1. Patent Search
  2. Details

PHOTO-SWITCHABLE CHEMICAL INDUCERS OF DIMERIZATION FOR CONTROL OF PROTEIN FUNCTION IN CELLS BY LIGHT

20200190067 ยท 2020-06-18

    Inventors

    Cpc classification

    International classification

    Abstract

    The present application refers to photo-switchable chemical inducers of dimerization for control of protein interactions in cells by light. A compound, a test system, methods and uses are disclosed how the invention can be applied in the investigation of intracellular protein interactions. The system is composed of a compound of the general formula (I) as the photo-caged dimerizer, with the ability to covalently bind to HaloTag and a high affinity binding to eDHFR, respectively. The system can be activated and deactivated selectively on illumination with light under different irradiation conditions.

    Claims

    1. A compound of general formula (I)
    Hal-(CH.sub.2).sub.6F.sup.1PF.sup.2-E (I) wherein Hal is selected from Cl, Br and I; E is selected from: ##STR00036## ##STR00037## wherein R.sup.1 and R.sup.2 are independently of each other selected from: H, CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7, -Ph, CH(CH.sub.3).sub.2, C.sub.4H.sub.9, CH.sub.2CH(CH.sub.3).sub.2, CH(CH.sub.3)C.sub.2H.sub.5, C(CH.sub.3).sub.3, C.sub.5H.sub.11, CH(CH.sub.3)C.sub.3H.sub.7, CH.sub.2CH(CH.sub.3)C.sub.2H.sub.5, CH(CH.sub.3)CH(CH.sub.3).sub.2, C(CH.sub.3).sub.2C.sub.2H.sub.5, CH.sub.2C(CH.sub.3).sub.3, CH(C.sub.2H.sub.5).sub.2, C.sub.2H.sub.4CH(CH.sub.3).sub.2, C.sub.6H.sub.13, C.sub.3H.sub.6CH(CH.sub.3).sub.2, C.sub.2H.sub.4CH(CH.sub.3)C.sub.2H.sub.5, CH(CH.sub.3)C.sub.4H.sub.9, CH.sub.2CH(CH.sub.3)C.sub.3H.sub.7, CH(CH.sub.3)CH.sub.2CH(CH.sub.3).sub.2, CH(CH.sub.3)CH(CH.sub.3)C.sub.2H.sub.5, CH.sub.2CH(CH.sub.3)CH(CH.sub.3).sub.2, CH.sub.2C(CH.sub.3).sub.2C.sub.2H.sub.5, C(CH.sub.3).sub.2C.sub.3H.sub.7, C(CH.sub.3).sub.2CH(CH.sub.3).sub.2, C.sub.2H.sub.4C(CH.sub.3).sub.3, CH(CH.sub.3)C(CH.sub.3).sub.3, CHCH.sub.2, CH.sub.2CHCH.sub.2, C(CH.sub.3)CH.sub.2, CHCHCH.sub.3, C.sub.2H.sub.4CHCH.sub.2, C.sub.7H.sub.15, C.sub.8H.sub.17, CH.sub.2CHCHCH.sub.3, CHCHC.sub.2H.sub.5, CH.sub.2C(CH.sub.3)CH.sub.2, CH(CH.sub.3)CHCH, CHC(CH.sub.3).sub.2, C(CH.sub.3)CHCH.sub.3, CHCHCHCH.sub.2, C.sub.3H.sub.6CHCH.sub.2, C.sub.2H.sub.4CHCHCH.sub.3, CH.sub.2CHCHC.sub.2H.sub.5, CHCHC.sub.3H.sub.7, CH.sub.2CHCHCHCH.sub.2, CHCHCHCHCH.sub.3, cyclo-C.sub.3H.sub.5, cyclo-C.sub.4H.sub.7, cyclo-C.sub.5H.sub.9, cyclo-C.sub.6H.sub.11; Y represents a bond, CH.sub.2, NHR.sup.34, O, S, C(O)O, OC(O), CO, NHC(O), C(O)NH, NR.sup.34C(O), C(O)NR.sup.34, NHC(S), or C(S)NH; C is selected from C1, C2, C3, C4, C5, C6, C7, C8, C9, and C10; ##STR00038## ##STR00039## P is selected from P1-, P2-, P3-, P4-, P5-, P6-, P7-, P8-, P9-, P10-, P11-, and P12-; wherein ##STR00040## ##STR00041## wherein if C is C1, P cannot be P1 or P2; wherein if C is C2, P cannot be P1 or P2; wherein if C is C3, P cannot be P3; wherein if C is C4, P cannot be P4 or P5; wherein if C is C5, P cannot be P4 or P5; wherein if C is C6, P cannot be P6 or P7; wherein if C is C7, P cannot be P8; wherein if C is C8, P cannot be P9; wherein if C is C9, P cannot be P10 or P11; wherein if C is C10, P cannot be P12; X.sup.1 is either ##STR00042## X.sup.4 is either ##STR00043## X.sup.2 and X.sup.5 are independently of each other selected from: O, S, NH, and NR.sup.32; X.sup.3 and X.sup.6 are independently of each other selected from: O, S, NH, and NR.sup.33; F.sup.1 is -A.sup.1-L.sup.A-B.sup.1 and F.sup.2 is -A.sup.2-L.sup.B-B.sup.2, wherein A.sup.1, A.sup.2, B.sup.1 and B.sup.2 represent independently of each other CH.sub.2, NH, O, S, CO, NHCO, CONH, NHCONH, OCO, OCOO, NHCOO, OCONH, NHCOCH.sub.2, CH.sub.2CONH and COO; L.sup.A and L.sup.B represent independently of each other (CH.sub.2).sub.m1, (CH.sub.2).sub.m2, (CH.sub.2).sub.m1CHR.sup.35(CH.sub.2).sub.m2, (CH.sub.2).sub.m1CR.sup.36R.sup.37(CH.sub.2).sub.m2, (C.sub.2H.sub.4O).sub.m1, (C.sub.2H.sub.4O).sub.m2, (OC.sub.2H.sub.4).sub.m1, (OC.sub.2H.sub.4).sub.m2, (CH.sub.2).sub.m5(C.sub.2H.sub.4O).sub.m6, (CH.sub.2).sub.m5(OC.sub.2H.sub.4).sub.m6, (C.sub.2H.sub.4O).sub.m5(CH.sub.2).sub.m6, (OC.sub.2H.sub.4).sub.m5(CH.sub.2).sub.m6, (CH.sub.2).sub.m7(C.sub.2H.sub.4O).sub.m8, (CH.sub.2).sub.m7(OC.sub.2H.sub.4).sub.m8, (C.sub.2H.sub.4O).sub.m7(CH.sub.2).sub.m8, (OC.sub.2H.sub.4).sub.m7(CH.sub.2).sub.m8, (CH.sub.2).sub.m1(C.sub.2H.sub.4O).sub.m2(CH.sub.2).sub.m5, (CH.sub.2).sub.m1(OC.sub.2H.sub.4).sub.m2(CH.sub.2).sub.m5, (CH.sub.2).sub.m6(C.sub.2H.sub.4O).sub.m7(CH.sub.2).sub.m8, (CH.sub.2).sub.m6(OC.sub.2H.sub.4).sub.m7(CH.sub.2).sub.m8, o-C.sub.6H.sub.4, -m-C.sub.6H.sub.4, -p-C.sub.6H.sub.4, ##STR00044## R.sup.3 to R.sup.14, R.sup.17 to R.sup.29 and R.sup.35 to R.sup.39 represent independently of each other H, FCl, Br, I, CF.sub.3, NH.sub.2, N(CH.sub.3).sub.2, N(C.sub.2H.sub.5).sub.2, ##STR00045## OH, OCH.sub.3, OC.sub.2H.sub.5, OC.sub.3H.sub.7, OCH.sub.2COOH, N(CH.sub.2COOH).sub.2, cyclo-C.sub.3H.sub.5, cyclo-C.sub.417, cyclo-C.sub.5H.sub.9, cyclo-C.sub.6H.sub.11, cyclo-C.sub.7H.sub.13, cyclo-C.sub.8H.sub.15, -Ph, CH.sub.2-Ph, CPh.sub.3, CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7, CH(CH.sub.3).sub.2, C.sub.4H.sub.9, CH.sub.2CH(CH.sub.3).sub.2, CH(CH.sub.3)C.sub.2H.sub.5, C(CH.sub.3).sub.3, C.sub.5H ii, CH(CH.sub.3)C.sub.3H.sub.7, CH.sub.2CH(CH.sub.3)C.sub.2H.sub.5, CH(CH.sub.3)CH(CH.sub.3).sub.2, C(CH.sub.3).sub.2C.sub.2H.sub.5, CH.sub.2C(CH.sub.3).sub.3, CH(C.sub.2H.sub.5).sub.2, C.sub.2H.sub.4CH(CH.sub.3).sub.2, C.sub.6H.sub.13, C.sub.3H.sub.6CH(CH.sub.3).sub.2, C.sub.2H.sub.4CH(CH.sub.3)C.sub.2H.sub.5, CH(CH.sub.3)C.sub.4H.sub.9, CH.sub.2CH(CH.sub.3)C.sub.3H.sub.7, CH(CH.sub.3)CH.sub.2CH(CH.sub.3).sub.2, CH(CH.sub.3)CH(CH.sub.3)C.sub.2H.sub.5, CH.sub.2CH(CH.sub.3)CH(CH.sub.3).sub.2, CH.sub.2C(CH.sub.3).sub.2C.sub.2H.sub.5, C(CH.sub.3).sub.2C.sub.3H.sub.7, C(CH.sub.3).sub.2CH(CH.sub.3).sub.2, C.sub.2H.sub.4C(CH.sub.3).sub.3, CH(CH.sub.3)C(CH.sub.3).sub.3, CHCH.sub.2, CH.sub.2CHCH.sub.2, C(CH.sub.3)CH.sub.2, CHCHCH.sub.3, C.sub.2H.sub.4CHCH.sub.2, C.sub.7H.sub.15, C.sub.8H.sub.17, CH.sub.2CHCHCH.sub.3, CHCHC.sub.2H.sub.5, CH.sub.2C(CH.sub.3)CH.sub.2, CH(CH.sub.3)CHCH, CHC(CH.sub.3).sub.2, C(CH.sub.3)CHCH.sub.3, CHCHCHCH.sub.2, C.sub.3H.sub.6CHCH.sub.2, C.sub.2H.sub.4CHCHCH.sub.3, CH.sub.2CHCHC.sub.2H.sub.5, CHCHC.sub.3H.sub.7, CH.sub.2CHCHCHCH.sub.2, CHCHCHCHCH.sub.3, CH.sub.2NH.sub.2, CH.sub.2OH, CH.sub.2SH, CH.sub.2CH.sub.2NH.sub.2, CH.sub.2CH.sub.2SH, C.sub.6H.sub.4OCH.sub.3, C.sub.6H.sub.4OH, CH.sub.2CH.sub.2OCH.sub.3, CH.sub.2CH.sub.2OH, CH.sub.2OCH.sub.3, CH.sub.2C.sub.6H.sub.4OCH.sub.3, CH.sub.2C.sub.6H.sub.4OH, or two neighbouring residues R.sup.3 to R.sup.12 and R.sup.17 to R.sup.25 form a benzo ring, or three neighbouring residues R.sup.3 to R.sup.12 and R.sup.17 to R.sup.25 form a ##STR00046## R.sup.15, R.sup.16, R.sup.30 to R.sup.34 represent independently of each other H, cyclo-C.sub.3H.sub.5, cyclo-C.sub.4H.sub.7, cyclo-C.sub.5H.sub.9, cyclo-C.sub.6H.sub.11, cyclo-C.sub.7H.sub.13, cyclo-C.sub.8H.sub.15, -Ph, CH.sub.2-Ph, CPh.sub.3, CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7, CH(CH.sub.3).sub.2, C.sub.4H.sub.9, CH.sub.2CH(CH.sub.3).sub.2, CH(CH.sub.3)C.sub.2H.sub.5, C(CH.sub.3).sub.3, C.sub.5H.sub.11, CH(CH.sub.3)C.sub.3H.sub.7, CH.sub.2CH(CH.sub.3)C.sub.2H.sub.5, CH(CH.sub.3)CH(CH.sub.3).sub.2, C(CH.sub.3).sub.2C.sub.2H.sub.5, CH.sub.2C(CH.sub.3).sub.3, CH(C.sub.2H.sub.5).sub.2, C.sub.2H.sub.4CH(CH.sub.3).sub.2, C.sub.6H.sub.13, C.sub.3H.sub.6CH(CH.sub.3).sub.2, C.sub.2H.sub.4CH(CH.sub.3)C.sub.2H.sub.5, CH(CH.sub.3)C.sub.4H.sub.9, CH.sub.2CH(CH.sub.3)C.sub.3H.sub.7, CH(CH.sub.3)CH.sub.2CH(CH.sub.3).sub.2, CH(CH.sub.3)CH(CH.sub.3)C.sub.2H.sub.5, CH.sub.2CH(CH.sub.3)CH(CH.sub.3).sub.2, CH.sub.2C(CH.sub.3).sub.2C.sub.2H.sub.5, C(CH.sub.3).sub.2C.sub.3H.sub.7, C(CH.sub.3).sub.2CH(CH.sub.3).sub.2, C.sub.2H.sub.4C(CH.sub.3).sub.3, CH(CH.sub.3)C(CH.sub.3).sub.3, CHCH.sub.2, CH.sub.2CHCH.sub.2, C(CH.sub.3)CH.sub.2, CHCHCH.sub.3, C.sub.2H.sub.4CHCH.sub.2, C.sub.7H.sub.15, C.sub.8H.sub.17, CH.sub.2CHCHCH.sub.3, CHCHC.sub.2H.sub.5, CH.sub.2C(CH.sub.3)CH.sub.2, CH(CH.sub.3)CHCH, CHC(CH.sub.3).sub.2, C(CH.sub.3)CHCH.sub.3, CHCHCHCH.sub.2, C.sub.3H.sub.6CHCH.sub.2, C.sub.2H.sub.4CHCHCH.sub.3, CH.sub.2CHCHC.sub.2H.sub.5, CHCHC.sub.3H.sub.7, CH.sub.2CHCHCHCH.sub.2, CHCHCHCHCH.sub.3, CH.sub.2NH.sub.2, CH.sub.2OH, CH.sub.2SH, CH.sub.2CH.sub.2NH.sub.2, CH.sub.2CH.sub.2SH, C.sub.6H.sub.4OCH.sub.3, C.sub.6H.sub.4OH, CH.sub.2CH.sub.2OCH.sub.3, CH.sub.2CH.sub.2OH, CH.sub.2OCH.sub.3, CH.sub.2C.sub.6H.sub.4OCH.sub.3, CH.sub.2C.sub.6H.sub.4OH, m1, m2, m5, m6, m7 and m8 represent independently of each other an integer from 1 to 20; m3 and m4 represent independently of each other an integer from 0 to 5.

    2. The compound according to claim 1 of general formula (I-A) ##STR00047## wherein E is selected from: ##STR00048## YC is ##STR00049## R.sup.3 is selected from NH.sub.2, N(CH.sub.3).sub.2, N(C.sub.2H.sub.5).sub.2, ##STR00050## and N(CH.sub.2COOH).sub.2; and wherein A.sup.1, L.sup.A, L.sup.B and B.sup.2 have the meanings as defined in claim 1.

    3. The compound according to claim 1 of general formula (I-B) ##STR00051## wherein E is selected from: ##STR00052## YC is ##STR00053## R.sup.3 is selected from NH.sub.2, N(CH.sub.3).sub.2, N(C.sub.2H.sub.5).sub.2, ##STR00054## and N(CH.sub.2COOH).sub.2; and wherein A.sup.1, L.sup.A, L.sup.B and B.sup.2 have the meanings as defined in claim 1.

    4. A chemo-optocenetic system for testing intracellular protein interaction in cells, comprising: a) the compound according to claim 1; b) fusion protein 1 comprising a test compound 1 and at least HaloTag; and c) fusion protein 2 comprising a test compound 2 and at least the TMP binding domain of a bacterial DHFR.

    5. The chemo-optocenetic system according to claim 4, wherein fusion protein 1 and/or fusion protein 2 comprise further a component for identification and/or purification of the fusion proteins and/or a targeting peptide or protein.

    6. The chemo-optocenetic system according to claim 4, wherein test compound 1 and test compound 2 are selected independently of each other among gene products, proteins, protein domains, peptides, polypeptides, glycopeptides, proteins with secondarily modified amino acids, peptides or proteins with protecting groups, saccharides, small molecules, lipids, polynucleotides, oligonucleic acids, DNA and RNA.

    7. The chemo-optocenetic system according to claim 4, wherein the bacterial DHFR is eDHFR.

    8. The chemo-optogenetic system according to claim 4, wherein the compound has the structure of formula (I-A) or (I-B).

    9. A method of using the chemo-optogenetic system according to claim 4, comprising testing the interactions of a test compound 1 with a test compound 2.

    10. A Method for testing intracellular protein interaction in cells, comprising the following steps: a) providing, transfecting and expressing the DNA sequence of a fusion protein 1 comprising a test compound 1 and at least HaloTag; b) providing, transfecting and expressing the DNA sequence of a fusion protein 2 comprising a test compound 2 and at least the TMP binding domain of a bacterial DHFR; c) adding compound according to claim 1 to cells and letting them pass the plasma membrane; d) activating and/or deactivating the compound according to claim 1 with light under irradiation condition A for activation and under irradiation condition B for deactivation; and e) determining the change in a selected test parameter system.

    11. The method according to claim 10, wherein the irradiation condition A corresponds to irradiation with an Argon laser at a wavelength of 458 nm and the irradiation condition B corresponds to irradiation with a laser diode at a wavelength of 405 nm.

    12. The method according to claim 10, wherein the irradiation condition A and the irradiation condition B correspond to irradiation with a laser diode at a wavelength of 405 nm and wherein the applied fluence of the laser under the irradiation condition A is lower than about 0.99 J/cm.sup.2 and the applied fluence of the laser under the irradiation condition B is higher than about 0.99 J/cm.sup.2.

    13. The method according to claim 10, wherein test compound 1 and test compound 2 are selected independently from one another among gene products, proteins, protein domains, peptides, polypeptides, glycopeptides, proteins with secondarily modified amino acids, peptides or proteins with protecting groups, saccharides, small molecules, lipids, polynucleotides, oligonucleic acids, DNA and RNA.

    14. An intermediate compound of the general formula (I-1-A) or (I-1-B), ##STR00055## wherein the moieties A.sup.1, B.sup.2, L.sup.A, L.sup.B, and E have the meanings as defined in claim 2 and wherein the residue YC present in the moiety E represents hydrogen (H).

    15. A kit, comprising a) the compound according to claim 1, b) the nucleotide sequences or the vectors including the nucleotide sequences coding for at least HaloTag and respectively a bacterial DHFR.

    Description

    FIGURES

    [0138] FIG. 1: UV-Vis absorption spectra of TMPPCCl (black) and 4-CmTMPPCCl (red). A solution of 50 M of the respective dimerizer in PBS buffer (pH 7.4, 0.5% DMSO) was subjected to UV-Vis absorption analysis (FIG. 1). 0.5% DMSO in PBS was used as the blank. TMPPCCl shows an absorption peak at 348.5 nm, which indicates the presence of the 2-nitrobenzyl photo-cleavable moiety. 4-CmTMPPCCl shows an additional absorption peak at 414.5 nm, which is attributed to the presence of the photo-caging diethylaminocoumarinyl group.

    [0139] FIG. 2: a) Chemical structure of the psCID, 4-CmTMPPCCl, featuring a chlorohexyl-ligand for covalent binding with HaloTag, a linker containing a photocleavable (PC) 2-nitrobenzyl module, and a diethylaminocoumarinyl-caged TMP ligand. [0140] b) Schematic view of 4-CmTMPPCCl for reversibly control of protein dimerization in a live cell using light. 4-CmTMPPCCl is readily cell permeable and first pre-localized to the protein of interest (POI) A fused with HaloTag. Afterwards, a first light illumination decages the diethylaminocoumarinyl group and exposes TMP ligand to binding with eDHFR-fused POI B. This step is called photoactivation (PA). Upon a second light illumination (e.g. using a different wavelength of light), the photocleavable linker is cleaved, leading to the dissociation of the dimerization process. This step is called photodeactivation (PD).

    [0141] FIG. 3: Reversible targeting to mitochondria using 4-CmTMPPCCl under the control of orthogonal illumination wavelengths. [0142] a) Schematic view of the assay. [0143] b) The two constructs used in the assay. [0144] c) HaloTag-EGFP-ActA (lower panel) is localized at mitochondria while mCherry-eDHFR (upper panel) is largely cytosolic before photoactivation (PA); herein, the C-terminal ActA peptide sequence (LILAMLAIGVFSLGAFIKIIQLRKNN) is responsive for targeting the protein to mitochondria; the whole cell was irradiated with increasing doses of 458 nm light, which gradually induced the targeting of mCherry-eDHFR from cytosol to mitochondria; afterwards, the dimerizer was photo-deactivated (PD) by applying increasing doses of 405 nm laser irradiation, which gradually reversed the mitochondria localization of mCherry-eDHFR. [0145] d) Enlarged images of three key frames in c). [0146] e) Pearson's correlation coefficient (PCC) analysis of the colocalization between mCherry and EGFP (n=10 cells).

    [0147] FIG. 4: Photoactivation and photodeactivation of the 4-CmTMPPCCl using a single wavelength of light (405 nm). [0148] a) Schematic view of the assay. [0149] b) The two constructs used in the assay. [0150] c) HeLa cells co-expressing mCherry-eDHFR and HaloTag-EGFP-ActA were illuminated with increasing doses of 405 nm light which gradually targeted mCherry-eDHFR from cytosol to mitochondria; after the irradiation dose exceeds one unit, higher density of 405 nm irradiation started to induce the dedimerization that relocates mCherry-eDHFR from mitochondria to cytosol. [0151] d) Enlarged images of three key frames in c). [0152] e) Pearson's correlation coefficient (PCC) analysis of the colocalization between mCherry and EGFP.

    [0153] FIG. 5: Spatiotemporal control of intracellular dynein-cargo motility by light using 4-CmTMPPCCl. The dynein binding domain of the dynein adaptor protein Bicaudal D2 (1-594), i.e. BicD2N, was reversibly targeted to Rab5a-localized early endosomes (EEs). Recruitment of BicD2N to EE recruits and activate cytoplasmic dynein and stimulate processive motility of EEs. [0154] a) Schematic view of the assay. Photoactivation allows the recruitment of BicD2N from cytosol to EEs to activate dynein. Subsequently, EEs start to migrate toward microtube organization center (MTOC) in the central part of the cell. After photodeactivation, BicD2N dissociates from EEs, leading to immediate disruption of cargo transport. [0155] b) The two constructs used in the assay. [0156] c) HeLa cells co-expressing BicD2N-Citrine-eDHFR and mCherry-HaloTag-Rab5a were treated with 4-CmTMPPCCl (1.0-1.2 M) and then washed before imaging. BicD2N was largely cytosolic (upper panels) while mCherry-HaloTag-Rab5a was located at EEs (lower panels) before PA (#1, 2). Upon PA, BicD2N-Citrine-eDHFR was recruited to EEs to initiate migration of Rab5a-labeled EEs to MTOC (#3, 4). Upon PD, BicD2N-Citrine-eDHFR rapidly dissociates from the cargo and diffuses to the cytosol within seconds, whereas EEs remain largely localized near MTOC (#5, 6). [0157] d) Analysis of the migration of a single EE: A single EE (indicated by the red triangle, located at the blue frame region in c) was almost steady before PA and started to migrate upon PA; its migration was disrupted upon PD. [0158] e) BicD2N was recruited to individual EEs after PA and dissociated from EEs after PD. [0159] f) The trajectory of the individual EE in d) before PA, after PA and after PD (10 interval between each dot).

    [0160] FIG. 6: Statistical analysis of EE migration rate before PA, after PA and after PD (n=13 vesicles).

    [0161] FIG. 7: UPLC chromatogram of compound 6 with a retention time of 2.65 min.

    EXAMPLES

    Example 1

    Synthesis of compound 6

    [0162] ##STR00031##

    [0163] Abbreviations: DMF: dimethylformamide, RT: room temperature, PC-linker: photocleavable linker, p-NPC: p-nitrophenyl chloroformate, DIEA: N,N-diisopropylethylamine, THF: tetrahydrofuran, ON: overnight, PEG: polyethylene glycol, TMP: trimethoprim, DCM: dichloromethane, Cm: diethylaminocoumarinyl group.

    Compound 3: (4-((21-Chloro-3,6,9,12,15-pentaoxahenicosyl)oxy)-5-methoxy-2-nitrophenyl)methanol

    [0164] ##STR00032##

    [0165] 4-(Hydroxymethyl)-2-methoxy-5-nitrophenol (250 mg, 0.906 mmol, from Chemspace # BBV-33813491) and Cs.sub.2CO.sub.3 (443 mg, 1.36 mmol) were combined in a two-necked round-bottom flask (RBF) and anhydrous DMF (3 m1) was injected under a flush of Ar. CI-PEO-OTs (600 mg, 1.18 mmol) was injected dropwise and the reaction solution was stirred under Ar at room temperature (RT) overnight (ON). After workup, the reaction mixture was purified by silica gel chromatography using EtOAc as the eluent. Approximately 463 mg yellowish viscous oil was obtained in a yield of 95%. .sup.1H-NMR (CDCl.sub.3, 400 MHz): 7.78 (s, 1H), 7.17 (s, 1H), 4.96 (s, 2H), 4.26 (t, J=4.88 Hz, 2H), 3.98 (s, 3H), 3.91 (t, J=4.6 Hz, 2H), 3.73 (m, 2H), 3.7-3.6 (m, 12H), 3.57 (m, 2H), 3.53 (t, J=6.72 Hz, 2H), 3.45 (t, J=6.64 Hz, 2H), 1.77 (p, J=7.88 Hz, 2H), 1.59 (p, J=7.56 Hz, 2H), 1.45 (m, 2H), 1.36 (m, 2H); .sup.13C-NMR (CDCl.sub.3, 400 MHz): 154.42, 147.33, 139.66, 132.57, 111.29, 110.27, 71.26, 70.97, 70.65, 70.62, 70.60, 70.12, 69.53, 69.11, 62.84, 56.41, 45.05, 32.56, 29.47, 26.71, 25.44; HRMS(ESI): C.sub.24H.sub.41ClNO.sub.10.sup.+ calcd. 538.2414, found 538.2426 [M+H].sup.+.

    Compound 4: 4-((21-Chloro-3,6,9,12,15-pentaoxahenicosyl)oxy)-5-methoxy-2-nitrobenzyl (4-nitrophenyl) carbonate

    [0166] ##STR00033##

    [0167] PC-linker (463 mg, 0.86 mmol) was dissolved in anhydrous THF (2.9 m1) under a flush of Ar. p-Nitrophenol chloroformate (208 mg, 1.03 mmol) and diisopropylethylamine (DIEA, 166 mg, 1.29 mmol) were added and the resultant reaction solution was stirred at RT for 6 h. Additional p-nitrophenol chloroformate (208 mg, 103 mmol) and DIEA (166 mg, 1.29 mmol) were added and the reaction solution was further stirred for 3 h to allow complete conversion of PC-linker intermediate. After workup, the reaction mixture was purified via silica gel chromatography to afford 538 mg light yellow oil as the amine-reactive chloroformate intermediate 4. LC-MS (C18, 254 nm, MeCN/H.sub.2O): t.sub.R 7.11 min, m/z 702.6 [M+H].sup.+ (C.sub.31H.sub.44ClN.sub.2O.sub.14.sup.+, calcd. 703.2476). .sup.1H-NMR (CDCl.sub.3, 400 MHz): 8.30 (d, J=9.12 Hz, 2H), 7.84 (s, 1H), 7.41 (d, J=9.12 Hz, 2H), 7.09 (s, 1H), 5.70 (s, 2H), 4.27 (t, J=4.92 Hz, 2H), 3.99 (s, 3H), 3.92 (t, J=4.52 Hz, 2H), 3.73 (m, 2H), 3.68 (m, 2H), 3.64-66 (m, 8H), 3.63 (m, 2H), 3.57 (m, 2H), 3.52 (t, J=6.68 Hz, 2H), 3.45 (t, J=6.64 Hz, 2H), 1.77 (p, J=7.64 Hz, 2H), 1.59 (p, J=7.36 Hz, 2H), 1.44 (m, 2H), 1.37 (m, 2H); .sup.13C-NMR (CDCl.sub.3, 400 MHz): 155.35, 154.09, 152.06, 148.16, 145.53, 139.83, 125.38, 125.24, 121.73, 110.86, 110.24, 77.20, 71.23, 70.93, 70.63, 70.57, 70.08, 69.42, 69.11, 67.72, 56.50, 45.04, 32.53, 29.43, 26.68, 25.40; DEPT135 (CDCl.sub.3, 400 MHz): (+) 71.23, 70.93, 70.63, 70.60, 70.57, 70.08, 69.42, 69.11, 67.72, 56.51, 45.04, 32.52, 29.43, 26.68, 25.40; () 125.38, 121.73, 110.87, 110.24, 56.51; HRMS (ESI): C.sub.31H.sub.44O.sub.14N.sub.2Cl.sup.+, calcd. 703.2476, found 703.2486 [M+H].sup.+.

    Compound 5: 4-((21-Chloro-3,6,9,12,15-pentaoxahenicosyl)oxy)-5-methoxy-2-nitrobenzyl (1-(4-((2,4-diaminopyrimidin-5-yl)methyl)-2,6-dimethoxyphenoxy)-2-oxo-7,10,13-trioxa-3-azahexadecan-16-yl)carbamate

    [0168] ##STR00034##

    [0169] In a typical reaction, TMP-PEG.sub.3-NH.sub.2.nTFA (61.4 mg, 0.114 mmol, 1.3 eq.) was dissolved in sat. Na.sub.2CO.sub.3 (1.44 m1) in a RBF equipped with a stir bar. A solution of Pc2-pNPF (61.8 mg, 0.088 mmol, 1.0 eq.) in THF (0.44 m1) was injected dropwise to the stirring solution under Ar. The reaction mixture was stirred at RT for 4 h. After workup, the reaction mixture was purified by silica gel chromatography to afford 25.6 mg yellowish solid as the product in a yield of 26%. .sup.1H-NMR (CDCl.sub.3, 500 MHz): 7.81 (t, J=5.10 Hz, 1H, CONH), 7.72 (s, 1H), 7.63 (s, 1H), 6.99 (s, 1H), 6.37 (s, 2H), 5.86 (t, J=5.45 Hz, 1H, OCONH), 5.44 (s, 2H, ArCH.sub.2OCONH), 5.31 (s, br, 2H, NH.sub.2), 5.10 (s, br, 2H, NH.sub.2), 4.45 (s, 2H, ArOCH.sub.2CONH), 4.21 (t, J=4.7 Hz, 2H, ArOCH.sub.2CH.sub.2O), 3.91 (s, 2H, ArCH.sub.2Ar)), 3.88 (t, J=4.6 Hz, 2H, ArOCH.sub.2CH.sub.2O), 3.80 (s, 3H, OCH.sub.3), 3.79 (s, 6H, (OCH.sub.3).sub.2), 3.71 (t, J=4.75 Hz, 2H, CH.sub.2Cl), 3.67-3.48 (m, 28H), 3.43 (t, J=6.65 Hz, 2H), 3.39 (m, 2H), 3.30 (m, 2H), 1.81 (m, 2H), 1.75 (m, 4H), 1.57 (m, 2H), 1.42 (m, 2H), 1.34 (m, 2H); .sup.13C-NMR (CDCl.sub.3, 500 MHz): 169.87, 162.87, 160.82, 155.99, 153.90, 153.90, 153.58, 152.51, 147.20, 139.43, 136.77, 135.38, 134.12, 133.27, 128.73, 126.09, 115.69, 110.32, 109.89, 106.39, 104.93, 104.88, 72.58, 71.17, 70.78, 70.50, 70.47, 70.41, 70.19, 70.03, 69.99, 69.38, 69.33, 68.89, 63.25, 60.83, 56.27, 56.09, 56.04, 45.04, 39.14, 36.39, 34.37, 32.47, 29.64, 29.36, 29.32, 26.63, 25.34; DEPT135 (CDCl.sub.3, 500 MHz): (+) 72.63, 71.22, 70.83, 70.55, 70.52, 70.24, 70.87, 70.05, 69.44, 69.39, 68.94, 63.31, 45.09, 39.20, 36.45, 34.42, 32.52, 29.42, 29.38, 26.68, 25.40; () 153.48, 126.14, 115.75, 110.37, 109.94, 104.99, 104.94, 60.88, 56.33, 56.14, 56.09; HRMS (ESI): C.sub.50H.sub.79CIN.sub.7O.sub.18.sup.+[M+H].sup.+, calcd. 1100.5165, found 5129.

    Compound 6: 4-((21-chloro-3,6,9,12,15-pentaoxahenicosyl)oxy)-5-methoxy-2-nitrobenzyl (1-(4-((2-amino-4-((((7-(diethylamino)-2-oxo-2H-chromen-4-yl)methoxy)carbonyl)amino)pyrimidin-5-yl)methyl)-2,6-dimethoxyphenoxy)-2-oxo-7,10,13-trioxa-3-azahexadecan-16-yl)carbamate

    [0170] ##STR00035##

    [0171] In a typical reaction, TMP-Pc2-CI (38.1 mg, 0.0346 mmol), and CoumCOCI (54.1 mg, 0.175 mmol) were combined in a dried small RBF equipped with a stir bar. Anhydrous DCM (0.876 m1) was injected and DIEA (116 l, 87.6 mg, 0.677 mmol) was added dropwise. The clear reaction solution was stirred at RT for 10 h. After workup, the reaction mixture residue was first purified by silica gel chromatography and then by preparative HPLC-MS (C18 column, 21 mm) to give 6.2 mg compound 6 as the target molecule (t.sub.R 7.9 min) in a yield of 13%. HRMS(ESI): C.sub.65H.sub.94O.sub.22N.sub.8Cl.sup.+, calcd. 1373.6166, found 1373.6176 [M+H].sup.+. U-HPLC/MS(ESI): t.sub.R 2.65 min, >99% purity.

    Example 2

    [0172] UV-Vis absorption spectra of TMPPCCl and 4-CmTMPPCCl.

    [0173] UV-Vis absorption spectra were recorded using SHIMADZU UV-2401PC UV-Vis Recording Spectrophotometer. A solution of 50 M of the respective dimerizer in PBS buffer (pH 7.4, 0.5% DMSO) was subjected to UV-Vis absorption analysis (FIG. 1). 0.5% DMSO in PBS was used as the blank. TMPPCCl shows an absorption peak at 348.5 nm, which indicates the presence of the 2-nitrobenzyl photo-cleavable moiety. 4-CmTMPPCCl shows an additional absorption peak at 414.5 nm, which is attributed to the presence of the photo-caging diethylaminocoumarinyl group.

    Example 3

    [0174] Reversible targeting to mitochondria using 4-CmTMPPCCl was controlled by orthogonal illumination wavelengths (FIG. 3). Imaging was carried out 24 h post transfection of HeLa cells using a 4-well or 8-well imaging chamber (SARSTEDT x-well slide) in Dulbecco's Modified Eagle Medium (gibco life technologies, REF: 21063-29) supplemented with additional 10% FBS, 1% sodium pyruvate, 1% NEAA and 1% penicillin-streptomycin at 37 C. under 5% CO.sub.2, using the Zeiss LSM 510 inverted scanning confocal microscope (EC Plan-NEOFLUAR, 40, oil, NA 1.3 objective) equipped with a 405 nm laser. HeLa cells were treated with psCID dimerizer at the concentration of 1.0-1.2 M for 30 min and then washed twice by PBS before imaging. The photoactivation step should be applied within 20-30 min after washing to achieve optimal result. In this patent, one unit of 405 nm light illumination dose is defined as 0.8 as/pixel irradiation at 80% power (150 W) using a 405 nm laser diode, while one unit of 458 nm light illumination dose is defined as 6.4 s/pixel irradiation with 50% output at 100% power applying the 458 nm Ar laser line, Both light illuminations are performed in a 512 pixel512 pixel image (56.3 m56.3 m). Light doses higher than one unit was achieved by proportionally increase the irradiation time while light dose less than one unit was achieved by proportionally reduce the laser power. Photoactivation was performed by applying increasing doses of 458 nm light irradiation while photodeactivation was achieved by applying increasing doses of 405 nm light within a defined region of photoactivation (ROP).

    Example 4

    [0175] Light-induced targeting to mitochondria can be tuned by application of different does of light illumination at 405 nm (FIG. 4). Photoactivation was performed by applying increasing dose ( 1/32 to 1 unit) of 405 nm light irradiation while photodeactivation was achieved by applying increasing doses (1 to 32 units) of 405 nm irradiation within a defined region of photoactivation (ROP).

    Example 5

    [0176] Reversible control of cytoplasmic dynein motor protein function for early endosome (EE) transport using the psCID system (FIG. 5). In this example, the motor binding domain of the dynein adaptor protein Bicaudal D2 (1-594), i.e. BicD2N, was reversibly targeted to Rab5a-localized early endosomes (EEs) using light. Recruitment of BicD2N to EE will recruit and activate cytoplasmic dynein and stimulate processive motility of EEs. Imaging, cell seeding, transfection, microscopy, photoactivation and photodeactivation were performed using the procedures described for FIG. 4.

    Example 6

    [0177] Statistical analysis of EE migration rate before PA, after PA and after PD (n=13 vesicles) (FIG. 6). Before PA and after PD, EEs show non-directional migration with an average migration speed of <10 nm/s. After PA, EEs show an enhanced and directional migration speed of >50 nm/s. Student's t-text reveals that migration speed change is significant after PA while the change of migration speed before PA and after PD is non-significant (n.s). The vesicles for chosen in this analysis are fully tractable during the migration process and the migration length is no less than 10 m.