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Quantitative conversion of indene to (1S,2R) indene oxide and (1S,2R)-indanpiol by combination of haloperoxidase bioconversion and chemical steps

A technology of dihydroindanediol and haloperoxide, applied in biochemical equipment and methods, microorganisms, organic chemistry, etc., can solve problems such as immature non-infectious virus particles

Inactive Publication Date: 2001-06-06
MERCK & CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, Kohl, N.E., et al. Proceedings of the American Academy of Sciences, 85, 4686 (1988) showed that genetic inactivation of the HIV-encoded protease results in the production of immature, non-infectious virions

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Example 1 Organisms and Culture Conditions

[0114] Fungal strains obtained from high-salt environments (such as deserts, swamps, seas, etc.) were maintained on YMA medium slant (composition gm / L: malt extract, 10; yeast extract, 4; glucose, 4; agar, 20 ; 10mL trace element solution; pH adjusted to 7.0). The trace element solution is made of FeSO 4 ·7H 2 O, 1.0; MnSO 4 ·H2 O, 1.0; CuCl 2 2H 2 O, 0.025; CaCl 2 2H 2 O, 0.10; H 3 BO 3 , 0.056; ZnSO 4 ·7H 2 O, 0.20; and (NH 4 ) 6 Mo 7 o 24 4H 2 O composition. Growth on medium slant (Ca, 1cm 2 ) was used to inoculate 250mL of KF seed medium containing 25mL (composition (gm / L): corn steep liquor, 5; tomato paste, 40; oat flour, 10; glucose, 10, 10mL trace element solution; pH adjusted to 6.8) Erlenmeyer flask. The seed culture was grown for 2 to 3 days at 28°C with shaking at 220 rpm. 1 mL of seed culture was used to inoculate 250 mL Erlenmeyer flasks each containing 25 mL of a modifi...

Embodiment 3

[0117] Example 3 Production of haloperoxidase in a 2-liter Erlenmeyer flask

[0118] Fungal strains identified in the screen as producing neutral haloperoxidase were subcultured in 2-liter Erlenmeyer flasks containing 500 mL of CFM medium. The Erlenmeyer flasks were inoculated with 20 mL of the 72-hour culture and grown on an orbital shaker at 180 rpm at 28°C. Samples (10 mL) were removed from the flasks and centrifuged at 3000 rpm for 15 minutes in a benchtop centrifuge. A volume of 5 mL of supernatant was mixed with 5 mL of phenol red solution and incubated at 25 °C. 20 μl volume of 0.3% H at 0, 1, 2 and 3 hours 2 o 2 The solution is added to the reactants. After incubation at 25°C for 24 hours, the absorbance of the test mixture was measured as previously described. One unit of haloperoxidase is defined as the amount of enzyme required to catalyze an increase of one OD unit at 595 nm using the aforementioned conditions.

Embodiment 4

[0119] Embodiment 4 enzyme preparation

[0120] Cultures identified in the phenol red screen as having neutral haloperoxidase activity were obtained by centrifuging the complete liquid medium and collecting the culture supernatant (approximately neutral pH). The concentrated preparation was obtained by adding ammonium sulfate (80% saturated) to the supernatant, redissolving the precipitate in 0.1 M phosphate buffer (pH 7), and filtering to remove insoluble melanin. Concentrated preparations typically give enzyme activity in a 3-5 fold increase when analyzed by the phenol red assay. Formulations were stored at 4°C until use.

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PUM

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Abstract

A process is disclosed that quantitatively bioconverts indene to (1S,2R)-indene oxide and (1S,2R)-indandiol, by the action of fungal haloperoxidase followed by various chemical step(s), e.g., adjusting the pH.

Description

Background of the invention [0001] This application is related to: Merck 18996, U.S.S.N. 08 / 059,038, filed May 7, 1993, 189961A, U.S.S.N. 08 / 235,576, filed April 29, 1994, Merck 19251, Merck 19114 and Merck 19115. [0002] The present invention relates to a method for synthesizing intermediates of compounds inhibiting protease encoded by human immunodeficiency virus (HIV), especially certain oligopeptide analog compounds, such as compound J in the following examples. These compounds are of value in the prevention of HIV infection, in the treatment of HIV infection and in the treatment of acquired immunodeficiency syndrome (AIDS) resulting therefrom. These compounds are also useful for inhibiting renin and other proteases. [0003] The present invention introduces the method that converts indene into chiral dichloroindene oxide, as shown in the following scheme: [0004] Program [0005] A retrovirus named Human Immunodeficiency Virus (HIV) is an etiologi...

Claims

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Application Information

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IPC IPC(8): C07D241/04C07D303/04C07D401/06C07D413/06C12P7/22C12P17/02
CPCY10S435/911C07D303/04C12P17/02C12P7/22C07D401/06C07D241/04C07D413/06
Inventor M·M·查尔特赖恩N·C·肯诺尔斯G·M·加尔里泰小R·C·奥勒温斯基T·R·维尔霍文张劲游
Owner MERCK & CO INC
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