Compositions and methods for detecting flavin levels

Abstract

Provided herein are compositions and methods for measuring the levels of flavins (e.g., in biological samples). In particular, provided herein are modified biotin-riboflavin conjugates and uses thereof (e.g., to measure flavin levels).

Description

[0001]UM-41536.601 COMPOSITIONS AND METHODS FOR DETECTING FLAVIN LEVELS PRIORITY STATEMENT This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 680,343, filed August 7, 2024, the entire contents of which are incorporated herein by reference for all purposes. FIELD OF THE DISCLOSURE Provided herein are compositions and methods for measuring the levels of flavins (e.g., in biological samples). In particular, provided herein are modified biotin-riboflavin conjugates and uses thereof (e.g., to measure flavin levels). BACKGROUND Vitamin deficiencies cause impaired health and disease, including stunted growth, mental deterioration and compromised cellular and tissue functioning. Riboflavin (Rf), a member of the B vitamin family, is a water-soluble vitamin that participates in energy metabolism processes via two coenzymes, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), in oxidized and reduced forms. Low levels of riboflavin have been associated with fatigue, mouth lesions, and growth and developmental abnormalities, and other diseases, and have been reported due to inadequate dietary riboflavin or to endocrine or genetic mutations impacting Rf uptake and transport. Riboflavin deficiency has not received the attention of many other micronutrients for several reasons. Riboflavin is found in many foods naturally and is often added to “fortified” breads and grain products. Deficiency I rare in some countries, but prevalent in countries or regions without supplementation or proper nutrition. Although flavins act in redox reactions, the role of flavins as “antioxidants” has not garnered the popular press attention awarded other micronutrients. The bright yellow color of urine following ingestion of riboflavin-enriched foods seemed to confirm that Rf deficiency was not of concern- at least in the USA. Recent reports indicate that riboflavin deficiency, known as ariboflavinosis, has been observed in at-risk groups, particularly young women, and that riboflavin deficiency can be correlated with mineral status. Numerous studies confirm riboflavin deficiencies particularly in UM-41536.601 mothers and infants; both rural and urban populations suffer. Some cancers such as HPV and gastric cancer are associated with lower blood levels of riboflavin. In addition, mental health issues have been liked to flavin status. Assays to identify Rf deficiency are needed. SUMMARY There is a lack of data on Rf deficiency because there is not a simple and inexpensive test to measure Rf in at risk individuals. Experiments described herein resulted in the design of an assay to detect the flavin status of individuals (e.g., so that dietary intervention can reduce deficiency and disease), along with other uses in research, screening, diagnostic, and therapeutic applications. The assay utilizes a riboflavin derivative able to interact selectively and simultaneously with riboflavin binding protein (RBP) and a detection system (e.g., avidin based detection system), at physiological pH. For example, in some embodiments, provided herein is a composition, comprising a structure of: wherein n is 1 R2 is selected from, for example, H alkyl, Cl, morpholine, or histidyl; R3and R4are independently selected UM-41536.601 from H or alkyl; and X and Y are independently selected from C or N. In some embodiments, the structure is . In some embodiments, n is 6. provide a composition, comprising a structure of: , wherein n is 1 to 8 (e.g., 5). the structure binds to a flavin binding protein, e.g., riboflavin binding protein (RBP). In some embodiments, the structure binds to avidin and / or streptavidin. In some embodiments, the structure binds simultaneously to both RBP and avidin. Also provided is a kit comprising a composition described herein. In some embodiments, the kit further comprises a flavin binding protein (e.g., RBP) and / or avidin. In some embodiments, the kit further comprises a solid support. The present disclosure is not limited to particular solid supports. Examples include but are not limited to plastic, metal, glass, paper, fabric, hydrogels, foam, microfluidic devices, capillaries, beads (e.g., magnetic or paramagnetic beads), a microtiter plate, a membrane (e.g., a nitrocellulose membrane), a microstructured polymer, or a sintered polymer. In some embodiments, the RBP or the avidin is bound to the solid support. Further embodiments provide a method of detecting a flavin in a sample, comprising: a) providing a detection system comprising avidin, a flavin binding protein (e.g., RBP), and a composition described herein; b) contacting the detection system with a sample under conditions such that a flavin in the sample competes with the composition for binding to RBP; and c) UM-41536.601 detecting the level of the flavin in the sample based on the amount of the composition bound to said RBP. The present disclosure is not limited to a particular detection system. Examples include but are not limited to, level of fluorescence quenching or the level of absorbance. In some embodiments, an anti-biotin antibody is utilized to detect a bound composition of the disclosure. In some embodiments, the RBP or the avidin is bound to a solid support and the method further comprises the step of washing the solid support to remove unbound components of the sample. In some embodiments, the sample is isolated from a subject (e.g., a subject suspected of having a flavin deficiency). Additional embodiments provide a method of treating a flavin deficiency in a subject, comprising a) determining the level of a flavin in a sample from a subject using an assay described herein; and b) administering supplemental flavin to a subject identified as having a flavin deficiency. In some embodiments, the method further comprises repeating the determining step at a later time point. Any number of different supplemental flavins find use in the methods described herein. Examples include but are not limited to, a flavin supplement, a flavin-containing food, a flavin- containing nutraceutical, or a flavin-containing pharmaceutical. In some embodiments, the supplemental flavin is administered orally or parentally. The present disclosure is not limited to the detection of specific flavin. Examples include but are not limited to, riboflavin, flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD). The present disclosure is not limited to a specific sample type. Examples include but are not limited to, blood, blood products, tissue, saliva, or urine. In some embodiments, the present disclosure provides the use of a composition or kit described herein in the treatment of a disease or condition or to detect a flavin level in a sample. Additional embodiments are described herein. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows a C6tag competition assay of HABA bound to Avidin. FIG. 2 shows a fluorescence quenching assay where increasing amounts of apo RBP were a) added to C6tag in a microplate assay, reading excitation / emission at 450 / 525 wavelength and b) added to N-(6’-hydroxyhexyl)isoalloxazine 4 at a concentration is 17.3 mM; 6-(7,8-dimethyl UM-41536.601 2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)hexyl-5-((3aS,4S,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl)pentanoate 5 at a concentration of 16.8 mM and riboflavin at a concentration of 23.4. FIG. 3a,b shows a demonstration of potential simultaneous binding of C6tag to Avidin RBP. FIG. 4 shows a schematic of vitamin-biotin conjugate and protein interactions. FIG. 5 shows NMR spectra of exemplary compounds. FIG. 6 shows NMR spectra of exemplary compounds. FIG. 7 shows a schematic of an exemplary method of detection of riboflavin in samples. FIG. 8. shows a1H NMR spectrum 6-(7,8-dimethyl-2,4-dioxo-3,4- dihydrobenzo[g] (2H)-yl)hexyl-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4- d]imidazol-4-yl)pentanoate (400MHz, DMSO). FIG. 9 shows a1H NMR spectrum 6-(7,8-dimethyl-2,4-dioxo-3,4- dihydrobenzo[g]pteridin-10(2H)-yl)hexyl-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4- d]imidazol-4-yl)pentanoate region 1-5 ppm (400MHz, DMSO). FIG. 10 shows a13C NMR spectrum 6-(7,8-dimethyl-2,4-dioxo-3,4- dihydrobenzo[g]pteridin-10(2H)-yl)hexyl-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4- d]imidazol-4-yl)pentanoate (400MHz, DMSO). FIG. 11 shows a UV Spectrum of 6-(7,8-dimethyl-2,4-dioxo-3,4- dihydrobenzo[g]pteridin-10(2H)-yl)hexyl-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4- d]imidazol-4-yl)pentanoate, 1 mM in DMSO. FIG. 12 shows a IR Spectrum of 6-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin- 10(2H)-yl)hexyl-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate. FIG. 13 shows a high resolution mass spectrum of 6-(7,8-dimethyl-2,4-dioxo-3,4- dihydrobenzo[g]pteridin-10(2H)-yl)hexyl-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4- d]imidazol-4-yl)pentanoate. FIG. 14 shows a mass spectrum fragmentation pattern of 6-(7,8-dimethyl-2,4-dioxo-3,4- dihydrobenzo[g]pteridin-10(2H)-yl)hexyl-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4- d]imidazol-4-yl)pentanoate. FIG. 15 shows a docking image of Rf versus C6-biotin tag binding to protein. FIG. 16 shows exemplary uses of Rf-biotin tags. UM-41536.601 DETAILED DESCRIPTION OF THE DISCLOSURE Provided herein are compositions and methods for measuring the levels of flavins (e.g., in biological samples). In particular, provided herein are modified biotin-riboflavin conjugates and uses thereof (e.g., to measure flavin levels). Definitions As used herein, the terms “detect,” “detecting,” or “detection” may describe either the general act of discovering or discerning or the specific observation of a composition. Detecting a composition may comprise determining the presence or absence of a composition. Detecting may comprise quantifying a composition. For example, detecting comprises determining the expression level of a composition. For example, the composition may comprise at least a portion of a flavin. Alternatively, or additionally, the composition may be a detectably labeled composition. As used herein, the term “subject” refers to any organisms that are treated or screened using the methods described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and most preferably includes humans. The term “diagnosed,” as used herein, refers to the recognition of a disease by its signs and symptoms, or genetic analysis, pathological analysis, histological analysis, and the like. The term “label” as used herein refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) effect, and that can be attached to a nucleic acid or protein. Labels include but are not limited to dyes; radiolabels such as32P; binding moieties such as biotin; haptens such as digoxgenin; luminogenic, phosphorescent or fluorogenic moieties; and fluorescent dyes alone or in combination with moieties that can suppress or shift emission spectra by fluorescence resonance energy transfer (FRET). Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. A label may be a charged moiety (positive or negative charge) or alternatively, may be charge neutral. Labels can include or consist of nucleic acid or protein sequence, so long as the sequence comprising the label is detectable. UM-41536.601 As used herein, the term “sample” is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids (e.g., blood, urine, saliva), solids, tissues, and gases. Biological samples include urine, urine supernatant, and urine cell pellet as well as blood products, such as plasma, serum and the like. Such examples are not however to be construed as limiting the sample types applicable to the present disclosure. The terms substituted, whether preceded by the term “optionally” or not, and substituent, as used herein, refer to the ability, as appreciated by one skilled in this art, to change one functional group for another functional group on a molecule, provided that the valency of all atoms is maintained. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Where substituent groups or linking groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., -CH2O- is equivalent to - OCH2-; -C(=O)O- is equivalent to -OC(=O)-; -OC(=O)NR- is equivalent to -NRC(=O)O-, and the like. When the term “independently selected” is used, the substituents being referred to (e.g., R groups, such as groups R1, R2, and the like, or variables, such as “m” and “n”), can be identical or different. For example, both R1and R2can be substituted alkyls, or R1can be hydrogen and R2 can be a substituted alkyl, and the like. The terms “a,” “an,” or “a(n),” when used in reference to a group of substituents herein, mean at least one. For example, where a compound is substituted with “an” alkyl or aryl, the compound is optionally substituted with at least one alkyl and / or at least one aryl. Moreover, where a moiety is substituted with an R substituent, the group may be referred to as “R- substituted.” Where a moiety is R-substituted, the moiety is substituted with at least one R substituent and each R substituent is optionally different. A named “R” or group will generally have the structure that is recognized in the art as corresponding to a group having that name, unless specified otherwise herein. For the purposes of illustration, certain representative “R” groups as set forth above are defined below. UM-41536.601 Unless otherwise explicitly defined, a “substituent group,” as used herein, includes a functional group selected from one or more of the following moieties, which are defined herein; The term hydrocarbon, as used herein, refers to any chemical group comprising hydrogen and carbon. The hydrocarbon may be substituted or unsubstituted. As would be known to one skilled in this art, all valencies must be satisfied in making any substitutions. The hydrocarbon may be unsaturated, saturated, branched, unbranched, cyclic, polycyclic, or heterocyclic. Illustrative hydrocarbons are further defined herein below and include, for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl, and the like. The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a univalent group derived from an alkane by removal of a hydrogen atom from any carbon atom –CnH2n+1. The groups derived by removal of a hydrogen atom from a terminal carbon atom of unbranched alkanes form a subclass of normal alkyl (n-alkyl) groups H(CH2)n. The groups RCH2, R2CH (R ≠ H), and R3C (R ≠ H) are primary, secondary and tertiary alkyl groups, respectively. An alkyl can be a straightchain (i.e., unbranched) or branched acyclic hydrocarbon having the number of carbon atoms designated (i.e., C1-10 means one to ten carbons, including 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbons). In particular embodiments, the term “alkyl” refers to C1-20 inclusive, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 carbons. Representative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, isopentyl, neopentyl, n- hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, and dodecyl. “Branched” refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. “Lower alkyl” refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a C1-8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, “alkyl” refers, in particular, to C1-8straight-chain alkyls. In other embodiments, “alkyl” refers, in particular, to C1-8 branched-chain alkyls. Alkyl groups can optionally be substituted (a “substituted alkyl”) with one or more alkyl group substituents, which can be the same or different. The term “alkyl group substituent” UM-41536.601 includes but is not limited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There can be optionally inserted along the alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), or aryl. Thus, as used herein, the term “substituted alkyl” includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, cyano, and mercapto. The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain having from 1 to 20 carbon atoms or heteroatoms or a cyclic hydrocarbon group having from 3 to 10 carbon atoms or heteroatoms, or combinations thereof, consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si and S, and wherein the nitrogen, phosphorus, and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, P and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH- CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)- CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)- CH3, O-CH3, -O-CH2-CH3, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. As described above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such as -C(O)NR’, -NR’R”, -OR’, -SR, -S(O)R, and / or –S(O2)R’. Where “heteroalkyl” is recited, followed by recitations of specific heteroalkyl groups, such as -NR’R or the like, it will be understood that the terms heteroalkyl and -NR’R” are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term “heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR'R” or the like. UM-41536.601 An unsaturated hydrocarbon has one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. Alkyl groups which are limited to hydrocarbon groups are termed “homoalkyl.” More particularly, the term “alkenyl” as used herein refers to a monovalent group derived from a C2-20inclusive straight or branched hydrocarbon moiety having at least one carbon-carbon double bond by the removal of a single hydrogen molecule. Alkenyl groups include, for example, ethenyl (i.e., vinyl), propenyl, butenyl, 1-methyl-2-buten-1-yl, pentenyl, hexenyl, octenyl, allenyl, and butadienyl. The term “cycloalkenyl” as used herein refers to a cyclic hydrocarbon containing at least one carbon-carbon double bond. Examples of cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl, cycloheptatrienyl, and cyclooctenyl. The term “alkynyl” as used herein refers to a monovalent group derived from a straight or branched C2-20 hydrocarbon of a designed number of carbon atoms containing at least one carbon-carbon triple bond. Examples of “alkynyl” include ethynyl, 2-propynyl (propargyl), 1- propynyl, pentynyl, hexynyl, and heptynyl groups, and the like. The term “alkylene” by itself or a part of another substituent refers to a straight or branched bivalent aliphatic hydrocarbon group derived from an alkyl group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkylene group can be straight, branched or cyclic. The alkylene group also can be optionally unsaturated and / or substituted with one or more “alkyl group substituents.” There can be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also referred to herein as “alkylaminoalkyl”), wherein the nitrogen substituent is alkyl as previously described. Exemplary alkylene groups include methylene (–CH2–); ethylene (–CH2–CH2–); propylene (–(CH2)3–); cyclohexylene (– C6H10–); –CH=CH–CH=CH–; –CH=CH–CH2–; -CH2CH2CH2CH2-, -CH2CH=CHCH2-, - CH2CsCCH2-, -CH2CH2CH(CH2CH2CH3)CH2-, -(CH2)q-N(R)-(CH2)r–, wherein each of q and r is independently an integer from 0 to about 20, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl (–O–CH2–O–); and UM-41536.601 ethylenedioxyl (-O-(CH2)2–O–). An alkylene group can have about 2 to about 3 carbon atoms and can further have 6-20 carbons. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being some embodiments of the present disclosure. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms. The term “heteroalkylene” by itself or as part of another substituent means a divalent group derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms also can occupy either or both of the chain termini (e.g., alkyleneoxo, alkylenedioxo, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)OR’- represents both -C(O)OR’- and –R’OC(O)-. A dashed line representing a bond in a cyclic ring structure indicates that the bond can be either present or absent in the ring. That is, a dashed line representing a bond in a cyclic ring structure indicates that the ring structure is selected from the group consisting of a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure. The symbol ( ) denotes the point of attachment of a moiety to the remainder ofthe molecule. When a named atom of an aromatic ring or a heterocyclic aromatic ring is defined as being “absent,” the named atom is replaced by a direct bond. Each of above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl, and “heterocycloalkyl”, “aryl,” “heteroaryl,” “phosphonate,” and “sulfonate” as well as their divalent derivatives) are meant to include both substituted and unsubstituted forms of the indicated group. Optional substituents for each type of group are provided below. As used herein, the term “acyl” refers to an organic acid group wherein the -OH of the carboxyl group has been replaced with another substituent and has the general formula RC(=O)-, wherein R is an alkyl, alkenyl, alkynyl, aryl, carbocylic, heterocyclic, or aromatic heterocyclic group as defined herein). As such, the term “acyl” specifically includes arylacyl groups, such as a 2-(furan-2-yl)acetyl)- and a 2-phenylacetyl group. Specific examples of acyl groups include UM-41536.601 acetyl and benzoyl. Acyl groups also are intended to include amides, -RC(=O)NR’, esters, -RC(=O)OR’, ketones, -RC(=O)R’, and aldehydes, -RC(=O)H. The term “carbonyl” refers to the –C(=O)– group, and can include an aldehyde group represented by the general formula R-C(=O)H. The term “cyano” refers to the -C≡N group. The terms “halo,” “halide,” or “halogen” as used herein refer to fluoro, chloro, bromo, and iodo groups. Additionally, terms such as “haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term “halo(C1-4)alkyl” is mean to include, but not be limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like. The term “hydroxyl” refers to the –OH group. The term “oxo” as used herein means an oxygen atom that is double bonded to a carbon atom or to another element. The term “nitro” refers to the –NO2 group. The term “thio” refers to a compound described previously herein wherein a carbon or oxygen atom is replaced by a sulfur atom. The term “sulfate” refers to the –SO4 group. More particularly, the term “sulfide” refers to compound having a group of the formula – SR. The term “sulfone” refers to compound having a sulfonyl group –S(O2)R. The term “sulfoxide” refers to a compound having a sulfinyl group –S(O)R The term ureido refers to a urea group of the formula –NH—CO—NH2. Detailed description Provided herein are compositions and methods for measuring the levels of flavins (e.g., in biological samples). In particular, provided herein are modified biotin-riboflavin conjugates and uses thereof (e.g., to measure flavin levels). The compositions and methods of the present disclosure solve a problem of significance to human health - allowing for inexpensive, accurate and rapid determination of riboflavin (Rf) status in animals (e.g., humans), especially those that might be at risk such as pregnant or nursing women, or children with insufficient nutrition, in animals for husbandry, in bacteria and organisms used to create flavins, or for tissues for organ transport and in ophthalmic solutions. UM-41536.601 In some embodiments, the compositions comprise conjugates configured to bind a flavin binding protein such as riboflavin binding protein (RBP) that further include a first member of a binding pair (e.g., a biotin-avidin binding pair) as well as methods of using the compositions. Currently, the methods available for determining Rf status in humans, or other sources such as milk or food, are expensive or require instrumentation not typically available in remote areas of the globe. Riboflavin (Rf) is a bright yellow, fluorescent and water soluble (barely) member of the B vitamin family. Riboflavin (Vitamin B2) is transformed to the active forms Flavin Mononucleotide (FMN) and Flavin Adenine Dinucleotide (FADH2), by a series of enzymatic reactions. FMN and FADH2 are cofactors critical in numerous redox reactions including metabolic pathways. In addition, they are required in conversion of many biological precursors to the active forms, such as the conversion of vitamin B6 to pyridoxal 5’-phosphate, and are bound by flavoproteins mediating gene expression. The recommended dietary intake of Rf for humans varies from 0.3 to 1.3 mg / day, depending on age and sex (US Food and Drug Administration). Physiological indicators of riboflavin deficiency include anemia, sores near and around the mouth, sore throat and problems with mucous membranes. n some embod men s, e presen d sc osure prov des a composition generated by covalently attaching a first member of a binding pair (e.g., biotin molecule) to the tail of UM-41536.601 riboflavin or riboflavin analog to generate a conjugate analog that binds to a flavin binding protein such as RBP, and also is able to bind simultaneously to a second member of a binding pair (e.g., an avidin detection system). This system can be used in an exchange / competition assay to measure the concentration of riboflavin and other flavins (e.g., FAD or FMN). For example, in some embodiments, provided herein is a composition, comprising a structure of: or Cl; R2 is selected from, for example, H alkyl, Cl, morpholine, or histidyl; R3 and R4 are independently selected from H or alkyl; and X and Y are independently selected from C or N. In some embodiments, the structure is some embodiments, n is 6. a composition, comprising a structure of: UM-41536.601 , wherein n is 1 to 8 (e.g., 5). Such compositions find use in (e.g., to alter the activity of flavins). Also provided is a kit comprising a composition described herein. In some embodiments, the kit further comprises riboflavin binding protein (RBP). In some embodiments, all the materials and reagents necessary, useful, or sufficient for detecting a flavin in a sample (e.g., obtained from a subject) are provided in the kit. In some embodiments, the kits include a component for containing the reagents in close confinement for commercial sale such as, e.g., plastic or glass containers into which the desired reagent are retained. Other containers suitable for conducting certain steps of the disclosed methods also may be provided. The compositions and methods described herein find use in a variety of research, screening, diagnostic, and therapeutic methods. In some embodiments, the present disclosure provides a method of detecting a flavin (e.g., riboflavin, flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) in a sample. One exemplary binding assay is shown in FIG. 4. The assay is based on a specific binding interaction of the flavin binding protein riboflavin binding protein (RBP) with analog compositions described herein. FIG. 4 shows a riboflavin-biotin analog molecule (B2-B7) bound to avidin via an avidin:biotin interaction. RBP binds to riboflavin in the analog. The presence or absence of a flavin (exemplified by riboflavin in FIG 4) in a sample is determined, for example, by its competition for binding to RBP with the analog. The assays of the present disclosure find use with any number of different avidin molecules, including both natural and synthetic avidins. Not limiting examples include avidin and streptavidin. For example, in some embodiments, the level of analog bound in the presence of a sample comprising a flavin is used to determine the level of the flavin in the sample. One method for such a determination is exemplified in FIG. 2. As shown in FIG. 2, binding of the analog to RBP results in fluorescence quenching. A decrease in the level of quenching in the presence of a sample is indicative of flavin in the sample out-competing the analog for binding to RBP. UM-41536.601 Other competition assays utilize a decrease in absorbance (See e.g., FIG. 1). Additional detection methodologies may be utilized for quantitating flavin levels in a sample and are specifically contemplated. For example, in some embodiments, a primary (e.g., anti-biotin) and / or secondary antibody is used to detect binding (e.g., as exemplified in FIG. 7). The present disclosure is not limited to a particular assay format. In some embodiments, assays are performed on a solid surface where one or more components of the assay (e.g., RBP or avidin) is attached to a solid surface. In some embodiments, assays performed on solid surfaces utilize a washing step prior to detection in order to remove unbound components of the sample. The present disclosure is not limited to particular solid surfaces. In some embodiments, the surface is solid, semi-solid, porous, or non-porous. In some embodiments, the support or solid surface is, for example, plastics, metal, glass, paper, fabric, hydrogels, foam, surfaces of sensors, electrodes, cantilevers, microfluidic device surfaces, inside capillaries, beads (e.g., paramagnetic bead), a microtiter plate, a membrane (e.g., nitrocellulose membrane), microstructured polymer, or sintered polymer. The present disclosure is not limited to particular sample types for use in detection assays. Examples include but are not limited to, blood, blood products, tissue (e.g., biopsy tissue), saliva, or urine. The methods described herein find use in a variety of applications. For example, in some embodiments, detection of flavins such as riboflavin is used in a screening or diagnostic assay to identify individuals with riboflavin deficiencies and / or monitor treatment of riboflavin deficiencies. For example, in some embodiments, treatment methods are provided that comprises the steps of performing an assay to detect the level of riboflavin in a sample from a subject, and administering supplemental riboflavin to a subject identified as deficient in riboflavin. In some embodiments, the assay is repeated one or more times during treatment and used to determine the need for additional treatment or when to cease treatment. In some embodiments, the assays described herein are used to isolate a flavin from a sample (e.g., in research and screening assays), for example, to isolate and identify new flavins. While the present disclosure is exemplified with assays for detection of flavins (e.g., in biological samples), the present disclosure is not limited to such embodiments. The compositions described herein find use in a variety of additional applications. Examples include but are not UM-41536.601 limited to, inhibiting a flavin activity (e.g., as a cancer treatment or as an antibiotic), detection of flavin binding sties on cells membranes or tissue surfaces, identifying flavin membrane receptors in cancerous tissues, altering gene regulation or protein synthesis via binding to nucleic acid polymers or aptamers (See e.g., Dmytruk, K.V., Ruchala, J., Fayura, L.R. et al. Efficient production of bacterial antibiotics aminoriboflavin and roseoflavin in eukaryotic microorganisms, yeasts. Microb Cell Fact 22, 132 (2023); Benore-Parsons, Marilee, (1986), Ph D Thesis, University of Delaware, The Transport of Riboflavin-binding Protein to the Hen Oocyte; Tsvetkova, Yoanna , PhD Thesis, (2018) Riboflavin-targeted nanomedicines for cancer imaging and drug delivery = Riboflavin-funktionalisierte Nanotherapeutika zur bildgebenden Darstellung von Krebs und für den Wirkstofftransport, DOI: 10.18154 / RWTH-2018-226246; Phelps, Mitch A. (2005) PhD Thesis, Ohio State, Novel approaches for characterizing the riboflavin transport and trafficking mechanism and its potential as a target in breast cancer; Crielaard S, Maassen R, Vosman T, Rempkens I, Velema WA. Affinity-Based Profiling of the Flavin Mononucleotide Riboswitch. J Am Chem Soc. 2022 Jun 15;144(23):10462-10470. doi: 10.1021 / jacs.2c02685. Epub 2022 Jun 6. PMID: 35666649; PMCID: PMC9204756; Xiangrui Li, Huan Lv, Wencan Luo, WenJia Yang, Linghong Kong, Qiujin Zhu, Lu Zeng, Recent advances in detection techniques for vitamin analysis: A comprehensive review, Food Chemistry: X, Volume 26, 2025, 102226, ISSN 2590-1575; Matnuri M, Zheng C, Sidik D, Bai G, Abdukerim M, Abdukadier A, Ahmat K, Ma Y, Eli M. Correlation analysis of riboflavin, RFT2 and Helicobater pylori in gastric carcinoma. Int J Clin Exp Pathol. 2015 Oct 1;8(10):13339-45. PMID: 26722538; PMCID: PMC4680483; each of which is herein incorporated by reference in its entirety), detection of flavin biding sites in nucleic acids (e.g., DNA or RNA) via streptavidin binding to the Rf-biotin tag of embodiments of the disclosure or a biotin binding antibody, and identification of novel flavin binding proteins (See e.g., FIG. 16). In some embodiments, therapeutic applications utilize pharmaceutical compositions. Depending on the specific conditions being treated, such agents may be formulated into liquid or solid dosage forms and administered systemically or locally. The agents may be delivered, for example, in a timed- or sustained-slow-release form as is known to those skilled in the art. Techniques for formulation and administration may be found in Remington: The Science and Practice of Pharmacy (20thed.) Lippincott, Williams & Wilkins (2000). Suitable routes may include oral, buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal, transmucosal, UM-41536.601 nasal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intra- articular, intra -sternal, intra-synovial, intra-hepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular injections or other modes of delivery. In some embodiments, treatments for flavin (e.g., riboflavin) deficiency comprises oral riboflavin supplements. Examples of commercially available riboflavin supplements include 25 mg, 50 mg, and 100 mg tablets. According to the National Institutes of Health, the recommended daily nutrient intake of riboflavin is 1.3 mg for men, 1.1 mg for women, 1.3 mg for male adolescents (age 14 to 18), and 1.0 mg for female adolescents (age 14 to 18). Recommendations are that pregnant women take 1.4 mg and breastfeeding women take 1.6 mg. For infants 0 to 6 months old, the dose is 0.3 mg; for 7 to 12 months, it is 0.4 mg; for 1 to 3 years, it is 0.5 mg; for 4 to 8 years old is 0.6 mg; and for 9 to 13 years is 0.9 mg. In some embodiments, dosages are increased or decreased based on riboflavin levels. In some embodiments, riboflavin supplements are administered with meals to increase absorption levels. In addition to a flavin supplement, a flavin-containing food, a flavin-containing nutraceutical, or a flavin-containing pharmaceutical may also be utilized. In some embodiments, the supplemental flavin is administered orally or parentally. In some embodiments, the assays described herein are used in industrial applications to monitor cleaning procedures. Riboflavin sticks to machinery or other industrial surfaces (e.g., in clean rooms) and provides a marker to aid in cleaning operations and to detect leaks. In such embodiments, a solution containing riboflavin is applied to surfaces and then visualized using an assay described herein. EXAMPLES The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only UM-41536.601 intended for the purposes of illustration, and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods. Example 1 Methods and Procedures of Analog Synthesis The following tags were synthesized and used to determine binding to RBP: C5 modified 5 carbon flavin derivative, MW—342 g / mol- C6 modified 6 carbon flavin derivative, MW—- 376 g / mol (Shown below) (FIG. 5) C6-tag 6 carbon flavin derivative bound to biotin tail via ester linkage MW—620 g / mol- (Shown below; FIG. 6) Scheme 1 C6-Biotin Conjugate Synthesis (n=6) Chemistry Reagents, materials, and solvents were purchased from MilliporeSigma, St. Louis, MO, USA, and used without further purification. Riboflavin derivative, N-(6′ -hydroxyhexyl) UM-41536.601 isoalloxazine, was synthesized based on a published procedure (Frier, C.; Dècout, J.L. Nucleotides and Flavin Method for Preparing New Flavin Derivatives: Synthesis of Flavin- Thymine-Oligonucleotide Adducts. J. Org. Chem 1997, 62, 3520–3528) while RF derivatives were synthesized following a modified published protocol ( Pittelkow, M.; Kamounah, F.S.; Boas, U.; Pederson, B.; Christensen, J.B. TFFH as an Excellent Reagent for Acylation of Alcohols, Thiols and Dithiocarbamates. Synthesis 2004, 15, 2485–2492).1H and13C NMR spectra were recorded on a Bruker Avance 400 MHz spectrometer using solvent peak as an internal reference, with chemical shifts expressed in ppm. The HRMS and fragmentation pattern was collected on a Waters Xevo G2-XS QTof mass spectrometer, Waters, Millford, MA, USA, with a flow injection method at 0.2 mL / min 95% methanol / 5% water, EI method in ion positive mode. Melting points were determined using a MelTemp apparatus. The UV-VIS data was collected on a Shimadzu UV 2600i, Shimadzu, Columbia, MD, USA, in the range 250–600 nm with a 1 cm quartz cell and a 1 mm wide slit on a solution of 0.074 mM of 5 in DMSO. The IR spectrum was acquired on an AGILENT CARY 630 FTIR spectrometer, Agilent, Santa Clara, CA, USA. Step 1: N-(6’-Hydroxyhexyl)-3,4-dimethylaniline. 3,4-dimethylaniline (9.33 g, 76 mmol), mixture of triethylamine (15 mL), and 6-chloro-1- hexanol (3.38 g, 24.73 mmol, 3.3 mL were refluxed overnight. After the mixture was cooled to room temperature, DCM (200 mL) was added, and the resulting solution was washed with NaHCO3(10%, 40.0 mL). The aqueous layer was extracted with DCM (2 x 100 mL). The combined organic layers were dried with magnesium sulfate and concentrated in vacuo. The brown residue was purified via column chromatography (eluent: DCM:methanol = 98:2) and recrystallized from hexanes to yield a light brown solid (2.57 g, 11.6 mmol, 47%), m.p.: 46-47. ºC,1HNMR (400MHz, CDCl3): δ 1.43-1.46 (m, 4H), 1.59-1.66 (m, 4H), 2.18 (s, 3H), 2.22 (s, 3H), 3.12 (t, 2H), 3.68 (t, 2H), 6.41 (d, 1H), 6.47 (s, 1H), 6.95 (d, 2H). Step 2: 6-((3,4-dimethylphenyl)(5-hydroxyhexyl)amino)pyrimidine-2,4(1H,3H)-dione N-(6’-Hydroxyhexyl)-3,4-dimethylaniline (2.19 g, 9.88 mmol) was refluxed under inert atmosphere in water-dioxane (1:1, 22 mL) for 20 minutes. 6-chlorouracil (0.50 g, 3.44 mmol) was added and the solution was refluxed overnight. After the mixture was cooled to room temperature, the pH was adjusted to 11 with NaOH solution (10%). The resulting solution was extracted with DCM (2 x 50 mL) to remove any unreacted material. The aqueous layer was UM-41536.601 acidified (pH = 3) with dil. HCl when a white precipitate formed. The solid was filtered and recrystallized from water to yield a white powder (0.77 g, 2.36 mmol, 68%), m.p.: 205-207 ºC;1HNMR ((CD3)2SO): δ 1.20-1.25 (m, 4H), 1.33-1.41 (m, 2H), 1.41-1.52 (m, 2H), 2.31 (s, 6H), 3. 36 (t, 2H), 3.58 (t, 2H), 4.09 (s, 1H), 4.32 (t, 1H), 6.96 (d, 1H), 7.04 (s, 1H, (7.22 (d, 1H), 10.09 (s, 1H), 10.34 (s, 1H). Step 3: 2-hydroxy-10-(5-hydroxyhexyl)-7,8-dimethyl-4-oxo-2,3,4,10- tetrahydrobenzo[g]pteridine 5-oxide 6-[N-(6’-Hydroxyhexyl)-3,4-xylidino]uracil (0.78 g, 2.39 mmol) was dissolved in acetic acid (6 mL) and sodium nitrite (0.79 g, 11.5 mmol) was added. The reaction mixture was stirred at room temperature, in the dark for 3 h. Water (4.0 mL) was added, and solution was kept overnight in the dark at room temperature. The solvents were removed in vacuo and the residue was washed with water, filtered, and recrystallized from ethyl acetate-ethanol (50:50) to produce a yellow solid (0.66g, 1.84 mmol, 77%): m.p. 221-224 ºC;1HNMR (400 MHz, (CD3)2SO) δ 1.35-1.46 (m, 6H), 1.71 (m, 2H), 2.40 (s, 3H), 2.49 (s, 3H), 3.41 (q, 2H), 4.38 (t, 1H), 4.53 (m, 2H), 7.79 (s, 1H), 8.09 (s, 1H), 11.03 (s, NH) Step 4: 10-(6-hydroxyhexyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione An aqueous solution of dithiothreitol (1.14 g, 7.38 mmol, 16 mL) was added to a suspension of isoalloxazine 5-oxide (0.56 g, 1.64 mmol) in ethanol (400 mL). The reaction mixture was stirred under reflux for 70 minutes while the solution became clear. The solvents were removed in vacuo and the resulting residue was recrystallized from ethanol to yield an orange powder (0.44 g, 1.28 mmol, 82%): m.p.: 272-274 ºC;1HNMR (400 MHz, (CD3)2SO): δ 1.25-1.46 (m, 6H), 1.72 (m, 2H), 2.40 (s, 3H), 2.49 (s, 3H), 3.41 (q, 2H), 4.38 (t, 1H), 4.53 (m, 2H), 7.78 (s, 1H), 7.91 (s, 1H), 11.29 (s, NH), elem.analysis: calc.: C(62.79), H(6.45), N(16.29), O(13.95); exp.: C( 62.16), H(6.60), N(16.09), O(14.02) Step 5: 6-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)hexyl 5- ((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate Triethylamine (0.65 g, 0.90 mL, 6.46 mmol) was added to suspension of biotin (0.35 g, 1.42 mmol) and tetramethylfluoroformamidinium hexafluorophosphate, TFFH, (1.33 mmol, 0.35 g) in DMF (12.0 mL) in ice. After the clear solution was stirred at room temperature for 50 minutes, 10-(6-hydroxyhexyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (0.44 g, 1.28 mmol) and DMAP (0.035g, 0.29 mmol) were added and the reaction mixture was stirred at room UM-41536.601 temperature overnight. The solution was washed with DMF (10 mL) and the solid removed. The filtrate was diluted with H2O (25 mL) and extracted with DCM (50 mL x 3). The combined organic layers were dried with magnesium sulfate and concentrated in vacuo. The residue was recrystallized from methanol / ether to yield an orange powder (0.31 g, 0.63 mmol, 49%), m.p.: 221-224 °C;1HNMR (400 MHz, (CD3)2SO) δ: 1.28-1.75 (m, 14H), 2.30 (t, 2H), 2.42 (s, 3H), 2.53 (s, 3H), 2.56 (d, 1H), 2.79 (m, 1H), 3.10 (m, 1H), 3.31 (m, 1H), 4.04 (t, 2H), 4.13 (m, 1H), 4.31 (m, 1H), 4.60 (t, 2H), 6.36 (s, NH), 6.47 (s, NH), 7.81 (s, 1H), 7.93 (s, 1H), 11.31 (s, NH), elem. analysis: calc: C (59.13), H(6.38), N(14.78), O(14.07), S(5.64) ; exp: C (58.45), H(6.14), N(15.22), O (13.29), S(6.35) Characterization The structure of the C6 derivative was verified by1H and13C NMR spectroscopy, mass spectrometry, UV-VIS spectroscopy, and elemental analysis (FIGs. 8-14). The1H NMR spectrum of displayed the N-H peaks from the ureido region at 6.35 and 6.43 and the flavin’s NH at 11.30 ppm. On the tetrahydrothiophene ring, the CH2 protons α to the S were identified as a doublet at 2.58 and a doublet of doublets at 2.80 ppm, consistent with the corresponding resonances in the1H NMR spectrum of free biotin. At the same time, the CH α to the S was observed at 3.08 ppm as a multiplet. The two CH protons neighboring the NH groups in the ureido bicycle appeared at 4.13 and 4.29 ppm, flanked by the triplets of the CH2-N and CH2O groups belonging to the riboflavin derivative tail. On the flavin moiety, the aromatic hydrogens and one methyl’s hydrogens were detected at 7.79 and 7.91 ppm, as well as 2.41 ppm, respectively; the other methyl’s hydrogens overlapped with the residual peak of the solvent, DMSO. The resonances observed in the13C NMR spectrum were assigned tentatively based on previous experimental data on flavins. The C=O ester’s signal at 173.4 ppm, together with the three amidic resonances at 156.1, 160.4, and 163.2 ppm, as well as the imide ones at 147.1 and 150.4 ppm, support the synthesis of the target compound. The aromatic carbon atoms were in the range 131.11–137.50 ppm, with only the resonance corresponding to carbon ortho to the methyl group at 116.54 ppm. The tetrahydrothiophene ring exhibited peaks at 55.9, 59.0, and 61.0 ppm, while the CH2O from the ester group was present at 64.09 ppm. The remaining aliphatic carbons were in the range 21.0–33.8 ppm. The CH2-N riboflavin derivative tail and the CH-S from the tetrahydrothiophene ring were UM-41536.601 obscured by the residual solvent peak. The presence of the methyl groups was indicated by peaks at 19.0 and 19.5 ppm. The C=O bonds were present in the IR spectrum around 1688 cm−1while the stretching vibrations corresponding to the N-H bonds showed at 3293 cm−1. The mass spectrum contained, in addition to the base peak at 569 (M + 1), two major peaks, m / z = 243 and 343, corresponding to biotin and 4 fragments, respectively, resulting from the cleavage of the ester bond. The UV spectrum of solution of 0.074 mM of the compound in DMSO exhibited the crucial peaks at 273 nm (ε = 28,500), 344 nm (ε = 7200), and 448 nm (ε = 12,000) that are characteristic of the flavin moiety, which were also observed in the control UV spectrum of Rf (0.065 mM solution in DMSO) 273 nm (ε = 24,300), 347 nm (ε = 5600), and 449 nm (ε = 8700). C5 Derivative Synthesis (n=5) Step 1: N-(5’-Hydroxypentyl)-3,4-dimethylaniline. A mixture of 3,4-dimethylaniline (9.14 g, 75 mmol), triethylamine (15 mL), and 5-chloro-1- pentanol (3.38 g, 25.94 mmol, 3.0 mL) were refluxed overnight. After the mixture was cooled to room temperature, DCM (200 mL) was added, and the resulting solution was washed with NaHCO3(10%, 40.0 mL). The aqueous layer was extracted with DCM (2 x 100 mL). The combined organic layers were dried with magnesium sulfate, concentrated in vacuo. The brown residue was purified via column chromatography (eluent: DCM:methanol = 98:2) and washed with hexanes to yield a light brown paste (2.14 g, 10.30 mmoles, 39%),1HNMR (400 MHz, CDCl3: δ 1.44-1.51 (m, 2H), 1.60-1.67 (m, 4H), 2.19 (s, 3H), 2.24 (s, 3H), 2.64 (br, NH), 3.13 (t, 2H), 3.58 (t, 1H), 3.67 (t, 2H), 6.43 (d, 1H), 6.49 (s, 1H), 6.97 (d, 1H). Step 2: 6-((3,4-dimethylphenyl)(5-hydroxypentyl)amino)pyrimidine-2,4(1H,3H)-dione N-(5’-Hydroxypentyl)-3,4-dimethylaniline (2.14 g, 10.30 mmol) was refluxed under inert atmosphere in water-dioxane (1:1, 22 mL) for 20 minutes. 6-chlorouracil (0.495 g, 3.38 mmol) was added and the solution was refluxed overnight. After the mixture was cooled to room temperature, the pH was adjusted to 11 with NaOH (10%). The resulting solution was extracted with DCM (2 x 50 mL) to remove any unreacted material. The aqueous layer was acidified (pH = 3 ) with dil. HCl when a white precipitate formed. The solid was filtered and recrystallized from water to yield a pale yellow powder (0.55 g, 1.73 mmol, 17%) m.p.: 225-229°C,1HNMR (400 UM-41536.601 MHz, (CD3)2SO) δ: 1.24 (m, 2H), 1.35 (m, 2H), 1.43 (m, 2H), 2.45 (s, 6H), 3.33 (m, 2H), 3.58 (t, 2H), 4.1(s, 1H), 4.35 (t, 1H) 6.97 (d, 1H), 6.98 (s, 1H), 7.21(d, 1H), 10.10 (s, 1H), 10.34 (s, 1H). Step 3: 2-hydroxy-10-(5-hydroxypentyl)-7,8-dimethyl-4-oxo-2,3,4,10- tetrahydrobenzo[g]pteridine 5-oxide 5-[N-(5’-Hydroxypentyl)-3,4-xylidino]uracil (0.502 g, 1.58 mmol) was dissolved in acetic acid ( 4.2 g, 69.9 mmol, 4 mL) while stirring. Sodium nitrite (0.534 g, 7.73 mmol) was added, and the reaction mixture was stirred at room temperature, in the dark for 3 h. Water (4.0 mL) was added and solution was kept in the dark at room temperature for additional 3 h. The solvents were removed in vacuo and the residue was washed with water, filtered, and recrystallized from ethyl acetate-ethanol (50:50) to produce a yellow solid (0.64 g, 1.85 mmol,), m.p.: 236-239°C;1HNMR (400 MHz, (CD3)2SO) δ 1.52 (m, 4H), 1.75 (m, 2H), 2.39 (s, 3H), 3.46 (t, 2H), 4.57 (t, 2H), 7.82 (s, 1H), 8.13 (s, 1H), 11. 04 (s, 1H). Step 4: 10-(5-hydroxypentyl)-7,8-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione An aqueous solution of dithiothreitol (1.14 g, 7.38 mmol, 16 mL) was added to a suspension of isoalloxazine 5-Oxide (0.56 g, 1.64 mmol) in ethanol (400 mL). The reaction mixture was stirred under reflux for 70 minutes while the solution became clear. The solvents were removed in vacuo and the resulting residue was recrystallized from ethanol to yield an orange powder (0.32 g, 0.99 mmol, 84%) m.p. 295-298°C:1HNMR (400 MHz, (CD3)2SO) δ 1.5 (m, 4H), 1.7 (m, 2H), 2.4 (s, 3H), 3.4 (m, 2H), 4.41 (t, 1H), 4.57 (t, 2H)7.79 (s, 1H), (7.89 (s, 1H), 11.30 (s, NH). Elemental analysis: calc.: C(61.80), H(6.71), N (16.96), O(14.53); exp: C (62.16), H(6.60), N(16.09), O(14.02). Example 2 Analog And Tag Binding To RBP and / or Avidin Spectroscopy measurements of analogs and analog binding to proteins RBP and avidin UV / Vis spectroscopy and measurements conducted on either a Genesis, Shimadzu, or Thermo Fisher nanodrop, and visible and fluorescence absorbance values obtained using a TECAN M2000. RBP was purified from egg white following a modification of the method by Miller and White (1986). RBP concentrations were determined using the Kozik equation, which adjusts for UM-41536.601 the presence of flavin, using a MW of 32,000 and ApoRBP Extinction coefficient of 49,000 L / mol cm; RBP has a MW of ~32,000, with heterogeneity in the glyco groups. Avidin (purchased) concentrations and MW were determined from the literature or product information. Analog and tag binding to RBP and / or avidin Analog Preparation for assays All analogs were synthesized, crystallized, dissolved in DMSO, and stored in the dark at room temperature prior to assay, at ~ 400uM. NMR data used to characterize structures. Samples were diluted in dH2O or buffer and stored in the dark at RT prior to use in assays. Analog Binding to apoRBP Riboflavin and other flavins bind to apoRiboflavin Binding Protein (apoRBP) within a pocket between a Trp and Tyr pocket (FIG. 15), quenching the inherent fluorescence. Proof of binding of analogs to apoRBP binding was verified by fluorescent quenching using a TECAN with fluorescence and absorbance detection. Results demonstrate saturable binding of riboflavin, the C5, C6, and C6tag by fluorescence quenching measuring Ex / Em at 450 / 526 nm (Figures 1 and 2). To demonstrate affinity of C6tag to apo RBP by dialysis, a mixture of apoRBP and C6tag were placed in a 3 mL Slide-a-lyzer dialysis cassette, MWCO 10,000, and dialyzed against three changes of dH2O over 3 days to remove excess unbound C6tag. Initial amounts added were 1 mL of 320uM apoRBP and 2 mL 390uM C6tag, for final conc of 107uM RBP and 260uM C6tag. The cassette retained solution was yellow, demonstrating RBP is saturated with C6tag. Analog Binding to Avidin Proof of binding to avidin was demonstrated by competition with HABA, an avidin ligand. The assay uses displacement of HABA by the competing ligand, by observing the colorimetric absorbance shift of bound and free HABA. The experiments were conducted with both in house assays and purchased premade kits (Sigma H2153, HABA / Avidin reagent) To demonstrate affinity of C6tag to avidin, a mixture of 1 mg / mL streptavidin (NEB N70215, estimated 16uM) and C6tag were placed in a 3 mL dialysis cassette, MWCO 10,000, and UM-41536.601 dialyzed against three changes of dH2O over 4 days. The retained solution was yellow, demonstrating avidin is saturated with C6tag. Proof of Binding of C6tag to Avidin and RBP Simultaneously To demonstrate the potential for simultaneous binding avidin was first saturated with C6tag using the HABA / avidin kit, and tag binding verified by change in absorbance. To this complex apoRBP was added, and the fluorescence of the tag was quenched. Fluorescent values and absorbance indicate that that the HABA did not rebind, demonstrating that both RBP and avidin were bound to the tag (Figure 3). Protein Samples Avidin and Streptavidin were purchased from Sigma or Thermo Fisher. HoloRiboflavin Binding Protein was purified in the lab from eggs, typically chicken egg white or yolk, following a modified procedure of Miller and White 1986. Purity determined by SDS PAGE versus standards, and the MW verified. The apo form is created by dialysis again 6mM HCL over several days and verified by absorbance and fluorescence measurements. UV / Vis Spectroscopy of Analogs All analogs were characterized by UV / Vis and fluorescence spectroscopy. All analogs had similar scans, but were less fluorescent than riboflavin. All publications, patents, patent applications and accession numbers mentioned in the above specification are herein incorporated by reference in their entirety. Although the disclosure has been described in connection with specific embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications and variations of the described compositions and methods of the disclosure will be apparent to those of ordinary skill in the art and are intended to be within the scope of the following claims.

Claims

UM-41536.601 CLAIMS We claim:

1. A composition, comprising a structure of:of H, alkyl, and Cl; R2 is selected from the group consisting of H, alkyl, Cl, morpholine, and histidyl; R3 and R4 are independently selected from the group consisting of H and alkyl; and X and Y are independently selected from the group consisting of C and N.

2. The composition of claim 1, wherein said structure isUM-41536.601 3. The composition of claim 1 or 2, wherein n is 6.

4. A composition, comprising a structure of: , wherein n is 1 to 8.

5. The composition of claim 4, wherein n is 5.

6. The composition of claim 5, wherein n is 6.

7. The composition of any one of the preceding claims, wherein said structure binds to riboflavin binding protein (RBP).

8. The composition of any one of the preceding claims, wherein said structure binds to avidin and streptavidin.

9. The composition of any one of claims 1 to 3, wherein said structure binds simultaneously to both RBP and avidin.

10. A kit, comprising the composition of any one of the preceding claims.

11. The kit of claim 10, wherein said kit further comprises a riboflavin binding protein (RBP) and / or avidin.

12. The kit of claim 10 or 11, wherein said kit further comprises a solid support.UM-41536.601 13. The kit of claim 12, wherein said solid support is selected from the group consisting of plastic, metal, glass, paper, fabric, hydrogels, foam, microfluidic devices, capillaries, beads, a microtiter plate, a membrane, a microstructured polymer, and a sintered polymer.

14. The kit of claim 13, wherein said bead is a magnetic or paramagnetic bead.

15. The kit of claim 13, wherein said membrane is a nitrocellulose membrane.

16. The kit of any one of claims 12 to 15, wherein said RBP or said avidin is bound to said solid support.

17. A method of detecting a flavin in a sample, comprising: a) providing a detection system comprising avidin, RBP, and the composition of any one of claims 1 to 9; b) contacting said detection system with a sample under conditions such that a flavin in said sample competes with said composition for binding to said RBP; and c) detecting the level of said flavin in said sample based on the amount of said composition bound to said RBP.

18. The method of claim 17, wherein said level of said composition bound to said RBP is detected based on the level of fluorescence quenching.

19. The method of claim 17, wherein said level of said composition bound to said RBP is detected based on the level of absorbance.

20. The method of any one of claims 17 to 19, wherein said RBP or said avidin is bound to a solid support.

21. The method of claim 20, wherein said method further comprises the step of washing said solid support to remove unbound components of said sample.UM-41536.601 22. The method of claim 17, wherein said level of said composition bound to said RBP is detected based on binding of an anti-biotin antibody to said composition.

23. The method of any one of claims 17 to 22, wherein said flavin is selected from the group consisting of riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD).

24. The method of any one of claims 17 to 23, wherein said sample is isolated from a subject.

25. The method of claim 24, wherein said subject is suspected of having riboflavin deficiency.

26. The method of any one of claims 17 to 25, wherein said sample is selected from the group consisting of blood, tissue, saliva, and urine.

27. A method of treating a flavin deficiency in a subject, comprising a) determining the level of a flavin in a sample from a subject using the method of claim 17; and b) administering a flavin supplement to a subject identified as having a flavin deficiency.

28. The method of claim 27, further comprising repeating said determining step at a later time point.

29. The method of claim 27 or 28, wherein said flavin supplement is selected from the group consisting of a flavin supplement, a flavin-containing food, a flavin-containing nutraceutical, or a flavin-containing pharmaceutical.

30. The method of claim 29, wherein said flavin supplement is administered orally or parentally.UM-41536.601 31. The method of any one of claims 27 to 30, wherein said flavin is selected from the group consisting of riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD).

32. Use of the composition of any one of claims 1 to 9 in the treatment of a disease or condition.

33. Use of the composition of any one of claims 1 to 9 to detect a flavin level in a sample.

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