WO2019211705A1 - Molecular probes for detection of mycobacteria - Google Patents

Molecular probes for detection of mycobacteria Download PDF

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Publication number
WO2019211705A1
WO2019211705A1 PCT/IB2019/053369 IB2019053369W WO2019211705A1 WO 2019211705 A1 WO2019211705 A1 WO 2019211705A1 IB 2019053369 W IB2019053369 W IB 2019053369W WO 2019211705 A1 WO2019211705 A1 WO 2019211705A1
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group
mycobacteria
molecule
detection
acid
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PCT/IB2019/053369
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French (fr)
Inventor
Avinash BAJAJ
Siddhi GUPTA
Deepakumar MISHRA
Sandeep Kumar
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Regional Centre For Biotechnology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J43/003—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton not condensed
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0052—Small organic molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria

Definitions

  • the present invention relates to synthesis, purification, and development of novel small molecules conjugated (Covalently/non-covalently) with fluorophore/imaging agent for detection of mycobacteria.
  • This invention further discloses the applications of these probes for selective detection of non-pathogenic, pathogenic, drug- sensitive, drug-resistant, live/dead, persistent, stationary, metabolically-inactive or any type of mycobacterial species.
  • This invention also covers the use of these probes to selectively detect mycobacteria among multi-microbial species, biofilms, intracellular mycobacteria; and mycobacteria in human/animal body fluids and tissues or any other media.
  • Mycobacterium is a genus of Actinobacteria, given its own family, the Mycobacteriaceae. Over 190 species are recognized in this genus. It includes pathogens known to cause serious diseases in mammals.
  • Mycobacterium tuberculosis is a species of pathogenic bacteria in the family Mycobacteriaceae and the causative agent of tuberculosis.
  • M. tuberculosis has an unusual, waxy coating on its cell surface primarily due to the presence of mycolic acids. The most frequently used diagnostic methods for tuberculosis are the tuberculin skin test, acid-fast staining, culture, and polymerase chain reaction.
  • Detection of mycobacteria has been a challenge because of the complex nature of these bacteria, and their ability to rapidly change their genomic nature.
  • Nucleic acids, protein and sugars of mycobacteria have been used to engineer molecular probes for easy detection of these bacteria in different samples and biological fluids.
  • Engineering of specific antibodies for a targeted antigen, or PCR based methods have been developed and approved for bacterial diagnostics.
  • rapid changes in genomic sequences, development of drug resistance in mycobacteria, poor sensitivity of these assays, and inability to test different strains could not provide success to these methods. Poor knowledge of structural features of polysaccharides and proteins present on these pathogenic bacteria also could not provide any major breakthrough for detection of mycobacteria.
  • Gastrointestinal Tuberculosis shows intricate physiological similarity with Crohn’s Disease thus causing clinical diagnostic difficulties.
  • Detection of mycobacteria additionally, provides further challenges due to increased burden of tuberculosis, slow growing nature of mycobacteria, and its ability to mutate its genomic structure; thereby pressing the need for a simple and affordable diagnostic tool for mycobacterial infections.
  • the primary object of this invention is to develop different structural classes of novel small molecules which can specifically bind to mycobacteria and lead to detection.
  • Another object of the invention is to synthesize small molecules that can selectively bind with mycobacteria for identification and detection.
  • the Invention provides methods to synthetically develop a set of novel small molecular derivatives to detect mycobacteria under in-vitro , in-vivo and clinical settings. These derivatives can selectively bind to mycobacterial species at very low concentrations and differentiate them in the presence of other gram negative and gram-positive species.
  • the invented molecules conjugated with a fluorophore allow specific detection of mycobacteria in planktonic forms, biofilm forms, intracellular, mouse and human tissues.
  • the invented molecules can selectively bind to pathogenic, non-pathogenic, metabolically inactive, drug sensitive as well as drug resistant mycobacteria.
  • the Invention provides a potential molecule to be used as a fluorescent probe or a sensor to differentiate between clinical cases of Gastrointestinal Tuberculosis and Crohn’s disease by binding and showing fluorescent signals in the Gastrointestinal Tuberculosis positive tissues.
  • the invention provides scope and methods to develop this molecule as a point-of-care diagnostic system (probe/assay/kit) for Tuberculosis detection and overcome the challenges of time consuming and inaccurate diagnostic methods.
  • Figure 1 illustrates general structures of small molecules for detection of mycobacteria.
  • Figure 2 illustrates synthesis of Cholic Acid-Fluorophore Conjugated Probes.
  • Reagents and reaction conditions (i) Propargyl amine, EDC.HC1, HOBt, DIPEA, DMF/DCM (1:3), RT, 24h; (ii) Chloroacetic anhydride, DMAP, DCM, RT, 3h; (iii) NBD-CH 2 -CH 2 -N 3 , CuS0 4 , Sodium Ascorbate, THF: DCM: H 2 0 (5:5:1), RT, l2h (iv) DMAP/TMA, DMF, 70°C, 48h.
  • Figure 3 illustrates synthesis of Deoxycholic Acid-Fluorophore Conjugated Probes.
  • Reagents and reaction conditions (v) Propargyl amine, EDC.HC1, HOBt, DIPEA, DMF/DCM (1:3), RT, 24h; (vi) Chloroacetic anhydride, DMAP, DCM, RT, 3h; (vii) NBD- CH 2 -CH 2 -N 3 , CUS0 4 , Sodium Ascorbate, THF: DCM: H 2 0 (5:5:1), RT, l2h (viii) DMAP, DMF, 70°C, 48h.
  • Figure 4 illustrates synthesis of Biotin Conjugated Probes. Reagents and reaction conditions: (ix) Biotin- Azide, CuS0 4 , Sodium Ascorbate, THF: MeOH: H 2 0 (2:2:1), RT, l2h (x) DMAP, DMF, 70°C, 48h.
  • Figure 5 illustrates synthesis of Alkyne Conjugated Probes. Reagents and reaction conditions: (xi) DMAP, DMF, 70°C, 48h.
  • Figure 6 illustrates synthesis of Azide Conjugated Probes. Reagents and reaction conditions: (xii) Azidoethyl amine.TFA, EDC.HC1, HOBt, DIPEA, DMF/DCM (1:3), RT, 24h; (xiii) Chloroacetic anhydride, DMAP, DCM, RT, 3h; (xiv) DMAP, DMF, 70°C, 48h.
  • Figure 7 illustrates confocal microscopic images of different mycobacteria, Gram-positive and Gram-negative bacterial strains after staining with Molecule 1 probe showing the selectivity of the probe for mycobacteria.
  • TRITC/DAPI is fluorescence of bacteria due to plasmid
  • FITC Molecule 1 Fluorescence
  • the Invention has used mCherry expressing M. smegmatis, M. bovis, S. typhimurium, and pCyPet (Blue Fluorescence) expressing E. coli and S. aureus for staining purposes.
  • Figure 8 illustrates confocal microscopic images of mcherry expressing M.
  • TRITC/DAPI is fluorescence of bacteria due to plasmid; FITC: Molecule 1 Fluorescence
  • Figure 9 illustrates confocal microscopic images of mcherry expressing M. smegmatis, pCyPet expressing E. coli and pCyPet expressing S. aureus biofilms after staining with Molecule 1 showing the selective staining of mcherry expressing M. smegmatis bio films.
  • TRITC/DAPI is fluorescence of bacteria due to plasmid
  • FITC Molecule 1 Fluorescence
  • Figure 10 illustrates confocal microscopic images of mCherry expressing- smegmatis and pCyPet expressing- E. coli biofilms after staining with Molecule 1 showing the selective staining of mcherry expressing M. smegmatis mycobacterial strains.
  • TRITC fluorescence of mycobacteria due to plasmid
  • FITC Molecule 1 Fluorescence
  • DAPI Fluorescence of pCypet E. coli
  • Figure 11 illustrates confocal microscopic images showing the positive staining of mycobacteria in Mycobacterium tuberculosis infected mice lung sections with Molecule 1 at 10 nM concentration. Hoechst 33258 was used to stain the cell nuclei. (FITC: Molecule 1 Fluorescence, TRITC: antibody Fluorescence, DAPI channel: Nuclear stain)
  • Figure 12 illustrates confocal microscopic images showing the positive staining of mycobacteria in Mycobacterium tuberculosis infected human tissues with Molecule 1 at 10 nM concentration.
  • Hoechst 33258 was used to stain nuclei, and mycobacteria specific antibody was used for confirmation.
  • TRITC fluorescence due to antibody staining
  • FITC Molecule 1 Fluorescence
  • DAPI Nuclear stain
  • Figure 13 illustrates confocal microscopic images showing the positive staining of mycobacteria in Mycobacterium tuberculosis infected Gastrointestinal TB tissues with
  • FIG. 14 Confocal microscopic images showing the positive staining of mycobacteria in Mycobacterium tuberculosis infected mice lung tissues with molecule 4 (Biotin-conjugate) folowed by incubation with Biotin-Quantum Dot (Qdot 655 Streptavidin Conjugate) at 10 nM concentration.
  • the Inventors have developed a novel small molecule for detection of mycobacteria, diagnosis of Tuberculosis and clinical differentiation of Gastrointestinal Tuberculosis with Crohn’s Disease.
  • Mycobacterial membranes of Gram-positive, Gram-negative, mycobacteria, and mammalian cells differ in their chemical nature.
  • Mycobacterial membranes are highly rigid and hydrophobic in nature due to presence of mycolic acids whereas Gram-negative and Gram-positive bacteria possess lipopolysaccharide and proteoglycan shielding on their outer surface. Therefore, to achieve the stated objectives, the hydrophobic nature of mycobacterial membranes was exploited to engineer selective mycobacterial targeting probes that help in easy identification of mycobacteria in tissue samples.
  • the interactions of these probes with cellular membranes involve the electrostatic interactions between probes and cell membranes involving dehydration; followed by hydrophobic interactions.
  • mycobacterial-specific probes were designed and the ability of these probes to detect mycobacteria was evaluated. These probes will specifically target the mycobacterial membrane, and will be able to stain drug- sensitive, drug-resistant, persistent, live/dead, metabolically active and inactive mycobacterial strains.
  • the present invention involves the use of small molecules that have ligands and a fluorophore/imaging reagent.
  • Ligands help in binding of the probe to mycobacteria selectively, and fluorophore/imaging agent helps in easy detection of the sample.
  • Fluorophore conjugated small molecules that can selectively bind with mycobacteria have been synthesized; and capable of being detected using flow cytometry and fluorescence microscopy.
  • Biotin conjugated small molecules that selectively bind with mycobacteria have also been synthesized. The selective binding of Biotin conjugated molecules can then be detected using Streptavidin- Fluorophore conjugates, or Streptavidin tagged quantum dots, or Streptavidin conjugated enzymatic assays.
  • the Invention has also synthesized Alkyne- or Azide conjugated small molecules that selectively bind with mycobacteria. Selective binding of these Alkyne or Azide-conjugated molecules can be detected using Copper catalysed or Copper free click chemistry using Azide or Alkyne conjugated fluorescent molecules, proteins or enzymes.
  • invention focuses on synthesis of fluorophore/imaging agent-tagged small molecules that has specific ligands and binds to mycobacteria described as Fluorophore/imaging agent-conjugated small molecules of general formula I and II.
  • P in formula I and II represents an imaging agent like nitrobenzoxadiazole, Rhodamine, Fluorescein isothiocyanate, Cyanine dyes or any dyes from Nitroso dyes, Indophenol dyes, Nitro dyes, Azine dyes, Azo dyes, Oxazine dyes, Azoic dyes, Thiazine dyes, Stilbene dyes, Carotenoid dyes, Lactone dyes, Diphenylmethane dyes, Aminoketone dyes, Triarylmethane dyes, Xanthene dyes, Anthraquinone dyes, Acridine dyes, Indigoid dyes, Quinoline dyes, Phthalocyanine dyes, Methine dyes, Thiazole dyes, Indamine dyes etc.
  • “P” can also be groups like alkyne, biotin, azide, A- hydroxy succinimide, maleimide, Naphthalimide or lipid molecule or hydrocarbon or any other chemical mo
  • Linker Ll and L2 represent a linker or spacer, which is an aromatic, aliphatic, alicyclic, small polymeric linker that covalently conjugates the deoxycholic acid (DCA) and cholic acid (CA) moieties and the ligand or imaging agent.
  • Linker Ll is covalently attached between DCA/CA with targeting ligand; and
  • Linker L2 is between DCA/CA and imaging agent.
  • Spacer/linker chemistry represents simple organic functional groups such as ester, amide, carbonate, carbamate, phosphate, phosphonate, phosphoramidate, amine, urea, thiourea, sulfonamide, ether, thioether, sulfoxide, sulfone, thioester, thioamide, disulfide, oxime, o-acyloxime, o- acyloxyalkyloxime, o-carbamoyloxime etc. It also includes small organic linker molecules include aliphatic polar molecules, polar aromatic compounds, as well as heterocyclic molecules.
  • the present invention preferentially uses few organic small linkers such as ethylene glycol, ethanolamine, 2-amino-ethylamine, 2-amino-ethanethiol, p-aminobenzoic acid, p-azidobenzoic acid, triazole etc.
  • Second part of invention deals with use of these probes for detection of mycobacteria by flow cytometry, and fluorescence microscopy.
  • These probes are able to selectively stain the pathogenic, non-pathogenic, drug- sensitive, drug-resistant, persistent, stationary mycobacterial strains alone or in a polymicrobial population in different media, buffers, animal and human biological samples.
  • reaction mixture was stirred at 0 °C for 3 hours. After the reaction completion, reaction mixture was diluted with dichloro methane (250 mL) and washed with saturated NaHC0 3 solution (3 X 200 mL) and brine (2 X 200 mL). Organic phase was dried over anhydrous Na 2 S0 4 , and solvent was reduced under vacuum. Crude mixture was purified by column chromatography on silica gel (230-400 mesh) using Petroleum Ether/Ethyl Acetate as eluent, to give a colorless solid (350 mg). Product 9 was characterized by 1H NMR spectroscopy.
  • Molecule 1 can stain >90% of the mycobacterial cells including Mycobacterium bovis (BCG), Mycobacterium smegmatis ( Msm ), Mycobacterium tuberculosis ( Mtb ). Molecule 1 is unable to stain Gram- negative strains ( Escherichia coli ( E . coli), or Gram-positive ( Staphylococcus aureus (S. aureus) as shown in Table 1
  • the Invention used the different bacterial strains and incubated these strains with Molecule 1. After this, cells were washed to remove the unbound molecule. Cell were then fixed with 4% paraformaldehyde (PFA). For Molecule 1, FITC channel was used. For imaging, 63X oil was the objective and Hyvolution mode was chosen to minimize the background.
  • the images of planktonic bacteria revealed a clear staining of mycobacterial strains (. Mycobacterium smegmatis ( Msm ); Mycobacterium bovis ( M . bovis (BCG)), Mycobacterium tuberculosis ( Mtb ); whereas Molecule 1 is unable to stain any of the Gram-positive (5. aureus) or Gram negative stains (. E . coli, S. typhimurium,) suggesting the selectivity of these probes for mycobacteria detection.
  • the Invention tested the ability of Molecule 1 to detect the mycobacteria (mCherry expressing M. smegmatis) selectively in presence of other Gram-negative (pCyPet expressing E. coli) or Gram-positive bacteria (pCyPet expressing S. aureus).
  • the process involves mixing of mCherry expressing M. smegmatis and pCyPet (Blue fluorescence) expressing E. coli or mCherry expressing M. smegmatis and pCyPet expressing S. aureus (Blue fluorescence) and stained with Molecule 1 (2nM).
  • Molecule 1 is able to selectively stain the mycobacteria species as co localization of green fluorescence of probe was observed with mcherry expressing M. smegmatis in both the co-culture studies with pCyPet expressing E. coli and pCyPet expressing S. aureus. Probe did not show any co-localization with pCyPet expressing E. coli or pCyPet expressing S. aureus.
  • the Invention tested the Molecule 1 for selective staining of mycobacterial bio films.
  • Bacterial biofilms using mCherry expressing M. smegmatis, pCyPet expressing E. coli and pCyPet expressing S. aureus were prepared separately and incubated with Molecule 1 (2 nM). Biofilms were washed and observed under confocal microscope. Confocal images showed the selective staining of mycobacterial mCherry expressing M. smegmatis bio films as co-localization of mCherry M. smegmatis with green fluorescent stain of Molecule 1 was observed. Molecule 1 does not stain pCyPet expressing E. coli and pCyPet expressing S. aureus biofilms.
  • the Invention then tested the ability of Molecule 1 for detection of mycobacteria in polymicrobial biofilms.
  • Polymicrobial biofilms were prepared using pCypet expressing E. coli and mcherry expressing M. smegmatis; and incubated with Molecule 1 (2nM). After washing off the probe, biofilms were observed under confocal microscope. It was observed that the co-localization of green fluorescence of Molecule 1 with mcherry expressing M. smegmatis happened whereas no co-localization was observed with pCypet expressing E. coli. Therefore, Molecule 1 can detect mycobacteria in poly-microbial biofilms.
  • the Invention then tested the Molecule 1 for detection of mycobacteria in infected mouse lung tissues.
  • Paraffin- fixed transverse lung sections of M. tuberculosis infected mice were obtained and paraffin wax was removed by microwave treatment and xylene washing. Gradient hydration was given using 90% and 80% ethanol and sections were stained with Molecule 1 (10 nM) concentration as well as Anti Mtb antibody, Ab905 (abeam) used in a 1:1000 dilution. Nuclei staining was done using Hoechst 33258, and finally, sections were fixed using 4% PFA and observed under confocal microscope. The staining of mycobacteria using Molecule 1 as green fluorescent bacteria clearly suggested that these probes can be used for detection of mycobacteria in tissue sections as well.
  • the ability of the molecule 1 to detect the mycobacteria in infected human tissues was tested. Sections of infected human tissue were stained with Molecule 1. Hoechst was used for staining of nuclei, and mycobacteria specific antibody, Ab905 (abeam) was used for the confirmation of mycobacteria. As shown in Figure 12, Molecule 1 can detect the presence of mycobacteria in human tissue sections (FITC Channel). Co-localization of antibody staining with Molecule 1 confirm the presence of mycobacteria in the sections. Example 12
  • the ability of the molecule 1 to differentiate between the human Gastrointestinal and Crohn’s Disease tissues was tested. Hoechst was used for staining of nuclei and mycobacteria specific antibody Ab905(Abcam) was used for the confirmation of mycobacteria. As shown in Figure 13, Molecule 1 can detect the presence of mycobacteria in TB sections but did not show presence of mycobacteria in Crohn’s disease sections. Presence of mycobacteria was confirmed only in the TB positive sections by the colocalization of antibody staining with Molecule 1.
  • molecule 4 Biotin-conjugate
  • the biotin labelled compound 4 was first incubated with the tissue sections, folowed by incubation with Streptavidin-Quantum Dot conjugate (1:5000) that emits fluorescence near infra-red region. Hoechst was used for staining of nuclei. As shown in Figure 14, molecule 4 can detect the presence of mycobacteria in mice lung tissues.

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Abstract

A modified small molecule of general formula (I) or (II) or various forms thereof which can be used for selective detection of non-pathogenic, pathogenic, drug-sensitive, drug-resistant, live/dead, persistent, stationary, metabolically-inactive or any type of mycobacterial species via different detection systems like probe, assay, kit, nanoparticles, sensors.

Description

‘MOLECULAR PROBES FOR DETECTION OF MYCOBACTERIA’
Field of the Invention
The present invention relates to synthesis, purification, and development of novel small molecules conjugated (Covalently/non-covalently) with fluorophore/imaging agent for detection of mycobacteria. This invention further discloses the applications of these probes for selective detection of non-pathogenic, pathogenic, drug- sensitive, drug-resistant, live/dead, persistent, stationary, metabolically-inactive or any type of mycobacterial species. This invention also covers the use of these probes to selectively detect mycobacteria among multi-microbial species, biofilms, intracellular mycobacteria; and mycobacteria in human/animal body fluids and tissues or any other media.
Background of the Invention
Mycobacterium is a genus of Actinobacteria, given its own family, the Mycobacteriaceae. Over 190 species are recognized in this genus. It includes pathogens known to cause serious diseases in mammals. Mycobacterium tuberculosis is a species of pathogenic bacteria in the family Mycobacteriaceae and the causative agent of tuberculosis. First discovered in 1882 by Robert Koch, M. tuberculosis has an unusual, waxy coating on its cell surface primarily due to the presence of mycolic acids. The most frequently used diagnostic methods for tuberculosis are the tuberculin skin test, acid-fast staining, culture, and polymerase chain reaction.
Detection of mycobacteria has been a challenge because of the complex nature of these bacteria, and their ability to rapidly change their genomic nature. Nucleic acids, protein and sugars of mycobacteria have been used to engineer molecular probes for easy detection of these bacteria in different samples and biological fluids. Engineering of specific antibodies for a targeted antigen, or PCR based methods have been developed and approved for bacterial diagnostics. However, rapid changes in genomic sequences, development of drug resistance in mycobacteria, poor sensitivity of these assays, and inability to test different strains could not provide success to these methods. Poor knowledge of structural features of polysaccharides and proteins present on these pathogenic bacteria also could not provide any major breakthrough for detection of mycobacteria. Moreover, Gastrointestinal Tuberculosis shows intricate physiological similarity with Crohn’s Disease thus causing clinical diagnostic difficulties. Detection of mycobacteria, additionally, provides further challenges due to increased burden of tuberculosis, slow growing nature of mycobacteria, and its ability to mutate its genomic structure; thereby pressing the need for a simple and affordable diagnostic tool for mycobacterial infections.
In order to facilitate rapid and precise diagnosis of mycobacterial infections, a novel diagnostic tool has been developed using novel small molecule conjugate complex.
Objects of the Invention
The primary object of this invention is to develop different structural classes of novel small molecules which can specifically bind to mycobacteria and lead to detection.
Another object of the invention is to synthesize small molecules that can selectively bind with mycobacteria for identification and detection.
It is yet another object of the invention to develop different structural assemblies of the non- invasive mycobacterial detection system in the forms of probes, assays, kits nanoparticles, sensors.
It is yet another objective of the invention to develop broad spectrum mycobacterial detection systems.
It is yet further objective to provide a means and method for rapid and accurate diagnosis of tuberculosis in subjects.
Summary of the Invention:
In an aspect, the Invention provides methods to synthetically develop a set of novel small molecular derivatives to detect mycobacteria under in-vitro , in-vivo and clinical settings. These derivatives can selectively bind to mycobacterial species at very low concentrations and differentiate them in the presence of other gram negative and gram-positive species. The invented molecules conjugated with a fluorophore allow specific detection of mycobacteria in planktonic forms, biofilm forms, intracellular, mouse and human tissues. The invented molecules can selectively bind to pathogenic, non-pathogenic, metabolically inactive, drug sensitive as well as drug resistant mycobacteria. In another aspect, the Invention provides a potential molecule to be used as a fluorescent probe or a sensor to differentiate between clinical cases of Gastrointestinal Tuberculosis and Crohn’s disease by binding and showing fluorescent signals in the Gastrointestinal Tuberculosis positive tissues. The invention provides scope and methods to develop this molecule as a point-of-care diagnostic system (probe/assay/kit) for Tuberculosis detection and overcome the challenges of time consuming and inaccurate diagnostic methods. Description of Figures:
Figure 1: illustrates general structures of small molecules for detection of mycobacteria.
Figure 2: illustrates synthesis of Cholic Acid-Fluorophore Conjugated Probes. Reagents and reaction conditions: (i) Propargyl amine, EDC.HC1, HOBt, DIPEA, DMF/DCM (1:3), RT, 24h; (ii) Chloroacetic anhydride, DMAP, DCM, RT, 3h; (iii) NBD-CH2-CH2-N3, CuS04, Sodium Ascorbate, THF: DCM: H20 (5:5:1), RT, l2h (iv) DMAP/TMA, DMF, 70°C, 48h.
Figure 3: illustrates synthesis of Deoxycholic Acid-Fluorophore Conjugated Probes.
Reagents and reaction conditions: (v) Propargyl amine, EDC.HC1, HOBt, DIPEA, DMF/DCM (1:3), RT, 24h; (vi) Chloroacetic anhydride, DMAP, DCM, RT, 3h; (vii) NBD- CH2-CH2-N3, CUS04, Sodium Ascorbate, THF: DCM: H20 (5:5:1), RT, l2h (viii) DMAP, DMF, 70°C, 48h.
Figure 4: illustrates synthesis of Biotin Conjugated Probes. Reagents and reaction conditions: (ix) Biotin- Azide, CuS04, Sodium Ascorbate, THF: MeOH: H20 (2:2:1), RT, l2h (x) DMAP, DMF, 70°C, 48h.
Figure 5: illustrates synthesis of Alkyne Conjugated Probes. Reagents and reaction conditions: (xi) DMAP, DMF, 70°C, 48h.
Figure 6: illustrates synthesis of Azide Conjugated Probes. Reagents and reaction conditions: (xii) Azidoethyl amine.TFA, EDC.HC1, HOBt, DIPEA, DMF/DCM (1:3), RT, 24h; (xiii) Chloroacetic anhydride, DMAP, DCM, RT, 3h; (xiv) DMAP, DMF, 70°C, 48h.
Figure 7: illustrates confocal microscopic images of different mycobacteria, Gram-positive and Gram-negative bacterial strains after staining with Molecule 1 probe showing the selectivity of the probe for mycobacteria. (TRITC/DAPI is fluorescence of bacteria due to plasmid; FITC: Molecule 1 Fluorescence). The Invention has used mCherry expressing M. smegmatis, M. bovis, S. typhimurium, and pCyPet (Blue Fluorescence) expressing E. coli and S. aureus for staining purposes. Figure 8: illustrates confocal microscopic images of mcherry expressing M. smegmatis and pCyPet expressing E. coli or pCyPet expressing S. aureus co-cultures after staining with Molecule 1 showing co-localization of mcherry expressing M. smegmatis with green fluorescent probe. (TRITC/DAPI is fluorescence of bacteria due to plasmid; FITC: Molecule 1 Fluorescence)
Figure 9: illustrates confocal microscopic images of mcherry expressing M. smegmatis, pCyPet expressing E. coli and pCyPet expressing S. aureus biofilms after staining with Molecule 1 showing the selective staining of mcherry expressing M. smegmatis bio films. (TRITC/DAPI is fluorescence of bacteria due to plasmid; FITC: Molecule 1 Fluorescence)
Figure 10: illustrates confocal microscopic images of mCherry expressing- smegmatis and pCyPet expressing- E. coli biofilms after staining with Molecule 1 showing the selective staining of mcherry expressing M. smegmatis mycobacterial strains. (TRITC is fluorescence of mycobacteria due to plasmid; FITC: Molecule 1 Fluorescence, DAPI: Fluorescence of pCypet E. coli)
Figure 11: illustrates confocal microscopic images showing the positive staining of mycobacteria in Mycobacterium tuberculosis infected mice lung sections with Molecule 1 at 10 nM concentration. Hoechst 33258 was used to stain the cell nuclei. (FITC: Molecule 1 Fluorescence, TRITC: antibody Fluorescence, DAPI channel: Nuclear stain)
Figure 12: illustrates confocal microscopic images showing the positive staining of mycobacteria in Mycobacterium tuberculosis infected human tissues with Molecule 1 at 10 nM concentration. Hoechst 33258 was used to stain nuclei, and mycobacteria specific antibody was used for confirmation. (TRITC is fluorescence due to antibody staining, FITC: Molecule 1 Fluorescence, DAPI: Nuclear stain)
Figure 13: illustrates confocal microscopic images showing the positive staining of mycobacteria in Mycobacterium tuberculosis infected Gastrointestinal TB tissues with
Molecule 1 at 10 nM concentration. CD refers to Crohn’s Disease. Hoechst 33258 was used to stain nuclei, and mycobacteria specific antibody was used for confirmation. (TRITC is fluorescence due to antibody staining, FITC: Molecule 1 Fluorescence, DAPI: Nuclear stain). Figure 14: Confocal microscopic images showing the positive staining of mycobacteria in Mycobacterium tuberculosis infected mice lung tissues with molecule 4 (Biotin-conjugate) folowed by incubation with Biotin-Quantum Dot (Qdot 655 Streptavidin Conjugate) at 10 nM concentration.
Detailed Description of the Invention:
With the above objects in mind, the Inventors have developed a novel small molecule for detection of mycobacteria, diagnosis of Tuberculosis and clinical differentiation of Gastrointestinal Tuberculosis with Crohn’s Disease.
It should be understood that the detailed description and specific examples, while indicating embodiments of the invention, are given by way of illustrative purpose only and not limitative to the disclosure in any way whatsoever.
Cellular membranes of Gram-positive, Gram-negative, mycobacteria, and mammalian cells differ in their chemical nature. Mycobacterial membranes are highly rigid and hydrophobic in nature due to presence of mycolic acids whereas Gram-negative and Gram-positive bacteria possess lipopolysaccharide and proteoglycan shielding on their outer surface. Therefore, to achieve the stated objectives, the hydrophobic nature of mycobacterial membranes was exploited to engineer selective mycobacterial targeting probes that help in easy identification of mycobacteria in tissue samples. The interactions of these probes with cellular membranes involve the electrostatic interactions between probes and cell membranes involving dehydration; followed by hydrophobic interactions. Therefore, engineered mycobacterial- specific probes were designed and the ability of these probes to detect mycobacteria was evaluated. These probes will specifically target the mycobacterial membrane, and will be able to stain drug- sensitive, drug-resistant, persistent, live/dead, metabolically active and inactive mycobacterial strains.
The present invention involves the use of small molecules that have ligands and a fluorophore/imaging reagent. Ligands help in binding of the probe to mycobacteria selectively, and fluorophore/imaging agent helps in easy detection of the sample. Fluorophore conjugated small molecules that can selectively bind with mycobacteria have been synthesized; and capable of being detected using flow cytometry and fluorescence microscopy. Biotin conjugated small molecules that selectively bind with mycobacteria have also been synthesized. The selective binding of Biotin conjugated molecules can then be detected using Streptavidin- Fluorophore conjugates, or Streptavidin tagged quantum dots, or Streptavidin conjugated enzymatic assays. The Invention has also synthesized Alkyne- or Azide conjugated small molecules that selectively bind with mycobacteria. Selective binding of these Alkyne or Azide-conjugated molecules can be detected using Copper catalysed or Copper free click chemistry using Azide or Alkyne conjugated fluorescent molecules, proteins or enzymes.
In first part, invention focuses on synthesis of fluorophore/imaging agent-tagged small molecules that has specific ligands and binds to mycobacteria described as Fluorophore/imaging agent-conjugated small molecules of general formula I and II. Modified Deoxycholic Acid (DCA) and Cholic Acid (CA) derivatives of general formulae (I and II).
In the embodiment“Ri” in formula I and II represents the different chemical moieties like peptides, sugars, aliphatic or aromatic groups, charged or uncharged groups.
In the embodiment, “P” in formula I and II represents an imaging agent like nitrobenzoxadiazole, Rhodamine, Fluorescein isothiocyanate, Cyanine dyes or any dyes from Nitroso dyes, Indophenol dyes, Nitro dyes, Azine dyes, Azo dyes, Oxazine dyes, Azoic dyes, Thiazine dyes, Stilbene dyes, Carotenoid dyes, Lactone dyes, Diphenylmethane dyes, Aminoketone dyes, Triarylmethane dyes, Xanthene dyes, Anthraquinone dyes, Acridine dyes, Indigoid dyes, Quinoline dyes, Phthalocyanine dyes, Methine dyes, Thiazole dyes, Indamine dyes etc. “P” can also be groups like alkyne, biotin, azide, A- hydroxy succinimide, maleimide, Naphthalimide or lipid molecule or hydrocarbon or any other chemical moiety that can be used for diagnostic purposes.
“Ll and L2” represent a linker or spacer, which is an aromatic, aliphatic, alicyclic, small polymeric linker that covalently conjugates the deoxycholic acid (DCA) and cholic acid (CA) moieties and the ligand or imaging agent. Linker Ll is covalently attached between DCA/CA with targeting ligand; and Linker L2 is between DCA/CA and imaging agent. Spacer/linker chemistry represents simple organic functional groups such as ester, amide, carbonate, carbamate, phosphate, phosphonate, phosphoramidate, amine, urea, thiourea, sulfonamide, ether, thioether, sulfoxide, sulfone, thioester, thioamide, disulfide, oxime, o-acyloxime, o- acyloxyalkyloxime, o-carbamoyloxime etc. It also includes small organic linker molecules include aliphatic polar molecules, polar aromatic compounds, as well as heterocyclic molecules. The present invention preferentially uses few organic small linkers such as ethylene glycol, ethanolamine, 2-amino-ethylamine, 2-amino-ethanethiol, p-aminobenzoic acid, p-azidobenzoic acid, triazole etc.
Second part of invention deals with use of these probes for detection of mycobacteria by flow cytometry, and fluorescence microscopy. These probes are able to selectively stain the pathogenic, non-pathogenic, drug- sensitive, drug-resistant, persistent, stationary mycobacterial strains alone or in a polymicrobial population in different media, buffers, animal and human biological samples.
The Invention is further described with the help of non-limiting examples:
Example 1
Synthesis of Cholic Acid-Fluorophore Conjugated Probes (Figure 2)
Synthesis of Cholic acid derived amphiphiles are mentioned below. Similar protocol can be used for other bile acids.
Synthesis of compound with formula 8: Cholic acid (7) (4g, 9.79 mmol), N- hydroxybenzotriazole (l.455g, 10.76 mmol), and EDC.HC1 (2.064g, 10.76 mmol) were dissolved in 40 mL of anhydrous DMF/DCM (1:3), and DIPEA (3.58 mL, 20.56 mmol) was added to it. Reaction mixture was stirred at room temperature for 15 minutes; and propargyl amine (0.94 mL, 16.15 mmol) was added drop wise followed by stirring at room temperature for 24 h. Solvents were removed under vacuum and reaction mixture was diluted with dichloromethane (250 mL) and washed with saturated NaHC03 solution (3 X 200 mL) and brine (2 X 200 mL). Organic phase was dried over anhydrous Na2S04, and concentrated under reduced pressure. Compound was purified by column chromatography on silica gel (230-400 mesh) using CLLCh/MeOH as eluent, to give a colorless solid (2.8 g). Compound 8 was characterized by 1H NMR spectroscopy. (1H NMR, 400MHz, CDCl3) d 0.68 (s, 1H), 0.89 (s, 4H), 0.95-2.30 (m, steroid) 3.43-3.48 (m, 1H), 3.85 (s, 1H), 3.97 (s, 1H), 4.04 (s, 2H), 5.83 (s, 1H). Synthesis of compound with formula 9: Compound 8 (300mg, 0.67 mmol) was dissolved in 20 ml anhydrous DCM; and DMAP (246 mg, 2.01 mmol) was added. The solution was cooled to 0 °C and solution of chloroacetic anhydride (403 mg, 2.35 mmol) in DCM was added dropwise. The reaction mixture was stirred at 0 °C for 3 hours. After the reaction completion, reaction mixture was diluted with dichloro methane (250 mL) and washed with saturated NaHC03 solution (3 X 200 mL) and brine (2 X 200 mL). Organic phase was dried over anhydrous Na2S04, and solvent was reduced under vacuum. Crude mixture was purified by column chromatography on silica gel (230-400 mesh) using Petroleum Ether/Ethyl Acetate as eluent, to give a colorless solid (350 mg). Product 9 was characterized by 1H NMR spectroscopy. (1H NMR, 400MHz, CDCl3) d 0.75 (s, 3H), 0.83 (d, / = 6Hz, 3H), 0.94 (s, 3H) 1.06-1.16 (m, 2H), 1.21-2.27 (m, steroid), 4.02-4.14 (m, 8H), 4.63-4.68 (m, 1H), 5.03 (s, 1H), 5.20 (s, 1H), 5.56 (s, 1H).
Synthesis of compound with formula 10: Compound 9 (200 mg, 0.295 mmol.) and NBD- CH2-CH2-N3 (67 mg, 0.27 mmol) was dissolved in DCM: THF (1:1); and Sodium Ascorbate (31 mg, 0.16 mmol) was added to this solution that was stirred for 5 min. Copper sulfate (40 mg, O.l6mmol) in 1 ml water was then added to reaction mixture and stirred for 12 hours at room temperature. After the reaction completion, the reaction mixture was passed through celite, and organic solvent removed under vacuum. The compound 10 was isolated by silica gel (230-400mesh) combi flash chromatography using Ethyl Acetate and Petroleum Ether as eluent. Compound was characterized by 1H NMR spectroscopy. (1H NMR, 400MHz, CDCl3) d 0.72-0.75 (m, 6H), 0.85-0.89 (m, 1H), 0.94 (m, 3H), 1.033-2.221 (m, steroid), 4.03 (s, 2H), 4.07 (s, 1H), 4.09-4.18 (m, 5H), 4.38-4.49 (m, 2H), 4.63-4.69 (m, 1H), 4.72-4.75 (m, 2H), 5.18 (s, 1H), 5.29 (s, 1H), 6.15 (d, / = 8 Hz, 1H), 6.34 (s, 1H), 7.67 (s, 1H), 8.43 (s, 1H).
Synthesis of Cholic Acid-NBD Conjugates (1, 2): Compound 10 (lOOmg; 1 equiv.) was taken in anhydrous Dimethylformamide; and respective Dimethylaminopyridine (DMAP)/Trimethylamine gas (excess) was added to it and reaction mixture was heated at 70 °C for 48 hours in ace pressure tube. After the completion of the reaction; the crude product was precipitated in ethyl acetate. The product was re-suspended in ethyl acetate and washed multiple times to give pure product. Compound was characterized by 1H NMR spectroscopy. CA-NBD-DMAP3 (1) (1H NMR, 400MHz, MeOD-d4) ό 0.80-0.82 (m, 6H), 1.00-2.30 (m, steroid), 4.09-4.12 (m, 1H), 4.26 (s, 1H), 4.75-4.82 (m, 2H) 5.13-5.21 (m, 2H), 6.01 (s, 1H), 7.04-7.11 (m, 6H), 7.83 (s, 1H), 8.24-8.29 (m, 6H), 8.44 (d, / = 9Hz, 1H).
CA-Click-NBD-TMAs (2) (1H NMR, 400MHz, D20) d 0.63-0.65 (m, 6H) 0.80-2.47 (m, steroid), 3.19 (s, 4H), 3.33-3.40 (m, 27H), 4.13-4.58 (m, 9H), 5.14 (s, 2H), 5.98 (s, 1H) 7.84 (d, / = 2Hz, 1H) 8.38 (s, 1H).
Synthesis of Deoxycholic Acid-Fluorophore Conjugated Probes (Figure 3)
Synthesis of compound with formula 12: Deoxycholic acid (11) (4g, 10.20 mmol), N- hydroxybenzotriazole (l.65g, 12.3 mmol), and EDC.HC1 (2.35g, 1.23 mmol) were dissolved in 40 mL of anhydrous DMF/DCM (1:3), and DIPEA (3.58 mL, 20.40 mmol) was added to it. Reaction mixture was stirred at room temperature for 15 minutes; and propargyl amine (1.3 mL, 20.40 mmol) was added drop wise followed by stirring at room temperature for 24 h. Solvents were removed under vacuum and reaction mixture was diluted with dichloromethane (250 mL) and washed with saturated NaHC03 solution (3 X 200 mL) and brine (2 X 200 mL). Organic phase was dried over anhydrous Na2S04, and solvent was evaporated under vacuum. Compound was purified by column chromatography on silica gel (230-400 mesh) using CH2Cl2/MeOH as eluent, to give a colorless solid (3.2 g). Compound 12 was characterized by 1H NMR spectroscopy. (1H NMR, 400MHz, CDCI3) d 0.68 (s, 3H), 0.84-1.93 (m, steroid) 2.08-2.16 (m, 1H), 2.23-2.31 (m, 2H), 3.61 (m, 1H), 3.98 (m, 1H), 4.06 (m, 2H), 5.58 (s, 1H).
Synthesis of compound with formula 13: Compound 12 (300mg, 0.698 mmol) was dissolved in 20 ml anhydrous DCM; and DMAP (171 mg, 1.40 mmol) was added. The solution was cooled to 0 °C and solution of chloroacetic anhydride (538 mg, 3.14 mmol) in DCM was added dropwise. The reaction mixture was stirred at 0 °C for 3 hours. After the reaction completion; reaction mixture was diluted with dichloromethane (250 mL) and washed with saturated NaHCCT solution (3 X 200 mL) and brine (2 X 200 mL). Organic phase was dried over anhydrous Na2S04, and solvent was reduced under vacuum. Crude mixture was purified by column chromatography on silica gel (230-400 mesh) using Petroleum Ether/Ethyl Acetate as eluent, to give a colorless solid (350 mg). Product 13 was characterized by 1H NMR spectroscopy. (1H NMR, 400MHz, CDCl3) d 0.75 (s, 3H), 0.83 (d, = 6Hz, 3H), 0.94 (s, 3H) 1.00-2.28 (m, Steroid), 4.03-4.08 (m, 6H), 4.79 (m, 1H), 5.20 (s, 1H), 5.59 (s, 1H).
Synthesis of compound with formula 14: Compound 13 (200 mg, 0.26 mmol.) and NBD- CH2-CH2-N3 (120 mg, 0.48 mmol) was dissolved in DCM: THF (1:1); and Sodium ascorbate (29 mg, 0.14 mmol) was added to this solution that was stirred for 5 min. Copper sulfate (36 mg, 0.14 mmol) in 1 ml water was then added to reaction mixture and stirred for 12 hours at room temperature. After the reaction completion, the reaction mixture was passed through celite, and organic solvent removed under vacuum. The compound 14 was isolated by silica gel (230-400mesh) combi flash chromatography using Ethyl Acetate and Petroleum Ether as eluent. Compound was characterized by 1H NMR spectroscopy. (1H NMR, 400MHz, CDCI3) S 0.68 (m, 3H), 0.71-2.20 (m, steroid), 3.95 (s, 2H), 4.21 (d, / = 5.72 2H), 4.33 (s, 2H), 4.40 (m, 2H), 4.67 (m, 3H), 5.05 (s, 1H), 6.34 (d, J = 8.8 Hz, 1H), 7.93 (s, 1H), 8.24 (t, / = 4 Hz, 1H), 8.48 (d, J =8.8 Hz, 1H), 9.44 (s, 1H).
Synthesis of Deoxycholic Acid-NBD Conjugate (3): Compound 14 (lOOmg; 1 equiv.) was taken in anhydrous Dimethylformamide; and respective 59 mg (4 equiv.) Dimethylaminopyridine (DMAP) was added to it and reaction mixture was heated at 70 °C for 48 hours in ace pressure tube. After the completion of the reaction; the crude product was precipitated in Ethyl acetate. The product was re-suspended in ethyl acetate and washed multiple times to give pure product. Compound was characterized by 1H NMR spectroscopy. DCA-NBD-DMAP3 (3) (1H NMR, 400MHz, DMSO-d6) S 0.63 (s, 3H), 0.71-2.09 (m, Steroid), 3.20-3.24 (m, 12H), 3.97 (s, 1H), 4.23 (d, 7 =5.4 Hz, 2H) 4.60-4.76 (m, 3H), 5.09 (s, 1H), 5.21-5.32 (m, 1H), 5.36-5.52 (m, 2H), 6.33 (s, 1H), 7.09-7.15 (m, 4H), 7.96 (s, 1H), 8.33-8.46(m, 5H), 9.49(s, 1H).
Example 2
Synthesis of Biotin Conjugated Probes (Figure 4):
Synthesis of compound with formula 15: Compound 9 (200 mg, 0.295 mmol.) and Biotin Azide (116 mg, 0.268 mmol) was dissolved in DCM: THF (1:1); and Sodium Ascorbate (26.5 mg, 0.134 mmol) was added to this solution that was stirred for 5 min. Copper sulfate (33.4 mg, 0.134 mmol) in 1 ml water was then added to reaction mixture and stirred for 12 hours at room temperature. The compound 15 was purified from crude mixture by silica gel (230-400mesh) combi flash chromatography using DCM/Methanol as eluent. Compound was characterized by 1H NMR spectroscopy. (1H NMR, 400MHz, DMSO-d6) d 0.61 (m, 3H), 0.70-2.18 (m, steroid), 2.97-2.98 (m, 2H), 3.16-3.17 (m, 1H), 4.04-4.07 (m, 1H), 4.23-4.30 (m, 10H), 4.54 (s, 1H), 4.86 (s, 1H), 5.04 (s, 1H), 5.76-5.81 (m, 1H), 6.35-6.54 (m, 2H), 8.08- 8.22 m, 4H), 8.40 (s, 1H), 8.75 (s, 1H).
Synthesis of Cholic Acid-Biotin Conjugate (4): Compound 15 (lOOmg; 1 equiv.) was dissolved in anhydrous Dimethylformamide; and 67mg (6 equiv.); and Dimethylaminopyridine (DMAP) was added to it and reaction mixture was heated at 70 °C for 48 hours in ace pressure tube. After the completion of the reaction; the crude product was precipitated in Ethyl acetate. The product was re-suspended in ethyl acetate and washed multiple times to give pure product. Compound was characterized by 1H NMR spectroscopy. CA-Biotin-DMAPs (4) (1H NMR, 400MHz, DMSO-d6) d 0.64 (s, 3H), 0.71-2.34 (m, Steroid), 2.73-2.89 (m, 1H), 2.97-3.05 (m, 1H), 3.18-3.23 (m, 18H) 3.45-3.46 (m, 2H), 4.01- 4.06 (m, 1H), 4.24-4.30 (m, 3H), 4.40 (s, 2H), 4.63 (s, 1H), 4.48(s, 1H), 5.13-5.26 (m, 2H), 5.45-5.63 (m, 2H) 5.83-5.95 (m, 1H), 6.37-6.41 (m, 1H), 6.97-7.18 (m, 6H), 8.09-8.25 (m, 5H), 8.40-8.62 (m, 6H), 8.79-8.84(m, 1H).
Example 3
Synthesis of Alkyne (Figure 5) Conjugated Probes:
Synthesis of Cholic Acid- Alkyne Conjugate (5): Compound 9 (lOOmg; 1 equiv.) was taken in anhydrous Dimethylformamide; and Dimethylaminopyridine (DMAP) (108 mg (6 equiv.)) was added to it and reaction mixture was heated at 70 °C for 48 hours in ace pressure tube. After the completion of the reaction; the crude product was precipitated in Ethyl acetate. The product was re-suspended in ethyl acetate and washed multiple times to give pure product. Compound was characterized by 1H NMR spectroscopy.
CA-PA-DMAPs (5) (1H NMR, 400MHz, DMSO-d6) d 0.71 (s, 3H), 0.76 (d, / = 6.4Hz, 3H), 0.92 (s, 3H) l.07-2.24(m, Steroid), 4.63 (m, 1H), 4.91 (s, 1H), 5.15 (s, 1H), 5.19-5.57 (m, 4H), 5.90 (m, 2H), 7.07-7.19 (m, 6H), 8.28(s, 1H), 8.39 (d, / = 6.5Hz, 2H), 8.61 (d, 4H). Example 4
Synthesis of Azide (Figure 6) Conjugated Probes:
Synthesis of Compound with formula 16: Cholic acid (7) (4g, 9.79 mmol), N- hydroxybenzotriazole (l.455g, 10.76 mmol), and EDC.HC1 (2.064g, 10.76 mmol) were dissolved in 40 mL of anhydrous DMF/DCM (1:3); and DIPEA (3.58 mL, 20.56 mmol) was added to it. Reaction mixture was stirred at room temperature for 15 minutes; and 2- Azidoethylamine.TFA (2.l5g, 10.76 mmol) in DCM (lOml) was added drop wise followed by stirring at room temperature for 24 h. Solvents were removed under vacuum and reaction mixture was diluted with dichloromethane (250 mL) and washed with saturated NaHC03 solution (3 X 200 mL) and brine (2 X 200 mL). Organic phase was dried over anhydrous Na2S04, and solvent was evaporated under vacuum. Residue was purified by column chromatography on silica gel (230-400 mesh) using CH2Cl2/MeOH as eluent, to give a colorless solid (2.8 g). Compound 16 was characterized by 1H NMR spectroscopy. (1H NMR, 400MHz, DMSO-de) S 0.57 (s, 3H), 0.80-2.26 (m, steroid) 2.98 (s, 1H), 3.15-3.23 (m, 2H), 3.30 (s, 1H), 3.56 (s, 4H), 3.60 (s, 1H), 3.77(s, 1H) 4.01 (s, 1H), 4.10 (s, 1H), 4.31 (s, 1H).
Synthesis of compound with formula 17: Compound 16 (300mg, 0.63 mmol) was dissolved in 20 ml anhydrous DCM; and DMAP (231 mg, 1.89 mmol) was added. The solution was cooled to 0 °C and solution of chloroacetic anhydride (645 mg, 3.78 mmol) in DCM was added dropwise. The reaction mixture was stirred at 0 °C for 3 hours. After the reaction completion; reaction mixture was diluted with dichloromethane (250 mL) and washed with saturated NaHCCT solution (3 X 200 mL) and brine (2 X 200 mL). Organic phase was dried over anhydrous Na2S04, and solvent was reduced under vacuum. Crude mixture was purified by column chromatography on silica gel (230-400 mesh) using Petroleum Ether/Ethyl Acetate as eluent, to give a colorless solid (350 mg). Product 17 was characterized by 1H NMR spectroscopy. (1H NMR, 400MHz, CDCl3) S 0.77 (s, 3H), 0.85-2.30 (m, Steroid), 3.45 (s, 4H), 4.04-4.15 (m, 4H), 4.68 (s, 1H), 5.05 (s, 1H), 5.22 (s, 1H), 5.77 (s, 1H).
Synthesis of Cholic Acid-Azide Conjugate (6): Compound 17 (100 mg; 0.14 mmol) was taken in anhydrous Dimethylformamide; and (154 mg, 0.85 mmol) Dimethylaminopyridine (DMAP) was added to it and reaction mixture was heated at 70 °C for 48 hours in ace pressure tube. After the completion of the reaction; the crude product was precipitated in Ethyl acetate. The product was re-suspended in ethyl acetate and washed multiple times to give pure product. Compound was characterized by 1H NMR spectroscopy.
CA-Azide-DMAP3 (6) (1H NMR, 400MHz, DMSO-d6) d 0.71 (s, 3H), 0.76 (d, / = 6Hz, 3H), 0.92 (s, 3H) 1.07-2.18 (m, Steroid), 3.21-3.24 (m, 20H), 4.63 (m, 1H), 4.91 (s, 1H), 5.15 (s, 1H), 5.20-5.63 (m, 4H), 5.83-5.98 (m, 2H), 7.07-7.20 (m, 6H), 8.37-8.39 (m, 2H), 8.59-8.62 (m, 4H).
Example 5
Detection and selectivity of probes for mycobacteria (Table 1):
Selectivity of the Molecule 1 was tested against different Gram-negative, Gram-positive and mycobacterial strains using flow cytometry studies at different concentrations of probe. Bacterial cultures were grown and adjusted till required CFU, harvested and fresh suspensions were passed through a 26-gauge needle for almost 7-8 times so as to obtain single cells. Thereafter, these single cell suspensions were incubated with Molecule 1 at tested concentrations. Cells were centrifuged and washed with PBS to remove the unbound probe, and analyzed by flow cytometry. Molecule 1 can stain >90% of the mycobacterial cells including Mycobacterium bovis (BCG), Mycobacterium smegmatis ( Msm ), Mycobacterium tuberculosis ( Mtb ). Molecule 1 is unable to stain Gram- negative strains ( Escherichia coli ( E . coli), or Gram-positive ( Staphylococcus aureus (S. aureus) as shown in Table 1
Figure imgf000014_0001
Example 6
Detection of mycobacteria by confocal microscopy (Figure 7):
The Invention used the different bacterial strains and incubated these strains with Molecule 1. After this, cells were washed to remove the unbound molecule. Cell were then fixed with 4% paraformaldehyde (PFA). For Molecule 1, FITC channel was used. For imaging, 63X oil was the objective and Hyvolution mode was chosen to minimize the background. The images of planktonic bacteria revealed a clear staining of mycobacterial strains (. Mycobacterium smegmatis ( Msm ); Mycobacterium bovis ( M . bovis (BCG)), Mycobacterium tuberculosis ( Mtb ); whereas Molecule 1 is unable to stain any of the Gram-positive (5. aureus) or Gram negative stains (. E . coli, S. typhimurium,) suggesting the selectivity of these probes for mycobacteria detection.
Example 7
Detection of mycobacteria in polymicrobial cultures (Figure 8)
The Invention tested the ability of Molecule 1 to detect the mycobacteria (mCherry expressing M. smegmatis) selectively in presence of other Gram-negative (pCyPet expressing E. coli) or Gram-positive bacteria (pCyPet expressing S. aureus). The process involves mixing of mCherry expressing M. smegmatis and pCyPet (Blue fluorescence) expressing E. coli or mCherry expressing M. smegmatis and pCyPet expressing S. aureus (Blue fluorescence) and stained with Molecule 1 (2nM). Two cultures grown separately in suitable antibiotic supplemented media were mixed in equal volumes and incubated with Molecule 1. As shown in Figure 8, Molecule 1 is able to selectively stain the mycobacteria species as co localization of green fluorescence of probe was observed with mcherry expressing M. smegmatis in both the co-culture studies with pCyPet expressing E. coli and pCyPet expressing S. aureus. Probe did not show any co-localization with pCyPet expressing E. coli or pCyPet expressing S. aureus.
Example 8
Detection of mycobacteria in biofilms (Figure 9)
The Invention tested the Molecule 1 for selective staining of mycobacterial bio films. Bacterial biofilms using mCherry expressing M. smegmatis, pCyPet expressing E. coli and pCyPet expressing S. aureus were prepared separately and incubated with Molecule 1 (2 nM). Biofilms were washed and observed under confocal microscope. Confocal images showed the selective staining of mycobacterial mCherry expressing M. smegmatis bio films as co-localization of mCherry M. smegmatis with green fluorescent stain of Molecule 1 was observed. Molecule 1 does not stain pCyPet expressing E. coli and pCyPet expressing S. aureus biofilms.
Example 9
Detection of mycobacteria in polymicrobial biofilms (Figure 10):
The Invention then tested the ability of Molecule 1 for detection of mycobacteria in polymicrobial biofilms. Polymicrobial biofilms were prepared using pCypet expressing E. coli and mcherry expressing M. smegmatis; and incubated with Molecule 1 (2nM). After washing off the probe, biofilms were observed under confocal microscope. It was observed that the co-localization of green fluorescence of Molecule 1 with mcherry expressing M. smegmatis happened whereas no co-localization was observed with pCypet expressing E. coli. Therefore, Molecule 1 can detect mycobacteria in poly-microbial biofilms.
Example 10
Detection of mycobacteria in infected mouse lung tissues (Figure 11)
The Invention then tested the Molecule 1 for detection of mycobacteria in infected mouse lung tissues. Paraffin- fixed transverse lung sections of M. tuberculosis infected mice were obtained and paraffin wax was removed by microwave treatment and xylene washing. Gradient hydration was given using 90% and 80% ethanol and sections were stained with Molecule 1 (10 nM) concentration as well as Anti Mtb antibody, Ab905 (abeam) used in a 1:1000 dilution. Nuclei staining was done using Hoechst 33258, and finally, sections were fixed using 4% PFA and observed under confocal microscope. The staining of mycobacteria using Molecule 1 as green fluorescent bacteria clearly suggested that these probes can be used for detection of mycobacteria in tissue sections as well.
Example 11
Detection of mycobacteria in human tissue sections (Figure 12)
The ability of the molecule 1 to detect the mycobacteria in infected human tissues was tested. Sections of infected human tissue were stained with Molecule 1. Hoechst was used for staining of nuclei, and mycobacteria specific antibody, Ab905 (abeam) was used for the confirmation of mycobacteria. As shown in Figure 12, Molecule 1 can detect the presence of mycobacteria in human tissue sections (FITC Channel). Co-localization of antibody staining with Molecule 1 confirm the presence of mycobacteria in the sections. Example 12
Differentiation between Gastrointestinal TB and Crohn’s Disease human tissue sections (Figure 13)
The ability of the molecule 1 to differentiate between the human Gastrointestinal and Crohn’s Disease tissues was tested. Hoechst was used for staining of nuclei and mycobacteria specific antibody Ab905(Abcam) was used for the confirmation of mycobacteria. As shown in Figure 13, Molecule 1 can detect the presence of mycobacteria in TB sections but did not show presence of mycobacteria in Crohn’s disease sections. Presence of mycobacteria was confirmed only in the TB positive sections by the colocalization of antibody staining with Molecule 1.
Example 13
Detection in mice lung tissue sections using Ml Biotin- Streptavidin Quantum Conjugate (Figure 14)
We then tested the ability of the molecule 4 (Biotin-conjugate) to detect mycobacteria in the M. tuberculosis infected mice lung tissues. The biotin labelled compound 4 was first incubated with the tissue sections, folowed by incubation with Streptavidin-Quantum Dot conjugate (1:5000) that emits fluorescence near infra-red region. Hoechst was used for staining of nuclei. As shown in Figure 14, molecule 4 can detect the presence of mycobacteria in mice lung tissues.

Claims

Claims:
1. A modified molecule of general formula (I) or (II) or structural forms thereof,
Figure imgf000018_0001
wherein:
Rl is natural or synthetic peptides, sugars, aliphatic or aromatic chemical moieties, charged or uncharged chemical moieties;
P is an imaging agent like nitrobenzoxadiazole, Rhodamine, Fluorescein isothiocyanate, Cyanine dyes or any dyes from Nitroso group, Indophenol group, Nitro group, Azine group, Azo group, Oxazine group, Azoic group, Thiazine group, Stilbene group, Carotenoid group, Lactone group, Diphenylmethane group, Aminoketone group, Triarylmethane group, Xanthene group, Anthraquinone group, Acridine group, Indigoid group, Quinoline group, Phthalocyanine group, Methine group, Thiazole group, Indamine group.; alkyne, biotin, azide, /V- hydroxy succinimide, maleimide, Naphthalimide or lipid molecule or hydrocarbon or any other chemical moiety; P can also be Biotin, Alkyne, Azide, Nitriloacetic acid, or any other agent that can be used for diagnostic purposes; or its further complexation with imaging agent for diagnosis applications.
Ll and L2 represent linkers or spacers and are independently selected from ester, amide, carbonate, carbamate, phosphate, phosphonate, phosphoramidate, amine, urea, thiourea, sulfonamide, ether, thioether, sulfoxide, sulfone, thioester, thioamide, disulfide, oxime, o-acyloxime, o-acyloxyalkyloxime, o-carbamoyloxime, ethylene glycol, ethanolamine, 2-amino-ethylamine, 2-amino-ethanethiol, p-aminobenzoic acid, p-azidobenzoic acid, triazole.
2. A process of preparation of modified molecule conjugate complex of general formula (I) or (II) wherein Rl, P, Ll and L2 are as defined hereinbefore, comprising the steps of - modifying the hydroxyl group by covalently or noncovalently linking it to a head group to form a microbe detection mechanism;
- modifying the acid by covalently or noncovalently linking it to a head group, fluorophore or an imaging agent to form a microbe detection mechanism;
-forming probe of the said molecule.
3. The modified molecule conjugate complex of Claim 1, wherein the conjugate is
Modified cholic acid of first part and nitrobenzoxadiazole
Modified deoxycholic acid of first part and nitrobenzoxadiazole
Modified acid of first part and biotin
Modified acid of first part and alkyne
Modified acid of first part and azide
4. A system for detection and identification of microorganisms comprising molecule (I) or (II).
5. The system claimed in Claim 4 wherein the microorganism belongs to Mycobacteriaceae family.
6. The system claimed in Claim 5 wherein the microorganism is Mycobacterium tuberculosis.
7. The system claimed in Claim 5 can detect pathogenic, non-pathogenic, drug- sensitive, drug-resistant, persistent, live/dead, metabolically active and inactive mycobacteria.
8. The system claimed in Claim 5 can detect pathogenic, non-pathogenic, drug- sensitive, drug-resistant, persistent, live/dead, metabolically active and inactive mycobacteria in presence of other microorganisms, mammalian cells, or other biological and non- biological materials.
9. The system claimed in Claim 5 can detect pathogenic, non-pathogenic, drug- sensitive, drug-resistant, persistent, live/dead, metabolically active and inactive mycobacterial strains in different animal or human biological fluids and tissues.
10. The system claimed in Claim 5 can be conjugated using covalently or non-covalent interactions to imaging agents like fluorescent proteins, kinases, phosphatases, galactosidases, or other enzymes for enzymatic assays.
11. The system claimed in Claim 5 can be conjugated using covalently or non-covalent interactions to imaging agents like gold nanoparticles, iron oxide nanoparticles, quantum dots or any other metallic or non-metallic or hybrid nanoparticles for detection purposes.
12. The system claimed in Claim 5 can be used as such or in nano/micro/ or any other particulate form for non-invasive detection of mycobacterial infections.
13. The system claimed in Claim 5 can be used for detection and differentiation of clinical mycobacterial infections from other diseases, Crohn’s disease or infections by other microorganisms.
14. Biotin-conjugated probe can be used in combination with Streptavidin-Fluorophore or Streptavidin-Quantum dot or Streptavidin-enzyme conjugates for detection of mycobacteria.
15. Alkyne or azide conjugated probe can be used in combination with Alkyne/azide- fluorphore or alkyne/azide-enzyme conjugates for detection of mycobacteria.
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