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].

International patent application no. PCT/GB2004/005140 discloses the covalent attachment of a biopolymer to a triarylmethyl derivative via an aromatic group adjacent to the central α-triarylmethyl carbon atom. The biopolymer-bound triarylmethyl derivatives have improved ionisability with respect to free biopolymer and allow for improved analysis of the biopolymer by mass spectrometry.

The compounds disclosed in PCT/GB2004/005140 typically employ a group comprising an ether oxygen atom bound directly to the α-triarylmethyl carbon atom capable of being cleaved from the carbon atom to form an ion. Examples of such groups include hydrocarbyloxy groups (e.g. ethoxy). However, the bond between the ether oxygen and the α-triarylmethyl carbon atom is often extremely sensitive to acidic conditions, which may be inconvenient when attaching a biopolymer to the triarylmethyl derivative by causing premature release of the leaving group prior to mass spectrometry analysis. There is therefore a need for improvements in triarylmethyl derivatives for assisting the analysis of biopolymers by mass spectrometry.

DISCLOSURE OF THE INVENTION

It has now been found that triarylmethyl derivatives having a thioether sulphur atom in place of the ether oxygen atom allow improvements in the analysis of biopolymers by mass spectrometry.

In particular, it has been discovered that the thioether-containing compounds of formula (Ha) below are more stable to acidic conditions compared with compounds employing a group comprising an ether oxygen atom bound directly to the α-triarylmethyl carbon atom. Advantageously, attachment of the biopolymer to the compounds of formula (Ha) may therefore be effected under acidic conditions without premature release of the leaving group prior to mass spectrometry analysis.

Furthermore, it has surprisingly been discovered that the compounds of formula (Ilia) below have improved ionisability, especially in LDI techniques absent a matrix, compared with compounds employing a group comprising an ether oxygen atom bound directly to the α-triarylmethyl carbon atom. Furthermore, the compounds of formula (Ilia), especially in LDI techniques absent a matrix, provide cleaner mass spectra allowing for superior analysis of the biopolymer.

The invention provides methods of forming ions from covalent or ionic compounds and solid supports.

Derivatised Biopolymers

The invention provides a method of forming an ion of formula (I):

(Ar ² ) _n —C— [V-(LM(M ¹ - B _P '} _p ) _q ] _m * (I) comprising the steps of:

(i) reacting a compound of the formula (Ha):

(Ar ² ) _n - C- [Ar ¹ - (L _M {M} _p ) _q ] _m

^X (Ha); 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):

(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 comprising a thioether sulphur atom bound directly to the α-carbon which is 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;

Ar ¹ is independently an aromatic group or an aromatic group substituted with one or more A; Ar ² is independently an aromatic group or an aromatic group substituted with one or more A; optionally wherein (a) 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 (b) 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;

A is independently a substituent;

L _M 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 compounds of formula (Ha) may optionally be purified after step (i).

The invention also provides biopolymer derivatives of the formula (Ilia), 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. Moreover, it has been discovered that the compounds of formula (Ilia) below have improved ionisability, especially in LDI techniques absent a matrix, compared with compounds employing a group comprising an ether oxygen bound directly to the α-triarylmethyl carbon atom. Furthermore, the compounds of formula (Ilia), especially in LDI techniques absent a matrix, provide cleaner, i.e. less cluttered, mass spectra allowing for superior analysis of the biopolymer.

The ions of formula (I) 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, 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 (Ha), as defined above. Compounds of formula (Ha) are more stable to acidic conditions compared with compounds employing a group comprising an ether oxygen bound directly to the α-triarylmethyl carbon atom. Advantageously, attachment of the biopolymer may therefore be effected under acidic conditions without premature release of the leaving group prior to mass spectrometry analysis. Compounds of formula (Ha) 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.

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):

(Ar ² )

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):

(Ar ² ) _n - C- [Ar ¹ - (LM(M ¹ - B _P '} _p ) _q ] m

^y (Vai)); and

(iia) 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); where:

Ar ¹ , Ar ² , Bp', L _M , M, M', n, m, p and q are as defined above; Ss is a solid support; and

C- - -Ss comprises a cleavable bond between C and Ss involving a thioether sulphur atom bound directly to the α-carbon atom.

The invention also provides a method of forming an ion of formula (I) comprising the steps of: (i) reacting a solid support of formula (IVaii), or (IVaiii):

(IVaii);

"Ar ² (ArV ₁ - C— [Ar ¹ - (L _M {M} _p ) _q ] _m

^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 (Vaii), or (Vaiii), respectively:

[Ar ¹ - (L _M (M ¹ - B _P '} _p ) _q ] _m (Vaiii); and either:

(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- - -Ar ¹ bond between the solid support and the Ar ¹ 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 — Ar ² bond between the solid support and the Ar ² group to form the ion of formula (I); where: X, Ar ¹ , Ar ² , Bp', L _M , M, M', n, m, p and q are as defined above;

Ss is a solid support;

Ss- - -Ar ¹ comprises a cleavable bond between Ar ¹ and Ss; and Ss- - -Ar ² comprises a cleavable bond between Ar ² and Ss-

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 forming an ion of formula (I) comprising the steps of: (i) reacting a solid support of formula (IVaiv):

(IVaiv); 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):

and

(iia) cleaving the C-X bond between X and the α-carbon atom to form the ion of formula

(I); where:

X, Ar ¹ , Ar ² , Bp', L _M , 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), or (Vaiv) may optionally be washed after step (i).

The invention also provides solid supports of the formulae (IVai), (IVaii), (IVaiii) and (IVaiv) as defined above. Similarly, the invention provides modified solid supports of the formulae (Vai), (Vaii), (Vaiii) and (Vaiv) 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 (Ha) or a solid support of formula (IVai), (IVaii), (IVaiii) or (IVaiv); (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 (Ha) or a solid support of formula (IVai), (IVaii), (IVaiii) or (IVaiv); (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 ¹ 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 B _P ' 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 (K _a ) of at least 10 ¹⁴ M ^"1 , preferably about 10 ¹⁵ 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, Ar ¹ and Ss, or Ar ² 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, Ar ¹ and Ss, or Ar ² and Ss, as appropriate, is cleaved.

L _M bound to Ar ¹ by more than one bond

The above-mentioned embodiments of the invention may also be provided in which L _M is bound to 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 Ar ¹ and L _M -

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.

Disclaimers Preferably, all embodiments of the invention (including products of formulae (Ha)) involving or relating to compounds wherein X is -S-succinimidyl or a nucleic acid comprising a thioether sulphur atom bound directly to the α-carbon (e.g. -S-oligonucleotide) are disclaimed.

Preferred Embodiments

Definition of C * 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:

(Ar ² ) - C— [A^-(LM(M ¹ - B _P '} _p ) _q ] _m

n, m, p and q

For the purposes of compounds of the invention having n-1 groups Ar ² , 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 = l, p = l and q = 1. The ion of formula (I) thus has the structure:

f Ar ²

Ar ² - C- A^-LMM'- Bp' _M 2_ ' ^i^ ,__ _β

^ or more preferably © and the compounds of formulae (Ha), (Ilia), (IVai), (IVaii), (IVaiii), (IVaiv), (Vai), (Vaii), (Vaiii) and (Vaiv) have the structures disclosed in table 1.

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 least 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 preferred biopolymers have a molecular mass oiqqq kDa or less, where qqq is 30 or less (e.g. 20, 10, 5). Preferably, the mass, m(IX), of the fragment (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 preferred 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 (Ha).

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. Preferred 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 -(NMe ₃ ) ^4" binding groups and the biopolymer has a net negative charge, or vice versa (i.e. the -(NMe ₃ ) ^4" 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 JV- and O- glycoproteins, amine groups (e.g. on lysine residues or at the N-terminus of a protein), hydroxyl (e.g. in β-hydroxyaspartate, β-hydroxyasparagine, 5-hydroxylysine, %-hydroxyproline), thiol, sulfhydryl, phosphoryl, sulfate, methyl, acetyl, formyl (e.g. on N-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: -NR2 e.g. -NHR, especially -NH ₂ ; -SR e.g. -SH; -OR e.g. -OH; -B(R)Y; -BY ₂ ; -C(R) ₂ Y; -C(R)Y ₂ ; -CY ₃ ; -Q=Z)Y e.g. -C(=O)Y; -Z-C(=Z)Y; -C(=Z)R e.g. -C(=Z)H, especially -C(=O)H; -C(R)(OH)OR; -C(R)(OR) ₂ ; -S(=O)Y; -Z-S(=O)Y; -S(=O) ₂ Y; -Z-S(=O) ₂ Y; -S(=O) ₃ Y; -Z-SC=O) ₃ Y; -P(=Z)(ZR)Y e.g. -P(^O)(OH)Y; -PC=Z)Y ₂ ; -Z-P(=Z)(ZR)Y; -Z-PC=Z)Y ₂ ; -PC=Z)(R)Y e.g. -PC=O)CH)Y; -Z-P(=Z)(R)Y; or -N=C(=Z) e.g. -N=CC=O).

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:

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-; -O-; -B(Y)-; -C(R)(Y)-; -CY ₂ -; -C(O)-; -C(OH)(OR)-; -C(OR) ₂ -.

Groups which may be formed synthetically on the biopolymer and which are bound to the

biopolymer by three bonds include C(Y)

Preferred groups include nucleophilic groups, either natural or synthetic, e.g.: -NR ₂ s.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 Bp.

Preferred examples of Y include halogen (preferably iodo), Ci-shydrocarbyloxy {e.g. Ci-salkoxy), Ci _-8 hydrocarbyloxy substituted with one or more A, C _1-8 heterohydrocarbyloxy, Ci _-S 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).

illa)

Thus, preferred reactive groups on the biopolymer are:

Other preferred examples of Y include -ZR. Particularly preferred examples of Y are -ZH (e.g. -OH or -NH ₂ ) and -Z-Ci. ₈ alkyl groups such as -NH-Ci-salkyl groups (e.g. -NHMe) and -O-Q-salkyl groups (e.g. -O-t-butyl). Thus, preferred reactive groups are -C(O)-NH-C _1-8 alkyl and -C(O)-O-C ₁ . salkyl (e.g. -C(O)-O-t-butyl).

Other preferred examples of Y include -Z-ZR. Particularly preferred examples include -NR-NR ₂ , especially -NH-NH ₂ , and -ONR ₂ , especially -O-NH ₂ .

Z is independently O, S or N(R). Preferred (=Z) is (=0).

R is independently H, Ci _-8 hydrocarbyl (e.g. C _1-8 alkyl) or C _1-8 hydrocarbyl substituted with one or more A.

R is preferably H. Other preferred reactive groups include -C(=0)Y, especially -C(=O)-O-succinimidyl and -C(=O)-O-(p-nitrophenyl).

In a further embodiment, the reactive group may be -Si(R) ₂ -Y, with Y being halo (e.g. chloro) being especially preferred. Preferred groups R in this embodiment are d-galkyl, especially methyl. A particularly preferred reactive group in this embodiment is -Si(Me) ₂ Cl. 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. Preferred diene groups are

and multivalent derivatives formally formed by removal of one or more hydrogen atoms, where A ¹ is -R ¹ or -Z ¹ R ¹ , where R ¹ and Z ¹ are defined below.

Preferred dienophile groups are -CR^CR^, -CR^qR^A ² , -CA ² ORS, -CA^C^A ² or -CA ² =CA ² ₂ , and multivalent derivatives formally formed by removal of one or more hydrogen atoms, where R ¹ is defined below and A ² is independently halogen, trihalomethyl, -NO ₂ , -CN,

-N ⁺ (R ¹ ^O ^" , -CO ₂ H, -CO ₂ R ¹ , -SO ₃ H, -SOR ¹ , -SO ₂ R ¹ , -SO ₃ R ¹ , -OCC=O)OR ¹ , -C(=0)H, -CO=O)R ¹ ,

-OCC=O)R ¹ , , -OCC=O)NR^, -N(R^CC=O)R ¹ , -CC=S)NR^, -NR ¹ CC=S)R ¹ , -SO ₂ NR^, -NR ¹ SO ₂ R ¹ ,

-N(R ¹ )C(=S)NR ¹ 2, or -N(R ¹ )SO ₂ NR ¹ ₂ , where R ¹ 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 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). Preferably, M is bound to L _M by one single bond.

Alternatively, or in addition, M is bound by more than one L _M , such L _M either being attached to the same or different Ar ¹ or Ar ² . In a preferred embodiment M is bound by more than one L _M from different Ar ¹ or Ar ² , e.g. :

Examples of group M bound to L _M by one bond include -NR ₂ e.g. -NHR, especially -NH ₂ ; -SR e.g. -SH; -OR e.g. -OH; -B(R)Y; -BY ₂ ; -C(R) ₂ Y; -C(R)Y ₂ ; -CY ₃ ; -C(=Z)Y e.g. -C(O)Y; -Z-CC=Z)Y; -C(=Z)R e.g. -C(=Z)H, especially -C(O)H; -C(R)(OH)OR; -C(R)(OR) ₂ ; -S(O)Y; -Z-S(O)Y; -S(O) ₂ Y; -Z-S(O) ₂ Y; -S(O) ₃ Y; -Z-S(O) ₃ Y; -P(=Z)(ZR)Y e.g. -P(O)(OH)Y; -P(=Z)Y ₂ ; -Z-P(=Z)(ZR)Y; -Z-P(=Z)Y ₂ ; -P(=Z)(R)Y e.g. -P(O)(H)Y; -Z-P(=Z)(R)Y; or -N=C(=Z) e.g. -N=C(O).

Another example of a group M bound to LM by one bond is -CN.

Other examples of 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: y N(IPr) ₂

^-CN Another example of group M bound to L _M 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, L _M may, for example, be a single bond.

Examples of group M bound to L _M by two bonds include -N(R)- e.g. -NH-; -S-; -O-; -B(Y)-; -C(R)(Y)-; -CY ₂ -; -C(=O)-; -C(OH)(OR)-; -C(OR) ₂ -.

Examples of group M bound to L _M by three bonds include C(Y)

Preferred groups M include electrophilic groups, especially those susceptible to SN ₂ substitution reactions, addition-elimination reactions and addition reactions, e.g. -B(R)Y; -BY ₂ ; -C(R) ₂ Y; -C(R)Y ₂ ; -CY ₃ ; -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(O)Y; -Z-S(=O)Y; -S(=O) ₂ Y; -Z-S(=O) ₂ Y; -S(O) ₃ Y; -Z-S(O) ₃ Y; -P(=Z)(ZR)Y e.g. -P(O)(OH)Y; -P(=Z)Y ₂ ; -Z-P(=Z)(ZR)Y; -Z-P(=Z)Y ₂ ; -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)-; -CY ₂ -; -C(O)-;

-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 preferred embodiment, the R groups are linked together to form a hydrocarbyl group, e.g. a group. A preferred example of group M in this embodiment is:

Another preferred group M is maleimido.

Y, Z and R are defined as above. Preferred 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 .

Preferred examples of Y include halogen (preferably iodo), C _1-8 hydrocarbyloxy (e.g. C^salkoxy),

Ci-βhydrocarbyloxy substituted with one or more A, Ci^heterohydrocarbyloxy,

Q-sheterohydrocarbyloxy 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).

illa) Thus, preferred groups M are:

Other preferred examples of Y include -ZR. Particularly preferred examples of Y are -ZH (e.g. -OH or -NH ₂ ) and -Z-Ci _-8 alkyl groups such as -NH-Cj.galkyl groups (e.g. -NHMe) and -O-Cμsalkyl groups (e.g. -O-t-butyl). Thus, preferred groups M are -C(O)-NH-Ci _-8 alkyl (e.g. -C(O)NHMe) and -C(O)-O-Ci _-8 alkyl (e.g. -C(O)-O-t-butyl).

Other preferred examples of Y include -Z-ZR. Particularly preferred examples include -NR-NR ₂ , especially -NH-NH ₂ , and -ONR ₂ , especially -0-NH ₂ .

Particularly preferred groups M include -C(=0)Y, especially -C(=O)-O-succinimidyl and -C(=O)-O-(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 especially methyl. A particularly preferred group M in this embodiment is -Si(Me) ₂ Cl.

In a further embodiment, M may be -C(Ar ² ) ₂ X. Preferred groups Ar ² and X are set out below. In this embodiment it is preferred that LM is a bond. A particularly preferred group M in this embodiment is:

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. Preferred diene groups are

and multivalent derivatives formally formed by removal of one or more hydrogen atoms, where A ¹ is -R ¹ or -Z ¹ R ¹ , where R ¹ and Z ¹ are defined below.

Preferred dienophile groups are -CR ¹ ^CR ¹ ;,, -CR^C^A ² , -CA^CR ¹ ^ -CA^C^A ² or -CA ² =CA ² 2, and multivalent derivatives formally formed by removal of one or more hydrogen atoms, where R ¹ is defined below and A ² is independently halogen, trihalomethyl, -NO ₂ , -CN, -N ⁺ (R ¹ ^O ^" , -CO ₂ H, -CO ₂ R ¹ , -SO ₃ H, -SOR ¹ , -SO ₂ R ¹ , -SO ₃ R ¹ , -0C(=0)0R ^] , -C(=O)H, -CC=O)R ¹ , -OC(^O)R ¹ , , -OCC=O)NR^, -NC^)CC=O)R ¹ , -CC=S)NR^, -NR ¹ CC=S)R ¹ , -SO ₂ NR ¹ _Z , -NR ¹ SO ₂ R ¹ , -N(R ¹ )C(=S)NR ¹ ₂ , or -N(R^SO ₂ NR ¹ ₂ , where R ¹ 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 and M

The reactive group on the biopolymer [shown as 'F' in the drawings] and the group M [shown as 'AFG' in the drawings] must be dependency selected in order to form the covalent linkage. For example, where the biopolymer includes the groups -NH ₂ , -OH or -SH, M will typically be -B(R)Y; -BY ₂ ; -C(R) ₂ Y; -C(R)Y ₂ ; -CY ₃ ; -CC=Z)Y e.g. -C(=0)Y; -Z-C(=Z)Y; -CC=Z)R e.g. -C(=Z)H, especially -C(=0)H; -C(R)(OH)OR; -C(R)(OR) ₂ ; -S(=O)Y; -Z-S(=O)Y; -S(=O) ₂ Y; -Z-S(=O) ₂ Y; -S(=O) ₃ Y; -Z-SC=O) ₃ Y; -P(=Z)(ZR)Y e.g. -P(=0)(0H)Y; -PC=Z)Y ₂ ; -Z-P(=Z)(ZR)Y; -Z-P(=Z)Y ₂ ; -PC=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)-; -CY ₂ -;

-C(=0)-; -C(OH)(OR)-; -C(OR) ₂ -; or — ^C 00 . M may also be -CN. In a preferred 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.

Preferred covalent linkages are those produced through the reaction of the following groups:

The covalent residue M ¹ -Bp' is the reaction product of M and Bp. B _P ^! 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 L _M -M'-B _P ¹ , including -C(R) ₂ Z-, -ZC(R) ₂ -, -C(=Z)Z-, -ZCC=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(=O)Z-, -ZS(O)-, -ZS(O)Z-, -SC=O) ₂ Z-, -ZSC=O) ₂ -, -ZSC=O) ₂ Z-, -SC=O) ₃ Z-, -ZSC=O) ₃ -, -ZSC=O) ₃ Z-, -P(=ZXZR)Z-, -ZP(=Z)(ZR)-, -ZPC=Z)CZR)Z-, -PC=Z)(R)Z-, -ZPC=Z)CR)-, -ZP(=Z)(R)Z-, -NH-CC=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- - -S _s 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.

Preferred 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 preferred group M" is:

LM

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 Ar ¹ by a single covalent bond (or more, as appropriate) and to link LM to the p instances of M (or M ¹ , 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 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 .

In an alternative embodiment, LM may be bound to Ls . 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 Ar ¹ -(L _M {M} _p ) _q or Ar'-(L _M (M'} _p ) _q , as appropriate, are -E ^M -, -(D ^M ) _t -, -(E ^M -D ^M ) _t -, -(D ^M -E ^M ) _r , -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 Q.ghydrocarbylene or C^shydrocarbylene substituted with one or more A. Preferred D ^M are C _]-8 alkylene, and Cμgalkynylene, especially Cμgalkylene and Ci-galkynylene, each optionally substituted with one or more A (preferably unsubstituted). A preferred substituent A is ² H. Preferred LM in the orientation Ar'-(L _M {M} _p ) _q or as appropriate, are: -CH ₂ CH ₂ -; -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 -.

E ^M , in the orientation Ar ¹ -(L _M {M} _p )q or Ar ¹ -(L _M {M'} _p ) _q , as appropriate, is independently -Z ^M -, -C(=Z ^M )-, -Z ^M C(=Z ^M )-, -C(=Z ^M )Z ^M -, -Z ^M C(=Z ^M )Z ^M -, -S(=O)-, -Z ^M S(=O)-, -S(=O)Z ^M -, -Z ^M S(=O)Z ^M -, -Si=O) ₂ -, -Z ^M S(=O) ₂ -, -S(=O) ₂ Z ^M -, -Z ^M S(=O) ₂ Z ^M -, where Z ^M is independently O, S or N(R ^M ) and where R ^M is independently H, Ci,ghydrocarbyl (e.g. Cμgalkyl) or substituted with one or more A. Preferably E ^M is, in the orientation Ar^(L _M (M) ₁ ,),, or -OC(=S)-, -C(=O)S-, -SC(=O)-, -S(O)-, -S(O) ₂ -, -NR ^M -, -C(=0)N(R ^M )-, -C(=S)N(R ^M )-, -N(R ^M )C(=0)-, -N(R ^M )C(=S)-, -S(=0)N(R ^M K -N(R ^M )S(=O>, -S(=0) ₂ N(R ^M )-, -N(R ^M )S(=O) ₂ -, -OC(=O)O-, -SC(=O)O-, -OC(=O)S-, -N(R ^M )C(=0)O, -OC(=O)N(R ^M )-, -N(R ^M )C(=0)N(R ^M )-, -N(R ^M )C(=S)N(R ^M )-, -N(R ^M )S(=0)N(R ^M )- or -N(R ^M )S(=0) ₂ N(R ^M )-.

Alternative groups E ^M to those defined above, in the orientation Ar ¹ -(L _M {M} _p ) _q or Ar ¹ -(L _M {M ^l } _p ) _q , as appropriate, are -Z ^M -Si(R ^M ) ₂ -Z ^M -, -Si(R ^M ) ₂ -Z ^M - and -Z ^M -Si(R ^M ) ₂ -. The group -Si(R ^M ) ₂ -Z ^M - is particularly preferred. Z ^M is preferably O. R ^M is preferably C _1-8 alkyl, preferably methyl. These groups E ^M are particularly preferred in the groups -(E ^M -D ^M )t-, especially when t=l and D ^M is Q-salkylene. The following group is especially preferred:

In addition to the above definition of D ^M , D ^M may also be Q-gheterohydrocarbylene or Ci.gheterohydrocarbylene substituted with one or more A. In this embodiment,

Q-gcycloheteroalkylene groups are particularly preferred, Thus, preferred L _M groups -D ^M -E ^M -D ^M - are, in the orientation Ar ¹ -(L _M {M} _p ) _q or Ar ¹ -(L _M {M'} _p ) _q , as appropriate, -Ci-8alkylene-C(O)-Ci-gcycloheteroalkylene (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. Preferably t = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

Preferably, LM 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. LM{M}I 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}).

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 -(D ^M ) _r , -(E ^M -D ^M ) _r , or -D ^M -(E ^M -D ^M ) _t -.

When group LM is -(D ^M )t-, t is preferably 1. D ^M is preferably Ci-salkylene, preferably methylene or ethylene.

When group L _M is -(E ^M -D ^M ) _t -, or -D ^M -(E ^M -D ^M ) _t -, E ^M is preferably (in the orientation Ar ¹ -(L _M {M} _p ) _q or Ar ¹ -(L _M {M'}p) _q , as appropriate), -C(=0)N(R ^M )- (e.g. -C(=O)NH-) or O (preferably O), and D ^M is preferably C^alkylene, preferably ethylene, propylene, butylene or pentylene (preferably ethylene or propylene), t is preferably 1. Especially preferred LM are, in the orientation Ar ¹ -(L _M {M} _p ) _q or Ar ¹ -(L _M {M ^l } _p ) _q , as appropriate,, -0-CH ₂ CH ₂ CH ₂ - and -0-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -.

Another preferred group -D ^M -(E ^M -D ^M ) _r is where D ^M is C _1-8 alkylene and t is 1. Preferred E ^M in this group, in the orientation Ar ¹ -(LM{M} _p ) _q or (especially -N(R ^M )C(=O)-, e.g. -N(Me)C(O)-) and -C(=Z ^M )Z ^M - (especially -C(=0)0-). Particularly preferred L _M groups are:

The group -(E ^M -D ^M ) _t - is preferred, a particularly preferred example of which is (in the orientation Ar ¹ -(L _M {M} _p ) _q or as appropriate) -CeO)NH-CH ₂ CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ CH ₂ -.

In an alternative embodiment it is preferred that L _M is a single covalent bond.

When Ar ² is phenyl, L _M is preferably provided in a position ortho or para to C*. When 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). Where C* is a cation, L _M is preferably an electron-donating group. Where C* is an anion, L _M is preferably an electron-withdrawing group.

Preferred examples of L _M are shown in figure 1OA and 1OB.

C- - -S _s> Ss- - -Ar ¹ and Ss- - -Ar ² Bonds

C — Ss, 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.

In addition, however, the C — Ss bond comprises a cleavable bond between C and Ss involving a thioether sulphur atom bound directly to the α-carbon atom, i.e. without any other intervening atoms between the thioether sulphur atom and the α-carbon atom, and the definitions of the bonds below are

applicable to the definition of the C- - -Ss bond provided they involve a thioether sulphur atom bound directly to the α-carbon atom.

Covalent Bonding

Where the bond is covalent, the bond may be direct {e.g. C-Ss, Ar'-Ss or Ar ² -Ss, respectively) or may be provided by a linker atom or group L ⁴ (e.g. C-L ⁴ -Ss, Ar^lΛSs or Ar ² -L ⁴ -Ss, respectively).

When L ⁴ is a linker group, preferred 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 ) _t ..-.

D ⁴ is independently C _1-8 hydrocarbylene or C _1-8 hydrocarbylene substituted with one or more A.

E ⁴ is, in the orientation C-L ⁴ -S _s , independently -Z ⁴ -, -C(=Z ⁴ )-, -Z ⁴ C(=Z ⁴ )-, -C(=Z ⁴ )Z ⁴ -, -Z ⁴ C(=Z ⁴ )Z ⁴ -, -S(=O)-, -Z ⁴ S(=O)-, -SC=O)Z ⁴ -, -Z ⁴ S(=O)Z ⁴ -, -S(=O) ₂ -, -Z ⁴ SC=O) ₂ -, -SC=O) ₂ Z ⁴ -, -Z ⁴ SC=O) ₂ Z ⁴ -, where

Z ⁴ is independently O, S or NCR ⁴ ), and where R ⁴ is independently H, C _1-8 hydrocarbyl {e.g. Ci _-8 alkyl) or C _1-8 hydrocarbyl substituted with one or more A. Preferably E ⁴ is, in the orientation C-L ⁴ -Ss, -0-,

-S-, -C(=o)-, -CC=O)O-, -CC=S)-, -CC=S)O-, -oc(=s)-, -C(=o)s-, -sc(=o)-, -SCO)-, -S(O) ₂ -,

-N(R ⁴ )-, -C(=0)N(R ⁴ )-, -C(=S)N(R ⁴ )-, -N(R ⁴ )C(=0>, -N(R ⁴ )C(=S)-, -S(=O)NCR ⁴ )-, -N(R ⁴ )S(=O>, -SC=O) ₂ N(R ⁴ )-, -N(R ⁴ )S(=O) ₂ -, -0C(=0)0-, -SC(=O)O-, -OC(=O)S-, -N(R ⁴ )C(=0)0-, -0C(=0)N(R ⁴ )-, -N(R ⁴ )CC=O)N(R ⁴ )-, -N(R ⁴ )CC=S)N(R ⁴ )-, -NCR ⁴ )SC=O)NCR ⁴ )- or - N(R ⁴ )S(=O) ₂ N(R ⁴ )-. 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 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.

When the solid support Ss is gold, L ⁴ is preferably covalently attached to the Ss by a sulphide or disulphide group. When the cleavable bond of C — Ss bond is covalent and direct, the Ss solid support comprises a thioether sulphur atom bound directly to α-carbon atom. When the bond is provided by a linker atom or group L ⁴ , L ⁴ comprises a thioether sulphur atom bound directly to the α-carbon atom, e.g. L ⁴ is

-E ⁴ -, -(E ⁴ -D ⁴ )f ■-, or -E ⁴ -(D ⁴ -E ⁴ )f-, as defined above but wherein the left hand -E ⁴ - is -S-. A preferred L ⁴ is this embodiment is -S-. Other preferred L ⁴ include a group comprising a thioether sulphur atom linked to the α-carbon atom and a secondary alcohol, e.g. a group of the formula:

-f-S— linker — / ^

wherein the linker is a hydrocarbylene group or a heterohydrocarbylene group. Preferred

hydrocarbylene groups include alkylenearylene groups (e.g. or

\=/ wherein the left hand side is attached to the sulphur atom) or alkylene groups

(e.g. propylene).

Ionic Bonding

Where the bond is ionic, the bond is typically direct (e.g. C* Ss*, where Ss* is a solid support counterion to C*).

Alternatively, it may be provided by binding groups, e.g. chelating ligands, present on C or Ss, Ar ¹ or Ss, or Ar ² 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, Ar ¹ or S _s , or Ar ² or S _s , respectively.

Typically, in order to form the hydrogen bond, one of C or S ₅ , Ar ¹ or Ss, or Ar ² 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, Ar ¹ or Ss, or Ar ² or S _s , 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 S _s , Ar ¹ or Ss, or Ar ² or S _s , as appropriate, will have a binding group comprising biotin, and the other of C or Ss, Ar ¹ or Ss, or Ar ² 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, -(NMe ₃ ) ^4" , 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, Ar ¹ or Ss, or Ar ² or Ss, respectively.

Typically, in order to form the van der Waals bond, at least one, but preferably both, of C or Ss, 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 preferred. Typically, 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 ^ ₁ where Ar ³ is independently defined the same as Ar ² and R ⁶ is H, Ci _-8 hydrocarbyl, Cj.g hydrocarbyl substituted by one or more A, Ci _-S heterohydrocarbyl or C _1-8 heterohydrocarbyl substituted by one or more A. A preferred binding group is tetrabenzofullerene (formula X).

(formula X)

Alternatively, the van der Waals bond may be direct.

Bond Cleavage

Preferably, 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.

Preferably, the compounds of formula (Ha) or (Ilia) or the solid supports of formula (IVai), (IVaii), (IVaiii) or (IVaiv) provide ions of formula (I) having a pK _r+ 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 irradiation or chemical ionisation.

The term 'irradiation' includes, for example, laser illumination, in particular as used in MALDI mass spectrometry. Laser light of about 340 run 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.

Although the compounds of the invention are relatively stable to acid to allow attachment of the biopolymer without ionisation of the compound, chemical ionisation can nevertheless be effected, for example, by treatment with more acidic and/or more selective acid or acidic matrices {e.g. acidic matrices used in MALDI analysis).

Preferably, the step of cleaving the C-X bond or C — Ss bond in the methods of the invention (and preferably all steps) are carried out in the absence of an acidic matrix {e.g. an acidic matrix used in MALDI analysis), and preferably in the absence of any matrix. Group X comprises a thioether sulphur atom bound directly to the α-carbon and is capable of being cleaved from the α-carbon atom to form an ion of formula (I).

Preferably, group X is sulfanyl, hydrocarbylsufanyl {e.g. especially Cμghydrocarbylsufanyl), hydrocarbylsufanyl {e.g. C _1-14 hydrocarbylsufanyl, especially Ci-shydrocarbylsufanyl) substituted with one or more A, heterohydrocarbylsufanyl {e.g. Ci-i ₄ heterohydrocarbylsufanyl, especially Ci-gheterohydrocarbylsufanyl) or heterohydrocarbylsufanyl {e.g. Ci-i ₄ heterohydrocarbylsufanyl, especially Ci-sheterohydrocarbylsufanyl) substituted with one or more A.

Preferred hydrocarbylsulfanyl groups include: alkylsulfanyl groups, e.g. alkylsulfanyl groups substituted by a hydroxyl group [especially a secondary hydroxyl group, e.g. -S(CH ₂ ) _S CH(OH)CHs)]; arylalkylsulfanyl groups, e.g. phenylalkylsulfanyl groups [especially benzylsulfanyl and phenylethylsulfanyl]; and arylsulfanyl groups, e.g. phenylsulfanyl. More preferred hydrocarbylsulfanyl groups are alkylsulfanyl groups and arylalkylsulfanyl groups, especially arylalkylsulfanyl groups.

Another preferred group X is an amidite linker comprising a thioether sulphur atom linked to the α-carbon atom and a phosphorylated secondary alcohol, e.g. of the formula:

-|~ S — linker — { Q

-CN wherein the linker is a hydrocarbylene group or a heterohydrocarbylene group. Preferred

hydrocarbylene groups include alkylenearylene groups {e.g. or

\=/ wherein the left hand side is attached to the sulphur atom) or alkylene groups {e.g. propylene).

In another embodiment X may be -S-succinimidyl.

C- - -S _& Ss- - -Ar ¹ or S ₅ - - -Ar ²

The C- - -Ss, 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. Preferably, the C- - -Ss, Ss- - -Ar ¹ or Ss- - -Ar ² bonds are cleavable by irradiation or chemical ionisation.

Where appropriate, 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 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 carried out substantially (e.g. seconds, minutes, hours or even days) after the first sub-step. Ar ¹ and Ar ²

Ar ²

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. Where aryl or substituted aryl, Ar ² is preferably Cό- ₃ o aryl or substituted aryl. Where heteroaryl or substituted heteroaryl, Ar ² is preferably Cβ-30 heteroaryl or substituted C6.30 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) ₁ -- 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, pyrrolizine, 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 fiiran, imidazole, isothiazole, isoxazole, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine and thiophene. The five-membered monovalent derivatives are particularly preferred, 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 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 Ar ² is phenyl, A is preferably provided in a position ortho or para to C*. When 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 preferred 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.

Preferred heteroaryl Ar ² groups, whether substituted or unsubstituted, are pyridyl, pyrrolyl, thienyl and furyl, especially thienyl.

A preferred Ar ² group is thiophenyl or thiophenyl substituted with one or more A. Unsubstituted thiophenyl is preferred. Examples of thiophenyl are thiophen-2-yl and thiophen-3-yl, with thiophen- 2-yl being especially preferred.

When substituted, Ar ² is preferably substituted by 1, 2 or 3 A. Ar ² is preferably:

When unsubstituted, Ar ² is preferably:

In another preferred embodiment, Ar ² is cyclopropyl or cyclopropyl substituted with one or more A. Unsubstituted cyclopropyl is preferred. 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

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 preferred 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 -

When q = 1, 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.

Where arylene or substituted arylene, Ar ¹ is preferably C _δ -30 arylene or substituted C ₆ - ₃₀ arylene. Where heteroarylene or substituted heteroarylene, Ar ¹ is preferably Cg^o heteroarylene or substituted C6-3 ₀ 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 L _M ) 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 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, pyrrolizine, 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, pyrrole, pyrrolizine and thiophene. The fϊve-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. When Ar ¹ is phenylene, A is preferably provided in a position ortho or para to C*. When 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).

When substituted, Ar ¹ is preferably substituted by 1, 2 or 3 A. When unsubstituted, preferred Ar ¹ are:

Preferred examples of group Ar ¹ are shown in figures 12A and 12B.

Combinations of Ar

Optionally 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.

When L ⁵ is a linker group, preferred linker groups are -E ⁵ -, -(D ⁵ )t'-, -(E ⁵ -D ⁵ )f-, -(D ⁵ -E ⁵ )f-, -E ⁵ -(D ⁵ -E ⁵ )f- or -D ⁵ -(E ⁵ -D ⁵ ) _t -.

D ⁵ is independently Ci-shydrocarbylene or Ci.ghydrocarbylene substituted with one or more A.

E ⁵ is independently -Z ⁵ -, -C(=Z ⁵ )-, -Z ⁵ C(=Z ⁵ )-, -C(=Z ⁵ )Z ⁵ -, -Z ⁵ C(=Z ⁵ )Z ⁵ -, -SO=O)-, -Z ⁵ S(=O)-, -S(=O)Z ⁵ -, -Z ⁵ S(=O)Z ⁵ -, -SC=O) ₂ -, -Z ⁵ SC-O) ₂ -, -SC=O) ₂ Z ⁵ -, -Z ⁵ SC=O) ₂ Z ⁵ -, where Z ⁵ is independently O, S or NCR ⁵ ) and where R ⁵ is independently H, d _-8 hydrocarbyl or C _1-8 hydrocarbyl substituted with one or more A. Preferably E ⁵ is -O-, -S-, -C(O)-, -C(=O)O-, -CC=S)-, -CC=S)O-, -OCO=S)-, -CC=O)S-, -SC(=O)-, -S(O)-, -S(O) ₂ -, -N(R ⁵ )-, -C(=0)N(R ⁵ )-, -C(=S)N(R ⁵ )-, -N(R ⁵ )C(=0>, -NCR ⁵ )CC=S)-, -S(=0)N(R ⁵ )-, -N(R ⁵ )S(=O)-, -SC=O) ₂ N(R ⁵ )-, -N(R ⁵ )S(=O) ₂ -, -OC(O)O-, -SCC=O)O-, -OCC=O)S-, -N(R ⁵ )C(=0)0-, -OCC=O)N(R ⁵ )-, -N(R ⁵ )C(=O)N(R ⁵ )-, -N(R ⁵ )C(=S)N(R ⁵ )-, -NCR ⁵ )SC=O)N(R ⁵ )- or -N(R ⁵ )S(=O) ₂ N(R ⁵ )-. 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 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. When L ⁵ is a linker group, a preferred L ⁵ is -NCR ⁵ )-.

In another embodiment in which L ⁵ is a linker group, L ⁵ is -S(=O)-.

When 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 preferred 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 preferred combinations of two Ar ² groups are:

In another embodiment, at least one L _M is linked to an atom or group L ⁵ . In this embodiment, 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 . In this embodiment, the L ⁵ group to which L _M is bound is preferably:

Ar'/Ar ² /

\

Ar'/Ar ² Preferred combinations of Ar ¹ and/or Ar ² in this embodiment are:

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, 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 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 _8-5O 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) ₁ - linking group, where r is one or more (e.g. 1, 2, 3, 4 or 5).

When two or three of the groups Ar ¹ and Ar ² together form a carbopolycyclic fused ring aromatic group, preferred 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 Ar ¹ and Ar ² 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, pyrrolizine, quinazoline, quinoline, quinolizine and quinoxaline.

Substitution of Ar ¹ and Ar ² — Anions and Cations

When C* is a cation, A is preferably an electron-donating group, including -R ¹ or -Z ¹ R ¹ , where R ¹ and Z ¹ are defined below. Preferably, R ¹ is more preferably 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. 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 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*. Thus, preferred groups Ar ¹ and Ar ² are as follows:

When C* is an anion, A is preferably an electron- withdrawing group, including halogen, trihalomethyl, -NO ₂ , -CN, -N ⁺ (R ¹ ^O ^" , -CO ₂ H, -CO ₂ R ¹ , -SO ₃ H, -SOR ¹ , -SO ₂ R ¹ , -SO ₃ R ¹ , -0C(=0)0R ¹ , -C(=O)H, -C(=0)R ¹ , -OC(^O)R ¹ , -C(=0)NH ₂ , -C(=0)NR ¹ ₂ , -N(R ¹ )C(=0)0R ¹ , -N(R ¹ )C(=0)NR ¹ ₂ , -0C(=0)NR ¹ ₂ , -N(R ¹ )C(=0)R ¹ , -C(=S)NR ¹ ₂ , -NR ¹ C(^S)R ¹ , -SO ₂ NR ¹ _Z , -NR ¹ SO ₂ R ¹ , -N(R ¹ )C(=S)NR ¹ ₂ , or -N(R ¹ ^O ₂ NR ¹ ;,, where R ¹ is defined below. When C* is an anion, A, 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 nieta 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. Preferred isotopes are ¹³ C, ¹⁸ O and ² H. When providing a series of compounds which differ only in their masses, ¹³ C and ¹⁸ O are particularly preferred 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 preferred 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) or (IVaiv) followed by cleavage of the support to leave compounds of formula (I). Solid supports of formulae (IVai), (IVaii), (IVaiii) or (IVaiv) 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) or (IVaiv) 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), (Ha), (Ilia), (IVai), (IVaii), (IVaiii), (IVaiv), (Vai), (Vaii), (Vaiii) and (Vaiv), 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 preferred 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 L _M , Ar ¹ and/or Ar ² . Preferably, the masses of the compounds of the invention are varied by varying A on the groups Ar ¹ and/or Ar ² .

In this aspect of invention, compounds of the invention advantageously comprise one or more of F or 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 O, preferably as a substituent A or a part thereof of the groups L _M , 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 O may also be used to provide a series of molecular mass labels having substantially identical shaped peaks on a mass spectrum, by varying the number Of ² H, ¹³ C and ¹⁸ O atoms present in the structure of the compounds. When providing a series of compounds which differ only in their masses, ¹³ C and ¹⁸ O are particularly preferred as ² H 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 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 .

Preferred 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 (Ha) or a solid support of formula (IVai), (IVaii), (IVaiii) or (IVaiv) at a time ti;

(ii) reacting a second sample comprising biopolymer Bp with a compound of formula (Ha) or a solid support of formula (IVai), (IVaii), (IVaiii) or (IVaiv) 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).

If levels of the biopolymer B _P decrease between times ti and t ₂ then there will be a decrease in detected ion; if levels of the biopolymer Bp increase between times ti and t ₂ 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 (Ha) or different solid supports of formula (IVai), (IVaii), (rVaiii) or (IVaiv) 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 ti and t ₂ differ only by a ¹ H to ¹⁹ F 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 the solid support of formula (IVai), (IVaii), (IVaiii) or (IVaiv) will fix the biopolymer to prevent it reacting further and the steps of providing and analysing the cations may be carried out at a later convenient time. Alternatively, if the reaction of the biopolymer with the compound of formula (Ha) or the solid support of formula (IVai), (IVaii), (IVaiii) or (IVaiv) 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 (Ha) or the solid support of formula (IVai), (IVaii), (IVaiii) or (IVaiv).

Compounds of Formula (Ila)

The compounds of formula (Ila) are available commercially or may be synthesised by known techniques.

Commercially available trityls, and derivatives and analogues thereof, may also be derivatised with the groups (L _M {M} _p ) _q by known techniques. Groups (L _M {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.

Compounds of formula (Ila) may be synthesised analogously to the synthetic routes disclosed in PCT/GB2004/005140, Chem. Soc. Rev. (2003) 32 p. 3-13 scheme 2 and "1. introduction" last two paragraphs, WO99/60007 and EP 1 506 959. The compounds of the invention may also be synthesised by the treatment of a halide (e.g. chloride) of a triarylmethyl derivative with an appropriate thiol.

Chemical Groups

The ions of the invention are stabilised by the resonance effect of the aromatic groups Ar ¹ and Ar ² . The term 'C ir 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 term '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 Ci _-J4 hydrocarbyl, more preferably C] _-S hydrocarbyl. Unless indicated explicitly otherwise, where combinations of groups are referred 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. Preferred hydrocarbylene are Cj _-I4 hydrocarbylene, more preferably Ci _-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 C ₃ . ₈ groups, preferably C _5-8 groups. The term 'alkyl' includes monovalent saturated hydrocarbyl groups. Preferred alkyl are Ci _-8 , more preferably C] _-4 alkyl such as methyl, ethyl, n-propyl, i-propyl or t-butyl groups.

Preferred cycloalkyl are C _5-8 cycloalkyl. The term 'alkoxy' means alky l-O- .

The term 'alkenyl' includes monovalent hydrocarbyl groups having at least one carbon-carbon double bond and preferably no carbon-carbon triple bonds. Preferred alkenyl are C _2-4 alkenyl.

The term 'alkynyl' includes monovalent hydrocarbyl groups having at least one carbon-carbon triple bond and preferably no carbon-carbon double bonds. Preferred alkynyl are C _2-4 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 C ₆ -C ₁₄ 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 C] _-4 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. Preferred alkenylene are C _2-4 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 C _2-4 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. Preferred arylene are Ce-Cuarylene. Other examples of arylene groups are divalent derivatives of aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, chrysene, coronene, fluoranthene, fluorene, as-m ^' dacene, s- indacene, indene, naphthalene, ovalene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene and rubicene.

The term 'heterohydrocarbyF 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— is replaced by

-CH= is replaced by -N=; or

-CH ₂ - is replaced by -O-, -S- or -Se-.

The term 'heteroalkyl' 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 'heteroalkenyP 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 'heteroaryP 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 Cs-^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, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, thiophene and xanthene. 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, pyrrolizine and thiophene. The five-membered monovalent derivatives are particularly preferred, i.e. the monovalent derivatives of furan, imidazole, isothiazole, isoxazole, pyrazole, pyrrole and thiophene.

The term '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 ₅ 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 Cs.^heteroarylene. 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 L _M ) of acridine, carbazole, yS-carboline, chromene, cinnoline, fiiran, imidazole, indazole, indole, indolizine, isobenzofϊiran, isochromene, isoindole, isoquinoline, isothiazole, isoxazole, naphthyridine, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, thiophene and xanthene. Preferred 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, pyrrole, pyrrolizine and thiophene. The five-membered divalent derivatives are particularly preferred, i.e. the divalent derivatives of furan, imidazole, isothiazole, isoxazole, pyrazole, pyrrole and thiophene. Substitution

A is independently a substituent, preferably a substituent S _ub ¹ . Alternatively, A may be ² H.

S _ub ¹ is independently halogen, trihalomethyl, -NO ₂ , -CN, -N ⁺ (R ¹ ^O ^" , -CO ₂ H, -CO ₂ R ¹ , -SO ₃ H ₅ -SOR ¹ , -SO ₂ R ¹ , -SO ₃ R ¹ , -Oq=O)OR ¹ , -C(-0)H, -C(=0)R ^! , -OC(^O)R ¹ , -NR' ₂ , -C(=0)NH ₂ , -Ct=O)NR ¹ _S , -SO ₂ NR ¹ Z, -NR ¹ SO ₂ R ¹ , -N(R ¹ )C(=S)NR ¹ ₂ , -N(R^SO ₂ NR ¹ _Z , -R ¹ or -Z ¹ R ¹ .

R ¹ is independently H, Ci-shydrocarbyl, Ci-ghydrocarbyl substituted with one or more S _Ub ² , Ci-sheterohydrocarbyl or Ci-gheterohydrocarbyl substituted with one or more S _ub ² .

S _ub ² is independently halogen, trihalomethyl, -NO ₂ , -CN ₅ -N ⁺ (C ₁ . ₆ alkyl) ₂ O ^" , -CO ₂ H, -CO ₂ C _1-6 alkyl, -SO ₃ H ₅ -SOC, _-6 alkyl, -SO ₂ C _1-6 alkyl, -SO ₃ C _1-6 alkyl ₅ -OC(=O)OC _1-6 alkyl ₅ -C(=0)H, -C(=O)C _1-6 alkyl,

-OC(=O)C _1-6 alkyl, -N(C _1-6 alkyl) ₂ , -C(=O)NH ₂ , -C(=O)N(Ci _-6 alkyl) ₂ ,

-N(C _1-6 alkyl)C(=O)O(C _1-6 alkyl), -N(C _1-6 alkyl)C(=O)C _1-6 alkyl, -C(=S)N(C _1-6 alkyl) ₂ , -N(C _1-6 alkyl)C(=S)C _1-6 alkyl, -SO ₂ N(C _1-6 alkyl) ₂₅ -N(C _1-6 alkyl)SO ₂ Ci _-6 alkyl, -N(Ci _-6 alkyl)C(=S)N(Ci. ₆ alkyl) ₂ , -N(Ci _-6 alkyl)SO ₂ N(C _1-6 alkyl) ₂ , C _1-6 alkyl or -Z^^alkyl.

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 Su _b ¹ or S _ut , 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 S _ub 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 — n = 2, m = I ₁ p - 1 and q = 1

Formula (Vaiv)

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the arrangement of the TLC plates in example 2.

Figure 2 shows the evolution of the trityl compounds of example 2 in 40% AcOH. Figure 3 shows the evolution of the trityl compounds of example 2 in 80% AcOH.

Figure 4 shows the stability of trimethoxytrityl (TMTt) compounds to standard acid solution used in DNA automatic synthesis after 1 min, 5 min and 18 h.

Figure 5 shows the stability of aliphatic TMTt thioether against 5%, 10% and 20% TFA solutions in MeOH, water and THF. Figures 6-9 show spectra of (MA)LDI MS analyses of TMTt ethers and thioethers. Figure 10 shows the arrangement of the TLC plates in example 4.

Figure 11 shows the TLC plates used for monitoring the degree of cleavage of the trityl-heteroatom (O or S) bond of the compounds of example 4 in DCA deblock solution and 40% and 80% AcOH solutions. Figure 12 shows the TLC plates used for monitoring the degree of cleavage of the trityl-heteroatom (O or S) bond of the compounds of example 4 in 5%, 10 % and 20% TFA solutions.

Figures 13-15 show spectra of (MA)LDI MS analyses of the compounds of example 5. Figure 16 shows the spotting pattern used on the gold plate of example 6.

Figure 17 shows the MALDI MS analysis (using DHB matrix) of the trityl ether and trityl thioether of example 6.

Figure 18 shows the LDI MS analysis (without matrix) of the trityl ether and trityl thioether of example 6.

Figures 19A and 19B show preferred examples of group L _M - Figures 2OA and 2OB show preferred examples of group M. Figures 21A and 21B show preferred examples of groups Ar ¹ and Ar ² . Figure 22 shows preferred examples of substituent group A.

MODES FOR CARRYING OUT THE INVENTION

Example 1 — Synthesis

Trityl ethers and thioethers were obtained by the treatment of monomethoxy (MMTt) or trimethoxy trityl (TMTt) chloride with the corresponding alcohols or thiols:

Example 2 — Comparison of Acid Stability of Trityl Ethers and Trityl Thioethers

In order to assess the acid-stability of the oxygen-trityl bond versus the sulphur-trityl bond equivalent trityl ethers and thioethers were subjected through identical acidic conditions and the time evolution of the trityl-heteroatom bond was monitored. Since they can display different behaviour, both aliphatic and aromatic trityl ethers and thioethers were monitored. The following compounds were analysed:

R = H, OMe

Because of its simplicity, it was decided that the most appropriate method to monitor the evolution of the trityl ethers and thioethers in acidic conditions would be thin layer chromatography (TLC).

Experimental 1x10 ^"2 M solutions of the trityl ethers and thioethers to be compared were prepared with THF solvent. The following solutions were then prepared:

solution 1 2 3 4

compound ζ_ ^- Q-MYTi 4 ^-S-MMT!

Code 23.32 23.21 23.16 23.19

IvIW 366.5 382.5 394.5 410.6

Weiglit (mg) 21.1 21.4 22.0 22.6

vol 5.757 5.595 5.577 5.504

solution 5 6 7 8

compound <( J)-O-TlVTTt \ rf— S-TMTt fx

Code 23.17 23.20 23.11 23.18

MW 426.5 442.6 454.56 470.6

Weight (mg) 14.3 22.7 25.0 28.7

vol 3.353 5.130 5.500 6.099

MMTt = monomethoxytrityl; TMTt = trimethoxytrityl

The solutions were tested in both 80% and 40% acetic acid by dissolving lOOμL of the solutions above in 400μL of 100% and 50% AcOH, respectively. The solutions of the trityl ethers and thioethers in acetic acid were spotted in a TLC plate, and the plate run using a mixture of hexane:ethyl acetate; 4:1, with a few drops of diisopropylethylamine (DIEA).

Results

The TLC plates were examined by UV and by exposing to trifluoroacetic acid (TFA) vapours. The evolution of hydrolysis of the trityl-heteroatom bond was then visually evaluated. The arrangement of the solutions on the TLC plate is shown in figure 1.

40% AcOH Solutions

The evolution of the trityl compounds in 40% AcOH can be followed in the TLC plates shown in figure 2. MMTt derivatives were stable to 40%AcOH whereas TMTt compounds were gradually cleaved. The following pattern in stability was found:

The trityl-sulphur bond is clearly more resistant to the acidic conditions than an equivalent trityl- oxygen bond. Aliphatic thioethers or ethers are more resistant than their aromatic analogues. 80% AcOH Solutions

The evolution of the trityl compounds in 80% AcOH can be followed in the TLC plates shown in figure 3.

The hydrolysis was significantly faster under these conditions, although the same pattern of stability could be observed. The stability of the aliphatic sulphur -TMTt bond was remarkably good, and after Ih the compound remained practically without being cleaved. Even after 24h, at least 50% of the starting thioether remained in solution.

Further Experiments

Given that the TMTt-sulphur bond was reasonably stable to acetic acid, TMTt compounds were tested against different acid solutions.

ABI (Applied Biosystems Inc) Acid

The stability of TMTt compounds to standard acid solution used in DNA automatic synthesis ('ABI acid') was evaluated. The results after lmin, 5min and 18h are shown in figure 4.

TMTt ethers and thiophenyl were readily cleaved. However, the aliphatic TMTt thioether was again outstandingly resistant to the acidic conditions, and even after 18h, most of the starting material remained uncleaved.

Aliphatic TMTt Thioether in TFA Solutions The aliphatic TMTt thioether was tested against 5%, 10% and 20% TFA solutions in MeOH, water and THF. The results are shown in figure 5.

Under the test conditions (room temperature, lOOμL of trityl solution in 500μL of acid solution) the trityl-sulphur bond was practically resistant to the 5% TFA solution, it was partially cleaved in 10% TFA and it was instantaneously cleaved in 20% TFA. There was not an appreciable difference between the use of THF or MeOH as a solvent. However, the aqueous solution behaviour deserves a comment.

When the trityl solution was added to the aqueous 5% TFA solution the thioether crashed out of solution. After hours the precipitate started to disappear. When 10% TFA was used, the solution acquired an instant orange colour and no precipitate was generated. This is an indication that the trityl thioether is temporally resistant to 5% aqueous TFA, whereas 10% aqueous TFA is strong enough to cleave the thioether, liberating the trimethoxy tritylium cation and the free sulphide.

Example 3 - (MA)LDI TOFMS Experiments

Trimethoxytrityl thioethers are more slowly cleaved under acidic conditions than the corresponding trimethoxytrityl ethers. However, taking into account the application of the solid support immobilised trimethoxy trityl as enhancers for the analysis of biopolymers, the behaviour of the sulphur-linked trityl tags under (MA)LDI conditions was verified.

Equimolar solutions of trimethoxytrityl ethers and thioethers were submitted for (MA)LDI analysis. The objective of the experiment was to obtain an estimate of the behaviour of both trimethoxytrityl

ethers and thioethers under LDI MS spectrometry, both with and without the assistance of matrix. The resulting spectra are shown in figures 6-9.

In all the cases, trimethoxytritylium cations from thioether samples produced peaks of a higher absolute intensity than cations from their counterpart ethers. In addition, when the samples were run without the assistance of matrix, the spectra from thioethers were evidently cleaner. This would appear to be an indication of a neater and more efficient liberation of the trityl tag in the thioethers.

Example 4 - Comparison ofDimethoxytrityl-0 (DMTt-O) and Trimethoxytrityl-S (TMTt-S)

From the previous experiments, it is evident that tritylated thiols are significantly more stable than their corresponding alcohols. However, in order to establish the behaviour of benzyl thiol and to compare how alkyloxy-DMTts compare with mercapto-TMTts, the following experiment was carried out.

Alkyloxy-DMTt was compared against the three variations of mercapto-TMTt systems: arylthio, benzylthio and alkylthio. In this manner, the relative stability of the tritylated thiols could be classified at the time that alkoxy-DMTt was compared against the collection of mercapto-TMTts. The following new model compounds were synthesised using standard protocols:

Experimental

1x10 ^"2 M solutions of the compared trityl ethers and thioethers were prepared using THF as a solvent.

The following table shows the preparation of the solutions used:

solution

compound -S-TMTt

,.DMTt Λ. ,TMTt

TMTt

Code 23.44 23.20 23.42 23.18

MW 424.6 442.6 456.6 470.6

Weight (tng) 36.7 33.9 23.5 26.1

vol 6.453 7.659 5.147 5.546

In order to obtain a comprehensive picture of the process, the acidolysis of the above solutions was evaluated in 6 different acidic solutions: dichloroacetic acid (DCA) deblock solution, 80% AcOH ₅ 40% AcOH, 5% TFA, 10% TFA and 20% TFA. In a typical experiment, the solutions of the trityl ethers and thioethers in the series of acidic solutions were capillary spotted in a TLC plate after certain intervals, and the plate run using a mixture of hexane:ethyl acetate; 4:1, with a few drops of DIEA.

Figure 10 shows the used layout of a TLC analysis.

The TLC plates were examined by UV and by exposing to TFA vapours. The evolution of hydrolysis of the trityl-heteroatom bond was then visually evaluated.

Analysis was carried out after controlled periods of time to determine the evolution of the trityl ethers and thioethers in the acidic solutions. Times (min): 5, 10, 20, 60, 100, 180, 240, 390, and 1440.

Results General Remarks

Alkyl and benzyl mercapto-TMTts are the most resistant species to the acid solutions. The stability of both compounds is similar and significantly higher than the stability of the alkoxy-DMTt and arylmercapto-TMTt.

DCA Deblock Solution and 40% and 80% AcOH Solutions Figure 11 shows the TLC plates used for monitoring the cleavage of the trityl-heteroatom (O or S) bond.

AlkoxyDMTt compound was practically instantaneously hydrolised by the deblock solution. ThiophenylTMTt was notably more resistant to this acidic solution. After Ih the sulphur compound was hydrolysed in ca 80%.

Alkylmercapto and benzylmercapto-TMTts were slowly deprotected, with an approximate 50% evolution after 24h.

Once more, two clearly different behaviours could be observed in acetic acid solutions. Alkylmercapto and benzylmercapto TMTts remained practically intact in the first 6 hours and the deprotection was just detectable after 24h (-20%).

The deprotection of alkoxyDMTt and thiophenylTMTt was faster. In 80% AcOH, both compounds reached a -50% of deprotection progress after 2h. In 40% AcOH the 50% of deprotection progress was reached after ~24h.

5%. 10% and 20% TFA Solutions Figure 12 shows the TLC plates used for monitoring the cleavage of the trityl-heteroatom (O or S) bond.

TFA solutions deprotected almost instantaneously the DMTt compound. ThiophenylTMTt was also deprotected quickly (in 5% TFA the deprotection was completed in 15min).

Consistently with previous experiments, alkylmercapto and benzylmercapto TMTts were surprisingly resistant to TFA -solution. In all three TFA solutions the deprotection was below 50% after Ih, above 50% in 2h and ca 80% after 24h.

Example 5 - (MA)LDI TOFMS Experiments

Trimethoxytrityl thioethers are more stable to acidic conditions than alkylic dimethoxytrityl ethers. In order to establish whether, despite its higher acid stability, TMTt thioethers would still give a higher response in (MA)LDI than DMTt ethers, the following experiments were carried out.

A series of binary equimolecular mixtures of alkylic DMTt ether with arylic, benzylic and alkylic TMTt thioethers, respectively, were prepared and analysed by MS with and without matrix. The results are shown in figures 13, 14 and 15.

The results are conclusive. Because TMTt cation is a better flyer than DMTt cation, because the sulphur-trityl bond is more easily cleaved than the corresponding oxygen-trityl bond, or because of a combination of the two, in all the spectra the peak corresponding to the TMTt cation was significantly bigger than the peak corresponding to the DMTt cation. The difference was still bigger in those spectra run without the assistance of matrix.

Example 6 - Comparison of the Ionisability of TrUyI Ethers and TrUyI Thioethers

C ₃₄ H ₄₅ NO ₄ S C ₃₂ H ₄₁ NO ₅

Exact Mass: 563.3069 Exact Mass: 519.2985

MoI. Wt.: 563.7904 MoI. Wt.: 519.6716

The following tags were prepared and were purified via preparative TLC and filtered using the Millex syringe driven filter to ensure no fine particles of silica were present.

1. Sulphur Linker, 1. Pyr, cat. DMAP, AcOH Oxygen Linker

2. THF, H ₂ N(CHa) ₄ CH ₃ 2. THF, H ₂ N(CH ₂ ) ₄ CH ₃

3. Prep. TLC 3. Prep. TLC

C ₃₄ H ₄₅ NO ₄ S C ₃₂ H ₄₁ NO ₅

Exact Mass: 563.3069 Exact Mass: 519.2985

MoI. Wt.: 563.7904 MoI. Wt.: 519.6716

Equimolar solutions of both tags were then prepared and spotted on to a gold plate with 64 wells as shown in figure 16. The trityl tag-mix (equimolar solution of both trityl tags) was spotted on wells Cl with matrix and C2 without matrix.

The samples were spotted and the plate allowed to dry. The plate was analyzed by MALDI-TOF using the Voyager Spec (ABI) with 5-dihydroxybenzoic acid (DHB) matrix and without DHB matrix.

linker

Trityl ethers are more easily observed in the presence of matrix. The acidic nature of DHB results in an initial cleavage of the trityl ether bond, therefore the trityl is already present in the form of a trityl cation, whereas the trityl thioether is resistant to DHB and is not cleaved (well Cl). The spectrum shown in figure 17 confirmed that the 201C tag on oxygen was the more intense peak (well Cl).

By removing matrix and analysing both the trityl ether and trityl thioether, it was observed that 201C on sulphur was more readily cleaved than the trityl ether (well C2). This is seen in the spectrum in figure 18.

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.