WO2007110624A2 - Reactif immobilise - Google Patents

Reactif immobilise Download PDF

Info

Publication number
WO2007110624A2
WO2007110624A2 PCT/GB2007/001089 GB2007001089W WO2007110624A2 WO 2007110624 A2 WO2007110624 A2 WO 2007110624A2 GB 2007001089 W GB2007001089 W GB 2007001089W WO 2007110624 A2 WO2007110624 A2 WO 2007110624A2
Authority
WO
WIPO (PCT)
Prior art keywords
immobilised
reagent
phosphorus
atom
compound
Prior art date
Application number
PCT/GB2007/001089
Other languages
English (en)
Other versions
WO2007110624A3 (fr
Inventor
Mark Bradley
Carole Bruckler
Original Assignee
University Court Of The University Ofedinburgh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Court Of The University Ofedinburgh filed Critical University Court Of The University Ofedinburgh
Publication of WO2007110624A2 publication Critical patent/WO2007110624A2/fr
Publication of WO2007110624A3 publication Critical patent/WO2007110624A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6564—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms
    • C07F9/6571—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms
    • C07F9/6574—Esters of oxyacids of phosphorus
    • C07F9/65744—Esters of oxyacids of phosphorus condensed with carbocyclic or heterocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids

Definitions

  • the present invention relates to an immobilised reagent useful for the preparation of phosphate compounds, in particular, for the preparation of triphosphate nucleosides.
  • the present invention also relates to the methods for making the immobilised reagent and its use in the preparation of phosphate compounds.
  • Nucleosides have been an area of interest since their discovery. As well as the four natural bases, there are numerous other nucleosides which are of interest due to their chemistry, enzymology and possible applications as drugs' and the general availability of their triphosphates is key to their successful application.
  • the triphosphates of fluorogenically labelled nuclesides are of great interest in the dideoxy sequencing method, which forms the basis of all DNA sequencing methods.”
  • Several procedures have been reported for the conversion of a nucleoside to its triphosphate. A comprehensive review of synthetically useful methods for triphosphate preparation was published by Burgess and Cook.”' Mofatt et al.
  • ⁇ v for example used DCC (dicyclohexyl carbodiimide) and morpholine to produce the phosphoroamidate, which reacts with bis-(tributylammonium) pyrophosphate to give the triphosphate, but di and tetraphosphates are formed readily through disproportionation reactions.
  • Tor et aV used phosphoryl chloride to form the monophosphate which was converted to its triphosphate with 1,1-carbonyldiimidazole and tributylammonium pyrophosphate.
  • Another method by Hovinen et aV 1 involves treatment with phosphoryltris(triazole) prior to reaction with bis-(tributylammonium) pyrophosphate.
  • nucleoside triphosphates This method is widely used for synthesis of nucleoside triphosphates and has also been applied to synthesis of modified triphosphate analogues such as ⁇ -thio,"” ⁇ - borano, 1 "” and ⁇ -thio- ⁇ -borano triphosphates.”
  • modified nucleosides are of biochemical interest for example because they are substrates for DNA polymerase enzymes and can be successfully incorportated into DNA, but are more resistant to exo- and endo-nuclease than phosphate diesters/"'
  • a second method based on the Ludwig-Eckstein method avoided the use of coupling a linker to the nucleoside, but is therefore only applicable for amino-nucleosides.
  • Schoetzau et a ⁇ . x ⁇ based their method on Staudinger chemistry, where an azide reacts with polystyrene-triphenylphosphine resin to immobilise the nucleoside, which is then phosphorylated and cleaved to yield the triphosphorylated amino-nucleoside (Scheme 3).
  • An object of the present invention is to mitigate and/or obviate the disadvantages of the prior art methods for synthesising phosphate compounds.
  • the present invention is based on immobilising a reagent useful in the synthesis of phosphates, for example, triphosphate compounds, and the present invention provides such immobilised reagent.
  • an immobilised cyclic phosphitylation or immobilised cyclic phosphorylation reagent there is provided an immobilised cyclic phosphitylation or immobilised cyclic phosphorylation reagent.
  • a phosphitylation reagent includes a phosphorus atom in oxidation state (III), which is available to react with a nucleophile to form a phosphite compound.
  • a phosphorylation reagent includes a phosphorus atom in oxidation state (V), which is available to react with a nucleophile to form a phosphate compound.
  • the reagent includes a phosphinyl or phosphoryl moiety.
  • the reagent is cyclic in the sense that the phosphorus atom forms part of a ring, thus, such reagents typically include a phosphorus atom in oxidation states (III) or (V) bonded within, and to, a ring system via two single bonds, with the remaining valency of the phosphorus atom being fulfilled by further covalent bonds and/or by forming a positively charged phosphorus atom.
  • the reagent will typically comprise the group according to formula (A):
  • X is a leaving group
  • P is a phosphorus (III) or phosphorus (V) atom
  • the ring may comprise a single ring or several fused rings.
  • remaining bonds/charges with respect to the phosphorus atom are not shown in the above drawing in respect of a P(V) atom for succinctness.
  • the charge/number of bonds on the P atom shall be present according to the phosphorus oxidation state, for example when P is P(V), the reagent may comprise the group according to formula (B):
  • X is a leaving group
  • P is a phosphorus (V) atom
  • the immobilised reagent may be generally represented according to formula (I):
  • SS represents a substrate to which the reagent is bound
  • L is an optional linker
  • P is as previously defined, and X is a leaving group.
  • the group Z is also bound to P.
  • the leaving group X is selected from the group consisting of halogens e.g. F, Br, I; imidazoles; triazoles; tetrazoles; amines (to provide phosphoroamidites), e.g. primary amines, secondary amines (e.g. morpholine and diisopropylamine in particular) and tertiary amines (e.g. anilines); activated alcohols, such as OTs, OMs and OTf etc.
  • X is a halogen atom.
  • the halogen is a chlorine atom
  • the reagent will comprise a phosphorochloridite (for P(III)) or a phosphorochloridate (for P(V)) moiety. Therefore, the present invention in particular provides an immobilised cyclic phosphorochloridite or phosphorochloridate compound.
  • the ring includes an aromatic group, and thus the immobilised reagent may be represented according to formula (II):
  • Ar is an unsubstituted or substituted aromatic or heteroaromatic group.
  • the aromatic group is an aryl group.
  • the aryl group is selected from phenyl, naphthyl or anthracyl group. Phenyl is most preferred.
  • the phosphorus atom is bonded together within the ring with two oxygen atoms to provide a compound according to formula (III):
  • the ring comprises a substituted or unsubstituted aliphatic ring, further optionally containing one or more heteroatoms.
  • the ring is a five, six or seven membered ring. A six membered ring is most preferred.
  • the immobilised reagent includes a salicyl moiety derivative, thus, there is preferably provided an immobilised reagent according to formula (IV):
  • X is a halogen atom, most preferably, a chlorine atom.
  • the reagent includes an immobilised salicyl phosphorochloridite compound or an immobilised salicyl phosphorochloridate compound.
  • the substrate may be any substrate to which the compound may be bound, and the compound may be attached to the substrate using methods readily available to the skilled person.
  • the compound may be attached to the substrate via a linker, in which case, in the compound according to formula (I), L will be present.
  • the linker may be provided as an integral component of the substrate, or the substrate may be modified to include a linker component, for example, either by forming the substrate in modified form, or by first providing the substrate and then modifying it to include a linker component.
  • the substrate may comprise substituent surface groups or may be derivatised to provide such groups, to enable a linker to be bonded to the substrate surface, or to enable the reagent to be bonded directly to the substrate surface.
  • the substrate comprises a polymeric material, and this may be provided in a linear or a crosslinked form.
  • suitable substrates include those chosen from polystyrene, e.g. microporous or macroporous polystyrene, modified polystyrene, e.g. ethylene grafted polystyrene and polystyrene-polyethyleneglycol co-polymers, controlled pore glass (CPG), and modified CPG.
  • Further substrates include those chosen from polymers based on polyethylene glycol
  • PEG polystyrene-PEG copolymer
  • Other polymers include those based solely on PEG chains.
  • Still further polymers include polyamide resins, which are co-polymers of dimethylacrylamide, bisacryloylethylenediamine and N-Boc-alanyl-N'- acryloylolhexamethylenediamine; polyamide-PEG copolymers known as PEGA resins and which are co-polymerised from dimethylacrylamide, bis-acrylamidopropyl-PEG and acrylamidopropyl-aminopropyl-PEG.
  • polymers comprising PEG components display free hydroxyl groups which enable the reagent to be bound to the substrate by reaction with the hydroxyl groups.
  • Further substrates include silica based resins, which are quite similar to CPG, but tend to have smaller pore sizes, smaller bead sizes and most importantly a higher number of surface hydroxyl groups, i.e more potential functionalisation sites.
  • Chiron SynPhaseTM Crowns and lanterns activated through gamma irradiation, or soluble polymers, such as linear or branched PEG polymers or dendrimers.
  • a preferred substrate is polystyrene, modified polystyrene, or a polystyrene- polyethyleneglycol co-polymer, any one of which may be at least partly crosslinked with a crosslinking agent.
  • the crosslinking agent is preferably divinyl benzene.
  • the amount of crosslinking may be expressed with reference to the weight amount of crosslinking agent in the polymer.
  • the amount of crosslinking agent may be from about 0.25% to about 80% by weight, preferably from about 0.5% to about
  • the substrate may be termed a resin, and this term is usually applied to substrates which are based on organic polymers.
  • the polymer may be provided in gel form or swollen in an organic or aqueous solvent.
  • the substrate may be provided in any of a number of physical/geometric forms such as particulate material, generally spherical particles, beads, granules, microporous solids, etc, which may be bound in unreactive polymeric matrices (plugs) or handled in polymeric meshes (teabags) etc.
  • particulate material generally spherical particles, beads, granules, microporous solids, etc, which may be bound in unreactive polymeric matrices (plugs) or handled in polymeric meshes (teabags) etc.
  • the particulate material may typically have a cross sectional measurement, e.g. a diameter, of from about 30 ⁇ m to about 400 ⁇ m, preferably from about 40 ⁇ m to about 350 ⁇ m, e.g. in the ranges of from about 40 ⁇ m to about 63 ⁇ m, from about 38 ⁇ m to about 75 ⁇ m, from about 75 ⁇ m to about 150 ⁇ m, from about 150 ⁇ m to about 300 ⁇ m or from about 150 ⁇ m to about 350 ⁇ m.
  • a cross sectional measurement e.g. a diameter, of from about 30 ⁇ m to about 400 ⁇ m, preferably from about 40 ⁇ m to about 350 ⁇ m, e.g. in the ranges of from about 40 ⁇ m to about 63 ⁇ m, from about 38 ⁇ m to about 75 ⁇ m, from about 75 ⁇ m to about 150 ⁇ m, from about 150 ⁇ m to about 300 ⁇ m or from about 150 ⁇ m to about 350 ⁇ m.
  • a range of from about 75 ⁇ m to about 150 ⁇ m is preferred for organic polymer-based substrates, which advantageously generally provides greater yields of the desired, cleaved, phosphate compound when compared with yields obtained from using a substrate having larger particle dimensions.
  • the substrate may be porous, i.e. contains a multitude of interconnecting pores which may form an array of interconnecting passagways throughout the substrate material.
  • porous materials may conveniently be described in terms of the pore size.
  • the pores tend to swell or shrink depending on the nature of the solvent and defining the pore size may not be appropriate.
  • rigid substrates e.g. macroporous substrates/resins
  • the pore sizes can generally be measured/defined.
  • Typical pore sizes may be chosen from about 4 ⁇ A to about 15 ⁇ A, preferably about 5 ⁇ A to about 12 ⁇ A. For example, about 6 ⁇ A, e.g. for silica substrates, or about 100A, e.g. for CPG substrates.
  • the substrate particulate size appears to influence the yield amount of product to a greater extent than a variation in the substrate type chosen.
  • the product yield obtained appears to be generally independent of the type of chemical groups at the resin surface, e.g. the linker moieties, but rather tends to be influenced by the resin bead size. This contrasts with silica-based substrates, which do not appear to show this effect.
  • the yield appears to depend on the ability/ease of the solution-phase molecules to diffuse into and out of the substrate pores.
  • longer diffusion times may cause lower yields due to incomplete formation of the immobilised reagent during its preparation due to incomplete diffusion of reagent-forming molecules into the internal substrate structure, and/or due to decomposition of the final product obtained by cleavage from the substrate which is required to diffuse out of the internal substrate structure before its isolation.
  • the linker L may be any assemblage of atoms suitable for joining the substrate to the compound, for example, typical linkers may comprise an alkylene, alkenylene or alkynylene moiety, each of which may be independently substituted or unsubstituted.
  • the linker may include one or more heteroatoms, such as O, N, S or P, a group of heteroatoms such as NN, NO, and/or one or more further moieties comprising at least one heteroatom such as CONH.
  • the linker may include an ether moiety, which may comprise more than one ether oxygen atom to provide a polyether moiety.
  • the linker is an alkylene ether, e.g. having the formula -((CH 2 ) X -O y - (CH 2 ) z ) n -O- wherein, n is a number selected from O, 1, 2, 3, 4, 5 or 6, and when n is not 0, y is a number selected from 0 or 1, and x and z are integers independently selected from 0, 1, 2, 3, 4, 5 or 6.
  • n 1, 2, 3 or 4
  • y is 0, x is 1, and z is 0, or alternatively, y is 1, x is 0 and z is 2.
  • n is 1, y is 0, x is 1, and z is 0, or alternatively, n is 4, y is 1, x is 0 and z is 2.
  • the alkylene ether linker described above may be part of a longer alkylene chain comprising part of the substrate material, or modified substrate material.
  • the substrate SS is a polystyrene support resin, such as a microporous polystyrene.
  • the present invention extends to the methods of preparation of the immobilised reagent, and such methods include providing and binding of the reagent to a substrate, or alternatively providing and binding a reagent precursor to a substrate and then subsequently transforming the bound precursor into the useable reagent.
  • a linker may be bound to the substrate prior to binding the reagent or the reagent precursor to the substrate via the linker, i.e. prior to binding the reagent or the reagent precursor to the bound linker.
  • the methods may include the steps of:
  • the substrate may be subjected to one or more reaction steps prior to step (2) to activate the substrate such that it becomes receptive to the binding of the linker.
  • step (2) may be dispensed with depending on the specific materials being used, such that the reagent precursor is directly bound to the substrate (or activated substrate) in step (3).
  • the linker may become introduced to the substrate and/or fully formed simultaneously on binding the reagent or reagent precursor to the substrate.
  • the reagent may be provided in a useable, but unbound form, and then bound to the linker or the substrate to provide a bound useable reagent without the need for further transformation into a useable reagent.
  • the substrate is polystyrene, most preferably, microporous polystyrene.
  • the polystyrene is derivatised with a handle for functionalisation prior to reaction with a reagent precursor compound.
  • the polystyrene may be derivatised with a moiety comprising a leaving group or a group which can be modified to become a leaving group in situ, to facilitate reaction in the method sequence to provide the useable bound reagent.
  • Such handles for functionalisation include, but are not limited to, benzylic positions with leaving groups such as the halogens, in particular fluoride, chloride, bromide and iodide, especially chloride; addionally hydroxymethyl and its activated derivations obtained by prior treatment with, for example, para-toluenesulonylchloride or mesylchloride or by in situ treatment with phosphines, such as PPh3 and PBu3, and azodicarboxylates, such as DIAD or TMAD, according to Mitsunobu's methods in the presence of the linker L and or Ar.
  • halogens in particular fluoride, chloride, bromide and iodide, especially chloride
  • addionally hydroxymethyl and its activated derivations obtained by prior treatment with, for example, para-toluenesulonylchloride or mesylchloride or by in situ treatment with phosphines, such as PPh
  • the reagent precursor compound is a benzoic acid, e.g., salicylic acid, derivative, for example, 2,3-dihydroxy benzoic acid, 2,4-dihydroxy benzoic acid, 2,5- dihydroxy benzoic acid and 2,6-dihydroxy benzoic acid, especially 2,4-dihydroxy benzoic acid and 2,5-dihydroxy benzoic acid.
  • a benzoic acid e.g., salicylic acid
  • derivative for example, 2,3-dihydroxy benzoic acid, 2,4-dihydroxy benzoic acid, 2,5- dihydroxy benzoic acid and 2,6-dihydroxy benzoic acid, especially 2,4-dihydroxy benzoic acid and 2,5-dihydroxy benzoic acid.
  • the immobilised precursor may then be provided for further transformation to introduce a phosphorus (III) atom or a phosphorus (V) atom, e.g. with phosphorus trichloride or phosphorus oxytrichloride or phosphorus thiochloride or thiophosphoryl chloride or phosphorus tribromide or other chlorination or bromination reagents, to provide the final useable immobilised phosphitylation reagent, e.g. a phosphorochloridite reagent or, with further oxidation, the immobilised phosphorylation reagent, e.g. a phosphorochoridate reagent. It will be appreciated that further transformation may introduce leaving groups other than chlorine or bromine.
  • the oxidation of the phosphorus (III) atom to a phosphorus (V) atom may be accomplished directly, or step-wise.
  • PL 3 Br bromine adducts
  • chlorine adducts (PL 3 Cl) + provided by treatment with CC14/H2O/NMM/Py/CH3CN or N- chlorosuccinimide.
  • the present invention also extends to the derivatised substrates provided at any of the stages in the preparation of the immobilised reagent.
  • the present invention further extends to the uses of the immobilised reagents described herein for the preparation of phosphorus-containing compounds.
  • the preparation may comprise the synthesis of a library of phosphate compounds.
  • an immobilised reagent provides a solid phase component to the reaction rather than a solely solution phase reaction in the preparation of the phosphate compounds, their precursors and their intermediates.
  • a solid phase reaction enables the provision of reaction conditions in which reaction efficiencies may be improved compared to solution phase reactions, thus providing the opportunity for higher product yields to be achieved, and further providing a means for high throughput production of the desired compounds.
  • the immobilised reagent may be provided in a molar excess compared to the reactant compounds which are added to the reagent.
  • Those reactant compounds may be scarce, thus, desirably a reaction with such compounds should attempt to maximally react all of the reactant compound without the need to use an excess of that compound, and the use of a solid phase reaction in which the reagent is immobilised, may provide this advantage.
  • the phosphate compounds may be prepared as mono-, di- and tri-phosphate compounds using the presently described immobilised reagent.
  • Typical preparative methods include:
  • the substrate which includes the immobilised reagent, may be contained in any suitable reaction vessel for the purpose of performing the reaction methods, and typically, the substrate may be provided as a column of material or polymer matrix with embedded beads or meshed bag containing beads through which reaction solutions comprising reactants may be flushed, washed, filtered etc.
  • the methods may also employ other techniques readily applied by the skilled person, such as providing a bed of substrate particles, for example the use of a fluidised reaction bed.
  • the method typically utilises the immobilised reagent which reacts with a starting compound to provide an intermediate immobilised activated phosphite or phosphate compound which may then be transformed into the desired final phosphate compound.
  • the starting compound typically comprises a nucleophilic component which reacts with the phosphorus atom of the immobilised reagent.
  • the starting compound typically comprises a hydroxyl moiety, and that moiety reacts with the reagent to provide the activated compound, and then through a series of chemical reactions, is transformed into a final phosphate compound.
  • the hydroxyl moiety may be a primary, secondary, tertiary or phenolic hydroxyl group, preferably a primary or secondary hydroxyl group, and especially a primary hyHroxyl moiety.
  • the reaction sequence may broadly follow the mechanism described by Ludwig and Eckstein in which the hydroxyl group of a hydroxyl-containing compound displaces the leaving group, e.g. chlorine atom, of the immobilised cyclic phosphinyl or phosphoryl reagent, e.g. the immobilised phosphorochloridite reagent.
  • the hydroxyl-containing compound becomes bound to the immobilised reagent to form an immobilised activated cyclic phosphite or phosphate.
  • This immobilised activated phosphite or phosphate may be stored as an intermediate for later use. Such intermediate advantageously allows the hydroxyl-containing compound to be stored in an activated, immobilised, condition ready for subsequent reaction on demand.
  • Subsequent reaction may be a cleavage reaction to provide a mono-phosphite or mono-phosphate compound.
  • the released cyclic compound may then be subsequently transformed to provide the final desired linear triphosphate compound, e.g. a triphosphate compound.
  • the method includes the step of introducing a phosphorus atom to a starting compound to form an immobilised precurser phosphorus-containing compound, and in subsequent step(s) a further desired number of phosphorus atoms e.g. one or preferably two, may be optionally introduced, whilst simultaneously cleaving the resultant compound from the immobilised reagent.
  • the immobilised activated phosphite or phosphate compounds react with pyrophosphate in the present methods, undergoing a double nucleophilic attack on the phosphorus atom to cleave and simultaneously provide the cyclic triphosphite or triphosphate compound in a single step.
  • the methods provide the cyclic compounds and cleave the compounds from the substrate in the same reaction.
  • the subsequent reaction is conducted from about 30 seconds to about 1 hour, preferably, from about 1 minute to about 30 minutes, most preferably, from about 5 minutes to about 20 minutes, especially from about 10 minutes to about 15 minutes.
  • the reaction/cleavage may be conducted in intervals, with each interval conducted for a period of time independently chosen from those listed above.
  • the reaction may be conducted using two to five interval periods, each one accompanied with removal and separation of the product from the substrate. Three interval periods is preferred, especially, three intervals of from about 5 minutes to 20 minutes.
  • the method may involve a number of optional reaction steps after coupling the hydroxyl-containing compound to the immobilised reagent.
  • the phosphorus (III) atom may be oxidised to a phosphorus (V) atom using an oxidising agent and the techniques described herein, whilst the hydroxyl-containing compound remains bound to the immobilised reagent.
  • the oxidation step may be performed after the desired, e.g. cyclic phosphorus-containing product, has been cleaved from the substrate.
  • the method of the present invention may be applied to the preparation of any desired phosphate compound, such as triphosphates, diphosphates, monophosphates, e.g. monophosphate triesters, monophosphate diesters and monophosphate monoesters, phosphonate esters, e.g. phosphonate diesters and phosphonate monoesters, phosphate monoester diamidates, phosphate monoester diamidites and phosphite triesters for example.
  • phosphate compound such as triphosphates, diphosphates, monophosphates, e.g. monophosphate triesters, monophosphate diesters and monophosphate monoesters, phosphonate esters, e.g. phosphonate diesters and phosphonate monoesters, phosphate monoester diamidates, phosphate monoester diamidites and phosphite triesters for example.
  • the method may be applied to compounds having several reactive groups to provide a regioselective reaction of such compounds, e.g. reaction at one particular type of hydroxyl group, e.g. a primary hydroxyl group, in a poly hydroxyl- containing compound.
  • the method is applicable to the preparation of nucleoside phosphate compounds, and in particular to nucleoside triphosphate compounds and analogues thereof.
  • the method is applicable to any nucleoside, in particular natural and non-natural nucleosides.
  • the method is applicable to ribonucleosides (N), deoxyribonucleosides (dN), dideoxynucleosides (ddN), dideoxydidehydronucleosides, acyclonucleosides, nucleosides with non-natural substituents on the ribose sugar, such as halogens, azido groups, unnatural stereochemistry or a combination of the above.
  • C cytidine
  • A adenosine
  • U uridine
  • G guanosine
  • G thymidine
  • T thymidine
  • I inosine
  • dC adenosine
  • dA uridine
  • U uridine
  • G guanosine
  • T thymidine
  • I inosine
  • dA 2'-deoxyadenosine
  • DU 2'-deoxyuridine
  • DG deoxyguanosine
  • DT 2'-deoxythymidine
  • dl 2',3'- dideoxycytidine
  • ddC 2',3'-dideoxyadenosine
  • ddU 2',3'-dideoxyuridine
  • ddT 2',3'-dideoxythymidine
  • ddl and their derivatives.
  • nucleosides that can be applied to Sanger-type dideoxysequencing. For this they are required to be as their triphosphates. Especially deoxynucleoside triphosphates (dNTPs) and dideoxynucleoside triphosphates (ddNTPs) are of interest, particularly if they have been modified on the purine or pyrimide base to incorporate a fluorophore label or a biotin label or the potential to introduce such a moiety after phosphorylation. Examples of such moieties include aminoalkyl, aminoalkenyl and aminoalkynyl linkers.
  • the method may also be applied to carbohydrates, e.g. sugars.
  • the present invention also extends to the immobilsed activated phosphite intermediate or immobolised phosphate intermediate compounds.
  • the invention provides a compound according to formula (V):
  • R may be selected from nucleosides, including ribonucleosides (N), deoxyribonucleosides (dN), dideoxynucleosides (ddN), dideoxydidehydronucleosides, acyclonucleosides, nucleosides with non-natural substituents on the ribose sugar, such as halogens, azido groups, and nucleosides with an unnatural stereochemistry or a combination of the above, or carbohydrates.
  • nucleosides including ribonucleosides (N), deoxyribonucleosides (dN), dideoxynucleosides (ddN), dideoxydidehydronucleosides, acyclonucleosides, nucleosides with non-natural substituents on the ribose sugar, such as halogens, azido groups, and nucleosides with an unnatural stereochemistry or a combination of the above, or carbohydrates.
  • Phosphorylated nucleoside compounds useful as pharmaceutically active compounds and/or prodrugs may be prepared by the methods according to the present invention, and the present invention further extends to the preparation of such compounds and to the compounds themselves.
  • the methods of the present invention may be used to prepare nucleoside phosphate compounds and analogues thereof, typically nucleoside triphosphate, monophosphate triesters, monophosphate diesters, monophosphate monoesters, phosphonate diesters, phosphonate monoesters, phosphate monoester diamidates, phosphate monoester diamidites compounds, any of which may find uses as pharmaceutically active agents, more specifically as prodrugs.
  • the methods may be applied to the preparation of compounds or their precursors to provide pro-drugs, which, following transformation within the body of the organism into an active molecule, may interfere with nucleic acid synthesis.
  • useful compounds include anti-viral compounds, useful as anti-HIV (Human Immunodeficiency Virus) compounds in the treatment of AIDS (Acquired Immunodeficiency Syndrome).
  • anti-viral compounds inelude 3'-azido-2',3'-dideoxythymidine (AZT) and (2',3'-didehydro-2',3'-dideoxythymidine (d4T).
  • Phosphorylated pro-drug compounds may be provided, for example, which are converted to a useable form inside the body of the organism being treated.
  • the methods of the present invention may also be applied to the preparation of modified triphosphate analogues such as ⁇ -thio, v " ⁇ -borano,*'" and ⁇ -thio- ⁇ -borano triphosphates.*' 1 '
  • modified nucleosides are of biochemical interest for example because they are substrates for DNA polymerase enzymes and can be successfully incorportated into DNA, but are more resistant to exo- and endo-nuclease than phosphate diesters/"'
  • the substrate used was a modified polystyrene, and the polymer-supported reagent was synthesised as detailed in Scheme 5.
  • the nucleoside was captured onto the resin and then released using pyrophosphate cleavage, followed by oxidation, hydrolysis and deprotection to yield the triphosphate in a 50 % yield after HPLC purification (Scheme 6).
  • Microporous polystyrene was thus used successfully as a support for the reagent. It was used to synthesise thymidine triphosphate 3 as its tetrakis [triethylammonium] salt in a 50 % yield from its 3'-O-acetyl protected analogue.
  • S: S8, X Se: Se powder; (e) ethylenediamine; (f) NH 3 /H 2 0, 1 h
  • the desired nucleoside 2 was isolated in a 37 % yield over two steps as out-lined below:
  • the propagylamino linked uridine (1) was converted to the mono-phosphonate (3) as shown in scheme 2.
  • Nucleoside 1 (40 mg, 95 ⁇ mol) was evaporated in vacuo from dry pyridine (1 mL), dried over P 2 O 5 in a desiccator for 1 h in vacuo and dissolved in dry pyridine (0.25 mL) and dry THF (0.75 mL). Resin (0.27 mmol, 3 eq) was swollen in dry THF (2 x 2 mL for 5 min) at 60 °C. PCl 3 (95 ⁇ L, 12 eq) was added to the resin in dry THF (4 mL) and the syringe was heated at 60 °C for 2 h. The resin was drained and rinsed with dry THF (2 x 2 mL for 5 min).
  • the N 2 flushed resin was treated with the nucleoside solution for 1 h at rt, drained and rinsed with dry THF (2 x 2 mL for 5 min). The resin was suspended with THF/H 2 O (1/1, 4 mL) for 10 min, then drained and evaporated in vacuo. The residue was purified by Prep HPLC eluted with an MeCN/0.1 M ammonium acetate gradient to yield a white hygroscopic powder (27 mg, 54 ⁇ mol, 57 % yield).
  • Supported linker (1 eq) was swollen in dry THF (2 x 2 mL for 5 min) at 60 °C.
  • PCl 3 (4 eq) was added to the resin in dry THF (4 mL) and the syringe was heated at 60 0 C for 2 h.
  • the resin was drained and rinsed with dry THF (2 x 2 mL for 5 min).
  • the nucleoside (2) was dissolved in dry pyridine/ dry THF (1/3 ) to load onto the resin and the loading was performed for 2 h.
  • the nucleoside was evaporated in vacuo from dry pyridine (1 mL), dried over P 2 O 5 in a desiccator for 1 h in vacuo before dissolving in dry pyridine and dry THF).
  • the N 2 flushed resin was treated with the nucleoside solution for 2 h at rt, drained and rinsed with dry THF (2 x 2 mL for 5 min).
  • the N 2 flushed resin was treated three times with 0.5 M bis (tributyl ammonium) pyrophosphate in DMF (800 ⁇ L, 1.5 eq) and Bu 3 N (255 ⁇ L, 4 eq) at rt for 10 min each.
  • the cleaved product was collected in an flask with 0.5 M I 2 /pyridine:H 2 O 98:2 (800 ⁇ L, 1.5 eq). 15 min after the last cleavage, excess iodine was destroyed by adding a few drops of a 5 % aqueous solution Of NaHSO 3 .
  • the reaction mixture was diluted with H 2 O (20 mL), stirred for 30 min and evaporated in vacuo. The residue was dissolved in concentrated ammonia (20 mL) and was left for 1 h the solution and the crude evaporated in vacuo.
  • the lyophilised residue was purified by Prep HPLC eluted with 0.1 M TEAB on a Cl 8 column. The fractions containing triphosphate were pooled and lyophilised to remove the buffer to yield an orange solid. 31 P NMR
  • Figure 2 shows the crude HPLC chromatogram for dUTP-6-FAM 5. Inspection of the HPLC chromatogram revealed a major peak a 9.52 min. As the HPLC chromatogram was run at identical conditions than for the corresponding phosphonate, it can be asserted with confidence then no phosphonates are present in the reaction mixture, which indicates that all precautions to prevent premature hydrolysis of the resin were successful. The peak was isolated by Prep HPLC and lyophilised to yield an orange solid. A 31 P NMR spectrum provided the confirmation that the triphosphate group was present by exhibiting the characteristic signals at -6, -11 and -22 ppm. The 31 P
  • the present invention generally provides an immobilised reagent as a polymer-supported reagent, in particular a reagent whose chemistry is well established and where the immobilised/supported reagent provides advantages over non- immobilised reagent/solution phase chemistry.
  • the methods of the present invention allow a solid-supported reagent to be used in excess without causing unwanted side-reactions.
  • Excess reagents can push sluggish reactions to completion without prolonging reaction times, and the reagents remain immobilised throughout the reaction and remain so afterwards, and thus do not contaminate the reaction.
  • by-products formed can simply be washed off while the compound of interest stays immobilised on the polymer.
  • the immobilised intermediate advantageously is released by a controlled cleavage procedure, and the final triphosphate compound is isolated with a single chromatography procedure.
  • the present invention is not limited solely to the preparation of triphosphate compounds, but is equally applicable to the preparation of mono- and di- phosphate compounds, monophosphate triesters, monophosphate diesters, monophosphate monoesters, phosphonate diesters, phosphonate monoesters, phosphate monoester diamidates, phosphate monoester diamidites and phosphite triesters.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Saccharide Compounds (AREA)

Abstract

L'invention concerne un réactif cyclique immobilisé de phosphitylation ou de phosphorylation, comprenant une fonction phosphinyle ou phosphoryle, l'atome de phosphore (III) ou l'atome de phosphore (V) étant respectivement lié à et dans un système cyclique.
PCT/GB2007/001089 2006-03-27 2007-03-26 Reactif immobilise WO2007110624A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0606038A GB0606038D0 (en) 2006-03-27 2006-03-27 Immobilised reagent
GB0606038.8 2006-03-27

Publications (2)

Publication Number Publication Date
WO2007110624A2 true WO2007110624A2 (fr) 2007-10-04
WO2007110624A3 WO2007110624A3 (fr) 2008-04-03

Family

ID=36384204

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2007/001089 WO2007110624A2 (fr) 2006-03-27 2007-03-26 Reactif immobilise

Country Status (2)

Country Link
GB (1) GB0606038D0 (fr)
WO (1) WO2007110624A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010102997A3 (fr) * 2009-03-09 2011-03-03 Freie Universität Berlin Composé modifié par des groupes phosphoramidate et/ou phosphonamide et son utilisation

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003008432A1 (fr) * 2001-07-16 2003-01-30 Isis Pharmaceuticals, Inc. Procede de preparation de nucleosides triphosphates modifies en position alpha et composes obtenus conformement a ce procede

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003008432A1 (fr) * 2001-07-16 2003-01-30 Isis Pharmaceuticals, Inc. Procede de preparation de nucleosides triphosphates modifies en position alpha et composes obtenus conformement a ce procede

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BURGESS, K. AND COOK, D.: "Synthesis of nucleoside triphosphates" CHEM. REV., vol. 100, 2000, pages 2047-2059, XP002464530 cited in the application *
PARANG, K. ET AL.: "Selective diphosphorylation, dithiodiphosphorylation, triphosphorylation, and trithiotriphosphorylation of unprotected carbohydrates and nucleosides" ORG. LETT., vol. 7, 2005, pages 5589-5592, XP002464531 cited in the application *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010102997A3 (fr) * 2009-03-09 2011-03-03 Freie Universität Berlin Composé modifié par des groupes phosphoramidate et/ou phosphonamide et son utilisation
US8575282B2 (en) 2009-03-09 2013-11-05 Freie Universitaet Berlin Compound modified by a phosphoramidate and/or phosphonamide group and use thereof

Also Published As

Publication number Publication date
GB0606038D0 (en) 2006-05-03
WO2007110624A3 (fr) 2008-04-03

Similar Documents

Publication Publication Date Title
JP2705913B2 (ja) インビトロにおけるオリゴヌクレオチド合成法
US4591614A (en) Preparation of oligodeoxyribonucleoside alkyl or arylphosphonates
JPH0631305B2 (ja) ヌクレオシド誘導体
JPH05507279A (ja) グリセロールジ―およびトリホスフェート誘導体の合成
JPH03501128A (ja) ヌクレオシドおよびポリヌクレオチドチオホスホラミダイトおよびホスホロジチオエイト化合物並びに方法
US5780617A (en) Synthesis of liponucleotides
JP5184621B2 (ja) α−リン酸擬似体を有するヌクレオチド
Sherstyuk et al. How to form a phosphate anhydride linkage in nucleotide derivatives
US8742094B2 (en) Method for the solid phase-based production of phosphate-bridged nucleoside conjugates
US8614312B2 (en) Method for preparing nucleotides and related analogues by synthesis on soluble substrate, and biological tools thus prepared
Dutta et al. New synthetic methods for phosphate labeling
JP2006501250A (ja) オリゴヌクレオチドの分離及び脱保護方法
WO2007110624A2 (fr) Reactif immobilise
EP2880044B1 (fr) Nouveau procédé pour la synthèse de polyphosphates
WO2005070946A1 (fr) Compose d'acide ribonucleique et procede de synthese en phase liquide de compose d'acide oligonucleique
CA1254887A (fr) 2'-desoxy-3'-phosphonylmethyl-nucleosides
US20110137023A1 (en) Method for producing phosphate-bridged nucleoside conjugates
JP2671446B2 (ja) イノシン及びグアノシンの混合リン酸化方法
WO2014114326A1 (fr) Procédé pour la synthèse à base de phase solide de conjugués de nucléoside à pont phosphate
Singh Development of novel p-amidites and their application in the synthesis of the most densely phosphorylated compounds
AU647164B2 (en) Synthesis of glycerol di- and triphosphate derivatives
JPS6363694A (ja) ヌクレオシドホスホロチオイットおよびその合成法
JPH0372235B2 (fr)
JPH027597B2 (fr)
PL175220B1 (pl) Sposób wytwarzania estrów fosforowych modyfikowanych P-chiralnych analogów nukleotydów

Legal Events

Date Code Title Description
NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07732150

Country of ref document: EP

Kind code of ref document: A2