, 2Me), 3.35 (s, 12H, 2NMe.sub.2), 3.47-3.58 (m, 2H, NiPr.sub.2), 4.27-4.37 (m, 1H, OAll), 4.52-4.61 (m, 1H, OAll), 5.09-5.14 (m, J.sub.HH=10.3 and 1.1 Hz, 1H, OAll), 5.19-5.26 (m, J.sub.HH=17.1 and 1.5 Hz, 1H, OAll), 5.75-5.86 (m, 1H, OAll), 6.75 (t, J.sub.HH=9.8 Hz, J.sub.HP=2.5 Hz, 2H.sub.ar), 7.11 (t, J.sub.HH=2.5 Hz, J.sub.HP=2.5 Hz, 2H.sub.ar), 8.84 (d, J.sub.HH=9.8 Hz, 2H.sub.ar) ppm. .sup.13C NMR (100 MHz, CDCl.sub.3, APT): =14.0 (+), 22.6 (+, d, J.sub.CP=3.1 Hz), 22.8 (+, d, J.sub.CP=2.7 Hz), 41.4 (+), 47.3 (+, d, J.sub.CP=6.0 Hz), 66.8 (, d, J.sub.CP=5.3 Hz), 111.2 (+), 112.3 (+), 118.6 (), 124.0 (, d, J.sub.CP=8.5 Hz), 129.0 (+), 132.6 (+, d, J.sub.CP=6.7 Hz), 138.6 (d, +, J.sub.CP=4.2 Hz), 155.4 (), 156.7 (, d, J.sub.CP=12.1 Hz) ppm. .sup.31P NMR (162 MHz, CDCl.sub.3): =22.3 ppm. MS (ESI): m/z (positive mode, rel. int., %)=496.4 (100) [MOTf].sup.+. HPLC: t.sub.R=15.7 min (96%), B/A=30/70-100/0 in 25 min, column 4250 mm, 1.2 mL/min, detection at 635 nm. UV/Vis (MeCN): .sub.max ()=698 nm (40789 M.sup.1cm.sup.1); fluorescence (MeCN): .sub.excit=700 nm, .sub.em=775 nm, .sub.fl=0.26. Standard: Rhodamine 800, CD.sub.fl=0.25 (EtOH).
6-[(Allyloxy)(diisopropylamino)phosphoryl]-1,11-bis(2-methoxyethyl)-13,13-dimethyl-1,2,3,4,6,8,9,10,11,13-decahydrobenzo[1,2-g:5,4-g]diquinolin-6-ylium trifluoroacetate (26)
(100) ##STR00102##
(101) Compound 26. Prepared similarly to compound 25 from the corresponding ketone. The product was isolated by column chromatography (30 g of SiO.sub.2, MeCN.fwdarw.MeCN/H.sub.2O 20:1.fwdarw..fwdarw.5:1.fwdarw.2:1). Green-colored fractions were collected, combined and evaporated. After additional reverse-phase column chromatography (20 g of RPSiO.sub.2, MeCN/H.sub.2O 1:1.fwdarw.5:1, then 1:10.fwdarw.1:5.fwdarw.1:1+0.1 v/v % of TFA) 4 mg (5%) of green powder was obtained. HPLC: t.sub.R=19.8 min (93%), B/A=30/70-100/0 in 25 min, column 4250 mm, 1.2 mL/min, detection at 254 nm.
EXAMPLE 6
Synthesis of Fluorescent Phosphorylated Si-pyronine Dyes and their Precursors
Methyl P-[3,7-bis(dimethylamino)-5,5-dimethyl-5,10-dihydrodibenzo[b,e]silin-10-yl]-N,N-diisopropylphosphonamidate (27)
(102) ##STR00103##
(103) Compound 27. Dimethyl N,N-diisopropylphosphoramidite (84 L, 0.366 mmol) was added to a solution of 3,7-bis(dimethylamino)-5,5-dimethyl-5,10-dihydrodibenzo[b,e]silinylium perchlorate [Koide, Y.; Urano, Y.; Hanaoka, K.; Terai, T.; Nagano, T. ACS Chem. Biol. 2011, 6(14), 600-608] (50 mg, 0.122 mmol) and tetrabutylammonium iodide (45 mg, 0.122 mmol) in MeCN (2 mL) and CH.sub.2Cl.sub.2 (2 mL), and the resulting mixture was stirred at rt for 20 min. The clear colorless solution was evaporated to dryness and the product was isolated by column chromatography (25 g of SiO.sub.2, gradient 33% to 50% EtOAc/hexane). The fraction containing the product were pooled, evaporated and dried in vacuo to yield 55 mg (92%) of the leuco dye as a viscous colorless oil. .sup.1H NMR (400 MHz, CD.sub.3CN): 7.18 (ddd, J=8.6, 3.7, 2.5 Hz, 2H), 6.97 (dd, J=16.1, 2.9 Hz, 2H), 6.73 (dddd, J=11.4, 8.5, 3.0, 0.9 Hz, 2H), 4.49 (d, J=24.2 Hz, 1H), 3.41 (dp, J=17.6, 6.7 Hz, 2H), 3.16 (d, J=10.6 Hz, 3H), 2.92 (s, 6H), 2.90 (s, 6H), 1.17 (d, J=6.6 Hz, 6H), 0.93 (d, J=6.7 Hz, 6H), 0.61 (s, 3H), 0.37 (s, 3H) ppm. .sup.13C NMR (101 MHz, CD.sub.3CN): 150.1 (d, J=3.0 Hz), 149.8 (d, J=2.9 Hz), 138.3 (d, J=4.8 Hz), 137.8 (d, J=4.6 Hz), 132.5 (d, J=8.5 Hz), 132.1 (d, J=5.4 Hz), 131.5 (d, J=6.9 Hz), 118.7 (d, J=3.4 Hz), 118.1 (d, J=3.2 Hz), 114.3 (d, J=3.0 Hz), 113.8 (d, J=3.3 Hz), 52.1 (d, J=120.9 Hz), 51.2 (d, J=7.6 Hz), 46.7 (d, J=3.6 Hz), 41.0 (d, J=1.1 Hz), 24.2, 22.6 (d, J=2.3 Hz), 0.2 (d, J=1.0 Hz), 0.1 (d, J=4.2 Hz) ppm. .sup.31P NMR (162 MHz, CD.sub.3CN): 31.20 ppm. MS (ESI): m/z (positive mode, rel. int., %)=488.3 (100) [M+H].sup.+. HRMS (C.sub.26H.sub.42N.sub.3O.sub.2PSi): m/z (positive mode)=488.2860 (found [M+H].sup.+), 488.2857 (calc.).
(104) Oxidation of the leuco dye (DDQ in CH.sub.2Cl.sub.2) as follows led to formation of an unstable silaxanthylium dye 27 (easily hydrolyzes to the starting 3,7-bis(dimethylamino)-5,5-dimethyl-5,10-dihydrodibenzo[b,e]silinylium). 22 mg (0.045 mmol) of the leuco dye was dissolved in CH.sub.2Cl.sub.2 (2 mL), and the solution was cooled to 78 C. A solution of DDQ (10 mg, 0.044 mmol) in CH.sub.2Cl.sub.2 (1 mL) was then added. The reaction mixture was allowed to warm up to rt and directly subjected to column chromatography (30 g of SiO.sub.2, MeCN.fwdarw.MeCN+0.1% v/v TFA.fwdarw.MeCN/H.sub.2O 40:1+0.1 v/v % of TFA), the green-colored fractions were pooled and evaporated. The residue was redissolved in cold water and freeze-dried to give 50 mg of the material.
EXAMPLE 7
Synthesis of Fluorescent Phosphorylated Benzanthrylium Dyes and their Precursors
1-Bromo-5-(Dimethylamino)Naphthalene (28a)
(105) ##STR00104##
(106) Compound 28a. To a suspension of 1-bromo-5-nitronaphthalene (2 g, 7.94 mmol) in EtOH (50 mL), a solution of NH.sub.4Cl (2.2 g, 41.1 mmol) in water (20 mL) was added, followed by iron powder (1.33 g, 23.82 mmol). The resulting mixture was stirred at 75 C. (bath temperature) for 1.5 h. Celite (3 g) was added, and the mixture was allowed to cool down to rt, diluted with CH.sub.2Cl.sub.2 (100 mL), filtered through a plug of Celite, washing with CH.sub.2Cl.sub.2 (150 mL). The filtrate was washed with brine and dried over Na.sub.2SO.sub.4. Upon evaporation of the filtrate, the crude material was redissolved in CH.sub.2Cl.sub.2 (20 mL), transferred on top of a 80 g SiO.sub.2 column, and ran with 20% to 80% EtOAc/hexane gradient. The fractions containing the product were evaporated to viscous light brown oil that quickly crystallized. Yield of 5-bromo-1-aminonaphthalene [West, R. W. J. Chem. Soc. 1925, 127, 494] 1.48 g (84%).
(107) 5-Bromo-1-aminonaphthalene (1.37 g, 6.17 mmol) was dissolved in trimethyl phosphate (760 L, 6.5 mmol) in a 50 mL round-bottom flask, equipped with an air condenser and a CaCl.sub.2 drying tube, the apparatus was flushed with nitrogen, and the mixture was heated at 200 C. (bath temperature) for 1.5 h. The flask was then allowed to cool below 100 C., 1 N NaOH (20 mL) was added, the resulting suspension was sonicated briefly and stirred at rt overnight. The mixture was diluted with brine, extracted with CH.sub.2Cl.sub.2 (350 mL), the combined extracts were dried over Na.sub.2SO.sub.4. The product was isolated by column chromatography (100 g of SiO.sub.2, gradient 10% to 50% CH.sub.2Cl.sub.2/hexane) to yield 1-bromo-5-(dimethylamino)naphthalene 28a [West, R. W. J. Chem. Soc. 1925, 127, 494] as a light-orange viscous oil (1.29 g, 84%). .sup.1H NMR (301 MHz, CDCl.sub.3): 8.26 (dt, J=8.6, 1.0 Hz, 1H), 7.95 (dt, J=8.6, 0.9 Hz, 1H), 7.78 (dt, J=7.4, 1.0 Hz, 1H), 7.51 (ddd, J=8.5, 7.5, 0.7 Hz, 1H), 7.32 (ddd, J=8.4, 7.3, 0.7 Hz, 1H), 7.14 (dd, J=7.6, 1.0 Hz, 1H), 2.90 (s, 6H) ppm. .sup.13C NMR (76 MHz, CDCl.sub.3): 151.3, 133.4, 130.4, 130.1, 127.3, 125.4, 124.3, 123.3, 122.0, 115.0, 45.5.
4-(Dimethylamino)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde (28b)
(108) ##STR00105##
(109) Compound 28b. 1,4-Dioxane (25 mL) was added to the solid 2-bromo-4-(dimethylamino)benzaldehyde [Meyer, W. E.; Tomcufcik, A. S.; Chan, P. S.; Emma, J. E. J. Med.Chem. 1984, 27, 1705-1710] (684 mg, 3 mmol), bis(pinacolato)diboron (840 mg, 3.3 mmol), KOAc (880 mg, 9 mmol) and Pd(dppf)Cl.sub.2 (66 mg, 0.09 mmol), the mixture was deoxygenated on a Schlenk line and stirred under N.sub.2 at 90 C. (bath temperature) for 4 h. Upon cooling to rt, the reaction mixture was filtered through a 1.5 cm pad of Celite, washing with EtOAc (100 mL). The filtrate was evaporated; the residue was dissolved in CH.sub.2Cl.sub.2, transferred on top of a 30 g SiO.sub.2 column and ran with 10% to 50% EtOAc/hexane gradient. The fractions containing the product were evaporated, and the residue was recrystallized from CH.sub.2Cl.sub.2-hexane (with cooling in 78 C. bath) to give 536 mg (65%) of 28b as light-orange crystals. .sup.1H NMR (500 MHz, CDCl.sub.3): 10.20 (s, 1H), 7.84 (d, J=8.8 Hz, 1H), 7.01 (d, J=2.7 Hz, 1H), 6.77 (dd, J=8.8, 2.7 Hz, 1H), 3.08 (s, 6H), 1.39 (s, 12H). .sup.13C NMR (126 MHz, CDCl.sub.3): 192.22, 192.20, 152.9, 130.8, 130.1, 117.3, 112.9, 84.3, 40.4, 25.1. MS (ESI): m/z (positive mode, rel. int., %)=244.1 (100), 298.2 (3) [M+Na].sup.+. HRMS (C.sub.15H.sub.22NO.sub.3B): m/z (positive mode)=298.1578 (found [M+Na].sup.+), 298.1588 (calc.).
4-(Dimethylamino)-2-[5-(dimethylamino)naphthalen-1-yl]benzaldehyde (28c)
(110) ##STR00106##
(111) Compound 28c. 1,4-Dioxane (15 mL) and water (3 mL) were added to a mixture of 28a (414 mg, 1.65 mmol), 28b (500 mg, 1.82 mmol), K.sub.2CO.sub.3 (455 mg, 3.3 mmol) and Pd(dppf)Cl.sub.2 (24 mg, 0.033 mmol), the mixture was deoxygenated on a Schlenk line and stirred under N.sub.2 at 100 C. (bath temperature) for 1 h. Upon cooling down to rt, the mixture was diluted with sat. aq. NaHCO.sub.3 (50 mL), extracted with EtOAc (340 mL), washed with brine and dried over Na.sub.2SO.sub.4. The product was isolated by column chromatography (40 g of SiO.sub.2, gradient 10% to 30% EtOAc/hexane) and dried in vacuo to yield 28c (524 mg, 99%) as a yellowish foam. .sup.1H NMR (400 MHz, CDCl.sub.3): 9.39 (s, 1H), 8.38 (d, J=8.5 Hz, 1H), 8.06 (d, J=8.9 Hz, 1H), 7.56 (dd, J=8.6, 6.9 Hz, 1H), 7.45 (d, J=6.9 Hz, 1H), 7.37-7.25 (m, 2H), 7.11 (dd, J=6.7, 1.8 Hz, 1H), 6.83 (dd, J=8.9, 2.7 Hz, 1H), 6.61 (d, J=2.7 Hz, 1H), 3.09 (s, 6H), 2.96 (s, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3): 190.4, 153.5, 151.0, 147.2, 137.2, 134.2, 129.0, 128.8, 127.5, 126.3, 124.4, 124.2, 124.1, 121.2, 114.2, 113.1, 111.0, 45.4, 40.1. MS (ESI): m/z (positive mode, rel. int., %)=319.2 (100) [M+H].sup.+, 341.2 (29) [M+Na].sup.+. HRMS (C.sub.21H.sub.22N.sub.2O): m/z (positive mode)=319.1810 (found [M+H].sup.+), 319.1805 (calc.).
4,10-Bis(dimethylamino)-7H-benzo[de]anthracenylium perchlorate (28d)
(112) ##STR00107##
(113) Compound 28d. A solution of 28c (440 mg, 1.38 mmol) in methanesulfonic acid (1 mL) was heated at 100 C. (bath temperature) overnight. The viscous mixture was diluted with methanesulfonic acid (2 mL) and poured into 150 mL of ice-water mixture, containing 5 g NaClO.sub.4. The resulting blue suspension was stirred until all ice melted; the dark solid was filtered off, washed with water and dried on filter. The crude solid was recrystallized from MeOH/CH.sub.2Cl.sub.2, adding hexane to complete precipitation, filtered off, washed with hexane and dried in vacuo. Small black crystals, yield 520 mg (94%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): 9.28 (d, J=8.0 Hz, 1H), 8.68 (d, J=8.0 Hz, 1H), 8.42 (s, 1H), 8.11 (d, J=9.5 Hz, 1H), 7.94 (t, J=8.1 Hz, 1H), 7.92 (d, J=9.1 Hz, 1H), 7.81 (d, J=2.4 Hz, 1H), 7.38 (d, J=9.5 Hz, 1H), 7.26 (dd, J=9.1, 2.4 Hz, 1H), 3.73 (s, 6H), 3.27 (s, 6H). .sup.13C NMR (101 MHz, DMSO-d.sub.6): 163.2, 153.0, 142.9, 141.6, 135.8, 133.7, 133.2, 131.2, 128.3, 126.2, 125.9, 122.3, 121.9, 120.4, 116.2, 115.5, 103.3, 46.3, 40.2. MS (ESI): m/z (positive mode, rel. int., %)=288.2 (100) [MCH.sub.3].sup.+, 301.2 (1) [M].sup.+. HRMS (C.sub.21H.sub.21N.sub.2): m/z (positive mode)=301.1690 (found [M].sup.+), 301.1699 (calc.). UV/Vis (MeOH): .sub.max (E) =646 nm (23000 M.sup.1cm.sup.1); fluorescence (MeOH): .sub.excit=550 nm, .sub.em=673 nm, .sub.fl=0.25. Standard: Atto 594, .sub.fl=0.85 (H.sub.2O).
7-[(Diisopropylamino)(methoxy)phosphoryl]-4,10-bis(dimethylamino)-7H-benzo[de]anthracen-7-ylium trifluoroacetate (28)
(114) ##STR00108##
(115) Compound 28. Dimethyl N,N-diisopropylphosphoramidite (172 L; 0.75 mmol) was added to a stirred solution of 28d (100 mg; 0.25 mmol) and tetrabutylammonium iodide (92 mg; 0.25 mmol) in dry CH.sub.2Cl.sub.2 (5 mL). The vial was flushed with argon and the blue suspension was stirred at RT for 30 min, turning into a clear brown solution. The mixture was evaporated to dryness, and the intermediate 7H-benz[de]anthracene adduct was isolated by column chromatography (25 g of SiO.sub.2, gradient 50% to 100% EtOAc.sub.2/hexane). The compound was used immediately in the next step.
(116) The material was dissolved in CH.sub.2Cl.sub.2 (10 mL), the solution was cooled in dry ice-acetone bath, and DDQ (57 mg; 0.105 mmol) in CH.sub.2Cl.sub.2 (3 mL) was added quickly dropwise. The resulting blue-green solution was allowed to warm up to RT, stirred for 15 min, followed by addition of trifluoroacetic acid (100 L). The mixture was evaporated to dryness, and the residue was subjected to column chromatography (35 g of SiO.sub.2, gradient 0% to 50% H.sub.2O/MeCN); the fractions containing the product were pooled, trifluoroacetic acid (200 L) was added, MeCN was evaporated (bath temperature 25 C.) and the aqueous solution was freeze-dried. The residue was dissolved in 1,4-dioxane (with addition of minimal amount of water to dissolve the solids), filtered through 0.2 M PTFE membrane filter and re-lyophilized. Blue solid, yield 88 mg (61%). .sup.1H NMR (400 MHz, CD.sub.3OD): 9.49 (d, J=10.3 Hz, 1H), 9.35 (d, J=8.2 Hz, 1H), 9.19 (d, J=9.8 Hz, 1H), 8.73 (d, J=7.9 Hz, 1H), 8.06 (t, J=8.0 Hz, 1H), 8.01 (t, J=2.0 Hz, 1H), 7.58 (d, J=10.3 Hz, 1H), 7.41 (dd, J=9.8, 2.7 Hz, 1H), 4.99 (d, J=1.7 Hz, 6H), 3.85 (s, 6H), 3.68 (d, J=11.5 Hz, 2H), 3.34 (s, 3H), 1.35 (dd, J=6.8, 3.2 Hz, 12H) ppm. .sup.19F NMR (376 MHz, CD.sub.3OD): 77.29 ppm. .sup.13C NMR (101 MHz, CD.sub.3OD): 165.9, 153.6, 143.1 (d, J=4.9 Hz), 138.9 (d, J=152.9 Hz), 137.8 (d, J=11.5 Hz), 135.3, 133.4 (d, J=4.2 Hz), 132.5, 131.4 (d, J=2.4 Hz), 128.3, 127.9 (d, J=13.7 Hz), 126.3 (d, J=9.4 Hz), 125.3 (d, J=9.1 Hz), 124.0 (d, J=1.2 Hz), 104.4 (d, J=1.2 Hz), 52.9 (d, J=5.9 Hz), 48.7 (d, J=5.8 Hz), 46.7, 40.5, 23.2 (d, J=2.8 Hz), 22.8 (d, J=2.8 Hz) ppm. .sup.31P NMR (162 MHz, CD.sub.3OD): 25.95 ppm. MS (ESI): m/z (positive mode, rel. int., %)=478.3 (100) [M].sup.+. HRMS (C.sub.28H.sub.32N.sub.3O.sub.2P): m/z (positive mode)=478.2621 (found [M].sup.+), 478.2618 (calc.). UV/Vis (PBS 7.4): .sub.max ()=644 nm (7600 M.sup.1cm.sup.1); fluorescence (PBS 7.4): .sub.excit=580 nm, .sub.em=782 nm, CD.sub.fl=0.04. Standard: Atto 594, .sub.fl=0.85 (H.sub.2O).
EXAMPLE 8
Synthesis of Phosphorylated BODIPY Dyes and their Precursors
10-(Diisopropoxyphosphoryl)-5,5-difluoro-1,3,7,9-tetramethyl-5H-dipyrrolo[1,2-c:2,1-f][1,3,2]diazaborinin-4-ium-5-uide (29)
(117) ##STR00109##
(118) Compound 29. A solution of 10-chloro-5,5-difluoro-1,3,7,9-tetramethyl-5H-dipyrrolo[1,2-c:2,1-f][1,3,2]diazaborinin-4-ium-5-uide [Leen, V.; Yuan, P.; Wang, L.; Boens, N.; Dehaen, W. Org. Lett. 2012, 14(24), 6150-6153] (20 mg; 0.07 mmol) in triisopropyl phosphite (0.5 mL) was stirred under argon at 100 C. for 30 min. After cooling down to room temperature, the reaction mixture was diluted with n-hexane (.sup.5 mL) and subjected to column chromatography (30 g of SiO.sub.2, hexane/EtOAc 1:1) to yield 28 mg (96%) of a violet solid. .sup.1H NMR (400 MHz, CDCl.sub.3): 6.10 (s, 2H.sub.Ar), 4.84 (d.hept, 2H, J.sub.HP=7.7 Hz, J.sub.HH=6.2 Hz, 2CH.sub.ipr), 2.51 (s, 6H, 2Me.sub.Ar), 2.46 (s, 6H, 2Me.sub.Ar), 1.40 (d, J.sub.HH=6.2 Hz, 6H, 2Me.sub.ipr), 1.28 (d, J.sub.HH=6.2 Hz, 6H, 2Me.sub.ipr). .sup.13C NMR (101 MHz, CDCl.sub.3): 157.6, 144.5, 137.10 (d, .sup.2J.sub.CP=13.9 Hz), 129.62 (d, .sup.1J.sub.CP=182.3 Hz), 123.5 (d, .sup.4J.sub.CP=2.4 Hz), 72.8 (d, .sup.2J.sub.CP=6.7 Hz), 24.0 (d, .sup.3J.sub.CP=4.4 Hz), 23.6 (d, .sup.3J.sub.CP=5.0 Hz), 16.6. .sup.19F NMR (376 MHz, CDCl.sub.3): =146.59 (app.q 1:1:1:1, J.sub.F-11B=32.2 Hz) ppm. .sup.31P NMR (162 MHz, CDCl.sub.3): =9.8 ppm. MS (ESI): m/z (positive mode, rel. int., %)=413.1 (100) [M+H].sup.+, 435.1 (93) [M+Na].sup.+.
EXAMPLE 9
Characterisation of Exemplary Compounds of the Present Invention
(119) General Materials and Methods
(120) Thin Layer Chromatography
(121) 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); leuco dyes were detected by staining with 1% DDQ in CH.sub.2Cl.sub.2.
(122) Column Chromatography
(123) 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 Isolera One system (Biotage AG, Sweden) with commercially available cartridges.
(124) Absorption Spectroscopy
(125) 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).
(126) Nuclear Magnetic Resonance (NMR)
(127) NMR Spectra were recorded on an Agilent 400MR DD2 spectrometer. 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.
(128) Mass-Spectrometry (MS)
(129) 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).
(130) High-Performance Liquid Chromatography (HPLC)
(131) 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 (unless 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 (unless stated otherwise). For isolation and purification of the acid sensitive dyes or their derivatives, acetonitrile aqueous 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).
(132) STED (Stimulated Emission Depletion) Microscopy
(133) STED and confocal counterpart images were acquired using the commercially available two-color STED 775 quad scanning microscope from Abberior Instruments (Gottingen, Germany) equipped with an Olympus IX83 microscope stand and an Olympus UplanSApo 100/1.4 OIL objective. Dyes were excited respective their excitation; the STED laser was pulsed at 775 nm. Imaging and image processing was done with ImSpector software, and the images are displayed as raw data.
(134) TABLE-US-00002 TABLE 2 Properties of exemplary novel compounds of the invention Absorption max., nm Emission Stability; (, max., nm Stokes fluorescence No. Compound M.sup.1cm.sup.1) (QY) shift, nm lifetime, ns 1
EXAMPLE 10
STED Optical Microscopy of Cells using Exemplary Novel Dyes of the Invention
(135) For these experiments, STED optical microscopy was performed as indicated above.
(136)
(137)