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WO2009067031A1 - Procédé pour la détection et/ou le dosage de la lovastatine estérase avec utilisation d'un réactif fluorogène/chromogène, lovastatine estérase isolée et/ou purifiée par ce procédé, ensemble pour la détection et/ou le dosage et utilisation du réactif fluorogène/chromogène pour la détection et/ou le dosage de la lovastatine estérase - Google Patents

Procédé pour la détection et/ou le dosage de la lovastatine estérase avec utilisation d'un réactif fluorogène/chromogène, lovastatine estérase isolée et/ou purifiée par ce procédé, ensemble pour la détection et/ou le dosage et utilisation du réactif fluorogène/chromogène pour la détection et/ou le dosage de la lovastatine estérase Download PDF

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Publication number
WO2009067031A1
WO2009067031A1 PCT/PL2008/000085 PL2008000085W WO2009067031A1 WO 2009067031 A1 WO2009067031 A1 WO 2009067031A1 PL 2008000085 W PL2008000085 W PL 2008000085W WO 2009067031 A1 WO2009067031 A1 WO 2009067031A1
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Prior art keywords
assay
detection
alkyl
enzymatic activity
fluorogenic
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PCT/PL2008/000085
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English (en)
Inventor
Ryszard Ostaszewski
Dominik Koszelewski
Waldemar Kurek
Olga Piotrowska
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Instytut Chemii Organicznej Polska Akademia Nauk
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Instytut Chemii Organicznej Polska Akademia Nauk
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
    • C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
    • C07D311/16—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted in position 7
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
    • C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
    • C07D311/18—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted otherwise than in position 3 or 7
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase

Definitions

  • the invention relates to a method for detection and/or assay of the lovastatin esterase enzymatic activity, with use of the fluorogenic/chromogenic reagent, lovastatin esterase isolated and/or purified using this method, an assembly for detection and/or assay of the lovastatin esterase enzymatic activity and use of the fluorogenic/chromogenic reagent for detection and/or assay of the lovastatin esterase enzymatic activity.
  • the lovastatin esterase enzyme independently of a source of origin, is an enzyme catalyzing the hydrolysis reaction of an ester bond in 2-methylbutyric acid side chain of lovastatin and its derivatives.
  • the enzyme may be obtained inter alia from Clonostachys compactiuscula culture, said fungus being deposited under No. ATCC 38009 or ATCC 74178.
  • the enzyme features by high selectivity with respect to a structure of lovastatin side chain and does not catalyze "the hydrolysis of another statin being structurally similar, i.e. simvastatin and its derivatives. Such capability is employed in the process for manufacturing of purified simvastatin.
  • the use of lovastatin esterase in the process of simvastatin synthesis is disclosed in U.S. patent No. 5,223,415.
  • Such assay technique and the type of substrate used render this method expensive.
  • the method cannot be applied for analyzing of small biological samples and for detection of an enzyme on a gel in a electrophoresis method.
  • the object of this invention is to present a method for detection and/or assay of lovastatin esterase enzymatic activity, with use of a fluorogenic/chromogenic reagent, lovastatin esterase isolated and/or purified using this method, an assembly for detection and/or assay of the lovastatin esterase enzymatic activity and use of the fluorogenic/chromogenic reagent for detection and/or assay of the lovastatin esterase enzymatic activity.
  • a method for detection and/or assay of the lovastatin esterase enzymatic activity, with use of the fluorogenic/chromogenic reagent, according to the invention is characterized in that, it comprises use of the fluorogenic/chromogenic reagent of formula II
  • R represents hydrogen or Ci-C 6 -alkyl
  • R' represents one or two substituents of the ring A independently selected from the group comprising: hydrogen, halogens, Ci-C 6 -alkyl, Ci-C 6 -alkenyl, Cj-C 6 -alkoxy, C]-C 6 -alkylcarboxy, d-C ⁇ -alkylcarbonyl, OH, CN, NO 2 ,
  • CONR R where each of R and R independently represents hydrogen, C 1 -C 6 alkyl or R 7 and R 8 taken together represent C 3 -C 6 alkylene substituent
  • NR 9 R 10 where each of R 9 and R 10 independently represents hydrogen, Ci-C 6 alkyl or R 9 and R 10 taken together represent C 3 -C 6 alkylene substituent, heteroaryl substituent having nitrogen atom as a ring atom and optionally an additional heteroatom selected from the group comprising O and S atoms, said heteroaryl substituent optionally fused with the benzene ring,
  • R" represents one or two or three substituents of the ring B independently selected from the group comprising hydrogen, halogens, Ci-C 6 -alkyl, Q-C ⁇ -alkenyl, d-C 6 -alkoxy, Ci-C 6 -alkylcarboxy, d-C 6 -alkylcarboxy-Ci-C 6 -alkyl, NO 2 , NR 11 R 12 where each of R 11 and R 12 independently represents hydrogen, Ci-C 6 alkyl or R 11 and R 12 taken together represent C 3 -C 6 alkylene substituent, by adding said reagent to a material of enzymatic activity and carrying out the hydrolysis reaction under enzyme action, followed by detection and/or assay of the liberated coumarin compound of formula III
  • a method comprises use of the fluorogenic/chromogenic reagent of formula II, wherein
  • R represents hydrogen or d-C 6 -alkyl
  • R' represents one or two substituents of the ring A independently selected from the group comprising hydrogen, halogens, d-C 6 -alkyl, Ci-C ⁇ -alkenyl, Ci-C 6 -alkoxy, Ci-C 6 - alkylcarboxy, Ci-Q-alkylcarbonyl, aminocarbonyl, OH, CN, NO 2 , NH 2 , - A -
  • R" represents one or two or three substituents of the ring B independently selected from the group comprising hydrogen, halogens, C!-C 6 -alkyl, Q-C ⁇ -alkenyl, CrC ⁇ -alkoxy, C i -C 6 -alky lcarboxy, C i -C ⁇ -alkylcarbonyl .
  • a method comprises use of the fluorogenic/chromogenic reagent of formula II, wherein
  • R represents hydrogen or Ci-Q-alkyl
  • R' represents one or two substituents of the ring A independently selected from the group comprising hydrogen, halogens, Q-C ⁇ -alkyl, Cj-C ⁇ -alkenyl, Q-C ⁇ -alkoxy
  • R" represents one or two or three substituents of the ring B independently selected from the group comprising hydrogen, halogens, Cj-C ⁇ -alkyl, Q-Co-alkenyl, CrC 6 -alkoxy.
  • a method comprises use of the fluorogenic/chromogenic reagent of formula II, wherein R represents methyl or C 3 -C 6 -alkyl, said reagent selected from a group comprising a racemic mixture, a mixture enriched in R enantiomer, a mixture enriched in S enantiomer, an optically pure S enantiomer and an optically pure R enantiomer.
  • a method comprises use of the fluorogenic/chromogenic reagent selected from the group comprising:
  • a method comprises use of the fluorogenic/chromogenic reagent selected from the group comprising:
  • the hydrolysis reaction is conducted in a buffered solution.
  • the hydrolysis reaction is conducted under a control of p ⁇ change and an adjustment of p ⁇ by use of a base. More preferably, the hydrolysis reaction is conducted at p ⁇ of 6-11.5, and even more preferably, the hydrolysis reaction is conducted at p ⁇ of 7.5-11. Most preferably, the hydrolysis reaction is conducted at p ⁇ of 8.5-10.5. Also preferably, the hydrolysis reaction is conducted at the temperature of 20°C-50°C. More preferably, the hydrolysis reaction is conducted at the temperature of 26°C-40°C.
  • the liberated coumarin compound of formula III is detected and/or assayed by means of a spectrophotometric method.
  • the liberated coumarin compound of formula III is detected and/or assayed by means of a fluorescent method. More preferably, the coumarin compound is qualitatively assayed and then the content/activity of lovastatin esterase is determined.
  • Lovastatin esterase is characterized in that, said esterase is isolated and/or purified using the method indicated above.
  • An assembly for detection and/or assay of lovastatin esterase enzymatic activity, according to the invention is characterized in that, it comprises the fluorogenic/chromogenic reagent of the formula II
  • R represents hydrogen or d-C 6 -alkyl
  • R' represents one or two substituents of the ring A independently selected from the group comprising: hydrogen, halogens, d-C 6 -alkyl, Ci-C 6 -alkenyl, d-C 6 -alkoxy, d-C 6 -alkylcarboxy, d-C 6 -alkylcarbonyl, OH, CN, NO 2 , CONR 7 R 8 where each of R 7 and R 8 independently represents hydrogen, Ci-C 6 alkyl, or R 7 and R 8 taken together represent d-C ⁇ alkylene substituent, NR 9 R 10 where each of R 9 and R 10 independently represents hydrogen, d-C ⁇ alkyl or R 9 and R 10 taken together represent C 3 -C 6 alkylene substituent, heteroaryl substituent having nitrogen atom as a ring atom and optionally an additional heteroatom selected from the group comprising O and S atoms, said heteroaryl substituent optionally fused with benzene ring,
  • R" represents one or two or three substituents of the ring B independently selected from the group comprising hydrogen, halogens, d-C 6 -alkyl, d-C 6 -alkenyl, d-C 6 -alkoxy, Ci-C 6 -alkylcarboxy, d-C 6 -alkylcarboxy-C,-C 6 -alkyl, NO 2 , NR 11 R 12 where R 11 and R 12 independently represents hydrogen, C!-C 6 alkyl or R 11 and R 12 taken together represent C 3 -C 6 alkylene substituent, and a compatible solvent.
  • R represents hydrogen or Cj-C 6 -alkyl
  • R' represents one or two substituents of the ring A independently selected from the group comprising: hydrogen, halogens, Q-C ⁇ -alkyl, Cj-C ⁇ -alkenyl, Q-C ⁇ -alkoxy, Ci-C ⁇ -alkylcarboxy, d-Ce-alkylcarbonyl, OH, CN, NO 2 ,
  • CONR 7 R 8 where each of R 7 and R 8 independently represents hydrogen, Ci-C 6 alkyl, or R and R taken together represent C 3 -C 6 alkylene substituent
  • NR 9 R 10 where each of R 9 and R 10 independently represents hydrogen, Ci-Qalkyl, or R 9 and R 10 taken together represent C 3 -C 6 alkylene substituent, heteroaryl substituent having nitrogen atom as a ring atom and optionally an additional heteroatom selected from the group comprising O and S atoms, said heteroaryl substituent optionally fused with benzene ring,
  • R" represents one or two or three substituents of the ring B independently selected from the group comprising hydrogen, halogens, Cj-C ⁇ -alkyl, d-C 6 -alkenyl, CpC ⁇ -alkoxy, Ci-C ⁇ -alkylcarboxy, d-Qj-alkylcarboxy-d-Ce-alkyl, NO 2 , NR 11 R 12 where each of R 11 and R 12 independently represents hydrogen, Cj-C 6 alkyl, or R 11 and R 12 taken together represent C 3 -C 6 alkylene substituent, is used for detection and/or assay of the lovastatin esterase enzymatic activity.
  • the fluorogenic/chromogenic reagent is used for detection of lovastatin esterase on a gel in a electrophoresis method.
  • the fluorogenic/chromogenic reagent is used for detection and/or assay of lovastatin esterase in an eluate in a chromatography method.
  • the fluorogenic/chromogenic reagent is used for detection and/or assay of lovastatin esterase in the method for manufacturing and/or purifying of simvastatin.
  • the activity of the enzyme has to be assayed and the purification has to be carried out to isolate the suitable fraction.
  • the method for detection and/or assay of the activity according to the invention is distinguished by high sensitivity, reproducibility and simplicity of experimen- tation. Due to the factj that a reaction product is a substance exhibiting strong absorption in the visible or UV region, and/or exhibiting strong UV fluorescence, the hydrolytic action of an enzyme is detected by means of optical detection methods (such as photometry, spectrophotometry, fluorescence, luminescence) assuring a high quality of assay.
  • fluorogenic/chromogenic reagent denotes a reagent of enzymatic reaction, where the fluorogenic/chromogenic reagent on treatment of the lovastatin esterase enzyme is cleaved to form fluorescent and/or chromatic compound, which may be detected by optical methods, especially by measurement of fluorescence and/or absorption in visible and UV region of light.
  • M represents a hydrogen atom, and also wherein M represents a metal cation or ammonium cation, i.e., the compound in the free-acid form or in the salt form, respectively, if not specified otherwise.
  • M represents a metal cation or ammonium cation, i.e., the compound in the free-acid form or in the salt form, respectively, if not specified otherwise.
  • the term "triol" may also comprise the lactone-diol form of the formula Id, if not specified otherwise.
  • lovastatin salt represents the compound of the formula Ia, wherein M represents a metal cation or an ammonium cation
  • sivastatin salt represents the compound of the formula Ib, wherein M represents a metal cation or an ammonium cation.
  • the fluorogenic/chromogenic reagent according to the invention is the compound of formula II wherein
  • R represents hydrogen or Cj-C 6 -alkyl
  • R' represents one or two substituents of the ring A independently selected from the group comprising: hydrogen, halogens, C!-C 6 -alkyl, Q-Co-alkenyl, CrQ-alkoxy, Cj-Q-alkylcarboxy, Ci-C ⁇ -alkylcarbonyl, OH, CN, NO 2 ,
  • CONR 7 R 8 where each of R 7 and R 8 independently represents hydrogen, Q-C ⁇ alkyl or R and R taken together represent C 3 -C 6 alkylene substituent, NR 9 R 10 where each of R 9 and R 10 independently represents hydrogen, C 1 -QaIlCyI or
  • R 9 and R 10 taken together represent C 3 -C 6 alkylene substituent, heteroaryl substituent having nitrogen atom as a ring atom and optionally an additional heteroatom selected from the group comprising O and S atoms, said heteroaryl substituent optionally fused with the benzene ring, R" represents one or two or three substituents of the ring B independently selected from the group comprising hydrogen, halogens, Ci-C 6 -alkyl, C]-C 6 -alkenyl, Cj-C 6 -alkoxy, C ⁇ -C 6 -alkylcarboxy, d-Q-alkylcarboxy-Ci-Ce-alkyl, NO 2 , NR 11 R 12 where each of R 11 and R 12 independently represents hydrogen, Ci-C 6 alkyl or R 11 and R 12 taken together represent C 3 -C 6 alkylene substituent.
  • R' represents one or two substituents of the ring A independently selected from the group comprising hydrogen, halogens, Ci-C ⁇ -alkyl, d-C ⁇ -alkenyl, Cj-C ⁇ -alkoxy, Ci-C ⁇ -alkylcarboxy, Cj-C ⁇ -alkylcarbonyl, aminocarbonyl, OH, CN, NO 2 , NH 2 , and R" represents one or two or three substituents of the ring B independently selected from the group comprising hydrogen, halogens, Ci-C ⁇ -alkyl, Ci-C 6 -alkenyl, Q-C ⁇ -alkoxy, Q-Q-alkylcarboxy, Cj-Co-alkylcarbonyl.
  • R' represents one or two substituents of the ring A independently selected from the group comprising hydrogen, halogens, Ci-C 6 -alkil, Ci-C 6 -alkenyl, Ci-C 6 - alkoxy
  • R" represents one or two or three substituents of the ring B independently selected from the group comprising hydrogen, halogens, Ci-C 6 -alkyl, Ci-C 6 -alkenyl, Q-Q-alkoxy.
  • the fluorogenic/chromogenic reagent may exist in a form of racemic mixture comprising R and S enantiomers.
  • the fluorogenic/chromogenic reagent used is selected from a group comprising a racemic mixture, a reagent enriched in R enantiomer, a reagent enriched in S enantiomer, an optically pure S enantiomer and an optically pure R enantiomer.
  • the fluorogenic/chromogenic reagent of formula II is used for detection and/or assay of lovastatin esterase activity in crude and purified biological preparations obtained from native and/or genetically modified organisms.
  • an enzyme catalyzed reaction illustrated on Scheme II,
  • the coumarin compounds of the formula III are produced (where R' and R" possess the meaning indicated above), which strongly absorb the radiation in the visible and/or UV region, and/or strongly fluoresce allowing the detection and/or assay of a liberated coumarin compound by means of optical methods. Therefore rapid qualitative and quantitative assay of the lovastatin esterase enzyme in test samples is possible. Especially, the detection and/or assay of corresponding coumarin compounds of formula III is conducted by means of spectrophotometric and/or spectrofluorimetric methods.
  • the assay may be performed for different biological preparations containing the enzyme of such activity, being of various origin, in aqueous solutions, in the course of an enzyme purification process, an immobilization process, in the course of analyze of test samples derived from the enzyme purification process following separation by means of medium pressure liquid chromatography and high performance liquid chromatography, inokulum culturing, a fermentation on a laboratory and industrial scale.
  • the compound of formula II as the reagent in a method for detection and/or assay of enzymatic activity, detecting of the lovastatin esterase enzyme on gels following a sample separation by an electrophoresis method is possible.
  • identification comprises lighting of a gel surface e.g. by UV light of a wavelength 366 run, to track a fluorescence band on a surface at location of a presence of an enzyme catalyzed reaction product, i.e. the coumarin compound of formula III.
  • Fig. 1 presents the change of fluorescence intensity (in relative measures) of the reagent governed by a kinetic catalytic effect of the enzyme versus time
  • Fig. 2 presents the change of enzyme specific activity versus pH, determined using said reagent.
  • LE enzyme represents the lovastatin esterase enzyme.
  • the fluorogenic/chromogenic reagents of the formula II are obtained in conventional manner, according to Scheme III, by reacting a substituted coumarin compound and an aliphatic carboxylic acid derivative of formula C 2 HsCH(R)COOH, where R represents hydrogen or CrC 6 -alkyl.
  • R represents hydrogen or CrC 6 -alkyl.
  • the derivative of 2-methylbutyric acid is used (R represents methyl).
  • an aliphatic carboxylic acid derivative is an activated acid form, such as acid halide, active ester, acid anhydride or mixed anhydride prepared in situ or separately prepared.
  • the reaction usually is carried out in aprotic solvents, e.g.
  • IR (KBr) cm '1 2971, 1758, 1732, 1615, 1388, 1263, 1148, 1131, 1108, 1017, 858.
  • the embodiment examples illustrate use of the compounds of formula V for rapid detection of LE enzyme in a biological material (employing the enzymatic reaction according to Scheme IV), for spectrophotometric assay of the enzymatic activity of LE enzyme in the samples obtained and in the process of enzyme purification.
  • Example 35 Use of the compound of formula V as the fluorogenic/chromogenic reagent for optical assay of lovastatin esterase
  • the lovastatin esterase enzyme (LE) is produced by fungus Clonostachys compactiuscula ATCC 38009. The biological preparation of fungus is prepared according to the protocol disclosed in U.S. patent No. 5,223,415.
  • the frozen homogenate is transferred to the centrifuge tubes and allowed to thaw.
  • the enzyme is extracted with the glicyne buffer (50 mM, pH 9.4, 4°C) of a volume being proportional to the weight of mycelium (50 mL). Following cellular material centrifugation (20 min., 14000 rpm, 4°C) the pellet is extracted once again to obtain 130 mL of supernatant, in total.
  • the concentration of protein is determined by a direct measurement of a solution absorbance or by Lowry method [Cwiczenia z biochemii (Exercises in biochemistry), ed. L. Klyszejko-Stefanowic, PWN, 2005]. The accurate protocol is listed below.
  • the concentration is red out from the standard curve formerly prepared for BSA protein (calf albumin) given by the equation
  • the protein extract Prior to the column chromatography purification, the protein extract (0.5 mL) is diluted 20-fold for assay; following purification the dilution of protein extract is not required, since the protein concentration is lowered. Each assay is repeated three-fold.
  • a sample of 15 ⁇ L is drawn.
  • the protein mass being present in a sample is determined by multiplication of the concentration and volume values.
  • a compound of formula V is added (50 ⁇ L of the solution in acetone, the concentration of 0.0 IM) to 1 mL of the supernatant obtained and the mixture is allowed to stay for 1 minute at ambient temperature.
  • the characteristic blue coloration is observed originated from the hydrolysis product being the compound of formula IV, this is observed for the assayed solution only, but not in the case of the blank sample.
  • Example 37 Use of compounds of formula V for spectrophotometric assay of the enzymatic activity of the LE enzyme-containing material - general method
  • the measurement assembly comprises a thermostatted reactor (30°C ⁇ l) of capacity 20 mL positioned on a magnetic stirrer. The stirring rate of 300 rpm is applied. The absorbance changes are recorded by the detector Shiniadzu SPD-6A.
  • Example 38 Use of compounds from Examples 1 and 2 as the fluorogenic/chromogenic reagents for the chromatic and spectrophotometric assays of lovastatin esterase
  • the specific activity for the reaction of zero order is calculated from linear relationship: concentration [nmol/mg]/time [min], as a gradient graph.
  • Example 40 Use of compounds from Examples 1 and 2 for qualitative and quantitative assay of the LE enzyme activity in the course of enzyme purification process - general method
  • the protein fraction obtained in Example 44 by. precipitation on salting with ammonium sulfate (the saturation range 40-80%), dissolved in the glicyne buffer (5 mL, 12.8 mg/mL), is loaded on the hydrophobic interaction column (HIC) packed with Phenyl- Sepharose (1.5 x 18 cm).
  • the proteins are eluted with the same glycine buffer of pH 9.4 (flow: 1.5 mL/min, pressure 69 kPa).
  • the changes of LE enzyme activity with respect to pH are determined with use of 2-methylbutyric acid 2-oxo-2H-chromen-7-yl ester from Example 1, according to the protocol described in Example 37.
  • the activity values are determined in the course of sequential adjustments of pH of a buffer used, from the value 7.8 to 9.9, and are presented in the form of bar diagram on Fig. 2.
  • the data obtained reveal the fact that the enzyme activity is the highest one at the pH value of 9.6.
  • Example 43 Use of the compounds from Examples 1 and 2 for LE enzymatic activity assay in the course of enzyme purification process by means of the polyacrylamide gel electrophoresis
  • the homogenate of cellular material is extracted with phosphorane buffer (20 mM, pH 7.8, 4°C, Tween 80 (0.1%), NaN 3 (0.01%), 50 mL) with careful mixing of the extract with a glass spatula in order to avoid formation of a foam.
  • the supernatant 1 is obtained.
  • the extraction of a pellet is repeated and the sequentially obtained supernatants are subjected to the determination of a protein concentration and specific activities, using the compound from Example 3 as the fluorogenic/chromogenic reagent.
  • Table 5 The results obtained are summarized in Table 5.
  • the protein concentration is determined by the direct measurement of solution absorbance or by Lowry method [Cwiczenia z biochemii (Exercises in biochemistry), ed. L. Klyszejko-Stefanowic, PWN, 2005). The accurate protocol is summarized below.
  • the fraction B is purified by the method of hydrophobic interaction chromatography (HIC) using column packed with Phenyl-Sepharose and eluting with phosphorane buffer (20 mM, pH 6.5, EDTA 5mM, NaCl 0.5 M; flow: 1.6 mL/min, pressure 10 psi, detector sensivity 0.1). Following washing out of the proteins non- adsorbed on the column, (0-15 minute period, UV 254 run), the eluent system is changed to water. The presence of the LE enzyme in the specific fractions is detected by an addition of the fluorogenic/chromogenic reagent solution (0.1 mL, 0.05 mol).
  • HIC hydrophobic interaction chromatography
  • the fraction collected is the fraction exhibiting the activity in the reaction with the fluorogenic/chromogenic reagent, and is featured by the retention time of 20 minutes and by the volume of 50 mL (fraction C).
  • the analysis of the fraction C activity leads to the conclusion, that the fraction C contains purified LE enzyme, in 8% yield (defined as a ratio of total activity of a given fraction to the total activity of the homogenate, and expressed in percent), of the purity level 25 (defined as a ratio of specific activity of a given fraction to specific activity of homogenate).
  • the HPLC analyses were performed on the liquid chromatograph apparatus Pro Star by Varian, equipped with ProStar 330 Photodiode Array Detector, Prostar 410 AutoSampler, ProStar 410 Solvent Delivery Module.
  • the separation of compounds was performed on the column RP LiChro Cart 55-4, packed with octadecylsilyl silica gel for chromatography R (RP- 18), 3 ⁇ m.
  • the elution of molecules was carried out using the solvent of the following content: acetonitrile (350 mL), orthophosphoric acid (0.375 mL), water (150 mL), at the flow speed 1.5 mL/min, at wavelength 238 nm.
  • the retention times of the consecutive components of the reaction mixture were as follows: 0.51 minutes - triol, 2.21 minutes - lovastatin, 3.22 minutes - simvastatin.

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Abstract

L'invention concerne un procédé pour la détection et/ou le dosage de l'activité enzymatique lovastatine estérase, avec l'utilisation du réactif fluorogène/chromogène, comprenant l'utilisation du réactif fluorogène/chromogène de formule II dans laquelle R représente hydrogène ou alkyle en C1-C6, R' représente un ou deux substituants du noyau A, R' représente un ou deux ou trois substituants du noyau B. Selon un procédé, le réactif est ajouté à un matériel d'activité enzymatique et la réaction d'hydrolyse sous action enzymatique est réalisée, en faisant suivre par la détection et/ou le dosage du composé coumarine libérée de formule III. L'invention concerne également une lovastatine estérase isolée et/ou purifiée à l'aide de ce procédé, un ensemble pour la détection et/ou le dosage de l'activité enzymatique lovastatine estérase, et une utilisation du réactif fluorogène/chromogène de formule II pour la détection et/ou le dosage de l'activité enzymatique lovastatine estérase.
PCT/PL2008/000085 2007-11-19 2008-11-18 Procédé pour la détection et/ou le dosage de la lovastatine estérase avec utilisation d'un réactif fluorogène/chromogène, lovastatine estérase isolée et/ou purifiée par ce procédé, ensemble pour la détection et/ou le dosage et utilisation du réactif fluorogène/chromogène pour la détection et/ou le dosage de la lovastatine estérase Ceased WO2009067031A1 (fr)

Applications Claiming Priority (2)

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PLP383818 2007-11-19
PL383818A PL212182B1 (pl) 2007-11-19 2007-11-19 Sposób wykrywania i/lub oznaczania aktywności enzymatycznej esterazy lowastatyny z użyciem substratu fluoro/chromogennego, zestaw do wykrywania i/lub oznaczania aktywności esterazy lowastatyny oraz zastosowanie substratu fluoro/chromogennego do pozaustrojowego wykrywania i/lub oznaczania aktywności enzymatycznej

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013003168A1 (fr) * 2011-06-27 2013-01-03 Merial Limited Dérivés insectifuges inédits de coumarine, leur synthèse et leurs procédés d'utilisation
WO2013044118A3 (fr) * 2011-09-23 2013-07-04 Merial Limited Modélisation indirecte de molécules répulsives inédites actives contre les insectes, les acariens et autres arthropodes
US9400273B1 (en) 2009-12-09 2016-07-26 Life Technologies Corporation 7-hydroxycoumarin-based cell-tracking reagents
CN111039910A (zh) * 2019-12-31 2020-04-21 云南大学 一种光引发的合成3-芳基黄酮或香豆素类化合物的方法及应用
CN117665174A (zh) * 2024-02-01 2024-03-08 巴中市产品质量检验检测中心 一种检测pde5和ssri的方法及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5223415A (en) * 1990-10-15 1993-06-29 Merck & Co., Inc. Biosynthetic production of 7-[1',2',6',7',8',8a'(R)-hexahydro-2'(S),6'(R)-dimethyl-8'(S)-hydroxy-1'(S)-naphthyl]-3(R),5(R)-dihydroxyheptanoic acid (triol acid)
US5859051A (en) * 1996-02-02 1999-01-12 Merck & Co., Inc. Antidiabetic agents
WO2005040107A2 (fr) * 2003-10-21 2005-05-06 Diversa Corporation Procedes pour fabriquer la simvastatine et leurs intermediaires

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5223415A (en) * 1990-10-15 1993-06-29 Merck & Co., Inc. Biosynthetic production of 7-[1',2',6',7',8',8a'(R)-hexahydro-2'(S),6'(R)-dimethyl-8'(S)-hydroxy-1'(S)-naphthyl]-3(R),5(R)-dihydroxyheptanoic acid (triol acid)
US5859051A (en) * 1996-02-02 1999-01-12 Merck & Co., Inc. Antidiabetic agents
WO2005040107A2 (fr) * 2003-10-21 2005-05-06 Diversa Corporation Procedes pour fabriquer la simvastatine et leurs intermediaires

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BABIAK PETER ET AL: "A high-throughput, low-volume enzyme assay on solid support.", ANALYTICAL CHEMISTRY 15 JAN 2005, vol. 77, no. 2, 15 January 2005 (2005-01-15), pages 373 - 377, XP002512505, ISSN: 0003-2700 *
KOLLER E ET AL: "SYNTHESES AND SPECTRAL PROPERTIES OF LONGWAVE ABSORBING AND FLUORESCING SUBSTRATES FOR THE DIRECT AND CONTINUOUS KINETIC ASSAY OF CARBOXYLESTERASES PHOSPHATASES AND SULFATASES", MONATSHEFTE FUER CHEMIE, vol. 116, no. 1, 1985, pages 65 - 75, XP002512506, ISSN: 0026-9247 *
OSTASZEWSKI ET AL.: "A new substrate for fluoresecent and spectrophotometric detection and assays of lovastatin esterase, plus a method to immobilize lovastatin esterase and the use of immobilisates in the biotechnological synthesis of simvastatin.", SELECTED RESEARCH FINDING OF AN INNOVATIVE NATURE 2007- BIOLOGICAL AND MEDICAL SCIENCES-INSTITUTES OF THE POLISH ACADEMY OF SCIENCES, January 2007 (2007-01-01), XP002512504, Retrieved from the Internet <URL:http://www.portalwiedzy.pan.pl/images/stories/pliki/publikacje/katalogi_osiagniec/selected_research_2007/pan_str62_82.pdf> [retrieved on 20090128] *
PARMAR V S ET AL: "Synthesis, Characterisation and In Vitro Anti-invasive Activity Screening of Polyphenolic and Heterocyclic Compounds", BIOORGANIC & MEDICINAL CHEMISTRY, ELSEVIER SCIENCE LTD, GB, vol. 11, no. 6, 1 January 2003 (2003-01-01), pages 913 - 929, XP002348797, ISSN: 0968-0896 *
SCHIMMEL TIMOTHY G ET AL: "Purification and characterization of a lovastatin esterase from Clonostachys compactiuscula", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 63, no. 4, 1997, pages 1307 - 1311, XP002512508, ISSN: 0099-2240 *
WOLFBEIS O S ET AL: "SYNTHESES ABSORPTION AND FLUORESCENCE SPECTRA OF 7 HYDROXY-3-PYRIDYLCOUMARINS THEIR ESTERS ETHERS AND QUATERNIZED DERIVATIVES", CHEMISCHE BERICHTE, vol. 118, no. 9, 1985, pages 3664 - 3672, XP002512507, ISSN: 0009-2940 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9400273B1 (en) 2009-12-09 2016-07-26 Life Technologies Corporation 7-hydroxycoumarin-based cell-tracking reagents
US11175204B1 (en) 2009-12-09 2021-11-16 Life Technologies Corporation 7-hydroxycoumarin-based cell-tracking reagents
WO2013003168A1 (fr) * 2011-06-27 2013-01-03 Merial Limited Dérivés insectifuges inédits de coumarine, leur synthèse et leurs procédés d'utilisation
WO2013044118A3 (fr) * 2011-09-23 2013-07-04 Merial Limited Modélisation indirecte de molécules répulsives inédites actives contre les insectes, les acariens et autres arthropodes
CN111039910A (zh) * 2019-12-31 2020-04-21 云南大学 一种光引发的合成3-芳基黄酮或香豆素类化合物的方法及应用
CN117665174A (zh) * 2024-02-01 2024-03-08 巴中市产品质量检验检测中心 一种检测pde5和ssri的方法及其应用
CN117665174B (zh) * 2024-02-01 2024-04-23 巴中市产品质量检验检测中心 一种检测pde5和ssri的方法及其应用

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