FLUOROGENIC CYANINE CARBAMATES

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of the earlier filing date of U.S. Provisional Application No. 63/165,511, filed March 24, 2021, which is incorporated by reference herein in its entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under project number Z01 ZIA BC011506 by the National Institutes of Health, National Cancer Institute. The government has certain rights in the invention.

FIELD

Fluorogenic cyanine carbamates are disclosed, as well as methods of making and using the Anorogenic cyanine carbamates.

BACKGROUND

Selectively activating a Auorescent signal is a powerful approach to interrogate biological processes. Fluorogenic probes are powerful tools with significant potential to non-invasively monitor enzymatic processes and other stimuli in real time in living organisms. A common tactic uses multi-chromophore systems that rely on quenching through Fluorescence Resonance Energy Transfer (FRET) and related mechanisms. Another approach uses Auorogenic probes, where the change in signal results from chemical transformations to the chromophore itself. The latter often provides improved tum-ON ratios and benefits from requiring only a single chromophore. The most broadly used Auorogenic chemistry is based on derivatization of the coumarin, rhodamine, and hybrid cyanine scaffolds, which absorb and emit light in the visible to far-red range. To carry out such experiments in living organisms, it would be desirable to have tum-ON probes that absorb and emit long wavelength near- infrared (NIR) light (>700 nm), which is less attenuated by tissue. However, only systems based on FRET pairs or self-quenching have routinely been applied in this range.

Antibody-drug conjugates (ADCs) are a rapidly emerging therapeutic platform. The chemical linker between the antibody and the drug payload plays an essential role in the efficacy and tolerability of these agents. New methods that quantitively assess cleavage efficiency in complex tissue settings could provide valuable insights into the ADC design process. ADC activity generally requires lysosomal processing of a linker domain to release the active payload. Consequently, the linker component should be stable in circulation, but selectively cleaved following target binding and internalization - a significant chemical challenge. ADCs are conventionally assessed by examining tumoricidal activity and toxicity profiling. While these methods are important benchmarks, they provide only indirect insights into the site and mechanism of drug release. Enzyme-linked immunosorbent assays (ELISAs) are also broadly employed, but only determine the blood-pool concentration and biodistribution of the antibody component. Radiolabeling methods can provide important insights regarding mAb localization, but are costly and do not directly report on the linker cleavage step. Optical imaging has the potential to provide critical insights to the ADC design and optimization process. Prior efforts using stimuli-responsive fluorophores with conventional visible wavelengths have quantified payload processing and internalization kinetics in cellular imaging experiments (Sorkin et al., cell Chem Biol 2019, 26(12):1643-1651e4; Lee et al., Bioconjug Chem 2018, 29(7):2468-2477); Knewtson et al., ACS Omega 2019, 4(7): 12955-19268). However, these probes are not suitable for applications in deep tissue due to the poor penetration depth of wavelengths in the visible region.

Thus, a need exists for Anorogenic probes comprising a single chromophore that has a high turn- ON ratio and emits NIR light.

SUMMARY

Embodiments of cyanine carbamates are disclosed as well as methods of making and using the cyanine carbamates. In some embodiments, the cyanine carbamate is a compound according to Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof: where L is substituted or unsubstituted C _n conjugated alkenyl, each carbon having sp ² hybridization, where n is 7, 3, 5, or 9; R ¹ is substituted aryl or substituted heteroaliphatic, and the bond between the nitrogen atom and -C(O)-O-CH2-R ¹ is cleavable by an enzyme or chemical trigger; Y ¹ and Y ² independently are C(R ^b )2, N(R ^C ), S, O, or Se, wherein each R ^b independently is aliphatic, H, deuterium, aryl, -(OCH2CH2) _X OH where x is an integer > 2, trityl, or a group comprising a conjugatable moiety, a targeting agent, or a drug, and each R ^c independently is H, deuterium, aliphatic, or heteroaliphatic; R ³ and R ⁴ independently are H, aliphatic, amino, a sulfonate-containing group, or a group comprising a conjugatable moiety, a targeting agent, or a drug, or R ³ and Y ¹ together with the atoms to which they are bound form a 6-membered aryl ring, and R ⁴ and Y ² together with the atoms to which they are bound form a 6-membered aryl ring; R ⁵ -R ¹⁰ independently are H, a sulfonate-containing group, aliphatic, heteroaliphatic, amino, or a group comprising a conjugatable moiety, a targeting agent, or a drug; and R ^x is absent, aliphatic, heteroaliphatic, aryl, heteroaryl, alkylaryl, alkylheteroaryl, or H, wherein if R ^x is present, the nitrogen to which R ^x is attached has a +1 charge and a counterion X’ is also present.

In some embodiments, , where R ² and R ² independently are aryl, heteroaryl, heterocycloaliphatic,

H, halo, -OR ^a , -NR ^a 2, or a group comprising a conjugatable moiety, a targeting agent, or a drug, where each R ^a independently is H, halo, alkyl, or aryl; and (i) R ¹¹ together with R ¹² and the atoms to which they are bound forms a 5-7 membered cycloaliphatic or heterocycloaliphatic ring and, if present, R ¹¹ is H, aliphatic, heteroaliphatic, or a group comprising a conjugatable moiety, a targeting agent, or a drug, or (ii) R ¹¹ together with R ¹² and the atoms to which they are bound forms a 5-7 membered cycloaliphatic or heterocycloaliphatic ring and R ¹¹ is H, aliphatic, heteroaliphatic, or a group comprising a conjugatable moiety, a targeting agent, or a drug or (iii) each of R ¹¹ , R ¹¹ , and R ¹² independently is H, aliphatic, heteroaliphatic, or a group comprising a conjugatable moiety, a targeting agent, or a drug, or (iv) R ¹¹ , R ¹¹ , and R ¹² together with the atoms to which they are bound form a fused cycloaliphatic or heterocycloaliphatic ring system, each ring comprising 5-7 members. In certain embodiments, L is and A' independently are absent, -CHR ^d -, -CH2CHR ¹ -, -CHR ^d CH2-, or -NR ^d -, where R ^d is H, alkyl, aryl, or a group comprising a conjugatable moiety, a targeting agent, or a drug. In any of the foregoing or following embodiments, the compound may have a structure according to Formula Ila:

In any of the foregoing or following embodiments, Y ¹ and Y ² independently may be C(R ^b )2, where each R ^b independently is aliphatic or aryl; R ² and R ² independently may be phenyl, H, halo, -OR ^a , -NR ^a 2, or a group comprising a conjugatable moiety, a targeting agent, or a drug; R ⁵ and R ⁸ independently may be -SO3", , , H, or -OCH3; A and A' independently may be -N(CH3)- or -CH2-; and R ³ , R ⁴ , R ⁶ , R ⁷ , R ⁹ , and R ¹⁰ may be H. In some embodiments, A or A' is -N(CH3)-. In some embodiments, Y ¹ and Y ² are -C(CH3)2-. In an independent embodiment, R ³ and Y ¹ together with the atoms to which they are bound form a 6-membered aryl ring; and R ⁴ and Y ² together with the atoms to which they are bound form a 6-membered aryl ring.

In some embodiments, a pharmaceutical composition comprise a cyanine carbamate as disclosed herein, or stereoisomer thereof, and a pharmaceutically acceptable carrier.

In some embodiments, a method of using a cyanine carbamate includes contacting a biological sample with a compound as disclosed herein; exposing the compound to an enzyme or a chemical trigger capable of cleaving -C(O)OCH2R ¹ from the compound to provide a cleaved compound; exposing the cleaved compound to a pH less than 8; subsequently exposing the cleaved compound to light having a wavelength greater than 700 nm; and detecting fluorescence from the cleaved compound. In some embodiments, the biological sample comprises the enzyme, and exposing the compound to the enzyme comprises contacting the biological sample comprising the enzyme with the compound; or the biological sample comprises the chemical trigger, and exposing the compound to the chemical trigger comprises contacting the biological sample comprising the chemical trigger with the compound; or exposing the compound to the enzyme or chemical trigger comprises adding the enzyme or the chemical trigger to the biological sample and the compound. In any of the foregoing or following embodiments, the biological sample may comprise an environment having a pH less than 7, thereby exposing the cleaved compound to the pH less than 7. In any of the foregoing or following embodiments, the biological sample may comprise, or be suspected of comprising, a target; the compound comprises a group comprising a targeting agent that binds to the target; and detecting fluorescence from the cleaved compound indicates presence of the target in the biological sample. In some embodiments, the targeting agent is an antibody.

In any of the foregoing or following embodiments, contacting the biological sample with the compound may be performed in vivo. In some embodiments, contacting the biological sample in vivo comprises administering the compound or a pharmaceutical composition comprising the compound to a subject; and detecting fluorescence from the cleaved compound is performed via in vivo imaging.

The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a reaction scheme showing cleavage of a cyanine carbamate (CyBam-N ) to produce a norcyanine (Sulfo-NorCy7) and subsequent indolenine protonation of the norcyanine (NorCy7-[H ⁺ ]).

FIG. 2 shows absorbance spectra of CyBam-Na, Sulfo-NorCy7, and Sulfo-NorCy7-[H ⁺ ].

FIGS. 3A and 3B show the effects of pH on absorbance (3A) and fluorescence (3B) spectra of CyBam-Na and Sulfo-NorCy7.

FIGS. 4A and 4B are graphs showing conversion of CyBam-Na to Sulfo-NorCy7 by reaction with triphenyl phosphine at pH 5.2 over time (4 A) and the increased fluorescence after the conversion occurred (4B).

FIGS. 5A-5F are graphs comparing properties of two xanthene cyanines. FIGS. 5A and 5B show the absorbance spectra at pH 7.2 (5 A) and 4.5 (5B). FIGS. 5C and 5D show the emission spectra at pH 7.2 (5C) and 4.5 (5D) with an excitation wavelength of 640 nm. FIGS. 5E and 5F show the emission spectra at pH 7.2 (5E) and 4.5 (5F) with an excitation wavelength of 690 nm.

FIGS. 6A-6C are graph showing fluorescence intensity of CyBam-y-Glu (20 pM) after incubation with increasing concentrations of y-glutamyl transpeptidase (GGT, 0-400 U/L) (6A), rate of activation in the presence of GGT (0-400 U/L in PBS (pH 7.4) for 30 minutes (6B), and kinetics of Anorogenic probe activations at different concentrations of CyBam-y-Glu (2.5-50 pM) in PBS (pH 7.4, 37 °C) with 100 U/L GGT (6C). Mean ± SD of Auorescent signal from three independent experiments. FIG. 7 is a graph showing activation of CyBam-y-Glu (20 pM) after incubation with GGT (100 U/L), leucine aminopeptidase (LAP; 800 U/L), pig-liver esterase (PLE; 800 U/L) at 37 °C for 30 mins in PBS pH 7.4 followed by pH adjustment to pH 5.2. Cathepsin B (CatB; 2.5 mg) was used in acetate buffer pH 5.2 for 30 mins at 37 °C.

FIG. 8 is a graph showing the inhibition of CyBam-y-Glu in the presence of inhibitors DON and GGsTop (1 mM). Mean ± SD of absorbance signal from at least three independent experiments.

FIG. 9 is a series of images showing cellular uptake of Sulfo-NorCy7 (20 pM, red channel) in SHIN-3 cells after 4 h incubation followed by organelle staining (green channel). The upper panels show uptake in mitochondria (Mitotracker Green) and the lower panels show uptake in lysosomes (Lysotracker Green).

FIG. 10 shows confocal images of fluorescence activation of CyBam-y-Glu (20 pM) and CyBam-N.C. (20 pM) showing in vitro uptake of CyBam-y-Glu in SHIN-3 cells.

FIG. 11 shows quantification of fluorescent signal after incubation of CyBam-y-Glu (20 mM) in presence of GGT inhibitors (DON, GGsTop) and CyBam-N.C. in SHIN-3 cells using flow cytometry. Geometric mean fluorescent intensity (± SD) of fluorescent signal in the cells is shown (n = 6 independent experiments).

FIG. 12 shows in vivo imaging of a SHIN-3-ZsGreen metastatic tumor model at 3h after injection with CyBam-y-Glu (30 nmol). Green and red pseudo colors are used to represent signal from the GFP and Cy7 channels, respectively. Fluorescent line graphs show correlation between fluorescent signal from GFP and Cy7 channel across the metastatic tumor in two different regions (A and B). Data points are displayed as mean ± SD, and the p-values were evaluated by student t-test (*** p-value < 0.001).

FIG. 13 shows brightfield, GFP channel, Cy7 channel, and merged images of the SHIN- 3 -Zs Green metastatic tumor model at Ih after injection with CyBam-y-Glu (30 nmol). Green and red pseudo colors are used to represent signal from the GFP and Cy7 channels respectively.

FIG. 14 shows brightfield, GFP channel, Cy7 channel, and merged images of the SHIN- 3 -Zs Green metastatic tumor model at 6h after injection with CyBam-y-Glu (30 nmol). Green and red pseudo colors are used to represent signal from the GFP and Cy7 channels respectively.

FIG. 15 shows brightfield and Cy7 channel images of mice 4 h after injection with CyBam-y-Glu and CyBam-N.C (25 nM). Signal from tumors, liver and background in mice are shown in red, blue and magenta dotted circles. Green pseudo colors are used to represent fluorescent signal from and Cy7 channel.

FIGS. 16A and 16B are graphs quantifying the fluorescence signal emitting from the tumors of FIG. 15 (16A) and tumor to background ratio in the mice (16B).

FIGS. 17A and 17B are graphs showing absorbance and fluorescence spectra of A-Me-NorCy7 (5 pM) in PBS pH 7.20 and acetate buffer pH 4.25 (ex. 690 nm) (17A), and determination of pKa in buffers ranging from pH 3.5 - pH 7.4 (17B).

FIG. 18 is graph showing flow cytometry quantification of in vitro uptake of norcyanine heptamethine cyanines (5 mM) in MDA-MB-468 after 1 h incubation.

FIGS. 19A-19F show confocal microscopy images of NorCy7 compounds (5 pM) in MDA-MB-468 cells after 6 h of incubation: A-Me-Pip-NorCy7 (19A), A-Me-NorCy7 (19B), H-NorCy7 (19C), Pip-NorCy7 (19D), OMe-NorCy7 (19E) and Sulfo-NorCy7 (19F). Nucleus and fluorescent signal from probes are shown in the left and center images, respectively. Confocal imaging carried out at 63X oil immersed lens with 1.4 NA. The images were processed with identical brightness/contrast using Fiji.

FIGS. 20A and 20B show organelle localization of N-Me-NorCy7 (5 pM; red channel) in MDA-MB-468 cells after 6 h incubation followed by organelle staining (green channel) with lysosome (Lysotracker Green; 20A) and mitochondria (Mitotracker Green; 20B). Nucleus stained using NucBlue (blue channel). Confocal imaging carried out at 63X oil immersed lens with 1.4 NA. The images were processed with identical brightness/ contrast using Fiji. Scale bar 10 pm.

FIG. 21 is a table calculating degree of labeling (DOL) for Pan-Sulfo-CyBam and Pan-N-Me CyBams cathepsin (VC) and non-cleavable (NC) linkers.

FIG. 22 shows chemical structures of two CyLBam antibody conjugates and two CyBam antibody conjugates.

FIG. 23 shows confocal images (63X) of Pan-VC-CyLBam, Pan-NC-CyLBam, Pan-VC-CyBam, and Pan-NC-CyBam in MDA-MB-468 (EGFR+) after 24 h incubation (23A). Fluorescent signal from the probe and nucleus (Hoechst) is shown in red and blue, respectively. Scale bar 10 pm.

FIG. 24 is a graph showing flow cytometry quantification of in vitro uptake in MDA-MB-468 (EGFR+) and MCF-7 (EGFR-) after 24 h incubation. All antibody conjugates were labeled at DOL 4. Geometric mean fluorescent intensity (± SD) of fluorescent signal in the cells is shown (n = 4 independent experiments; -10,000 cells counted). FIG. 25 is confocal microscopy images of Pan-VC-CyLBam (A) Pan-NC-CyLBam (B), Pan-VC-CyBam (C) and Pan-NC-CyBam (D) probes in MDA-MB-468 cells. The probes were incubated for 24 h (50 pg; DOL 4). Nucleus and Fluorescent signal from probes are pseudo colored in blue and red respectively. Confocal imaging carried out at 63X oil immersed lens with 1.4 NA. The images were processed with identical brightness/contrast using Fiji. Scale bar 10 pm.

FIG. 26 is confocal microscopy images of Pan-VC-CyBam probe (50 pg; DOL 4) in MDA-MB-468 (A) and MCF-7 (B) cells after 24 h of incubation. Nucleus and Fluorescent signal from probes are pseudo colored in blue and red respectively. Confocal imaging carried out at 63X oil immersed lens with 1.4 NA. The signal in red channel is enhanced to visualize the fluorescent signal from the probe. The images were processed with identical brightness/contrast using Fiji. Scale bar 8 pm.

FIG. 27 is confocal microscopy images of Pan-VC-CyLBam probe (50 pg; DOL 4) in MDA-MB-468 (A) and MCF-7 cells (B); and Pan-NC-CyLBam probe (50 pg; DOL 4) in MDA-MB-468 (C) and MCF-7 cells (D) after 24 h of incubation. Nucleus and Fluorescent signal from probes are pseudo colored in blue and red respectively. Confocal imaging carried out at 63X oil immersed lens with 1.4 NA. The images were processed with identical brightness/contrast using Fiji. Scale bar 10 pm.

FIGS. 28A and 28B are in vivo imaging system fluorescent images 48 h post injection for probes (200 pg; DOL 4) intravenously injected in a xenograft model of female athymic nude mice implanted with MDA-MB-468 tumors (n = 3; tumors are highlighted in dotted circles) (28A); and a graph showing tumor-to-background ratios, the fluorescent signal of the tumor relative to an equal area in the neck measured at different time intervals (4, 48 and 168 h) (28B). Data points are displayed as mean ± SD, and the p-values were evaluated by the Student’s t-test (*** p-value < 0.001, **** p-value < 0.0001).

FIG. 29 shows side view in vivo imaging following intravenous injection (200 pg in 100 pL PBS pH 7.2) of Pan-VC-CyBam (DOL 4) (upper images) and Pan-NC-CyBam (DOL 4) (lower images) in mice containing MDA-MB-468 xenograft tumors. Mice were imaged 4, 24, 48, 72 and 168 h time points. Tumors are shown in dotted circle. Images were captured at (ex/em filter 745/800 nm).

FIG. 30 shows ventral view in vivo imaging following intravenous injection (200 pg in 100 pL PBS pH 7.2) of Pan-VC-CyBam (DOL 4) (upper images) and Pan-NC-CyBam (DOL 4) (lower images) in mice containing MDA-MB-468 xenograft tumors. Mice were imaged 4, 24, 48, 72 and 168 h time points. Tumors are shown in dotted circle. Images were captured at (ex/em filter 745/800 nm).

FIG. 31 shows dorsal view in vivo imaging following intravenous injection (200 pg in 100 pL PBS pH 7.2) of Pan-VC-CyBam (DOL 4) (upper images) and Pan-NC-CyBam (DOL 4) (lower images) in mice containing MDA-MB-468 xenograft tumors. Mice were imaged 4, 24, 48, 72 and 168 h time points. Tumors are shown in dotted circle. Images were captured at (ex/em filter 745/800 nm).

FIG. 32 shows side view in vivo imaging following intravenous injection (200 pg in 100 pL PBS pH 7.2) of Pan-VC-CyLBam (DOL 4) (upper images) and Pan-NC-CyLBam (DOL 4) (lower images) in mice containing MDA-MB-468 xenograft tumors. Mice were imaged 4, 24, 48, 72 and 168 h time points. Tumors are shown in dotted circle. Images were captured at (ex/em filter 710/760 nm).

FIG. 33 shows ventral view in vivo imaging following intravenous injection (200 pg in 100 pL PBS pH 7.2) of Pan-VC-CyLBam (DOL 4) (upper images) and Pan-NC-CyLBam (DOL 4) (lower images) in mice containing MDA-MB-468 xenograft tumors. Mice were imaged 4, 24, 48, 72 and 168 h time points. Tumors are shown in dotted circle. Images were captured at (ex/em filter 710/760 nm).

FIG. 34 shows dorsal view in vivo imaging following intravenous injection (200 pg in 100 pL PBS pH 7.2) of Pan-VC-CyLBam (DOL 4) (upper images) and Pan-NC-CyLBam (DOL 4) (lower images) in mice containing MDA-MB-468 xenograft tumors. Mice were imaged 4, 24, 48, 72 and 168 h time points. Tumors are shown in dotted circle. Images were captured at (ex/em filter 710/760 nm).

FIG. 35 shows chemical structures of four Pan-CyLBam conjugates - Pan-AA-CyLBam, Pan-S,S-CyLBam and Pan-S,SMe2-CyLBam.

FIG. 36 is a table showing calculation of DOL of Pan-CyLBam conjugates (Pan-CyLBam: VC, AA, S,S, S,SMe2 and NC) with cathepsin and glutathione (GSH) cleavable linkers.

FIG. 37 is a graph showing quantification of in vitro uptake of the Pan-CyLBam conjugates (50 pg; DOL 4) in MDA-MB-468 (EGFR+) and MCF-7 (EGFR-) after 24 h incubation. Geometric mean fluorescent intensity (± SD) of fluorescent signal in the cells is shown (n = 4 independent experiments; -10,000 cells counted).

FIG. 38 is a graph showing quantification of fluorescent signal in MDA-MB-231 cells after 6 and 24 h incubation with the Pan-CyLBam conjugates (50 pg). Geometric mean fluorescent intensity (± SD) of fluorescent signal in the cells is shown (n = 3 independent experiments; -10,000 cells counted).

FIG. 39 shows fluorescent images following injection of the Pan-CyLBam conjugates (100 pg; DOL 4) in female athymic nude mice (n = 5) with MDA-MB-468 tumors at 48 h time point. Tumors are highlighted in dotted white circles.

FIG. 40 is a graph showing quantification of tumor-to-background ratio 48 hours post injection in the mice of FIG. 39.

FIG. 41 is a graph showing quantification of fluorescent signal of Pan-AA-CyLBam from liver and tumor in the mice of FIG. 39. Tumor-to-liver ratio (TLR) is depicted in insert. Data points are displayed as mean ± SD, and the p- values were evaluated by the Student’s t-test (** p- value < 0.01, *** p-value < 0.001).

FIG. 42 shows ventral view in vivo imaging of mice at 4, 24 and 48 h after intravenous injection of Pan- CyLBam conjugates (100 pg in 100 pL PBS pH 7.2). Pan-VC-CyLBam (row A), Pan-AA-CyLBam (row B), Pan-S,S -CyLBam (row C), Pan-S,SMe2-CyLBam (row D), and Pan-NC-CyLBam (row E). Images were captured at (ex/em filter 710/760 nm).

FIG. 43 shows side view in vivo imaging of mice at 4, 24 and 48 h after intravenous injection of Pan- CyLBam conjugates (100 pg in 100 pL PBS pH 7.2). Pan-VC-CyLBam (row A), Pan-AA-CyLBam (row B), Pan-S,S -CyLBam (row C), Pan-S,SMe2-CyLBam (row D), and Pan-NC-CyLBam (row E). Images were captured at (ex/em filter 710/760 nm).

FIG. 44 shows dorsal view in vivo imaging of mice at 4, 24 and 48 h after intravenous injection of Pan- CyLBam conjugates (100 pg in 100 pL PBS pH 7.2). Pan-VC-CyLBam (row A), Pan-AA-CyLBam (row B), Pan-S,S -CyLBam (row C), Pan-S,SMe2-CyLBam (row D), and Pan-NC-CyLBam (row E). Images were captured at (ex/em filter 710/760 nm).

FIG. 45 shows ex vivo imaging of mice at 48 h after intravenous injection of Pan-CyLBam conjugates (100 pg in 100 pL PBS pH 7.2). Pan-VC-CyLBam (row A), Pan-AA-CyLBam (row B), Pan-S,S-CyLBam (row C), Pan-S,SMe2-CyLBam (row D), and Pan-NC-CyLBam (row E). Images were captured at (ex/em filter 710/760 nm).

FIGS. 46A and 46B are graphs showing ex vivo quantification of Pan-VC-CyLBam, Pan-AA-CyLBam, Pan-S,S- CyLBam, Pan-S,SMe2-CyLBam, and Pan-NC-CyLBam. tumor to liver ratio (46 A) and tumor to muscle ratio (46B). The p-values were evaluated by Student’s t-test (ns p- value > 0.05, * p-value < 0.05, *** p-value < 0.001).

FIG. 47 is a table showing calculation of DOL of m276-SL and IgG-CyLBams with cathepsin and GSH cleavable linkers. FIG. 48 is a graph showing quantification of cellular uptake of m276-SL-S,SMe2-CyLBam, m276-SL-VC-CyLBam and m276-SL-NC-CyLBam (80 mg; DOL 4) in JIMT-1 cells following 24 h incubation. Geometric mean fluorescent intensity (± SD) of fluorescent signal in the cells is shown (n = 4 independent experiments; -10,000 cells counted).

FIG. 49 is a graph showing quantification of cellular uptake of m276-SL and IgG conjugated with VC-CyLBam, S,SMe2-CyLBam and NC-CyLBam linkers (80 pg; DOL 4) in JIMT-1 after 24 h incubation. Geometric mean fluorescent intensity ( ± SD) of fluorescent signal in the cells is shown (n = 4 independent experiments; -10,000 cells counted).

FIG. 50 is a graph showing tumor-to-background ratios at different time intervals after intravenous injection of m276-SL-S,SMe2-CyLBam and m276-SL- VC-CyLBam (100 pg; DOL - 4) in female athymic nude mice bearing orthotopic JIMT-1 tumors (n = 5).

FIG. 51 is fluorescence images of the mice of FIG. 50 72 h post injection. Tumors are highlighted in dotted circles.

FIG. 52 shows in vivo imaging of mice intravenously injected with m276-SL-VC-CyLBam (100 pg in 100 pL PBS pH 7.2) at different orientations, side view (panel A), dorsal view (panel B), and ventral view (panel C). The mice were imaged at 4, 24, 48, 72 and 168 h. Images were captured at (ex/em filter 710/760 nm).

FIG. 53 shows in vivo imaging of mice intravenously injected with m276-SL-S,SMe2-CyLBam (100 pg in 100 pL PBS pH 7.2) at different orientations, side view (panel A), dorsal view (panel B), and ventral view (panel C). The mice were imaged at 4, 24, 48, 72 and 168 h. Images were captured at (ex/em filter 710/760 nm).

FIGS. 54A-54C are graphs quantifying fluorescent signal from Pan-VC-CyLBam and Pan-NC-CyLBam (ex/em 710/760 nm): total fluorescent signal (54A), tumor to liver ratio (54B), and liver to background ratio (54C).

DETAILED DESCRIPTION

Fluorogenic cyanine carbamates are disclosed. Methods of making and using the compounds also are disclosed. The compounds include a carbamate group that may be cleaved by an enzyme or chemical trigger to produce a corresponding pH-responsive norcyanine. Following cleavage of the carbamate group, fluorescence intensity of the compound increases in a near-neutral or acidic environment (e.g., when the cleaved compound is protonated) when irradiated with targeted application of an effective quantity of light having a selected wavelength and a selected intensity to induce fluorescence, preferably in the near-infrared range. Fluorogenic tum-ON probes that use near-infrared (NIR) wavelengths (-700-900 nm) have significant potential to provide insight into dynamics and localization of biological phenomena in complex tissue settings. Disclosed herein are fluorogenic cyanine carbamates that exhibit exceptional turn-ON ratios. In some embodiments, untargeted compounds enable in vivo imaging in a metastatic tumor model. In certain embodiments, mAb-conjugated variants provide a real-time, quantitative approach to determine the site and extent of antibody-drug-conjugate linker cleavage in complex model organisms. In particular implementations, inclusion of a basic amine in the cyanine carbamate molecule dramatically improves cellular photon output, likely due to enhanced lysosomal uptake and retention.

I. Definitions and Abbreviations

The following explanations of terms and abbreviations are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, “comprising” means “including” and the singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise. The term “or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise.

Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the disclosure are apparent from the following detailed description and the claims.

The disclosure of numerical ranges should be understood as referring to each discrete point within the range, inclusive of endpoints, unless otherwise noted. Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims are to be understood as being modified by the term “about.” Accordingly, unless otherwise implicitly or explicitly indicated, or unless the context is properly understood by a person of ordinary skill in the art to have a more definitive construction, the numerical parameters set forth are approximations that may depend on the desired properties sought and/or limits of detection under standard test conditions/methods as known to those of ordinary skill in the art. When directly and explicitly distinguishing embodiments from discussed prior art, the embodiment numbers are not approximates unless the word “about” is recited.

Although there are alternatives for various components, parameters, operating conditions, etc. set forth herein, that does not mean that those alternatives are necessarily equivalent and/or perform equally well. Nor does it mean that the alternatives are listed in a preferred order unless stated otherwise.

Definitions of common terms in chemistry may be found in Richard J. Lewis, Sr. (ed.), Hawley’s Condensed Chemical Dictionary, published by John Wiley & Sons, Inc., 2016 (ISBN 978-1-118-13515-0). Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and other similar references.

In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

Aliphatic: A substantially hydrocarbon-based compound, or a radical thereof (e.g., Cetin, for a hexane radical), including alkanes, alkenes, alkynes, including cyclic versions thereof (cycloaliphatic), and further including straight- and branched-chain arrangements, and all stereo and position isomers as well. Unless expressly stated otherwise, an aliphatic group contains from one to twenty-five carbon atoms; for example, from one to fifteen, from one to ten, from one to six, or from one to four carbon atoms. An aliphatic chain may be substituted or unsubstituted. Unless otherwise specified, an aliphatic group can either be unsubstituted or substituted. An aliphatic group can be substituted with one or more substituents (up to two substituents for each methylene carbon in an aliphatic chain, or up to one substituent for each carbon of a -C=C- double bond in an aliphatic chain, or up to one substituent for a carbon of a terminal methine group). Exemplary substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, acyl, aldehyde, amide, amino, aminoalkyl, aryl, arylalkyl, carboxyl, cyano, cycloalkyl, dialkylamino, halo, haloaliphatic, heteroaliphatic, heteroaryl, heterocycloaliphatic, hydroxyl, oxo, sulfonamide, sulfhydryl, thioalkoxy, or other functionality.

Alkyl: A hydrocarbon group having a saturated carbon chain. The chain may be branched, unbranched, or cyclic (cycloalkyl). Unless otherwise specified, the term alkyl encompasses substituted and unsubstituted alkyl. Alkylaryl: An alkyl radical substituted with a terminal aryl group. One nonlimiting example of an alkylaryl group is -(CH2) _n -aryl, such as benzyl. Unless otherwise specified, the aryl group may be substituted or unsubstituted. The alkyl radical may include substituents in addition to the aryl group.

Amino: A chemical functional group -N(R)R' where R and R' are independently hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, alkylsulfano, or other functionality. A “primary amino” group is -NH2. “Mono-substituted amino” means a radical -N(H)R substituted as above and includes, e.g., methylamino, (l-methylethyl)amino, phenylamino, and the like. “Di-substituted amino” means a radical -N(R)R' substituted as above and includes, e.g., dimethylamino, methylethylamino, di(l-methylethyl)amino, and the like.

Antibody: A protein (or protein complex) that includes one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. In avian and reptilian species, IgY antibodies are equivalent to mammalian IgG.

The basic immunoglobulin (antibody) structural unit is generally a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” (about 50-70 kDa) chain. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms “variable light chain” (VL) and “variable heavy chain” (Vn) refer, respectively, to these light and heavy chains.

The structure of IgY antibodies is similar to the structure of mammalian IgG, with two heavy ("nu" chains; approximately 67-70 kDa) and two light chains (22-30 kDa). The molecular weight of an IgY molecule is about 180 kDa, but it often runs as a smear on gels due to the presence of about 3% carbohydrate. Heavy chains (H) of IgY antibodies are composed of four constant domains and one variable domain, which contains the antigen-binding site.

As used herein, the term “antibodies” includes intact immunoglobulins as well as a number of well-characterized fragments. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs) that bind to target protein (or epitope within a protein or fusion protein) would also be specific binding agents for that protein (or epitope). These antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab’, the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab’ fragments are obtained per antibody molecule; (3) (Fab’)2, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab’)2, a dimer of two Fab’ fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single chain antibody, a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Methods of making these fragments are routine (see, for example, Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999). As used herein, the term “antibodies” includes antibodies comprising one or more unnatural (i.e., non-naturally occurring) amino acids (e.g., p-acetyl-phenylalanine) to facilitate site-specific conjugation.

Antibodies for use in the methods of this disclosure can be monoclonal or polyclonal, and for example specifically bind a target such as the target antigen. Merely by way of example, monoclonal antibodies can be prepared from murine hybridomas according to the classical method of Kohler and Milstein (Nature 256:495-97, 1975) or derivative methods thereof. Detailed procedures for monoclonal antibody production are described in Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999.

Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. As used herein, a “target antigen” is an antigen (including an epitope of the antigen) that is recognized and bound by a targeting agent. “Specific binding” does not require exclusive binding. In some embodiments, the antigen is obtained from a cell or tissue extract. In some embodiments, the target antigen is an antigen on a tumor cell. An antigen need not be a full-length protein. Antigens contemplated for use include any immunogenic fragments of a protein, such as any antigens having at least one epitope that can be specifically bound by an antibody.

Aryl: A monovalent aromatic carbocyclic group of, unless specified otherwise, from 6 to 15 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings in which at least one ring is aromatic (e.g., quinoline, indole, benzodioxole, and the like), provided that the point of attachment is through an atom of an aromatic portion of the aryl group and the aromatic portion at the point of attachment contains only carbons in the aromatic ring. If any aromatic ring portion contains a heteroatom, the group is a heteroaryl and not an aryl. Aryl groups are monocyclic, bicyclic, tricyclic or tetracyclic. Unless otherwise specified, the term aryl encompasses substituted and unsubstituted aryl.

Biological sample: As used herein, a “biological sample” refers to a sample obtained from a subject (such as a human or veterinary subject) or other type of organism, such as a plant, bacteria or insect. Biological samples from a subject include, but are not limited to, cells, tissue, serum, blood, plasma, urine, saliva, cerebral spinal fluid (CSF) or other bodily fluid. In particular examples of the method disclosed herein, the biological sample is a tissue sample.

Chemical trigger: As used herein, the term “chemical trigger” refers to a non-enzyme compound capable of cleaving a carbamate group from a Anorogenic cyanine carbamate compound as disclosed herein. For example, triphenylphosphine is capable of cleaving a -C(O)-O-CH2-C6HSN3 group from a cyanine carbamate compound as disclosed herein.

Conjugatable moiety: A portion of a molecule that allows the molecule to be conjugated (i.e., coupled or bound) to another molecule, e.g., to a drug or targeting agent such as an antibody.

Drug: As used herein, the term “drug” refers to a substance which has a physiological effect when administered to a subject, and is intended for use in the treatment, mitigation, cure, prevention, or diagnosis of disease or used to otherwise enhance physical or mental well-being. The term “small molecule drug” refers to a drug having a molecular weight < 1,000 Daltons.

Effective amount: As used herein, the term “effective amount” refers to an amount sufficient for detection in a biological sample, e.g., by Auorescence.

Enzyme: A protein molecule that is capable of catalyzing a chemical reaction. For example, galactosidase is an enzyme capable of hydrolyzing P-galactosides into monosaccharides through the breaking of a glycosidic bond.

Halogen: The terms halogen and halo refer to Auorine, chlorine, bromine, iodine, and radicals thereof.

Heteroaliphatic: An aliphatic compound or group having at least one heteroatom, i.e., one or more carbon atoms has been replaced with an atom having at least one lone pair of electrons, typically nitrogen, oxygen, phosphorus, silicon, or sulfur. Unless otherwise specified, heteroaliphatic compounds or groups may be substituted or unsubstituted, branched or unbranched, cyclic (heterocycloaliphatic) or acyclic, and include "heterocycle", "heterocyclyl", "heterocycloaliphatic", or "heterocyclic" groups.

Heteroalkylaryl: As used in the term heteroalkylaryl refers to a heteroalkyl radical substituted with a terminal aryl group. Unless otherwise specified, the aryl group may be substituted or unsubstituted. The heteroalkyl radical may include substituents in addition to the aryl group.

Heteroaryl: An aromatic compound or group having at least one heteroatom, i.e., one or more carbon atoms in the ring has been replaced with an atom having at least one lone pair of electrons, typically nitrogen, oxygen, phosphorus, silicon, or sulfur. Unless otherwise specified, the term heteroaryl encompasses substituted and unsubstituted heteroaryl.

Ligand: A molecule that binds to a receptor, having a biological effect.

Near-infrared (near-IR, NIR): Wavelengths within the range of 650-2500 nm. NIR wavelengths for optical imaging of tissue may be divided into three windows - NIR-I, 650-950 nm; NIR-II, 1000-1350 nm (sometimes defined as 1000-1700 nm); and NIR-III, 1600-1870 nm. Unless otherwise specified, the terms “near-infrared” and “NIR” as used herein refer to wavelengths within the range of 650-1350 nm.

Norcyanine: As used herein, the term “norcyanine” refers to the product following cleavage of the carbamate group from a cyanine carbamate compound.

Pharmaceutically acceptable carrier: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 ^st Edition (2005), describes compositions and formulations suitable for pharmaceutical delivery of one or more targeting agent-drug conjugates as disclosed herein.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In some examples, the pharmaceutically acceptable carrier may be sterile to be suitable for administration to a subject (for example, by parenteral, intramuscular, or subcutaneous injection). In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. Pharmaceutically acceptable salt: A biologically compatible salt of a disclosed conjugate, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, and the like. Pharmaceutically acceptable acid addition salts are those salts that retain the biological effectiveness of the free bases while formed by acid partners that are not biologically or otherwise undesirable, e.g., inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, as well as organic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, -lol uenesul Ionic acid, salicylic acid and the like. Pharmaceutically acceptable base addition salts include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Exemplary salts are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. (See, for example, S. M. Berge, et al., “Pharmaceutical Salts,” J. Pharrn. Sci., 1977; 66:1-19, which is incorporated herein by reference.)

Specific binding partner: A member of a pair of molecules that interact by means of specific, non-covalent interactions that depend on the three-dimensional structures of the molecules involved. Exemplary pairs of specific binding partners include antigen/antibody, hapten/antibody, receptor/ligand, nucleic acid strand/complementary nucleic acid strand, substrate/enzyme, inhibitor/enzyme, carbohydrate/lectin, biotin/avidin (such as biotin/streptavidin), and virus/cellular receptor. Substituent: An atom or group of atoms that replaces another atom in a molecule as the result of a reaction. The term "substituent" typically refers to an atom or group of atoms that replaces a hydrogen atom, or two hydrogen atoms if the substituent is attached via a double bond, on a parent hydrocarbon chain or ring. The term “substituent” may also cover groups of atoms having multiple points of attachment to the molecule, e.g., the substituent replaces two or more hydrogen atoms on a parent hydrocarbon chain or ring. In such instances, the substituent, unless otherwise specified, may be attached in any spatial orientation to the parent hydrocarbon chain or ring. Exemplary substituents include, for instance, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, acyl, aldehyde, amido, amino, aminoalkyl, aryl, arylalkyl, arylamino, carbonate, carboxyl, cyano, cycloalkyl, dialkylamino, halo, haloaliphatic (e.g., haloalkyl), haloalkoxy, heteroaliphatic, heteroaryl, heterocycloaliphatic, hydroxyl, isocyano, isothiocyano, oxo, sulfonamide, sulfhydryl, thio, and thioalkoxy groups.

Substituted: A fundamental compound, such as an aryl or aliphatic compound, or a radical thereof, having coupled thereto one or more substituents, each substituent typically replacing a hydrogen atom on the fundamental compound. Solely by way of example and without limitation, a substituted aryl compound may have an aliphatic group coupled to the closed ring of the aryl base, such as with toluene. Again solely by way of example and without limitation, a long-chain hydrocarbon may have a hydroxyl group bonded thereto.

Sulfonate-containing group: A group including SO3". The term sulfonate-containing group includes -SO3" and -RSO3" groups, where R is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

Target: An intended molecule to which a disclosed conjugate comprising a targeting agent is capable of specifically binding. Examples of targets include proteins and nucleic acid sequences present in tissue samples. A target area is an area in which a target molecule is located or potentially located.

Targeting agent: An agent that promotes preferential or targeted delivery to a target site, for example, a targeted location in a subject’s body, such as a specific organ, organelle, physiologic system, tissue, or site of pathology such as a tumor, area of infection, or area of tissue injury. Targeting agents function by a variety of mechanisms, such as selective concentration in a target site or by binding to a specific binding partner. Suitable targeting agents include, but are not limited to, proteins, polypeptides, peptides, glycoproteins and other glycoslyated molecules, oligonucleotides, phospholipids, lipoproteins, alkaloids, steroids, and nanoparticles. Exemplary targeting agents include antibodies, antibody fragments, affibodies, aptamers, albumin, cytokines, lymphokines, growth factors, hormones, enzymes, immune modulators, receptor proteins, antisense oligonucleotides, peptides, avidin, nanobodies (small (15kDa) antigen-binding VHH fragments derived from heavy chain only antibodies present in camelids and cartilaginous fishes), nanoparticles, and the like. Particularly useful targeting agents are antibodies, peptides, nanobodies, nucleic acid sequences, and receptor ligands, although any pair of specific binding partners can be readily employed for this purpose.

II. Fluorogenic Cyanine Carbamates

Fluorogenic cyanine carbamate compounds are disclosed. The compounds include a carbamate group that may be cleaved by an enzyme or chemical trigger. Following cleavage of the carbamate group, the compound is fluorescent in an acidic environment (i.e., when the cleaved compound is protonated) when irradiated with targeted application of an effective quantity of light having a selected wavelength and a selected intensity to induce fluorescence.

In some embodiments, the fluorogenic cyanine carbamate has a structure according to Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof:

The bond indicated by ” is an optional bond, which is present when R ^x is present and absent when R ^x is absent. L is substituted or unsubstituted conjugated alkenyl, each carbon having sp ² hybridization. R ¹ is substituted aryl or substituted heteroaliphatic, and the bond between the nitrogen atom and -C(O)-O-CH2-R ¹ is cleavable by an enzyme or chemical trigger. Y ¹ and Y ² independently are C(R ^b )2, N(R ^C ), S, O, or Se, wherein each R ^b independently is aliphatic, H, deuterium, aryl, -(OCfFCfFjxOH where x is an integer > 2, trityl, or a group comprising a conjugatable moiety, a targeting agent, or a drug, and each R ^c independently is H, deuterium, aliphatic, or heteroaliphatic. R ³ and R ⁴ independently are H, aliphatic, amino, a sulfonate-containing group, or a group comprising a conjugatable moiety, a targeting agent, or a drug, or R ³ and Y ¹ together with the atoms to which they are bound form a 6-membered aryl ring, and R ⁴ and Y ² together with the atoms to which they are bound form a 6-membered aryl ring. R ⁵ -R ¹⁰ independently are H, a sulfonate-containing group, aliphatic, heteroaliphatic, amino, or a group comprising a conjugatable moiety, a targeting agent, or a drug. R ^x is absent, aliphatic, heteroaliphatic, aryl, heteroaryl, alkylaryl, alkylheteroaryl, or H, wherein if R ^x is present, the nitrogen to which R ^x is attached has a +1 charge and a counterion X’ is also present. Suitable counterions include, but are not limited to, halide (fluoride, chloride, iodide, or bromide), hydroxy, carboxylate (e.g., formate, acetate), nitrate, hydrogen sulfate, hydrogen carbonate, dihydrogen phosphate, and hexafluorophosphate, among others.

In any of the foregoing or following embodiments, the recited groups may be substituted or unsubstituted, unless otherwise specified. For example, the term “aryl” encompasses both substituted and unsubstituted aryl groups. In some implementations, the substituent is further substituted. The substituent may be substituted with any substituent that does not interfere with cleavage of the carbamate group and/or near-infrared fluorescence of the cleaved compound. Substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, acyl, aldehyde, amido, amino, aminoalkyl, aryl, arylalkyl, arylamino, carbonate, carboxyl, cyano, cycloalkyl, dialkylamino, halo, haloaliphatic (e.g., haloalkyl), haloalkoxy, heteroaliphatic, heteroaryl, heterocycloaliphatic, hydroxyl, isocyano, isothiocyano, oxo, sulfonamide, sulfhydryl, thio, and thioalkoxy groups, and combinations thereof. For instance, in one nonlimiting example, R ¹ is a substituted aryl group wherein the substituent is a substituted heteroaliphatic chain, e.g.,

L is substituted or unsubstituted conjugated alkenyl, each carbon having sp ² hybridization.

In some embodiments, L is substituted or unsubstituted C _n conjugated alkenyl where n is 3, 5, 7, or

9. In certain implementations, n is 7. In certain embodiments, L is

In any of the foregoing or following embodiments, R ² and R ² independently are aryl, heteroaryl, heterocycloaliphatic, H, halo, -OR ^a , -NR ^a 2, or a group comprising a conjugatable moiety, a targeting agent, or a drug. Each R ^a independently is H, halo, alkyl, or aryl.

In any of the foregoing or following embodiments, when L contains R ¹¹ and R ¹² , but does not include R ¹¹ , then (i) R ¹¹ and R ¹² independently are H, aliphatic, heteroaliphatic, or a group comprising a conjugatable moiety, a targeting agent, or a drug, or (ii) R ¹¹ and R ¹² together with the atoms to which they are bound form a 5-7 membered cycloaliphatic or heterocycloaliphatic ring. In any of the foregoing or following embodiments, when L contains R ¹¹ , R ¹¹ , and R ¹² , then (i) R ¹¹ together with R ¹² and the atoms to which they are bound forms a 5-7 membered cycloaliphatic or heterocycloaliphatic ring and R ¹¹ is H, aliphatic, heteroaliphatic, or a group comprising a conjugatable moiety, a targeting agent, or a drug, or (ii) R ¹¹ together with R ¹² and the atoms to which they are bound form a 5-7 membered cycloaliphatic or heterocycloaliphatic ring and R ¹¹ is H, aliphatic, heteroaliphatic, or a group comprising a conjugatable moiety, a targeting agent, or a drug or (iii) each of R ¹¹ , R ¹¹ , and R ¹² independently is H, aliphatic, heteroaliphatic, or a group comprising a conjugatable moiety, a targeting agent, or a drug, or (iv) R ¹¹ , R ¹¹ , and R ¹² together with the atoms to which they are bound form a fused cycloaliphatic or heterocycloaliphatic ring system, each ring comprising 5-7 members

In any of the foregoing or following embodiments, L may be

-CHR ^d -, -CH2CHR ^d -, -CHR ^d CH2-, or -NR ^d -, where R ^d is H, alkyl, aryl, or a group comprising a conjugatable moiety, a targeting agent, or a drug. In some embodiments, A and A' independently are -CHR ^d - or -NR ^d -. In certain implementations, A and A' independently are -CH2- or -N(CH3)-. When A and/or A' is -CH2-, the compound may hereinafter be referred to as a CyBam compound. When A and/or A' is -N(CH3)-, the compound may hereinafter be referred to as a CyLBam compound. In some embodiments, a CyLBam exhibits improved cellular permeability and lysosomal accumulation in vivo. Advantageously, presence of -N(CH3)- at A and/or A' dramatically improves cellular photon output, likely due to enhanced lysosomal uptake and retention.

In some implementations, A and/or A' may comprise a targeting agent or a conjugatable moiety. In certain embodiments, R ^d is -(CH2) _m -R ^e , -(CH2) _m CH(NH2)-R ^e , -(CH2) _m C(O)R ^e , -(CH ₂ ) _m N(H)R ^e , -(CH ₂ ) _m N(H)C(O)R ^e , -(CH ₂ ) _m C(O)N(H)R ^e , -(CH ₂ ) _m C(O)SR ^e , -C(O)R ^e , -C(O)N(H)R ^e , -C(O)N(H)(CH2CH ₂ O)n(CH2)mC(O)R ^e , -N(H)C(O)R ^e , -N(H)R ^e , or -SR ^e , where m is an integer > 1. R ^e is a targeting agent, -OH, -COOH, a phosphoramidite,

In some embodiments, the Anorogenic cyanine carbamate has a structure according to any one of Formulas II-IV, or a stereoisomer or pharmaceutically acceptable salt thereof:

R'-R ¹² , R ² , R ¹¹ , R ^X , Y ¹ , and Y ² are as previously defined. In some implementations, R ^x is absent.

If R ^x is present, then the nitrogen to which R ^x is attached has a +1 charge and a counterion X’ is also present. In some implementations, R ¹¹ and/or R ¹¹ together with R ¹² and the atoms to which they are bound forms a 5-7 membered cycloaliphatic or heterocycloaliphatic ring, and the Anorogenic cyanine carbamate has a structure according to formula Ila, Va, Vb, or Vc, or a stereoisomer or pharmaceutically acceptable salt thereof.

R^-R ¹¹ , R ² , R ¹¹ , R ^x , A, A', Y ¹ , and Y ² are as previously defined. In some embodiments, A and A' independently are -CHR ^d - or -NR ^d -, where R ^d is H or alkyl. In certain implementations, A and A' independently are -CH2- or -N(CH3)-. In some implementations, R ^x is absent. If R ^x is present, then the nitrogen to which R ^x is attached has a +1 charge and a counterion X’ is also present.

In any of the foregoing or following embodiments, Y ¹ and Y ² independently may be C(R ^b )2, N(R ^C ), S, O, or Se, wherein each R ^b independently is H, deuterium, aliphatic, aryl, -(OCH2CH2) _X OH where x is an integer > 2, trityl, or a group comprising a conjugatable moiety, a targeting agent, or a drug, and each R ^c independently is H, deuterium, aliphatic, or heteroaliphatic. In some embodiments, Y ¹ and Y ² independently are C(R ^b )2, where each R ^b independently is aliphatic or aryl. In some implementations, each R ^b independently is aliphatic, such as C1-C5 alkyl. In certain embodiments, Y ¹ and Y ² are -C(CH3)2-.

In any of the foregoing or following embodiments, R ³ and R ⁴ independently may be H, aliphatic, amino, a sulfonate-containing group, or a group comprising a conjugatable moiety, a targeting agent, or a drug, or R ³ and Y ¹ together with the atoms to which they are bound may form a 6-membered aryl ring, and R ⁴ and Y ² together with the atoms to which they are bound may form a 6-membered aryl ring. In some embodiments, R ³ and R ⁴ are H.

In some implementations, R ³ and Y ¹ together with the atoms to which they are bound form a 6-membered aryl ring, and R ⁴ and Y ² together with the atoms to which they are bound form a 6-membered aryl ring. In such embodiments, the Anorogenic cyanine carbamate may have a structure according to any one of Formulas lib, Illa, IVa, or Vd, or a stereoisomer or pharmaceutically acceptable salt thereof:

R ¹ , R ² , R ² , R ⁵ -R ¹² , R ¹¹ , and R ^x are as previously defined. R ¹³ -R ¹⁸ independently are H, aliphatic, amino, a sulfonate-containing group, or a group comprising a conjugatable moiety, a targeting agent, or a drug. In some implementations, R ¹¹ and/or R ¹¹ together with R ¹² and the atoms to which they are bound forms a 5-7 membered cycloaliphatic or heterocycloaliphatic ring, and the Anorogenic cyanine carbamate has a structure according to formula lie, Ve, Vf, or Vg , or a stereoisomer or pharmaceutically acceptable salt thereof.

R ¹ , R ² , R ² , R ⁵ -R ⁿ , R ¹¹ , R ¹³ -R ¹⁸ , R ^X , A, and A' are as previously defined.

In any of the foregoing or following embodiments, R ² and R ² independently are aryl, heteroaryl, heterocycloaliphatic, H, halo, -OR ^a , -NR ^a 2, or a group comprising a conjugatable moiety, a targeting agent, or a drug, where each R ^a independently is H, halo, alkyl, or aryl. In some implementations, R ² and/or R ² is aryl. In certain embodiments, R ² and/or R ² is unsubstituted phenyl. In some implementations, R ² and/or R ² is phenyl substituted with -(CH ₂ ) _P CO ₂ H where p is an integer from 0 to 10 (e.g., -CO ₂ H or -C ₂ H4CO ₂ H), an aryl group, or a group comprising a conjugatable moiety, a targeting agent (e.g., an antibody, a peptide, or a nanobody), or a drug. In some embodiments, the substituent is para to the attachment of R ² and/or R ² to the remainder of the molecule. In some embodiments, R ² and/or R ² is H, halo (e.g., chloro), -OR ^a , or -NR ^a ₂ , where each R ^a independently is H, halo, alkyl, or aryl. In some implementations, when R ² and R ² are both present, one of R ² and R ² is H and the other of R ² and R ² is aryl, heteroaryl, heterocycloaliphatic, H, halo, -OR ^a , -NR ^a ₂ , or a group comprising a conjugatable moiety, a targeting agent, or a drug. or a targeting agent, where y is an integer > 1.

In any of the foregoing or following embodiments, R ² and/or R ² may be unsubstituted or substituted phenyl as previously described above, H, halo, -OR ^a , -NR ^a ₂ , or a group comprising a conjugatable moiety, a targeting agent, or a drug. In any of the foregoing or following embodiments, R ⁵ and R ⁸ independently may be H or a sulfonate-containing group, such as -SO3" or

In any of the foregoing or following embodiments where the Anorogenic cyanine carbonate comprises a ring comprising A, a ring comprising A', or rings comprising A and A', A and A' independently may be -CH ₂ - or -N(CH3)-. In some implementations, R ³ , R ⁴ , R ⁶ , R ⁷ , R ⁹ , R ¹⁰ , and R ¹³ -R ¹⁸ are H. If present, R ¹¹ or R ¹¹ may also be H.

In any of the foregoing or following embodiments, R ⁵ -R ¹⁰ independently may be H, a sulfonate-containing group, aliphatic, heteroaliphatic, amino, or a group comprising a conjugatable moiety, a targeting agent, or a drug. In any of the foregoing or following embodiments, R ⁵ and R ⁸ independently may be H, a sulfonate-containing group, or heteroahphatic. In some embodiments, i, n /~ » f~ \

, ₈ “Hr ^_ “ ’

R ³ and R ⁸ independently are -SO3", ⁰ , ⁰ , H, or -OCH3. In some embodiments, R ³ , R ⁴ , R ⁶ , R ⁷ , R ⁹ , and R ¹⁰ are H. In certain implementations, R ³ , R ⁴ , R ⁶ , R ⁷ , R ⁹ , R ¹⁰ , and R ¹³ -R ¹⁸ are H.

In any of the foregoing or following embodiments, R ^x may be absent, aliphatic, heteroaliphatic, aryl, heteroaryl, or H, wherein if R ^x is present, the nitrogen to which R ^x is attached has a +1 charge and a counterion X’ is also present. In some embodiments, R ^x is absent. In some implementations, R ^x is aliphatic, heteroaliphatic, aryl, heteroaryl, alkylaryl, alkylheteroaryl, or H; in such implementations, the nitrogen to which R ^x is attached has a +1 charge and the counterion X" is present. In certain implementations, R ^x is alkyl, heteroalkyl, alkenyl, heteroalkenyl, aryl, heteroaryl, or H. For example, R ^x may be H, C1-C10 straight, branched, or cyclo alkyl or alkenyl, C2-C10 straight, branched, or cyclo heteroalkyl or heteroalkenyl, phenyl, pyridinyl, pyrrolyl, pyrimidinyl, pyrazolyl, imidazolyl, isoxazolyl, oxazolyl, furyl, or thiophenyl. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl (2-methylpropyl), sec-butyl (butan-2-yl), tert-butyl, and the like. Exemplary heteroalkyl groups include, but are not limited to, alkoxy groups (methoxy, ethoxy, propoxy, cyclopropoxy, cyclobutoxy, and the like), as well as groups including nitrogen or sulfur heteroatoms. Exemplary cycloalkyl and cycloalkenyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexenyl. Exemplary cycloheteroalkyl groups include, but are not limited to, piperidinyl, pyrrolidinyl, piperazinyl, furyl, tetrahydrofuryl, and morpholinyl groups.

In certain embodiments, the Anorogenic cyanine carbamate has a structure according to Formula IIA where R ¹ and R ^x are as previously defined, R ² is substituted or unsubstituted phenyl; A is -CH2- or -N(CH ₃ )-; Y ¹ and Y ² are C(CH ₃ ) ₂ ; R ³ , R ⁴ , R ⁶ , R ⁷ , R ⁹ , and R ¹⁰ are H; and R ⁵ and R ⁸ are -SO3", , , H, or -OCH3. In certain implementations, the compound has a structure according to any one of Formulas Ild-IIi, or a stereoisomer or pharmaceutically acceptable salt thereof, where R ¹ and R ¹³ are as previously defined. In certain implementations, R ^x is absent.

In any of the foregoing or following embodiments, R ^x may be absent, and the Anorogenic cyanine carbamate may have a formula as shown in Table 1, wherein R^-R ¹⁸ , R ² , R ¹¹ , Y ¹ , Y ² , A, and A' are as previously defined.

Table 1

Advantageously, the carbamate group, -C(O)OCH ₂ R ¹ , is cleavable by one or more enzymes or chemical triggers. R ¹ may be chosen to provide selectivity for a particular enzyme or chemical trigger. In any of the foregoing or following embodiments, R ¹ may be substituted aryl or substituted heteroaliphatic. In some embodiments, R ¹ is substituted phenyl or substituted heteroaliphatic. In some implementations, R ¹ is

-N ₃ , -NO ₂ , a heterocycle, -O-heterocycloaliphatic, -O-heteroaliphatic, -O-aliphatic, -O-heteroalkylaryl, -O-alkylaryl, or -O-aryl, where R' is aliphatic and each R" independently is H or aliphatic. R ^s is aliphatic, heteroaliphatic, heterocycloaliphatic, alkylaryl, or heteroalkylaryl. R ^h is aliphatic. In any of the foregoing recitations, the heterocycle, heterocycloaliphatic, heteroaliphatic, alkyl, aryl, aliphatic, or heteroaliphatic portions may be substituted or unsubstituted. Substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, acyl, aldehyde, amido, amino, aminoalkyl, aryl, arylalkyl, arylamino, carbonate, carboxyl, cyano, cycloalkyl, dialkylamino, halo, haloaliphatic (e.g., haloalkyl), haloalkoxy, heteroaliphatic, heteroaryl, heterocycloaliphatic, hydroxyl, isocyano, isothiocyano, oxo, sulfonamide, sulfhydryl, thio, and thioalkoxy groups, conjugatable groups, targeting agents, and combinations thereof. Aliphatic, heteroaliphatic, and alkyl moieties may be straight or branched chains. In one embodiment, -C(O)OCH ₂ R ¹ may not be cleavable, and the compound may be utilized as a nonfluorescent control compound.

Several exemplary R ¹ groups are shown in Table 2.

Table 2

In any of the foregoing or following embodiments, one of R ¹ -R ^{1 s} , R ² ’ A, A', Y ¹ , or Y ² may comprise a group comprising a targeting agent. In some embodiments, R ² , R ² , A, or A' comprises the group comprising the targeting agent. In certain implementations, R ¹ comprises the targeting agent, and the targeting agent thus is cleaved from the compound upon exposure to the enzyme or chemical trigger. In any of the foregoing or following embodiments, the targeting agent may be an antibody, a peptide, or a nanobody. In some implementations, the targeting agent is an antibody. In certain examples, the antibody is panitumumab.

In any of the foregoing or following embodiments, one of R ² -R ¹⁸ , R ² , R ¹¹ , A, A', Y ¹ , or Y ² may comprise a group comprising a drug. Exemplary groups comprising a drug include, but are not limited to, groups having a formula -Li-C(O)-X ¹ -drug, where Li is a linker moiety or is absent and X ¹ is O, N(H), or N(CH3). In one embodiment, Li is absent. In another embodiment, Li is O. In an independent embodiment, Li is aryl or heteroaryl substituted with at least one substituent comprising a substituted or unsubstituted aliphatic or heteroaliphatic moiety, wherein the aryl or heteroaryl ring is the site of attachment to the remainder of the conformationally restricted cyanine fluorophore and the substituent is bonded to the -C(O)-X ¹ -drug moiety. In some embodiments, the group comprising a drug is: independently are H, alkyl, -NO2, -NR*2, -NR ¹ , alkoxy, or sulfonate, wherein each R ¹ independently is H, halo, or alkyl. In certain embodiments, R ¹⁹ -R ²⁶ are H. In some examples, the group comprising a drug is -C(O)-X ¹ -Drug. The drug can be any drug capable of conjugation to the remainder of the group. In some embodiments, the drug is a small-molecule drug, e.g., a drug having a molecular weight < 1,000 Daltons. In certain embodiments, the drug moiety is an anti-cancer drug.

Exemplary compounds include, but are not limited to:

where Ab is an antibody.

III. Pharmaceutical Compositions

This disclosure also includes pharmaceutical compositions comprising at least one Anorogenic cyanine carbamate compound as disclosed herein. Some embodiments of the pharmaceutical compositions include a pharmaceutically acceptable carrier and at least one fluorogenic cyanine carbamate. Useful pharmaceutically acceptable carriers and excipients are known in the art.

The pharmaceutical compositions comprising one or more fluorogenic cyanine carbamates may be formulated in a variety of ways depending, for example, on the mode of administration and/or on the location to be imaged. Parenteral formulations may comprise injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like. Excipients may include, for example, nonionic solubilizers, such as Cremophor®, or proteins, such as human serum albumin or plasma preparations. If desired, the pharmaceutical composition to be administered may also contain non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate.

The form of the pharmaceutical composition will be determined by the mode of administration chosen. Embodiments of the disclosed pharmaceutical compositions may take a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, transdermal, rectal, vaginal, etc., or a form suitable for administration by inhalation or insufflation. Generally, embodiments of the disclosed pharmaceutical compositions will be administered by injection, systemically, or orally.

Useful injectable preparations include sterile suspensions, solutions or emulsions of the active compound(s) in aqueous or oily vehicles. The compositions may also contain formulating agents, such as suspending, stabilizing and/or dispersing agent. The formulations for injection may be presented in unit dosage form, e.g., in ampules or in multidose containers, and may contain added preservatives. The composition may take such forms as suspension, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. For example, parenteral administration may be done by bolus injection or continuous infusion. Alternatively, the fluorogenic cyanine carbamate may be in powder form for reconstitution with a suitable vehicle, e.g. sterile water, before use.

Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal, oral or pulmonary administration.

Oral formulations may be liquid (e.g., syrups, solutions or suspensions), or solid (e.g., powder, tablets, or capsules). Oral formulations may be coupled with targeting ligands for crossing the endothelial barrier. Some fluorogenic cyanine carbamate formulations may be dried, e.g., by spray-drying with a disaccharide, to form Anorogenic cyanine carbamate powders. Solid compositions prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, mannitol, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well known in the art with, for example, sugars, films or enteric coatings. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.

Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups or suspensions. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, Cremophor® or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, preservatives, Aavoring, coloring and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated to give controlled release of the Auorophore, as is well known.

For rectal and vaginal routes of administration, the Anorogenic cyanine carbamate(s) may be formulated as solutions (for retention enemas) suppositories or ointments containing conventional suppository bases such as cocoa butter or other glycerides.

For nasal administration or administration by inhalation or insufAation, the Anorogenic cyanine carbamate(s) can be conveniently delivered in the form of an aerosol spray or mist from pressurized packs or a nebulizer with the use of a suitable propellant, e.g., dichlorodiAuoromethane, trichloro Auoromethane, dichlorotetraAuoroethane, Auorocarbons, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

Certain embodiments of the pharmaceutical compositions comprising Anorogenic cyanine carbamates as described herein may be formulated in unit dosage form suitable for individual administration of precise dosages. The pharmaceutical compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the Anorogenic cyanine carbamate. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The amount of Anorogenic cyanine carbamate administered will depend at least in part on the subject being treated, the target (e.g., the size, location, and characteristics of a tumor), and the manner of administration, and may be determined as is known to those skilled in the art of pharmaceutical composition and/or contrast agent administration. Within these bounds, the formulation to be administered will contain a quantity of the Anorogenic cyanine carbamate disclosed herein in an amount effective to enable visualization of the Anorogenic cyanine carbamate by suitable means after administration to the subject. In certain embodiments, the Anorogenic cyanine carbamate comprises a drug bound to the molecule, and the formulation to be administered will contain a quantity of the drug bound to the Anorogenic cyanine carbamate effective to provide a therapeutically effective dose of the drug to the subject being treated.

In some embodiments, the pharmaceutical composition includes a second agent other than the Anorogenic cyanine carbamate. The second agent may be, for example, an anti-tumor agent or an angiogenesis inhibitor.

IV. Uses

Embodiments of the disclosed Anorogenic cyanine carbamates are suitable for in vivo, ex vivo, or in vitro use. Advantageously, following cleavage of the carbamate group, the compound is Auorescent in an acidic or neutral pH environment when irradiated with targeted application of an effective quantity of light having a selected wavelength and a selected intensity to induce Auorescence.

In some embodiments, a method of using a Anorogenic cyanine carbamate as disclosed herein includes contacting a biological sample with the compound, exposing the compound to an enzyme or a chemical trigger capable of cleaving -C(O)OCH2R ¹ from the compound to provide a cleaved compound (hereinafter referred to as a “norcyanine”), exposing the norcyanine to a pH less than 8, subsequently exposing the norcyanine to light having a wavelength greater than 700 nm, and detecting Auorescence from the norcyanine. In some embodiments, the cyanine carbamate does not include an R ^x substituent, and the norcyanine is exposed to a pH less than 7 to provide a protonated norcyanine. Both the initial compound and the norcyanine exhibit minimal absorbance and emission in the near-infrared range. However, exposing the norcyanine to a pH less than 8, or a pH less than 7, provides a shift in absorbance and dramatically increased Auorescence signal in the NIR range. In some embodiments, at a pH less than 8 or a pH less than 7, the norcyanine has a Auorescence intensity at least 100X greater than the cyanine carbamate or norcyanine, such as a Auorescence intensity that is at least 125X greater, at least 150X greater, or even at least 170X greater than the cyanine carbamate or the norcyamne at a pH greater than 8 or a pH greater than 7. In certain implementations, fluorescence may be detected at pH ranges near neutral, e.g., pH 7-8, or 7-7.5. However, protonation provides a dramatic increase in fluorescence when the compound does not include an R ^x substituent.

In some embodiments, the cyanine carbamate is a CyBam (a cyanine carbamate wherein the linker includes a 6-membered aliphatic ring where A or A' is -CH2-) or a CyLBam (a cyanine carbamate wherein the linker includes a 6-membered heteroaliphatic ring where A or A' is -N(CH3)-) as disclosed herein. In some embodiments, a CyLBam exhibits improved cellular permeability and lysosomal accumulation in vivo.

One generalized reaction scheme showing the carbamate group cleavage and subsequent indolenine protonation is shown below:

(protonated norcyanine)

In some embodiments, the cyanine carbamate has an absorbance maximum of 400-450 nm, whereas the resulting norcyanine has an absorbance maximum of 700-800 nm.

In one specific example, the cyanine carbamate is:

Cleavage of the carbamate group shifts Abs to 520 nm, and subsequent indolenine protonation further shifts Abs to 755 nm. The protonated norcyanine has a 170-fold increase in fluorescence signal (Xmax = -775 nm, xcitation = 710 nm) compared to the original compound.

The carbamate group is cleaved in situ by an enzyme or a chemical trigger. In one embodiment, the biological sample comprises the enzyme, and exposing the cyanine carbamate to the enzyme comprises contacting the biological sample comprising the enzyme with the cyanine carbamate. In an independent embodiment, the biological sample comprises the chemical trigger, and exposing the cyanine carbamate to the chemical trigger comprises contacting the biological sample comprising the chemical trigger with the cyanine carbamate. In another independent embodiment, exposing the cyanine carbamate to the enzyme or chemical trigger comprises adding the enzyme or the chemical trigger to the biological sample and the cyanine carbamate.

The cleaved compound, or norcyanine, is exposed to a pH less than 8, such as pH less than 7. In some embodiments, the norcyanine does not include an R ^x substituent and the norcyanine is protonated by exposure to a pH less than 7. In some implementations, the biological sample comprises an environment having a pH less than 7, thereby exposing the norcyanine to the pH less than 7. In certain implementations, the pH of the biological sample may be reduced, e.g., with a mild acid, to provide expose the compound to a pH less than 8 or a pH less than 7. In other implementations, sufficient fluorescence may be detectable at a neutral pH, e.g., a pH of 7-8 or 7-7.5.

In any of the foregoing or following embodiments, subsequently exposing the cleaved compound, or norcyanine, to light having a wavelength greater than 700 nm and detecting fluorescence from the norcyanine may comprise irradiating the biological sample, or a targeted portion of a subject comprising the biological sample, with targeted application of a quantity of light having a wavelength in the visible, far-red, or near-infrared range and a selected intensity, wherein the quantity of light is sufficient to produce fluorescence of the norcyanine, and detecting any fluorescence emitted by the norcyanine. Advantageously, the light has a wavelength at or near a maximum absorption wavelength of the norcyanine in an environment having a pH less than 8 or a pH less than 7. In some embodiments, the light has a wavelength at or near a maximum absorption wavelength of the protonated norcyanine. For example, the biological sample may be irradiated with light having a wavelength within a range of 600 nm to 2500 nm, such as from 600-900 nm, or 600-700 nm. In some embodiments, the light source is a laser. Suitable light intensities may range from 1 mW/cm ² to 1000 mW/cm ² , such as 1-750 mW/cm ² or 300-700 mW/cm ² , depending on the target site and method of application. Near-infrared light sources can be obtained from commercial sources, including Thorlabs (Newton, NJ), Laser Components, USA (Hudson, NH), ProPho tonix (Salem, NH) and others. In some embodiments, the effective quantity of far-red or NIR light is 10-250 J, such as 10-200 J, 10-150 J, or 10-100 J. In some embodiments, visualization may include techniques such as fluoroscopy, single-molecule localization microscopy (SMLM), photo-activated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), direct stochastic optical reconstruction microscopy (dSTORM), biplane imaging (BP), temporal radial-aperture based intensity estimation (TRABI), fluorescence resonance energy transfer (FRET), and combinations thereof.

In any of the foregoing or following embodiments, the carbamate group may be selected to provide desired stimuli-dependent information. In some embodiments, R ¹ is selected to provide cleavage of the carbamate group by a particular enzyme or chemical trigger. For example, a glutamate-substituted compound can be activated by y-glutamyl transpeptidase (GGT) with high selectivity in vitro and in a metastatic model of ovarian cancer. Table 3 provides several exemplary combinations of enzyme and/or chemical triggers and suitable R ¹ groups for those triggers. Table 3

In any of the foregoing or following embodiments, the biological sample may comprise, or be suspected of comprising a target. In such embodiments, the cyanine carbamate may comprise a group comprising a targeting agent that binds to the target, and detecting fluorescence from the norcyanine indicates presence of the target in the biological sample. In some embodiments, the targeting agent is an antibody, a nanobody, or a peptide. In certain implementations, the targeting agent is an antibody. In some examples, the antibody is panitumumab.

In any of the foregoing or following embodiments, contacting the biological sample with the cyanine carbamate may be performed in vivo. In some embodiments, contacting the biological sample in vivo comprises administering the cyanine carbamate or a pharmaceutical composition comprising the cyanine carbamate to a subject, and detecting fluorescence from the norcyanine is performed via in vivo imaging. The amount of the cyanine carbamate administered may be an amount effective to provide detectable fluorescence from the norcyanine. Administration is performed by any suitable method, e.g., intravenous, intra-arterial, intramuscular, intratumoral, or subcutaneous injection, or oral, intranasal, or sublingual administration.

A suitable period of time for cleavage of the carbamate group from the cyanine carbamate is allowed to elapse. The suitable period of time may further include time for and subsequent protonation of the norcyanine. In some embodiments, the period of time ranges from a few minutes to several days. For instance, the period of time may be from 15 minutes to 7 days, such as from 30 minutes to 48 hours, 30 minutes to 24 hours, 30 minutes to 12 hours, 1 hour to 12 hours, or 3 hours to 12 hours.

The norcyanine is subsequently irradiated by targeted application of a quantity of light having a wavelength in the visible, far-red, or near-infrared range and a selected intensity to a target area of the subject, wherein the quantity of light is sufficient to excite the protonated cleaved compound. When irradiating a target area (e.g., an area proximate a tumor), the effective quantity of far-red or NIR light may be 1-250 J/cm ² , such as 1-250 J/cm ² , such as 5-250 J/cm ² , 10-250 J/cm ² , 10-200 J/cm ² , 10-150 J/cm ² , 10-100 J/cm ² , or 30-100 J/cm ² . Any fluorescence from the protonated cleaved compound in the targeted portion of the subject is detected, thereby diagnosing the subject as having the condition.

In any of the foregoing or following embodiments, certain cells within the biological sample may express enzymes or chemical triggers capable of cleaving the carbamate group from the compound. The norcyanine then may be taken up by the cell and protonated intracellularly. In one non-limiting example, the cleaved compound Sulfo-NorCy7 (below) may be taken up by ovarian cancer cells with strong lysosomal localization, and subsequently protonated in the lysosome. The protonated Sulfo-NorCy7 is detectable by excitation at 710 nm and subsequent fluorescence detection at 760-800 nm. Detection of the protonated compound may diagnose the subject as having ovarian cancer. Sulfo-NorCy7

In certain embodiments, the subject has, or is suspected of having, a cancer characterized in part by overexpression of y-glutamyl transpeptidase, and R ¹ of the compound is . Cancers characterized in part by overexpression of y-glutamyl transpeptidase include, for example, ovarian cancer, cervical cancer, liver cancer, colon cancer, gastric cancer, astrocytic glioma, soft tissue sarcoma, melanoma, and leukemia.

In addition to diagnostic uses, embodiments of the disclosed compounds may be used in theranostic procedures and/or for research uses. For example, embodiments of the disclosed compounds may be used as activatable probes for in vivo imaging, including for optically guided surgical procedures. Additionally, the compounds may be used to non-invasively monitor enzymatic processes and other stimuli in real time in living organisms. When conjugated to a targeting agent, the compounds may also be useful as probes for detecting enzymatic activity at a particular cell type or location of interest. If the cell or location of interest does not express an enzyme or chemical trigger capable of cleaving the carbamate group from the compound, an amount of an effective enzyme or chemical trigger may be administered to the location of interest after the compound comprising the targeting agent has bound to the target cell or location of interest, thereby inducing cleavage of the carbamate group so that the cleaved compound may be protonated and visualized.

V. Representative Embodiments

Certain representative embodiments are exemplified in the following numbered paragraphs.

1. A compound according to Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof: where R ¹ is substituted aryl or substituted heteroaliphatic; R ² is aryl, heteroaryl, heterocycloaliphatic, H, halo, -OR ^a , -NR ^a 2, or a group comprising a conjugatable moiety, a targeting agent, or a drug, where each R ^a independently is H, halo, alkyl, or aryl; Y ¹ and Y ² independently are C(R ^b )2, N(R ^C ), S, O, or Se, wherein each R ^b independently is H, deuterium, aliphatic, aryl, -(OCFFCFbjxOH where x is an integer > 2, trityl, or a group comprising a conjugatable moiety, a targeting agent, or a drug, and each R ^c independently is H, deuterium, aliphatic, or heteroaliphatic; R ³ and R ⁴ independently are H, aliphatic, amino, a sulfonate-containing group, or a group comprising a conjugatable moiety, a targeting agent, or a drug, or R ³ and Y ¹ together with the atoms to which they are bound form a 6-membered aryl ring, and R ⁴ and Y ² together with the atoms to which they are bound form a 6-membered aryl ring;

R ⁵ -R ¹⁰ independently are H, aliphatic, amino, a sulfonate-containing group, or a group comprising a conjugatable moiety, a targeting agent, or a drug; and R ¹¹ and R ¹² independently are H, aliphatic, heteroaliphatic, or a group comprising a conjugatable moiety, a targeting agent, or a drug, or R ¹¹ and R ¹² together with the atoms to which they are bound form a 5-7 membered cycloaliphatic or heterocycloaliphatic ring.

2. The compound of paragraph 1, where the compound has a structure according to Formula IA: where , alkyl, aryl, or a group comprising a conjugatable moiety, a targeting agent, or a drug.

3. The compound of paragraph 2, where: Y ¹ and Y ² independently are C(R ^b )2, where each R ^b independently is aliphatic or aryl; R ² is, H, halo, -OR ^a , -NR ^a 2, or a group comprising a conjugatable moiety, a targeting agent, or a drug; R ⁵ and R ⁸ independently are -SO3", are H.

4. The compound of paragraph 3, where Y ¹ and Y ² are -C(CH ₃ )2-.

5. The compound of paragraph 4, having a structure according to Formula IB, IC, or

ID:

6. The compound of paragraph 1, where the compound has a structure according to Formula II: where R ¹³ -R ¹⁸ independently are H, aliphatic, amino, a sulfonate-containing group, or a group comprising a conjugatable moiety, a targeting agent, or a drug.

7. The compound of paragraph 6, where the compound has a structure according to Formula II A:

absent, -CHR ^d -, -CH ₂ CHR ^d -, -CHR ^d CH ₂ -, or -NR ^d -, where R ^d is H, aliphatic, aryl, or a group comprising a conjugatable moiety, a targeting agent, or a drug.

8. The compound of paragraph 7, where: R ² is phenyl, H, halo, -OR ^a , -NR ^a ₂ , or a group comprising a conjugatable moiety, a targeting agent, or a drug; R ⁵ and R ⁸ are -SO3", s an integer > 1 ; and m is an integer > 1.

10. The compound of any one of paragraphs 1-9, wherein R ¹ is substituted phenyl or substituted heteroaliphatic.

11. The compound of paragraph 10, wherein: R ¹ is , or , -O-alkylaryl, or -O-aryl, where R' is aliphatic and each R" independently is H or aliphatic; R ^s is aliphatic, heteroaliphatic, heterocycloaliphatic, alkylaryl, or heteroalkylaryl; and R ^h is aliphatic.

12. The compound of paragraph 1, wherein R ¹ is

13. The compound of any one of paragraphs 1-12, wherein: R ² is unsubstituted phenyl, , R ^h , H, Cl, -OR ^a , or -NR ^a ₂ ; R ^h is -(CH ₂ ) _m -R ^e , -(CH ₂ ) _m CH(NH ₂ )-R ^e , -(CH ₂ ) _m C(O)R ^e , -(CH ₂ ) _m N(H)R ^e , -(CH ₂ ) _m N(H)C(O)R ^e , -(CH ₂ ) _m C(O)N(H)R ^e , -(CH ₂ ) _m C(O)SR ^e , -C(O)R ^e , -N(H)C(O)R ^e , -N(H)R ^e , or -SR ^e ; R ^e is -OH, phosphoramidite, or a targeting agent, where y is an integer > 1 ; and m is an integer > 1.

14. The compound of paragraph 13, wherein R ² is unsubstituted phenyl.

15. The compound of any one of paragraphs 1-11 or 13, wherein one of R ² -R ¹⁸ , A, Y ¹ , or Y ² comprises a group comprising a targeting agent.

16. The compound of paragraph 15, wherein R ² or A comprises the group comprising the targeting agent.

17. The compound of paragraph 15 or paragraph 16, wherein the targeting agent comprises an antibody.

18. The compound of paragraph 1, wherein the compound is:

19. A pharmaceutical composition comprising a compound, or stereoisomer thereof, according to any one of paragraphs 1-18, and a pharmaceutically acceptable carrier.

20. A method, comprising: contacting a biological sample with a compound according to any one of paragraphs 1-18; exposing the compound to an enzyme or a chemical trigger capable of cleaving -C(O)OCH ₂ R ¹ from the compound to provide a cleaved compound; exposing the cleaved compound to a pH less than 7 to provide a protonated cleaved compound; subsequently exposing the protonated cleaved compound to light having a wavelength greater than 700 nm; and detecting fluorescence from the protonated cleaved compound.

21. The method of paragraph 20, wherein: the biological sample comprises the enzyme, and exposing the compound to the enzyme comprises contacting the biological sample comprising the enzyme with the compound; or the biological sample comprises the chemical trigger, and exposing the compound to the chemical trigger comprises contacting the biological sample comprising the chemical trigger with the compound; exposing the compound to the enzyme or chemical trigger comprises adding the enzyme or the chemical trigger to the biological sample and the compound.

22. The method of paragraph 20 or paragraph 21, wherein the biological sample comprises an environment having a pH less than 7, thereby exposing the cleaved compound to the pH less than 7.

23. The method of any one of paragraphs 20-22, wherein the enzyme or chemical trigger and R ¹ are selected from the following combinations: carboxy esterase (CES 1, 2) and acetylcholinesterase (ACHe) and butyrylcholinesterase (BCHe) and aminopeptidase N and cathepsin B and cathepsin S and cathepsin L and cathepsin K and pantetheinase (Vanin- 1) and caspase 3/7 and caspase 8 and trysin and chymotrypsin and alkaline phosphatase and fibroblast activating protein and dipeptidyl peptidase 4 and furin and galactosidase and granzymes and nuraminidase and leucine aminopeptidase and legumain and neutrophil elastase and gamma glutamyltranspeptidase (GGT) and tyrosinase and lysyl oxidase and

NAD(P)H:quinone oxidoreductase isozyme 1 and monoamine oxidase A or B and cytochormide P450 and nitro reductase and hydrogen peroxide and super oxide and hydrogen sulfide or triphenyl phosphine and hypochlorous acid and peroxynitrite and carbon monoxide and selenol and thiophenol and glutathione, cysteine, or homocysteine and

24. The method of any one of paragraphs 20-23, wherein: the biological sample comprises, or is suspected of comprising, a target; the compound comprises a group comprising a targeting agent that binds to the target; and detecting fluorescence from the protonated cleaved compound indicates presence of the target in the biological sample. 25. The method of paragraph 24, wherein the targeting agent is an antibody.

26. The method of any one of paragraphs 20-25, wherein contacting the biological sample with the compound is performed in vivo.

27. The method of paragraph 26, wherein: contacting the biological sample in vivo comprises administering the compound or a pharmaceutical composition comprising the compound to a subject; and detecting fluorescence from the protonated cleaved compound is performed via in vivo imaging.

28. The method of paragraph 27, wherein the subject has, or is suspected of having, a cancer characterized in part by overexpression of y-glutamyl transpeptidase, and R ¹ of the compound is 29. The method of paragraph 28, wherein the cancer comprises ovarian cancer, cervical cancer, liver cancer, colon cancer, gastric cancer, astrocytic glioma, soft tissue sarcoma, melanoma, or leukemia.

30. The method of any one of paragraphs 20-29, wherein the protonated cleaved compound has a fluorescence intensity at least 100X greater than the compound or cleaved compound.

VI. Examples

General Procedures

Synthesis. All reactions were carried out under an argon atmosphere. Reagents were purchased at a high commercial quality (typically 97 % or higher) and used without further purification, unless otherwise stated. ^! H NMR and ¹³ C NMR spectra were recorded on Bruker spectrometers (at 400 or 500 MHz or at 100 or 125 MHz) and are reported relative to deuterated solvent signals (CDC1 ₃ : ¹ H NMR = 7.24, ¹³ C NMR = 77.0, MeOD: ¹ H NMR = 3.30, ¹³ C NMR = 49.0, DMSO-d ₆ : ¹ H NMR = 2.50, ¹³ C NMR = 39.5). The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, dd = double doublet, dt = double triplet, dq = double quartet, m = multiplet, br = broad. Electrospray ionization mass spectrometry (ESI-MS) data were collected on triple-stage quadrupoleinstrument in a positive mode. Flash column chromatography was performed using reversed phase (100 A, 20- 40-micron particle size, RediSep® Rf Gold® Reversed-phase Cl 8 or C18Aq) and silica on a CombiFlash® Rf 200i (Teledyne Isco, Inc.). LC/MS was performed using a Shimadzu LCMS-2020 SingleQuadrupole utilizing a Kinetex 2.6 pm C18 100 A (2.1 x 50 mm) column obtained from Phenomenex, Inc(Torrance, CA). Runs employed a gradient of 0-90% MeCN/H2O with 0.1% formic acid over 4.5 min at a flow rate of 0.2 mL/min. Preparatory HPLC was performed on Waters HPLC equipped with Waters 515 HPLC Pump, Waters 2998 Photodiode Array Detector, Waters 2545 Binary Gradient Module and Waters 2767 Sample Manager. The HPLC was installed with Luna 10 pm C18 column (100 x 30 mm) and each run employed 10-95% or 60-95% CH ₃ CN/H ₂ O gradient with 0.1% TFA over 10 mins. Absorbance curves were obtained on a Shimadzu UV-2550 spectrophotometer operated by UVProbe 2.32 software. Fluorescence traces were recorded on a PTI QuantaMaster steady state spectrofluorometer operated by FelixGX 4.2.2 software, with 5 nm excitation and emission slit widths, and a 0.1 s integration rate. Analytical LC analyses were collected from Agilent 1260 Infinity Quaternary LC module using Poroshell 120 EC-C18 2.7 pM (4.6 x 50 mm) column in 5-95% CH ₃ CN/water gradient with 0.1% formic acid over 25 minutes. Microplates were read on Synergy Mx microplate reader. All statistical analyses were carried out by Graphpad Prism version 9.0 (Graphpad Software).

Absorbance and Fluorescence Measurements. Absorbance curves were obtained on a Shimadzu UV- 2550 spectrophotometer operated by UVProbe 2.32 software. Fluorescence traces were recorded on a PTIQuantaMaster steady-state spectrofluorometer operated by FelixGX 4.2.2 software, with 5 nm excitation and emission slit widths, and a 0.1 s integration rate.

Determination of Molar Absorption Coefficients. Molar absorption coefficients (e) were determined in PBS (pH 7.2) or acetate buffer (pH 4.5) using Beer’s law, from plots of absorbance vs. concentration (1 - 10 pM). Spectra were recorded with disposable micro-UV-Cuvette holder with 10 mm path length, with absorbance at the highest concentration < 0.20. Three independent readings were taken at each concentration.

Absolute Fluorescence Quantum Yields. Absolute fluorescence quantum yields (OF) were measured in PBS (pH 7.2), acetate buffer (pH 4.5), or methanol + 0.1% formic acid using a Horiba fluorimeter QM-8075-11-C equipped with R928 PMT point detector and an integrating sphere to determine photons absorbed and emitted by a sample. Probe was excited at 710 nm and emission was collected from 730-850 nm or 730-900 nm. Measurements were carried out a concentration with optical density of less than 0.1 in buffer or solvent and self-absorption corrections were performed using the instrument software.

Determination of pKa. Absorption and emission spectra were recorded in different buffers (pH 3.00 to pH 10.0) using a spectrophotometer and fluorometer, respectively. Following buffers were used in the study: Citrate buffers (pH 3.05 - 3.50), acetate buffers (pH 3.75 to 5.75) and PBS (20 mM, pH 6.00 - 10.0).

Kinetics of release by Staudinger reaction. CyBam-N (10 pM) and PPh (10 eq) added in PBS: MeOH (1:1) at pH 7.4 or pH 5.2. A spectral scan reading was taken after every 5 min for 1 h. monitored at different intervals using microplate reader (300 - 800 nm). Three independent experiments were carried out.

GGT probe. All the assays were conducted at 37 °C for 30 mins unless otherwise stated. The pH was adjusted to pH 5.2 after completion of the assay as described below. The absorbance was measured using plate reader and fluorescence was measured using a fluorimeter.

1. Kinetics of Activation. CyBam-y-Glu (20 pM) was dissolved PBS buffer (IX, pH = 7.4) at increasing concentration of GGT (0 - 800 U/L) at 37 °C. KM was calculated after incubating CyBam-y-Glu (2.5 - 50 pM) with 100 U/L. 2. Inhibition of GGT. 6-Diazo-5-oxo-L-norleucine (DON; 1 mM) and GGsTop (0.5 mM) were incubated with GGT (100 U/L) for 1 h prior to addition of CyBam-y-Glu (20 pM).

3. Specificity against GGT. CyBam-y-Glu (20 pM) was incubated with GGT (100 U/L), LAP (leucine amino peptidase; 800 U/L) and PLE (pig liver esterase; 800 U/L).

4. pH and cell culture media stability. CyBam-y-Glu (20 pM) was incubated with DMEM with 10% FBS, 100% FBS and pH (4.5, 5.5, 6.5 and 7.5) for 18 h. The stability of the compound was monitored by observing the absorbance of the probe every 1 h.

5. Activity at different pH. Different pH (6.0, 6.5, 7.0, 7.5 and 8.0) was generated using Gibco PBS (IX). The probes were incubated for 30 mins with GGT (100 U/L).

Fluorescent Imaging in Live Cells. Briefly, 25,000 cells were seeded on Greiner Bio-One CELLview™ Cell Culture Slides and allowed to adhere overnight.

Fluorescent Imaging in Live Cells of NorCy7 Compounds: 15,000 cells/well (MDA-MB-468) were seeded on Greiner Bio-One CELLview™ Cell Culture Slides (10 compartments and allowed to adhere overnight. Cells in NorCy7 (5 pM) were incubated with for 6 h in media (with FBS), followed by wash with DPBS (twice). Nucleus was stained with NucBlue™ Live Ready Probes (Invitrogen; 1 drop/ 500 pL) for 15 min. Live cell microscopy was carried out in DMEM phenol red free media. The images were captured using two different lasers: nucleus (405, blue channel) and NorCy7 library (700 nm, red channel).

Subcellular Localization. Sulfo-NorCy7 (20 pM) in RPMI media was incubated for 4 h followed by addition of Lysosome (Lysotracker Green DND 26; Invitrogen), Mitochondria (Mitotracker Green FM; Invitrogen) for 1 h. Afterwards, the cells were washed with PBS (pH 7.4) and nucleus was stained with NucBlue™ Live Ready Probes (Invitrogen) for 15 mins. Following concentrations of probes were used in the study: Mitotracker Green (500 nM), Lysotracker Green (75 nM) and NucBlue (1 drop/500 pL). Live cell microscopy was carried out in DMEM phenol red free media. The images were captured using three different lasers: nucleus (405, blue channel), mitochondria and lysosome (488 nm, green channel) and Sulfo-NorCy7 (640 nm, red channel).

2V-Me-NorCy7 (10 pM) in DMEM/10%FBS media was incubated for 6 h followed by addition of Lysosome (Lysotracker Green DND 26; Invitrogen), Mitochondria (Mitotracker Green FM; Invitrogen) for 1 h. Afterwards, the cells were washed with DPBS (pH 7.2) and nucleus was stained with NucBlue™ Live Ready Probes (Invitrogen) for 15 mins. Following concentrations of probes were used in the study: Mitotracker Green (500 nM), Lysotracker Green (75 nM) and NucBlue (1 drop/500 pL). Live cell microscopy was carried out in DMEM phenol red free media. The images were captured using three different lasers: nucleus (405, blue channel), mitochondria and lysosome (488 nm, green channel) and 2V-Me-NorCy7 (640 nm, red channel).

Competition experiment. The cells were divided in three groups: Group 1 contained untreated cells, Group 2 contained blocked probes and Group 3 contained nonselective and non-cleavable probe. Group 2 was treated with DON (1 mM) and GGsTop (1 mM), 1 h prior to study. CyBam-y-Glu (20 pM) was incubated in Group 1 and 2, whereas CyBam-N.C was incubated in Group 3. After 3 h the cells were washed, and nucleus was stained as described above. The media was changed to HBSS for live cell study.

Flow Cytometry Analysis. 300,000 cells/well were seeded on 12 well plate (Coming Costar). The cells were treated with compounds, described as below, follow by cleavage using cell dissociation free buffer and spun down at 200 ref using centrifuge. The cells were suspended in DMEM, phenol red free and live cells counting was performed on flow cytometer. Geometric mean fluorescence intensity was measured at least in three independent trials (at least 10,000 cells counted). Flow cytometry data was processed using FlowJo. CyBam-y-Glu: The cells were divided in three groups: Group 1 was untreated, Group 2 contained blocked probes and Group 3 contained nonselective and non-cleavable probe. Group 2 was treated with DON (1 mM) and GGsTop (1 mM), 1 h prior to study. CyBam- y-Glu (20 pM) was incubated in Group 1 and 2, whereas CyBam-N.C was incubated in Group 3. After 3 h the cells were washed twice with PBS (pH 7.4).

Enzyme and Protein Assays, y- Glutamyltranspeptidase from equine kidney (Type VI, 5-12 units/mg solid), leucine aminopeptidase, microsomal from porcine kidney (Type IV-S, 10-40 units/mg protein), Cathepsin B from human liver and Esterase from porcine liver (>15 units/mg solid) were purchased from Millipore Sigma.

Cytotoxicity Assay. 5000 SHIN-3 cells/well were seeded on 96 well plate and allowed to adhere overnight. Stock solutions of Sulfo-NorCy7and CyBam-y-Glu (20 mM in DMSO) were diluted with protein-free medium (PFHM-II) to make desired final concentrations varying from 0.01 to 80 pM. The cells were incubated with the desired concentration for 72h. The cell viabilities were calculated using alamarBlue assay. Briefly, 10 pL of alamarBlue™ Cell Viability Reagent (Invitrogen) was incubated for 1 h and fluorescence was measured with excitation at 560 nm and emission wavelength at 590 nm with a Microplate Reader. The viability of each cell line in response to the treatment with tested compounds was calculated as: % dead cells = 100 - (OD treated/OD control) x 100.

5000 cells/well (MDA-MB-468 and MCF-7) were seeded on 96 well plate and allowed to adhere overnight. Stock solutions (10 mM in DMSO) of all six compounds in NorCy7 library were diluted in media for each respective cell lime to make desired final concentrations varying of 1 , 5 and 10 pM. The cells were incubated with the desired concentration for 24h. The cell viabilities were calculated using alamarBlue assay. Briefly, 10 pL of alamarBlue™ Cell Viability Reagent (Invitrogen) was incubated for 1 h and fluorescence was measured with excitation at 560 nm and emission wavelength at 590 nm with a Microplate Reader. The viability of each cell line in response to the treatment with tested compounds was calculated as: % dead cells = 100 - (OD treated/OD control) x 100.

ROS generation. PC-3 cells (250,000) were seeded in 12 well plate (Coming Costar) and allowed to adhere overnight. The cells were divided in five groups: Group 1 - doxorubicin (20 pM), Group 2 CyBam- B(OHji (20 pM), Group 3 CyBam-P(OPh)2 (20 pM), Group 4 coincubation of CyBam-B(OH)2 (20 pM) + doxorubicin (20 pM) and Group 5 coincubation of CyBam-P(OPh)2 (20 pM) + doxorubicin (20 pM). The groups were incubated with compounds for 12 hours. Afterwards, cells were washed with PBS twice, cleaved from plate using cell dissociation free buffer and spun down at 200 ref using centrifuge. Geometric mean fluorescence intensity was measured in four independent trials (-10,000 cells counted). Flow cytometry data was processed using FlowJo.

Cellular assays. SHIN-3 cells were generously provided by Dr. Hisataka Kobayashi. MDA-MB-468 and PC-3 were bought from ATCC. SHIN-3 cells were cultured in Roswell Park Memorial Institute media (RPMI) containing 10% FBS and 1% PS. MDA-MB-468 cells were grown in Dulbecco's Modified Eagle'smedium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-dtreptomycin (PS). PC-3 cells were grown in F-12K media containing 10% FBS and 1%PS. Cells were grown in a cell culture incubator at 37°C in a humidified atmosphere containing 5% CO ₂ . Cells were grown in T-75 or T-175 culture flask till 70% confluency before splitting into next passage.

MDA-MB-468, MDA-MB-231 and JIMT-1 cells were grown in Dulbecco's Modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (PS). MCF-7 cells were grown in Eagle's Minimal Essential Medium (EMEM) containing 10% fetal bovine serum (FBS), 1% penicillin- streptomycin (PS) and 1% L-glutamine. Cells were grown in a cell culture incubator at 37 °C in a humidified atmosphere containing 5% CO ₂ . Cells were grown in T-75 or T-175 culture flask till 70% confluency before splitting into next passage following trypsinization with 5% trypsin-EDTA. The cells were evaluated for molecular testing of biological materials by animal health diagnostic laboratory at Frederick National Laboratory for Cancer Research. The results confirmed the absence of the following agents within the cells: Ectromelia virus (ECT), Mouse rotavirus (EDIM), Lymphocytic coriomeningitis virus (LCMV), Lactic dehydrogenase elevating virus (LDHV), Mouse adenovirus (MAD), Mouse cytomegalovirus (MCMV), Mouse hepatitis virus (MHV), Mouse norovirus (MNV), Mouse parvovirus (MPV), Minute virus of mice (MVM), Mycoplasma spp. (MYCO), Polyoma virus (POLY), Pneumonia virus of mice (PVM), Reovirus 3 (REO3), Sendai virus (SEN), Theiler's murine encephalomyelitis virus (TMEV).

Flow Cytometry was performed at Flow Cytometry Core (CCR) using BD LSRII. The fluorescence signal in the near infrared region was excited using 633 nm laser (18 mW) and emission detected on APC-Cy7 detectors (780/60). The flow cytometry data was processed using FlowJo software. Geometric mean fluorescent intensity was calculated in APC-Cy7 channel.

Confocal imaging was carried out at Optical Microscopy and Image Analysis Lab (OMAL) on Andor spinning disk confocal microscope on Leica DMi8 base. DAPI was imaged at Blue channel (excitation 405 nm, emission 450/50 nm) and Cy7 fluorophores were images at red channel (excitation 640 or 710 nm, emission 810/90). The images were taken at 63x/1.4 oil immersed objective. The images were processed using Fiji software. The images were taken at 63x/1.4 oil immersed objective. The images were processed using Fiji software.

SHIN-3 metastatic tumor model. Animal experiments were approved by the Institutional Animal Care and Use Committee of the NIH and conducted in compliance with the Guide for the Care and Use of Laboratory Animal Resources and the National Research Council. The tumors were implanted by intraperitoneal injection of 2 x 10 ⁶ SHIN- 3 -Zs Green cells suspended in 300pl of PBS into female athymicnude mice (Athymic NCr-nu/nu, strain #553). Experiments with tumor-bearing mice were performed afterl5 days of implantation of SHIN-3-ZsGreen models, when disseminated peritoneal implants grew to about 1 mm in size. 100 |1M of 300 |1L (30 nmol) was injected intraperitoneally, and mice were sacrificed at 1, 3 and 6 h time interval. Acquisition for GFP signal (ZsGreen): Excitation: band-pass filter from 445 - 490 nm; Emission long-pass filter over 515 nm; Acquisition: 500 - 720 nm in 10 nm steps. Acquisition for Cy7 channel signal (probe): Excitation: band-pass filter from 710 - 760 nm; Emission long- pass filter over 800 nm; Acquisition: 780 - 950 nm in 10 nm steps. Fluorescence images were automatically acquired in 10 nm increments with constant exposure. The images, which consisted of an autofluorescence spectrum and the spectra from GFP and Cy7 channel, were reconstructed using Maestro software, based on their unique spectral patterns.

In vivo studies: Studies were performed according to Animal Care and Use committee guidelines at Frederick National Laboratory for Cancer Research (Frederick, MD). Frederick National Laboratory for Cancer Research is accredited by American Association for Accreditation of Laboratory Animal Care (AAALAC) International and follows the Public Health Service Policy for the Care and use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the “Guidefor Care and Use of Laboratory Animals” (National Research Council; 2011; National Academies Press; Washington, D.C.).

Fluorescence was longitudinally monitored employing the IVIS spectrum imager (PerkinElmer Inc, Waltham, MA). Images were acquired and processed using Living Image software. Mice body temperature were maintained constant at 37 °C during the imaging procedure with a heated pad located under the anesthesia induction chamber, imaging table, and post procedure recovery cage. All mice were anesthetized in the induction chamber with 3% isoflurane with filtered (0.2 pm) air at 1 liter/minute flow rate for 3-4 minutes and then modified for imaging to 2% with 02 as a carrier with a flow rate of 1 liter/minute. Static 2D images were acquired with the following parameters: excitation filter 745 ± 15 nm, emission filter 800 ± 10 nm, f/stop2, medium binning (8x8) and auto exposure (typically 1-60 seconds or 1-120 seconds). 4-7- week-old female athymic nude mice were purchased from Charles River Laboratories International, Inc. (Frederick, MD).

MDA-MB-468 tumor model. 5xl0 ⁶ human breast cancer cells (MDA-MB-468) in 100 pL of Hanks Balanced Salt Solution (HBSS) were subcutaneously injected in the right flank of the mice. Tumors were monitored daily until they reach 4-6 mm in the longest diameter or 25-35 mm ³ . In vivo studies were initiated 10 days post cell injection of the mice.

JIMT-1 tumor model. 5xl0 ⁶ human breast cancer cells (JIMT-1) in 100 pL of MatrigekPBS (1:1) were injected subcutaneously in the inguinal memory fat pad of mice. Tumors were monitored daily until they reached 200-250 mm ³ . In vivo studies were initiated 21 days post cell injection.

Data Analysis Living Image software was used for image analysis. Using white light images, the tumors were identified, and regions of interest (ROI) were drawn over the tumor, liver, and ear (used as background) to quantify in vivo data. Fitted ROIs were drawn over each organ to quantify ex vivo data. Total radiance efficiency within each ROI was normalized by the corresponding area of the ROI. This normalized radiance efficiency was used to calculate ratios (tumor to background, tumor to liver and liver to background). Statistics (unpaired 2-tail Student’s t-test) were carried out using Prism 9.

Panitumumab, m276-SL and IgG conjugation. 1 mg of 25 mg/mL of antibody and 10X of CyBam and CyLBams (10 mM stock in DMSO) was added to 300 pL of PBS (pH 7.2) in 1.5 mL Eppendorf tube. The mixture was covered in aluminum foil and stirred for 2 h on orbital shaker. The reaction mixture was eluted through a pH 7.4 PBS equilibrated Zeba spin DS column (7K MWCO, Thermo Fisher Scientific) to remove unreacted free dye. This was followed by buffer exchange and further purification using slide- A-Lyzer MINI Dialysis Devices (10K MWCO, 0.5mL, Thermofisher) in PBS (IX, pH 7.4). The buffer was replaced twice, at 1 and 3 h time interval. Purified conjugate was collected in 0.5 mL Eppendorf. The antibody conjugates were concentrated using Amicon® Ultra Centrifugal Filter Devices (10k MWCO; Millipore Sigma) by centrifuging at 10,000 rom for 10 mins. The degree of labeling (DOL; dye to antibody ratio) was determined by measuring absorption at 280 nm and 430 nm. A correction factor of 0.03 at 280 nm was used in the calculations. DOL and protein concentrations were measured by the following equations. The antibody conjugates were stored at 4 °C. No changes in absorbance spectra were monitored over 28 days.

Eq. 1. DOL = (A43o/£dye)/[(A28O - 0.03 X A430)]/ Sprotein

Eq. 2. protein cone (mg/mL) = [(A280 - 0.03 x A430)/ ^protein] x MW _pr otein x dilution factor Fluorescent Imaging in Live Cells with Pan Conjugates: 15,000 cells/well (MDA-MB-468 or MCF-7) were seeded on Greiner Bio-One CELLview™ Cell Culture Slides (10 compartments and allowed to adhere overnight. Compounds in Panitumumab conjugates (50 pg; DOL 4) were incubated with for 24 h in respective media (with FBS), followed by wash with DPBS (twice). Nucleus was stained with NucBlue™ Live Ready Probes (Invitrogen; 1 drop/ 500 pL) for 15 min. Live cell microscopy was carried out in DMEM phenol red free media. The images were captured using two different lasers: nucleus (405, blue channel) and Cy7 (640 nm, red channel).

Flow Cytometry Analysis of Panitumumab, m276-SL and IgG Conjugates. 300,000 cells/well were seeded on 12 well plate (Corning Costar). The cells were treated with Panitumumab conjugates (50 pg; DOL 4), m276-SL or IgG (80 pg; DOL 4) for 6 and 24 h (as required), followed by wash with cold DPBS (twice). The cells were cleaved using Gibco™ Cell Dissociation Buffer, enzyme- free, PBS. and were centrifuged at 200 ref using Eppendorf centrifuge 5424R. The cells were resuspended in cold DPBS and cells were counted on flow cytometer. Geometric mean fluorescence intensity was measured at least in three independent trials (~ 10,000 cells counted).

Flow Cytometry Analysis of NorCy7 compounds. 300,000 cells/well were seeded on 12 well plate (Coming Costar). The cells were treated with NorCy7 library (5 pM) for 1, 3 and 6 h, followed by wash with cold DPBS (twice). The cells were cleaved using Gibco™ Cell Dissociation Buffer, enzyme-free, PBS. and were centrifuged at 200 ref using Eppendorf centrifuge 5425. The cells were resuspended in cold DPBS and cells were counted on flow cytometer. Geometric mean fluorescence intensity was measured at least in three independent trials (~ 10,000 cells counted).

Example 1

Compound Synthesis

Sulfo-NorCy7, was prepared as shown below.

Compound SI. To a 20 mL microwave vial, Vilsmeier reagent (400 mg, 1.11 mmol), phenyl boronic acid (269.6 mg, 2.22 mmol) and K3PO4 (236.2 mg, 2.11 mmol) were added sequentially. Pd(PPh3)4 (45mg, 0.038 mmol) was added in glove box and the vial was sealed. Subsequently, degassed DMF:H2O (5:1) was added to the solution and heated at 90 °C for 18 h. The solvent was removed and filtered through celite to remove inorganic compounds. Crude mixture was taken forward to next step.

Sulfo-NorCy7. Compounds S2 was synthesized according to literature procedure (Park et al., Bioconjug Chem 2012, 23(3):350-362). To a 100 mL round bottom flask, SI (800 mg, 2.19 mmol), S2 (1.1 g, 4.39 mmol) and sodium acetate (NaOAc, 360 mg, 4.39 mmol) were added sequentially in 20 mL of absolute ethanol. The reaction mixture was heated at 70 °C for 6 h. The flask was cooled on ice to room temperature and solvent was removed. The crude reaction mixture was purified on reversed phase chromatography (0-50% MeCN:H ₂ O). (320 mg, 21%). ^! H NMR (500 MHz, DMSO) 57.62 - 7.55 (m, 5H), 7.53 (dd, J = 8.0, 1.6 Hz, 2H), 7.21 (dd, J = 6.4, 2.9 Hz, 2H), 7.07 - 6.97 (m, 4H), 6.10 (d, J = 14.1 Hz, 2H), 2.59 (t, J = 6.3 Hz, 4H), 1.92 (p, J = 6.2 Hz, 2H), 1.23 (s, 12H). ¹³ C NMR (126 MHz, DMSO) 5 160.8, 144.7, 141.7, 140.2, 138.4, 134.5, 130.7, 130.4, 130.1, 128.9, 128.7, 127.8, 126.6, 120.4, 116.7, 114.4, 111.0, 49.05, 36.25, 26.91, 25.01, 21.48. HRMS (ESI) calculated for C36H37N2O6S2 [M+H] ⁺ 657.2088; observed: 657.2094.

The dye exhibited two forms in biologically relevant conditions, an unprotonated quenched form, Sulfo-NorCy7 (Z _a bs = 520 nm @ pH 7.4) and a protonated fluorescent form, Sulfo- NorCy7-[H ⁺ ] (Z _a bs = 755 nm @ pH 4.5), with a pKa of 5.2. After examining several conditions, it was determined that exposure of Sulfo-NorCy7 and 4-nitrophenyl carbonates to NaH or CS2CO3 in DMF afforded the corresponding carbamate products.

CyBam-Ns was prepared from Sulfo-NorCy7 and 1 in reasonable yield following purification by reversed-phase chromatography (Scheme 1).

Scheme 1

Compound 1 was synthesized according to a known procedure (van Brakel et al. , Bioconjug Chem 2008, 19(3):714-718). To a 5 mL microwave vial, Sulfo-NorCy7 (20 mg, 0.030 mmol), NaH (1.4 mg, 0.061 mmol) were added sequentially to dry DMF (1.5 mF) and stirred for 1 h. S3 (19 mg, 0.060 mmol) was dissolved in dry DMF (1.5 mL) and added dropwise over 10 mins. Reaction progress was monitored by LC/MS. After 2 h, the reaction was complete by LC/MS and was quenched using H2O (1 mL). The resulting mixture was purified by reversed phase HPLC (60-95% MeCN:H2O, 0.1% TFA) to give CyBam-Ns as purple amorphous solid (12.5 mg, 50%). ¹ H NMR (500 MHz, DMSO) 57.79 (d, J = 1.6 Hz, 1H), 7.70 - 7.65 (m, 2H), 7.64 - 7.58 (m, 5H), 7.47 - 7.44 (m, 2H), 7.41 (d, J = 1.8 Hz, 1H), 7.30 (d, J = 13.1 Hz, 1H), 7.22 (td, J = 7.5, 7.0, 1.8 Hz, 4H), 6.70 (d, J = 15.8 Hz, 1H), 6.36 (d, J = 13.0 Hz, 1H), 5.38 (s, 2H), 2.61 (t, J = 6.2 Hz, 2H), 2.30 (t, J = 6.2 Hz, 2H), 1.86 - 1.80 (m, 2H), 1.29 (s, 6H), 1.09 (s, 6H). ¹³ C NMR (125 MHz, DMSO) 5 183.4, 153.8, 152.2, 148.1, 145.2, 143.5, 140.2, 139.5, 138.5, 138.3, 136.8, 133.8, 132.3, 131.5, 130.0, 129.1, 128.6, 128.5, 126.8, 125.9, 120.5, 119.9, 119.8, 119.6, 119.2, 116.8, 115.7, 115.0, 114.5, 108.4, 68.38, 52.01, 45.09, 29.03, 26.91, 24.90, 24.74, 24.68, 21.39. HRMS (ESI) calculated for C44H42N5O8S2 [M+H] ⁺ 832.2468; observed: 832.2469.

Compound S4. Compound S3 was prepared according to a known procedure (Huang et al., J Med Chem 2017, 60(21):8847-8857). To a 20 mL microwave vial, S3 (100 mg, 0.20 mmol) and 4-nitrophenyl chloroformate (45 mg, 0.22 mmol) were added sequentially to dry THF (10 mL) and stirred on ice for 30 mins. Triethylamine (57 pL, 0.40 mmol) was added dropwise, and the solution was stirred for 12 h. The resulting white precipitate were filtered, and the solvent was evaporated. The product was purified by normal phase chromatography (ethyl acetate (EtO Ac) .’hexanes; 0 - 100%, 24 g SiCh). Product containing fractions were combined, evaporated, and triturated with ether to give compound S4 as a white amorphous solid (100 mg, 77% yield). ¹ H NMR (500 MHz, CDC13) 5 8.33 - 8.20 (m, 4H), 7.81 - 7.71 (m, 3H), 7.60 (dt, J = 17.1, 8.7 Hz, 5H), 7.39 (q, J = 8.8, 7.0 Hz, 4H), 7.31 (d, J = 7.2 Hz, 2H), 5.61 (d, J = 8.1 Hz, 2H), 5.24 (s, 2H), 4.51 - 4.37 (m, 4H), 4.20 (t, J = 6.8 Hz, 2H), 3.76 (s, 3H), 3.49 (d, J = 4.2 Hz, 1H), 2.38 (dt, J = 28.9, 10.2 Hz, 4H), 2.17 (s, 2H), 1.99 - 1.88 (m, 2H). ¹³ C NMR (125 MHz, CDC1 ₃ ) 5 172.2, 170.3, 155.5, 152.4, 145.3, 143.7, 143.4, 141.3, 129.8, 127.8, 127.1, 127.0, 125.3, 125.0, 124.9, 121.7, 120.1, 120.0, 119.7, 70.68, 67.21, 53.14, 52.80, 47.18, 33.90, 30.01. HRMS (ESI) calculated for C35H32N3O10 [M+H] ⁺ 654.2082; observed: 654.2096.

CyBam-v-Glu: To a 5 mL microwave vial, Sulfo-NorCy7 (30 mg, 0.046 mmol) and CS2CO3 (30 mg, 0.092) were added sequentially to dry DMF (2 mL) and stirred for 3 h. Compound S4 (60 mg, 0.092 mmol) was dissolved in dry DMF (2 mL) and added dropwise over 10 mins. The reaction mixture was monitored at different intervals using LC/MS. After 3 h, the reaction was quenched using H2O (1 mL). The crude product concentrated and then dissolved in 20% piperidine in DMF (1 mL). After 1 h, the solvent was evaporated and LiOH (1.1 mg; 0.046 mmol) was added to THF:H2O (1:1) on ice. The reaction was monitored by LC/MS. After 1 h the reaction was complete and the crude mixture was purified by reversed phase HPLC (60-95% MeCN:H2O, 0.1% TFA) to provide CyBam- y -Glu as a purple amorphous solid (13 mg, 29%). ¹ H NMR (500 MHz, DMSO) 5 8.29 (d, J = 5.4 Hz, 3H), 7.67 (t, J = 8.5 Hz, 3H), 7.58 - 7.54 (m, 3H), 7.50 (d, J = 8.6 Hz, 2H), 7.42 (d, J = 1.8 Hz, 1H), 7.38 (d, J = 1.8 Hz, 1H), 7.37 (s, 1H), 7.28 (s, 1H), 7.25 (s, 1H), 7.19 (d, J = 1.7 Hz, 1H), 7.17 (t, J = 1.5 Hz, 1H), 6.61 (d, J = 16.1 Hz, 2H), 6.14 (d, J = 12.8 Hz, 2H), 5.32 (s, 2H), 2.37 (t, J = 1.9 Hz, 1H), 2.28 (t, J = 5.9 Hz, 3H), 2.15 - 2.08 (m, 4H), 1.81 (s, 3H), 1.13 (d, J = 42.3 Hz, 12H). ¹³ C NMR (125 MHz, DMSO) 5 184.1, 171.3, 170.3, 152.3, 145.1, 139.9, 139.6, 139.1, 138.5, 136.9, 133.5, 130.3, 130.1, 130.1, 128.9, 128.1, 126.1, 120.3, 119.8, 119.5, 117.9, 115.6, 114.9, 113.2, 108.5, 68.62, 52.16, 52.00, 44.71, 31.99, 28.87, 26.08, 24.99, 24.19. HRMS (ESI) calculated for C49H51N4O11S2 [M+H] ⁺ 935.2992; observed: 935.2990.

Compound S6. Compound S5 was synthesized according to known procedure (Blaser et al., Helvetica Chimica Acta 2001, 84(7):2119-2131). To 20 mL microwave vial, S5 (500 mg, 1.99 mmol) and 4-nitrophenyl chloroformate (400 mg, 2.38 mmol) were added sequentially in dry THF (10 mL) and stirred on ice for 30 mins. Triethylamine (550 pL, 4.00 mmol) was added dropwise and the solution was stirred for 12 h. White precipitate were filtered, and the solvent was evaporated. The crude mixture redissolved in CH2CI2 and purified by normal phase silica column (EtOAcm-hexane; 0 - 100%, 24 g SiCL) to give S7 white color product (620 mg, 74% yield). ^! H NMR (500 MHz, DMSO) 5 10.01 (s, 1H), 8.32 (d, J = 8.6 Hz, 2H), 7.67 - 7.52 (m, 5H), 7.38 (dd, J = 24.2, 8.3 Hz, 3H), 5.24 (s, 2H), 4.71 (s, 1H), 3.60 (s, 4H), 2.51 (s, 3H), 1.84 (s, 3H). ¹³ C NMR (125 MHz, DMSO) 5 173.52, 171.22, 155.76, 152.43, 145.64, 140.17, 129.93, 129.43, 125.88, 123.09, 119.49, 119.41, 70.77, 51.75, 46.70, 35.70, 33.08, 20.77. HRMS (ESI) calculated for C20H21N2O6 [M+H] ⁺ 417.1288; observed: 417.1292.

CyBam-N.C. To a 5 mL microwave vial, Sulfo-NorCy7 (30 mg, 0.046 mmol) and NaH (2.1 mg, 0.092) were added sequentially in dry DMF (2 mL) and stirred for 30 min. Compound S7 (38.2 mg, 0.092 mmol) was dissolved in dry DMF (2 mL) and added dropwise over 10 mins. The reaction mixture was monitored at different intervals using LC/MS. After 1 h, the reaction was quenched using H2O (1 mL). The crude mixture was concentrated. The resulting mixture was dissolved in THF:H2O (1:1), cooled to 4 °C, and LiOH (1.1 mg; 0.046 mmol) was added . The reaction monitored by LC/MS. After 1 h the reaction was complete. The crude mixture was purified by reversed phase HPLC (60-95% MeCN:H2O, 0.1% TFA) to provide CyBam-N.C as purple amorphous solid (8.0 mg, 17%). ¹ H NMR (500 MHz, DMSO) 57.62 (dd, J = 8.6, 6.4 Hz, 3H), 7.52 (dd, J = 9.5, 1.6 Hz, 2H), 7.50 - 7.43 (m, 5H), 7.43 - 7.39 (m, 2H), 7.36 (dd, J = 8.5, 1.8 Hz, 1H), 7.30 (d, J = 1.8 Hz, 1H), 7.27 (d, J = 8.1 Hz, 2H), 7.12 - 7.06 (m, 4H), 6.52 (d, J = 16.1 Hz, 3H), 6.03 (d, J = 12.8 Hz, 2H), 5.23 (s, 2H), 2.32 (t, J = 7.4 Hz, 2H), 2.23 (t, J = 7.4 Hz, 2H), 2.13 (d, J = 5.4 Hz, 2H), 1.79 - 1.74 (m, 2H), 1.70 (d, J = 6.6 Hz, 3H), 1.08 (s, 5H), 1.00 (s, 5H). ¹³ C NMR (125 MHz, DMSO) 5 184.1, 174.6, 171.3, 158.5, 152.3, 145.1, 140.2, 139.7, 139.3, 138.5, 137.0, 133.4, 130.4, 130.2, 129.8, 128.9, 128.0, 126.0, 125.8, 119.8, 119.5, 119.4, 118.4, 114.9, 108.5, 68.69, 52.19, 44.68, 35.92, 33.42, 28.86, 24.95, 24.07, 20.94. HRMS (ESI) calculated for C49H50N3O11S2 [M+H] ⁺ 920.2809; observed: 920.2880.

Compound S8 (reported in J Mater Chem B 2017, 5:5278-5283, Chem Commun 2020, 56:5819-5822, and Dyes and Pigments 2004, 61:103-107, without synthetic details). Compound S7 was synthesized according to known procedure (Wolf et al., J Org Chem 2020, 85(15):9751-9760). To a 20 mL microwave vial S7 (327.8 mg, 0.93 mmol) and Vilsmeier reagent (150 mg, 0.46 mmol) were added sequentially in dry ethanol (20 mL). The solution was heated at 70 °C for 6 h. The reaction was cooled, and solvent was evaporated. The crude mixture was redissolved in H2O and purified over reversed phase chromatography (0-50% MeCN:H2O) to provide S8 as green amorphous solid (150 mg, 38%). ' H NMR (500 MHz, DMSO) 5 8.26 (d, 7= 13.9 Hz, 2H), 7.81 (d, 7 = 1.6 Hz, 2H), 7.66 (dd, 7= 8.2, 1.6 Hz, 2H), 7.40 (d, 7= 8.4 Hz, 2H), 6.34 (d, 7 = 14.1 Hz, 2H), 4.22 (t, 7= 7.3 Hz, 4H), 2.71 (t, 7= 6.0 Hz, 4H), 2.21 (t, 7 = 7.3 Hz, 4H), 1.90 - 1.82 (m, 2H), 1.74 (p, 7= 7.5, 7.0 Hz, 5H), 1.68 (s, 12H), 1.56 (p, 7 = 7.4 Hz, 4H), 1.41 - 1.35 (m, 4H). ¹³ C NMR (125 MHz, DMSO) 5 174.8, 172.8, 148.4, 145.9, 143.3, 142.5, 140.9, 127.0, 126.7, 120.3, 111.1, 102.4, 49.47, 44.22, 33.92, 27.87, 27.21, 26.29, 26.08, 24.66, 20.82. LRMS: calculated for C42H53CIN2O10S2 [M+H] ⁺ 843.2; observed: 843.2.

Compound S9. To a 10 mL microwave vial resorcinol (26mg, 0.24 mmol) and K2CO3 (49.2, 0.36 mmol) were added sequentially in dry DMF (5 mL). The solution was stirred at 25 °C for 15 mins followed by addition of S8 (100 mg, 0.11 mmol). The solution was first stirred at 25 °C for 30 mins and then heated at 60 °C for 1.5 h. The reaction was cooled, and solvent was evaporated. The crude mixture was redissolved in H2O and purified over reversed phase chromatography (0-50% MeCN:H2O) to provide S9 as blue amorphous solid (30.1 mg, 47%). ^! H NMR (500 MHz, DMSO) 5 8.57 (d, J = 14.7 Hz, 2H), 7.74 (dd, J = 8.3, 1.6 Hz, 1H), 7.58 (s, 2H), 7.50 (d, 7 = 8.5 Hz, 1H), 6.96 (d, 7= 2.4 Hz, 1H), 6.86 (dd, 7= 8.5, 2.2 Hz, 1H), 6.59 (d, 7 = 4.0 Hz, 1H), 6.49 (d, 7 = 14.8 Hz, 2H), 4.37 (t, 7 = 7.3 Hz, 2H), 2.74 - 2.68 (m, 3H), 2.68 - 2.63 (m, 2H), 2.21 (t, 7 = 7.3 Hz, 2H), 1.84 (t, 7= 6.0 Hz, 2H), 1.75 (s, 6H), 1.59 - 1.52 (m, 2H), 1.45 - 1.35 (m, 3H). ¹³ C NMR (125 MHz, DMSO) 5 174.8, 161.7, 154.6, 147.5, 141.9, 141.7, 135.1, 129.7, 126.8, 126.3, 120.4, 114.9, 114.4, 112.6, 102.4, 50.60, 44.94, 33.86, 28.76, 27.94, 27.49, 26.00, 24.62, 20.45. HRMS (ESI) calculated for C31H34NO7S [M+H] ⁺ 564.2046; observed: 564.2050.

Compound S10. To a 5 mL microwave vial S9 (10 mg, 0.021 mmol) was dissolved in dry DMF (2 mL). Acetyl chloride (2.49 pL, 0.035 mmol) and El N (7.41 pL. 0.053 mmol) were added sequentially and solution stirred at 25 °C for 1 h. The crude mixture was purified by reversed phase Prep HPLC (10-95% MeCN:H2O,0.1% TFA) to provide S10 as blue amorphous solid (6.2 mg, 8.85 (d, 7 = 15.0 Hz, 1H), 8.08 (d, 7 = 1.6 Hz, 1H), 8.02 (dd, 7= 8.4, 1.7 Hz, 1H), 7.66 (dd, 7 = 8.4, 1.5 Hz, 1H), 7.58 (d, 7 = 8.4 Hz, 1H), 7.45 - 7.40 (m, 2H), 7.15 (dd, 7 = 8.5, 2.2 Hz, 1H), 6.63 (d, 7 = 14.9 Hz, 2H), 4.43 (t, 7 = 7.5 Hz, 2H), 2.84 (t, 7 = 5.9 Hz, 2H), 2.77 (t, 7= 6.1 Hz, 2H), 2.37 (s, 3H), 2.01 (s, 1H), 2.00 - 1.94 (m, 4H), 1.87 (s, 6H), 1.78 - 1.71 (m, 3H), 1.58 - 1.52 (m, 3H). ¹³ C NMR (125 MHz, MeOD) 5 179.4, 175.6, 169.1, 161.2,

146.9, 144.5, 142.2, 132.4, 129.9, 128.1, 127.02, 120.2, 119.7, 119.3, 115.1, 112.6, 109.5, 104.8, 50.94, 45.02, 33.07, 28.83, 27.07, 26.73, 25.82, 24.14, 23.59, 20.08, 19.50. HRMS (ESI) calculated for C33H36NO8S [M+H] ⁺ 606.2155; observed: 606.2156. CyBam-B(OH)2. Compound Sil was synthesized according to a known procedure Skarbek et al., Bioorg Chem 2019, 91:103158). To a 5 mL microwave vial, Sulfo-NorCy7 (30 mg, 0.045 mmol), NaH (2.1 mg, 0.091 mmol) were added sequentially to dry DMF (1.5 mL) and stirred for 1 h. Sil (36.5 mg, 0.091 mmol) was dissolved in dry DMF (1.5 mL) and added dropwise over 10 mins. Reaction progress was monitored by LC/MS. After 2 h, the reaction was complete by LC/MS and was quenched using H2O (1 mL). The resulting mixture was purified by reversed phase HPLC (60- 95% MeCN:H2O, 0.1% TFA) to give CyBam-B(OH)2 as purple amorphous solid (13.5 mg, 37%). NMR (500 MHz, MeOD) 57.81 (dd, J = 9.9, 1.6 Hz, 2H), 7.68 (dd, J = 8.4, 3.2 Hz, 3H), 7.53 - 7.45 (m, 6H), 7.40 (d, J = 7.9 Hz, 2H), 7.35 (d, J = 8.1 Hz, 1H), 7.30 (d, J = 13.1 Hz, 1H), 7.26 - 7.23 (m, 1H), 7.20 (dd, J = 8.1, 6.5 Hz, 2H), 7.04 (dd, J = 6.6, 2.9 Hz, 2H), 6.53 (d, J = 15.6 Hz, 1H), 6.45 (d, J = 13.0 Hz, 1H), 5.29 (s, 2H), 2.45 (t, J = 6.2 Hz, 2H), 2.19 (t, J = 6.2 Hz, 2H), 1.72 (t, J = 6.2 Hz, 2H), 1.21 (s, 6H), 1.03 (s, 6H). ¹³ C NMR (125 MHz, MeOD) 5 184.5, 158.6,

155.0, 153.1, 152.1, 145.0, 142.8, 141.2, 140.9, 140.7, 139.2, 138.0, 137.0, 136.5, 136.0, 134.1,

134.0, 133.9, 133.5, 133.2, 129.5, 128.5, 128.3, 128.0, 127.1, 127.0, 126.3, 125.6, 120.4, 119.6,

115.2, 114.3, 109.6, 108.4, 68.87, 66.76, 51.60, 45.09, 38.99, 28.22, 24.41, 24.38, 24.35, 20.99.

HRMS (ESI) calculated for C44H44BN2O10S2 [M+H] ⁺ 835.2552; observed: 835.2533.

Compound S13. Compound S12 was synthesized according to known procedure (Chem Commun (Camb) 2017, 53(10): 1653- 1656). To 20 mL microwave vial, S5 (250 mg, 0.77 mmol) and 4-nitrophenyl chloroformate (171 mg, 0.85 mmol) were added sequentially in dry THF (10 mL) and stirred on ice for 30 mins. Triethylamine (600 pL, 1.54 mmol) was added dropwise and the solution was stirred for 12 h. The solvent was removed, the crude mixture redissolved in CH2CI2 and purified by normal phase silica column (EtOAcm-hexane; 0 - 100%, 24 g SiCh) to give S7 white color product (320 mg, 84% yield). ^! H NMR (500 MHz, MeOD) 5 8.31 - 8.27 (m, 1H), 7.93 (ddt, J = 12.7, 7.1, 1.4 Hz, 4H), 7.66 - 7.62 (m, 2H), 7.56 (td, J = 7.5, 3.7 Hz, 4H), 7.46 - 7.38 (m, 4H), 7.27 (dd, J = 8.7, 1.4 Hz, 2H), 5.23 (s, 2H). ¹³ C NMR (125 MHz, MeOD) 5 155.6, 152.4, 151.0, 150.94, 145.4, 132.8, 132.8, 131.8, 131.5, 131.4, 130.3, 129.9, 129.2, 128.7, 128.7, 128.6,

128.5, 124.8, 121.8, 120.7, 120.6, 69.61. HRMS (ESI) calculated for C26H21NO7P [M+H] ⁺ 490.1057; observed: 490.1050.

CyBam-P(OPh)2. To a 5 mL microwave vial, Sulfo-NorCy7 (30 mg, 0.045 mmol), NaH (2.1 mg, 0.091 mmol) were added sequentially to dry DMF (1.5 mL) and stirred for 1 h. S13 (45 mg, 0.091 mmol) was dissolved in dry DMF (1.5 mL) and added dropwise over 10 mins. Reaction progress was monitored by LC/MS. After 2 h, the reaction was complete by LC/MS and was quenched using H2O (1 mL). The resulting mixture was purified by reversed phase HPLC (60-95% MeCN:H2O, 0.1% TFA) to give CyBam-P(OPh)2 as purple amorphous solid (15.5 mg, 38%). ' H NMR (500 MHz, MeOD) 57.85 - 7.80 (m, 6H), 7.72 - 7.67 (m, 1H), 7.60 - 7.57 (m, 2H), 7.56 - 7.51 (m, 3H), 7.50 (dd, J = 6.1, 4.2 Hz, 4H), 7.45 (td, J = 7.6, 3.6 Hz, 5H), 7.37 - 7.33 (m, 2H), 7.23 (dd, J = 8.5, 1.3 Hz, 2H), 7.12 - 7.09 (m, 2H), 6.57 (d, J = 15.6 Hz, 1H), 6.49 (d, J = 13.0 Hz, 1H), 5.24 (s, 2H), 2.51 (t, J = 6.1 Hz, 2H), 2.31 (t, J = 6.2 Hz, 2H), 1.83 - 1.76 (m, 2H), 1.26 (s, 6H), 1.05 (s, 6H). ¹³ C NMR (125 MHz, MeOD) 5 184.6, 155.2, 153.1, 151.9, 151.2, 151.1, 145.0, 142.8, 141.3, 140.8, 140.7, 139.1, 138.0, 137.0, 136.2, 134.1, 132.9, 132.9, 131.9, 131.8, 131.8,

131.5, 131.4, 130.9, 130.8, 130.7, 130.3, 129.6, 129.2, 128.7, 128.7, 128.6, 128.5, 128.3, 128.3,

128.2, 127.0, 125.6, 120.8, 120.8, 120.4, 119.6, 115.1, 114.2, 109.4, 108.3, 68.00, 51.67, 45.06,

28.17, 24.44, 24.35, 21.02. HRMS (ESI) calculated for C56H52N2O10PS ’ [M+H] ⁺ 1007.2828; observed: 1007.2796.

Synthesis of NorCy7s, Sulfo-NorCy7, Pip-NorCy7, /V-Me-Pip-NorCy, H-NorCy7, and OMe-NorCy7

Compound S2. Compound SI was synthesized according to literature procedure (Reynolds, et al., J Org Chem 1977, 42(5): 885-888). To a 20 mL microwave vial, SI (400 mg, 2.32 mmol), phenylboronic acid (562.7 mg, 4.65 mmol) and K3PO4 (493.6 mg, 2.32 mmol) were added sequentially. Pd(PPh3)4 (95 mg, 0.081 mmol) was added in glove box and the vial was sealed under nitrogen. Subsequently, degassed DMF:H2O (5:1; 12 mL) was added to the solution and heated at 90 °C for 18 h. The solvent was removed and filtered through celite to remove inorganic compounds. Crude mixture was taken forward to next step.

Compound S3, S4 and SulfoNorCy7 were synthesized according to a previous published procedure (Usama et al., JACS 2021, 143(150:5674-5679).

Compound S5. To a 20 mL microwave vial compound S3 (1200 mg, 5.04 mmol) and

POCh (1.4 mL, 15.1 mmol) were refluxed for 2 h. The reaction was cooled on ice for 15 mins and n-hexane (10 mL) was added to remove excess POCI3. The supernatant was removed carefully. The red thick red liquid was dried under high vacuum overnight to get solid compound (S5). The crude mixture was carried into to the next steps without purification.

Compound S6. To a 20 mL microwave vial compound S5 (500 mg; 1.94) was dissolved in MeCN (10 mL) and cooled down to 5 °C on ice for 15 mins. Piperidine (960 pL; 9.73 mmol) and EtsN (1 mL) were added sequentially on ice. The reaction mixture was stirred on ice for 15 mins, followed by at room temperature for 1 h. The solvent was removed, and crude reaction mixture passed through celite to remove salts. The crude was purified by normal phase chromatography (0-10% MeOFPCFhCh) to give S6 as mustard yellow solid (495 mg, 84% over two steps). Rf value = 0.6 ( ) 57.82 (t, J = 1.1 Hz, 1H), 7.68 - 7.64 (m, 2H), 2.88 (t, J = 5.5 Hz, 4H), 2.28 (s, 3H), 1.56 - 1.51 (m, 4H), 1.38 - 1.32 (m, 3H), 1.31 (s, 6H).

¹³ C NMR (125 MHz, DMSO) 5 193.1, 157.6, 147.4, 132.3, 128.2, 121.5, 120.1, 54.41, 47.03, 25.12, 23.32, 22.53, 15.90. HRMS (ESI) calculated for C16H23N2O2S [M+H] ⁺ 307.1475; observed: 307.1467. Compound S7. To a 20 mL microwave vial compound S5 (1000 mg; 3.89 mmol) was dissolved in MeCN (10 mL) and cooled down to 5 °C on ice for 15 mins. 1 -methylpiperazine (2.15 mL; 19.5 mmol) and Et3N (2 mL) were added sequentially on ice. The reaction mixture was stirred on ice for 15 mins, followed by at room temperature for 1 h. The solvent was removed, and crude reaction mixture passed through celite to remove salts. The crude was purified by normal phase chromatography (0-10% MeOH:CH2C12) to give S7 as mustard yellow solid (500 mg, 84% over two steps). Rf value = 0.3 (10% MeOH/DCM). NMR (400 MHz, DMSO) 57.81 (d, J = 1.6 Hz, 1H), 7.69 - 7.62 (m, 2H), 2.87 (s, 4H), 2.32 (t, J = 4.9 Hz, 4H), 2.27 (s, 3H), 2.09 (s, 3H), 1.30 (s, 6H). 13C NMR (101 MHz, DMSO) 5 193.23, 157.75, 147.47, 131.56, 128.47, 121.64, 120.15, 54.43, 53.90, 46.22, 45.71, 22.52, 15.90. HRMS (ESI) calculated for C16H24N3O2S [M+H] ⁺ 322.1584; observed: 322.1571.

Pip-NorCy7. To a 20 mL microwave vial, S6 (1.75 g, 3.51 mmol) S2 (250 mg, 1.10 mmol) and TsOH (603.72 mg, 3.51 mmol) were added sequentially in 20 mL of absolute ethanol. The reaction mixture was heated at 70 °C for 18 h. The flask was cooled on ice to room temperature and solvent was removed. The crude reaction mixture was purified on normal phase chromatography (0-10% MeOH:CH2C12) to give Pip- NorCy7 (240 mg, 27%). Rf value = 0.3 (10% MeOH/DCM). ¹ H NMR (500 MHz, MeOD) 57.64 - 7.57 (m, 4H), 7.52 (qd, J = 4.9, 1.7 Hz, 3H), 7.21 - 7.13 (m, 3H), 7.13 - 7.06 (m, 3H), 6.12 (d, J = 14.0 Hz, 2H), 3.23 (p, J = 1.6 Hz, 5H), 2.86 (t, J = 5.5 Hz, 8H), 2.61 (t, J = 6.2 Hz, 4H), 1.98 - 1.90 (m, 2H), 1.39 - 1.31 (m, 5H), 1.26 (s, 12H). ¹³ C NMR (125 MHz, MeOD) 5 173.2, 163.1, 147.2, 145.4, 141.4, 138.2, 132.8, 130.9, 128.9, 128.4, 124.5, 121.8, 111.2, 48.85, 46.76, 25.94, 24.95, 24.68, 23.13, 21.11. HRMS (ESI) calculated for C46H55N4O4S2 [M+H] ⁺ 791.3659; observed: 791.3687.

/V-Me-Pip-NorCy7. To a 20 mL microwave vial, S7 (1.5 g, 4.67 mmol) S2 (500 mg, 2.34 mmol) and TsOH (1.2 g, 7.1 mmol) were added sequentially in 20 mL of absolute ethanol. The reaction mixture was heated at 70 °C for 18 h. The flask was cooled on ice to room temperature and solvent was removed. The crude reaction mixture was purified on normal phase chromatography (0-20% MeOH:CH2C12) to give -Me-pip- NorCy7 (410 mg, 20%). Rf value = 0.2 (10% MeOH/DCM). MHz, MeOD) 57.75 (dd, J = 8.2, 1.8 Hz, 3H), 7.65 - 7.58 (m, 3H), 7.31 - 7.27 (m, 3H), 7.27 - 7.19 (m, 4H), 6.25 (d, J = 14.0 Hz, 2H), 3.98 - 3.86 (m, 4H), 3.61 - 3.52 (m, 4H), 3.23 (s, 4H), 2.90 (s, 6H), 2.72 (t, J = 6.2 Hz, 4H), 2.07 - 2.00 (m, 3H), 1.37 (s, 12H). ¹³ C NMR (125 MHz, MeOD) 5 163.7, 146.0, 141.7, 138.1, 133.2, 129.9, 129.3, 129.2, 128.4,

128.3, 121.9, 117.1, 114.8, 111.5, 52.54, 48.86, 43.31, 41.99, 25.91, 24.67, 21.06. HRMS (ESI) calculated for C46H57N2O6S2 [M+H] ⁺ 821.3877; observed: 821.3878.

24% yield

Compound S8. In a 500 mL round bottom flask, DMF/CH2CI2 (18 mL/18 mL) was cooled down on ice for 20 mins. POCI3 (16.29 mL, 174.1 mmol) was added dropwise over 10 mins to the solution and reaction mixture was stirred for 1 h on ice. N-Methyl-4-piperidone (5.43 mL, 44.24 mmol) was added dropwise over 10 mins and the solution was heated at 70 °C for 6 h. The excess POCh was quenched by adding distilled H2O (20 mL) dropwise on ice. The solvent was removed on rotavap. Ethanol (20 mL) and aniline (6 mL) were premixed and poured in the solution. Thick slurry solution was stirred at room temperature for 30 mins followed by addition of cone. HC1 (7 mL). The precipitate is filtered and washed with ethyl acetate and ether to give compound S8 as a reddish black solid (8.2 g, 70% yield). ¹ H NMR (400 MHz, DMSO) 57.46 - 7.34 (m, 15H), 7.20 (t, J = 7.1 Hz, 3H), 4.65 (s, 4H), 4.21 (s, 4H), 2.99 (s, 6H). ¹³ C NMR (100 MHz, DMSO) 5 186.8, 145.2, 141.2, 137.7, 130.2, 130.1, 130.1, 128.4, 123.8, 123.7, 118.04 116.7, 116.7, 99.07, 50.94, 50.31, 41.78, 41.68. LRMS (ESI) calculated for CI ₄ HI ₆ C1N ₂ O and C20H21CIN3 [M+H] ⁺ 263.0 and 338.1; observed: 263.1 and 338.2.

Compound S9. To a 20 mL microwave vial, S8 (400 mg, 1.51 mmol), phenyl boronic acid (371 mg, 3.04 mmol) and K3PO4 (637 mg, 3.01 mmol) were added sequentially. Pd(PPh3) ₄ (61 mg, 0.042 mmol) was added in glove box and the vial was sealed under nitrogen. Subsequently, degassed DMF:H2O (5:1; 12 mL) was added to the solution and heated at 90 °C for 18 h. The solvent was removed and filtered through celite to remove inorganic compounds. The crude reaction mixture was purified on normal phase chromatography (0-15% MeOH:CH ₂ Cl ₂ ) to give compound S9 (110 mg, 24%). Rf value = 0.4 (10% MeOH/DCM). ' H NMR (500 MHz, DMSO) 5 7.50 - 7.39 (m, 4H), 7.29 - 7.20 (m, 4H), 7.20 - 7.14 (m, 2H), 7.07 (t, J = 7.4 Hz, 1H), 6.81 (d, J = 7.8 Hz, 3H), 6.68 (d, J = 8.0 Hz, 2H), 6.21 (d, J = 11.9 Hz, 1H), 4.11 (d, J = 5.3 Hz, 1H), 3.46 (d, J = 27.2 Hz, 4H), 3.18 (d, J = 5.0 Hz, 3H). ¹³ C NMR (125 MHz, DMSO) 5 158.9, 153.3, 149.1,

142.8, 136.7, 130.7, 130.5, 129.9, 129.9, 129.8, 129.7, 129.5, 128.7, 128.3, 125.9, 125.2, 120.9,

120.8, 116.2, 114.5, 113.2, 53.70, 53.06, 49.07, 45.81. LRMS (ESI) calculated for C20H21N2O and C26H26N3 [M+H] ⁺ 305.1 and 380.2; observed: 305.2 and 380.3.

/V-Me-NorCy7. To a 20 mL microwave vial, S9 (110 mg, 0.36 mmol) 2,3,3-trimethylindolenine (174 mg, 1.08 mmol) and TsOH (1.2 g, 7.1 mmol) were added sequentially in 15 mL of absolute ethanol. The reaction mixture was heated at 70 °C for 18 h. The flask was cooled on ice to room temperature and solvent was removed. The crude reaction mixture was purified on normal phase chromatography (0-20% MeOH:CH ₂ Cl ₂ ). (140 mg, 75%). ^! H NMR (500 MHz, MeOD) 57.70 - 7.66 (m, 3H), 7.41 (d, J = 7.5 Hz, 2H), 7.37 - 7.34 (m, 2H), 7.34 - 7.30 (m, 2H), 7.28 (d, J = 5.5 Hz, 2H), 7.21 (t, J = 7.5 Hz, 2H), 7.15 (d, J = 7.9 Hz, 2H), 6.07 (d, J = 14.6 Hz, 1H), 4.69 (d, J = 14.1 Hz, 2H), 4.20 (d, J = 14.7 Hz, 2H), 3.27 (s, 3H), 1.37 (s, 12H). ¹³ C NMR (125 MHz, MeOD) 5 176.8, 156.1, 144.9, 140.9, 140.7, 135.6, 129.8, 129.0, 128.8, 128.4, 124.8, 122.2, 118.4, 117.1, 114.8, 111.9, 51.26, 49.61, 41.83, 25.49. HRMS (ESI) calculated for C36H38N3 [M+H] ⁺ 512.3060; observed: 512.3071.

H-NorCy7. To a 20 mL microwave vial, S2 (200 mg, 0.93 mmol) 2,3,3-trimethylindolenine (594 mg, 3.73 mmol) and TsOH (643 mg, 3.73 mmol) were added sequentially in 15 mL of absolute ethanol. The reaction mixture was heated at 70 °C for 18 h. The flask was cooled on ice to room temperature and solvent was removed. The crude reaction mixture was purified on normal phase chromatography (0-10% MeOH:CH2Ch) to give a green amorphous solid (143 mg, 77%). Rf value = 0.3 (10% MeOH/DCM). ¹ H NMR (500 MHz, MeOD) 57.65 - 7.58 (m, 4H), 7.33 (d, J = 7.4 Hz, 3H), 7.26 (dd, J = 7.4, 5.0 Hz, 4H), 7.17 (s, 2H), 7.13 (d, J = 7.5 Hz, 2H), 7.05 (d, J = 7.8 Hz, 2H), 2.67 (t, J = 5.1 Hz, 4H), 2.05 - 1.99 (m, 2H), 1.30 (s, 12H). ¹³ C NMR (125 MHz, MeOD) 5 141.3, 140.5, 138.7, 130.6, 129.8, 129.6, 128.2, 128.1, 127.9, 123.7, 122.3, 122.1, 117.8, 115.5, 111.6, 110.9, 54.41, 42.37, 26.09, 25.62, 24.62, 21.20, 17.29, 15.86, 11.74. HRMS (ESI) calculated for C36H37N2 [M+H] ⁺ 497.2951; observed: 497.2949.

OMe-NorCy7. To a 20 mL microwave vial, S2 (250 mg, 1.11 mmol) 2,3,3-trimethyl-5-methoxy-3h-indole (664 mg, 3.51 mmol) and TsOH (604 mg, 3.51 mmol) were added sequentially in 15 mL of absolute ethanol. The reaction mixture was heated at 70 °C for 18 h. The flask was cooled on ice to room temperature and solvent was removed. The crude reaction mixture was purified on normal phase chromatography (0-10% MeOH:CH2C12) to give OME-NorCy7 as green amorphous solid (220 mg, 33%). Rf value = 0.2 (10% MeOH/DCM). ¹ H NMR (500 MHz, DMSO) 57.52 - 7.46 (m, 3H), 7.14 - 7.10 (m, 2H), 7.03 (d, J = 2.6 Hz, 2H), 6.95 (d, 7= 8.6 Hz, 2H), 6.85 (d, 7= 14.3 Hz, 2H), 6.75 (dd, 7 = 8.6, 2.6 Hz, 2H), 5.97 (d, 7 = 14.3 Hz, 2H), 3.66 (s, 6H), 2.48 (t, 7= 6.1 Hz, 4H), 1.84 (q, 7 = 6.0 Hz, 2H), 1.12 (s, 12H). ¹³ C NMR (125 MHz, DMSO) 5 157.2, 138.9, 135.4, 130.1, 129.4, 128.8, 128.3, 113.76, 112.6, 109.8, 56.10, 49.24, 27.00, 24.88, 21.55. HRMS (ESI) calculated for C38H41N2O2 [M+H] ⁺ 557.3163; observed:

557.3158.

VC-CyBam. To a 10 mL microwave vial Sulfo-NorCy7 (60 mg, 0.091 mmol) and CS2CO3 (59.44 mg, 0.183) were added sequentially to dry DMF (3 mL) and stirred for 1 h. Subsequently,

Boc-Val-Cit-PABA- PNB (140 mg, 0.183 mmol, obtained from BroadPharm, Cat:BP-24175) was dissolved in dry DMF (3 mL) and added dropwise over 15 mins. The reaction mixture was stirred overnight. The reaction mixture was monitored at different intervals using LC/MS. After 6 h, the reaction was quenched using H2O (1 mL). The crude product concentrated and then dissolved in TFA:CH2Ch (1 mL) on ice. After 1 h, the solvent was evaporated, followed by addition of glutaric anhydride (4.30 mg, 0.037 mmol) and DIPEA (7.45 pL, 0.056 mmol) in dry DMF. The reaction mixture was stirred at room temperature. The reaction was monitored by LC/MS. After 3 h the reaction was complete. The reaction mixture was purified by reversed phase HPLC (10-95% MeCN:H2O, 0.1% TFA) to give VC-CyBam as amorphous purple solid (28 mg, 30% yield). ' H NMR (500 MHz, DMSO) 5 8.08 (d, J = 7.6 Hz, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.64 (d, J = 8.5 Hz, 2H), 7.55 (d, J = 1.6 Hz, 1H), 7.49 (dd, J = 7.8, 1.8 Hz, 2H), 7.45 - 7.42 (m, 2H), 7.37 (dd, J = 8.5, 1.8 Hz, 1H), 7.32 - 7.28 (m, 2H), 7.13 (d, J = 4.0 Hz, 1H), 7.09 (dt, J = 6.0, 1.5 Hz, 2H), 6.54 (d, J = 16.0 Hz, 2H), 6.05 (d, J = 13.0 Hz, 2H), 5.23 (s, 1H), 4.40 - 4.35 (m, 2H), 3.33 (d, J = 5.6 Hz, 3H), 3.29 (t, J = 5.5 Hz, 3H), 2.82 (s, 1H), 2.66 (s, 2H), 2.22 (t, J = 7.4 Hz, 2H), 2.15 (dt, J = 14.9, 7.4 Hz, 7H), 2.10 - 2.05 (m, 2H), 1.92 (dq, J = 12.8, 6.6 Hz, 2H), 1.49 (dd, J = 7.4, 4.1 Hz, 2H), 1.39 (dq, J = 6.4, 3.1, 2.7 Hz, 3H), 1.33 (dd, J = 6.9, 3.1 Hz, 3H), 1.20 - 0.98 (m, 12H), 0.83 - 0.75 (m, 6H). ¹³ C NMR (125 MHz, DMSO) 5 184.2, 174.8, 174.7, 174.6, 172.4, 171.8, 171.2, 170.2,

162.8, 159.6, 152.3, 145.1, 139.9, 139.8, 139.2, 138.6, 137.1, 133.5, 130.5, 130.1, 130.0, 128.6,

120.6, 119.7, 119.5, 118.3, 115.9, 114.9, 113.6, 58.08, 53.64, 52.12, 46.25, 44.74, 36.26, 34.74,

33.50, 33.47, 33.21, 31.98, 31.24, 30.88, 28.89, 26.56, 25.79, 24.54, 24.22, 21.31, 20.83, 20.42,

19.72, 18.68. HRMS (ESI) calculated for C60H70N7O14S2 [M+H] ⁺ 1176.4417; observed: 1176.4434.

VC-CyBam-NHS. To a 10 mL microwave vial, VC-CyBam (10 mg, 0.0085 mmol), TSTU (7.7 mg, 0.025 mmol) and DIPEA (4.4 pL, 0.025 mmol) were added sequentially to dry DMF (3 mL). The progress of the reaction was monitored by LC/MS. After 1 h, the reaction was complete. The reaction mixture was purified by reversed phase HPLC (10-95% MeCN:H2O, 0.1% TFA) to give VC-CyBam-NHS as amorphous purple solid (8.6 mg, 80% yield). HRMS (ESI) calculated for C64H73N8O16S2 [M+H] ⁺ 1273.4580; observed: 1273.4579.

NC-CyBam-NHS. Compound NC-CyBam was synthesized according to our reported procedure. ² To a 10 mL microwave vial, NC-CyBam (5.0 mg, 0.0065 mmol), TSTU (5.9 mg, 0.019 mmol) and DIPEA (2.5 pL, 0.019 mmol) were added sequentially to dry DMF (3 mL). The progress of the reaction was monitored by LC/MS. After 1 h, the reaction was complete. The reaction mixture was purified by reversed phase HPLC (10-95% MeCN:H2O, 0.1% TFA) to give N.C-CyBam-NHS as amorphous purple solid (4.1 mg, 72% yield). HRMS (ESI) calculated for C53H53N4O13S2 [M+H] ⁺ 1017.3045; observed: 1017.3031. VC-CyLBam. To a 10 mL microwave vial 2V-Me-NorCy7 (30 mg, 0.059 mmol) and

CS2CO3 (38 mg, 0.11 mmol) were added sequentially to dry DMF (2 mL) and stirred for 1 h. Subsequently, Boc-Val-Cit-PABA- PNB (140 mg, 0.18 mmol) was dissolved in dry DMF (2 mL) and added dropwise over 15 mins. The reaction mixture was stirred overnight. The reaction mixture was monitored using LC/MS and quenched using H2O (1 mL). The crude product concentrated and then dissolved in TFA:CH2C12 (1 mL) on ice. After 1 h, the solvent was evaporated, followed by addition of glutaric anhydride (2.5 mg, 0.022 mmol) and DIPEA (5.7 pL, 0.033 mmol) in dry DMF. The reaction mixture was stirred at room temperature and was monitored by LC/MS. After 3 h the reaction was complete. The reaction mixture was purified by reversed phase HPLC (10-95% MeCN:H2O, 0.1% TFA) to give VC-CyLBam as amorphous dark blue solid (7.9 mg, 60% yield). ^J H NMR (500 MHz, MeOD) 5 8.04 - 7.96 (m, 1H), 7.92 (d, J = 8.2 Hz, 1H), 7.80 - 7.74 (m, 1H), 7.74 - 7.59 (m, 4H), 7.55 - 7.47 (m, 3H), 7.44 (t, J = 7.0 Hz, 1H), 7.38 (ddd, J = 19.0, 10.7, 5.6 Hz, 3H), 7.32 (d, J = 8.6 Hz, 1H), 7.30 - 7.22 (m, 2H), 7.21 - 7.15 (m, 1H), 6.95 (t, J = 20.2 Hz, 2H), 6.72 (t, J = 16.7 Hz, 1H), 5.39 (d, J = 11.5 Hz, 1H), 5.32 (d, J = 11.4 Hz, 1H), 5.24 (dd, J = 11.4, 8.8 Hz, 1H), 4.85 - 4.72 (m, 2H), 4.65 (t, J = 14.7 Hz, 2H), 4.58 - 4.53 (m, 1H), 4.51 (dp, J = 13.4,

4.5, 3.5 Hz, 1H), 4.26 (d, J = 6.2 Hz, 1H), 4.18 (dd, J = 10.3, 7.3 Hz, 2H), 4.00 (d, J = 14.4 Hz, 1H), 3.17 (dq, J = 14.6, 7.2 Hz, 3H), 3.07 (s, 1H), 3.02 (s, 1H), 2.96 (s, 1H), 2.46 - 2.38 (m, 3H), 2.38 - 2.28 (m, 3H), 2.01 - 1.87 (m, 4H), 1.69 - 1.54 (m, 4H), 1.40 - 1.28 (m, 6H), 1.22 (d, J = 5.0 Hz, 2H), 1.13 - 1.03 (m, 5H), 1.03 - 0.89 (m, 4H). ¹³ C NMR (125 MHz, MeOD) 5 183.6, 175.7, 175.5,

175.5, 175.1, 174.6, 174.5, 174.4, 173.3, 173.1, 172.8, 171.9, 171.4, 163.5, 152.2, 144.7, 144.6,

140.9, 139.9, 139.6, 138.8, 138.7, 135.3, 135.2, 133.6, 133.4, 130.1, 129.8, 129.5, 129.1, 128.9,

128.5, 127.7, 127.5, 127.2, 124.6, 122.6, 122.5, 122.1, 121.8, 121.5, 121.4, 119.8, 119.5, 117.5,

117.3, 115.8, 114.9, 112.6, 105.1, 104.9, 60.2, 60.1, 59.78, 59.18, 54.50, 53.90, 53.80, 53.73, 52.28, 45.86, 45.77, 38.54, 35.59, 34.21, 33.81, 32.75, 32.71, 32.64, 32.42, 30.20, 30.11, 30.06, 29.90,

28.78, 28.62, 28.56, 28.29, 28.19, 26.99, 23.15, 20.80, 20.22, 19.89, 18.49, 18.47, 18.44, 18.41,

17.80, 17.68, 17.57, 17.37. HRMS (ESI) calculated for CeoHviNsOs [M+H] ⁺ /2 516.2731; observed: 516.2740.

VC-CyLBam-NHS. To a 10 mL microwave vial, VC-CyLBam (8.0 mg, 0.0077 mmol), TSTU (4.7 mg, 0.015 mmol) and DIPEA (4.05 pL, 0.023 mmol) were added sequentially to dry DMF (3 mL). The progress of the reaction was monitored by LC/MS. After 1 h, the reaction was complete. The reaction mixture was purified by reversed phase HPLC (10-95% MeCN:H2O, 0.1% TFA) to give VC-CyLBam-NHS as amorphous dark blue solid (6.5 mg, 75% yield). LRMS (ESI) calculated for C64H74N9O10 [M+H] ⁺ 1128.5; observed: 1128.4.

Compound S10. To a 100 mL round bottom flask 5-(tert-Butoxy)-5-oxopentanoic acid

(840 mg, 4.46 mmol) and HATU (1.69 g, 4.46 mmol) were added sequentially in dry THF and stirred on ice for 10 mins. Subsequently, 4-aminobenzyl alcohol (500 mg. 4.06 mmol) and DIPEA (1.4 mL, 8.1 mmol) were sequentially added to the reaction mixture and stirred for 12 h. Afterwards, the solvent was evaporated, and crude mixture was dissolved in EtOAc. The organic layer was washed twice with 1 N HC1, sat. NaHCO and brine. The organic layer was dried over Na2SO4 and dried to get product as yellow fluffy power (1.11 g, 82%). ^! H NMR (500 MHz, DMSO) 57.56 - 7.51 (m, 2H), 7.24 - 7.19 (m, 2H), 5.09 (t, J = 5.7 Hz, 1H), 4.43 (d, J = 5.7 Hz, 2H), 2.70 (s, 1H), 2.33 (t, J = 7.4 Hz, 2H), 2.25 (t, J = 7.4 Hz, 2H), 1.80 (q, J = 7.4 Hz, 2H), 1.41 (s, 9H). ¹³ C NMR (125 MHz, DMSO) 5 170.3, 168.8, 136.2, 135.4, 125.2, 117.1, 77.90, 60.96, 53.26, 33.53, 32.45, 26.12, 18.85. HRMS (ESI) calculated for C18H28N3O5 [M+H] ⁺ 366.2023; observed: 366.2031.

Compound Sil. To a 20 mL microwave vial, S10 (300 mg, 1.02 mmol) and 4-nitrophenyl chloroformate (308 mg, 1.53 mmol) were added sequentially to dry THF (15 mL) and stirred on ice for 30 mins. Triethylamine (285 pL, 0.40 mmol) was added dropwise, and the solution was stirred for 12 h. The resulting white precipitate was filtered, and the solvent was evaporated. The product was purified by normal phase chromatography (EtO Ac .’hexanes; 0 - 100%) to give compound Sil as a white amorphous solid (276 mg, 58% yield). Rf value = 0.5 (1:1 EtOAc/n-hexane). ^! H NMR (500 MHz, DMSO) 5 8.35 - 8.30 (m, 2H), 7.67 - 7.62 (m, 2H), 7.61 - 7.56 (m, 2H), 7.43 - 7.38 (m, 2H), 5.24 (s, 2H), 2.36 (t, J = 7.4 Hz, 2H), 2.26 (t, J = 7.4 Hz, 2H), 1.84 - 1.77 (m, 2H), 1.41 (s, 9H). ¹³ C NMR (125 MHz, DMSO) 5 170.3, 169.1, 153.6, 150.3, 143.5, 138.1, 127.8, 127.3, 123.6, 120.9, 117.3, 77.91, 68.63, 33.56, 32.40, 26.11, 18.76. HRMS (ESI) calculated for C25H31N4O9 [M+H] ⁺ 531.2086; observed: 531.2089.

Compound S12. To a 10 mL microwave vial 2V-Me-NorCy7 (30 mg, 0.059 mmol) and CS2CO3 (38.23 mg, 0.11) were added sequentially to dry DMF (2 mL) and stirred for 1 h. Subsequently, compound Sil (53 mg, 0.12 mmol) was dissolved in dry DMF (2 mL) and added dropwise over 15 mins. The reaction mixture was stirred overnight. The reaction mixture was monitored using LC/MS and quenched using H2O (1 mL). The reaction mixture was purified by reversed phase HPLC (60-95% MeCN:H2O, 0.1% TFA) to give S12 as amorphous dark blue solid (18 mg, 41% yield). ¹ H NMR (500 MHz, DMSO) 57.73 (d, J = 8.4 Hz, 1H), 7.71 - 7.57 (m, 5H), 7.53 - 7.48 (m, 1H), 7.48 - 7.35 (m, 6H), 7.35 - 7.26 (m, 2H), 7.26 - 7.18 (m, 2H), 7.18 - 7.11 (m, 2H), 6.62 (d, J = 16.4 Hz, 1H), 6.55 (d, J = 13.2 Hz, 1H), 5.37 (d, J = 1.7 Hz, 1H), 5.28 (d, J = 11.7 Hz, 1H), 4.65 (dd, J = 17.8, 4.6 Hz, 2H), 4.39 (d, J = 14.4 Hz, 1H), 4.22 (d, J = 14.4 Hz, 1H), 3.18 (s, 2H), 2.36 (td, J = 7.4, 3.8 Hz, 3H), 2.28 - 2.23 (m, 2H), 1.83 - 1.76 (m, 3H), 1.42 - 1.37 (m, 12H), 1.16 (dd, J = 30.0, 8.8 Hz, 8H). ¹³ C NMR (125 MHz, DMSO) 5 182.9, 174.6, 172.4, 172.4,

171.6, 171.3, 155.5, 153.3, 152.3, 146.8, 142.9, 141.7, 140.1, 139.3, 139.1, 136.1, 134.3, 134.1,

132.8, 130.5, 130.1, 129.9, 129.9, 129.8, 129.4, 129.1, 129.1, 128.3, 128.3, 126.3, 124.9, 124.3,

123.3, 123.1, 122.1, 121.8, 120.5, 120.1, 119.8, 119.6, 119.5, 119.4, 117.4, 116.1, 115.1, 112.8,

105.7, 80.04, 68.66, 57.58, 56.71, 52.48, 50.05, 45.56, 45.33, 35.78, 35.71, 34.54, 34.47, 34.44,

31.76, 29.24, 29.00, 28.23, 28.22, 27.51, 23.46, 23.43, 23.36, 20.92, 20.88. HRMS (ESI) calculated for C53H59N4O5 [M+H] ⁺ 831.4; observed: 831.4.

NC-CyLBam. To a 10 mL microwave vial compound S12 was dissolved in TFA:CH2C12 (1 mL) on ice and stirred for 1 h. Afterwards the solvent was evaporated and crude product was purified over reversed phase HPLC (60-95% MeCN:H2O, 0.1% TFA) to give NC-CyLBam as amorphous dark blue solid (11.5 mg, 62% yield). ^! H NMR (500 MHz, DMSO) 57.62 (d, J = 8.2 Hz, 2H), 7.60 - 7.48 (m, 6H), 7.41 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 7.7 Hz, 1H), 7.31 (t, J = 7.8 Hz, 3H), 7.29 - 7.22 (m, 3H), 7.18 (td, J = 7.5, 1.3 Hz, 1H), 7.13 - 7.08 (m, 3H), 7.06 - 6.99 (m, 2H), 6.51 (d, J = 16.4 Hz, 1H), 6.44 (d, J = 13.1 Hz, 1H), 5.25 (d, J = 11.8 Hz, 1H), 5.19 (s, 1H), 4.55 (d, J = 7.6 Hz, 2H), 3.07 (s, 2H), 2.27 (td, J = 7.4, 3.5 Hz, 4H), 2.18 (dt, J = 10.6, 7.3 Hz, 4H), 1.71 (q, J = 7.4 Hz, 4H), 1.09 - 1.03 (m, 8H). ¹³ C NMR (125 MHz, DMSO) 5 182.9, 174.6, 174.6, 171.7, 171.4, 155.4, 153.5, 152.3, 146.9, 142.9, 141.7, 140.1, 139.3, 139.1, 136.1, 134.1, 132.7, 130.5,

130.1, 129.9, 129.8, 129.4, 129.1, 128.3, 128.3, 126.3, 124.9, 124.3, 123.3, 123.2, 122.1, 121.8,

120.6, 120.2, 119.6, 119.4, 117.5, 116.1, 115.2, 105.7, 68.66, 57.51, 56.79, 52.49, 50.09, 45.33,

35.92, 35.85, 33.43, 33.43, 33.35, 29.24, 29.01, 23.42, 23.35, 20.85, 20.83. HRMS (ESI) calculated for C49H51N4O5 [M+H] ⁺ 775.3854; observed: 775.3864.

NC-CyLBam-NHS. To a 10 mL microwave vial, NC-CyLBam (6 mg, 0.0077 mmol), TSTU (4.7 mg, 0.015 mmol) and DIPEA (4.05 pL, 0.023 mmol) were added sequentially to dry DMF (3 mL). The progress of the reaction was monitored by LC/MS. After 1 h, the reaction was complete. The reaction mixture was purified by reversed phase HPLC (10-95% MeCN:H2O, 0.1% TFA) to give NC-CyLBam-NHS as amorphous dark blue solid (6.5 mg, 86% yield). LRMS (ESI) calculated for C53H54N5O7 [M+H] ⁺ 871.39; observed: 871.4.

Compound S13. To a 100 mL round bottom flask Boc-Ala-Ala-OH (1.0 g, 3.84 mmol, obtained from Chemlmpex: Cat 04505) and HATU (1.64 g, 4.23 mmol) were added sequentially in dry THF and stirred on ice for 10 mins. Subsequently, 4-aminobenzyl alcohol (568.3 mg. 4.61 mmol) and DIPEA (1.25 mL, 7.69 mmol) were sequentially added to the reaction mixture and stirred for 12 h. Afterwards, the solvent was evaporated, and crude mixture was dissolved in EtOAc. The organic layer was washed twice with 1 N HC1, sat. NaHCO and brine. The organic layer was dried over Na2SC>4 and dried to get compound S13 as yellow fluffy power (1.18 g, 85%). ^J H NMR (500 MHz, DMSO) 57.57 - 7.52 (m, 2H), 7.26 - 7.22 (m, 2H), 5.11 (t, J = 5.7 Hz, 1H), 4.44 (d, J = 5.8 Hz, 2H), 3.99 (t, J = 7.2 Hz, 1H), 1.38 (s, 9H), 1.31 (d, J = 7.1 Hz, 3H), 1.19 (d, J = 7.1 Hz, 3H). ¹³ C NMR (125 MHz, DMSO) 5 173.01, 171.44, 155.67, 137.95, 127.35, 119.37, 78.58, 63.03, 50.16, 49.32, 28.66, 18.74, 18.47. HRMS (ESI) calculated for C18H28N3O5 [M+H] ⁺ 366.2023; observed: 366.2031.

Compound S14. To a 20 mL microwave vial, S13 (200 mg, 0.54 mmol) and 4-nitrophenyl chloroformate (160 mg, 0.82 mmol) were added sequentially to dry THF (15 mL) and stirred on ice for 30 mins. Triethylamine (153 pL, 1.09 mmol) was added dropwise, and the solution was stirred for 12 h. The resulting white precipitate was filtered, and the solvent was evaporated. The product was purified by normal phase chromatography (MeOH:CH2Ci2; 0 - 10%) to give compound S14 as a white amorphous solid (276 mg, 77% yield). Rf value = 0.4 (10% MeOH/DCM). ^! H NMR (500 MHz, DMSO) 5 10.06 (s, 1H), 8.35 - 8.29 (m, 2H), 8.03 (d, J = 7.2 Hz, 1H), 7.65 (d, J = 8.5 Hz, 2H), 7.60 - 7.55 (m, 2H), 7.43 (d, J = 8.3 Hz, 2H), 6.99 (d, J = 7.4 Hz, 1H), 5.25 (s, 2H), 4.41 (t, J = 7.1 Hz, 1H), 4.00 (t, J = 7.3 Hz, 1H), 1.39 (s, 9H), 1.32 (d, J = 7.1 Hz, 4H). ¹³ C NMR (125 MHz, DMSO) 5 173.1, 171.8, 155.8, 155.7, 152.4, 145.6, 139.8, 129.9, 129.8, 125.9, 123.1, 119.6, 119.5, 78.57, 70.71, 65.39, 50.10, 49.42, 28.67, 18.62, 18.48, 15.65. HRMS (ESI) calculated for C25H31N4O9 [M+H] ⁺ 531.2086; observed: 531.2089.

Compound S15. To a 10 mL microwave vial 2V-Me-NorCy7 (30 mg, 0.059 mmol) and CS2CO3 (38 mg, 0.11) were added sequentially to dry DMF (2 mL) and stirred for 1 h. Subsequently, compound S14 (62.23 mg, 0.12 mmol) was dissolved in dry DMF (2 mL) and added dropwise over 15 mins. The reaction mixture was stirred overnight. The reaction mixture was monitored using LC/MS and quenched using H2O (1 mL). The reaction mixture was purified by reversed phase HPLC (10-95% MeCN:H2O, 0.1% TFA) to give S15 as amorphous dark blue solid (26 mg, 52% yield). ¹ H NMR (500 MHz, DMSO) 5 8.12 - 8.03 (m, 1H), 7.73 (q, J = 10.7, 9.4 Hz, 2H), 7.64 (dq, J = 19.4, 7.9, 6.8 Hz, 3H), 7.52 (d, J = 7.4 Hz, 1H), 7.48 - 7.33 (m, 5H), 7.29 (q, J =

7.2 Hz, 2H), 7.21 (p, J = 7.6, 7.0 Hz, 2H), 7.12 (q, J = 7.7, 6.2 Hz, 1H), 6.98 (t, J = 12.1 Hz, 1H), 6.62 (d, J = 16.6 Hz, 1H), 6.55 (d, J = 13.2 Hz, 1H), 5.36 (d, J = 11.6 Hz, 1H), 5.30 (d, J = 12.9 Hz, 1H), 4.72 - 4.59 (m, 3H), 4.39 (q, J = 14.0, 13.5 Hz, 2H), 4.01 (q, J = 8.5, 8.0 Hz, 2H), 3.67 - 3.59 (m, 1H), 3.15 (dq, J = 14.5, 3.7 Hz, 2H), 1.38 (s, 5H), 1.33 (dd, J = 18.0, 6.7 Hz, 9H), 1.26 (q, J =

7.3 Hz, 6H), 1.22 - 1.13 (m, 9H). ¹³ C NMR (125 MHz, DMSO) 5 182.8, 173.2, 173.1, 172.2,

171.9, 155.7, 155.4, 153.8, 152.3, 146.9, 142.6, 141.5, 139.7, 139.3, 139.1, 136.1, 134.2, 130.5,

130.2, 130.1, 129.9, 129.4, 129.1, 128.3, 128.2, 126.7, 126.3, 124.9, 124.3, 123.4, 123.3, 122.2,

122.1, 120.7, 120.5, 120.4, 119.7, 119.6, 119.5, 118.1, 116.1, 115.6, 113.3, 78.61, 78.58, 78.55,

68.64, 54.02, 52.47, 50.13, 49.49, 45.33, 45.31, 42.27, 29.23, 29.03, 28.66, 28.64, 28.61, 23.46,

23.43, 23.36, 18.57, 18.53, 18.49, 18.46, 18.41, 17.19, 12.91 HRMS (ESI) calculated for C55H62N6O6 [M+H] ⁺ 903.4804; observed: 903.4830. AA-CyLBam. To a 10 mL microwave vial compound S15 (15 mg, 0.019 mmol) was dissolved in TFA:CH2C12 (1 mL) on ice and stirred for 1 h. Afterwards the solvent was evaporated, followed by addition of glutaric anhydride (4.3 mg, 0.037 mmol) and DIPEA (0.5 pL, 0.03 mmol) in dry DMF (2 mL). The reaction mixture was stirred at room temperature and was monitored by LC/MS. After 3 h the reaction was complete. The reaction mixture was purified by reversed phase HPLC (10-95% MeCN:H2O, 0.1% TFA) to give AA-CyLBam as amorphous dark blue solid (10.2 mg, 58% yield). ^J H NMR (500 MHz, DMSO) 5 8.05 (dd, J = 7.0, 2.5 Hz, 1H), 8.02 (d, J = 7.2 Hz, 1H), 7.95 (ddd, J = 15.5, 8.0, 4.4 Hz, 2H), 7.65 (t, J = 8.6 Hz, 2H), 7.59 - 7.53 (m, 3H), 7.53 - 7.48 (m, 1H), 7.41 (q, J = 3.1 Hz, 1H), 7.34 - 7.28 (m, 3H), 7.28 - 7.22 (m, 3H), 7.19 - 7.14 (m, 1H), 7.10 (q, J = 7.8 Hz, 2H), 7.01 (dt, J = 16.3, 6.1 Hz, 2H), 6.50 (d, J = 16.5 Hz, 1H), 6.43 (dd, J =

13.1, 4.2 Hz, 1H), 5.23 (dd, J = 11.8, 7.0 Hz, 1H), 4.27 (qd, J = 7.2, 4.4 Hz, 4H), 4.17 - 4.12 (m,

2H), 3.03 (d, J = 16.3 Hz, 2H), 2.13 - 2.02 (m, 6H), 1.60 (dt, J = 11.6, 7.4 Hz, 4H), 1.24 - 1.17 (m, 5H), 1.15 - 1.02 (m, 12H). ¹³ C NMR (125 MHz, DMSO) 5 182.8, 174.7, 174.6, 174.6, 172.9, 172.8, 172.8, 172.6, 172.5, 172.4, 172.3, 172.3, 172.2, 172.1, 171.8, 162.7, 155.4, 155.3, 153.6,

152.2, 146.9, 142.7, 141.4, 139.6, 139.6, 139.4, 139.3, 139.1, 136.1, 134.3, 134.1, 132.6, 130.5,

130.1, 130.1, 129.9, 129.4, 129.2, 129.1, 128.3, 128.2, 126.2, 124.9, 124.3, 124.2, 123.3, 123.1,

122.1, 122.1, 120.6, 120.3, 120.1, 119.7, 119.6, 119.4, 117.7, 116.1, 115.3, 113.1, 105.6, 68.66,

52.47, 49.68, 49.64, 49.54, 48.87, 48.80, 48.77, 48.65, 45.35, 45.32, 36.25, 34.62, 34.60, 34.55,

34.45, 33.45, 33.42, 33.39, 31.24, 29.24, 29.02, 28.87, 23.46, 23.43, 23.37, 23.34, 22.97, 21.03,

21.00, 20.92, 18.52, 18.38, 18.35, 18.33, 18.28, 18.21, 18.09. LRMS (ESI) calculated for

C55H59N6O7 [M+H] ⁺ 915.4 observed: 915.3.

AA-CyLBam-NHS. To a 10 mL microwave vial, AA-CyLBam (10 mg, 0.014 mmol), TSTU (8.7 mg, 0.028 mmol) and DIPEA (4.50 pL, 0.028 mmol) were added sequentially to dry DMF (3 mL). The progress of the reaction was monitored by LC/MS. After 1 h, the reaction was complete. The reaction mixture was purified by reversed phase HPLC (10-95% MeCN:H2O, 0.1% TFA) to give AA-CyLBam-NHS as amorphous dark blue solid (6.8 mg, 70% yield). LRMS (ESI) calculated for C59H64N7O9 [M+H] ⁺ 1014.3; observed: 1014.4.

M-Me-!torCy7

Compound S17. Compound S16 was synthesized according to reported literature procedures (Suma et al., JACS 2017, 139(11):4009-4018; Skakuj et al., JACS 2018, 140(4): 1227-1230). To a 10 mL microwave vial 2V-Me-NorCy7 (30 mg, 0.059 mmol) and CS2CO3 (38 mg, 0.11) were added sequentially to dry DMF (2 mL) and stirred for 1 h. Subsequently, compound S16 (41 mg, 0.12 mmol) was dissolved in dry DMF (2 mL) and added dropwise over 15 mins. The reaction mixture was stirred overnight. The reaction mixture was monitored using LC/MS and quenched using H2O (1 mL). The reaction mixture was purified by reversed phase HPLC (10-95% MeCN:H2O, 0.1% TFA) to give S17 as amorphous dark blue solid (26 mg, 60% yield). ^J H NMR (500 MHz, DMSO) 5 8.42 (dt, J = 4.9, 1.3 Hz, 2H), 8.37 (dt, J = 4.9, 1.4 Hz, 1H), 7.74 (td, J = 7.8, 1.9 Hz, 2H), 7.69 (q, J = 1.7 Hz, 2H), 7.56 (dt, J = 9.6, 1.3 Hz, 3H), 7.38 - 7.34 (m, 2H), 7.28 (dd, J = 7.5, 1.4 Hz, 1H), 7.24 - 7.20 (m, 3H), 7.12 (dd, J = 7.4, 1.1 Hz, 1H), 7.08 - 7.03 (m, 2H), 6.61 (d, J = 16.3 Hz, 1H), 6.31 (d, J = 13.1 Hz, 1H), 4.52 (t, J = 5.9 Hz, 3H), 4.21 - 4.02 (m, 4H), 3.29 (s, 2H), 3.06 (s, 3H), 1.09 (d, J = 28.3 Hz, 12H). ¹³ C NMR (125 MHz, DMSO) 5

182.9, 157.8, 154.1, 152.1, 150.1, 150.1, 146.8, 143.0, 138.6, 138.2, 136.6, 134.4, 130.4, 129.4,

128.9, 128.2, 126.5, 125.4, 124.9, 122.2, 120.1, 120.1, 117.6, 115.3, 106.4, 64.69, 52.44, 45.14, 42.66, 36.90, 29.07, 23.45. LRMS (ESI) calculated for C44H45N4O2S2 [M+H] ⁺ 725.2; observed: 725.3.

5.5-CyLBam. To a 10 mL microwave vial S17 (10 mg, 0.013 mmol) and

3-mercaptopropionic acid (2.31 pL, 0.027) were added sequentially to dry DMF (2 mL) and stirred for 2 h. The reaction mixture was monitored using LC/MS and purified by reversed phase HPLC (10-95% MeCN:H ₂ O, 0.1% TFA) to give S,S-CyLBam as amorphous dark blue solid (8.6 mg, 86% yield). ¹ H NMR (500 MHz, DMSO) 57.76 (d, J = 8.0 Hz, 1H), 7.67 - 7.64 (m, 2H), 7.60 (s, 1H), 7.44 (d, J = 7.6 Hz, 1H), 7.41 (dd, J = 7.4, 1.3 Hz, 1H), 7.36 - 7.32 (m, 2H), 7.30 - 7.24 (m, 3H), 7.22 - 7.18 (m, 1H), 7.14 - 7.09 (m, 2H), 7.06 (s, 1H), 6.68 (d, J = 16.3 Hz, 2H), 6.38 (d, J = 13.2 Hz, 2H), 4.61 (t, J = 6.1 Hz, 2H), 4.25 - 4.13 (m, 4H), 3.22 (t, J = 6.1 Hz, 2H), 3.14 (s, 3H), 2.94 (t, J = 7.0 Hz, 2H), 2.66 (t, J = 7.0 Hz, 2H), 1.16 (d, J = 28.1 Hz, 12H). ¹³ C NMR (125 MHz, DMSO) 5 182.9, 173.1, 162.7, 154.1, 153.7, 152.2, 146.9, 142.7, 139.2, 139.1, 136.6, 136.1, 134.1, 130.1, 130.2, 130.1, 129.4, 128.9, 128.3, 128.1, 126.6, 126.5, 126.1, 125.4, 124.9, 122.9, 122.1,

120.6, 120.5, 120.4, 118.1, 116.3, 116.2, 115.7, 113.4, 106.3, 64.75, 52.44, 51.28, 50.79, 45.13,

42.68, 36.25, 36.22, 34.02, 33.57, 33.49, 31.24, 29.05, 23.43. HRMS (ESI) calculated for

C45H52N3O4S2 [M+H] ⁺ 762.3394; observed: 762.3389.

5.5-CyLBam-NHS. To a 10 mL microwave vial, S,S-CyLBam (5 mg, 0.007 mmol), TSTU (8.7 mg, 0.014 mmol) and DIPEA (2.40 pL, 0.014 mmol) were added sequentially to dry DMF (2 mL). The progress of the reaction was monitored by LC/MS. After 2 h, the reaction was complete. The reaction mixture was purified by reversed phase HPLC (10-95% MeCN:H2O, 0.1% TFA) to give S,S- -Me-CyBam-NHS as amorphous dark blue solid (4.5 mg, 84% yield). HRMS (ESI) calculated for C42H46N3O4S2 [M+H] ⁺ 720.2924; observed: 720.2935.

S,SMe2-CyLBam. To a 10 mL microwave vial S17 (10 mg, 0.013 mmol) and 4-mercapto-4- methylpentanoic acid (2.31 pL, 0.027 obtained from BroadPharm, Cat:BP-22000) were added sequentially to dry DMF (2 mL) and stirred for 2 h. The reaction mixture was monitored using LC/MS and purified by reversed phase HPLC (10-95% MeCN:H2O, 0.1% TFA) to give S,SMe2-CyLBam as amorphous dark blue solid (23 mg, 70% yield). ^! H NMR (500 MHz, DMSO) 57.78 - 7.76 (m, 1H), 7.66 (td, J = 7.3, 1.5 Hz, 2H), 7.62 - 7.59 (m, 1H), 7.46 - 7.41 (m, 2H), 7.36 - 7.32 (m, 2H), 7.31 - 7.24 (m, 3H), 7.23 - 7.18 (m, 2H), 7.14 - 7.10 (m, 1H), 7.07 (s, 1H), 6.67 (s, 1H), 6.40 (d, J = 1.8 Hz, 1H), 4.59 (t, J = 6.0 Hz, 3H), 4.24 - 4.14 (m, 3H), 3.18 (t, J = 6.1 Hz, 2H), 3.14 (s, 3H), 2.39 - 2.34 (m, 1H), 2.34 - 2.25 (m, 2H), 2.10 - 2.05 (m, 1H), 1.88 - 1.83 (m, 1H), 1.83 - 1.73 (m, 2H), 1.31 (s, 2H), 1.26 (d, J = 15.6 Hz, 6H), 1.21 (d, J = 12.6 Hz, 6H), 1.14 (s, 5H). ¹³ C NMR (125 MHz, DMSO) 5 182.9, 174.9, 174.7, 154.2, 153.3, 152.2, 146.8, 143.0, 139.2, 139.1, 136.6, 134.5, 130.4, 130.1, 129.4, 128.9, 128.3, 128.2, 126.5, 126.3, 126.2, 125.4, 124.9, 122.9, 122.1, 120.4, 120.1, 119.8, 117.5, 116.36, 115.2, 112.8, 106.3, 65.04, 56.82, 52.44, 51.27, 51.13, 51.00, 50.77, 45.15, 44.21, 42.68, 42.64, 42.28, 41.18, 37.78, 35.96, 32.55, 30.72, 30.52, 30.05, 29.05, 28.75, 28.13, 27.53, 27.45, 23.45. LRMS (ESI) calculated for C45H52N3O4S2 [M+H] ⁺ 762.3; observed: 762.3.

S,SMe2-CyLBam-NHS. To a 10 mL microwave vial, S,SMe2-CyLBam (5 mg, 0.007 mmol), TSTU (8.7 mg, 0.014 mmol) and DIPEA (2.40 pL, 0.014 mmol) were added sequentially to dry DMF (2 mF). The progress of the reaction was monitored by LC/MS. After 2 h, the reaction was complete. The reaction mixture was purified by reversed phase HPLC (10-95% MeCN:H2O, 0.1% TFA) to give S,SMe2-CyLBam- NHS as amorphous dark blue solid (4.1 mg, 75% yield). HRMS (ESI) calculated for C42H46N3O4S2 [M+H] ⁺ 859.3; observed: 859.3.

Example 2 Characterization ofCyBam Ns

A disulfonated heptamethine norcyanine, CyBam-Ns, was synthesized as described in

Example 1 and shown below:

CyBam -Ns allowed examination of the tum-ON chemistry using a chemical trigger. Azide reduction, involving aza-ylide formation and hydrolysis, was hypothesized to initiate 1,6 elimination and carbamic acid hydrolysis to result in unmasking of the pH-sensitive norcyanine (FIG. 1). CyBam-Ns exhibited minimal absorbance and emission in the NIR range at either neutral (pH 7.2) or acidic pH (pH 4.5) (FIG. 2). Examining the absorbance profiles of CyBam-Ns and Sulfo-NorCy7 at both neutral and acidic pH revealed three readily distinguishable species as shown in FIG. 2. FIGS. 3A and 3B show the absorbance (300-800 nm) and fluorescence (720-850 nm) spectra, respectively, with 710 nm excitation of CyBam-Ns (10 pm) and Sulfo-NorCy7 (10 pm) at pH 7.2 (PBS) and pH 4.5 (acetate buffer) The pKa of Sulfo-NorCy7 was determined as 5.4 by absorbance and 5.2 by fluorescence. The photophysical properties of the three species are summarized in Table 4. The stability of CyBam-Ns at physiological pH values ranging from 4.5-7.5 and in serum was evaluated and little degradation (< 5%) was observed over 24 h. Table 4

The fluorogenic response of CyBam-N was investigated by incubating with triphenyl phosphine (PPI13) at pH 5.2 (FIGS. 4A and 4B). The reaction of CyBam-N (10 mM; 1 eq) and PPI13 (100 mM; 10 eq) in PBS:MeOH (1:1), pH 5.2, was monitored at 5-minute intervals (n = 3). Complete conversion to Sulfo-NorCy7-[H ⁺ ] occurred within 60 mins of PPI13 addition. Rapid conversion to Sulfo-NorCy7-[H ⁺ ] was observed (FIG. 4A) with a dramatic 170-fold increase in the fluorescence signal (FIG. 4B). This reaction could also be carried out at neutral pH to provide the neutral form Sulfo-NorCy7.

The magnitude of the turn-ON response of CyBams was compared to xanthene cyanines, which are far-red probes that have been broadly employed for in vivo fluorogenic imaging. A sulfonated, acetylated variant, compound S10, was prepared as described in Example 1 and compared to the xanthene cyanine, compound S9. The absorbance spectra of both compounds are shown at pH 7.2 (PBS, FIG. 5A) and pH 4.5 (acetate buffer, FIG. 5B). Compound S9 exhibited turn-ON ratios of 15 and 1.5 over compound S10 were obtained at pH 7.2 (FIG. 5C) and pH 4.5 (FIG. 5D), respectively, with an excitation of 640 nm. Compound S9 exhibited tum-ON ratios of 13 and 8 over compound S10 were obtained at pH 7.2 (FIG. 5E) and pH 4.5 (FIG. 5F), respectively, with an excitation of 690 nm. The critical distinction is the absorption profile in the OFF state. The acetylated xanthene cyanine exhibited substantial long-wavelength absorption, which leads to significant emission from the quenched state (FIG. 5A). In contrast, CyBams exhibit minimal absorption in the NIR region at either neutral or acidic pHs. Example 3

Cellular and In Vivo Imaging with CyBam-y-Glu and Sulfo-NorCy7

To investigate the utility of CyBams for cellular and in vivo imaging, a validated Anorogenic trigger with significant translational potential was used. y-Glutamyl transpeptidase (GGT) is a cell-surface-bound enzyme involved in maintaining cellular glutathione (GSH) and cysteine homeostasis (Wickham et al., Anal Biochem 2011, 414(2):208-214). Additionally, it has been shown to act as a biomarker of several malignant tumors (including liver, cervical, and ovarian) and overexpression of GGT has been correlated with metastases (Schafer et al., Acta Oncol 2001, 40(4):529-535; Yao et al., Cancer 2000, 88(4):761-769; Luo et al., Oncotarget 2017, 8(4):67651-67662; Hanigan, et al., Cancer Res 1994, 54(l):286-290). A cleavable glutamate was installed on CyBam-v-Glu as described in Example 1.

After confirming the stability of CyBam-y-Glu, the probe was evaluated in enzymatic assays. A GGT-dependent increase in Auorescent signal was observed (FIG. 6) with a Michaelis constant (KM = 16 mM) similar to those obtained with other GGT probes. GGT converts CyBam-y-Glu to the corresponding norcyanine via 1,6-elimination. CyBam-y-Glu was determined to be specifically activated by GGT and did not show any significant signal when incubated with representative proteases and esterases (FIG. 7). The selectivity of CyBam-y-Glu was determined using established GGT inhibitors. GGT (100 U/L) was pre-blocked with covalent inhibitors DON and GGsTop (1 mM) for 1 h in PBS (pH. 7.4) followed by incubation with CyBam-y-Glu (20 pM) at 37 °C. A 60% and 80% decrease in Auorescent signal in the presence of DON and GGsTop, respectively, was observed, confirming the selectivity of the probe (FIG. 8). CyBam-y-Glu and the corresponding non-cleavable variant CyBam-N.C were examined in cellular assays and in vivo imaging experiments. A SHIN-3 ovarian cancer cell line that has been previously shown to overexpress GGT was used (Urano et al., Sci Transl Med 2011, 3(110): 1 lOral 19). CyBam-y-Glu exhibited minimal toxicity, demonstrated by incubating SHIN-3 cells with 1-40 pM of the compound for 72 h. Cell viability was measured as described in the experimental procedures. Sulfo-NorCy7 (Example 2) also was evaluated and found to have minimal toxicity.

Sulfo-NorCy7 exhibited significant cellular uptake in SHIN-3 cells with strong lysosomal localization, consistent with prior results and the requirement for chromophore protonation (FIG. 9). The upper panels show uptake in mitochondria (Mitotracker Green) and the lower panels show uptake in lysosomes (Lysotracker Green). Overlap in the green and red channel was analyzed using fluorescent line graph in the green and the red channel. Nucleus stained using NucBlue (blue channel). Confocal imaging carried out at 63X oil immersed lens with 1.4 NA. The images were processed with identical brightness/ contrast using Fiji.

The cellular activation and selectivity of CyBam-y-Glu with and without incubation with GGT inhibitors was evaluated. Using confocal microscopy and flow cytometry, a strong fluorescent signal was observed in cells treated with CyBam-y-Glu. By contrast, minimal fluorescence signal was observed in cells that were treated with either CyBam-N.C or preincubated with GGT inhibitors (FIGS. 10 and 11). FIG. 10 shows confocal images of fluorescence activation of CyBam-y-Glu (20 mM) and CyBam-N.C (20 mM). Fluorescent signal from the probe and nucleus (Hoechst) is shown in red and blue, respectively. Images were taken using a 63X oil immersed lens (N.A 1.4). FIG. 11 shows quantification of fluorescent signal after incubation of CyBam-y-Glu (20 mM) in presence of GGT inhibitors (DON, GGsTop) and CyBam-N.C. in SHIN-3 cells using flow cytometry.

CyBam-y-Glu was evaluated in a metastatic tumor model of ovarian cancer. This model entails intraperitoneal injection of SHIN-3-ZsGreen cells, resulting in formation of a significant primary tumor in the greater omentum and locally disseminated metastases (Urano et al., Sci Transl Med 2011, 3(110: 1 lOral 19). CyBam-y-Glu (30 nmol) was injected intraperitoneally in mice which were euthanized after 1, 3, and 6 h and both the primary tumor and local metastases were imaged. Excellent colocalization between CyBam-y-Glu and the ZsGreen signal suggests that the probe was activated and taken up selectively by tumor cells, with significant signal at all three time points (FIGS. 12-14). FIG. 12 shows imaging of the SHIN-3-ZsGreen metastatic tumor model at 3h after injection with CyBam-y-Glu (30 nmol). Green and red pseudo colors are used to represent signal from the GFP and Cy7 channels, respectively. The fluorescent line graphs show correlation between fluorescent signal from GFP and Cy7 channel across the metastatic tumor in two different regions (A and B). FIGS. 13 and 14 show brightfield, GFP channel, Cy7 channel, and merged images of a SHIN-3-ZsGreen metastatic tumor model at Ih and 6 h, respectively, after injection with CyBam-y-Glu (30 nmol). After 1 h, the mouse was euthanized, and tumors (omental cake and metastatic) were imaged. GFP ex 445 - 490 nm, em 500 - 720 nm; Cy7 ex 710 - 760 nm, em 780 - 950 nm. Green and red pseudo colors are used to represent signal from the GFP and Cy7 channels respectively. These results indicate that CyBams have significant potential for use as activatable probes for in vivo imaging, including for optically guided surgical procedures. The extensive optical instrumentation in place for heptamethine cyanines makes this prospect more enticing.

CyBam-y-Glu and CyBam-N.C (25 nM) were injected (in sterile IX PBS pH 7.4) intravenously in mice containing MDA-MB-468 xenograft tumors. Mice were imaged after 4 h in bright field and Cy7 channel (ex/em filter 745/800 nm) (FIG. 15). Signal from tumors, liver and background in mice are shown in red, blue and magenta dotted circles. Green pseudo colors are used to represent fluorescent signal from and Cy7 channel. FIGS. 16A and 16B show quantification of fluorescent signal emitting from tumors (16A) and tumor to background ratio in mice by drawing ROIs (16B); p-values were evaluated by student t-test (*** p-value < 0.001).

The data shows that CyBam-y-Glu has a turn-ON ratio that dramatically exceeds those found with existing far-red Anorogenic probes, particularly in acidic conditions. Unlike existing far-red Anorogenic probes, the cationic chromophore is only formed through cleavage of the carbamate group and subsequent protonation of the resulting norcyanine, leading to an exceptional turn-ON ratio (>150X). The CyBam is selectively activated upon protonation in the lysosome.

Example 4 Optimization of Norcyanine Signal

While the sulfonated norcyanine, Sulfo-NorCy7 can provide high contrast imaging, relatively high concentrations (20 - 40 mM) were required for sufficient in vitro signal. This requirement might be problematic for mAb targeted imaging because probe concentrations are intrinsically limited by antigen levels. Microscopy studies indicated that the cellular signal of Sulfo-NorCy7 was predominately from the lysosome - an observation consistent with probe protonation and a pX _a between 4 and 5. Poor lysosomal accumulation might be responsible for the modest cellular fluorescence of this probe. It was hypothesized that improving cellular permeability and, perhaps, introducing a lysosomal targeting element could improve the fluorescent output.

To approach this problem, a small panel of chemically diverse norcyanines was designed and synthesized. These compounds contain a C4’-phenyl-substituent appended to the polymethine chromophore, which has been found to improve synthetic accessibility, photostability, and serum stability. To test the question of cell permeability alone, two hydrophobic derivatives, OMe-NorCy7 and H-Nor-Cy7 were included. Commonly used lysotracker probes contain tertiary amines, suggesting this functional group can promote lysosomal targeting (Xu et al., Angew Chem Int Ed Engl 2016, 55(44):13658-13699; Zhu et al., Acc Chem Res 2016, 49(10):2115-2126; Choi et al., Molecules 2021, 26(1)). To determine whether might apply to norcyanines, we prepared indolenine-substituted sulfonamide derivatives modified with both the hydrophobic piperidine (Pip-NorCy7) and the tertiary amine-containing X-Me piperazine (N-Me-Pip-NorCy7) substituents. Another “lysotracker-like” derivative, N-Me-NorCy7, in which the central ring system contains an A-methyl substituent also was prepared. The syntheses are described in Example 1. The photophysical properties and cellular uptake of the probes was investigated. All six probes exhibited absorbance and emission maxima above 700 nm, similar extinction coefficients and quantum yields, and pk'ys between 4.4 and 5.2 (see, e.g., FIGS. 17A and 17B). The results are summarized in Table 5.

Table 5 a acetate buffer (pH 4.5); ^b MeOH:acetate buffer pH 4.5 (2:1); ^c MeOH:acetate buffer pH 3.75 (2:1); dMeOH + 0.1% formic acid for all compounds.

The NorCy7 series was determined to have minimal toxicity in MDA-MB-468 and MCF-7 cells. Signal in MDA-MB-468 cells was quantified at 1, 3 and 6 h time points with flow cytometry using the APC-Cy7 channel. At all three time points, the uptake of the six norcyanines follows the rank order: 2V-Me-NorCy7 > -Me-Pip-NorCy7 » Pip-NorCy7, OMe-NorCy7, H-Nor-Cy7 » Sulfo-NorCy7. FIG. 18 shows flow cytometry quantification of in vitro uptake of the compounds (5 mM) in MDA-MB-468 after 1 h incubation. Geometric mean fluorescent intensity (+ SD) of fluorescent signal in the cells is shown (n = 4 independent experiments; -10,000 cells counted). The 2V-Me-NorCy7 and -Me-Pip-NorCy7 exhibited 187-fold and 72-fold higher uptake, respectively, in MDA-MB-468 cells after only 1 h incubation compared to previously used Sulfo-NorCy7. The fluorescent signal remained constant at the 1 , 3 and 6 h time points suggesting efficient uptake and high retention of tertiary amine substituted norcyanines (2V-Me-NorCy7 and 2V-Me-Pip-NorCy7). In contrast, the signal from Sulfo-NorCy7 increased steadily overtime and was ~3-fold higher after 6 h relative to 1 h. Using confocal microscopy, the subcellular localization of the 2V-Me-NorCy7 was confirmed to be lysosomal, with dramatically higher signal than SulfoNorCy7 (FIGS. 19A-19F). FIGS. 20A and 20B show organelle localization of 2V-Me-NorCy7 (5 pM; red channel) in MDA-MB-468 cells after 6 h incubation followed by organelle staining (green channel) with lysosome (Lysotracker Green; 20A) and mitochondria (Mitotracker Green; 20B). Nucleus stained using NucBlue (blue channel). Confocal imaging carried out at 63X oil immersed lens with 1.4 NA. The images were processed with identical brightness/ contrast using Fiji. Scale bar 10 pm. The relatively modest signal of the three hydrophobic derivatives, Pip-NorCy7, OMe-NorCy7, and H-NorCy7, indicates that the tertiary amine, the lysotracker-mimicking feature, is important for fluorescent signal enhancement. Overall, this data indicates that incorporation of a single basic amine dramatically enhances cellular uptake and lysosomal localization of norcyanines. The enhanced cellular signal and synthetic accessibility led the selection of 2V-Me-NorCy7 to compare to the previously described Sulfo-NorCy7 in developing activable mAb conjugates.

Example 5 Norcyanine-mAb Conjugates and Targeting

The impact of norcyanine modification on the signal of mAb conjugates was assessed. 2V-Me-NorCy7 was converted to the cyanine lysosome-targeting carbamate (CyLBam) variant by attaching a cathepsin cleavable dipeptide (valine-citrulline, VC) and a non-cleavable (NC) glutaric anhydride linker substituted with a lysine reactive NHS ester (see Example 1 for synthesis details). As a comparison, Sulfo-NorCy7 was converted to the corresponding CyBam molecules. The four probes were conjugated to the FDA-approved anti-Epidermal Growth Factor Receptor (EGFR) antibody Panitumumab (Pan) at pH 7.4 (degree of labeling (DOL) ~4; FIG. 21). The resulting conjugates were purified by both spin column and dialysis to ensure that no free small molecule remained. The inventors’ prior work had shown that similar conjugates maintain the in vitro and in vivo targeting properties of the parent mAb (Gorka et al., Acc Chem Res 2018, 51 (12) :3226-3235 ; Usama et al., Curr Opin Chem Biol 2021, 63:38-45).

The series of Pan probes was compared in cellular assays. The four conjugates, Pan-VC-CyLBam, Pan-NC-CyLBam, Pan-VC-CyBam, Pan-NC-CyBam, (FIG. 22) were tested in MDA-MB-468 (EGFR+) and MCF-7 (EGFR-) cells (Mamor et al., Cancer Res 2003, 63(12):3154-61 ; Bhattacharyya et al. , Medchemcomm 2014, 5(9): 1337- 1346). The fluorescent signal emitting from Pan-VC-CyLBam was 50-fold higher than Pan-VC-CyBam after incubation with MDA-MB-468 cells for 24 h (FIGS. 23, 24). Validating the role of receptor- mediated uptake, the fluorescent signal of both probes was lower in MCF-7 cells (by 7.5-fold for the CyLBam and 4.5-fold for the CyBam (FIG. 24). As expected, the non-cleavable probes, Pan-NC-CyLBam and Pan-NC-CyBam, did not exhibit any significant fluorescent signal in either cell line. Confocal microscopy confirmed the trends observed by flow cytometry, as well as the lysosomal uptake of the released norcyanine (FIGS. 25-27).

The conjugates (200 mg) were injected intravenously into female athymic nude mice with MDA-MB-468 xenograft tumors (25-35 mm ³ ). The mice were imaged at 4, 24, 48, 72, and 168 h post-injection using an In Vivo Imaging System (IVIS) (FIG. 28A and FIGS. 29-34). After 24 h, a strong fluorescent signal was observed in tumors injected with Pan-VC-CyLBam and the signal persisted over 168 h, with tumor-to-background ratios (TBRs) between 4 and 5 (FIG. 28B). In contrast, the Pan-VC-CyBam group had lower TBRs (1-2) at all time points - similar to the two non-cleavable probes, Pan-NC-CyLBam and Pan-NC-CyBam. Significant liver signal was observed with the two probes containing the protease cleavable linker (Pan-VC-CyLBam and Pan-VC-CyBam) at early time points (4 h and 24 h), and the fluorescent signal decreased afterwards.

The selective hepatic signal of Pan-VC-CyBam and its time-dependent disappearance suggests that the released Sulfo-NorCy7 is rapidly cleared through hepatobiliary pathways. Thus, the reduced tumor signal observed with this probe likely reflects lower tumor retention (compared to V-Me-NorCy7) and subsequent clearance. Overall, these results suggest that the improved cellular retention of V-Me-NorCy7, first observed in vitro, extends to an in vivo setting.

Example 6 Quantitative Comparison of Antibody-Drug-Conjugate Linkers

A small panel of probes was designed to compare two commonly used cathepsin-cleavable peptide linkers, valine-citrulline, Pan-VC-CyLBam (Example 5, FIG. 22) and alanine-alanine, Pan-AA-CyLBam, and two disulfides, one hindered gem-dimethyl-substituted variant, Pan-S,SMe2-CyLBam and one primary disulfide, Pan-S,S-CyLBam (FIGS. 35, 36). As a control, the non-cleavable probe, Pan-NC-CyLBam (Example 5, FIG. 22), was also employed.

The signals of the panel of conjugates were examined in cells expressing variable levels of EGFR. The fluorescent signal from linker cleavage was quantified using flow cytometry at 6 and 24 h post-incubation (FIG. 37). At the 6 h time point, Pan-S,S-CyLBam exhibited the highest fluorescent signal in EGFR+ MDA-MB-468 cells. The other three cleavable probes had only modest fluorescent signal after 6 h, but the signal increased substantially after 24 h. Pan-VC-CyLBam had the highest fluorescent signal among the cleavable probes after 24 h, indicating the most extensive linker cleavage. All 5 conjugates exhibited low levels of probe signal at 6 and 24 h in the EGFR- cell lines (MCF-7 and MDA-MB-231, FIGS. 37, 38), albeit with slightly higher signal with the Pan-S,S-CyLBam probe. Significantly, if these data were used in isolation, it would be difficult to differentiate these linkers and assess the optimal agent for further study.

With the goal of investigating differences between these linkers in an organismal context, the NIR spectroscopic properties of these probes was then harnessed for in vivo imaging. The full series of probes (100 mg dose, half the dose of the studies above), were administered to female athymic nude mice implanted with MDA-MB-468 tumors and imaged at regular intervals up to 48 h (FIG. 39). With the disulfide probes, Pan-S,SMe2-CyLBam and Pan-S,S-CyLBam, significant tumor signal was not observed, and the TBR was similar to the non-cleavable Pan-NC-CyLBam control. In contrast, strong tumor signal was observed from the cathepsin-sensitive probes, Pan-VC-CyLBam and Pan-AA-CyLBam (FIGS. 40-44), with the TBR reaching as high as 4.5 after 48 h. These studies indicated that cathepsin-cleavable linkers provide dramatically higher tumor activation relative to hindered or non-hindered disulfides. Importantly, as only modest differences are observed using the cellular methods described above, this key distinction was only apparent with in vivo imaging.

In addition to tumor uptake, these data also provide significant insight into off-target cleavage pathways. As observed above, conjugates with cleavable linkers exhibit significant liver signal at early time points (4 and 24 h, FIG. 42). While likely due to a combination of liver uptake/cleavage and probe clearance through hepatobiliary pathways, this approach can provide quantitative insight into off target cleavage. With Pan-AA-CyLBam as an example, the liver signal decreased overtime but increased in the tumor (FIG. 41), which leads to an increase in the tumor-to-liver ratio over time (FIG. 41, inset). The in vivo imaging results were corroborated with an ex vivo assessment, performed 48 h post injection. As expected, the TMR (tumor-to-muscle ratio) revealed an identical trend to that observed in vivo (FIGS. 45, 46A, 46B).

Finally, to test the generality of the approach, CyLBams were tested against an alternative cancer target. The cell surface protein CD276, also known as B7 homolog H3 (B7H3), is overexpressed in both tumor cells and tumor vasculature of multiple solid tumor types (Qin et al. , Onco Targets Ther 2013, 6:1667-73; Zang et al., Mod Pathol 2010, 23(8): 1104-12; Brunner et al., Gynecol Oncol 2012, 124(1): 105-11; Seaman et al., Cancer Cell 2007, 11(6):539-54). VC and S,SMe2 linkers were selected for comparison. The CD276 targeting antibody, m276-SL, reported previously, and non-cleavable NC control m276-SL conjugates were prepared, as well as a non-binding mAb IgG control (DOL 4, FIG. 47). In initial in vitro testing using a CD276+ JIMT-1 triple negative breast cancer cell line, both cleavable m276-SL conjugates showed high levels of cleavage, with little activation using the control non-binding IgG antibody (FIGS. 48, 49).

Protease-cleavable m276-SL-VC-CyLBam and the disulfide-cleavable m276-SL-S,SMe2-CyLBam conjugates were compared in JIMT-1 tumors (200-250 mm ³ ) grown orthotopically in the mammary fat pad. As observed in the studies above, only the protease-cleavable m276-SL-VC-CyLBam conjugate exhibited a TBR greater than 5 (5.2 after 48 h), while the disulfide m276-SL-S,SMe2-CyLBam probe exhibited much lower tumor signal (FIGS. 50-53, 54A-54C).

These initial studies in this model serve to both validate the generality of the CyBam imaging strategy, as well as the observation that protease-cleavable linkers outperform disulfide linkers in solid tumor models.

The mode of action of ADCs is complex. Optimal efficacy depends on target binding, internalization, and, finally, lysosomal catabolism to initiate payload release. While the linker domain plays a central role in determining the efficacy and selectivity of ADCs, it is challenging to directly assess the site and extent of linker cleavage using conventional methods. This is an important issue because off-target cleavage of ADCs contributes significantly to ADC toxicity (Masters et al., Invest New Drugs 2018, 36:121-135). The optical imaging approach detailed here provides a general means to analyze linker chemistry across cellular, tissue, and body-wide scales. This work identified probes suitable for mAb-targeted activatable imaging. Installation of a tertiary amine into the norcyanine framework dramatically improved cellular and in vivo signal compared to sulfonated CyBams, likely due to enhanced lysosomal uptake and retention. It is likely that these “lysotracker-like” norcyanines and the resulting CyLBam probes will have applications in other settings, including their use as activity-based sensing agents.

These examples have shown that quantitative comparisons of linker chemistries can be obtained by varying the linker component. In addition to linker chemistry, the properties of the payload molecule (e.g. charge, hydrophobicity) are also important aspects of tumor targeting. While the norcyanine probe is a hydrophobic, sp ² -rich small molecule, so too are many ADC payloads. Further variations of the cyanine component could provide additional insights into the role of pay load properties on tumor and normal tissue distributions. CyLBam imaging could provide insights that directly inform the development of ADCs. These studies have suggested significant differences between protease cleavable dipeptides and disulfides in terms of tumor cleavage, which is likely due to instability of disulfides in circulation. Prior studies looking at various cysteine mutants have found the site of mAb labeling can have dramatic effects on disulfide stability (Ohri et al., Bioconjug Chem 2018, 29(2):473-485). This approach is well positioned to investigate these and related homogenous labeling strategies.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.