Note: Descriptions are shown in the official language in which they were submitted.
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 ~
6 D
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~ ."
WO92/17S07 PCT/GB92/00571
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 ~
12
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
~ ~ .
17
SIJ~STl?ATE REACTION/ENZY~ DSM DSM D5M
6372 6371 6373
, 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~
18
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.
. . '.
. ,'','.'
PREPARATIVE EXAMPLE 1
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
-23
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 ¦ ~
26
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
27
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
29
EXAMPLE 2
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
~ - .
31
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
32
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~ ~
EXAMPLE 6
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
36
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
37
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.
', .
EXAMPLE 9
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.
EXAMPLE 10
. --
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
41
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 ~
IN VITRO ACTIVITY 2ROFIEE
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 .
.~ . ...
- TABLE 2
~ , , .
~ 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~
'
. ,
.
. , . :. .
.
. , '-,.:
:, '
~ .
: `
~ , '.
~,. ` :. " ,' .'', ,': . ,.''' ,.'':'' "'.' '.'' . ~ ` ' ' '; ;~ .. .. , . '' .. ' , .. .. '
