# Patent

## Title
Reactive labelling compounds and uses thereof

## Abstract

Provided are azido-BODIPY compounds of formula (I), cyclooctyne-based fluorogenic probes of formula (IV), and activity-based probes of formula (VI). These compounds undergo azide-alkyne cycloadditions (AAC) with to form triazolyl products. The provided compounds are useful for detection and imaging of alkyne-, or azide-containing molecules. Methods for detection and imaging biomolecules using compounds of the present disclosure are disclosed.

## Background
RELATED APPLICATION
This application is a continuation-in-part of, and claims the benefit of priority of International Application Serial No. PCT/US2015/022977, filed Mar. 27, 2015, which claims the benefit of priority to U.S. provisional application Ser. No. 61/971,313, filed on Mar. 27, 2014. The contents of which is incorporated herein by reference.


FIELD
The present disclosure relates to the field of triazole formation of cyclooctyne-fused fluorogenic probes, azido-BODIPY compounds, and fluorescently-cleaved probes for the diagnosis and imaging of alkyne- or azide-containing biomolecules. The present disclosure relates to a fluorescence enhancement strategy upon azide-alkyne cycloadditions (AAC).
BACKGROUND
Copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) has gained widespread use in chemical biology for applications such as labeling of biomolecules in complex mixtures and imaging of fixed cells and tissues. (Kolb, et al., Angew. Chem. Int. Ed. 2001, 40, 2004; Rostovtsev, et al., Angew. Chem. Int. Ed. 2002, 41, 2596; Wu and Fokin, Aldrichimica Acta 2007, 40, 7.) Incorporation of fluorescent probes into proteins, DNA, RNA, lipids and glycans within their native cellular environments provides opportunities for imaging and understanding their roles in vivo. (Best, Biochemistry 2009, 48, 6571.)
For example, glycans in protein are displayed on the cell surface with implications in numerous physiological and pathological processes. Aberrant glycosylation on the surface of diseased cells is often observed in pathological conditions, such as inflammation and cancer metastasis. In particular, altered terminal sialylation and fucosylation, which are believed to result from changes in expression locations and levels of sialyltransferases and fucosyltransferases, are associated with tumor malignancy. The ability to explore the biological information content of glycans as biomarkers of cancer, attached to either proteins or lipids, has become a major course of glycomics research. (Hsu, et al., Proc. Nat. Acad. Sci. U.S.A., 2007, 104, 2614; Sawa, et al., Proc. Nat. Acad. Sci. U.S.A., 2006, 103, 12371.)
Analysis of changes in glycosylation patterns in living systems is now possible. (Prescher and Bertozzi, Nat. Chem. Bio. 2005, 1, 13.) Metabolic incorporation of an unnatural carbohydrate containing unique functional group that acts as a bioorthogonal chemical reporter into the cell biosynthetic machinery initiates the process. The modified glycan is then processed and constructed on the cell surface. Subsequent reaction with a detectable fluorescent probe equipped with a complementary bioorthogonal functional group enables detection of the incorporated unnatural glycan. (Sletten and Bertozzi, Angew. Chem. Int. Ed. 2009, 48, 2.)
The concept of bioorthogonal chemical reporter has been applied to proteomic analysis of glycosylation in proteins and chemical remodeling of cell surfaces in living systems. Bioorthogonal chemical reactions have also been used for other applications, such as protein labeling, activity-based protein folding, protein targets identification, posttranslational modifications, and cell proliferation monitoring. Labeling of specific functional groups on living cell via bioorthogonal chemical reporter strategies have become increasingly powerful in cell biology. In the past few years, a tremendous progress has been made in bioorthogonal chemistry, especially that shows biocompatibility and selectivity in living systems. These approaches are often based on cycloadditions as ideal bioorthogonal reactions because of their intrinsic selectivity and tunable electronics. However, there are still many challenges facing the field, particularly from the perspective of cellular and organismal applications. For example, most bioorthogonal reporter strategies entail multistep procedures that use fluorophroe-labeled reactant partners, which often cause high background fluorescent noise that is difficult to remove from intracellular environments or tissues. In addition, these methods require high concentrations of reagents and catalysts in order to achieve detectable signals.
Some recent efforts have been focused on the design of non- or weak fluorescent probe upon CuAAC reactions with non-fluorescent alkynes or azides, which can ligate to afford a highly fluorescent triazole complex (FIG. 2). (Zhou and Fahrni, J. Am. Chem. Soc. 2004, 126, 8862; Sivakumar, et al., Org. Lett. 2004, 24, 4603; Sawa, et al., Proc. Nat. Acad. Sci. U.S.A., 2006, 103, 12371; Xie, et al., Tetrahedron 2008, 64, 2906; Li, et al., Org. Lett. 2009, 11, 3008; Le Droumaguet, et al., Chem. Soc. Rev. 2010, 39, 1223; Qi, et al, Bioconjugate Chem. 2011, 22, 1758; Chao, et al., Sci. China Chemistry 2012, 55, 125. Herner, et al., Org. Biomol. Chem. 2013, 11, 3297.) This type of CuAAC reaction occurring in high efficiency would have broad applications in the emerging field of cell biology and functional proteomics due to the distinct fluorescence properties in formation of the triazole without background fluorescent noise of the starting materials. However, these azido- and alkynyl-functionalized probes usually require excitation in the UV region and emit blue light with poor quantum yield in aqueous solution; such optical properties are not ideal for biological applications.
The distinct fluorescence enhancement induced by highly efficient CuAAC reactions would have broad applications in the emerging field of cell biology and functional proteomics (Le Droumaguet, C.; Wang, C.; Wang, Q. Chem. Soc. Rev. 2010, 39, 1233-1239; Sawa, M.; Hsu, T.-L.; Itoh, T.; Sugiyama, M.; Hanson, S. R.; Vogt, P. K.; Wong, C.-H. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 12371-12376, Shie, J.-J.; Liu, Y.-C.; Lee, Y.-M.; Lim, C.; Fang, J.-M.; Wong, C.-H. J. Am. Chem. Soc. 2014, 136, 9953-9961, Hsu, T.-L.; Hanson, S. R.; Kishikawa, K.; Wang, S.-K.; Sawa, M.; Wong, C.-H. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2614-2619, Tsai, C.-S.; Liu, P.-Y.; Yen, H.-Y.; Hsu, T.-L.; Wong C.-H. Chem. Commun. 2010, 46, 5575-5577). However, the toxicity of Cu(I) has hindered the use of CuAAC in living systems.
To circumvent the cytotoxicity problem associated with the metal catalyst, the ring strain-promoted azide-alkyne cycloadditions (SPAAC) have been developed as an alternative strategy (Jewett, J. C.; Bertozzi, C. R. Chem. Soc. Rev. 2010, 39, 1272-1279, Debets, M. F.; van Berkel, S. S.; Dommerholt, J.; Dirks, A. T. J.; Rutjes, F. P. J. T.; van Delft, F. L. Acc. Chem. Res. 2011, 44, 805-815). A cyclooctyne moiety is often incorporated as a stem structure into the SPAAC reagents, such as difluorinated cyclooctynes (DIFO) and the derivatives (Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. J. Am. Chem. Soc. 2004, 126, 15046-15047, Codelli, J. A.; Baskin, J. M.; Agard, N. J.; Bertozzi, C. R. J. Am. Chem. Soc. 2008, 130, 11486-11493). To increase the ring strain, the cyclooctyne moiety can be fused with other rings to give SPAAC reagents with higher reactivity, such as dibenzylcyclooctyne (DIBO) (Ning, X.; Guo, J.; Wolfert, M. A.; Boons, G.-J. Angew. Chem. Int. Ed. 2008, 47, 2253-2255, Poloukhtine, A. A.; Mbua, N. E.; Wolfert, M. A.; Boons, G.-J.; Popik, V. V. J. Am. Chem. Soc. 2009, 131, 15769-15777, Stöckmann, H.; Neves, A. A.; Stairs, S.; Ireland-Zecchini, H.; Brindle, K. M.; Leeper, F. J. Chem. Sci. 2011, 2, 932-936, Friscourt, F.; Ledin, P. A.; Mbua, N. E.; Flanagan-Steet, H. R.; Wolfert, M. A.; Steet, R.; Boons, G.-J. J. Am. Chem. Soc. 2012, 134, 5381-5389) diarylazacyclooctynone (BARAC) (Jewett, J. C.; Sletten, E. M.; Bertozzi, C. R. J. Am. Chem. Soc. 2010, 132, 3688-3690) and bicyclononynes (BCN) (Dommerholt, J.; Schmidt, S.; Temming, R.; Hendriks, L. J. A.; Rutjes, F. P. J. T.; van Hest, J. C. M.; Lefeber, D. J.; Friedl, P.; van Delft, F. L. Angew. Chem. Int. Ed. 2010, 49, 9422-9425). Tetramethylthiacycloheptyne (TMTH) bearing a contracted seven-membered ring also exhibits reactivity in cycloaddition reactions with azides (de Almeida, G.; Sletten, E. M.; Nakamura, H.; Palaniappan, K. K.; Bertozzi, C. R. Angew. Chem. Int. Ed. 2012, 51, 2443-2447, King, M., Baati, R.; Wagner, A. Chem. Commun. 2012, 48, 9308-9309). Two cyclooctyne-based fluorogenic probes, CoumBARAC (Jewett, J. C.; Bertozzi, C. R. Org. Lett. 2011, 13, 5937-5939) and Fl-DIBO (Friscourt, F.; Fahrni, C. J.; Boons, G.-J. J. Am. Chem. Soc. 2012, 134, 18809-18815) have been described by the Bertozzi and Boons groups, respectively.
4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (also known as BODIPY) dyes are a type of popular fluorophores for many biological applications. BODIPY dyes have numerous advantages including great chemical and photophysical stability, relatively high molar absorption coefficients and fluorescence quantum yields (Φfl), excitation/emission wavelengths in the visible spectral region (longer than 500 nm), and narrow emission bandwidths with high peak intensities. (Loudet and Burgess, Chem. Rev. 2007, 107, 4891; Ulrich et al., Angew. Chem. Int. Ed. 2008, 47, 1184; Boens, et al., Chem. Soc. Rev. 2012, 41, 1130; Kamkaew, et al, Chem. Soc. Rev. 2013, 42, 77.)
Some azido-BODIPY derivatives have been developed for fluorescent labeling upon CuAAC reactions. (Li, et al., J. Org. Chem. 2008, 73, 1963.) Specifically, the low fluorescence 3-azido-BODIPY derivatives have been shown to undergo a CuAAC reaction to give the corresponding triazole with enhanced fluorescence. Although the triazole product provided a 300-fold increased emission compared to azido-BODIPY, it exhibited a low fluorescence quantum yield (Φfl<0.03) and the unreacted azido-BODIPY compound is unstable and fails to react with alkynyl biomolecules under physiological conditions, making it is incompatible with many biological applications. (Wang, et al., Sci. China Chemistry 2012, 55, 125; Chauhan, et al. Tetrahedron Lett. 2014, 55, 244.)
SUMMARY OF THE INVENTION
Accordingly, there is a need for new design of molecular probes pertinent to cell environments for cell labeling, detecting and/or visualizing the localization of biomolecules in cells.
Accordingly, the present disclosure relates to a new series of azido-BODIPY compounds containing a green-emitting BODIPY scaffold pertinent to cell environments. The BODIPY scaffold is used as a starting module for its appealing synthetic and fluorescent features. Exemplary BODIPYs are easily modified at the 8-position. Arylation at this position has no substantial influence on absorption and emission wavelengths because the aryl moiety and BODIPY core are twisted and conjugation uncoupled.
These exemplary compounds are useful for labeling with alkyne-functionalized proteins without washing processes, and are suitable for visualizing the localization of alkyne-tagged glycosyl conjugates in cells by confocal microscopy. Furthermore, the alkynyl-saccharide modified cells can be lysed and analyzed on SDS-PAGE by using AzBOCEt labeling for direct detection of the probe-labeled glycoproteins without enrichment.
The present disclosure also relates to cyclooctyne-based fluorogenic probes which are capable of reacting to alkyne-functionalized moieties. In some aspects, the cyclooctyne-based fluorogenic probes can be present in a cell. In some aspects, the cyclooctyne-based fluorogenic probes can be used to detect azide-glycoconjugates in a cell.
The present disclosure also relates to the dual-imaging of both azido-functionalized glycoconjugates and alkynyl-functinonalized glyconjugates by contacting a sample with azido-BODIPY compounds of formula (I) and/or cyclooctyne-based fluorogenic probes of formula (IV) in a dual imaging mode.
The present disclosure also relates to measuring the activity of enzymes using probes designed to form a covalent bond with the active site of an enzyme wherein said probes comprise an alkyne moiety for further detection by an azide-containing fluorogenic probe. The enzyme can be a sialidase. The fluorogenic sialidase probe can be based on 3-fluorosialyl fluoride as a mechanism-based inhibitor.
Accordingly, the present disclosure relates to exemplary novel azido-BODIPY compounds of formula (I) that undergo azide-alkyne cycloadditions (AAC). The azide-alkyne cycloadditions (AAC) can be strain or catalyst (metal or organic) promoted. In some embodiments, the catalyst is a metal catalyst. In certain embodiments, the metal catalyst is copper(I).
Exemplary azido-BODIPY compounds described herein can react with alkyne compounds to give stable triazole products with enhanced fluorescence to facilitate the detection. The provided exemplary compounds represent a significant advance in cell-imaging without washing processes and are applicable to direct in-gel detection of the alkyne-tagged glycoproteins from cell lysates after SDSPAGE.
One aspect of the present disclosure relates to an azido-BODIPY compound of Formula (I):




or a pharmaceutically acceptable salt, solvate, or hydrate thereof, and in which G1, G2, G3, G4a, G4b, G5, G6, G7 and G8 and n are as described herein.

In another aspect, the present disclosure provides synthetic methods for preparation of azido-BODIPY compounds. The present disclosure also demonstrates that the azido-BODIPY compounds described herein can react with organic alkynes to form triazole products with enhanced fluorescence.
In another aspect, the present disclosure provides a triazolyl-BODIPY compound of Formula (III):




or a pharmaceutically acceptable salt, solvate, or hydrate thereof, and in which G1, G2, G3, G4a, G4b, G5, G6, G7, G8 and n are as described herein. Target molecule includes, but not limited to, a biomolecule such as a DNA, RNA, protein and glycan.

The azide-alkyne cycloadditions (AAC) can be strain or catalyst (metal or organic) promoted. In some embodiments, the catalyst is a metal catalyst. In certain embodiments, the metal catalyst is copper(I).
In yet another aspect, present disclosure relates to methods for detecting and/or imaging biomolecules.
In certain embodiments, the present disclosure provides a method for imaging an alkyne-containing molecule, the method comprising

    
    
        (a) incubating a compound as described herein with a sample containing the alkyne-containing molecule under conditions allowing for ligation of the compound to an alkyne group of the molecule to form a triazole product; and
        (b) detecting a fluorescent signal released from the triazole product.
    
    


In certain embodiments, the present disclosure provides a method for detecting an alkyne-containing molecule in a sample, the method comprising:

    
    
        (a) contacting a compound as described herein to a sample suspected of having an alkyne-containing molecule;
        (b) detecting a level of a fluorescent signal released from the sample, and
        (c) determining presence of the alkyne-containing molecule in the sample,

wherein an enhanced fluorescent signal as compared to a level of the fluorescent signal in the absence of the molecule indicates presence of the alkyne-containing molecule.

In another aspect, the present disclosure provides a cyclooctyne-based fluorogenic probe of Formula (IV):

    
    






or a pharmaceutically acceptable salt, solvate, or hydrate thereof, and wherein:

each instance of G9, G10, G11, G12, G13, G14, G15, G16, G17 and G18 is independently hydrogen, optionally substituted C1-6 alkyl, optionally substituted C1-6 alkenyl, optionally halogen, optionally nitroso, optionally substituted C1-6 alkynyl, optionally substituted aryl, optionally substituted acyl, —ORA, —CH2ORA, —OC(O)RA, —SRA, —N(RB)2, —N(RA)C(O)RA, —C(O)N(RB)2, —CN, —NO2, —C(O)RA, —C(O)ORA, —S(O)RA, —SO2RA, —SO3RA, —SO2N(RB)2, and —NHSO2RB;
each instance of R is independently selected from hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted acyl, —ORA, —CH2ORA, —OC(O)RA, —SRA, —N(RB)2, —N(RA)C(O)RA, —C(O)N(RB)2, —CN, —NO2, —C(O)RA, —C(O)ORA, —S(O)RA, —SO2RA, —SO3RA, —SO2N(RB)2, and —NHSO2RB;
each RA is independently selected from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl; and
each RB is independently selected from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl, or two RB taken together with the intervening nitrogen form a heterocycle;
each instance of G9 and G10 is hydrogen, fluoro, chloro, bromo, iodo, nitroso, alkyl, alkoxy, aryloxy, or alkynyl, and
wherein alkyl and alkoxy groups are unbranched, saturated, and have 1-4 carbon atoms; aryl groups and aryl groups of aryloxy can be either carbocyclic aryl or heterocyclic aryl; carbocyclic aryl groups have a total of 6-20 carbon atoms, including carbon atoms of substituents; heterocyclic aryl groups have a total of 5-20 carbon atoms, including carbon atoms of substituents; carboalkoxy groups are alkyl esters of a carboxylic acid wherein alkyl groups are as defined above; each alkyl, aryl, alkoxy, aryloxy, benzo, and carboalkoxy, independently, may be unsubstituted or substituted with one or more substituent; alkyl substituents are halo, hydroxyl, amino, or aryl; aryl substituents are halo, hydroxyl, amino, alkyl, aryl, nitro, or carboxyl; and halo substituents are fluoro or chloro;
m is 0 or 1;
n is 0, 1, 2, 3, or 4.
In certain embodiments, cell permeable fluorogenic probes with improved reactivity can be prepared by implementation of additional ring strain or electron-withdrawing substituents to the cyclooctyne moiety. Additional ring strain could be achieved by, for example, altering the given formula from a cyclooctyne ring to a cycloheptyne ring.
In certain embodiments, the present disclosure provides a method for imaging an azide-containing molecule, the method comprising

    
    
        (a) incubating a compound of formula (IV) with a sample containing the azide-containing molecule under conditions allowing for ligation of the compound to an azide group of the molecule to form a triazole product; and
        (b) detecting a fluorescent signal released from the triazole product.
    
    


In certain embodiments, the present disclosure provides a method for detecting an azide-containing molecule in a sample, the method comprising:

    
    
        (a) contacting a compound of formula (IV) to a sample suspected of having an azide-containing molecule;
        (b) detecting a level of a fluorescent signal released from the sample, and
        (c) determining presence of the azide-containing molecule in the sample,

wherein an enhanced fluorescent signal as compared to a level of the fluorescent signal in the absence of the molecule indicates presence of the azide-containing molecule.

    
    


In another aspect, this disclosure relates to compounds of formula (V):




wherein L is selected from the group consisting of halogen, alkoxy, phenoxy, pentafluorophenoxy, 4-nitrophenoxy, umbelliferyl, alkanoate, benzoate, triflate, mesylate, or tosylate.

In certain embodiments of formula V or IV, L can be a halogen independently including or excluding F, Cl, Br, or I. In certain other embodiments, L is Cl, Br, or I. In certain embodiments, L is halogen but not F.
In certain embodiments, the present disclosure provides a method for imaging the active site of a sialidase enzyme, the method comprising:
(a) contacting a compound of formula (VI):




wherein L is selected from the group consisting of halogen, F, Cl, Br, I, alkoxy, phenoxy, pentafluorophenoxy, 4-nitrophenoxy, umbelliferyl coumarin oxide, alkanoate, benzoate, triflate, mesylate, or tosylate;

    
    
        with a sample suspected of comprising the sialidase enzyme under conditions for ligation of the compound to the active site of the sialidase enzyme to form a covalent bond product,
        (b) contacting the covalent bond product with an azide-containing fluorogenic probe as described herein to form a fluorogenic triazole product,
        (c) measuring a fluorescent signal released from the triazole product.
    
    


In certain embodiments, the present disclosure provides a method for detecting the active site of a sialidase enzyme in a sample, comprising:

    
    
        (a) contacting the compound 601:
    
    






with a sample suspected of having an sialidase enzyme molecule;

    
    
        (b) measuring the level of a fluorescent signal released from the coumadin in the sample mixture, and
        (c) determining the presence of the sialidase enzyme molecule in the sample, wherein an enhanced fluorescent signal as compared to a level of the fluorescent signal in the absence of the molecule indicates presence of the azide-containing molecule.
    
    





BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the structures of azido-substituted BODIPY derivatives Az1-Az8 used in the fluorescence screening through the CuAAC reactions.
FIG. 2 shows the fluorescence screening of CuAAC reactions of Az1-Az8, giving the corresponding triazole derivatives T1-T8, in a microtier plate with UV lamp excitation (λex=365 nm). Compounds Az1 to Az8 and T1 to T8 are sorted in an increasing order of electron density of the aryl moiety.
FIG. 3A, 3B, 3C shows the structures of amino-BODIPY Am10, azido-BODIPYs Az2, Az9-Az11 and the corresponding triazolyl-BODIPYs T2, T9-T11 obtained by CuAAC reactions with 4-pentyn-1-ol.
FIG. 4 shows the absorption and normalized emission spectra of triazolyl-BODIPY T10, azido-BODIPY Az10 and amino-BODIPY Am10 in ethanol solution (12 μM) at 25° C. Inset: the images of T10, Az10 and Am10 in ethanol solution (120 μM). The change of yellow Az10 solution to green T10 solution was apparent.
FIG. 5 shows alkyne-functionalized BSA labeling with AzBOCEt (Az10). The gel was analyzed by fluorescence imaging (λex=488 nm; λem=526 nm). The total protein content was revealed by Coomassie blue stain.
FIG. 6A, 6B, 6C shows the cell fluorescence labeling with AzBOCEt (Az10) and imaging by confocal microscopy. (A) Illustration of the cell labeling experiments using Ac4ManNAl, Ac4GalNAl and Az10. CL1-5 cells were incubated with 100 μM Ac4ManNAl, Ac4GalNAl or control sugars (Ac4ManNAc and Ac4GalNAc) for 3 days, and then treated with 0.1 μM Az10 for 1 h under CuAAC conditions. (B) Fluorescence, bright field and overlaid images. Scale bar: 75 μm. (C) Localization of the expressed glycosyl conjugates in CL1-5 cells. These glycosyl conjugates were labeled with fluorogenic probe Az10 (green), anti-GRASP65 (a Golgi marker) followed by Cy3 conjugated anti-rabbit (red), and Hoechst (blue, a nucleus marker). Scale bar: 10 μm.
FIG. 7 shows the direct in-gel fluorescence detection of alkyne-tagged glycoproteins using CuAAC with AzBOCEt (Az10) from cell lysates. The gel was analyzed by fluorescence imaging)λex=488 nm; λem=526 nm) and Coomassie blue staining to reveal the total protein content.
FIG. 8A, 8B shows the cell fluorescence labeling with AzBOCEt (Az10) and imaging by confocal microscopy. The CL1-5 cells were incubated with Ac4GlcNAl or Ac4GlcNAc for 3 days and then treated with 0.1 μM Az10 for 1 h under CuAAC conditions. (A) Fluorescence, bright field and overlaid images. Scale bar: 75 μm. (B) Localization of the expressed glucosyl conjugates in CL1-5 lung cells. These glucosyl conjugates were labeled with fluorogenic probe Az10 (green), anti-GRASP65 (a Golgi marker) followed by Cy3 conjugated anti-rabbit (red), and Hoechst (blue, a nucleus marker). Scale bar: 10 μm
FIG. 9 shows Fluorescence imaging of glycan trafficking using AzBOCEt of CuAAC in cells. CL1-5 cells were incubated with 500 μM Ac4ManNAl for 1 h and subsequently washed with PBS buffer to remove excess Ac4ManNAl. The sugar-treated cells were incubated in culture medium for 1 h, 7 h, 14 h and 21 h, and then labeled with 0.1 μM Az10 for 1 h under CuAAC conditions, respectively.
FIG. 10A, 10B, 10C shows a SPAAC-based fluoresence-forming probe, a comparative probe, and the triazole product of the comparative probe.
FIG. 11A, 11B shows structures of sialidase probes.
FIG. 12 shows the general structure of an activity-based probe.
FIG. 13 shows the mechanism and transition state of sialidase enzymes.
FIG. 14A, 14B shows the x-ray crystal structure of sialidase active site.
FIG. 15 shows fluorogenic reactions for the identification of sialidases.
FIG. 16A, 16B shows the absorbance and emission spectra and time-course during reaction of fluorogenic probes.
FIG. 17 shows a plot of the inverse flourogenic probe concentration as a function of reaction time of 101 and a model azide molecule.
FIG. 18 shows a plot of the inverse flourogenic probe concentration as a function of reaction time of 101 and a model azide-containing glycan. Plot of 1/[101] vs. time for the reaction of compound 101 and N-azidoacetylmannosamine in a solution of CD3OD-D2O (5:1, v/v) as monitored by 1H-NMR.
FIG. 19 shows time-lapse fluorescence and overlaid images of living CL1-5 cells incubated with 200 μM of Ac4ManNAz and labeled with 100 μM of probe 101 under no-wash and no-fixation conditions.
FIG. 20 shows localization of probe-labeled sialyl glycoconjugates in CL1-5 cells as visualized by confocal microscopy.
FIG. 21 shows high contrast fluorescence imaging of glycoconjugates trafficking using coumOCT (101) of SPAAC in cells.
FIG. 22 shows time-lapse fluorescence and overlaid images of living CL1-5 cells incubated with 200 μM of Ac4ManNAz and labeled with 100 μM of probe 101.
FIG. 23 shows fluorescence imaging of glycoconjugates trafficking using coumOCT (101) of SPAAC in cells.
FIG. 24A, 24B shows dual fluorescence labeling in cells with coumOCT (1), AzBOCEt and imaging by confocal microscopy. Dual fluorescence labeling in cells with coumOCT (1), AzBOCEt and imaging by confocal microscopy. (A) Illustration of the cell labeling experiments using Ac4ManNAl, Ac4GlcNAz, 101 and AzBOCEt. CL1-5 cells were incubated with 100 μM of Ac4ManNAl and Ac4GlcNAz or control sugars (Ac4ManNAc and Ac4GlcNAc) for 3 days, which were treated with 100 μM of probe 101 for 0.5 h under SPAAC conditions, and then incubated with 0.1 μM of AzBOCEt for 1 h under CuAAC conditions. (B) Dual fluorescence imaging in CL1-5 cells. These glycoconjugates were labeled with probe 101 (cyan) for azido-containing glycoconjugates and AzBOCEt (green) for alkyne-containing glycoconjugates. (Scale bar: 10 μm)
FIG. 25 shows 1H-NMR spectrum in CDCl3 of compound 101 and 3-mercaptopropionic acid.
FIG. 26 shows 1H-NMR spectrum in CDCl3 of compound 111 and 3-mercaptopropionic acid.



DETAILED DESCRIPTION OF THE INVENTION
Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.
Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various stereoisomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures using methods including chiral high pressure liquid chromatography (HPLC), and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, N.Y., 1962); and Wilen, S. H. Tables of Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind. 1972). The present disclosure additionally encompasses compounds as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.
When a range of values is listed, it is intended to encompass each value and sub range within the range. For example “C1-6 alkyl” is intended to encompass, C1, C2, C3, C4, C5, C6, C1-6, C1-5, C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5, and C5-6 alkyl.
As used herein, “alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 10 carbon atoms (“C1-10 alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C1-8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C1-7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C1-6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C1-5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C1-4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1-2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C1 alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-6 alkyl”). Examples of C1-6 alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8) and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents. In certain embodiments, the alkyl group is an unsubstituted C1-10 alkyl (e.g., —CH3). In certain embodiments, the alkyl group is a substituted C1-10 alkyl.
As used herein, “alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 10 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). In some embodiments, an alkenyl group has 2 to 9 carbon atoms (“C2-9 alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C2-8 alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C2-7 alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C2-6 alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C2-5 alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C2-4 alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C2-3 alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C2 alkenyl”). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C2-4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-6 alkenyl groups include the aforementioned C2-4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like. Unless otherwise specified, each instance of an alkenyl group is independently unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents. In certain embodiments, the alkenyl group is an unsubstituted C2-10 alkenyl. In certain embodiments, the alkenyl group is a substituted C2-10 alkenyl.
As used herein, “alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 10 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (“C2-10 alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C2-9 alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C2-8 alkynyl”). In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C2-7 alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C2-6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C2-5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C2-4 alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C2-3 alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C2 alkynyl”). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C2-4 alkynyl groups include, without limitation, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Examples of C2-6 alkenyl groups include the aforementioned C2-4 alkynyl groups as well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl include heptynyl (C7), octynyl (C8), and the like. Unless otherwise specified, each instance of an alkynyl group is independently unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents. In certain embodiments, the alkynyl group is an unsubstituted C2-10 alkynyl. In certain embodiments, the alkynyl group is a substituted C2-10 alkynyl.
As used herein, “carbocyclyl” or “carbocyclic” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C3-10 carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms (“C3-8 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (“C3-7 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C3-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 4 to 6 ring carbon atoms (“C4-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms (“C5-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms (“C5-10 carbocyclyl”). Exemplary C3-6 carbocyclyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Exemplary C3-8 carbocyclyl groups include, without limitation, the aforementioned C3-6 carbocyclyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptanyl (C7), bicyclo[2.2.2]octanyl (C8), and the like. Exemplary C3-10 carbocyclyl groups include, without limitation, the aforementioned C3-8 carbocyclyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C10), spiro[4.5]decanyl (C10), and the like. As the foregoing examples illustrate, in certain embodiments, the carbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can be saturated or can contain one or more carbon-carbon double or triple bonds. “Carbocyclyl” also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system. Unless otherwise specified, each instance of a carbocyclyl group is independently unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a “substituted carbocyclyl”) with one or more substituents. In certain embodiments, the carbocyclyl group is an unsubstituted C3-10 carbocyclyl. In certain embodiments, the carbocyclyl group is a substituted C3-10 carbocyclyl.
As used herein, “heterocyclyl” or “heterocyclic” refers to a radical of a 3- to 14-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“3-14 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or polycyclic (e.g., a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”) or tricyclic system (“tricyclic heterocyclyl”)), and can be saturated or can contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each instance of heterocyclyl is independently unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a “substituted heterocyclyl”) with one or more substituents. In certain embodiments, the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3-14 membered heterocyclyl.
As used herein, “aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-14 aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C6 aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms (“C14 aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents. In certain embodiments, the aryl group is an unsubstituted C6-14 aryl. In certain embodiments, the aryl group is a substituted C6-14 aryl.
As used herein, “heteroaryl” refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5-14 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system. Polycyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl).
As understood from the above, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, are, in certain embodiments, optionally substituted. Optionally substituted refers to a group which may be substituted or unsubstituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or “unsubstituted” heteroalkyl, “substituted” or “unsubstituted” heteroalkenyl, “substituted” or “unsubstituted” heteroalkynyl, “substituted” or “unsubstituted” carbocyclyl, “substituted” or “unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or “unsubstituted” heteroaryl group). In general, the term “substituted” means that at least one hydrogen present on a group is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position. The term “substituted” is contemplated to include substitution with all permissible substituents of organic compounds, any of the substituents described herein that results in the formation of a stable compound. The present present disclosure contemplates any and all such combinations in order to arrive at a stable compound. For purposes of this present disclosure, heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.
Exemplary carbon atom substituents include, but are not limited to, halogen, —CN, —NO2, —N3, —SO2H, —SO3H, —OH, —ORaa, —ON(Rbb)2, —N(Rbb)2, —N(Rbb)3+X−, —N(ORcc)Rbb, —SH, —SRaa, —SSRcc, —C(═O)Raa, —CO2H, —CHO, —C(ORcc)2, —CO2Raa, —OC(═O)Raa, —OCO2Raa, —C(═O)N(Rbb)2, —OC(═O)N(Rbb)2, —NRbbC(═O)Raa, —NRbbCO2Raa, —NRbbC(═O)N(Rbb)2, —C(═NRbb)Raa, —C(═NRbb)ORaa, —OC(═NRbb)Raa, —OC(═NRbb)ORaa, —C(═NRbb)N(Rbb)2, —OC(═NRbb)N(Rbb)2, —NRbbC(═NRbb)N(Rbb)2, —C(═O)NRbbSO2Raa, —NRbbSO2Raa, —SO2N(Rbb)2, —SO2Raa, —SO2ORaa, —OSO2Raa, —S(═O)Raa, —OS(═O)Raa, —Si(Raa)3, —OSi(Raa)3—C(═S)N(Rbb)2, —C(═O)SRaa, —C(═S)SRaa, —SC(═S)SRaa, —SC(═O)SRaa, —OC(═O)SRaa, —SC(═O)ORaa, —SC(═O)Raa, —P(═O)2Raa, —OP(═O)2Raa, —P(═O)(Raa)2, —OP(═O)(Raa)2, —OP(═O)(ORcc)2, —P(═O)2N(Rbb)2, —OP(═O)2N(Rbb)2, —P(═O)(NRbb)2, —OP(═O)(NRbb)2, —NRbbP(═O)(ORcc)2, —NRbbP(═O)(NRbb)2, —P(Rcc)2, —P(Rcc)3, —OP(Rcc)2, —OP(Rcc)3, —B(Raa)2, —B(ORcc)2, —BRaa(ORcc), C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 heteroalkyl, C2-10 heteroalkenyl, C2-10heteroalkynyl, C3-14 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;
or two geminal hydrogens on a carbon atom are replaced with the group ═O, ═S, ═NN(Rbb)2, ═NNRbbC(═O)Raa, ═NNRbbC(═O)ORaa, ═NNRbbS(═O)2Raa, ═NRbb, or ═NORcc;
each instance of Raa is, independently, selected from C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 heteroalkyl, C2-10 heteroalkenyl, C2-10heteroalkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two Raa groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;
each instance of Rbb is, independently, selected from hydrogen, —OH, —ORaa, —N(Rcc)2, —CN, —C(═O)Raa, —C(═O)N(Rcc)2, —CO2Raa, —SO2Raa, —C(═NRcc)ORaa, —C(═NRcc)N(Rcc)2, —SO2N(Rcc)2, —SO2Rcc, —SO2ORcc, —SORaa, —C(═S)N(Rcc)2, —C(═O)SRcc, —C(═S)SRcc, —P(═O)2Raa, —P(═O)(Raa)2, —P(═O)2N(Rcc)2, —P(═O)(NRcc)2, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 heteroalkyl, C2-10 heteroalkenyl, C2-10heteroalkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two Rbb groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;
each instance of Rcc is, independently, selected from hydrogen, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 heteroalkyl, C2-10 heteroalkenyl, C2-10 heteroalkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two Rcc groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;
each instance of Rdd is, independently, selected from halogen, —CN, —NO2, —N3, —SO2H, —SO3H, —OH, —ORee, —ON(Rff)2, —N(Rff)2, —N(Rff)3+X−, —N(ORee)Rff, —SH, —SRee, —SSRee, —C(═O)Ree, —CO2H, —CO2Ree, —OC(═O)Ree, —OCO2Ree, —C(═O)N(Rff)2, —OC(═O)N(Rff)2, —NRffC(═O)Ree, —NRffCO2Ree, —NRffC(═O)N(Rff)2, —C(═NRff)ORee, —OC(═NRff)Ree, —OC(═NRff)ORee, —C(═NRff)N(Rff)2, —OC(═NRff)N(Rff)2, —NRffC(═NRff)N(Rff)2, —NRffSO2Ree, —SO2N(Rff)2, —SO2Ree, —SO2ORee, —OSO2Ree, —S(═O)Ree, —Si(Ree)3, —OSi(Ree)3, —C(═S)N(Rff)2, —C(═O)SRee, —C(═S)SRee, —SC(═S)SRee, —P(═O)2Ree, —P(═O)(Ree)2, —OP(═O)(Ree)2, —OP(═O)(ORee)2, C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 heteroalkyl, C2-6 heteroalkenyl, C2-6heteroalkynyl, C3-10 carbocyclyl, 3-10 membered heterocyclyl, C6-10 aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups, or two geminal Rdd substituents can be joined to form ═O or ═S;
each instance of Ree is, independently, selected from C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 heteroalkyl, C2-6 heteroalkenyl, C2-6 heteroalkynyl, C3-10 carbocyclyl, C6-10 aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups;
each instance of Rff is, independently, selected from hydrogen, C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 heteroalkyl, C2-6 heteroalkenyl, C2-6heteroalkynyl, C3-10 carbocyclyl, 3-10 membered heterocyclyl, C6-10 aryl and 5-10 membered heteroaryl, or two Rff groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups; and
each instance of Rgg is, independently, halogen, —CN, —NO2, —N3, —SO2H, —SO3H, —OH, —OC1-6 alkyl, —ON(C1-6 alkyl)2, —N(C1-6 alkyl)2, —N(C1-6 alkyl)3+X−, —NH(C1-6 alkyl)2+X−, —NH2(C1-6 alkyl)+X−, —NH3+X−, —N(OC1-6 alkyl)(C1-6 alkyl), —N(OH)(C1-6 alkyl), —NH(OH), —SH, —SC1-6 alkyl, —SS(C1-6 alkyl), —C(═O)(C1-6 alkyl), —CO2H, —CO2(C1-6 alkyl), —OC(═O)(C1-6 alkyl), —OCO2(C1-6 alkyl), —C(═O)NH2, —C(═O)N(C1-6 alkyl)2, —OC(═O)NH(C1-6 alkyl), —NHC(═O)(C1-6 alkyl), —N(C1-6 alkyl)C(═O)(C1-6 alkyl), —NHCO2(C1-6 alkyl), —NHC(═O)N(C1-6 alkyl)2, —NHC(═O)NH(C1-6 alkyl), —NHC(═O)NH2, —C(═NH)O(C1-6 alkyl), —OC(═NH)(C1-6 alkyl), —OC(═NH)OC1-6 alkyl, —C(═NH)N(C1-6 alkyl)2, —C(═NH)NH(C1-6 alkyl), —C(═NH)NH2, —OC(═NH)N(C1-6 alkyl)2, —OC(NH)NH(C1-6 alkyl), —OC(NH)NH2, —NHC(NH)N(C1-6 alkyl)2, —NHC(═NH)NH2, —NHSO2(C1-6 alkyl), —SO2N(C1-6 alkyl)2, —SO2NH(C1-6 alkyl), —SO2NH2, —SO2C1-6 alkyl, —SO2OC1-6 alkyl, —OSO2C1-6 alkyl, —SOC1-6 alkyl, —Si(C1-6 alkyl)3, —OSi(C1-6 alkyl)3-C(═S)N(C1-6 alkyl)2, C(═S)NH(C1-6 alkyl), C(═S)NH2, —C(═O)S(C1-6 alkyl), —C(═S)SC1-6 alkyl, —SC(═S)SC1-6 alkyl, —P(═O)2(C1-6 alkyl), —P(═O)(C1-6 alkyl)2, —OP(═O)(C1-6 alkyl)2, —OP(═O)(OC1-6 alkyl)2, C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 heteroalkyl, C2-6 heteroalkenyl, C2-6heteroalkynyl, C3-10 carbocyclyl, C6-10 aryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl; or two geminal Rgg substituents can be joined to form ═O or ═S; wherein X− is a counterion.
As used herein, the term “halo” or “halogen” refers to fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine (bromo, —Br), or iodine (iodo, —I). In certain embodiments, L can independently include or exclude F, Cl, Br, or I.
In certain embodiments, the substituent present on the nitrogen atom is an nitrogen protecting group (also referred to herein as an “amino protecting group”). Nitrogen protecting groups include, but are not limited to, —OH, —ORaa, —N(Rcc)2, —C(═O)Raa, —C(═O)N(Rcc)2, —CO2R, —SO2R, —C(═NRcc)Raa, —C(═NRcc)ORaa, —C(═NRcc)N(Rcc)2, —SO2N(Rcc)2, —SO2Rcc, —SO2ORcc, —SORaa, —C(═S)N(Rcc)2, —C(═O)SRcc, —C(═S)SRcc, C1-10 alkyl (e.g., aralkyl, heteroaralkyl), C2-10 alkenyl, C2-10 alkynyl, C1-10 heteroalkyl, C2-10 heteroalkenyl, C2-10 heteroalkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl groups, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups, and wherein Raa, Rbb, Rcc and Rdd are as defined herein. Nitrogen protecting groups include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
For example, nitrogen protecting groups such as amide groups (e.g., —C(═O)Raa) include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N′-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.
Nitrogen protecting groups such as carbamate groups (e.g., —C(═O)ORaa) include, but are not limited to, methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and 4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate.
Nitrogen protecting groups such as sulfonamide groups (e.g., —S(═O)2Raa) include, but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.
Other nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacyl derivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine derivative, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-p-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivative, N-diphenylborinic acid derivative, N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).
In certain embodiments, the substituent present on an oxygen atom is an oxygen protecting group (also referred to herein as an “hydroxyl protecting group”). Oxygen protecting groups include, but are not limited to, —Raa, —N(Rbb)2, —C(═O)SRaa, —C(═O)Raa, —CO2Raa, —C(═O)N(Rbb)2, —C(═NRbb)Raa, —C(═NRbb)ORaa, —C(═NRbb)N(Rbb)2, —S(═O)Raa, —SO2Raa, —Si(Raa)3, —P(Rcc)2, —P(Rcc)3, —P(═O)2Raa, —P(═O)(Raa)2, —P(═O)(ORcc)2, —P(═O)2N(Rbb)2, and —P(═O)(NRbb)2, wherein Raa, Rbb, and Rcc are as defined herein. Oxygen protecting groups include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
Exemplary oxygen protecting groups include, but are not limited to, methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxyl)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl, 4,4′,4′′-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4′,4′′-tris(levulinoyloxyphenyl)methyl, 4,4′,4′′-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4′,4′′-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), isobutyl carbonate, vinyl carbonate, allyl carbonate, t-butyl carbonate (BOC), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzyl carbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate, p-nitrobenzyl carbonate, S-benzyl thiocarbonate, 4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts).
In certain embodiments, the substituent present on an sulfur atom is a sulfur protecting group (also referred to as a “thiol protecting group”). Sulfur protecting groups include, but are not limited to, —Raa, —N(Rbb)2, —C(═O)SRaa, —C(═O)Raa, —CO2Raa, —C(═O)N(Rbb)2, —C(═NRbb)Raa, —C(═NRbb)ORaa, —C(═NRbb)N(Rbb)2, —S(═O)Raa, —SO2Raa, —Si(Raa)3, —P(Rcc)2, —P(Rcc)3, —P(═O)2Raa, —P(═O)(Raa)2, —P(═O)(ORcc)2, —P(═O)2N(Rbb)2, and —P(═O)(NRbb)2, wherein Raa, Rbb, and Rcc are as defined herein. Sulfur protecting groups include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
As used herein, when two entities are “conjugated” or “ligated” to one another, they are linked by a direct or indirect covalent or non-covalent interaction. In certain embodiments, the association is covalent. In other embodiments, the association is non-covalent. Non-covalent interactions include hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetic interactions, electrostatic interactions, etc. In certain embodiments, two entities are covalently connected, optionally through a linker group.
As used herein, the term “salt” refers to any and all salts, including pharmaceutically acceptable salt which refers to those salts within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio (see Berge et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19). Examples of pharmaceutically acceptable, nontoxic acid salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.
A “subject” to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) and/or other non-human animals, for example, mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs) and birds (e.g., commercially relevant birds such as chickens, ducks, geese, and/or turkeys). In certain embodiments, the animal is a mammal. The animal may be a male or female and at any stage of development. A non-human animal may be a transgenic animal.
As used herein, the terms “condition,” “disease,” and “disorder” are used interchangeably.
As used herein “inhibition”, “inhibiting”, “inhibit” and “inhibitor”, and the like, refer to the ability of a compound to reduce, slow, halt, or prevent the activity of a particular biological process.
As used herein, the term “cell” present disclosure is meant to encompass eukaryotic and prokaryotic cells of any genus or species, with mammalian cells being of particular interest. “Cell” is also meant to encompass both normal cells and diseased cells, e.g., cancerous cells. In certain embodiments, the cells described herein are living cells.
As used herein the term “sample” includes any chemical sample or biological sample. Chemical sample refers to any chemical mixtures or chemical compounds. Biological sample includes, without limitation, cell cultures or extracts thereof; biopsied material obtained from an animal (e.g., mammal) or extracts thereof; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof. For example, the term “biological sample” refers to any solid or fluid sample obtained from, excreted by or secreted by any living organism, including single-celled microorganisms (such as bacteria and yeasts) and multicellular organisms (such as plants and animals, for instance a vertebrate or a mammal, and in particular a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated). The biological sample can be in any form, including a solid material such as a tissue, cells, a cell pellet, a cell extract, cell homogenates, or cell fractions; or a biopsy, or a biological fluid. The biological fluid may be obtained from any site (e.g. blood, saliva (or a mouth wash containing buccal cells), tears, plasma, serum, urine, bile, cerebrospinal fluid, amniotic fluid, peritoneal fluid, and pleural fluid, or cells therefrom, aqueous or vitreous humor, or any bodily secretion), a transudate, an exudate (e.g. fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (e.g. a normal joint or a joint affected by disease such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis). The biological sample can be obtained from any organ or tissue (including a biopsy or autopsy specimen) or may comprise cells (whether primary cells or cultured cells) or medium conditioned by any cell, tissue or organ. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes. Biological samples also include mixtures of biological molecules including proteins, lipids, carbohydrates and nucleic acids generated by partial or complete fractionation of cell or tissue homogenates. Although the sample is preferably taken from a human subject, biological samples may be from any animal, plant, bacteria, virus, yeast, etc. The term animal, as used herein, refers to humans as well as non-human animals, at any stage of development, including, for example, mammals, birds, reptiles, amphibians, fish, worms and single cells. Cell cultures and live tissue samples are considered to be pluralities of animals. In certain exemplary embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). An animal may be a transgenic animal or a human clone. If desired, the biological sample may be subjected to preliminary processing, including preliminary separation techniques.
The term “physiological conditions” is meant to encompass those conditions compatible with living cells, e.g., predominantly aqueous conditions of a temperature, pH, salinity, etc. that are compatible with living cells.
The present disclosure is based on the design and chemical synthesis of a new series of reactive labelling compounds. The compounds can be azido-BODIPY compounds containing a green-emitting BODIPY scaffold pertinent to cell environments. The BODIPY scaffold is used as a starting module for its appealing synthetic and fluorescent features. BODIPYs are easily modified at the 8-position. Arylation at this position has no substantial influence on absorption and emission wavelengths because the aryl moiety and BODIPY core are twisted and conjugation uncoupled.
Described herein are azido-BODIPY compounds that undergo azide-alkyne cycloadditions (AAC) in the presence of catalysts with alkyne-containing molecules to form triazolyl products that exhibit enhanced fluorescence to facilitate the detection of the molecules. The azido-BODIPY compounds represent an advance in cell-imaging without washing processes and are applicable to direct in-gel detection of the alkyne-tagged glycoproteins from cell lysates after SDS-PAGE.
The reactive labelling compounds can also be cyclooctyne-based fluorogenic probes. The cyclooctyne-based fluorogenic probes can further comprise cyclooctynes linked to a coumarin moiety. Also, described herein are cyclooctyne-based fluorogenic probes compounds that undergo azide-alkyne cycloadditions (AAC) in the presence of catalysts with azide-containing molecules to form triazolyl products that exhibit enhanced fluorescence to facilitate the detection of the molecules.
Described herein are methods of using both azido-BODIPY and cyclooctyne-based fluorogenic probes for imaging azido-containing glycoconjugates and alkynyl-containing glycoconjugates in living cells.
Described herein are compounds and methods of using said compounds for conjugation to the active site of enzymes. In some embodiments, the enzyme is a sialidase enzyme. In some embodiments, the compounds are alkynyl-containing compounds. In some embodiments, the compounds form a covalent bond with the active site of the sialidase enzyme.
Azido-BODIPY Compounds
An azido-BODIPY compound is the Formula (I):




or a pharmaceutically acceptable salt, solvate, or hydrate thereof, and wherein:

each instance of G1, G2, G3, G5, G6, G7 and G8 is independently hydrogen, optionally substituted C1-6 alkyl, optionally substituted C1-6 alkenyl, optionally substituted C1-6 alkynyl, optionally substituted aryl, optionally substituted acyl, —ORA, —CH2ORA, —OC(O)RA, —SRA, —N(RB)2, —N(RA)C(O)RA, —C(O)N(RB)2, —CN, —NO2, —C(O)RA, —C(O)ORA, —S(O)RA, —SO2RA, —SO3RA, —SO2N(RB)2, and —NHSO2RB;
each instance of R is independently selected from hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted acyl, —ORA, —CH2ORA, —OC(O)RA, —SRA, —N(RB)2, —N(RA)C(O)RA, —C(O)N(RB)2, —CN, —NO2, —C(O)RA, —C(O)ORA, —S(O)RA, —SO2RA, —SO3RA, —SO2N(RB)2, and —NHSO2RB;
each RA is independently selected from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl; and
each RB is independently selected from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl, or two RB taken together with the intervening nitrogen form a heterocycle.
each instance of G4a and G4b is fluoro, alkyl, alkoxy, aryloxy, or alkynyl, and wherein alkyl and alkoxy groups are unbranched, saturated, and have 1-4 carbon atoms; aryl groups and aryl groups of aryloxy can be either carbocyclic aryl or heterocyclic aryl; carbocyclic aryl groups have a total of 6-20 carbon atoms, including carbon atoms of substituents; heterocyclic aryl groups have a total of 5-20 carbon atoms, including carbon atoms of substituents; carboalkoxy groups are alkyl esters of a carboxylic acid wherein alkyl groups are as defined above; each alkyl, aryl, alkoxy, aryloxy, benzo, and carboalkoxy, independently, may be unsubstituted or substituted with one or more substituent; alkyl substituents are halo, hydroxyl, amino, or aryl; aryl substituents are halo, hydroxyl, amino, alkyl, aryl, nitro, or carboxyl; and halo substituents are fluoro or chloro;
n is 0, 1, 2, 3, or 4.
As generally defined herein, G1 is independently selected from hydrogen, halogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORA, —CH2ORA, —OC(O)RC, —SRA, —N(RB)2, —N(RA)C(O)RC, —C(O)N(RB)2, —CN, —NO2, —C(O)RC, —C(O)ORA, —S(O)RC, —SO2RA, —SO2N(RB)2, ═O, ═NOH, ═N—ORA, ═N—NH2, ═N—NHRA, ═N—N(RB)2, and —NHSO2RA. In certain embodiments, G1 is H. In certain embodiments, G1 is halogen. In certain embodiments, G1 is optionally substituted C1-C6 alkyl. In certain embodiments, G1 is methyl, ethyl, or n-propyl. In certain embodiments, G1 is —ORA, wherein RA is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or an oxygen protecting group when attached to an oxygen, or a sulfur protecting group when attaching to sulfur. In certain embodiments, G1 is —OH. In certain embodiments, G1 is —ORA, wherein RA is optionally substituted C1-C6 alkyl. In certain embodiments, G1 is —OCH3 or —OC2H5. In certain embodiments, G1 is —ORA, wherein RA is an oxygen protecting group. In certain embodiments, G1 is —N(RB)2, wherein each instance of RB is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl, or a nitrogen protecting group, or two RB taken together with the intervening nitrogen form a heterocycle. In certain embodiments, G1 is NH2. In certain embodiments, G1 is NHRB, wherein RB is optionally substituted C1-C6 alkyl. In certain embodiments, G1 is NHCH3 or NHC2H5. In certain embodiments, G1 is NHRB, wherein RB is a nitrogen protecting group.
As generally defined herein, G2 is independently selected from hydrogen, halogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORA, —CH2ORA, —OC(O)RC, —SRA, —N(RB)2, —N(RA)C(O)RC, —C(O)N(RB)2, —CN, —NO2, —C(O)RC, —C(O)ORA, —S(O)RC, —SO2RA, —SO2N(RB)2, ═O, ═NOH, ═N—ORA, ═N—NH2, ═N—NHRA, ═N—N(RB)2, and —NHSO2RA. In certain embodiments, G2 is H. In certain embodiments, G2 is halogen. In certain embodiments, G2 is optionally substituted C1-C6 alkyl. In certain embodiments, G2 is methyl, ethyl, or n-propyl. In certain embodiments, G7 is —ORA, wherein RA is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, or an oxygen protecting group when attached to an oxygen, or a sulfur protecting group when attaching to sulfur. In certain embodiments, G2 is —OH. In certain embodiments, G2 is —ORA, wherein RA is optionally substituted C1-C6 alkyl. In certain embodiments, G2 is —OCH3 or —OC2H5. In certain embodiments, G2 is —ORA, wherein RA is an oxygen protecting group. In certain embodiments, G2 is —N(RB)2, wherein each instance of RB is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl, or a nitrogen protecting group, or two RB taken together with the intervening nitrogen form a heterocycle. In certain embodiments, G2 is NH2. In certain embodiments, G2 is NHRB, wherein RB is optionally substituted C1-C6 alkyl. In certain embodiments, G2 is NHCH3 or NHC2H5. In certain embodiments, G2 is NHRB, wherein RB is a nitrogen protecting group.
As generally defined herein, G3 is independently selected from hydrogen, halogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORA, —CH2ORA, —OC(O)RC, —SRA, —N(RB)2, —N(RA)C(O)RC, —C(O)N(RB)2, —CN, —NO2, —C(O)RC, —C(O)ORA, —S(O)RC, —SO2RA, —SO2N(RB)2, ═O, ═NOH, ═N—ORA, ═N—NH2, ═N—NHRA, ═N—N(RB)2, and —NHSO2RA. In certain embodiments, G3 is H. In certain embodiments, G3 is halogen. In certain embodiments, G3 is optionally substituted C1-C6 alkyl. In certain embodiments, G3 is methyl, ethyl, or n-propyl. In certain embodiments, G7 is —ORA, wherein RA is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, or an oxygen protecting group when attached to an oxygen, or a sulfur protecting group when attaching to sulfur. In certain embodiments, G3 is —OH. In certain embodiments, G3 is —ORA, wherein RA is optionally substituted C1-C6 alkyl. In certain embodiments, G3 is —OCH3 or —OC2H5. In certain embodiments, G3 is —ORA, wherein RA is an oxygen protecting group. In certain embodiments, G3 is —N(RB)2, wherein each instance of RB is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl, or a nitrogen protecting group, or two RB taken together with the intervening nitrogen form a heterocycle. In certain embodiments, G3 is NH2. In certain embodiments, G3 is NHRB, wherein RB is optionally substituted C1-C6 alkyl. In certain embodiments, G3 is NHCH3 or NHC2H5. In certain embodiments, G3 is NHRB, wherein RB is a nitrogen protecting group.
As generally defined herein, G5 is independently selected from hydrogen, halogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORA, —CH2ORA, —OC(O)RC, —SRA, —N(RB)2, —N(RA)C(O)RC, —C(O)N(RB)2, —CN, —NO2, —C(O)RC, —C(O)ORA, —S(O)RC, —SO2RA, —SO2N(RB)2, ═O, ═NOH, ═N—ORA, ═N—NH2, ═N—NHRA, ═N—N(RB)2, and —NHSO2RA. In certain embodiments, G5 is H. In certain embodiments, G5 is halogen. In certain embodiments, G5 is optionally substituted C1-C6 alkyl. In certain embodiments, G5 is methyl, ethyl, or n-propyl. In certain embodiments, G5 is —ORA, wherein RA is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, or an oxygen protecting group when attached to an oxygen, or a sulfur protecting group when attaching to sulfur. In certain embodiments, G5 is —OH. In certain embodiments, G5 is —ORA, wherein RA is optionally substituted C1-C6 alkyl. In certain embodiments, G5 is —OCH3 or —OC2H5. In certain embodiments, G5 is —ORA, wherein RA is an oxygen protecting group. In certain embodiments, G5 is —N(RB)2, wherein each instance of RB is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl, or a nitrogen protecting group, or two RB taken together with the intervening nitrogen form a heterocycle. In certain embodiments, G5 is NH2. In certain embodiments, G5 is NHRB, wherein RB is optionally substituted C1-C6 alkyl. In certain embodiments, G5 is NHCH3 or NHC2H5. In certain embodiments, G5 is NHRB, wherein RB is a nitrogen protecting group.
As generally defined herein, G6 is independently selected from hydrogen, halogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORA, —CH2ORA, —OC(O)RC, —SRA, —N(RB)2, —N(RA)C(O)RC, —C(O)N(RB)2, —CN, —NO2, —C(O)RC, —C(O)ORA, —S(O)RC, —SO2RA, —SO2N(RB)2, ═O, ═NOH, ═N—ORA, ═N—NH2, ═N—NHRA, ═N—N(RB)2, and —NHSO2RA. In certain embodiments, G6 is H. In certain embodiments, G6 is halogen. In certain embodiments, G6 is optionally substituted C1-C6 alkyl. In certain embodiments, G6 is methyl, ethyl, or n-propyl. In certain embodiments, G6 is —ORA, wherein RA is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, or an oxygen protecting group when attached to an oxygen, or a sulfur protecting group when attaching to sulfur. In certain embodiments, G6 is —OH. In certain embodiments, G6 is —ORA, wherein RA is optionally substituted C1-C6 alkyl. In certain embodiments, G6 is —OCH3 or —OC2H5. In certain embodiments, G6 is —ORA, wherein RA is an oxygen protecting group. In certain embodiments, G6 is —N(RB)2, wherein each instance of RB is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl, or a nitrogen protecting group, or two RB taken together with the intervening nitrogen form a heterocycle. In certain embodiments, G6 is NH2. In certain embodiments, G6 is NHRB, wherein RB is optionally substituted C1-C6 alkyl. In certain embodiments, G6 is NHCH3 or NHC2H5. In certain embodiments, G6 is NHRB, wherein RB is a nitrogen protecting group.
As generally defined herein, G7 is independently selected from hydrogen, halogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORA, —CH2ORA, —OC(O)RC, —SRA, —N(RB)2, —N(RA)C(O)RC, —C(O)N(RB)2, —CN, —NO2, —C(O)RC, —C(O)ORA, —S(O)RC, —SO2RA, —SO2N(RB)2, ═O, ═NOH, ═N—ORA, ═N—NH2, ═N—NHRA, ═N—N(RB)2, and —NHSO2RA. In certain embodiments, G7 is H. In certain embodiments, G7 is halogen. In certain embodiments, G7 is optionally substituted C1-C6 alkyl. In certain embodiments, G7 is methyl, ethyl, or n-propyl. In certain embodiments, G7 is —ORA, wherein RA is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, or an oxygen protecting group when attached to an oxygen, or a sulfur protecting group when attaching to sulfur. In certain embodiments, G7 is —OH. In certain embodiments, G7 is —ORA, wherein RA is optionally substituted C1-C6 alkyl. In certain embodiments, G7 is —OCH3 or —OC2H5. In certain embodiments, G7 is —ORA, wherein RA is an oxygen protecting group. In certain embodiments, G7 is —N(RB)2, wherein each instance of RB is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl, or a nitrogen protecting group, or two RB taken together with the intervening nitrogen form a heterocycle. In certain embodiments, G7 is NH2. In certain embodiments, G7 is NHRB, wherein RB is optionally substituted C1-C6 alkyl. In certain embodiments, G7 is NHCH3 or NHC2H5. In certain embodiments, G7 is NHRB, wherein RB is a nitrogen protecting group.
As generally defined herein, G8 is independently selected from hydrogen, halogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORA, —CH2ORA, —OC(O)RC, —SRA, —N(RB)2, —N(RA)C(O)RC, —C(O)N(RB)2, —CN, —NO2, —C(O)RC, —C(O)ORA, —S(O)RC, —SO2RA, —SO2N(RB)2, ═O, ═NOH, ═N—ORA, ═N—NH2, ═N—NHRA, ═N—N(RB)2, and —NHSO2RA. In certain embodiments, G8 is H. In certain embodiments, G8 is halogen. In certain embodiments, G8 is optionally substituted C1-C6 alkyl. In certain embodiments, G8 is methyl, ethyl, or n-propyl. In certain embodiments, G8 is —ORA, wherein RA is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, or an oxygen protecting group when attached to an oxygen, or a sulfur protecting group when attaching to sulfur. In certain embodiments, G8 is —OH. In certain embodiments, G8 is —ORA, wherein RA is optionally substituted C1-C6 alkyl. In certain embodiments, G8 is —OCH3 or —OC2H5. In certain embodiments, G8 is —ORA, wherein RA is an oxygen protecting group. In certain embodiments, G8 is —N(RB)2, wherein each instance of RB is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl, or a nitrogen protecting group, or two RB taken together with the intervening nitrogen form a heterocycle. In certain embodiments, G8 is NH2. In certain embodiments, G8 is NHRB, wherein RB is optionally substituted C1-C6 alkyl. In certain embodiments, G8 is NHCH3 or NHC2H5. In certain embodiments, G8 is NHRB, wherein RB is a nitrogen protecting group.
In certain embodiments, two of G8 groups adjacent to each other are taken together with their intervening atoms to form an optionally substituted carbocycle or heterocycle. In certain embodiments, two of G8 groups adjacent to each other are taken together with their intervening atoms to form an optionally substituted carbocycle. In certain embodiments, two of G8 groups adjacent to each other are taken together with their intervening atoms to form an optionally substituted 5-membered carbocycle. In certain embodiments, two of G8 groups adjacent to each other are taken together with their intervening atoms to form an optionally substituted 6-membered carbocycle. In certain embodiments, two of G8 groups adjacent to each other are taken together with their intervening atoms to form an optionally substituted phenyl. In certain embodiments, two of G8 groups adjacent to each other are taken together with their intervening atoms to form an unsubstituted phenyl. In certain embodiments, two of G8 groups adjacent to each other are taken together with their intervening atoms to form an optionally substituted heterocycle. In certain embodiments, two of G8 groups adjacent to each other are taken together with their intervening atoms to form an optionally substituted 5-membered heterocycle with one heteroatom of S, N, or O. In certain embodiments, two of G8 groups adjacent to each other are taken together with their intervening atoms to form an optionally substituted 5-membered heterocycle with two heteroatom each independently selected from the group of S, N, and O. In certain embodiments, two of G8 groups adjacent to each other are taken together with their intervening atoms to form an optionally substituted 6-membered carbocycle with one heteroatom of S, N, or O. In certain embodiments, two of G8 groups adjacent to each other are taken together with their intervening atoms to form an optionally substituted 6-membered carbocycle with two heteroatoms each independently selected from the group consisting of S, N, and O.
In some embodiments, at least two of G1, G3, G5 and G7 are C1-C6 alkyl. In certain embodiments, at least two of G1, G3, G5 and G7 is methyl, ethyl, or n-propyl. In some embodiments, each of G1, G3, G5 and G7 is C1-C6 alkyl. In certain embodiments, each of G1, G3, G5 and G7 is methyl, ethyl, or n-propyl. In certain embodiments, each of G1, G3, G5 and G7 is methyl. In certain embodiments, the present disclosure is directed to a compound of Formula (II):




or a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein G2, G4a, G4b, G6, G8 and n are as described herein.

In certain embodiments,




is of the formula:





As used herein, each instance of R8 is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or an oxygen protecting group. In certain embodiments, R8 is hydrogen. In certain embodiments, R8 is optionally substituted C1-C6 alkyl. In certain embodiments, R8 is methyl or ethyl. In certain embodiments, R8 is an oxygen protecting group.

In certain embodiments,




is of the formula:








As used herein, each instance of R9 is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or an oxygen protecting group when attached to an oxygen. In certain embodiments, R9 is hydrogen. In certain embodiments, R9 is optionally substituted C1-C6 alkyl. In certain embodiments, R9 is methyl or ethyl. In certain embodiments, R9 is an oxygen protecting group, each instance of G4a and G4b is fluoro, alkyl, alkoxy, aryloxy, or alkynyl,

In certain embodiments, a substituent may contribute to optical isomerism and/or stereo isomerism of a compound. Salts, solvates, hydrates, and prodrug forms of a compound are also of interest. All such forms are embraced by the present disclosure. Thus the compounds described herein include salts, solvates, hydrates, prodrug, and isomer forms thereof, including the pharmaceutically acceptable salts, solvates, hydrates, prodrugs, and isomers thereof. In certain embodiments, a compound may be a metabolized into a pharmaceutically active derivative.
Exemplary compounds of formulae (I) and (II) are shown in Table 1:







TABLE 1





Exemplary compounds of formulae (I) and (II).


























 












 












 












 












 












 












 












 












 












 












 












 












 












 












 












 












 












 












 












 












 












 












 












 












 












 












 












 












 












 












 



















Exemplary Azido-BODIPY compounds were used to detect alkyne compounds, including the alkynyl-annexed biomolecules. After AAC reaction with an alkyne compound, the formation of triazole ring releases the fluorescence quenching, and results in a fluorescence-enhancing phenomenon. Compounds T2 and T9-T11 are examples of such type of probes that undergo AAC with azides to give fluorescent triazole products.
Alkyne-Containing Molecules
Alkyne-containing molecule described in the disclosure is of the formula:




Exemplary alkyne-containing target molecules include, but are not limited to, amino acids and amino acid residues, polypeptides (including peptides and proteins), sugars or sugar residues, and the like, which contain or are modified to contain at least one alkyne.
Exemplary target molecule includes, but not limited to, a biomolecule such as DNA, RNA, protein or glycan, and it can be naturally occurring, or may be synthetically or recombinantly produced, and may be isolated, substantially purified, or present within the native milieu of the unmodified molecule upon which the alkyne-containing target molecule is based (e.g., on a cell surface or within a cell, including within a host animal, e.g., a mammalian animal, such as a murine host (e.g., rat, mouse), hamster, canine, feline, bovine, swine, and the like). In some embodiments, the target molecule is present in vitro in a cell-free reaction. In other embodiments, the target molecule is present in a cell and/or displayed on the surface of a cell. In many embodiments of interest, the target molecule is in a living cell; on the surface of a living cell; in a living organism, e.g., in a living multicellular organism. Suitable living cells include cells that are part of a living multicellular organism; cells isolated from a multicellular organism; immortalized cell lines; and the like.
Where the target molecule is a polypeptide, the polypeptide may be composed of D-amino acids, L-amino acids, or both, and may be further modified, either naturally, synthetically, or recombinantly, to include other moieties. For example, the target polypeptide may be a lipoprotein, a glycoprotein, or other such modified protein.
In general, the target molecule useful comprises at least one alkyne for reaction with modified azido-BODIPY according to the disclosure, but may comprise 2 or more, 3 or more, 5 or more, 10 or more alkynes. The number of alkynes that may be present in a suitable target molecule will vary according to the intended application of the final product of the reaction, the nature of the target molecule itself, and other considerations which will be readily apparent to the ordinarily skilled artisan in practicing the disclosure as disclosed herein.
This embodiment of the disclosure is particularly useful in modification of a target molecule in vivo. In this embodiment, the target substrate is modified to comprise an alkynyl group at the point at which linkage to the modified azido-BODIPY is desired. For example, where the target substrate is a polypeptide, the polypeptide is modified to contain an N-terminal alkyne. Where the target substrate is a glycoprotein, a sugar residue of the glycoprotein can be modified to contain an alkyne. A target molecule that is unmodified with an alkyne, but that is to be modified with an alkyne, is referred to herein as a “target substrate.” A target molecule that is modified with an alkyne is referred to herein as “an alkyne-modified target molecule” or “an alkyne-containing target molecule.”
The target substrate can be generated in vitro and then introduced into the cell (e.g., via microinjection, liposome or lipofectin-mediated delivery, electroporation, etc.), which methods will vary according to the nature of the substrate to be targeted for modification and can be readily and appropriately selected by the ordinarily skilled artisan. The final target substrate can also be generated in vivo by exploiting a host cell's natural biosynthetic machinery. For example, the cell can be provided with a biocompatible alkyne-derivative of a substrate for synthesis of the desired target molecule, which substrate is processed by the cell to provide an alkyne-derivative of the desired final target substrate. For example, where the target substrate is a cell surface glycoprotein, the cell can be provided with an alkyne derivative of a sugar residue found within the glycoprotein, which is subsequently processed by the cell through natural biosynthetic processes to produce a modified glycoprotein having at least one modified sugar moiety comprising an accessible alkyne group.
The target substrate can also be produced in vivo using methods. For example, unnatural amino acids having alkynes can be incorporated into recombinant polypeptides expressed in E. coli (see, e.g., Kiick et al. (2000) Tetrahedron 56:9487). Such recombinantly produced polypeptides can be selectively reacted with a modified azido-BODIPY according to the disclosure.
In one example, an alkyne group is incorporated into the target molecule by providing a cell (e.g., a eukaryotic cell that glycosylates biopolymers such as proteins) with a synthetic building block for the desired biopolymer target substrate. For example, the cell can be provided with a sugar molecule comprising an alkyne group to provide for incorporation of the alkyne group in a glycoprotein. In some embodiments, the glycoprotein is expressed on the cell surface. Alternatively, the alkyne group can be incorporated into an amino acid, which is subsequently incorporated into a peptide or polypeptide synthesized by the cell. Several methods are available for incorporating unnatural building blocks into biopolymers; one need not be restricted to cell surface oligosaccharides as target molecules. See, e.g., vanHest et al. (1998) FEBS Lett. 428:68; and Nowak et al. (1995) Science 268:439.
In one embodiment, the target molecule is a carbohydrate-containing molecule (e.g., a glycoprotein; a polysaccharide; etc.), and an alkyne group is introduced into the target molecule using a synthetic substrate. In some embodiments, the synthetic substrate is an alkyne derivative of a sugar utilized in production of a glycosylated molecule. In some embodiments, the synthetic substrate is an alkyne derivative of a sugar utilized in production of a cell surface molecule, e.g., in the glycoprotein biosynthetic pathway. For example, the host cell can be provided with a synthetic sialic acid alkynyl-derivative, which is incorporated into the pathway for sialic acid biosynthesis, eventually resulting in the incorporation of the synthetic sugar residue in glycoproteins. In some embodiments, the glycoproteins are displayed on the cell surface.
In one example, the synthetic substrate is an alkynyl derivative of mannosamine of the general formula:




where n is from 0 to 7, generally from 1 to 4, more usually 1 to 2, and R1, R2, R3, and R4 are independently hydrogen or acetyl. In some embodiments, the substrate is N-3-butynoylmannosamine (n=0) or an acetylated derivative thereof, or N-4-pentynoylmannosamine (n=1) or an acetylated form thereof.

In another embodiment, the synthetic substrate is an alkynyl sugar derivative of a general formula of, for example:




either of which can be incorporated into the sialic acid biosynthesis pathway, and where n is from 1 to 6, generally from 1 to 4, more usually 1 to 2, and R2, R3, and R4 are independently hydrogen or acetyl.

In another embodiment, the synthetic substrate is an alkynyl sugar derivative of a general formula of, for example:




where R1, R2, R3, and R4 are independently hydrogen or acetyl, and where the synthetic substrate is incorporated into biosynthetic pathways involving fucose.

In another embodiment, the synthetic substrate is an alkynyl sugar derivative of a general formula of, for example:




where n is from 1 to 6, generally from 1 to 4, more usually 1 to 2, and R1, R2, R3, and R4 are independently hydrogen or acetyl, and which is incorporated into biosynthetic pathways involving galactose.

In some embodiments, a subject method is used to modify the surface of a cell. Thus, in one aspect, the disclosure features a method of modifying the surface of cell in vitro or in vivo. The method generally involves reacting an alkyne group in a target molecule that comprises an alkynyl group with a modified azido-BODIPY to provide for chemoselective ligation at the cell surface. In many embodiments, the method comprises modifying a target molecule on a cell surface with an alkynyl group; and reacting the alkynyl group in the target molecule with a modified azido-BODIPY. For example, as described above, an alkynyl sugar is provided to a living cell, which alkynyl sugar is incorporated into a glycoprotein that is displayed on the cell surface.
Subject modified azido-BODIPY compounds, and subject modification methods, are useful in a variety of applications, including research applications and diagnostic applications.
In some embodiments, subject modified azido-BODIPY compounds, and subject modification methods, are useful in research applications. Applications of interest include research applications, e.g., exploring functional and physical characteristics of a receptor; proteomics; metabolomics; and the like. Research applications also include drug discovery or other screening applications.
Proteomics aims to detect, identify, and quantify proteins to obtain biologically relevant information. Metabolomics is the detection, identification, and quantification of metabolites and other small molecules such as lipids and carbohydrates. Fiehn (2001) Comparative and Functional Genomics 2:155-168; and U.S. Pat. No. 6,873,914.
Drug discovery applications include, but are not limited to, identifying agents that inhibit cancer cell viability and/or growth. Thus, in some embodiments, the instant disclosure provides methods of identifying an agent that inhibits cancer cell viability and/or growth. The methods generally involve modifying a component of the cell to comprise a first reactive partner comprising an alkyne; contacting the cell, in the presence of a test agent, with a second reactive partner comprising a modified azido-BODIPY, the contacting being under physiological conditions; where the contacting results in reaction between the alkynyl group of the first reactive partner and the azido-BODIPY compound of the second reactive partner, thereby synthetically and covalently modifying the cellular component; and determining the effect, if any, of the test agent on the level of modification of the cell with the second reactive partner.
Where the cancer cell is one that produces a higher amount of a carbohydrate than a normal (non-cancerous) cell of the same cell type, the method provides for identifying an agent that reduces growth and/or viability of the cancerous cell.
Applications of interest also include diagnostic applications, e.g., for detection of cancer; and the like, where a subject modified cycloalkyne comprising a detectable label is used to label an alkyne-modified target molecule, e.g., an alkyne-labeled target molecule present on a cancer cell. Applications of interest also include therapeutic applications, where a drug or other therapeutic agent is delivered to an alkyne-modified target molecule, using a subject modified azido-BODIPY compound that comprises a covalently linked drug or other therapeutic agent.
Certain embodiments of the present disclosure are used for in vivo imaging, e.g., to determine the metabolic or other state of a cell in an organism, e.g., an individual. As one non-limiting example, a subject method can be applied to in vivo imaging of cancer cells in an individual (e.g., a mammal, including rodents, lagomorphs, felines, canines, equines, bovines, ovines, caprines, non-human primates, and humans).
One exemplary, non-limiting application of a subject azide-alkyne cycloaddition is in the detection of metabolic change in cells that occur as they alter their phenotype. As one example, altered glycosylation patterns are a hallmark of the tumor phenotype, consisting of both the under- and over-expression of naturally-occurring glycans as well as the presentation of glycans normally restricted to expression during embryonic development. Examples of common antigens associated with transformed cells are sialyl Lewis a, sialyl Lewis x, sialyl T, sialyl Tn, and polysialic acid (PSA). Jørgensen et al. (1995) Cancer Res. 55, 1817-1819; Sell (1990) Hum. Pathology 21, 1003-1019; Taki et al. (1988) J. Biochem. 103, 998-1003; Gabius (1988) Angew. Chem. Int. Ed. Engl. 27, 1267-1276; Feizi (1991) Trends Biochem. Sci. 16, 84-86; Taylor-Papadimitriou and Epenetos (1994) Trends Biotech. 12, 227-233; Hakomori and Zhang (1997) Chem. Biol. 4, 97-104; Dohi et al. (1994) Cancer 73, 1552. These antigens share an important feature—they each contain terminal sialic acid. PSA is a homopolymer of sialic acid residues up to 50 units in length. Elevated levels of sialic acid are highly correlated with the transformed phenotype in many cancers, including gastric (Dohi et al. (1994) Cancer 73, 1552; and Yamashita et al. (1995) J. Natl. Cancer Inst. 87, 441-446), colon (Yamashita et al. (1995) J. Natl. Cancer Inst. 87, 441-446; Hanski et al. (1995) Cancer Res. 55, 928-933; Hanski et al. (1993) Cancer Res. 53, 4082-4088; Yang et al. (1994) Glycobiology 4, 873-884; Saitoh et al. (1992) J. Biol. Chem. 267, 5700-5711), pancreatic (Sawada et al. (1994) Int. J. Cancer 57, 901-907), liver (Sawada et al. (1994) J. Biol. Chem. 269, 1425-1431), lung (Weibel et al. (1988) Cancer Res. 48, 4318-4323), prostate (Jørgensen et al. (1995) Cancer Res. 55, 1817-1819), kidney (Roth et al. (1988) Proc. Natl. Acad. Sci. USA 85, 2999-3000), and breast cancers (Cho et al. (1994) Cancer Res. 54, 6302-6305), as well as several types of leukemia (Joshi et al. (1987) Cancer Res. 47, 3551-3557; Altevogt et al. (1983) Cancer Res. 43, 5138-5144; Okada et al. (1994) Cancer 73, 1811-1816). A strong correlation between the level of cell surface sialic acid and metastatic potential has also been observed in several different tumor types (Kakeji et al. (1995) Brit. J. Cancer 71, 191-195; Takano et al. (1994) Glycobiology 4, 665-674). The collective display of multiple sialylated antigens on a single cancer cell can account for the fact that so many different tumor types share the high sialic acid phenotype without necessarily expressing an identical complement of antigens (Roth et al. (1988) supra). Consequently, diagnostic or therapeutic strategies that target cells on the basis of sialic acid levels have broad applicability to many cancers.
Introduction and incorporation of unnatural alkynylsugars (ManNAl, GalNAl) into living animals provides for detection of changes in metabolic state. Via the attachment of the appropriate epitope tag, the modified azido-BODIPY compound labels these cells in a living organism, and consequently detects changes in metabolic state. Early detection of tumorigenic cells and subsequent intervention reduces the severity and increases survival rates for cancer patients.
Triazolyl-BODIPY Compounds
A triazolyl-BODIPY compound is formed by reacting an azido-BODIPY compound with an alkyne-containing molecule via azide-alkyne cycloadditions (AAC). The triazolyl-BODIPY compound exhibits enhanced fluorescence compared to the corresponding azido-BODIPY compound. Triazolyl-BODIPY compound described in the disclosure is of Formula (III):




or a pharmaceutically acceptable salt, solvate, or hydrate thereof,

wherein:

each instance of G1, G2, G3, G5, G6, G7 and G8 is independently hydrogen, optionally substituted C1-6 alkyl, optionally substituted C1-6 alkenyl, optionally substituted C1-6 alkynyl, optionally substituted aryl, optionally substituted acyl, —ORA, —CH2ORA, —OC(O)RA, —SRA, —N(RB)2, —N(RA)C(O)RA, —C(O)N(RB)2, —CN, —NO2, —C(O)RA, —C(O)ORA, —S(O)RA, —SO2RA, —SO3RA, —SO2N(RB)2, and —NHSO2RB;
each instance of R is independently selected from hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted acyl, —ORA, —CH2ORA, —OC(O)RA, —SRA, —N(RB)2, —N(RA)C(O)RA, —C(O)N(RB)2, —CN, —NO2, —C(O)RA, —C(O)ORA, —S(O)RA, —SO2RA, —SO3RA, —SO2N(RB)2, and —NHSO2RB;
each RA is independently selected from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl; and
each RB is independently selected from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl, or two RB taken together with the intervening nitrogen form a heterocycle.
each instance of G4a and G4b is fluoro, alkyl, alkoxy, aryloxy, or alkynyl, and
wherein alkyl and alkoxy groups are unbranched, saturated, and have 1-4 carbon atoms; aryl groups and aryl groups of aryloxy can be either carbocyclic aryl or heterocyclic aryl; carbocyclic aryl groups have a total of 6-20 carbon atoms, including carbon atoms of substituents; heterocyclic aryl groups have a total of 5-20 carbon atoms, including carbon atoms of substituents; carboalkoxy groups are alkyl esters of a carboxylic acid wherein alkyl groups are as defined above; each alkyl, aryl, alkoxy, aryloxy, benzo, and carboalkoxy, independently, may be unsubstituted or substituted with one or more substituent; alkyl substituents are halo, hydroxyl, amino, or aryl; aryl substituents are halo, hydroxyl, amino, alkyl, aryl, nitro, or carboxyl; and halo substituents are fluoro or chloro;
n is 0, 1, 2, 3, or 4.
As generally defined herein, G1 is independently selected from hydrogen, halogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORA, —CH2ORA, —OC(O)RC, —SRA, —N(RB)2, —N(RA)C(O)RC, —C(O)N(RB)2, —CN, —NO2, —C(O)RC, —C(O)ORA, —S(O)RC, —SO2RA, —SO2N(RB)2, ═O, ═NOH, ═N—ORA, ═N—NH2, ═N—NHRA, ═N—N(RB)2, and —NHSO2RA. In certain embodiments, G1 is H. In certain embodiments, G1 is halogen. In certain embodiments, G1 is optionally substituted C1-C6 alkyl. In certain embodiments, G1 is methyl, ethyl, or n-propyl. In certain embodiments, G1 is —ORA, wherein RA is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or an oxygen protecting group when attached to an oxygen, or a sulfur protecting group when attaching to sulfur. In certain embodiments, G1 is —OH. In certain embodiments, G1 is —ORA, wherein RA is optionally substituted C1-C6 alkyl. In certain embodiments, G1 is —OCH3 or —OC2H5. In certain embodiments, G1 is —ORA, wherein RA is an oxygen protecting group. In certain embodiments, G1 is —N(RB)2, wherein each instance of RB is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl, or a nitrogen protecting group, or two RB taken together with the intervening nitrogen form a heterocycle. In certain embodiments, G1 is NH2. In certain embodiments, G1 is NHRB, wherein RB is optionally substituted C1-C6 alkyl. In certain embodiments, G1 is NHCH3 or NHC2H5. In certain embodiments, G1 is NHRB, wherein RB is a nitrogen protecting group.
As generally defined herein, G2 is independently selected from hydrogen, halogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORA, —CH2ORA, —OC(O)RC, —SRA, —N(RB)2, —N(RA)C(O)RC, —C(O)N(RB)2, —CN, —NO2, —C(O)RC, —C(O)ORA, —S(O)RC, —SO2RA, —SO2N(RB)2, ═O, ═NOH, ═N—ORA, ═N—NH2, ═N—NHRA, ═N—N(RB)2, and —NHSO2RA. In certain embodiments, G2 is H. In certain embodiments, G2 is halogen. In certain embodiments, G2 is optionally substituted C1-C6 alkyl. In certain embodiments, G2 is methyl, ethyl, or n-propyl. In certain embodiments, G7 is —ORA, wherein RA is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, or an oxygen protecting group when attached to an oxygen, or a sulfur protecting group when attaching to sulfur. In certain embodiments, G2 is —OH. In certain embodiments, G2 is —ORA, wherein RA is optionally substituted C1-C6 alkyl. In certain embodiments, G2 is —OCH3 or —OC2H5. In certain embodiments, G2 is —ORA, wherein RA is an oxygen protecting group. In certain embodiments, G2 is —N(RB)2, wherein each instance of RB is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl, or a nitrogen protecting group, or two RB taken together with the intervening nitrogen form a heterocycle. In certain embodiments, G2 is NH2. In certain embodiments, G2 is NHRB, wherein RB is optionally substituted C1-C6 alkyl. In certain embodiments, G2 is NHCH3 or NHC2H5. In certain embodiments, G2 is NHRB, wherein RB is a nitrogen protecting group.
As generally defined herein, G3 is independently selected from hydrogen, halogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORA, —CH2ORA, —OC(O)RC, —SRA, —N(RB)2, —N(RA)C(O)RC, —C(O)N(RB)2, —CN, —NO2, —C(O)RC, —C(O)ORA, —S(O)RC, —SO2RA, —SO2N(RB)2, ═O, ═NOH, ═N—ORA, ═N—NH2, ═N—NHRA, ═N—N(RB)2, and —NHSO2RA. In certain embodiments, G3 is H. In certain embodiments, G3 is halogen. In certain embodiments, G3 is optionally substituted C1-C6 alkyl. In certain embodiments, G3 is methyl, ethyl, or n-propyl. In certain embodiments, G7 is —ORA, wherein RA is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, or an oxygen protecting group when attached to an oxygen, or a sulfur protecting group when attaching to sulfur. In certain embodiments, G3 is —OH. In certain embodiments, G3 is —ORA, wherein RA is optionally substituted C1-C6 alkyl. In certain embodiments, G3 is —OCH3 or —OC2H5. In certain embodiments, G3 is —ORA, wherein RA is an oxygen protecting group. In certain embodiments, G3 is —N(RB)2, wherein each instance of RB is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl, or a nitrogen protecting group, or two RB taken together with the intervening nitrogen form a heterocycle. In certain embodiments, G3 is NH2. In certain embodiments, G3 is NHRB, wherein RB is optionally substituted C1-C6 alkyl. In certain embodiments, G3 is NHCH3 or NHC2H5. In certain embodiments, G3 is NHRB, wherein RB is a nitrogen protecting group.
As generally defined herein, G5 is independently selected from hydrogen, halogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORA, —CH2ORA, —OC(O)RC, —SRA, —N(RB)2, —N(RA)C(O)RC, —C(O)N(RB)2, —CN, —NO2, —C(O)RC, —C(O)ORA, —S(O)RC, —SO2RA, —SO2N(RB)2, ═O, ═NOH, ═N—ORA, ═N—NH2, ═N—NHRA, ═N—N(RB)2, and —NHSO2RA. In certain embodiments, G5 is H. In certain embodiments, G5 is halogen. In certain embodiments, G5 is optionally substituted C1-C6 alkyl. In certain embodiments, G5 is methyl, ethyl, or n-propyl. In certain embodiments, G5 is —ORA, wherein RA is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, or an oxygen protecting group when attached to an oxygen, or a sulfur protecting group when attaching to sulfur. In certain embodiments, G5 is —OH. In certain embodiments, G5 is —ORA, wherein RA is optionally substituted C1-C6 alkyl. In certain embodiments, G5 is —OCH3 or —OC2H5. In certain embodiments, G5 is —ORA, wherein RA is an oxygen protecting group. In certain embodiments, G5 is —N(RB)2, wherein each instance of RB is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl, or a nitrogen protecting group, or two RB taken together with the intervening nitrogen form a heterocycle. In certain embodiments, G5 is NH2. In certain embodiments, G5 is NHRB, wherein RB is optionally substituted C1-C6 alkyl. In certain embodiments, G5 is NHCH3 or NHC2H5. In certain embodiments, G5 is NHRB, wherein RB is a nitrogen protecting group.
As generally defined herein, G6 is independently selected from hydrogen, halogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORA, —CH2ORA, —OC(O)RC, —SRA, —N(RB)2, —N(RA)C(O)RC, —C(O)N(RB)2, —CN, —NO2, —C(O)RC, —C(O)ORA, —S(O)RC, —SO2RA, —SO2N(RB)2, ═O, ═NOH, ═N—ORA, ═N—NH2, ═N—NHRA, ═N—N(RB)2, and —NHSO2RA. In certain embodiments, G6 is H. In certain embodiments, G6 is halogen. In certain embodiments, G6 is optionally substituted C1-C6 alkyl. In certain embodiments, G6 is methyl, ethyl, or n-propyl. In certain embodiments, G6 is —ORA, wherein RA is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, or an oxygen protecting group when attached to an oxygen, or a sulfur protecting group when attaching to sulfur. In certain embodiments, G6 is —OH. In certain embodiments, G6 is —ORA, wherein RA is optionally substituted C1-C6 alkyl. In certain embodiments, G6 is —OCH3 or —OC2H5. In certain embodiments, G6 is —ORA, wherein RA is an oxygen protecting group. In certain embodiments, G6 is —N(RB)2, wherein each instance of RB is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl, or a nitrogen protecting group, or two RB taken together with the intervening nitrogen form a heterocycle. In certain embodiments, G6 is NH2. In certain embodiments, G6 is NHRB, wherein RB is optionally substituted C1-C6 alkyl. In certain embodiments, G6 is NHCH3 or NHC2H5. In certain embodiments, G6 is NHRB, wherein RB is a nitrogen protecting group.
As generally defined herein, G7 is independently selected from hydrogen, halogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORA, —CH2ORA, —OC(O)RC, —SRA, —N(RB)2, —N(RA)C(O)RC, —C(O)N(RB)2, —CN, —NO2, —C(O)RC, —C(O)ORA, —S(O)RC, —SO2RA, —SO2N(RB)2, ═O, ═NOH, ═N—ORA, ═N—NH2, ═N—NHRA, ═N—N(RB)2, and —NHSO2RA. In certain embodiments, G7 is H. In certain embodiments, G7 is halogen. In certain embodiments, G7 is optionally substituted C1-C6 alkyl. In certain embodiments, G7 is methyl, ethyl, or n-propyl. In certain embodiments, G7 is —ORA, wherein RA is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, or an oxygen protecting group when attached to an oxygen, or a sulfur protecting group when attaching to sulfur. In certain embodiments, G7 is —OH. In certain embodiments, G7 is —ORA, wherein RA is optionally substituted C1-C6 alkyl. In certain embodiments, G7 is —OCH3 or —OC2H5. In certain embodiments, G7 is —ORA, wherein RA is an oxygen protecting group. In certain embodiments, G7 is —N(RB)2, wherein each instance of RB is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl, or a nitrogen protecting group, or two RB taken together with the intervening nitrogen form a heterocycle. In certain embodiments, G7 is NH2. In certain embodiments, G7 is NHRB, wherein RB is optionally substituted C1-C6 alkyl. In certain embodiments, G7 is NHCH3 or NHC2H5. In certain embodiments, G7 is NHRB, wherein RB is a nitrogen protecting group.
As generally defined herein, G8 is independently selected from hydrogen, halogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORA, —CH2ORA, —OC(O)RC, —SRA, —N(RB)2, —N(RA)C(O)RC, —C(O)N(RB)2, —CN, —NO2, —C(O)RC, —C(O)ORA, —S(O)RC, —SO2RA, —SO2N(RB)2, ═O, ═NOH, ═N—ORA, ═N—NH2, ═N—NHRA, ═N—N(RB)2, and —NHSO2RA. In certain embodiments, G8 is H. In certain embodiments, G8 is halogen. In certain embodiments, G8 is optionally substituted C1-C6 alkyl. In certain embodiments, G8 is methyl, ethyl, or n-propyl. In certain embodiments, G8 is —ORA, wherein RA is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, or an oxygen protecting group when attached to an oxygen, or a sulfur protecting group when attaching to sulfur. In certain embodiments, G8 is —OH. In certain embodiments, G8 is —ORA, wherein RA is optionally substituted C1-C6 alkyl. In certain embodiments, G8 is —OCH3 or —OC2H5. In certain embodiments, G8 is —ORA, wherein RA is an oxygen protecting group. In certain embodiments, G8 is —N(RB)2, wherein each instance of RB is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl, or a nitrogen protecting group, or two RB taken together with the intervening nitrogen form a heterocycle. In certain embodiments, G8 is NH2. In certain embodiments, G8 is NHRB, wherein RB is optionally substituted C1-C6 alkyl. In certain embodiments, G8 is NHCH3 or NHC2H5. In certain embodiments, G8 is NHRB, wherein RB is a nitrogen protecting group.
In certain embodiments, two of G8 groups adjacent to each other are taken together with their intervening atoms to form an optionally substituted carbocycle or heterocycle. In certain embodiments, two of G8 groups adjacent to each other are taken together with their intervening atoms to form an optionally substituted carbocycle. In certain embodiments, two of G8 groups adjacent to each other are taken together with their intervening atoms to form an optionally substituted 5-membered carbocycle. In certain embodiments, two of G8 groups adjacent to each other are taken together with their intervening atoms to form an optionally substituted 6-membered carbocycle. In certain embodiments, two of G8 groups adjacent to each other are taken together with their intervening atoms to form an optionally substituted phenyl. In certain embodiments, two of G8 groups adjacent to each other are taken together with their intervening atoms to form an unsubstituted phenyl. In certain embodiments, two of G8 groups adjacent to each other are taken together with their intervening atoms to form an optionally substituted heterocycle. In certain embodiments, two of G8 groups adjacent to each other are taken together with their intervening atoms to form an optionally substituted 5-membered heterocycle with one heteroatom of S, N, or O. In certain embodiments, two of G8 groups adjacent to each other are taken together with their intervening atoms to form an optionally substituted 5-membered heterocycle with two heteroatom each independently selected from the group of S, N, and O. In certain embodiments, two of G8 groups adjacent to each other are taken together with their intervening atoms to form an optionally substituted 6-membered carbocycle with one heteroatom of S, N, or O. In certain embodiments, two of G8 groups adjacent to each other are taken together with their intervening atoms to form an optionally substituted 6-membered carbocycle with two heteroatoms each independently selected from the group consisting of S, N, and O.
The efficacy of one or more embodiments of the disclosure are set forth in the description below. Other features or advantages of the present disclosure will be apparent from the following drawings and detailed description of several embodiments, and also from the appending claims.
Cyclooctyne-based Fluorogenic Probes
A cyclooctyne-based fluorogenic probe is the Formula (IV):




or a pharmaceutically acceptable salt, solvate, or hydrate thereof, and wherein:

each instance of G9, G10, G11, G12, G13, G14, G15, G16, G17 and G18 is independently hydrogen, optionally substituted C1-6 alkyl, optionally substituted C1-6 alkenyl, optionally halogen, optionally nitroso, optionally substituted C1-6 alkynyl, optionally substituted aryl, optionally substituted acyl, —ORA, —CH2ORA, —OC(O)RA, —SRA, —N(RB)2, —N(RA)C(O)RA, —C(O)N(RB)2, —CN, —NO2, —C(O)RA, —C(O)ORA, —S(O)RA, —SO2RA, —SO3RA, —SO2N(RB)2, and —NHSO2RB;
each instance of R is independently selected from hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted acyl, —ORA, —CH2ORA, —OC(O)RA, —SRA, —N(RB)2, —N(RA)C(O)RA, —C(O)N(RB)2, —CN, —NO2, —C(O)RA, —C(O)ORA, S(O)RA, —SO2RA, —SO3RA, —SO2N(RB)2, and —NHSO2RB;
each RA is independently selected from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl; and
each RB is independently selected from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl, or two RB taken together with the intervening nitrogen form a heterocycle;
each instance of G9 and G10 is hydrogen, fluoro, chloro, bromo, iodo, nitroso, alkyl, alkoxy, aryloxy, or alkynyl, and
wherein alkyl and alkoxy groups are unbranched, saturated, and have 1-4 carbon atoms; aryl groups and aryl groups of aryloxy can be either carbocyclic aryl or heterocyclic aryl; carbocyclic aryl groups have a total of 6-20 carbon atoms, including carbon atoms of substituents; heterocyclic aryl groups have a total of 5-20 carbon atoms, including carbon atoms of substituents; carboalkoxy groups are alkyl esters of a carboxylic acid wherein alkyl groups are as defined above; each alkyl, aryl, alkoxy, aryloxy, benzo, and carboalkoxy, independently, may be unsubstituted or substituted with one or more substituent; alkyl substituents are halo, hydroxyl, amino, or aryl; aryl substituents are halo, hydroxyl, amino, alkyl, aryl, nitro, or carboxyl; and halo substituents are fluoro or chloro;
m is 0 or 1;
As generally defined herein, G9 is independently selected from hydrogen, halogen, nitroso, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORA, —CH2ORA, —OC(O)RC, —SRA, —N(RB)2, —N(RA)C(O)RC, —C(O)N(RB)2, —CN, —NO2, —C(O)RC, —C(O)ORA, —S(O)RC, —SO2RA, —SO2N(RB)2, ═O, ═NOH, ═N—ORA, ═N—NH2, ═N—NHRA, ═N—N(RB)2, and —NHSO2RA. In certain embodiments, G9 is H. In certain embodiments, G9 is halogen. In certain embodiments, G9 is optionally substituted C1-C6 alkyl. In certain embodiments, G1 is methyl, ethyl, or n-propyl. In certain embodiments, G9 is —ORA, wherein RA is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or an oxygen protecting group when attached to an oxygen, or a sulfur protecting group when attaching to sulfur. In certain embodiments, G9 is fluoro.
As generally defined herein, G10 is independently selected from hydrogen, halogen, nitroso, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —ORA, —CH2ORA, —OC(O)RA, —SRA, —N(RB)2, —N(RA)C(O)RA, —C(O)N(RB)2, —CN, —NO2, —C(O)RA, —C(O)ORA, —S(O)RA, —SO2RA, —SO2N(RB)2, ═O, ═NOH, ═N—ORA, ═N—NH2, ═N—NHRA, ═N—N(RB)2, and —NHSO2RA, wherein RA is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, or an oxygen protecting group when attached to an oxygen, or a sulfur protecting group when attaching to sulfur, wherein each instance of RB is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl, or a nitrogen protecting group, or two RB taken together with the intervening nitrogen form a heterocycle. In certain embodiments, G10 is fluoro.
In certain embodiments, the present disclosure is directed to a compound of 101:




A new fluorescence-forming probe 101, designed by fusing cyclooctyne with a coumarin fluorophore is described herein for the real-time imaging of azido-containing glycoconjugates in living cells under no-wash and no-fixation as well as catalyst-free conditions. This probe is cell-permeable with low cytotoxicity and generating fluorescence after triazole formation, thus enabling intracellular imaging of glycoprotein localization and trafficking with good fluorescent signal. Moreover, combination of 101 and azido-BODIPY probes described herein allows detection of two different metabolically incorporated azido- and alkyne-containing glycoconjugates, respectively, upon triazole formations in live cells in a dual-labeling experiment.
The low fluorescence 7-alkynylcoumarin 102 has been shown to undergo a CuAAC reaction to give triazole 103 with enhanced fluorescence due to the electron-donating property of the triazole ring.6c Thus, incorporation of a cyclooctyne to the coumarin moiety 101 may decrease fluorescence, but the SPAAC reaction of 101 with azides can give highly fluorescent triazole products for sensitive detection. In this disclosure, we report a SPAAC-based fluorescence-forming probe 101, (FIG. 10), namely coumOCT, that can be used for time-course imaging of azido-tagged glycans in living cells under no-wash and no-fixation conditions.




FIG. 10. Design of the SPAAC-based fluorescence-forming probe 101, showing high fluorescence upon triazole formation for imaging of azido-containing glycoconjugates in living cells. For comparison, 7-alkynylcoumarin 102 is a weakly fluorescent probe that undergoes a CuAAC reaction with azide to form a highly fluorescent triazole 103.
Activity-based Enzyme Probes
This disclosure describes a series of activity-based sialidase probes, DFSA (501), and CoumFSA (601) prepared from 3-fluorosialyl fluoride as the mechanism-based inhibitor and by incorporating an alkyne group for reporter ligation. DFSA (501) is an active-site inactivator of all tested sialidases. In this report, we have described the chemical synthesis of activity-based sialidase probes, CoumFSA (601), and DFSA (501).


















In some embodiments, a series of activity-based enzyme probes can be synthesized by varying the leaving group L added to compound 206. As shown in Scheme 700, any selection of a salt (the sodium salt is as shown, but any cation counterion can be used) of a weak acid can be used as an acceptable leaving group. For example, L can be alkoxy, phenoxy, pentafluorophenoxy, 4-nitrophenoxy, coumarin, alkanoate, benzoate, triflate, mesylate, or tosylate.




FIG. 12 depicts a general structure of an activity-based enzyme probe (ABP). The ABP contains a reactive group, a spacer or binding group, and a reporter tag. The reactive group is designed based on the catalytic mechanism of the enzyme targets, and it can contain a electrophile that can react with a nucleophilic residue in the enzyme active site to form covalent adduct. A variety of reporter tags can be used for enzyme visualization and enrichment, including fluorophores and biotin, as well as “clickable” handles, such as azides and acetylenes.
This disclosure describes the design of an activity-based enzyme probe specifically for sialidases. 3-Fluorosialyl fluoride inactivates wild-type trans-sialidases in a time-dependent manner according to the kinetic model. Because of the strong electron-withdrawing group, the fluorine can destabilize the positively changed oxo-carbenium ion like transition state (FIG. 13), thereby slowing both the formation and hydrolysis of the covalent intermediate.




The inventors have recognized that the crystal structure of the sialidase enzyme supports a neuraminidase-covalent complex with 3-fluorosialyl fluoride which can be further modified to incorporate novel chemical functionality into the active site. As shown in FIG. 14, the Tyr406 in the neurminidase (sialidase) active site performs a nucleophilic attack on the C-2 on the 3-fluorosialyl fluoride substrate (Nat. Commun. 2013, 4, 1491). Accordingly, modification of the C-2 with leaving groups will yield a covalent bond between the Tyr406 and the 3-fluorosialyl fluoride substrate. Further modification of the 3-fluorosialyl fluoride substrate to include an alkyl functional group enables the covalently-bound substrate to be detected by azide-containing fluorogenic probes, such as those compounds described herein. FIG. 14. The crystal structure of the neurminidase (sialidase) active site with the 3-fluorosialyl fluoride substrate.
In some embodiments, the activity based probes (ABP) described herein can be used to detect and identify sialidase enzymes. As depicted in FIG. 26, the ABP can react with the active site of the sialidase forming a covalent bond. The cell can optionally be lysed. The ABP can be further reacted with an azide-containing reporter molecule (for example, the azide-containing fluorogenic probes described herein) to form a triazole complex. The triazole complex can then be detected (e.g. by mass-spectroscopy, fluorescence, luminescence, absorption spectroscopy). FIG. 15. Fluorogenic reactions for identification of sialidases.
EXAMPLES
Example 1
Synthesis of Azido-BODIPY Compounds
Materials
All the reagents were commercially available and used without further purification unless indicated otherwise. All solvents were anhydrous grade unless indicated otherwise. All non-aqueous reactions were carried out in oven-dried glassware under a slightly positive pressure of argon unless otherwise noted. Reactions were magnetically stirred and monitored by thin-layer chromatography on silica gel. Column chromatography was performed on silica gel of 40-63 μm particle size. Yields are reported for spectroscopically pure compounds.
Instruments
Melting points were recorded on an Electrothermal MEL-TEMP® 1101D melting point apparatus and were not corrected. NMR spectra were recorded on Bruker AVANCE 600 spectrometer (600 MHz). Chemical shifts are given in δ values relative to tetramethylsilane (TMS); coupling constants J are given in Hz. Internal standards were CDCl3 (δH=7.24) for 1H-NMR spectra, CDCl3 (δc=77.0) for 13C-NMR spectra. The splitting patterns are reported as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad) and dd (double of doublets). High resolution ESI mass spectra were recorded on a Bruker Daltonics spectrometer. Absorbance spectra were recorded on a Perkin Elmer Lambda 35 UV-Visible spectrophotometer. Fluorescence spectra were recorded on an AMINCO-Bowman Series 2 luminescence spectrometer. All photos were collected on a Leica TCS-SP5 confocal laser-scanning microscope.
A convenient route for the synthesis of azido-BODIPY compounds Az1, Az2, Az3, Az4, Az5, Az6, Az7 and Az8 is disclosed herein. The preparation made use of the key nitro intermediates 1, 2, 3, 4, 5, 6, 7 and 8 which were effectively converted into the corresponding azido-BODIPY compounds. The structure of azido-BODIPY compounds Az1, Az2, Az3, Az4, Az5, Az6, Az7 and Az8 is shown in FIG. 1. Reagents and steps in the synthetic route are as follows.
Step 1. The acid-catalyzed condensation of 2,4-dimethyl-3-ethylpyrrole with substituted nitrobenzaldehydes, followed by oxidation with DDQ in mild conditions, gave dipyrromethene intermediates, which were treated with BF3.OEt2 to yield the corresponding nitro-BODIPYs 1-8.




Step 2. According to the previously reported method, the amino-BODIPYs Am1-Am8 were obtained in reasonable yields by reduction of the nitro-BODIPYs with hydrazine in the presence of 10% Pd/C. On treatment with triflyl azide (TfN3) in mild conditions, the amino-BODIPYs were converted to the target azido-BODIPYs Az1-Az8. (Li, L.; Han, J.; Nguyen, B.; Burgess, K. J. Org. Chem. 2008, 73, 1963-1970.)




A convenient route for the synthesis of azido-BODIPY compounds Az9, Az10 and Az11 is disclosed herein. The preparation made use of the key nitro intermediates 9, 10 and 11 which were effectively converted into the corresponding azido-BODIPY compounds. The structure of azido-BODIPY compounds Az9, Az10 and Az11 is shown in FIG. 3. Reagents and steps in the synthetic route are as follows.
Step 1. The acid-catalyzed condensation of 2,4-dimethyl-3-substituted pyrroles with 2-methoxy-5-nitrobenzaldehyde, followed by oxidation with DDQ in mild conditions, gave dipyrromethene intermediates, which were treated with BF3.OEt2 to yield the corresponding nitro-BODIPYs 9-11.




Step 2. According to the previously reported method, the amino-BODIPYs Am9-Am11 were obtained in reasonable yields by reduction of the nitro-BODIPYs with hydrazine in the presence of 10% Pd/C. On treatment with triflyl azide (TfN3) in mild conditions, the amino-BODIPYs were converted to the target azido-BODIPYs Az9-Az11. (Li, L.; Han, J.; Nguyen, B.; Burgess, K. J. Org. Chem. 2008, 73, 1963-1970.)




Example 2
Synthesis of Triazolyl-BODIPY Compounds
A convenient route for the synthesis of triazolyl-BODIPY compounds T2, T9, T10 and T11 is disclosed herein. The structure of triazolyl-BODIPY compounds T2, T9, T10 and T11 is shown in FIG. 3. Reagents and step in the synthetic route is as follows.
The triazolyl-BODIPY compounds T2, T9, T10 and T11 were obtained in reasonable yields by 1,3-dipolar cycloaddition of the azido-BODIPY compounds Az2, Az9, Az10 and Az11 with 4-pentyn-1-ol in CuAAC conditions containing CuSO4, sodium ascorbate and a tris-triazole ligand prepared from tripropargylamine and ethyl azidoacetate. (Zhou, Z.; Fahrni, C. J. Am. Chem. Soc. 2004, 126, 8862-8863).




General Synthetic Procedures and Product Characterization of Nitro-BODIPYs

Substituted nitrobenzaldehyde (3 mmol) and 3-substituted 2,4-dimethylpyrrole (6 mmol) were dissolved in anhydrous CH2Cl2 (400 mL) under an Ar atmosphere. TFA (1 drop) was added, and the resulting solution was stirred at room temperature overnight. After complete consumption of the reactants in 12-18 h as shown by TLC analysis, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 3 mmol) was added in one portion. The reaction mixture was stirred at room temperature for 1 h, and then washed with brine (400 mL). The organic fraction was dried over MgSO4, filtered, and concentrated. The crude compound was purified on a short alumina oxide column (CH2Cl2) to afford brown solids of dipyrromethene. The crude dipyrromethene product and N,N-diisopropylethylamine (DIEA) (40 mmol) were dissolved in anhydrous toluene (150 mL) and stirred at room temperature for 10 min. BF3.OEt2 (55 mmol) was added slowly, and stirring was continued for 1 h. The reaction mixture was washed with water (3×50 mL) and brine (50 mL), dried over MgSO4, filtered, and concentrated. The crude product was purified by column chromatography on silica gel by elution with CH2Cl2/hexane or EtOAc/hexane to afford the corresponding nitro-BODIPY product 1-11.
2,6-Diethyl-4,4-difluoro-8-(3-nitrophenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (1)




Compound 1 was prepared from 2,4-dimethyl-3-ethylpyrrole and 3-nitrobenzaldehyde in 15% yield for three steps. C23H26BF2N3O2, dark red solids, mp 246-248° C.; TLC (EtOAc/hexane, 1:4) Rf=0.38; 1H NMR (600 MHz, CDCl3) δ 8.36-8.34 (1 H, m), 8.20 (1 H, t, J=1.7 Hz), 7.70-7.64 (2 H, m), 2.52 (6 H, s), 2.28 (4 H, q, J=7.6 Hz), 1.23 (6 H, s), 0.96 (6 H, t, J=7.6 Hz); 13C NMR (150 MHz, CDCl3) δ 155.0, 148.6, 137.6, 136.3, 134.8, 133.5, 130.4, 130.2, 123.89, 123.85, 17.0, 14.5, 12.6, 12.1; HRMS calcd for C23H27BF2N3O2: 426.2164, found: m/z 426.2167 [M+H]+.
2,6-Diethyl-4,4-difluoro-8-(2-methoxy-5-nitrophenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (2)




Compound 2 was prepared from 2,4-dimethyl-3-ethylpyrrole and 2-methoxy-5-nitrobenzaldehyde in 51% yield for three steps. C24H28BF2N3O3, dark red solids, mp 210-212° C.; TLC (EtOAc/hexane, 1:4) Rf=0.33; 1H NMR (600 MHz, CDCl3) δ 8.42 (1 H, dd, J=9.1, 2.5 Hz), 8.15 (1 H, d, J=2.5 Hz), 7.11 (1 H, d, J=9.1 Hz), 3.93 (3 H, s), 2.56 (6 H, s), 2.32 (4 , q, J=7.6 Hz), 1.36 (6 H, s), 1.02 (6 H, t, J=7.6 Hz); 13C NMR (150 MHz, CDCl3) δ 162.0, 154.4, 141.8, 137.1, 133.5, 133.0, 130.4, 126.8, 126.1, 125.6, 110.9, 56.6, 17.0, 14.6, 12.6, 11.5; HRMS calcd for C24H29BF2N3O3: 456.2270, found: m/z 456.2267 [M+H]+.
2,6-Diethyl-4,4-difluoro-8-(3,4-dimethoxy-5-nitrophenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (3)




Compound 3 was prepared from 2,4-dimethyl-3-ethylpyrrole and 3,4-dimethoxy-5-nitrobenzaldehyde in 45% yield for three steps. C25H30BF2N3O4, dark red solids, mp 190-192° C.; TLC (EtOAc/hexane, 1:3) Rf=0.5; 1H NMR (600 MHz, CDCl3) δ 7.28 (1 H, s), 7.02 (1 H, s), 4.04 (3 H, s), 3.89 (3 H, s), 2.51 (6 H, s), 2.30 (4 H, q, J=7.5 Hz), 1.38 (6 H, s), 0.98 (6 H, t, J=7.5 Hz); 13C NMR (150 MHz, CDCl3) δ 154.9, 145.3, 143.2, 137.7, 136.4, 133.4, 131.4, 130.4, 116.16, 116.12, 62.5, 56.8, 17.0, 14.5, 12.6, 12.0; HRMS calcd for C25H31BF2N3O4: 486.2376, found: m/z 486.2377 [M+H]+.
2,6-Diethyl-4,4-difluoro-8-(2,3-dimethoxy-5-nitrophenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (4)




Compound 4 was prepared from 2,4-dimethyl-3-ethylpyrrole and 2,3-dimethoxy-5-nitrobenzaldehyde in 41% yield for three steps. C25H30BF2N3O4, dark red solids, mp 185-187° C.; TLC (EtOAc/hexane, 1:4) Rf=0.35; 1H NMR (600 MHz, CDCl3) δ 7.88 (1 H, d, J=2.3 Hz), 7.76 (1 H, d, J=2.3 Hz), 4.00 (3 H, s), 3.83 (3 H, s), 2.51 (6 H, s), 2.29 (4 H, q, J=7.6 Hz), 1.38 (6 H, s), 0.97 (6 H, t, J=7.6 Hz); 13C NMR (150 MHz, CDCl3) δ 154.5, 152.8, 151.8, 143.8, 137.1, 133.4, 133.2, 130.3, 129.5, 117.8, 108.1, 60.8, 56.4, 17.0, 14.6, 12.6, 11.6; HRMS calcd for C25H31BF2N3O4: 486.2376, found: m/z 486.2378 [M+H]+.
2,6-Diethyl-4,4-difluoro-8-(2,5-dimethoxy-3-nitrophenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (5)




Compound 5 was prepared from 2,4-dimethyl-3-ethylpyrrole and 2,5-dimethoxy-3-nitrobenzaldehyde in 36% yield for three steps. C25H30BF2N3O4, dark red solids, mp 181-183° C.; TLC (CH2Cl2/hexane, 3:7) Rf=0.5; 1H NMR (600 MHz, CDCl3) δ 7.41 (1 H, d, J=3.1 Hz), 6.98 (1 H, d, J=3.1 Hz), 3.83 (3 H, s), 3.71 (3 H, s), 2.52 (6 H, s), 2.30 (4 H, q, J=7.6 Hz), 1.44 (6 H, s), 0.98 (6 H, t, J=7.6 Hz); 13C NMR (150 MHz, CDCl3) δ 155.6, 154.8, 145.0, 144.3, 137.3, 133.4, 133.2, 132.9, 130.3, 120.7, 110.4, 62.2, 56.2, 17.1, 14.6, 12.6, 11.7; HRMS calcd for C25H31BF2N3O4: 486.2376, found: m/z 486.2378 [M+H]+.
2,6-Diethyl-4,4-difluoro-8-(2-morpholino-5-nitrophenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (6)




Compound 6 was prepared from 2,4-dimethyl-3-ethylpyrrole and 2-morpholino-5-nitrobenzaldehyde in 38% yield for three steps. C27H33BF2N4O3, dark red solids, mp 179-181° C.; TLC (EtOAc/hexane, 3:7) Rf=0.25; 1H NMR (600 MHz, CDCl3) δ 8.24 (1 H, dd, J=9.1, 2.6 Hz), 8.02 (1 H, d, J=2.6 Hz), 6.96 (1 H, d, J=9.1 Hz), 3.84 (4 H, t, J=4.3 Hz), 3.24 (4 H, t, J=4.3 Hz), 2.52 (6 H, s), 2.31 (4 H, q, J=7.6 Hz), 1.42 (6 H, s), 0.99 (6 H, t, J=7.6 Hz); 13C NMR (150 MHz, CDCl3) δ 155.1, 154.9, 141.1, 137.1, 136.0, 133.5, 129.8, 127.4, 126.2, 126.1, 117.4, 66.6, 50.4, 17.1, 14.6, 12.6, 12.1; HRMS calcd for C27H34BF2N4O3: 511.2692, found: m/z 511.2695 [M+H]+.
2,6-Diethyl-4,4-difluoro-8-[2-(4-methylpiperazino)-5-nitrophenyl]-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (7)




Compound 7 was prepared from 2,4-dimethyl-3-ethylpyrrole and 2-(4-methylpiperazino)-5-nitrobenzaldehyde in 48% yield for three steps. C28H36BF2N5O2, dark red solids, mp 175-177° C.; TLC (EtOAc/hexane, 3:7) Rf=0.25; 1H NMR (600 MHz, CDCl3) δ 8.21 (1 H, dd, J=9.2, 2.8 Hz), 8.00 (1 H, d, J=2.8 Hz), 6.93 (1 H, d, J=9.2 Hz), 3.29 (4 H, t, J=4.7 Hz), 2.52 (6 H, s), 2.32 (4 H, q, J=7.6 Hz), 2.19 (4 H, t, J=4.7 Hz), 2.15 (3 H, s), 1.42 (6 H, s), 0.99 (6 H, t, J=7.6 Hz); 13C NMR (150 MHz, CDCl3) δ 155.1, 154.6, 140.6, 137.2, 136.4, 133.4, 129.9, 127.4, 126.1, 125.5, 117.5, 54.7, 49.8, 45.9, 17.1, 14.7, 12.6, 12.1; HRMS calcd for C28H37BF2N5O2: 524.3008, found: m/z 524.3010 [M+H]+.
2,6-Diethyl-4,4-difluoro-8-(2-piperidino-5-nitrophenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (8)




Compound 8 was prepared from 2,4-dimethyl-3-ethylpyrrole and 2-piperidino-5-nitrobenzaldehyde in 40% yield for three steps. C28H35BF2N4O2, dark red solids, mp 201-203° C.; TLC (EtOAc/hexane, 1:9) Rf=0.38; 1H NMR (600 MHz, CDCl3) δ 8.19 (1 H, dd, J=9.2, 2.8 Hz), 7.97 (1 H, d, J=2.8 Hz), 6.91 (1 H, d, J=9.2 Hz), 3.23 (4 H, t, J=5.3 Hz), 2.52 (6 H, s), 2.30 (4 H, q, J=7.6 Hz), 1.48-1.44 (2 H, m), 1.43 (6 H, s), 1.37-1.34 (4 H, m), 0.98 (6 H, t, J=7.6 Hz); 13C NMR (150 MHz, CDCl3) δ 156.0, 154.3, 139.9, 137.2, 137.0, 133.2, 130.0, 127.5, 126.1, 125.1, 117.3, 51.3, 25.8, 23.9, 17.1, 14.6, 12.6, 12.1; HRMS calcd for C28H36BF2N4O2: 509.2899, found: m/z 509.2901 [M+H]+.
4,4-Difluoro-8-(2-methoxy-5-nitrophenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (9)




Compound 9 was prepared from 2,4-dimethylpyrrole and 2-methoxy-5-nitrobenzaldehyde in 39% yield for three steps. C20H20BF2N3O3, dark red solids, mp 178-180° C.; TLC (EtOAc/hexane, 1:4) Rf=0.31; 1H NMR (600 MHz, CDCl3) δ 8.42 (1 H, dd, J=9.1, 2.7 Hz), 8.17 (1 H, d, J=2.7 Hz), 7.12 (1 H, d, J=9.1 Hz), 6.02 (2 H, s), 3.93 (3 H, s), 2.59 (6 H, s), 1.46 (6 H, s); 13C NMR (150 MHz, CDCl3) δ 161.7, 156.1, 141.9, 135.0, 131.1, 126.9, 126.0, 124.8, 121.5, 118.9, 111.0, 56.7, 14.6, 14.2; HRMS calcd for C20H21BF2N3O3: 400.1644, found: m/z 400.1640 [M+H]+.
2,6-Diethoxycarbonyl-4,4-difluoro-8-(2-methoxy-5-nitrophenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (10)




Compound 10 was prepared from 2,4-dimethy-4-ethoxycarbonylpyrrole and 2-methoxy-5-nitrobenzaldehyde in 60% yield for three steps. C26H28BF2N3O7, orange-red solids, mp 181-183° C.; TLC (CH2Cl2/hexane, 7:3) Rf=0.18; 1H NMR (600 MHz, CDCl3) δ 8.43 (1 H, dd, J=9.2, 2.8 Hz), 8.09 (1 H, d, J=2.8 Hz), 7.11 (1 H, d, J=9.2 Hz), 4.26 (4 H, q, J=7.1 Hz), 3.89 (3 H, s), 2.82 (6 H, s), 1.71 (6 H, s), 1.31 (6 H, t, J=7.1 Hz); 13C NMR (150 MHz, CDCl3) δ 164.0, 161.4, 160.0, 146.4, 142.2, 139.2, 131.1, 127.6, 125.5, 124.3, 122.7, 111.3, 60.3, 56.8, 15.1, 14.2, 13.2; HRMS calcd for C26H29BF2N3O7: 544.2067, found: m/z 544.2061 [M+H]+.
2,6-Dicyano-4,4-difluoro-8-(2-methoxy-5-nitrophenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (11)




Compound 11 was prepared from 3-cyano-2,4-dimethylpyrroleS7 and 2-methoxy-5-nitrobenzaldehyde in 13% Yield for three steps. C22H18BF2N5O3, red solids, mp 278-280° C.; TLC (CH2Cl2/hexane, 7:3) Rf=0.18; 1H NMR (600 MHz, CDCl3) δ 8.49 (1 H, dd, J=9.1, 2.6 Hz), 8.10 (1 H, d, J=2.6 Hz), 7.19 (1 H, d, J=9.1 Hz), 3.93 (3 H, s), 2.72 (6 H, s), 1.63 (6 H, s); 13C NMR (150 MHz, CDCl3) δ 160.9, 160.2, 148.2, 142.2, 140.6, 131.2, 128.3, 125.1, 122.2, 113.2, 111.8, 106.8, 57.1, 13.9, 13.7; HRMS calcd for C22H19BF2N5O3: 450.1549, found: m/z 450.1546 [M+H]+.
General Synthetic Procedures and Product Characterization of Azido-BODIPYs (Az1-Az11)
A solution of nitro-BODIPY 1-11 (0.5 mmol) in a mixed solvent of EtOH (20 mL) and THF (20 mL) was purged with Ar for 10 min. Hydrazine monohydrate (0.3 mL) and 10% Pd/C (60 mg, 0.1 equiv) were added. The reaction mixture was heated to reflux for 30 min, and then Pd/C was removed by vacuum filtration. After evaporation of the solvent, the residue was purified by column chromatography on silica gel to afford red solids of amino-BODIPY. The crude product of amino-BODIPY Am1-Am11 was dissolved in CH2Cl2 (20 mL) in a 50 mL round-bottomed flask. Triethylamine (Et3N, 1.5 mmol) and a solution of CuSO4 (25 μmol in 0.1 mL of water) were added to the flask. A solution of freshly prepared triflyl azide (TfN3) (1.5 mmol in 3 mL of CH2Cl2) was then added, and the mixture was brought to homogeneity by adding methanol (0.5 mL). After stirring for 3 h at room temperature, the mixture was poured into saturated aqueous NaHCO3 (10 mL) and extracted with CH2Cl2 (3×10 mL). The combined organic extracts were dried over MgSO4, filtered, and concentrated. The residue was purified by column chromatography on silica gel by elution with CH2Cl2/hexane, EtOAc/hexane or MeOH/CH2Cl2 to afford the corresponding azido-BODIPY product Az1-Az11.
8-(5-Amino-2-methoxyphenyl)-2,6-diethoxycarbonyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (Am10)




Compound Am10 was prepared from compound 10 in 83% yield. C26H30BF2N3O5, dark red solids, mp 188-190° C.; TLC (EtOAc/hexane, 3:7) Rf=0.23; 1H NMR (600 MHz, CDCl3) δ 6.82-6.77 (2 H, m), 6.45 (1 H, d, J=2.5 Hz), 4.25 (4 H, q, J=7.1 Hz), 3.66 (3 H, s), 3.52 (2 H, br s), 2.79 (6 H, s), 1.82 (6 H, s), 1.30 (6 H, t, J=7.1 Hz); 13C NMR (150 MHz, CDCl3) δ 164.4, 158.9, 148.8, 147.3, 143.4, 141.4, 131.5, 123.7, 122.0, 117.4, 115.6, 112.8, 60.1, 56.0, 14.9, 14.3, 12.9; HRMS calcd for C26H31BF2N3O5: 514.2325, found: m/z 514.2327 [M+H]+.
8-(3-Azidophenyl)-2,6-diethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (Az1)




Compound Az1 was prepared from compound 1 in 47% yield for two steps. C23H26BF2N5, dark red solids, mp 151-153° C. (dec.); TLC (EtOAc/hexane, 1:9) Rf=0.38; 1H NMR (600 MHz, CDCl3) δ 8.01 (1 H, d, J=7.9 Hz), 7.85 (1 H, s), 7.75 (1 H, t, J=7.7 Hz), 7.65 (1 H, d, J=7.1 Hz), 2.52 (6 H, s), 2.27 (4 H, q, J=7.4 Hz), 1.22 (6 H, s), 0.95 (6 H, t, J=7.4 Hz); 13C NMR (150 MHz, CDCl3) δ 165.0, 154.7, 137.7, 137.4, 137.1, 135.3, 133.3, 130.5, 130.2, 121.2, 121.0, 17.0, 14.5, 12.6, 12.1; HRMS calcd for C23H27BF2N5: 422.2328, found: m/z 422.2330 [M+H]+.
8-(5-Azido-2-methoxyphenyl)-2,6-diethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (Az2)




Compound Az2 was prepared from compound 2 in 59% yield for two steps. C24H28BF2N5O, dark red solids, mp 163-165° C. (dec.); TLC (EtOAc/hexane, 1:4) Rf=0.52; 1H NMR (600 MHz, CDCl3) δ 7.08 (1 H, d, J=8.7 Hz), 6.95 (1 H, d, J=8.7 Hz), 6.84 (1 H, s), 3.74 (3 H, s), 2.50 (6 H, s), 2.28 (4 H, q, J=7.4 Hz), 1.36 (6 H, s), 0.97 (6 H, t, J=7.4 Hz); 13C NMR (150 MHz, CDCl3) δ 154.1, 153.7, 137.6, 135.5, 133.4, 132.6, 130.6, 126.2, 120.5, 112.4, 56.0, 17.1, 14.6, 12.5, 11.2; HRMS calcd for C24H29BF2N5O: 452.2433, found: m/z 452.2429 [M+H]+.
8-(3-Azido-4,5-dimethoxyphenyl)-2,6-diethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a, 4a-diaza-s-indacene (Az3)




Compound Az3 was prepared from compound 3 in 51% yield for two steps. C25H30BF2N5O2, orange-red solids, mp 156-158° C. (dec.); TLC (EtOAc/hexane, 1:4) Rf=0.41; 1H NMR (600 MHz, CDCl3) δ 7.09 (1 H, d, J=1.4 Hz), 5.85 (1 H, d, J=1.4 Hz), 4.64 (3 H, s), 3.95 (3 H, s), 2.49 (6 H, s), 2.26 (4 H, q, J=7.5 Hz), 1.26 (6 H, s), 0.94 (6 H, t, J=7.5 Hz); 13C NMR (150 MHz, CDCl3) δ 160.4, 155.2, 154.56, 154.53, 137.6, 137.3, 133.1, 130.4, 128.4, 118.2, 100.6, 65.6, 56.9, 17.0, 14.5, 12.5, 12.0; HRMS calcd for C25H31BF2N5O2: 482.2539, found: m/z 482.2542[M+H]+.
8-(5-Azido-2,3-dimethoxyphenyl)-2,6-diethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (Az4)




Compound Az4 was prepared from compound 4 in 54% yield for two steps. C25H30BF2N5O2, dark red solids, mp 158-160° C. (dec.); TLC (EtOAc/hexane, 1:4) Rf=0.48; 1H NMR (600 MHz, CDCl3) δ 8.18 (1 H, s), 6.82 (1 H, s), 3.94 (3 H, s), 3.88 (3 H, s), 2.54 (6 H, s), 2.29 (4 H, q, J=7.6 Hz), 1.43 (6 H, s), 0.97 (6 H, t, J=7.6 Hz); 13C NMR (150 MHz, CDCl3) δ 163.1, 154.4, 153.1, 152.9, 137.3, 134.1, 133.2, 130.6, 129.3, 125.1, 97.6, 60.8, 56.1, 17.1, 14.6, 12.6, 11.7; HRMS calcd for C25H31BF2N5O2: 482.2539, found: m/z 482.2541 [M+H]+.
8-(3-Azido-2,5-dimethoxyphenyl)-2,6-diethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (Az5)




Compound Az5 was prepared from compound 5 in 54% yield for two steps. C25H30BF2N5O2, dark red solids, mp 150-152° C. (dec.); TLC (CH2Cl2/hexane, 1:4) Rf=0.45; 1H NMR (600 MHz, CDCl3) δ 7.16 (1 H, d, J=3.2 Hz), 5.81 (1 H, d, J=3.2 Hz), 4.42 (3 H, s), 3.72 (3 H, s), 2.55 (6 H, s), 2.32 (4 H, q, J=7.6 Hz), 1.52 (6 H, s), 0.99 (6 H, t, J=7.6 Hz); 13C NMR (150 MHz, CDCl3) δ 160.0, 156.7, 154.7, 154.4, 137.3, 134.1, 133.1, 133.0, 130.6, 126.0, 90.7, 66.1, 55.8, 17.1, 14.6, 12.6, 11.8; HRMS calcd for C25H31BF2N5O2: 482.2539, found: m/z 482.2543 [M+H]+.
8-(5-Azido-2-morpholinophenyl)-2,6-diethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (Az6)




Compound Az6 was prepared from compound 6 in 62% yield for two steps. C27H33BF2N6O, dark red oil; TLC (EtOAc/hexane, 1:4) Rf=0.4; 1H NMR (600 MHz, CDCl3) δ 7.06 (1 H, dd, J=8.7, 2.7 Hz), 7.01 (1 H, d, J=8.7 Hz), 6.81 (1 H, d, J=2.7 Hz), 3.48 (4 H, t, J=4.4 Hz), 2.94 (4 H, t, J=4.4 Hz), 2.51 (6 H, s), 2.30 (4 H, q, J=7.6 Hz), 1.39 (6 H, s), 0.97 (6 H, t, J=7.6 Hz); 13C NMR (150 MHz, CDCl3) δ 153.8, 147.3, 137.4, 134.9, 132.9, 131.0, 130.2, 121.1, 120.6, 120.5, 67.1, 51.6, 17.1, 14.6, 12.6, 11.7; HRMS calcd for C27H34BF2N6O: 507.2855, found: m/z 507.2858 [M+H]+.
8-[5-Azido-2-(4-methylpiperazino)phenyl]-2,6-diethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (Az7)




Compound Az7 was prepared from compound 7 in 72% yield for two steps. C28H36BF2N7, dark red foam; TLC (MeOH/CH2Cl2, 1:19) Rf=0.33; 1H NMR (600 MHz, CDCl3) δ 7.06-7.01 (2 H, m), 6.80 (1 H, s), 2.99 (4 H, br s), 2.51 (6 H, s), 2.32-2.26 (8 H, m), 2.18 (3 H, s), 1.38 (6 H, s), 0.97 (6 H, t, J=7.4 Hz); 13C NMR (150 MHz, CDCl3) δ 153.8, 146.8, 137.4, 135.3, 133.0, 131.2, 130.2, 121.4, 121.0, 120.5, 54.7, 50.1, 45.0, 17.1, 14.7, 12.6, 11.7; HRMS calcd for C28H37BF2N7: 520.3172, found: m/z 520.3177 [M+H]+.
8-(5-Azido-2-piperidinophenyl)-2,6-diethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (Az8)




Compound Az8 was prepared from compound 8 in 69% yield for two steps. C28H35BF2N6, dark red oil; TLC (EtOAc/hexane, 1:4) Rf=0.67; 1H NMR (600 MHz, CDCl3) δ 7.07 (1 H, dd, J=8.7, 2.6 Hz), 7.03 (1 H, d, J=8.7 Hz), 6.82 (1 H, d, J=2.6 Hz), 2.94 (4 H, t, J=5.2 Hz), 2.56 (6 H, s), 2.34 (4 H, q, J=7.6 Hz), 1.44 (6 H, s), 1.43-1.38 (6 H, m), 1.02 (6 H, t, J=7.6 Hz); 13C NMR (150 MHz, CDCl3) δ 153.3, 148.9, 138.2, 137.5, 133.8, 132.6, 130.7, 130.4, 121.0, 120.8, 120.3, 52.6, 26.2, 24.1, 17.1, 14.6, 12.5, 11.7; HRMS calcd for C28H36BF2N6: 505.3063, found: m/z 505.3066 [M+H]+.
8-(5-Azido-2-methoxyphenyl)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (Az9)




Compound Az9 was prepared from compound 9 in 71% yield for two steps. C20H20BF2N5O, dark red solids, mp 152-154° C. (dec.); TLC (EtOAc/hexane, 1:4) Rf=0.51; 1H NMR (600 MHz, CDCl3) δ 7.08 (1 H, d, J=8.7 Hz), 6.96 (1 H, d, J=8.7 Hz), 6.84 (1 H, s), 5.95 (2 H, s), 3.74 (3 H, s), 2.53 (6 H, s), 1.46 (6 H, s); 13C NMR (150 MHz, CDCl3) δ 155.4, 153.8, 142.3, 137.1, 133.5, 131.2, 125.3, 121.1, 120.7, 120.3, 112.5, 56.0, 14.6, 13.9; HRMS calcd for C20H21BF2N5O: 396.1807, found: m/z 396.1805 [M+H]+.
8-(5-Azido-2-methoxyphenyl)-2,6-diethoxycarbonyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (Az10)




Compound Az10 was prepared from compound Am10 in 58% yield. C26H28BF2N5O5, red solids, mp 86-88° C.; TLC (EtOAc/hexane, 3:7) Rf=0.41; 1H NMR (600 MHz, CDCl3) δ 7.14 (1 H, dd, J=8.8, 2.8 Hz), 6.99 (1 H, d, J=8.8 Hz), 6.79 (1 H, d, J=2.8 Hz), 4.26 (4 H, q, J=7.1 Hz), 3.75 (3 H, s), 2.80 (6 H, s), 1.76 (6 H, s), 1.31 (6 H, t, J=7.1 Hz); 13C NMR (150 MHz, CDCl3) δ 164.3, 159.4, 153.5, 146.9, 141.4, 134.2, 131.3, 124.7, 122.3, 121.4, 119.8, 112.7, 60.2, 56.1, 15.0, 14.3, 13.0; HRMS calcd for C26H29BF2N5O5: 540.2230, found: m/z 540.2227 [M+H]+.
8-(5-Azido-2-methoxyphenyl)-2,6-dicyano-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (Az11)




Compound Az11 was prepared from compound 11 in 61% yield for two steps. C22H18BF2N7O, red solids, mp 141-143° C. (dec.); TLC (CH2Cl2/hexane, 7:3)Rf=0.31; 1H NMR (600 MHz, CDCl3) δ 7.21 (1 H, dd, J=8.9, 2.3 Hz), 7.06 (1 H, d, J=8.9 Hz), 6.77 (1 H, d, J=2.3 Hz), 3.77 (3 H, s), 2.70 (6 H, s), 1.67 (6 H, s); 13C NMR (150 MHz, CDCl3) δ 158.6, 151.9, 147.7, 141.8, 133.6, 130.4, 121.6, 121.2, 118.2, 112.5, 112.1, 105.4, 55.3, 12.9, 12.4; HRMS calcd for C22H19BF2N7O: 446.1712, found: m/z 446.1714 [M+H]+.
Procedure for Copper(I)-Catalyzed Azide-Alkyne Cycloaddition Reaction of Azido-BODIPY with 4-Pentyn-1-ol
Azido-BODIPY (Az2 or Az9-Az11, 0.1 mmol) and 4-pentyn-1-ol (0.1 mmol) were dissolved in THF (5 mL). A freshly prepared 1 M solution of sodium ascorbate (0.2 mmol in 0.2 mL of water) was added, followed by addition of copper (II) pentahydrate (0.005 mmol in 0.1 mL of water). The mixture was brought to homogeneity by adding methanol (0.5 mL), and then stirred for 12 h at room temperature. TLC monitoring showed complete consumption of the reactants in 12 h. After evaporation of the solvents, the residue was purified by column chromatography on silica gel by elution with EtOAc/hexane to afford the corresponding triazole product T2 and T9-T11.
2,6-Diethyl-4,4-difluoro-8-{3-[4-(3-hydroxypropyl)-1H-1,2,3-triazol-1-yl]-2-methoxyphenyl}-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (T2)




Compound T2 was prepared from compound Az2 in 71% yield. C29H36BF2N5O2, red solids, mp 180-182° C.; TLC (EtOAc/hexane, 1:1) Rf=0.15; 1H NMR (600 MHz, CDCl3) δ 7.89 (1 H, dd, J=8.9, 2.7 Hz), 7.66 (1 H, s), 7.46 (1 H, d, J=2.7 Hz), 7.10 (1 H, J=8.9 Hz), 3.83 (3 H, s), 3.73 (2 H, t, J=6.1 Hz), 2.88 (2 H, t, J=7.3 Hz), 2.51 (6 H, s), 2.28 (4 H, q, J=7.5 Hz), 1.98-1.96 (2 H, m), 1.38 (6 H, s), 0.97 (6 H, t, J=7.5 Hz); 13C NMR (150 MHz, CDCl3) δ 156.8, 154.0, 148.3, 137.5, 134.7, 132.8, 131.3, 130.5, 125.9, 122.6, 121.9, 119.1, 112.0, 61.8, 56.1, 31.8, 22.0, 17.0, 14.6, 12.5, 11.4; HRMS calcd for C29H37BF2N5O2: 536.3008, found: m/z 536.3005 [M+H]+.
4,4-Difluoro-8-{3-[4-(3-hydroxypropyl)-1H-1,2,3-triazol-1-yl]-2-methoxyphenyl}-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (T9)




Compound T9 was prepared from compound Az9 in 79% yield. C25H28BF2N5O2, red solids, mp 165-167° C.; TLC (EtOAc/hexane, 7:3) Rf=0.23; 1H NMR (600 MHz, CDCl3) δ 7.88 (1 H, d, J=8.8 Hz), 7.66 (1 H, s), 7.49 (1 H, s), 7.11 (1 H, d, J=8.8 Hz), 5.97 (2 H, s), 3.83 (3 H, s), 3.73 (2 H, q, J=5.3 Hz), 2.88 (2 H, t, J=7.3 Hz), 2.54 (6 H, s), 1.99-1.95 (2 H, m), 1.47 (6 H, s); 13C NMR (150 MHz, CDCl3) δ 156.5, 155.7, 148.4, 142.2, 136.3, 131.3, 131.2, 125.0, 122.7, 121.7, 121.2, 119.0, 112.1, 61.7, 56.1, 31.8, 22.0, 14.6, 14.1; HRMS calcd for C25H29BF2N5O2: 480.2382, found: m/z 480.2379 [M+H]+.
2,6-Diethoxycarbonyl-4,4-difluoro-8-{3-[4-(3-hydroxypropyl)-1H-1,2,3-triazol-1-yl]-2-methoxypheny}-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (T10)




Compound T10 was prepared from compound Az10 in 90% yield. C31H36BF2N5O6, red solids, mp 157-159° C.; TLC (EtOAc/hexane, 4:1) Rf=0.32; 1H NMR (600 MHz, CDCl3) δ 7.91 (1 H, dd, J=8.9, 2.6 Hz), 7.67 (1 H, s), 7.49 (1 H, d, J=2.6 Hz), 7.14 (1 H, d, J=8.9 Hz), 4.26 (4 H, q, J=7.1 Hz), 3.84 (3 H, s), 3.73 (2 H, q, J=5.8 Hz), 2.88 (2 H, t, J=7.3 Hz), 2.81 (6 H, s), 1.99-1.94 (2 H, m), 1.79 (6 H, s), 1.31 (6 H, t, J=7.1 Hz); 13C NMR (150 MHz, CDCl3) δ 164.2, 159.6, 156.3, 148.5, 146.7, 140.6, 131.7, 131.3, 124.5, 123.3, 122.5, 121.4, 119.0, 112.3, 61.6, 60.3, 56.3, 31.8, 21.9, 15.0, 14.2, 13.1; HRMS calcd for C31H37BF2N5O6: 624.2805, found: m/z 624.2801 [M+H]+.
2,6-Dicyano-4,4-difluoro-8-{3-[4-(3-hydroxypropyl)-1H-1,2,3-triazol-1-yl]-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (T11)




Compound T11 was prepared from compound Az11 in 78% yield. C27H26BF2N7O2, red solids, mp 175-177° C. (dec.); TLC (EtOAc) Rf=0.42; 1H NMR (600 MHz, CDCl3) δ 7.90 (1 H, dd, J=9.0, 2.3 Hz), 7.71 (1 H, s), 7.58 (1 H, d, J=2.3 Hz), 7.20 (1 H, d, J=9.0 Hz), 3.86 (3 H, s), 3.73 (2 H, t, J=6.1 Hz), 2.89 (2 H, t, J=7.3 Hz), 2.71 (6 H, s), 1.99-1.95 (2 H, m), 1.69 (6 H, s); 13C NMR (150 MHz, CDCl3) δ 159.9, 155.7, 148.7, 148.6, 142.0, 131.9, 131.4, 123.7, 122.5, 121.0, 118.9, 113.4, 112.6, 106.5, 61.7, 56.5, 31.8, 22.0, 13.9, 13.6; HRMS calcd for C27H27BF2N7O2: 530.2287, found: m/z 530.2289 [M+H]+.
Example 3
Detecting and Imaging Biomolecules
Spectroscopic Measurements
All spectroscopic measurements of the amino-BODIPY Am10, the azido-BODIPYs Az2 and Az9-Az11 as well as the corresponding triazolyl-BODIPYs T2 and T9-T11 were performed in ethanol using a cuvette with 1-cm path length at 25±0.1° C. All solutions were degassed under argon for several minutes prior to measurements. For each experiment, the slit width was 2.0 nm for both excitation and emission. The absorbance spectra were measured within an absorbance range of 0.07 to 0.7 (l=10 cm). Fluorescence quantum yield measurements were performed on a fluorometer and UV-Vis instrument. Relative quantum efficiencies were obtained by comparing the areas under corrected emission spectrum. The reported quantum yield was calculated as an average of 4 points according to the following equation:

Φsample=Φstandard(Astandard/Asample)(Fsample/Fstandard)(nsample/nstandard)2 

where “Φ” is the quantum yield, “A” is the absorbance at the excitation frequency, “F” is the integrated area under the emission curve, and “n” is the refractive index of the solvent used. Fluorescein (Φf=0.85) in 0.1 M aqueous NaOH and Rhodamine 6G (Φf=0.95) in ethanol are fluorescence standards. (Parker, C. A.; Rees, W. T. Analyst 1960, 85, 587-600; Kubin, R. F.; Fletcher, A. N. J. Luminescence 1982, 27, 455-462.)

Procedure of Fluorescence Screening of CuAAC Reaction in Microtiter Plate

A 96-well black-bottom microtiter plate was used for the experiments, and fluorescence measurements were made using a Molecular Devices Spectramax M5 spectrometer. In the upper row, each well contained 200 μL of azido-BODIPY (Az1-Az8) (15 μM) in EtOH/water (1:1). In the bottom row, the overall volume in each well was 200 μL containing a solution of azido-BODIPY (Az1-Az8) (15 μM), 4-pentyn-1-ol (75 μM), CuSO4 (150 μM), sodium ascorbate (300 μM) and tris-triazole ligand (150 μM) in EtOH/water (1:1). The plate was incubated for 6 h at room temperature as monitored by TLC or MS analysis, and then fluorescence measurements (λex=488 nm) were taken in situ. The formation of the fluorescent or non-fluorescent triazole compounds could be discerned upon irradiation at 365 nm with a UV lamp (FIG. 2).
Protein Labeling with AzBOCEt (Az10)
For the protein labeling experiments, 60 μg/mL alkynyl-functionalized BSA and unmodified BSA in 90:10 pH 7.4 PBS/DMSO were incubated with 100 μM tris-triazole ligand, 1 mM CuSO4, 2 mM freshly prepared sodium ascorbate, and 0 to 100 μM Az10 at room temperature for 1 h in the dark. Each mixture (30 μL) was mixed with 10 μL (4×) SDS loading dye containing 5% β-mercaptoethanol, and 40 μL of each was gradually loaded onto 4 to 12% Bis-Tris gel. The gel was run for 2.5 h at 100 V. The gel was imaged using a Typhoon 9400 Variable Mode Imager (Amersham BioScience) (λex=488 nm; λem=526 nm) and stained with Coomassie blue (FIG. 5).
Microscopic Analysis of Fluorescence Labeling in Cells
CL1-5 cells were seeded on chamber slide (1×104 cells/1 mL per well), and incubated in culture medium (RPMI-1640 supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM L-glutamine and 1 mM sodium pyruvate) with 100 μM of respective alkynyl-sugar or control for 3 days. The alkynyl-sugars include peracetylated alkynyl-N-acetylmannosamine (Ac4ManNAl), peracetylated alkynyl-N-acetylgalactosamine (Ac4GalNAl) and peracetylated alkynyl-N-acetylglucosamine (Ac4GlcNAl). The control sugars include peracetylated N-acetylmannosamine (Ac4ManNAc), peracetylated N-acetylgalactosamine (Ac4GalNAc) and peracetylated N-acetylglucosamine (Ac4GlcNAc).
For detecting the labeling of AzBOCEt (Az10) to sugar analogs treated cells, The sugar-treated cells were washed with PBS, fixed with 3% paraformaldehyde in PBS at room temperature for 20 min, permeablized with 0.2% Triton X-100 in PBS at room temperature for 20 min, and blocked with 3% bovine serum albumin in PBS at room temperature for 30 min. To observe the fluorescence-labeled alkyne-tagged glycosyl conjugates in cells, the cells were incubated with 0.1 μM AzBOCEt (Az10), 100 μM tris-triazole ligand, 1 mM CuSO4, and 2 mM sodium ascorbate in PBS buffer with 50% ethanol at room temperature for 1 h. Fluorescence images of cells were acquired at 496-nm excitation by using confocal laser-scanning microscope. To identify the location of alkynyl-glycans, the probe-labeled cells were further stained with anti-GRASP65 followed by Cy3-conjugated anti-rabbit for Golgi, and Hoechst for nucleus (FIG. 6).
Detection and Identification of alkyne-Tagged Sialyl Glycoproteins in Cell Extracts
Cell extracts (20 μg) harvested from CL1-5 cells treated with 100 M Ac4ManNAc or Ac4ManNAl for 3 days were incubated with indicated concentration of Az10, 100 μM tris-triazole ligand,S3 1 mM CuSO4, and 2 mM sodium ascorbate at room temperature for 1 h to proceed CuAAC reaction. The reacted extracts were further separated by SDS-PAGE and the fluorescence signals were detected with the Typhoon 9400 Variable Mode Imager (Amersham BioScience) (λex=488 nm; λem=526 nm). The protein bands stained with Coomassie blue represented the loading control (FIG. 7).
The indicated 12 bands containing proteins labeled with fluorescence signal were excised and cut into small pieces. The gel pieces were destained with 50 mM NH4HCO3/CH3CN 1:1 (v/v), and then rehydrated with 10 mM dithiothreitol (DTT) in 25 mM NH4HCO3 at 56° C. for 45 min. The excess DTT was removed and 55 mM iodoacetamide (IAA) in 25 mM NH4HCO3 was added at room temperature for 30 min in the dark for alkylation. The excess IAA was removed and the gels were washed twice with 50 mM NH4HCO3/CH3CN 1:1 (v/v), and dried with CH3CN after being dried using a vacuum centrifuge. Freshly prepared trypsin solution (5 ng/μL in 25 mM NH4HCO3, 40 μL) was added to each gel pieces, and gel pieces were warmed to 37° C. for 18 h. The digested peptides were extracted twice with aqueous solution of 1% trifluoracetic acid (TFA) containing 50% CH3CN (50 μL) under sonication for 10 min. The combined extracts and washes were concentrated using a vacuum centrifuge for 3 h to remove the volatiles. The digested peptides were analyzed and identified with a hybrid linear ion trap quadrupole fourier-transfer (LTQ-FT) mass spectrometer as shown in Table 2.







TABLE 2







Sialyl glycoproteins identified by mass spectrometry in cell


extracts. Proteins are tabulated by accession number and number


of peptides (# of Pep.) found for that proteins are listed.
















Accession



Band
No.
Gene name
Protein name
number
# of Pep.















1
1-1
IGF2R
Cation-independent mannose-6-
IPI00289819
13





phosphate receptor




4
4-1
COLGALT1
Procollagen galactosyltransferase 1
IPI00168262
16



4-2
PRKCSH
Glucosidase 2 subunit beta
IPI00026154
19






IPI00792916




4-3
FKBP10
Peptidyl-prolyl cis-trans isomerase
IPI00303300
22





FKBP10





4-4
SLC1A1
Neutral amino acid transporter B(0)
IPI00019472
6






IPI00922487







IPI00922776




4-5
RPN1
Ribophorin-1
IPI00025874
1


5
5-1
P4HA1
Prolyl 4-hydroxylase subunit
IPI00009923
14





alpha-1
IPI00218682







IPI00916535




5-2
OIT3
Oncoprotein-induced transcript 3
IPI00328215
4





protein
IPI00855752



6
6-1
NCLN
Nicalin
IPI00470649
4






IPI00607732



7
7-1
ERO1L
ERO1-like protein alpha
IPI00386755
4


8
8-1
NCEH1
Neutral cholesterol ester hydrolase
IPI00002230
4





1
IPI00790972







IPI00924788




8-2
SERPINH1
Serpin H1
IPI00032140
3



8-3
SIGLEC7
Sialic acid-binding Ig-like lectin 7
IPI00004288
2






IPI00220858







IPI00220860







IPI00220862



9
9-1
ERLIN1
Erlin-1
IPI00007940
18



9-2
DNAJB11
DnaJ homolog subfamily B
IPI00074870
9





member 11





9-3
AHSG
Alpha-2-HS-glycoprotein
IPI00953689
6



9-4
RCN1
Reticulocalbin-1
IPI00015842
1



9-5
IMPAD1
Inositol monophosphatase 3
IPI00787853
2



9-6
IKBIP
Inhibitor of nuclear factor kappa-B
IPI00043598
4





kinase- interacting protein
IPI00401792







IPI00797136







IPI00896464



10
10-1 
CTSL
Cathepsin L1
IPI00012887
7



10-2 
TOR1B
Torsin-1B
IPI00023137
1


11
11-1 
SUMF2
Sulfatase-modifying factor 2
IPI00334513
5






IPI00334514







IPI00334516







IPI00783919







IPI00939930




11-2 
CNPY3
Protein canopy homolog 3
IPI00398366
4






IPI00551062




11-3 
PPT1
Palmitoyl-protein thioesterase 1
IPI00002412
2



11-4 
SSR1
Translocon-associated protein
IPI00301021
2





subunit alpha
IPI00449669



12
12-1 
PSMA1
Proteasome subunit alpha type-1
IPI00016832
30






IPI00472442




12-2 
CTXZ
Cathepsin Z
IPI00002745
3









Microscopic Analysis of Dual Fluorescence Labeling in Cells

CL1-5 cells were seeded on chamber slide (2.5×104 cells/0.5 mL per wells) and incubated in culture medium (RPMI-1640 supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM L-glutamine and 1 mM sodium pyruvate) for 3 days with either 100 μM of alkynyl-sugar (peracetylated alkynyl-N-acetylmannosamine, Ac4ManNAl or azido-sugar (peracetylated azido-N-acetylglucosamine, Ac4GlcNAz) or both or without sugar as negative control. Cells were washed three times with PBS and then incubated with 100 μM of coumOCT in PBS with 10% DMSO for 30 min at 37° C. After three washes with PBS with 10% DMSO, followed by fixed with 3% paraformaldehyde in PBS at room temperature for 20 min, the cells were incubated with 0.1 μM of AzBOCEt (Az10), 100 μM of ligand, 1 mM of CuSO4, and 2 mM of sodium ascorbate in PBS with 50% ethanol at room temperature for 1 h and fluorescence images of cell were carried out using a confocal microscope (TCS-SP5-MP-SMD, Leica) (FIG. 9).
Example 4
Synthesis of Cyclooctyne-Functionalized Fluorescent Probe 101
Scheme 100 shows the synthesis of compound 101 using 1-benzosuberone as the starting material. According to the previously reported procedure,12 1-benzosuberone was subject to regioselective nitration at 8-position. The nitro group was then reduced, followed by diazotization and hydroxylation under acid conditions to give alcohol 104. After protection of the hydroxyl group as a benzyl ether, the cyclic ketone 105 underwent a ring expansion by treatment with TMS-diazomethane in the presence of BF3 .OEt2 to afford the cyclooctanone product 106 in 73% yield. Reduction of the carbonyl group with NaBH4, followed by silylation, gave the silyl ether 107 in 96% yield. The benzyl group in 107 was removed by hydrogenation, and the phenol intermediate was treated with excess paraformaldehyde in the presence of Et3N and MgCl2 to form the salicylaldehyde 108. The coumarin scaffold in 109 was constructed by treatment of 108 with freshly prepared ketenylidenetriphenylphosphosphorane. After desilylation and oxidation, ketone 110 was obtained in 78% yield. The carbonyl group in compound 110 was converted to enol triflate, which was subsequently treated with a strong base NaHMDS to render an elimination reaction, giving the coumOCT probe 101.







Reagents and conditions: (a) conc. H2SO4, KNO3, 0° C., 1.5 h, 72%; (b) Sn, conc. HCl, C2H5OH, reflux, 50 min, 82%; (c) 10% aq. H2SO4, NaNO2, 0° C. to rt, 72 h, 76%; (d) BnBr, K2CO3, DMF, rt, 24 h, 98%; (e) TMSCHN2, BF3.OEt2, CH2Cl2, 0° C., 12 h, 73%; (f) NaBH4, CH3OH, 0° C., 1 h; (g) TIPSOTf, 2,6-lutidine, CH2Cl2, rt, 1 h, 96% for two steps; (h) H2, Pd/C, CH3OH, EtOAc, 1 h; (i) paraformaldehyde, MgCl2, Et3N, CH3CN, reflux, 12 h, 87% for two steps; (j) Ph3P═C═C═O, toluene, 90° C., 1.5 h, 83%; (k) TBAF, THF, 0° C. to rt, 1 h; (1) (COCl)2, DMSO, Et3N, CH2Cl2, −78° C. to rt, 1 h, 78% for two steps; (m) NaHMDS, Tf2NPh, −78° C. to rt, 2 h, 44%; (n) BnN3 (1.5 equiv.), CH3CN, rt, 2 h, 95%; (o)N-azidoacetylmannosamine (1.5 equiv.), MeOH, H2O, rt, 2 h, 92%.
3-Nitro-6,7,8,9-tetrahydrobenzocyclohepten-5-one (A)




A solution of 1-benzosuberone (4.0 g, 25 mmol) in concentrated H2SO4 (28 mL) was cooled at 0° C., and a solution of KNO3 (2.8 g, 27.7 mmol) in concentrated H2SO4 (7.5 mL) was added dropwise over a period of 30 min. The mixture was stirred for additional 1 h at 0° C., and then poured into crushed ice. The precipitate was filtered, washed with water and air-dried to yield a yellow solid. The crude product was purified by column chromatography on silica gel (EtOAc/hexane, 1:4) to afford the pure nitro product A (3.69 g, 72%). C11H11NO3, white needles, mp 90-92° C. (lit.S2 mp 89-90° C.); TLC (EtOAc/hexane, 1:4) Rf=0.31; 1H NMR (600 MHz, CDCl3) δ 8.53 (1 H, d, J=2.5 Hz), 8.22 (1 H, dd, J=8.3, 2.5 Hz), 7.37 (1 H, d, J=8.3 Hz), 3.01 (2 H, t, J=6.4 Hz), 2.77 (2 H, t, J=6.1 Hz), 1.94-1.90 (2 H, m), 1.85-1.81 (2 H, m); 13C NMR (150 MHz, CDCl3) δ 203.4, 148.0, 147.0, 139.8, 131.0, 126.2, 123.9, 40.4, 32.4, 24.7, 20.5; HRMS calcd for C11H12NO3: 206.0812, found: m/z 206.0814 [M+H]+.
3-Amino-6,7,8,9-tetrahydrobenzocyclohepten-5-one (B)




A mixture of nitro compound A (2.05 g, 10 mmol) and Sn (8.31 g, 70 mmol) in concentrated HCl (45 mL) and ethanol (25 mL) was heated at reflux for 50 min. The mixture was cooled to room temperature, and basified with 30% NaOH aqueous solution. The mixture was filtered through a pad of Celite, and washed with ethanol. The filtrate was extracted with EtOAc (5×50 mL). The combined organic extracts were washed with brine (100 mL), dried over MgSO4, filtered, and concentrated under reduced pressure to afford the analytically pure amino product B (1.44 g, 82%). C11H13NO, yellowish solid, mp 102-104° C. (lit.S2 mp 103-105° C.); TLC (EtOAc/hexane, 3:7) Rf=0.29; 1H NMR (600 MHz, CDCl3) δ 7.02 (1 H, d, J=2.6 Hz), 6.96 (1 H, d, J=8.0 Hz), 6.72 (1 H, dd, J=8.0, 2.6 Hz), 3.65 (2 H, br s, NH), 2.79 (2 H, t, J=5.5 Hz), 2.67 (2 H, t, J=6.6 Hz), 1.81-1.74 (4 H, m); 13C NMR (150 MHz, CDCl3) δ 206.2, 144.9, 139.3, 131.6, 130.7, 118.8, 114.5, 40.8, 31.5, 25.4, 20.9; HRMS calcd for C11H14NO: 176.1070, found: m/z 176.1069 [M+H]+.
3-Hydroxy-6,7,8,9-tetrahydrobenzocyclohepten-5-one (104)




A cold (0° C.) solution of amino compound B (1.45 g, 8.3 mmol) in H2SO4 (40 mL of 10% aqueous solution) was cautiously added an aqueous solution of NaNO2 (687 mg, 9.96 mmol) in water (3 mL). The reaction mixture was stirred for 30 min at 0° C., and then sulfamic acid was added to destroy excess nitrous acid. The suspension was filtered and the filtrate was poured into a 10% aqueous solution of H2SO4 (100 mL) and toluene (50 mL). The mixture was stirred for 3 days at room temperature. The layers were then separated and the aqueous layer was extracted with EtoAc (5×30 mL). The combined organic extracts were washed with brine (100 mL), dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc/hexane, 1:4) to afford the analytically pure alcohol product 104 (1.11 g, 76%). C11H12O2, yellow solid, mp 98-100° C. (lit.S3 mp 96-99° C.); TLC (EtOAc/hexane, 3:7) Rf=0.37; 1H NMR (600 MHz, CDCl3) δ 7.33 (1 H, d, J=2.8 Hz), 7.07 (1 H, d, J=8.2 Hz), 6.94 (1 H, dd, J=8.2, 2.8 Hz), 6.27 (1 H, s, OH), 2.84 (2 H, t, J=5.7 Hz), 2.72 (2 H, t, J=6.4 Hz), 1.86-1.76 (4 H, m); 13C NMR (150 MHz, CDCl3) δ 206.7, 154.7, 139.2, 133.9, 131.3, 119.8, 114.9, 40.8, 31.6, 25.3, 20.8; HRMS calcd for C11H13O2: 177.0910, found: m/z 177.0911 [M+H]+.
3-Benzyloxy-6,7,8,9-tetrahydrobenzocyclohepten-5-one (105)




A solution of alcohol compound 104 (1.25 g, 7.1 mmol) in anhydrous DMF (10 mL) was treated with benzyl bromide (1 mL, 8.4 mmol) and potassium carbonate (2.1 g, 15.2 mmol). The suspension was vigorously stirred for 24 h at room temperature. The mixture was poured into water (20 mL) and extracted with Et2O (4×30 mL), The combined organic extracts were washed with water (3×20 mL) and brine (100 mL), dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc/hexane, 1:9) to afford the pure benzyloxy product 105 (1.85 g, 98%). C18H18O2, pale yellow oil; TLC (EtOAc/hexane, 1:9) Rf=0.37; 1H NMR (600 MHz, CDCl3) δ 7.41 (2 H, d, J=7.2 Hz), 7.37-7.35 (3 H, m), 7.31 (1 H, d, J=7.4 Hz), 7.10 (1 H, d, J=8.3 Hz), 7.02 (1 H, dd, J=8.3, 2.9 Hz), 5.06 (2 H, s), 2.86 (2 H, t, J=5.8 Hz), 2.71 (2 H, t, J=6.2 Hz), 1.84-1.78 (4 H, m); 13C NMR (150 MHz, CDCl3) δ 205.6, 157.4, 139.5, 136.7, 134.2, 131.0, 128.5 (2×), 127.9, 127.5 (2×), 119.7, 113.2, 70.1, 40.7, 31.6, 25.3, 20.8; HRMS calcd for C18H19O2: 267.1380, found: m/z 267.1383 [M+H]+.
3-Benzyloxy-7,8,9,10-tetrahydro-5H-benzocycloocten-6-one (106)




A stirred solution of (trimethylsilyl)diazomethane (5 mL, ca. 2 M solution in hexane, 10 mmol) in CH2Cl2 (10 mL) was added dropwise over a period of 1 h to a stirred solution of compound 105 (1.6 g, 6 mmol) and BF3.OEt2 (820 μL, 10 mmol) in CH2Cl2 (20 mL) at 0° C. The mixture was stirred for 12 h at 0° C., and then poured into crushed ice. The aqueous layer was extracted with CH2Cl2 (3×20 mL). The combined organic extracts were washed with brine (50 mL), dried over MgSO4, and concentrated to give an orange oil that was purified by column chromatography on silica gel (EtOAc/hexane, 1:19) to afford pure cyclooctanone product 106 (1.23 g, 73%). C19H20O2, colorless oil; TLC (EtOAc/hexane, 1:9) Rf=0.29; 1H NMR (600 MHz, CDCl3) δ 7.40 (2 H, d, J=7.4 Hz), 7.37-7.34 (2 H, m), 7.31-7.29 (1 H, m), 7.09 (1 H, d, J=8.4 Hz), 6.84 (1 H, dd, J=8.4, 2.7 Hz), 6.75 (1 H, d, J=2.7 Hz), 5.01 (2 H, s), 3.72 (2 H, s), 2.74 (2 H, t, J=5.8 Hz), 2.31 (2 H, t, J=5.3 Hz), 1.81-1.77 (2 H, m), 1.72-1.68 (2 H, m); 13C NMR (150 MHz, CDCl3) δ 211.8, 157.4, 136.9, 134.7, 133.4, 131.2, 128.5 (2×), 127.9, 127.5 (2×), 116.0, 114.4, 70.0, 48.8, 41.0, 32.3, 31.5, 24.7; HRMS calcd for C19H21O2: 281.1536, found: m/z 281.1539 [M+H]+.
3-Benzyloxy-6-triisopropylsilyloxy-5,6,7,8,9,10-hexahydrobenzocyclooctene (107)




A cold (0° C.) solution of compound 106 (4.8 g, 17.1 mmol) in methanol (40 mL) was treated with NaBH4 (970 mg, 25.7 mmol). The mixture was stirred for 1 h at the 0° C., and then concentrated under reduced pressure. The residue was dissolved in CH2Cl2 (80 mL), and washed with 1 M HCl aqueous solution (50 mL) and brine (50 mL). The organic layer was dried over MgSO4, filtered, and concentrated to provide a crude alcohol product as colorless foam (4.8 g), which was used in the next step without further purification.
The above-prepared alcohol (4.8 g, 17.0 mmol) and 2,6-lutidine (8 mL, 68.7 mmol) were dissolved in anhydrous CH2Cl2 (50 mL), and cooled to 0° C. Triisopropylsilyl trifluoromethanesulfonate (9.2 mL 34.2 mmol) was added dropwise over a period of 3 min to the mixture. The mixture was stirred for 1 h at room temperature, and then diluted with CH2Cl2 (100 mL). The solution was washed with saturated aqueous NaHCO3 (50 mL), 1 M HCl aqueous solution (50 mL), and brine (50 mL). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (EtOAc/hexane, 1:9) to afford the pure silyl ether product 107 (7.2 g, 96% for two steps). C28H42O2Si, colorless syrup; TLC (EtOAc/hexane, 1:9) Rf=0.51; 1H NMR (600 MHz, CDCl3) δ 7.41 (2 H, d, J=7.4 Hz), 7.37-7.34 (2 H, m), 7.31-7.28 (1 H, m), 6.99 (1 H, dd, J=6.6, 2.5 Hz), 6.75-6.74 (2 H, m), 5.01 (2 H, s), 3.96-3.93 (1 H, m), 2.91-2.83 (2 H, m), 2.77-2.72 (1 H, m), 2.65-2.61 (1 H, m), 1.76-1.72 (1 H, m), 1.71-1.64 (1 H, m), 1.50-1.41 (3 H, m), 1.18-1.15 (1 H, m), 1.07-1.05 (21 H, m); 13C NMR (150 MHz, CDCl3) δ 156.8, 138.5, 137.3, 134.1, 130.1, 128.5 (2×), 127.8, 127.5 (2×), 116.7, 112.5, 73.8, 70.0, 40.9, 34.5, 32.3, 32.0, 20.8, 18.2 (6×), 12.4 (3×); HRMS calcd for C28H43O2Si: 439.3032, found: m/z 439.3022 [M+H]+.
3-Hydroxy-6-triisopropylsilyloxy-5,6,7,8,9,10-hexahydrobenzocycloocten-2-carboxaldehyde (108)




A solution of compound 107 (7.1 g, 16.2 mmol) in methanol (50 mL) and EtOAc (20 mL) was treated with Pd/C (100 mg) under an atmosphere of hydrogen. After stirring for 1 h, the mixture was filtered through Celite, and rinsed with EtOAc. The filtrate was concentrated under reduced pressure to give a light brown syrup (5.6 g), which was dissolved in anhydrous acetonitrile (150 mL) and treated with anhydrous MgCl2 (4.64 g, 48.6 mmol), triethylamine (13.5 mL, 97.2 mmol) and paraformaldehyde (4.86 g, 162 mmol). The suspension was heated at reflux for 12 h. The mixture was cooled to room temperature, and the deep-yellow suspension was acidified with a 1 M HCl aqueous solution (200 mL). The solution was extracted with EtOAc (5×150 mL). The combined organic extracts were washed with brine (200 mL), dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc/hexane, 1:19) to afford the pure salicylaldehyde product 108 (5.3 g, 87% for two steps). C22H36O3Si, pale yellow syrup; TLC (EtOAc/hexane, 1:9) Rf=0.71; 1H NMR (600 MHz, CDCl3) δ 10.77 (1 H, s), 9.79 (1 H, s), 7.24 (1 H, s), 6.76 (1 H, s), 4.03-3.99 (1 H, m), 2.94-2.88 (2 H, m), 2.81-2.76 (1 H, m), 2.71-2.67 (1 H, m), 1.80-1.75 (1 H, m), 1.72-1.66 (1 H, m), 1.52-1.46 (2 H, m), 1.43-1.38 (1 H, m), 1.24-1.16 (1 H, m), 1.07-1.05 (21 H, m); 13C NMR (150 MHz, CDCl3) δ 195.9, 159.5, 148.2, 133.8, 133.7, 119.5, 118.8, 73.2, 41.0, 34.4, 32.2, 32.1, 20.6, 18.1 (6×), 12.4 (3×); HRMS calcd for C22H37O3Si: 377.2506, found: m/z 377.2511 [M+H]+.
6,7,8,9,10,11-Hexahydro-10-triisopropylsilyloxy-cycloocta[g]chromen-2(2H)-one (109)




Preparation of ketenylidenetriphenylphosphorane: A stirred solution of carbethoxymethylenetriphenylphosphorane (10 g, 30 mmol) in anhydrous toluene (200 mL) was added dropwise a solution of sodium hexamethyldisilazide (17.5 mL, 2 M solution in THF, 35 mmol) at 0° C. Once the addition was complete, the mixture was heated at 60° C. for 24 h. The reaction was then allowed to cool to room temperature and filtered out. The filtrate was concentrated under reduced pressure, and then poured into ether (200 mL). The precipitate was filtered, washed with ether, and air-dried to afford ketenylidenetriphenylphosphorane (5.8 g, 64%) as pale yellow solids.
A stirred solution of salicylaldehyde 108 (4.3 g, 11.42 mmol) in anhydrous toluene (100 mL) was added the fresh prepared ketenylidenetriphenylphosphosphorane (5.2 g, 17.2 mmol) at room temperature. The mixture was heated at 90° C. for 1.5 h, and then concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (EtOAc/hexane, 1:9) to afford the pure coumarin product 109 (3.8 g, 83%). C24H36O3Si, colorless solid, mp 103-105° C.; TLC (EtOAc/hexane, 1:9) Rf=0.25; 1H NMR (600 MHz, CDCl3) δ 7.61 (1 H, d, J=9.5 Hz), 7.18 (1 H, s), 7.10 (1 H, s), 6.33 (1 H, d, J=9.5 Hz), 4.02 (1 H, dd, J=11.9, 5.5 Hz), 3.00-2.94 (2 H, m), 2.86-2.81 (1 H, m), 2.76-2.72 (1 H, m), 1.80-1.73 (1 H, m), 1.69-1.64 (1 H, m), 1.60-1.55 (1 H, m), 1.47-1.43 (2 H, m), 1.20-1.13 (1 H, m), 1.11-1.03 (21 H, m); 13C NMR (150 MHz, CDCl3) δ 161.3, 152.3, 143.3, 142.4, 138.4, 127.8, 118.1, 117.3, 115.7, 73.3, 40.8, 34.5, 32.4, 32.0, 20.6, 18.1 (6×), 12.4 (3×); HRMS calcd for C24H37O3Si: 401.2506, found: m/z 401.2511 [M+H]+.
6,7,8,9-Tetrahydro-1H-10-oxo-cycloocta[g]chromen-2(2H)-one (110)




A cold (0° C.) solution of compound 109 (3.0 g, 7.5 mmol) in THF (20 mL) was treated with a solution of tetrabutylammonium fluoride (10 mL, 1 M solution in THF, 10 mmol). After stirring for 1 h at room temperature, the mixture was concentrated under reduced pressure. The residual oil was filtered through a short pad of silica gel (EtOAc/hexane, 1:4) and the filtrate was concentrated to give a colorless solid (1.67 g).
A solution of DMSO (1.5 mL, 21.2 mmol) in anhydrous CH2Cl2 (10 mL) was added dropwise to a stirred solution of oxalyl chloride (0.89 mL, 10.3 mmol) in anhydrous CH2Cl2 (10 mL) at −78° C. under an atmosphere of nitrogen. The mixture was stirred for 30 min at −78° C., and the above-prepared alcohol (1.67 g) in anhydrous CH2Cl2 (10 mL) was added dropwise. The mixture was stirred for additional 30 min at −78° C., and triethylamine (7.1 mL, 50.4 mmol) was added. The mixture was allowed to warm to 0° C. for 30 min, and then poured into water (40 mL). The aqueous layer was extracted with CH2Cl2 (5×50 mL). The combined organic extracts were washed with water (100 mL) and brine (100 mL), dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc/hexane, 1:4) to afford the desired product 110 (1.45 g, 78% for two steps). C15H14O3, colorless solid, mp 127-129° C.; TLC (EtOAc/hexane, 3:7) Rf=0.32; 1H NMR (600 MHz, CDCl3) δ 7.63 (1 H, d, J=9.5 Hz), 7.29 (1 H, s), 7.09 (1 H, s), 6.34 (1 H, d, J=9.5 Hz), 3.82 (2 H, s), 2.84 (2 H, t, J=5.7 Hz), 2.31 (2 H, t, J=5.6 Hz), 1.86-1.84 (2 H, m), 1.73-1.71 (2 H, m); 13C NMR (150 MHz, CDCl3) δ 209.9, 160.7, 152.6, 142.8, 138.2, 137.7, 129.0, 118.4, 117.7, 116.6, 48.8, 41.1, 32.3, 31.4, 24.5; HRMS calcd for C15H15O3: 243.1016, found: m/z 243.1016 [M+H]+.
6,7,8,9-Tetrahydro-10,11-didehydro-cycloocta[g]chromen-2(2H)-one (101)




A cold (−78° C.) solution of compound 110 (245 mg, 1 mmol) and N-phenyl bis(trifluoromethanesulfonimide) (393 mg, 1.1 mmol) in anhydrous THF (10 mL) was added a solution of sodium hexamethyldisilazide (0.55 mL, 2 M solution in THF, 1.1 mmol) via syringe over a period of 5 min. The mixture was stirred for 1 h at −78° C., and another batch of sodium hexamethyldisilazide (0.55 mL, 2 M solution in THF, 1.1 mmol) was added. The mixture was allowed to warm to 0° C., stirred for additional 1 h, and then quenched with methanol (1 mL). The mixture was concentrated under reduced pressure to give a yellow syrup, which was purified by column chromatography on silica gel (EtOAc/hexane, 1:9) to afford the target product 101 (72 mg, 44%). C15H12O2, light-yellow solid, mp 98-100° C.; TLC (EtOAc/hexane, 3:7) Rf=0.42; 1H NMR (600 MHz, CDCl3) δ 7.60 (1 H, d, J=9.5 Hz), 7.23 (1 H, s), 7.06 (1 H, s), 6.35 (1 H, d, J=9.5 Hz), 2.81 (2 H, br s), 2.61 (2 H, t, J=56.7 Hz), 2.13 (2 H, br s), 1.74 (2 H, br s); 13C NMR (150 MHz, CDCl3) δ 161.1, 152.6, 147.4, 143.3, 128.6, 127.1, 117.5, 117.4, 116.1, 113.9, 92.5, 38.3, 33.5, 25.5, 20.5; HRMS calcd for C15H13O2: 225.0910, found: m/z 225.0910 [M+H]+.
10-Benzyl-6,7,8,9-tetrahydro-cyclooctatriazolo[5,4-g]chromen-2(2H)-one (111)




A solution of compound 101 (50 mg, 0.22 mmol) in CH3CN (5 mL) was treated with benzyl azide (44 μL, 0.33 mmol). After stirring for 2 h at room temperature, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (EtOAc/hexane, 3:7) to afford the desired triazole product 111 (75 mg, 95%). C22H19N3O2, colorless solid, mp 60-62° C.; TLC (EtOAc/hexane, 1:1) Rf=0.35; 1H NMR (600 MHz, CDCl3) δ 7.75 (1 H, s), 7.65 (1 H, d, J=9.4 Hz), 7.37-7.32 (3 H, m), 7.24 (1 H, s), 7.20-7.19 (2 H, m), 6.39 (1 H, d, J=9.4 Hz), 5.50 (2 H, s), 2.69-2.67 (2 H, m), 2.65-2.63 (2 H, m), 1.79-1.75 (2 H, m), 1.67-1.64 (2 H, m); 13C NMR (150 MHz, CDCl3) δ 161.0, 152.4, 143.1, 136.1, 135.3, 135.0, 134.4, 129.0 (2×), 128.7, 128.4, 128.1, 127.1 (2×), 126.9, 116.7, 116.4, 51.9, 30.8, 30.6, 23.9, 20.0; HRMS calcd for C22H20N3O2: 358.1550, found: m/z 358.1548 [M+H]+.
N-[2-(11-Oxo-4,6,7,11-tetrahydrochromeno[7′,6′:3,4]cycloocta[1,2-d][1,2,3]triazol-3(5H)-yl)]acetamido-2-deoxy-α,β-D-mannopyranose (112)




A solution of compound 101 (50 mg, 0.22 mmol) in MeOH (5 mL) and water (1 mL) was treated with N-azidoacetylmannosamine (142 mg, 0.33 mmol). After stirring for 2 h at room temperature, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (MeOH/CH2Cl2, 1:9) to afford the desired triazole product 112 (98 mg, 92%). C23H26N4O8, colorless solid, mp 170-172° C. (dec.); TLC (MeOH/CH2Cl2, 1:8) Rf=0.25; 1H NMR (600 MHz, DMSO-d6) δ 8.04 (1 H, d, J=9.5 Hz),7.59 (1 H, s), 7.52 (1 H, s), 6.48 (1 H, d, J=9.5 Hz), 5.15 (1 H, d, J=2.9 Hz), 5.11 (1 H, d, J=6.9 Hz), 4.98-4.85 (1 H, m), 4.89-4.81 (2 H, m), 4.44-4.38 (1 H, m), 3.61-3.59 (3 H, m), 3.53-3.45 (2 H, m), 3.42-3.37 (1 H, m), 3.16-3.14 (2 H, m), 2.86-2.82 (2 H, m), 2.72-2.70 (2 H, m), 1.78 (2 H, br s), 1.61 (2 H, br t, J=5.5 Hz); 13C NMR (150 MHz, DMSO-d6) δ 165.2, 160.1, 151.9, 143.8, 140.6, 137.0, 135.9, 134.7, 128.8, 118.6, 116.1, 115.2, 92.5, 90.4, 72.9, 72.1, 71.0, 70.5, 68.2, 67.5, 61.5, 61.0, 54.7, 54.2, 50.0, 49.9, 30.6, 29.9, 22.8, 19.6; HRMS calcd for C23H27N4O8: 487.1829, found: m/z 487.1827 [M+H]+.
Example 5
Measuring Reaction Scope and Kinetics of Cyclooctyne-fused Fluorogenic Probe Using on a Model Substrate
To evaluate the feasibility of compound 101 as a reagent for the SPAAC-based fluorescence labeling, its reaction scope and kinetics using benzyl azide as a model substrate was first studied. The SPAAC reaction of 101 with benzyl azide in acetonitrile was completed in 2 h at room temperature to give triazole 111 in 95% yield (Scheme 100). The reactivity of 101 with benzyl azide (1:1) in CD3CN was determined by integration of multiple chemical shifts in the 1H-NMR spectrum to yield a second-order rate constant of 0.012 M−1s−1 at 25° C. We also observed the cycloaddition reaction between 101 and N-azidoacetylmannosamine (ManNAz), proceeding similarly to give triazole 112 with a second-order rate constant of 0.010 M−1s−1 at 25° C. in a solution of CD3OD-D2O (5:1, v/v) (FIGS. 17 and 18).
Table 3 shows the absorption and fluorescence data of 101, 111 and 112 recorded under simulated physiological conditions (PBS buffer containing 10% DMSO, pH 7.4). Formation of triazoles 111 and 112 were accompanied by a significant increase in fluorescence intensity with a large Stokes shift into a standard range for coumarin emission (FIG. 16a). Upon excitation at 330 nm, probe 101 produced a weak emission band centered at 405 nm with a low quantum yield (Φf=0.011), whereas both triazoles 111 and 112 exhibited a strong fluorescence at 435 nm with a quantum yield of 0.23 and 0.21, respectively.
To probe the SPAAC reaction under the conditions that would be more typical for biomolecule labeling, we investigated the fluorescence response and time course for the reaction of 101 with ManNAz. The experiments indicated that more than 90% of ManNAz was consumed in 40 min, and the fluorescence intensity reached a plateau in less than 1 h (FIG. 16b).







TABLE 3







Spectroscopic properties of probe 101 and triazoles 111, 112













Absorption
ε
Emission
Stokes shift




(λmax, nm)
(M−1cm−1)[a]
(λmax, nm)
(cm−1)
Φf[b]















101
336
7800
405
5070
0.011


111
328
12200
435
7500
0.23


112
330
10800
435
7320
0.21





[a]Extinction coefficient; measured at 340 nm for 101, and at 330 nm for 111 and 112.


[b]Fluorescence quantum yield; using quinine sulfate (Φf = 0.54 ± 0.03) as standard.






FIG. 16 describes (a) Absorption and Fluorescence emission spectra (λex=330 nm) of 101 (black), 111 (blue) and 112 (red) (45 μM, PBS buffer containing 10% DMSO, pH 7.4). (b) Time course of normalized fluorescence intensity at 435 nm (λex=330 nm) for the ligation reaction of 101 (30 μM) with N-azidoacetylmannosamine (30 μM) in PBS buffer containing 10% DMSO.
Part A: Compound 101 and benzyl azide were predissolved in CD3CN, and then mixed at equimolar concentration of 20 mM. The reaction was monitored by 1H-NMR analysis over a period of 1 h. The concentration of each component was determined, based on the concentration of initial compound 101, by integration at multiple chemical shifts in the 1H-NMR spectrum. By plotting 1/[101](M−1) vs. time (sec), a second order rate constant in unit of M−1s−1 was determined using linear regression analysis. This procedure was repeated 3 times with a concentrated of 20 mM to afford a rate constant of 0.012 M−1s−1 at 25° C. (FIG. 17).
FIG. 17 describes a Plot of 1/[101] vs. time for the reaction of compound 101 and benzyl azide in CD3CN as monitored by 1H-NMR.
Part B: Compound 101 and N-azidoacetylmannosamine were predissolved in CD3OD/D2O (5:1, v/v), and then mixed at equimolar concentration of 20 mM. The reaction was monitored by 1H-NMR analysis over a period of 1 h. The concentration of each component was determined, based on the concentration of initial compound 101, by integration at multiple chemical shifts in the 1H-NMR spectrum. By plotting 1/[101](M1) vs. time (sec), a second order rate constant in unit of M−1s−1 was determined using linear regression analysis. This procedure was repeated 3 times with a concentrated of 20 mM to afford a rate constant of 0.010 M−1s−1 at 25° C. (FIG. 18).
FIG. 18 describes a Plot of 1/[101] vs. time for the reaction of compound 101 and N-azidoacetylmannosamine in a solution of CD3OD-D2O (5:1, v/v) as monitored by 1H-NMR.
Example 6
Live Cell Imaging of Stained Samples Using Cyclooctyne-fused Fluorogenic Probe 101
The performance of 101 in live cell imaging was evaluated. For this purpose, highly sialylated lung cancer cells, CL1-5, were cultured in the presence of peracetylated N-azidoacetylmannosamine (Ac4ManNAz) for 3 days to metabolically produce the azido-sialic acid expressed cells. As a negative control, CL1-5 cells were grown in the presence of peracetylated N-acetylmannosamine (Ac4ManNAc). A time course experiment was conducted by exposing the cells at 30-min intervals under no-wash and no-fixation conditions (FIG. 19, FIG. 22). The Ac4ManNAz treated cells showed a time-dependent increase of fluorescence intensity (upper row: cyan, bottom row: blue) and then reached saturation for 1.5 h incubation. In contrast, the control cells exhibited almost no fluorescence staining, supporting that background labeling is negligible. Furthermore, the localization of azido-containing glycoconjugates in living cells was visualized by confocal microscopy. The cells labeled by probe 101 were subsequently stained with anti-GRASP65 followed by FITC-conjugated anti-rabbit for Golgi and propidium iodide (PI, a nuclei marker). The blue fluorescent signal derived from the coumarin probe apparently showed in Ac4ManNAz treated cells without addition of Ac4ManNAc (FIG. 20). The labeled sialylated glycoconjugates were visualized in the cytosol using coumOCT probe (blue fluorescence), and significantly overlapped with the Golgi apparatus (green staining), but not in the nucleus (red staining).
FIG. 19 describes Time-lapse fluorescence and overlaid images of living CL1-5 cells incubated with 200 μM of Ac4ManNAz and labeled with 100 μM of probe 101 under no-wash and no-fixation conditions: fluorescence image of cells (upper row) and bright field overlaid image of cells (bottom row). Control: cells incubated with Ac4ManNAc. (Scale bar: 10 μm)
FIG. 20 describes localization of probe-labeled sialyl glycoconjugates in CL1-5 cells as visualized by confocal microscopy. Cells incubated with 200 μM of Ac4ManNAz or Ac4ManNAc were labeled with 100 μM of 101 (blue) and stained with anti-GRASP65 followed by FITC-conjugated anti-rabbit (for Golgi, green) and propidium iodide (for nucleus, red). (Scale bar: 10 μm)
Surprisingly, the blue fluorescence was observed not only on the cell surface but also inside the cells in the time-lapse experiment. We therefore examined whether coumOCT 101 could be a cell-permeable probe that could be utilized for direct intracellular labeling in live cells. Toward this goal, CL1-5 cells were incubated with Ac4ManNAz for 1 h and subsequently removed excess Ac4ManNAz. We then performed the imaging to monitor the trafficking of the sialylated glycoconjugates. As shown in FIG. 21, the sialylated glycoconjugates were readily imaged by using coumOCT and significantly overlapped with the Golgi apparatus (red) at initial stage. However, the appearance of blue fluorescence signal was detected on the cell surface after 5 h. The intensity of fluorescence increased over time, and reached saturation at 8 h (FIG. 21 and FIG. 23). Our results indicate that coumOCT is not only a cell-permeable probe but also specific for direct labeling of endogenous azido-bearing glycoconjugates in live cells.
FIG. 21 describes fluorescence imaging of glycoconjugates trafficking using coumOCT (1) of SPAAC in cells. CL1-5 cells were incubated with 500 μM of Ac4ManNAz for 1 h and subsequently washed with PBS buffer to remove excess Ac4ManNAz. The sugar-treated cells were incubated in culture medium for 3 h, 5 h and 8 h, and then labeled with 100 M of probe 101 for 0.5 h under SPAAC conditions, respectively. The Golgi was labeled with anti-GRASP65 followed by Cy3-conjugated anti-rabbit. (Scale bar: 5 μm)
Time Course Measurements by Fluorescence Spectroscopy
A solution of probe 101 (0.075 μmol) and N-azidoacetylmannosamine (0.075 μmol) in a mixture of 10% DMSO in PBS buffer (2.5 mL) was incubated at 37° C. The fluorescence emission intensity at 435 nm upon excitation at 330 nm was monitored in 5 min intervals. For each point the fluorescence intensity was measured over a period of 5 sec and averaged over a total of 3 points. In a control experiment the same conditions were used except that N-acetylmannosamine (0.075 μmol) was added to the solution.
Time-Lapse Microscopic Analysis of Fluorescence Labeling in Live Cells
To observe the fluorescence labeled azido-glycoconjugates in cells, CL1-5 cells were seeded on chamber slide (2.5×104 cells/0.5 mL per wells) and incubated in culture medium (RPMI-1640 supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM L-glutamine and 1 mM sodium pyruvate) with either 200 μM of control sugar (peracetylated N-acetylmannosamine, Ac4ManNAc) or azido-sugar (peracetylated N-azidoacetylmannosamine, Ac4ManNAz) for 3 days.
For Time-lapse imaging of live cells, experiments were carried out using a confocal microscope (TCS-SP5-MP-SMD, Leica) equipped with an incubator to keep the cells in culture conditions. Prewashed cells were incubated with 100 μM of probe 101 in PBS with 10% DMSO and fluorescence imaging of live cells from the previous experiment over 1.5 h. The images were acquired at 450 nm emission and in 5-min intervals.
For comparing the localization of azido-glycoconjugates, probe-labeled cells were washed with PBS, fixed with 3% paraformaldehyde in PBS at room temperature for 20 min, permeablized with 0.2% Triton X-100 in PBS at room temperature for 20 min, and blocked with 3% bovine serum albumin in PBS at room temperature for 30 min. Cells were stained with anti-GRASP65 followed by FITC-conjugated anti-rabbit for Golgi, and propidium iodide (PI) for nucleus.
FIG. 22 describes time-lapse fluorescence and overlaid images of living CL1-5 cells incubated with 200 μM of Ac4ManNAz and labeled with 100 μM of probe 101 under no-wash and no-fixation conditions: fluorescence image of cells (upper row) and bright field overlaid image of cells (bottom row). Control: cells incubated with Ac4ManNAc. (Scale bar: 25 μm)
Microscopic Analysis of Sialylconjugates Trafficking by Fluorescence Labeling in Live Cells
To observe the fluorescence labeled sialylconjugates in cells dependent on different time, CL1-5 cells were seeded on chamber slide (2.5×104 cells/0.5 mL per wells) and incubated in culture medium (RPMI-1640 supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM L-glutamine and 1 mM sodium pyruvate) with 500 μM of azido-sugar (peracetylated N-azidoacetylmannosamine, Ac4ManNAz) for 1 h. Cells were washed three times with PBS and then incubated in culture medium. After 3 h, 5 h and 8 h, cells were incubated with 100 μM of coumOCT (101) in PBS with 10% DMSO for 30 min at 37° C. For fluorescence imaging of live cells, experiments were carried out using a confocal microscope (TCS-SP5-MP-SMD, Leica) equipped with an incubator to keep the cells in culture conditions.
For comparing the localization of sialylconjugates, probe-labeled cells were washed with PBS, fixed with 3% paraformaldehyde in PBS at room temperature for 20 min, permeablized with 0.2% Triton X-100 in PBS at room temperature for 20 min, and blocked with 3% bovine serum albumin in PBS at room temperature for 30 min. Cells were further stained with anti-GRASP65 followed by Cy3-conjugated anti-rabbit for Golgi.
FIG. 23 describes fluorescence imaging of glycoconjugates trafficking using coumOCT (101) of SPAAC in cells. CL1-5 cells were incubated with 500 μM of Ac4ManNAz for 1 h and subsequently washed with PBS buffer to remove excess Ac4ManNAz. The sugar-treated cells were incubated in culture medium for 3 h, 5 h and 8 h, and then labeled with 100 μM of probe 101 for 0.5 h under SPAAC conditions, respectively. The Golgi was labeled with anti-GRASP65 followed by Cy3-conjugated anti-rabbit. (Scale bar: 10 μm)
Example 7
Dual-labeling Experiment
A dual-labeling experiment was performed using SPAAC in combination with CuAAC for the concurrent fluorescence labeling of two different metabolically incorporated glycoconjugates. Peracetylated N-acetylmannosamine (Ac4ManNAc) and peracetylated N-acetylglucosamine (Ac4GlcNAc) were employed as the control sugars. ManNAc is converted metabolically to sialic acid that is found as a terminal monosaccharide of glycoproteins and glycolipids in cell, whereas GlcNAz is an internal monosaccharide that abundant in the N- and O-linked glycans produced in the endoplasmic reticulum and Golgi. CL1-5 cells were incubated in the presence of both alkyne-containing sugar (Ac4ManNAl) and azido-containing sugar (Ac4GlcNAz) for three days. The cells were stained by SPAAC with coumOCT (for azido sugars), followed by CuAAC with AzBOCEt6j (for alkynyl sugars), and investigated by confocal microscopy (FIG. 24).
The cells treated both azido- and alkynyl-sugars showed a distinct pattern (FIG. 24a, 24b, 24c) in both fluorescence channels, whereas omission of either sugar resulted in no labeling in the corresponding channel (FIG. 24d, 24e, 24f and FIG. 24g, 24h, 24i). Interestingly, the imaging experiments revealed that ManNAl and GlcNAz labeled species exhibited considerably at the same localization, possibly in the Golgi. In addition, the expressed alkynyl-labeled sialylated glycoconjugates were observed clearly on the cell surface and partially in the cytosol on AzBOCEt labeling, whereas the GlcNAz-labeled glycans were shown only in the cytosol using coumOCT probe. Although sialic acid is usually attached to N- and O-linked glycans by terminal glycosylation, incorporation of unnatural GlcNAz to glycoproteins may affect the specificity and efficiency of glycosyltransferase for further glycosylations. Thus, the immature aberrant glycoproteins may be transported to cytosol for degradation. These findings validate that coumOCT and AzBOCEt can be employed as fluorescence-forming probes for simultaneous detection of azido- and alkyne-labeled metabolically incorporated glycoconjugates within a single cell by SPAAC and CuAAC triazole formation chemistries.
FIG. 24A, 24B shows dual fluorescence labeling in cells with coumOCT (1), AzBOCEt and imaging by confocal microscopy. (A) Illustration of the cell labeling experiments using Ac4ManNAl, Ac4GlcNAz, 101 and AzBOCEt. CL1-5 cells were incubated with 100 μM of Ac4ManNAl and Ac4GlcNAz or control sugars (Ac4ManNAc and Ac4GlcNAc) for 3 days, which were treated with 100 μM of probe 101 for 0.5 h under SPAAC conditions, and then incubated with 0.1 μM of AzBOCEt for 1 h under CuAAC conditions. (B) Dual fluorescence imaging in CL1-5 cells. These glycoconjugates were labeled with probe 101 (cyan) for azido-containing glycoconjugates and AzBOCEt (green) for alkyne-containing glycoconjugates. (Scale bar: 10 μm)
Microscopic Analysis of Dual Fluorescence Labeling in Cells
CL1-5 cells were seeded on chamber slide (2.5×104 cells/0.5 mL per wells) and incubated in culture medium (RPMI-1640 supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM L-glutamine and 1 mM sodium pyruvate) for 3 days with either 100 μM of alkynyl-sugar (peracetylated alkynyl-N-acetylmannosamine, Ac4ManNAl or azido-sugar (peracetylated N-azidoacetylglucosamine, Ac4GlcNAz) or both or without sugar as negative control. Cells were washed three times with PBS and then incubated with 100 μM of coumOCT (101) in PBS with 10% DMSO for 30 min at 37° C. After three washes with PBS with 10% DMSO, followed by fixed with 3% paraformaldehyde in PBS at room temperature for 20 min, the cells were incubated with 0.1 μM of AzBOCEt, 100 μM of ligand, 1 mM of CuSO4, and 2 mM of sodium ascorbate in PBS with 50% ethanol at room temperature for 1 h and fluorescence images of cell were carried out using a confocal microscope (TCS-SP5-MP-SMD, Leica).
Example 8
Stability of Compound (101) and Compound (111) in Presence of 3-Mercaptopropionic Acid
A potential problematic side reaction of cyclooctynes is the addition with proteinic or endogenous thiols to the corresponding vinyl sulfides. To test whether potential non-specific stainings of 101 and the corresponding triazole 111 may occur due to additions of the SH groups to the triple bond or double bond by proteinic or endogenous thiols, the experiment was performed by incubation of 101 and 111 with 3-mercaptopropionic acid in CDCl3 (FIGS. 25 and 26). The NMR spectral analyses suggested that 101 and 111 were inactive to the thiol-yne or thiol-ene addition with 3-mercaptopropionic acid. These results show that the application of 101 as a fluorescence-forming probe allows the surprising detection of metabolically incorporated glycoconjugates in living cells without non-specific staining by thiol-yne addition, and the formed triazoles would not undergo thiol-ene addition to quench fluorescence.
A solution of compound 101 and compound 111 (25 mM in CDCl3) was incubated with a solution of 3-mercaptopropionic acid (32 mM in CDCl3) at room temperature (25° C.). The reaction was monitored by 1H-NMR analysis over a period of 24 h. 1H-NMR analysis suggested that compound 101 and compound 111 have high stability in the presence of thiols due to no substitution effect on compound 101 and compound 111 with 3-mercaptopropionic acid by 1H-NMR analysis (FIG. 25 and FIG. 26).
FIG. 25 show 1H-NMR spectrum in CDCl3 of compound 101 and 3-mercaptopropionic acid alone, and 1H-NMR spectrum of compound 101 was treated with 3-mercaptopropionic acid at 25° C. for 0 h, 2 h and 24 h.
FIG. 26 shows 1H-NMR spectrum in CDCl3 of compound 111 and 3-mercaptopropionic acid alone, and 1H-NMR spectrum of compound 111 was treated with 3-mercaptopropionic acid at 25° C. for 24 h.
This disclosure describes a new SPAAC-based fluorescence-forming probe coumOCT (101) for real-time imaging in living cells under no-wash and no-fixation conditions.
The SPAAC reactions of 101 with benzyl azide and N-azidoacetylmannosamine proceeded with a rate constant of 0.012 M−1s−1 in CD3CN and 0.010 M−1s−1 in a solution of CD3OD-D2O (5:1, v/v), respectively. The triazole products 111 and 112 showed 20-fold increase in quantum yield (Φf=0.23 and Φf=0.21) compared to unreacted 101. Furthermore, this disclosure establishes that 101 is a fluorescence turn-on probe for imaging azido-containing glycoconjugates in living cells. The SPAAC reaction is spontaneous and no washing steps are needed. Moreover, probe 101 is nontoxic and cell-permeable with no problem of background labeling, which allows the simultaneous labeling of two different sugars in combination with AzBOCEt under CuAAC. This disclosure represents a significant advance in cell imaging, and should be potentially applicable to real-time detection of biochemical events in vivo.
Example 9
Synthesis of CoumFSA (601)
Sialic acid (Neu5Ac) was treated with acetyl chloride to give the chloride intermediate in more than 90% yield according to 1H NMR analysis. The crude product was dissolved in pyridine and heated to 50° C., followed by concentration and trituration to remove pyridine hydrochloride, giving the glycal 201 with 78% overall yield from sialic acid. Neu5Boc2en 203 was synthesized from compound 201 by treating it with Boc2O to produce N-acetyl-N-Boc protected product 202, which was deacetyled under Zemplén condition followed by acetylation provided N-Boc protected compound 203. Bromohydroxylation of 203 with N-bromosuccinimide (NBS) and water in MeCN at 80° C. gave the bromohydrins 204a and 204b (91%; 204a/204b=3.1:1). Treatment of 204a with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) gave the epoxide 205 (86%). Glycosyl bromide 206 with a hydroxyl group at C-3 was prepared from 205. The alpha-glycoside 207 was obtained in 52% yield with sodium methylumbelliferone in anhydrous DMF. After removal of the Boc group of 207 with trifluoroacetic acid (TFA) in water, the obtained amine salt was treated with 4-pentynoic acid in the presence of base DIPEA and coupling reagent HBTU in DMF to afford the alkynyl product 208. The fluoro compound 209 was carried out by conversion of 208 into the triflate allowed reaction with tris(dimethylamino)sulfonium difluorotrimethylsilicate (TASF) in refluxing THF. Deprotection of 209 under alkaline condition produced CoumFSA (601) in 75% yield after purification on a reverse-phase column.
Example 10
Synthesis of DFSA (501)
Fluorohydroxylation of compound 203 with Selectfluor in aqueous MeNO2 at room temperature gave the fluorohydrins 210a and 210b (59%; 210a/210b=1.3:1). Treatment of 210a with diethylaminosulfur trifluoride (DAST) gave the difluoro compounds 211a and 211b (75%; 211a/211b=5.3:1). After removal of the Boc group of 211a with trifluoroacetic acid (TFA) in water, the obtained amine salt was treated with 4-pentynoic acid in the presence of base DIPEA and coupling reagent HBTU in DMF to afford the alkynyl product 212. Deprotection of 212 under alkaline condition produced DFSA (501) in 55% yield after purification on a reverse-phase column.
In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The present disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
Furthermore, the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the present disclosure, or aspects of the present disclosure, is/are referred to as comprising particular elements and/or features, certain embodiments of the present disclosure or aspects of the present disclosure consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the present disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the present disclosure can be excluded from any claim, for any reason, whether or not related to the existence of prior art.
Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present present disclosure, as defined in the following claims.
REFERENCES
Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. “A stepwise Huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes.” Angew. Chem. Int. Ed. 2002, 41, 2596-2599.
Zhou, Z.; Fahrni, C. J. A “Fluorogenic probe for the copper(I)-catalyzed azide-alkyne ligation reaction: Modulation of the fluorescence emission via 3(n,π*)-1(π,π*) Inversion.” J. Am. Chem. Soc. 2004, 126, 8862-8863.
Sivakumar, K.; Xie, F.; Cash, B. M.; Long, S.; Barnhill, H. N.; Wang, Q. “A fluorogenic 1,3-dipolar cycloaddition reaction of 3-azidocoumarins and acetylenes.” Org. Lett. 2004, 24, 4603-4606.
Prescher, J. A.; Bertozzi, C. R. “Chemistry in living systems.” Nat. Chem. Biol. 2005, 1, 13-21.
Sawa, M.; Hsu, T.-L.; Itoh, T.; Sugiyama, M.; Hanson, S. R.; Vogt, P. K.; Wong, C.-H. “Glycoproteomic probes for fluorescent imaging of fucosylated glycans in vivo.” Proc. Nat. Acad. Sci. U.S.A., 2006, 103, 12371-12376.
Hsu, T.-L.; Hanson, S. R.; Kishikawa, K.; Wang, S.-K.; Sawa, M.; Wong, C.-H. “Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells.” Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2614-2619.
Loudet, A.; Burgess, K. “BODIPY dyes and their derivatives: syntheses and spectroscopic properties.” Chem. Rev. 2007, 107, 4891-4932.
Wu, P.; Fokin, V. V. “Catalytic azide-alkyne cycloaddition: reactivity and Applications.” Aldrichim. Acta 2007, 40, 7-17.
Ulrich, G.; Ziessel, R.; Harriman, A. “The chemistry of fluorescent bodipy dyes: Versatility unsurpassed.” Angew. Chem. Int. Ed. 2008, 47, 1184-1201.
Li, L.; Han, J.; Nguyen, B.; Burgess, K. “Syntheses and spectral properties of functionalized, water-soluble BODIPY derivatives.” J. Org. Chem. 2008, 73, 1963-1970.
Xie, F.; Sivakumar, K.; Zeng, Q. B.; Bruckman, M. A.; Hodges, B.; Wang, Q. “A fluorogenic ‘click’ reaction of azidoanthracene derivatives.” Tetrahedron 2008, 64, 2906-2914.
Baskin, J. M.; Amacher, S. L.; Bertozzi, C. R. “In vivo imaging of membrane-associated glycans in developing zebrafish.” Science 2008, 320, 664-667.
Best, M. D. “Click chemistry and bioorthogonal reactions: unprecedented selectivity in the labeling of biological molecules.” Biochemistry 2009, 48, 6571-6584.
Sletten, E. M.; Bertozzi, C. R. “Bioorthogonal chemistry: fishing for selectivity in a sea of Functionality.” Angew. Chem. Int. Ed. 2009, 48, 6974-6998.
Li, J.; Hu, M.; Yao, S. Q. “Rapid synthesis, screening, and identification of xanthone and xanthene-based fluorophores using click chemistry.” Org. Lett. 2009, 11, 3008-3011.
Le Droumaguet, C.; Wang, C.; Wang, Q. “Fluorogenic click reaction.” Chem. Soc. Rev. 2010, 39, 1233-1239.
Qi, J.; Han, M.-S.; Chang, Y.-C.; Tung, C.-H. “Developing visible fluorogenic ‘click-on’ dyes for cellular imaging.” Bioconjugate Chem. 2011, 22, 1758-1762.
Chao, W.; Fang, X.; Nisaraporn, S.; Jian, S.; Qian, W. “Tuning the optical properties of BODIPY dye through Cu(I) catalyzed azide-alkyne cycloaddition (CuAAC) reaction.” Sci. China Chemistry 2012, 55, 125-130.
Boens, N.; Leen, V.; Dehaen, W. “Fluorescent indicators based on BODIPY.” Chem. Soc. Rev. 2012, 41, 1130-1172.
Shieh, P.; Hangauer, M. J.; Bertozzi, C. R. “Fluorogenic azidofluoresceins for biological imaging.” J. Am. Chem. Soc. 2012, 134, 17428-17431.
Kamkaew, A.; Lim, S. H.; Lee, H. B.; Kiew, L. V.; Chung, L. Y.; Burgess, K. “BODIPY dyes in photodynamic therapy.” Chem. Soc. Rev. 2013, 42, 77-88.
Herner, A.; Nikić, I.; Kállay, M.; Lemke, E. A.; Kele, P. “A new family of bioorthogonally applicable fluorogenic labels.” Org. Biomol. Chem. 2013, 11, 3297-3306.
Chauhan, D. P.; Saha, T.; Lahiri, M.; Talukdar, P. “BODIPY based ‘click on’ fluorogenic dyes: application in live cell imaging.” Tetrahedron Lett. 2014, 55, 244-247.

## Claims

1. A compound of Formula (I):




or a pharmaceutically acceptable salt thereof,
wherein:
B is boron;
each instance of G1, G2,G3, G5, G6, G7 and G8 is independently hydrogen, optionally substituted C1-6 alkyl, optionally substituted C1-6 alkenyl, optionally substituted C1-6 alkynyl, optionally substituted aryl, optionally substituted acyl, —ORA, —CH2ORA, —OC(O)RA, —SRA, —N(RB)2, —N(RA)C(O)RA, —C(O)N(RB)2, —CN, —NO2, —C(O)RA, —C(O)ORA, —S(O)RA, —SO2RA, —SO3RA, —SO2N(RB)2, and —NHSO2RB;
each RA is independently selected from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl;
each RB is independently selected from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl, or two RB taken together with the intervening nitrogen form a heterocycle;
each instance of G4a and G4b is fluoro, alkyl, alkoxy, aryloxy, or alkynyl;
wherein alkyl and alkoxy groups are unbranched, saturated, and have 1-4 carbon atoms; aryl groups and aryl groups of aryloxy can be either carbocyclic aryl or heterocyclic aryl; carbocyclic aryl groups have a total of 6-20 carbon atoms, including carbon atoms of substituents; heterocyclic aryl groups have a total of 5-20 carbon atoms, including carbon atoms of substituents; carboalkoxy groups are alkyl esters of a carboxylic acid wherein alkyl groups are as defined above; each alkyl, aryl, alkoxy, aryloxy, benzo, and carboalkoxy, independently, may be unsubstituted or substituted with one or more substituent; alkyl substituents are halo, hydroxyl, amino, or aryl; aryl substituents are halo, hydroxyl, amino, alkyl, aryl, nitro, or carboxyl; and halo substituents are fluoro or chloro; and
n is 0, 1, 2, 3, or 4.

2. The compound of claim 1, wherein G1 and G3 are methyl.

3. The compound of claim 1, wherein G5 and G7 are methyl.

4. The compound of any one of claims 1-3, wherein the compound is of Formula (II):




or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

5. The compound of claim 4, wherein the compound is described by a formula selected from Table 1.

6. The compound of claim 4, wherein the compound is selected from the group consisting of:

7. A method for imaging an alkyne-containing molecule comprising
(a) contacting a compound of claim 1 with a sample containing the alkyne-containing molecule under conditions for ligation of the compound to an alkyne group of the molecule to form a triazole product; and
(b) measuring a fluorescent signal released from the triazole product.

8. The method of claim 7, wherein the contacting step is carried out in the presence of a metal catalyst.

9. The method of claim 8, wherein the metal catalyst is copper(I).

10. The method of claim 7, wherein the compound is covalently linked to the alkyne group.

11. The method of claim 7, wherein the sample contains cells and the alkyne-containing molecule is located on the cell surface or inside the cells.

12. The method of claim 7, wherein the compound is described by a formula selected from Table 1.

13. The method of claim 7, wherein the compound is selected from a compound according to claim 6.

14. The method of any one of claims 7-11, wherein the alkyne-containing molecule is a biomolecule.

15. The method of claim 14, wherein the biomolecule is a DNA, RNA, protein or glycan.

16. The method of claim 14, wherein the biomolecule is located on or near the surface of a cell.

17. The method of claim 14, wherein the biomolecule is an intracellular biomolecule.

18. A method for detecting an alkyne-containing molecule in a sample, comprising:
(a) contacting the compound of claim 1 with a sample suspected of having an alkyne-containing molecule;
(b) measuring the level of a fluorescent signal released from the sample mixture, and
(c) determining the presence of the alkyne-containing molecule in the sample, wherein an enhanced fluorescent signal as compared to a level of the fluorescent signal in the absence of the molecule indicates presence of the alkyne-containing molecule.

19. The method of claim 18, wherein the contacting step is carried out in the presence of a metal catalyst.

20. The method of claim 19, wherein the metal catalyst is copper(I).

21. The method of any one of claims 18-20, wherein the sample contains cells and the alkyne-containing molecule is located on or near the surface of a cell or inside a cell.

22. The method of claim 18, wherein the compound is described by a formula selected from Table 1.

23. The method of claim 18, wherein the compound is selected from a compound according to claim 6.

24. The method of claim 18, wherein the alkyne-containing molecule is a biomolecule.

25. The method of claim 24, wherein the biomolecule is a DNA, RNA, protein or glycan.

26. The method of claim 24, wherein the biomolecule is located on or near the surface of a cell.

27. The method of claim 24, wherein the biomolecule is an intracellular biomolecule.

28. The method of claim 7 or 18 wherein the method comprises the use of the compound for dual fluorescence imaging in a cell.