EP1095052A1 - Silicium-enthaltende linker für nukleinsäure-massmarker - Google Patents

Silicium-enthaltende linker für nukleinsäure-massmarker

Info

Publication number
EP1095052A1
EP1095052A1 EP99933026A EP99933026A EP1095052A1 EP 1095052 A1 EP1095052 A1 EP 1095052A1 EP 99933026 A EP99933026 A EP 99933026A EP 99933026 A EP99933026 A EP 99933026A EP 1095052 A1 EP1095052 A1 EP 1095052A1
Authority
EP
European Patent Office
Prior art keywords
compound
analyte
compound according
mass
metal ion
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.)
Withdrawn
Application number
EP99933026A
Other languages
English (en)
French (fr)
Inventor
Günter Schmidt
Andrew Hugin Thompson
Robert Alexander Walker Johnstone
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.)
Xzillion GmbH and Co KG
Original Assignee
Brax Group Ltd
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
Application filed by Brax Group Ltd filed Critical Brax Group Ltd
Priority to EP02021105A priority Critical patent/EP1275655A1/de
Publication of EP1095052A1 publication Critical patent/EP1095052A1/de
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00—Compounds containing boron, silicon or a metal, e.g. chelates or vitamin B12
    • 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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6872—Methods for sequencing involving mass spectrometry
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures

Definitions

  • This invention concerns compounds which comprise mass markers for detection by mass spectrometry.
  • the invention relates to methods for characterising nucleic acids or other molecules by labelling with markers that are cleavably detachable from their associated nucleic acid and that are detectable by mass spectrometry.
  • this invention relates to improved methods of detaching mass labels from their associated nucleic acids or other molecules of interest.
  • PCT/GB98/00127 describes arrays of cleavable labels that are detectable by mass spectrometry which identify the sequence of a covalently linked nucleic acid probe. These mass labels have a number of advantages over other methods of analysing nucleic acids. At present commercially favoured systems are based on fluorescent labelling of DNA. Fluorescent labelling schemes permit the labelling of a relatively small number of molecules simultaneously, typically 4 labels can be used simultaneously and possibly up to eight. However the costs of the detection apparatus and the difficulties of analysing the resultant signals limit the number of labels that can be used simultaneously in a fluorescence detection scheme.
  • An advantage of using mass labels is the possibility of generating large numbers of labels which have discrete peaks in a mass spectrum allowing similar numbers of distinct molecular species to be labelled simultaneously. Fluorescent dyes are expensive to synthesise whereas mass labels can comprise relatively simple polymers permitting combinatorial synthesis of large numbers of labels at low cost.
  • a feature of the mass labelling techniques disclosed in PCT/GB98/00127 is the need for linker groups that covalently link a mass marker to its corresponding nucleic acid. These linkers must permit the mass marker to be separated from its nucleic acid prior to detection within a mass spectrometer. It is desirable that the cleavage of the label from its nucleic acid be performed in-line with a mass spectrometer, possibly after some in-line pre-fractionation step such as capillary electrophoresis. It is also desirable that this in-line cleavage step does not require a complex interface with the mass spectrometer to enable this step to occur. Ideally linkers should cleave at some predetermined point within existing instruments without any modification to the instrument beyond changes of normal operating parameters
  • N comprises a nucleic acid
  • R.1 and R.2 are substituents selected such that when the compound reacts with an electron donating moiety, either N or M cleaves from the Si atom in preference to R.1 and RA
  • R.1 and R ⁇ are substituents as defined below,
  • the invention additionally provides use of a linker group in the characterisation of an analyte, to attach a reporter group to the analyte, wherein the linker group is cleavable and has the following formula
  • Rl and R ⁇ are substituents as defined below
  • Figure 1 depicts the mechanism of cleavage of a linker used in the present invention, by means of a primary amine Cleavage takes place via a five-co-ordinate intermediate to produce two possible products
  • the mass spectrum of the charged products is measured
  • Figure 2 shows the negative ion mass spectrum of FT23 at 0 35 pmol/ ⁇ l This spectrum shows a very clean molecular ion less 1 proton at m/z 729 4,
  • Figure 3 shows the positive ion mass spectrum of FT23 at 0 35 pmol/ ⁇ l This spectrum shows a strong protonated molecular ion peak at m/z 731 5 and a second stronger peak at m/z 753 3 corresponding to a sodium adduct of FT23,
  • Figure 4 shows the negative ion mass spectrum of FT23 at 0 35 pmol/ ⁇ l in the presence of a PCR product The molecular ion is not detectable over the background peaks,
  • Figure 5 shows the positive ion mass spectrum of FT23 at 0 35 pmol/ ⁇ l in the presence of a PCR product
  • This spectrum does not show any of the protonated molecular ion peak at m/z 731 4 or the sodium adduct peak at m/z 753 3
  • Two additional peaks of significance appear in this spectrum
  • One peak at m/z 739 2 and a second peak at m/z 755 2 These peaks are believed to be end-products of a novel gas phase cleavage reaction discussed below
  • the two peaks corresponding to gas phase cleavage products are the only major ion peaks in this spectrum, and
  • Figure 6 shows a reaction mechanism for ammonia reacting with a TBDMS protective group used m the present invention
  • the analyte is not particularly limited and can be any analyte or molecule of interest, such as a nucleic acid or other molecule
  • the analyte comprises a biological molecule
  • the biological molecule is selected from a protein, a polypeptide, an ammo acid, a nucleic acid (e g.
  • the analyte comprises a nucleotide, oligonucleotide or nucleic acid
  • the nucleotide, oligonucleotide or nucleic acid may be natural, or may be modified by modifying a base, sugar and/or backbone of the nucleotide, oligonucleotide or nucleic acid
  • the analyte is a nucleic acid, and may be any type of nucleic acid
  • the nucleic acid is of a type as defined above
  • R and R ⁇ are not especially limited It is preferred that R* and R ⁇ are selected such that their bond energies to Si are greater than the bond energy of N and/or M to Si to ensure that when the compound is reacted with an electron donating moiety either N or M cleaves from the Si atom in preference to R* and R ⁇ , and/or Rl and R ⁇ are selected such that their ste ⁇ c bulk is sufficient to ensure that when the compound is reacted with an electron donating moiety either N or M
  • R! and R ⁇ are each independently a hydrogen atom, a halogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted aryl group It is particularly preferred that R ⁇ and R ⁇ are each independently fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl or phenyl groups
  • substituents may be introduced at the positions R* and R ⁇ including fluorine, chlo ⁇ ne and other halogens, methyl, ethyl and other alkyl groups
  • Phenyl groups may also be approp ⁇ ate
  • substituents at R! and R ⁇ should be stable during synthesis of the marker, during incorporation of the mass label into an oligonucleotide in an automated synthesiser and under mass spectrometry
  • a wide variety of groups have these properties and may be incorporated into the linker at these positions It may also be desirable in some embodiments to choose substituents which change the solubility of the linker and alter the rigidity of the linker
  • a covalent linkage is formed in attaching the analyte and/or the reporter group to the cleavable linker
  • the covalent linkage is not particularly limited provided that the analyte and/or reporter group can readily be attached to the cleavable linker using reactive functionalities attached to the linker and the analyte Table 1 below lists some reactive functionalities that may be reacted together to generate a covalent linkage between two entities Any of the functionalities listed below could be used to form the compounds used in the present invention to permit the linker to be attached to an analyte (such as a nucleic acid or protein) for detection (e g by mass spectrometry) If desired, a reactive functionality can be used to introduce a further linking group with a further reactive functionality
  • a short alkyl linkage is appropriate to link the mass marker to the linker, although a wide variety of linkages are available which can be used to link a mass marker to a linker
  • the reporter group used in the present invention is not especially limited and may be any group, provided that it is readily detectable and can se related to an analyte to identify the analyte.
  • the reporter group is a mass marker, that is detectable by mass spectrometry.
  • Other approp ⁇ ate reporters include fluorophores, radiolabels, chemiluminescent labels, and electron capture labels
  • the reporter group comprises a mass marker
  • mass markers disclosed in PCT/GB98/00127, PCT/GB98/03842, GB 9815166 5 and GB 9826159 7 can be employed.
  • PCT/GB98/00127 and PCT/GB98/03842 disclose poly-ether mass markers which are thermally stable, chemically inert and fragmentation resistant compounds, and which can be substituted with a variety of groups to alter properties such as solubility and charge
  • mass markers are also preferred for use in the present invention and the content of this application is incorporated by reference GB 9826159 7 discloses markers which comprise two components, which may be poly-ethers, which are analysed by selected reaction monitoring
  • GB 9815166 5 discloses mass markers that bind metal ions, which are also preferred markers for use with this invention
  • Reporter groups that can be detected by more than one detection means may also be desirable as with, for example, a fluorescent marker that incorporate
  • the oligoether or polyether may be a substituted or unsubstituted oligo- or poly-arylether
  • the oligoether or polyether preferably comp ⁇ ses one or more fluorine atom or methyl group substituents, or one or more ⁇ H or 13( ⁇ j tsotopic substituents
  • the mass marker comp ⁇ ses a metal ion-binding moiety
  • the metal ion-binding moiety comprises a porphy ⁇ n, a crown ether, hexahistidine, or a multidentate hgand
  • the metal ion-binding moiety is a bidentate gand or is EDTA
  • the metal ion-binding moiety may be bound to a monovalent, divalent or t ⁇ valent metal ion
  • the metal ion is not especially limited
  • Prefe ⁇ ed metal ions include a transition metal ion, or a metal ion of group IA, IIA or IIIA of the periodic table Particularly preferred metal ions are N ⁇ 2+ , L ⁇ + , Na + , K + , Mg 2+ , Ca 2+ , Sr 2+ , Ba 2+ , or Al 3+
  • the presence of a metal ion on the mass marker increases the sensitivity of detection
  • Mass labels and their linkers can be attached to a nucleic acid molecule at a number of locations in the nucleic acid
  • the 5' hydroxyl of the sugar is the most readily accessible
  • Other favoured positions for modifications are on the base at the 5' position in the py ⁇ midines and the 7' and 8' positions in the pu ⁇ nes These would all be appropriate positions to attach a cleavable mass with the linker of this invention
  • the cleavable linker used in this invention may be cleaved in the ion source of a mass spectrometer by ammonia
  • this invention is not limited to the use of ammonia
  • Most amines are be capable of separating the mass marker from its cognate oligonucleotide and other nucleophiles may also be used
  • Figure 2 shows the negative ion mass spectrum of FT23 at 0 35 pmol/ ⁇ l This spectrum shows a very clean molecular ion less 1 proton at m/z 729 4
  • Figure 3 shows the positive ion mass spectrum of FT23 at 0 35 pmol/ ⁇ l This spectrum shows a strong protonated molecular ion peak at m/z 731 5 and a second stronger peak at m/z 753 3 corresponding to a sodium adduct of FT23
  • the poly-ether mass label with the succinate linker is behaving in a similar manner to a crown ether and is binding strongly to sodium to generate these sodium adducts
  • Figure 4 shows the negative ion mass spectrum of FT23 at 0 35 pmol/ ⁇ l in the presence of a PCR product The molecular ion is not detectable over the background peaks
  • Figure 5 shows the positive ion mass spectrum of FT23 at 0 35 pmol/ ⁇ l in the presence of a PCR product
  • This spectrum does not show any of the protonated molecular ion peak at m/z 731 4 or the sodium adduct peak at m/z 753 3
  • Two additional peaks of significance appear in this spectrum
  • One peak at m/z 739 2 and a second peak at m/z 755 2 These peaks are believed to be end-products of a novel gas phase cleavage reaction discussed below
  • the two peaks corresponding to gas phase cleavage products are the only major ion peaks in this spectrum
  • FT23 is a thymidine derivative which has been protected on the 5' hydroxyl using a tert-butyl dimethylsilyl (TBDMS) protective group
  • TBDMS tert-butyl dimethylsilyl
  • Figure 1 shows how this chemistry can be adapted for use as a gas phase cleavable linker as discussed above.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
EP99933026A 1998-07-13 1999-07-13 Silicium-enthaltende linker für nukleinsäure-massmarker Withdrawn EP1095052A1 (de)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP02021105A EP1275655A1 (de) 1998-07-13 1999-07-13 Silicium enthaltende Linker für Nukleinsäure-Massmarker

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GBGB9815164.0A GB9815164D0 (en) 1998-07-13 1998-07-13 Compounds for mass spectrometry
GB9815164 1998-07-13
PCT/GB1999/002247 WO2000002894A1 (en) 1998-07-13 1999-07-13 Silicon-containing linkers for nucleic acid mass markers

Related Child Applications (1)

Application Number Title Priority Date Filing Date
EP02021105A Division EP1275655A1 (de) 1998-07-13 1999-07-13 Silicium enthaltende Linker für Nukleinsäure-Massmarker

Publications (1)

Publication Number Publication Date
EP1095052A1 true EP1095052A1 (de) 2001-05-02

Family

ID=10835418

Family Applications (2)

Application Number Title Priority Date Filing Date
EP02021105A Withdrawn EP1275655A1 (de) 1998-07-13 1999-07-13 Silicium enthaltende Linker für Nukleinsäure-Massmarker
EP99933026A Withdrawn EP1095052A1 (de) 1998-07-13 1999-07-13 Silicium-enthaltende linker für nukleinsäure-massmarker

Family Applications Before (1)

Application Number Title Priority Date Filing Date
EP02021105A Withdrawn EP1275655A1 (de) 1998-07-13 1999-07-13 Silicium enthaltende Linker für Nukleinsäure-Massmarker

Country Status (7)

Country Link
EP (2) EP1275655A1 (de)
JP (1) JP2002520330A (de)
AU (1) AU772514B2 (de)
CA (2) CA2337207A1 (de)
GB (2) GB9815164D0 (de)
NZ (1) NZ509516A (de)
WO (1) WO2000002894A1 (de)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170089915A1 (en) * 2015-09-30 2017-03-30 Agilent Technologies, Inc. Methods of analyte derivatization and enhanced soft ionization

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PT850320E (pt) * 1996-01-23 2000-05-31 Rapigene Inc Metodos para a deteccao da ligacao num par de ligantes com sensibilidade aumentada
JP3884087B2 (ja) * 1997-01-15 2007-02-21 イクスツィリオン ゲゼルシャフト ミット ベシュレンクテル ハフツング ウント コンパニー コマンディトゲゼルシャフト 質量標識結合ハイブリダイゼーションプローブ
US6410225B1 (en) * 1997-06-27 2002-06-25 Yale University Purification of oligomers
AU734636B2 (en) * 1997-07-11 2001-06-21 Xzillion Gmbh & Co. Kg Characterising nucleic acids
WO1999005319A2 (en) * 1997-07-22 1999-02-04 Rapigene, Inc. Methods and compounds for analyzing nucleic acids by mass spectrometry
GB9823646D0 (en) * 1997-12-19 1998-12-23 Brax Genomics Ltd Compounds for mass spectrometry

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0002894A1 *

Also Published As

Publication number Publication date
GB9815164D0 (en) 1998-09-09
WO2000002894A1 (en) 2000-01-20
GB2340237B (en) 2003-10-08
GB9916401D0 (en) 1999-09-15
AU4920499A (en) 2000-02-01
GB2340237A (en) 2000-02-16
AU772514B2 (en) 2004-04-29
CA2337207A1 (en) 2000-01-20
EP1275655A1 (de) 2003-01-15
CA2385970A1 (en) 2000-01-20
JP2002520330A (ja) 2002-07-09
NZ509516A (en) 2003-01-31

Similar Documents

Publication Publication Date Title
US6287780B1 (en) Compounds for mass spectrometry comprising nucleic acid bases and aryl ether mass markers
Stawinski et al. Arylsulfonyltetrazoles, new coupling reagents and further improvements in the triester method for the synthesis of deoxyribooligonucleotides1
KR101032008B1 (ko) 폴리뉴클레오티드 표지 시약
US4997928A (en) Fluorescent reagents for the preparation of 5'-tagged oligonucleotides
EP1095053B1 (de) Arylsulfon-linker zur massenspektrometrischen analyse
JP2002515588A (ja) 試薬及び方法
EP0466773A1 (de) Kumarinderivate zur verwendung als nukleotidvernetzungsreagenzien.
JPH01250393A (ja) アミノ誘導化されたオリゴヌクレオチドの合成
EP2484671A1 (de) Fluoreszierendes molekül und verfahren zum nachweis einer zielnukleinsäure
AU601361B2 (en) Hybridisation method and probes therefor
US7541455B2 (en) Microwave mediated synthesis of nucleic acid probes
EP1791981A2 (de) Verfahren zur verwendung von schwefelnukleophilen als verbesserte alternativen zu natriumhydrogensulfit für die analyse methylierter dna
WO2000002894A1 (en) Silicon-containing linkers for nucleic acid mass markers
EP1308452B1 (de) Oligonukleotidmarkierungsreagenzien auf der Basis von azyklischen Nukleosiden, und deren derivatisierte Konjugate
JP4779147B2 (ja) ヌクレオチド誘導体及びその利用
JP4963015B2 (ja) ヌクレオチド誘導体及びその利用
JP4467930B2 (ja) ヌクレオチド標識試薬およびそれが導入されたオリゴヌクレオチド誘導体
CA2098187A1 (en) Methods for labelling oligonucleotides
AU2011254065A1 (en) Methods of using sulfur nucleophiles as improved alternatives to sodium bisulfite for methylated DNA analysis
JP2009153381A (ja) オリゴヌクレオチド固定化固相担体

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20010206

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: XZILLION GMBH & CO.KG

17Q First examination report despatched

Effective date: 20011102

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20020923