Chemiluminescent array-based assay for rapid identification of bacteria and method of use

Abstract

The present invention provides a method for rapid detection and identification of a pathogenic bacteria in an aqueous medium, by creating a unique enzymatic activity fingerprint of said bacteria using an array of chemiluminescent probes each composed of a phenyl-dioxetane luminophore masked by a caging group cleavable by a bacterial analyte or enzyme; and a computing system for implementing said method.

Description

CHEMILUMINESCENT ARRAY-BASED ASSAY FOR RAPID IDENTIFICATIONOF BACTERIA AND METHOD OF USETECHNICAL FIELD

[0001] The present invention provides a method for rapid classification and identification of a pathogenic bacteria in an aqueous medium, using an array of chemiluminescent probes each composed of a phenyl-dioxetane luminophore masked by a caging group cleavable by a bacterial analyte or enzyme; and a computing system for implementing said method.BACKGROUND ART

[0002] Accurately identifying microorganisms and comprehensively characterizing their diverse attributes in a reproducible, rapid, and cost-effective manner presents a substantial challenge (Maugeri et al., 2019). Rapid and precise identification and characterization of pathogens are crucial to ensure accurate diagnosis, effective treatment, and the prevention of outbreaks in clinical, industrial, and agricultural settings.

[0003] Clinical microbiology laboratories endeavor to attain precise bacterial identification and determine drug susceptibility profiles. This empowers physicians to make informed decisions swiftly regarding optimal treatment options. The progression of the COVID-19 pandemic has highlighted the pivotal role of research and clinical microbiology laboratories in uncovering the sources and modes of transmission of outbreaks. This essential information is paramount for effectively controlling infectious diseases and averting their widespread dissemination and recurrence.

[0004] In recent decades, the field of pathogen identification has experienced significant advancement through the introduction of innovative analytical methods reliant on modern technologies and equipment. Among these advancements, next-generation sequencing has transformed microbial diagnostics by enabling unbiased and hypothesis-free detection of a wide spectrum of microbial pathogens, both common and rare, without the necessity for prior culture growth. Another method gaining increasing prominence in clinical microbiology laboratories is mass spectrometry-based techniques, with a particular focus on matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). This method has demonstrated its effectiveness in identifying pathogenic bacteria, including microaerobes, anaerobes, mycobacteria, and fungi. Its extensive and robust database, coupled with exceptional performance, guarantees rapid and precise outcomeswithout the necessity for highly specialized laboratory personnel to oversee the procedure. Nonetheless, it is worth noting that this technology has limitations; it can solely identify isolates if the measured spectrum contains peptide mass fingerprints of the pathogen found in a given database, which can diminish its effectiveness when dealing with newly emerging pathogens. It is also important to highlight that molecular microbiology methods like 16S sequencing and MALDI-TOF do not offer insights into specific pathogenic strain properties, such as drug resistance or virulence levels. Finally, the widespread adoption of sequencing and mass spectrometry technologies faces obstacles due to their substantial instrumentation costs and infrastructure requirements. These requirements include a stable power supply, maintenance by qualified technicians, a dust-free environment, and a stable climate. As a result, a significant percentage of the world's population still faces barriers to accessing these advanced technologies.

[0005] Traditional practices, such as assessing Gram stain and colony morphology, utilizing acid-fast stains, and conducting spot indole oxidase tests, continue to be widely used around the globe. Nonetheless, owing to the immense diversity of the microbiome and the evolving comprehension of microbiological pathogenicity, these methods frequently lack the precision needed for definitive pathogen identification. Conventional identification processes, following streaking procedures, are time-consuming, spanning from hours to days, contingent upon the type of pathogen involved. As an illustration, diagnosing infectious diarrhea via bacterial culture and identification in stool samples usually demands 3-5 days, rendering it a complex and expensive procedure with limited applicability for point-of-care treatments.

[0006] To tackle these challenges, numerous clinical microbiology labs have adopted FDA- or EMA-approved biochemical kits, i.e., the Analytical Profile Index (API), for the identification of bacterial pathogens (Smith et al., 1972). These kits entail a series of complementary tests conducted sequentially, guided by a flowchart-based identification procedure. The API identification process is initiated with Gram staining, an oxidase test, and a fermentation test. Depending on the initial findings, the appropriate API kit is chosen for the ultimate identification and categorization of the pathogen. As an example, in the API 20E kit, a plastic strip featuring twenty mini-test chambers containing dehydrated media is utilized, with specific probes employed for each mini-test. The metabolic activities of the introduced organisms, including processes such as carbohydrate fermentation or protein catabolism, trigger color changes during incubation. These color changes are subsequentlymatched with profile numbers in a commercial codebook (or online resource) to ascertain bacterial species identification. This colorimetric identification process usually necessitates an incubation period of 18-24 hours, although certain tests may extend beyond this timeframe. Another widely employed method for identifying microbial pathogens involves assessing carbon-source utilization profiles. In this approach, a kit featuring a 96-well plate containing diverse carbon sources is utilized. Like the API method, carbon source profiling is time-consuming, owing to the essential incubation period for obtaining discernible results and the necessity of choosing the suitable growth conditions for the unidentified bacterial pathogen within the sample.

[0007] To address the challenges of existing identification methodologies, researchers around the world, including ourselves, have developed a remarkably sensitive and expeditious method for classifying and characterizing bacterial pathogens using chemiluminescent agents (Roth-Konforti et al., 2019; Babin et al., 2021). In contrast to fluorescence, chemiluminescence presents distinct advantages, including the absence of the need for external light irradiation. This characteristic leads to an exceptionally minimal background signal and heightened sensitivity, setting it apart from fluorescence-based and colorimetric assays.

[0008] A fascinating category of chemiluminescent compounds, referred to as triggerable phenoxy-dioxetanes, has drawn specific attention (Fig. 1A). This feature enables the linkage of light emission to the actions of specific analytes by including a suitable phenol-protecting group as a triggering substrate. Nevertheless, despite their promise, the limited emission intensity of these dioxetane compounds in aqueous solutions has rendered them unsuitable for utilization in biological assays conducted in such settings (Schaap et al., 1987). To overcome this hurdle, one of our groups has achieved a significant breakthrough. It was found that incorporating an acrylate substituent at the ortho position of a phenoxy- adamantyl-l,2-dioxetane diminishes the water-quenching effect on the excited benzoate intermediate (Fig. IB) (Green et al., 2017). Furthermore, this alteration enhances the lightemission intensity of the chemiluminescence luminophore by up to 3 orders of magnitude in comparison to the original phenoxy-dioxetanes (Fig. 1C). This pivotal advancement has empowered researchers to employ chemiluminescence probes in aqueous solutions without the necessity for additives, thereby broadening their scope for potential applications in biological studies.

[0009] WO 2019 / 224338 discloses a chemiluminescent probes consisting of a phenoxy- adamantyl-l,2-dioxetane based on the one disclosed in Green el al. (2017), wherein the pi- system of the central aromatic ring is extended by conjugating a cyclic or heterocyclic structure, formed based on the carbon atoms at the meta and either ortho or para positions of said phenoxy-adamantyl-l,2-dioxetane, rather than incorporating an acrylate substituent at the ortho position of said phenoxy-adamantyl-l,2-dioxetane.

[0010] WO 2024 / 147125 discloses additional phenoxy 1,2-dioxetane luminophores having a spiro-cyclobutyl structural motif, in which the adamantyl linked to the 1,2- dioxetane is replaced by an optionally substituted 3-4 membered carbocyclyl or heterocyclyl such as cyclobutyl, oxetan-3-yl, azetidine-3-yl, and thietane-3-yl. The chemiluminescent probes disclosed are said to be superior to the parent phenoxy-adamantyl-l,2-dioxetanes, with a higher signal-to-noise ratio and detection sensitivity.

[0011] US Provisional Application No. 63 / 564,881, filed March 13, 2024 (of the present applicant), discloses further phenoxy 1,2-dioxetane luminophores, said to have a significant chemiexcitation acceleration effect of dioxetanes induced by spiro-fused six-member rings with inductive electron- withdrawing properties. Specifically disclosed are 1,2-dioxetane luminophores in which the adamantyl linked to the 1,2-dioxetane is replaced by an optionally substituted 6 membered carbocyclyl or heterocyclyl such as cyclohexyl, tetrahydropyranyl (oxanyl) or dioxanyl, tetrahydrothiopyranyl (thianyl), 1-oxidotetrahydrothiopyranyl or 1,1- dioxidotetrahydrothiopyranyl, and piperidinyl or piperidinyl- 1 -oxide, but excluding unsubstituted cyclohexyl.SUMMARY OF INVENTION

[0012] The present invention relates to a novel and general approach for the rapid classification and identification of bacterial pathogens, and in fact harnesses the power of ultra-sensitive chemiluminescent probes to create unique enzymatic activity fingerprints. These fingerprints offer a rapid and accurate tool for characterizing key bacterial enzymatic activities that can be used for the identification of diverse bacterial pathogens. Notably, the enzymatic activity fingerprints of unidentified bacteria provide crucial insights into their primary enzymatic activities, which is pivotal for the characterization and documentation of such bacteria.

[0013] In one aspect, disclosed herein is a method for detection and identification of a bacteria in an aqueous medium, comprising:(i) providing an array comprising a plurality of chemiluminescent probes, each composed of a phenyl-dioxetane luminophore masked by a caging group cleavable by a bacterial analyte or enzyme, wherein each one of said phenyl-dioxetane luminophores is masked with a caging group cleavable by a different bacterial analyte or enzyme, and upon exposure to said bacterial analyte or enzyme, said caging group is cleaved from said phenyl-dioxetane luminophore thereby generating an unstable phenolate-dioxetane compound, which is then decomposed through a chemiexcitation process to produce an excited intermediate that decays to its ground-state through emission of light;(ii) applying said aqueous medium to each one of said plurality of chemiluminescent probes of said array (i.e., exposing each one of said plurality of chemiluminescent probes of said array to said aqueous medium);(iii) detecting the emission of light from each one of said chemiluminescent probes, thereby generating a normalized metabolic activity profile for said bacteria, said normalized metabolic activity profile representing the quantity and intensity of emission of light detected from each one of the chemiluminescent probes; and(iv) applying via a processor one or more data analysis methods, e.g., statistical methods and / or machine learning models, on said normalized metabolic activity profile to identify said bacteria.

[0014] In certain embodiments, the phenyl-dioxetane luminophores composing said array each independently is a compound of formula I as defined hereinbelow and according to any one of the embodiments referred to.

[0015] In another aspect, disclosed herein is a computing system comprising: at least one processor; and at least one memory communicatively coupled to the at least one processor comprising computer-readable instructions that when executed by the at least one processor cause the computing system to implement a method for detection and identification of a bacteria in an aqueous medium, the method comprising:(i) providing an array comprising a plurality of chemiluminescent probes, each composed of a phenyl-dioxetane luminophore masked by a caging group cleavable by bacterial analyte or enzyme,wherein each one of said phenyl-dioxetane luminophores is masked with a caging group cleavable by a different bacterial analyte or enzyme and upon exposure to said bacterial analyte or enzyme, said caging group is cleaved from said phenyl-dioxetane luminophore thereby generating an unstable phenolate-dioxetane compound, which is then decomposed through a chemiexcitation process to produce an excited intermediate that decays to its ground-state through emission of light;(ii) applying said aqueous medium to each one of said plurality of chemiluminescent probes of said array (i.e., exposing each one of said plurality of chemiluminescent probes of said array to said aqueous medium);(iii) detecting the emission of light from each one of said chemiluminescent probes, thereby generating a normalized metabolic activity profile for said bacteria, said normalized metabolic activity profile representing the quantity and intensity of emission of light detected from each one of the chemiluminescent probes; and(iv) applying via said at least one processor one or more data analysis methods, e.g., statistical methods and / or machine learning models, on said normalized metabolic activity profile to identify said bacteria.BRIEF DESCRIPTION OF DRAWINGS

[0016] Figs. 1A-1C show the structures and chemiexcitation pathways of Schaap’s dioxetane-based probes (1A) and ortho-substituted phenoxy- 1,2-dioxetane probes (IB); as well as the chemical structure, chemiluminescent kinetic profile, and total light emitted (TLE) by a phosphatase-triggered chemiluminescent probe (10 μM ) with or without commercial alkaline phosphatase (ALP; 1 U / mL) in PBS at pH 7.4, 0.1% DMSO, 37°C (1C) (ES, enzymatic substrate; EWG, electron-withdrawing group; S / N, signal-to-noise).

[0017] Figs. 2A-2C show the structures of the chemiluminescent (CL)-, fluorescent (FL)- , and colorimetric (CM)-phosphatase probes (2A); and the limit of detection (LOD) of said phosphatase probes (in concentration of 10 μM , 10 μM , and 100 μM , respectively) in the presence of a 5-fold serial dilution of (2B) commercial alkaline phosphatase (ALP; 0.1-2.56 x 10’7U / mL) and (2C) S. aureus ATCC 29213 (0.4-2.56 x 10’5OD6oo) in PBS pH 7.4, 0.1% DMSO, 37°C.

[0018] Figs. 3A-3B show the general synthetic pathway for the preparation of the chemiluminescent probes disclosed (3A); and the substrate specificity evaluation of the 10 bacterial enzymatic activity sensing probes (10 μM) in the presence of the commercially available probe activating enzymes / / -glucosidase (10 U / mL), β-glucuronidase (1 U / mL), β- galactosidase (1 U / mL), pyroglutamyl-peptidase I (0.05 mg / mL), alkaline phosphatase (1 U / mL), aminopeptidase-M (1 U / mL), nitroreductase (1 mg / mL, 100μM NADH), NAD(P)H quinone oxidoreductase (NQO1; 0.8 mg / mL, 100 μM NADH), / / -lactamase (2 U / mL), and penicillin-G amidase (PGA; 1 U / mL) (3B).

[0019] Figs. 4A-4B show profiling of the bacterial enzymatic activities with chemiluminescent probes. (4A) Schematic representation depicting the bacterial identification process, exemplified by the chemiluminescent enzymatic profile of Streptococcus pyogenes ATCC 14289. (4B) Application of the 1-N-N method to analyze the database, successfully distinguishing between all 29 strains in the panel (TLE, total light emission; CI, clinical isolate).

[0020] Figs. 5A-5B show evaluation of the enzymatic activity fingerprint of bacterial mixtures. (5A) Log total light emission profiles of the chemiluminescent array of E. coli ATCC 25922 (upper) and S. aureus MRSA ATCC 33592 (lower). (5B) Log total light emission fingerprints of the chemiluminescent array of E. coli ATCC 25922 and S. aureus MRSA ATCC 33592 mixtures 1:1, 9:1 and 1:9 (upper, middle and lower, respectively).

[0021] Figs. 6A-6C show several species in the panel can be distinguished based on increased activation of one or two chemiluminescent probes in the array. (6A) TLE by theβ-glucuronidase probe 2; (6B) TLE by the phosphatase probe 5; and (6C) scatter plot of the TLE by the pyroglutamyl peptidase probe 4 and leucine aminopeptidase probe 6 from the 29 strains of bacteria. The reported results are the mean values derived from a minimum of three independent experiments, each performed in triplicate.

[0022] Figs. 7A-7D show top three most similar strains for four unknown bacteria that were not included in the original panel: (7A) S. aureus ATCC 33591 classified with the highest resemblance as S. aureus, (7B) E. coli clinical isolate-2 (CI-2) classified with the highest resemblance as E. coli, and (7C) P. aeruginosa PAO1 classified with the highest resemblance as P. aeruginosa. (7D) L. monocytogenes ATCC 19115 belonging to a species not represented in the database was classified with the highest resemblance as S. aureus MRSA 43300.DETAILED DESCRIPTION

[0023] In one aspect, the present invention relates to a method for detection and identification of a bacteria, also referred to herein as "an unknown bacteria”, in an aqueous medium, said method comprising:(i) providing an array comprising a plurality of chemiluminescent probes, each composed of a phenyl-dioxetane luminophore masked by a caging group cleavable by a bacterial analyte or enzyme, wherein each one of said phenyl-dioxetane luminophores is masked with a caging group cleavable by a different bacterial analyte or enzyme, and upon exposure to said bacterial analyte or enzyme, said caging group is cleaved from said phenyl-dioxetane luminophore thereby generating an unstable phenolate-dioxetane compound, which is then decomposed through a chemiexcitation process to produce an excited intermediate that decays to its ground-state through emission of light;(ii) applying said aqueous medium to each one of said plurality of chemiluminescent probes of said array (i.e., exposing each one of said plurality of chemiluminescent probes of said array to said aqueous medium);(iii) detecting the emission of light from each one of said chemiluminescent probes, thereby generating a normalized metabolic activity profile for said bacteria, said normalized metabolic activity profile representing the quantity and intensity of emission of light detected from each one of the chemiluminescent probes; and(iv) applying via a processor one or more data analysis methods, e.g., statistical methods and / or machine learning models, on said normalized metabolic activity profile to identify said bacteria.

[0024] In certain embodiments, the detection of the emission of light from each one of said chemiluminescent probes in step (iii) of the method disclosed herein comprises using a plate reader, e.g., over about 10, 20, 30, 40, 50, 60, 75, or 90 minutes. The term “plate reader”, also known as a microplate reader or microplate photometer, refers to an instrument used to detect biological, chemical or physical events of samples in microtiter plates. Sample reactions can be assayed in 1-1536 well format microtiter plates, wherein the most common microplate format used in academic research laboratories or clinical diagnostic laboratories is 96-well with a typical reaction volume between 100 and 200 pL per well.

[0025] According to the method disclosed herein, in order to identify the unknown bacteria, the readout of the chemiluminescent probe array representing the quantity and intensity of emission of light detected from each one of the chemiluminescent probes, more specifically the normalized metabolic activity profile generated in step (iii), may be analyzed using any suitable data analysis method. For example, said normalized metabolic activity profile may be subjected in step (iv) to one or more statistical methods; one or more machine learning models, e.g., machine learning models previously trained by receiving a plurality of normalized metabolic activity profiles of identified bacteria; or a combination of one or more statistical methods and one or more machine learning models.

[0026] Examples of statistical methods that may be applied on the normalized metabolic activity profile to identify said bacteria include, without being limited to, principal component analysis (PCA), hierarchical cluster analysis (HCA; also known as “hierarchical clustering”), centroid-based cluster analysis, density-based cluster analysis, distributionbased cluster analysis, constraint-based cluster analysis, fuzzy clustering cluster analysis, and factor analysis.

[0027] Examples of machine learning models that may be applied on the normalized metabolic activity profile so as to identify the unknown bacteria include, without limiting, unsupervised machine learning clustering methods such as K-Means algorithm, Mean-Shift Algorithm, DBSCAN algorithm, Expectation-Maximization Clustering using Gaussian Mixture Models, and Agglomerative Hierarchical algorithm; supervised machine learning classification methods such as Logistic Regression, Naive Bayes, K-Nearest Neighbors, Decision Tree, and Support Vector Machines; semi-supervised machine learning models; and deep learning classification methods such as Convolutional Neural Networks (CNNs), Long Short Term Memory Networks (LSTMs), Recurrent Neural Networks (RNNs), Generative Adversarial Networks (GANs), Radial Basis Function Networks (RBFNs), Multilayer Perceptrons (MLPs), Self Organizing Maps (SOMs), Deep Belief Networks (DBNs), Restricted Boltzmann Machines (RBMs), and Autoencoders.

[0028] In certain embodiments, the identification of said bacteria by the method of the present invention includes a confidence level, indicating the probability with which the estimation of said identification is true. Such a confidence level may be up to 80%, or higher than 80%, e.g., about 85%, about 90%, about 92%, about 94%, about 96%, about 98%, about 99%, or 100%.

[0029] The method of the present invention is aimed at detecting and identifying with a high confidence level, e.g., with a confidence level of about 90%, about 92%, about 94%, about 96%, about 98%, about 99%, or 100%, an unknown bacteria present in an aqueous medium. The aqueous medium subjected to said method is prepared according to the following two-step procedure. First, a sample from a medium to be analyzed, i.e., a medium suspected as being contaminated by said unknown bacteria, is taken and cultured under appropriate conditions and using a bacterial growth media, preferably an enriched bacterial growth media, e.g., in Petri dishes, to allow bacterial colonies, each consisting of a group of genetically identical bacterial cells derived from the same mother cell, to develop. Then, i.e., after bacterial colonies have been developed, an inoculum is taken from a particular colony to be identified and cultured in an aqueous medium comprising, e.g., PBS and a bacterial growth medium, to allow reproduction of the cultured bacteria in said aqueous medium. The medium to be analyzed may be any medium including solid, semi-solid and liquid mediums. In certain embodiments, the medium analyzed is a biological sample, i.e., a tissue biopsy sample; a bodily fluid such as an amniotic fluid, aqueous humour, vitreous humour, bile, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), endolymph, perilymph, female ejaculate, gastric juice, mucus, peritoneal fluid, saliva, sebum (skin oil), semen, sweat, tears, vaginal secretion, vomit and urine; or a bodily fluid-based solution, i.e., an aqueous solution in which a bodily fluid is dissolved. In other embodiments, the medium analyzed is a dairy product such as milk including, e.g., cow milk, goat milk, sheep milk, and camel milk. In further embodiments, the medium analyzed is wastewater including industrial wastewater.

[0030] The procedure by which the aqueous medium is prepared is aimed at guaranteeing that said medium contains a sole bacterial species to be identified, and that the normalized metabolic activity profile generated in step (iii) of the method, representing the quantity and intensity of emission of light detected from each one of the chemiluminescent probes after application of said aqueous medium, indeed represents a particular specific bacterium to be identified.

[0031] The method disclosed herein is global, and may thus be used so as to detect and identifiy any type of bacteria. In certain embodiments, the bacteria detected and identified in the aqueous medium is a Gram-positive bacteria, and in other embodiments, said bacteria is a Gram-negative bacteria. Examples of possible bacteria to be detected and identified by the method disclosed include, without limiting, a Salmonella such as S. enlerica; a Listeriasuch as L. monocytogenes; a Staphylococcus such as S. aureus, S. epidermidis, S. aureus, and methicillin-resistant S. aureus (MRSA); E. coli; a carbapenem-resistant bacteria such as Pseudomonas aeruginosa and Klebsiella pneumonia; a Campylobacter such as C. jejuni, C. coli, and C. lari; a Bacillus such as B. cereus; a Clostridium such as C. perfringens and C. difficile; a Mycobacterium such as M. tuberculosis and M. smegmatis; a Streptococcus such as S. pneumonia, S. agalactiae and S. pyogenes; a Pseudomonas such as P. aeruginosa; an Enterococcus such as E. faecalis; a vancomycin-resistant Enterococcus (VRE); a Neisseria such as N. meningitides; Haemophilus influenza; Helicobacter pylori; Burkholderia cepacian; a Propionibacterium such as P. acnes; a Citrobacter; Cronobacter sakazakii; Geobacillus stearothermophilus; a Vibrio; a Legionella such as L. pneumophilia; an apyrase-containing bacterium such as Shigella; Coliform; an extended- spectrum betalactamase (ESBL)-producing enterobacterium; and a Prevotella.

[0032] In certain embodiments, the bacterial analyte or enzyme capable of cleaving the caging group masking a particular phenyl-dioxetane luminophore composing the array provided in step (i) of the method disclosed each independently is a glycosidase, protease, alkaline phosphatase (ALP), or an antibiotic resistance-conferring enzyme such as a P- lactamase, amidase (e.g., penicillin-G amidase), transferase (e.g., acyltransferase, phosphotransferase, and nucleotidyltransferase), glycoside hydrolase (e.g., P-galactosidase, β-glucuronidase, and β-glucosidase), pyroglutamyl-peptidase, leucine aminopeptidase, nitro-reductase, N-acetyl hydrolase, and NAD(P)H quinone oxidoreductase 1.

[0033] The term “phenyl-dioxetane”, as used herein, refers to a compound comprising the moiety wherein RAis a linear or branched alkyl, or cycloalkyl; andRBand Rceach independently is a branched alkyl or cycloalkyl, or RBand Rctogether with the carbon atom to which they are attached form a carbocyclic or heterocyclic ring, which may be fused, spiro or bridged cyclic or polycyclic ring.

[0034] The phenyl-dioxetane luminophores composing the array provided in step (i) of the method, according to any one of the embodiments above, each independently may bear a π* acceptor group at either the ortho or para position to the phenol moiety; or alternatively comprise a cyclic or heterocyclic structure which is conjugated to the phenolic ring and extends the pi-system of said ring, i.e., comprises a pi-system that conjugates with the pi-system of the phenolic ring. Suitable such phenyl-dioxetane luminophores may be based on those disclosed, e.g., in WO 2017 / 130191, WO 2018 / 216013, WO 2019 / 224338, WO 2024 / 147125, and US Provisional Application No. 63 / 564,881 of the present applicant. The phenyl-dioxetane luminophores composing said array may be identical or different, regardless of the specific caging group masking each one of them (which is cleavable by a different bacterial analyte or enzyme, and thus structurally different by definition). In particular embodiments, the phenyl-dioxetane luminophores composing said array are identical, and in other embodiments, said phenyl-dioxetane luminophores are different.

[0035] In certain embodiments, the phenyl-dioxetane luminophore composing said array each independently is a compound of formula I:whereinR1is selected from a linear or branched (C1-C18 / alkyl, or (Cs-Cv / cycloalkyl;R2and R3each independently is selected from a branched (C3-Cis)alkyl or (C3- Cvjcycloalkyl, or R2 and R3 together with the carbon atom to which they are attached form a carbocyclic or heterocyclic ring, which may be fused, spiro or bridged cyclic or polycyclic ring;R4, R5and R6each independently is selected from H, an electron acceptor group such as halogen, -NO2, -CN, -COOR9, -C(=O)R9, and -SO2R9, and a π* acceptor group of the formula -CH=CH-E, wherein E is (a) -CN, -COOH, -COO(Ci-Ci8)alkyl optionally interrupted in the alkylene chain with one or more -O- groups, or -COO(C2-C18)alkenyl such as -COO-allyl; or (b) 4-pyridinyl, methylpyridinium-4-yl, 3,3-dimethyl-3H-indolyl, or 1,3,3- trimethyl-3H-indol-l-ium-2-yl, optionally substituted at one or more of the carbon atoms of the aromatic or heteroaromatic ring with a substituent each independently selected from halogen, -CN, -COOH, -COO(C1-C18)alkyl, and -C(O)(C1-C18)alkyl; or one of R5and R6, together with R7, forms an optionally substituted cyclic or heterocyclic structure that extends the pi-system of the central aromatic ring, and the other one of R5and R6is H;R7is H, or together with one of R5and R6forms said optionally substituted cyclic or heterocyclic structure;R8is said caging group selected from an enzyme-labile group and a boron- containing group having the formula -B(Z)(Z') or -B(Z")3- Kat+;R9each independently is H, (Ci-Ci8)alkyl, or (C6-Ci4)aryl;Z and Z' each independently is selected from -OR10and -O’ Kat+;Z" is selected from F, Cl, Br, and I;R10is selected from H, (Ci-C4)alkyl, (C2-C4)alkenyl, (C2-C4)alkynyl, (C2- C4)heteroalkyl, (C2-C4)heteroalkenyl, (C2-C4)heteroalkynyl, (C6-Cio)aryl, (C5- C6)heteroaryl, (C6-Cio)aralykl, and (C6-Cio)heteroaralkyl, or two R10together with their intervening atoms (i.e., the boron atom and the two oxygen atoms linked thereto) form a 5- to 7-membered optionally substituted heterocyclic ring, preferably a saturated optionally substituted heterocyclic ring;Kat+each independently is an organic cation, or inorganic cation such as an alkali metal cation, e.g., sodium or potassium ion;L is absent or is a linker of the formula LI, L2 or L3:optionally substituted at the aromatic ring with one or more substituents each independently selected from (Ci-Ci8)alkyl and (C3-C7)cycloalkyl, wherein M is absent or is -O-, -NH-, - N(C1-C8)alkyl-, or -N+((C1-C8)alkyl)2-, and the asterisk represents the point of attachment to the group Y ; andY is absent or is -O-, provided that when R8is said enzyme-labile group, Y is -O-; and when R8is said boron-containing group, Y is absent, and either L is absent or L is said linker wherein M is absent.

[0036] The term "alkyl" typically means a linear or branched hydrocarbyl having, e.g., 1- 18 carbon atoms and includes methyl, ethyl, n-propyl, isopropyl, n-butyl, sec -butyl, isobutyl, tert-butyl, n-pentyl, isoamyl, neopentyl, 2,2-dimethylpropyl, n-hexyl, n-heptyl, n-octyl, n- nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, and the like. Preferred are (C1-C8)alkyl groups, more preferably (Ci-C4)alkyl groups, mostpreferably methyl, ethyl, n-propyl, isopropyl, and tert-butyl. The alkyl may optionally be substituted with one or more groups each independently selected from halogen, -COR, - COOR, -OCOOR, -OC ON(R)2, -CN, -NO2, -SR, -OR, -N(R)2, -CON(R)2, -SO2R, -SO3R or -S(=O)R, wherein R is H or unsubstituted (C1-C8)alkyl.

[0037] The term "alkylene" refers to a hydrocarbylene, i.e., a divalent group of the formula CnH2n wherein n is an integer of at least 1, derived from a saturated linear or branched aliphatic chain having, e.g., 1-18 carbon atoms, by removal of two hydrogen atoms. Examples of alkylenes include, without being limited to, methylene, ethylene, propylene, butylene, 2-methylpropylene, pentylene, 2-methylbutylene, hexylene, 2-methylpentylene, 3- methylpentylene, 2,3-dimethylbutylene, heptylene, octylene, n-tridecanylene, n- tetradecanylene, n-pentadecanylene, n-hexadecanylene, n-heptadecanylene, n- octadecanylene, n-nonadecanylene, icosanylene, henicosanylene, docosanylene, tricosanylene, tetracosanylene, pentacosanylene, and the like. Preferred are (C1-C8)alkylene or (Ci-C6)alkylene, more preferably (Ci-C4)alkylene, most preferably methylene, ethylene, propylene, or butylene. The term “alkylene chain” refers to a linear alkylene group of the formula -(CH2)n- wherein n is an integer of at least 2, derived from a saturated linear aliphatic chain having at least 2 carbon atoms, after removal of a hydrogen atom from each one of the terminal carbon atoms, but may further refer to such a linear chain being a part of an alkyl group.

[0038] The terms "alkenyl" and "alkynyl" typically mean linear or branched hydrocarbyls having 2-18, e.g., 2-8, carbon atoms and at least one double or triple bond, respectively, and include ethenyl, propenyl, 3-buten-l-yl, 2-ethenylbutyl, 3-octen-l-yl, 3-nonenyl, 3 -decenyl, and the like, and propynyl, 2-butyn-l-yl, 3-pentyn-l-yl, 3-hexynyl, 3-octynyl, 4-decynyl, and the like. The alkenyl and alkynyl defined herein may optionally be substituted with one or more groups each independently selected from halogen, -COR, -COOR, -OCOOR, - OCON(R)2, -CN, -NO2, -SR, -OR, -N(R)2, -CON(R)2, -SO2R, -SO3R or -S(=O)R, wherein Ris H or unsubstituted (C1-C8)alkyl.

[0039] The terms "heteroalkyl," "heteroalkenyl," and "heteroalkynyl", as used herein, refer to the corresponding hydrocarbyl (i.e., alkyl, alkenyl, and alkynyl), which contains one or more heteroatoms each independently selected from O, N, and S within the backbone residue; thus, at least one carbon atom of a corresponding alkyl, alkenyl, or alkynyl is replaced by one of the specified heteroatoms to form a heteroalkyl, heteroalkenyl, orheteroalkynyl group. Preferred are (C2-C4)heteroalkyls, (C2-C4)heteroalkenyls, and (C2- C4)heteroalkynyls.

[0040] The term “aliphatic ring” or “carbocyclic ring” used herein interchangeably refers to a mono-, bi-, or poly-cyclic non-aromatic hydrocarbon having, e.g., 3-12, but preferably 3-10, carbon atoms. The carbocyclic ring may be saturated, such as cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, adamantane, and the like; or unsaturated, i.e., having at least one double bond, such as cyclopropene, cyclobutene, cyclopentene, cyclohexene, and the like. The carbocyclic ring may be substituted as defined herein with at least one group each independently selected from alkyl optionally substituted with at least one aryl group, aryl, and an electron withdrawing group, and may also be oxidized at a carbon atom thereof. The term "cycloalkyl" means a mono- or bicyclic saturated hydrocarbyl group having, e.g., 3-10 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, and the like, that may be substituted, e.g., by one or more alkyl groups.

[0041] The term "heterocyclic ring" denotes a mono-, bi-, or poly-cyclic non-aromatic ring of 4-12, but preferably 3-8, atoms and consisting of at least one carbon atom and at least one heteroatom selected from oxygen, nitrogen, and sulfur (optionally oxidized), which may be saturated or unsaturated, i.e., containing at least one unsaturated bond. The heterocyclic ring may be substituted as defined herein with at least one group each independently selected from alkyl optionally substituted with at least one aryl group, aryl, and an electron withdrawing group, and may also be oxidized at either a carbon atom or a heteroatom thereof (in cases said heteroatom is sulfur, it may also be dioxidized). Non-limiting examples of heterocyclic rings include oxetane, azetidine, thietane, 1-oxidothietan, 1,1-dioxidothietan, pyrrolidine, piperidine, morpholine, thiomorpholine, piperazine, oxazolidine, thiazolidine, imidazolidine, oxazoline, thiazoline, imidazoline, dioxole, dioxolane, dihydrooxadiazole, pyran, dihydropyran, tetrahydropyran, thiopyran, dihydrothiopyran, tetrahydrothiopyran, 1- oxidotetrahydrothiopyran, 1,1-dioxidotetrahydrothiopyran, tetrahydrofuran, pyrazolidine, pyrazoline, tetrahydropyrimidine, dihydro triazole, tetrahydrotriazole, azepane, dihydropyridine, tetrahydropyridine, and the like. The term "heterocyclyl" as used herein refers to a univalent group derived from a heterocyclic ring by removal of hydrogen from any of the ring atoms.

[0042] The term “aromatic ring” as used herein denotes an aromatic carbocyclic ring having, e.g., 6-14 carbon atoms, and consisting of a single ring or multiple rings eithercondensed or linked by a covalent bond. Non-limiting examples of aromatic rings include benzene, naphthalene, anthracene, naphthacene, phenanthrene, pyrene, chrysene, tetracene, and triphenylene. The aromatic ring may optionally be substituted by at least one group each independently selected from halogen, (C1-C8)alkyl, -O-(C1-C8)alkyl, -COO(C1-C8)alkyl, - CN, and -NO2. The term "aryl" denotes a univalent aromatic carbocyclic group derived from an aromatic ring by removal of hydrogen atom from any of the ring atoms.

[0043] The term “heteroaromatic ring” as used herein refers to a mono-, bi-, or poly-cyclic aromatic ring having, e.g., 4-12 atoms, and consisting of at least one carbon atom and at least one heteroatom selected from nitrogen, oxygen, sulfur (optionally oxidized). Preferred are 5- or 6-membered heteroaromatic rings. Non-limiting examples of heteroaromatic rings include thiophene, imidazole, pyridine, furan, pymole, oxazole, thiazole, purine, indole, pyrrole, pyrazine, isoquinoline, pyrazole, isoxazole, thiazole, isothiazole, pyrazine, pyrimidine, pyridazine, carbazole. The term “heteroaryl” as used herein refers to a univalent group derived from a heteroaromatic ring as defined herein by removal of hydrogen atom from any of the ring atoms. Examples of mono-cyclic heteroaryls include, without being limited to, pyrrolyl, furyl, thienyl, thiazinyl, pyrazolyl, pyrazinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, 1,2,3-triazinyl, 1,3,4-triazinyl, and 1,3,5-triazinyl. Polycyclic heteroaryl radicals are preferably composed of two rings such as, but not limited to, benzofuryl, isobenzofuryl, benzothienyl, indolyl, quinolinyl, isoquinolinyl, imidazo[l,2-a]pyridyl, benzimidazolyl, benzthiazolyl, benzoxazolyl, pyrido[l,2-a]pyrimidinyl and 1,3-benzodioxinyl. The heteroaryl may optionally be substituted, at any position of the ring, with at least one group each independently selected from halogen, (C1-C8)alkyl, -O-(Ci-C8)alkyl, -COO(C1-C8)alkyl, -CN, and -NO2. It should be understood that when a polycyclic heteroaryl is substituted, the substitution may be in any of the carbocyclic and / or heterocyclic rings.

[0044] The term "halogen" as used herein refers to a halogen and includes fluoro, chloro, bromo, and iodo, but it is preferably fluoro or chloro.

[0045] The term “electron withdrawing group” as used herein refers to a group reducing electron density in a molecule through the carbon atom to which it is bonded. Non-limiting examples of such groups include halogen, preferably fluoro and chloro, -NO2, -CN, =0, - C(=O)OR, -C(=O)R, -C(=O)NR2, -0-(Ci-C8)alkylene-(C6-Cio)aryl, and -SO2R, wherein R each independently may be, e.g., hydrogen, linear or branched (Ci-Ci8)alkyl, or (C6-C14aryl. Particular such electron acceptor groups include fluoro, chloro, -NO2, -CN, -C(=O)OR, -C(=O)R, -C(=0)NR2, -O-Cfk-phenyl (benzyloxy), and -SO2R, wherein R each independently is H, (Ci-C4)alkyl, or phenyl.

[0046] The term "electron acceptor group" as used herein refers to a group of atoms with a high electron affinity. Non-limiting examples of such groups include halogen, preferably fluoro and chloro, -NO2, -CN, -C(=O)R, -C(=O)OR, -C(=O)NR2, and -SO2R, wherein R each independently may be, e.g., hydrogen, linear or branched (Ci-Ci8)alkyl, or (C6-Ci4)aryl. Particular such electron acceptor groups include halogen, -NO2, -CN, -C(=O)R, -C(=O)OR, and -SO2R, wherein R each independently is H, -(Ci-C4)alkyl, or phenyl.

[0047] The term "K* acceptor group" as used herein refers to any group containing a π* acceptor system capable of accepting electrons. Specific such groups are those of the formula -CH=CH-E, wherein E is (a) -CN, -COOH, -COO(Ci-Ci8)alkyl optionally interrupted in the alkylene chain with one or more -O- groups, or -COO(C2-Cis)alkenyl such as -COO-allyl; or (b) 4-pyridinyl, methylpyridinium-4-yl, 3,3-dimethyl-3H-indolyl, or l,3,3-trimethyl-3H- indol-l-ium-2-yl (see Table 1), optionally substituted at one or more of the carbon atoms of the aromatic or heteroaromatic ring with a substituent each independently selected from halogen, -CN, -COOH, -COOR, or -C(O)R, wherein R is -(C1-C18)alkyl.Table 1: Certain π* acceptor groups of the formula -CH=CH-E (names refer to group E)3,3-dimethyl-3H- l,3,3-trimethyl-3H-4-pyridinyT methylpyridinium-4-yT indolyl* indol-l-ium-2-yrOptionally substituted at one or more of the carbon atoms of the aromatic or heteroaromatic ring with a substituent each independently selected from halogen, -CN, -COOH, -COO(Ci-Cis)alkyl, and -C(O)(Ci-Ci8)alkyl.

[0048] In certain embodiments, the phenyl-dioxetane luminophore composing said array each independently is a compound of the formula I, wherein R1is a linear or branched (Ci- Cs)alkyl, preferably (Ci-C4)alkyl, more preferably methyl, ethyl, n-propyl, isopropyl, or tertbutyl.

[0049] The primary function of groups R2and R3is to sterically protect the dioxetane moiety of the compound of formula I. In certain embodiments, the phenyl-dioxetane luminophore composing said array each independently is a compound of the formula I,wherein R2and R3each independently is a branched (C3-Ci8)alkyl or (C3-C7)cycloalkyl. In other embodiments, R2and R3together with the carbon atom to which they are attached form a carbocyclic or heterocyclic ring.

[0050] In certain particular embodiments, R2and R3together with the carbon atom to which they are attached form a carbocyclic ring selected from propellane; a bicyclus defined by the formula [A.B.l]pentane, [A.B.l]hexane, [A.B.l]heptane, [A.B.l]octane, [A.B.l]nonane, [A.B.l]decane, [A.B.l]undecane, or [A.B.l]dodecane, wherein A and B are independently selected from 1, 2, 3, 4, and 5; and adamantane. Preferred such embodiments are those wherein R2and R3together with the carbon atom to which they are attached form adamantyl.

[0051] In other particular such embodiments, R2and R3together with the carbon atom to which they are attached form a 3-4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from (C1-C8)alkyl optionally substituted with at least one (C6-Cio)aryl group, (C6-Cio)aryl, or an electron withdrawing group such as halogen, -NO2, -CN, =0, -COOR11, -C(=O)R12, -O-(C1-C8)alkylene-(C6- Cio)aryl, and -SO2R11, wherein two adjacent carbon atoms of said carbocyclic ring optionally form an additional 3-8 membered carbocyclic, heterocyclic, or aromatic ring, optionally substituted with at least one group each independently selected from halogen and (Ci- Cisjalkyl; two non-adjacent carbon atoms of said carbocyclic ring optionally form bicyclo [l.l.l]pentan-2-yl, or bicyclo[2.1.1]hexan-5-yl; or two alkyl groups attached to the same carbon atom of said carbocyclic ring, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic or heterocyclic ring, wherein R11is H, (Ci- Cisjalkyl, or (C6-C14)aryl; and R12is H, (C1-C18jalkyl, (C6-C14)aryl, or halogen.

[0052] In more particular such embodiments, R2and R3together with the carbon atom to which they are attached form: (a) cyclobutyl, optionally substituted with one or more groups each independently selected from (Ci-C4)alkyl optionally substituted with one or two (C6- Cio)aryl groups, (C6)aryl, =0, -COOR11, -C(=O)R12, and -0-(Ci-C4)alkylene-(C6-Cio)aryl, wherein two adjacent carbon atoms of said cyclobutyl optionally form 3-membered carbocyclic ring or 6-membered aromatic ring; two non-adjacent carbon atoms of said carbocyclic ring optionally from bicyclo [l.l.l]pentan-2-yl; or two alkyl groups attached to the same carbon atom of the cyclobutyl, together with said carbon atom, optionally form an additional 4-membered carbocyclic or heterocyclic ring, said heterocyclic ring being elected from oxetane or azetidine optionally substituted at the nitrogen atom with one or two (Ci-C4)alkyl groups; (b) oxetan-3-yl optionally substituted with one or more (Ci-C4)alkyl groups; (c) azetidine- 3 -yl optionally substituted at the nitrogen atom with one or two groups each independently selected from (Ci-C4)alkyl optionally substituted with one or two (Ce- Cio)aryl groups, -COOR11, -C(=O)R12, and -SO2R11; or (d) thietane-3-yl optionally oxidized or dioxidized at the sulfur atom, wherein R11is H, (Ci-C4)alkyl, or (C6-Cio)aryl such as phenyl; and R12is H, (Ci-C4)alkyl, (C6-Cio)aryl such as phenyl, or halogen. Preferred such embodiments are those wherein R2and R3together with the carbon atom to which they are attached form cyclobutyl, 2,2-dimethylcyclobutyl, 2,2,4-trimethylcyclobutyl, 2, 2,4,4- tetramethylcyclobutyl, 3 -phenylcyclobutyl, 3-benzyloxycyclobutyl, 3-carboxycyclobutyl, 3-carbomethoxycyclobutyl, bicyclo[4.2.0]octa-l(6),2,4-trien-7-yl, bicyclo[l.l.l]pentan-2- yl, bicyclo[2.1.1]hexan-5-yl, spiro[3.3]heptan-2-yl, 5-oxo-2-oxaspiro[3.3]heptan-6-yl, 6- oxo-2-oxaspiro[3.3]heptan-5-yl, 2-methyl-5-oxo-2-azaspiro[3.3]heptan-6-yl, 2-methyl-6- oxo-2-azaspiro[3.3]heptan-5-yl, 2,2-dimethyl-5-oxo-2-azaspiro[3.3]heptan-2-ium-6-yl, 2,2- dimethyl-6-oxo-2-azaspiro[3.3]heptan-2-ium-5-yl, oxetan-3-yl, 2,2-dimethyloxetan-3-yl, 2,2,4-trimethyloxetan-3-yl, 2,2,4,4-tetramethyloxetan-3-yl, azetidin-3-yl, 1-methylazetidin- 3-yl, l-ethylazetidin-3-yl, l-phenylazetidin-3-yl, l-diphenylmethyl-azetidin-3-yl, 1,1- dimethylazetidin-3-ylium, l-(methoxycarbonyl)azetidin-3-yl, 1 -(tert-butoxycarbonyl) azetidin-3-yl, l-acetylazetidin-3-yl, 1 -(phenylsulfonyl) azetidin-3-yl, thietan-3-yl, 1- oxidothietan-3-yl, or l,l-dioxidothietan-3-yl (see Table 2).

[0053] In further particular such embodiments, R2and R3together with the carbon atom to which they are attached form a 6 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from (C1-C8)alkyl optionally substituted with at least one (C6-Cio)aryl group, (C6-Cio)aryl, or an electron withdrawing group such as halogen, -NO2, -CN, =0, -COOR11, -C(=O)R12, -O-(Ci-C8)alkylene-(C6- Cio)aryl, and -SO2R11, wherein two non-adjacent carbon atoms of said carbocyclic or heterocyclic ring optionally from an additional 3-4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from halogen and (Ci-Ci8)alkyl; or two alkyl groups attached to the same carbon atom of said carbocyclic or heterocyclic ring, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic or heterocyclic ring, wherein R11is H, (Ci-Ci8)alkyl, or (C6-Ci4)aryl; and R12is H, (Ci-Ci8)alkyl, (C6-Ci4)aryl, or halogen.Table 2: Specific 3-4 membered carbocyclic and heterocyclic rings that may be formed by R2and R3together with the carbon atom to which they are attached

[0054] In more particular such embodiments, R2and R3together with the carbon atom to which they are attached form: (a) cyclohexyl optionally substituted with one or more groups each independently selected from (Ci-C4)alkyl optionally substituted with one or two (Ce- Cio)aryl groups, (C6)aryl, halogen, =0, -COOR11, -C(=O)R12, and -O-(Ci-C4)alkylene-(C6-Cio)aryl, wherein two non-adjacent carbon atoms of said cyclohexyl optionally from an additional 3-4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from halogen and (Ci-Ci8)alkyl; or two alkyl groups attached to the same carbon atom of said cyclohexyl, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic or heterocyclic ring; (b) tetrahydropyranyl (oxanyl) or dioxanyl, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (Ci-C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, (C6)aryl, halogen, =0, -C00R11, - C(=0)R12, and -0-(Ci-C4)alkylene-(C6-Cio)aryl; (c) tetrahydrothiopyranyl (thianyl), 1- oxidotetrahydrothiopyranyl or 1,1-dioxidotetrahydrothiopyranyl, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (Ci-C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, -C00R11, - C(=0)R12, and -SO2R11; or (d) piperidinyl or piperidinyl- 1 -oxide, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (Ci-C4)alkyl optionally substituted with one or two (C6-C10)aryl groups, (C6)aryl, halogen, =0, -C00R11, -C(=0)R12, and -0-(Ci-C4)alkylene-(C6-Cio)aryl, said piperidinyl being further optionally substituted at the nitrogen atom with one or two groups each independently selected from (Ci-C4)alkyl, (C3-C7)cycloalkyl, and (C6-Cio)aryl, wherein R11is H, (Ci- C4)alkyl, or (C6-Cio)aryl; and R12is H, (Ci-C4)alkyl, (C6-Cio)aryl, or halogen. Preferred such embodiments are those wherein R2and R3together with the carbon atom to which they are attached form cyclohexyl, 3-carboxycyclohexyl, 3 -carbomethoxy cyclohexyl (3- (methoxycarbonyl)cyclohexyl), 3-carboethoxycyclohexyl (3-(ethoxycarbonyl)cyclohexyl), 4-carboxycyclohexyl, 4-carbomethoxycyclohexyl (4-(methoxycarbonyl)cyclohexyl), 4- carboethoxycyclohexyl (4-(ethoxycarbonyl)cyclohexyl), (chlorocarbonyl)cyclohexyl, (bromocarbonyl)cyclohexyl, fluorocyclohexyl, difluorocyclohexyl, 3,3-difluorocyclohexyl, 4,4-difluorocyclohexyl, chlorocyclohexyl, dichlorocyclohexyl, 3,3-dichlorocyclohexyl, 4,4- dichlorocyclohexyl, cyanocyclohexyl, 3 -cyanocyclohexyl, 4-cyanocyclohexyl, nitrocyclohexyl, 3 -nitrocyclohexyl, 4-nitrocyclohexyl, 3 -tetrahydropyranyl, 4- tetrahydropyranyl, l,3-dioxan-5-yl, 2,2-dimethyl-l,3-dioxan-5-yl, 4-tetrahydrothiopyranyl, l-oxido-4-tetrahydrothiopyranyl, or l,l-dioxido-4-tetrahydrothiopyranyl (see Table 3).Table 3: Specific 6-membered carbocyclic and heterocyclic rings that may be formed by R2and R3together with the carbon atom to which they are attached

[0055] In certain embodiments, the phenyl-dioxetane luminophore composing said array each independently is a compound of the formula I, wherein (i) R4, R5and R6each independently is selected from H, halogen, preferably chlorine, -CN, and a π* acceptor group of the formula -CH=CH-E as defined above; or (ii) R4is selected from H, halogen, preferably chlorine, and -CN; one of R5and R6is H; and the other one of R5and R6, together with R7, forms a 6-membered structure selected fromthat extends the pi-system of the central aromatic ring, wherein R13is (Ci-Ci2)alkyl, preferably (Ci-C4)alkyl; and R14each indepednetly is selected from -CN, -NO2, (C2-Ci2)alkenyl, preferably (C2- C4)alkenyl, aryl, -COOR15, -COO- Kat+, and -C(O)N(R15)2, wherein R15each independently is H or (Ci-Ci2)alkyl, preferably (Ci-C4)alkyl, or the two R15together with the nitrogen atom to which they are attached form a heterocyclic ring.

[0056] In certain particular such embodiments, one of R4, R5and R6is a π* acceptor group of the formula -CH=CH-E, and the other two of R4, R5and R6each independently is H orhalogen, preferably chlorine, i.e., (i) R4and R5each independently is H or halogen; and R6is a π* acceptor group of the formula -CH=CH-E; (ii) R4and R6each independently is H or halogen; and R5is a π* acceptor group of the formula -CH=CH-E; or (iii) R5and R6each independently is H or halogen; and R4is a π* acceptor group of the formula -CH=CH-E. In preferred such embodiments, R4and R6each independently is H or halogen; and R5is a π* acceptor group of the formula -CH=CH-E, more preferably R4is halogen; R5is a π* acceptor group of the formula -CH=CH-E; and R6is H. Particular such embodiments, are those wherein E is -CN; -COOH; -COO(C1-C8)alkyl, preferably -COO(C1-C4)alkyl such as - COOCH3 and -COOC(CH3)3; or -COO(C2-C8)alkenyl such as -COOCH=CH-CH3.

[0057] In other particular such embodiments, R4, R5and R6each independently is H or halogen, preferably chlorine, i.e., none of R4, R5and R6is a π* acceptor group. In preferred such embodiments, R4is halogen, and R5and R6each is H.

[0058] In further particular such embodiments, R4is selected from H, halogen, preferably chlorine, and -CN; one of R5and R6, preferably R6, together with R7, forms a 6-membered structure selected fromand the other one of R5and R6is H. Preferred such 6-membered structures are those wherein R13is (Ci- C4)alkyl; and R14is selected from -CN, -NO2, (C2-C4)alkenyl, phenyl, -COOR15, -COO- Kat+, and -C(O)N(R15)2, wherein R15each independently is H or CH3. In particular such embodiments, R4is H or halogen; R5is H; and R6together with R7forms said 6-membered structure. In preferred such embodiments, the 6-membered structure formed is

[0059] In certain embodiments, the phenyl-dioxetane luminophore composing said array each independently is a compound of the formula I, wherein L is absent or is a linker of theformula LI, L2 or L3, preferably LI, wherein M is absent or is selected from -O-, -NH-, and -N+(CH3)2-.

[0060] In certain embodiments, the phenyl-dioxetane luminophore composing said array each independently is a compound of the formula I, wherein R1is a linear or branched (Ci- C8)alkyl, e.g., (Ci-C4)alkyl such as methyl, ethyl, n-propyl, isopropyl, and tert-butyl; R2and R3together with the carbon atom to which they are attached form a carbocyclic or heterocyclic ring such as (i) adamantyl; (ii) a 3-4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from (C1-C8)alkyl optionally substituted with at least one (C6-Cio)aryl group, (C6-Cio)aryl, or an electron withdrawing group such as halogen, -NO2, -CN, =0, -COOR11, -C(=O)R12, -O-(Ci- C8)alkylene-(C6-Cio)aryl, and -SO2R11, wherein two adjacent carbon atoms of said carbocyclic ring optionally form an additional 3-8 membered carbocyclic, heterocyclic, or aromatic ring, optionally substituted with at least one group each independently selected from halogen and (C1-C18jalkyl; two non-adjacent carbon atoms of said carbocyclic ring optionally from bicyclo[l.l.l]pentan-2-yl, or bicyclo[2.1.1]hexan-5-yl; or two alkyl groups attached to the same carbon atom of said carbocyclic ring, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic or heterocyclic ring; or (iii) a 6 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from (C1-C8)alkyl optionally substituted with at least one (Ce- Cio)aryl group, (C6-Cio)aryl, or an electron withdrawing group such as halogen, -NO2, -CN, =0, -COOR11, -C(=O)R12, -0-(Ci-C8)alkylene-(C6-Cio)aryl, and -SO2R11, wherein two non- adjacent carbon atoms of said carbocyclic or heterocyclic ring optionally from an additional 3-4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from halogen and (C i-Cis)alkyl; or two alkyl groups attached to the same carbon atom of said carbocyclic or heterocyclic ring, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic or heterocyclic ring; either R4, R5and R6each independently is selected from H, halogen, preferably chlorine, -CN, and a π* acceptor group of the formula -CH=CH-E; or R4is selected from H, halogen, preferably chlorine, and -CN; one of R5and R6is H; and the other one of R5and R6, together with R7,selected from -CN, -NO2, (C2-Ci2)alkenyl, aryl, -COOR15, -COO Kat+, and -C(O)N(R15)2, wherein R15is H or (Ci-Ci2)alkyl, or the two R15together with the nitrogen atom to which they are attached form a heterocyclic ring; R11each independently is H, (Ci-Ci8)alkyl, or (C6-Ci4)aryl; R12each independently is H, (Ci-Ci8)alkyl, (C6-C14)aryl, or halogen; and L is either absent, or is a linker of the formula LI, L2 or L3, preferably LI, wherein M is absent or is -O-, -NH-, or -N+(CH3)2-.

[0061] In particular such embodiments, said penyl-dioxetane luminophore each independently is a compound of the formula I, wherein R1is methyl; and R2and R3together with the carbon atom to which they are attached form: (i) adamantyl; (ii) cyclobutyl optionally substituted with one or more groups each independently selected from (Ci- C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, (C6)aryl, =0, -COOR11, -C(=O)R12, and -0-(C1-C4)alkylene-(C6-Cio)aryl, wherein two adjacent carbon atoms of said cyclobutyl optionally form 3-membered carbocyclic ring or 6-membered aromatic ring; two non-adjacent carbon atoms of said carbocyclic ring optionally from bicyclo[l.l.l]pentan-2- yl; or two alkyl groups attached to the same carbon atom of the cyclobutyl, together with said carbon atom, optionally form an additional 4-membered carbocyclic or heterocyclic ring, said heterocyclic ring being elected from oxetane or azetidine optionally substituted at the nitrogen atom with one or two (C1-C4) alkyl groups; (iii) oxetan-3-yl optionally substituted with one or more (Ci-C4)alkyl groups; (iv) azetidine- 3 -yl optionally substituted at the nitrogen atom with one or two groups each independently selected from (Ci-C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, -COOR11, -C(=O)R12, and - SO2R11; (v) thietane-3-yl optionally oxidized or dioxidized at the sulfur atom; (vi)cyclohexyl optionally substituted with one or more groups each independently selected from (Ci-C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, (C6)aryl, halogen, =0, -C00R11, -C(=O)R12, and -0-(Ci-C4)alkylene-(C6-Cio)aryl, wherein two non-adjacent carbon atoms of said cyclohexyl optionally from an additional 3-4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from halogen and (Ci-Ci8)alkyl; or two alkyl groups attached to the same carbon atom of said cyclohexyl, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic or heterocyclic ring; (vii) tetrahydropyranyl or dioxanyl, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (Ci-C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, (C6)aryl, halogen, =0, -C00R11, -C(=0)R12, and -0-(Ci-C4)alkylene-(C6-Cio)aryl; (viii) tetrahydrothiopyranyl, 1-oxidotetrahydrothiopyranyl or 1,1-dioxidotetrahydrothiopyranyl, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (Ci-C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, -C00R11, -C(=0)R12, and -SO2R11; or (ix) piperidinyl or piperidinyl-1 -oxide, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (Ci-C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, (C6)aryl, halogen, =0, -C00R11, -C(=0)R12, and -0-(Ci-C4)alkylene-(C6-Cio)aryl, said piperidinyl being further optionally substituted at the nitrogen atom with one or two groups each independently selected from (Ci-C4)alkyl, (C3-C7)cycloalkyl, and (C6-Cio)aryl, wherein R11each independently is H, (Ci-C4)alkyl, or (C6-Cio)aryl; and R12each independently is H, (Ci-C4)alkyl, (C6-Cio)aryl, or halogen.

[0062] In more particular such embodiments, said penyl-dioxetane luminophore each independently is a compound of the formula I, wherein R1is methyl; and R2and R3together with the carbon atom to which they are attached form adamantyl, cyclobutyl, 2,2- dimethylcyclobutyl, 2,2,4-trimethylcyclobutyl, 2,2,4,4-tetramethylcyclobutyl, 3- phenylcyclobutyl, 3-benzyloxycyclobutyl, 3-carboxycyclobutyl, 3- carbomethoxycyclobutyl, bicyclo[4.2.0]octa-l(6),2,4-trien-7-yl, bicyclo[l.l.l]pentan-2-yl, bicyclo [2. l.l]hexan-5-yl, spiro[3.3]heptan-2-yl, 5-oxo-2-oxaspiro[3.3]heptan-6-yl, 6-oxo- 2-oxaspiro[3.3]heptan-5-yl, 2-methyl-5-oxo-2-azaspiro[3.3]heptan-6-yl, 2-methyl-6-oxo-2- azaspiro[3.3]heptan-5-yl, 2,2-dimethyl-5-oxo-2-azaspiro[3.3]heptan-2-ium-6-yl, 2,2- dimethyl-6-oxo-2-azaspiro[3.3]heptan-2-ium-5-yl, oxetan-3-yl, 2,2-dimethyloxetan-3-yl, 2,2,4-trimethyloxetan-3-yl, 2,2,4,4-tetramethyloxetan-3-yl, azetidin-3-yl, 1-methylazetidin-3-yl, l-ethylazetidin-3-yl, l-phenylazetidin-3-yl, l-diphenylmethyl-azetidin-3-yl, 1,1- dimethy lazetidin- 3 -y lium, 1 -(methoxy c arbony 1) azetidin- 3 -y 1, 1 - (tert- butoxycarbonyl)azetidin-3-yl, l-acetylazetidin-3-yl, 1 -(phenylsulfonyl) azetidin-3-yl, thietan-3-yl, l-oxidothietan-3-yl, l,l-dioxidothietan-3-yl; cyclohexyl, 3-carboxycyclohexyl, 3-carbomethoxycyclohexyl, 3-carboethoxycyclohexyl, 4-carboxycyclohexyl, 4- carbomethoxycyclohexyl, 4-carboethoxycyclohexyl, (chlorocarbonyl)cyclohexyl, (bromocarbonyl)cyclohexyl, fluorocyclohexyl, difluorocyclohexyl, 3,3-difluorocyclohexyl, 4,4-difluorocyclohexyl, chlorocyclohexyl, dichlorocyclohexyl, 3,3-dichlorocyclohexyl, 4,4- dichlorocyclohexyl, cyanocyclohexyl, 3 -cyanocyclohexyl, 4-cyanocyclohexyl, nitrocyclohexyl, 3 -nitrocyclohexyl, 4-nitrocyclohexyl, 3 -tetrahydropyranyl, 4- tetrahydropyranyl, l,3-dioxan-5-yl, 2,2-dimethyl-l,3-dioxan-5-yl, 4-tetrahydrothiopyranyl, 1 -oxido-4-tetrahydrothiopyranyl, or 1 , 1 -dioxido-4-tetrahydrothiopyranyl.

[0063] In certain of those embodiments defined hereinabove, one of R4, R5and R6is a π* acceptor group of the formula -CH=CH-E, and the other two of R4, R5and R6each independently is H or halogen, preferably chlorine, i.e., (i) R4and R5each independently is H or halogen; and R6is a π* acceptor group of the formula -CH=CH-E; (ii) R4and R6each independently is H or halogen; and R5is a π* acceptor group of the formula -CH=CH-E; or (iii) R5and R6each independently is H or halogen; and R4is a π* acceptor group of the formula -CH=CH-E. In preferred such embodiments, R4and R6each independently is H or halogen; and R5is a π* acceptor group of the formula -CH=CH-E, more preferably R4is halogen; R5is a π* acceptor group of the formula -CH=CH-E; and R6is H. Particular such embodiments, are those wherein E is -CN; -COOH; -COO(Ci-C8)alkyl, preferably - COO(C1-C4)alkyl such as -COOCH3 and -COOC(CH3)3; or -COO(C2-C8)alkenyl such as - COOCH=CH-CH3.

[0064] In other of those embodiments defined hereinabove, R4, R5and R6each independently is H or halogen, preferably chlorine, i.e., none of R4, R5and R6is a it* acceptor group. In preferred such embodiments, R4is halogen, and R5and R6each is H.

[0065] In further of those embodiments defined hereinabove, R4is selected from H, halogen, preferably chlorine, and -CN; one of R5and R6, preferably R6, together with R7, forms a 6-membered structure selected fromone of R5and R6is H. Preferred such 6-membered structures are those wherein R13is (Ci- C4)alkyl; and R14is selected from -CN, -NO2, (C2-C4)alkenyl, phenyl, -COOR15, -COO- Kat+, and -C(O)N(R15)2, wherein R15each independently is H or CH3. In particular such embodiments, R4is H or halogen; R5is H; and R6together with R7forms said 6-membered structure. In preferred such embodiments, the 6-membered structure formed

[0066] The term "caging group" as used herein denotes a group, also referred to herein as a “triggering substrate”, masking a phenyl-dioxetane luminophore and cleavable by a bacterial analyte or enzyme such as those specifically listed above. Such a caging group may be, without being limited to, an enzyme cleavable group (also referred to herein as “enzyme- labile group”), i.e., a group that can be removed (at least in part) or modified by means of a specific enzyme, e.g., a monosaccharide moiety linked through a carbon atom thereof or an amino acid residue linked via amide bond through the a-carboxyl group thereof, or a boron- containing group of the formula -B(Z)(Z') or -B(Z")3- Kat+, wherein Z, Z’ and Z" are each as defined herein, and Kat+is a cation as defined herein, preferably an alkali metal (e.g., sodium or potassium) cation.

[0067] According to the method of the present invention, each one of the phenyl-dioxetane luminophores composing the array provided in step (i) is masked with a different triggering substrate, i.e., a caging group cleavable by a different bacterial analyte or enzyme, and thus generates an unstable phenolate-dioxetane compound (which is then decomposed through a chemiexcitation process to produce an excited intermediate that decays to its ground-state through emission of light) upon exposure to a different bacterial analyte or enzyme. Depending on the actual number of chemiluminescent probes composing the array utilized, said array is thus capable of “sensing”, i.e., reacting (by means of emission of light) to thepresence of, dozens or even hundreds of different bacterial analytes or enzymes. The quantity and intensity of emission of light detected over time, e.g., over about 5, 10, 15, 20, 25, 30, or 45 minutes, from each one of the chemiluminescent probes for which emission of light is detected, indicate the amount of the specific bacterial analyte or enzyme, which was the trigger for said emission, in the aqueous solution tested.

[0068] This way, after applying the unknown bacteria-containing aqueous medium to each one of the chemiluminescent probes of said array, and detecting the emission of light from each one of said chemiluminescent probes, a normalized metabolic activity profile of said bacteria is generated. Said normalized metabolic activity profile represents the quantity and intensity of emission of light detected from each one of the chemiluminescent probes, and in fact constitutes a “barcode” of said unknown bacteria, indicating which bacterial analytes or enzymes are produced and secreted by said bacteria and thus present in the aqueous medium, and in which quantities. The “barcode” generated is then compared to a reference bank, or subjected to one or more statistical methods and / or deep learning models, so as to identify said unknown bacteria.

[0069] In certain embodiments, the caging group masking a phenyl-dioxetane luminophore composing the array provided in step (i), e.g., R8in the compound of the formula I, according to any one of the embodiments above, each independently is a boron- containing group of the formula -B(Z)(Z') or -B(Z"):f Kat+as defined above, or an enzyme- labile group selected from the groups listed in Table 4.Table 4: Certain enzyme-labile groups for use as caging groups according to the method of the present invention (in all cases except for those marked with asterisk, the name given refers to the whole molecule rather than the group shown)

[0070] In specific embodiments, the phenyl-dioxetane luminophore composing said array each independently is a compound of the formula I, wherein R1is methyl; R2and R3together with the carbon atom to which they are attached form adamantyl, cyclobutyl, 3- phenylcyclobutyl, 3-benzyloxycyclobutyl, 3-carbomethoxycyclobutyl, 2,2- dimethylcyclobutyl, 2,2,4,4-tetramethylcyclobutyl, 3 -carboxy cyclobutyl, bicyclo[4.2.0]octa-l(6),2,4-trien-7-yl, spiro[3.3]heptan-2-yl, oxetan-3-yl, 2, 2,4,4- tetramethyloxetan-3-yl, l-acetylazetidin-3-yl, l-(terZ-butoxycarbonyl)azetidin-3-yl, 1- (methoxycarbonyl)azetidin-3-yl, l-diphenylmethyl-azetidin-3-yl, cyclohexyl, 4- carbomethoxycyclohexyl, 4-tetrahydropyranyl, 3 -tetrahydropyranyl, 4,4- difluorocyclohexyl, 2,2-dimethyl-l,3-dioxan-5-yl, or l,l-dioxido-4-tetrahydrothiopyranyl;R4is halogen; R6is H; R5is a π* acceptor group of the formula -CH=CH-E, wherein E is - COOH, -COO(Ci-C4)alkyl such as -COOCH3 and -COOC(CH3)3, or -COO(C2-C4)alkenyl such as -COOCH=CH-CH3; and (i) L is LI wherein M is absent; and R8is P-glucoside, P- glucuronide, P-galactoside, L-pyroglutamyl-amide, phosphate ester, leucine amide, nitro, A^,3-dimethyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa- 1 ,4-dien- 1 -yl)butanamide (herein also referred to also as “quinone”), phenylacetamide, or acetamide; or (ii) L is absent; and R8is R8is ((6R,7R)-2-carboxy-8-oxo-7-(2-phenylacetamido)-5-thia-l-azabicyclo[4.2.0]oct-2-en- 3-yl)methyl (herein also referred to as “lactame”), or 3,4-dihydroxybutyl. More specific such phenyl-dioxetane luminophores exemplified herein are those wherein R1is methyl; R2and R3together with the carbon atom to which they are attached form adamantyl; R4is chlorine; R5is a π* acceptor group of the formula -CH=CH-E, wherein E is -COOH; R6is H; and (a) L is LI wherein M is absent; and R8is P-glucoside (herein identified “Probe 1”); (b) L is LI wherein M is absent; and R8is P-glucuronide (herein identified “Probe 2”); (c) L is LI wherein M is absent; and R8is P-galactoside (herein identified “Probe 3”y (d) L is LI wherein M is absent; and R8is L-pyroglutamyl-amide (herein identified “Probe 4”); (e) L is LI wherein M is absent; and R8is phosphate ester (herein identified “Probe 5”); (f) L is LI wherein M is absent; and R8is leucine amide (herein identified “Probe 6”), (g) L is LI wherein M is absent; and R8is nitro (herein identified “Probe 7'y (h) L is LI wherein M is absent; and R8is A^,3-dimethyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-l,4-dien-l- yl)butanamide (herein identified “Probe 8”); (i) L is absent; and R8is ((6R,7R)-2-carboxy- 8-oxo-7-(2-phenylacetamido)-5-thia-l-azabicyclo[4.2.0]oct-2-en-3-yl)methyl (herein identified “Probe 9”); (j) L is LI wherein M is absent; and R8is phenylacetamide (herein identified “Probe 10”), (k) L is absent; and R8is 3,4-dihydroxybutyl (herein identified “Probe 11”); or (1) L is LI wherein M is absent; and R8is acetamide (herein identified “Probe 12”) (Table 5).Table 5: Specific Probes exemplified herein

[0071] In another aspect, the present invention provides a computing system comprising:at least one processor; and at least one memory communicatively coupled to the at least one processor comprising computer-readable instructions that when executed by the at least one processor cause the computing system to implement a method for detection and identification of a bacteria, also referred to herein as "an unknown bacteria”, in an aqueous medium, the method comprising:(v) providing an array comprising a plurality of chemiluminescent probes, each composed of a phenyl-dioxetane luminophore masked by a caging group cleavable by bacterial analyte or enzyme, wherein each one of said phenyl-dioxetane luminophores is masked with a caging group cleavable by a different bacterial analyte or enzyme and upon exposure to said bacterial analyte or enzyme, said caging group is cleaved from said phenyl-dioxetane luminophore thereby generating an unstable phenolate-dioxetane compound, which is then decomposed through a chemiexcitation process to produce an excited intermediate that decays to its ground-state through emission of light;(vi) applying said aqueous medium to each one of said plurality of chemiluminescent probes of said array (i.e., exposing each one of said plurality of chemiluminescent probes of said array to said aqueous medium);(vii) detecting the emission of light from each one of said chemiluminescent probes, thereby generating a normalized metabolic activity profile for said bacteria, said normalized metabolic activity profile representing the quantity and intensity of emission of light detected from each one of the chemiluminescent probes; and(viii) applying via said at least one processor one or more data analysis methods, e.g., statistical methods and / or machine learning models, on said normalized metabolic activity profile to identify said bacteria.

[0072] The emission of light from each one of the chemiluminescent probes composing said array can be detected utilizing any technique or procedure known in the art.

[0073] Optical molecular imaging is a promising technique that provides a high degree of sensitivity and specificity in tumor margin detection. Furthermore, existing clinical applications have proven that optical molecular imaging is a powerful intraoperative tool for guiding surgeons performing precision procedures, thus enabling radical resection andimproved survival rates. An example of a clinically approved instrument for minimally invasive surgical procedures under fluorescence guidance is the da Vinci Surgical System. This instrument is featured with a 3D HD vision system for a clear and magnified view inside a patient's body and allows surgeons to perform complex and routine procedures through a few small openings, similar to traditional laparoscopy. In addition, the following systems have already been applied in surgeries for breast cancer, liver metastases and bypassing graft surgery: The Hamamatsu's Photodynamic Eye (PDE™), Artemis™ and Novadaq SPY™ (Novadaq Technologies Inc., Toronto, Canada) (Chi et al., 2014). Several existing intraoperative NIR fluorescence molecular imaging systems were evaluated in clinical trials, including Fluobeam®, FLARE™ and GXMI Navigator. They have played an important role in operation convenience, improving image assessment and increasing detection depth.

[0074] In recent years, there has been a great progress in the development of cameras and lasers for optical fluorescence imaging in the IR range. In parallel, there is a vast clinical use of low MW organic dyes such as ICG and methylene blue for determining cardiac output, hepatic function and liver blood flow, and for ophthalmic angiography. In 2015, the fluorescence imaging system, Xiralite®, gained FDA approval for visualization of microcirculation in the hands (for inflammation and perfusion-related disorders).

[0075] The invention will now be illustrated by the following non-limiting Examples.EXAMPLESStudy 1. Enzymatic activity profiling using an ultrasensitive array of chemiluminescent probes for bacterial classification and characterizationMATERIALS AND METHODSAbbreviations

[0076] ACN, acetonitrile; AgiO, silver oxide; CHCh, chloroform; DCM, dichloromethane; DIPEA, AW-diisopropylcthylaminc; DMBA, dimethyl barbituric acid; DMF, A,V-di methyl formamide; DMSO, dimethylsulfoxide; EEDQ, A-ethoxycarbonyl-2- ethoxy-l,2-dihydroquinoline; EtOAc, ethyl acetate; Hex, hexane; iPrOH, isopropyl alcohol; K2CO3, potassium carbonate; LiOH, lithium hydroxide; MB, methylene blue; MeOH, methanol; NH4CI, ammonium chloride; NaHCO.v sodium bicarbonate; NarSrO.v sodium thiosulfate; NaiSCh, sodium sulfate; NaBH4, sodium borohydride; Nal, sodiumiodide; PBra, phosphorus tribromide; Pd(PPh3)4, tetrakis(triphenylphosphine)palladium; THE, tetrahydrofuran; TLC, thin-layer chromatography; TMS-C1, trimethylsilyl chloride.General methods

[0077] All reactions requiring anhydrous conditions were performed under an Argon atmosphere. All reactions were carried out at room temperature unless stated otherwise. Chemicals and solvents were either A.R. grade or purified by standard techniques. Thin- layer chromatography (TLC): silica gel plates Merck 60 F254: compounds were visualized by irradiation with UV light. Column chromatography (FC): silica gel Merck 60 (particle size 0.040-0.063 mm), eluent given in parentheses. Reverse-phase high-pressure liquid chromatography (RP-HPLC): C18 5u, 250x4.6mm, eluent given in parentheses. Preparative RP-HPLC: C18 5u, 250x21mm, eluent given in parentheses.XH-NMR spectra were measured using Bruker Avance operated at 400MHz.13C-NMR spectra were measured using Bruker Avance operated at 100 MHz. Chemical shifts were reported in ppm on the 6 scale relative to a residual solvent (CDCL: 6=7.26 forXH-NMR and 77.16 for13C-NMR, DMSO- dr>: 6=2.50 for ’ H-NMR and 39.52 for13C-NMR). Multiplicities are reported with the following abbreviations: br, broad; s, singlet; d, doublet; t, triplet; dt, doublet of triplets; dd, doublet of doublets; ddd, doublet of doublet of doublets; qd, quartet of doublets; m, multiplet; eq, equatorial; ax, axial. Coupling constants (J) are given in Hertz. Mass spectra were measured on Waters Xevo TQD. Chemiluminescence was recorded on Molecular Devices Spectramax iD3. Fluorescence was recorded on Tecan infinite 200 Pro. All chemicals, unless otherwise stated, were obtained from commercial sources. Light irradiation for photochemical reactions: LED PAR38 lamp (19W, 3000K).

[0078] Synthesis of nitroreductase Probe 7 and NAD(P)H quinone oxidoreductuse- 1 Probe 8 were carried out according to known procedures (Ryan et al., 2019; Son et al., 2019).B-slucosidase Probe 1

[0079] Compound 1. As depicted in Scheme 1, phenol enol ether b (Hananya et al., 2019) (40 mg, 0.10 mmol, 1.2 eq.) and K2CO3 (21 mg, 0.15 mmol, 1.5 eq.) were dissolved in DMF (1 mL). The solution was stirred for 5 minutes before the addition of compound a (Gnaim et al., 2019) (44 mg, 0.08 mmol, 1 eq.). The reaction mixture was stirred at room temperature and monitored by TLC (EtOAc / Hex mixture). Upon completion, the reaction mixture was diluted with EtOAc (100 mL) and washed with 0.1M HC1 (50 mL) and brine (50 mL). Theorganic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc: Hex 6:4). Compound 1 was obtained as a white solid (61 mg, 72% yield). MS (ES-): m / z calc, for C43H49CIO14: 824.28; found: 847.7 [M+Na]+.1H NMR (400 MHz, CDCI3) 67.94 (d, 7=16.2 Hz, 1H), 7.46 (d, 7=8.3 Hz, 3H), 7.09 (d, 7=8.0 Hz, 1H), 7.03 (d, 7=8.6 Hz, 2H), 6.47 (d, 7=16.2 Hz, 1H), 5.33-5.28 (m, 1H), 5.23-5.14 (m, 7=9.8 Hz, 1H), 5.14-5.07 (m, 1H), 4.96 (d, 7=3.5 Hz, 2H), 4.37-4.27 (m, 1H), 4.19 (dd, 7=12.3, 2.4 Hz, 1H), 3.89 (ddd, 7=10.0, 5.2, 2.4 Hz, 1H), 3.82 (s, 3H), 3.34 (s, 3H), 3.30 (d, 7=4.3 Hz, 1H), 2.11 (s, 3H), 2.09 (s, 4H), 2.07 (s, 3H), 2.06 (s, 3H), 2.01-1.67 (m, 13H).13C NMR (100 MHz, CDCI3) 6 170.6, 170.3, 169.4, 169.3, 167.1, 157.1, 153.7, 139.4, 138.8, 138.2, 132.5, 131.1, 130.4, 127.8, 125.0, 119.9, 117.1, 99.2, 75.6, 72.7, 72.1, 71.2, 68.3, 61.9, 57.3, 51.8, 39.2, 39.0, 38.6, 37.0, 32.9, 29.7, 28.3, 28.2, 20.7, 20.6.

[0080] Compound 2. Compound 1 (61 mg, 0.07 mmol, 1 eq.) and LiOH (18 mg, 0.7 mmol, 10 eq.) were dissolved in 1 mL solution of THF: H2O (4:1). The reaction mixture was stirred at 40°C and monitored by RP-HPLC. Upon completion, the solvent was concentrated under reduced pressure, and the product was purified by preparative RP-HPLC (mobile phase: acetonitrile in H2O containing 0.1% TFA; gradient from 70 to 100%; flow rate: 20 mL / min; retention time: 6 min). Compound 2 was obtained as a white solid (50 mg, 77% yield). MS (ES+): m / z calc, for C34H39CIO10: 642.22; found: 641.6 [M-H]“1H NMR (400 MHz, CDCI3) 8 7.94 (d, 7=16.2 Hz, 1H), 7.46 (d, 7=8.3 Hz, 3H), 7.09 (d, 7=8.0 Hz, 1H), 7.03 (d, 7=8.6 Hz, 2H), 6.47 (d, 7=16.2 Hz, 1H), 5.33-5.28 (m, 1H), 5.23-5.14 (m, 7=9.8 Hz, 1H), 5.14-5.07 (m, 1H), 4.96 (d, 7=3.5 Hz, 2H), 4.37-4.27 (m, 1H), 4.19 (dd, 7=12.3, 2.4 Hz, 1H), 3.89 (ddd, 7=10.0, 5.2, 2.4 Hz, 1H), 3.82 (s, 3H), 3.34 (s, 3H), 3.30 (d, 7=4.3 Hz, 1H), 2.11 (s, 3H), 2.09 (s, 4H), 2.07 (s, 3H), 2.06 (s, 3H), 2.01-1.67 (m, 13H).13C NMR (100 MHz, CDCI3) 6170.6, 170.3, 169.4, 169.3, 167.1, 157.1, 153.7, 139.4, 138.8, 138.2, 132.5, 131.1, 130.3, 127.8, 125. 1, 119.9, 117.1, 99.2, 75.6, 72.7, 72.1, 71.2, 68.3, 61.9, 57.3, 51.8, 39.2, 39.0,38.6, 37.0, 32.9, 29.7, 28.3, 28.2, 20.7, 20.6.

[0081] P-glucosidase Probe 1. Compound 2 (10 mg, 0.02 mmol) and a catalytic amount of methylene blue (~1 mg) were dissolved in 10 mL of DCM. Oxygen was bubbled through the solution while irradiating with yellow light. The reaction was monitored by RP-HPLC. Upon completion, the solvent was concentrated under reduced pressure, and the product was purified by preparative RP-HPLC (mobile phase: acetonitrile in H2O containing 0.1% TFA; gradient from 70 to 100%; flow rate: 20 mL / min; retention time: 9 min). / / -GlucosidaseProbe 1 was obtained as a white solid (8.3 mg, 79% yield). MS (ES-): m / z calc, for C34H39C1O12: 674.21; found: 673.6 [M-H]“ ’ H NMR (400 MHz, CDCI3) 6 7.61 (d, 7=16.1 Hz, 1H), 7.34 (d, 7=8.1 Hz, 1H), 7.24 (d, 7=8.3 Hz, 2H), 7.05-6.94 (m, 7=8.9 Hz, 3H), 6.16 (d, 7=6.1 Hz, 1H), 5.00-4.84 (m, 7=24.8, 6.6 Hz, 3H), 3.91 (d, 7=10.7 Hz, 1H), 3.86-3.54 (m, 7=21.4, 12.8, 6.7 Hz, 9H), 3.53-3.44 (m, 7=8.5 Hz, 1H), 3.30 (s, 3H), 3.24 (s, 1H), 2.05 (s, 1H), 1.97-1.60 (m, 12H).13C NMR (100 MHz, CDCI3) 6 169.1, 157.4, 153.6, 139.7, 139.4, 138.0, 132.6, 130.9, 130.1, 129.6, 127.7, 124. 8, 119.4, 116.7, 100.4, 76.2, 76.0, 73.2, 69.5, 61.3, 57.2, 39.1, 39.0, 38.6, 37.0, 32.9, 29.7, 28.3, 28.1. β-glucuronidase Probe 2

[0082] Compound 3. As depicted in Scheme 2, Ag2O (145 mg, 0.63 mmol, 2.5 eq.) was added to acetobromo-a-D-glucuronic acid methyl ester (100 mg, 0.25 mmol, 1 eq.) and 4- hydroxybenzaldehyde (34 mg, 0.28 mmol, 1.1 eq.) dissolved in ACN. The reaction mixture was stirred at room temperature and monitored by TLC. Upon completion, ACN was evaporated under reduced pressure and the crude product was purified by column chromatography on silica gel (EtOAc: Hex 3:7) to afford compound 3 (82 mg, 82% yield) as an off-white solid. ’ H NMR (400 MHz, CDCI3) 6 9.89 (s, 1H), 7.83 (d, 7=8.8 Hz, 2H), 7.09 (d, 7=8.7 Hz, 2H), 5.38-5.32 (m, 2H), 5.30-5.27 (m, 2H), 4.28-4.21 (m, 1H), 3.74-3.67 (m, 4H), 2.06-2.00 (m, 12H).13C NMR (100 MHz, CDCI3) 6 190.9, 170.2, 169.5, 169.3, 166.9, 161.2, 132.0, 117.0, 98.1, 72.8, 71.7, 71.0, 69.0, 53.2, 20.7. TheXH-NMR and13C- NMR data are in agreement with the literature values (Zhang et al., 2012).

[0083] Compound 4. Compound 3 (35 mg, 0.08 mmol, 1 eq.) was dissolved in 1 ml of MeOH and DCM (1:1) and cooled to 0°C. NaBH4 (3.4 mg, 0.09 mmol, 1.1 eq.) was added to the reaction mixture and the reaction was allowed to warm up to room temperature. The reaction mixture was stirred at room temperature and monitored by TLC (EtOAc: Hex 6:4). Upon completion, the reaction mixture was diluted with EtOAc (100 mL) and washed with NH4CI (50 mL) and brine (50 mL). The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude mixture was reacted without further purification. The crude was dissolved in DCM followed by dropwise addition of PB13 (14 pL, 0.15mmol, 1.8 eq.). The reaction was monitored by TLC (EtOAc: Hex 6:4). Upon completion, the reaction mixture was diluted with EtOAc, and washed with saturated NaHCOs followed by brine. The organic layer was separated, dried over Na2SO4, and filtered and the solvent was evaporated under reduced pressure, the crude product was purified bycolumn chromatography on silica gel (EtOAc: Hex 6:4) to afford compound 4 (70 mg, 65% yield) a yellow oil.1H NMR (400 MHz, CDC13) 6 7.32 (d, 7=8.0 Hz, 2H), 6.95 (d, 7=8.0 Hz, 2H), 5.38-5.24 (m, 3H), 5.15 (d, 7=7.1 Hz, 1H), 4.47 (s, 1H), 4.18 (d, 7=8.0 Hz, 1H), 3.72 (s, 3H), 2.04 (s, 9H).13C NMR (101 MHz, CDCI3) 6 170.2, 169.5, 169.4, 167.0, 156.7, 133.1, 130.6, 117.4, 99.0, 72.8, 71.9, 71.2, 69.2, 53.1, 33.2, 29.8, 20.7.

[0084] Compound 5. Phenol enol ether b (20 mg, 0.04 mmol, 1 eq.) and K2CO3 (9 mg, 0.06 mmol, 1.5 eq.) were dissolved in DMF (0.5 mL). The solution was stirred for 5 minutes before compound 4 (16 mg, 0.04 mmol, 1 eq.) was added. The reaction mixture was stirred at room temperature and monitored by TLC (EtOAc: Hex 6:4). Upon completion, the reaction mixture was diluted with EtOAc (100 mL) and washed with saturated NH4CI (50 mL) and brine (50 mL). The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude mixture was reacted without further purification. The crude and LiOH (9.5 mg, 0.39 mmol, 10 eq.) were dissolved in 1 mL solution of THF: H2O (4:1). The reaction mixture was stirred at 60°C and monitored by RP-HPLC. Upon completion, the solvent was concentrated under reduced pressure and the product was purified by preparative RP-HPLC (mobile phase: acetonitrile in H2O containing 0.1% TFA; gradient from 30 to 100%; flow rate: 20 mL / min; retention time: 7 min). Compound 5 was obtained as a white solid (14 mg, 55% yield). MS (ES+): m / z calc, for C34H37CIO11: 656.20; found: 679.5 [M+Na]+.1H NMR (400 MHz, CDCI3) 6 7.56- .45 (m, 7=16.0 Hz, 1H), 7.23 (s, 1H), 7.12-7.04 (m, 2H), 6.99-6.87 (m, 7=7.0 Hz, 3H), 6.08 (d, 7=15.9 Hz, 2H), 5.05-4.92 (m, 1H), 4.83-4.62 (m, 2H), 4.24-4.07 (m, 1H), 3.97-3.73 (m, 3H), 3.27-3.16 (m, 4H), 2.04- 1.97 (m, 2H), 1.93-1.56 (m, 12H).13C NMR (100 MHz, CDCI3) 6 172.5, 169.9, 157.4, 153.7, 140.4, 139.5, 138.3, 132.6, 131.2, 130.4, 130.0, 129.7, 127.8, 124.9, 119.1, 117.1, 100.5, 76.3, 75.6, 74.4, 72.9, 71.5, 57.3, 39.2, 39.14, 38.8, 38.7, 38.7, 37.1, 33.0, 29.8, 28.3.

[0085] P-glucuronidase Probe 2. Compound 5 (10 mg, 0.02 mmol) and a catalytic amount of methylene blue (~1 mg) were dissolved in 10 mL of DCM. Oxygen was bubbled through the solution while irradiating with yellow light. The reaction was monitored by RP-HPLC. Upon completion, the solvent was concentrated under reduced pressure and the product was purified by preparative RP-HPLC (mobile phase: acetonitrile in H2O containing 0.1% TFA; gradient from 30 to 100%; flow rate: 20 mL / min; retention time: 5 min). P-Glucuronidase Probe 2 was obtained as a white solid (6 mg, 60% yield). MS (ES-): m / z calc, for C34H37CIO13: 688.19; found: 687.3 [M-H]’ • MS (ES+): m / z calc, for C34H37CIO13: 688.19; found: 711.5 [M-Na]+.XH NMR (400 MHz, DMSO) 8 7.93 (d, 7=8.3 Hz, 1H), 7.79-7.72 (m,2H), 7.38 (d, 7=7.0 Hz, 2H), 7.01 (d, 7=8.4 Hz, 2H), 6.64 (d, 7=16.1 Hz, 1H), 5.49-5.24 (m, 2H), 5.03 (d, 7=7.2 Hz, 1H), 4.86 (s, 2H), 3.94-3.82 (m, 2H), 3.09 (s, 3H), 2.87 (s, 1H), 2.22 (d, 7=11.2 Hz, lH), 2.01 (s, 1H), 1.87 (d, 7=15.3 Hz, 2H), 1.73-1.51 (m, 8H), 1.46 (d, 7=11.1 Hz, 1H), 1.32 (d, 7=11.9 Hz, 1H), 1.24-1.15 (m, 2H).13C NMR (100 MHz, DMSO) 8 170.7,167.7, 157.8, 154.2, 137.3, 134.6, 131.9, 130.8, 130.8, 129.8, 129.00 127.5, 126.7, 123.6, 116.7, 111.8, 100.5, 96.0, 76.3, 76.0, 73.5, 71.9, 50.0, 46.9, 39.7, 39.5, 36.5, 33.7, 32.2, 31.7,31.5. 27.4, 26.1, 25.8. β-salactosidase Probe 3

[0086] As depicted in Scheme 3, Compound c (10 mg, 0.02 mmol) and a catalytic amount of methylene blue (~1 mg) were dissolved in 10 mL of DCM. Oxygen was bubbled through the solution while irradiating with yellow light. The reaction was monitored by RP-HPLC. Upon completion, the solvent was concentrated under reduced pressure and the product was purified by preparative RP-HPLC (mobile phase: acetonitrile in H2O containing 0.1% TFA; gradient from 70 to 100%; flow rate: 20 mL / min; retention time: 5 min). ^-Galactosidase Probe 3 was obtained as a white solid (9.0 mg, 86% yield). MS (ES+): m / z calc, for C34H39CIO12: 674.21; found:675.6 [M-H]+, 697.6 [M-Na]+.XH NMR (400 MHz, DMSO) 8 7.95 (d, 7=8.4 Hz, 1H), 7.83-7.72 (m, 7=12.2, 6.2 Hz, 2H), 7.38 (d, 7=7.6 Hz, 2H), 7.04 (d, 7=8.6 Hz, 2H), 6.65 (d, 7=16.1 Hz, 1H), 4.93-4.79 (m, 3H), 3.70 (d, 7=3.3 Hz, 2H), 3.61- 3.38 (m, 6H), 3.12 (s, 3H), 2.89 (s, 1H), 2.24 (d, 7=11.9 Hz, 1H), 1.91 (s, 1H), 1.77-1.15 (m, 12H).13C NMR (100 MHz, DMSO) 8 167.6, 158.2, 154.0, 137.2, 134.5, 131.8, 130.6, 129.3,128.8. 127.3. 126.5. 123.4, 116.6, 111.7, 101.4, 95.9, 75.9, 73.7, 70.7, 68.6, 60.8, 49.9, 36.3,33.7, 33.5, 32.3, 32.1, 31.6, 31.3, 26.0, 25.6.Pyroslutamy / l aminopeptidase Probe 4

[0087] Compound 6. As depicted in Scheme 4, 4-amino benzyl alcohol (262 mg, 2.13 mmol, 1.1 eq.) and L-pyroglutamic acid (250 mg, 1.93 mmol, 1 eq.) were dissolved in 3 ml of THF followed by the addition of EEDQ (957 mg, 3.87 mmol, 2 eq.). The reaction mixture was stirred at room temperature and monitored by TLC (EtOAc: Hex mixture). Upon completion, the reaction mixture was filtered and washed three times with Et20, to afford compound 6 (400 mg, 88% yield) as a white solid. ’H NMR (400 MHz, DMSO) 8 10.00 (s, 1H), 7.88 (s, 1H), 7.56 (d, 7=8.5 Hz, 2H), 7.25 (d, 7=8.6 Hz, 2H), 5.10 (t, 7=5.7 Hz, 1H), 4.43 (d, 7=5.6 Hz, 2H), 4.18 (dd, 7=8.5, 4.4 Hz, 1H), 2.41-1.91 (m, 4H).13C NMR (100 MHz, DMSO) 8 177.9, 171.6, 138.1, 137.9, 127.4, 119.6, 63.0, 56.8, 29.7, 25.8.

[0088] Compound 7. Compound 6 (400 mg, 1.71 mmol, 1 eq.) was dissolved in 4 ml of ACN and cooled to 0°C. Sodium Iodide (764 mg, 5.13 mmol, 3 eq.) was added followed by the rapid addition of TMS-C1 (647 pl, 5.13 mmol, 3 eq.). The reaction was allowed to warm up to room temperature and monitored by TLC (EtOAc: Hex mixture). Upon completion, the reaction mixture was diluted with EtOAc, and washed with saturated Na2S20s followed by brine. The organic layer was separated, dried over Na2SO4, filtered and the solvent was evaporated under reduced pressure, the crude product was reacted without further purification.

[0089] Phenol enol ether b (Hananya et al. , 2019) (796 mg, 2.05 mmol, 1.2 eq.) and K2CO3 (354 mg, 2.56 mmol, 1.5 eq.) were dissolved in DMF (5 mL). The solution was stirred for 5 minutes before crude product was added. The reaction mixture was stirred at room temperature and monitored by TLC (EtOAc:Hex mixture). Upon completion, the reaction mixture was diluted with EtOAc (100 mL) and washed with 0.1M HC1 (50 mL) and brine (50 mL). The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc: Hex mixture) to afford compound 7 (650 mg, 63% yield) as a white solid. MS (ES+): m / z calc, for C34H37CIN2O6: 604.23; found:627.6 [M+Na]+.1H NMR (400 MHz, CDC13) 8 8.89 (s, 1H), 7.88 (d, 7=16.2 Hz, 1H), 7.64 (d, J=8.4 Hz, 2H), 7.49 (s, 1H), 7.45-7.38 (m, 7=8.2, 4.1 Hz, 3H), 7.08 (d, 7=8.0 Hz, 1H), 6.42 (d, 7=16.2 Hz, 1H), 4.95 (d, 7=3.7 Hz, 2H), 4.32- 4.26 (m, 7=8.4, 4.9 Hz, 1H), 3.79 (s, 3H), 3.32 (s, 3H), 3.28 (s, 1H), 2.60-2.25 (m, 4H), 2.08 (s, 1H), 2.00-1.65 (m, 12H).13C NMR (100 MHz, CDCI3) 6 179.6, 170.5, 167.2, 153.7, 139.4, 138.9, 138.2, 137.9, 132.5, 132.3, 129.8, 129.7, 127.8, 125.1, 120.1, 119.8, 75.7, 57.9, 57.3, 51.8, 39.2, 39.0, 38.6, 37.0, 32.9, 29.7, 29.5, 28.3, 28.2, 25.8.

[0090] Compound 8. Compound 7 (100 mg, 0.16 mmol, 1 eq.) and LiOH (39 mg, 1.6 mmol, 10 eq.) were dissolved in 1 mL solution of THF: H2O (4:1). The reaction mixture was stirred at room temperature and monitored by RP-HPLC. Upon completion, the solvent was concentrated under reduced pressure and the product was purified by preparative RP-HPLC (mobile phase: acetonitrile in H2O containing 0.1% TFA; gradient from 70 to 100%; flow rate: 20 mL / min; retention time: 7.5 min). Compound 8 was obtained as a white solid (59 mg, 61% yield). MS (ES+): m / z calc, for C33H35C1N2O6: 590.22; found: 613.5 [M+Na]+.1H NMR (400 MHz, DMSO) 8 10.10 (s, 1H), 7.86 (s, 1H), 7.76 (d, 7=7.1 Hz, 1H), 7.73 (s, 1H), 7.61 (d, 7=8.5 Hz, 2H), 7.36 (d, 7=8.5 Hz, 2H), 7.08 (d, 7=8.0 Hz, 1H), 6.55 (d, 7=16.1 Hz,1H), 4.93 (d, 7=16.3 Hz, 2H), 4.17 (dd, 7=8.5, 4.3 Hz, 1H), 3.20 (s, 3H), 3.16 (s, 1H), 2.51- 2.43 (m, 2H), 2.37-2.27 (m, 1H), 2.23-2.09 (m, 2H), 1.99-1.63 (m, 13H).13C NMR (100 MHz, CDC13) 6 170.8, 168.9, 153.4, 139.3, 138.0, 137. 9, 132.6, 132.0, 130.0, 129.7, 127.8, 124.9, 120.1, 119.8, 76.0, 57.6, 57.2, 39.1, 39.0, 38.6, 37.0, 32.9, 29.7, 29.4, 28.3, 28.1, 25.4

[0091] Pyroglutamyl aminopeptidase Probe 4. Compound 8 (10 mg, 0.02 mmol) and a catalytic amount of methylene blue (~1 mg) were dissolved in 10 mL of DCM. Oxygen was bubbled through the solution while irradiating with yellow light. The reaction was monitored by RP-HPLC. Upon completion, the solvent was concentrated under reduced pressure, and the product was purified by preparative RP-HPLC (mobile phase: acetonitrile in H2O containing 0.1% TFA; gradient from 70 to 100%; flow rate: 20 mL / min; retention time: 6 min). Pyroglutamyl aminopeptidase Probe 4 was obtained as a white solid (8.8 mg, 84% yield). MS (ES+): m / z calc, for C33H35C1N2O8: 622.21; found: 645.5 [M-H]+.1H NMR (400 MHz, CDCI3) 6 8.81 (s, 1H), 7.97-7.89 (m, 1H), 7.66 (dd, 7=26.2, 16.1 Hz, 1H), 7.56 (t, 7=6.5 Hz, 2H), 7.19 (dd, 7=12.2, 8.4 Hz, 2H), 6.27 (dd, 7=21.5, 16.1 Hz, 1H), 5.06 (dd, J=31.5, 11.4 Hz, 1H), 4.85 (dd, 7=39.4, 11.3 Hz, 1H), 4.42 (s, 1H), 3.28 (d, 7=4.8 Hz, 3H), 3.06 (s, 1H), 2.67-2.26 (m, 4H), 2.04 (s, 1H), 1.94-1.24 (m, 12H).13C NMR (100 MHz, CDCI3) 6 181.3, 181.2, 171.0, 169.7, 139.8, 139.7, 138.0, 135.4, 132.1, 131.9, 131.6, 130.4, 130.3, 128.9, 127.7, 124.8, 120.0, 111.7, 96.4, 58.5, 49.8, 36.6, 33.9, 33.6, 32.7, 32.23, 31.6, 29.6, 26.1, 25.9, 25.5Phosphatase Probe 5

[0092] Compound 9. As depicted in Scheme 5, compound d (100 mg, 0.35 mmol, 1 eq.) was dissolved in 1 ml DCM and cooled to 0°C followed by dropwise addition of PB13 (60 pl, 0.63 mmol, 1.8 eq.). The reaction was monitored by TLC (EtOAc: Hex mixture). Upon completion, the reaction mixture was diluted with EtOAc, and washed with saturated NaHCOs followed by brine. The organic layer was separated, dried over Na2SO4, filtered and the solvent was evaporated under reduced pressure, the crude product was purified by column chromatography on silica gel (EtOAc: Hex gradient) to afford compound 9 (51 mg, 41% yield) as a yellow oil. MS (ES+): m / z calc, for C13H16BrO4P: 346.00; found: 371.2 [M- H]+. ’ H NMR (400 MHz, CDCI3) 6 7.26 (d, 7=8.5 Hz, 2H), 7.12 (d, 7=7.8 Hz, 2H), 5.94- 5.81 (m, 2H), 5.33 (dd, 7=17.1, 1.4 Hz, 2H), 5.22 (dd, 7=10.4, 1.1 Hz, 2H), 4.63-4.52 (m, 6H).13C NMR (100 MHz, CDCI3) 6 132.0, 131.9, 130.5, 120.4, 120.4, 118.8, 69.0, 68.9,32.6.

[0093] Compound 10. Phenol enol ether e (Green et al. , 2017) (51 mg, 0.12 mmol, 1.2 eq) and K2CO3 (25 mg, 0.18 mmol, 1.5 eq) were dissolved in DMF (1 mL). The solution was stirred for 5 minutes before adding compound 9 (42 mg, 0.12 mmol, 1 eq). The reaction mixture was stirred at room temperature and monitored by TLC (EtOAc / Hex mixture). Upon completion, the reaction mixture was diluted with EtOAc (100 mL) and washed with 0.1M HC1 (50 mL) and brine (50 mL). The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc: Hex gradient). Compound 10 was obtained as a white solid (83 mg, 81% yield). MS (ES+): m / z calc, for C37H42C1O8P: 680.23; found: 703.6 [M+H]+.1H NMR (400 MHz, CDCI3) 67.96 (d, 7=16.2 Hz, 1H), 7.49-7.43 (m, 3H), 7.23 (d, 7=7.8 Hz, 2H), 7.08 (d, 7=8.0 Hz, 1H), 6.48 (d, 7=16.2 Hz, 1H), 6.05-5.88 (m, 3H), 5.41- 5.34 (m, 3H), 5.32-5.24 (m, 3H), 4.96 (d, 7=3.9 Hz, 2H), 4.71 (dt, 7=5.7, 1.2 Hz, 2H), 4.68- 4.61 (m, 4H), 3.32 (s, 3H), 3.27 (s, 1H), 2.07 (s, 1H), 1.99-1.63 (m, 12H).13C NMR (100 MHz, CDCI3) 6 166.2, 153.6, 150.8, 150.7, 139.4, 138.8, 138.3, 132.9, 132.5, 132.2, 132.1, 132.0, 130.3, 129.8, 129.5, 127.9, 125.1, 120.2, 120.1, 118.7, 118.3, 75.3, 68.9, 68.8, 65.3, 57.3, 39.2, 39.0, 38.6, 37.0, 32.9, 29.7, 28.3, 28.2.

[0094] Phosphatase Probe 5. Compound 10 (30 mg, 0.4 mmol, 1 eq.) was dissolved in DCM (2 ml), followed by the addition of DMBA (20 mg, 0.13 mmol, 3 eq.) and tetrakis(triphenylphosphine)palladium(5.1 mg, 0.004 mmol, 0.1 eq.). The reaction was stirred at room temperature and monitored by RP-HPLC. Upon completion, additional DCM was added (8 mL), followed by the addition of a catalytic amount of methylene blue. Then, oxygen was bubbled through the solution while irradiating with yellow light. The reaction was monitored by RP-HPLC. Upon completion, the solvent was concentrated under reduced pressure, and the product was purified by preparative RP-HPLC (mobile phase: acetonitrile in H2O containing 0.1% TFA; gradient from 70 to 100%; flow rate: 20 mL / min; retention time: 5 min). Phosphatase Probe 5 was obtained as a white solid (9.4 mg, 36% yield). MS (ES-): m / z calc, for C28H3oC1010P: 592.13; found: 591.4 [M-H]“. ’ H NMR (400 MHz, DMSO) 87.97 (d, 7=8.4 Hz, 1H), 7.82-7.74 (m, 2H), 7.46 (d, 7=8.5 Hz, 2H), 7.19 (dd, 7=8.6, 1.0 Hz, 2H), 6.66 (d, 7=16.1 Hz, 1H), 4.89 (d, 7=5.0 Hz, 2H), 3.13 (s, 3H), 2.89 (s, 1H), 2.25 (d, 7=12.7 Hz, 1H), 1.92 (s, 1H), 1.78-1.31 (m, 11H).13C NMR (100 MHz, DMSO) 6 168.4, 163.6, 155.0, 154.9, 153.9, 140.4, 135.6, 133.9, 132.8, 130.4, 129.09, 128.7, 127.2, 126.1,120.2, 112.1, 111.7, 95.8, 76.1, 49.7, 36.3, 33.8, 33.5, 32.3, 32.12, 31.6, 31.3, 29.9, 29.34,27.4, 27.1, 26.0, 25.7.31P NMR (162 MHz, CDC13) 6 -6.32.Leucine aminopeptidase Probe 6

[0095] As depicted in Scheme 6, compound f (Wang et al., 2022) (25 mg, 0.04 mmol, 1 eq) and LiOH (10 mg, 0.41 mmol, 10 eq) were dissolved in 1 mL solution of THF: H2O (4:1). The reaction mixture was stirred at room temperature and monitored by RP-HPLC. Upon completion, the reaction mixture was diluted with EtOAc (100 mL) and washed with 0.1M HC1 (50 mL) and brine (50 mL). The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was reacted without further purification, and a catalytic amount of methylene blue (~1 mg) was dissolved in 10 mL of DCM. Oxygen was bubbled through the solution while irradiating with yellow light. The reaction was monitored by RP-HPLC. Upon completion, the solvent was concentrated under reduced pressure and the product was purified by preparative RP-HPLC (mobile phase: acetonitrile in H2O containing 0.1% TFA; gradient from 50 to 100%; flow rate: 20 mL / min; retention time: 11 min). Leucine aminopeptidase Probe 6 was obtained as a white solid (19 mg, 80% yield). MS (ES-): m / z calc, for C34H41C1N2O7: 624.26; found:625.7 [M-H]“. ’ H NMR (400 MHz, DMSO) 8 10.59 (s, 1H), 8.24 (s, 2H), 7.94 (d, 7=8.4 Hz, 1H), 7.83-7.70 (m, 2H), 7.62 (d, 7=8.4 Hz, 2H), 7.43 (d, 7=8.4 Hz, 2H), 6.63 (d, 7=16.1 Hz, 1H), 4.89 (dd, 7=20.5, 10.8 Hz, 2H), 3.91 (s, 1H), 3.10 (s, 3H), 2.87 (s, 1H), 2.21 (d, 7=11.5 Hz, 1H), 1.90 (s, 1H), 1.78-1.50 (m, 11H), 1.45 (d, 7=11.8 Hz, 1H), 1.31 (d, 7=4.3 Hz, 1H), 1.27-1.11 (m, 2H), 0.92 (d, 7=2.4 Hz, 6H).13C NMR (100 MHz, 5% MeOD in CDCI3) 6 170.0, 168.6,154.4, 140.4, 137.3, 135.6, 132.6, 131.8, 130.3, 129.0, 127.6, 124.9, 121.0, 119.9, 111.7,96.4, 53.5, 49.7, 40.5, 36.6, 33.9, 33.6, 32.6, 32.2, 31.5, 29.7, 26.1, 25.8, 24.4, 22.2, 22.1. β-lactamase Probe 9

[0096] Compound 11. As depicted in Scheme 7, phenol enol ether e (57 mg, 0.14 mmol, 1.2 eq.) and K2CO3 (24 mg, 0.17 mmol, 1.5 eq.) were dissolved in DMF (1 mL). The solution was stirred for 5 minutes followed by the addition of compound i (Zhao et al., 2006) (66 mg, 0.11 mmol, 1 eq.). The reaction mixture was stirred at room temperature and monitored by TLC (EtOAc / Hex mixture). Upon completion, the reaction mixture was diluted with EtOAc (100 mL) and washed with 0.1M HC1 (50 mL) and brine (50 mL). The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc: Hex gradient). Compound 11was obtained as a white solid (75 mg, 88% yield). MS (ES+): m / z calc, for C48H49CIN2O9S: 864.28; found: 865.8 [M+H]+, 887.8 [M+Na]+.[HNMR (400 MHz, DMSO) 59.13 (d, 7=8.3 Hz, 1H), 7.81 (d, 7=8.0 Hz, 1H), 7.32-7.17 (m, 8H), 7.12 (d, 7=8.1 Hz, 1H), 6.85 (d, 7=8.7 Hz, 2H), 6.71 (d, 7=16.1 Hz, 1H), 5.97 (ddt, 7=17.2, 10.7, 5.5 Hz, 1H), 5.71 (dd, 7=7.9, 5.0 Hz, 1H), 5.33 (dd, 7=17.2, 1.6 Hz, 1H), 5.23 (dd, 7=10.4, 1.4 Hz, 1H), 5.12 (d, 7=4.9 Hz, 1H), 5.03 (d, 7=6.0 Hz, 2H), 4.92-4.83 (m, 1H), 4.74-4.69 (m, 1H), 4.69-4.66 (m, 2H), 3.92 (d, 7=18.1 Hz, 1H), 3.81 (d, 7=18.2 Hz, 1H), 3.71 (s, 3H), 3.56 (d, 7=13.9 Hz, 1H), 3.48 (d, 7=13.9 Hz, 1H), 3.19 (s, 3H), 3.16 (s, 1H), 1.96-1.59 (m, 13H).13C NMR (100 MHz, CDC13) 8 171.1, 166.4, 164.8, 161.2, 159.9, 138.7, 138.3, 133.7, 132.1, 129.4, 129.2, 128.7, 128.1, 127.7, 126.7, 125.0, 120.4, 118.5, 113.9, 72.6, 67.9, 65.4, 59.2, 57.3, 57.3, 55.3, 43.4, 39.1, 38.6, 37.0, 32.9, 29.7, 28.3, 26.5.

[0097] P-Lactamase Probe 9. Compound 11 (75 mg, 0.08 mmol, 1 eq.) was dissolved in DCM (2 ml), followed by the addition of DMBA (40 mg, 0.26 mmol, 3 eq.) and tetrakis(triphenylphosphine)palladium (10 mg, 0.008 mmol, 0.1 eq.). The reaction was stirred at room temperature and monitored by RP-HPLC. Upon completion, additional DCM was added (8 mL), followed by the addition of a catalytic amount of methylene blue. Then, oxygen was bubbled through the solution while irradiating with yellow light. The reaction was monitored by RP-HPLC. Upon completion, anisole (75 mL, 0.8 mmol, 10 eq.) and TFA (200 mL, 2% v / v) were added to the reaction mixture. The reaction was monitored by RP- HPLC. Upon completion, the solvent was concentrated under reduced pressure and the product was purified by preparative RP-HPLC (mobile phase: acetonitrile in H2O containing 0.1% TFA; gradient from 70 to 100%; flow rate: 20 mL / min; retention time: 8 min), [>- Lactamase Probe 9 was obtained as a white solid (12 mg, 18% yield). MS (ES-): m / z calc, for C37H37ClN2O10S: 736.19; found:735.6 [M-H] .^ NMR (400 MHz, DMSO) 8 9.13 (dd, 7=8.3, 2.8 Hz, 1H), 7.94 (dd, 7=8.4, 2.4 Hz, 1H), 7.83-7.70 (m, 2H), 7.33-7.26 (m, 3H), 7.25- .21 (m, 1H), 7.13-7.04 (m, 1H), 6.87-6.79 (m, 1H), 6.64 (dd, 7=16.1, 1.3 Hz, 1H), 5.75-5.65 (m, 1H), 5.13 (dd, 7=16.8, 4.9 Hz, 1H), 4.87 (t, 7=10.5 Hz, 1H), 4.77-4.61 (m, 1H), 3.99- 3.85 (m, 1H), 3.77 (s, 1H), 3.70 (d, 7=2.0 Hz, 1H), 3.63-3.43 (m, 2H), 3.12 (s, 3H), 2.88 (s, 1H), 2.29-2.16 (m, 1H), 1.90 (s, 1H), 1.77-1.43 (m, 8H), 1.34 (d, 7=6.8 Hz, 1H), 1.26-1.12 (m, 2H).13C NMR (100 MHz, CDCI3) 8 171.1, 166.4, 164.8, 161.2, 159.9, 138.7, 138.3, 133.7, 132.1, 130.6, 129.4, 129.2, 128.1, 127.7, 126.7, 125.0, 120.4, 118.5, 113.9, 72.6, 67.9, 65.4, 59.3, 57.3, 55.3, 43.4, 39.1, 38.6, 37.0, 32.9, 29.7, 28.34, 26.5.Penicillin-G amidase Probe 10

[0098] Compound 12. As depicted in Scheme 8, phenol enol ether b (125 mg, 0.27 mmol, 1.2 eq.) and K2CO3 (47 mg, 0.34 mmol, 1.5 eq.) were dissolved in DMF (1 mL). The solution was stirred for 5 minutes followed by the addition of compound] (60 mg, 0.23 mmol, 1 eq.). The reaction mixture was stirred at room temperature and monitored by TLC (EtOAc / Hex mixture). Upon completion, the reaction mixture was diluted with EtOAc (100 mL) and washed with 0.1M HC1 (50 mL) and brine (50 mL). The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc: Hex gradient). Compound 12 was obtained as a white solid (122 mg, 88% yield). MS (+): m / z calc, for C37H38C1NO5: 611.24; found: 634.6 [M+H]+.1H NMR (400 MHz, CDCI3) 6 7.90 (d, 7=16.2 Hz, 1H), 7.48-7.32 (m, 10H), 7.25 (s, 1H), 7.07 (d, 7=8.0 Hz, 1H), 6.44 (d, 7=16.2 Hz, 1H), 4.96 (d, 7=4.8 Hz, 2H), 3.79 (s, 3H), 3.75 (s, 2H), 3.32 (s, 3H), 3.29 (s, 1H), 2.07 (s, 1H), 2.02-1.65 (m, 12H).13C NMR (100 MHz, CDCI3) 6 169.1, 167.1, 153.6, 139.4, 138.9, 138.1, 138.0, 134.4, 132.4, 131.9, 129.8, 129.7, 129.5, 129.3, 127.8, 127.7, 125.1, 119.9, 119.6, 75.7, 68.0, 57.2, 51.8, 44.9, 39.2, 39.0, 38.6, 37.1, 32.9, 30.3, 29.7, 28.4, 28.2, 25.6.

[0099] Compound 13. Compound 12 (50 mg, 0.08 mmol, 1 eq.) and LiOH (20 mg, 0.8 mmol, 10 eq.) were dissolved in 1 mL solution of THF: H2O (4:1). The reaction mixture was stirred at room temperature and monitored by RP-HPLC. Upon completion, the solvent was concentrated under reduced pressure, and the product was purified by preparative RP-HPLC (mobile phase: acetonitrile in H2O containing 0.1% TFA; gradient from 70 to 100%; flow rate: 20 mL / min; retention time: 13.5 min). Compound 13 was obtained as a white solid (43 mg, 88% yield). MS (ES+): m / z calc, for C36H36C1NO5: 597.23; found: 620.5 [M+Na]+.XH NMR (400 MHz, DMSO) 8 10.22 (s, 1H), 7.75 (dd, 7=12.1, 6.1 Hz, 2H), 7.58 (d, 7=8.5 Hz, 2H), 7.33 (d, 7=8.6 Hz, 2H), 7.30 (d, 7=1.9 Hz, 2H), 7.08 (d, 7=8.0 Hz, 1H), 6.55 (d, 7=16.1 Hz, 1H), 4.91 (d, 7=18.4 Hz, 2H), 3.62 (s, 2H), 3.19 (s, 3H), 3.16 (s, 1H), 2.48 (d, 7=1.8 Hz, 4H), 1.94-1.82 (m, 5H), 1.80-1.58 (m, 8H).13C NMR (100 MHz, CDCI3) 6 171.0, 169.5, 153.7, 140.8, 139.4, 138.5, 138.0, 134.3, 132.6, 131.9, 129.9, 129.5, 129.2, 127.8, 127.7, 125.1, 120.0, 119.2, 75.9, 57.3, 44.7, 39.2, 39.0, 38.6, 37.1, 32.1, 29.7, 29.3, 28.2.

[0100] Penicillin-G amidase Probe 10. Compound 13 (50 mg, 0.08 mmol) and a catalytic amount of methylene blue (~1 mg) were dissolved in 10 mL of DCM. Oxygen was bubbled through the solution while irradiating with yellow light. The reaction was monitored by RP-HPLC. Upon completion, the solvent was concentrated under reduced pressure and the product was purified by preparative RP-HPLC (mobile phase: acetonitrile in H2O containing 0.1% TFA; gradient from 70 to 100%; flow rate: 20 mL / min; retention time: 11 min). Penicillin-G amidase Probe 10 was obtained as a white solid (35 mg, 68% yield). MS (ES+): m / z calc, for C36H36C1NO7: 629.22; found: 652.6 [M+Na]+.1H NMR (400 MHz, CDCI3) 6 7.96-7.85 (m, 2H), 7.57 (d, 7=8.9 Hz, 2H), 7.48 (d, 7=8.3 Hz, 2H), 7.42-7.30 (m, 6H), 6.40 (d, 7=16.1 Hz, 1H), 4.93 (d, 7=2.8 Hz, 2H), 3.74 (s, 2H), 3.25 (s, 3H), 3.05 (s, 1H), 2.34 (d, J=12.1 Hz, 1H), 2.02 (s, 1H), 1.94-1.57 (m, 8H), 1.49 (d, 7=11.1 Hz, 1H), 1.36 (s, 2H).13C NMR (101 MHz, CDCI3) 6 170.8, 169.7, 140.2, 138.2, 135.6, 134.3, 131.6, 131.5, 130.0, 129.5, 129.2, 129.0, 127.8, 127.7, 125.3, 120.3, 120.1, 96.4, 76.1, 49.8, 44.7, 36.6, 33.9, 33.6, 32.6, 32.2, 31.6, 31.5, 26.2, 25.8.Periodate oxidative-cleavase Probe 11

[0101] Compound 14. As depicted in Scheme 9, compound g (Taber et al., 2002) (13 mg, 0.05 mmol, 1.0 eq) was added to a solution of phenolic enol-ether b (23 mg, 0.06 mmol, 1.2 eq.) and K2CO3 (8.5 mg, 0.06 mmol, 1.2 eq.) in DMF (1 mL). The reaction was stirred at 40°C and monitored by TLC. Upon completion, the mixture was diluted with EtOAc (100 mL) and washed with 0.5M HC1 (50 mL). The organic layer was separated, washed with brine, dried over Na2SO4 and evaporated under reduced pressure. The crude residue was dissolved in 5 ml THF : H2O (4:1) followed by the addition of NaOH (20 mg, 0.5 mmol, 10 eq.). The reaction mixture was stirred for 2 hours at 50°C and monitored by RP-HPLC. Upon completion, the mixture was diluted with EtOAc (100 mL) and washed with 0.5M HC1 (50 mL). The organic layer was separated, washed with brine, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc: Hex gradient) to afford compound 14 (19 mg, 60% yield) as an off-white solid. ’ H NMR (400 MHz, CDCI3) 6 7.68 (d, 7=16.1 Hz, 1H), 7.42 (d, 7=8.0 Hz, 1H), 6.98 (d, 7=8.0 Hz, 1H), 6.47 (d, 7=16.1 Hz, 1H), 4.44-4.35 (m, 1H), 4.19 (dd, 7=8.1, 6.0 Hz, 1H), 4.08-3.92 (m, 2H), 3.26 (s, 3H), 3.20 (s, 1H), 2.14-2.07 (m, 2H), 2.03 (s, 1H), 1.96-1.58 (m, 13H), 1.35 (d, 7=19.0 Hz, 6H).13C NMR (100 MHz, CDCI3) 6 173.3, 153.6, 139.4, 136.9, 134.9, 132.5, 130.3, 129.2, 127.6, 125.9, 124.8, 118.0, 116.1, 109.8, 73.7, 71.1, 69.5, 57.1, 39.0, 38.9, 38.6, 38.5, 37.0, 33.8, 32.8, 29.6, 26.7, 25.4.

[0102] Periodate oxidative cleavage Probe 11. Compound 14 (10 mg, 0.05 mmol) and a catalytic amount of methylene blue were dissolved in 10 mL of DCM. Then, oxygen wasbubbled through the solution while irradiating with yellow light. The reaction was monitored by RP-HPLC. Upon completion, the solvent was concentrated under reduced pressure. The crude product was dissolved in MeOH (3 mL) followed by the addition of H2O (3 ml, 1% TFA). Once the acetonide's hydrolysis terminated (monitored by RP-HPLC) the solvent was concentrated under reduced pressure and the product was purified by preparative RP-HPLC (mobile phase: acetonitrile in H2O containing 0.1% TFA; gradient from 50 to 100%; flow rate: 20 mL / min; retention time: 9 min). Periodate oxidative-cleavage Probe 11 was obtained as a white solid (4.6 mg, 47% yield). MS (ES-): m / z calc, for C25H31C1O8: 494.17; found: 493.6 [M-H]’. ’ H NMR (400 MHz, CDCI3) 6 8.13 (d, 7=16.2 Hz, 1H), 7.94 (d, 7=8.3 Hz, 1H), 7.63 (d, 7=8.4 Hz, 1H), 6.55 (d, 7=16.2 Hz, 1H), 4.37 (s, 1H), 4.17-4.02 (m, 3H), 3.83 (d, 7=10.5 Hz, 1H), 3.65 (d, 7=7.6 Hz, 1H), 3.23 (d, 7=6.0 Hz, 3H), 3.04 (s, 1H), 2.30 (s, 1H), 2.12-1.95 (m, 2H), 1.75 (m, 9H), 1.48 (d, 7=12.8 Hz, 1H), 1.40-1.27 (m, 2H).13C NMR (100 MHz, CDCI3) 6 170.8, 154.7, 140.3, 135.8, 130.6, 129.0, 127.7, 125.0, 120.2, 111.6, 96.4, 70.9, 69.0, 67.0, 49.7, 36. 6, 33.9, 33.6, 32.8, 32.6, 32.2, 31.6, 31.5, 26.1, 25.8.N -acetyl hydrolase Probe 12

[0103] Compound 15. As depicted in Scheme 10, phenol enol ether b (46 mg, 0.12 mmol, 1 eq.) and K2CO3 (24 mg, 0.17 mmol, 1.5 eq.) were dissolved in DMF (0.5 mL). The solution was stirred for 5 minutes before adding compound h (27 mg, 0.12 mmol, 1 eq.). The reaction mixture was stirred at room temperature and monitored by TLC (EtOAc: Hex mixture). Upon completion, the reaction mixture was diluted with EtOAc (100 mL) and washed with saturated NH4CI (50 mL) and brine (50 mL). The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude mixture was reacted without further purification. The crude and LiOH (9.5 mg, 0.39 mmol, 10 eq.) were dissolved in 1 mL solution of THF: H2O (4:1). The reaction mixture was stirred at 50 °C and monitored by RP-HPLC. Upon completion, the solvent was concentrated under reduced pressure, and the product was purified by preparative RP-HPLC (mobile phase: acetonitrile in H2O containing 0.1% TFA; gradient from 70 to 100%; flow rate: 20 mL / min; retention time: 10 min). Compound 15 was obtained as a white solid (36 mg, 58% yield). MS (ES+): m / z calc, for C30H32ClNO5: 521.20; found: 544.5 [M+Na]+. ’ H NMR (400 MHz, 5% MeOD in CDCI3) 6 7.92 (dd, 7=20.5, 12.2 Hz, 2H), 7.81 (s, 1H), 7.56 (d, 7=8.2 Hz, 3H), 7.39 (d, 7=8.2 Hz, 2H), 6.39 (d, 7=16.1 Hz, 1H), 4.94 (d, 7=2.6 Hz, 3H), 3.26 (s, 3H), 3.06 (s, 1H), 2.36 (d, 7=12.2 Hz, 1H), 2.20 (s, 3H), 2.03 (s, 1H), 1.79 (ddd, 7=55.0, 37.2, 15.0 Hz, 8H), 1.50 (d, 7=12.7Hz, 1H), 1.37 (s, 2H).13C NMR (100 MHz, 5% MeOD in CDC13) 8 169.5, 169.0, 153.5,139.5, 139.3, 138.4, 138.0, 132.6, 131.5, 129.9, 129.7, 127. 8, 125.0, 120.0, 119.8, 76.0,57.2, 39.1, 39.0, 38.6, 37.0, 32.9, 29.7, 28.3, 28.2, 24.1.

[0104] N-acetyl hydrolase Probe 12. Compound 15 (10 mg, 0.02 mmol) and a catalytic amount of methylene blue (~1 mg) were dissolved in 10 mL of DCM. Oxygen was bubbled through the solution while irradiating with yellow light. The reaction was monitored by RP- HPLC. Upon completion, the solvent was concentrated under reduced pressure, and the product was purified by preparative RP-HPLC (mobile phase: acetonitrile in H2O containing 0.1% TFA; gradient from 70 to 100%; flow rate: 20 mL / min; retention time: 8 min). N- acetyl hydrolase Probe 12 was obtained as a white solid (8.7 mg, 82% yield). MS (ES+): m / z calc, for C30H32ClNO7: 553.19; found:576.5 [M-H]+. ’ H NMR (400 MHz, CDCI3) 67.92 (dd, 7=20.5, 12.2 Hz, 2H), 7.81 (s, 1H), 7.57 (t, 7=8.2 Hz, 3H), 7.39 (d, 7=8.2 Hz, 2H), 6.39 (d, 7=16.1 Hz, 1H), 5.00-4.89 (m, 2H), 3.26 (d, 7=6.4 Hz, 3H), 3.06 (s, 1H), 2.36 (d, 7=12.2 Hz, 1H), 2.20 (s, 3H), 2.03 (s, 1H), 1.97-1.24 (m, 12H).13C NMR (100 MHz, CDCI3) 6170.6, 169.1, 154.1, 140.2, 138.5, 135.6, 131.6, 131.4, 130.2, 129.0, 127.8, 125.2, 120.2,120.2, 111.7, 96.4, 76.16, 49.8, 36.6, 33.9, 33.6, 32.6, 32.2, 31.6, 31.5, 26.2, 25.8, 24.5.Fluorescent phosphatase probe ( Compound 17)

[0105] Compound 16. As depicted in Scheme 11, umbelliferone (12 mg, 0.07 mmol, 1.2 eq.) and K2CO3 (12 mg, 0.09 mmol, 1.5 eq.) were dissolved in DMF (1 mL). The solution was stirred for 5 minutes followed by the addition of compound 9 (21 mg, 0.06 mmol, 1 eq.). The reaction mixture was stirred at room temperature and monitored by TLC (EtOAc / Hex mixture). Upon completion, the reaction mixture was diluted with EtOAc (100 mL) and washed with 0.1M HC1 (50 mL) and brine (50 mL). The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc: Hex gradient). Compound 16 was obtained as a yellow oil (18 mg, 73% yield). ’H NMR (400 MHz, DMSO) 8 7.99 (d, 7=9.5 Hz, 1H), 7.64 (d, 7=8.6 Hz, 1H), 7.52 (d, 7=8.5 Hz, 2H), 7.25 (dd, 7=8.6, 1.0 Hz, 2H), 7.08 (d, 7=2.4 Hz, 1H), 7.02 (dd, 7=8.6, 2.4 Hz, 1H), 6.29 (d, 7=9.5 Hz, 1H), 6.01-5.87 (m, 2H), 5.36 (dd, 7=17.2, 1.6 Hz, 2H), 5.25 (dd, 7=10.5, 1.4 Hz, 2H), 5.21 (s, 2H), 4.64 (ddt, 7=8.4, 5.4, 1.4 Hz, 4H).13C NMR (100 MHz, CDCI3) 8 161.7, 161.3, 155.8, 143.50, 132.7, 131.9, 131.9, 129.1, 128.9, 120.4, 120.3, 118.8, 113.25, 112.8, 101.97, 69.8, 69.0, 68.9.31P NMR (162 MHz, DMSO) 8 -6.27.

[0106] Compound 17. Compound 16 (18 mg, 0.04 mmol, 1 eq.) was dissolved in DCM (2 ml), followed by the addition of DMBA (21 mg, 0.13 mmol, 3 eq.) and tetrakis(triphenylphosphine)palladium(5.1 mg, 0.004 mmol, 0.1 eq.). The reaction was stirred at room temperature and monitored by RP-HPLC. Upon completion, the solvent was concentrated under reduced pressure, and the product was purified by preparative RP-HPLC (mobile phase: acetonitrile in H2O containing 0.1% TFA; gradient from 30 to 100%; flow rate: 20 mL / min; retention time: 5 min). Compound 17 was obtained as a white solid (9.8 mg, 67% yield). MS (ES-): m / z calc, for CI6H13O7P: 348.04; found: 347.3 [M-H]’. ’ H NMR (400 MHz, MeOD) 8 7.89 (d, 7=9.5 Hz, 1H), 7.55 (d, 7=8.4 Hz, 1H), 7.48 (d, 7=8.5 Hz, 2H), 7.25 (dd, 7=8.6, 1.1 Hz, 2H), 7.03-6.96 (m, 2H), 6.26 (d, 7=9.5 Hz, 1H), 5.18 (s, 2H), 1.31 (s, 1H).13C NMR (100 MHz, MeOD) 6 162.2, 162.0, 155.8, 151.4, 144.4, 132.8, 129.2, 128.9, 120.3, 113.2, 112.3, 101.5, 69.7.31P NMR (162 MHz, MeOD) 6 -5.24Colorimetric phosphatase probe ( Compound 19)

[0107] Compound 18. As depicted in Scheme 12, 4-nitrophenol (9.7 mg, 0.07 mmol, 1.2 eq.) and K2CO3 (12 mg, 0.09 mmol, 1.5 eq.) were dissolved in DMF (1 mL). The solution was stirred for 5 minutes followed by the addition of compound 9 (21 mg, 0.06 mmol, 1 eq.). The reaction mixture was stirred at room temperature and monitored by TLC (EtOAc / Hex mixture). Upon completion, the reaction mixture was diluted with EtOAc (100 mL) and washed with 0.1M HC1 (50 mL) and brine (50 mL). The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc: Hex gradient). Compound 18 was obtained as a yellow oil (22 mg, 78% yield). ’ H NMR (400 MHz, CDCI3) 6 8.26-8.14 (m, 2H), 7.39 (dd, 7=18.4, 6.4 Hz, 2H), 7.32-7.22 (m, 2H), 7.07-6.96 (m, 2H), 5.96 (ddd, 7=22.6, 10.8, 5.6 Hz, 2H), 5.38 (dt, 7=14.7, 7.3 Hz, 2H), 5.27 (dt, 7=2.2, 6.1 Hz, 2H), 5.14 (s, 2H), 4.66 (tt, 7=17.1, 6.3 Hz, 4H).13C NMR (100 MHz, CDCI3) 6 163.5, 150.7, 150.7, 141.8, 132.3, 132.0, 132.0, 129.0, 126.0, 120.5, 120.4, 118. 8, 114.8, 70.0, 68.9, 68.9.

[0108] Compound 19. Compound 18 (16 mg, 0.04 mmol, 1 eq.) was dissolved in DCM (2 ml), followed by the addition of DMBA (21 mg, 0.13 mmol, 3 eq.) and tetrakis(triphenylphosphine)palladium(5.1 mg, 0.004 mmol, 0.1 eq.). The reaction was stirred at room temperature and monitored by RP-HPLC. Upon completion, the solvent was concentrated under reduced pressure, and the product was purified by preparative RP-HPLC (mobile phase: acetonitrile in H2O containing 0.1% TFA; gradient from 30 to 100%; flowrate: 20 mL / min; retention time: 7 min). Compound 19 was obtained as a white solid (8.7 mg, 67% yield). MS (ES-): m / z calc, for C13H12NO7P: 325.04; found: 324.2 [M-H]“.1H NMR (400 MHz, 10% MeOD in CDC13) 6 8.22 (d, 7=9.3 Hz, 2H), 7.47 (d, 7=8.5 Hz, 2H), 7.26 (d, 7=7.7 Hz, 2H), 7.16 (d, 7=9.3 Hz, 2H), 5.21 (s, 2H).13C NMR (100 MHz, 10% MeOD in CDCI3) 6 163.7, 151.4, 141.7, 131.8, 129.0, 126.0, 120.7, 114.9, 70.2, 50.0, 49.8, 49.6, 49.4, 49.2, 49.0, 48.8.31P NMR (162 MHz, 10% MeOD in CDCI3) 6 -4.77 (s).Biological evaluation: general information and procedures

[0109] List of bacteria strains. The bacterial strains used are listed in Table 6.LOD experiments procedure

[0110] Staphylococcus aureus ATCC 29213 was incubated in lysogeny broth (LB) at 37°C for 18 hours under aerobic conditions. The initial culture was rinsed with PBS (centrifuged at 5000 rpm, 10 minutes), and the resulting bacterial pellet was resuspended in 4 mL of PBS to facilitate a 1:5 dilution experiment. For the subsequent procedure, a 96-well plate was utilized, with each well initially loaded with 50 pL of the phosphatase chemiluminescent probe 5 [20 pM, 0.1% DMSO v / v in PBS pH 7.4], phosphatase colorimetric probe [compound 17, 100 pM, 0.1% DMSO v / v in PBS pH 7.4], or phosphatase fluorescence probe [compound 19, 20 pM, 0.1% DMSO v / v in PBS pH 7.4]. Subsequently, 50pL of bacterial aliquot was introduced into each well, marking the commencement of the 1:5 dilution experiment, which was initiated with an ODeoo of 0.4. The ensuing chemiluminescence signal was monitored over 30 minutes of incubation at 37°C using a Molecular Devices Spectramax i3x. Simultaneously, the fluorescence signal was recorded during the same timeframe at the same temperature using a Tec an Infinite 200 Pro.Enzymatic substrate specificity experiments

[0111] The specificity of each chemiluminescent probe was assessed in the presence of ten commercially available recombinant enzymes. The chemiluminescent intensity of each probe [10 mM in PBS (7.4 pH), 0.1% DMSO, 37°C] was measured in conjunction with the following commercially available recombinant enzymes: / / -glucosidase from almonds [10 U / mL], / / -glucuronidase from E.coli [1 U / mL], / / -galactosidase from E.coli [1 U / mL], pyroglutamyl-peptidase I from E.coli [0.05 mg / mL], alkaline phosphatase from bovine intestinal mucosa [1 U / mL], aminopeptidasse-M from porcine kidney [1 U / mL], nitroreductase from E.coli [1 mg / mL, lOOmM], NADH NQO1 (from E.coli) [0.8 mg / mL,lOOmM NADH], / / -lactamase from E.coli [2 U / mL], penicillin g-amidase from E.coli [1 U / mL].Table 6. Bacteria strains* Supplemented BHI with hemin and nicotinamide adenine dinucleotide.Chemiluminescent measurements of bacterial enzymatic activity fingerprint

[0112] All bacterial strains were cultured in lysogeny broth (LB) / brain heart infusion broth (BHI) at either 37°C or 30°C for 18 hours under aerobic conditions. Subsequently, the initial culture was subjected to a PBS wash (centrifuged at 5000 rpm, 10 minutes), and the bacterialpellet obtained was reconstituted in 4 mL of PBS, aiming for an ODeoo of 0.8. Following this, a 96-well plate was utilized, and each well was pre-loaded with 50 pL of each chemiluminescent probe [20 mM, 0.1% DMSO v / v in PBS pH 7.4]. Next, 50 pL of bacterial aliquot was introduced into each well, bringing the final ODeoo to 0.4. The resultant chemiluminescence signal was monitored using a Molecular Devices Spectramax iD3 over the course of 1 hour of incubation at 37 °C.Chemiluminescent measurements of bacterial enzymatic activity fingerprint inbacterial mixing

[0113] Staphylococcus aureus MRS A ATCC 33592 and Escherichia coli ATCC 25922 29213 were each cultured in LB at 37°C for 18 hours under aerobic conditions. The initial cuiltures of both bacteria were subjected to a PBS wash (centrifuged at 5000 rpm, 10 minutes), and the bacterial pellets obtained were reconstituted in 4 mL of PBS, aiming for an ODeoo of 0.8. Following this, a 96-well plate was utilized, and each well was pre-loaded with 50 pL of each chemiluminescent probe [20 mM, 0.1% DMSO v / v in PBS pH 7.4]. Next, 50 pL of bacteria mixture aliquot (1:1, 9:1, 1:9 of E. coli, MRSA respectively) was introduced into each well. The resultant chemiluminescence signal was monitored using a Molecular Devices Spectramax iD3 over the course of 1 hour of incubation at 37°C.Computational methods

[0114] General methods. The 1-N-N classification method was executed using MATLAB's K-Nearest Neighbors (KNN) algorithm, where K was set to 1, and the Euclidean distance metric was employed for similarity measurement. Furthermore, MATLAB's built- in Principal Component Analysis (PCA) implementation was utilized for visualization, primarily aimed at dimensionality reduction of the dataset. To comprehensively evaluate the efficacy of our approach, the MATLAB KNN classifier was applied to all 4094 potential probe combinations, with K set to 1 and the Euclidean distance metric. This enabled measurement of classification accuracy across all probe combinations, facilitating a robust assessment of the method's performance

[0115] Data acquisition and processing. The data acquisition process involved meticulous aggregation from multiple sources. For each measurement, raw data were gathered by evaluating three distinct wells harboring the target bacteria, contrasted with three control wells that lacked bacteria and served as background references. The processing of these raw data entailed a series of steps. Initially, the mean light intensity value wascalculated across measurements from the three wells containing bacteria and similarly for the control wells. Following this, the light intensity was integrated over a one-hour interval in the presence and absence of bacteria. A crucial stage involved subtracting the logarithm of TLE gathered from the control wells (termed "Control") from the logarithm of the TLE gathered from the bacteria-containing wells. To ensure consistency, any negative log ratios were set to zero. This process culminated in the generation of a 12-dimensional vector for each strain, with entries corresponding to the distinct probes in our array.

[0116] 1 -N-N analysis. 1-N-N Analysis computational analysis of data involved the manipulation of 12-dimensional vectors containing background- subtracted log-intensity data. To accomplish this, the K-Nearest Neighbors (KNN) algorithm was employed. Specifically, the MATLAB implementation of KNN was utilized with K set to 1. In congruence with this choice, the Euclidean distance was adopted as the metric to facilitate pairwise comparisons between data points. The KNN algorithm facilitates informed labeling of unknown data points within the test set. This is achieved by assigning to each unknown data point the label of its nearest counterpart within the training set. To ensure the robustness and reliability of outcomes, the dataset of enzymatic profiles underwent a randomized split, resulting in two distinct data sets: the training set encompassing 72% of the data and the test set comprising the remaining 28%. This partitioning strategy is designed to enhance precision and dependability in the classification results for log-intensity data. To guarantee comprehensive coverage and robust evaluation, it is important to note that the partitioning strategy employed also ensures that each bacterium within the dataset is included in both the training and test set, thereby allowing for a thorough assessment of the classification performance. By employing this approach, effective categorization of new data points is achieved through their proximity to reference points within the training set.Details of the chi-squaredresemblance ranking and test.

[0117] Definition of the resemblance test. The methodology employed in this study focuses on the characterization and classification of bacterial strains based on their enzymatic profiles within a 12-dimensional space. It is grounded in the assumption that these profiles conform to Gaussian distributions. To assess the degree of similarity between an unfamiliar bacterial strain and those cataloged within our dataset, we employ a probabilitycentered approach. Central to this approach is the Chi-Squaredmetric, a statistical measure that quantifies deviations from reference statistics and is crucial for evaluating theresemblance of unknown bacteria to those within a reference dataset. The degrees of freedom for the / 2statistic is set to 12, corresponding to the 12 dimensions of the enzymatic profile space. This systematic methodology serves as the foundation for probabilistic classification and allows for the identification and categorization of novel bacteria based on their enzymatic profiles.

[0118] Data retrieval. Enzymatic profile data for known bacterial species and the enzymatic profile of an unknown bacterium are retrieved from a structured Excel dataset.

[0119] Parameter extraction. For each known bacterial species, mean vectors and variances are computed to characterize their respective Gaussian distributions. These parameters are pivotal for subsequent analyses.

[0120] Calculation of Chi-Squared statistics. The Chi-Squared statistic is computed by centering and normalizing the mean vectors and variances of the known bacterial species and the unknown bacterium. Careful precautions are taken to prevent division by zero.

[0121] Hypothesis testing. To determine the extent of dissimilarity between the unknown bacterium and the reference species, Chi-Squared statistics and associated p-values are calculated. These p-values are essential for hypothesis testing.

[0122] Probabilistic classification. The Chi-Squared statistics are used to rank the similarity between the unknown bacterium and the reference species. This ranking provides a quantitative measure of resemblance.

[0123] Statistical analysis. Data are presented as means (three or more replicates) ± SD (error bars). All statistical analyses were performed using the two-tailed Student’s t-test unless otherwise indicated. Differences were considered significant at p<0.05.RESULTS

[0124] Chemiluminescent probes outperform in the signal-to-noise ratio and limit of detection compared to fluorescent and colorimetric probes. To explore the advantages of developing an array of chemiluminescent probes in comparison to fluorescent or colorimetric probes, we evaluated three related probes for their ability to detect specific enzymatic activity. In each of these probes, the same phosphatase-triggering substrate initiates specific light-emitting reactions. These reactions activate one of the three optical signaling mechanisms: ortho acrylate- substituted phenoxy-adamantyl-l,2-dioxetane (for chemiluminescence), 7-hydroxy-coumarin (for fluorescence), or a para- nitropheno 1 dye (for colorimetry) (Fig. 2A). Phosphatase catalyzed cleavable of the phosphate headgroupsubstrate results in the formation of a phenolate functional group. This is followed by a subsequent disassembly phase characterized by a 1,6-elimination reaction, leading to the liberation of a quinone-methide molecule. Concurrently, this reaction triggers the emission of the respective optical signal, whether it be chemiluminescent, fluorescent, or colorimetric, as part of the reporting mechanism.

[0125] Activation of the probes with recombinant alkaline phosphatase revealed that the phosphatase limit-of-detection (LOD) value of the chemiluminescent probe was ~25-fold lower than that of the fluorescent probe, which was in turn ~ 125-fold lower than that of the colorimetric probe (Fig. 2B). We next compared the performance of the three optical probes in the presence of bacteria. Phosphatase activity was measured while incubating each of the three probes in PBS, pH=7.4, buffer containing cells of Staphylococcus aureus (ATCC 29213). The LOD value for the chemiluminescent probe was ~25-fold lower than that of the fluorescent probe, which was in turn ~25-fold lower than that of the colorimetric probe (Fig. 2C). Moreover, the signal-to-noise ratio of the chemiluminescent probe was ~70-fold higher than that of the fluorescent probe and -673 -fold higher than that of the colorimetric probe; hence, chemiluminescence detection based on a phenoxy-adamantyl-l,2-dioxetane unit was chosen as the detection method for the designed array of enzymatic activity sensors.

[0126] Design and synthesis of an array of chemiluminescent probes for sensing catabolic bacterial enzymes with high substrate specificity. Taking advantage of the chemiluminescent phosphatase probe's heightened sensitivity, we constructed an array comprised of 12 chemiluminescent probes (full synthesis described in the Materials and Methods section hereinabove). This array consists of 10 distinct triggers, each representing a substrate of a different prevalent bacterial enzymatic activity. We also included two control probes: an A- acetyl hydrolase probe for non-specific A-dcacctylasc detection and a vicinal - diol oxidative-cleavage probe commonly activated by periodate. The periodate oxidativecleavage probe is unresponsive to bacterial activation and served as a negative control.

[0127] Seven of the 12 chemiluminescent probes were designed especially for this study (probes 1, 2, 4, 9, 10, 11, and 12); ^-galactosidase probe 3 is the water-soluble carboxylic acid variant of the acrylonitrile or methyl ester ^-galactosidase probes previously described (Green et al., 2017); Phosphatase probe 5 is an analogue that contains a self-immolative spacer and an orthochloride with improved sensitivity compared to the previously published version of this chemiluminescent probe (Green et al., 2017); Probe 6 for leucineaminopeptidase is the water-soluble carboxylic acid version of previously reported amide and methyl ester versions of this probe (Wang et al., 2022; Gunduz et al., 2023); Nitroreductase probe 7 and NAD(P)H quinone oxidoreductase 1 -activated probe 8 were previously reported (Son et al., 2019; Ryan et al., 2019).

[0128] We proceeded to investigate the chemiluminescent probes' specificity as substrates for the target enzymes, assessing them across a panel of recombinant enzymes corresponding to the 10 sensed activities (Fig. 3B). The probes within the array exhibited favorable selectivity as substrates for their designated enzymes with a modest background for some of the probes (Fig. 3B). Finally, to assess the stability of the probes within the array, we examined their behavior under assay conditions (PBS 100 mM, pH 7.4, 0.1% DMSO, 37°C, 60 min). The probes demonstrated satisfactory stability, with the Mactamase probe exhibiting the least stability (~1% decomposition) and the ^-galactosidase probe showcasing the highest stability, with a decomposition of less than 0.00001%. Additionally, DMSO stock solutions of the probes stored at -20°C remained unchanged for over a year.

[0129] Array of chemiluminescent probes yields distinct enzymatic activity profiles, enabling differentiation between bacterial species and strains within a Species. Employing an array of 12 chemiluminescent probes, we characterized enzymatic activity profiles across a panel encompassing 29 strains representing nine Gram-positive and seven Gram-negative species (Table 6). This panel included prominent bacterial pathogens and representative strains of the the ESKAPE group.

[0130] Of note, the presence or absence, expression level, and intracellular or extracellular localization of individual enzymes in bacteria are all determined by a complex interplay of factors, including the species and strain, growth conditions, and even cell cycle stage. These factors collectively shape the overall enzymatic activity profile, measured by the chemiluminescent array. Moreover, the chemiluminescent probes are incubated with intact cells, meaning that the permeability or exclusion of probes from the intracellular environment plays a crucial role in shaping the measured enzymatic activity fingerprint. The negatively charged chemiluminescent unit (carboxylic acid) and certain triggers (i.e., phosphate or glucuronic acid) can hinder or even completely abrogate probe membrane permeability or uptake depending on the specific bacterial species or strain. Consequently, a significant portion of the signal obtained using the current probes likely originates fromthe cell surface and secreted enzymes, rather than those located within the cells of the bacteria in the panel.

[0131] The streamlined process, taking approximately 1.5 hours to complete, is outlined in Fig. 4A. This procedure consistently yielded reproducible enzymatic activity fingerprints for each strain, culminating in a comprehensive database comprising of 12-dimensional vectors that encapsulate the total emitted light from each probe over the course of the experiment. We assessed the logarithm of the ratio between the total emitted light and the background light, which we have identified as a background- subtracted value for enzymatic activity. Each strain displayed a distinct enzymatic activity fingerprint based on this metric, underscoring the array's potential for bacterial species classification and even strain differentiation.

[0132] For an initial assessment of the differentiation among the bacterial species in the panel, principal component analysis (PCA) was conducted, resulting in effective clustering among all bacterial species in the panel. Subsequently, a quantitative analysis of the resulting database was carried out using the first nearest-neighbor method (1-N-N) based on enzymatic activity log-ratios within the data space (illustrated in Figure 4A) (Cover et al., 1967). This proposed methodology provides a direct and efficient means for bacterial identification through examination of chemiluminescent probe data. Application of the 1-N- N analysis to the 12-probe enzymatic activity fingerprint revealed distinct differentiation among all 29 diverse bacterial strains within our panel (see Figure 4B).

[0133] We explored the impact of the assay duration on species identification accuracy. A 15 and 30 min chemiluminescence (CL) measurement yielded an accuracy of 89.6% for both. Extending the measurement to 45 and 60 min improved accuracy to 93.1 and 100%, respectively. To assess if comparable accuracy could be achieved with fewer probes, we analyzed the variance in the response of each probe across all bacterial strains in our panel. Intriguingly, the selection of the six most variable probes (5, 4, 2, 7, 3, and 6) proved sufficient to differentiate all 29 diverse bacterial strains in the tested panel with an accuracy of 89.6%.

[0134] We subsequently evaluated the performance of the chemiluminescent array-based process against the commercial API method, employing two relevant kits: the API20E for E. coll ATCC 25922, E. coll ATCC 9637, and E. coll ATCC BAA-2452, and the API Staph 20 for S. aureus ATCC 29213. The API method yielded identification results similar to those of our method. However, it necessitated distinct API kits for each bacterial species, and theidentification was limited to the species level. Moreover, the API-based assay required 24 h to complete. In contrast, the chemiluminescent array-based method allows for universal strain-level identification within a 90 min time frame.

[0135] To investigate the utility of the chemiluminescent array in characterizing the enzymatic activity fingerprint of samples containing more than one species, we measured the chemiluminescent fingerprint of mixed samples featuring varying ratios of two bacterial species. We chose to focus on mixtures of E. coli and S. aureus, a coinfection frequently observed in the blood of COVID- 19 patients (Russell etal., 2021). A previous study reported a microbiological investigations on 8649 (17.7%) out of 48,902 COVID-19 patients, with clinically significant results for 1107 patients. The subsequent analysis revealed E. coli and S. aureus as the most common causative agents of bloodstream infections in hospitalized COVID- 19 patients. The chemiluminescent measurements of samples with varying bacterial ratios of the two bacteria aimed to determine whether the resulting fingerprints accurately reflected the combined enzymatic activity of both bacteria or whether they revealed a novel fingerprint unrelated to either individual species.

[0136] Notably, the fingerprints exhibited signals from probes activated by both species as well as probes predominantly activated by either E. coli or S. aureus (Figure 5). The intensity of the chemiluminescent signal from probes activated by the characteristic dominant activity of each species nonlinearly intensified with the respective ratio of each bacterium in the mixture. These findings underscore the efficacy of the chemiluminescent array method for characterizing the enzymatic activity fingerprints of samples containing more than one bacterial species. These findings underscore the versatility and potential of the chemiluminescent array of probes in studying and characterizing more complex microorganism systems, such as entire microbiomes by exploring their distinctive enzymatic activity fingerprints.

[0137] Through comparative analyses of array TLEs across the various bacteria in the panel, several unexpected discoveries emerged. Notably, when examining the mean P- glucuronidase activity across all E. coli strains in the panel, a significantly higher value was observed compared to those of other bacteria (as demonstrated in Fig. 6A). This elevated activity facilitated the differentiation of E. coli strains within the panel based on p- glucuronidase activity alone. This is in agreement with prior reports that designate P- glucuronidase as a marker for E. coli presence. Harking back to the mid-1970s, Kilian and Bulow scrutinized clinical E. coli isolates, discovering that around 97% exhibit p~glucuronidase production, while the majority of other coliform bacteria do not. A total of 460 humans, 105 cows, and 55 horses E. coli isolates were tested. The results showed 95.5% ^-glucuronidase-positive isolates in 24 h and 99.5% positive after 28 h of incubation.

[0138] Similarly, the TLE of phosphatase probe 5 was significantly higher for the S. pyogenes strains in the panel than for all other strains (Fig. 6B). S. pyogenes, also known as Group A Streptococcus, is a group of Gram-positive bacteria that can be carried in human throats or skin, and it is responsible for more than 500,000 deaths worldwide annually. The conventional tests for the identification of S. pyogenes in clinical samples involve blood agar plates that are screened for the presence of / / -hemolytic colonies. The typical appearance of S. pyogenes colonies after 24 hours of incubation at 35-37°C is dome-shaped with a smooth or moist surface and clear margins. It is noteworthy that automated bacterial identification by MALDLTOF has limitations in distinguishing among closely related taxa within group A syreptococci, to which S. pyogenes belongs. Several point-of-care tests for the detection of S. pyogenes in throat swabs using rapid automated PCR technology have received FDA clearance to date, yet the use of PCR for diagnosing streptococcal throat infections remains low. The high activity detected by chemiluminescent probe 5 suggests that testing for high levels of phosphatase could potentially provide an affordable substitute for the detection of S. pyogenes.

[0139] Pseudomonas aeruginosa is yet another bacterium which can be easily identified using less than the 12 probes in the array. Prior studies have indicated that P. aeruginosa exhibits pyroglutamyl aminopeptidase activity. Interestingly, using solely the pyroglutamyl aminopeptidase probe yielded a high success rate (-90%) in distinguishing the three P. aeruginosa strains from all the other bacteria within our panel. Moreover, a dual-probe strategy involving pyroglutamyl aminopeptidase and leucine aminopeptidase achieved -97% identification accuracy (as illustrated in Fig. 6C).

[0140] Next, we investigate the capability of the chemiluminescence-based enzymatic activity fingerprinting method to classify strains of bacteria that were not part of the database, even though they belong to species already represented. Specifically, we focused on three strains: S. aureus ATCC 33591, an E. coli clinical isolate, and P. aeruginosa. To assess their similarity to known strains, we adopted a statistical approach, which involved representing each strain in the original data set as a Gaussian cloud within the 12- dimensional space corresponding to log-ratios of the TLE from each probe. Thisrepresentation facilitated the ranking of the similarity of new bacteria to existing strains in our data set and enabled the statistical testing of the hypothesis that they are identical.

[0141] Using the / 2metric, we conducted a thorough comparison of the enzymatic activity fingerprints of the selected strains with those in our panel. The results of this analysis are summarized in Fig. 7. Notably, the statistical findings consistently demonstrated that all three examined bacteria exhibit the nearest resemblance to known panel bacteria from the same species. It is important to emphasize that in all cases, the p-values resulting from the %2test indicated a rejection of the hypothesis that the unknown bacteria were present in the database. Instead, our analysis consistently highlighted the closest strains from the same bacterial families as the most resembling. The tested S. aureus ATCC 33591 showed the highest resemblance to S. aureus ATCC 35556, while the E. coli clinical isolate closely resembled E. coli ATCC 25922. Similarly, the enzymatic profile of the P. aeruginosa PAO1 demonstrated a resemblance to P. aeruginosa ATCC 27853. These results demonstrate the effectiveness of the chemiluminescence-based enzymatic fingerprinting method in categorizing and identifying unknown bacteria.

[0142] Finally, we further expanded the scope of our investigation by introducing a bacterium strain belonging to a species not originally represented in the database: the Grampositive pathogen Listeria monocytogenes ATCC 19115. This strain, like all others tested previously, displayed a distinct enzymatic fingerprint. The / 2 metric score indicated that its closest match within the database was the Gram-positive S. aureus ATCC 43300 (Fig. 7D). Of note, the proximity between the enzymatic profiles of L. monocytogenes ATCC 19115 and S. aureus ATCC 43300, as measured by the / 2 metric score, was not as high as the proximity observed between the three unknown strains (belonging to species already represented in the database) and their closest counterparts in the database. Notably, both L. monocytogenes and S. aureus are Gram-positive pathogens. This illustrates that while unknowns not represented in the panel cannot be directly identified, their enzymatic activity fingerprint offers valuable insights that, in turn, can be utilized to identify close resemblances to other bacteria.CONCLUSIONS

[0143] In summary, our study introduces a robust and cost-effective method for the identification and characterization of bacteria, addressing the challenges posed by labor- intensive protocols and expensive and difficult-to-maintain equipment. This approach holdspromise for widespread adoption in microbiology laboratories worldwide. The development of a 12-probe array of highly sensitive and enzyme- selective chemiluminescent probes enables the rapid acquisition of baqcteria’s enzymatic activity fingerprints in 90 minutes. In contrat to methods confined to database comparisons, when encountering bacteria or samples containing multiple bacteria not represented in the enzymatic fingerprint database, the enzymatic activity profile of the unknown can still yield valuable information. This information sheds light on key enzymatic activity similarities found in other species. By expediting bacterial identification and characterization while offering essential information about their primary enzymatic activities, our method can also suggest potential new approaches for combination therapy using enzyme inhibitors. In summary, the method presented represents a significant advancement in rapid and highly sensitive diagnosis, offering potential applications in fundamental research, clinical settings, and industry.SCHEMESScheme 1. Synthesis of β-glucosidase Probe 1Scheme 2. Synthesis of β-glucurinidase Probe 2Scheme 3. Synthesis of ^-galactosidase Probe 3Scheme 4. Synthesis of pyroglutamyl aminopeptidase Probe 4Scheme 5. Synthesis of phosphatase Probe 5Scheme 6. Synthesis of leucine aminopeptidase Probe 6Scheme 7. Synthesis of ^-lactamase Probe 9Scheme 8. Synthesis of penicillin-G amidase Probe 10Scheme 9. Synthesis of periodate oxidative cleavage Probe 11Periodate oxidative-cleavage probe 11Scheme 10. Synthesis of N-acetyl hydrolase ProbeScheme 11. Synthesis of fluorescent phosphatase probe (Compound 17)Scheme 12. Synthesis of calorimetric phosphatase probe (Compound 19)REFERENCESBabin, B.M., et al., Chemiluminescent protease probe for rapid, sensitive, and inexpensive detection of live mycobacterium tuberculosis. ACS Cent. Sci., 2021, 7, 803-814 Cover, T.M.; Hart, P.E., Nearest neighbor pattern classification. IEEE Trans. Inf.Theory., 1967, 13(1), 21-27Gnaim, S., et al., Light emission enhancement by supramolecular complexation of chemiluminescence probes designed for bioimaging. Chemical Science, 2019, 10(10), 2945- 2955Green, O., et al., Opening a gateway for chemiluminescence cell imaging: distinctive methodology for design of bright chemiluminescent dioxetane probes. ACS Cent. Sci., 2017, 3, 349-358Gunduz, H., et al., Directing chemiluminescent dioxetanes to mitochondria: A cationic luminophore enables in vitro and in vivo detection of cancer cells upon enzymatic activation. Sens. Actuators B Chem., 2023, 383, 133574Hananya, N., et al., Rapid chemiexcitation of phenoxy-dioxetane luminophores yields ultrasensitive chemiluminescence assays. Chemical Science., 2019, 10(5), 1380-1385Maugeri, G., et al., Identification and antibiotic -susceptibility profiling of infectious bacterial agents: A review of current and future trends. Biotechnol. J., 2019, 14, 1700750Roth-Konforti, M., et al., Ultrasensitive detection of Salmonella and Listeria monocytogenes by small-molecule chemiluminescence probes. Angew. Chem. Int. Ed., 2019, 58, 10361-10367Russell, C.D., et al., Co-infections, secondary infections, and antimicrobial use in patients hospitalised with COVID-19 during the first pandemic wave from the ISARIC WHO CCP-UK study: a multicentre, prospective cohort study. Lancet Microbe., 2021, 2(8), e354-e365Ryan, L.S., et al., Kinetics-based measurement of hypoxia in living cells and animals using an acetoxymethyl ester chemiluminescent probe. ACS Sens., 2019, 4(5), 1391-1398Schaap, A.P., et al., Chemical and enzymatic triggering of 1,2-dioxetanes .2: Fluoride-induced chemiluminescence from tert-butyldimethylsilyloxy-substituted dioxetanes. Tetrahedron Lett., 1987, 28, 1155-1158Smith, P.B., et al., API system: a multitube micromethod for identification of Enterobacteriaceae. Appl. Microbiol., 1972, 24, 449-452Son, S . , et al. , Chemiluminescent probe for the in vitro and in vivo imaging of cancers over-expressing NQ01. Angew. Chem. Int. Ed., 2019, 58(6), 1739-1743Taber, D.F., et al., Synthesis of the eight enantiomerically pure diastereomers of the 12-F-2-isoprostanes. J. Am. Chem. Soc., 2002, 124(44), 13121-13126Wang, B.Q., et al., A highly selective and sensitive chemiluminescent probe for leucine aminopeptidase detection in vitro, in vivo and in human liver cancer tissue. Chem. Sci., 2022, 13(8), 2324-2330Zhang, H., et al., Self-powered microscale pumps based on analyte-initiated epolymerization reactions. Angew. Chem. Int. Ed., 2012, 51(10), 2400-2404Zhao, G.Y., et al., Syntheses and studies of quinolone-cephalosporins as potential anti-tuberculosis agents. Bioorg. Med. Chem. Eett., 2006, 16(21), 5534-5537

Claims

CLAIMS1. A method for detection and identification of a bacteria in an aqueous medium, said method comprising:(i) providing an array comprising a plurality of chemiluminescent probes, each composed of a phenyl-dioxetane luminophore masked by a caging group cleavable by a bacterial analyte or enzyme, wherein each one of said phenyl-dioxetane luminophores is masked with a caging group cleavable by a different bacterial analyte or enzyme, and upon exposure to said bacterial analyte or enzyme, said caging group is cleaved from said phenyl-dioxetane luminophore thereby generating an unstable phenolate - dioxetane compound, which is then decomposed through a chemiexcitation process to produce an excited intermediate that decays to its ground-state through emission of light;(ii) applying said aqueous medium to each one of said chemiluminescent probes of said array;(iii) detecting the emission of light from each one of said chemiluminescent probes, thereby generating a normalized metabolic activity profile for said bacteria, said normalized metabolic activity profile representing the quantity and intensity of emission of light detected from each one of the chemiluminescent probes; and(iv) applying via a processor one or more statistical methods and / or machine learning models on said normalized metabolic activity profile to identify said bacteria.

2. The method of claim 1, wherein the detection of the emission of light from each one of said chemiluminescent probes comprises using a plate reader, e.g., over about 10, 20, 30, 60, 75, or 90 minutes.

3. The method of claim 1, wherein: said one or more statistical methods include principal component analysis (PCA), hierarchical cluster analysis (HCA), centroid-based cluster analysis, density-based cluster analysis, distribution-based cluster analysis, constraint-based cluster analysis, fuzzy clustering cluster analysis, and / or factor analysis; and / orsaid one or more machine learning models each independently is selected from an unsupervised machine learning clustering method such as K-Means algorithm, Mean-Shift Algorithm, DBSCAN algorithm, Expectation-Maximization Clustering using Gaussian Mixture Models, and Agglomerative Hierarchical algorithm; a supervised machine learning classification method such as Logistic Regression, Naive Bayes, K-Nearest Neighbors, Decision Tree, and Support Vector Machines; a semi- supervised machine learning model; and a deep learning classification method such as Convolutional Neural Networks (CNNs), Long Short Term Memory Networks (LSTMs), Recurrent Neural Networks (RNNs), Generative Adversarial Networks (GANs), Radial Basis Function Networks (RBFNs), Multilayer Perceptrons (MLPs), Self Organizing Maps (SOMs), Deep Belief Networks (DBNs), Restricted Boltzmann Machines (RBMs), and Autoencoders.

4. The method of claim 1, wherein the identification of said bacteria includes a confidence level.

5. The method of claim 1, wherein said aqueous medium comprises phosphate-buffered saline (PBS, pH~7.4) and / or a bacterial growth medium.

6. The method of claim 1, wherein said bacteria is a Gram-positive bacteria or Gramnegative bacteria.

7. The method of claim 1, wherein said bacteria is selected from a Salmonella such as S. enterica', a Listeria such as L. monocytogenes', a Staphylococcus such as S. aureus, S. epidermidis, S. aureus, and methicillin-resistant S. aureus (MRSA); E. coli; a carbapenem- resistant bacteria such as Pseudomonas aeruginosa and Klebsiella pneumonia', a Campylobacter such as C. jejuni, C. coli, and C. lari; a Bacillus such as B. cereus; a Clostridium such as C. perfringens and C. difficile', a Mycobacterium such as M. tuberculosis and M. smegmatis; a Streptococcus such as S. pneumonia, S. agalactiae and S. pyogenes', a Pseudomonas such as P. aeruginosa; an Enterococcus such as E. faecalis; a vancomycin- resistant Enterococcus (VRE); a Neisseria such as N. meningitides; Haemophilus influenza Helicobacter pylori; Burkholderia cepacian; a Propionibacterium such as P. acnes; a Citrobacter; Cronobacter sakazakii; Geobacillus stearothermophilus; a Vibrio; a Legionella such as L. pneumophilia; an apyrase-containing bacterium such as Shigella; Coliform; an extended- spectrum beta-lactamase (ESBL)-producing enterobacterium; and a Prevotella.

8. The method of claim 1, wherein said bacterial analyte or enzyme is selected from a glycosidase, protease, alkaline phosphata.se, and an antibiotic resistance-conferring enzyme such as a p- lactamase, amidase (e.g., penicillin- G amidase), transferase (e.g., acyltransferase, phosphotransferase, and nucleotidyltransferase), glycoside hydrolase (e.g., p -galactosidase, p-glucuronidase, and p-glucosidase), pyroglutamyl -peptidase, leucine aminopeptidase, nitro-reductase, N-acetyl hydrolase, and NAD(P)H quinone oxidoreductase 1.

9. The method of any one of claims 1-8, wherein said phenyl-dioxetane luminophore each independently is a compound of formula I:whereinR1is selected from a linear or branched (C1-C18)alkyl, or (C3-C7lcycloalkyl;R2and R3each independently is selected from a branched (C3-C18)alkyl or (C3- C7)cycloalkyl, or R2 and R3 together with the carbon atom to which they are attached form a carbocyclic or heterocyclic ring;R4, R5and R6each independently is selected from H, an electron acceptor group such as halogen, -NO2, -CN, -C00R9, -C(=O)R9, and -SO2R9, and a π* acceptor group of the formula -CH=CH-E, wherein E is (a) -CN, -C00H, -COO(C1-C18)alkyl optionally interrupted in the alkylene chain with one or more -O- groups, or -COO(C2-C18)alkenyl such as -COO-allyl; or (b) 4-pyridinyl, methylpyridinium-4-yl, 3,3-dimethyl-3H-indolyl, or 1,3,3- trimethyl-3H-indol-l-ium-2-yl, optionally substituted at one or more of the carbon atoms of the aromatic or heteroaromatic ring with a substituent each independently selected from halogen, -CN, -C00H, -COO(C1-C18)alkyl, and -C(O)(C1-C18)alkyl; or one of R5and R6, together with R7, forms an optionally substituted cyclic or heterocyclic structure that extends the pi-system of the central aromatic ring, and the other one of R5and R6is H;R7is H, or together with one of R5and R6forms said optionally substituted cyclic or heterocyclic structure;R8is said caging group selected from an enzyme-labile group and a boron-containing group having the formula -B(Z)(Z') or -B(Z”)3“ Kat+;R9each independently is H, (Ci-Ci8)alkyl, or (C6-Ci4)aryl;Z and Z' each independently is selected from -OR10and -O’ Kat+;Z" is selected from F, Cl, Br, and I;R10is selected from H, (Ci-C4)alkyl, (C2-C4)alkenyl, (C2-C4)alkynyl, (C2- C4)heteroalkyl, (C2-C4)heteroalkenyl, (C2-C4)heteroalkynyl, (C6-Cio)aryl, (C5- C6)heteroaryl, (C6-Cio)aralykl, and (C6-Cio)heteroaralkyl, or two R10together with their intervening atoms form a 5- to 7-membered optionally substituted heterocyclic ring, preferably a saturated optionally substituted heterocyclic ring;Kat+each independently is an organic cation, or inorganic cation such as an alkali metal cation;L is absent or is a linker of the formula LI, L2 or L3:optionally substituted at the aromatic ring with one or more substituents each independently selected from (Ci-Ci8)alkyl and (C3-C7)cycloalkyl, wherein M is absent or is -O-, -NH-, - N(C1-C8)alkyl-, or -N+((C1-C8)alkyl)2-, and the asterisk represents the point of attachment to the group Y ; andY is absent or is -O-, provided that when R8is said enzyme-labile group, Y is -O-; and when R8is said boron-containing group, Y is absent, and either L is absent or L is said linker wherein M is absent.

10. The method of claim 9, wherein R1is a linear or branched (C1-C8)alkyl.

11. The method of claim 10, wherein R1is methyl.

12. The method of claim 9, wherein R2and R3together with the carbon atom to which they are attached form:(i) adamantyl;(ii) a 3-4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from (C1-C8)alkyl optionally substituted with at least one (C6-Cio)aryl group, (C6-Cio)aryl, or an electron withdrawing group such as halogen, -NO2, -CN, =0, -C00R11, -C(=0)R12, - O-(Ci-C8)alkylene-(C6-Cio)aryl, and -SO2R11, wherein two adjacent carbon atoms of said carbocyclic ring optionally form an additional 3-8 membered carbocyclic, heterocyclic, or aromatic ring, optionally substituted with at least one group each independently selected from halogen and (C1-C18)alkyl; two non-adjacent carbon atoms of said carbocyclic ring optionally form bicyclo [l.l.l]pentan-2-yl, or bicyclo[2.1.1]hexan-5-yl; or two alkyl groups attached to the same carbon atom of said carbocyclic ring, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic or heterocyclic ring; or(iii) a 6 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from (C1-C8)alkyl optionally substituted with at least one (C6-Cio)aryl group, (C6-Cio)aryl, or an electron withdrawing group such as halogen, -NO2, -CN, =0, -C00R11, -C(=0)R12, - O-(Ci-C8)alkylene-(C6-Cio)aryl, and -SO2R11, wherein two non-adjacent carbon atoms of said carbocyclic or heterocyclic ring optionally from an additional 3-4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from halogen and (Ci-Ci8)alkyl; or two alkyl groups attached to the same carbon atom of said carbocyclic or heterocyclic ring, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic or heterocyclic ring, whereinR11each independently is H, (C1-C18jalkyl, or (C6-C14)aryl; andR12each independently is H, (C1-C18jalkyl, (C6-C14)aryl, or halogen.

13. The method of claim 12, wherein R2and R3together with the carbon atom to which they are attached form:(i) adamantyl;(ii) cyclobutyl optionally substituted with one or more groups each independently selected from (Ci-C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, (C6)aryl, =0, -C00R11, -C(=O)R12, and -O-(Ci-C4)alkylene-(C6- Cio)aryl, wherein two adjacent carbon atoms of said cyclobutyl optionally form 3-membered carbocyclic ring or 6-membered aromatic ring; two non-adjacent carbon atoms of said carbocyclic ring optionally from bicyclo [1.1.1 ]pentan-2- yl; or two alkyl groups attached to the same carbon atom of the cyclobutyl, together with said carbon atom, optionally form an additional 4-membered carbocyclic or heterocyclic ring, said heterocyclic ring being elected from oxetane or azetidine optionally substituted at the nitrogen atom with one or two (Ci-C4)alkyl groups;(iii) oxetan-3-yl optionally substituted with one or more (Ci-C4)alkyl groups;(iv) azetidine-3-yl optionally substituted at the nitrogen atom with one or two groups each independently selected from (Ci-C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, -C00R11, -C(=0)R12, and -SO2R11;(v) thietane-3-yl optionally oxidized or dioxidized at the sulfur atom;(vi) cyclohexyl optionally substituted with one or more groups each independently selected from (Ci-C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, (Cejaryl, halogen, =0, -C00R11, -C(=0)R12, and -0-(Ci-C4)alkylene- (C6-Cio)aryl, wherein two non-adjacent carbon atoms of said cyclohexyl optionally from an additional 3-4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from halogen and (C1-C18jalkyl; or two alkyl groups attached to the same carbon atom of said cyclohexyl, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic or heterocyclic ring;(vii) tetrahydropyranyl (oxanyl) or dioxanyl, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (Ci-C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, (Cejaryl, halogen, =0, -C00R11, -C(=0)R12, and -O-(Ci-C4)alkylene-(C6- Ciojaryl;(viii) tetrahydrothiopyranyl (thianyl), 1-oxidotetrahydrothiopyranyl or 1,1- dioxidotetrahydrothiopyranyl, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (Ci-C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, -COOR11, -C(=O)R12, and -SO2R11; or(ix) piperidinyl or piperidinyl-1 -oxide, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (Ci- C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, (C6)aryl, halogen, =0, -COOR11, -C(=0)R12, and -0-(Ci-C4)alkylene-(C6-Cio)aryl, said piperidinyl being further optionally substituted at the nitrogen atom with one or two groups each independently selected from (Ci-C4)alkyl, (C3- C7)cycloalkyl, and (C6-Cio)aryl, whereinR11each independently is H, (Ci-C4)alkyl, or (C6-Cio)aryl; andR12each independently is H, (Ci-C4)alkyl, (C6-Cio)aryl, or halogen.

14. The method of claim 13, wherein R2and R3together with the carbon atom to which they are attached form adamantyl, cyclobutyl, 2,2-dimethylcyclobutyl, 2,2,4- trimethylcyclobutyl, 2,2,4,4-tetramethylcyclobutyl, 3 -phenylcyclobutyl, 3- benzyloxycyclobutyl, 3-carboxycyclobutyl, 3-carbomethoxycyclobutyl, bicyclo[4.2.0]octa- l(6),2,4-trien-7-yl, bicyclo[l.l.l]pentan-2-yl, bicyclo [2. l.l]hexan-5-yl, spiro[3.3]heptan-2- yl, 5-oxo-2-oxaspiro[3.3]heptan-6-yl, 6-oxo-2-oxaspiro[3.3]heptan-5-yl, 2-methyl-5-oxo-2- azaspiro[3.3]heptan-6-yl, 2-methyl-6-oxo-2-azaspiro[3.3]heptan-5-yl, 2,2-dimethyl-5-oxo- 2-azaspiro[3.3]heptan-2-ium-6-yl, 2,2-dimethyl-6-oxo-2-azaspiro[3.3]heptan-2-ium-5-yl, oxetan-3-yl, 2,2-dimethyloxetan-3-yl, 2,2,4-trimethyloxetan-3-yl, 2,2,4,4- tetramethyloxetan-3-yl, azetidin-3-yl, l-methylazetidin-3-yl, l-ethylazetidin-3-yl, 1- phenylazetidin-3-yl, l-diphenylmethyl-azetidin-3-yl, l,l-dimethylazetidin-3-ylium, 1- (methoxycarbonyl)azetidin-3-yl, l-(terZ-butoxycarbonyl)azetidin-3-yl, l-acetylazetidin-3- yl, 1 -(phenylsulfonyl) azetidin-3-yl, thietan-3-yl, l-oxidothietan-3-yl, l,l-dioxidothietan-3- yl, cyclohexyl, 3 -carboxy cyclohexyl, 3 -carbomethoxy cyclohexyl, 3- carboethoxycyclohexyl, 4-carboxycyclohexyl, 4-carbomethoxycyclohexyl, 4- carboethoxycyclohexyl, (chlorocarbonyl)cyclohexyl, (bromocarbonyl)cyclohexyl, fluorocyclohexyl, difluorocyclohexyl, 3 ,3 -difluorocyclohexyl, 4.4-difluorocyclohexyl, chlorocyclohexyl, dichlorocyclohexyl, 3 ,3 -dichlorocyclohexyl, 4.4-dichlorocyclohexyl, cyanocyclohexyl, 3 -cyanocyclohexyl, 4-cyanocyclohexyl, nitrocyclohexyl, 3- nitrocyclohexyl, 4-nitrocyclohexyl, 3 -tetrahydropyranyl, 4-tetrahydropyranyl, l,3-dioxan-5-yl, 2,2-dimethyl-l,3-dioxan-5-yl, 4-tetrahydrothiopyranyl, l-oxido-4-tetrahydrothiopyranyl, or 1 , 1 -dioxido-4-tetrahydrothiopyranyl.

15. The method of claim 9, wherein:(i) R4, R5and R6each independently is selected from H, halogen, preferably chlorine, -CN, and a π* acceptor group of the formula -CH=CH-E; or(ii) R4is selected from H, halogen, preferably chlorine, and -CN; one of R5and R6is H; and the other one of R5and R6, together with R7, forms a 6-memberedis selected from -CN, -NO2, (C2-Ci2)alkenyl, aryl, -COOR15, -COO- Kat+, and -C(O)N(R15)2, wherein R15is H or (C1-C12)alkyl, or the two R15together with the nitrogen atom to which they are attached form a heterocyclic ring.

16. The method of claim 15, wherein:(i) R4and R5each independently is H or halogen; and R6is a π* acceptor group of the formula -CH=CH-E;(ii) R4and R6each independently is H or halogen; and R5is a π* acceptor group of the formula -CH=CH-E; or(iii) R5and R6each independently is H or halogen; and R4is a π* acceptor group of the formula -CH=CH-E.

17. The method of claim 16, wherein R4and R6each independently is H or halogen; andR5is a π* acceptor group of the formula -CH=CH-E.

18. The method of claim 17, wherein R4is halogen; R6is H; and R5is a π* acceptor group of the formula -CH=CH-E.

19. The method of any one of claims 15-18, wherein E is -CN, -COOH, -COO(C1- C8)alkyl, or -COO(C2-C8)alkenyl.

20. The method of claim 19, wherein E is -CN, -COOH, -COO(Ci-C4)alkyl such as - COOCH3and -COOC(CH3)3, or -COO(C2-C4)alkenyl such as -COOCH=CH-CH3.

21. The method of claim 15, wherein R4, R5and R6each independently is H or halogen.

22. The method of claim 21, wherein R4is halogen, and R5and R6each is H.

23. The method of claim 15, wherein R4is selected from H, halogen and -CN; R5is H; and R6together with R7forms a 6-membered structure selected from24. The method of claim 23, wherein R4is H or halogen; R5is H; and R6together withR7forms said 6-membered structure.

25. The method of claim 9, wherein L is absent or is a linker of the formula L1, L2 or L3, preferably L1, wherein M is absent or is -O-, -NH-, or -N+(CH3)2-.

26. The method of claim 9, wherein:R1is a linear or branched (C1-C8)alkyl;R2and R3together with the carbon atom to which they are attached form:(i) adamantyl;(ii) a 3-4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from (C1-C8)alkyl optionally substituted with at least one (C6-Cio)aryl group, (C6-Cio)aryl, or an electron withdrawing group such as halogen, -NO2, -CN, =0, -C00R11, -C(=0)R12, - O-(Ci-C8)alkylene-(C6-Cio)aryl, and -SO2R11, wherein two adjacent carbon atoms of said carbocyclic ring optionally form an additional 3-8 membered carbocyclic, heterocyclic, or aromatic ring, optionally substituted with at least one group each independently selected from halogen and (C1-C18)alkyl; two non-adjacent carbon atoms of said carbocyclic ring optionally from bicyclo[l.l.l]pentan-2-yl, or bicyclo[2.1.1]hexan-5-yl; or two alkyl groups attached to the same carbon atom of said carbocyclic ring, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic or heterocyclic ring; or(iii) a 6 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from (C1-C8)alkyl optionally substituted with at least one (C6-Cio)aryl group, (C6-Cio)aryl, or an electron withdrawing group such as halogen, -NO2, -CN, =0, -C00R11, -C(=0)R12, - 0-(Ci-C8)alkylene-(C6-Cio)aryl, and -SO2R11, wherein two non-adjacent carbon atoms of said carbocyclic or heterocyclic ring optionally from an additional 3-4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from halogen and (Ci-Ci8)alkyl; or two alkyl groups attached to the same carbon atom of said carbocyclic or heterocyclic ring, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic or heterocyclic ring; either(iv) R4, R5and R6each independently is selected from H, halogen, preferably chlorine, -CN, and a π* acceptor group of the formula -CH=CH-E; or(v) R4is selected from H, halogen, preferably chlorine, and -CN; one of R5and R6is H; and the other one of R5and R6, together with R7, forms a 6-memberedstructure selected fromand, wherein R13is (Ci-Ci2)alkyl; and R14is selected from -CN,-NO2, (C2-Ci2)alkenyl, aryl, -C00R15, -COO- Kat+, and -C(O)N(R15)2, wherein R15is H or (Ci-Ci2)alkyl, or the two R15together with the nitrogen atom to which they are attached form a heterocyclic ring;R11each independently is H, (C1-C18jalkyl, or (C6-C14)jaryl;R12each independently is H, (C1-C18jalkyl, (C6-C14)jaryl, or halogen; andL is absent or is a linker of the formula LI, L2 or L3, preferably LI, wherein M is absent or is -0-, -NH-, or -N+(CH3)2-.

27. The method of claim 26, whereinR1is methyl; andR2and R3together with the carbon atom to which they are attached form:(i) adamantyl;(ii) cyclobutyl optionally substituted with one or more groups each independently selected from (Ci-C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, (C6)aryl, =0, -C00R11, -C(=0)R12, and -O-(Ci-C4)alkylene-(C6- Cio)aryl, wherein two adjacent carbon atoms of said cyclobutyl optionally form 3-membered carbocyclic ring or 6-membered aromatic ring; two non-adjacent carbon atoms of said carbocyclic ring optionally from bicyclo [1.1.1 ]pentan-2- yl; or two alkyl groups attached to the same carbon atom of the cyclobutyl, together with said carbon atom, optionally form an additional 4-membered carbocyclic or heterocyclic ring, said heterocyclic ring being elected fromoxetane or azetidine optionally substituted at the nitrogen atom with one or two (Ci-C4)alkyl groups;(iii) oxetan-3-yl optionally substituted with one or more (Ci-C4)alkyl groups;(iv) azetidine-3-yl optionally substituted at the nitrogen atom with one or two groups each independently selected from (Ci-C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, -COOR11, -C(=O)R12, and -SO2R11;(v) thietane-3-yl optionally oxidized or dioxidized at the sulfur atom;(vi) cyclohexyl optionally substituted with one or more groups each independently selected from (Ci-C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, (C6)aryl, halogen, =0, -COOR11, -C(=O)R12, and -0-(Ci-C4)alkylene- (C6-Cio)aryl, wherein two non-adjacent carbon atoms of said cyclohexyl optionally from an additional 3-4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from halogen and (Ci-Ci8)alkyl; or two alkyl groups attached to the same carbon atom of said cyclohexyl, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic or heterocyclic ring;(vii) tetrahydropyranyl (oxanyl) or dioxanyl, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (Ci-C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, (C6)aryl, halogen, =0, -COOR11, -C(=0)R12, and -O-(Ci-C4)alkylene-(C6- Cio)aryl;(viii) tetrahydrothiopyranyl (thianyl), 1-oxidotetrahydrothiopyranyl or 1,1- dioxidotetrahydrothiopyranyl, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (Ci- C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, -COOR11, -C(=0)R12, and -SO2R11; or(ix) piperidinyl or piperidinyl- 1 -oxide, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (Ci- C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, (C6)aryl, halogen, =0, -COOR11, -C(=0)R12, and -0-(Ci-C4)alkylene-(C6-Cio)aryl, said piperidinyl being further optionally substituted at the nitrogen atom with one or two groups each independently selected from (Ci-C4)alkyl, (C3- C7)cycloalkyl, and (C6-Cio)aryl,whereinR11each independently is H, (Ci-C4)alkyl, or (C6-Cio)aryl; andR12each independently is H, (Ci-C4)alkyl, (C6-Cio)aryl, or halogen.

28. The method of claim 27, wherein R2and R3together with the carbon atom to which they are attached form adamantyl, cyclobutyl, 2,2-dimethylcyclobutyl, 2,2,4- trimethylcyclobutyl, 2,2,4,4-tetramethylcyclobutyl, 3 -phenylcyclobutyl, 3- benzyloxycyclobutyl, 3-carboxycyclobutyl, 3-carbomethoxycyclobutyl, bicyclo[4.2.0]octa- l(6),2,4-trien-7-yl, bicyclo[l.l.l]pentan-2-yl, bicyclo [2. l.l]hexan-5-yl, spiro[3.3]heptan-2- yl, 5-oxo-2-oxaspiro[3.3]heptan-6-yl, 6-oxo-2-oxaspiro[3.3]heptan-5-yl, 2-methyl-5-oxo-2- azaspiro[3.3]heptan-6-yl, 2-methyl-6-oxo-2-azaspiro[3.3]heptan-5-yl, 2,2-dimethyl-5-oxo-2-azaspiro[3.3]heptan-2-ium-6-yl, 2,2-dimethyl-6-oxo-2-azaspiro[3.3]heptan-2-ium-5-yl, oxetan-3-yl, 2,2-dimethyloxetan-3-yl, 2,2,4-trimethyloxetan-3-yl, 2, 2,4,4- tetramethyloxetan-3-yl, azetidin-3-yl, l-methylazetidin-3-yl, l-ethylazetidin-3-yl, 1- phenylazetidin-3-yl, l-diphenylmethyl-azetidin-3-yl, l,l-dimethylazetidin-3-ylium, 1-(methoxycarbonyl)azetidin-3-yl, l-(terZ-butoxycarbonyl)azetidin-3-yl, l-acetylazetidin-3- yl, 1 -(phenylsulfonyl) azetidin-3-yl, thietan-3-yl, l-oxidothietan-3-yl, l,l-dioxidothietan-3- yl, cyclohexyl, 3 -carboxy cyclohexyl, 3 -carbomethoxy cyclohexyl, 3- carboethoxycyclohexyl, 4-carboxycyclohexyl, 4-carbomethoxycyclohexyl, 4- carboethoxycyclohexyl, (chlorocarbonyl)cyclohexyl, (bromocarbonyl)cyclohexyl, fluorocyclohexyl, difluorocyclohexyl, 3 ,3 -difluorocyclohexyl, 4.4-difluorocyclohexyl, chlorocyclohexyl, dichlorocyclohexyl, 3 ,3 -dichlorocyclohexyl, 4.4-dichlorocyclohexyl, cyanocyclohexyl, 3 -cyanocyclohexyl, 4-cyanocyclohexyl, nitrocyclohexyl, 3- nitrocyclohexyl, 4-nitrocyclohexyl, 3 -tetrahydropyranyl, 4-tetrahydropyranyl, l,3-dioxan-5- yl, 2,2-dimethyl-l,3-dioxan-5-yl, 4-tetrahydrothiopyranyl, l-oxido-4-tetrahydrothiopyranyl, or 1 , 1 -dioxido-4-tetrahydrothiopyranyl.

29. The method of claim 28, wherein:(i) R4and R5each independently is H or halogen; and R6is a π* acceptor group of the formula -CH=CH-E;(ii) R4and R6each independently is H or halogen; and R5is a π* acceptor group of the formula -CH=CH-E; or(iii) R5and R6each independently is H or halogen; and R4is a π* acceptor group of the formula -CH=CH-E.

30. The method of claim 29, wherein R4and R6each independently is H or halogen; and R5is a π* acceptor group of the formula -CH=CH-E.

31. The method of claim 30, wherein R4is halogen; R6is H; and R5is a π* acceptor group of the formula -CH=CH-E.

32. The method of any one of claims 26-31, wherein E is -CN, -COOH, -COO(Ci- C8)alkyl, or -COO(C2-C8)alkenyl.

33. The method of claim 32, wherein E is -CN, -COOH, -COO(Ci-C4)alkyl such as - COOCH3and -COOC(CH3)3, or -COO(C2-C4)alkenyl such as -COOCH=CH-CH3.

34. The method of claim 28, wherein R4, R5and R6each independently is H or halogen.

35. The method of claim 34, wherein R4is halogen, and R5and R6each is H.

36. The method of claim 28, wherein R4is selected from H, halogen and -CN; R5is H; and R6together with R7forms a 6-membered structure selected from37. The method of claim 36, wherein R4is H or halogen; R5is H; and R6together with R7forms said 6-membered structure.

38. The method of any one of claims 9-37, wherein said enzyme-labile group each independently is selected from:

39. The method of claim 38, wherein:R1is methyl;R2and R3together with the carbon atom to which they are attached form adamantyl, cyclobutyl, 3 -phenylcyclobutyl, 3-benzyloxycyclobutyl, 3-carbomethoxycyclobutyl, 2,2- dimethylcyclobutyl, 2,2,4,4-tetramethylcyclobutyl, 3 -carboxy cyclobutyl, bicyclo[4.2.0]octa-l(6),2,4-trien-7-yl, spiro[3.3]heptan-2-yl, oxetan-3-yl, 2, 2,4,4- tetramethyloxetan-3-yl, l-acetylazetidin-3-yl, l-(terZ-butoxycarbonyl)azetidin-3-yl, 1- (methoxycarbonyl)azetidin-3-yl, or l-diphenylmethyl-azetidin-3-yl, cyclohexyl, 4- carbomethoxycyclohexyl, 4-tetrahydropyranyl, 3 -tetrahydropyranyl, 4,4- difluorocyclohexyl, 2,2-dimethyl-l,3-dioxan-5-yl, or l,l-dioxido-4-tetrahydrothiopyranyl;R4is halogen;R6is H;R5is a π* acceptor group of the formula -CH=CH-E, wherein E is -COOH, -COO(Ci- C4)alkyl such as -COOCH3 and -COOC(CH3)3, or -COO(C2-C4)alkenyl such as - COOCH=CH-CH3; and(i) L is LI wherein M is absent; and R8is β-glucoside, β-glucuronide, P- galactoside, L-pyroglutamyl- amide, phosphate ester, leucine amide, nitro, N,3- dimethyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-l,4-dien-l-yl)butanamide, phenylacetamide, or acetamide; or(ii) L is absent; and R8is ((6R,7R)-2-carboxy-8-oxo-7-(2-phenylacetamido)-5- thia-l-azabicyclo[4.2.0]oct-2-en-3-yl)methyl, or 3,4-dihydroxybutyl.

40. The method of claim 39, wherein R2and R3together with the carbon atom to which they are attached form adamantyl; R4is chlorine; and R5is a it* acceptor group of the formula -CH=CH-E, wherein E is -COOH.

41. A computing system comprising: at least one processor; and at least one memory communicatively coupled to the at least one processor comprising computer-readable instructions that when executed by the at least one processor cause the computing system to implement a method for identification of a bacteria in an aqueous medium, the method comprising:(i) providing an array comprising a plurality of chemiluminescent probes, each composed of a phenyl-dioxetane luminophore masked by a caging group cleavable by bacterial analyte or enzyme. wherein each one of said phenyl-dioxetane luminophores is masked with a caging group cleavable by a different bacterial analyte or enzyme and upon exposure to said bacterial analyte or enzyme, said caging group is cleaved from said phenyl-dioxetane luminophore thereby generating an unstable phenolate - dioxetane compound, which is then decomposed through a chemiexcitation process to produce an excited intermediate that decays to its ground-state through emission of light;(ii) applying said aqueous medium to each one of said chemiluminescent probes of said array;(iii) detecting the emission of light from each one of said chemiluminescent probes, thereby generating a normalized metabolic activity profile for said bacteria, said normalized metabolic activity profile representing the quantity and intensity of emission of light detected from each one of the chemiluminescent probes; and(iv) applying via said at least one processor one or more statistical methods and / or machine learning models on said normalized metabolic activity profile to identify said bacteria,

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