Bioorthogonal urine test

Abstract

Disclosed herein are a bioorthogonal urinary reporter for cancer detection, a bioorthogonal urinary reporter for treatment-associated kidney injury, a bioorthogonal fluorescence indicator, and kits of parts thereof. Also disclosed herein are methods for detecting cancer in a subject, for detecting treatment-associated kidney injury in a subject, and for cancer detection and for detecting treatment-associated kidney injury.

Description

[0001] BIOORTHOGONAL URINE TEST

[0002] Field of Invention

[0003] The present disclosure generally relates to urine tests, and more particularly relates to biorthogonal urine tests for detecting cancers and treatment-associated kidney injuries.

[0004] Background

[0005] The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0006] The use of antineoplastic agents remains the first-line cancer treatment and has led to overall improvement in the prognosis of patient cohorts with diverse malignancies. However, the toxic and even fetal complications of these antineoplastics are frequently underestimated; these adverse events not only severely compromise the patients' wellbeing and survival, but also impair the patients' ability to receive and tolerate recommended dosages during the schedule of treatment. Therefore, concurrent monitoring of treatment efficacy and associated adverse events throughout the treatment journey is important to improve the prognosis and overall outcomes of patients with cancer. In contrast to multi-omics analyses that require expensive instrumentations, specialized personnel, and tedious sample preparation, urine test is easy- to-perform and cost-effective, offering a non-invasive and convenient approach for point-of- care (POC) diagnosis and prognosis. However, because of the very limited number and low concentration of biomolecules available in urine, the dynamic and longitudinal urine test to evaluate both the efficacy and safety profiles of cancer treatment has yet to be explored.

[0007] Bioorthogonal chemistry can proceed rapidly and selectively in biological environments, and act as a powerful biochemical tool for both fundamental and preclinical research. In particular, bioorthogonal chemistry has been used for in vivo optical imaging and diagnosis. It often starts by administrating an antibody or ligand with bioorthogonal handle as the targeting molecule to bind onto the biomolecule-of-interest; followed by administration of a fluorophore with the corresponding bioorthogonal handle as the imaging probe, in-situ bioorthogonal reaction can occur for optical signalling. As compared to single-step imaging using antibody / ligand- conjugated fluorophores, this two-step pre-targeting approach has the advantage to exert the time difference between administration of targeting molecule and imaging agent on optimizing the accumulation and clearance of the targeting molecule at diseases to improve the delivery of imaging probe. However, such a two-step approach also leads to a long detection time (often > 26 hours / h). Moreover, as the bioorthogonal reaction occurs as long as the targeting molecule encounters the imaging probe, the signal can be non-specifically detected in healthy tissues, compromising detection specificity. In the aspect of in vivo detection, optical imaging is difficult to detect deep-seated diseases due to the light scattering with surrounding tissues and shallow light penetration.

[0008] Therefore, to overcome at least one of the aforementioned problems, there exists a need for new bioorthonogal in vivo diagnostic tools.

[0009] Summary of Invention

[0010] Aspects and embodiments of the invention are provided in the following numbered clauses.

[0011] 1. A bioorthogonal urinary reporter for cancer detection (BURC) having formula I:

[0012] [A]-[B-C]n' where:

[0013] A is a polymeric material selected from: _ the squiggly line represents the point of attachment to the rest of the molecule, optionally wherein n’ is from 3 to 160;

[0014] B represents a cancer-associated biomarker-responsive peptide selected from:

[0015] where peptide structures are drawn from N-terminal to C-terminal, Ri denotes the point of attachment to A, and R2 denotes the point of attachment to C;

[0016] 5 where C a renal-clearable moiety is selected from:

[0017]

[0018] X is from 1 to 11 , where the squiggly line is the point of attachment to the rest of the molecule, or a pharmaceutically acceptable salt or solvate thereof. 2. The BURc according to Clause 1 , wherein the BURc is: optionally where n is from 100 to 160, or a pharmaceutically acceptable salt or solvate thereof. 3. A bioorthogonal urinary reporter for treatment-associated kidney injury of formula II:

[0019] [D]a-[E]-[F] II where a is 1 or 2; where D is a renal clearance moiety selected from:

[0020] where PEG represents a polyethylene glycol chain having a number average molecular weight of from 1 ,000 to 5,000 Daltons; m is from 10 to 100; n” is from 20 to 130; the squiggly line represents the point of attachment to the rest of the molecule;

[0021] E is a bioorthogonal chemistry-based reporter moiety selected from:

[0022] where R represents a point of attachment to F and Ri represents a point of attachment to D;

[0023] F is a biomarker-cleavable moiety selected from: where the squiggly line represents the point of attachment to the rest of the molecule, or a pharmaceutically-acceptable salt or solvate thereof. 4. The bioorthogonal urinary reporter for treatment-associated kidney injury according to Clause 1 , wherein the bioorthogonal urinary reporter for treatment-associated kidney injury is:

[0024] , where PEG is a polyethylene glycol moiety having a number average molecular weight of about 2 kDa, or a pharmaceutically-acceptable salt or solvate thereof. 5. A bioorthogonal fluorescence indicator selected from: where Ri represents H or a group selected from: and X represents halogen atoms (F / CI / Br / l) , or a pharmaceutically-acceptable salt or solvate thereof.

[0025] 6. A kit of parts for cancer detection, comprising:

[0026] (a) a bioorthogonal urinary reporter for cancer detection (BURC) according to Clause 1 or Clause 2, or a pharmaceutically-acceptable salt or solvate thereof; and

[0027] (b) a bioorthogonal fluorescence indicator according to Clause 5 or a pharmaceutically- acceptable salt or solvate thereof.

[0028] 7. The kit of parts according to Clause 6, wherein the bioorthogonal urinary reporter for cancer detection (BURC) is the compound according to Clause 2, or a pharmaceutically- acceptable salt or solvate thereof.

[0029] 8. The kit of parts according to Clause 6 or Clause 7, wherein the bioorthogonal

[0030] 9. A kit of parts for detecting treatment-associated kidney injury, comprising:

[0031] (i) a bioorthogonal urinary reporter for treatment-associated kidney injury according to Clause 3 or Clause 4 or a pharmaceutically-acceptable salt or solvate thereof;

[0032] (ii) a bioorthogonal fluorescence indicator according to Clause 5 or a pharmaceutically- acceptable salt or solvate thereof. 10. The kit of parts according to Clause 9, wherein the bioorthogonal urinary reporter for treatment-associated kidney injury is the compound according to Clause 4, or a pharmaceutically-acceptable salt or solvate thereof.

[0033] 11. The kit of parts according to Clause 9 or Clause 10, wherein the bioorthogonal

[0034] I ° o N^.NAO. fluorescence indicator is

[0035] 12. A kit of parts for cancer detection and for detecting treatment-associated kidney injury, comprising:

[0036] (ai) the kit of parts according to any one of Clauses 6 to 8; and

[0037] (aii) the kit of parts according to any one of Clauses 9 to 11.

[0038] 13. A method for detecting cancer in a subject, the method comprising the steps of:

[0039] (bi) administering to a subject suspected of having cancer, a bioorthogonal urinary reporter for cancer detection (BURC), or pharmaceutically acceptable salt thereof, according to Clause 1 or Clause 2 and, after a period of time, collecting urine from said subject; and

[0040] (bii) adding a bioorthogonal fluorescence indicator configured to react with an activated renal clearable compound released from the bioorthogonal urinary reporter for cancer detection (BURC) and detecting the presence of cancer if fluorescence is detected due to reaction of the activated renal clearable compound with the bioorthogonal fluorescence indicator.

[0041] 14. The method according to Clause 13, wherein the bioorthogonal urinary reporter for cancer detection (BURC) is the compound according to Clause 2, or a pharmaceutically- acceptable salt or solvate thereof.

[0042] 15. The method according to Clause 13 or Clause 14, wherein the bioorthogonal fluorescence indicator i 16. A method for detecting treatment-associated kidney injury in a subject, the method comprising the steps of:

[0043] (ci) administering to a subject suspected of having treatment-associated kidney injury, a bioorthogonal urinary reporter for detecting treatment-associated kidney injury, or pharmaceutically acceptable salt thereof, according to Clause 3 or Clause 4 and, after a period of time, collecting urine from said subject; and

[0044] (cii) adding a bioorthogonal fluorescence indicator configured to react with an activated renal clearable compound released from the bioorthogonal urinary reporter for treatment- associated kidney injury and detecting the presence of renal injury if fluorescence is detected due to reaction of the activated renal clearable compound with the bioorthogonal fluorescence indicator.

[0045] 17. The method according to Clause 16, wherein the bioorthogonal urinary reporter for treatment-associated kidney injury is the compound according to Clause 4, or a pharmaceutically-acceptable salt or solvate thereof.

[0046] 18. The method according to Clause 16 or Clause 17, wherein the bioorthogonal i o OYN_ .NAO.

[0047] 1fluorescence indicator is

[0048] 19. A method for cancer detection and for detecting treatment-associated kidney injury, comprising:

[0049] (di) the method according to any one of Clauses 13 to 15; and

[0050] (dii) the method according to any one of Clauses 16 to 18.

[0051] Drawings

[0052] Fig. 1 depicts design and mechanisms of Bioorthogonal Optical Urine Test (BOUT) for concurrent monitoring of cancer therapy efficacy and antineoplastics-induced kidney injury (AIKI). a, Mechanism of BOUT, which consists of (i) in vivo BURs activation in the presence of biomarkers associated with lung cancers and AIKI, (ii) the subsequent ex vivo BFI urineincubation and handphone-facilitated fluorescence (FL) signal read-out. b, Evaluation of overall patient performance by calculation of BOUT index, and the potential applications of BOUT index for dosage optimization and precision medicine of cancer treatment, c, Chemical structures of (i) cancer reporter BURc and its activation by CTSB, the activated BURc subsequently reacts with BFIc to produce activated BFIc with turned-on FL at 450 nm; and (ii) AIKI reporter BURK and its activation by NAG, the activated BURK subsequently reacts with BFIK to produce activated BFIK with turned-on FL at 590 nm. n = 100-160, m = 45.

[0053] Fig. 2 depicts in vitro sensing evaluation and live cell imaging, a, FL spectra (Aex= 360nm) of BFIc (10 pM) in the absence or presence of the incubation mixture ultrafiltrate of BURc (10 pM) only, or BURc and CTSB (0.5 pg) in buffer solutions at 37 °C, and spectrum of the BURc and CTSB incubation mixture, b, High-performance liquid chromatography (HPLC) traces of pure BFIc (1stpanel), the incubation mixture of BFIc and BURc incubation ultrafiltrate in the absence (2ndpanel) or presence (3rdpanel) of CTSB (0.5 pg), and HPLC traces of activated BFIc (lower panel), c, The FL (A+sonm) changes of BFIc in the presence of the incubation mixture ultrafiltrate of BURc with different enzymes. Two-tailed Student's t-test; enzyme incubation groups versus the control group. Insets: the representative images acquired with handphone- assisted device, d, FL spectra (Aex= 540nm) of BURK (10 pM) and BFIK (10 pM) in the absence or presence of NAG (10 mU), pure BFIK, and the incubation mixture of BURK and NAG in buffer solutions at 37 °C. e, HPLC traces of pure BFIK(1stpanel), the incubation mixture of BURK and BFIK in the absence (2ndpanel) or presence (3rdpanel) of NAG (10 mU), and traces of activated BFIK(lower panel), f, The FL (Assonm) changes of BURK and BFIKafter incubation with different enzymes. Insets: the representative bright-field and FL images, the FL images were acquired at 590 nm with excitation at 545 nm using IVIS spectrum imaging system. Two- tailed Student's t-test; enzyme incubation groups versus the control group, g, Left: FL images of A549 cells, 4T1 cells, and NIH3T3 cells with different treatments. From top to bottom: untreated cells, cells treated with BFIc (10 pM, 1 h), and cells pretreated with BURc (50 pM, 3 h) before BFIc incubation. Pseudo-green signals are from activated BFIc. Right: mean FL enhancement of single cells or culture medium ultrafiltrate from different groups. Two-tailed Student's t-test; A549 cells, 4T1 cells versus NIH3T3 cells under the same treatment, h, Left: FL images of HK-2 cells, A549 cells, and NDF cells with different treatments. From top to bottom: untreated cells, cells treated with BFIK (10 pM, 1 h), cells pretreated BURK (15 pM, 3 h) before BFIK incubation. Pseudo-blue and pseudo-red signals indicate the cell nucleus stained with Hoechst and signals from activated BFIK, respectively. Right: mean FL enhancement of single cells. Two-tailed Student's t-test; HK-2 cells versus A549, NDF cells under the same treatment. The data in (c, f-h) were presented as mean ± s.d. n = 3 independent experiments. CTSB, cathepsin B; Casp3, caspase-3; MMP2, matrix metalloproteinase 2; uPA, urokinase-type plasminogen activator; GGT, y-glutamyl transferase; NTR, nitroreductase; AAP, alanine aminopeptidase; NAG, N-acetyl-p- glucosaminidase. Fig. 3 depicts optical properties and sensing characterizations of BUR / BFI pairs, a, UV / vis absorption spectrum of BFIc (10 pM) in the absence or presence of BURc (10 pM). b-c, FL signal changes (450 nm) of BFIc (50 nM) with different concentrations of activated BURc (0 - 200nM) in urine and buffer (1 xPBS w 5% DMSO, pH = 7.4). d, Time course of FL changes of BFIc (10pM) upon addition of activated BURc (20 pM) in buffer (1 xPBS w 5% DMSO, pH = 7.4). e, UV / vis absorption spectrum of BURK (10 pM) in the absence or presence of NAG (10 mU). f-g, FL signal changes (590 nm) of BFIK (10 nM) with different concentrations of activated BURK (0 - 50 nM) in urine and buffer (1 xPBS w 5% DMSO, pH = 7.4). h, Time course of FL changes of BFIK (10 pM) upon addition of activated BURK (20 pM) in buffer (1xPBS w 5% DMSO, pH = 7.4). Data in (b-c, f-g) were presented as mean ± s.d. (n = 3). Both the detection sensitivity and reaction kinetics of BUR / BFI pairs were comparable in buffer and urine.

[0054] Fig. 4 depicts LC-MS characterizations of activated (a) BFIc and (b) BFIK after BFIs incubation with respective BURs and biomarkers.

[0055] Fig. 5 depicts biomarker sensing characterizations and enzyme kinetic studies of BUR / BFI pairs, a, BURc (100nM) was incubated with different concentrations of CTSB (0, 0.05, 0.1 , 0.2, 0.5, 1 ng / mL) for 2 h, and the incubation mixture ultrafiltrate was incubated with BFIC(100 nM) for 1 h before FL measurement, b, The optical signal (OD450) readouts with different concentrations of CTSB (0 - 10 ng / mL) measured with CTSB ELISA kit. c, Enzyme kinetic studies of CTSB (0.5 pg) towards BURc (1 - 40 pM). d, BURK (100 nM) was incubated with different concentrations of NAG (0, 0.1, 0.2, 0.5, 1 , 2 mU / mL) for 2 h, and the enzyme cleavage was terminated by addition of methanol. BFIK (100 nM) was then added into the incubation mixture for 1 h before FL measurement, e, The optical signal (OD450) readouts with different concentrations of NAG (0 - 25 ng / mL) measured with NAG ELISA kit. f, Enzyme kinetic studies of NAG (10 mU) towards BURK (1 - 120 pM). Data in (a-f) were presented as mean ± s.d. (n = 3).

[0056] Fig. 6 depicts cytotoxicity studies of (a) BURc, (b) BURK, (C) BURCM, or (d) BURKA by MTS assay in 4T1 , A549, NIH3T3, HK-2, and NDF cell lines. Data were presented as mean ± s.d. (n = 5).

[0057] Fig. 7 depicts cell imaging with pre-incubation of enzyme inhibitors, a, Left: FL images of A549 cells, 4T1 cells, and NIH3T3 cells with pre-treatment of CTSB inhibitor (CA-074, 50 pM, 3 h). From top to bottom: untreated cells, cells treated with BFIc (10 pM, 1 h), and cells pretreated with BURc (50 pM, 3 h) before BFIc incubation. Pseudo-green signals are from activated BFIc. Right: mean FL enhancement of single cells or culture medium ultrafiltrate from different groups, b, Left: FL images of HK-2 cells, A549 cells, and NDF cells with pre-treatment of NAG inhibitor (nagstatin, 50 pM, 3 h). From top to bottom: untreated cells, cells treated with BFIK (10 pM, 1 h), cells pretreated BURK (15 pM, 3 h) before BFIK incubation. Pseudo-blue and pseudo-red signals indicate the cell nucleus stained with Hoechst and signals from activated BFIK, respectively. Right: mean FL enhancement of single cells. The data in (a-b) were presented as mean ± s.d. n = 3 independent experiments.

[0058] Fig. 8 depicts pharmacokinetic studies and excretion of BURs. a-b, Probe in blood (% ID g-1) decay (a) and renal clearance efficiencies (b) of BURc or activated BURc with a constant dosage (6 pmol azide / kg bw) as a function of time after i t. injection into living mice, c, HPLC traces of excreted components in the urine samples at 3 h p.i. of (activated) BURc, and pure (activated) BURc for comparison, d-e, Quantitative analysis of residual (d) BURc or (e) activated BURc in major organs (gray bar) and excreted into urine (red bar) 24 h p.i. f-g, Quantitative studies of (f) probe in blood (% ID g1) decay, and (g) renal clearance efficiencies of (activated) BURK (4.5 pmol / kg bw) as a function of time after i t. injection into living mice, h, HPLC traces of pure BURK, excreted components in the urine samples at3 h p.i., and activated BURK for comparison, i, Quantitative analysis of residual (activated) BURK in major organs and excreted into urine 24 h p.i.. Data in (a-b,d-g,i) were presented as mean ± s.d., (n = 3).

[0059] Fig. 9 depicts biocompatibility studies of BURs. Representative H&E staining of major organs including heart, liver, spleen, lung, and kidney from mice at 24 h after i t. injection of BURc (7.5 mg / kg bw), BURK (12.5 mg / kg bw), BURCM (10.5 mg / kg bw), BURKA (12 mg / kg bw), or saline as the control group. Scale bar = 100 pm.

[0060] Fig. 10 depicts comparison of pharmacokinetic studies of BURK between intravenous (i.v.) or intratracheal (i t.) administration, a, Schematic illustration of i.v. or i.t. injection, b-c, Quantitative studies of (b) Probe in blood (% ID g1) decay, and (c) renal clearance efficiencies of BURK (12.5 mg / kg bw) as a function of time post-injection after i.v. or i t. injection into living mice, d, HPLC quantitative analysis of residual BURK in major organs and excreted into urine 24 h after i.v. injection. Data in (b-d) were presented as mean ± s.d. (n = 3).

[0061] Fig. 11 depicts probe stability in urine. HPLC traces of (activated) BURc and (activated) BURK after incubation with mice urine for different time (6 and 24 h), and HPLC traces of pure compounds for comparison.

[0062] Fig. 12 depicts BOUT for sensitive detection of orthotopic lung cancer, a, Timeline for development of orthotopic lung tumor mouse model, BURc injection and optical urine test, b, BOUTc changes at different tumor post-implantation time (mean ± s.d., n = 3). The control groups were healthy BALB / c mice or mice treated with CA-074 (CTSB inhibitor). The dashed line indicates the average BOUTc when BFIc was incubated with blank urine (urine from healthy mice without BURc injection). Two-tailed Student's t-test; tumor-implantation groups versus the control group, c-d, The changes of serum levels of (c) CYFRA21-1 and (d) CEA at different tumor post-implantation time (mean ± s.d., n = 3). Two-tailed Student's t-test; tumorimplantation groups versus the control group, e, ROC curves for the exclusion analysis (tumorbearing mice with obvious lung nodules [n = 26] versus healthy mice [n = 5]). f, Correlation between BOUTc and tumor area (mm2). Adj. R-Square = 0.96. Light grey: 95% prediction band, dark grey: 95% confidence band. Three samples from week 2 post-tumor implantation are highlighted in red. g, Representative Hematoxylin & Eosin (H&E) staining of whole lung tissues at different tumor post-implantation time.

[0063] Fig. 13 depicts representative immunofluorescence staining of lung from mice at different tumour post-implantation time. For lung tissues, red, green, and blue signals are from anti- CTSB staining, anti-pan Keratin staining, and Hoechst for nuclei staining. Scale bar = 100 pm. For quantification, data was presented as mean ± s.d. (n = 3). Quantification showed that the CTSB MFI increased from week 0 to 4 and reached the plateau, as the size of tumour nodules grew large beyond the pictures for quantification.

[0064] Fig. 14 depicts correlation studies between serum biomarker levels and BOUTs changes, a, Correlation between mice serum CEA (ng / mL) and BOUTc in orthotopic lung cancer mice model. Pearson’s r = 0.81. Adj. R-Square = 0.65. b, Correlation between mice serum CYFRA21-1 (ng / mL) and BOUTc in orthotopic lung cancer mice model. Pearson’s r = 0.82. Adj. R-Square = 0.73. c, Correlation between mice blood urea nitrogen (mg / dL) and BOUTK in AIKI mice model. Pearson’s r = 0.92. Adj. R-Square = 0.84. d, Correlation between mice serum creatinine (ng / .L) and BOUTK in AIKI mice model. Pearson’s r = 0.86. Adj. R-Square = 0.73. Light grey: 95% prediction band, dark grey: 95% confidence band.

[0065] Fig. 15 depicts BOUT for sensitive detection of AIKI. a, Timeline for development of cisplatin- induced kidney injury mouse model, BURK injection, and optical urine test, b, BOUTK changes at different tdpt (0 to 96 h). The control groups were healthy BALB / c mice or mice treated with Nagstatin (NAG inhibitor). The dashed line indicates the average BOUTK when BFIK was incubated with blank urine (urine from healthy mice without BURK injection). Two-tailed Student's t-test; cisplatin-challenged groups versus the control group, c-d, Changes of serum biomarkers (c) BUN, and (d) creatinine at different tdpt. Two-tailed Student's t-test; cisplatin- challenged groups versus the control group, e, ROC curves for the exclusion analysis (mice with observed renal injuries [n = 6] versus mice without renal injuries [n = 12]). f, Quantitative studies of urinary NAG levels (ng / mL) with ELISA assay at different tdpt (0 to 96 h). g, Representative H&E staining of kidney tissues from different tdpt- Stars, arrowheads, and arrows indicate formation of hyaline casts in the kidney tubules, renal cell debris and loss of brush border, respectively. Data in (b-d, f) are presented as mean ± s.d. (n = 3).

[0066] Fig. 16 depicts BOUT for precision cancer therapy diagnostics, a, Timeline of applying BOUT for monitoring cancer therapy via optical urine test, b-c, Quantifications of (b) BOUTc, and (c) BOUTK changes for different treatment groups in cancer treatment regimen (mean ± s.d., n = 3). Two-tailed Student's t-test; tumor-implantation groups with different treatments versus the control group, d, Survival curves for mice from different treatment groups using the Kaplan- Meier method (n = 5). e, Changes of body weight for mice from different treatment groups (mean ± s.d., n = 5). f, ROC curves for the exclusion analysis (tumor-bearing mice with obvious lung nodules [n = 27] versus healthy mice [n = 26]). g, ROC curves for the exclusion analysis (mice with observed renal injuries [n = 12] versus mice without renal injuries [n = 42]). h, Confusion matrix to visualize the five-fold cross-validation of a BOUT-based multiclass SVM algorithm to distinguish among all four states of interest, i, Tumor inhibition (BOUTC‘1) and toxicity effects (BOUTK) for different treatment groups at 3rddosage, j, BOUT index for different treatment groups at 3rddosage. Data in (i-j) are presented as mean ± s.d. (n = 3). k, Correlation between mice weight gain and BOUT index. Adj. R-Square = 0.64. 1, Correlation between mice lifetime and BOUT index. Adj. R-Square = 0.57. Light grey: 95% prediction band, dark grey: 95% confidence band.

[0067] Fig. 17 depicts quantification of mice orthotopic lung tumour size, correlation studies with BOUTc, and BOUT index, a, Orthotopic lung tumour area (mm2) for different treatment groups throughout the cancer treatment regimen. Data were presented as mean ± s.d., n = 3. G1 : Saline, G2: PEM / CIS2, G3: PEM / CIS5, G4: PEM / CIS10. b-c, Correlation studies between mice tumour size and BOUTc, BOUT index, b, Adj. R-Square = 0.53. c, Adj. R-Square = 0.41 . Light grey: 95% prediction band, dark grey: 95% confidence band.

[0068] Fig. 18 depicts changes of serum biomarkers (a) CYFRA21-1 , (b) CEA, (c) creatinine, and (d) BUN for different treatment groups throughout the cancer treatment regimen. Data were presented as mean ± s.d., n = 3. G1 : Saline, G2: PEM / CIS2, G3: PEM / CIS5, G4: PEM / CIS10.

[0069] Fig. 19 depicts representative H&E staining of (a) lung and (b) kidney from mice in different treatment groups throughout the cancer treatment regimen. Mice from G1-G2 at 4thdosage is not shown here due to dropped viability. For lung tissues, tumour nodules are circled with dotted line. For kidney tissues, stars and arrowheads indicate formation of hyaline casts in the kidney tubules, and renal cell debris, respectively. Scale bar = 100 pm. G1 : Saline, G2: PEM / CIS2, G3: PEM / CIS5, G4: PEM / CIS10.

[0070] Fig. 20 depicts representative immunofluorescence staining of (a) lung and (b) kidney from mice in different treatment groups throughout the cancer treatment regimen. Mice from G1- G2 at 4thdosage is not shown here due to dropped viability. For lung tissues, red, green, and blue signals are from anti-CTSB staining, anti-pan Keratin staining, and Hoechst for nuclei staining. For kidney tissues, green and blue signals are from anti-caspase3 staining, and Hoechst for nuclei staining. Scale bar = 100 pm. G1: Saline, G2: PEM / CIS2, G3: PEM / CIS5, G4: PEM / CIS10.

[0071] Fig. 21 depicts evaluation of overall health status and differentiation with BOUT index, a, BOUT index changes for different treatment groups in the treatment journey, b, Principle Component Analyses to differentiate mice among all four states based on histological studies: healthy mice, mice with observed lung tumour nodules, mice with observed renal injuries, and mice with both observed lung tumour nodules and renal injuries. G1 : Saline, G2: PEM / CIS2, G3: PEM / CIS5, G4: PEM / CIS10.

[0072] Fig. 22 depicts correlation studies between mice viability, lifetime, weight gain and BOUTC, BOUTK, BOUT index, a, Adj. R-Square = 0.43. b, Adj. R-Square = 0.03. c, Adj. R-Square = 0.62. d, Adj. R-Square = 0.03. e, Adj. R-Square = 0.05. f, Adj. R-Square = 0.57. g, Adj. R- Square = 0.11. h, Adj. R-Square = 0.15. I, Adj. R-Square = 0.64. Light grey: 95% prediction band, dark grey: 95% confidence band. Figs. 16k, I are shown here as (f, I) for better comparison.

[0073] Fig. 23 depicts a, chemical structures of cancer reporter BURCM and its activation by MMP-3, the activated BURCM subsequently reacts with BFICM to produce activated BFICM with turned- on FL at 710 nm. b, FL spectra (Aex= 690nm) of BFICM (10 pM) in the absence or presence of the incubation mixture ultrafiltrate of BURCM (10 pM) only, or BURCM and MMP-3 (0.5 pg) in buffer solutions at 37 °C, and spectrum of the BURc and MMP-3 incubation mixture, c, HPLC traces of pure BFICM (1stpanel), the incubation mixture of BFICM and BURCM incubation ultrafiltrate in the absence (2ndpanel) or presence (3rdpanel) of MMP-3 (0.5 pg), and HPLC traces of activated BFICM (lower panel), d, The FL (A?ionm) changes of BFICM in the presence of the incubation mixture ultrafiltrate of BURC with different enzymes. Two-tailed Student's t- test; enzyme incubation groups versus the control group, e, Enzyme kinetic studies of MMP- 3 (0.2 pg) towards BURCM (1 - 40 pM). f, Time course of FL changes of BFICM (10 pM) upon addition of activated BURCM (20 pM) in buffer (1 xPBS w 5% DMSO, pH = 7.4). g, FL signal changes (710 nm) of BFICM (50 nM) with different concentrations of activated BURCM (0 - 200 nM) in buffer (I xPBS w 5% DMSO, pH = 7.4). h, Left: FL images of A549 cells, 4T1 cells, and NIH3T3 cells with different treatments. From top to bottom: untreated cells, cells treated with BFICM (10 pM, 1h), and cells pretreated with BURC (50 pM, 3 h) before BFI CM incubation. Pseudo-red, blue signals are from activated BFICM and the cell nucleus stained with Hoechst. Right: mean FL enhancement of single cells or culture medium ultrafiltrate from different groups. Two-tailed Student's t-test; A549 cells, 4T1 cells versus NIH3T3 cells under the same treatment. The data in (d-h) were presented as mean ± s.d. n = 3 independent experiments. Another cancer reporter, BURCM, was developed to detect MMP3, which is a biomarker in lung cancer. The molecular design of BURCM was similar to BURCM, by replacing the CTSB- cleavable peptide with MMP3-cleavable peptide sequence. The sensing capability of BURCM / BFICM to detect MMP3 was validated both in vitro and in living cells.

[0074] Fig. 24 depicts a, chemical structures of AIKI reporter BURKA and its activation by ALP, the activated BURKA subsequently reacts with BFIK to produce activated BFIK with turned-on FL at 590 nm. b, FL spectra (Aex= 540 nm) of BURKA (10 pM) and BFIK(10 pM) in the absence or presence of ALP (20 mU), pure BFIK, and the incubation mixture of BURKA and ALP in buffer solutions at 37 °C. c, HPLC traces of pure BFIK(1stpanel), the incubation mixture of BFIKand BURKA in the absence (2ndpanel) or presence (3rdpanel) of ALP (20 mU), and HPLC traces of activated BFIK (lower panel), d, The FL (Asgonm) changes of BURKA and BFIKafter incubation with different enzymes. Two-tailed Student's t-test; enzyme incubation groups versus the control group, e, BURKA (100 nM) was incubated with different concentrations of ALP (0 - 200 ng / mL) for 2 h, and the enzyme cleavage was terminated by addition of methanol. BFIK (100nM) was then added into the incubation mixture for 1 h before FL measurements, Enzyme kinetic studies of ALP (20 mU) towards BURKA (1 - 120 pM). g, Left: FL images of HK-2 cells, A549 cells, and NDF cells with different treatments. From top to bottom: untreated cells, cells treated with BFIK (10 pM, 1 h), cells pretreated BURKA (15 pM, 3h) before BFIK incubation. Pseudo-blue and pseudo-red signals indicate the cell nucleus stained with Hoechst and signals from activated BFIK, respectively. Right: mean FL enhancement of single cells. Two- tailed Student's t-test; HK-2 cells versus A549, NDF cells under the same treatment. The data in (d-g) were presented as mean ± s.d. n = 3 independent experiments.

[0075] Fig. 25 depicts a-b, probe in blood (% ID g1) decay (a) and renal clearance efficiencies (b) of BURKA (4.5 pmol / kg bw) as a function of time after i.t. injection into living mice, c, Quantitative analysis of residual (activated) BURKA in major organs and excreted into urine 24 h p.i.. d, Timeline for development of cisplatin-induced kidney injury mouse model, BURKA injection, and optical urine test, e, BOUTKA changes at different tdpt (0 to 96 h). The control groups were healthy BALB / c mice. The dashed line indicates the average BOUTK when BF was incubated with blank urine (urine from healthy mice without BURK injection). Two-tailed Student's t-test; cisplatin-challenged groups versus the control group, f, Correlation between mice serum creatinine (ng / L) and BOUTKA in AIKI mice model. Pearson’s r = 0.78. Light grey: 95% prediction band, dark grey: 95% confidence band. Data in (a-c,e) were presented as mean±s.d., (n = 3).

[0076] Fig. 26 depicts pharmacokinetic studies and excretion of BURCM. a-b, Probe in blood (% ID g-1) decay (a) and renal clearance efficiencies (b) of BURCM or activated BURCM with a constant dosage (6 mol N3 / kg bw) as a function of time after i.t. injection into living mice, c-d, Quantitative analysis of residual (c) BURCM or (d) activated BURCM in major organs and excreted into urine 24 h p.i. The MMP3-activatable renal clearance of BURC was demonstrated in living mice. The data in (a-d) were presented as mean ± s.d. n = 3 independent experiments.

[0077] Fig. 27 depicts a, timeline of applying BOUT for monitoring cancer therapy via optical urine test, b-c, Quantifications of (b) BOUTCM, and (c) BOUTKchanges for different treatment groups in cancer treatment regimen (mean ± s.d., n = 3). Two-tailed Student's t-test; tumorimplantation groups with different treatments versus the control group, d, Survival curves for mice from different treatment groups using the Kaplan-Meier method (n = 5). e, Changes of body weight for mice from different treatment groups (mean ± s.d., n = 5). f, Changes of tumor area (mm2) for mice from different treatment groups, g, ROC curves for the exclusion analysis (tumor-bearing mice with obvious lung nodules [n = 23] versus healthy mice [n = 23]). h, ROC curves for the exclusion analysis (mice with observed renal injuries [n = 13] versus mice without renal injuries [n = 31]). i, Tumor inhibition (BOUTCM'1) and toxicity effects (BOUTK) for different treatment groups at 3rddosage, j, BOUTM index for different treatment groups at 3rddosage, k, Correlation between mice tumour area and BOUTCM. Adj. R-Square =0.54. I, Correlation between mice tumour area and BOUTM index. Adj. R-Square =0.24. m, Correlation between weight gain and BOUTM index. Adj. R-Square = 0.42. n, Correlation between mice viability and BOUTM index. Adj. R-Square = 0.44. o, Correlation between mice lifetime and BOUTM index. Adj. R-Square = 0.71. Light grey: 95% prediction band, dark grey: 95% confidence band. The pair of BOUTCM / BOUTK was applied for concurrent detection of cancer and treatment-associated kidney injury in cancer therapy. The A549 tumor-bearing BALB / c nude mice were treated weekly with saline (G1) or an FDA-approved chemodrug (doxorubicin) at different dosages of 2 (G2), 5 (G3), or 10 (G4) mg / kg for four weeks. Quantification in AUC of ROC revealed that both BOUTCM and BOUTK exhibited high diagnostic accuracy than serum biomarkers in the respective detection of orthotopic lung cancer and Al KI. BOUTCM showed strong positive correlation with tumor size (Pearson’s r = 0.74). Evaluating the overall treatment outcomes (BOUTM index = BOUTCM' BOUTK) showed that mice in G3 had outstanding treatment outcomes out of four groups. The BOUTM index also showed strong positive correlation with mice weight gain (Pearson’s r = 0.66), total lifetime (Pearson’s r = 0.85), and viability (Pearson’s r = 0.70).

[0078] Fig. 28 depicts a-b, representative immunofluorescence staining of (a) lung and (b) kidney from mice in different treatment groups throughout the cancer treatment regimen. Mice from G1 at 4thdosage is not shown here due to dropped viability. For lung tissues, red, green, and blue signals are from anti-MMP3 staining, anti-pan Keratin staining, and Hoechst for nuclei staining. For kidney tissues, green and blue signals are from anti-caspase3 staining, and Hoechst for nuclei staining. Scale bar = 100 pm. c-d, Representative H&E staining of (c) lung and (d) kidney from mice in different treatment groups throughout the cancer treatment regimen. Mice from G1 at 4thdosage is not shown here due to dropped viability. For lung tissues, tumour nodules are circled with dotted line. For kidney tissues, stars, arrowheads, arrows, and crosses indicate formation of hyaline casts in the kidney tubules, renal cell debris, mesangium expansion, and glomerular hypertrophy, respectively. Scale bar = 100 pm. G1: Saline, G2: DOX2, G3: DOX5, G4: DOX10.

[0079] Fig. 29 depicts handphone-assisted BOUT for point-of-care (POC) patient monitoring, a, Schematic illustration of POC monitoring of cancer treatment and Al KI. Upon inhalation of BURs, the mice urine was collected and incubated with BFIs. The FL signals from activated BFIs are determined via handphone-assisted signal readout. The results are applied for therapeutic monitoring to improve treatment outcomes, b-c, Correlation studies between POC (b) BOUTc or (c) BOUTK signals and Horiba FL signals when BFIc or BFIK (10pM) were incubated with different concentrations of activated BURs (0-50 pM). Light grey: 95% prediction band, dark grey: 95% confidence band, d-e, POC (d) BOUTc or (e) BOUTK for urine specimens from healthy mice, and mice from different treatment groups at 3rddosage (mean ± s.d., n = 3). Two-tailed Student's t-test; tumor-implantation groups with different treatments versus the control group. Insets: representative images of urine specimens acquired with handphone-assisted POC device, f, POC BOUT index for mice from different treatment groups at 3rddosage.

[0080] Fig. 30 depicts BUR / BFI sensing characterizations with handphone-assisted BOUT, a, Representative images of BFIc (10 pM) after incubation with different concentrations of activated BURc (0 - 50 pM), or PBS buffer as the control. The images were acquired with handphone-assisted portable device, b, Representative images of BFIK (10 pM) after incubation with different concentrations of activated BU K (0 - 50 pM), or PBS buffer as the control. The images were acquired with handphone-assisted portable device, c, POC BOUTc signal quantifications from (a), d, POC BOUTK signal quantifications from (b).

[0081] Fig. 31 depicts synthetic routes for (a) BFIc, (b) activated BURc, and (c) BURc. Reagents and conditions: (i) N,N’-Dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), dichloromethane (CH2CI2), 0 °C to r.t., 12 h; (ii) hexafluorophosphate benzotriazole tetramethyl uronium (HBTU), N,N-diisopropylethylamine (DIEA), tetrahydrofuran (THF), 12 h; (iii) trifluoroacetic acid (TFA) / CH2Cl2 (30% v / v), 0 °C, 3 h; (iv) HBTU, DIEA, THF, 12 h; (v) Piperidine / DMF (5% v / v), 2 h; (vi) HBTU, DIEA, THF, 12 h; (vii) Piperidine / DMF (5% v / v), 2 h; (viii) Hyaluronic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N- hydroxysuccinimide (NHS), DIEA, dimethylformamide (DMF) / H2O (v / v = 1:1), 48 h. n = 100- 160.

[0082] Fig. 32 depicts synthetic routes for (a) BFIK and (b) activated BURK. Reagents and conditions:

[0083] (i) N,N’-disuccinimidyl carbonate (DSC), Et3N, acetonitrile (CH3CN) / CH2CI2(v / v = 1:1), 18 h;

[0084] (ii) Triphosgene, EUN, CH2CI2 / THF (v / v = 1 :1), 0 °C to r.t., 2h; (iii) / V-Boc- / V, / V- dimethylethylenediamine, CH2CI2; (iv) TFA / CH2CI2(30% v / v), r.t., 1 h; (v) TCO-NHS, Et3N, CH2CI2, 0 °C, 1 h; (vi) NaN3, DMF, 75 °C, overnight; (vii) 3-mercaptopropionic acid, hydrazine monohydrate, 60 °C, 24 h, NaNO2; (viii) 1 ,4-diazidobutane, CuSO4.5H2O, sodium ascorbate, tris-hydroxypropyltriazolylmethylamine (THPTA), dimethyl sulfoxide (DMSO) / H2<D (v / v = 4); (ix) DBCO-PEG2k, H2O, 2 h. m = 45.

[0085] Fig. 33 depicts synthetic route for BURK. Reagents and conditions: (i) 4-hydroxybenzaldehyde, Ag2O, Nal, CH3CN, 12 h; (ii) NaBH4, CH3OH, 0 °C, 2 h; (iii) PBr3, CH2CI2, 0 °C, 2 h; (iv) thiorea dioxide, DMF / H2O (v / v = 10), 95 °C, 4 h; (v)dihvTz-Alky, CH3CN / THF (v / v = 1 :2), overnight; (vi) K2CO3, CH3OH, 30 min; (vii) 1 ,4-diazidobutane, CuSO4.5H2O, sodium ascorbate, THPTA, DMSO / H2O (v / v = 4); (viii) DBCO-PEG2k, H2O, 2 h. m = 45.

[0086] Fig. 34 depicts synthetic routes for (a) BFICM, (b) activated BURCM, and (c) BURCM. Reagents and conditions: (i) DCC, DMAP, CH2CI2, 0 °C to r.t., 12 h; (ii) HBTU, DIEA, THF, 12 h; (iii) TFA / CH2CI2 (50% v / v), 0 °C, 2 h; (iv) Piperidine / DMF (5% v / v), 2 h; (v) HBTU, DIEA, THF, 12 h; (vi) Piperidine / DMF (5% v / v), 2 h; (vii) Hyaluronic acid, EDC, NHS, DIEA, DMF / H2O (v / v = 2:1), 48 h. n = 100-160; (viii) TFA / CH2CI2 (50% v / v), 0 °C, 2 h. Fig. 35 depicts synthetic routes for BURKA. Reagents and conditions: (I) Diethyl chlorophosphite, NaH, THF, r.t., 24 h; (ii) CBr4, P(Ph)s, r.t., 24 h; (iii) thiorea dioxide, DMF / H2O (v / v = 10), 95 °C, 4 h; (iv)dihn z-Alky, CH3CN / THF (v / v = 1:2), overnight; (v) 1 ,4-diazidobutane, CUSO4.5H2O, sodium ascorbate, THPTA, DMSO / H2O (v / v = 4); (vi) TMSBr, CH2CI2, r.t., 2 h; (vii) DBCO-PEG2k, H2O, 2 h. m = 45.

[0087] Fig. 36 depicts the design of Bioorthogonal Urinary Reporter for cancer detection (BURc).

[0088] Description

[0089] It has been surprisingly found that a hybrid Bioorthogonal Optical Urine Test (BOUT) could be applied for concurrent monitoring of cancer treatment efficacy and associated adverse events. In general, BOUT consists of: (i) an in vivo Bioorthogonal Urinary Reporter (BUR) to detect the level of disease biomarkers for cancer or antineoplastic-induced kidney injury; and (ii) an ex vivo Bioorthogonal Fluorescence Indicator (BFI) to convert the biomarker levels to fluorescence signals.

[0090] Thus, in a first aspect of the invention, there is provided a bioorthogonal urinary reporter for cancer detection (BURC) having formula I:

[0091] [A]-[B-C]n’ where:

[0092] A is a polymeric material selected from: the squiggly line represents the point of attachment to the rest of the molecule, optionally wherein n’ is from 3 to 160;

[0093] B represents a cancer-associated biomarker-responsive peptide selected from:

[0094] where peptide structures are drawn from N-terminal to C-terminal, Ri denotes the point of attachment to A, and R2denotes the point of attachment to C;

[0095] X is from 1 to 11 , where the squiggly line is the point of attachment to the rest of the molecule, or a pharmaceutically acceptable salt or solvate thereof.

[0096] The word “comprising” refers herein may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components / features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of’ or the phrase “consists essentially of’ or synonyms thereof and vice versa.

[0097] The phrase, “consists essentially of’ and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

[0098] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an oxygen carrier” includes mixtures of two or more such oxygen carriers, reference to “the catalyst” includes mixtures of two or more such catalysts, and the like.

[0099] As discussed above, bioorthogonal chemistry may be applied in in vivo optical imaging and diagnosis. Without wishing to be bound by theory, bioorthogonal chemistry is worked by administering an antibody or ligand with bioorthogonal handle as the targeting molecule to bind onto the biomolecule-of-interest, followed by administering of a fluorophore with the corresponding bioorthogonal handle as the imaging probe, such that in-situ bioorthogonal reaction can occur for optical signalling.

[0100] When used herein, the term “bioorthogonal urinary reporter (BUR) for cancer detection” refers to compounds that can detect cancer biomarkers in vivo and convert such biomarkers levels to an amount of clickable BURs excreted in urine.

[0101] The term “cancer” will be understood by those skilled in the art to include conditions such as, but not limited to, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain tumours, CNS tumours, breast cancer, Castleman disease, cervical cancer, colon cancer, rectum cancer, endometrial cancer, esophagus cancer, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal cancer, hypopharyngeal cancer, leukemia (e.g. acute lymphocytic, acute myeloid, chronic lymphocytic, chronic myeloid, chronic myelomonocytic), liver cancer, lung cancer (e.g. small cell or nonsmall cell), lung carcinoid tumour, lymphoma (e.g. of the skin), malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity cancer, paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumours, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer (basal and squamous cell, melanoma, Merkel cell), small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumour.

[0102] In some embodiments that may be mentioned herein, the bioorthogonal urinary reporter for cancer detection (BURC) may have formula I:

[0103] [A]-[B-C]n I where:

[0104] A is a polymeric material selected from:

[0105] the squiggly line represents the point of attachment to the rest of the molecule, wherein n’ is from 3-160;

[0106] B represents a cancer-associated biomarker-responsive peptide selected from:

[0107] where peptide structures are drawn from N-terminal to C-terminal, Ri denotes the point of attachment to A, and R2 denotes the point of attachment to C;

[0108] X is from 1 to 11 , where the squiggly line is the point of attachment to the rest of the molecule, or a pharmaceutically acceptable salt or solvate thereof.

[0109] In some embodiments that may be mentioned herein, the cancer detected may be lung cancer.

[0110] In some embodiments of the first aspect of the invention that may be mentioned herein, the BURc may be:

[0111] optionally where n is from 100 to 160, or a pharmaceutically acceptable salt or solvate thereof. In some embodiments of the first aspect of the invention that may be mentioned herein, the

[0112] BURc may be: where n is from 100 to 160, or a pharmaceutically acceptable salt or solvate thereof. In a second aspect of the invention, there is provided a bioorthogonal urinary reporter for treatment-associated kidney injury of formula II:

[0113] [D]a-[E]-[F] II where a is 1 or 2; where D is a renal clearance moiety selected from: where PEG represents a polyethylene glycol chain having a number average molecular weight of from 1 ,000 to 5,000 Daltons; m is from 10 to 100; n” is from 20 to 130; the squiggly line represents the point of attachment to the rest of the molecule; E is a bioorthogonal chemistry-based reporter moiety selected from: where R represents a point of attachment to F and i represents a point of attachment to D;

[0114] F is a biomarker-cleavable moiety selected from: where the squiggly line represents the point of attachment to the rest of the molecule, or a pharmaceutically-acceptable salt or solvate thereof.

[0115] When used herein, the term “bioorthogonal urinary reporter for treatment-associated kidney injury” refers to compounds that can detect treatment-associated kidney injury.

[0116] The term “treatment-associated kidney injury” will be understood by those skilled in the art to include conditions such as, but not limited to, antineoplastic-induced kidney injury (Al KI). AIKI is associated with side effects of drugs including doxorubicin, cisplatin, oxaliplatin, carboplatin, cyclophosphamide, interferon-alfa, methotrexate, and mitomycin-C.

[0117] In some embodiments of the second aspect of the invention that may be mentioned herein, the bioorthogonal urinary reporter for treatment-associated kidney injury may be of formula II:

[0118] [D]a-[E]-[F] where a is 1 or 2; where D is a renal clearance moiety selected from:

[0119] where PEG represents a polyethylene glycol chain having a number average molecular weight of from 1 ,000 to 5,000 Daltons; m is from 10 to 100; n” is from 20 to 130; the squiggly line represents the point of attachment to the rest of the molecule;

[0120] E is a bioorthogonal chemistry-based reporter moiety selected from:

[0121] where R represents a point of attachment to F and i represents a point of attachment to D;

[0122] F is a biomarker-cleavable moiety selected from: where the squiggly line represents the point of attachment to the rest of the molecule, or a pharmaceutically-acceptable salt or solvate thereof. In some embodiments of the second aspect of the invention that may be mentioned herein, the bioorthogonal urinary reporter for treatment-associated kidney injury may be:

[0123]

[0124] , where PEG is a polyethylene glycol moiety having a number average molecular weight of about 2 kDa, or a pharmaceutically-acceptable salt or solvate thereof. In a third aspect of the invention, there is provided a bioorthogonal fluorescence indicator selected from:

[0125] where Ri represents H or a group selected from: and X represents halogen atoms (F / CI / Br / l), or a pharmaceutically-acceptable salt or solvate thereof.

[0126] In a fourth aspect of the invention, there is provided a kit of parts for cancer detection, comprising:

[0127] (a) a bioorthogonal urinary reporter for cancer detection (BURC) according to the first aspect of the invention, or a pharmaceutically-acceptable salt or solvate thereof; and

[0128] (b) a bioorthogonal fluorescence indicator according to the third aspect of the invention or a pharmaceutically-acceptable salt or solvate thereof.

[0129] In some embodiments of the fourth aspect of the invention that may be mentioned herein, the bioorthogonal urinary reporter for cancer detection (BURC) may be:

[0130] or a pharmaceutically acceptable salt or solvate thereof.

[0131] In some embodiments of the fourth aspect of the invention that may be mentioned herein, the bioorthogonal fluorescence indicator may

[0132] In a fifth aspect of the invention, there is provided a kit of parts for detecting treatment- associated kidney injury, comprising:

[0133] (i) a bioorthogonal urinary reporter for treatment-associated kidney injury according to the second aspect of the invention or a pharmaceutically-acceptable salt or solvate thereof;

[0134] (ii) a bioorthogonal fluorescence indicator according to the third aspect of the invention or a pharmaceutically-acceptable salt or solvate thereof.

[0135] In some embodiments of the fifth aspect of the invention that may be mentioned herein, the bioorthogonal urinary reporter for treatment-associated kidney injury may be:

[0136] , where PEG is a polyethylene glycol moiety having a number average molecular weight of about 2 kDa, or a pharmaceutically-acceptable salt or solvate thereof.

[0137] In some embodiments of the fifth aspect of the invention that may be mentioned herein, the bioorthogonal fluorescence indicator may be

[0138] In a sixth aspect of the invention, there is provided a kit of parts for cancer detection and for detecting treatment-associated kidney injury, comprising:

[0139] (ai) the kit of parts according to the fourth aspect of the invention; and

[0140] (aii) the kit of parts according to the fifth aspect of the invention.

[0141] In a seventh aspect of the invention, there is provided a method for detecting cancer in a subject, the method comprising the steps of:

[0142] (bi) administering to a subject suspected of having cancer, a bioorthogonal urinary reporter for cancer detection (BURC), or pharmaceutically acceptable salt thereof, according to the first aspect of the invention and, after a period of time, collecting urine from said subject; and (bii) adding a bioorthogonal fluorescence indicator configured to react with an activated renal clearable compound released from the bioorthogonal urinary reporter for cancer detection (BURC) and detecting the presence of cancer if fluorescence is detected due to reaction of the activated renal clearable compound with the bioorthogonal fluorescence indicator. When used herein, the term “bioorthogonal fluorescence indicator” refers to compounds that can detect clickable BURs excreted in urine and convert the levels of clickable BU s in urine to fluorescence signals that can be qualitatively or quantitatively determined. In some embodiments of the seventh aspect of the invention that may be mentioned herein, the bioorthogonal urinary reporter for cancer detection (BURC) may be: or a pharmaceutically acceptable salt or solvate thereof. In some embodiments of the seventh aspect of the invention that may be mentioned herein, the bioorthogonal fluorescence indicator may

[0143] In an eighth aspect of the invention, there is provided a method for detecting treatment- associated kidney injury in a subject, the method comprising the steps of: (ci) administering to a subject suspected of having treatment-associated kidney injury, a bioorthogonal urinary reporter for detecting treatment-associated kidney injury, or pharmaceutically acceptable salt thereof, according to the second aspect of the invention and, after a period of time, collecting urine from said subject; and (cii) adding a bioorthogonal fluorescence indicator configured to react with an activated renal clearable compound released from the bioorthogonal urinary reporter for treatment- associated kidney injury and detecting the presence of renal injury if fluorescence is detected due to reaction of the activated renal clearable compound with the bioorthogonal fluorescence indicator.

[0144] In some embodiments of the eighth aspect of the invention that may be mentioned herein, the bioorthogonal urinary reporter for treatment-associated kidney injury may be:

[0145] , where PEG is a polyethylene glycol moiety having a number average molecular weight of about 2 kDa, or a pharmaceutically-acceptable salt or solvate thereof.

[0146] In some embodiments of the eighth aspect of the invention that may be mentioned herein, the bioorthogonal fluorescence indicator may be

[0147] In a ninth aspect of the invention, there is provided a method for cancer detection and for detecting treatment-associated kidney injury, comprising:

[0148] (di) the method according to the seventh aspect of the invention; and (dii) the method according to the eighth aspect of the invention.

[0149] References herein (in any aspect or embodiment of the invention) to compounds of formula I, or II includes references to such compounds perse, to tautomers of such compounds, as well as to pharmaceutically acceptable salts or solvates, or pharmaceutically functional derivatives of such compounds. Pharmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of formula I or II with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound of formula I or II in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.

[0150] Examples of pharmaceutically acceptable salts include acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, or preferably, potassium and calcium.

[0151] Examples of acid addition salts include acid addition salts formed with acetic, 2,2- dichloroacetic, adipic, alginic, aryl sulphonic acids (e.g. benzenesulphonic, naphthalene-2- sulphonic, naphthalene-1 ,5-disulphonic and p-toluenesulphonic), ascorbic (e.g. L-ascorbic), L-aspartic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulphonic, (+)- (1 S)-camphor-10-sulphonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulphuric, ethane-1,2-disulphonic, ethanesulphonic, 2-hydroxyethanesulphonic, formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic and (±)-DL-lactic), lactobionic, maleic, malic (e.g. (-)-L-malic), malonic, (±)-DL-mandelic, metaphosphoric, methanesulphonic, 1- hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic, tartaric (e.g.(+)-L-tartaric), thiocyanic, undecylenic and valeric acids.

[0152] Particular examples of salts are salts derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.

[0153] As mentioned above, also encompassed by formula I or II are any solvates of the compounds and their salts. Preferred solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent). Examples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulphoxide. Solvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography.

[0154] The solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and di hydrates.

[0155] For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn et a!., Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.

[0156] “Pharmaceutically functional derivatives” of compounds of formula I or II as defined herein includes ester derivatives and / or derivatives that have, or provide for, the same biological function and / or activity as any relevant compound of the invention. Thus, for the purposes of this invention, the term also includes prodrugs of compounds of formula I or II.

[0157] The term “prodrug” of a relevant compound of formula I or II includes any compound that, following oral or parenteral administration, is metabolised in vivo to form that compound in an experimentally-detectable amount, and within a predetermined time (e g. within a dosing interval of between 6 and 24 hours (i.e. once to four times daily)).

[0158] Prodrugs of compounds of formula I or II may be prepared by modifying functional groups present on the compound in such a way that the modifications are cleaved, in vivo when such prodrug is administered to a mammalian subject. The modifications typically are achieved by synthesizing the parent compound with a prodrug substituent. Prodrugs include compounds of formula I or II wherein a hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group in a compound of formula I is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group, respectively.

[0159] Examples of prodrugs include, but are not limited to, esters and carbamates of hydroxyl functional groups, esters groups of carboxyl functional groups, N-acyl derivatives and N- Mannich bases. General information on prodrugs may be found e.g. in Bundegaard, H.

[0160] “Design of Prodrugs” p. 1-92, Elsevier, New York-Oxford (1985).

[0161] Compounds of formula I, as well as pharmaceutically acceptable salts, solvates and pharmaceutically functional derivatives of such compounds are, for the sake of brevity, hereinafter referred to together as the “compounds of formula I”. The same applies for “compounds of formula II”

[0162] Compounds of formula I or II may contain double bonds and may thus exist as E (entgegeri) and Z (zusammeri) geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention.

[0163] Compounds of formula I or II may exist as regioisomers and may also exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention.

[0164] Compounds of formula I or II may contain one or more asymmetric carbon atoms and may therefore exhibit optical and / or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation (i.e. a 'chiral pool’ method), by reaction of the appropriate starting material with a ‘chiral auxiliary’ which can subsequently be removed at a suitable stage, by derivatisation (i.e. a resolution, including a dynamic resolution), for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means such as chromatography, or by reaction with an appropriate chiral reagent or chiral catalyst all under conditions known to the skilled person. All stereoisomers and mixtures thereof are included within the scope of the invention.

[0165] It will be appreciated that the above definitions may also be applied to the bioorthogonal fluorescence indicators disclosed herein too.

[0166] For the avoidance of doubt, in the context of the present invention, the term “treatment” includes references to therapeutic or palliative treatment of patients in need of such treatment, as well as to the prophylactic treatment and / or diagnosis of patients which are susceptible to the relevant disease states. The terms “patient” and “patients” include references to mammalian (e g. human) patients. As used herein the terms "subject" or "patient" are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some embodiments, the subject is a subject in need of treatment or a subject with a disease or disorder. However, in other embodiments, the subject can be a normal subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.

[0167] The term “effective amount” refers to an amount of a compound, which confers a therapeutic effect on the treated patient (e.g. sufficient to treat or prevent the disease). The effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. the subject gives an indication of or feels an effect).

[0168] Further embodiments of the invention that may be mentioned include those in which the compound of formula I or II is isotopically labelled. However, other, particular embodiments of the invention that may be mentioned include those in which the compound of formula I or II is not isotopically labelled.

[0169] The term "isotopically labelled", when used herein includes references to compounds of formula I in which there is a non-natural isotope (or a non-natural distribution of isotopes) at one or more positions in the compound. References herein to "one or more positions in the compound" will be understood by those skilled in the art to refer to one or more of the atoms of the compound of formula I or II. Thus, the term "isotopically labelled" includes references to compounds of formula I or II that are isotopically enriched at one or more positions in the compound.

[0170] The isotopic labelling or enrichment of the compound of formula I or II may be with a radioactive or non-radioactive isotope of any of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, bromine and / or iodine. Particular isotopes that may be mentioned in this respect include2H,3H,11C,13C,14C,13N,15N,15O,17O,180,35S,18F,37CI,77Br,82Br and125l).

[0171] When the compound of formula I or II is labelled or enriched with a radioactive or nonradioactive isotope, compounds of formula I or II that may be mentioned include those in which at least one atom in the compound displays an isotopic distribution in which a radioactive or non-radioactive isotope of the atom in question is present in levels at least 10% (e.g. from 10% to 5000%, particularly from 50% to 1000% and more particularly from 100% to 500%) above the natural level of that radioactive or non-radioactive isotope.

[0172] It will be appreciated that the current invention may include the application of two or more compounds to a subject. In such cases, these compounds may be administered sequentially, simultaneously or concomitantly. When used herein, the term “administered sequentially, simultaneously or concomitantly” includes references to: administration of separate pharmaceutical formulations (one containing the compound of formula I and one or more others containing the compound of formula II etc.); and administration of a single pharmaceutical formulation containing the compound of formula I and the the compound of formula II etc.

[0173] The combination product described above provides for the administration of component (A) in conjunction with component (B), and may thus be presented either as separate formulations, wherein at least one of those formulations comprises component (A) and at least one comprises component (B) , or may be presented (i.e. formulated) as a combined preparation (i.e. presented as a single formulation including component (A) and component (B)).

[0174] Compounds of formula I or II may be administered by any suitable route, but may particularly be administered orally, intravenously, intramuscularly, cutaneously, subcutaneously, transmucosally (e.g. sublingually or buccally), rectally, transdermally, nasally, pulmonarily (e.g. tracheally or bronchially), topically, by any other parenteral route, in the form of a pharmaceutical preparation comprising the compound in a pharmaceutically acceptable dosage form. Particular modes of administration that may be mentioned include oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal administration.

[0175] Compounds of formula I or II will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. Suitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e g. Langer, Science (1990) 249, 1527.

[0176] Otherwise, the preparation of suitable formulations may be achieved routinely by the skilled person using routine techniques and / or in accordance with standard and / or accepted pharmaceutical practice.

[0177] The amount of compound of formula I or II in any pharmaceutical formulation used in accordance with the present invention will depend on various factors, such as the severity of the condition to be treated, the particular patient to be treated, as well as the compound(s) which is / are employed. In any event, the amount of compound of formula I or II in the formulation may be determined routinely by the skilled person.

[0178] For example, a solid oral composition such as a tablet or capsule may contain from 1 to 99 % (w / w) active ingredient; from 0 to 99% (w / w) diluent or filler; from 0 to 20% (w / w) of a disintegrant; from 0 to 5% (w / w) of a lubricant; from 0 to 5% (w / w) of a flow aid; from 0 to 50% (w / w) of a granulating agent or binder; from 0 to 5% (w / w) of an antioxidant; and from 0 to 5% (w / w) of a pigment. A controlled release tablet may in addition contain from 0 to 90 % (w / w) of a release-controlling polymer.

[0179] A parenteral formulation (such as a solution or suspension for injection or a solution for infusion) may contain from 1 to 50 % (w / w) active ingredient; and from 50% (w / w) to 99% (w / w) of a liquid or semisolid carrier or vehicle (e.g. a solvent such as water); and 0-20% (w / w) of one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.

[0180] Depending on the disorder, and the patient, to be diagnosed, as well as the route of administration, compounds of formula I or II may be administered at varying diagnostically effective doses to a patient in need thereof.

[0181] However, the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a diagnostic response in the mammal over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being diagnosed, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage / severity of the disease.

[0182] Administration may be continuous or intermittent (e.g. by bolus injection). The dosage may also be determined by the timing and frequency of administration. In the case of oral or parenteral administration the dosage can vary from about 0.01 mg to about 1000 mg per day of a compound of formula I.

[0183] In any event, the medical practitioner, or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient. The above- mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

[0184] As noted herein, the compounds of formula I and II, together with the bioorthogonal fluorescence indicators mentioned herein may be used for the diagnosis of cancer and / or kidney injury due to therapeutic treatment. These techniques may make use of visualisation methods. Visualising methods that may be mentioned include spectroscopic detection methods (e g. fluorescence detection, magnetic resonance imaging, etc.) or, when the compound of formula I is isotopically labelled or enriched with a radioisotope (such as3H,11C,35S,18F, or125l), radioactivity detection methods (e.g. alpha-, beta- or gamma-detection by standard autoradiography, phosphor or scintillation methods known to those skilled in the art, or positron emission tomography (which latter method may be employed, for example, when the compound of formula I is isotopically labelled or enriched with11C, or, particularly,18F)).

[0185] Therefore, there is disclosed herein a bioorthogonal urine test (BOUT), comprising:

[0186] (a) a Bioorthogonal Urinary Reporter (BUR) molecule; and

[0187] (b) a Bioorthogonal Fluorescence Indicator (BFI) molecule;

[0188] (c) wherein the BUR molecule comprises a caged clickable handle or a caged renal-clearable clickable handle for detecting in vivo cancer biomarkers or kidney injury biomarkers;

[0189] (d) wherein the clickability or renal clearance capability of the BUR molecule is only activated in the presence of the cancer biomarkers or kidney injury biomarkers; and

[0190] (e) wherein the BFI molecule comprises a corresponding handle that can react with the activated BUR molecule and convert into fluorescence signals for ex vivo readout. In some embodiments that may be mentioned herein, BURs may be administered into a cancer-bearing subject under cancer treatment regimen and in the presence of biomarkers at disease site, the clickability or renal clearance capability is activated. The activated BUR fragments are then efficiently excreted in urine, which can be reacted with subsequently added BFIs to turn on its fluorescence for ex vivo readout.

[0191] As will be appreciated, BOUT may be used for monitoring the efficacy of a cancer treatment and / or detecting antineoplastic-induced kidney injury (Al KI) associated with the cancer treatment. In some embodiments that may be mentioned herein, the two BUR / BFI pairs may be employed concurrently to monitor the efficacy of a cancer treatment and detect antineoplastic-induced kidney injury (AIKI) associated with the cancer treatment simultaneously.

[0192] Further aspects and embodiments of the invention will now be discussed by reference to the following non-limiting examples.

[0193] Examples

[0194] Materials

[0195] All commercial reagents were purchased from Sigma Aldrich and used without further purification, unless indicated otherwise. Sodium hyaluronate (Cat. No. HA40K-1) was purchased from Lifecore Biomedical. TCO-OH axial (Cat. No. SC-8013) was purchased from SiChem GmbH. DBCO-PEG2K (Cat. No. BP-23949) was purchased from Broad Pharm. N- Boc- / \ / ,A / '-dimethylethylene diamine was purchased from Enamine. 2-acetamido-3,4,6-tri-O- acetyl-2-deoxy-or-D-glucopyranosyl chloride, N, A / -Diisopropylethyleneamine (DIEA) was purchased from TCI. 2-chlorotrityl chloride polystyrene resin, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc- Met-OH, Fmoc-Arg(Boc)2-OH, 1-hydroxybenzotriazole hydrate (HOBT), 3- [bis(dimethylamino)methyliumyl]-3H-benzotriazol-1-oxide hexafluorophosphate (HBTU) were purchased from Sangon Biotech for solid-phase peptide synthesis (SPPS) synthesis. Cathepsin B (Cat. No. C6286), A / -acetyl-p-D-glucosaminidase (Cat. No. A6805) and - glutamyl transferase (Cat. No. G2262) were purchased from Sigma-Aldrich. Matrix metalloproteinase-2 (MMP2), Matrix metalloproteinase-3 (MMP3), furin, urokinase (uPA), caspase-3 and alanyl aminopeptidase (AAP) were purchased from R&D Systems. ELISA kits for Carcinoembryonic Antigen (Cat. No. SEA150Mu), Cytokeratin Fragment Antigen 21-1 (Cat. No. SEB246Hu) were purchased from Cloud-Clone Corp. Creatinine assay kit (Cat. No. MAK079-1 KT) was purchased from Sigma-Aldrich. QuantiChrom™ urea assay kit (Cat. No. DIUR-100) was purchased from Bioassay Systems. Mouse NAGase(N-Acetyl Beta-D- Glucosaminidase) ELISA Kit (Cat. No. EM1225) was purchased from FineTest. Mouse Cathepsin B ELISA Kit (Cat. No. ab119585) was purchased from Abeam. Cleaved Caspase- 3 rabbit mAb (5A1 E), cathepsin B rabbit mAb (D1C7Y) were purchased from Cell Signaling Technology. Pan-Cytokeratin antibody (sc-81714), MMP-3 Antibody (sc-21732) was purchased from Santa Cruz. Alexa Fluor™ 488-conjugated goat anti-mouse IgG (H+L), Alexa Fluor™ 647-conjugated goat anti-rabbit IgG (H+L) were purchased from ThermoFisher Scientific.

[0196] Thin-layer chromatography (TLC) on pre-coated silica plates (Merck 60 F254 nm, 250 pm thickness) was used to monitor reaction progress. UV light or appropriate staining (e g phosphomolybdic acid stain / PMA, basic KMnC ) was used to visualize the spots on TLC plates. Column chromatography was carried out using 200 or 400 mesh silica gel (Silicycle). Proton, carbon, phosphorus, and fluorine magnetic resonance spectra (1H NMR,13C NMR,31P NMR, and19F NMR) were carried out on a Bruker ACF-400 MHz NMR spectrometer (1H NMR at 400 MHz,13C NMR at 100 MHz,31P NMR at 162 MHz, and19F NMR at 376 MHz). Electrospray ionization-mass spectrometry (ESI-MS) spectra were carried out on a Thermo Finnigan Polaris Q quadrupole ion trap mass spectrometer (ThermoFisher Corporation) equipped with a standard ESI source. High-performance liquid chromatography (HPLC) analyses were carried out on an Agilent 1260 system equipped with a G1311B pump, UV detector and an Agilent Zorbax SB-C18 RP (9.4 x 250 mm) column, with methanol (0.1 % trifluoroacetic acid (TFA)) and water (0.1% TFA) as the eluent. HPLC purification was done on an Agilent 1260 gradient preparative system equipped with a G1361A pump, UV detection, and an Agilent Zorbax SB-C18 RP (21.2 x 150 mm) column, with methanol (0.1% TFA) and water (0.1% TFA) as the eluent. UV / vis spectra were measured on a Shimadzu UV-2450 spectrophotometer. Fluorescence measurements were performed on a Fluorolog 3-TCSPC spectrofluorometer (Horiba Jobin Yvon). For intratracheal injection, IV catheters (pen-type, 22 gauge) were purchased from MediLab. Blood samples were collected using heparinized capillary tubes (Paul Marienfeld, Germany). Urine samples were collected with metabolic cages (Lab Products Inc, USA).

[0197] Statistical

[0198] Statistical comparisons between the two groups were determined by Student's t-test (two- tailed, unpaired). For all tests, p < 0.05 was considered as statistically significant. All statistical calculations were performed using GraphPad Prism v.6 (GraphPad Software Inc., CA, USA). Example 1. Bioorthogonal Optical Urine Test (BOUT)

[0199] The development of BOUT for concurrent monitoring of efficacy and safety profiles throughout cancer treatment regimen is reported herein (Fig. 1a). BOUT contains Bioorthogonal Urinary Reporter (BUR) and Bioorthogonal Fluorescence (FL) Indicator (BFI) for in vivo detection and ex vivo bioorthogonal reaction, respectively. BUR is designed to detect disease biomarkers in vivo and convert biomarkers level to an amount of clickable BURs excreted in urine. BFI is designed to convert the amount of urinary BURs to signal readout via FL turn-on responses. Such a hybrid approach not only ensures the high specificity for disease detection but also circumvents the issue of shallow tissue penetration in optical imaging by performing optical readout in urine.

[0200] To detect antineoplastic-induced kidney injury (AIKI), inverse electron-demand Diels-Alder (I EDDA) reaction between tetrazine (BURK) and trans-cyclooctenes (BFIK) was chosen as the bioorthogonal reaction. The clickability of BURK is initially masked by a biomarker-cleavable group and only triggered by N-acetyl-p-D-glucosaminidase (NAG), a biomarker associated with kidney tubular injury. Meanwhile, to achieve kidney specificity, BURK is designed to have high renal clearance efficiency. The excreted BURK in the urine with turned-on clickability thus reflects the level of NAG in the kidneys and is subsequently reacted with BFIKvia I EDDA reaction for FL turn-on readout.

[0201] Example 2. Syntheses

[0202] As shown in Fig. 1c, the cancer reporter, BURc, consists of a perfluoroaryl azide unit, a CTSB- cleavable peptide, and a hyaluronic acid backbone. It was prepared by conjugating 4-azido- 2,3,5,6-tetrafluorobenzoic acid to the C-terminal of CTSB-cleavable peptide sequence (Gly- Phe-Leu-Gly-Gly) via a short PEG chain. The N-terminal of the peptide was then conjugated to the reactive carboxyl group on hyaluronic acid, and the conjugation of one azide per disaccharide repeat unit was confirmed by1H NMR (Supplementary information). Upon CTSB cleavage, the activated BURc, a PEGylated perfluoroaryl azide, is cleaved off from the hyaluronic acid backbone. To sensitively detect kidney injury, BURK is designed as a PEGylated phenyl-substituted dihydrotetrazine with the secondary amine caged by a NAG- cleavable N-acetyl-|3-D-glucosaminide. BURK was synthesized by firstly preparing p- formylphenyl N-acetyl-glucosamine by a phase-transfer catalytic system, followed by aldehyde reduction and bromination before conjugating to a phenyl-substituted dihydrotetrazine with an alkyne functional group. Pure compound BURK was then obtained after deacetylation and conjugation to DBCO-PEG via click reaction. In the presence of NAG, the terminal glycosidic bond of BURK is cleaved, leading to formation of clickable tetrazine after a rapid oxidation reaction. BFIs are constructed based on the corresponding bioorthogonal handles, and BFIc (BFIK) is designed by caging the hydroxyl group of coumarin (resorufin) by triphenylphosphine (trans-cyclooctenes). BFIc was prepared by conjugating 2- (diphenylphosphanyl)benzoic acid to coumarin via carbodiimide crosslinker, and BFIK was prepared according to Mancuso, F. et al., ChemPlusChem 85, 1669-1675 (2020).

[0203] Synthesis of BFIc

[0204] Coumarin (40 mg, 0.224 mmol) was dissolved in 2.0 mL anhydrous CH2CI2 and cooled to 0 °C, and a solution of 2-(diphenylphosphino)benzoic acid (75 mg, 0.247 mmol), dicyclodexylcarbodiimide (60 mg, 0.292 mmol), 4-dimethylaminopyridine (3.5 mg, 0.292 mmol) in 2.0 mL anhydrous CH2CI2 was added dropwise. The solution was allowed to stir at room temperature for 12 h and monitored by TLC. Upon completion, the solvent was removed in vacuo and purification by column chromatography using 0-30% ethyl acetate: hexanes to afford pure BFIc as a white solid (93 mg, 90%).

[0205] 1H NMR (400 MHz, CDC -di) 5: 8.13 (d, 2H, J = 8Hz), 7.66 (d, 1 H, J = 8Hz), 7.42 (m, 1H), 7.38 (m, 7H), 7.36 (m, 3H), 7.26 (s, 1H), 7.21 (m, 1 H), 7.20 (m, 1 H), 6.30 (s, 1 H), 2.46 (s, 3H).13C NMR (100 MHz, CDCI3-C / 7) 5: 164.64, 160.53, 154.12, 152.95, 151.87, 141.89, 141.61 , 137.36, 137.26, 134.49, 134.16, 133.96, 132.89, 131.52, 128.96, 128.68, 128.61 , 128.38, 125.21, 120.00, 118.20, 117.85, 114.52, 110.54, 34.93, 25.47, 24.71 , 18.73.31P NMR (162 MHz, CDC -dj): 6: -3.72 (s, 1 H). HR-MS (ESI): calcd for [M+H]+C29H21O4P: 465.1250; found: 465.1207.

[0206] Synthesis ofNH2PEG7-N3 f-boc-amido-PEGy-amine (500 mg, 1.07 mmol), 4-azido-2,3,5,6-tetrafluorobenzoic acid (300 mg, 1.28 mmol), HBTU (608 mg, 1.60 mmol) and DIEA (372 pL, 2.14 mmol) were dissolved in anhydrous THF and allowed to stir for 12h at room temperature. Upon completion, the reaction mixture was dissolved in EtOAc, washed with water and brine. The organic extracts were dried over Na2SC>4 and concentrated under reduced pressure. The residue was then dissolved in 30%TFA / DCM (5 ml_) and stirred at 0 °C. The reaction was continuously monitored by TLC, and upon completion, the mixture was concentrated under reduced pressure. Preparative HPLC purification was then carried out to afford pureNH2PEG7-N3 as colourless liquid (560 mg, 90%).

[0207] 1H NMR (400 MHz, DMSO-cy 5: 8.99 (t, J = 4Hz, 1 H), 7.78 (s, 2H), 3.60 (t, J = 6Hz, 2H), 3.57 (m, 4H), 3.52 (m, 23H), 3.41 (q, J = 5.33Hz, 2H), 2.99 (m, 2H).13C NMR (100 MHz, DMSO- de) 6: 159.01 (q, J = 35Hz, CF3COOH), 157.55, 144.55, 142.09, 141.66, 139.20, 116.36 (q, J = 283Hz, CF3COOH), 70.20, 70.08, 69.01 , 67.08.19F NMR (376 MHz, Methanol-^) 6: -144.10, -152.70. HR-MS (ESI): calcd for [M+H]+C23H35F4N5O8: 586.2495; found: 586.2506.

[0208] Synthesis of Activated BURC.

[0209] NH2PEG7-N3(200 mg, 0.34 mmol), Fmoc-Gly-OH (121 mg, 0.41 mmol), HoBT (92 mg, 0.68 mmol), HBTU (258 mg, 0.68 mmol), DIEA (116 pL, 0.68 mmol) was dissolved in anhydrous THF and allowed to stir for 12h at room temperature. Upon completion, the reaction mixture was dissolved in EtOAc, washed with water and brine. The organic extracts were dried over Na2SO4 and concentrated under reduced pressure. The crude product was then directly used in the deprotection step and dissolved in 5% piperidine / DMF (5 mL). The mixture was stirred at room temperature for 2 h and continuously monitored by TLC. Upon completion, the mixture was concentrated under reduced pressure and underwent preparative HPLC purification to obtain pure activated BURc as colourless liquid (197 mg, 90%).

[0210] 1H NMR (400 MHz, Methanol-ck) 6: 3.75 (s, 2H), 3.66 (m, 27H), 3.59 (m, 4H), 3.43 (t, J = 4Hz, 2H).13C NMR (100 MHz, DMSO-c / 6) 5: 166.37, 143.87, 140.80, 128.57, 127.90, 125.70, 120.84, 70.21 , 70.09, 69.29, 69.00, 58.86, 53.88, 43.18. HR-MS (ESI): calcd for [M+H]+C25H38F4N6O9: 643.2709; found: 643.2744.

[0211] Peptide / V-Fmoc-Gly-Phe-Leu-Gly-Gly-OH (3.10 g, 4.6 mmol) was synthesised by SPPS.

[0212] 1H NMR (400 MHz, Methanol-c / 4) 6: 7.82 (d, J = 8Hz, 2H), 7.67 (m, 2H), 7.41 (t, J = 8Hz, 2H), 7.33 (t, J = 8Hz, 2H), 7.24 (m, 5H), 4.65 (m, 1 H), 4.30 (m, 4H), 3.86 (m, 6H), 3.16 (m, 1H), 2.97 (m, 1 H), 1.63 (m, 3H), 0.90 (m, 6H).13C NMR (100 MHz, Methanol-^) 6: 173.55, 172.52, 171.39, 171.14, 170.47, 157.77, 143.86, 143.79, 141.19, 136.66, 128.97, 128.18, 127.44, 126.80, 126.48, 124.88, 124.83, 119.57, 66.96, 54.69, 52.40, 43.61 , 42.07, 40.40, 39.70, 36.92, 24.29, 22.09, 20.52. HR-MS (ESI): calcd for [M+H]+C36H4iN5O8: 672.3028; found: 672.3086.

[0213] Synthesis ofFmocG

[0214] NH2PEG7-N3(200 mg, 0.34 mmol), peptide / V-Fmoc-Gly-Phe-Leu-Gly-Gly-OH (275 mg, 0.41 mmol), HoBT (92 mg, 0.68 mmol), HBTU (258 mg, 0.68 mmol), and DIEA (116 pL, 0.68 mmol) was dissolved in anhydrous THF and allowed to stir for 12 h at room temperature. Upon completion, the reaction mixture was extracted with EtOAc, washed with water and brine. The organic extracts were dried over Na2SO4 and concentrated under reduced pressure, the crude product was used directly in the next step.

[0215] HR-MS (ESI): calcd for [M+H]+C59H74F4N10O15: 1239.5344; found: 1239.5798.

[0216] Synthesis ofNH2GFL

[0217] The crude productFmocGFLGG-PEG7-N3was dissolved in 5% piperidine / DMF (5 mL) and stirred at room temperature for 2 h with continuous TLC monitoring. Upon completion, the mixture was concentrated under reduced pressure. PureNH2GFLGG-PEG7-N3was obtained as white solid after preparative HPLC purification (310 mg, 90%).1H NMR (400 MHz, DMSO-cfe) 6: 8.99 (t, J = 4Hz, 1 H), 8.62 (d, J = 8Hz, 1 H), 8.38 (d, J = 8Hz, 1H), 8.08 (m, 2H), 7.90 (t, J = 4Hz, 1 H), 7.26 (m, 4H), 7.21 (m, 1 H), 4.66 (m, 1 H), 4.33 (q, J = 8Hz, 1H), 3.74 (d, 4H), 3.70 (d, 2H), 3.51 (m, 26H), 3.41 (t, J = 6Hz, 4H), 3.22 (q, J = 5.33Hz, 2H), 3.06 (m, 1 H), 2.74 (m, 1 H), 1.61 (m, 1H), 1.51 (t, J = 8Hz, 2H), 0.90 (dd, J = 20, 4Hz, 6H).13C NMR (100 MHz, DMSO-cfe) 6: 172.66, 171.17, 169.39, 169.18, 166.16, 157.51 , 137.93, 129.69, 128.56, 126.83, 70.24, 70.20, 70.17, 70.12, 70.06, 69.41 , 69.04, 54.47, 51.70, 42.52, 42.41 , 41.18, 39.04, 38.27, 24.62, 23.47, 22.10. HR-MS (ESI): calcd for [M+H]+C44H64F4N10O13: 1017.4663; found: 1017.4949.

[0218] Sodium hyaluronate (500 mg, 0.01 mmol) was dissolved in DI Water and stirred at 0 °C, Dowex® 50WX8-400 ion-exchange resin was slowly added into the reaction mixture to adjust the pH to 5-6. The mixture was filtered and the filtrate was collected, hyaluronic acid was obtained after lyophilization (350 mg, 74%). A mixture of hyaluronic acid (10 mg, 0.026 mmol disaccharide repeats), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride / EDC (192 mg, 0.052 mmol), and N-Hydroxysuccinimide / NHS (115 mg, 0.052 mmmol) are dissolved and stirred in 3 ml_ deionized H2O at room temperature. After stirring for 30 min, a mixture of pure productNH2GFLGG-PEG?-N3 (53 mg, 0.052 mmol) and DIEA (22 pL, 0.13 mmol) in 3 mL DMF was added in dropwise. The reaction mixture was stirred for 48 h, and dialysed against deionized H2O for 96 h before pure BURc was obtained after lyophilization (30 mg, 90%).

[0219] 1H NMR (400 MHz, D2O) 6: 7.30 (m, 5H), 4.40 (m, 5H), 3.87 (s, 3H), 3.14-3.75 (m, 47H), 1.96 (s, 6H), 0.82 (dd, J = 20, 4Hz, 6H).13C NMR (100 MHz, D2O) 5: 174.61 , 172.98, 171.60, 171.24, 166.81, 135.95, 129.23, 128.83, 127.31 , 103.14, 100.74, 82.48, 80.00, 75.41 , 73.62, 72.36, 69.59, 68.76, 68.51 , 55.03, 54.39, 52.39, 42.68, 42.56, 38.98, 38.75, 25.01 , 24.13, 22.46, 22.09, 20.79, 17.74, 16.27, 12.13.

[0220] Synthesis of BFIK

[0221] Synthesis of BFIKwas conducted according to Mancuso, F. et al., ChemPlusChem 85, 1669- 1675 (2020).

[0222] 1H NMR and13C NMR corresponded with reported literature.1H NMR (400 MHz, CDCh-d) 6: 7.83 (m, 1H), 7.50 (d, J = 8Hz, 1H), 7.22 (m, 2H), 6.94 (d, J = 8Hz, 1 H), 6.41 (s, 1 H), 5.80 (m, 1H), 5.58 (m, 1 H), 5.40 (s, 1 H), 3.49 - 3.67 (m, 4H (retainers)), 3.18 & 3.10 (2s, 3H total (rotamers)), 3.04 - 3.07 (m, 3H), 2.95 (m, 1 H), 2.02 (m, 3H), 1 .90 (m, 1 H), 1 .72 (m, 2H), 1 .47 (m, 1 H), 1.14 (m, 1H), 0.84 (m, 1H).13C NMR (100 MHz, Methanol-^) 6: 188.40, 145.78, 136.63, 135.57, 132.79, 132.56, 132.20, 132.16, 130.87, 130.79, 120.85, 111.02, 110.76, 107.26, 75.80, 41.68, 37.05, 36.77, 33.06, 31.82, 30.74, 28.11 , 26.90, 26.15, 23.72. HR-MS (ESI): ealed for [M+H]+C26H29N3O6: 480.2129; found: 480.2096.

[0223] 1,4-dibromobutane (5 mL, 43 mmol) was dissolved in 15 mL DMF, followed by addition of NaNs (8.2 g, 126 mmol). The reaction was stirred at 75 °C overnight. Upon completion, the reaction was diluted with DCM and filtered. The filtrate was washed with water, brine, dried with Na2SO4 and evaporated under reduced pressure to afford a pale yellow oil. The crude product was used immediately in the next step without further purification.

[0224] 1H NMR (400 MHz, CDCb-di) 6: 3.34-3.31 (m, 4H), 1.70-1.67 (m, 4H).13C NMR (100 MHz, CDC -di) 6: 51.07, 26.31.

[0225] Benzonitrile (0.8 mL, 8 mmol), 4-pentynenitrile (2.88 mL, 32 mmol), and 3-mercaptopropionic acid (0.34 mL, 4 mmol) were in an ice bath. Next, hydrazine monohydrate (64-65%, 6.2 mL) was added dropwise. The reaction mixture was stirred at 60 °C for 24 h. Upon completion, the reaction was added into 100 mL of ice water. Sodium nitrite (2 g, 120 mmol) dissolved in 50 mL of water was slowly added into the reaction mixture, followed by slow addition of 1M HCI until effervescence ceased. The reaction mixture was extracted with CH2CI2, washed with water and brine. The organic extracts were combined, dried over Na2SC and concentrated under reduced pressure. The residue was purified by column chromatography using 30%-50% CH2Cl2 / hexanes to afford pure Tz-Alk as a pink solid (820 mg, 51%).

[0226] 1H NMR (400 MHz, Methanol-d4) 6: 8.59-8.57 (m, 2H), 7.69-7.63 (m, 3H), 3.58 (t, J = 7.3 Hz, 2H), 2.95 (td, J= 7.3 Hz, J= 2.6 Hz, 2H), 2.31 (t, J= 2.6 Hz, 1 H).13C NMR (100 MHz, Methanol- d4) 6: 168.39, 164.35, 132.28, 132.01, 128.96, 127.50, 81.61, 69.67, 33.47, 16.11. HR-MS (ESI): calcd for [M+H]+C12H10N4: 211.0978; found: 211.0838.

[0227] Synthesis of Tz-Az

[0228] Tz-Alk (21 mg, 0.10 mmol) and 1,4-diazobutane (70 mg, 0.50 mmol) was first dissolved in 4 mL DMSO. THPTA (22 mg, 0.05 mmol) and CuSC StW (25 mg, 0.10 mmol) were premixed in 500 pL water before addition. The reaction was purged with nitrogen gas for 10 mins. Sodium ascorbate (35 mg, 0.20 mmol) dissolved in 500 pL of water was next added. The reaction was purged with nitrogen gas for another 10 mins and allowed to stir at room temperature for 1 h. Upon completion, preparative HPLC purification was carried out to afford pure Tz-Az as a yellow gum (20 mg, 69%).

[0229] 1H NMR (400 MHz, Methanol-^) 6: 8.57 (d, J = 6.7 Hz, 2H), 7.83 (s, 1 H), 7.70-7.62 (m, 3H), 4.40 (t, J = 6.9 Hz, 2H), 3.73 (t, J = 7.4 Hz, 2H), 3.45 (t, J = 7.4 Hz, 2H), 3.33-3.29 (m, 2H), 1.95 (q, J = 7.0, 2H), 1.56-1.49 (m, J = 6.88 Hz, 2H).13C NMR (100 MHz, Methanol-^) 6: 168.84, 164.18, 145.80, 132.24, 132.01 , 128.94, 127.47, 122.39, 50.35, 49.33, 33.89, 27.09, 25.41 , 22.93. HR-MS (ESI): calcd for [M+H]+CI6HI8NI0: 351.1789; found: 351.1709.

[0230] Synthesis of Activated BURK

[0231]

[0232] Tz-Az (50 mg, 0.076 mmol) and DBCO-PEG2K (226 mg, 0.113 mmol) were dissolved in 2 mL H2O and allowed to stir at room temperature for 2 h. After completion, the resulting mixture was purified by preparative HPLC to afford pure activated BURK as a pink gum (181 mg, 89%).

[0233] 1H NMR (400 MHz, Methanol-cf4) 6: 8.58-8.52 (m, 2H), 8.02-7.97 (m, 1 H), 7.72-7.59 (m, 4H), 7.56-7.47 (m, 2H), 7.43-7.29 (m, 4H), 7.18 (d, J = 7.2 Hz, 1 H), 4.50-4.34 (m, 4H), 3.83-3.74 (m, 4H), 3.63 (s, 200H), 3.56-3.54 (m, 5H), 3.50-3.47 (m, 5H), 3.37 (s, 3H), 3.28-3.37 (m, 2H), 2.13-2.09 (m, 2H).13C NMR (100 MHz, Methanol-c ) 3: 172.93, 172.69, 172.49, 171.96, 171.35, 168.79, 164.21 , 158.75, 158.32, 158.20, 157.78, 157.37, 156.95, 151.37, 148.14,

[0234] 145.37, 144.61 , 143.68, 142.78, 142.39, 141.16, 140.21 , 135.54, 134.62, 133.75, 133.47,

[0235] 132.68, 132.44, 132.38, 132.18, 132.04, 131.70, 131.62, 131.27, 131.15, 130.97, 129.75,

[0236] 129.70, 129.65, 129.52, 129.37, 129.29, 129.15, 129.08, 128.69, 128.55, 128.28, 128.19,

[0237] 127.98, 127.91 , 127.57, 127.55, 127.45, 127.08, 126.96, 126.89, 126.78, 125.14, 123.87,

[0238] 123.27, 122.99, 122.21 , 118.89, 116.64, 116.06, 113.80, 113.24, 110.41 , 77.06, 72.46, 72.30, 71.57, 70.16, 69.96, 69.17, 69.12, 67.87, 63.27, 60.85, 57.76, 55.30, 54.90, 53.69, 53.47, 53.24, 53.01 , 52.79, 52.36, 50.92, 49.66, 39.04, 33.69, 30.53, 30.11 , 30.01 , 29.46, 29.33, 28.83, 28.00, 27.11 , 26.43, 25.50, 22.63. MALDI-TOF MS found: 2,300-2,600.

[0239] Synthesis of0AcNAG-PABA

[0240] 4-hydroxybenzaldehyde (2.00 g, 16.4 mmol) and 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D- glucopyranosyl chloride (3.00 g, 8.2 mmol) were dissolved in 80 mL dry CH3CN. Next, Nal (2.46 g, 16.4 mmol) and Ag2O (3.77 g, 16.4 mmol) were added. The mixture was stirred at room temperature for 12 h. The reaction mixture was diluted with CH2CI2 and filtered. The filtrate was collected, washed with water and brine, dried over anhydrous Na2SC>4 and concentrated under reduced pressure. The residue was suspended in 20 mL of EtOH, sonicated and filtered to afford pureOAcNAG-PABA as a white solid (1.4 g, 38%).

[0241] 1H NMR (400 MHz, CDCh-d;) 6: 9.91 (s, 1 H), 7.83 (d, J = 8Hz, 2H), 7.10 (d, J = 8Hz, 2H), 5.86 (d, J = 8Hz, 1 H), 5.44-5.49 (m, 2H), 5.15 (t, J = 10Hz, 1 H), 4.28 (dd, J = 12, 4Hz, 1H), 4.13-4.19 (m, 2H), 3.94-3.98 (m, 1 H). 2.06-2.08 (m, 9H), 1.95 (s, 3H).13C NMR (100 MHz, CDC -d,) 0: 190.90, 170.94, 170.66, 170.63, 169.53, 161.64, 131.92, 131.78, 116.88, 97.99, 72.38, 71.87, 68.54, 62.22, 54.90, 23.44, 20.82, 20.75. HR-MS (ESI): calcd for [M+H]+C21 H25NO10: 452.1551 ; found: 452.1508.

[0242] OAcNAG-PABA (677 mg, 1.50 mmol) in 50 mL anhydrous MeOH was cooled in an ice bath. NaBH4(0.11 g, 3.00 mmol) was added slowly to the solution. The reaction was stirred for 2 h at 0 °C, quenched with saturated NH4CI and extracted with CH2CI2. The organic layer was dried over Na2SO4 and evaporated under reduced pressure to affordOAGNAG-OH as a white solid (462 mg, 68%), which was directly used in the next step without further purification.OAcNAG-OH (0.18 g, 0.40 mmol) was dissolved in 8 mL anhydrous THF and cooled in an ice bath. PBrs (0.08 mL, 0.80 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 2 h. Upon completion, the reaction was quenched with saturated NaHCOs at 0 °C and extracted with CH2CI2. The combined organic extracts were washed with saturated NaHCOs, water, followed by brine. The organic layer was dried with Na2SC>4 and evaporated under reduced pressure to afford a white solid. The crude product was used immediately in the next step without further purification.

[0243] Thiourea dioxide (64 mg, 0.6 mmol) was added to a solution of Tz-Alk (84 mg, 0.4 mmol) in 1.5 mL of DMF:H2O (10:1) at room temperature under argon. The reaction mixture was stirred in an oil bath at 95 °C for 4 h. Upon completion, the solvent was removed under reduced pressure. The yellow residue was dried under high vacuum and used directly in the next step.

[0244] 0AcNAG-Br (25.8 mg, 0.05 mmol) anddih',Tz-Alky (19.8 mg, 0.1 mmol) were dissolved in 3 mL ACN / THF (1 :2) and allowed to stir at room temperature overnight. The reaction was purified by preparative HPLC to affordOAoNAG-Tz-Alk as a pale-yellow gum (52%: Isomer 1 ; 16%: Isomer 2).

[0245] 1H NMR (400 MHz, Chloroform-d) 5: 7.84 (d, J = 7.3 Hz, 2H), 7.62-7.59 (m, 1H), 7.52-7.24 (m, 2H), 7.32-7.30 (m, 1 H), 7.28-7.24 (m, 2H), 7.02-7.00 (d, J = 7.6 Hz, 1 H), 6.71 (s, 1 H), 5.35- 5.28 (m, 2H), 5.16 (t, J = 9.6 Hz, 1 H), 4.33-4.26 (m, 4H), 4.20-4.17 (m, 1 H), 3.92 (d, J = 7.76 Hz, 1H), 2.62 - 2.61 (m, 2H), 2.51-2.50 (m, 2H), 2.16 (s, 1 H), 2.07 (s, 3H), 2.05 (s, 6H), 2.01 (s, 3H).13C NMR (100 MHz, Chloroform-d) 6: 172.75, 170.84, 170.67, 169.60, 162.50, 160.92, 160.56, 157.74, 154.52, 133.05, 130.81 , 129.37, 128.14, 128.04, 117.45, 117.14, 114.26, 99.18, 80.17, 72.37, 72.00, 71.70, 68.42, 62.06, 58.19, 54.30, 27.91 , 22.69, 20.64, 20.55, 15.60. HR-MS (ESI): calcd for [M+H]+C33H37N5O9: 648.2664; found: 648.2726.

[0246] Synthesis of NAG-Tz-Alk

[0247] 0AcNAG-Tz-Alk (64.7 mg, 0.10 mmol) was dissolved in 2 mL anhydrous MeOH followed by the addition of K2CO3 (55.2 mg, 0.40 mmol). The reaction was allowed to stir at room temperature for 30 mins. Upon completion, preparative HPLC purification was carried out to afford pure

[0248] NAG-Tz-Alk as a yellow gum (45.6 mg, 90%).

[0249] 1H NMR (400 MHz, Methanol-d4) 5: 7.82 (d, J = 7.4 Hz, 1 H), 7.64-7.60 (m, 1 H), 7.56-7.53 (m, 2H), 7.32-7.30 (d, J = 7.6 Hz, 2H), 7.07 (d, J = 7.5 Hz, 2H), 5.11 (d, J = 8.0 Hz, 1 H), 4.29 (s, 2H), 3.99-3.87 (m, 2H), 3.75-3.71 (m, 1 H), 3.62 (t, J = 9.5 Hz, 1 H), 3.47-3.42 (m, 2H), 2.59- 2.56 (m, 5H), 2.00 (s, 3H).13C NMR (100 MHz, Methanol-d4) 6: 172.57, 162.51, 158.10, 154.32, 132.30, 130.53, 128.95, 127.97, 127.81 , 116.80, 99.25, 79.86, 76.86, 74.24, 71.51 , 70.56, 61.22, 57.98, 56.04, 27.96, 21.65, 14.95. HR-MS (ESI): calcd for [M+H]+C27H31N5O6: 522.2347; found: 522.2347.

[0250] Synthesis of NAG-Tz-Az

[0251] NAG-Tz-Alk (52 mg, 0.10 mmol) and 1 ,4-diazobutane (70 mg, 0.50 mmol) was first dissolved in 3 ml_ DMSO. THPTA (22 mg, 0.05 mmol) and CuSO4'5H2O (25 mg, 0.10 mmol) were premixed in 500 pL water before addition. The reaction was purged with nitrogen gas for 10 mins. Sodium ascorbate (35 mg, 0.20 mmol) dissolved in 500 pL of water was next added. The reaction was purged with nitrogen gas for another 10 min and allowed to stir at room temperature for 1 h. Upon completion, preparative HPLC purification was carried out to afford pure NAG-Tz-Az as a yellow gum (49 mg, 75%).

[0252] 1H NMR (400 MHz, Methanol-c / 4) 0: 7.87 (d, J = 7.2 Hz, 1 H), 7.78 (s, 1 H), 7.64-7.57 (m, 1H), 7.56-7.54 (m, 2H), 7.28 (d, J = 8.4 Hz, 1 H), 7.08 (d, J = 8.4 Hz, 2H), 5.11 (d, J = 8.0 Hz, 1H), 4.43 (t, J = 6.9 Hz, 2H), 4.28 (s, 2H), 3.95-3.87 (m, 2H), 3.74-3.70 (m, 1 H), 3.64-3.59 (m, 1 H), 3.49-3.42 (m, 2H), 3.34-3.33 (m, 2H), 3.00 (t, J = 7.3 Hz, 1 H), 2.77 (t, J= 7.3 Hz, 2H), 1.99 (s, 3H), 1.97-1.95 (m, 2H), 1.60-1.53 (m, 2H).13C NMR (100 MHz, Methanol-^) 5: 172.63, 158.20, 132.54, 130.78, 129.01 , 128.79, 127.76, 116.82, 99.29, 76.86, 74.29, 70.53, 61.18, 58.22, 55.96, 50.36, 49.52, 28.17, 27.12, 25.45, 21.68, 21.10. HR-MS (ESI): calcd for [M+H]+C3IH39NHOS: 662.3158; found: 622.3240.

[0253] Synthesis of BU K

[0254]

[0255] NAG-Tz-Az (50 mg, 0.076 mmol) and DBCO-PEG2K (226 mg, 0.113 mmol) were dissolved in 2 ml_ H2O and allowed to stir at room temperature for 2 h. After completion, the resulting mixture was purified by preparative HPLC to afford pure BURK as a yellow gum (181 mg, 89%).

[0256] 1H NMR (400 MHz, Methanol-d4) 6: 7.90-7.84 (m, 2H), 7.67-7.62 (m, 2H), 7.61-7.59 (m, 2H), 7.56-7.55 (m, 3H), 7.41-7.37 (m, 3H), 7.30-7.28 (m, 3H), 7.21-7.19 (m, 1 H), 7.11-7.05 (m, 2H),

[0257] 5.14 (d, J = 7.5 Hz, 1 H), 4.77 (d, J = 11.7 Hz, 1 H), 4.59-4.49 (m, 2H), 4.43-4.36 (m, 3H), 4.29 (s, 2H), 3.95-3.90 (m, 2H), 3.82-3.81 (m, 2H), 3.65 (s, 200H), 3.55-3.54 (m, 3H), 3.48-3.42 (m, 2H), 3.37 (s, 3H), 3.29-3.28 (m, 2H), 3.03-3.01 (m, 2H), 2.82-2.80 (m, 2H), 2.11-2.10 (m, 2H), 1.99 (s, 3H), 1.76-1.70 (m, 2H).13C NMR (100 MHz, Methanol-^) 6: 173.08, 172.81, 172.52, 172.19, 171.47, 164.45, 158.89, 158.47, 158.23, 157.79, 157.37, 156.96, 154.54, 144.67,

[0258] 143.99, 142.80, 141.09, 140.14, 135.42, 134.74, 133.69, 132.71, 132.06, 131.94, 131.73,

[0259] 131.16, 131.03, 130.84, 129.79, 129.68, 129.54, 129.43, 129.09, 128.72, 127.92, 127.75,

[0260] 127.51, 126.99, 126.80, 123.86, 122.70, 118.88, 116.87, 116.05, 113.83, 113.22, 110.39,

[0261] 99.20, 76.94, 74.26, 72.43, 71.55, 70.14, 69.93, 69.09, 67.88, 61.26, 57.73, 56.02, 51.00,

[0262] 49.35, 39.05, 30.38, 30.16, 29.47, 28.21 , 26.66, 26.42, 21.71. MALDI-TOF MS found: 2,600- 2,900.

[0263] Synthesis of CvOH

[0264] Hemicyanine dye CyOH was prepared according to Yuan, L. et al., J. Am. Chem. Soc. 134, 13510-13523 (2012).

[0265] 1H NMR (400 MHz, CDCk-d,): 6: 8.15 (d, J = 12Hz, 1 H), 7.30 (m, 3H), 7.22 (d, J = 12Hz, 1H), 7.09 (t, J = 8Hz, 1 H), 6.88 (m, 2H), 6.71 (s, 1 H), 5.65 (d, J = 12Hz, 1 H), 3.41 (s, 3H), 2.68 (t, J = 6Hz, 2H), 2.62 (t, J = 6Hz, 2H), 1.90 (m, 2H), 1.68 (s, 6H). HR-MS (ESI): calcd for [M]+C2SH2SNO2: 384.1958; found: 384.1966.

[0266] Synthesis of BFICM

[0267] CyOH (20 mg, 0.05 mmol) was dissolved in 2.0 ml_ anhydrous CH2CI2 and cooled to 0 °C, and a solution of 2-(diphenylphosphino)benzoic acid (17 mg, 0.057 mmol), dicyclodexylcarbodiimide (13 mg, 0.065 mmol), 4-dimethylaminopyridine (0.8 mg, 0.065 mmol) in 2.0 mL anhydrous CH2CI2 was added dropwise. The solution was allowed to stir at room temperature for 12 h and monitored by TLC. Upon completion, the solvent was removed in vacuo and purification by column chromatography using 0-30% methanokdichloromethane to afford pure BFICM as a purple solid (30 mg, 90%).

[0268] 1H NMR (400 MHz, CDCh-cf;) 6: 8.64 (d, J = 12Hz, 1 H), 8.30 (m, 1 H), 8.14 (d, J = 6Hz, 1H), 7.88 (m, 1 H), 7.70 (m, 1 H), 7.61 (m, 1 H), 7.53 (m, 3H), 7.38-7.43 (m, 8H), 7.17 (m, 1 H)„ 7.12 (m, 1 H), 7.05 (m, 2H), 4.27 (s, 3H), 2.93 (t, 2H), 2.74 (t, 2H), 1.96 (s, 2H), 1.78 (s, 6H), 1.25 (s, 2H).13C NMR (100 MHz, DMSO-cfe) 6: 179.29, 164.48, 158.98, 153.01 , 152.82,, 145.72,

[0269] 145.52, 142.79, 142.64, 135.99, 135.88, 134.21 , 134.01 , 133.66, 133.47, 130.85, 130.44,

[0270] 130.38, 130.21 , 130.05, 129.57, 129.50, 129.33, 129.01 , 128.74, 128.17, 123.05, 120.13,

[0271] 119.73, 114.61 , 114.24, 110.53, 107.22, 51.19, 33.56, 29.16, 27.45, 23.99, 20.30.31P NMR

[0272] (162 MHz, CDCh-cG): 6: -4.61 (s, 1 H). HR-MS (ESI): calcd for [M]+C45H39NO3P: 672.2662; found: 672.2663.

[0273] Peptide / \Z-Fmoc-Tyr(tBu)-Trp(Boc)-Met-Arg(Boc)2-Gly-OH (2.60 g, 2 mmol) was synthesised by SPPS.1H NMR (400 MHz, Methanol-^) 6: 8.07 (d, J = 4Hz, 2H), 7.78 (d, J = 8Hz, 2H), 7.59 (m, 3H), 7.54 (s, 1 H), 7.40 (m, 2H), 7.22 - 7.38 (m, 5H), 7.08 (d, J = 8Hz, 2H), 6.82 (d, J = 8Hz, 2H), 4.72 (s, 2H), 4.49 (m, 2H), 4.26 - 4.32 (m, 2H), 4.11 - 4.15 (m, 3H), 3.87 (d, J = 18Hz, 3H), 3.74 (m, 1 H), 3.62 (m, 1 H), 3.25 (m, 1 H), 3.15 (m, 2H), 2.46 (q, J = 8Hz, 2H), 2.07 (m, 2H), 2.02 (s, 3H), 1.61 - 1.93 (m, 18H), 1.23 (m, 18H).13C NMR (100 MHz, Methanol-ck) 3: 172.91, 172.35, 171.91 , 171.18, 156.83, 153.86, 149.58, 143.72, 141.12, 135.32, 132.12, 130.27, 129.52, 127.37, 126.77, 124.90, 124.10, 123.74, 122.39, 119.49, 118.82, 115.69, 114.76, 83.50, 78.07, 66.80, 56.65, 53.41 , 52.42, 40.45, 37.01 , 31.19, 29.50, 27.76, 27.02, 26.79, 26.65, 13.84. HR-MS (ESI): calcd for [M+H]+C67H87N9O15S: 1290.6115; found: 1290.6121.

[0274] Synthesis of Activa

[0275] NH2PEG7-N3(60 mg, 0.1 mmol), Fmoc-YWMRG-OH (193 mg, 0.15 mmol), HoBT (27 mg, 0.2 mmol), HBTU (76 mg, 0.2 mmol), DIEA (34 pL, 0.2 mmol) was dissolved in anhydrous THF and allowed to stir for 12h at room temperature. Upon completion, the reaction mixture was dissolved in EtOAc, washed with water and brine. The organic extracts were dried over Na2SO4 and concentrated under reduced pressure. The crude product was then directly used in the deprotection step and dissolved in 50% TFA / DCM (3 ml_) and was allowed to stir at 0 °C for 2 h. The reaction was monitored by TLC, and upon completion, the mixture was concentrated under reduced pressure, dissolved with EtOAc, and then washed with saturated NaHCO3, water, followed by brine. The organic layer was dried with Na2SO4 and evaporated under reduced pressure to afford a white solid. The crude product was then dissolved in 5% piperidine / DMF (5 mL) and stirred at room temperature for 2 h, as continuously monitored by TLC. Upon completion, the mixture was concentrated under reduced pressure and underwent preparative HPLC purification to obtain pure activated BURCM as colourless liquid (115 mg, 90%).

[0276] 1H NMR (400 MHz, DMSO-cfe) 6: 8.97 (m, 1 H), 8.75 (d, J = 8Hz, 1 H), 8.42 (d, J = 8Hz, 1 H), 7.97 (m, 4H), 7.85 (m, 1 H), 7.68 (d, J = 8Hz, 1 H), 7.34 (d, J = 8Hz, 1 H), 7.20 (s, 1 H), 6.99 - 7.06 (m, 4H), 6.70 (d, J = 8Hz, 2H), 4.68 (m, 1 H), 4.41 (m, 3H), 3.92 (m, 3H), 3.68 (s, 2H), 3.50 - 3.53 (m, 26H), 3.42 (m, 5H), 3.18 - 3.24 (m, 4H), 3.01 (m, 2H), 2.82 (m, 1 H), 2.46 (m, 2H), 2.04 (s, 3H), 1.96 (m, 1 H), 1.85 (m, 1H).13C NMR (100 MHz, DMSO-cfe) 3: 171.72, 171.54, 169.09, 168.57, 158.85, 157.56, 157.06, 144.57, 142.12, 139.21, 136.60, 131.03, 127.69, 125.02, 124.28, 121.40, 118.97, 118.72, 115.84, 112.95, 111.77, 110.06, 70.23, 70.13, 70.06, 69.42, 69.03, 54.14, 53.92, 52.66, 42.46, 36.65, 32.16, 29.99, 28.13, 15.05. HR-MS (ESI): calcd for [M+Na]+C5SH78F4Ni4Oi4S: 1301.5371 ; found: 1301.5332.

[0277] Synthesis of Fmoc-PY

[0278] Peptide A / -Fmoc-Pro-Tyr(tBu)-Ala-Tyr(tBu)-Trp(Boc)-Met-Arg(Boc)2-Gly-OH (2.51 g, 1.50 mmol) was synthesised by SPPS.

[0279] 1H N MR (400 MHz, DMSO-cfe) 5: 7.93 - 8.16 (m, 6H), 7.82 (m, 5H), 7.60 (m, 2H), 7.49 (m, 1 H),

[0280] 7.44 (s, 1 H), 7.33 (q, J = 8Hz, 2H), 7.22 (m, 3H), 7.14 (m, 1 H), 6.96 (m, 4H), 6.67 (m, 3H),

[0281] 6.45 (d, J = 8Hz, 1 H), 4.49 - 4.57 (m, 2H), 4.19 - 4.32 (m, 5H), 4.02 - 4.10 (m, 3H), 3.91 (m, 1H), 3.59 - 3.76 (m, 4H), 3.52 (m, 3H), 3.02 (m, 2H), 2.81 - 2.86 (m, 4H), 2.65 (t, J = 12Hz, 3H), 2.37 - 2.42 (m, 9H), 1 .93 (m, 7H), 1 .68 (m, 4H), 1 .49 (s, 9H), 1.32 - 1 .38 (m, 3H), 1 .01 - 1.21 (m, 18H), 0.96 (s, 6H), 0.78 - 0.81 (m, 3H).13C NMR (100 MHz, DMSO-cfe) 6: 171.72, 171.20, 171.08, 170.90, 170.78, 153.39, 149.07, 143.81 , 140.81, 134.68, 132.52, 132.10, 130.35, 129.64, 127.74, 127.65, 127.19, 125.29, 124.28, 123.35, 123.27, 123.03, 122.44, 120.19, 120.03, 119.56, 119.51 , 116.34, 114.62, 83.45, 77.51 , 77.29, 67.05, 66.72, 60.25, 59.62, 53.62, 52.27, 51.88, 47.16, 46.61 , 40.65, 36.83, 32.06, 31.17, 29.59, 29.35, 28.51 , 28.33, 27.67, 25.15, 23.74, 22.75, 18.09, 17.74, 14.61. HR-MS (ESI): calcd for [M+H]+CasHiisNizOigS: 1677.8273; found: 1677.8289.

[0282] Synthesis of NH2PEG?-N3 (200 mg, 0.34 mmol), peptide / V-Fmoc-Pro-Tyr(tBu)-Ala-Tyr(tBu)-Trp(Boc)-Met- Arg(Boc)2-Gly-OH (680 mg, 0.41 mmol), HoBT (92 mg, 0.68 mmol), HBTU (258 mg, 0.68 mmol), and DIEA (116 pL, 0.68 mmol) was dissolved in anhydrous THF and allowed to stir for 12h at room temperature. Upon completion, the reaction mixture was concentrated under reduced pressure, and pureFmocPYAYWMRG-PEG7-N3 was obtained as white solid after preparative HPLC purification (530 mg, 70%).

[0283] 1H NMR (400 MHz, DMSO-de) 6: 8.98 (t, J = 6 Hz, 1 H), 8.01 - 8.31 (m, 7H), 7.86 - 7.91 (m, 5H), 7.64 - 7.71 (m, 2H), 7.57 (m, 1 H), 7.54 (s, 1 H), 7.40 (d, 2H), 7.31 (m, 3H), 7.23 (m, 1H), 7.04 (m, 4H), 6.77 (m, 3H), 6.54 (d, J = 8Hz, 1 H), 4.56 - 4.67 (m, 2H), 4.25 - 4.42 (m, 5H), 4.09 - 4.21 (m, 4H), 3.95 - 4.03 (m, 2H), 3.69 (m, 2H), 3.49 - 3.62 (m, 28H), 3.23 (d, J = 4Hz, 2H), 3.12 (m, 2H), 2.91 (m, 4H), 2.73 (m, 2H), 2.42 (m, 3H), 1.97 - 2.01 (m, 6H), 1.63 - 1.82 (m, 5H), 1.57 (s, 12H), 1.44 (m, 2H), 1.39 (m, 2H), 1.23 - 1.28 (m, 3H), 1.10 - 1.20 (m, 18H), 1.05 (m, 6H), 0.87 (m, 3H).13C NMR (100 MHz, DMSO-cfe) 6: 172.44, 172.21 , 171.51 , 171.34, 169.09, 167.45, 157.53, 153.86, 149.50, 144.23, 141.22, 135.16, 132.91, 132.51 , 132.20, 132.04, 130.77, 130.05, 129.11 , 128.17, 127.61 , 125.71 , 125.59, 124.71, 124.50, 123.68, 123.45, 122.87, 120.60, 120.45, 119.92, 116.79, 115.07, 112.89, 83.87, 77.94, 70.24, 69.43, 69.05, 67.90, 67.41, 67.18, 60.75,, 60.09, 54.10, 52.70, 48.89, 48.60, 47.58, 47.09, 46.91 , 39.00, 38.57, 37.29, 37.17, 36.45, 32.36, 32.12, 31.61 , 30.27, 29.93, 28.95, 28.78, 28.44, 28.11 , 24.19, 23.73, 23.17, 22.84, 18.51 , 18.10, 15.05, 14.32, 11.25.

[0284] Synthesis ofN

[0285] The crude product F^^PYA YWMRG-PEG -NSVJSS dissolved in 5% piperidine / DMF (5 ml_) and stirred at room temperature for 2 h with continuous TLC monitoring. Upon completion, the mixture was the reaction mixture was extracted with EtOAc, washed with water and brine. The organic extracts were dried over Na2SC>4 and concentrated under reduced pressure. The crude productNH2PYAYWMRG-PEG?-N3 was directly used in next step without further purification.

[0286] Synthesis of BURCM

[0287] Sodium hyaluronate (500 mg, 0.01 mmol) was dissolved in DI Water and stirred at 0 °C, Dowex® 50WX8-400 ion-exchange resin was slowly added into the reaction mixture to adjust the pH to 5-6. The mixture was filtered and the filtrate was collected, hyaluronic acid was obtained after lyophilization (350 mg, 74%). A mixture of hyaluronic acid (10 mg, 0.026 mmol disaccharide repeats), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride / EDC (192 mg, 0.052 mmol), N-Hydroxysuccinimide / NHS (115 mg, 0.052 mmmol) are dissolved and stirred in 3 mL deionized H2O at room temperature. After stirring for 30 min, a mixture of productNH2PYAYWMRG-PEG7-N3(105 mg, 0.052 mmol) and DIEA (22 pL, 0.13 mmol) in 6 mL DMF was added in dropwise. The reaction mixture was stirred for 48 h, and dialysed against deionized H2O for 96 h before lyophilization. The obtained cruide product (white solid) was then dissolved in 50% TFA / DCM (3 mL) and was allowed to stir at 0 °C for 2 h. Upon completion, the mixture was concentrated under reduced pressure, dialysed against deionized H2O for 96 h and pure BURCM (36 mg, 70%) was obtained after lyophilization.

[0288] 1H NMR (400 MHz, D2O) 0: 6.80 - 7.06 (m, 13H), 4.51 - 4.55 (m, 7H), 3.75 - 3.90 (m, 18H), 3.53 - 3.60 (m, 28H), 3.35 (m, 6H), 3.15 (m, 6H), 2.91 (m, 4H), 2.72 (s, 2H), 2.01 (s, 12H), 1.46 (m, 4H), 1.21 (m, 3H).

[0289] EtALP-Br (32.3 mg, 0.1 mmol) anddihyTz-Alky (40 mg, 0.2 mmol) were dissolved in 3 mL ACN / THF (1 :2) and allowed to stir at room temperature under N2 environment overnight. The reaction was purified by preparative HPLC to affordEtALP-Tz-Alk as a pale-yellow gum (43%: Isomer 1 ; 10%: Isomer 2).

[0290] 1H NMR (400 MHz, Chloroform-d) 6: 7.45-7.53 (m, 5H), 7.29 (m, 2H), 7.16 (s, 2H), 7.42 (m, 2H), 4.21 (qu, J = 7Hz, 4H), 2.47 (m, 4H), 2.04 (s, 1H), 1.35 (t, J = 6Hz, 6H).13C NMR (100 MHz, Chloroform-d) 3: 150.32, 131.75, 129.63, 129.14, 128.71 , 120.14, 115.34, 77.22, 70.60, 64.91, 55.67, 28.81, 16.02, 15.49. HR-MS (ESI): calcd for [M+Na]+C23H27N4O4PNa: 477.1662; found: 477.1664.

[0291] Synthesis ofEtALP-Tz-Az

[0292] EtALP-Tz-Alk (45 mg, 0.10 mmol) and 1 ,4-diazobutane (70 mg, 0.50 mmol) was first dissolved in 3 ml_ DMSO. THPTA (22 mg, 0.05 mmol) and CuSO4-5H2O (25 mg, 0.10 mmol) were premixed in 500 uL water before addition. The reaction was purged with nitrogen gas for 10 mins. Sodium ascorbate (35 mg, 0.20 mmol) dissolved in 500 L of water was next added. The reaction was purged with nitrogen gas for another 10 mins and allowed to stir at room temperature for 1 h. Upon completion, preparative HPLC purification was carried out to afford pureEtALP-Tz-Az as a yellow gum (41 mg, 70%).

[0293] 1H NMR (400 MHz, Chloroform-d) 6: 7.69 (d, J = 8Hz, 2H), 7.53-7.55 (m, 1H), 7.49-7.53 (m, 3H), 7.31 (d, J = 8Hz, 2H), 7.17 (d, J = 8Hz, 2H), 4.33 (s, 2H), 4.22 (m, 4H), 3.31 (t, J = 6Hz, 3H), 2.98 (s, 1 H), 2.74 (s, 1H), 1.97 (m, 2H), 1.56 (m, 2H), 1.36 (m, 8H), 1.25 (s, 2H).13C NMR (100 MHz, Chloroform-d) 3: 150.94, 132.29, 130.71 , 130.12, 129.16, 128.14, 120.33, 65.00, 50.58, 50.07, 28.32, 27.41 , 27.25, 25.74, 16.03. HR-MS (ESI): calcd for [M+Na]+C27H35Nio04PNa: 617.2473; found: 617.2474.

[0294] Synthesis of ALP-Tz-Az

[0295] To a solution ofEtALP-Tz-Az (30 mg, 0.05 mmol) in dry CH2CI2 (5 mL) was added bromotrimethylsilane (68 pL, 0.5 mmol) dropwise at room temperature. The reaction mixture was allowed to stir at room temperature under argon for 2 h and quenched by addition of 2 mL MeOH. The reaction was purified by preparative HPLC to afford ALP-Tz-Alk as a paleyellow gum (8.1 mg, 30%).

[0296] 1H NMR (400 MHz, Methanol-ck) 6: 7.89-7.93 (m, 5H), 7.60 (t, J = 6Hz, 2H), 7.51 (t, J = 8Hz, 3H), 3.11 (m, 4H), 2.70 (m, 2H), 2.02 (m, 2H), 1.60 (m, 2H), 1.31 (m, 4H).13C NMR (100 MHz, Methanol-cL) 5: 132.15, 130.54, 129.20, 128.32, 120.34, 57.14, 50.60, 29.68, 27.30, 25.77. HR-MS (ESI): calcd for [M+Na]+C23H27Nio04PNa: 561.1847; found: 561.1850.

[0297] Synthesis of BURKA

[0298] ALP-Tz-Az (5 mg, 0.01 mmol) and DBCO-PEG2K (20 mg, 0.01 mmol) were dissolved in 2 mL H2O and allowed to stir at room temperature for 2 h. After completion, pure BURKA was obtained as a yellow gum after lyophilization (24.5 mg, 99%).1H NMR (400 MHz, Methanol-^) 6: 7.88-7.92 (m, 1 H), 7.60-7.68 (m, 5H), 7.48-7.52 (m, 5H), 7.27-7.39 (m, 6H), 3.65 (s, 200H), 3.26 (m, 1 H), 3.09-3.15 (m, 1 H), 2.70-2.74 (m, 2H), 2.36- 2.40 (m, 2H), 2.15-2.40 (m, 2H), 2.02-2.03 (m, 2H), 1.31-1.35 (m, 4H).

[0299] Example 3. In vitro detections

[0300] To investigate the optical properties and sensing capability of BUR / BFI pairs, the absorption and FL spectra were measured in the absence or presence of their respective biomarkers.

[0301] Preparation of stock solutions

[0302] BURc, BURK, and activated BURc, activated BURK were dissolved in distilled water to obtain a 5 mM stock solution. BFIc, BFIK were freshly dissolved in DMSO to obtain a 1 mM stock solution. Enzyme stock solutions of cathepsin B (CTSB), -glutamyl transferase (GGT), alkaline phosphatase (ALP), furin, urokinase (uPA), matrix metalloproteinase-2 (MMP2), nitroreductase, caspase-3, alanyl aminopeptidase (AAP), p-galactosidase and p-N- acetylglucosaminidase (NAG) were prepared in distilled water.

[0303] In vitro enzyme incubation

[0304] CTSB (5 pg / mL) and BURc (10 pM) were incubated in 100 pL Tris buffer (20 mM Tris, pH = 7.4, 150 mM NaCI, 1 mM EDTA, 1 mM DTT) at 37 °C. MMP-3 (5 pg / mL) was pre-activated according to protocol (R. K. Harrison et al., Biochem. 31 , 10757 (1992)), before incubation with BURCM (10 pM) in 100 pL Tris buffer (50 mM Tris, pH = 7.5, 10 mM CaCh, 150 mM NaCI, 0.05% (w / v) Brij-35) at 37 °C. After 2 h, the enzyme incubation mixture was transferred to an ultrafiltration tube with molecular cutoff of 6 kDa. After centrifugation at 6,000 rpm for 30 min, the ultrafiltrate was collected and incubated with BFIC(10 pM) for 1 h at 37 °C before UV / vis and FL measurements. NAG (100 mU / mL) and BURK (10 pM) were incubated in 100 pL 1x PBS buffer (pH = 7.4) at 37 °C for 2 h, before 10 pM BFIK was added. ALP (200 mU / mL) and BURKA (10 pM) were incubated in 100 pL Tris buffer (200 mM Tris-HCI, pH = 9.5, 1 mM MgCh) at 37 °C for 2 h, before 10 pM BFIK was added. The mixture was allowed to be further incubated for 1 h before UV / vis and FL measurements of the solutions.

[0305] Enzyme kinetic studies

[0306] Various concentrations of BURc (1 , 2, 4, 6, 8, 10, 20, 40 pM), BURCM (1 , 2, 4, 6, 8, 10, 20, 40 pM), BURK (1, 5, 10, 15, 20, 40, 80, 120 pM), or BURKA (1 , 5, 10, 15, 20, 40, 80, 120 pM), were incubated with CTSB (5 pg / mL), pre-activated MMP-3 (2 pg / mL), NAG (100 mU / mL), or ALP (200 mU / mL) at 37 °C for 30 min in a 100 pL system of Tris buffer (20 mM Tris, pH = 7.4, 150 mM NaCI, 1 mM EDTA, 1 mM DTT), Tris buffer (50 mM Tris, pH = 7.5, 10 mM CaCI2, 150 mM NaCI, 0.05% (w / v) Brij-35), 1x PBS buffer (pH = 7.4), or Tris buffer (200 mM Tris-HCI, pH = 9.5, 1 mM MgCl2), respectively. For BURc and BURCM, the enzyme incubation mixture underwent ultrafiltration and the ultrafiltrate was collected and incubated with BFIc (10 pM) for 1 h before FL measurements of the solutions. A calibration curve of FL signals was plotted against different coumarin concentrations, and the percentage of BFIc conversion was subsequently calculated. For BURK and BURKA, the enzyme cleavage was quenched with methanol before quantitative HPLC analyses. The initial reaction velocity was calculated, plotted against the respective BUR concentrations, and fitted to a Michaelis-Menten curve. The kinetic parameters were calculated using Michaelis-Menten equation: V = V maxX [S] / (Km+ [S]), where V is initial velocity, and [S] is substrate concentration.

[0307] In vitro selectivity studies

[0308] BURc, BURCM, BURK, or BURKA (10 pM) were incubated with the indicated enzymes including CTSB, GGT, furin, uPA, MMP2, nitroreductase, caspase-3, p-Gal, AAP (0.5 pg), ALP (20 mU) and NAG (10 mU) in 100 pL respective enzyme incubation buffers at 37 °C for 2 h. The BURc incubation mixture was then transferred to ultrafiltration tubes and underwent centrifugation at 6,000 rpm for 30 min. Subsequently, BFICor BFIK(10 pM) was incubated with the ultrafiltrate of BURC / BURCM incubation mixture, or BURK / BURKA incubation mixture, respectively. The mixtures were incubated for another 1 h before FL measurement.

[0309] Cell Culture and Confocal Laser Scanning Microscopy (CLSM) Cell Imaging

[0310] Cell lines HK-2, A549, 3T3, 4T1 and NDF cells were cultured in culture medium stated on ATCC website. Cells were maintained in a humidified environment containing 5% CO2 and 95% air at 37 °C, and culture media were replaced every two to three days. For CLSM cell imaging, HK-2, A549, 3T3, 4T1 and NDF cells (6 x 104cells in 1 mL) were seeded in ibidi p- slide 8 well imaging dishes and incubated overnight to reach 80% confluency. Cells were randomly divided into three groups, and cells were treated with saline for the control group. For BFI-treated group, cells were incubated with BFIs (10 pM) for 1 h. For BUR / BFI-treated group, cells were pre-incubated with BURC / BURCM (50 pM) or BURK / BURKA (15 pM) for 3 h before BFI incubation for 1 h. Cells were washed for three times between treatment for different drugs. For BURC< ) / BFIC, cell culture supernatant was collected for all three groups before BFI incubation, and the supernatant ultrafiltrate was incubated with BFIc (10 pM, 1 h) before FL measurement. For BURK / BFIK, the cells were stained with Hoechst 33342 (NucBlue Live ReadyProbes Reagent, Thermo Fisher) for nuclei. Cell FL images were acquired on a Laser Scanning Microscope LSM800 (Zeiss). The excitation and emission wavelengths for cell imaging were 405 / 410-470 nm for both BFIc and Hoechst, 561 / 580-630 nm for BFIK. Imaged software was utilized to remove signal background and quantify cellular FL intensity. Cytotoxicity Assay

[0311] Cell lines HK-2, A549, 3T3, 4T1 and NDF cells were seeded in 96-well plates (104cells per well) and cultured overnight. Different concentrations (0, 10, 20, 50 and 100 M) of BURC / BURCM or BURK / BURKA were added to the cell culture medium and incubated for 24 h, followed by addition of MTS reagent (Promega Cat. no. G3581 , 100 mL, 0.1 mg / mL) for 4 h. The absorbance of MTS at 490 nm was measured by using a microplate reader. The cytotoxic effects (VR) of BURs were assessed using the following equation: VR = A / Ao * 100%, where A and Ao are the absorbance of the experimental group and control group, respectively. The assays were performed in five sets for each concentration.

[0312] Results and discussion

[0313] As shown in Fig. 2a, minimal FL signals were observed for BFIc, because the optically tunable hydroxyl group of coumarin was initially caged by the triphenylphosphine group to diminish the electron-donating ability of the oxygen atom. As the bioorthogonal handle of BURc (perfluoroaryl azide) is always reactive, the enzyme incubation mixture was filtrated to obtain the PEGylated perfluoroaryl azide fragments released from BURc via CTSB cleavage; the ultrafiltrate was then incubated with BFIc, and the Staudinger reaction with BFIChappened, leading to spontaneous generation of activated BFICwith a 27-fold FL enhancement at 450 nm. For BFIK, it was barely fluorescent at its intrinsic state, as the optically tunable hydroxyl group of resorufin was initially caged by the bioorthogonal handle (frans-cyclooctenes) (Fig. 2d). Upon treatment of BFIK with the incubation mixture of BURK and NAG where BURK had activated clickability from NAG cleavage, the I EDDA reaction took place to generate activated BFIK, and a 47-fold FL enhancement at 590 nm. In contrast, the mixture of BFIK and BURK showed minimum fluorescence due to the masked clickability of BURK.

[0314] To investigate the optical properties and sensing capability of BUR / BFI pairs, the absorption and FL spectra were measured in the absence or presence of their respective biomarkers. As shown in Fig. 2a, minimal FL signals were observed for BFIc, because the optically tunable hydroxyl group of coumarin was initially caged by the triphenylphosphine group to diminish the electron-donating ability of the oxygen atom. As the bioorthogonal handle of BURc (perfluoroaryl azide) is always reactive, the enzyme incubation mixture was filtrated to obtain the PEGylated perfluoroaryl azide fragments released from BURc via CTSB cleavage; the ultrafiltrate was then incubated with BFIc, and the Staudinger reaction with BFIc happened, leading to spontaneous generation of activated BFIc with a 27-fold FL enhancement at 450 nm. For BFIK, it was barely fluorescent at its intrinsic state, as the optically tunable hydroxyl group of resorufin was initially caged by the bioorthogonal handle (frans-cyclooctenes) (Fig. 2d). Upon treatment of BFIK with the incubation mixture of BURK and NAG where BURK had activated clickability from NAG cleavage, the I EDDA reaction took place to generate activated BFIK, and a 47-fold FL enhancement at 590 nm. In contrast, the mixture of BFIK and BURK showed minimum fluorescence due to the masked clickability of BU K.

[0315] Dynamic monitoring indicates that the reactions between activated BURs and BFIs completed within 30 minutes, and a gradual increase in fluorescence intensities was observed with biomarker-activated BURs concentrations, with the limits of detection (LCDs) determined to be 5.30 (5.80) and 0.70 (4.00) nM for BURc and BU K in buffer solution (mice urine), respectively (Fig. 3). Besides, HPLC and liquid chromatography-mass spectrometry (LC-MS) studies confirmed that BFIs were totally converted into activated BFIs in the presence of corresponding BURs and biomarkers (Figs. 2b, e and 4). The BUR / BFI pairs increased FL with biomarker concentrations, with the LCDs of 0.05 ng / mL and 0.47 ng / mL determined for CTSB and NAG respectively, and such detection sensitivities are slightly higher than the in vitro detection limits of CTSB (0.17 ng / mL) and NAG (1.02 ng / mL) by ELISA assays (Fig. 5). The catalytic efficiencies of CTSB and NAG towards BURs were calculated to be 2.17 x 102and 1.98 x 104M’1S’1, respectively. Besides, the BUR / BFI pairs showed negligible FL changes in the presence of other interfering enzymes, confirming their high specificity (Figs. 2c, f).

[0316] The BUR / BFI pairs were applied for imaging endogenous biomarkers in living cells after confirming their low cytotoxicity (Fig. 6). BURc was incubated with CTSB-expressing carcinoma epithelial cells including both lung carcinoma cells (A549) and mammary carcinoma cells (4T1), and the control, mouse embryonic fibroblasts (NIH3T3); both cells and supernatant ultrafiltrates were collected for BFIc incubation. However, because only the CTSB-activated BURc released the small PEGylated perfluoroaryl azide fragments that could be collected in the ultrafiltrate of cell supernatant, the supernatant ultrafiltrates of A549 and 4T1 cells showed 3.0, 2.7-fold higher FL signals than that of NIH3T3 cells. The detection specificity was further confirmed by the FL signals comparable to the control groups for cells pre-treated with CTSB inhibitors (CA-074) (Fig. 7). Moreover, BURK was incubated with NAG-expressing kidney proximal tubule epithelial cells (HK-2), while both normal dermal fibroblasts (NDF) and A549 were used as the control (Fig. 2h). After further incubation with BFIK, HK-2 cells showed 7.8, 6.4-fold higher FL signals than NDF and A549 cells, respectively; yet no statistically significant FL enhancement was observed for HK-2 cells pre-treated with NAG inhibitor (Fig. 7). In BFIK- treated cells, some background fluorescence signals from BFIK were observed as compared to saline-treated cells. These results confirmed the feasibility of the BUR / BFI pairs to specifically detect endogenous CTSB and NAG in living cells. Example 4. In vivo pharmacokinetics and excretion

[0317] Biocompatibility, in vivo biodistribution and renal clearance efficiency studies

[0318] Animal experiments were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of the Nanyang Technological University-Institutional Animal Care and Use Committee (NTU-IACUC) and approved by the Institutional Animal Care and Use Committee (IACUC) for Animal Experiment, Singapore. Adult female BALB / c nude mice (18 - 20 g) received intratracheal injection (i.t.) with 25 pL BURC(M>, activated BURC(M>, BURK<A), or activated BURK(A>, and immediately placed in metabolic cages. Urine was collected at 3, 6 and 24 h post-injection, centrifuged at 4,500 r.p.m. for 10 min and filtered by a 0.22 pm syringe filter. After 24 h urine collection, the mice were dissected to collect major organs. The collected organs were randomly divided into two groups for biocompatibility studies and in vivo biodistribution studies. For biocompatibility studies, organs are cut into sections for histological studies. For studies of in vivo biodistribution, organs were homogenized in 1 mL PBS buffer and centrifuged at 4,500 r.p.m. for 15 min to remove insoluble components. For (activated) BURc, (activated) BURCM and activated BURK, the collected urine (20 pL) or tissue homogenates (50 pL) were incubated with BFICor BFIK(10 pM) in 100 pL buffer (1x PBS buffer w 5% DMSO, pH = 7.4) for 1 h before FL measurement. For BURK, the collected urine (20 pL) or tissue homogenates (50 pL) were incubated with NAG (100 mU / mL, 2 h) before further incubation with BFIK (10 pM, 1 h) and FL measurement. A calibration curve of FL signals was plotted against different coumarin / resorufin concentrations, and the percentage of BFIC / BFIK conversion was subsequently calculated. The amounts of BUR in excreted urine or major organs were calculated as percentages of injected dosage.

[0319] BFI urine-incubation and signal readout

[0320] The collected urine was directly used in BFI incubation without further treatment. The collected urine (50 pL) was incubated with BFIc or BFIK (10 pM) in 100 pL buffer solution (1x PBS buffer w 5% DMSO, pH = 7.4) for 1 h before FL measurement.

[0321] Pharmacokinetic studies

[0322] Adult female BALB / c nude mice (16 - 18 g) received intratracheal injection (i.t.) with 25 pL BURc (7.5 mg / kg bw), activated BURc (3.5 mg / kg bw), or BURK (12.5 mg / kg bw) and blood sample (20 pL) was collected from tail vein at 1 , 4, 9, 16, 25, 35, 55, 75, 95, and 120 min postinjection. Blood was sampled in heparinized capillary tubes and stored in an ice box to prevent clotting before centrifugation at 3,500 r.p.m. for 10 min. For BURK, the obtained blood serum (2 pL) was pre-incubated with NAG (100 mU / mL, 2 h). For other probes, the obtained blood serum (2 pL) were incubated with BFIc or BFIK (10 pM) for 1 h before FL measurement. A calibration curve of FL signals was plotted against different coumarin / resorufin concentrations, and the percentage of BFIC / BFIK conversion was subsequently calculated. The amounts of BUR in blood serum were calculated as percentages of injected dosage per gram (%ID g_1) and plotted against post-injection time. The points were then fitted by a bi-exponential decay curve to estimate elimination half-life (t ).

[0323] Intratracheal administration of BURs and urinalysis

[0324] Healthy, tumor-bearing, or kidney-injured BALB / c nude mice were anesthetized by continuous flow of 2-3% isoflurane. Subsequently, mice received i.t. administration of BURc (7.5 mg / kg bw) or BURK (12.5 mg / kg bw) through a catheter, and immediately placed in metabolic cages. Urine was collected at 3 h post-injection, centrifuged at 4,500 r.p.m. for 10 min and filtered by a 0.22 pm syringe filter.

[0325] Histology

[0326] All tissues were fixed with 4% paraformaldehyde (PFA), dehydrated in ethanol solution, embedded in paraffin and cut into sections for histological staining. The paraffin was removed by xylene washing and tissue sections from major organs (5 pm thick) underwent Hematoxylin & Eosin (H&E) staining, followed by washing with distilled water. The stained sections were examined using a Nikon ECLIPSE 80i microscope.

[0327] Results and discussion

[0328] BURs were tested in living mice for pharmacokinetics and biodistribution studies. To demonstrate CTSB-activatable renal clearance, both BURc and activated BURc were injected into living mice via intratracheal (i t.) instillation; their pharmacokinetics and urinary excretion were then compared with a constant injection dosage (6 pmol azide / kg body weight / bw). BURc and activated BURc showed similar pharmacokinetic behaviors, where a rapid absorption was observed within 25 min post-injection (p.i.), followed by elimination with a half-life of 26 (12) min for BURc (activated BURc) (Fig. 8a). At 24 h p.i., up-to 67% injected dosage (ID) of activated BURc was urinary-excreted, yet less than 10% ID of BURc was cleared into urine (Fig. 8b). HPLC analyses identified some activated BURc in the urine from BURc-treated mice, probably due to the basal CTSB expression in healthy lung tissues (Fig. 8c). However, when mice were pre-treated with CTSB inhibitor, traces of activated BURc became absent in the urine from BURc-treated mice. Besides, the activated BURc in excreted urine had the same retention time as the pure compound, indicating minimal in vivo metabolism and urinary excretion in an intact form. To study the nonspecific accumulation of BURs in vital organs, mice were dissected at 24 h p.i., and all vital organs were subsequently harvested and homogenized. The tissue extracts were then incubated with BFIs, and the residual amount of BURs in major organs were quantitatively determined by FL measurement. For BURc, liver contained approximately 58% ID, while other organs contained 0.5 - 5% ID; yet activated BURc showed minimum organ accumulation in liver (4.3% ID) and intestine (3.4% ID), with negligible amounts in other organs (Figs. 8d,e).

[0329] Following i t. injection, BURK (activated BURK) underwent an absorption phase within 35 (31) min p.i., and subsequent elimination with a half-life of 28 (30) min (Fig. 8f). At 24 h p.i., up-to 75% (84%) ID of BURK (activated BURK) was urinary-excreted, and the residual amount of BURK (activated BURK) in major organs were quantitatively determined to be 8% (2%) ID in lung, 5% (4%) ID in liver, 4% (2%) ID in kidney, and negligible amount in other organs (Figs. 8g, i). HPLC analysis confirmed that in healthy mice, urinary-excreted BURK had the same retention time as the pure compound, while traces of activated BURK were absent (Fig. 8h). Histological studies of major organs revealed normal tissue morphology, demonstrating minimal toxicity of BURs (Fig. 9). Besides, the intratracheally injected-BURK showed higher bioavailability, similarly high renal clearance efficiency, and minimal organ accumulation as compared to intravenously injected-BURK (Fig. 10). Furthermore, HPLC studies revealed that both BURs and their activated forms remained stable after incubation with mice urine for 24 hours (Fig. 11).

[0330] Example 5. Cancer detection

[0331] To monitor cancer treatment efficacy, a different bioorthogonal reaction, Staudinger reaction between different BUR / BFI pairs is employed to assure duplex disease detection. Because the reactivity between perfluoroaryl azide on BURc and triphenylphosphine group on BFIc is always on, BURc is designed to specifically react with a cancer-related biomarker, Cathepsin B (CTSB), to release the renal-clearable bioorthogonal handle (perfluoroaryl azide fragment) into urine. In the follow-up ex vivo bioorthogonal reaction, the excreted BURc in the urine is reacted with BFIc via Staudinger reaction for FL turn-on readout.

[0332] The urinary FL enhancement from BFIc (BFIK) in tumor-bearing mice as compared to healthy controls is defined as BOUTc (BOUTK). AS the BOUT signals are associated with the levels of disease-specific biomarkers, BOUTc and BOUTK reveals the degree of cancer growth and kidney injury (Fig. 1b), respectively. Thereafter, the BOUT index is defined as the ratio of BOUTc'1and BOUTK to reveal the overall treatment efficacy-safety profile, providing precision intervention during cancer therapy.

[0333] Establishment of orthotopic lung cancer model Adult male BALB / c nude mice (28 - 30 g) was anesthetized by continuous flow of 2-3% isoflurane. A549 cells (5 x 106cells) were resuspended in 25 pL PBS and administered to the murine lung through a catheter. For the control group, nude mice received administration of 25 pL PBS. For inhibitor treatment group, healthy or tumor-bearing mice received i.t. injection of CA-047 (10 mg / kg) twice for two days before BOUT.

[0334] Chemotherapy for lung cancer treatment

[0335] At seven days after establishing orthotopic lung cancer model, tumor-bearing mice were randomly divided into four groups for chemotherapy that lasts for four weeks. Mice from G1 received weekly i.v. injection of saline. Mice from G2-G4 received weekly i.v. injection of different doxorubicin dosages (2, 5, and 10 mg / kg bw). Mice weight and survival were continuously monitored for 35 days. One day after chemotherapy, mice received i.t. administration of BUR cocktail (10.5 mg / kg bw BURCM and 12.5 mg / kg bw BURK in 50 pL) and immediately placed in metabolic cages. Urine was collected at 3 h post-injection for BFI urineincubation and signal readout. After urine collection, one mice was randomly selected from each group, and the mice was dissected for histological studies of lung and kidney tissues by following the protocol in Example 4.

[0336] Immunofluorescence staining

[0337] Major organs and graft tissues were fixed with 4% PFA, dehydrated with graded sucrose (30%), embedded with OCT, and frozen sectioned at 10 pm thick by a cryostat. The section slides were dried at room temperature for 60 min, washed with PBST (PBS with 0.1% Triton X-100) for three times, blocked by 3% BSA solution at room temperature for 1 h, washed with PBS for three times, incubated with anti-Caspase-3, anti-Cathepsin B, anti-MMP3, or anti-pan Keratin antibodies overnight at 4 °C, washed with PBS for three times, and labelled by goat anti-mouse AF488-conjugated IgG H&L or anti-rabbit IgG AF647-conjugated IgG H&L at room temperature for 1 h. Nuclei were stained with Hoechst. Fluorescent images were visualized and captured using LSM800 confocal laser scanning microscope.

[0338] Results and discussion

[0339] BOUT was applied to detect endogenous biomarker CTSB in an orthotopic lung cancer model (Fig. 12a). BALB / c nude mice received i.t. inoculation of human lung adenocarcinoma cells (A549), and healthy BALB / c nude mice or inhibitor-treated mice were used as the control. At different time points post-tumor implantation (1 , 2, 3, 4, 5, and 6 weeks), they received i.t. injection of BURc, followed by a timed urine collection at 3 h p.i. Subsequently, the urine specimens were incubated with BFIc before direct FL measurement, and the ratio of urine FL from the tumor-bearing mice to that from the control mice was defined as BOUTc (Fig. 12b). At 2 weeks post-implantation, statistically significant enhancement of BOUTc (1 17) was observed, and BOUTc continued increasing afterwards and reached 2.77 at 6 weeks postimplantation, which coincided with elevated CTSB expression as evidenced by immunofluorescence staining (Fig. 13). In contrast, BOUTc from tumor-bearing mice that were treated with CTSB inhibitor (CA-074) did not show statistically significant enhancement.

[0340] The detection time point of clinical assays used to measure serum biomarkers was determined for comparison. The earliest detection time point with statistically significant enhancement was observed at 5 weeks post-implantation for carcinoembryonic antigen / CEA and cytokeratin-19 fragments / CYFRA21-1, showing 2.05, 2.78-fold enhancement, respectively (Figs. 12c, d). The changes of BOUTc post-implantation showed positive correlations with elevations of CEA (Pearson’s r = 0.81) and CYFRA21-1 (Pearson’s r = 0.82) levels (Fig. 14). The diagnostic accuracy of BOUTc was then compared against clinical serum biomarkers via receiver operating characteristic (ROC) exclusion analysis, and quantification of area under curve (AUG) revealed that BOUTc showed the highest predictive power (AUG = 0.98), followed by serum CEA (AUC = 0.80) and CYFRA21-1 (AUC = 0.78) (Fig. 12e). Whole lung section imaging revealed that an average 0.11 , 2.01 mm2tumor nodules were observed in 1 , 2 weeks post-implantation, respectively; and the tumor size continued increasing in the subsequent weeks (Fig. 12g). Correlation studies revealed a positive correlation existed between BOUTc changes and observed tumor area with Pearson's r = 0.98 (Fig. 12f). These results demonstrated that BOUTc detected orthotopic lung tumor at 2 weeks post-implantation with detection limit of ~2 mm2(diameter~1.2 mm), and such detection time point was 3 weeks earlier than detection time points for elevated serum biomarkers (5 weeks post-implantation).

[0341] Example 6. Detection of antineoplastics-induced kidney injury

[0342] Establishment of cisplatin-induced kidney injury model

[0343] Cisplatin (1 mg / mL in 0.9% NaCI solution) was injected once intraperitoneally into Adult female BALB / c nude mice (18 -20 g) at a drug dosage of 15 mg / kg body weight. Age-matched control BALB / c mice were injected with an equal volume of isotonic saline. For inhibitor treatment group, mice were pre-treated with nagstatin (5 mg / kg) before cisplatin challenge.

[0344] Results and discussion

[0345] BOUT was applied to detect disease biomarker NAG in a cisplatin-induced kidney injury model. BALB / c nude mice were challenged with cisplatin (an antineoplastic drug) at a reported nephrotoxic dosage (intraperitoneal injection, 15 mg / kg bw) (Fig. 15a) (Perse, M. & Veceric- Haler, Z., BioMed Res. Int. 2018, 1462802 (2018)). At different drug post-treatment time points (tdpt = 6, 9, 12, 24, 48, 72, and 96 h), mice received i t. injection of BUR and urine was collected within 3 h p.i.. The collected urine was incubated with BFIK for FL measurements, and the ratio of urine FL from the cisplatin-challenged mice to that from the control mice was defined as BOUTK (Fig. 15b). At tdpt = 9 h, statistically significant enhancement of BOUTK (1 -52) was observed, and BOUTK continued increasing at subsequent time points. However, BOUTK didn't show statistically significant enhancement for mice pre-treated with NAG inhibitor (Nagstatin) before cisplatin challenge. Besides, the detection time points of clinical assays measuring serum biomarkers (serum creatinine / sCr, blood urea nitrogen / BUN) and histological studies to evaluate kidney tissue injuries were determined (Figs. 15c, d). At tdpt = 48 h, statistically significant elevations were observed for sCr (1.40-fold) and BUN (1 39-fold). In addition, at tdpt = 72 h, kidney tissue injuries such as renal cell debris, loss of brush border, and hyaline casts were observed (Fig. 15g). The BOUTK changes at different tdpt showed positive correlation with sCr (Pearson’s r = 0.86) and BUN (Pearson’s r = 0.92) levels (Fig. 14). Besides, ROC exclusion analysis suggested that BOUTK (AUG = 1.0) exhibited higher diagnostic accuracy than sCr (AUC = 0.96) and BUN (AUG = 0.97) (Fig. 15e). The NAG ELISA assays showed statistically significant elevations at tdpt = 12 h (Fig. 15f). Therefore, BOUT outperformed NAG ELISA assays in detecting renal NAG, this could be explained by both enzymatically catalyzed signal amplification of BOUTK and dilution effects of kidney NAG after excretion into urine. These results validated that BOUTK detected AKI at 9 h post-cisplatin treatment, which was 3 h earlier than detection time point for NAG ELISA assay (tdpt = 12 h), 39 h earlier than detection time points for elevated serum biomarkers (tdpt = 48 h), and 63 h earlier than kidney tissue injury (tdpt = 72 h).

[0346] Example 7. Concurrent monitoring of efficacy and safety profiles

[0347] Combinational chemotherapy for lung cancer treatment

[0348] Adult female BALB / c nude mice (20 - 25 g) was anesthetized by continuous flow of 2-3% isoflurane. A549 cells (5 x 106cells) were resuspended in 25 pL PBS and administered to the murine lung through a catheter. For the control group, nude mice received administration of 25 pL PBS. At seven days after establishing orthotopic lung cancer model, tumor-bearing mice were randomly divided into four groups for combinational chemotherapy that lasts for four weeks. Mice from G1 received weekly i.p. injection of saline. Mice from G2-G4 received weekly i.p. injection of pemetrexed (100 mg / kg bw) and different cisplatin dosages (2, 5, and 10 mg / kg bw). Mice weight and survival were continuously monitored for 35 days. One day after chemotherapy, mice received i t. administration of BUR cocktail (7.5 mg / kg bw BURc and 12.5 mg / kg bw BURK in 50 pL) and immediately placed in metabolic cages. Urine was collected at 3 h post-injection for BFI urine-incubation and signal readout. After urine collection, one mice was randomly selected from each group, and the mice was dissected for histological studies of lung and kidney tissues.

[0349] Results and discussion

[0350] BOUT was applied for concurrent detection of cancer and treatment-associated kidney injury in cancer therapy (Fig. 16a). After BALB / c nude mice received i.t. inoculation of human lung adenocarcinoma cells (A549) for seven days, they were treated weekly with saline (G1) or combinational chemotherapy including pemetrexed / PEM (dosage: 100 mg / kg) and cis pl atin / C I S at different dosages of 2 (G2), 5 (G3), or 10 (G4) mg / kg for four weeks. PEM / CIS is a combinational chemotherapy commonly employed for lung cancer treatment in the clinical settings. To evaluate the efficacy of combinational chemotherapy, the mice weight and survival were continuously monitored for 35 days. One day post-drug treatment, a cocktail of BURc and BURK was i.t. injected into living mice, and a subsequent urine collection was carried out at 3 h p.i.. The collected urine specimens were incubated with a BFI cocktail (BFICand BFIK) for 1 h before FL measurement. Post-urine collection, mice were dissected to conduct histological studies for lung and kidney tissues. Drug treatment and urine test were not carried out at 4thdosage for G1-G2 due to dropped viability.

[0351] To evaluate cancer treatment efficacy and treatment-associated kidney injury, BOUTCand BOUTK from different treatment groups were quantitatively studied. The first statistically significant enhancement of BOUTc was respectively observed at 2nd(1.77-fold), 2nd(1.51-fold), 4th(1.31 -fold) and 4thdosage (1.33-fold) for GI to G4, and it continued increasing at 3rddosage for G1-G2 (Fig. 16b). Meanwhile, the first statistically significant enhancement of BOUTK was observed at 3rd(1.93-fold), 3rd(1.64-fold), 2nd(1.74-fold) and 1stdosage (1.96-fold) for G1 to G4, respectively (Fig. 16c). A positive correlation was observed between BOUTc changes and observed tumor area with Pearson's r = 0.73 (Fig. 17). The first detection time points of preclinical assays for lung cancer-related serum biomarkers (CEA, CYFRA21-1) were observed at the 2nddosage for all treatment groups; and assay kits to detect kidney injury- related serum biomarkers (sCr, BUN) showed statistically significant elevations from 1stdosage onwards for G4 only (Fig. 18). Furthermore, histological studies revealed that obvious lung tumor nodules were firstly observed at 2nddosage for G1-G2, 3rddosage for G3-G4, and the tumor area continued increasing at subsequent time points (Figs. 19-20). Meanwhile, histological evidence of kidney injuries was observed at 4thdosage for G3, 3rdand 4thdosage for G4. Thus, BOUT achieved sensitive detection and detected the onset of lung tumor growth and Al KI at an early time point, which was comparable to or earlier than elevations of serum biomarkers and histological evidence. The diagnostic accuracy of BOUT was then compared against serum biomarkers in the respective detection of orthotopic lung cancer and Al KI. As shown in Figs. 16f,g, quantification of AUC revealed that both BOUTc and BOUTK exhibited higher detection accuracy (AUC = 1.0) than serum cancer biomarkers (CYFRA21-1 AUC = 0.97; CEA AUC = 0.93) and serum kidney injury biomarkers (BUN AUC = 0.95; sCr AUC = 0.94). To demonstrate the capability of BOUT to distinguish patient disease states, a machine learning algorithm for support vector machine (SVM) classifier was created for diagnostic purposes. The BOUT panel (BOUTc and BOUTK) was applied to differentiate mice among all four states based on histological studies: healthy mice, mice with observed lung tumor nodules, mice with observed renal injuries, and mice with both observed lung tumor nodules and renal injuries; 70% of data was used for training and 30% for prediction. As shown in Fig. 16h, the SVM classifier proved that BOUT achieved patient segregation with high accuracy, which was also confirmed with principal component analysis (Fig. 21).

[0352] After confirming the detection accuracy of BOUTs (BOUTc and BOUTK) towards orthotopic lung cancer and AIKI respectively, the tumor inhibition effects, and organ toxicity could be respectively reflected by BOUTc'1and BOUTK (Fig. 16i). The safe CIS dosage in combinational therapy is determined to be between ED50 (median effective dosage = 3.2 mg / kg) and TD50 (median toxic dosage = 7.5 mg / kg). This coincides with mice experiments, as mice in G3 (CIS dosage = 5 mg / kg) exhibited prolonged survivals and minimum weight loss among four groups (Figs. 16d,e). Besides, quantitative study of the overall treatment outcomes by benefit-cost ratio (BOUT index = BOUTC'VBOUTK) showed that mice in G3 had outstanding treatment outcomes out of four groups (Fig. 16j and 21). The BOUT index also showed strong positive correlation with mice weight gain (Pearson’s r = 0.70), total lifetime (Pearson’s r = 0.74), and viability (Pearson’s r = 0.80), yet BOUTc or BOUTK alone failed to show strong correlation with these crucial factors (Figs. 16k, I and 22). Besides, the BOUT index showed negative correlation with observed tumor area (Pearson's r = -0.65).

[0353] Last but not least, BOUT provides a general platform for biomarker detection, which was demonstrated by developing BOUTCM and BOUTKA to detect matrix metalloproteinase-3 (MMP-3) and alkaline phosphatase (ALP), which are other biomarkers highly expressed in lung cancer and antineoplastic-induced kidney injury, respectively (Figs. 23-26). The pair of BOUTCM / BOUTK was then applied for concurrent monitoring of efficacy and safety profiles in tumor-bearing mice receiving different dosages of doxorubicin (a FDA-approved chemodrug for lung cancer). While the tumor inhibition effects and treatment-associated chronic kidney diseases could be measured by BOUTCM'1and BOUTK, the BOUTM index (BOUTCM'VBOUTK) reflected the overall treatment outcomes, showing positive correlation with mice weight gain (Pearson's r = 0.66), viability (Pearson's r = 0.70), and total lifetime (Pearson's r = 0.85) (Figs. 27-28). These results suggested that both BOUT and BOUTM indexes could be potentially applied for evaluation of drug therapeutic index and examination of an overall efficacy-safety profiles in the cancer treatment regimen.

[0354] Example 8. Handphone-assisted BOUT for point-of-care patient monitoring

[0355] With advantages of high availability, affordability, rapid and easy-to-use, POC testing has revolutionized clinical diagnosis by empowering patients to monitor their health status more frequently in the convenience of their home. Thus, the potential of using BOUT for handphone- assisted detection was tested.

[0356] Handphone-assisted signal readout

[0357] The collected urine at 3rddosage in combinational chemotherapy (50 pL) was incubated with BFIc or BFIK(10 pM) in 100 pL buffer solution (1x PBS buffer w 5% DMSO, pH = 7.4) for 1 h before handphone-assisted signal readout. The portable device was implemented on an IOS- driven smartphone and mobile phone camera with 12 MP resolution was employed to capture images. For BFIC, an external UV lamp (365 nm) was used for excitation, and images were acquired by smartphone in a dark room. For BFIK, an excitation filter (550 ± 2 nm, 120 mm diameter) and an emission filter (590 ± 2 nm, 120 mm diameter) are collocated with phone LED and camera, respectively. After putting the samples inside the chamber, images are directly captured using smartphone and no external light sources are required. The signals in acquired images were analysed and quantitatively determined with Imaged.

[0358] Results and discussion

[0359] The handphone-assisted signal readout is facilitated by a portable module with excitation / emission filters, which could be installed onto handphone by a simple plug-and-play assembly (Fig. 29a). To test the accuracy of POC signal readout, BFIs were titrated with different concentrations of activated BURs, the POC signal readouts were then correlated with the spectrometer FL signals from these samples, and positive correlations with Pearson's r = 0.91-0.94 were found (Figs. 29b, c and 30). Then, the module was applied to test the urine samples from different treatment groups at 3rddosage (Fig. 16a), and the ratio of urine POC signal from different treatment groups to that from healthy mice was defined as POC BOUTc (BOUTK). AS shown in Figs. 29d,e, statistically significant enhancement of POC BOUTc was observed for G1 (2.02-fold), and G2 (1.38-fold). Meanwhile, the statistically significant enhancement of POC BOUTK was observed for G4 (2.24-fold), and the other groups showed no statistically significant difference from healthy mice. To quantitatively study the overall treatment outcomes, POC BOUT index (POC BOUTc' POC BOUTK) was calculated and mice from G3 exhibited the highest POC BOUT index among all four groups (Fig. 29f), which coincided with optical urinalysis results from spectrometer FL signals (Fig. 16j). Hence, with advantages of high detection accuracy, portability, rapid and easy-to-use, BOUT can be potentially used for POC testing to monitor patients' efficacy-safety profiles during the cancer treatment regimen.

[0360] Comparative Example 1 Table 1. Comparison of imaging components and diagnostic time in bioorthogonal chemistrybased in vivo detection.

[0361] ^■ Targeting molecule and imaging agent are administered via intravenous injection, unless otherwise stated.[blThe diagnostic time is presented as the time gap between two administrations + imaging period. Comparative Example 2

[0362] Table 2. Comparison of enzyme detection sensitivities in commercial assays and preclinical studies.

[0363] N.R.: No report.

[0364] Comparative Example 3 Table 3. Comparison of lung tumour detection limit in clinical in vivo / ex vivo approaches.

[0365] N.R.: No report.

[0366] Comparative Example 4 Table 4. Comparison of detection time point for cisplatin-induced kidney injury (15 mg / kg unless otherwise stated) in preclinical in vivo / ex vivo approaches. [a|Mice were treated with cisplatin at a dosage of 20 mg / kg bw.[blMice were treated with cisplatin at a dosage of 12 mg / kg bw.

[0367] Conclusion

[0368] Bioorthogonal chemistry holds great promise for in vivo optical imaging and diagnosis. However, the current approaches face the challenges such as long detection time (due to the requirements of injection of two components and in-situ in vivo reaction), poor specificity (due to the biomarker-independent reaction), and incapability to detect deep-seated diseases (due to the shallow optical penetration). In contrast, BOUT solved these issues by delivering only one bioorthogonal handle (BUR) into the living body to react with the biomarkers-of-interest and convert the level of biomarkers into the reactive reporters in urine for further ex vivo bioorthogonal reaction and signal readout. This hybrid in vivo / ex vivo detection approach eliminates the need of in-situ bioorthogonal reaction at disease site, significantly reducing the diagnostic time from >26 h for the two-step pre-targeting approach to 4 h (Table 1). BOUT also showed at least 2-fold higher detection sensitivity towards biomarker-of-interests than traditional biomarker-activatable fluorescent probes or ELISA assays in solution (Table 2). As the detection specificity is determined by biomarker-triggered clickability or renal clearable ability of bioorthogonal handles (BURs) injected into the living body, BOUTCor BOUTK achieved high diagnostic accuracies in mice bearing orthotopic lung cancer or AIKI where respective biomarkers-of-interest are highly expressed. Owing to the ex vivo optical readout in urine, BOUT bypasses the optical tissue limitation to detect deep-seated diseases. Meanwhile, utilization of intrinsically non-fluorescent fluorophores caged by bioorthogonal handles to react with the corresponding bioorthogonal handles excreted in urine resulted in the FL turn-on signal readout, which further ensures high signal to background ratio and thus high sensitivity.

[0369] Urine test for dynamic and longitudinal monitoring of cancer treatment efficacy and safety profiles are highly desired but remain challenging due to lack of suitable urinary biomarkers. On the basis of "in vivo"-“ex vivo" bioorthogonal chemistry approach, BOUT led to the ultrasensitive and specific urine test of orthotopic lung cancer and AIKI in mouse models. In orthotopic lung cancer mice model, BOUTc detected orthotopic lung tumor at two weeks posttumor implantation with tumor diameter down to ~1.2 mm; such detection time point was at least 3 weeks earlier than elevations of serum biomarkers (CEA, CYFRA21-1). Besides, the detection sensitivity of BOUTc surpassed the detection limit of clinical approaches to detect lung cancer, such as low-dose computed tomography (2-5 mm), white light bronchoscopy (-5 mm) or chest radiography (1 cm) (Table 3). In AIKI mice model, BOUTK detected the disease onset at 9 h post-cisplatin treatment, which was 3 h earlier than NAG ELISA assay, 39 h earlier than elevations of serum biomarkers (creatinine, BUN), and 63 h earlier than histological injuries or other literature reports (Table 4). Besides, AUC quantification of ROC exclusion analysis showed that BOUT has the highest predictive power (BOUTC / BOUTK AUC = 0.98 / 1.00) than lung cancer-related serum biomarkers (0.78, 0.80) or kidney injury-related serum biomarkers (0.96, 0.97), proving its superb specificity. In concurrent monitoring of treatment efficacy and safety profiles during cancer combinational chemotherapy, the BOUT index (BOUTC VBOUTK) provides a comprehensive assessment of the overall health status of mice throughout the treatment journey. This is confirmed by the positive correlation between BOUT index and weight gain (Pearson’s r = 0.70), lifetime (Pearson’s r = 0.74), and viability (Pearson’s r = 0.80), and similar trends are also observed for handphone-assisted BOUT.

[0370] A first kind of bioorthogonal chemistry-based urine test (BOUT) for concurrent monitoring of cancer treatment efficacy and treatment-associated kidney injury in cancer treatment regimen has been reported. Such a hybrid approach takes advantage of biomarker-activated bioorthogonal reactivity or renal clearance and bioorthogonal-triggered fluorescence turn-on to possess high specificity, superb sensitivity and rapid detection. Coupled with handphone- assisted optical readout, BOUT provides a non-invasive way for high-throughput drug screening in preclinical studies, and holds potential for convenient monitoring of the treatment efficacy-safety profile during cancer therapy in the POC settings. Moreover, BOUT serves as a general platform for duplex biomarker detection, provides opportunities for real-time monitoring of multiple physiopathological processes through convenient urine tests with no tissue penetration limit. The present disclosure thus not only opens a new approach towards urine tests but also facilitates the application of bioorthogonal chemistry in disease diagnosis.

Claims

Claims1. A bioorthogonal urinary reporter for cancer detection (BURC) having formula I:[A]-[B-C]n’ where:A is a polymeric material selected from:the squiggly line represents the point of attachment to the rest of the molecule, optionally wherein n’ is from 3 to 160;B represents a cancer-associated biomarker-responsive peptide selected from:where peptide structures are drawn from N-terminal to C-terminal, Ri denotes the point of attachment to A, and R2 denotes the point of attachment to C;X is from 1 to 11 , where the squiggly line is the point of attachment to the rest of the molecule, or a pharmaceutically acceptable salt or solvate thereof.

2. The BURc according to Claim 1 , wherein the BURCis:optionally where n is from 100 to 160, or a pharmaceutically acceptable salt or solvate thereof.

3. A bioorthogonal urinary reporter for treatment-associated kidney injury of formula II:[D]a-[E]-[F] where a is 1 or 2; where D is a renal clearance moiety selected from:where PEG represents a polyethylene glycol chain having a number average molecular weight of from 1 ,000 to 5,000 Daltons; m is from 10 to 100; n” is from 20 to 130; the squiggly line represents the point of attachment to the rest of the molecule;E is a bioorthogonal chemistry-based reporter moiety selected from:where R represents a point of attachment to F and i represents a point of attachment to D;F is a biomarker-cleavable moiety selected from:where the squiggly line represents the point of attachment to the rest of the molecule, or a pharmaceutically-acceptable salt or solvate thereof.

4. The bioorthogonal urinary reporter for treatment-associated kidney injury according to Claim 1, wherein the bioorthogonal urinary reporter for treatment-associated kidney injury is:, where PEG is a polyethylene glycol moiety having a number average molecular weight of about 2 kDa, or a pharmaceutically-acceptable salt or solvate thereof.

5. A bioorthogonal fluorescence indicator selected from:where Ri represents H or a group selected from:and X represents halogen atoms (F / CI / Br / l) , or a pharmaceutically-acceptable salt or solvate thereof.

6. A kit of parts for cancer detection, comprising:(a) a bioorthogonal urinary reporter for cancer detection (BURC) according to Claim 1 or Claim 2, or a pharmaceutically-acceptable salt or solvate thereof; and(b) a bioorthogonal fluorescence indicator according to Claim 5 or a pharmaceutically- acceptable salt or solvate thereof.

7. The kit of parts according to Claim 6, wherein the bioorthogonal urinary reporter for cancer detection (BURC) is the compound according to Claim 2, or a pharmaceutically- acceptable salt or solvate thereof.

8. The kit of parts according to Claim 6 or Claim 7, wherein the bioorthogonal fluorescence indicator i9. A kit of parts for detecting treatment-associated kidney injury, comprising:(i) a bioorthogonal urinary reporter for treatment-associated kidney injury according to Claim 3 or Claim 4 or a pharmaceutically-acceptable salt or solvate thereof;(ii) a bioorthogonal fluorescence indicator according to Claim 5 or a pharmaceutically- acceptable salt or solvate thereof.

10. The kit of parts according to Claim 9, wherein the bioorthogonal urinary reporter for treatment-associated kidney injury is the compound according to Claim 4, or a pharmaceutically-acceptable salt or solvate thereof.

11. The kit of parts according to Claim 9 or Claim 10, wherein the bioorthogonal °Y^ INA °0.i fluorescence indicator is12. A kit of parts for cancer detection and for detecting treatment-associated kidney injury, comprising:(ai) the kit of parts according to any one of Claims 6 to 8; and(aii) the kit of parts according to any one of Claims 9 to 11 .

13. A method for detecting cancer in a subject, the method comprising the steps of:(bi) administering to a subject suspected of having cancer, a bioorthogonal urinary reporter for cancer detection (BURC), or pharmaceutically acceptable salt thereof, according to Claim 1 or Claim 2 and, after a period of time, collecting urine from said subject; and(bii) adding a bioorthogonal fluorescence indicator configured to react with an activated renal clearable compound released from the bioorthogonal urinary reporter for cancer detection (BURC) and detecting the presence of cancer if fluorescence is detected due to reaction of the activated renal clearable compound with the bioorthogonal fluorescence indicator.

14. The method according to Claim 13, wherein the bioorthogonal urinary reporter for cancer detection (BURC) is the compound according to Claim 2, or a pharmaceutically- acceptable salt or solvate thereof.

15. The method according to Claim 13 or Claim 14, wherein the bioorthogonal fluorescence indicator i16. A method for detecting treatment-associated kidney injury in a subject, the method comprising the steps of:(ci) administering to a subject suspected of having treatment-associated kidney injury, a bioorthogonal urinary reporter for detecting treatment-associated kidney injury, or pharmaceutically acceptable salt thereof, according to Claim 3 or Claim 4 and, after a period of time, collecting urine from said subject; and(cii) adding a bioorthogonal fluorescence indicator configured to react with an activated renal clearable compound released from the bioorthogonal urinary reporter for treatment- associated kidney injury and detecting the presence of renal injury if fluorescence is detected due to reaction of the activated renal clearable compound with the bioorthogonal fluorescence indicator.

17. The method according to Claim 16, wherein the bioorthogonal urinary reporter for treatment-associated kidney injury is the compound according to Claim 4, or a pharmaceutically-acceptable salt or solvate thereof.

18. The method according to Claim 16 or Claim 17, wherein the bioorthogonal fluorescence indicator is19. A method for cancer detection and for detecting treatment-associated kidney injury, comprising:(di) the method according to any one of Claims 13 to 15; and(dii) the method according to any one of Claims 16 to 18.

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