CAGING-GROUP-FREE PHOTOACTIVATABLE FLUORESCENT DYES AND THEIR USE

Abstract

The invention relates to novel caging-group-free photactivatable fluorescent dyes having the structural formula (I) as well as to the corresponding photoactivated fluorescent dyes having the structural formula (II). The invention further relates to the use of the photoactivatable compounds as such or after photoactivation, in particular as fluorescent tags, analytical reagents and labels in optical microscopy, imaging techniques, protein tracking, nucleic acid labeling, glycan analysis, capillary electrophoresis, flow cytometry or as a component of biosensors, or as analytical tools or reporters in microfluidic devices or nanofluidic circuitry.

##STR00001##

Claims

1. A compound, in particular a photoactivatable fluorescent dye, having the structural formula I: ##STR00112## wherein: R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7 and R.sup.8, independently of each other are selected from H, halogen, SO.sub.3H, CO.sub.2H, CN, NO.sub.2, CO.sub.2R, SO.sub.2R with R in CO.sub.2R or SO.sub.2R being selected from C.sub.1 to C.sub.4 unsubstituted alkyl , and an unsubstituted or substituted moiety, in particular an unsubstituted or halogen-, amino-, hydroxyl-, SO.sub.3H and/or carboxyl-substituted moiety, which is selected from C.sub.1-C.sub.20 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne, C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof; and where the substituents R.sup.6 and R.sup.7, taken together with the atoms to which they are bound, may form a 5-8 membered ring structure; and/or where the substituents R.sup.7 and R.sup.8, taken together with the atoms to which they are bound, may form a 5-8 membered ring structure; R.sup.9, R.sup.10, R.sup.11, R.sup.12 are: a. independently selected from H, unsubstituted and substituted C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.8 acyl, C.sub.1-C.sub.8 alkoxycarbonyl, and C.sub.7-C.sub.12 alkylaryl, and unsubstituted phenyl or phenyl substituted by unsubstituted alkyl, halogen, alkoxy, NO.sub.2, CO.sub.2H, CO.sub.2R and/or CONR.sup.2 with each R in CO.sub.2R or CONR.sup.2 being selected independently from C.sub.1 to C.sub.4 unsubstituted alkyl-; or b. R.sup.9 together with R.sup.10 and a nitrogen atom to which they are bound, and/or R.sup.11 together with R.sup.12 and a nitrogen atom to which they are bound form a 3-7 membered ring structure; or c. R.sup.9 and/or R.sup.11 are independently selected from H and unsubstituted and substituted C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, and C.sub.7-C.sub.12 alkylaryl; and R.sup.10 together with R.sup.2 or R.sup.3 and the atoms to which they are bound form a 5-7 membered ring structure, and/or R.sup.12 together with R.sup.4 or R.sup.5 and the atoms to which they are bound form a 5-7 membered ring structure; d. R.sup.9 together with R.sup.2 and the atoms to which they are bound form a 5-7 membered ring structure, and/or R.sup.10 together with R.sup.3 and the atoms to which they are bound form a 5-7 membered ring structure, and/or R.sup.11 together with R.sup.4 and the atoms to which they are bound form a 5-7 membered ring structure, and/or R.sup.12 together with R.sup.5 and the atoms to which they are bound form a 5-7 membered ring structure; X is independently selected from: a. O or S atom or SO.sub.2 group; b. NR.sup.13 or P(O)R.sup.13 group, where R.sup.13 is selected from H, unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.1-C.sub.20 alkoxycarbonyl, C.sub.2-C.sub.20 acyl, C.sub.2-C.sub.20 alkylsulfonyl, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof; c. SiR.sup.14R.sup.15 or GeR.sup.14R.sup.15 group, where R.sup.14 and R.sup.15 are each independently selected from unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof, or where both substituents R.sup.14 and R.sup.15, taken together with the Si or Ge to which they are attached, form a 4-7 membered ring structure; d. CR.sup.16R.sup.17 group, where R.sup.16 and R.sup.17 are each independently selected from H, F, CF.sub.3, CN, COR.sup.18, CO.sub.2R.sup.18, SO.sub.2R.sup.18, CONR.sup.18R.sup.19where R.sup.18 and R.sup.19 in COR.sup.18, CO.sub.2R.sup.18, SO.sub.2R.sup.18, and CONR.sup.18R.sup.19 are each independently selected from unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof, unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof, or where both substituents R.sup.16 and R.sup.17, taken together with the C atom to which they are attached, form a 4-7 membered ring structure; Y is independently selected from: a. O or S atom; b. NR.sup.20 group, where R.sup.20 is selected from H, unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 acyl, C.sub.2-C.sub.20 alkylsulfonyl, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof; c. CR.sup.21R.sup.22 group, where R.sup.21 and R.sup.22 are each independently selected from H, F, CF.sub.3, CN, COR.sup.23, CO.sub.2R.sup.23, SO.sub.2R.sup.23, CONR.sup.23R.sup.24where R.sup.23 and R.sup.24 in COR.sup.23, CO.sub.2R.sup.23, SO.sub.2R.sup.23, CONR.sup.23R.sup.24 are each independently selected from unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, phenyl and 5- or 6-membered ring heteroaryl, or a combination thereof, unsubstituted and substituted C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20 alkylyne and C.sub.7-C.sub.20 alkylaryl, unsubstituted and substituted phenyl, unsubstituted and substituted 5- or 6-membered ring heteroaryl, or a combination thereof, or where both substituents R.sup.21 and R.sup.22, taken together, form a 4-7 membered ring structure.

2. A compound, in particular a fluorescent dye, which has the structural formula II and is obtainable by irradiation with UV, visible or infrared light through a one-photon absorption process or a multiphoton absorption process from any of the compounds of general formula I of claim 1: ##STR00113## where R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12, X and Y are defined as in claim 1.

3. The compound according to claim 1, wherein the compound is covalently linked, particularly through any one of substituents R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11 and R.sup.12 or through any of the groups X and Y, to a binding moiety M selected from: a. a moiety selectively attachable by covalent bond to a protein or nucleic acid, particularly a moiety able to form an ester bond, an ether bond, an amide or thioamide bond, a sulfide or disulfide bond, a carbon-carbon bond, a carbon-nitrogen bond such as a Schiff base, or a moiety able to react in a click-chemistry reaction with a corresponding functional group of a protein or nucleic acid, more particularly selected from COCHCH.sub.2, SO.sub.2CHCH.sub.2, COCH.sub.2I, COCCH, NCS, CONHS or another active ester, biotin, an azide or a tetrazine moiety, a diazoalkane or diazoketone moiety, a diazirine moiety, an alkyne, a strained alkyne such as bicyclo[6.1.0]nonyne moiety or cyclooctyne moiety, a strained alkene such as trans-cyclooctene moiety or norbornene moiety, a maleimide; or from b. a substrate of a haloalkane halotransferase, particularly a 1-chlorohexyl moiety as exemplarily shown below: ##STR00114## or from c. a substrate of O.sup.6-alkylguanine-DNA-alkyltransferase, particularly a (substituted)(6-benzylguanine, O.sup.2-benzylcytosine or 4-benzyloxy-6-chloropyrimidine-2-amine moiety as exemplarily shown below: ##STR00115## or from d. a substrate of dihydrofolate reductase, particularly a 4-demethyltrimethoprim moiety as exemplarily shown below: ##STR00116## or from e. a moiety capable of selectively interacting non-covalently with a biomolecule, particularly a protein or nucleic acid, wherein said moiety and said biomolecule form a complex having a dissociation constant k.sub.D of 10.sup.6 mol/L or less, more particularly, M is selected from de-N-Boc-docetaxel, de-N-Boc-cabazitaxel, de-N-Boc-larotaxel or another taxol derivative, a phalloidin derivative, a jasplakinolide derivative, a bis-benzimide DNA stain, pepstatin A or triphenylphosphonium, e.g. as shown below: ##STR00117## ##STR00118## f. or wherein M is an oligonucleotide having a sequence length between 10 and 40 nucleotides; g. or wherein M is a lipid, particularly a sphingosine derivative such as a ceramide, or a phospholipid such as dioleoylphosphatidylethanolamine (DOPE) or dipalmitoylphosphatidylethanolamine (DPPE), or a fatty acid.

4. The compound according to claim 1, having one of the structural formulas I-1-I-32 or II-1-II-32: ##STR00119## ##STR00120## ##STR00121## ##STR00122## ##STR00123## ##STR00124## ##STR00125## ##STR00126## ##STR00127## ##STR00128## ##STR00129## ##STR00130## ##STR00131## ##STR00132## ##STR00133## ##STR00134## wherein any one of substituents R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12 or one of the substituents R.sup.13, R.sup.14, R.sup.15, R.sup.16, R.sup.17, R.sup.18, R.sup.19, R.sup.20, R.sup.21, R.sup.22, if present, independently of any other is H or a moiety having a molecular weight between 15 and 1500 Da; particularly wherein: a) the substituents R.sup.9, R.sup.10, R.sup.11, R.sup.12 are selected from H and methyl, or any of the substituents-NR.sup.9R.sup.10 and NR.sup.11R.sup.12 represents an azetidine ring, and b) one of substituents R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8 or one of the R.sup.13, R.sup.14, R.sup.15, R.sup.16, R.sup.17, R.sup.18, R.sup.19, R.sup.20, R.sup.21, R.sup.22, if present, is H or a moiety having a molecular weight between 15 and 1500 Da, and c) the other substituents R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 are selected from H and F, and d) the other substituents R.sup.6, R.sup.7, R.sup.8 are selected from H and methyl, and e) the other substituents R.sup.13, R.sup.14, R.sup.15, if present, are selected from methyl, ethyl, isopropyl or phenyl, f) the other substituents R.sup.16, R.sup.17 if present, are methyl, g) the other substituents R.sup.20, R.sup.21, R.sup.22, if present, are selected from H and methyl.

5. The compound according to claim 4, wherein said moiety having a molecular weight between 15 and 1500 Da is characterized by a general formula-L-M, wherein L is a linker covalently connecting the compound of structure I-1-I-32 or II-1-II-32 to the binding moiety M as defined above, and L is a covalent bond or a linker consisting of 1 to 50 atoms having an atomic weight of 12 or higher(in addition to the number of hydrogen atoms required to satisfy the valence rules), particularly wherein said moiety having a molecular weight between 15 and 1500 Da is characterized by a general formula
-L.sub.m.sup.A1-L.sub.m.sup.J1-L.sub.n.sup.A2-L.sub.n.sup.J2,-L.sub.p.sup.A3-L.sub.p.sup.J3,-L.sub.q.sup.A4,-L.sub.q.sup.J4,-M.sub.s, 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 cycloalkyl, (CH.sub.2CH.sub.2O), with r being an integer from 1 to 20, alkylaryl, alkylaryl-alkyl, and unsubstituted or alkyl-, halogen-, amino-, alkylamino-, imido-, nitro-, hydroxyl-, oxyalkyl-, carbonyl-, carboxyl-, sulfonyl- 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 NRC(O), C(O)N(R), NRC(O)O, OC(O)N(R), C(R)N, NC(R), C(O), OC(O), C(O)O, N(R), O, P(O)(OR), P(O)(OR)O, OP(O)(OR), OP(O)(OR)O, S, SO, SO.sub.2, SO.sub.2N(R), N(R) SO.sub.2N(R), N(R) SO.sub.2 with R selected from H and unsubstituted or amino-, hydroxyl-, carboxyl, sulfonate or fluoro substituted C.sub.1 to C.sub.6 alkyl, particularly when R is selected from H and methyl; m, m, n, n, p, p, q, q and s independently from each other are selected from 0 and 1, and M has the meaning defined above.

6. The compound according to claim 4, wherein said moiety is represented by one of the following structures: ##STR00135## ##STR00136## ##STR00137## ##STR00138##

7. The compound according to claim 1, wherein a. R.sup.9 and R.sup.10, and/or R.sup.11 and R.sup.12, are independently selected from H, unsubstituted and amino-, hydroxy-, carboxy-, sulfonate- and/or fluoro-substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 acyl, C.sub.1-C.sub.4 alkoxycarbonyl, including tert-butyloxycarbonyl or Boc group, and C.sub.3-C.sub.6 cycloalkyl, particularly R.sup.9 and R.sup.10, and/or R.sup.11 and R.sup.12, are independently selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, allyl and CH.sub.2CF.sub.3, b. R.sup.9 together with R.sup.10, and/or R.sup.9 together with R.sup.10, are independently forming an unsubstituted or alkyl-, amino-, hydroxy-, carboxy-, sulfonate- 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, (CH.sub.2).sub.2SO.sub.2 (CH.sub.2).sub.2 or (CH.sub.2).sub.2NR.sup.23 (CH.sub.2).sub.2 with R.sup.23 being selected from H and unsubstituted C.sub.1 to C.sub.4 alkyl, particularly methyl; c. R.sup.9 and/or R.sup.11 are independently selected from H, unsubstituted and alkyl-substituted, particularly methyl-substituted, amino-, hydroxy-, carboxy-, sulfonate- and/or fluoro-substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 acyl, C.sub.1-C.sub.4 alkoxycarbonyl, including tert-butyloxycarbonyl or Boc group, and C.sub.3-C.sub.6 cycloalkyl, and R.sup.10 together with R.sup.2 or R.sup.3, and/or R.sup.12 together with R.sup.4 or R.sup.5, is an alkyl or heteroalkyl bridge selected from (CH.sub.2).sub.2, (CH.sub.2).sub.3, CH.sub.2CHCH or (CH.sub.2).sub.4- or CH.sub.2O, CH.sub.2NR, CH.sub.2S, CH.sub.2SO.sub.2, (CH.sub.2).sub.2O, (CH.sub.2).sub.2NR, (CH.sub.2).sub.2S, (CH.sub.2).sub.2SO.sub.2, CH.sub.2OCH.sub.2, CH.sub.2NR, CH.sub.2SCH.sub.2, CH.sub.2SO.sub.2CH.sub.2, with R selected from H and unsubstituted or amino-, hydroxyl-, carboxyl, sulfonate- or fluoro-substituted C.sub.1 to C.sub.6 alkyl, particularly when R is selected from H and methyl-, and a mono- or dimethyl-substituted derivative of any one of the foregoing alkyl or heteroalkyl bridge moieties; d. R.sup.10 and/or R.sup.11 are independently selected from H, unsubstituted and alkyl-, substituted, particularly methyl-substituted-, amino-, hydroxy-, carboxy-, sulfonate- and/or fluoro-substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 acyl, C.sub.1-C.sub.4 alkoxycarbonyl, including tert-butyloxycarbonyl or Boc group, and C.sub.3-C.sub.6 cycloalkyl, and R.sup.9 together with R.sup.2, and/or R.sup.12 together with R.sup.5, form a fused annular structure according to any one of the following substructures: ##STR00139## e. R.sup.9 and/or R.sup.12 are independently selected from H, unsubstituted and alkyl-substituted, particularly methyl-substituted, amino-, hydroxy-, carboxy-, sulfonate- and/or fluoro-substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 acyl, C.sub.1-C.sub.4 alkoxycarbonyl, including tert-butyloxycarbonyl or Boc group, and C.sub.3-C.sub.6 cycloalkyl, and R.sup.10 together with R.sup.3, and/or R.sup.11 together with R.sup.4, form a fused annular structure according to any one of the following substructures: ##STR00140## f. R.sup.9 together with R.sup.2, and R.sup.10 together with R.sup.3, and/or R.sup.12 together with R.sup.5, and R.sup.11 together with R.sup.4, form a fused biannular structure according to any one of the following substructures: ##STR00141##

8. The compound according to claim 1, wherein R.sup.1 is structurally identical to the substituent-CR.sup.6CR.sup.7R.sup.8, in particular when the substituents R.sup.2 and R.sup.5 are structurally identical, and/or the substituents-NR.sup.9R.sup.10 and NR.sup.11R.sup.12 are structurally identical, and/or the substituents R.sup.3 and R.sup.4 are structurally identical;

9. The compound according to claim 1, wherein: R.sup.1 is H, and/or R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are independently selected from H, halogen, CN, and/or R.sup.9, R.sup.10, R.sup.11 and R.sup.12 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 Ro together with R.sup.10 together with the N atom to which they are bound, and R.sup.11 together with R.sup.12 together with the N atom to which they are bound form an unsubstituted or methyl-, hydroxy-, methoxy-, or halogen-substituted aziridine, azetidine, pyrrolidine, piperidine, piperazine, morpholine, thiomorpholine-S,S-dioxide, and/or R.sup.13, R.sup.14, R.sup.15, if present, are selected from methyl, ethyl, isopropyl or phenyl, R.sup.16, R.sup.17, if present, are methyl, one of the substituents R.sup.6, R.sup.7, R.sup.8 and R.sup.20, R.sup.21, R.sup.22, (if present, is selected from a) unsubstituted or amino-, hydroxyl-, carboxyl- and/or halogen-substituted C.sub.2 to C.sub.12 alkyl or C.sub.3 to C.sub.7 cycloalkyl; or b)-L.sub.m.sup.A1-L.sub.m.sup.J1-L.sub.n.sup.A2, -L.sub.n.sup.J2-L.sub.p.sup.A3-L.sub.p.sup.J3-L.sub.q.sup.A4-L.sub.q.sup.J4-M.sub.s, wherein L.sup.A1, L.sup.A2, L.sup.A3, L.sup.A4, L.sup.J1, L.sup.J2, L.sup.J3, L.sup.J4 m, m, n, n, p, p, q, q, s and M have the definitions recited above, and the other substituents R.sup.6, R.sup.7, R.sup.8 and R.sup.20, R.sup.21, R.sup.22, if present, are selected from H or methyl.

10. The compound according to claim 1, wherein the substituents NR.sup.9R.sup.10 and/or NR.sup.11R.sup.12 are represented by one of the following structures, particularly when the substituents NR.sup.9R.sup.10 and NR.sup.11R.sup.12 are structurally identical: ##STR00142##

11. The compound according to claim 1, wherein the fragment CR.sup.6CR.sup.7R.sup.8 is represented by one of the following structures: ##STR00143## ##STR00144##

12. The compound according to claim 1, wherein the substituent Y is represented by one of the following structures: ##STR00145## ##STR00146##

13. The compound according to any one of the preceding claim 1, wherein the group X is represented by one of the following structures: ##STR00147##

14. The compound according to claim 1 which is selected from the group of compounds below: ##STR00148## ##STR00149## ##STR00150## ##STR00151## ##STR00152## ##STR00153## ##STR00154## ##STR00155##

15. The compound of claim 1 in the 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.

16. 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, such as amine, thiol, carboxylic acid, aldehyde, alcohol, aromatic compound, heterocycle, e.g. tetrazine, alkyne, alkene including strained and bicyclic alkenes, e. g. trans-cyclooctene, cyclopropene and norbornene derivatives, organic azide, dye, amino acid, amino acid residue coupled to any chemical entity, peptide, protein, in particular enzymes and immunoglobulins, antibody, single-domain antibody, carbohydrate including a carbohydrate residue attached to a protein, nucleic acid, toxin, lipid, virus, virus-like particle, biotin and its derivatives, a chemical tag, a recognition unit, etc.

17. A method of using the compounds or compositions according to claim 1 or of their conjugates as photoactivatable fluorescent dyes.

18. A method of using the compounds or compositions according to claim 1 in a method of staining a biological sample, in particular whole organisms, mammalian and non-mammalian cells including insect, plant, fungi, bacteria cells and viral particles.

19. A method of using the compounds or compositions according to claim 1 or of their conjugates as such or after photoactivation for tracking and monitoring dynamic processes in a sample or an object, or tracking and monitoring the behavior of single molecules within a sample or an object; in particular wherein changes in the shape, dimensions and/or the intensity of the fluorescence signal obtained after photoactivation of the compounds or compositions or of their conjugates correspond to changes of the sample or object or of its environment.

20. A method of using the compounds or compositions according to claim 1 as components in inorganic, bio-inorganic, organic or macromolecular composites as materials for optical memories, data storage, photo-lithography, photo-activatable paints and inks.

21. A method of using the compounds or compositions according to claim 1 or of their conjugates as such or after photoactivation as fluorescent tags, analytical reagents and labels in optical microscopy, imaging techniques, protein tracking, nucleic acid labeling, glycan analysis, capillary electrophoresis, flow cytometry or as a component of biosensors, or as analytical tools or reporters in microfluidic devices or nanofluidic circuitry.

22. The method according to claim 21, wherein the compounds, compositions or conjugates as such or after photoactivation are used as energy donors or acceptors (reporters) in applications based on fluorescence energy transfer (FRET) process or as energy acceptors (reporters) in applications based on bioluminescence resonance energy transfer (BRET) process.

23. The method according to claim 21, wherein the optical microscopy and imaging methods comprise single molecule switching techniques (SMS: diffraction unlimited optical resolution achieved by recording the fluorescence signals of single molecules, reversibly or irreversibly switched between emitting and non-emitting states, such as single molecule localization microscopy [SMLM], photoactivation localization microscopy [PALM, PALMIRA, fPALM], stochastic optical reconstruction microscopy [STORM], minimal photon fluxes [MINFLUX] or their parallelized implementations, fluorescence correlation spectroscopy [FCS], fluorescence recovery after photobleaching [FRAP], fluorescence lifetime imaging [FLIM], stimulated emission depletion microscopy [STED], including FastRESCue STED.

24. The method according to claim 21, wherein additional color multiplexing is achieved by using the compounds, compositions or conjugates as such or after photoactivation together with any other fluorescent dyes in a single sample or object under study, or wherein the controlled photoactivation of spatiotemporal subpopulations of molecules of the compounds, compositions or conjugates allows imaging with the photoactivated fluorophore molecules while protecting the remaining photoactivatable fluorophores from photobleaching.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0146] FIG. 1 shows absorption (A) and emission (B) changes during photo-induced activation of compound 13 with violet light (405 nm) in an aqueous buffered solution at pH 7 (phosphate buffer, 100 mM); HPLC 2D-maps of absorption spectra vs. retention time for samples of the solution before (C) and after (D) the photo-induced activation; and chromatograms (E) of these samples at the wavelengths corresponding to the respective absorption maxima.

[0147] FIG. 2 shows photo-fatigue resistance of compound 20 and established commercial fluorophores to excitation light (530 nm) in an aqueous buffered solution at pH 7 (phosphate buffer, 100 mM).

[0148] FIG. 3 shows live-cell dual-color/channel confocal imaging of vimentin filaments labelled with compound 20-Halo (A & C) and tubulin labelled with 6-SiR-CTX (B & D) in U2OS cells. Images were recorded before (A-B) and after photo-activation (C-D) with 405 nm light.

[0149] FIG. 4 shows live-cell confocal (A) and STED (B) image of vimentin filaments labelled with compound 20-Halo in U2OS cells. The compound was pre-activated by irradiation with a 405 nm laser. (C) The same samples was imaged in a confocal microscope, before pre-activation (top half of the image), and a 2-photon activation laser was switched on approximately in the middle of the scanning (bottom half of the image, as indicated by the arrows).

[0150] FIG. 5 shows live-cell single-color/channel confocal imaging U2OS cells stably expressing Vimentin-HaloTag fusion construct labelled with compound 20-Halo and Abberior Live 560-Tubulin. Sequential imaging was performed before photo-activation (A), after photo-bleaching of Abberior Live 560-Tubulin (B), and after photo-activation of compound 20-Halo (C). Absorption and emission spectra of Abberior Live 560-Tubulin (marked as AL-560) and 20-Halo (after photoactivation) is shown (D).

[0151] FIG. 6 shows Single Molecule Localization Microscopy superresolution images of nuclear pore complexes (A-C) and microtubules (D) in COS7 cells. Samples were fixed and immunolabelled with a primary antibody against NUP-98 from rabbit and an anti-rabbit secondary antibody labelled with 5-NHS (A-C) or a primary antibody against alpha-tubulin from mouse and an anti-mouse secondary labelled with 9-NHS (D).

[0152] FIG. 7 shows confocal imaging of a fixed Cos7 cells immunolabeled with anti-alpha-tubulin primary antibody from mouse and anti-clathrin primary antibody from rabbit, and anti-mouse secondary antibody labelled with 20-NHS and anti-rabbit secondary antibody labelled with 5NHS. Sequential imaging was performed before photoactivation (A, B), after low photoactivation dose selective for 20 (C, D), and after high photoactivation dose sufficient to convert compound 5 (E, F).

[0153] FIG. 8 shows fluorescence patterning in a polymer film (PVA) doped with compound 5. Selected areas were irradiated with light of 405 nm. Confocal images of the same area were recorded in a before (A) and after photo-patterning (B), and in a wide-field fluorescence microscope (C), only of the patterned structure.

[0154] The present invention is further illustrated by the following specific but non-limiting examples.

Example 1

Synthesis of the Starting Materials, Photoactivatable Compounds and Photoactivatable Labels

[0155] ##STR00050##

[0156] Compound B1 was prepared according to the method reported in S. Bera, X. Hu. Angew. Chem. Int. Ed. 2019, 58 (39), 13854-13859. In a dried 10 mL crimp-top tube (a 2-5 mL Biotage microwave vial was used), compound 1 (1 g, 6 mmol; prepared according to the literature procedure: E. Bomal et al. Chem. Eur. J. 2020, 26 (41), 8907-8915) and Schwartz's reagent (zirconocene chloride hydride; 155 mg, 0.6 mmol, 10 mol %) were placed, and the contents of the vial were degassed on a Schlenk line. Pinacolborane (HBpin; 950 L, 6.55 mmol, 1.1 equiv) was then injected followed by triethylamine (84 L, 0.6 mmol, 10 mol %), the vial was placed in a 60 C. oil bath and the reaction mixture was stirred for 24 h. The mixture was then cooled down to rt, diluted with diethyl ether and filtered through a 2 cm plug of silica, washing with diethyl ether (50 mL). The filtrate was evaporated and the product was isolated by flash chromatography on Biotage Isolera system (40 g RediSep Rf cartridge, gradient 0% to 20% ethyl acetate/hexane) to give colorless oil, yield 1.28 g (72%).

[0157] .sup.1H NMR (400 MHZ, CDCl.sub.3): 6.60 (dt, J=18.0, 6.4 Hz, 1H), 5.45 (dt, J=18.0, 1.6 Hz, 1H), 2.26-2.14 (m, 4H), 1.72 (p, J=7.6 Hz, 2H), 1.44 (s, 9H), 1.27 (s, 12H).

[0158] .sup.13C NMR (101 MHz, CDCl.sub.3): 173.0, 153.4, 119.6 (br.), 83.2, 80.2, 35.2, 35.1, 28.2, 24.9, 23.7. HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.16H.sub.29BO.sub.4 297.2235; Found 297.2231.

##STR00051##

[0159] Compound 3. In a 25 mL round-bottom flask, the mixture of compound A1 (382 mg, 1.35 mmol; known compound: P. Horvath et al. J. Org. Chem. 2015, 80 (3), 1299-1311), bis(pinacolato) diboron (344 mg, 1.35 mmol, 1 equiv), [Ir (cod)(OMe)].sub.2 (45 mg, 0.068 mmol, 5 mol %) and ligand L1 (8-(diisopropylsilyl) quinoline; known compound: B. Ghaffari et al. J. Am. Chem. Soc. 2014, 136, 14345-14348)(33 mg, 0.135 mmol, 10 mol %) in dry n-octane (13 mL) was degassed on a Schlenk line and stirred at 120 C. for 22 h. On cooling, the reaction mixture was diluted with dichloromethane and filtered through a plug of Celite, washing with dichloromethane. The filtrate was evaporated to dryness in a 50 mL round-bottom flask and the obtained crude 2 was used directly in the next step.

[0160] To the residue of crude compound 2, potassium fluoride (313 mg, 5.40 mmol, 4 equiv), copper (II) bromide (903 mg, 4.05 mmol, 3 equiv) were added, followed by DMSO (12 mL), water (1.2 mL) and pyridine (2.2 mL, 27 mmol, 20 equiv). The reaction mixture was stirred at 80 C. for 30 min. Upon cooling to rt, the reaction mixture was diluted with ethyl acetate and poured into water (100 mL). The product was extracted with ethyl acetate (350 mL), the combined extracts were washed with brine (50 mL) and dried over Na.sub.2SO.sub.4. The product was isolated by flash chromatography on Biotage Isolera system (40 g RediSep Rf cartridge, gradient 0% to 20% ethyl acetate/dichloromethane) to give 327 mg (67%) of 3 as yellow solid.

[0161] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.08 (d, J=9.0 Hz, 1H), 6.86 (d, J=2.6 Hz, 1H), 6.64 (dd, J=9.0, 2.5 Hz, 1H), 6.40 (d, J=2.6 Hz, 1H), 6.35 (d, J=2.5 Hz, 1H), 3.06 (s, 6H), 3.03 (s, 6H).

[0162] .sup.13C NMR (101 MHz, CDCl.sub.3): 174.1, 159.0, 157.0, 154.4, 152.9, 128.1, 122.4, 115.6, 112.2, 109.5, 109.2, 97.7, 96.5, 40.3, 40.1.

[0163] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.17H.sub.17BrN.sub.2O.sub.2 361.0546; Found 361.0546.

##STR00052##

[0164] Compound 4. In a 10 ml tube, compound 3 (36 mg, 0.10 mmol), compound B1 (44 mg, 0.15 mmol), K.sub.2CO.sub.3 (18 mg, 0.13 mmol) and Pd(dppf) Cl.sub.2.Math.CH.sub.2Cl.sub.2 (4.1 mg, 5 mol, 5 mol %) were loaded. Dioxane (1.0 mL) and water (0.2 mL) were added. The mixture was sparged with argon for 30 min, and the tube sealed and stirred at 80 C. for 14 h. Upon cooling, the reaction mixture was diluted with ethyl acetate (10 mL) and washed with sat. aq. NH.sub.4Cl (10 mL), and brine (10 ml). The organics were dried over Na.sub.2SO.sub.4, filtered, evaporated. The product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 0% to 50% ethyl acetate/hexane) and freeze-dried from dioxane to yield 40 mg (89%) of 4 as a pale yellow solid.

[0165] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.08 (d, J=9.0 Hz, 1H), 7.91 (d, J=15.7 Hz, 1H), 6.66 (dd, J=9.0, 2.4 Hz, 1H), 6.63 (dd, J=2.6, 0.6 Hz, 1H), 6.42 (dd, J=5.2, 2.5 Hz, 2H), 6.01 (dt, J=15.5, 6.9 Hz, 1H), 3.09 (s, 6H), 3.08 (s, 6H), 2.39-2.28 (m, 4H), 1.90-1.78 (m, 2H), 1.45 (s, 9H).

[0166] .sup.13C NMR (101 MHz, CDCl.sub.3): 176.6, 173.3, 159.2, 157.2, 154.1, 153.0, 142.3, 132.4, 131.3, 127.7, 113.0, 109.0, 107.7, 97.0, 96.5, 80.0, 40.2, 40.1, 35.3, 32.5, 28.2, 25.0.

[0167] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.27H.sub.34N.sub.2O.sub.4: 451.2591, found: 451.2590.

##STR00053##

[0168] Compound 5. To a solution of compound 4 (34 mg; 0.075 mmol) in CH.sub.2Cl.sub.2 (600 L) trifluoroacetic acid (200 L) was added dropwise. The resulting reaction mixture was stirred for 30 minutes at rt, protected from light. The volatiles were removed in vacuo by coevaporation with toluene (310 ml). The product was freeze-dried from dioxane to give 30 mg (100%, remainder dioxane) of 5 as a pale yellow solid.

[0169] .sup.1H NMR (400 MHZ, DMSO-d.sub.6): 12.02 (s, 1H), 7.85 (d, J=3.7 Hz, 1H), 7.82 (d, J=3.0 Hz, 1H), 6.75 (dd, J=9.0, 2.4 Hz, 1H), 6.68 (d, J=2.5 Hz, 1H), 6.49-6.45 (m, 2H), 6.10 (dt, J=15.7, 6.7 Hz, 1H), 3.06 (s, 6H), 3.05 (s, 6H), 2.32 (t, J=7.4 Hz, 2H), 2.23 (q, J=6.8 Hz, 2H), 1.73 (p, J=7.4 Hz, 2H).

[0170] .sup.13C NMR (101 MHz, DMSO-d.sub.6): 174.9, 174.5, 158.6, 156.5, 154.1, 152.8, 141.0, 131.4, 131.1, 127.0, 111.8, 109.2, 107.9, 107.0, 96.6, 96.0, 39.6, 33.2, 32.0, 24.1.

[0171] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.23H.sub.26N.sub.2O.sub.4: 395.1965, found: 395.1961.

##STR00054##

[0172] Compound 5-NHS. In an amber vial, compound 5 (23 mg, 0.058 mmol) and TSTU (N,N,N, N-tetramethyl-O(N-succinimidyl) uronium tetrafluoroborate; 35 mg, 0.12 mmol) were dissolved in DMF (500 L) and 2,6-lutidine (62 mg, 0.58 mmol) was added and stirred for 2 h at rt. The volatiles were removed in vacuo. The product was isolated by flash chromatography on a Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 20% to 100% ethyl acetate/hexane) and freeze-dried from 1,4-dioxane to give 5-NHS as an off-white solid. Yield 24 mg (81%, remainder dioxane).

[0173] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.07 (d, J=9.0 Hz, 1H), 7.94 (d, J=15.7 Hz, 1H), 6.67 (dd, J=9.0, 2.5 Hz, 1H), 6.62 (dd, J=2.6, 0.6 Hz, 1H), 6.44 (d, J=2.6 Hz, 1H), 6.42 (d, J=2.4 Hz, 1H), 5.99 (dt, J=15.6, 6.8 Hz, 1H), 3.10 (s, 6H), 3.08 (s, 6H), 2.83 (s, 4H), 2.74 (dd, J=8.1, 7.2 Hz, 2H), 2.48-2.39 (m, 2H), 2.02 (p, J=7.6 Hz, 2H).

[0174] .sup.13C NMR (101 MHZ, CDCl.sub.3): 176.6, 169.1, 168.7, 159.2, 157.2, 154.2, 153.0, 142.0, 133.3, 129.9, 127.7, 112.9, 109.0, 107.8, 97.1, 96.5, 67.1, 40.2, 40.1, 32.0, 30.6, 25.6, 24.4.

[0175] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.27H.sub.29N.sub.3O.sub.6: 492.2129, found: 492.2130.

##STR00055##

[0176] Compound 5-Halo. In an amber vial, compound 5-NHS (9.9 mg, 0.02 mmol) and HaloTag (02) Amine (6.7 mg, 0.03 mmol) were dissolved in DMF (100 L). DIPEA (N,N-diisopropylethylamine; 12.9 mg, 0.1 mmol) was added and stirred for 3 hours at rt. The volatiles were removed in vacuo. The product was isolated by flash chromatography on a Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 0% to 100% A/B, A: 100% ethyl acetate, B: 2% methanol-98% ethyl acetate) and freeze-dried from dioxane to yield 9.7 mg (81%) of 5-Halo as an off-white solid.

[0177] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.33H.sub.46ClN.sub.3O.sub.5: 600.3199, found: 600.3202.

##STR00056##

[0178] Compound 5-CTX. In an amber vial, compound 5-NHS (3.0 mg, 6.1 mol) and H.sub.2N-CTX.Math.HCO.sub.2H [A. N. Butkevich et al. ACS Chem. Biol. 2018, 13 (2), 475-480](6.3 mg, 8.0 mol, 1.3 equiv) were dissolved in DMF (100 L). DIPEA (30 L) was added, and the reaction mixture was at rt for 1.5 h. The solvents were removed in vacuo, and the product the product was isolated by preparative HPLC (Interchim Uptisphere Strategy PhC4 25021.2 mm 5 m, solvent flow rate 18 mL/min, gradient 40% to 85% A: B, A-acetonitrile+0.1% (v/v) formic acid, B-water+0.1% (v/v) formic acid) and freeze-dried from dioxane to give 3.0 mg (44%) of 5-CTX as yellowish solid.

[0179] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.63H.sub.73N.sub.3O.sub.15: 1112.5114, found: 1112.5113.

##STR00057##

[0180] Compound 6. In a 10 ml tube, the mixture of compound A2 (92 mg, 0.30 mmol; known compound: [A. N. Butkevich et al. Angew. Chem. Int. Ed., 2016, 55 (10), 3290-3294]), bis(pinacolato) diboron (85 mg, 0.33 mmol, 1.1 equiv), [Ir (cod)(OMe)].sub.2 (12 mg, 0.018 mmol, 5 mol %), triphenylarsine (10 mg, 0.033 mmol, 10 mol %) in dry n-octane (3 mL) was degassed on a Schlenk line and stirred at 120 C. for 18 h. On cooling, the reaction mixture was diluted with dichloromethane and filtered through a plug of Celite, washing with dichloromethane. The filtrate was evaporated to dryness and the product was isolated by flash chromatography on a Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 0% to 10% ethyl acetate/dichloromethane) and freeze-dried from dioxane to 59 mg (45%) of 6 as an orange solid.

[0181] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.20 (d, J=8.9 Hz, 1H), 6.85 (d, J=2.2 Hz, 1H), 6.76 (d, J=2.5 Hz, 1H), 6.70 (dd, J=8.9, 2.5 Hz, 1H), 6.61 (d, J=2.2 Hz, 1H), 3.13 (s, 6H), 3.12 (s, 6H), 1.62 (s, 6H), 1.47 (s, 12H).

[0182] .sup.13C NMR (101 MHZ, CDCl.sub.3): 182.6, 155.2, 154.6, 153.9, 150.6, 130.2, 123.3, 116.0, 113.3, 110.4, 108.3, 107.3, 81.8, 40.5, 40.2, 38.9, 33.2, 25.3.

[0183] HRMS (ESI) m/z: [M].sup.+ Calcd for C.sub.20H.sub.24BN.sub.2O.sub.2: 335.1929, found: 335.1928-corresponds to the pinacol ester hydrolysis product (6a).

##STR00058##

[0184] Compound 7. In a 10 mL round-bottom flask, compound 6 (59 mg, 0.14 mmol), potassium fluoride (33 mg, 0.56 mmol, 4 equiv), copper (II) bromide (94 mg, 0.42 mmol, 3 equiv) were placed, followed by addition of DMSO (1.2 mL), water (120 L) and pyridine (220 L, 2.7 mmol, 20 equiv). The reaction mixture was stirred at 80 C. for 30 min. Upon cooling to rt, the mixture was diluted with ethyl acetate and poured into water (10 mL). The product was extracted with ethyl acetate (310 mL), the combined extracts were washed with brine (50 mL) and dried over Na.sub.2SO.sub.4. The product was isolated by flash chromatography on Biotage Isolera system (25 g Interchim SiHP 30 m cartridge, gradient 0% to 10% ethyl acetate/dichloromethane and freeze-dried from 1,4-dioxane to give 39 mg (72%) of 7 as yellow solid.

[0185] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.22 (d, J=8.8 Hz, 1H), 7.00 (d, J=2.6 Hz, 1H), 6.79 (d, J=2.6 Hz, 1H), 6.75 (dd, J=8.9, 2.5 Hz, 1H), 6.70 (d, J=2.5 Hz, 1H), 3.09 (s, 6H), 3.08 (s, 6H), 1.70 (s, 6H).

[0186] .sup.13C NMR (101 MHz, CDCl.sub.3): 180.4, 154.3, 153.0, 151.8, 150.3, 129.6, 124.0, 120.9, 118.0, 117.5, 111.0, 108.2, 106.8, 40.2, 40.0, 38.9, 34.1.

[0187] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.20H.sub.23BrN.sub.2O: 387.1067, found: 387.1066.

##STR00059##

[0188] Compound 8. In a 10 ml tube, compound 7 (33 mg, 0.085 mmol), compound B1 (37 mg, 0.13 mmol, 1.5 equiv), K.sub.2CO.sub.3 (20 mg, 0.14 mmol, 1.6 equiv) and Pd(dppf) Cl.sub.2.Math.CH.sub.2Cl.sub.2 (3.4 mg, 4.2 mol, 5 mol %) were loaded. Dioxane (1.0 mL) and water (0.2 mL) were added. The mixture was sparged with argon for 30 min, the tube was sealed and the reaction mixture stirred at 80 C. for 17 h. Upon cooling, the reaction mixture was diluted with ethyl acetate (10 mL) and washed with sat. aq. NH.sub.4Cl (10 mL) and brine (10 ml). The organics were dried over Na.sub.2SO.sub.4, filtered, evaporated. The product was isolated by flash chromatography on a Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 0% to 50% ethyl acetate/hexane) and freeze-dried from dioxane to yield 23 mg (58%) of 8 as a yellow solid.

[0189] .sup.1H NMR (400 MHz, CDCl.sub.3): 8.19 (d, J=8.5 Hz, 1H), 7.64 (d, J=15.6 Hz, 1H), 6.79-6.71 (m, 3H), 6.67 (dd, J=2.7, 0.6 Hz, 1H), 5.90 (dt, J=15.4, 6.9 Hz, 1H), 3.11 (s, 6H), 3.09 (s, 6H), 2.38-2.30 (m, 4H), 1.85 (p, J=7.6 Hz, 2H), 1.71 (s, 6H), 1.45 (s, 9H).

[0190] .sup.13C NMR (101 MHZ, CDCl.sub.3): 182.9, 173.4, 153.5, 152.8, 151.9, 151.1, 143.1, 134.8, 129.3, 129.1, 121.6, 117.8, 111.2, 110.9, 107.9, 107.2, 79.9, 40.2, 40.1, 38.8, 35.3, 34.2, 32.5, 28.2, 25.1.

[0191] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.30H.sub.40N.sub.2O.sub.3: 477.3112, found: 477.3112.

##STR00060##

[0192] Compound 9. To a solution of compound 8 (17.5 mg, 0.075 mmol) in CH.sub.2Cl.sub.2 (300 L), trifluoroacetic acid (100 L) was added dropwise. The resulting reaction mixture was stirred for 60 minutes at rt, protected from light. The volatiles were removed in vacuo by coevaporation with toluene (310 ml). The product was isolated by flash chromatography on a Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 20% to 100% ethyl acetate/hexane) and freeze-dried from dioxane to yield 16.5 mg (100%, remainder dioxane) of 9 as a yellow solid.

[0193] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.19 (d, J=8.5 Hz, 1H), 7.64 (d, J=15.6 Hz, 1H), 6.79-6.71 (m, 3H), 6.67 (dd, J=2.7, 0.6 Hz, 1H), 5.90 (dt, J=15.4, 6.9 Hz, 1H), 3.11 (s, 6H), 3.09 (s, 6H), 2.38-2.30 (m, 4H), 1.85 (p, J=7.6 Hz, 2H), 1.71 (s, 6H), 1.45 (s, 9H).

[0194] .sup.13C NMR (101 MHz, CDCl.sub.3): 153.5, 152.8, 151.9, 151.1, 143.1, 134.8, 129.3, 129.1, 111.2, 110.9, 107.9, 107.2, 40.2, 40.1, 38.8, 35.3, 34.2, 32.5, 28.2, 25.1.

[0195] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.26H.sub.32N.sub.2O.sub.3: 421.2486, found: 421.2486.

##STR00061##

[0196] Compound 9-NHS. In an amber vial, compound 9 (8.6 mg, 0.020 mmol) and TSTU (12 mg, 0.04 mmol) were dissolved in DMF (200 L). 2,6-Lutidine (21 mg, 0.20 mmol) was added and the reaction mixture was stirred for 3 h at rt. The volatiles were removed in vacuo. The product was isolated by flash chromatography on a Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 20% to 80% ethyl acetate/hexane) and freeze-dried from dioxane to yield 9.2 mg (89%) of 9-NHS as a pale yellow solid.

[0197] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.30H.sub.35N.sub.3O.sub.5: 518.2649, found: 518.2647.

##STR00062##

[0198] Compound 9-Halo. To a solution of compound 9-NHS (7.2 mg, 0.014 mmol) in DMF (100 L) was added HaloTag (02) Amine (4.7 mg, 0.021 mmol) and DIPEA (9.0 mg, 0.070 mmol), and the mixture stirred for 2 h at rt. The volatiles were removed in vacuo. The product was isolated by flash chromatography on a Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, 100% ethyl acetate) and freeze-dried from dioxane to yield 5.2 mg (59%) of 9-Halo as an orange oil.

[0199] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.36H.sub.52ClN.sub.3O.sub.4: 626.3719, found: 626.3714.

##STR00063##

[0200] Compound 10. In a 25 mL round-bottom flask, the mixture of compound A3 (324 mg, 1 mmol; known compound: [A. N. Butkevich et al. J. Am. Chem. Soc. 2017, 139, 12378-12381]), bis(pinacolato) diboron (280 mg, 1.1 mmol, 1.1 equiv), [Ir (cod)(OMe)].sub.2 (33 mg, 0.05 mmol, 5 mol %) and triphenylarsine (31 mg, 0.01 mmol, 10 mol %) in dry n-octane (10 mL) was degassed on a Schlenk line and stirred at 120 C. for 22 h. On cooling, the reaction mixture was evaporated on Celite and the product was isolated by flash chromatography on Biotage Isolera system (10 g Biotage Sfr Duo cartridge, gradient 0% to 100% A: B, A=20% ethyl acetate in dichloromethane, B=dichloromethane) and freeze-dried from dioxane to give 322 mg (72%) of 10 as an orange solid.

[0201] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.38 (d, J=9.0 Hz, 1H), 7.02 (d, J=2.5 Hz, 1H), 6.80 (d, J=2.7 Hz, 1H), 6.75 (dd, J=9.1, 2.7 Hz, 1H), 6.72 (br.s, 1H), 3.14 (s, 12H), 1.43 (s, 12H), 0.39 (s, 6H).

[0202] .sup.13C NMR (101 MHZ, CDCl.sub.3): 187.5, 153.4, 153.3, 145.9, 139.2, 133.8, 122.4, 115.6, 115.1, 114.7, 112.3, 80.5, 40.5, 40.1, 25.7,-1.2.

[0203] HRMS (ESI) m/z: [M].sup.+ Calcd for C.sub.19H.sub.24BN.sub.2O.sub.2Si 351.1698; Found 351.1693-corresponds to the pinacol ester hydrolysis product (10a):

##STR00064##

[0204] Compound 11. In a 25 mL round-bottom flask, compound 10 (259 mg, 0.58 mmol), potassium fluoride (135 mg, 2.32 mmol, 4 equiv), copper (II) bromide (388 mg, 1.74 mmol, 3 equiv) were placed, followed by addition of DMSO (5 mL), water (500 L) and pyridine (940 L, 11.6 mmol, 20 equiv). The reaction mixture was stirred at 80 C. for 30 min. Upon cooling to rt, the mixture was diluted with ethyl acetate and poured into water (80 mL). The product was extracted with ethyl acetate (430 mL), the combined extracts were washed with brine (50 mL) and dried over Na.sub.2SO.sub.4. The product was isolated by flash chromatography on Biotage Isolera system (25 g Interchim SiHP 30 m cartridge, gradient 0% to 100% A/B, A: 10% ethyl acetate in dichloromethane, B: dichloromethane) and freeze-dried from dioxane to give 212 mg (91%) of 11 as yellow solid.

[0205] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.20 (d, J=8.9 Hz, 1H), 7.06 (d, J=2.7 Hz, 1H), 6.83 (dd, J=9.0, 2.8 Hz, 1H), 6.76 (br.d, J=2.8 Hz, 1H), 6.74 (d, J=2.7 Hz, 1H), 3.07 (s, 6H), 3.05 (s, 6H), 0.46 (s, 6H).

[0206] .sup.13C NMR (101 MHz, CDCl.sub.3): 186.3, 151.2, 150.9, 142.3, 138.4, 131.5, 127.9, 125.3, 120.1, 114.1, 113.8, 113.6, 40.3, 40.0,-1.0.

[0207] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.19H.sub.23BrN.sub.2OSi 403.0836; Found 403.0830.

##STR00065##

[0208] Compound 12. In a 10 ml tube, compound 11 (40 mg, 0.10 mmol), potassium vinyltrifluoroborate (15 mg, 0.11 mmol, 1.1 equiv), K.sub.2CO.sub.3 (22 mg, 0.16 mmol, 1.6 equiv) and Pd(dppf) Cl.sub.2.Math.CH.sub.2Cl.sub.2 (4.1 mg, 5.0 mol, 5 mol %) were loaded. Dioxane (1.0 mL) and water (0.2 mL) were added. The mixture was sparged with argon for 30 min, and the tube sealed and stirred at 80 C. for 17 h. Upon cooling, the reaction mixture was diluted with ethyl acetate (10 mL) and washed with sat. aq. NH.sub.4Cl (10 mL) and brine (10 mL). The organics were dried over Na.sub.2SO.sub.4, filtered, evaporated. The product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 0% to 30% ethyl acetate/hexane) and freeze-dried from 1,4-dioxane to yield 31 mg (87%) of 12 as a yellow solid.

[0209] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.25 (d, J=8.9 Hz, 1H), 7.58 (dd, J=17.2, 10.7 Hz, 1H), 6.82 (dd, J=9.0, 2.8 Hz, 1H), 6.80-6.75 (m, 3H), 5.44 (dd, J=17.2, 1.8 Hz, 1H), 5.24 (dd, J=10.8, 1.8 Hz, 1H), 3.10 (s, 6H), 3.07 (s, 6H), 0.46 (s, 6H).

[0210] .sup.13C NMR (101 MHZ, CDCl.sub.3): 187.7, 151.2, 150.8, 144.1, 141.8, 141.3, 139.0, 131.9, 131.3, 128.4, 114.5, 113.9, 113.7, 113.3, 112.5, 40.1, 40.0.

[0211] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.21H.sub.27N.sub.2OSi: 351.1887, found: 351.1888.

##STR00066##

[0212] Compound 13. In a 10 ml tube, compound 11 (40 mg, 0.10 mmol), trans-1-propenylboronic acid pinacol ester (18 mg, 0.11 mmol, 1.1 equiv), K.sub.2CO.sub.3 (18 mg, 0.13 mmol, 1.3 equiv) and Pd(dppf) Cl.sub.2.Math.CH.sub.2Cl.sub.2 (4.1 mg, 5.0 mol, 5 mol %) were loaded. Dioxane (1.0 mL) and water (0.2 mL) were added. The mixture was sparged with argon for 30 min, the tube was sealed and the mixture was stirred at 80 C. for 3 h. Upon cooling, the reaction mixture was diluted with ethyl acetate (10 mL) and washed with sat. aq. NH.sub.4Cl (10 mL) and brine (10 mL). The organics were dried over Na.sub.2SO.sub.4, filtered, evaporated. The product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 0% to 30% ethyl acetate/hexane) and freeze-dried from dioxane to yield 16 mg (43%) of 13 as a yellow solid.

[0213] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.25 (d, J=8.9 Hz, 1H), 7.33-7.26 (m, 1H), 6.82 (dd, J=9.0, 2.8 Hz, 1H), 6.75 (dt, J=5.4, 2.8 Hz, 3H), 5.93 (dq, J=15.4, 6.6 Hz, 1H), 3.09 (s, 6H), 3.07 (s, 6H), 1.95 (dd, J=6.6, 1.7 Hz, 3H), 0.45 (s, 6H).

[0214] .sup.13C NMR (101 MHz, CDCl.sub.3): 187.9, 151.1, 150.7, 143.9, 141.2, 138.9, 135.3, 132.2, 131.3, 124.6, 114.1, 113.9, 113.7, 113.3, 40.1, 40.0, 18.7,-1.0.

[0215] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.22H.sub.28N.sub.2OSi: 365.2044, found: 365.2044.

##STR00067##

[0216] Compound 14. In a 10 ml tube, compound 11 (40 mg, 0.10 mmol), 2-methyl-1-propenylboronic acid pinacol ester (20 mg, 0.11 mmol, 1.1 equiv), K.sub.2CO.sub.3 (18 mg, 0.13 mmol, 1.3 equiv) and Pd(dppf) Cl.sub.2.Math.CH.sub.2Cl.sub.2 (4.1 mg, 5.0 mol, 5 mol %) were loaded. Dioxane (1.0 mL) and water (0.2 mL) were added. The mixture was sparged with argon for 30 min, the tube was sealed and the mixture was stirred at 80 C. for 3 h. Upon cooling, the reaction mixture was diluted with ethyl acetate (10 mL) and washed with sat. aq. NH.sub.4Cl (10 mL) and brine (10 mL). The organics were dried over Na.sub.2SO.sub.4, filtered, evaporated. The product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 0% to 30% ethyl acetate/hexane) and freeze-dried from dioxane to yield 28 mg (74%) of 14 as a yellow solid.

[0217] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.26 (d, J=9.0 Hz, 1H), 6.89-6.85 (m, 1H), 6.81 (dd, J=8.9, 2.8 Hz, 1H), 6.75 (dd, J=10.3, 2.8 Hz, 2H), 6.60 (dd, J=2.9, 0.8 Hz, 1H), 3.07 (d, J=0.9 Hz, 12H), 1.97 (d, J=1.4 Hz, 3H), 1.74 (d, J=1.5 Hz, 3H), 0.46 (s, 6H).

[0218] .sup.13C NMR (101 MHz, CDCl.sub.3): 187.3, 151.0, 150.1, 143.4, 141.3, 139.0, 132.0, 131.4, 129.7, 129.6, 128.7, 117.0, 113.7, 113.5, 113.2, 67.1, 40.1, 40.0, 26.3, 19.5,-0.9.

[0219] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.23H.sub.31N.sub.2OSi: 379.2200, found: 379.2200.

##STR00068##

[0220] Compound 15. In a 10 ml tube, compound 12 (40 mg, 0.10 mmol), isopropenylboronic acid pinacol ester (18 mg, 0.11 mmol, 1.1 equiv), K.sub.2CO.sub.3 (20 mg, 0.14 mmol, 1.4 equiv) and Pd(dppf) Cl.sub.2.Math.CH.sub.2Cl.sub.2 (4.1 mg, 5.0 mol, 5 mol %) were loaded. Dioxane (1.0 mL) and water (0.2 mL) were added. The mixture was sparged with argon for 30 min, and the tube was sealed and the mixture was stirred at 80 C. for 18 h. Upon cooling, the reaction mixture was diluted with ethyl acetate (10 mL) and washed with sat. aq. NH.sub.4Cl (10 mL) and brine (10 mL). The organics were dried over Na.sub.2SO.sub.4, filtered, evaporated. The product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 0% to 30% ethyl acetate/hexane) and freeze-dried from dioxane to yield 29 mg (81%) of 15 as a light orange solid.

[0221] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.26 (d, J=8.9 Hz, 1H), 6.82 (dd, J=8.9, 2.8 Hz, 1H), 6.76 (dd, J=8.3, 2.8 Hz, 2H), 6.57 (d, J=2.8 Hz, 1H), 5.01 (dq, J=2.9, 1.4 Hz, 1H), 4.80 (dd, J=2.2, 0.9 Hz, 1H), 3.08 (s, 6H), 3.07 (s, 6H), 2.12 (dd, J=1.4, 0.8 Hz, 3H), 0.47 (s, 6H).

[0222] .sup.13C NMR (101 MHZ, CDCl.sub.3): 186.6, 151.9, 151.2, 150.6, 149.3, 141.4, 139.0, 131.8, 131.3, 128.0, 115.5, 113.8, 113.8, 113.3, 109.4, 40.1, 40.0, 24.5,-0.9.

[0223] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.22H.sub.28N.sub.2OSi: 365.2044, found: 365.2045.

##STR00069##

[0224] Compound 16. In a 10 ml tube, compound 11 (40 mg, 0.10 mmol), 1-cyclopentenylboronic acid (21 mg, 0.11 mmol, 1.1 equiv), K.sub.2CO.sub.3 (18 mg, 0.13 mmol, 1.3 equiv) and Pd(dppf) Cl.sub.2.Math.CH.sub.2Cl.sub.2 (4.1 mg, 5.0 mol, 5 mol %) were loaded. Dioxane (1.0 mL) and water (0.2 mL) were added. The mixture was sparged with argon for 30 min, and the tube was sealed and the mixture was stirred at 80 C. for 18 h. Upon cooling, the reaction mixture was diluted with ethyl acetate (10 mL) and washed with sat. aq. NH.sub.4Cl (10 mL) and brine (10 mL). The organics were dried over Na.sub.2SO.sub.4, filtered, evaporated. The product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 0% to 30% ethyl acetate/hexane) and freeze-dried from dioxane to yield 23 mg (59%) of 16 as yellow solid.

[0225] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.20 (d, J=8.8 Hz, 1H), 6.81 (dd, J=8.9, 2.8 Hz, 1H), 6.76 (dd, J=13.2, 2.8 Hz, 2H), 6.60 (d, J=2.8 Hz, 1H), 5.58 (p, J=2.0 Hz, 1H), 3.07 (s, 6H), 3.07 (s, 6H), 2.60-2.50 (m, 4H), 2.08 (tt, J=8.0, 6.7 Hz, 2H), 0.46 (s, 6H).

[0226] .sup.13C NMR (101 MHZ, CDCl.sub.3): 187.1, 151.1, 150.5, 149.8, 144.2, 141.0, 138.9, 132.2, 131.1, 128.9, 123.7, 115.8, 113.8, 113.8, 113.2, 40.1, 40.0, 36.5, 33.2, 24.6,-1.0.

[0227] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.24H.sub.30N.sub.2OSi: 391.2227, found: 392.2225.

##STR00070##

[0228] Compound 17. In a 10 ml tube, compound 11 (40 mg, 0.10 mmol), 1-cyclohexen-1-ylboronic acid pinacol ester (23 mg, 0.11 mmol, 1.1 equiv), K.sub.2CO.sub.3 (18 mg, 0.13 mmol, 1.3 equiv) and Pd(dppf) Cl.sub.2.Math.CH.sub.2Cl.sub.2 (4.1 mg, 5.0 mol, 5 mol %) were loaded. Dioxane (1.0 mL) and water (0.2 mL) were added. The mixture was sparged with argon for 30 min, and the tube was sealed and the mixture was stirred at 80 C. for 18 h. The reaction mixture was diluted with ethyl acetate (10 mL) and washed with sat. aq. NH.sub.4Cl (10 mL) and brine (10 mL). The organics were dried over Na.sub.2SO.sub.4, filtered, evaporated. The product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 0% to 40% ethyl acetate/hexane) and freeze-dried from dioxane to yield 17 mg (42%) of 17 as a light yellow solid.

[0229] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.21 (d, J=8.9 Hz, 1H), 6.81 (dd, J=8.9, 2.8 Hz, 1H), 6.77 (d, J=2.7 Hz, 1H), 6.73 (d, J=2.9 Hz, 1H), 6.53 (d, J=2.9 Hz, 1H), 5.50 (tt, J=3.7, 1.5 Hz, 1H), 3.08 (s, 6H), 3.07 (s, 6H), 2.26-2.13 (m, 4H), 1.88-1.79 (m, 2H), 1.78-1.68 (m, 2H), 0.46 (s, 6H).

[0230] .sup.13C NMR (101 MHz, CDCl.sub.3): 187.0, 151.0, 150.6, 149.8, 144.5, 141.1, 138.9, 132.3, 131.2, 120.3, 116.0, 113.8, 113.5, 113.2, 40.1, 40.0, 30.5, 25.6, 23.5, 22.4,-0.9.

[0231] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.25H.sub.32N.sub.2OSi: 405.2357, found: 405.2356.

##STR00071##

[0232] Compound 18. In a 10 ml tube, compound 11 (80 mg, 0.20 mmol), Pd(OAc).sub.2 (2.2 mg, 0.01 mmol, 5 mol %), triphenylphosphine (5.3 mg, 0.020 mol, 10 mol %), and toluene (2 mL) were degassed on a Schlenk line. tert-Butyl acrylate (128 mg, 1.0 mmol, 5 equiv) and triethylamine (30 mg, 0.30 mmol, 1.5 equiv) were added, the tube was sealed and the mixture was stirred at 120 C. for 24 h. The reaction mixture was diluted with ethyl acetate and poured into sat. aq. NH.sub.4Cl (20 mL), and extracted with ethyl acetate (310 ml). The combined organics were washed with brine (50 mL), dried over Na.sub.2SO.sub.4, filtered, and evaporated. The product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 0% to 50% ethyl acetate/hexane) and freeze-dried from dioxane to yield 41 mg (45%) of 18 as a yellow solid.

[0233] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.40 (d, J=15.6 Hz, 1H), 8.29 (d, J=9.0 Hz, 1H), 6.86-6.82 (m, 1H), 6.81 (d, J=2.8 Hz, 1H), 6.76 (d, J=2.7 Hz, 1H), 6.72 (d, J=2.7 Hz, 1H), 6.02 (d, J=15.5 Hz, 1H), 3.10 (s, 6H), 3.08 (s, 6H), 1.56 (s, 9H), 0.47 (s, 6H).

[0234] .sup.13C NMR (101 MHZ, CDCl.sub.3): 186.8, 166.8, 151.3, 150.6, 149.7, 141.6, 141.2, 139.0, 131.6, 131.1, 119.1, 115.4, 114.1, 113.7, 113.3, 80.0, 40.1, 40.0, 28.3,-1.0.

[0235] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.26H.sub.34N.sub.2O.sub.3Si: 451.2411, found: 451.2411.

##STR00072##

[0236] Compound 19. In a 100 mL round bottom flask, compound 11 (200 mg, 0.50 mmol), compound B1 (220 mg, 0.75 mmol, 1.5 equiv), K.sub.2CO.sub.3 (104 mg, 0.75 mmol, 1.5 equiv) and Pd(dppf) Cl.sub.2.Math.CH.sub.2Cl.sub.2 (20 mg, 25 mol, 5 mol %) were loaded. Dioxane (10 mL) and water (2 mL) were added. The mixture was sparged with argon for 30 min and then stirred at 90 C. for 3 h. Upon cooling, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with sat. aq. NH.sub.4Cl (50 mL) and brine (50 mL). The organics were dried over Na.sub.2SO.sub.4, filtered, evaporated. The product was isolated by flash chromatography on Biotage Isolera system (25 g Interchim SiHP 30 m cartridge, gradient 0% to 40% ethyl acetate/hexane) and freeze-dried from dioxane to yield 195 mg (79%) of 19 as a yellow solid.

[0237] .sup.1H NMR (400 MHz, CDCl.sub.3): 8.23 (d, J=8.9 Hz, 1H), 7.31-7.26 (m, 1H), 6.81 (dd, J=9.0, 2.8 Hz, 1H), 6.77-6.72 (m, 3H), 5.89 (dt, J=15.4, 6.9 Hz, 1H), 3.09 (s, 6H), 3.07 (s, 6H), 2.37-2.28 (m, 4H), 1.83 (p, J=7.6 Hz, 2H), 1.45 (s, 9H), 0.45 (s, 6H).

[0238] .sup.13C NMR (101 MHz, CDCl.sub.3): 173.4, 151.1, 150.7, 143.5, 141.1, 138.9, 134.9, 132.3, 131.2, 128.8, 114.2, 113.74, 113.68, 113.2, 80.0, 40.1, 40.0, 35.2, 32.5, 28.2, 25.1,-1.0.

[0239] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.29H.sub.42N.sub.2O.sub.3Si: 495.3037, found: 495.3032.

##STR00073##

[0240] Compound 20. To a solution of compound 19 (195 mg, 0.40 mmol) in CH.sub.2Cl.sub.2 (3.0 ml), trifluoroacetic acid (1.0 mL) was added dropwise. The resulting reaction mixture was stirred for 60 minutes at rt, protected from light. The volatiles were removed in vacuo by coevaporation with toluene (310 ml). The product was isolated by flash chromatography on a Biotage Isolera system (25 g Interchim SiHP 30 m cartridge, gradient 10% to 50% ethyl acetate/hexane) and freeze-dried from 1,4-dioxane to yield 172 mg (99%) of 20 as an orange solid.

[0241] .sup.1H NMR (400 MHZ, DMSO-d.sub.6): 12.03 (s, 1H), 7.98 (d, J=8.9 Hz, 1H), 7.16 (dt, J=15.5, 1.5 Hz, 1H), 6.89-6.80 (m, 3H), 6.73 (d, J=2.8 Hz, 1H), 5.88 (dt, J=15.5, 6.8 Hz, 1H), 3.06 (s, 6H), 3.03 (s, 6H), 2.33 (t, J=7.4 Hz, 2H), 2.21 (q, J=6.8 Hz, 2H), 1.71 (p, J=7.4 Hz, 2H), 0.43 (s, 6H).

[0242] .sup.13C NMR (101 MHZ, DMSO-d.sub.6): 186.5, 174.5, 150.9, 150.4, 142.4, 140.7, 138.3, 134.0, 131.0, 130.4, 128.4, 126.9, 114.3, 113.8, 113.0, 112.7, 39.5, 39.4, 33.1, 31.8, 24.2,-1.2.

[0243] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.25H.sub.32N.sub.2O.sub.3Si: 437.2255, found: 437.2257.

##STR00074##

[0244] Compound 20-NHS. In an amber vial, compound 20 (8.0 mg, 0.018 mmol) and TSTU (11 mg, 0.036 mmol, 2 equiv) were dissolved in DMF (200 L). 2,6-Lutidine (19 mg, 0.18 mmol) was added, and the reaction mixture was stirred for 3 h at rt. The volatiles were removed in vacuo. The product was isolated by flash chromatography on a Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 20% to 80% ethyl acetate/hexane) and freeze-dried from dioxane to yield 7.5 mg (78%) of 20-NHS as a beige solid.

[0245] .sup.1H NMR (400 MHZ, DMSO-d.sub.6): 7.99 (d, J=8.9 Hz, 1H), 7.21-7.12 (m, 1H), 6.88-6.80 (m, 3H), 6.73 (d, J=2.8 Hz, 1H), 5.88 (dt, J=15.5, 6.8 Hz, 1H), 3.06 (s, 6H), 3.03 (s, 6H), 2.85-2.78 (m, 6H), 2.30 (q, J=7.2, 6.5 Hz, 2H), 1.85 (p, J=7.4 Hz, 2H), 0.43 (s, 6H).

[0246] .sup.13C NMR (101 MHZ, DMSO-d.sub.6) 170.2, 169.0, 150.9, 150.5, 142.3, 140.7, 138.4, 134.7, 130.9, 130.5, 127.5, 126.9, 114.4, 113.8, 113.0, 112.8, 31.2, 30.3, 29.6, 25.4, 24.1,-1.2.

[0247] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.29H.sub.35N.sub.3O.sub.5Si: 534.2419, found: 534.2415.

##STR00075##

[0248] Compound 20-Halo. In an amber vial, compound 20 (22 mg, 0.050 mmol), HaloTag (02) Amine (17 mg, 0.075 mmol, 1.5 equiv), and HATU (1-[bis(dimethylamino) methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; 29 mg, 0.075 mmol, 1.5 equiv) were dissolved in DMF (200 L). DIPEA (32 mg, 0.25 mmol, 5 equiv) was added and the mixture stirred for 2 h at rt. The volatiles were removed in vacuo. The product was isolated by flash chromatography on a Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, 100% ethyl acetate) and freeze-dried from dioxane to yield 20-Halo as a yellow solid. Yield 33 mg (84%, remainder dioxane).

[0249] .sup.1H NMR (400 MHZ, DMSO-d.sub.6): 7.99 (d, J=8.9 Hz, 1H), 7.92 (t, J=5.6 Hz, 1H), 7.16-7.09 (m, 1H), 6.89-6.79 (m, 3H), 6.71 (d, J=2.8 Hz, 1H), 5.84 (dt, J=15.5, 6.9 Hz, 1H), 3.57 (t, J=6.6 Hz, 2H), 3.48-3.44 (m, 2H), 3.44-3.39 (m, 4H), 3.31-3.27 (m, 4H), 3.24 (q, J=5.8 Hz, 2H), 3.06 (s, 6H), 3.03 (s, 6H), 2.22-2.13 (m, 4H), 1.75-1.60 (m, 4H), 1.46-1.37 (m, 2H), 1.37-1.10 (m, 6H), 0.43 (s, 6H).

[0250] .sup.13C NMR (101 MHZ, DMSO-d.sub.6): 186.7, 172.2, 151.1, 150.6, 142.7, 140.9, 138.5, 134.4, 131.0, 130.6, 128.5, 126.9, 114.4, 113.9, 113.1, 112.9, 70.2, 69.6, 69.4, 69.2, 45.3, 38.6, 34.7, 32.0, 31.9, 29.0, 26.1, 25.1, 24.9, 1.1.

[0251] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.35H.sub.52ClN.sub.3O.sub.4Si: 642.3488, found: 642.3487.

##STR00076##

[0252] Compound 20-BG. In an amber vial, compound 20 (10.4 mg, 0.024 mmol) and TSTU (14 mg, 0.048 mmol, 2 equiv) were dissolved in DMF (240 L). DIPEA (31 mg, 0.24 mmol) was added, and the mixture was stirred for 30 min at rt. 6-((4-(Aminomethyl) benzyl) oxy)-7H-purin-2-amine (20.0 mg, 0.036 mmol, 1.5 equiv) was then added, and the reaction mixture was stirred at rt for 30 min. The volatiles were removed in vacuo. The product was isolated by flash chromatography on a Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 0% to 10% methanol/dichloromethane) and freeze-dried from dioxane to yield 15.1 mg (92%) of 20-BG as a yellow solid.

[0253] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.38H.sub.44N.sub.8O.sub.3Si: 689.3378, found: 689.3378.

##STR00077##

[0254] Compound 21-Maleimide. In an amber vial, compound 20 (22 mg, 0.050 mmol) and TSTU (30 mg, 0.10 mmol) were dissolved in DMF (500 L) and DIPEA (64 mg, 0.25 mmol) was added and stirred for 60 min. at rt. 1-(1-Aminoethyl) maleimide hydrochloride (18 mg, 0.10 mmol) was added and the reaction was stirred at rt for 60 min. The volatiles were removed in vacuo. The product was isolated by flash chromatography on a Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 50% to 100% ethyl acetate/hexane) and freeze-dried from dioxane to yield 18.8 mg (67%) of 20-Maleimide as a yellow solid.

[0255] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.24 (t, J=5.9 Hz, 1H), 8.04 (d, J=9.0 Hz, 1H), 7.01 (d, J=15.5 Hz, 1H), 6.85 (dd, J=9.0, 2.8 Hz, 1H), 6.76 (d, J=2.7 Hz, 1H), 6.75 (d, J=2.8 Hz, 1H), 6.71 (d, J=2.8 Hz, 1H), 6.50 (s, 2H), 5.67 (dt, J=15.4, 7.2 Hz, 1H), 3.78 (dd, J=6.6, 4.6 Hz, 2H), 3.68-3.58 (m, 2H), 3.10 (s, 12H), 2.41-2.33 (m, 2H), 2.31-2.21 (m, 2H), 1.93-1.82 (m, 2H), 0.46 (s, 6H).

[0256] .sup.13C NMR (101 MHz, CDCl.sub.3): 188.1, 175.3, 171.0, 151.4, 151.1, 144.2, 141.8, 139.5, 136.8, 134.1, 131.5, 131.4, 128.7, 127.7, 114.3, 114.1, 113.9, 113.5, 40.2, 40.1, 38.5, 38.1, 34.3, 31.2, 24.8,-0.8.

[0257] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.31H.sub.38N.sub.4O.sub.4Si: 559.2735, found: 559.2735.

##STR00078##

[0258] Compound 20-Tz. In an amber vial, compound 20 (12.0 mg, 0.027 mmol) and TSTU (16 mg, 0.053 mmol, 2 equiv) were dissolved in DMF (250 L). DIPEA (33 mg, 0.25 mmol) was added, and the mixture was stirred for 30 min at rt. 4-(1,2,4,5-Tetrazin-3-yl) benzylamine hydrochloride (Sigma-Aldrich, Cat. Nr. 761591; 9.0 mg, 0.040 mmol, 1.5 equiv) and additional DIPEA (8.2 mg, 0.06 mmol, 2.2 equiv) were added, and the reaction was stirred at rt overnight. The volatiles were removed in vacuo. The product was isolated by flash chromatography on a Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 50% to 100% ethyl acetate/hexane) and freeze-dried from dioxane to yield 7.0 mg (43%) of 20-Tz as an orange solid.

[0259] .sup.1H NMR (400 MHZ, DMSO-d.sub.6): 10.57 (s, 1H), 8.52 (t, J=6.0 Hz, 1H), 8.45 (d, J=8.4 Hz, 2H), 7.93 (d, J=8.9 Hz, 1H), 7.56 (d, J=8.4 Hz, 2H), 7.15 (d, J=15.6 Hz, 1H), 6.84 (t, J=3.0 Hz, 2H), 6.79 (dd, J=9.0, 2.8 Hz, 1H), 6.73 (d, J=2.8 Hz, 1H), 5.88 (dt, J=15.5, 6.9 Hz, 1H), 4.45 (d, J=5.8 Hz, 2H), 3.30 (s, 6H), 3.05 (s, 6H), 3.02 (s, 7H), 2.32 (t, J=7.2 Hz, 2H), 2.22 (q, J=6.8 Hz, 2H), 1.78 (p, J=7.3 Hz, 2H), 0.42 (s, 6H).

[0260] .sup.13C NMR (101 MHz, DMSO-d.sub.6): 186.7, 172.4, 165.4, 158.1, 151.0, 150.6, 145.1, 142.6, 140.9, 138.5, 134.3, 131.0, 130.6, 130.3, 128.6, 128.1, 127.8, 126.9, 114.5, 113.9, 113.0, 112.9, 41.9, 39.3, 39.4, 34.7, 32.0, 25.1,-1.1.

[0261] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.34H.sub.39N.sub.7O.sub.2Si: 606.3007, found: 606.3007.

##STR00079##

[0262] Compound 20-CTX. In an amber vial, a solution of HATU (13 mg, 0.035 mmol, 1.5 equiv) in DMF (50 L) was added to the mixture of compound 20 (10 mg, 0.023 mmol), H.sub.2N-CTX.Math.HCO.sub.2H [A. N. Butkevich et al. ACS Chem. Biol. 2018, 13 (2), 475-480](23 mg, 0.03 mmol, 1.3 equiv) in DIPEA (100 L) and DMF (100 L). The reaction mixture was stirred at rt for 1 h. The solvents were removed in vacuo, and the product the product was isolated by preparative HPLC (Interchim Uptisphere Strategy PhC4 25021.2 mm 5 m, solvent flow rate 18 mL/min, gradient 50% to 90% A: B, A-acetonitrile+0.1% (v/v) formic acid, B-water+0.1% (v/v) formic acid) and freeze-dried from dioxane to give 17 mg (64%) of yellow solid.

[0263] .sup.1H NMR (400 MHZ, acetone-d.sub.6): 8.27 (d, J=9.0 Hz, 1H), 8.19 (d, J=9.0 Hz, 1H), 8.14-8.08 (m, 2H), 7.68-7.62 (m, 1H), 7.62-7.52 (m, 4H), 7.43-7.34 (m, 3H), 7.32-7.26 (m, 1H), 6.96 (t, J=3.1 Hz, 2H), 6.81 (dd, J=9.1, 2.8 Hz, 1H), 6.77 (d, J=2.8 Hz, 1H), 6.23 (td, J=9.2, 1.8 Hz, 1H), 5.78-5.62 (m, 3H), 4.99 (d, J=7.2 Hz, 1H), 4.95 (dd, J=9.7, 2.0 Hz, 1H), 4.84 (s, 1H), 4.82-4.77 (m, 1H), 4.18-4.11 (m, 2H), 3.93 (dd, J=10.7, 6.5 Hz, 1H), 3.86 (d, J=7.0 Hz, 1H), 3.77 (s, 1H), 3.39 (s, 3H), 3.29 (s, 3H), 3.12 (s, 6H), 3.10 (s, 6H), 2.82 (s, 3H), 2.71 (ddd, J=14.1, 9.7, 6.5 Hz, 1H), 2.57-2.39 (m, 2H), 2.37 (s, 3H), 2.32-2.12 (m, 3H), 1.98 (d, J=1.5 Hz, 3H), 1.92-1.70 (m, 2H), 1.67 (s, 3H), 1.66-1.57 (m, 1H), 1.41 (s, 1H), 1.21 (s, 3H), 1.18 (s, 3H), 0.47 (s, 6H).

[0264] .sup.13C NMR (101 MHz, acetone-d.sub.6): 205.5, 188.4, 174.0, 173.9, 173.5, 171.1, 166.6, 152.4, 152.0, 145.1, 142.5, 140.8, 140.0, 139.6, 137.6, 136.6, 134.0, 132.5, 132.4, 131.3, 130.9, 129.4, 129.3, 128.9, 128.4, 128.3, 128.2, 115.4, 114.9, 114.6, 114.0, 84.8, 83.6, 82.0, 81.7, 78.7, 78.6, 76.7, 75.7, 74.9, 74.8, 72.3, 67.6, 57.6, 57.4, 57.2, 56.4, 48.1, 44.30, 44.28, 40.1, 40.0, 36.8, 34.9, 32.9, 32.1, 27.4, 25.9, 23.0, 21.9, 14.8, 10.8, 0.86, 0.88.

[0265] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.65H.sub.79N.sub.3O.sub.14Si 1154.5404; Found 1154.5407.

##STR00080##

[0266] Compound 20-Phalloidin. In an amber vial, compound 20-NHS (2.0 mg, 3.8 mol, 2.9 equiv) and Lys.sup.7-Phalloidin (trifluoroacetate salt, Bachem Cat. Nr. H-7636; 1 mg, 1.3 mol) were dissolved in DMF (200 L). DIPEA (90 L) was added, and the reaction mixture was at rt for 18 h. The solvents were removed in vacuo, and the product the product was isolated by preparative HPLC (Interchim Uptisphere Strategy PhC4 25021.2 mm 5 m, solvent flow rate 18 mL/min, gradient 40% to 85% A: B, A-acetonitrile+0.1% (v/v) formic acid, B-water+0.1% (v/v) formic acid) and freeze-dried from dioxane to give 1.5 mg (97%) of 20-Phalloidin as light pink solid.

[0267] HRMS (ESI) m/z: [M+2H].sup.2+ Calcd for C.sub.60H.sub.79N.sub.11O.sub.11SSi: 595.7798, found: 595.7786.

##STR00081##

[0268] Compound 21. In a 10 mL round-bottom flask, compound 11 (80 mg, 0.20 mmol), compound B2 [N. L. Reed et al. Org. Lett. 2018, 20, 7345-7350](88 mg, 0.26 mmol, 1.3 equiv), Pd(dppf) Cl.sub.2.Math.CH.sub.2Cl.sub.2 (8.2 mg, 0.01 mmol, 5 mol %) and K.sub.2CO.sub.3 (82 mg, 0.6 mmol, 3 equiv) were placed, dioxane (1.7 mL) and water (0.3 mL) were added, the mixture was degassed and stirred at 80 C. for 6 h. On cooling, the reaction mixture was diluted with ethyl acetate and poured into brine (50 mL), extracted with ethyl acetate (325 mL), and the combined extracts were dried over Na.sub.2SO.sub.4. The product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 20% to 80% ethyl acetate/hexane) and freeze-dried from dioxane to give 101 mg (97%) of 21 as yellow solid.

[0269] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.23 (d, J=8.9 Hz, 1H), 7.25 (d, J=15.5 Hz, 1H), 6.81 (dd, J=9.0, 2.8 Hz, 1H), 6.78-6.73 (m, 3H), 5.88 (dt, J=15.5, 6.8 Hz, 1H), 4.84 (br.s, 1H), 3.19 (q, J=6.5 Hz, 2H), 3.09 (s, 6H), 3.07 (s, 6H), 2.36-2.27 (m, 2H), 1.68-1.53 (m, 4H), 1.44 (s, 9H), 0.45 (s, 6H).

[0270] .sup.13C NMR (101 MHZ, CDCl.sub.3): 188.1, 156.3, 151.2, 150.8, 143.8, 141.3, 139.1, 134.8, 132.3, 131.4, 129.3, 128.4, 114.3, 113.9, 113.8, 113.4, 40.6, 40.2, 40.1, 32.6, 29.4, 28.6, 26.7,-0.8.

[0271] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.30H.sub.43N.sub.3O.sub.3Si 522.3146; Found 522.3143.

##STR00082##

[0272] Compound 21-Pepstatin. To a solution of Pepstatin A (19 mg, 27.6 mol, 1.2 equiv) in the mixture of DIPEA (40 L) and DMSO (800 L), a solution of TSTU (N,N,N,N-tetramethyl-O(N-succinimidyl) uronium tetrafluoroborate; 10 mg, 33 mol, 1.2 equiv relative to Pepstatin A) in DMSO (100 L) was added, and the reaction mixture was stirred at rt for 1.5 h to give the solution of Pepstatin A NHS ester in DMSO.

[0273] Separately, in a 10 mL round-bottom flask, a solution of compound 22 (12 mg, 23 mol) in dichloromethane (3 mL) and trifluoroacetic acid (300 L) was stirred at rt for 30 min. The mixture was then diluted with toluene (2 mL), evaporated to dryness and chased twice with 3 mL toluene. Afterwards, the solution of Pepstatin A NHS ester in DMSO was added to the dry residue followed by additional DIPEA (50 L). The reaction mixture was stirred at rt for 1 h, excess DIPEA was evaporated and the product was isolated from the crude mixture by preparative HPLC (Interchim Uptisphere Strategy PhC4 25021.2 mm 5 m, solvent flow rate 18 mL/min, gradient 40% to 90% A: B, A-acetonitrile+0.1% (v/v) formic acid, B-water+0.1% (v/v) formic acid) and freeze-dried from dioxane-water to give 13 mg (52%) of light yellow solid.

[0274] .sup.1H NMR (400 MHZ, DMSO-d.sub.6): 8.00-7.89 (m, 2H), 7.85-7.71 (m, 2H), 7.47 (d, J=8.8 Hz, 1H), 7.33 (d, J=9.1 Hz, 1H), 7.17 (dt, J=15.5, 1.4 Hz, 1H), 6.88-6.81 (m, 3H), 6.72 (d, J=2.7 Hz, 1H), 5.88 (dt, J=15.5, 6.8 Hz, 1H), 4.87 (d, J=5.1 Hz, 1H), 4.83 (d, J=4.9 Hz, 1H), 4.25 (p, J=7.1 Hz, 1H), 4.18 (dd, J=8.8, 7.3 Hz, 1H), 4.13 (dd, J=8.9, 7.2 Hz, 1H), 3.88-3.73 (m, 4H), 3.15-3.04 (m, 1H), 3.05 (s, 6H), 3.03 (s, 6H), 2.19 (q, J=6.8 Hz, 2H), 2.15-2.00 (m, 5H), 2.01-1.87 (m, 3H), 1.60-1.43 (m, 6H), 1.41-1.30 (m, 1H), 1.28-1.17 (m, 5H), 0.89-0.75 (m, 28H), 0.43 (s, 6H).

[0275] .sup.13C NMR (101 MHz, DMSO-d.sub.6): 186.7, 172.2, 171.6, 171.1, 170.8, 170.70, 170.65, 162.3, 151.0, 150.5, 142.5, 140.8, 138.4, 133.6, 131.1, 130.6, 129.2, 127.0, 114.4, 113.9, 113.1, 112.7, 69.2, 69.0, 58.0, 57.8, 50.7, 50.5, 48.3, 44.4, 38.6, 38.4, 35.8, 34.4, 32.3, 30.8, 30.4, 30.3, 30.1, 28.8, 26.4, 25.7, 24.2, 23.5, 23.3, 22.3, 21.9, 21.6, 21.1, 19.30, 19.25, 18.4, 18.3, 18.2,-1.1.

[0276] HRMS (ESI) m/z: [M+2H].sup.2+ Calcd for C.sub.59H.sub.96N.sub.8O.sub.9Si 545.3608; Found 545.3599.

##STR00083##

[0277] Compound 21-TPP. A suspension of 4-(carboxybutyl) triphenylphosphonium bromide (44 mg, 0.1 mmol) in dry dichloromethane (1 mL) was cooled in ice-water bath, and oxalyl chloride (85 L, 1.0 mmol, 10 equiv) was added. The mixture was allowed to warm up to rt and stirred for 15 min. The resulting clear light-yellow solution was evaporated to dryness and chased with dry dichloromethane (1 mL). The residue was dissolved in dry dichloromethane (1 mL) to give 0.1 M solution of TPP-C.sub.4COCl.

[0278] Separately, in a 10 mL round-bottom flask, a solution of compound 21 (12 mg, 23 mol) in dichloromethane (3 mL) and trifluoroacetic acid (300 L) was stirred at rt for 30 min. The mixture was then diluted with toluene (2 mL), evaporated to dryness and chased twice with 3 mL toluene.

[0279] Afterwards, the residue was dissolved in dry dichloromethane (1 mL), and DIPEA (30 L) and the solution of TPP-C.sub.4COCl (350 L of 0.1 M in dichloromethane, 35 mol) were added. The reaction mixture was stirred at rt for 1 h, the solvents were evaporated and the product was isolated from the residue by preparative HPLC (12 g Interchim Uptisphere Strategy PhC4 25021.2 mm 5 m, solvent flow rate 18 mL/min, gradient 40% to 90% A: B, A-acetonitrile+0.1% (v/v) TFA, B-water+0.1% (v/v) TFA) and freeze-dried from dioxane to give 20 mg (98%) of light pink viscous oil (trifluoroacetate salt).

[0280] .sup.1H NMR (400 MHZ, DMSO-d.sub.6): 7.97 (d, J=8.9 Hz, 1H), 7.92-7.85 (m, 3H), 7.82-7.71 (m, 12H), 7.16 (dt, J=15.5, 1.5 Hz, 1H), 6.87 (d, J=2.7 Hz, 1H), 6.85 (d, J=2.8 Hz, 1H), 6.82 (dd, J=9.0, 2.7 Hz, 1H), 6.71 (d, J=2.8 Hz, 1H), 5.87 (dt, J=15.5, 6.8 Hz, 1H), 3.62-3.51 (m, 2H), 3.05 (s, 6H), 3.03 (s, 6H), 2.21-2.07 (m, 4H), 1.71 (p, J=7.3 Hz, 2H), 1.58-1.46 (m, 2H), 1.45-1.39 (m, 4H), 0.43 (s, 6H).

[0281] .sup.19F NMR (376 MHZ, DMSO-d.sub.6): -74.5.

[0282] .sup.31P NMR (162 MHZ, DMSO-d.sub.6): 23.9.

[0283] .sup.13C NMR (101 MHz, DMSO-d.sub.6): 186.7, 171.3, 158.1 (q, .sup.2J.sub.C-F=35.1 Hz), 151.0, 150.5, 142.5, 140.8, 138.5, 134.92, 134.89, 133.64, 133.59, 133.5, 131.1, 130.5, 130.3, 130.2, 128.1 (q, .sup.1J.sub.C-F=213.7 Hz), 118.9, 118.1, 117.5, 114.6, 114.5, 114.0, 113.15, 112.7, 38.3, 34.4, 32.2, 30.4, 28.8, 26.31, 26.25, 26.1, 21.4, 21.3, 20.2, 19.7,-1.1 (CP multiplets were not interpreted).

[0284] HRMS (ESI) m/z: [M+H].sup.2+ Calcd for C.sub.48H.sub.58N.sub.3O.sub.2PSi 383.7012; Found 383.7008.

##STR00084##

[0285] Compound 22. In a 10 mL tube, compound A4 [S. Shen et al., RSC Adv., 2017, 7 (18) 10922-10927](86 mg, 0.25 mmol), bis(pinacolato) diboron (70 mg, 0.28 mmol, 1.1 equiv), (1,5-cyclooctadiene)(methoxy) iridium (I) dimer (8.2 mg, 0.012 mmol, 5 mol %), triphenylarsine (7.7 mg, 0.025 mmol, 10 mol %) in dry n-octane (2.5 mL) was degassed on a Schlenk line and stirred at 120 C. for 15 h. The reaction mixture was diluted with dichloromethane and evaporated onto Celite. The product was isolated by flash chromatography on a Biotage Isolera system (25 g Interchim SiHP 30 m cartridge, gradient 0% to 20% ethyl acetate/dichloromethane) and freeze-dried from 1,4-dioxane to yield 70 mg (80%) of 22 as a yellow solid.

[0286] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.35 (d, J=8.8 Hz, 1H), 6.66 (d, J=2.2 Hz, 1H), 6.45 (d, J=2.4 Hz, 1H), 6.41 (dd, J=8.7, 2.5 Hz, 1H), 6.33 (d, J=2.2 Hz, 1H), 4.18-4.03 (m, 8H), 2.53-2.36 (m, 4H), 1.41 (s, 12H), 0.35 (s, 6H).

[0287] .sup.13C NMR (101 MHz, CDCl.sub.3): 187.4, 153.7, 153.4, 145.7, 139.1, 133.7, 131.5, 122.6, 114.0, 113.2, 113.0, 110.7, 80.3, 51.3, 51.2, 25.6, 16.44, 16.36,-1.4.

[0288] HRMS (ESI) m/z: [M].sup.+ Calcd for C.sub.21H.sub.24BN.sub.2O.sub.2Si: 375.1699, found: 375.1701-corresponds to the pinacol ester hydrolysis product (22a):

##STR00085##

[0289] Compound 23. In a 25 ml flask, compound 22 (64 mg, 0.13 mmol), copper (II) bromide (90 mg, 0.40 mmol, 3 equiv) and potassium fluoride (31 mg, 0.54 mmol, 4 equiv) were loaded. DMSO (5 mL), water (500 L) and pyridine (210 mg, 2.7 mmol, 20 equiv) were added, and the reaction mixture was stirred at 80 C. for 45 minutes. The reaction mixture was then quenched with water (20 mL) and extracted with ethyl acetate (320 mL). The combined organics were washed with brine (75 mL), dried over Na.sub.2SO.sub.4, filtered, and evaporated. The product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 0% to 10% ethyl acetate/dichloromethane) and freeze-dried from dioxane to yield 46 mg (83%) of 23 as a yellow solid.

[0290] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.15 (d, J=8.6 Hz, 1H), 6.75 (d, J=2.4 Hz, 1H), 6.51 (dd, J=8.7, 2.5 Hz, 1H), 6.46-6.40 (m, 2H), 4.01 (td, J=7.3, 2.0 Hz, 8H), 2.50-2.35 (m, 4H), 0.43 (s, 6H).

[0291] .sup.13C NMR (101 MHZ, CDCl.sub.3): 186.6, 152.4, 151.8, 142.2, 138.0, 132.8, 131.1, 128.6, 124.8, 118.8, 112.8, 112.4, 112.3, 51.8, 51.7, 16.7, 16.6,-1.3.

[0292] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.21H.sub.23BrN.sub.2OSi: 427.0836, found: 427.0833.

##STR00086##

[0293] Compound 24. In a 10 ml tube, compound 23 (43 mg, 0.10 mmol), compound B1 (44 mg, 0.15 mmol), K.sub.2CO.sub.3 (21 mg, 0.15 mmol) and Pd(dppf) Cl.sub.2.Math.CH.sub.2Cl.sub.2 (4.1 mg, 5.0 mol, 5 mol %) were loaded. Dioxane (1.0 mL) and water (0.2 mL) were added. The mixture was sparged with argon for 30 min, and the tube was sealed and the mixture was stirred at 80 C. for 18 h. The reaction mixture was quenched with sat. aq. NH.sub.4Cl (10 mL), and extracted with ethyl acetate (310 mL). The combined organics were washed with brine (50 mL), dried over Na.sub.2SO.sub.4, filtered, and evaporated. The product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 0% to 30% ethyl acetate/hexane) and freeze-dried from dioxane to yield 37 mg (72%) of 24 as yellow solid.

[0294] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.17 (d, J=8.7 Hz, 1H), 7.21 (dt, J=15.6, 1.5 Hz, 1H), 6.50 (dd, J=8.7, 2.6 Hz, 1H), 6.48-6.41 (m, 3H), 5.88 (dt, J=15.4, 6.9 Hz, 1H), 4.06-3.96 (m, 8H), 2.49-2.36 (m, 4H), 2.36-2.25 (m, 4H), 1.82 (p, J=7.6 Hz, 2H), 1.45 (s, 9H), 0.42 (s, 6H).

[0295] .sup.13C NMR (101 MHz, CDCl.sub.3): 188.3, 173.3, 152.3, 151.9, 143.2, 140.9, 138.7, 134.3, 133.2, 131.0, 129.1, 129.0, 113.1, 112.6, 112.4, 112.2, 79.9, 51.82, 51.81, 35.2, 32.5, 28.2, 25.1, 16.7,-1.2.

[0296] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.31H.sub.40N.sub.2O.sub.3Si: 517.2881, found: 517.2879.

##STR00087##

[0297] Compound 25. To a solution of compound 24 (32 mg; 0.062 mmol) in CH.sub.2Cl.sub.2 (600 L) trifluoroacetic acid (200 L) was added dropwise. The resulting reaction mixture was stirred for 60 minutes at rt, protected from light. The volatiles were removed in vacuo by coevaporation with toluene (310 ml). The product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 20% to 80% ethyl acetate/hexane) and freeze-dried from 1,4-dioxane to yield 19 mg (67%) of 25 as yellow solid.

[0298] .sup.1H NMR (400 MHZ, DMSO-d.sub.6): 12.01 (s, 1H), 7.95 (d, J=8.7 Hz, 1H), 7.09 (d, J=15.5 Hz, 1H), 6.55 (d, J=2.5 Hz, 1H), 6.53 (d, J=2.5 Hz, 1H), 6.50 (dd, J=8.7, 2.5 Hz, 1H), 6.42 (d, J=2.5 Hz, 1H), 5.85 (dt, J=15.5, 6.8 Hz, 1H), 4.02-3.90 (m, 8H), 2.41-2.27 (m, 6H), 2.19 (q, J=6.7 Hz, 2H), 1.70 (p, J=7.4 Hz, 2H), 0.40 (s, 6H).

[0299] .sup.13C NMR (101 MHz, DMSO-d.sub.6): 186.9, 174.5, 152.2, 151.8, 142.3, 140.5, 138.2, 133.6, 132.0, 130.4, 128.7, 127.8, 113.3, 112.8, 112.0, 111.5, 51.5, 51.4, 33.1, 31.9, 24.3, 16.2,-1.3.

[0300] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.27H.sub.32N.sub.2O.sub.3Si: 461.2255, found: 461.2253.

##STR00088##

[0301] Compound 27-Maleimide. In an amber vial, compound 27 (8.4 mg, 0.018 mmol) and TSTU (12 mg, 0.036 mmol) were dissolved in DMF (180 L) and DIPEA (23 mg, 0.18 mmol) was added and stirred for 1.5 hours at rt, then 1-(2-aminoethyl) maleimide hydrochloride was added and the reaction mixture was stirred for 1.5 hours at rt. The volatiles were removed in vacuo. The product was isolated by flash chromatography on a Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 50% to 100% ethyl acetate/hexane) and freeze-dried from 1,4-dioxane to yield 6.7 mg (89%) of 27-Maleimide as a yellow solid.

[0302] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.01 (d, J=8.7 Hz, 2H), 6.97 (d, J=15.4 Hz, 1H), 6.54 (dd, J=8.7, 2.5 Hz, 1H), 6.50 (s, 2H), 6.46 (d, J=2.5 Hz, 1H), 6.43 (d, J=2.5 Hz, 1H), 6.40 (d, J=2.5 Hz, 1H), 5.66 (dt, J=15.4, 7.2 Hz, 1H), 4.03 (t, J=7.3 Hz, 8H), 3.80-3.75 (m, 2H), 3.62 (q, J=5.8 Hz, 2H), 2.44 (p, J=7.3 Hz, 4H), 2.37-2.30 (m, 2H), 2.25 (q, J=6.7, 6.3 Hz, 2H), 1.92-1.82 (m, 2H), 0.43 (s, 6H).

[0303] .sup.13C NMR (101 MHZ, CDCl.sub.3): 174.4, 170.9, 152.4, 152.0, 143.8, 141.4, 139.1, 136.1, 133.9, 132.3, 131.0, 128.7, 113.0, 112.7, 112.6, 112.2, 51.8, 51.7, 38.2, 38.1, 34.3, 31.1, 24.7, 16.7,-1.1.

[0304] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.33H.sub.38N.sub.4O.sub.4Si: 583.2735, found: 583.2733.

##STR00089##

[0305] Compound 26. In a dried 10 mL crimp-top tube (a 2-5 ml Biotage microwave vial was used), compound A5 (134 mg, 0.25 mmol; prepared according to the literature procedure: S. Jia et al. ACS Chem. Biol. 2018, 13 (7), 1844-1852), RuPhos Pd G4 (32 mg, 37.5 mol, 15 mol %), RuPhos (18 mg, 37.5 mol, 15 mol %) and Cs.sub.2CO.sub.3 (245 mg, 0.75 mmol, 3 equiv) were placed, and the contents of the vial were degassed on a Schlenk line. Anhydrous dioxane (1 mL) and 2,2,2-trifluoroethylamine (1 mL) were then injected, the vial was placed in a 80 C. oil bath and the reaction mixture was stirred for 18 h. The mixture was then cooled down to rt, diluted with dichloromethane and filtered through a 1 cm plug of Celite, washing with dichloromethane and ethyl acetate (30 mL each). The filtrate was evaporated on silica and the product was isolated by flash chromatography on Biotage Isolera system (24 g Interchim SiHP 30 m cartridge, gradient 20% to 80% ethyl acetate/hexane) and freeze-dried from dioxane to give light yellow solid containing 1 equiv of dioxane. Yield 125 mg (96%).

[0306] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.38-8.34 (m, 2H), 6.85-6.80 (m, 4H), 4.44 (t, J=6.8 Hz, 2H), 3.88 (qd, J=8.7, 6.8 Hz, 4H), 0.44 (s, 6H).

[0307] .sup.19F NMR (376 MHZ, CDCl.sub.3): -72.1.

[0308] .sup.13C NMR (101 MHZ, CDCl.sub.3): 185.2, 148.5, 141.0, 132.5, 132.2, 124.9 (q, .sup.1J.sub.C-F=280.1 Hz), 116.0, 114.3, 45.35 (q, .sup.2J.sub.C-F=34.2 Hz), -1.3.

[0309] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.19H.sub.18F.sub.6N.sub.2OSi 433.1165; Found 433.1165.

##STR00090##

[0310] Compound 27. In a 10 mL round-bottom flask, the mixture of compound 26 (60 mg, 0.139 mmol), bis(pinacolato) diboron (29 mg, 0.153 mmol, 1.1 equiv), [Ir (cod)(OMe)].sub.2 (4.6 mg, 6.95 mol, 5 mol %) and triphenylarsine (4.3 mg, 13.9 mol, 10 mol %) in dry n-octane (2 mL) was degassed on a Schlenk line and stirred at 120 C. for 22 h. On cooling, the reaction mixture was diluted with dichloromethane, evaporated on Celite and the product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 2% to 50% ethyl acetate: dichloromethane) and freeze-dried from dioxane to give 59 mg (76%) of 27 as bright yellow solid.

[0311] .sup.1H NMR (400 MHZ, DMSO-ds): 8.09 (d, J=8.9 Hz, 1H), 7.54 (t, J=6.9 Hz, 1H), 7.20 (t, J=7.0 Hz, 1H), 7.18 (d, J=2.4 Hz, 1H), 7.04 (d, J=2.3 Hz, 1H), 6.99 (dd, J=8.9, 2.4 Hz, 1H), 6.84 (d, J=2.3 Hz, 1H), 4.27-4.08 (m, 2H), 1.25 (s, 12H), 0.42 (s, 6H).

[0312] .sup.19F NMR (376 MHZ, DMSO-d.sub.6): -70.4.

[0313] .sup.13C NMR (101 MHz, DMSO-d.sub.6): 185.8, 152.5, 151.5, 145.2, 139.5, 132.1, 131.8, 125.6 (q, .sup.1J.sub.C-F=281.3 Hz), 125.5 (q, .sup.1J.sub.C-F=281.0 Hz), 123.2, 116.8, 115.8, 115.4, 114.1, 79.9, 43.2 (q, .sup.2J.sub.C-F=32.7 Hz), 43.0 (d, .sup.2J.sub.C-F=32.3 Hz), 25.3,-1.7.

[0314] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.25H.sub.29BF.sub.6N.sub.2O.sub.3Si 559.2022; Found 559.2023.

##STR00091##

[0315] Compound 28. In a 10 mL round-bottom flask, compound 27 (53 mg, 0.095 mmol), potassium fluoride (22 mg, 0.380 mmol, 4 equiv), copper (II) bromide (64 mg, 0.285 mmol, 3 equiv) were placed, followed by addition of DMSO (1 mL), water (100 L) and pyridine (155 L, 1.90 mmol, 20 equiv). The reaction mixture was stirred at 80 C. for 30 min. Upon cooling to rt, the mixture was diluted with ethyl acetate and poured into water (80 mL). The product was extracted with ethyl acetate (325 mL), the combined extracts were washed with brine (50 mL) and dried over Na.sub.2SO.sub.4. The product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 10% to 60% ethyl acetate: hexane) and freeze-dried from dioxane to give 50 mg (95%, contains 0.5 equiv. dioxane) of 28 as yellow solid.

[0316] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.13 (d, J=8.6 Hz, 1H), 7.08 (d, J=2.5 Hz, 1H), 6.81 (dd, J=8.6, 2.6 Hz, 1H), 6.79 (d, J=2.5 Hz, 1H), 6.77 (d, J=2.6 Hz, 1H), 4.40 (t, J=7.0 Hz, 1H), 4.37 (t, J=6.9 Hz, 1H), 3.92-3.79 (m, 4H), 0.44 (s, 6H).

[0317] .sup.19F NMR (376 MHZ, CDCl.sub.3): -72.06,-72.13.

[0318] .sup.13C NMR (101 MHz, CDCl.sub.3): 186.6, 148.2, 147.8, 142.7, 138.7, 134.9, 131.8, 131.1, 125.3, 124.9 (q, .sup.1J.sub.C-F=280.2 Hz), 124.7 (q, .sup.1J.sub.C-F=280.2 Hz), 120.9, 115.7, 115.4, 114.4, 45.4 (q, .sup.2J.sub.C-F=34.0 Hz), 45.2 (q, .sup.2J.sub.C-F=34.4 Hz), -1.4.

[0319] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.19H.sub.17BrF.sub.6N.sub.2OSi 513.0252; Found 513.0250.

##STR00092##

[0320] Compound 29. In a 10 mL tube, compounds 28 (46 mg, 0.090 mmol), B3 (52 mg, 0.15 mmol, 1.6 equiv; known compound: Wang et al. Tet. Lett., 2005, 46 (50), 8777-8780), K.sub.2CO.sub.3 (16 mg, 0.12 mmol, 3 equiv) and Pd(dppf) Cl.sub.2.Math.CH.sub.2Cl.sub.2 (3.7 mg, 4.5 mol, 5 mol %) were loaded. Dioxane (900 L) and water (180 L) were added. The mixture was sparged with argon for 30 min, and the tube was sealed and the mixture was stirred at 80 C. for 18 h. The reaction mixture was quenched with sat. aq. NH.sub.4Cl (10 mL) and extracted with ethyl acetate (310 mL). The combined organics were washed with brine (50 mL), dried over Na.sub.2SO.sub.4, filtered, and evaporated. The product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m, gradient 0% to 20% ethyl acetate/hexane) and freeze-dried from dioxane to yield 50 mg (86%) of 29 as an orange oil.

[0321] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.16 (dd, J=8.1, 1.0 Hz, 1H), 7.17 (dt, J=15.7, 1.5 Hz, 1H), 6.82-6.72 (m, 4H), 5.94 (dt, J=15.5, 6.9 Hz, 1H), 4.33-4.26 (m, 2H), 3.94-3.82 (m, 4H), 3.66 (t, J=6.4 Hz, 2H), 2.30 (qd, J=7.1, 1.5 Hz, 2H), 1.67-1.50 (m, 4H), 0.90 (s, 9H), 0.43 (s, 6H), 0.06 (s, 6H).

[0322] .sup.13C NMR (101 MHZ, CDCl.sub.3): 188.2, 147.8, 147.3, 143.9, 141.4, 139.2, 135.3, 132.7, 131.5, 131.3, 124.9 (q, .sup.1J.sub.C-F=280.2 Hz), 115.3, 114.4, 114.1, 63.2, 45.34 (q, .sup.2J.sub.C-F=34.0 Hz), 45.20 (q, .sup.2J.sub.C-F=33.8 Hz), 45.06, 32.9, 32.6, 26.0, 25.8, 18.4,-1.4,-5.2.

[0323] .sup.19F NMR (376 MHZ, CDCl.sub.3): -72.09,-72.17.

[0324] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.31H.sub.42F.sub.6N.sub.2O.sub.2Si.sub.2: 645.2762, found: 645.2762.

##STR00093##

[0325] Compound 30. To a solution of compound 29 (43 mg, 0.066 mmol) in THF (660 L), tetrabutylammonium fluoride trihydrate (TBAF; 19 mg, 0.073 mmol) was added. After stirring for 6 hr. at rt, sat. aq. NH.sub.4Cl (10 mL) was added and the reaction mixture was extracted with ethyl acetate (310 mL). The combined extracts were dried over Na.sub.2SO.sub.4, filtered, evaporated, the product was isolated by flash chromatography on a Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 20% to 80% ethyl acetate/hexane) and freeze-dried from 1,4-dioxane to yield 28 mg (80%) of 30 as a beige solid.

[0326] .sup.1H NMR (400 MHZ, DMSO-d.sub.6): 7.91 (d, J=8.8 Hz, 1H), 7.10 (d, J=15.8 Hz, 1H), 6.97 (d, J=2.5 Hz, 1H), 6.94 (d, J=2.6 Hz, 1H), 6.91-6.83 (m, 4H), 5.88 (dt, J=15.5, 6.8 Hz, 1H), 4.37 (t, J=5.1 Hz, 1H), 4.19-4.02 (m, 4H), 3.44 (q, J=5.8 Hz, 2H), 2.23-2.14 (m, 2H), 1.56-1.45 (m, 4H), 0.39 (s, 6H).

[0327] .sup.13C NMR (101 MHZ, DMSO-d.sub.6): 186.8, 149.4, 148.9, 142.6, 140.8, 138.5, 132.8, 130.6, 129.8, 128.5, 127.1, 124.3, 115.0, 114.7, 113.8, 113.6, 60.6, 43.3 (q, J=32.3 Hz), 43.2 (q, J=32.7 Hz), 32.4, 32.1, 25.5,-1.4.

[0328] .sup.19F NMR (376 MHZ, DMSO-d.sub.6) -70.40,-70.48.

[0329] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.25H.sub.28F.sub.6N.sub.2O.sub.2Si: 531.1897, found: 531.1893.

##STR00094##

[0330] Compound 31. In a 10 mL round-bottom flask, the mixture of compound A6 [G. Lukinaviius et al. J. Am. Chem. Soc. 2016, 138 (30), 9365-9368](174 mg, 0.50 mmol), bis(pinacolato) diboron (140 mg, 0.55 mmol, 1.1 equiv), [Ir (cod)(OMe)].sub.2 (16.6 mg, 0.025 mmol, 5 mol %) and triphenylarsine (15.3 mg, 0.05 mmol, 10 mol %) in dry n-octane (3.5 mL) was degassed on a Schlenk line and stirred at 120 C. for 22 h. On cooling, the reaction mixture was diluted with dichloromethane, evaporated on Celite and the product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 5% to 50% ethyl acetate/dichloromethane) and freeze-dried from dioxane to give 164 mg (69%) of 31 as orange-red solid.

[0331] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.12 (t, J=1.1 Hz, 1H), 6.49 (s, 1H), 6.39 (s, 1H), 3.58 (dd, J=9.0, 8.1 Hz, 2H), 3.53 (dd, J=9.0, 8.1 Hz, 2H), 3.20 (dd, J=9.0, 8.1 Hz, 2H), 3.06 (ddd, J=9.0, 8.1, 1.1 Hz, 2H), 2.95 (s, 6H), 2.90 (s, 6H), 1.41 (br.s, 12H), 0.36 (s, 6H).

[0332] .sup.13C NMR (101 MHz, CDCl.sub.3): 186.3, 156.5, 156.3, 146.5, 139.6, 134.1, 133.5, 131.4, 127.4, 124.1, 109.0, 108.3, 80.3, 54.9, 54.5, 34.3, 33.9, 27.60, 27.56, 26.2 (br), 25.8 (br), -1.3.

[0333] HRMS (ESI) m/z: [M].sup.+ Calcd for C.sub.21H.sub.24BN.sub.2O.sub.2Si 375.1699; Found 375.1694-corresponds to the pinacol ester hydrolysis product (31a):

##STR00095##

[0334] Compound 32. In a 100 mL round-bottom flask, hydrogen peroxide (1 ml of 30% aq. solution) was added to the mixture of compound 31 (217 mg, 0.46 mmol), potassium fluoride (133 mg, 2.30 mmol, 5 equiv) and sodium hydroxide (1 mL of 1 N aq. solution, 1 mmol, 2 equiv) 16.6 mg, 0.025 mmol, 5 mol %) in 2-methoxyethanol (22 mL). The reaction mixture was stirred at 100 C. for 30 min, cooled down to rt and the second portion of hydrogen peroxide (1 ml of 30% aq. solution) was added. After stirring for further 15 min at 100 C., the mixture was cooled down, diluted with ethyl acetate (50 mL) and poured into brine. The aqueous phase was adjusted to pH 3-4 with 1 N HCl and it was extracted with ethyl acetate (330 mL). The combined organic layers were washed with aq. Na.sub.2S.sub.2O.sub.3, brine and dried over Na.sub.2SO.sub.4. The filtrate was evaporated on silica, the product was isolated by flash chromatography on Biotage Isolera system (24 g Interchim SiHP 30 m cartridge, gradient 10% to 70% ethyl acetate/hexane) and freeze-dried from dioxane to give 36 mg (21%) of 32 as orange-yellow solid.

[0335] .sup.1H NMR (400 MHZ, CDCl.sub.3): 14.80 (s, 1H), 8.21 (t, J=1.3 Hz, 1H), 6.48 (s, 1H), 6.20 (s, 1H), 3.54 (t, J=8.6 Hz, 2H), 3.49 (t, J=8.6 Hz, 2H), 3.08-3.00 (m, 4H), 2.91 (s, 3H), 2.90 (s, 3H), 0.42 (s, 6H).

[0336] .sup.13C NMR (101 MHz, CDCl.sub.3): 189.7, 161.7, 157.8, 155.1, 143.1, 140.8, 132.2, 130.6, 125.9, 115.20, 115.15, 108.1, 103.3, 55.4, 54.8, 34.5, 34.4, 28.1, 24.9,-0.7.

[0337] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.21H.sub.24N.sub.2O.sub.2Si 365.1680; Found 365.1678.

##STR00096##

[0338] Compound 33. A solution of compound 32 (31 mg, 84.9 mol) and pyridine (27 L, 340 mol, 4 equiv) in dry dichloromethane (4 mL) was cooled in ice-water bath, and trifluoromethanesulfonic anhydride (127 L of 1 M solution in dichloromethane, 127 mol, 1.5 equiv) was added dropwise. The solution has gradually turned green. After 30 min, the reaction mixture was quenched by addition of sat. aq. NaHCO.sub.3 (20 mL) and extracted with dichloromethane (320 mL). The combined extracts were washed with brine and dried over Na.sub.2SO.sub.4. The product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 10% to 80% ethyl acetate/hexane) and freeze-dried from dioxane to give 23 mg (55%) of 33 as green-yellow solid.

[0339] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.02 (t, J=1.3 Hz, 1H), 6.45 (s, 1H), 6.43 (s, 1H), 3.55 (t, J=8.4 Hz, 2H), 3.46 (t, J=8.4 Hz, 2H), 3.15 (t, J=8.4 Hz, 2H), 3.03 (td, J=8.4, 1.3 Hz, 2H), 2.91 (s, 3H), 2.88 (s, 3H), 0.45 (s, 6H).

[0340] .sup.13C NMR (101 MHZ, CDCl.sub.3): 185.8, 156.6, 154.9, 146.5, 143.6, 137.9, 133.4, 132.7, 126.1, 125.0, 124.0, 118.8 (q, .sup.1J.sub.C-F=320.4 Hz), 107.6, 107.5, 55.0, 54.9, 34.8, 34.4, 28.2, 26.2,-1.1.

[0341] .sup.19F NMR (376 MHz, CDCl.sub.3): -74.3.

[0342] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.22H.sub.23F.sub.3N.sub.2O.sub.4SSi 497.1173; Found 497.1169.

##STR00097##

[0343] Compound 34. In a 25 mL flask, compound 33 (23 mg, 0.046 mmol), compound B1 (20 mg, 0.069 mmol, 1.5 equiv), Cs.sub.2CO.sub.3 (30 mg, 0.092 mmol, 2 equiv), Pd.sub.2 (dba) 3 (4.2 mg, 4.6 mol, 10 mol %), and XPhos (4.4 mg, 9.2 mol, 20 mol %) were loaded. Dry acetonitrile (1.0 mL) was added, the mixture was degassed and stirred at 80 C. for 1 h. Upon cooling, the reaction mixture was quenched with sat. aq. NH.sub.4Cl (10 mL) and extracted with ethyl acetate (310 mL). The combined organics were washed with brine (50 mL), dried over Na.sub.2SO.sub.4, filtered, and evaporated. The product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 0% to 40% ethyl acetate/hexane) and freeze-dried from dioxane to yield 12 mg (50%) of 34 as yellow solid.

[0344] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.01 (d, J=1.2 Hz, 1H), 6.89 (dt, J=16.0, 1.4 Hz, 1H), 6.47 (d, J=5.8 Hz, 2H), 5.57 (dt, J=16.1, 6.9 Hz, 1H), 3.46-3.35 (m, 4H), 3.06 (t, J=8.2 Hz, 2H), 3.01 (t, J=8.1 Hz, 2H), 2.88 (s, 3H), 2.87 (s, 3H), 2.38-2.25 (m, 4H), 1.80 (p, J=7.5 Hz, 2H), 1.45 (s, 9H), 0.43 (s, 6H).

[0345] .sup.13C NMR (101 MHz, CDCl.sub.3): 188.2, 173.3, 154.4, 154.3, 138.2, 134.4, 132.2, 131.8, 131.3, 129.7, 125.8, 107.5, 79.9, 55.3, 55.0, 35.1, 35.0, 34.9, 32.7, 29.3, 28.16, 28.15, 25.2,-1.1.

[0346] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.31H.sub.40N.sub.2O.sub.3Si: 517.2881, found: 517.2879.

##STR00098##

[0347] Compound 35. To a solution of compound 34 (11 mg, 0.021 mmol) in CH.sub.2Cl.sub.2 (1.0 mL), trifluoroacetic acid (200 L) was added dropwise. The resulting reaction mixture was stirred for 1 h at rt, protected from light. The volatiles were removed in vacuo by coevaporation with toluene (310 ml). The product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 0% to 10% methanol/dichloromethane) and freeze-dried from dioxane to yield 9.0 mg (92%) of 35 as a yellow solid.

[0348] .sup.1H NMR (400 MHZ, DMSO-d.sub.6): 12.02 (br, s, 1H), 7.76 (s, 1H), 6.76 (d, J=16.2 Hz, 1H), 6.65 (s, 1H), 6.63 (s, 1H), 5.49 (dt, J=16.1, 6.9 Hz, 1H), 3.45-3.26 (m, 4H), 3.01-2.91 (m, 4H), 2.84 (s, 3H), 2.84 (s, 3H), 2.33 (t, J=7.5 Hz, 2H), 2.18 (q, J=7.0 Hz, 2H), 1.68 (p, J=7.4 Hz, 2H), 0.39 (s, 6H).

[0349] .sup.13C NMR (101 MHZ, DMSO-d.sub.6): 186.7, 174.6, 154.4, 154.3, 140.2, 137.6, 137.3, 133.3, 131.9, 131.5, 130.8, 129.8, 129.2, 125.0, 107.7, 54.5, 54.3, 34.6, 34.4, 33.1, 32.2, 28.8, 27.6, 24.5,-1.2.

[0350] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.27H.sub.32N.sub.2O.sub.3Si: 461.2255, found: 461.2252.

##STR00099##

[0351] Compound 37. In a 25 mL round-bottom flask, the mixture of compound A7 (200 mg, 0.61 mmol; known compound: J. Liu et al. ACS Appl. Mater. Interfaces 2016, 8, 22953-22962), bis(pinacolato) diboron (308 mg, 1.21 mmol, 2 equiv), [Ir (cod)(OMe)].sub.2 (40 mg, 0.061 mmol, 10 mol %) and ligand L1 (8-(diisopropylsilyl) quinoline; known compound: B. Ghaffari et al. J. Am. Chem. Soc. 2014, 136, 14345-14348)(30 mg, 0.121 mmol, 20 mol %) in dry n-octane (8 mL) was degassed on a Schlenk line and stirred at 120 C. for 22 h. On cooling, the reaction mixture was diluted with dichloromethane and evaporated to dryness in a 50 mL round-bottom flask and the obtained crude 36 was used directly in the next step.

[0352] To the residue of crude compound 36, potassium fluoride (141 mg, 2.42 mmol, 4 equiv) and copper (II) bromide (405 mg, 1.82 mmol, 3 equiv) were added, followed by DMSO (6 mL), water (600 L) and pyridine (980 L, 12.1 mmol, 20 equiv). The reaction mixture was stirred at 80 C. for 30 min. Upon cooling to rt, the reaction mixture was diluted with ethyl acetate and poured into 0.5 N HCl (100 mL). The product was extracted with ethyl acetate (430 mL), the combined extracts were washed with water, brine (50 mL each) and dried over Na.sub.2SO.sub.4. The product was isolated by flash chromatography on Biotage Isolera system (25 g Interchim SiHP 30 m cartridge, gradient 0% to 10% ethyl acetate/dichloromethane) to give 79 mg (32%) of 37 as yellow solid.

[0353] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.13 (d, J=9.0 Hz, 1H), 7.31 (d, J=2.7 Hz, 1H), 7.17 (d, J=2.6 Hz, 1H), 7.10 (d, J=2.7 Hz, 1H), 6.87 (dd, J=9.0, 2.6 Hz, 1H), 3.15 (s, 12H).

[0354] .sup.13C NMR (101 MHz, CDCl.sub.3) 175.8, 152.8, 151.9, 144.3, 140.5, 131.5, 125.3, 121.2, 120.9, 117.1, 115.1, 105.4, 103.7, 40.34, 40.26.

[0355] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.17H.sub.17BrN.sub.2O.sub.3S 409.0216; Found 409.0217.

##STR00100##

[0356] Compound 38. In a 10 mL tube, compound 37 (41 mg, 0.10 mmol), compound B1 (44 mg, 0.15 mmol, 1.5 equiv), K.sub.2CO.sub.3 (28 mg, 0.20 mmol, 2 equiv) and Pd(dppf) Cl.sub.2.Math.CH.sub.2Cl.sub.2 (4.1 mg, 5.0 mol, 5 mol %) were loaded. Dioxane (1.0 mL) and water (0.2 mL) were added. The mixture was sparged with argon for 30 min, and the tube was sealed and the mixture was stirred at 80 C. for 1 h. Upon cooling, sat. aq. NH.sub.4Cl (10 mL) was added and extracted with ethyl acetate (310 ml). The combined organics were washed with brine (50 mL), dried over Na.sub.2SO.sub.4, filtered, and evaporated. The product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 20% to 80% ethyl acetate/hexane) and freeze-dried from dioxane to yield 44 mg (88%) of 38 as yellow solid.

[0357] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.11 (d, J=9.0 Hz, 1H), 7.42 (dt, J=15.5, 1.5 Hz, 1H), 7.26 (d, J=3.0 Hz, 1H), 7.19 (d, J=2.7 Hz, 1H), 6.86 (dd, J=9.0, 2.7 Hz, 1H), 6.78 (d, J=2.8 Hz, 1H), 5.98 (dt, J=15.5, 6.9 Hz, 1H), 3.17 (s, 6H), 3.14 (s, 6H), 2.38-2.28 (m, 4H), 1.84 (p, J=7.7 Hz, 2H), 1.45 (s, 9H).

[0358] .sup.13C NMR (101 MHz, CDCl.sub.3): 177.8, 173.1, 152.6, 151.9, 144.6, 143.7, 140.6, 132.42, 132.40, 131.1, 120.8, 116.6, 114.9, 114.4, 104.6, 103.6, 80.1, 40.2, 40.1, 35.1, 32.5, 28.2, 24.7.

[0359] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.27H.sub.34N.sub.2O.sub.5S: 499.2261, found: 499.2262.

##STR00101##

[0360] Compound 39. To a solution of compound 38 (38 mg, 0.062 mmol) in CH.sub.2Cl.sub.2 (1 mL) trifluoroacetic acid (200 L) was added dropwise. The resulting reaction mixture was stirred for 60 minutes at rt, protected from light. The volatiles were removed in vacuo by coevaporation with toluene (310 ml). The product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 0% to 10% methanol/dichloromethane) and freeze-dried from 1,4-dioxane to yield 24 mg (67%) of 39 as yellow solid.

[0361] .sup.1H NMR (400 MHZ, DMSO-d.sub.6): 12.03 (br s, 1H), 7.95 (d, J=8.2 Hz, 1H), 7.31 (d, J=15.6 Hz, 1H), 7.12 (d, J=2.6 Hz, 1H), 7.04 (dd, J=8.9, 1.7 Hz, 2H), 6.89 (s, 1H), 6.16 (dt, J=14.0, 6.7 Hz, 1H), 3.15 (s, 6H), 3.12 (s, 6H), 2.32 (t, J=7.4 Hz, 2H), 2.24 (q, J=6.9 Hz, 2H), 1.72 (p, J=7.4 Hz, 2H).

[0362] .sup.13C NMR (101 MHZ, DMSO-d.sub.6): 177.3, 174.5, 152.4, 151.6, 143.30, 143.26, 140.1, 133.1, 130.7, 130.6, 119.8, 115.3, 115.2, 113.6, 103.8, 102.7, 39.6, 39.5, 33.2, 32.0, 23.9.

[0363] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.23H.sub.26N.sub.2OSS: 443.1635, found: 443.1634.

##STR00102##

[0364] Compound 39-Halo. In an amber vial, compound 39 (10 mg, 0.023 mmol), HaloTag (02) Amine (9.3 mg, 0.042 mmol), and HATU (18 mg, 0.047 mmol) were dissolved in DMF (200 L) and DIPEA (32 mg, 0.25 mmol) was added and stirred for 2 h at rt. The volatiles were removed in vacuo. The product was isolated by flash chromatography on a Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, eluting with ethyl acetate) and freeze-dried from dioxane to yield 11 mg (74%) 39-Halo as a yellow solid.

[0365] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.33H.sub.46ClN.sub.3O.sub.6S: 648.2869, found: 648.2864.

##STR00103##

[0366] Compound 40. In a 100 mL round-bottom flask, 3,3-dimethylacryloyl chloride (0.5 mL, 4.4 mmol, 1.1 equiv) was added to a suspension of methyl 6-aminohexanoate hydrochloride (728 mg, 4 mmol) in dry dichloromethane (40 mL), cooled in ice-water bath. Pyridine (0.75 mL, 8.8 mmol, 2.2 equiv) was then added dropwise, the reaction mixture was warmed up to rt and stirred for 2.5 h. The resulting yellowish solution was then poured into sat. aq. NaHCO.sub.3 (50 mL), extracted with dichloromethane (330 mL); the combined extracts were washed with 0.1 N HCl, water and brine (50 mL each) and dried over Na.sub.2SO.sub.4. The product was isolated by flash chromatography on Biotage Isolera system (40g RediSep Rf cartridge, gradient 30% to 100% ethyl acetate/hexane) and dried in vacuo to give 40 as colorless oil, yield 852 mg (94%).

[0367] .sup.1H NMR (400 MHZ, CDCl.sub.3): 5.54 (hept, J=1.3 Hz, 1H), 5.42 (br.s, 1H), 3.67 (s, 3H), 3.28 (td, J=7.1, 5.9 Hz, 2H), 2.32 (t, J=7.4 Hz, 2H), 2.15 (d, J=1.3 Hz, 3H), 1.83 (d, J=1.3 Hz, 3H), 1.71-1.60 (m, 2H), 1.58-1.48 (m, 2H), 1.43-1.31 (m, 2H).

[0368] .sup.13C NMR (101 MHZ, CDCl.sub.3): 174.2, 167.1, 150.7, 118.7, 51.6, 39.0, 34.0, 29.5, 27.2, 26.6, 24.7, 19.9.

[0369] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.12H.sub.21NO.sub.3 228.1594; Found 228.1591.

##STR00104##

[0370] Compound 41. A solution of bromine (0.19 mL, 3.7 mmol, 1 equiv) in CCl.sub.4 (2 mL) was added dropwise to a solution of 40 (843 mg, 3.7 mmol) in CCl.sub.4 (2 mL), cooled in ice-water bath. The reaction mixture was warmed up to rt, stirred for 1.5 h, the solvents were evaporated and the residue was chased with dichloromethane (20 mL). The resulting light orange oil of the crude dibromide was dissolved in dichloromethane (5 mL), cooled in ice-water bath, and triethylamine (1 mL, 7.4 mmol, 2 equiv) in dichloromethane (1.5 mL) was added dropwise. The cold bath was removed, and the reaction mixture was left stirring at rt for 3 days. It was then poured into water (100 mL), extracted with dichloromethane (330 mL), the combined extracts were washed with brine (50 mL each) and dried over Na.sub.2SO.sub.4. The product was isolated by flash chromatography on Biotage Isolera system (40g RediSep Rf cartridge, gradient 20% to 80% ethyl acetate/hexane) and dried in vacuo to give 41 as yellowish oil, yield 817 mg (72%).

[0371] .sup.1H NMR (400 MHZ, CDCl.sub.3): 6.18 (br.s, 1H), 3.67 (s, 3H), 3.31 (td, J=7.1, 5.9 Hz, 2H),), 2.33 (t, J=7.4 Hz, 2H), 2.11 (s, 3H), 1.98 (s, 3H), 1.71-1.62 (m, 2H), 1.62-1.52 (m, 2H), 1.43-1.33 (m, 2H).

[0372] .sup.13C NMR (101 MHZ, CDCl.sub.3): 174.1, 164.8, 143.5, 111.1, 51.7, 39.9, 34.0, 29.2, 26.51, 26.48, 24.6, 22.9.

[0373] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.12H.sub.20BrNO.sub.3 306.0699; Found 306.0697.

##STR00105##

[0374] Compound 42. In a dried 10 mL crimp-top tube (a 2-5 mL Biotage microwave vial was used), the mixture of compound A7 (15 mg, 45.5 mol), bis(pinacolato) diboron (23 mg, 91 mol, 2 equiv), [Ir (cod)(OMe)].sub.2 (3 mg, 4.55 mol, 10 mol %) and ligand L1 (8-(diisopropylsilyl) quinoline; 2.2 mg, 9.1 mol, 20 mol %) in dry n-octane (0.6 mL) was degassed on a Schlenk line and stirred at 120 C. for 24 h. On cooling, the reaction mixture was diluted with dichloromethane, filtered through a short plug of Celite, washed with dichloromethane; the filtrate was concentrated, transferred into another 10 mL crimp-top tube and dried. Compound 41 (21 mg, 68 mol, 1.5 equiv), Pd(dppf) Cl.sub.2.Math.CH.sub.2Cl.sub.2 (1.8 mg, 2.25 mol, 5 mol %) and K.sub.2CO.sub.3 (12 mg, 90 mol, 2 equiv) were added followed by dioxane (500 L) and water (100 L), the mixture was degassed and stirred at 80 C. for 19 h. The product was isolated by preparative HPLC (Interchim Uptisphere Strategy PhC4 25021.2 mm 5 m, solvent flow rate 18 mL/min, gradient 35% to 75% A/B, A: acetonitrile+0.1% (v/v) formic acid, B: water+0.1% (v/v) formic acid) and freeze-dried from dioxane to give 14 mg (56%) of 42 as yellow solid.

[0375] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.02 (d, J=9.0 Hz, 1H), 7.60 (br.t, J=5.9 Hz, 1H), 7.26 (d, J=2.8 Hz, 1H), 7.22 (d, J=2.7 Hz, 1H), 6.85 (dd, J=9.0, 2.7 Hz, 1H), 6.76 (d, J=2.8 Hz, 1H), 3.64 (s, 3H), 3.43-3.31 (m, 1H), 3.20-3.10 (m, 1H), 3.16 (s, 6H), 3.15 (s, 6H), 2.26 (t, J=7.6 Hz, 2H), 1.90 (s, 3H), 1.67-1.48 (m, 4H), 1.38-1.30 (m, 2H), 1.30 (s, 3H).

[0376] .sup.13C NMR (101 MHz, CDCl.sub.3): 178.2, 174.2, 170.3, 152.9, 152.2, 143.6, 142.6, 141.3, 133.6, 131.8, 130.9, 120.6, 118.0, 117.2, 114.8, 104.8, 104.2, 51.6, 40.4, 40.3, 39.3, 34.1, 29.4, 26.6, 24.7, 21.4, 20.6.

[0377] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.29H.sub.37N.sub.3O.sub.6S 556.2476; Found 556.2472.

##STR00106##

[0378] Compound 43. A solution of LiOH.Math.H.sub.2O (9 mg, 216 mol, 10 equiv) in water (100 L) was added to the solution of compound 42 (12 mg, 21.6 mol) in tetrahydrofuran (500 L) and methanol (100 L), the mixture was sonicated briefly and stirred vigorously at rt for 2 h. Acetic acid (50 L) was then added, and the mixture was evaporated to dryness. The product was isolated from the residue by preparative HPLC (Interchim Uptisphere Strategy PhC4 25021.2 mm 5 m, solvent flow rate 18 mL/min, gradient 30% to 70% A/B, A: acetonitrile+0.1% (v/v) formic acid, B: water +0.1% (v/v) formic acid) and freeze-dried from dioxane to give 11 mg (94%) of 43 as yellow solid.

[0379] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.02 (d, J=9.0 Hz, 1H), 7.70 (t, J=5.9 Hz, 1H), 7.26 (d, J=2.8 Hz, 1H), 7.22 (d, J=2.7 Hz, 1H), 6.85 (dd, J=9.0, 2.7 Hz, 1H), 6.77 (d, J=2.8 Hz, 1H), 3.40-3.28 (m, 1H), 3.27-3.17 (m, 1H), 3.16 (s, 6H), 3.15 (s, 6H), 2.27 (t, J=7.4 Hz, 2H), 1.90 (s, 3H), 1.66-1.47 (m, 4H), 1.38-1.27 (m, 2H), 1.29 (s, 3H).

[0380] .sup.13C NMR (101 MHz, CDCl.sub.3): 178.2, 177.6, 170.5, 152.9, 152.2, 143.4, 142.6, 141.1, 133.3, 132.2, 131.0, 120.5, 117.8, 117.2, 114.9, 104.9, 104.2, 40.4, 40.3, 39.2, 33.8, 29.2, 26.4, 24.4, 21.5, 20.6.

[0381] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.28H.sub.35N.sub.3O.sub.6S 542.2319; Found 542.2315.

##STR00107##

[0382] Compound 45. Compound 12 (20 mg, 0.057 mmol) was dissolved in dichloromethane (500 L). Trifluoromethanesulfonic anhydride solution (86 l of 1 M in dichloromethane, 0.086 mmol, 1.5 equiv) was added dropwise, and the solution stirred for 20 min at rt. The resulting blue solution was transferred dropwise to a stirring solution of tert-butyl 6-aminohexanoate (21.3 mg, 0.114 mmol, 2 equiv) and 2,6-lutidine (30.5 mg, 0.285 mmol) in dichloromethane (500 L), cooled in an ice-water bath. An additional rinse of dichloromethane (500 L) was used to ensure complete transfer. The solution was stirred for 30 min, then sat. aq. NaHCO.sub.3 (10 mL) was added and the reaction mixture was extracted with ethyl acetate (310 mL). The combined extracts were washed with brine (50 mL), dried over Na.sub.2SO.sub.4, filtered, evaporated, the product was isolated by flash chromatography on a Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, 20 to 100% ethyl acetate/hexane) and freeze-dried from 1,4-dioxane to yield 20 mg (66%) of 44 as a brown oil which was used directly in the next step.

[0383] To a solution of compound 44 (20 mg, 0.038 mmol) in dichloromethane (300 L), trifluoroacetic acid (100 L) was added dropwise. The reaction mixture was stirred for 45 minutes at rt, protected from light. The volatiles were removed in vacuo by coevaporation with toluene (310 ml) and freeze-dried from dioxane to yield 22 mg (100%, or 66% over 2 steps) of 45 as an orange solid (mixture of (E)- and (Z)-isomers of the imine in 2:1 ratio).

[0384] .sup.1H NMR, major (E)-isomer (400 MHz, DMSO-d.sub.6): 11.99 (br.s, 1H), 11.54 (d, J=8.1 Hz, 1H), 7.76 (d, J=8.8 Hz, 1H), 7.06 (d, J=2.6 Hz, 1H), 7.04 (d, J=2.7 Hz, 1H), 6.96 (d, J=2.6 Hz, 1H), 6.91 (dd, J=8.8, 2.7 Hz, 1H), 6.74 (dd, J=17.3, 10.9 Hz, 1H), 5.88 (dd, J=17.3, 0.9 Hz, 1H), 5.45 (dd, J=10.9, 0.9 Hz, 1H), 3.40-3.20 (m, 2H), 3.10 (s, 6H), 3.07 (s, 6H), 2.05 (t, J=7.3 Hz, 2H), 1.65-1.52 (m, 2H), 1.36-1.18 (m, 4H), 0.68 (s, 3H), 0.32 (s, 3H).

[0385] .sup.13C NMR, major (E)-isomer (101 MHz, DMSO-d.sub.6): 175.5, 173.8, 151.3, 150.9, 139.4, 137.7, 137.6, 134.5, 128.6, 125.6, 124.6, 118.3, 115.8, 115.3, 111.9, 109.0, 49.0, 39.4, 39.3, 32.9, 26.9, 25.2, 23.4,-0.5,-5.2.

[0386] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.27H.sub.37N.sub.3O.sub.2Si: 464.2728, found: 464.2725.

##STR00108##

[0387] Compound 46. In a 25 mL flask, compound 11 (200 mg, 0.50 mmol), compound B3 (255 mg, 0.75 mmol, 1.5 equiv), K.sub.2CO.sub.3 (104 mg, 0.75 mmol, 1.5 equiv) and Pd(dppf) Cl.sub.2.Math.CH.sub.2Cl.sub.2 (21 mg, 25 mol, 5 mol %) were loaded. Dioxane (5.0 mL) and water (1.0 mL) were added. The mixture was sparged with argon for 30 min, and the reaction was stirred at 80 C. for 3 h. Upon cooling, the reaction was diluted with ethyl acetate (20 mL) and washed with sat. aq. NH.sub.4Cl (20 mL), brine (20 mL). The organics were dried over Na.sub.2SO.sub.4, filtered, and evaporated. The product was isolated by flash chromatography on Biotage Isolera system (40 g RediSep Rf cartridge, gradient 0% to 30% ethyl acetate/hexane) and freeze-dried from dioxane to yield 193 mg (72%) of 46 as yellow solid.

[0388] .sup.1H NMR (400 MHZ, CDCl.sub.3): 8.23 (d, J=8.9 Hz, 1H), 7.26 (s, 1H), 6.82 (dd, J=8.9, 2.8 Hz, 1H), 6.78-6.75 (m, 2H), 6.74 (d, J=2.8 Hz, 1H), 5.93 (dt, J=15.4, 6.8 Hz, 1H), 3.66 (t, J=6.4 Hz, 2H), 3.09 (s, 6H), 3.07 (s, 6H), 2.31 (td, J=7.0, 5.5 Hz, 2H), 1.68-1.53 (m, 4H), 0.90 (s, 9H), 0.45 (s, 6H), 0.06 (s, 6H).

[0389] .sup.13C NMR (101 MHz, CDCl.sub.3): 188.0, 151.1, 150.7, 143.7, 141.1, 138.9, 134.1, 132.3, 131.2, 129.9, 128.3, 114.1, 113.71, 113.67, 113.2, 63.3, 40.1, 40.0, 32.9, 32.7, 26.0, 25.9, 18.4,-1.0,-5.2.

[0390] HRMS (ESI) m/z: [M+H].sup.+ C.sub.31H.sub.48N.sub.2O.sub.2Si.sub.2: 537.3327, found: 537.3327.

##STR00109##

[0391] Compound 48. Compound 46 (17 mg, 0.031 mmol) was dissolved in dichloromethane (500 L). Trifluoromethanesulfonic anhydride solution (47 L of 1 M in dichloromethane, 0.047 mmol, 1.5 equiv) was added dropwise, and the solution stirred for 20 min at rt. The resulting blue solution was transferred dropwise to a stirring solution of methylamine (93 L of 2 M in THF, 0.186 mmol, 6 equiv) and 2,6-lutidine (33 mg, 0.31 mmol, 10 equiv) in dichloromethane (500 L) cooled in an ice-water bath. An additional rinse of dichloromethane (500 L) was used to ensure complete transfer. The solution was stirred for 30 min, then sat. aq. NaHCO.sub.3 (10 mL) was added and the reaction mixture was extracted with ethyl acetate (310 mL). The combined extracts were washed with brine (50 mL), dried over Na.sub.2SO.sub.4, filtered, evaporated and the crude 47 was used directly in the next step without purification.

[0392] The crude 47 was dissolved in THF (500 l) and tetrabutylammonium fluoride trihydrate (TBAF; 14 mg, 0.044 mmol, 1.4 equiv) was added and stirred at rt for 4 hours. The reaction was quenched with addition sat. aq. NH.sub.4Cl (10 mL), and extracted with ethyl acetate (310 mL). The combined organics were washed with brine, dried over Na.sub.2SO.sub.4, filtered, and evaporated. The product was isolated by preparative HPLC (Interchim Uptisphere Strategy PhC4 25021.2 mm 5 m, solvent flow rate 18 mL/min, gradient 25% to 65% A/B, A: acetonitrile+0.1% (v/v) formic acid, B: water +0.1% (v/v) formic acid) to give 6.6 mg (49%) of 48 as an orange solid (mixture of (E)- and (Z)-isomers of the imine in 4:1 ratio).

[0393] .sup.1H NMR, major (E)-isomer (400 MHZ, DMSO-d.sub.6): 7.41 (d, J=8.5 Hz, 1H), 6.88 (d, J=2.6 Hz, 1H), 6.83 (d, J=2.8 Hz, 1H), 6.81 (d, J=2.5 Hz, 1H), 6.74 (dd, J=8.5, 2.8 Hz, 1H), 6.25 (dt, J=15.9, 6.7 Hz, 1H), 6.10 (d, J=15.9 Hz, 1H), 4.38 (br.s, 1H), 3.47-3.39 (m, 2H), 3.04 (s, 3H), 2.96 (s, 6H), 2.92 (s, 6H), 2.21-2.10 (m, 2H), 1.50-1.43 (m, 4H), 0.59 (s, 3H), 0.17 (s, 3H).

[0394] .sup.13C NMR, major (E)-isomer (101 MHz, DMSO-d.sub.6): 167.3, 149.3, 138.1, 138.0, 135.4, 135.2, 131.7, 128.9, 128.1, 126.3, 115.5, 114.6, 113.0, 108.5, 60.5, 41.3, 39.9, 39.7, 32.6, 32.2, 25.4,-0.4,-5.3.

[0395] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.26H.sub.37N.sub.3OSi: 436.2779, found: 436.2777.

##STR00110##

[0396] Compound 50. To a solution of compound 46 (34.6 mg, 0.064 mmol) in anhydrous THF (2.0 mL) under argon, a solution of methyllithium (0.20 ml of 1.6 M in hexane, 0.32 mmol, 5 equiv) was added dropwise and stirred at rt for 30 minutes. The reaction was quenched by slow addition of sat. aq. NH.sub.4Cl (10 mL) and extracted with ethyl acetate (310 mL). The combined organics were washed with brine (50 mL), dried over Na.sub.2SO.sub.4, filtered, and evaporated. The product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 0% to 40% ethyl acetate/hexane) and freeze-dried from 1,4-dioxane to yield a mixture of 49 and 49a as a colourless oil.

[0397] This mixture was dissolved in THF (1 ml), and tetrabutylammonium fluoride trihydrate (TBAF; 23 mg, 0.073 mmol, 1.1 equiv) was added and stirred at rt for 4 h. The reaction was quenched with addition sat. aq. NH.sub.4Cl (10 mL), and extracted with ethyl acetate (310 mL). The combined organics were washed with brine, dried over Na.sub.2SO.sub.4, filtered, and evaporated. The product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 m cartridge, gradient 20% to 80% ethyl acetate/hexane) and freeze-dried from dioxane to yield 14.1 mg of 46 (52%) as a red oil.

[0398] .sup.1H NMR (400 MHZ, DMSO-d.sub.6): 7.41 (d, J=8.6 Hz, 1H), 6.87 (dd, J=7.6, 2.8 Hz, 2H), 6.81 (d, J=2.7 Hz, 1H), 6.78-6.69 (m, 2H), 6.10 (dt, J=15.7, 6.9 Hz, 1H), 5.50 (d, J=2.1 Hz, 1H), 5.01 (d, J=2.2 Hz, 1H), 4.37 (t, J=5.2 Hz, 1H), 3.48-3.39 (m, 2H), 2.94 (s, 6H), 2.91 (s, 6H), 2.23-2.14 (m, 2H), 1.53-1.45 (m, 4H), 0.36 (s, 6H).

[0399] .sup.13C NMR (101 MHz, DMSO-d.sub.6): 148.9, 148.4, 145.0, 136.3, 136.0, 135.0, 134.3, 132.6, 131.1, 129.6, 126.6, 116.6, 115.6, 115.2, 113.6, 111.3, 60.6, 40.20, 40.16, 32.5, 32.1, 25.4,-2.9.

[0400] HRMS (ESI) m/z: [M+H].sup.+ Calcd for C.sub.26H.sub.36N.sub.2OSi 421.2670; Found 421.2668.

##STR00111##

[0401] Compound 51. In a 10 mL round-bottom flask, trifluoromethanesulfonic anhydride (43 L of 1 M solution in dichloromethane, 43 mol, 1.5 equiv) was added to the solution of 12 (10 mg, 28.5 mol) in dry dichloromethane (0.5 mL), cooled in ice-water bath. After stirring for 20 min at 0 C., the solution was transferred into the mixture of triisopropylsilanethiol (TIPS-SH; 12 L, 57 mol, 2 equiv) and 2,6-lutidine (17 L, 143 mol, 5 equiv) in dry dichloromethane (0.5 mL). The resulting blue solution was stirred at rt for 18 h, during which time the color of the mixture changed to violet. The solvents were evaporated and the product was isolated from the residue by preparative HPLC (Interchim Uptisphere Strategy PhC4 25021.2 mm 5 m, solvent flow rate 18 mL/min, gradient 40% to 80% A/B, A: acetonitrile+0.1% (v/v) formic acid, B: water+0.1% (v/v) formic acid) to give 1.4 mg (12%) of 51 as dark blue film (formate salt).

[0402] HRMS (ESI) m/z: [M].sup.+ Calcd for C.sub.21H.sub.27N.sub.2SSi 367.1659; Found 367.1657.

Example 2

Characterisation of Exemplary Compounds of the Present Invention

General Materials and Methods

[0403] All chemical reagents (TCI, Sigma-Aldrich, Alfa Aesar) and dry solvents for synthesis (over molecular sieves, AcroSeal package, Acros Organics) were purchased from reputable suppliers and were used as received without further purification. The products were lyophilized from a suitable solvent system using Alpha 2-4 LDplus freeze-dryer (Martin Christ Gefriertrocknungsanlagen GmbH).

Thin Layer Chromatography

[0404] 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. 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), unless indicated otherwise.

Flash Chromatography

[0405] Preparative flash chromatography was performed with an automated Isolera One system with Spektra package (Biotage AG) using commercially available cartridges of suitable size as indicated (RediSep Rf series from Teledyne ISCO, Puriflash Silica HP 30 m series from Interchim).

Nuclear Magnetic Resonance (NMR)

[0406] NMR spectra (.sup.1H, .sup.13C {.sup.1H}, .sup.19F) were recorded on a Bruker DPX 400 spectrometer. All spectra are referenced to tetramethylsilane as an internal standard (=0.00 ppm). Multiplicities of the signals are described as follows: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet or overlap of non-equivalent resonances; br=broad signal. 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 (J.sub.H-H are always listed as J without indices).

Mass-Spectrometry (MS)

[0407] Low resolution mass spectra (100-1500 m/z) with electro-spray ionization (ESI) were obtained on a Shimadzu LC-MS system described below. High resolution mass spectra (HRMS) were obtained on a maxis II ETD (Bruker) with electrospray ionization (ESI) at the Mass Spectrometry Core facility of the Max-Planck Institute for Medical Research (Heidelberg, Germany).

High-Performance Liquid Chromatography (HPLC)

[0408] Analytical liquid chromatography-mass spectrometry was performed on an LC-MS system (Shimadzu): 2 LC-20AD HPLC pumps with DGU-20A3R solvent degassing unit, SIL-20ACHT autosampler, CTO-20AC column oven, SPD-M30A diode array detector and CBM-20A communication bus module, integrated with CAMAG TLC-MS interface 2 and LCMS-2020 spectrometer with electrospray ionization (ESI, 100-1500 m/z). Analytical column: Hypersil GOLD 502.1 mm 1.9 m, standard conditions: sample volume 1-2 L, solvent flow rate 0.5 mL/min, column temperature 30 C. General method: isocratic 95:5 A: B over 2 min, then gradient 95:5-0:100 A: B over 5 min, then isocratic 0:100 A: B over 2 min; solvent A=water+0.1% v/v HCO.sub.2H, solvent B=acetonitrile+0.1% v/v HCO.sub.2H.

[0409] Preparative high-performance liquid chromatography was performed on a Bchi Reveleris Prep system using the suitable preparative columns and conditions as indicated for individual preparations. Method scouting was performed on a HPLC system (Shimadzu): 2 LC-20AD HPLC pumps with DGU-20A3R solvent degassing unit, CTO-20AC column oven equipped with a manual injector with a 20 L sample loop, SPD-M20A diode array detector, RF-20A fluorescence detector and CBM-20A communication bus module; or on a Dionex Ultimate 3000 UPLC system: LPG-3400SD pump, WPS-3000SL autosampler, TCC-3000SD column compartment with 27-port 6-position valves and DAD-3000RS diode array detector. The test runs were performed on analytical columns with matching phases (HPLC: Interchim 2504.6 mm 10 m C.sub.18HQ, Interchim 2504.6 mm 5 m PhC4, solvent flow rate 1.2 mL/min; UPLC: Interchim C.sub.18HQ or PhC4 752.1 mm 2.2 m, ThermoFisher Hypersil GOLD 1002.1 mm 1.9 m, solvent flow rate 0.5 mL/min).

STED (Stimulated Emission Depletion) Microscopy

[0410] One- and multiphoton-activated STED and confocal counterpart images were acquired using two Abberior Expert Line (Abberior Instruments GmbH, Gttingen, Germany) fluorescence microscopes built on a motorized inverted microscope IX83 (Olympus, Tokyo, Japan), and equipped with a 100/1.40 or a 60/1.42 oil immersion objective lenses (Olympus). One of the microscopes is equipped with pulsed STED lasers at 595 nm and 775 nm shaped by Spatial Light Modulators (SLMs), and with 355 nm, 405 nm, 485 nm, 561 nm, and 640 nm excitation lasers. The other microscope is equipped with pulsed STED lasers at 655 nm and 775 nm, and with 520 nm, 561 nm, 640 nm, and multiphoton (Chameleon Vision II, Coherent, Santa Clara, USA) excitation lasers. The multiphoton laser is tunable in the 680 nm-1080 nm range. Spectral detection is performed in both cases with avalanche photodiodes at spectral windows adjusted for each particular fluorophore.

[0411] Imaging and image processing was done with ImSpector software, and all images are displayed as raw data.

Superresolution Single Molecule Localization Microscopy (SMLM)

[0412] Images were acquired on a custom-built setup [K. Uno et al. Proc. Nat. Acad. Sci. 2021, 118 (14), e2100165118], equipped with a 473 nm (500 mW), a 532 nm (1 W) and a 560 nm (1 W) laser for excitation, a 405 nm (300 mW) laser for activation, a back illuminated EMCCD camera (Andor iXon 897/512512 sensor), and a Leica HCX PL APO CS 100/1.46 oil lens. Emission light was separated from the excitation and activation light with proper combination of a dichroic mirror and a suppression filter for each excitation laser. A movable mirror was used to switch between wide field, highly inclined and laminated optical sheet (HILO) and total internal reflection fluorescence (TIRF) illumination modes. Images were acquired with a 10-20 ms exposure time, and actual excitation laser powers in the back focal plane of ca. 50-400 mW, depending on the dye and sample properties. The 405 nm activation laser was incorporated as 100-500 us pulses, in between frames, with a power of 0.001-1 mW in the back focal plane.

[0413] All images were analyzed and processed using the ThunderSTORM plugin [M. Ovesn et al. Bioinformatics, 2014, 30 (16), 2389-2390] on ImageJ (version 1.52p).

Example 3

Photolysis of the Exemplary Novel Compounds of the Invention and Chemometric Analysis of the Photoactivation and Photobleaching Reaction Kinetics

[0414] Solutions in phosphate buffer (100 mM, pH=7.0)(1.66 g mL.sup.1) were irradiated in a previously described home-built setup [K. Uno et al. Adv. Opt. Mat. 2019, 7, 1801746] with a 405 nm LED source (M405L3, Thorlabs Inc.) in combination with a 10 nm bandpass filter (FB405-10, Thorlabs Inc.). During the irradiation, samples were maintained at 20 C. and continuously stirred. The absorption and emission of irradiated solutions was monitored at desired irradiation intervals. For such purpose, excitation was performed with an LED emitting at a wavelength suitable for each compound (e.g. 470 nm or 505 nm).

[0415] FIG. 1 shows absorption (A) and emission (B) changes during photo-induced activation of compound 13 with violet light (405 nm) in an aqueous buffered solution at pH 7 (phosphate buffer, 100 mM); HPLC 2D-maps of absorption spectra vs. retention time for samples of the solution before (C) and after (D) the photo-induced activation; and chromatograms (E) of these samples at the wavelengths corresponding to the respective absorption maxima.

[0416] FIG. 2 shows the photo-fatigue resistance of compound 20 and established commercial fluorophores in an aqueous buffered solution at pH 7 (phosphate buffer, 100 mM). All compounds were irradiated with a green LED (Thorlabs, model M530L4, Nominal Wavelength 530 nm) under identical conditions and at similar initial concentrations. The measurement was performed as previously described in [A. N. Butkevich et al. J. Am. Chem. Soc. 2019, 141 (2), 981-989].

Example 4

Live Cell Optical Microscopy of Cells Using Exemplary Novel Dyes of the Invention with Self-Labelling Enzymes

[0417] Stocks solutions of BG-(O.sup.6-benzylguanine, SNAPtag ligand) or HaloTag (O.sup.2)-derivatives (HaloTag ligand) of the dyes of the present invention (e.g. 5-Halo, 9-Halo, 20-Halo, and 20-BG) were prepared in DMSO (ca. 1 mM). U2OS cells that stably expressed Vimentin-HaloTag or Vimentin-SNAP [Ratz et al. Sci. Rep. 2015, 5, 9592; Butkevich et al. ACS Chem. Biol. 2018, 13 (2), 475-480] were grown for 12-72 h on glass coverslips. Cells were incubated for 30 min to overnight (depending on the dye and experiment) with the respective fluorescent ligands diluted from DMSO stock solutions with culture medium (without phenol red) to a final concentration of 10-500 nM. Cells were washed with cell culture medium for ca. 15-30 minutes; then the medium was changed for fresh media for imaging. If necessary, the cells were co-stained with the always-on dyes (6-SiR-CTX [J. Buceviius et al., Chem. Sci., 2020, 11, 7313-7323] or Abberior Live 560-Tubulin). Samples were imaged by confocal or single molecule localization superresolution microscopy.

[0418] FIG. 3 shows live-cell dual-color/channel confocal imaging of vimentin filaments labelled with compound 20-Halo (A & C) and tubulin labelled with 6-SiR-CTX (B & D) in U2OS cells. Images were recorded before (A-B) and after photo-activation (C-D) with 405 nm light. Images A & C and B & D are shown on the same intensity scale, respectively. Green/orange channel (A & C): 561 nm excitation; 574-626 nm detection. Red channel (B & D): 640 nm excitation; 663-800 nm detection.

[0419] FIG. 4 shows live-cell confocal (A) and STED (B) image of vimentin filaments labelled with compound 20-Halo in U2OS cells. The compound was pre-activated by irradiation with a 405 nm laser. (C) The same samples was imaged in a confocal microscope, before pre-activation (top half of the image), and a 2-photon activation laser was switched on approximately in the middle of the scanning (bottom half of the image, as indicated by the arrows). The activation rate of selected areas on the same sample was calculated (mono-exponential fitting) by activation with variable powers of a one-photon activation laser (365 nm) or a two-photon activation laser (810 nm). The lines represent fittings to a linear or a quadratic function for one- and two-photon activation, respectively.

Example 5

Single Detection Channel Two-Color Multiplexing by Photoactivation

[0420] U2OS cells that stably expressed vimentin-HaloTag were labelled with 20-Halo and Abberior Live 560-Tubulin as described above. Sequential imaging was performed on a confocal setup before photoactivation (see FIG. 5, A), after photobleaching of Abberior Live 560-Tubulin with high power of 561 nm light (FIG. 5, B), and after photoactivation of 20-Halo with a 405 nm activation (FIG. 5, C). All images were acquired with the same excitation laser, 561 nm, and detection channel, 574 nm-626 nm range (FIG. 5, D). A single-channel, pseudo two-color (multiplexed) image of two different targets may be obtained from overlaying FIGS. 5A & 5C.

Example 6

Optical Microscopy of Cells Using Exemplary Novel Dyes of the Invention Coupled to Antibody

[0421] Amino-reactive NHS-esters of the present dyes were coupled to secondary antibodies (product #111-005-003 or 115-005-003, Jackson ImmunoResearch Europe Ltd.) using a standard coupling protocol. In brief, the reactive dye (e.g. 5-NHS, 9-NHS, or 20-NHS) was dissolved in anhydrous DMSO (ca. 2 mg/ml), and mixed with 0.5-1 mg antibody in a proportion of 5-10 equivalents (dye/protein). The pH of the solution was adjusted to 8.4, and stirred for 1 h in the dark. The mixture was purified using a size exclusion column (PD 10, GE Healthcare).

[0422] Cells were grown for 12-72 h on glass coverslips and then washed twice with PBS (pH 7.4), and then fixed with either methanol (MeOH) or paraformaldehyde (PFA) depending on the favored method for the chosen antibodies or the imaging structures. For MeOH fixation, the samples were treated with MeOH previously cooled to 20 C. for 5 min, and finally washed twice with PBS. PFA fixation was performed with a 4% formaldehyde solution in PBS at room temperature for 20 min, washed twice with PBS, and then treated with a quenching solution (0.1 M NH.sub.4Cl and 0.1 M Glycine in PBS) for 5 min at room temperature. To reduce unspecific binding blocking buffer (2% BSA in PBS) was added and incubated for 30-60 min at room temperature. For samples fixed witFA, Triton 100-X was added to the blocking buffer to a concentration of 0.1%. The coverslips were overlaid with the primary antibody solution in blocking buffer and incubated in a humid chamber for 1 h at room temperature, or overnight at 4 C. Following this, the coverslips were washed with blocking buffer (35 min). The coverslips were then incubated with the secondary antibody in blocking buffer in a humid chamber for 45-60 min at room temperature. Lastly, coverslips were washed with blocking buffer (35 min), with PBS (15 min) and mounted with PBS or Mowiol, and sealed with nail polish or a two-component silicone resin (Picodent Twinsil, Picodent Dental-Produktions-und Vertriebs-GmbH). Samples were imaged by confocal, STED, or single molecule localization microscopy (SMLM).

[0423] FIG. 6 shows Single Molecule Localization Microscopy superresolution images of nuclear pore complexes (A-C) and microtubules (D) in COS7 cells. Samples were fixed and immunolabelled with a primary antibody against NUP-98 from rabbit and an anti-rabbit secondary antibody labelled with 5-NHS (A-C) or a primary antibody against alpha-tubulin from mouse and an anti-mouse secondary labelled with 9-NHS (D). Figure B is a magnified area from A displayed in a dotted box, and C are selected single nuclear pore complexes indicated in B.

Example 7

Color Multiplexing of Two Photoactivatable Dyes by Photoactivation Kinetics

[0424] Cos7 cells were fixed with MeOH and immunolabeled as described above with a mixture of anti-clathrin primary antibody (from rabbit) and anti-alpha tubulin primary antibody (from mouse), and then with a mixture of anti-rabbit secondary antibody labelled with 5-NHS and anti-mouse secondary antibody labelled with 20-NHS. Two-color imaging was performed simultaneously (line-by-line) in two channels with excitation at 485 nm and detection in a 500-551 nm window (FIG. 7B, D, F) and excitation at 561 nm and detection in a 571-691 nm window (FIG. 7A, C, E) for compounds 5 and 20, respectively. Sequential imaging was performed on a confocal setup before photoactivation (see FIG. 7A,B), after photoactivation of 20 with low activation laser dose (FIG. 7C,D), and after photoactivation of 5 with higher activation laser dose sufficient to convert compound 5 (FIG. 7E, F) to obtain two-color image of two photoactivatable dyes multiplexed by photoactivation kinetics.

[0425] Green/orange channel (A, C & E): 561 nm excitation; 571-691 nm detection. Blue/Green channel (B, D & F): 485 nm excitation; 500-551 nm detection. Images corresponding to each channel are shown on the same intensity scale, respectively.

Example 8

Photopatterning of a Polymer Matrix with an Exemplary Compound of the Present Invention

[0426] A polymer film composed of polyvinyl alcohol (PVA) and compound 5 was spin-coated on a cover slide from an aqueous mixture of PVA (2% w/v) and of compound 5 (0.05 mg/ml). The film was placed face-down towards a microscopy slide and fixed with nail polish. Photopatterning was performed on a confocal setup, where select areas were irradiated with light of 405 nm until full activation (i.e. fluorescence reached a plateau). Images of the film before and after patterning was recorded with the same fluorescence microscope (excitation 561 nm/detection 571-691 nm), see FIG. 8A-B, and in a wide-field configuration, with a commercial Cy3 filter-cube (FIG. 8C).