JP2914405B2 - Method for producing labeled porphyrin compound - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention
^{The present} invention relates to a method for producing a porphyrin into which ¹³ C has been introduced.
Porphyrins labeled with ¹³ C are useful for studying porphyrin and porphyrinogen metabolism in vivo and for diagnosing porphyria, lead poisoning, hepatic dysfunction, etc., and only specific sites are labeled with ¹³ C. The obtained porphyrins are particularly useful for studying metabolic pathways of porphyrins.

[0002]

BACKGROUND ART Rhodopseudomonas as microorganisms which produce porphyrins (Phodopseudomonas) microorganisms and Propionibacterium (Propionibacterium) a microorganism belonging to the genus [U.Ya.Bykhouskii et, Biokhim.Mikrobiol., 23 (6) ,
725-736, 1987]. In addition,
No. 21,520 and JP-B-63-19156, methods for producing uroporphyrin III and coproporphyrin III using microorganisms of the genus Arthrobacter are known.

[0003] However, it is not clear from which carbon atom of the porphyrins each carbon of the raw material or precursor for the production is derived, and therefore a method for producing a porphyrin compound in which a specific site is labeled with ¹³ C is not known. Had not been.

[0004]

Accordingly, an object of the present invention is to provide a novel method for producing porphyrins whose specific site is labeled with ¹³ C.

[0005]

Means for Solving the Problems The present inventors have conducted various studies in order to solve the above-mentioned problems. As a result, 5-aminolevulinic acid is used as a precursor of porphyrins when fermentatively producing porphyrins by a microorganism of the genus Arthrobacter. Is added to the medium, a carbon atom at a specific site of the 5-aminolevulinic acid molecule is introduced into a specific site of the porphyrin molecule, that is, 5-aminolevulinic acid in which only a specific carbon atom is labeled with ¹³ C. Was found to synthesize porphyrin labeled only with a specific site with ¹³ C, and based on this finding, the present invention was completed.

Accordingly, the present invention provides the following formulas (I), (II) and (II)
I) or (IV):

[0007]

Embedded image

[0008]

Embedded image

[0009]

Embedded image

[0010]

Embedded image

[In the formula, the symbols x, *, ▲, Δ, and ● each represent a carbon atom ¹³ C or a naturally occurring carbon atom, and the carbon atom represented by any one of these symbols is ¹³ C Wherein the carbon atom represented by the other symbol is a naturally occurring carbon atom and R represents hydrogen or a lower alkyl group or a salt-forming cation. A method comprising the steps of converting a microorganism belonging to the genus Arthrobacter having the ability to produce a porphyrin compound into a microorganism of the following formula (V):

[0012]

Embedded image

[Wherein the symbols x, *, ，, ■ and ● represent a carbon atom ¹³ C or a naturally occurring carbon atom, and the carbon atom represented by any one of these symbols is ¹³ C
The carbon atom represented by the other symbol when is a naturally occurring carbon atom and is of the above formula (I), (II)
The carbon in the formula (V) represented by the same symbol as the symbol representing ¹³ C among the symbols in (III) or (IV) is ¹³ C]. A method of collecting a desired porphyrin compound from the culture or isolating it as a lower alkyl ester.

[0014]

DETAILED DESCRIPTION OF THE INVENTION According to the present invention, the symbol ×, *, ▲, △ or ● of 5-aminolevulinic acid represented by the formula (V) is used.
When cultured porphyrin producing microorganism as described later in a medium containing the 5-aminolevulinic acid with ¹³ C as either of the carbon atoms represented in, among the symbols in formula (V) representing the levulinic acid ¹³ Formula (I), (II), (III) or (IV) represented by the same symbol as C
Porphyrins with ¹³ C at the middle site are produced.
Therefore, it is possible to obtain porphyrins corresponding specific sites were labeled with ¹³ C by using 5-aminolevulinic acid alone is labeled with ¹³ C specific site.

In the above formula, formula (I) represents uroporphyrin I or an ester thereof, formula (II) represents uroporphyrin III or an ester thereof, formula (III) represents coproporphyrin I or an ester thereof, and Formula (IV) represents coproporphyrin III or an ester thereof. Since the microorganism of the genus Arthrobacter produces porphyrins in the form of a free compound or a salt thereof, when producing an ester of a porphyrin in which R is a lower alkyl group, any step after the microbial production of the free compound. According to a conventional method. Examples of the ester-forming alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group,
A linear or branched pentyl group and the like can be mentioned. Examples of the salt-forming cation R include a sodium ion, a potassium ion, and an ammonium ion.

The microorganism used in the present invention may be any microorganism which belongs to Arthrobacter and has the ability to produce uroporphyrin.
Is not limited. In addition, any of strains isolated from nature, already owned strains, and mutant strains obtained based on these strains can be used. Specific examples of porphyrin-producing strains that can be used in the present invention include Arthrobacter hyalinus ,
Arthrobacter Guromihorumisu (Arthrobacter gl
obiformis ) and Arthrobacter penses ( Arthro)
bacter pascens ), and mutants thereof.

The Arthrobacter hirinas is a publicly-available free deposit deposited at the Institute of Microbial Industry and Technology (FRI) of the National Institute of Advanced Industrial Science and Technology as Microorganisms Bacteria No. 3125 and also as the American Type Culture Collection ATCC 31263. It is a strain. Details of the mycological properties are described, for example, in JP-B-52-94498. or,
Mutant strain 5 methyl-D of Arthrobacter hirinas
The L-tryptophan resistance strain has been deposited with the Research Institute of Microbial Industry and Technology (FRI) of the National Institute of Advanced Industrial Science and Technology (Microorganism Receiving No. 5256). In addition, a mutant coproporphyrin III resistant strain of Arthrobacter hyalinas has been deposited with the Research Institute of Microbial Industry and Technology (FRI) of the National Institute of Advanced Industrial Science and Technology (FRI No. 5259).

The Earthlobacter gloviformis is:
IFO No. 12137 for Fermentation Research Institute and ATC for American Type Culture Collection
This is a freely separable known strain deposited as C8010, and its bacteriological properties are described, for example, in Bergey's Manual.
of Determinative Bacteriology 8th edition.

Further, the above-mentioned Arthrobacter paciens was sent to the Fermentation Research Institute as IFO No. 12139,
Also, it is a freely segregable known strain deposited with the American Type Culture Collection as ATCC 13346, and details of its bacteriological properties are described, for example, in Be.
It is described in rgey's Manual of Determinative Bacteriology 8th edition. In addition, the mutant L-tryptophan-resistant strain of Arthrobacter paciens was obtained from FRI, Japan.
No. 5257).

The above mutant or variant strain can be obtained from the parent strain by known means such as irradiation with ultraviolet rays and irradiation with cobalt 60. For example, the irradiated strain is cultured on a porphyrin production inhibitor-containing mineral-agar medium,
If necessary, irradiation and culturing can be repeated to obtain a porphyrin-producing strain with an increased ability. The culture can be performed under culture conditions known for the parent strain. The bacteriological properties of the resulting mutant or variant strain are substantially the same as those of the parent strain, differing in that uroporphyrin III-producing ability is improved over the parent strain.

According to the present invention, a porphyrin-producing bacterium belonging to the genus Arthrobacter as described above is cultured in a medium,
Porphyrins can be produced by directly or indirectly collecting porphyrin from the obtained culture, for example, via an ester intermediate. Furthermore, L
-The porphyrin production can be further improved by using a cystine-containing medium or a Mg ⁺⁺ -containing medium.

In carrying out the method of the present invention, a medium containing a carbon source, a nitrogen source, inorganic salts and the like can be used as the above-mentioned medium. The medium may also contain L-cystine, Mg ⁺⁺ , antifoam and other components. Examples of such carbon sources include, for example, carbon sources such as sugars, alcohols, hydrocarbons, and bran. Examples of the nitrogen source include, for example, nitrogen sources such as corn steep liquor, yeast extract, meat extract, peptone, fish meal, ammonium salts, nitrates, urea, and further inorganic salts include, for example, Phosphates, magnesium salts, zinc salts, calcium salts, manganese salts, molybdenum salts, copper salts and the like can be mentioned.

The composition of the medium can be changed as appropriate, and can be added as needed during the culture. For example, when using alcohols as a carbon source, the production of porphyrins can be increased by adding alcohols when the residual concentration of alcohols is low, or at or around the start of porphyrin production. In culturing, it is preferable that iron salts are not substantially present in the medium.

In the present invention, a slant medium is suitable for daily storage of porphyrin-producing bacteria, and a liquid medium is preferable for production culture. Table 1 shows examples of these preferable medium compositions excluding the carbon source such as glucose and isopropanol.

Table 1 Composition of slant medium and production medium (per 200 mL) ─ Component Slant medium Production medium 培 地 NH ₄ NO ₃ 0.6g 0 _{.6g Na 2 HPO 4 · 12H 2} O 0.3g 0.3g KH 2 PO 4 0.08g 0.08g MgSO 4 · 7H 2 O 0.1g 1.0g FeSO 4 · 7H 2 O 2.0mg ZnSO 4 · 7H ₂ O 2.0 mg 2.0 mg CaCl ₂ .6H ₂ O 2.0 mg CuSO ₄ .5H ₂ O 10 μg 10 μg MoO ₃ 2 μg 2 μg Yeast extract 0.2 g 0.2 g Peptone 0.6 g L-cystine 0.12 g CaCO ₃ 1.0g agar 3g ───────────────────────────────────

In the method of the present invention, the specific site is ¹³
The porphyrin-producing bacteria are cultured in a medium containing 5-aminolevulinic acid labeled with C. In this case, 5-aminolevulinic acid labeled with ¹³ C can be used as such. , Or as a mixture with unlabeled 5-aminolevulinic acid. In each case, 5-aminolevulinic acid means not only free 5-aminolevulinic acid but also a derivative such as a salt thereof.

The amount of 5-aminolevulinic acid to be added varies depending on the strain used, culture conditions, medium composition and the like, but is generally in the range of 0.01 g / L to 3 g / L, preferably 0.03 g / L. 0.6 g / L, more preferably 0.05 g / L to 0.2 g / L,
And for example, it is 0.1 g / L. The amount of 5-aminolevulinic acid to be added can be easily determined experimentally so as to be most suitable under various conditions.

It is preferable to add 5-aminolevulinic acid to the medium from the beginning of the production culture, but it can also be added at an appropriate time after the start of the culture, for example, at the time when the production bacteria have grown to some extent. Also, 5-aminolevulinic acid is
It can be added to the medium at once, or it can be added continuously or intermittently during culture. In the present invention, any of the sites of symbols represented by the formula (V) is ¹³
Using 5-aminolevulinic acid labeled with C,
These 1, 2, 3, 4 or 5 positions are labeled with ¹³ C.
-Aminolevulinic acid can be produced (or obtained) from, for example, ¹³ C-labeled bromoacetic acid, Meldrum's acid or glycine. That is, [1- ¹³ C] 5-aminolevulinic acid and [2- ¹³ C] 5-aminolevulinic acid are
^{From 13} C-bromoacetic acid, [3- ¹³ C] 5-aminolevulinic acid is from ¹³ C-meldrum acid, and [4- ¹³ C] 5-aminolevulinic acid and [5- ¹³ C] 5-aminolevulinic acid are ¹³
It can be manufactured (or obtained) from C-glycine. [References: Katsuyuki Kurumaya, Takao Okazaki,
NobuoSeido, Yuzuru Akasaka, Yoshiki Kawajiri, and
Masahiro Kajiwara, Journal of Labeled Compounds
andRadiopharmaceuticals, XXVII, No.2, 217-235 (198
9)]

In the method of the present invention, the culture is carried out under aerobic conditions such as shaking or aeration with stirring, but it is preferable to employ relatively low aeration conditions. Generally, a temperature of about 20 ° C. to about 40 ° C. is used as the culture temperature, and a pH condition of about pH 4 to about 9.5 can be employed. The culturing time is usually about 2 to 20 days, and can be appropriately changed according to the selection of other culturing conditions.

In carrying out the method of the present invention, L
-Porphyrin production can be further increased by including cystine and / or Mg ⁺⁺ . As the compound that gives Mg ⁺⁺ , various water-soluble magnesium compounds can be used, and examples thereof include magnesium compounds such as magnesium sulfate, magnesium chloride, magnesium nitrate, and magnesium acetate.
The amount of L-cystine and / or Mg ⁺⁺ can be appropriately selected.
The use amount of cystine, for example, the use amount of Mg ⁺⁺ such as 0.1 g to 10 g can be exemplified.

Porphyrins accumulate in the obtained culture, for example, a culture solution. The porphyrin produced from the culture can be collected by various means. For example,
Porphyrins can be extracted and collected from the culture at a high yield by using acetic acid ethyl acetate or other suitable organic solvent. At the time of extraction, it is usual to extract from a culture solution from which cells and other solids have been separated and removed. Further, for example, the pH of the culture solution separated from the cells and other solids,
The porphyrins can be isoelectrically precipitated by adjusting the porphyrin concentration to about 1.6 to 3.6, and a porphyrin concentrate can be obtained by an appropriate solid-liquid separation means such as filtration or centrifugation.

The concentrate or extract obtained as described above is purified, for example, by subjecting the contained porphyrins to methyl esterification using sulfuric acid methanol and, if necessary, purifying it by column chromatography using alumina. And porphyrins in the form of methyl esters. Alternatively, the porphyrins can be collected by subjecting the concentrate or extract to liquid chromatography treatment, for example, high-performance liquid chromatography using a styrene-divinylbenzene gel column. When the porphyrins are collected in the form of esters, they can be converted to salts by hydrolysis if desired. For example, esters of uroporphyrin III can be converted to, for example, toluene, pyridine, dichloroethane, trichloroethane,
When dissolved in an organic solvent such as tetrahydrofuran or dimethyl sulfoxide, and a small amount of alkali is added thereto and heated, an alkali salt of uroporphyrin III precipitates. The precipitate can be collected by filtration, for example, and recovered in the form of a salt. Furthermore, the ester can be hydrolyzed with a mineral acid and then neutralized with an appropriate alkali to recover the precipitated alkali salt of uroporphyrin III.

The specifically exemplified production strains used in the present invention produce high proportions of coproporphyrin III and uroporphyrin III, but other porphyrins, namely uroporphyrin I and coproporphyrin I.
Also produce. Therefore, the target porphyrin compound can be obtained in the process of purification after fermentation production.

[0034]

Next, the present invention will be described more specifically with reference to examples. Example 1 Arthrobacter hirinas (Microtechnical Laboratory No. 5259) cultured on a slant to which one drop of isopropanol was added.
No.) per 1 L of ion exchange pure water, 10 mL of isopropanol, 1.0 g of yeast extract, 3.0 g of peptone, 3.0 g of ammonium nitrate, 0.4 g of monopotassium phosphate, 1.5 g of disodium phosphate, 5.0 g of magnesium sulfate, Manganese sulfate 10mg, zinc sulfate 10mg, copper sulfate 200μ
g, molybdenum trioxide 10 μg, calcium carbonate 5.0
g, 0.6 g of L-cystine and [1- ¹³ C] 5-aminolevulinic acid [ ¹³ C at the position indicated by × in the formula (V).
A sterilized medium 2 containing 0.1 g
Inoculate a 500 mL Erlenmeyer flask containing
Shaking culture was performed at 0 ° C. for one week. Labeled with accumulated regioselective ¹³ C in culture medium the coproporphyrin III of this time; the concentration of [compound having ¹³ C to the position indicated by the × of the formula (IV) shown as IV ×] is 105 mg / L Uroporphyrin labeled regioselectively with ¹³ C
The concentration of III [compound having ¹³ C at the position indicated by x in the formula (II); indicated as IIx] was 318 mg / L.

1 L of the culture was centrifuged (10,000 G for 10 minutes), and the supernatant was recovered. 2 g of ion exchange resin (DEAE-Sephadex A25) was added to the supernatant to adsorb porphyrin. This was filtered, and the ion exchange resin was recovered and freeze-dried. This was immersed in 95 mL of methanol in an ice bath, 5 mL of concentrated sulfuric acid was added, and the mixture was stored in a dark place for about 12 hours to methylate porphyrins. This was filtered, porphyrin methyl ester was confirmed in the filtrate, and the pH was adjusted to 7.0 by adding aqueous ammonia in an ice bath. This was transferred to a separating funnel, and 100 mL of methylene chloride was added to separate an organic layer.

The extraction using the separating funnel was repeated three times. Next, the organic layer was dried over anhydrous magnesium and methylene chloride was distilled off under reduced pressure. The extract is purified by column chromatography using silica gel (mesh 300),
¹³ C-labeled uroporphyrin III (II) by recrystallization using chloroform and methanol
*) 303 mg of octamethyl ester crystals were obtained.

[0037] showed the analysis results of ¹³ C-NMR of labeled resulting regioselective ¹³ C during this the uroporphyrin III (II ×) octamethyl ester in FIG. Also F
The spectrum of AB-MASS is shown in FIG. Meanwhile, ^13
13 CN of uroporphyrin III octamethyl ester synthesized using 5-aminolevulinic acid not labeled with C
FIG. 1 shows the results of the MR analysis. As a result, it was confirmed that ¹³ C was introduced into a predetermined site of the uroporphyrin III molecule.

On the other hand, at the stage of column chromatography using silica gel, coproporphyrin III (IV *) tetramethyl ester labeled regioselectively with ¹³ C was isolated, and the same as that for uroporphyrin III (II ×) was isolated. Thus, 102 mg of coproporphyrin III (IV ×) tetramethyl ester was obtained. It was also confirmed that ¹³ C was introduced at a predetermined site in the coproporphyrin molecule in the same manner as described above.

[0039] Instead of Example 2 ^[1-13 C] 5-aminolevulinic acid [2-
[ ¹³ C] 5-Aminolevulinic acid [compound having ¹³ C at the position indicated by * in the formula (V)] was used for culturing and purification in the same manner as in Example 1. As a result, uroporphyrin II regioselectively labeled with ¹³ C was obtained.
304 mg of I octamethyl ester was obtained. Position selective
¹³ C-labeled uroporphyrin III [* of formula (II)
Figure 4 shows the II indicated as *] analysis of ¹³ C-NMR of octamethyl ester; compound with ¹³ C at the position indicated by the.

In the same manner as in Example 1, coproporphyrin III regioselectively labeled with ¹³ C [formula (IV)
A compound having ¹³ C at the * position indicated by *; IV *]
To give 109 mg of tetramethyl ester. FIG. 5 shows the ¹³ C-NMR spectrum of compound VI * tetramethyl ester.

[0041] Instead of Example 3 ^[1-13 C] 5-aminolevulinic acid [3-
[ ^13C ] 5-Aminolevulinic acid (compound having ^{13C at the} position indicated by ▲ in formula (V)) was cultured and purified in exactly the same manner as in Example 1. As a result, uroporphyrin III regioselectively labeled with ¹³ C [compound having ¹³ C at the position indicated by ▲ in formula (II); indicated by IIII]
309 mg of octamethyl ester was obtained. Position selective
¹³ C-labeled with uroporphyrin III a (II ▲) analysis of ¹³ C-NMR of octamethyl ester shown in FIG.

Further, in the same manner as in Example 1, coproporphyrin III labeled with regioselectively ¹³ C [Formula (IV)
A compound having ¹³ C at the position indicated by ▲; 107 mg of tetramethyl ester of IV ▲] was obtained.

[0043] Instead of Example 4 ^[1-13 C] 5-aminolevulinic acid [4-
[ ¹³ C] 5-Aminolevulinic acid (compound having ¹³ C at the position indicated by ■ in formula (V)) was used for culturing and purification in exactly the same manner as in Example 1. As a result, uroporphyrin III regioselectively labeled with ¹³ C [compound having ¹³ C at the position indicated by ■ in formula (II); indicated by II ■]
315 mg of octamethylester was obtained. Position selective
¹³ C-labeled with uroporphyrin III a (II ■) analysis of ¹³ C-NMR of octamethyl ester shown in FIG.

In the same manner as in Example 1, coproporphyrin III regioselectively labeled with ¹³ C [formula (IV)
Of a compound having ¹³ C at position ■ of the compound (IV)], 108 mg of tetramethyl ester was obtained.

Example 5 [1- ¹³ C] Instead of 5-aminolevulinic acid,
¹³ C] 5-aminolevulinic acid [ ^{13 at} the position of ● in formula (V)
Culture and purification were carried out in exactly the same manner as in Example 1 except that the compound having C was used. As a result, uroporphyrin III labeled with regioselectively ¹³ C; to give 308mg of octamethyl ester [Compound having ¹³ C at the position of ● of formula (II) II shown in ●]. Of uroporphyrin III (II ●) octamethyl ester regioselectively labeled with ¹³ C
FIG. 8 shows the results of ¹³ C-NMR analysis.

In the same manner as in the first embodiment, ¹³ C
104 mg of coproporphyrin III [compound having ¹³ C at position of ● in formula (IV); indicated by IV ●] was obtained.

[Brief description of the drawings]

FIG. 1 shows unlabeled uroporphyrin III.
1 shows a ¹³ C-NMR spectrum of octamethyl ester.

FIG. 2 shows labeled uroporphyrin III (II
X) ¹³ C-NMR spectrum of octamethyl ester is shown.

FIG. 3 shows labeled uroporphyrin III (II
X) shows the FAB-MASS spectrum of octamethyl ester.

FIG. 4 shows labeled uroporphyrin III (II
*) Shows ¹³ C-NMR spectrum of octamethyl ester.

FIG. 5 shows labeled coproporphyrin III (IV
*) Shows ¹³ C-NMR spectrum of tetramethyl ester.

FIG. 6 shows labeled uroporphyrin III (II
▲) shows the ¹³ C-NMR spectrum of octamethyl ester.

FIG. 7 shows labeled uroporphyrin III (II
(3) shows the ¹³ C-NMR spectrum of octamethyl ester.

FIG. 8 shows labeled uroporphyrin III (II ●).
1 shows a ¹³ C-NMR spectrum of octamethyl ester.

Claims (1)

(57) [Claims] (1) The following formula (I), (II), (III) or (I
V): Embedded image Embedded image Embedded image [Wherein the symbols ×, *, ▲, △, and ● each represent a carbon atom ¹³ C or a naturally occurring carbon atom, and when the carbon atom represented by any one of these symbols is ¹³ C, Wherein the carbon atom represented by the other symbol is a naturally occurring carbon atom, and R represents hydrogen or a lower alkyl group or a salt-forming cation. The Arthrobacter
A microorganism belonging to the genus and capable of producing a porphyrin compound is represented by the following formula (V): [Wherein the symbols x, *, ▲, △ and ● represent a carbon atom ¹³ C or a naturally occurring carbon atom, and when the carbon atom represented by any one of these symbols is ¹³ C, The carbon atom represented by the symbol is a naturally occurring carbon atom, and is represented by the same symbol as the symbol representing ¹³ C among the symbols in the above formulas (I), (II), (III) or (IV). Carbon in the formula (V) is ¹³ C], and the porphyrin compound of interest is collected from the culture and the porphyrin compound is recovered as a lower alkyl ester. A method characterized by separating.