U.S. patent application number 10/047882 was filed with the patent office on 2002-05-23 for high yield s-nitrosylation process.
Invention is credited to Herrmann, Robert A., Knapp, David.
Application Number | 20020062020 10/047882 |
Document ID | / |
Family ID | 24587901 |
Filed Date | 2002-05-23 |
United States Patent
Application |
20020062020 |
Kind Code |
A1 |
Herrmann, Robert A. ; et
al. |
May 23, 2002 |
High yield S-nitrosylation process
Abstract
A method for producing a S-nitrosylated species is provided. The
method comprises: (a) providing a deoxygenated, alkaline aqueous
solution comprising a thiol and a nitrite-bearing species; (b)
acidifying the solution by adding acid to the solution while
concurrently mixing the solution (e.g., by vigorously stirring the
solution) to produce the S-nitrosylated species; and (c) isolating
the S-nitrosylated species. The nitrite-bearing species can be, for
example, an inorganic nitrite, such as an alkali metal nitrite, or
an organic nitrite, such as an alkyl nitrite (e.g., ethyl nitrite,
amyl nitrite, isobutyl nitrite or t-butyl nitrite). The thiol is
preferably a thiol-containing polysaccharide, a thiol-containing
lipoprotein, a thiol-containing amino acid or a thiol-containing
protein, and more preferably a thiol-containing polysaccharide such
as thiolated cyclodextrin. In many preferred embodiments, the
S-nitrosylated species is insoluble in the acidified solution,
precipitating upon formation. The S-nitrosylated species can be
isolated, for example, by a process in which the precipitate is
removed from the solution (e.g., by centrifugation) and the aqueous
solvent remaining in the precipitate is sublimated (e.g., by
freezing the precipitate and subjecting it to a vacuum). The
isolated S-nitrosylated product is preferably protected from heat,
light, moisture and oxygen.
Inventors: |
Herrmann, Robert A.;
(Boston, MA) ; Knapp, David; (Wellesley,
MA) |
Correspondence
Address: |
MAYER, FORTKORT & WILLIAMS, PC
251 NORTH AVENUE WEST
2ND FLOOR
WESTFIELD
NJ
07090
US
|
Family ID: |
24587901 |
Appl. No.: |
10/047882 |
Filed: |
January 14, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10047882 |
Jan 14, 2002 |
|
|
|
09645171 |
Aug 24, 2000 |
|
|
|
Current U.S.
Class: |
536/54 ; 530/350;
558/488 |
Current CPC
Class: |
C07C 381/00 20130101;
C08B 37/00 20130101; C08B 37/0012 20130101 |
Class at
Publication: |
536/54 ; 530/350;
558/488 |
International
Class: |
C08B 037/00; C07C
381/00 |
Claims
1. A method for producing an S-nitrosylated species comprising:
providing a deoxygenated, alkaline aqueous solution comprising a
thiol and a nitrite-bearing species; acidifying said solution by
adding acid to said solution while concurrently mixing said
solution to produce said S-nitrosylated species; and isolating said
S-nitrosylated species.
2. The method of claim 1, wherein said aqueous solution is rendered
alkaline by adding an alkali metal hydroxide.
3. The method of claim 1, wherein said solution is deoxygenated by
drawing a vacuum.
4. The method of claim 1, wherein said solution is deoxygenated by
purging with an inert gas.
5. The method of claim 1, wherein said solution is deoxygenated by
first drawing a vacuum and subsequently purging with an inert
gas.
6. The method of claim 5, wherein said inert gas is nitrogen.
7. The method of claim 1, wherein 1 to 2 equivalents of
nitrite-bearing species are provided per thiol equivalent.
8. The method of claim 1, wherein said nitrite-bearing species is
an inorganic nitrite.
9. The method of claim 8, wherein said inorganic nitrite is an
alkali metal nitrite.
10. The method of claim 1, wherein said nitrite-bearing species is
an organic nitrite.
11. The method of claim 10, wherein said organic nitrite is an
alkyl nitrite.
12. The method of claim 11, wherein said alkyl nitrite is selected
from ethyl nitrite, amyl nitrite, isobutyl nitrite and t-butyl
nitrite.
13. The method of claim 1, wherein said acid is added to said
solution in an amount effective to provide at least 70% of
theoretical yield within at least 1 hour.
14. The method of claim 1, wherein said acid is an inorganic
acid.
15. The method of claim 14, wherein said inorganic acid is selected
from hydrochloric acid, phosphoric acid and sulfuric acid.
16. The method of claim 1, wherein said acid is an organic
acid.
17. The method of claim 16, wherein said organic acid is acetic
acid.
18. The method of claim 1, wherein said thiol is selected from a
thiol-containing polysaccharide, a thiol-containing lipoprotein, a
thiol-containing amino acid and a thiol-containing protein.
19. The method of claim 18, wherein said thiol-containing
polysaccharide is thiolated cyclodextrin.
20. The method of claim 1, wherein said mixing is carried out by
stirring said solution.
21. The method of claim 1, wherein said S-nitrosylated species is
insoluble in said acidified solution and forms a precipitate.
22. The method of claim 21, wherein the S-nitrosylated species is
isolated by a process comprising removing said precipitate from
said solution by centrifugation.
23. The method of claim 21, wherein the S-nitrosylated species is
isolated by a process comprising: removing said precipitate from
solution and sublimating aqueous solvent remaining in said
precipitate.
24. The method of claim 23, wherein said sublimating step comprises
freezing said precipitate and subjecting said precipitate to a
vacuum.
25. The method of claim 1, wherein said isolated S-nitrosylated
product is subsequently protected from heat, light, moisture and
oxygen.
26. A method for producing an S-nitrosylated cyclodextrin species
comprising: providing a deoxygenated, alkaline aqueous solution
comprising a thiolated cyclodextrin and a nitrite-bearing species
selected from an alkali metal nitrite and an alkyl nitrite;
acidifying said solution by adding acid to said solution while
concurrently mixing said solution to produce an S-nitrosylated
cyclodextrin species precipitate; and removing said precipitate
from said solution.
27. The method of claim 26, wherein 1 to 2 equivalents of
nitrite-bearing species are provided per thiol equivalent.
28. The method of claim 26, further comprising sublimating aqueous
solvent remaining in said precipitate after removal from said
solution.
29. The method of claim 28, wherein said sublimating step comprises
freezing said precipitate and subjecting said precipitate to a
vacuum.
30. The method of claim 28, wherein said sublimated precipitate is
subsequently protected from heat, light, moisture and oxygen.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to high-yield methods for
producing S-nitrosylated compounds.
BACKGROUND OF THE INVENTION
[0002] S-nitrosylated compounds are compounds with one or more
--S--NO groups. An --S--NO group is also referred to in the art as
a sulfonyl nitrite, a thionitrous acid ester, an S-nitrosothiol or
a thionitrite.
[0003] Various S-nitrosylated species are known and include:
[0004] a) Certain S-nitrosylated polysaccharides, such as
S-nitrosylated starch, cellulose, alginic acid, K-carrageenan,
fucoidin, and cyclodextrins (e.g., .alpha.-cyclodextrin,
.beta.-cyclodextrin and .gamma.-cyclodextrin).
[0005] b) Certain lipoproteins substituted at the S-moiety with a
nitric oxide moiety. Examples are chylomicron, a chylomicron
remnant particle, a very low-density lipoprotein, a low-density
lipoprotein, an intermediate-density lipoprotein, a high-density
lipoprotein and a lipoprotein (a).
[0006] c) Certain S-nitrosylated polypeptides and proteins, such as
neuropeptides, tissue-type plasminogen activator, streptokinase,
urokinase, BSA, immunoglobulin (e.g., IgG, IgM, IgA, IgD, IgE),
hemoglobin, myoglobin and cathepsin.
[0007] d) Certain S-nitroso amino acids and their derivatives (many
of which are ACE inhibitors) including S-nitroso-N-acetylcysteine,
S-nitroso-glutathione, S-nitroso-cysteine,
S-nitroso-gamma-methyl-L-homoc- ysteine, S-nitroso-L-homocysteine,
S-nitroso-gamma-thio-L-leucine, S-nitroso-delta-thio-L-leucine,
S-nitroso-captopril, N-acetyl-S-nitroso-D-cysteinyl-L-proline,
N-acetyl-S-nitroso-D, L-cysteinyl-L-proline,
1-[5-guanidino-2-(S-nitroso)mercaptomethyl-pentano- yl]-L-proline,
1-[5-amino-2-(S-nitroso)mercaptomethyl-pentanoyl]-4-hydroxy-
-L-proline,
1-[5-guanidino-2-(S-nitroso)mercaptomethyl-pentanoyl]-4-hydrox-
y-L-proline,
1-[2-aminomethyl-3(S-nitroso)-mercaptomethyl-pentanoyl]-L-pro-
line, and S-nitroso-L-cysteinyl-L-proline.
[0008] e) Certain other S-nitrosylated compounds such as
S-nitroso-penicillamine, S-nitroso-N-acetylpenicillamine, and
Y(CH.sub.2).sub.xSNO, where x is 2-20 and Y can be --H, --SH,
fluoro, C.sub.1-C.sub.6 alkoxy, cyano, carboxamido, C.sub.3-C.sub.6
cycloalkyl, aralkoxy, C.sub.2-C.sub.6 alkylsulfinyl, arylthio,
C.sub.1-C.sub.6 alkylamino, C.sub.2-C.sub.15 dialkylamino, hydroxy,
carbamoyl, C.sub.1-C.sub.6 N-alkylcarbamoyl, C.sub.2-C.sub.15
N,N-dialkylcarbamoyl, amino, hydroxyl, carboxyl, hydrogen, nitro or
aryl; wherein aryl includes benzyl, naphthyl, and anthracenyl
groups.
[0009] Several of these S-nitrosothiol species have been noted for
their platelet inhibition and/or thrombolytic characteristics, as
well as their ability to relax skeletal muscle and smooth muscle,
including vascular smooth muscle (vasodilation), airway smooth
muscle, gastrointestinal smooth muscle, corpus cavernosum smooth
muscle, bladder smooth muscle, and uterine smooth muscle. For
additional information, see, e.g., U.S. Pat. Nos. 5,770,645,
5,583,101, 863,890, 5,612,314, 5,648,393, 6,057,367 or U.S. Pat.
Nos. 5,380,758, 5,593,876, 5,574,068 or U.S. Pat. No. 5,385,937,
the disclosures of which are hereby incorporated by reference in
their entireties.
[0010] U.S. Pat. No. 5,770,645, the entire disclosure of which is
incorporated by reference immediately above, teaches that compounds
with one or more free nucleophilic groups, such as polysaccharides
that have been provided with pendant thiol groups, can be reacted
with a nitrosylating agent under conditions suitable for
nitrosylating the free thiol groups. Nitrosylating agents disclosed
as suitable include acidic nitrite, nitrosyl chloride, compounds
comprising an S-nitroso group (S-nitroso-N-acetyl-D,L-penicillamine
(SNAP), S-nitrosoglutathione (SNOG),
N-acetyl-S-nitrosopenicillaminyl-S-nitrosopenicillamine,
S-nitrosocysteine, S-nitrosothioglycerol, S-nitrosodithiothreitol
and S-nitrosomercaptoethanol), an organic nitrite (e.g. ethyl
nitrite, isobutyl nitrite, and amyl nitrite), peroxynitrites,
nitrosonium salts (e.g. nitrosyl hydrogen sulfate), oxadiazoles
(e.g. 4-phenyl-3-furoxancarbonitrile) and the like. For more
information, see U.S. Pat. No. 5,770,645.
SUMMARY OF THE INVENTION
[0011] According to an embodiment of the invention, a method for
producing a S-nitrosylated species is provided. The method
comprises: (a) providing a deoxygenated, alkaline aqueous solution
comprising a thiol and a nitrite-bearing species; (b) acidifying
the solution by adding acid to the solution while concurrently
mixing the solution (e.g., by vigorously stirring the solution) to
produce the S-nitrosylated species; and (c) isolating the
S-nitrosylated species.
[0012] The aqueous solution can be rendered alkaline, for example,
by adding an alkali metal hydroxide.
[0013] The solution can be deoxygenated, for example, by drawing a
vacuum, by purging with an inert gas (such as nitrogen), or by
first drawing a vacuum followed by purging with an inert gas.
[0014] The nitrite-bearing species can be, for example, an
inorganic nitrite, such as an alkali metal nitrite, or an organic
nitrite, such as an alkyl nitrite (e.g., ethyl nitrite, amyl
nitrite, isobutyl nitrite or t-butyl nitrite).
[0015] The thiol is preferably a thiol-containing polysaccharide, a
thiol-containing lipoprotein, a thiol-containing amino acid or a
thiol-containing protein, and more preferably a thiol-containing
polysaccharide such as thiolated cyclodextrin.
[0016] Preferably, 1 to 2 equivalents of nitrite-bearing species
are provided per thiol equivalent.
[0017] The acid can be, for example, an inorganic acid, such as
hydrochloric acid, phosphoric acid or sulfuric acid, or an organic
acid, such as acetic acid. Preferably, the acid is added to the
solution in an amount effective to provide at least 70% of
theoretical yield within at least 1 hour.
[0018] The S-nitrosylated species is preferably insoluble in the
acidified solution, precipitating upon formation. The
S-nitrosylated species can be isolated, for example, by a process
in which the precipitate is removed from the solution (e.g., by
centrifugation) and the aqueous solvent remaining in the
precipitate is sublimated (e.g., by freezing the precipitate and
subjecting it to a vacuum).
[0019] The isolated S-nitrosylated product is preferably protected
from heat, light, moisture and oxygen.
[0020] In one particularly preferred embodiment of the invention, a
method for producing an S-nitrosylated cyclodextrin species is
provided. The method comprises: (a) providing a deoxygenated,
alkaline aqueous solution comprising a thiolated cyclodextrin and a
nitrite-bearing species selected from an alkali metal nitrite and
an alkyl nitrite; (b) acidifying the solution by adding acid to the
solution, while concurrently mixing the solution, to produce an
S-nitrosylated cyclodextrin species precipitate; and (c) removing
the precipitate from the solution. 1 to 2 equivalents of
nitrite-bearing species are preferably provided per thiol
equivalent. Moreover, the method preferably comprises sublimating
any aqueous solvent remaining in the precipitate after removal from
the solution (e.g., by freezing the precipitate and subjecting it
to a vacuum). It is also preferable to protect the sublimated
S-nitrosylated cyclodextrin species precipitate from heat, light,
moisture and oxygen.
[0021] One advantage of the present invention is that
S-nitrosylated species are produced under conditions that minimize
exposure to environmental conditions that result in premature
breakdown and NO release.
[0022] Another advantage of the present invention is that thiol
species can be nitrosylated in high yield.
[0023] Yet another advantage of the present invention is that a
S-nitrosylated species can be produced in the form of a fine
powder, eliminating the need for crushing or otherwise milling the
product.
[0024] These and other embodiments and advantages of the present
invention will become readily apparent to those of ordinary skill
in the art upon review of the Detailed Description and Claims to
follow.
DETAILED DESCRIPTION OF THE INVENTION
[0025] According to one aspect of the invention, S-nitrosylated
species are produced by first providing a deoxygenated, alkaline
aqueous solution comprising a thiol and a nitrite-bearing species,
and subsequently acidifying this solution by adding acid with
concurrent vigorous mixing.
[0026] Essentially any nitrite-bearing species that is soluble in
basic solution and is capable of nitrosylating free thiol groups
under acidic conditions can be used. Preferred nitrite-bearing
species include organic nitrites and inorganic nitrites. Preferred
organic nitrites are alkyl nitrites, more preferably
C.sub.1-C.sub.8 linear or branched alkyl nitrites, such as ethyl
nitrite, amyl nitrite, isobutyl nitrite and t-butyl nitrite.
Preferred inorganic nitrites are alkali metal nitrites such as
sodium nitrite, lithium nitrite, and potassium nitrite.
[0027] A wide range of base-soluble thiol species can be
S-nitrosylated in accordance with the present invention, so long as
any undesirable side reactions are kept to a minimum. Several
S-nitrosylated products of thiol species are discussed in the
Background section above. Preferred thiol species include
thiol-containing polysaccharides, thiol-containing lipoproteins,
thiol-containing amino acids and thiol-containing proteins.
[0028] In some embodiments of the present invention, it is
desirable to provide a species with thiol groups for subsequent
S-nitrosylation. For example, a thiolated species can be formed
from a species having one or more pendant nucleophilic groups, such
as alcohols or amines. These pendant nucleophilic groups can be
converted to pendant thiol groups by methods known in the art, such
as those disclosed in Gaddell and Defaye, Angew. Chem. Int. Ed.
Engl. 30: 78 (1991) and Rojas et al., J. Am. Chem. Soc. 117: 336
(1995), the teachings of which are hereby incorporated into this
application by reference.
[0029] As a specific example, polysaccharides typically do not have
thiol groups, but do have pendant alcohol groups. For instance,
particularly preferred polysaccharides for the practice of the
invention include cyclodextrins such as: 1
[0030] In accordance with the present invention, prior to
S-nitrosylation, the polysaccharide is first converted to a
polythiolated polysaccharide using, for example, the methods
disclosed above. In these methods, primary alcohols are thiolated
preferentially over secondary alcohols. In some preferred
embodiments, a sufficient excess of thiolating reagent is used to
form perthiolated polysaccharides. Polysaccharides are
"perthiolated" when all of primary alcohols have been converted to
thiol groups. As seen from the above, alpha-cyclodextrin has six
primary alcohols, beta-cyclodextrin has seven primary alcohols and
gamma-cyclodextrin has eight primary alcohols. Hence, perthiolated
alpha-cyclodextrin has six thiol groups, perthiolated
beta-cyclodextrin has seven thiol groups and perthiolated
gamma-cyclodextrin has eight thiol groups.
[0031] Further details and a specific procedure in which
beta-cyclodextrin can be converted to
per-(6-deoxy-6-thio)-beta-cyclodextrin are found in U.S. Pat. No.
5,770,645 (see, inter alia, Examples 1 and 2).
[0032] U.S. Pat. No. 5,770,645 also teaches that a polythiolated
species can be prepared by reacting a polyhydroxylated species, and
preferably the primary alcohol groups of the polyhydroxylated
species, with a reagent that adds a moiety containing a free thiol
or protected thiol to the alcohol. In one example the
polysaccharide is reacted with a bis isocyanatoalkyldisulfide
followed by reduction to functionalize the alcohol as shown in
Structural Formula (I): 2
[0033] Conditions for carrying out this reaction are found in
Cellulose and its Derivatives, Fukamota, Yamada and Tonami, Eds.
(John Wiley & Sons), Chapter 40, (1985) the teachings of which
are incorporated herein by this route is shown in Structural
Formula (II): 3
[0034] Once obtained, the desired thiol and nitrite-bearing species
are combined in an alkaline aqueous solution.
[0035] Preferably, the solution is provided with 1 to 2 equivalents
of nitrite for each thiol equivalent. The thiol bearing species is
not necessarily fully nitrosylated using this range of nitrite. For
example, in the case of perthiolated beta-cyclodextrin, which
contains seven thiol groups per molecule, this range typically
provides on the order of about one S-nitroso group per cyclodextrin
molecule.
[0036] In general, the solution should be sufficiently alkaline to
prevent premature and significant nitrosylation of the thiol groups
and to prevent precipitation of material under non-ideal
conditions. Essentially any basic material can be used to render
the solution alkaline. Alkali metal hydroxides are preferred, most
preferably lithium, potassium or sodium hydroxide.
[0037] While the solution should be sufficiently alkaline to
prevent significant nitrosylation of the thiol groups, additional
base can be added beyond this point, if necessary to assist in
dissolving the thiol species. The appropriate pH to achieve each of
these objectives will vary depending, for example, on the thiol
species and nitrite-bearing species selected, but it can be readily
be determined by those of ordinary skill in the art.
[0038] The order of addition of thiol, nitrite-bearing species and
base is unimportant, so long as the solution is sufficiently
alkaline at the point where the thiol becomes associated with the
nitrite-bearing species in solution. For example, (1) the thiol can
first be dissolved in the basic solution, followed by the
nitrite-bearing species, (2) the nitrite-bearing species can first
be dissolved in the basic solution, followed by the thiol species,
(3) the nitrite-bearing species and the thiol can be dissolved in
the basic solution at the same time, and so forth.
[0039] As a specific example, a 0.1N NaOH solution (having a pH of
about 13) can be used as a starting solution, with thiol and the
nitrite-bearing species added in turn.
[0040] Degradation of nitrosylated thiols (i.e., the release of NO
and reversion to the thiol form) is accelerated upon exposure to
oxygen, moisture, heat and/or light. Hence, in accordance with the
present invention, it is desirable to reduce exposure to these
conditions during production and storage to the greatest extent
possible.
[0041] In this connection and in accordance with the present
invention, the solution is deoxygenated prior to the formation of
the S-nitrosylated species (i.e., prior to acidification of the
alkaline solution containing the thiol species and the
nitrite-bearing species). As specific examples, prior to
acidification, (1) the basic solution can first be deoxygenated,
followed by dissolution of the thiol species and nitrite-bearing
species in the same (2) the thiol species and nitrite-bearing
species can first be dissolved in the basic solution, followed by
deoxygenation, (3) the thiol species can first be dissolved in the
basic solution, followed by deoxygenation and dissolution of the
nitrite-bearing species, (4) the nitrite-bearing species can be
dissolved in the basic solution, followed by deoxygenation and
dissolution of the thiol species, and so forth.
[0042] A given solution can be deoxygenated by using any of several
known methods. For example, the solution can be placed in a closed
container followed by (1) drawing a vacuum, (2) purging the
overhead space with an inert gas, (3) bubbling the solution with an
inert gas, (4) heating to near boiling, and so forth. In one
preferred technique, the solution is deoxygenated by first drawing
a vacuum. Then, the vacuum is eliminated by purging the overhead
space with an inert gas. Preferred inert gases for the above
applications include nitrogen and argon.
[0043] Once a deoxygenated, basic aqueous solution of the thiol
species and the nitrite-bearing species is obtained, the solution
is subsequently acidified, preferably accompanied by vigorous
mixing.
[0044] The acid is preferably added to the solution in an amount
effective to provide at least 70% of the theoretical yield within
24 hours, more preferably within 1 hour, most preferably 10
minutes. The precise pH required to achieve this outcome will vary,
depending on the thiol species and nitrite-bearing species
selected, and it can be readily determined by those of ordinary
skill in the art.
[0045] Essentially any acid can be used for this purpose so long as
undesirable side reactions are kept to a minimum. Preferred acids
for the practice of the present invention include both inorganic
acids, such as hydrochloric acid, phosphoric acid and sulfuric
acid, and organic acids, such as acetic acid.
[0046] Vigorous mixing is carried out to minimize concentration
gradients in the solution while the nitrosylation reaction takes
place. Mixing can be realized by known methods that include
contacting the fluid with a moving member such as a stir bar or
paddle, directing the fluid into a stationary member such as a
baffle, and so forth.
[0047] Various benefits can be garnered by mixing the solution,
particularly where the reaction proceeds very quickly.
[0048] For instance, where multiple reaction sites are available,
product uniformity is enhanced. As a specific example, perthiolated
beta-cyclodextrin has seven available thiol sites for reaction.
Based on the reaction stoichiometry, however, only about one of
these sites is nitrosylated on average. While not wishing to be
bound by theory, it is believed that since this reaction proceeds
rapidly, if concentration gradients are allowed to persist in the
solution after the acid is added even for a short period of time,
the beta-cyclodextrin molecules in the more acidic environments
will have more nitrosylated sites (due to more favorable reaction
kinetics) than those in less acidic environments.
[0049] As another example, where the resulting S-nitrosylated
species is insoluble in the acidified solution and precipitates
from solution upon formation, a finer precipitate product is
typically obtained by vigorously mixing during acid addition than
would otherwise be obtained in the absence of such mixing.
[0050] Each of the above procedures of the present invention is
preferably conducted at a temperature within the range of from
about 0.degree. C. to about 50.degree. C., more preferably at room
temperature.
[0051] After the S-nitrosylated species is formed, it is preferably
isolated and dried to provide the product in powder form. In
preferred embodiments of the invention, the S-nitrosylated species
precipitates from solution upon formation.
[0052] In particularly preferred embodiments of the present
invention, this precipitate is first isolated in wet form from the
aqueous solvent (e.g., by centrifugation), washed in deionized
water and remaining solvent in the wet sample removed by
sublimation (e.g., lyophilization or freeze drying). The
sublimation process acts to protect the sample from degradation
(e.g., because moisture and oxygen are removed), and it promotes
the formation of a very fine powder. As a specific example, where
the precipitate is S-nitrosylated cyclodextrin, and where the
sample is allowed to dry in air, substantial clumps form. In
contrast, where the precipitate is dried by sublimation, a fine
powder is produced, and conditions are maintained which are less
prone to degrade the final product.
[0053] Preferably, the wet precipitate is sublimated by first
freezing it (e.g., by cooling the sample to -70 .degree. C. or by
immersion in liquid nitrogen). The frozen precipitate is then
placed in a container, and a vacuum is drawn, whereupon the aqueous
solvent is sublimated from the frozen precipitate.
[0054] Once a dry product is formed, steps are preferably taken to
avert premature product degradation. For example, the product is
preferably kept in a cool and dark place, such as a refrigerator or
freezer. For additional protection, the environment surrounding the
sample is preferably an inert, water-free environment, and is more
preferably a vacuum environment or a desiccated, inert gas
environment.
EXAMPLE
[0055] 4 grams of beta-cyclodextrin thiol are initially provided.
(Beta-cyclodextrin thiol can be prepared for example, using the
procedures of Examples 1 and 2 of U.S. Pat. No. 5,770,645, the
entire disclosure of which is incorporated by reference.) Then, a
300 ml portion of 0.1 N NaOH (which has a pH of approximately 13.0)
is added to the beta-cyclodextrin thiol. After mixing to dissolve
the cyclodextrin thiol, the solution is filtered through a 0.45
micron filter. 1.6 grams of sodium nitrite (NaNO.sub.2) are then
added. After mixing to dissolve the sodium nitrite, the solution is
divided into 25 ml aliquots. For each aliquot, the following
procedure is performed:
[0056] 1. The solution is degassed by applying a vacuum, after
which the removed gas is either replaced with nitrogen or the
solution is kept under vacuum.
[0057] 2. Under nitrogen or vacuum, 300 ml of 0.1 N HCl are quickly
injected into the solution accompanied by vigorous stirring.
[0058] 3. The mixture is reacted for 5 minutes, over which time a
pink precipitate is formed.
[0059] 4. The mixture is then centrifuged at 4000 rpm (approx. 800
.times. g) for 10 minutes.
[0060] 5. The supernatant is removed.
[0061] 6. The precipitate is washed by suspending it in degassed
de-ionized water, followed by centrifugation and recovery of the
precipitate.
[0062] 7. After repeating the prior washing step three times, the
recovered precipitate is immediately frozen in liquid nitrogen for
5 minutes.
[0063] 8. The frozen material is then lyophilized (sublimated under
vacuum) until dry (typically over a period of several days).
[0064] 9. Once dry, the material is stored under nitrogen at
-20.degree. C.
[0065] Although various embodiments are specifically illustrated
and described herein, it will be appreciated that modifications and
variations of the present invention are covered by the above
teachings and are within the purview of the appended claims without
departing from the spirit and intended scope of the invention.
* * * * *