JPH0259151B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to pterin derivatives useful for measuring pteridine derivatives in vivo. Pteridine derivatives, represented by folic acid, have recently been shown to play a role as coenzymes in amino acid metabolism, and knowing their quantitative changes in the body is important from both basic and clinical medical perspectives to understand various pathological conditions such as enzyme deficiency diseases. It has come to be considered indispensable for analysis and diagnosis. Particularly recently, a relationship between phenylketonuria and biopterin has been reported (Ann, Neurol.,
3, 224-230 (1978), The New England
Journal of Medicine, 299 , 673–679 (1978) and Clinica Chimica Acta, 93 , 251–262
(1979)), pterin or 2-amino-4
- Quantitative changes of hydroxypteridines in the body have begun to attract attention. The present inventor is an enzyme immunoassay (hereinafter referred to as
biopterin (2-amino-4-hydroxy-6-(L-erythro-1,2-dihydroxypropyl) pteridine) and neopterin (2-amino-4-hydroxy-6-(L-erythro-1,2 ,3-trihydroxypropyl)
We planned to develop a method for measuring pterins, mainly pteridine, and produced pterin derivatives represented by the following general formula. (In the formula, R ₆ and R ₇ each mean H, lower alkyl or hydroxy lower alkyl group, and R means H, maleimidobenzoyl, enzyme-labeled maleimidobenzoyl, or maleimidobenzoyl condensed with a protein or polypeptide.) This compound can basically be produced by a method exemplified by the following reaction formula. That is, 4-hydroxy-2-methylthiopteridine () (Bull.Chem.Soc.Jpn., 53 ,
2344-2347 (1980)) is heated with ethylenediamine to obtain the 2-(2-aminoethyl)amino substituted product (). This compound is one of the compounds represented by the general formula (), and when it is further modified by the method shown below, the formula ()
A pterin derivative in which R is replaced with something other than hydrogen is obtained. Compound (), that is, a pterin derivative in which m-maleimidobenzoyl is introduced into the terminal amino group at the 2-position, can be obtained by preparing compound () by a known method (J.
Biochem., 79 , 233-236 (1976)). When this compound () is further reacted with a protein (or polypeptide) having a mercapto group, such as bovine serum albumin (BSA), in a phosphate buffer, the corresponding protein (or polypeptide) condensate () is obtained. . This one is EIA
It is also useful as an immunizing antigen for creating antiserum for radioimmunoassay. The product can be confirmed by measuring the fluorescence intensity of the gel chromatography elution fraction of the reaction solution and the ultraviolet absorption of the protein at 275 nm. In addition, compound () was added to β-D in a phosphate buffer.
- When reacted with an enzyme such as galactosidase or glucose oxidase, an enzyme label () is obtained. Since this reaction can be carried out under mild conditions of neutrality and room temperature, and there is little loss of enzyme activity, these labeled substances can be used as labeled antigens in EIA. It is appropriate that the enzyme to be bound has an SH group that is not involved in the activity, but
Currently, β-D- has a high detection sensitivity for enzyme activity.
Galactosidase is most preferred. It is appropriate to confirm the product by measuring the fluorescence intensity and enzyme activity of the gel chromatography elution fraction of the reaction solution. Antigens and labeled antigens for producing antibodies are those in which the substituents represented by R ₆ and R ₇ in formula () are the same, depending on the substance to be measured. For example, biopterin
In EIA, R ₆ is dihydroxypropyl and R ₇
A substance in which is H is used. When a substance in which a protein or the like is bound to a hapten through a maleimidobenzoyl group is used as an antigen for producing antibodies, there is a possibility that antibodies against the maleimidobenzoyl group, which is the bridge part, will be produced; Using a labeled antigen with the same bridge
When EIA is performed, the labeled pterin and the measured pterin are not completely replaced in the antigen-antibody reaction, which may reduce the sensitivity of the measurement. Therefore,
In actual EIA, it is usually preferable to use combinations of labeled antigens and antibody-producing antigens with different bridge portion structures. However, since the phenomenon that is considered to be the effect of antibodies on bridge parts does not necessarily occur all the time, it may be possible to perform EIA using the same combination of bridges. To obtain antibodies using condensates of pterins and proteins, for example, suspend the antigen in Freund's adjuvant and inject it into mammals (rabbits, guinea pigs, sheep, goats, etc.). After the appropriate booster immunization, blood is collected and blood cells are removed to obtain antiserum. For EIA, samples such as urine are pretreated as necessary. For example, in the EIA of biopterin, it is preferable to oxidize coexisting reduced dihydrobiopterin and tetrahydrobiopterin to produce biopterin before subjecting it to the assay. EIA is an antigen produced by mixing an antiserum diluted to an appropriate concentration, a buffer containing a specimen or a standard, and a labeled antigen, such as the compound shown in Example 4 later, and incubating the mixture at an appropriate temperature, such as room temperature. After separating the antibodies using a BF separation method such as the two-antibody method, the enzyme substrate is applied to the precipitate, the fluorescence intensity of the resulting product is measured, and a labeling curve is created from the values for the standard. Determine the amount of pterins in the sample. Example 1 2-(2-aminoethyl)amino-4-hydroxy-6-(L-erythro-1,2-dihydroxypropyl)pteridine (:R ₆ =CH
(OH)CH(OH)CH ₃ , R ₇ =H) 4-hydroxy-6-[L-erythro-1,2
-Dihydroxypropyl]-2-Methylthiopteridine 500mg was dissolved in ethylenediamine 5ml,
The mixture was heated at 110° C. for 4 hours under a nitrogen atmosphere. After the reaction solution was dried under reduced pressure, the residue was applied to a Florisil column and eluted with ammonia water having a concentration gradient of 0 to 3%. Of each fraction, only the fluorescent portion was collected and chromatographed again using a Dowex 50W column (solvent: 0-3% aqueous ammonia).
The eluate was concentrated under reduced pressure until it became jelly-like, and then left in the refrigerator for several days to crystallize. The obtained crystals (220 mg) were pale yellow needle-like crystals that turned brown at 235°C.
Completely decomposed at 245°C. Example 2 N-[2-[4-hydroxy-6-(L-erythro-1,2-dihydroxypropyl)-2-
pteridinyl]aminoethyl]-m-maleimidobenzamide () 2-(2-aminoethyl)amino-4- of Example 1
Hydroxy-6-(L-erythro-1,2-dihydroxypropyl)pteridine 100mg N,N-
After dissolving in 10 ml of dimethylformamide, 120 mg of m-maleimidobenzoyl N-hydroxysuccimide ester (MBS) was added and stirred at 25°C for 2.5 hours. After adding 40 ml of water to the reaction solution and extracting and removing unnecessary components with dichloromethane (40 ml x 3), the aqueous layer was concentrated under reduced pressure until almost all the solvent was removed. 1 ml of tetrahydrofuran was added to this mixture and cooled to obtain 80 mg of the target compound as a solid. This product was used in the next reaction (Example 3 and Example 4) without purification. Example 3 Biopterin-bovine serum albumin condensate (:
Protein = BSA) Add the above phosphate buffer solution (10 mg/1 ml) of bovine serum albumin (BSA) to a 0.1 M phosphate buffer (PH7.0) solution (1 mg/0.5 ml) of the biopterin derivative () of Example 2. After adding and stirring at 25℃ for 1.5 hours,
The product was separated and purified by Sepharose 6B column chromatography using 0.05M phosphate buffer (PH7.0) as the eluent. At this time, the elution site predicted from the molecular weight of the biopterin-BSA condensate, the biopterin-based fluorescence intensity (excited at 360 nm, measured at 430 nm) in each fraction of the eluate, and the BSA-based ultraviolet absorption at 257 nm were evaluated. By tracking and measuring, we confirmed and collected fractions of the target product. Example 4 Biopterin-β-D-galactosidase labeled product (enzyme = β-D-galactosidase) E. Coli.
The above phosphate buffer solution of β-D-galactosidase [EC3.2.1.23] obtained from
After stirring at 25°C for 2 hours, the product was separated by Sepharose 6B column chromatography using 0.05M phosphate buffer (PH7.0) as the eluent. At this time, the fluorescence intensity of the eluate was measured in the same manner as in Example 3, and the β-galactosidase activity was also measured to confirm the progress of the reaction. Enzyme activity was measured as follows. That is, 4-methylumbelliferyl-β-
D-galacside 0.01M phosphate buffer (PH7.0)
Add each fraction obtained by the above chromatography to 50 μl of the solution (prepared to 1 mg/30 ml) and add 0.1% BSA.
100 μl of a 50-fold dilution with 0.01M phosphate buffer (PH7.0) was added and incubated at 37°C for 10 minutes. Enzyme activity was determined by adding 3 ml of 0.1M glycine buffer (PH10.3) to each reaction solution and measuring the fluorescence intensity at 440 nm (excitation at 360 nm). As a result, the activity was 40% of the original enzyme. It was confirmed that a labeled compound () having the following properties was obtained. Example 5 A 0.02M phosphate buffer (PH7.4) containing 0.3% BSA was used for EIA assay of biopterin. In addition, 2-(5-carboxypentylamino)-4 is used as an antigen for antibody production.
-Hydroxy-6-(L-erythro-1,2-dihydroxypropyl)pteridine/BSA condensate (-NH ₂ at the 2nd position of biopterin is -NH-
_Antiserum (T. _Nagatsu et.
al, Proc. Japan Acad., 55 , Ser. B, 317 (1979))
It was used. 100 μl of phosphate buffer and 100 μl of 25,000-fold diluted antiserum
Example 4
10,000 purified fractions of the compound () obtained in
100 μl of the diluted solution was added and left at 4° C. for 1 hour. To this was added 100 μl of anti-rabbit IgG as a second antibody and left at room temperature for 10 minutes. Next, 100 μl of 4% dextran T-70 (manufactured by Pharmacia) was added, mixed vigorously, and centrifuged at 4° C. and 3000 rpm for 10 minutes.
After aspirating the supernatant, add 100 μl of buffer and
0.1mM β-methyl-umbelliferyl-β-D
- 50 μl of the above buffer solution containing 1 mM magnesium chloride of galactoside was added and incubated at 37° C. for 10 minutes. 0.1M glycine buffer (PH10.3) for each
After adding 2.5 ml to stop the reaction, at 4°C.
It was centrifuged at 3000 rpm for 10 minutes, and the fluorescence intensity of the supernatant was measured (excitation: 330 nm, measurement: 450 nm). A standard curve prepared based on the measured values for standard biopterin is shown in the drawing. In the drawing, the vertical axis "BN/B ₀ N%" indicates the rate of change in fluorescence intensity. Here, B ₀ , B and N have the following meanings. B ₀ : Fluorescence intensity when biopterin concentration is 0 B: Fluorescence intensity at each biopterin concentration N: Nonspecific fluorescence intensity of sample without antiserum added (blank value)

[Brief explanation of the drawing]

The drawing is a standard curve.

Claims (1)

[Claims] 1 formula (In the formula, R ₆ and R ₇ each mean H, lower alkyl or hydroxy lower alkyl group, and R
means H, maleimidobenzoyl or enzyme-labeled maleimidobenzoyl group. ) A pterin derivative represented by 2. The pterin derivative according to claim 1, wherein R is an enzyme-labeled m-maleimidobenzoyl group. 3 R ₆ is a dihydroxypropyl group, R ₇ is H, and R is β-D-galactosidase labeled m
- The pterin derivative according to claim 1, which is maleimidobenzoyl.