Classifications

• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
  • C09B15/00—Acridine dyes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/531—Production of immunochemical test materials
  • G01N33/532—Production of labelled immunochemicals
  • G01N33/533—Production of labelled immunochemicals with fluorescent label

Definitions

• R3 is hydrogen.
• the compound e.g., a compound for conjugating with an analyte or binding partner of an analyte such as a peptide, a protein, or a macromolecule including an antibody
• the compound may have the structure of formula (IV): wherein RFG is a reactive functional group for conjugating to the analyte or binding partner for an analyte, L is absent (i.e., it is a bond) or a linker, and ⁇ is a chemiluminescent acridinium comprising the structure: wherein “ R1 is hydrogen, –R, –X, –R L –X b , –L1–R, –L1–X b , –Z, –R L –Z, –L1–Z, or –R L –L1–R L –Z; R 2 and R 3 are independently selected from hydrogen, –R, an electron donating group
• R1 is –R, –X b , –R L –X b , –L1–R, –L1–X b , –Z, –R L –Z, –L 1 –Z, or –R L –L 1 –R L –Z.
• the compound for forming a conjugate is selected from:
• Methods for forming a conjugate are provided.
• the method may comprise reacting the compound for forming a conjugate with an analyte or binding partner for an analyte (e.g., an antibody) to form a conjugate.
• an analyte e.g., an antibody
• a reagent is provided for the detection of an analyte comprising a detectable conjugate having a chemiluminescent acridinium with a heterocycle fused to the acridinium ring structure.
• the detectable conjugate may comprise one or more (e.g., one, two) zwitterionic functional groups.
• FIG.2 provides the light emission kinetics of acridinium esters HEGAE (1), HQYAE (2) and ADOAEs D to L (7-15).
• ADOAE F (9), ADOAE H (11), ADOAE J (13), and ADOAE L (15) each have faster chemiluminescent reaction kinetics than the other tested compounds.
• FIG. 3 (3A-3D) provides structures of comparative acridiniums (FIG. 3A) and exemplary ADOAEs (FIGS.3B-3D) described and used in the syntheses, measurements, and analyses provided herein.
• DETAILED DESCRIPTION [0022] For convenience, certain terms employed in the specification, including the examples and appended claims, are collected here.
• any compound disclosed herein which has one or more chiral centers may be in the form of a racemic mixture with respect to each chiral center, or may exist as pure or substantially pure (e.g., great than 98% ee) R or S enantiomers with respect to each chiral center, or may exist as mixtures of R and S enantiomers with respect to each chiral center, wherein the mixture comprises an enantiomeric excess of one or the other configurations, for example an enantiomeric excess (of R or S) of more than 60% or more than 70% or more than 80% or more than 90%, or more than 95%, or more than 98%, or more than 99% enantiomeric excess.
• any chiral center may be in the “S” or “R” configurations.
• the description of compounds herein is limited by principles of chemical bonding. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding such as regard to valencies, and to give compounds which are not inherently unstable. For example, any carbon atom will be bonded to two, three, or four other atoms, consistent with the four valence electrons of carbon.
• Any atom can be optionally substituted, e.g., by one or more substituents.
• alkyl groups include without limitation methyl, ethyl, n-propyl, isopropyl, and tert-butyl.
• Any alkyl group referenced herein e.g., R, R’, R”, L1, L C , R L , R C , R1, R2, R3, R4, R5, R6, R7, R8) may have from 1-35 carbon atoms.
• alkyl groups will have from 1-20 or from 1-12 or from 1-8 or from 1-6 or from 1-3 carbon atoms, including for example, embodiments having one, two, three, four, five, six, seven, eight, nine, or ten carbon atoms.
• Alkyl groups may be lower alkyl (e.g., C1-C4 alkyl).
• An alkyl group substituted with one or more heteroatoms may include heteroalkyl groups such as amino groups (e.g., alkylamino, dialkylamino), alkoxy groups, or haloalkyl groups).
• Haloalkyl groups are typically alkyl groups where at least one hydrogen atom is replaced by halo. In some embodiments, more than one hydrogen atom (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) are replaced by halo. In these embodiments, the hydrogen atoms can each be replaced by the same halogen (e.g., fluoro) or the hydrogen atoms can be replaced by a combination of different halogens (e.g., fluoro and chloro).
• haloalkoxy and halothioalkoxy refer to —O(haloalkyl) and – S(haloalkyl), respectively.
• sulfhydryl refers to —SH.
• hydroxyl employed alone or in combination with other terms, refers to a group of formula – OH. Any alkoxy, thioalkoxy, or haloalkoxy group referenced herein (e.g., R, R’, R”, L 1 , L C , R L , R C , R1, R2, R3, R4, R5, R6, R7, R8) may have from 1-35 carbon atoms.
• alkoxy, thioalkoxy, or haloalkoxy groups will have from 1-20 or from 1-12 or from 1-8 or from 1-6 or from 1-3 carbon atoms, including for example, embodiments having one, two, three, four, five, six, seven, eight, nine, or ten carbon atoms.
• Alkoxy groups may be lower alkoxy (e.g., C 1 -C 4 alkoxy).
• Aralkyl groups typically refers to groups where an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. One of the carbons of the alkyl moiety serves as the point of attachment of the aralkyl group to another moiety.
• Any ring or chain atom can be optionally substituted, e.g., by one or more substituents.
• aralkyl include benzyl, 2-phenylethyl, and 3-phenylpropyl groups.
• An aralalkyl group substituted with one or more heteroatoms may include heteroarylalkyl groups such as amino groups (e.g., arylamino), aryloxy groups, or haloarylalkyl groups).
• alkenyl may refer to a straight or branched hydrocarbon chain containing the indicated number of carbon atoms and having one or more carbon-carbon double bonds.
• Alkenyl groups can include, e.g., vinyl, allyl, 1-butenyl, and 2-hexenyl.
• One of the double bond carbons can optionally be the point of attachment of the alkenyl substituent.
• Any alkenyl group referenced herein e.g., R, R’, R”, L1, L C , R L , R C , R1, R2, R3, R4, R5, R6, R7, R8) may have from 1-35 carbon atoms.
• alkenyl groups will have from 1-20 or from 1-12 or from 1-8 or from 1-6 or from 1-3 carbon atoms, including for example, embodiments having one, two, three, four, five, six, seven, eight, nine, or ten carbon atoms.
• An alkenyl group substituted with one or more heteroatoms may include heteroalkenyl groups such as amino groups (e.g., alkenylamino, alkenylalkylamino), alkenyloxy groups, or haloalkenyl groups).
• heterocyclyl groups can include, e.g., tetrahydrofuryl, tetrahydropyranyl, piperidyl (piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolidinyl.
• heterocyclic ring containing from 5-6 ring atoms, wherein from 1-2 of the ring atoms is independently selected from N, NH, N(C 1 -C 6 alkyl), NC(O)(C 1 -C 6 alkyl), O, and S; and wherein said heterocyclic ring is optionally substituted with from 1-3 independently selected R” would include (but not be limited to) tetrahydrofuryl, tetrahydropyranyl, piperidyl (piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolidinyl.
• heterocycloalkenyl typically refers to partially unsaturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon groups having one or more (e.g., 1-4) heteroatom ring atoms independently selected from O, N (it is understood that one or two additional groups may be present to complete the nitrogen valence and/or form a salt), or S.
• a ring carbon (e.g., saturated or unsaturated) or heteroatom can be the point of attachment of the heterocycloalkenyl substituent. Any atom can be optionally substituted, e.g., by one or more substituents.
• Heterocycloalkenyl groups can include, e.g., dihydropyridyl, tetrahydropyridyl, dihydropyranyl, 4,5-dihydrooxazolyl, 4,5-dihydro-1H-imidazolyl, 1,2,5,6-tetrahydro- pyrimidinyl, and 5,6-dihydro-2H-[1,3]oxazinyl.
• Cycloalkyl groups may be fully saturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon groups. Any atom can be optionally substituted, e.g., by one or more substituents.
• Cycloalkyl moieties can include, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and norbornyl (bicycle[2.2.1]heptyl).
• a cycloalkyl group substituted with one or more heteroatoms may include heterocycloalkyl groups such as oxiranyl, oxetanyl, azetidinyl, aziridinyl, furanyl, pyranyl, pyrrolidinyl, piperidinyl, thiiranyl, thietanyl, tetrahydrothiphenyl, thiopyranyl, or halocycloakyl.
• Cycloalkenyl groups may be partially unsaturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon groups.
• a cycloalkenyl group substituted with one or more heteroatoms may include heterocycloalkenyl groups such as oxiranyl, oxetanyl, azetidinyl, aziridinyl, furanyl, pyranyl, pyrrolidinyl, piperidinyl, thiiranyl, thietanyl, tetrahydrothiphenyl, thiopyranyl, or halocycloalkenyl.
• Aryl groups are often aromatic monocyclic, bicyclic (2 fused rings), or tricyclic (3 fused rings), or polycyclic (> 3 fused rings) hydrocarbon ring system.
• One or more ring atoms can be optionally substituted, e.g., by one or more substituents.
• Aryl moieties include, e.g., phenyl and naphthyl.
• a cycloalkenyl group substituted with one or more heteroatoms may include heteroaryl groups or haloaryl groups.
• heteroaryl groups include, but are not limited to, 2H-pyrrolyl, 3H-indolyl, 4H- quinolizinyl, acridinyl, benzo[b]thienyl, benzothiazolyl, ⁇ -carbolinyl, carbazolyl, coumarinyl, chromenyl, cinnolinyl, dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl, indazolyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, oxazolyl, perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phen
• substituted may refer to a group “substituted” on a hydrocarbon (e.g., an alkyl, haloalkyl, cycloalkyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group) at any atom of that group, replacing one or more atoms therein.
• a hydrocarbon e.g., an alkyl, haloalkyl, cycloalkyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group
• the substituent(s) on a group are independently any one single, or any combination of two or more of the permissible atoms or groups of atoms delineated for that substituent.
• a substituent may itself be substituted with any one of the above substituents.
• the phrase “optionally substituted” means unsubstituted (e.g., substituted with an H) or substituted. It is understood that substitution at a given atom is limited by valency. Common substituents include halo (e.g.
• substituted means that a hydrogen and/or carbon atom is removed and replaced by a substituent (e.g., a common substituent).
• a substituent e.g., a common substituent.
• a substituent (radical) prefix names such as alkyl without the modifier “optionally substituted” or “substituted” is understood to mean that the particular substituent is unsubstituted.
• haloalkyl without the modifier “optionally substituted” or “substituted” is still understood to mean an alkyl group, in which at least one hydrogen atom is replaced by halo and any other associated substitutions as necessary.
• the compound may have the structure of Formula (Ia): wherein “ R1 is hydrogen, –R, –X b , –R L –X b , –L1–R, –L1–X b , –Z, –R L –Z, –L1–Z, or –R L –L1–R L –Z; R2 and R3 are independently selected from hydrogen, –R, an electron donating group, or –Z, or R 2 and R 3 together form a 5-10 membered fused heterocyclyl group; R4 is independently at each occurrence selected from hydrogen and –R (e.g., R’, R N , lower alkyl such as C 1 -C 4 alkyl); ⁇ is S, O, or N; Y is selected from –R, –L 1 –R, –R L –Z, –L 1 –R L –Z, or in the case where ⁇ is O or S then Y is absent
• ⁇ is S, O, or N.
• R1 is–R, –X b , –R L –X b , –L1–R, –L1–X b , –Z, –R L –Z, –L1–Z, or –R L –L1–R L –Z.
• R1 is–R, –X b , –R L –X b , –L1–R, –L1–X b , –Z, –R L –Z, –L1–Z, or –R L –L1–R L –Z and ⁇ is S, O, or N.
• R L is independently at each occurrence a C1-20 bivalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., substituted with 1-10 heteroatoms, substituted with 1-10 substituents); and R’ is hydrogen or a C1-10 alkyl.
• a C1-20 bivalent hydrocarbon radical e.g., alkyl, alkenyl, aryl, alkynyl, arylalkyl
• R’ is hydrogen or a C1-10 alkyl.
• the reactive functional group may be selected from: COOH.
• the compound may have the structure of Formula (IVa): wherein “j” is 1, 2, 34, 5, or 6; R 1 is hydrogen, –R, –X b , –R L –X b , –L 1 –R, –L 1 –X b , –Z, –R L –Z, –L 1 –Z, or –R L –L 1 –R L –Z; R2 and R3 are independently selected from hydrogen, –R, an electron donating group, or –Z, or R 2 and R 3 together form a 5-10 membered fused heterocyclyl group; R 4 is independently at each occurrence selected from hydrogen and –R (e.g., R’, R N , lower alkyl such as C1-C4 alkyl); ⁇ is S, O, or N; Y is selected from –R, –L1–R, –R L –Z, –L1
• ⁇ is S, O, or N.
• R 1 is–R, –X b , –R L –X b , –L 1 –R, –L 1 –X b , –Z, –R L –Z, –L 1 –Z, or –R L –L1–R L –Z.
• R1 is–R, –X b , –R L –X b , –L1–R, –L1–X b , –Z, –R L –Z, –L1–Z, or –R L –L 1 –R L –Z and ⁇ is S, O, or N.
• each of R 4 in Formula (IVa) may be hydrogen.
• “ ” is 2 or 3.
• the compounds for forming the conjugates may have the relevant groups (e.g., ⁇ , Y, Y’, Y”, R, R’, R”, L 1 , L C , R L , R C , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , X, X a , X b ) as described herein.
• the compound may have the structure of formula (V), (Va), (Vb), (Vc), (Vd), (Ve), (VIa), (VIb), (VIc), (VId), or (VIe):
• the compound is selected from:
• the counterion may be selected from CH 3 SO 4 -, FSO 3 -, CF 3 SO4-, C 4 F 9 SO 4 - , CH3C6H4SO3-, halide (e.g., Cl-, F-, Br-), CF3COO-, CH3COO-, or NO3-.
• R1 is methyl, ethyl, propyl, or isopropyl.
• the acridinium compound may be zwitterionic by via covalent attachment to an anion.
• R1 may comprises –R L – X and –X is sulfonate (–SO ⁇ ).
• R 1 is –R L –X and –X is sulfonate (–SO ⁇ ).
• R1 is –R L –X or –L8–Z.
• L8 is –S(O)2–NH– or – (CH2)1-3–S(O)2–NH–.
• R1 may comprise a sulfopropyl group (–(CH2)3–SO ⁇ ).
• R 1 is sulfopropyl.
• the chemiluminescent acridinium ⁇ is an acridinium ester.
• ⁇ may have the structure:
• ⁇ is S, O, or N; Y is selected from –R, –L 1 –R, –R L –Z, –L 1 –R L –Z, or in the case where ⁇ is O or S then Y is absent; Y’ is either absent (i.e.
• ⁇ is S, O, or N and/or R1 is –R, –X b , –R L –X b , –L1– R, –L 1 –X b , –Z, –R L –Z, –L 1 –Z, or –R L –L 1 –R L –Z.
• ⁇ may be a chemiluminescent acridinium comprising the structure: wherein “h” is 1, 2, 3, 4, 5, or 6.
• ⁇ is a chemiluminescent acridinium comprising the structure: .
• R 9 -R 14 are independently selected at each occurrence a methyl group or a group – (CH2CH2O)aCH3, where a is an integer from 1 to 5.
• R2 and R3 are independently at each occurrence hydrogen, alkyl (e.g., methyl, ethyl, propyl, isopropyl), or alkoxy (e.g., methoxy, ethoxy, propoxy, or isopropoxy).
• R2 and R3 are each hydrogen.
• R 2 or R 3 is hydrogen and the other of R 2 or R 3 is alkoxy or an electron donating group.
• the detectable conjugate or compound for forming a detectable conjugate may comprise a chemiluminescent acridinium sulfonamide.
• ⁇ in the conjugate or compound for forming a detectable conjugate may have the structure of formula (IIa), (IIb), (IIc), (IId), or (IIe): wherein “h” is 1, 2, 3, 4, 5, or 6;
• R 15 is independently at each occurrence hydrogen, halogen, or R.
• R L has the structure: wherein R 5 -R 8 are independently C 1-35 alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, or amino.
• R7 and R8 are each hydrogen and R5 and R6 are each methyl.
• ⁇ comprises two flanking methyl groups on a phenolic ester to stabilize the bond as disclosed in Law et al. Journal of Bioluminescence and Chemiluminescence 4: 88-89 (1989), hereby incorporated by reference in its entirety.
• ⁇ has the structure:
• the reactive functional group may comprise (or be) an isothiocyanate, isocyantate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, maleimide, imidoester, carbodiimide, anhydride, fluorophenyl ester, or combinations thereof.
• the reactive functional group labels the analyte or binding partner therefor through acylation or alkylation.
• the covalent linkage between A and ⁇ (e.g., L) or RFG and ⁇ (e.g., L) may comprise (or be) a divalent C1-20 alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted with up to 20 heteroatoms (e.g., N, O, S, P, Cl, F, Br).
• L comprises a zwitterionic linker.
• L may have the structure –L C –(Z L )z–, wherein z is 0 or 1.
• the compound for forming a conjugate may have the structure: [0070] Exemplary compounds for forming the conjugates are disclosed in Table 1.
• the detectable conjugate is formed by reacting a compound (e.g., a compound of Formula (IV), (Va), (Vb), (Vc), (Vd), (Ve), (VIa), (VIb), a compound from Table 1) with an analyte, binding partner thereof, or derivatized version of the foregoing capable of reacting with a reactive functional group.
• a compound e.g., a compound of Formula (IV), (Va), (Vb), (Vc), (Vd), (Ve), (VIa), (VIb), a compound from Table 1
• ADO 2,3- cyclic alkylenedioxy
• the conjugate may have the formula:
• the increase in quantum yield from acridinium esters is one of several advantageous properties for the presently disclosed acridinium compounds which exhibit higher quantum yield relative to other acridinium compounds.
• Chemiluminescence may be measured in relative light units (RLU) on a luminometer.
• Quantum yields of acridinium may be measured as the amount of chemiluminescence (RLU) per mole of acridinium.
• Table 2 provides exemplary characterizations of the compounds for several of the measured species (the dashed double bond indicates the fusing to the acridinium ring) Table 2 [0079]
• the compounds may also be characterized by their light output or signal to noise ratio in a chemiluminescent assay.
• the compound may be characterized by a thyroid stimulating hormone assay having a larger signal to noise ratio as compared to HQYAE or TSPAE, acridinium conjugates having the similar as shown in U.S. Pat. Nos. 7,309,615 and 7,785,904, which are each hereby incorporated by reference in their entirety and particularly in relation to quantum yields of TSPAE and HQYAE.
• the compounds may be characterized as having a relative signal to noise ratio as compared to HQYAE or TSPAE (e.g., in a TSH assay) of more than 1 (e.g., from 1 to 2).
• a higher signal to noise ratio for the same amount of analyte is an indicator of better assay sensitivity.
• the compounds can be prepared from commercially available starting materials, compounds known in the literature, or readily prepared intermediates, by employing standard synthetic methods (in addition to those provided herein). Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be readily obtained from the relevant scientific literature or from standard textbooks in the field.
• the reactive functional group may by NH 2 , or N-hydroxysuccinimidyl ester (“NHS”), or maleimide derived.
• the compound e.g., a compound for conjugating with an analyte or binding partner of an analyte such as a peptide, a protein, or a macromolecule including an antibody
• the compound may have the structure of formula (IV): ( IV) wherein RFG is a reactive functional group for conjugating to the analyte or binding partner for an analyte, L is absent (i.e., it is a bond) or a linker, and ⁇ is a chemiluminescent acridinium comprising the structure: wherein “ R 1 is hydrogen, –R, –X, –R L –X b , –L 1 –R, –L 1 –X b , –Z, –R L –Z, –L 1 –Z, or –R L –L
• the concentration of the analyte in an unknown sample can then be calculated by comparing the signal generated by an unknown sample containing the macromolecular analyte, with the dose-response curve.
• the methodology of the attachment of binding molecules such as antibodies to solid phases typically involves a mixing of the requisite components to induce attachment.
• an antibody can be covalently attached to a particle containing amines on its surface by using a cross-linking molecule such as glutaraldehyde.
• the attachment may also be non- covalent and may involve simple adsorption of the binding molecule to the surface of the solid phase, such as polystyrene beads and microtiter plate.
• a reagent may be provided for the detection of an analyte comprising a chemiluminescent acridinium compound bound the analyte or binding partner.
• the reagent may have a concentration of detectable conjugate of less than 10 -3 M.
• the reagent may have a concentration of less than 10 -3 M (e.g., 10 -15 M to 10 -3 M chemiluminescent acridinium compound.
• the compound is provided in a reagent which further comprises a buffer.
• the sample derived from a mammal e.g., human
• the sample comprises saliva and/or blood and/or serum.
• the sample is saliva and/or blood and/or serum.
• the sample to be analyzed is subjected to a pretreatment to release analyte from endogenous binding substances such as, for example, plasma or serum proteins that bind the analyte.
• the release of the analyte from endogenous binding substances may be carried out, for example, by addition of a digestion agent or a releasing agent or a combination of a digestion agent and a releasing agent used sequentially.
• the digestion agent is one that breaks down the endogenous binding substances so that they can no longer bind the analyte.
• the conditions for conducting an assay on a portion of a sample in accordance with the principles described herein may include carrying out the assay in an aqueous buffered medium at a moderate pH, generally that which provides optimum assay sensitivity.
• the aqueous medium may be solely water or may include from 0.1 to 40 % by volume of a cosolvent.
• the pH for the medium may be in the range of 4 to 11, or 5 to 10, or 6.5 to 9.5, or 7 to 8.
• the medium may comprise proteins (e.g., albumins), organic solvents (e.g., formamide), quaternary ammonium salts, polyanions (e.g., dextran sulfate), binding enhancers (e.g., polyalkylene glycols), polysaccharides (e.g., dextran, trehalose), and combinations thereof.
• Triggering the chemiluminescence of the analogs may be performed by the addition chemiluminescent triggering reagents.
• the chemiluminescent triggering reagents may be acidic or basic. Multiple chemiluminescent triggering reagents may be added sequentially.
• Example 1 Quantum Yield [0102] Comparative chemiluminescence quantum yields were measured for new structures of acridinium esters. The increase in quantum yield from acridinium esters is one of several advantageous properties for acridinium esters exhibiting higher quantum yields relative to those exhibiting lower quantum yields. Chemiluminescence was measured in relative light units (RLU) on a luminometer. Quantum yields of acridinium esters are measured as the amount of chemiluminescence (RLU) per mole of acridinium ester.
• RLU relative light units
• Quantum yield is therefore the amount of observable chemiluminescence for a defined mass of acridinium ester.
• An increase of quantum yield from an acridinium ester increases its likelihood of detection particularly at diminishingly low masses of acridinium ester, for example, with low doses of analyte in immunoassays.
• An increase in the quantum yield of acridinium ester might consequently increase the sensitivity of immunoassays employing high quantum yield acridinium ester.
• Relative quantum yields were calculated as the ratio with that for HEGAE as the denominator. Relative quantum yields with values greater than one indicate enhancement of quantum yield with respect to HEGAE.
• Table 3 lists the relative quantum yields of novel ADO acridinium esters of the present invention, along with HEGAE and HQYAE for comparison.
• Table 3 [0104] Compounds ADOAE A (4) and ADOAE C (6) are structurally very similar. The only difference is that the former contains a five-membered ADO ring, and the latter has six- membered ADO ring at their respective 2- and 3- positions. However, the quantum yield of ADOAE A (4) is only 1/5 of that of ADOAE C (6).
• Example 2 Light Emission Wavelength [0105] The light emission spectra of new compounds were measured using PR-740 FSSS Spectro camera, which is capable of measuring light emission intensity over a wavelength range of 380 – 780 nm. The emission wavelengths of HEGAE and HQYAE were measured for comparison.
• the emission wavelength maxima ( ⁇ max) of the new acridinium esters are found to be 440 – 450 nm, which is longer than that of HEGAE (425 nm) and shorter than that of HQYAE (475 nm).
• All acridinium compounds were diluted with DMF and prepared in a 1 mg/mL stock solution.20 ⁇ L of aliquot was placed in a glass tube and further diluted with 250 ⁇ L of DMF. Next, 300 ⁇ L of flash reagent 1 was added to the sample and the glass tube was placed in front of the PR-740 FSSS Spectro camera. The emission spectrums of all compounds were recorded after the addition of 300 ⁇ L of flash reagent 2 in a 5 second time window.
• Emission spectra are given in FIGS.1A-E.
• Table 4 provides the measured ⁇ max for several acridinium esters of the present disclosure.
• the property of fast light emission kinetics is suitable for a short cycle of light detection, which is desirable for high throughput instruments.
• Thyroid Stimulating Hormone (TSH) assays [0109] AntiTSH-mAb conjugates of new acridinium esters 7-11 and 13-15 were prepared along with TSPAE (3) for comparison. TSPAE (3) is one of the best high quantum yield AEs used in assays having similar light output to HQYAE from U.S. Pat. Nos.
• Relative light units for each tested compound were measured at 10 different standards—each having a known concentration of TSH.
• TSH3-UL Lite reagent buffer 0.3mg/mL.
• Commercially available TSH3-UL reagents (REF 06491072 lot 332) were used for the study.
• the anti-FITC solid phase and FITC ancillary reagent from lot 332 were recovered and paired with each TSH-AE Lite reagent. The reagents were then assayed on ADVIA Centaur XPT (Equipment ID: B1072).
• the system automatically performs the following actions: • Dispenses 100 ⁇ L of sample (standards) into a cuvette. • Dispenses 50 ⁇ L of Ancillary Reagent and 50 ⁇ L of Lite Reagent and incubates for 2.75 minutes at 37°C. • Dispenses 200 ⁇ L of Solid Phase and incubates for 5.5 minutes at 37°C. • Separates, aspirates, and washes the cuvettes with Wash 1. • Dispenses 300 ⁇ L each of Acid Reagent (flash reagent 1) and Base Reagent (flash reagent 2) to initiate the chemiluminescent reaction. • Internal TSH3-UL Master Curve Standards lot 19031 were used as sample and mean RLUs calculated.
• chemiluminescence Stability is one of several advantageous properties of acridinium esters used as labels in immunoassays, which ensures that assay-derived clinical data do not change and become invalid over the lifetime of test kits.
• the main mechanism of chemiluminescence instability of acridinium esters in aqueous solution is hydrolysis of the phenolic ester by hydroxide anion and other nucleophiles.
• High quantum yield acridinium esters such as HQYAE and TSPAE contain two hydrophilic alkoxy groups at 2-and 7- positions and have been observed to be less stable than the un- substituted acridinium esters, due presumably to additional mechanisms of chemiluminescence instability.
• Comparative chemiluminescence stabilities were measured for new ADO acridinium esters conjugated to anti-hTSH monoclonal antibody through N-hydroxysuccinimide activation of the acridinium esters benzoic acid groups. The rate of chemiluminescence instability of acridinium esters was measured by the loss of chemiluminescence over a set period under conditions approximating the expected storage and handling conditions of assay test kits.
• Chemiluminescence reactions were initiated in the cuvettes with the sequential addition of 0.30 mL of flash reagent 1 followed 60s later by the addition of 0.30 mL of flash reagent 2.
• the chemiluminescence acquisition time was the nominal 3.500s.
• the dark count time was 2.000s.
• Chemiluminescence was reported as net chemiluminescence being the gross chemiluminescence minus adjusted dark counts.
• the residual chemiluminescence percentages were calculated and tabulated in relation to the initial chemiluminescence from the means of five replicates gathered from each time point. [0118] Two sets of experiments were performed.
• Tables 9 and 10 show the stabilities of several presentative ADO acridinium esters at three pH conditions (pH 6, 7, and 8) at 4°C (common reagent storage temperature, Table 9) and 37°C (accelerated temperature, Table 10) over 33 days for the first set of experiments and over 35 days for the second set of experiments. All ADOAEs showed better chemiluminescence stability than TSPAE at nominal 4°C storage. At 37°C where the instability of acridinium esters was accelerated by elevated temperature, all new ADOAEs revealed significantly better chemiluminescence stability than TSPAE. This is particularly apparent at pH 7 and pH 8; at this pH range most immunoassays are performed.
• the N-Arylation of 5-Methoxy-isatin was done on a 2 g scale with 12 (2.1g, 11mmol) using NaH (11 mmol) as a base and CuI (22 mmol) as a coupling agent in 8 h at 150 o C in DMF.
• the LC/MS analysis showed that the 70% N-Arylisatin proceeded with further rearrangement to Acridine 9-carboxylic acid (4C).
• DMF was removed from the reaction mixture with a high vacuum at 60 o C, added 10% KOH solution, and reflux continued for 2 h at 120 o C.
• the LCMS analysis confirmed the reaction intermediate was completely converted to 4C.
• N-sulfopropanation of 4E (50 mg, 0.11 mmol) was done in a microwave reactor at 155 o C with 10 eq. of 1,3-propane sultone in an ionic liquid 1-butyl-3- methylimidazolium hexafluorophosphate [BMIM][PF6] and 2,6-Di-tert-butylpyridine used as a base.
• BMIM][PF6] and 2,6-Di-tert-butylpyridine used as a base.
• the 60% of the reaction was completed in 6 h, at this stage 2 N HCl was added to the reaction mixture and continued stirring at 120 o C for 2 h afforded ADOAE A (4) with 14% overall yield.
• the active NHS-ester synthesis was prepared for protein conjugation.
• ADOAE A (9 mg, 0.015 mmol) was treated with TSTU and N, N-Diisopropylethylamine in DMF for 30 min at room temperature obtained the final ADOAE B (5) as a yellow solid.
• the synthesis of ADOAE C (6) and ADOAE D (7) started from the starting material 5-Methoxyisatin (4B) and Bromo derivative 6A.
• Example 8 Synthesis of ADOAE E (8) [0121] The synthesis of ADOAE E (8) synthesis began with an intermediate Acridine 9-ester (6C). The methyl ether cleavage of the 6C (400 mg, 0.084 mmol) was done using 10 eq BBr 3 (1M, CH2Cl2) at 0 o C in 5 h, obtained the hydroxy derivative 8A in 75.5% yield.
• reaction mixture was purified on prep-HPLC and the prep-HPLC fractions were lyophilized for 48 h to obtain the amine compound 9A with 79% yield.
• the reaction mixture was directly purified on HPLC, after the lyophilization of prep fractions to get the 6.2 mg of ADOAE F (9) as a yellow-colored compound.
• Second step the methyl ester hydrolysis was done with 2N HCl at 120 o C in 2 h and the acid compound was purified by using prep-HPLC, obtained 19 mg of the 10D in 20% yield.
• the HPLC purified material 10D (6mg, 0.001 mmol) was treated with 3 eq of TSTU and 2 eq of N, N-diisopropylethylamine in DMF. After 30 min, the reaction was purified on the prep-HPLC and obtained the 8 mg of ADOAE G (10).
• ADOAE H (11) started from the acid derivative 10D.
• the acid derivative (10 mg, 0.016 mmol) was coupled with the HEG diamine (13.4 mg, 0.048 mmol) via the acid-activated with TSTU followed by amide formation with HEG-diamine.
• the reaction was completed in 30 min.
• the crude product was directly purified on preparative- HPLC, obtained 2 mg of the terminal amine 11A.
• the crude product was purified on preparative-HPLC yielding the 2 mg of ADOAE H (11) in 64% yield.
• ADOAE I (12) and J (13) started from the commercially available 5-Methoxyisatin.
• the 5-Methoxyisatin 4B (2g, 13.6 mmol) was N-arylated with Bromo 12A (1 g, 5.64 mmol) using CuI, NaH in dry DMF for 12h at 150 o C, but here the maximum reaction (90%) further proceeded and rearranged to Acridine-9-carboxylic acid.
• DMF was removed from the reaction mixture under reduced pressure at 60 o C and the crude mixture was refluxed for 30 min with 10% KOH (10 mL). The crude acid product was acidified with conc.
• Second step the acridine 9- carboxylate 12C (100 mg, 0.205 mmol) was N-alkylated with 10 equivalents of 1,3-propane sultone in a microwave reactor, this reaction was monitored on LCMS, N-alkylation went to complete in 8 h.
• Second step the methyl ester hydrolysis was done with 2N HCl (10 mL) in a microwave reactor at 105 o C in 2 h and purified the acid compound by using prep-HPLC, obtained Acridinium NSP-DMAE-acid 12D in 17% yield.
• the NHS-ester intermediate mixture was transferred to a stirred mixture of Diamino-PEG6 and 4 eq of DIPEA in DMF at room temperature. After 2h, the mixture was purified on HPLC yielding the 3 mg of HEG-amine 13A in a 26% yield.
• Example 12 Synthesis of ADOAE K (14) and L (15) [0127]
• the synthesis of ADOAE K (14) began from the known commercially available isatin (14A) and 6-Bromo-1,4-benzo dioxane 6A.
• the Isatin (2g, 13.6 mmol) was N-arylation with Bromo 6A (4.36 g, 20.4 mmol) using CuI, NaH in dry DMF at150 o C in 12 h, but here the maximum reaction was further proceeded and completely rearranged to Acridine-9-carboxylic acid.
• DMF was removed from the reaction mixture under reduced pressure at 60 o C, and the crude acid product was acidified with conc.
• Second step the acridine 9-carboxylate 14C (120 mg, 0.127 mmol) was N-alkylated with 10 equivalents of 1,3-propane sultone in a microwave reactor, this reaction was monitored on LCMS, N-alkylation went to complete in 8 h.
• Second step the methyl ester hydrolysis was done with 2N HCl (10 mL) in a microwave reactor at 110 o C for 2 h. The resulted crude product was filtered through the sintered funnel and filtrate was purified on prep-HPLC, obtained Acridinium NSP-AE-acid 14D in 56% yield.
• the HEG-Amine synthesis was carried out from Acid 14D (30 mg, 0.054 mmol) in two steps.
• Step 1 20 mg of the acid derivative treated with TSTU (24 mg, 0.082 mmol) in DMF with 2 eg of DIPEA base, reaction completed in 30 min and it was confirmed by LCMS. At this stage, 50% of the reaction mixture was directly purified on prep-HPLC and isolated 6mg of ADOAE K (14).
• Step 2 the remaining 50% of the reaction mixture (step 1) reacted with Diamino-PEG6 and 4 eq of DIPEA in DMF. The mixture was purified on HPLC and lyophilized after the prep fraction was obtained pure material of 5mg of HEG-amine (15A).
• Step 1 The AE-acid 3 mg of each compound was separately activated with 2 eq of TSTU in DMF with 2 eq of DIPEA, and the reaction was completed in 30 min by LCMS mass analysis. At this stage, the NHS-activated mixture(AE-NHS ester) was transferred dropwise to a stirred mixture of 1.5 eq of HEG-diamine and DIPEA (2 eq) in DMF at 0 o C.
• Step 2 2 eq of (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) Sulfo-SMCC was added to the reaction mixture, raised the temperature to 40 o C, and the reaction was completed in 1 h by LCMS mass analysis. The resulting crude mixtures were directly purified on prep- HPLC.
• ADOAE R (21) The synthesis of ADOAE R (21) started from the acid derivative 8B.
• the acid derivative (5 mg, 0.009 mmol) was coupled with the HEG diamine (3.64 mg, 0.013 mmol) via the acid-activated with TSTU followed by amide formation with HEG-diamine.
• the reaction was completed in 30 min.
• the crude product was directly purified on preparative-HPLC and obtained 3 mg of the terminal amine 21A in a 43% yield.
• the final acridinium ester- maleimidylcyclohexanecarboxylate (AE-MCC) synthesis was done by reacting 21A (3 mg.
• the labeling reactions were transferred into an Amicon Ultra-4 30kDa molecular weight cut-off filter and diluted with 3 mL of deionized water.
• the filter was centrifuged at 5000 ⁇ G for 10 min to reduce the volume to ⁇ 0.2 mL. This process was repeated four more times.
• the final conjugate in ⁇ 0.2 mL was brought to 1 mL in total with deionized water to give a 2 mg/mL solution.
• the AE-antiTSH mAb protein concentrations were determined with a micro BCA assay.
• the acridinium ester incorporation onto the antiTSH mAb was measured through MALDI-TOF mass spectroscopy.

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