High yield S-nitrosylation process

Details High yield S-nitrosylation process High yield S-nitrosylation process

2003-05-13

2004-07-13

Wilson, James O. (Department: 1623)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

Synthesis of peptides

C530S345000, C530S402000, C530S408000, C530S409000, C536S112000, C536S124000

Reexamination Certificate

active

06762282

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to high-yield methods for producing S-nitrosylated compounds.
BACKGROUND OF THE INVENTION
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.
Various S-nitrosylated species are known and include:
a) Certain S-nitrosylated polysaccharides, such as S-nitrosylated starch, cellulose, alginic acid, K-carrageenan, fucoidin, and cyclodextrins (e.g., &agr;-cyclodextrin, &bgr;-cyclodextrin and &ggr;-cyclodextrin).
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).
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.
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-homocysteine, 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-pentanoyl]-L-proline, 1-[5-amino-2-(S-nitroso) mercaptomethyl-pentanoyl]-4-hydroxy-L-proline, 1-[5-guanidino-2-(S-nitroso)mercaptomethyl-pentanoyl]-4-hydroxy-L-proline, 1-[2-aminomethyl-3(S-nitroso)-mercaptomethyl-pentanoyl]-L-proline, and S-nitroso-L-cysteinyl-L-proline.
e) Certain other S-nitrosylated compounds such as S-nitroso-penicillamine, S-nitroso-N-acetylpenicillamine, and Y(CH
2
)
x
SNO, where x is 2-20 and Y can be —H, —SH, fluoro, C
1
-C
6
alkoxy, cyano, carboxamido, C
3
-C
6
cycloalkyl, aralkoxy, C
2
-C
6
alkylsulfinyl, arylthio, C
1
-C
6
alkylamino, C
2
-C
15
dialkylamino, hydroxy, carbamoyl, C
1
-C
6
N-alkylcarbamoyl, C
2
-C
15
N,N-dialkylcarbamoyl, amino, hydroxyl, carboxyl, hydrogen, nitro or aryl; wherein aryl includes benzyl, naphthyl, and anthracenyl groups.
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. No. 5,770,645, U.S. Pat. No. 5,583,101, U.S. Pat. No. 5,863,890, U.S. Pat. No. 5,612,314, U.S. Pat. No. 5,648,393, U.S. Pat. No. 6,057,367 or U.S. Pat. No. 5,380,758, U.S. Pat. No. 5,593,876, U.S. Pat. No. 5,574,068 or U.S. Pat. No. 5,385,937, the disclosures of which are hereby incorporated by reference in their entireties.
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
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.
The aqueous solution can be rendered alkaline, for example, by adding an alkali metal hydroxide.
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.
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.
Preferably, 1 to 2 equivalents of nitrite-bearing species are provided per thiol equivalent.
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.
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).
The isolated S-nitrosylated product is preferably protected from heat, light, moisture and oxygen.
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.
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.
Another advantage of the present invention is that thiol species can be nitrosylated in high yield.
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.
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
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

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