Classifications

• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B70/00—Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
  • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
  • C07C229/34—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton containing six-membered aromatic rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
  • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
  • C07C237/22—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
  • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
  • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D211/60—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
  • C07D211/62—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals attached in position 4
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
  • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
  • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D211/60—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
  • C07D211/62—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals attached in position 4
  • C07D211/64—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals attached in position 4 having an aryl radical as the second substituent in position 4
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/06—Phosphorus compounds without P—C bonds
  • C07F9/08—Esters of oxyacids of phosphorus
  • C07F9/141—Esters of phosphorous acids
  • C07F9/1414—Esters of phosphorous acids with arylalkanols
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/06—Phosphorus compounds without P—C bonds
  • C07F9/22—Amides of acids of phosphorus
  • C07F9/24—Esteramides
  • C07F9/2404—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic
  • C07F9/2429—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic of arylalkanols
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
  • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
  • C12P41/007—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures by reactions involving acyl derivatives of racemic amines

Definitions

• the present invention relates generally to enantiomerically-enriched compounds, specifically enantiomerically-enriched compounds having one or more photocleavable bonds, as well as methods relating thereto, including synthetic methods.
• CMSTs cleavable mass spectrometry tags
• SNP single nucleotide polymorphism
• the present invention provides advantageous tags, including tags detectable by mass spectrometry, and methods of their use, as disclosed more fully herein.
• the present invention affords enantiomerically-enriched compounds having photocleavable bonds, and methods for preparing labeled molecules with enantiomerically-enriched photocleavable tags.
• the present invention provides advantages including the ability to obtain a single distinct product in those instances where there is a need to isolate members of a plurality of structurally similar tagged molecules, as well as further related advantages as disclosed more fully herein.
• the present invention provides a compound having the formula (I) or (II):
• R 1 is selected from halogen and organic moieties
• R 2 and R are independently selected from hydrogen and organic moieties having a mass greater than 15 Daltons where R and R can together form a carbonyl group or may be joined together within a cyclic structure
• Z is an (n+l)-valent atom excluding carbon where n is an integer greater than 0
• R is independently selected from hydrogen, halogen, and organic moieties having a mass greater than 15 Daltons, with the proviso that at least one R 4 (namely R 4a ) is an organic moiety having a mass greater than 100 Daltons
• R 5 at each occurrence is independently either halogen or an organic moiety having a mass of less than 500 Daltons
• Z is nitrogen; R 4a is detectable by mass spectrometry; R 2 and R 3 are each hydrogen; R 1 comprises synthetic or natural biological material (e.g., nucleic acid, protein or saccharide); and/or R 4a has a mass of less than 10,000 Daltons and a molecular formula of C
• R 4a is detectable by mass spectrometry
• R 2 and R 3 are each hydrogen
• R 1 comprises synthetic or natural biological material (e.g., nucleic acid, protein or saccharide); and/or R 4a has a mass of less than 10,000 Daltons and a molecular formula of C
• R 4a has the formula T 2 -(J-T 3 -) P -; wherein T 2 is an organic moiety formed from carbon and one or more of hydrogen, fluoride, iodide, oxygen, nitrogen, sulfur and phosphorus, having a mass of 15 to 500 Daltons; T 3 is an organic moiety formed from carbon and one or more of hydrogen, fluoride, iodide, oxygen, nitrogen, sulfur and phosphorus, having a mass of 50 to 1000 Daltons; J is a direct bond or a functional group selected from amide, ester, amine, sulfide, ether, thioester, disulfide, thioether, urea, thiourea, carbamate, thiocarbamate, Schiff base, reduced Schiff base, imine, oxime, hydrazone, phosphate, phosphonate, phosphoramide, phosphonamide, sulfonate, sulfonamide or carbon-carbon bond; and p is an organic moiety formed
• R a has a formula comprising:
• G is (CH 2 ) ⁇ - 6 wherein a hydrogen on one and only one of the
• T 2 and T 4 are organic moieties of the formula C ⁇ -25 No- 9 ⁇ 0 - 9 So- 3 Po- H ⁇ F ⁇ I ⁇ wherein the sum of ⁇ , ⁇ and ⁇ is sufficient to satisfy the otherwise unsatisfied valencies of the C, N, O, S and P atoms;
• Amide is — N— C — or — C— N — ;
• R is hydrogen or C 0 alkyl;
• w is an
• the invention further provides a composition comprising compounds of formulae (1) and/or (II) wherein the formula (I):formula (II) molar ratio in the composition is within the range of 95:5 to 100:0 or within the range of 5:95 to 0: 100.
• the invention further provides a process for providing an enantiomerically-enriched compound comprising contacting a compound of the formula:
• Z is selected from oxygen, nitrogen and sulfur, where R 6 is hydrogen when Z is oxygen or sulfur, and when Z is nitrogen then R 6 is selected from hydrogen and -C 22 hydrocarbon and two R groups are bonded to Z;
• R 5 at each occurrence is independently either halogen or an organic moiety having a mass of less than 500 Daltons; m is selected from 0, 1, 2, 3 and 4;
• the compound to be acted upon in the process has the formula:
• R at each occurrence is independently either halogen or an organic moiety having a mass of less than 500 Daltons; m is selected from 0, 1, 2, 3 and 4; R is hydrogen or an organic moiety; and the compound is contacted with an enzyme to provide an enantiomerically-enriched compound of the formula:
• the compound to be acted upon in the process has the formula:
• the compound to be acted upon in the process has the formula:
• R 5 at each occurrence is independently either halogen or an organic moiety having a mass of less than 500 Daltons; m is selected from 0, 1 , 2, 3 and 4; R 7 is hydrogen or an organic moiety; and the compound is contacted with a chiral acid, to provide an enantiomerically-enriched salt of the formula:
• the compound to be acted upon in the process has the formula: wherein R ⁇ at each occurrence is independently either halogen or an organic moiety having a mass of less than 500 Daltons; m is selected from 0, 1 , 2, 3 and 4; and the compound is contacted with a chiral amine, to provide an enantiomerically enriched salt of the formula:
• the compound to be acted upon in the process has the formula:
• R at each occurrence is independently either halogen or an organic moiety having a mass of less than 500 Daltons; m is selected from 0, 1, 2, 3 and 4; and the compound is contacted with hydrogen in the presence of a chiral hydrogenation catalyst, to provide an enantiomerically-enriched compound of the formula:
• the invention provides compounds of the formula
• the invention provides compounds of the formula
• the phosphoramidite moiety of R 1 1 has the formula -O-P(OR 13 )(N(R l ) 2 ) wherein each of R 13 and R 14 is independently selected from an alkyl group or a substituted alkyl group having one or more substituents selected from halogen and cyano, and two R 14 groups may be bonded together to form a heterocycloalkyl group with the nitrogen of the phosphoramidite.
• the present invention provides tags suitable for coupling to nucleic acids or other molecules of interest.
• the tags When coupled to DNA, the tags provide a means to achieve a high- throughput genotyping system.
• the linker between the tag and the molecule of interest should completely cleave under conditions that do not cause tag fragmentation.
• the tag preferably yields one peak per oligonucleotide injected, and gives optimal signal in terms of ion current.
• the tag is preferably stable to PCR conditions, HPLC, and other manipulations used in assay formats. Reagents to introduce tags to a molecule of interest should proceed reproducibly and in good yield. In a preferred approach to meeting these objectives, a modular structure was developed that allows CMST components to be optimized independently.
• the components of a CMST preferably include a photolabile linker, a mass spectrometry sensitivity enhancer (MSSE), and a variable mass unit (VMU), all connected together through a scaffold.
• MSSE mass spectrometry sensitivity enhancer
• VMU variable mass unit
• the present invention provides tagged molecules of interest, precursors to tagged molecules of interest, and methods for generated the tagged molecules and precursors thereto, wherein the tag is enantiomerically-enriched. By using enantiomerically-enriched precursors, the purification of the reaction product of the precursor and the molecule of interest, in order to afford purified tagged molecule of interest, is facilitated.
• the present invention provides a compound having the formula (I) or (II):
• R is selected from halogen and organic moieties; R and R are independently selected from hydrogen and organic moieties having a mass greater than 15 Daltons where R " and R can together form a carbonyl group or may be joined together within a cyclic structure; Z is an (n+l)-valent atom excluding carbon where n is an integer greater than 0; R 4 is independently selected from hydrogen, halogen, and organic moieties having a mass greater than 15 Daltons, with the proviso that at least one R 4 is an organic moiety having a mass greater than 100 Daltons; R 5 at each occurrence is independently either halogen or an organic moiety having a mass of less than 500 Daltons; and m is selected from 0, 1, 2, 3 and 4.
• the compound of the invention is enantiomerically enriched, such that if compounds of both formulae (I) and (II) are present in an admixture, the molar formula (I): formula (II) ratio in the admixture is other than 50:50.
• the compound of the invention includes a 2-nitrophenyl group having, at the 1 -position of the phenyl ring, a substituted methyl group, where the carbon atom of the methyl group will be referred to herein as either Cl or the benzylic carbon atom.
• the C 1 atom is directly bonded to a hydrogen atom (not shown), a carbon atom (referred to herein as C2) and a non-carbon atom.
• compounds of the invention are photocleavable. That is, compounds of the invention will respond to contact with certain electromagnetic radiation by undergoing a cleavage reaction whereby the substituted Z atom separates from Cl as shown in the following structures, where indicates a bond that is photolytically unstable.
• C 1 is directly bonded to only one atom that is not a carbon or hydrogen atom
• compounds of the invention undergo a selective photocleavage reaction. That is, the Cl-Z bond can be selectively cleaved by appropriate photolytic conditions, leaving the other C 1 bonds largely if not entirely intact. This selective cleavage can occur so long as Z is not a carbon atom.
• Z is selected from oxygen, nitrogen and sulfur. Each of oxygen, nitrogen, and sulfur is a preferred atom for position Z.
• any one or more of the hydrogen atoms of the phenyl ring may be replaced with an equal number of R 5 groups.
• compounds of the invention may have a number ("m") of R 5 groups, where m is an integer selected from 0, 1, 2, 3 and 4.
• the desired photocleavage chemistry is largely independent of the nature of the R 5 group, so that R 5 may be an inorganic group, e.g., halogen, or an organic group. Typically, an organic R group is not too large, and accordingly has a mass of less than about 500 Daltons.
• Preferred R 5 groups are selected from C)-C 22 hydrocarbon groups.
• m is equal to zero, so that the 2-nitrophenyl ring has four hydrogen substituents.
• the resulting Z-containing residue which derived from a compound of the invention will be referred to herein as a tag.
• the tag is, or includes a detectable moiety, where this term refers to a moiety that can be detected, and preferably characterized, by an analytical method.
• Representative analytical methods include spectrometric or potentiometric methods.
• Representative spectrometric methods include mass spectrometry, infrared spectrometry, ultraviolet spectrometry and fluorescent spectrometry.
• a representative potentiometric method is potentiostatic amperometry.
• At least one R 4 group is an organic moiety having a mass greater than 100 Daltons.
• This R 4 group will be referred to herein as the first R 4 group (or R 4a ). That tags should have masses greater than 100 Daltons is desirable for a number of reasons. When the tags have masses of less than 100 Daltons, it may be difficult to identify the tags. For instance, where the tags are identified and characterized by mass spectrometry, tags of molecular weight less than 100 Daltons may be difficult to distinguish from the background noise inherent in the operation of a mass spectrometer.
• the present invention provides compositions containing a number of non-identical compounds of the invention, where the non-identical compounds contain non-identical tags that may be distinguished from one another by an analytical method.
• the tags In order to distinguish a number of non-identical tags, the tags must be sufficiently different from one another that the analytical method can detect the difference.
• the tags In order to obtain a suitable number of tags that are distinct by, e.g., potentiometry, the tags must contain a number of atoms that can be bonded together in non-identical ways which are analytically distinct.
• tags have a mass of less than 100 Daltons, there are an unduly limiting number of analytically distinct tags. Accordingly, to allow sufficient variability in structure, and to allow easy and effective detection, the tags should have masses in excess of 100 Daltons.
• the identity of the other R 4 groups in a compound of the invention may be selected to achieve one or more goals.
• a second or more R 4 (e.g., R 4b , R 4c , etc.) group may impart information supplementing the information derived from the first R 4 group.
• a second or more R 4 group also preferably has a mass of at least 100 Daltons.
• the second or more R 4 groups do no more than satisfy the otherwise unsatisfied valence state of Cl , not interfere with the photocleavage reaction or detection of the tag, and be synthetically easy to form.
• the second or more R 4 groups might be hydrogen or simple hydrocarbon groups.
• the second or more R 4 groups, if present, is/are hydrogen.
• R 1 is selected from halogen and organic moieties, while R and R are independently selected from hydrogen and organic moieties having a mass greater than 15 Daltons.
• compounds of the invention include a detectable moiety where detection, and optionally characterization of the detectable moiety provides information about the compound of the invention.
• the R 1 group contains the feature of the compound about which information is desired.
• R 1 may include biological material from an individual, and the tag is characteristic of that individual and/or the specific biological material.
• the R group may contain a particular synthetic nucleic acid, peptide or saccharide sequence, which is uniquely identified by the tag.
• R 1 is a reactive chemical group which can undergo a chemical reaction whereby biologically-derived material may be photocleavably linked to a tag.
• R 1 may be a halogen atom which may be displaced in a chemical reaction by biological material or derivatized biological material.
• the R 2 and R groups are independently selected from hydrogen and organic moieties having a mass greater than 15 Daltons. Typically, the R 2 and R 3 groups are selected with a view to their impact on the chemistry selected for joining R 1 to Cl, and/or for their impact on the photocleavage reaction whereby the tag is separated from C 1. When each of R 2 and R 3 is hydrogen, then neither R 2 nor R 3 adversely impacts the photocleavage reaction, and typically they are compatible with
• R and R are preferably
• R and R could be groups other than hydrogen.
• R and R together might form a carbonyl group with C 1.
• R and R might, together with C 1 , form a cyclic structure.
• either of R 2 or R 3 might be an organic moiety of mass greater than 15 Daltons, e.g., a C]-C 22 hydrocarbon group.
• hydrocarbon groups do not interfere with the desired photocleavage reaction, and do not interfere with R C I coupling chemistry.
• Another feature of a compound of the invention is that the Cl atom is chiral. As seen from the formulae (I) and (II), the nature of the substituents on Cl necessarily require that Cl is chiral. In addition, if compounds of formulae (I) and (II) are in admixture, the molar ratio of the formula (I) compounds to the formula (II) compounds is other than 50:50. In a preferred embodiment of the invention, compounds of formula (I) are not in admixture with compounds of formula (II), and vice versa.
• the molar ratio of the formula (I) compounds to the formula (II) compounds is preferably in excess of 95:5, or 96:4, or 97:3, or 98:2, or 99:1, or in excess of 99.5:0.5, or vice versa (i.e., the molar ratio of the formula (II) compounds to the formula (I) compounds is preferably in excess of 95:5, or 96:4, or 97:3, or 98:2, or 99: 1, or in excess of 99.5:0.5).
• compositions containing a plurality of compounds of formulae (I) or (II) which, because of their isomeric constitution, may be more readily separated from one another than would otherwise be the case. This enhanced ability to distinguish differently tagged compounds allows the formation of compositions that could otherwise not be characterized.
• the present invention addresses this problem by providing compounds of formula (I) free of, or effectively free of, compounds of formula (II).
• the present invention is directed to a process for providing an enantiomerically-enriched compound using a specified agent. More specifically, the process acts on a compound of the formula:
• Z is selected from oxygen, nitrogen and sulfur; where R 6 is hydrogen when Z is oxygen or sulfur, and when Z is nitrogen then R 6 is selected from hydrogen and C t -C 2 hydrocarbon and two R 6 groups are bonded to Z; R 5 at each occurrence is independently either halogen or an organic moiety having a mass of less than 500 Daltons; m is selected from 0, 1, 2, 3 and 4; R 7 is hydrogen or an organic moiety, and the bond represented by both a solid and dashed line represents either a single or double bond.
• the inventive process contacts a compound as identified above with an agent.
• the compound is contacted with the agent so as to provide an enantiomerically-enriched compound of the formula:
• Molecules with photocleavably attached tags which may be provided in optically-enriched form according to the present invention, as well as methods that may be practiced with compounds and compositions of the present invention, are set forth in, for example, PCT International Publication Nos. WO 99/05319; WO 97/27331 ; WO 97/27327; WO 97/27325; and WO 95/04160.
• the compounds of the present invention may be substituted for the tagged compounds disclosed in these four publications, as well as other methods and assays where molecules having photocleavably linked tags are utilized.
• the following examples are set forth as a means of illustrating the present invention and are not to be construed as a limitation thereon.
• the chemical reactants and reagents were of standard commercial grade, obtained from commercial supply houses such as Aldrich (Milwaukee, WI; www.sigma-aldrich.com), Fluka (a division of Aldrich) and Lancaster Synthesis, Inc. (Windham, NH; http://www.lancaster.co.uk).
• the initial filtrate was basified to pH 8 and extracted first with the DCM washings collected above; and secondly with fresh DCM.
• the organic layers were combined, dried and evaporated to give the amino ethyl ester (23 g, 80%o yield).
• the enantiomeric excess (e.e.) of both the amino acid and amino ethyl ester were determined by cHPLC and were both in excess of 98% enantiomerically pure.
• the tags described herein are preferably conjugated to a molecule of interest through a photolabile bond.
• a preferred photolabile bond is present within an o-nitrobenzyl group (see, e.g., (a) Greenberg, M.M.; Gilmore, J.L. J. Org. Chem. 1994, 59, 746-753; (b) Yoo, D.J.; Greenberg, M.M. J. Org. Chem. 1995, 60, 3358-3364; and (c) Venkatesan, H.; Greenberg, M.M. J. Org. Chem. 1996, 61, 525-529).
• the intramolecular photoredox reaction in an o-nitrobenzyl group (see, e.g., Pillai, V.N.R. Synthesis 1980, 1-26) enables the tag to cleave from the oligonucleotide quickly under neutral conditions (six second exposure with a 254nm Hg lamp) and with no fragmentation.
• the synthetic route begins (Scheme 1) with esterification of the photosensitive linker 3-amino-3-(2-nitrophenyl) propionic acid, (ANP, 1, see, e.g., Brown, B.B.; Wagner, D.S.; Geysen, H.M. Molecular Diversity 1995, 1, 4-12; and (b) Rodebaugh, R.; Fraser-Reid, B.; Geysen, H.M. Tetrahedron Letters 1997, 38, 7653- 7656) to give the ethyl ester hydrochloride (2) in 84%> yield.
• ABP 1, see, e.g., Brown, B.B.; Wagner, D.S.; Geysen, H.M. Molecular Diversity 1995, 1, 4-12; and (b) Rodebaugh, R.; Fraser-Reid, B.; Geysen, H.M. Tetrahedron Letters 1997, 38, 7653- 7656
• Lysine provided a very suitable scaffold for the modular approach to the synthesis of the tags.
• the two amine and one carboxylic acid groups present in lysine provided handles for robust peptide chemistries with resulting amide linkages between the tag components.
• Peptides have a predictable behavior in mass spectrometry and are also relatively stable to 254 nm light.
• Perfluorinated aromatics may be used as electrophoric labels for analytical purposes in negative ion mode mass spectrometry (see, e.g., (a) Abdel-Baky, S.; Klempier, N.; Giese, R.W. Tetrahedron 1990, 46, 5859; (b) Abdel-Baky, S.; Allam, K.; Giese, R.W. Anal. Chem. 1993, 65, 498-499; (c) Trainor, T; Giese, R.W.; Vouros, P. Journal of Chromatography 1988, 452, 369-376; and (d) Saha, M.; Saha, J.; Giese, R.W.
• INA hydrochloride was coupled to 7, using EDAC and triethylamine, to give crude Alloc-protected structure 8, which was deprotected with diethylamine, triphenylphosphine, and palladium acetate.
• the resulting core structure (9) was crystallized from the reaction mixture and recovered by filtration in 95% yield as a yellow solid.
• VMUs variable mass units
• the masses of the VMUs are spaced a minimum of 4 a.m.u. apart, to minimize isotopic overlap between tags.
• the molecular weights of the VMUs ranged from 90-298 a.m.u.
• VMUs did not have the following properties or features: (1) functionalities incompatible with the synthetic sequence, such as esters; (2) elements with multiple isotopes (Cl, Br, S); (3) functionalities that might lead to competing photoprocesses (iodides, acyl- and aryl-phenones); (4) racemic acids, and (5) lack of commercial availability.
• VMUs were coupled to 9, using HATU and N-methyl mo ⁇ holine. After purification by column chromatography, the CMST ethyl ester (10) was recovered in variable yields. Saponification of 10 with NaOH gave the CMST acid (11) in quantitative yield.
• TFP TFA tetrafluorophenyl trifluoroacetate
• the tetrafluorophenyl active ester was chosen because of easily removed side products and relative stability and compatibility towards oligonucleotide conjugation conditions.
• the CMST TFP esters were conjugated to 5'-aminohexyl-tailed oligonucleotides (obtained from TriLink Biotechnology, San Diego, CA) according to the method of Lukhtanov et al. (see, e.g., Lukhtanov, E.A.; Kutyavin, I.V.; Gamper, H.B.; Meyer, R.B. Jr. Bioconjug. Chem. 1995, 4, 418-426).
• the number of tags was further expanded by the incremental use of a tyrosine derivative which functioned as a gross mass adjuster (GMA, and more specifically N-Boc-O-ethyl-L-tyrosine, which can be ordered commercially from Bachem California (Torrance, CA) or prepared by alkylation of N-Boc-L-Tyrosine-OMe with iodoethane and cesium carbonate in 86%o yield, followed by hydrolysis with NaOH in quantitative) which increases the molecular weight of the cleaved tag by 191 a.m.u. and allows the set of VMUs to be re-used for higher molecular weight tags.
• GMA gross mass adjuster
• the VMUs were coupled in the same manner as before, resulting in additional sets of tags (14). Using four units of the mass adjuster, a total of nearly 200 tags may be synthesized.

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• Medicinal Chemistry (AREA)
• Analytical Chemistry (AREA)
• Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)

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Citations (4)

• 2000
  • 2000-08-31 KR KR1020027002645A patent/KR20020037042A/ko not_active Application Discontinuation
  • 2000-08-31 CN CN00812153A patent/CN1371356A/zh active Pending
  • 2000-08-31 BR BR0013024-9A patent/BR0013024A/pt not_active Application Discontinuation
  • 2000-08-31 MX MXPA02002265A patent/MXPA02002265A/es unknown
  • 2000-08-31 WO PCT/US2000/024347 patent/WO2001016090A1/en not_active Application Discontinuation
  • 2000-08-31 CA CA002384191A patent/CA2384191A1/en not_active Abandoned
  • 2000-08-31 JP JP2001519660A patent/JP2003508378A/ja not_active Withdrawn
  • 2000-08-31 AU AU73490/00A patent/AU7349000A/en not_active Abandoned
  • 2000-08-31 EP EP00961549A patent/EP1208079A1/en not_active Withdrawn

Patent Citations (4)

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