WO2017078623A1

WO2017078623A1 - Background-free fluorescent probes for live cell imaging

Info

Publication number: WO2017078623A1
Application number: PCT/SG2016/050536
Authority: WO
Inventors: Young-Tae Chang; Samira Husen ALAMUDI; Rudrakanta SATAPATHY; Dongdong SU
Original assignee: National University Of Singapore; Agency For Science, Technology And Research
Priority date: 2015-11-02
Filing date: 2016-11-01
Publication date: 2017-05-11
Also published as: WO2017078623A9

Abstract

BODIPY (boron-dipyrromethene) containing fluorescent compounds for use in live cell intracellular imaging are described. Methods of producing the compounds, methods of labeling tagged organelles, and methods of live cell intracellular imaging using the compounds/probes are described herein.

Description

BACKGROUND-FREE FLUORESCENT PROBES FOR LIVE CELL IMAGING

BACKGROUND OF THE INVENTION

[0001] Labeling a specific biomolecule in the native state of a living cell, particularly with functional synthetic fluorescent probes, is a powerful strategy to achieve an in-depth understanding of its roles and functions. In addition to being nontoxic and specific, the desirable probes for intracellular labeling should also be highly cell permeable with low nonspecific interactions with cellular components to provide high signal-to-noise ratio.

Traditional probes, either do not efficiently cross the cell membrane and/or leave nonspecific intracellular background, resulting in limited applicability for live cell labeling (Jung, D. et al, Mol. BioSyst., 9, 862-872 (2013)).

SUMMARY OF THE INVENTION

[0002] The present invention is based on the discovery that fluorescence labelling of an intracellular biomolecules (e.g., proteins) and/or intracellular target organelles (e.g., mitochondria, lysosome, golgi apparatus) can be achieved by the use of selective fluorescent compounds. Accordingly, in one embodiment, the invention provides a novel class of BODIPY (boron-dipyrromethene) probes of the formula (I) described herein. The compounds described herein are highly cell-permeable and have low background

fluorescence. The compounds of the present invention are also synthesized through synthesis schemes described herein.

[0003] The probes of the present invention (for example, CO- 1 , CO-2, COA- 1 , COC- 1 , AzG-1, AzA-1, AzC-1, AzC-2 and AzR-1) are able to specifically label intracellular organelles and proteins expressing azide and/or cyclooctyne reporter analogs in live cells through strain -promoted alkyne-azide cycloaddition (SPAAC).

[0004] The invention also provides, in additional embodiments, a method of labeling azide and/or cyclooctyne -tagged organelles. The method generally comprises the steps of adding to one or more cells an azide and/or cyclooctyne-containing organelle-localizing reporter, thereby producing one or more azide and/or cyclooctyne-tagged organelles. The probes of the present invention are added to the same one or more cells described above to covalently label the azide and/or cyclooctyne-tagged organelles described above. The azide and/or cyclooctyne-containing organelle-localizing reporters are described herein. [0005] The invention further provides, in other embodiments, a method of live cell intracellular imaging. The method generally comprises the steps of adding the probes of the present invention to one or more live cells comprising one or more tagged organelles described herein. The washed cells are then subjected to imaging using techniques well known in the art (e.g., confocal fluorescence imaging). In a particular embodiment, the method further comprises monitoring one or more dynamic processes (e.g., cell movement, intracellular organelle mobility, cell cycle and distribution of proteins) in the live cells.

[0006] The probes of the present invention have shown their capability of effectively labeling organelle-localizing reporters described herein, regardless of the reporters' cellular localization. The probes have also shown outstanding physical properties, such as high photo stability and narrow emission bandwidth. The compositions and methods described herein provide a significant new tool for live cell intracellular imaging.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1A shows the cellular retention of CO-1 and CO-2 in CHO cells. Cells were stained with probes at 1 μΜ final concentration for 30 min. The probe was observed to enter the cells and leave after washing leaving a clear fluorescence background (green signal from FITC channel). Scale bar, 20 μπι.

[0008] FIG. IB shows the cellular retention of CO-1 and CO-2 in U-2 OS cells. Cells were stained with probes at 1 μΜ final concentration for 30 min. The probe was observed to enter the cells and leave after washing leaving a clear fluorescence background (green signal from FITC channel). Scale bar, 20 μπι.

[0009] FIG.2 shows the absorption and emission spectra of CO- 1 and CO-2. Absorbance and fluorescence emission were measured in DMSO at 10 uM of compound concentration.

[0010] FIG. 3 is a schematic illustration of the covalent labeling of azide-tagged organelles using CO-1 or CO-2 in live cells. Azide-containing organelle-localizing reporters were added to the cells to tag respective organelles. Afterwards, organelles displaying azide were labeled with the probes.

[0011] FIGS. 4A-4B: Live cell imaging with CO- 1 (FIG. 4A) and CO-2 (FIG. 4B) via SPAAC. Fluorescence imaging of mitochondria, lysosome and golgi apparatus in U-2 OS cells labeled with CO- 1 and CO-2. Cells were incubated with TPP-Az, Morph-Az or Sphingo-Az in culture media at 37 °C for 1 hr with 2 μΜ CO-1 and CO-2 separately and followed by counterstaining with organelle trackers. The merged images contain superimposed images of Hoechst, CO-1 or CO-2 and organelle trackers. Scale bar, 15 μιη.

[0012] FIGS. 5A-5B: Photostability analysis of CO-1 and CO-2. 10 μΜ CO-1 and CO-2 solution in PBS buffer (pH 7.4) containing 1% DMSO were placed in a 96-well plates. (FIG. 5A) Fluorescence measurement were recorded every 30 seconds interval for a total period of 12 hours (Ex/Em = 490/520) under a xenon flashlamp. (FIG. 5B) Photostability test under high intensity UV lamp (Blak Ray, 100W, 365 nm). Plates were irradiated for 10 minutes up to 2.5 hours at 10 cm distance. Values are represented as means (n=3) and fitted to a nonlinear regression one -phase exponential decay (GraphPad Prism 5.0).

[0013] FIGS. 6A-6B: Absorption/emission spectra and cellular retention of COA-1. (FIG. 6A) Absorbance and fluorescence emission were measured in DMSO at 10 uM of COA-1 concentration. (FIG. 6B) In cellular retention test, U-2 OS cells were stained with COA-1 probe at 3 μΜ final concentration for 30 min. The probe was observed to enter the cells and leave after washing, leaving a clear fluorescence background (blue signal from DAPI channel).

[0014] FIGS. 7A-7B: Absorption/emission spectra and cellular retention of AzA-1. (FIG. 7 A) Absorbance and fluorescence emission were measured in DMSO at 10 μΜ of AzA-1 concentration. (FIG. 7B) In cellular retention test, U-2 OS cells were stained with AzA-1 probe at 3 μΜ final concentration for 30 min. The probe was observed to enter the cells and leave after washing leaving a clear fluorescence background (blue signal from DAPI channel).

[0015] FIGS. 8A-8B: Absorption/emission spectra and cellular retention of AzC-1. (FIG. 8A) Absorbance and fluorescence emission were measured in DMSO at 10 μΜ of AzC-1 concentration. (FIG. 8B) In cellular retention test, U-2 OS cells were stained with AzC-1 probe at 3 μΜ final concentration for 30 min. The probe was observed to enter the cells and leave after washing leaving a clear fluorescence background (yellow signal from TRITC channel).

[0016] FIGS. 9A-9B: Absorption/emission spectra and cellular retention of AzG-1. (FIG. 9A) Absorbance and fluorescence emission were measured in DMSO at 10 μΜ of AzG-1 concentration. (FIG. 9B) In cellular retention test, U-2 OS cells were stained with AzG-1 probe at 3 μΜ final concentration for 30 min. The probe was observed to enter the cells and leave after washing leaving a clear fluorescence background (green signal from FITC channel).

[0017] FIG. 10: AzG-1 labels molecules in mitochondria. To target mitochondria, U-2 OS cells were pretreated with TPP-BCN (an analogue of triphenylphosphonium bearing cyclooctyne moiety which accumulates in mitochondria) before being labelled with 10 μΜ AzG-1. Cell images show that AzG-1 specifically labelled mitochondria.

[0018] The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0019] A description of example embodiments of the invention follows.

Compounds of the invention

[0020] The present in ention is directed to a compound of formula (I):

wherein Ri-R_7i L and R' are as defined below. It is understood that the invention

encompasses all combinations of the substituent variables {i.e., Ri-R₇ L R' etc.) defined herein_.

Ri is H, C₁-C4 alkyl or

wherein n is a whole number selected from 1 to 4 and R4 is

and each R_2, R_3, R₆ and R₇ is independently selected from H or C₁-C₄ alkyl;

R5 is H, C₁-C₄ alkyl or

wherein n is a whole number selected from 1 to 4; and

L is a linear C₁-C₁₀ alkylene, wherein one or more methylene groups in the C₁-C₁₀ alkylene is optionally and independently replaced by -N(R')C(0)-, -C(O)-, -O-C(O)-, -0-, -N(R , -C(0)N(R'K -N(R')C(0)0-, or -N(R')C(0)N(R')-, wherein each R' is independently hydrogen or Ci_C₃ alkyl, and each carbon in the C₁-C₁₀ alkylene is optionally and

independently substituted by one or two Ci_C₃ alkyl groups.

each R_2, R_3, R₆ and R₇ is independently selected from H or Ci-C₄ alkyl;

L is a linear Ci-Cio alkylene, wherein one or more methylene groups in the Ci-Cio alkyli optionally and independently replaced by -N(R')C(0)-, -C(O)-, -O-C(O)-, -N(R')-, -0-, -C(0)N(R')-, -N(R')C(0)0-, or -N(R')C(0)N(R')-, wherein each R' is independently hydrogen or C₁-C₃ alkyl, and each carbon in the C₁-C₁₀ alkylene is optionally and independently substituted by one or two C₁-C₃ alkyl groups.

[0022] In one aspect of the first embodiment,

and each R_2, R_3, R₆ and R₇ is independently selected from H or Ci-C₄ alkyl; R₅ is H, C₁-C4 alkyl or

and

L is a linear C₁-C₁₀ alkylene, C₁-C9 alkylene, Ci-C₈ alkylene, C₁-C₇ alkylene or Ci-C₆ alkylene, wherein one or more methylene groups in the C₁-C₁₀ alkylene, C₁-C9 alkylene, Ci-Cg alkylene, C₁-C₇ alkylene or Ci-C₆ alkylene is optionally and independently replaced by -N(R')C(0)-, -C(O)-, -O-C(O)-, -N(R')-, -0-, -C(0)N(R')-, -N(R')C(0)0-, or

-N(R')C(0)N(R')-, wherein each R' is independently hydrogen or Ci_C₃ alkyl, and each carbon in the C₁-C₁₀ alkylene, C₁-C9 alkylene, Ci-C₈ alkylene, C₁-C₇ alkylene or Ci-C₆ alkylene is optionally and independently substituted by one or two Ci_C₃ alkyl groups.

[0023] In a particular embodiment of the one aspect of the first embodiment described above, the one or more methylene groups in the C₁-C₁₀ alkylene, C₁-C9 alkylene, Ci-C₈ alkylene, C₁-C₇ alkylene, or Ci-C₆ alkylene described above, is optionally and independently replaced by one or more of -N(R')C(0)-, -C(0)N(R , -N(R and -C(O)- groups. In a different embodiment of the one aspect of the first embodiment described above, the one or more methylene groups in the

C₁-C₁₀ alkylene, C₁-C9 alkylene, Ci-C₈ alkylene, C₁-C₇ alkylene, or Ci-C₆ alkylene described above, is optionally and independently replaced by one or more of N(R')C(0)-, -C(0)N(R')-, -N(R')- and -C(O)- groups. In another embodiment of the one aspect of the first embodiment described above, the one or more methylene groups in the Ci-C₆ alkylene described above, is optionally and independently replaced by one or more of N(R')C(0)-, -C(0)N(R')-, and -C(O)- groups. In yet another embodiment of the one aspect of the first embodiment described above, the one or more methylene groups in the Ci-C₆ alkylene described above, is optionally and independently replaced by one or more of N(R')- and -C(O)- groups. [0024] In certain embodiments, the present invention provides compounds having the structural formula selected from a group comprising:

[0025] A second embodiment of the present invention is a compound represented by structural formula (I):

wherein R1-R7, L and R' are as defined below.

wherein n is a whole number

each R₂ R_3i R₆ and R₇ is independently selected from H or Ci-C₄ alkyl;

R5 is H, Q-C₄ alkyl or

wherein n is a whole number selected from 1 to 4; and

L is a linear C1-C10 alkylene, wherein one or more methylene groups in the C 1-C10 alkylene is optionally and independently replaced by -N(R')C(0)-, -C(O)-, -O-C(O)-, -N(R')-, -0-, -C(0)N(R')-, -N(R')C(0)0-, or -N(R')C(0)N(R')-, wherein each R' is independently hydrogen or Ci_C₃ alkyl, and each carbon in the C1-C10 alkylene is optionally and

independently substituted by one or two Ci_C₃ alkyl groups.

[0026] In one aspect of the second embodiment of the present invention,

each R₂ R_3i R₆ and R₇ is independently selected from H or Ci-C₄ alkyl; and

L is a linear Ci-Cio alkylene, C₁-C9 alkylene, Ci-C₈ alkylene, or Ci-C₇ alkylene, wherein one or more methylene groups in the C₁-C₁₀ alkylene, C₁-C9 alkylene, Ci-C₈ alkylene, or Ci-C₇ alkylene is optionally and independently replaced by -N(R')C(0)-, -C(O)-, -O-C(O)-, -N(R , -0-, -C(0)N(R')-, -N(R')C(0)0-, or -N(R')C(0)N(R , wherein each R' is independently hydrogen or Ci_C₃ alkyl, and each carbon in the C₁-C₁₀ alkylene, C₁-C9 alkylene, Ci-C₈ alkylene, or Ci-C₇ alkylene is optionally and independently substituted by one or two Ci_C₃ alkyl groups.

[0027] In a particular embodiment of the one aspect of the second embodiment described above, the one or more methylene groups in the C₁-C₁₀ alkylene, C₁-C9 alkylene, Ci-C₈ alkylene, or Ci-C₇ alkylene described above, is optionally and independently replaced by one or more of -N(R')C(0)-, -C(0)N(R and -C(O)- groups. In a different embodiment of the one aspect of the second embodiment described above, the one or more methylene groups in the

C₁-C₁₀ alkylene, C₁-C9 alkylene, Ci-C₈ alkylene, or Ci-C₇ alkylene described above, is optionally and independently replaced by one or more of N(R')C(0)-, -C(0)N(R')-, -N(R')- and -C(0)-groups. In another embodiment of the one aspect of the second embodiment described above, the one or more methylene groups in the C₁-C₇ alkylene described above, is optionally and independently replaced by one or more of N(R')C(0)-, -C(0)N(R')-, and -C(O)- groups. In yet another embodiment of the one aspect of the second embodiment described above, the one or more methylene groups in the C₁-C₇ alkylene described above, is optionally and independently replaced by one or more of N(R')- and -C(O)- groups.

[0028] In certain embodiments, the present invention provides compounds having the structural formula selected from a group comprising:

[0029] The compounds of the present invention can also be referred to as "probes". The probes of the present invention specifically label intracellular organelles or proteins expressing azide in live cells through strain-promoted alkyne-azide cycloaddition (SPAAC) regardless of their cellular localization. The protocol for SPAAC is described in Agard, N. J et.al, J. Am. Chem. Soc. 126, 15046-15047 (2004), which has been incorporated herein as a reference in its entirety. Cellular influx/efflux of the unbound probe occurs rapidly with minimal background.

[0030] The probes of the present invention have several advantages. The probes of the present invention have shown their capability of labeling azide and/or cyclooctyne reporters effectively, regardless of the reporters' cellular localization. The probes have also shown outstanding physical properties, such as high photostability and narrow emission bandwidth. [0031] Of the probes of formula (I), CO- 1 , CO-2, COA- 1 and COC- 1 in particular are highly cell-permeable and have low fluorescence background. As will be shown below, CO- 1, CO-2, COA- 1 and COC- 1 are able to specifically label intracellular organelles and proteins expressing azide in live cells through SPAAC. The synthesis of CO-1, CO-2, COA-1 and COC-1 can be seen in Scheme 1-4, respectively, described in Example 1.

[0032] Of the probes of formula (I), AzG- 1 , AzA- 1 , AzC- 1 , AzC-2 and AzR- 1 in particular are highly cell -permeable and have low fluorescence background. As will be shown below, AzG- 1, AzA- 1 and AzC- 1 are able to specifically label intracellular organelles and proteins expressing azide in live cells through SPAAC. The synthesis of AzA-1 and AzC-1 can be seen in Scheme 5 and Scheme 6, respectively, described in Example 1.

Methods described in the invention

[0033] The compounds of the present invention are especially useful as fluorescent probes for labeling biological samples. In one aspect, the invention features a method of labeling a cell (e.g., live cell) and/or an organelle in a cell (e.g., mitochondria, golgi apparatus and lysosome) and/or azide containing reporter molecule described herein (also referred to herein as "azide reporters" and "azide-containing organelle-localizing reporters"). The method comprises adding to one or more cells an azide-containing organelle-localizing reporter described herein, thereby producing one or more azide-tagged organelles. The method further comprises adding the compound/probes of the present invention described herein (e.g., compound of formula (I)) to the cells, thereby covalently labeling the azide- tagged organelles. In another aspect, the invention features a method of labeling a cell (e.g., live cell) and/or an organelle in a cell (e.g., mitochondria, golgi apparatus and lysosome) and/or cyclooctyne containing reporter molecule described herein (also referred to herein as "cyclooctyne reporters" and "cyclooctyne-containing organelle-localizing reporters"). In certain embodiments of the method of labeling, the azide and/or cycloctyne-containing organelle-localizing reporter binds and/or covalently attaches to the organelle in a cell (e.g., mitochondria, golgi apparatus and lysosome) via strain-promoted alkyne-azide cycloaddition (SPAAC). In a particular embodiment of the method of labeling described above, the azide- containing organelle-localizing reporter is selected from a group comprising one or more of

and a probe of formula (I), such as CO- 1, CO-2, COA-1 or COC-1, is added to the cells comprising one or more azide-tagged organelles described above and the organelle is one or more of mitochondria, lysosome and golgi apparatus of a live cell. In a different embodiment of the method of labeling described above, the cyclooctyne-containing organelle-localizing reporter is

and a probe of formula (I), such as AzG- 1, AzA- 1, AzC- 1, AzC-2 or AzR-1, is added to the cells comprising the cyclooctyne-tagged organelles described above and the organelle is one or more of mitochondria, lysosome and golgi apparatus of a live cell.

[0034] Various azide -bearing reporters (also referred to herein as "azide reporters" and "azide-containing organelle-localizing reporters") were synthesized. TPP-Az (Scheme 7), a triphenylphosphonium analogue which accumulates in mitochondria; Morph-Az (Scheme 8), an azide derivative with morpholine moiety as a directing group for lysosome and Sphingo- Az (Scheme 9), a ceramide analogue to target golgi apparatus, were synthesized as shown in Example 1.

[0035] Also provided herein are methods of live cell intracellular imaging. The method comprises adding the compound/probes of the present invention described herein (e.g., compound of formula (I)) to live cells comprising at least one azide-tagged organelle (e.g., Azide- tagged mitochondria, golgi apparatus and/or lysosome). The method further comprises imaging the washed cells to detect fluorescence signals from the reaction of the compound/probes of the present invention described herein with the one or more azide- tagged organelles described above. In a particular embodiment of the method of live cell intracellular imaging described above, a probe of formula (I), such as CO-1, CO-2, COA-1 or COC-1, is added to one or more of the azide-tagged organelles comprising azide-tagged mitochondria, lysosome and/or golgi apparatus of a live cell. In a different embodiment of the method of live cell intracellular imaging described above, a probe of formula (I), such as AzG-1, AzA-1, AzC-1, AzC-2 or AzR-1, is added to one or more of the azide-tagged organelles comprising azide-tagged mitochondria, lysosome and/or golgi apparatus of a live cell. Methods of visualizing a live cell (e.g., one or more azide-tagged organelles comprising azide-tagged mitochondria, lysosome and/or golgi apparatus) include, but not limited to, fluorescence microscopy. Fluorescence microscopy methods for measuring a fluorescence signal of a compound of Formula (I) (e.g., fluorescence signal resulting from the reaction of the compound/probes of the present invention described herein with the one or more azide-tagged organelles described above) include general fluorescence microscopy, confocal microscopy/imaging, two photon microscopy and superresolution (e.g., STROM) microscopy. Such methods are well known to a person skilled in the art. In a particular embodiment, the imaging in the methods of live cell intracellular imaging is time lapse imaging. In a different embodiment of the methods of live cell intracellular imaging, the imaging is done by time lapse imaging and the method further comprises monitoring one or more dynamic processes in the live cells. Examples of dynamic processes in the live cells without limitation include cell movement, intracellular organelle mobility, cell cycle and distribution of proteins. The methods of time lapse imaging and the methods of monitor dynamic processes in the live cells are described in Min, K. A. et al., Adv. Sci. (Weinh) 2, pii 1500025 (2015) and Fu, D. et al, Nat. Chem. 6, 614-622 (2014), which have been incorporated herein as references in their entirety.

Definitions

[0036] As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a

biomolecule" can include a plurality of biomolecules. Further, the plurality can comprise more than one of the same biomolecule or a plurality of different biomolecules.

[0037] The term "alkyl" as used herein means a straight- or branched-chain hydrocarbon radical; in one aspect, having from one to ten carbon atoms, and includes, for example, and without being limited thereto, methyl, ethyl, propyl, isopropyl, t-butyl and the like. Thus, "(Ci- C₆) alkyl" means a radical having from 1 to 6 carbon atoms in a linear or branched arrangement. "(Ci-C₆)alkyl" includes, for example, methyl, ethyl, propyl, iso-propyl, n- butyl, tert-butyl, pentyl and hexyl.

[0038] The term "alkylene" as used herein means a bivalent branched or unbranched saturated hydrocarbon radical; in one aspect, having one to ten carbon atoms, and includes, for example, and without being limited thereto, methylene, ethylene, n-propylene, n-butylene and the like. Thus, "(Ci-C₆) alkylene" means a divalent saturated aliphatic radical having from 1-6 carbon atoms in a linear arrangement, e.g., -[(CH₂)_n]-, where n is an integer from 1 to 6. "(Ci-Ce) alkylene" includes methylene, ethylene, propylene, butylene, pentylene and hexylene. Alternatively, "(Ci-C₆)alkylene" means a divalent saturated radical having from 1-6 carbon atoms in a branched arrangement, for example:

-[(CH₂CH₂CH₂CH₂CH(CH₃)]-, -[(CH₂CH₂CH₂CH₂C(CH₃)₂]-, -[(CH₂C(CH₃)₂CH(CH₃))]-, and the like. The term " methylene" as used herein means -[(CH₂)i]-.

[0039] The symbol represents the point of attachment of the R groups (e.g., R₁-R7) to the compound of formula (I):

[0040] "L," as used herein denotes a linear C₁-C₁₀ alkylene, Q-C9 alkylene, Q-Q alkylene, C₁-C₇ alkylene or Ci-C₆ alkylene, wherein one or more methylene groups in the C₁-C₁₀ alkylene, C₁-C9 alkylene, Ci-C₈ alkylene, C₁-C₇ alkylene or Ci-C₆ alkylene is optionally and independently replaced by -N(R')C(0)-, -C(O)-, -O-C(O)-, -N(R')-, -0-, -C(0)N(R'K -N(R')C(0)0-, or -N(R')C(0)N(R')-. When a chemical formula denoting L is used, it should be read to include all possible modes of linkage and the linkages is bidirectional. For example, if "L" is Ci-C₆ alkylene wherein one or more methylene groups in the Ci-C₆ alkylene replaced by -N(R')C(0)-, then "-N(R')C(0)-"denotes both -N(R')C(0)- and -C(0)N(R')-.

[0041] Compounds of this invention include those described generally above, and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0042] Unless specified otherwise within this specification, the nomenclature used in this specification generally follows the examples and rules stated in Nomenclature of Organic Chemistry, Sections A, B, C, D, E, F, and H, Pergamon Press, Oxford, 1979, which is incorporated by reference herein for its exemplary chemical structure names and rules on naming chemical structures. Optionally, a name of a compound may be generated using a chemical naming program: ACD/ChemSketch, Version 5.09/September 2001, Advanced Chemistry Development, Inc., Toronto, Canada.

[0043] Compounds of the present invention may have asymmetric centers, chiral axes, and chiral planes (e.g., as described in: E. L. Eliel and S. H. Wilen, Stereo-chemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 1119-1190), and occur as racemates, racemic mixtures, and as individual diastereomers or enantiomers, with all possible isomers and mixtures thereof, including optical isomers, being included in the present invention.

[0044] Generally, reference to a certain element such as hydrogen or H is meant to include, if appropriate, all isotopes of that element, for example, deuterium and tritium for hydrogen.

[0045] As described herein, compounds of the invention may contain "optionally substituted" moieties. In general, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term "stable", as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0046] It is understood that substituents and substitution patterns on the compounds of the invention may be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, as long as a stable structure results.

[0047] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. The term "stereoisomers" is a general term for all isomers of the individual molecules that differ only in the orientation of their atoms in space. It includes mirror image isomers (enantiomers), geometric (cis/trans) isomers and isomers of compounds with more than one chiral center that are not mirror images of one another (diastereomers). Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention.

[0048] The term "Minimal background" as used here refers to the fluorescence value when the RR value (Retaining Ratio value, which is a measure of per cent ratio of the average fluorescence intensity of after-washing over before-washing of cell images) of the cell image is < 5%.

[0049] The terms "sticky" and "hydrophobic" refer to a condition where a probe (e.g., any compound of the current invention) has high non-specific affinity to different molecules/organelles in cells. As a result of this non-specific binding, the probe that is "sticky/hydrophobic" cannot be washed out of the cells.

[0050] The term "tag" as used herein may refer to a biological or chemical material, such as an organelle localizing reporter of the present invention, that can readily be attached to and has an affinity for a target organelle and/or and/or biomolecule and/or protein. The term "to produce one or more azide or cyclooctyne-tagged organelles" as used herein refers to an action or process of coupling an organelle localizing reporter of the present invention to, or incorporating an organelle localizing reporter of the present invention within the specified organelle (e.g., mitochondria). The coupling may be a direct coupling (via covalent interactions) or indirect coupling (via non covalent interactions, for example, hydrophobic or ionic).

EXEMPLIFCATION

Reagents and Instrumentation:

[0051] All the chemicals and solvents were purchased from Sigma Aldrich, Alfa Aesar, Fluka, MERCK, Tocris or Acros, and used without further purification. Normal phase purifications were carried out using Merck Silica Gel 60 (particle size: 0.040-0.063 mm, 230- 400 mesh). Analytical characterization was performed on a HPLC-MS (Agilent- 1200 series) with a DAD detector and a single quadrupole mass spectrometer (6130 series) with an ESI probe. ¹H-NMR and ¹³C-NMR spectra were recorded on Bruker Avance 300 MHz NMR spectrometers, and chemical shifts are expressed in parts per million (ppm) and coupling constants are reported as a J value in Hertz (Hz). High resolution mass spectrometry

(HRMS) data was recorded on a Micro mass VG 7035 (Mass Spectrometry Laboratory at National University of Singapore (NUS)). Spectroscopic and quantum yield data were measured on spectroscopic measurements, performed on a fluorometer and UV/VIS instrument, Synergy 4 of Bioteck Company. The slit width was 1 nm for both excitation and emission, and the data analysis was performed using GraphPrism 5.0. Tests and Procedures

[0052] Cellular retention and efflux characteristics test: Influx and efflux profile of the probe set was tested in two types of mammalian cells, U-2 OS and CHO. U-2 OS, a human osteosarcoma cell line, and CHO, a Chinese hamster ovary cell line, were cultured in

Dulbecco's modified eagle's medium (DMEM) (Invitrogen, CA, USA) supplemented with fetal bovine serum (10%) and penicillin-streptomycin (1%). Materials used in the cell culture were purchased from Invitrogen. U-2 OS and CHO cells were seeded onto 96-well plate in growth media at 37°C in the presence of 5% C0₂ and were then allowed to attach and grow to 70-80% confluence. Probe was dissolved in DMSO to make the 1 mM solution, and stored in -20 °C. Prior to experiment, the growth media was aspirated and replaced by 200 μL· fresh growth media containing probes in final concentration of 1 μΜ and nuclei dye Hoechst33342. Plates were incubated for 30 minutes at 37 °C then were imaged using ImageXpress Micro™ cellular imaging system (Molecular Device) with 10^χ objective lens. Immediately after image acquisition, the cells were washed with fresh growth media, and transferred back to a 37 °C cell incubator for further incubation. After 10 minutes, cells were again imaged (after washing image, AW). The first imaging step allowed in-flux

measurement of before washing image (BW), while the second imaging step allowed out-flux measurement of after washing image (AW). Images of two regions per well were acquired.

[0053] Sphingo-Az stock solution preparation: BODIPY TR ceramide were prepared according to manufacturer protocol. Sphingo-Az was prepared as a form of complex with BSA similar to the preparation of BODIPY TR ceramide. Solid Sphingo-Az was dissolved in chloroform:ethanol (19: 1 v/v) of 1 mM stock solution. The stock solution was dried and redissolved in 200

anol. This solution was then added to 10 mL of HBBS/BSA solution (HBSS + 10 mM HEPES pH 7.4 + 0.34 mg/mL of defatted BSA) on a vortex mixer to give 5 μΜ Sphingo-Az^M BSA stock solution. This solution can then be stored at -20 °C.

[0054] Confocal microscopes: Confocal imaging experiments were performed on an inverted Nikon A1R+ confocal laser microscope system using 562/672/405 nm lasers with Plan Apo TIRF 100X DiC oil H H2 objectives (Nikon Instruments Inc, Japan). Image processing and overlay analysis were performed using NIS Elements 3.10 software (Nikon Instruments Inc, Japan). Example 1: Synthesis of the compounds of the invention:

[0055] The synthesis of CO-1, CO-2, COA-1 and COC-1 can be seen below in Scheme 1-4, respectively, while the synthesis of AzA-1 and AzC-1 can be seen below in Scheme 5 and Scheme 6, respectively.

heme 1: Synthesis of CO-1

Synthetic Procedures Associated With Scheme 1

[0056] Synthesis of Compound B (10-(2-(3-carboxypropanamido)ethyl)-5,5-difluoro-7,9- dimethyl-5H-dipyrrolo[l,2-c:2 ',1 '-f][l,3,2]diazaborinin-4-ium-5-uide) Compound A was prepared according to the reported procedure (Vendrell, M. et al. Chem. Commun., 47, 8424- 8426 (2011)). Compound A (20 mg, 0.04 mmol) was dissolved in DCM (dichloromethane) (1 mL). To it, DBU (l,8-diazabicyclo[5.4.0]undec-7-ene) (6.27 mg, 6.1 iL, 0.04 μιηοΐ) was added drop wise and the reaction was stirred for 30 minutes at room temperature. To the reaction mixture, succinic anhydride (8mg, 0.08 mmol) was added and stirred overnight at room temperature. The reaction mixture was evaporated and crude product was purified by column chromatography (MeOH:DCM = 1: 10). Product was obtained as red solid (10.9 mg, 75.4%). The 1H NMR chemical shift data for compound B in Schemel is 1H NMR (CDC1₃, 300 MHz): δ 7.61 (s, 1H), 7.12 (d, j= 3 Hz, 1H), 6.46 (d, j= 3 Hz, 2H), 6.19

(s, 1H), 3.19 (t, j= 6 Hz, 2H), 2.68-2.63 (m, 4H), 2.58 (s, 3H), 2.46 (s, 3H), 2.43 (t, j= 6 Hz, 2H). EI-MS (m/z): Calcd for C₁₇H₂₀BF₂N₃O₃ 363.15; found 362.1 (M-H).

[0057] Synthesis and Characterization of CO-1: Compound B (5 mg, 0.013 mmol), bicyclo[6.1.0]non-4-yn-9-ylmethanol (2.47 mg, 0.016 mmol), EDCI (l-ethyl-3-(3- dimethylaminopropyl)carbodiimide) (5.25 mg, 0.027 mmol) and DMAP (4- dimethylaminopyridine) (0.8 mg, 0.006 mmol) were dissolved together in DCM (0.2 mL) and stirred for 12 hours at room temperature. Water (0.5 mL) was added to the reaction mixture and the organic layer was extracted in DCM. The crude was purified by column

chromatography (MeOH:DCM = 0.5 : 10). Product was obtained as red solid (5.1 mg, 76.3%). The 1H NMR chemical shift data for compound CO-1 in Scheme 1 is 1H NMR (CDCI₃, 300 MHz): δ 7.61 (s, 1H), 7.15 (d, j= 3 Hz, 1H), 6.45 (dd, j= 6 Hz, j= 3 Hz, 2H), 6.19 (s, 1H), 4.19 (d, j= 9 Hz, 2H), 3.19 (t, j= 7.5 Hz, 2H), 2.66 (m, 2H), 2.58 (s, 3H), 2.47 (s, 3H), 2.43 (t, j= 6 Hz, 2H), 2.30-2.22 (m, 4 H), 1.61-1.52 (m, 2H), 1.31—1.25 (m, 3H), 0.90- 0.85 (m, 2H). The ¹³C NMR chemical shift data for compound CO-1 in Scheme 1 is 13C NMR (75 MHz, CDC13) 5172.9, 161.1, 145.1, 142.5, 138.2, 130.8, 129.9,129.6, 128.7, 124.1, 123.7, 115.9,114.0, 98.8, 98.7, 62.7, 59.9, 41.3, 31.8, 30.9, 29.6, 22.6, 21.4, 21.3, 20.1, 20.0, 17.3, 16.1,14.0. The HRMS: m/z calcd for C₂₇H₃₁BF₂N₃O₃ (M-H)^" 494.2437, found

494.2424. _a em = 495/510 nm and quantum yield =0.66 (Tetramethyl Bodipy as standard), extinction co-efficient of CO-1 = 66,668 M^"1 cm ¹ measured in DMSO.

Scheme 2: Synthesis of CO-2

Synthetic Procedures Associated With Scheme 2

[0058] Synthesis of Compound C (l-(3,5-dimethyl-lH-pyrrol-2-yl)-4-morpholino butane- 1, 4-dione): 4-(3, 5-dimethyl-lH-pyrrol-2-yl)-4-oxobutanoic acid (0.6 g, 3.07 mmol), morpholine (0.53 g, 6.1 mmol), and HBTU (3-[Bis(dimethylamino)methyliumyl]-3H- benzotriazol- 1 -oxide hexafluorophosphate) (1.4 g, 3.68 mmol) were dissolved in dry THF (tetrahydrofuran). DIEA (N,N-diisopropylethylamine) (1.6 mL, 9.21 mmol) was added to it and the reaction mixture was stirred for 4 hours under nitrogen atmosphere. Then the solvent was evaporated in rota vapour. Crude was dissolved in ethyl acetate and washed with water two times. Organic layer was dried over Na₂S0₄ and evaporated over rotary evaporator. Crude was purified by column chromatography (EA: Hexane = 1: 4) to obtain pure product as a yellowish white solid (0.62 g, 78%). The 1H NMR chemical shift data for compound C in Scheme 2 is 1H NMR (300 MHz, CDC1₃) δ 9.42 (s, 1H), 5.80 (s, 1H), 3.66-3.55 (m, 8H), 3.08 (t, J = 6.6 Hz, 2H), 2.72 (t, J = 6.3 Hz, 2H), 2.36 (s, 3H), 2.23 (s, 3H). The ^1JC NMR chemical shift data for compound C in Scheme 2 is ¹³C NMR (75 MHz, CDC1₃) δ 187.55, 170.81, 128.01, 112.70, 77.41, 76.98, 76.56, 66.77, 66.51, 45.81, 42.05, 34.11, 26.90, 14.45, 12.91. The EI-MS (m/z): Calcd for Ci₄H₂₀N₂O₃ 264.1; found 265.1 (M+H).

[0059] Synthesis of Compound D (ethyl 3-(lH-pyrrol-2-yl)propanoate) This compound was synthesized as reported (GieBler, K. et al. European Journal of Organic Chemistry, 19, 3611-3620 (2010); and Cuevas-Yanez, E. et al. Tetrahedron, 60, 1505-1511 (2004)).

[0060] Synthesis and Characterization of CO-2: 3-(2-carboxyethyl)-5,5-difluoro-7, 9-dimethyl-10-(3-morpholino-3-oxopropyl)-5H-dipyrrolo[l,2-c:2', -f][l,3,2]diazaborinin- 4-ium-5-uide (10 mg, 0.023 mmol), bicyclo[6.1.0]non-4-yn-9-ylmethanol (4.16 mg, 0.027 mmol), EDCI (8.81 mg, 0.046 mmol) and DMAP (1.4 mg, 0.011 mmol) were dissolved together in DCM (0.2 mL) and stirred for 8 hours at room temperature. Water (0.5 mL) was added to the reaction mixture and the organic layer was extracted in DCM. The crude was purified by column chromatography (EA: Hexane = 3: 5). Product was obtained as red solid (11.1 mg, 85.6%). The 1H NMR chemical shift data for compound CO-2 in Scheme 2 is 1H NMR (300 MHz, CDC1₃) δ 7.06 (d, J = 4.1 Hz, 1H), 6.28 (d, J = 4.1 Hz, 1H), 6.13 (s, 1H), 4.19 (d, J = 8.2 Hz, 2H), 3.61 - 3.49 (m, 4H), 3.34-3.25 (m, 8H), 2.75 (t, J = 6.0 Hz, 2H), 2.66 (t, J = 6.0 Hz, 2H), 2.54 (s, 3H), 2.40 (s, 3H), 2.24 - 2.20 (m, 4H), 1.61 - 1.52 (m, 2H), 1.40-1.28 (m, 2H), 1.24 (bs, 1H), 0.97 - 0.87 (m, 2H). The ¹³C NMR chemical shift data for compound CO-2 in Scheme 2 is ¹³C NMR (75 MHz, CDC1₃) δ 172.56, 169.41, 158.70, 156.04, 143.93, 142.83, 133.77, 132.34, 130.21, 124.98, 122.87, 116.17, 98.72, 66.61, 66.29, 62.49, 45.82, 42.14, 35.06, 33.43, 28.98, 24.62, 23.79, 21.31, 20.13, 17.32, 15.96, 14.72. The HRMS: m/z calcd for C₃iH₃₇BF₂N₃0₄ (M-H)^~ 564.2856, found 564.2867. _h k_sm = 500/515 nm and quantum yield =0.83 (Tetramethyl Bodipy as standard), extinction co-efficient of CO-2 = 83,333 M^"1 cm ^_1 measured in DMSO. cheme 3: Synthesis of COA-1

Synthetic Procedures Associated With Scheme 3

[0061] Synthesis of Compound E: 5,5-difluoro-10-(methylthio)-5H-414,514-dipyrrolo[l, 2-c:2',l'-f][l,3,2]diazaborinine (5mg, 0.02 mmol), 3-aminopropanoic acid (2.7 mg, 0.03 mmol) and TEA were dissolved together in acetonitrile:H₂0 (9: 1, 1 mL) and stirred for 2 hours at room temperature. After remove the solvent, the crude was purified by column chromatography (DCM: MeOH = 20:1). Product was obtained as orange solid.

[0062] Synthesis of COA-1: Compound E (5 mg, 0.018 mmol), bicyclo[6.1.0]non-4-yn-9 -ylmethanol (3.3 mg, 0.022 mmol), EDCI (5.2 mg, 0.027 mmol) and DMAP (1 mg, 0.009 mmol) were dissolved together in DCM (2 mL) and stirred for 8 hours at room temperature. Water (0.5 mL) was added to the reaction mixture and the organic layer was extracted in DCM. The crude was purified by column chromatography (DCM:MeOH= 99: 1). Product was obtained as orange solid. Other COA compounds follow similar synthesis scheme. Scheme 4: Synthesis of COC-1

Synthetic Procedures Associated With Scheme 4

[0063] Synthesis of Compound F : 2,5-dioxopyrrolidin-l-yl 5,5-difluoro-3,7-dimethyl-5H- 414,514-dipyrrolo[l,2-c:2',r-f][l,3,2]diazaborinine-10-carboxylate (10 mg, 0.028 mmol) and 3-aminopropanoic acid (4.9 mg, 0.055 mmol) were dissolved together in acetonitrile:H₂0 (9: 1, 10 mL) and stirred for 2 hours at room temperature. After remove the solvent, the crude was purified by column chromatography (DCM: MeOH = 20: 1). Product was obtained as orange solid.

[0064] Synthesis of COC-1: Compound F (2 mg, 0.006 mmol), bicyclo[6.1.0]non-4-yn-9 -ylmethanol (1.1 mg, 0.007 mmol), EDCI (1.7 mg, 0.009 mmol) and DMAP (0.37 mg, 0.003 mmol) were dissolved together in DCM (1 mL) and stirred for 8 hours at room temperature. Water (0.5 mL) was added to the reaction mixture and the organic layer was extracted in DCM. The crude was purified by column chromatography (DCM:MeOH= 99: 1). Product was obtained as orange solid. Other COC compounds follow similar synthesis scheme.

Scheme 5: S nthesis of AzA-1

Synthetic Procedures Associated With Scheme 5

[0065] Synthesis ofAzA-1: 5,5-difluoro-10-(methylthio)-5H-414,514-dipyrrolo[l,2-c:2', l'-f][l,3,2]diazaborinine (3 mg, 0.013 mmol), 3-azidopropan-l-amine (1.4 mg, 0.014 mmol) and TEA were dissolved together in acetonitrile (1 mL) and stirred for 2 hours at room temperature. After remove the solvent, the crude was purified by column chromatography (DCM). Product was obtained as orange solid. Other AZA compounds follow similar synthesis scheme. Scheme 6: S nthesis ofAzC-1

Synthetic Procedures Associated With Scheme 6

[0066] Synthesis of Compound AzC-1 : 2,5-dioxopyrrolidin-l-yl 5,5-difluoro-3,7- dimethyl-5H-414,514-dipyrrolo[l,2-c:2^1'-f][l,3,2]diazaborinine-10-carboxylate (10 mg, 0.028 mmol) and 3-azidopropan-l-amine (2.8 mg, 0.028 mmol) were dissolved together in acetonitrile 2 mL and stirred for 2 hours at room temperature. After remove the solvent, the crude was purified by column chromatography (DCM: MeOH = 20: 1). Product was obtained as orange solid. Other AzC compounds follow similar synthesis scheme.

[0067] The synthesis of azide-bearing reporters TPP-Az, Morph-Az and Sphingo-Az can be seen below in Scheme 7-9, respectively.

Scheme 7: Synthesis of TPP-Az

TPP-Az

Synthetic Procedures Associated With Scheme 7

[0068] Synthesis of(5-carboxypentyl)triphenylphosphonium bromide:

Triphenylphosphine (20 mg, 0.076 mmol) and 6-bromohexanoic acid (14.8 mg, 0.076 mmol) were dissolved in dry toluene (0.2 mL). The reaction mixture was refluxed over 72 hours. The solution was concentrated. The residue was washed consecutively with benzene (3 x 1 mL), hexane (1 mL), and Et₂0 (2 x 1 mL). The crystalline white solid was dried to give the pure product (28 mg, 97.5 %). 1H NMR (300 MHz, CDC1₃) δ 7.80-7.68 (m, 15H), 3.58 (bs, 2H), 2.34-2.32 (m,2H), 1.63-1.57 (m, 6H). EI-MS (m z): Calcd for C₂₄H₂₆0₂P⁺ 377.16; found 377.1

[0069] Synthesis of TPP-Az ((6-((4-azidophenyl)amino)-6- oxohexyl)triphenylphosphonium bromide): (5-carboxypentyl)triphenylphosphonium bromide (10 mg, 0.026 mmol), 4-azidobenzenaminium chloride (4.5 mg, 0.026 mmol), and HBTU (20 mg, 0.052 mmol) were dissolved in a mixture of solvents (dry DMF (0.05 mL) and DCM (0.1 mL). To the reaction mixture, 7 of DIE A was added and the reaction mixture was stirred overnight at room temperature. Solvent was evaporated and the crude was purified by column chromatography (EA: Hexane = 1: 2). Product was obtained as white solid (9.9 mg, 77.7 %). 1H NMR (DMSO-d6, 300 MHz): 7.83-7.70 (m, 15H), 7.63 (d, j= 9Hz, 2H), 7.07 (d, j= 9 Hz, 2H), 3.62 (bs, 2H), 2.30-2.25 (m, 2H), 1.61-1.54 (m, 6H). ¹³C NMR (75 MHz, DMSO-d6) 5 171.29, 136.94, 135.23, 133.99, 130.67, 120.85, 119.72, 119.47, 118.33, 36.14, 31.13, 24.64, 18.43, 17.08. HRMS: m/z calculated for C₃oH₃oN₄OP⁺ 493.2152, found

493.2153.

ylphosphonium analogue cyclooctyne -bearing reporters {e.g., TPP-

were also prepared by a similar synthesis scheme shown in Scheme 7.

Scheme 8: Synthesis of Morph-Az

Synthetic Procedures Associated With Scheme 8

[0071] Synthesis of(6-amino-N-(2-morpholinoethyl)-2-naphthamide): 6-amino-2- naphthoic acid (25 mg, 0.13 mmol), 2-morpholinoethanamine (69.5 mg, 70 μί, 0.53 mmol), and HBTU (98.5 mg, 0.26 mmol) were dissolved together in DCM. To the stirred solution, N,N-Diisopropylethylamine (16.77 mg, 22.6 μί, 0.13 mmol) was added drop wise. The reaction mixture was stirred at room temperature for 4 hours. The solvent was evaporated in rota vapour and the crude was obtained. The crude was purified by column chromatography using EA as eluent. Product was obtained as brown semi solid (21.24 mg, 54.6%). 1H NMR (CDCI₃, 300 MHz): δ 7.21 (d, IH, j= 3 Hz), 7.78-7.73 (m, 2H), 7.65 (d, j= 9 Hz, IH), 7.73- 7.00 (m, 2H), 3.79 (t, j= 4.5 Hz, 4H), 3.65 (q, j= 6.0 Hz, 2H), 2.68 (t, j= 6.0 Hz 2H), 2.58(t, j= 4.5 Hz, 4H). ¹³C NMR (75 MHz, CDCI₃) δ 167.69, 145.89, 136.50, 130.38, 128.29, 127.52, 126.73, 125.94, 123.96, 118.81, 107.84, 66.81, 57.03, 53.27, 36.00. EI-MS (m/z): Calcd for C₁₇H₂₁N₃O₂, 299.16; found 300.0 (M+H).

[0072] Synthesis ofMorph-Az ((9H-fluoren-9-yl)methyl(5-azido-l-((6-((2- morpholinoethyl)carbamoyl)naphthalen-2-yl)amino)-l-oxopentan-2-yl)carbamate):

Compound B (20 mg, 0.066 mmol), 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5- azidopentanoic acid (38 mg, 0.01 mmol), and HBTU (35 mg, 0.092 mmol) were dissolved together in DCM. To the stirred solution, N,N-Diisopropylethylamine (8.5 mg, 11.5 μί, 0.066 mmol) was added drop wise. The reaction mixture was stirred overnight at room temperature. The solvent was evaporated in rota vapour and the crude was obtained. The crude was purified by column chromatography using (MeOH:DCM = 1 : 10). Product was obtained as white solid (18.5 mg, 42.5%). 1H NMR (CDCI₃, 300 MHz): δ 8.94 (s, IH), 8.28 (s, IH), 8.20 (s, IH), 7.80-7.69 (m, 5H), 7.63-7.59 (m, 2H), 7.44-7.39 (m, 3H), 7.33-7.29 (m, 2H), 5.81 (d, j= 9 Hz, IH), 4.53-4.49 (m, 2H), 4.26 (t, j= 7.5 Hz, IH), 3.88 (t, j= 4.5 Hz, 4H), 3.77-3.73 (m, 2H), 3.42-3.35 (m, 2H), 2.92-2.85 (m, 2H), 2.84-2.74 (m, 4H), 2.07-1.65 (m, 4H). ¹³C NMR (75 MHz, CDC1₃) δ. 171.69, 166.71, 156.47, 144.23, 144.13, 141.09, 138.40, 135.11, 130.00, 129.90, 129.14, 128.00, 127.73, 127.68, 127.59, 127.42,125.69, 125.06, 121.02, 120.47, 115.34, 66.12, 66.06, 55.54, 50.74, 47.06, 35.48, 31.62, 29.38, 26.96, 25.49, 22.43. HRMS: m/z calcd for C₃₇H₃9N₇O₅ (M+H)^" 661.3085, found 661.3046. Scheme 9: Synthesis ofSphingo-Az

Synthetic Procedures Associated With Scheme 9

[0073] Synthesis of (E)-2-bromo-N-(l ,3-dihydroxyoctadec-4-en-2-yl)acetamide:

Sphingosine (20 mg, 0.066 mmol) was dissolved in acetonitrile (30 mL). To this solution, 100 mL of saturated sodium bicarbonate solution was added. The solution was kept in ice bath and then bromoacetyl chloride (42 mL, 0.266 mmol) was added slowly and reaction was allowed to stir at room temperature for 30 min. The reaction was monitored by TLC using 20% Ethyl acetate/Hexane system. Upon completion of the reaction, solvent was removed under reduced pressure to dryness. Then around 20 mL of Ethyl acetate was added and then the organic layer was washed with water, brine and collected, dried over sodium sulphate and then evaporated to obtain 25 mg of white solid crude. Crude was directly carried forward for the next reaction.

[0074] Synthesis of Sphingo-Az ((E)-2-azido-N-( 1 ,3-dihydroxyoctadec-4-en-2-yl) acetamide): Crude compound 2 (25 mg, 0.047 mmol) was dissolved in of DMSO (10 mL) and then sodium azide (15 mg, 0.23 mmol) was added to the solution. The reaction mixture was allowed to stir at room temperature for 4 hours. The reaction was monitored by TLC using 20% Ethylacetate/Hexane system. Upon completion of the reaction, water (20 mL) was added slowly to the reaction mixture followed by Ethyl acetate (20 mL). The organic layer was extracted out and then dried over sodium sulphate and then evaporated to obtain white solid crude which was then purified by column chromatography (EA: Hexane = 1: 9).

Product was obtained as white solid (12.1 mg, 67.5%). 1H NMR (300 MHz, CDC1₃) δ 6.70 (d, = 8.0 Hz, 1H), 5.87 - 5.70 (m, 1H), 5.49 (dd, = 15.5, 6.2 Hz, 1H), 4.40 (d, = 4.9 Hz, 1H), 4.29 - 4.24 (m, 1H), 4.00 (s, 1H), 3.89 (s, 1H), 2.09 - 2.02 (m, 2H), 1.36 - 1.25 (m, 22H), 0.87 (t, = 6.7 Hz, 3H). ¹³C NMR (75 MHz, CDCI₃) δ 166.92, 135.50, 127.63, 72.64, 63.74, 52.59, 52.46, 50.31, 31.83, 29.59, 29.51, 29.39, 29.26, 29.12, 28.98, 22.60, 14.02. HRMS: m/z calcd for C₂oH₃₈N₄0₃ 405.2836 (M+Na)⁺, found 405.2847.

Example 2: Cellular retention and efflux characteristics of the probes :

[0075] Characteristics of CO-1, CO-2 and COA-1 probes: Screening was conducted to study cell permeability and nonspecific background properties of the CO-1, CO-2 and COA-1 probes. Probes were separately added into live CHO (FIG 1A) and U-2 OS (FIG IB) cell lines. Cells were stained separately with CO-1 and CO-2 probes at 1 μΜ final concentration for 30 min. U-2 OS (FIG 8B) cells were also stained separately with COA-1 probe at 3 μΜ final concentration for 30 min. Cell images were taken after 30 minutes to check the cell permeability by ImageXpress Micro™ cellular imaging system (Molecular Device). These cell lines were chosen due to their flexibility in genetic manipulation, adaptability to various culture conditions, and versatility in wide biomedical research such as gene expression, toxicity screening, recombinant protein and live cell imaging. The cells were then washed with growth media, and after another 10 minutes, second image was taken, to check the complete washing out of the probes. The first and second imaging facilitated influx (before washing, BW) and efflux (after washing, AW) measurements, respectively. Automated quantitative intensity-based image analysis was then performed for influx/efflux analysis using MetaXpress High Content Image software. Result showed that CO-1, CO-2 and COA- 1 are cell -permeable and have low nonspecific binding group as they enter the cell and leave after washing leaving a clear fluorescence background (green signal from FITC channel, Scale bar, 20 μιη in FIGS. 1A-1B and blue signal from DAPI channel, Scale bar, 20 μιη in FIG 6B).

[0076] Characteristics ofAzA-1, AzC-1 andAzG-1 probes: Screening was conducted to study cell permeability and nonspecific background properties of the AzA-1, AzC-1 and AzG-1 probes as described above. Probes were separately added into live U-2 OS (FIGS. 7B, 8B and 9B) cells. Cells were stained separately with the AzA-1, AzC-1 and AzG-lprobes at 3 uM final concentration for 30 min. Result showed that AzA-1, AzC-1 and AzG-1 probes are cell-permeable and have low nonspecific binding group as they enter the cell and leave after washing leaving a clear fluorescence background (blue signal for AzA-1 from DAPI channel in FIG. 7B, yellow signal for AzC-1 from TRITC channel, in FIG. 8B and green signal for AzG-1 from FITC channel, in FIG. 9B). Example 3: Absorbance and emission characteristics of the probes :

[0077] Spectral characteristics of CO-1, CO-2 and COA-1 probes: Absorbance and fluorescence emission were measured in DMSO at 10 uM of compound/probe concentration. Absorbance and emission spectra of CO-l(FIG. 2), CO-2 (FIG. 2) and COA-l(FIG. 6A) probes were visualized on most standard fluorescence microscope.

[0078] Spectral characteristics ofAzA-1, AzC-1 andAzG-1 probes: Absorbance and fluorescence emission were measured in DMSO at 10 uM of compound/probe concentration. Absorbance and emission spectra of AzA-1 (FIG. 9A), AzC-1 (FIG. 10A) and AzG-1 (FIG. 11 A) probes were visualized on most standard fluorescence microscope.

Example 4: Photostability analysis of CO-1 and CO-2:

[0079] Photostability measurements were performed by placing 10 μΜ solution of the compound of the present invention {e.g., CO-1 or CO-2), in PBS buffer (pH 7.4) containing 1% DMSO in 96-well plates. Fluorescence measurement were recorded every 30 seconds interval for a total period of 12 hours (Ex/Em = 490/520) under a xenon flashlamp. To simulate stringent conditions, photostability test was done under high intensity UV lamp (Blak Ray, 100W, 365 nm). Plates were irradiated for 10 minutes up to 2.5 hours at 10 cm distance. Values are represented in FIGS. 5A-5B as means (n=3) and fitted to a non-linear regression one -phase exponential decay (GraphPad Prism 5.0). These results demonstrate that the probes tested were photostable. In contrast to fluorescent proteins which easily undergo photochemical oxidation and are easily bleached under live-cell imaging condition, CO-1 and CO-2 exhibited superior photostability even under strong light source condition (FIGS. 5A-5B), enabling their use for extensive time lapse imaging to monitor dynamic processes in live cells or in vivo, including but not limited to changes in the distribution of target proteins, cell movement tracking, intracellular organelle mobility and cell cycle.

Example 5: Intracellular imaging in live cells using probes of the present invention:

[0080] Intracellular imaging in live cells using CO-1 and CO-2 probes: Live cell imaging with CO-1 and CO-2 was performed in U-2 OS cells. For Mitochondrial imaging in live cells, U-2 OS cells were treated with 5 μΜ TPP-Az, in culture media for 2 hrs at 37 °C. After incubation, cells were washed and added with 2 μΜ of the compound of the present invention {e.g., CO-1 or CO-2). Labeling was allowed to proceed 1 hr at 37 °C in the incubator chamber. Following incubation, cells were treated with nuclei dye Hoechst33342 (1 μ /μί) and MitoTracker Red (1 μΜ). After counterstaining, cells were washed 3 times with culture media and imaged using Nikon A1R+ confocal laser microscope system. For Lysosomal imaging in live cells, U-2 OS cells were treated with 5 μΜ Morph-Az, in culture media for 1 hr at 37 °C. After incubation, cells were washed and added with 2 μΜ of the compound of the present invention (e.g., CO-1 or CO-2), then proceed for 1 hr incubation at 37 °C. Following incubation, cells were treated with nuclei dye Hoechst33342 (1 μ /μί) and LysoTracker Red (1 μΜ). After counterstaining, cells were washed 3 times with culture media and imaged using Nikon A1R+ confocal laser microscope system. For imaging Golgi apparatus in live cells, U-2 OS cells were treated with 5 μΜ Sphingo-Az/ BSA and 5 μΜ BODIPY TR ceramide/BSA in HBSS/HEPES for 30 min at 4 °C. Afterwards, cells were washed with cold culture media and further incubated for 30 min at 37 °C. After incubation, cells were washed and incubated with 2 μΜ of the compound of the present invention (e.g., CO-1 or CO-2) in culture media for 1 hr at 37 °C. Following incubation, cells were treated with nuclei dye Hoechst33342 (1 μg/μL). After counterstaining, cells were washed 3 times with culture media and imaged using Nikon A1R+ confocal laser microscope system. The merged images contain superimposed images of Hoechst, CO-1 or CO-2 and organelle trackers. Scale bar, 15 μτη. Results showed that both probes, CO-1 (FIG. 4A) or CO-2 (FIG. 4B), are able to label mitochondria, golgi apparatus and lysosome in live cells with a clear background. As shown in FIGS. 4A-4B, CO-1 and CO-2 specifically labeled the azide- tagged intracellular organelles in live cells and their signal are colocalized with respective organelle probe trackers. Clean fluorescence background was observed due to washable property of the unreacted CO-1 and CO-2.

[0081] Intracellular imaging in live cells using AzG-1 probe: Live cell imaging with AzG-1 probe was performed in U-2 OS cells as described above. For Mitochondrial imaging in live cells, U-2 OS cells were pretreated with TPP-BCN (an analogue of

triphenylphosphonium bearing cyclooctyne moiety which accumulates in mitochondria) before being labelled with 10 μΜ AzG-1. As shown in FIG. 10, AzG-1 specifically labelled mitochondria and its signals are colocalized with the MitoTracker red. Clean fluorescence background was observed due to washable property of the unreacted AzG-1.

[0082] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0083] While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

What is claimed is:

1. A compound having a structural formula (I):

wherein

Ri is

or , and R₄ is H or Ci-C₄ alkyl; or

Ri is H, C₁-C4 alkyl or

wherein n is a whole number selected from 1 to 4 and R₄ is

; and

R₂ is H or Ci-C₄ alkyl; R₃ is H or Ci-C₄ alkyl;

R₅ is H, C1-C4 alkyl or

wherein n is a whole number selected from 1 to 4;

R₆ is H or Ci-C₄ alkyl; R₇ is H or Q-C₄ alkyl, and

L is a linear C1-C10 alkylene, wherein one or more methylene groups in the Q-Qo alkylene is optionally and independently replaced by -N(R')C(0)-, -C(O)-, -O-C(O)- -N(R , -0-, -C(0)N(R , -N(R')C(0)0-, or -N(R')C(0)N(R')-, wherein each R' is independently hydrogen or Ci_C₃ alkyl, and each carbon in the C1-C10 alkylene is optionally and independently substituted by one or two Ci_C₃ alkyl groups.

The compound of claim 1, wherein

Ri is H, C₁-C4 alkyl or

wherein n is a whole number selected from 1 to 4 and R4 is

The compound of claim 2, wherein L is a linear C₁-C₁₀ alkylene, wherein one or more methylene groups in the Q-Qo alkylene is optionally and independently replaced by one or more of -N(R')C(0)-, -C(O)-, -O-C(O)- and -N(R')--

The compound of claim 3, wherein L is a linear Ci-C₆ alkylene, wherein one or more methylene groups in the Ci-C₆ alkylene is optionally and independently replaced by one or more of -N(R')C(0)-, -C(O)-, -O-C(O)- and -N(R')--

The compound of claim 4, wherein one or more methylene groups in the Q-C₆ alkylene is optionally and independently replaced by one or more of -N(R')C(0)- and

-C(O)-.

The compound of claim 4, wherein one or more methylene groups in the Ci-C₆ alkylene is optionally and independently replaced by one or more of -N(R')- and

-C(O)-. The compound of claim 4, wherein

and R₄ is H or methyl; or

The compound of claim 1, wherein

. L,

N₃

R, is and R4 is H or Ci-C₄ alkyl; or wherein n is a whole number

9. The compound of claim 8, wherein L is a linear Ci-Cio alkylene, wherein one or more methylene groups in the Ci-Cio alkylene is optionally and independently replaced by one or more of -N(R')C(0)-, -C(O)-, -O-C(O)-, and -N(R .

10. The compound of claim 9, wherein L is a linear C₁-C₇ alkylene, wherein one or more methylene groups in the C₁-C₇ alkylene is optionally and independently replaced by one or more of N(R')C(0)-, -C(O)-, -O-C(O)-, and -N(R')-.

11. The compound of claim 10, wherein one or more methylene groups in the C₁-C₇ alkylene is optionally and independently replaced by -N(R')C(0)-.

12. The compound of claim 10, wherein one or more methylene groups in the C₁-C7 alkylene is optionally and independently replaced by -N(R')-.

13. The compound of claim 10, wherein

H or methyl; or

The compound of claim 7, having a structural formula:

The compound of claim 13, having a structural formula:

A method of labeling tagged organelles, the method comprising:

adding to one or more cells an azide or cyclooctyne-containing organelle- localizing reporter, thereby producing one or more tagged organelles; and

adding the compound of any one of claims 1-15 to the cells, thereby covalently labeling the tagged organelles. The method of claim 16, wherein the azide or cyclooctyne-containing -containing organelle-localizing reporter is

18. The method of claim 16, wherein the organelle is one or more of mitochondria,

lysosome and golgi apparatus.

19. A method of live cell intracellular imaging, the method comprising:

adding the compound of any one claims 1-15 to one or more live cells comprising at least one tagged organelle;

washing the cells; and

conducting confocal fluorescence imaging of the washed cells.

20. The method of claim 19, wherein the imaging is time lapse imaging and the method further comprises monitoring one or more dynamic processes in the live cells, wherein the one or more dynamic processes are cell movement, intracellular organelle mobility, cell cycle and distribution of proteins.