Claims
- 1. Sphingosine-1-phosphate essentially free of L-threo isomer.
- 2. Sphingosine-1-phosphate essentially free of L-threo isomer synthesized chemically by a method comprising:
- (1) protecting the C1 and C3 hydroxyl groups of sphingosine (SPN);
- (2) deprotecting the C1 hydroxyl group of SPN;
- (3) phosphorylating the C1 hydroxyl group of SPN; and
- (4) deprotecting the C3 hydroxyl group of SPN; to produce sphingosine-1-phosphate essentially free of L-threo isomer.
- 3. The sphingosine-1-phosphate essentially free of L-threo isomer of claim 2, wherein said method comprises:
- (A) esterifying the C-3 hydroxyl group of compound 1' to give compound 2' ##STR1## wherein X is a moiety that protects the amine group formed in step (B), (B) selectively deprotecting the C-1 hydroxyl group of said compound 2' to give compound 3' ##STR2## (C) phosphorylating the C-1 hydroxyl group of said compound 3', followed by, hydrolyzing to give compound 4' ##STR3## (D) deprotecting the C-3 hydroxyl group and the amino group, respectively, of said compound 4' to give compound 1, which is said sphingosine-1-phosphate essentially free of L-threo isomer.
- 4. The sphingosine-1-phosphate of claim 3, wherein:
- (1) X is N-tert-butyloxycarbonyl;
- (2) said esterifying step (A) comprises treating said compound 1' with pivaloyl chloride in dry pyridine;
- (3) said deprotecting step (B) comprises treating said compound 2' with p-toluenesulfonic acid in methanol;
- (4) said phosphorylating and hydrolyzing step (C) comprises treating said compound 3' with:
- (a) phosphorous oxychloride in the presence of triethylamine and CH.sub.2 Cl.sub.2, and then
- (b) 1N HCl in CHCl.sub.3 ; and
- (5) said deprotecting step (D) comprises treating said compound 4' with:
- (a) tetrabutylammonium hydroxide in aqueous dioxane, AMBERLITE IR-120, water and then
- (b) trifluoroacetic acid and CH.sub.2 Cl.sub.2.
- 5. N,N-dimethylsphingosine-1-phosphate essentially free of L-threo isomer chemically synthesized by a method comprising:
- (A) esterifying the C-3 hydroxyl group of compound 1' to give compound 2' ##STR4## wherein X is a moiety that protects the amino group formed in step (B), (B) selectively deprotecting the C-1 hydroxyl group of said compound 2' to give compound 3' ##STR5## (C) eliminating the protecting moiety X from said compound 3' and then subjecting the product to reductive methylation to give compound 3.sup.a ##STR6## (D) phosphorylating the C-1 hydroxyl group of said compound 3.sup.a followed by hydrolyzing to give compound 3.sup.b ##STR7## (E) deprotecting the C-3 hydroxyl group to give compound 2, which is said N,N-dimethyl-sphingosine-1-phosphate.
- 6. The N,N-dimethylsphingosine-1-phosphate of claim 5, wherein:
- (1) X is N-tert-butyloxycarbonyl,
- (2) said esterifying step (A) comprises treating said compound 1' with pivaloyl chloride in dry pyridine;
- (3) said deprotecting step (B) comprises treating said compound 2' with p-toluenesulfonic acid in methanol;
- (4) said eliminating and reductive methylation step (c) comprises treating said compound 3' with:
- (a) trifluoroacetic acid in CH.sub.2 Cl.sub.2, and then
- (b) 37% CH.sub.2 O and NaCNBH.sub.3 in sodium acetate aqueous buffer;
- (5) said phosphorylating and hydrolyzing step (D) comprises treating said compound 3.sup.a with:
- (a) phosphorous oxychloride in the presence of triethanolamine and CH.sub.2 Cl.sub.2, and then
- (b) 1N HCl in CHCl.sub.2 ; and
- (6) said deprotecting step (E) comprises treating said compound 3.sup.b with tetrabutylammonium hydroxide in aqueous dioxane.
- 7. N,N,N-trimethylsphingosine-1-phosphate essentially free of L-threo isomer chemically synthesized by a method comprising:
- (A) esterifying the C-3 hydroxyl group of compound 1' to give compound 2' ##STR8## wherein X is a moiety that protects the amino group formed in step (B), (B) selectively deprotecting the C-1 hydroxyl group of said compound 2' to give compound 3' ##STR9## (C) eliminating the protecting moiety X from said compound 3' and then subjecting the product to reductive methylation to give compound 3.sup.a ##STR10## (D) permethylating said compound 3.sup.a followed by treating with a basic anion exchange resin to give compound 3.sup.h ##STR11## (E) phosphorylating the primary hydroxyl group of said compound 3.sup.h followed by hydrolyzing and then deprotecting the C-3 hydroxyl to give compound 3, which is said N,N,N-trimethyl-sphingosine-1-phosphate.
- 8. The N,N,N-trimethylsphingosine-1-phosphate of claim 7, wherein:
- (1) X is N-tert-butyloxycarbonyl;
- (2) said esterifying step (A) comprises treating said compound 1' with pivaloyl chloride in dry pyridine;
- (3) said deprotecting step (B) comprises treating said compound 2' with p-toluenesulfonic acid in methanol;
- (4) said eliminating and reductive methylation step (C) comprises treating said compound 3' with:
- (a) trifluoroacetic acid in CH.sub.2 Cl.sub.2, and then
- (b) 37% CH.sub.2 O and NaCNBH.sub.3 in sodium acetate aqueous buffer;
- (5) said permethylating step (D) comprises treating said compound 3.sup.a with:
- (a) CH.sub.3 I in NaHCO.sub.3 and CHCl.sub.3, and then
- (b) DOWEX 1.times.2 (Cl.sup.-), and
- (6) said phosphorylating, hydrolyzing, and deprotecting step (E) comprises treating said compound 3.sup.h with:
- (a) POCl.sub.3 in triethyl amine and CH.sub.2 Cl.sub.2, then
- (b) 1N HCl and CHCl.sub.3, and then
- (c) tetrabutylammonium hydroxide in aqueous dioxane.
- 9. N-acylsphingosine-1-phosphate or N-acetylsphingosine-1-phosphate essentially free of L-threo isomer chemically synthesized by a method comprising:
- (A) esterifying the C-3 hydroxyl group of compound 1' to give compound 2' ##STR12## wherein X is a moiety that protects the amino group formed in step (B), (B) selectively deprotecting the C-1 hydroxyl group of said compound 2' to give compound 3' ##STR13## (C) eliminating the protecting moiety X, then acylating or acetylating the unprotected amino group to give compound 3.sup.i ##STR14## wherein Ac represents acyl or acetyl, (D) phosphorylating the C-1 hydroxyl group of said compound 3.sup.i, followed by hydrolyzing to give compound 3.sup.j ##STR15## (E) deprotecting the C-3 hydroxyl group to give compound 4, which is said N-acylsphingosine-1-phosphate or N-acetylsphingosine-1-phosphate.
- 10. The N-acylsphingosine-1-phosphate or N-acetylsphingosine-1-phosphate of claim 9, wherein:
- (1) X is N-tert-butyloxycarbonyl;
- (2) said esterifying step (A) comprises treating said compound 1' with pivaloyl chloride in dry pyridine;
- (3) said deprotecting step (B) comprises treating said compound 2' with p-toluenesulfonic acid in methanol;
- (4) said eliminating and acylating or acetylating step (C) comprises treating said compound 3' with:
- (a) trifluoroacetic acid in CH.sub.2 Cl.sub.2, and then
- (b) CH.sub.3 (CH.sub.2).sub.n COCl, wherein n is 0 to 22, in aqueous 55% K.sub.2 CO.sub.3 tetrahydrofuran;
- (5) said phosphorylating and hydrolyzing step (D) comprises treating said compound 3.sup.i with:
- (a) phosphorous oxychloride in the presence of triethylamine and CH.sub.2 Cl.sub.2, and then
- (b) 1N HCl in CHCl.sub.3 ; and
- (6) said deprotecting step (E) comprises treating said compound 3.sup.j with tetrabutylammonium hydroxide in dioxane.
Parent Case Info
This is a divisional of U.S. Ser. No. 08/104,504 filed 9 Aug. 1993, now U.S. Pat. No. 5,391,800, which is a divisional of U.S. Ser. No. 07/863,179 filed 3 Apr. 1992, now U.S. Pat. No. 5,260,288.
US Referenced Citations (1)
Number |
Name |
Date |
Kind |
5110987 |
Liotta et al. |
May 1992 |
|
Non-Patent Literature Citations (5)
Entry |
"Enzymatic Quantification of Sphingosine in the Picomole Range in Cultured Cells", Van Veldhoven, et al., Analytical Biochemistry 183, 177-189 (1989). |
Chemical Abstracts 92 (5), No. 41295 (1980). |
A.S. Bushnev et al., Bioorg. Khim. 5 (9), 1381-1384 (1979). |
Stoffel, W. et al. Hoppe-Seyler's Z. Physiol. Chem. 351, 635-642 (1970). |
Van Veldhoven, P.P. et al. Journal of Lipid Research, vol. 30, pp. 611-616 (1989). |
Divisions (2)
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Number |
Date |
Country |
Parent |
104504 |
Aug 1993 |
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Parent |
863179 |
Apr 1992 |
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