CA2107124A1 - Anticoagulants and processes for preparing such - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The present invention relates to anticoagulants prepared from the K5 saccharide of E. coli, which have good activity and which can be mass produced.

Description

W092/17507 21 Q 7 ~ 2 l~ PCT~GB92/00571 ANTICOAGULANTS AND PROCESSES FOR PR~PARING SUCH

F~ld of the In~entioa The present invention relates to multimeric compounds useful as anticoagulants, as well as to processe3 for their production. :

Prior.Ar~

Enzymatically modified poly~accharide~, con~isting of alternating D-glucuronic acid and N-acetyl-D-glucosamine units, have been exten~ively inYestigated in relation to the biosynthe~i~ o~ heparin and heparan sulphate (see, for instance ~HEPARIN - Chemical and biological properties, clinical application~n, D. Lane and U. :
Lindahl Editor~, published by Edward Arnold, pages : ;
159-l90, 19~9; and U. hindahl ~ al., TIBS, ll, May 1986, page 221). Such enzymic modi~ications in~olve the N-deacetylation of the glucosamine unit~, the subsequent N-~ulphation o~ the resulting free amino groups, C5-epimerisation of the D-glucuronate residues to : .
h-iduronate resldues, and O-sulphation at variou~
p~sitions (primarily at C-2 of the iduronic acids and .: .
C-6 of the glucosamine units). Additio~al enzymatic :~
O~sulphation may also a~fect the OH groups at the .: . -3-p~ition of the glucosamine residues~ : :
- . .. , -To date, it has only been p~s9ible to perform this se~uence of enzymatic ~tep o~ a ~icroscale basis, ~
suitable only ~or:experime~tal pu~poses, to mimic what .~.
happe~ in ~ammalian mast cell~ during the biosynthesis ;
o_ heparin and heparan sulphate. The chemieal and .
: ~ biolo~ical differences between heparin and heparan ~: .

, .
",.",.

W092/l750~ 21 Q 712 ~ PCT~CB92/0057l sulphate are illustrated in B. Casu et al., Arz.
Forsch., 33, 135, (1983).

The literature also describes methods for the N-deacetylation of N-acetylhexosamine residues present in polysaccharid~ molecules (L. Thunberg ~t al., Carbohydrate Res., lOQ, 393, [19~2] and Shaklee Qt al., 3iochem. J., 217, 187 [1984]), as well as procedures for N- and 0-sulphation (Levy et al., Proc. Soc. Exp. Biol.
Med., 109, 901 [1962]).

EP-A-333243 discloses compounds resulting from extensi~e sulphation of a K5 saccharide isolated from E. coli strain9.

Summary of the Inv~atiQn It is an object to pro~ide compounds prepared from K5 which have useful anticoagula~tJantithrombotic activity, and which can be produced on a lar~er scale than i9 provided by the art.

It is a further object to pro~ide anticoagulants/
antithrombotics prepar~d from the K5 saccharide of ~. coli, thereby allowing ma9S production o~ products having particularly good acti~ity.

The present invention provide~ deacetylated ~5 . coli saccharide, wherein th~ deacetylation amounts ~o at least 35~ of the acetyl group9 of naturally occur~ing KS.
: :
Briçf De~riptiQ~ ~he D~awi~

The accompanying drawing9 ~Fiyurèg 1 to 32) are the NMR ~pectra of ~arious compound9 oî the in~ention.
. . -"'~92/17507 210 7 ~ PCT/CB92/00~71 De~ailed Descrip~ion of the_In~ention The invention further provides the modified K5 saccharide as defined above, wherein sulphate groups are substituted in all, or sub~tantially all, of the positions on K5 which ha~e be~n deacetylated. Such positions include tho~e which would nonmally be expected to be acetylated, particularly the amine groups of the glucosamine, especially D-gluco~amine, residue~.

The invention also provides a modified K5 a~ defined above, wherein at least some of ~he glucuronic acid residue~ are epimeriRed to the L-iduronic acid residues.
. ~
The invention also provides modified K5 as defined, wherein at leas~ ~ome of the free hydroxyl groups, especially those in the 6- po~ition of the glucosamine acid residues and/or, where appropriate, those in the 2-position of the iduronic acid re~idues, are sulphated, prefexably to an extent of at lea~t 25~. ;

The invention further provides a saccharide or deri~ative thereof, comprising s~bsta~ially units of glucuronic acid and glucosamine, especially where such units alternate, modified as defined abo~e for K5. ~ -. ., : .. . .
The present invention further provides any of the modified compound~ as defined, wherein at least some of the residues are 3-O-sulphated.
~-..
The present invention also provides the use of the cQmpounds of the invention in therapy.
. ~
The present invention further provides use of any of the compounds of the invention in the manufacture of a medicament for the treatmen~ or preven~ion of condicions ~requiring antithrombotic or anticoagulant activity.
, : ' : ' ' ,'., ':.' ~JO92/17507 2 i a 7 ~ ~J 1~ PCT/GB92/00571 The present inve~tion also provideg a process ~or the preparation o~ any of the compounds descri~ed above which process comprise~ one or more of the following steps:

a) subjecting the appropriate starting material to an N-deacetylation process;

b) sulphating free NH2 groups, particularly where they are produced by a) above; ~;

c) epimerising the product~ of b) such that at least some o~ the D-glucuronic acid re~idues are transformed into L-iduronic acid re~idues; and . . .
d) sulphating at least some of the free hydroxy groups in any result1ng compound.
.
Preferably, ~aid proce~s comprises ~ubjecting polysaccharides of different molecular weights ,~.
(hereinafter referred to a~ ~S ~accharide3), extracted from certain E. coli strain~, to a ~eque~ce of chemical and en2ymatic passages, which c~n ~chematically be illu~trated as follows:

a) the K5 saccharides, which consist essentially of an alter~ate linear ~equence of D-glucuro~ic acid and N-acetyl-D-glucosamine are subjected to a chemical N-deac~tylation process;
.~ .

b~ the ree NH~ groupY of the products obtained under ~ -~) are 3ulphated by means of appropria~e sulphati~g agents;

c)~ the products obtained under b) are incubated with D-glucuronyl-~-~iduronyl-CS-epimera~e, extracted ~rom bovine liver, to tran~form a certain amount of the ~ :
D-glucuronic acid re~idues into L-iduronic acid resldues; ~ :
, : :': ' ., , . ..

~ W092/17507 PCT/GB92/00571 ~ 2 ~ 0 ~

d) the products obtained under c) are reacted with suitable sulphating agents, thereby to substitute a certain amount of the hydrogen of free hydroxy groups in the polysaccharide chain by sulphate groups.

As preferred embodiments, the present lnvention provides novel polysaccharides consisting of alternate seauences of uronic acids a~d glucosamine residues, characterised by the fact that they have a percent ' content o~ N-sulphated groups varying from about 35 to about 100, a percent content of N-acetylated groups varying from about 0 to about 65, a percent content of L-iduronic acids ~arying from about 10 to about 25, a minimal percent conte~t of 6-O-sulphated groups of about 25, the compounds being further characteri~ed in that : the remaining uronic acids are essentially D-glucuronic acid re~idues. Such com~ounds may be prepared by the ^~ :
following process:
: ., j a) a K5 saccharide, essentially consisting of an j alternate linear sequence of D-glucuronic acid and N-acetyl-D-glucosamine re~idu s i5 treated with a mixture of hydrazine/hydrazine sulphate, for from ~:
about 30 minutes to aboue 6 hours, at a temperature comprised between ab~ut 80 and about 110C;

b) the compounds obtained under a) are ~reated with a sulphating agent selected from ehe complexes between sulphur trioxide and nitrogen organic containing ,:
bases, at a temperature between about 45 and abouc 65CC, for a period of time up to a maximum of 2~ -hours; . :
.
c) the compounds o~tained under b), which are : polysaccharides essentially consisting of alter~ating D-glucuronic acid and D-glucosamine residues containing ace~ylamino and sulphamino .
-. .
: ,- ..

:j~ ' ..... .
: . . .

WO92/17507 210 ~ :L 2 ~31 pcr/G~92/oo57l ~

groups irl various proportion~ are ~ubjec~ed e~ ehe action of ~he enzyme D-glucuronyl-L-iduronyl-CS-epimerase, at about room cemperature, ror a period :;
of time up to a maximum of two day~;

d) the compounds obtained under c) are converted in~o the corresponding salts of organic nicrogen containing bases and are subcequently treated with a sulphating agent ~elected from the complexes between sulphur trioxide and the organic nitrogen containing bases, in an inert organic solvent, a~ a cemperature -~
comprised between about -5 and ~O~C, or a period ~ t time up to a maximum of 24 hour~
... . .
the procPss being further characterised in that the i ., compound~ obtained under d) may optionally undergo the sulphation procedure of qtep b).

The above process preferably further comprises subjecting the products as originally defined co che action of 3-0-sulphotran~ferase.

The present inventio~ ~urther provldes R5 saccharides essentially consisti~g of aIternate linear se~uence9 o D-glucuronic acid and N-acetyl- ~ :
D-glùcosamine re~idue~ represented by the follawing ~or~lula:

,.

0~ ~C~C~3 .
. ~ .
.~
. . .

I: ~.

.' ' '~' , . .i ' . ' ,, ... , 1 " .,.. ,. " .. ,., .' ,, ... . . , , ' . ', ' ' " ' , . ' I ,, ~ . .. ..

~ WO92/17507 2 1~ 71~ ~ PCT/GB92/00571 -~:-having an average molecular weight ~arying from about r1000 to about 100000 Daltons or more, and showing characteristic signals in the 13C-NMR spectrum at 104, 98 and 55 ppm, and also provides polysaccharides Pssentially consisting of alternate sequences of D-glucuroniC acid and D-glucosamine units, characterised in that they contai~ from about 3S to about 100~ of N-sulphated groups, from about 0 to about 65% of : . .
N-acetylated groups and showing characteristic slgnals in the 13C-NMR spectrum at 104, 60 and 24 ppm.

Other polysaccharides of the invention include .. ~.
polysaccharides essentially consi~ting of alternate sequences of D-glucuronic acid and D-glucosamine units, characteri~ed in that they contain from about 35 to about 100% ~f N- ulphated groups, from about 0 to about 65~ of N-acetylated groups, a minimal percent content of 6-O-~ulphated group~ of about 25, the compound being : . .
further characterised by a ratio of sulphate group~/
carboxylic groups varying from about 1.0 to about 2.7, optical rotation varying from ab~ut 155 to about +65 and having an af f inity f or antithrombin III. These compounds may be prepared by the above processes in the appropriate sequences, such as a) and b), a), b~ and d) and the li~e. :

The novel polysacchar1des obtained according to this ..
se~uence, a~ well as the intermediates of each reaction step, can be recovered as free acids or in the form of their salt3, ~uch as their mineral alkali salts, including the sodium, potassium, calcium or magnesium :
~alts, from which, in turn, the compou~ds E~E ~ can be prepared by treatment with mineral or organic acids, for ~example. ~

The combination of chemical and enzymatic steps of ~ :
the presen~ invention is new, and has not previously , :,', ~- , ... ...

WO92J17507 PCT/CB92/00571 ~
21~712ll ;

been perfonmed starting ~rom polysaccharides of bacterial origin.

According to ~tep a) above, K5 saccharides, which generally have molecular weights in the range of from about 1000 to ahou~ 100000 Dalton or more, dete~tined by HPLC, may be treated with hydrazine co~taining hydrazine sulphate, preferably about lO~ by weight of hydrazine sulphate, preferably i~ a sealed tube, for a period of time suitably varyi~g from about 30 minutes to about 6 hours, at a temperature which may be between about 80 and about 110C, for example.

By this procedure, a certain percentage of ~he N-acetyl groups of the glucosamine u~its i~ removed and, according ~o ~tep b~, the resulting compounds are treated with suita~le ~ulphati~g agent~ in order to transform the free amino groups into sulphamino groups.
Suitable sulphatin~ agents may be selected from ehe complexes of sulphur trioxide and ni~rogen-containing .
arganic base~, such as tri-(Cl 4alkyl)amine.sulphur trioxide, pyridine.~ulphur trioxide and analogues thereof. It is generally prePerred, but not essential, to u3e the a~hydrous age~t, as the presence of eve~ a small amol~nt of water may affect the ~ature of the fi~al product. Other ~ulphating agents capable of introducing a~ SO3 group onto the desired position also fall within the ~cope of the invention.

The N-sulpha~ion reaction i9 preferably per.ormed at a temperature betwee~ about 45 and 65C and, depending -on the period of time for which ~he reaction iq performed, N-sulphatio~ i9 either more or le~s e~ten~ive. In general, from a~out 6 to about 24 hour~
are su~icient for the majority of the free amino groups ~
to be` sulphated. `
.. . .
~ . '"'.''" .' . ~
~ ~ ;'"'"', '' ' ' " . , ~ ' ': . '.,, ' " ,` ' ' .',`. i'. :: , ' . , ' ' ', ~ ' ' ' " ' ' "'~': ' ' ' " ' ' ' . ' ' :
.,. , : ' ::.'' . ' ' ,.:: '~ ': ,'. . . ' ', " , , : ' ., ', ' , . '" . ' ' , :, : . ., ,: . : .

~:40 92J17~07 210 l 12 -~ PCr/GB92/00571 ~- g ~.
, .
The resulting polysaccharides, which generally consist essentially of alter~ating D-glucuronic acids and D-glucosamine units containing acetylamino and sulphamino groups in various proportions, may the~ be subjected to an enzymatic treatment, for example, :
according to step c) above, in order to epimerise a certain proportion of the D-glucuronic acid residues of the polysaccharide chain into L-iduronic acid residues.
The epimerisation is most preferably achieved by means o~ the enzyme D-glucuronyl-L-iduronyl-c~-epimerase, o~tainable from bovine liver following the procedure of H. Prihar et al., (Biochemistry, 19, 495 ~1980]).

I~ preferred practice, the polysaccharides obtained under b) are incubated wlth the enzyme, at room temperature, under co~ditions which will be apparent to those s~illed in the art, for a period of time of from, say, a few hours up to two day~. Again, depe~ding on the type o~ substrate employed and the i~cubation cime, poly~accharides having different degrees of conversion of D-glucuro~ic acid re~idues into L-iduronic acid : .:
residues can be obtained. 5tep d) may be performed ~;
su~stantially as de~cribed by A. Ogamo et al., (Carbohydrate Res., 12~, 165 ~1989]), or as illuserated below. .
.~
The polysaccharides obtained under c) are advantageou~ly first converted into the corresponding salts of organic nitrogen containing ba~e3 such as, for :
instance, the trimethylamine, triethylami~e or tributylamine ~alts, a~d are subsequently treated with .:
suitable sulphating agent~, such as tho~e emp}oyed for the N-sulphaeion of 3tep b). The reaction is preferably ~:
carried out in the presence of an anhydrous, iner~
organic sol~ent such a~, for in~eance, dimethylformamide, dimethylace~amide, dimethylsulphoxide or mixtures :.
thereof. ~ :
. I ' .-~
. ;
L~ ."

21~o~ ~. ?, '~ ~ !~
The degree of O-sulphation depends on the subserat~s ~.
employed, as well as the reaction conditions. : _ For the purposes of the present invention, this passage is run f or a period of time of up to 24 hours, .
at a temperature between about -5 and about 60~C. T

Generally, firom about 5 to about 20 equivalents by weight of the predetermined sulphating agent, calculated over the amount o~ the N-deacetylated-N-sulphated K5 saccharide, are employed. 7 Partial N-desulphation may occur during the course 3 of this reaction. If desired, the product of step d) q can be subjected to the same N-~ulphation procedure as described in step b). ~

The polysaccharides thus prepared may be recovered J
according to techniques known in the art, such as by dialysi~ of ehe reaction mixture and subsequent lyophili~ation ofi the dialysed ~olution, and may be characterised by 13C-NMR and 1H-NMR spectroscopy, which is capable of pro~iding specific f ingerprints of ~
the glyco~aminoglycans ~A. S. Perlin, Mechods o~
Carbohydrate Chemistry, 7 [1976], 94; L. Ayotte et al., Carb. Res. ~19a0], 145, 267). Other characteri~ation techniques, such as HPLC, may also ad~antageously be 1 employed. j More specifically, lH-NMR spectra allow the ;
identification and quantification o~ the ~on-sulphated 3 L-iduronic and D-glucuronic acid residue~ by the 3ignals at 5.35 ppm and 4.55 ppm of the spectra reported in Figures S, 7 through 11 and 16 ~9ee ~. Casu in "HEPARIN, I
Chemical and biological propertiesn, published by Edward Arnold, Ed's D. Lane and U. Lindahl, 25-49 [1986]).
Il .
,:
- D~ .;.

92/17507 ~ 2 1 0 7 1 2 '~ PCT/GB92/0057l . E~
.. IE 11 Other minor signal~ are detectable in the 13C-NMR
spectra aasociated with end residues, that is, those o~
the reducing anomeric carbons at 90-95 and 95-98 ppm (Table 1 in A~ S. Perlin and ~. Casu; The ~ Polysaccharides, Vol 1, Academic Press, New York [1982~, i 133) and those of the unsaturated terminal uronic acid residues at 110 ppm (B. Casu et al., Biochem. J. 187, 599 ~1981]; R. Casu, Nouv. Rev. Fr. Haematol, 26, 211 [19843; J. R. Linhardt, J. 3iol. Chem. 261, 1444 [lsa6] ) .
.
The relative percentages o~ D-glucuronic acids and L-iduronic acids may al~o be determlned by paper chromAtography of the disaccharides obtained by deaminative cleavage of the C5-epimerised polysaccharides, according to the procedure described by J. Jacobsson et al., E3iochem. J., 1?2, 77 (1979). The relevant chromatograms are shown in Figure 13.
.-¦ The analyses of the NMR spectra and of the paper chromatograms indicate that the no~el polysaccharides of ;~
the present invention have a pexcent coneent of N-sulphated groups varying ~rom about 35 to about 100%, a p~rce~t conte~t o~ N-acetylated yroups varyin~ from about O to about 65~, a pexcent content of L-iduronic ;
acids, calculated over the total uronic acids, comprised between about 10 and about 25%, and a minimal content of 6-0-sulphated groups of about 25%.
:
It will be apparent eo those skilled in the art ~hat those polysaccharidè~ having lower percentages of N-sulphated group~, higher percentage of N-acetylated groups, a per cent concent of iduro~ic acids higher than 25~ and a minimal per cent content of 6-0-sulphated groups lower than 25~ can also be prepared according ~o the above pr~ce3~es. Said compound~, as well a~ the ! ~ corresponding ineermediate~ in the various reaction . ' .

~ ~ ' ' ' ,.

WO 92tl7507 PCl'~G~92/00~;71 2 ~. a rl ~, 2 ~

step~, fall within the scope ~ the present invention. ~_ As stated above, these novel polysaccharides display interesting and use~ul biological properties, particularly as antithrombotics and anticoagulants, and activities of par~icular compounds of the inven~ion are ~.
given in accompanying Examples 14 and 15.

The compounds of the invention may be administered one or more times per day in unitary injectable dosages varying from about 30 to about 300 m~, for example.

The present invention particularly provides a $
product which can be manufactured on an economically viable scale. It concerns all the aspects applicable on an industrial scale, acsociated with the use of the y product~, re~ulting from the i~ve~tion for human th~rapeutic applications such as antithrombotic and anticoagulant agents. For this purpose the compounds that are the object of the present invention may be formulated ~y conventional technique~ using suitable excipients and other such ingredients for pharmaceutical composition~ suitable for parenteral admi~iseration, for example.

Examples of formulationY ~or parenteral admlni9tratio~ include ~terile solutions contai~ed in ampoules, and may also contain substance~ to render the solution isotonic with bodily fluids, for example.

The compounds obtalned as the i~termediates in each of the various sceps of the process of the invention are, in general, isolated and characteri~ed, but can also be used as such in the subsequent tra~sfo~mations.
If they are characteri~ed, thi~ i~ made by lH-NMR and 13~ NMR spectroscopy, or any other appropriate means, as illustraced above for the end polysaccharides.
~ , ...
~' :

c~_ ~ ' ',~ ' ' . " ' ' ~ ~ ~2/17507 ~ 2 1 B 7 1 2 ~ PCT/~B92/U0~71 Thus, ~ar lnstance, the substances prepared in ste~
b~ may be polysaccharides consisting essentially of alternating D-glucuronic acid9 and D-glucosamine residues, containing ~rom about 35 to about lOO~ o~
M-sulphated groups and from about O ~o about 65~ of N-acetylated groups. Their 13C-NMR specera show -characteristic signals at 104 ppm, typical of the D-glucuronic acids, ag well as characceristic 5ignals at 60 ppm and 24 ppm, typical of the N-sulphated and the N-acetylated groups (Figures 4, 5 and 7 through ll).
,,:.
Other minor signal5 detectable in the above spectra are evident at lO9 and 103 ppm, and are as90ciated with the terminal uronic acid residues (Figures 4, 5 and f).

1 The i~t~rmediate3 of the variou~ reaction steps pos3ess antithrombotic and anticoagula~t propertie~, and fall within the ~cope of the pre~ent inve~tion. -In particular, the polysaccharides obtained by step b) may be further ~bjected to O-sulphation, performed subs~antially as described for step d). ~
,:~.. .
Again, occasional partial N-de9ulphation may occur duri~g the course of this reaction. If de9ired, the O-sulphation can be followed by an N-resulphation carried out a9 degcribed above; It ha9 been found that the resulting comp~u~dg, gurpri8ingly, poggeg~ affinity for antithrombin III. Thi9 result ig suxpriging, as the presence of L-iduronic acid residue9 wa9 preYiously considered to be e9sential for activity. Accordingly, these compounds which have not bee~ epimeri5 d but whlch exhibit an a~finity for antithrombin III form a particularly preferred feature of the in~e~tion.

This cla~s a~ pclysaccharide9 may be characterised by ha~ins al~ernating D-glucuronic acid a~d , , :

:. :: ,.~ , : - .. . ...

WO92~17507 21 D 71~ ~I PCT/GB92/0057l D-glucosamine residues, a per cent conten~. o~ ~
N-sulphated groups varying from about 35 to about 100, a ~
per cent content o~ N-acetylated groups varying from ~ ~.
about 0 to about 65 and a minimal per cent content of .
6-O-sulphated groups of Z5.

This, again, may be shown by 13C-NMR spectra (c.f.
Figures 17 through 32), with characteristic signals at 60 and 69 ppm, typical of the N- and 6-O-sulphated ~
~roups of the D-glucosamine residues. E~ -These compounds are further characterised by a ratio ~;
of sulphate groups/carboxylic groups varying from about ~.
1.0 to about 2.7 and optical rotation varying from about ~ :
+55 to about ~65~. Again, it will be apparent to those ~killed in the art that those polysaccharides having . ~:
alternati~g D-glucoro~i~ acid aAd D-glucosamine residues, a~d Surther having lower percentages of N-sulphated group~, higher percentages o~ N-acetylated groups, a per cent content of 6-O-sulphated groups lower . .: :
than 25 and affi~ity for Antithrombin III can also be .
prepared and fall within ~he scope of the present ....
invention. ~ ~ .
:: ' .
Thus, it will be appreciaced that the present in~ention provides a wide ra~ge of useful polysaccharide~, as well as convenient methods for their preparation. !: -.. .
Extractive procedures for obtaining nacural poly~accharides are o~te~ ~ediouc and expensive, and .. ..
various ~ractionation and de-polymerisation ~echniques for obtaining the purified native 9ub9ta~ce5 are not ... - ..
always capable of providing reproducible products. ¦ ~ :

These drawbacks, including eho9e of relying on crude ~animal ex~racts (which run the ri3ks inheren~ in animal :,'' '.' ,:
I .
~* :~.'..' , ~ 92/17507 ~ ~ lU ~. ~' PCT/GB92/00571 ~ . ,! ~ 15 illness, epidemics and 90 on), are overcome by the present invenrlon as microorganisms provide a practically unlimited source of starting materials, and :' t~_ can be kept under carefully controlled conditions while _ still synthesi3ing the desired products in bulk.

The compounds of the invention, optionally including those which are additionally N-sulphated, may serve as the starting materials for a subsequent enzymatic ~ reaction, by virtue of which certain hydroxy groups at ; the 3-position of the D-glucosamine residues are con~erted into the corresponding O-sulphated groups. :
Such compounds are also defined above.

j Such reactio~ are preferably carried out in the r presence of the enzyme 3-O-~ulphotransferase, which may t be prepared as described in Preparative Example 1, below.

The poly~accharideq may then be incubated with the ;. enzyme under conditions known in the ar~, to allow 3-O-sulphation.

t The K5 ~accharides employed as ehe starting , materials in the present invention may be prepared by j culti~ating serains of Esçhe~l~hia sQli, under aerobic ; conditions, in a suitable ~ermentation medium. It has ! been ~ound that, while the nature of the resulting saccharide cannot be exactly predicted, higher levels of carbon source, especially glucose, tends to give rise to j higher molecular weight forms of K5 polysaccharide.
.
5trai~3 of ~. Ç~li which can be employed for the purposes of the prese~t inve~tion are those exhibiting the presence of the ~5 cap~ular polysaccharide antigen, i a~d are available with ~e~eral differe~t source~, : including the American Type Culture Collection and the .: International E~cherichia Centre of the Statens Serum , ~ L

., ,., . . , ..... .. . .. .... . ; ,.. ~ , . .. . . . .. . .. . , ., . , . . .. ~. . . .. . .. . . .. .

i W O 92/17507 ~ 210 7 l162 ~ PC'r/GB92/00s7~

.Institut of Copenhagen, Denmark. ~
. ' ....................... '~
Other E. coli stxai~s which may be employed for the ~
purpo~es of the present in~ention are again available , with several different strain collections or are of clinical isolation, mainly ~rom pyelonephritis and urinary tract infections. They may be characterised via .~. ~
API SYSTEM 20 ~ and as strains showing the presence of . - ~.
the K5 capsular polysaccharide antigen.according to W. ~
Nimmich et al., Z. Gesamte Hyg., 35(10), 583 (~9B9), or .
D. S. Dupte çt al., Sem. Microbiol. Letters, 14, 75 ~1982). Some of these clinically isolated E. coli strai~s have been deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-3300, Germany, on February 27, 1991, under the provisions o~ the ~udap~3t Treaty. These strains were . .
assigned the accesi3ion number3 DSM 6371, DSM 6372 and ..
DSM 6373. By way of example their characteristics are .
repor~ed below:
'.' ',, ~. ~ .-' . :,'. ., '' .
: ,: ' ' t :
' : ' ' "

,' ''':
':, : :: ... . :

': ''.'~
~: ; ' , ; ' :-. : ~

~O ~2tl7507 2 10 712 ~ PCI/CB92/0057l ~ ~ .

SIJ~STl?ATE REACTION/ENZY~ DSM DSM D5M

, ONPG O-nitrophenyl-. 3-galactoside ~-galactosidase + + +
, ADH arginine arginine dehydrolase -LDC lysine lysine decarboxylase + + +
ODC ornithine. ornithine -. decarboxylase - +
CIT sodium citrate citrate utilisation - - -H2S sodium thio-sulphate production of H2S
URE urea urease TDA tryptophan tryptophan dèaminase -IND tryptophan indole production + + +
VP sodium pyru~ate ace~oin production ~; GEL Rohn gelatine gelatina~e GLU glucose fermentation + + +
MAN mannitol fermentation-oxidation + + +
~ INO inositol ~erme~tation-s oxidation ~ SOR sorbitol do + + +
F RHA rham~ose do + + +
SA~ saccharose do - + +
MEL meliblose do + - +
AMY amyydaline do ARA arabinose do + +
OX filter paper cytochrome oxidase NO3 glucose tube NO2 production + + +
NO2 glucose tube reductiOn o~ N2 to N2 MOB APIM
!. (micro~cope~ mobility + + +
MAC MacConkey culture in + + +
OF medium ~ermentation glucose (API OF) (under oil) + + +
OF glucose (API OF) fermentation (air) + + +
, po~itive - - negative do = same as preceding example These E. coli strains showed the presence of the KS
capsular polysaccharide antigen, detenmined as described above. The ~ trains useful for the pre~ent ~:~
invent-on may be maintained on 5t ~dard agar (Merck I), on Loeb agar, or on any medium suitable for E. coli. :
':
.. .
Preparation of K5 and cultivation of E. coli is illustrated below in Preparative Example 2.

SUE35rlTUTE SHE.FT

WO92/17507 PCT/GB92/00571 ~ l ` 2~ ~712l~

The following Examples and Preparative Examples ar~
provided only for the purpose of better illustrating ~he present in~ention, and are no~ to be co~strued as limiting the present invention in any way.
. . '.
. ,'','.' Isolatio~-~f-3-o-s~l-ph-Q~ransfQrase 3-0-sulphotran3ferase may be prepared fram Furth mast cell tumour~ extracted from mice available at the Swedi3h U~iversity of Agricultural Scie~ces, The ~iomedical Centre, ~ox 575 s-751 23 Uppsala, Sweden. . .
Furth mase cell tumourC can be developed in normal mice strains, a~ described by J. Furth et al., Proc. Soc.
Exp. ~iol., 95, ~24 (1957). A specifio preparation is j .
as follows. I
` I :,';~. :' .
Mastocytoms t~mours ~about 70g of tissue) were homogenised in 200 ml of 0.05 M Tris-l~ Triton X-100, ~ -pH 7.4, contai~ing protease inhibitors: lO ~g~ml of :
pep~tatin, 2 mM EDTA and 1 mM of PMSF (Sigma Chemical Co). :-'~
Th~ homogenate was gently stirred for 1 hour at 4C .:-and was t~en centrifuged at 100000 x g for 1 hour. The supernatant was pas ed through a gla~s fibre filter and subjected to the following purification proeocol~

1) Hepari~-Sepharose, 31 ml colum~, which was flrst wa hed with buf~er A (0.05 M Tris - 0.1~ Triton X-100, p~ 7.4, 1 ~g/ml o~ pep~tati~i 2 mM EDI~, 20~ of glycerol co~taining 0.15 M NaCl), and was then eluced :: : ~ using a linear gradient of 0.15 - 1.0 M NaC1 in buff~r -.
A.~ Eluent fractions of 4 ml were assayed for 1~
1~

~- .
~ ~92~17507 ~ 210 712 4 PCTt~B92~00~7l ,~
~ 19 ._ .
~- O-sulphotransferase ac~ivity substantially ~ollowing the --- procedure described by Ja~sson et al., 3iochem. J., 14~, - 49 (1975). O-De~ulphated heparin was used as a sulphate acceptor. Ten ~g of acceptor, 5 uCi of 35S PAPS
and enzyme protein in a total ~olume o~ 100 ~l of 50 mM HEPES, 10 mM MnC12, 10 mM MgC12, 5 m~ CaC12, 3.5 ~M NaF, 1~ TRITON X-100 , pH 7.4, were incubated at 37C for 30 minutes. The reactions were terminated by the addition of 400 ~l of ethanol, containing 1.3%
sodium acetate, along wich 0.4 mg o~ carrier heparin, and the samples were left at -20C overnight. After ~t centrifugation (13000 rpm for 10 minutes) the supernatant3 were discarded and the pellets were dissol~ed in 100 ~l of water.
~ 35 i The S-labeled poly~accharide wa~ separated from residual unincorpora~ed label by ce~trifugation through Sephadex, as ~ollow~. Syri~ges (Scm x 0.9cm i.~.) were packed with Sephadex G-25, ~uperfine grade, equilibrated with 0.2 M NH4HCO3, and w~re then centrifuged at 2000 rpm for 5 minutes and suspended in conical : centrifuge tube~, to eIiminate most of the liquid. The samples (100 ~1) were applied to packed and centrifuged ~yringes, which wer~ then again centrifuged ~ in the same manner. The labeled polysaccharides were I recovered in the effluents collected in the tubes, i whereas low mo}ecular weight labeled compou~ds were retai~ed by the gels. The effluents were analysed by scintillatio~ photometry. The a~eive fractions were ; pooled and dialysed against buffer A contai~ing 0.15 M
i NaCl.

.
~ 2) Sepharose Blue, 41 ml column, flow ra~e 15 ml/hour.
i ~ ~Following applicatio~ o the sa~mple, the coIumn was washed with buffer A cantaining 0.15 M NaCl and again eluted with a linear sal~ gradient from 0.15 to 1.0 M
; NaCl to ob~ain the last por~io~ of the enzy~atic '~
~ :

W~92/17507 ~ 210 ~12 'I , CT/GB92/~571 ~ v activity. Fractions of 5 ml were assayed for 0-sulphotransferase acti~ity as described above. The active fractions were pooLed, concentrated to about lO ml and dialysed against buffer A - 0.075 M NaCl. To the sample was subsequently added 3-mercaptoethanol to 12 mM concentration.

The obtained degree of purification was lS0 fold, with a~ apparent lO0~ reco~ery of 0-sulphotransferase activity. Tha sample obtained ~y the purification through Sepharose Blue contained demonstrable glucosaminyl 6-0-sulphotran~ferase, iduro~osyl 7 2-0-sulphotransferase and glucosaminyl 3-0-sulphotransferase.

. . .
, ,. ,"
PARA~IVE EXAMPLE 2 . . . , ' ' .
C~ ~ation of E co~i and Production of K5 Culti~ation Media -,. .

For producing the ~5 saccharides, E. ~oli may be cultivated under aerobic conditions in an aqueous 1i nutrie~t mediu~ containing assimilable 30urces of: ~ -carbon; ~itrogen; a~d inorganic salts. The culture medium may be any one of a number of nutrient media employed in the fermentation art, and the composition thereof may be adjusted or modified according to the experlence of the skilled technician, in order to obtain XS 9accharideg of the desired average molecular weight a~ well a~ improve the yield3 of the ferme~tation. The preferred carbo~ ~ources are glucose, ma~ose, galactose and peptone. Preferred nitrogen sources are ammonia, nitrates, soybean meal, pe~tone, meat extract, yeasc ex~race, tryptone and amino acids. Among the preferred " ' . . - . .
,. .~ ' :" ': ' .

l ~ ~92/17~07 ~ PCT/CB92/00511 ~ 2 1 0 7 .~
,~
inorganic salts which are incorporated in the culture media are the customary soluble salts capable of yielding sodium, potassium, iron, zinc, cobalt, magnesium, calcium, ammonium, chlorlde, carbonate, phosphate, hydrogenphospha~e, dihydrogenphosphate and .~ nitrate anions.
f _ .
Ordinarily, the K5 saccharide-producing strains are pre-cultured in a shake fla9k, the~ the cultures are put into jar fermenters for the production of ~ubs~antial quantities of the a~ove saccharides. A typical representative fermentation medium, which can also be employed for the preculture, ha~ the following composition for 1 litre:
K2HPO4 3.6 g 2P4 1.2 g Casamino acid 20 g Sodium citrate dihydrate 0.s g Ammo~ium ~ulphate 1 g ~
Glucose 4 g -Mg504 0.15 g ! The dialysable part of 100 g of t yeast extract in 100 ml o~ water (dialysi~ against water, cut-off o~ the men~rane: 15000 D) 1 litre .
The p~ of the medium is 7.2.
'~
Fçrmentation ': -a) Pre-cul~ - One loop of E. SQli from a Loeb agar plate i~ suspended in 5 ml o Merck Standard 1 medium and incubated for 6 h~urs, at a pH of about 7.2, at 37~C. The mix~ure wa~ then put into 700 ml of the above culture medium and incubated overnight under the same conditions. ;-~'' ', ', '', . . . .

W09~l7s07 2la~l22~ PCT/GB92/0057~

b) F~me~a~io~ - The fermentation i9 carried out under aerobic conditions at an e~fective p~ of 6.8 for a period o~ time ~arying from about 1 to 10 hours, at 37C.
. . ..
A jar fermenter co~taining 10 litres o~ the medium:
. ,.
K2HPO4 36 g KH2P4 12 g Casamino acid . 200 g ~ :
Sodium citrate dihydrate 5 g Ammonium sulphate 10 g Glucose 40 g MgSO4 1.5 g t ', ' .
The dialyi3ab1e part of lOOOg of ~ .yeas~ extract in 1 li~re of water (dialysis against water, cut-o f of the membrane: 15000 D) 101 a i9 employed. :

Analogouq ferme~tation media, con~aining glucose or other carbon source~ in amounts lower than those ~. . :
indicated, or up to a~out 5~ w/v, can also .. .
advantageously be employed.
'''''.'~
. ' ,' . ~' . .
~xtraction of_K5 :, .
E~tracei~ - The K5 ~accharides are recovered from I ::
t~e fermentation media as under b), according to the .
procedure described by W. Vann ~t ~1., Eur. J. ~iochem., Ll~, 359 (19~1), or as follows.
:., : :The fermentation broth is ce~trifuged for abouc 35 1 -; minutes:at 5200 rpm. The obcained sediments are suspended in ab~ut 800 ml of phoqphate buf~er and the :
'., , , . .
..

~ ~n2~175~7 ~ 2 1 n 7 1 2 1l PCT/GBg2/00571 re~ulting suspension ls centrifuged for lS minutes at 8000 rpm, and the supernatant is removed. The sediment is suspended in 800 ml of SO mM Tris 5 mM EDTA buffer pH 7.3 (prewarmed at ~7C) and the suspension is kept for 30 minutes at 37C in a water bath. The suspension is then centri~uged at 9000 rpm ~or 20 minutes. The supernatant i9 remo~ed and the extraction is repeated three times.

The supernatants of the Tris/EDTA^extractions are com~ined and added with an aqueous 10~ ~olutian o~
CET~VhON ~Trade mark) until no further precipitation is obser~ed and the muxture i9 kept overnight at room temperature. The mixture i9 ce~erifuged at 20C and ~000 rpm for 10 minute~, the precipita~e is dissolved in about 10-20 ml of aqueous 1 ~ NaCl, the solution diluted with at least 8 vo}um~s o ethanol, centrifuged. The solid, consisting of the crude KS sac~haride, i5 ~-collected and redissolved in about 10-20 ml of 1 M NaCl and at leas~ ~ volume3 o~ ethanol added. The precipitate is collected by centrifugatio~, dissolved in water and dialysed in a dialysis bag with a cu~-off of 3500 Dalton for 24 hours. The dialysed solution is cen~rifuged or 20 minutes at 9000 rpm at 4C and the precipita~e i9 discarded. The obtained supernatant, con~i3ting o puri~ied K5 saccharide, is freeze-dried. ~ --~ ipopoly~accharides are removed by dissolving in water the obtained purified K5 saccharide to a concentration of 2-3% and centrifuging the obtained so}ution at 45000 rpm for 4 hours at 4~C. The presence of traces of residual lipopolysacch~ride does not affect the processing of tha starting materials. In order to remove E. ÇQli ribonucleic acid, the resulting solution is treated for 24 hours with ribonuclease (Sigmaj in phosphate buf~er containing MgC12 ~10 mM) and then dialysed again~t deionised water wich a dia1ysis bag WO92/17507 21 Q 7 ~ PCTJ~B92/0057 24 .
having a cut-of~ of 3500 Dalton for 24 hours and lyophilised. This procedure yields about 0.8-lg per ~
10 1. culture. -. .
Representative K5 saccharides prepared according to ..
the above procedures are as follows.
. . ,:

A) KS saccharide having an average molecular weight of about S000 Daltons determined by HPLC. This KS: .
saccharide has the C-NMR spectrum reported i~ Figure 1, showing the typical major signals of D-glucuronic acids at 104 ppm, a~d of N-acetyl-D-glucosamine units at .
55 and 98 ppm, as taught by W. Vann e~. ~1., in Eur. J.
~i~chem., 116, 359 (1981), and typical minor signals at 110 and 1a3 ppm, aqcribable to terml~al residues derived ~rom D-glucuronic acid, as taught by B Ca~u et al., .:.
9iochem. J., 197, 599 (1981). ..
:':
~) ~5 saccharide ~aving an average molecular weight of about 50000 DaLton, as determined by HPLC. This K5 ~:
saccharide has the 13C-NMR 3pectrum shown in Figure 2, :.
showing the typical major signal~ of D-glucuronic acids at 104 ppm and of N-acetyl-D-glucosamine u~its at 55 and :~
98 ppm CJ gS saccharlde ha~ing an average molecular weight of about 100000 Dalt~n, as determlned by HPLC. The .
13C-NMR spectrum of this R5 saccharide i9 shown in F~gure 3. Typica7 major signals of D-glucuronic acids and N-acetyl-D-glucosamine uni~s are at 104 ppm and at 55 and 98 ppm respectively. .

The above detenminations confirm that the starting saccharide~ have the following repeating disaccharide unit:

:~ , '' ; ' ~ , ~

: . . - - : . : . :: - . ~ . . : :: : .
:. : - . .: ~ ., . . :: , . :; . ;; . .: ~ , : ,: . , . :
.: :; -. i. . , . ,: .- ,.

~ ~l ~ ~092tl7~07 PCT/GB92/00~7l 'i ~E 2~ 1 ~ 7 ~ 2 l~

J~La.
OH ~HCOGH3 L`_ _ n ~ :
,.~ .
.. . . .:
The 13C-NMR and lK-NMR spectra of the compounds prepared accor~ing to the multi-step processe~ o~ the prese~e invention, as well as thoqe o~ the start~ng ~
saccharides, wer~ recorded from ~olutions in D2O wich a ~ruker CXP-300 spectromecer, wich the excep~ion o the : - -compound of Example 8D, whose 13C-NMR spect~um was ~ -recorded from a ~olution in D2O with a ~ruker spectrometer AMX500.
. . .
Decenmination ~1~ HPLC of the molecular weight3 of the K5 saccharides was perfonmed using Supero e ~ (Trade mark~ and Superose 12 (Trade mark) column~ equilibra~ed with 1 M NaCl in 0.05 M T~IS-HCl pH a. buffer. The c purity (polysaccharid~ content) of the compounds eF the .
inve~tion and the relevant intermediate3 was asi~ayed by the carbazole method (T. Bitcer and H.M. MUiE, Anal.
Biochem., ~, 330 [1962]). The degree oE purity was generally higher thau 90%. The optical roca~ion3 wer~ ' -determined at ro~m temperature at a concentration ~f 1% - i.
in water, with a JASCO DIP370 p~larimeter. The :
deeenmiaed values were subseque~tly corrected, taking into account ehe purity degree o~ the ~est sample.
.

: . . ..
, ~: ... ..
' ..:

.
: . ~,. , ,:

W09~tl7507 2 i Q 71~ ~1 PCT/GB92/00$71 ¦ ~

EX~PLE 1 : ~... .
Prepaxation of N-dçacetylat~d-N-sulphat~d polysaccharides from K5-saccharide A (steps a) and b)) :' ,.
A) In a vial, lOO mg of K5 saccharide A and 138 mg o~
hydrazine sulphate were dissolved in l.3a ml of hydrazine. The solution was frozen by placing tne vial into liquid nitrogen, while keeping the solution under a nitrogen atmosphere. The vial wa~ then sealed and slowly brought to room tempera~ure, then heated for 5 hours at 96C. The vial was then re-frozen with liquid ~, nitrogen, opened, and 910wly brou~ht to room temperature. The solution was poured into a round-bottom ves~el, washing the vial out witn 5 ml of ~oluene.

The solu~ion was conce~trated under reduced pressure and the operation was repeated twice (each with 20 ml of tolueue), to evaporate of~ (together with toluene) most of the hydrazine. The residue was then added to 50 ml of distilled wa~er and the resulting solution brought to neutrality by means of a~ueous 37~ hydrochloric acid, then dialysed for 5 days throuyh a 3S00 Dalton cut-off membrane, against sodium chloride ~olutions and water T
~2 x 2 1, 0.5 M NaCl the first day, 2 x 2 1, 0.2 M NaCl the second day, 2 x 2 1, 0.1 M NaCl the third day, 2 x 2 1, H20 the fourth and fifth day). The solution was the~ concen~rated u~der reduced pressure, and di~solved in 65 ml of distilled water. ' -The pH of the obtained solution wa3 adjusted to 9 by ¦ -the addition o~ solid sodium bicarbonate, and the temperature raised to 55C. At this temperature, under co~tinuous stirring, 65 ml of the adduct o~
crimethylamine.sulphur trioxide wa9 added to the solution, which was kept at thi9 temperature for one . ~, ' ~ , '~;':'' .
~ ~ ' ~ ' .' : : .: ~ , ~ - i .; .. ~ . -.. - .. : . , . - . . . ,. : : , ~ ~4/17507 2 1 0 7 ~ ~ '1 PCT/GBg2/00571 hour, then a further 65 ml of the same adduct was added, ~nd the whole was reacted for an additional 5 hours.
The solution was dialysed ~gai~st aqueous solutions of sodium chloride of decreasing concentrations and water as described above. The dialy~ed solution was finally freeze-dried and 80 mg of a product, having the 3C-MMR spectrum of Figure 4, were obtained.

The product shows the following characteristic signals:
. .
~' stronq ~ianals:

60 ppm, N-sulphated groups 62 ppm, unsubs~ituted 6-hydroxyl groups of glucosami~e residueis 9~ ppm, glucosamine residue~ -104 ppm, glucuronic acid residues weak si~nal~:

109 and 103 ppm, terminal glucuronic acid residues ..
ve~Ey w~ q~ala:

24 ppm, residual N-acetylated groups.
~' .:, The perce~t content of N-acetylated group~, determined by l~C-NMR from the ratio of the area of the signal at 24 ppm to that of the area o~ the signals o~ all anomeric (C-1) carbon~ in the 100-110 ppm region, wa~ about 5. The per cent content of N-qulphated groups wa~ about 95. No signals ascribable to free -NH2 groups were o~served.

~)~ Following the same procedure of the foregoiny preparation, starti~g from 100 mg of ~5 saccharide A and ~ -. , ' . ' ::::
L

W092/~7507 210 712 !1 PCT/GB92/U0571 limiting to 3 hours the reaction with hydrazine -sulphate/hydrazine, 77 mg of a product were obtained, having a percent content of N-acetylated groups of c~out ~ :
15, determined by 13C-NMR (Figure 5) as for the product of Ex. lA, and a percent content o~ W-sulphated groups of abou~ 85. The 13C-NMR speccrum of Figure S .
shows the following characteristic signa~s~

24 and 55 ppm (weak) : N-acetylated groups 60 ppm ( trong) : N-sulphated ~roups 62 ppm (strong) : unsubstituted 6-hydroxy groups ~ .
of D-glucosamine residues ~ -98 ppm (strong) : D-glucosamine residues t 104 ppm (s~rong) : D-glucuronic acid residues 103 and 109 ppm (weak) : terminal D-glucuronic acid residues No signals of free -NX2 are pre~ent.

C) Following the same procedure for the preparation of t the product o~ Example lA, starti~g from 100 mg of K5 :
~accharide A) and limiting to one hour the reaction with hydrazi~e sulphate/hydrazi~e, 75 mg of a product were obtained, having a percent content of N-acetylated groups of about 30, as determined by 1H NMR (Figure 6) from the ratio of the area of the signal of the ~-acetylated groups at 2.1 ppm and the total area of the ~ignals of the a~omeric hydrogens (between 5 and 6 ppm), ' .~
and the 13C-NMR spectrum of Figure 7, showing the same t ~ ~.
characteristic signals of the compound of Example la.
The compound has a percent content of N-sulpha~ed groups -;
of about 70. No ~ignals characterisitic of free -NH2 group~ are present.

... i .

~: : ''~'"
- : ' ' ~1 ~ '.','',' ,.
.. . . . . . .. ., . , . . . . ~ . , ... ~ .. ,, ..

: ~092/17507 210 I ~ 2 ~ PCTtGB92/00571 Pre~aration of an N-deacetylated-N-sul~hated polysaccharide from K5-saccharide ~ (steps a) and b)) Starting from 100 mg of K5 saccharide B, and ~ollowing the same procedure described for the preparation o~ the product of Example lA, carrying out the reaction with hydrazine sulphate/hydrazine for 5 hours, 75 mg of a product were obtained ha~ing a percent content of N-acetylated groups le59 than 5, detenmined by 13C-MMR (Pigure 8) as for the product of Example lA, and a percene content of N-sulphated groups higher than g5. ~he 13C-NMR spectrum o Figure a shows the same characteristic signals of the compound of Example lA. No signals charac~eristic of free NX2 groups are present.

, . .
~: ' ': .'' .
. EXAMPL~
,, , Pre~aration o~ ~-d~acetylat~d,-N-sul~at~,d : .
~oly~a~chari~s~ m K5-saccharide ~ (step~ a) and b)) : ~:

The following polysaccharides were prepared, starting from 100 mg of K5 saccharide C), following the ~ame procedure illustrated ~or the preparation of Example lA, with different times for the reaction with hydrazine sulphate/hydrazine.
. .
! A) Reaction time with hydrazine sulphate/hydrazine: S ~ -hours~ Yields: 85 mg of a product ha~ing a percen~
content of N-acetylated groups o~ about 2, and a percent content of N-sulphaCed groups of about 98, decermined as ~abo~e described by ~he 13C-NMR (Figure 9). The ~ ~-: ~ ~ spectrum hows the same characteristic signals as ~he .,.
:::

:
LP:

~ . ~; .. ,. . . .. .- . . . ... ... ... . . .. . . .. .. .

W092~17507 21 Q ~ PCT/GB92/00571 ~:, compound of Example lA. ~
y,.
B) Reaction time with hydrazine sulphate/hydrazine: 2., hours. Yields: ao mg of a product ha~ing a percene content o~ N-acetylated groups o~ about ~3, a percent ~- -content of N-sulphated groups of about 77, determined as above described by the 13C-NMR spectrum of Figure 10. ~.
The spectrum shows the same characteristic signals as the ~ompound of Example la.

C) Reaction time with hydrazine sulphate/hydrazine: 80 minutes. Yield 75 mg o~ a product having a percent ~ :
content of N-acetylated groups of about 48, a percent content of N-sulphated groups o~ about 52, determined as described above by 13C-NMR spectrum of Figure 11. The spectrum shows the same characteristic signals as the compound of Example lC. t t The NMR spectra of the polysaccharides prepared in Example~ 1, 2 and 3 clearly indicate that no o~her modification of the structure of the starting K5 saccharides has occurred.
.
E :`
: ' X~MPLE 4 ~, ~,' .
p~Qaration-of ~ p~ ed-N-sul~h~ted polysacchar~des t~e~ c !
, A) 10 mg of the product of Example lA were incubated with 8 mg of a preparation obtained ~rom bo~ine liver as described by H. Prihar (s~ee above) co~taining the enzyme : : D-glucuronyl-L-iduronyl-C5-epimerase, in 1 ml of 0.05 M
Hepe~, pH 7.4, containing 50 mM potas~ium chloride, }5 mM EDT~ and 1% of Triton X-100 . The mixture was . :
kept at room temperature for 2 days, then the desired :: ~ .
.' . ~, ...
.

;, ~ : ~: : ' ' ' , : : .
: . : ', ' .- :. ' ~ . ~ : .

~ ~092/17507 21 Q 712 ~ PCT/CB92/0057l ~ - .

e~imerised product was isolated by ion-exchange chromatography on DEAE cellulose. 7 mg of product were ~ obtained.

t- The low-field region of the H-NMR spectrum (Figure 12) clearly show~ signals at 4.7 and 4.95 ppm typical o~ the L-iduronic acid residues (Perlin A. S. in ~ Methods in Carbohydrate Chemistry Vol. VII, Academic i Dress, New York ~1976], p 94), the area of which ~ corresponds to 18~ o~ the total area o~ uronic acids. A
f similar result wa~ obtained by paper chromatograph (Figure 13), performed as described by J. Jacobsson et al. (see above), from which a percent content of L-iduronic acids of 18 was determined.
, . ....
B) 10 mg of the product obtai~d in Example 2 were epimerised following the procedure of A) abo~e.
Seven mg of product were obtained. The lH-NMR
spectrum (Figure 14) shows ~ignal~ typical of h-iduronic acid re~idues at 4.7 and 4.95 ppm, the area o~ which corresponds to 22~ of the tocal area oS uronic acids.

Figure 13 refers to the paper chromatograph, performed a3 described above, rom which a percent content oS L-iduronic acids o~ 20 is detenmined.
.
C) 10 mg of the product obtained in Example 3C were epimerised following the procedure of A) above. 7 mg of product were obtained having the paper chromatogram of I Figure 13, from which a percent content of L-iduronic acid~ of 19 was detenmined.

The N~ spectra of the polysaccharides prepared in i ExampLes 4A and 4~ clearly indicate that no other - ~ structural modification than the CS-epimerisation has occurred. In view of the starti~g 3ubs~ra~e and the epimerisa~ion procedure, neither ha~ any other : . . .:, L
~ : , W092/l75~7 21 Q 71~? ~ PCT/GB92/00~71 structural modificatio~ cccurred for the product of Example 4C. F,~

EXAMPLE S

Preparati~n of 0-sulphated-N-s~lphat~d-C5-~imerised ~lysaccha~id 5 ~step d~
. ' A) 100 mg of the product prepared in Example 4A were . 1.
dissolved in 20 ml of water and pa3sed through an Amberlite (Registered Trade Mark) IR-120 H+ column at room temperature. The column wa~ subsequently washed with 20 ml ~f water. The eluates were collected and ¦
brought to pH 5.5 with a 10~ solution of tributylamine ~ :
in ethanol. The exce~s of tributylamine was extracted I :
three time~ with 40 ml each of diethyl ether and the aqueous solution containing the tributylamine salt o~
the product of Example 4A was freeze^dried. 100 mg of the re~ulting pro~uct were dissolved i~ 32 ml of .
a~hydrou~ dimethyl~ormamide, 765 mg of the adduct pyridine.3ulphur trioxide dissolved i~ 15 ml of anhydrous dimethylform~mide was added to the solution a~d the resulting mixture was kept at 0C for 1 hour.
~n equal volu~e of water was added, ~he solution was brought to pH 9 with aqueous 4~ sodium hydroxide and the whole was dialysed agai~s~ aqueou~ solutio~s of sodium chloride o~ decrea~ing co~centrations and water, as de~cribed in Example lA.
..
The dialy~ed solution was freeze-drled and the resulting product, upon transformatio~ into the correspondi~g tributylami~e 9alt, was reacted under the same conditions of thè present Example. 180 mg of a product were obtained, which was fi~ally reacted with the trimethylamine.sulphur trioxide adduct as described in Example lA
' ~ .
~: ~ , ,, ~

~ W092/l7507 PCT~GB92/0057l ~ 210712~
: r ~ 3 ao mg of the title compound were o~tained, having the C-NMR spectrum shown in Figure 15, showing characterlstiC signal9 at 60 ppm (strong): N-sulphated groups; 62 ppm (strong): unsubstituted 6-hydroxy groups . o~ D-glucosamine re~idues; 69 ppm (medium):
6-0-sulphaCed groups; 98 ppm (strong): glucosamine residues; 99 ppm (medium): non-suiphated ~-iduronic acid residues; 104 ppm (strong): D-glucuronic acid residues.
~, .
The product has a minimum percent content of 6-0-sulphated groups o~ 52, determined from the area of the same signal at 69 ppm.

~) Following the procedure of Example 5A above, and star~ing with 100 mg of the product o~ Example 4B, 160 mg of a product were obtained ha~ing ~he 13C-NMR -spectrum of Figure 16, and ~howi~g the ~ame characteristic signals as the compound prepared under Example SA. :
~ .
The product has a minimum percent contene of 6-0-sulphated groups o~ 44, determined from the area of .
the signal at 69 ppm C) Followi~g the procedure of Example 5A above, and . .:
starting with S mg of the product o~ Example 4C, 9 mg of :.
a product were obtai~ed.
. .
In ~iew of the ~tarting substrate and the sulphation proceduxe, no other ~tructural modification has occurred ~or the product of Example SC.
. .
1 '".: ':
-:, -:.: -: ., , ' . ,. ' : , ''"" ''' W092/17507 PC~/GB92/00571 ` 2~Q7~2~ ~

Prepara~ion of a non-epimerised N- and O-sul~hated ~olysaccharide ~, 100 mg of the product prepared in Example lA were ~-dissolved in 20 ml of water and passed through an ~r Amberlite (Registered Trade Mark) IR-120 H column at iT
room te~perature. The column was subseouently washed with 20 ml of water. The eluates were collected and ~-brought to p~ 5.5 with 3 ml of a solution of tributylamine (10~ in ethanol). The excess of cributylami~e was extracted (three times) with 40 ml of ethyl ether, and the solution containing the tri~utylamine salt o~ the product of Example lA was freeze-dried.

180 ms of the obtained salt were dis~olved in 32 ml ~ -of anhydrous dimethylformamide. To this solution 765 mg of anhydrou~ pyridine.sulphur trioxide adduct, dis~ol~ed - -in 15 ml of anhydrous dimethylformamide, were added and the mixture was kept 1 hour at 0C. The reaction mixture waC combined with an equal ~olume of water and the pH broughC to 7 0 with aqueous 4S NaOH. The mixture was then dialysed against NaCl solution~ of decreasing concentrations as described in Example lA.

Th~ dialy ed solution wa3 freeze-dried, and the re~ulting product upon transformation into the corresponding tributylamine salt, was reacted under the same condition~ of the present Example. 90 mg of a product were obtain_d, which was treated with ~he adduct ~trimethylamine.sulphur ~rioxide under the same condition~ of Example lA. A producc wa~ obtained, ~ having a minimum~percenc con~en~ of 6-0-9ulphated groups : ~ of 35~determined~from the area of the signal at 69 ppm ; in~ehe 13C-NMR ~pectrum (Figure 17), a~d having the ~ ~ same characteristic signals as the product of ExamDle 1~.
: -, . '':.',' _ ~

~ ~092~17507 2 1 ~ 7 1 2 ll PCT/GB92/0057l . EXAMPLE 7 ~ ' ,.
Preparation o~ non-epimerised N- and O-sulphated polysaccha~ides The tributylamine salt of the compound of Example lA
was prepared as described in Example 6, and 1ao mg of this salt were dissolved in 32 ml of anhydrous dimethylformamide. The solution was cooled to 0C and 765 ml of a solution of anhydrou~ pyridine.sulphur trioxide prepared at 0C in 15 ml of anhydrous dimethylformamide added. The resulting mixture was kept ~ hour ac 0C and was subsequently mixed with an equal volume of distilled water. The pH was adjusted co 9 by mean~ of 4~ sodium hydroxide and 4 ~olumes of ethanol saturated with sodium acetate were added. The mixture was kept overnight at 4~C, whereby a precipitate was obtained, which was di solved in 50 ml of distilled water. The resulting ~olution waq dialysed against distilled water for 3 days t3 x 2 l each day, cut-off 14000 D~ and finally free~e-dried.

The following compounds wer~ obtained in re8petitions of the above procedure:
,:, .' ' :' A) Yield: 85 mg. The compound showed a percent content of N-sulphated groups of about 95 and a percent concent of 6-O-sulphated groups of about 100 tsignal9 at 60 and 69 ppm, respecti~ely, in the 13C-NMR spectrum of Figure 18), a ratio SO3/COO o~ about 2.1, and an `~
~]D f +55 4 ' 'i.~
}) Yield: 94 mg. The compound ~howed a percent content of N-sulphated and 6-O-sulphated group~ similar tO that of Compound 7A (signals at 60 and 69 ppm, respectively, in the 13C-NMR spectrum of Figure 19), and a ratio SO3/COO of about 2.2.
~., ! ':
. ,, ,` ' ,''' .'.'''~."~''," . ' . ' WO92/17507 2 ~ 0 7 ~ PCT/GB9t/0057 C) Yield: 82 mg. The compound showed a percent content o~ N-sulphated and 6-O-sulphated groups similar to that of Compound 7A (signals a~ 60 and 69 ppm, respectively, in the 13C-NMR spectrum o~ Figure 20), and a ratio SO3/COO of about 2.2.

D) Yield: 95 mg. The compound showed a percent content of N-sulphated groups of about 95 and a percent content .
of 6-O-sulphated groups of about 85 (sig~als at 60 and 69 ppm, respecti~ely, in the 13C-NMR spectrum of Figure 21), and a ratio SO3/COO of about 1.8.
..' . ' . ' ' ' ''.

EXAMPLE ~

PreE~ara ~ im~i~çd N-Tand_O-sul~hated olysaccha~ld~3 The procedure of Example 7 was repeated, using the tributylamine salt o~ the compound of Example lA as teh : -starting material, and keeping the mixture of the two solutions at 0C for different ~mes. The following compounds were pxepared in repetitions of the procedure: . .

A) Yield: 30 mg. Reaction time: 20 minutes. The compound showed a percent content of N-sulphated groups o~ about 95 a~d a percent content of 6-O-sulphated groups of about 70 (signalis at 60 and 69 ppm, respectively, in the 3C-NMR spectrum of Figure 22), and a ratio SO3/COO of about 1.6. .
.
3) Yield: 92 mg. Reaction time: 40 minutes. The compound 3howed a percent ~ontent o N-sulphated groups .
of about 95 and a percent content of 6-O-sulphated :groups of ~bout 75 (signals ac 60 and 69 ppm, : reipecti~ely, in the 13C-NMR ~pectrum of Figure 23), a~d a ratio SO3/COO of about 1.7.
, ' : ~ ' ' ,. " ' i~ L

i... . ~ ~ " ., . .. ., , , , ,, , "

~ ~092/17507 21~ 712 ~ PCT/GB92/00571 C) Yield: 90 mg. Reaction time: 4 hours. The compound ~ showed a percent content of N-sulphated and 6-O-sulphated groups similar to that of Compound 7A
(signals at 6~ and 69 ppm, respectively, ln the 3C NMR spectrum of Figure 24), a ratio SO3/C00 of about 2.3, and an ~]D of +62.4.

i D) Yield: 95 mg. Reaction time: 5.5 hours. An N-resulphation was performed as described in Example lA. The compou~d i~howed a percent content of N-sulphated and 5-O-sulphated groups similar to that of Compound 7A (signals at 60 and 69 ppm, respectively, in the 13C-NMR spectrum of Figure 25), a ratio SO3/COO o~ about 2.52.

', .

Pre~araSlg~ on-e~imeri~e~ N- a_d Q-sulpha~ed po~ys~ccha~i~es . .
The procedure of Example 7 was repeated, again using the tri~utylamine salt of the oomp~und of Example lA as the starting material, and using pyridine.sulphur trioxide adduct not prepared to be anhydrous. The ~ollowing compounds were prepared in repetition~i of the above procedure:
' A) Yield: a3 mg. The compound showed a percent content of N-sulphated a~d 6-O-~ulphated group~ similar eo that of Compound 7A (qignals at 60 and 69 ppm, respec~ively, in the 13C-NMR spectrum of Figure 26), a ratio S03~COO o~ about 1.9, and an [~]D of +56.~.

~) Yield: 75 mg. The compound showed a percent content of N-sulphated and 6-O-sulphated groups similar to tha~
.' ., :,'.. .
, z ~ .. ..

WQ92/17~07 PCT/GB92/00571 ~
2~ 12~ ~
3a of Compou~d 7A (signals at 60 and 69 ppm, respectively, in the ~3C-NMR spectrum o~ Figure 2~), a ratio SO3/COO of about 1.9, and an [~]D of +56.2.

C) Yield: 70 mg. The compound showed a oercen~ content of N-sulphated and 6-O-sulphated groups similar co ~ha~
o~ Compound 7A (signals at 60 and 69 ppm, respectively, in ~he 13C-NMR spectrum of Figure 28), a ratio SO3/COO of about 2.05, and an [~]D of +56.7.

. --Pre~aticn of a non-epimerised N- and O-s~l~hated pol~açc~ride ., The tributylamine ~alt of the compound of Example lA
was prepared as described in Example 6, and 180 mg of this salt were dissolved in 32 ml of anydrous dimethylformamide a~ room tempera~ure. Keeping the solution at room t~mperature, a solution of 1.53g of ~he anhydrous adduct pyridine . sulphur trioxide prepared in 30 ml o~ anhydrou~ dimethylSormamide at room tempera~ure was added. The resulting mixture was kept at room t~mperature for 5 . 5 hours, and was 3ubsequently mixed with an equal colume of di~tilled water. The pH was adjusted to 9 by means of 4~ sodium hydroxidei and the compou~d w~s recovered as described in Example 7. The resulting material wa3 subjected to N-resulphation, per~omed as described in ExamDle lA. The reaction mixtura wa~ diluted wi~h 4 ~olumes of ethanol satura~ed wiCh sodium acetate and the end product was recovered as described in Example 7. j -A percent content of N-sulphated and 6-O-~ulphated groups similar co thac o~ Compound 7a was detected (sig~al3 ae 60 and 69 ppm, respectively, in the . ~ , ::
,r ~50~ 21~ 71~ .1 C:MMR spectrum of Figure 29), with a ratio S03/C00 of about 2.5, and an [~]D f +59.4. Yield: 86 mg.
j '.
. . .
~ .
EXAMPL~

Preparation of a non-ePim~rised N- and 0-sul~hated ~ ooly~accharide $
The procedure of Example 10 wa~ repeated, but wherein ~he two solutions were ~repared at 55C, and mixed and kept at this ~emperature for 2~ houxs. The obtained compcund showed a percent content of N-sulphated and 6-0-sulphated group~ similar to that of Compound 7A (signals at 60 and 69 ppm, re~pecti~ely, in -:
the C-NMR spectrum of.Figure 30), and a ratio .
S03/C00 of about 2.7. Yield 77 mg. . .
~ , . .
....
. ' -EXAMPLF~12 . - .... .
pren~Eaeio~ o~ ~on-eoimerised N- and 0-sul~hated ~olYsacch~rid~

The tri~utylamine salts of the compounds of Example ..
3A, 3B and 3C were prepared as described in Example 6.
These salts were treated with the anh~drous adduct of .:
pyridine.sulphur trioxide, and recovered from the ~ ...
reaction medium, as described in Example 7. The ~: -ollowing compounds were prepared in repetitions of ehe ::
above~procedure. ~. ;
:. :
A) Yield: 80 mg. The starting ~ribucylamine salt was -.
: that of the compou~d of Example 3A. The compound showed .. . ~ :
.- . ~
. .... ..
~ ' ' ' '; , :

'~
WO92/17~07 PCT/~B92/00571 " 21~7~2~ -a ratio SO3/COO of about 2.

~) Yield: 72 mg. The s~arting tributylamine salt was that of the compound of Example 3B. The compound showed a percent co~tent of N-sulphated groups o~ about 77 and a percent content o~ 6-O-sulphated graups of about 100 (signals at 60 and 69 ppm, respeccively, in the ~.
13C-NMR spectrum of Figure 31), and a ratio SO3/COO of about 1.8.

C) Yield: 90 mg. The starting tributylamine salt was that of the compound o~ Example 3C. The compound showed a percent content of N-sulphated groups of about 52 and a percent coneent of 6-O-sulphated groups of about 100 ~signals at 60 a~d 69 ppm, respectively, in the 13C-NMR spectrum o~ Figure 32), and a ratio SO3/COO of about 1.5.

~AMPLE 13 E~z~m~i_1~Q:59L~ha~i9~
,...
2 ml o~ a reaction mixture were prepared using the following ingredieuts: 2 mg of the compound of Example S~; 0.4 ml of an enzyme preparation prepared according to the procedure of Preparative Example 1 (corresponding to about 1.6 mg of protein); 1 mM PAPS in 0.05 M Hepes;
10 mM MnC12 - 5 mM CaC12 - 10 mM MgC12 - 3.5 ~M
NaF - ~0.5 - 1~) Triton X-100, pH 7.4. The reaction mixture was incubated for 2 hour~ at 37C, then the , :
reaction wa~ terminated b~ heating at 100C for 2 minutes. The denatured proteins were removed by centrifugacion and the supernatant pa99ed through a ~ :
column (0.8 x lOOcm) of Sephadex G-15 equilibrated wi~h O.2 M H4NN03. The eluted ma~erial wa~ freeze-dried.
" ~
L ~
. . .

I~ ~' ~ 2/17507 ~ 21~ 712 11 PCT/GB92/00~71 A sample (500 ~g) of the material th~s prepared, dissolved in 500 ~l o~ 50 mM T_is-0.SM NaCl, pH 7.4, was applied to a 3 ml column of antithrombin III -Sepharose, equilibrated in the same bufrer. The column was then eluced with 50 mM Tris-2 M NaCl, oH 7.4. The eluate contained 10~ of the total material. -~
~, ,"
. ..
.
~1 EXAMPLE 14 .
Activity of Compounds of the Invention The acti~ity of certain compounds of the invention was evaluated 1n vitro using the following tests:
.,:
a) Anti-Xa activity, performed as described by A. N.
Teien et al., Thrombosis Res., 8, 413 ~1976);
.:
b) Heparin Cofactor II activity, performed as described by D. Dupoy et al., Thromb. Haem., 60(2), 237 (1988);
'.".,~ .'...
c) APTT, performed substantially as described by W. N.
~ell ~ al., Nature, 174, 8~0 (1954), but diluting the plasma sample 1:1 with saline; and d) TT, performed substantially as described by C. Eika at al., Lance II, 376 ~1972), but diluting the plasma sample 1:1 with saline.

The results obtained are 3ummarised in the following ta~le: ;
.. - . .
... :

.,~

, -.

, .

" ~ "" ';',~ ,'",,,'~,";'"' ,";

WO92tl7507 PCT/GB92/00571 ¦
2 10 112 ~
..
TA3LE 1 ~

COMPOUND Anti-Xa HCII APTT TT
OF IC50* ICS0~ IC200**-C2~0~*
EXAMPLE .

SA l' 0.4 20 4 5~ 56 0.46 39 13 .

5C 59.7 0.05 3.55 1.2 conce~tration neceqsary to i~hibit 50~ (~s/ml) concentration necessary to double the coagulation :
time (~g/ml) ,~
The result~ are indicative of the clinical use~ulness of the polysaccharides of thi invention as :~
antithrombotic and anticoagulant agents. - :~
.." ~ ' '''' ':
; "' -:
Pxoperties of various non-epimerlsed, N-, O-sulphated compound~ of the Examples are Table 2 below.
, -'.'" '' ' , .
! ~:
:
.. : : :.:
.- .
, .
' ',.''-.

' I ~ ' ' ' ' .' " :' '' .'` . '~ ';. "'. ''. i"" .' ', ,: ,, ,, ",1 , . , , ~ , ' , .. . . . .. .

~ ~ :

W~ ~2/17507 ~ 2 l~ 712 ~ PCT/GB92/00571 43 .
.~ . ...

~ , , .
~ I~ VITRO ACTIVITY PROFILE
~ ' ' :. .
COMPOUND Anti-Xa HCII APTT TT
OF IC50~ IC50~ IC200~ IC200*t EXAMPLE
, 6 5.7 0.26 8 2.5 . ~ 7A 5.06 0.2S
7C 5.4 0.5l 8C 2.7 0.s7 8D 2.2 0.05 - - ::
9~ 8.7 0.31 9C 8.2 0.3S
l.7 0.99 - - .: -ll 6.1 0.62 - - .
. . :,~ ....
concentration necessary to inhibit S0~ (~g/ml) concentration necessary to double t~e coagulacion time (~g/m1~

' . , .

. , . :. .
.
. , '-,.:

:, ' ~ .

: `
~ , '.

~,. ` :. " ,' .'', ,': . ,.''' ,.'':'' "'.' '.'' . ~ ` ' ' '; ;~ .. .. , . '' .. ' , .. .. '

Claims (9)

1. A saccharide consisting of alternating uronic acid and D-glucosamine residues, characterised in that the uronic acid residues consist of about 10 to about 25%
L-iduronic acid residues, the remainder consisting essentially of D-glucuronic acid residues, the saccharide being from about 35 to about 100%
N-sulphated and between about 0 and about 65%
N-acetylated.

2. A saccharide consisting of alternating uronic acid and D-glucosamine residues, characterised in that essentially all of the D-glucosamine residues are N-sulphated.

3. A saccharide according to claim 2, which has affinity for antithrombin III.

4. A saccharide according to any preceding claim, which is at least 25% 6-O-sulphated.

5. A saccharide according to any preceding claim, which is derived from K5 E. coli saccharide.

6. A modified K5 E. coli saccharide characterised in that essentially all of the D-glucosamine units are N-deacetylated.

7. A modified K5 E. coli saccharide according to claim 6, wherein essentially all of the free amino groups of the D-glucosamine units are sulphated.

8. A process for the preparation of a compound according to claim 1, wherein said compound is further characterised in that it is at least 25% 6-O-sulphated and is derived from K5 E. coli saccharide, which process comprises the following steps:

a) subjecting K5 E. coli saccharide to an N-deacetylation process to give at least 35% free NH2 groups;

b) sulphating said free NH2 groups produced by step a) above;

c) epimerising the product of step b) such that at least 10% of said D-glucuronic acid residues are transformed into L-iduronic acid residues; and d) sulphating at least 25% of the free hydroxy groups of the product of step c).

9. A process for the preparation of a compound according to claim 2, wherein said compound is further characterised in that it is at least 25% 6-O-sulphated and is derived from K5 E. coli saccharide, which process comprises the following steps:

a) subjecting K5 E. coli saccharide to an N-deacetylation process to give essentially 100% free NH2 groups;

b) sulphating said free NH2 groups produced by step a) above;

c) sulphating at least 25% of the free hydroxy groups of the product of step c).