WO2015035487A1

WO2015035487A1 - Method and kit for synthesizing phenolphthalein diphosphate tetrasodium

Info

Publication number: WO2015035487A1
Application number: PCT/BR2014/000328
Authority: WO
Inventors: Claudio Cerqueira Lopes; Rosangela Sabbatini CAPELLA; André Luiz Mazzei ALBERT; Paulo Roberto Miguel FRAGAS
Original assignee: Universidade Federal Do Rio De Janeiro - Ufrj
Priority date: 2013-09-11
Filing date: 2014-09-11
Publication date: 2015-03-19
Also published as: BR102013023221B1; BR102013023221A2

Abstract

The present invention relates to a method for synthesizing the compound phenolphthalein diphosphate tetrasodium using triethylamine. More specifically, the compound according to the present invention makes it possible to identify in loco the acid phosphatase enzyme present in semen stains, and is applicable to cases of denunciation of the crime of rape.

Description

"PHENOLPHALEINE SYNTHESIS PROCESS Tetrasodium bisphosphate and KIT"

FIELD OF INVENTION

The present invention relates to a process for the synthesis of the tetrasodium phenolphthalein bisphosphate compound.

More specifically, the compound of the present invention allows for the in situ identification of the acid phosphatase enzyme present in semen spots applicable to cases of rape allegations.

BACKGROUND OF THE INVENTION

In cases of rape crime, where there are no witnesses, detecting the presence of human semen is a helpful factor in forensic investigations to confirm the nature of the crime, which combined with criminal body examinations and laboratory analyzes to verify in the collected material. in the victim's body, containing the presence of the intact male gamete and finally the confirmation of the aggressor assumption indicated by the victim through the DNA examination of this material obtained. Collection of material in or around the female genital region or even anal region for the presence of semen may also include investigation through biochemical analyzes based on the detection of seminal fluid metabolites such as: the amino acid choline, prostaglandin-E and the zinc element, abundantly found in seminal fluid, as well as the detection of enzymatic activity among them the enzyme gamma glutamyl transpepidase and the enzyme acid phosphatase.

Identification of seminal patches is often of great value in forensic practice, particularly in cases of rape, sexual assault, sexual homicide or even adultery. One of the main purposes of investigations of sexual offenses in forensic laboratories is to analyze the stains or any other biological material taken from both the offender and the victim or to analyze stains found on tissues, linings, or any other possible evidence of the offense to verify the presence. of semen, with the purpose of proving the crime and offering justice to the defendant conviction (DU MONT, PATNIS; 2000).

The most recommended biochemical techniques for routine forensic analysis in rape cases include cytological verification of the presence of intact male gametes, acid phosphatase enzyme (APA) activity, and more recently PSA enzyme detection. Notably, a major test for localization and characterization of the seminal spot includes detection of the prostate acid phosphatase enzyme.

Acid phosphatase is an enzyme that is secreted by the prostate gland in seminal fluid and is present in all portions of the ejaculate. Its concentrations in seminal fluid are up to 400 times higher than those found in any other body fluid.

This test is useful for identifying seminal patches in the absence of sperm, since the number of individuals with aspermia is increasing due to the popularization of vasectomy surgeries and, moreover, those with oligospermia and azoospermia are the most prevalent ones. cases of sexual abuse crimes compared with normal fertile adult men (SAFERSTEIN; 2001) (McCLOSKY, MUSCILLO, NOORDEWIER; 1975).

The acid phosphatase test seems to be a more reliable test for characterizing a seminal spot in rape cases, because even in cases of ejaculation in individuals with aspermia the acid phosphatase enzyme content will give comparatively high results as compared to individuals. sexually healthy (BRAUNER, GALLILI; 1993).

Some reagents may be employed in the qualitative detection and quantitative determination of the acid phosphatase enzyme. All of these reagents act in a similar way, they are basically molecules used as pH indicators that change in their structural form as the pH of the medium assuming a generally chromophoric ion form. These substances are modified in their structures having one or more phosphate groups attached to an oxygen atom in the phenolic part of the molecule and these bonds are stable and selectively ruptured through the action of the acid phosphatase enzyme releasing the chemical phosphate P0 ₄ ^{3 "} or HP04. ^{2 "} and the indicator in its ionic form which usually have a definite color for a given value or pH range.

The most common reagents used for the determination of the acid phosphatase enzyme are phenolphthalein bisphosphate tetrasodium, thymolphthalein monophosphate disodium and naphthyl amine monophosphate disodium, all of these substances are phosphate derivatives of classical pH indicators, usually employed in the form of sodium salt to favor Solubility in water, solvent commonly used in biological analysis.

Of these reagents, the handling with phenolphthalein bisphosphate tetrasodium is more efficient due to its ease of visualization than colorless while buffered at pH = 5 going directly to the ionic form of this indicator that shows pink coloration to pH 8.3 when in the presence of a coloring agent, in this case a solution of an inorganic base, due to the low toxicity of phenolphthalein released in the medium, by the low cost of this reagent compared to others used.

In this process two purification steps are required due to the use of pyridine. In the first stage part of the pyridine is eliminated in the treatment phase of the tetrachlorinated product with water and the second stage consists of successive extractions with ethyl ether until the complete removal of this toxic reagent.

Furthermore, the research group of the present invention has carried out this synthesis proposed by the prior art, finding yields of less than 10% of the desired product.

Accordingly, said process does not address any type of formulation containing phenolphthalein tetrasodium phosphate useful in detecting the acid phosphatase enzyme.

US3,331,857 describes a process for synthesizing phenolphthalein monophosphoric acid also useful in determining acid phosphatase.

Further, US2999793 describes dry preparation for the determination of phosphatases comprising an alkali metal or phenolphthalein phosphate alkaline earth metal salt and a buffer to maintain a pH of 9-1.

The tetrasodium phenolphthalein bisphosphate compound is obtained by a classical route proposed by Huggins and Talalay in 1945. The major drawback of this process is the use of pyridine as a base, as this reagent has high toxicity and provides a final product with high concentrations of sodium ethoxide, a condition that makes it impossible to use this product in the quantitative assessment of acid phosphatase in various forensic applications such as: rape crime scenes, semen quantification in samples of this biological matrix in cattle, horses, pigs, goats and insect larvae, such as blowflies of the species Chrysomia albiceps, etc.

Thus, there remains a demand for a new synthetic route for the phenolphthalein bisphosphate tetrasodium compound, where it is possible to reduce toxicity levels in good yields and economically viable.

SUMMARY OF THE INVENTION

The present invention relates to a tetrasodium phenophthalein bisphosphate synthesis process using triethylamine useful in the on-site identification of acid phosphatase enzyme, present in semen spots, applicable to cases of allegations of rape crimes.

The process of the present invention is capable of generating a higher purity end product and is still economically viable as it uses commercially available reagents.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 - Synthesis of tetrasodium phenolphthalein bisphosphate (I).

Figure 2 - Hydrogen nuclear magnetic resonance spectrum of phenolphthalein bisphosphate tetrasodium commercially obtained from Sigma Aldrich Co.

Figure 3 - Hydrogen nuclear magnetic resonance spectrum of the phenolphthalein bisphosphate tetrasodium synthesized and purified by the process of the present invention.

Figure 4 - Carbon Nuclear 13 magnetic resonance spectrum of phenolphthalein tetrasodium bisphosphate commercially obtained from Sigma Aldrich Co.

Figure 5 - Carbon nuclear 13 magnetic resonance spectrum of phenolphthalein bisphosphate tetrasodium synthesized and purified by the process of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a tetrasodium phenophthalein bisphosphate synthesis process using triethylamine.

This process has a high yield and purity far superior to the methods proposed by the prior art, which do not address the yield or the purity of the obtained product.

The reagents used in the original route published by Huggins and Talalay in 1945 are of high toxicity, presenting the final product with many aggregate impurities. The commercial product had an impurity content of approximately 20%.

Advantageously, the method described in this invention is capable of producing the tetrasodium phenolphthalein bisphosphate reagent in 75% yields of high purity.

In this same context, the phenolphthalein bisphosphate tetrasodium synthesized in this work as an analytical standard does not contain sodium ethoxide, a condition that enables the use of this product in the quantitative evaluation of acid phosphatase in various forensic applications, such as: in scenes of crimes of rape, semen quantification in samples of this biological matrix in cattle, horses, pigs, goats and insect larvae, such as Chrysomia albiceps blowflies, etc.

The process of the present invention is based on the reaction of phenolphthalein with triethylamine and phosphorus oxychloride and is generally described by the following steps:

(i) Mixing phenolphthalein with phosphorus oxychloride and dichloromethane;

(ii) ^" addition of triethylamine to the reaction mixture;

(iii) Evaporation of dichloromethane;

(iv) Addition of sodium hydroxide;

(v) acidification of the solution;

(vi) Addition of sodium ethoxide;

(vii) Purification.

(viii) Re-precipitation

The reaction is conducted at a maximum of 4 ° C. However, despite the low temperature the reaction mixture advantageously does not have high viscosity.

Generally the synthesis process of the present invention is carried out according to the procedure outlined below.

Experimental procedure

In a 50 g beaker, 8.0 grams of phenolphthalein was weighed, with the aid of the funnel for solids the mass was transferred into a tritubulated flask immersed in crushed ice and coarse salt bath.

Then 12.5 mL of dichloromethane and 6.3 mL of phosphorus oxychloride were added. The phenolphthalein-dichloromethane-containing reaction mixture was stirred vigorously and a uniform suspension was obtained at this stage.

A constant pressure addition funnel containing 6.3 mL triethylamine was coupled to the tritubulated flask, then added, so that the reaction temperature did not exceed 4 ° C.

The reaction should be stirred at a maximum of 4 ° C for a period of 6 hours.

The progress of the reaction can be assessed by taking small aliquots of the reaction mixture, dripping on a touch plate followed by adding small amounts of a 10% NaOH solution. While this test has a pink color, the reaction was kept at rest and at room temperature, this test should be repeated if a negative result, ie, colorless against 10% NaOH solution was observed.

The next step was to remove dichloromethane by evaporation, then 100 mL of ice-cold distilled water was added at about 4 ° C with constant stirring.

After all HCl vapor release, a yellowish amber-like precipitated mass is formed.

The next step was to add under stirring 40% NaOH solution until the entire precipitate was resolubilized and the solution may turn slightly pink due to the small amount of unreacted phenyphthalein. This solution was filtered with activated charcoal.

The next step is to acidify the aqueous solution (phenaphthalene bisphosphate) with a mineral acid at pH 1.00, preferably concentrated HCl, strongly acidic pH, pH 1.00 verified with Merck universal indicator paper. acidic, like a mass that resembles brown-colored chewing gum.

Then the mass was separated from the supernatant liquid and in a 250 mL beaker, the entire mass of acid-phenphenalphenephalate bisphosphate was dissolved with as little freshly distilled Vetec PA grade methanol as possible, adding a few drops of Vetec PA grade formamide to increase solubility. , the amount of formamide is empirical.

1.0 mol.L-1 sodium ethoxide solution was added under stirring until all precipitation was complete.

The tetrasodium phenyphthalein bisphosphate precipitate was filtered off.

The tetrasodium phenyphthalein bisphosphate was washed repeatedly with small portions of ethyl alcohol and then with dry ether, this sequence was repeated until the solid was almost dry.

The tetrasodium phenyphthalein bisphosphate was dissolved with as little methanol / formamide as possible (5: 1) and then ethanol was slowly added to re-precipitate the purified tetrasodium phenyphthalein bisphosphate. After filtration separation the tetrasodium phenyphthalein bisphosphate was kept in a vacuum desiccator.

When kept in a dry, dark and refrigerated place, under these conditions the tetrasodium phenyphthalein bisphosphate is stable. In addition, this invention provides a kit containing tetrasodium phenyphthalein bisphosphate, useful in identifying and quantifying acid phosphatase enzyme.

The compound obtained by the process of the present invention acts chemically when in contact with the acid phosphatase enzyme under appropriate conditions, by cleaving the PO bonds, releasing in the medium an equimolar amount of indicator that is directly proportional to the enzyme activity, a fact that allows to determine quantitatively by instrumental techniques of optical spectrometry (absorption of the chromophore formed in UV or visible) the enzyme content in the medium.

The tetrasodium phenyphthalein bisphosphate exhibits ease of viewing ranging from colorless, while buffered at pH = 5, to pink coloration to pH 8.3 when in the presence of a coloring agent, in this case a solution of an inorganic base.

Thus, the present invention uses as a developing solution 1.0 mol.L-1 ammonium hydroxide solution which has the ability to promote sufficient basic character to obtain the chromophore ionic form of phenyphthalein after identification in tissue samples the presence of the acid phosphatase enzyme, for example, this base evaporates and does not keep residue in the original matrix as in the case of sodium hydroxide or sodium carbonate that may come into contact with the biological sample causing some change in its property, This is not the case with ammonium hydroxide, which greatly facilitates the storage of such samples as control. In typical cases of garments with shades close to the pink, which is similar to the chenophorine form of chromophore where the detection of the enzyme by this method can be masked, it was decided to use a staining solution consisting of copper sulfate solution with concentration 0.5 mol.L-1 which when mixed with the pink tone of the phenyphthalein assumes a contrasting lilac tone with the original colorings allowing its visualization.

This phosphatase enzyme determination kit consists of:

(1) active agent;

(2) developer solution;

(3) coloring enhancing solution;

(4) buffer solution;

(5) stabilizing solution. The five reagents are more precisely: (1) phenolphthalein tetrasodium bisphosphate solution 760 mg.L ^"1 , (2) NH ₄ OH developer solution 1.0 mol.L-1 .e (3) CuS0 staining enhancing solution ₄ 0, 1 mol L-1 - (4) buffer solution sodium acetate / acetic acid adjusted to pH 5 and (5) stabilizing solution: NaCl / 1 NaF, 0 mol L-1.

Comparative Tests

Hydrogen and carbon nuclear magnetic resonance spectrometry 13 proves to be an excellent technique for structural recognition and even for checking the purity of a given substance since the nuclear magnetic resonance spectrum of a pure chemical compound is unique and constant. For nuclear magnetic resonance spectra to be performed on equipment of different manufacturers, only the resolution of the spectrum can vary, but not the chemical shifts, hydrogen integration, or carbon numbers of the molecule given the identity of a molecule.

It was purchased as standard to match the tetrasodium phenolphthalein bisphosphate synthesized product marketed by Sigma Aldrich CO., Lot number 128k5307 / ec272-326-3 wgk3.

The hydrogen nuclear magnetic resonance spectrum obtained for this compound (Figure 2) using deuterated water as a solvent showed a triplet at 1.25 ppm and a quadruple at 3.15 ppm characteristic for the ethyl group from co-precipitation of sodium ethoxide used in the step of obtaining the tetrasodium form of phenolphthalein bisphosphate and present in this compound.

The hydrogen nuclear magnetic resonance spectrum (Figure 2) of tetrasodium phenolphthalein bisphosphate containing 60 milligrams of commercially purchased sample from Sigma Aldrich Co. clearly shows the presence of co-precipitated sodium ethoxide as an impurity along with the amount of tetrasodium phenolphthalein bisphosphate significant.

The product obtained by the process of the present invention is completely free of this aggregate impurity as verified by the nuclear magnetic resonance technique (Figure 3) which gives it greater reliability in the quantitative determinations for which it is intended.

On the other hand, the nuclear magnetic resonance spectrum (Figure 3) of the same compound synthesized by the proposed process via reaction with triethylamine after purification, did not show after recrystallization with absolute ethanol. the presence of the same impurity, which leads to a chemically similar product but of high purity since it has no detectable sodium ethoxide content by this sensitive technique.

A full knowledge of the purity of this class of reagents for biochemical analysis of human interest is of utmost importance, requiring a quality control of the purchased product beforehand and whether or not the purity is complete to allow the conclusion of accurate and reliable results.

The carbon-13 nuclear magnetic resonance spectrum of the tetrasodium phenyphthalein bisphosphate containing 60 milligrams of commercial sample (Figure 4) also clearly shows the presence of co-precipitated sodium ethoxide as impurity along with the significant amount of tetrasodium phenyphthalein bisphosphate when purchased with the product obtained by the process of this invention (Figure 5).

Therefore, the present invention has as its main innovative feature the selection of the triethylamine reagent for use in the preparation of tetrasodium phenyphthalein bisphosphate for the determination of the acid phosphatase enzyme.

In addition, the tetrasodium phenyphthalein bisphosphate synthesized in this work as an analytical standard does not contain sodium ethoxide, a condition that enables the use of this product in the quantitative evaluation of acid phosphatase in various forensic applications, such as: in rape crime scenes , semen quantification in samples of this biological matrix in cattle, horses, pigs, goats and insect larvae, such as Chrysomia albiceps blowflies, etc.

It should be noted, however, that the examples and embodiments presented herein are for illustrative purposes only and are not, therefore, limiting to the invention. It will be apparent to those skilled in the art that other concentrations may be employed without departing from the scope of the invention.

Claims

1. Tetrasodium phenolphthalein bisphosphate synthesis process for the determination of acid phosphatase enzyme characterized by the use of triethylamine.

Tetrasodium phenolphthalein bisphosphate synthesis process according to claim 1, characterized by the steps:

(i) Mixing phenolphthalein with phosphorus oxychloride and dichloromethane;

(ii) adding triethylamine to the reaction mixture;

0 (Ni) Evaporation of dichloromethane;

(iv) Addition of sodium hydroxide;

(v) acidification of the solution;

(vi) Addition of sodium ethoxide;

(vii) Purification.

5 (viii) Re-precipitation

Tetrasodium phenolphthalein bisphosphate synthesis process according to Claims 1 to 2, characterized in that the reaction temperature is at most 4 ° C.

Tetrasodium phenolphthalein bisphosphate synthesis process according to Claims 1 to 3, characterized in that the reaction is kept under constant stirring at a temperature of at most 4 ° C for at least 6 hours.

Tetrasodium phenolphthalein bisphosphate synthesis process according to Claims 1 to 4, characterized in that the acidification occurs with mineral acids with pH 1.0.

Tetrasodium phenolphthalein bisphosphate synthesis process according to Claim 5, characterized in that the acid is preferably hydrochloric acid.

Tetrasodium phenolphthalein bisphosphate 1 synthesis process according to Claims 1 to 6, characterized in that it uses 1.0 mol / l sodium ethoxide.

Tetrasodium phenolphthalein bisphosphate synthesis process according to Claims 1 to 7, characterized in that the purification is carried out with ethyl alcohol, then with dry ether, and then methanol / formamide (5: 1).

9. Acid phosphatase enzyme determination kit comprising: (i) active agent;

(ii) buffer solution;

(iii) stabilizing solution;

(iv) developer solution;

(v) staining fortifying solution.

A kit according to claim 9, characterized in that the active agent is 760mg / L phenaphthalene tetrasodium phosphate, buffer solution is pH 5 sodium acetate / acetic acid, stabilizer solution is NaCl / NaF 1.0 mol / L, developing solution is 1 mol / L NH OH and CuS0 ₄ 0.1 mol / L staining fortifying solution.

Kit according to Claim 10, characterized in that it is in the form of spray, aerosol, dropper and solution.