CELL-PERMEABLE FLUOROGENIC FLUOROPHORES
20220404242 · 2022-12-22
Assignee
Inventors
Cpc classification
G01N1/30
PHYSICS
C09B11/24
CHEMISTRY; METALLURGY
International classification
G01N1/30
PHYSICS
C09B11/24
CHEMISTRY; METALLURGY
Abstract
The present invention relates to rhodamine-type fluorophores being present in two states, a cell-permeable non-florescent form and a fluorescent form. The present invention also relates to use of the fluorophores in staining and live cell fluorescence imaging. The compounds have general formula (10) or the general formula (10′).
Claims
1. A compound of the general formula (10) or the general formula (10′) ##STR00082## wherein R.sup.1 is selected from H and F, particularly R.sup.1 is H; R.sup.2 and R.sup.3 can be any moiety, particularly R.sup.2 and R.sup.3 are independently selected from H, substituted or unsubstituted C.sub.1 to C.sub.4 alkyl optionally forming a bridge to the substituent designated Y, and a moiety having a molecular weight between 15 and 250 u (g/mol); X is selected from O, S, Se, TeO, POR.sup.X, POOR.sup.X, SO.sub.2, NR.sup.X, CR.sup.X.sub.2, SiR.sup.X.sub.2, GeR.sup.X.sub.2, and SnR.sup.X.sub.2, with each R.sup.X being independently selected from H and an unsubstituted or substituted (particularly unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy-substituted) moiety selected from C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, C.sub.7-C.sub.12 alkylaryl, phenyl and 5- or 6-membered heteroaryl, or two R.sup.X moieties form a four-, five-, six- or seven-membered unsubstituted or amino-, hydroxy- and/or halogen substituted alkyl ring; Y is OH or NR.sup.Y1R.sup.Y2, with R.sup.Y1, and R.sup.Y2 each independently selected from H, an unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy-substituted hydrocarbon moiety selected from C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.4 acyl, C.sub.7-C.sub.12 alkylaryl, an unsubstituted phenyl or a phenyl substituted by any one or several of the following substituents: unsubstituted C.sub.1-C.sub.4 alkyl, halogen, C.sub.1-C.sub.4 oxyalkyl, COOH, COOR.sup.YC, CONR.sup.YC.sub.2, with R.sup.YC being selected from H and unsubstituted or amino- or hydroxy-substituted C.sub.1-C.sub.8 alkyl; or R.sup.Y1 and R.sup.Y2 together are a C.sub.3-C.sub.6 unsubstituted or hydroxy-, amino-, halogen-, alkoxy- and/or carboxy-substituted alkyl forming a 4-7-membered ring structure with Y; or one of R.sup.Y1 and R.sup.Y2, or both R.sup.Y1 and R.sup.Y2, together with R.sup.2 and/or R.sup.3, respectively, form an unsubstituted or hydroxy-, amino-, halogen-, carboxy- and/or aryl-substituted 4-7-membered alkyl or alkylene ring; a) A and B together with the atoms that they are covalently coupled to form a phenyl ring or 5- or 6-membered heteroaryl ring, wherein the ring is unsubstituted or substituted by one of NR.sup.N1R.sup.N2— and OH and wherein the ring is optionally further by R.sup.4, R.sup.5 and/or R.sup.6, wherein R.sup.N1 and R.sup.N2 have the same meanings as R.sup.Y1, and R.sup.Y2 above, R.sup.4 has the same meaning as R.sup.3, R.sup.5 has the same meaning as R.sup.1, and R.sup.6 has the same meaning as R2 particularly A and B are a moiety of the general formula (50) ##STR00083## wherein R.sup.4 and G are independently selected from H, substituted or unsubstituted C.sub.1 to C.sub.4 alkyl optionally forming a bridge to the substituent designated E, and a moiety having a molecular weight between 15 and 250 u (g/mol); R.sup.5 is selected from H and F, particularly R.sup.5 is H; E is OH or NR.sup.EG1R.sup.EG2, wherein R.sup.EG1 and R.sup.EG2 have the same meanings as R.sup.Y1, and R.sup.Y2 above, respectively, wherein optionally one of R.sup.EG1 and R.sup.EG2, or both R.sup.EG1 and R.sup.EG2, together with R.sup.4 and/or G, respectively, form an unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy-substituted 4-7-membered alkyl ring; b) A is an unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy-substituted conjugated carbocycle or heterocycle having one, two or three rings, particularly A is an aromatic or heteroaromatic ring system, more particularly A is selected from an unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy-substituted aryl, and heteroaryl, most particularly A is a substituted (particularly hydroxy-, amino-, halogen-, and/or carboxy-substituted) coumarin; and B is selected from H (or D, F) and a moiety having a molecular weight between 15 and 250 u (g/mol); U and V together with the atoms they are covalently coupled to form an unsubstituted or substituted 5-7 membered ring, wherein the substitution is selected from one or several substituents independently selected from OH, SH, amine [particularly NR.sup.UV.sub.2]halogen, CN, NC, CNO, NCO, CNS, NCS, unsubstituted C.sub.1-C.sub.4 O-alkyl, C.sub.1-C.sub.4 S-alkyl, O-aryl, S-aryl, NO.sub.2, CHO, CONR.sup.UV.sub.2, COOR.sup.UV, COO-aryl, COO-alkylaryl (particularly benzyl), PO.sub.3H, PO.sub.3R.sup.UV, SO.sub.3H, SO.sub.3R.sup.UV and SO.sub.2R.sup.UV, with R.sup.UV being selected from H, and C.sub.1-C.sub.4 unsubstituted alkyl and the 5-7 membered ring is selected from cycloalkyl, aryl, heteroaryl, particularly U and V form an unsubstituted or substituted 5-membered heteroaryl or phenyl, or U is selected from H, D and F and V is selected from H, D, F, and C.sub.1 to C.sub.4 unsubstituted or amino-, hydroxy- or halogen substituted alkyl, Z is selected from CN, CHO, COR.sup.Z, COOR.sup.Z, and CONR.sup.Z.sub.2, wherein each R.sup.Z is independently selected from H, unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy-substituted C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.4 acyl, C.sub.7-C.sub.12 alkylaryl, unsubstituted phenyl or phenyl substituted by any one or several of the following substituents: unsubstituted C.sub.1-C.sub.4 alkyl, halogen, C.sub.1-C.sub.4 oxyalkyl, COOR.sup.C, CONR.sup.C.sub.2, with R.sup.C being selected from H, C.sub.1-C.sub.8 alkyl; particularly R.sup.Z is selected from H, unsubstituted C.sub.1-C.sub.4 alkyl, and alkylaryl (particularly benzyl); or SO.sub.2R.sup.S with R.sup.S being selected from NH.sub.2, NHR.sup.SN, NR.sup.SN.sub.2, with each R.sup.SN independently selected from a unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy-substituted moiety selected from C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.4 acyl, C.sub.7-C.sub.12 alkylaryl, or from unsubstituted phenyl or phenyl substituted by any one or several of the following substituents: unsubstituted C.sub.1-C.sub.4 alkyl, halogen, O—C.sub.1-C.sub.4 alkyl, COOH, COOR.sup.C, CONR.sup.C.sub.2, with R.sup.C being selected from H and C.sub.1-C.sub.8 alkyl; an unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy-substituted moiety selected from C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.4 acyl, C.sub.7-C.sub.12 alkylaryl, unsubstituted phenyl or phenyl substituted by any one or several of the following substituents: unsubstituted C.sub.1-C.sub.4 alkyl, halogen, O—C.sub.1-C.sub.4 alkyl, COOH, COOR.sup.SC, CONR.sup.SC.sub.2, with R.sup.SC being selected from H and C.sub.1-C.sub.8 alkyl; particularly R.sup.SC is unsubstituted or fluorinated alkyl; OR.sup.SO, with R.sup.SO selected from a unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy-substituted moiety selected from C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.4 acyl, C.sub.7-C.sub.12 alkylaryl, or from unsubstituted phenyl or phenyl substituted by any one or several of the following substituents: unsubstituted C.sub.1-C.sub.4 alkyl, halogen, O—C.sub.1-C.sub.4 alkyl, COOH, COOR.sup.C, CONR.sup.C.sub.2, with R.sup.C being selected from H and C.sub.1-C.sub.8 alkyl; CH.sub.2R.sup.F, CHR.sup.F.sub.2, and CR.sup.F.sub.3, with R.sup.F selected from CH.sub.2F, CHF.sub.2, CF.sub.3, CH.sub.2Cl, and CHCl.sub.2, CCl.sub.3, particularly with R.sup.F being CF.sub.3; H CH.sub.2COOR.sup.K1 or CH.sub.2CONHR.sup.K1 with R.sup.K1 selected from H, and unsubstituted or halogen-substituted C.sub.1 to C.sub.4 alkyl; wherein optionally, the compound is covalently linked to a binding moiety M capable of attaching the compound to a biomolecule, with the proviso that the following compounds are not claimed: ##STR00084## with R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 and G are H, and Y and E are NEt.sub.2 and Z is CONH.sub.2 or Z is CONHC.sub.6H.sub.5; or Z is CN or Z is selected from CH.sub.2CF.sub.3, CH.sub.2CHF.sub.2, and CH.sub.2CH.sub.2F, or Z is selected from CH.sub.2COOCH.sub.3 and CH.sub.2COOH, Z is H, or Y and E are NMe.sub.2 and Z is selected from CH.sub.2COOCH.sub.3 and CH.sub.2COOH or Y and E are NH.sub.2 and Z is selected from CH.sub.2COOH and CH.sub.2COOCH.sub.3 or Y is OH and E is ═O (for formula 10) or E is OH (for formula 10′), and Z is C(═O)(CH.sub.3)C.sub.6H.sub.5; or R.sup.1, R.sup.3, R.sup.4, and R.sup.5 are H, R.sup.2 and G are methyl, Y and E are NHEt and Z is CH.sub.2COOH or CH.sub.2COOCH.sub.3 or the compound is of formula (301), (302), (303), or (304) ##STR00085##
2. The compound according to claim 1, wherein the compound is covalently linked (particularly through any one of substituents A, B, R.sup.2, R.sup.3, R.sup.4, R.sup.EG1, R.sup.EG2, R.sup.EG, G, R.sup.X, R.sup.Y, R.sup.Y2, R.sup.Y, U, V, R.sup.Z, R.sup.Ph, R.sup.S, R.sup.K1) to M, and M is selected from: a. a moiety selectively attachable by a covalent or quasi-covalent bond to a protein or a nucleic acid under conditions prevailing in cell culture or inside of a living cell, particularly a moiety able to form an ester bond, an ether bond, an amide bond, a disulfide bond, a Schiff' base, or a moiety able to react in a click-chemistry reaction, more particularly a moiety selected from —COCHCH.sub.2, —CO—NHS, biotin, an azide or ethyne moiety, a tetrazine moiety, a (bicyclo[6.1.0]nonyne) moiety, a cyclooctyne moiety, a transcyclooctene moiety and a maleimide, or a moiety employed for specific protein labelling, b. a substrate of an O.sup.6-alkylguanine-DNA-alkyltransferase or a functional variant thereof, particularly a 6-[(4-methylenephenyl)methoxy]-9H-purin-2-amine moiety of formula (71), or a pyrimidine derivative thereof, particularly a moiety of formula (72), or a moiety of formula (73), ##STR00086## c. a substrate of a haloalkane halotransferase, particularly a 1-chlorohexyl moiety as exemplarily shown below; ##STR00087## or from d. a substrate of dihydrofolate reductase, particularly the moiety: ##STR00088## e. a moiety capable of selectively interacting non-covalently with a biomolecule (particularly a protein or nucleic acid) under conditions prevailing in a living cell, wherein said moiety and said biomolecule form a complex having a dissociation constant k.sub.D of 10.sup.−4 mol/l or less, particularly wherein M has a molecular mass of more than 160 u but less than 1000 u, particularly less than 700 u, more particularly less than 500 u, and M comprises up to five hydrogen bond donators, up to ten hydrogen bond acceptors; more particularly, M is selected from taxol, jasplaklinolide, a bis-benzimide DNA stain, pepstatin A and triphenylphosphonium; or M is an oligonucleotide having a sequence length of 10 to 40 nucleotides. f. a lipid, particularly a lipid selected from a ceramide derivative, a glyceride, or a fatty acid.
3. The compound according to claim 2, wherein the compound is connected to said binding moiety M through a covalent bond or a linker moiety L consisting of 1 to 50 atoms having an atomic weight of 12 or higher, particularly wherein L is a moiety described by a formula -L.sup.A1.sub.n-L.sup.J1.sub.n′-L.sup.A2.sub.m-L.sup.J2.sub.m′-L.sup.A3.sub.p-L.sup.J3.sub.p′-L.sup.A4.sub.q-L.sup.J4.sub.q′-, wherein L.sup.A1, L.sup.A2, L.sup.A3 and L.sup.A4 independently of each other are selected from C.sub.1 to C.sub.12 unsubstituted or amino-, hydroxyl-, carboxyl- or fluoro-substituted alkyl or C.sub.3 to C.sub.7 cycloalkyl, (CH.sub.2—CH.sub.2—O).sub.r or (CH.sub.2—CH(OH)—CH.sub.2—O).sub.r with r being an integer from 1 to 20, alkylaryl, alkylaryl-alkyl, and unsubstituted or C.sub.1-C.sub.4 alkyl-, halogen-, amino-, C.sub.1-C.sub.4 alkylamino-, imido-, nitro-, hydroxyl-, C.sub.1-C.sub.4 oxyalkyl-, carbonyl-, carboxyl-, sulfuryl- and/or sulfoxyl-substituted aryl or heteroaryl, L.sup.J1, L.sup.J2, L.sup.J3 and L.sup.J4 independently of each other are selected from —NR.sup.N5C(O)—, —C(O)N(R.sup.N5)—, —CN—, —NC—, —CO—, —OC(O)—, —C(O)O—, —NR.sup.N5—, —O—, —P(OOH)—, —OP(OOH)—, —P(OOH)O—, —OP(OOH)O—, —OP(OOH)O—, —S—, —SO—, SO.sub.2—, with R.sup.N5 selected from H and unsubstituted or amino-, hydroxyl-, carboxyl or fluoro-substituted C.sub.1 to C.sub.6 alkyl, particularly R.sup.N5 is selected from H and unsubstituted C.sub.1 to C.sub.3 alkyl; n, n′, m, m′, p, p′, q, q′ and s independently from each other are selected from 0 and 1.
4. The compound according to claim 3, wherein L is -L.sup.A1-L.sup.J1-L.sup.A2.sub.m-L.sup.J2.sub.m′-L.sup.A3.sub.p, wherein L.sup.A1, L.sup.A2 and L.sup.A3 are independently selected from unsubstituted, amino-, hydroxyl-, carboxyl- or fluoro substituted C.sub.1 to C.sub.6 alkyl or C.sub.3 to C.sub.6 cycloalkyl, (CH.sub.2—CH.sub.2—O).sub.r or (CH.sub.2—CH(OH)—CH.sub.2—O).sub.r with r being an integer from 1 to 4, and L.sup.J1 and L.sup.J2 are selected independently from —NR.sup.N5C(O)—, —C(O)N(R.sup.N5), —CN—, —NC—, —CO—, —OC(O)—, —C(O)O—, NR.sup.N5—, —O—, and —S—, and m, m′ and p independently from each other are selected from 0 and 1.
5. The compound according to claim 1, wherein the compound is connected to said binding moiety M through one of substituents A, R.sup.2, R.sup.3, R.sup.4, G, or a phenyl moiety formed by U and V.
6. The compound according to claim 1, wherein any one of substituents R.sup.2, R.sup.3, R.sup.4, G and R.sup.X independently of any other is selected from H and unsubstituted or fluoro-, amino-, hydroxyl-, SO.sub.3H— and/or carboxyl substituted C.sub.1 to C.sub.4 alkyl, C.sub.1 to C.sub.4 alkenyl or C.sub.1 to C.sub.4 alkynyl, wherein optionally one of R.sup.2, R.sup.3, R.sup.4, G and R.sup.X is linked to a moiety M, particularly wherein M is defined by claim 2, more particularly wherein M is linked to the compound by a moiety L as defined in claim 3 or 4.
7. The compound according to claim 1, wherein A is a moiety of the general formula (60) ##STR00089## wherein R.sup.A is selected from H, OH, NR.sup.Y1R.sup.Y2, with R.sup.Y1, and R.sup.Y2 as defined in claim 1, or one of R.sup.Y1, and R.sup.Y2, or R.sup.A itself, is L-M, with L as defined in claim 3 or 4, and M as defined in claim 2.
8. The compound according to claim 1, wherein B is selected from H, D and F.
9. The compound according to claim 1, wherein Z is selected from CN and CONR.sup.Z.sub.2 wherein each R.sup.Z is independently selected from H, unsubstituted C.sub.1-C.sub.4 alkyl and alkylaryl (particularly benzyl); or SO.sub.2R.sup.S with R.sup.S being selected from NR.sup.SN.sub.2, with each R.sup.SN being independently selected from H, unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy-substituted moiety selected from C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.4 acyl, C.sub.7-C.sub.12 alkylaryl, unsubstituted phenyl or phenyl substituted by any one or several of the following substituents: unsubstituted C.sub.1-C.sub.4 alkyl, halogen, O—C.sub.1-C.sub.4 alkyl, COOH, COOR.sup.C, CONR.sup.C.sub.2, with R.sup.C being selected from H and C.sub.1-C.sub.8 alkyl; unsubstituted or perfluorinated alkyl; particularly Z is SO.sub.2R.sup.S with R.sup.S being selected from NR.sup.SN.sub.2, with each R.sup.SN being independently selected from H, unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy-substituted C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.4 acyl, C.sub.7-C.sub.12 alkylaryl, unsubstituted phenyl or phenyl substituted by any one or several of the following substituents: unsubstituted C.sub.1-C.sub.4 alkyl, halogen, O—C.sub.1-C.sub.4 alkyl, COOH, COOR.sup.C, CONR.sup.C.sub.2, with R.sup.C being selected from H and C.sub.1-C.sub.8 alkyl; unsubstituted or perfluorinated alkyl.
10. The compound according to claim 1, wherein U and V form an unsubstituted or substituted phenyl, wherein one or several substituents of phenyl are independently selected from OH, SH, amine [particularly NR.sup.UV.sub.2], halogen, CN, NC, CNO, NCO, CNS, NCS, unsubstituted C.sub.1-C.sub.4 O-alkyl, C.sub.1-C.sub.4 S-alkyl, O-aryl, S-aryl, NO.sub.2, CHO, CONR.sup.UV.sub.2, COOR.sup.UV, COO-aryl, COO-alkylaryl (particularly benzyl), PO.sub.3H, PO.sub.3R.sup.UV, SO.sub.3H, SO.sub.3R.sup.UV and SO.sub.2R.sup.UV, with R.sup.UV being selected from H, and C.sub.1-C.sub.4 unsubstituted alkyl, or wherein U and V form a phenyl substituted with a moiety L-M, wherein M has the meaning as specified in claim 2, and L has the meaning as specified in claim 3 or 4.
11. The compound according to claim 1, wherein each R.sup.X is independently selected from unsubstituted or hydroxyl-, amino- or halogen-substituted C.sub.1 to C.sub.4 alkyl, C.sub.2 to C.sub.4 alkenyl or C.sub.2 to C.sub.4 alkynyl, unsubstituted or hydroxyl-, amino- or halogen-substituted C.sub.4 to C.sub.6 cycloalkyl or unsubstituted or hydroxyl-, alkyoxy-, amino- or halogen-substituted phenyl, particularly wherein R.sup.X is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and phenyl, more particularly wherein both R.sup.X are the same for X selected from C, Si, Ge, and Sn.
12. The compound according to claim 1, wherein Y is NR.sup.Y1R.sup.Y2 and E is NR.sup.EG1R.sup.EG2 and a. R.sup.Y1 and R.sup.Y2, and/or R.sup.EG1 and R.sup.EG2, are independently selected from H, unsubstituted and amino-, hydroxy-, carboxy- and/or fluoro-substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 acyl, and C.sub.3-C.sub.6 cycloalkyl, particularly R.sup.Y1 and R.sup.Y2, and/or R.sup.EG1 and R.sup.EG2, are independently selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl and CH.sub.2CF.sub.3, more particularly wherein R.sup.Y1, R.sup.Y2, R.sup.EG1 and R.sup.EG2 are the same and are selected from H or unsubstituted C.sub.1-C.sub.4 alkyl and C.sub.3-C.sub.6 cycloalkyl; b. R.sup.Y1 together with R.sup.Y2, and/or R.sup.EG1 together with R.sup.EG2 form a ring and are an unsubstituted or alkyl-, amino-, hydroxy-, carboxy- and/or fluoro-substituted C.sub.3-C.sub.6 alkyl, particularly —(CH.sub.2).sub.3—, —(CH.sub.2).sub.4—, —(CH.sub.2).sub.5—, —(CH.sub.2).sub.2O(CH.sub.2).sub.2— or —(CH.sub.2).sub.2NR.sup.NN(CH.sub.2).sub.2— with R.sup.NN being selected from H and unsubstituted C.sub.1 to C.sub.4 alkyl; c. R.sup.Y1 and/or R.sup.EG1 are independently selected from H, unsubstituted and alkyl- (particularly methyl-), amino-, hydroxy-, carboxy- and/or fluoro-substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 acyl, and C.sub.3-C.sub.6 cycloalkyl, and R.sup.Y2 together with R.sup.2 or R.sup.3, and/or R.sup.EG2 together with G or R.sup.4, is an alkyl or heteroalkyl bridge selected from —(CH.sub.2).sub.2—, —(CH.sub.2).sub.3—, —CH.sub.2CH═CH— or —(CH.sub.2).sub.4— or —CH.sub.2—O—, —CH.sub.2—NR.sup.5—, —CH.sub.2—S—, —CH.sub.2—Se—, —(CH.sub.2).sub.2O—, —(CH.sub.2).sub.2NR.sup.N—, —(CH.sub.2).sub.2S—, —(CH.sub.2).sub.2Se—, —CH.sub.2—O—CH.sub.2—, —CH.sub.2NR.sup.5—, —CH.sub.2S—CH.sub.2—, —CH.sub.2—Se—CH.sub.2—, —CH.sub.2-(1,2)phenyl-, and a mono- or dimethyl substituted derivative of any one of the foregoing alkyl or heteroalkyl bridge moieties; d. R.sup.Y1 and/or R.sup.EG1 are independently selected from H, unsubstituted and alkyl- (particularly methyl-), amino-, hydroxy-, carboxy- and/or fluoro-substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 acyl, and C.sub.3-C.sub.6 cycloalkyl, and R.sup.Y2 together with R.sup.2, and/or R.sup.EG2 together with G, form an cyclic structure according to any one of substructures (41) to (44) or (51) to (54): ##STR00090## wherein R.sup.11, R.sup.12, R.sup.13, R.sup.14, R.sup.15 and R.sup.16 are selected from H, unsubstituted or hydroxyl-, amino-, carboxyl-, sulfoxyl- or halogen-substituted C.sub.1 to C.sub.4 alkyl, halogen, SO.sub.3R′, COOR′, CONR′.sub.2 with R′ selected from H and unsubstituted C.sub.1 to C.sub.4 alkyl; and R.sup.17 is selected from H unsubstituted or hydroxyl-, amino-, carboxyl-, sulfoxyl- or halogen-substituted C.sub.1 to C.sub.4 alkyl, halogen, NO.sub.2, CN, SO.sub.3R′, COOR′, CONR′.sub.2 with R′ selected from H and unsubstituted C.sub.1 to C.sub.4 alkyl; particularly wherein R.sup.11, R.sup.12, R.sup.13, R.sup.14, R.sup.15 and R.sup.16 are selected from H, methyl, CH.sub.2—SO.sub.3H, Cl and F, and R.sup.1 and R.sup.3 can have any of the meanings given herein; or e. R.sup.Y1 together with R.sup.3, and R.sup.Y2 together with R.sup.2, and/or R.sup.EG1 together with R.sup.4, and R.sup.EG2 together with G, form a bi-cyclic structure according to any one of substructures (45) to (47) or (55) to (57): ##STR00091## wherein R.sup.11, R.sup.12, R.sup.13, and R.sup.15 are selected from H, unsubstituted or hydroxyl-, amino-, carboxyl-, sulfoxyl- or halogen-substituted C.sub.1 to C.sub.4 alkyl, halogen, SO.sub.3R′, COOR′, CONR′.sub.2 with R′ selected from H and unsubstituted C.sub.1 to C.sub.4 alkyl; particularly wherein R.sup.11, R.sup.12, R.sup.13, and R.sup.15 are selected from H, methyl, CH.sub.2—SO.sub.3H, Cl and F, and R.sup.1 and R.sup.5 can have any of the meanings given herein; or f. R.sup.Y2 and/or R.sup.EG2 are independently selected from H, unsubstituted and alkyl- (particularly methyl-), amino-, hydroxy-, carboxy- and/or fluoro-substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 acyl, and C.sub.3-C.sub.6 cycloalkyl, and R.sup.Y1 together with R.sup.3, and/or R.sup.EG1 together with R.sup.4, form an cyclic structure according to any one of substructures (48) to (49) or (58) to (59): ##STR00092## wherein R.sup.1 and R.sup.5 can have any of the meanings given herein.
13. The compound according to claim 12, wherein R.sup.Y1 together with R.sup.Y2, and/or R.sup.EG1 together with R.sup.EG2 are —(CH.sub.2).sub.3—, —CH.sub.2CHFCH.sub.2—, —CH.sub.2CF.sub.2CH.sub.2—, —CH.sub.2CH(CH.sub.3)CH.sub.2—, —CH.sub.2C(CH.sub.3).sub.2CH.sub.2—, CH.sub.2CH(CN)CH.sub.2—, CH.sub.2CH(COOH)CH.sub.2—, CH.sub.2CH(CH.sub.2COOH)CH.sub.2—, —CH.sub.2CH(OCH.sub.3)CH.sub.2— and —CH.sub.2CH(N(CH.sub.3).sub.2)CH.sub.2—, particularly wherein the substituent is the same for R.sup.Y1 with R.sup.Y2, and R.sup.EG1 with R.sup.EG2.
14. The compound according to claim 12, wherein R.sup.Y1 together with R.sup.3, and R.sup.Y2 together with R.sup.2, and/or R.sup.EG1 together with R.sup.4, and R.sup.EG2 together with G, form a bi-cyclic structure according to any one of substructures (45) to (47) or (55) to (57), and Z is selected from CH.sub.2R.sup.F, CHR.sup.F.sub.2, and CR.sup.F.sub.3, with R.sup.F selected from CH.sub.2F, CHF.sub.2, CF.sub.3, CH.sub.2Cl, and CHCl.sub.2, CCl.sub.3, particularly with R.sup.F being CF.sub.3; H CH.sub.2COOR.sup.K1 or CH.sub.2CONHR.sup.K1 with R.sup.K1 selected from H, and unsubstituted or halogen-substituted C.sub.1 to C.sub.4 alkyl particularly wherein X is O.
15. The compound according to claim 1, wherein Y is NR.sup.Y1R.sup.Y2 and E is NR.sup.EG1R.sup.EG2, R.sup.Y1, R.sup.Y2, R.sup.EG1 and R.sup.EG2 are unsubstituted or halogen-substituted C.sub.1-C.sub.4 alkyl, particularly methyl, ethyl or CH.sub.2CF.sub.3; optionally, R.sup.2, R.sup.3,R.sup.4 and G can be halogen (particularly F or Cl); and Z is selected from CH.sub.2R.sup.F, CHR.sup.F.sub.2, and CR.sup.F.sub.3, with R.sup.F selected from CH.sub.2F, CHF.sub.2, CF.sub.3, CH.sub.2Cl, and CHCl.sub.2, CCl.sub.3, particularly with R.sup.F being CF.sub.3; H CH.sub.2COOR.sup.K1 with R.sup.K1 selected from H, and unsubstituted or halogen-substituted C.sub.1 to C.sub.4 alkyl; particularly wherein X is O.
16. The compound according to claim 1, wherein the compound is of the general formula (30) or (30′) ##STR00093## wherein R.sup.A, B, U and V have the meanings as defined in claim 1, Y is NR.sup.Y1R.sup.Y2, R.sup.Y1 and R.sup.Y2 are unsubstituted or halogen-substituted C.sub.1-C.sub.4 alkyl, particularly methyl, ethyl or CH.sub.2CF.sub.3; optionally, R.sup.2 and R.sup.3, can be halogen (particularly F or Cl); and Z is selected from CH.sub.2R.sup.F, CHR.sup.F.sub.2, and CR.sup.F.sub.3, with R.sup.F selected from CH.sub.2F, CHF.sub.2, CF.sub.3, CH.sub.2Cl, and CHCl.sub.2, CCl.sub.3, particularly with R.sup.F being CF.sub.3; H CH.sub.2COOR.sup.K1 with R.sup.K1 selected from H, and unsubstituted or halogen-substituted C.sub.1 to C.sub.4 alkyl; particularly wherein X is O.
17. The compound according to claim 1, wherein the compound is of the general formula (20) or (20′) ##STR00094## wherein Y is NR.sup.Y1R.sup.Y2 and E is NR.sup.EG1R.sup.EG2, wherein R.sup.Y1, R.sup.Y2, R.sup.EG1 and R.sup.EG2 are individually unsubstituted or amino-, hydroxyl- or halogen-substituted C.sub.1 to C.sub.4 alkyl or C.sub.3 to C.sub.6 cycloalkyl, or R.sup.Y1 together with R.sup.Y2, and R.sup.EG1 together with R.sup.EG2 together with the N form an unsubstituted or methyl-, ethyl-propyl-, or halogen-substituted aziridine, pyrrolidine, piperidine, piperazine or morpholine, and/or R.sup.1 and R.sup.5 are H, and/or R.sup.2, R.sup.3, R.sup.4 and G are independently selected from H, halogen, SO.sub.3H, and unsubstituted and amino-, hydroxy-, carboxy-, SO.sub.3H—, and/or halogen-substituted C.sub.1-C.sub.4 alkyl, CO.sub.2H, CO.sub.2R, SO.sub.2R with R being selected from C.sub.1 to C.sub.4 unsubstituted alkyl, and/or X is selected from O, CR.sup.X.sub.2, SiR.sup.X.sub.2, particularly wherein X is O, and/or each R.sup.X is independently selected from unsubstituted or halogen-substituted C.sub.1 to C.sub.4 alkyl or C.sub.3 to C.sub.6 cycloalkyl and phenyl, and/or U and V have the meaning defined in claim 9, and/or Z has the meaning as defined in claim 1, particularly the meaning as defined in claim 8, and wherein optionally, one of G, R.sup.2, R.sup.3, R.sup.4, or a phenyl linking U and V bears a moiety M as defined in claim 2, linked to the compound by a covalent bond or a linker L, with L as defined as in claim 3 or 4.
18. The compound according to claim 1, wherein the compound is of the general formula (30) or (30′) ##STR00095## wherein R.sup.A has the meaning as defined in claim 7 and B is H, and/or Y is NR.sup.Y1R.sup.Y2, wherein R.sup.Y1 and R.sup.Y2 are individually unsubstituted or amino-, hydroxyl- or halogen-substituted C.sub.1 to C.sub.4 alkyl or C.sub.3 to C.sub.6 cycloalkyl, or R together with R.sup.Y2, and R.sup.EG1 together with R.sup.EG2 together with the N form an unsubstituted or methyl-, ethyl- propyl-, or halogen-substituted aziridine, pyrrolidine, piperidine, piperazine or morpholine, and/or R.sup.1 is H, and/or R.sup.2 and R.sup.3 are independently selected from H, halogen, SO.sub.3H, and unsubstituted and amino-, hydroxy-, carboxy-, SO.sub.3H—, and/or halogen-substituted C.sub.1-C.sub.4 alkyl, CO.sub.2H, CO.sub.2R, SO.sub.2R with R being selected from C.sub.1 to C.sub.4 unsubstituted alkyl, and/or X is selected from O, CR.sup.X.sub.2, SiR.sup.X.sub.2, particularly wherein X is O, and/or each R.sup.X is independently selected from unsubstituted or halogen-substituted C.sub.1 to C.sub.4 alkyl or C.sub.3 to C.sub.6 cycloalkyl and phenyl, and/or U and V have the meaning defined in claim 10, and/or Z has the meaning as defined in claim 1, particularly the meaning as defined in claim 9, and wherein optionally, one of R.sup.2, R.sup.3, R.sup.A, or a phenyl linking U and V bears a moiety M as defined in claim 2, linked to the compound by a covalent bond or a linker L, with L as defined as in claim 3 or 4.
19. The compound according to claim 17, wherein R.sup.2, R.sup.3, R.sup.4, and G, are H, or one of R.sup.1, R.sup.2, R.sup.3, R.sup.4, G, and R.sup.5 is L.sup.A1.sub.n-L.sup.J1.sub.n′-L.sup.A2.sub.m-L.sup.J2.sub.m′-L.sup.A3.sub.pL.sup.J3.sub.p′-L.sup.A4.sub.q-L.sup.J4.sub.q′-M.sub.s, wherein L.sup.A1 . . . 4, L.sup.J1 . . . 4, n, n′ . . . q′, s and M have the definitions recited above each R.sup.X is C.sub.1 to C.sub.4 alkyl or phenyl, Y is NR.sup.Y1R.sup.Y2 and E is NR.sup.EG1R.sup.EG2; and R.sup.Y1, R.sup.Y2, R.sup.EG1 and R.sup.EG2 are individually unsubstituted or amino-, hydroxyl- or fluoro substituted C.sub.1 to C.sub.4 alkyl, or R.sup.Y1 together with R.sup.Y2, and R.sup.EG1 together with R.sup.EG2 form a ring and are —(CH.sub.2).sub.3—, —(CH.sub.2).sub.4—, —(CH.sub.2).sub.5—, —(CH.sub.2).sub.2O(CH.sub.2).sub.2— or —(CH.sub.2).sub.2NH(CH.sub.2).sub.2—.
20. The compound according to claim 1, wherein R.sup.2 and G are F or Cl.
21. (canceled)
22. A method to stain a sample, said method comprising the steps of: a. contacting the sample with a compound according to claim 1 or being characterized by one of the following combinations ##STR00096## with R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 and G are H, and Y and E are NEt.sub.2 and Z is CONH.sub.2 or Z is CONHC.sub.6H.sub.5; or Z is CN or Z is selected from CH.sub.2CF.sub.3, CH.sub.2CHF.sub.2, and CH.sub.2CH.sub.2F, or Z is selected from CH.sub.2COOCH.sub.3 and CH.sub.2COOH, Z is H, or Y and E are NMe.sub.2 and Z is selected from CH.sub.2COOCH.sub.3 and CH.sub.2COOH or Y and E are NH.sub.2 and Z is selected from CH.sub.2COOH and CH.sub.2COOCH.sub.3 or Y is OH and E is ═O (for formula 10) or E is OH (for formula 10′), and Z is C(═O)(CH.sub.3)C.sub.6H.sub.5; or R.sup.1, R.sup.3, R.sup.4, and R.sup.5 are H, R.sup.2 and G are methyl, Y and E are NHEt and Z is CH.sub.2COOH or CH.sub.2COOCH.sub.3 or the compound is of formula (301), (302), (303) or (304) ##STR00097## b. illuminating the sample with light of a wavelength ranging from 400-800 nm; c. recording the presence and location of said compound in said sample by illuminating the sample with light of an appropriate excitation wavelength and recording light emitted from said sample, particularly at an appropriate emission wavelength λ, more particularly a λ close to the maximum of the emission spectrum of 400-800 nm.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0220]
[0221]
[0222] RhB-CH3: compound 201 with Z=methyl; RhB-Ben: 201, Z=benzyl; RhB-CN: 201, Z=CN; RhB-SCH3 (115), RhB-SNH2 (116), RhB-SNMe2 (117), RhB: Rhodamine B, RhB-CONH2 (118), WS1-UREA (124), WS1-CN (121), WS1: compound 202 with N—Z=OH; WS1-SO3 (123), WS1-SNH2 (122), WS1-SCH3 (120), WS1-SCF3 (119), TMRCOOH (6-Carboxytetramethylrhodamine), TMRCNCOOH (132), TMRSCH3COOH (133), TMRSNH2COOH (134), TMRSNMe2COOH (135), CPYCOOH (6-Carboxycarbopyronine), CPYCNCOOH (136), CPYSCH3COOH (137), CPYSNH2COOH (138), CPYSNMe2COOH (139).
[0223]
[0224]
[0225]
[0226]
[0227]
[0228]
[0229]
[0230]
[0231]
[0232]
[0233]
[0234]
[0235]
[0236]
[0237]
[0238]
[0239]
[0240]
[0241]
[0242]
[0243]
[0244]
[0245]
[0246]
[0247]
[0248]
[0249]
[0250]
[0251]
[0252]
EXAMPLES
[0253]
[0254] The following compounds with moieties for Z were synthesized according to scaffold 201:
[0255] RhB-CH3 Z=—CH.sub.3
[0256] RhB-Ben Z=—C.sub.6H.sub.5
[0257] RhB-CN Z=—CN
[0258] RhB-SCH3 (115) Z=—SO.sub.2CH.sub.3
[0259] RhB-SNH2 (116) Z=—SO.sub.2NH.sub.2
[0260] RhB-SNMe2 (117) Z=—SO.sub.2N(CH.sub.3).sub.2
[0261] RhB-CONH2 (118) Z=—CONH.sub.2.
[0262] For compound 202 the following moieties for Z were synthesized:
[0263] WS1-SCF3 (119) Z=—SO.sub.2CF.sub.3
[0264] WS1-SCH3 (120) Z=—SO.sub.2CH.sub.3
[0265] WS1-CN (121) Z=—CN
[0266] WS1-SNH2 (122) Z=—SO.sub.2NH.sub.2
[0267] WS1-SO (123) Z=—SO.sub.3
[0268] WS1-UREA (124) Z=—CONH.sub.2
[0269] WS1-SCH2-C3-SNAP (125) Z=SO.sub.2CH.sub.2CH.sub.2CH.sub.2CO—R.sub.2, R.sub.2=1
[0270] WS1-SCH2-C3-HALO (126) Z=SO.sub.2CH.sub.2CH.sub.2CH.sub.2CO—R.sub.2, R.sub.2=2.
[0271] The following compounds with moieties for Z, X and R.sub.1-R.sub.4 were synthesized according to scaffold 203:
[0272] RhB-SNH2-SNAP (146) X=O, Z=SO.sub.2NH.sub.2, R.sub.1=R.sub.2=CH.sub.2CH.sub.3, R.sub.3=H, R.sub.4=1
[0273] RhB-SNH2-HALO (147) X=O, Z=SO.sub.2NH.sub.2, R.sub.1=R.sub.2=CH.sub.2CH.sub.3, R.sub.3=H, R.sub.4=2
[0274] RhB-SNMe2-SNAP (148) X=O, Z=SO.sub.2N(CH.sub.3).sub.2, R.sub.1=R.sub.2=CH.sub.2CH.sub.3, R.sub.3=H, R.sub.4=1
[0275] RhB-SNMe2-HALO (149) X=O, Z=SO.sub.2N(CH.sub.3).sub.2, R.sub.1=R.sub.2=CH.sub.2CH.sub.3, R.sub.3=H, R.sub.4=2
[0276] RhB-CONH2-HALO (175) X=O, Z=CONH.sub.2, R.sub.1=R.sub.2=CH.sub.2CH.sub.3, R.sub.3=H, R.sub.4=2
[0277] R110-SNMe2-SNAP (173) X=O, Z=SO.sub.2N(CH.sub.3).sub.2, R.sub.1=R.sub.2=H, R.sub.3=H, R.sub.4=1
[0278] R110-SNMe2-HALO (174) X=O, Z=SO.sub.2N(CH.sub.3).sub.2, R.sub.1=R.sub.2=H, R.sub.3=H, R.sub.4=2
[0279] TMR-CN-SNAP (152) X=O, Z=CN, R.sub.1=R.sub.2=CH.sub.3, R.sub.3=H, R.sub.4=1
[0280] TMR-CN-HALO (153) X=O, Z=CN, R.sub.1=R.sub.2=CH.sub.3, R.sub.3=H, R.sub.4=2
[0281] TMR-SCH3-SNAP (154) X=O, Z=SO.sub.2CH.sub.3, R.sub.1=R.sub.2=CH.sub.3, R.sub.3=H, R.sub.4=1
[0282] TMR-SCH3-HALO (155) X=O, Z=SO.sub.2CH.sub.3, R.sub.1=R.sub.2=CH.sub.3, R.sub.3=H, R.sub.4=2
[0283] TMR-SNH2-SNAP (156) X=O, Z=SO.sub.2NH.sub.2, R.sub.1=R.sub.2=CH.sub.3, R.sub.3=H, R.sub.4=1
[0284] TMR-SNH2-HALO (157) X=O, Z=SO.sub.2NH.sub.2, R.sub.1=R.sub.2=CH.sub.3, R.sub.3=H, R.sub.4=2
[0285] TMR-SNMe2-SNAP (158) X=O, Z=SO.sub.2N(CH.sub.3).sub.2, R.sub.1=R.sub.2=CH.sub.3, R.sub.3=H, R.sub.4=1
[0286] TMR-SNMe2-HALO (159) X=O, Z=SO.sub.2N(CH.sub.3).sub.2, R.sub.1=R.sub.2=CH.sub.3, R.sub.3=H, R.sub.4=2
[0287] TMR-SNMe2-Actin (169) X=O, Z=SO.sub.2N(CH.sub.3).sub.2, R.sub.1=R.sub.2=CH.sub.3, R.sub.3=H, R.sub.4=3
[0288] TMR-SNMe2-Tubulin (172) X=O, Z=SO.sub.2N(CH.sub.3).sub.2, R.sub.1=R.sub.2=CH.sub.3, R.sub.3=H, R.sub.4=4
[0289] CPY-CN-SNAP (160) X=C(CH.sub.3).sub.2, Z=CN, R.sub.1=R.sub.2=CH.sub.3, R.sub.3=H, R.sub.4=1
[0290] CPY-CN-HALO (161) X=C(CH.sub.3).sub.2, Z=CN, R.sub.1=R.sub.2=CH.sub.3, R.sub.3=H, R.sub.4=2
[0291] CPY-SCH3-SNAP (162) X=C(CH.sub.3).sub.2, Z=SO.sub.2CH.sub.3, R.sub.1=R.sub.2=CH.sub.3, R.sub.3=H, R.sub.4=1
[0292] CPY-SCH3-HALO (163) X=C(CH.sub.3).sub.2, Z=SO.sub.2CH.sub.3, R.sub.1=R.sub.2=CH.sub.3, R.sub.3=H, R.sub.4=2
[0293] CPY-SNH2-SNAP (164) X=C(CH.sub.3).sub.2, Z=SO.sub.2NH.sub.2, R.sub.1=R.sub.2=CH.sub.3, R.sub.3=H, R.sub.4=1
[0294] CPY-SNH2-HALO (165) X=C(CH.sub.3).sub.2, Z=SO.sub.2NH.sub.2, R.sub.1=R.sub.2=CH.sub.3, R.sub.3=H, R.sub.4=2
[0295] CPY-SNMe2-SNAP (166) X=C(CH.sub.3).sub.2, Z=SO.sub.2N(CH.sub.3).sub.2, R.sub.1=R.sub.2=CH.sub.3, R.sub.3=H, R.sub.4=1
[0296] CPY-SNMe2-HALO (167) X=C(CH.sub.3).sub.2, Z=SO.sub.2N(CH.sub.3).sub.2, R.sub.1=R.sub.2=CH.sub.3, R.sub.3=H, R.sub.4=2
[0297] CPY-CN-Actin (170) X=C(CH.sub.3).sub.2, Z=CN, R.sub.1=R.sub.2=CH.sub.3, R.sub.3=H, R.sub.4=3
[0298] CPY-SCH3-Actin (171) X=C(CH.sub.3).sub.2, Z=SO.sub.2CH.sub.3, R.sub.1=R.sub.2=CH.sub.3, R.sub.3=H, R.sub.4=3
[0299] SiR700-CN-HALO (168) X=Si(CH.sub.3).sub.2, Z=CN, R.sub.1=CH.sub.3, R.sub.2-R.sub.3=CH.sub.2—CH.sub.2, R.sub.4=2.
[0300] The following compounds with moieties for Z and R.sub.2— were synthesized according to scaffold 204:
[0301] WS1-CN-SNAP (176) Z=CN, R.sub.2=1
[0302] WS1-CN-HALO (177) Z=CN, R.sub.2=2
[0303] WS1-CN-TMP (178) Z=CN, R.sub.2=5
[0304] WS1-SCH3-HALO (179) Z=SO.sub.2CH.sub.3, R.sub.2=2
[0305] WS1-SCH3-TMP (180) Z=SO.sub.2CH.sub.3, R.sub.2=5
[0306] WS1-SNH2-HALO (181) Z=SO.sub.2NH.sub.2, R.sub.2=2
[0307] WS1-SNH2-TMP (182) Z=SO.sub.2NH.sub.2, R.sub.2=5
[0308] WS1-SNH2-TMP-C6 (183) Z=SO.sub.2NH.sub.2, R.sub.2=6
[0309] WS1-SNH2-TMP-C8 (184) Z=SO.sub.2NH.sub.2, R.sub.2=7.
[0310] The following compounds with moieties for R.sub.2 were synthesized according to scaffold 205:
[0311] WS1-SNH2-TMP-PEG2BG (191) n=2, R.sub.2=5
[0312] WS1-SNH2-TMP-PEG5BG (192) n=2, R.sub.2=5.
[0313] The following compounds with moieties for R.sub.2 were synthesized according to scaffold 206:
[0314] TMR-SNAP (150) R.sub.4=1
[0315] TMR-HALO (151) R.sub.4=2
with R.sub.4 depicted in
[0316] From
[0317]
[0318]
[0319]
[0320] The cell image of
Example 1: Designing Cell-Permeable and Fluorogenic Rhodamine Derivatives
[0321] The inventors' efforts to improve the permeability and fluorogenicity of rhodamine derivatives for live-cell imaging focused on increasing the propensity of rhodamines to form the spirocyclic, non-fluorescent form without affecting the spectroscopic properties of the parental molecule. The inventors initially considered replacing the carboxylic acid responsible for formation of the spirolactone with an amide. However, such rhodamine derivatives have been reported to strongly favor spirolactams at physiological pH, making them unsuitable for live-cell imaging. The inventors therefore attempted to destabilize the spirolactam by attaching different electron-withdrawing groups to the underlying amides. Specifically, the inventors converted 6-carboxytetramethylrhodamine (6-TAMRA), a widely used fluorophore with good photophysical properties, into the corresponding acyl cyanamide (132), acyl sulfonamide (133) and acyl sulfamide (134 and 135), (
Example 2: Cell-Permeable and Fluorogenic TAMRA Derivatives for No-Wash Live-Cell Imaging
[0322] Self-labeling protein tags such as SNAP-tag and HaloTag are an efficient approach to attach synthetic fluorophores to proteins in living cells. The main challenge when using these tags for live-cell imaging is the low cell permeability of most fluorescent probes used for labeling and high background signal resulting from their unspecific binding. SNAP-tag fusion proteins can be specifically labeled with different fluorophores using appropriate O.sup.6-benzlyguanine derivatives. A 6-TAMRA based probe (150) for SNAP-tag has been previously introduced for live-cell imaging, but its low cell-permeability and lack of fluorogenicity requires the use of high concentrations (5 μM) and repeated washing steps to remove excess and non-specifically bound probe. To address these problem, the inventors coupled fluorophores 132-135 to O.sup.6-benzylguanine to obtain probes 152, 154, 156, and 158. In vitro characterization of the spectroscopic properties of these probes in the presence and absence of SNAP-tag showed that in particular probe 158 containing the N,N-dimethylsulfamide group (in the following abbreviated as MaP555-SNAP) showed a significant increase in absorbance and fluorescence upon binding to SNAP-tag: the absorbance at 555 nm increases 15-fold and the intensity of fluorescence emission at 580 nm increases 21-fold. The inventors then examined the performance of probes 150, 152, 154, 156, and 158 in live-cell imaging, in which U2OS cells stably expressing a nuclear localized SNAP-Halo fusion protein were co-cultured with regular U2OS cells not expressing any protein tags. The performance of the probes in the in vitro tests correlated also with their performance in live-cell imaging: MaP555-SNAP even at a very low concentration (250 nM) allowed to perform wash-free labeling with excellent nuclei to cytosol signal ratio (F.sub.nuc/F.sub.cyt=15), while labeling with control probe 150 under identical conditions only lead to a barely detectable signal. Furthermore, the kinetics of labeling of nuclear localized SNAP-tag with MaP555-SNAP were much faster than those of control probe 6. The inventors then prepared the corresponding 6-TAMRA-based probes 151, 153, 155, 157, and 159 for HaloTag. As for SNAP-tag, the probe with the largest enhancement in absorbance (16-fold) and fluorescence intensity (35-fold) in in vitro assays was based on the N,N-dimethylsulfamide derivative 159 (abbreviated as MaP555-Halo in the following). In no-wash live-cell imaging experiments, MaP555-Halo furthermore showed much lower background signal than control probe 151, as most of MaP555-Halo exist as the non-fluorescent spirolactam prior to binding to HaloTag. Furthermore, the intracellular kinetics of labeling of nuclear HaloTag with MaP555-Halo were about two-fold faster than with control probe 151. The excellent performance of MaP555-SNAP and MaP555-Halo in live-cell imaging experiments support the inventors' assumption that establishing a dynamic equilibrium between the fluorescent zwitterion and the more hydrophobic spirolactam enables the generation of highly cell-permeable and fluorogenic probes for live-cell imaging.
[0323] Fluorescent probes for live-cell imaging of cytoskeletal structures have become important tools in the life sciences. In particular, the far-red, SiR-based probes for F-actin (SiR-actin) and microtubules (SiR-tubulin) have become popular as they are fluorogenic and enable no-wash imaging with little background signal. SiR-actin and SiR-tubulin are based on SiR linked to the F-actin-binder jasplakinolide and microtubule-binder docetaxel, respectively. Binding to their targets shifts for both probes the equilibrium between the cell-permeable spirolactone and the fluorescent zwitterion towards the fluorescent form. To generate fluorescent stains for F-actin and microtubule in different colors, the inventors coupled jasplakinolide and docetaxel to 6-TAMRA and its N,N-dimethylsulfamide derivative (probes 169 and 172, named MaP555-actin and MaP555-tubulin in the following). As expected, the 6-TAMRA derivatives of jasplakinolide and docetaxel did not allow to perform live-cell imaging of F-actin and microtubules in U2OS cells, presumably because these probes predominantly exist as zwitterions with low permeability. In contrast, MaP555-actin and MaP555-tubulin enabled no-wash, high-contrast staining of F-actin and microtubules in U2OS cells. The specificity of MaP555-actin and MaP555-tubulin was further confirmed by co-staining with SiR-actin and SiR-tubulin. The performance of both probes can be rationalized by their fluorogenicity: In in vitro assays, MaP555-actin showed a large increase in absorbance (26-fold) and fluorescence intensity (107-fold) upon binding to F-actin.
[0324] Similarly, binding of MaP555-tubulin to microtubules caused a high increase in absorbance (5.8-fold) and fluorescence (11-fold).
Example 3: Extension of the Strategy to Other Rhodamine Derivatives
[0325] Encouraged by the outstanding performance of these new TAMRA-based probes, the inventors extended their design strategy to other commonly used rhodamine derivatives with wavelengths ranging from cyan to near-infrared. Rhodamine 110 (R110) is a classic fluorophore emitting cyan fluorescence with good photostability and photophysical properties. However, its low D.sub.50 value of 15 and the presence of two polar NH.sub.2 groups results in low cell permeability of R110-based probes. Live-cell imaging of R110-based probes thus often requires high concentrations, long incubation times, and tedious washing steps. The introduction of N,N-dimethylsulfamide into R110 to yield fluorophore MaP510 shifted its D.sub.50 from 15 to 70. The corresponding probe for HaloTag, MaP510-Halo showed a large increase response in absorbance and fluorescence (14-fold and 11-fold, respectively) upon binding to HaloTag in vitro. In contrast, the regular R110-based HaloTag probe showed no significant fluorogenicity. In addition, MaP510-Halo even at a low concentration (250 nM) resulted in fast staining (<30 min) of nuclear localized HaloTag in U2OS cells with high signal to background ratio (18-fold), whereas the control compound showed a much lower signal to background ratio (2.5-fold) under these conditions.
[0326] The inventors next applied this strategy to carbopyronine, an orange fluorophore whose brightness and photostability make it an attractive choice for confocal and superresolution microscopy. Introduction of the cyanamide, sulfonamide and the two sulfamides into carbopyronine, allowed the inventors to generate HaloTag probes 161, 163, 165, and 167. All four probes showed large increases in fluorescence intensities upon binding to HaloTag, with values ranging from 100- to a 1000-fold. In contrast, the regular carbopyronine HaloTag probe only showed a 3.8-fold increase in fluorescence upon HaloTag binding. The observed 1000-fold increase in fluorescence intensity of probe 167 (in the following named MaP618-Halo) and its brightness sets this probe apart from other fluorogenic HaloTag substrates reported previously. The outstanding fluorogenicity of MaP618-Halo and the other carbopyronine-based probes 161, 163, 165, and 167 make them powerful probe for live-cell imaging: no-wash live-cell imaging of U2OS cells expressing nuclear localized HaloTag showed bright nuclei with extremely low unspecific extranuclear fluorescence (F.sub.nuc./F.sub.cyt.=58). In contrast, labeling with the regular carbopyronine probe resulted in significantly higher unspecific extranuclear fluorescence (F.sub.nuc./F.sub.cyt.=6.8). Furthermore, labeling with MaP618-Halo was very rapid and reached saturation within 5 min.
[0327] In addition, the inventors coupled the acyl sulfonamide of carbopyronine to jasplakinolide, yielding the actin probe MaP618-actin (171). MaP618-actin possesses orange fluorescence (λ.sub.ex/λ.sub.em: 618/635 nm) and its absorbance and fluorescence intensity increase 122-fold and 449-fold upon incubation with F-actin. MaP618-actin is thus about four times more fluorogenic than the previously described SiR-actin. Micrographs of live U2OS cells incubated with 500 nM of MaP618-actin for 1 h without any washing steps clearly reveal F-actin structures, which were also verified by colocalization with SiR-actin. Overall, these data clearly demonstrate the potential of MaP618-actin for live-cell imaging of F-actin.
[0328] Fluorophores with absorption and emission wavelength in the NIR windows are attractive choices for live-cell and in vivo imaging because of the reduced autofluorescence, deep tissue penetration and decreased phototoxicity at this wavelength. The inventors have previously described the NIR probe silicon-rhodamine 700 (SiR700). SiR700 possesses fluorogenic properties and SiR700-based probes have been successfully used for live-cell imaging of microtubule, F-actin and lysosomes in no-wash live-cell imaging. However, a SiR700-based probe for HaloTag showed only modest fluorogenicity and relatively high background signal in live-cell imaging. To solve this problem, the inventors incorporated the acyl cyanamide into SiR700, which exists predominantly as the spirolactam in aqueous solution, was then used to prepare a probe for HaloTag (MaP700-Halo, 168). Incubation of MaP700-Halo with HaloTag resulted in a dramatic increase in absorbance (150-fold) and fluorescence intensity (650-fold). Most importantly, U2OS cells incubated with 250 nM of MaP700-Halo and imaged without any washing steps displayed bright nuclear fluorescence and negligible background (F.sub.nuc./F.sub.cyt.=47). Furthermore, labeling of nuclear localized HaloTag in U2OS cells with MaP700-Halo (250 nM) was completed within 10 min. These features make MaP700-Halo an appealing probe for live-cell and in vivo imaging.
[0329] The experiments described above demonstrate how the introduction of acyl amides into rhodamine derivatives can be used to dramatically increase both their fluorogenicity and cell-permeability. The availability of different acyl amides that vary in their propensities for spirolactam formation facilitates for a given target the design of fluorescent probe that has the right balance between cell permeability, fluorogenicity and brightness.
Example 4: Applications in No-Wash, Multicolour Confocal and STED Microscopy
[0330] Mechanistic studies of most biological processes require the simultaneous imaging of multiple biomolecules and biochemical activities. Synthetic fluorescent probes for multi-colour, live-cell imaging need to be spectrally distinguishable, cell-permeable and ideally should be suitable for no-wash imaging. However, due to the paucity of suitable fluorescent probes that fulfil these requirements, very few no-wash, multicolour, live-cell imaging experiments have been reported so far. The cell-permeability and fluorogenicity of the here introduced MaP510, MaP555, MaP618 and MaP700 probes make them attractive candidates for no-wash, multicolour microscopy. In proof-of-principle experiments, U2OS cells stably expressing mitochondrial localized Cox8-Halo-SNAP were incubated with Hoechst (0.2 μg/mL), MaP555-tubulin (1 μM), MaP618-actin (500 nM), and MaP700-Halo (168, 250 nM) for 1.5 h and imaged directly without any washing steps. In addition, time-lapse movies enabled us to follow dynamic changes of the labelled structures. To underscore the potential of the inventors' MaP probes for multicolour imaging, various other combinations of fluorescent probes for no-wash, live-cell confocal imaging were successfully tested.
[0331] Stimulated Emission Depletion (STED) microscopy is a powerful tool to image biological structures in living cells on the nanoscale. As for conventional microscopy, the impact of live-cell STED nanoscopy is limited by the number of available fluorescent probes. The spectroscopic properties of the fluorophores on which the inventors' MaP probes are based are all compatible with STED nanoscopy and the inventors therefore investigated their performance in such experiments. The inventors first utilized MaP555-tubulin to image microtubules in live U2OS cells, using a 660 nm STED beam. In these experiments, U2OS cells were incubated with 1 μM of MaP555-tubulin for 1 h and subsequently imaged without any washing steps. The images showed both peripheral microtubules and the microtubules of the centrosomes. The apparent diameter of peripheral microtubules determined in these experiments was 39.5±10 nm, a value which is similar to the one obtained with SiR-tubulin. These images also provided a detailed view of the structure of the centriole, the structure around which the centrosome is assembled. Centrioles are cylindrical structures composed of nine triplets of microtubules, with the triplets forming the outer ring of the cylinder. The observed intensity maxima along the ring of the cylinder clearly revealed the nine-fold symmetry of the centriole. To the inventors' knowledge, it is the first time that the individual microtubule triplets are resolved in no-wash, live-cell imaging experiments. Similar to MaP555-tubulin, MaP555-actin also allowed to image the actin cytoskeleton in U2OS cells with nanoscale resolution.
[0332] The inventors previously used live-cell STED nanoscopy and SiR-actin to image the periodic arrangement of the actin subcortical cytoskeleton along the neurites of hippocampal neurons. To increase the signal-to-noise ratio in these experiments, excess probe needs to be removed through a washing step. Using MaP618-actin, the inventors were able to directly image this subcortical actin structure under no-wash conditions. The inventors attribute the increased signal-to-noise ratio of MaP618-actin relative to that of SiR-actin to its increased fluorogenicity.
[0333] Having a set of spectrally orthogonal and highly fluorogenic probes at hand, the inventors next tested their performance in no-wash, multicolour, live-cell STED nanoscopy. U2OS stably expressing vimentin-HaloTag were incubated with MaP510-Halo (green), MaP555-tubulin (red), and MaP618-actin (magenta) for 2 h and imaged directly. In these experiments, MaP555-tubulin and MaP618-actin were simultaneously imaged using a 775 nm depletion laser. Subsequently, MaP510-Halo was imaged using a 595 nm depletion laser. In this way, three-color, no-wash STED images at sub-diffraction resolution and without any cross-talk between the different channels were obtained.
[0334] Overall, these experiments clearly highlight the potential of the compounds shown herein for live-cell nanoscopy.
Description of Syntheses
[0335] ##STR00025## ##STR00026##
Step i.
[0336] The fluorophores were synthesized based on the reported method (Kvach et al. Bioconjug Chem 20, 1673-1682 (2009), Brem et al. The Journal of Physical Chemistry C 121, 15310-15317 (2017), Liu et al. Analyst 138, 2654-2660 (2013)). The fluorophores (1.0 eq) were dissolved in 3 mL DMF, and (2.0 eq) K.sub.2CO.sub.3 and 2.0 eq Et.sub.3N were added. Then allyl bromide (1.5 eq) was added slowly in the presence of ice bath. Then the mixture was stirred for 2 h at r.t. It was then diluted with water and extracted with CH.sub.2Cl.sub.2 (2×). The combined organics were washed with brine, dried (MgSO4), filtered, and concentrated in vacuo. The mixture was purified by flash chromatography on silica gel.
Step ii
[0337] Method 1 for NH.sub.2SONMe.sub.2, NH.sub.2SO.sub.2CH.sub.3, and NH.sub.2CN
[0338] The fluorophores (1.0 eq) were dissolved in dry 5 mL DCM. POCl.sub.3 (20 eq.) was added and refluxed for 2-3 h. DCM and POCl.sub.3 was removed by rotary evaporator and solid was obtained. Sulfonamide (10.0 eq.) and DIEPA (5.0 eq) were dissolved in dry 2-3 mL ACN. Then the mixture solution was poured into the solid with stirring. 4 mL more ACN was added and the reaction was stirred at r.t. The ACN was removed by rotary evaporator after 2 h. The mixture was washed by DCM and water. The combined organics were washed with brine, dried (MgSO.sub.4), filtered, and concentrated in vacuo.
Method 2 for NH.sub.2SO.sub.2NH.sub.2
[0339] The fluorophores (1.0 eq) and BOP (2.0 eq.), sulfonamide (10.0 eq.) and DIEPA (5 eq.) were dissolved in DMF/DCM (v/v=4/1). The mixture was stirred at r.t overnight. LCMS was used to check the reaction. The mixture was washed by DCM and water. The combined organics were washed with brine, dried (MgSO.sub.4), filtered, and concentrated in vacuo.
Step iii
[0340] The fluorophores (1.0 eq), 1,3-dimethylbarbituric acid (5.0 eq.) and tetrakis(triphenyl-phosphine)palladium(0) (5.0 eq) were dissolved in methanol/DCM (5/1) and stirred at r.t for 1 h. The mixture was purified by pre-HPLC.
Step iv
[0341] The fluorophores (1.0 eq) and BOP (1.2 eq), and DIEPA (2 eq) were dissolved in 2 mL DMF. O.sup.6-Benzylguanine or HaloTag(O2)amine (1.2 eq.) were added and stirred at r.t for 20 min.
##STR00027##
Step ii
[0342] Method 1 for NH.sub.2SONMe.sub.2, NH.sub.2SO.sub.2CH.sub.3, and NH.sub.2CN
[0343] The fluorophores (1.0 eq) were dissolved in dry 5 mL DCM. POCl.sub.3 (20 eq.) was added and refluxed for 2-3 h. DCM and POCl.sub.3 was removed by rotary evaporator and solid was obtained. Sulfonamide (10.0 eq.) and DIEPA (5.0 eq) were dissolved in dry 2-3 mL ACN. Then the mixture solution was poured into the solid with stirring. 4 mL more ACN was added and the reaction was stirred at r.t. The ACN was removed by rotary evaporator after 2 h. The mixture was washed by DCM and water. The combined organics were washed with brine, dried (MgSO.sub.4), filtered, and concentrated in vacuo. The mixture was purified by Pre-HPLC.
Method 2 for NH.sub.2SO.sub.2NH.sub.2.
[0344] The fluorophores (1.0 eq) and BOP (2.0 eq.), sulfonamide (10.0 eq.) and DIEPA (5 eq.) were dissolved in DMF/DCM (v/v=4/1). The mixture was stirred at r.t overnight. LCMS was used to check the reaction. The mixture was washed by DCM and water. The combined organics were washed with brine, dried (MgSO.sub.4), filtered, and concentrated in vacuo. The mixture was purified by Pre-HPLC.
##STR00028##
[0345] .sup.1H NMR (400 MHz, CD.sub.3OD/CDCl.sub.3) δ 8.13 (d, J=7.6 Hz, 1H), 7.75 (dt, J=23.7, 7.2 Hz, 2H), 7.26 (d, J=7.6 Hz, 1H), 6.80 (d, J=8.8 Hz, 2H), 6.62 (d, J=2.5 Hz, 2H), 6.57 (dd, J=9.2, 2.6 Hz, 2H), 3.54 (q, J=7.1 Hz, 8H), 3.01 (s, 3H), 1.33 (t, J=7.1 Hz, 12H). .sup.13C NMR (101 MHz, CD.sub.3OD/CDCl.sub.3) δ 168.60, 154.14, 150.22, 149.76, 134.44, 129.67, 129.14, 128.76, 125.50, 124.66, 108.72, 106.83, 97.45, 44.45, 40.97, 12.05. HRMS (ESI): m/z calc. for C.sub.29H.sub.33N.sub.3O.sub.4S 520.2265; found 520.2271, [M+H].sup.+
##STR00029##
[0346] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.99 (dd, J=7.5, 1.4 Hz, 1H), 7.89-7.74 (m, 2H), 7.45 (d, J=7.4 Hz, 1H), 7.17 (d, J=9.4 Hz, 2H), 7.02 (dd, J=9.3, 2.4 Hz, 2H), 6.97 (d, J=2.4 Hz, 2H), 3.68 (q, J=7.1 Hz, 8H), 1.30 (t, J=7.1 Hz, 12H). .sup.13C NMR (101 MHz, CD.sub.3OD_SPE) δ 167.56, 158.79, 134.61, 133.54, 132.31, 131.33, 129.22, 122.23, 119.33, 116.44, 115.20, 114.85, 113.54, 97.94, 47.09, 12.70. HRMS (ESI): m/z calc. for C.sub.28H.sub.32N.sub.4O.sub.4S 521.2217; found 521.2215, [M+H].sup.+
##STR00030##
[0347] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.91 (d, J=7.6 Hz, 1H), 7.52 (dt, J=24.2, 7.4 Hz, 2H), 7.11 (d, J=7.6 Hz, 1H), 6.53 (d, J=8.8 Hz, 2H), 6.41 (s, 2H), 6.27 (d, J=9.1 Hz, 2H), 3.33 (q, J=7.1 Hz, 8H), 2.72 (s, 6H), 1.15 (t, J=7.0 Hz, 13H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 167.39, 153.93, 153.63, 148.97, 134.52, 128.74, 128.60, 128.32, 124.85, 123.43, 107.36, 106.25, 97.81, 69.19, 44.32, 37.87, 12.62. HRMS (ESI): m/z calc. for C.sub.30H.sub.36N.sub.4O.sub.4S 549.2530; found 549.2536, [M+H].sup.+
##STR00031##
##STR00032## ##STR00033##
[0348] Yield: 90%. .sup.1H NMR (400 MHz, Chloroform-d) δ 8.33-8.19 (m, 1H), 8.09 (dd, J=7.9, 4.0 Hz, 1H), 7.83 (s, 1H), 6.62 (dd, J=8.8, 4.0 Hz, 2H), 6.50 (d, J=3.1 Hz, 2H), 6.44-6.33 (m, 2H), 6.04-5.84 (m, 1H), 5.35 (d, J=17.1 Hz, 1H), 5.26 (d, J=10.5 Hz, 1H), 4.76 (d, J=5.6 Hz, 2H), 2.98 (s, 12H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 168.88, 165.05, 153.36, 152.58, 135.70, 132.05, 131.73, 130.74, 128.97, 125.91, 125.45, 119.29, 109.16, 106.72, 98.45, 66.44, 40.33. HRMS (ESI): m/z calc. for C.sub.28H.sub.26N.sub.2O.sub.5 471.1914; found 471.1916, [M+H].sup.+
##STR00034##
[0349] .sup.1H NMR (400 MHz, Chloroform-d) δ 8.37-8.13 (m, 2H), 7.78 (s, 1H), 7.12 (d, J=2.5 Hz, 2H), 6.81 (d, J=9.0 Hz, 2H), 6.67 (dd, J=9.2, 2.4 Hz, 2H), 5.99 (ddt, J=16.6, 11.3, 5.9 Hz, 1H), 5.44-5.32 (m, 1H), 5.29 (d, J=10.4 Hz, 1H), 4.80 (d, J=5.8 Hz, 2H), 3.15 (s, 12H), 1.84 (s, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 167.37, 164.89, 153.21, 152.95, 134.09, 133.84, 131.61, 130.40, 129.03, 128.34, 121.47, 119.19, 113.10, 111.13, 66.40, 41.26, 40.63, 35.43, 32.28. HRMS (ESI): m/z calc. for C.sub.31H.sub.32N.sub.2O.sub.4 497.2435; found 497.2441, [M+H].sup.+
##STR00035##
[0350] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.16 (q, J=8.2 Hz, 2H), 7.51 (s, 1H), 6.71 (d, J=9.3 Hz, 2H), 6.52 (d, J=7.0 Hz, 4H), 2.96 (s, 12H). .sup.13C NMR (101 MHz, DMSO-d.sub.6) δ 165.80, 165.46, 152.25, 151.99, 137.65, 130.56, 129.11, 128.30, 125.20, 124.84, 118.08, 109.58, 106.91, 102.95, 98.06, 54.91, 48.60. HRMS (ESI): m/z calc. for C.sub.26H.sub.22N.sub.4O.sub.4 455.1714; found 455.1714, [M+H].sup.+
##STR00036##
[0351] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.41 (d, J=8.1 Hz, 1H), 8.09 (d, J=8.1 Hz, 1H), 8.04 (s, 1H), 7.16 (d, J=9.4 Hz, 2H), 7.04 (dd, J=9.5, 2.3 Hz, 2H), 6.96 (d, J=2.4 Hz, 2H), 3.30 (s, 12H), 2.96 (d, J=1.3 Hz, 3H). .sup.13C NMR (101 MHz, DMSO) δ 166.39, 166.17, 154.02, 152.50, 151.72, 137.52, 130.84, 130.47, 128.50, 125.13, 124.92, 109.05, 106.21, 98.73, 68.62, 55.38, 49.06, 42.44. HRMS (ESI): m/z calc. for C.sub.26H.sub.25N.sub.3O.sub.6S 508, 1537; found 508.1540, [M+H].sup.+
##STR00037##
[0352] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.09 (dd, J=8.0, 1.4 Hz, 1H), 8.02 (d, J=8.0 Hz, 1H), 7.70 (s, 2H), 7.34 (s, 1H), 6.53 (d, J=8.6 Hz, 2H), 6.41 (d, J=8.0 Hz, 4H), 2.92 (s, 12H). .sup.13C NMR (101 MHz, DMSO) δ 166.51, 165.74, 154.38, 152.34, 151.47, 136.94, 131.18, 130.23, 128.42, 125.00, 124.35, 109.08, 106.80, 98.79, 67.96, 40.28. HRMS (ESI): m/z calc. for C.sub.25H.sub.24N.sub.4O.sub.6S 509, 1489; found 509, 1489, [M+H].sup.+
##STR00038##
[0353] 1H NMR (400 MHz, DMSO-d6) δ 8.12 (d, J=8.1 Hz, 1H), 8.04 (d, J=8.0 Hz, 1H), 7.38 (s, 1H), 6.56 (d, J=8.6 Hz, 2H), 6.42 (d, J=11.3 Hz, 4H), 2.93 (s, 12H). .sup.13C NMR (101 MHz, DMSO) δ 166.04, 165.61, 153.71, 152.45, 151.12, 136.70, 130.75, 129.89, 128.58, 124.92, 124.10, 108.32, 106.21, 98.24, 68.10, 54.92, 37.50. HRMS (ESI): m/z calc. for C.sub.27H.sub.28N.sub.4O.sub.6S 537, 1802; found 537.1802, [M+H].sup.+
##STR00039##
[0354] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.14 (s, 2H), 7.32 (s, 1H), 6.92 (s, 2H), 6.63 (d, J=9.0 Hz, 2H), 6.58 (d, J=8.7 Hz, 2H), 2.97 (s, 12H), 1.84 (s, 3H), 1.79 (s, 3H). .sup.13C NMR (101 MHz, DMSO) δ 166.32, 165.79, 154.40, 150.55, 146.38, 137.66, 130.08, 128.49, 128.05, 125.13, 124.40, 114.82, 112.67, 109.45, 106.83, 71.15, 37.66, 35.36, 34.65. HRMS (ESI): m/z calc. for C.sub.29H.sub.28N.sub.4O.sub.3 481.2234; found 481.2236, [M+H].sup.+
##STR00040##
[0355] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.13-7.97 (m, 2H), 7.12 (s, 1H), 7.00 (s, 2H), 6.77-6.63 (m, 4H), 3.12 (d, J=1.7 Hz, 3H), 2.97 (d, J=1.8 Hz, 12H), 1.83 (s, 3H), 1.76 (s, 3H). .sup.13C NMR (101 MHz, DMSO) δ 166.69, 165.85, 155.43, 149.06, 144.85, 136.94, 129.43, 129.33, 127.38, 124.73, 123.95, 119.31, 112.91, 111.01, 71.05, 42.24, 40.63, 37.62, 35.82, 32.67. HRMS (ESI): m/z calc. for C.sub.29H.sub.31N.sub.3O.sub.5S 534.2057; found 534.2059, [M+H].sup.+
##STR00041##
[0356] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.00 (s, 2H), 7.08 (s, 1H), 7.01 (s, 2H), 6.67 (d, J=9.1 Hz, 2H), 6.62 (d, J=8.6 Hz, 2H), 2.97 (s, 12H), 1.85 (s, 3H), 1.74 (s, 3H). .sup.13C NMR (101 MHz, DMSO) δ 166.60, 166.43, 155.85, 149.17, 145.02, 136.70, 130.30, 129.66, 127.71, 124.69, 124.40, 113.66, 111.65, 71.24, 41.25, 38.02, 36.00, 33.69. HRMS (ESI): m/z calc. for C.sub.28H.sub.30N.sub.4O.sub.5S 535.2010; found 536.2041, [M+H].sup.+
##STR00042##
[0357] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.02 (s, 2H), 7.10 (s, 1H), 6.97 (s, 2H), 6.63 (s, 4H), 2.96 (d, J=1.6 Hz, 12H), 2.71 (d, J=1.7 Hz, 6H), 1.81 (s, 3H), 1.78 (s, 3H). .sup.13C NMR (101 MHz, DMSO) δ 166.71, 165.97, 155.76, 149.07, 145.28, 136.52, 129.74, 129.26, 128.22, 124.34, 124.25, 112.41, 110.64, 70.98, 40.58, 37.59, 35.97, 32.73. HRMS (ESI): m/z calc. for C.sub.30H.sub.34N.sub.4O.sub.5S 563.2323; found 563.2324, [M+H].sup.+
##STR00043##
[0358] 1H NMR (400 MHz, DMSO-d6) δ 8.16-8.04 (m, 2H), 7.38 (s, 1H), 6.79 (d, J=2.3 Hz, 2H), 6.58 (s, 2H), 3.25 (dt, J=26.5, 8.6 Hz, 4H), 2.88-2.66 (m, 1 OH), 0.57 (d, J=2.3 Hz, 3H), 0.53 (d, J=2.3 Hz, 3H). .sup.13C NMR (101 MHz, DMSO) δ 166.90, 165.74, 155.11, 152.58, 137.54, 134.03, 133.52, 129.79, 128.99, 127.19, 125.67, 123.97, 122.72, 109.66, 106.89, 75.11, 54.65, 35.00, 27.95, 0.30, −0.23. HRMS (ESI): m/z calc. for C.sub.30H.sub.28N.sub.4O.sub.3Si 521.2003; found 521.2002, [M+H].sup.+
##STR00044##
[0359] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.11 (d, J=8.1 Hz, 1H), 8.00 (d, J=8.0 Hz, 1H), 7.41 (s, 1H), 6.48-6.31 (m, 4H), 6.25 (d, J=8.5 Hz, 2H), 2.62 (s, 6H).
##STR00045## ##STR00046## ##STR00047## ##STR00048##
[0360] 1H NMR (400 MHz, DMSO-d6) δ 8.50 (s, 1H), 8.20-8.09 (m, 2H), 7.61 (s, 1H), 7.46 (d, J=7.8 Hz, 2H), 7.29 (d, J=7.8 Hz, 2H), 6.68 (d, J=8.6 Hz, 2H), 6.51 (d, J=8.0 Hz, 4H), 5.50 (s, 2H), 4.40 (d, J=5.9 Hz, 2H), 2.95 (s, 12H). HRMS (ESI): m/z calc. for C.sub.39H.sub.34N.sub.10O.sub.4 354, 1455; found 354, 1457, [M+2H].sup.2+
##STR00049##
[0361] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.12 (d, J=1.1 Hz, 2H), 7.68 (d, J=1.2 Hz, 1H), 6.87 (d, J=9.1 Hz, 2H), 6.77 (dd, J=9.1, 2.6 Hz, 2H), 6.73 (d, J=2.5 Hz, 2H), 3.61-3.55 (m, 4H), 3.51 (dt, J=5.6, 2.0 Hz, 6H), 3.39 (t, J=6.5 Hz, 2H), 3.13 (s, 12H), 1.73-1.66 (m, 2H), 1.50-1.44 (m, 2H), 1.41-1.35 (m, 2H), 1.32-1.25 (m, 2H). HRMS (ESI): m/z calc. for C.sub.36H.sub.42ClN.sub.5O.sub.5 660, 2947; found 660.2954, [M+H].sup.+
##STR00050##
[0362] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.29 (s, 1H), 8.17 (dd, J=8.2, 1.7 Hz, 1H), 8.05 (d, J=8.2 Hz, 1H), 7.79 (d, J=1.7 Hz, 1H), 7.48 (d, J=7.8 Hz, 2H), 7.36 (d, J=7.9 Hz, 2H), 6.99 (d, J=9.2 Hz, 2H), 6.84 (dd, J=9.2, 2.6 Hz, 2H), 6.80 (d, J=2.5 Hz, 2H), 5.60 (s, 2H), 4.55 (s, 2H), 3.19 (s, 12H), 2.93 (s, 3H). HRMS (ESI): m/z calc. for C.sub.39H.sub.37N.sub.9O.sub.6S 380.6367; found 380.6369, [M+2H].sup.2+
##STR00051##
[0363] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.18 (d, J=8.3 Hz, 1H), 8.08 (d, J=8.1 Hz, 1H), 7.76 (s, 1H), 6.99 (d, J=9.3 Hz, 2H), 6.84 (d, J=9.2 Hz, 2H), 6.79 (s, 2H), 3.62 (dd, J=11.4, 5.2 Hz, 4H), 3.54 (dd, J=11.2, 5.6 Hz, 6H), 3.41 (d, J=6.7 Hz, 2H), 2.95 (s, 3H), 1.72 (p, J=7.0 Hz, 2H), 1.51 (p, J=7.0 Hz, 2H), 1.40 (q, J=7.2, 6.7 Hz, 2H), 1.32 (p, J=7.7 Hz, 2H). HRMS (ESI): m/z calc. for C.sub.36H.sub.44ClN.sub.5O.sub.7S 713.2770; found 713.2768, [M+H].sup.+
##STR00052##
[0364] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.29 (d, J=1.6 Hz, 1H), 8.18 (d, J=8.3 Hz, 1H), 8.02 (dd, J=8.1, 1.7 Hz, 1H), 7.80 (s, 1H), 7.52-7.46 (m, 2H), 7.37 (d, J=7.8 Hz, 2H), 7.04 (d, J=9.3 Hz, 2H), 6.89 (d, J=9.4 Hz, 2H), 6.84 (s, 2H), 5.60 (s, 2H), 4.56 (s, 2H), 3.22 (s, 12H). HRMS (ESI): m/z calc. for C.sub.38H.sub.36N.sub.10O.sub.6S 381.1343; found 381.1342, [M+2H].sup.2+
##STR00053##
[0365] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.17 (d, J=8.1 Hz, 1H), 8.03 (dd, J=8.2, 1.7 Hz, 1H), 7.77 (s, 1H), 7.03 (s, 2H), 6.86 (d, J=23.2 Hz, 4H), 3.67-3.58 (m, 4H), 3.57-3.49 (m, 6H), 3.41 (td, J=6.6, 1.6 Hz, 2H), 3.21 (s, 12H), 1.76-1.65 (m, 2H), 1.49 (p, J=7.2 Hz, 2H), 1.39 (h, J=6.7, 6.1 Hz, 2H), 1.32 (q, J=7.6 Hz, 2H). HRMS (ESI): m/z calc. for C.sub.35H.sub.44ClN.sub.5O.sub.7S 714.2723; found 714.2724, [M+H].sup.+
##STR00054##
[0366] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.53 (s, 1H), 8.11 (d, J=8.1 Hz, 1H), 8.01 (d, J=7.9 Hz, 1H), 7.46 (d, J=6.9 Hz, 3H), 7.28 (d, J=7.8 Hz, 2H), 6.52 (d, J=8.7 Hz, 2H), 6.39 (d, J=8.1 Hz, 4H), 5.50 (s, 2H), 4.38 (d, J=5.7 Hz, 2H), 2.91 (s, 12H), 2.62 (s, 6H). HRMS (ESI): m/z calc. for C.sub.40H.sub.40N.sub.10O.sub.6S 395.1499; found 395.1499, [M+2H].sup.2+
##STR00055##
[0367] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.06 (d, J=8.0 Hz, 1H), 7.98 (dd, J=8.2, 1.7 Hz, 1H), 7.44 (s, 1H), 6.52 (dd, J=8.6, 1.6 Hz, 2H), 6.39 (dd, J=9.3, 1.9 Hz, 4H), 3.59 (td, J=6.6, 1.7 Hz, 2H), 3.43-3.37 (m, 6H), 3.33-3.25 (m, 4H), 2.92 (d, J=1.6 Hz, 12H), 2.62 (d, J=1.6 Hz, 6H), 1.71-1.60 (m, 2H), 1.40 (p, J=7.2 Hz, 2H), 1.36-1.28 (m, 2H), 1.24 (q, J=7.9 Hz, 2H). HRMS (ESI): m/z calc. for C.sub.37H.sub.48ClN.sub.5O.sub.7S 742.3036; found 742.3031, [M+H].sup.+
##STR00056##
[0368] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.35 (d, J=1.8 Hz, 1H), 8.11 (dd, J=8.0, 1.7 Hz, 1H), 8.07 (d, J=8.1 Hz, 1H), 7.46 (dd, J=8.2, 1.8 Hz, 2H), 7.41 (s, 1H), 7.34-7.28 (m, 2H), 7.11 (d, J=2.3 Hz, 2H), 6.82-6.75 (m, 2H), 6.65 (dd, J=8.9, 1.7 Hz, 2H), 5.61 (d, J=1.8 Hz, 2H), 4.48 (d, J=4.8 Hz, 2H), 3.04 (d, J=1.7 Hz, 12H), 1.90 (d, J=1.8 Hz, 3H), 1.85 (d, J=1.8 Hz, 3H). HRMS (ESI): m/z calc. for C.sub.42H.sub.40N.sub.10O.sub.3 367.1715; found 367.1717, [M+2H].sup.2+
##STR00057##
[0369] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.11 (d, J=7.7 Hz, 1H), 8.06 (d, J=8.0 Hz, 1H), 7.38 (s, 1H), 7.17 (d, J=2.7 Hz, 2H), 6.84 (dt, J=9.1, 2.1 Hz, 2H), 6.68 (dd, J=8.8, 1.7 Hz, 2H), 3.54 (dd, J=5.8, 2.3 Hz, 4H), 3.51-3.43 (m, 6H), 3.40-3.35 (m, 2H), 3.06 (d, J=1.6 Hz, 12H), 1.92 (d, J=1.7 Hz, 3H), 1.86 (s, 3H), 1.69 (p, J=6.9 Hz, 2H), 1.47 (p, J=7.0 Hz, 2H), 1.41-1.34 (m, 2H), 1.30 (q, J=6.7, 4.8 Hz, 2H). HRMS (ESI): m/z calc. for C.sub.39H.sub.48ClN.sub.5O.sub.4 686.3468; found 686.3471, [M+H].sup.+
##STR00058##
[0370] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.32 (d, J=1.7 Hz, 1H), 8.11-8.03 (m, 1H), 7.96 (d, J=7.9 Hz, 1H), 7.51-7.42 (m, 2H), 7.29 (d, J=7.4 Hz, 2H), 7.22 (t, J=2.4 Hz, 3H), 6.88-6.82 (m, 2H), 6.80 (dd, J=8.8, 1.7 Hz, 2H), 5.60 (s, 2H), 4.46 (d, J=4.7 Hz, 2H), 3.35 (d, J=1.7 Hz, 3H), 3.07 (d, J=1.7 Hz, 12H), 1.89 (s, 3H), 1.84 (d, J=1.7 Hz, 3H). HRMS (ESI): m/z calc. for C.sub.42H.sub.43N.sub.9O.sub.5S 393.6627; found 393.6628, [M+2H].sup.2+
##STR00059##
[0371] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.07 (d, J=8.0 Hz, 1H), 7.97 (d, J=8.0 Hz, 1H), 7.35 (s, 2H), 7.18 (s, 1H), 6.95 (d, J=8.8 Hz, 2H), 6.87 (d, J=9.1 Hz, 2H), 3.54 (d, J=5.4 Hz, 4H), 3.50 (d, J=5.3 Hz, 4H), 3.44 (t, J=5.1 Hz, 2H), 3.39 (t, J=6.6 Hz, 2H), 3.13 (d, J=4.5 Hz, 12H), 3.08 (s, 3H), 1.93 (s, 3H), 1.85 (s, 3H), 1.70 (p, J=7.2 Hz, 2H), 1.48 (h, J=6.5, 6.1 Hz, 2H), 1.39 (h, J=6.7, 6.3 Hz, 2H), 1.30 (td, J=8.3, 7.9, 3.9 Hz, 2H). HRMS (ESI): m/z calc. for C.sub.39H.sub.51ClN.sub.4O.sub.6S 739.3291; found 739.3291, [M+H].sup.+
##STR00060##
[0372] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.30 (d, J=1.6 Hz, 1H), 8.04 (d, J=8.1 Hz, 1H), 7.93 (d, J=8.1 Hz, 1H), 7.48-7.42 (m, 2H), 7.28 (d, J=6.8 Hz, 4H), 7.16 (s, 1H), 6.89 (d, J=8.6 Hz, 2H), 6.80 (d, J=9.0 Hz, 2H), 5.59 (s, 2H), 4.45 (d, J=4.8 Hz, 2H), 3.08 (d, J=1.7 Hz, 12H), 1.91 (s, 3H), 1.83 (s, 3H). HRMS (ESI): m/z calc. for C.sub.41H.sub.42N.sub.10O.sub.5S 787.3133; found 787.3133, [M+H].sup.+
##STR00061##
[0373] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.04 (dd, J=8.1, 1.7 Hz, 1H), 7.93 (d, J=8.1 Hz, 1H), 7.31 (s, 2H), 7.14 (s, 1H), 6.91 (d, J=8.9 Hz, 2H), 6.83 (d, J=8.7 Hz, 2H), 3.58-3.53 (m, 4H), 3.53-3.48 (m, 4H), 3.45 (q, J=5.8 Hz, 2H), 3.40 (dd, J=5.9, 1.5 Hz, 2H), 3.10 (s, 12H), 1.95 (d, J=1.7 Hz, 3H), 1.85 (d, J=1.9 Hz, 3H), 1.70 (p, J=6.5 Hz, 2H), 1.49 (p, J=6.6 Hz, 2H), 1.40 (h, J=7.2, 6.1 Hz, 2H), 1.33 (d, J=8.1 Hz, 2H). HRMS (ESI): m/z calc. for C.sub.38H.sub.50ClN.sub.5O.sub.6S 740.3243; found 740.3246, [M+H].sup.+
##STR00062##
[0374] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.97 (d, J=8.1 Hz, 1H), 7.91 (d, J=8.1 Hz, 1H), 7.87 (d, J=1.6 Hz, 1H), 7.41 (d, J=7.3 Hz, 2H), 7.24 (d, J=7.5 Hz, 2H), 7.17 (s, 1H), 6.91 (d, J=2.4 Hz, 2H), 6.64-6.55 (m, 4H), 5.49 (s, 2H), 4.43 (d, J=4.1 Hz, 2H), 2.93 (d, J=1.8 Hz, 12H), 2.73 (d, J=1.7 Hz, 6H), 1.83 (s, 3H), 1.82 (s, 3H). HRMS (ESI): m/z calc. for C.sub.43H.sub.46N.sub.10O.sub.5S 408.1759; found 408.1762, [M+2H].sup.2+
##STR00063##
[0375] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.03 (d, J=8.2 Hz, 1H), 7.96 (d, J=8.1 Hz, 1H), 7.42 (s, 2H), 7.15 (s, 1H), 6.99 (d, J=8.9 Hz, 2H), 6.86 (d, J=8.8 Hz, 2H), 3.54 (d, J=5.0 Hz, 4H), 3.52-3.48 (m, 4H), 3.45 (d, J=5.6 Hz, 2H), 3.41-3.37 (m, 2H), 3.14 (s, 12H), 2.75 (s, 6H), 1.92 (s, 3H), 1.89 (s, 3H), 1.70 (p, J=6.9 Hz, 2H), 1.49 (p, J=6.8 Hz, 2H), 1.40 (h, J=6.7, 6.0 Hz, 2H), 1.34-1.28 (m, 2H). HRMS (ESI): m/z calc. for C.sub.49H.sub.54ClN.sub.5O.sub.6S 768.3556; found 768.3557, [M+H].sup.+
##STR00064##
[0376] .sup.1H NMR (400 MHz, Chloroform-d) δ 8.05 (dd, J=8.0, 2.2 Hz, 1H), 7.88 (dd, J=8.1, 2.0 Hz, 1H), 7.36 (s, 1H), 6.59 (d, J=2.3 Hz, 2H), 6.48 (s, 2H), 3.60 (dt, J=11.6, 4.0 Hz, 6H), 3.54 (dt, J=6.9, 3.5 Hz, 4H), 3.40 (dt, J=8.7, 4.4 Hz, 2H), 3.34 (t, J=8.5 Hz, 4H), 2.95-2.70 (m, 10H), 1.77 (q, J=7.4 Hz, 2H), 1.54 (p, J=6.8, 6.3 Hz, 2H), 1.44 (p, J=7.8 Hz, 2H), 1.38-1.31 (m, 2H), 0.60 (d, J=2.2 Hz, 3H), 0.57 (d, J=2.3 Hz, 3H). HRMS (ESI): m/z calc. for C.sub.40H.sub.46ClN.sub.5O.sub.4Si 726.3237; found 726.3238, [M+H].sup.+
##STR00065##
[0377] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.34 (d, J=8.6 Hz, 1H), 8.18 (d, J=8.2 Hz, 1H), 8.01-7.96 (m, 1H), 7.86 (s, 1H), 7.55 (d, J=7.8 Hz, 1H), 7.26 (d, J=8.0 Hz, 1H), 7.17-6.80 (m, 11H), 6.74-6.67 (m, 2H), 5.58 (t, J=7.9 Hz, 1H), 5.20 (d, J=10.7 Hz, 1H), 5.00 (s, 1H), 4.73 (s, 1H), 3.40 (t, J=7.0 Hz, 2H), 3.24 (s, 12H), 3.13 (d, J=8.1 Hz, 2H), 3.06 (d, J=2.0 Hz, 3H), 2.97 (dt, J=14.0, 7.1 Hz, 1H), 2.88 (dt, J=13.4, 6.9 Hz, 1H), 2.68 (d, J=5.1 Hz, 1H), 2.58 (d, J=2.0 Hz, 6H), 2.25 (t, J=13.1 Hz, 1H), 2.16 (t, J=7.6 Hz, 2H), 1.86 (q, J=5.8 Hz, 3H), 1.62 (q, J=7.0, 6.1 Hz, 4H), 1.59-1.53 (m, 1H), 1.50 (s, 3H), 1.39 (q, J=8.3, 6.9 Hz, 3H), 1.30 (d, J=14.1 Hz, 3H), 1.22-1.10 (m, 5H), 1.08-1.02 (m, 3H), 1.01-0.84 (m, 3H), 0.78 (d, J=9.2 Hz, 1H). HRMS (ESI): m/z calc. for C.sub.71H.sub.88N.sub.10O.sub.12S 653.3225; found 625.3222, [M+2H].sup.2+
##STR00066##
[0378] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.38 (d, J=8.6 Hz, 1H), 8.16-8.09 (m, 1H), 8.09-8.03 (m, 1H), 7.59 (d, J=7.9 Hz, 1H), 7.37 (d, J=2.1 Hz, 1H), 7.32-7.21 (m, 3H), 7.04 (dd, J=13.1, 5.9 Hz, 4H), 7.00-6.94 (m, 1H), 6.93-6.84 (m, 2H), 6.77-6.66 (m, 4H), 5.63 (t, J=8.1 Hz, 1H), 5.24 (s, 1H), 5.03 (s, 1H), 4.74 (s, 1H), 3.26 (d, J=6.0 Hz, 2H), 3.17 (d, J=8.4 Hz, 2H), 3.09 (s, 15H), 3.01-2.84 (m, 2H), 2.77-2.64 (m, 2H), 2.60 (s, 1H), 2.26 (t, J=12.8 Hz, 1H), 2.11 (t, J=7.4 Hz, 2H), 1.95-1.91 (m, 3H), 1.90 (s, 2H), 1.87 (d, J=2.4 Hz, 3H), 1.59 (q, J=7.7 Hz, 3H), 1.52 (s, 5H), 1.45-1.38 (m, 1H), 1.31 (s, 3H), 1.23-1.13 (m, 5H), 1.06 (d, J=6.5 Hz, 3H), 1.02-0.94 (m, 2H), 0.93-0.87 (m, 1H), 0.80 (d, J=8.6 Hz, 1H). HRMS (ESI): m/z calc. for C.sub.73H.sub.88N.sub.10O.sub.9 625.3441; found 625.3440, [M+2H].sup.2+
##STR00067##
[0379] .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.37 (d, J=8.6 Hz, 1H), 8.07 (dd, J=8.0, 2.1 Hz, 1H), 7.95 (d, J=7.9 Hz, 1H), 7.58 (d, J=8.1 Hz, 1H), 7.40 (s, 2H), 7.31-7.24 (m, 1H), 7.17 (s, 1H), 7.08-7.00 (m, 4H), 6.96 (tt, J=7.7, 4.9, 4.3 Hz, 3H), 6.88 (d, J=8.7 Hz, 2H), 6.75-6.66 (m, 2H), 5.62 (t, J=8.1 Hz, 1H), 5.23 (d, J=10.0 Hz, 1H), 5.01 (d, J=7.3 Hz, 1H), 4.74 (d, J=5.7 Hz, 1H), 3.27-3.21 (m, 2H), 3.16 (d, J=8.2 Hz, 2H), 3.12 (d, J=2.7 Hz, 12H), 3.08 (d, J=2.2 Hz, 6H), 3.02-2.83 (m, 3H), 2.76-2.64 (m, 2H), 2.59 (d, J=9.1 Hz, 1H), 2.25 (t, J=12.9 Hz, 1H), 2.10 (t, J=7.8 Hz, 2H), 1.92 (d, J=2.2 Hz, 3H), 1.88 (d, J=8.4 Hz, 3H), 1.85 (d, J=2.2 Hz, 3H), 1.57 (q, J=7.0 Hz, 3H), 1.51 (s, 3H), 1.48 (d, J=7.5 Hz, 1H), 1.40 (dt, J=14.3, 6.9 Hz, 1H), 1.28 (q, J=8.0 Hz, 2H), 1.16 (dd, J=6.4, 2.1 Hz, 5H), 1.05 (dd, J=6.8, 2.1 Hz, 3H), 0.98 (q, J=6.6 Hz, 2H), 0.94-0.86 (m, 1H), 0.79 (d, J=8.8 Hz, 1H). HRMS (ESI): m/z calc. for C.sub.73H.sub.91N.sub.9O.sub.11S 651.8352; found 651.8350, [M+2H].sup.2+
##STR00068##
[0380] .sup.1H NMR (400 MHz, Methanol-d.sub.4) b 8.31 (s, 1H), 8.21-8.13 (m, 1H), 8.01 (dd, J=8.1, 0.6 Hz, 1H), 7.76 (s, 1H), 7.54-7.47 (m, 2H), 7.38 (d, J=8.1 Hz, 2H), 6.91 (s, 2H), 6.77 (d, J=2.1 Hz, 2H), 6.72 (s, 2H), 5.62 (s, 2H), 4.56 (s, 2H), 2.63 (s, 6H).
##STR00069## ##STR00070##
##STR00071## ##STR00072##
Step v
[0381] Methyl 4-((4-formyl-3-hydroxyphenyl)(methyl)amino)butanoate was synthesized based on reference (Lin et al., Chemistry 19, 2531-2538 (2013)). Methyl 4-((4-formyl-3-hydroxyphenyl)(methyl)amino)butanoate (1 eq.) and ethyl acetoacetate (1.5 eq.) were dissolved in EtOH and refluxed for 2 h. Then the reaction mixture was cooled to room temperature and the precipitate was filtered. The yellow solid methyl 4-((3-acetyl-2-oxo-2H-chromen-7-yl)(methyl)amino)butanoate was used in the next step without further purification.
Step vi
[0382] Methyl 4-((3-acetyl-2-oxo-2H-chromen-7-yl)(methyl)amino)butanoate (1 eq.) and 4-Dimethylamino-2-hydroxy-2′,4′(5′)-dicarboxy-benzophenones (1.5 eq.) (Kvach et al. Bioconjug Chem 20, 1673-1682 (2009)) were dissolved in conc. H2SO4 (5 mL) and stirred at 90° C. for 48 h. After cooling to room temperature, the solution was added ice then 70% perchloric acid, filtered, and washed with water to afford crude fluorophore semi-rhodamine derivatives.
[0383] The semi-rhodamine was dissolved in 3 mL DMF, and (2.0 eq) K.sub.2CO.sub.3 and 2.0 eq Et.sub.3N were added. Then allyl bromide (2.5 eq) was added slowly and stirred for 2 h at room temperature. It was then diluted with water and extracted with CH.sub.2Cl.sub.2 (2×). The combined organics were washed with brine, dried (MgSO4), filtered, and concentrated in vacuo. The mixture was purified by flash chromatography on silica gel. Compound 16 was obtained based on the above steps ii-iii.
Step vii
[0384] The fluorophores (1.0 eq) and BOP (1.2 eq), and DIEPA (2 eq) were dissolved in 2 mL DMF in ice bath. Polyethylene glycol-containing O.sup.6-benzylguanine (1.0 eq.).sup.8 was added and stirred at 0° C. for 20 min. The mixture was purified through prep.-HPLC.
[0385] Compound WS1-SNH2-TMP-PEG2BG and WS1-SNH2-TMP-PEG5BG was synthesized based on the above step iv.
##STR00073##
UV-Vis and Fluorescence Spectroscopy
[0386] Fluorescent and fluorogenic molecules for spectroscopy were prepared as stock solutions in DMSO and diluted such that the DMSO concentration did not exceed 1% (vol/vol). Spectroscopy was performed using 96-well plate (Thermo Fisher) with optical bottom. All measurements were taken at ambient temperature (25±2° C.). Absorption and fluorescence spectra were recorded on Spark® microplate reader (Tecan). Maximum absorption wavelength (λabs), and maximum emission wavelength (λem) were taken in 10 mM HEPES, pH 7.3 buffer unless otherwise noted; reported values for ε are averages (n=3). Normalized spectra are shown for clarity.
Measurements of UV Absorbance Spectra in Water-Dioxane Mixtures
[0387] Solutions of 5 μM Rhodamine B, RhB-CH3 (201, Z=CH.sub.3), RhB-Ben (201, Z=CH.sub.2Ph), RhB-CN (201, Z=CN), RhB-SCH3 (115), RhB-SNH2 (116), RhB-SNMe.sub.2 (117), RhB-CONH2 (118), WS1-SCF3, (119) WS1-SCH3 (120), WS1-CN (121), WS1-SNH2 (122), WS1-SO (123), WS1-UREA (124) in water-dioxane mixtures containing 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90% of dioxane (by volume) were prepared. The absorbance spectra were recorded using a Spark® microplate reader (Tecan). The spectra were integrated from 400 to 700 nm for Rhodamine B-based fluorophores and from 350 to 750 nm for WS1-based fluorophores using the software Prism 7. Normalized integrals were plotted against dielectric constant of water-dioxane mixture.
Measurement of Increase in Absorbance and Fluorescence.
[0388] SNAP-HaloTag protein were used as a 85 μM-200 μM solution in 75 mM NaCl, 50 mM TRIS-HCl, pH 7.4 with 50% v/v glycerol (TBS-glycerol). SNAPTag and HaloTag based fluorogenic probes were diluted to 1.0-2.0 μM in 10 mM HEPES, pH 7.3. Then an aliquot of SNAPTag/HaloTag protein (1.5-2.0 equiv) was added and the resulting mixture was incubated until consistent absorbance signal was observed (˜60 min).
[0389] TMP-containing fluorophores were diluted to 1.0-2.5 μM in HEPES buffer (10 mM, pH 7.3, 0.1% Triton X-100). Protein ecDHFR and DHFR-2 used as a 200-400 μM solution in 75 mM NaCl, 50 mM TRIS-HCl, pH 7.4 with 50% v/v glycerol (TBS-glycerol) were added and mixed with fluorophores for 10-30 min.
[0390] SDS (10% in 10 mM HEPES) was added with dilution of 100 folds as a control group.
[0391] Absorbance and fluorescence measurements were performed in in 96-well plate (Thermos Fisher) with optical bottom.
Generation of U2OS FlpIn Halo-SNAP-NLS Cell Lines
[0392] The Flp-In™ System (ThermoFisher Scientific) was used to generated U2OS FlpIn Halo-SNAP-NLS expressing cells. pcDNA5-FRT-Halo-SNAP-NLS and pOG44 were co-transfected into the host cell line U2OS FLpIn (Malecki et al. Molecular and Cellular Biology 26, 4642 (2006)). Homologous recombination between the FRT sites in pcDNA5-FRT-Halo-SNAP-NLS and on the host cell chromosome, catalysed by the Flp recombinase expressed from pOG44, produced the U2OS FlpIn Halo-SNAP-NLS stably expressing cells.
Live-Cell Confocal Imaging
[0393] U2OS FlpIn Halo-SNAP-NLS expressing cells were grown in 96-well plate (Thermos Fisher) with optical bottom at 37° C. in a humidified 5% (vol/vol) CO2 environment. 50-500 nM probes and 2 μM verapamil were added and incubated for 1 h in DMEM media (Evrogen) containing 10% FBS. The cells were directly used to image without washing. Meanwhile, another group was imaged after being washed 2 times for 5 min with 1× phosphate buffered saline (PBS, pH 7.4) and DMEM supplemented with 10% FBS. Imaging was performed using Leica TCS SP8 confocal microscope. TMR- and Rhodamine B—, and Rhodamine 110-based probes were excited at 540 nm and the fluorescence signal was collected from 560-700 nm. Semi-Rhodamine-based probes were excited at 650 nm and the fluorescence signal was excited from 670-760 nm. Images were processed with Fiji (http://fiji.sc/wiki/index.php/Fiji) to obtain MIP (maximum intensity projections)
Abbreviations:
[0394] ACN, acetonitrile; BOP, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate; DCM, dichloromethane; DIEPA, diisopropylethylamine; DMF, dimethylformamide; LCMS, liquid chromatography-mass spectrometry; NBS, N-bromosuccinimide; PBS, phosphate buffered saline; r.t., room temperature; secBuLi secondary butyl lithium
TABLE-US-00001 TABLE 1 Photophysical properties of TAMRA derivatives. ε/ε.sup.protein/ε.sup.SDS Z λ.sub.abs/λ.sub.em (M.sup.−1cm.sup.−1) Φ D.sub.50 F/F.sub.0 F.sub.nuc./F.sub.cyt. 6-TAMRA O 550.sup.a/578.sup.a 84,000.sup.a 0.43.sup.a/0.39.sup.c 12.sup.d — — 132 NCN 552.sup.a/580.sup.a 89,000.sup.a 0.45.sup.a/0.45.sup.c 32.sup.d — — 133 NSO.sub.2CH.sub.3 553.sup.a/580.sup.a 89,000.sup.a 0.43.sup.a/0.46.sup.c 44.sup.d — — 134 NSO.sub.2NH.sub.2 552.sup.a/580.sup.a 83,000.sup.a 0.44.sup.a/0.44.sup.c 57.sup.d — — 135 NSO.sub.2N(CH.sub.3).sub.2 553.sup.a/580.sup.a 88,000.sup.a 0.4.sup.a/0.43.sup.c 58.sup.d — — 150 O 555.sup.b/578.sup.b 22,000.sup.a/66,000.sup.b/82,000.sup.c 0.42.sup.b/0.61.sup.c — 7.9 ± 0.2.sup.e 1.8 ± 0.1.sup.f 152 NCN 558.sup.b/580.sup.b 18,000.sup.a/68,000.sup.b/82,000.sup.c 0.33.sup.b/0.58.sup.c — 18 ± 2.6.sup.e 8.1 ± 0.9.sup.f 154 NSO.sub.2CH.sub.3 558.sup.b/580.sup.b 15,000.sup.a/65,000.sup.b/86,000.sup.c 0.38.sup.b/0.65.sup.c — 9.7 ± 0.1.sup.e 7.9 ± 0.9.sup.f 156 NSO.sub.2NH.sub.2 557.sup.b/578.sup.b 9,800.sup.a/57,000.sup.b/89,000.sup.c 0.46.sup.b/0.62.sup.c — 13 ± 2.4.sup.e 9.8 ± 0.8.sup.f 158, NSO.sub.2N(CH.sub.3).sub.2 556.sup.b/576.sup.b 3,500.sup.a/54,000.sup.b/88,000.sup.c 0.46.sup.b/0.61.sup.c — 21 ± 3.2.sup.e 15 ± 2.9.sup.f MaP555- SNAP 151 O 555.sup.b/578.sup.b 58,000.sup.a/82,000.sup.b/87,000.sup.c 0.51.sup.b/0.68.sup.c — 1.8 ± 0.1.sup.e 9.4 ± 1.7.sup.f 153 NCN 557.sup.b/580.sup.b 15,000.sup.a/71,000.sup.b/79,000.sup.c 0.46.sup.b/0.71.sup.c — 7.3 ± 0.8.sup.e 10 ± 0.4.sup.f 155 NSO.sub.2CH.sub.3 559.sup.b/580.sup.b 40,000.sup.a/85,000.sup.b/95,000.sup.c 0.56.sup.b/0.66.sup.c — 3.7 ± 0.4.sup.e 12 ± 2.6.sup.f 157 NSO.sub.2NH.sub.2 558.sup.b/580.sup.b 9,600.sup.a/87,000.sup.b/91,000.sup.c 0.56.sup.b/0.65.sup.c — 19 ± 3.2.sup.e 14 ± 0.7.sup.f 159, NSO.sub.2N(CH.sub.3).sub.2 558.sup.b/578.sup.b 5,200.sup.a/87,000.sup.b/92,000.sup.c 0.54.sup.b/0.63.sup.c — 35 ± 2.3.sup.e 18 ± 2.9.sup.f MaP555- Halo 169, NSO.sub.2N(CH.sub.3).sub.2 558.sup.g/580.sup.g 3,600.sup.i/95,000.sup.g/103,000.sup.j 0.57.sup.g/ 0.49.sup.j — 107 ± 9.6.sup.m — MaP555- actin 172, NSO.sub.2N(CH.sub.3).sub.2 559.sup.h/582.sup.h 13,000.sup.k/75,000.sup.h/84,000.sup.l 0.53.sup.h/0.41.sup.l — 11 ± 0.3.sup.n — MaP555- tubulin .sup.aHEPES buffer (pH 7.3), .sup.bbinding with SNAP-Halo-tag, .sup.c0.1% SDS in HEPES buffer (pH 7.3), .sup.ddioxane-H.sub.2O mixture (v/v: 90/10 −10/90), .sup.eratio of fluorescence intensities at 580 nm in the presence and absence of SNAP-Halo-tag, .sup.faverage ratio between nuclear signal (U2OS FlpIn Halo-SNAP-NLS expressing cells) and cytosol signal (normal U2OS cell), .sup.gbinding with actin, .sup.hbinding with tubulin, .sup.igeneral actin buffer, .sup.j0.2% SDS in general actin buffer, .sup.kgeneral tubulin buffer, .sup.l0.2% SDS in general tubulin buffer, .sup.mratio of fluorescence intensities at 580 nm in the presence and absence of actin, .sup.nratio of fluorescence intensities at 580 nm in the presence and absence of tubulin. Error bars show ± s.e.m.
TABLE-US-00002 TABLE 2 Photophysical properties of R110 derivatives. ε/ε.sup.protein/ε.sup.SDS Z λ.sub.abs/λ.sub.em (M.sup.−1 cm.sup.−1) Φ D.sub.50 F/F.sub.0 F.sub.nuc./F.sub.cyt. 21 NSO.sub.2N(CH.sub.3).sub.2 502.sup.a/526.sup.a 74,000.sup.a 0.68.sup.a/0.69.sup.c 70.sup.d — — 174, NSO.sub.2N(CH.sub.3).sub.2 510.sup.b/531.sup.b 4,500.sup.a/61,000.sup.b/ 0.97.sup.b/0.95.sup.c — 11 ± 1.8.sup.e 18 ± 2.9.sup.f MaP510- 73,000.sup.c Halo .sup.aHEPES buffer (pH 7.3), .sup.bbinding with SNAP-Halo-tag, .sup.c0.1% SDS in HEPES buffer (pH 7.3), .sup.ddioxane-H.sub.2O mixture (v/v: 90/10-10/90), .sup.eratio of fluorescence intensities at 530 nm in the presence and absence of SNAP-Halo-tag, .sup.faverage ratio between nuclear signal (U2OS Flpln Halo-SNAP-NLS expressing cells) and cytosol signal (normal U2OS cell). Error bars show ± s.e.m
TABLE-US-00003 TABLE 3 Photophysical properties of CPY derivatives. ε/ε.sup.protein/ε.sup.SDS Z λ.sub.abs/λ.sub.em (M.sup.−1 cm.sup.−1) Φ D.sub.50 F/F.sub.0 F.sub.nuc./F.sub.cyt. 24 O 610.sup.a/635.sup.a 113,000.sup.a 0,50.sup.b/0.51.sup.c 38.sup.d — — 25 NCN 612.sup.a/638.sup.a 68,800.sup.a 0,50.sup.b/0.49.sup.c 72.sup.d — — 26 NSO.sub.2CH.sub.3 612.sup.a/636.sup.a 30,600.sup.a 0,50.sup.b/0.57.sup.c >75.sup.d — — 27 NSO.sub.2NH.sub.2 612.sup.a/— 2,000.sup.a —/— >75.sup.d — — 28 NSO.sub.2N(CH.sub.3).sub.2 612.sup.a/— 1,000.sup.a —/— >75.sup.d — — 161 NCN 618.sup.b/635.sup.b 1,500.sup.a/144,000.sup.b/ 0.53.sup.b/0.72.sup.c — 112 ± 3.8.sup.e 19 ± 1.2.sup.f 70,000.sup.c 163 NSO.sub.2CH.sub.3 618.sup.b/635.sup.b 1,600.sup.a/121,000.sup.b/ 0.50.sup.b/0.76.sup.c — 179 ± 14.sup.e 24 ± 1.8.sup.f 45,000.sup.c 165 NSO.sub.2NH.sub.2 618.sup.b/635.sup.b 440.sup.a/101,000.sup.b/ 0.58.sup.b/— — 611 ± 42.sup.e 30 ± 4.5.sup.f 8,300.sup.c 167 NSO.sub.2N(CH.sub.3).sub.2 618.sup.b/635.sup.b 260.sup.a/107,000.sup.b/ 0.61.sup.b/— — 1000 ± 44.sup.e 58 ± 3.9.sup.f 7,200.sup.c 171 NSO.sub.2CH.sub.3 618.sup.g/635.sup.g 500.sup.h/61,000.sup.g/ 0.78.sup.g/0.71.sup.i — 449 ± 6.8.sup.j — 24,000.sup.i .sup.aHEPES buffer (pH 7.3), .sup.bbinding with SNAP-Halo-tag, .sup.c0.1% SDS in HEPES buffer (pH 7.3), .sup.ddioxane-H.sub.2O mixture (v/v: 90/10-10/90), .sup.eratio of fluorescence intensities at 635 nm in the presence and absence of SNAP-Halo-tag, .sup.faverage ratio between nuclear signal (U2OS Flpln Halo-SNAP-NLS expressing cells) and cytosol signal (normal U2OS cell), .sup.gbinding with actin, .sup.hgeneral actin buffer, .sup.i0.2% SDS in general actin buffer, .sup.jratio of fluorescence intensities at 635 nm in the presence and absence of actin. Error bars show ± s.e.m.
TABLE-US-00004 TABLE 4 Photophysical properties of SiR700 derivatives. ε/ε.sup.protein/ε.sup.SDS Z λ.sub.abs/λ.sub.em (M.sup.−1 cm.sup.−1) Φ D.sub.50 F/F.sub.0 F.sub.nuc./F.sub.cyt. 35 O 694.sup.a/718.sup.a 104,000.sup.a 0.17.sup.b/ 0.16.sup.c 54.sup.d — — 140 NCN 698.sup.a/720.sup.a 8,200.sup.a —/— >75.sup.d — — 37 O 695.sup.b/718.sup.b 38,000.sup.a/83,000.sup.b/ 0.27.sup.b/0.25.sup.c — 7.5 ± 0.2.sup.e 6.7 ± 1.5.sup.f 85,000.sup.c 168 NCN 700.sup.b/720.sup.b 340.sup.a/52,000.sup.b/ 0.24.sup.b/0.18.sup.c — 650 ± 5.8.sup.e 47 ± 3.4.sup.f 12,000.sup.c .sup.aHEPES buffer (pH 7.3), .sup.bbinding with SNAP-HALO-tag, .sup.c0.1% SDS in HEPES buffer (pH 7.3), .sup.ddioxane-H.sub.2O mixture (v/v: 90/10-10/90), .sup.eratio of fluorescence intensities at 720 nm in the presence and absence of SNAP-Halo-tag, .sup.faverage ratio between nuclear signal (U2OS Flpln Halo-SNAP-NLS expressing cells) and cytosol signal (normal U2OS cell). Error bars show ± s.e.m.