JPH11310594A - Synthesis of peptide and protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain a peptide and a protein by reacting an amino acid, a peptide, a protein, etc., as a raw material in a supercritical fluid of such as carbon dioxide or the like, a high-pressure gas, a liquid or a mixed fluid of a solvent, a gas, etc., by using a supporting medium. SOLUTION: In synthesizing a peptide, a protein or a compound containing them by using an amino acid, a peptide, a protein and a chemical species containing them in the structure as a raw material in a supercritical fluid of such as carbon dioxide or the like, a high-pressure gas, a liquid or a mixed fluid or them, another solvent and a chemical species such as a gas or the like by using a supporting medium such as a polymer or the like, at least one of pressure, a temperature and a composition used is controlled so that the properties of a peptide, a protein and a compound containing them to be synthesized are controlled to efficiently synthesize the objective peptide and protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for synthesizing peptides, proteins and compounds containing them, and relates to a method for synthesizing supercritical fluids such as carbon dioxide, high-pressure gas and liquid, and chemical species such as these and other solvents and gases. The present invention relates to a method for synthesizing a compound containing a peptide and a protein in a mixed fluid using a support such as a polymer.

[0002]

2. Description of the Related Art With the development of biotechnology in recent years, attention has been paid to physiologically active substances such as peptides and proteins contained in natural products. However, physiologically active substances in natural products are often contained in compounds having extremely similar physicochemical properties, and depending on the target substance, it is often not possible to perform appropriate high concentration.

In that case, a method using an appropriate synthetic chemistry technique is an effective means. In particular, the process of industrially producing large quantities of peptides and proteins that have been used as food and industrial materials for a long time is regarded as important, and many synthetic methods have been reported (Izumiya et al., “Peptide Synthesis”, Maruzen (1975)).

[0004] In general, methods for synthesizing peptides and proteins are classified into a liquid phase method and a solid phase method. What is the liquid phase method?
A method for synthesizing amino acids and peptides as raw materials for peptides and proteins in a solvent in an organic solvent, which enables the synthesis of a high-purity target substance, but is difficult depending on the difficulty of mass production or the target substance. It is necessary to select a suitable solvent and reaction system.

On the other hand, a solid phase method (RBMerrifield, J. Amer.
Hem. Soc., 85, 2149 (1963)) is a method for synthesizing peptides and proteins by sequentially introducing amino acids into a polymer carrier such as styrene. The solid phase method is used industrially because the synthesis method is easier than the liquid phase method and mass production is possible. However, in the solid phase method, since a large amount of residues are generated in the synthesis process, it is necessary to combine an appropriate purification method in order to recover a high-purity target substance as in the case of using the liquid phase method. In addition, high-performance liquid chromatography is often used as a laboratory-level purification method.
It is not practical because harmful organic solvents are used and recovery from the eluent is difficult.

[0006] These liquid phase method and solid phase method are generally
Since the reaction is performed in an organic solvent such as dimethylformamide, very large energy is required to recover the target substance from such a reaction medium. Further VOC
There have been many problems in the treatment of the solvent that evaporates in the desolvation step due to problems such as regulations.

On the other hand, unlike a conventional synthesis reaction in an organic solvent, a method of using a supercritical fluid or a high-pressure gas as a reaction medium has attracted attention. In particular, carbon dioxide used as a supercritical fluid is generally harmless and has a critical temperature of 30.
Since the temperature is 4.2K, which is close to room temperature, many research reports have been made. By using a supercritical fluid as a reaction medium, many studies on enzyme reactions and non-catalytic reactions in supercritical carbon dioxide have been carried out because the reaction can be controlled only by pressure operation and there is no problem with residual solvents. Is being done.

For example, a radical polymerization reaction of an acrylic ester having a fluoroalkyl group in supercritical carbon dioxide (DeSimone et al., Science, 265, 356 (1994)), a noncatalytic hydrogenation reaction (Jessop et al., Nature) , 368, 231 (19
94)), enzyme reaction (O. Aaltonen and M. Rantakyla, CHEMT
ECH, 4, 240 (1991)).

[0009] However, there has been no report that a supercritical fluid such as carbon dioxide, a high-pressure gas or the like has been applied to a method for synthesizing peptides and proteins which are ecologically related substances and compounds containing them.

[0010]

DISCLOSURE OF THE INVENTION The present invention relates to peptides, proteins and compounds containing them, which have been carried out on a polymeric support in an organic solvent which does not adversely affect the environment and usually adversely affect the environment. To provide a method for obtaining peptides, proteins and compounds containing them by conducting a synthesis reaction of a compound in a supercritical fluid such as carbon dioxide, a high-pressure gas, a liquid, or a mixed fluid of these and other chemical species such as a solvent and a gas. The purpose is to do.

[0011]

SUMMARY OF THE INVENTION The gist of the present invention is to provide a method for preparing a supercritical fluid such as carbon dioxide, a high-pressure gas, a liquid and the like from an amino acid, a peptide, a protein and a chemical species containing the same in the structure. Synthesis of peptides and proteins using a resin support in a mixed fluid of chemical species such as solvents and gases.

[0012] The gist of the present invention is that in the above method,
In some cases, by controlling at least one of the working pressure, temperature and composition, the properties of the peptides and proteins to be synthesized and the compounds containing them are controlled.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The amino acids and peptides used as raw materials in the present invention and the chemical species containing them in the structure are compounds containing at least one amino acid and peptide which are constituents of peptides and proteins. means.

The amino acids and peptides used as the starting materials and the amino acids and peptides contained in the chemical species containing them in the structure are not particularly limited, and are generally used as food additives or industrial materials. And the like. Examples include amino acids such as alanine (Ala), phenylalanine (Phe), leucine (Leu), aspartic acid (Asp), serine (Ser), tyrosine (Tyr), and glycine (Gly), and peptides composed of these amino acids. be able to.

In the present invention, peptides and proteins are dissolved in a mixed fluid of a supercritical fluid, a high-pressure gas, a liquid, and a chemical species such as those and other solvents and gases. The supercritical fluid, high-pressure gas, and liquid are not particularly limited, and are compounds known to have utility as supercritical fluids such as carbon dioxide, methane, ethane, propane, ethylene, and ammonia. The following additives can be added to the above-mentioned supercritical fluid, high-pressure gas, and liquid as long as the novelty of the present invention is not impaired: alcohols; for example, methanol, ethanol, 1-propanol and other aliphatic alcohols Ketones; for example, acetone, methyl ethyl ketone, cyclohexanone and other aliphatic ketones, aromatic hydrocarbons; for example, benzene, toluene, xylene, tetrahydrafuran, dimethylformamide and other aromatic solvents.

The chemical species of the above-mentioned supercritical fluid, high-pressure gas, liquid, and other solvents and gases are different from dimethylformamide and tetrahydrafuran, and include amino acids and peptides after the reaction and compounds containing them. There is no need to worry about the environment because it is easy to recover without deactivation.

One of the features of the present invention is to synthesize peptides, proteins and compounds containing them in a supercritical fluid. Furthermore, one of the characteristic features of the present invention is to synthesize peptides, proteins, and compounds containing them in a high-pressure gas or a liquid at a pressure higher than normal pressure, which does not become a supercritical fluid in relation to temperature and pressure. .

In the present invention, a peptide, a protein and a supercritical fluid such as carbon dioxide, a high-pressure gas and a liquid, and a support such as a polymer are used in a mixed fluid of a chemical species such as a solvent and a gas. Compounds containing them are synthesized. As the synthesis device used in the present invention, for example, a device using a supercritical fluid as shown in FIG. 1 can be used. The synthesizing device includes a boosting section from the cylinder 1 to the stop valve V2 and a synthesizing section downstream thereof.

The pressurizing section has two pressurizing pumps 4A and 4B for a carbon dioxide as a supercritical fluid or the like and an additive solvent. The cylinder 1 is a pump 4A for pressurizing liquid carbon dioxide.
A siphon-type carbon dioxide cylinder was used to send the gas to the factory. The drying tube 2 was placed between the cylinder and the pump in order to remove water in the liquid carbon dioxide sent from the cylinder.
Drying tube specifications are material SUS316, maximum working pressure 20MP
a, inner diameter 35.5 mm, length 310 mm. Also,
Molecular sieve 5A (1/16 inch Pellet) manufactured by GL Science Co., Ltd. was used as a desiccant. The liquid carbon dioxide from which water has been removed by the drying tube is cooled by ethylene glycol kept at about −5 ° C. by a cooling unit 5 (BL-22 manufactured by Yamato Scientific Co., Ltd.) and sent to a pressure increasing pump 4A (gas supply pump). Can be In this apparatus, liquid carbon dioxide is used as the solvent. However, the substance used as the solvent can be appropriately changed by replacing the cylinder of the desired substance with the cylinder 1.

Supply pumps 4A and 4 for gas and additive solvent
B is a plunger pump for high pressure manufactured by JASCO Corporation
880-PU (maximum pressure 350 kgf · cm ² , normal pressure 300 kgf · cm ² , flow rate 0.01 to 10.0 ml · min ⁻¹ ) was used. A cooling unit is mounted on the head of the pump 4A to prevent the vaporization of liquid carbon dioxide. The additive solvent was placed in the sample bottle 17 and installed upstream of the additive solvent pump 4B. The added solvent liquid sent out by the pump 4B is a stop valve V
3 and before the stop valve V2, it is mixed with the pressurized carbon dioxide and supplied to the column separation unit. Further, a stop valve V4 was provided for discharging the additive solvent. As a stop valve, a bonnet integrated flow control stop valve SS-OKS2BKB made by Whity was used. Further, in order to prevent impurities such as dust from being mixed between the drying tube 2 and the cooling unit 5, FT4-10 type manufactured by GL Science Co., Ltd. was used as the filter 3. The average pore size of the filter is about 10 μm.

The pressure in the system is set to an arbitrary pressure by the expansion valve 12. The expansion valve used was 880-81 manufactured by JASCO Corporation. This valve can control the pressure in the system at ± 0.1 MPa, and the maximum working pressure is 350 kgf · cm 2. The pressure inside the system is a Bourdon-type pressure gauge 5A (GL Science Co., Ltd.)
LCG-350; maximum operating pressure 34.3 MPa). This pressure gauge was provided with an upper limit contact output terminal, and was set so that the power supply of the pressure increasing pump 4A was turned off at a specified pressure. Also,
This pressure gauge was verified by using the economy pressure gauge PE-3 manufactured by Shisoken Co., Ltd.
3-A (strain gauge type, accuracy ± 0.3%) was used.

In order to control the pressure of the boosting section, a stop valve V2 was provided between the boosting section and the synthesizing section. The stop valve is GL Science's 2 Way Valve 02-0
120 (maximum operating pressure 98.0 MPa) was used. In addition, a safety valve 6 was provided between the booster and the synthesizer to ensure safety. The safety valve is a Nupro spring type, and is adjusted and verified so that the pressure in the system operates at 34.3 MPa. The pipe used was a 1/16 inch stainless steel pipe (material: SUS316, outer diameter 1.588 mm, inner diameter 0.8 mm).

The synthesis column 10 is provided with an air oven 1 in the apparatus.
6. The internal volume of the air bath is 20 liters, and the temperature controller DB1000 manufactured by Chino Co., Ltd.
The temperature can be controlled at the measured temperature ± 0.1K. For the temperature measurement part, a platinum resistance thermometer 1TPF483 made by Chino was used. The liquid carbon dioxide and the eluent supplied from the pressure increasing section are sent to the preheating column 8.

The column is filled with glass beads, and 9-fluoromethoxycarbonyl-phenylalanine-alcohol resin (Fmoc-Phe-Alko Resin, Watanabe Chemical Co., Ltd.) in which 9-fluoromethoxycarbonyl is supported on styrene resin
1 g) was added.

The preheating column 8 is for heating a solvent (carbon dioxide or the like) to an equilibrium temperature to make it a supercritical fluid. A 1/16 inch stainless steel tube (about 4 m long) having a diameter of 55 mm and a length of 140 mm is used. Deform into a spiral shape,
It was installed in a thermostat. The carbon dioxide which has been converted into a supercritical fluid by the preheating column 8 is mixed with the added solvent together with the hexagonal valves 7 and 9.
(Rhodine injector 7125 manufactured by JASCO Corporation: maximum working pressure of 490 kgf · cm ² ), and introduced into the synthesis column 10 (stainless steel column 954R manufactured by JASCO Corporation). In addition, the column 10 was installed in the six-way valve 9, and the pipes could be washed by switching the six-way valve 9 except during the synthesis operation using the synthesis column 10. Also, stop valves V5 and V6 were installed to discharge the pressure in the column.

As the stop valves V5 and V6, a bonnet-integrated flow control stop valve SS-OKS2BKB made by Whity was used. The pressure in the column was measured with a pressure gauge 13
And adjusted to the set pressure by the expansion valve 12. The pressure gauge is of the Bourdon type, using LCG-350 (maximum working pressure: 34.3 MPa) manufactured by GL Sciences Co., Ltd. The pressure gauge is verified by an economy pressure gauge PE-3 manufactured by Shisoken Co., Ltd.
3-A (strain gauge type, accuracy: ± 0.3% FS, FS: kgf · cm ^-2 ) was used. Further, a safety valve 6 was installed on the upstream side of the synthesis column 10 for the purpose of preventing explosion due to pressure increase in the column.
The safety valve 6 has a NUPRO spring type, 177-R3AKI-G
The pressure in the system was adjusted and operated so as to operate at 34.3 MPa. The supercritical fluid (carbon dioxide) in which the sample was dissolved was discharged out of the system using the expansion valve 12.

The flow rate of the supercritical fluid was adjusted by the expansion valve 12, and the pressure was reduced. Further, a heater was installed in the expansion valve 12 in order to prevent the sample from condensing due to the pressure reduction and the generation of dry ice due to the supercritical fluid (carbon dioxide). Further, by making the outlet of the tube a vibrating type, it is possible to prevent the tube from being clogged by the precipitate. After the pressure was reduced by the expansion valve 12, the precipitated sample was collected by the trap 14. The flow rate of the supercritical fluid was measured by the flow meter 15. As a flow meter, an integrating wet gas meter W-NK-0.5B (measurement accuracy 0.1 ml) of Shinagawa Seiki Co., Ltd. was used. In addition, peptides and proteins synthesized in the synthesis column 10 and compounds containing them are supplied with a high-pressure ultraviolet detector 11 (UV-9) manufactured by JASCO Corporation.
70) can also be used for detection.

[0028]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples. In the following examples, a supercritical chromatograph configured as described above (Super-20 manufactured by JASCO Corporation) was used.
0) was used.

Example 1 Leucine-phenylalanine (Leu-phenylalanine) which is a dipeptide synthesized using the above apparatus
Examples for synthesizing Phe) are shown below.

The high-pressure cell was filled with glass beads, and 9-fluoromethoxycarbonyl-phenylalanine-containing styrene resin carrying 9-fluoromethoxycarbonyl was used.
Alco resin (Fmoc-Phe-Alko Resin, Watanabe Chemical Co., Ltd.)
1 g), V2 was opened, carbon dioxide was introduced into the cell, and the pressure inside the cell was set to 20 MPa. At this time, the upper limit pressure was adjusted by the expansion valve 12. At this time, the air bath 1
6 was adjusted to 35 ° C., and the expansion valve 12 was adjusted to 80 ° C.

Thereafter, in order to deprotect 9-fluoromethoxycarbonyl, 6 ml of a 20% piperidine / dimethylformamide solution was introduced into the high-pressure cell using the pump 4B. Further, in order to cause a coupling reaction of leucine with phenylalanine, a 5% leucine / dimethylformamide solution was introduced into the high-pressure cell using pump 4B.

To separate the synthesized leucine-phenylalanine from the resin, 10 ml of a 20% piperidine / dimethylformamide solution and 0.1 ml of a 0.1% phenol / trifluoroacetic acid solution were introduced using a pump 4B. Thereafter, leucine-phenylalanine synthesized from the expansion valve 12 was recovered.

Leucine synthesized in supercritical carbon dioxide
Phenylalanine was analyzed using high performance liquid chromatography. The analysis results are shown in FIG. Leucine-phenylalanine synthesized from FIG. 2 was confirmed to have a peak at the same retention time as the leucine-phenylalanine standard reagent, indicating that the peptide leucine-phenylalanine was synthesized in supercritical carbon dioxide. Was.

Example 2 As in Example 1, leucine-
The synthesis of phenylalanine was performed at a pressure of 0.1 to 19.8 MPa.
It went in the range of. FIG. 3 shows the relationship between yield and pressure of leucine-phenylalanine, which is a dipeptide synthesized at a pressure in the range of 0.1 to 19.8 MPa. As can be seen from the figure, the higher the pressure during the synthesis, the higher the yield is obtained, which is preferable from the viewpoint of economic efficiency. In this example, the synthesis was performed over the critical pressure of carbon dioxide. However, it was confirmed that leucine-phenylalanine can be synthesized even in the state of a high-pressure gas of about 7.4 MPa (73 atm) or less, which is the critical pressure of carbon dioxide. it can.

Example 3 As in Example 1, leucine-
The synthesis of phenylalanine was carried out at a dimethylformamide concentration of 20-80 wt. % In a mixed fluid of supercritical carbon dioxide and dimethylformamide. FIG. 4 shows a dimethylformamide concentration of 20 to 80 wt. 5 shows the relationship between the yield of the dipeptide leucine-phenylalanine synthesized in the range of% and pressure. This figure shows that adding dimethylformamide to supercritical carbon dioxide gives a higher conversion. In addition, the dimethylformamide concentration was adjusted to 50 wt. % Is preferable in terms of economic efficiency.

Example 4 As in Example 1, leucine-phenylalanine was synthesized using supercritical ethane and supercritical ammonia instead of supercritical carbon dioxide. The reactants synthesized using supercritical ethane and supercritical ammonia were analyzed using high performance liquid chromatography. As a result, the same peak as in the case of synthesis using supercritical carbon dioxide was obtained, indicating that leucine-phenylalanine could be synthesized. The supercritical ethane and ammonia have a dimethylformamide concentration of 50 wt. %
Is preferably set from the viewpoint of economic efficiency.

Example 5 An example of synthesizing trileucine-phenylalanine (Leu-Leu-Leu-Phe) is described below.

A high-pressure cell was filled with glass beads, and 9-fluoromethoxycarbonyl-phenylalanine-containing styrene resin carrying 9-fluoromethoxycarbonyl was used.
Alco resin (Fmoc-Phe-Alko Resin Watanabe Chemical Co., Ltd.)
Was added, V2 was opened, carbon dioxide was introduced into the cell, and the pressure in the cell was set to 20 MPa. At this time, the upper limit pressure was adjusted by the expansion valve 12. At this time, 3
The temperature of the expansion valve 12 was adjusted to 5 ° C and the temperature of the expansion valve 12 to 80 ° C.

Thereafter, in order to deprotect 9-fluoromethoxycarbonyl, 6 ml of a 20% piperidine / dimethylformamide solution was introduced into the high-pressure cell using the pump 4B. Further, in order to cause a coupling reaction of leucine with phenylalanine, a 5% solution of 9-fluoromethoxycarbonyl-leucine / dimethylformamide hydroxide was introduced into the high-pressure cell using pump 4B.

In order to couple leucine to leucine-phenylalanine formed on the resin in the high-pressure cell at this time, pump 4B was used to deprotect 9-fluoromethoxycarbonyl-leucine. Then, 6 ml of a 20% piperidine / dimethylformamide solution was introduced into the high-pressure cell. Then, pump 4
Using B, a 5% solution of 9-fluoromethoxycarbonyl-leucine hydroxide / dimethylformamide was introduced into a high-pressure cell to perform a coupling reaction. By repeating the same operation several times, trileucine-phenylalanine was synthesized on a styrene resin.

In order to separate the synthesized trileucine-phenylalanine from the resin, 10 ml of a 20% piperidine / dimethylformamide solution and 0.1% phenol / trifluoroacetic acid solution were introduced by using a pump 4B. Thereafter, trileucine-phenylalanine synthesized from the expansion valve 12 was recovered.

Trileucine-phenylalanine synthesized in supercritical carbon dioxide was analyzed using high performance liquid chromatography. The analysis results are shown in FIG. The peak of trileucine-phenylalanine synthesized from FIG. 5 could be confirmed at a retention time equivalent to that of the standard reagent of trileucine-phenylalanine, and thus, the peptide trileucine-phenylalanine could be synthesized in supercritical carbon dioxide. It has been shown.

Example 6 An example is shown below for the case of synthesizing the peptide alanine-glycine-phenylalanine (Ala-Gly-Phe).

9-fluoromethoxycarbonyl-glycine-phenylalanine-alco-resin (Fmoc-Gly-Phe-Alko Resin, Watanabe Chemical Co., Ltd.) in which glass beads are filled in a high-pressure cell and 9-fluoromethoxycarbonyl is supported on styrene resin 1) was added, and V2 was opened to introduce carbon dioxide into the cell, and the internal pressure of the cell was set to 20 MPa. At this time, the upper limit pressure was adjusted by the expansion valve 12. At this time,
The temperature of the air bath was adjusted to 35 ° C, and the temperature of the expansion valve 12 was adjusted to 80 ° C.

Thereafter, in order to deprotect 9-fluoromethoxycarbonyl, 6 ml of a 20% piperidine / dimethylformamide solution was introduced into the high-pressure cell using the pump 4B. Further, in order to cause a coupling reaction between alanine (Ala) and glycine-phenylalanine (Gly-Phe), a 5% leucine / dimethylformamide solution was introduced into the high-pressure cell using the pump 4B.

To separate the synthesized alanine-glycine-phenylalanine from the resin, 10 ml each of a 20% piperidine / dimethylformamide and 0.1% phenol / trifluoroacetic acid solution were introduced using a pump 4B. Thereafter, alanine-glycine-phenylalanine synthesized from the expansion valve 12 was recovered.

Alanine synthesized in supercritical carbon dioxide
Glycine-phenylalanine was analyzed using high performance liquid chromatography. Synthesized alanine-glycine-
The peak of phenylalanine was confirmed at the same retention time as the standard reagent of alanine-glycine-phenylalanine, indicating that the peptide alanine-glycine-phenylalanine could be synthesized in supercritical carbon dioxide.

[0048]

According to the present study, according to the present invention, a support such as a polymer is used in a supercritical fluid such as carbon dioxide, a high-pressure gas, a liquid, and a mixed fluid of these and other chemicals such as a solvent and a gas. , Peptides, proteins and compounds containing them. In addition, the amount of the organic solvent can be reduced, and the properties of the peptide and protein to be synthesized and the compound containing them can be controlled by controlling the working pressure, temperature and composition.

[Brief description of the drawings]

FIG. 1 shows a prototype for synthesizing peptides and proteins using a support such as a polymer in a supercritical fluid, a high-pressure gas, a liquid, or a mixed fluid of a chemical species such as those and other solvents and gases according to the present invention. It is a figure showing an experimental device.

FIG. 2 is a high-performance liquid chromatogram of leucine-phenylalanine (Leu-Phe), a peptide synthesized in supercritical carbon dioxide at 35 ° C. and 20 MPa.

FIG. 3. Synthetic leucine when pressure is changed.
It is a relationship between the yield of phenylalanine (Leu-Phe) and pressure.

FIG. 4 shows the relationship between the yield of leucine-phenylalanine (Leu-Phe) synthesized in a mixed fluid of supercritical carbon dioxide and dimethylformamide and the composition of the mixed fluid.

FIG. 5 Trileucine-phenylalanine (Leu-Leu-Le) synthesized in supercritical carbon dioxide at 35 ° C. and 20 MPa.
u-Phe) is a high performance liquid chromatogram.

[Explanation of symbols]

 1: cylinder 2: drying tube 3: filter 4A, 4B: liquid sending pump 5: cooler 6: safety valve 7: 6-way valve 8: residual heat tube 9: 6-way valve 10: synthesis column 11: ultraviolet detector 12: expansion valve 13 : Pressure gauge 14: Trap 15: Flow meter 16: Air bath 17: Sample bottle V1 to V6: Stop valve

Claims (2)

[Claims] 1. An amino acid, a peptide, a protein and a chemical species containing them in a structure as a raw material, a supercritical fluid such as carbon dioxide, a high-pressure gas, a liquid, and a mixture thereof with a chemical species such as another solvent or gas. A method for synthesizing peptides, proteins and compounds containing them in a fluid using a support such as a polymer. 2. In synthesizing peptides, proteins and compounds containing them, the properties of the peptides, proteins and compounds containing them are controlled by controlling at least one of working pressure, temperature and composition. The method according to claim 1, wherein: