CA2141301A1 - Esterification process - Google Patents
Esterification processInfo
- Publication number
- CA2141301A1 CA2141301A1 CA002141301A CA2141301A CA2141301A1 CA 2141301 A1 CA2141301 A1 CA 2141301A1 CA 002141301 A CA002141301 A CA 002141301A CA 2141301 A CA2141301 A CA 2141301A CA 2141301 A1 CA2141301 A1 CA 2141301A1
- Authority
- CA
- Canada
- Prior art keywords
- oxyacid
- amine
- mixture
- water
- reaction mixture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H11/00—Compounds containing saccharide radicals esterified by inorganic acids; Metal salts thereof
- C07H11/04—Phosphates; Phosphites; Polyphosphates
-
- 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 System
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/582—Recycling of unreacted starting or intermediate materials
Abstract
An esterification or amidation process comprises reacting a hydroxylated compound or an organic amine with an oxyacid or its anion capable of accepting a lone pair of electrons. The reaction has the advantage that it is carried out under mild condi-tions in a single step and enables phosphate and other esters of organic compounds (particularly mono- and oligo-saccharides) to be prepared easily.
Description
,.. .
ESTE~IFTCATION ~ROCESS
The present invention reiates ~o a process for the esterification cf hydroxylated compounds.
c Esters, particularly phosphate esters, are widespread in nature and occupy a central role in biochemical processes. Of special interest are carbohydrate phosphates which are used extensively for studying 'O biosynthetic pathways, and inositol phospnates which act as secondary cellular messengers. However, because these compounds are often present at low concentration and are usually difficult to isolate in pure form, it is often necessary to synthesise them chemically.
A number of pharmaceutical products possess ester functionalities. Examples include betamethasone, which is used in the treatment of asthma, and men~;ol, which is used for haemorrhage control. 30th of these compounds are orthophosphates. Phosphate esters are structural features of several bacterial polysaccharides which are used in vaccines to protect against diseases such as pneumonia and meningitis; synthetic antigens with this type of structu~e have, in recent years, been prepared with a view to using them as synthetic vaccines. It is therefore clear that the introduction of the ester functional group is necessary for the preparation of a number cf pharmaceutical products.
~0 Another =rea in which esters have proved to be useful is in detailed biochemical studies, lncluding studying the erfects ~f s~ruc ural modificatlons Gn -he biological actlvity of various compounds. Such studies include pharm~colo~lcal ~_sting oI new ~ruas and therapeuti~
.w~94/024g5 ~14 ~-3 - Pcr/GBg3/0l545 agents and also investigations into the mechanism of diseases including cancer. In s~udies such as these, i t is often par~ic~lariy useful to use radioactive esters into which a raaioactive isotope of one of the a~oms nas _ been incorporated.
It is often impossible to obtain the required ester compounds by any other method than chemical synthesis but, in fact, the chemical synthesis of esters has, in ;0 the past, proved to be extremely difficult. Direct methods for the synthesis of esters are available and an example of such a method is the esterification of a hydroxylated compound (HC) using phosphoric acid or polypAospAoric acid (MacDonald, The Carboh~drates, lA, 254 (1972) W. Pigman and D. Horton (Eds). Ar~e~ic Press, New York). However, the utility of this method is limited because many HCs are sensitive to the reaction condit~ons which are, of course, strongly acidic and often the products undergo further reaction to form 0 modified compounds such as cyclic esters or anhydrides.
This route cannot therefore be used for most compounds.
Indirect methods of chemical synthesis have also been devised and include those described in the artic~e entitled "Synthesis of Some Low Molecular Carbohydrate Esters of Biological Significance" by Lindh, Chemical Communications, UniversitY of Stockholm, (1988), No 8.
Further examples are given in the articie by Ozaki et al, _ Chem. Soc. ~erkin ~rans. _, il992), 729-737. Both of ~hese articles demons~rate 'he complexity of known synthetic _outes to pAospAate esters. Indeed, the latter desc-ibes a twelve step svnthesis of the ?roduct~
:nc lainq severai steps wnic.. _nvolve chromatograpnic separati~ns and aivinq an overai: yieid of less than 2~.
W094/0~95 214 1 3 D I PCT/GB9~/01545 -Clearly, therefore, existing methods for the production of esters are far from ideal and one serious problem is that the extreme reac~ion conditions necessary for direct esterification lead to unwanted side reactions of both starting materials and products. Attempts have been made to o~ercome this by using indirect methods of chemical synthesis but these methods usuaily involve a number of steps selectiveiy to expose specific hydroxyl groups, a number of further steps to introduce the ester ana iO finally a series of deprotection steps. This strategy requires the presence of compatible functional groups in the molecule during the synthesis, which can be limiting.
Furthermore, the large number of steps will reduce the overall yield of the final product and often involves the use of large quantities of expensive reagents.
There is therefore a need for a simple and cost effective method of esterifying hydroxyl groups. The present invention is based on the discovery that ester formation can be promoted remarkably easily in solutions containing a hydroxylated compound and an oxyacid salt.
Certain superficially similar processes have previously been appiied to starches, for ood purposes, as in US-A-2884413, US-A-2865762, US-A-2884412 and US-A-2961440.
However, ;hese proposals date from the late 1950s or eariy ~960s, and the applica~ility of modified methods _or other hydroxyiated compounds does not seem to have Deen appreciated in the intervening 30 to 35 years.
~o Acc~r~ing to a first as~ect o. the invention, there ls F-C~lGe~ 8 Frocess for ~he este-Lfication of :-ya--xvla~ed c-m~ound (~C) o~her .han a starch, or Ihe ~m ~a.ion of an or~ani- amlne, c~.arac~e~ised in that ~he W094/0~95 2 1 4 1 3 0 I PCT/GB93/01~5 process ccm~rises -eacting the HC or amine with an oxyacid, sxvacid an~on or a mixture thereof wherein the oxyacid or its anion is capable of accept ng a lone pair of elect-ons ~rcm ~he oxygen of the HC or -.itrogen of the amine.
In the context of the present invention, the term "oxyacid~' inciuaes wlthin its scope hetero-oxyacids in which one or more cf the oxygen atoms have been replaced '0 by a hetero atom, in particular one such as nitrogen, sulphur or a halogen atom. Examples of hetero-oxyacids include thiophosDhates, halophosphates and phosphonitriles.
lS The term "hydroxyiated compound" refers to any compound containing a hydroxyl group or an alkoxy or phenoxy anion. Preferred hydroxylated compounds are hydroxylated organic compounas although inorganic compounds, for example oxyacids may De used. Exampies of hydroxylated organic compounds include sugars, proteins, glycoproleins, peptides, glycopeptides and giycoconlugates and other molecules having sugar functionality, amino acids, alditols, cyclitols, phosphate esters and carboxylic acids.
The condit ons of the process of the present invention are auite surprisingly mild and one part cular advantage of this is that, because of the mild pH conditions, it is not necessary tc protect ~and subseauentlv to deprotect) ~0 other func-ional ~rou~s of the HC. This means that ~he overali number ~f steps in the Frocess is reduced ana that t~.erefore ~he overaii yield c. the final prcauct is lncreased, t~us lnc-easin~ the cost-effec~iveness o the ester~ 2t~-n p~ccess. ~urther~ore ! tr.e m1ld reaction W094/0~9~ ~ 1 4 1 3~ I PCT/GB93/01~5 _ conditions aiso -nsure that the reaction s suitable for nearly any star-_ng material and that .Aere is little likelihood of -roducts undergoing unwanted further reaction.
-The oxyacid may ~e an oxyacid of an element M, which iscapable of 'orming trimeric oxyacids of the structure:
:0 ~YnHm)`~h/ \M' (YnHm) I\M/I
~YnoHm ) wherein:
M is an eiec~ron accepting element;
X is oxvgen cr a hetero-atom such as sulphur or a substitutea r.itrogen atom;
Y is oxvgen, hydrogen or a hetero atom such as sulphur cr a substituted nitrogen atom;
n is 0 or : and m is 0 to 2, depending on the valency of M.
Examples of eiements M which are capable of forming trimeric oxyacias include phosphorus, boron and silicon.
Usually, the -itrogen atom will be substituted by hydrogen or an aikyl group.
- V ?hosphorus is _~pable of forming .he metaphosphate oxyacid. Recause of electron deficiency at the phosphorus atcm, the ion tends to polymerise and may fcrm ~he followina trimer:
WO 94/0249~ 2 1 ~7/ 1 3 0 I Pcr/GBg3/01545 o~ ~o\ ~o OH/ ¦ ¦ OH
\p/
O~ \OH
Alternative s~r~ctures for metaphosphate are cyclic :3 tetramers or chain structures.
Similariy, boron forms an oxyacid having the following structure:
OH\ /O\ /OH
\B/
and silicon forms an oxyacid as follows:
OH~ ~ O~ ~ OH
OH/ I ¦ \ OH
si \
, 0 OH/ OH
~owever, the aDil}.y of an element to form this.type or t-imeri- struc-ure is merely an indica~ion that one or more ^f its oxvaci~s will be capable of ~ccepting a lone W094/02495 214 13~ I PCT/GB93/01~
pair cf electrons.
One phosphorus oxyacid ~hich has been found to be particularly sui~able for use in esterification reactions according ~o the present invention is metaphosphoric acid and one suggestion for the reaction mechanism for esterification using a metaphosphate is as follows:
o~S~ J P--OH ~ P--O t ~H;
OH O O
O
--h ~ ~ _ HO_ LOR + H+
~ o O
~R O ~ OR + H+
¦ ¦ ~ H
~ a o~
This reaction mechanism ~ndicates that it is the metapnospnate monomer rather than the trimer which is the reactive species. ~owever, the usefulness of .he O inventi~n s no~ dependent ~n the accuracy or otherwise of this reac~loR~ mecnanism.
~he eaulvaien~ ~onomers of the boron and siiicon oxyan~r.s a-e _s f~ilcws:
WO9~ g5 ~ ~ PCT/GB93/01~5 O OH
..1 si ~~
:, O~ 11 and it s like~y that -hey will undergo similar reactions to the phosphate oxyacids and anions.
0 -It is not always necessary to use a salt of the oxyacid anion which forms the type of monomer structure shown above. For examDie, -n the case of phosphorus, although metaphosphate salts are preferred, other oxyacid salts such as orthopnospnate, diphosphate, triphosphate, lS polyphospnate, ?hosphonate, phosphinate, peroxyphosphate, and hypophospAate salts or their free acids may also be used. If the reactive species for the formation of orthophosphate esters is indeed the metaphosphate monomer, the -eactivity of ortho-, di-, tri- and ~0 polyphosphate oxyacids can be explained by the fact that in solution these oxyacids and their anions will be in equilibrium with the metaphosphate species via which the reaction appears to ?roceed. Furthermore, when these other phosphates are used, metaphosphate is continuously reacting with the HC and so the equilibrium with the other phosDhates will ~e driven to the metaphosphate side ailowing the reaction tO continue. An analogous applies to the othe- species listed above.
~0 Other phosphcrus ~ased oxyacids which have been found to be useful ~n t~e present invention are hetero-oxyacids such as thiophcspr.ate, haloDhosDhates and phosphon~trilic comDounds .
W094/0~95 ~14 1~/ PCT/GB93~01~5 The cation of the oxyacid salt will be sucA as to ensure that the salt is soluble in the solvent in which the reaction is carriea out. In aoueous solution, 'he cation may be an alkali or aikaline earth metal such as sodium, potassium or ~agnesium. For certain solvents, particularly organic solvents, larger cations such as lanth~ntlm, caesium or less ionic species such as lithium and ~mmn~i um may be more appropriate. In aqueous and in other solvents, the cation may be chosen because of its catalytic effect on the reaction. Preferably, the counter ion will be chosen so that the salt does not have an inappropriate pK value, particularly bearing in mind the preferred FH cperating conditions which will be discussed in more detail below.
The reaction can be carried out in both aqueous and organic solvents. When an organic solvent is used it is preferred that -_ is a highly polar solvent such as dimethyl sulphoxiae ~DMSO).
In general, the --ocess consists of the following steps:
(a) providing a reaction mixture comprising the HC or amine; an oxyac ~ or oxyacid salt capable of producing in solution a species capable of accepting a lone pair of electrons f~om the oxygen of the HC or nitrogen of the amine; ana from 0 to 30~ (w/w) water based on the weight of the ~C or amine;
(b) allowing th~ :~C or amine and the oxvacid species to 3 react to form a F~oauc. mixture containing an oxyacid ester of the H. or amine; and optionallv lc) either li) at ~east par~ially recovering the ester ~~om the proauc~ mixture or (ii) furthe- reacting the ester in si t~ tD ~rm a aeslred compound cr mi~ture.
ESTE~IFTCATION ~ROCESS
The present invention reiates ~o a process for the esterification cf hydroxylated compounds.
c Esters, particularly phosphate esters, are widespread in nature and occupy a central role in biochemical processes. Of special interest are carbohydrate phosphates which are used extensively for studying 'O biosynthetic pathways, and inositol phospnates which act as secondary cellular messengers. However, because these compounds are often present at low concentration and are usually difficult to isolate in pure form, it is often necessary to synthesise them chemically.
A number of pharmaceutical products possess ester functionalities. Examples include betamethasone, which is used in the treatment of asthma, and men~;ol, which is used for haemorrhage control. 30th of these compounds are orthophosphates. Phosphate esters are structural features of several bacterial polysaccharides which are used in vaccines to protect against diseases such as pneumonia and meningitis; synthetic antigens with this type of structu~e have, in recent years, been prepared with a view to using them as synthetic vaccines. It is therefore clear that the introduction of the ester functional group is necessary for the preparation of a number cf pharmaceutical products.
~0 Another =rea in which esters have proved to be useful is in detailed biochemical studies, lncluding studying the erfects ~f s~ruc ural modificatlons Gn -he biological actlvity of various compounds. Such studies include pharm~colo~lcal ~_sting oI new ~ruas and therapeuti~
.w~94/024g5 ~14 ~-3 - Pcr/GBg3/0l545 agents and also investigations into the mechanism of diseases including cancer. In s~udies such as these, i t is often par~ic~lariy useful to use radioactive esters into which a raaioactive isotope of one of the a~oms nas _ been incorporated.
It is often impossible to obtain the required ester compounds by any other method than chemical synthesis but, in fact, the chemical synthesis of esters has, in ;0 the past, proved to be extremely difficult. Direct methods for the synthesis of esters are available and an example of such a method is the esterification of a hydroxylated compound (HC) using phosphoric acid or polypAospAoric acid (MacDonald, The Carboh~drates, lA, 254 (1972) W. Pigman and D. Horton (Eds). Ar~e~ic Press, New York). However, the utility of this method is limited because many HCs are sensitive to the reaction condit~ons which are, of course, strongly acidic and often the products undergo further reaction to form 0 modified compounds such as cyclic esters or anhydrides.
This route cannot therefore be used for most compounds.
Indirect methods of chemical synthesis have also been devised and include those described in the artic~e entitled "Synthesis of Some Low Molecular Carbohydrate Esters of Biological Significance" by Lindh, Chemical Communications, UniversitY of Stockholm, (1988), No 8.
Further examples are given in the articie by Ozaki et al, _ Chem. Soc. ~erkin ~rans. _, il992), 729-737. Both of ~hese articles demons~rate 'he complexity of known synthetic _outes to pAospAate esters. Indeed, the latter desc-ibes a twelve step svnthesis of the ?roduct~
:nc lainq severai steps wnic.. _nvolve chromatograpnic separati~ns and aivinq an overai: yieid of less than 2~.
W094/0~95 214 1 3 D I PCT/GB9~/01545 -Clearly, therefore, existing methods for the production of esters are far from ideal and one serious problem is that the extreme reac~ion conditions necessary for direct esterification lead to unwanted side reactions of both starting materials and products. Attempts have been made to o~ercome this by using indirect methods of chemical synthesis but these methods usuaily involve a number of steps selectiveiy to expose specific hydroxyl groups, a number of further steps to introduce the ester ana iO finally a series of deprotection steps. This strategy requires the presence of compatible functional groups in the molecule during the synthesis, which can be limiting.
Furthermore, the large number of steps will reduce the overall yield of the final product and often involves the use of large quantities of expensive reagents.
There is therefore a need for a simple and cost effective method of esterifying hydroxyl groups. The present invention is based on the discovery that ester formation can be promoted remarkably easily in solutions containing a hydroxylated compound and an oxyacid salt.
Certain superficially similar processes have previously been appiied to starches, for ood purposes, as in US-A-2884413, US-A-2865762, US-A-2884412 and US-A-2961440.
However, ;hese proposals date from the late 1950s or eariy ~960s, and the applica~ility of modified methods _or other hydroxyiated compounds does not seem to have Deen appreciated in the intervening 30 to 35 years.
~o Acc~r~ing to a first as~ect o. the invention, there ls F-C~lGe~ 8 Frocess for ~he este-Lfication of :-ya--xvla~ed c-m~ound (~C) o~her .han a starch, or Ihe ~m ~a.ion of an or~ani- amlne, c~.arac~e~ised in that ~he W094/0~95 2 1 4 1 3 0 I PCT/GB93/01~5 process ccm~rises -eacting the HC or amine with an oxyacid, sxvacid an~on or a mixture thereof wherein the oxyacid or its anion is capable of accept ng a lone pair of elect-ons ~rcm ~he oxygen of the HC or -.itrogen of the amine.
In the context of the present invention, the term "oxyacid~' inciuaes wlthin its scope hetero-oxyacids in which one or more cf the oxygen atoms have been replaced '0 by a hetero atom, in particular one such as nitrogen, sulphur or a halogen atom. Examples of hetero-oxyacids include thiophosDhates, halophosphates and phosphonitriles.
lS The term "hydroxyiated compound" refers to any compound containing a hydroxyl group or an alkoxy or phenoxy anion. Preferred hydroxylated compounds are hydroxylated organic compounas although inorganic compounds, for example oxyacids may De used. Exampies of hydroxylated organic compounds include sugars, proteins, glycoproleins, peptides, glycopeptides and giycoconlugates and other molecules having sugar functionality, amino acids, alditols, cyclitols, phosphate esters and carboxylic acids.
The condit ons of the process of the present invention are auite surprisingly mild and one part cular advantage of this is that, because of the mild pH conditions, it is not necessary tc protect ~and subseauentlv to deprotect) ~0 other func-ional ~rou~s of the HC. This means that ~he overali number ~f steps in the Frocess is reduced ana that t~.erefore ~he overaii yield c. the final prcauct is lncreased, t~us lnc-easin~ the cost-effec~iveness o the ester~ 2t~-n p~ccess. ~urther~ore ! tr.e m1ld reaction W094/0~9~ ~ 1 4 1 3~ I PCT/GB93/01~5 _ conditions aiso -nsure that the reaction s suitable for nearly any star-_ng material and that .Aere is little likelihood of -roducts undergoing unwanted further reaction.
-The oxyacid may ~e an oxyacid of an element M, which iscapable of 'orming trimeric oxyacids of the structure:
:0 ~YnHm)`~h/ \M' (YnHm) I\M/I
~YnoHm ) wherein:
M is an eiec~ron accepting element;
X is oxvgen cr a hetero-atom such as sulphur or a substitutea r.itrogen atom;
Y is oxvgen, hydrogen or a hetero atom such as sulphur cr a substituted nitrogen atom;
n is 0 or : and m is 0 to 2, depending on the valency of M.
Examples of eiements M which are capable of forming trimeric oxyacias include phosphorus, boron and silicon.
Usually, the -itrogen atom will be substituted by hydrogen or an aikyl group.
- V ?hosphorus is _~pable of forming .he metaphosphate oxyacid. Recause of electron deficiency at the phosphorus atcm, the ion tends to polymerise and may fcrm ~he followina trimer:
WO 94/0249~ 2 1 ~7/ 1 3 0 I Pcr/GBg3/01545 o~ ~o\ ~o OH/ ¦ ¦ OH
\p/
O~ \OH
Alternative s~r~ctures for metaphosphate are cyclic :3 tetramers or chain structures.
Similariy, boron forms an oxyacid having the following structure:
OH\ /O\ /OH
\B/
and silicon forms an oxyacid as follows:
OH~ ~ O~ ~ OH
OH/ I ¦ \ OH
si \
, 0 OH/ OH
~owever, the aDil}.y of an element to form this.type or t-imeri- struc-ure is merely an indica~ion that one or more ^f its oxvaci~s will be capable of ~ccepting a lone W094/02495 214 13~ I PCT/GB93/01~
pair cf electrons.
One phosphorus oxyacid ~hich has been found to be particularly sui~able for use in esterification reactions according ~o the present invention is metaphosphoric acid and one suggestion for the reaction mechanism for esterification using a metaphosphate is as follows:
o~S~ J P--OH ~ P--O t ~H;
OH O O
O
--h ~ ~ _ HO_ LOR + H+
~ o O
~R O ~ OR + H+
¦ ¦ ~ H
~ a o~
This reaction mechanism ~ndicates that it is the metapnospnate monomer rather than the trimer which is the reactive species. ~owever, the usefulness of .he O inventi~n s no~ dependent ~n the accuracy or otherwise of this reac~loR~ mecnanism.
~he eaulvaien~ ~onomers of the boron and siiicon oxyan~r.s a-e _s f~ilcws:
WO9~ g5 ~ ~ PCT/GB93/01~5 O OH
..1 si ~~
:, O~ 11 and it s like~y that -hey will undergo similar reactions to the phosphate oxyacids and anions.
0 -It is not always necessary to use a salt of the oxyacid anion which forms the type of monomer structure shown above. For examDie, -n the case of phosphorus, although metaphosphate salts are preferred, other oxyacid salts such as orthopnospnate, diphosphate, triphosphate, lS polyphospnate, ?hosphonate, phosphinate, peroxyphosphate, and hypophospAate salts or their free acids may also be used. If the reactive species for the formation of orthophosphate esters is indeed the metaphosphate monomer, the -eactivity of ortho-, di-, tri- and ~0 polyphosphate oxyacids can be explained by the fact that in solution these oxyacids and their anions will be in equilibrium with the metaphosphate species via which the reaction appears to ?roceed. Furthermore, when these other phosphates are used, metaphosphate is continuously reacting with the HC and so the equilibrium with the other phosDhates will ~e driven to the metaphosphate side ailowing the reaction tO continue. An analogous applies to the othe- species listed above.
~0 Other phosphcrus ~ased oxyacids which have been found to be useful ~n t~e present invention are hetero-oxyacids such as thiophcspr.ate, haloDhosDhates and phosphon~trilic comDounds .
W094/0~95 ~14 1~/ PCT/GB93~01~5 The cation of the oxyacid salt will be sucA as to ensure that the salt is soluble in the solvent in which the reaction is carriea out. In aoueous solution, 'he cation may be an alkali or aikaline earth metal such as sodium, potassium or ~agnesium. For certain solvents, particularly organic solvents, larger cations such as lanth~ntlm, caesium or less ionic species such as lithium and ~mmn~i um may be more appropriate. In aqueous and in other solvents, the cation may be chosen because of its catalytic effect on the reaction. Preferably, the counter ion will be chosen so that the salt does not have an inappropriate pK value, particularly bearing in mind the preferred FH cperating conditions which will be discussed in more detail below.
The reaction can be carried out in both aqueous and organic solvents. When an organic solvent is used it is preferred that -_ is a highly polar solvent such as dimethyl sulphoxiae ~DMSO).
In general, the --ocess consists of the following steps:
(a) providing a reaction mixture comprising the HC or amine; an oxyac ~ or oxyacid salt capable of producing in solution a species capable of accepting a lone pair of electrons f~om the oxygen of the HC or nitrogen of the amine; ana from 0 to 30~ (w/w) water based on the weight of the ~C or amine;
(b) allowing th~ :~C or amine and the oxvacid species to 3 react to form a F~oauc. mixture containing an oxyacid ester of the H. or amine; and optionallv lc) either li) at ~east par~ially recovering the ester ~~om the proauc~ mixture or (ii) furthe- reacting the ester in si t~ tD ~rm a aeslred compound cr mi~ture.
2~4 1 ~1 W094/02~g5 PCT/GB93/01545 ~ne methoa 'or _repari-.g G suitaDle reaction mixture -s _o prepare an aaueous soiu__cn cf the ~C or amine and the oxyaci~ sait and subseauently to remove ~ater rom ~he solution. Since water -s a product of the esterificatisn - reac~ion, it is clear f-cm application of Le Chateiier's ?rincipie that the Leu.o~al of water f_om the mixture will ~end to shif~ .he eauilibrium in favour of ester .ormation and therefore, -his method of carrying out the -eaction is part_cuiar~y ~referred.
.his method of 'ormina the reaction mixture works particularly well if the water in the ~:C/oxyacid salt soiution is initiaily present in considerable excess (for example, at ~east 2, 5, 10, 20, 50 or 100 fold (on a :_ weicht basis). ~uring .:~e water ~e-,~oval step, most of ~he water is removed, so as to leave, say, less than one ~art (for example f_om 0.1 to 30~ w/w), based on the start~ng amount of ~C -r amine. As will be recocnised by ~hose skiiled in the ar-, a balance has to be struck ~~ between considerations c- yieid, which favour eu.~dl of a large amount of water, and considerations of time and energy expenditure, which favours the c~nverse. The optimal piace to draw _~.e h~l ~nce wiil doubtless vary with the nature of the reactants and the operator~s 2, process conditions.
~e wa~er can ~e removea -y any aDpropria~e physicai or _hemical me~hod. rxamp~es cf sui.able physical methods, ~rich are ~ene-~ily ~re~er_ed, are evaporation, -vapora~ic. unae- reduce~ pressure ~nd ~reeze drylng.
Conslder .i~ns of energy consumpel~n and tlme will often ~_c-_,e .he best ln any p rti~ular circums~ances.
A~ ~Lt_rnatL~- ~e~hct f~r prepGrlng ~he --acti~r. ~l~urG
W094/0~95 - 214 1 3~ l PCT/GB93/01~
is to dissolve the HC _r amine and the oxyacid or oxyacid salt in an organic soivent which may also contain water in an amount of up tO 3 0~ (W/W) based on the starting amount of HC or amine.
It is important that ;he oxyacid salt is soluble in the chosen solvent and therefore, a polar solvent such as dimethylsulphoxide snould generally be used.
It has been found that in many cases the process is more efficient if the reaction mixture contains water in an amount of from 0.1 to 1~ w/w of the HC.
The reaction can be carried out at almost any pH but a preferred pH range is from 1 to 9 and the best results are obtained when the pH is from 2 to 6. However, one advantage of the reacti~n is that it does work well under mild conditions of pH, for example from pH 4.5 to 6 and this can be particularly important when the HC or the product ester is sensitive to highly acidic conditions.
For reactions cf amines, the pH will preferably be greater than 7 in order to ensure that a free amine exists in the reaction mixture. Generally, for reaction of amines, the pH will be between 7.5 and 9, although use of pH of 10 or even higher may be necessary in some cases.
Any hydroxylated compound may be esterified or any amine amidated by this methoa but, in particular, the method is _0 sui~aDle lor the este~~fication of organic compounds such as: sugar~, -articularly non-reducing sugars such as trehaiose and suc-ose ~nd reduclng-sugars such as lactose and maitose; ?rotei~s; giycoproteins, oeptides, glycooeo~ldes ana clycocon~ugates and other ~oiecules 21~1 ~ 3~ i W094~0~gS ~ PCT/GB93/01545 having sugar functicnalityi amino acids, partic~iarly, serine and threonine; and other organic compounds such as alditols and cycli.ols, carboxylic acids, organic oxyacids, particulariy phosphates, and compounds containing hydroxyi groups or alkoxy or phenoxy anions.
The invention has application to polysaccharides other than starch, oligosaccharides (say with 3 to 10 monosaccharide units) and di- and mono-sacchariaes.
Inorganic hydroxylatea compounds which may be esterified by the method of ~he invention include oxyacids themselves and, for example, the esterification reaction of the invention is of use for the synthesis of di-, tri-and poly-phosphates.
In general, the reaction is most successful for primary and secondary UCs and amines, although it is possible to esterify tertiary derivatives, particularly _f the subst.tuents are not __o bulky.
After the f~rmation cf .he starting mixture, it may then be further treatea .o m~ i se the esterification reaction. In some cases, it may be preferable to heat the reaction mixture tcr a time between 0.1 hour and 40 days at a temperature of from 20 to 200C, preferably So to 100C. The heatir.a may be carried out in a sealed system, parricularly if a high temperature is used and will increase the rate cf the esterification reaction.
Since wate- is a proauct of the esterification process, _emovai or wa~er ~rom t~e system as the reaction proceeas will cause ~he eauil blium tO be shifted so _hat more ester is fcrme¢ frcm ~he reaction. One way ~f removing the water wouid e ~ heat the mixture ln an unseale¢
svs~em allcwln~ wate- ~ eva~orate.
W094/024~ ~ 14 13 ~ ~ PCT/GB93iO1545 Once the reaction is complete, the ester or amidate products may be isolated by any suitable method: by ion ~xch~nge chromatography, for example. If more than one reaction product is oDtained, it is generally easy to separate the different products by chromatography.
Alternatively, the ester may be left in the resulting mixture and treatea or allowed to react further to produce a co---yound, or mixture, of choice.
The process of the present invention has made it possible to obtain new esters and amidates which could not be prepared by any previously known route. In particular, it is now possible to prepare esters of acid-labile sugars such as sucrose and trehalose and therefore, in further aspects of -he in~ention, there are pro~ided trehalose-2-phospAate, ~rehalose-3-phosphate, trehalose-4-phosphate and a number of mono-orthophosphates of sucrose. These compounds are new and could only have been prepared by ?reviously known methods with considerable aiff~culty and cost.
The invention is further illustrated by the following examples. The examples refer to the accompanying drawings, in which:
2~
FIGURE 1 is a 'H nmr spectrum of trehalose-6-phosphate obtained from an orthophosphate salt by the meehod of the present invention;
'O -IGURE 2 is a ~H nmr spectrum of trehalose-2-phosphate _btained from an cr~.ophosphate salt by the method of ~he present in~ent~on;
FIG'~RE ~ is G 'H nmr spectrum or t_ehalose-4-phosphate 2 1~ /3~
W094~/02495 PCT/GB93/01~5 obtained from an ort;opnosphate salt by the methoa ~- -.he present invention;
-IGURE 4 is a iH nmr spect~um of trehalose-3-pnosphate obtained from an orthophosphate salt by the method of the present invention;
FIGURE 5 is a HPLC _on-exchange chromatogram oDtained from a sample of ~,a'-trehalose treated with sodium iO phosphate according ~o the method of the ?resen~
invention.
I
FIGURE 6 is a lH nmr spectrum of trehalose-3-ohospAate obtained from a metapnosphate salt by the method of the present invention.
~IGURE 7 is a lH nmr spectrum of trehalose-6-ohospnate obtained from a metaphosphate salt by the method of ~he present invention.
~xample l The preparation of the four isomeric mono-orthopilospAates of ~,a'-trehaiose.
An aaueous solution of ~,a'-trehalose dihydrate (l.Og, 2.5 mmole) in sodium ?hosphate buffer solution pH 5.5 (O.lM, lO0 ml), in wnich the water is present in an approximately ~00 fcld excess, on a weight basls, 0 com~ared to the sugar, is frozen to -78C ~solid C0~) and is then f-eeze-dried to give a ~reparation with a mois~ure content (determined by Karl-Fischer tL~rationj of 4._% w/w with respect ~o treAalose. The prepara.l~n, closed to the atmosp~.e~e, is heatea a. 56C for lO aavs W094/0~95 2 1~ ~ /01545 after ~hich it is reconst-tuted in deionised water (lO0 mi) and the solution aDplied to an anion-~ch~nged c~lumn, 120 x 2 cm, 3io-Rad AG 1 resin which nad been washed successively with 1~ sodium hydroxide, water, lM
sodium acetate, water and is therefore in the acetate form. The expression Bio-Rad AG 1 is a trade mark. The column was first washed with water to remove unreacted trehalose (for recycling if so desired) and any other un~ound material, and .hen eluted with a 0.2 to 0.8M
~mmo~ ium acetate aqueous solution gradient to give fractions containing th~ isomeric trehalose mono-orthophosphates separated from inorganic orthophosphate.
The fractions cont~in;ng the trehalose phosphates were then analysed on an analytical ion-~xch~nge column (Dionex BioLC PA 100) and the fractions containing each of the four pure isomers were combined and their solutions freeze-dried to give as analytically pure amorphous solids, ~rehalose monophosphates as their ammonium salts. The expression Dionex BioLC PA iOO is a .rade mark. Fraction , 0.085 g (6.6%), fraction 2, 0.035g ~2.7%), fraction 3, 0.041g (3.1%), fraction 4, 0.027% (2.1%). Total 0.188g (14.6~). All four products gave, on treatment with the enzyme alkaline phosphates, ~,~'-trehalose (identified by HPLC ion-~x~h~nge chromatograpAy) and inorganic orthophosphate (identified colorimetrically using ammonium molybdate).
Elemental analysis data:
.his method of 'ormina the reaction mixture works particularly well if the water in the ~:C/oxyacid salt soiution is initiaily present in considerable excess (for example, at ~east 2, 5, 10, 20, 50 or 100 fold (on a :_ weicht basis). ~uring .:~e water ~e-,~oval step, most of ~he water is removed, so as to leave, say, less than one ~art (for example f_om 0.1 to 30~ w/w), based on the start~ng amount of ~C -r amine. As will be recocnised by ~hose skiiled in the ar-, a balance has to be struck ~~ between considerations c- yieid, which favour eu.~dl of a large amount of water, and considerations of time and energy expenditure, which favours the c~nverse. The optimal piace to draw _~.e h~l ~nce wiil doubtless vary with the nature of the reactants and the operator~s 2, process conditions.
~e wa~er can ~e removea -y any aDpropria~e physicai or _hemical me~hod. rxamp~es cf sui.able physical methods, ~rich are ~ene-~ily ~re~er_ed, are evaporation, -vapora~ic. unae- reduce~ pressure ~nd ~reeze drylng.
Conslder .i~ns of energy consumpel~n and tlme will often ~_c-_,e .he best ln any p rti~ular circums~ances.
A~ ~Lt_rnatL~- ~e~hct f~r prepGrlng ~he --acti~r. ~l~urG
W094/0~95 - 214 1 3~ l PCT/GB93/01~
is to dissolve the HC _r amine and the oxyacid or oxyacid salt in an organic soivent which may also contain water in an amount of up tO 3 0~ (W/W) based on the starting amount of HC or amine.
It is important that ;he oxyacid salt is soluble in the chosen solvent and therefore, a polar solvent such as dimethylsulphoxide snould generally be used.
It has been found that in many cases the process is more efficient if the reaction mixture contains water in an amount of from 0.1 to 1~ w/w of the HC.
The reaction can be carried out at almost any pH but a preferred pH range is from 1 to 9 and the best results are obtained when the pH is from 2 to 6. However, one advantage of the reacti~n is that it does work well under mild conditions of pH, for example from pH 4.5 to 6 and this can be particularly important when the HC or the product ester is sensitive to highly acidic conditions.
For reactions cf amines, the pH will preferably be greater than 7 in order to ensure that a free amine exists in the reaction mixture. Generally, for reaction of amines, the pH will be between 7.5 and 9, although use of pH of 10 or even higher may be necessary in some cases.
Any hydroxylated compound may be esterified or any amine amidated by this methoa but, in particular, the method is _0 sui~aDle lor the este~~fication of organic compounds such as: sugar~, -articularly non-reducing sugars such as trehaiose and suc-ose ~nd reduclng-sugars such as lactose and maitose; ?rotei~s; giycoproteins, oeptides, glycooeo~ldes ana clycocon~ugates and other ~oiecules 21~1 ~ 3~ i W094~0~gS ~ PCT/GB93/01545 having sugar functicnalityi amino acids, partic~iarly, serine and threonine; and other organic compounds such as alditols and cycli.ols, carboxylic acids, organic oxyacids, particulariy phosphates, and compounds containing hydroxyi groups or alkoxy or phenoxy anions.
The invention has application to polysaccharides other than starch, oligosaccharides (say with 3 to 10 monosaccharide units) and di- and mono-sacchariaes.
Inorganic hydroxylatea compounds which may be esterified by the method of ~he invention include oxyacids themselves and, for example, the esterification reaction of the invention is of use for the synthesis of di-, tri-and poly-phosphates.
In general, the reaction is most successful for primary and secondary UCs and amines, although it is possible to esterify tertiary derivatives, particularly _f the subst.tuents are not __o bulky.
After the f~rmation cf .he starting mixture, it may then be further treatea .o m~ i se the esterification reaction. In some cases, it may be preferable to heat the reaction mixture tcr a time between 0.1 hour and 40 days at a temperature of from 20 to 200C, preferably So to 100C. The heatir.a may be carried out in a sealed system, parricularly if a high temperature is used and will increase the rate cf the esterification reaction.
Since wate- is a proauct of the esterification process, _emovai or wa~er ~rom t~e system as the reaction proceeas will cause ~he eauil blium tO be shifted so _hat more ester is fcrme¢ frcm ~he reaction. One way ~f removing the water wouid e ~ heat the mixture ln an unseale¢
svs~em allcwln~ wate- ~ eva~orate.
W094/024~ ~ 14 13 ~ ~ PCT/GB93iO1545 Once the reaction is complete, the ester or amidate products may be isolated by any suitable method: by ion ~xch~nge chromatography, for example. If more than one reaction product is oDtained, it is generally easy to separate the different products by chromatography.
Alternatively, the ester may be left in the resulting mixture and treatea or allowed to react further to produce a co---yound, or mixture, of choice.
The process of the present invention has made it possible to obtain new esters and amidates which could not be prepared by any previously known route. In particular, it is now possible to prepare esters of acid-labile sugars such as sucrose and trehalose and therefore, in further aspects of -he in~ention, there are pro~ided trehalose-2-phospAate, ~rehalose-3-phosphate, trehalose-4-phosphate and a number of mono-orthophosphates of sucrose. These compounds are new and could only have been prepared by ?reviously known methods with considerable aiff~culty and cost.
The invention is further illustrated by the following examples. The examples refer to the accompanying drawings, in which:
2~
FIGURE 1 is a 'H nmr spectrum of trehalose-6-phosphate obtained from an orthophosphate salt by the meehod of the present invention;
'O -IGURE 2 is a ~H nmr spectrum of trehalose-2-phosphate _btained from an cr~.ophosphate salt by the method of ~he present in~ent~on;
FIG'~RE ~ is G 'H nmr spectrum or t_ehalose-4-phosphate 2 1~ /3~
W094~/02495 PCT/GB93/01~5 obtained from an ort;opnosphate salt by the methoa ~- -.he present invention;
-IGURE 4 is a iH nmr spect~um of trehalose-3-pnosphate obtained from an orthophosphate salt by the method of the present invention;
FIGURE 5 is a HPLC _on-exchange chromatogram oDtained from a sample of ~,a'-trehalose treated with sodium iO phosphate according ~o the method of the ?resen~
invention.
I
FIGURE 6 is a lH nmr spectrum of trehalose-3-ohospAate obtained from a metapnosphate salt by the method of the present invention.
~IGURE 7 is a lH nmr spectrum of trehalose-6-ohospnate obtained from a metaphosphate salt by the method of ~he present invention.
~xample l The preparation of the four isomeric mono-orthopilospAates of ~,a'-trehaiose.
An aaueous solution of ~,a'-trehalose dihydrate (l.Og, 2.5 mmole) in sodium ?hosphate buffer solution pH 5.5 (O.lM, lO0 ml), in wnich the water is present in an approximately ~00 fcld excess, on a weight basls, 0 com~ared to the sugar, is frozen to -78C ~solid C0~) and is then f-eeze-dried to give a ~reparation with a mois~ure content (determined by Karl-Fischer tL~rationj of 4._% w/w with respect ~o treAalose. The prepara.l~n, closed to the atmosp~.e~e, is heatea a. 56C for lO aavs W094/0~95 2 1~ ~ /01545 after ~hich it is reconst-tuted in deionised water (lO0 mi) and the solution aDplied to an anion-~ch~nged c~lumn, 120 x 2 cm, 3io-Rad AG 1 resin which nad been washed successively with 1~ sodium hydroxide, water, lM
sodium acetate, water and is therefore in the acetate form. The expression Bio-Rad AG 1 is a trade mark. The column was first washed with water to remove unreacted trehalose (for recycling if so desired) and any other un~ound material, and .hen eluted with a 0.2 to 0.8M
~mmo~ ium acetate aqueous solution gradient to give fractions containing th~ isomeric trehalose mono-orthophosphates separated from inorganic orthophosphate.
The fractions cont~in;ng the trehalose phosphates were then analysed on an analytical ion-~xch~nge column (Dionex BioLC PA 100) and the fractions containing each of the four pure isomers were combined and their solutions freeze-dried to give as analytically pure amorphous solids, ~rehalose monophosphates as their ammonium salts. The expression Dionex BioLC PA iOO is a .rade mark. Fraction , 0.085 g (6.6%), fraction 2, 0.035g ~2.7%), fraction 3, 0.041g (3.1%), fraction 4, 0.027% (2.1%). Total 0.188g (14.6~). All four products gave, on treatment with the enzyme alkaline phosphates, ~,~'-trehalose (identified by HPLC ion-~x~h~nge chromatograpAy) and inorganic orthophosphate (identified colorimetrically using ammonium molybdate).
Elemental analysis data:
3 Frac~icn 1, ~ound C 31.55%, H 6.62%, N 5.81%, P 6.45%.
Fracticn a, ~cund C 31.22%, H 6.26%, N 5.97%, P 6.39%.
Fracticn 3, ~cund C ~1.52%, H 6.55%, N 6.20%, P 5.55%.
~racti_n 4, ~und C ~1.37%, H 6.32%, N 5.92%, P ~.89~.
21L1130~
W094/0~9~ PCT/GB93/01545 C,2~.9N2P~OI4 requires C 31.-8~, ~ 6.40~, N 6.14~, P 6.79%.
NMR data. The proton nmr spectra of the products present in fractions 1, 2, 3 and are shown in Figures 1 .o 4 and are consistent with the 6-, 2-, 4- and ~he 3-pAosphates respectively, i.e structures 1, 2, 3 and ~
shown in the 'igures. ~igure ' is identicai to the proton nmr spectr~m of trehalose-6-phosphate - data from ~0 Sigma Chemical Co. Confirmation of these structures was also obtained from 13C and 31p nmr spectroscopy.
~xam~le 2 Using conaitions similar to .hose of Example ~, ~ut modified as shown in the six entries (a) to ~f) 'or _xample 2 in Table 1, phosphate esters of ~,~'-t-ehaiose were prepared from a,~ -ehalose.
Exam~le 3 The eight isomeric mono-orthophosphates of sucrose can be prepared by a similar proceàure to that of Example 1, using ~he conditions shown in Table 1. Of major note in this case, however, is the 'act that sucrose is an e~t-emely acid labiie disaccharide and consequently ls not ~me~hie ~o direc- phosphorylation with phospnor~c acid. As with 'hree o- the trehaiose phospAates above, 3~ the majority of the sucrose isomeric esters are new ~ompounds ana, furthermore, would be very difficul~ _o p~epare usir.~ existin~ me~hods.
W094/02495 214 13~ / PCT/GB93/01545 - Exam~les ~ to 7 In an analogous m~ner a-glycerophosphate;
serine phosphate;
~-cyclodextrin t~iosulphates; and serine thiophospAate, 0 were aiso prepared using the conditions shown in Table l.
Further experiments were carried out to investigate the effects or sodium phosphate buffer on several .~Cs. ~he experiments were carried out in solutions of pH 5.5, 7.0 or 8.' and all gave, after heating at 56C for several days, products which co-eluted with carbohydrate monophosphate esters on ion-exchange chromatography. The results of these experiments are shown in Table l.
~0 A number of experimen~s have also been carriea out ~o investigate the effects of other oxyacid salt buffers on several HCs at pH 2.0-6Ø After heating at ~56C for several days "ester-type" products are observed on ion-exchanae chromatograpAy. The oxyacid buffers which were used include those de-ived from boric, thiophosphoric, phosphonic and phosphinic acids.
WO 94/02495 ` ~- - PCI-/GB93/01545 C~ ~
O U~ ~ N t` ~ ~ m 0 tD m m ~ m m a~ m 0 ~ 1~ ~ r ~ m CD
~ .
o ' U~
O O ~ 1~1 0 0 ~ O ~0 O O O N 111 ~ ~1 0 ~- ~1 m ~ ~ ~ ~ o~ o r o ~ ~ ~ o - ~~ ~ ~r ~ r~ Or~l ~ O ~ m u~
Ul O ~ O ~1 Ul O ~ O rl U~ U~ Ul Ul m ul o ...... ... ... ... . .. .. ..
r~ o u7 ~ r 0 u~
~
0~ u~ 0~ .o~ u ~.a u ~ u 0.a ~s.a o~
_l -I n~
, _ _ r ~/ ' ' ' ' ' I ' _' ' O O O O ~ 00 0 o I _ _I _ _ I ~~ I _ ~ _ -O O
~ ~
t~ r `.D
O
Il~ O ~1 ~ O O
C ~ ~ ~
a ~ c ~ ~ ~ .
O
D.
o ~ r~- ~ Ul ~D r ro Z
SUBSTITUTE SHEET
WO 94/02495 21 g 3 01 PCI`/GB93/01545 19 ' .
-O O ~ 1 Dl . 0 o 0 ~J g 1~ Q r ~ ~ ~ ~ ~ ~ r~ S ~ ~ D~
D~ 1 N t' ~ O
, ~ E
O ~ ,.1 ~ ~ ~ '` ~ O
~ r~ O ~D m . ~ ~
m ~ o~ --o ~ ~ ~ o n r o nJ
---- ~ ~ 0 o o In _ ~
.~ ~ r~ o~ ~ ~ n o ~ O O O O ~ O N nJ
.. .. .. .
O O O 0~0 0 0 o D , "
r '.~
" C ''I O
~ ~ ~ n~ D ~, ,' ~ o o o u~ u~ r r m r ~ o O c ~:
0 ~
D, 1~1 ~C ' ~ ~
0 ,.
0 .11 0 .a 0.a 0~ s 0 ,., rl Dl 0 D' ~ Dl nJ .a E
,, , n, , ~ ' ' ~ O ~ 'r L~
~ ~ ' aJ C a~ E
..-- _ _ _ _ -- ~1 ' I I
r-l O O O O O O ~ ~ ~ -E~ .' . ~ ..
r _~,, L~ , o ,~ W
.u o -- a u ~,.
. a~ - .t _c.~ ~E O
-- -- C ' ' o ~
o ~ o,~ ~ ~ ~ ~ . Il Z r~, 1 r ~ r~ r ~ W _ ~ r~,~
SUBSTITUTE SHEET
~2`il~i301 , ~
W094/02495 - ~-~ PCT/GB93/01545 It can herefcre be seen that the esterification methoa of the inven~ion is extremely flexible and can be used to esteri-y a iarge -ange of compounds under relati~eiy mild conditions. In addition, the starting materials can be recycled so that increased amounts of product can be o~tained.
Exam~le 8 o Formation of phosphate esters from preparations of trehalose and sodium metarhosphate.
The procedure of Example l was repeated except that ~,a'-trehalose dihydrate (l.0 g, 2.6 mmol) was dissolved in a lS solution of sodium metaphosphate (O.l M, lO0 ml). The preparation is frozen to -78 as before, freeze aried, closed to the atmosphere and heated at 56C for either S
days or '2 days. The reaction was repeated at different pH values and the results are shown in Table 2 below.
^o W094/02495 21113 ~ I PCT/GB93/0l545 TA~3LE 2 pH (inltial) ~ monophosphate % monophosphate 5 days i2 days 1.9 32 34(7) 3.0 18 23(6) 3.5 15 20(4) 4.0 13 19(4) 4.5 17 15(4) 5.0 13 16(4) 5.5 13 17(4) o 6.0 15 12(3) 6.7 11 7.2 9 8.0 8.5 3.3 3.4 9.9 2.6' after 7 days The _igures in parentheses are the percentages of 2 o diphosphates formed.
The percentage of phosphate shown in Table 2 is determined from detector response and is therefore an underestimate, ?ossibly by a factor as high as 2.
T~e amount of phosphate formea will depend ~pon the moistur~ content and on the temoerature at wnich the CtiQn LS car-ied cut. In seve-ai seoarate ex~eriments car-~ea cut at higher t-moerature DUt 'or shorter times, -~ 3 the yield cf pnosphates i-.creasea to about 40% based on 21$1301 W0~4/024~ PCT/GB93/01545 detectc~ ~esponse. Again, ~his is an underestimate and the ~rue value may be as much as twice the aetect~
response vaiue.
Exam~le c Reaction of a,a~-trehalose i~ DNSO.
o a) with orthophosphate ~,a'-lrehaiose dihydrate (1 part), disodium hydrocen crthophospAate and sodium dihydrogen orthophosphate ~4 par~s), (the salts mixed in proportions so as to produce a pH cf 5.5 when in aqueous solution) were a-dded to ary DMSO ~100 parts). The mixture was heated at 80C for ' days, and the DMSO was then el~-oved by lyophiiisation.
The _esidue was dissolved in water (100 parts) and analysed chromatographically (ion-exch~nge). The mixture ^o containea about 1% of trehalose phosphates. ~he -eiatively low yield is probably due to the _oor solubilily of the sodium salts in DMSO.
b) with met~h~sphate ,5 ~-~'-T-enalose dihydrate (1 part), sodium metapnospAate/metaphosphoric acid (4 parts), mixed so as ~o gi~e a pH of 2 if in aqueous solution, were addea t~
dry DMSO \100 parts) and the mixt~re heated at 80C for ~o ~ days. ~he DMSO was removed and the residue analysed as descri~ed above. The mixture c~ntained 1.5% of ~-enalose ~nosprat2s. T~e reLativeiy lcw yleld is probably due ~e ~c~r solubilit~ of ~he sodium salt in DMSO,~
Fracticn a, ~cund C 31.22%, H 6.26%, N 5.97%, P 6.39%.
Fracticn 3, ~cund C ~1.52%, H 6.55%, N 6.20%, P 5.55%.
~racti_n 4, ~und C ~1.37%, H 6.32%, N 5.92%, P ~.89~.
21L1130~
W094/0~9~ PCT/GB93/01545 C,2~.9N2P~OI4 requires C 31.-8~, ~ 6.40~, N 6.14~, P 6.79%.
NMR data. The proton nmr spectra of the products present in fractions 1, 2, 3 and are shown in Figures 1 .o 4 and are consistent with the 6-, 2-, 4- and ~he 3-pAosphates respectively, i.e structures 1, 2, 3 and ~
shown in the 'igures. ~igure ' is identicai to the proton nmr spectr~m of trehalose-6-phosphate - data from ~0 Sigma Chemical Co. Confirmation of these structures was also obtained from 13C and 31p nmr spectroscopy.
~xam~le 2 Using conaitions similar to .hose of Example ~, ~ut modified as shown in the six entries (a) to ~f) 'or _xample 2 in Table 1, phosphate esters of ~,~'-t-ehaiose were prepared from a,~ -ehalose.
Exam~le 3 The eight isomeric mono-orthophosphates of sucrose can be prepared by a similar proceàure to that of Example 1, using ~he conditions shown in Table 1. Of major note in this case, however, is the 'act that sucrose is an e~t-emely acid labiie disaccharide and consequently ls not ~me~hie ~o direc- phosphorylation with phospnor~c acid. As with 'hree o- the trehaiose phospAates above, 3~ the majority of the sucrose isomeric esters are new ~ompounds ana, furthermore, would be very difficul~ _o p~epare usir.~ existin~ me~hods.
W094/02495 214 13~ / PCT/GB93/01545 - Exam~les ~ to 7 In an analogous m~ner a-glycerophosphate;
serine phosphate;
~-cyclodextrin t~iosulphates; and serine thiophospAate, 0 were aiso prepared using the conditions shown in Table l.
Further experiments were carried out to investigate the effects or sodium phosphate buffer on several .~Cs. ~he experiments were carried out in solutions of pH 5.5, 7.0 or 8.' and all gave, after heating at 56C for several days, products which co-eluted with carbohydrate monophosphate esters on ion-exchange chromatography. The results of these experiments are shown in Table l.
~0 A number of experimen~s have also been carriea out ~o investigate the effects of other oxyacid salt buffers on several HCs at pH 2.0-6Ø After heating at ~56C for several days "ester-type" products are observed on ion-exchanae chromatograpAy. The oxyacid buffers which were used include those de-ived from boric, thiophosphoric, phosphonic and phosphinic acids.
WO 94/02495 ` ~- - PCI-/GB93/01545 C~ ~
O U~ ~ N t` ~ ~ m 0 tD m m ~ m m a~ m 0 ~ 1~ ~ r ~ m CD
~ .
o ' U~
O O ~ 1~1 0 0 ~ O ~0 O O O N 111 ~ ~1 0 ~- ~1 m ~ ~ ~ ~ o~ o r o ~ ~ ~ o - ~~ ~ ~r ~ r~ Or~l ~ O ~ m u~
Ul O ~ O ~1 Ul O ~ O rl U~ U~ Ul Ul m ul o ...... ... ... ... . .. .. ..
r~ o u7 ~ r 0 u~
~
0~ u~ 0~ .o~ u ~.a u ~ u 0.a ~s.a o~
_l -I n~
, _ _ r ~/ ' ' ' ' ' I ' _' ' O O O O ~ 00 0 o I _ _I _ _ I ~~ I _ ~ _ -O O
~ ~
t~ r `.D
O
Il~ O ~1 ~ O O
C ~ ~ ~
a ~ c ~ ~ ~ .
O
D.
o ~ r~- ~ Ul ~D r ro Z
SUBSTITUTE SHEET
WO 94/02495 21 g 3 01 PCI`/GB93/01545 19 ' .
-O O ~ 1 Dl . 0 o 0 ~J g 1~ Q r ~ ~ ~ ~ ~ ~ r~ S ~ ~ D~
D~ 1 N t' ~ O
, ~ E
O ~ ,.1 ~ ~ ~ '` ~ O
~ r~ O ~D m . ~ ~
m ~ o~ --o ~ ~ ~ o n r o nJ
---- ~ ~ 0 o o In _ ~
.~ ~ r~ o~ ~ ~ n o ~ O O O O ~ O N nJ
.. .. .. .
O O O 0~0 0 0 o D , "
r '.~
" C ''I O
~ ~ ~ n~ D ~, ,' ~ o o o u~ u~ r r m r ~ o O c ~:
0 ~
D, 1~1 ~C ' ~ ~
0 ,.
0 .11 0 .a 0.a 0~ s 0 ,., rl Dl 0 D' ~ Dl nJ .a E
,, , n, , ~ ' ' ~ O ~ 'r L~
~ ~ ' aJ C a~ E
..-- _ _ _ _ -- ~1 ' I I
r-l O O O O O O ~ ~ ~ -E~ .' . ~ ..
r _~,, L~ , o ,~ W
.u o -- a u ~,.
. a~ - .t _c.~ ~E O
-- -- C ' ' o ~
o ~ o,~ ~ ~ ~ ~ . Il Z r~, 1 r ~ r~ r ~ W _ ~ r~,~
SUBSTITUTE SHEET
~2`il~i301 , ~
W094/02495 - ~-~ PCT/GB93/01545 It can herefcre be seen that the esterification methoa of the inven~ion is extremely flexible and can be used to esteri-y a iarge -ange of compounds under relati~eiy mild conditions. In addition, the starting materials can be recycled so that increased amounts of product can be o~tained.
Exam~le 8 o Formation of phosphate esters from preparations of trehalose and sodium metarhosphate.
The procedure of Example l was repeated except that ~,a'-trehalose dihydrate (l.0 g, 2.6 mmol) was dissolved in a lS solution of sodium metaphosphate (O.l M, lO0 ml). The preparation is frozen to -78 as before, freeze aried, closed to the atmosphere and heated at 56C for either S
days or '2 days. The reaction was repeated at different pH values and the results are shown in Table 2 below.
^o W094/02495 21113 ~ I PCT/GB93/0l545 TA~3LE 2 pH (inltial) ~ monophosphate % monophosphate 5 days i2 days 1.9 32 34(7) 3.0 18 23(6) 3.5 15 20(4) 4.0 13 19(4) 4.5 17 15(4) 5.0 13 16(4) 5.5 13 17(4) o 6.0 15 12(3) 6.7 11 7.2 9 8.0 8.5 3.3 3.4 9.9 2.6' after 7 days The _igures in parentheses are the percentages of 2 o diphosphates formed.
The percentage of phosphate shown in Table 2 is determined from detector response and is therefore an underestimate, ?ossibly by a factor as high as 2.
T~e amount of phosphate formea will depend ~pon the moistur~ content and on the temoerature at wnich the CtiQn LS car-ied cut. In seve-ai seoarate ex~eriments car-~ea cut at higher t-moerature DUt 'or shorter times, -~ 3 the yield cf pnosphates i-.creasea to about 40% based on 21$1301 W0~4/024~ PCT/GB93/01545 detectc~ ~esponse. Again, ~his is an underestimate and the ~rue value may be as much as twice the aetect~
response vaiue.
Exam~le c Reaction of a,a~-trehalose i~ DNSO.
o a) with orthophosphate ~,a'-lrehaiose dihydrate (1 part), disodium hydrocen crthophospAate and sodium dihydrogen orthophosphate ~4 par~s), (the salts mixed in proportions so as to produce a pH cf 5.5 when in aqueous solution) were a-dded to ary DMSO ~100 parts). The mixture was heated at 80C for ' days, and the DMSO was then el~-oved by lyophiiisation.
The _esidue was dissolved in water (100 parts) and analysed chromatographically (ion-exch~nge). The mixture ^o containea about 1% of trehalose phosphates. ~he -eiatively low yield is probably due to the _oor solubilily of the sodium salts in DMSO.
b) with met~h~sphate ,5 ~-~'-T-enalose dihydrate (1 part), sodium metapnospAate/metaphosphoric acid (4 parts), mixed so as ~o gi~e a pH of 2 if in aqueous solution, were addea t~
dry DMSO \100 parts) and the mixt~re heated at 80C for ~o ~ days. ~he DMSO was removed and the residue analysed as descri~ed above. The mixture c~ntained 1.5% of ~-enalose ~nosprat2s. T~e reLativeiy lcw yleld is probably due ~e ~c~r solubilit~ of ~he sodium salt in DMSO,~
Claims (22)
1. A process for the esterification of a hydroxylated compound (HC) other than a starch, or the amidation of an organic amine, characterised in that the process comprises reacting the HC or amine with an oxyacid, oxyacid anion or a mixture thereof wherein the oxyacid or its anion is capable of accepting a lone pair of electrons from the oxygen of the HC or nitrogen of the amine.
2. A process as claimed in claim 1, wherein the oxyacid is an oxyacid of an element M, which is capable of forming trimeric oxyacids of the structure:
wherein:
M is an electron accepting element;
X is oxygen or a hetero-atom such as sulphur or a substituted nitrogen atom;
Y is oxygen, hydrogen or a hetero atom such as sulphur or a substituted nitrogen atom;
n is 0 or 1 and m is 0 to 2, depending on the valency of M.
wherein:
M is an electron accepting element;
X is oxygen or a hetero-atom such as sulphur or a substituted nitrogen atom;
Y is oxygen, hydrogen or a hetero atom such as sulphur or a substituted nitrogen atom;
n is 0 or 1 and m is 0 to 2, depending on the valency of M.
3. A process as claimed in claim 1 or claim 2, wherein M is phosphorous, boron or silicon.
4. A process as claimed in any one of claims 1 to 3, wherein the oxyacid species is derived from a metaphosphate, orthophosphate, diphosphate, triphosphate, polyphosphate, phosphonate, phosphinate, peroxyphosphate, hypophosphate, thiophosphate, halophosphate, phosphonitrilic, borate or silicate salt or the free acid thereof.
5. A process as claimed in any one of claims 1 to 4, comprising:
(a) providing a reaction mixture comprising the HC or amine; an oxyacid or oxyacid salt capable of producing in solution a species capable of accepting a lone pair of electrons from the oxygen of the HC or nitrogen of the amine; and from 0 to 30% (w/w) water based on the weight of the HC or amine;
(b) allowing the HC or amine and the oxyacid species to react to form a product mixture containing an oxyacid ester of the HC or amine; and optionally (c) either (i) at least partially recovering the ester from the product mixture or (ii) further reacting the ester in situ to form a desired compound or mixture.
(a) providing a reaction mixture comprising the HC or amine; an oxyacid or oxyacid salt capable of producing in solution a species capable of accepting a lone pair of electrons from the oxygen of the HC or nitrogen of the amine; and from 0 to 30% (w/w) water based on the weight of the HC or amine;
(b) allowing the HC or amine and the oxyacid species to react to form a product mixture containing an oxyacid ester of the HC or amine; and optionally (c) either (i) at least partially recovering the ester from the product mixture or (ii) further reacting the ester in situ to form a desired compound or mixture.
6. A process as claimed in claim 5, wherein the reaction mixture is prepared by forming an aqueous solution of the HC or amine and the oxyacid salt and removing water from the solution to form the starting mixture.
7. A process as claimed in claim 6, wherein the water is initially present in at least 2 fold excess, on a weight basis, compared to the starting amount of HC or amine.
8. A process as claimed in claim 6 or claim 7, wherein the water is removed by evaporation, evaporation under reduced pressure or freeze-drying.
9. A process as claimed in claim 5, wherein the reaction mixture is prepared by dissolving the HC or amine and the oxyacid salt in an organic solvent, wherein the organic solvent contains water in an amount of from 0 to 30% (w/w) based on the amount of HC or amine.
10. A process as claimed in claim 9, wherein the solvent is dimethyl sulphoxide.
11. A process as claimed in any one of claims 1 to 10, wherein the amount of water in the reaction mixture is not greater than 15% (w/w) based on the weight of the HC.
12. A process as claimed in any one of claims 1 to 11, wherein the pH of the mixture is from 1 to 9.
13. A process as claimed in claim 12 wherein the pH of the mixture is from 2 to 6.
14. A process as claimed in any one of the preceding claims wherein the HC comprises a sugar, a protein, a glycoprotein, a peptide, a glycopeptide, a glycoconjugate, an amino acid, an alditol or a cyclitol.
15. A process as claimed in claim 14, wherein the sugar is a reducing sugar.
16. A process as claimed in claim 14, wherein the sugar is a non-reducing sugar.
17. A process as claimed in claim 14, wherein the amino acid is serine or threonine.
18. A process as claimed in any one of claims 1 to 17, including the step of heating the mixture for from 0.1 hours to 40 days at a temperature from 20° to 200°C.
19. A process as claimed in claim 18, wherein the temperature is from 50° to 100°C.
20. A product mixture obtainable by a process as claimed in any one of claims 5 to 19.
21. A reaction mixture obtainable by a process as claimed in any one of claims 5 to 19.
21. A reaction mixture obtainable by a process as claimed in any one of claims 5 to 19.
21. A reaction mixture containing a hydroxylated compound or an organic amine, an oxyacid or oxyacid salt capable of producing, in solution, an oxyacid species which is capable of accepting a lone pair of electrons and water, wherein the water is present in an amount of from 0.1 to 30% (w/w) based on the weight of the HC or amine.
22. Trehalose-2-phosphate;
trehalose-4-phosphate;
trehalose-3-phosphate; or a mono-orthophosphate of sucrose.
trehalose-4-phosphate;
trehalose-3-phosphate; or a mono-orthophosphate of sucrose.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB929216026A GB9216026D0 (en) | 1992-07-28 | 1992-07-28 | Esterification process |
| GB9216026.6 | 1992-07-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2141301A1 true CA2141301A1 (en) | 1994-02-03 |
Family
ID=10719437
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002141301A Abandoned CA2141301A1 (en) | 1992-07-28 | 1993-07-22 | Esterification process |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP0652886A1 (en) |
| JP (1) | JPH07509457A (en) |
| AU (1) | AU4714793A (en) |
| CA (1) | CA2141301A1 (en) |
| FI (1) | FI950381A0 (en) |
| GB (1) | GB9216026D0 (en) |
| NO (1) | NO950321L (en) |
| WO (1) | WO1994002495A1 (en) |
| ZA (1) | ZA935408B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6146886A (en) | 1994-08-19 | 2000-11-14 | Ribozyme Pharmaceuticals, Inc. | RNA polymerase III-based expression of therapeutic RNAs |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2884412A (en) * | 1953-09-04 | 1959-04-28 | Int Minerals & Chem Corp | Phosphate-modified starches and preparation |
| US2865762A (en) * | 1954-09-16 | 1958-12-23 | Int Minerals & Chem Corp | Pudding mix |
| US2961440A (en) * | 1956-01-23 | 1960-11-22 | Corn Products Co | Process for preparing inorganic starch esters |
| US2884413A (en) * | 1956-04-06 | 1959-04-28 | Corn Products Co | Orthophosphate esters of starch |
| JPS62174094A (en) * | 1985-12-16 | 1987-07-30 | Ss Pharmaceut Co Ltd | Alpha, alpha-trehalose derivative and production thereof |
-
1992
- 1992-07-28 GB GB929216026A patent/GB9216026D0/en active Pending
-
1993
- 1993-07-22 CA CA002141301A patent/CA2141301A1/en not_active Abandoned
- 1993-07-22 AU AU47147/93A patent/AU4714793A/en not_active Abandoned
- 1993-07-22 EP EP93917889A patent/EP0652886A1/en not_active Withdrawn
- 1993-07-22 JP JP6504297A patent/JPH07509457A/en active Pending
- 1993-07-22 WO PCT/GB1993/001545 patent/WO1994002495A1/en not_active Application Discontinuation
- 1993-07-27 ZA ZA935408A patent/ZA935408B/en unknown
-
1995
- 1995-01-27 NO NO950321A patent/NO950321L/en unknown
- 1995-01-27 FI FI950381A patent/FI950381A0/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| AU4714793A (en) | 1994-02-14 |
| GB9216026D0 (en) | 1992-09-09 |
| NO950321D0 (en) | 1995-01-27 |
| NO950321L (en) | 1995-01-27 |
| EP0652886A1 (en) | 1995-05-17 |
| ZA935408B (en) | 1995-01-27 |
| JPH07509457A (en) | 1995-10-19 |
| FI950381A (en) | 1995-01-27 |
| WO1994002495A1 (en) | 1994-02-03 |
| FI950381A0 (en) | 1995-01-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Khorana | Carbodiimides. Part V. 1 A Novel Synthesis of Adenosine Di-and Triphosphate and P1, P2-Diadenosine-5'-pyrophosphate | |
| Turner et al. | Studies on Polynucleotides. VI. 1 Experiments on the Chemical Polymerization of Mononucleotides. Oligonucleotides Derived from Thymidine-3'Phosphate2 | |
| Moffatt et al. | Nucleoside Polyphosphates. VIII. 1 New and Improved Syntheses of Uridine Diphosphate Glucose and Flavin Adenine Dinucleotide Using Nucleoside-5'Phosphoramidates2 | |
| Weimann et al. | Studies on Polynucleotides. XVII. 1 On the Mechanism of Internucleotide Bond Synthesis by the Carbodiimide Method 2 | |
| Takanami | Analysis of the 5′-terminal nucleotide sequences of ribonucleic acids: I. The 5′-termini of Escherichia coli ribosomal RNA | |
| Danilov et al. | A simple procedure for preparing dolichyl monophosphate by the use of POCl3 | |
| Esquerré-Tugayé et al. | Effect of a fungal disease on extensin, the plant cell wall glycoprotein | |
| Weigel et al. | Preparation of 6-aminohexyl D-aldopyranosides | |
| Cook et al. | Nucleoside S-alkyl phosphorothioates. II. Preparation and chemical and enzymic properties | |
| LE DUR et al. | Novel Type of Endotoxin Structure Present in Bordetella pertussis: Isolation of Two Different Polysaccharides Bound to Lipid A | |
| Handschuh et al. | The effect of Mg 2+ and Ca 2+ on urea-catalyzed phosphorylation reactions | |
| Yamanaka et al. | Condensation of oligoglycines with trimeta-and tetrametaphosphate in aqueous solutions | |
| CA2141301A1 (en) | Esterification process | |
| SAWAI et al. | Synthesis of 2'-5'linked Oligouridylates in aqueous medium using the Pd2+ Ion | |
| Chambers et al. | Nucleoside Polyphosphates. V. 1 Syntheses of Guanosine 5'-Di-and Triphosphates | |
| Chambers et al. | Synthesis of Cytidine 5'-Diphosphate and Guanosine 5'-Diphosphate1 | |
| Tarelli et al. | The preparation of the four monophosphates of α, α′-trehalose from trehalose and sodium phosphate | |
| Richard et al. | Stereochemical course of phosphoanhydride synthesis | |
| Yount | [56] Adenylylimidodiphosphate and guanylylimidodiphosphate | |
| Bennett et al. | Guanosine tetraphosphate and its analogs. Chemical synthesis of guanosine 3', 5'-dipyrophosphate, deoxyguanosine 3', 5'-dipyrophosphate, guanosine 2', 5'-bis (methylenediphosphonate), and guanosine 3', 5'bis (methylenediphosphonate) | |
| Ikehara et al. | Studies on Coenzyme Analogs. XV. A Novel Phosphorylating Agent, P-Diphenyl-, P'-Morpholino Pyrophosphorochloridate. | |
| SANEYOSHI et al. | Synthetic Nucleosides and Nucleotides. VI. On the Several Routes for the Syntheses of 4-Thiouridylic Acid Homologues | |
| Ikehara et al. | Studies of Nucleosides and Nucleotides. XXI. A New Synthesis of Thymidine 5'-Triphosphate and the Use of P1, P2-Di-(2-cyanoethyl) pyrophosphate in the Nucleoside Triphosphate Synthesis. | |
| Chung et al. | Syntheses of all regioisomers of myo-inositol bisphosphate | |
| Szabó | Phosphorylated sugars. Part 25. Synthesis and behaviour in acidic media of 2-acetamido-2-deoxy-D-glucose 4-and 6-phosphates and of a ‘Lipid A’analogue |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Dead |