Compounds, complexes, and methods useful for detecting and/or treating bacterial pathogens
10969387 · 2021-04-06
Assignee
Inventors
Cpc classification
C12Q1/18
CHEMISTRY; METALLURGY
C07C235/60
CHEMISTRY; METALLURGY
C07C237/22
CHEMISTRY; METALLURGY
C12Q1/04
CHEMISTRY; METALLURGY
International classification
C12Q1/18
CHEMISTRY; METALLURGY
C07F5/00
CHEMISTRY; METALLURGY
Abstract
Compounds and complexes that can be useful as enterobactin probes not necessary are disclosed herein. Methods of detecting bacteria and/or methods of determining susceptibility of bacteria to an antibiotic using such compounds and complexes are also disclosed herein.
Claims
1. A compound of formula I, II or III: ##STR00017## wherein: each X independently represents H and the vicinal Y represents H or -L-B; or each Y independently represents H and the vicinal X represents H or -L-B; each Z independently represents H or -L-B; each L independently represents an organic linking group; each B independently represents —OR, —NR.sub.2, —SR, —C(O)OR, —C(O)NR.sub.2, —S(O)R, or —SO.sub.2R; each R independently represents H or an organic group; each b is independently 0 or 1; each c is independently 0 or 1; and x=0 to 3; with the proviso that at least one of X, Y, or Z is present and represents -L-B; and wherein at least one R is an organic group, said organic group comprising a group selected from the group consisting of a dye, a Lanthanide complex, a magnetic resonance imaging (MM) contrast agent, a positron emission tomography (PET) agent, a gold nanoparticle, a silver nanoparticle, a quantum dot, an antibiotic or an antibacterial drug, an antimicrobial peptide, a polymer, a dendrimer, an ionophore, and combinations thereof.
2. The compound of claim 1, wherein each organic linking group L independently represents a straight-chain group of the formula —(CH.sub.2).sub.n— or —(CH.sub.2—CH.sub.2—O).sub.n—, wherein n is 1 to 8.
3. The compound of claim 1, wherein each organic linking group L independently represents a branched chain dendrimer that contains one or more of C, N, S, and O.
4. A compound of formula II, ##STR00018## wherein: each Q independently represents H or —(CH.sub.2).sub.n-D, wherein n is 1 to 8; each D independently represents —OR, —NR.sub.2, —SR, or —C(O)OR; each R independently represents H or an organic group; and each d is independently 2 to 5; with the proviso that at least one Q represents —(CH.sub.2).sub.n-D; and wherein at least one R is an organic group, said organic group comprising a group selected from the group consisting of a dye, a Lanthanide complex, a magnetic resonance imaging (MRI) contrast agent, a positron emission tomography (PET) agent, a gold nanoparticle, a silver nanoparticle, a quantum dot, an antibiotic or an antibacterial drug, an antimicrobial peptide, a polymer, a dendrimer, and ionophore, and combinations thereof.
5. A compound of formula III: ##STR00019## wherein: each A independently represents —OR, —NR.sub.2, —SR, or —C(O)OR; each R independently represents H or an organic group; each n is independently 2 to 5; each e is independently 0 or 1; each f is independently 2 or 3; each g is independently 0 or 1; and x=0 to 3; with the proviso that at least one A is present and wherein at least one R is an organic group comprising a group selected from the group consisting of a dye, a Lanthanide complex, a magnetic resonance imaging (MM) contrast agent, a positron emission tomography (PET) agent, a gold nanoparticle, a silver nanoparticle, a quantum dot, an antibiotic or an antibacterial drug, an antimicrobial peptide, a polymer, a dendrimer, an ionophore, and combinations thereof.
6. A complex of a compound of claim 1 selected from the group consisting of a Ga(III) complex, an Fe(III) complex, an Al(III) complex, a V(IV) complex, a Zn(II) complex, an Y(III) complex, a Zr(VI) complex, a Cu(II) complex, and combinations thereof.
7. A complex of a compound of claim 4 selected from the group consisting of a Ga(III) complex, an Fe(III) complex, an Al(III) complex, a V(IV) complex, a Zn(II) complex, an Y(III) complex, a Zr(VI) complex, a Cu(II) complex, and combinations thereof.
8. A complex of a compound of claim 5 selected from the group consisting of a Ga(III) complex, an Fe(III) complex, an Al(III) complex, a V(IV) complex, a Zn(II) complex, an Y(III) complex, a Zr(VI) complex, a Cu(II) complex, and combinations thereof.
9. A method of detecting bacteria comprising: contacting a probe comprising a compound of formula I, II or III: ##STR00020## wherein: each X independently represents H and the vicinal Y represents H or -L-B; or each Y independently represents H and the vicinal X represents H or -L-B; each Z independently represents H or -L-B; each L independently represents an organic linking group; each B independently represents —OR, —NR.sub.2, —SR, —C(O)OR, —C(O)NR.sub.2, —S(O)R, or —SO.sub.2R; each R independently represents H or an organic group; each b is independently 0 or 1; each c is independently 0 or 1; and x=0 to 3; with the proviso that at least one of X, Y, or Z is present and represents -L-B; ##STR00021## wherein: each Q independently represents H or —(CH.sub.2).sub.n-D, wherein n is 1 to 8; each D independently represents —OR, —NR.sub.2, —SR, or —C(O)OR; each R independently represents H or an organic group; and each d is independently 2 to 5; with the proviso that at least one Q represents —(CH.sub.2).sub.n-D; or ##STR00022## wherein: each A independently represents —OR, —NR.sub.2, —SR, or —C(O)OR; each R independently represents H or an organic group; each n is independently 2 to 5; each e is independently 0 or 1; each f is independently 2 or 3; each g is independently 0 or 1; and x=0 to 3; with the proviso that at least one A is present, with a sample comprising a component selected from the group consisting of a bodily fluid, an isolated colony, a culture, and combinations thereof, under conditions effective for the probe to complex Fe(III), Al(III), V(IV), Zn(II), Y(III), Zr(IV), and/or Cu(II) present in the bodily fluid, the isolated colony, and/or the culture; and detecting the presence of Fe(III), Al(III), V(IV), Zn(II), Y(III), Zr(IV), and/or Cu(II)-probe complex to indicate the presence of bacteria in the bodily fluid, the isolated colony, and/or the culture; wherein for each of a compound of formula I, II or III, at least one R is an organic group, said organic group comprises a group selected from the group consisting of a dye, a Lanthanide complex, a magnetic resonance imaging (MRI) contrast agent, a positron emission tomography (PET) agent, a gold nanoparticle, a silver nanoparticle, a quantum dot, an antibiotic or an antibacterial drug, an antimicrobial peptide, a polymer, a dendrimer, an ionophore, and combinations thereof.
10. The method of claim 9 further comprising determining the concentration of the Fe(III), ARM), V(IV), Zn(II), Y(III), Zr(IV), and/or Cu(II)-probe complex to indicate the concentration of bacteria in the bodily fluid, the isolated colony, and/or the culture, wherein the concentration of the Fe(III), ARM), V(IV), Zn(II), Y(III), Zr(IV), and/or Cu(II)-probe complex is determined by a technique selected from the group consisting of fluorescence, positron emission tomography (PET), magnetic resonance imaging (MM), field microscopy, colorimetry, electrochemistry, mass spectrometry (MS), fluorescence spectroscopy, and combinations thereof.
11. The method of claim 9, wherein conditions effective for the probes and/or metal-probe complexes to be recognized by bacteria comprise contacting the probe and the sample for 1 minute to 1 hour at 10° C. to 40° C.
12. A method of detecting bacteria comprising: contacting a probe comprising a compound of formula I, II or III: ##STR00023## wherein: each X independently represents H and the vicinal Y represents H or -L-B; or each Y independently represents H and the vicinal X represents H or -L-B; each Z independently represents H or -L-B; each L independently represents an organic linking group; each B independently represents —OR, —NR.sub.2, —SR, —C(O)OR, —C(O)NR.sub.2, —S(O)R, or —SO.sub.2R; each R independently represents H or an organic group; each b is independently 0 or 1; each c is independently 0 or 1; and x=0 to 3; with the proviso that at least one of X, Y, or Z is present and represents -L-B; ##STR00024## wherein: each Q independently represents H or —(CH.sub.2).sub.n-D, wherein n is 1 to 8; each D independently represents —OR, —NR.sub.2, —SR, or —C(O)OR; each R independently represents H or an organic group; and each d is independently 2 to 5; with the proviso that at least one Q represents —(CH.sub.2).sub.n-D; or ##STR00025## wherein: each A independently represents —OR, —NR.sub.2, —SR, or —C(O)OR; each R independently represents H or an organic group; each n is independently 2 to 5; each e is independently 0 or 1; each f is independently 2 or 3; each g is independently 0 or 1; and x=0 to 3; with the proviso that at least one A is present, wherein for each of a compound of formula I, II or III, at least one R is an organic group, said organic group comprising a group selected from the group consisting of a dye, a Lanthanide complex, a magnetic resonance imaging (MRI) contrast agent, a positron emission tomography (PET) agent, a gold nanoparticle, a silver nanoparticle, a quantum dot, an antibiotic or an antibacterial drug, an antimicrobial peptide, a polymer, a dendrimer, an ionophore, and combinations thereof; with a sample comprising a bodily fluid, under conditions effective for the probe to complex Fe(III), Al(III), V(IV), Zn(II), Y(III), Zr(IV), and/or Cu(II) present in the bodily fluid; and detecting the presence of Fe(III), Al(III), V(IV), Zn(II), Y(III), Zr(IV), and/or Cu(II)-probe complex to indicate the presence of bacteria in the bodily fluid, wherein the sample comprising the bodily fluid is selected from the group consisting of urine, blood, cerebrospinal fluid (CSF), pleural fluid, a bacteria culture, an isolated colony reconstituted in media, and combinations thereof.
13. The method of claim 9, wherein the detected bacteria is Gram-negative or Gram-positive.
14. A method of determining susceptibility of bacteria to an antibiotic comprising: treating a sample comprising a component selected from the group consisting of a bodily fluid, a bacteria culture, a single colony reconstituted in media with an antibiotic, and combinations thereof; contacting a probe comprising a compound of formula I, II or III: ##STR00026## wherein: each X independently represents H and the vicinal Y represents H or -L-B; or each Y independently represents H and the vicinal X represents H or -L-B; each Z independently represents H or -L-B; each L independently represents an organic linking group; each B independently represents —OR, —NR.sub.2, —SR, —C(O)OR, —C(O)NR.sub.2, —S(O)R, or —SO.sub.2R; each R independently represents H or an organic group; each b is independently 0 or 1; each c is independently 0 or 1; and x=0 to 3; with the proviso that at least one of X, Y, or Z is present and represents -L-B; ##STR00027## wherein: each Q independently represents H or —(CH.sub.2).sub.n-D, wherein n is 1 to 8; each D independently represents —OR, —NR.sub.2, —SR, or —C(O)OR; each R independently represents H or an organic group; and each d is independently 2 to 5; with the proviso that at least one Q represents —(CH.sub.2).sub.n-D; or ##STR00028## wherein: each A independently represents —OR, —NR.sub.2, —SR, or —C(O)OR; each R independently represents H or an organic group; each n is independently 2 to 5; each e is independently 0 or 1; each f is independently 2 or 3; each g is independently 0 or 1; and x=0 to 3; with the proviso that at least one A is present, wherein for each of a compound of formula I, II or III, at least one R is an organic group, said organic group comprising a group selected from the group consisting of a dye, a Lanthanide complex, a magnetic resonance imaging (MRI) contrast agent, a positron emission tomography (PET) agent, a gold nanoparticle, a silver nanoparticle, a quantum dot, an antibiotic or an antibacterial drug, an antimicrobial peptide, a polymer, a dendrimer, an ionophore, and combinations thereof; with the sample comprising the bodily fluid, the isolated colony, and/or the culture under conditions effective for the probe to complex Fe(III); and determining the change in concentration of the compound or complex to indicate the concentration of bacteria in the bodily fluid, the bacteria culture, or the isolated colony after treatment with the antibiotic, wherein the difference between the initial concentration of bacteria and the concentration of bacteria in the bodily fluid, the bacteria culture, or the isolated colony after treatment with the antibiotic is an indication of the susceptibility of the bacteria to the antibiotic.
15. The method of claim 14, wherein the concentration of the Fe(III), Al(III), V(IV), Zn(II), Y(III), Zr(IV), and/or Cu(II)-probe complex is determined by a technique selected from the group consisting of fluorescence, positron emission tomography (PET), magnetic resonance imaging (MRI), field microscopy, colorimetry, electrochemistry, mass spectrometry (MS), fluorescence spectroscopy, and combinations thereof.
16. A compound selected from the group consisting of: ##STR00029## ##STR00030## ##STR00031##
17. The method of claim 9, wherein the presence of the Fe(III), V(IV), Zn(II), Y(III), Zr(IV), and/or Cu(II)-probe complex is detected by a technique selected from the group consisting of fluorescence, positron emission tomography (PET), magnetic resonance imaging (MRI), field microscopy, colorimetry, electrochemistry, mass spectrometry (MS), fluorescence spectroscopy, and combinations thereof.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
(34) Siderophores as Markers of Bacterial Virulence
(35) Iron is an essential element for all but one known organism, including pathogenic bacteria. The oxidized form is insoluble, while the reduced form is highly toxic, and the human body efficiently chelates both forms by iron and heme-carrier proteins. Consequently, the free ferric ion concentration available to bacteria in serum is maintained at approximately 10.sup.−24M (Raymond et al., Proceedings of the National Academy of Sciences. 2003; 100(7):3584-8). In response to the paradox of approximately 10.sup.−6 M concentration required for microbial growth (Braun et al., Trends in Biochemical Sciences. 1999; 24(3):104-9), and the negligible amount of free iron available in vivo, bacteria have developed intricate systems to sequester iron from their environment. Of these, during infection, Gram-negative pathogens rely primarily on siderophores to acquire their iron. Siderophores are small molecule iron chelators excreted by microorganisms to sequester iron from their environment and bring it into bacteria. Siderophores have been identified as important virulence factors for every type of pathogenic Gram-negative bacteria (Caza et al., Frontiers in Cellular and Infection Microbiology. 2013; 3. doi: 10.3389/fcimb.2013.00080; Beceiro et al., Clinical Microbiology Reviews. 2013; 26(2):185-230). As discussed herein, siderophores may have great potential for the development of novel technology for bacterial detection, a potential that is yet untapped.
(36) All clinical isolates of the Enterobacteriaceae, including E. coli (Raymond et al., Proceedings of the National Academy of Sciences. 2003; 100(7):3584-8, Watts et al., Infect Immun. 2012; 80(1):333-44), Klebsiella (El Fertas-Aissani et al., Pathol Biol. 2013; 61(5):209-16), and Enterobacter species (Mokracka et al., FEMS Immunol Med Microbiol. 2004; 40(1):51-5), the microorganisms that cause greater than 90% of cases of urinary tract infection (UTI), synthesize and take up the catechol-based siderophore enterobactin (
(37) Importantly, urine samples are not completely sterile. Low concentration of bacteria present in the sample is not indicative of UTI. The most commonly used criterion for defining significant bacteriuria is the presence of ≥10.sup.5 cfu per mL of urine (Stamm et al., N Engl J Med. 1982; 307(8):463-8; Wilson et al., Clinical Infectious Diseases. 2004; 38(8):1150-8). For practical diagnosis of UTI, it is therefore important not only to be able to detect the presence of bacteria, but also to be able to determine the concentration of the bacteria. This is a distinct advantage of fluorescence assays over gene-based assays: not only can they indicate the concentration of the bacteria, but they can also be tuned to determine the concentration of bacteria between 10.sup.3 and 10.sup.7 CFU/mL, as needed for the accurate diagnosis of UTI.
(38) Detection of E. coli with a Fluorescent Siderophore Probe
(39) The structural parameters of enterobactin necessary for its recognition by the outer-membrane receptor FepA and ATP-dependent uptake by E. coli and other bacteria are known. While modification of the trilactone backbone does not inhibit uptake of the siderophore, modification of the catecholamide moieties does (Raymond et al., Proceedings of the National Academy of Sciences. 2003; 100(7):3584-8). Among the derivatives studied, TREN-CAM is recognized by FepA and actively taken up by E. coli. (Thulasiraman et al., J Bacteriol. 1998; 180(24):6689-96). Herein we have investigated modifying the natural product further by incorporating a chemical handle, such as a carboxylic acid, on the TREN backbone, an dextended TREN backbone, or a linear cap. The carboxylic acid would in turn enable further conjugation of a detection group or a drug, effectively changing the siderophore into an effective vector. This strategy exploits the siderophore uptake system of bacteria, which are key to their virulence, as a gate into the bacterial envelope. With this in mind, we designed and synthesized entero-light (
(40) Following the protocol detailed below (Sample Processing for bacteria detection by fluorescence in T media), we determined that our class of probes can detect pathogenic Gram-negative bacteria such as E. coli, K pneumoniae, Salmonella, A. baumannii, B. subtilus, and S. aureus with substantial turn-on. In each case, at least a four-fold increase in fluorescence intensity was observed in the presence of bacteria. This protocol required little steps: a short incubation followed by a simple centrifugal wash. (Importantly, this washing step can be eliminated with turn-on probes such as Ga-2,2-LiCAM-GluCAM-light switch—example 6 below). Comparison of the different examples of probes indicate that Ga-3,3-LiCAM-GluCAM gives a higher turn-on. Notably, when not conjugated to an enterobactin analogue, the OREGON GREEN fluorescent dye does not accumulate in bacteria. This control experiment highlights the benefits of incorporating our enterobactin targeting vectors in the design of probes for bacteria imaging.
(41) Further comparison of the Ga(III), Fe(III) and apo (non-metallated) probe indicate that, although not necessary, the gallium(III) and iron(III) probes yield greater response. It is known that only metal complexes of siderophore are recognized by the enterobactin receptor FepA. Although the incubation media, T-media, is deprived of iron, it is not absolutely iron-free. The apo probe efficientlchelates out any residual iron from the media, and it is this iron complex that is taken up by the bacteria. Moreover, the iron probe exhibits a lower response than the gallium probe. This observation is likely due either to partial quenching of the fluorescence of the OREGON GREEN dye by the iron(III) ion, or by lower uptake of the Fe(III) probe than the Ga(III) one. This is predicted on the basis that, as opposed to the Fe(III) probe, uptake of the Ga(III) complex would not trigger the Fur (ferric iron uptake regulator) receptor. In any case, this result confirms that the gallium complexes presented are recognized by the FepA receptor. This conclusion is further supported by the observation that the uptake of the Ga(III), Fe(III), and unmetallated 3,3-LiCAM-GluCAM-OG probe decreases substantially if the media is supplemented by iron. Importantly, varying the incubation time indicates that the probes are taken up rapidly and efficiently in as little as 10 minutes. The protocol can thus be shortened to include a brief 10 minute incubation time (as opposed to a 20 minute incubation time).
(42) Importantly, this class of probe is not limited to the detection of bacteria by fluorescence. As shown in
(43) Compounds and Complexes
(44) In one embodiment, the present disclosure provides a compound of the formula:
(45) ##STR00004##
wherein: each X independently represents H and the vicinal Y represents H or -L-B; or each Y independently represents H and the vicinal X represents H or -L-B; each Z independently represents H or -L-B; each L independently represents an organic linking group; each B independently represents —OR, —NR.sub.2, —SR, —C(O)OR, —C(O)NR.sub.2, —S(O)R, or —SO.sub.2R; each R independently represents H or an organic group; each b is independently 0 or 1; each c is independently 0 or 1; and x=0 to 3; with the proviso that at least one of X, Y, or Z is present and represents -L-B. In some embodiments, each R can independently represent an organic moiety.
(46) In some embodiments, each organic linking group L can independently represent a straight-chain group of the formula —(CH.sub.2).sub.n— or —(CH.sub.2—CH.sub.2—O).sub.n—, wherein n is 1 to 8. In certain embodiments, each organic linking group L can independently represent a branched chain dendrimer that contains one or more of C, N, S, and O. In some certain embodiments, each organic linking group L can independently represent an organic moiety.
(47) In another embodiment, the present disclosure provides a compound of the formula:
(48) ##STR00005##
wherein: each Q independently represents H or —(CH.sub.2).sub.n-D; each D independently represents —OR, —NR.sub.2, —SR, or —C(O)OR; each R independently represents H or an organic group; and each d is independently 2 to 5; with the proviso that at least one Q represents —(CH.sub.2).sub.n-D. In some embodiments, each R can independently represent an organic moiety.
(49) In another embodiment, the present disclosure provides a compound of the formula:
(50) ##STR00006##
wherein: each A independently represents —OR, —NR.sub.2, —SR, or —C(O)OR; each R independently represents H or an organic group; each n is independently 2 to 5; each e is independently 0 or 1; each f is independently 2 to 3; each g is independently 0 or 1; and x=0 to 3; with the proviso that at least one A is present. In some embodiments, each R can independently represent an organic moiety.
(51) In some embodiments of compounds of Formula I, Formula II, and/or Formula III, R can be an organic group including a group selected from the group consisting of a dye, a Lanthanide complex, a magnetic resonance imaging (MRI) contrast agent, a positron emission tomography (PET) agent, a gold nanoparticle, a silver nanoparticle, a quantum dot, an antibiotic or an antibacterial drug, an antimicrobial peptide, a polymer, a dendrimer, an ionophore, and combinations thereof.
(52) For embodiments in which for compounds of Formula I, Formula II, and/or Formula III R represents an organic group including a dye, a wide variety of dyes can be used including, for example, fluorescent dyes such as fluorescein dyes, rhodamine dyes, thiozole orange, ethidium dyes, Alexa fluor dyes, coumarin dyes, cyanine dyes, SYBR dyes, SYTO dyes, acridine dyes, propidium dyes, TOPRO, TOTO, YOYO, YOPRO, BOBO, and BODIPY dyes. Exemplary dyes include, but are not limited to, dyes available under the trade designation ALEXA FLUOR from Thermo Fisher Scientific, dyes available under the trade designation TAMRA from Thermo Fisher Scientific, dyes available under the trade designation OREGON GREEN from Thermo Fisher Scientific, dyes available under the trade designation SYBR from Thermo Fisher Scientific, dyes available under the trade designation CY from Thermo Fisher Scientific, dyes available under the trade designation BODIPY from Thermo Fisher Scientific, dyes available under the trade designation PACIFIC from Thermo Fisher Scientific (e.g., PACIFIC GREEN, PACIFIC BLUE, AND PACIFIC ORANGE), coumarin dyes, DNA stain, and quantum dots available under the trade designation Qdot from Thermo Fisher Scientific.
(53) For embodiments in which for compounds of Formula I, Formula II, and/or Formula III R represents an organic group including a positron emission tomography (PET) agent, a wide variety of a PET agents can be used. Exemplary PET agents include, but are not limited to, .sup.18F probes such as charged chelated aluminum fluoride complexes; .sup.18F probes such as conjugated .sup.18FDG, .sup.64Cu probes such as charged chelated copper complexes; .sup.67Ga and .sup.68Ga probes such as conjugated chelated gallium complexes; .sup.94mTc probes such as chelated technetium complexes, and .sup.89Zr probes such as chelated zirconium complexes.
(54) For embodiments in which for compounds of Formula I, Formula II, and/or Formula III R represents an organic group including an antiobiotic or an antibacterial drug, a wide variety of antibiotics and/or antibacterial drugs can be used. Exemplary antibiotics and/or antibacterial drugs include, but are not limited to, polyether antibiotics such as monensin; β-lactam antibiotics such as penicillins, amoxicillin, cephalosporins, and cephalexin; antibacterial drugs such as salinomycin; gramicidin; ciprofloxacin; metronidazole; sulfamethoxazole; trimethopin; levoflaxin; polymyxins; rifamycins; lipiarmycins; quinolones; sulfonamides; macrolide antibiotics such as azithromycin; lincosamides such as clindamycin; tetracyclines such as doxycycline and tigecycline; daptomycin; oxazolidinone antibiotics; and coumarin antibiotics such as aminocoumarin.
(55) For embodiments in which for compounds of Formula I, Formula II, and/or Formula III R represents an organic group including an ionophore, a wide variety of ionophores can be used. Exemplary ionophores include, but are not limited to, channel compounds, modified hydraphiles, and modified calixarenes such as ionomycin and A23187; and crown ethers.
(56) In another aspect, the present disclosure provides a complex of a compound of Formula I, Formula II, and/or Formula III selected from the group consisting of a Ga(III) complex, an Fe(III) complex, an ARM) complex, a V(IV) complex, a Zn(II) complex, an Y(III) complex, a Zr(VI) complex, a Cu(II), and combinations thereof.
(57) Methods of Detecting Bacteria
(58) In another aspect, the present disclosure provides a method of detecting bacteria including: contacting a probe including a compound or complex as disclosed herein with a sample including a component selected from the group consisting of a bodily fluid, an isolated colony, a culture, and combinations thereof, under conditions effective for the probe to complex Fe(III), Al(III), V(IV), Zn(II), Y(III), Zr(IV), and/or Cu(II) present in the bodily fluid, the isolated colony, and/or the culture; and detecting the presence of Fe(III), ARM), V(IV), Zn(II), Y(III), Zr(IV), and/or Cu(II)-probe complex to indicate the presence of bacteria in the bodily fluid, the isolated colony, and/or the culture.
(59) In some embodiments, the presence of the Fe(III), Al(III), V(IV), Zn(II), Y(III), Zr(IV), and/or Cu(II)-probe complex is detected by a technique selected from the group consisting of fluorescence, positron emission tomography (PET), magnetic resonance imaging (MM), field microscopy, colorimetry, electrochemistry, mass spectrometry (MS), fluorescence spectroscopy, and combinations thereof.
(60) In certain embodiments, the method further includes determining the concentration of the Fe(III), ARM), V(IV), Zn(II), Y(III), Zr(IV), and/or Cu(II)-probe complex to indicate the concentration of bacteria in the bodily fluid, the isolated colony, and/or the culture. For example, the concentration of the Fe(III), Al(III), V(IV), Zn(II), Y(III), Zr(IV), and/or Cu(II)-probe complex can be determined by a technique selected from the group consisting of fluorescence, positron emission tomography (PET), magnetic resonance imaging (MM), field microscopy, colorimetry, electrochemistry, mass spectrometry (MS), fluorescence spectroscopy, and combinations thereof.
(61) Exemplary conditions for the probes and/or metal-probe complexes to be recognized by bacteria include contacting the probe and the sample for 1 minute to 1 hour at 10° C. to 40° C.
(62) A wide variety of bodily fluids can be used with the methods for detecting bacteria disclosed herein. Exemplary bodily fluids include, but are not limited to, urine, blood, cerebrospinal fluid (CSF), pleural fluid, a bacteria culture, an isolated colony reconstituted in media, and combinations thereof.
(63) In some embodiments, the methods disclosed herein can detect Gram-negative or Gram-positive bacteria.
(64) A wide variety of Gram-negative bacteria can be detected by the methods disclosed herein. Exemplary Gram-negative bacteria that can be detected by the methods disclosed herein include, but are not limited to, Enterobacteriaceae, Mycobacteria tuberculosis, Acinetobacter baumannii, Pseudomonas aeruginosa, Salmonella sp., Escherichia coli, Yersinia pestis, Yersinia enterocolotica, Shigella sp., Proteus mirabilis, Klebsiella oxytoca, Klebsiella pseumoniae Enterobacter, Neisseria meningitidis, Neisseria gonorrhea, Serratia marcescens, and combinations thereof.
(65) A wide variety of Gram-positive bacteria can be detected by the methods disclosed herein. Exemplary Gram-positive bacteria that can be detected by the methods disclosed herein include, but are not limited to, Gram positive Staphylococcus aureus, Enterococcus sp, Streptococcus sp, or a combination thereof.
(66) Methods of Determining the Susceptibility of Bacteria to an Antibiotic
(67) In another aspect, the present disclosure provides a method of determining susceptibility of bacteria to an antibiotic including: treating a sample including a component selected from the group consisting of a bodily fluid, a bacteria culture, a single colony reconstituted in media with an antibiotic, and combinations thereof contacting a probe including a compound or complex of Formula I, Formula II, and/or Formula III with the sample including the bodily fluid, the isolated colony, and/or the culture under conditions effective for the probe to complex Fe(III); and determining the change in concentration of the compound or complex to indicate the concentration of bacteria in the bodily fluid, the bacteria culture, or the isolated colony after treatment with the antibiotic, wherein the difference between the initial concentration of bacteria and the concentration of bacteria in the bodily fluid, the bacteria culture, or the isolated colony after treatment with the antibiotic is an indication of the susceptibility of the bacteria to the antibiotic.
(68) In some embodiments, the method can further include determining the initial concentration of the bacteria in the bodily fluid, the bacteria culture, or the single colony by a method disclosed herein above. In certain embodiments, the concentration of the Fe(III), ARM), V(IV), Zn(II), Y(III), Zr(IV), and/or Cu(II)-probe complex can be determined by a technique selected from the group consisting of fluorescence, positron emission tomography (PET), magnetic resonance imaging (MRI), field microscopy, colorimetry, electrochemistry, mass spectrometry (MS), fluorescence spectroscopy, and combinations thereof.
(69) Exemplary conditions for the probes and/or metal-probe complexes to be recognized by bacteria include contacting the probe and the sample for 1 minute to 1 hour at 10° C. to 40° C.
(70) A wide variety of bodily fluids can be used with the methods for detecting bacteria disclosed herein. Exemplary bodily fluids include, but are not limited to, urine, blood, cerebrospinal fluid (CSF), pleural fluid, a bacteria culture, an isolated colony reconstituted in media, and combinations thereof.
(71) In some embodiments, the methods disclosed herein can detect Gram-negative or Gram-positive bacteria.
(72) A wide variety of Gram-negative bacteria can be detected by the methods disclosed herein. Exemplary Gram-negative bacteria that can be detected by the methods disclosed herein include, but are not limited to, Enterobacteriaceae, Mycobacteria tuberculosis, Acinetobacter baumannii, Pseudomonas aeruginosa, Salmonella sp., Escherichia coli, Yersinia pestis, Yersinia enterocolotica, Shigella sp., Proteus mirabilis, Klebsiella oxytoca, Klebsiella pseumoniae Enterobacter, Neisseria meningitidis, Neisseria gonorrhea, Serratia marcescens, and combinations thereof.
(73) A wide variety of Gram-positive bacteria can be detected by the methods disclosed herein. Exemplary Gram-positive bacteria that can be detected by the methods disclosed herein include, but are not limited to, Gram positive Staphylococcus aureus, Enterococcus sp, Streptococcus sp, or a combination thereof.
(74) Methods of Treating a Disease Caused by Bacteria
(75) In another aspect, the present disclosure provides a method of treating a patient having a disease caused by bacteria comprising: administering to the patient under conditions effective to treat the disease a probe comprising a compound or complex as described herein, wherein at least one R represents an organic group comprising an antiobiotic.
Additional Illustrative Embodiments
Embodiment A: Ligand 1 and its Complexes with Ga and Fe Structure of Ligand
(76) ##STR00007##
wherein A can be linear or branched, with linear including alkane or PEG chains with or without the presence of N (nitrogen), with alkane defined as a succession of methylenes (CH.sub.2).sub.n where n=1,2,3,4,5,6,7,8; with PEG defined as a succession of (CH.sub.2—CH.sub.2—O).sub.n where n=1,2,3,4,5,6,7,8; wherein branched is defined as a dendrimer that contains one or more of C, N, and O; wherein B is N, O or S; wherein R is selected from the group consisting of H, an organic group; a dye (e.g., ALEXA FLUOR dyes, TAMRA dyes, OREGON GREEN dyes), Lanthanide complexes, Mill contrast agents (Gd-DTPA, Gd-DOTA, Gd-DO3A); PET agents; gold nanoparticles; silver nanoparticles; quantum dots; antibiotics, antimicrobial peptides; polymers, dendrimers, and ionophores.
(77) Exemplary species of Embodiment A include ligands and complexes of the formulas illustrated in
Embodiment B
(78) ##STR00008##
wherein A can be linear or branched, with linear including alkane or PEG chains with or without the presence of N (nitrogen), with alkane defined as a succession of methylenes (CH2).sub.n where n=1,2,3,4,5,6,7,8; with PEG defined as a succession of (CH.sub.2—CH.sub.2—O).sub.n where n=1,2,3,4,5,6,7,8; wherein branched is defined as a dendrimer that contains one or more of C, N, and O; wherein B is N, O or S; wherein R is selected from the group consisting of H, an organic group; a dye (e.g., ALEXA FLUOR dyes, TAMRA dyes, OREGON GREEN dyes), Lanthanide complexes, MRI contrast agents (Gd-DTPA, Gd-DOTA, Gd-DO3A); PET agents; gold nanoparticles; silver nanoparticles; quantum dots; antibiotics, antimicrobial peptides; polymers, dendrimers, and ionophores.
(79) Exemplary species of Embodiment B include ligands and complexes of the formulas illustrated in
(80) The present disclosure is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the disclosure as set forth herein.
EXAMPLES
(81) Results
(82) General Consideration
(83) Unless otherwise noted, starting materials were obtained from commercial suppliers and used without further purification. Water was distilled and further purified by a Millipore cartridge system (resistivity 18×10.sup.6Ω). .sup.1H NMR spectra were recorded on a Varian 300 at 300 MHz or on a Bruker AX 400 at 400 MHz and .sup.13C NMR spectra were recorded on a Varian 300 at 75 MHz at the Le Claire-Dow Characterization Facility of the Department of Chemistry of the University of Minnesota; the solvent residual peak was used as an internal reference. .sup.1H NMR data are reported as follows: chemical shift (δ, ppm), multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet), integration, coupling constant (Hz). Mass spectra (LR=low resolution; HR=high resolution; ESI MS=electrospray ionization mass spectrometry) were recorded on a Bruker BioTOF II at the Waters Center for Innovation for Mass Spectrometry Facility at the Department of Chemistry at the University of Minnesota, Twin-Cities. Analytical HPLC was performed on a Variant pro star instrument (Agilent, Santa Clara, Calif.) equipped with a diode array detector and thermostat set at 25° C., and a Zorbax Eclipse XDB-C18 column (9.4×250 mm, 5 μm, Agilent, Santa Clara, Calif.). The mobile phase of a binary gradient (Method 1: 2 minutes 2% B, 13 minutes 2 to 80% B, 2 minutes, 80% B, 3 minutes 80 to 100% B, 1 minute 100% B, Method 2: 2 minutes 5% B, 8 minutes 5 to 80% B, 3 minutes 80% B, 3 minutes 90 to 100% B, 1 minute 100%, 3 minutes 100 to 5% B 2 minutes 5% B or Method 2: 10-60% B, Method 4: 2 minutes 15% B, 20 minutes 15 to 100% B, 3 minutes, 100% B, 4 minutes 100 to 15% B, 1 minute) where A is water and B is acetonitrile at a flow rate of 1 mL/minutes was used for analytical and preparative HPLC Varian Cary Eclipse fluorescence spectrometer with temperature controlled cells, and plate reader. 384 black plate square wells, flat bottom microplates available under the trade designation CORNING 3577 from Corning.
Example 1: Ga-TREN-CAM-C2-OG
(84) ##STR00009##
(85) The synthesis of TREN-CAM-C2-OG was performed in 8 steps as illustrated in
(86) A Mitsunobu reaction converted the alcohol to an iodo derivative 2b (Mandal et al., J Med Chem 2009, 52 (19), 6126-6141). The tripodal protected cap amine (3) was then obtained by alkylation of the di-Z-protected diethylendiamine (DiZDETA) in DMF using potassium carbonate as a base. Note that the nature of the base affects the product distribution ratio, with potassium carbonate increasing the yield of the desired compound while reducing the cyclization of the iodo-derivative Glu(O.sup.tBu)I on itself. Higher yields of the TREN cap were obtained using the iodo derivative of glutamic acid, as opposed to the bromo, chloro or triflate ones. The Z protective groups of the TREN cap were then removed by hydrogenation. The protected ligand (5) was obtained by acylation of the tripodal free amine (4) with CAM(Bn.sub.2) acid.
(87) The synthetic siderophore was deprotected in acid conditions and then complexed with gallium in neutral conditions. TREN-CAM-C2-OG (8) is obtained after coupling with OREGON GREEN dye.
(88) 2a: Z-Glu(O.sup.tBu)OH
(89) N,N′-diisopropylcarbodiimide (DIC, 1.38 g, 6.70 mmol, 1.13 eq.) was added to a solution of Z-Glu(O.sup.tBu)COOH (1, 2.00 g, 5.93 mmol, 1.00 eq.) in ethyl acetate (20 mL), resulting in a precipitate. 2-Mercaptopyridine (0.720 g, 6.52 mmol, 1.10 eq.) was then added to the reaction mixture which turned yellow. After 4 hours of stirring at room temperature a precipitate was filtered out and the filtrate was concentrated under reduced pressure. The oily residue (0.300 g) was dissolved in 1,4-dioxane (10 mL) and cooled to 0° C. NaBH.sub.4 (0.800 g, 21.0 mmol, 3.60 eq.) was added to the reaction mixture which was stirred for 30 minutes at 0° C. The reaction was monitored by thin layer chromatograph (TLC). The reaction mixture was then quenched with a 2% KHSO.sub.4 (aq) solution and extracted with diethylether (3×100 mL). The organic phases were combined and washed with a 5% HCO.sub.3 (aq) and dried with MgSO.sub.4 (s). The solvent was removed under reduced pressure and the residual colorless oil was purified by flash chromatography over silica using a mixture of 30% ethyl acetate in hexanes as the eluent, yielding 2a as a white sticky solid (1.20 g, 62.8%).
(90) [M+Na].sup.+ 346 m/z, [2M+Na].sup.+ 669.3 m/z.
(91) .sup.1H NMR (300 MHz, CDCl.sub.3): (δ, ppm): 1.43; (s, 9H), 1.87-1.78; (m, 2H), 2.35-2.30; (m, 2H), 3.69-3.54; (m, 3H), 5.08; (s, 2H), 5.25; (d, NH, J=9.0 Hz), 7.37-7.26; (m, 5H).
(92) .sup.13C NMR (75 MHz, CDCl.sub.3): (δ, ppm): 26.55, 28.73, 32.66, 53.65, 65.39, 67.51, 81.59, 128.60, 128.82, 129.22, 137.05, 157.27, 174.00.
(93) 2b: Z-Glu(O.sup.tBu)I
(94) Iodine (2.82 g, 11.1 mmol, 3.00 eq) was added to a solution of triphenyl phosphine (2.92 g, 11.1 mmol, 3.00 eq.) and imidazole (1.26 g, 18.6 mmol, 5.00 eq.) in anhydrous dichloromethane (100 mL). The reaction mixture was stirred under N.sub.2 (g) for 30 minutes after which Z-Glu(O.sup.tBu)OH (2a, 1.20 g, 3.71 mmol, 1.00 eq.) was added. The reaction mixture was then stirred for 4 hours at room temperature.
(95) The solvent was removed under reduced pressure and the residue was purified by flash chromatography over silica with 35% ethyl acetate in hexane as the eluent yielding 2b as a white sticky solid (1.17 g, 72.5%).
(96) ESI-MS m/z=456.0 ([M+Na].sup.+) (Calcd. 456.0), 472.0 m/z ([M+K].sup.+) (Calcd. 472.1).
(97) .sup.1H NMR (300 MHz, CDCl.sub.3) (δ, ppm)=1.40; (s, 9H), 1.82; (m, 2H), 2.28; (m, 2H), 3.28; (m, 1H), 3.36; (m, 1H), 3.50; (m, 1H), 5.06; (s, 2H), 5.17; (d, NH, J=9.0 Hz), 7.31; (m, 5H).
(98) .sup.13C NMR (75 MHz, CDCl.sub.3) (δ, ppm)=14.87, 28.73, 30.37, 32.49, 51.13, 67.51, 81.46, 128.74, 128.84, 129.20, 136.97, 156.39, 173.01.
(99) 3: Z.sub.3-TREN-C2 (O.sup.tBu)
(100) Di-Z-DETA (500 mg, 1.35 mmol, 1.00 eq.) and K.sub.2CO.sub.3 (279 mg, 2.02 mmol, 1.50 eq) were dissolved in DMF (2 mL) and heated to 80° C. for 30 minutes. Z-Glu(O.sup.tBu)I (2b, 583 mg, 1.35 mmol, 1.00 eq) was diluted in DMF (2 mL) and added to the reaction mixture, which was heated at 80° C. for 15 hours. The solvent was removed under reduced pressure. The residue was diluted in dichloromethane (10 mL) and washed with water (2×10 mL). The organic phase was then dried with sodium sulfate. The residue was purified over reverse phase (C18) resin using a gradient of CH.sub.3CN in water to yield the protected TREN cap 3 as an oil. The solvent was removed under reduced pressure. The residue was diluted in dichloromethane (10 mL) and washed with water (2×10 mL). The organic phase was then dried with sodium sulfate. The residue was purified by flash chromatography (silica) using a gradient of (0 to 4%) methanol in DCM (0.163 g, 18%).
(101) ESI-MS m/z=[M+H].sup.+ 677.4 m/z (Calcd. 677.3), [M+K].sup.+ 715 m/z (Calcd. 715).
(102) .sup.1H NMR (400 MHz, CDCl.sub.3) (δ, ppm)=1.40; (s, 9H), 1.43; (t, 2H, J=8.0), 2.28-2.26; (m, 2H), 2.35; (m, 1H) 2.48-2.45; (m, 3H), 2.60; (m, 2H), 3.22; (m, 4H), 5.11; (m, 1H), 5.30; (s, 6H), 7.26; (m, 15H).
(103) .sup.13C NMR (400 MHz, CDCl.sub.3) (δ, ppm)=172.9, 162.7, 157.1, 141.17, 136.7, 136.5, 128.7, 128.5, 128.4, 128.4, 128.1, 128.0, 127.9, 127.8, 127.4, 126.9, 80.6, 79.5, 77.3, 64.99, 53.4, 50.5, 36.5, 29.7, 28.5, 28.0.
(104) 4: TREN-C2-(O.sup.tBu)
(105) A speck of Pd/C was added to a solution of Z.sub.3-TREN-C2 (O.sup.tBu) (3, 0.250 g, 0.369 mmol) in methanol (1 mL). The reaction mixture was placed in a Parr hydrogenator which was charged with H.sub.2 (g) to 50 psi. The Pd/C was filtered and the solvents removed under reduced pressure to yield the cap 4 as an oil (119 mg, quantitative).
(106) ESI-MS m/z=275.2 m/z ([M+H].sup.+) (Calcd. 275.4).
(107) .sup.1H NMR (400 MHz, CD.sub.3OD) (δ, ppm)=1.45; (s, 9H), 1.86-1.93; (m, 4H), 2.17; (m, 1H), 2.55; (m, 3H), 2.74; (m, 4H), 2.97; (m, 2H).
(108) .sup.13C NMR (100.62 MHz, CD.sub.3OD) δ 172.12, 80.79, 56.75, 51.38, 50.84, 49.17, 36.98, 34.09, 30.79, 29.51, 27.01 ppm.
(109) 5: TREN-C2-CAM(Bn)
(110) TREN C2 (4, 20.0 mg, 0.073 mmol, 1 eq) and CAM thiaz (95.0 mg, 0.218 mmol 3 eq), were dissolved in DCM (7 mL) and DIPEA (100 mg, 0.774 mmol, 10.6 eq) for 5 days. The solvent was removed and the residue purified by flash chromatography silica and a gradient of methanol in DCM (26.0 mg, 29.1%).
(111) ESI-MS m/z=[M+H].sup.+ 1223.5 m/z (Calcd. 1223.5) [M+Na].sup.+ 1245.5 m/z (Calcd. 1245.5).
(112) .sup.1H NMR (CDCl.sub.3, 400 MHz): δ 7.77-7.03; (m, 39H), 5.12; (m, 12H), 4.0; (m, 1H), 3.29-2.09; (m, 11H), 1.70; (m, 1H), 1.43; (m, 2H), 1.38; (s, 9H).
(113) .sup.13C NMR (100.62 MHz, CD.sub.3OD): δ 172.4, 165.5, 151.7, 151.5, 146.8, 146.7, 136.6, 136.6, 136.5, 136.4, 129.3, 128.9, 128.8, 128.8, 128.7, 128.7, 128.6, 128.6, 128.5, 128.4, 128.2, 128.1, 127.8, 127.6, 127.5, 125.0, 124.5, 124.2, 124.1, 123.2, 123.0, 119.0, 116.9, 116.7, 80.1, 77.2, 76.2, 76.1, 71.6, 71.3, 71.2, 58.2, 53.4, 50.9, 37.1, 32.2, 29.7, 28.0.
(114) 6: TREN-C2-CAM
(115) TREN-C2-CAM(Bn) (5, 10.3 mg, 84.2 μmol) was diluted in AcOH (1 ml) and HCl (1 mL). The solution was stirred for 4 days. The solvent was removed using the schlenk line. DCM (2 mL) was added then removed with rotavap twice. Then methanol (2 mL) was added and removed with the rotavap (3.19 mg, 60%).
(116) ESI-MS m/z=[M+H].sup.+ 627.5 m/z (Calcd. 627.2) [M+(MeOH).sub.2].sup.+ 691.5 m/z (Calcd. 691.3),) [M+Na].sup.+ 649.5 m/z (Calcd. 649.2).
(117) .sup.1H NMR MeOD 400 MHz: δ 7.35; (3H, dd, 1.2 Hz, 8.0 Hz), 7.01; (3H, dd, 1.6 Hz, 8.0 Hz), 6.74; (3H, t, 8.0 Hz), 4.65; (1H, m), 3.85-3.55; (4H, m), 3.24-2.6; (4H, m), 2.46-2.1; (2H, m), 1.8-1.45; (2H, m). .sup.13C NMR MeOD 400 MHz: δ 181.9, 172.5, 150.3, 145.5, 120.4, 120.1, 118.3, 112.7, 61.19, 59.96, 46.11, 23.96, 17.3.
(118) A UV-Visible absorbance spectrum of TREN-C2-CAM is illustrated in
(119) 7: Ga-TREN-C2-CAM
(120) A solution of Ga(acac).sub.3 (6, 1.0 mg, 2.8 μmol 1 eq) in methanol (0.5 ml) was added to a solution of TREN-C2-CAM (1.7 mg, 2.8 μmol, 1 eq). pH was adjusted to 7 with a drop of KOH in methanol 0.5 M. (1.3 mg, 75%).
(121) 8: Ga-TREN-CAM-C2-OG
(122) Ga-TREN-C2-CAM (7, 1.3 mg, 2.1 mmol, 1 eq), OREGON GREEN (OG) dye (1.0 mg, 2.1 mmol, 1 eq) 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (0.5 mg, 2.4 mmol, 1. Eq.) were dissolved in DMF (1 mL). Reaction was stirred at room temperature for two days. The solvent was speed vacuumed and the residue was purified by C18 using a gradient of CH.sub.3CN in water. Fraction two led to a 30 μM solution (3.6 ml).
(123) A UV-visible spectrum of the final probe, Ga-TREN-C2-CAM-OG is illustrated in
Example 2: Ga-TREN-GlyCAM-GluCAM-OG
(124) ##STR00010##
(125) The synthesis of Ga-TREN-GlyCAM-GluCAM-OG (22) is illustrated in
(126) 16: Gly-CAM(Bn)
(127) CAM(Bn) (502 mg, 1.50 mmol) and NHS (208 mg, 1.81 mmol) were disolved in 5 mL of 1,4-dioxane. The crude obtained was cooled down to 11° C., and then DIC (238.5 mg, 1.89 mmol) was added. The system was magnetically stirred at room temperature for 24 hours. Next, a mixture of NaHCO.sub.3 (120 mg, 1.43 mmol) and Glycine (134 mg, 1.79 mmol) disolved in 2 mL of 1,4-dioxane were added to the crude. The suspension obtained was magnetically stirred at room temperature under N.sub.2 gas. After 22 hours, 6 mL of water were added. 3 hours later, the solvent was reduced to ⅓ its original volume. Then, HCl 1 M was dropwise added until pH 2 was reached. The product was extracted form the crude via liquid-liquid extraction using DCM and water as solvents. The organic phase was collected and dried over Na.sub.2SO.sub.4, the solvent evaporated and the yellowish oil applied to a silica gel column and subsequently eluted with MeOH (1-8%) in DCM. (249.5 mg, 42%).
(128) .sup.1H-NMR (400 MHz, CD.sub.2Cl.sub.2) δ 4.05; (d, 2H, CH.sub.2), 5.13; (s, 2H, CH.sub.2), 5.16; (s, 2H, CH.sub.2), 7.17; (m, 2H, ArH), 7.28; (m, 3H, ArH), 7.39; (m, 5H, ArH), 7.48; (m, 2H, ArH), 7.63; (dd, 1H, ArH), 8.48; (t, 1H, NH).
(129) 17: Glu-CAM(Bn)
(130) A solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 343 mg, 7.63 mmol, 1.20 eq.) in 1,4-dioxane (2.2 mL) was added dropwise to a solution of CAM(Bn.sub.2) (500 mg, 1.49 mmol, 1.00 eq.) and N-hydroxysuccinimide (NHS, 206 mg, 1.79 mmol, 1.20 eq.) in 1,4-dioxane (2 mL) and cooled to 4° C. The solution was allowed to return to room temperature and stirred at room temperature for 16 hours. The crude reaction mixture was filtered. A solution of glutamic-γ-tertbutylester (395 mg, 1.94 mmol, 1.30 eq.) and sodium bicarbonate (163 mg, 1.94 mmol, 1.30 eq.) in water (8 mL) was added to the yellowish filtrate. The reaction mixture was stirred at room temperature for 47 hours. The reaction mixtue was then concentrated under reduced pressure and the pH was ajusted to 2 by addition of an aqueous solution of HCl (1 M). A white precipitate was formed and collected by filtration. The precipitate was purified by flash chormatography over silica using a gradient of 1% to 5% methanol in dichloromethane yielding the product 17 as a white sticky powder (363 mg, 46.7%).
(131) .sup.1H NMR (400 MHz, CD.sub.3OD) (δ, ppm)=1.42 (s, 9H), 1.84; (m, 1H), 2.23; (m, 1H), 2.25; (m, 2H), 4.59; (m, 1H), 5.22; (s, 4H), 7.53-7.16; (m, 13H).
(132) 18: TREN-Glu-CAM(Bn)
(133) Glu-CAM(Bn) (17, 311 mg, 0.600 mmol) was dissolved in 10 mL of 1,4-dioxane, followed by the addition of NHS (83.4 mg, 0.720 mmol). EDC.HCl (140 mg, 0.730 mmol) dissolved in 10 mL of 1,4-dioxane was then dropwise added under N.sub.2 gas atmosphere to the mixture Glu-CAM(Bn)/NHS which had been previously cooled down to 9° C. The crude of the reaction was allowed to reach room temperature (25-27° C.) and then magnetically stirred under N.sub.2 gas for 27 hours. Next, the crude obtained was slowly added via canula to a mixture of TREN (511 mg, 3.49 mmol) and DIPEA (1.36 g, 10.5 mmol) in 6 mL of 1,4-dioxane. The solution obtained was magnetically stirred at room temperature for 65 hours and after that time the solvent was evaporated. The yellowish oil obtained was dissolved in 70 mL of DCM and then washed three times with water (20 mL per wash). The organic phase was collected and dried over Na.sub.2SO.sub.4. The solvent was evaporated, and then the yellowish oil observed (mixed with a white solid) was isolated by extracting the oil in a minimum amount of DCM. Finally, the DCM is evaporated and the yellowish oil was recovered (0.3345 g, 86%).
(134) .sup.1H-NMIR (400 MHz, MeOD): δ 1.44; (s, 9H, CH.sub.3), 1.74; (m, 1H, CH.sub.2), 2.06; (m, 1H, CH.sub.2), 2.25; (m, 2H, CH.sub.2), 2.50-2.82; (m, 12H, CH.sub.2), 3.18-3.59; (m, 4H, NH.sub.2), 4.62; (m, 1H, CH), 5.11-5.27; (m, 12H, CH.sub.2), 7.13-7.50; (m, 13H, ArH), 7.67; (m, 1H, NH), 8.57; (dd, 1H, NH).
(135) MS (ESI-): m/z ([M−H].sup.− calcd 646.36 obsd 646.3, 647.3, 648.3.
(136) 19: TREN-Glu-CAM(Bn) Gly-CAM(Bn)
(137) CAM(Bn)-Gly (16, 90.3 mg, 0.230 mmol) and NHS (34.9 mg, 0.300 mmol) were dissolved in 2 mL of 1,4-dioxane at room temperature. Once everything was dissolved, the solution obtained was cooled down until it reached 13° C. Next, EDC.HCl (58.9 mg, 0.310 mmol) dissolved in 2 mL of 1,4-dioxane, was added dropwise. Once all the EDC.HCl was added, the system was allowed to slowly warm up until it reached room temperature and then magnetically stirred for 96 hours under N.sub.2 atmosphere. The dioxane was then evaporated and the yellowish oil obtained was mixed with DIPEA (149 mg, 1.15 mmol) and TREN-[Glu-O-t-Butyl-CAM(Bn)] (18, 53.8 mg, 0.0800 mmol) in 3 mL of dichloromethane. The resulting mixture was magnetically stirred at room temperature for 73 hours under nitrogen atmosphere. The crude obtained was diluted in a total of 30 mL of DCM and then washed with 10 mL of water (three times). The organic phase was collected and dried over Na.sub.2SO.sub.4, then the DCM was evaporated under reduced pressure and the yellowish oil obtained was applied to a silica gel column and eluted with MeOH (0-5%) in dichloromethane (19.0 mg, 17%).
(138) .sup.1H-NMR (400 MHz, CD.sub.2Cl.sub.2) δ 1.36; (m, 9H, CH.sub.3), 1.69; (m, 1H, CH.sub.2), 1.94; (m, 1H, CH.sub.2), 2.16; (m, 2H, CH.sub.2), 2.40-2.58; (m, 6H, CH.sub.2), 2.97-3.40; (m, 6H, CH.sub.2), 3.89; (m, 4H, CH.sub.2), 4.49; (m, 1H, CH), 5.04-5.17; (m, 12H, CH.sub.2), 7.05-7.60; (m, 39H, ArH), 8.49; (m, 3H, NH).
(139) .sup.13C-NMR (100 MHz, CD.sub.2Cl.sub.2) δ 27.78, 31.82, 38.22, 43.65, 54.43, 71.21, 76.00, 76.08, 80.09, 117.02, 122.63, 122.71, 124.27, 127.18, 127.80, 127.93, 128.16, 128.22, 128.40, 128.44, 128.47, 128.57, 128.59, 129.19, 136.37, 136.48, 136.50, 136.58, 146.87, 151.96, 165.38, 165.71, 169.24, 171.38, 171.84.
(140) MS (ESI+): m/z [M+H].sup.+ calcd 1394.6 obsd 1394.7, [M+Na].sup.+ calcd 1416.6 obsd 1416.7.
(141) 20: TREN-[Glu-CAM][Gly-CAM].sub.2
(142) TREN-[Glu-O-t-Butyl-CAM(Bn)][Gly-CAM(Bn)]2(19, 15.8 mg, 0.0110 mmol) was dissolved first in 1 mL of glacial acetic acid. Once everything was dissolved, 1 mL of HCl 37% was added. The reaction mixture obtained was magnetically stirred at room temperature for 27 hours under nitrogen atmosphere. After this time, the solvent was evaporated and the yellowish paste obtained was mixed with 2 mL of methanol that was subsequently rota-evaporated (this process was repeated three times) (8.1 mg, 90%).
(143) .sup.1H-NMR (400 MHz, MeOD) δ 2.08; (m, 1H, CH.sub.2), 2.24; (m, 1H, CH.sub.2), 2.45; (m, 2H, CH.sub.2), 3.47-3.69; (m, 12H, CH.sub.2), 4.06; (m, 4H, CH.sub.2), 4.57; (m, 1H, CH), 6.73; (m, 3H, ArH), 6.94; (td, 3H, ArH), 7.28; (m, 3H, ArH).
(144) MS (ESI-): m/z [M−H].sup.− calcd 796.28 obsd 796.3, 797.3.
(145) 21: Ga-TREN-[Glu-CAM][Gly-CAM].sub.2
(146) TREN-[Glu-CAM] [[Gly-CAM].sub.2 (20, 6.3 mg, 79 μmol) was dissolved in 500 μL of MeOH yielding a yellowish solution. Next, the reaction flask was degassed and 500 μL of a methanolic solution of NaOH 15.5 mM (77.5 μmol) were added. The pinkish solution obtained was magnetically stirred at room temperature for a few minutes. Then, Ga(acac).sub.3 (3.1 mg, 85 μmol) dissolved in 500 μL of MeOH was added. The crude obtained from the reaction was degassed and magnetically stirred at room temperature for 18 hours. After this time, the volume of the crude obtained was reduced to 1/10 its original volume, and then 1 mL of ether was added. The precipitated metal complex was isolated by centrifugation; the supernatant obtained was discarded and the solid obtained was isolated and dried under reduced pressure at 40° C. (6.2 mg, 91%).
(147) UV-visible spectra of TREN-[Glu-CAM]-[Gly-CAM].sub.2 (-) and Ga-TREN-[Glu-O-t-Butyl-CAM]-[Gly-CAM].sub.2 (---) in TRIS buffer pH 7.4 (94 mM and 90 mM respectively) are illustrated in
(148) 22: Ga-TREN-GlyCAM-GluCAM-OG
(149) Ga-TREN-[Glu-O-t-Butyl-CAM][Gly-CAM].sub.2 (21, 1.8 mg, 18 μmol), OREGON GREEN 488 Isomer 5 dye (1.1 mg, 22 μmol), EDC.HCl (0.5 mg, 26. μmol) and DMAP (catalytic amount) were dissolved in 1200 μL of anhydrous DMF. The reaction mixture obtained was shook at room temperature for 13 days. Next, the DMF was evaporated and the orange solid obtained was resuspended in a mixture of DMF 18% (v/v) and MeOH 7% (v/v) in water, then applied to a C-18 reversed phase silica gel column and eluted with acetonitrile (10-60%) in water. Yield: 1.3%
(150) UV-visible spectra of Ga-TREN-GlyCAM-GluCAM-OG (λ.sub.max 508) and OREGON GREEN 488 CADAVERINE dye (λ.sub.max 494) in TRIS buffer pH 9.06 are illustrated in
Example 3: Ga-TREN-GluCAM-OG
(151) ##STR00011##
(152) The synthesis of Ga-TREN-GluCAM-OG (26) is illustrated in
(153) 23: TREN-tris-Glu-CAM(Bn)
(154) Glu-CAM(Bn) (17, 287.4 mg, 0.550 mmol) and NHS (78.0 mg, 0.68 mmol) were dissolved in 7 mL of 1,4-dioxane under N.sub.2 gas. Then, the reaction flask was cooled down to 12° C., and EDC.HCl (129 mg, 0.670 mmol) was added. The crude obtained from the reaction was magnetically stirred at room temperature under N.sub.2 atmosphere for 19 hours. Next, DIPEA (338 mg, 2.62 mmol) and TREN (6.9 mg, 0.050 mmol) were mixed and added to the crude. The reaction was magnetically stirred at room temperature under N.sub.2 gas for 19 hours and after this time, a second portion of TREN (9.4 mg, 0.060 mmol) was added. After 1 hour, the final portion of TREN (8.3 mg, 0.057 mmol) was added and the crude was magnetically stirred for 17 additional hours. The solvent of the crude was evaporated under reduced pressure, the yellowish oil obtained was dissolved in 40 mL of DCM and then washed with 30 mL of water (this process was carried out three times). The organic phase was collected and dried over Na.sub.2SO.sub.4 overnight. Then, the solvent was evaporated under reduced pressure yielding a yellowish sticky oil. The oil was applied to a column and then eluted with a gradient of MeOH (0-7%) in DCM (121.0 mg, 44%).
(155) .sup.1H-NMR (400 MHz, CD.sub.2Cl.sub.2): δ 1.39; (s, 27H, CH.sub.3), 1.73; (m, 3H, CH.sub.2), 1.99; (m, 3H, CH.sub.2), 2.18; (m, 6H, CH.sub.2), 2.38-3.44; (m, 12H, CH.sub.2), 4.57; (m, 3H, CH), 5.10; (m, 12H, CH.sub.2), 7.02-7.56; (m, 39H, ArH), 7.58-7.73; (m, 3H, NH), 8.51; (m, 3H, NH).
(156) MS (ESI+): m/z [M+Na].sup.+ calcd 1672.79 obsd 1673.1.
(157) 24: TREN-Glu-CAM
(158) TREN-[Glu-CAM(Bn)]3 (23, 121 mg, 0.0732 mmol) was dissolved in 1 mL of glacial acetic acid. Once everything was dissolved, 1 mL of concentrated HCl was added to the crude of reaction. The crude was magnetically stirred under N.sub.2 atmosphere for 16 hours. After this time, the solvent was removed under vacuum. The yellowish oil obtained was dried 4 hours under vacuum (72.0 mg, 100%).
(159) .sup.1H-NMR (400 MHz, MeOD): δ 2.09; (s, 3H, CH.sub.2), 2.24; (s, 3H, CH.sub.2), 2.48; (m, 6H, CH.sub.2), 3.48-3.65; (m, 12H, CH.sub.2), 4.55; (s, 3H, CH), 6.74; (m, 3H, ArH), 6.95; (m, 3H, ArH), 7.31; (m, 3H, ArH).
(160) 25: Ga-TREN-tris-Glu-CAM
(161) TREN-[Glu-CAM]3 (5.1 mg, 5.2 μmol) was dissolved in 500 μL MeOH and mixed with 31 μL of methanolic KOH 0.5M (15.5 μtmol). Next, Ga(acac).sub.3 (1.9 mg, 5.2 μmol) was added. After 24 hours shaking at room temperature, the solvent was evaporated and then the off-white solid was resuspeded in 0.2 mL of MeOH, Then, 1 mL of ether was added, the solution obtained was shaken and the suspension obtained was centrifuged for 5 minutes at 12000 rpm. The supernatant was removed and the off-white solid was collected. (3.8 mg, 64%).
(162) UV-visible spectra of fraction Ga-TREN-Glu-CAM (λ.sub.max 340) and TREN-Glu-CAM (λ.sub.max 324) in TRIS buffer 93 mM pH 7.45 are illustrated in
(163) 26: Ga-TREN-GluCAM-OG
(164) Ga-TREN-[Glu-CAM]3 (2.7 mg, 2.4 mol), OREGON GREEN CADAVERINE 488 dye (1.2 mg, 2.4 mol), EDC.HCl (0.6 mg, 3 mol) and DMAP (catalytic amount) were dissolved in 1 mL of anhydrous DMF. The orange crude obtained was shaken at room temperature for 5 days. After this time, the solvent was evaporated under reduced pressure. The orange solid obtained was dissolved in a mixture of 6.6% ACN and 3.3% of DMF, applied to a C-18 reversed-phase silica column and then eluted with 100 mL 6.6% ACN and 3.3% DMF in water, then 50 mL 20% ACN, 50 mL 50% ACN, 50 mL 60% ACN, 50 mL 80% ACN, 50 mL MeOH, 50 mL H.sub.2O, 50 mL 20% ACN, 50 mL 20% ACN, 50 mL 50% ACN, 50 mL H.sub.2O and 100 mL MeOH respectively. Yield: 2.2%
(165) A UV-visible absorbance spectrum of Ga-TREN-GluCAM-OG (36) in TRIS buffer pH 9.06 is illustrated in
Example 4: Ga-3,3-LiCAM-GluCAM-OG
(166) ##STR00012##
(167) The synthesis of Ga-3,3-LiCAM-GluCAM-OG (36) is illustrated in
(168) 32: 3,3-Li-bis-CAM(Bn)
(169) CAM(Bn2)-thiaz was synthesized as previously reported (Allred et al., ACS Chem Biol 2013, 8 (9), 1882-1887). A solution of dipropylenetriamine (DPTA, 28.0 mg, 0.219 mmol, 1.00 eq) and triethylamine (5 drops) in dichloromethane (5 mL) was added to a solution of CAM(Bn2)-thiaz (200 mg, 0.450 mmol, 2.10 eq). The reaction mixture was stirred at room temperature for 26 hours. The solvent was removed under reduced pressure and the residue dissolved in dichloromethane (100 mL) and extracted with water (3×100 mL). The organic phase was dried with sodium sulfate. Removal of the solvent under reduced pressure yielded the intermediate as an off white foam that was used immediately in the next step.
(170) .sup.1H NMR (400 MHz, CDCl.sub.3) (δ, ppm)=7.95; (m, 2H), 7.39-7.19; (m, 20H), 7.05; (m, 4H), 5.06; (s, 4H), 4.98; (s, 4H), 3.24; (dt, 2H, J.sub.1=6.8 Hz, J.sub.2=5.6 Hz), 2.35; (td, 4H, J.sub.1=7.0 Hz, J.sub.2=5.6 Hz), 1.42; (tt, J.sub.1=6.8 Hz, J.sub.2=6.8 Hz).
(171) 33: Symmetric-3,3-Li-Glu-CAM(Bn)
(172) Diisopropylethylamine (DIPEA, 35.0 mg, 0.275 mmol, 1.20 eq.) was added to a solution of the intermediate 32 (175 mg, 0.229 mmol, 1.00 eq.), Glu-CAM(Bn) (142 mg, 0.275 mmol, 1.00 eq.) and O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU, 88.0 mg, 0.275 mmol, 1.20 eq.) in DCM (50 mL). The reaction mixture was stirred at room temperature for 48 hours. The solvent was removed under reduced pressure and the residue dissolved in dichloromethane (100 mL) and washed with mQ water (3×100 mL). The organic phase was dried with sodium sulfate, the solvent removed under reduced pressure and the residue purified by flash chromatography over silica using a gradient of 1-5% methanol in dichloromethane. The solvent were removed under reduced pressure yielding the protected ligand 33 as a white foamy solid (162 mg, 56.0% over the last two steps).
(173) .sup.1H NMR (400 MHz, CDCl.sub.3) (δ, ppm)=8.51; (d, 1H, J=8), 8.00; (m, 2H), 7.66; (m, 3H), 7.48-7.08; (m, 36H), 5.23-5.02; (m, 13H), 3.43-3.18; (m, 8H), 2.24-1.56; (m, 8H), 1.41; (s, 9H).
(174) .sup.13C NMR(400 MHz, CDCl.sub.3) (δ, ppm)=172.0, 171.7, 169.16, 165.5, 165.0, 151.8, 151.7, 151.6, 146.9, 146.8, 146.7, 136.5, 136.4, 136.2, 129.1, 128.8, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2,1 128.1, 128.0, 127.9, 127.8, 127.6, 127.5, 127.3, 127.1, 124.3, 124.2, 124.1, 123.2, 123.1, 123.0, 80.5, 76.2, 76.0, 75.8, 71.4, 71.2, 70.7, 48.7, 46.1, 45.3, 43.4, 41.9, 31.1, 29.0, 28.2, 27.57, 27.34.
(175) ESI-MS m/z=[M+Na].sup.+1287.7 m/z (Calcd. 1287.5)
(176) 34: Symmetric-3,3-Li-Glu-CAM
(177) The protected ligand symmetric-3,3-Li-Glu-CAM(Bn) (33, 90.0 mg, 71.0 μmol) was dissolved in 6 M HCl (aq) (1 mL) and acetic acid (1 mL). The reaction mixture was stirred for 18 h at room temperature after which the solvent was removed under reduced pressure. The residue was dissolved in CH.sub.3OH/water and lyophilized to yield the deprotected ligand 34 as a white solid (4.70 mg, 9.88%).
(178) .sup.1H NMR (400 MHz, CDCl.sub.3) (δ, ppm)=7.74-7.34; (m, 3H), 6.95; (m, 3H), 6.73; (m, 3H), 4.72; (m, 1H), 4.32; (t, 1H, J=8), 3.96; (t, 1H, J=8), 3.78-3.458; (m, 4H), 3.26-3.11; (m, 4H), 2.49; (m, 2H), 2.63; (m, 1H), 2.16; (m, 1H), 2.04; (m, 2H).
(179) .sup.13C NMR (400 MHz, MeOD) (δ, ppm)=173.6, 173.5, 170.7, 169.9, 148, 9, 148.6, 146.0, 145.9, 118.5, 118.2, 117.9, 117.5, 117.2, 115.8, 115.2, 51.8, 51.5, 50.8, 45.3, 35.7, 29.7, 26.3, 26.1.
(180) 35: Ga-Symmetric-3,3-Li-Glu-CAM
(181) The deprotected ligand symmetric-3,3-Li-Glu-CAM (34, 4.7 mg, 7.3 μmol, 1.0 eq) was dissolved in CH.sub.3OH (2 mL). Ga(acac).sub.3 (2.7 mg, 7.3 μmol, 1.0 eq) was added to the reaction mixture. A solution of 0.5 M KOH (aq) (0.1 mL) was added to the reaction mixture to adjust the pH to 7. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure. Addition of diethylether to the reaction mixture resulted in the formation of a precipitate that was isolated by centrifugation. The slightly pink solid was re-suspended in diethtylether and centrifuged and dried in vacuo (3.7 mg, 62.7%).
(182) UV-visible absorbance spectra of symmetric-3,3-Li-Glu-CAM (34) (λ.sub.max 322) and Ga-3,3-LiCAM-GluCAM-OG (35) (λ.sub.max 332) in Tris buffer, pH 7.4 are illustrated in
(183) 36: Ga-3,3-LiCAM-GluCAM-OG
(184) Ga-symmetric-3,3-Li-Glu-CAM (35, 1.47 mg, 1.36 μmol, 1.00 eq), and OREGON GREEN CADAVERINE dye (1.00 mg, 2.01 μmol, 1.00 eq) were dissolved in DMF (2 mL). Dimethylaminopyridine (DMAP, one crystal) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 0.462 mg, 2.41 μmol, 1.20 eq.) were added to the reaction mixture that was further stirred at room temperature for 15 hours. The solvent was removed under reduced pressure and the red residue purified by HPLC using method 4.
(185) A UV-visible absorbance spectra of Ga-3,3-LiCAM-GluCAM-OG (36) in illustrated in
Example 5: Ga-2,2-LiCAM-GluCAM-OG
(186) ##STR00013##
(187) The synthesis of Ga-2,2-LiCAM-GluCAM-OG is illustrated in
(188) 38: 2,2-Li-[CAM(Bn)].sub.2-[Glu-CAM(Bn)]
(189) 2,2-Li-CAM(Bn) (37, 241 mg, 0.330 mmol), Glu-CAM(Bn) (17, 235 mg, 0.452 mmol) and TBTU (154 mg, 0.480 mmol) were disolved in 5 mL of dichloromethane. DIPEA (98 mg, 0.76 mmol) was added to the crude. The yellowish solution obtained was magnetically stirred under N.sub.2 atmosphere for 96 hours. The crude was diluted in 30 mL of DCM and then washed with NaHCO.sub.3 4.6% three times. The organic phase was collected, dried over Na.sub.2SO.sub.4 and the solvent evaporated under reduced pressure. The yellowish oil obtained was purified using the combiflash system using a gradient of MeOH in DCM. 46.4 mg recovered (yield: 7%)
(190) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 1.41; (d, 9H), 1.66; (m, 1H), 1.95; (m, 1H), 2.24; (m, 2H), 3.25-3.70; (m, 8H), 5.04-5-24; (m, 13H) 7.06-7.74; (m, 39H), 8.05-8-16; (m, 2H8.58-8.68; (m, 1H).
(191) .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 17.47, 28.11, 29.78, 31.15, 37.74, 38.46, 45.83, 46.96, 48.92, 71.32, 80.42, 116.93, 117.14, 123.14, 123.40, 124.28, 127.02, 127.65, 127.94, 128.41, 128.70, 128.95, 129.27, 136.55, 146.78, 147.05, 151.79, 164.90, 165.69, 165.96, 171.98, 172.48.
(192) 39: 2,2-Li-[CAM].sub.2-[Glu-CAM]
(193) 2,2-Li-[CAM(Bn)]2-[Glu-CAM(Bn)] (42.7 mg, 34.5 μmol) was dissolved in 1 mL of HCl 37% and 1 mL of glacial acetic acid and magnetically stirred at room temperature under N.sub.2 gas for 17 hours. The solvent was evaporated and then fresh HCl 37% and glacial AcOH were added. The reaction was allowed to run for 24 hours under the same conditions described above. Next, the solvent was evaporated and the solid obtained was dissolved in MeOH and then shook off for 5 days under H.sub.2 gas in presence of Pd/C catalyst (Degussa Type). The crude obtained was filtrated through Celite 545 powder. The volume of the crude obtained was condensed to 0.4 mL and then 1 mL of ether was added. The suspension observed was centrifuged, the supernatant was removed and the solid recovered (process carried out three times). Next, the purple solid obtained was dissolved in 10 mL of a mixture (50/50) of MeOH and HCl 0.1 M, and then washed three times with hexanes. The aqueous phase was recovered and the solvent evaporated to yield 8.3 mg (yield: 36%) of a yellowish/brownish compound
(194) .sup.1H-NMR (400 MHz, MeOD) δ 2.13; (m, 1H), 2.32; (m, 1H), 2.49; (m, 2H), 3.49-3.89; (m, 9H), 4.70; (dd, 1H), 6.46-7.32; (m, 9H).
(195) 40: Ga-2,2-Li-[CAM].sub.2-[Glu-CAM]
(196) 2,2-Li-CAM-Glu-CAM (4.6 mg, 7.1 μmol) was dissolved in MeOH and mixed with 43 μL of methanolic KOH 0.5M (22 μmol). Next, Ga(acac).sub.3 (2.6 mg, 7.0 μmol) was added and the crude obtained was rotated at room temperature for 15 hours. After this time, the volume of the crude was reduced to 1/10 of its original volume. Then, 1 mL of ether was added, the solution obtained was shaken and the suspension obtained was centrifuged for 5 minutes at 12000 rpm. The supernatant was removed and the brownish solid was collected. This process was carried out three times and at the end 6 mg of a brownish solid were recovered (yield: 102%).
(197) UV-visible spectra of 2,2-Li-[CAM]-[Glu-CAM] (λ.sub.max 324) and Ga-2,2-Li-[CAM]-[Glu-CAM] (λ.sub.max 329) in TRIS buffer pH 7.45 and DMF 2% (v/v) are illustrated in
41 Ga-2,2-LiCAM-GluCAM-OG
(198) Ga-2,2-Li-CAM-Glu-CAM (1.9 mg, 2.3 μmol), OREGON GREEN 488 CADAVERINE dye (1.1 mg, 2.2 μmol), ED.HCl (0.7 mg, 4 μmol) and DMAP (catalytic amount) were dissolved in 1 mL of anhydrous DMF. The solution obtained was rotated at room temperature for 65 hours. After this time, the DMF was evaporated and the solid obtained re-suspended in a mixture of ACN 20% and DMF 7% in water. Next, the sample was applied to a C-18 reversed phase silica column and eluted with 100 mL of ACN 20%-DMF 7% in water, 50 mL of ACN 50%-DMF 7% in water, 50 mL of ACN 60%-DMF 5% in water and 50 mL of ACN 80%-DMF 7% in water. Yield: 2.3%.
(199) UV-visible spectra of Ga-2,2-LiCAM-GluCAM-OG (λ.sub.max 504) and OREGON GREEN 488 dye (λ.sub.max 494) in TRIS buffer, pH 9.06 are illustrated in
Example 6: Ga-3,3-LiCAM-GluCAM-Light-Switch
(200) ##STR00014##
(201) The synthesis of Ga-3,3-LiCAM-GluCAM-light-switch is illustrated in
(202) 42: Lepidine (0.100 g, 0.698 mmol, 1.00 eq) and N-(b-iodoethyl)trifluoror acetamide (0.540 g, 2.00 mmol, 2.90 eq) were diluted in dry toluene (1 ml) and refluxed overnight under N.sub.2. All compounds were soluble and without color. After 2 hours the mixture turned orange and heterogeneous. The reaction mixture was refluxed overnight. The yellow precipitate was rinsed with toluene (2×5 mL) then ether (5 mL). The yield obtained was over 100 percent (0.290 mg). Toluene was observed in .sup.1H NMR.
(203) .sup.1H NMR 400 MHz, (MeOD δ ppm): 9.20; (1H, d, 6.0 Hz), 8.63; (2H, m), 8.32; (1H, m), 8.11; (1H, m), 8.01; (1H, d, 5.6 Hz), 5.24; (2H, t, 4.8 Hz), 4.39; (2H, t, 4.8 Hz), 3.11; (3H, s).
(204) 43: 3-methylbenzothiazole-2-thione (0.107 g, 0.590 mmol, 1.00 eq.) was diluted in Mel (0.100 g, 0.704 mmol, 1.20 eq) and DCM (1 mL). The reaction mixture was stirred at 50° C. for 4 hours. The precipitate was filtered and washed with acetone (52.0 mg, 48.1%).
(205) .sup.1H NMR 400 MHz, (MeOD δ ppm): 8.27; (1H, d, 8.0 Hz), 8.12; (1H, d, 8.3 Hz), 7.88; (1H, t, 8.8 Hz), 7.75; (1H, t, 7.6 Hz), 4.19; (3H, s).
(206) 44: 42 (86.0 mg, 0.303 mmol, 1.00 eq) and 43 (60 mg, 0.303 mmol, 1.00 eq) was diluted under N.sub.2 in anhydrous ethanol (2 ml). The solution turned yellow and was heterogeneous. The reaction mixture was stirred at 65° C. for 5 minutes to allow full solubilization of both solids. Trimethyl amine (61.4 mg, 0.607 mmol, 2.00 eq) was added and the solution turned blood red immediately. The reaction was left to return to room temperature then ether (5 mL) was added to the solution to precipitate the red solid. The dye was filtered out, and then recrystallized with acetone and ether leading to 76 mg (58.4%).
(207) .sup.1H NMR 400 MHz (MeOD δ ppm): 8.70; (1H, d, 8.0 Hz), 8.29; (1H, d, 7.2 Hz), 8.15; (1H, d, 8.8 Hz), 8.00, (1H, t, 7.2 Hz), 7.94; (1H, d, 8.0 Hz), 7.79; (1H, t, 8.0 Hz), 7.72; (1H, d, 8.0 Hz), 7.65; (1H, t, 7.6 Hz), 7.48; (2H, m), 6.99; (1H, s), 4.77; (2H, t, 6.0 Hz), 4.06; (3H, s), 3.86; (2H, t, 6.0 Hz).
(208) 45: Protected turn on dye (30 mg) was diluted in 0.6 ml of concentrated HCl and heated for 1 hour at 65° C. KOH was added until a red precipitate formed. The red solid was filtered and washed with ether. Proton NMR was busy and .sup.19F still showed a signal. The reaction was run again using the same conditions of equivalency but this time for longer and for 3 hours. After the same work up .sup.19F NMR showed no signal and the .sup.1H NMR was less busy even though not an ideal spectrum.
(209) 46: Ga-3,3-LiCAM-GluCAM-light-switch. Ga-3,3-Li-CAM-Glu-CAM (35, 17.5 mg, 0.025 mmol, 1.00 eq), dye (45, 8.28 mg, 0.024 mmol, 1.00 eq) and EDC (5.66 mg, 0.029 mmol, 1.20 eq.) were solubilized in DMF (1.5 mL). A catalytic amount of DMAP was added. The reaction was stirred overnight. The product was obtained after removal of the solvents under reduced pressure.
Example 7: Fe-3,3-LiCAM-GluCAM-Tb
(210) ##STR00015##
(211) The synthesis of Fe-3,3-LiCAM-GluCAM-Tb is illustrated in
(212) 50: Fe-3,3-Li-CAM-Glu-CAM-DO3A
(213) Fe-3,3-Li-CAM-Glu-Cam (0.9 mg, 0.00125 mmol, 1 eq) was diluted in DMF (0.2 mL) and EDC (0.286 mg, 0.0015 mmol, 1.2 eq). Aminobutyl-DOTA, 4HCl, HPF.sub.4 (Macrocyclic, 0.96 mg, 0.0125 mmol, 1 eq) was diluted in DIPEA (0.8 mg, 0.00625 mmol, 5 eq) and DMF (0.2 mL) and added to the red iron complex solution. The reaction mixture turns purple and was stirred for 2 hours. Solvent were removed and water was added to the residue which was purified by HPLC.
(214) 51: Fe-3,3-LiCAM-GluCAM-Tb
(215) Fe-3,3-Li-CAM-Glu-CAM-Do3A was solubilized in methanol and terbium was added. Then a drop of pyridine was added. The reaction mixture was stirred at reflux for 3 days, with regular addition of NaOH (aq) to maintain the pH around 8. Removal of the solvents under reduced pressure yielded the Tb probe 51.
Example 8: AuNP @ Fe-3,3-LiCAM-GluCAM
(216) ##STR00016##
(217) The synthesis of AuNP@Fe-3,3-LiCAM-GluCAM is illustrated in
(218) 52: Fe-3,3-Li-CAM-Glu-CAM
(219) A drop of methanolic KOH (0.5M) was added to a solution of 3,3-Li CAM 4 (3.30 mg, 4.90 mmol, 1 eq) and Fe(acac).sub.3 (1.74 mg, 4.90 mmol, 1 eq.) in methanol (0.5 mL). After 3 hours the complex was precipitated by addition of ether. A red solid was recovered after centrifugation. The red solid was washed twice with ether.
(220) UV-visible spectra of 3,3-Li-CAM-GLu CAM (λ.sub.max 329) and Fe-3,3-Li-CAM-GluCAM (λ.sub.max 331) in Tris buffer, pH 7 are illustrated in
(221) Synthesis of Citrate Capped AuNP
(222) A 1.00 w/w % solution of sodium citrate (stock 1) was made by dissolving 1.14 g of Na.sub.3-citrate dehydrate into 99.9 g of deionized water available under the trade designation MILLI-Q water from Millipore Corporation. A 0.01 w/w % solution of gold chloride (stock 2) was made by dissolving 0.100 g of a 30 w/w % HAuCl.sub.4 into 300 g of MILLI-Q water. AuNP were made by boiling and stirring 50 mL of stock 2 and pipetting in 1 mL of stock 2. This solution was kept at a boil for 10 minutes to ensure the reaction went to completion. DLS indicates that the average diameter of these particles is 16.0 nm with an average zeta potential of −55.5 mV.
(223) TEM of Citrate-caped AuNP frame are illustrated in
(224) AuN @ PEG: 25 mL of the 21/24 nm AuNP solution was mixed with NH.sub.3Cl-PEG-SH (2000 mw, 5.0 mg, 2.5 μmol) for 2 hours. The crude reaction mixture was then filtered on 10 kDa centrifugal filters and washed with MILLI-Q water three times. The remaining AuNPs were then re-suspended in MILLI-Q water.
(225) Size distribution of Au NP and Au NP @ PEG obtained from dynamic light scattering are illustrated in
(226) AuNP @ Fe-3,3-LiCAM-GluCAM: 53
(227) The PEGylated AuNPs(b) were mixed with Fe.sup.III-5-(Bis(3-(2,3-dihydroxybenzamido)propyl)amino)-4-(2,3-dihydroxybenzamido)-5-oxopentanoic acid (1 mg, 1.31 μmol) dissolved in DMF (0.25 mL), DMAP (0.03 mg, 0.25 μmol), TBTU (1 mg, 3 μmol), and DIPEA (0.3 mg, 3 μmol). This reaction as allowed to run overnight, the solution was centrifuged through 10 kDa filters and washed with MILLI-Q water three times. The resulting solution was re-suspended in MILLI-Q water for analysis by TEM and DLS.
(228) A TEM of AuNP @ Fe-3,3-LiCAM-GluCAM is illustrated in
(229) UV-visible spectra of AuNP @ Fe-3,3-LiCAM-GluCAM (λ.sub.max 320, 527), Fe-3,3-LiCAM-GluCAM (λ.sub.max 334, 525), and the citrate capped Au nanoparticles (λ.sub.max 522) are illustrated in
(230) Preparation of T-Media
(231) The reagents described in Table 1 were dissolved in 1 L of mQ water. The pH of the solution is fixed at 7.4 by using HCl 1N.
(232) TABLE-US-00001 TABLE 1 Biological media, inorganic salts and aminoacids used for the preparation of T-media solution. Compound Amount Compound Amount NaCl 6.0714 g Leucine 58.2 mg KCl 3.7034 g Proline 60.0 mg CaCl.sub.2 141.3 mg Tryptophan 51.0 mg MgCl.sub.2•6H.sub.2O 112.1 mg Thiamine 7.5 mg hydrochloride NH.sub.4Cl 122.5 mg Glucose 2.0128 g TRIS 12.0313 g Casoaminoacids 55.1 mg Na.sub.2SO.sub.4 157.2 mg KH.sub.2PO.sub.4 32.7 mg
The resulting solution is filtered through a 20 μm Nalgene filter.
Preparation of LB-Broth Media
(233) 5.0416 g of LB-Broth powder reagent were dissolved in 250 mL of mQ water. The resulting solution was autoclavated for 20 minutes.
(234) Agar Plates
(235) 2.5365 g of LB Broth powder and 0.9055 g of nutrient agar were dissolved in 64 mL mQ water. The solution obtained was autoclavated for 20 minutes. The liquid recovered was poured into the agar plates. The agar plates prepared were allowed to dry and solidify at room temperature.
(236) Sample Processing for Bacteria Detection by Fluorescence in T Media Experiments with Bacteria Genus
(237) Four Days are Needed to do a Run
(238) Day 1: Sub bacteria onto agar
(239) Day 2 pm: Sub bacteria into LB broth
(240) Day 3: Testing
(241) Day 4: Quantitative culture results
(242) Bacteria Information
(243) 1. Klebsiella pneumoniae ATCC 10031 (IDRL 4078)
(244) 2. Acinetobacter baumanii (IDRL 9919)
(245) 3. Salmonella species non typhi (IDRL 4247)
(246) 4. Staphylococcus aureus ATCC 29213 (IDRL 4307)
(247) 5. Bacillus subtilis ATCC 6633 (IDRL 3766)
(248) Procedure
(249) 1. Bacteria is prepared a. Sub bacteria onto blood agar and incubate overnight b. Select several colonies and grow overnight in LB media at 37° C. in room air on orbital shaker c. 100 μl of overnight culture is placed into 100 ml T media, incubated for 5 hours (E. coli, Salmonella, A. baumannii, Klebsiella pneumoniae) or 10 hours (B. subtillus, S. aureus) d. Quantitate using 1:10 dilution and plating 100 μl of dilutions onto blood agar 2. Prepare the samples in 1.5 microcentrifuge tubes, vortex, in triplicate a. For each bacteria i. 1 ml bacteria+25 μl Tris ii. 1 ml bacteria+25 μl Probe b. For each run include controls i. 1 ml T media+25 μl Tris ii. 1 ml T media+25 μl Probe 3. Incubate for 20 minutes on orbital shaker at 37° C. room air 4. Centrifuge at room temperature at 15,000 rpm for 5 minutes 5. Remove supernatant by pipet (pellet may not be visible, remove all fluid carefully) 6. Resuspend pellet in 125 μl T media and vortex 7. Centrifuge at room temperature at 15,000 rpm for 5 min 8. Remove supernatant by pipet (pellet may not be visible, remove all fluid carefully) 9. Resuspend pellet in 125 μl T media and vortex 10. Pipet 100 μl of each sample into 384 black deep well plate (make sure no bubbles) 11. Let sit for 5 minutes 12. Measure in fluorometer at 485 nm (swipe 20 nm) emission at 510-540 nm (swipe 10 nm) and using 800 volt 13. Save results into an excel file. 14. Wash plate and sonicate in ethanol 15. 24+ hours count colonies on quantitative cultures
(250) Inclusivity and an increase in fluorescence intensity after incubation of different genus of bacteria with Ga-3,3-LiCAM-GluCAM-OG is illustrated in
(251) The effect of probe chemical structure on response can be seen in
(252) The effect of metal ion on probe uptake can be seen in
(253) The effect of bacteria concentration can be seen as an increase in fluorescence intensity in
(254) The effect of the concentration of iron in the media on probe uptake can be seen in an increase in fluorescence intensity in
(255) The effect of incubation time can be seen in an increase in fluorescence intensity in
(256) An epifluorescence image of 0.5 mL of E. coli (ATCC 25922) at 10.sup.7 cfu/mL incubated with 1 μM Ga-TREN-CAM-C2-6FAM for 5 minutes and filtered on an isopore membrane filter (0.2 μm pore size) is illustrated in
(257) Sample Processing for Bacteria Detection by Cytomass in T Media Experiments with Bacteria Genus
(258) 1. Bacteria is prepared a. Sub bacteria onto blood agar and incubate overnight b. Select several colonies and grow overnight in LB media at 37° C. in room air on orbital shaker c. 100 μl of overnight culture is placed into 100 ml T media, incubated for 5 hours (E. coli, Salmonella, A. baumannii, Klebsiella pneumoniae) or 10 hours (B. subtillus, S. aureus) d. Quantitate using 1:10 dilution and plating 100 μl of dilutions onto blood agar 2. Prepare the samples in 1.5 microcentrifuge tubes, vortex, in triplicate a. For each bacteria i. 1 ml bacteria+25 μl Tris ii. 1 ml bacteria+25 μl Probe b. For each run include controls i. 1 ml T media+25 μl Tris ii. 1 ml T media+25 μl Probe 3. Incubate for 20 minutes on orbital shaker at 37° C. room air 4. Centrifuge at room temperature at 15,000 rpm for 5 minutes 5. Remove supernatant by pipet (pellet may not be visible, remove all fluid carefully) 6. Resuspend pellet in 125 μl T media and vortex 7. Centrifuge at room temperature at 15,000 rpm for 5 min 8. Remove supernatant by pipet (pellet may not be visible, remove all fluid carefully) 9. Re-suspend pellet in 125 μl T media and vortex 10. The palettes were spin down (1000 RCF, 4° C., 5 minutes). 11. The supernatant was removed and the palettes were suspended in 4% formaldehyde in PBS. 12. The palettes were incubated 15 minutes at 37° C. and then centrifuged (1000 RCF, 4° C., 5 minutes). 13. The filtrate was removed and the palettes were re-suspended in PBS and spin down (1000 RCF, 4° C., 5 minutes). This later action was repeated twice. 14. After the last wash the palettes was suspended in MQ water and the data were acquire in CytOF
(259) Cytomas data is illustrated in
(260) The complete disclosure of all patents, patent applications, and publications, and electronically available material (e.g., GenBank amino acid and nucleotide sequence submissions; and protein data bank (pdb) submissions) cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. This disclosure is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the disclosure defined by the claims.