Mass tags for quantitative analyses

Abstract

This invention pertains to methods, mixtures, kits and/or compositions for the determination of analytes by mass analysis using unique labeling reagents or sets of unique labeling reagents. The labeling reagents can be isomeric or isobaric and can be used to produce mixtures suitable for multiplex analysis of the labeled analytes.

1 . A kit comprising at least two different compounds represented by the following formula:  
       
         
           
           
               
               
           
         
       
       or a salt form and/or hydrate form thereof, wherein independently for each different compound: 
 RG is a nucleophilic group or an electrophilic group, or a reaction product of an analyte with a nucleophilic group = or an electrophilic group;  
 r and t are both 0 or one of r and t is 1 and the other is 0;  
 S′ is a cleavable linker coupled to a solid support or an affinity ligand;  
 X and Y are each a bond, wherein X couples an atom or an optional substituent of each of RP and LK to thereby link RP to LK, and Y couples an atom or an optional substituent of LK to RG;  
 RP and LK are each optionally and independently substituted, wherein 
 RP and LK are each independently a heteroaryl or heterocycloalkyl, or a linear or branched aliphatic or heteroaliphatic group substituted or interrupted with a heteroaryl or heterocycloalkyl group; or  
 LK is a linking moiety and RP is a tertiary amine, a 4-9 membered nitrogenous heteroaryl or heterocycloalkyl bonded at a ring nitrogen to X, a 5-6 membered arylmethylene, a 5-6 membered heteroarylmethylene, or a 5-6 membered heterocycloalkyl; and  
 RP has a unique gross mass for each compound, and LK has a unique gross mass for each compound that compensates for the difference in unique gross mass between the RP for each compound such that the aggregate gross mass of the RP and LK for each compound is the same,  
 
 provided that; RP and LK are not both selected from the group consisting of amino acids, nucleotides, oligonucleotides, peptides, and proteins; and when t is 0, the group RP is not an optionally substituted 5, 6 or 7 membered heterocycloalkyl comprising a ring nitrogen atom that is N-alkylated with a substituted or unsubstituted moiety of the formula —C(J) 2 -LK′— such that LK′ is —C(O)—, —C(S)—, —C(NH)—, or —C(NRz)-, wherein Rz is an alkyl group comprising one to eight carbon atoms which may optionally contain a heteroatom or optionally substituted aryl group wherein the carbon atoms of the alkyl and aryl groups independently comprise linked hydrogen, deuterium and/or fluorine atoms and each J is the same or different and is H, deuterium (D), Rz, ORz, SRz, NHRz, N(Rz) 2 , fluorine, chlorine, bromine or iodine.  
 
     
     
         2 . The kit of  claim 1 , wherein all compounds of the kit are isobaric.  
     
     
         3 . The kit of  claim 2 , wherein the compounds are isobaric isomers.  
     
     
         4 . The kit of  claim 2 , wherein the compounds are isobaric isotopologues.  
     
     
         5 . The kit of  claim 1 , where each compound of the kit comprises a unique isotopically coded reporter.  
     
     
         6 . The kit of  claim 1 , wherein r and t are both 0.  
     
     
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         10 . The kit of  claim 6 , wherein at least one compound is represented by structural formula V:  
         RP 5 —X-LK 5 —Y—RG  V  
       wherein: 
 RP 5  is a reporter group and LK 5  is a linking moiety represented by structural formula E:  
                     
 wherein each n, independently, is an integer from 1 to 3; and  
 each R, independently, is H, D, an alkyl, a heteroalkyl, an aryl, a heteroaryl, or a halo group.  
 
     
     
         11 . The kit of  claim 10 , wherein at least one compound is represented by structural formula D:  
       
         
           
           
               
               
           
         
       
     
     
         12 . The kit of  claim 6 , wherein at least one compound is represented by structural formula I:  
         RP 1 —X-LK 1 —Y—RG  I  
       wherein: 
 RP 1  is a reporter group represented by structural formula A:  
                     
 wherein,  
 Ring A is aromatic;  
 each Z is independently CH, CR 2 , or N, provided that no more than two Z groups are N;  
 n is 1 or 2;  
 each R 2  is independently selected from hydrogen, deuterium, —OH, halogen, —CN, —NO 2 , alkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, heteroalkyl, heterocycloalkyl, —R 3 , or -T-R 3 ;  
 each R 3  is independently hydrogen, deuterium, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroalkyl, heterocycloalkyl, heteroaryl, or heteroaralkyl;  
 T is —O—, —NR 4 —, —S—, —C(O)—, —S(O)—, —SO 2 —, —NR 4 C(O)—, —C(O)NR 4 —, —NR 4 SO 2 —, —SO 2 NR 4 —, —C(O)O—, —OC(O)—, —NR 4 C(O)O—, or —OC(O)NR 4 —; 
 each R 4  is independently hydrogen, deuterium, alkyl, heteroalkyl, aryl, or aralkyl;  
 LK 1  is a linking moiety;  
 X is a bond between an atom of the reporter and LK 1 ; and  
 Y is a bond between an atom of the linker and an atom of RG,  
 wherein at least one of RP 1  and LK 1  is isotopically enriched with one or more heavy atom isotopes.  
 
 
     
     
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         29 . The kit of  claim 6 , wherein at least one compound is represented by Structural Formula III:  
         RP 3 —X-LK 3 —Y—RG  III  
       wherein: 
 RP 3  is a reporter group represented by structural formula C:  
                     
 wherein,  
 each of R x  and R y  is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, or heteroalkyl, wherein optional substituents for R x  and R y  are independently selected from hydrogen, deuterium, —OH, halogen, —CN, —NO 2 , —R 3 , -T-R 3 , ribose, deoxyribose or phosphate, or R x  and R y  are taken together to form a Ring C′:  
                     
 wherein,  
 ring C′ is heteroaryl or heterocycloalkyl, wherein the substituents for Ring C′ are independently hydrogen, deuterium, —OH, halogen, —CN, —NO 2 , alkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, heteroalkyl, —R 3 , -T-R 3 , ribose, deoxyribose or phosphate;  
 each R 3  is independently hydrogen, deuterium, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroalkyl, heterocycloalkyl, heteroaryl, or heteroaralkyl;  
 T is —O—, —NR 4 —, —S—, —C(O)—, —S(O)—, —SO 2 —, —NR 4 C(O)—, —C(O)NR 4 —, —NR 4 SO 2 —, —SO 2 NR 4 —, —C(O)O—, —OC(O)—, —NR 4 C(O)O—, or —OC(O)NR 4 —;  
 each R 4  is independently hydrogen, deuterium, alkyl, heteroalkyl, aryl, or aralkyl;  
 LK 3  is a linking moiety, provided that when R x  and R y  are taken together to form Ring C′, then the ring nitrogen that links R x  and R y  is linked to a group other than a substituted or unsubstituted moiety of the formula —C(J) 2 -LK′— such that LK′ is —C(O)—, —C(S)—, —C(NH)—, or —C(NRz)-, wherein Rz is is an alkyl group comprising one to eight carbon atoms which may optionally contain a heteroatom or optionally substituted aryl group wherein the carbon atoms of the alkyl and aryl groups independently comprise linked hydrogen, deuterium and/or fluorine atoms and J is the same or different and is H, deuterium (D), Rz, ORz, SRz, NHRz, N(Rz) 2 , fluorine, chlorine, bromine or iodine;  
 X is a bond between an atom of the reporter and LK 3 ; and  
 Y is a bond between an atom of the linker and an atom of RG,  
 wherein at least one of RP 3  and LK 3  is isotopically enriched with one or more heavy atom isotopes.  
 
     
     
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         34 . The kit of  claim 6 , wherein RP comprises an optionally substituted piperazinyl and LK is an aryl or cycloalkyl, or a linear or branched aliphatic or heteroaliphatic group substituted or interrupted with an aryl or cycloalkyl.  
     
     
         35 . A kit comprising a compound represented by the Structural Formula VI:  
         RP 6 —X-LK 6 —Y—RG  VI  or a salt form and/or hydrate form thereof,    wherein    RP 6  and LK 6  are each independently a heteroaryl or heterocycloalkyl, or a linear or branched aliphatic or heteroaliphatic group substituted or interrupted with a heteroaryl or heterocycloalkyl, wherein 
 at least one of RP 6  or LK 6  comprises an optionally substituted nucleobase, or a linear or branched aliphatic or heteroaliphatic group substituted or interrupted with an optionally substituted nucleobase;  
   optional substituents for RP 6  and LK 6  are independently selected from hydrogen, deuterium, —OH, halogen, —CN, —NO 2 , alkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, heteroalkyl, heterocycloalkyl, —R 3 , -T-R 3 , ribose, deoxyribose, or phosphate;    each R 3  is independently hydrogen, deuterium, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroalkyl, heterocycloalkyl, heteroaryl, or heteroaralkyl;    T is —O—, —NR 4 —, —S—, —C(O)—, —S(O)—, —SO 2 —, —NR 4 C(O)—, —C(O)NR 4 —, —NR 4 SO 2 —, —SO 2 NR 4 —, —C(O)O—, —OC(O)—, —NR 4 C(O)O—, or —OC(O)NR 4 —;    each R 4  is independently hydrogen, deuterium, alkyl, heteroalkyl, aryl, or aralkyl;    X is a bond between an atom of the reporter and LK 6 ; and    Y is a bond between an atom of the linker and an atom of RG,    wherein at least one of RP 6  and LK 6  is isotopically enriched with one or more heavy atom isotopes.    
     
     
         36 . The kit of  claim 35 , wherein only LK 6  is a nucleobase.  
     
     
         37 . The kit of  claim 6 , wherein at least one compound is represented by Structural Formula IV:  
         RP 4 —X-LK 4 —Y—RG  IV  
       wherein 
 RP 4  and LK 4  are each independently a heteroaryl or heterocycloalkyl, or a linear or branched aliphatic or heteroaliphatic group substituted or interrupted with a heteroaryl or heterocycloalkyl;  
 optional substituents for RP 4  and LK 4  are independently selected from hydrogen, deuterium, —OH, halogen, —CN, —NO 2 , alkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, heteroalkyl, heterocycloalkyl, —R 3 , -T-R 3 , ribose, deoxyribose, or phosphate;  
 each R 3  is independently hydrogen, deuterium, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroalkyl, heterocycloalkyl, heteroaryl, or heteroaralkyl;  
 T is —O—, —NR 4 —, —S—, —C(O)—, —S(O)—, —SO 2 —, —NR 4 C(O)—, —C(O)NR 4 —, —NR 4 SO 2 —, —SO 2 NR 4 —, —C(O)O—, —OC(O)—, —NR 4 C(O)O—, or —OC(O)NR 4 —;  
 each R 4  is independently hydrogen, deuterium, alkyl, heteroalkyl, aryl, or aralkyl;  
 X is a bond between an atom of the reporter and LK 4 ; and  
 Y is a bond between an atom of the linker and an atom of RG,  
 wherein at least one of RP 4  and LK 4  is isotopically enriched with one or more heavy atom isotopes;  
 provided that if RP 4  is a heterocycloalkyl, the heterocycloalkyl is not a 5, 6 or 7 membered heterocycloalkyl comprising a ring nitrogen atom that is N-alkylated with a substituted or unsubstituted moiety of the formula —C(J) 2 -LK′— such that LK′ is —C(O)—, —C(S)—, —C(NH)—, or —C(NRz)-, wherein Rz is an alkyl group comprising one to eight carbon atoms which may optionally contain a heteroatom or optionally substituted aryl group wherein the carbon atoms of the alkyl and aryl groups independently comprise linked hydrogen, deuterium and/or fluorine atoms and J is the same or different and is H, deuterium (D), Rz, ORz, SRz, NHRz, N(Rz) 2 , fluorine, chlorine, bromine or iodine.  
 
     
     
         38 . The kit of  claim 37 , wherein, the heteroaryl or heterocycloalkyl groups in RP 4  and LK 4  are each independently selected from optionally substituted imidazolyl, furyl, pyrrolyl, thienyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridazinyl, quinolyl, isoquinolinyl, indazolyl, benzoxazolyl, benzisooxazolyl, benzofuryl, benzothiazolyl, indolizinyl, imidazopyridinyl, pyrazolyl, triazolyl, isothiazolyl, oxazolyl, tetrazolyl, benzimidazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, benzoxadiazolyl, indolyl, tetrahydroindolyl, azaindolyl, imidazopyridyl, quinazolinyl, purinyl, pyrrolo[2,3]pyrimidyl, pyrazolo[3,4]pyrimidyl, benzo(b)thienyl, morpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, and thiomorpholinyl.  
     
     
         39 . The kit of  claim 38 , wherein for at least one compound, at least one of RP 4  or LK 4  comprises an optionally substituted piperazinyl, or a linear or branched aliphatic or heteroaliphatic group substituted or interrupted with piperazinyl.  
     
     
         40 . The kit of  claim 39 , wherein for at least one compound, RP 4  comprises an optionally substituted piperazinyl.  
     
     
         41 . The kit of  claim 38 , wherein at least one of RP 4  or LK 4  comprises an optionally substituted nucleobase, or a linear or branched aliphatic or heteroaliphatic group substituted or interrupted with an optionally substituted nucleobase.  
     
     
         42 . The kit of  claim 41 , wherein LK 4  comprises an optionally substituted nucleobase, or a linear or branched aliphatic or heteroaliphatic group substituted or interrupted with an optionally substituted nucleobase.  
     
     
         43 . The kit of  claim 42 , wherein for at least one compound, LK 4  comprises an optionally substituted nucleobase.  
     
     
         44 . The kit of  claim 43 , wherein the nucleobase is an optionally substituted 9H-purin-6-amine (adenine), 2-amino-1H-purin-6(9H)-one (guanine), 4-aminopyrimidin-2(1H)-one (cytosine), 5-methylpyrimidine-2,4(1H,3H)-dione (thymine) or pyrimidine-2,4(1H,3H)-dione (uracil).  
     
     
         45 . The kit of  claim 44 , wherein RP 4  comprises an optionally substituted piperazinyl.  
     
     
         46 . The kit of  claim 45 , wherein at least one compound is represented by a structural formula selected from:  
       
         
           
           
               
               
           
         
       
       wherein, 
 R 5  is —C(J) 2 -C(O)—, —C(J) 2 -C(S)—, —C(J) 2 -C(NH)—, or —C(J) 2 -C(NR)—, wherein 
 R z  is an alkyl group comprising one to eight carbon atoms that may optionally contain a heteroatom or optionally substituted aryl group wherein the carbon atoms of the alkyl and aryl groups independently comprise linked hydrogen, deuterium and/or fluorine atoms; and  
 each J is the same or different and is H, deuterium (D), Rz, ORz, SRz, NHRz, N(Rz) 2 , fluorine, chlorine, bromine or iodine  
 
 R 6  and R7 are each independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, heteroalkyl, heterocycloalkyl, —R 3 , -T-R 3 , ribose, deoxyribose, or phosphate; wherein 
 each R 3  is independently hydrogen, deuterium, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroalkyl, heterocycloalkyl, heteroaryl, or heteroaralkyl;  
 
 R 8  and R 9  are each independently H, deuterium (D), fluorine, chlorine, bromine, iodine, or a halogenated alkyl.  
 
     
     
         47 . The kit of  claim 46 , wherein at least one compound is:  
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         an isotopologue thereof.  
       
     
     
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         58 . A mixture comprising a plurality of labeled analytes represented by the following formula:  
       
         
           
           
               
               
           
         
       
       or a salt form and/or hydrate form thereof, wherein independently for each labeled analyte: 
 r and t are both 0 or one of r and t is I and the other is 0;  
 S′ is a cleavable linker coupled to a solid support or an affinity ligand;  
 X and Y are each a bond, wherein X couples an atom or an optional substituent of each of RP and LK to thereby link RP to LK, and Y couples an atom or an optional substituent of LK to -Analyte;  
 RP and LK are each optionally and independently substituted, wherein 
 RP and LK are are each independently a heteroaryl or heterocycloalkyl, or a linear or branched aliphatic or heteroaliphatic group substituted or interrupted with a heteroaryl or heterocycloalkyl; or  
 LK is a linking moiety and RP is a tertiary amine, a 4-9 membered nitrogenous heteroaryl or heterocycloalkyl bonded at a ring nitrogen to X, a 5-6 membered arylmethylene, a 5-6 membered heteroarylmethylene, or a 5-6 membered heterocycloalkyl; and  
 
 RP has a unique gross mass for each labeled analyte, and LK has a unique gross mass for each labeled analyte that compensates for the difference in unique gross mass between the RP for each labeled analyte such that the aggregate gross mass of the RP and LK for each labeled analyte is the same,  
 provided that; RP and LK are not both selected from the group consisting of naturally occurring amino acids, nucleotides, oligonucleotides, peptides, and proteins; and when t is 0, the group RP is not an optionally substituted 5, 6 or 7 membered heterocycloalkyl comprising a ring nitrogen atom that is N-alkylated with a substituted or unsubstituted moiety of the formula —C(J) 2 -LK′— such that LK′ is —C(O)—, —C(S)—, —C(NH)—, or —C(NRz)-, wherein Rz is an alkyl group comprising one to eight carbon atoms which may optionally contain a heteroatom or optionally substituted aryl group wherein the carbon atoms of the alkyl and aryl groups independently comprise linked hydrogen, deuterium and/or fluorine atoms and each J is the same or different and is H, deuterium (D), Rz, ORz, SRz, NHRz, N(Rz) 2 , fluorine, chlorine, bromine or iodine.  
 
     
     
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         61 . An isotopically enriched compound represented by the following formula:  
         RP—X-LK—Y—RG  
       or a salt form and/or hydrate form thereof, wherein: 
 RG is a nucleophilic group or an electrophilic group, or a reaction product of an analyte with a nucleophilic group or an electrophilic group;  
 X and Y are each a bond, wherein X couples an atom or an optional substituent of each of RP and LK to thereby link RP to LK, and Y couples an atom or an optional substituent of LK to RG;  
 RP and LK are each optionally and independently substituted, wherein 
 RP and LK are are each independently a heteroaryl or heterocycloalkyl, or a linear or branched aliphatic or heteroaliphatic group substituted or interrupted with a heteroaryl or heterocycloalkyl; or  
 LK is a linking moiety and RP is a tertiary amine, a 4-9 membered nitrogenous heteroaryl or heterocycloalkyl bonded at a ring nitrogen to X, a 5-6 membered arylmethylene, a 5-6 membered heteroarylmethylene, or a 5-6 membered heterocycloalkyl; and  
 
 at least two atoms of the compound are isotopically enriched with a heavy atom isotope,  
 provided that; RP and LK are not both selected from the group consisting of naturally occurring amino acids, nucleotides, oligonucleotides, peptides, and proteins; and when t is 0, the group RP is not an optionally substituted 5, 6 or 7 membered heterocycloalkyl comprising a ring nitrogen atom that is N-alkylated with a substituted or unsubstituted moiety of the formula —C(J) 2 -LK′— such that LK′ is —C(O)—, —C(S)—, —C(NH)—, or —C(NRz)-, wherein Rz is an alkyl group comprising one to eight carbon atoms which may optionally contain a heteroatom or optionally substituted aryl group wherein the carbon atoms of the alkyl and aryl groups independently comprise linked hydrogen, deuterium and/or fluorine atoms and each J is the same or different and is H, deuterium (D), Rz, ORz, SRz, NHRz, N(Rz) 2 , fluorine, chlorine, bromine or iodine.  
 
     
     
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         65 . A method comprising: 
 a) reacting two or more samples, each sample comprising one or more analytes, with a different labeling reagent to thereby produce two or more differently labeled samples each comprising one or more labeled analytes, wherein the labeling reagents are represented by the formula:                          or a salt form and/or hydrate form thereof, wherein independently for each labeling reagent:    RG is a nucleophilic group or an electrophilic group;    r and t are both 0 or one of r and t is 1 and the other is 0;    S′ is a cleavable linker coupled to a solid support or an affinity ligand;    X and Y are each a bond, wherein X couples an atom or an optional substituent of each of RP and LK to thereby link RP to LK, and Y couples an atom or an optional substituent of LK to RG;    RP and LK are each optionally and independently substituted, wherein 
 RP and LK are are each independently a heteroaryl or heterocycloalkyl, or a linear or branched aliphatic or heteroaliphatic group substituted or interrupted with a heteroaryl or heterocycloalkyl; or  
 LK is a linking moiety and RP is a tertiary amine, a 4-9 membered nitrogenous heteroaryl or heterocycloalkyl bonded at a ring nitrogen to X, a 5-6 membered arylmethylene, a 5-6 membered heteroarylmethylene, or a 5-6 membered heterocycloalkyl; and  
   RP has a unique gross mass for each labeled analyte, and LK has a unique gross mass for each labeled analyte that compensates for the difference in unique gross mass between the RP for each labeled analyte such that the aggregate gross mass of the RP and LK for each labeled analyte is the same; and    b) mixing two or more of the labeled samples, or a portion thereof, and optionally one or more calibration standards to thereby produce the mixture, 
 provided that; RP and LK are not both selected from the group consisting of naturally occurring amino acids, nucleotides, oligonucleotides, peptides, and proteins; and when t is 0, the group RP is not an optionally substituted 5, 6 or 7 membered heterocycloalkyl comprising a ring nitrogen atom that is N-alkylated with a substituted or unsubstituted moiety of the formula —C(J) 2 -LK′— such that LK′ is —C(O)—, —C(S)—, —C(NH)—, or —C(NRz)-, wherein Rz is an alkyl group comprising one to eight carbon atoms which may optionally contain a heteroatom or optionally substituted aryl group wherein the carbon atoms of the alkyl and aryl groups independently comprise linked hydrogen, deuterium and/or fluorine atoms and each J is the same or different and is H, deuterium (D), Rz, ORz, SRz, NHRz, N(Rz) 2 , fluorine, chlorine, bromine or iodine.  
   
     
     
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