Methods for detecting mycobacteria with solvatochromic dye conjugates
11884956 ยท 2024-01-30
Assignee
Inventors
- Carolyn R. Bertozzi (Stanford, CA, US)
- Mireille Kamariza (Stanford, CA, US)
- Peyton Shieh (La Jolla, CA, US)
Cpc classification
C12Q1/04
CHEMISTRY; METALLURGY
International classification
Abstract
A series of carbohydrate-dye conjugates, as well as a method for detection of pathogenic or other organisms (e.g., bacteria) using the same are provided. The carbohydrate-dye conjugate can be enzymatically incorporated into live and active (viable) bacteria of interest for facile detection of said bacteria. The conjugate incorporation is achieved by utilizing one or more of the enzymes that are endogenous to the bacteria of interest, which can incorporate the conjugate via the conjugate's carbohydrate. A detectable signal is produced by the conjugate's dye only upon incorporation into the bacteria of interest, due to the changes in the dye's local environment upon incorporation. The conjugate may be metabolically incorporated into the fatty outer membrane of a bacterial cell wall, which provides a distinctly hydrophobic environment for the conjugate's dye, causing it to produce a detectable signal.
Claims
1. A method for detecting metabolically active bacteria of interest in a sample, the method comprising: contacting the sample with an aqueous solution having an excess of a trehalose-solvatochromic dye conjugate that comprises: a trehalose moiety, and a solvatochromic dye linked to the trehalose moiety, wherein the solvatochromic dye is one of: 4-DMAP, 4-DMN, 6-DMN, Nile Red, 3-HC, 3-MC, PRODAN, Anthradan, NBD or a derivative thereof; and detecting a spectroscopic signal from the solvatochromic dye moiety of the trehalose-solvatochromic dye conjugate, the spectroscopic signal indicating the uptake of the trehalose-solvatochromic dye conjugate by the metabolically active bacteria of interest in the sample; wherein the detecting step is performed without removal of the excess of trehalose-solvatochromic dye conjugate in the sample, and wherein the indicative spectroscopic signal is readily detectable over the total spectroscopic signal provided by the excess of trehalose-solvatochromic dye conjugate remaining in the aqueous solution of the sample.
2. The method of claim 1, wherein the outer cell membrane of the bacteria of interest is a mycomembrane rich in hydrophobic mycolates.
3. The method of claim 2, wherein the mycomembrane mycolates include trehalose mycolates.
4. The method of claim 1, wherein the bacteria of interest is capable of metabolic uptake of trehalose.
5. The method of claim 1, wherein the bacteria of interest metabolically uptakes trehalose with high specificity.
6. The method of claim 1, wherein the bacteria of interest possess acyl transferase antigen 85 (Ag85) protein complex capable of trehalose mycolylation, thereby promoting the uptake of trehalose by the bacteria of interest.
7. The method of claim 1, wherein the bacteria of interest belong to Actinobacteria phylum.
8. The method of claim 7, wherein the bacteria of interest are mycobacteria or corynebacteria.
9. The method of claim 1, wherein, the trehalose moiety of the conjugate is a trehalose analog.
10. The method of claim 1, wherein the trehalose-solvatochromic dye conjugate has formula: ##STR00040## wherein one and only one of Z.sup.1-Z.sup.7 is a linked solvatochromic dye and the rest of Z.sup.1-Z.sup.7 are OH.
11. The method of claim 10, wherein the trehalose-solvatochromic dye conjugate has formula: ##STR00041## wherein one and only one of Z.sup.1-Z.sup.4 is a linked solvatochromic dye and the rest of Z.sup.1-Z.sup.4 are OH.
12. The method of claim 11, wherein Z.sup.1 is the linked solvatochromic dye.
13. The method of claims 1, wherein the sample is a biological sample.
14. The method of claim 13, wherein the biological sample is selected from the group of: sputum, blood, serum, plasma, urine, bronchoalveolar lavage fluid, buccal swab, and tissue samples.
15. The method of claim 14, wherein the biological sample is a sample from a subject suspected of having, or at risk of infection with the bacteria of interest.
16. The method of claim 15, wherein the biological sample is a sample from a subject suspected of having, or at risk of infection with Mycobacterium tuberculosis.
17. The method of claim 1, wherein the sample is an environmental sample.
18. The method of claim 17, wherein the sample is taken from an environment selected from the group of: air, water, or inanimate surface.
19. The method of claim 17, wherein the environmental sample is a sample known or suspected to have contact with subject or subjects suspected of having, or at risk of infection with the bacteria of interest.
20. The method of claim 17, wherein the environmental sample is a sample known or suspected to have contact with subject or subjects suspected of having, or at risk of infection with Mycobacterium tuberculosis.
21. The method of claim 1, wherein the bacteria of interest is one or more selected from the group of: M. tuberculosis, M. avium (or M. avium-intracellulare), M. leprae (particularly M. leprae infection leading to tuberculoid leprosy), M. kansasii, M. fortuitum, M. chelonae, M. absecessus, M. marinum, M. Nocardia, M. xenopi, M. simiae, M. szulgai, M. scrofulaceum, M. malmoense, M. terrae-nonchromogenicum complex, M. haemophilum, M. genavense, M. celatum, M. interjectum, M. confluentis, M. triplex, M. lentiflavum, M. branderi, M. conspicuum, M. cookii, M. asiaticum, M. marinum, M. gordonae, M. fortuitum, M. chelonae-abscessu, and M. mucogenicum.
22. The method of claim 1, wherein the metabolically active bacteria of interest is selected from the group of: Mycobacterium tuberculosis, Mycobacterium leprae, or Corynebacterium diphtheriae.
23. The method of claim 1, wherein the bacteria of interest is present extracellularly.
24. The method of claim 1, wherein the bacteria of interest is present inside a eukaryotic cell.
25. The method of claim 1, wherein the detecting step is performed without washing the sample subsequent to the contacting step and prior to the detecting step.
26. The method of claim 1, wherein the spectroscopic signal fluorescence intensity is enhanced approximately 700-fold.
27. The method of claim 1, wherein the solvatochromic dye is linked to the trehalose moiety as shown in the following formula without optional linker L: ##STR00042##
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) These and other features and advantages of the present invention will be better understood by reference to the following detailed description when considered in conjunction with the accompanying data and figures, wherein:
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DETAILED DISCLOSURE
(20) The embodiments of the invention described herein are not intended to be exhaustive or to limit the invention to precise forms disclosed. Rather, the embodiments selected for description have been chosen to enable one skilled in the art to practice the invention.
(21) Turning now to the figures and schemes, a series of carbohydrate-dye conjugates is described, as well as a method for detection of pathogenic or other organisms (e.g., bacteria) using the same. In many embodiments, a carbohydrate-dye conjugate is selected such that it can be enzymatically incorporated into live and active (viable) bacteria of interest for facile detection of said bacteria. In many embodiments, the conjugate incorporation is achieved by utilizing one or more of the enzymes that are endogenous to the bacteria of interest, which can incorporate the conjugate via the conjugate's carbohydrate. In many embodiments, a detectable signal is produced by the conjugate's dye only upon incorporation into the bacteria of interest, due to the changes in the dye's local environment upon incorporation. In many embodiments, the conjugate is metabolically incorporated into the fatty outer membrane of a bacterial cell wall, which provides a distinctly hydrophobic environment for the conjugate's dye, causing it to produce a detectable signal. To this end, in many embodiments, the conjugate's carbohydrate is chosen to facilitate the conjugate's metabolic uptake and incorporation into the cell wall of viable bacteria of interest, while conjugate's dye is chosen to efficiently and reliably report such successful incorporation events. In many embodiments, the bacteria of interest are actinobacteria, and, more specifically, mycobacteria, characterized by exceptionally hydrophobic outer cell membranemycomembrane.
(22) Glycolipids featuring long fatty mycolic acids are known to be an abundant and essential component of bacterial mycomembranes (
(23) Furthermore, trehalose mycolates are thought to be unique to the Actinobacteria phylum, which includes pathogenic mycobacteria and corynebacteria, but not canonical gram-positive or gram-negative organisms, nor human hosts. In support of this notion, it has been shown that Ag85A-C protein homologs, the enzymes known to be responsible for mycolylation of trehalose, are largely restricted to the Actinomycetales order of bacteria. To this end,
(24) TABLE-US-00001 TABLE 1 [Brevibacterium] flavum Actinoalloteichus hymeniacidonis Actinoalloteichus sp. Actinoplanes friuliensis Actinoplanes sp. Actinosynnema mirum Alloactinosynnema sp. Amycolatopsis japonica Amycolatopsis lurida Amycolatopsis mediterranei Amycolatopsis orientalis Amycolicicoccus subflavus Arthrobacter sp. Brachybacterium faecium Brachybacterium sp. Brevibacterium linens Corynebacteriales bacterium Corynebacterium ammoniagenes Corynebacterium aquilae Corynebacterium argentoratense Corynebacterium atypicum Corynebacterium aurimucosum Corynebacterium callunae Corynebacterium camporealensis Corynebacterium casei Corynebacterium deserti Corynebacterium diphtheriae Corynebacterium doosanense Corynebacterium efficiens Corynebacterium epidermidicanis Corynebacterium falsenii Corynebacterium flavescens Corynebacterium frankenforstense Corynebacterium glutamicum Corynebacterium glyciniphilum Corynebacterium halotolerans Corynebacterium humireducens Corynebacterium imitans Corynebacterium jeikeium Corynebacterium kroppenstedtii Corynebacterium kutscheri Corynebacterium lactis Corynebacterium marinum Corynebacterium maris Corynebacterium mustelae Corynebacterium phocae Corynebacterium pseudotuberculosis Corynebacterium resistens Corynebacterium simulans Corynebacterium singulare Corynebacterium sp. Corynebacterium sphenisci Corynebacterium stationis Corynebacterium terpenotabidum Corynebacterium testudinoris Corynebacterium ulcerans Corynebacterium urealyticum Corynebacterium ureicelerivorans Corynebacterium uterequi Corynebacterium variabile Corynebacterium vitaeruminis Curtobacterium sp. Cutibacterium avidum Dermabacter vaginalis Dietzia timorensis Frankia alni Frankia sp. Gordonia bronchialis Gordonia polyisoprenivorans Gordonia sp. Gordonia terraee Jonesia denitrificans Kibdelosporangium phytohabitans Kocuria rhizophila Kutzneria albida Luteipulveratus mongoliensis Micromonospora sp. Mycobacterium abscessus Mycobacterium africanum Mycobacterium avium Mycobacterium bovis Mycobacterium canetti Mycobacterium chelonae Mycobacterium chimaera Mycobacterium chubuense Mycobacterium fortuitum Mycobacterium gilvum Mycobacterium goodii Mycobacterium haemophilum Mycobacterium immunogenum Mycobacterium indicus pranii Mycobacterium intracellulare Mycobacterium kansasii Mycobacterium leprae Mycobacterium liflandii Mycobacterium marinum Mycobacterium massiliense Mycobacterium microti Mycobacterium neoaurum Mycobacterium phlei Mycobacterium rhodesiae Mycobacterium sinense Mycobacterium smegmatis Mycobacterium sp. Mycobacterium tuberculosis Mycobacterium ulcerans Mycobacterium vaccae Mycobacterium vanbaalenii Nocardia brasiliensis Nocardia cyriacigeorgica Nocardia farcinica Nocardia nova Nocardia seriolae Nocardia soli Nocardiopsis alba Propionibacterium avidum Pseudonocardia Rhodococcus aetherivorans Rhodococcus equi Rhodococcus erythropolis Rhodococcus fascians Rhodococcus jostii Rhodococcus opacus Rhodococcus pyridinivorans Rhodococcus sp. Saccharomonospora viridis Saccharopolyspora erythraea Salinispora arenicola Segniliparus rotundus Streptomyces albus Streptomyces ambofaciens Streptomyces ambofaciens Streptomyces bingchenggensis Streptomyces cattleya Streptomyces coelicolor Streptomyces globisporus Streptomyces lincolnensis Streptomyces lividans Streptomyces lydicus Streptomyces pactum Streptomyces parvulus Streptomyces pratensis Streptomyces reticuli Streptomyces sampsonii Streptomyces scabiei Streptomyces silaceus Streptomyces sp. Streptomyces venezuelae Streptomyces violaceusniger Streptosporangium roseum Thermomonospora curvata Tsukamurella paurometabola Verrucosispora maris
(25) Moreover, it has been shown that modifying trehalose sugar with various functionalities, such as fluorine, azide, and alkyne, as well as larger fluorophore-functionalized derivatives, does not prevent its metabolic incorporation into the mycobacterial outer membrane as trehalose mycolates (
(26) Furthermore, although any fluorescently labeled trehalose analog can be used for detection of viable actinobacteria in theory, the necessity of removing unmetabolized and, thus, unincorporated into bacterial cell wall probe to eliminate background fluorescence is a major impediment in practice. Therefore, in many embodiments, trehalose probe whose fluorescence signal is specifically activated by metabolic incorporation into the mycomembrane is used to detect live actinobacteria. More specifically, trehalose probes used for detection of actinobacteria, according to many embodiments of the invention, are conjugated to environmentally sensitive solvatochromic dyes, such as, for example, 4-N,N-dimethylamino-1,8-naphthalimide (DMN), which exhibit a dramatic fluorescence turn-on when transitioned from aqueous to hydrophobic solvents. (G. Loving, B. Imperiali, A versatile amino acid analogue of the solvatochromic fluorophore 4-N,N-dimethylamino-1,8-naphthalimide: a powerful tool for the study of dynamic protein interactions, J. Am. Chem Soc. 130, 13630-13638 (2008). G. Loving, B. Imperiali, Thiol-reactive derivatives of the solvatochromic 4-N,N-dimethylamino-1,8-naphthalimide fluorophore: a highly sensitive toolset for the detection of biomolecular interactions, Bioconjug. Chem. 20, 2133-2141 (2009). B. N. Goguen, G. S. Loving, B. Imperiali, Development of a fluorogenic sensor for activated Cdc42, Bioorg. Med. Chem. Lett. 21, 5058-5061 (2011). The disclosures of which are incorporated herein by reference.) As such, according to many embodiments, metabolic mycolylation of carbohydrate-solvatochromic dye conjugate by Ag85, or another enzyme with similar mycolylation abilities, followed by subsequent integration of the resulting mycolate into the hydrophobic mycomembrane, activates dye's fluorescence and enables detection of live actinobacteria (including Mtb) cells without the need to wash away unmetabolized probe. One specific example of the overall detection process using trehalose-DMN conjugate (DMN-Tre) is illustrated in
(27) Solvatochromic Dye Conjugates
(28) Aspects of the present disclosure include solvatochromic dye-carbohydrate conjugates. Specifically, in many embodiments, the conjugate of interest is a single molecule, wherein a carbohydrate moiety is covalently linked to a solvatochromic dye. As such, in many embodiments, the carbohydrate moiety imparts the capability of metabolic uptake on the subject conjugate. Accordingly, in many embodiments, the carbohydrate moiety is derived from or structurally analogous to a naturally occurring carbohydrate of the target bacteria. In some embodiments, the carbohydrate moiety can act as a substrate for a lipid transferase enzyme of interest, whereby a hydroxyl group of the carbohydrate is esterified by the enzyme with a fatty acid lipid. In some embodiments, the lipid transferase enzyme is endogenous to the target bacteria, including actinobacteria, such as, for example, mycobacteria. Furthermore, it will be understood that, depending on the faithfulness and functionality/structure/size tolerance of the endogenous enzyme or enzymes involved in the conjugate's metabolic uptake by the target, the carbohydrate moiety can be further decorated, or otherwise altered, with additional chemical or structural features, as needed to enhance conjugate's overall efficiency and reliability as a probe.
(29) Carbohydrate Moiety
(30) In some embodiments carbohydrate moiety of interest includes 1 to 6 monosaccharide units, optionally glycosidically linked. In other embodiments, the carbohydrate moiety has 1 to 4 monosaccharide units. In many embodiments, the carbohydrate moiety is selected from monosaccharides and disaccharides. In some embodiments, at least one of the monosaccharide units, in some cases, a terminal monosaccharide unit of the carbohydrate, comprises a six-membered ring. In some instances, the carbohydrate moiety is a disaccharide in which both monosaccharide units have 6-membered rings.
(31) In some embodiments, the carbohydrate moiety is a trehalose, or a derivative, or an analog thereof. As used here a trehalose moiety is a carbohydrate that is structurally analogous to trehalose, and hence can interact with the target enzyme active sites, and provide selectivity for and uptake into mycobacteria. Trehalose is found in the outermost portion of the mycobacterial membrane, along with the glycolipids trehalose monomycolate (TMM) and trehalose dimycolate (TDM), as depicted in
(32) ##STR00011##
(33) In certain instances, the carbohydrate moiety is an isomeric form of trehalose, such as , -trehalose, also termed neotrehalose or , -trehalose, also termed isotrehalose, or a hydrate thereof. In certain instances, the carbohydrate moiety is a trehalose oligosaccharide. As used herein, the term trehalose oligosaccharide refers to an oligosaccharide (e.g., a trisaccharide or tetrasaccharide) that includes trehalose as the base component with one, two or more optional additional sugars attached to it. In some cases, a trehalose oligosaccharide is the disaccharide trehalose. Any convenient sugars (e.g., monosaccharides or disaccharides) may be utilized in the subject trehalose oligosaccharides, including but not limited to, Glc, Galf, Gal, LGal, Man, All, LAll, Gul, Lido, Tal, Ribf, Rib, Araf, Ara, LAraf, Lara, Xyl, Lyx, and the like. In some instances, the trehalose oligosaccharide is selected from Glc1-4Trehalose, Glc1-6Trehalose, Glc1-6Glc1-6Trehalose, Gal1-6Gal1-6Trehalose, and a trehalose with an 1-6Gal on one of its glucose residues and an 1-4Glc on the other glucose residue. Unless indicated otherwise, all monosaccharide codes described herein have their standard meaning. For example, Glc refers to D-glucose and Gal refers to D-galactose. In some cases, a trehalose oligosaccharide moiety includes an oligosaccharide including 5 or more units, such as 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 100 or more, 200 or more, or even more sugar units containing trehalose.
(34) In some embodiments, the carbohydrate moiety is a derivatized version of any convenient carbohydrate. Herein, a derivative of a carbohydrate is a molecule which is based on a naturally occurring carbohydrate, with one or more of the hydroxyl or hydrogen atoms being replaced with other chemical moieties (herein derivative groups), which derivative groups do not substantially affect the ability of the probe molecule to engage with the active sites of the target enzymes (e.g., Ag85A, B or C), and hence which do not prevent incorporation of the carbohydrate-dye conjugate into mycobacteria. Such derivative groups in some cases do not inhibit the reactions which relate to incorporation of the conjugate into the actinobacteria or mycobacteria. In addition, such derivative groups are, in some embodiments, selected so as not to introduce instabilities into the probe molecule, for example by providing two anionic and/or nucleophilic groups on the same carbon atom, examples being two hydroxide groups, a hydroxide and ether group, or a hydroxide and halide group on the same carbon atom. In some embodiments, the carbohydrates or derivatives thereof have no more than one such derivative group per carbon atom of the carbohydrate substrate molecule. Examples of derivative groups include: halides, amino, substituted amino, optionally substituted linear or branched alkyl, alkenyl or alkynyl group, and optionally substituted aryl or heteroaryl group.
(35) In some embodiments, the carbohydrate-dye conjugate has formula (I):
(36) ##STR00012##
wherein: Z is a carbohydrate or carbohydrate derivative of 1 to 5 monosaccharide units (e.g., 1 or 2 monosaccharide units) connected to C1 via a bridging group or heteroatom (X) in either an or configuration; R.sup.1 is H, alkyl or substituted alkyl; R.sup.2 to R.sup.9 are each independently H, OH, alkoxy, substituted alkoxy, amino, substituted amino, halogen (e.g., fluoro), thiol, alkylthio, substituted alkylthio or a linked solvatochromic dye. In certain instances of formula (I), X is O. In certain instances of formula (I), X is NH. In certain instances of formula (I), Z is a monosaccharide or monosaccharide derivative.
(37) In some embodiments, the carbohydrate-dye conjugate has the structure of formula (II):
(38) ##STR00013##
wherein R.sup.1 is H, alkyl or substituted alkyl; one of Z.sup.1-Z.sup.7 is a linked solvatochromic dye and the rest of Z.sup.1-Z.sup.7 are independently selected from H, OH, alkoxy, substituted alkoxy, amino, substituted amino, halogen (e.g., fluoro), thiol, alkylthio, substituted alkylthio. In certain instances of formula (II), one and only one of Z.sup.1-Z.sup.7 is a linked solvatochromic dye and the rest of Z.sup.1-Z.sup.7 are OH. In certain embodiments of formula (II), Z.sup.1 is the linked solvatochromic dye. In certain embodiments of formula (II), Z.sup.2 is the linked solvatochromic dye. In certain embodiments of formula (II), Z.sup.3 is the linked solvatochromic dye. In certain embodiments of formula (II), Z.sup.4 is the linked solvatochromic dye. In certain embodiments of formula (II), Z.sup.5 is the linked solvatochromic dye. In certain embodiments of formula (II), Z.sup.6 is the linked solvatochromic dye. In certain embodiments of formula (II), Z.sup.7 is the linked solvatochromic dye. In some instances of formula (II), one and only one of Z.sup.1-Z.sup.7 is a linked solvatochromic dye and the rest of Z.sup.1-Z.sup.7 are OH. In certain embodiments of formula (II), R.sup.1 is H. In certain embodiments of formula (II), R.sup.1 is alkyl (e.g., a lower alkyl). In certain embodiments of formula (II), R.sup.1 is substituted alkyl.
(39) In some instances of formula (II), the carbohydrate-dye conjugate has formula (III):
(40) ##STR00014##
wherein R.sup.1 is H, alkyl or substituted alkyl; one and only one of Z.sup.1-Z.sup.4 is a linked solvatochromic dye and the rest of Z.sup.1-Z.sup.4 are independently selected from H, OH, alkoxy, substituted alkoxy, amino, substituted amino, halogen (e.g., fluoro), thiol, alkylthio, substituted alkylthio. In some instances of formula (III), one and only one of Z.sup.1-Z.sup.4 is a linked solvatochromic dye and the rest of Z.sup.1-Z.sup.4 are OH. In certain embodiments of formula (III), Z.sup.1 is the linked solvatochromic dye. In certain embodiments of formula (III), Z.sup.2 is the linked solvatochromic dye. In certain embodiments of formula (III), Z.sup.3 is the linked solvatochromic dye. In certain embodiments of formula (III), Z.sup.4 is the linked solvatochromic dye. In certain embodiments of formula (III), R.sup.1 is H. In certain embodiments of formula (III), R.sup.1 is alkyl (e.g., a lower alkyl). In certain embodiments of formula (III), R.sup.1 is substituted alkyl.
(41) certain embodiments of formulae (II) and (III), the carbohydrate-dye conjugate has formula (IV):
(42) ##STR00015##
wherein L is an optional linker, Dye is a solvatochromic dye, and R.sup.1 is H, alkyl or a substituted alkyl.
(43) In certain embodiments of formulae (II) and (III), the carbohydrate-dye conjugate has formula (V):
(44) ##STR00016##
wherein L is an optional linker, Dye is a solvatochromic dye, and R.sup.1 is H, alkyl or a substituted alkyl.
(45) certain embodiments of formulae (II) and (III), the carbohydrate-dye conjugate has formula (VI):
(46) ##STR00017##
wherein L is an optional linker, Dye is a solvatochromic dye, and R.sup.1 is H, alkyl or a substituted alkyl.
(47) In certain embodiments of formulae (II) and (III), the carbohydrate-dye conjugate has formula (VII):
(48) ##STR00018##
wherein L is an optional linker, Dye is a solvatochromic dye, and R.sup.1 is H, alkyl or a substituted alkyl.
(49) In certain embodiments of formula (II), the carbohydrate-dye conjugate has formula (VIII):
(50) ##STR00019##
wherein L is an optional linker, Dye is a solvatochromic dye, and R.sup.1 is H, alkyl or a substituted alkyl.
(51) In certain embodiments of formula (II), the carbohydrate-dye conjugate has formula (IX):
(52) ##STR00020##
wherein L is an optional linker, Dye is a solvatochromic dye, and R.sup.1 is H, alkyl or a substituted alkyl.
(53) In certain embodiments of formula (II), the carbohydrate-dye conjugate has formula (X):
(54) ##STR00021##
wherein L is an optional linker, Dye is a solvatochromic dye, and R.sup.1 is H, alkyl or a substituted alkyl
(55) In certain embodiments of formulae (IV) to (X), R.sup.1 is H. In certain embodiments of formulae (IV) to (X), R.sup.1 is alkyl (e.g., a lower alkyl, such as methyl). In certain embodiments of formulae (IV) to (X), R.sup.1 is substituted alkyl. In certain embodiments of formulae (IV) to (X), L is NH-L.sup.1- where L.sup.1 is a linker such as an alkyl or substituted alkyl. In certain embodiments of formulae (IV) to (X), L comprises NHCO. In certain embodiments of formulae (IV) to (X), L comprises NHC(X)NH where X is O or S. In certain embodiments of formulae (IV) to (X), L comprises NHC(O)O. In certain embodiments of formulae (IV) to (X), L is an alkyl or substituted alkyl linker.
(56) Solvatochromic Dye Moiety
(57) As summarized above, the color and/or transparency of the solvatochromic dye depends upon the immediate environment of the dye. Solvent polarity, as well as hydrogen bonding and other environmental factors, plays a significant role in determining the ground state and excited state energy levels that in turn determine the color and/or transparency of the dye. As used herein, the term solvatochromic dye refers to a detectable dye molecule that exhibits a detectable change in a spectroscopic property due to a change in solvent polarity. The spectroscopic property change can include a change in color (i.e., a wavelength shift of maximum absorption or emission wavelength) with increasing solvent polarity. In some cases, the spectroscopic property change can include a change in molar absorptivity or quantum yield with increasing solvent polarity.
(58) Any convenient solvatochromic dye can be adapted for use in the subject carbohydrate-dye conjugates. The solvatochromic dye can be linked to a carbohydrate moiety (e.g., as described above) at any convenient positions of the dye and the carbohydrate moiety to produce a carbohydrate-dye conjugate that is capable of uptake by a target bacterial cell. Solvatochromic dyes of interest include, but are not limited to, merocyanine dyes, Reichardt's dye, 1-Docosyl-4-(4-hydroxystyryl)-pyridinium bromide, 2,6-dichloro-4-(2,4,6-triphenyl-N-pyridinio)-phenolate, 1-(4-hydroxyphenol)-2,4,6-triphenylpyridinium hydroxide, other pyridinium N-phenoxide betaines, zwitterionic dyes, 4-[2-N-substituted-1,4-hydropyridin-4-ylidine)ethylidene]cyclohexa-2,5-di-en-1-one, red pyrazolone dyes, azomethine dyes, indoaniline dyes, diazamerocyanine dyes, and mixtures thereof and those dyes described by MacDonald et al. in U.S. Pat. No. 7,829,181, the disclosure of which is herein incorporated by reference. Zwitterionic dyes (or chromogens) are dyes in which formal positive and negative charges are contained within a contiguous pi-electron system. Other solvatochromic dyes include, but are not limited to, pyrene, 4-dicyanmethylene-2-methyl-6-(p-dimethyl-aminostyryl)-4H-pyran; 6-propionyl-2-(dimethylamino) naphthalene; 9-(diethyl-amino)-5H-benzo[a]phenoxazin-5-one; phenol blue; stilbazolium dyes; coumarin dyes; ketocyanine dyes; thymol blue, congo red, methyl orange, bromocresol green, methyl red, bromocresol purple, bromothymol blue, cresol red, phenolphthalein, seminaphthofluorescein (SNAFL) dyes, seminaphtharhodafluor (SNARF) dyes, 8-hydroxypyrene-1,3,6-trisulfonic acid, fluorescein and its derivatives, oregon green, and a variety of dyes mostly used as laser dyes including rhodamine dyes, styryl dyes, cyanine dyes, and a large variety of other dyes. Still other solvatochromic dyes may include indigo, 4-dimethylaminophthalimide (4-DMAP), 6-dimethylaminonaphthalimide (6-DMN), 4-dicyanmethylene-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran (DCM); 6-propionyl-2-(dimethylamino)-naphthalene (PRODAN); 9-(diethylamino)-5H-benzo[a]phenox-azin-5-one (Nile Red); 4-(dicyanovinyl)julolidine (DCVJ); phenol blue; stilbazolium dyes; coumarin dyes; ketocyanine dyes; N,N-dimethyl-4-nitroaniline (NDMNA) and N-methyl-2-nitroaniline (NM2NA); Nile blue; 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS), Anthradan, 7-Nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), NBD-TMA ([2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium), dapoxylbutylsulfonamide (DBS) and other dapoxyl analogs. Other suitable dyes, according to some embodiments, include, but are not limited to: 4-[2-N-substituted-(1,4-hydropyridin-4-ylidine)ethylidene]cyclohexa-2,5-dien-1-one, red pyrazolone dyes, azomethine dyes, indoaniline dyes, and mixtures thereof.
(59) In some embodiments of formulae (I)-(X), the linked solvatochromic dye of the carbohydrate-dye conjugate is of formula (XI):
(60) ##STR00022##
wherein: B is an optional fused 5 or 6-membered aryl or heteroaryl ring, optionally further substituted with one or more Z substituents independently selected from H, alkyl, substituted alkyl, halogen (e.g., chloro or fluoro), hydroxyl, amino, substituted amino, cyano, nitro, carboxy, alkoxy, substituted alkoxy; Y is one or more substituents independently selected from H, alkyl, substituted alkyl, halogen (e.g., chloro or fluoro), hydroxyl, amino, substituted amino, cyano, nitro, alkoxy and substituted alkoxy, wherein at least one Y or Z is NR.sub.2; R.sup.1 is selected from H, alkyl and substituted alkyl; and wherein at least one Z, Y or R.sup.1 is linked to the carbohydrate moiety of the conjugate via an optional linker. In certain embodiments of formula (XI), R.sup.1 is linked to the carbohydrate moiety of the conjugate.
(61) In certain instances of formula (XI), the carbohydrate-dye conjugate has the following structure:
(62) ##STR00023##
where L is an optional linker and R.sup.1 is as defined above.
(63) In some embodiments of formulae (I)-(X), the linked solvatochromic dye of the carbohydrate-dye conjugate is of formula (XII):
(64) ##STR00024##
wherein: A is an aryl or heteroaryl system comprising up to three, 5 or 6-membered rings, optionally further substituted with one or more Z substituents independently selected from H, alkyl, substituted alkyl, halogen (e.g., chloro or fluoro), hydroxyl, amino, substituted amino, cyano and nitro; n is 0 or 1; each R and R.sup.21 is independently selected from H, alkyl and substituted alkyl; and R.sup.28R.sup.31 are each independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, halogen (e.g., chloro or fluoro), hydroxyl, amino, substituted amino, cyano, and nitro; wherein at least one of Z, R.sup.21 and R.sup.28-R.sup.31 is linked to the carbohydrate moiety of the conjugate via an optional linker.
(65) In certain instances of formula (XII), the carbohydrate-dye conjugate has the following structure:
(66) ##STR00025##
where L is an optional linker and R.sup.1 is as defined above.
(67) In some embodiments of formulae (I)-(X), the linked solvatochromic dye of the carbohydrate-dye conjugate is of formula (XIII)
(68) ##STR00026##
wherein: R.sup.11, R.sup.15 and R.sup.16 are independently selected from H, alkyl and substituted alkyl; and R.sup.12-R.sup.14, R.sup.17-R.sup.19 are each independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, halogen (e.g., chloro or fluoro), hydroxyl, amino, substituted amino, cyano and nitro; wherein at least one of R.sup.11, R.sup.12-R.sup.14, R.sup.17-R.sup.19 is linked to the carbohydrate moiety of the conjugate via an optional linker.
(69) In certain instances of formula (XIII), the carbohydrate-dye conjugate has the following structure:
(70) ##STR00027##
where L is an optional linker and R.sup.1 is as defined above.
(71) In some embodiments of formulae (I)-(X), the linked solvatochromic dye of the carbohydrate-dye conjugate is of formula (XIV):
(72) ##STR00028##
wherein: B is an optional fused 5 or 6-membered aryl or heteroaryl ring, optionally further substituted with one or more Z substituents independently selected from H, alkyl, substituted alkyl, halogen (e.g., chloro or fluoro), hydroxyl, amino, substituted amino, cyano, nitro, carboxy, alkoxy, substituted alkoxy; Y is one or more substituents independently selected from H, alkyl, substituted alkyl, halogen (e.g., chloro or fluoro), hydroxyl, amino, substituted amino, cyano, nitro, alkoxy and substituted alkoxy, R.sup.1 is selected from H, alkyl and substituted alkyl; and wherein at least one of R.sup.1, Y and Z is linked to the carbohydrate moiety of the conjugate via an optional linker. In some cases, at least one Y or Z is NR.sub.2.
(73) In some embodiments of formulae (I)-(X), the linked solvatochromic dye of the carbohydrate-dye conjugate is of formula (XV):
(74) ##STR00029##
wherein: X is O, S or NR.sup.1; Y is one or more substituents independently selected from H, alkyl, substituted alkyl, halogen (e.g., chloro or fluoro), hydroxyl, amino, substituted amino, cyano, nitro, alkoxy and substituted alkoxy; each R.sup.1 is independently selected from H, alkyl and substituted alkyl; and wherein at least one of R.sup.1 and Y is linked to the carbohydrate moiety of the conjugate via an optional linker.
(75) In some embodiments of formulae (I)-(XV), the linked solvatochromic dye is of one of formula (XVI)-(XXV):
(76) ##STR00030## ##STR00031## ##STR00032##
wherein R.sup.1, R.sup.5, R.sup.6, R.sup.11, R.sup.15, R.sup.16, R.sup.21, R.sup.25, R.sup.26, R.sup.33-R.sup.35, R.sup.36, R.sup.51, R.sup.60 and R.sup.61 are independently selected from H, alkyl and substituted alkyl; and R.sup.2-R.sup.4, R.sup.7-R.sup.9, R.sup.12-R.sup.14, R.sup.17-R.sup.19, R.sup.22-R.sup.24, R.sup.27-R.sup.32, R.sup.37-R.sup.40 and R.sup.52-R.sup.59 are each independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, halogen (e.g., chloro or fluoro), hydroxyl, amino, substituted amino, cyano and nitro; wherein at least one of R.sup.1-R.sup.61 is linked to the carbohydrate of the conjugate via an optional linker.
(77) certain embodiments of formulae (I)-(XXV), the solvatochromic dye is selected from one of the following structures:
(78) ##STR00033##
wherein each R is independently H, an alkyl or a substituted alkyl, and the solvatochromic dye is linked to the carbohydrate moiety via any convenient position. In certain instances, each R is independently H or a lower alkyl such as methyl or ethyl
(79) In certain embodiments of formulae (I)-(XXV), the solvatochromic dye is selected from one of the following structures:
(80) ##STR00034##
wherein each R is independently H, an alkyl or a substituted alkyl, and the solvatochromic dye is linked to the carbohydrate moiety via any convenient position. In certain instances, each R is independently H or a lower alkyl such as methyl or ethyl.
(81) In certain embodiments of formulae (I)-(XXV), the solvatochromic dye is selected from one of the following structures:
(82) ##STR00035## ##STR00036##
wherein each R is independently H, an alkyl or a substituted alkyl, and the solvatochromic dye is linked to the carbohydrate moiety via any convenient position. In certain instances, each R is independently H or a lower alkyl such as methyl or ethyl.
(83) In certain embodiments of formulae (I)-(XXV), the solvatochromic dye is selected from one of the following structures:
(84) ##STR00037##
wherein each R is independently H, an alkyl or a substituted alkyl, and the solvatochromic dye is linked to the carbohydrate moiety via any convenient position. In certain instances, each R is independently H or a lower alkyl such as methyl or ethyl.
(85) In certain embodiments of formulae (I)-(XXV), the solvatochromic dye has the following structure:
(86) ##STR00038##
wherein each R is independently H, an alkyl or a substituted alkyl, and the solvatochromic dye is linked to the carbohydrate moiety via any convenient position. In certain instances, one R is linked to the carbohydrate moiety.
(87) In certain instances of formula (I)-(X), the linked solvatochromic dye is selected from 4-DMAP, 4-DMN, 6-DMN, Nile Red, 3-HC, 3-MC, PRODAN, Anthradan, NBD and derivatives thereof. In certain instances of formula (I)-(X), the linked solvatochromic dye is 4-DMAP. In certain instances of formula (I)-(X), the linked solvatochromic dye is 4-DMN or 6-DMN. In certain instances of formula (I)-(X), the linked solvatochromic dye is Nile Red. In certain instances of formula (I)-(X), the linked solvatochromic dye is 3-HC. In certain instances of formula (I)-(X), the linked solvatochromic dye is PRODAN. In certain instances of formula (I)-(X), the linked solvatochromic dye is Anthradan. In certain instances of formula (I)-(X), the linked solvatochromic dye is NBD.
(88) In certain instances of formula (I)-(X), the linked solvatochromic dye is selected from one of the following structures:
(89) ##STR00039##
or an analog thereof, wherein the solvatochromic dye is linked to the carbohydrate at any available position (e.g., the R position).
In addition,
Methods of Detecting Bacteria
(90) The present disclosure provides methods of detecting live bacteria in a biological sample. In many embodiments, the methods generally involve: a) contacting a biological sample to be tested (e.g., a biological sample containing, or suspected of containing, a live bacterial cell of interest, with a carbohydrate-solvatochromic dye conjugate (e.g., as described herein); and b) detecting a spectroscopic signal from the dye due to the changes in the dye's local environment upon interaction with the tested sample. In many embodiments, the solvatochromic dyes used in the detection methods are fluorogenic and the spectroscopic signal to be detected is fluorescence. In many embodiments, the dye's fluorescence is turned on (enhanced) to produce a detectable signal by the interactions with the target, while remaining undetectable in the absence of target encounters. In many embodiments, the bacteria of interest for detection are actinobacteria, such as mycobacteria.
(91) The detection of a spectroscopic signal, such as, for example, fluorescence, from the solvatochromic dye of the conjugate indicates the presence of the target metabolically active bacterial cell in the sample, since target cells will metabolically label the conjugate leading to uptake of the labelled conjugate into the bacterial cell wall, where the solvatochromic dye exhibits a change in spectroscopic properties. In some instances, the target bacterial cell includes a cell wall comprising mycolic acid. In certain cases, the fluorogenic carbohydrate-solvatochromic dye conjugate is a trehalose-solvatochromic dye conjugate (e.g., as described herein), which is metabolically labelled with a mycolic acid of the target bacterial cell. In certain embodiments, the live bacterial cell is a target mycobacterium.
(92) A method of the present disclosure provides for detection of as few as 10.sup.4 target live bacterial cells (e.g., mycobacteria) in a sample, such as fewer than 510.sup.3 fewer than 10.sup.3, fewer than 510.sup.2, fewer than 10.sup.2, fewer than 50, or fewer than 10 target bacterial cells (e.g., mycobacteria) in the sample. In some cases, the subject method provides for detection of 510.sup.3/mL target live bacterial cells or less, such as 10.sup.3/mL or less, 510.sup.2/mL or less, 10.sup.2/mL or less, 50/mL or less, or 10/mL or less, mycobacteria in a biological sample.
(93) In some instances, the subject method provides for detection of 500 colony forming units (CFU) of a target mycobacteria per mL or less, such as 200 CFU/mL or less, 100 CFU/mL or less, 90 CFU/mL or less, 80 CFU/mL or less, 70 CFU/mL or less, 60 CFU/mL or less, 50 CFU/mL or less, 40 CFU/mL or less, 30 CFU/mL or less, 20 CFU/mL or less, 10 CFU/mL or less, 8 CFU/mL or less, 6 CFU/mL or less, 5 CFU/mL or less, 4 CFU/mL or less, 3 CFU/mL or less, 2 CFU/mL or less, or even less. Any convenient methods can be utilized to detect bacterial cells and colony forming units of bacterial cells of interest in a sample, e.g., microscopy methods, colorimetric bacterial cell assays and the like.
(94) The subject methods can be utilized to identify and distinguish metabolically active mycobacteria in a biological sample from non-metabolically active mycobacteria and other microorganisms. The subject methods can be utilized to identify and distinguish target mycobacteria from non-target mycobacteria. Non-target mycobacteria can include microorganisms that are not actinobacteria or mycobacteria, which may be metabolically active or not.
(95) Mycobacteria that can be detected using a method of the present disclosure include any convenient mycobacterial species. Mycobacteria of interest includes but are not limited to those mycobacteria provided in Table 1, including, M. tuberculosis, M. avium (or M. avium-intracellulare), M. leprae (particularly M. leprae infection leading to tuberculoid leprosy), M. kansasii, M. fortuitum, M. chelonae, M. absecessus, M. marinum, M. Nocardia, M. xenopi, M. simiae, M. szulgai, M. scrofulaceum, M. malmoense, M. terrae-nonchromogenicum complex, M. haemophilum, M. genavense, M. celatum, M. interjectum, M. confluentis, M. triplex, M. lentiflavum, M. branderi, M. conspicuum, M. cookii, M. asiaticum, M. marinum M. gordonae, M. fortuitum, M. chelonae-abscessu, and M. mucogenicum. While detection of mycobacteria in biological samples from humans is of interest, detection of mycobacteria biological samples from non-human subjects is also of interest. For example, M. avium causes lymphadenitis in slaughter pigs; M. paratuberculosis infection causes paratuberculosis, a tuberculosis-like disease that can result in great production losses in cattle, sheep and goats; and M. bovis is carried by cattle and can cause a tuberculin-like infection in humans. Thus, in some cases, the biological sample is obtained from a pig; and a method of the present disclosure detects M. avium in the biological sample. In some cases, the biological sample is obtained from a cow, a sheep, or a goat; and a method of the present disclosure detects M. paratuberculosis in the biological sample. Mycobacteria of interest include animal and plant pathogens carrying the mycolic acid membrane. In some cases, bacteria can be targeted that are part of the Corynebacterine suborder. In certain instances, the subject methods can be adapted to detect a bacterium of the Corynebacterium family, such as Corynebacterium glutamicum or Corynebacterium diphtherie.
(96) In some embodiments, the biological sample is obtained from a human; and a method of the present disclosure detects M. tuberculosis in the biological sample. In some cases, the individual has a human immunodeficiency virus (HIV) infection and in addition has, or is suspected of having or being at risk of infection with, TB. In some cases, the individual is living in an area in which TB is endemic. In some cases, the individual is military personnel. In some cases, the individual is incarcerated (e.g., living in a prison). In some cases, the individual is immunocompromised.
(97) A biological sample encompasses a variety of sample types obtained from an individual or a group of individuals, directly (e.g., drawn or swabbed from a body) or indirectly (e.g. from the individual's or group's environment, such as air, water, or an inanimate surface), and can be used in a diagnostic or monitoring assay. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. It will be understood that air or water samples, or solid inanimate surfaces or objects, such as, for example, air or water filters, room walls or windows, or laboratory equipment, that can potentially contain the biological sample of interest (via, for example, direct contact) are also included. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as polynucleotides. The term biological sample encompasses a clinical sample, and includes cells in culture, cell supernatants, cell lysates, blood, serum, plasma, biological fluid, buccal swab (e.g., cheek swab) and tissue samples. Biological fluids include sputum, buccal swab saliva, cerebrospinal fluid, urine, bronchoalveolar lavage fluid, and the like. In some embodiments, the sample is a sputum sample. In other embodiments, the sample is a buccal swab (e.g., cheek swab). In yet other embodiments, the sample is air filter.
(98) In general, the subject methods are performed in vitro. Any convenient method may be used to contact the sample with the conjugate. In some instances, the sample is contacted with the conjugate under conditions in which the conjugate is taken up into the cell wall of the bacterial cell, if present. In some cases, for metabolic labelling and uptake of the conjugate to the target bacterial cell, an appropriate solution may be used that maintains the viability of the cells. The solution may be a balanced salt solution, e.g., normal saline, PBS, Hank's balanced salt solution, etc., conveniently supplemented with fetal calf serum, human platelet lysate or other factors, in conjunction with an acceptable buffer at low concentration, such as from 5-25 mM. In some other embodiments, a sample of interested dispersed in air or water is contacted with the detecting conjugate by passing the said sample through an air or water filter having the conjugate on its sample contacting surface.
(99) The temperature at which contacting the sample with the solvatochromic dye-sugar conjugate is performed may vary, and in some instances may range from 5 C. to 50 C., such as from 10 C. to 40 C., 15 C. to 40 C., 20 C. to 40 C., e.g., 20 C., 25 C., 30 C., 35 C. or 37 C. In some instances, the temperature at which contacting takes place is selected to be compatible with the biological activity or viability of the target bacteria, e.g., target mycobacteria, and/or other components of the sample. In certain instances, the temperature is 25 C., 30 C., 35 C. or 37 C. In certain cases, the temperature at which the contacting takes place is room temperature (e.g., 25 C., 30 C., 35 C. or 37 C.). Any convenient incubation time for incubating the contacted sample may be selected to allow for the incorporation of a desirable amount of the conjugate into the target bacterial cell, and in some instances, may be 1 minute (min) or more, such as 2 min or more, 10 min or more, 30 min or more, 1 hour or more, 2 hours or more, 6 hours or more, 12 hours or more, or even 24 hours or more.
(100) The subject methods provide for a method that does not require a washing step, e.g., where cells in the sample are immobilized and/or washing prior to detection to remove excess dye reagents that can cause background signals. In the subject methods detection can be performed directly on the contact sample.
(101) In some instances, the sample can be diluted prior to detection. Dilution can be performed in any convenient buffer to a known volume to assist in qualitative or quantitative detection of the target bacterial cells in the sample.
(102) Detecting fluorescence may include exciting a fluorescent dye (e.g., the fluorogenic solvatochromic dye of the conjugate) with one or more lasers, and subsequently detecting fluorescence emission from the dye using one or more optical detectors. In some embodiments, the methods further include counting, sorting, or counting and sorting a labeled microorganism. The solvatochromic dyes may be detected and uniquely identified by exposing them to excitation light and measuring the fluorescence produced in one or more detection channels, as desired. The excitation light may be from one or more light sources and may be either narrow or broadband. Examples of excitation light sources include lasers, light emitting diodes, and lamps, including mercury or xenon lamps, arc lamps, a flash lamp, incandescent bulb or any other light source suitable for excitation of fluorescence. Fluorescence emitted in detection channels used to identify the solvatochromic dye and components associated therewith may be measured following excitation with a single light source, or may be measured separately following excitation with distinct light sources. The solvatochromic dyes may be selected such that the dyes are excitable by the excitation light sources of interest that are used. Any convenient methods of microscopy can be utilized in the detecting step of the subject methods.
(103) In some embodiments, detection involves a qualitative determination of the presence of target bacterial cells in the sample. In certain instances, the target bacterial cell is one that has infected a cell of the host. A qualitative determination can include observing a characteristic fluorescence from components of an infected cell, e.g., from the cell wall of a target bacterial cell. In some cases, detection can involve a quantitative determination of the number of target bacterial cells in the sample. A quantitative determination can include counting the number of cells (e.g., per sample, per unit volume, mL) that are positively fluorescent versus a negative control cell. In certain instances, a negative control cell may exhibit some minor background fluorescence that can be easily distinguished from that of a labelled target cell.
(104) Monitoring Bacterial Cells During Treatment for Infection
(105) As used herein, the terms treatment, treating, and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease (e.g., mycobacterial infection) or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease (e.g., mycobacterial infection) and/or adverse effect attributable to the disease (e.g., mycobacterial infection). As used herein, the terms treating, treatment, therapeutic, or therapy do not necessarily mean total cure or abolition of the disease or condition. Any alleviation of any undesired signs or symptoms of a disease or condition, to any extent can be considered treatment and/or therapy. Furthermore, treatment may include acts that may worsen the patient's overall feeling of well-being or appearance. Treatment, as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease (e.g., mycobacterial infection) from occurring in a subject which may be predisposed to the disease (e.g., mycobacterial infection) but has not yet been diagnosed as having it; (b) inhibiting the disease (e.g., mycobacterial infection), i.e., arresting its development; and (c) relieving the disease (e.g., TB infection), i.e., causing regression of the disease (e.g., mycobacterial infection).
(106) Aspects of the present disclosure include methods of detecting and monitoring target bacterial cells in a multitude of samples taken at intervals from a subject during treatment or prevention of an infection of the subject. In some embodiments, the subject is infected with a mycobacteria (e.g., as described herein, such as Mycobacterium tuberculosis). Such individuals may be tested at one or a plurality of time points, including, without limitation, at the time of diagnosis, prior to, during and/or after anti-infective treatment; and at various time points to monitor disease progression. As such, aspects of the method include collecting one or more samples from a subject suffering from a bacterial infection before, during and/or after treatment (e.g., administration of an anti-infective agent to the subject). In some cases, sampling the subject before treatment to detect target bacteria according to the subject methods further includes diagnosis of an infection in the subject. For example, in some cases, a biological sample is obtained from an individual at a first time point, and target cells are detected in the biological sample; a biological sample is obtained from the individual at a second time point, and target cells are detected in the biological sample; and the number of target cells detected in the biological sample from the second time point is compared to the number of target cells detected in the biological sample from the first time point, where the second time point is after the first time point (e.g., one hour to 4 hours, 4 hours to 8 hours, 8 hours to 24 hours, 1 day to 1 week, 1 week to 1 month, 1 month to 6 months, 6 months to 1 year, or more than 1 year, after the first time point).
(107) Aspects of the subject method include quantitating the target metabolically active bacterial cells in the sample, so that the levels of the cells over time can be monitored to assess the effectiveness of the treatment method. As such, the collection of the sample can be performed before treatment and at one or more time points during treatment or after treatment. Depending on the results of the monitoring, the anti-infective treatment method can be adjusted to provide for a desirable therapeutic outcome, e.g., the treatment dosage or regimen can be altered according to a change in the amount of target metabolically active bacterial cells that is quantitated in the sample over time.
(108) In some embodiments, the method includes administering to a subject in need thereof a therapeutically effective amount of anti-infective agent, e.g., an anti-mycotic agent, as guided by the findings of the treatment monitoring methods according to the embodiments of this application. In certain instances, the subject is suffering from an HIV infection in addition to the target bacterial infection, and exhibits relatively low numbers of target bacterial cells in samples. The subject methods provide for detection of target cells in such samples.
(109) Determining Bacteria's Drug Sensitivity/Resistance
(110) Aspects of the subject methods include rapid detection of drug-resistant bacteria. Accordingly, in some embodiments, the subject conjugates can be used to discriminate bacteria that are resistant to a treatment, from bacteria that are successfully targeted by the treatment. In many such embodiments, a sample of interest is split into a multitude of samples according to the number of available treatment plans. Next, each of the new samples is treated according to its assigned treatment plan, while being subjected to the conjugate probe at predetermined intervals. In many embodiments, the sample resistant to treatment and, thus, maintaining the vitality of its bacteria, will continue to uptake the conjugate and produce a detectable signal. In contrast, the sample sensitive to the assigned treatment will not be able to produce a measurable signal, as its ability to metabolically uptake the conjugate is compromised in the absence of live bacteria. The described herein method of testing for drug resistance is a significant improvement, including in speed, over the current practices for determining drug susceptibility of, for example, clinical Mtb isolates, which typically rely on PCR-based methods (testing for known resistance genes) or lengthy (up to 6 weeks) culturing of samples in the presence of drugs. In stark contrast, in some examples studying mycobacteria resistance to TB drug cocktail according to the embodiments of the instant application, it was found that DMN-Tre labeling is affected by drug action within hours, and the results of such a microscopy test could, therefore, be available on the same day as sample collection.
(111) In some cases, the subject methods include detecting and/or identifying drug-resistant bacterial cells, e.g., in a sample from a subject undergoing treatment. The treatment regimen or type of anti-infective agent can be adjusted according to the type and number of drug-resistant bacteria that are detected or identified. As such, the subject methods can be performed in conjunction with a method of treating an infective condition.
(112) Therapeutic Utilities
(113) The subject compositions and methods can be employed in a variety of diagnostic, research and therapeutic applications. The subject methods compositions and methods find use in any applications where the identification of a target bacterial cell is of interest, such as target bacterial cells that are capable of metabolically labelling and uptake of the subject carbohydrate-dye conjugates.
(114) Diagnostic applications of interest include, but are not limited to, practicing the subject methods of detection to provide diagnosis of an infection. Therapeutic applications of interest include, but are not limited to, monitoring the progress of anti-infective treatment, prior to, during and/or after anti-infective treatment, and selecting and/or adjusting a desired treatment regimen or type of anti-infective agent based on the detection of drug-resistant bacterial cells. In some embodiments, the subject methods find use in the direct detection of pathogenic bacteria, such as Mtb, in the sample of a patient, or the monitoring of pathogenic bacteria levels in samples of a patient being treated for an infection. In other embodiments, the subject methods find use in determining the sensitivity of a target bacteria or organism to a panel of possible therapeutics.
EXAMPLARY EMBODIMENTS
(115) The following examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.
(116) The following examples, demonstrating various features and advantages of the mycobacteria detection methods according to the embodiments of the present invention, primarily rely on trehalose conjugated to the solvatochromic dye 4-N,N-dimethylamino-1,8-naphthalimide (DMN), a reagent termed here as DMN-Tre. In many embodiments, DMN-Tre is metabolically incorporated into mycomembranes where it undergoes a dramatic enhancement in fluorescence that enables detection of mycobacteria in TB patient sputum samples in under an hour (according to the process schematically depicted in
Example 1. Detection of Mycobacteria with DMN-Tre Conjugate
(117) First, DMN-Tre conjugate was synthesized, as well as two control compounds, DMN-glucose (DMN-Glc) and a non-fluorogenic analog 6-fluorescein-trehalose (6-FITre), as depicted in
(118) A time course to determine the labeling kinetics was also performed. Msmeg and Cg harvested during their exponential growth phase were incubated as above and imaged at various time points. As shown in
Example 2. DMN-Tre Conjugate Selectivity for Mycobacteria
(119) DMN-Tre's potential as a diagnostic tool, including for diagnosis of TB, depends on its selectivity for mycobacteria among other bacterial species. Therefore, to test its selectivity, canonical gram-negative and gram-positive organisms (Bacillus subtilis (Bs), Escherichia coli (Ec), Listeria monocytogenes (Lm), and Staphylococcus aureus (Sa)) were incubated with DMN-Tre.
Example 3. Metabolic Nature of DMN-Tre Conjugate Uptake by Mycobacteria
(120) A series of experiments were performed to confirm that DMN-Tre labeling results from metabolic conversion to trehalose mycolates within the mycomembrane rather than nonspecific insertion into the mycomembrane. First, flow cytometry was used to confirm that Msmeg exhibit significant fluorescence over background within 20 min (
(121) Current microscopy-based methods for TB diagnosis cannot distinguish live from dead mycobacteria. Given that DMN-Tre labeling appears to be of metabolic nature and depend on mycomembrane biosynthesis, it was hypothesized that the methods of the present disclosure would be specific for live bacteria. Indeed, labeling was abrogated by heat killing Msmeg (
Example 4. Use of DMN-Tre Conjugate in Drug Resistance Tests
(122) The effect of TB drug treatment on labeling was evaluated. To this end, Msmeg cells treated for 3 hours with a cocktail of ethambutol, rifampicin, isoniazid and SQ109, each at a dose at or above reported minimum inhibitory concentrations (MICs) to induce cell killing, lost all detectable labeling with DMN-Tre (
Example 5. Detection of Pathogenic Mtb with DMN-Tre Conjugate
(123) Having established the parameters and mechanistic basis of DMN-Tre labeling with Msmeg, attention was focused on studies with pathogenic Mtb. Liquid cultures of Mtb (H37Rv) were visibly labeled with DMN-Tre (
(124) Furthermore, to gain insight into the potential clinical utility of DMN-Tre, detection of Mtb cells in TB-positive patient sputum samples was attempted. Sputum samples from 16 treatment-nave patients, who were TB positive, were obtained by either smear microscopy or GeneXpert. The samples were decontaminated using a standard N-acetyl-L-cysteine-sodium hydroxide (NALC/NaOH) treatment and then incubated with DMN-Tre (
(125) In summary, DMN-Tre labeling appears to be unique in its combination of attributes. Unlike classic methods for detecting Mtb cells by microscopy, DMN-Tre labeling specifically targets a pathway in mycomembrane biosynthesis and therefore reports both on bacterial identity and vitality. The unique solvatochromic property of DMN-Tre enables rapid Mtb imaging without cumbersome washing steps. When combined, these features enable the detection of live Mtb in samples as complex as patient sputum.
(126) DMN-Tre's unique mode of fluorescence activation allows for an operationally simple procedurea single incubation step. Notably, it was found that DMN-Tre is very stable on the bench or in shipping containers for weeks at room temperature, and even in aqueous solution at 37 C. Thus, the DMN-Tre labeling procedure may translate well both to research and clinical applications in low-resource environments.
Experimental Materials and Methods
(127) Metabolic Labeling of Bacteria with DMN-Tre, DMN-Glc or 6-FITre
(128) Cultures of M. smegmatis (Msmeg) or mutants were made by inoculation of a single colony from an agar plate into 1 milliliter (mL) 7H9 liquid medium supplemented with 10% (v/v) oleate-albumin-dextrose-catalase enrichment (BBL Middlebrook OADC, catalog no. 212351), 0.5% (v/v) glycerol, and 0.05% (w/v) Tween 80 (Sigma, P1754) with or without antibiotic (if necessary) in a 5-mL culture tube (FisherSci, 14-959-11B). Cultures of C. glutamicum (Cg), B. subtilis (Bs), E. coli (Ec), L. monocytogenes (Lm) and S. aureus (Sa) were generated by inoculation of a single colony from an agar plate into 1 mL Luria Broth (Invitrogen, 12795-084) liquid medium in a 5-mL culture tube. Cultures of M. tuberculosis (Mtb) were made by inoculation of a 1-mL frozen stock into 50 mL Middlebrook 7H9 liquid medium supplemented with 10% (v/v) oleate-albumin-dextrose-catalase enrichment (BBL Middlebrook OADC, 212351), 0.5% (v/v) glycerol, and 0.05% (w/v) Tween 80 (Sigma, P1754) in a roller bottle or a tissue culture flask. Cultures were grown to OD.sub.600=0.5 to begin experiments. Bacterial cultures were mixed with DMN-Tre (
(129) Flow Cytometry
(130) Following metabolic labeling (and after fixation for Mtb cells), cells were harvested by centrifugation (3 min, 3,300g), washed (2500 microliter (L) PBS) and resuspended in 300 L PBS. Fluorescence measurements were taken in 5-mL culture tubes (FisherSci, 14-959A) suitable for flow cytometry. Data collection was performed on a BD LSR II.UV instrument in the shared FACS Facility at Stanford University (NIH S10 Shared Instrument Grant (S10RR027431-01)). This instrument is equipped with a 405 nm violet laser and 488 nm blue laser (for Aqua Amine and FITC channels, respectively) both used to detect DMN-Tre fluorescence. Fluorescence data was obtained for 100,000 cells per sample and processed using FlowJo (Tree Star) software. Experiments were conducted in three biological replicates.
(131) Fluorescence Microscopy
(132) Following metabolic labeling (and fixation for Mtb), 6 L of cell suspension were spotted onto slides, covered with coverslips and sealed with adhesive. Microscopy was performed on a Nikon A1R confocal microscope equipped with a Plan Fluor 60 Oil immersion NA 1.30 objective. This instrument is equipped with a 405 nm violet laser, 488 nm blue laser and 561 nm green laser (for Aqua Amine, FITC/GFP and RFP channels, respectively). NIS-Elements AR software (Nikon, Inc.) was used to process images. All image acquisition and processing was executed under identical conditions for control and test samples.
(133) Absorbance and Fluorescence Measurements of DMN-Tre
(134) One L of 10 mM DMN-Tre in H2O was added to 1 mL of mixtures of dioxane and water at different ratios. Absorbance spectra were recorded on a Varian Cary 50 UV-Visible spectrophotometer. Fluorescence spectra were recorded on a Photon Technology International Quanta Master 4 L-format scanning spectrofluorometer equipped with an LPS-220B 75-W xenon lamp and power supply, an A-1010B lamp housing with an integrated igniter, a switchable 814 photon counting/analog photomultiplier detection unit, and an MD5020 motor driver. Measurements were made in 1 cm0.4 cm quartz cuvettes with a total sample volume of 1 mL.
(135) No-Wash Imaging of Msmeg, Cg, and M. marinum by DMN-Tre and 6-FITre
(136) Msmeg or Cg was grown to an OD.sub.600 of 0.5 from a single colony (37 C. for Msmeg; 30 C. for Cg). To 500 L of culture was added either 5 L of 10 mM DMN-Tre or 10 mM 6-FITre in H2O. The bacteria were incubated for another 1 h (Msmeg) or 2 h (Cg), then placed under a coverslip and imaged directly. M. marinum (Mm) was stored as an OD 0.5 stock in 50% glycerol/50% 7H9+OADC. 1 mL of this frozen culture was thawed and the bacteria pelleted (3 min, 3,300g) and resuspended in 2 mL of 7H9 liquid medium supplemented with 10% (v/v) oleate-albumin-dextrose-catalase enrichment (BBL Middlebrook OADC, 212351), 0.5% (v/v) glycerol, and 0.05% (w/v) Tween 80 (Sigma, P1754). The bacteria were incubated at 33 C. overnight (16 h). To 500 L of Mm culture was added 5 L of 10 mM DMN-Tre or 10 mM 6-FITre in H2O. The bacteria were incubated at 33 C. for 6 h, then placed under a coverslip and imaged without washing.
(137) No-Wash Labeling of Msmeg and Cg Over Time
(138) Msmeg or Cg cells were grown to an OD.sub.600 of 0.5 from a single colony as above. To 200 L of culture was added 2 L of 10 mM DMN-Tre in H2O. Aliquots were taken from this culture at the indicated time points, immediately placed under a coverslip, and imaged.
(139) Labeling of Non-Mycomembrane Bearing Bacteria
(140) Lm, Bs, Ec, and Sa were grown from single colonies at 37 C. overnight. The bacteria were diluted to an OD.sub.600 of 0.4. To 1 mL aliquots of these bacteria, 10 L of 10 mM DMN-Tre was added to reach a final concentration of 100 M. The cells were then incubated at 37 C. for 2 h. Aliquots were taken and the cells were imaged under a coverslip. As controls, cells from the same OD.sub.600=0.4 culture, without the addition of DMN-Tre, were also imaged.
(141) Selective Labeling of Msmeg in the Presence of Other Bacterial Species.
(142) Bacteria (Lm, Bs, Ec, and Sa) were grown overnight with shaking in media as noted above. The bacteria were diluted to OD.sub.600=0.5, then 500 L of each culture were mixed together to create 2 mL of mixed bacteria. One mL of Msmeg expressing mCherry was grown from a single colony overnight to OD.sub.600 of 0.5. Both cultures were pelleted by centrifugation (3 min, 3,300g) and resuspended in an identical volume of LB medium. To 540 L of mix, 60 L of Msmeg was added. Lastly, this final mixture of bacteria containing Msmeg and the four other non-mycomembrane bearing bacterial species was split into two 300 L aliquots. To each group, 0.3 L of 2 mg/mL Hoechst DNA stain (Thermo Fisher Scientific, 62249) was added to stain all the bacteria. To one of the two aliquots, 3 L of 10 mM DMN-Tre were added, while no DMN-Tre was added to the other portion. The two aliquots were incubated with shaking at 37 C. for 1 h before a sample was taken out and imaged without washing.
(143) Trehalose Competition of DMN-Tre Labeling in M. smegmatis
(144) Msmeg was grown to OD.sub.600=0.4 from a single colony as noted above. Then, the bacteria were divided into 100 L aliquots. To these aliquots was added 1 L of 10 mM DMN-Tre and 1 L of 0, 10 or 100 mM trehalose in water. The bacteria were grown for another 1 hour, washed twice with PBS, resuspended in PBS, and examined by flow cytometry and microscopy.
(145) Ebselen Inhibition Studies
(146) Msmeg was grown to OD.sub.600=0.4 from a single colony as noted above. Then, 500 L aliquots of bacterial culture were incubated with 25, 50 or 100 g/mL ebselen (Sigma-Aldrich, 60940-34-3) for 3 h. To these pre-treated samples was added 5 L 10 mM DMN-Tre. The cultures were grown for another 1 h, washed twice with PBS, resuspended in PBS, and examined by flow cytometry and microscopy.
(147) Growth Phase Studies
(148) Msmeg was grown from a single colony to OD.sub.600=0.5 or OD.sub.600>2. Then, 500 L aliquots of bacterial culture were incubated with 5 L 10 mM DMN-Tre for 30 minutes, washed twice with PBS, resuspended in PBS, and examined by flow cytometry and microscopy.
(149) DMN-Tre Labeling of Drug-Treated Msmeg and Mtb
(150) Msmeg was grown to OD.sub.600=0.4 from a single colony as noted above. Then, 500 L aliquots of bacterial culture were incubated with control or drug cocktail (cocktail contents: 1 g/mL ethambutol, 0.2 g/mL rifampicin, 10 g/mL SQ109 and 10 g/mL isoniazid in 7H9 media) for 3 h in a 37 C. atmospheric incubator. To these pre-treated samples was added 5 L 10 mM DMN-Tre. The cultures were grown for another 30 minutes, washed twice with PBS, resuspended in PBS, and examined by flow cytometry and microscopy.
(151) For Mtb, cultures were made by inoculation of a 1-mL frozen stock into 50 mL Middlebrook 7H9 liquid medium supplemented with 10% (v/v) oleate-albumin-dextrose-catalase enrichment (BBL Middlebrook OADC, 212351), 0.5% (v/v) glycerol, and 0.05% (w/v) Tween 80 (Sigma, P1754) in a roller bottle. Cells were grown to OD.sub.600=0.5 to begin experiments. Five hundred L aliquots of bacterial culture were incubated with control or drug cocktail (cocktail contents: 1 g/mL ethambutol, 0.2 g/mL rifampicin, 10 g/mL SQ109 and 10 g/mL isoniazid in 7H9 media) for 3 h in a 37 C. atmospheric incubator followed by incubation with 100 M DMN-Tre overnight (16 h). Labeled cells were harvested by centrifugation (10 min, 3,000g) then fixed in an equal volume of 2.5% glutaraldehyde. The cells were incubated at room temperature for 1 h, with occasional rotation of the tube to ensure sterilization of all internal surfaces prior to fluorescence and flow cytometry analysis.
(152) Patient Recruitment and Sputum Sample Collection
(153) Ethics approval for the study was provided by the University of the Witwatersrand Human Research Ethics Committee (clearance number: M110833). Participants for the study were approached at primary health care clinics; those willing to participate were then asked to visit the study clinic where informed consent was administered. Thereafter, a spot or overnight sputum sample was collected and transported to the lab for processing. Sputum was decontaminated by addition of an equal volume of 2.9% sodium citrate and 4% sodium hydroxide (NalC/NaOH), followed by incubation at room temperature for 20 minutes. Thereafter, the bacterial cells were harvested at 3900g for 10 min and washed with 4.5 mL of 0.01 M phosphate buffered saline (PBS), pH 7.4 followed by resuspension in 2 mL Middlebrook 7H9 media supplemented with 0.5% Tween and OADC (oleic acid, albumin, dextrose, catalase; Becton Dickinson, South Africa). To disperse clumps, cells were vortexed briefly in the presence of 2 mm glass beads.
(154) Microscopy Analysis of Mtb in Sputum Samples
(155) Sputum samples from TB patients were isolated as described above. To 0.1 mL aliquots of these samples, 10 L of 10 mM DMN-Tre was added to reach a final concentration of 1 mM. Samples were then incubated at 37 C. for the indicated times. Samples were fixed in a final concentration of 2.5% glutaraldehyde, and incubated at room temperature for 1 h, with occasional rotation of the tube to ensure sterilization of all internal surfaces. Prior to imaging, samples were resuspended in 30 l of 1PBS.
(156) Auramine Vs DMN-Tre Smear Test
(157) Auramine smear was performed according to standard conditions for kit. Briefly, NalC-NaOH-decontaminated sputum sample was smeared onto the slide and heat-fixed (heating block, 95 C., 5-10 min). Smears were then treated with Auramine O for 5 min, de-stained and then counter-stained before viewing in the FITC/GFP channel of a Zeiss Observer Z1-inverted fluorescence microscope.
(158) For DMN-Tre labeling, the same sample was stained as follows: 100 l of sample was incubated with 1 mM DMN-trehalose for 30 minutes at 37 C. followed by fixing in 2.5% glutaraldehyde for 1.5 h. Samples were then re-suspended in 30 l 1PBS, of which 20 l was mounted on a 2% agarose pad for viewing in the FITC and DIC channels of a Zeiss Observer Z1-inverted fluorescence microscope.
(159) Colony-Forming Units (CFU) Plating.
(160) To determine CFU, cells were treated as needed and ten-fold serial dilutions were produced in complete 7H9 medium and plated on Middlebrook 7H10 agar plates (Difco, 262710) containing 10% OADC and 0.05% glycerol (v/v) without antibiotics. CFU were counted after 3 days (for Msmeg) and 14-17 days (for Mtb) of incubation at 37 C. in a humidified incubator.
(161) Purification of C. glutamicum Trehalose Glycolipids
(162) C. glutamicum cultures (200 mL) were incubated with 0.1 mM DMN-Tre or left untreated until stationary phase. Bacterial cell pellets were washed twice with PBS before subjecting to organic extraction. Cell wall glycolipids were isolated by sequentially extracting with 2:1, 1:1, 1:2 MeOH:CH.sub.3Cl mixtures, followed by concentration of organic extracts. Partial purification was achieved by preparative TLC (Analtech, 2020 cm, 1 mm thickness) developed with 65:25:4 CHCl.sub.3:MeOH:H.sub.2O. Purity of glycolipid fractions was monitored by HPTLC (Uniplate HPTLC-GHL, 55 cm, 150 m thickness), imaged with typhoon scanner (Amersham Biosciences, Typhoon 9410) to detect fluorescently labeled glycolipids and stained with 5% H.sub.2SO.sub.4 in MeOH with charring for standard glycolipid visualization.
(163) Validation of DMN-Tre Labeled Glycolipids by Mass Spectrometry
(164) C. glutamicum trehalose glycolipids were isolated as described above. Glycolipid samples were dissolved in 200 L 2:1 CHCl.sub.3:MeOH (HPLC grade) and filtered through a 0.45 m PVDF membrane. Mass spectra were acquired on a Waters Q-tof Premier quadrupole time-of-flight mass spectrometer equipped with a nanoelectrospray ionization (nanoESI) source, located in the QB3/Chemistry Mass Spectrometry Facility at the University of California, Berkeley. Ions were formed in the positive ion mode from pulled borosilicate glass nanoESI tips. Mass spectra were recorded over the range, mass-to-charge ratio (m/z)=100 to 4000. Data acquisition was controlled using MassLynx software (version 4.1, Waters). Tandem mass spectrometry was performed on three precursor ions using collision induced dissociation (CID) fragmentation to show loss of lipid tails from DMN-Tre labeled mycolates.
(165) Axenic MTB Culture Time-Course Experiment
(166) The laboratory strain of Mycobacterium tuberculosis, H37Rv, was grown axenically to an optical density (600 nm) of approximately 0.5 (i.e. exponential growth phase). Five equal volumes of 200 l, assigned to a time-course points at 30 min, 1 h, 2 h, 3 h and overnight, were stained with 1 mM DMN-trehalose at 37 C. Three independent biological replicates for each time-point were prepared. Unlabeled samples of M. tuberculosis served as a control. Following incubation, samples were analyzed by flow cytometry using a CytoFlex (Beckman Coulter). Samples were aliquoted into separate wells of a 96-well, flat-bottomed culture plate. The flow cytometer was calibrated using Daily Fluorophore QC beads (Beckman Coulter). Two gates were constructed using signal detected with the violet laser (KO525A channel). One corresponded to unlabeled M. tuberculosis while the other corresponded to all cells positive for DMN-trehalose. Flow cytometry data was for 50000 events or for 2 min, whichever variable was true first (according to the acquisition setting outlined below). To prevent carry-over, wells containing water separated each sample which was run for 2 min. DMN-trehalose staining was plotted as a percentage of total events.
(167) Synthetic Procedures
(168) All chemical reagents obtained from commercial suppliers were used without further purification. Reversed-phase HPLC was performed on a Varian Pro Star system with a Varian UV-Vis detector model 345 (210, 254 nm) on a Dynamax Microsorb C-18 preparative column (21.4250 mm) at a flow rate of 20 mL/min. NMR spectra were obtained on a Varian INOVA-600 spectrometer at ambient temperature at the Stanford Department of Chemistry NMR Facility.
(169) DMN-Trehalose (3)
(170) 6-Amino trehalose (1) (31 mg, 0.091 mmol) and 4-N,N-dimethylaminonaphthalic anhydride (2) (22 mg, 0.091 mmol, 1 equiv.) were added to a flask along with 2 mL of absolute ethanol. To the mixture was added 20 mg of sodium bicarbonate. The yellow suspension was heated to 85 C. under a nitrogen atmosphere and stirred for 6 h. The now red-orange solution was then concentrated by rotary evaporation and the suspension was dissolved in 10 mL of H.sub.2O, filtered through a plug of cotton, and reconcentrated. The remaining orange residue was purified by reversed-phased HPLC (5-80% MeCN in H.sub.2O) and lyophilized to yield DMN-Tre (3) (22.1 mg, 0.039 mmol, 43%) as a bright orange solid.
(171) .sup.1H NMR (600 MHz, D.sub.2O) 8.21 (d, J=8.5 Hz, 1H), 8.13 (d, J=7.3 Hz, 1H), 7.95 (d, J=8.4 Hz, 1H), 7.46 (t, J=7.9 Hz, 1H), 6.90 (d, J=8.6 Hz, 1H), 4.97 (d, J=3.8 Hz, 1H), 4.53 (d, J=3.7 Hz, 1H), 4.23 (d, J=6.2 Hz, 2H), 4.03 (dt, J=12.3, 6.4 Hz, 1H), 3.79 (t, J=9.4 Hz, 1H), 3.73-3.61 (m, 4H), 3.56 (dd, J=12.1, 5.4 Hz, 1H), 3.44 (t, J=9.5 Hz, 1H), 3.18 (t, J=9.5 Hz, 1H), 3.07 (m, 1H), 3.07 (s, 6H).
(172) .sup.13C NMR (151 MHz, 5% CD.sub.3OD in D.sub.2O) 166.16, 165.34, 157.94, 134.16, 133.31, 132.29, 130.33, 125.19, 124.05, 121.44, 113.21, 111.71, 94.03, 93.86, 93.82, 73.84, 73.62, 73.47, 73.03, 72.08, 71.94, 70.61, 70.19, 61.53, 44.93, 41.80. HRMS (ESI-TOF, m/z): calcd for C.sub.26H.sub.32N.sub.2O.sub.12 [M+Na].sup.+ 587.1847, found 587.1849.
(173) DMN-Glucose (5)
(174) 6-Amino-6-deoxy methyl glucoside (4) (38 mg, 0.20 mmol) and 4-N,N-dimethylaminonaphthalic anhydride (2) (48 mg, 0.20 mmol, 1 equiv.) were added to a flask along with 4 mL of absolute ethanol. To the suspension was added 40 mg of sodium bicarbonate. The orange suspension was then heated to 85 C. under a nitrogen atmosphere and stirred for 6 h. The clear red-orange solution was then concentrated, dissolved in 3 mL of H.sub.2O, and filtered through a plug of cotton. The orange solution was then purified by reversed-phase HPLC (5-80% MeCN in H.sub.2O) and lyophilized to yield DMN-Glc (5) (46.5 mg, 0.12 mmol, 56%) as a bright orange solid.
(175) .sup.1H NMR (600 MHz, D.sub.2O) 8.37-8.24 (m, 1H), 8.21 (t, J=5.9 Hz, 1H), 8.03 (t, J=6.2 Hz, 1H), 7.53 (d, J=4.0 Hz, 1H), 7.03-6.90 (m, 1H), 4.58 (d, J=3.5 Hz, 1H), 4.32 (t, J=11.4 Hz, 1H), 4.23 (d, J=13.7 Hz, 1H), 3.88 (t, J=10.0 Hz, 1H), 3.63 (t, J=9.3 Hz, 1H), 3.57 (dd, J=9.7, 3.8 Hz, 1H), 3.43 (t, J=9.4 Hz, 1H), 3.10 (d, J=2.7 Hz, 6H), 2.90 (s, 3H).
(176) .sup.13C NMR (151 MHz, 5% CD.sub.3OD in D.sub.2O) 166.07, 165.18, 157.79, 134.05, 133.28, 132.14, 130.24, 124.99, 123.76, 121.21, 112.94, 111.29, 99.90, 74.10, 73.84, 72.24, 69.07, 55.20, 44.86, 41.99. HRMS (ESI-TOF, m/z): calcd for C.sub.21H.sub.24N.sub.2O.sub.7 [M+Na].sup.+ 439.1476, found 439.1472.
DOCTRINE OF EQUIVALENTS
(177) This description of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form described, and many modifications and variations are possible considering the teaching above. The embodiments were chosen and described to best explain the principles of the invention and its practical applications. This description will enable others skilled in the art to best utilize and practice the invention in various embodiments and with various modifications that are best suited for a specific need. The scope of the invention is defined by the following claims.