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  • C12Q2304/00—Chemical means of detecting microorganisms

Definitions

• the present invention relates to dioxetane compounds, their use for the detection of presence or absence, quantification and identification of microorganisms including bacteria, bacterial fragments (e.g., LPS, endotoxin), viruses, fungi as well as other pathogens by means of chemiluminescent indication of action of metabolic, reagent or reference enzymes on suitable molecular probes, indication of hydrogen peroxide resulting from enzymatic oxidation of microbial metabolites or nutrients by reagent enzymes or detection of inorganic phosphate playing roles of nutrient, substrate, metabolic product or by-product of action by a reagent enzyme.
• microorganisms including bacteria, bacterial fragments (e.g., LPS, endotoxin), viruses, fungi as well as other pathogens by means of chemiluminescent indication of action of metabolic, reagent or reference enzymes on suitable molecular probes, indication of hydrogen peroxide resulting from enzymatic oxidation of m
• chemiluminescence Due to their superior sensitivity and higher signal-to-noise ratio compared to methods based on fluorescence or coloration, detection methods based on chemiluminescence are particularly favored for the detection of microorganisms, in particular bacteria.
• S/N signal-to-noise ratios
• S/N signal-to-noise ratios
• D-luciferin is a bioluminogenic compound that is oxidized by luciferase (an oxidoreductase) in the presence of molecular oxygen, ATP and magnesium to a metastable intermediate which in turn decays to emit blue-green light.
• D-luciferin can be masked with enzyme labile groups, restricting light emission in the presence of luciferase to situations where also the enzyme acting on the enzyme labile group is present.
• luciferin-6-O-beta-D-galactopyranoside have reported the detection of coliform bacteria with D-luciferin-6-O-beta-D-galactopyranoside as luminogenic substrate (Masuda-Nishimura et al.
• chemiluminescent dioxetane probes suitable for use under aqueous conditions, which are masked with enzyme- labile groups and suitable for the detection of enzyme activities. [0006] However, the successful use of chemiluminescent dioxetane probes in the field of microbiology for the detection of specific microorganisms has not been reported so far.
• chemiluminescent probes for detecting microorganisms under“real-life” conditions is a particularly difficult challenge due to complex interactions between the probe, the enzyme of the microorganism required for removing enzyme-labile protecting group, and the environment of the chemiluminescent reaction (i.e. the medium comprising the microorganisms).
• a suitable chemiluminescent probe for the detection of microorganisms must (i) be non- toxic for the microorganism to be detected, (ii) have a high stability in aqueous medium, (iii) be capable of generating a strong chemiluminescent signal in a given medium, and (iv) be able to reach the site of the enzyme that removes the enzyme- labile protecting group (e.g., the periplasmic space, the outside or inside surface of the inner membrane of gram-negative bacteria, the cytosol etc.).
• the chemiluminescent probes for the detection of microorganism should be inexpensive and convenient to use.
• a chemiluminescent dioxetane probe is suitable for the“real-life” detection of a microorganism (in aqueous media) cannot be derived or predicted from its chemical structure. Even if a probe shows good performance under“laboratory” conditions, it may be unsuitable for“real-life” applications. Thus, extensive experiments are required for determining whether a specific probe is suitable for the detection of microorganisms under“real- life” conditions.
• the luciferase-luciferin system is the only efficient“real-life” (bio)lumiscencent means available for the detection of microorganisms.
• this system has a number of drawbacks.
• it is a complex multi-component system (standard composition: 1. pro- luciferin enzyme substrate (e.g., luciferin-beta-D-galactopyranoside), 2. luciferase, 3. bovine serum albumin, 4. ATP, 5. EDTA, 6. D/L-cysteine, 7. MgSO 4 , 8. sodium pyrophosphate), which is generally more complex than a one-component system.
• the luciferase-luciferin system requires the use of luciferase, which makes its use quite costly and limits shelf-life due to the notorious instability of commercially available luciferase.
• chemiluminescence is preferred over bioluminescence as it shows a sensitivity that is about 10 to 100 times higher than that achieved with bioluminescence, in particular the bioluminescent luciferase-luciferin system, and, particularly important, allows for a remarkably simple and straightforward application, in particular compared to very complex bioluminescent luciferase-luciferin system.
• OBJECT OF THE INVENTION it is the object of the present invention to provide probes as well as methods for the detection of presence or absence, quantification and identification of microorganisms including bacteria, bacterial fragments (e.g., LPS, endotoxin), viruses, fungi as well as other pathogens that overcome the disadvantages of commonly applied luciferase-luciferin systems, in particular have a significantly increased sensitivity and are easier to use than a luciferase-luciferin based system.
• chemiluminescent probes should be provided that can be used to detect the presence or absence, quantification and identification of microorganisms including bacteria, bacterial fragments (e.g., LPS, endotoxin), viruses, fungi as well as other pathogens by means of chemiluminescent indication, e.g. chemiluminescent indication of action of metabolic, reagent or reference enzymes on suitable molecular probes, indication of hydrogen peroxide resulting from enzymatic oxidation of microbial metabolites or nutrients by reagent enzymes or detection of inorganic phosphate playing roles of nutrient, substrate, metabolic product or by-product of action by a reagent enzyme.
• chemiluminescent indication e.g. chemiluminescent indication of action of metabolic, reagent or reference enzymes on suitable molecular probes, indication of hydrogen peroxide resulting from enzymatic oxidation of microbial metabolites or nutrients by reagent enzymes or detection of inorganic phosphat
• the dioxetane compounds according to the present invention are highly efficient probes that enable the detection of presence or absence, quantification and identification of microorganisms including bacteria, bacterial fragments (e.g., LPS, endotoxin), viruses, fungi as well as other pathogens in a more sensitive and simpler way as the commonly applied luciferase-luciferin system.
• the present invention provides a compound of Formula I
• R4 is selected from the group consisting of -OH, -O- Kat+ , optionally substituted C1-C4 alkyl, optionally substituted C2-C4 heteroalkyl, optionally substituted C2-C4 alkenyl, optionally substituted C2-C4 heteroalkenyl, optionally substituted C2-C4 alkynyl, optionally substituted C2-C4 heteroalkynyl, optionally substituted C5-C6 aryl, optionally substituted C5-C6 heteroaryl, optionally substituted C6-C10 aralykl, and optionally substituted C6-C10 heteroaralkyl,
• R5 is selected from the group consisting of -H, optionally substituted C1-C4 alkyl, optionally substituted C2-C4 heteroalkyl, optionally substituted C2-C4 alkenyl, optionally substituted C2-C4 heteroalkenyl, optionally substituted C2-C4 alkynyl, optionally substituted C2-C4 heteroalkynyl, optionally substituted C5-C6 aryl, optionally substituted C5-C6 heteroaryl, optionally substituted C6-C10 aralykl, and optionally substituted C6-C10 heteroaralkyl, or
• Z'' is selected from F, Cl, Br, I, preferably Z'' is F;
• Kat+ is an organic or anorganic cation, preferably an alkali metal cation
• L is a self-immolative linker group which, upon acting of an analyte on the analyte- responsive group R1 , is released from the remainder part of the compound of Formula I, wherein L is optionally functionalized with a peptide, preferably a cell penetrating peptide, an endolysine or a protein;
• R1 is an enzyme-labile group
• n is 1 and m is 1 or n is 0 and m is 1, and if R1 is -B(Z)(Z') or , n and m are both 0 or both 1;
• RA and RC are independently selected from H, F, Cl, Br, I, CF3 and R2-Q-, preferably from H, Cl and R2-Q, or one of RA and RC together with RB forms an optionally substituted cyclic or heterocyclic structure that extends the pi-system of the central aromatic ring and the other one is H or R2-Q-; provided that RA and RC are not both H; RB is H or forms together with one of RA and RC said optionally substituted cyclic or heterocyclic structure; Q is group comprising a pi-system that is conjugated with the pi-system of the central aromatic ring of the compound of Formula I; R2 is a group selected from cyano, nitro, sulfoxide, sulfon, optionally substituted aryl, optionally substituted alkenyl, , carbonyl, carbonyl having the structure , amide, amide having the structure ,
• Y is H, an optionally substituted C1-C12 alkyl or an alkali metal ion, wherein Y’ and Y’’ are independently selected from H, and optionally substituted C1- C12 alkyl or together with the nitrogen atom form an optionally substituted heterocyclic structure, preferably an optionally substituted maleimide group; and R3 is H, F, Cl, Br, I, CF3 or R2-Q-; provided that at least one of RA and RC, preferably RA , is H, F, Cl, Br, I or CF 3 , preferably Cl, if R3 is R2-Q-; RD is selected from a linear or branched C1-C18 alkyl or C3-C7 cycloalkyl;
• RE and RF are independently selected from a branched C3-C18 alkyl or C3-C7 cycloalkyl, or RE and RF together with the carbon atom to which they are attached form an optionally substituted fused, spiro or polycyclic ring.
• the present invention is directed to the use of a compound of Formula I for the detection of a target analyte (e.g., hydrogen peroxide), a target microorganism or a target metabolite, preferably a microorganism, e.g. a bacterium and to a method for the detection of a target analyte, a target microorganism or a target metabolite.
• a target analyte e.g., hydrogen peroxide
• a target microorganism or a target metabolite preferably a microorganism, e.g. a bacterium
• a target analyte e.g., hydrogen peroxide
• a target microorganism or a target metabolite e.g. a bacterium
• the present invention is directed to the use of a compound of Formula I for the detection of growth substrates, nutrients, and/or metabolites by enzymatic oxidation of said growth substrates, nutrients, and metabolites and to a method for the detection of growth substrates, nutrients, and/or metabolites by enzymatic oxidation of said growth substrates, nutrients, and metabolites.
• the present invention is directed to the use of a compound of Formula I for the detection of bacterial endotoxins using limulus factor C and to a method for the detection of bacterial endotoxins using limulus factor C.
• the present invention is directed to the use of a compound of Formula I for testing of pasteurization of dairy products and to a method of testing of pasteurization of dairy products.
• the present invention is directed to the use of a compound of Formula I for testing of antibiotic resistance in microorganisms and to a method for testing of antibiotic resistance in microorganisms.
• the present invention is directed to the use of a compound of Formula I for the detection of inorganic phosphate and to a method for the detection of inorganic phosphate.
• the present invention is directed to the use of a compound of Formula I for monitoring of a sterilization process, in particular through detection of alpha-D-glucosidase activity of the indicator microorganism Geobacillus stearothermophilus, and to a method for monitoring of a sterilization process, in particular by detecting alpha-D-glucosidase activity of the indicator microorganism Geobacillus stearothermophilus.
• the present invention is directed to the use of a compound of Formula I for endpoint and online detection of antibiotic resistance of bacteria and for antibiotic susceptibility testing as well as to a method for endpoint and online detection of antibiotic resistance of bacteria and for antibiotic susceptibility testing.
• the present invention is based on the surprising finding that, although a luciferase-luciferin-based system shows a number of drawbacks (as set out above), such a system is currently the only“real-life” bio- or chemiluminescent system available for the detection of microorganisms.
• the inventors of the present invention have surprisingly found that dioxetane compounds of Formula I are highly efficient probes for detecting microorganisms.
• dioxetane compounds of Formula I are chemiluminescent even in aqueous media and show a remarkably high sensitivity when used for the detection of microorganisms, which is significantly higher than that of a commonly applied luciferase-luciferin system.
• important properties of the inventive compounds e.g. the membrane-permeability and/or solubility, can be modified by varying the substituent R2 , if present.
• the dioxetane compounds of Formula I are stable in aqueous media and in particular stable in microbial growth media.
• dioxetane- based compounds of Formula I allow for an easy, straight-forward, cheap and reliable detection of microorganisms.
• the compounds can simply be added to the medium comprising the microorganism as they are without the need for any further compounds.
• dioxetane compounds of Formula I are superior to the commonly-used luciferase-luciferin-based system.
• the present invention relates to a compound of Formula I
• R4 is selected from the group consisting of -OH, -O- Kat+ , optionally substituted C1-C4 alkyl, optionally substituted C2-C4 heteroalkyl, optionally substituted C2-C4 alkenyl, optionally substituted C2-C4 heteroalkenyl, optionally substituted C2-C4 alkynyl, optionally substituted C2-C4 heteroalkynyl, optionally substituted C5-C6 aryl, optionally substituted C5-C6 heteroaryl, optionally substituted C6-C10 aralykl, and optionally substituted C6-C10 heteroaralkyl,
• R5 is selected from the group consisting of -H, optionally substituted C1-C4 alkyl, optionally substituted C2-C4 heteroalkyl, optionally substituted C2-C4 alkenyl, optionally substituted C2-C4 heteroalkenyl, optionally substituted C2-C4 alkynyl, optionally substituted C2-C4 heteroalkynyl, optionally substituted C5-C6 aryl, optionally substituted C5-C6 heteroaryl, optionally substituted C6-C10 aralykl, and optionally substituted C6-C10 heteroaralkyl, or
• Z'' is selected from F, Cl, Br, I, preferably Z'' is F;
• Kat+ is an organic or anorganic cation, preferably an alkali metal cation;
• L is a self-immolative linker group which, upon acting of an analyte on the analyte- responsive group R1 , is released from the remainder part of the compound of Formula I, wherein L is optionally functionalized with a peptide, preferably a cell penetrating peptide, an endolysine or a protein;
• R1 is an enzyme-labile group
• n is 1 and m is 1 or n is 0 and m is 1, and 1;
• RA and RC are independently selected from H, F, Cl, Br, CF 3 and R2-Q-, preferably from H, Cl and R2-Q-, or one of RA and RC together with RB forms an optionally substituted cyclic or heterocyclic structure that extends the pi-system of the central aromatic ring and the other one is H or R2-Q-; provided that RA and RC are not both H; RB is H or forms together with one of RA and RC said optionally substituted cyclic or heterocyclic structure; Q is group comprising a pi-system that is conjugated with the pi-system of the central aromatic ring of the compound of Formula I; R2 is a group selected from cyano, nitro, sulfoxide, sulfon, optionally substituted aryl, optionally substituted alkenyl, , carbonyl, carbonyl having the structure
• Y is H, an optionally substituted C1-C12 alkyl or an alkali metal ion, wherein Y’ and Y’’ are independently selected from H, and optionally substituted C1- C12 alkyl or together with the nitrogen atom form an optionally substituted heterocyclic structure, preferably an optionally substituted maleimide group; and R3 is H, F, Cl, Br, I, CF3 or R2-Q-; provided that at least one of RA and RC, preferably RA , is H, F, Cl, Br, I or CF 3 , preferably Cl, if R3 is R2-Q-; RD is selected from a linear or branched C1-C18 alkyl or C3-C7 cycloalkyl;
• RE and RF are independently selected from a branched C3-C18 alkyl or C3-C7 cycloalkyl, or RE and RF together with the carbon atom to which they are attached form an optionally substituted fused, spiro or polycyclic ring.
• alkyl refers to a linear or branched hydrocarbon radical and includes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, n-pentyl and so on.
• the term“C 1 -C 12 alkyl” refers to an“alkyl” having 1 to 12 carbon atoms.
• alkenyl refers to a linear or branched hydrocarbon radical having one or more carbon-carbon double bonds.
• alkynyl refers to a linear or branched hydrocarbon radical having one or more carbon-carbon triple bonds.
• heteroalkyl refers to the corresponding hydrocarbyl (alkyl, alkenyl, and alkynyl) group, which contain one or more O, S or N heteroatoms or combinations thereof within the backbone residue; thus, at least one carbon atom of a corresponding alkyl, alkenyl, or alkynyl group is replaced by one of the specified heteroatoms to form a heteroalkyl, heteroalkenyl, or heteroalkynyl group.
• aryl refers to an aromatic carbocyclic group consisting of a single ring or condensed multiple rings such as, but not limited to, phenyl, naphthyl, phenanthryl, and biphenyl.
• The“aryl” may be substituted or unsubstituted.
• heteroaryl refers to an aromatic group containing at least one heteroatom (i.e. an atom different from carbon or hydrogen, e.g. N, S, O, P, Se, Te, preferably N, S, O, P) as a ring member.
• dioxetane compound refers to a compound
• analyte-responsive group refers to a group that can be removed (at least in part) or modified by means of a specific analyte, wherein removal or modification is such that luminescence is triggered.
• n 1 (i.e., linker group L is present)
• the action of an analyte on the analyte-responsive group transforms the analyte-responsive group R1 into a -OH or -NH 2 moiety (when L comprises group X, the moiety“X-R1” is preferably transformed to a -OH or -NH2 moiety) such that elimination of the linker group“L”, e.g.1,6-elimination, is triggered.
• n 0 (i.e., linker group L is not present)
• the action of an analyte on the analyte-responsive group R1 transforms the analyte-responsive group R1 to -OH or another acidic group (when m is 1, the moiety“O-R1“ is transformed to - OH or another acidic group).
• the term“enzyme-labile group”, as used herein, refers to a group that can be removed (at least in part) or modified by means of a specific enzyme.
• target analyte, a target microorganism or a target metabolite refers to an analyte, microorganism or metabolite that is to be detected by means of a compound Formula I.
• RA and RC are independently selected from H and R2-Q-, or one of RA and RC together with RB forms an optionally substituted cyclic or heterocyclic structure that extends the pi-system of the central aromatic ring and the other one is H or R2-Q-, and R3 is H, F, Cl, Br, I.
• one of RA and RC together with RB forms an optionally substituted cyclic or heterocyclic structure that extends the pi- system of the central aromatic ring and the other one is H or R2-Q-.
• one of RA and RC is R2-Q- and the other one is H.
• RA is R2-Q- and RC is H.
• RC is R2-Q- and RA is H.
• Q is a group comprising a pi-system that is conjugated with the pi-system of the central aromatic ring of the compound of Formula I.
• group Q (potentially together with group R2 attached thereto) can be used to tailor design the luminescence of the compound of Formula I.
• group Q may influence the emission wavelength, the kinetics of emission (flash emission vs. glow emission) and the quantum yield.
• Q is selected from the group
• q and r are selected from the group consisting of 1, 2, 3, 4, 5, and 6, preferably q and r are 1.
• one of RA and RC together with RB forms an optionally substituted cyclic or heterocyclic structure that extends the pi- system of the central aromatic ring.
• the other one of RA and RC is R2-Q or H, preferably H.“Extends the pi-system of the central aromatic ring” means that the optionally substituted cyclic or heterocyclic structure comprises a pi-system that conjugates with the pi-system of the central aromatic ring.
• the optionally substituted cyclic or heterocyclic structure formed by one of RA and RC together with RB is a 6-membered ring. More preferably, said 6-
• membered ring is selected from the group consisting of , ,
• RG is selected from a substituted or unsubstituted C1-C12 alkyl and wherein R2 is as defined above and below. It is understood that the above moieties may be connected to RA and RB or RC and RB in any way. This means that a compound of Formula I, wherein RA together with RB
• R3 substituent R3
• RE and RF together with the carbon atom to which they are attached form an optionally substituted fused, spiro or bridged cyclic or polycyclic ring.
• said optionally substituted fused, spiro or bridged cyclic or polycyclic ring is selected from an optionally substituted propellane; an optionally substituted bicyclus defined by the formula [A.B.1]pentane, [A.B.1]hexane, [A.B.1]heptane, [A.B.1]octane, [A.B.1]nonane, [A.B.1]decane, [A.B.1]undecane, [A.B.1]dodecane, wherein A and B are independently selected from 1, 2, 3, 4, and 5; or optionally substituted adamantine. More preferably, RE and RF together with the carbon atom to which they are attached preferably form an optionally substituted
• RD is methyl, ethyl, or isopropyl.
• RD is methyl.
• Particularly preferred compounds are selected from the group consisting of
• the compound of Formula I is represented by Formula I'
• R1 is an enzyme-labile group.
• R1 is selected from the group consisting of acetyl, butyryl, octanoyl, nonanoyl, myo-inositol phosphoryl, phosphoryl, an amino acidyl group, L- pyroglutamic acidyl, a di-peptidyl group, a tri-peptidyl group, beta-D- galactopyranosidyl, alpha-D-galactopyranosidyl, alpha-D-glucopyranosidyl, beta-D- glucopyranosidyl, beta-D-glucuronyl, beta-D-glucuronyl sodium salt, n-acetyl-beta-D- galactosaminidyl, N-acetylneuraminidyl, cellobiosidyl, alpha-D-ribofuranosidy
• cephalosporins of generation 1 to 5 are selected from cefacteril, cefradin, cefroxadin, cefaloglycin, cefaclor, cefalexin, cefadroxil, cefatrizin, cefazedon, cefapirin, ceftezol, cefazolin, cefezaflur, cefalotin, cefaloridin, cefalonium, wherein the carbon atom next to the carbon atom to which the carboxylic acid group is attached is used to bind the cephalosporin the compound of Formula I,
• Preferred carbapenems are selected from , ,
• a particularly preferred carbapenem is the moiety , preferably
• R1 is Ac-QLQ-, Ac-FQLQ-, Ac- EFQLQ-, or Ac-DEFQLQ-. These groups are suitable for the detection of norovirus.
• R1 is a group having the formula -B(Z)(Z') or , wherein preferably at least one of Z and Z' is OR5, more preferably both of Z and Z' are OR5, or wherein the group having the formula -B(Z)(Z') is preferably selected from the group consisting of -B(OH) 2 ,
• the linker group“L” has several advantages that were not known before the present invention was made. One the one hand, it leads to a better hydrolysis- stability of the compound of Formula I, which is particularly important, because the compound of Formula I is preferably used in aqueous media. On the other hand, it leads to a good availability of group R1 by sterically distancing the group R1 from the remainder part of the compound of Formula I. Better (hydrolysis) stability leads to a less unspecific hydrolysis, thereby to a lower background, which leads to a better signal to noise ratio and, consequently, to a higher sensitivity.
• L is a self-immolative group that, upon acting of an analyte on the analyte-responsive group R1 (thereby leading to an at least partial removal or modification of the analyte-responsive group R1), is released from the remainder part of the compound of Formula I.
• L is the preferred moiety for attachment of a peptide (preferably a cell penetrating peptide), an endolysine or a protein to the compound of Formula I.
• a peptide preferably a cell penetrating peptide
• functionalization of L with these groups does not quench luminescence of a compound of Formula I.
• the linker L is generally cleaved off from the remainder part of the compound of Formula I before luminescence is triggered.
• L is functionalized with a peptide (preferably a cell penetrating peptide), an endolysine or a protein.
• L is not functionalized with a peptide (preferably a cell penetrating peptide), an endolysine or a protein.
• L is selected from the group consisting ,
• each of these linkers may be functionalized with a peptide, preferably a cell penetrating peptide, an endolysine or a protein and wherein
• X is -O-, -N+(RG) 2 -, preferably -N+(CH 3 ) 2 -, or -NH-, wherein X is absent if R1 is -B(Z)(Z') or -NO2, X' is selected from S, O, NH, and NRG; and X is connected to R1.
• L is N
• X is -N+(CH 3 ) 2 - when R1 is preferably
• X is -O-. According to another preferred embodiment, X is -NH-. [0064] According to a preferred embodiment, n is 1 and m is 1. According to another preferred embodiment, n is 0 and m is 1. [0065] According to a preferred embodiment, R2 is a water-solubilizing group.
• R2 is selected from the group consisting of cyano and , wherein Y is H or an optionally substituted C1-C12 alkyl or an alkali metal ion, wherein the alkali metal ion is preferably sodium or potassium and the optionally substituted C 1 -C 12 alkyl is preferably methyl, ethyl propyl, isopropyl, butyl, isobutyl, or tert-butyl.
• R2 is .
• Y is -H, an optionally substituted C 1 -C 12 alkyl or an alkali metal ion, wherein the alkali metal ion is preferably sodium or potassium and the optionally substituted C 1 -C 12 alkyl is preferably methyl, ethyl propyl, isopropyl, butyl, isobutyl, or tert-butyl.
• Y is -H, or an optionally substituted C 1 -C 12 alkyl, wherein the C 1 -C 12 alkyl is preferably methyl, ethyl propyl, isopropyl, butyl, isobutyl, or tert-butyl. More preferably, Y is -H or methyl. Even more preferably, R2 is -COOH or -COO- . [0068] According to a preferred embodiment, R3 is selected from the group consisting H and Cl, preferably Cl. [0069] Preferred compounds of Formula I are selected from the group consisting of compounds of Formula II, IIa, III, IIIa, IV, IVa, V, Va, VI, VIa, VIb, VIc, VII and VIIa:
• Y is H, an optionally substituted C1-C12 alkyl or an alkali metal ion. Particularly preferred compounds are those where Y is H.
• the compound of Formula II, in particular IIa has been proven to be particularly suitable for the detection of Salmonella, in particular Salmonella enterica.
• the compound of Formula III, in particular IIIb has been proven to be particularly suitable for the detection of Listeria in particular Listeria monocytogenes.
• the compound of Formula IV, in particular IVa has been proven to be particularly suitable for the detection of Staphylococcus aureus.
• the compound of Formula V, in particular Vb has been proven to be particularly suitable for the detection of coliform and E. coli.
• the compound of Formula VI in particular VIc, has been proven to be particularly suitable for the detection of H 2 O 2 , irrespective of its origin.
• the compounds of Formula VII and VIII preferably VIIb and VIIIa, have been shown to be particularly suitable for distinguishing carbapenem resistant bacteria from carbapenem sensitive bacteria.
• group R1 it has been found that when group R1 is present, the compound of Formula I is stable even in aqueous media and no photons are emitted.
• R1 Removal (at least in part) or modification of the R1 group by means of an interaction with an analyte generates an unstable species, which decomposes through a chemiexcitation process to yield in an excited intermediate, which in turn decays to its ground state through an emission of a photon.
• the analyte-responsive group R1 restricts light emission to situations where also an analyte acting on the analyte-responsive group R1 is present.
• R1 is an enzyme-labile group
• the compound of Formula I is suitable for the detection of an enzyme and, consequently, for the detection of a microorganism expressing this enzyme.
• said enzyme- labile group R1 Upon contact with the enzyme, said enzyme- labile group R1 is removed (at least in part) or modified, whereupon the self- immolative linker, if present, is removed from the remainder part of the compound of Formula I and an unstable species is formed, which then decomposes through a chemiexcitation process to yield in an excited intermediate, which in turn decays to its ground state through an emission of a photon.
• R1 is an enzyme-labile group
• R1 is responsive to only one specific enzyme, such that a microorganism expressing this enzyme may be detected in the presence of other microorganisms not expressing this enzyme. In this way, it is for example possible to specifically detect Salmonella (e.g.
• Exemplary analyte-responsive groups R1 that (among others) may be used in the present invention and the respective target analytes, the target microorganisms and target metabolites are shown in Table 1. Further analyte-responsive groups R1 , the respective analytes, target analytes/target microorganisms/target metabolites are discernible from, e.g., Orenga et al, Journal of Microbiological Methods , 79, 2009, 139-155; and Varadi et al., Che. Soc. Rev., 2017, 46, 4818-4832. [0080] Table 1
• amino acidyl refers to an amino acid moiety that is bound to the remainder part of the dioxetane compound by means of its carboxylic acid group.
• an amino acid comprises more than one carboxylic acid group
• each of said carboxylic acid groups may bind the amino acid to the remainder part of the dioxetane compound.
• an amino acid comprises more than one carboxylic acid group
• it is bound to the remainder part of the dioxetane compound by means of its alpha-carboxylic acid group.
• R1 is an amino acidyl group or a di- or tri-peptidyl group and X is -NH-, the carboxylic acid group of the amino acidyl group or the di- or tri-peptidyl group (which is involved in bond formation), together with the -NH- group forms an amide group (-CONH-).
• Preferred amino acidyl groups are alanyl (A-), preferably L-alanyl, pyroglutamic acidyl, preferably L-pyroglutamic acidyl, argininyl (R-), asparaginyl (N-), aspartic acidyl (D-), cysteinyl (C-), glutaminyl (Q-), glutamic acidyl (E-), glycinyl (G-), histidinyl (H-), isoleucinyl (I-), leucinyl (L-), lysinyl (K-), methioninyl (M-), phenylalanyl (F-), prolinyl (P-), serinyl (S-), threoninyl (T-), tryptophanyl (W-), tyrosinyl (Y-), and valinyl (V-).
• Particularly preferred amino acidyl groups are L-alanyl, L-pyroglutamic acidyl, L-leucinyl, or b-alanyl.
• Preferred tri-peptidyl groups are Boc-Val-Pro-Argininyl, Boc-Asp(OBzl)-Pro- Argininyl, and SucOMe-Arg-Pro-Tyrosinyl (SucOMe-RPY-).
• R1 is myo-inositol phosphoryl
• the compound of Formula I is particularly suitable for detecting a microorganism expressing Phosphatidylinositol- specific phospholipase C (PI-PLC), e.g.
• the compound of Formula I is particularly suitable for detecting a microorganism expressing C8 esterase, e.g. Salmonella, in particular Salmonella enterica.
• the compound of Formula I is particularly suitable for detecting a microorganism expressing a phosphatase, e.g. S. aureus, which is a major carrier of antibiotic resistance.
• R1 is an amino acidyl group, preferably L-alanyl or L-pyroglutamic acidyl, a di-peptidyl group or a tri-peptidyl group (in this case X is preferably -NH-).
• R1 is an amino acidyl group.
• R1 is myo-inositol phosphoryl,
• Particularly preferred compounds are compounds of Formula I, wherein the substituents and variables are defined as follows (the other substituents are defined as set out above) (see Table A; If L is present, stands for
• the present invention relates to the use of a compound of Formula I as described in the first aspect for the detection of a target analyte (e.g. hydrogen peroxide) / target microorganism / target metabolite (irrespective of its origin). More preferably, the present invention relates to the use of a compound of Formula I as described in the first aspect for the detection of a target microorganism, more preferably a pathogenic microorganism, even more preferably, a bacterium, virus or fungi.
• a target analyte e.g. hydrogen peroxide
• target microorganism e.g. hydrogen peroxide
• target metabolite e.g. hydrogen peroxide
• the present invention relates to the use of a compound of Formula I as described in the first aspect for the detection of a target microorganism, more preferably a pathogenic microorganism, even more preferably, a bacterium, virus or fungi.
• the present invention relates to the use of a compound of Formula I as described in the first aspect for the detection of presence or absence, quantification and identification of microorganisms including bacteria, bacterial fragments (e.g., LPS, endotoxin), viruses, fungi as well as other pathogens.
• microorganisms including bacteria, bacterial fragments (e.g., LPS, endotoxin), viruses, fungi as well as other pathogens.
• the present invention relates to the use of a compound of Formula I as described in the first aspect for the detection of presence or absence, quantification and identification of microorganisms including bacteria, bacterial fragments (e.g., LPS, endotoxin), viruses, fungi as well as other pathogens by means of chemiluminescent indication of action of metabolic, reagent or reference enzymes on suitable molecular probes, indication of hydrogen peroxide resulting from enzymatic oxidation of microbial metabolites or nutrients by reagent enzymes or detection of inorganic phosphate playing roles of nutrient, substrate, metabolic product or by-product of action by a reagent enzyme.
• microorganisms including bacteria, bacterial fragments (e.g., LPS, endotoxin), viruses, fungi as well as other pathogens by means of chemiluminescent indication of action of metabolic, reagent or reference enzymes on suitable molecular probes, indication of hydrogen peroxide resulting from enzymatic oxid
• the microorganism is selected from the group consisting of Salmonella; Salmonella enterica; Listeria, preferably, Listeria monocytogenes; S. aureus; E. coli; carbapenem-resistant bacteria, preferably Pseudomonas aeruginosa, and Klebsiella pneumonia; Campylobacter jejuni; C. coli; C. lari; Bacillus; Staphylococcus; Clostridium; Mycobacterium tuberculosis; Clostridium perfringens; S.
• the microorganism is selected from the group consisting of Salmonella, Salmonella enterica, Listeria,
• the compound of Formula I allows for a remarkably easy, straight-forward and reliable detection of microorganisms, because the compound of Formula I can simply be added as it is to a microorganism-containing medium without the need for any further compounds or for an additional preparation of the medium.
• This is a huge advantage over the commonly applied luciferase- luciferin system, which requires the use of multiple compounds, one of which, i.e. luciferase, is quite costly and limits shelf-life due to notorious instability.
• the compound of Formula I is stable in aqueous media and has a sensitivity that is significantly higher than that of a luciferase-luciferin system. (see Examples 9 and 10).
• the compound of Formula I can be added to the microorganism (one or more), in particular to a microorganism- containing medium (preferably aqueous), in solid form or in solution.
• a microorganism- containing medium preferably aqueous
• the compound of Formula I in solution preferably DMSO solution, is preferred for the following reason: Due to the high sensitivity (significantly higher than that of a luciferase-luciferin system), only a very small amount of the compound of Formula I is required.
• the compound of Formula I is used in an amount of less than 0.1 ⁇ g, preferably less than 0.09 ⁇ g, more preferably less than 0.08 ⁇ g, even more preferably less than 0.07 ⁇ g, even more preferably less than 0.065 ⁇ g, even more preferably 0.04 to 0.06 ⁇ g, even more preferably 0.045 to 0.055 ⁇ g, most preferably about 0.05 ⁇ g.
• using the compound of Formula I in solution, particularly DMSO solution allows for an easy determination of the correct quantity added to the microorganism-containing medium by means of aliquotation from a stock solution of a known concentration.
• the compound of Formula I is highly stable in DMSO solution (several months at room temperature and years at 4°C).
• the compound of Formula I is used for the detection of a target analyte (e.g. hydrogen peroxide) / target microorganism / target metabolite, preferably a microorganism, wherein the group R1 is responsive to an analyte, in particular an enzyme expressed by said microorganism. Whether group R1 is responsive to a specific analyte, e.g.
• the microorganism is Salmonella, preferably Salmonella enterica, Listeria, preferably Listeria monocytogenes, S. aureus or E. coli.
• the microorganism is Salmonella, preferably Salmonella enterica, and the compound is a compound of Formula II:
• Y is -H, an optionally substituted C 1 -C 12 alkyl or an alkali metal ion, wherein the alkali metal ion is preferably sodium or potassium and the optionally substituted C 1 -C 12 alkyl is preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl.
• Y is -H, or an optionally substituted C1-C12 alkyl, wherein the optionally substituted C 1 -C 12 alkyl is preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl. More preferably, Y is -H or methyl.
• a compound of Formula IIa is used.
• the microorganism is Salmonella, preferably Salmonella enterica, and the compound is a compound of Formula IIa.
• the microorganism is Listeria, preferably Listeria monocytogenes
• the compound is a compound of Formula III: (Formula III), wherein Y is -H, an optionally substituted C1-C12 alkyl or an alkali metal ion, wherein the alkali metal ion is preferably sodium or potassium and the optionally substituted C 1 -C 12 alkyl is preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl.
• Y is -H, or an optionally substituted C 1 -C 12 alkyl, wherein the optionally substituted C 1 -C 12 alkyl is preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl. More preferably, Y is -H or methyl.
• a compound of Formula IIIa or IIIb, more preferably IIIb is used.
• the microorganism is Listeria, preferably Listeria monocytogenes, and the compound is a compound of Formula IIIb.
• the microorganism is S. aureus and the compound is a compound of Formula IV:
• Y is -H, an optionally substituted C 1 -C 12 alkyl or an alkali metal ion, wherein the alkali metal ion is preferably sodium or potassium and the optionally substituted C 1 -C 12 alkyl is preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl.
• Y is -H, or an optionally substituted C 1 -C 12 alkyl, wherein the optionally substituted C 1 -C 12 alkyl is preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl. More preferably, Y is -H or methyl.
• a compound of Formula IVa is used.
• the microorganism is S. aureus and the compound is a compound of Formula IVa.
• the microorganism is E. coli and the compound is a compound of Formula V:
• Y is -H, or an optionally substituted C1-C12 alkyl, wherein the optionally substituted C 1 -C 12 alkyl is preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl. More preferably, Y is -H or methyl.
• a compound of Formula Va or Vb more preferably Vb, is used.
• the microorganism is E. coli and the compound is a compound of Formula Vb.
• the target analyte / target microorganism / target metabolite is hydrogen peroxide and the compound of Formula I is a compound of Formula VI, VIa, VIb, or VIc.
• growth substrates and metabolites such as glucose can be detected directly in supernatants of microbial cultures by chemiluminescence when using a compound of Formula I, wherein R1 is -B(Z)(Z’) as defined in the first aspect and the other substituents are as defined in the first aspect (preferably a compound of Formula VI, VIa, VIb, or VIc), in combination with suitable hydrogen peroxide-releasing enzymes (e.g. oxidases).
• a compound of Formula VI in particular a compound of Formula VIa, VIb, or VIc, most preferably VIc, or related compounds are used.
• the microorganism are carbapenem-resistant bacteria, e.g., Pseudomonas aeruginosa or Klebsiella pneumonia, and the compound is a compound of Formula VII or VIII
• Y is -H, or an optionally substituted C 1 -C 12 alkyl, wherein the optionally substituted C 1 -C 12 alkyl is preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl. More preferably, Y is -H or methyl.
• a compound of Formula VIIa, VIIb, or VIIIa, more preferably VIIb or VIIIa, even more preferably VIIIa is used.
• the microorganisms are carbapenem-resistant Pseudomonas aeruginosa and Klebsiella pneumonia and the compound is a compound of Formula VIIb or VIIIa.
• target analytes / target microorganisms / target metabolites as well as specific compounds of Formula I, in particular groups R1 , suitable for their detection are discernible from the first aspect of the invention.
• the present invention relates to a method for the detection of a target analyte (e.g. hydrogen peroxide) / target microorganism / target metabolite.
• a target analyte e.g. hydrogen peroxide
• the present invention relates to a method for the detection of a target microorganism.
• the present invention relates to a method for the detection of presence or absence, quantification and identification of microorganisms including bacteria, bacterial fragments (e.g., LPS, endotoxin), viruses, fungi as well as other pathogens.
• the present invention relates to a method for the detection of presence or absence, quantification and identification of microorganisms including bacteria, bacterial fragments (e.g., LPS, endotoxin), viruses, fungi as well as other pathogens by means of chemiluminescent indication of action of metabolic, reagent or reference enzymes on suitable molecular probes, indication of hydrogen peroxide resulting from enzymatic oxidation of microbial metabolites or nutrients by reagent enzymes or detection of inorganic phosphate playing roles of nutrient, substrate, metabolic product or by-product of action by a reagent enzyme.
• the method comprises the steps of a) providing a medium comprising one or more target analytes (e.g. hydrogen peroxide) / target microorganisms / target metabolites, b) adding a compound of Formula I as described in the first embodiment to the medium so that the compound of Formula I emits light, and c) detecting the emitted light.
• step b which is connected to a linker by means of a -N+(CH 3 ) 2 - moiety (see above) then also an oxidizing agent (preferably hydrogen peroxide) is preferably added in step b).
• an oxidizing agent preferably hydrogen peroxide
• opening of the beta-lactam ring by means of a carbapenemase does not directly lead to the generation of an emissive species. Rather, oxidation of the sulfide moiety is required to initiate a Hofmann elimination reaction which finally leads to self-immolation of the linker and, thus, to the generation of an emissive species.
• the method comprises an additional step of lysis, which may be carried out between steps a) and b) or in step b) together with or after adding a compound of Formula I to the medium.
• a compound of Formula I such as ethanol or another suitable solvent mixture (preferably in an amount of 15%) may be added to the medium.
• intracellular enzymes are released into the medium and can be detected by means of a compound of Formula I.
• a selective lysis reagent such as phages, peptides, proteins (in particular endolysins and derivatives thereof) may be added to the medium.
• Said selective lysis reagents lead to the release of intracellular enzymes of only specific cells (i.e., cells that are responsive to a respective selective lysis reagent).
• said selective lysis reagents represent, in addition to the detected enzyme, a further selection criterion, which prevents false-positive results and increases specificity. This applies, in principle, also to all further aspects disclosed herein.
• a further example of increasing specificity is the use of antibody-based capturing methods. For example, antibody-functionalized magnetic beads may be added after steps a) and b). Thus, specific cells may be collected/separated from other cells, which increases specificity.
• the medium is an aqueous medium.
• the target analyte / target microorganism / target metabolite is a microorganism.
• the microorganism is a microorganism disclosed in Table 1.
• the microorganism is selected from the group consisting of Salmonella; Salmonella enterica; Listeria, preferably, Listeria monocytogenes; S. aureus; E.
• coli carbapenem-resistant bacteria, preferably Pseudomonas aeruginosa, and Klebsiella pneumonia; Campylobacter jejuni; C. coli; C. lari; Bacillus; Staphylococcus; Clostridium; Mycobacterium tuberculosis; Clostridium perfringens; S.
• agalactiae Candida spp.; Gram negative bacteria, yeast, molds, Pseudomonas aeruginosa, Enterococci, Streptococcus pyogenes; Citrobacter, Coliform; Cronobacter sakazakii; MRSA, VRE, Geobacillus stearothermophilus; Listeria spp., ESBL producing enterobacteria; Vibrio; Clostridium difficile; Candida albicans; Prevotella; Shigella, a microorganism containing apyrase, preferably Shigella; Legionella pneumophilia; and a virus of the Caliciviridae family, preferably a Lagovirus, a Norovirus, a Sapovirus, a Nebovirus, a Recovirus, more preferably a Norovirus.
• the microorganism is selected from the group consisting of Salmonella, Salmonella enterica, Listeria, preferably, Listeria monocytogenes, S. aureus, E. coli, and carbapenem-resistant bacteria, preferably Pseudomonas aeruginosa and Klebsiella pneumonia.
• step b) less than 0.1 ⁇ g of the compound of Formula I are added in step b), more preferably, less than 0.09 ⁇ g, even more preferably less than 0.08 ⁇ g, even more preferably less than 0.07 ⁇ g, even more preferably less than 0.065 ⁇ g, even more preferably 0.04 to 0.06 ⁇ g, even more preferably 0.045 to 0.055 ⁇ g, most preferably about 0.05 ⁇ g.
• the compound added to the medium in step b) is present in DMSO solution.
• the total amount of the compound of Formula I added in step b) can simply be determined by means of aliquotation from a stock solution of a known concentration.
• the final concentration of the compound in the medium after step b) is 2-50 ⁇ M, preferably 2-40 ⁇ M, more preferably 2-30 ⁇ M, more preferably 5-20 ⁇ M, more preferably 8-15 ⁇ M, even more preferably 9-11 ⁇ M, most preferably about 10 ⁇ M.
• the microorganism is a pathogenic microorganism, more preferably a bacterium, even more preferably Salmonella, Listeria or S. aureus.
• the microorganism is Salmonella, preferably Salmonella enterica, and the compound is a compound of Formula II:
• Y is -H, an optionally substituted C1-C12 alkyl or an alkali metal ion, wherein the alkali metal ion is preferably sodium or potassium and the optionally substituted C 1 -C 12 alkyl is preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl.
• Y is -H, or an optionally substituted C 1 -C 12 alkyl, wherein the optionally substituted C 1 -C 12 alkyl is preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl. More preferably, Y is -H or methyl.
• the microorganism is Salmonella, preferably Salmonella enterica, and the compound is a compound of Formula IIa.
• the microorganism is Listeria, preferably Listeria monocytogenes, and the compound is a compound of Formula III:
• Y is -H, an optionally substituted C 1 -C 12 alkyl or an alkali metal ion, wherein the alkali metal ion is preferably sodium or potassium and the optionally substituted C 1 -C 12 alkyl is preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl.
• Y is -H, or an optionally substituted C1-C12 alkyl, wherein the optionally substituted C 1 -C 12 alkyl is preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl. More preferably, Y is -H or methyl.
• the microorganism is Listeria, preferably Listeria monocytogenes, and the compound is a compound of Formula IIIa or IIIb, preferably IIIb.
• the microorganism is S. aureus and the compound is a compound of Formula IV:
• Y is -H, an optionally substituted C1-C12 alkyl or an alkali metal ion, wherein the alkali metal ion is preferably sodium or potassium and the optionally substituted C 1 -C 12 alkyl is preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl.
• Y is -H, or an optionally substituted C 1 -C 12 alkyl, wherein the optionally substituted C 1 -C 12 alkyl is preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl. More preferably, Y is -H or methyl.
• the microorganism is S. aureus and the compound is a compound of Formula IVa.
• the microorganism is E.
• the compound is a compound of Formula V: (Formula V) wherein Y is -H, an optionally substituted C 1 -C 12 alkyl or an alkali metal ion, wherein the alkali metal ion is preferably sodium or potassium and the optionally substituted C1-C12 alkyl is preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl.
• Y is -H, or an optionally substituted C 1 -C 12 alkyl, wherein the optionally substituted C 1 -C 12 alkyl is preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl. More preferably, Y is -H or methyl.
• the microorganism is E. coli and the compound is a compound of Formula Va or Vb, preferably Vb.
• the microorganism are carbapenem-resistant bacteria, preferably Pseudomonas aeruginosa and Klebsiella pneumonia, and the compound is a compound of Formula VII or VIII
• Y is -H, or an optionally substituted C 1 -C 12 alkyl, wherein the optionally substituted C 1 -C 12 alkyl is preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl. More preferably, Y is -H or methyl.
• the microorganisms are carbapenem-resistant Pseudomonas aeruginosa and Klebsiella pneumonia and the compound is a compound of Formula VIIa, VIIb or VIIIa, preferably VIIIa.
• the medium comprises more than one microorganism and one of said microorganisms leads to an at least 10-fold, preferably at least 20-fold, higher light emission than one or more other ones of said microorganisms present in the medium.
• the present invention relates to the use of a compound of Formula I as described in the first aspect for the detection of growth substrates, nutrients, and/or metabolites by enzymatic oxidation of said growth substrates, nutrients, and metabolites.
• the detection of growth substrates, nutrient and metabolites allows for the indirect detection of pathogens.
• the growth substrates, nutrients, and/or metabolites are detected by contacting them with an enzyme that oxidizes the growth substrates, nutrients, and/or metabolites and thereby produces hydrogen peroxide.
• the growth substrates, nutrients, or metabolites are indirectly detected by detecting the hydrogen peroxide produced by an enzyme acting on the growth substrates, nutrients, or metabolites.
• the nutrient is a carbohydrate or an amino acid and the enzyme is a corresponding oxidase.
• the nutrient is glucose and the enzyme is glucose oxidase; or the nutrient is a D-amino acid and the enzyme is D- amino acid oxide (DAO); or the nutrient is D-aspartic acid and the enzyme is D- aspartate oxidase.
• the metabolite is histamine and the enzyme is diaminooxidase.
• R1 is preferably -B(Z)(Z’) or as defined in the first aspect and the other substituents are also as defined in the first aspect.
• n is 0. In another embodiment, n is 1.
• Particularly preferred compounds are those disclosed in Table A, wherein .
• the compound of Formula I can be used solid form or in solution. However, using the compound of Formula I in solution, preferably DMSO solution, is preferred for the reasons set out above.
• the compound of Formula I is used in an amount of less than 0.1 ⁇ g, preferably less than 0.09 ⁇ g, more preferably less than 0.08 ⁇ g, even more preferably less than 0.07 ⁇ g, even more preferably less than 0.065 ⁇ g, even more preferably 0.04 to 0.06 ⁇ g, even more preferably 0.045 to 0.055 ⁇ g, most preferably about 0.05 ⁇ g.
• the compound is used in a final concentration of 1 to 100 ⁇ M, preferably 5 to 80 ⁇ M, more preferably 10 to 70 ⁇ M, more preferably 20 to 60 ⁇ M, more preferably 30 to 50 ⁇ M, even more preferably 35 to 45 ⁇ M.
• the compound is used in a final concentration of 10 to 500 ⁇ M, preferably 10 to 250 ⁇ M, more preferably 10 to 50 ⁇ M.
• the medium is a prokaryotic cell-comprising medium, a prokaryotic culture supernatant, an eukaryotic cell-comprising medium, an eukaryotic culture supernatant, blood serum or whole blood.
• the present invention relates to a method for the detection of growth substrates, nutrients, and/or metabolites by enzymatic oxidation of said growth substrates, nutrients, and metabolites.
• the method comprises the steps of a) providing a medium comprising a growth substrate, nutrient, and/or metabolite capable of being oxidized by an enzyme, b) (b1) adding an enzyme capable of oxidizing the growth substrate, nutrient, and/or metabolite and thereby producing hydrogen peroxide, (b2) adding a compound of Formula I, wherein R1 is -B(Z)(Z’) and the other substituents are described in the first aspect, to the medium so that the compound of Formula I emits light upon contact with hydrogen peroxide, wherein steps (b1) and (b2) may be performed simultaneously or subsequently, and c) detecting the emitted light.
• the medium is an aqueous medium.
• the medium is a prokaryotic cell-comprising medium, a prokaryotic culture supernatant, an eukaryotic cell-comprising medium, an eukaryotic culture supernatant, blood serum or whole blood.
• the nutrient is a carbohydrate or an amino acid and the enzyme is a corresponding oxidase.
• the nutrient is glucose and the enzyme is glucose oxidase; or the nutrient is a D-amino acid and the enzyme is D- amino acid oxide (DAO); or the nutrient is D-aspartic acid and the enzyme is D- aspartate oxidase.
• the metabolite is histamine and the enzyme is diaminooxidase.
• Ethanol or another suitable solvent or solvent mixture preferably in an amount of 15%, may be added in any of steps a) and b) (including substeps b1 and b2) or between or after steps a) and b).
• other lysis reagents may alternatively be used.
• the compound of Formula I can be used in solid form or in solution. However, using the compound of Formula I in solution, preferably DMSO solution, is preferred for the reasons set out above.
• the compound of Formula I is added in an amount of less than 0.1 ⁇ g, preferably less than 0.09 ⁇ g, more preferably less than 0.08 ⁇ g, even more preferably less than 0.07 ⁇ g, even more preferably less than 0.065 ⁇ g, even more preferably 0.04 to 0.06 ⁇ g, even more preferably 0.045 to 0.055 ⁇ g, most preferably about 0.05 ⁇ g.
• the compound of Formula I is added such that the final concentration of the compound in the medium is 2-50 ⁇ M, preferably 2-40 ⁇ M, more preferably 2-30 ⁇ M, more preferably 5-20 ⁇ M, more preferably 8-15 ⁇ M, even more preferably 9-11 ⁇ M, most preferably about 10 ⁇ M.
• the compound of Formula I is added such that the final concentration of the compound in the medium is 1 to 100 ⁇ M, preferably 5 to 80 ⁇ M, more preferably 10 to 70 ⁇ M, more preferably 20 to 60 ⁇ M, more preferably 30 to 50 ⁇ M, even more preferably 35 to 45 ⁇ M.
• the present invention relates to the use of a compound of Formula I as defined in the first aspect for the detection of bacterial endotoxins via detection of limulus Factor C.
• group R1 is responsive/labile towards limulus Factor C.
• R1 is Boc-Val-Pro-Argininyl or Boc-Asp(OBzl)-Pro-Argininyl and the other substituents are as defined in the first aspect.
• the compound of Formula may be used in solid form or in solution, preferably DMSO solution.
• the compound of Formula I is used in solution, preferably in DMSO solution.
• the compound of Formula I is used in an amount of less than 0.1 ⁇ g, preferably less than 0.09 ⁇ g, more preferably less than 0.08 ⁇ g, even more preferably less than 0.07 ⁇ g, even more preferably less than 0.065 ⁇ g, even more preferably 0.04 to 0.06 ⁇ g, even more preferably 0.045 to 0.055 ⁇ g, most preferably about 0.05 ⁇ g.
• the present invention relates to a method for the detection of bacterial endotoxins via detection of limulus factor C, wherein a compound of Formula I and limulus factor C are added to an endotoxin-comprising medium.
• R1 is Boc-Val-Pro-Argininyl or Boc-Asp(OBzl)-Pro-Argininyl and the other substituents are as defined in the first aspect.
• the compound is a compound of Formula I as disclosed in Table A, wherein R1 is Boc-Val-Pro-Argininyl or Boc-Asp(OBzl)-Pro-Argininyl.
• the peptidase activity of limulus factor C is activated and this peptidase activity cleaves R1 from the remainder part of the compound of Formula I resulting in chemiluminescence. In the absence of the respective endotoxin, there is no peptidase activity.
• the medium used in the method is an aqueous medium.
• Ethanol or another suitable solvent or solvent mixture preferably in an amount of 15%, may be added to the medium. As described above, other lysis reagents may alternatively be used.
• the compound of Formula I can be used solid form or in solution.
• the compound of Formula I is used in an amount of less than 0.1 ⁇ g, preferably less than 0.09 ⁇ g, more preferably less than 0.08 ⁇ g, even more preferably less than 0.07 ⁇ g, even more preferably less than 0.065 ⁇ g, even more preferably 0.04 to 0.06 ⁇ g, even more preferably 0.045 to 0.055 ⁇ g, most preferably about 0.05 ⁇ g.
• the final concentration of the compound of Formula I in the medium is 2-50 ⁇ M, preferably 2-40 ⁇ M, more preferably 2-30 ⁇ M, more preferably 5- 20 ⁇ M, more preferably 8-15 ⁇ M, even more preferably 9-11 ⁇ M, most preferably about 10 ⁇ M.
• the present invention relates to the use of a compound of Formula I as described in the first aspect for testing of pasteurization of dairy products, e.g. milk.
• the used compound is a compound of Formula I, wherein R1 is phosphoryl and the other substituents are as described in the first aspect.
• the compound of Formula I is used in an amount of less than 0.1 ⁇ g, preferably less than 0.09 ⁇ g, more preferably less than 0.08 ⁇ g, even more preferably less than 0.07 ⁇ g, even more preferably less than 0.065 ⁇ g, even more preferably 0.04 to 0.06 ⁇ g, even more preferably 0.045 to 0.055 ⁇ g, most preferably about 0.05 ⁇ g.
• the compound is used in a final concentration of 1 to 100 ⁇ M, preferably 5 to 80 ⁇ M, more preferably 10 to 70 ⁇ M, more preferably 20 to 60 ⁇ M, more preferably 30 to 50 ⁇ M, even more preferably 35 to 45 ⁇ M.
• the compound of Formula I is used in a final concentration of 1 to 50 ⁇ M, preferably 5 to 40 ⁇ M, more preferably 10 to 30 ⁇ M, more preferably 15 to 25 ⁇ M, more preferably 18 to 22 ⁇ M, more preferably 19 to 21 ⁇ M.
• the present invention relates to a method of testing pasteurization of dairy products, preferably milk.
• the method comprises the steps of a) providing a dairy product medium, preferably milk, b) pasteurization, c) adding a compound of Formula I as described in the first embodiment, wherein R1 is phosphoryl, to the medium, optionally in combination with a buffer, so that the compound of Formula I emits light, and d) detecting the emitted light.
• a dairy product medium preferably milk
• b) pasteurization c) adding a compound of Formula I as described in the first embodiment, wherein R1 is phosphoryl, to the medium, optionally in combination with a buffer, so that the compound of Formula I emits light
• d detecting the emitted light.
• Phosphatase which is naturally present in a dairy product, degrades during sterilization. Thus, if sterilization is successful, no light emission is detected as no active phosphatase is present in the dairy product.
• the compound added to the medium in step b) is present in DMSO solution.
• the compound of Formula I is added to the medium such that it is present in a final concentration of 2-50 ⁇ M, preferably 2-40 ⁇ M, more preferably 2- 30 ⁇ M, more preferably 5-20 ⁇ M, more preferably 8-15 ⁇ M, even more preferably 9- 11 ⁇ M, most preferably about 10 ⁇ M.
• the compound of Formula I is added to the medium such that it is present in a final concentration of 1 to 50 ⁇ M, preferably 5 to 40 ⁇ M, more preferably 10 to 30 ⁇ M, more preferably 15 to 25 ⁇ M, more preferably 18 to 22 ⁇ M, more preferably 19 to 21 ⁇ M.
• less than 0.1 ⁇ g of the compound of Formula I are added to the medium, more preferably, less than 0.09 ⁇ g, even more preferably less than 0.08 ⁇ g, even more preferably less than 0.07 ⁇ g, even more preferably less than 0.065 ⁇ g, even more preferably 0.04 to 0.06 ⁇ g, even more preferably 0.045 to 0.055 ⁇ g, most preferably about 0.05 ⁇ g.
• the compound added to the medium in step b) is present in DMSO solution.
• the total amount of the compound of Formula I added in step b) can simply be determined by means of aliquotation from a stock solution of a known concentration.
• the present invention relates to the use of a compound of Formula I for testing antibiotic resistance in microorganisms.
• R1 is a beta-lactamase-labile group as defined in the first aspect, preferably a beta-lactam antibiotic, more preferably a penicillin, a cephalosporin of generation 1 to 5, a cephamycin, or a carbapenem, and the other substituents are as defined in the first aspect.
• the compound of Formula I is as defined in Table A, wherein R1 is the preferred depicted carbapenem moiety.
• the microorganism is selected from the group consisting of Salmonella; Salmonella enterica; Listeria, preferably, Listeria monocytogenes; S. aureus; E. coli; carbapenem-resistant bacteria, preferably Pseudomonas aeruginosa, and Klebsiella pneumonia; Campylobacter jejuni; C. coli; C. lari; Bacillus; Staphylococcus; Clostridium; Mycobacterium tuberculosis; Clostridium perfringens; S.
• agalactiae Candida spp.; Gram negative bacteria, yeast, molds, Pseudomonas aeruginosa, Enterococci, Streptococcus pyogenes; Citrobacter, Coliform; Cronobacter sakazakii; MRSA, VRE, Geobacillus stearothermophilus; Listeria spp., ESBL producing enterobacteria; Vibrio; Clostridium difficile; Candida albicans; Prevotella; Shigella, Legionella pneumophilia; and a virus of the Caliciviridae family, preferably a Lagovirus, a Norovirus, a Sapovirus, a Nebovirus, a Recovirus, more preferably a Norovirus.
• the microorganism is selected from the group consisting of Salmonella; Salmonella enterica; Listeria, preferably, Listeria monocytogenes; S. aureus; E. coli; carbapenem-resistant bacteria, preferably Pseudomonas aeruginosa, and Klebsiella pneumonia; Campylobacter jejuni; C. coli; C. lari; Bacillus; Staphylococcus; Clostridium; Mycobacterium tuberculosis; Clostridium perfringens; S.
• the microorganisms are selected from Pseudomonas aeruginosa and Klebsiella pneumoniae.
• the compound may be used in solid form or in solution. However, using the compound of Formula I in solution, preferably DMSO solution, is preferred. [00195] Preferably, the compound of Formula I is used in an amount of less than 0.1 ⁇ g, preferably less than 0.09 ⁇ g, more preferably less than 0.08 ⁇ g, even more preferably less than 0.07 ⁇ g, even more preferably less than 0.065 ⁇ g, even more preferably 0.04 to 0.06 ⁇ g, even more preferably 0.045 to 0.055 ⁇ g, most preferably about 0.05 ⁇ g.
• the compound is used in a final concentration of 1 to 100 ⁇ M, preferably 5 to 80 ⁇ M, more preferably 10 to 70 ⁇ M, more preferably 20 to 60 ⁇ M, more preferably 30 to 50 ⁇ M, even more preferably 35 to 45 ⁇ M.
• the compound is used in a final concentration of about 1 to 50 ⁇ M, preferably 2 to 40 ⁇ M, more preferably 3 to 30 ⁇ M, more preferably 4 to 20 ⁇ M, more preferably 5 to 15 ⁇ M, more preferably 7 to 13 ⁇ M, more preferably 9 to 11 ⁇ M.
• the present invention relates to a method for testing of antibiotic, in particular beta-lactam antibiotic such as a penicillin, a cephalosporin, a cephamycin, or a carbapenem, resistance in microorganisms.
• beta-lactam antibiotic such as a penicillin, a cephalosporin, a cephamycin, or a carbapenem
• the method comprises the steps of a) providing a medium comprising one or more microorganisms b) adding a compound of Formula I, wherein R1 is a beta- lactamase-labile group, preferably a beta-lactam antibiotic such as a penicillin, a cephalosporin of generation 1 to 5, a cephamycin, or a carbapenem and the other substituents are as defined in the first aspect, to the medium so that the compound of Formula I emits light when antibiotic, preferably a beta-lactam antibiotic such as a penicillin, a cephalosporin, a cephamycin or a carbapenem, resistant microorganisms are present in the medium, and c) detecting the emitted light.
• R1 is a beta- lactamase-labile group, preferably a beta-lactam antibiotic such as a penicillin, a cephalosporin of generation 1 to 5, a cephamycin, or a carbapenem and the other substituents are as
• the compound of Formula I is as defined in Table A, wherein R1 is carbapenemyl.
• the medium is an aqueous medium.
• Ethanol or another suitable solvent or solvent mixture preferably in an amount of 15%, may be added in any of steps a) and b) or between or after steps a) and b). As described above, other lysis reagents may alternatively be used.
• the compound of Formula I is added to the medium such that it is present in a final concentration of 2-50 ⁇ M, preferably 2-40 ⁇ M, more preferably 2-30 ⁇ M, more preferably 5-20 ⁇ M, more preferably 8-15 ⁇ M, even more preferably 9-11 ⁇ M, most preferably about 10 ⁇ M.
• the compound of Formula I is added to the medium such that it is present in a final concentration of 1 to 50 ⁇ M, preferably 2 to 40 ⁇ M, more preferably 3 to 30 ⁇ M, more preferably 4 to 20 ⁇ M, more preferably 5 to 15 ⁇ M, more preferably 7 to 13 ⁇ M, more preferably 9 to 11 ⁇ M.
• the compound added to the medium in step b) is present in DMSO solution.
• the total amount of the compound of Formula I added in step b) can simply be determined by means of aliquotation from a stock solution of a known concentration.
• the method is suitable for distinguishing antibiotic resistant microorganisms from antibiotic sensitive microorganisms as light emission will only occur for antibiotic resistant microorganisms.
• the present invention relates to the use of a compound of Formula I for the detection of inorganic phosphate, preferably inorganic phosphate produced by enzymatic reactions.
• R1 is oxalylester and the other substituents are as defined in the first aspect.
• the inorganic phosphate is produced by apyrase, preferably Shigella apyrase. In this case, the compound is suitable for detecting Shigella, one of the leading bacterial causes of diarrhea worldwide.
• the compound of Formula I is used in an amount of less than 0.1 ⁇ g, preferably less than 0.09 ⁇ g, more preferably less than 0.08 ⁇ g, even more preferably less than 0.07 ⁇ g, even more preferably less than 0.065 ⁇ g, even more preferably 0.04 to 0.06 ⁇ g, even more preferably 0.045 to 0.055 ⁇ g, most preferably about 0.05 ⁇ g.
• the compound may be used in in solid form or in solution. However, using the compound of Formula I in solution, preferably DMSO solution, is preferred.
• the compound is used in a final concentration of 1 to 100 ⁇ M, preferably 5 to 80 ⁇ M, more preferably 10 to 70 ⁇ M, more preferably 20 to 60 ⁇ M, more preferably 30 to 50 ⁇ M, even more preferably 35 to 45 ⁇ M.
• the present invention relates to a method for detecting inorganic phosphate, preferably inorganic phosphate produced by enzymatic reactions.
• the method comprises the steps of a) providing a medium comprising inorganic phosphate b) adding a compound of Formula I, wherein R1 is oxalylester and the other substituents are as defined in the first aspect, to the medium so that the compound of Formula I emits light, and c) detecting the emitted light.
• the compound of Formula I is as defined in Table A, wherein R1 is oxalylester.
• the inorganic phosphate comprised in the medium is produced by apyrase, preferably Shigella apyrase.
• the medium in step a) comprises apyrase, preferably Shigella apyrase. It is particularly preferred that the medium in step a) comprises Shigella.
• the medium is an aqueous medium.
• Ethanol or another suitable solvent or solvent mixture preferably in an amount of 15%, may be added in any of steps a) and b) or between or after steps a) and b). As described above, other lysis reagents may alternatively be used.
• less than 0.1 ⁇ g of the compound of Formula I are, more preferably, less than 0.09 ⁇ g, even more preferably less than 0.08 ⁇ g, even more preferably less than 0.07 ⁇ g, even more preferably less than 0.065 ⁇ g, even more preferably 0.04 to 0.06 ⁇ g, even more preferably 0.045 to 0.055 ⁇ g, most preferably about 0.05 ⁇ g.
• the compound added to the medium in step b) is present in DMSO solution.
• the final concentration of the compound in the medium is 2-50 ⁇ M, preferably 2-40 ⁇ M, more preferably 2-30 ⁇ M, more preferably 5-20 ⁇ M, more preferably 8-15 ⁇ M, even more preferably 9-11 ⁇ M, most preferably about 10 ⁇ M.
• the present invention relates to the use of a compound of Formula I for monitoring of a sterilization process, preferably through detection of alpha-D- glucosidase activity of the indicator microorganism Geobacillus stearothermophilus.
• Geobacillus stearothermophilus produces alpha-glucosidase, which, however, is inactivated upon sterilization.
• R1 is alpha-D-glucopyranosidyl and the other substituents are as defined in the first aspect.
• the compound of Formula I is used in an amount of less than 0.1 ⁇ g, preferably less than 0.09 ⁇ g, more preferably less than 0.08 ⁇ g, even more preferably less than 0.07 ⁇ g, even more preferably less than 0.065 ⁇ g, even more preferably 0.04 to 0.06 ⁇ g, even more preferably 0.045 to 0.055 ⁇ g, most preferably about 0.05 ⁇ g.
• the compound of Formula I may be used in solid form or in solution. However, using the compound of Formula I in solution, preferably DMSO solution, is preferred.
• the compound is used in a final concentration of 1 to 100 ⁇ M, preferably 5 to 80 ⁇ M, more preferably 10 to 70 ⁇ M, more preferably 20 to 60 ⁇ M, more preferably 30 to 50 ⁇ M, even more preferably 35 to 45 ⁇ M.
• the present invention relates a method for monitoring of a sterilization process, preferably through detection of alpha-D-glucosidase activity of the indicator microorganism Geobacillus stearothermophilus.
• the method comprises the steps of a1) providing a medium comprising a microorganism that, under normal conditions, produces alpha-glucosidase, preferably Geobacillus stearothermophilus, a2) sterilizing the medium, b) adding a compound of Formula I, wherein R1 is alpha-D-glucopyranosidyl and the other substituents are as defined in the first aspect, to the medium and c) detecting the emitted light, if any.
• the compound of Formula I is as defined in Table A, wherein R1 is alpha-D-glucopyranosidyl and the other substituents are as defined in the first aspect.
• the medium is an aqueous medium.
• Ethanol or another suitable solvent or solvent mixture preferably in an amount of 15%, may be added in or after step b).
• other lysis reagents may alternatively be used.
• less than 0.1 ⁇ g of the compound of Formula I are, more preferably, less than 0.09 ⁇ g, even more preferably less than 0.08 ⁇ g, even more preferably less than 0.07 ⁇ g, even more preferably less than 0.065 ⁇ g, even more preferably 0.04 to 0.06 ⁇ g, even more preferably 0.045 to 0.055 ⁇ g, most preferably about 0.05 ⁇ g.
• the compound added to the medium is present in DMSO solution.
• the final concentration of the compound in the medium is 2-50 ⁇ M, preferably 2-40 ⁇ M, more preferably 2-30 ⁇ M, more preferably 5-20 ⁇ M, more preferably 8-15 ⁇ M, even more preferably 9-11 ⁇ M, most preferably about 10 ⁇ M.
• the present invention relates to the use of a compound of Formula I as described in the first aspect for endpoint and online detection of antibiotic resistance of bacteria and for antibiotic susceptibility testing.
• the compound may be added to a bacteria- and antibiotic- containing medium at the beginning of cultivation or at the time of measurement. Antibiotic resistant bacteria will multiply even in the presence of the antibiotic, which will lead to light emission of the compound of Formula I by means of interaction of group R1 with the respective bacteria (in particular an enzyme or the like thereof) (cf., Example 8).
• R1 which group R1 may be used for said endpoint and online detection of antibiotic resistance of bacteria and for antibiotic susceptibility testing, is discernible from Table 1.
• a compound of Formula I, wherein R1 is phosphoryl may be used for endpoint and online detection of antibiotic resistance of Staphylococcus aureus, in particular MRSA, Clostridium perfringens, S. agalactiae, or Candida and for antibiotic susceptibility testing thereof.
• a compound of Formula I, wherein R1 is L-pyroglutamic acidyl may be used for endpoint and online detection of antibiotic resistance of Enterococci, Streptococcus pyogenes, or Citrobacter and for antibiotic susceptibility testing thereof.
• a compound of Formula I wherein R1 is beta-D-galactopyranosidyl, may be used for endpoint and online detection of antibiotic resistance of Coliform, or E. coli and for antibiotic susceptibility testing thereof.
• the compound of Formula I is used in an amount of less than 0.1 ⁇ g, preferably less than 0.09 ⁇ g, more preferably less than 0.08 ⁇ g, even more preferably less than 0.07 ⁇ g, even more preferably less than 0.065 ⁇ g, even more preferably 0.04 to 0.06 ⁇ g, even more preferably 0.045 to 0.055 ⁇ g, most preferably about 0.05 ⁇ g.
• the compound of Formula I may be used in solid form or in solution. However, using the compound of Formula I in solution, preferably DMSO solution, is preferred. [00241] Preferably, the compound is used in a final concentration of 1 to 100 ⁇ M, preferably 5 to 80 ⁇ M, more preferably 10 to 70 ⁇ M, more preferably 20 to 60 ⁇ M, more preferably 30 to 50 ⁇ M, even more preferably 35 to 45 ⁇ M. [00242] Further, the present invention relates a method for endpoint and online detection of antibiotic resistance of bacteria and for antibiotic susceptibility testing.
• the method comprises the steps of a) providing a medium comprising a microorganism, preferably bacteria, b) adding an antibiotic, c) adding a compound of Formula I, wherein R1 is responsive towards said microorganism, in particular an enzyme produced thereby, wherein step c) may be performed before, together with or after step b), d) detecting the emitted light, if any.
• step c) may be performed before, together with or after step b
• step d) detecting the emitted light if any.
• less than 0.1 ⁇ g of the compound of Formula I are, more preferably, less than 0.09 ⁇ g, even more preferably less than 0.08 ⁇ g, even more preferably less than 0.07 ⁇ g, even more preferably less than 0.065 ⁇ g, even more preferably 0.04 to 0.06 ⁇ g, even more preferably 0.045 to 0.055 ⁇ g, most preferably about 0.05 ⁇ g.
• the compound added to the medium is present in DMSO solution.
• the final concentration of the compound in the medium is 2-50 ⁇ M, preferably 2-40 ⁇ M, more preferably 2-30 ⁇ M, more preferably 5-20 ⁇ M, more preferably 8-15 ⁇ M, even more preferably 9-11 ⁇ M, most preferably about 10 ⁇ M.
• the medium is an aqueous medium.
• Ethanol or another suitable solvent or solvent mixture preferably in an amount of 15%, may be added in or after step b). As described above, other lysis reagents may alternatively be used.
• the present invention will now be further illustrated by the following, non- limiting example.
• 1,2:4,5-Di-O-isopropylidene-myo-inositol 250 mg, 0.96 mmol, 1eq
• Imidazole 98 mg, 1.44 mmol, 1.5 eq
• dry pyridine 3 ml
• t-Butyldiphenylsilyl chloride 275 ⁇ l, 1.06 mmol, 1.1 eq
• TLC 50:50 EtOAc:Hex
• ester 4 (1.34 g, 0.00345 mol) in DMF (9 mL) (colorless solution), was cooled to 0 °C (yellow suspension), K 2 CO 3 (1.2 eq., 0.00413 mol, 0.57 g) was added and the mixture was stirred at 0 °C. After 10 min a solution of bromide 3 (1 eq., 0.00345 mol, 1.20 g) in DMF (8 mL) was added and the resulting mixture (yellow- orange suspension) was stirred at RT.
• ester 5 (1.9 g, 0.0029 mol) in CH 3 CN (29 mL) was added pyrrolidine (6.2 eq., 0.0018 mol, 1.28 g, 1.5 mL), Ph3P (0.20 eq., 0.00059 mol, 0.15 g) and Pd(PPh 3 ) 4 (0.05 eq., 0.000145 mol, 0.168 g) and the resulting mixture was stirred at RT. After 0.5 h the reaction mixture was concentrated in vacuo yielding pale-yellow oil. The crude mixture containing ester 6 was taken for the next step (Me-ester hydrolysis) without further purification. [00267]
• Ester 2 (0.00644 mol, 2.51 g) was dissolved in DMF (50 mL) and the resulting mixture was cooled to 0 °C.
• K 2 CO 3 (1.2 eq., 0.00773 mol, 1.07 g) was added at 0 °C and the mixture was stirred at 0 °.
• a solution of bromide 1 (1.2 eq., 0.00773 mol, 4.00 g) in DMF (14 mL) was added and the resulting mixture was stirred at RT.
• Ester 3 (0.00300 mol, 2.48 g) was dissolved in a mixture of THF (60 mL) and H 2 O (15 mL) and LiOH (3.9 eq., 0.0117 mol, 0.280 g) was added. The resulting mixture was stirred at RT. After 24 h to the mixture was added saturated NH 4 Cl (80 mL) and the mixture was extracted with EtOAc (3-times 60 mL), the combined organics were washed with brine (60 mL), dried (MgSO 4 ) and concentrated in vacuo yielding a pale- yellow oil. This residue was purified by column chromatography (eluent EtOAc to EtOAc-MeOH 8:1) affording ester 4 (1.33 g, 70%) as an off-white solid. [00271]
• Triflate 2 (0.00925 mol, 4.50 g), B2Pin2 (2 eq., 0.0185 mol, 4.96 g), KOAc (3 eq., 0.0278 mol, 2.72 g) and Pd(dppf) 2 Cl 2 (0.2 eq., 0.00185 mol, 1.35 g) were placed in a round-bottom flask, the content was placed under argon (2 vacuum-argon cycles) and dry dioxane (40 mL) was added. The resulting mixture was degassed (2 vacuum- argon cycles) and stirred at reflux under argon. After 45 min the reaction mixture (dark brown mixture) was concentrated in vacuo. This mixture was purified by column chromatography affording boronate 3 (3.83 g, 83%) as an off-white solid. [00274]
• Synthesis Example 7 Synthesis of a D-luciferin-spacer-caprylate [00283] 500ml 3-necked flask was charged with 10g of p-cresol (92,5 mmol / 1 eq.) followed by dry DCM (200 mL). Mixture was cooled in ice-water cooling bath. To this solution Pyridine (10,24 g / 12,9 mmol / 1,4 eq.) was added. Capryloyl chloride (18 g / 110,7 mmol / 1,2 eq.) (dissolved in 50ml of dry DCM) was added dropwise from dropping funnel keeping temperature of reaction mixture below 12°C. After 15 minutes cooling bath was removed and mixture was stirred at ambient temperature.
• Example 1 Enzymatic assay of pig liver esterase activity with Compound IIa
• Compound IIa was added to a final concentration of 62.5 ⁇ M to phosphate- buffered saline (pH 7.4) containing 10% v/v dimethyl sulfoxide.
• the assay mix was pre-incubated for 20 min at room temperature, then 1:5 v/v of pig liver esterase solutions containing varying enzyme concentrations were added and luminescence was recorded for 20 min at room temperature with a SpectraMax M5 reader in luminescence mode.
• Assays were performed in a black microtiter plate with a total liquid volume of 0.25 mL.
• Example 2 Detection of Salmonella enterica with Compound IIa
• Citrobacter freundii ATCC 8090 C.f., C8E negative
• Escherichia coli ATCC 25922 E.c., C8E negative
• Salmonella Typhimurium D
• ATCC 14028 S.T., Salmonella enterica ssp. I ser. Typhimurium, C8E positive
• Salmonella Enteritidis D
• ATCC 13076 S.E.1, Salmonella enterica ssp. enterica ser.
• Salmonella Enteritidis, C8E positive and Salmonella Enteritidis RKI 05/07992 were cultivated in Nutrient Broth (5 g/l peptone, 5 g/l NaCl, 2 g/L yeast extract, 1 g/l beef extract, pH 7.4) for 17 h and then serially diluted in sterile saline (0.9% NaCl). Similar cell concentrations of all strains were inoculated in triplicate test tubes with Nutrient Broth. In addition, further dilutions of S.E. 2 cells in sterile saline were inoculated in triplicate test tubes.
• Example 3 Enzymatic assay of PI-PLC activity with Compound IIIa
• Compound IIIa was added to a final concentration of 10 ⁇ M to phosphate- buffered saline (pH 7.4) containing 1% v/v dimethyl sulfoxide. Varying concentrations of phosphatidylinositol-specific phospholipase C (PI-PLC) were added and luminescence was recorded at room temperature (Table 4). Assays were performed in a white microtiter plate with 0.25 mL total liquid volume. Concentration of PI-PLC and maximal RLU (relative light unit) values within a 20 min measurement period exhibited a positive correlation over two orders of magnitude.
• PI-PLC phosphatidylinositol-specific phospholipase C
• Example 4 Detection of Listeria monocytogenes with Compound IIIa
• Listeria monocytogenes ATCC 7644 (L.m. 1, PI-PLC positive), Listeria monocytogenes (4b) ATCC 19115 (L.m.
• Example 5 Comparison of detection of Salmonella enterica with Compound IIa and 4- Methylumbelliferyl caprylate (Figure 2) [00305] Salmonella Enteritidis RKI 05/07992 was cultivated in Nutrient Broth (5 g/l peptone, 5 g/l NaCl, 2 g/L yeast extract, 1 g/l beef extract, pH 7.4) for 20 h (37°C, 150 rpm) and then serially diluted in phosphate buffered saline.
• Nutrient Broth 5 g/l peptone, 5 g/l NaCl, 2 g/L yeast extract, 1 g/l beef extract, pH 7.4
• Example 6 Detection of Staphylococcus aureus with Compound IVa
• Staphylococcus aureus ATCC 29213 S.a., phosphatase positive
• Staphylococcus haemolyticus RKI 06-01354 Staphylococcus haemolyticus RKI 06-01354
• Nutrient Broth 5 g/l peptone, 5 g/l NaCl, 2 g/L yeast extract, 1 g/l beef extract, pH 7.4
• sterile saline 0.9% NaCl
• Example 7 Monitoring of pasteurization of milk with compound IVa
• UHT whole milk (milk 1) and pasteurized whole milk (milk 2) were obtained from a Swiss super market.
• Alkaline phosphatase (AP) from calf intestine was added to milk samples at a final concentration of 0.5 U/mL.
• Triplicate milk samples in polypropylene test tubes spikeked with alkaline phosphatase were heated for 20 min at 70°C in a water bath, similar reference samples were kept at room temperature.
• Methicillin-resistant Staphylococcus aureus ATCC 33592 (MRSA), a strain which is resistant to penicillin derivatives such as methicillin and oxacillin, and methicillin-susceptible Staphylococcus aureus ATCC 29213 (MSSA) were cultivated overnight at 37°C in nutrient broth (5 g/L peptone, 5 g/L sodium chloride, 2 g/L yeast extract, 1 g/L meat extract, pH 7.4) and diluted in sterile saline. MRSA and MSSA strains were inoculated at approx.106 CFU/mL in nutrient broth containing compound IVa at a final concentration of 10 ⁇ M.
• MRSA Methicillin-resistant Staphylococcus aureus ATCC 33592
• MSSA methicillin-susceptible Staphylococcus aureus ATCC 29213
• oxacillin 20 mg/L final concentration
• Tube cultures were incubated at 37°C and 150 rpm.
• a sterile control (nutrient broth with 10 ⁇ M of compound IVa) was incubated in parallel.
• Luminescence of culture samples (0.2 mL in a white microtiter plate) was recorded at regular intervals for 6 h with a SpectraMax M5 plate reader ( Figure 4: Luminescence development in cultures of MRSA and MSSA in the presence and absence of the antibiotic oxacillin).
• Example 9 Detection of Escherichia coli with compound Va and comparison to D- Luciferin-6-O-beta-D-galactopyranoside
• Escherichia coli ATCC 25922 E.c., beta-galactosidase positive
• Salmonella Enteritidis RKI 05/07992 S.E., beta-galactosidase negative
• Diluted cell suspensions were inoculated 1:100 v/v in test tubes with either full-strength LB medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) or LB medium diluted 1:20 v/v with ultrapure water (LB1:20). All media contained 1 mM IPTG for induction of beta-galactosidase. Sterile saline was added to negative control tubes. After 6 h of cultivation at 37°C and 150 rpm, samples (0.255 mL) were withdrawn from cultures and mixed with 45 ⁇ L stock solution of compound Va in ethanol in a white microtiter plate.
• culture samples (45 ⁇ L) were transferred to a microtiter plate and mixed with 5 ⁇ L of 10x lysis reagent which contained 2% w/v dodecyl-trimethylammonium bromide, 1 mM D-Luciferin-6-O-beta-D-galactopyranoside (commercially available, Biosynth Cat. No. L-8600) and 10 mM magnesium chloride. Lysis and reaction with D-Luciferin-6-O- beta-D-galactopyranoside was carried out for 1 h at 37°C. Then 0.2 mL of luciferase assay mix was added (5:1 v/v).
• the assay mix contained 62.5 mM Tris acetate (pH 7.8), 12.5 mM magnesium sulfate, 2.5 g/L bovine serum albumin, 7.5 mM D/L- cysteine, 1.25 mM ethylene diamine tetraacetate, 25 ⁇ M sodium pyrophosphate, 10 g/L cyclodextrin, 1.25 mM adenosine 5-triphosphate and 13.1 ⁇ g/mL commercially available Photinus pyralis luciferase (recombinant). Light emission was recorded with a SpectraMax M5 plate reader. The mean relative light units (RLU) of the first two minutes of measurement are shown in Table VIb.
• RLU relative light units
• Table VIb Luminescence of Escherichia coli culture samples after lysis in the presence of Luciferin-6-O-beta-D-galactopyranoside and addition of luciferase assay mix
• Example 10 Detection of Salmonella enterica with D-luciferin-6-O-phenyl-caprylate
• D-luciferin-6-O-phenyl-caprylate was synthesized according to procedures known to persons skilled in the art.
• Salmonella Enteritidis RKI 05/07992 S.E., Salmonella enterica ssp. enterica ser. Enteritidis, C8 esterase positive
• Diluted cell suspensions were inoculated 1:100 v/v in test tubes with Nutrient Broth containing 0.1 mM D-luciferin-6-O-phenyl-caprylate. After 6.5 h of cultivation at 37°C and 150 rpm, samples (50 ⁇ L) were withdrawn from cultures and mixed with 0.2 mL luciferase assay mix (composition described in example 9) in a microtiter plate. Use of a cell lysis reagent such as dodecyl trimethyl ammonium bromide was not required (data not shown). Light emission was recorded with a plate reader equipped with a luminescence detection system.
• Table VII Luminescence of Salmonella enterica cultures samples containing D- luciferin-6-O-phenyl-caprylate after mixing with luciferase assay mix
• Example 11 Detection of glucose in culture supernatant of Escherichia coli using
• Escherichia coli ATCC 25922 was cultivated in Nutrient Broth (5 g/L peptone, 5 g/L NaCl, 2 g/L yeast extract, 1 g/L yeast extract, pH h 7.4) for 16 h. Cells were separated from culture supernatant by centrifugation (13’000 x g, 2 min). Varying concentrations of glucose were added to culture supernatant from 100-fold concentrated stock solutions in water, a similar amount of water was added to the negative control. A second negative control also contained 5 mM glucose, but no glucose oxidase.
• Example 12 Detection of carbapenem-resistant bacteria with compound VIIa
• Carbapenem resistant bacterial strains Pseudomonas aeruginosa RKI 48/09 (IMP-2) (P.a.-ImpR) and Klebsiella pneumoniae RKI 92/08 (KPC-2) (K.p.-ImpR) were cultivated on trypticase soy agar supplemented with 8 mg/L and 4 mg/mL imipenem, respectively.
• Carbapenem sensitive strain Escherichia coli ATCC 25922 (E.c.) was cultivated on trypticase soy agar.
• Example 13 Luminescent properties of compounds comprising different substituents RA ,
• Compounds A, B and C represent the (luminescent) species that is formed from a respective compound of Formula I upon interaction of R1 with an analyte.
• the chemiluminescence kinetic profiles of Compounds A and B are compared in Figure 6A (insert: zoom on first 2 hours); the total light emitted of Compounds A and B is compared in Figure 6B.
• the chemiluminescence kinetic profiles of Compounds A and C are compared in Figure 7A; the total light emitted of Compounds A and C is compared in Figure 7B.

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