RU2024538C1 - METHOD OF SYNTHESIS OF D-2- (6-β-D- GALACTOPYRANOSYLBENZTHIAZOLYL-2) -4,5-DIHYDROTHIAZOLYL -4-CARBOXYLIC ACID - Google Patents

Abstract

FIELD: organic chemistry, carbohydrates. SUBSTANCE: synthesis is carried out by reaction of 2,3,4,6- tetra-O- acetyl -b-D-galactopyranosyl -fluoride with 2-cyano-6- trimethylsilylhydroxybenzthiazole in the medium of nonpolar organic solvent (benzene) in the presence of boron trifluoride etherate with isolation and purification of intermediate 6-(2-cyanobenzthiazolyl) -2,3,4,6-tetra -O-acetyl-b -D-galacto- pyranoside by chromatography following by its deacetylation and condensation with D-cysteine. Deacetylation product can be purified by chromatography and crystallization methods. New method ensures to prepare the end product from available reagents with good yield (61%) and provides high sensitivity of b-galactosidase activity detection by bioluminescent method. EFFECT: development of new method of synthesis of substance indicated above. 1 dwg

Description

используемого в качестве субстрата для определения ферментативной активности β -галактозидазы, который может найти применение в генетической инженерии, иммуноферментном анализе и в ДНК-зондах.The invention relates to carbohydrates and heterocyclic compounds, in particular to a new method for producing β-D-galactoside D-luciferin of the formula I

used as a substrate for determining the enzymatic activity of β-galactosidase, which can be used in genetic engineering, enzyme immunoassay and in DNA probes.

 Known bioluminescent method for the detection of β-galactosidase activity [1]. The best substrate in this method is o-nitrophenyl-β-D-galactopyranoside, which is cleaved by the enzyme to form o-nitrophenol and galactose. Galactose is determined by a complex multistage detection system, which includes three consecutive enzymatic reactions requiring the use of enzyme substrates such as CAD (nicotinamide adenine dinucleotide), FMN (flavin mononucleotide), and long-chain aldehyde. All this complicates and increases the cost of analysis.

где R остаток фосфорной, серной или аминокислоты. В патенте [2] упоминаются соединения, где R - углеводный остаток, в частности, - β -D-галактопиранозил, предназначенные для биолюминесцентного обнаружения гидролаз (фосфатаз, сульфатаз, протеиназ и гликозидаз). Однако, способ получения соединения формулы I и метод его использования не описаны.Derivatives of luciferin with an esterified phenolic group of the general formula

where R is the remainder of phosphoric, sulfuric or amino acids. In the patent [2], compounds are mentioned where R is a carbohydrate residue, in particular β-D-galactopyranosyl intended for bioluminescent detection of hydrolases (phosphatases, sulfatases, proteinases and glycosidases). However, the method of obtaining the compounds of formula I and the method of its use are not described.

Недостатком этого способа является то, что в качестве исходного вещества используется дорогостоящий люциферин. Кроме того, люциферин имеет две функциональные группы, по которым может протекать реакция взаимодействия - фенольный гидроксил, и карбокси-группу. Именно по этой причине выходы производных люциферина по фенольной гидроксильной группе низки и составляют, например, в случае фосфатов 29%. Существенным недостатком является также то, что в реакционных смесях присутствует не вошедший в реакцию люциферин, от которого чрезвычайно трудно освободиться и который мешает биолюминесцентному определению, создавая высокий фон.The method of obtaining an analogue in structure - luciferyl phosphate is based on the interaction of D-luciferin with phosphorus oxychloride and the subsequent hydrolysis of P-Cl bonds in accordance with the following synthesis scheme [3]

The disadvantage of this method is that expensive luciferin is used as the starting material. In addition, luciferin has two functional groups through which an interaction reaction can occur - phenolic hydroxyl, and a carboxy group. For this reason, the yields of luciferin derivatives with respect to the phenolic hydroxyl group are low and, for example, make up 29% in the case of phosphates. A significant drawback is that in the reaction mixtures there is unreacted luciferin, which is extremely difficult to free from and which interferes with bioluminescent determination, creating a high background.

 The aim of the invention is to develop a new method for producing the compounds of formula I, which allows to obtain the target product with high sensitivity and high sensitivity when detecting the enzymatic activity of β-galactosidase.

 The proposed method for obtaining the compounds of formula I is that 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl fluoride is reacted with 2-cyan-6-trimethylsilyloxybenzothiazole in a non-polar organic solvent such as benzene, in the presence of boron trifluoride etherate, an intermediate 6- (2-cyanobenzothiazolyl (2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside) is isolated and chromatographed, followed by deacetylation and condensation with D cysteine.

+

___→

Cпособ осуществляют следующим образом.The synthesis scheme of the proposed method is as follows:

+

___ →

The method is as follows.

 The reaction of 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl fluoride with 2-cyan-6-hydroxybenzothiazole trimethylsilyl ether gives galactopyranoside (IV) in 77% yield. Deacetylated galactoside (V) is obtained in 86% yield by deprotecting acetyl groups from derivative (IV) by the action of catalytic amounts of sodium methylate in methanol. Compound (V) is converted to the target galactoside of D-luciferin by condensation with D-cysteine in an aqueous methanol medium using sodium carbonate as a condensing agent. Compound (I) is isolated from the reaction mixture in 61% yield by crystallization. The use of 2-cyan-6-trimethylsilyloxybenzothiazole as the starting compound, rather than luciferin, as in the analogue method has several advantages.

In particular, in the proposed method, derivatives IV and V can easily be purified by chromatography and crystallization from possible impurities of 2-cyan-6-hydroxy-benzothiazole, from which free luciferin can be formed at the final stage of synthesis, which interferes with the bioluminescent detection of β-galactosidase. Secondly, -2- (6 ^l- β -galactopyranosylbenzothiazolyl-2) -4,5-dihydrothiazolyl-4-carboxylic acid (I) uses not expensive D-luciferin, and its predecessor - 2-cyano-6-hydroxybenzothiazole, which is cheaper and more stable.

The synthesis of compound (III) is carried out by silylation of 2-cyan-6-hydroxybenzothiazole with a mixture of hexamethyldisilazane-trimethylchlorosilane under heating. The interaction of fluoride (II) with derivative (III) is carried out in benzene using boron trifluoride etherate as a condensing agent at room temperature. The resulting compounds are characterized by melting points and specific rotation. Their structure was proved using ¹³ C NMR spectroscopy. Thus, in the spectrum of compound (IV) there are necessary signals of the galactopyranosyl residue bound by a β-glycosidic bond, signals of acetyl groups, CN groups and aromatic carbon atoms of benzothiazole. In the spectrum of compound (V), on the contrary, there are no signals of acetyl groups, there are the required signals of the CN group, benzothiazole residue and deacetylated β-D-galactopyranoside. In the spectrum of galactoside D-luciferin (I) there is no CN-group signal, there are 6 signals — β-D-galactopyranoside and 11 signals corresponding to the D-luciferin residue.

 PRI me R 1. Synthesis of β-D-galactoside D-luciferin (I).

Synthesis of 2-cyan-6-trimethylsilyloxybenzothiazole (III). A mixture of 0.15 g of 2-cyan-6-hydroxybenzothiazole, 2.0 ml of hexamethyldisilazane and 1 ml of trimethylchlorosilane is heated under reflux. After about 0.5 hours, the mixture becomes homogeneous, a precipitate of ammonium chloride is precipitated in the refrigerator. This precipitate was carefully removed, the excess silylating mixture was distilled off under reduced pressure, and the residue was crystallized from hexane. 0.17 g (81%) of compound (III) is isolated, mp. 68-69 ^about S.

Synthesis of 6- (2-cyanobenzothiazolyl) -2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (IV). To a solution of 0.25 g (1.01 mmol) of ether (III) and 0.40 g (1.15 mmol) of compound (II) in 2.0 ml of abs. a solution of 0.13 ml (1.0 mmol) of boron trifluoride etherate in 1 ml of benzene is added. The addition is carried out dropwise and with stirring. After the addition is complete (5-10 min), the mixture is stirred at room temperature for 4 hours. It is diluted with chloroform, washed with water, chromatographed on Silpearl sorbent (Czechoslovakia) in an ether-benzene 1: 3 solvent system. 0.39 g (77%) of compound (IV) is isolated. After crystallization from ethanol, so pl. 160-161 ^{° C,} [α] _D +2 ^o (c 0.5, chloroform).

¹³ C NMR (CDCl ₃ , δ, ppm): 99.6 (C1); 68.5 (C2); 70.7 (C3); 68.8 (C4); 71.7 (C ₅ ); 61.4 (C ₆ ) - signals of the galactopyranose residue, 20.6-20.7 (CH ₃ ); 169.3-170.2 (CH ₃ CO); 112.9 (CN group); 107.9; 119.4; 126.2; 137.5; 149.0; 157.7 - signals of the carbon atoms of benzothiazole.

Synthesis of 6- (2-cyanobenzthiazolyl) -β-D-galactopyranoside (V). To a mixture of 0.21 g of compound (IV) and 6 ml of abs. methanol add 5 drops of 1 N. a solution of sodium methylate in absolute methanol. After 1 h, neutralize KRS-2p cation exchanger and evaporate. 0.12 g (86%) of compound (V) was isolated by crystallization from 4 ml of ethanol, mp. 188-190 ^{° C,} [α] _D -56 ^o (c 0.5, pyridine), ¹³ C NMR (pyridine-d-5): 102,9 (C1); 72.0 (C2); 75.2 (C3); 70.1 (C4); 77.9 (C5); 62.4 (C6) - signals of the galactopyranosyl residue; 114.0 - (CN groups); 108.1; 119.9; 125.8; 137.9; 147.8; 158.9; 170,2- signals of the benzothiazole nucleus.

Synthesis of D-2- (6-β-D-galactopyranosylbenzothiazolyl-2) -4,5-dihydrothiazolyl-4-carboxylic acid (I) - - β-D-galactopyranoside D-luciferin. A mixture of 0.04 g of compound (V), 0.02 g of D-cysteine hydrochloride (2-amino-3-mercaptopropionic acid hydrochloride) in a mixture of 1 ml of methanol and 1 ml of water is stirred under argon flow. Add 0.025 g of sodium carbonate and continue stirring for 1.5 hours. Neutralize 0.2 N. HCl until acidic and left at a temperature of 5-6 ^{° C} for 10-15 hours Isolate 0.032 g (61%) of crystalline β-D-galactopyranoside D-luciferin firefly (I), which is a white solid soluble in water, so pl. 250-260 ^{° C} (dec.). When thin-layer chromatography on siloufol (Czechoslovakia) in the ethyl acetate-methanol system (3: 1) R _f = 0.25 (relative to luciferin).

¹³ C NMR (dimethyl sulfoxide -d ₆ ): 101.4 (C1); 73.3 (C2); 75.8 (C3); 70.3 (C4); 78.2 (C5); 60.4 (C6) - signals of the β-D-galactopyranose residue; 34.8; 68.1; 108.3; 118.1; 124.7; 136.8; 147.8; 157.0; 158.6; 164.4; 171.2 - signals of the D-luciferin residue.

 UV spectrum: three absorption maxima: at 330, 260 and 220 nm (0.1 M phosphate buffer, pH 7.3).

 Fluorescence spectra: maximum fluorescence at 435 nm (excitation at 355 nm, 0.1 M Tris-acetate buffer, pH 7.8). The luciferin content in the resulting substance is 0.13%.

 PRI me R 2. The synthesis of β-D-galactoside D-luciferin is carried out according to example 1, however, 6- (2-cyanobenzthiazolyl) -β-D-galactopyranoside (compound V) is subjected to further purification immediately before condensation with D-cysteine column chromatography on silica gel Silpearl (Czechoslovakia) in an organic solvent system of ethyl acetate-ethanol, followed by crystallization from ethanol. The resulting β-D-galactopyranoside of D-luciferin contains 0.02% D-luciferin.

 PRI me R 3. The use of β-D-galactoside D-luciferin as a substrate for bioluminescent determination of β-galactosidase activity.

, где С_х - неизвестная концентрация люциферина; N_x - разбавление реакционной смеси с неизвестной концентрацией люциферина в люминометрической кювете; С - концентрация люциферина в растворе внутреннего стандарта в кювете; N_c - разбавление раствора внутреннего стандарта в кювете.To 1.0 ml of a 0.8 mM solution of luciferin β-D-galactoside in 0.1 M phosphate buffer, pH 7.3, containing 1 mM MgSO ₄ , 0.1 M NaCl, 1-20 ml of β-galactosidase solution is added in the same buffer. The mixture was incubated at ³⁷ ° C for 100 minutes, then 20 ul withdrawn reaction mixture and the concentration in the mixture of luciferin bioluminescent method as described below. To do this, 0.7 ml of 0.1 M Tris-acetate buffer, pH 7.8, containing 2 mM EDTA, 10 mM MgSO ₄ , 0.3 mM ATP solution in the same buffer, 10 μl firefly luciferase solution is placed in a luminometric cell with a specific activity of 10 ⁸ -10 ⁹ conventional units / ml (1 conventional unit corresponds to the intensity of bioluminescence 10 ⁹ quantum / sec). The cuvette is placed in the cuvette compartment of the luminometer, the background glow (I _background ) is measured, 20 μl of the reaction mixture with an unknown concentration of luciferin is injected (see above), and the intensity of luminescence I _{1 is} measured. Then add 10 μl of a solution of luciferin with a known concentration ("internal standard") and measure the intensity of luminescence I ₂ . The unknown concentration of luciferin in the reaction mixture is calculated by the formula
C _x =

where C _x is the unknown concentration of luciferin; N _x - dilution of the reaction mixture with an unknown concentration of luciferin in the luminometric cell; C is the concentration of luciferin in the solution of the internal standard in the cuvette; N _c - dilution of the internal standard solution in the cuvette.

The activity of β-galactosidase is proportional to the concentration of luciferin formed by the action of this enzyme on β-D-galactoside of D-luciferin for 100 minutes. A calibration graph for determining β-galactosidase by the bioluminescent method using β-D-galactoside D-luciferin as a substrate of β-galactosidase is shown in the drawing, where [luciferin] ₁₀₀ is the concentration of luciferin, μM after 100 min of incubation of the substrate with β-galactosidase. Conditions: 0.1 M phosphate buffer, pH 7.3, containing 1 mm MgSO ₄ , 0.1 M NaCl; 0.8 mm β-D-galactoside D-luciferin, 37 ^about . The preparation of β-D-galactoside D-luciferin contains 0.02% D-luciferin. The detection limit of β-galactosidase by this method is 10 ^-16 M, that is, it is not inferior in sensitivity to the bioluminescent detection method of β-galactosidase described in [1]. Moreover, the detection technique is simplified by the use of not three enzymes, but one.

 Thus, the proposed new method for producing β-D-galactoside D-luciferin of the formula (I) allows to obtain the product from the available reagents, in good yield. Moreover, a feature of the proposed method is the ability to purify the compounds of formula (I) in at least two preliminary stages, which ensures high sensitivity in the detection of β-galactosidase by the bioluminescent method.

Claims (2)

1. METHOD FOR PRODUCING D-2 (6-β-D-GALACTOPYRANOSYLBENZTHIAZOLYL-2) -4,5-DIHYDROTHIAZOLYL-4-CARBOXYLIC ACID of the formula

characterized in that 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl fluoride is reacted with 2-cyan-6-trimethylsilyloxybenzothiazole in a non-polar organic solvent such as benzene in the presence of boron trifluoride ether in as a condensing reagent with isolation and purification by chromatography of the intermediate 6- (2-cyanobenzothiazolyl) -2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside of the formula

followed by its deacetylation and condensation with D-cysteine.  2. The method according to p. 1, characterized in that the deacetylation product is subjected to purification by chromatography and crystallization.

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