WO2005057207A1 - Trityl derivatives for enhancing mass spectrometry - Google Patents

Trityl derivatives for enhancing mass spectrometry Download PDF

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Publication number
WO2005057207A1
WO2005057207A1 PCT/GB2004/005140 GB2004005140W WO2005057207A1 WO 2005057207 A1 WO2005057207 A1 WO 2005057207A1 GB 2004005140 W GB2004005140 W GB 2004005140W WO 2005057207 A1 WO2005057207 A1 WO 2005057207A1
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Prior art keywords
formula
biopolymer
group
independently
ion
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PCT/GB2004/005140
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French (fr)
Inventor
Mikhail Sergeevich Shchepinov
Edwin Mellor Southern
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Oxford Gene Technology Ip Limited
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Priority claimed from GB0328414A external-priority patent/GB0328414D0/en
Priority claimed from GB0421110A external-priority patent/GB0421110D0/en
Priority claimed from EP04104605A external-priority patent/EP1506959A3/en
Application filed by Oxford Gene Technology Ip Limited filed Critical Oxford Gene Technology Ip Limited
Priority to EP04805960A priority Critical patent/EP1700116A1/en
Publication of WO2005057207A1 publication Critical patent/WO2005057207A1/en
Priority to US11/892,372 priority patent/US20080248584A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C205/00—Compounds containing nitro groups bound to a carbon skeleton
    • C07C205/39—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by esterified hydroxy groups
    • C07C205/42—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by esterified hydroxy groups having nitro groups or esterified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
    • C07C205/43—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by esterified hydroxy groups having nitro groups or esterified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton to carbon atoms of the same non-condensed six-membered aromatic ring or to carbon atoms of six-membered aromatic rings being part of the same condensed ring system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C215/00—Compounds containing amino and hydroxy groups bound to the same carbon skeleton
    • C07C215/02—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C215/22—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being unsaturated
    • C07C215/28—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being unsaturated and containing six-membered aromatic rings
    • C07C215/30—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being unsaturated and containing six-membered aromatic rings containing hydroxy groups and carbon atoms of six-membered aromatic rings bound to the same carbon atom of the carbon skeleton
    • C07C215/32—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being unsaturated and containing six-membered aromatic rings containing hydroxy groups and carbon atoms of six-membered aromatic rings bound to the same carbon atom of the carbon skeleton containing hydroxy groups and carbon atoms of two six-membered aromatic rings bound to the same carbon atom of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/42—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C331/00—Derivatives of thiocyanic acid or of isothiocyanic acid
    • C07C331/16—Isothiocyanates
    • C07C331/28—Isothiocyanates having isothiocyanate groups bound to carbon atoms of six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C43/00—Ethers; Compounds having groups, groups or groups
    • C07C43/02—Ethers
    • C07C43/20—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring
    • C07C43/23—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring containing hydroxy or O-metal groups
    • 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
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
    • C07D207/34—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two 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
    • C07D207/36—Oxygen or sulfur atoms
    • C07D207/40—2,5-Pyrrolidine-diones
    • C07D207/416—2,5-Pyrrolidine-diones 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 other ring carbon atoms
    • 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
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/44—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members
    • C07D207/444—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5
    • C07D207/448—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide
    • C07D207/452—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide with hydrocarbon radicals, substituted by hetero atoms, directly attached to the ring nitrogen atom
    • 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
    • C07D211/40—Oxygen atoms
    • C07D211/44—Oxygen atoms attached in position 4
    • C07D211/46—Oxygen atoms attached in position 4 having a hydrogen atom as the second substituent in position 4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
    • C07D333/06—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to the ring carbon atoms
    • C07D333/14—Radicals substituted by singly bound hetero atoms other than halogen
    • C07D333/16—Radicals substituted by singly bound hetero atoms other than halogen by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D335/00—Heterocyclic compounds containing six-membered rings having one sulfur atom as the only ring hetero atom
    • C07D335/04—Heterocyclic compounds containing six-membered rings having one sulfur atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
    • C07D335/10—Dibenzothiopyrans; Hydrogenated dibenzothiopyrans
    • C07D335/12—Thioxanthenes
    • C07D335/14—Thioxanthenes 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 in position 9
    • C07D335/16—Oxygen atoms, e.g. thioxanthones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/08—Bridged systems
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/02—Systems containing only non-condensed rings with a three-membered ring

Definitions

  • This invention relates to derivatised biopolymers and ions obtainable therefrom.
  • the invention further relates to compounds and solid supports useful for producing the derivatised biopolymers and ions of the invention.
  • Mass spectrometry is a versatile analytical technique possessing excellent detection range and speed of detection with respect to High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Infra-Red (IR) and Nuclear Magnetic Resonance (NMR).
  • HPLC High Performance Liquid Chromatography
  • GC Gas Chromatography
  • IR Infra-Red
  • NMR Nuclear Magnetic Resonance
  • the prior art attaches the polymer to the ⁇ -triphenylmethyl carbon atom through a non-aromatic linker.
  • the biopolymer is attached to the ⁇ -triarylmethyl carbon atom via an aromatic group adjacent to the central carbon atom. Consequently, ionisation of the prior art derivatives results in separation of the triphenylmethyl derivative and the biopolymer, whereas according to the present invention the biopolymer remains bound to the trityl derivative on ionisation, thereby allowing analysis of the biopolymer by mass spectrometry.
  • the invention provides methods of forming ions from covalent or ionic compounds and solid substrates.
  • the invention provides a method of forming an ion of formula (I): comprising the steps of: (i) reacting a compound of the formula (Ila):
  • the invention further provides a method of forming an ion of formula (I), comprising the steps of: (i) reacting a compound of the formula (lib): (Ar 2 ) n - C- [Ar 1 — (L M ⁇ M ⁇ p ) q ] m -AT X * (lib); with a biopolymer, Bp, having at least one group capable of reacting with M to form a covalent linkage, to provide a biopolymer derivative of the formula (Illb): (Ar 2 ) n - C- [Ar 1 - (L M ⁇ M'— B P ' ⁇ p ) q ] m X * (Illb); and dissociating X* from the derivative of formula (Illb), to form the ion of formula (I); where: X*is a counter-ion to C*; and C*, M, Bp', M', Ar 1 , Ar 2 , L M , n,
  • the compounds of formulae (Ila) or (lib) may optionally be purified after step (i).
  • the invention also provides biopolymer derivatives of the formula (Ilia) or (Illb), as defined above.
  • the biopolymer derivatives of the invention have enhanced ionisability with respect to free biopolymer, B P .
  • the biopolymer derivatives may not require a matrix (e.g. as used in MALDI-MS) in order to elicit ionisation, although a matrix may help to enhance ionisation.
  • ionisation may be obtained without requiring acid treatment, in particular by direct laser illumination.
  • the invention also provides ions of formula (I), as defined above. These ions are stabilised by the resonance effect of the aromatic groups Ar 1 and Ar 2 . Electron- withdrawing groups, when C* is an anion, or electron-donating groups, when C* is a cation, may optionally be provided on Ar 1 and/or Ar 2 to assist this resonance effect. Consequently, the biopolymer derivatives of the invention readily form ions of formula (I) relative to the native biopolymer, B P .
  • the ions of formula (I) are generally only ever seen on a mass spectrum with a single charge, which is advantageous since it reduces cluttering of the mass spectrum.
  • the invention also provides compounds of the formula (Ila) and (lib), as defined above. As mentioned above, these compounds are useful for forming ions of formula (I). As the difference in the molecular mass of the ions of formula (I) and that of the free biopolymer can be accurately calculated, the derivatised compounds of the invention allow analysis of the biopolymer B P , which may be otherwise difficult or impossible to analyse using known mass spectrometrical techniques.
  • compositions of the invention include more uniformity of the signal intensity between different analytes (useful for quantitative studies) and similar desorption properties between compounds with different, but close, masses, so that techniques such as isotope coded affinity tagging (ICAT) can be employed with the compounds of the invention.
  • ICAT isotope coded affinity tagging
  • the homogeneous methods of the invention are particularly appropriate for small molecules, e.g. amines.
  • Solid Supports e.g. amines.
  • the ions of formula (I) may also be formed using a derivatised solid support.
  • the invention therefore provides a method of forming an ion of formula (I) comprising the steps of: (i) reacting a solid support of formula (IVai), (IVaii), or (IVaiii):
  • the cleavable bond of C- - -Ss, Ss- - -Ar 1 or Ss- - -Ar 2 may be a covalent, ionic, hydrogen, dipole-dipole or van der Waals bond.
  • the invention further provides a method of fo ⁇ ning an ion of formula (I) comprising the steps of: (i) reacting a solid support of formula (IVbii) or (IVbiii):
  • the invention further provides a method of forming an ion of formula (I) comprising the steps of: (i) reacting a solid support of formula (IVaiv) or (IVbiv):
  • the solid support is bound to a part of group M" which does not go on to form the residue M'.
  • the derivatised biopolymer will be released from the solid support during the derivativisation step and an additional step of cleaving the biopolymer from the solid support is not required.
  • the modified solid supports of formulae (Vai), (Vaii), (Vaiii), (Vaiv), (Vbii), (Vbiii) or (Vbiv) may optionally be washed after step (i).
  • the invention also provides solid supports of the formulae (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) and (IVbiv), as defined above.
  • the invention provides modified solid supports of the formulae (Vai), (Vaii), (Vaiii), (Vaiv), (Vbii), (Vbiii), and (Vbiv), as defined above.
  • heterogeneous methods of the invention are particularly appropriate for synthetic biopolymers, e.g. oligonucleotides, peptides and carbohydrates.
  • the invention also provides a method for analysing a biopolymer, B P , comprising the steps of: (i) reacting the biopolymer Bp with a compound of formula (Ila) or (lib) or a solid support of formula (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) or (IVbiv); (ii) providing an ion of formula (I); and (iii) analysing the ion of formula (I) by mass spectrometry.
  • the biopolymer will typically have been obtained using a preparative or analytical process. For example, it may have been purified using various separation methods (e.g. 1 -dimensional or 2-dimensional, reverse-phase or normal-phase separation, by e.g. chromatography or electrophoresis) and the separation may be based on any of a number of characteristics (e.g. isoelectric point, molecular weight, charge, hydrophobicity, etc.). Typical methods include 2D SDS-PAGE , 2D liquid chromatography (e.g. Multidimensional Protein Identification Technology, MudPIT, or 2D HPLC methods). The separation method can preferably interface directly with the mass spectrometer.
  • various separation methods e.g. 1 -dimensional or 2-dimensional, reverse-phase or normal-phase separation, by e.g. chromatography or electrophoresis
  • Typical methods include 2D SDS-PAGE , 2D liquid chromatography (e.g. Multidimensional Protein Identification Technology, MudPIT, or 2D HPLC methods).
  • a particularly preferred method involves 2D-PAGE of a biopolymer, or mixture of biopolymers, selection of a spot of interest in the electrophoretogram, and then derivatisation and analysis of that spot using the techniques of the invention.
  • the biopolymer may be proteolytically digested prior to its analysis (typically within the PAGE gel, but optionally digested after extraction from the gel) and/or may itself be the product of a proteolytic digest.
  • the invention also provides, in a method for analysing a biopolymer, Bp, the improvement consisting of: (i) reacting a biopolymer, Bp with a compound of formula (Ila) or (lib) or a solid support of formula (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) or (IVbiv); (ii) providing an ion of formula (I); and (iii) analysing the ion by mass spectrometry.
  • the analysis by mass spectrometry is carried out in a spectrometer which is suitable for MALDI-TOF spectrometry.
  • the ion source may be a matrix-assisted laser desorption ionisation (MALDI), an electrospray ionisation (ESI) ion source, a Fast-Atom Bombardment (FAB) ion source.
  • MALDI matrix-assisted laser desorption ionisation
  • ESI electrospray ionisation
  • FAB Fast-Atom Bombardment
  • the ion source is a MALDI ion source.
  • the MALDI ion source may be traditional MALDI source (under vacuum) or may be an atmospheric pressure MALDI (AP-MALDI) source.
  • MALDI is a preferred ionisation method, although the use of a matrix is generally not required
  • the mass analyser may be a time of flight (TOF), quadrupole time of flight (Q-TOF), ion trap (IT), quadrupole ion trap (Q-IT), triple quadrupole (QQQ) Ion Trap or Time-Of- Flight Time-Of-Flight (TOFTOF) or Fourier transform ion cyclotron resonance (FTICR) mass analyser.
  • TOF time of flight
  • Q-TOF quadrupole time of flight
  • Ion trap Ion trap
  • Q-IT quadrupole ion trap
  • QQQQ triple quadrupole
  • Ion Trap or Time-Of- Flight Time-Of-Flight (TOFTOF) or Fourier transform ion cyclotron resonance (FTICR) mass analyser.
  • TOFTOF Time-Of- Flight Time-Of-Flight
  • FTICR Fourier transform ion cyclotron resonance
  • the mass analyser is a TOF mass analyser.
  • the non-covalent bond may be direct between M' and B P ' or may be provided by one or more binding groups present on M' and/or B P '.
  • Preferred non-covalent bonds are those having an association constant (K a ) of at least 10 14 M "1 , preferably about 10 15 M "1 .
  • K a association constant
  • one of M' and Bp' will have a binding group comprising biotin
  • the other of M' and B P ' will have a binding group comprising avidin or streptavidin.
  • the compounds of the invention comprise a non-covalent bond between M' and Bp' and a cleavable bond between C and Ss, Ar 1 and Ss, or Ar 2 and S s
  • these bonds are differentially cleavable.
  • the non-covalent bond between M' and B P ' is not cleaved under conditions which the cleavable bond between C and Ss, Ar 1 and Ss, or Ar 2 and Ss, as appropriate, is cleaved.
  • Ar 1 by more than one covalent bond (e.g. 2 or 3 bonds) which are either single, double or triple covalent bonds, or one or more multiple bonds (e.g. double or triple covalent bonds). All the other features of the invention are the same except the groups which relate to the bond or bonds between
  • the present invention may be used for ionising any molecule or complex of molecules which requires mass spectrum analysis.
  • the above-mentioned embodiments of the invention may also be provided in which B P is replaced by any molecule or complex having at least one group capable of reacting with M to form a covalent linkage. All the other features of the invention are the same, except group M is group capable of reacting with the molecule to be analysed.
  • Examples of other molecules which may be analysed in the present invention include non-biological polymers (e.g. synthetic polyesters, polyamides and polycarbonates), petrochemicals and small molecules (e.g. alkanes, alkenes, amines, alcohols, esters and amides). Amines are particularly preferred.
  • PNA peptide nucleic acid
  • all embodiments of the invention including products of formulae (I), (Ila), (lib), (Ilia), (Illb), (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii), (IVbiv), (Vai), (Vaii), (Vaiv), (Vbii), (Vbiii) and (Vbiv), methods of forming an ion of formula (I) and methods of analysis) involving or relating to the compound of formula (XI) are disclaimed.
  • C* bears a single positive charge such that ions of the invention are cations and the ion of formula (I) has the following structure:
  • n may not be less than 1.
  • p 1, 2 or 3.
  • p 1.
  • q 1 , 2 or 3.
  • q 1.
  • biopolymer' includes polymers found in biological samples, including polypeptides, polysaccharides, and polynucleotides (e.g. DNA or RNA).
  • Polypeptides may be simple copolymers of amino acids, or they may include post-translational modifications e.g. glycosylation, lipidation, phosphorylation, etc.
  • Polynucleotides may be single-stranded (in whole or in part), double-stranded (in whole or in part), DNA/RNA hybrids, etc.
  • RNA may be mRNA, rRNA or tRNA.
  • Biopolymers for use in the invention comprise two or more monomers, which may be the same or different as each other.
  • Preferred biopolymers comprise at eas pp monomers, where pp is 5 or more (e.g. 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250). More preferred biopolymers comprise ppp or fewer monomers where ppp is 300 or less (e.g. 200, 100, 50).
  • Biopolymers may have a molecular mass of at least qq kDa, where qq - 0.5 or more (e.g. 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, etc.).
  • Preferred biopolymers are those having a molecular mass within the range of detection of a mass spectrometer. More prefened biopolymers have a molecular mass of qqq kDa or less, where qqq is 30 or less (e.g. 20, 10, 5).
  • (Ar 2 ) — C— [Ar 1 -(L M ⁇ M' ⁇ p ) q ] m * (IX) of the cation of formula (I) is significantly less than the mass, m(B P '), of the biopolymer residue B P '.
  • the ratio m(Bp') / m(IX) is preferably more than nn, where nn is at least 2 (e.g. 3, 4, 5, 10, 100, 1000, etc.).
  • the invention is suitable for use with purified biopolymers or mixtures of biopolymers.
  • a pure recombinant protein could be derivatised and analysed by MS, or biopolymers within a cellular lysate or extract could be derivatives and then analysed.
  • Preferred biopolymers are polypeptides. Particularly prefened biopolymers are polypeptides formed after proteolytic digestion of a protein.
  • Biopolymers bound to solid supports In preferred embodiments of the invention the biopolymer is bound to a solid support such that it is cleavable from the solid support at least once it has been derivatised by a compound of the invention. Bp is thus derivatised in situ while bound to the support, and is then released.
  • this aspect of the invention is particular relevant to methods involving compounds of formulae (Ila) and (lib).
  • the biopolymer may be bound to the solid support by a covalent, ionic, hydrogen, dipole-dipole or van der Waals bond (also known as a dispersion bond or a London forces bond).
  • the covalent, ionic, hydrogen, dipole-dipole or van der Waals bond may be direct between the biopolymer and the solid support or may be provided by one or more binding groups present on the biopolymer and/or solid support. Prefened groups are non-covalent groups.
  • the solid support is provided with -(NMe 3 ) + binding groups and the biopolymer has a net negative charge, or vice versa (i.e. the -(NMe 3 ) + is on the biopolymer).
  • the solid support is provided with anions such as carboxylate, phosphate or sulphate, or anions formed from acid groups, and the biopolymer (e.g. a histone) has a net positive charge, or vice versa.
  • the biopolymers have at least one reactive group capable of reacting with M to form a covalent linkage.
  • groups typically include naturally occurring groups and groups formed synthetically on the biopolymer.
  • Naturally occurring groups include lipid groups of lipoproteins (e.g. myristoyl, glycosylphosphatidylinositol, ethanolamine phosphoglycerol, palmitate, stearate, S- or N- or O-acyl groups, lipoic acid, isoprenyl, geranylgeranyl, farnesyl, etc.), amide, carbohydrate groups of N- and O- glycoproteins, amine groups (e.g. on lysine residues or at the ⁇ -terminus of a protein), hydroxyl (e.g.
  • lipoproteins e.g. myristoyl, glycosylphosphatidylinositol, ethanolamine phosphoglycerol, palmitate, stearate, S- or N- or O-acyl groups, lipoic acid, isoprenyl, geranylgeranyl, farnesyl, etc.
  • amide carbohydrate groups of N- and O- glycoproteins
  • the reactive group is bound to the biopolymer by one or more covalent bonds (e.g. 2 or 3 bonds), which are either single, double or triple covalent bonds (preferably single bonds).
  • the reactive group is bound to the biopolymer by one single bond.
  • Another group which may be formed naturally or synthetically on the biopolymer and which is bound to the biopolymer by one bond is -CN.
  • Other groups which may be formed naturally or synthetically on the biopolymer and which are bound to the biopolymer by one bond are: -P(ZR)Y e.g. -P(OH)Y; -PY 2 ; -Z-P(ZR)Y; -Z-PY 2 ; -P(R)Y e.g. -P(H)Y; -Z-P(R)Y.
  • a particularly preferred group is -Z-P(ZR)Y, especially a phosphoramidite group:
  • a group which may be formed naturally or synthetically on the biopolymer and which is bound to the biopolymer by one bond is -Y.
  • the reactive group is halo (especially iodo)
  • the reactive group may be bound to an aliphatic or aromatic carbon.
  • Groups which may be formed synthetically on the biopolymer and which are bound to the biopolymer by three bonds include .
  • Preferred groups include nucleophilic groups, either natural or synthetic, e.g.: -NR 2 e.g. -NHR, especially -NH 2 ; -SR e.g. -SH; -OR e.g. -OH; -N(R)- e.g. -NH-; -S-; and -0-.
  • the groups -NH 2 , -SH and -OH are particularly preferred.
  • Another preferred reactive group is maleimidyl:
  • Y is independently a leaving group, including groups capable of leaving in an SN 2 substitution reaction or being eliminated in an addition-elimination reaction with the reactive group of the biopolymer B P .
  • Y include halogen (preferably iodo), C 1-8 hydrocarbyloxy (e.g. C 1-8 alkoxy), C 1-8 hydrocarbyloxy substituted with one or more A, C 1-8 heterohydrocarbyloxy, C 1-8 heterohydrocarbyloxy substituted with one or more A, mesyl, tosyl, pentafluorophenyl, -O-succinimidyl (formula VII) or a sulfo sodium salt thereof (sulfoNHS - formula Vila), -S-succinimidyl, or phenyloxy substituted with one or more A e.g. p-nitrophenyloxy (formula VIII) or pentafluorophenoxy (formula Villa).
  • halogen preferably iodo
  • C 1-8 hydrocarbyloxy e.g. C 1-8 alkoxy
  • C 1-8 hydrocarbyloxy substituted with one or more A C 1-8 heterohydr
  • prefened reactive group on the biopolymer are:
  • Y include -ZR.
  • Particularly preferred examples of Y are -ZH (e.g. -OH or -NH 2 ) and -Z-C 1-8 alkyl groups such as -NH-C 1-8 alkyl groups (e.g. -NHMe) and -0-C ⁇ -8 alkyl groups (e.g. -O-t-butyl).
  • preferred reactive groups are -C(0)-NH-C 1-8 alkyl and -C(0)-0-C 1- 8 alkyl (e.g. -C(O)-O-t-butyl).
  • Y include -Z-ZR.
  • Particularly preferred examples include -NR-NR 2 , especially -NH-NH 2 , and -ONR 2 , especially -0-NH 2 .
  • R is independently H, C ⁇ -8 hydrocarbyl (e.g. C 1-8 alkyl) or C 1-8 hydrocarbyl substituted with one or more A.
  • R is preferably H.
  • the reactive group may be -Si(R) 2 -Y, with Y being halo (e.g. chloro) being especially prefened.
  • Prefened groups R in this embodiment are C 1-8 alkyl, especially methyl.
  • a particularly prefened reactive group in this embodiment is -Si(Me) 2 Cl.
  • groups which may be formed naturally or synthetically on the biopolymer include groups capable of reacting in a cycloaddition reaction, especially a Diels-Alder reaction.
  • the reactive group on the biopolymer is either a diene or a dienophile.
  • Prefened diene groups are
  • a 1 is -R 1 or -Z'R 1 , where R 1 and Z 1 are defined below.
  • the group M is capable of reacting with the reactive group of the biopolymer, B P , to form a covalent linkage.
  • Group 'M' is shown as 'AFG' in the drawings.
  • the group M is bound to L M by one or more covalent bonds (e.g. 2 or 3 bonds, especially 2 such — L M M as ⁇ - — ), which are either single, double or triple covalent bonds (preferably single bonds).
  • M is bound to L M by one single bond.
  • M is bound by more than one L , such L M either being attached to the same or different Ar 1 or Ar 2 .
  • M is bound by more than one L M from different Ar 1 or Ar 2 , e.g. :
  • Another example of a group M bound to L M by one bond is -CN.
  • group M bound to L M by one bond are -P(ZR)Y e.g. -P(OH)Y; -PY 2 ; -Z-P(ZR)Y; -Z-PY 2 ; -P(R)Y e.g. -P(H)Y; -Z-P(R)Y.
  • a particularly preferred group M is -Z-P(ZR)Y, especially a phosphoramidite group: N(iPr) 2 _>-P o — , — CN
  • group M bound to L M by one bond is -Y.
  • M may be bound to an aliphatic or aromatic carbon.
  • M is halo (e.g. iodo) and is bound to an aromatic carbon
  • L M may, for example, be a single bond.
  • Examples of group M bound to L by three bonds include . Y )
  • Another preferred electrophilic group M is -CN.
  • group M are orthoesters, e.g. -C(OR) 3 .
  • the R groups are linked together to form a hydrocarbyl group, e.g. a C 1-8 alkyl group.
  • a preferred example of group M in this embodiment is:
  • Another prefened group M is maleimido.
  • Prefened Y groups when present on M are those capable of leaving in an SN 2 substitution reaction or being eliminated in an addition-elimination reaction with the reactive group of the biopolymer B P .
  • Y include halogen (preferably iodo), C 1-8 hydrocarbyloxy (e.g. C 1-8 alkoxy), C 1-8 hydrocarbyloxy substituted with one or more A, C 1-8 heterohydrocarbyloxy, C 1-8 heterohydrocarbyloxy substituted with one or more A, mesyl, tosyl, pentafluorophenyl, -O-succinimidyl (formula VII) or a sulfo sodium salt thereof (sulfoNHS - formula Vila), -S-succinimidyl, or phenyloxy substituted with one or more A e.g. p-nitrophenyloxy (formula VIII) or pentafluorophenoxy (formula Villa).
  • halogen preferably iodo
  • C 1-8 hydrocarbyloxy e.g. C 1-8 alkoxy
  • C 1-8 hydrocarbyloxy substituted with one or more A C 1-8 heterohydr
  • Y include -ZR.
  • Particularly prefened examples of Y are -ZH (e.g. -OH or -NH 2 ) and -Z-C 1-8 alkyl groups such as -NH-C 1-8 alkyl groups (e.g. -NHMe) and -0-C 1-8 alkyl groups (e.g. -O-t-butyl).
  • prefened groups M are -C(0)-NH-C 1-8 alkyl (e.g. -C(O)NHMe) and -C(0)-0-C 1-8 alkyl (e.g. -C(O)-O-t-butyl).
  • Other preferred examples of Y include -Z-ZR.
  • Particularly prefened examples include -NR-NR 2 , especially -NH-NH 2 , and -ONR 2 , especially -0-NH 2 .
  • M may be -Si(R) 2 -Y, with Y being halo (e.g. chloro) being especially preferred.
  • Preferred groups R in this embodiment are C 1-8 alkyl, especially methyl.
  • a particularly prefened group M in this embodiment is -Si(Me) Cl.
  • M may be -C(Ar 2 ) 2 X.
  • Prefened groups Ar 2 and X are set out below. In this embodiment it is prefened that L M is a bond.
  • a particularly prefened group M in this embodiment is:
  • groups M include groups capable of reacting in a cycloaddition reaction, especially a Diels- Alder reaction.
  • the reactive group on the biopolymer is either a diene or a dienophile.
  • Prefened diene groups are
  • a particularly preferred dienophile group is maleimidyl.
  • Preferred examples of group M are shown in figures 1 IA and 1 IB.
  • the reactive group on the biopolymer [shown as 'F' in the drawings] and the group M [shown as 'AFG' in the drawings] must be dependently selected in order to form the covalent linkage.
  • M may also be -CN.
  • one of the reactive group on the biopolymer and group M is a maleimidyl and the other will be a -SH group.
  • one of the reactive group on the biopolymer and group M will typically be a diene and the other will be a dienophile.
  • Prefened covalent linkages are those produced through the reaction of the following groups:
  • the covalent residue M'-B P ' is the reaction product of M and B P .
  • B P ' will generally be the same as B P except that instead of the reactive group, B P ' will have a residue of the reactive group covalently bound to the residue M'.
  • M" is the same as M except that the group S s is bound to a portion of M which does not form part of M'. Thus, M" is a residue of M formable by the conjugation of M and S s . However, M" need not necessarily be formed by the conjugation of M and Ss.
  • M"- - -Ss comprises a covalent, ionic, dipole-dipole, hydrogen, or van der Waals bond.
  • the covalent, ionic, hydrogen, dipole-dipole or van der Waals bond may be direct between M" and S s or may be provided by one or more binding groups present on M" and/or Ss.
  • Prefened groups M" are groups M having a leaving group, wherein the group Ss is bound to the leaving group, e.g. groups M mentioned above having a leaving group Y, wherein the group Ss is bound to the leaving group Y.
  • a particularly prefened group M" is:
  • group L M is a linker atom or group, it has a sufficient number of linking covalent bonds to link L M to the group Ar 1 by a single covalent bond (or more, as appropriate) and to link L M to the p instances of M (or M', as appropriate) groups (which may be attached to L M by one or more bonds).
  • the group L M may be directly bound to the aromatic part of Ar 1 , bound to one or more of the substituents A of Ar 1 , or both. Preferably, L M is bound directly to the aromatic part of Ar 1 .
  • L M may be bound to L 5 .
  • preferred linker atoms are O or S, particularly O.
  • preferred linker groups in the orientation Ar 1 -(L M ⁇ M ⁇ p ) q or Ar'- L M lM' ⁇ , as appropriate, are -E M -, -(D M ) t -, -(E M -D M ) t -, -(D M -E M ) t -, -E M -(D M -E M ) t - or -D M -(E M -D M )t ⁇ , where a sufficient number of linking covalent bonds, in addition to the covalent bonds at the chain termini shown, are provided on groups E M and D M for linking the p instances of M (or M') groups.
  • D M is independently C 1-8 hydrocarbylene or C 1-8 hydrocarbylene substituted with one or more A.
  • Prefened D M are C ⁇ -8 alkylene, C 1-8 alkenylene and C 1-8 alkynylene, especially C 1-8 alkylene and C 1-8 alkynylene, each optionally substituted with one or more A (preferably unsubstituted).
  • a prefened substituent A is 2 H.
  • Prefened L M in the orientation Ar 1 -(L M ⁇ M ⁇ p ) q or Ar 1 -(L M ⁇ M' ⁇ p ) q are: -CH 2 CH 2 -; -C ⁇ C-CH 2 CH 2 CH 2 -; -(CH 2 ) 5 -; -CD 2 CD 2 CH 2 CH 2 CH 2 -; -C ⁇ C-CH 2 - and -CH 2 CH 2 CH 2 -.
  • D M may also be C 1-8 heterohydrocarbylene or C 1-8 heterohydrocarbylene substituted with one or more A.
  • -scycloheteroalkylene groups are particularly preferred.
  • preferred L M groups -D M -E M -D M - are, in the orientation Ar 1 -(LM ⁇ M ⁇ p ) q or as appropriate, -C 1-8 alkylene-C(0)-C 1-8 cycloheteroalkylene (preferably where the hetero atom is N and is bound to the carboxy), especially:
  • t 1 or more, e.g. from 1 to 50, lto 40, 1 to 30, 1 to 20 or 1 to 10.
  • t 1, 2, 3, 4, 5, 6, 7, 8,
  • L M links one group M (or M') to Ar 1 , M (or M') is linked to L M by a single covalent bond and therefore no additional bonds are required (e.g. L ⁇ M ⁇ ! may be -E M - ⁇ M ⁇ , -(D M ) t - ⁇ M ⁇ , -(E M -D M )t- ⁇ M ⁇ , -(D M -E M ) t - ⁇ M ⁇ , -E M -(D M -E M ) t - ⁇ M ⁇ or -D M -(E M -D M ) t - ⁇ M ⁇ ).
  • L M includes a group which also falls within the definition of group M
  • the group M is preferably more reactive than the group included in L M -
  • L M is preferably -(D M ) t -, -(E M -D M ) t -,
  • D M is preferably C 1-8 alkylene, preferably methylene or ethylene.
  • E M is preferably (in the orientation or or O (preferably O)
  • D M is preferably C ⁇ -8 alkylene, preferably ethylene, propylene, butylene or pentylene (preferably ethylene or propylene).
  • t is preferably 1.
  • Ar ⁇ M fMj ⁇ q or Ar 1 -(L M ⁇ M' ⁇ p ) q are, as appropriate,, -0-CH 2 CH 2 CH 2 - and -0-CH 2 CH 2 CH 2 CH 2 CH 2 -.
  • E M is C 1-8 alkylene and t is 1.
  • Particularly prefened L M groups are:
  • the group -(E M -D M )t- is prefened, a particularly preferred example of which is (in the orientation or Ar 1 -(L M ⁇ M , ⁇ p ) q , as appropriate)
  • L M is a single covalent bond.
  • L M is preferably provided in a position ortho or para to C*.
  • Ar 2 is other than phenyl, L M is preferably attached to an atom which bears the charge in at least one of the resonance structures of the ions of formula (I).
  • L M is preferably an electron-donating group.
  • C* is an anion
  • L M is preferably an electron- withdrawing group.
  • Prefened examples of L M are shown in figure 10A and 10B.
  • C- - -S s , Ss- - -Ar 1 and Ss- - -Ar 2 comprise a cleavable covalent, ionic, hydrogen, dipole-dipole or van der Waals bond (also known as a dispersion bond or a London forces bond).
  • the covalent, ionic, hydrogen, dipole-dipole or van der Waals bond may be direct between C and Ss, Ar 1 and Ss, or Ar 2 and Ss, or may be provided by one or more binding groups present on C and/or Ss, Ar 1 and/or Ss, or Ar 2 and/or Ss, respectively.
  • the bond may be direct (e.g. C-Ss, A ⁇ -Ss or Ai ⁇ -Ss, respectively) or may be provided by a linker atom or group L 4 (e.g. C-L 4 -Ss, Ar 1 -L 4 -S s or Ar-L 4 -Ss, respectively).
  • prefened linker groups are -E 4 -, -(D 4 ) t »-, -(E 4 -D 4 )t"-, -(D 4 -E 4 )t"-, -E 4 -(D 4 -E 4 ) t - or -D 4 -(E 4 -D 4 ) r -.
  • D 4 is independently C 1-8 hydrocarbylene or C ⁇ -8 hydrocarbylene substituted with one or more A.
  • t" 1 or more, e.g. from 1 to 50, lto 40, 1 to 30, 1 to 20 or 1 to 10.
  • t" 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • L 4 includes a group which also falls within the definition of group M
  • the group M is preferably more reactive than the group included in L 5 .
  • L 4 is preferably a linker atom, preferably O or S, particularly O.
  • L 4 is preferably covalently attached to the Ss by a sulphide or disulphide group. Ionic Bonding
  • the bond is typically direct (e.g. C* S s *, where Ss* is a solid support counterionto C*).
  • binding groups e.g. chelating ligands, present on C or Ss, Ar 1 or Ss, or Ar 2 or Ss, respectively.
  • the chelating ligand is typically only present on Ss and chelates with C*.
  • Suitable chelating ligands which can bind anions include polyamines and cryptands.
  • Suitable chelating ligands which can bind cations include polyacidic compounds (e.g. EDTA) and crown ethers.
  • the bond is usually provided by binding groups present on C or Ss, Ar 1 or Ss, or Ar 2 or Ss, respectively.
  • one of C or S s , Ar 1 or Ss, or Ar 2 or Ss will have a binding group bearing one or more hydroxy, amino or thio hydrogen atoms
  • the other of C or Ss, Ar 1 or Ss, or Ar 2 or Ss, respectively will have a binding group bearing an atom having one or more lone pair of electrons (e.g. an oxygen, sulphur or nitrogen atom).
  • one of C or Ss, Ar 1 or S s , or Ar 2 or Ss, as appropriate, will have a binding group comprising biotin, and the other of C or Ss, Ar 1 or Ss, or Ar 2 or S s , respectively, will have a binding group comprising avidin or streptavidin.
  • the hydrogen bond may be direct.
  • the bond is a dipole-dipole bond, it may be formed between permanent dipoles or between a permanent dipole and an induced dipole.
  • one of Ss and the compound of the invention has a permanent dipole and the other of Ss and the compound of the invention has an induced dipole or a permanent dipole, the attraction between the dipoles forming a dipole-dipole bond.
  • Ss comprises binding groups (e.g. acid groups, -(NMe 3 ) + , carboxy, carboxylate, phosphate or sulphate groups) which produce a dipole at the surface of the solid support to bind the compound of the invention.
  • binding groups e.g. acid groups, -(NMe 3 ) + , carboxy, carboxylate, phosphate or sulphate groups
  • the bonding is usually provided by binding groups present on C or Ss, Ar 1 or Ss, or Ar 2 or Ss, respectively.
  • the bonding is usually provided by binding groups present on C or Ss, Ar 1 or Ss, or Ar 2 or Ss, respectively.
  • at least one, but preferably both, of C or S s , Ar 1 or Ss, or Ar 2 or Ss, as appropriate, will have a hydrocarbyl or heterohydrocarbyl group (usually a large hydrocarbyl group having at least ten carbon atoms up to about 50 carbon atoms), optionally substituted with one or more A.
  • Polyfluorinated hydrocarbyl and heterohydrocarbyl groups are particularly prefened.
  • the hydrocarbyl or heterohydrocarbyl groups are aryl or heteroaryl groups or groups of the formula -C(R 6 ) 2 Ar 3 , -C(R 6 )(Ar 3 ) 2 or -C(Ar 3 ) 3; where Ar 3 is independently defined the same as Ar 2 and R 6 is H, C 1-8 hydrocarbyl, C ⁇ -8 hydrocarbyl substituted by one or more A, C 1-8 heterohydrocarbyl or C 1-8 heterohydrocarbyl substituted by one or more A.
  • a prefened binding group is tetrabenzofullerene (formula X).
  • the van der Waals bond may be direct.
  • the ions of formula (I) have a pK r+ value of at least zz, where zz is 0 or more (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). More preferably, zz is 1 or more, still more preferably 2 or more, still more preferably 3 or more.
  • the compounds of formula (Ila), (lib), (Ilia) or (Illb) or the solid supports of formula (IVai), (IVaii), (IVaiii), (IVbii), (IVbiii), (IVaiv) or (IVbiv) provide ions of formula (I') having a pK r+ value of at least zz, where zz is defined above.
  • the C-X bonds are cleavable by irradiation, electron bombardment, electrospray, fast atom bombardment (FAB), inductively coupled plasma (ICP) or chemical ionisation.
  • the C-X bonds are cleavable by inadiation or chemical ionisation.
  • 'irradiation' includes, for example, laser illumination, in particular as used in MALDI mass spectrometry.
  • Laser light of about 340 nm is particularly preferred because it is typically used in MALDI mass spectrometers.
  • 'electron bombardment' includes, for example, bombardment with electrons having energy of about 70 ev.
  • Chemical ionisation can be effected, for example, by treatment with acid or acidic matrices (e.g. acidic matrices used in MALDI analysis).
  • group X is halogen, hydroxy, C 1-8 hydrocarbyloxy, C ⁇ -8 hydrocarbyloxy substituted with one or more A, C ⁇ -8 heterohydrocarbyloxy, C 1-8 heterohydrocarbyloxy substituted with one or more A, mesyl, tosyl, pentafluorophenyl, -O-succinimidyl -S-succinimidyl, or phenyloxy substituted with one or more A e.g. p-nitrophenyloxy.
  • the groups pentafluorophenyl, -O-succinimidyl, -S-succinimidyl, and p-nitrophenyloxy are particularly preferred.
  • Particularly prefened groups X are halogen, hydroxy, C 1-8 hydrocarbyloxy. Especially preferred groups are hydroxy, ethoxy and chloro groups.
  • prefened groups X are alkyl ethers, e.g. :
  • Group X may also be a -Q-oligonucleotide, where Q is O, S or N(R), where R is H, C 1-8 hydrocarbyl or C ⁇ -8 hydrocarbyl substituted with one or more A.
  • Q is preferably O.
  • Group X may also be a nucleoside, preferably where the nucleoside is bound via its 5' end, e.g. :
  • B P is an antibody (particularly where it is a monoclonal antibody that recognises a tumour-associated antigen)
  • X is not: or, optionally, X is not any other 2,6-diaminopurine nucleoside prodrug group.
  • X is not H. If X is H, preferably at least one of Ar 1 and Ar 2 is polycyclic, heterocyclic or unsubstituted. Prefened examples of group X are shown in figure 13.
  • X* is any counterion for forming salts with compounds of the invention.
  • X* includes ions having single charges and multiple charges. Typically ions having multiple charges will be associated with an appropriate number of compounds of formula (lib), (Illb), (IVbii), (IVbiii), (IVbiv), (Vbii), (Vbiii) or (Vbiv) in order balance the charge. Ions having multiple charges include doubly charged ions (e.g. S0 4 2" ) and triply charged ions. X* preferably has a single charge.
  • the counterion X* may be dissociated from the derivative of formula (lib), (Illb), (IVbii), (IVbiii), (IVbiv), (Vbii), (Vbiii) or (Vbiv) by inadiation, electron bombardment, electrospray, fast atom bombardment (FAB), inductively coupled plasma (ICP) or chemical ionisation.
  • the counterion X* may be dissociated by inadiation.
  • X* is preferably FT 1" .
  • X* is preferably, BF 6 “ or C10 4 " .
  • X* is an anion
  • the C- - -S s , Ss- - -Ar 1 or Ss Ar 2 bonds are cleavable by irradiation, electron bombardment, electrospray, fast atom bombardment (FAB), inductively coupled plasma (ICP) or chemical ionisation.
  • FAB fast atom bombardment
  • ICP inductively coupled plasma
  • the C- - -Ss, Ss- - -Ar 1 or Ss- - -Ar 2 bonds are cleavable by irradiation or chemical ionisation.
  • the C- - -Ss, Ss- - -Ar 1 or Ss- - -Ar 2 bonds may be cleaved simultaneously or sequentially with the cleaving of the C-X bond or the dissociation of X*, as appropriate, by selection of suitable cleaving/dissociating conditions.
  • the C- - -Ss bond in the solid support of formula (Vai) may be cleaved in sub-steps of step (iia) so that in a first sub-step a residue X (where X is the leaving group defined above) is provided and in a second subsequent sub-step the C-X bond is cleaved thereby forming the ion of formula (I).
  • the second sub-step may be canied out substantially (e.g. seconds, minutes, hours or even days) after the first sub-step.
  • Ar 2 is independently an aromatic group or an aromatic group substituted with one or more A and is preferably independently cyclopropyl, cyclopropyl substituted with one or more A, aryl, aryl substituted with one or more A, heteroaryl, or heteroaryl substituted with one or more A.
  • Ar 2 is preferably C 6-30 aryl or substituted C 6-3 o aryl. Where heteroaryl or substituted heteroaryl, Ar 2 is preferably C 6-3 o heteroaryl or substituted C 6-3 o heteroaryl.
  • monocyclic aromatic groups e.g. phenyl or pyridyl
  • fused polycyclic aromatic groups e.g. napthyl, such as 1-napthyl or 2-napthyl
  • aryl groups are monovalent derivatives of aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, chrysene, coronene, fluoranthene, fluorene, ⁇ s-indacene, s- indacene, indene, naphthalene, ovalene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene and rubicene, which groups may be optionally substituted by one or more A.
  • heteroaryl groups are monovalent derivatives of acridine, carbazole, ?-carboline, chromene, cinnoline, furan, imidazole, indazole, indole, indolizine, isobenzofuran, isochromene, isoindole, isoquinoline, isothiazole, isoxazole, naphthyridine, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pynolizine, quinazoline, quinoline, quinolizine, quinoxaline, thiophene and xanthene, which groups may be optionally substituted by one or more A.
  • Preferred heteroaryl groups are five- and six-membered monovalent derivatives, such as the monovalent derivatives of furan, imidazole, isothiazole, isoxazole, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pynolizine and thiophene.
  • the five-membered monovalent derivatives are particularly prefened, i.e. the monovalent derivatives of furan, imidazole, isothiazole, isoxazole, pyrazole, pyrrole and thiophene.
  • the heteroaryl groups may be attached to the remainder of the compound by any carbon or hetero (e.g. nitrogen) atom.
  • Ar 2 is preferably C 6-30 aryl substituted by one or more A, preferably phenyl or napthyl (e.g. 1-napthyl or 2-napthyl, especially 2-napthyl) substituted by one or more A, more preferably phenyl substituted by one or more A.
  • A is preferably provided in a position ortho or para to C*.
  • Ar 2 is other than phenyl, A is preferably attached to an atom which bears the charge in at least one of the resonance structures of the ions of formula (I). Fused polycyclic aromatic groups, optionally substituted with one or more A, are particularly preferred.
  • a particularly prefened Ar 2 is unsubstituted pyrenyl or pyrenyl substituted with one or more A. Unsubstituted pyrenyl is preferred.
  • the pyrenyl group may be 1 -pyrenyl, 2-pyrenyl or 4-pyrenyl.
  • Prefened heteroaryl Ar 2 groups, whether substituted or unsubstituted, are pyridyl, pyrrolyl, thienyl and furyl, especially thienyl.
  • a prefened Ar 2 group is thiophenyl or thiophenyl substituted with one or more A. Unsubstituted thiophenyl is prefened. Examples of thiophenyl are thiophen-2-yl and thiophen-3-yl, with thiophen- 2-yl being especially prefened.
  • Ar 2 is preferably substituted by 1, 2 or 3 A.
  • Ar 2 is preferably:
  • Ar 2 is preferably:
  • Ar 2 is cyclopropyl or cyclopropyl substituted with one or more A. Unsubstituted cyclopropyl is prefened. One or more, preferably one, of Ar 2 may be cyclopropyl.
  • Preferred examples of group Ar 2 are shown in figures 12A and 12B.
  • Ar 1 is independently an aromatic group or an aromatic group substituted with one or more A.
  • the definition of Ar 1 is the same as Ar 2 (as defined above), except that the valency of the group Ar 1 is adapted to accommodate the q instances of the linker L M .
  • Preferred Ar 2 groups are also prefened Ar 1 groups, (as defined above), except that the valency of the group Ar 1 is adapted to accommodate the q instances of the linker L M .
  • Ar 1 is a divalent radical and is preferably independently cyclopropylene, cyclopropylene substituted with one or more A, arylene, arylene substituted with one or more A, heteroarylene, or heteroarylene substituted with one or more A.
  • arylene or substituted arylene Ar 1 is preferably C 6-3 o arylene or substituted C 6-3 o arylene.
  • heteroarylene or substituted heteroarylene Ar 1 is preferably C 6-30 heteroarylene or substituted C 6-3 o heteroarylene.
  • monocyclic aromatic groups e.g. phenylene or pyridylene
  • fused polycyclic aromatic groups e.g. napthylene
  • arylene groups are polyvalent derivatives (where the valency is adapted to accommodate the q instances of the linker L M ) of aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, chrysene, coronene, fluoranthene, fluorene, ⁇ s-indacene, s-indacene, indene, naphthalene, ovalene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene and rubicene, which groups may be optionally substituted by one or more A.
  • heteroarylene groups are polyvalent derivatives (where the valency is adapted to accommodate the q instances of the linker L M ) of acridine, carbazole, ?-carboline, chromene, cinnoline, furan, imidazole, indazole, indole, indolizine, isobenzofuran, isochromene, isoindole, isoquinoline, isothiazole, isoxazole, naphthyridine, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pynolizine, quinazoline, quinoline, quinolizine, quinoxaline, thiophene and xanthene, which groups may be optionally substituted by one or more A.
  • Preferred heteroaryl groups are five- and six- membered polyvalent derivatives, such as the polyvalent derivatives of furan, imidazole, isothiazole, isoxazole, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pynole, pynolizine and thiophene.
  • the five-membered polyvalent derivatives are particularly preferred, i.e. the polyvalent derivatives of furan, imidazole, isothiazole, isoxazole, pyrazole, pyrrole and thiophene.
  • the heteroaryl groups may be attached to the remainder of the compound by any carbon or hetero (e.g. nitrogen) atom.
  • Ar 1 is preferably C 6-3 oarylene substituted by one or more A, preferably phenylene or napthylene substituted by one or more A, more preferably phenylene substituted by one or more A.
  • A is preferably provided in a position ortho or para to C*.
  • Ar 1 is other than phenylene, A is preferably attached to an atom which bears the charge in at least one of the resonance structures of the ions of formula (I).
  • Ar 1 is preferably substituted by 1, 2 or 3 A.
  • prefened Ar 1 When unsubstituted, prefened Ar 1 are:
  • Preferred examples of group Ar 1 are shown in figures 12A and 12B.
  • two or three of the groups Ar 1 and Ar 2 are linked together by one or more L 5 , where L 5 is independently a single bond or a linker atom or group; and/or two or three of the groups Ar 1 and Ar 2 together form an aromatic group or an aromatic group substituted with one or more A.
  • preferred linker groups are -E 5 -, -(D 5 ) t '-, -(E 5 -D 5 )t'- 5 -(D 5 -E 5 ) t >-, -E 5 -(D 5 -E 5 )f- or -D 5 -(E 5 -D 5 ) t .-.
  • D 5 is independently C 1-8 hydrocarbylene or C 1-8 hydrocarbylene substituted with one or more A.
  • t' 1 or more, e.g. from 1 to 50, lto 40, 1 to 30, 1 to 20 or 1 to 10.
  • t' 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • L 5 includes an atom or group which also falls within the definition of group M
  • the group M is preferably more reactive than the group included in L 5 .
  • L 5 is preferably a linker atom, preferably O or S, particularly O.
  • L 5 is a linker group
  • a preferred L 5 is -N(R 5 )-.
  • two of the groups Ar 1 and Ar 2 are linked together by one or more (e.g. 2, 3 or 4) L 5 , they are preferably linked together by one L 5 , preferably O.
  • Preferred combinations of Ar are two Ar 2 (e.g. two Ar 2 phenyl groups) linked together by one L 5 (e.g. O or S).
  • Particularly prefened combinations of Ar are two Ar 2 phenyl groups, optionally substituted by one or more A (preferably unsubstituted), linked together by one L 5 (e.g. O or S), where is L 5 is ortho to C* with respect to both phenyl groups.
  • Especially prefened combinations of two Ar 2 groups are:
  • At least one L is linked to an atom or group L 5 .
  • the preferred L 5 mentioned above are, where appropriate, modified to remove substituents R 5 in order to accommodate L M , e.g. the R 5 substituent of the group -N(R 5 )- is replaced by L M -
  • the L 5 group to which L M is bound is preferably: ArVAr 2
  • Prefened combinations of Ar 1 and/or Ar 2 in this embodiment are:
  • the aromatic group may be a carbocyclic aromatic group or a carbocyclic aromatic group in which one or more carbon atoms are each replaced by a hetero atom.
  • the aromatic group in which one or more carbon atoms are each replaced by a hetero atom up to three carbons are so replaced, preferably up to two carbon atoms, more preferably one carbon atom.
  • Preferred hetero atoms are O, Se, S or N, more preferably O, S or N.
  • aromatic groups Ar 1 and Ar 2 When two or three of the groups Ar 1 and Ar 2 together form an aromatic group or an aromatic group substituted with one or more A, preferred aromatic groups are C 8-50 aromatic groups.
  • monocyclic aromatic groups e.g. radicals of suitable valency derived from benzene
  • fused polycyclic aromatic groups e.g. radicals of suitable valency derived from napthalene
  • prefened groups are radicals of suitable valency obtained from napthalene, anthracene or phenanthracene, chrysene, aceanthrylene, acenaphthylene, acephenanthrylene, azulene, fluoranthene, fluorene, ⁇ s-indacene, s-indacene, indene, phenalene, and pleiadene.
  • preferred groups are radicals of suitable polyvalency obtained from acridine, carbazole, ⁇ -carboline, chromene, cinnoline, indole, indolizine, isobenzofuran, isochromene, isoindole, isoquinoline, naphthyridine, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pynolizine, quinazoline, quinoline, quinolizine and quinoxaline.
  • A is preferably an electron-donating group, including -R 1 or -Z'R 1 , where R 1 and Z 1 are defined below.
  • R 1 is C 1-8 hydrocarbyl, more preferably C 1-8 alkyl, especially methyl.
  • Z 1 is preferably O, S or NR 1 .
  • R 1 may be substituted with one or more S ut , 2 , but is preferably unsubstituted.
  • A is preferably -OMe, -SMe, -N(Me) 2 or Me.
  • C* when C* is a cation, A, when an electron-donating group, is preferably provided (especially in relation to Ar 1 or Ar 2 being phenyl) in a position ortho or para to C*, preferably para. Furthermore, when C* is a cation, A, when an electron-withdrawing group (e.g. F), is preferably provided (especially in relation to Ar 1 or Ar 2 being phenyl) in a position meta to C*.
  • prefened groups Ar 1 and Ar 2 are as follows:
  • C* when an electron-withdrawing group, is preferably provided (especially in relation to Ar 1 or Ar 2 being phenyl) in a position ortho or para to C*, preferably para. Furthermore, when C* is an anion, A, when an electron-donating group, is preferably provided (especially in relation to Ar 1 or Ar 2 being phenyl) in a position meta to C*.
  • the group A may also comprise one or more isotopes of the atoms making up group A (e.g. example 60), thus, as discussed in more detail below, allowing the masses of the compounds of the invention to be varied.
  • Prefened isotopes are 13 C, 18 0 and 2 H.
  • 13 C and 18 0 are particularly prefened as 2 H atoms may cause a substantial change in the chemical properties of the compound due to the kinetic isotope effect.
  • Solid Supports 'Solid supports' for use with the invention include polymer beads, metals, resins, columns, surfaces (including porous surfaces) and plates (e.g. mass-spectrometry plates).
  • the solid support is preferably one suitable for use in a mass spectrometer, such that the invention can be conveniently accommodated into existing MS apparatus. Ionisation plates from mass spectrometers are thus prefened solid supports, e.g. gold, glass-coated or plastic-coated plates. Solid gold supports are particularly preferred.
  • Resins or columns are particularly useful for receiving solutions of biopolymers (purified or mixtures).
  • a cellular lysate could be passed through such a column of formula (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) or (IVbiv) followed by cleavage of the support to leave compounds of formula (I).
  • Solid supports of formulae (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) or (IVbiv) will generally present exposed groups M capable of reacting with a biopolymer, B P .
  • ions preferably have a predictable mass to charge (m/e) ratio. If a biopolymer reacts with more than one M group, however, then it will carry more than one positive charge once ionised, and its m/e ratio will decrease.
  • the groups M are arranged such that any biopolymer molecule will covalently link with only a single group M. Consequently, each biopolymer will, on ionisation, carry a single positive charge and thus have a predictable mass to charge ratio.
  • the surface density of the solid supports of (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) or (IVbiv) will be provided so that a biopolymer molecule can only covalently link with one group M and thus to prevent the formation of multiply derivatised biopolymers. Varying the mass of compounds of the invention
  • the masses of the compounds of the invention can be varied via L M , Ar 1 and/or Ar 2 .
  • the masses of the compounds of the invention are varied by varying A on the groups Ar 1 and/or Ar 2 .
  • compounds of the invention advantageously comprise one or more of F or
  • F and I as substituents A of the groups Ar 1 , Ar 2 or Ar 3 .
  • F and I each only have one naturally occurring isotope, 19 F and 127 I respectively, and thus by varying the number of F and I atoms present in the structure of the compounds, can provide a series of molecular mass labels having substantially identical shaped peaks on a mass spectrum.
  • Compounds of the invention may also include one or more 2 H atoms, preferably as a substituent A or a part thereof of the groups L M , Ar 1 , Ar 2 or Ar 3 (in particular L M ), in order to vary the masses of the compounds of the invention.
  • the compounds of the invention may include isotopes of 13 C and 18 0, prefererably as a substituent A or a part thereof of the groups LM, Ar 1 , Ar 2 or Ar 3 (in particular Ar 1 , Ar 2 or Ar 3 ), in order to vary the masses of the compounds of the invention.
  • Compounds comprising 2 H, 13 C and 18 0 may also be used to provide a series of molecular mass labels having substantially identical shaped peaks on a mass spectrum, by varing the number of 2 H, 13 C and 18 0 atoms present in the structure of the compounds.
  • 13 C and 18 0 are particularly prefened as 2 H atoms may cause a substantial change in the chemical properties of the compound due to the kinetic isotope effect.
  • one or more of Ar 1 and Ar 2 may be substituted by one or more dendrimer radicals of appropriate valency, either as substituent A or group L M .
  • Prefened dendrimer radicals are the radicals obtained from the dendrimers of US 6,455,071 and PAMAM dendrimers.
  • the compounds of the invention may advantageously be used in the method of analysing a biopolymer disclosed herein, in particular in a method for following a reaction involving a biopolymer, Bp, since the abundance of a species of may be determined by mass spectrometry by measuring the intensity of the relevant peak in an obtained mass spectrum.
  • a method for analysing biopolymer B P comprising the steps of: (i) reacting a first sample comprising biopolymer B P with a compound of formula (Ila) or (lib) or a solid support of formula (IVai), (IVaii), (IVaiv), (IVbii), (IVbiii) or (IVbiv) at a time t ⁇ (ii) reacting a second sample comprising biopolymer B P with a compound of formula (Ila) or (lib) or a solid support of formula (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) or (IVbiv) at a later time t 2 ; (iii) preparing and analysing cations of formula (I) from the first and second samples; and (iv) comparing the results of the analysis from step (iii).
  • the reaction of the biopolymer with the compound of formula (Ila) or (lib) or the solid support of formula (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) or (IVbiv) will fix the biopolymer to prevent it reacting further and the steps of providing and analysing the cations may be canied out at a later convenient time.
  • Preferred compounds of formula (Ila), (lib) and (IVai) are:
  • the ions of the invention are stabilised by the resonance effect of the aromatic groups Ar 1 and Ar 2 .
  • the term 'C- is a carbon atom bearing a single positive charge or a single negative charge' therefore not only includes structures having the charge localised on the carbon atom but also resonance structures in which the charge is delocalised from the carbon atom.
  • 'linker atom or group' includes any divalent atom or divalent group.
  • the te ⁇ n 'aromatic group' includes quasi and/or pseudo-aromatic groups, e.g. cyclopropyl and cyclopropylene groups.
  • 'halogen' includes fluorine, chlorine, bromine and iodine.
  • hydrocarbyl' includes linear, branched or cyclic monovalent groups consisting of carbon and hydrogen.
  • Hydrocarbyl groups thus include alkyl, alkenyl and alkynyl groups, cycloalkyl (including polycycloalkyl), cycloalkenyl and aryl groups and combinations thereof, e.g. alkylcycloalkyl, alkylpolycycloalkyl, alkylaryl, alkenylaryl, cycloalkylaryl, cycloalkenylaryl, cycloalkylalkyl, polycycloalkylalkyl, arylalkyl, arylalkenyl, arylcycloalkyl and arylcycloalkenyl groups.
  • Preferred hydrocarbyl are C 1-14 hydrocarbyl, more preferably C 1-8 hydrocarbyl.
  • hydrocarbylene' includes linear, branched or cyclic divalent groups consisting of carbon and hydrogen formally made by the removal of two hydrogen atoms from the same or different
  • Hydrocarbylene groups thus include alkylene, alkenylene and alkynylene groups, cycloalkylene (including polycycloalkylene), cycloalkenylene and arylene groups and combinations thereof, e.g. alkylenecycloalkylene, alkylenepolycycloalkylene, alkylenearylene, alkenylenearylene, cycloalkylenealkylene, polycycloalkylenealkylene, arylenealkylene and arylenealkenylene groups.
  • Prefened hydrocarbylene are C 1-1 hydrocarbylene, more preferably C ⁇ -8 hydrocarbylene.
  • hydrocarbyloxy means hydrocarbyl-O-.
  • 'alkyl', 'alkylene', 'alkenyl', 'alkenylene', 'alkynyl', or 'alkynylene' are used herein to refer to both straight, cyclic and branched chain forms. Cyclic groups include C 3-8 groups, preferably C 5-8 groups.
  • the term 'alkyl' includes monovalent saturated hydrocarbyl groups. Prefened alkyl are C 1-8 , more preferably C 1-4 alkyl such as methyl, ethyl, n-propyl, i-propyl or t-butyl groups.
  • Prefened cycloalkyl are C 5-8 cycloalkyl.
  • alkoxy means alkyl-O-.
  • alkenyl' includes monovalent hydrocarbyl groups having at least one carbon-carbon double bond and preferably no carbon-carbon triple bonds. Prefened alkenyl are C 2-4 alkenyl.
  • alkynyl' includes monovalent hydrocarbyl groups having at least one carbon-carbon triple bond and preferably no carbon-carbon double bonds.
  • Prefened alkynyl are C 2-4 alkynyl.
  • 'aryl' includes monovalent aromatic groups, such as phenyl or naphthyl.
  • the aryl groups may be monocyclic or polycyclic fused ring aromatic groups.
  • Preferred aryl are C 6 -C 1 aryl.
  • aryl groups are monovalent derivatives of aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, chrysene, coronene, fluoranthene, fluorene, ⁇ s-indacene, s- indacene, indene, naphthalene, ovalene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene and rubicene.
  • alkylene' includes divalent saturated hydrocarbylene groups.
  • Preferred alkylene are C ⁇ - alkylene such as methylene, ethylene, n-propylene, i-propylene or t-butylene groups.
  • Preferred cycloalkylene are Cs -8 cycloalkylene.
  • alkenylene' includes divalent hydrocarbylene groups having at least one carbon-carbon double bond and preferably no carbon-carbon triple bonds. Prefened alkenylene are C 2-4 alkenylene.
  • alkynylene' includes divalent hydrocarbylene groups having at least one carbon-carbon triple bond and preferably no carbon-carbon double bonds.
  • Preferred alkynylene are C 2-4 alkynylene.
  • 'arylene' includes divalent aromatic groups, such phenylene or naphthylene.
  • the arylene groups may be monocyclic or polycyclic fused ring aromatic groups.
  • Prefened arylene are C 6 -C 14 arylene.
  • arylene groups are divalent derivatives of aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, chrysene, coronene, fluoranthene, fluorene, ⁇ s-indacene, s- indacene, indene, naphthalene, ovalene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene and rubicene.
  • heterohydrocarbyl' includes hydrocarbyl groups in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O, S or N.
  • Heterohydrocarbyl groups thus include heteroalkyl, heteroalkenyl and heteroalkynyl groups, cycloheteroalkyl (including polycycloheteroalkyl), cycloheteroalkenyl and heteroaryl groups and combinations thereof, e.g.
  • heteroalkylcycloalkyl alkylcycloheteroalkyl, heteroalkylpolycycloalkyl, alkylpolycycloheteroalkyl, heteroalkylaryl, alkylheteroaryl, heteroalkenylaryl, alkenylheteroaryl, cycloheteroalkylaryl, cycloalkylheteroaryl, heterocycloalkenylaryl, cycloalkenylheteroaryl, cycloalkylheteroalkyl, cycloheteroalkylalkyl, polycycloalkylheteroalkyl, polycycloheteroalkylalkyl, arylheteroalkyl, heteroarylalkyl, arylheteroalkenyl, heteroarylalkenyl, arylcycloheteroalkyl, heteroarylcycloalkyl, arylheteroalkenyl, heteroarylalkenyl, ary
  • heterohydrocarbylene' includes hydrocarbylene groups in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O, S or N.
  • Heterohydrocarbylene groups thus include heteroalkylene, heteroalkenylene and heteroalkynylene groups, cycloheteroalkylene (including polycycloheteroalkylene), cycloheteroalkenylene and heteroarylene groups and combinations thereof, e.g.
  • heteroalkylenecycloalkylene alkylenecycloheteroalkylene, heteroalkylenepolycycloalkylene, alkylenepolycycloheteroalkylene, heteroalkylenearylene, alkyleneheteroarylene, heteroalkenylenearylene, alkenyleneheteroarylene, cycloalkyleneheteroalkylene, cycloheteroalkylenealkylene, polycycloalkyleneheteroalkylene, polycycloheteroalkylenealkylene, aryleneheteroalkylene, heteroarylenealkylene, aryleneheteroalkenylene, heteroarylenealkenylene groups.
  • the heterohydrocarbylene groups may be attached to the remainder of the compound by any carbon or hetero (e.g. nitrogen) atom.
  • -CH 2 - is replaced by -0-, -S- or -Se-.
  • 'heteroalkyP includes alkyl groups in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O, S or N.
  • heteroalkenyl' includes alkenyl groups in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O, S or N.
  • heteroalkynyl' includes alkynyl groups in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O, S or N.
  • 'heteroaryl' includes aryl groups in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O, S or N.
  • Preferred heteroaryl are C 5-1 heteroaryl. Examples of heteroaryl are pyridyl, pyrrolyl, thienyl or furyl.
  • heteroaryl groups are monovalent derivatives of acridine, carbazole, ?-carboline, chromene, cinnoline, furan, imidazole, indazole, indole, indolizine, isobenzofuran, isochromene, isoindole, isoquinoline, isothiazole, isoxazole, naphthyridine, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pynolizine, quinazoline, quinoline, quinolizine, quinoxaline, thiophene and xanthene.
  • Prefened heteroaryl groups are five- and six-membered monovalent derivatives, such as the monovalent derivatives of furan, imidazole, isothiazole, isoxazole, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pynolizine and thiophene.
  • the five-membered monovalent derivatives are particularly preferred, i.e. the monovalent derivatives of furan, imidazole, isothiazole, isoxazole, pyrazole, pynole and thiophene.
  • the te ⁇ n 'heteroalkylene' includes alkylene groups in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O, S or N.
  • heteroalkenylene' includes alkenylene groups in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O, S or N.
  • heteroalkynylene' include alkynylene groups in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O, S or N.
  • heteroarylene' includes arylene groups in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O, S or N.
  • Preferred heteroarylene are C5 -l4 heteroarylene. Examples of heteroarylene are pyridylene, pyrrolylene, thienylene or furylene.
  • heteroarylene groups are divalent derivatives (where the valency is adapted to accommodate the q instances of the linker L M ) of acridine, carbazole, ?-carboline, chromene, cinnoline, furan, imidazole, indazole, indole, indolizine, isobenzofuran, isochromene, isoindole, isoquinoline, isothiazole, isoxazole, naphthyridine, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pynole, pynolizine, quinazoline, quinoline, quinolizine, quinoxaline, thiophene and xanthene.
  • Prefened heteroarylene groups are five- and six-membered divalent derivatives, such as the divalent derivatives of furan, imidazole, isothiazole, isoxazole, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pynole, pynolizine and thiophene.
  • the five-membered divalent derivatives are particularly prefened, i.e. the divalent derivatives of furan, imidazole, isothiazole, isoxazole, pyrazole, pynole and thiophene.
  • A is independently a substituent, preferably a substituent S Ub 0 Alternatively, A may be 2 H.
  • R 1 is independently H, C 1-8 hydrocarbyl, C 1-8 hydrocarbyl substituted with one or more S Ub 2 , C 1-8 heterohydrocarbyl or C 1-8 heterohydrocarbyl substituted with one or more S Ub 2 .
  • molecular mass labels of the invention will generally comprise 1 or more, typically between 1 and 100 (e.g. 1 to 50, preferably 1 to 20) substituents S u b 1 or S ub 2 , typically F or I, in order to vary the masses of the molecular mass labels.
  • A may optionally be a monovalent dendrimer radical or a monovalent dendrimer radical substituted with one or more substituents S ub 1 .
  • composition comprising X may consist exclusively of X or may include something additional e.g. X + Y.
  • Figure 1 demonstrates conceptually the effect of the signal on a mass spectrum by a compound of formula (Ila) or (lib) of the invention.
  • Free biopolymer such as a peptide
  • Figure 2 shows the steps of biopolymer with a compound of formula (IVai).
  • the derivativisation of a biopolymer with a compound of the invention can be canied out more conveniently by utilising the solid phase-based format, whereby the compound is temporarily covalently attached to a solid support.
  • the solid support can be a resin, a surface or a porous surface.
  • the solid support may be a mass-spectrometry sample plate, which dramatically decreases the sample preparation time. Both gold, glass- and plastic-coated plates are compatible with this approach.
  • Figure 3 shows the steps of 'reverse' biopolymer derivativisation on a covalent solid support whereby the release of the biopolymer derivative happens simultaneously with the derivativisation process. The process is applicable M groups involving leaving groups.
  • Figure 4 shows the steps of biopolymer derivativisation on an ionic solid support.
  • Figure 5 shows of the steps of solid support-assisted biopolymer derivativisation.
  • FIG. 6 shows the mass spectrum obtained when analysing an Gly-Gly-O-acyl dipeptide conjugated with a trityl compound of the invention.
  • Figure 7 shows the mass spectrum obtained when analysing a conjugate of a peptide with a trityl compound of the invention.
  • Figure 8 compared the mass spectra of a BSA digest without (8A) and with (8B) labelling.
  • Figure 9 shows the mass spectrum obtained when analysing a mixture of trityl-labelled amines.
  • Figures 10A and 10B show preferred examples of group L M .
  • Figures 11 A and 1 IB show prefened examples of group M.
  • Figures 12A and 12B shows prefened examples of groups Ar 1 and Ar 2 .
  • Figure 13 shows preferred examples of groups X and X*.
  • Figure 14 shows prefened examples of substituent group A.
  • the solid supports were Tenta Gel Macrobeads OH and NH 2 , 280-320 microns, Rapp Polymer.
  • (MA)LDI-TOF mass-spectra were recorded on a PE-ABI VoyagerTM Elite Reflectron Delayed Extraction Instrument.
  • TLC were canied out with Merck silica gel (Kieselgel 60 F 25 precoated plates and Kieselgel 60 0.040-0.063 mm).
  • HPLC was canied out on a Waters system (Milford, MA, USA).
  • Phosphoroamidite couplings were canied out in an ABI 394 DNA/RNA synthesiser. Chemicals and solvents were from Sigma/Aldrich/Fluka (USA), and BDH/Merck.
  • Example 1 Conjugation of a trityl tag (in solution phase) with solid support-bound biopolymer
  • a 15mer poly-T oligonucleotide was synthesised on an ABI 394 DNA synthesiser using a T CPG support according to standard protocols of phosphoramidite chemistry on 0.2 ⁇ mol scale. After the last coupling, a MMTr-protected 'aminolink' phosphoramidite (Glen Res., USA) was added to a growing chain and deprotected using standard deblocker (2% DCA in DCM).
  • the column was removed from the synthesiser, and after 10 min wash with acetonitrile it was attached to two 5 ml syringes and washed with a 0.1M solution of NHS-activated 4,4'-dimethoxy-4"-carboxyethyl trityl for 10 min at RT.
  • the column was then washed with (3 x 10 ml) acetonitrile, placed on a DNA synthesiser and deprotected with ammonia according to standard protocols.
  • the residue obtained after the evaporation was dissolved in 0.1 ml of 2M LiC10 4 and precipitated from cold acetone (1.5 ml). The precipitate was washed with 0.5ml of acetone and dried.
  • a solution of NHS-activated 4,4'-dimethoxy-4"-carboxyethyl trityl (0.1M) in THF/dioxane (1:1) was mixed with a solution (0.5-1M) of an amine or of a mixture of amines (for example, propyl amine, butyl amine, pentyl amine, hexyl amine and phenethyl amine), typically 10 ml of a solution of an activated trityl with 5 ml of an amine solution.
  • the mixtures were purified on prep-TLC (2mm-thick glass plates with UV254 indicator, Analtech/Aldrich-Sigma), typically in chloroform with 0.5% triethylamine.
  • the areas containing the desired products were scratched off the plate, and the conjugates or the mixtures thereof were eluted using same solvent with 2-5% MeOH, filtered through a layer of glass wool, evaporated and dried.
  • Example 3 Homogenous conjugation of a nhs-activated trityl with polymeric ligands
  • a peptide, an oligonucleotide, or any other biopolymer containing a (primary) amino group is dissolved in a mixture of water and acetonitrile depending on its solubility, typically 20-50% of water in CH 3 CN.
  • Non-aminogroup-containing buffers ie. 50 mM sodium phosphate, 0.15 M NaCl, pH 7.2, or a bicarbonate buffer, but an additional desalting step may then need to be introduced to cut off the metal ions prior to mass-spectrometry
  • solvents may be used such as THF, DMSO, etc.
  • a solution of NHS-activated 4,4'-dimethoxy-4"-carboxyethyl trityl in acetonitrile or THF is added in approx. 5-10 times excess compared to an amine component. Conjugation usually reaches the maximum yield over 2-4 hours of reaction time.
  • the conjugate formed can be analysed by MS directly, or after HPLC-purification.
  • a Solid Phase-Immobilised NHS-Activated Trityl Tag was prepared by either method 1 or method 2.
  • Method 1 A NHS-Activated 4,4'-dimethoxy-4"-carboxyethyl trityl tag was covalently attached to hydroxyl groups of 200 ⁇ m Rapp Polymer beads by shaking the suspension of 100 mg of the resin in 5 ml of 0.1 M solution of trityl chloride tag in dry pyridine at +4°C for 3 hours and then washing the resin with pyridine and acetonitrile and drying in vacuo.
  • a 5'-tritylated thymidine phosphoramidite was prepared from NHS-activated 4,4'- dimethoxy-4"-carboxyethyl trityl chloride in a standard way [M.J. Gait, Oligonucleotide Synthesis: A Practical Approach, IRL, Oxford, 1984].
  • the Rapp Polymer beads (2 x 40 mg) were placed in two 1 micromol scale DNA synthesis columns (Glen Res.). The first column was coupled with the said phosphoramidite on an ABI DNA synthesiser using manual supply of reagents (0.1M solution of a phosphoramidite and other standard phosphoramidite synthesis reagents) with a coupling step of 15 min.
  • the second column was first derivatised with a trebler phosphoramidite (Glen Res.) according to the manufacturer's protocols and then coupled with the trityl tag-containing phosphoramidite as described for the first column. Both columns were excessively washed with acetonitrile.
  • a trebler phosphoramidite Galy Res.
  • the trityl loading of the solid supports produced by either method was determined spectrophotometrically (absorbance measurements at 490nm) to be 0.21 mmol/g for a straight attachment and 0.39 mmol/g for a tritylation on top of the trebling synthon. (The hydroxyl group loading of the Rapp polymer used was 0.25mmol/g).
  • a mixture of compounds to be labelled (typically peptides) is added, typically in a mixture of 20-50% water in acetonitrile. After incubation, with occasional shaking, for 60-120 min the resin is washed with several volumes of the same solvent, and the conjugated products are cleaved off the resin, typically by adding 0.5-2% TFA in appropriate solvent. The collected sample is then analysed by MS.
  • compounds to be labelled typically peptides
  • Figure 6 shows the mass spectrum obtained from a compound of the invention comprising a derivatised Gly-Gly-O-acyl dipeptide biopolymer.
  • Figure 7 shows the mass spectrum obtained from a compound of the invention comprising a derivatised peptide biopolymer.
  • the free peptide had a molecular weight of 310.
  • the fragment of formula (VI), in which the derivatised peptide has been lost, is observed at the peak at the molecular weight 375.0. Significantly, there is only a very small peak at molecular weight 310, where a peak conesponding to the free biopolymer would be found. The relative size of the peaks at 665.0 and 310 thus demonstrate the significantly improved ionisability of the compounds of the invention compared with free biopolymer.
  • Example 7 Spectral improvement by trityls Three proteins (BSA, ⁇ -casein and ADH) were digested with trypsin and the resulting peptides analysed by MALDI-TOF mass spectrometry with or without derivatisation. The number of peptides identified for each protein is shown below. The theoretical total number of peptides that would be produced by trypsin digestion of each protein was calculated in silico and is shown in the second column the table below.
  • Derivatisation of peptides with trityl groups of the invention thus improves detection, as a significantly larger number of peptides was detected for each of the three proteins when derivatisation was used. Furthermore, protein identification by mass fingerprinting can be improved.
  • Example 8 BSA fragmentation and mass spectrometry Bovine serum albumin (BSA) was digested with trypsin and analysed by MALDI-TOF. The resulting spectrum is shown in Figure 8A. The experiment was repeated, but the peptide mixture was labelled with a dimethoxytrityl label after trypsin digestion. The spectrum in Figure 8B shows the dramatic increase in visible ions due to the trityl label. Four specific peptides have been highlighted in both spectra.
  • BSA Bovine serum albumin
  • Example 9 Mass spectrometry of amines
  • a solution of NHS-activated 4,4'-dimethoxy-4"-carboxyethyl trityl (0.1M) in THF/dioxane (1 :1) was mixed with a solution (0.5- 1M) of an amine or of a mixture of amines (for example, propyl amine, butyl amine, pentyl amine, hexyl amine and phenethyl amine), typically 10 ml of a solution of an activated trityl with 5 ml of an amine solution.
  • a solution of an activated trityl typically 10 ml of a solution of an activated trityl with 5 ml of an amine solution.

Abstract

The present invention provides a method of forming an ion of formula (I) comprising the steps of: (i) reacting a compound of the formula (IIa); with a biopolymer, BP, having at least one group capable of reacting with M to form a covalent linkage, to provide a biopolymer derivative of the formula (IIIa); and (ii) cleaving the C-X bond between X and the a-carbon atom of the derivative of formula (IIIa) to form the ion of formula (I); where: C * is a carbon atom bearing a single positive charge or a single negative charge; and X is a group capable of being cleaved from the a-carbon atom to form an ion of formula (I). The biopolymer derivatives of the invention have enhanced ionisability with respect to free biopolymer (BP) enabling improved analysis of the biopolymer using mass spectrometry.

Description

TRITYL DERIVATIVES FOR ENHANCING MASS SPECTROMETRY
All documents cited herein are incorporated by reference in their entirety.
TECHNICAL FIELD
This invention relates to derivatised biopolymers and ions obtainable therefrom. The invention further relates to compounds and solid supports useful for producing the derivatised biopolymers and ions of the invention.
BACKGROUND OF THE INVENTION
Mass spectrometry is a versatile analytical technique possessing excellent detection range and speed of detection with respect to High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Infra-Red (IR) and Nuclear Magnetic Resonance (NMR).
However, many biopolymers, such as carbohydrates and proteins, are difficult to analyse using mass spectrometry due to significant difficulties in ionising the biopolymer, even using Matrix Assisted Laser Desorption/Ionisation Time Of Flight (MALDI-TOF) techniques. Despite the considerable resolving power of 2D-PAGE, this technology has fallen far short of the ultimate goal of displaying the whole proteome in a single experiment, as many proteins are resistance to 2D-PAGE analysis (e.g those with low or high molecular masses, membrane proteins, proteins with extreme isoelectric points, etc.). Many proteins are thus invisible to 2-D PAGE [Cravatt & Sorensen (2000) Current Opinion in Chemical Biology vol. 4, p. 663-668].
There is thus a need for improvements in mass spectrometry analysis of biopolymers. DISCLOSURE OF THE INVENTION
It has now been found that covalent attachment of trityl derivatives to biopolymers can improve the ionisation properties of the biopolymer. The ions (formula (I) below) formed by ionisation of the derivatised biopolymers are particularly suitable for mass spectrometry analysis, and biopolymers derivatised as specified in formulae (Ilia) and (Illb) below can be readily ionised. Whereas triphenylmethyl derivatives covalently attached to certain biopolymers (e.g. DNA) are known in the prior art [e.g. Chem. Soc. Rev. (2003) 32, p. 3-13], the prior art attaches the polymer to the α-triphenylmethyl carbon atom through a non-aromatic linker. In contrast, under the present invention the biopolymer is attached to the α-triarylmethyl carbon atom via an aromatic group adjacent to the central carbon atom. Consequently, ionisation of the prior art derivatives results in separation of the triphenylmethyl derivative and the biopolymer, whereas according to the present invention the biopolymer remains bound to the trityl derivative on ionisation, thereby allowing analysis of the biopolymer by mass spectrometry.
The invention provides methods of forming ions from covalent or ionic compounds and solid substrates. Derivatised Biopolymers
The invention provides a method of forming an ion of formula (I):
Figure imgf000003_0001
comprising the steps of: (i) reacting a compound of the formula (Ila):
Figure imgf000003_0002
with a biopolymer, Bp, having at least one group capable of reacting with M to form a covalent linkage, to provide a biopolymer derivative of the formula (Ilia):
Figure imgf000003_0003
(ii) cleaving the C — X bond between X and the α-carbon atom of the derivative of formula (Ilia) to form the ion of formula (I); where: C* is a carbon atom bearing a single positive charge or a single negative charge; X is a group capable of being cleaved from the α-carbon atom to form an ion of formula (I); M is independently a group capable of reacting with BP to form the covalent linkage; Bp' is independently the biopolymer residue of BP produced on formation of the covalent linkage; M' is independently the residue of M produced on formation of the covalent linkage; Ar1 is independently an aromatic group or an aromatic group substituted with one or more A; Ar2 is independently an aromatic group or an aromatic group substituted with one or more A; optionally wherein (a) two or three of the groups Ar1 and Ar2 are linked together by one or more L5, where L5 is independently a single bond or a linker atom or group; and/or (b) two or three of the groups Ar1 and Ar2 together form an aromatic group or an aromatic group substituted with one or more A; A is independently a substituent; LM is independently a single bond or a linker atom or group; n = 0, 1 or 2 and m = 1, 2, or 3, provided the sum of n+m = 3; p independently = 1 or more; and q independently = 1 or more. The invention further provides a method of forming an ion of formula (I), comprising the steps of: (i) reacting a compound of the formula (lib): (Ar2)n- C- [Ar1— (LM{M}p)q]m -AT X* (lib); with a biopolymer, Bp, having at least one group capable of reacting with M to form a covalent linkage, to provide a biopolymer derivative of the formula (Illb): (Ar2)n- C- [Ar1- (LM{M'— BP'}p)q]m X* (Illb); and dissociating X* from the derivative of formula (Illb), to form the ion of formula (I); where: X*is a counter-ion to C*; and C*, M, Bp', M', Ar1, Ar2, LM, n, m, p and q are as defined above.
The compounds of formulae (Ila) or (lib) may optionally be purified after step (i).
The invention also provides biopolymer derivatives of the formula (Ilia) or (Illb), as defined above. The biopolymer derivatives of the invention have enhanced ionisability with respect to free biopolymer, BP. Advantageously, the biopolymer derivatives may not require a matrix (e.g. as used in MALDI-MS) in order to elicit ionisation, although a matrix may help to enhance ionisation. Preferably, ionisation may be obtained without requiring acid treatment, in particular by direct laser illumination.
The invention also provides ions of formula (I), as defined above. These ions are stabilised by the resonance effect of the aromatic groups Ar1 and Ar2. Electron- withdrawing groups, when C* is an anion, or electron-donating groups, when C* is a cation, may optionally be provided on Ar1 and/or Ar2 to assist this resonance effect. Consequently, the biopolymer derivatives of the invention readily form ions of formula (I) relative to the native biopolymer, BP.
The ions of formula (I) are generally only ever seen on a mass spectrum with a single charge, which is advantageous since it reduces cluttering of the mass spectrum.
The invention also provides compounds of the formula (Ila) and (lib), as defined above. As mentioned above, these compounds are useful for forming ions of formula (I). As the difference in the molecular mass of the ions of formula (I) and that of the free biopolymer can be accurately calculated, the derivatised compounds of the invention allow analysis of the biopolymer BP, which may be otherwise difficult or impossible to analyse using known mass spectrometrical techniques.
Other advantageous features of the compounds of the invention include more uniformity of the signal intensity between different analytes (useful for quantitative studies) and similar desorption properties between compounds with different, but close, masses, so that techniques such as isotope coded affinity tagging (ICAT) can be employed with the compounds of the invention.
The homogeneous methods of the invention are particularly appropriate for small molecules, e.g. amines. Solid Supports
The ions of formula (I) may also be formed using a derivatised solid support.
The invention therefore provides a method of forming an ion of formula (I) comprising the steps of: (i) reacting a solid support of formula (IVai), (IVaii), or (IVaiii):
Figure imgf000005_0001
(IVaii);
Figure imgf000005_0002
(IVaiii); with a biopolymer, Bp, having at least one group capable of reacting with M to form a covalent linkage, to provide a modified solid support of the formula (Vai), (Vaii), or (Vaiii), respectively:
Figure imgf000005_0003
Figure imgf000006_0001
(Vaiii); and either: (iia) for modified solid supports of formula (Vai) cleaving the C-Ss bond between the α-carbon atom of the modified solid support of formula (Vai) and the solid support Ss to form the ion of formula (I) ; (iib) for modified solid supports of formula (Vaii), either simultaneously or sequentially, cleaving the C-X bond between X and the α-carbon atom and cleaving the Ss- - -Ar1 bond between the solid support and the Ar1 group to form the ion of formula (I); or (iic) for modified solid supports of formula (Vaiii), either simultaneously or sequentially, cleaving the C-X bond between X and the α-carbon atom and cleaving the Ss — Ar2 bond between the solid support and the Ar2 group to form the ion of formula (I); where: X, Ar1, Ar2, BP', LM, M, M', n, m, p and q are as defined above; Ss is a solid support; C- - -Ss comprises a cleavable bond between C and Ss; Ss- - -Ar1 comprises a cleavable bond between Ar1 and Ss; and Ss- - -Ar2 comprises a cleavable bond between Ar2 and Ss. The cleavable bond of C- - -Ss, Ss- - -Ar1 or Ss- - -Ar2 may be a covalent, ionic, hydrogen, dipole-dipole or van der Waals bond. The invention further provides a method of foπning an ion of formula (I) comprising the steps of: (i) reacting a solid support of formula (IVbii) or (IVbiii):
Figure imgf000006_0002
X* (IVbii);
Figure imgf000006_0003
r2 (Ar2)^— O-EAr1— (LMlM J*
X* (IVbiii); with a biopolymer, BP, having at least one group capable of reacting with M to form a covalent linkage, to provide a modified solid support of the formula (Vbii) or (Vbiii), respectively:
Figure imgf000007_0001
X* (Vbii);
[Ar1— (LM{M'-BP'}p)q]m
Figure imgf000007_0002
X* (Vbiii); and either: (iia) for modified solid supports of formula (Vbii), either simultaneously or sequentially, dissociating X* from the derivative of formula (Vbii) and cleaving the Ss- - -Ar1 bond between the solid support and the Ar1 group to form an ion of formula (I); or (iib) for modified solid supports of formula (Vbiii), either simultaneously or sequentially, dissociating X* from the derivative of formula (Vbiii) and cleaving the Ss- - -Ar2 bond between the solid support and the Ar2 group to form an ion of formula (I); where: X*, Ar1, Ar2, BP', LM, M, M', n, m, p, q, Ss, C- - -Ss, Ss- - -Ar1 and Ss- - -Ar2 are as defined above.
The invention further provides a method of forming an ion of formula (I) comprising the steps of: (i) reacting a solid support of formula (IVaiv) or (IVbiv):
Figure imgf000007_0003
(IVaiv);
Figure imgf000007_0004
X* (IVbiv); with a biopolymer, BP, having at least one group capable of reacting with M to form a covalent linkage, to provide a modified solid support of the formula (Vaiv) or (Vbiv), respectively:
Figure imgf000008_0001
X* (Vbiv); and either: (iia) for modified solid supports of formula (Vaiv), cleaving the C-X bond between X and the α-carbon atom to form the ion of formula (I); or (iib) for modified solid supports of formula (Vbiv), dissociating X* from the derivative of formula (Vbiv) to form the ion of formula (I); where: X, X*, Ar1, Ar2, Bp', LM, M, M', p, q, n, m, and Ss are as defined above; M"- - -Ss comprises a bond between M" and Ss; and M" is the same as M except that Ss is bound to a portion of M which does not form part of M'. In this embodiment of the invention, the solid support is bound to a part of group M" which does not go on to form the residue M'. Thus, the derivatised biopolymer will be released from the solid support during the derivativisation step and an additional step of cleaving the biopolymer from the solid support is not required.
The modified solid supports of formulae (Vai), (Vaii), (Vaiii), (Vaiv), (Vbii), (Vbiii) or (Vbiv) may optionally be washed after step (i).
The invention also provides solid supports of the formulae (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) and (IVbiv), as defined above. Similarly, the invention provides modified solid supports of the formulae (Vai), (Vaii), (Vaiii), (Vaiv), (Vbii), (Vbiii), and (Vbiv), as defined above.
The heterogeneous methods of the invention are particularly appropriate for synthetic biopolymers, e.g. oligonucleotides, peptides and carbohydrates. Methods of Analysis
The invention also provides a method for analysing a biopolymer, BP, comprising the steps of: (i) reacting the biopolymer Bp with a compound of formula (Ila) or (lib) or a solid support of formula (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) or (IVbiv); (ii) providing an ion of formula (I); and (iii) analysing the ion of formula (I) by mass spectrometry.
The biopolymer will typically have been obtained using a preparative or analytical process. For example, it may have been purified using various separation methods (e.g. 1 -dimensional or 2-dimensional, reverse-phase or normal-phase separation, by e.g. chromatography or electrophoresis) and the separation may be based on any of a number of characteristics (e.g. isoelectric point, molecular weight, charge, hydrophobicity, etc.). Typical methods include 2D SDS-PAGE , 2D liquid chromatography (e.g. Multidimensional Protein Identification Technology, MudPIT, or 2D HPLC methods). The separation method can preferably interface directly with the mass spectrometer.
Known analytical techniques can thus be adapted or improved by the method of the invention. A particularly preferred method involves 2D-PAGE of a biopolymer, or mixture of biopolymers, selection of a spot of interest in the electrophoretogram, and then derivatisation and analysis of that spot using the techniques of the invention. The biopolymer may be proteolytically digested prior to its analysis (typically within the PAGE gel, but optionally digested after extraction from the gel) and/or may itself be the product of a proteolytic digest. The invention also provides, in a method for analysing a biopolymer, Bp, the improvement consisting of: (i) reacting a biopolymer, Bp with a compound of formula (Ila) or (lib) or a solid support of formula (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) or (IVbiv); (ii) providing an ion of formula (I); and (iii) analysing the ion by mass spectrometry.
Typically, the analysis by mass spectrometry is carried out in a spectrometer which is suitable for MALDI-TOF spectrometry.
In the spectrometer, the ion source may be a matrix-assisted laser desorption ionisation (MALDI), an electrospray ionisation (ESI) ion source, a Fast-Atom Bombardment (FAB) ion source. Preferably, the ion source is a MALDI ion source. The MALDI ion source may be traditional MALDI source (under vacuum) or may be an atmospheric pressure MALDI (AP-MALDI) source. MALDI is a preferred ionisation method, although the use of a matrix is generally not required
In the spectrometer, the mass analyser may be a time of flight (TOF), quadrupole time of flight (Q-TOF), ion trap (IT), quadrupole ion trap (Q-IT), triple quadrupole (QQQ) Ion Trap or Time-Of- Flight Time-Of-Flight (TOFTOF) or Fourier transform ion cyclotron resonance (FTICR) mass analyser. Preferably, the mass analyser is a TOF mass analyser. Preferably, the mass spectrometer is a MALDI-TOF mass spectrometer. Further Embodiments
M' bound to Bp' by a non-covalent linker
The above-mentioned embodiments of the invention may also be provided in which M' is bound to Bp' by a non-covalent bond. All the other features of the invention are the same except the groups which relate to the non-covalent bond between M' and BP'.
The non-covalent bond may be direct between M' and BP' or may be provided by one or more binding groups present on M' and/or BP'.
Preferred non-covalent bonds are those having an association constant (Ka) of at least 1014 M"1, preferably about 1015 M"1. In preferred embodiment, one of M' and Bp' will have a binding group comprising biotin, and the other of M' and BP' will have a binding group comprising avidin or streptavidin.
Preferably, when the compounds of the invention comprise a non-covalent bond between M' and Bp' and a cleavable bond between C and Ss, Ar1 and Ss, or Ar2 and Ss, these bonds are differentially cleavable. More preferably, the non-covalent bond between M' and BP' is not cleaved under conditions which the cleavable bond between C and Ss, Ar1 and Ss, or Ar2 and Ss, as appropriate, is cleaved.
LM bound to Ar1 by more than one bond
The above-mentioned embodiments of the invention may also be provided in which LM is bound to
Ar1 by more than one covalent bond (e.g. 2 or 3 bonds) which are either single, double or triple covalent bonds, or one or more multiple bonds (e.g. double or triple covalent bonds). All the other features of the invention are the same except the groups which relate to the bond or bonds between
Figure imgf000010_0001
Ionisation of Compounds other than Biopolymers
In addition to biopolymers, the present invention may be used for ionising any molecule or complex of molecules which requires mass spectrum analysis. Thus, the above-mentioned embodiments of the invention may also be provided in which BP is replaced by any molecule or complex having at least one group capable of reacting with M to form a covalent linkage. All the other features of the invention are the same, except group M is group capable of reacting with the molecule to be analysed. Examples of other molecules which may be analysed in the present invention include non-biological polymers (e.g. synthetic polyesters, polyamides and polycarbonates), petrochemicals and small molecules (e.g. alkanes, alkenes, amines, alcohols, esters and amides). Amines are particularly preferred. Examples of complexes which may be analysed in the present invention include double- and triple- stranded RNA, DNA and/or peptide nucleic acid (PNA) complexes, enzyme/substrate complexes, multimeric proteins (e.g. dimers, trimers, tetramers, pentamers, etc.), virions, etc.
Preferably, when the compound to be ionised is not a biopolymer, all embodiments of the invention (including products of formulae (I), (Ila), (lib), (Ilia), (Illb), (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii), (IVbiv), (Vai), (Vaii), (Vaiii), (Vaiv), (Vbii), (Vbiii) and (Vbiv), methods of forming an ion of formula (I) and methods of analysis) involving or relating to the compound of formula (XI) are disclaimed.
Figure imgf000011_0001
Disclaimers
Preferably, all embodiments of the invention (including products of formulae (I) and (Ila)) involving or relating to the compound of formula (XI) are disclaimed
solid
Figure imgf000011_0002
Preferably, all embodiments of the invention (including products of formulae (I) and (Ila)) involving or relating to the compound of formula (XIa) are disclaimed
Figure imgf000011_0003
Preferably, all embodiments of the invention (including products of formulae (I) and (Ila)) involving or relating to the compound of formula (Xlb) are disclaimed.
Figure imgf000012_0001
Preferably, all embodiments of the invention (including products of formulae (I) and (Ila)) involving or relating to the compound of formula (XIc) are disclaimed
Figure imgf000012_0002
Preferably, all embodiments of the invention (including products of formulae (I) and (Ila)) involving or relating to the compound of formula (Xld) are disclaimed
Figure imgf000012_0003
Preferably, all embodiments of the invention (including products of formulae (I) and (Ila)) involving or relating to the compound of formula (Xle) are disclaimed
Figure imgf000013_0001
Preferably, all embodiments of the invention (including products of foπnulae (I) and (Ila)) involving or relating to the compound of formula (Xle) are disclaimed
Figure imgf000013_0002
Preferably, all embodiments of the invention (including products of foπnulae (I) and (Ila)) involving or relating to the compound of formula (Xlg-j) are disclaimed
Figure imgf000013_0003
Ar = p-anisyl
Figure imgf000013_0004
Preferably, all embodiments of the invention (including products of formulae (I) and (Ila)) involving or relating to the compound of formula (Xlk-n) are disclaimed
Figure imgf000014_0001
Ar = p-anisyl
Figure imgf000014_0003
Preferred Embodiments Definition ofC *
Preferably, C* bears a single positive charge such that ions of the invention are cations and the ion of formula (I) has the following structure:
(Ar2) — C— [Ar1-(LM{M'- BP'}p)q]m Θ and the compounds of formulae (lib), (Illb), (IVbii), (IVbiii), (IVbiv), (Vbii), (Vbiii) and (Vbiv) have the structures disclosed in table 1.
n, m, p and q
For the purposes of compounds of the invention having n-1 groups Ar2, n may not be less than 1.
Preferably n = 2 and m = 1.
Preferably p = 1, 2 or 3. Preferably p = 1. Preferably q = 1 , 2 or 3. Preferably q = 1.
Preferably n = 2, m = 1, p = 1 and q = 1. The ion of formula (I) thus has the structure:
Figure imgf000014_0002
and the compounds of formulae (Ila), (lib), (Ilia), (Illb), (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii), (IVbiv), (Vai), (Vaii), (Vaiii), (Vaiv), (Vbii), (Vbiii) and (Vbiv) have the structures disclosed in table 2. Biopolymers
The term 'biopolymer' includes polymers found in biological samples, including polypeptides, polysaccharides, and polynucleotides (e.g. DNA or RNA). Polypeptides may be simple copolymers of amino acids, or they may include post-translational modifications e.g. glycosylation, lipidation, phosphorylation, etc. Polynucleotides may be single-stranded (in whole or in part), double-stranded (in whole or in part), DNA/RNA hybrids, etc. RNA may be mRNA, rRNA or tRNA.
Advantageous biopolymers are those which do not readily form a molecular ion in known MALDI-TOF MS techniques, especially those which do not form a molecular ion on illumination of laser light at 340 nm. Biopolymers for use in the invention comprise two or more monomers, which may be the same or different as each other. Preferred biopolymers comprise at eas pp monomers, where pp is 5 or more (e.g. 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250). More preferred biopolymers comprise ppp or fewer monomers where ppp is 300 or less (e.g. 200, 100, 50).
Biopolymers may have a molecular mass of at least qq kDa, where qq - 0.5 or more (e.g. 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, etc.). Preferred biopolymers are those having a molecular mass within the range of detection of a mass spectrometer. More prefened biopolymers have a molecular mass of qqq kDa or less, where qqq is 30 or less (e.g. 20, 10, 5).
Preferably, the mass, m(IX), of the fragment (IX)
(Ar2) — C— [Ar1-(LM{M'}p)q]m * (IX) of the cation of formula (I) is significantly less than the mass, m(BP'), of the biopolymer residue BP'. For example the ratio m(Bp') / m(IX) is preferably more than nn, where nn is at least 2 (e.g. 3, 4, 5, 10, 100, 1000, etc.).
The invention is suitable for use with purified biopolymers or mixtures of biopolymers. For example, a pure recombinant protein could be derivatised and analysed by MS, or biopolymers within a cellular lysate or extract could be derivatives and then analysed.
Preferred biopolymers are polypeptides. Particularly prefened biopolymers are polypeptides formed after proteolytic digestion of a protein.
Biopolymers bound to solid supports In preferred embodiments of the invention the biopolymer is bound to a solid support such that it is cleavable from the solid support at least once it has been derivatised by a compound of the invention. Bp is thus derivatised in situ while bound to the support, and is then released. As the biopolymer is bound to the solid support, this aspect of the invention is particular relevant to methods involving compounds of formulae (Ila) and (lib). The biopolymer may be bound to the solid support by a covalent, ionic, hydrogen, dipole-dipole or van der Waals bond (also known as a dispersion bond or a London forces bond). The covalent, ionic, hydrogen, dipole-dipole or van der Waals bond may be direct between the biopolymer and the solid support or may be provided by one or more binding groups present on the biopolymer and/or solid support. Prefened groups are non-covalent groups.
Examples of groups which can form these types of bond, and methods for cleaving these types of bond, are set out below in connection with C- - -Ss bonds, etc.
In a particularly preferred embodiment, the solid support is provided with -(NMe3)+ binding groups and the biopolymer has a net negative charge, or vice versa (i.e. the -(NMe3)+ is on the biopolymer). In other preferred embodiments, the solid support is provided with anions such as carboxylate, phosphate or sulphate, or anions formed from acid groups, and the biopolymer (e.g. a histone) has a net positive charge, or vice versa.
Reactivity with group M
The biopolymers have at least one reactive group capable of reacting with M to form a covalent linkage. Such groups typically include naturally occurring groups and groups formed synthetically on the biopolymer.
Naturally occurring groups include lipid groups of lipoproteins (e.g. myristoyl, glycosylphosphatidylinositol, ethanolamine phosphoglycerol, palmitate, stearate, S- or N- or O-acyl groups, lipoic acid, isoprenyl, geranylgeranyl, farnesyl, etc.), amide, carbohydrate groups of N- and O- glycoproteins, amine groups (e.g. on lysine residues or at the Ν-terminus of a protein), hydroxyl (e.g. in β-hydroxyaspartate, β-hydroxyasparagine, 5-hydroxylysine, 3/4-hydroxyproline), thiol, sulfhydryl, phosphoryl, sulfate, methyl, acetyl, formyl (e.g. on Ν-terminal methionines from prokaryotes), phenyl, indolyl, guanidyl, hydroxyl, phosphate, methylthio, ADP-ribosyl etc.
The reactive group is bound to the biopolymer by one or more covalent bonds (e.g. 2 or 3 bonds), which are either single, double or triple covalent bonds (preferably single bonds). Preferably, the reactive group is bound to the biopolymer by one single bond.
Groups which may be formed naturally or synthetically on the biopolymer and which are bound to the biopolymer by one bond include: -ΝR2 e.g. -NHR, especially -NH2; -SR e.g. -SH; -OR e.g. -OH; -B(R)Y; -BY2; -C(R)2Y; -C(R)Y2; -CY3; -C(=Z)Y e.g. -C(=0)Y; -Z-C(=Z)Y; -C(=Z)R e.g. -C(=Z)H, especially -C(=0)H; -C(R)(OH)OR; -C(R)(OR)2; -S(=0)Y; -Z-S(=0)Y; -S(=0)2Y; -Z-S(=0)2Y; -S(=0)3Y; -Z-S(=0)3Y; -P(=Z)(ZR)Y e.g. -P(=0)(OH)Y; -P(=Z)Y2; -Z-P(=Z)(ZR)Y; -Z-P(=Z)Y2; -P(=Z)(R)Y e.g. -P(=0)(H)Y; -Z-P(=Z)(R)Y; or -N=C(=Z) e.g. -N=C(=0).
Another group which may be formed naturally or synthetically on the biopolymer and which is bound to the biopolymer by one bond is -CN. Other groups which may be formed naturally or synthetically on the biopolymer and which are bound to the biopolymer by one bond are: -P(ZR)Y e.g. -P(OH)Y; -PY2; -Z-P(ZR)Y; -Z-PY2; -P(R)Y e.g. -P(H)Y; -Z-P(R)Y. A particularly preferred group is -Z-P(ZR)Y, especially a phosphoramidite group:
Figure imgf000017_0001
Another example of a group which may be formed naturally or synthetically on the biopolymer and which is bound to the biopolymer by one bond is -Y. In particular, when the reactive group is halo (especially iodo), the reactive group may be bound to an aliphatic or aromatic carbon.
Groups which may be formed synthetically on the biopolymer and which are bound to the biopolymer by two bonds include -N(R)- e.g. -NH-; -S-; -0-; -B(Y)-; -C(R)(Y)-; -CY2-; -C(=O)-; -C(OH)(OR)-; -C(OR)2-.
Groups which may be formed synthetically on the biopolymer and which are bound to the
Figure imgf000017_0002
biopolymer by three bonds include .
Preferred groups include nucleophilic groups, either natural or synthetic, e.g.: -NR2 e.g. -NHR, especially -NH2; -SR e.g. -SH; -OR e.g. -OH; -N(R)- e.g. -NH-; -S-; and -0-. The groups -NH2, -SH and -OH are particularly preferred.
Another preferred reactive group is maleimidyl:
Figure imgf000017_0003
Y is independently a leaving group, including groups capable of leaving in an SN2 substitution reaction or being eliminated in an addition-elimination reaction with the reactive group of the biopolymer BP.
Preferred examples of Y include halogen (preferably iodo), C1-8hydrocarbyloxy (e.g. C1-8alkoxy), C1-8hydrocarbyloxy substituted with one or more A, C1-8heterohydrocarbyloxy, C1-8heterohydrocarbyloxy substituted with one or more A, mesyl, tosyl, pentafluorophenyl, -O-succinimidyl (formula VII) or a sulfo sodium salt thereof (sulfoNHS - formula Vila), -S-succinimidyl, or phenyloxy substituted with one or more A e.g. p-nitrophenyloxy (formula VIII) or pentafluorophenoxy (formula Villa).
Figure imgf000018_0001
Thus, prefened reactive group on the biopolymer are:
Figure imgf000018_0002
Other prefened examples of Y include -ZR. Particularly preferred examples of Y are -ZH (e.g. -OH or -NH2) and -Z-C1-8alkyl groups such as -NH-C1-8alkyl groups (e.g. -NHMe) and -0-Cι-8alkyl groups (e.g. -O-t-butyl). Thus, preferred reactive groups are -C(0)-NH-C1-8alkyl and -C(0)-0-C1- 8alkyl (e.g. -C(O)-O-t-butyl).
Other preferred examples of Y include -Z-ZR. Particularly preferred examples include -NR-NR2, especially -NH-NH2, and -ONR2, especially -0-NH2.
Z is independently O, S orN(R). Preferred (=Z) is (=0). R is independently H, Cι-8hydrocarbyl (e.g. C1-8alkyl) or C1-8hydrocarbyl substituted with one or more A.
R is preferably H. Other preferred reactive groups include -C(=0)Y, especially -C(=0)-0-succinimidyl and -C(=0)-0-(p-nitrophenyl).
In a further embodiment, the reactive group may be -Si(R)2-Y, with Y being halo (e.g. chloro) being especially prefened. Prefened groups R in this embodiment are C1-8alkyl, especially methyl. A particularly prefened reactive group in this embodiment is -Si(Me)2Cl.
Other groups which may be formed naturally or synthetically on the biopolymer include groups capable of reacting in a cycloaddition reaction, especially a Diels-Alder reaction.
In the case of Diels-Alder reactions, the reactive group on the biopolymer is either a diene or a dienophile. Prefened diene groups are
Figure imgf000019_0001
and multivalent derivatives formally formed by removal of one or more hydrogen atoms, where A1 is -R1 or -Z'R1, where R1 and Z1 are defined below.
Prefened dienophile groups are -CR^CR^, -CR1=C(R1)A2, -CA2=CR1 2, -CA^C^A2 or -CA2=CA2 2, and multivalent derivatives formally formed by removal of one or more hydrogen atoms, where R1 is defined below and A2 is independently halogen, trihalomethyl, -N02, -CN, -N^R^O', -C02H, -COsR1, -S03H, -SOR1, -SOaR1, -SO3R1, -OC(=0)OR1, -C(=O)H, -C(=0)R1, -OC(=0)R1, , -OC(=0)NR1 2, -N(R')C(=0)R1, -C(=S)NR1 2, -NR1C(=S)R1, -S02NR1 2, -MR1SO2R1, -N(R1)C(=S)NR1 2, or -N(R1)S02NR1 2, where R1 is defined below. A particularly preferred dienophile group is maleimidyl. Group M
The group M is capable of reacting with the reactive group of the biopolymer, BP, to form a covalent linkage. [Group 'M' is shown as 'AFG' in the drawings].
The group M is bound to LM by one or more covalent bonds (e.g. 2 or 3 bonds, especially 2 such — LM M as ^- — ), which are either single, double or triple covalent bonds (preferably single bonds). Preferably, M is bound to LM by one single bond.
Alternatively, or in addition, M is bound by more than one L , such LM either being attached to the same or different Ar1 or Ar2. In a preferred embodiment M is bound by more than one LM from different Ar1 or Ar2, e.g. :
Figure imgf000020_0001
Examples of group M bound to LM by one bond include -NR2 e.g. -NHR, especially -NH ; -SR e.g. -SH; -OR e.g. -OH; -B(R)Y; -BY2; -C(R)2Y; -C(R)Y2; -CY3; -C(=Z)Y e.g. -C(=0)Y; -Z-C(=Z)Y; -C(=Z)R e.g. -C(=Z)H, especially -C(=0)H; -C(R)(OH)OR; -C(R)(OR)2; -S(=0)Y; -Z-S(=0)Y; -S(=0)2Y; -Z-S(=0)2Y; -S(=0)3Y; -Z-S(=0)3Y; -P(=Z)(ZR)Y e.g. -P(=0)(OH)Y; -P(=Z)Y2; -Z-P(=Z)(ZR)Y; -Z-P(=Z)Y2; -P(=Z)(R)Y e.g. -P(=0)(H)Y; -Z-P(=Z)(R)Y; or -N=C(=Z) e.g. -N=C(=0).
Another example of a group M bound to LM by one bond is -CN.
Other examples of group M bound to LM by one bond are -P(ZR)Y e.g. -P(OH)Y; -PY2; -Z-P(ZR)Y; -Z-PY2; -P(R)Y e.g. -P(H)Y; -Z-P(R)Y. A particularly preferred group M is -Z-P(ZR)Y, especially a phosphoramidite group: N(iPr)2 _>-P o — , — CN
Another example of group M bound to LM by one bond is -Y. In particular, when group M is halo (especially iodo), M may be bound to an aliphatic or aromatic carbon. When M is halo (e.g. iodo) and is bound to an aromatic carbon, LM may, for example, be a single bond.
Examples of group M bound to LM by two bonds include -N(R)- e.g. -NH-; -S-; -0-; -B(Y)-; -C(R)(Y)-; -CY2-; -C(=0)-; -C(OH)(OR)-; -C(OR)2-.
Examples of group M bound to L by three bonds include . Y )
Preferred groups M include electrophilic groups, especially those susceptible to SN2 substitution reactions, addition-elimination reactions and addition reactions, e.g. -B(R)Y; -BY2; -C(R)2Y; -C(R)Y2; -CY3; -C(=Z)Y e.g. -C(=0)Y; -Z-C(=Z)Y; -C(=Z)R e.g. -C(=Z)H, especially -C(=0)H; -C(R)(OH)OR; -C(R)(OR)2; -S(=0)Y; -Z-S(=0)Y; -S(=0)2Y; -Z-S(=0)2Y; -S(=0)3Y; -Z-S(=0)3Y; -P(=Z)(ZR)Y e.g. -P(=0)(OH)Y; -P(=Z)Y2; -Z-P(=Z)(ZR)Y; -Z-P(=Z)Y2; -P(=Z)(R)Y e.g. -P(=0)(R)Y; -Z-P(=Z)(H)Y; -N=C(=Z) e.g. -N=C(=O); -B(Y)-; -C(R)(Y)-; -CY2-; -C(=0)-;
-C(OH)(OR)-; -C(OR)2-; or C(Y) .
Another preferred electrophilic group M is -CN.
Still further preferred examples of group M are orthoesters, e.g. -C(OR)3. In a prefened embodiment, the R groups are linked together to form a hydrocarbyl group, e.g. a C1-8alkyl group. A preferred example of group M in this embodiment is:
Figure imgf000021_0001
Another prefened group M is maleimido.
Y, Z and R are defined as above. Prefened Y groups when present on M are those capable of leaving in an SN2 substitution reaction or being eliminated in an addition-elimination reaction with the reactive group of the biopolymer BP.
Preferred examples of Y include halogen (preferably iodo), C1-8hydrocarbyloxy (e.g. C1-8alkoxy), C1-8hydrocarbyloxy substituted with one or more A, C1-8heterohydrocarbyloxy, C1-8heterohydrocarbyloxy substituted with one or more A, mesyl, tosyl, pentafluorophenyl, -O-succinimidyl (formula VII) or a sulfo sodium salt thereof (sulfoNHS - formula Vila), -S-succinimidyl, or phenyloxy substituted with one or more A e.g. p-nitrophenyloxy (formula VIII) or pentafluorophenoxy (formula Villa).
Figure imgf000021_0002
(VIII). (Villa)
Thus, preferred groups M are:
Figure imgf000021_0003
Other preferred examples of Y include -ZR. Particularly prefened examples of Y are -ZH (e.g. -OH or -NH2) and -Z-C1-8alkyl groups such as -NH-C1-8alkyl groups (e.g. -NHMe) and -0-C1-8alkyl groups (e.g. -O-t-butyl). Thus, prefened groups M are -C(0)-NH-C1-8alkyl (e.g. -C(O)NHMe) and -C(0)-0-C1-8alkyl (e.g. -C(O)-O-t-butyl). Other preferred examples of Y include -Z-ZR. Particularly prefened examples include -NR-NR2, especially -NH-NH2, and -ONR2, especially -0-NH2.
Particularly prefened groups M include -C(=0)Y, especially -C(=0)-0-succinimidyl and -C(=0)-0-(p-nitrophenyl). In a further embodiment, M may be -Si(R)2-Y, with Y being halo (e.g. chloro) being especially preferred. Preferred groups R in this embodiment are C1-8alkyl, especially methyl. A particularly prefened group M in this embodiment is -Si(Me) Cl.
In a further embodiment, M may be -C(Ar2)2X. Prefened groups Ar2 and X are set out below. In this embodiment it is prefened that LM is a bond. A particularly prefened group M in this embodiment is:
Figure imgf000022_0001
Other groups M include groups capable of reacting in a cycloaddition reaction, especially a Diels- Alder reaction.
In the case of Diels-Alder reactions, the reactive group on the biopolymer is either a diene or a dienophile. Prefened diene groups are
Figure imgf000022_0002
and multivalent derivatives formally formed by removal of one or more hydrogen atoms, where A1 is
-R or -Z R , where R and Z are defined below.
Preferred dienophile groups are -CR^CR^, -CR1=C(R1)A2, -CA^CR^, -CA2=C(R!)A2 or -CA2=CA2 2, and multivalent derivatives formally formed by removal of one or more hydrogen atoms, where R1 is defined below and A2 is independently halogen, trihalomethyl, -N02, -CN, -N^R^O", -C02H, -COaR1, -S03H, -SOR1, -SOzR1, -SOsR1, -OC(=0)OR1, -C(=0)H, -C(=0)R1, -OC(=0)R!, , -OC(=0)NR1 2, -Nt ^CHJtR1, -C(=S)NR1 2, -NR1C(=S)R1, -S02NR1 2, -NR^R1, -N(R1)C(=S)NR1 2, or -N(R1)S02NR1 2, where R1 is defined below. A particularly preferred dienophile group is maleimidyl.
Preferred examples of group M are shown in figures 1 IA and 1 IB.
Matching Bp andM
The reactive group on the biopolymer [shown as 'F' in the drawings] and the group M [shown as 'AFG' in the drawings] must be dependently selected in order to form the covalent linkage. For example, where the biopolymer includes the groups -NH2, -OH or -SH, M will typically be -B(R)Y; -BY2; -C(R)2Y; -C(R)Y2; -CY3; -C(=Z)Y e.g. -C(=0)Y; -Z-C(=Z)Y; -C(-Z)R e.g. -C(=Z)H, especially -C(=0)H; -C(R)(OH)OR; -C(R)(OR)2; -S(=0)Y; -Z-S(=0)Y; -S(=0)2Y; -Z-S(=0)2Y; -S(=0)3Y; -Z-S(=O)3Y; -P(=Z)(ZR)Y e.g. -P(=O)(OH)Y; -P(=Z)Y2; -Z-P(=Z)(ZR)Y; -Z-P(=Z)Y2; -P(=Z)(R)Y e.g. -P(=0)(H)Y; -Z-P(=Z)(R)Y; -N=C(=Z) e.g. -N-C(=0); -B(Y)-; -C(R)(Y)-; -CY2-;
-C(=0)-; -C(OH)(OR)-; -C(OR)2-; or — C 0 . M may also be -CN. In a prefened embodiment, one of the reactive group on the biopolymer and group M is a maleimidyl and the other will be a -SH group. Alternatively, when the covalent linkage is to be formed by a Diels Alder reaction, one of the reactive group on the biopolymer and group M will typically be a diene and the other will be a dienophile. Prefened covalent linkages are those produced through the reaction of the following groups:
Figure imgf000023_0001
The covalent residue M'-BP' is the reaction product of M and BP. BP' will generally be the same as BP except that instead of the reactive group, BP' will have a residue of the reactive group covalently bound to the residue M'. Depending on the choice of the reactive group and the choice of M, M' and the residue of the reactive group will typically form linkages, in the orientation LM-M'-BP', including -C(R)2Z-, -ZC(R)2-, -C(=Z)Z-, -ZC(=Z)-, -ZC(=Z)Z-, -C(OH)(R)Z-, -ZC(OH)(R)~, -C(R)(OR)Z-, -ZC(R)(OR)-, -C(R)(OR)Z-, -ZC(R)(OR)-, -S(=0)Z-, -ZS(=0)-, -ZS(=0)Z-, -S(=0)2Z-, -ZS(=0)2-, -ZS(=0)2Z-, -S(=0)3Z-, -ZS(=0)3-, -ZS(=0)3Z-, -P(=Z)(ZR)Z-, -ZP(=Z)(ZR)-, -ZP(=Z)(ZR)Z-, -P(=Z)(R)Z-, -ZP(=Z)(R)-, -ZP(=Z)(R)Z-, -NH-C(=Z)-Z-, where Z and R are as defined above. Group M"
M" is the same as M except that the group Ss is bound to a portion of M which does not form part of M'. Thus, M" is a residue of M formable by the conjugation of M and Ss. However, M" need not necessarily be formed by the conjugation of M and Ss. M"- - -Ss comprises a covalent, ionic, dipole-dipole, hydrogen, or van der Waals bond. The covalent, ionic, hydrogen, dipole-dipole or van der Waals bond may be direct between M" and Ss or may be provided by one or more binding groups present on M" and/or Ss.
Examples of groups which can form these types of bond, and methods for cleaving these types of bond, are set out below in connection with C- - -Ss bonds, etc. This embodiment of the invention is advantageous, since the derivativisation of the biopolymer will also release the derivatised biopolymer from the solid support. Thus, an additional step of cleaving the biopolymer from the solid support is not required.
Prefened groups M" are groups M having a leaving group, wherein the group Ss is bound to the leaving group, e.g. groups M mentioned above having a leaving group Y, wherein the group Ss is bound to the leaving group Y.
A particularly prefened group M" is:
Figure imgf000024_0001
Where the group LM is a linker atom or group, it has a sufficient number of linking covalent bonds to link LM to the group Ar1 by a single covalent bond (or more, as appropriate) and to link LM to the p instances of M (or M', as appropriate) groups (which may be attached to LM by one or more bonds).
The group LM may be directly bound to the aromatic part of Ar1, bound to one or more of the substituents A of Ar1, or both. Preferably, LM is bound directly to the aromatic part of Ar1.
In an alternative embodiment, LM may be bound to L5. When LM is a linker atom, preferred linker atoms are O or S, particularly O.
When LM is a linker group, preferred linker groups, in the orientation Ar1-(LM{M}p)q or Ar'- LMlM'}^, as appropriate, are -EM-, -(DM)t-, -(EM-DM)t-, -(DM-EM)t-, -EM-(DM-EM)t- or -DM-(EM-DM)t~, where a sufficient number of linking covalent bonds, in addition to the covalent bonds at the chain termini shown, are provided on groups EM and DM for linking the p instances of M (or M') groups. DM is independently C1-8hydrocarbylene or C1-8hydrocarbylene substituted with one or more A. Prefened DM are Cι-8alkylene, C1-8alkenylene and C1-8alkynylene, especially C1-8alkylene and C1-8alkynylene, each optionally substituted with one or more A (preferably unsubstituted). A prefened substituent A is 2H. Prefened LM in the orientation Ar1-(LM{M}p)q or Ar1-(LM{M'}p)q, as appropriate, are: -CH2CH2-; -C≡C-CH2CH2CH2-; -(CH2)5-; -CD2CD2CH2CH2CH2-; -C≡C-CH2- and -CH2CH2CH2-.
EM, in the orientation Ar1-(LM{M}p)q or Ar1-(LM{M'}p)q, as appropriate, is independently -ZM-, -C(=ZM)-, -ZMC(=ZM)-, -C(=ZM)ZM-, -ZMC(=ZM)ZM-, -S(=O)-, -ZMS(=0)-, -S(=0)ZM-, -ZMS(=0)ZM-, -S(=0)2-, -ZMS(=0)2-, -S(=0)2ZM-, -ZMS(=0)2ZM-, where ZM is independently O, S or N(RM) and where RM is independently H, Cι-8hydrocarbyl (e.g. C1-8alkyl) or C1-8hydrocarbyl substituted with one or more A. Preferably EM is, in the orientation Ar1-(LM{M}p)q or Ar1-(LM{M,}p)q, as appropriate, -0-, -S-, -C(=0)-, -C(=0)0-, -C(=S)-, -C(=S)0-, -OC(=S)-, -C(=0)S-, -SC(=0)-, -S(O)-, -S(0)2-, -NRM-, -C(=0)N(RM)-, -C(=S)N(RM)-, -N(RM)C(=0)-, -N(RM)C(=S)-, -S(=0)N(RM)-, -N(RM)S(=0)-, -S(=0)2N(RM)-, -N(RM)S(=0)2-, -OC(=0)0-, -SC(=0)0-, -OC(=0)S-, -N(RM)C(=0)0-, -OC(=0)N(RM)-, -N(RM)C(=0)N(RM)-, -N(RM)C(=S)N(RM)-, -N(RM)S(=0)N(RM)- or -N(RM)S(=0)2N(RM)-.
Alternative groups EM to those defined above, in the orientation
Figure imgf000025_0001
as appropriate, are -ZM-Si(RM)2-ZM-, -Si(RM)2-ZM- and -ZM-Si(RM)2-. The group -Si(RM)2-ZM- is particularly prefened. ZM is preferably O. RM is preferably C1-8alkyl, preferably methyl. These groups EM are particularly prefened in the groups -(EM-DM)f, especially when t=l and DM is C1-8alkylene. The following group is especially prefened:
Figure imgf000025_0002
In addition to the above definition of DM, DM may also be C1-8heterohydrocarbylene or C1-8heterohydrocarbylene substituted with one or more A. In this embodiment, -scycloheteroalkylene groups are particularly preferred,
Figure imgf000025_0003
Thus, preferred LM groups -DM-EM-DM- are, in the orientation Ar1-(LM{M}p)q or
Figure imgf000025_0004
as appropriate, -C1-8alkylene-C(0)-C1-8cycloheteroalkylene (preferably where the hetero atom is N and is bound to the carboxy), especially:
Figure imgf000025_0005
t = 1 or more, e.g. from 1 to 50, lto 40, 1 to 30, 1 to 20 or 1 to 10. Preferably t = 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10. Preferably, LM links one group M (or M') to Ar1, M (or M') is linked to LM by a single covalent bond and therefore no additional bonds are required (e.g. L {M}! may be -EM-{M}, -(DM)t-{M}, -(EM-DM)t-{M}, -(DM-EM)t-{M}, -EM-(DM-EM)t-{M} or -DM-(EM-DM)t-{M}).
Where LM includes a group which also falls within the definition of group M, the group M is preferably more reactive than the group included in LM-
LM is preferably -(DM)t-, -(EM-DM)t-,
Figure imgf000026_0001
When group LM is -(DM)t-, t is preferably 1. DM is preferably C1-8alkylene, preferably methylene or ethylene.
When group LM is -(EM-DM)t-, or -DM-(EM-DM)t-, EM is preferably (in the orientation
Figure imgf000026_0002
or
Figure imgf000026_0003
or O (preferably O), and DM is preferably Cι-8alkylene, preferably ethylene, propylene, butylene or pentylene (preferably ethylene or propylene). t is preferably 1. Especially prefened LM are, in the orientation Ar^^MfMj^q or Ar1-(LM{M'}p)q, as appropriate,, -0-CH2CH2CH2- and -0-CH2CH2CH2CH2CH2-.
Another prefened group -DM-(EM-DM)t- is where DM is C1-8alkylene and t is 1. Preferred EM in this group, in the orientation Arl-( u{M}p)q or Arl-(LuWl'}p)q, as appropriate, are -ZMC(=ZM)- (especially -N(RM)C(=0)-, e.g. -N(Me)C(=0)-) and -C(=ZM)ZM- (especially -C(=0)0-). Particularly prefened LM groups are:
Figure imgf000026_0004
The group -(EM-DM)t- is prefened, a particularly preferred example of which is (in the orientation
Figure imgf000026_0005
or Ar1-(LM{M,}p)q, as appropriate)
-C(=0)NH-CH2CH2CH2-0-CH2CH2-0-CH2CH2-0-CH2CH2CH2-.
In an alternative embodiment it is preferred that LM is a single covalent bond. When Ar2 is phenyl, LM is preferably provided in a position ortho or para to C*. When Ar2 is other than phenyl, LM is preferably attached to an atom which bears the charge in at least one of the resonance structures of the ions of formula (I).
Where C* is a cation, LM is preferably an electron-donating group. Where C* is an anion, LM is preferably an electron- withdrawing group.
Prefened examples of LM are shown in figure 10A and 10B.
C- - -Ss, Ss- - -Ar1 and Ss- - -Ar2 Bonds
C- - -Ss, Ss- - -Ar1 and Ss- - -Ar2 comprise a cleavable covalent, ionic, hydrogen, dipole-dipole or van der Waals bond (also known as a dispersion bond or a London forces bond). The covalent, ionic, hydrogen, dipole-dipole or van der Waals bond may be direct between C and Ss, Ar1 and Ss, or Ar2 and Ss, or may be provided by one or more binding groups present on C and/or Ss, Ar1 and/or Ss, or Ar2 and/or Ss, respectively.
Covalent Bonding
Where the bond is covalent, the bond may be direct (e.g. C-Ss, A^-Ss or Ai^-Ss, respectively) or may be provided by a linker atom or group L4 (e.g. C-L4-Ss, Ar1-L4-Ss or Ar-L4-Ss, respectively).
When L4 is a linker group, prefened linker groups are -E4-, -(D4)t»-, -(E4-D4)t"-, -(D4-E4)t"-, -E4-(D4-E4)t- or -D4-(E4-D4)r-.
D4 is independently C1-8hydrocarbylene or Cι-8hydrocarbylene substituted with one or more A.
E4 is, in the orientation C-L4-Ss, independently -Z4-, -C(=Z4)-, -Z4C(=Z4)-, -C(=Z4)Z4-, -Z4C(=Z4)Z4-, -S(=0)-, -Z4S(=0)-, -S(=0)Z4-, -Z4S(=0)Z4-, -S(=0)2-, -Z4S(=0)2-, -S(=0)2Z4-, -Z4S(=0)2Z4-, where Z4 is independently O, S or N(R4), and where R4 is independently H, C1-8hydrocarbyl (e.g. C)-8alkyl) or C1-8hydrocarbyl substituted with one or more A. Preferably E4 is, in the orientation C-L4-Ss, -0-, -S-, -C(=0)-, -C(=0)0-, -C(=S)-, -C(=S)0-, -OC(=S)-, -C(=0)S-, -SC(=0)-, -S(O)-, -S(0)2-, -N(R4)-, -C(=0)N(R4)-, -C(=S)N(R4)-, -N(R4)C(=0)-, -N(R4)C(=S)-, -S(-0)N(R4)-, -N(R4)S(=0)-, -S(=0)2N(R4)-, -N(R4)S(=0)2-, -OC(=0)0-, -SC(=0)0-, -0C(O)S-, -N(R4)C(=0)0-, -OC(=0)N(R4)-, -N(R )C(=0)N(R4)-, -N(R4)C(=S)N(R4)-, -N(R4)S(=0)N(R4)- or - N(R4)S(=0)2N(R4)-. t" = 1 or more, e.g. from 1 to 50, lto 40, 1 to 30, 1 to 20 or 1 to 10. Preferably t" = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Where L4 includes a group which also falls within the definition of group M, the group M is preferably more reactive than the group included in L5.
L4 is preferably a linker atom, preferably O or S, particularly O.
When the solid support Ss is gold, L4 is preferably covalently attached to the Ss by a sulphide or disulphide group. Ionic Bonding
Where the bond is ionic, the bond is typically direct (e.g. C* Ss*, where Ss* is a solid support counterionto C*).
Alternatively, it may be provided by binding groups, e.g. chelating ligands, present on C or Ss, Ar1 or Ss, or Ar2 or Ss, respectively. In the case of C- - -Ss bonds, the chelating ligand is typically only present on Ss and chelates with C*.
Suitable chelating ligands which can bind anions include polyamines and cryptands.
Suitable chelating ligands which can bind cations include polyacidic compounds (e.g. EDTA) and crown ethers.
Hydrogen Bonding
Where the bond is a hydrogen bond, the bond is usually provided by binding groups present on C or Ss, Ar1 or Ss, or Ar2 or Ss, respectively.
Typically, in order to form the hydrogen bond, one of C or Ss, Ar1 or Ss, or Ar2 or Ss, as appropriate, will have a binding group bearing one or more hydroxy, amino or thio hydrogen atoms, and the other of C or Ss, Ar1 or Ss, or Ar2 or Ss, respectively, will have a binding group bearing an atom having one or more lone pair of electrons (e.g. an oxygen, sulphur or nitrogen atom). Preferably, one of C or Ss, Ar1 or Ss, or Ar2 or Ss, as appropriate, will have a binding group comprising biotin, and the other of C or Ss, Ar1 or Ss, or Ar2 or Ss, respectively, will have a binding group comprising avidin or streptavidin. Alternatively, the hydrogen bond may be direct.
Dipole-Dipole Bonding
Where the bond is a dipole-dipole bond, it may be formed between permanent dipoles or between a permanent dipole and an induced dipole.
Typically, in order to form the dipole-dipole bond, one of Ss and the compound of the invention has a permanent dipole and the other of Ss and the compound of the invention has an induced dipole or a permanent dipole, the attraction between the dipoles forming a dipole-dipole bond.
Preferably, Ss comprises binding groups (e.g. acid groups, -(NMe3)+, carboxy, carboxylate, phosphate or sulphate groups) which produce a dipole at the surface of the solid support to bind the compound of the invention.
Van der Waals Bonding
Where the bond is a van der Waals bond, the bonding is usually provided by binding groups present on C or Ss, Ar1 or Ss, or Ar2 or Ss, respectively. Typically, in order to form the van der Waals bond, at least one, but preferably both, of C or Ss, Ar1 or Ss, or Ar2 or Ss, as appropriate, will have a hydrocarbyl or heterohydrocarbyl group (usually a large hydrocarbyl group having at least ten carbon atoms up to about 50 carbon atoms), optionally substituted with one or more A. Polyfluorinated hydrocarbyl and heterohydrocarbyl groups are particularly prefened. Typically, the hydrocarbyl or heterohydrocarbyl groups are aryl or heteroaryl groups or groups of the formula -C(R6)2Ar3, -C(R6)(Ar3)2 or -C(Ar3)3; where Ar3 is independently defined the same as Ar2 and R6 is H, C1-8 hydrocarbyl, Cι-8 hydrocarbyl substituted by one or more A, C1-8 heterohydrocarbyl or C1-8 heterohydrocarbyl substituted by one or more A.
A prefened binding group is tetrabenzofullerene (formula X).
(formula X)
Figure imgf000029_0001
Alternatively, the van der Waals bond may be direct.
Bond Cleavage
Preferably, the ions of formula (I) have a pKr+ value of at least zz, where zz is 0 or more (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). More preferably, zz is 1 or more, still more preferably 2 or more, still more preferably 3 or more.
Preferably, the compounds of formula (Ila), (lib), (Ilia) or (Illb) or the solid supports of formula (IVai), (IVaii), (IVaiii), (IVbii), (IVbiii), (IVaiv) or (IVbiv) provide ions of formula (I') having a pKr+ value of at least zz, where zz is defined above.
C-XBonds
The C-X bonds are cleavable by irradiation, electron bombardment, electrospray, fast atom bombardment (FAB), inductively coupled plasma (ICP) or chemical ionisation. Preferably, the C-X bonds are cleavable by inadiation or chemical ionisation.
The term 'irradiation' includes, for example, laser illumination, in particular as used in MALDI mass spectrometry. Laser light of about 340 nm is particularly preferred because it is typically used in MALDI mass spectrometers.
The term 'electron bombardment' includes, for example, bombardment with electrons having energy of about 70 ev.
Chemical ionisation can be effected, for example, by treatment with acid or acidic matrices (e.g. acidic matrices used in MALDI analysis). Preferably group X is halogen, hydroxy, C1-8hydrocarbyloxy, Cι-8hydrocarbyloxy substituted with one or more A, Cι-8heterohydrocarbyloxy, C1-8heterohydrocarbyloxy substituted with one or more A, mesyl, tosyl, pentafluorophenyl, -O-succinimidyl -S-succinimidyl, or phenyloxy substituted with one or more A e.g. p-nitrophenyloxy. The groups pentafluorophenyl, -O-succinimidyl, -S-succinimidyl, and p-nitrophenyloxy are particularly preferred.
Particularly prefened groups X are halogen, hydroxy, C1-8hydrocarbyloxy. Especially preferred groups are hydroxy, ethoxy and chloro groups.
Other prefened groups X are alkyl ethers, e.g. :
Figure imgf000030_0001
Group X may also be a -Q-oligonucleotide, where Q is O, S or N(R), where R is H, C1-8hydrocarbyl or Cι-8hydrocarbyl substituted with one or more A. Q is preferably O.
Group X may also be a nucleoside, preferably where the nucleoside is bound via its 5' end, e.g. :
Figure imgf000030_0002
In some embodiments of the invention, where BP is an antibody (particularly where it is a monoclonal antibody that recognises a tumour-associated antigen), X is not:
Figure imgf000031_0001
or, optionally, X is not any other 2,6-diaminopurine nucleoside prodrug group.
In some embodiments of the invention, X is not H. If X is H, preferably at least one of Ar1 and Ar2 is polycyclic, heterocyclic or unsubstituted. Prefened examples of group X are shown in figure 13.
Ionic C *X* Bonds
X* is any counterion for forming salts with compounds of the invention.
X* includes ions having single charges and multiple charges. Typically ions having multiple charges will be associated with an appropriate number of compounds of formula (lib), (Illb), (IVbii), (IVbiii), (IVbiv), (Vbii), (Vbiii) or (Vbiv) in order balance the charge. Ions having multiple charges include doubly charged ions (e.g. S04 2") and triply charged ions. X* preferably has a single charge.
The counterion X* may be dissociated from the derivative of formula (lib), (Illb), (IVbii), (IVbiii), (IVbiv), (Vbii), (Vbiii) or (Vbiv) by inadiation, electron bombardment, electrospray, fast atom bombardment (FAB), inductively coupled plasma (ICP) or chemical ionisation. Preferably, the counterion X* may be dissociated by inadiation.
When X* is a cation, X* is preferably FT1".
When X* is an anion, X* is preferably, BF6 " or C104 ".
It is preferred that X* is an anion.
Preferred examples of group X* are shown in figure 13.
C- - -Ss, Ss- - -Ar1 or Ss- - -Ar2
The C- - -Ss, Ss- - -Ar1 or Ss Ar2 bonds are cleavable by irradiation, electron bombardment, electrospray, fast atom bombardment (FAB), inductively coupled plasma (ICP) or chemical ionisation. Preferably, the C- - -Ss, Ss- - -Ar1 or Ss- - -Ar2 bonds are cleavable by irradiation or chemical ionisation. Where appropriate, the C- - -Ss, Ss- - -Ar1 or Ss- - -Ar2 bonds may be cleaved simultaneously or sequentially with the cleaving of the C-X bond or the dissociation of X*, as appropriate, by selection of suitable cleaving/dissociating conditions.
In one embodiment of the invention, the C- - -Ss bond in the solid support of formula (Vai) may be cleaved in sub-steps of step (iia) so that in a first sub-step a residue X (where X is the leaving group defined above) is provided and in a second subsequent sub-step the C-X bond is cleaved thereby forming the ion of formula (I). If desired, the second sub-step may be canied out substantially (e.g. seconds, minutes, hours or even days) after the first sub-step.
Ar1 andAr2 Ar2
Ar2 is independently an aromatic group or an aromatic group substituted with one or more A and is preferably independently cyclopropyl, cyclopropyl substituted with one or more A, aryl, aryl substituted with one or more A, heteroaryl, or heteroaryl substituted with one or more A.
Where aryl or substituted aryl, Ar2 is preferably C6-30 aryl or substituted C6-3o aryl. Where heteroaryl or substituted heteroaryl, Ar2 is preferably C6-3o heteroaryl or substituted C6-3o heteroaryl.
Examples of aryl and heteroaryl are monocyclic aromatic groups (e.g. phenyl or pyridyl), fused polycyclic aromatic groups (e.g. napthyl, such as 1-napthyl or 2-napthyl) and unfused polycyclic aromatic groups (e.g. monocyclic or fused polycyclic aromatic groups linked by a single bond, a double bond, or by a -(CH=CH)r- linking group, where r is one or more (e.g. 1, 2, 3, 4 or 5). Other examples of aryl groups are monovalent derivatives of aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, chrysene, coronene, fluoranthene, fluorene, αs-indacene, s- indacene, indene, naphthalene, ovalene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene and rubicene, which groups may be optionally substituted by one or more A.
Other examples of heteroaryl groups are monovalent derivatives of acridine, carbazole, ?-carboline, chromene, cinnoline, furan, imidazole, indazole, indole, indolizine, isobenzofuran, isochromene, isoindole, isoquinoline, isothiazole, isoxazole, naphthyridine, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pynolizine, quinazoline, quinoline, quinolizine, quinoxaline, thiophene and xanthene, which groups may be optionally substituted by one or more A. Preferred heteroaryl groups are five- and six-membered monovalent derivatives, such as the monovalent derivatives of furan, imidazole, isothiazole, isoxazole, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pynolizine and thiophene. The five-membered monovalent derivatives are particularly prefened, i.e. the monovalent derivatives of furan, imidazole, isothiazole, isoxazole, pyrazole, pyrrole and thiophene. The heteroaryl groups may be attached to the remainder of the compound by any carbon or hetero (e.g. nitrogen) atom.
Ar2 is preferably C6-30aryl substituted by one or more A, preferably phenyl or napthyl (e.g. 1-napthyl or 2-napthyl, especially 2-napthyl) substituted by one or more A, more preferably phenyl substituted by one or more A. When Ar2 is phenyl, A is preferably provided in a position ortho or para to C*. When Ar2 is other than phenyl, A is preferably attached to an atom which bears the charge in at least one of the resonance structures of the ions of formula (I). Fused polycyclic aromatic groups, optionally substituted with one or more A, are particularly preferred.
A particularly prefened Ar2 is unsubstituted pyrenyl or pyrenyl substituted with one or more A. Unsubstituted pyrenyl is preferred. The pyrenyl group may be 1 -pyrenyl, 2-pyrenyl or 4-pyrenyl. Prefened heteroaryl Ar2 groups, whether substituted or unsubstituted, are pyridyl, pyrrolyl, thienyl and furyl, especially thienyl.
A prefened Ar2 group is thiophenyl or thiophenyl substituted with one or more A. Unsubstituted thiophenyl is prefened. Examples of thiophenyl are thiophen-2-yl and thiophen-3-yl, with thiophen- 2-yl being especially prefened. When substituted, Ar2 is preferably substituted by 1, 2 or 3 A. Ar2 is preferably:
Figure imgf000033_0001
When unsubstituted, Ar2 is preferably:
Figure imgf000033_0002
In another prefened embodiment, Ar2 is cyclopropyl or cyclopropyl substituted with one or more A. Unsubstituted cyclopropyl is prefened. One or more, preferably one, of Ar2 may be cyclopropyl.
Preferred examples of group Ar2 are shown in figures 12A and 12B.
Ar1
Ar1 is independently an aromatic group or an aromatic group substituted with one or more A. The definition of Ar1 is the same as Ar2 (as defined above), except that the valency of the group Ar1 is adapted to accommodate the q instances of the linker LM. Preferred Ar2 groups are also prefened Ar1 groups, (as defined above), except that the valency of the group Ar1 is adapted to accommodate the q instances of the linker LM.
When q = 1, Ar1 is a divalent radical and is preferably independently cyclopropylene, cyclopropylene substituted with one or more A, arylene, arylene substituted with one or more A, heteroarylene, or heteroarylene substituted with one or more A. Where arylene or substituted arylene, Ar1 is preferably C6-3o arylene or substituted C6-3o arylene. Where heteroarylene or substituted heteroarylene, Ar1 is preferably C6-30 heteroarylene or substituted C6-3o heteroarylene.
Examples of arylene and heteroarylene are monocyclic aromatic groups (e.g. phenylene or pyridylene), fused polycyclic aromatic groups (e.g. napthylene) and unfused polycyclic aromatic groups (e.g. monocyclic or fused polycyclic aromatic groups linked by a single bond, a double bond, or by a -(CH=CH)r- linking group, where r is one or more (e.g. 1, 2, 3, 4 or 5).
Other examples of arylene groups are polyvalent derivatives (where the valency is adapted to accommodate the q instances of the linker LM) of aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, chrysene, coronene, fluoranthene, fluorene, αs-indacene, s-indacene, indene, naphthalene, ovalene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene and rubicene, which groups may be optionally substituted by one or more A.
Other examples of heteroarylene groups are polyvalent derivatives (where the valency is adapted to accommodate the q instances of the linker LM) of acridine, carbazole, ?-carboline, chromene, cinnoline, furan, imidazole, indazole, indole, indolizine, isobenzofuran, isochromene, isoindole, isoquinoline, isothiazole, isoxazole, naphthyridine, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pynolizine, quinazoline, quinoline, quinolizine, quinoxaline, thiophene and xanthene, which groups may be optionally substituted by one or more A. Preferred heteroaryl groups are five- and six- membered polyvalent derivatives, such as the polyvalent derivatives of furan, imidazole, isothiazole, isoxazole, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pynole, pynolizine and thiophene. The five-membered polyvalent derivatives are particularly preferred, i.e. the polyvalent derivatives of furan, imidazole, isothiazole, isoxazole, pyrazole, pyrrole and thiophene. The heteroaryl groups may be attached to the remainder of the compound by any carbon or hetero (e.g. nitrogen) atom.
Ar1 is preferably C6-3oarylene substituted by one or more A, preferably phenylene or napthylene substituted by one or more A, more preferably phenylene substituted by one or more A. When Ar1 is phenylene, A is preferably provided in a position ortho or para to C*. When Ar1 is other than phenylene, A is preferably attached to an atom which bears the charge in at least one of the resonance structures of the ions of formula (I).
When substituted, Ar1 is preferably substituted by 1, 2 or 3 A.
When unsubstituted, prefened Ar1 are:
Figure imgf000035_0001
Preferred examples of group Ar1 are shown in figures 12A and 12B.
Combinations ofAr
Optionally two or three of the groups Ar1 and Ar2 are linked together by one or more L5, where L5 is independently a single bond or a linker atom or group; and/or two or three of the groups Ar1 and Ar2 together form an aromatic group or an aromatic group substituted with one or more A.
When L5 is a linker group, preferred linker groups are -E5-, -(D5)t'-, -(E5-D5)t'-5 -(D5-E5)t>-, -E5-(D5-E5)f- or -D5-(E5-D5)t.-.
D5 is independently C1-8hydrocarbylene or C1-8hydrocarbylene substituted with one or more A. E5 is independently -Z5-, -C(=Z5)-, -Z5C(=Z5)-, -C(=Z5)Z5-, -Z5C(=Z5)Z5-, -S(=0)-, -Z5S(=0)-, -S(=0)Z5-, -Z5S(=0)Z5-, -S(=0)2-, -Z5S(=0)2-, -S(=0)2Zs-, -Z5S(=0)2Z5-, where Z5 is independently O, S or N(R5) and where R5 is independently H, Cι-8hydrocarbyl or C1-8hydrocarbyl substituted with one or more A. Preferably E5 is -0-, -S-, -C(=0)-, -C(-0)0-, -C(=S)-, -C(=S)0-, -OC(=S)-, -C(=0)S-, -SC(=0)-, -S(O)-, -S(0)2-, -N(R5)-, -C(=0)N(R5)-, -C(=S)N(R5)-, -N(R5)C(=0)-, -N(R5)C(=S)-, -S(=0)N(R5)-, -N(R5)S(-0)-, -S(=0)2N(R5)-, -N(R5)S(=0)2-, -OC(=0)0-, -SC(=0)0-, -OC(=0)S-, -N(R5)C(=0)0-, -OC(=0)N(R5)-, -N(R5)C(=0)N(R5)-, -N(R5)C(=S)N(R5)-, -N(R5)S(=0)N(R5)- or -N(R5)S(=0)2N(R5)-. t' = 1 or more, e.g. from 1 to 50, lto 40, 1 to 30, 1 to 20 or 1 to 10. Preferably t' = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Most preferably t-1. Where L5 includes an atom or group which also falls within the definition of group M, the group M is preferably more reactive than the group included in L5.
L5 is preferably a linker atom, preferably O or S, particularly O.
When L5 is a linker group, a preferred L5 is -N(R5)-.
In another embodiment in which L5 is a linker group, L5 is -S(=0)-. When two of the groups Ar1 and Ar2 are linked together by one or more (e.g. 2, 3 or 4) L5, they are preferably linked together by one L5, preferably O.
Preferred combinations of Ar are two Ar2 (e.g. two Ar2 phenyl groups) linked together by one L5 (e.g. O or S). Particularly prefened combinations of Ar are two Ar2 phenyl groups, optionally substituted by one or more A (preferably unsubstituted), linked together by one L5 (e.g. O or S), where is L5 is ortho to C* with respect to both phenyl groups. Especially prefened combinations of two Ar2 groups are:
Figure imgf000036_0001
In another embodiment, at least one L is linked to an atom or group L5. In this embodiment, the preferred L5 mentioned above are, where appropriate, modified to remove substituents R5 in order to accommodate LM, e.g. the R5 substituent of the group -N(R5)- is replaced by LM- In this embodiment, the L5 group to which LM is bound is preferably: ArVAr2
Figure imgf000036_0002
Prefened combinations of Ar1 and/or Ar2 in this embodiment are:
Figure imgf000036_0003
When two or three of the groups Ar1 and Ar2 together form an aromatic group or an aromatic group substituted with one or more A, the aromatic group may be a carbocyclic aromatic group or a carbocyclic aromatic group in which one or more carbon atoms are each replaced by a hetero atom. Typically, in an aromatic group in which one or more carbon atoms are each replaced by a hetero atom, up to three carbons are so replaced, preferably up to two carbon atoms, more preferably one carbon atom.
Preferred hetero atoms are O, Se, S or N, more preferably O, S or N.
When two or three of the groups Ar1 and Ar2 together form an aromatic group or an aromatic group substituted with one or more A, preferred aromatic groups are C8-50 aromatic groups.
The aromatic groups may be monocyclic aromatic groups (e.g. radicals of suitable valency derived from benzene), fused polycyclic aromatic groups (e.g. radicals of suitable valency derived from napthalene) and unfused polycyclic aromatic groups (e.g. monocyclic or fused polycyclic aromatic groups linked by a single bond, a double bond, or by a -(CH=CH)r- linking group, where r is one or more (e.g. 1, 2, 3, 4 or 5). When two or three of the groups Ar1 and Ar2 together form a carbopolycyclic fused ring aromatic group, prefened groups are radicals of suitable valency obtained from napthalene, anthracene or phenanthracene, chrysene, aceanthrylene, acenaphthylene, acephenanthrylene, azulene, fluoranthene, fluorene, αs-indacene, s-indacene, indene, phenalene, and pleiadene. When two or three of the groups Ar1 and Ar2 together form a carbopolycyclic fused ring aromatic group in which one or more carbon atoms are each replaced by a hetero atom, preferred groups are radicals of suitable polyvalency obtained from acridine, carbazole, β-carboline, chromene, cinnoline, indole, indolizine, isobenzofuran, isochromene, isoindole, isoquinoline, naphthyridine, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pynolizine, quinazoline, quinoline, quinolizine and quinoxaline.
Substitution ofAr1 and Ar2 — Anions and Cations
When C* is a cation, A is preferably an electron-donating group, including -R1 or -Z'R1, where R1 and Z1 are defined below. Preferably, R1 is C1-8hydrocarbyl, more preferably C1-8alkyl, especially methyl. Z1 is preferably O, S or NR1. R1 may be substituted with one or more Sut,2, but is preferably unsubstituted. When C* is a cation, A is preferably -OMe, -SMe, -N(Me)2 or Me. When C* is a cation, A, when an electron-donating group, is preferably provided (especially in relation to Ar1 or Ar2 being phenyl) in a position ortho or para to C*, preferably para. Furthermore, when C* is a cation, A, when an electron-withdrawing group (e.g. F), is preferably provided (especially in relation to Ar1 or Ar2 being phenyl) in a position meta to C*. Thus, prefened groups Ar1 and Ar2 are as follows:
Figure imgf000037_0001
When C* is an anion, A is preferably an electron- withdrawing group, including halogen, trihalomethyl, -N02, -CN, -N+(R1)2O_, -C02H, -COaR1, -S03H, -SOR1, -SOaR1, -SOsR1, -OC(=0)OR1, -C(=0)H, -C(=0)R1, -OC(=0)R1, -C(=0)NH2, -C(=0)NR1 2, -N(R1)C(=0)OR1, -N(R1)C(=0)NR1 2, -OC(=0)NR1 2, -N(R1)C(=0)R1, -C(=S)NR1 2, -NR1C(=S)R1, -SC^NR^, -NR'SQjR1, -N(R1)C(=S)NR1 2, or -N(R1)S02 R1 , where R1 is defined below. When C* is an anion, A, when an electron-withdrawing group, is preferably provided (especially in relation to Ar1 or Ar2 being phenyl) in a position ortho or para to C*, preferably para. Furthermore, when C* is an anion, A, when an electron-donating group, is preferably provided (especially in relation to Ar1 or Ar2 being phenyl) in a position meta to C*.
The group A may also comprise one or more isotopes of the atoms making up group A (e.g. example 60), thus, as discussed in more detail below, allowing the masses of the compounds of the invention to be varied. Prefened isotopes are 13C, 180 and 2H. When providing a series of compounds which differ only in their masses, 13C and 180 are particularly prefened as 2H atoms may cause a substantial change in the chemical properties of the compound due to the kinetic isotope effect.
Solid Supports 'Solid supports' for use with the invention include polymer beads, metals, resins, columns, surfaces (including porous surfaces) and plates (e.g. mass-spectrometry plates).
The solid support is preferably one suitable for use in a mass spectrometer, such that the invention can be conveniently accommodated into existing MS apparatus. Ionisation plates from mass spectrometers are thus prefened solid supports, e.g. gold, glass-coated or plastic-coated plates. Solid gold supports are particularly preferred.
Resins or columns, such as those used in affinity chromatography and the like, are particularly useful for receiving solutions of biopolymers (purified or mixtures). For example, a cellular lysate could be passed through such a column of formula (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) or (IVbiv) followed by cleavage of the support to leave compounds of formula (I). Solid supports of formulae (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) or (IVbiv) will generally present exposed groups M capable of reacting with a biopolymer, BP. For MS analysis, ions preferably have a predictable mass to charge (m/e) ratio. If a biopolymer reacts with more than one M group, however, then it will carry more than one positive charge once ionised, and its m/e ratio will decrease. Advantageously, therefore, the groups M are arranged such that any biopolymer molecule will covalently link with only a single group M. Consequently, each biopolymer will, on ionisation, carry a single positive charge and thus have a predictable mass to charge ratio.
Typically, the surface density of the solid supports of (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) or (IVbiv) will be provided so that a biopolymer molecule can only covalently link with one group M and thus to prevent the formation of multiply derivatised biopolymers. Varying the mass of compounds of the invention
Within the general formulae (I), (Ila), (lib), (Ilia), (Illb), (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii), (IVbiv), (Vai), (Vaii), (Vaiii), (Vaiv), (Vbii), (Vbiii) and (Vbiv), there is much scope for variation. There is thus much scope of variation in the mass of these compounds. In some embodiments of the invention, it is prefened to use a series of two or more (e.g. 2, 3, 4, 5, 6 or more) compounds with different and defined molecular masses.
The masses of the compounds of the invention can be varied via LM, Ar1 and/or Ar2. Preferably, the masses of the compounds of the invention are varied by varying A on the groups Ar1 and/or Ar2.
In this aspect of invention, compounds of the invention advantageously comprise one or more of F or
I as substituents A of the groups Ar1, Ar2 or Ar3. F and I each only have one naturally occurring isotope, 19F and 127I respectively, and thus by varying the number of F and I atoms present in the structure of the compounds, can provide a series of molecular mass labels having substantially identical shaped peaks on a mass spectrum.
Compounds of the invention may also include one or more 2H atoms, preferably as a substituent A or a part thereof of the groups LM, Ar1, Ar2 or Ar3 (in particular LM), in order to vary the masses of the compounds of the invention. The compounds of the invention may include isotopes of 13C and 180, prefererably as a substituent A or a part thereof of the groups LM, Ar1, Ar2 or Ar3 (in particular Ar1, Ar2 or Ar3), in order to vary the masses of the compounds of the invention. Compounds comprising 2H, 13C and 180 may also be used to provide a series of molecular mass labels having substantially identical shaped peaks on a mass spectrum, by varing the number of 2H, 13C and 180 atoms present in the structure of the compounds. When providing a series of compounds which differ only in their masses, 13C and 180 are particularly prefened as 2H atoms may cause a substantial change in the chemical properties of the compound due to the kinetic isotope effect.
In order to increase the molecular mass of the compounds of the invention and to increase the number of available sites for substitution by A, especially F and I, one or more of Ar1 and Ar2 may be substituted by one or more dendrimer radicals of appropriate valency, either as substituent A or group LM.
Prefened dendrimer radicals are the radicals obtained from the dendrimers of US 6,455,071 and PAMAM dendrimers.
The compounds of the invention may advantageously be used in the method of analysing a biopolymer disclosed herein, in particular in a method for following a reaction involving a biopolymer, Bp, since the abundance of a species of may be determined by mass spectrometry by measuring the intensity of the relevant peak in an obtained mass spectrum.
Specifically, there is provided a method for analysing biopolymer BP, comprising the steps of: (i) reacting a first sample comprising biopolymer BP with a compound of formula (Ila) or (lib) or a solid support of formula (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) or (IVbiv) at a time t^ (ii) reacting a second sample comprising biopolymer BP with a compound of formula (Ila) or (lib) or a solid support of formula (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) or (IVbiv) at a later time t2; (iii) preparing and analysing cations of formula (I) from the first and second samples; and (iv) comparing the results of the analysis from step (iii).
If levels of the biopolymer Bp decrease between times t] and t2 then there will be a decrease in detected ion; if levels of the biopolymer Bp increase between times t and t2 then there will be an increase in detected ion. The effects of stimuli on transcription and/or translation can therefore be monitored. Advantageously, different compounds of formula (Ila) or (lib) or different solid supports of formula (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) or (IVbiv) are used at different times in order to facilitate simultaneous and parallel analysis of the first and second samples. For example, if the two compounds used at times t] and t differ only by a 1H to 19F substitution then the relative abundance of Bp at the two times can be determined by comparing peaks separated by 18 units.
Advantageously, the reaction of the biopolymer with the compound of formula (Ila) or (lib) or the solid support of formula (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) or (IVbiv) will fix the biopolymer to prevent it reacting further and the steps of providing and analysing the cations may be canied out at a later convenient time. Alternatively, if the reaction of the biopolymer with the compound of formula (Ila) or (lib) or the solid support of formula (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) or (IVbiv) does not quench the reaction of the biopolymer being followed, a cation of formula (I) from the reaction product of step (i) or step (v) should be obtained as soon as possible after reaction of the biopolymer with the compound of formula (Ila) or (lib) or the solid support of formula (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) or (IVbiv). Compounds of Formulae (Ila) and (lib)
The compounds of formulae (Ila) or (lib) are available commercially or may be synthesised by known techniques.
Commercially available compounds of formulae (Ila) or (lib) are disclosed, for example in the Molecular Probes Catalogue, 2002. Commercially available trityls, and derivatives and analogues thereof, may also be derivatised with the groups (LM{M}p)q by known techniques.
Methods for synthesis of compounds of formula (Ila) or (lib) useful in the present invention are described in Chem. Soc. Rev. (2003) 32, p. 3-13, scheme 2 and "1. introduction", last two paragraphs. Groups (LM{M}p)q are usually introduced into the intermediates and the compounds are then assembled using the appropriate pathways. Alternatively, the groups (LM-{M}p)q may be added after assembly of the aromatic groups and α-carbon of the compounds.
Methods for synthesis of compounds of formulae (Ila) or (lib) are also described in WO99/60007.
Further methods for synthesising the compounds of formulae (Ila) or (lib) are described in European patent application 04 104 605.3.
Preferred compounds of formula (Ila), (lib) and (IVai) are:
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000042_0002
Chemical Groups
The ions of the invention are stabilised by the resonance effect of the aromatic groups Ar1 and Ar2. The term 'C- is a carbon atom bearing a single positive charge or a single negative charge' therefore not only includes structures having the charge localised on the carbon atom but also resonance structures in which the charge is delocalised from the carbon atom.
The term 'linker atom or group' includes any divalent atom or divalent group.
The teπn 'aromatic group' includes quasi and/or pseudo-aromatic groups, e.g. cyclopropyl and cyclopropylene groups.
The term 'halogen' includes fluorine, chlorine, bromine and iodine.
The term 'hydrocarbyl' includes linear, branched or cyclic monovalent groups consisting of carbon and hydrogen. Hydrocarbyl groups thus include alkyl, alkenyl and alkynyl groups, cycloalkyl (including polycycloalkyl), cycloalkenyl and aryl groups and combinations thereof, e.g. alkylcycloalkyl, alkylpolycycloalkyl, alkylaryl, alkenylaryl, cycloalkylaryl, cycloalkenylaryl, cycloalkylalkyl, polycycloalkylalkyl, arylalkyl, arylalkenyl, arylcycloalkyl and arylcycloalkenyl groups. Preferred hydrocarbyl are C1-14 hydrocarbyl, more preferably C1-8 hydrocarbyl.
Unless indicated explicitly otherwise, where combinations of groups are refened to herein as one moiety, e.g. arylalkyl, the last mentioned group contains the atom by which the moiety is attached to the rest of the molecule.
The term 'hydrocarbylene' includes linear, branched or cyclic divalent groups consisting of carbon and hydrogen formally made by the removal of two hydrogen atoms from the same or different
(preferably different) skeletal atoms of the group. Hydrocarbylene groups thus include alkylene, alkenylene and alkynylene groups, cycloalkylene (including polycycloalkylene), cycloalkenylene and arylene groups and combinations thereof, e.g. alkylenecycloalkylene, alkylenepolycycloalkylene, alkylenearylene, alkenylenearylene, cycloalkylenealkylene, polycycloalkylenealkylene, arylenealkylene and arylenealkenylene groups. Prefened hydrocarbylene are C1-1 hydrocarbylene, more preferably Cι-8 hydrocarbylene.
The term 'hydrocarbyloxy' means hydrocarbyl-O-.
The terms 'alkyl', 'alkylene', 'alkenyl', 'alkenylene', 'alkynyl', or 'alkynylene' are used herein to refer to both straight, cyclic and branched chain forms. Cyclic groups include C3-8 groups, preferably C5-8 groups. The term 'alkyl' includes monovalent saturated hydrocarbyl groups. Prefened alkyl are C1-8, more preferably C1-4 alkyl such as methyl, ethyl, n-propyl, i-propyl or t-butyl groups.
Prefened cycloalkyl are C5-8 cycloalkyl.
The term 'alkoxy' means alkyl-O-.
The term 'alkenyl' includes monovalent hydrocarbyl groups having at least one carbon-carbon double bond and preferably no carbon-carbon triple bonds. Prefened alkenyl are C2-4 alkenyl.
The term 'alkynyl' includes monovalent hydrocarbyl groups having at least one carbon-carbon triple bond and preferably no carbon-carbon double bonds. Prefened alkynyl are C2-4 alkynyl.
The term 'aryl' includes monovalent aromatic groups, such as phenyl or naphthyl. In general, the aryl groups may be monocyclic or polycyclic fused ring aromatic groups. Preferred aryl are C6-C1 aryl. Other examples of aryl groups are monovalent derivatives of aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, chrysene, coronene, fluoranthene, fluorene, αs-indacene, s- indacene, indene, naphthalene, ovalene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene and rubicene.
The term 'alkylene' includes divalent saturated hydrocarbylene groups. Preferred alkylene are Cι- alkylene such as methylene, ethylene, n-propylene, i-propylene or t-butylene groups.
Preferred cycloalkylene are Cs-8 cycloalkylene.
The term 'alkenylene' includes divalent hydrocarbylene groups having at least one carbon-carbon double bond and preferably no carbon-carbon triple bonds. Prefened alkenylene are C2-4 alkenylene.
The term 'alkynylene' includes divalent hydrocarbylene groups having at least one carbon-carbon triple bond and preferably no carbon-carbon double bonds. Preferred alkynylene are C2-4 alkynylene.
The term 'arylene' includes divalent aromatic groups, such phenylene or naphthylene. In general, the arylene groups may be monocyclic or polycyclic fused ring aromatic groups. Prefened arylene are C6-C14arylene. Other examples of arylene groups are divalent derivatives of aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, chrysene, coronene, fluoranthene, fluorene, αs-indacene, s- indacene, indene, naphthalene, ovalene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene and rubicene. The term 'heterohydrocarbyl' includes hydrocarbyl groups in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O, S or N. Heterohydrocarbyl groups thus include heteroalkyl, heteroalkenyl and heteroalkynyl groups, cycloheteroalkyl (including polycycloheteroalkyl), cycloheteroalkenyl and heteroaryl groups and combinations thereof, e.g. heteroalkylcycloalkyl, alkylcycloheteroalkyl, heteroalkylpolycycloalkyl, alkylpolycycloheteroalkyl, heteroalkylaryl, alkylheteroaryl, heteroalkenylaryl, alkenylheteroaryl, cycloheteroalkylaryl, cycloalkylheteroaryl, heterocycloalkenylaryl, cycloalkenylheteroaryl, cycloalkylheteroalkyl, cycloheteroalkylalkyl, polycycloalkylheteroalkyl, polycycloheteroalkylalkyl, arylheteroalkyl, heteroarylalkyl, arylheteroalkenyl, heteroarylalkenyl, arylcycloheteroalkyl, heteroarylcycloalkyl, arylheterocycloalkenyl and heteroarylcycloalkenyl groups. The heterohydrocarbyl groups may be attached to the remainder of the compound by any carbon or hetero (e.g. nitrogen) atom.
The term 'heterohydrocarbylene' includes hydrocarbylene groups in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O, S or N. Heterohydrocarbylene groups thus include heteroalkylene, heteroalkenylene and heteroalkynylene groups, cycloheteroalkylene (including polycycloheteroalkylene), cycloheteroalkenylene and heteroarylene groups and combinations thereof, e.g. heteroalkylenecycloalkylene, alkylenecycloheteroalkylene, heteroalkylenepolycycloalkylene, alkylenepolycycloheteroalkylene, heteroalkylenearylene, alkyleneheteroarylene, heteroalkenylenearylene, alkenyleneheteroarylene, cycloalkyleneheteroalkylene, cycloheteroalkylenealkylene, polycycloalkyleneheteroalkylene, polycycloheteroalkylenealkylene, aryleneheteroalkylene, heteroarylenealkylene, aryleneheteroalkenylene, heteroarylenealkenylene groups. The heterohydrocarbylene groups may be attached to the remainder of the compound by any carbon or hetero (e.g. nitrogen) atom.
Where reference is made to a carbon atom of a hydrocarbyl or other group being replaced by an O, S, Se or N atom, what is intended is that: — CH— — N — I is replaced by I
-CH= is replaced by -N=; or
-CH2- is replaced by -0-, -S- or -Se-. The term 'heteroalkyP includes alkyl groups in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O, S or N.
The term 'heteroalkenyl' includes alkenyl groups in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O, S or N.
The term 'heteroalkynyl' includes alkynyl groups in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O, S or N. The term 'heteroaryl' includes aryl groups in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O, S or N. Preferred heteroaryl are C5-1 heteroaryl. Examples of heteroaryl are pyridyl, pyrrolyl, thienyl or furyl.
Other examples of heteroaryl groups are monovalent derivatives of acridine, carbazole, ?-carboline, chromene, cinnoline, furan, imidazole, indazole, indole, indolizine, isobenzofuran, isochromene, isoindole, isoquinoline, isothiazole, isoxazole, naphthyridine, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pynolizine, quinazoline, quinoline, quinolizine, quinoxaline, thiophene and xanthene. Prefened heteroaryl groups are five- and six-membered monovalent derivatives, such as the monovalent derivatives of furan, imidazole, isothiazole, isoxazole, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pynolizine and thiophene. The five-membered monovalent derivatives are particularly preferred, i.e. the monovalent derivatives of furan, imidazole, isothiazole, isoxazole, pyrazole, pynole and thiophene.
The teπn 'heteroalkylene' includes alkylene groups in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O, S or N.
The term 'heteroalkenylene' includes alkenylene groups in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O, S or N. The term 'heteroalkynylene' include alkynylene groups in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O, S or N.
The term 'heteroarylene' includes arylene groups in which up to three carbon atoms, preferably up to two carbon atoms, more preferably one carbon atom, are each replaced independently by O, S, Se or N, preferably O, S or N. Preferred heteroarylene are C5-l4heteroarylene. Examples of heteroarylene are pyridylene, pyrrolylene, thienylene or furylene. Other examples of heteroarylene groups are divalent derivatives (where the valency is adapted to accommodate the q instances of the linker LM) of acridine, carbazole, ?-carboline, chromene, cinnoline, furan, imidazole, indazole, indole, indolizine, isobenzofuran, isochromene, isoindole, isoquinoline, isothiazole, isoxazole, naphthyridine, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pynole, pynolizine, quinazoline, quinoline, quinolizine, quinoxaline, thiophene and xanthene. Prefened heteroarylene groups are five- and six-membered divalent derivatives, such as the divalent derivatives of furan, imidazole, isothiazole, isoxazole, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pynole, pynolizine and thiophene. The five-membered divalent derivatives are particularly prefened, i.e. the divalent derivatives of furan, imidazole, isothiazole, isoxazole, pyrazole, pynole and thiophene.
Substitution
A is independently a substituent, preferably a substituent SUb0 Alternatively, A may be 2H.
Su 1 is independently halogen, trihalomethyl, -N02, -CN, -N^R^O", -C02H, -COsR1, -S03H, -SOR1, -SOzR1, -SOsR1, -OC(=0)OR1, -C(=0)H, -C(=0)R1, -OC(=0)R1, -NR^, -C(=0)NH2, -C(=0)NR1 2, -N(R1)C(=0)OR1, -N(R1)C(=0)NR1 2, -OC(=0)NR1 2, -N(R1)C(=0)R1, -C(=S)NR1 2, -NR1C(=S)R1, -S02NR12, -NR'SQjR1, -N(R1)C(=S)NR1 2, -NCR^SOzNR * -R1 or -Z1^.
Z s O, S, Se orNR0
R1 is independently H, C1-8hydrocarbyl, C1-8hydrocarbyl substituted with one or more SUb 2, C1-8heterohydrocarbyl or C1-8heterohydrocarbyl substituted with one or more SUb 2.
Sub 2 is independently halogen, trihalomethyl, -N02, -CN, -N+(C1-6alkyl)20', -C02H, -C02Ci-6alkyl, -S03H, -SOC1-6alkyl, -S02C1-6alkyl, -S03C1-6alkyl, -OC(=0)OC1-6alkyl, -C(=0)H, -C(=0)C1-6alkyl, -OC(=0)C1-6alkyl, -N(C1-6alkyl)2, -C(=0)NH2, -C(=0)N(C1-6alkyl)2,
-N(C1-6alkyl)C(=0)0(C1-6alkyl), -N(C1-6alkyl)C(=0)N(C1-6alkyl)2, -OC(=0)N(C1-6alkyl)2, -N(C1-6alkyl)C(=0)C1-6alkyl, -C(=S)N(C1-6alkyl)2, -N(C1-6alkyl)C(=S)C1-6alkyl, -S02N(C1-6alkyl)2, -N(C1-6alkyl)S02C1-6alkyl, -N(Cι-6alkyl)C(=S)N(C1-6alkyl)2, -N(C1-6alkyl)S02N(C1-6alkyl)2, C1-6alkyl
Figure imgf000046_0001
Where reference is made to a substituted group, the substituents are preferably from 1 to 5 in number, most preferably 1. However, molecular mass labels of the invention will generally comprise 1 or more, typically between 1 and 100 (e.g. 1 to 50, preferably 1 to 20) substituents Sub1 or Sub 2, typically F or I, in order to vary the masses of the molecular mass labels.
Preferred examples of substituent A are shown in figure 14. Miscellaneous
A may optionally be a monovalent dendrimer radical or a monovalent dendrimer radical substituted with one or more substituents Sub 1.
General The term "comprising" means "including" as well as "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y.
The term "about" in relation to a numerical value x means, for example, x+10%.
The word "substantially" does not exclude "completely" e.g. a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from the definition of the invention.
Tables
Table 1 — C* is a cation
Figure imgf000047_0001
Figure imgf000048_0001
Table 2 — n = 2, m = 1, p = 1 and q = 1
Figure imgf000048_0002
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 demonstrates conceptually the effect of the signal on a mass spectrum by a compound of formula (Ila) or (lib) of the invention. Free biopolymer, such as a peptide, has poorer desorption properties characterised by a smaller peak on the left of mass-spectrum whereas desorption improves when the same molecule is conjugated to a compound of the invention. Figure 2 shows the steps of biopolymer with a compound of formula (IVai). The derivativisation of a biopolymer with a compound of the invention can be canied out more conveniently by utilising the solid phase-based format, whereby the compound is temporarily covalently attached to a solid support. This eliminates all the separation steps associated with homogenous approach as the only additional step required would be a washing step. The solid support can be a resin, a surface or a porous surface. Alternatively, the solid support may be a mass-spectrometry sample plate, which dramatically decreases the sample preparation time. Both gold, glass- and plastic-coated plates are compatible with this approach. Figure 3 shows the steps of 'reverse' biopolymer derivativisation on a covalent solid support whereby the release of the biopolymer derivative happens simultaneously with the derivativisation process. The process is applicable M groups involving leaving groups. Figure 4 shows the steps of biopolymer derivativisation on an ionic solid support. Figure 5 shows of the steps of solid support-assisted biopolymer derivativisation. The biopolymer is first trapped onto a solid support and then labelled with a compound of formula (Ila) or (lib). An advantage of this technique is that a preliminary sample enrichment occurs, since not all of the biopolymer in the sample will stick to the solid support surface. Figure 6 shows the mass spectrum obtained when analysing an Gly-Gly-O-acyl dipeptide conjugated with a trityl compound of the invention. Figure 7 shows the mass spectrum obtained when analysing a conjugate of a peptide with a trityl compound of the invention. Figure 8 compared the mass spectra of a BSA digest without (8A) and with (8B) labelling. Figure 9 shows the mass spectrum obtained when analysing a mixture of trityl-labelled amines. Figures 10A and 10B show preferred examples of group LM. Figures 11 A and 1 IB show prefened examples of group M. Figures 12A and 12B shows prefened examples of groups Ar1 and Ar2. Figure 13 shows preferred examples of groups X and X*. Figure 14 shows prefened examples of substituent group A.
MODES FOR CARRYING OUT THE INVENTION Materials and Methods
The solid supports were Tenta Gel Macrobeads OH and NH2, 280-320 microns, Rapp Polymer. (MA)LDI-TOF mass-spectra were recorded on a PE-ABI Voyager™ Elite Reflectron Delayed Extraction Instrument. TLC were canied out with Merck silica gel (Kieselgel 60 F25 precoated plates and Kieselgel 60 0.040-0.063 mm). HPLC was canied out on a Waters system (Milford, MA, USA). Phosphoroamidite couplings were canied out in an ABI 394 DNA/RNA synthesiser. Chemicals and solvents were from Sigma/Aldrich/Fluka (USA), and BDH/Merck.
Example 1 — Conjugation of a trityl tag (in solution phase) with solid support-bound biopolymer
A 15mer poly-T oligonucleotide was synthesised on an ABI 394 DNA synthesiser using a T CPG support according to standard protocols of phosphoramidite chemistry on 0.2 μmol scale. After the last coupling, a MMTr-protected 'aminolink' phosphoramidite (Glen Res., USA) was added to a growing chain and deprotected using standard deblocker (2% DCA in DCM). The column was removed from the synthesiser, and after 10 min wash with acetonitrile it was attached to two 5 ml syringes and washed with a 0.1M solution of NHS-activated 4,4'-dimethoxy-4"-carboxyethyl trityl for 10 min at RT. The column was then washed with (3 x 10 ml) acetonitrile, placed on a DNA synthesiser and deprotected with ammonia according to standard protocols. The residue obtained after the evaporation was dissolved in 0.1 ml of 2M LiC104 and precipitated from cold acetone (1.5 ml). The precipitate was washed with 0.5ml of acetone and dried.
Example 2 — Homogenous conjugation of a trityl with non-polymeric ligands
A solution of NHS-activated 4,4'-dimethoxy-4"-carboxyethyl trityl (0.1M) in THF/dioxane (1:1) was mixed with a solution (0.5-1M) of an amine or of a mixture of amines (for example, propyl amine, butyl amine, pentyl amine, hexyl amine and phenethyl amine), typically 10 ml of a solution of an activated trityl with 5 ml of an amine solution. The mixtures were purified on prep-TLC (2mm-thick glass plates with UV254 indicator, Analtech/Aldrich-Sigma), typically in chloroform with 0.5% triethylamine. The areas containing the desired products were scratched off the plate, and the conjugates or the mixtures thereof were eluted using same solvent with 2-5% MeOH, filtered through a layer of glass wool, evaporated and dried.
Example 3 — Homogenous conjugation of a nhs-activated trityl with polymeric ligands
A peptide, an oligonucleotide, or any other biopolymer containing a (primary) amino group, is dissolved in a mixture of water and acetonitrile depending on its solubility, typically 20-50% of water in CH3CN. Non-aminogroup-containing buffers (ie. 50 mM sodium phosphate, 0.15 M NaCl, pH 7.2, or a bicarbonate buffer, but an additional desalting step may then need to be introduced to cut off the metal ions prior to mass-spectrometry) can be used to keep the pH at between 7-9. For particularly poorly soluble ligands other solvents may be used such as THF, DMSO, etc.
A solution of NHS-activated 4,4'-dimethoxy-4"-carboxyethyl trityl in acetonitrile or THF is added in approx. 5-10 times excess compared to an amine component. Conjugation usually reaches the maximum yield over 2-4 hours of reaction time. The conjugate formed can be analysed by MS directly, or after HPLC-purification.
Example 4 — Conjugation of a solid phase-immobilised nhs-activated trityl tag with a ligand
A Solid Phase-Immobilised NHS-Activated Trityl Tag was prepared by either method 1 or method 2.
Method 1: A NHS-Activated 4,4'-dimethoxy-4"-carboxyethyl trityl tag was covalently attached to hydroxyl groups of 200 μm Rapp Polymer beads by shaking the suspension of 100 mg of the resin in 5 ml of 0.1 M solution of trityl chloride tag in dry pyridine at +4°C for 3 hours and then washing the resin with pyridine and acetonitrile and drying in vacuo.
Method 2. A 5'-tritylated thymidine phosphoramidite was prepared from NHS-activated 4,4'- dimethoxy-4"-carboxyethyl trityl chloride in a standard way [M.J. Gait, Oligonucleotide Synthesis: A Practical Approach, IRL, Oxford, 1984]. The Rapp Polymer beads (2 x 40 mg) were placed in two 1 micromol scale DNA synthesis columns (Glen Res.). The first column was coupled with the said phosphoramidite on an ABI DNA synthesiser using manual supply of reagents (0.1M solution of a phosphoramidite and other standard phosphoramidite synthesis reagents) with a coupling step of 15 min. The second column was first derivatised with a trebler phosphoramidite (Glen Res.) according to the manufacturer's protocols and then coupled with the trityl tag-containing phosphoramidite as described for the first column. Both columns were excessively washed with acetonitrile.
The trityl loading of the solid supports produced by either method was determined spectrophotometrically (absorbance measurements at 490nm) to be 0.21 mmol/g for a straight attachment and 0.39 mmol/g for a tritylation on top of the trebling synthon. (The hydroxyl group loading of the Rapp polymer used was 0.25mmol/g).
To the solid support prepared as described above, a mixture of compounds to be labelled (typically peptides) is added, typically in a mixture of 20-50% water in acetonitrile. After incubation, with occasional shaking, for 60-120 min the resin is washed with several volumes of the same solvent, and the conjugated products are cleaved off the resin, typically by adding 0.5-2% TFA in appropriate solvent. The collected sample is then analysed by MS.
Example 5 — Mass spectrometry analysis of a derivatised Gly-Gly dipeptide
Figure 6 shows the mass spectrum obtained from a compound of the invention comprising a derivatised Gly-Gly-O-acyl dipeptide biopolymer.
The ion of formula (I) containing the derivatised Gly-Gly-O-acyl biopolymer is observed at the peak at molecular weight 516.5. There was no peak corresponding to the free dipeptide. The fragment of formula (VI), in which the derivatised Gly-Gly-O-acyl biopolymer has been lost, is observed at the peak at the molecular weight 374.6.
Example 6 — Mass spectrometry analysis of a derivatised peptide
Figure 7 shows the mass spectrum obtained from a compound of the invention comprising a derivatised peptide biopolymer. The free peptide had a molecular weight of 310.
The ion of formula (I) containing the derivatised peptide biopolymer is observed at the peak at molecular weight 665.0.
The fragment of formula (VI), in which the derivatised peptide has been lost, is observed at the peak at the molecular weight 375.0. Significantly, there is only a very small peak at molecular weight 310, where a peak conesponding to the free biopolymer would be found. The relative size of the peaks at 665.0 and 310 thus demonstrate the significantly improved ionisability of the compounds of the invention compared with free biopolymer.
Example 7 — Spectral improvement by trityls Three proteins (BSA, β-casein and ADH) were digested with trypsin and the resulting peptides analysed by MALDI-TOF mass spectrometry with or without derivatisation. The number of peptides identified for each protein is shown below. The theoretical total number of peptides that would be produced by trypsin digestion of each protein was calculated in silico and is shown in the second column the table below.
Figure imgf000054_0001
+ The number of theoretical peptides for each protein was generated assuming one missed cleavage and disregarding di- and mono-amino acids generated. * Score is -10*Log(P), where P is the probability that the observed match is a random event. Protein scores greater than 63 are significant (p<0.05).
Derivatisation of peptides with trityl groups of the invention thus improves detection, as a significantly larger number of peptides was detected for each of the three proteins when derivatisation was used. Furthermore, protein identification by mass fingerprinting can be improved.
Taking β-casein as an example, the number of detectable fragments more than tripled, and the derivatised spectrum allowed a MASCOT-based identification which was not previously possible.
Example 8 — BSA fragmentation and mass spectrometry Bovine serum albumin (BSA) was digested with trypsin and analysed by MALDI-TOF. The resulting spectrum is shown in Figure 8A. The experiment was repeated, but the peptide mixture was labelled with a dimethoxytrityl label after trypsin digestion. The spectrum in Figure 8B shows the dramatic increase in visible ions due to the trityl label. Four specific peptides have been highlighted in both spectra.
Example 9 — Mass spectrometry of amines A solution of NHS-activated 4,4'-dimethoxy-4"-carboxyethyl trityl (0.1M) in THF/dioxane (1 :1) was mixed with a solution (0.5- 1M) of an amine or of a mixture of amines (for example, propyl amine, butyl amine, pentyl amine, hexyl amine and phenethyl amine), typically 10 ml of a solution of an activated trityl with 5 ml of an amine solution. The mixtures were purified on prep-TLC (2mm-thick glass plates with UV254 indicator, Analtech/Aldrich-Sigma), typically in chloroform with 0.5% triethylamine. The areas containing the desired products were scratched off the plate, and the conjugates or the mixtures thereof were eluted using same solvent with 2-5% MeOH, filtered through a layer of glass wool, evaporated and dried. Figure 9 shows a spectrum obtained in this way.
It will be understood that the invention is described above by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

Claims

CLAIMS 1. A method of forming an ion of formula (I) : (Ar2) — C— [AT LMIM'- BP'}p)q]m * (I) comprising the steps of: (i) reacting a compound of the formula (Ila):
Figure imgf000056_0001
with a biopolymer, BP, having at least one group capable of reacting with M to form a covalent linkage, to provide a biopolymer derivative of the formula (Ilia): (Ar2)n- C- [Ar1- (LM{M'- BP'}p)q]m X (Ilia); and (ii) cleaving the C — X bond between X and the α-carbon atom of the derivative of formula (Ilia) to form the ion of formula (I); where: C* is a carbon atom bearing a single positive charge or a single negative charge; X is a group capable of being cleaved from the α-carbon atom to form an ion of foπnula (I); M is independently a group capable of reacting with Bp to form the covalent linkage; Bp' is independently the biopolymer residue of BP produced on formation of the covalent linkage; M' is independently the residue of M produced on formation of the covalent linkage; Ar1 is independently an aromatic group or an aromatic group substituted with one or more A; Ar2 is independently an aromatic group or an aromatic group substituted with one or more A; optionally wherein (a) two or three of the groups Ar1 and Ar2 are linked together by one or more L5, where L5 is independently a single bond or a linker atom or group; and/or (b) two or three of the groups Ar1 and Ar2 together form an aromatic group or an aromatic group substituted with one or more A; A is independently a substituent; LM is independently a single bond or a linker atom or group; n = 0, 1 or 2 and m = 1, 2, or 3, provided the sum of n+m = 3; p independently = 1 or more; and q independently = 1 or more.
2. A method of forming an ion of formula (I), comprising the steps of: (i) reacting a compound of the foπnula (lib):
Figure imgf000057_0001
X* (lib); with a biopolymer, Bp, having at least one group capable of reacting with M to form a covalent linkage, to provide a biopolymer derivative of the formula (Illb): (Ar2)n- C- [ Ar1- (LM{M'— BP'}p)q]m
X* (Illb); and dissociating X* from the derivative of formula (Illb), to form the ion of formula (I); where: X*is a counter-ion to C*; and C*, M, Bp', M', Ar1, Ar2, LM, n, m, p and q are as defined in claim 1.
3. A biopolymer derivative of the formula (Ilia).
4. A biopolymer derivative of the formula (Illb).
5. An ion of formula (I).
6. A compound of the formula (Ila).
7. A compound of the formula (lib).
8. A method of forming an ion of formula (I) comprising the steps of: (i) reacting a solid support of formula (IVai), (IVaii), or (IVaiii):
Figure imgf000058_0001
(IVaii);
Figure imgf000058_0002
r2 (Ar2)n.i— C— [Ar1— (LM{M>p)Jm X (IVaiii); with a biopolymer, BP, having at least one group capable of reacting with M to form a covalent linkage, to provide a modified solid support of the formula (Vai), (Vaii), or (Vaiii), respectively:
Figure imgf000058_0003
and either: (iia) for modified solid supports of formula (Vai) cleaving the C-Ss bond between the α-carbon atom of the modified solid support of formula (Vai) and the solid support Ss to form the ion of formula (I); (iib) for modified solid supports of formula (Vaii), either simultaneously or sequentially, cleaving the C-X bond between X and the α-carbon atom and cleaving the SS Arl bond between the solid support and the Arl group to form the ion of formula (I); or (iic) for modified solid supports of formula (Vaiii), either simultaneously or sequentially, cleaving the C-X bond between X and the α-carbon atom and cleaving the SS Ar2 bond between the solid support and the Ar2 group to form the ion of formula (I); where: X, Ar1, Ar2, BP', LM, M, M', n, m, p and q are as defined in claim 1; Ss is a solid support; C- - -Ss comprises a cleavable bond between C and Ss; Ss- - -Ar1 comprises a cleavable bond between Ar1 and Ss; and Ss- - -Ar2 comprises a cleavable bond between Ar2 and Ss.
9. A method of forming an ion of formula (I) comprising the steps of: (i) reacting a solid support of formula (IVbii) or (IVbiii):
Figure imgf000059_0001
X* (IVbii);
Figure imgf000059_0002
X* (IVbiii); with a biopolymer, BP, having at least one group capable of reacting with M to form a covalent linkage, to provide a modified solid support of the formula (Vbii) or (Vbiii), respectively:
Figure imgf000059_0003
X* (Vbii);
Figure imgf000060_0001
X* (Vbiii); and either: (iia) for modified solid supports of formula (Vbii), either simultaneously or sequentially, dissociating X* from the derivative of formula (Vbii) and cleaving the Ss- - -Ar1 bond between the solid support and the Ar1 group to form an ion of formula (I); or (iib) for modified solid supports of formula (Vbiii), either simultaneously or sequentially, dissociating X* from the derivative of formula (Vbiii) and cleaving the Ss- - -Ar2 bond between the solid support and the Ar2 group to form an ion of formula (I); where: X*, Ar1, Ar2, BP', LM, M, M', n, m, p, q, Ss, C- - -Ss, Ss- - -Ar1 and Ss- - -Ar2 are as defined in claim 8.
10. A method of forming an ion of formula (I) comprising the steps of: (i) reacting a solid support of formula (IVaiv) or (IVbiv):
Figure imgf000060_0002
(IVaiv);
Figure imgf000060_0003
X* (IVbiv); with a biopolymer, BP, having at least one group capable of reacting with M to form a covalent linkage, to provide a modified solid support of the formula (Vaiv) or (Vbiv), respectively:
Figure imgf000061_0001
X* (Vbiv); and either: (iia) for modified solid supports of formula (Vaiv), cleaving the C-X bond between X and the α-carbon atom to form the ion of formula (I); or (iib) for modified solid supports of formula (Vbiv), dissociating X* from the derivative of formula (Vbiv) to form the ion of formula (I); where: X, X*, Ar1, Ar2, BP', LM, M, M', p, q, n, m, and Ss are as defined in claims 8 and 9; M"- - -Ss comprises a bond between M" and Ss; and M" is the same as M except that Ss is bound to a portion of M which does not form part of M'.
11. A solid support of the formula (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) or (IVbiv).
12. A modified solid support of the formula (Vai), (Vaii), (Vaiii), (Vaiv), (Vbii), (Vbiii) or (Vbiv).
13. A method of any of claims 8-10 or a product of claim 11 or 12 wherein the biopolymer is a synthetic biopolymer.
14. A method or product of claim 13 wherein the synthetic biopolymer is an oligonucleotide, a peptide or a carbohydrate.
15. A method for analysing a biopolymer, BP, comprising the steps of: (i) reacting the biopolymer BP with a compound of formula (Ila) or (lib) or a solid support of formula (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) or (IVbiv); (ii) providing an ion of formula (I); and (iii) analysing the ion of formula (I) by mass spectrometry.
16. In a method for analysing a biopolymer, Bp, the improvement consisting of: (i) reacting a biopolymer, Bp with a compound of formula (Ila) or (lib) or a solid support of formula (IVai), (IVaii), (IVaiii), (IVaiv), (IVbii), (IVbiii) or (IVbiv); (ii) providing an ion of foπnula (I); and (iii) analysing the ion by mass spectrometry.
17. A method of claim 15 or claim 16 wherein the analysis by mass spectrometry is canied out in a spectrometer which is suitable for MALDI-TOF spectrometry.
18. A method of any of claims 1, 2, 8-10 or 13-17 or a product of any of claims 3-7, 11 or 12, wherein C* bears a single positive charge, such that the ions of formulae (I), (lib) and (Illb) have the structures:
Figure imgf000062_0001
19. A method of any of claims 1, 2, 8-10 or 13-18 or a product of any of claims 3-7, 11, 12 or 18 wherein n = 2 and m = 1.
20. A method of any of claims 1, 2, 8-10 or 13-19 or a product of any of claims 3-7, 11, 12, 18 or 19 wherein p = 1, 2 or 3.
21. A method of any of claims 1, 2, 8-10 or 13-20 or a product of any of claims 3-7, 11, 12 or
18-20 wherein p = 1.
22. A method of any of claims 1, 2, 8-10 or 13-21 or a product of any of claims 3-7, 11, 12 or 18-21 wherein q = 1, 2 or 3.
23. A method of any of claims 1, 2, 8-10 or 13-22 or a product of any of claims 3-7, 11, 12 or 18-22 wherein q = 1.
24. A method of any of claims 1, 2, 8-10 or 13-23 or a product of any of claims 3-7, 11, 12 or 18-23 wherein n = 2, m = 1, p = 1 and q = 1, such that the ion of formula (I) has the structure:
Figure imgf000063_0001
25. A method of any of claims 1, 2, 8-10 or 13-24 or a product of any of claims 3-7, 11, 12 or 18-24 wherein the biopolymer is a polymer found in biological samples.
26. A method or product of claim 25 wherein the biopolymer is a polypeptide, polysaccharide, or polynucleotide.
27. A method or product of claim 26 wherein the biopolymer is a polypeptide.
28. A method or product of any of claims 25-27 wherein the biopolymer does not readily form a molecular ion on illumination of laser light at 340 nm.
29. A method of any of claims 1, 2, 8-10 or 13-28 or a product of any of claims 3-7, 11, 12 or 18-28 wherein the ratio m(Bp') / m(IX) is more than 2, where m(IX) is the mass of the fragment (LX)
(Ar2) — C— [A^-CL^M},,).,].,, * (IX) of the cation of formula (I) and m(BP') is the mass of the biopolymer residue Bp'.
30. A method of any of claims 1, 2, 8-10 or 13-29 or a product of any of claims 3-7, 11, 12 or 18-29 wherein M is: -NR2; -SR; -OR; -B(R)Y; -BY2; -C(R)2Y; -C(R)Y2; -CY3; -C(=Z)Y; -Z-C(=Z)Y; -C(=Z)R; -C(R)(OH)OR; -C(R)(OR)2; -S(=0)Y; -Z-S(=0)Y; -S(-0)2Y; -Z-S(=0)2Y; -S(=0)3Y; -Z-S(=0)3Y; -P(=Z)(ZR)Y; -P(=Z)Y2; -Z-P(=Z)(ZR)Y; -Z-P(=Z)Y2; -P(=Z)(R)Y; -Z-P(=Z)(R)Y; or -N=C(=Z), where Y is independently a leaving group, Z is independently O, S or N(R) and R is independently H, C1-8hydrocarbyl or C1-8hydrocarbyl substituted with one or more A.
31. A method of any of claims 1, 2, 8-10 or 13-29 or a product of any of claims 3-7, 11, 12 or 18-29 wherein M is: -N(R)-; -S-; -0-; -B(Y)-; -C(R)(Y)-; -CY2-; -C(=0)-; -C(OH)(OR)-; or -C(OR)2-, where Y is independently a leaving group and R is independently H, Cι-8hydrocarbyl or C1-8hydrocarbyl substituted with one or more A.
32. A method of any of claims 1, 2, 8-10 or 13-29 or a product of any of claims 3-7, 11, 12 or
18-29 wherein M is: .Y) ^ where Y is a leaving group.
33. A method of any of claims 1, 2, 8-10 or 13-29 or a product of any of claims 3-7, 11, 12 or 18-29 wherein the covalent linkage is selected from those produced through the reaction of one the
following groups: -CO-NH-; biotin-(strept)avidin;
Figure imgf000064_0001
; or -NH-CS-NH-
34. A method of any of claims 1, 2, 8-10 or 13-33 or a product of any of claims 3-7, 11, 12 or 18-33 wherein LM is O or S.
35. A method of any of claims 1, 2, 8-10 or 13-33 or a product of any of claims 3-7, 11, 12 or 18-33 wherein LM is -EM-, -(DM)t-, -(EM-DM)t-, -(DM-EM)t-, -EM-(DM-EM)t- or -DM-(EM-DM)t- (in the orientation Ar1-(LM{M}p)q or Ar1-(LM{M'}p)q, as appropriate), where: a sufficient number of linking covalent bonds, in addition to the covalent bonds at the chain termini shown, are provided on groups EM and DM for linking the p instances of M (or M') groups; DM is independently C1-8hydrocarbylene or C1-8hydrocarbylene substituted with one or more A; EM (in the orientation Ar1-(LM{M}p)q or Ar1-(LM{M'}p)q, as appropriate) is independently -ZM-,
-C(=ZM)-, -ZMC(=ZM)-, -C(=ZM)ZM-, -ZMC(=ZM)ZM-, -S(=0)-, -ZMS(=0)-, -S(=0)ZM-, -ZMS(=0)ZM-, -S(=0)2-, -ZMS(=0)2-, -S(=0)2ZM-, -ZMS(=0)2Z -, where ZM is independently 0, S or N(RM) and where RM is independently H, Cι-8hydrocarbyl (e.g. Cι-8alkyl) or C1-8hydrocarbyl substituted with one or more A; and t = 1 or more.
36. A method of any of claims 1, 8, 10 or 13-35 or a product of any of claims 3, 6, 11, 12 or
18-35 wherein the group X is halogen, hydroxy, C1-8hydrocarbyloxy, C1-8hydrocarbyloxy substituted with one or more A, Cι-8heterohydrocarbyloxy, C1-8heterohydrocarbyloxy substituted with one or more A, mesyl, tosyl, pentafluorophenyl, -O-succinimidyl -S-succinimidyl, or phenyloxy substituted with one or more A.
37. A method of any of claims 1, 2, 8-10 or 13-36 or a product of any of claims 3-7, 11, 12 or 18-36 wherein Ar2 is independently cyclopropyl, cyclopropyl substituted with one or more A, aryl, aryl substituted with one or more A, heteroaryl, or heteroaryl substituted with one or more A.
38. A method of any of claims 1, 2, 8-10 or 13-37 or a product of any of claims 3-7, 11, 12 or 18-37 wherein Ar2 is
Figure imgf000065_0001
39. A method of any of claims 1, 2, 8-10 or 13-38 or a product of any of claims 3-7, 11, 12 or 18-38 wherein Ar1 is independently cyclopropylene, cyclopropylene substituted with one or more A, arylene, arylene substituted with one or more A, heteroarylene, or heteroarylene substituted with one or more A.
40. A method of any of claims 1, 2, 8-10 or 13-39 or a product of any of claims 3-7, 11, 12 or
18-39 wherein Ar1 is
Figure imgf000066_0001
41. A method of any of claims 1, 2, 8-10 or 13-40 or a product of any of claims 3-7, 11, 12 or 18-40 wherein L5 is O or S.
42. A method of any of claims 1, 2, 8-10 or 13-40 or a product of any of claims 3-7, 11, 12 or 18-40 wherein L5 is -E5-, -(D5)t-, -(E5-D5)t-, -(D5-E5)t-, -E5-(D5-E5)t- or -D5-(E5-D5)t*-, where: D5 is independently Cι-8hydrocarbylene or Cι-8hydrocarbylene substituted with one or more
A; E5 is independently -Z5-, -C(=Z5)-, -Z5C(=Z5)-, -C(=Z5)Z5-, -Z5C(=Z5)Z5-, -S(=0)-, -Z5S(=0 , -S(=0)Z5-, -Z5S(=0)Z5-, -S(=0)2-, -Z5S(=0)2-, -S(=0)2Z5-, -Z5S(=0)2Z5-, where Z5 is independently O, S or N(R5) and where R5 is independently H, C1-8hydrocarbyl or Cμ8hydrocarbyl substituted with one or more A; and t' = 1 or more.
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