Note: Descriptions are shown in the official language in which they were submitted.
The present invention relates to a
process for the depolymerization and sulfatation of po.
saccharides.
Sulfated polysaccharides are compounds
having a great importance in cosmetic~ textile, alimentary
and pharmaceutical industry. More particularly their
use is recommended in ~revention of venous throm~osis
(I.B. Jacques, Pharmacological Reviews, 1979r 31r 99-166)~
Besides r low molecular weiyht sulfated
polvsaccharides have been proposed as antithrombotic
non-anticoagulant agents, thus involving a weak hemorraglc
risk (D.P. Thomas, Seminars in Hematology, 1978, 15,
1-17).
Low molecular weight sulfated poly-
saccharid2s are obtained by sulfatation or low molecularweight polysaccharides. The sulfatation is generally
carried Ollt by treatment with chlorosulfonic acid in
~yridine (~.L. Wol rom et al., J. Am. Chem. Soc. lg53,
7S, 1519) or with adducts of sulrur trioxide (sulfuric
anhydride) with aprotic solvents (P.L. Whilster, W.W.
Spencer, Methods Carbohydrate Chem., 1964, ~ 297-298;
R.L. Whilstex~ i~id. , 1972, ~, 426-~29).
The low molecular weight polysaccharides
are generally obtained by fractionating a whole of species
~5 with various molecular weights or by controlled de~oly-
merisation of non-fract.ionated ~olysaccharides ~with
nitrous acid~
Ho~ever, the known sulfatation processes
~resent some disadvantages, particularly due to the
operating conditions and to the di~ficulty o controlling
the reaction.
The depolymerisation processes, on the
other hand, also present the disadvantage of giving a certain
percent of inactive products.
In the case of N-sulfated polysaccharides
~,". ..
-- 2
such as heparin, the depolymerisation processes also
involve a hydrolysis of said N-sulfated group, essential
to the biological activity of heparin.
It has now surprisingly been found that by reacting a
polysaccharide with a mixture of sulfuric acid and chloro-
sulfonic acid both a depolymerisation and a sul:Eatation
take place concurrently. This finding is particularly
surprising, especially because it has also been found that
the sulfatation is always total on the possibly present
primary hydroxy groups.
In accordance with the present invention, therefore,
there is provided a process for the depolymerisation and
sulfatation of polysaccharides, which comprises reacting
said polysaccharide with a mixture of sulfuric acid and
chlorosulfonic acid.
In the mixture, the two acids are concentrated;
preferably their concentration is at least 95% by weight.
The ra-tio of the two acids is highly variable and may
go from traces of chlorosulfonic acid in sulfuric acid up
to a ratio sulfuric acid:chlorosulfonic acid 4:1 by volume.
Advantageously, the ratio sulfuric acid/chlorosulfonic
acid varies between 4:1 and 1:1, a ratio of about 2:1
being particularly preferred.
The reaction temperature and the concentration of the
starting product in the sulfuric acid/chlorosulfonic acid
mixture may vary according to the nature of the substrate.
F'or example, the poor solubility of cellulose suggests
more elevated dilutions, whereas, in the case of chitosan,
it is possible to use a higher concen-tration and to carry
out the reac-tion at a relatively low temperature.
Generally, the reaction temperature may vary between
-20 and +40C; after a period varying from some minutes to
2 hours, according to the reaction temperature, the reac-
tion is complete and the depolymerized and sulfated poly-
sacchar-
ide is isolated according to the conventional techniques,
for example by neutralization and dialysis, by chroma-
tographv or by lyophilisation.
The depolymerized and sulfated polysaccharide mav
also be isolated by pouring the reaction mixture in a
solvent wherein the end product is insoluble, ror example
in a non-polar, aprotic solvent such as diethvl ether,
by filtering the precipitate which forms and purifying
it according to the techniques known in the sugars
chemistrY
The depolymerized and sulrated ?ol~rsaccharides may
further be isolated as alkali metal salts tnereof
according to the usual methods, for exemple by lyophili-
sation or by evaporation under reduced pressure, and
characterized according to the ~nown physicochemical
methods.
Other salts, such as the calcium salt, may be ob-
tained starting from the alkaline salts, preferably from
the sodium salt, by exchange reaction with the appropriate
salt, for example with a calcium salt, by optionall~
using an ion exchange resin.
In the case of a startin~ polysaccharide having a
very high polymerization degree, for exam~le in the case
of chitosan, chitin or cellulose, it is advantageous to
submit said starting product to a previous depolymerisation
according to known methods, for example by treatment
with nitrous acid. The product thus ~reviouslv partially
depolymerized can be further depolymerized and sulfated
according to the ~rocess of the present invention.
The starting polysaccharide having a very high mole~
cular weight may also be submitted to the process of the
present invention twice. In such a case it is not even
necessarv to isolate the depolymeri~ed ?roduct; a further
amount of the sulfuric acid/chlorosulfonic acid mi~ture
can be added to the reaction mixture, for example after
the first hour. Surprisingly, this ~rocedure does no
involve any degradation or further sulfatation. For
example, in the case of cellulose a com~ound de~olymerized
and totally sulfated in the 6-position, i.e. on the primary
hydroxy group, is obtained accordin~ to this procedure.
The process of the present invention may be carried
out on the known polysaccharides. Suitable starting
materials are heparin, he~aransulfates, chitosan,
chitin, cellulose, starch, guaran, the chondroitinsulfates,
the ~olyxylans, inulin, dermatansulfate, ~.eratan, the
mannans, scleroglucan, the galactomannans, the dextrans,
the galactans, xanthan.
The process of the present invention is advantageous
for its selectivity and conveniences in handling.
In the case of heparin, Cor examole, there is
obtained a depolymeri~ed and "supersulfated" heparin ha~ing
a molecular weight of from 2000 and 9000 and a sulfatat on
degree higher than tha~ of the starting heparin. In
this depolymerized and "suDersulfated" henarin, all
Of the ~rimary hydroxy groups are sulfated.
In the case of chitosan, the reaction with a sul-
furic acid/chlorcsulfonic acid mixture according to the
present invention provides a chitosan with a depoly-
mersation degree which is unknown because the molecular
weisht, as that of the starting compound, is too high,
but which is suooposecl to be depolymerized. The primary
hydro~y groups of this compound is selectively sulfated,
without anv variation on the secondary hydroxy grou~
or on the free amino grou~.
In additlon, according to the process of the
present invention it is possible to control the sulfatation
-- 5
degree by suitably varying -the reaction temperature and/or
time. For example, in the case of chitosan a~ain, it is
possible to obtain a chitosan having a sulfatation degree,
selective in the 6-position, higher than ~ero, which can
arrive up to 1.
Cellulose, starch and chitin behave as chitosan.
Chondroitinsulfate and dermatansulfate behave as
heparin.
In the case of guaran, it is possible to obtain
depolymerized guaranes having a sulfate group on the
primary hydroxy group of D-mannose.
The depolymerisation degree varies according to the
molecular weight of the starting product and the stability.
In the case o-f cellulose and starch, depolymerized
and sulfated products having a higher depolymerisation
degree are obtained.
Chondroitinsulfate and dermatansulfate are less
stable and the depolymerisation may go up to three- and
tetrasaccharides.
Generally, the depolymerisation degree may be con-
trolled by suitably modifying the sulfuric acid/chlorosul-
fonic acid ratio, the reac-tion -time as well as the concen-
tratlon of the starting product in the mixture of the two
acids.
The following examples illustrate the invention
without, however, limiting it. In the following Examples,
reference is made to the accompanying drawings, wherein
Figures 1 to 10 are graphical representations of electro-
phoresis determinations in hydrochloric acid, in which the
migration is a function of the sulfatation degree.
EXAMPLE 1
. .
To a mixture of 20 ml of 9S% sulfuric acid and 10 ml
of chlorosulfonic acid, cooled to a temperature between -4
and 0C, there is added 1 g of heparin from pig intestinal
35 mucosa (PROQUIFIN*, lot 7926-7935, code number : D-212)
having a sulfatation degree of 1.95 and a molecular weight
13500, then it is stirred for 1 hour at -the same tempera-
ture. After further 60 minutes at room temperature, the
* - Trademark
-- 6 --
mixture is poured into 500 ml of cold diethyl ether (-4 -to
4C), the precipitate is fil-tered and washed with cold
diethyl ether. The product thus obtained is dissolved in
water, neutralized with 0.5 N sodium hydroxide and dialyzed
against distilled water in membranes at 3500 D (THOMAS
DIALYZED TUBING* 3787-H47, 11 mm diameter). Thus a desal~
ting is obtained as well as the ellmination of low molecu-
lar fragments. By slow evaporation under reduced pressure,
a depolymerized and supersulfated sodium heparin (code
number : AH-16) is obtained in 93% yield by weight, as a
powder having the following characteristics:
- M.W.: ~ 6000
- Elemental analysis:S:12.93%; C:18.48%; H:3.30%; N:1.76%
- Sulfatation degree (SO3 /COO ):3.0
- IR spectrum:broad band in the region 1300-1200 cm 1,
characteristic of the sulfate groups
- Electrophoresis in hydrochloric acid: with this technique,
the migration is function of -the sulfatation degree. Fig.
1 shows the significant increasing of the electrophoretic
migration of the depolymerized and supersulfated heparin
compared with the starting heparin.
- Barium acetate electrophoresis: Fig. 2 shows that
depolymerized and supersulfated heparin as a "slow-moving"
electrophoretic characteristic, differently from the
starting heparin containing both "slow-moving" and "fast
moving" components.
- 13C-NMR spectrum: Fig. 3 shows the comparison between
the spectrum of the starting heparin and that of depoly-
merized and supersulfated heparin. In the spectrum of the
new low molecular weight heparin new signals appear, due
to the effect of the depolymerisation and of the in-troduc-
tion of additional sulfate groups as well as to the disap-
pearance of the 6-OH signal. The depolymerized and super-
sulfated heparin thus obtained shows a sulfatation degree
which is 53% higher than that of starting heparin without
any significant decarboxylation.
EXAMPLE 2
To a mixture of 10 ml of 98% sulfuric acid and 5 ml
* - Trademark
~ J
g~
-- 7
of chlorosulfonic acid, cooled to a temperature between -4
and 0C, there are added 500 mg of a high molecular weigh-t
fraction (M.W. 16500, code number:D-212/B), obtained by
precipitation with ethanol and having a sulfatation degree
(SO3/COO ) of 2, of heparin PROQUIFIN, lot 7926-7935. The
mixture is left to stand 1 hour at room temperature, then
it is poured into 250 ml of cold diethyl ether (-10 to
4C) and filtered; the precipitate thus obtained is dis-
solved in water, the solution is neutralized with 0.5 N
sodium hydroxide and dialysed against distilled water in
membranes at 3500 D (THOMAS DIALYZER TUBING 3787-H47, 11
mm diameter), in order to eliminate the salts and the
smallest size reaction products. By evaporation under
reduced pressure, a depolymerized and supersulfated sodium
heparin (code number:AH-18) is obtained in 60% yield. The
product has the following characteristics:
- M.W. : 3000-5000
- Elemental analysis:S:13.56%; C:1~.03%, H:3.00% N:1.70%
- Sulfatation degree (SO3/COO ):2.6
20 - IR spectrum: broad band in the region 1300-1200 cm 1,
characteristic of the sulfate groups.
- Barium acetate electrophoresis: Fig. 4 indicates that
AH- 18 show "slow-moving" components only, whereas the
starting product also shows "fast-moving" components.
EXAMPLE 3
.
To a mixture of 10 ml of 98% sulfuric acid and 5 ml
of chlorosulfonic acid, cooled to a temperature between -4
and 0C, there are added 500 mg of sodium heparin from pig
intestinal mucosa (PROQVIFIN, lot 7926-7935, code number:
D-212) having a sulfatation degree (SO3/COO ) of 1.95.
The mixture is left to stand 1 hour at room tempera-
ture, then it is poured into 250 ml of cold diethyl ether
(-10 to 4C), and afterwards treated as described in
Examples 1 and 2. Thus, a depolymerized and supersulfated
sodium heparin (code number:A~-l9) is obtained in 90%
yield. The product has the following characteristics:
- M.W. :~ 6000
- Sulfatation degree (SO3/COO ):3.0
3~
- IR spectrum: broad band in -the region 1300-1200 cm
characteristic of the sulfate groups
- Barium acetate electrophoresis: Fig. 5 indicates that
AH-l9 shows "slow-moving" components only, whereas the
starting product also shows ~fast moving" components.
EXAMPLE 4
To a mixture of 10 ml of 98% sulfuric acid and 5 ml
of 95% chlorosulfonic acid, cooled to a temperature be-
tween -4 and 0C, there are added 500 mg of a mean molecu-
10 lar weight heparin fraction (M.W.~ 10000, code number:
D-212/A), obtained by fractionation with ethanol of hepar-
in PROQUIFIN, lot 7926-7935, said fraction having a sul-
fatation degree (SO3/COO ) of 1.5
$
and a barium acetate electroohoretic pattern which
shows a very important "fast moving" comronent. The
mixture is left to stand 1 hour under stirring at room
temperature, then it is poured into 250 ml of cold
diethyl ether (-10 to 4C), and afterwards treated
as described in Examrles 1 and 2. Thus, a deoolvmerized
and suoersulfated sodium her,arin (code number : AH-17)
is obtained having the following charasteristics :
- M.W.: 3000-5000
- Elemental analysis: S : 12.70%; C : 17.24~; H : 3.10~;
N :1.67~
- Sul~atation degree (S03/COO ) : 2.S
- XR spectr~ : broad band in the region 1300-1~00 cm
characteristic of the sulfate grouos
- ~arium acetate electrophoresis : Fig. 6 indicates
that AH ~17, compared to the starting herarin fraction,
shows a "slow-moving" com?onent onlyn
EXAMPLE 5.
To a mixture of 20 ml of 95~ sulfuric
acid and 10 ml of 93~ chlorosulfonic acid, cooled to
a temnerature between -4 and 0C, there is added 1 g
of heparin from pig intestinal mucosa (PROQUIFIN,
lot 7926-7935, code number o D-212) having a
5ulratation degree of 1.95, then the reaction mixture
is stirred 1 hour at room temperature. The mixture is
poured into 500 ml of cold diethyl ether (-4 to 4C), the
precipitate is filtered and ~ashed with cold diethvl
ether. The product thus obtained is dissolved in 0.1
M calcium chloride a~ueous solution, then O.5 ~ calcium
hydroxide is added thereto up to pH 8. The solution
is dialysed against 500 ml of 0~1 ~I calcium chloride
solution and then against distilled water. By slow
evaporation under reduced pressure, a calcium salt of
a depolymerized and su~ersulfated herJarin is obtained
~5 as a white powder.
EXAMPLES 6 _o 10.
To a mixture o. 10 ml of 98% sulfuric
acid and 5 ml of chlorosulfonic acid, cooled to a
temperature between -4 and 0C, there are added
500 mg of heparin from pig intestinal mucosa
(PROQUIE'IN, lot 7926-7935, cocle number : D-212) having
a sulfatation degree of 1.95 and a molecular weight
13500~ By operating as descri~ed in Example 1, a
depolymerized,and supersul'ated heparin (code number:
AH-104) is obtained, in 98~ yield.
The same procedure and conditions are
followed in 4 parallel experiments in which the same
starting heparin is used~ There are obtained the
products desiynated by -their code numbers AH-103,
AH-105, AH-106 and AH-107. The characteristics of the
products thus obtained as well as those of the 2roduct
coded AH 104 are given in Table IV.
TABLE IV
_ . , ,, . . . I
Ex. Product Elemental Analysis Sulfatation Yield
bv
S~ C% H% N%
deg.ree weight
_ _ _ _
6 AH-104 14.54 15.42 2.84 1.43 2.9 + 0.1 98%
7 AH - 103 14.63 15.53 2.76 1.43 2.8 + 0.1 89
8 AH-105 14.48 15.43 2.61 1.44 3.0 ~ 0.1 67%
9 AH-106 14.54 15.53 2.81 1.46 2.8 + 0.1 96%
AH-107 14.12 15.65 2.80 1.40 3.0 + 0.1 ~ 77
- Molecular weight : ~6000 for the 5 products
- IR spectrum : the 5 products show a spectrum identical
to that of compound AH-16 described in Exam~le 1.
- Electrophoresis in hydrochloric acid: the electro-
phoretic profiles are indentical to those of Eig. 1
for both the starting heparin and the 5 products ,;
- Barium acetate electroohoresis : the electrophoretic
.
profiles are identical to those of Fig. 2 for both the
starting heparin and the 5 products, apart from the fact
that the traces relative to the 5 products do not show the
background noise - caused by a temporary defect of the
tracing pen or of the paper - observed in the horizontal
cart of the graph of Fig. 2 relative to AH-16
- 13C-NMR spectrum: the 5 products and the s-tarting
compound present the same spectra as those given in Fig.
3.
The 5 compounds thus obtained are identical each
other and identical to the compound described in Example 1
as well.
EXAMPLES 11 to 14
15 In 4 parallel experiments, -to a mixture of 10 ml of
98% sulfuric acid and 5 ml of chlorosulfonic acid, cooled
to -4 - 0C, there are added 500 mg of previously lyophil-
ized heparin from pig intestinal mucosa (DIOSYNTH* batch
CH/N 665, code number:D-479), having a sulfatation degree
(SO3/COO ) of 2.1 and a molecular weight of about 11000.
The reaction mixture is left to stand 1 hour at 0C, then
it is poured into 250 ml of diethyl ether previously
cooled (between -10C and +4C). By operating as des-
cribed in Example 1 the products of Table V are ob-tained.
TABLE V
, __ _ _ _
Ex. Product Ele! lental Analysis Sulfatation Yield
S% C% H% N% degree by weigh-t
__ ___ _ ._
11 AH-108 14.88 15.29 2.52 1.47 3.1 + 0.1 90%
30 12 AH-109 14.43 15.48 2.72 1.44 3.0 + 0.1 106%
13 AH-110 14.45 15.72 2.76 1.50 2.9 + 0.1 65%
14 AH-lll 14.55 15.08 2.60 1.41 3.0 ~ 0.1 23%
_ . _ __ .......... ___ . _ . .
- Molecular weight: 6000 for the 4 products
- IR spectrum:~ broad band between 1300 and 1200 cm 1,
characteristlc of the sulfate group
- Electrophoresis in hydrochloric acid: Fig. 7 shows the
traces of the starting heparin D-479 and of one of the 4
* - Trademark
~ ... .
- 12 -
samples obtained in the different experiments (AH-108).
The traces of the other three compounds are identical.
This figure evidences the significant increase of the
electrophoretic migration of the depolymerized and super-
sulfated heparin in comparison with the starting heparin
- Barium acetate electrophoresis: Fig. 8 shows the traces
of the starting heparin D-479 and of AH-108. It results
that the depolymerized and supersulfated heparin has a
"slow-moving" electrophoretic profile, unlike the starting
heparin containing ~slow-moving~ components as well as
"fast-moving" components. The traces of the products
AH-109, AH-110 and AH-lll are identical to that of AH-108.
EXAMPLE 15
To a mixture of 10 ml of 98% sulfuric acid and 5 ml
of chlorosulfonic acid, cooled to a temperature between -4
and 0C, there are added 500 mg of heparin from pig intes-
tinal mucosa (TEROPMON*, batch 575/018, code number:D-98)
having a sulfatation degree of 1.8 and a molecular weight
135.00. By operating as described in Example 1, a depoly-
merized and supersulfated heparin is obtained, in 75%yield. The product has the following characteristics:
- M.W.: ~v 6000
- Elemental analysis:S:13.90%; C:15.75%; H:2.96%; N:1.48%
- Sulfatation degree (SO3/COO ):2.8 + 0.1
- IR spectrum: broad band in the region 1300-1200 cm 1,
characteristic of the sulfate groups
- Electrophoresis in hydrochloric acid: Fig. 9 shows the
traces of the starting heparin D-98 and of -the product
AH-118. A significant increasing of the electrophoretic
migration of AH-L18 compared with the starting heparin
D-98, may be observed. Fig. 9 shows also that the com-
pound AH-118 possesses a photodensitometric outline analo-
gous to those of compounds AH-16 (Example 1, Fig. 1) and
AH-17 (Example 4, Fig. 6) whereas the starting heparin
D-98 appears very heterogeneous and completely different
from the starting heparins utiliæed in Examples 1 and 4.
- Barium acetate electrophoresis: Fig. 10 indica-tes that
AH-118 shows a "slow-moving" electrophoretic characteristic
* - Trademark
,;'~ :`
, . . .
~,
~2~ r f~
- 13 -
which is different from that of the starting heparin D-98
showing both "slow-moving~ and "fast-moving~ components.
Fig. 10 also confirms that data of Flg. 9 and moreover
surprisingly shows that compound AH-118 is not signifi-
cantly different from AH-108 of Example 11, though the
starting heparins are quite different.
..s~.. .
14
EXAM~LE 16.
. ~
To a mixture of 20 ml of 95% sul~uric acid
lO ml of chlorosulfonic acid, ~reviously cooled to
0-4C, there are added 500 mg of ch1tosan ANIC, lot
116. The mi~ture is stirred at the same tem~erature
for about 1 ho~r, then it is ~oured into ~reviouslv
cooled diethvl ether. The ?recipitate ~Jhich forms is
filtered and neutralized with a ~otassium carhonate
solution. After a dial~sis in THO~S DIALYZER T~BI~JG
at 8500 D, a chitosan 6--sulate is obtained, havinc
the followins characteristics :
- Substitution degree (conductime'ric ~ethod): l
- IR s~ect~-um : ~road band in the reaion 1300-1200 cm 1,
characteristic of the sulfate ~rou~s
- 13C~N.~R sectru~ : disa~earance of the sianal o~ the
~rimar-~r h~tdxoxy ~rou~ and a~nearance of the sinal
relatin~ to the sulfate arou~
EX.~IE~LE 17.
To a mixture of 20 ml of 95~ sulFuric acid
and 10 ml of 98~ chlorosulfonis acid, cooled to a
temDerature between -4 and 0C, there is added l a
o~ chitosan ~NIC, lot 116. The reactlon mixture is
left ~o stand 30 minutes at room temoerature, then
it ls ?ouxed into 500 ml of ~reviousl-y cooled d~ethyl
ether. After filtration, the ~recipitate is ~Jashed
in water and neutralized with a solution of 0.5 ~ sodium
h~,~dro~ide, then it i.s dialvzed a~ainst distilled
water in membranes at 8000D ~TH0~1AS DIALV~E~ TUBI~
and eva~orated under reduced pressure. Thus, a
chitosan 5-sul~ate is obtained in 90~ yield. The
~roduct has the follo~7ing characteristics :
- Substitution degree ~conductimetric method~ : 0.5,
namely, 50~ only of the hvdrox~ gro1m in 6 ~osition
has been sulfated
- IR s~ectrum : broad band in the region 1300-1200 cm l,
- 15 -
characteristic of the sulfate groups
- 13C-NMR spectrum: diminution of the slgnal relating to
the primary hydroxy group and apparatus of the signal
relating to the sulfate group.
EXAMPLE 18
.
a) To a solution of 1 g of chitosan ANIC, lot 116, in 50
ml of 30% acetic acid, there are added 2.3 ml of 0.5 M
nitrous acid, prepared from 10 ml of 0.5 M barium nitrite
monohydrate and 10 ml of 0.5 M sulfuric acid. The mixture
is stirred 12 hours at room temperature, concentrated
under reduced pressure and treated with acetone. The
precipi-tate which forms is filtered, washed with acetone,
dried, dissolved in water and treated with 30 ml of sodium
borohydride. After 12 hours at room temperature, the
excess of sodium borohydride is destroyed with AMBERLITE*
IP 120 H+ and the boric acid is eliminated by evaporation
under reduced pressure in the presence of methanol. Thus
a depolymerized chitosan is obtained, having a molecular
weight much lower than that of the starting chitosan.
b) To a mixture of 20 ml of 95% sulfuric acid and 10 ml
of 98% chlorosulfonic acid, cooled to a temperature be-
tween -4 and O~C, there is added 1 g of depolymerized
chitosan, described hereinabove. The reaction mixture is
left to stand 1 hour at room temperature, then it is
poured into 250 ml of previously cooled diethyl ether; the
precipitate which forms is filtered and washed with cold
diethyl ether. The product is dissolved in water and
neu-tralized with a 0.5 M sodium hydroxide solution. After
desalting by chromatography on Sephadex* G25, a depolymer-
ized chitosan 6-sulfate is obtained in a 90% yield. The
product has the following characteristics:
- Substitution degree:l
* - Trademarks
~.,,1-
16
- IR s~ectrum : broad band in the resion 1300-1200 cm 1,
characteristic of the sulfate grou~s
- 13C-NMR spectrum : disa~pearance o~ the signal
relating to the primarv hydroxv group and appearance
of a ne~l signal due to the sulfate grou~.
EXAMPLE 19.
To a mixture of 20 ml of 95~ sulfuric acid
and 10 ml of 98~ chlorosulfonic acid, cooled to 0-4C,
there is added 1 g of cellulose microcristalline
(M.W. 20000~. The reaction mixture is left 1 hour under
stirring, then additional 30 ml of the mixture sulfuric
acid:chlorosulfonic acid 2:1 are added thereto. After
30 minutes, the mixture is ~oured into 500 ml of cold
diethyl ether, then it is filtered, the preci~itate
is washed with diethyl ether and disssolved in water.
By neutralization with a 0.5 M sodium hydroxide
solu~ion, dialysis in membranes at 3500 D (TUO~S
DIALYZE?~ TUBING 3787~H 47, 11 m~ diameter) and evapora-
tion under ~ressure, a cellulose 6-sulfate is ohtained,
with a 36go yield Of dialvsable fraction and 29~ vield
of non-dialysable fraction. The ~roduct has the follow-
ing characteristics :
- Substitution degree (conductimetric metho~
- IR s~ectrum : broad band in the region 1300-1200 cm 1,
~5 characteristic of the sulfate grou~s
- Molecular weight : 3400
E~PLES 20 to 22.
In three parallel ex~eriments, 500 mg of
heparin from ~ig intestinal mucosa (DIOSYNTH, batch
CH/N 665, code n. D-479) having a sulfatation degree
(S03/COo ) 2.1 and a molecular ~eight 11000 are added
to 15 ml of a mixture of 98g~ sulfuric acid and 98
chlorosulfonic acid in the following ratios :
Example 20 : 1:4
Example 21 ~
Exam~le 22 : 4:1
- 17 -
By operating as described in Example 1, three depolymer-
ized and supersulfated heparins, having the characteris-
tics given in Table VI, are obtained.
TABLE VI
Example Sulfatation Molecular Yield
(code NO) degree weight % by weight
2.5 4000 89.9
10 (AH-67)
21 2.5 3800 86.8
(AH-65)
22 2.8 4500 77.4
(AH-68)
EXAMPLE 23
__
To a mlxture of 10 ml of 98% sulfuric acid and 5 ml
of 98% chlorosulfonic acid, at the temperature of 0 - 4C,
1 g of guaran (AGOGUM* F-90, lot 433) is added. After 1
hour at *he same temperature, by operating as described in
Example 1, a guaran 6-sulfate is isolated as sodium salt
(code No . AH-102). The product has the following charac-
teristics:
- Substitution degree (conductimetric method):l
- IR spectrum: broad band in the region between 1300 and
1200 cm 1, characteristic of the sulfate group.
Yleld: 26% by weight
EXAMPLE 24
To a mixture of 20 ml of 95% sulfuric acid and 10 ml
30 of 98% chlorosulfonic acid, cooled to 0 - 4C, 1 g of
chitin (SIGMA, lot 12 F-7060) is added. The reaction
mixture is left to stand 1 hour at the same temperature.
Then, by operating as described in Example 1, after dial-
ysis and evaporation under reduced pressure a chitin
6-sulfate is ob-tained and isolated as sodium salt (code
No. AH-50). The product has the following characteristics:
- Substitution degree (conductimetric method):l
- IR spectrum: broad band in the region between 1300 and
* - Trademark
- 18 -
1200 cm 1, characteristic of the sulfate group.
- 13C-NMR spectrum:disappearance of the signal of the
primary hydroxy group and appearance of the signal of
sulfate groups.
Yield: 70% by weight
EXAMPLE 25
To a mixture of 15 ml of 95% sulfuric acid: 98%
chlorosulfonic acid 2:1, cooled to 0 - 4C, there are
added 500 mg of chondroitinsulfate TAKEDA* (lot BB-185,
No. Code:D-267) having a molecular weight 22000 and con-
taining 18% of moisture. After 1 hour at room temperature,
the mixture is poured into 500 ml of cold diethyl ether~
The precipitate which forms is dissolved in water, the
solution is neutralised with 0.5 M sodium hydroxide, then
it is dialyzed in tubes at 3500 D (THOMAS DIALYZER TUBING,
diameter 15 mm). By evaporating under reduced pressure a
depolymerized and supersulfa-ted chondroitinsulfate (code
No. AH 69) having the fbllowing characteristics is obtained;
- IR spectrum: broad band in the region 1300-1200 cm 1,
characteristic of the sulfate groups
- Molecular weight: 2000
EXAMPLE 26
To a mixture of 10 ml of 98% sulfuric acid and 5 ml
of 98% chlorosulfonic acid, there is added 500 ml of
dermatansulfate OPOCRIN* (lot 7-8 HF) having a molecular
weight 27000 and a substitution degree (SO3/COO ):1. By
operating as described in Example 25, a depolymerized and
supersulfated dermatansulfate (code No. AH-79) is obtained.
The product has the following characteristics:
* - Trademarks
~2~
19
- Substitution de~ree (S03/C00 ), conductimetric
method ) : 2.8
- IR s~ectrum broad band in the resion between 1300
and 1200 c~ 1, characteristic of sulfate grou~s
- Molecular weight 2000.
