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Synthesis of phosphocholine ester derivatives and conjugates thereof

a technology of phosphocholine ester and phosphocholine ester, which is applied in the field of synthesizing phosphocholine ester derivatives and conjugates, can solve the problems that antibodies are not found to be protective against i>s. pneumoniae, and achieve the effect of preventing microorganism infections and being more efficient and cost-effectiv

Inactive Publication Date: 2008-07-24
UNITED STATES OF AMERICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides methods for making phosphocholine conjugates and vaccines that can generate an immune response to PC in mammals. The methods involve contacting phosphocholine with a compound to form a phosphocholine conjugate, and then replacing R1 with a different compound to form the desired phosphocholine conjugate. The methods can produce the desired phosphocholine conjugate in no more than 2 steps. The phosphocholine conjugates can be used to generate an immune response to PC in mammals. The invention also includes the products formed by the methods.

Problems solved by technology

A commonly utilized hapten PC, p-diazophenylphosphocholine (DPPC), has been conjugated to protein antigens to produce high affinity phenylphosphocholine-(PPC) specific antibodies; however, unfortunately, these antibodies are not found to be protective against S. pneumoniae.

Method used

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  • Synthesis of phosphocholine ester derivatives and conjugates thereof
  • Synthesis of phosphocholine ester derivatives and conjugates thereof
  • Synthesis of phosphocholine ester derivatives and conjugates thereof

Examples

Experimental program
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Effect test

example 1

Step 1: Synthesis of 6-(O-Phosphocholine)Hydroxyhexanoic Acid

[0070]A sample of 6-bromohexanoic acid is combined with a catalytic amount of 1 8-crown-6 in formamide. To this combination, an equal molar amount of phosphorylcholine chloride calcium salt tetrahydrate is added. The resulting mixture is heated to 110-120° C. for about 3 hours. After incubation, the mixture is cooled to room temperature and is applied to a silica gel column (Emerck: 230-400 mesh silica gel; d2.5 cm×L35 cm). The product, 6- (O-phosphocholine)hydroxyhexanoic acid, is eluted from the silica gel column with methanol:water (4:1) solution.

Step 2: Synthesis of P-Nitrophenyl-6-(O-Phosphocholine)Hydroxyhexanoate

[0071]A sample of 6-phosphocholinehexanoic acid in dimethylformamide is stirred at room temperature for 5 min. To this mixture, approximately a three fold molar excess of p-nitrophenyl trifluoroacetate is added. While stirring the resulting mixture, approximately a 2 fold molar excess of 2,6-lutidine is adde...

example 2

[0073]Synthesis of 4-Nitrophenyl-6-(O-Phosphocholine)Hydroxyhexanoate from Ethyl 6-Bromohexanoate

Step 1: Synthesis of Ethyl 6-Phosphocholinehexanoic Acid

[0074]A sample of 9.1 g (41.2 mmol) of ethyl 6-bromohexanoate was combined with 1 .1 g (4.1 mmol) of 18-crown-6 in 40 ml of formamide. To this combination, 9.2 g (41.2 mmol) of phosphorylcholine chloride calcium salt tetrahydrate was added. The resulting mixture was heated to 110-120 ° C. for about 3 hours. After incubation, the mixture was cooled to room temperature and applied to a silica gel column (Emerck: 230-400 mesh silica gel; d2.5 cm×L35 cm). The product was eluted from the silica gel column with methanol:water (4:1) solution, which yielded 10 grams of ethyl 6-phosphocholinehexanoate with a percentage yield of 74%.

Step 2: Deprotection of Ethyl 6-Phosphocholinehexanoate to Yield 6-Phosphocholine Hexanoic Acid

[0075]A sample of 2.0 g (6.15 mmol) of ethyl 6-phosphocholinehexnoate was combined with 3.2 g (12.3 mmol) of tetrabuty...

example 3

Conjugation of PC Haptens

[0078]EPC haptens are conjugated to keyhole limpet hemocyanin (KLH) (400,000 used as MW) dissolved in borate buffered saline (BBS), pH 8.5 at 10 mg / ml. EPC is dissolved in dry acetonitrile (100 mg / ml) just prior to addition to the KLH. Hapten and KLH are mixed overnight at 4° C. and then dialyzed to remove unbound hapten and the released p-nitrophenylate. Alternatively, the PC-KLH conjugate can be purified by gel exclusion chromatography on a Sephadex G-25 column. The conjugation efficiency is estimated by determining the phosphate bound to protein according to the method described in Ames, B. N., et al. (1960) J Biol. Chem. 235:769.

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Abstract

Aspects of the present invention include methods of synthesizing phosphocholine analogues and the phosphocholine conjugates formed therefrom and their use in preventing infections caused by microorganisms.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]The application claims the benefit of U.S. Provisional Application No. 60 / 664,716 filed Mar. 23, 2005, which is hereby incorporated by reference in its entirety.STATEMENT OF FEDERALLY SPONSORED RESEARCH[0002]The U.S. Government may have certain rights in the invention described herein, which was made in part with funds from NIH Contract No. 263-02-D-0053.FIELD OF THE INVENTION[0003]The present invention relates to methods of synthesizing phosphocholine ester derivatives useful for, inter alia, protection against pathogenic microorganisms.BACKGROUND OF THE INVENTION[0004]Phosphocholine conjugates have been studied extensively because phosphocholine (PC) is thought to play a vital role in the pathogenesis of pathogenic microorganisms, such as Streptococcus pneumoniae. Some reports suggest that by binding to the platelet activating factor receptor on epithelial and endothelial cells, PC facilitates transport of S. pneumoniae into the blood a...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/00C07F9/02
CPCC07F9/091A61K39/00Y02A50/30
Inventor REZANKA, LOUIS J.
Owner UNITED STATES OF AMERICA
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