GLUCOSYL ESTERS FOR INFECTION SCREENING

Abstract

Compositions and methods of use for a glucosyl ester or a salt or solvate thereof, to detect and/or measure LE activity in a sample are disclosed.

Claims

1. A glucosyl ester having a chemical formula of Formula I or a salt or solvate thereof: ##STR00012## wherein, X is O, CR.sub.3R.sub.4 or NR.sub.5, and wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are independently hydrogen, a C.sub.1 to C.sub.8 alkyl, a substituted C.sub.1 to C.sub.8 alkyl, a C.sub.3 to C.sub.7 cycloalkyl, a substituted C.sub.3 to C.sub.7 cycloalkyl, a heteroalkyl, a substituted heteroalkyl, a heterocycle, a substituted heterocycle, an aryl, a substituted aryl, a fused aryl, a substituted fused aryl, a heteroaryl, or a substituted heteroaryl.

2. The glucosyl ester of claim 1, wherein X is CR.sub.3R.sub.4, R.sub.1, R.sub.3, and R.sub.4 are independently hydrogen, or a C.sub.1 to C.sub.8 alkyl, and R.sub.2 is an aryl or a substituted aryl.

3. The glucosyl ester of claim 2, wherein R.sub.1 is methyl, R.sub.3 and R.sub.4 are independently hydrogen or methyl, and R.sub.2 is a substituted aryl.

4. The glucosyl ester of claim 1 having the chemical formula of formula II ##STR00013## wherein Ts is tosyl group.

5. The glucosyl ester of claim 1 having the chemical formula of formula III ##STR00014## wherein Ts is tosyl group.

6. The glucosyl ester of claim 1, wherein X is NR.sub.5, R.sub.1 and R.sub.5 are independently hydrogen, or a C.sub.1 to C.sub.8 alkyl, and R.sub.2 is an aryl or a substituted aryl.

7. The glucosyl ester of claim 1 having the chemical formula of formula IV ##STR00015## wherein Ts is tosyl group.

8. The glucosyl ester of claim 1 having the chemical formula of formula V ##STR00016## wherein Ts is tosyl group.

9. A method for detecting leukocyte esterase (LE) activity in a sample comprising: contacting the sample with a glucosyl ester or a salt or solvate thereof of claim 1 forming a test sample; and detecting cleavage of the glucosyl ester by LE in the test sample.

10. The method of claim 9, wherein cleavage of the glucosyl ester by LE is determined by measuring concentration of glucose liberated in the test sample by cleavage of the glucosyl ester by LE.

11. The method of claim 10, wherein the glucose in the test sample is measured by electrochemical detection.

12. The method of claim 9, wherein the initial glucosyl ester concentration in the test sample is above 5 mg/L.

13. A method for treating an infection in a subject, the method comprising: contacting a sample obtained from the subject with a glucosyl ester or a salt or solvate thereof of claim 1 to form a test sample; determining glucose concentration in the test sample; and administering a treatment of the infection to the subject if the glucose concentration in the test sample is elevated with respect to a non-infected control.

14. The method of claim 13, wherein the subject is a human.

15. The method of claim 13, wherein the sample is a blood, plasma, serum, tears, urine, synovial (joint) fluid or saliva sample.

16. The method of claim 13, wherein the infection is a urinary tract infection, or periprosthetic joint infection.

17. A composition comprising the glucosyl ester of claim 1.

18. A kit comprising the glucosyl ester of claim 1.

19. The kit of claim 18, further comprising a glucose detection component.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

[0036] FIGS. 1A-1D. Examples of a compounds of Formula I. (FIG. 1A) (2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl 4-{[(2S)-2-(4-methylbenzenesulfonamido)propanoyl]oxy}butanoate (CIDD-0150156). (FIG. 1B) (2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl 2,2-dimethyl-4-{[(2S)-2-(4-methylbenzenesulfonamido)propanoyl]oxy}butanoate (CIDD-0150181). (FIG. 1C) 2-[({[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}carbonyl)amino]ethyl (2S)-2-(4-methylbenzenesulfonamido)propanoate (CIDD-0150161). (FIG. 1D) 2-[methyl({[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}carbonyl)amino]ethyl (2S)-2-(4-methylbenzenesulfonamido)propanoate (CIDD-0150157).

[0037] FIG. 2. The Accu-Check glucose test strip inserted in a DropSens electronic holder ready to be contacted with a small (˜10 μL) drop of analyzed bodily fluid placed on a Petri dish.

[0038] FIGS. 3A-3B. (FIG. 3A) The charge Q flowing through a commercial glucose test strip that was contacted at time t=0 s with a pH 7.40 PBS solution containing (a) 0, (b) 1.0, (c) 10, (d) 20, (e) 50, (f) 75, and (g) 100 μM glucose. The Q values were obtained by integrating amperograms (0.15 V), which were recorded with a laboratory potentiostat, between t=2 and 22 s. Potential, 0.15 V vs. strip's counter/reference electrode. (FIG. 3B) The calibration plot based on data in panel A. The ΔQ=Q.sub.(glucose)−Q.sub.(no glucose), where Q.sub.(glucose) and Q.sub.(no glucose) were the charges measured in the presence and absence of glucose in a solution.

[0039] FIG. 4. Dependence of signal ΔQ vs. incubation time for a pH 7.40 PBS solution containing both 25 μg L.sup.−1 LE and 5.5 mM ester α. The ΔQ=Q.sub.(ester)−Q.sub.(no ester), where Q.sub.(ester) and Q.sub.(no ester) are the charges flowing through a glucose strip in contact with an ester-incubated solution and ester-free solution, respectively. Potential, 0.15 V vs. glucose strip's counter/reference electrode.

[0040] FIGS. 5A-5B. The charge Q flowing through a commercial glucose test strip that was contacted at time t=0 s with human (FIG. 5A) synovial fluid containing (a) 0, (b) 50, (c) 110, (d) 250 and (e) 800 μg L.sup.−1 LE, and (FIG. 5B) urine containing (a) 0, (b) 25, (c) 80, (d) 150 and (e) 500 μg L.sup.−1 LE. The Q values were obtained by integrating amperograms (0.15 V), which were recorded with a laboratory potentiostat, between t=2 and 22 s. The synovial fluid and urine samples were mixed with a stock solution of ester α (5% sample dilution; final ester concentration, 5.5 mM) and incubated for 5 min before contacting them with a glucose strip. Potential, 0.15 V vs. strip's reference/counter electrode.

[0041] FIG. 6. Dependence of signal ΔQ on LE concentration in human synovial fluid (red circles) and urine (black squares) samples. The samples were incubated with 5.5 mM ester α for 5 min. The ΔQ=Q.sub.(ester)−Q.sub.(no ester), where Q.sub.(ester) and Q.sub.(no ester) are the charges flowing through a glucose strip in contact with an ester-incubated solution and ester-free solution, respectively. The Q values were obtained by integrating amperograms (0.15 V), which were recorded with a laboratory potentiostat, between t=2 and 22 s. The error bars (±SD) for points at <110 μg L.sup.−1 LE were smaller than markers. The different intensities of purple show a response of commercial colorimetric LE test strips to increasing concentration of LE (from trace to +, ++, +++).

[0042] FIGS. 7A-7B. (FIG. 7A) The charge Q flowing through a commercial glucose test strip that was contacted at time t=0 s with the glucose-loaded (18 mM) urine containing (a) 0, (b) 25, (c) 80, (d) 150 and (e) 500 μg L.sup.−1 LE. The Q values were obtained by integrating amperograms (0.15 V), which were recorded with a laboratory potentiostat, between t=2 and 22 s. The urine samples were mixed with a stock solution of ester α (5% sample dilution; final ester concentration, 5.5 mM) and incubated for 5 min before contacting them with a glucose strip. (FIG. 7B) Dependence of signal ΔQ on LE concentration in urine samples that contained glucose at 0 (black squares) and 18 mM (white squares, data from panel A). The different intensities of purple show a response of commercial colorimetric LE test strips to increasing concentration of LE (from trace to +, ++, +++).

[0043] FIG. 8. Dependence of signal ΔQ on LE concentration in human urine that was incubated for 5 min with ester α (black squares) or 10 min with ester γ (red triangles). The ΔQ=Q.sub.(ester)−Q.sub.(no ester), where Q.sub.(ester) and Q.sub.(no ester) are the charges flowing through a glucose strip in contact with an ester-incubated solution and ester-free solution, respectively. The amperograms (0.15 V) were recorded with a laboratory potentiostat and integrated between t=2 and t=22 s to obtain Q values. The different intensities of purple show a response of commercial colorimetric LE test strips to increasing concentration of LE (from trace to +, ++, +++).

DETAILED DESCRIPTION OF THE INVENTION

[0044] Embodiments of the present invention include compounds, devices, and/or diagnostic methods for detecting or evaluating the presence of infection or inflammation in humans or animals. These methods can include measuring the amount of leukocyte esterase (LE) present in a sample obtained from the human or animal being tested or diagnosed. Amount of leukocyte esterase (LE) present in the sample can be measured by contacting the sample with a glucosyl ester described herein to obtain a test sample and measuring concentration of glucose liberated in the test sample by cleavage of the glucosyl ester by LE. Certain embodiments of the present invention provides a relatively high-resolution quantification of a degree of infection in a biological fluid irrespective of their state (opacity, color). The biological fluid can be blood, plasma, serum, tears, urine, synovial (joint) fluid or saliva or other bodily fluids and effusions for infections. The glucosyl esters described herein are substrates for LE and release glucose in the presence of active enzyme leukocyte esterase (LE), which is a proxy for the presence of leukocytes and the marker of common infections. Synthesis of such esters, including their structural requirements, and the coupling of their enzymatic reactions to commercial glucose test strips is described herein.

A. Glucosyl Esters

[0045] Certain embodiments are directed to glucosyl ester of Formula I as substrates for LE. In certain aspects the glucosyl ester can be utilized in an assay of enzymatic activity of leukocyte esterase (LE) for the rapid and accurate diagnosis of the presence and extent of infection in human and animal samples.

[0046] Compounds of Formula I or their salts such as pharmaceutically acceptable salts or solvates thereof, can be prepared according to reaction Scheme 1, 2, and 3 below. Isolation and purification of the products is accomplished by standard procedures, which are known to a chemist of ordinary skill. The following schemes and examples provide examples of the processes for making compounds of Formula I. It is to be understood, however, that the invention, as fully described herein and as recited in the claims, is not intended to be limited by the details of the following examples. Unless otherwise noted, n and R.sub.1 through R.sub.5 are defined as above.

[0047] Referring to scheme 1, a compound of the formula 1 is reacted with chloroacetyl chloride in the presence of a base such as pyridine in a solvent such as dichloromethane at a temperature from 0° C. to room temperature for a time from 1 hour to 24 hours. The resulting compound, 2, is treated with hydrazine acetate in a solvent such as dimethylformamide at 0° C. for a time from 1 hour to 2 hours. The resulting compound, 3, is coupled with reactive components such as trichloroacetonitrile or 4-nitrophenyl chloroformate in the presence of base such as 1,8-diazabicyclo[5.4.0]undec-7-ene or triethylamine in a solvent such as dichloromethane, at a temperature from 0° C. to room temperature for a time from 1 hour to 24 hours. The resulting glycosyl donors 4 and 5 are reacted with the glycosyl acceptors such as compounds 9 and 12 as described in Scheme 2.

##STR00006##

[0048] Referring to Scheme 2, a compound of the formula 6 is reacted with substituted hydroxyl reagent such as compound 7 or 10 in the presence of a carboxylic acid activating reagent such as EDCI and DMAP in a solvent such as dichloromethane at a temperature from 0° C. to room temperature for a time from 1 hour to 24 hours. The resulting compound, 8 and 11, is individually treated with an organic/inorganic acid, such as TFA or hydrochloric acid, in a solvent such as ether, dioxane, or dichloromethane, at a temperature from 0° C. to room temperature for a time from 1 hour to 24 hours. In some aspects, R.sub.1 can be H or a C.sub.1 to C.sub.8 alkyl. In some particular aspects, R.sub.1 can be methyl. In some aspects, R.sub.2 can be aryl or substitutes aryl. In some particular aspects, R.sub.2 can be pC.sub.6H.sub.4CH.sub.3. In some aspects, R.sub.2 and R.sub.3 can be independently H or a C.sub.1 to C.sub.8 alkyl. In some particular aspects, R.sub.2 and R.sub.3 can be independently H or methyl. In some particular aspects, R.sub.2 and R.sub.3 can be H or methyl. In some aspects, R.sub.5 can be H or a C.sub.1 to C.sub.8 alkyl. In some particular aspects, R.sub.5 can be H or methyl. In some particular aspects, n can be 1. The resulting compounds, 9 and 12, are reacted with the glycosyl donors such as compounds 4 and 5 as described in Scheme 1.

##STR00007##

[0049] Referring to Scheme 3, a compound of the formula 5 is reacted with a glycosyl acceptor such as compounds 9 in the presence of an organic base such as DIPEA or triethylamine in a solvent such as dichloromethane at a temperature from 0° C. to room temperature for a time from 1 hour to 24 hours to form a compound having the chemical formula of Formula I where X is NR.sub.5. Alternatively, a compound of the formula 4 is reacted with a glycosyl acceptor such as compounds 12 in the presence of an organic acid such as TMSOTf and molecular sieves in a solvent such as dichloromethane at a temperature from 0° C. to room temperature for a time from 1 hour to 24 hours to form a compound having the chemical formula of Formula I where X is CR.sub.3R.sub.4. Finally, removal of the MCA protecting groups is accomplished in the presence of a tertiary amine or an organic amine base such as DIPEA or pyridine in a solvent such as water at a temperature from 0° C. to room temperature for a time from 1 hour to 24 hours.

##STR00008##

[0050] Finally, salts such as pharmaceutically acceptable salts of compounds of Formula I may be prepared by one or more of three methods: (i) by reacting the compound of Formula I with the desired acid; (ii) by removing an acid-labile protecting group from a suitable precursor of the compound of Formula I or by ring-opening a suitable cyclic precursor, for example, a lactone or lactam, using the desired acid or base; or (iii) by converting one salt of the compound of Formula I to another by reaction with an appropriate acid or base or by means of a suitable ion exchange column.

[0051] Pharmaceutically acceptable salts of the compounds of Formula I include the acid or base addition salts thereof. All three reactions are typically carried out in solution. The resulting salt may precipitate out and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionization in the resulting salt may vary from completely ionized to almost non-ionized. Suitable non-toxic, acid-addition pharmaceutically acceptable salts include, but are not limited to, the acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mandelates mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, salicylate, saccharate, stearate, succinate, sulfonate, stannate, tartrate, tosylate, trifluoroacetate and xinofoate salts.

[0052] Suitable non-toxic, base-addition pharmaceutically acceptable salts include, but are not limited to aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts. For a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002).

[0053] Included within the scope of the present invention are all stereoisomers, geometric isomers and tautomeric forms of the compounds of Formula I, including compounds exhibiting more than one type of isomerism, and mixtures of one or more thereof.

[0054] The present invention can include all pharmaceutically acceptable isotopically-labelled compounds of Formula I wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number which predominates in nature.

B. Chemical Definitions

[0055] Various chemical definitions related to such compounds are provided as follows.

[0056] As used herein, “predominantly one enantiomer” means that the compound contains at least 85% of one enantiomer, or more preferably at least 90% of one enantiomer, or even more preferably at least 95% of one enantiomer, or most preferably at least 99% of one enantiomer. Similarly, the phrase “substantially free from other optical isomers” means that the composition contains at most 5% of another enantiomer or diastereomer, more preferably 2% of another enantiomer or diastereomer, and most preferably 1% of another enantiomer or diastereomer.

[0057] As used herein, the term “water soluble” or “hydrophilic” means that the compound dissolves in water. In the context of a LE assay, “water soluble” is the minimum concentration of LE substrate that generates a measurable amount of current from the LE+substrate reaction, which can be as low as 1 micromole/liter.

[0058] As used herein, the term “nitro” means —NO.sub.2; the term “halo” or “halogenated” designates —F, —Cl, —Br or —I; the term “mercapto” means —SH; the term “cyano” means —CN; the term “azido” means —N.sub.3; the term “silyl” means —SiH.sub.3, and the term “hydroxyl” means —OH.

[0059] The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a linear (i.e. unbranched) or branched carbon chain, which may be fully saturated, mono- or polyunsaturated. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Saturated alkyl groups include those having one or more carbon-carbon double bonds (alkenyl) and those having one or more carbon-carbon triple bonds (alkynyl). The groups, —CH.sub.3 (Me), —CH.sub.2CH.sub.3 (Et), —CH.sub.2CH.sub.2CH.sub.3 (n-Pr), —CH(CH.sub.3).sub.2(iso-Pr), —CH.sub.2CH.sub.2CH.sub.2CH.sub.3 (n-Bu), —CH(CH.sub.3)CH.sub.2CH.sub.3 (sec-butyl), —CH.sub.2CH(CH.sub.3).sub.2(iso-butyl), —C(CH.sub.3).sub.3(tert-butyl), —CH.sub.2C(CH.sub.3).sub.3 (neo-pentyl), are all non-limiting examples of alkyl groups. C.sub.1 alkyl refers to alkyl group with n carbon atoms, e.g. C.sub.1 refers to methyl.

[0060] The term “halogenated alkyl” means a straight-chain or branched saturated monovalent hydrocarbon group of one to twelve carbon atoms, wherein at least one of the carbon atoms is replaced by a halogen atom (e.g. fluoromethyl, 1-bromo-ethyl, 2-chloro-pentyl, 6-iodo-hexyl, and the like).

[0061] The term “heteroalkyl” by itself or in combination with another term, means, unless otherwise stated, a linear or branched chain having at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, S, P, and Si. In certain embodiments, the heteroatoms are selected from the group consisting of 0 and N. The heteroatom(s) may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Up to two heteroatoms may be consecutive. The following groups are all non-limiting examples of heteroalkyl groups: trifluoromethyl, —CH.sub.2F, —CH.sub.2Cl, —CH.sub.2Br, —CH.sub.2OH, —CH.sub.2OCH.sub.3, —CH.sub.2OCH.sub.2CF.sub.3, —CH.sub.2OC(O)CH.sub.3, —CH.sub.2NH.sub.2, —CH.sub.2NHCH.sub.3, —CH.sub.2N(CH.sub.3).sub.2, —CH.sub.2CH.sub.2Cl, —CH.sub.2CH.sub.2OH, CH.sub.2CH.sub.2OC(O)CH.sub.3, —CH.sub.2CH.sub.2NHCO.sub.2C(CH.sub.3).sub.3, and —CH.sub.2Si(CH.sub.3).sub.3.

[0062] The terms “cycloalkyl” and “heterocycle,” by themselves or in combination with other terms, means cyclic versions of “alkyl” and “heteroalkyl”, respectively. Additionally, for heterocycle, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. C.sub.1 cycloalkyl refers to cycloalkyl group with n carbon atoms, e.g C.sub.3 refers to cyclopentyl.

[0063] The term “aryl” means a polyunsaturated, aromatic, hydrocarbon substituent. Aryl groups can be monocyclic or polycyclic (e.g., 2 to 3 rings that are fused together or linked covalently). The term “heteroaryl” refers to an aryl group that contains one to four heteroatoms selected from N, O, and S. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.

[0064] Various groups are described herein as substituted or unsubstituted (i.e., optionally substituted). Optionally substituted groups may include one or more substituents independently selected from: halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, oxo, carbamoyl, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, alkoxy, alkylthio, alkylamino, (alkyl).sub.2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. In certain aspects the optional substituents may be further substituted with one or more substituents independently selected from: halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, unsubstituted alkyl, unsubstituted heteroalkyl, alkoxy, alkylthio, alkylamino, (alkyl).sub.2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, unsubstituted cycloalkyl, unsubstituted heterocyclyl, unsubstituted aryl, or unsubstituted heteroaryl. Exemplary optional substituents include, but are not limited to: —OH, oxo (═O), —Cl, —F, Br, C.sub.1-4alkyl, phenyl, benzyl, —NH.sub.2, —NH(C.sub.1-4alkyl), —N(C.sub.1-4alkyl).sub.2, —NO.sub.2, —S(C.sub.1-4alkyl), —SO.sub.2(C.sub.1-4alkyl), —CO.sub.2(C.sub.1-4alkyl), and —O(C.sub.1-4alkyl). In certain aspects a heterocycle or a heteroaryl is optionally substituted with 1 to 3 halogens, C.sub.1 to C.sub.3 alkyl, OH, NH.sub.2, C(O)NH.sub.2, CO.sub.2H, CH.sub.2OH and —C(O)NHR′, C(O)NR′.sub.2, OR′, CH.sub.2OR′, NHR′ or N(R′).sub.2, where in R′ is C.sub.1 to C.sub.3 alkyl or halogenated alkyl. In other aspects a cycloakyl can be optionally substituted with 1 to 3 halogens, C.sub.1 to C.sub.3 alkyl, OH, NH.sub.2, C(O)NH.sub.2, CO.sub.2H, CH.sub.2OH, C(O)NHR″, C(O)NR″.sub.2, OR″ CH.sub.2OR″, NHR″, —N(R″).sub.2 or combinations thereof, where R″ is C.sub.1 to C.sub.3 alkyl or halogenated alkyl.

[0065] The term “alkoxy” means a group having the structure —OR′, where R′ is an optionally substituted alkyl or cycloalkyl group. The term “heteroalkoxy” similarly means a group having the structure —OR, where R is a heteroalkyl or heterocyclyl.

[0066] The term “amino” means a group having the structure —NR′R″, where R′ and R″ are independently hydrogen or an optionally substituted alkyl, heteroalkyl, cycloalkyl, or heterocyclyl group. The term “amino” includes primary, secondary, and tertiary amines.

[0067] The term “oxo” as used herein means an oxygen that is double bonded to a carbon atom.

[0068] The term “alkylsulfonyl” as used herein means a moiety having the formula —S(O.sub.2)—R′, where R′ is an alkyl group. R′ may have a specified number of carbons (e.g. “C.sub.1-4 alkylsulfonyl”).

[0069] The term “pharmaceutically acceptable salts,” as used herein, refers to salts of compounds of this invention that are substantially non-toxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of a compound of this invention with an inorganic or organic acid, or an organic base, depending on the substituents present on the compounds of the invention.

[0070] Non-limiting examples of inorganic acids which may be used to prepare pharmaceutically acceptable salts include: hydrochloric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid and the like. Examples of organic acids which may be used to prepare pharmaceutically acceptable salts include: aliphatic mono- and dicarboxylic acids, such as oxalic acid, carbonic acid, citric acid, succinic acid, phenyl-heteroatom-substituted alkanoic acids, aliphatic and aromatic sulfuric acids and the like. Pharmaceutically acceptable salts prepared from inorganic or organic acids thus include hydrochloride, hydrobromide, nitrate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, hydroiodide, hydro fluoride, acetate, propionate, formate, oxalate, citrate, lactate, p-toluenesulfonate, methanesulfonate, maleate, and the like.

[0071] Suitable pharmaceutically acceptable salts may also be formed by reacting the agents of the invention with an organic base such as methylamine, ethylamine, ethanolamine, lysine, ornithine and the like. Pharmaceutically acceptable salts include the salts formed between carboxylate or sulfonate groups found on some of the compounds of this invention and inorganic cations, such as sodium, potassium, ammonium, or calcium, or such organic cations as isopropylammonium, trimethylammonium, tetramethylammonium, and imidazolium.

[0072] It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable.

[0073] Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, Selection and Use by Stahl and Wermuth (Wiley-VCH, 2002) which is incorporated herein by reference.

[0074] An “isomer” of a first compound is a separate compound in which each molecule contains the same constituent atoms as the first compound, but where the configuration of those atoms in three dimensions differs. Unless otherwise specified, the compounds described herein are meant to encompass their isomers as well. A “stereoisomer” is an isomer in which the same atoms are bonded to the same other atoms, but where the configuration of those atoms in three dimensions differs. “Enantiomers” are stereoisomers that are mirror images of each other, like left and right hands. “Diastereomers” are stereoisomers that are not enantiomers.

C. Leukocyte Esterase Assay

[0075] The glucosyl esters described herein can function as substrates for LE, and can get cleaved by LE to release glucose, reaction (1). Amount of glucose released can be proportional to the LE activity:

[00001]$\begin{array}{cc}\mathrm{glucosyl}\mathrm{ester}\stackrel{\mathrm{LE}}{.fwdarw.}\mathrm{glucose}& \left(1\right)\end{array}$

In some aspects, LE activity in a sample can be measured by contacting the sample with a glucosyl ester described herein to form a test sample and measuring concentration of glucose released in the test sample by cleavage of the glucosyl ester by LE. The glucose released can be detected with a glucose detecting component. In some aspects, concentration of the glucose released can be measured by a glucose test strip, by measuring electrical charge flowing through the glucose test strip. In some aspects, the test sample can be contacted with a glucose test strip, such as a known glucose test strip, and charge flowing through the glucose test strip can be measured to obtain glucose concentration in the test sample. In some aspects, the charge flowing through the glucose test strip can be measured with a potentiostat or a glucometer.

[0076] Glucosyl ester described herein can be incorporated into diagnostic products or kits. The diagnostic products can be used for detecting LE activity by detecting glucose. In certain aspects, the diagnostic products can include at least one compound or agent useful in detecting the presence and/or concentration of glucose. The term “compound or agent useful in detecting the presence of glucose”, as used herein, refers to a compound, composition, or combination thereof that is changed by presence and/or concentration of glucose.

[0077] Diagnostic kits can be useful for detecting LE activity by detecting glucose. In certain aspects kits can include a device or apparatus or product for collecting a sample such as a biological fluid from a human or an animal being tested or diagnosed, and assay or assay device for measuring the amount of glucose released in the sample after the sample is contacted with a glucosyl ester described herein.

[0078] The phrase “device or apparatus for collecting a sample”, as used herein, means any device or apparatus or product which is useful for removing a sample of fluid, tissue, or cells from a human or animal being tested or diagnosed without adversely affecting the ability to detect the presence of leukocyte esterase activity in the sample. Non-limiting examples of such devices include swabs, pipettes, syringes, absorbent tapes, absorbent gauzes, absorbent strips, scoops, suction bulbs, and aspirators. A kit can include one or more diagnostic products described herein.

[0079] In certain aspects the kits can be manufactured such that the sample collecting device and the assay device are separate components in the kits. The kit can include optional components to be used with the kits (e.g. test tubes for diluting samples in; bottles containing dilution fluid for diluting samples; instruction sheets; etc.) that can be combined into one package. An example of such a package is a box which is shrink wrapped with plastic.

EXAMPLES

[0080] The following examples as well as the figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

Glucosyl Ester Substrates for Enzyme Leukocyte Esterase (LE)

[0081] Reagents and Solutions. The esters a (CIDD-0150156, MW: 491.51 g mol.sup.−1), R (CIDD-0150181, MW: 506.52 g mol.sup.−1), γ (CIDD-0150161, MW: 492.5 g mol.sup.−1), and κ (CIDD-0150157, MW:519.56 g mol.sup.−1) were synthesized in the Center for Innovative Drug Discovery (CIDD) at the University of Texas at San Antonio.

[0082] The human leukocyte suspension in 154 mM NaCl solution (cat. No. MBS173116, 0.0867 mg mL.sup.−1 leukocyte esterase (LE) protein, 4×10.sup.8 WBC mL.sup.−1) was purchased from MyBioSource (San Diego, Calif.). The suspension was diluted 10 times with a 90:10 vol. % mixture of 50 mM phosphate buffer saline (PBS, pH 7.40, 154 mM NaCl) solution and dimethylsulfoxide, left for 10 min to lyse the leukocytes chemically, sonicated for 30 s with a Q125 Qsonica probe (20% power) to further lyse them mechanically, and centrifuged at 15 000×g for 30 min. The resulting supernatant that contained LE was stored at −20° C.

[0083] The human synovial fluid (DLS0092658) was purchased from Discovery Life Sciences (Los Osos, Calif.) and stored at −70° C. when not in use. The clean-catch midstream portion of human urine (˜15.0 mL) was collected early in the morning in a 50.0-mL sterile centrifuge tube (Fisher Scientific, Pittsburgh, Pa.) centrifuged at 600×g for 5 min, and the supernatant was stored at −20° C. The synovial fluid and urine were spiked with known amount of LE before the analysis.

[0084] The glucose test strips (ACCU-CHECK Aviva Plus, Roche Diabetes Care Inc., Indianapolis, Ind.) and colorimetric urinary infection LE test strips (Siemens Multistix 10 SG) were purchased locally. The correlation between the color zones of colorimetric LE strips (trace, +, ++, +++) and the concentration of LE was established by using a PBS solution, which was spiked with a known amount of LE (μg L.sup.−1).

[0085] Electrochemical Measurements. The glucose strip was inserted into an electronic holder (DropSens, Llanera, Spain), which was then connected to a CHE 832B potentiostat (other potentiostats could also be used). The strip was used in a 2-electrode mode with the counter and reference electrodes connected together as a single cathode and a working electrode serving as an anode (the strip's fill-detector electrode was not used). The potential applied to a working electrode was equal to 0.15 V vs. strip's reference/counter electrode.

[0086] The 0.15 V potential was applied to a glucose strip, which was then contacted at time t=0 s with a small drop of sample (˜10 μL) that was placed on a Petri dish (FIG. 2). The amperogram obtained in such a way was integrated between t=2 and t=22 s to determine the charge Q flowing through the strip. Two charges were always determined: Q.sub.(ester) for a sample incubated with 5.5 mM ester for 5 min, and Q.sub.(no ester) for an original sample without ester. The difference in charge ΔQ [=Q.sub.(ester)−Q.sub.(no ester)] was proportional to the enzymatic activity of LE in a sample.

[0087] General procedures. All operations were carried out at room or ambient temperature, that is, in the range of 18-25° C.; evaporation of solvent was carried out using a rotary evaporator under reduced pressure with a bath of up to 60° C.; reactions were monitored by thin layer chromatography (tlc) and reaction times are given for illustration only; melting points (m.p.) given are uncorrected (polymorphism may result in different melting points); structure and purity of all isolated compounds were assured by at least one of the following techniques: tlc (Merck silica gel 60 F-254 precoated plates), high performance liquid chromatography (HPLC), mass spectrometry, nuclear magnetic resonance (NMR) or infrared spectroscopy (IR). Yields are given for illustrative purposes only. Flash column chromatography was carried out using Merck silica gel 60 (230-400 mesh ASTM). Low-resolution mass spectral data (EI) were obtained on platform 1: an Agilent 1290 series HPLC system (Method 1) comprised of binary pumps, degasser and UV detector, equipped with an auto-sampler that is coupled with Agilent 6150 mass spectrometer; platform 2: a Thermo Scientific Vanquish UHPLC system. The general Liquid Chromatography parameters were as follows using solvent A (0.10% formic acid in water) and solvent B (0.00% formic acid in acetonitrile): Method 1: analysis was performed on a Zorbax Eclipse Plus C18 column with dimension of 2.1×50 mm. The flow rate was 0.7 ml/minute running a gradient of 5% to 95% solvent B in 5 minutes and hold at 95% solvent B for 2 minutes. Method 2: analysis was performed on a Hypersil GOLD C18 column with dimension of 2.1×100 mm. The flow rate was 1.0 ml/minute running a gradient of 5% to 95% solvent B in 0.8 minutes and hold at 95% solvent B for 0.4 minutes. The ionization type for the mass detector of the mass spectrophotometer was atmospheric pressure electrospray in the positive ion mode with a fragmentor voltage of 50 volts. NMR data was determined at 400 MHz (Agilent DD2 400 MHz spectrometer) using deuterated chloroform (99.8% D), methanol (99.8% D) or dimethylsulfoxide (99.9% D) as solvent unless indicated otherwise, relative to tetramethylsilane (TMS) as internal standard in parts per million (ppm); conventional abbreviations used are: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broad, etc.

[0088] The following abbreviations are used:

K.sub.2CO.sub.3: potassium carbonate; MeCN: acetonitrile; NaOH: sodium hydroxide; MeOH: methanol; SOCl.sub.2: thionyl chloride; DMF: dimethylformamide; CH.sub.2Cl.sub.2: dichloromethane; THF: tetrahydrofuran; Et.sub.3N: triethylamine; Pd/C: palladium on activated carbon; EtOH: ethanol; NaHCO.sub.3: sodium bicarbonate; HCl: hydrogen chloride; EtOAc: ethyl acetate; Na.sub.2SO.sub.4: sodium sulfate; MeCN: acetonitrile; MCA: methylchloroacetate; DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene; EDCI: N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride; DMAP: 4-(Dimethylamino)pyridine; TFA: Trifluoroacetic acid; DIPEA: N,N-Diisopropylethylamine; TMSOTf: Trimethylsilyl trifluoromethanesulfonate.

##STR00009##

[0089] Referring to Scheme 1 above, a compound of the formula 1 is reacted with chloroacetyl chloride in the presence of a base such as pyridine in a solvent such as dichloromethane at a temperature from 0° C. to room temperature for a time from 1 hour to 24 hours. The resulting compound, 2, is treated with hydrazine acetate in a solvent such as dimethylformamide at 0° C. for a time from 1 hour to 2 hours. The resulting compound, 3, is coupled with reactive components such as trichloroacetonitrile or 4-nitrophenyl chloroformate in the presence of base such as 1,8-diazabicyclo[5.4.0]undec-7-ene or triethylamine in a solvent such as dichloromethane, at a temperature from 0° C. to room temperature for a time from 1 hour to 24 hours. The resulting glycosyl donors 4 and 5 are reacted with the glycosyl acceptors such as compounds 9 and 12 as described in Scheme 2.

[0090] Oenta(chloroacetyl)glucose 2: A solution of β-D-glucose (5.0 g, 27.7 mmol, 1 equiv) in dry CH.sub.2Cl.sub.2 (110 ml) and dry pyridine (14 ml) at 0° C. was added a solution of chloroacetyl chloride (31.3 g, 277 mol, 10 equiv) in dry CH.sub.2Cl.sub.2 (45 ml) through an addition funnel over a period of 2 hours. The resulting slurry turned from bright orange/red colors to yellowish homogeneous solution after stirring at ambient temperate for 24 hours. The reaction mixture was quenched with 1N HCl (aq.) (100 ml) and the mixture was extracted with CH.sub.2Cl.sub.2 (2×50 ml). The combined organic extracts were washed with saturated NaHCO.sub.3 (aq.) (100 ml), brine (100 ml) and were drived over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The crude material was purified by Biotage flash chromatography (gradient elution, 0 to 35% EtOAc in hexanes) to obtain the title compound 2 (15.2 g, 98%) as viscous yellow oil. .sup.1H NMR matched the one reported by Y. Zhu, J. Ralph, Tetrahedron Letters, 2011, 52, 3729-3731.

[0091] 2,3,4,6-tetra-O-chloroacetylglucose 3: A solution of β-D-glucose pentamethylchloroacetate (2) (1.3 g, 2.31 mmol, 1 equiv) in DMF (4 ml) at 0° C. was added hydrazine acetate (255 mg, 2.77 mmol, 1.2 equiv). The reaction mixture was stirred at this temperature for 2 hours. Upon reaction completion based on TLC analysis (50% EtOAc in hexanes), the reaction was diluted with EtOAc (20 ml) and washed with H.sub.2O (20 ml), and the organic extracts were washed with brine and dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The crude material was purified by Biotage flash chromatography (gradient elution, 0 to 35% EtOAc in hexanes) to obtain the title compound 3 (883 mg, 79%) as colorless foams, .sup.1H NMR matched the one reported by Y. Zhu, J. Ralph, Tetrahedron Letters, 2011, 52, 3729-3731.

[0092] 2,3,4,6-tetra-O-chloroacetylglucosyl trichloroacetimidate 4: A solution of the 2,3,4,6-tetra-O-chloroacetylglucose (3) (1.82 g, 3.74 mmol, 1 equiv) in dry CH.sub.2Cl.sub.2 (12 ml) at 0° C. was added trichloroacetonitrile (5.4 g, 37.4 mmol, 10 equiv) and catalytic amount of DBU (85 mg, 0.561 mmol, 0.15 equiv). The reaction was left stirring at this temperature and gradually raised to ambient temperature for overnight. The reaction mixture was concentrated and the resulting residue was purified by Biotage flash chromatography (gradient elution, 0 to 20% EtOAc in hexanes) to obtain the title compound 4 (2.06 g, 88%) as yellow viscous oil, which was used as is.

[0093] 2,3,4,6-tetra-O-chloroacetylglucosyl 4-nitrophenylcarbonate 5: A solution of 2,3,4,6-tetra-O-chloroacetylglucose (3) (1.1 g, 2.26 mmol, 1 equiv) in dry CH.sub.2Cl.sub.2 (12 ml) at 0° C. was added triethylamine (343 mg, 3.39 mmol, 1.5 equiv) and 4-nitrophenyl chloroformate (547 mg, 2.71 mmol, 1.2 equiv). The reaction was raised to ambient temperature and stirred for 1 hour. The reaction mixture was concentrated and the resulting residue was purified by Biotage flash chromatography (gradient elution, 0 to 35% EtOAc in hexanes) to obtain the title compound 5 (1.2 g, 81%) as yellow viscous oil, which was used as is.

##STR00010##

[0094] Referring to Scheme 2 above, a compound of the formula 6 is reacted with substituted hydroxyl reagent such as compound 7a/b or 10a/b in the presence of a carboxylic acid activating reagent such as EDCI and DMAP in a solvent such as dichloromethane at a temperature from 0° C. to room temperature for a time from 1 hour to 24 hours. R depicted in scheme 2 is individually selected from groups such as hydrogen, methyl and other alkyl substituents. The resulting compound, 8a/b and 11a/b, is individually treated with an organic/inorganic acid, such as TFA or hydrochloric acid, in a solvent such as ether, dioxane, or dichloromethane, at a temperature from 0° C. to room temperature for a time from 1 hour to 24 hours. The resulting compounds, 9a/b and 12a/b, are reacted with the glycosyl donors such as compounds 4 and 5 as described in Scheme 1.

[0095] 2-((tert-butoxycarbonyl)(methyl)amino)ethyl tosyl-L-alaninate 8a: A solution of 6 (1 g, 5.70 mmol, 1 equiv) and N-methyl-N-boc-glycinol 7a (R=methyl) (1.45 g, 5.99 mmol, 1.05 equiv) in dry CH.sub.2Cl.sub.2 (15 ml) was added DMAP (350 mg, 2.86 mmol, 0.5 equiv) and EDCI (1.20 g, 6.27 mmol, 1.1 equiv), and the resulting reaction was stirred at room temperature for 24 hours. The reaction was concentrated and purified by Biotage flash chromatography (gradient elution, 0 to 40% EtOAc in hexanes) to obtain the title compound 8a (R=methyl) (1.40 g, 58%) as colorless oil. .sup.1H NMR (400 MHz, Chloroform-d) δ 7.73 (d, J=8.3 Hz, 2H), 7.29 (d, J=8.2 Hz, 2H), 4.04 (m, 2H), 3.97 (pent, J=7.8 Hz, 1H), 3.44-3.29 (br, 3H). 2.83 (s, 3H), 2.42 (s, 3H), 1.45 (s, 9H), 1.39 (d, J=7.2 Hz, 3H). LCMS (m/z) 301.1 (M-Boc); RT (Method 2, std LCMS method), 1.08 min.

[0096] Compound 8b (R=hydrogen) was prepared using the reaction condition described above with the corresponding N-boc-glycinol 7b (R=hydrogen) to give the title compound 8b (R=hydrogen) (1.61 g, 64%) as colorless oil. .sup.1H NMR (400 MHz, Chloroform-d) δ 7.74 (d, J=8.2 Hz, 2H), 7.30 (d, J=8.2 Hz, 2H), 5.21 (m, 1H), 4.69 (br, 1H), 4.04-3.95 (m, 3H), 3.27 (m, 2H), 2.42 (s, 3H), 1.45 (s, 9H), 1.38 (d, J=7.2 Hz, 3H). LCMS (m/z) 287.2 (M-Boc); RT (Method 2, std LCMS method), 1.25 min.

[0097] Compound 11a (R=methyl) was prepared using the reaction condition described above with the corresponding tert-butyl-4-hydroxy-2,2-dimethylbutanoate 10a (R=methyl) to give the title compound 11a (R=methyl) (1.11 g, 68%) as colorless oil. .sup.1H NMR (400 MHz, Chloroform-d) δ 7.72 (d, J=8.2 Hz, 2H), 7.29 (d, J=8.2 Hz, 2H), 5.18 (m, 1H), 3.95 (m, 3H), 2.41 (s, 3H), 1.70 (m, 2H), 1.43 (s, 9H), 1.37 (d, J=7.1 Hz, 3H), 1.11 (s, 6H).

[0098] Compound 11b (R=hydrogen) was prepared using the reaction condition described above with the corresponding tert-butyl 4-hydroxybutanoate 10b (R=hydrogen) to give the title compound 11b (R=hydrogen) (1.96 g, 91%) as colorless oil. .sup.1H NMR (400 MHz, Chloroform-d) δ 7.72 (d, J=8.2 Hz, 2H), 7.30 (d, J=8.0 Hz, 2H), 5.19 (d, J=8.2 Hz, 1H), 4.01-3.92 (m, 3H), 2.42 (s, 3H), 2.20 (t, J=7.4 Hz, 2H), 1.80 (pent, J=7.2 Hz, 2H), 1.45 (s, 9H), 1.39 (d, J=7.2 Hz, 3H).

[0099] 2-(methylamino)ethyl tosyl-L-alaninate 9a: A solution of 8a (1.40 g, 3.49 mmol, 1 equiv) in dry CH.sub.2Cl.sub.2 (10 ml) was added HCl (2.6 ml, 10.48 mmol, 3 equiv, 4M solution in dioxane), and the resulting reaction was stirred at room temperature for 3 hours. The reaction was concentrated and triturated with methanol and diethyl ether, the resulting precipitates were collected through filtration to give the title compound 9a (R=methyl) as hydrochloride salt (1.08 g, 92%). .sup.1H NMR (400 MHz, DMSO-d6) δ 8.86 (br, 2H), 8.28 (br, 1H), 7.67 (d, J=8.2 Hz, 2H), 7.40 (d, J=8.0 Hz, 2H), 4.11 (m, 1H), 4.02 (m, 1H), 3.92 (m, 1H), 3.07 (m, 2H), 2.56 (s, 3H), 2.39 (s, 3H), 1.20 (d, J=7.2 Hz, 3H). LCMS (m/z) 301.2 (M+1); RT (Method 2, std LCMS method), 0.73 min.

[0100] Compound 9b (R=hydrogen) was prepared using the reaction condition described above to give the title compound 9b (R=hydrogen) (1.3 g, 92%) as hydrochloride salt. .sup.1H NMR (400 MHz, DMSO-d6) δ 8.28 (br, 4H), 7.68 (d, J=8.0 Hz, 2H), 7.39 (d, J=8.1 Hz, 2H), 4.05 (m, 1H), 3.94 (m, 2H), 2.94 (m, 2H), 2.38 (s, 3H), 1.21 (d, J=7.2 Hz, 3H). LCMS (m/z) 287.1 (M+1); RT (Method 2, std LCMS method), 0.74 min.

[0101] 2,2-dimethyl-4-((tosyl-L-alanyl)oxy)butanoic acid 12a: A solution of 11a (1.11 g, 2.68 mmol, 1 equiv) in CH.sub.2Cl.sub.2 (9 ml) was added TFA/CH.sub.2Cl.sub.2 (v:v/1:1, 9 ml) at 0° C., and the resulting reaction was raised to room temperature and stirred for 3 hours. The reaction was concentrated and purified by Biotage flash chromatography (gradient elution, 0 to 75% EtOAc in hexanes) to obtain the title compound 12a (R=methyl) (930 mg, 97%) as colorless gel. .sup.1H NMR (400 MHz, Chloroform-d) δ 7.74 (d, J=8.2 Hz, 2H), 7.30 (d, J=8.1 Hz, 2H), 5.38 (d, J=8.8 Hz, 1H), 4.09-3.96 (m, 3H), 2.42 (s, 3H), 1.96-1.82 (m, 2H), 1.32 (d, J=7.2 Hz, 3H), 1.24 (s, 3H), 1.21 (d, 3H). LCMS (m/z) 358.1 (M+1); RT (Method 2, std LCMS method), 1.15 min.

[0102] Compound 12b (R=hydrogen) was prepared using the reaction condition described above to give the title compound 12b (R=hydrogen) (1.46 g, 87%) as colorless gel. .sup.1H NMR (400 MHz, Chloroform-d) δ 7.74 (d, J=8.2 Hz, 2H), 7.30 (d, J=8.4 Hz, 2H), 5.35 (br, 1H), 5.28 (d, J=8.6 Hz, 1H), 4.05-3.95 (m, 3H), 2.42 (s, 3H), 2.37 (t, J=7.2 Hz, 2H), 1.94-1.84 (m, 2H), 1.37 (d, J=7.2 Hz, 3H). LCMS (m/z) 330.1 (M+1); RT (Method 2, std LCMS method), 1.02 min.

##STR00011##

[0103] Referring to Scheme 3 above, a compound of the formula 5 is reacted with the glycosyl acceptor such as compounds 9a/b in the presence of an organic base such as DIPEA or triethylamine in a solvent such as dichloromethane at a temperature from 0° C. to room temperature for a time from 1 hour to 24 hours. Alternatively, a compound of the formula 4 is reacted with the glycosyl acceptor such as compounds 12a/b in the presence of an organic acid such as TMSOTf and molecular sieves in a solvent such as dichloromethane at a temperature from 0° C. to room temperature for a time from 1 hour to 24 hours. R depicted in scheme 3 is individually selected from groups such as hydrogen, methyl and other alkyl substituents. Finally, removal of the MCA protecting groups is accomplished in the presence of a tertiary amine or an organic amine base such as DIPEA or pyridine in a solvent such as water at a temperature from 0° C. to room temperature for a time from 1 hour to 24 hours.

[0104] 2-(methyl((((3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)-tetrahydro-2H-pyran-2-yl)oxy)carbonyl)amino)ethyl tosyl-L-alaninate 13a (CIDD-0150157): A solution of 5 (475 mg, 0.73 mmol, 1 equiv) and 9a (R=methyl) (245 mg, 0.73 mmol, 1 equiv) in dry CH.sub.2Cl.sub.2 (5 ml) was added DIPEA (188 mg, 1.46 mmol, 2 equiv), and the resulting reaction was stirred at room temperature for 24 hours. The reaction was concentrated and added pyridine/water (v:v/1:1, 10 ml), and the reaction mixture was stirred at room temperature for 48 hours. The reaction was concentrated and azerotropically removing the residual water by co-evaporating with toluene for couple times under reduced pressure, and the material was purified by Biotage flash chromatography (gradient elution, 0 to 25% MeOH in CH.sub.2Cl.sub.2) to obtain the title compound 13a (CIDD-0150157, R═CH.sub.3, glucosyl ester κ) (225 mg, 50%) as colorless gel. .sup.1H NMR (400 MHz, Methanol-d4) δ 7.72 (d, J=8.1 Hz, 2H), 7.37 (d, J=8.0 Hz, 2H), 5.99 (d, J=3.3 Hz, OH), 4.14-3.91 (m, 3H), 3.78-3.66 (m, 3H), 3.64-3.41 (m, 5H), 2.43 (s, 3H), 1.37 (d, J=6.4 Hz, 3H), 1.28 (d, J=6.8 Hz, 3H). LCMS (m/z) 505.8 (M−1); RT (Method 2, std LCMS method), 0.80 min

[0105] Compound 13b (CIDD-0150161, R═H, glucosyl ester γ) was prepared using the reaction condition described above with the corresponding compound 9b (R=hydrogen) to give the title compound 13b (R=hydrogen) (222 mg, 62%) as colorless foams. .sup.1H NMR (400 MHz, Methanol-d4) δ 7.72 (d, J=7.9 Hz, 2H), 7.37 (d, J=7.9 Hz, 2H), 5.97 (d, J=3.4 Hz, 1H), 3.97-3.82 (m, 4H), 3.77-3.62 (m, 4H), 3.27-3.20 (m, 3H), 2.42 (s, 3H), 1.29 (d, J=7.2 Hz, 3H). LCMS (m/z) 491.7 (M−1); RT (Method 2, std LCMS method), 0.79 min

[0106] (2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-vi 2,2-dimethyl-4-((tosyl-L-alanyl)oxy)butanoate 14a (CIDD-0150181): To a flame-dried round-bottom flask with activated 4A molecular sieves was charged with compound 4 (705 mg, 1.12 mmol, 1 equiv) and 12a (R=methyl) (440 mg, 1.23 mmol, 1.1 equiv) and dissolved the mixture in dry CH.sub.2Cl.sub.2 (12 ml). The mixture was cooled to 0° C. and added TMSOTf (50 mg, 0.22 mmol, 0.2 equiv), and the reaction was stirred at this temperature gradually raised to room temperature without replenishing the cooling bath for the next 24 hours. The reaction was quenched by adding saturated NaHCO.sub.3 (aq.) (20 ml) and extracted with CH.sub.2Cl.sub.2 (2×20 ml). The combined organic extracts were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The crude material was diluted with pyridine/water (v:v/1:1, 10 ml), and the reaction mixture was stirred at room temperature for 48 hours. The reaction was concentrated and azerotropically removing the residual water by co-evaporating with toluene for couple times under reduced pressure, and the material was purified by Biotage flash chromatography (gradient elution, 0 to 18% MeOH in CH.sub.2Cl.sub.2) to obtain the title compound 14a (CIDD-0150181, R═CH.sub.3, glucosyl ester β) (327 mg, 56%) as colorless foam. .sup.1H NMR (400 MHz, Methanol-d4) δ 7.72 (d, J=8.3 Hz, 2H), 7.36 (d, J=8.0 Hz, 2H), 5.45 (d, J=8.0 Hz, 1H), 4.00-3.78 (m, 4H), 3.76-3.64 (m, 1H), 3.45-3.34 (m, 4H), 2.43 (s, 3H), 1.86-1.71 (m, 2H), 1.27 (d, J=7.2 Hz, 3H), 1.21 (s, 3H), 1.20 (s, 3H). LCMS (m/z) 518.8 (M−1); RT (Method 2, std LCMS method), 0.92 min

[0107] Compound 14b (CIDD-0150156, R═H, glucosyl ester α) was prepared using the reaction condition described above with the corresponding compound 12b (R=hydrogen) to give the title compound 14b (R=hydrogen) (126 mg, 41%) as colorless foam. .sup.1H NMR (400 MHz, Methanol-d4) δ 7.71 (d, J=8.2 Hz, 2H), 7.37 (d, J=8.1 Hz, 2H), 5.49 (d, J=8.2 Hz, 1H), 3.98-3.82 (m, 4H), 3.68 (dd, J=12.0, 4.9 Hz, 1H), 3.45-3.35 (m, 4H), 2.42 (s, 3H), 2.43-2.37 (m, 2H), 1.81 (pent, J=6.9 Hz, 2H), 1.29 (d, J=7.2 Hz, 3H). LCMS (m/z) 490.8 (M−1); RT (Method 2, std LCMS method), 0.85 min

[0108] Electrochemical Testing. The four glucosyl esters α, β, γ, and κ (Scheme 3) were tested for the determination of LE in synovial (joint) fluid and urine samples:

[00002]$\begin{array}{cc}\mathrm{ester}\alpha ,\beta ,\gamma ,\mathrm{or}\kappa \stackrel{\mathrm{LE}}{.fwdarw.}\mathrm{glucose}& \left(1\right)\end{array}$

[0109] The samples were incubated with each ester to release glucose in the direct proportion to the enzymatic activity of LE in a sample. The released glucose was then detected at a commercial glucose test strip. A laboratory potentiostat was used to read the glucose strip. The strip was inserted in an electronic holder to facilitate the measurements (FIG. 2). Such a strip-potentiostat measuring system provided a much lower limit of glucose detection (5.0 μM, S/N=3; FIG. 3A) and a linear range at least up to 100 μM glucose (FIG. 3B), which allowed for a much shorter (minutes) incubation times (FIG. 4).

[0110] FIG. 5 shows the chronocoulometric plots that were recorded with the strip-potentiostat system after only a 5-min sample incubation with ester α. The charge flowing through a glucose strip in contact with a LE-free synovial fluid sample (panel A, trace a) was much larger than the corresponding charge for urine (panel B, trace a). Despite this difference, the rise in the LE content of either sample caused the increase in a charge flowing through the contacting glucose test strip.

[0111] FIG. 6 shows the direct correlation between the glucose strip's signal (ΔQ) and the concentration of LE in synovial fluid and urine. It illustrates several useful features including the: [0112] a. distinction of different LE contents within a one-color zone of a colorimetric LE strip [0113] b. short sample incubation time (e.g. 5 min instead of hours) [0114] c. clinically relevant LE range from 25 up to at least 800 μg L.sup.1 [0115] d. one convergent calibration plot for different real-life samples [0116] e. linear “signal vs. concentration” plot due to the prevalence of zero-order kinetics [0117] f. quantitative assessment of infection irrespective of sample transparency (opaque, colored, bloodied)

[0118] FIG. 7A shows the chronocoulometric plots that were recorded for urine samples containing very high concentration of native glucose (18 mM). As predicted, the charge Q flowing through a glucose strip in contact with such a sample was very high (trace a). Nevertheless, the ester-glucose strip system could detect the extra glucose produced by LE present in such samples (traces b-e). FIG. 7B shows that the proposed differential measurement based on ΔQ could reliably account for the presence of native glucose in a sample as indicated by the overlap of data points recorded in the presence and absence of glucose in a sample.

[0119] Analysis of LE with Ester γ. When compared to α, the ester γ required a longer incubation time (10 min) to yield enough glucose (reaction 1) to be above the detection limit of the potentiostat/glucose strip system (5 μM). FIG. 8 shows that γ also gave a ˜10% lower calibration slope for LE in urine. The longer sample incubation time and lower calibration slope could be ascribed to the slower kinetics of the enzymatic reaction of LE with ester γ. The slower kinetics could be attributed to the more proteolytically stable carbamate functionality of ester γ (Scheme 3).

[0120] The LE-triggered release of glucose from the glucosyl esters provides a relatively rapid method for the screening of human synovial (joint) fluid and urine samples for infections. Such esters, together with commercial glucose strips and a laboratory potentiostat, offer a superior signal resolution and the ability to quantify an infection enzyme LE in real-life samples regardless of their state (opaque, colored, bloodied) and the presence of potentially interfering species including native glucose. Such a screening of patient samples has a potential to facilitate clinical decisions about the use of antibiotics e.g. in the case of urinary tract and periprosthetic joint infections; the latter being one of the most devastating and costly complications of joint reconstruction/replacement (Wang et. al., Med. Sci. Monit. 2017, 23, 353-358; Kurtz et al., J. Arthroplasty 2012, 27, 61-65; Peel et al., J. Hosp. Infect. 2013, 85, 213-219.