US20050148771A1 - Active esters of N-substituted piperazine acetic acids, including isotopically enriched versions thereof - Google Patents

Active esters of N-substituted piperazine acetic acids, including isotopically enriched versions thereof Download PDF

Info

Publication number
US20050148771A1
US20050148771A1 US10/751,354 US75135404A US2005148771A1 US 20050148771 A1 US20050148771 A1 US 20050148771A1 US 75135404 A US75135404 A US 75135404A US 2005148771 A1 US2005148771 A1 US 2005148771A1
Authority
US
United States
Prior art keywords
compound
group
substituted
acetic acid
alkyl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/751,354
Other languages
English (en)
Inventor
Subhakar Dey
Darryl Pappin
Subhasish Purkayastha
Sasi Pillai
James Coull
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
DH Technologies Pte Ltd
Applied Biosystems LLC
Applied Biosystems Inc
Original Assignee
Applera Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to US10/751,354 priority Critical patent/US20050148771A1/en
Priority to US10/751,388 priority patent/US7307169B2/en
Priority claimed from US10/751,353 external-priority patent/US20050147982A1/en
Priority claimed from US10/751,387 external-priority patent/US7355045B2/en
Application filed by Applera Corp filed Critical Applera Corp
Assigned to APPLERA CORPORATION reassignment APPLERA CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: COULL, JAMES M., DEY, SUBHAKAR, PAPPIN, DARRYL J.C., PILLAI, SASI, PURKAYASTHA, SUBHASISH
Priority claimed from US10/822,639 external-priority patent/US20050147985A1/en
Priority claimed from US10/852,730 external-priority patent/US20050148087A1/en
Priority to AT05705033T priority patent/ATE479669T1/de
Priority to DE602005023260T priority patent/DE602005023260D1/de
Priority to JP2006547623A priority patent/JP4832312B2/ja
Priority to AU2005205522A priority patent/AU2005205522A1/en
Priority to PCT/US2005/000223 priority patent/WO2005068446A1/en
Priority to CA2552304A priority patent/CA2552304C/en
Priority to EP05705033A priority patent/EP1701945B1/de
Priority to EP10008184.3A priority patent/EP2251334B1/de
Publication of US20050148771A1 publication Critical patent/US20050148771A1/en
Priority to US12/001,734 priority patent/US7932388B2/en
Assigned to BANK OF AMERICA, N.A, AS COLLATERAL AGENT reassignment BANK OF AMERICA, N.A, AS COLLATERAL AGENT SECURITY AGREEMENT Assignors: APPLIED BIOSYSTEMS, LLC
Priority to US12/398,920 priority patent/US8569304B2/en
Assigned to APPLIED BIOSYSTEMS, LLC reassignment APPLIED BIOSYSTEMS, LLC MERGER (SEE DOCUMENT FOR DETAILS). Assignors: APPLIED BIOSYSTEMS INC.
Assigned to APPLIED BIOSYSTEMS INC. reassignment APPLIED BIOSYSTEMS INC. MERGER (SEE DOCUMENT FOR DETAILS). Assignors: APPLIED BIOSYSTEMS INC.
Assigned to APPLIED BIOSYSTEMS INC. reassignment APPLIED BIOSYSTEMS INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: APPLERA CORPORATION
Assigned to DH TECHNOLOGIES PTE. LTD. reassignment DH TECHNOLOGIES PTE. LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: APPLIED BIOSYSTEMS, LLC
Assigned to APPLIED BIOSYSTEMS, LLC reassignment APPLIED BIOSYSTEMS, LLC MERGER (SEE DOCUMENT FOR DETAILS). Assignors: APPLIED BIOSYSTEMS INC.
Assigned to APPLIED BIOSYSTEMS INC. reassignment APPLIED BIOSYSTEMS INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: APPLERA CORPORATION
Assigned to APPLIED BIOSYSTEMS, LLC reassignment APPLIED BIOSYSTEMS, LLC MERGER (SEE DOCUMENT FOR DETAILS). Assignors: APPLIED BIOSYSTEMS INC.
Priority to JP2010142103A priority patent/JP2010217197A/ja
Assigned to APPLIED BIOSYSTEMS, INC. reassignment APPLIED BIOSYSTEMS, INC. LIEN RELEASE Assignors: BANK OF AMERICA, N.A.
Assigned to APPLIED BIOSYSTEMS, LLC reassignment APPLIED BIOSYSTEMS, LLC CORRECTIVE ASSIGNMENT TO CORRECT THE RECEIVING PARTY NAME PREVIOUSLY RECORDED AT REEL: 030182 FRAME: 0677. ASSIGNOR(S) HEREBY CONFIRMS THE RELEASE OF SECURITY INTEREST. Assignors: BANK OF AMERICA, N.A.
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/46—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
    • C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
    • C07D241/06—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having one or two double bonds between ring members or between ring members and non-ring members
    • C07D241/08—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having one or two double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D253/00—Heterocyclic compounds containing six-membered rings having three nitrogen atoms as the only ring hetero atoms, not provided for by group C07D251/00
    • C07D253/08—Heterocyclic compounds containing six-membered rings having three nitrogen atoms as the only ring hetero atoms, not provided for by group C07D251/00 condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/02—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms containing only hydrogen and carbon atoms in addition to the ring hetero elements
    • C07D295/027—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms containing only hydrogen and carbon atoms in addition to the ring hetero elements containing only one hetero ring
    • C07D295/03—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms containing only hydrogen and carbon atoms in addition to the ring hetero elements containing only one hetero ring with the ring nitrogen atoms directly attached to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/14—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D295/145—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/15—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain

Definitions

  • this invention pertains to active esters of N-substituted piperazine acetic acid, including isotopically enriched versions thereof. In some embodiments, this invention pertains to methods for the preparation of active esters of N-substituted piperazine acetic acid, including isotopically enriched versions thereof.
  • this invention pertains to active esters of N-substituted piperazine acetic acid, including isotopically enriched versions thereof. In some embodiments, this invention pertains to methods for the preparation of active esters of N-substituted piperazine acetic acid, including isotopically enriched versions thereof. Active esters are well known in peptide synthesis and refer to certain esters that are easily reacted with an amine of an amino acid under conditions commonly used in peptide synthesis (For a discussion of active esters please see: Innovation And Perspectives In Solid Phase Synthesis, Editor: Roger Epton, SPCC (UK) Ltd, Birmingham, 1990).
  • the active esters of N-substituted piperazine acetic acid can be used as labeling reagents.
  • a set of isobaric labeling reagents can be prepared.
  • the set of isobaric labeling reagents can be used to label analytes, such as peptides, proteins, amino acids, oligonucleotides, DNA, RNA, lipids, carbohydrates, steroids, small molecules and the like.
  • the labeled analytes can be mixed together and analyzed simultaneously in a mass spectrometer.
  • each of the isobaric labeling reagents can be designed to result in the generation of a unique “signature ion” when analyzed in a mass spectrometer (MS)
  • labeled components of the mixture associated with each of the labeling reagents, and by implication components of each labeling reaction used to produce the mixture can be deconvoluted.
  • Deconvolution can include determining the relative and/or absolute amount of one or more labeled components in each of the individual samples that were labeled and combined to form the mixture.
  • the N-substituted piperazine acetic acid active esters described herein therefore can be powerful tools for analyte analysis, including but not limited to multiplex proteomic analysis.
  • FIG. 1 is an illustration of a synthetic scheme for the synthesis of N-methyl piperazines.
  • FIG. 2A is an illustration of a synthetic scheme for the synthesis of N-methyl piperazine acetic acids.
  • FIG. 2B is an illustration of another synthetic scheme for the synthesis of N-methyl piperazine acetic acids.
  • FIG. 2C is an illustration of yet another synthetic scheme for the synthesis of N-methyl piperazine acetic acids.
  • FIG. 3A is an illustration of a synthetic scheme for the synthesis of 18 O labeled N-methyl piperazine acetic acids.
  • FIG. 3B is an illustration of another synthetic scheme for the synthesis of 18 O labeled N-methyl piperazine acetic acids.
  • FIG. 4A is an illustration of a synthetic scheme for the synthesis of various active esters of N-methyl piperazine acetic acid.
  • FIG. 4B is an illustration of another synthetic scheme for the synthesis of various active esters of N-methyl piperazine acetic acid.
  • FIG. 4C is an illustration of yet another synthetic scheme for the synthesis of various active esters of N-methyl piperazine acetic acid.
  • FIG. 4D is an illustration of still another synthetic scheme for the synthesis of various active esters of N-methyl piperazine acetic acid.
  • FIG. 5A is an illustration of the heavy atom isotope incorporation pathway for the preparation of four isobaric N-methyl piperazine acetic acids.
  • FIG. 5B is an illustration of the labeling and fragmentation of peptides using four isobaric N-methyl piperazine acetic acid active ester labeling reagents.
  • analyte refers to a molecule of interest that may be determined.
  • analytes include, but are not limited to, proteins, peptides, nucleic acids (both DNA or RNA), carbohydrates, lipids, steroids and other small molecules with a molecular weight of less than 1500 Daltons (Da).
  • the source of the analyte, or the sample comprising the analyte is not a limitation as it can come from any source.
  • the analyte or analytes can be natural or synthetic.
  • sources for the analyte, or the sample comprising the analyte include cells or tissues, or cultures (or subcultures) thereof.
  • Non-limiting examples of analyte sources include, but are not limited to, crude or processed cell lysates, body fluids, tissue extracts, cell extracts or fractions (or portions) from a separations process such as a chromatographic separation, a 1D electrophoretic separation, a 2D electrophoretic separation or a capillary electrophoretic separation.
  • Body fluids include, but are not limited to, blood, urine, feces, spinal fluid, cerebral fluid, amniotic fluid, lymph fluid or a fluid from a glandular secretion.
  • processed cell lysate we mean that the cell lysate is treated, in addition to the treatments needed to lyse the cell, to thereby perform additional processing of the collected material.
  • the sample can be a cell lysate comprising one or more analytes that are peptides formed by treatment of the cell lysate with a proteolytic enzyme to thereby digest precursor peptides and/or proteins.
  • esters refers to both an ester and/or a thioester.
  • fragmentation refers to the breaking of a covalent bond.
  • fragment refers to a product of fragmentation (noun) or the operation of causing fragmentation (verb).
  • isotopically enriched means that a compound (e.g. labeling reagent) has been enriched synthetically with one or more heavy atom isotopes (e.g. stable isotopes such as Deuterium, 13 C, 15 N, 18 O, 37 Cl or 81 Br). Because isotopic enrichment is not 100% effective, there can be impurities of the compound that are of lesser states of enrichment and these will have a lower mass. Likewise, because of over-enrichment (undesired enrichment) and because of natural isotopic abundance, there can be impurities of greater mass.
  • heavy atom isotopes e.g. stable isotopes such as Deuterium, 13 C, 15 N, 18 O, 37 Cl or 81 Br.
  • labeling reagent refers to a moiety suitable to mark an analyte for determination.
  • label is synonymous with the terms tag and mark and other equivalent terms and phrases.
  • a labeled analyte can be referred to as a tagged analyte or a marked analyte.
  • natural isotopic abundance refers to the level (or distribution) of one or more isotopes found in a compound based upon the natural prevalence of an isotope or isotopes in nature.
  • a natural compound obtained from living plant matter will typically contain about 0.6% 13 C.
  • this invention pertains to a method for the production of isotopically enriched N-substituted piperazines, and the N-substituted piperazines themselves.
  • a partially protected amino acid can be condensed with an N-substituted amino acid ester wherein at least one of the two amino acids comprises a heavy atom isotope such as, for example, 18 O, 15 N, 13 C, 81 Br, 37 Cl or deuterium.
  • any side chain reactive groups can be protected as they would be for the condensation of amino acids to form peptides.
  • the condensation chemistry can be chosen from the various methods known for condensing amino acids. These include, but are not limited to, the use of carbodiimides (e.g. dicyclohexylcarbodiimide, DCC), active esters, mixed anhydride formation and the like.
  • the partially protected amino acid comprises an amine-protecting group (N-protecting group), such as tert-butyloxycarbonyl (t-boc); a well-known protecting group in peptide synthesis.
  • N-protecting group such as tert-butyloxycarbonyl (t-boc); a well-known protecting group in peptide synthesis.
  • the partially protect amino acid can comprise a side chain protecting where the amino acid comprises a reactive side chain moiety.
  • the amino acid can be any natural amino acid (e.g. glycine, alanine, lysine) or non-natural amino acid of basic structure: wherein Pg can be the N-protecting group.
  • Each group Z can be independently hydrogen, deuterium, fluorine, chlorine, bromine, iodine, an amino acid side chain, a straight chain or branched C1-C6 alkyl group that may optionally contain a substituted or unsubstituted aryl group wherein the carbon atoms of the alkyl and aryl groups each independently comprise linked hydrogen, deuterium or fluorine atoms, a straight chain or branched C1-C6 alkyl ether group that may optionally contain a substituted or unsubstituted aryl group wherein the carbon atoms of the alkyl and aryl groups each independently comprise linked hydrogen, deuterium or fluorine atoms or a straight chain or branched C1-C6 alkoxy group that may optionally contain a substituted or unsubstituted aryl group wherein the carbon atoms of the alkyl and aryl groups each independently comprise linked hydrogen, deuterium or fluorine atoms.
  • each Z is independently hydrogen, methyl or methoxy. In some embodiments, each Z is hydrogen, deuterium, fluorine, chlorine, bromine or iodine.
  • An alkyl ether group as used herein, can include one or more polyethylene glycol substituents. Similarly, the alkoxy group, as used herein, can comprise ether and/or polyethylene glycol substituents.
  • the N-protecting group can be an acid labile protecting group.
  • the N-protecting group can be a base labile protecting group.
  • the N-substituted amino acid ester can be any natural amino acid (e.g. glycine, alanine, lysine) or non-natural amino acid of basic structure: wherein Z is previously defined above.
  • the group Y can be a straight chain or branched C1-C6 alkyl group or a straight chain or branched C1-C6 alkyl ether group wherein the carbon atoms of the alkyl group or alkyl ether group each independently comprise linked hydrogen, deuterium or fluorine atoms.
  • Y is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl.
  • the group R can be a straight chain or branched C1-C6 alkyl group or a substituted or unsubstituted phenyl group, wherein the carbon atoms of the alkyl group or phenyl group each independently comprise linked hydrogen, deuterium or fluorine atoms.
  • the N-substituted amino acid ester is the ester (e.g. methyl or ethyl) of sarcosine, which is an ester of N-methyl glycine.
  • Every possible permutation of 15 N or 13 C labeled glycine is commercially available.
  • other natural amino acids are commercially available with one or more incorporated heavy atom isotopes.
  • glycine, and other amino acids, comprising one or more heavy atom isotopes are commercially available, these amino acids can be easily incorporated into the procedure for the production of N-substituted piperazines.
  • the amino acids comprising heavy atom isotopes can be N-protected using procedures well-known in peptide chemistry.
  • the amino acids can be N-protected with a 9-fluorenylmethoxycarbonyl (Fmoc) group or a t-boc group.
  • the amino acids comprising heavy atom isotopes can be N-alkylated and converted to an ester of the amino acid using well-known procedures.
  • heavy atom isotope containing starting materials for the preparation of N-substituted piperazines, as described herein, are either commercially available, or can be easily prepared from commercially available amino acids using no more than routine experimentation.
  • the two amino acids can be condensed to thereby produce an N-protected peptide dimer as an ester.
  • the N-protected peptide dimer ester can comprise one or more heavy atom isotopes via the incorporation of the one or more amino acids comprising one or more heavy atom isotopes.
  • the N-protected peptide dimer ester can comprise one heavy atom isotope, two heavy atom isotopes, three heavy atom isotopes, four heavy atom isotopes, five heavy atom isotopes or six heavy atom isotopes.
  • the N-protected peptide dimer ester can have the general formula: wherein Pg, R, Y and Z are previously defined.
  • the N-protected peptide dimer ester can then be cyclized to form a 6-membered cyclic dione.
  • Cyclization proceeds by removing the N-protecting group of the N-protected peptide dimer ester and driving the reaction of the deprotected amine with the ester group.
  • the reaction can be carried out under basic conditions and can be heated to speed production of the product.
  • the product of the cyclization can have the general formula: wherein Y and Z are previously defined.
  • the ketone groups of the cyclic dione can then be reduced to form the desired N-substituted piperazine comprising one or more heavy atom isotopes.
  • the reduction can be performed using a reducing agent, such as lithium aluminum hydride (LAH) or Red-Al (Sigma-Aldrich).
  • LAH lithium aluminum hydride
  • Red-Al Red-Al
  • the product in some embodiments being a volatile oil, can be optionally temporarily modified (e.g. protected) to aid in isolation.
  • piperazine comprises two basic nitrogen atoms
  • the product can, in some embodiments, be isolated as a mono or bis-acid salt.
  • the N-substituted piperazine comprising one or more heavy atom isotopes can be isolated as a mono-TFA salt, a mono-HCl salt, a bis-TFA salt or a bis-HCl salt.
  • FIG. 1 illustrates the application of the aforementioned general procedure to the production of N-methyl piperazine.
  • Examples 1-4 describe the application of the illustrated procedure to the production of three different N-methyl piperazines each comprising 1-3 heavy atom isotopes.
  • t-boc protected glycine (1) is condensed with sarcosine methyl ester (2) to thereby produce the dipeptide (3).
  • the t-boc group is removed and the dipeptide is cyclized to the cyclic dione (4).
  • the ketone groups of the dione are then reduced to produce N-methyl piperazine.
  • the N-methyl piperazine product can either be transiently protected (5) or can be obtained directly from the reduction (6).
  • the product can also be obtained as a salt (e.g. TFA salt (Z) or HCl (8)) of an acid.
  • N-substituted piperazine compounds unlabeled or labeled with one or more heavy atom isotopes
  • the present invention contemplates all possible isotopically enriched N-substituted piperazine compound comprising one or more heavy atom isotopes of the general formula: including all possible salt forms thereof, wherein Y and Z are previously defined.
  • this invention pertains to methods for the production of isotopically enriched N-substituted piperazine acetic as well as the isotopically enriched N-substituted piperazine acetic acids.
  • an N-substituted piperazine can be reacted with a halo acetic acid moiety comprising one or more heavy atom isotopes.
  • halo refers to the halogens, chlorine, bromine and iodine.
  • an N-substituted piperazine comprising one or more heavy atom isotopes can be reacted with a halo acetic acid moiety.
  • an N-substituted piperazine comprising one or more heavy atom isotopes can be reacted with a halo acetic acid moiety comprising one or more heavy atom isotopes.
  • the heavy atom isotopes found in the product N-substituted piperazine acetic acids can be introduced by way of the piperazine, by way of the halo acetic acid moiety or by way of both the piperazine and the halo acetic acid moiety.
  • 18 O can also be introduced into the carboxylic acid moiety of an N-substituted piperazine acetic acid by way of exchange with H 2 18 O.
  • N-substituted piperazines e.g. N-methyl and N-ethyl piperazine
  • Section I above describes the preparation of N-substituted piperazine comprising one or more heavy atoms from commercially available amino acids.
  • light and heavy by heavy we mean that the compound has been isotopically enriched with one or more heavy atom isotopes
  • N-substituted piperazine can be used to produce the N-substituted piperazine acetic acids comprising one or more heavy atom isotopes.
  • halo acetic acid moieties are commercially available.
  • the halo acetic acid moiety to be reacted with the N-substituted piperazine can be purchased as the carboxylic acid or as an ester of the carboxylic acid (e.g. the methyl ester, ethyl ester or phenyl ester). If only the carboxylic acid is available and the ester is desired, the ester can be prepared using well-known esterification methods. If only the ester is available and the carboxylic acid is desired, the ester can be hydrolyzed to produce the carboxylic acid.
  • Either the carboxylic acid or the ester can be used in the alkylation reaction provided that an additional equivalent of base is required if the carboxylic acid is used. If the ester is used to perform the alkylation, the product ester can be hydrolyzed to produce the N-substituted piperazine acetic acid.
  • General structures for the carboxylic acid and the ester compounds that can be used to alkylate N-substituted piperazines are: wherein Z and R are defined above.
  • Hal is a halogen (Cl, Br or I) and X is oxygen (O) or sulfur (S). In some embodiments, X is 16 O or 18 O.
  • One or more of the atoms of the halo acetic acid compound can be a heavy atom isotope.
  • the alkylation of an N-substituted piperazine with a halo acetic acid moiety proceeds under basic conditions.
  • the base need only be strong enough to deprotonate piperazine but can be selected to not substantially react with the halo acetic acid moiety.
  • two or more equivalents of N-substituted piperazine can be used, as N-substituted piperazine is a base. If it is desirable to use only one equivalent of N-substituted piperazine (for example, when the N-substituted piperazine is labeled with one or more heavy atom isotopes and is therefore valuable), other bases can be used.
  • Suitable bases include, but are not limited to, hindered bases such as triethylamine (Et 3 N) and diisopropylethyamine (DIEPA). Other suitable bases in sodium carbonate and potassium carbonate. Hindered bases are a good choice because they do not react substantially with the halo acetic acid moiety.
  • a solid phase base such as 1,5,7-Triazabicyclo[4.4.0]dec-5-ene (TBD) bound to polystyrene crosslinked with 2% DVB, Capacity (base): ⁇ 2.6 mmol/g (ss-TBD, Fluka, P/N 90603) can also be used (See FIG. 2B ).
  • a solid phase base has the advantage that it is easily, and completely, removed from the product by filtration once the alkylation reaction has been completed. Accordingly, the resulting product is not contaminated with salt of the base.
  • the reaction can produce a product of the general formula: or a salt thereof, wherein X, Y and Z have been previously defined.
  • One or more atoms of the N-substituted piperazine acetic acid can be a heavy atom isotope.
  • the reaction will produce an ester of the general formula: or a salt thereof, wherein R, X, Y and Z have been previously defined.
  • One or more atoms of the N-substituted piperazine acetic acid ester can be a heavy atom isotope.
  • the N-substituted piperazine acetic acid ester can be converted to the N-substituted piperazine acetic acid of general formula: by hydrolysis of the ester.
  • base can be added as required to induce the hydrolysis of the ester to the carboxylic acid, but in some embodiments it will not be required.
  • Hydrolysis can also be performed under aqueous acidic conditions.
  • N-substituted piperazine acetic acid is zwitterionic. Because it comprises a carboxylic acid group (or thio acid group) and two basic nitrogen atoms, it can exist in at least four different forms. It can exist completely deprotonated as its carboxylate anion. It can exist as its mono protonated zwitterion. It can exist as a monobasic salt (e.g. mono-TFA or mono-HCl salt). It can also exist as its dibasic salt (e.g. bis-TFA or bis-HCl salt). The state of protonation of the product is a function of the conditions under which it was isolated. All protonation states of N-substituted piperazine acetic acid are contemplated as embodiments of the present invention.
  • FIGS. 2A and 2B With reference to FIGS. 2A and 2B , as well as Examples 5 and 6, respectively, the production of two different isotopically enriched N-methyl piperazine acetic acid compounds is described.
  • FIG. 2A and Example 5 two equivalents of commercially available unlabeled N-methyl piperazine is reacted with ethyl bromoacetate to produce a N-methyl piperazine acetic acid compound comprising two 13 C atoms. Because N-methyl piperazine is basic, hydrolysis of the ethyl ester proceeded by merely heating the compound in an aqueous solution.
  • the starting piperazine is a bis-TFA salt of 15 N labeled N-methyl piperazine.
  • Acid salts of the piperazine base can be alkylated so long as sufficient base is added to the reaction to deprotonate piperazine.
  • the ethyl bromoacetate is 13 C labeled. Because both the piperazine and acetic acid reactants comprise heavy atom isotopes, a solid phase base was chosen so that only one equivalent of each reactant was required to produce the product.
  • hydrolysis of the ethyl ester proceeded by mere heating the compound in an aqueous solution.
  • the N-substituted piperazine acetic acid can be assembled on a solid support.
  • the halo acetic acid moiety as a carboxylic acid, can be reached with trityl chloride resin to thereby produce a support bound halo acetic acid.
  • the support bound halo acetic acid can then be treated with the desired N-substituted piperazine (e.g. N-methyl piperazine) under basic conditions to thereby produce the N-substituted piperazine acetic acid.
  • the desired N-substituted piperazine e.g. N-methyl piperazine
  • Isotopically enriched N-methyl piperazine and halo acetic acid moieties can be used, including 18 O labeled compounds although 18 O labeling can involve special considerations and is discussed in more detail below.
  • a heavy atom isotope can be incorporated at virtually any position of the N-substituted piperazine acetic acid, including 18 O incorporation that will be discussed in more detail below. Consequently, the present invention contemplates all possible isotopically enriched N-substituted piperazine acetic acids comprising one or more heavy atom isotopes of the general formula: including all possible salt forms thereof. III. Incorporation of 18 O Into N-Substituted Piperazine Acetic Acids
  • this invention pertains to methods for the incorporation of 18 O into N-substituted piperazine acetic acids as well as to the 18 O labeled N-substituted piperazine acetic acids themselves.
  • incorporation of 18 O is not substantially different as compared with the methods described for the preparation of isotopically labeled N-substituted piperazine acetic acids in Section II, above.
  • incorporation of 18 O is substantially different and takes advantage of the very caveat that creates some concern about the methods previously discussed.
  • the 18 O labeled N-substituted piperazine acetic acid was obtained by alkylation with an appropriately 18 O labeled halo acetic acid moiety.
  • the procedure is essentially as outlined in Section II, above except that an acid labile ester of the halo acetic acid was used in the alkylation reaction.
  • the halo acetic acid moiety comprised the formula: wherein Hal is previously defined and R′ is an acid labile ester group, including but not limited to tert-butyldimethylsilyl or t-boc.
  • the tert-butyldimethylsilyl (TBDMS) ester of ( 18 O) 2 bromoacetic acid (14) was used in the alkylation reaction.
  • This ester was prepared using 18 O labeled bromoacetic acid (13), obtained as a custom order from Cambridge Isotope Laboratory, Inc., and TBDMS-CN.
  • the TBDMS ester of N-methyl piperazine acetic acid (15) was the product of the alkylation with N-methyl piperazine.
  • the TBDMS ester was selected so that it could be converted to the acid chloride with, for example, oxalyl chloride thereby avoiding the requirement for any water and the possible exchange of 18 O with 16 O.
  • the alkylation to produce N-substituted piperazine acetic acid proceeded as described in Section II, above and the 18 O was later incorporated.
  • 18 O could be incorporated into the carboxylic acid group of any N-substituted piperazine acetic acid by treatment of the N-substituted piperazine acetic acid with H 2 18 O under acidic conditions.
  • FIG. 3B and Example 9 it was found that 18 O could be incorporated into the carboxylic acid group of any N-substituted piperazine acetic acid by treatment of the N-substituted piperazine acetic acid with H 2 18 O under acidic conditions.
  • the isotopic purity of the product could be increased by repeated cycles of treatment with H 2 18 O under acidic conditions.
  • the higher the state of enrichment of the H 2 18 O the fewer cycles required to produce highly 18 O enriched N-substituted pipe razine acetic acid.
  • H 2 18 O of 99% purity was used, the isotopic enrichment of N-substituted piperazine acetic acid was typically 96% after two cycles. Because this exchange was performed under acidic conditions, the product was easily isolated as the bis-acid salt of N-substituted piperazine acetic acid (e.g. the bis-TFA or bis-HCl salt).
  • the present invention contemplates all possible isotopically enriched N-substituted piperazine acetic acids comprising one or more heavy atom isotopes of the general formula: including all possible salt forms thereof.
  • this invention pertains to methods for the preparation of active esters of N-substituted piperazine acetic acid, including isotopically enriched versions thereof, as well as the N-substituted piperazine acetic acid esters themselves, and isotopically enriched versions thereof.
  • the active ester can be any active ester.
  • the active ester can be formed using an alcohol or thiol of the following formula: wherein X is O or S, but preferably O.
  • the active ester can be formed using an alcohol or thiol of the following formula: wherein X is O or S, but preferably O.
  • the active ester can be prepared through an intermediary imidazolide.
  • an N-substituted piperazine acetic acid ester including isotopically enriched versions thereof, can be converted to the imidazolide.
  • the imidazolide so prepared can then be reacted with the alcohol of choice to thereby produce the active ester of the selected alcohol.
  • the imidazolide (20) was then reacted with either 2,2,2-trifluorethanol or 1,1,1,3,3,3-hexafluoro-2-propanol to produce the desired active ester of N-methyl piperazine acetic acid (21) or (22), respectively as a bis-acid salt.
  • the active ester can be prepared by conversion of the N-substituted piperazine acetic acid, including isotopically enriched versions thereof, to an acid chloride followed by subsequent reaction of the acid chloride with the alcohol of choice to thereby produce the active ester of the selected alcohol.
  • N-methyl piperazine acetic acid is treated with oxalyl chloride to produce the acid chloride (23).
  • the acid chloride is then treated with either of NHP or NHS and solid phase base to thereby produce the active ester (24) or (25), respectively as the free piperazine base (not as an acid salt).
  • FIG. 4B also illustrates the application of oxalyl chloride to the production of the pentafluorophenyl (Pfp) ester (26) wherein a solution phase base (e.g. triethylamine) is used.
  • a solution phase base e.g. triethylamine
  • the reaction proceeded well with the solution phase base but the hydrochloride salt of the base proved difficult to remove.
  • Application of the solid phase base avoids this caveat.
  • the active ester can be prepared by treatment of the N-substituted piperazine acetic acid, including isotopically enriched versions thereof, with a trihalooacetate ester of the alcohol that is desired to form the active ester of the N-substituted piperazine acetic acid.
  • halo refers to fluorine, chlorine, bromine and iodine but preferably to fluorine and chlorine.
  • the trihalooacetate ester has the general formula: wherein Hal refers to a halogen (fluorine, chlorine, bromine or iodine) and LG refers to the leaving group alcohol.
  • the leaving group (LG) of the trihaloacetate esters can have the following general formula: wherein X is O or S, but preferably O. Active esters of N-methyl piperazine acetic acid comprising these leaving groups (LG) were successfully prepared using the identified trifluoroacetate esters (where X is O).
  • This procedure can be applied to the N-substituted piperazine acetic acids whether they are the acid salt or the zwitterion form.
  • the N-substituted piperazine acetic acids can be reacted with the triholoacetate ester of the alcohol to thereby produce the active ester of the N-substituted piperazine acetic acid.
  • a base that can deprotonate the basic nitrogen atoms of piperazine ring can be added to the reaction as need to induce formation of the product when the starting material is an acid salt of N-substituted piperazine acetic acid.
  • the active ester of the N-substituted piperazine acetic acid can itself be isolated as the mono-acid salt or the di-acid salt.
  • the product can be: or a salt thereof, wherein X, Y and Z are previously defined.
  • the group LG is the leaving group of the active ester that is displaced by the reactive group of an analyte to be labeled; in essence the leaving group is the alcohol used to form the active ester.
  • trihaloacetate esters are commercially available.
  • the trifluoracetate esters of pentafluorphenol and 4-nitrophenol can be purchased from commercial sources. However, the others can be obtained by reacting the desired alcohol with trihaloacetic anhydride.
  • the trifluoroacetate esters of Pcp, Dhbt, NHS, 3-NP and NHP were prepared by reacting the respective alcohol with trifluoracetic anhydride. The general procedure for such reactions can be found in Example 12.
  • Other alcohols that can be used to produce trihaloacetate esters suitable for the formation of other active esters can also be used.
  • FIG. 4C illustrates the production of the 114 and 115 labeling reagents as the NHS ester. Accordingly, the procedure was successfully applied to the production of isotopically enriched active esters of N-substituted piperazine acetic acids. These active ester reagents were produced as the bis-HCl salts from the bis-HCl salts of the piperazine base.
  • FIG. 4D illustrates the production of numerous other active esters of N-methyl piperazine acetic acid that were produced using this generic process. As will be appreciated by the ordinary practitioner, this procedure is generic and robust and can be applied to the production of numerous other active esters of a plethora of N-substituted piperazine acetic acid derivatives.
  • FIG. 5A illustrates the general pathway taken to the production of a set of four isobaric labeling reagents identified as 114, 115, 116 and 117. These designations are based upon the “signature ion” each reagent produces upon fragmentation in a mass spectrometer ( FIG. 5B ).
  • the “signature ion” can be used to deconvolute information associated with different samples in a multiplex assay as discussed in the Introduction.
  • the pathways illustrated in FIG. 5A utilize the procedures set forth above for the production of N-substituted piperazine acetic acids, and active esters thereof.
  • suitable isotopically labeled glycines were used in the preparation of suitable isotopically labeled N-substituted piperazines (e.g. N-methyl piperazines).
  • the labeled and unlabeled N-methyl piperazines can be treated with isotopically labeled bromoacetic acid derivatives, with or without subsequent 18 O enrichment to thereby produce the N-methyl piperazine acetic acid compounds of desired structure.
  • These suitably labeled N-methyl piperazine acetic acid compounds were used as labeling reagents; in the present case by conversion to an active ester for coupling with analytes such as peptides.
  • All four of the labeling reagents (114, 115, 116 and 117) were produced as NHS esters. All four reagents were used to label peptides, including peptides (analytes) obtained from digested protein. The set of reagents (two or more of them), were shown to be suitable for the multiplex analysis, including proteome analysis, as described in copending and co-owned application Ser. No. 60/443,612, incorporated herein by reference.
  • two or more samples containing digested peptides as the analyte each sample being labeled with one of the isobaric labeling reagents (114, 115, 116 or 117), were mixed to form a mixture that was analyzed in a tandem mass spectrometer.
  • selected ions of a particular mass representing a mixture of fragment ions of the same analyte labeled with two or more different isobaric labels, were subjected to dissociative energy causing fragmentation of the selected ions.
  • the selected ions, and the fragments thereof, were then re-analyzed in the mass spectrometer wherein signature ions of the isobaric labeling reagents used to label the analytes, as well as daughter ions of the analyte, were observed.
  • the various N-substituted piperazines, N-substituted piperazine acetic acids and active esters of N-substituted piperazine acetic acid can be prepared with starting materials of greater than 80 percent isotopic purity of for each heavy atom isotope.
  • the isotopic purity can be greater than 93 percent for each heavy atom isotope in some starting materials. In other starting materials the isotopic purity can be greater than 96 percent for each heavy atom isotope. In still other starting materials the isotopic purity can be greater than 98 percent for each heavy atom isotope.
  • this invention pertains to N-substituted piperazines, N-substituted piperazine acetic acids and/or active esters of N-substituted piperazine acetic acid having an isotopic purity of at least 80 percent for each heavy atom isotope.
  • this invention pertains to N-substituted piperazines, N-substituted piperazine acetic acids and/or active esters of N-substituted piperazine acetic acid having an isotopic purity of at least 93 percent for each heavy atom isotope. In still some other embodiments, this invention pertains to N-substituted piperazines, N-substituted piperazine acetic acids and/or active esters of N-substituted piperazine acetic acid having an isotopic purity of at least 96 percent for each heavy atom isotope.
  • this invention pertains to N-substituted piperazines, N-substituted piperazine acetic acids and/or active esters of N-substituted piperazine acetic acid having an isotopic purity of at least 98 percent for each heavy atom isotope.
  • Isotopically enriched starting materials were generally obtained from either Isotec (a Sigma-Aldrich company) or Cambridge Isotope Laboratories (Andover, Mass.). Generally, the most highly enriched starting materials were obtained and used in the production of the isotopically enriched piperazine derivatives.
  • the state of isotopic enrichment of starting materials is a choice which the ordinary practitioner will appreciate strikes a balance between the price of the starting materials (wherein the higher the state of isotopic enrichment, the higher the price) and requirement for purity of the enriched isotopes in the final product. Accordingly, the ordinary practitioner will appreciate that the most practical method of synthesis of isotopically enriched compounds may not always proceed through the most common synthetic routes. Indeed, there may be two or more different routes to the different isotopic variants of the same compound. Thus, for some reactions and/or compounds described herein, various synthetic routes have been undertaken and are therefore discussed below. Certain advantages and caveats pertaining to these routes are also discussed.
  • N-methyl piperazine (a.k.a. 1-methyl piperazine) is commercially available from a variety of sources. However, no source for any type of isotopically enriched N-methyl piperazine (as a stock item) could be found. It was determined however that suitably protected glycine and sarcosine could be condensed, cyclized and the product (a diketone) could be reduced to thereby produce N-methyl piperazine (See FIG. 1 ). Furthermore, it was determined that all possible permutations of 15 N and 13 C isotopically labeled glycine, as well as partially protected versions thereof (e.g.
  • t-boc protected amino acids were commercially available from sources such as Isotec or Cambridge Isotope Laboratory, Inc. Accordingly, this appeared to be a promising route to various N-methyl piperazine compounds comprising one or more heavy atoms. Because appropriately protected (t-boc) isotopically enriched glycines and suitably protected sarcosine can be purchased from commercial sources and because the protection of amino acids, such as glycine and sarcosine, are well-known, this discussion of the synthetic route to N-methyl piperazine begins with the suitably protected amino acids ( FIG. 1 ).
  • Sarcosine is commercially available as either the methyl or the ethyl ester. Either can be used in the condensation reaction.
  • reaction was stirred overnight. The reaction was monitored by thin layer chromatography (TLC). If t-boc-glycine was still present, additional DCC in DCM was added. When the reaction was determined to be complete, the solids were filtered off and the cake was rinsed with DCM. The product containing solution was then evaporated to dryness.
  • TLC thin layer chromatography
  • the product was purified by silica gel chromatography using a column packed in 50% ethyl acetate (EtOAc)/hexane. A small amount of the 50% EtOAc/hexane solution was used to dissolve/suspend the dried down product (not all will dissolve). This solution/slurry was loaded onto the packed column. The column was eluted 50% EtOAc/hexane to obtain the minimally retained product. Product containing fractions were evaporated to provide an oil, speckled oil, or flaky solid (materials that are higher in heavy atom isotope content appeared to exhibit more characteristics of a solid).
  • EtOAc ethyl acetate
  • reaction mixture was removed, diluted with water, and the pH of the solution was determined. If the pH was below 8, more potassium carbonate was added. Once the pH was confirmed to be greater than 8, the reaction was allowed to reflux overnight. The warm reaction mixture was then passed through a plug of Celite to remove the excess salts. The cake was rinsed twice with anhydrous ethanol. The filtrate was transferred to a larger flask and stripped to dryness. The product foam was redissolved in 9:1 ethyl acetate-methanol and passed through a plug of silica-gel. The silica-gel was then washed with ⁇ 4 column volumes of 9:1 ethyl acetate-methanol. All fractions were evaporated to dryness.
  • a saturated solution of sodium sulfate was prepared. Tetrahydrofuran (THF) (4 mL per mmol starting material based upon the material used in Example 2) was added to the diketopiperazine formed using the procedure of Example 2.
  • THF Tetrahydrofuran
  • LAH solution 1M LiAlH 4 in THF
  • the reaction was heated to reflux for 4 hours. After the reaction was complete, the solution was cooled to room temperature and the remaining LAH was quenched with the very slow addition of saturated aqueous sodium sulfate (1 ⁇ 4 the volume of the LAH solution added). The reaction appeared as a gray suspension.
  • TFA was added to the product residue ( ⁇ 2 mL/g starting material) to form a free flowing solution.
  • the TFA solution was transferred to a centrifuge tube and diethyl ether was added to precipitate the product salt.
  • the solution was mixed using a vortex.
  • the solution was then centrifuged and the supernatant decanted to collect the precipitate.
  • the filtrate was then washed 1 time with ether by resuspending the product, vortexing, and re-centrifugation. Product was dried under low vacuum to remove residual ether.
  • Example 2 The product of the procedure of Example 2 was dissolved in anhydrous THF (5 mL per mmol SM) in a multi-neck RBF fitted with condenser, addition funnel and argon (Ar) inlet. To this solution was added 3 equivalent of the LAH solution slowly through a dropping funnel at RT under Ar. Vigorous hydrogen evolution was observed at the beginning. After addition, the cloudy solution was heated to reflux for 3 hours. TLC was used to determine when the reaction was complete (disappearance of starting material (SM), 10% MeOH/DCM TLC developing solvent, PMA as visualizer).
  • SM starting material
  • PMA as visualizer
  • FIG. 5A illustrates the pathway for the synthetic incorporation of heavy atom isotopes into four isobaric labeling reagents referred to herein as 114, 115, 116 and 117.
  • certain of the heavy atom isotopes can be incorporated by the choice of the commercially available isotopically labeled glycine used in the production of the N-methyl piperazine.
  • Certain other heavy atoms can be incorporated during the alkylation reaction based upon the choice of the commercially available bromoacetic acid.
  • the 18 O can be incorporated through an efficient exchange using 18 O labeled water.
  • the labeling reagents are designated 114, 115, 116 and 117 based upon the mass of the fragment that forms a signature ion in the mass spectrometer (see: FIG. 5A and FIG. 5B ) once the reagent has been fragmented by the application of dissociative energy.
  • scheme A is useful for producing the N-methyl piperazine acetic acid as a zwitterion and not as a salt (e.g. mono or bis TFA or HCl salt).
  • scheme B is useful since it requires the use of only one equivalent of N-methyl piperazine for the production of the N-methyl piperazine acetic acid thereby foreclosing the waste of the valuable isotopically labeled starting material.
  • scheme C is useful for alkylations involving the isotopically labeled bromoacetic acid, particularly the 18 O labeled bromoacetic acid as it was expected to reduce the occurrence of 18 O scrambling (or exchange with 16 O from residual water).
  • This procedure utilizes unlabeled N-methyl piperazine. This procedure is useful for producing the zwitterion of N-methyl piperazine acetic acid.
  • the product can also be isolated as the mono or bis-HCl or mono or bis-TFA salt by treatment with the appropriate acid prior to or subsequent to its isolation as described above.
  • the product can also be isolated as the bis-HCl or bis-TFA salt by treatment with the appropriate acid prior to or subsequent to its isolation as described above.
  • N-methyl piperazine (0.57 mL, 5 mmol) in DMF (5 mL) for 30 minutes and then washed with DMF and dichloromethane (3 ⁇ 4 mL each).
  • N-MPA N-methyl piperazine
  • the N-MPA so generated on resin was cleaved with a 25% solution of TFA in dichloromethane (10 mL for 5 min)) and resin was washed with the same solution (2 ⁇ 5 mL). After evaporation of TFA, the product was precipitated and washed with ether (388 mg, 99% yield, bis TFA salt).
  • the product could also be isolated as its bis-HCl salt if HCl was used to cleave the product from the support rather than TFA.
  • Other acids could also be used for the cleavage reaction and product would be the salt of the acid used.
  • the product was used without further purification in the production of active ester of the N-methyl piperazine acetic acid.
  • the bis-TFA salt was also produced using the above-described procedure wherein TFA was substituted for HCl.
  • N-methyl piperazine phenyl ester was prepared by the alkylation procedures described above (See FIGS. 2A and 2B ) wherein phenyl bromoacetate is substituted for ethyl bromoacetate.
  • N-methyl piperazine acetic acid (N-MPAA) (79 mg, 0.5 mmol) in DCM (25 mL) was added a solution of oxalyl chloride (4 mL, 0.8 mmol, 2.0 M solution in DCM) over 10 minute at room temperature. After another 30 minutes of reaction, solvent and excess reagent were removed under reduced pressure to give a white solid (23).
  • a solution of NHS (57 mg, 0.5 mmol) in DCM (25 mL) was added to the solid followed by ss-TBD (390 mg, 1 mmol, 2.6 mmol/g). The resulting solution was sonicated for 5 minute when all solid dissolved.
  • the ss-TBD resin was removed by filtration and solvent was evaporated to yield a white foam (97% yield). Product was characterized by ES-MS as before.
  • N-methyl piperazine acetic acids N-MPAAs
  • conversion of the N-methyl piperazine acetic acids (N-MPAAs) to their active esters via the trifluoracetate ester is typically a two-step process. Except for the rare case where the reagent is commercially available (See: Table 1), the first step involves the preparation of a reagent for esterifying the acetic acid. The second step involves reacting the esterifying reagent with the N-methyl piperazine acetic acid to produce the active ester.
  • Various active esters were produced and tested for the aqueous labeling of peptides. Though the NHS ester proved to be quite useful for this application, other esters may prove useful in other applications. Nevertheless, this method of producing the active esters proved to be quite robust and generally applicable across a wide variety of compounds.
  • FIG. 4B illustrates 7 different active esters that were produced using the same generic procedure.
  • Trifluoroacetic anhydride (4.9 mL, 4 ⁇ 8.68 mmol (2.5-4 equivalents is typically used) was added to N-hydroxysuccinimide (NHS) (1 g, 8.69 mmol) and stirred under argon for 1-2 h to produce a homogeneous reaction mixture. Excess reagent and by-product CF 3 COOH were removed under reduced pressure (rotary evaporation). The product was obtained as white solid in quantitative yield. The solid was dried under high vacuum for 3-4 h and stored under argon (Ar) or nitrogen (N 2 ) gas.
  • the active ester product was precipitated as dihydrochloride salt by the addition of a solution by addition of HCl solution in dioxane (4 M, 50% volume of the reaction) followed by washing with THF, ethyl acetate and hexanes. In other cases the product was isolated from the reaction as the mono TFA salt. Addition of TFA could be performed if the bis-TFA salt was desired.
  • the trifluroacetate ester reagent can be reacted with the zwitterion of N-methyl piperazine acetic acid and well as with a mono salt or bis salt (e.g. mono-HCl salt, mono-TFA salt, bis-HCl salt or bis-TFA salt) of the N-methyl piperazine acetic acid.
  • a mono salt or bis salt e.g. mono-HCl salt, mono-TFA salt, bis-HCl salt or bis-TFA salt
  • DIPEA diisoproplyethylamine

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
US10/751,354 2004-01-05 2004-01-05 Active esters of N-substituted piperazine acetic acids, including isotopically enriched versions thereof Abandoned US20050148771A1 (en)

Priority Applications (13)

Application Number Priority Date Filing Date Title
US10/751,354 US20050148771A1 (en) 2004-01-05 2004-01-05 Active esters of N-substituted piperazine acetic acids, including isotopically enriched versions thereof
US10/751,388 US7307169B2 (en) 2004-01-05 2004-01-05 Isotopically enriched N-substituted piperazines and methods for the preparation thereof
EP10008184.3A EP2251334B1 (de) 2004-01-05 2005-01-05 Markierungsreagentien und markierte Analyten
AT05705033T ATE479669T1 (de) 2004-01-05 2005-01-05 Markierungsreagentien, markierte analyten, einschliesslich mischungen davon, und davon abgeleitete fragmentionen und verfahren zu deren analyse
EP05705033A EP1701945B1 (de) 2004-01-05 2005-01-05 Markierungsreagentien, markierte analyten, einschliesslich mischungen davon, und davon abgeleitete fragmentionen und verfahren zu deren analyse
CA2552304A CA2552304C (en) 2004-01-05 2005-01-05 Labeling reagents, labeled analytes, including mixtures thereof, and fragment ions derived therefrom and methods for the analysis thereof
PCT/US2005/000223 WO2005068446A1 (en) 2004-01-05 2005-01-05 Labeling reagents, labeled analytes, including mixtures thereof, and fragment ions derived therefrom and methods for the analysis thereof
AU2005205522A AU2005205522A1 (en) 2004-01-05 2005-01-05 Labeling reagents, labeled analytes, including mixtures thereof, and fragment ions derived therefrom and methods for the analysis thereof
DE602005023260T DE602005023260D1 (de) 2004-01-05 2005-01-05 Markierungsreagentien, markierte analyten, einschliesslich mischungen davon, und davon abgeleitete fragmentionen und verfahren zu deren analyse
JP2006547623A JP4832312B2 (ja) 2004-01-05 2005-01-05 標識試薬および標識化分析物の混合物を含む、標識試薬、標識化分析物、およびそれらに由来するフラグメントイオン、ならびにそれらの分析方法
US12/001,734 US7932388B2 (en) 2004-01-05 2007-12-11 Isotopically enriched N-substituted piperazines and methods for the preparation thereof
US12/398,920 US8569304B2 (en) 2004-01-05 2009-03-05 Active esters of N-substituted piperazine acetic acids, including isotopically enriched versions thereof
JP2010142103A JP2010217197A (ja) 2004-01-05 2010-06-22 標識試薬および標識化分析物の混合物を含む、標識試薬、標識化分析物、およびそれらに由来するフラグメントイオン、ならびにそれらの分析方法

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US10/751,353 US20050147982A1 (en) 2004-01-05 2004-01-05 Mixtures of isobarically labeled analytes and fragments ions derived therefrom
US10/751,387 US7355045B2 (en) 2004-01-05 2004-01-05 Isotopically enriched N-substituted piperazine acetic acids and methods for the preparation thereof
US10/751,354 US20050148771A1 (en) 2004-01-05 2004-01-05 Active esters of N-substituted piperazine acetic acids, including isotopically enriched versions thereof
US10/751,388 US7307169B2 (en) 2004-01-05 2004-01-05 Isotopically enriched N-substituted piperazines and methods for the preparation thereof
US10/822,639 US20050147985A1 (en) 2004-01-05 2004-04-12 Mixtures of isobarically labeled analytes and fragments ions derived therefrom
US10/852,730 US20050148087A1 (en) 2004-01-05 2004-05-24 Isobarically labeled analytes and fragment ions derived therefrom

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/398,920 Continuation US8569304B2 (en) 2004-01-05 2009-03-05 Active esters of N-substituted piperazine acetic acids, including isotopically enriched versions thereof

Publications (1)

Publication Number Publication Date
US20050148771A1 true US20050148771A1 (en) 2005-07-07

Family

ID=34799954

Family Applications (4)

Application Number Title Priority Date Filing Date
US10/751,388 Active 2025-02-17 US7307169B2 (en) 2004-01-05 2004-01-05 Isotopically enriched N-substituted piperazines and methods for the preparation thereof
US10/751,354 Abandoned US20050148771A1 (en) 2004-01-05 2004-01-05 Active esters of N-substituted piperazine acetic acids, including isotopically enriched versions thereof
US12/001,734 Active 2025-07-17 US7932388B2 (en) 2004-01-05 2007-12-11 Isotopically enriched N-substituted piperazines and methods for the preparation thereof
US12/398,920 Active 2025-10-04 US8569304B2 (en) 2004-01-05 2009-03-05 Active esters of N-substituted piperazine acetic acids, including isotopically enriched versions thereof

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US10/751,388 Active 2025-02-17 US7307169B2 (en) 2004-01-05 2004-01-05 Isotopically enriched N-substituted piperazines and methods for the preparation thereof

Family Applications After (2)

Application Number Title Priority Date Filing Date
US12/001,734 Active 2025-07-17 US7932388B2 (en) 2004-01-05 2007-12-11 Isotopically enriched N-substituted piperazines and methods for the preparation thereof
US12/398,920 Active 2025-10-04 US8569304B2 (en) 2004-01-05 2009-03-05 Active esters of N-substituted piperazine acetic acids, including isotopically enriched versions thereof

Country Status (7)

Country Link
US (4) US7307169B2 (de)
EP (2) EP2251334B1 (de)
AT (1) ATE479669T1 (de)
AU (1) AU2005205522A1 (de)
CA (1) CA2552304C (de)
DE (1) DE602005023260D1 (de)
WO (1) WO2005068446A1 (de)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040220412A1 (en) * 2003-01-30 2004-11-04 Apple Corporation Compositions and kits pertaining to analyte determination
WO2006017208A1 (en) * 2004-07-12 2006-02-16 Applera Corporation Mass tags for quantitative analyses
US20060078960A1 (en) * 2004-05-19 2006-04-13 Hunter Christie L Expression quantification using mass spectrometry
US20060183238A1 (en) * 2005-02-09 2006-08-17 Applera Corporation Amine-containing compound analysis methods
US20070037286A1 (en) * 2005-02-09 2007-02-15 Subhasish Purkayastha Thyroxine-containing compound analysis methods
US20070048752A1 (en) * 2004-07-12 2007-03-01 Applera Corporation Mass tags for quantitative analyses
US20070054345A1 (en) * 2004-05-19 2007-03-08 Hunter Christie L Expression quantification using mass spectrometry
WO2007087534A2 (en) 2006-01-24 2007-08-02 Applera Corporation Methods, mixtures, kits and compositions pertaining to analyte determination
US8492163B2 (en) 2011-01-31 2013-07-23 Dh Technologies Development Pte. Ltd. Methods, mixtures, kits and compositions pertaining to analyte determination
EP2438448A4 (de) * 2009-05-31 2015-05-06 Dh Technologies Dev Pte Ltd Spezifische analyse von keton- und aldehydanalyten mithilfe von strategien zur kennzeichnung von reagenzienverbindungen sowie eines massenspektrometrie-arbeitsablaufs
US11453621B2 (en) * 2015-12-11 2022-09-27 Electrophoretics Limited Isobaric mass labels having n',n'-dimeihyl piperazine-2-carboxylic acid reporter moieties

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2542941C (en) * 2003-11-26 2013-02-12 Applera Corporation Analysis of mass spectral data in the quiet zones and label selection therefor
EP1916527A1 (de) * 2003-11-26 2008-04-30 Applera Corporation Analyse von Massenspektraldaten in ruhigen Bereichen
US7307169B2 (en) 2004-01-05 2007-12-11 Applera Corporation Isotopically enriched N-substituted piperazines and methods for the preparation thereof
US20050148087A1 (en) 2004-01-05 2005-07-07 Applera Corporation Isobarically labeled analytes and fragment ions derived therefrom
WO2005085869A2 (en) 2004-03-01 2005-09-15 Applera Corporation Determination of analyte characteristics based upon binding properties
US20080206737A1 (en) * 2004-05-19 2008-08-28 Hunter Christie L Expression quantification using mass spectrometry
US8236565B2 (en) 2006-05-26 2012-08-07 Dh Technologies Development Pte. Ltd. Tagging reagents and methods for hydroxylated compounds
US7906341B2 (en) 2006-06-30 2011-03-15 Dh Technologies Development Pte, Ltd. Methods, mixtures, kits and compositions pertaining to analyte determination
US8362242B2 (en) 2006-06-30 2013-01-29 Dh Technologies Development Pte. Ltd. Analyte determination utilizing mass tagging reagents comprising a non-encoded detectable label
KR101081053B1 (ko) 2008-07-18 2011-11-07 포항공과대학교 산학협력단 가변질량 라벨링제와 이를 이용한 아미노산 서열 및 단백질정량 동시 분석방법
US9678083B2 (en) * 2009-10-14 2017-06-13 Dana-Farber Cancer Institute, Inc. Protected amine labels and use in detecting analytes
CA2784495A1 (en) * 2009-12-15 2011-06-23 Dh Technologies Development Pte. Ltd. Analysis of amino acids and amine-containing compounds using tagging reagents and lc-ms workflow
KR101229704B1 (ko) 2010-08-23 2013-02-05 포항공과대학교 산학협력단 동중체로 표지된 단백질 및 펩티드를 큰 질량의 ｙ－타입 이온을 이용하여 정량분석하는 방법
US8809012B2 (en) 2010-08-23 2014-08-19 Postach Academy-Industry Foundation Labeling agent and methods for simultaneous sequencing and quantification of multiple peptides and proteins using the same
US20120329661A1 (en) * 2011-05-18 2012-12-27 Guo Zengkui Isotope Labeling-Assisted Quantification (iLAQ) of Biological Compounds
JP6272834B2 (ja) 2012-05-18 2018-01-31 ディーエイチ テクノロジーズ デベロップメント プライベート リミテッド サイト特異的誘導とｌｃ／ｍｓ／ｍｓワークフローとを使用した脳腱黄色腫症バイオマーカーのパネルの解析
US9835629B2 (en) 2012-05-18 2017-12-05 Pierce Biotechnology, Inc. Methods and reagents for biomolecule labeling, enrichment and gentle elution
CN113514531B (zh) * 2021-04-27 2022-10-25 清华大学 一种化合物的碎片离子预测方法及应用

Citations (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3860581A (en) * 1972-12-27 1975-01-14 American Home Prod Preparation of 1,4-benzodiazepines
US5705610A (en) * 1990-05-15 1998-01-06 Chiron Corporation Method and apparatus for biopolymer synthesis
US5780232A (en) * 1996-05-28 1998-07-14 Atom Sciences, Inc. DNA sequencing, mapping, and diagnostic processes using hybridization and stable isotope labels of DNA
US5800992A (en) * 1989-06-07 1998-09-01 Fodor; Stephen P.A. Method of detecting nucleic acids
US6027890A (en) * 1996-01-23 2000-02-22 Rapigene, Inc. Methods and compositions for enhancing sensitivity in the analysis of biological-based assays
US6156527A (en) * 1997-01-23 2000-12-05 Brax Group Limited Characterizing polypeptides
US6270976B1 (en) * 1998-09-15 2001-08-07 Brax Group Limited Characterizing nucleic acid by mass spectrometry
US6287780B1 (en) * 1997-12-19 2001-09-11 Brax Group Limited Compounds for mass spectrometry comprising nucleic acid bases and aryl ether mass markers
US6312893B1 (en) * 1996-01-23 2001-11-06 Qiagen Genomics, Inc. Methods and compositions for determining the sequence of nucleic acid molecules
US6319476B1 (en) * 1999-03-02 2001-11-20 Perseptive Biosystems, Inc. Microfluidic connector
US6329180B1 (en) * 1996-09-13 2001-12-11 Alex M. Garvin Genetic analysis using peptide tagged in-vitro synthesized proteins
US6403309B1 (en) * 1999-03-19 2002-06-11 Valigen (Us), Inc. Methods for detection of nucleic acid polymorphisms using peptide-labeled oligonucleotides and antibody arrays
US6428956B1 (en) * 1998-03-02 2002-08-06 Isis Pharmaceuticals, Inc. Mass spectrometric methods for biomolecular screening
US6472156B1 (en) * 1999-08-30 2002-10-29 The University Of Utah Homogeneous multiplex hybridization analysis by color and Tm
US6475807B1 (en) * 1996-04-08 2002-11-05 Smithkline Beecham Corporation Mass-based encoding and qualitative analysis of combinatorial libraries
US6613508B1 (en) * 1996-01-23 2003-09-02 Qiagen Genomics, Inc. Methods and compositions for analyzing nucleic acid molecules utilizing sizing techniques
US6629040B1 (en) * 1999-03-19 2003-09-30 University Of Washington Isotope distribution encoded tags for protein identification
US6750061B2 (en) * 1992-05-29 2004-06-15 The Rockefeller University Method and product for the sequence determination of peptides using a mass spectrometer
US6824981B2 (en) * 2000-08-11 2004-11-30 Agilix Corporation Ultra-sensitive detection systems using alterable peptide tags
US7045296B2 (en) * 2001-05-08 2006-05-16 Applera Corporation Process for analyzing protein samples

Family Cites Families (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE1932995A1 (de) * 1969-06-28 1971-01-07 Bosch Gmbh Robert Steuereinrichtung zum Steuern eines ersten Druckmittelstromes in Abhaengigkeit eines zweiten Druckmittelstromes
DE2851953A1 (de) * 1978-12-01 1980-06-19 Thomae Gmbh Dr K Neue spectinomycylamine, verfahren zu ihrer herstellung und diese enthaltenden arzneimittel
AU5940186A (en) 1985-07-05 1987-01-08 Institut Fur Pflanzenschutzforschung 2, 6 - dimethyl - morpholine quartenary compounds
US4761415A (en) 1986-08-28 1988-08-02 Smithkline Beckman Corporation Dopamine-β-hydroxylase inhibitors
JPH01125357A (ja) 1987-11-06 1989-05-17 Dainippon Pharmaceut Co Ltd トリペプチドの誘導体
GB9300883D0 (en) 1993-01-18 1993-03-10 Pfizer Ltd Antiparasitic agents
US6495314B1 (en) 1995-09-29 2002-12-17 The Scripps Research Institute Process for characterizing proteins
JP2001501825A (ja) 1996-10-04 2001-02-13 ダコ アクティーゼルスカブ マイコバクテリア（Ｍｙｃｏｂａｃｔｅｒｉａ）の検出のための新規プローブ
ATE340869T1 (de) 1997-01-15 2006-10-15 Xzillion Gmbh & Co Kg Massenmarkierte hybridisierungssonden
NZ501919A (en) 1997-07-22 2001-11-30 Qiagen Genomics Inc Methods for analyzing nucleic acid molecules utilizing mass spectroscopy readable tags
JP4296608B2 (ja) * 1997-08-27 2009-07-15 田辺三菱製薬株式会社 Ｐｐａｒのアゴニスト及びアンタゴニストのスクリーニング方法
ATE344835T1 (de) 1997-09-23 2006-11-15 Sarissa Inc Antisense-oligonukleotide gegen thymidylatsynthase
ATE536422T1 (de) 1998-08-25 2011-12-15 Univ Washington Schnelle quantitative analyse von proteinen oder proteinfunktionen in komplexen gemischen
GB0006141D0 (en) 2000-03-14 2000-05-03 Brax Group Ltd Mass labels
AU2001259586A1 (en) 2000-05-05 2001-11-20 Agilix Corporation Highly multiplexed reporter carrier systems
GB0115581D0 (en) 2001-06-26 2001-08-15 Glaxo Group Ltd Method of mass spectometry
WO2003025576A2 (en) 2001-09-14 2003-03-27 Xzillion Gmbh & Co. Kg Mass labels
EP1446665A2 (de) 2001-11-09 2004-08-18 Bayer HealthCare AG Isotopencodierte affinitätsmarker 3
AU2003224674A1 (en) 2002-03-11 2003-09-29 Hk Pharmaceuticals, Inc. Compounds and methods for analyzing the proteome
US7473535B2 (en) 2002-08-20 2009-01-06 The Institute For Systems Biology Chemical reagents and methods for detection and quantification of proteins in complex mixtures
US20040219685A1 (en) 2003-01-30 2004-11-04 Applera Corporation Methods and mixtures pertaining to analyte determination using electrophilic labeling reagents
GB0306756D0 (en) 2003-03-24 2003-04-30 Xzillion Gmbh & Co Kg Mass labels
US7355045B2 (en) * 2004-01-05 2008-04-08 Applera Corporation Isotopically enriched N-substituted piperazine acetic acids and methods for the preparation thereof
US7307169B2 (en) 2004-01-05 2007-12-11 Applera Corporation Isotopically enriched N-substituted piperazines and methods for the preparation thereof

Patent Citations (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3860581A (en) * 1972-12-27 1975-01-14 American Home Prod Preparation of 1,4-benzodiazepines
US5800992A (en) * 1989-06-07 1998-09-01 Fodor; Stephen P.A. Method of detecting nucleic acids
US5705610A (en) * 1990-05-15 1998-01-06 Chiron Corporation Method and apparatus for biopolymer synthesis
US6750061B2 (en) * 1992-05-29 2004-06-15 The Rockefeller University Method and product for the sequence determination of peptides using a mass spectrometer
US6027890A (en) * 1996-01-23 2000-02-22 Rapigene, Inc. Methods and compositions for enhancing sensitivity in the analysis of biological-based assays
US6613508B1 (en) * 1996-01-23 2003-09-02 Qiagen Genomics, Inc. Methods and compositions for analyzing nucleic acid molecules utilizing sizing techniques
US20030077595A1 (en) * 1996-01-23 2003-04-24 Qiagen Genomics, Inc. Methods and compositions for enhancing sensitivity in the analysis of biological-based assays
US6312893B1 (en) * 1996-01-23 2001-11-06 Qiagen Genomics, Inc. Methods and compositions for determining the sequence of nucleic acid molecules
US6475807B1 (en) * 1996-04-08 2002-11-05 Smithkline Beecham Corporation Mass-based encoding and qualitative analysis of combinatorial libraries
US5780232A (en) * 1996-05-28 1998-07-14 Atom Sciences, Inc. DNA sequencing, mapping, and diagnostic processes using hybridization and stable isotope labels of DNA
US6329180B1 (en) * 1996-09-13 2001-12-11 Alex M. Garvin Genetic analysis using peptide tagged in-vitro synthesized proteins
US6156527A (en) * 1997-01-23 2000-12-05 Brax Group Limited Characterizing polypeptides
US6287780B1 (en) * 1997-12-19 2001-09-11 Brax Group Limited Compounds for mass spectrometry comprising nucleic acid bases and aryl ether mass markers
US6428956B1 (en) * 1998-03-02 2002-08-06 Isis Pharmaceuticals, Inc. Mass spectrometric methods for biomolecular screening
US6270976B1 (en) * 1998-09-15 2001-08-07 Brax Group Limited Characterizing nucleic acid by mass spectrometry
US6319476B1 (en) * 1999-03-02 2001-11-20 Perseptive Biosystems, Inc. Microfluidic connector
US6403309B1 (en) * 1999-03-19 2002-06-11 Valigen (Us), Inc. Methods for detection of nucleic acid polymorphisms using peptide-labeled oligonucleotides and antibody arrays
US6629040B1 (en) * 1999-03-19 2003-09-30 University Of Washington Isotope distribution encoded tags for protein identification
US6472156B1 (en) * 1999-08-30 2002-10-29 The University Of Utah Homogeneous multiplex hybridization analysis by color and Tm
US6824981B2 (en) * 2000-08-11 2004-11-30 Agilix Corporation Ultra-sensitive detection systems using alterable peptide tags
US7045296B2 (en) * 2001-05-08 2006-05-16 Applera Corporation Process for analyzing protein samples

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060105416A1 (en) * 2003-01-30 2006-05-18 Applera Corporation Methods, compositions and kits pertaining to analyte determination
US20040220412A1 (en) * 2003-01-30 2004-11-04 Apple Corporation Compositions and kits pertaining to analyte determination
US7195751B2 (en) * 2003-01-30 2007-03-27 Applera Corporation Compositions and kits pertaining to analyte determination
US20100227352A1 (en) * 2004-05-19 2010-09-09 Life Technologies Corporation Expression Quantification Using Mass Spectrometry
US20060078960A1 (en) * 2004-05-19 2006-04-13 Hunter Christie L Expression quantification using mass spectrometry
US8633031B2 (en) 2004-05-19 2014-01-21 Dh Technologies Development Pte. Ltd. Expression quantification using mass spectrometry
US20070054345A1 (en) * 2004-05-19 2007-03-08 Hunter Christie L Expression quantification using mass spectrometry
WO2006017208A1 (en) * 2004-07-12 2006-02-16 Applera Corporation Mass tags for quantitative analyses
US20060172319A1 (en) * 2004-07-12 2006-08-03 Applera Corporation Mass tags for quantitative analyses
US20070048752A1 (en) * 2004-07-12 2007-03-01 Applera Corporation Mass tags for quantitative analyses
US8501498B2 (en) 2004-07-12 2013-08-06 Dh Technologies Development Pte. Ltd. Mass tags for quantitative analyses
US20100311175A1 (en) * 2004-07-12 2010-12-09 Life Technologies Corporation Mass tags for quantitative analyses
US20060183238A1 (en) * 2005-02-09 2006-08-17 Applera Corporation Amine-containing compound analysis methods
US20100136703A1 (en) * 2005-02-09 2010-06-03 Life Technologies Corporation Thyroxine-Containing Compound Analysis Methods
US8569071B2 (en) 2005-02-09 2013-10-29 Dh Technologies Development Pte. Ltd. Amine-containing compound analysis methods
US20070037286A1 (en) * 2005-02-09 2007-02-15 Subhasish Purkayastha Thyroxine-containing compound analysis methods
WO2007087534A2 (en) 2006-01-24 2007-08-02 Applera Corporation Methods, mixtures, kits and compositions pertaining to analyte determination
EP2438448A4 (de) * 2009-05-31 2015-05-06 Dh Technologies Dev Pte Ltd Spezifische analyse von keton- und aldehydanalyten mithilfe von strategien zur kennzeichnung von reagenzienverbindungen sowie eines massenspektrometrie-arbeitsablaufs
US9500622B2 (en) 2009-05-31 2016-11-22 Dh Technologies Development Pte. Ltd. Specific analysis of ketone and aldehyde analytes using reagent compounds, labeling strategies, and mass spectrometry workflow
US10094809B2 (en) 2009-05-31 2018-10-09 Dh Technologies Development Pte. Ltd. Specific analysis of ketone and aldehyde analytes using reagent compounds, labeling strategies, and mass spectrometry workflow
US8492163B2 (en) 2011-01-31 2013-07-23 Dh Technologies Development Pte. Ltd. Methods, mixtures, kits and compositions pertaining to analyte determination
US11453621B2 (en) * 2015-12-11 2022-09-27 Electrophoretics Limited Isobaric mass labels having n',n'-dimeihyl piperazine-2-carboxylic acid reporter moieties

Also Published As

Publication number Publication date
US7932388B2 (en) 2011-04-26
US20050148773A1 (en) 2005-07-07
US20080114169A1 (en) 2008-05-15
EP1701945A1 (de) 2006-09-20
AU2005205522A1 (en) 2005-07-28
US8569304B2 (en) 2013-10-29
CA2552304A1 (en) 2005-07-28
US20090227791A1 (en) 2009-09-10
DE602005023260D1 (de) 2010-10-14
US7307169B2 (en) 2007-12-11
EP2251334B1 (de) 2013-08-14
EP1701945B1 (de) 2010-09-01
WO2005068446A1 (en) 2005-07-28
CA2552304C (en) 2012-03-27
ATE479669T1 (de) 2010-09-15
EP2251334A1 (de) 2010-11-17

Similar Documents

Publication Publication Date Title
US8569304B2 (en) Active esters of N-substituted piperazine acetic acids, including isotopically enriched versions thereof
US7355045B2 (en) Isotopically enriched N-substituted piperazine acetic acids and methods for the preparation thereof
US8273706B2 (en) Isobarically labeled analytes and fragment ions derived therefrom
EP1984726B1 (de) Verfahren, mixturen, kits und zusammensetzungen bezüglich der analytbestimmung
US20050147982A1 (en) Mixtures of isobarically labeled analytes and fragments ions derived therefrom
Reed et al. Acridine-and cholesterol-derivatized solid supports for improved synthesis of 3'-modified oligonucleotides
JP3692299B2 (ja) 質量分析のためのアリールスルフォンリンカー
US20120116079A1 (en) Phosphonate fluorescent dyes and conjugates
US20050147985A1 (en) Mixtures of isobarically labeled analytes and fragments ions derived therefrom
CN101421609A (zh) 与分析物测定相关的方法、混合物、试剂盒和组合物
Kachalova et al. A New and Efficient Method for Synthesis of 5′‐Conjugates of Oligonucleotides through Amide‐Bond Formation on Solid Phase
WO2007128874A1 (en) Biomolecule labeling reactants based on azacycloalkanes and conjugates derived thereof
Zhou et al. Synthesis of oligonucleotides containing site-specific carcinogen adducts. Preparation of the 2-cyanoethyl N, N-diisopropylphosphoramidite of N-(2'-deoxyguanosin-8-yl)-2-(acetylamino) fluorene with Fmoc as the base-protecting group
CN116514795A (zh) 3CLpro蛋白酶抑制剂的制备方法
JP2010217197A (ja) 標識試薬および標識化分析物の混合物を含む、標識試薬、標識化分析物、およびそれらに由来するフラグメントイオン、ならびにそれらの分析方法
Cebon et al. New DNA modification strategies involving oxime formation
EP1846378A2 (de) Kationische lipide zur transfektion von nukleinsäuren
Far et al. A novel phosphoramidite for the synthesis of α-oxo aldehyde-modified oligodeoxynucleotides
Alnabari et al. New quinone-amino acid conjugates linked via a vinylic spacer

Legal Events

Date Code Title Description
AS Assignment

Owner name: APPLERA CORPORATION, MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:DEY, SUBHAKAR;PAPPIN, DARRYL J.C.;PURKAYASTHA, SUBHASISH;AND OTHERS;REEL/FRAME:014510/0969

Effective date: 20040331

AS Assignment

Owner name: BANK OF AMERICA, N.A, AS COLLATERAL AGENT, WASHING

Free format text: SECURITY AGREEMENT;ASSIGNOR:APPLIED BIOSYSTEMS, LLC;REEL/FRAME:021976/0001

Effective date: 20081121

Owner name: BANK OF AMERICA, N.A, AS COLLATERAL AGENT,WASHINGT

Free format text: SECURITY AGREEMENT;ASSIGNOR:APPLIED BIOSYSTEMS, LLC;REEL/FRAME:021976/0001

Effective date: 20081121

AS Assignment

Owner name: APPLIED BIOSYSTEMS, LLC, CALIFORNIA

Free format text: MERGER;ASSIGNOR:APPLIED BIOSYSTEMS INC.;REEL/FRAME:022410/0649

Effective date: 20081121

Owner name: APPLIED BIOSYSTEMS INC., CALIFORNIA

Free format text: CHANGE OF NAME;ASSIGNOR:APPLERA CORPORATION;REEL/FRAME:022410/0636

Effective date: 20080630

Owner name: APPLIED BIOSYSTEMS INC., CALIFORNIA

Free format text: MERGER;ASSIGNOR:APPLIED BIOSYSTEMS INC.;REEL/FRAME:022410/0642

Effective date: 20081121

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION

AS Assignment

Owner name: DH TECHNOLOGIES PTE. LTD.,SINGAPORE

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:APPLIED BIOSYSTEMS, LLC;REEL/FRAME:023937/0854

Effective date: 20100129

Owner name: DH TECHNOLOGIES PTE. LTD., SINGAPORE

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:APPLIED BIOSYSTEMS, LLC;REEL/FRAME:023937/0854

Effective date: 20100129

AS Assignment

Owner name: APPLIED BIOSYSTEMS INC.,CALIFORNIA

Free format text: CHANGE OF NAME;ASSIGNOR:APPLERA CORPORATION;REEL/FRAME:023994/0538

Effective date: 20080701

Owner name: APPLIED BIOSYSTEMS, LLC,CALIFORNIA

Free format text: MERGER;ASSIGNOR:APPLIED BIOSYSTEMS INC.;REEL/FRAME:023994/0587

Effective date: 20081121

Owner name: APPLIED BIOSYSTEMS, LLC,CALIFORNIA

Free format text: MERGER;ASSIGNOR:APPLIED BIOSYSTEMS INC.;REEL/FRAME:023985/0801

Effective date: 20081121

Owner name: APPLIED BIOSYSTEMS, LLC, CALIFORNIA

Free format text: MERGER;ASSIGNOR:APPLIED BIOSYSTEMS INC.;REEL/FRAME:023985/0801

Effective date: 20081121

Owner name: APPLIED BIOSYSTEMS INC., CALIFORNIA

Free format text: CHANGE OF NAME;ASSIGNOR:APPLERA CORPORATION;REEL/FRAME:023994/0538

Effective date: 20080701

Owner name: APPLIED BIOSYSTEMS, LLC, CALIFORNIA

Free format text: MERGER;ASSIGNOR:APPLIED BIOSYSTEMS INC.;REEL/FRAME:023994/0587

Effective date: 20081121

AS Assignment

Owner name: APPLIED BIOSYSTEMS, INC., CALIFORNIA

Free format text: LIEN RELEASE;ASSIGNOR:BANK OF AMERICA, N.A.;REEL/FRAME:030182/0677

Effective date: 20100528

AS Assignment

Owner name: APPLIED BIOSYSTEMS, LLC, CALIFORNIA

Free format text: CORRECTIVE ASSIGNMENT TO CORRECT THE RECEIVING PARTY NAME PREVIOUSLY RECORDED AT REEL: 030182 FRAME: 0707. ASSIGNOR(S) HEREBY CONFIRMS THE RELEASE OF SECURITY INTEREST;ASSIGNOR:BANK OF AMERICA, N.A.;REEL/FRAME:038007/0115

Effective date: 20100528

Owner name: APPLIED BIOSYSTEMS, LLC, CALIFORNIA

Free format text: CORRECTIVE ASSIGNMENT TO CORRECT THE RECEIVING PARTY NAME PREVIOUSLY RECORDED AT REEL: 030182 FRAME: 0677. ASSIGNOR(S) HEREBY CONFIRMS THE RELEASE OF SECURITY INTEREST;ASSIGNOR:BANK OF AMERICA, N.A.;REEL/FRAME:038007/0115

Effective date: 20100528