Field of the Invention

The present invention relates to multimeric compounds useful as anticoagulants, as well as to processes for their production.

Prior Art

Enzymatically modified polysaccharides, consisting of alternating D-glucuronic acid and N-acetyl-D-glucosamine units, have been extensively investigated in relation to the biosynthesis of heparin and heparan sulphate (see, for instance "HEPARIN - Chemical and biological properties, clinical applications", D. Lane and U. Lindahl Editors, published by Edward Arnold, pages 159-190, 1989; and U. Lindahl et al. , TIBS, 11, May 1986, page 221) . Such enzy ic modifications involve the N-deacetylation of the glucosamine units, the subsequent N-sulphation of the resulting free amino groups, C5-epimerisation of the D-glucuronate residues to L-iduronate residues, and O-sulphation at various positions (primarily at C-2 of the iduronic acids and C-6 of the glucosamine units) . Additional enzymatic O-sulphation may also affect the OH groups at the 3-position of the glucosamine residues.

To date, it has only been possible to perform this sequence of enzymatic steps on a microscale basis, suitable only for experimental purposes, to mimic what happens in mammalian mast cells during the biosynthesis of heparin. and heparan sulphate. The chemical and biological differences between heparin and heparan

sulphate are illustrated in B. Casu gt al., Arz. Forsch., 33_, 135, (1983).

The literature also describes methods for the N-deacetylation of N-acetylhexosamine residues present in polysaccharide molecules (L. Thunberg et ^' al. , Carbohydrate Res., 100. 393, [1982] and Sha lee et. al. , Biochem. J., 217. 187 [1984]), as well as procedures for N- and O-sulphation (Levy et al. , Proc. Soc. Exp. Biol. Med., 109 _. , 901 [1962] ) .

EP-A-333243 discloses compounds resulting from extensive sulphation of a 5 saccharide isolated from E. coli strains.

Summary of the Invention

It is an object to provide compounds prepared from K5 which have useful anticoagulant/antithrombotic activity, and which can be produced on a larger scale than is provided by the art.

It is a further object to provide anticoagulants/ antithrombotics prepared from the K5 saccharide of E. coli. thereby allowing mass production of products having particularly good activity.

The present invention provides deacetylated K5 E. coli saccharide, wherein the deacetylation amounts to at least 35% of the acetyl groups of naturally occurring K5.

Brief Description of the Drawings

The accompanying drawings (Figures 1 to 32) are the NMR spectra of various compounds of the invention.

Detailed Description of the Invention

The invention further provides the modified K5 saccharide as defined above, wherein sulphate groups are substituted in all, or substantially all, of the positions on K5 which have been deacetylated. Such positions include those which would normally be expected to be acetylated, particularly the amine groups of the glucosamine, especially D-glucosamine, residues.

The invention also provides a modified K5 as defined above, wherein at least some of the glucuronic acid residues are epimerised to the L-iduronic acid residues.

The invention also provides modified K5 as defined, wherein at least some of the free hydroxyl groups, especially those in the 6- position of the glucosamine acid residues and/or, where appropriate, those in the 2- position of the iduronic acid residues, are sulphated, preferably to an extent of at least 25%.

The invention further provides a saccharide or derivative thereof, comprising substantially units of glucuronic acid and glucosamine, especially where such units alternate, modified as defined above for K5.

The present invention further provides any of the modified compounds as defined, wherein at least some of the residues are 3-0-sulphated.

The present invention also provides the use of the compounds 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 treatment or prevention of conditions requiring antithrombotic or anticoagulant activity.

The present invention also provides a process for the preparation of any of the compounds described above, which process comprises one or more of the following steps:

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

b) sulphating free NH ₂ groups, particularly where they are produced by a) above;

c) epimerising the products of b) such that at least some of the D-glucuronic acid residues are transformed into L-iduronic acid residues; and

d) sulphating at least some of the free hydroxy groups in any resulting compound.

Preferably, said process comprises subjecting polysaccharides of different molecular weights (hereinafter referred to as K5 saccharides) , extracted from certain E. coli strains, to a sequence of chemical and enzymatic passages, which can schematically be illustrated as follows:

a) the K5 saccharides, which consist essentially of an alternate linear sequence of D-glucuronic acid and

N-acetyl-D-glucosamine are subjected to a chemical N-deacetylation process;

b) the free NH ₂ groups of the products obtained under a) are sulphated by means of appropriate sulphating agents;

c) the products obtained under b) are incubated with D-glucuronyl-L-iduronyl-C5-epimerase, extracted from bovine liver, to transform a certain amount of the D-glucuronic acid residues into L-iduronic acid residues;

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 invention provides novel polysaccharides consisting of alternate sequences of uronic acids and glucosamine residues, characterised by the fact that they have a percent content of 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 varying from about 10 to about 25, a minimal percent content of 6-0-sulphated groups of about 25, the compounds being further characterised in that the remaining uronic acids are essentially D-glucuronic acid residues. Such compounds may be prepared by the following process:

a) a K5 saccharide, essentially consisting of an alternate linear sequence of D-glucuronic acid and N-acetyl-D-glucosamine residues is treated with a mixture of hydrazine/hydrazine sulphate, for from about 30 minutes to about 6 hours, at a temperature comprised between about 80 and about 110°C;

b) the compounds obtained under a) are treated with a sulphating agent selected from the complexes between sulphur trioxide and nitrogen organic containing bases, at a temperature between about 45 and about 65°C, for a period of time up to a maximum of 24 hours;

c) the compounds obtained under b) , which are polysaccharides essentially consisting of alternating D-glucuronic acid and D-glucosamine residues containing acetylamino and sulphamino

groups in various proportions are subjected to the action of the enzyme D-glucuronyl-L-iduronyl-C5- epimerase, at about room temperature, for a period of time up to a maximum of two days;

d) the compounds obtained under c) are converted into the corresponding salts of organic nitrogen containing bases and are subsequently treated with a sulphating agent selected from the complexes between sulphur trioxide and the organic nitrogen containing bases, in an inert organic solvent, at a temperature comprised between about -5 and 60°C, for a period of time up to a maximum of 24 hours;

the process being further characterised in that the compounds obtained under d) may optionally undergo the sulphation procedure of step b) .

The above process preferably further comprises subjecting the products as originally defined to the action of 3-O-sulpho ransferase

The present invention further provides K5 saccharides essentially consisting of alternate linear sequences of D-glucuronic acid and N-acetyl- D-glucosamine residues represented by the following formula:

having an average molecular weight varying from about

1000 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 essentially consisting of alternate sequences of

D-glucuronic acid and D-glucosamine units, ^' characterised in that they contain from about 35 to about 100% of

N-sulphated groups, from about 0 to about 65% of

N-acetylated groups and showing characteristic signals in the 13C-NMR spectrum at 104, 60 and 24 ppm.

Other polysaccharides of the invention include polysaccharides essentially consisting of alternate sequences of D-glucuronic acid and D-glucosamine units, characterised in that they contain from about 35 to about 100% of N-sulphated groups, from about 0 to about 65% of N-acetylated groups, a minimal percent content of 6-0-sulphated groups of about 25, the compound being further characterised by a ratio of sulphate groups/ carboxylic groups varying from about 1.0 to about 2.7, optical rotation varying from about +55° to about +65° and having an affinity for 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 like.

The novel polysaccharides obtained according to this sequence, as well as the intermediates of each reaction step, can be recovered as free acids or in the form of their salts, such as their mineral alkali salts, including the sodium, potassium, calcium or magnesium salts, from which, in turn, the compounds per se can be prepared by treatment with mineral or organic acids, for example.

The combination of chemical and enzymatic steps of the present invention is new, and has not previously

been performed starting from polysaccharides of bacterial origin.

According to step a) above, K5 saccharides, which generally have molecular weights in the range of from about 1000 to about 100000 Dalton or more, determined by HPLC, may be treated with hydrazine containing hydrazine sulphate, preferably about 10% by weight of hydrazine sulphate, preferably in a sealed tube, for a period of time suitably varying from about 30 minutes to about 6 hours, at a temperature which may be between about 80 and about 110°C, for example.

By this procedure, a certain percentage of the N-acetyl groups of the glucosamine units is removed and, according to step b) , the resulting compounds are treated with suitable sulphating agents in order to transform the free amino groups into sulphamino groups. Suitable sulphating agents may be selected from the complexes of sulphur trioxide and nitrogen-containing organic bases, such as tri- (C- alkyl)amine.sulphur trioxide, pyridine.sulphur trioxide and analogues thereof. It is generally preferred, but not essential, to use the anhydrous agent, as the presence of even a small amount of water may affect the nature of the final product. Other sulphating agents capable of introducing an S0 _g - group onto the desired position also fall within the scope of the invention.

The N-sulphation reaction is preferably performed at a temperature between about 45 and 65°C and, depending on the period of time for which the reaction is performed, N-sulphation is either more or less extensive. In general, from about 6 to about 24 hours are sufficient for the majority of the free amino groups to be sulphated.

The resulting polysaccharides, which generally consist essentially of alternating D-glucuronic acids and D-glucosamine units containing acetylamino and sulphamino groups in various proportions, may then 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 of the enzyme D-glucuronyl-L-iduronyl-C5-epimerase, obtainable from bovine liver following the procedure of H. Prihar et al. , (Biochemistry, 19 _. , 495 [1980]).

In preferred practice, the polysaccharides obtained under b) are incubated with the enzyme, at room temperature, under conditions which will be apparent to those skilled in the art, for a period of time of from, say, a few hours up to two days. Again, depending on the type of substrate employed and the incubation time, polysaccharides having different degrees of conversion of D-glucuronic acid residues into L-iduronic acid residues can be obtained. Step d) may be performed substantially as described by A. Ogamo e_t al. , (Carbohydrate Res., 193. 165 [1989]), or as illustrated below.

The polysaccharides obtained under c) are advantageously first converted into the corresponding salts of organic nitrogen containing bases such as, for instance, the trimethylamine, triethylamine or tributylamine salts, and are subsequently treated with suitable sulphating agents, such as those employed for the N-sulphation of step b) . The reaction is preferably carried out in the presence of an anhydrous, inert, organic solvent such as, for instance, dimethylformamide, dimethylacetamide, dimethylsulphoxide or mixtures thereof.

The degree of O-sulphation depends on the substrates employed, as well as the reaction conditions.

For the purposes of the present invention, this passage is run for a period of time of up to 24 hours, at a temperature between about -5 and about 60°C.

Generally, from about 5 to about 20 equivalents by weight of the predetermined sulphating agent, calculated over the amount of the N-deacetylated-N-sulphated K5 saccharide, are employed.

Partial N-desulphation may occur during the course of this reaction. If desired, the product of step d) can be subjected to the same N-sulphation procedure as described in step b) .

The polysaccharides thus prepared may be recovered according to techniques known in the art, such as by dialysis of the reaction mixture and subsequent lyophilisation of the dialysed solution, and may be characterised by 13C-NMR and 1H-NMR spectroscopy, which is capable of providing specific fingerprints of the glycosaminoglycans (A. S. Perlin, Methods of

Carbohydrate Chemistry, 1_ [1976], 94; L. Ayotte et al.,

Carb. Res. [1980], 145. 267). Other characterisation techniques, such as HPLC, may also advantageously be employed.

More specifically, H-NMR spectra allow the identification and quantification of the non-sulphated L-iduronic and D-glucuronic acid residues by the signals at 5.35 ppm and 4.55 ppm of the spectra reported in Figures 5, 7 through 11 and 16 (see B. Casu in "HEPARIN, Chemical and biological properties", published by Edward Arnold, Ed's D. Lane and U. Lindahl, 25-49 [1986]).

Other minor signals are detectable in the 13C-NMR spectra associated with end residues, that is, those of the reducing anomeric carbons at 90-95 and 95-98 ppm

(Table 1 in A. S. Perlin and B. Casu; The

Polysaccharides, Vol 1, Academic Press, New York [1982] ,

133) and those of the unsaturated terminal ^' uronic acid residues at 110 ppm (B. Casu et al. , Biochem. J. 187,

599 [1981] ; B. Casu, Nouv. Rev. Fr. Haematol, 26 _. , 211

[1984] ; J. R. Linhardt, J. Biol. Chem. 261. 14448

[1986] ) .

The relative percentages of D-glucuronic acids and L-iduronic acids may also be determined 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. , Biochem. J. , 179, 77 (1979) . The relevant chromatograms are shown in Figure 13.

The analyses of the NMR spectra and of the paper chromatograms indicate that the novel polysaccharides of the present invention have a percent content of 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, 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 to those skilled in the art that those polysaccharides having lower percentages of N-sulphated groups, higher percentages of N-acetylated groups, a per cent content of iduronic acids higher than 25% and a minimal per cent content of 6-0-sulphated groups lower than 25% can also be prepared according to the above processes. Said compounds, as well as the corresponding intermediates in the various reaction

steps, fall within the scope of the present invention.

As stated above, these novel polysaccharides display interesting and useful biological properties, particularly as antithrombotics and anticoagulants, and activities of particular compounds of the invention 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 mg, 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, associated with the use of the products, resulting from the invention for human therapeutic applications such as an ithrombotic and anticoagulant agents. For this purpose the compounds that are the object of the present invention may be formulated by conventional techniques using suitable excipients and other such ingredients for pharmaceutical compositions suitable for parenteral administration, for example.

Examples of formulations for parenteral administration include sterile solutions contained in ampoules, and may also contain substances to render the solution isotonic with bodily fluids, for example.

The compounds obtained as the intermediates in each of the various steps of the process of the invention are, in general, isolated and characterised, but can also be used as such in the subsequent transformations.

I Iff they are characterised, this is made by H-NMR and 13, C-NMR spectroscopy, or any other appropriate means, as illustrated above for the end polysaccharides.

Thus, for instance, the substances prepared in step b) may be polysaccharides consisting essentially of alternating D-glucuronic acids and D-glucosamine residues, containing from about 35 to about 100% of

N-sulphated groups and from about 0 to about 65% of N-acetylated groups. Their 13C-NMR spectra show characteristic signals at 104 ppm, typical of the

D-glucuronic acids, as well as characteristic signals at

60 ppm and 24 ppm, typical of the N-sulphated and the

N-acetylated groups (Figures 4, 5 and 7 through 11) .

Other minor signals detectable in the above spectra are evident at 109 and 103 ppm, and are associated with the terminal uronic acid residues (Figures 4, 5 and 7) .

The intermediates of the various reaction steps possess antithrombotic and anticoagulant properties, and fall within the scope of the present invention.

In particular, the polysaccharides obtained by step b) may be further subjected to O-sulphation, performed substantially as described for step d) .

Again, occasional partial N-desulphation may occur during the course of this reaction. If desired, the O-sulphation can be followed by an N-resulphation carried out as described above. It has been found that the resulting compounds, surprisingly, possess affinity for antithrombin III. This result is surprising, as the presence of L-iduronic acid residues was previously considered to be essential for activity. Accordingly, these compounds which have not been epimerised but which exhibit an affinity for antithrombin III form a particularly preferred feature of the invention.

This class of polysaccharides may be characterised by having alternating D-glucuronic acid and

D-glucosamine residues, a per cent content of N-sulphated groups varying from about 35 to about 100, a per cent content of N-acetylated groups varying from about 0 to about 65 and a minimal per cent content of 6-0-sulphated groups of 25.

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 6-0-sulphated groups of the D-glucosamine residues.

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 skilled in the art that those polysaccharides having alternating D-glucoronic acid and D-glucosamine residues, and further having lower percentages of N-sulphated groups, higher percentages of N-acetylated groups, a per cent content of 6-0-sulphated groups lower than 25 and affinity for Antithrombin III can also be prepared and fall within the scope of the present invention.

Thus, it will be appreciated that the present invention provides a wide range of useful polysaccharides, as well as convenient methods for their preparation.

Extractive procedures for obtaining natural polysaccharides are often tedious and expensive, and various fractionation and de-polymerisation techniques for obtaining the purified native substances are not always capable of providing reproducible products.

These drawbacks, including those of relying on crude animal extracts (which run the risks inherent in animal

illness, epidemics and so on) , are overcome by the present invention as microorganisms provide a practically unlimited source of starting materials, and can be kept under carefully controlled conditions while still synthesising 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 converted into the corresponding 0-sulphated groups. Such compounds are also defined above.

Such reactions are preferably carried out in the presence of the enzyme 3-0-sulphotransferase, which may be prepared as described in Preparative Example 1, below.

The polysaccharides may then be incubated with the enzyme under conditions known in the art, to allow 3-O-sulphation.

The K5 saccharides employed as the starting materials in the present invention may be prepared by cultivating strains of Escherichia coli, under aerobic conditions, in a suitable fermentation medium. It has been found that, while the nature of the resulting saccharide cannot be exactly predicted, higher levels of carbon source, especially glucose, tends to give rise to higher molecular weight forms of K5 polysaccharide.

Strains of E. coli which can be employed for the purposes of the present invention are those exhibiting the presence of the K5 capsular polysaccharide antigen, and are available with several different sources, including the American Type Culture Collection and the International Escherichia Centre of the Statens Serum

Institut of Copenhagen, Denmark.

Other E. coli strains which may be employed for the purposes of the present invention are again available with several different strain collections or are of clinical isolation, mainly from pyelonephritis and urinary tract infections. They may be characterised via API SYSTEM 20 ε and as strains showing the presence of the K5 capsular polysaccharide antigen according to . Ni mich et al.. Z. Gesamte Hyg., 15.(10), 583 (1989), or D. S. Dupte et al.. , Sem. Microbiol. Letters, 14, 75 (1982) . Some of these clinically isolated E. coli strains have been deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-3300, Germany, on February 27, 1991, under .the provisions of the Budapest Treaty. These strains were assigned the accession numbers DSM 6371, DSM 6372 and DSM 6373. By way of example their characteristics are reported below:

TEST SUBSTRATE REACTION/ENZYME

β-galactosidase arginine dehydrolase lysine decarboxylase ornithine decarboxylase citrate utilisation production of H ₂ S urease tryptophan deaminase indole production acetoin production gelatinase fermentation fermentation- oxidation fermentation- oxidation do + do + + do + + do do do cytochrome oxidase NO2 production reduction of N0 ₂ to N ₂ mobility + + culture in + + + fermentation (under oil) fermentation (air)

+ = positive = negative do = same as preceding exampl

These E. coli strains showed the presence of the K5 capsular polysaccharide antigen, determined as described above. The E. coli strains useful for the present invention may be maintained on standard 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.

SUBSTITUTE SHEET

The following Examples and Preparative Examples are provided only for the purpose of better illustrating the present invention, and are not to be construed as limiting the present invention in any way.

PREPARATIVE EXAMPLE 1

Isolation of 3-O-Sulphotransferase

3-0-sulphotransferase may be prepared from Furth mast cell tumours extracted from mice available at the Swedish University of Agricultural Sciences, The Biomedical Centre, Box 575 s-751 23 Uppsala, Sweden. Furth mast cell tumours can be developed in normal mice strains, as described by J. Furth et al. , Proc. Soc. Exp. Biol., 95., 824 (1957). A specific preparation is as follows.

Mastocytoma tumours (about 70g of tissue) were homogenised in 200 ml of 0.05 M Tris-1% Triton X-100, pH 7.4, containing protease inhibitors: 10 μg/ml of pepstatin, 2 mM EDTA and 1 mM of PMSF (Sigma Chemical Co) .

The homogenate was gently stirred for 1 hour at 4°C and was then centrifuged at 100000 x g for 1 hour. The supernatant was passed through a glass fibre filter and subjected to the following purification protocol:

1) Heparin-Sepharose, 31 ml column, which was first washed with buffer A (0.05 M Tris - 0.1% Triton X-100, pH 7.4, 1 μg/ml of pepstatin, 2 mM EDTA, 20% of glycerol containing 0.15 M NaCl), and was then eluted using a linear gradient of 0.15 - 1.0 M NaCl in buffer A. Eluent fractions of 4 ml were assayed for

O-sulphotransferase activity substantially following the procedure described by Jansson et. al.. , Biochem. J. , 149 ,

49 (1975) . O-Desulphated heparin was used as a sulphate acceptor. Ten μg of acceptor, 5 μCi of 35S PAPS and enzyme protein in a total volume of 100 μl of

50 mM HEPES, 10 mM MnCl ₂ , 10 mM MgCl ₂ , 5 mM CaCl ₂ , 3.5 μM NaF, 1% TRITON X-100 ^R , pH 7.4, were incubated at 37°C for 30 minutes. The reactions were terminated by the addition of 400 μl of ethanol, containing 1.3% sodium acetate, along with 0.4 mg of carrier heparin, and the samples were left at -20°C overnight. After centrifugation (13000 rpm for 10 minutes) the supernatants were discarded and the pellets were dissolved in 100 μl of water.

The 35S-labeled polysaccharide was separated from residual unincorporated label by centrifugation through

Sephadex, as follows. Syringes (5cm x 0.9cm i.d.) were packed with Sephadex G-25, superfine grade, equilibrated with 0.2 M NH.HC0 ₃ , and were then centrifuged at

2000 rpm for 5 minutes and suspended in conical centrifuge tubes, to eliminate most of the liquid. The samples (100 μl) were applied to packed and centrifuged syringes, which were then again centrifuged in the same manner. The labeled polysaccharides were recovered in the effluents collected in the tubes, whereas low molecular weight labeled compounds were retained by the gels. The effluents were analysed by scintillation photometry. The active fractions were pooled and dialysed against buffer A containing 0.15 M

NaCl.

2) Sepharose Blue, 41 ml column, flow rate 15 ml/hour. Following application of the sample, the column was washed with buffer A containing 0.15 M NaCl and again eluted with a linear salt gradient from 0.15 to 1.0 M NaCl to obtain the last portion of the enzymatic

activity. Fractions of 5 ml were assayed for O-sulphotransferase activity as described above. The active fractions were pooled, concentrated to about 10 ml and dialysed against buffer A - 0.075 M NaCl. To the sample was subsequently added β-mercaptoethanol to 12 mM concentration.

The obtained degree of purification was 150 fold, with an apparent 100% recovery of O-sulphotransferase activity. The sample obtained by the purification through Sepharose Blue contained demonstrable glucosaminyl 6-O-sulphotransferase, iduronosyl 2-O-sulphotransferase and glucosaminyl 3-0-sulphotransferase.

PREPARATIVE EXAMPLE 2

Cultivation of E. coli and Production of K5

Cultivation Media

For producing the K5 saccharides, E. coli may be cultivated under aerobic conditions in an aqueous nutrient medium containing assimilable sources of: carbon; nitrogen; and 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 experience of the skilled technician, in order to obtain K5 saccharides of the desired average molecular weight as well as improve the yields of the fermentation. The preferred carbon sources are glucose, mannose, galactose and peptone. Preferred nitrogen sources are ammonia, nitrates, soybean meal, peptone, meat extract, yeast extract, tryptone and amino acids. Among the preferred

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, chloride, carbonate, phosphate, hydrogenphosphate, dihydrogenphosphate and nitrate anions.

Ordinarily, the K5 saccharide-producing strains are pre-cultured in a shake flask, then the cultures are put into jar fermenters for the production of substantial quantities of the above saccharides. A typical representative fermentation medium, which can also be employed for the preculture, has the following composition for 1 litre:

K ₂ HP0 ₄ 3.6 g

KH ₂ P0 ₄ 1.2 g

Casamino acid 20 g

Sodium citrate dihydrate 0.5 g

Ammonium sulphate 1 g

Glucose 4 g

MgS0 ₄ 0.15 g

The dialysable part of 100 g of yeast extract in 100 ml of water (dialysis against water, cut-off of the membrane: 15000 D) 1 litre

The pH of the medium is 7.2.

Fermentation

a) Pre-culture - One loop of E. coli from a Loeb agar plate is suspended in 5 ml of Merck Standard 1 medium and incubated for 6 hours, at a pH of about 7.2, at 37°C. The mixture was then put into 700 ml of the above culture medium and incubated overnight under the same conditions.

b) Fermentation - The fermentation is carried out under aerobic conditions at an effective pH of 6.8 for a period of time varying from about 1 to 10 hours, at 37°C.

A jar fermenter containing 10 litres of the medium:

K ₂ HP0 ₄ KH ₂ P0 ₄

Casamino acid Sodium citrate dihydrate Ammonium sulphate Glucose MgS0 ₄

The dialysable part of lOOOg of yeast extract in 1 litre of water (dialysis against water, cut-off of the membrane: 15000 D) 10 1

is employed.

Analogous fermentation media, containing glucose or other carbon sources in amounts lower than those indicated, or up to about 5% w/v, can also advantageously be employed.

Extraction of K5

Extraction - The K5 saccharides are recovered from the fermentation media as under b) , according to the procedure described by W. Vann et al. , Eur. J. Biochem. 116, 359 (1981) , or as follows.

The fermentation broth is centrifuged for about 35 minutes at 5200 rpm. The obtained sediments are suspended in about 800 ml of phosphate buffer and the

resulting suspension is centrifuged for 15 minutes at 8000 rpm, and the supernatant is removed. The sediment is suspended in 800 ml of 50 mM Tris 5 mM EDTA buffer pH 7.3 (prewarmed at 37°C) and the suspension is kept for 30 minutes at 37°C in a water bath. The suspension is then centrifuged at 9000 rpm for 20 minutes. The supernatant is removed and the extraction is repeated three times.

The supernatants of the Tris/EDTA-extractions are combined and added with an aqueous 10% solution of CETAVLON (Trade mark) until no further precipitation is observed and the mixture is kept overnight at room temperature. The mixture is centrifuged at 20°C and 8000 rpm for 10 minutes, the precipitate is dissolved in about 10-20 ml of aqueous 1 M NaCl, the solution diluted with at least 8 volumes of ethanol, centrifuged. The solid, consisting of the crude K5 saccharide, is collected and redissolved in about 10-20 ml of 1 M NaCl and at least 8 volumes of ethanol added. The precipitate is collected by centrifugation, dissolved in water and dialysed in a dialysis bag with a cut-off of 3500 Dalton for 24 hours. The dialysed solution is centrifuged for 20 minutes at 9000 rpm at 4°C and the precipitate is discarded. The obtained supernatant, consisting of purified K5 saccharide, is freeze-dried.

Lipopolysaccharides are removed by dissolving in water the obtained purified K5 saccharide to a concentration of 2-3% and centrifuging the obtained solution at 45000 rpm for 4 hours at 4°C. The presence of traces of residual lipopolysaccharide does not affect the processing of the starting materials. In order to remove E. coli ribonucleic acid, the resulting solution is treated for 24 hours with ribonuclease (Sigma) in phosphate buffer containing MgCl ₂ (10 mM) and then dialysed against deionised water with a dialysis bag

having a cut-off 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) K5 saccharide having an average molecular weight of about 5000 Daltons determined by HPLC. This K5 saccharide has the 13C-NMR spectrum reported in Figure l, showing the typical major signals of D-glucuronic acids at 104 ppm, and of N-acetyl-D-glucosamine units at

55 and 98 ppm, as taught by W. Vann e al. , in Eur. J.

Biochem., 116, 359 (1981) , and typical minor signals at

110 and 103 ppm, ascribable to terminal residues derived from D-glucuronic acid, as taught by B Casu et al. ,

Biochem. J. , 19_7, 599 (1981) .

B) K5 saccharide having an average molecular weight of about 50000 Dalton, as determined by HPLC. This K5 saccharide has the 13C-NMR spectrum shown in Figure 2, showing the typical major signals of D-glucuronic acids at 104 ppm and of N-acetyl-D-glucosamine units at 55 and

98 ppm

C) K5 saccharide having an average molecular weight of about 100000 Dalton, as determined by HPLC. The

13 C-NMR spectrum of this K5 saccharide is shown m

Figure 3. Typical major signals of D-glucuronic acids and N-acetyl-D-glucosamine units are at 104 ppm and at

55 and 98 ppm respectively.

The above determinations confirm that the starting K5 saccharides have the following repeating disaccharide unit:

The 13C-NMR and 1H-NMR spectra of the compounds prepared according to the multi-step processes of the present invention, as well as those of the starting K5 saccharides, were recorded from solutions in D_0 with a Bruker CXP-300 spectrometer, with the exception of the compound of Example 8D, whose C-NMR spectrum was recorded from a solution in D O with a Bruker spectrometer AMX500.

Determination via HPLC of the molecular weights of the K5 saccharides was performed using Superose 6 (Trade mark) and Superose 12 (Trade mark) columns equilibrated with 1 M NaCl in 0.05 M TRIS-HC1 pH 8 buffer. The purity (polysaccharide content) of the compounds of the invention and the relevant intermediates was assayed by the carbazole method (T. Bitter and H.M. Muir, Anal. Biochem., 4., 330 [1962]). The degree of purity was generally higher than 90%. The optical rotations were determined at room temperature at a concentration of 1% in water, with a JASCO DIP370 polarimeter. The determined values were subsequently corrected, taking into account the purity degree of the test sample.

EXAMPLE 1

Preparation of N-deacetylated-N-sulphated polysaccharides from K5-saccharide A (steps a) and b) )

A) In a vial, 100 mg of K5 saccharide A and 138 mg of hydrazine sulphate were dissolved in 1.38 ml of hydrazine. The solution was frozen by placing the vial into liquid nitrogen, while keeping the solution under a nitrogen atmosphere. The vial was then sealed and slowly brought to room temperature, then heated for 5 hours at 96°C. The vial was then re-frozen with liquid nitrogen, opened, and slowly brought to room temperature. The solution was poured into a round-bottom vessel, washing the vial out with 5 ml of toluene.

The solution was concentrated under reduced pressure and the operation was repeated twice (each with 20 ml of toluene) , to evaporate off (together with toluene) most of the hydrazine. The residue was then added to 50 ml of distilled water and the resulting solution brought to neutrality by means of aqueous 37% hydrochloric acid, then dialysed for 5 days through a 3500 Dalton cut-off membrane, against sodium chloride solutions and water (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 2 1, H ₂ 0 the fourth and fifth day) . The solution was then concentrated under reduced pressure, and dissolved in 65 ml of distilled water.

The pH of the obtained solution was adjusted to 9 by the addition of solid sodium bicarbonate, and the temperature raised to 55°C. At this temperature, under continuous stirring, 65 ml of the adduct of trimethylamine.sulphur trioxide was added to the solution, which was kept at this temperature for one

hour, then a further 65 ml of the same adduct was added, and the whole was reacted for an additional 5 hours. The solution was dialysed against aqueous solutions of sodium chloride of decreasing concentrations and water as described above. The dialysed solution was finally freeze-dried and 80 mg of a product, having the C-NMR spectrum of Figure 4, were obtained.

The product shows the following characteristic signals:

strong signals:

60 ppm, N-sulphated groups

62 ppm, unsubstituted 6-hydroxyl groups of glucosamine residues 98 ppm, glucosamine residues 104 ppm, glucuronic acid residues

weak signals:

109 and 103 ppm, terminal glucuronic acid residues

very weak signals:

24 ppm, residual N-acetylated groups.

The percent content of N-acetylated groups, determined by C-NMR from the ratio of the area of the signal at 24 ppm to that of the area of the signals of all anomeric (C-1) carbons in the 100-110 ppm region, was about 5. The per cent content of N-sulphated groups was about 95. No signals ascribable to free -NH ₂ groups were observed.

B) Following the same procedure of the foregoing preparation, starting from 100 mg of K5 saccharide A and

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 about 15, determined by 13C-NMR (Figure 5) as for the product of Ex. IA, and a percent content of N-sulphated groups of about 85. The C-NMR spectrum ^' of Figure 5 shows the following characteristic signals:

24 and 55 ppm (weak) : N-acetylated groups

60 ppm (strong) : N-sulphated groups

62 ppm (strong) : unsubstituted 6-hydroxy groups of D-glucosamine residues 98 ppm (strong) : D-glucosamine residues 104 ppm (strong) : D-glucuronic acid residues 103 and 109 ppm (weak) : terminal D-glucuronic acid -residues No signals of free - H ₂ are present.

C) Following the same procedure for the preparation of the product of Example IA, starting from 100 mg of 5 saccharide A) and limiting to one hour the reaction with hydrazine sulphate/hydrazine, 75 mg of a product were obtained, having a percent content of N-acetylated groups of about 30, as determined by H NMR (Figure 6) from the ratio of the area of the signal of the N-acetylated groups at 2.1 ppm and the total area of the signals of the anomeric hydrogens (between 5 and 6 ppm) , and the 13C-NMR spectrum of Figure 7, showing the same characteristic signals of the compound of Example IB.

The compound has a percent content of N-sulphated groups of about 70. No signals characterisitic of free - H ₂ groups are present.

EXAMPLE 2

Preparation of an N-deacetylated-N-sulphated polysaccharide from K5-saccharide B (steps a) and b) )

Starting from 100 mg of K5 saccharide B, and following the same procedure described for the preparation of the product of Example IA, carrying out the reaction with hydrazine sulphate/hydrazine for 5 hours, 75 mg of a product were obtained having a percent content of N-acetylated groups less than 5, determined by 13C-NMR (Figure 8) as for the product of Example

IA, and a percent content of N-sulphated groups higher than 95. The 13C-NMR spectrum of Figure 8 shows the same characteristic signals of the compound of Example

IA. No signals characteristic of free NH» groups are present.

EXAMPLE 3

Preparation of N-deacetylated-N-sulphated polysaccharides from K5-saccharide C (steps a) and b) )

The following polysaccharides were prepared, starting from 100 mg of K5 saccharide C) , following the same procedure illustrated for the preparation of Example IA, with different times for the reaction with hydrazine sulphate/hydrazine.

A) Reaction time with hydrazine sulphate/hydrazine: 5 hours. Yields: 85 mg of a product having a percent content of N-acetylated groups of about 2, and a percent content of N-sulphated groups of about 98, determined as above described by the 13C-NMR (Figure 9) . The spectrum shows the same characteristic signals as the

compound of Example IA.

B) Reaction time with hydrazine sulphate/hydrazine: 2.5 hours. Yields: 80 mg of a product having a percent content of N-acetylated groups of about 23, 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 compound of Example IB.

C) Reaction time with hydrazine sulphate/hydrazine: 80 minutes. Yield 75 mg of a product having a percent content of N-acetylated groups of about 48, a percent content of N-sulphated groups of 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 IC.

The NMR spectra of the polysaccharides prepared in Examples 1, 2 and 3 clearly indicate that no other modification of the structure of the starting 5 saccharides has occurred.

EXAMPLE 4

Preparation of C5-epimerised-N-sulphated polysaccharides (step c)

A) 10 mg of the product of Example IA were incubated with 8 mg of a preparation obtained from bovine liver as described by H. Prihar (see above) containing the enzyme D-glucuronyl-L-iduronyl-C5-epimerase, in 1 ml of 0.05 M Hepes, pH 7.4, containing 50 mM potassium chloride, 15 mM EDTA and 1% of Triton X-100 . The mixture was kept at room temperature for 2 days, then the desired

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

The low-field region of the H-NMR spectrum (Figure 12) clearly shows signals at 4.7 and 4.95 ppm typical of the L-iduronic acid residues (Perlin A. S. in Methods in Carbohydrate Chemistry Vol. VII, Academic Press, New York [1976] , p 94) , the area of which corresponds to 18% of the total area of uronic acids. A similar result was 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 obtained in Example 2 were epimerised following the procedure of A) above. Seven mg of product were obtained. The H-NMR spectrum (Figure 14) shows signals typical of L-iduronic acid residues at 4.7 and 4.95 ppm, the area of which corresponds to 22% of the total area of uronic acids.

Figure 13 refers to the paper chromatograph, performed as described above, from which a percent content of L-iduronic acids of 20 is determined.

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 Figure 13, from which a percent content of L-iduronic acids of 19 was determined.

The NMR spectra of the polysaccharides prepared in Examples 4A and 4B clearly indicate that no other structural modification than the C5-epimerisation has occurred. In view of the starting substrate and the epimerisation procedure, neither has any other

structural modification occurred for the product of Example 4C.

EXAMPLE 5

Preparation of O-sulphated-N-sulphated-C5-epimerised polysaccharides (step d)

A) 100 mg of the product prepared in Example 4A were dissolved in 20 ml of water and passed through an Amberlite (Registered Trade Mark) IR-120 H ⁺ column at room temperature. The column was subsequently washed with 20 ml of water. The eluates were collected and brought to pH 5.5 with a 10% solution of tributylamine in ethanol. The excess of tributylamine was extracted three times with 40 ml each of diethyl ether and the aqueous solution containing the tributylamine salt of the product of Example 4A was freeze-dried. 100 mg of the resulting product were dissolved in 32 ml of anhydrous dimethylformamide, 765 mg of the adduct pyridine.sulphur trioxide dissolved in 15 ml of anhydrous dimethylformamide was added to the solution and the resulting mixture was kept at 0°C for 1 hour. An equal volume of water was added, the solution was brought to pH 9 with aqueous 4% sodium hydroxide and the whole was dialysed against aqueous solutions of sodium chloride of decreasing concentrations and water, as described in Example IA.

The dialysed solution was freeze-dried and the resulting product, upon transformation into the corresponding tributylamine salt, was reacted under the same conditions of the present Example. 180 mg of a product were obtained, which was finally reacted with the trimethylamine.sulphur trioxide adduct as described in Example IA

180 mg of the title compound were obtained, having the 13C-NMR spectrum shown m Figure 15, showing characteristic signals at 60 ppm (strong) : N-sulphated groups; 62 ppm (strong) : unsubstituted 6-hydroxy groups of D-glucosamine residues; 69 ppm (medium) :

6-0-sulphated groups; 98 ppm (strong) : glucosamine residues; 99 ppm (medium) : non-sulphated L-iduronic acid residues; 104 ppm (strong) : D-glucuronic acid residues.

The product has a minimum percent content of 6-0-sulphated groups of 52, determined from the area of the same signal at 69 ppm.

B) Following the procedure of Example 5A above, and starting with 100 mg of the product of Example 4B, 160 mg of a product were obtained having the C-NMR spectrum of Figure 16, and showing the same characteristic signals as the compound prepared under Example 5A.

The product has a minimum percent content of 6-0-sulphated groups of 44, determined from the area of the signal at 69 ppm

C) Following the procedure of Example 5A above, and starting with 5 mg of the product of Example 4C, 9 mg of a product were obtained.

In view of the starting substrate and the sulphation procedure, no other structural modification has occurred for the product of Example 5C.

EXAMPLE 6

Preparation of a non-epimerised N- and 0-sulphated polysaccharide

100 mg of the product prepared in Example IA were dissolved in 20 ml of water and passed through an Amberlite (Registered Trade Mark) IR-120 H ⁺ column at room temperature. The column was subsequently washed with 20 ml of water. The eluates were collected and brought to pH 5.5 with 3 ml of a solution of tributylamine (10% in ethanol) . The excess of tributylamine was extracted (three times) with 40 ml of ethyl ether, and the solution containing the tributylamine salt of the product of Example IA was freeze-dried.

180 mg of the obtained salt were dissolved in 32 ml of anhydrous dimethylformamide. To this solution 765 mg of anhydrous pyridine.sulphur trioxide adduct, dissolved in 15 ml of anhydrous dimethylformamide, were added and the mixture was kept 1 hour at 0°C. The reaction mixture was combined with an equal volume of water and the pH brought to 7.0 with aqueous 4% NaOH. The mixture was then dialyse against NaCl solutions of decreasing concentrations as described in Example IA.

The dialysed solution was freeze-dried, and the resulting product upon transformation into the corresponding tributylamine salt, was reacted under the same conditions of the present Example. 90 mg of a product were obtained, which was treated with the adduct trimethylamine.sulphur trioxide under the same conditions of Example IA. A product was obtained, having a minimum percent content of 6-0-sulphated groups of 35 determined from the area of the signal at 69 ppm in the 13C-NMR spectrum (Figure 17) , and having the same characteristic signals as the product of Example IA.

EXAMPLE 7

Preparation of non-epimerised N- and 0-sulphated polysaccharides

The tributylamine salt of the compound of Example IA was prepared as described in Example 6, and 180 mg of this salt were dissolved in 32 ml of anhydrous dimethylformamide. The solution was cooled to 0°C and 765 ml of a solution of anhydrous pyridine.sulphur trioxide prepared at 0°C in 15 ml of anhydrous dimethylformamide added. The resulting mixture was kept 1 hour at 0°C and was subsequently mixed with an equal volume of distilled water. The pH was adjusted to 9 by means of 4% sodium hydroxide and 4 volumes of ethanol saturated with sodium acetate were added. The mixture was kept overnight at 4°C, whereby a precipitate was obtained, which was dissolved in 50 ml of distilled water. The resulting solution was dialysed against distilled water for 3 days (3 x 2 1 each day, cut-off 14000 D) and finally freeze-dried.

The following compounds were obtained in reδpetitions of the above procedure:

A) Yield: 85 mg. The compound showed a percent content of N-sulphated groups of about 95 and a percent content of 6-0-sulphated groups of about 100 (signals at 60 and 69 ppm, respectively, in the 13C-NMR spectrum of

Figure 18), a ratio Sθ ^" /COθ ^" of about 2.1, and an

[ ] _D Of +55.4°.

B) Yield: 94 mg. The compound showed a percent content of N-sulphated and 6-0-sulphated groups similar to that of Compound 7A (signals at 60 and 69 ppm, respectively, in the 13C-NMR spectrum of Figure 19) , and a ratio

SO ^" /COO ^" of about 2.2.

C) Yield: 82 mg. The compound showed a percent content of N-sulphated and 6-0-sulphated groups similar to that of Compound 7A (signals at 60 and 69 ppm, respectively, m the 13C-NMR spectrum of Figure 20) , and a ratio

SO ^" /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 6-0-sulphated groups of about 85 (signals at 60 and 69 ppm, respectively, in the 13C-NMR spectrum of

Figure 21) , and a ratio Sθ ^" /COθ ^" of about 1.8.

EXAMPLE 8

Preparation of non-epimerised N- and 0-sulphated polysaccharides

The procedure of Example 7 was repeated, using the tributylamine salt of the compound of Example IA as teh starting material, and keeping the mixture of the two solutions at 0°C for different times. The following compounds were prepared in repetitions of the procedure:

A) Yield: 80 mg. Reaction time: 20 minutes. The compound showed a percent content of N-sulphated groups of about 95 and a percent content of 6-0-sulphated groups of about 70 (signals at 60 and 69 ppm,

13 respectively, in the C-NMR spectrum of Figure 22) , and a ratio Sθ ^" /COθ ^" of about 1.6.

B) Yield: 92 mg. Reaction time: 40 minutes. The compound showed a percent content of N-sulphated groups of about 95 and a percent content of 6-0-sulphated groups of about 75 (signals at 60 and 69 ppm, respectively, in the C-NMR spectrum of Figure 23) , and a ratio SO ^" /COO ^" of about 1.7.

C) Yield: 90 mg. Reaction time: 4 hours. The compound showed a percent content of N-sulphated and

6-0-sulphated groups similar to that of Compound 7A

(signals at 60 and 69 ppm, respectively, in the

13 C-NMR spectrum of Figure 24) , a ratio

SO ^" /COO ^" of about 2.3, and an [ ] of +62.4°.

D) Yield: 95 mg. Reaction time: 5.5 hours. An N-resulphation was performed as described in Example IA. The compound showed a percent content of N-sulphated and 6-0-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

Sθ ^" /COθ ^" of about 2.52.

EXAMPLE 9

Preparation of non-epimerised N- and 0-sulphated polysaccharides

The procedure of Example 7 was repeated, again using the tributylamine salt of the compound of Example IA as the starting material, and using pyridine.sulphur trioxide adduct not prepared to be anhydrous. The following compounds were prepared in repetitions of the above procedure:

A) Yield: 83 mg. The compound showed a percent content of N-sulphated and 6-0-sulphated groups similar to that of Compound 7A (signals at 60 and 69 ppm, respectively, in the 13C-NMR spectrum of Figure 26) , a ratio SO ^" /C00 ^" of about 1.9, and an [ ] _D of +56.4°.

B) Yield: 75 mg. The compound showed a percent content of N-sulphated and 6-0-sulphated groups similar to that

of Compound 7A (signals at 60 and 69 ppm, respectively, in the 13C-NMR spectrum of Figure 27) , a ratio

SO ^~ /COO ^~ of about 1.9, and an [α] _D of +56.2°.

C) Yield: 70 mg. The compound showed a percent content of N-sulphated and 6-0-sulphated groups similar to that of Compound 7A (signals at 60 and 69 ppm, respectively, m the 13C-NMR spectrum of Figure 28) , a ratio

SO ^" /COO ^" of about 2.05, and an [α]_ of +56.7°.

EXAMPLE 10

Preparation of a non-epimerised N- and 0-sulphated polysaccharide

The tributylamine salt of the compound of Example IA was prepared as described in Example 6, and 180 mg of this salt were dissolved in 32 ml of anydrous dimethylformamide at room temperature. Keeping the solution at room temperature, a solution of 1.53g of the anhydrous adduct pyridine.sulphur trioxide prepared in 30 ml of anhydrous dimethylformamide at room temperature was added. The resulting mixture was kept at room temperature for 5.5 hours, and was subsequently mixed with an equal colume of distilled water. The pH was adjusted to 9 by means of 4% sodium hydroxide, and the compound was recovered as described in Example 7. The resulting material was subjected to N-resulphation, perfomed as described in Example IA. The reaction mixture was diluted with 4 volumes of ethanol saturated with sodium acetate and the end product was recovered as described in Example 7.

A percent content of N-sulphated and 6-0-sulphated groups similar to that of Compound 7a was detected (signals at 60 and 69 ppm, respectively, in the

13C-NMR spectrum of Figure 29) , with a ratio

S0 ^" /C00 ^" of about 2.5, and an [α] of

+59.4° . Yield: 86 mg.

EXAMPLE 11

Preparation of a non-epimerised N- and 0-sulphated polysaccharide

The procedure of Example 10 was repeated, but wherein the two solutions were prepared at 55°C, and mixed and kept at this temperature for 24 hours. The obtained compound showed a percent content of N-sulphated and 6-0-sulphated groups similar to that of

Compound 7A (signals at 60 and 69 ppm, respectively, in the 13C-NMR spectrum of Figure 30) , and a ratio

SO ^" /COO ^" of about 2.7. Yield 77 mg.

EXAMPLE 12

Preparation of non-epimerised N- and 0-sulphated polysaccharides

The tributylamine salts of the compounds of Example 3A, 3B and 3C were prepared as described in Example 6. These salts were treated with the anhydrous adduct of pyridine.sulphur trioxide, and recovered from the reaction medium, as described in Example 7. The following compounds were prepared in repetitions of the above procedure.

A) Yield: 80 mg. The starting tributylamine salt was that of the compound of Example 3A. The compound showed

a ratio S0 ^~ /C00 ^" of about 2.

B) Yield: 72 mg. The starting tributylamine salt was that of the compound of Example 3B. The compound showed a percent content of N-sulphated groups of about 77 and a percent content of 6-0-sulphated groups of about 100

(signals at 60 and 69 ppm, respectively, in the

13 C-NMR spectrum of Figure 31) , and a ratio

SO ^" /COO ^" of about 1.8.

C) Yield: 90 mg. The starting tributylamine salt was that of the compound of Example 3C. The compound showed a percent content of N-sulphated groups of about 52 and a percent content of 6-0-sulphated groups of about 100

(signals at 60 and 69 ppm, respectively, in the

13 C-NMR spectrum of Figure 32) , and a ratio

SO ^" /COO ^" of about 1.5.

EXAMPLE 13

Enzymatic 3-O-sulphation

2 ml of a reaction mixture were prepared using the following ingredients: 2 mg of the compound of Example 5B; 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 MnCl ₂ - 5 mM CaCl ₂ - 10 mM MgCl ₂ - 3.5 μM NaF - (0.5 - 1%) Triton X-100, pH 7.4. The reaction mixture was incubated for 2 hours at 37°C, then the reaction was terminated by heating at 100°C for 2 minutes. The denatured proteins were removed by centrifugation and the supernatant passed through a column (0.8 x 100cm) of Sephadex G-15 equilibrated with 0.2 M H NNO- . The eluted material was freeze-dried.

A sample (500 μg) of the material thus prepared, dissolved in 500 μl of 50 mM Tris-0.5M NaCl, pH 7.4, was applied to a 3 ml column of antithrombin III - Sepharose, equilibrated in the same buffer. The column was then eluted with 50 mM Tris-2 M NaCl, pH 7.4. The eluate contained 10% of the total material.

EXAMPLE 14

Activity of Compounds of the Invention

The activity of certain compounds of the invention was evaluated in vitro using the following tests:

a) Anti-Xa activity, performed as described by A. N. Teien et al. , Thrombosis Res., J3, 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. Bell et al. , Nature, 174. 880 (1954) , but diluting the plasma sample 1:1 with saline; and

d) TT, performed substantially as described by C. Eika et al.. Lance II, 376 (1972) , but diluting the plasma sample 1:1 with saline.

The results obtained are summarised in the following table:

5A 11 0.4 20

5B 56 0.46 39 13

5C 59.7 0.05 3.55 1.2

* concentration necessary to inhibit 50% (μg/ml) ** concentration necessary to double the coagulation time (μg/ml)

The results are indicative of the clinical usefulness of the polysaccharides of this invention as antithrombotic and anticoagulant agents.

EXAMPLE 15

Properties of various non-epimerised, N-, 0-sulphated compounds of the Examples are Table 2 below.

* concentration necessary to inhibit 50% (μg/ml) ** concentration necessary to double the coagulation time (μg/ml)