CA2445293C - Labelled .gamma.-cyano-.alpha.-aminobutyric acid compounds - Google Patents

Abstract

The invention relates to labelled compounds consisting of .gamma.-cyano-.alpha.-aminobutyric acid, salts thereof or derivatives thereof having a protecting group, wherein the carbon atom of the cyano group is labelled with 11C.
The invention also relates to labelled compounds, consisting of amino acids of formula (4), salts thereof or derivatives thereof having a protecting group:

Description

LABELLED -y-CYANO-u-AMINOBUTYRIC ACID COMPOUNDS

This application is a division of Canadian Patent Application No. 2,172,455 filed on March 22, 1996 and relating to labelled compounds and a method for manufacturing these compounds. More particularly, the invention of the parent application relates to compounds labelled with radionuclide such as positron nuclide or stable isotope. These labelled compounds are useful for imaging tumors and brain.
Physiological, pharmacological or biochemical processes of extra-trace substances have conventionally been traced in vivo by using 'various labelled compounds in many methods.

As one of the methods using such labelled compounds, Positron Emission Tomography (PET) method is now attracting the general attention, which consists of synthesizing a positron-labelled compound using a positron decay nuclide prepared in a cyclotron, administrating the compound into body and imaging the behavior of the compound by means of PET. The positron nuclide can label various metabolites or drugs without causing any change in the structure thereof, because it mainly comprises elements constituting an organism such as carbon, nitrogen and oxygen. In addition, because of the characteristics of the released annihilation radiation, the PET permits measurement of the physiological, pharmacological and biochemical processes of extra-trace substances in vivo at a very high sensitivity and a very low concentration, thus providing information very useful for clinical purposes. An example is the diagnosis of tumor by means of PET using positron nuclide. It is generally believed that glycometabolism, amino acid metabolism, fat metabolism and nucleic acid metabolism exasperate more in tumor cells than in normal ones. Since these metabolisms in tumor directly represent viability of tumor and the status of proliferation thereof, trials have been made to diagnose tumor by using a compound available by labelling sugar or amino acid with positron nuclide. Under these circumstances, 11C-L-methionine is now popularly employed for positron diagnosis of tumor.
Currently, 2,4-diaminobutyric acid (DABA), L-glutamine and L-glutamic acids are expected to serve as specific tumor markers.These amino acids are found to be incorporated into human or rat gliomacyte, and because of the preferential antitumor activity, they are further expected, not only as tumor markers, but also as new therapeutic drugs.
Actually, however, a labelled compound used in the PET method should have a short half-life of positron nuclide (for example, 20.4 minutes for 11C), and for clinical purposes, purity, specific radioactivity and ultimate quantity of radiation must satisfy clinical requirements. It is however very difficult to manufacture a positron-labelled compound which satisfies these requirements.
Exposure of the operator during synthesis of the labelled compound is another problem. As a labelling method permitting rapid operation and giving a high radiochemical yield sufficient to meet practical requirements has not as yet been established, progress of the PET method has not been satisfactory in terms of application for biological observation and diagnosis, for example, imaging of tumor or brain.
For DABA, asparagine, aspartic acid, L-glutamine and L-glutamic acids, expected because of the favorable characteristics, for example, positron labelling has been very difficult for these reasons.
As to the difficulty of labelling, this is also the case with labelling with (3-decay nuclide or other radioactive isotope, or further, with a stable isotope, in addition to the case with positron nuclide.
It is an object of the invention of the parent application to provide novel compounds labelled with radionuclide or stable isotope which are achievable as a rapid labelling giving a high radiochemical yield, and labelled compounds which are a synthetic intermediate thereof.

It is another object of the invention of the parent application to provide a method for manufacturing the above-mentioned labelled compounds.
According to a first aspect of the invention of the parent application, there is provided P-cyano-L-alanine, salts thereof or derivatives thereof having a protecting group, wherein the carbon atom of the cyano group is labelled with radionuclide or stable isotope.

According to a second aspect of the invention of the parent application, there is provided a method for manufacturing the labelled compounds described in the first aspect of the invention of the parent application, which comprises reacting an amino acid of formula (1), a salt thereof or a derivative thereof having a protecting group with a cyanic compound having a cyano group labelled with radionuclide or stable isotope, in the presence of a thermostable P-cyano-L-alanine synthase:
Rl-CH2-CH-COOH (1) I

where Rl is hydrogen atom, halogen atom, a hydrocarbon group, an oxygen-containing group or a sulfur-containing group.
According to a third aspect described in the invention of the parent application, there is provided labelled compounds which are amino acids of formula (2), salts thereof or derivatives thereof having a protecting group:

1 (2) where R2 represents -*CONH2 (asparagine), -*COOH(asparatic acid), -*CH2-NH2 (DABA), and *C is carbon labelled with radionuclide or stable isotope.

Labelled compounds of the above formula (2) wherein R2 is -I1CONH2, -11COOH or -11CH2NH2 are particularly preferred.

According to a fourth aspect described in the invention of the parent application, there is provided a method for manufacturing the labelled compounds described in the third aspect of the invention of the parent application, which comprises organically or enzymatically synthesizing the labelled compounds, salts thereof or derivatives thereof having a protecting group by using, as an intermediate, a labelled compound described in the first aspect of the invention of the parent application or a labelled compound manufactured by the method described in the second aspect of the invention of the parent application.

The invention of the present divisional application is directed to labelled compounds whicli are y-cyano-a-aminobutyric acid, salts thereof or derivatives thereof having a protecting group, wherein the carbon atom of the cyano group is labelled with radionuclide or stable isotope. Labelled compounds wherein the carbon atom of the cyano group is labelled with "C are particularly preferred.

The invention of the present divisional application also provides, in another aspect thereof, a method for manufacturing the labelled compounds just described above, which comprises reacting an amino acid expressed by the following formula (3), a salt thereof or a derivative thereof having a protecting group with a cyanic compound having a cyano group labelled with radionuclide or stable isotope in the presence of a thermostable y-cyano-a-aminobutyric acid synthase:

Rl - CH2 - CH2 - CH - COOH
1 (3) where R, is hydrogen atom, halogen atom, a hydrocarbon group, an oxygen-containing group or a sulfur-containing group.

The invention of the present divisional application also provides, in a further aspect thereof labelled compounds which are amino acids expressed by the following formula (4), salts thereof or derivatives thereof having a protecting group:

1 (4) where R2 represents -*CONHz (glutamine), or -*COOH (glutamic acid), and *C is carbon labelled with radionuclide or stable isotope.

Labelled compounds wherein R2 is "CONH2 or "COOH are particularly preferred.
The invention of the present divisional application also provides, in still another aspect thereof a method for manufacturing the labelled compounds just described above, which comprises organically or enzymatically synthesizing the labelled compounds, salts thereof or the derivatives thereof having a protecting group by using, as an intennediate, y-cyano-a-aminobutyric acid, a salt thereof or a derivative thereof having a protecting group, wherein the carbon atom of the cyano group is labelled with radionuclide or sable isotope, or a labelled compound manufactured by a method which comprises reacting an amino acid expressed by the following formula (3), a salt thereof or a derivative thereof having a protecting group with a cyanic compound having a cyano group labelled with radionuclide or stable isotope in the presence of a thermostable y-cyano-a-aminobutyric acid synthase:

Rl - CH2 - CH2 - CH - COOH
1 (3) where Rl is hydrogen atom, halogen atom, a hydrocarbon group, an oxygen-containing group or a sulfur-containing group.
In the accompanying drawings:

Fig. 1 is the result of HPLC analysis showing that the reaction product of Example 2 is P-cyanic alanine.

Fig. 2 is the result of HPLC analysis showing that the reaction product of Example 2 is P-cyanic alanine labelled with 11C.

Fig. 3 is the mass-analysis spectrum for the reaction product of Example 2.
Fig. 4 is the result of HPLC analysis showing that the reaction product of Example 3 is DABA.
Fig. 5 is the result of HPLC analysis showing that the reaction product of Example 3 is DABA labelled with "C.

Fig. 6 is the result of HPLC analysis showing that the "C-labelled DABA of Example 3 is L-form.
Fig. 7 shows the uptake of "C-labelled DABA of Example 3 into gliomacyte.

Fig. 8 illustrates an optimum pH of thermostable y-cyano-a-aminobutyric acid synthase.
Fig. 9 shows a pH stability thereof.

Fig. 10 shows an optimum temperature thereof.
Fig. 11 shows temperature stability thereof.
Fig. 12 illustrates a subunit molecular weight of thermostable y-cyano-a-aminobutyric acid synthase by SDS-PAGE.

Fig. 13 shows a graph of molecular weights of the enzyme by gel filtration.
Fig. 14 shows the result of absorption spectral analysis of purified thermostable y-cyano-a-aminobutyric acid synthase.

Fig. 15 is the result of HPLC analysis showing that the reaction product of Example 7 is L-glutamic acid.
Fig. 16 is the result of HPLC analysis showing that the reaction product of Example 7 is L-glutamic acid labelled with C.

Fig. 17 is the result of HPLC analysis showing that the "C-labelled glutamic acid of Example 7 is L-form.

Both the labelled R-cyano-alanine, salts thereof or protective derivatives thereof (hereinafter referred to as the "labelled P-cyano-L-alanine compounds") and the labelled y-cyano-a-aminobutyric acid, salts thereof or protected derivatives thereof (hereinafter referred to as the "labelled y-cyano-a-aminobutyric acid compounds") are novel compounds provided by the invention.
The labelled P-cyano-L-alanine compounds are represented by formula (5) and the labelled y-cyano-a-aminobutyric acid compounds are represented by formula (6):
N*C-CH2-CH-COOH
(5) N *C - CH2 - CH2 - CH - COOH
1 (6) These compounds as shown in the formulae (5) and (6) are characterized in that the cyano group carbon atom (*C) is labelled with radionuclide or stable isotope. The terms "radionuclide" and "stable isotope"
using for labelling are denoted as having wide-range definitions including positron nuclide 11C, radioactive isotope 14C, and further, stable isotope 13C. What should be noted about these labels, particularly in the invention, is that compounds labelled with positron nuclide 11C are provided. These compounds are very useful as synthesis intermediates of labelled amino acid compounds manufactured through conversion of their (3-cyano group or y-cyano group. Because of the possibility of administering in animal body, these labelled amino acid compounds can be used in the application of the PET method.

Free amino groups or carboxyl groups may of course be present in the form of salts of acid or alkali, or may be derivatives protected by a conventional amino acid, or by any of various protecting groups in peptide synthesis. All such cases are included in the labelled 0-cyano-L-alanine compounds and the labelled y-cyano-a-aminobutyric acid compounds of the invention.

Now, the following paragraphs describe the methods for manufacturing the labelled (3-cyano-L-alanine compounds and the y-cyano-a-aminobutyric acid compounds, respectively, and the labelled amino acid compounds synthesized by using these labelled compounds as synthesis intermediates.

Manufacture of labelled P-cyano-L-alanine compounds.

The labelled P-cyano-L-alanine compounds of the invention can be manufactured by reacting an amino acid as expressed by the above-mentioned formula (1) or a salt thereof or a protected derivative thereof, with a cyanic compound of which cyano group carbon is labelled with radionuclide or stable isotope, in the presence of R-cyano-L-alanine synthase. It is needless to mention that it can be manufactured through conventional chemical synthesis.
When manufacturing by the use of an enzyme, it is possible to use (3-cyano-L-alanine synthase derived from a bacterium selected from the group consisting of, for example, Acinetobacter, Aerobacler, Agrobacterium, Arthrobacter, Bacillus, Brevibacterium, Cellulomanas, Corynebacterium, Enterobacter, Erwinia, Escherichia, Flavobacterium, Hafnia, Micrococcus, Mycobacterium, Nocardia, Pseudomonas, Rhodococcus, Salmonella, Serratia, and Staphylococcus.These bacteria can more specifically be shown in Table 1.

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p ~ N M M It Ser L-serine O-M- Ser O-methyl-L-serine Cys L-cysteine O-P-Ser O-phosphoryl-L-serine (3-Cl-ALa 0-chloro-L-alanine O-A-Ser O-scetyl-L-serine In the manufacturing method of the invention, it is preferable to use aP-cyano-L-alanine synthase derived from a bacterium of Bacillus, or more specifically, an enzyme isolated from, for example, Bacillus stearothermophilus.
Particularly, Bacillus stearothermophilus CN3 is the most preferable for the invention. This strain was isolated by the present inventors from a natural source and deposited on August 8, 1994 to National Institute of Bioscience and Human Technology under a deposit number of FERM BP-4773.

Actually, as a synthase from these bacteria, a thermostable 0-cyano-L-alanine synthase having the following properties can be presented:

(1) action: generating 0-cyano-L-alanine from O-acetyl-L-serine and a cyanic compound;
(2) optimum pH: 7.0 to 9.0;
(3) stable pH: 6.0 to 10.0;
(4) optimum temperature: 40 to 50 C;
(5) thermostability: stable up to 70 C when holding at pH 7.5 for minutes;
(6) molecular weight: 60,000 to 80,000 with gel filtration.
This enzyme is manufacturable by culturing a thermophilic Bacillus 25 on a(3-cyano-L-alanine synthase producing medium, and then isolating the target P-cyano-L-alanine synthase from the cultured bacterium. In this process, the strain would be used preferably. This enzyme requires, for example, pyridoxal phosphate as a coenzyme, and applicable substrates include O-acetyl-L-serine, L-cystine, L-serine, O-methyl-L-serine, O-phosphoryl-L-serine, O-succinyl-L-30 serine and (3-chloro-L-alanine.

In the reaction of the compounds of formula (1) using the synthase above, the substituent of the formula (1) compounds may more specifically be -0-alkyl group, -0-phosphoryl group, or halogen atom, and the cyanic compound may be prussic acid (CN-), NaCN or KCN of which carbon is labelled.
The labelled cyanic compound is available, in the case of prussic acid labelled with positron nuclide 11 C, by for example reducing 11 CO2 prepared in a cyclotron into 11 CH4, and reacting it with ammonia in the presence of platinum (Pt) catalyst at a high temperature of about 1,000 C, just as in the ordinary prussic acid synthesis. The P-cyano-L-alanine compounds of which cyano group carbon is labelled with positron nuclide "C can be manufactured by reacting one of the cyanic compounds with an amino acid represented by formula (1) in an aqueous medium in the presence of the above-mentioned synthase. The P-cyano-L-alanine compounds labelled with 13C and 14C are similarly produced.

Manufacture of labelled y-cyano-a-aminobutyric acid compounds.

The labelled y-cyano-a-aminobutyric acid compounds of the invention can be manufactured by reacting an amino acid represented by formula (3), a salt thereof or a protected derivative thereof, with a cyanic compound of which cyano group carbon is labelled with radionuclide or stable isotope, in the presence of y-cyano-a-aminobutyric acid synthase. It is of course manufacturable also through chemical synthesis.

The thermostable y-cyano-a-aminobutyric acid synthase, when manufacturing by the use of an enzyme, may be one available by isolating from a thermophilic Bacillus, or more specifically, for example, may be one obtained from Bacillus stearothermophilus CN3 strain (FERM BP-4773).

Actually, a thermostable y-cyano-a-aminobutyric acid synthase having the following properties may be presented as an example of the enzyme for the above-mentioned reaction:

(1) action: producing y-cyano-a-aminobutyric acid from O-acetyl-L-homoserine and cyanic compound;
(2) optimum pH: 7.5 to 8.5;

(3) stable pH: 6.0 to 10.5;
(4) optimum temperature: 55 to 65 C;
(5) thermostability: stable up to 65 C when holding at pH of 7.5 for 30 minutes;
(6) molecular weight: about 180,000 with gel filtration.
This enzyme can be manufactured, for example, by culturing Bacillus stearothermophilus CN3 strain on a y-cyano-a-aminobutyric acid synthase producing medium, and then, isolating the target enzyme. This enzyme requires, for example, pyridoxal phosphate as a coenzyme, and applicable substrates include O-acetyl-L-homoserine, or L-homocystine.
For example, in the reaction of the formula (3) compounds using the synthase above, the substituent Rl of these formula (3) compounds may more specifically be -0-acyl group, -0-alkyl group, -0-phosphoryl group, or halogen atom, and the cyanic compound be prussic acid (CN-) , NaCN or KCN of which carbon is labelled. In the case of prussic acid labelled with positron nuclide 11C, the labelled cyanic compound can be obtained by reducing 11 COZ prepared in a cyclotron into 11CH4, and reacting it with ammonia at a high temperature of about 1,000 C in the presence of a platinum (Pt) catalyst, just as in the ordinary prussic acid synthesis. The y-cyano-a-aminobutyric acid compound of which cyano group carbon is labelled with positron nuclide 11 C can be manufactured by reacting this cyanic compound with the above-mentioned formula (3) amino acid in the presence of said synthase.

Similarly, there is available the y-cyano-a-aminobutyric acid compound labelled with 13C or 14C.
Manufacture of labelled amino acids.
The labelled amino acid compounds of the invention are manufacturable by using the above-mentioned labelled P-cyano-L-alanine compounds or labelled y-cyano-a-aminobutyric acid compounds as intermediates.
It is possible, for example, to convert cyano group into amino acid through a reduction reaction, and cyano group into amide acid or carboxyl group through a hydrolysis reaction. More specifically, the above-mentioned formula (2) or (4) labelled amino acids are manufacturable by reduction or decomposition under various conditions, and further, the labelled amino acid compound, salts thereof or protected derivatives thereof by the conventional method.

The reduction reaction is made possible by a method based on Raney nickel or Raney cobalt, or any of the various means including the conventional methods such as one using NaBH4 or other reducing agent. This is also the case with the hydrolysis reaction. By the application of any of these means including the enzyme method, for example, the following labelled amino acids are synthesized from the labelled (3-cyano-L-alanine compounds:
L-2,4-diaminobutyric acid (DABA):

NH2 - *CH2 - CH2 - CH - COOH

y-aminobutyric acid (GABA):
NH2 -*CH2 - CH2 - CH2 - COOH
L-asparagine:

NH2 - *C - CH2 - CH - COOH
I - I I

L-aspartic acid:
HO - *C - CH2 - CH - COOH
II I

The following labelled amino acid compounds are for example manufactured from the labelled y-cyano-a-aminobutyric acid compounds:

L-glutamine:

NH2 -*C - CH2 - CH2 - CH - COOH
I I I

L-glutamic acid:
HO -*C - CH2 - CHZ - CH - COOH
II I

The labelled amino acid compounds thus synthesized can be combined, for example, with a biopolymer such as peptide or protein through substitution of amino acid residue or addition of other amino acid residue.
The invention permits, as described above, easy radiochemical labelling or stable isotope labelling with positron nuclide "C or the like through substitution or addition reaction of the amino acid and a cyanic compound in the presence of a specific synthase. Particularly, the findings that bacteria of Bacillus can produce an enzyme for this reaction make it possible, in the present invention, to achieve labelling not only with positron nuclide 11C, but also with a radioisotope such as 14C or a stable isotope such as 13C.
Labelling of various amino acids makes a great contribution to observation and diagnosis by the PET method as well as to NMR diagnosis and biochemical research on metabolism. Although a method of labelling amino group or carboxyl group of amino acid with an isotope has conventionally been known, these groups were easily metabolized in vivo, so that it was impossible to trace the mother nucleus of amino acid. The present invention makes it possible to label carbon which is hard to metabolize, and now permits very easy tracing of the mother nucleus. It is possible to label the mother nucleus with a(3-decaying radioisotope 3H or 14C through chemical synthesis consuming a long period of time. However, because radiation does not run through the body when using these isotopes, the position of a labelled compound in vivo cannot be detected from outside the body. On the other hand, 11C nuclide, which P+ decays and releases y rays upon hitting negatrons inside cells and tissues, can be detected from outside the body and therefore permits tracing distribution and localization of a labelled compound administered in vivo from outside. As it is possible to trace behavior of the labelled compound in vivo while comparing between before and after treatment or with clinical effect, it is very useful for diagnosis and medical treatment of diseases.
The invention will now be described in further detail by means of examples.

A thermostable (3-cyano-L-alanine synthase was prepared as follows.
A culture medium comprising 1% polypepton, 0.25% yeast extract, 0.1% glycerol, 0.1% (NH4)2SO4, 0.05% MgSO4 7H20, and 0.1% K2HPO4 (pH: 7.2) was poured into two large test tubes by equal amounts of 8 ml, sterilized at 120 C for 20 minutes, and cooled. Then, Bacillus stearothermophilus CN3 strain (No. FERM BP-4773) was inoculated in an amount of one platinum spoon, and after culture at 60 C for 24 hours, the product was used as a basic medium.
An antifoaming agent (made by Asahi Denka Company, ADEK.ANOLr KG-126) in an amount of 0.01% (VN) was added to a culture medium having the same composition as above. The resultant medium in an amount of 1.6 1 was placed in a jar fermenter having a volume of 2 1, and after sterilization at 120 C for 20 minutes and cooling, 16 ml of the above-mentioned basic medium (for two test tubes) were inoculated. Culture was thus conducted at 60 C for 27 hours under stirring conditions including a volume of aeration of 1.6 1/minute and a stirring velocity of 300 rpm, and the resultant medium was used as the preculture medium.
Then, a culture comprising 1% polypepton, 0.25% yeast extract, 0.1% glycerol, 0.1% (NH4)2SO4, 0.05% MgSO4 7H20, 0.1% K2HPO4, and 0.1% L-serine (pH: 7.2) in an amount of 160 1 was placed in a jar fermenter having a volume of 200 1, sterilized at 120 C for 30 minutes, cooled, and then, 1.6 1 of the Trade-mark above-mentioned preculture medium were inoculated to conduct culture at 60 C
for 24 hours under stirring conditions including a volume of aeration of 1201/minute and a stirring velocity of 200 rpm. After culturing, bacteria were collected through sharpless centrifugal separation.
The resultant bacteria were equally divided into eight (each in an amount of 20 1), and were each suspended in an appropriate amount of potassium phosphate buffer solution (10 mM, pH: 8.0, containing 0.1 mM dithiothreitol) to subject to cryopreservation at -80 C. This was used for the subsequent manufacture of enzyme by defrosting.

An amount of 60 1 of the frozen bacteria was suspended to give a total amount of about 2,000 ml, and the bacteria were crushed on a DYNO-MILL' (made by WAB company). The crushed solution was centrifugally separated to obtain 2,100 ml of cell-free extract by removing residual bacteria.
Ammonium sulfate was added to this cell-free extract to achieve 40% saturation. After holding for a night, precipitate was removed by centrifugal separation, and ammonium sulfate was added again to the resultant supernatant to achieve 90% saturation. The saturated supernatant was held for 5 hours and a precipitate was obtained by centrifugal separation. The precipitate thus obtained was dissolved in a 10 mM potassium phosphate buffer solution (pH: 8.0) containing 0.1 mM dithiothreitol, and was desalted with a buffer solution of the same composition by the use of a dialysis membrane. Ethanol previously cooled to -80 C was added to the thus desalted solution in an amount of 1,065 ml to achieve an ultimate concentration of 70%, and a precipitate was obtained through centrifugal separation. The resultant precipitate was suspended in a 10 mM

potassium phosphate buffer solution (pH: 8.0) containing 0.1 mM dithiothreitol and was subjected to a heat treatment at 70 C for 30 minutes. After removing precipitate through centrifugal separation, the supernatant in an amount of 1,024 ml was passed through a DEAE-cellurofine A-500 colunm (6.0 cm diameter x 18 cm length) previously equilibrated with a 10 mM potassium phosphate buffer Trade-mark solution (pH: 8.0) containing 0.1 mM dithiothreitol for adsorption of enzyme.
After washing with a buffer solution having the same composition, the enzyme was eluted by gradient elution from the 10 mM potassium phosphate buffer solution (pH: 8.0) containing 0.1 mM dithiothreitol to a 100 mM potassium phosphate buffer solution (pH: 8.0) containing 0.1 mM dithiothreitol and 0.5 M
NaCl to collect an active fraction.

Ammonium sulfate was added to this active fraction so as to achieve 80% saturation, and after holding for a night, a precipitate was obtained through centrifugal separation. This precipitate was dissolved in a 10 mM potassium phosphate buffer solution (pH: 8.0) containing 0.1 mM dithiothreitol, and subjected to an adjusted electrophoresis (7.5% polyacrylamide gel). After electrophoresis, an active portion in the gel was cut out, milled, and an enzyme was extracted by means of a 10 mM potassium phosphate buffer solution (pH: 8.0) containing 0.1 mM dithiothreitol. Ammonium sulfate was added to this active fraction so as to achieve 30% saturation, passed through a Phenyl SEPHAROSE* CL-4B column (2.5 cm diameter x 12 cm length) previously equilibrated with 30% saturated ammonium sulfate and a 10 mM potassium phosphate buffer solution (pH: 8.0) containing 0.1 mM dithiothreitol for adsorption of an enzyme. After washing with a buffer solution having the same composition, the enzyme was eluted by gradient elution from the 30% saturated ammonium sulfate and the 10 mM potassium phosphate buffer solution (pH: 8.0) containing 0.1 mM dithiothreitol to a 10 mM potassium phosphate buffer solution (pH: 8.0) containing 0.1 mM dithiothreitol to collect an active fraction.
Ammonium sulfate was added to this active fraction so as to achieve 30%

saturation, and the saturated fraction was passed through an Octyl SEPHAROSE
CL-4B column (1.5 cm diameter x 10 cm length) previously equilibrated with 30% saturated ammonium sulfate and a 10 mM potassium phosphate buffer solution (pH: 8.0) containing 0.1 mM dithiothreitol, for adsorption of an enzyme.
After washing with a buffer solution having the same composition, an active ' Trade-mark fraction was collected by eluting the enzyme by the gradient elution method from the 30% saturated ammonium sulfate and the 10 mM potassium phosphate buffer solution (pH: 8.0) containing 0.1 mM dithiothreitol to a 10 mM potassium phosphate buffer solution (pH: 8.0) containing 0.1 mM dithiothreitol. The enzyme preparation thus obtained was confirmed to be single in terms of electrophoresis.
For the process of acquiring enzyme as described above, enzyme activity, yield and the like of each step are shown in Table 2. The term "Unit" as used in Table 2 is defined as the enzyme activity of generating P-cyano-L-alanine in an amount of 1 mol during one minute, as measured by the activity measuring method shown in Table 3.

b O (~ ~-r 00 ct N O
y~ ~' t~ l~ ~3 N =--+ r-+ O~
.,~

.-~ C- 01 0 O ~ O o0 ~ N N 00 bA
u E~ ~c -0 00 Q1 ~O ~O
u CA "p 00 a1 C.
O M ~ ~
N ~
t~.
~
~
~
O
N N O O O O~
Rt ~o 1-4 p*N v~ 00 ~ y. d' N ~ (y O~

h CA
.~
y p0 p0 o tn tn v N ~ ~ I~ ~O M N N
~

a) Cd ~
~
~
v z w x Qd Z a,~U o~U o =-+ N M ~O [~ 00 O O O O
~ O o O
o a ;d o w cd O U
M
~ ~~ ~~ ~. v 0 N N N C>

,=-. = w W 't C>

to o ~ x ~~, =~ 'b ~ ' cd ~~ ~ '~a a U w x U
~
P4 rA
o r.
co a o ~ ~ x o The resultant enzyme, having 0-cyano-L-alanine synthetic activity from O-acetyl-L-serine and cyanic compound, had the following properties:
(1) thermostability: stable at temperatures of up to 70 C (20 mM
potassium phosphate buffer solution, pH: 7.5, heat treatment for 30 minutes);
(2) optimum temperature: 45 C (20 mM potassium phosphate buffer solution, pH: 7.5);
(3) pH stability: stable at pH 6 to 10 (20 mM buffer solution, treatment at 60 C for 30 minutes);
(4) optimum pH: pH 8.0 (20 mM potassium phosphate buffer solution);
(5) molecular weight: 70,000 (gel filtration);
(6) subunit molecular weight: 34,000 (SDS-PAGE); and (7) number of subunits: 2.

H11CN was prepared by reducing 11C02, having positron nuclide 11C
prepared in cyclotron, at 400 C in a mixed atmosphere of H2 and N2 in the presence of Ni into 11CH4, and contact-reacting the resulting 11CH4 with ammonia using platinum (Pt) as a catalyst at a temperature of 1,000 C. The resultant in the form of a mixed gas was passed through a 50% H2SO4 solution in an amount of 1.5 ml to remove residual ammonia, further brought into contact with P205 to remove ammonia, and H11CN was trapped with 50 mM KOH in an amount of 350 l.
O-acetyl-L-serine was mixed, together with the 0-cyano-L-alanine synthase obtained in Example 1, into this H11CN aqueous solution, and was reacted at the room temperature.
The product was analyzed under the following conditions:
Column: Beckman C-18 SPHERISORB* (4.6 x 250 mm);
Eluent: 10 mM Potassium phosphate buffer (pH: 4.6);
Trade-mark Flowrate: 0.75 ml/min;
Detection: UV 220 nm and Radiodetector;
Temperature: Room temperature; and Injection volume: 10-20 1.
The results of analysis with UV 220 nm and radiodetector are shown in Figs. 1 and 2. It was confirmed from these results that the reaction product is 0-cyano-L-alanine from the comparison with the retention time of standard, and the presence of cyano group having positron nuclide ~ 1C was also confirmed.
Fig. 3 which illustrates a quantitative analysis spectrum permitted confirmation as well, together with the results shown in Figs. 1 and 2, of the fact that the reaction product was R-cyano-L-alanine.

Reducing agents CoBr2 and NaBH4 were added to the labelled P-cyano-L-alanine obtained in Example 2 for reduction. Then, after filtration (0.2 pm), 6 M hydrochloric acid in an amount of 500 l was added and the mixture was filtered through a 0.2 m filter to remove protein and collect an enzyme, which was then purified with HPLC.
The product was analyzed under the following HPLC conditions:
Column: Beckman CX (4.6 x 250 mm);
Eluent: 10 mM Potassium phosphate buffer (pH: 4.6);
Flowrate: 2 ml/min.;
Detection: UV 220 nm and Radiodetector;
Temperature: Room temperature; and Injection volume: 10-40 l.
The results of analysis using UV 220 nm and Radiodetector are shown in Figs. 4 and 5. These results show that the reaction product was L-2,4-diaminobutyric acid (L-DABA) labelled with 11C. This compound had a radiochemical purity of at least 96% and a radiochemical yield within a range of from 30 to 40%.

Fig. 6 illustrates values of analysis based on LN 340 nm and Radiodetector carried out for identification of L-DABA and D-DABA. It is thus proved that the DABA enzymatically synthesized in the present invention is of the L-form. The chart (a) of Fig. 6 shows a racemi authentic sample of DABA as converted into a derivative to perform HPLC analysis. D- and L-forms were converted into derivatives with reference to the method of Marfey P.
(Carlsberg Res. Commun. 49,591, 1984).
The chart (b) of Fig. 6 also demonstrates that the enzymatically synthesized DABA is of L-form.

Biological applicability of the 11C-labelled L-DABA obtained in Example 3 was evaluated. The relationship between concentration of the L-DABA
added to culture medium of rat glioma and uptake of radioactivity into the glioma cells in a given duration was investigated in a medium having an amino acid content close to the biological one, and for control, in a physiological saline buffered with phosphoric acid.
The results are shown in Fig. 7. From the results, it is known that the uptake of 11C-labelled L-DABA was dependent on the concentration of it in culture medium, and has properties as a satisfactory labelling substance applicable to biological bodies.

A thermostable y-cyano-a-aminobutyric acid synthase was prepared as follows.

Dry bouillon medium NISSUI' for general bacteria (made by Nissui Seiyaku Company) was poured into four test tubes (2.2 cm diameter x 19.5 cm length), sterilized at 120 C for 20 minutes and cooled. Then, Bacillus stearothermophilus CN3 strain was inoculated to the cooled medium by an amount of one platinum dose.

' Trade-mark A basic culture medium was prepared by shake-culturing the inoculated medium at 58 C for 18 hours. A medium (pH: 7.2) comprising 1%
soluble starch, 0.5% yeast extract, 0.05% MgSO4 7H20, 0.1% KZHPO4, 0.001%
FeSO4 7H20, and 0.1 % L-glutamine was poured into four culturing flasks having a volume of 21 each in an amount of 400 ml. After sterilization at 120 C for 20 minutes and cooling, the above-mentioned basic culture medium in an amount of 16 ml (in the four test tubes) was inoculated by an amount of 4 ml to each of the flasks, and the inoculated medium was shake-cultured at 58 C for 18 hours to prepare a preculture medium. Then, a medium prepared by adding an antifoaming agent ADEKANOL LG126 (made by Asahi Denka Company) in an amount of 0.01% (W/V) to a medium having the same composition as above.The resultant medium in an amount of 160 1 was placed in a jar fermenter having a volume of 200 1. After sterilization at 120 C for 20 minutes and cooling, the above-mentioned preculture medium in an amount of 1.6 1 was inoculated and culturing was carried out at 58 C for 18 hours under conditions including a flowrate of aeration of 160 1/minute and a stirring velocity of 200 rpm. After the completion of culture, bacteria were collected through sharpless.
The resultant bacteria in an amount of 660 g was suspended in a potassium phosphate buffer solution (20 mM, pH: 7.5, containing 0.1 mM
dithiothreitol) so as to achieve a total amount of 2.5 1, and the suspension was milled in a DYNO-MILL (made by WAB Company). The milled solution was subjected to centrifugal separation to remove bacterial residue, and a cell-free extract in an amount of 2,799 ml was obtained. The thus obtained cell-free extract was held at 60 C for 30 minutes, and produced precipitate was removed by centrifugal separation to give a supematant.
Ammonium sulfate was added to this supernatant so as to achieve 40% saturation, and the saturated supematant was held for a night. The precipitate was removed by centrifugal separation. Ammonium sulfate was added again to the resultant supematant to achieve 90% saturation, and the mixture was held for a night, thus resulting in a precipitate. The precipitate was dissolved in a 20 mM

potassium phosphate buffer solution (pH: 7.5) containing 0.1 mM dithiothreitol and 0.01 mM pyridoxal phosphate, and desalted by this buffer solution with the use of a dialysis membrane. The desalted solution was passed through a previously equilibrated DEAE-cellurofine A-500 column (8 cm diameter x 22 cm length) for adsorption of an enzyme. After washing with a 100 mM potassium phosphate buffer solution (pH: 7.5) containing 0.1 mM dithiothreitol and 0.01 mM
pyridoxal phosphate, the enzyme was eluted by the gradient elution method from this buffer solution to a 100 mM potassium phosphate buffer solution (pH: 7.5) containing 0.1 mM dithiothreitol, 0.01 mM pyridoxal phosphate and 0.4 M KCL, thus collecting an active fraction.
Then, ammonium sulfate was added to the resultant active fraction to achieve 60% saturation, and after holding for a night, produced precipitate was removed through centrifugal separation. Ammonium sulfate was added again to the supematant thus obtained to achieve 75% saturation, which was held for a night, and a precipitate was obtained through centrifugal separation. This precipitate was dissolved in 30% saturated ammonium sulfate and 20 mM
potassium phosphate buffer solution (pH: 7.5) containing 0.1 mM dithiothreitol and 0.01 mM pyridoxal phosphate, and the resultant solution was passed through a Phenyl-TOYOPAL* 650S column (2.5 cm diameter x 8.5 cm length) previously equilibrated by the above-mentioned buffer solution to adsorb the enzyme.
After washing with this buffer solution, the enzyme was eluted from this buffer solution to a 20 mM potassium phosphate buffer solution (pH: 7.5) containing 0.1 mM
dithiothreitol and 0.01 mM pyridoxal phosphate by the gradient elution method and an active fraction was collected.
Ammonium sulfate was added to the thus collected active fraction so as'to achieve 80% saturation, and after holding for a night, a precipitate was obtained by centrifugal separation. This precipitate was dissolved in a 50 mM
sodium phosphate buffer solution (pH: 7.5) containing 0.1 mM dithiothreitol and " Trade-mark 0.2 M NaCI. The solution was then applied to a SEPHACRYL* S-200HR column (2.0 cm diameter x 106 cm length) previously equilibrated with the above-mentioned buffer solution, and an active fraction was collected by eluting enzyme with this buffer solution.
Ammonium sulfate was added to the collected active fraction so as to achieve 80% saturation, and after holding for a night, a precipitate was obtained through centrifugal separation. This precipitate was dissolved in a 100 mM
sodium phosphate buffer solution (pH: 7.0) containing 0.2 M NaCI, and the solution was poured at a flow rate of 0.7 ml/minute as a mobile phase into a TSK
gel-G3000SW column (0.75 cm diameter x 60 cm length) for HPLC to take out the active fraction. The resultant enzyme was electrophoretically homogeneous, having a specific activity of 147 U/mg.
Total activity, total protein, specific activity, purifying magnifications and yield of the enzyme obtained in the above-mentioned extraction and purifying steps were as shown in Table 4. Enzyme activity was measured by incubating a reaction solution (total amount: 200 l) comprising 10 l 1 M potassium phosphate buffer solution (pH: 7.5) (ultimate concentration:
50 mM), 100 l 10 mM O-acetyl-L-homoserine (ultimate concentration: 5 mM), l 100 mM potassium cyanide (ultimate concentration: 10 mM), 20 10.8 mM

20 pyridoxal phosphate (ultimate concentration: 0.08 mM), and 50 l enzyme solution at 45 C for 10 minutes, discontinuing the reaction by placing the mixture in boiling water bath for two minutes, then subjecting a supematant centrifugally separated at 10,000 rpm for five minutes to HPLC, and measuring y-cyano-a-aminobutyric acid produced through the enzymatic reaction.
As the unit for enzyme activity, the enzyme activity of producing I
mol y-cyano-a-aminobutyric acid in a minute under the following conditions was defined as a unit. The conditions for HPLC was:

Trade-mark Column: INERTSIL# ODS-2 (4.6 mm inside diameter x 250 mm;
made by G.L. Science Company), and Eluent: 20 mM sodium phosphate buffer solution (pH 6.8)/ acetonitrile (85:15).

' Trade-mark 'd O r! ON CN 'r?
00 It kn .,..i b w ~ N ~ M N O
M ~G M M
M -+ C~ V 1 \O ~!1 .,., ['- ~ N t- 00 V'1 N ~
N O O r~
v~ C O O N

~
~

C~
~ O 0o O O 00 ~ ~, M M N M N
O
E~

O 0 O C>
O Nt 01 F C~ CN ~ N 00 O
H b o ~
C) O U
sz, a~i ~ ; ~ p C~ ~ o U x p~ G a c~ v~ E-4 ~
O
=-~ c i ri d v~ oo Further, for the y-cyano-a-amino-butyric acid synthase obtained, tests were carried out on optimum pH, stable pH, optimum temperature, thermo-stability and molecular weight.
1. Optimum pH:
Enzyme activity was measured by replacing the buffer of reaction solution for measuring activity in the enzyme activity measuring method described above with MES (pH: 6.0 to 7.0), KPB (pH: 6.0 to 8.0), MOPS (pH: 6.5 to 7.5), Tris-HCl (pH: 7.5 to 9.0), and NH4C1-NH4OH (pH: 8.5 to 10.0). The results are as shown in Fig. 8: the optimum pH of this y-cyano-a-aminobutyric acid synthase was found to be within a range of from 7.5 to 8.5.
2. Stable pH:

The y-cyano-a-aminobutyric acid synthase was dissolved in various mM concentration buffer solutions, i.e., citric acid/sodium citrate (pH: 3.5 to 5.5), MES (pH: 6.0 to 7.0), KPB (pH: 6.0 to 8.0), Tris-HCl (pH: 7.5 to 9.0), 15 NH4C1-NH4OH (pH: 8.5 to 10.0), and glycine/KCl-KOH (pH: 10.0 to 10.5), respectively, and residual activity after holding at 60 C for 30 minutes was measured. The results are as shown in Fig. 9: the stable pH for this y-cyano-a-aminobutyric acid synthase was found to be within a range of from 6.0 to 10.5.
3. Optimum temperature:
20 The y-cyano-a-aminobutyric acid synthase was dissolved in a 20 mM potassium phosphate buffer solution (pH: 7.5), and enzyme activity was measured within a temperature range of from 30 C to 70 C by the enzyme activity measuring method described above. The results are as shown in Fig. 10: the optimum temperature for this y-cyano-a-aminobutyric acid synthase was found to be within a range of from 55 to 65 C.

4. Thermostability:
The y-cyano-a-aminobutyric acid synthase was dissolved in a 20 mM potassium phosphate buffer solution (pH: 7.5), and after holding at each of various temperatures of from 45 C to 90 C or 30 minutes, residual activity was measured. The results are as shown in Fig. 11: the y-cyano-a-aminobutyric acid synthase was found to have a very high thermostability, and to be stable at temperatures up to 65 C.
5. Molecular weight:
The molecular weight of the thermostable y-cyano-a-aminobutyric acid synthase was measured by gel filtration and SDS-PAGE. The results are as shown in Figs. 12 and 13: the molecular weight was confirmed to be about 43 kDa (Fig. 12) as measured by the SDS-PAGE, and about 180 kDa (Fig. 13) as measured by gel filtration.
6. Absorption spectrum:
The thermostable y-cyano-a-aminobutyric acid synthase was dissolved in a 20 mM potassium phosphate (pH: 7.5) containing 0.1 mM
dithiothreitol and 0.01 mM pyridoxal phosphate, and absorption spectrum was measured on this solution by means of a U-3200 type spectrophotometer (made by Hitachi Ltd.). The results are as shown in Fig. 14: for the thermostable y-cyano-a-aminobutyric acid synthase of the present invention, absorption was observed within a range of from 410 to 440 nm, which is intrinsic to an enzyme utilizing pyridoxal phosphate as coenzyme.

H"CN was manufactured by reducing 11 C containing positron nuclide 11C prepared in cyclotron into 11CH4 at 400 C in the presence of Ni in a mixed atmosphere of H2 and N2, and contact reacting the resultant "CH4 with ammonia at a temperature of 1,000 C in the presence of a platinum (Pt) catalyst.
This process was based on a known method (Iwata et al. Appl. Radiat. 38, 97, 1987). Then, H"CN in the form of a mixed gas was passed through a 50% H2SO4 solution in an amount of 1.5 ml to remove residual ammonia, and after further removing ammonia by bringing same into contact with P205, H11 CN was trapped with 50 mM KOH in an amount of 350 l.
Then, 250 l 200 mM K2HPO4, 10 l 10 mM pyridoxal phosphate (PLP), 110 l 25 mM O-acetyl-L-homoserine (OAHS) dissolved in 100 mM
K2 HPO4, and y-cyano-a-aminobutyric acid synthase (GCAs) obtained in Example 5 were added to this trapped H11CN, and the mixture was subjected to enzymatic reaction at 65 C for 10 minutes.
The reaction solution was analyzed with UV 220 nm and a radiodetector by means of HPLC. This reaction product showed the same retention time as that of the standard y-cyano-a-aminobutyric acid, and was confirmed to be labelled with positron nuclide "C. The y-cyano-a-aminobutyric acid synthesized by the enzymatic reaction had a radiochemical yield (corrected decay value) of 93.99%.

NaOH of 2.5 M was added to a reaction solution containing the y-cyano-a-aminobutyric acid of which cyano group carbon being labelled with positron nuclide 11C, as obtained in Example 6, and temperature of the mixture was raised to 135 C. After the lapse of 15 minutes, the reaction solution was mixed with 8 ml 50 mM NaH2PO4, and the resultant mixture was passed through an anion exchange resin (800 mg AGI-x8 200-400 mesh hydroxide form). After washing with 50 mM NaH2PO4 in an amount of 6 ml, the reaction product was eluted with 150 mM NaH2PO4 (pH adjusted to 2.8 with phosphoric acid), and the elute was received in a receptacle containing 150 l 8.5% phosphoric acid. The contents were passed through a sterilized filter having a pore diameter of 0.2 pm for collection in bial. The product was germ-free, and no exothermic substance was detected.
Figs. 15 and 16 illustrate the results of HPLC analysis carried out by admixing standard glutamic acid to the above-mentioned reaction product. The analysis shown in Figs. 15 (a) and (b) was carried out under the following conditions:
Column: LC-NH2 4.6 x 250 mm 5 m;
Eluent: 10 mM KHZPO4/CH3CN, linear gradient 15/85 to 80/20, 0-7 min.;
Flow rate: 1 mUmin.;
Detection: UV 220 nm and Radiodetector; and Temperature: Room temperature.

The conditions for analysis of Figs. 16 (a) and (b) were as follows:
Column: Beckman CX 4.6 x 250 mm;
Eluent: 10 mM KH2PO4/CH3CN (95/5);
Flow rate: 2 ml/min.;
Detection: UV 210 nm and Radiodetector; and Temperature: Room temperature.
As is clear from these results of analysis, peaks were in agreement between the labelled compound and the standard glutamic acid under different conditions using two different columns. It was thus confirmed that the above-mentioned reaction product was 11C-labelled glutamic acid.
The resultant "C-labelled glutamic acid had a radiochemical yield (decay corrected value) of about 50% and a radiochemical purity of at least 95%.
Fig. 17 shows the results of measurement of optical purity of the "C-labelled glutamic acid, in which (a) is a chart of HPLC in the case where a racemi standard glutamic acid is converted into a derivative, and (b) gives the result of analysis carried out by similarly converting the enzymatically synthesized "C-labelled glutamic acid into a derivative. The analytic conditions were as follows:
Column: Beckman ODS (C-18) 4.6 x 250 mm 5 m;
Eluent: 0.05 M ammonium formate, (pH: 3.5)/Methanol, linear gradient 55/45 to 40/60, 0-6 min.;
Flow rate: 2 ml/min.;
Detection: UV 340 nm and Radiodetector; and Temperature: Room temperature.
It was confirmed from these results that the 11C-labelled glutamic acid was of the L-form. The method for conversion into derivative of glutamic acid was in accordance with Marfey's et al (Determination of enantiomenic excess: Determination of D-amino acid. II. Use of a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene, Marfey P. Cawrlsberg Res. Commun., 49, 591, 1984).

Claims (9)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. Labelled compounds consisting of .gamma.-cyano-.alpha.-aminobutyric acid, salts thereof or derivatives thereof having a protecting group, wherein the carbon atom of the cyano group is labelled with 11C.

2. A method for manufacturing the labelled compounds of claim 1, which comprises reacting an amino acid of formula (3), a salt thereof or a derivative thereof having a protecting group:

wherein, R1 is a hydrogen atom, a halogen atom, a hydrocarbon group, an oxygen-containing group or a sulfur-containing group, with a cyanic compound having a cyano group carbon labelled with 11C in the presence of a thermostable .gamma.-cyano-.alpha.-aminobutyric acid synthase.

3. The method of claim 2, wherein said thermostable .gamma.-cyano-.alpha.-aminobutyric acid synthase is an enzyme isolated from a bacterium of Bacillus.

4. The method of claim 3, wherein said thermostable .gamma.-cyano-.alpha.-aminobutyric acid synthase has the following properties:

(1) action : synthesizing .gamma.-cyano-.alpha.-aminobutyric acid from O-acetyl-L-homoserine and a cyanic compound;
(2) pH: 7.5 to 8.5;
(3) stable pH: 6.0 to 10.5;

(4) optimum temperature : 55 to 65°C;

(5) thermostability: stable at temperatures of up to 65°C when holding at pH 7.5 for 30 minutes; and (6) molecular weight: about 180,000 with gel filtration.

5. The method of claim 3 or 4, wherein said bacterium is Bacillus stearothermophilus CN3 deposited at National Institute of Bioscience and Human Technology deposit No. FERM BP-4773.

6. The method of any one of claims 2 to 5, wherein R1 is -O-acyl group, -O-alkyl group, -O-phosphoryl group or halogen atom.

7. Labelled compounds consisting of amino acids of formula (4), salts thereof or derivatives thereof having a protecting group:

wherein R2 is -11CONH2, or -11COOH.

8. Labelled compounds as defined in claim 7 combined with a biopolymer or a derivative thereof.

9. A method for manufacturing the labelled compounds of claim 7, comprising the step of organically or enzymatically synthesizing said labelled compounds by using, as an intermediate, .gamma.-cyano-.alpha.-aminobutyric acid, a salt thereof or a derivative thereof having a protecting group, of which cyano group carbon is labelled with 11C.