doi.org/10.26434/chemrxiv.11888214.v1

Towards Personalized Medicine: Photoacoustic Imaging EnablesCompanion Diagnosis and Targeted Treatment of Lung CancerMelissa Lucero, Jefferson Chan

Submitted date: 23/02/2020 • Posted date: 24/02/2020Licence: CC BY-NC-ND 4.0Citation information: Lucero, Melissa; Chan, Jefferson (2020): Towards Personalized Medicine:Photoacoustic Imaging Enables Companion Diagnosis and Targeted Treatment of Lung Cancer. ChemRxiv.Preprint. https://doi.org/10.26434/chemrxiv.11888214.v1

Companion diagnostics (CDx) represent a new frontier in personalized medicine that promises to improvetreatment outcomes by matching therapies to patients. Currently, these tests are limited in scope and cannotreport on real-time changes associated with disease progression and remediation. To address this, we havedeveloped the first photoacoustic imaging-based CDx (PACDx) for the selective detection of elevatedglutathione (GSH) in lung cancer. Since GSH is abundant in most cells, it was essential to tune the reactivityof the benzenesulfonyl-based trigger to distinguish between normal and pathological states. Moreover, wedesigned a matching prodrug, PARx, that utilizes the same mechanism to release both a chemotherapeutic(Gemcitabine) and a PA readout. We demonstrate that PARx can inhibit tumor growth while sparing all othertissue from off target toxicity in a A549 lung cancer xenograft model. We envision that this work will establish anew standard for personalized medicine by employing a unique imaging-based approach.

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Towards personalized medicine: Photoacoustic imaging enables companion diagnosis and

targeted treatment of lung cancer

Melissa Y. Lucero and Jefferson Chan*

Department of Chemistry and Beckman Institute for Advanced Science and Technology, University of

Illinois at Urbana-Champaign, Urbana, IL 61801, United States

Abstract

Companion diagnostics (CDx) represent a new frontier in personalized medicine that promises to improve

treatment outcomes by matching therapies to patients. Currently, these tests are limited in scope and

cannot report on real-time changes associated with disease progression and remediation. To address this,

we have developed the first photoacoustic imaging-based CDx (PACDx) for the selective detection of

elevated glutathione (GSH) in lung cancer. Since GSH is abundant in most cells, it was essential to tune

the reactivity of the benzenesulfonyl-based trigger to distinguish between normal and pathological states.

Moreover, we designed a matching prodrug, PARx, that utilizes the same mechanism to release both a

chemotherapeutic (Gemcitabine) and a PA readout. We demonstrate that PARx can inhibit tumor growth

while sparing all other tissue from off target toxicity in a A549 lung cancer xenograft model. We envision

that this work will establish a new standard for personalized medicine by employing a unique imaging-based

approach.

Introduction

The Centers for Disease Control and Prevention estimates that there will be over 1.8 million new cases of

cancer in the United States in 2020. Despite recent breakthroughs in cancer treatment that include new

FDA approved drugs and immunotherapy, over a third of these patients will not survive. Lung cancer is

expected be responsible for the majority of these deaths.1 Disease heterogeneity and interpatient variability

are major contributors for the poor efficacy and safety of common treatment regimens. Indeed, it is not

uncommon for physicians to rely almost entirely on a trial and error approach when making therapeutic

decisions.2 A new frontier in personalized medicine that aims to overcome this major challenge is the

application of companion diagnostics (CDx).3 A CDx is a test that can provide essential information for

determining which patients may benefit from a particular drug by detecting biomarkers critical for drug

2

activation.4 For instance, a CDx for Herceptin (an immunotherapy for breast cancer) can identify the

presence and expression levels of the target receptor, HER2, in a patient’s tumor. However, most FDA-

approved CDx are limited to in vitro testing which cannot account for factors such as biochemical changes

that occur as the disease progresses. In order to provide accurate, real-time information, it is crucial to

develop new molecular imaging-based CDx for aberrant cancer properties. It is noteworthy that most

imaging probes targeting cancer have been developed for optical-based methods (e.g., fluorescence) which

can only image in the millimeter depth range due to scattering and attenuation of light.5 Photoacoustic (PA)

imaging, on the other hand, is a noninvasive modality that utilizes near-infrared (NIR) light to induce the

generation of ultrasound. Because ultrasound scatters 103 times less than light in biological tissues, high

resolution images (tens of microns) can be obtained in the centimeter range.6 PA imaging has already been

employed clinically for the detection of breast cancer,7 thyroid cancer,8 inflammatory arthritis,9 and

scleroderma10 in label-free studies. Recently, our group11,12 and others have expanded the scope of PA

imaging through the development of acoustogenic probes. Select examples include imaging agents for the

detection of metal ion dysregulation,13-16 hypoxia,17-20 proteases,21,22 and signaling molecules such as nitric

oxide23,24 and hydrogen sulfide.25 By leveraging our expertise in probe design and molecular imaging, we

now report the development of the first PA imaging-based CDx (PACDx), as well as a matching

Gemcitabine-based prodrug (PARx) that utilizes the same activation mechanism to target lung cancer in a

murine model system.

Results

The design of PACDx features two main components: 1) a near-infrared dye26 capable of generating a

strong PA signal upon irradiation and 2) a benzenesulfonyl trigger for detecting glutathione (GSH) via

nucleophilic aromatic substitution (SNAr) chemistry (Figure 1a). As the most abundant biological thiol in the

body, GSH is essential for maintaining redox homeostasis and detoxifying xenobiotics.27 Consequently,

aberrant changes in the cellular levels of GSH are correlated with a variety of pathologies such as cancer.28

Among the various cancer types, non-small cell lung carcinoma has the largest change in GSH levels for

both patient derived tissue samples (up to 4-fold)29 and human cell lines (up to 7-fold),30,31 relative to healthy

tissue and non-cancerous cell lines, respectively. With this in mind, we chose to develop a CDx assay for

3

lung cancer based on the detection of GSH via PA imaging. This represents a significant and exciting

challenge because existing GSH-responsive triggers based on disulfide exchange32 and SNAr33 chemistry

are generally too reactive to distinguish GSH levels in lesions versus healthy tissue, let alone different

cancer types. For instance, installation of the 2,4-dinitrobenzenesulfonate trigger onto our PA platform

afforded a probe that was fully activated with only 100 µM GSH (~10-fold less than physiological levels)

(Figure 1b). For this reason, this probe could not distinguish GSH levels across several mammalian cell

lines (Figure S1). We hypothesized that increasing the electron density on the benzenesulfonyl core could

attenuate the SNAr reaction with GSH and thus, afford a new trigger with the appropriate reactivity between

0.1 to 10 mM. To this end, we synthesized a panel of fourteen probes, from which two promising candidates

were identified (Figures S2-4). Specifically, replacement of the 4-nitro group with a trifluoromethyl

substituent (compound 3) decreased the reactivity towards low concentrations of GSH (100 µM, 1 h)

(Figure 1c); however, it was still too reactive in the presence of physiological concentrations (1 mM GSH,

11.9-fold turn-on response after 1 h). This result indicates there would be significant false positives if the

probe was taken up by healthy tissue. Reducing the reactivity further by substituting the 2-nitro moiety with

a fluoro group resulted in highly optimized trigger (Figure 1d). Activation of the resulting probe, PACDx,

under physiological conditions was completely attenuated. On the other hand, a dose-dependent signal

increase up to 31.6-fold was observed when PACDx was incubated with 10 mM GSH for 1 h. Prior to

activation, PACDx does not absorb strongly within the PA window (680 - 950 nm); however, treatment with

GSH induces a bathochromic shift into the NIR region (λmax = 690 nm) (Figure 1e). Irradiation at this

wavelength yields the strongest PA response (Figure 1f). A dose-dependent PA signal enhancement (R2

= 0.99) was observed when PACDx was treated with increasing concentrations of GSH (Figure 1g and

1h). In contrast, when PACDx was incubated with biologically relevant concentrations of cysteine (200 µM)

and homocysteine (100 µM) there was no notable reactivity which further demonstrates the exceptional

selectivity exhibited by our probe (Figure S5).

Next, we turned our attention to evaluating the performance of PACDx in live cells using confocal

microscopy. Of note, PACDx activation can be conveniently monitored in both PA and fluorescent modes

due to its multimodal imaging capabilities. In addition to examining PACDx in A549 lung cancer cells, we

also imaged U87 glioblastoma cells (a brain cancer cell line) and HEK 293 cells (a non-cancerous cell line)

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which are expected to have low GSH levels. When A549 cells were incubated with PACDx, we observed a

highly fluorescent cytosolic and mitochondrial staining pattern (Figure 2a). In contrast, when A549 cells

were pretreated with N-ethylmaleimide (NEM), a reagent used to reduce the levels of intracellular thiols,

the fluorescence signal was ~50% dimmer (Figure 2b). To confirm that the decrease in intensity was due

to GSH depletion, we treated a third set of A549 cells with a non-responsive probe featuring an electron

rich 4-methoxybenzensulfonyl trigger (Ctrl-PACDx, Figure S6). As anticipated, these cells were almost

completely non-fluorescent (Figure 2c). For further validation of GSH detection, U87 cells were treated

with PACDx where we observe a similar trend comparable to A549 cell imaging (Figure S7). Of note,

probes featuring this trigger typically serve as substrates for glutathione S-transferase (GST), an abundant

cytosolic enzyme that catalyzes the conjugation of GSH to electrophilic centers.34,35 Since the expression

of GST may vary in cancer, reactivity with this enzyme can confound imaging results. Interestingly, when

A549 cells (and U87 cells for further validation) were pretreated with ethacrynic acid,36 a potent reversible

inhibitor of human GST, prior to staining with PACDx, no effect on probe activation was observed (Figure

S8). This data suggests that the SNAr reaction between GSH and the trigger is not dependent on GST

enzymatic activity. For PACDx to function as an effective CDx, it is essential for it to be able to accurately

differentiate the relative GSH levels in lung cancer cells compared to other cancer types, as well as healthy

cells. To our delight, the intensity of PACDx in A549 cells was indeed the highest relative to U87 and HEK

293 cells (Figure 2e). However, it is important to note that the signal from the U87 cells were significantly

lower than that of the HEK 293 cells. To account for possible variations in probe uptake and retention that

may be responsible for this result, we incubated PACDx with cell lysates from each cell line and obtained

results consistent with what was observed in intact cells (Figure S9). This suggests that the intracellular

levels of GSH is highest in A549 cells and lowest in U87 cells. This was confirmed with the established

Ellman’s assay which allows for accurate quantification of GSH levels in cell lysates (Figure 2f).37

Having successfully developed PACDx for the selective detection of elevated GSH levels in lung cancer

cells, we turned our attention to designing a prodrug invoking the same GSH-mediated chemistry. We

rationalized it would be possible to append a chemotherapeutic to the PACDx core by strategically installing

a hydroxymethyl handle ortho to the phenolic alcohol. Upon removal of the 2-fluoro-4-nitrobenzenesulfonyl

trigger, the resultant phenolate intermediate can fragment via a 1,4-elimination pathway to release the drug

5

and the corresponding dye (HD-CH2OH) for PA imaging (Figure 3a). We selected to append Gemcitabine,

an FDA-approved drug, because it is commonly used to treat non-small cell lung cancer through the

inhibition of DNA synthesis.38,39 However, as with many chemotherapeutics, Gemcitabine indiscriminately

targets any rapidly dividing cell in the body which results to adverse effects such as myelosuppression and

liver toxicity.40 Masking the primary alcohol with a variety of capping groups have led to the development

of Gemcitabine-based prodrugs displaying attenuated cytotoxicity until they are activated.41 Although we

could have directly modified Gemcitabine with our new 2-fluoro-4-nitrobenzenesulfonyl trigger, we wanted

to leverage the PA imaging capabilities of the resulting prodrug (herein named PARx) to monitor drug

release in real-time. PARx was synthesized starting from the sequential reduction of 2,4-

dihydroxybenzaldehyde and TBS protection to afford 16 in 61% yield. In situ deprotection of the phenolic

alcohols with sodium hydride facilitated a nucleophilic substitution and retro-Knoevenagel sequence with

Cy7-Cl to obtain 17. The GSH-responsive trigger was installed, and the primary alcohol was deprotected

under acidic conditions to yield 18 (Ctrl-PARx-2) in 46% over 2-steps. Finally, a chloroformate intermediate

was generated using phosgene which was reacted with Gemcitabine to obtain PARx (Supporting

information).

With PARx in hand, we first evaluated whether attaching Gemcitabine would alter the PA imaging properties

of the dye scaffold, as well as the responsiveness of the trigger to GSH. The wavelength of maximum

absorption (λmax = 700 nm) of HD-CH2OH was red-shifted by ~10 nm relative to 1 (Figure 1e and 3b).

However, the wavelength associated with the strongest PA signal (690 nm) was identical for both dyes

(Figure 1f and 3c). We observed that irradiation of PARx consistently yielded a more intense PA signal,

presumably due to the larger PA brightness value (ɛ × (1- ΦFl)).20 Importantly, the response to GSH was

not affected as PARx could also be activated in a dose-dependent fashion (Figures 3d and S10). We also

demonstrated that PARx displayed exceptional selectivity against a panel of metal ions, amino acids,

reductants, reactive nitrogen species, reactive oxygen species, metabolic liver enzymes, and competing

thiols (Figure 3e). Next, we employed MS and NMR analyses to confirm that GSH mediates the release of

Gemcitabine (Figures S11 and S12). To further support these results, we prepared a control compound

(Ctrl-PARx-1) that is equipped with an unreactive 4-methoxybenzenesulfonyl trigger (Figure S13). MS

analysis reveals that both the trigger and carbonate linkage of Ctrl-PARX-1 were stable in the presence of

6

10 mM GSH for at least 1 h (Figure S14). Moreover, when subjected to live cells, PARx could readily

distinguish GSH levels in A549, U87, and HEK293 cells (Figures 3f and S15). Lastly, we assessed the

cytotoxicity of PARx in A549 cells using the MTT assay. Our results indicate that PARx exhibits dose-

dependent toxicity that is comparable to free Gemcitabine. On the contrary, when we treated cells with

either Ctrl-PARx-1 or Ctrl-PARx-2, a second control reagent lacking Gemcitabine, the cytotoxicity was

significantly attenuated (Figure 3g). Repeating the corresponding MTT assays in U87 cells clearly

establishes that PARx requires the elevated levels of GSH found in A549 cells to effectively mediate the

release of Gemcitabine.

To evaluate the in vivo efficacy of the PACDx and PARx pair, we established a A549 xenograft model of

lung cancer. After the tumors had grown to a volume of ~100 mm3 we administered PACDx via intratumor

injection. Irradiation at 680 nm (the optimal in vivo wavelength) at the 1 h time point resulted in an increase

in the PA signal by ~1.5-fold relative to the control flank which does not bare a tumor. These results are

consistent with our in vitro findings and clearly demonstrate that our trigger can distinguish normal and

pathological levels of GSH in live animals. Next, we introduced PARx via systemic administration (i.e.,

retroorbital injection) to determine its biodistribution profile, as well as potential off-target cytotoxic effects.

We harvested tissue from vital organs 1 h post-injection for PA imaging analysis. The PA signals from the

heart, kidneys, liver and spleen were not statistically different between the treatment and control groups

(Figure 4a and 4b). In contrast, the PA intensity of the tumor tissue was 1.2-fold higher for the PARx treated

animals suggesting that activation and subsequent release of Gemcitabine was selective in tumors. The

results from our PA imaging experiments suggest PARx will not have off-target toxicity. To asses this, we

performed histological staining which revealed that there was minimal toxicity to the heart, kidneys, liver,

and spleen in PARx treated animals (Figure 4c). However, PARx was highly toxic in tumor tissue as

indicated by the decrease in the number of nuclei and size of the tumor cells. In addition, TUNEL staining

revealed that the poor morphology observed was due to apoptosis induced by the release of Gemcitabine

from PARx (Figure 4d).

Encouraged by these promising results, we set forth to determine whether PARx could function as an

effective prodrug for the selective, GSH-mediated killing of lung cancer cells. Over a 21-day period, PARx

7

was administered to A549 xenografts via intratumoral injection every 7 days. PA imaging was performed 1

h after treatment to monitor Gemcitabine release (Figure S16). Compared to control tumors (Figure 5a

and 5d), the PA signal was higher in PARx-treated tumors which indicates turnover of the 2-fluoro-4-

nitrobenzenesulfonyl trigger (Figure 5b and 5d). Owing to the release of Gemcitabine, tumor growth was

fully attenuated when treated with PARx (Figure 5e). On the contrary, tumors grew up to ~600 mm3 in size

(6-fold increase) when animals received a saline vehicle control. To evaluate the systemic compatibility of

PARx, we retro-orbitally administered PARx every 7 days over the course of a 21-day period (Figures 5e).

The inhibition of tumor growth using this dosing regimen was nearly identical to the results obtained via

intratumoral administration. During the same time period, we monitored the animal’s body weight as a

measure of general toxicity over the course of treatment. Under no treatment conditions did we observe

any loss of weight (Figure 5f) or change in behavior.42 To further challenge the prodrug approach, we

increased the dosing frequency of PARx from once every 7 days to once every 3 days (Figure S17). We

hypothesized that we would not see any adverse effects, especially in the liver where severe damage is

common with free Gemcitabine. Gratifyingly, we did not observe a change in the body weight (Figure S18)

or behavior of the animals, nor did we detect any damage to the liver of the treated animals.

Discussion

The identification of disease biomarkers and the subsequent use of this information to guide

therapeutic decision-making is a hallmark of personalized medicine. In this study, we have developed the

first PA imaging-based CDx for the detection of GSH in lung cancer. A major advantage of employing an

imaging-based approach rather than a conventional in vitro testing strategy, is the unique ability to visualize

changes that occur during disease progression in real time. It is noteworthy that GSH is typically not

considered to be an ideal cancer biomarker, even though it is elevated by up to 7-fold in lung cancer,

because current sensing strategies are too insensitive to distinguish between normal and pathological

levels, especially in vivo . For instance, we demonstrate that when the common 2,4-dinitrobenzenesulfonyl

trigger is installed onto our PA platform, the resulting probe is fully activated within minutes with as little as

100 µM GSH. This proves difficult for use in living systems (e.g., live cells and animals) where GSH is

present in all tissue at high concentrations (> 1 mM). By rationally modulating the structure and electronics

8

of the benzenesulfonyl trigger we have fine-tuned the SNAr reactivity enabling us to develop PACDx which

can reliably differentiate GSH in the 0.1 to 10 mM concentration range. Although several fluorescent probes

have been developed using an analogous approach, they detect GSH indirectly via GST activity.43 In

contrast, we demonstrate PACDx is directly sensing GSH since inhibition GST had no effect on probe

activation, presumably because PACDx is not a viable substrate for GST. This eliminates a potentially

confounding variable since GST expression can vary across cancer types and is known to change

throughout tumor progression.

In this study we also developed a highly effective prodrug (PARx) that utilizes the same GSH-mediated

activation mechanism to selectively release Gemcitabine from a PA imaging dye. Although we could have

directly installed the new trigger onto Gemcitabine, the ability to perform PA imaging provides us with a

powerful handle to monitor and confirm drug delivery. We envision further exploiting this robust design since

other drugs, as well as biologically relevant analytes can be appended for targeted delivery. With regards

to Gemcitabine, it is an FDA-approved drug and one of the first line treatment options for various cancers

including lung cancer. Unfortunately, it is characterized by rapid metabolism, poor bioavailability and low

tumor uptake. As a result, frequent doses must be administered to ensure a therapeutic response. However,

high levels of this drug result in adverse effects such as myelosuppression and severe liver damage. Since,

PARx is only activated in lung cancer cells we were able to demonstrate through different dosing regimens

(every 7 days versus every 3 days for 21 days) that PARx did not display any off-target toxicity. For instance,

histological analysis of tissue samples harvested after the final dosing was identical to the non-treated

control tissue. Lastly, PARx does not damage the liver because it is not processed by liver enzymes, as

demonstrated by an in vitro assay where it was incubated with RLMs that contain a variety of cytochrome

P450 enzymes.

In conclusion, this study showcases the potential use of PA imaging-based CDx to detect critical biomarkers

for effective personalized therapy. In current studies we are employing PACDx and PA imaging to stratify

animals based on GSH levels. This will allow us to predict, prior to treatment, which group of animals will

have the greatest therapeutic efficacy when treated with PARx. On another important note, PA monitoring

of drug release may also aid in the drug development process since confirmation of drug delivery in real-

time is a major challenge. However, in the case of PARx, our in vitro assays demonstrate that the intensity

9

of the PA response correlates with the amount of drug being released. We envision that this work will

establish a new standard for co-developed PA imaging-based CDx and safe prodrugs that both employ the

same selective activation mechanism.

Acknowledgements

This work was supported the National Institutes of Health (R35GM133581). M.Y.L acknowledges the Alfred

P. Sloan Foundation for financial support. Major funding for the 500 MHz Bruker CryoProbeTM was

provided by the Roy J. Carver Charitable Trust (Muscatine, Iowa; Grant No. 15-4521) to the School of

Chemical Sciences NMR Lab. The Q-Tof Ultima mass spectrometer was purchased in part with a grant

from the National Science Foundation, Division of Biological Infrastructure (DBI-0100085). We also

acknowledge the Core Facilities at the Carl R. Woese Institute for Genomic Biology for access to the Zeiss

LSM 700 confocal microscope and corresponding software. M.Y.L. thanks Ms. Hailey Knox for initial animal

training and assistance with the NanoZoomer. We thank Mr. Thomas Bearrood and Ms. Chelsea Anorma

for assistance with initial confocal imaging experiments, Mr. Lucas Akin for aid with mass spectrometry

experiments, and Prof. Sayeepriyadarshini Anakk and Ms. Angela Dean for help with interpreting results

from H&E staining experiments.

Author contributions

M.Y.L. performed all experiments in this study that include chemical synthesis, in vitro characterization,

cellular studies, tumor model studies, in vivo imaging, and sample preparation for ex vivo analysis. M.Y.L.

and J.C. analyzed the data and prepared the manuscript. J.C. conceived the project with intellectual

contributions from M.Y.L.

10

Figure 1. a) General schematic for GSH-responsive photoacoustic companion diagnostics. Dose-

dependent activation of b) 2,4-dinitrobenzenesulfonyl HD, c) 4-trifluoromethyl-2-nitrobenzenesulfonyl HD;

and d) 2-fluoro-4-nitrobenzenesulfonyl HD (PACDx) where [GSH] = 0.1 – 10 mM. Error bars = SD (n = 3).

e) Absorbance profile of 5 µM PACDx before (black line) and after (red line) treatment with 10 mM GSH at

37 °C for 1 h (pH 7.4, 70% PBS/MeCN). f) PA spectra of 50 µM turned over HD dye in 70% PBS/MeCN. g)

PA signal (n = 3) and h) PA images of PACDx (50 µM, 70 % PBS/MeCN, pH 7.4, 37 °C for 1 h) in response

to GSH.

11

Figure 2. Confocal microscopy image representing a) A549 cells treated with 5 µM PACDx for 1 h at 37

ºC, b) A549 cells pre-treated with 1 mM NEM then incubated with 5 µM PACDx for 1 h at 37 ºC, and c)

A549 cells treated with 5 µM Ctrl-PACDx for 1 h at 37 ºC. Scale bar represents 20 µm. d) Normalized

fluorescence intensity obtained from cell imaging under conditions represented in a-c. Error bars = SD (n ≥

3). e) Normalized fluorescence intensity obtained from cell imaging A549, U87, and HEK293 cells with 5

µM PACDx for 1 h at 37 ºC. Error bars = SD (n ≥ 3). f) Normalized [GSH] obtained from Ellman’s assay.

Error bars = SD (n = 2). Statistical analysis was performed using Student’s t-test, ***: p < 0.001, relative to

A549.

12

Figure 3. a) Reaction scheme of PARx with GSH to release HD-CH2OH and Gemcitabine. b) Normalized

absorbance profile of 5 µM PARx before (black line) and after (red line) treatment with 10 mM GSH at 37

°C for 1 h (pH 7.4, 70% PBS/MeCN). c) PA spectra of 50 µM turned over HD-CH2OH dye in 70%

PBS/MeCN. d) PA images of PARx in response to GSH. e) Reactivity of PARx with biologically relevant

metals, amino acids, rat liver microsomes, ascorbic acid, RNS, ROS, and thiols after 1 h incubation at 37

°C. f) Normalized fluorescence intensity obtained from cell imaging A549, U87, and HEK293 cells with 5

µM PARx for 1 h at 37 ºC. Error bars = SD (n ≥ 3). g) Cell viability assay using various concentrations of

Gemcitabine, PARx, and Ctrl-PARx-2 after 48 h incubation with A549 cells.

13

Figure 4. a) Representative ex vivo PA images of heart, kidneys, liver, spleen and tumor after systemic

injection of PARx (400 µM, 10% DMSO/PBS, retro-orbital injection) or vehicle. Scale bar represents 2 mm.

b) Normalized PA signal (relative to a reference probe-independent PA signal) after systemic injection of

PARx or vehicle. Error bars = SD (n = 3). c) H&E staining of heart, kidney, liver, spleen, and tumor tissue

from PARx-treated and untreated A549 xenografts. d) TUNEL staining of tumor tissue from PARx-treated

and untreated A549 xenografts. Brown staining indicates apoptotic cell death. Scale bar represents 100

µm.

14

Figure 5. PA images of tumors after a) treatment with vehicle (10%DMSO/PBS), b) intratumoral injection

of PARx (100 µM, 10% DMSO/PBS), or c) retro-orbital injection of PARx (400 µM, 10% DMSO/PBS). Scale

bar represents 2 mm. d) Normalized PA signal after treatment with vehicle or PARx. a: intratumoral injection.

b: retro-orbital injection. Error bars = SD (n = 3, tumor volume < 200 mm3). e) Average tumor volume after

treatment with vehicle, intratumoral, or retro-orbital injection of PARx over 21 days. f) Average body weight

after treatment with vehicle, intratumoral, or retro-orbital injection of PARx over 21 days. g) Representative

tumor photographs that were treated with vehicle, intratumoral, or retro-orbital injection of PARx. Statistical

analysis was performed using Student’s t-test, **: p < 0.01, *: p < 0.05.

15

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download fileview on ChemRxivPACDx ChemRxiv Final.pdf (1.34 MiB)

Towards personalized medicine: Photoacoustic imaging enables companion diagnosis and

targeted treatment of lung cancer

Melissa Y. Lucero and Jefferson Chan*

Department of Chemistry and Beckman Institute for Advanced Science and Technology, University of

Illinois at Urbana-Champaign, Urbana, IL 61801, United States

Materials. Materials were purchased from commercial vendors and used without further purification. All

deuterated solvents were purchased from Cambridge Isotope Laboratories. Acetone, ammonium chloride,

dichloromethane, dimethyl sulfoxide, glacial acetic acid, phosphate saline buffer (Corning), Matrigel

(Corning), sodium bicarbonate, sodium carbonate, sodium chloride, sodium hydroxide were purchased

from Thermo Fisher Scientific. Anhydrous methanol and conc. hydrochloric acid were purchased from

Macron Fine Chemicals. Acetic anhydride, anhydrous dichloromethane, anhydrous dimethylformamide,

sodium hydride, phosgene (15 wt. % in toluene), β-Nicotinamide adenine dinucleotide 2′-phosphate

reduced tetrasodium salt hydrate (NADPH), formaldehyde (37 % w/w in water), glutathione (reduced),

hexanes, L-ascorbic acid, L-cysteine, L-phenylalanine, L-arginine, glycine, L-lysine, rat liver microsomes

(pooled, male), and trypan blue powder were purchased from Millipore-Sigma Aldrich. Potassium iodide,

2,4-dihydroxybenzaldehyde and sodium sulfate (anhydrous) were purchased from Oakwood Chemicals.

Gemcitabine HCl was purchased from AK Scientific. N-ethylmaleimide was purchased from Pierce

Chemical Company. A549, HEK 293, and U87 cells were purchased from ATCC. Cells were generally

incubated at 37 °C under 5% CO2.

Instruments and Software. 1H and 13C NMR spectra were acquired on the Carver B500 spectrometer.

The following abbreviations were used to describe coupling constants: singlet (s), doublet (d), triplet (t), or

multiplet (m). Spectra were visualized and analyzed using MestReNova (version 10.0) and referenced to

trace non-deuterated solvent. High-resolution mass spectra were acquired on a Waters Q-TOF Ultima ESI

mass spectrometer or a Waters Synapt G2-Si ESI/LC-MS spectrometer. Ultraviolet-visible spectroscopy

was performed on a Cary 60. Fluorescence spectra were acquired on a QuantaMaster400 scanning

spectrofluorometer. Ultraviolet-visible spectroscopy and fluorimetry was performed with a micro

fluorescence quartz cuvette (Science Outlet). Cells were visualized on an EVOS FL epifluorescence

microscope and cellular imaging was performed using a Zeiss LSM 700 confocal microscope. SpectraMax

M2 plate reader was used for cell viability assays. Confocal images were analyzed using Fiji1. Data was

analyzed using Microsoft Excel. PA imaging was performed using an Endra Nexus 128+ photoacoustic

tomography system. PA images were analyzed using Horos software. Reported values correspond to mean

PA signals in regions of interest (ROIs) of equal area. Statistical calculations (Student’s t-test) were

performed using Microsoft Excel.

Synthetic Procedures. Thin-layer chromatography (TLC) was performed on glass-backed TLC plates

precoated with silica gel containing an UV254 fluorescent indicator (Macherey-Nagel). TLC’s were

visualized with a 254/365 nm UV hand-held lamp (UVP). Flash silica gel chromatography was performed

using 0.04 – 0.063 mm 60 M silica (Macherey-Nagel). All glassware used under anhydrous reaction

conditions were flame-dried under vacuum and cooled immediately before use.

Synthesis of 1. Compound 1 was synthesized by following a published protocol2.

General synthesis of 2-14. To a solution of 1 and the appropriate benzenesulfonyl chlorides (4.0 eq.) in

CH2Cl2 was added Cs2CO3 (0.5 eq.). The reaction was monitored by TLC. After stirring at room temperature,

the reaction was concentrated under reduced pressure to afford the crude residue which was purified via

flash chromatography on a silica column (1:19 v/v MeOH:CH2Cl2) to afford compounds 2-14 as purple films.

O O

NCs2CO3

CH2Cl2

2-14

O OH

NR1

SO Cl

O

Cmpd = R1, R22 = NO2, NO23 = CF3, NO24 = H, NO25 = NO2, H 6 = CN, H7 = F, F8 = CF3, H9 = Cl, H10 = Br, H11 = F, H12 = H, H13 = CH3, H14 = OCH3, H

R2

R1

S OOR2

1

Compound 2. 1H NMR (500 MHz, CDCl3) δ 8.68 (t, J = 1.7 Hz, 1H), 8.64 – 8.58 (m, 2H), 8.37 (ddd, J =

14.0, 8.6, 1.3 Hz, 2H), 8.28 (dt, J = 8.7, 1.7 Hz, 1H), 8.25 (t, J = 1.7 Hz, 1H), 7.54 (t, J = 7.7 Hz, 2H), 7.48

(t, J = 7.1 Hz, 2H), 7.33 (dd, J = 8.5, 1.2 Hz, 1H), 7.21 (d, J = 2.2 Hz, 1H), 7.03 (dt, J = 8.4, 1.7 Hz, 1H),

6.98 (s, 1H), 6.75 (d, J = 15.2 Hz, 1H), 4.60 (q, J = 7.2 Hz, 2H), 2.77 (t, J = 6.1 Hz, 2H), 2.71 (t, J = 6.1 Hz,

2H), 1.92 (p, J = 6.1 Hz, 2H), 1.81 (s, 6H), 1.56 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 178.71,

158.45, 152.99, 151.14, 149.37, 148.93, 148.25, 147.46, 146.56, 145.39, 142.51, 140.86, 134.24, 133.19,

131.90, 129.45, 129.02, 128.60, 128.37, 127.10, 125.28, 122.63, 121.72, 120.46, 118.48, 118.24, 116.02,

113.38, 110.11, 107.25, 51.34, 41.82, 29.71, 29.58, 27.78, 24.14, 20.08, 13.20.

Compound 3. 1H NMR (500 MHz, CDCl3) δ 8.60 (d, J = 14.4 Hz, 1H), 8.30 (d, J = 8.0 Hz, 2H), 8.10 (s,

2H), 7.69 (dd, J = 8.3, 1.8 Hz, 1H), 7.64 (s, 1H), 7.48 (ddt, J = 23.6, 14.6, 7.2 Hz, 4H), 7.36 (d, J = 8.3 Hz,

1H), 7.18 (s, 1H), 7.01 (d, J = 9.8 Hz, 2H), 6.73 (d, J = 14.8 Hz, 1H), 4.59 (s, 2H), 2.74 (s, 2H), 2.67 (d, J =

6.0 Hz, 2H), 1.86 (d, J = 11.6 Hz, 2H), 1.79 (s, 6H), 1.53 (d, J = 6.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ

178.61, 158.62, 152.94, 149.51, 148.72, 148.29, 146.51, 143.11, 142.50, 140.85, 133.51, 131.62, 131.50,

131.26, 129.77, 129.45, 128.80, 128.31, 127.48, 127.45, 123.88, 122.62, 122.41, 122.38, 121.64, 119.93,

119.90, 119.87, 118.60, 115.91, 113.47, 110.09, 107.11, 51.28, 41.87, 29.71, 29.54, 27.83, 24.18, 20.09,

13.26.

Compound 4. 1H NMR (500 MHz, CDCl3) δ 8.57 (d, J = 15.2 Hz, 1H), 8.17 (dd, J = 7.9, 1.4 Hz, 1H), 8.04

(dd, J = 8.0, 1.3 Hz, 1H), 7.93 (td, J = 7.7, 1.4 Hz, 1H), 7.88 (dd, J = 7.9, 1.4 Hz, 1H), 7.79 (td, J = 7.7, 1.5

Hz, 1H), 7.54 – 7.48 (m, 3H), 7.45 (ddt, J = 9.0, 3.7, 2.6 Hz, 2H), 7.37 (dtd, J = 15.0, 7.9, 1.5 Hz, 2H), 7.30

(d, J = 8.5 Hz, 1H), 7.14 (d, J = 2.2 Hz, 1H), 6.99 (dd, J = 8.4, 2.3 Hz, 1H), 6.97 (s, 1H), 6.81 (d, J = 15.4

Hz, 1H), 4.64 (q, J = 7.3 Hz, 2H), 2.77 (t, J = 6.1 Hz, 2H), 2.66 (d, J = 5.8 Hz, 2H), 1.86 (t, J = 6.2 Hz, 2H),

1.78 (s, 6H), 1.53 (t, J = 7.3 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 178.58, 158.43, 152.89, 149.82, 148.59,

148.40, 146.42, 142.49, 140.92, 139.54, 136.31, 132.64, 132.15, 131.62, 130.89, 130.79, 130.17, 129.60,

129.42, 129.13, 128.41, 128.20, 127.78, 125.09, 122.53, 121.40, 118.70, 116.08, 113.51, 110.08, 107.43,

51.22, 41.87, 29.71, 29.56, 27.83, 24.11, 20.09, 13.28.

Compound 5. 1H NMR (500 MHz, CDCl3) δ 8.57 (d, J = 14.9 Hz, 1H), 8.44 (d, J = 8.5 Hz, 2H), 8.13 (dd, J

= 12.0, 8.6 Hz, 4H), 8.05 (d, J = 8.8 Hz, 2H), 7.54 (t, J = 7.1 Hz, 2H), 7.49 (t, J = 7.7 Hz, 2H), 7.28 (s, 1H),

7.05 (d, J = 2.2 Hz, 1H), 6.97 (s, 1H), 6.83 (d, J = 15.0 Hz, 1H), 6.74 (dd, J = 8.4, 2.1 Hz, 1H), 4.67 (d, J =

7.1 Hz, 2H), 2.79 (s, 2H), 2.69 (d, J = 6.5 Hz, 2H), 1.87 (s, 2H), 1.79 (s, 6H), 1.57 (t, J = 6.6 Hz, 3H). 13C

NMR (125 MHz, CDCl3) δ 178.64, 158.44, 153.40, 152.93, 151.27, 149.98, 147.85, 146.42, 142.45, 140.84,

140.63, 131.61, 130.05, 129.54, 129.12, 128.45, 128.38, 127.46, 124.65, 123.19, 122.62, 121.38, 118.59,

115.98, 113.46, 110.25, 107.44, 51.25, 41.94, 29.71, 29.58, 27.83, 24.19, 20.08, 13.34.

Compound 6. 1H NMR (500 MHz, CDCl3) δ 8.57 (d, J = 15.2 Hz, 1H), 8.08 – 8.03 (m, 2H), 8.01 (d, J = 8.2

Hz, 2H), 7.94 – 7.89 (m, 2H), 7.59 – 7.45 (m, 6H), 7.28 (d, J = 8.5 Hz, 1H), 7.04 – 6.98 (m, 2H), 6.81 (d, J

= 15.3 Hz, 1H), 6.73 (dd, J = 8.4, 2.3 Hz, 1H), 4.66 (q, J = 7.3 Hz, 2H), 2.77 (t, J = 6.1 Hz, 2H), 2.68 (t, J =

6.2 Hz, 2H), 1.86 (p, J = 6.1 Hz, 2H), 1.80 (s, 6H), 1.56 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ

178.60, 158.55, 152.92, 151.67, 150.03, 146.43, 142.44, 140.82, 139.13, 133.22, 132.46, 131.81, 129.53,

129.30, 129.26, 128.42, 127.08, 122.67, 121.34, 118.90, 118.69, 118.45, 116.80, 115.94, 113.43, 112.22,

110.24, 107.28, 51.26, 41.87, 29.71, 29.56, 27.86, 24.17, 20.07, 13.32.

Compound 7. 1H NMR (500 MHz, CDCl3) δ 8.57 (d, J = 14.9 Hz, 1H), 8.01 – 7.84 (m, 2H), 7.57 – 7.42 (m,

4H), 7.31 (d, J = 8.4 Hz, 1H), 7.13 – 7.04 (m, 3H), 7.00 (s, 1H), 6.92 (d, J = 15.2 Hz, 1H), 6.87 (dd, J = 8.5,

2.2 Hz, 1H), 6.72 (dtd, J = 16.0, 9.4, 8.8, 2.4 Hz, 2H), 4.74 (d, J = 7.5 Hz, 2H), 2.82 (t, J = 5.8 Hz, 2H), 2.68

(t, J = 6.0 Hz, 2H), 1.92 – 1.84 (m, 2H), 1.79 (s, 6H), 1.55 (t, J = 6.5 Hz, 3H).13C NMR (125 MHz, CDCl3) δ

178.52, 158.49, 152.89, 149.95, 146.40, 142.44, 140.92, 133.42, 133.34, 131.55, 131.16, 130.88, 129.50,

129.23, 128.43, 128.24, 122.46, 121.30, 119.76, 119.68, 118.46, 116.18, 113.59, 112.63 (dd, J = 22.3, 3.8

Hz), 110.00, 109.81 (d, J = 20.8 Hz), 107.66, 106.67 – 105.98 (t), 104.13 (t, J = 26.0 Hz). 51.14, 42.07,

29.71, 29.60, 27.89, 24.28, 20.12, 13.40.

Compound 8. 1H NMR (500 MHz, CDCl3) δ 8.56 (d, J = 15.1 Hz, 1H), 8.04 (dd, J = 19.9, 8.1 Hz, 4H), 7.87

(d, J = 8.1 Hz, 2H), 7.51 (dd, J = 7.7, 4.0 Hz, 4H), 7.49 – 7.43 (m, 2H), 7.28 (d, J = 8.5 Hz, 1H), 7.05 – 6.99

(m, 2H), 6.82 (d, J = 15.2 Hz, 1H), 6.73 (dd, J = 8.4, 2.2 Hz, 1H), 4.66 (q, J = 7.2 Hz, 2H), 2.76 (t, J = 6.0

Hz, 2H), 2.67 (t, J = 6.0 Hz, 2H), 1.85 (t, J = 6.0 Hz, 2H), 1.77 (s, 6H), 1.54 (t, J = 6.7 Hz, 3H).13C NMR

(125 MHz, CDCl3) δ 178.52, 158.64, 152.90, 150.73, 150.15, 146.43, 142.42, 140.85, 138.65, 131.48,

129.52, 129.44, 129.15, 128.43, 128.33, 126.71, 126.69, 126.66, 126.63, 126.60, 124.87, 124.84, 124.81,

124.78, 122.52, 121.27, 118.75, 115.96, 113.49, 110.19, 107.25, 51.17, 41.88, 29.70, 29.54, 27.83, 24.16,

20.08, 13.33.

Compound 9. 1H NMR (500 MHz, CDCl3) δ 8.56 (d, J = 15.0 Hz, 1H), 7.88 – 7.81 (m, 3H), 7.57 (d, J = 8.4

Hz, 2H), 7.55 – 7.43 (m, 4H), 7.26 (d, J = 8.5 Hz, 3H), 7.24 – 7.20 (m, 1H), 6.99 (t, J = 2.4 Hz, 2H), 6.91 (d,

J = 15.2 Hz, 1H), 6.71 (dd, J = 8.4, 2.1 Hz, 1H), 4.73 (q, J = 7.1 Hz, 2H), 2.80 (d, J = 6.0 Hz, 2H), 2.67 (t, J

= 6.0 Hz, 2H), 1.87 (t, J = 6.1 Hz, 2H), 1.78 (s, 6H), 1.58 – 1.52 (m, 3H).13C NMR (125 MHz, CDCl3) δ

178.53, 158.52, 152.89, 150.31, 146.42, 146.07, 142.46, 141.61, 140.93, 134.43, 133.46, 131.51, 129.98,

129.85, 129.51, 129.24, 128.27, 128.25, 127.86, 127.77, 122.48, 121.17, 118.82, 116.16, 113.56, 110.24,

107.67, 51.14, 42.06, 29.71, 29.61, 27.91, 24.27, 20.14, 13.43.

Compound 10. 1H NMR (500 MHz, CDCl3) δ 8.55 (d, J = 15.2 Hz, 1H), 7.81 – 7.70 (m, 6H), 7.56 – 7.50

(m, 2H), 7.50 – 7.43 (m, 2H), 7.41 – 7.35 (m, 2H), 7.27 (s, 1H), 7.02 – 6.97 (m, 2H), 6.88 (d, J = 15.3 Hz,

1H), 6.72 (dd, J = 8.4, 2.2 Hz, 1H), 4.70 (q, J = 7.2 Hz, 2H), 2.79 (t, J = 6.0 Hz, 2H), 2.67 (t, J = 6.1 Hz, 2H),

1.86 (t, J = 6.1 Hz, 2H), 1.78 (s, 6H), 1.54 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 178.51, 158.54,

152.88, 150.31, 146.49, 146.40, 142.45, 140.92, 134.00, 132.84, 131.48, 130.74, 130.22, 129.97, 129.51,

129.29, 128.81, 128.29, 128.25, 128.14, 122.84, 122.48, 121.17, 118.82, 116.09, 113.55, 110.22, 107.53,

51.15, 41.95, 29.71, 29.58, 27.90, 24.19, 20.11, 13.37.

Compound 11. 1H NMR (500 MHz, CDCl3) δ 8.57 (d, J = 15.0 Hz, 1H), 7.91 (ddd, J = 11.5, 8.6, 5.3 Hz,

3H), 7.55 – 7.49 (m, 3H), 7.47 (dt, J = 7.6, 5.7 Hz, 1H), 7.31 – 7.25 (m, 5H), 7.02 (s, 1H), 6.98 (d, J = 2.2

Hz, 1H), 6.96 – 6.84 (m, 2H), 6.71 (dd, J = 8.4, 2.1 Hz, 1H), 4.72 (q, J = 7.0 Hz, 2H), 2.82 (t, J = 5.8 Hz,

2H), 2.68 (t, J = 5.8 Hz, 2H), 1.88 (p, J = 5.2 Hz, 2H), 1.78 (s, 6H), 1.55 (t, J = 6.6 Hz, 3H).13C NMR (125

MHz, CDCl3) δ 178.47, 167.30, 165.25, 158.71, 152.84, 150.36, 146.46, 142.40, 140.88, 131.57, 131.50,

131.40, 130.96, 130.93, 129.51, 128.45, 128.38, 128.32, 128.24, 122.52, 121.11, 118.91, 117.02, 116.84,

116.06, 114.47, 114.30, 113.50, 110.26, 107.37, 51.13, 42.00, 29.70, 29.58, 27.89, 24.26, 20.14, 13.39.

Compound 12. 1H NMR (500 MHz, CDCl3) δ 8.57 (d, J = 15.2 Hz, 1H), 7.91 – 7.86 (m, 2H), 7.77 – 7.69

(m, 1H), 7.59 (t, J = 7.9 Hz, 2H), 7.55 – 7.50 (m, 3H), 7.46 (ddd, J = 6.9, 5.5, 3.0 Hz, 1H), 7.06 – 6.99 (m,

2H), 6.95 (d, J = 15.2 Hz, 1H), 6.68 (dd, J = 8.4, 2.2 Hz, 1H), 4.80 (q, J = 7.3 Hz, 2H), 2.86 (t, J = 6.1 Hz,

2H), 2.69 (t, J = 5.6 Hz, 2H), 1.91 (t, J = 6.1 Hz, 2H), 1.78 (s, 6H), 1.55 (t, J = 7.2 Hz, 3H). 13C NMR (125

MHz, CDCl3) δ 178.43, 158.87, 152.85, 150.57, 146.50, 142.39, 140.91, 134.94, 134.85, 131.32, 129.70,

129.52, 129.49, 128.51, 128.25, 128.21, 122.52, 121.00, 118.95, 116.12, 113.50, 110.35, 107.38, 103.06,

51.10, 42.12, 29.70, 29.58, 27.97, 24.43, 20.16, 13.41.

Compound 13. 1H NMR (500 MHz, CDCl3) δ 8.60 (d, J = 15.2 Hz, 1H), 7.79 – 7.74 (m, 2H), 7.57 – 7.50

(m, 3H), 7.50 – 7.43 (m, 1H), 7.39 – 7.35 (m, 2H), 7.24 (d, J = 8.4 Hz, 1H), 7.11 – 7.06 (m, 1H), 7.00 (d, J

= 14.7 Hz, 2H), 6.66 (dd, J = 8.4, 2.2 Hz, 1H), 4.81 (q, J = 7.3 Hz, 2H), 2.89 (t, J = 6.1 Hz, 2H), 2.71 (t, J =

5.4 Hz, 2H), 2.47 (s, 3H), 1.93 (dd, J = 8.6, 3.9 Hz, 2H), 1.80 (s, 6H), 1.56 (t, J = 7.2 Hz, 3H).13C NMR (125

MHz, CDCl3) δ 178.46, 158.82, 152.88, 150.67, 146.53, 146.08, 142.43, 140.95, 131.93, 131.35, 130.04,

129.53, 129.48, 128.53, 128.14, 128.08, 122.45, 120.90, 118.86, 116.26, 113.43, 110.44, 107.54, 51.08,

42.07, 29.71, 29.63, 27.96, 24.35, 21.83, 20.21, 13.40.

Compound 14. 1H NMR (500 MHz, CDCl3) δ 8.58 (d, J = 15.1 Hz, 1H), 7.82 – 7.75 (m, 2H), 7.52 (d, J =

3.7 Hz, 3H), 7.45 (dq, J = 10.9, 4.3 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.09 (s, 1H), 7.05 – 6.98 (m, 3H), 6.89

(d, J = 15.2 Hz, 1H), 6.68 (dd, J = 8.4, 2.2 Hz, 1H), 4.77 (q, J = 7.1 Hz, 2H), 3.88 (s, 3H), 2.84 (t, J = 6.0

Hz, 2H), 2.70 (t, J = 5.9 Hz, 2H), 1.90 (p, J = 5.9 Hz, 2H), 1.77 (s, 6H), 1.54 (t, J = 7.0 Hz, 3H).13C NMR

(125 MHz, CDCl3) δ 178.32, 164.56, 159.16, 152.85, 150.81, 146.53, 142.35, 140.89, 131.11, 130.85,

130.16, 129.52, 128.35, 128.17, 125.99, 122.53, 120.88, 119.15, 115.98, 114.67, 113.45, 110.38, 107.01,

55.99, 51.07, 42.07, 29.69, 29.55, 27.96, 20.16, 13.38.

Synthesis of 15. To a solution of 1 (30.0 mg, 0.075 mmol, 1.0 eq.) in CH2Cl2 (5 mL) was added 2-fluoro-

4-nitrobenzenesulfonyl chloride (60.0 mg, 0.25 mmol, 3.3 eq.) and triethylamine (10 µL, 0.075 mmol, 1.0

eq.). The color of reaction rapidly changed from blue to purple and reaction was complete by TLC. The

reaction was concentrated under reduced pressure to afford the crude residue which was purified via flash

chromatography on a silica column (1:9 v/v MeOH:CH2Cl2) to afford 15 (30.0 mg, 0.05 mmol, 60% yield) as

a purple film. 1H NMR (500 MHz, CDCl3) δ 8.59 (d, J = 15.2 Hz, 1H), 8.23 (dd, J = 8.6, 2.2 Hz, 1H), 8.21 –

8.12 (m, 2H), 7.58 – 7.45 (m, 4H), 7.37 (d, J = 8.5 Hz, 1H), 7.13 (d, J = 2.3 Hz, 1H), 7.08 (s, 1H), 6.91 (dd,

J = 8.4, 2.3 Hz, 1H), 6.83 (d, J = 15.2 Hz, 1H), 4.75 (q, J = 7.4 Hz, 2H), 2.88 (t, J = 6.1 Hz, 2H), 2.75 – 2.69

(m, 2H), 1.93 (p, J = 6.2 Hz, 2H), 1.81 (s, 6H), 1.57 (t, J = 7.3 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 178.47,

158.77, 152.97, 149.57, 146.42, 142.41, 140.83, 132.85, 131.73, 129.54, 129.37, 128.70, 128.40, 122.65,

121.60, 119.90, 119.87, 119.00, 118.29, 116.20, 113.62, 113.48, 113.42, 109.97, 107.60, 51.24, 42.87,

29.70, 29.56, 27.95, 25.11, 20.02, 13.48.

O

NTEA

CH2Cl2

15

O OH

NO2N F

SO

ClO

O OSF

NO2

O

1 60%

O O

N

O

O

O

HO

ON

ON

NH2

FF

SO OF

NO2

HO OH

O O OH

N

OTBS

F

NO2SO

OCl

O O

N

OH

SO O

NO2

F1) Phosgene, THF, 0

oC

2) Gemcitabine, DIPEADMF, 0 oC

TBSO OTBS

OTBS

1) NaBH4, THF

2) TBS-Cl, Imidazole, DMF

TEA, DCM

2) aq. HCl, MeOH

Cy7-Cl, NaH

DMF

1)

16 17

18 19

61%

46% 15%

Synthesis of 16. To a solution of 2,4-dihydroxybenzaldehyde (5.0 g, 36.0 mmol, 1.0 eq.) in anhydrous THF

(25 mL) was added NaBH4 (1.4 g, 36.0 mmol, 1.0 eq.). The reaction was stirred at room temperature. The

reaction was then treated with sat. NH4Cl (1 mL), filtered to remove solids and the filtrate was concentrated

under reduced pressure to obtain crude 4-(hydroxymethyl)benzene-1,3-diol as a white solid which was

used in the subsequent step without purification. The crude intermediate was dissolved in anhydrous DMF

(25 mL) and treated with imidazole (9.7 g, 140.0 mmol, 3.89 eq.) and TBS-Cl (22.0 g, 140.0 mmol, 3.89

eq.). After stirring overnight at room temperature, the reaction was transferred to a separatory funnel, diluted

with EtOAc (200 mL), and washed with brine (3×). The aqueous fractions were back-extracted with EtOAc

(1×) and the combined organic fractions were dried (Na2SO4), filtered and concentrated under reduced

pressure to afford the crude residue which was purified via flash chromatography on a silica column (1:9

v/v EtOAc:Hex) to afford 16 as a colorless liquid (10.5 g, 21.7 mmol, 61.0 % yield). 1H NMR (500 MHz,

CDCl3) δ 7.37 (d, J = 8.3 Hz, 1H), 6.58 (dd, J = 8.3, 2.3 Hz, 1H), 6.40 (d, J = 2.3 Hz, 1H), 4.80 (s, 2H), 1.12

(s, 9H), 1.09 (s, 9H), 1.05 (s, 9H), 0.33 (s, 6H), 0.29 (s, 6H), 0.19 (s, 6H). 13C NMR (125 MHz, CDCl3) δ

155.37, 152.98, 128.22, 125.46, 113.29, 110.69, 60.71, 26.37, 26.09, 26.07, 18.75, 18.54, -3.90, -4.06, -

4.93.

Synthesis of 17. A solution of 16 (10.3 g, 21.3 mmol, 2.0 eq.) in anhydrous DMF (30 mL) was cooled to 0

°C in an ice-bath. NaH (60% oil dispersion, 853.3 mg, 21.3 mmol, 2.0 eq.) was added portion-wise and

stirred until all solids were dissolved, typically yielding a deep orange solution. Cy7-Cl (5.46 g, 10.7 mmol,

1.0 eq.) was added and stirred overnight at room temperature. The reaction was concentrated to near

dryness by flowing air over the flask at room temperature. The crude residue was dissolved in CH2Cl2 (100

mL), treated with sat. NH4Cl (50 mL), transferred to a separatory funnel, and washed with brine (3×). The

organic fraction was dried (Na2SO4), filtered and concentrated under reduced pressure. The crude residue

was triturated with Et2O to afford a dark blue solid after vacuum filtration. Of note, the solid was a mixture

of compound 17 and the deprotected benzyl alcohol dye (NMR reported as 17-1) which was used without

further purification (5.64 g). 1H NMR (500 MHz, DMSO-d6) δ 8.55 (d, J = 14.8 Hz, 1H), 7.79 – 7.73 (m, 1H),

7.71 – 7.60 (m, 2H), 7.55 – 7.50 (m, 2H), 7.46 – 7.37 (m, 1H), 6.93 (s, 1H), 6.47 (d, J = 14.9 Hz, 1H), 4.72

– 4.68 (m, 2H), 4.39 (q, J = 7.2 Hz, 2H), 2.77 – 2.65 (m, 4H), 1.83 (p, J = 6.2 Hz, 2H), 1.74 (s, 6H), 1.37 (t,

J = 7.2 Hz, 3H), 0.94 (s, 9H), 0.11 (s, 6H). 13C NMR (125 MHz, DMSO-d6) δ 178.29, 163.88, 161.55, 155.87,

146.75, 144.39, 143.91, 137.90, 131.54, 130.33, 129.09, 128.55, 128.40, 125.48, 116.94, 116.47, 115.17,

105.32, 103.85, 62.06, 52.66, 30.14, 28.56, 28.50, 22.72, 20.81, 15.19, -2.63.

Synthesis of 18. To a solution of 17 (360.0 mg, 0.66 mmol, 1.0 eq., values obtained by assuming the solid

only contained 17) in CH2Cl2 (10 mL) was added 2-fluoro-4-nitrobenzenesulfonyl chloride (180 mg, 0.71

mmol, 1.13 eq.) and triethylamine (92 µL, 0.66 mmol, 1.0 eq.). The color of the reaction changed from blue

to purple and reaction was determined to be complete by TLC. The reaction was concentrated under

reduced pressure. The crude residue was dissolved in 1:1 v/v MeOH:CH2Cl2 (300 mL) and treated with 2

M aq. HCl (30 mL). After the reaction was complete it was transferred to a separatory funnel, treated with

brine (200 mL), and extracted with CH2Cl2 (3×). The combined organic fractions were dried (Na2SO4),

filtered and concentrated under reduced pressure to afford the crude residue which was purified via flash

chromatography on a silica column (1:9 v/v MeOH:CH2Cl2) to afford 18 as a deep purple film (190.0 mg,

0.30 mmol, 45.5% yield). 1H NMR (500 MHz, CDCl3) δ 8.57 (d, J = 15.0 Hz, 1H), 8.25 – 8.13 (m, 3H), 7.99

(dd, J = 8.5, 6.9 Hz, 1H), 7.82 (dd, J = 8.5, 2.1 Hz, 1H), 7.74 (dd, J = 9.0, 2.1 Hz, 1H), 7.68 (s, 1H), 7.52

(ddd, J = 20.7, 7.5, 1.3 Hz, 2H), 7.46 (td, J = 7.9, 2.6 Hz, 2H), 7.11 (s, 1H), 7.07 (s, 1H), 6.57 (d, J = 15.1

Hz, 1H), 4.54 – 4.45 (m, 4H), 2.68 (q, J = 6.3 Hz, 4H), 1.87 (p, J = 6.3 Hz, 2H), 1.79 (s, 6H), 1.54 (t, J = 7.2

Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 178.08, 159.80, 152.50, 151.40, 148.59, 147.04, 146.48, 142.20,

140.76, 132.96, 132.53, 131.40, 130.72, 130.46, 129.47, 128.47, 128.21, 122.69, 121.01, 119.92, 118.31,

115.33, 113.81, 113.61, 113.00, 111.85, 111.63, 109.32, 105.47, 58.14, 51.12, 41.37, 29.38, 27.88, 24.15,

20.08, 13.05.

Synthesis of 19 (PARx). To a solution of 18 (28.0 mg, 0.044 mmol, 1.0 eq.) in anhydrous THF (15 mL) at

0 °C under N2 was added phosgene (15 wt. % in toluene, 39.8 µL, 0.58 mmol, 13.2 eq.). Reaction was

stirred at room temperature for 3 hours, then N2 was bubbled through reaction for 20 minutes to obtain the

chloroformate intermedia. The chloroformate solution was added to a solution of gemcitabine (117 mg, 0.44

mmol, 10.0 eq.) and DIPEA (77.2 µL, 0.44 mmol, 10.0 eq.) in DMF (30 mL) at 0 °C. After 2 hours, the

reaction was treated with sat. NH4Cl and extracted with CH2Cl2. The collected organic fraction was washed

with brine, then dried over Na2SO4, filtered and concentrated under reduced pressure to afford the crude

residue which was purified via flash chromatography on a silica column (1:9 v/v MeOH:CH2Cl2) to afford 19

(6.0 mg, 0.007 mmol, 15% yield) as a purple film. 1H NMR (500 MHz, CD3OD) δ 8.69 (d, J = 15.2 Hz, 1H),

8.43 (dd, J = 9.5, 2.0 Hz, 1H), 8.36 – 8.24 (m, 3H), 7.75 – 7.65 (m, 4H), 7.65 – 7.59 (m, 1H), 7.59 – 7.52

(m, 1H), 7.22 (d, J = 15.4 Hz, 3H), 6.69 (d, J = 15.3 Hz, 1H), 6.27 – 6.21 (m, 1H), 5.20 (s, 2H), 4.50 (q, J =

7.2 Hz, 2H), 4.30 (td, J = 12.0, 8.4 Hz, 1H), 4.24 – 4.17 (m, 1H), 4.01 – 3.93 (m, 2H), 3.85 – 3.76 (m, 1H),

2.75 (dt, J = 24.6, 6.1 Hz, 4H), 1.93 (p, J = 6.2 Hz, 2H), 1.79 (s, 6H), 1.51 (t, J = 7.3 Hz, 3H). 13C NMR (125

MHz, CD3OD) δ 179.24, 167.94, 163.77, 158.54, 152.67, 147.98, 146.50, 142.83, 140.84, 132.49, 131.67,

131.01, 129.22, 129.11, 128.96, 128.46, 128.21, 125.84, 122.59, 121.46, 119.90, 115.07, 113.82, 113.61,

113.29, 109.55, 106.28, 67.71, 61.32, 58.86, 51.31, 40.86, 38.78, 30.23, 29.35, 29.00, 28.74, 26.28, 23.55,

22.63, 19.96, 13.00, 11.83, 10.00.

O O

N

O

O

O

HO

ON

ON

NH2

FF

SO O

OMeOMeS

O

OCl

O O

N

OH

SO O

OMe

1) Phosgene, THF, 0oC

2) Gemcitabine, DIPEADMF, 0oC

TEA, DCM

2) aq. HCl, MeOH

1)

20 21

O OH

N

OTBS

17 5.9% 7.2%

Synthesis of 20. To a solution of 17 (2.0 g, 3.68 mmol, 1.0 eq.) in CH2Cl2 was added 4-

methoxybenzenesulfonyl chloride (373 mg, 3.68 mmol, 1.0 eq.) and triethylamine (514 µL, 3.68 mmol, 1.0

eq.). Color of reaction rapidly changed from blue to purple and reaction was complete by TLC. The reaction

was concentrated under reduced pressure to afford the crude residue which was purified via flash

chromatography on a silica column (1:9 v/v MeOH:CH2Cl2) to obtain a mixture of 20 and the TBS protected

form of 20. The mixture was dissolved in CH2Cl2 (100 mL) and treated with 1 M HCl (50 mL, 1:1 v/v

MeOH:H2O) and stirred for 2 hours at room temperature. The reaction was washed 3 times with brine. The

combined organic fractions were dried (Na2SO4), filtered and concentrated under reduced pressure to afford

the crude residue which was purified via flash chromatography on a silica column (1:9 v/v MeOH:CH2Cl2)

to afford 20 (130 mg, 0.2 mmol, 5.9% yield) as a purple film. 1H NMR (500 MHz, CDCl3) δ 8.54 (d, J = 14.6

Hz, 1H), 7.86 – 7.70 (m, 3H), 7.48 (dt, J = 33.2, 7.5 Hz, 4H), 7.13 – 6.99 (m, 4H), 6.71 (d, J = 14.9 Hz, 1H),

4.64 (s, 2H), 4.40 (s, 2H), 3.89 (s, 3H), 2.79 – 2.57 (m, 4H), 1.86 (s, 2H), 1.78 (s, 6H), 1.53 (d, J = 6.6 Hz,

3H). 13C NMR (125 MHz, CDCl3) δ 177.78, 164.69, 160.07, 151.40, 147.86, 146.30, 142.19, 140.93, 134.00,

131.88, 130.62, 130.23, 129.41, 128.16, 127.89, 126.16, 122.57, 120.46, 115.51, 114.94, 112.96, 109.35,

105.75, 57.75, 56.05, 50.92, 29.71, 29.43, 28.04, 20.17, 13.17.

Synthesis of 21 (Ctrl-PARx-1). To a solution of 20 (31.0 mg, 0.052 mmol, 1.0 eq.) in anhydrous THF (15

mL) at 0 °C under N2 was added phosgene (15 wt. % in toluene, 46.5 µL, 0.67 mmol, 13.0 eq.). Reaction

was stirred at room temperature for 3 hours, then N2 was bubbled through the reaction for 20 minutes to

obtain the chloroformate. The chloroformate solution was added to a solution of gemcitabine (136 mg, 0.52

mmol, 10.0 eq.) and DIPEA (90.2 µL, 0.52 mmol, 10.0 eq.) in DMF (25 mL) at 0 °C. After 2 hours, the

reaction was treated with sat. NH4Cl and extracted with CH2Cl2. The collected organic fraction was washed

with brine, then dried over Na2SO4, filtered and concentrated under reduced pressure to afford the crude

residue which was purified via flash chromatography on a silica column (1:9 v/v MeOH: CH2Cl2) to afford

21 (3.3 mg, 0.003 mmol, 7.2% yield) as a purple film. 1H NMR (500 MHz, CD3OD) δ 8.71 (d, J = 15.2 Hz,

1H), 8.32 (d, J = 7.6 Hz, 1H), 7.88 (dd, J = 9.6, 2.7 Hz, 2H), 7.75 – 7.49 (m, 6H), 7.27 (d, J = 7.5 Hz, 1H),

7.20 (s, 1H), 7.17 – 7.11 (m, 3H), 6.67 (d, J = 15.2 Hz, 1H), 6.28 – 6.20 (m, 1H), 5.47 (s, 1H), 5.03 (s, 2H),

4.48 (q, J = 7.3 Hz, 2H), 4.29 (d, J = 8.7 Hz, 1H), 4.19 (dd, J = 5.7, 2.2 Hz, 1H), 4.00 – 3.92 (m, 2H), 3.85

(s, 3H), 3.83 – 3.76 (m, 1H), 2.78 – 2.69 (m, 4H), 1.92 (s, 2H), 1.81 (s, 6H), 1.50 (t, J = 7.2 Hz, 3H). 13C

NMR (125 MHz, CD3OD) δ 179.10, 167.93, 165.07, 163.86, 158.92, 152.56, 148.80, 146.51, 142.78,

140.88, 131.20, 131.00, 130.71, 129.49, 129.08, 128.82, 128.46, 128.09, 126.23, 125.99, 122.59, 120.85,

114.90, 114.71, 113.20, 109.79, 105.93, 95.61, 67.70, 61.32, 58.86, 55.21, 51.29, 40.80, 38.78, 30.23,

28.96, 28.74, 26.41, 23.56, 22.63, 20.01, 12.99, 11.81, 10.00.

In vitro buffer preparation. To 70% PBS/MeCN buffer solutions were added reduced GSH for final

concentrations of 0, 0.1, 1, 2.5, 5, and 10 mM. Adjustments to pH were made via addition of 1M HCl or 1

M NaOH. pH values were determined using a Mettler-Toledo SevenCompact pH meter calibrated using

pH 4.0, 7.0 and 10.0 standard buffers at 25 °C.

Selectivity studies. The initial absorbance (400–800 nm) was measured before the addition of amino

acids (2.5 mM), reductants (2.5 mM), thiols (2.5 mM Cys and Hcy, 10 mM GSH), reactive metals (2.5 mM),

oxygen (50 µM), and nitrogen species (50 µM). After addition, the reaction was sealed and incubated at 37

°C for 1 h. Final measurements were recorded, and relative turn-on was determined by change in

absorption at λmax. All metal solutions were prepared in water from their chloride salt. Superoxide anion was

added as a solution of potassium superoxide in DMSO. NO was generated in situ from a solution of

MAHMA-NONOate in degassed 10 mM potassium hydroxide. Peroxynitrite was synthesized according to

a literature report3. All other analytes were prepared by dilution from commercially available sources.

Microsome assay. The initial absorbance of 10 µM PARx with 10 µL rat liver microsomes in 0.1 M

potassium phosphate buffer (pH 7.4) was measured. After addition of 50 µM NADPH, the reaction was

incubated at 37 °C for 1h. The final absorbance was measured after quenching with acetonitrile.

Live cell imaging. A549 cells were plated in a poly-lysine coated microwell plate in Ham’s F12-K (10%

FBS) media for 24 h. A549 cells were then incubated with or without 1 mM N-ethylmaleimide (NEM) in 1%

DMSO:PBS or culture medium, respectively, for 30 min at 37 °C. After removing NEM, NEM-treated and

untreated cells were further incubated with PACDx (5 μM, 1% DMSO:DMEM) for 1 h at 37 °C. Another set

of cells were incubated with ctrl-PACDx (5 μM, 1% DMSO:DMEM) for 1 h at 37 °C. Cells were also imaged

after incubation with PARx (5 μM, 1% DMSO:DMEM) for 1 h at 37 °C.

U87 cells were plated in a poly-lysine coated microwell plate in EMEM (10% FBS) media for 24 h. U87 cells

were then incubated with or without 1 mM NEM in 1% DMSO:PBS or culture medium, respectively, for 30

min at 37 °C. After removing NEM, NEM-treated and untreated cells were further incubated with PACDx (5

μM, 1% DMSO:DMEM) for 1 h at 37 °C. Another set of cells were incubated with ctrl-PACDx (5 μM, 1%

DMSO:DMEM) for 1 h at 37 °C. Cells were also imaged after incubation with PARx (5 μM, 1%

DMSO:DMEM) for 1 h at 37 °C.

HEK293 cells were plated in a poly-lysine coated microwell plate in EMEM (10% FBS) media for 24 h.

HEK293 cells were then incubated with PACDx (5 μM, 1% DMSO:DMEM) for 1 h at 37 °C. Another set of

cells were incubated with PARx (5 μM, 1% DMSO:DMEM) for 1 h at 37 °C.

All cell imaging experiments were performed using Zeiss LSM700 confocal microscope with an excitation

filter of 639 nm. Imaging results were quantified relative to fluorescent intensity for each relevant control

experiment using Fiji.

Cytotoxicity assay. A549 cells were plated (5x104 cells/well) in a 24-well plate in Ham’s F12-K (10% FBS)

media for 24 h. A549 cells were then incubated with 0.0, 0.1, 5.0, 10.0, and 25 μM PARx, Ctrl-PARx-2, or

gemcitabine HCl at 37 °C in serum free DMEM. After 48 h, media was replaced with 5 mg/mL MTT reagent

(1:20 in PBS), and incubated for 1 h at 37 °C. Cells were then lysed using 500 μL DMSO, transferred to a

96-well plate and absorbances were measured using a plate reader at 555 nm. Viability was measured

relative to absorbance of control wells.

Tissue phantom preparation. Tissue phantoms were prepared by suspending agarose LE in a

solution of 2% milk (1 mL) and deionized water (39 mL). The suspension was heated in a microwave

until a viscous, translucent gel was produced. The hot gel was poured into a custom Teflon mold containing

two copper tubes and cooled at 4 °C for at least 2 hours. After cooling, the copper tubes were removed and

the gel was removed from the mold, yielding a tissue phantom with two parallel channels for the placement

of FEP tubes containing sample solutions.

Live-subject statement. All animal experiments were performed with the approval of the Institutional

Animal Care and Use Committee (IACUC) of the University of Illinois at Urbana–Champaign, following

the principles outlined by the American Physiological Society on research animal use.

A549 xenograft model. 4 to 5-week-old Nu/J mice were subcutaneously injected with 5x106 cells (50 μL,

1:1 PBS:Matrigel) in the lower right flank. Tumors were measured using calipers and volumes were

calculated using the equation: V= (W2*L)/2.4 Tumors were grown to 100 mm3 before being treated with

PARx or vehicle.

In vivo imaging. Intratumoral route: Tumor-bearing mice were anesthetized using isoflurane (1.5 - 2%). A

50 μL solution of PARx (100 μM) in saline containing 10% DMSO was administered via intratumoral

injection. PA images of tumor-bearing flanks were acquired immediately following injection and 60 minutes

post-injection. Images were acquired at 680 nm using continuous mode with a 6 second rotation time.

Retro-orbital route: Tumor-bearing mice were anesthetized using isoflurane (1.5 - 2%). A 150 μL solution

of PARx (400 μM) in saline containing 10% DMSO was administered via retro-orbital injection. PA images

of tumor-bearing flanks were acquired immediately following injection and 60 minutes post-injection.

Images were acquired at 680 nm using continuous mode with a 6 second rotation time.

Tumor growth inhibition. Intratumoral route: Tumor-bearing mice were anesthetized using isoflurane (1.5

- 2%). A 50 μL solution of PARx (100 μM) in saline containing 10% DMSO was administered via intratumoral

injection. Injections were performed once a week for 3 weeks and tumors growth was monitored over 3

weeks.

Retro-orbital route: Tumor-bearing mice were anesthetized using isoflurane (1.5 - 2%). A 150 μL solution

of PARx (400 μM) in saline containing 10% DMSO was administered via retro-orbital injection. Injections

were performed once a week for 3 weeks and tumors were measured every 3 days. For the higher

frequency treatment, injections were performed, and tumors were measured every 3 days for 3 weeks.

Tissue histology. 10% neutral buffered formalin was made with the following: 10 mL 37-40% formaldehyde

solution, 90 mL DI water, 400 mg sodium phosphate monobasic, 650 mg sodium phosphate dibasic. To

prepare tissue for histology staining, mice were sacrificed, and the liver, kidneys, spleen, heart, and tumor

were immediately dissected. Each tissue was placed immediately in cooled 10 mL freshly made 10%

formalin. Tissues were measured to be approximately 5 mm in thickness or less. Kidneys were slightly

punctured rather than sliced for better fixative penetration. Other larger tissues were sliced. Tissues were

fixed for at least 48 hours at 4 °C. Tissues were then transferred to 10 mL 70% EtOH. Tissue samples were

submitted to the histology lab at the College of Veterinary Medicine at UIUC for H&E and TUNEL staining.

Supplementary Figure 1. Representative confocal images of a) A549, b) U87, and c) HEK293 cells treated

with 5 µM 2 (dinitrobenzenesulfonyl trigger). Scale bar represents 20 µm. d) Normalized fluorescence

intensity obtained from cell imaging represented in a-c. Error bars = SD (n ≥ 3).

ORS OO

NO2

NO2

ORSO O

NO2

ORS OO

NO2

ORSO O

Br

ORSO O

Cl

ORSO O

F

ORSO O

CN

ORSO O

CF3

ORSO O

F

F

ORSO O

ORS OO

CF3

NO2

ORS OO

NO2

F

ORSO O

ORSO O

OMe

Supplementary Figure 2. Chemical structures of the panel of PA probes featuring different 2,4-

disubstituted, 2-monosubstituted, and 4-monosubstituted, and unsubstituted benzenesulfonyl triggers for

GSH detection.

Supplementary Figure 3. Absorbance spectra of each probe in Figure S1 after incubation with 1 mM GSH

(70% PBS/MeCN, pH 7.4, 37 °C). Top row (from left to right): Compound 2, 3, PACDx, 4. Second row (from

left to right): Compound 5, 6, 7, 8, 9. Third row (from left to right): Compound 10, 11, 12, 13, 14. Black line

represents initial time point and red line represents the 1 h time point.

Supplementary Figure 4. Absorbance spectra of probes after incubation with 10 mM GSH (70%

PBS/MeCN, pH 7.4, 37 °C). Top row (from left to right): Compound PACDx, 4, 5. Second row (from left to

right): Compound 6, 7, 8. Third row (from left to right): Compound 9, 10, 11, and 13. Black line represents

initial time point and red line represents the 1 h time point.

Supplementary Figure 5. Fold change in absorbance after 1 h incubation of PACDx with 200 µM Cys,

100 µM Hcy, and 2.5 mM GSH (70% PBS/MeCN, pH, 7.4, 37 °C). Error bars = SD (n = 3).

Supplementary Figure 6. a) PA signal and b) PA images of Ctrl-PACDx in response to GSH after 1 h

(70% PBS/MeCN, pH 7.4, 37 °C). Error bars = SD (n = 3).

Supplementary Figure 7. a) Representative confocal image of U87 cells treated with 5 µM PACDx. b)

Representative confocal image of U87 cells pre-treated with 1 mM NEM and incubated with 5 µM PACDx.

c) Representative confocal image of U87 cells treated with 5 µM Ctrl-PACDx. d) Normalized fluorescence

intensity obtained from cell imaging under conditions represented in a-c. Error bars = SD (n ≥ 3).

Supplementary Figure 8. a) Representative confocal image of A549 cells treated with 5 µM PARx. b)

Representative confocal image of A549 cells pre-treated with 100 µM ethacrynic acid and incubated with

5 µM PARx. c) Representative confocal image of U87 cells treated with 5 µM PARx. d) Representative

confocal image of U87 cells pre-treated with 100 µM ethacrynic acid and incubated with 5 µM PARx.

Scale bar represents 20 µm. e) Normalized fluorescence intensity obtained from imaging A549 and U87

cells with and without EA. Error bars = SD (n ≥ 3).

Supplementary Figure 9. Normalized fluorescence intensity obtained from treating A549, U87, and

HEK293 cell lysates with 5 µM PARx. Excitation wavelength used was 690 nm. Error bars = SD (n = 3).

Supplementary Figure 10. a) Normalized PA signal and b) PA images of PARx in response to GSH after

1 h (70% PBS/MeCN, pH 7.4, 37 °C). Error bars = SD (n = 3).

Supplementary Figure 11. ESI-MS spectra of PARx treated with 10 mM GSH.

Supplementary Figure 12. 19F NMR indicating the release of gemcitabine from a) PARx after incubation

with 10 mM GSH. b) 19F NMR of gemcitabine in D2O/CD3CN.

Supplementary Figure 13. a) PA signal and b) PA images of Ctrl-PARx in response to GSH after 1 h

(70% PBS/MeCN, pH 7.4, 37 °C). Error bars = SD (n = 3).

Supplementary Figure 14. ESI-MS spectra of Ctrl-PARx-1 treated with 10 mM GSH.

Supplementary Figure 15. Representative confocal images of a) A549, b) U87, and c) HEK293 cells

treated with 5 µM PARx. Scale bar represents 20 µm. d) Normalized fluorescence intensity obtained from

cell imaging represented in a-c. Error bars = SD (n ≥ 3).

Supplementary Figure 16. Representative PA images of A549 tumors before and 1 hour after a)

intratumoral and b) retro-orbital injection. Scale bar represents 2 mm.

Supplementary Figure 17. Monitoring tumor volume growth after retro-orbital injection of PARx every 3

days for 21 days. Vehicle included for comparison. Error bars = SD (n ≥ 3).

Supplementary Figure 18. Monitoring body weight after retro-orbital injection of PARx every 3 days for 21

days. Vehicle included for comparison. Error bars = SD (n ≥ 3).

Compound 2: 1H NMR (500 MHz, CDCl3)

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)

3.65

5.83

2.10

1.94

1.99

1.88

0.94

0.92

1.05

0.96

1.03

2.03

1.98

0.91

1.01

1.93

1.87

0.98

1.55

1.56

1.57

1.81

1.92

1.93

2.70

2.71

2.72

2.76

2.77

2.78

4.59

4.61

6.73

6.76

6.98

7.04

7.21

7.21

7.32

7.33

7.34

7.34

7.47

7.48

7.49

7.52

7.54

7.55

8.24

8.25

8.25

8.29

8.29

8.35

8.35

8.37

8.37

8.38

8.38

8.39

8.40

8.59

8.60

8.60

8.62

8.62

8.68

8.68

8.69

Compound 2: 13C NMR (125 MHz, CDCl3)

-10010203040506070809010011012013014015016017018019020021020f1 (ppm)

13.2

020

.08

24.1

427

.78

29.5

829

.71

41.8

2

51.3

410

7.25

110.

1111

3.38

116.

0211

8.24

118.

4812

0.46

121.

7212

2.63

125.

2812

7.10

128.

3712

8.60

129.

0212

9.45

131.

9013

3.19

134.

2414

0.86

142.

5114

5.39

146.

5614

7.46

148.

2514

8.93

149.

3715

1.14

152.

9915

8.45

178.

71

O O

N

SO

O

NO2

OS

O

O

O2N NO2

NO2

Compound 3: 1H NMR (500 MHz, CDCl3)

0.0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.51.0f1 (ppm)

3.21

5.68

2.45

2.01

1.93

1.72

0.81

1.82

0.90

0.96

4.38

1.12

1.20

1.95

1.98

0.89

1.52

1.53

1.55

1.79

1.85

1.87

2.66

2.68

2.69

2.74

4.59

6.72

6.75

7.00

7.02

7.18

7.35

7.37

7.43

7.45

7.46

7.48

7.49

7.50

7.51

7.53

7.64

7.68

7.69

7.70

7.70

8.10

8.29

8.31

8.58

8.61

Compound 3: 13C NMR (125 MHz, CDCl3)

-10010203040506070809010011012013014015016017018019020021020f1 (ppm)

13.2

620

.09

24.1

827

.83

29.5

429

.71

41.8

751

.28

107.

1111

0.09

113.

4711

5.91

118.

6011

9.87

119.

9011

9.93

121.

6412

2.38

122.

4112

2.62

123.

8812

7.45

127.

4812

8.31

128.

8012

9.45

129.

7713

1.26

131.

5013

1.62

133.

5114

0.85

142.

5014

3.11

146.

5114

8.29

148.

7214

9.51

152.

9415

8.62

178.

61

O O

N

SO

O

CF3

NO2OS

O

O

CF3

NO2

Compound 4: 1H NMR (500 MHz, CDCl3)

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)

3.19

5.79

2.16

1.94

1.98

1.85

1.04

0.78

1.01

0.89

0.99

2.01

2.22

2.84

1.19

1.19

1.07

0.94

0.99

0.93

1.78

1.85

1.86

1.88

2.66

2.77

4.63

4.65

6.79

6.83

6.97

6.98

6.98

7.00

7.00

7.13

7.14

7.26

7.29

7.31

7.35

7.37

7.37

7.38

7.39

7.43

7.44

7.45

7.45

7.46

7.48

7.49

7.49

7.49

7.51

7.52

7.53

7.79

7.79

7.89

7.89

7.91

7.93

8.03

8.03

8.04

8.05

8.16

8.16

8.18

8.18

8.56

8.59

Compound 4: 13C NMR (125 MHz, CDCl3)

-10010203040506070809010011012013014015016017018019020021020f1 (ppm)

13.2

820

.09

24.1

127

.83

29.5

629

.71

41.8

751

.22

107.

4311

0.08

113.

5111

6.08

118.

7012

1.40

122.

5312

5.09

127.

7812

8.20

128.

4112

9.13

129.

4212

9.60

130.

1713

0.79

130.

8913

1.62

132.

1513

2.64

136.

3113

9.54

140.

9214

2.49

146.

4214

8.40

148.

5914

9.82

152.

8915

8.43

178.

58

O O

N

SO

OOS

O

O

NO2

NO2

Compound 5: 1H NMR (500 MHz, CDCl3)

0.0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.51.0f1 (ppm)

3.32

5.92

1.79

2.02

1.69

1.70

1.03

0.99

1.01

1.04

0.75

2.28

2.08

2.08

4.14

2.13

1.02

1.55

1.57

1.58

1.79

1.87

2.68

2.70

2.79

4.67

4.68

6.73

6.73

6.74

6.75

6.82

6.85

6.97

7.05

7.06

7.28

7.47

7.49

7.50

7.52

7.54

7.55

8.04

8.06

8.11

8.13

8.14

8.15

8.43

8.45

8.55

8.58

Compound 5: 13C NMR (125 MHz, CDCl3)

-10010203040506070809010011012013014015016017018019020021020f1 (ppm)

13.3

420

.08

24.1

927

.83

29.5

829

.71

41.9

4

51.2

5

107.

4411

0.25

113.

4611

5.98

118.

5912

1.38

122.

6212

3.19

124.

6512

7.46

128.

3812

8.45

129.

1212

9.54

130.

0513

1.61

140.

6314

0.84

142.

4514

6.42

147.

8514

9.98

151.

2715

2.93

153.

4015

8.44

178.

64

O O

N

SO

O

NO2

OS

O

O

O2N

Compound 6: 1H NMR (500 MHz, CDCl3)

0.0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)

3.21

5.94

2.14

1.97

1.92

1.94

0.98

1.00

1.95

1.08

6.01

2.02

1.88

1.97

0.98

0.06

0.89

0.91

1.54

1.56

1.57

1.80

1.84

1.85

1.86

1.87

2.67

2.68

2.70

2.76

2.77

2.78

4.64

4.65

4.67

4.68

6.72

6.73

6.74

6.74

6.80

6.83

7.00

7.02

7.03

7.27

7.29

7.47

7.48

7.48

7.49

7.52

7.53

7.55

7.56

7.58

7.90

7.91

7.92

7.92

8.00

8.02

8.05

8.05

8.06

8.06

8.56

8.59

Compound 6: 13C NMR (125 MHz, CDCl3)

-10010203040506070809010011012013014015016017018019020021020f1 (ppm)

13.3

220

.07

24.1

727

.86

29.5

629

.71

41.8

7

51.2

6

107.

2811

0.24

112.

2211

3.43

115.

9411

6.80

118.

4511

8.69

118.

9012

1.34

122.

6712

7.08

128.

4212

9.26

129.

3012

9.53

131.

8113

2.46

133.

2213

9.13

140.

8214

2.44

146.

4315

0.03

151.

6715

2.92

158.

5517

8.60

O O

N

SO

O

CN

OS

O

O

NC

Compound 7: 1H NMR (500 MHz, CDCl3)

0.0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.51.0f1 (ppm)

3.34

6.05

2.34

1.98

1.94

1.87

1.49

1.09

0.92

1.02

3.11

1.03

4.14

1.71

0.97

1.54

1.55

1.57

1.79

1.86

1.87

1.88

2.66

2.68

2.69

2.81

2.82

2.83

4.73

4.75

6.70

6.71

6.72

6.73

6.73

6.74

6.86

6.86

6.88

6.88

6.90

6.93

7.00

7.05

7.06

7.07

7.08

7.08

7.09

7.09

7.11

7.30

7.32

7.46

7.47

7.49

7.51

7.51

7.53

7.89

7.89

7.90

7.91

7.94

7.94

7.96

8.56

8.59

Compound 7: 13C NMR (125 MHz, CDCl3)

-10010203040506070809010011012013014015016017018019020021020f1 (ppm)

13.4

020

.12

24.2

827

.89

29.6

029

.71

42.0

7

51.1

4

103.

9210

4.13

104.

3410

6.11

106.

3210

6.51

107.

6610

9.72

109.

8911

0.00

112.

5311

2.55

112.

7011

2.73

113.

5911

6.18

118.

4611

9.68

119.

7612

1.30

122.

4612

8.24

128.

4312

9.23

129.

5013

0.88

131.

1613

1.55

133.

3413

3.42

140.

9214

2.44

146.

4014

9.95

152.

8915

8.49

178.

52

O O

N

SO

O

FF

OS

O

O

F F

Compound 8: 1H NMR (500 MHz, CDCl3)

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.51.0f1 (ppm)

3.18

5.70

2.01

2.07

2.00

1.92

1.04

1.01

2.05

0.96

2.14

3.96

2.13

3.78

1.03

1.53

1.54

1.55

1.77

1.83

1.85

1.86

2.65

2.67

2.68

2.75

2.76

2.77

4.64

4.65

4.67

4.68

6.72

6.73

6.74

6.74

6.80

6.83

6.85

7.01

7.03

7.03

7.27

7.29

7.45

7.46

7.47

7.48

7.48

7.50

7.51

7.52

7.53

7.86

7.87

8.01

8.03

8.05

8.07

8.55

8.58

Compound 8: 13C NMR (125 MHz, CDCl3)

-10010203040506070809010011012013014015016017018019020021020f1 (ppm)

13.3

320

.08

24.1

627

.83

29.5

429

.70

41.8

851

.17

107.

2511

0.19

113.

4911

5.96

118.

7512

1.27

122.

5212

4.78

124.

8112

4.84

124.

8712

6.60

126.

6312

6.66

126.

6912

6.71

128.

3312

8.43

129.

1512

9.44

129.

5213

1.48

138.

6514

0.85

142.

4214

6.43

150.

1515

0.73

152.

9015

8.64

178.

52

O O

N

SO

O

CF3

OS

O

O

CF3

Compound 9: 1H NMR (500 MHz, CDCl3)

0.0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.51.0f1 (ppm)

3.22

5.89

2.34

1.88

1.77

1.76

0.95

0.91

1.90

1.45

2.49

4.13

2.02

3.36

0.94

1.54

1.55

1.56

1.78

1.85

1.87

1.88

2.66

2.67

2.69

2.80

2.81

2.82

4.72

4.74

6.70

6.71

6.72

6.72

6.90

6.93

6.98

6.99

6.99

7.21

7.21

7.22

7.23

7.23

7.24

7.26

7.27

7.45

7.46

7.46

7.48

7.49

7.51

7.52

7.54

7.56

7.57

7.58

7.83

7.84

7.84

7.84

7.85

7.85

7.86

7.86

8.54

8.57

Compound 9: 13C NMR (125 MHz, CDCl3)

-10010203040506070809010011012013014015016017018019020021020f1 (ppm)

13.4

320

.14

24.2

727

.91

29.6

129

.71

42.0

6

51.1

4

107.

6711

0.24

113.

5611

6.16

118.

8212

1.17

122.

4812

7.77

127.

8612

8.25

128.

2712

9.24

129.

5112

9.85

129.

9813

1.51

133.

4613

4.43

140.

9314

1.61

142.

4614

6.07

146.

4215

0.31

152.

8915

8.52

178.

53

Compound 10: 1H NMR (500 MHz, CDCl3)

O O

N

SO

O

Cl

OS

O

O

Cl

0.0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.51.0f1 (ppm)

3.33

6.12

2.26

2.01

1.94

1.94

0.99

0.98

1.98

0.76

1.63

2.12

2.16

5.77

1.01

1.53

1.54

1.56

1.78

1.84

1.86

1.87

2.66

2.67

2.68

2.78

2.79

2.80

4.69

4.71

6.71

6.71

6.72

6.73

6.86

6.89

6.99

6.99

7.00

7.27

7.37

7.37

7.38

7.39

7.46

7.46

7.47

7.47

7.48

7.49

7.51

7.51

7.52

7.52

7.72

7.73

7.74

7.74

7.75

7.76

7.76

7.77

7.77

7.78

7.79

7.79

8.54

8.57

Compound 10: 13C NMR (125 MHz, CDCl3)

-10010203040506070809010011012013014015016017018019020021020f1 (ppm)

13.3

720

.11

24.1

927

.90

29.5

829

.71

41.9

5

51.1

5

107.

5311

0.22

113.

5511

6.09

118.

8212

1.17

122.

4812

2.84

128.

1412

8.25

128.

2912

8.81

129.

2912

9.51

129.

9713

0.22

130.

7413

1.48

132.

8413

4.00

140.

9214

2.45

146.

4014

6.49

150.

3115

2.88

158.

5417

8.51

O O

N

SO

O

Br

OS

O

O

Br

Compound 11: 1H NMR (500 MHz, CDCl3)

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)

3.17

5.97

2.39

1.95

1.75

1.80

1.00

2.01

1.00

0.99

5.00

1.37

2.74

2.88

0.97

1.53

1.55

1.56

1.78

1.87

1.89

1.90

2.67

2.68

2.70

2.80

2.82

2.83

4.72

4.73

6.70

6.71

6.72

6.72

6.87

6.90

6.91

6.91

6.92

6.94

6.97

6.98

7.02

7.28

7.29

7.29

7.46

7.46

7.47

7.48

7.49

7.50

7.51

7.52

7.52

7.52

7.53

7.54

7.89

7.90

7.90

7.91

7.91

7.92

7.93

7.94

8.55

8.58

Compound 11: 13C NMR (125 MHz, CDCl3)

-10010203040506070809010011012013014015016017018019020021020f1 (ppm)

13.3

920

.14

24.2

627

.89

29.5

829

.70

42.0

051

.13

107.

3711

0.26

113.

5011

4.30

114.

4711

6.06

116.

8411

7.02

118.

9112

1.11

122.

5212

8.24

128.

3212

8.38

128.

4512

9.51

130.

9313

0.96

131.

4013

1.50

131.

5714

0.88

142.

4014

6.46

150.

3615

2.84

158.

7116

5.25

167.

30

178.

47

O O

N

SO

OOS

O

O

FF

Compound 12: 1H NMR (500 MHz, CDCl3)

0.0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)

3.21

5.96

2.23

2.12

1.96

1.95

1.01

1.03

2.04

1.11

3.13

2.10

1.09

2.02

1.04

1.54

1.55

1.57

1.78

1.90

1.91

1.92

2.68

2.69

2.71

2.85

2.86

2.87

4.79

4.80

6.67

6.67

6.68

6.69

6.93

6.96

7.01

7.01

7.04

7.26

7.46

7.46

7.46

7.47

7.48

7.52

7.52

7.53

7.53

7.54

7.57

7.58

7.59

7.60

7.61

7.71

7.71

7.72

7.73

7.74

7.87

7.88

7.88

7.89

7.89

7.89

8.56

8.59

Compound 12: 13C NMR (125 MHz, CDCl3)

-10010203040506070809010011012013014015016017018019020021020f1 (ppm)

13.4

120

.16

24.4

327

.97

29.5

829

.70

42.1

2

51.1

0

103.

0610

7.38

110.

3511

3.50

116.

1211

8.95

121.

0012

2.52

128.

2112

8.25

128.

5112

9.49

129.

5212

9.70

131.

3213

4.85

134.

9414

0.91

142.

3914

6.50

150.

5715

2.85

158.

8717

8.43

O O

N

SO

O

I

Compound 13: 1H NMR (500 MHz, CDCl3)

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)

3.29

6.01

2.22

3.04

1.96

1.90

1.88

0.94

1.89

0.98

0.97

1.99

1.30

2.76

1.92

0.97

1.55

1.56

1.58

1.80

1.92

1.93

1.95

2.47

2.70

2.71

2.72

2.88

2.89

2.90

4.80

4.81

6.65

6.65

6.67

6.67

6.98

7.01

7.08

7.08

7.09

7.23

7.25

7.36

7.36

7.36

7.37

7.38

7.38

7.46

7.47

7.48

7.48

7.49

7.51

7.51

7.52

7.52

7.52

7.53

7.53

7.54

7.54

7.76

7.76

7.77

7.77

8.58

8.62

Compound 13: 13C NMR (125 MHz, CDCl3)

-10010203040506070809010011012013014015016017018019020021020f1 (ppm)

13.4

020

.21

21.8

324

.35

27.9

629

.63

29.7

1

42.0

7

51.0

8

107.

5411

0.44

113.

4311

6.26

118.

8612

0.90

122.

4512

8.08

128.

1412

8.53

129.

4812

9.53

130.

0413

1.35

131.

9314

0.95

142.

4314

6.08

146.

5315

0.67

152.

8815

8.82

178.

46

O O

N

SO

OI

Compound 14: 1H NMR (500 MHz, CDCl3)

0.0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.51.0f1 (ppm)

3.15

5.69

2.18

1.95

1.88

2.97

1.87

0.99

1.01

3.07

0.96

1.01

1.20

3.09

2.02

0.98

1.53

1.54

1.56

1.77

1.88

1.89

1.90

1.92

1.93

2.69

2.70

2.71

2.83

2.84

2.85

3.88

4.75

4.76

4.78

4.79

6.67

6.67

6.69

6.69

6.87

6.90

7.00

7.00

7.01

7.02

7.02

7.03

7.09

7.29

7.31

7.43

7.44

7.45

7.45

7.46

7.46

7.50

7.51

7.52

7.53

7.77

7.77

7.78

7.79

7.79

7.80

8.56

8.59

Compound 14: 13C NMR (125 MHz, CDCl3)

-10010203040506070809010011012013014015016017018019020021020f1 (ppm)

13.3

820

.16

27.9

629

.55

29.6

9

42.0

7

51.0

755

.99

107.

0111

0.38

113.

4511

4.67

115.

9811

9.15

120.

8812

2.53

125.

9912

8.17

128.

3512

9.52

130.

1613

0.85

131.

1114

0.89

142.

3514

6.53

150.

8115

2.85

159.

1616

4.56

178.

32

O O

N

SO

O

OMe

I

Compound 15: 1H NMR (500 MHz, CDCl3)

0.0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.51.0f1 (ppm)

3.23

5.83

1.97

1.99

1.93

1.95

0.97

1.04

0.98

1.00

1.06

4.14

1.89

1.00

1.02

1.55

1.57

1.58

1.81

1.91

1.93

1.94

2.70

2.71

2.72

2.73

2.73

2.87

2.88

2.90

4.74

4.75

6.82

6.85

6.90

6.90

6.91

6.92

7.08

7.13

7.13

7.36

7.38

7.48

7.48

7.49

7.50

7.50

7.52

7.52

7.54

7.54

7.56

7.56

8.14

8.15

8.16

8.17

8.18

8.19

8.20

8.21

8.22

8.22

8.23

8.24

8.57

8.60

Compound 15: 13C NMR (125 MHz, CDCl3)

-10010203040506070809010011012013014015016017018019020021020f1 (ppm)

13.4

820

.02

25.1

127

.95

29.5

629

.70

42.8

7

51.2

4

107.

6010

9.97

113.

4211

3.48

113.

6211

6.20

118.

2911

9.00

119.

8711

9.90

121.

6012

2.65

128.

4012

8.70

129.

3712

9.54

131.

7313

2.85

140.

8314

2.41

146.

4214

9.57

152.

9715

8.77

178.

47

O O

N

SO

O

F NO2

I

Compound 16: 1H NMR (500 MHz, CDCl3)

0.0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.51.0f1 (ppm)

5.85

6.06

6.06

8.70

9.47

8.85

1.96

1.06

1.03

0.97

0.19

0.29

0.33

1.05

1.09

1.12

4.80

6.39

6.40

6.57

6.58

6.59

6.59

7.36

7.38

Compound 16: 13C NMR (125 MHz, CDCl3)

-1010203040506070809010011012013014015016017018019020021020f1 (ppm)

-4.9

3-4

.06

-3.9

0

18.5

418

.75

26.0

726

.09

26.3

7

60.7

1

110.

6911

3.29

125.

4612

8.22

152.

9815

5.37

TBSO OTBS

OTBS

Compound 17: 1H NMR (500 MHz, DMSO-d6)

0.0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.51.0f1 (ppm)

5.98

9.08

3.08

5.83

2.21

4.01

1.94

1.77

1.11

1.22

1.39

1.89

2.17

1.06

0.92

0.11

0.94

1.35

1.37

1.38

1.74

1.81

1.82

1.83

1.85

1.86

2.50

2.51

2.51

2.67

2.68

2.70

2.72

2.73

2.74

2.75

3.34

4.37

4.38

4.40

4.41

4.70

4.70

6.46

6.49

6.93

7.40

7.42

7.43

7.44

7.46

7.46

7.51

7.51

7.52

7.52

7.52

7.54

7.54

7.62

7.64

7.67

7.75

7.75

7.77

7.77

8.54

8.57

Compound 17: 13C NMR (125 MHz, DMSO-d6)

-10010203040506070809010011012013014015016017018019020021020f1 (ppm)

-2.6

315

.19

20.8

122

.72

28.5

028

.56

30.1

441

.71

41.8

842

.05

42.2

142

.38

42.5

542

.71

52.6

6

62.0

6

103.

8510

5.32

115.

1711

6.47

116.

9412

5.48

128.

4012

8.55

129.

0913

0.33

131.

5413

7.90

143.

9114

4.39

146.

7515

5.87

161.

5516

3.88

178.

29

O OH

N

OTBS

Compound 17-1: 1H NMR (500 MHz, 1:10 CD3OD:CDCl3)

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.51.0f1 (ppm)

3.00

6.06

2.03

1.84

1.89

2.17

1.58

0.82

0.82

1.08

1.17

2.89

1.96

0.81

1.31

1.32

1.34

1.63

1.80

1.81

1.83

1.84

1.85

2.53

2.54

2.56

2.63

2.65

2.66

3.24

3.24

3.92

3.94

3.95

3.96

4.60

5.79

5.82

6.67

6.96

6.98

7.10

7.11

7.13

7.25

7.25

7.26

CD

Cl3

7.27

7.28

7.30

7.32

7.35

8.30

8.33

Compound 17-1: 13C NMR (125 MHz, 1:10 CD3OD:CDCl3)

-100102030405060708090100110120130140150160170180190200210f1 (ppm)

11.6

520

.57

24.1

228

.08

28.2

929

.44

38.8

1

60.9

6

97.2

210

2.40

109.

5211

4.70

114.

7912

1.63

122.

2412

4.52

126.

5012

8.45

132.

2313

9.20

139.

6014

0.45

141.

70

156.

4916

2.34

170.

68

O OH

N

OH

Compound 18: 1H NMR (500 MHz, CDCl3)

0.0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.51.0f1 (ppm)

3.13

5.75

2.07

3.78

4.17

0.94

0.95

1.00

2.18

2.09

0.91

1.05

1.03

1.05

2.92

0.98

1.56

1.57

1.58

1.81

1.88

1.89

1.91

2.68

2.70

2.71

2.72

4.51

4.53

4.55

6.58

6.61

7.09

7.13

7.46

7.48

7.48

7.50

7.50

7.52

7.52

7.53

7.54

7.56

7.56

7.58

7.58

7.71

7.75

7.76

7.77

7.77

7.84

7.84

7.86

7.86

8.00

8.01

8.02

8.18

8.18

8.19

8.20

8.21

8.22

8.23

8.23

8.58

8.61

Compound 18: 13C NMR (125 MHz, CDCl3)

-10010203040506070809010011012013014015016017018019020021020f1 (ppm)

13.0

520

.08

24.1

527

.88

29.3

8

41.3

7

51.1

258

.14

105.

4710

9.32

111.

6311

1.85

113.

0011

3.61

113.

8111

5.33

118.

3111

9.92

121.

0112

2.69

128.

2112

8.47

129.

4713

0.46

130.

7213

1.40

132.

5313

2.96

140.

7614

2.20

146.

4814

7.04

148.

5915

1.40

152.

5015

9.80

178.

08

Compound 19: 1H NMR (500 MHz, CD3OD)

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.51.0f1 (ppm)

3.43

6.03

2.04

3.89

0.99

2.01

1.18

1.04

1.85

2.04

0.94

0.85

2.71

1.36

1.49

3.54

3.01

0.93

1.00

1.50

1.51

1.53

1.79

1.92

1.93

2.71

2.72

2.73

2.76

2.77

3.30

3.30

3.30

3.31

3.95

3.96

3.96

3.97

4.20

4.21

4.49

4.50

4.85

5.20

6.68

6.71

7.21

7.24

7.55

7.55

7.57

7.59

7.59

7.61

7.65

7.67

7.68

7.69

7.70

7.72

7.72

7.73

7.74

8.28

8.29

8.30

8.30

8.31

8.32

8.33

8.68

8.71

Compound 19: 13C NMR (125 MHz, CD3OD)

-10010203040506070809010011012013014015016017018019020021020f1 (ppm)

10.0

011

.83

13.0

019

.96

22.6

323

.55

26.2

828

.74

29.0

029

.35

30.2

338

.78

40.8

651

.31

58.8

661

.32

67.7

110

6.28

109.

5511

3.29

113.

6111

3.82

115.

0711

9.90

121.

4612

2.59

125.

8412

8.21

128.

4612

8.96

129.

1112

9.22

131.

0113

1.67

132.

4914

0.84

142.

8314

6.50

147.

9815

2.67

158.

5416

3.77

167.

9417

9.24

O O

N

SO O

NO2

F

O O

O

NH2N

O

NO

HO FF

Compound 20: 1H NMR (500 MHz, CDCl3)

0.0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)

2.84

6.10

1.95

3.81

3.23

1.98

1.71

1.02

4.26

4.23

3.30

1.00

1.53

1.54

1.55

1.78

1.86

2.62

2.63

2.64

2.73

3.89

4.40

4.64

6.70

6.73

7.01

7.03

7.05

7.08

7.26

7.44

7.45

7.47

7.50

7.52

7.53

7.76

7.78

7.80

8.53

8.56

Compound 20: 13C NMR (125 MHz, CDCl3)

-10010203040506070809010011012013014015016017018019020021020f1 (ppm)

13.1

720

.17

28.0

429

.43

29.7

1

50.9

256

.05

57.7

5

105.

7510

9.35

112.

9611

4.94

115.

5112

0.46

122.

5712

6.16

127.

8912

8.16

129.

4113

0.23

130.

6213

1.88

134.

0014

0.93

142.

1914

6.30

147.

8615

1.40

160.

0716

4.69

177.

78

O O

N

SO O

OMe

OH

Compound 21: 1H NMR (500 MHz, CD3OD)

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.51.0f1 (ppm)

3.43

5.82

1.96

3.76

1.22

2.94

1.92

1.16

0.85

2.02

1.61

1.42

0.85

0.87

2.80

1.01

0.96

6.42

2.07

0.94

0.97

1.48

1.50

1.52

1.81

1.81

1.92

2.71

2.73

2.76

3.28

3.29

3.29

3.30

3.85

3.94

3.96

4.19

4.20

4.20

4.47

4.49

4.85

5.03

5.47

6.66

6.69

7.13

7.13

7.14

7.15

7.20

7.53

7.55

7.56

7.58

7.59

7.60

7.60

7.61

7.65

7.67

7.68

7.71

7.71

7.73

7.86

7.87

7.88

7.88

8.32

8.33

8.69

8.72

Compound 21: 13C NMR (125 MHz, CD3OD)

-10010203040506070809010011012013014015016017018019020021020f1 (ppm)

10.0

011

.81

12.9

920

.01

22.6

323

.56

26.4

128

.74

28.9

630

.23

38.7

851

.29

55.2

158

.86

61.3

267

.70

95.6

110

5.93

109.

7911

3.20

114.

7111

4.90

120.

8512

2.59

125.

9912

6.23

128.

0912

8.46

128.

8212

9.08

129.

4913

0.71

131.

0013

1.20

140.

8814

2.78

146.

5114

8.80

152.

5615

8.92

163.

8616

5.07

167.

9317

9.10

O O

N

SO O

OMe

O O

O

NH2N

O

NO

HO FF

Supplementary References

1. Schindelin, J. et al. Nat. Methods. 2012, 9, 676-682

2. Yuan, L. et al. J. Am. Chem. Soc. 2012, 134, 13510–13523

3. R. M. U. Anal. Biochem. 2006, 354, 165–168

4. Faustino-Rocha, A. et al. Lab Anim. 2013, 42, 217–224

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