FLUOROGENIC PROBES FOR MEDIUM CHAIN ACYL-COA DEHYDROGENASE (MCAD)

This application claims benefit of U.S. Provisional Application No. 60/709,081, filed August 16, 2005, the contents of which are hereby incorporated by reference.

Throughout this application, various publications are referenced by complete citation in parentheses . The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

Background of the Invention

Enzyme-substrate specificity is a basic assumption in biochemistry, however examples have emerged where an enzyme acts on a molecule that is structurally different than the natural substrate. This ability to accept multiple substrates is termed "enzyme promiscuity." In fact, on close examination, many metabolic pathways have built-in flexibility where the enzymes metabolize an array of substrates. This promiscuity is essential to a number of pathways such as xenobiotic metabolism and fatty acid metabolism.

Mitochondria β-oxidation is a four-step cycle, that results in the loss of a two carbon unit (acetyl -CoA) , Fig. 1 (1, 2) . The promiscuity of this cycle was first shown when Knoop fed dogs ω-labeled fatty acids and found they were metabolized (3) . Each enzyme of the cycle can accommodate a variety of chain lengths, which allows

amino acid conservation. ' For example, the complete oxidation of palmitoyl-CoA (Ci _S H ₃ iO-CoA) requires seven "turns" of the cycle. If each step required a new enzyme, twenty-eight enzymes would be involved. Instead, overlapping specificity has evolved, thus a set of four enzymes can carry out the first six "turns" and a second set of four is required for the last cleavage. Only eight enzymes must be synthesized, rather than twenty- eight .

Acyl Coenzyme A Dehydrogenases (ACADs) , the first enzymes in β-oxidation, catalyze the α, β-dehydrogenation of acyl -CoAs, accompanied by the reduction of a bound FAD. The ACAD family is composed of nine isozymes, five of which are involved in fatty acid metabolism (4) . Medium- chain acyl-Coa dehydrogenase (MCAD) was once known as "general" due to its ability to act on a wide variety of chain lengths, Fig. 2. However, when an error occurs in MCAD' s genetic code it prohibits fatty acids from being metabolized completely and ultimately results in death.

If detected in the first months of life, symptoms can be prevented by avoidance of fasting and a regulated diet.

Over a hundred cases of MCAD deficiency have been reported since it first was identified in 1982 (5, 6) . The occurrence rate has been calculated to be 1:15,000 newborns in the US, similar to phenylketonuria (7) . The disease results in a build-up of octanoic acid and octanoyl-carnitine, which can be identified by tandem mass spectrometry. However, this is a costly technique and second confirmation is still needed. A simple activity based fluorescent assay would be ideal for identifying this disease. Further, MCAD controls β- oxidation, thus if its activity could be used to monitor the flux through the system, it could be used to detect

complex metabolic diseases such as diabetes or metabolic syndrome (8) .

Summary of the Invention

One embodiment of this invention provides a compound of the structure :

wherein one of R ¹ and R ⁶ is H and the other is - C ₂ H ₂ C(O)OR ⁸ , -C ₂ H ₄ C(O)OR ⁸ , -C ₂ H ₄ C(O)OH, -C ₂ H ₂ C(O)OH, - C ₂ H ₄ C (0) -coenzyme A, -C ₂ H ₂ C (0) -coenzyme A, or X where X is a halide, and where R ⁸ is alkyl, benzyl or aryl , and wherein R ² , R ³ , and R ⁷ are H, and

R ⁵ is H or alkyl, and R ⁴ is H, -N(C ₂ H ₅ ) ₂ ,

N(CHs) ₂ , -OH, aryl, or -OR ⁹ , wherein R ⁹ is alkyl, or

R ⁴ and R ⁵ are joined to form an aryl, heterocyclic or heteroaromatic ring, or wherein R ² and R ⁴ each form an aryl ring with R ³ so as to form a tetracyclic structure, and R ⁵ and R ⁷ are H, or wherein R ² is H, and R ³ and R ⁴ form a aryl, heterocyclic or heteroaromatic ring, and R ⁵ and R ⁷ are H, or R ⁵ and R ⁷ form a aryl, heterocyclic or heteroaromatic ring, and R ³ and R ⁴ are H, and wherein R ² , R ³ , R ⁵ and R ⁷ are H, and R ⁴ is hydroxyl ,

wherein in each heterocyclic or heteroaromatic ring the heteroatom is N, S or 0, or a salt thereof.

Brief Description of the Figures

Figure 1: β-Oxidation cycle. The four enzymes involved in β-oxidation are: acyl-CoA dehydrogenase (ACAD), enoyl-CoA hydratase (ECH), L-3-hydroxyacyl-CoA dehydrogenase (HCAD), and thiolase.

Figure 2: Substrate flexibility of MCAD. Long chain acyl- CoA dehydrogenase (LCAD) serves fatty acids from C ₈ -C _2O -

Medium chain acyl-CoA dehydrogenase (MCAD) is active with

C ₆ -Ci ₆ fatty acids. Short chain acyl-CoA dehydrogenase

(SCAD) is active with C ₄ -C ₆ fatty acids.

Figure 3: Design for a MCAD fluorescent reporter. MCAD action results in extending the π-conjugation of a fluorophore, causing in a change in the emission profile.

Figures 4A-4B: Potential substrates for MCAD. 4A: Fluorescent spectra of probes 1 (solid) and 3 (dashed) (λ _ex =340nm) . 4B : Fluorescent spectra of probes 2 (solid) and 4 (dashed) (λ _ex =390nm) . Fluorescence is measured in arbitrary unit (AU) for all fluorescent spectra (Figs. 4A, 4B, 6, 10, 11, and 12) .

Figure 5: Possible substrates for MCAD.

Figures 6A-6B : 6A: Fluorescent spectra of probes 7 (solid) and 11 (dashed) (λ _ex =350nm) . 6B : Fluorescent spectra of probes 9 and 12 (λ _ex =356nm) .

Figure 7 : Specific activity (units/mg) for straight chain and phenyl derivatives with rMCAD and 50μM substrate.

Figure 8: Competitive substrate assays with butanoyl-CoA, isovaleryl-CoA, octanoyl-CoA, and palmitoyl-CoA; and the fluorescent substrates 1, 7, and 9. Assays were carried out in 96-well plate with 50μM competitive substrate, lOμM fluorescent substrate, and rat liver homogenate as the enzyme source .

Figure 9 : Linearity of activity of probes with homogenate protein.

Figure 10: Fluorescence Spectra of Probe 1. Probes 1 (solid) and 3 (dashed) were excited at 340 nm, fluorescence emission spectra were recorded with 50 μM solutions in 100 mM potassium phosphate buffer (pH 8.0) .

Figure 11: Fluorescence Spectra of Probe 7. Probes 7 (solid) and 11 (dashed) were excited at 350 nm, fluorescence emission spectra were recorded with 50 μM solutions in 100 mM potassium phosphate buffer (pH 8.0) .

Figure 12: Fluorescence Spectra of Probe 11. Probes 11 (solid) and 12 (dashed) were excited at 356 nm, fluorescence emission spectra were recorded with 50 μM solutions in 100 mM potassium phosphate buffer (pH 8.0) .

Figure 13: Competitive substrate assay for probe 1 ^* .

Figure 14: Competitive substrate assay for probe 7 ^* .

Figure 15: Competitive substrate assay for probe 9 ^* .

*Zero activity means there was a decrease in fluorescence .

Figure 16: Scheme for synthesis of probes.

Figure 17: Scheme for synthesis of probes.

Figure 18: Scheme for synthesis of probes.

Figure 19: Structure of Coenzyme A as attached to probes. The arrow represents the bond attached to the probes at either the R ¹ or R ⁶ position via the sulfur atom.

Figure 20: Activity of probe 1 with MCAD deficient and control cell lines. All cell lines were obtained from Coriell Cell Repository, Camden, NJ. Activity of probe was determined as stated in materials and methods.

Figure 21: Average activity of probe 1 with MCAD deficient fibroblasts and normal fibroblasts (*significant p=0.0001).

Detailed Description

Abbreviations used in the specification: ACAD - Acyl-CoA dehydrogenase

LCAD - long chain acyl-CoA dehydrogenase MCAD - Medium chain acyl-CoA dehydrogenase CoA - Coenzyme A

As shown in Fig. 19, coenzyme A contains an S atom which is part of the coenzyme. For clarity regarding point of attachment however, the compounds in the specification and claims have been drawn to show an attachment through the sulfur atom to the coenzyme A, i.e. as -S-CoA, even though S is part of the coenzyme.

In one embodiment, this invention provides compounds of the general structure :

which can be used to monitor the activity of medium-chain acyl-CoA dehydrogenase by following change in fluorescence of the starting material or product (that resulting from dehydrogenation of the general structure above) . Specifically the following compounds (probes 1, 7, 9) act as nonphysiological substrates for MCAD and the activity of MCAD can be monitored by measuring the presence of product (3, 11, 12) fluorometrically.

11 12

This invention provides a compound having the structure:

wherein one of R ¹ and R ⁶ is H and the other is C ₂ H ₂ C(O)OR ⁸ , -C ₂ H ₄ C(O)OR ⁸ , -C ₂ H ₄ C(O)OH, -C ₂ H ₂ C(O)OH, C ₂ H ₄ C (0) -coenzyme A, -C ₂ H ₂ C (0) -coenzyme A, or X where X is a halide, and where R ⁸ is alkyl, benzyl or aryl, and wherein R ² , R ³ , and R ⁷ are H, and R ⁵ is H or alkyl, and R ⁴ is H, -N(C ₂ H ₅ ) ₂ , -N(CH ₃ ) ₂ , - OH, aryl, or -OR ⁹ , wherein R ⁹ is alkyl, or R ⁴ and R ⁵ are joined to form an aryl, heterocyclic or heteroaromatic ring, or wherein R ² and R ⁴ each form an aryl ring with R ³ so as to form a tetracyclic structure, and R ⁵ and R ⁷ are H, or wherein R ² is H, and R ³ and R ⁴ form a aryl, heterocyclic or heteroaromatic ring, and R ⁵ and R ⁷ are H, or R ⁵ and R ⁷ form a aryl, heterocyclic or heteroaromatic ring, and R ³ and R ⁴ are H, or wherein R ² , R ³ , R ⁵ and R ⁷ are H, and R ⁴ is hydroxyl,

wherein in each heterocyclic or heteroaromatic ring the heteroatom is N, S or 0,

or a salt thereof.

This invention provides the instant compound wherein R ⁸ is a C _x -C ₇ alkyl or wherein R ⁸ is aryl and is a phenyl. This invention provides the instant compound wherein R ¹ is - C ₂ H ₄ C (0) -coenzyme A or -C ₂ H ₂ C (0) -coenzyme A. In one embodiment R ¹ is -C ₂ H ₄ C (0) -coenzyme A.

This invention provides the instant compound, wherein R ¹ is -C ₂ H ₄ C (0) -coenzyme A, wherein R ² , R ³ and R ^s are H, and wherein R ⁴ is H or -OR ⁹ , wherein R ⁹ is alkyl, and R ⁵ is H, or wherein R ⁴ and R ⁵ are joined to form an aryl ring, or a salt or stereoisomer thereof.

This invention provides the instant compound wherein R ⁹ is alkyl. In one embodiment the alkyl is a branched alkyl. In another embodiment the alkyl is -CH ₃ .

This invention provides the instant compound, having the following structure:

This invention provides the instant compound, wherein R ¹ is -C ₂ H ₂ C(O) -coenzyme A, or has one of the following structures :

This invention provides the instant compound, wherein one of R ¹ and R ^s is H and the other is -C ₂ H ₄ C(O)- coenzyme A; and wherein R ² and R ³ are H, and R ⁴ is -N (C ₂ H ₅ ) ₂ , -N(CH ₃ ) ₂ , or -OH ₇ and R ⁵ is H, or wherein R ² and R ⁴ each form an aryl ring with R ³ so as to form a tetracyclic structure, or a salt thereof.

This invention provides the instant compound, having the structure :

This invention provides the instant compound, wherein one of R ¹ and R ⁶ is H and the other is

C ₂ H ₂ C(O)OR ⁸ , -C ₂ H ₄ C(O)O R ⁸ , -C ₂ H ₄ C (0) OH, or -C ₂ H ₂ C (0) OH ^;

and wherein R ² and R ³ are H, and R ⁴ is H, -N(C ₂ H ₅ ) ₂ , -N(CH ₃ ) ₂ , -OH, or -OR ⁷ , wherein R ⁷ is alkyl, and wherein R ⁵ is H, or wherein R ⁴ and R ⁵ are joined to form an aryl ring, or wherein R ² and R ⁴ each form an aryl ring with R ³ so as to form a tetracyclic structure or a salt thereof .

This invention provides the instant compound, having the structure:

This invention also provides a process for making the instant compound, comprising: a) exposing a compound having the structure:

to a compound having the structure :

in the presence of a suitable base, (PPh ₃ ) ₂ PdCl ₂ , and a suitable solvent so as to produce a compound having the structure :

treating the product of step a) so as to produce a compound having the structure :

c) flushing the product of step b) with argon, and dissolving in dry THF and exposing to triethylamine and ethylchloroformate ; exposing the product of step c) to coenzyme A, NaHCO ₃ in degassed water and dry THF, acidifying the product of step d) and removing the THF so as to produce the instant compound.

In one embodiment the process further comprises the step of purifying the probe after step e) by HPLC. In embodiments the suitable solvent is N-Methylpyrrolidone (NMP) and the suitable base is NaHCO ₃ . In one embodiment the product of step a) is treated by exposing it to KOSiMe ₃ in ether, and then dissolving in acetic acid and water. In differing embodiments, treating the product of step a) comprises exposing it to LiOH in THF and water, or to Li and NH ₃ , or to NaO ₂ in DMSO, or to Potassium t- butoxide in DMSO, or to KSCN in DMF, or to NaSEt in DMF, or to LiSeCH ₃ in DMF.

This invention also provides a process for making the instant compound: a) exposing a compound having the structure:

to methyl (triphenylphosphoranylidene) acetate and THF under argon so as to produce a compound having the structure :

treating the product of step a) so as to produce a compound having the structure :

c) flushing the product of step b) with argon, and dissolving in dry THF and exposing to triethylamine and ethylchloroformate ; exposing the product of step c) to coenzyme A, NaHCO ₃ in degassed water and dry THF, acidifying the product of step d) and removing the THF so as to produce the compound.

In one embodiment the process further comprises the step of purifying the probe after step e) by HPLC. In one embodiment, the product of step a) is treated by exposing it to KOSiMe3 in ether, and then dissolving in acetic acid and water. In differing embodiments treating the product of step a) comprises exposing it to LiOH in THF and water, or to Li and NH ₃ , or to NaO ₂ in DMSO, or to Potassium t-butoxide in DMSO, or to KSCN in DMF, or to NaSEt in DMF, or to LiSeCH ₃ in DMF.

This invention also provides a process for making the instant compound comprising: a) exposing Ar-Br, wherein Ar is:

or

to a compound having the structure :

in the presence of a suitable base, (PPh ₃ ) ₂ PdCl ₂ , and a suitable solvent, so as to produce a compound having the structure :

where AR is as defined above, b) treating the product of step a) so as to produce a compound having the structure :

where AR is as defined above, c) flushing the product of step b) with argon, and dissolving in dry THF and exposing to triethylamine and ethylchloroformate ; d) exposing the product of step c) to coenzyme A, NaHCO ₃ in degassed water and dry THF, acidifying the product of step d) and removing the THF so as to produce the compound.

In embodiments the suitable solvent is N- Methylpyrrolidone (NMP) and the suitable base is NaHCO ₃ . In one embodiment the product of step a) is treated by exposing it to KOSiMe ₃ in ether, and then dissolving in acetic acid and water. In differing embodiments treating the product of step a) comprises exposing it to LiOH in THF and water, or to Li and NH ₃ , or to NaO ₂ in DMSO, or to Potassium t-butoxide in DMSO, or to KSCN in DMF, or to NaSEt in DMF, or to LiSeCH ₃ in DMF.

This invention also provides a process for making the instant compound comprising: a) exposing a compound having the structure:

wherein Ar is :

to Pd/C under H ₂ at about 40 psi so as to produce compound having the structure :

where Ar is defined as above, b) exposing the product of step a) to KOSiMe ₃ in ether and then AcOH in water so as to produce a compound having the structure :

where Ar is defined as above, c) flushing the product of step b) with argon, and dissolving in dry THF and exposing to triethylamine and ethylchloroforraate; d) exposing the product of step c) to coenzyme A, NaHCO ₃ in degassed water and dry THF, e) acidifying the product of step d) and removing the THF so as to produce the compound.

This invention also provides a process for making the instant compound comprising: a) exposing a compound having the structure:

wherein Ar is :

or

to KOSiMe ₃ in ether and then AcOH in water so as to produce a compound having the structure :

where Ar is defined as above, b) flushing the product of step b) with argon, and dissolving in dry THF and exposing to triethylamine and ethylchloroformate; c) exposing the product of step c) to coenzyme A, NaHCO ₃ in degassed water and dry THF, e) acidifying the product of step d) and removing the THF so as to produce the compound.

In further embodiments the processes further comprise the step of purifying the compound after step e) by HPLC.

This invention provides a compound has the structure:



This invention also provides a method identifying an active mammalian Medium Chain Acyl-Coenzyme A Dehydrogenase in a sample comprising: providing a sample derived from a mammal; contacting the sample with a compound which undergoes a detectable change in fluorescence emission maxima when dehydrogenated in the presence of a mammalian Medium Chain Acyl-Coenzyme A Dehydrogenase under conditions permitting dehydrogenation of the compound in the

presence of the human Medium Chain Acyl-Coenzyme A Dehydrogenase; detecting a change in fluorescence emission maxima of the compound; wherein a change in the fluorescence emission maxima detected in step c) indicates an active mammalian Medium Chain Acyl -Coenzyme A Dehydrogenase in the sample.

In differing embodiments of this invention the mammal (as the MCAD source) is a human, a rate, a monkey, or a mouse. In addition, the "mammalian MCAD" may be a recombinant MCAD from a mammal such as a human or a rate which has been produced by another species e.g. a bacterium such as E. CoIi.

In one embodiment the fluorescence emission maxima shifts to a longer wavelength. In one embodiment the fluorescence emission maxima is measured at about 460nm to about 560nm under conditions comprising excitation of the compound at about 275nm to about 390nm. In one embodiment the fluorescence emission maxima is measured at about 425nm to about 650nm under conditions comprising excitation at about 300nm to about 420nm of the compound.

In embodiments of the method the compound has the

structure :

This invention provides a method of diagnosing a subject as suffering from a disease associated with a human MCAD deficiency or with reduced β-oxidation of acyl-CoA, comprising: a) obtaining a sample from the subject; b) contacting the sample with a compound which undergoes a detectable change in fluorescence emission maxima when dehydrogenated in the presence of a human Medium Chain Acyl-Coenzyme A Dehydrogenase under conditions permitting dehydrogenation of the compound in the presence of the human Medium Chain Acyl -Coenzyme A Dehydrogenase ; c) detecting a change in the fluorescence emission maxima of the sample; and d) comparing the change in fluorescence emission maxima detected in step c) with a reference standard, wherein an change fluorescence emission maxima detected in step c) less than that of the reference standard indicates that the subject is suffering from a disease associated with a human MCAD deficiency or with reduced β-oxidation of acyl -CoA.

In one embodiment the detectable change in fluorescence emission maxima is a shift to a longer wavelength.

This invention provides a method of diagnosing a subject as suffering from a disease associated with a human MCAD deficiency or with reduced β-oxidation of acyl-CoA: a) obtaining a sample from the subject; b) contacting the sample with a compound which undergoes a detectable change in fluorescence when dehydrogenated in the presence of a human Medium Chain Acyl-Coenzyme A Dehydrogenase under conditions permitting dehydrogenation of the compound in the presence of the human Medium Chain Acyl-Coenzyme A Dehydrogenase; c) detecting a change in the fluorescence the sample; and d) comparing the change in fluorescence detected in step c) with a reference standard, wherein a fluorescence detected in step c) less than that of the reference standard indicates that the subject is suffering from a disease associated with a human MCAD deficiency or with reduced β-oxidation of acyl-CoA.

In embodiments the detectable change in fluorescence is an increase in fluorescence, the disease is MCAD deficiency, and the fluorescence emission maxima is measured at about 460nm to about 560nm under conditions comprising excitation of the compound at about 275nm to about 390nm. In one embodiment the fluorescence is measured under conditions comprising excitation of the compound at about 275nm to about 375nm.

In an embodiment of the method the compound has the structure :

In embodiments of the method the disease is associated with reduced β-oxidation of acyl-CoA, and in further embodiments is diabetes or metabolic syndrome.

This invention provides the instant methods wherein the sample is blood, a derivative of blood, leukocytes, a plasma sample, a lymph sample, a muscle biopsy sample, a muscle biopsy sample derivative, a liver biopsy sample or liver biopsy derivative.

This invention provides the instant methods, wherein the conditions permitting dehydrogenation of the compound

comprise the presence of an oxidant. In one embodiment the oxidant is flavin adenine dinucleotide .

This invention provides a method of inhibiting the physiological activity of MCAD in a solution comprising exposing the MCAD in the solution to an effective amount of a non-physiological substrate of MCAD, so as to thereby inhibit the physiological activity of the MCAD.

This invention provides the instant methods wherein the non-physiological substrate is one of the instant compounds .

As used herein, "fluorescence emission maxima" of a compound means the wavelength (s) at which maximum fluorescence occurs as measured by any suitable fluorimetric device, e.g. a fluorometer, when the compound is excited with light of a predetermined excitation wavelength (s) . Accordingly, a "shift in fluorescence emission maxima" means a shift to a longer wavelength at which maximum fluorescence occurs.

As used herein, a "change in fluorescence emission intensity" of a compound ^' means an increase in the measured level of fluorescence of the compound when the compound is excited with light of a predetermined excitation wavelength (s) , or more specifically the emission intensity is directly proportional to brightness. In this case, brightness = (ε) (φ) , where ε is the extinction coefficient at which the quantum yield is measured and φ is the quantum yield.

As used herein, "reference standard" means a normalized value obtained form a standardized sample, and in the case of fluorescence means the normalized fluorescence

measured form a sample obtained from a subject without an MCAD deficiency or without impaired MCAD activity, or other standardized sample, as measured by a parallel assay with the same steps and conditions to which the tested sample is being subjected.

The compounds of the present invention, especially probes 1, 7, and 9 are non-physiological substrates of MCAD and therefore may be employed as competitive substrates of MCAD. As used herein, a "competitive substrate" in relation to an enzyme is a substance capable of binding to the enzyme's active site in place of the physiological substrate and being converted to product .

As used herein, "diagnosing" a MCAD deficiency, a disease associated with such, or a disease associated with impaired reduced β-oxidation of acyl-Coenzyme A, means identifying a cell, a tissue, or a sample as having impaired MCAD enzyme activity below the level of activity of that enzyme in a non-pathological or non-diseased cell, tissue or sample.

As used herein, the "physiological activity" of MCAD is the dehydrogenation of naturally occurring MCAD substrates, medium chain Acyl-CoA as found in situ for example. This activity may be inhibited, for example, by providing non-physiological substrates to the enzyme.

As used herein, "Coenzyme A" has the structure shown in Fig. 19.. The arrow represents the bond attached to the probes or other compounds at either the R ¹ or R ⁶ position via sulfur.

As used herein, a "salt" is salt of the instant compounds which has been modified by making acid or base salts of

the compounds. The salt can be pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxcylic acids. The salts can be made using an organic or inorganic acid. Such acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like. Carboxylate salts are the alkaline earth metal salts, sodium, potassium or lithium.

As used herein, a "reduction" when pertaining to fluorescence can mean either a blue shift in emission wavelength or a decrease in fluorescence emission intensity.

A "change" in fluorescence can mean either a red or blue shift in emission wavelength or an increase in fluorescence emission intensity.

As used herein, "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. Thus, C _x -C _n as in "Cχ-C _n alkyl" is defined to include groups having 1, 2, .... , n-1 or n carbons in a linear or branched arrangement, and specifically includes methyl , ethyl , propyl , butyl , pentyl , hexyl , and so on . For example, Ci-C ₆ , as in "Ci-C ₆ alkyl" is defined to include individual moieties having 1, 2, 3, 4, 5, or 6 carbons in a linear or branched arrangement. "Alkoxy" represents an alkyl moiety of indicated number of carbon atoms which is attached to the core through an oxygen bridge .

The term "cycloalkyl" shall mean cyclic rings of alkanes of three to eight total carbon atoms, or any number within this range (i.e., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl) .

If no number of carbon atoms is specified, the term "alkenyl" refers to a non-aromatic hydrocarbon radical, straight or branched, containing at least 1 carbon to carbon double bond, and up to the maximum possible number of non-aromatic carbon-carbon double bonds may be present. If the number of carbon atoms is specified, e.g. "C ₂ -C _n " alkenyl, each member of the numeric range is disclosed individually as discussed above. Thus, for example, "C ₂ -Cg alkenyl" means an alkenyl radical having 2, 3, 4, 5, or 6 carbon atoms, and 1, 2, 3, 4, or 5 carbon-carbon double bonds respectively. Alkenyl groups include ethenyl, propenyl , butenyl and cyclohexenyl .

The term "cycloalkenyl" shall mean cyclic rings of 3 to 10 carbon atoms and at least 1 carbon to carbon double bond (i.e., cyclopropenyl, cyclobutenyl , cyclopenentyl, cyclohexenyl, cycloheptenyl or cycloocentyl) .

The term "alkynyl" refers to a hydrocarbon radical straight or branched, containing at least 1 carbon to carbon triple bond, and up to the maximum possible number of non-aromatic carbon-carbon triple bonds may be present. Thus, "C ₂ -C ₆ alkynyl" means an alkynyl radical radical having 2, 3, 4, 5, or 6 carbon atoms, and for example 1 carbon-carbon triple bond, or having 4 or 5 carbon atoms, and up to 2 carbon-carbon triple bonds, or having 6 carbon atoms, and up to 3 carbon-carbon triple bonds . Alkynyl groups include ethynyl , propynyl and butynyl . As described above with respect to alkyl, the straight or branched portion of the alkynyl group may

contain triple bonds and may be substituted if a substituted alkynyl group is indicated.

As used herein, "aryl" is intended to mean any stable monocyclic, bicyclic or tricyclic carbon ring of up to 10 atoms in each ring, wherein at least one ring is aromatic. Examples of such aryl elements include phenyl, naphthyl, tetrahydro-naphthyl , indanyl, biphenyl , phenanthryl, anthryl or acenaphthyl . In cases where the aryl substituent is bicyclic and one ring is non- aromatic, it is understood that attachment is via the aromatic ring .

The term "heteroaryl" ¹ , as used herein, represents a stable monocyclic or bicyclic ring of up to 10 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of 0, N and S. Heteroaryl • groups within the scope of this definition include but are not limited to: benzoimidazolyl, benzofuranyl , benzofurazanyl, benzopyrazolyl , benzotriazolyl , benzothiophenyl , benzoxazolyl , carbazolyl, carbolinyl, cinnolinyl, furanyl , indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl , isoindolyl, isoquinolyl, isothiazolyl , isoxazolyl, naphthpyridinyl , oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl , pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl , quinoxalinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, aziridinyl,

1, 4-dioxanyl, hexahydroazepinyl, dihydrobenzoimidazolyl , dihydrobenzofuranyl , dihydrobenzothiophenyl , dihydrobenzoxazolyl , dihydrofuranyl , dihydroimidazolyl, dihydroindolyl , dihydroisooxazolyl , dihydroisothiazolyl, dihydrooxadiazolyl , dihydrooxazolyl , dihydropyrazinyl ,

dihydropyrazolyl , dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl , dihydroquinolinyl , dihydrotetrazolyl, dihydrothiadiazolyl , dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl , dihydroazetidinyl , methylenedioxybenzoyl, tetrahydrofuranyl, tetrahydrothienyl, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl , benzothiazolyl, benzoxazolyl , isoxazolyl, isothiazolyl , furanyl , thienyl , benzothienyl, benzofuranyl , quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl , pyrimidinyl, pyrrolyl , tetra- hydroquinoline . In cases where the heteroaryl substituent is bicyclic and one ring is non-aromatic or contains no heteroatoms, it is understood that attachment is via the aromatic ring or via the heteroatom containing ring, respectively. If the heteroaryl contains nitrogen atoms, it is understood that the corresponding N-oxides thereof are also encompassed by this definition.

"Halo" or "halogen" as used herein is intended to include chloro, fluoro, bromo and iodo.

The term "heterocycle" or "heterocyclyl" as used herein is intended to mean a 5- to 10-membered nonaromatic ring containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S, and includes bicyclic groups. "Heterocyclyl" therefore includes, but is not limited to the following: imidazolyl, piperazinyl, piperidinyl , pyrrolidinyl , morpholinyl, thiomorpholinyl , tetrahydropyranyl, dihydropiperidinyl , tetrahydrothiophenyl and the like. If the heterocycle contains a nitrogen, it is understood that the corresponding N-oxides thereof are also encompassed by this definition.

The alkyl, alkenyl, alkynyl, cycloalkyl, aryl , heteroaryl and heterocyclyl substituents may be substituted or unsubstituted, unless specifically defined otherwise. For example, a (C1-C7) alkyl may be substituted with one or more substituents selected from OH, oxo, halogen, alkoxy, dialkylamino, or heterocyclyl, such as morpholinyl, piperidinyl, and so on. In the compounds of the present invention, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl and heteroaryl groups can be further substituted by replacing one or more hydrogen atoms by alternative non-hydrogen groups. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.

The term "substituted" shall be deemed to include multiple degrees of substitution by a named substitutent . Where multiple substituent moieties are disclosed or claimed, the substituted compound can be independently substituted by one or more of the disclosed or claimed substituent moieties, singly or plurally. By independently substituted, it is meant that the (two or more) substituents can be the same or different.

It is understood that substituents and substitution patterns on the compounds of the instant invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the ^' art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results .

In choosing compounds of the present invention, one of ordinary skill in the art will recognize that the various substituents, i.e. R ¹ , R ² , and R ³ , are to be chosen in conformity with well-known principles of chemical structure connectivity.

The methods of the present invention when pertaining to cells, and samples derived or purified therefrom, including enzyme containing fractions, may be performed in vitro. The methods of diagnosis may, in different embodiments, be performed in vivo, in situ, or in vitro.

Experimental Details

The length of a fluorophore' s π-conjugation often determines its fluorescent properties. As MCAD catalysis results in double bond formation, the π-conjugation of a fluorophore is extended if ω-substituted propionyl-CoA derivatives serve as substrates, Fig. 3. Depending on the electronics of the fluorophore, MCAD action would result in a switch of fluorescence.

In order to function as a fluorescent reporter, probes have to meet two criteria: 1) dehydrogenation has to result in a fluorescent increase or switch, and 2) the probe must serve as substrates for MCAD. We started with two simple systems: 3- (2-naphthyl) propionyl-CoA and 3(6- dimethylamino-2-naphthyl)propionyl-CoA. The substituents were chosen because similarly sized molecules have been shown to act as MCAD substrates, such as, 3 -indole propionoyl-CoA (9) , 2 -fury1 propionyl-CoA (10) , and

[ ^99m Tc-MAMA-HA] (11, 12), a technetium chelated hexanoic

acid. Although some of these probes can be monitored spectrophotometrically, none of the existing substrates are fluorescent . As naphthalene is not the ideal fluorophore, the addition of a dimethylamino group was found to increase the fluorescence, similar tσ that seen in the fluorophore PRODAN (13) . Upon examination of their fluorescent properties, both probes were fluoromorphic, Fig. 4. When excited at 340 nm, probe 1 has a different emission maximum, than its product 3, with a three-fold increase in fluorescence at 492 nm. Similarly, dehydrogenation of 2 resulted in a 190 nm shift in fluorescence and a twenty-fold increase in fluorescence at the emission maxima of probe 4. Accordingly, probe criterion 1) had been met, now the probes had to be substrates.

The activity of 1 and 2 with isolated rat MCAD (rMCAD) (gift of Horst Schulz, CUNY-CCNY) was tested by monitoring the reduction of the external electron acceptor (DCPIP/PMS) spectrophometrically (14) . Probe 1 was active with MCAD, while 2 was not. Although 1 met both criteria it was determined that a better fluorescent substrate for MCAD could be designed.

It was hypothesized that 2 was not active due to the polarity of the amine functionality, and so a variety of substituents were chosen that would test this, as well as give other clues into the active site of the enzyme. Probes 5-10 (see Fig. 5) were synthesized to test: 1) linearity (probe 5) , 2) polarity tolerance (probes 6, 7, and 8) , and 3) size limitations (probes 9 and 10) , but at the same time optimizing the fluorescence. New substrates 5-10 were tested with rMCAD as above, except ferricenium hexafluorophosphate ¹¹⁵¹ (FePF ₆ ) was used as the electron acceptor (DCPIP/PMS was found to inhibit the fluorescence

of 1 and 3) . MCAD was active with 7 and .9, while the others led to no significant reduction of FePF ₆ . As shown in Fig. 6, the dehydrogenation of 7 to 11 resulted in an eighty-fold increase in fluorescence at 510 nm (λ _ex =350nm) , while 9 to 12 gave a fluorescent switch of 150 nm, also with an eighty-fold fluorescent increase at 567 nm (λ _ex =356nm) . Probes 6 and 8 supported the hypothesis that polarity prevents access to the enzyme pocket. Likewise, the odd shape of 5 and the diameter of 10 must prohibit these molecules from binding, because there is a small canal that the substrate must fit though in order to access the binding pocket (4) .

With three fluorescent substrates in hand, an assay was developed to measure the activity of MCAD by monitoring the formation of product by fluorescence. The kinetic parameters of probes 1, 7, and 9 are shown in Table 1, with rMCAD, as well as pig MCAD (pMCAD) (gift of Jung-Ja Kim, Medical College of Wisconsin) . All three probes have K _M ^app on the same order of magnitude as the optimum natural substrate octanoyl-CoA. As expected, probe 7 has the highest K _M ^app with both rMCAD and pMCAD, possibly due to the polarity of the methoxy group disrupting the binding. Substrate 1 has the largest k _cat of 370±10 and 150±4 min ^"1 for rMCAD and pMCAD, respectively, however this is still significantly slower than octanoyl-CoA. The fluorescent assay was validated by a spectrophotometric assay, in which the formation of 3 was followed at 325nm which gave results similar to those in Table 1 with a K _M ^app = 2.6+0.7 μM and k _cat = 310+60 min ^"1 . The activity of the substrate drops with each additional phenyl ring, which bears a striking similarity to the chain length activity relationship of regular fatty acids. 3-Phenylpropionyl- CoA (PPA) was synthesized and it along with 1, 9,

octanoyl-CoA, stearoyl-CoA, and palmitoyl-CoA were assayed with rMCAD. In the case of the regular fatty acids, FePF ₆ was monitored. As shown in Fig. 7, if the "tail" of octanoyl curls it can be made to look like PPA and these two of similar specific activities. Likewise, stearoyl-CoA and palmitoyl-CoA can curl to look like 1 and 9, respectively, with similar drops in their specific activities .

Table 1 : Kinetic Parameters of Probes

K _M ^app k, cat (μM) (min ^"x ) rMCAD [21] 4 . 0 2142

Octanoyl -CoA pMCAD [22] 2 .3+0 . 1 1176

Homogenate 2.2+0.3

5 rMCAD 6.01+0.7 16.1+0 .3 J pMCAD 14.5+1.5 12.3+0 .5

7 Homogenate 3.7+0.4 - rMCAD 1.1+0.2 10.4+0 .3 pMCAD 4.5±0.5 13.1+2

9 Homogenate 1.9+0.4 -

As described previously, there are five straight chain ACAD isozymes with overlapping specificity. To test if the probes can act as substrates toward these other enzymes, competitive substrate assays were performed using rat liver homogenate as the enzyme source since it would contain all soluble ACADs, including short-chain (SCAD) , medium-chain (MCAD) and long-chain (LCAD) acyl- CoA dehydrogenase. Butanoyl-CoA, octanoyl-CoA, palmitoyl- CoA, and isovaleroyl-CoA were used as competitive substrates for SCAD, MCAD, LCAD, and isovaleroyl acyl-CoA dehydrogenase (iVAD) , respectively. The results of the inhibition study with the 1, 7, and 9 are shown in Figure 8. With butanoyl-CoA and isovaleroyl-CoA there was no significant inhibition, showing that the probes were not substrates for SCAD and iVAD. However, there was significant inhibition with octanoyl-CoA. The addition of palmitoyl-CoA also led to inhibition of the formation of

product, however palmitoyl-CoA is a substrate for MCAD, as well as LCAD. Palmitoyl-CoA binds tighter to MCAD than to LCAD (K _M = 4 μM vs. 780 μM) , however it is more active towards LCAD (SA = 14.5 u/mg for LCAD vs SA = 0.23 u/mg for MCAD) , ^C1] thus palmiotyl-CoA was inhibiting MCAD, as well as LCAD. To further test that the probe was specific for MCAD, the activities of 1, 7, and 9 were tested with isolated pig SCAD (pSCAD) and human LCAD (hLCAD) (gifts of Jung-Ja Kim, Medical College of Wisconsin) . All three probes showed no significant activity with pSCAD or hLCAD, meaning the probes are not substrates for LCAD.

To examine the sensitivity and stability of 1, 7, and 9, their activity was assayed with rat liver homongenates . As shown in Fig. 9, their activity is linear with protein concentration, suggesting a catalytic reaction is taking place. There was a small amount of product formed with 1 and 7 when the external electron acceptor was omitted from the reaction with homogenate . This can be explained by having endogenous electron acceptors in the homogenate or from oxidase activity with MCAD (9, 10) . When the FePF ₆ was omitted from the assays with purified protein, the formation of product (oxidase activity) with 1 was ≤0.001% of the dehydrogenase activity with both rMCAD and pMCAD. Probe 7 showed as much as 0.03% of the dehydrogenase activity with pMCAD.

Since MCAD is the first enzyme of the β-oxidation cycle, the probes could continue through the cycle, with the product of the MCAD assay being consumed by the next enzyme, enoyl-CoA hydratase (ECH) . To test for this possibility, 3, 11, and 12 were exposed to the homogenates . The decrease in fluorescence was similar to that in absence of homogenate. Probes 3 and 11 are subject to photobleaching, while 12 is not. However,

photobieaching does not significantly effect the results of the assay, because the results are similar when conducting a continuous assay or an endpoint assay.

Discussion

The β-oxidation pathway is linked to several diseases, including diabetes and metabolic syndrome (16-18) . MCAD deficiency is a genetic disorder that appears in 1:15,000 newborns in the United States. The ability to measure MCAD activity with small amounts of enzyme is a must. The fluorogenic substrates are twenty-five times more sensitive than existing methods (19) when homogenate serves as the enzyme source. As little as 0.39 μg of homogenate protein is required to measure MCAD activity when the assay is extended to one hour.

Using enzyme promiscuity as a guide, a new method for monitoring the activity of MCAD by measuring the increase in fluorescence with fluorogenic substrates 1, 7, or 9 is described. In the development of a fluorogenic reporter described here we also probed the flexibility of the MCAD active site was also investigated. It was found that although MCAD can accept many substrates, substituents must be nonpolar and linear in shape. This model can serve as a guide for developing probes for the remainder of the β-oxidation pathway.

As β-oxidation has been linked to multiple diseases, it is essential to be able to measure the flux through the system efficiently. This continuous fluorescent assay is twenty-five times more sensitive than the current method of detection, and we are currently investigating its application to intact tissue. As probes 1, 7, and 9 can act as substrates for both rat and pig MCAD, and MCADs are well conserved evolutionary, (20) these probes should

serve as general fluorogenic substrates for all mammalian MCAD enzymes, including human.

Materials and Methods

¹ H and ¹³ C NMR spectra were recorded on Bruker 300 Fourier transform NMR spectrometers. Spectra were recorded in CDCl ₃ solutions referenced to solvent residual peak, unless otherwise indicated. Low Resolution Mass Spectra were obtained on a JOEL JMS-HXIlO HF mass spectrometer. Flash chromatography was performed on SILICYCLE silica gel (230-400 mesh) . All chemicals were purchased from Aldrich or Sigma and used as received. All reactions were monitored by Thin Layer Chromatography.

Ultraviolet spectra were measured on a Molecular Devices SpectraMax Plus 384 UV/VIS spectrophotometer. Fluorescence measurements were taken on a Jobin Yvon Fluorolog fluorescence spectrofluorometer . Fluorescent measurements with 96-well plates were performed by the MicroMax 384 connected to a Jobin Yvon Fluorolog through F-3000 fiber optic cables.

Synthesis of Probes

General scheme for probe synthesis are set forth in Figs. 16, 17, and 18.

General Procedure for the Synthesis of Methyl 3-aryl- acrylates from Aryl-bromides, Exemplified for Methyl 3-

(6-dimethylamino-2-naphthyl) -acrylate (2A). 2-Bromo-6- dimethylamino naphthalene (1.0 g, 4.0 mmol) , prepared according to literature (23), NaHCCb (0.4Og, 4.8 mmol),

(PPh ₃ ) ₂ PdCl2 (0.050 g, 0.072 mmol), methylacrylate (0.43 mL, 4.8 mmol), and 1-methyl-2 -pyrrolidone (10 mL) were

added to a sealed tube, placed under an Argon atmosphere, and heated at 130 ⁰ C for 15h. The tube was cooled and the contents were poured into water (20 mL) . The solids were collected by filtration and dissolved in CH2CI2, washed with brine, dried (MgSO*) , and filtered through a celite pad. Recrystallization from hexanes resulted in 0.67 g of methyl 3- (6-dimethylamino-2-naphthyl) -acrylate (2A) (66% yield) . NMR ¹ H (300 MHz, CDCIs) δ pptn: 7.83 (d, J=15.9 Hz, IH), 7.78 (s, IH), 7.71 (d, J=9 Hz, IH), 7.60 (q, J=8.7 Hz, 2H), 7.15 (dd, Ji=9 Hz, J ₂ =2.2 Hz, IH), 6.88 (d, J=2.2 Hz, IH), 6.41 (d, J=15.9 Hz, IH), 3.85 (s,3H) , 3.10 (s, 6H) . NMR "C ((300 MHz, CDCl ₃ ) δ ppm: 168.36, 149.84, 145.98, 136.55, 130.49, 130.06, 128.42, 127.23, 124.41, 116.79, 115.69, 106.43, 51.94, 41.00. LRMS (FAB) : 256 (Ci ₆ Hi ₇ NO ₂ , M+H) .

General Procedure for the Synthesis of Methyl 3-aryl- acrylates from Aryl-aldehydes. Exemplified by Methyl 3- (6-methoxy-2 -naphthyl) -acrylate (7A). See Fig. 17. A solution of 6-methoxy naphthaldehyde (0.5 g, 2.6 mmol) and methyl (triphenylphosphoranylidene) acetate (0.97 g, 2.9 mmol) in THF (1OmL) was refluxed overnight under an argon atmosphere. Once the solution cooled, the solvent was evaporated and the remaining solids were treated with 20 mL diethyl ether. After stirring for 2 hours, the undissolved solids were removed. The filtrate was concentrated and purified by column chromatography using silica gel and hexanes-EtOAc 9:1 to obtain 0.58 g (92%) of methyl 3- (6-methoxy-2-naphthyl) -acrylate (7A). NMR ¹ H (300 MHz, CDCIs) δ ppm:

7.94-7.80 (m, 4H) 7.65 (dd, .5 Hz, IH), 7.2-7.14 (m, 2H), 6.52 (d, J=15.9 Hz, IH), 3.96 (s, 3H), 3.85 (s, 3H)

NMR ¹³ C ((300 MHz, CDCl ₃ ) δ ppm: 168.12, 159.22, 145.54, 136.10, 130.54, 130.22, 130.15, 129.05, 127.90, 124.56, 119.90, 117.13, 106.34, 55.79, 52.10 LRMS (FAB) : 243 (Ci ₅ Hi ₄ O ₃ , M+H) .

General Procedure for the Synthesis of 3-Aryl-propionic

Acids from Methyl 3-aryl-Acrylates . Exemplified for 3- (6-

Dimethylamino-2-naphthyl) -propionic Acid (2C). See Fig.

18. Methyl 3- (6-dimethylamino-2-naphthyl) -acrylate (2A)

(0.67 g, 2.63 mmol) and a catalytic amount of 10% Pd/C (0.0060 g) were combined and hydrogenated at 40 psi for 3.5 h. The reaction mixture was filtered through celite, and concentrated to yield 0.65 g pure methyl 3- (6- dimethylamino-2-naphthyl) -propionate (2B) (97% yield) . NMR ¹ H (300 MHz, CDCla) δ ppm: 7.67 (d, J=9 Hz, IH), 7.62 (d, J=8.4 Hz, IH), 7.51 (s, IH), 7.25 (dd, Ji=8.4 Hz, J ₂ =I.5 Hz, IH), 7.18 (dd, Ji=9 Hz, J ₂ =2.7 Hz, IH), 6.92 (d, J=2.1 Hz, IH), 3.71 (s, 3H), 3.12 (t, J=6.9 Hz, 2H), 3.07 (s, 6H), 2.73 (t, J=6.9, 2H) NMR "C ((300 MHz, CDCl ₃ ) δ ppm: 174.0, 148.85, 134.48, 133.99, 128.69, 127.69, 127.36, 126.89, 126.46, 117.16, 106.97, 52.03, 41.43, 36.32, 31.37 LRMS (FAB) : 258 (Ci ₆ Hi ₉ NO ₂ , M+H) .

3- (6 -Dimethylamino-2 -naphthyl) -propionic acid (2C) Methyl 3- (6-dimethylamino-2 -naphthyl) -propionate (2B) (0.65 g, 2.5 mmol) was added to a slurry of potassiumtrimethylsilanoate (0.33 g, 2.5 mmol) in 10 mL dry diethyl ether. The reaction mixture stirred under argon atmosphere for 20 h at RT. The resulting solid was

filtered, washed with ether and dried. The solid was then dissolved in a small amount (~1 mL) of acetic acid and diluted with 70 mL water. After stirring for 1 h, the precipitate was collected to yield 0.34 g of 3- (6- dimethylamino-2-naphthyl) -propionic acid (2C) (63% yield) .

NMR ¹ H (300 MHz, D ₂ O) δ ppm:

7.72-7.64 (m, 2H), 7.56 (s, IH), 7.33-7.24 (m, 2H), 7.18 (s, IH), 2.94 (t, J=7.8 Hz, 2H), 2.85 (s, 6H), 2.49 (t, J=7.8 Hz, 2H)

NMR ¹³ C ((300 MHz, D2O) δ ppm:

179.26, 148.30, 134.78, 133.85, 128.89, 127.94, 127.79,

127.15, 126.51, 117.42, 108.23,

41.90, 36.15, 31.05 LRMS (FAB) : 244 (C15H17NO2, M+H) .

3- (2-Naphthyl) -propionoic acid (1C) was synthesized from

3- (2-naphthyl) -acrylic acid by hydrogenation as above for

2B. NMR ¹ H (300 MHz, DMSO-dff) δ ppm: d 12.20 (s, IH), 7.94-7.89 (m, 3H), 7.79 (s, IH), 7.58-

7.69 (m, 3H), 3.10 (t, J=7.7 Hz, 2H), 2.7 (t, J=7.7 Hz,

2H)

NMR ¹³ C ((300 MHz, DMSO-ds) δ ppm: 179.40, 144.15, 138.72, 137.26, 133.35, 133.07, 132.93,

132.85, 131.69, 131.92, 130.92, 40.71, 36.11

LRMS (FAB) : 201 (C13H12O2, M+H) .

Methyl 3- (6-methoxy-2-naphthyl) -propionate (7B). NMR ¹ H (300 MHz, CDCl ₃ ) δ ppm:

7.94-7.79 (m, 4H), 7.71 (dd, Hz, IH), 7.27-7.21 (m, 2H), 6.59 (d, J=15.9 Hz, IH), 4.03 (s, 3H), 3.92 (s, 3H) . NMR ¹³ C ((300 MHz, CDCl ₃ ) δ ppm:

173.85, 157.74, 136.07, 133.59, 129.41, 127.86, 127.40, 126.73, 119.25, 106.01, 55.70,52.07, 36.18, 31.33. LRMS (FAB) : 245 (Ci ₅ Hi ₄ O ₃ , M+H) .

3- (6-Methoxy-2-n.aph.thyl) -propionic acid (7C)

NMR ¹ H (300 MHz, DMSO- dε) δ ppm:

12.18 (s, IH) , 7.81-7.70 (m, 3H) , 7.44-7.18 (m, 3H) , 3.93 (s, 3H) , 3.04 (t, J=6.9 Hz, 2H) , 2.70 (t, J=6.9 Hz, 2H) NMR ¹³ C ((300 MHz, DMSO-de) δ ppm: 174.67, 157.70, 136.85, 133.70, 129.68, 129.41, 128.47, 127.56, 126.82, 119.36, 106.62, 55.98, 36.11, 31.21 LRMS (FAB) : 231 (Ci ₄ Hi ₄ O ₃ , M+H) .

3- (6-Methoxy-2-naphthyl) -acrylic acid (7D) NMR ¹ H (300 MHz, DMSO- de) δ ppm:

12.38 (sb, IH), 7.90-7.85 (m, 3H), 7.77 (d, J=16.2 Hz, IH), 7.43 (d, J=2.4 Hz, IH), 7.27 (dd, Ji=8.9 Hz, J ₂ =2.1 Hz, IH), 6.66 (d, J=15.9 Hz, IH), 3.97 (s, 3H) NMR ¹³ C ((300 MHz, DMSO-ds) δ ppm: 168.60, 159.23, 145.01, 136.17, 130.93, 130.45, 130.36, 129.10, 128.26, 125.41, 120.06, 119.15, 107.10, 56.18 LRMS (FAB) : 289 (Ci ₄ Hi ₂ O ₃ , M+H) .

Methyl 3- (2 -anthracene) -acrylate (9A) was prepared from 2 -anthracene carboxylic acid (24) using

Method B.

NMR ¹ H (300 MHz, CDCl ₃ ) δ ppm:

8.43 (d, J=15 Hz, 2H) , 8.09-7.99 (m, 4H) , 7.91 (d, J-15.9

Hz, IH) , 7.66 (d, J=15, IH) , 7.53-7.50 (m, 2H) , 6.59 (d, J=15.9 Hz, IH) , 3.87 (s, 3H)

NMR "C ((300 MHz, CDCl ₃ ) δ ppm:

179.26, 156.61, 144.25, 143.87, 143.50, 143.19, 142.99,

140.81, 139.99, 139.92, 139.24, 138.01, 137.88, 137.60,

134.06, 129.55, 63.44

LRMS (FAB) : 263 (Ci ₈ Hi ₃ O ₂ , M+H) .

2 -Anthracene acrylic acid (9D)

NMR ¹ H (300 MHz, DMSO-de) δ ppm: 12.44 (s, IH), 8.76 (d, J=8.4 Hz, 2H), 8.31 (s, IH), 8.10-8.07 (m, 2H), 7.88-7.78 (m, 2H), 7.56-7.53 (m, 2H), 6.71 (d, J=15.6, IH) NMR ¹³ C ((300 MHz, DMSO-ds) δ ppm:

168.53, 144.78, 132.85, 132.51, 132.44, 132.17, 131.81, 129.78, 129.07, 128.98, 128.18, 127.15, 127.00, 126.88, 123.80, 120.45 LRMS (FAB) : 249 (C17H12O2, M+H) .

Methyl 3- (2 -anthracene) -propionate (9B) was prepared from methyl 3- (2 -anthracene) -acrylate (9A) according to literature procedure (25) . Methyl 3- (2-anthracence) - acrylate (50 mg, 0.17 mmol) was hydrogenated at 10 psi over 10% Pd/C (4 mg) in 20 mL anhydrous ethanol for 4.5 h. This was filtered through celite and concentrated. The crude product was dissolved in benzene (5 mL) and o- chloranil (42 mg, 0.17 mmol) was added. This mixture was refluxed, under argon for 3 h. After the reaction cooled it was washed with water, concentrated, and purified by column chromatography on silica gel with Hexanes/EtOAc 95:5 to yield 43 mg (87%) . NMR ¹ H (300 MHz, CDCls) δ ppm:

8.39 (d, J=Il.1 Hz, 2H), 8.01-7.94 (m, 3H), 7.79 (s, IH), 7.49-7.43 (m, 2H), 7.33 (d, J=8.7 Hz, IH), 3.72 (s, 3H), 3.18 (t, J=7.7 Hz, 2H), 2.80 (t, J=7.7 Hz), 2H NMR "C ((300 MHz, CDCl ₃ ) δ ppm:

173.75, 137.79, 132.31, 132.22, 131.89, 131.03, 128.87, 128.58, 128.50, 127.33, 126.46, 126.41, 126.06, 125.78, 125.55, 52.08, 35.70, 31.68

LRMS (FAB) : 265 (Ci ₈ Hi ₆ O ₂ , M+H) .

3- (2 -anthracene) -propionic acid (9C)

NMR ¹ H (300 MHz, DMSO-de) δ ppm: 12.18 (s, IH), 8.49 (d, J=15.3 Hz, 2H), 8.07-7.99 (m, 3H), 7.82 (s, IH), 7.50-7.41 (m, 3H), 3.03 (t, J=7.5 Hz, 2H)), 2.69 (t, J=7.5Hz, 2H) NMR ¹³ C ((300 MHz, DMSO-de) δ ppm:

174.64, 138.94, 132.23, 131.76, 131.02, 128.95, 128.90, 128.80, 128.23, 126.61, 126.47, 126.35, 126.15, 126.13, 35.62, 31.55 LRMS (FAB) : 251 (Ci ₇ Hi ₄ O ₂ , M+H) .

2-Bromo-6-diethylamino naphthalene A mixture of 6-bromo- 2-naphthol (10.0 g, 44.8 mmol) , Na ₂ S ₂ Os (16.1 g, 85.1 mmol) , NaOH (6.9 g, 224 mmol), and 40 mL water were combined in a sealed tube. Ethylamine hydrochloride (16.2 g, 224 mmol) was quickly added, the tube was sealed, and heated at 14O ⁰ C for 5 days. After cooling, the mixture was poured into 80 mL NaOH (2M) . The precipitate was collected and used without purification in the next step. 2-Bromo-6-ethylamino naphthalene (4.31 g, 17.2 mmol), and sodiumtriacetoxyborohydride (3.65 g, 17.2 mmol) were added to a flask and flushed with argon. CH ₂ Cl ₂ (100 mL) , acetaldehyde (1.93 mL, 17.2 mmol), and acetic acid (0.99 mL, 17.2 mmol) were added to the flask. The reaction stirred for 6 h, after which a second equivalent of sodiumtriacetoxyborohydride (3.65 g, 17.2 mmol) and acetaldehyde (1.93 mL, 17.2 mmol) were added, this stirred for 12 h. The reaction was diluted with CH ₂ Cl ₂ (100 mL) , washed with a saturated solution of NaHCO3, water, and dried (MgSO4) .

Purification by column chromatography on silica gel with hexanes-EtOAc 95:5 gave 2-bromo-6-diethylamino naphthalene in 66% yield. NMR ¹ H (300 MHz, CDCl ₃ ) δ ppm: 7.88 (s, IH), 7.67 (d, J=9.0 Hz, IH), 7.56 (d, J=9.0 Hz, IH), 7.48 (d, J=9.0 Hz, IH), 7.18

(d, J=9 Hz, IH), 6.91 (d, J=I.5 Hz, IH), 3.56 (q, J=6.9 Hz, 4H), 1.33 (t, J=6.9 Hz, 6Hz) NMR ¹³ C (300 MHz, CDCl ₃ ) δ ppm: 146.48, 134.23, 129.70, 129.65, 128.40, 127.96, 127.62, 117.04, 114.79, 105.47, 44.91, 13.06 LRMS (FAB) : 278 (Ci ₄ HiSBrN, M+H) .

Methyl 3- (6-diethylamino-2-naphthyl) -acrylate (6A) was synthesized using Method A from 2-bromo-6-diethylamino naphthalene .

NMR ¹ H (300 MHz, CDCl ₃ ) δ ppm:

7.83-7.67 (m, 3H), 7.56-7.52 (m, 2H) , 7.10-7.04 (m, IH), 6.83 (s, IH) , 6.47 (d, J=15.8 Hz, IH) , 3.83 (s, 3H) , 3.50

(q, J=6.9 Hz, 4H) , 1.26 (t, J=6.9 Hz, 6H)

NMR "C ((300 MHz, CDCl ₃ ) δ ppm:

168.44, 147.35, 146.12, 136.95, 130.55, 130.28, 127.83,

126.92, 125.93, 124.32, 116.35, 115.23, 105.33, 51.91, 44.94, 13.10

LRMS (FAB) : 284 (C18H21NO2, M+H) .

Methyl 3- (6-diethylamino-2-naphthyl) -propionate (6B)

NMR ¹ H (300 MHz, CDCl ₃ ) δ ppm: 7.62 (d, J=9.0 Hz, IH) , 7.56 (d, J=8.4 Hz, IH) , 7.47 (s, IH) , 7.44 (dd, 0 Hz, J ₂ =2.4 Hz, IH), 6.87 (d, J=2.4 Hz, IH) , 3.69 (s, 3H) , 3.47 (q, J=7.0 Hz, 4H) , 3.07 (t, J=8.1 Hz, 2H) , 2.71 (t, J=8.1 Hz, 2H) , 1.24 (t, J=7.0 Hz, 6H)

NMR ¹³ C ( (300 MHz, CDCl ₃ ) δ ppm:

174.02, 145.99, 132.34, 133.86, 128.87, 127.61, 126.75, 126.61, 126.45, 116.71, 105.99, 52.07, 44.99, 36.38, 31.38, 13.06 LRMS (FAB) : 286 (Ci ₈ H ₂₃ NO ₂ , M+H) .

3- (6-Diethylamino-2-naphthyl) -propionic Acid (6C)

NMR ¹ H (300 MHz, MeOD-A) δ ppm:

7.61 (d, J=9.1 Hz, IH) , 7.54 (d, J=8.4 Hz, IH) , 7.47 (s, IH) , 7.21 (d, J=8.4 Hz, IH) , 7.12 (dd, Ji=9.1 Hz, J ₂ =2.2

Hz, IH) , 6.02 (s, IH) , 3.45 (q, J=7.0 Hz, 4H) , 3.01 (t,

J=7.8 Hz, 2H) , 2.66 (t, J=7.'8 Hz, 2H) , 1.19 (t, J=7.0 Hz,

6H)

NMR ¹³ C ( (300 MHz, MeOD-d<0 d ppm: 175.92, 134.41, 134.37, 145.56, 128.50, 127.42, 127.16,

126.36, 125.93, 116.72, 106.85, 44.89, 36.04, 31.08,

11.89

LRMS (FAB) : 272 (C17H21NO2, M+H) .

3- (1-Naphthyl) -propionic acid (5C) was synthesized by hydrogenation of 3- (1-naphthyl) -acrylic acid. NMR ¹ H (300 MHz, DMSO-de) δ ppm:

8.13 (d, J=8.1 Hz, IH), 7.99 (d, J=7.8 Hz, IH), 7.85 (d, J=7.8 Hz, IH), 7.66-7.56 (m, 2H), 7.52-7.44 (m, 2H), 3.39 (t, J=7.5, 2H), 2.76 (t, J=7.5, 2H) NMR "C (300 MHz, DMSO-de) δ ppm:

174.69, 137.63, 134.26, 132.07, 129.51, 127.57, 127.01, 126. GO, 126.50, 124.30, 35.47, 28.27 LRMS (FAB) : 201 (Ci ₃ Hi ₂ O ₂ , M+H) .

Benzyl 3- (6-hydroxy-2-naphthyl) -acylate was synthesized starting from 6-bromo-2-naphthol, substituting benzyl acrylate for methyl acrylate, using Method A . NMR ¹ H (300 MHz, CDCl ₃ ) δ ppm:

10.03 (s, IH) , 8.07 (s, IH) , 7.81-7.76 (m, 3H) , 7.69 (d, J=8.7 Hz, IH) , 7.45-7.31 (m, 5H) ,

7.13-7.09 (m, 2H) , 6.71 (d, J=15.9 Hz, IH) , 5.23 (s, 2H) NMR "C ( (300 MHz, CDCl ₃ ) δ ppm: 171.92, 162.49, 150.95, 141.97, 141.39, 135.96, 134.10, 133.69, 132.97, 132.41, 129.79, 124.91, 121.87, 114.64, 71.12 LRMS (FAB) : 229 (Ci ₄ Hi ₂ O ₃ , M+H) .

3- (6-hydroxy-2-naphthyl) -propionic acid (8C) was synthesized from catalytic hydrogenation of benzyl 3- (6- hydroxy-2-naphthyl) -acrylate.

NMR ¹ H (300 MHz, MeOD-A) δ ppm:

7.64 (d, J=8.7 Hz, IH) , 7.58-7.56 (m, 2H) , 7.27 (dd, Ji=8.4 Hz, J ₂ =I.8 Hz, IH) , 7.07-7.02 (m, 2H) , 3.37-3.32

(m, IH) , 3.03 (t, J=8.4 Hz, 2H) , 2.68 (t, J=8.4 Hz, 2H)

NMR "C ((300 MHz, MeOD-A) δ ppm:

175.86, 155.02, 135.48, 134.05, 129.02, 127.31, 126.43,

126.26, 118.27, 108.73, 35.77, 30.98 LRMS (FAB) : 217 (Ci ₃ Hi ₂ O ₃ , M+H) .

Methyl 3- (2 -pyrene) -acrylate (10A) was synthesized starting from 2-bromopyrene using Method A

NMR ¹ H (300 MHz, CDCl ₃ ) δ ppm: 8.82 (d, J=15.7 Hz, IH) , 8.45 (d, J=9.3 Hz, IH) , 8.25-

8.16 (m, 4H) , 8.13-8.09 (m, 2H) ,

8.05-8.0 (m, 2H) , 6.70 (d, J=15.7 Hz, IH) , 3.93 (s, 3H)

NMR ¹³ C ( (300 MHz, CDCl ₃ ) δ ppm:

167.99, 141.96, 133.08, 131.67, 131.05, 130.10, 128.96, 128.49, 127.69, 126.66, 126.39,

126.19, 125.42, 125.36 125.24, 124.50, 122.81, 120.13,

52.27, 30.14

LRMS (FAB) : 287 (C ₂₀ Hi ₄ O ₂ , M+H) .

Methyl (2 -pyrene) -propionate (10B)

NMR ¹ H (300 MHz, CDCl ₃ ) δ ppm:

8.27 (d, J=9.3 Hz, IH) , 8.19-8.10 (m, 4H) , 8.03-7.98 (m,

3H) , 7.90 (d, J=7.8 Hz, IH) , 3.74 (s, 3H) , 3.72 (t, J=8.1 Hz, 2H) , 2.90 (t, J=8.1 Hz, 2H)

NMR "C ( (300 MHz, CDCIs) δ ppm:

185.07, 146.19, 143.10, 142.55, 141.90, 140.29, 139.33,

139.16, 138.70, 138.58, 137.60, 136.76, 136.61, 134.58,

63.42, 47.61, 40.37 LRMS (FAB) : 289 (C20H1SO2, M+H) .

3- (2 -Pyrene) -propionic acid (10C)

NMR ¹ H (300 MHz, CDCl ₃ ) δ ppm:

8.16 (d, J=7.2 Hz, 2H) , 8.10-7.94 (m, 7) , 3.70 (t, J=8.2

Hz, 2H) , 2.89 (t, J=8.2 Hz, 2H) NMR ¹³ C ( (300 MHz, CDCl ₃ ) δ ppm:

175.07, 147.29, 145.62, 142.73, 140.84, 140.36, 139.37,

139.05, 138.65, 137.49, 136.21, 42.91, 39.54

LRMS (FAB) : 275 (Ci ₉ Hi ₄ O ₂ , M+H) .

Preparation of Coenzyme A Derivatives:

The coenzyme A derivatives were prepared using the mixed anhydride method as reported by Fong and Schulz (26) .

Briefly, a solution of coenzyme A (lOmg, 0.013mmol), and NaHCO ₃ in degassed water (3mL) was prepared and dry THF

(2mL) was added. The carboxylic acid (0.067 mmol) was added to a second flask and flushed with argon. It was then dissolved in dry THF (2mL) and triethylamine (0.067 mmol) and ethylchloroformate (0.067 mmol) were added. After ten minutes the reaction was quickly filtered through a pipette with a glass wool plug. This was added to the coenzyme A solution over 10 minutes, adding extra water to prevent phase separation. After 1 hour the

solution was acidified to pH 3 and the THF was removed under reduced pressure .

The solutions were purified by HPLC using water (with 0.1% TFA) and acetonitrile while monitoring at 256nm. Fractions containing product were determined by MS or fluorescence and the acetonitrile was removed under reduced pressure. Compounds were found to be greater than 95% pure by HPLC. Concentration of coenzyme A derivatives was determined as previously reported (27) . The thioester bond was quantitatively cleaved with IM hydroxylamine (pH 7) and the free thiols were reacted with 5,5'- dithiobis (2-nitrobenzoic acid) . The absorbance in 100 mM potassium phosphate buffer (pH 8) was recorded at 412 nm (ε=13,700 M ^-1 Cm ^"1 ) . CoA-derivatives were stored in water at -20°C and the concentration was checked periodically for degradation. The derivatives are stable for over a month at -20 ⁰ C, even with repeated thawing.

Photochemical Characterization

Fluorescence Spectra of Selected Probes 1, 7, and 9: Compounds 1 and 3 were excited at 340 nm, compounds 7 and 11 were excited at 350 nm, and compounds 9 and 12 were excited at 356nm, (see Figs. 10, 11 and 12) . Fluorescence emission spectra were recorded with 50 μM solutions in 100 mM potassium phosphate buffer (pH 8.0.

Photophysical Characterization

Extinction coefficients reported are the average of triplicate measurements of the lowest energy wavelength transition at three different concentrations.

Fluorescence quantum yields are the average of three independent quantum yield determinations and are determined by excitation at 260 or 350 nm using 9, 10- diphenylanthracene in cyclohexane (28), see Table 2.

Table 2: Photophysical Properties of Probes.

Saturated Unsaturated

φ φ

(nm) (M- ¹ Cm ^"1 ) (nm) (nm) (M ^-1 Cm ^"1 ) (nm)

260 28000+6000 436 0.026 ^b 3 3 ^~ 25 69000+8000 492 0.021* 232 17000+4000 - - 11 345 26000±400 510 0.043 ^E 256 9300+200 410 0.134 ^a 12 350 18000+6000 567 0.014 ^ε * In 100 mM potassium phosphate buffer (pH 7.2). ^a relative to 9, 10-diphenyl anthracene as a standard (excited at 350 nm) ; ^h relative to 9, 10-diphenyl anthracene as a standard (excited at 260 nm) .

Protocols for Enzymatic Assays

A general method for assaying ACADs is disclosed in Frerman et al . Biochemical Medicine 33:38-44 (1985), which is hereby incorporated by reference.

Procedure for Enzymatic Screening of Probes 1, 2, 5-10: Rat medium-chain acyl-CoA dehydrogenase (MCAD) was provided by Professor Horst Schulz (City University of New York) ; pig MCAD, pig short-chain acyl-CoA dehydrogenase (SCAD) , and human long-chain acyl-CoA dehydrogenase (LCAD) were provided by Professor Jung-Ja Kim (Medical College of Wisconsin) . Activity was checked by DCPIP/PMS assay (29) using butanoyl-CoA, octanoyl-CoA, or palmitoyl-CoA as the substrate for SCAD, MCAD, and LCAD respectively. Enzymatic assays were performed in

duplicate on probes using the DCPIP/PMS assay or ferricenium hexafluorophosphate assay (30) according to the following protocol. For the DCPIP/PMS assay was prepared: a buffer of 2 , 6-dichloroindolephenol (28 μM) , N-ethyl maleimide (0.2 mM) , and 10OmM potassium phosphate buffer pH 7.6 (Buffer A) . This was stored in an amber bottle, under argon. To initiate the assay, substrate

(50μl) was added to a quartz cuvette which contained

Buffer A, KCN 8.2 μL of 2.93mg/mL, 0.5-6 μL of enzyme (concentrations of 2-7 mg/mL) , with a total volume of assay was 730 μL . The absorbance decrease at 600 nm was recorded for 3 minutes (Rate A) . Then 35 μL of 20 mg/mL phenazine methosulfate was added and the absorbance decrease at 600 nm was recorded (Rate B) . A control experiment at each concentration of enzyme was run as above, omitting substrate. The rate was determined as (ε ⁶⁰⁰ 21,300 M ^-1 Cm ^"1 ) :

Rate = Rate B - Rate A - control

For FerPFg assay a solution of FerPF ₆ in 10 mM HCl was prepared daily and the concentration was determined by measuring the absorbance at 617 nm (ε ⁶¹⁷ 410 M ^-1 Cm ^"1 ) . The assay was started by addition of 0.5-6μL enzyme (concentrations 2-7mg/ml) to a quartz cuvette ^' containing Buffer B (700 μL; potassium phosphate buffer (10OmM, pH7.2) with O.lmM EDTA), FerPF ₆ (200 μM) , and substrate (50 μM) . The absorbance decrease was monitored at 300 nm (ε ³⁰⁰ 4,300 M ^-1 Cm ^"1 ) for three minutes. The rate was taken as this rate minus a control when substrate was omitted. In some instances an increase in absorbance was observed, due to the product absorbing at 300 nm. Inactive probes were considered those that did not give a significant

rate by either of the above assays . The formation of product was corroborated by HPLC analysis.

Determination of Steady State Kinetic Parameters for MCADs

Michaelis Menten constant (K _m ) and catalytic rate (k _cat ) of the fluorogenic substrates were determined as follows. To a STARNA semi-micro fluorimeter cell (equipped with a stir bar and with 4 polished walls) was added (i) Buffer

B (final volume 700 μL) , (ii) FerPF _s (200 μM) , (iii) substrate (to achieve assay concentrations of 5K _M to Ka/S) and (iv) 2 μL of diluted MCAD or homogenate (1:2 to 1:20, depending on the kinetics of a particular isozyme's reduction of a substrate) . Fluorescence arising from the formation of product was then monitored over the course of 3 minutes (Excitation and emission band pass slits both at 4 nm, lamp 750 V, λ _exc 350 for probes 1 and 7,

356 nm for 9, λ _em 492, 510, or 567 for 1, 7, and 9 respectively) . The rate of product formation, expressed in units of nanomoles per minute, was calculated according to previously published procedures (31) :

nstxl initial rate = — t (D where F _t and F ₀ represent the fluorescence at times t and 0 minutes, n _st is the nanomoles of product in a known concentration of product, and F _ξt is the fluorescence resulting from n _st of product. Kinetic parameters were approximated by GraFit (Erithacus Software, Surrey, UK) nonlinear regression analysis program to fit the untransformed data to a hyperbolic function as originally described. Reported enzymatic kinetic parameters are the

average of three independent determinations from three different preparations of enzyme. Validity of fluorescent assay was confirmed by a chromogenic assay where the formation of product was monitored spectrophotometrically (according to Table 1) . Kinetic parameters for chromogenic and fluorescent assays were similar. For example, for probe 1 with rMCAD K _cat was found to be 311 +/- 60 min ^"1 by the UV assay, while the fluorescent assay yielded a K _cat of 370 =/- 10 min ^"1 .

Preparation of Tissue Homogenate

Tissue homogenate was prepared as already reported (29) . Briefly, rat liver (gift of Horst Schulz, City University of New York) , was minced and then homogenized at 0°C with 5 vol of isolation buffer (mannitol (210 μM) , sucrose (70 μM) , Tris (10 μM) and EDTA (O.lmM), adjusted to pH 7.4 with HCl) for 2 minutes. This was treated with Triton X- 100 (0.2% v/v) and stirred at 0°C for 15 minutes. After, being clarified by centrifugation (12,00Og for 10 min at 4 ⁰ C) , the resultant clear homogenate was assayed for MCAD as above .

Competitive Substrate Assays

Competitive substrate assays were carried out as described above for FerPF _e , except in a black 96-well plate with a total volume of 200μL. To a black 96-well plate were added (i) Buffer B (total volume 200 μL) , (ii) FerPFg (200 μM) , (iii) butanoyl-CoA, octanoyl-CoA, or palmitoyl-CoA (50 μM) , and (iv) homogenate (87 μg protein) . After 30 seconds 1, 7, or 9 (10 μM) was added and the fluorescence was monitored for 3 minutes .

Fibroblast MCAD Assay

Medium-chain Acyl-CoA Dehydrogenase (MCAD) deficiency is an inborn error of fatty acid metabolism. It was first identified in 1982 (5 and 6) and has been estimated to occur in 1:15,000 births (7). MCAD deficiency is characterized by fasting intolerance, recurrent life- threatening episodes of hypoglycemic coma, and dicarboxylic aciduria, usually presented in the first two years of life. Screening programs utilizing tandem mass spectrometry have had wide spread implementation in recent years, however in some locations cost is often prohibitive. Even when MCAD deficiency is identified a confirmation test is needed.

MCAD dehydrogenates probes 1, 7, and 9 to form the unsaturated product 4, 11 and 12 respectively, along with the reduction of FAD to FADH ₂ . The conversion of probe to its product is accompanied by a change in the probe's fluorescent profile. Probes 1 and 9 are fluoromorphic, with the starting material and product having different fluorescent profiles. Probe 7 is fluorogenic with only the unsaturated product, 11, being fluorescent. Due to the change in fluorescence as the reaction proceeds, the MCAD activity can be quantitated. The probes can also differentiate between MCAD-deficient and normal human cells.

Hereinabove, a fluorescent assay for MCAD in vitro based on a fluoromorphic MCAD substrate is described. This method can also be applied living cells, and is exemplified here as applied to cultured fibroblasts.

Materials and Methods

Normal human fibroblasts and MCAD deficient fibroblasts were obtained from Coriell Cell Repository, Coriell

Institute for Medical Research, Camden NJ. The identification and characterization of the MCAD deficient cell lines have been reported elsewhere (5,6) . To compare the current results to those of previous studies, some of the same control cells lines that others have used,

(GM05565, GM05659, GM00038, GMOOOlO, GM00041) (31, 33) were employed. Probe 1 was used for further testing based on it having a much greater k _cat than probes 7 and 9, which leads to it being more sensitive to smaller amounts of enzyme .

Fibroblast cultures were grown in MEM medium supplemented with 10% FBS. Monolayers were harvested in Isolation Buffer (mannitol (210 μM) , sucrose (70 μM) , Tris (10 μM) and EDTA (0.1 mM) , adjusted to pH 7.4 with HCl) with cell scraper. Pelleted cells were either used immediately or frozen at -80 ⁰ C. Tissue homogenates were prepared as previously reported (29) . Briefly, the cell pellet was suspended in isolation buffer containing 0.2% (v/v) Triton-X 100. This was homogenized using a hand tissue grinder and vortexed (2xl5sec) . After being clarified by centrifugation (10,000xg for lOmin at 4°C) , the resultant clear homogenate was assayed for MCAD activity. Protein concentrations were determined by the Bradford method.

To a 96-well plate was added (1) buffer (0.1KPi, pH 7.2 with O.lmM EDTA), (2) FerPF ₆ (200μM) , (3) Probe 1, and (4) homogenate (144μg total protein) . The fluorescence was followed for 30 minutes, λ _ex =340 nm, λ _em = 492 nm. All assays were done in triplicate with at least three different passages from each cell line.

Results

The fibroblasts were made into protein homogenates and assayed for activity in a high-throughput 96-well plate format . The results from these assays for the various cell lines are shown in Fig. 20. The MCAD-deficient cells lines ranged in activity from 0.056 to 1.2 nmol/hr/mg protein, whereas the control cells lines had activities of 3.1 to 7.0 nmol/hr/mg protein. The average activity of the MCAD- deficient and control cells lines was 0.35+0.5 and 5.0+0.7 nmol/hr/mg protein, respectively. The two groups of cells were found to have activities that are significantly different (student T's test, p=0.0001) (see Fig. 21) . Thus, it was confirmed that use of probe 1 differentiates normal and MCAD-deficient cells. Each of probes 7 and 9 are also used in place of probe 1 in this assay to differentiate between normal and MCAD-deficient cells.

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