The method of producing peptides and device for its implementation

 

(57) Abstract:

The essence of the invention: a method of producing peptides by synthesis from a solution containing as components of amino acids, in which the pre at least part of the components, for example, by changing the pH of the solution was transferred to ionized state, and then the solution is subjected to the simultaneous impact of cross between an electric field and a magnetic field having a fixed and variable components, and the frequency of the variable component of the magnetic field corresponds to the natural frequency cycloidal motion of the components of the solution, and a device for implementing the method. 2 S. and 2 C.p. f-crystals, 1 Il.

The invention relates to biology, more precisely to a process for the production of peptides and devices for their implementation.

A method of obtaining peptides and a number of peptide hormones, enzymes and immune modulators by extraction from biological material (for example, A. Lehninger, "Biochemistry", Worth Rublishers, N. J. 1972).

The method is labor-intensive, the yield of the final product is 20-60% of the process of separating the final product from a variety of molecular impurities contained would obtain the peptide products of the synthesis of amino acids or by hydrolysis of the longer protein and peptide sequences under the action of proteolytic enzymes (Structural basis of action of peptide and protein immunomodulators, Ed. G. I. Chipens, Riga. Zinatne, 1990; Ovchinnikov, Y. A. Bioorganic chemistry, M. Education, 1987).

However due to the complexity of the control-oriented synthesis of the peptide sequence and complicated purification of the final product from enzymes and related impurity peptides the application of these methods is currently limited.

A method of obtaining a peptide and protein products through genetic engineering (albert A. Bray & Dlewis j and other Molecular biology. M. Mir, 1986). The method is targeted integration into the genome of the bacterium cell specific gene that encodes the synthesis of a given peptide or protein. Modified in this way, the cell begins to synthesize the required final product.

The application of this method is limited due to the complexity of the process, as well as the need for purification of the final products from other biological compounds, which are products of the metabolism of the bacteria.

The most widely currently used method of producing peptides by chemical synthesis of the peptide sequence ("The peptides, Analysis, Synthesis, Biology". Edited by Erhard Gross, Johahnes Meienhofed", Academic Press, N. Y. San Franc. London, 1979). Based spasivnenie amino acids to the growing peptide chain (Merrifield R. B. "Automated Synthesis of Peptides", Science, 1965, p. 150, 178-185).

Method methodologically complex, and involves constant monitoring of the content of amino acids in the reaction mixture. The need for intermediate purification allocated synthesized from a mixture of molecules of intermediate peptides for subsequent return them to the reaction mixture for further synthesis leads to a significant increase in the duration of the process of synthesis of the final product. Due to the fact that the way the reagents condensing agents, necessary to implement the reactions of formation of peptide bonds can react with groups of amino acids present in the mixture, but not participating in the formation of the desired peptide bonds, such groups of amino acids chemically "block", and then removed from the reaction mixture. For these reasons, described the synthesis of peptides is a multistage: block-condensation-release requires complex tooling, labor-intensive. On average, the synthesis of one peptide bond has a duration of about 3 hours, and the yield of the final product in one cycle is 50-90% of the dry matter of the original product.

A device for the synthesis of peptide the military means for feeding and selection of a solution of amino acids, dosed feed solution further components required for the implementation of the directed synthesis, and means to provide filtration of the solution in the process of synthesis(Merrifield, R. B. "Automated Synthesis of Peptides", Scince, 1965, p. 150, 178-185).

The basis of the invention is to create a method of producing peptides such physico-chemical conditions, which would increase the number of free ions of amino acids in the reaction mixture, increase of activity and to streamline the movement for the most effective use of fusion energy, as well as a device for implementing this method.

When creating a method of producing peptides was based on the principle of selectivity managed dehydration and activation of amino acid molecules in solution when combined impact on the solution of electric and magnetic fields in terms of a managed solution.

The problem is solved in that in the method of producing peptides by synthesis from a solution containing as components of amino acids, previously at least part of the components is transferred to the ionized state, and then the solution is subjected to simultaneous exposure screenname components, the frequency of the variable component corresponds to the natural frequency cycloidal motion components of the solution.

However the translation components in the ionized state is performed by changing the pH of the solution.

The task is solved in that the device for obtaining peptides containing the capacity to host the solution, provided with a means for feeding and selection of a solution with pH Adjuster solution and a source of a magnetic field formed by the two covering the container and placed coaxially one within the other magnetic coils, one of which is connected to a source of a constant and the other with a source of alternating current, and inside the tank has two connected to the current source electrode of a biologically inert material and located so that the generated electric field and the magnetic field is reduced among themselves and in this capacity made thermostatted.

For a better understanding of the proposed method let us consider first embodiment of the device proposed for its implementation, which is illustrated by a drawing.

Apparatus for producing peptides by the method according to the invention contains nl provided with means 2 for conveying solution from the connected with the tank 1 tank 3 and the means 4 for selecting the solution from the tank 1, connected to the tank 5 of the final product. 2 and 4 provide the application and selection solution using known means, for example by means of a pneumatic pump.

Capacity 1 performed thermally, for example by placing inside the tank 1 thermostat 6, for example, with a liquid coolant, such as water, feed rate and the temperature of which is regulated by the control block 7.

In the tank 1 is installed, the sensor 8 pH of the solution, coupled with the pH Adjuster solution containing block 9 management and capacity for 10 acid and 11 for lye.

The container 1 is placed inside arranged coaxially one within the other magnetic coils 12 and 13 of any known construction, the magnetic coil 12 is connected to the source 14 of direct current, and the magnetic coil 13 to the source 15 AC.

Inside the tank 1 set electrodes 16 of biologically inert material, such as gold or platinum, is connected to the source 17 DC.

Arrangement of electrodes 16 and the magnetic coils 12 and 13 provides the direction vectors of the electric and magnetic fields as follows: that creates the effect of a cross between elector the device may be surrounded by a protective screen 18, made for example of permalloy.

To ensure synchronous operation of the device according to the set program control device can be automated.

The proposed method of producing peptides is carried out in the proposed device, as follows.

Into the container 1 serves the original solution containing as components of amino acids, selected depending on the desired final product.

The original solution in the tank 1 is heated to the temperature necessary to start the process of ionization of amino acids.

In the original solution adjustable feed acid and alkali from the tanks 3 and 5 is set to the pH required for transfer in ionized state of the amino acids used for the synthesis of the initial a pair of a given peptide sequence.

To avoid making the end product more organic impurities, when adjusting the pH of the solution using an inorganic acid and alkali, for example, hydrochloric acid and caustic soda.

Then create an electric field by applying voltage to the electrodes 16 within the tank 1 and create a constant and alternating magnetic field by applying a DC and Panstwowa frequency cycloidal movement initially synthesized molecules of the components of the solution.

The solution is exposed to these fields for 15 minutes, then measure the pH of the solution and measure its flow in the solution is necessary dose of acid or alkali, ensuring that the pH required for the synthesis of the next component.

Then the solution is again subjected to the action of electric and magnetic fields by typing in the frequency of the variable component of the magnetic field harmonics own cycloidal movement of molecules attached component.

The process continues as described above to obtain a set of synthesized products of peptides of a given peptide sequence.

It is known that the level of the electric field strength should not exceed the values at which the insulation of the electrodes in the solution layer surrounding ions of solvent counterions, preventing the movement of ions dissolved components, in this case amino acids, in solution between the electrodes.

For example, used in the method of the electrodes of platinum or gold level of the electric field strength is from 0.1 to 10 V/m

It is known that in a magnetic field with the magnitude of induction of a constant magnetic field y component of the motion, that increases the likelihood of chaotic behavior of molecules in solution. If the value is greater than 100 T processes dissipation (dissipation) of energy by the movement of ions in solution increase faster than the processes of absorption by molecules the energy of the electromagnetic field required for synthesis, so in the way that an optimal range of values induction B0selected in the range from 10 to 100 t

The magnitude of induction of an alternating magnetic field is from 10 to 200 NT, and the electric signal of the alternating current creates an alternating magnetic field must contain frequency harmonics corresponding to its own cycloalkyl frequencies ionized molecules of the components of the solution, participating in the reaction. The determination of the values of cyclonic frequencies is carried out by the known formula

< / BR>
where q and m are the charge and mass of the ion component;

B0induction of a constant magnetic field.

The combination of the simultaneous effect of above fields on the components of the solution with heating and ionization of the solution creates the most favorable conditions for the flow of process of formation of peptide bonds.

It was established experimentally that such a large impact on amino acids is of Alakul peptides the time of exposure to electric and magnetic fields increase, pre-translating consistently attachable components in the ionized state by changing the pH of the solution, and add it to an electrical signal applied to the coil an alternating magnetic field, the frequency of the harmonic corresponding to its own cycloidal frequency attachable component.

At the end of the synthesis process, the solution is cooled and using a hand-held device in the vessel, select the final product.

Quantitative and qualitative characteristics of the final product was determined by chemical and physical peptides according to their types based on the data measured optical density of the peptide, as well as known methods of determining the amount of dry matter.

This implementation of the method of producing peptides can increase the number of free ions of amino acids in solution, activate it for the most effective synthesis, as well as by streamlining the movement of molecules of components in the solution to reduce the energy losses of the system.

The method is one that does not require intermediate purification of the components of the solution from impurities. The amount of impurities in the final product is low. The yield of the final product with the place about 20 minutes

Example 1. Obtaining peptide

NH2glutamic acid tryptophan (timogen).

For the synthesis of peptide as starting product solution was used amino acid glutamic acid (Glu) and tryptophan (Try) in distilled water: 210-5M dry matter of each amino acid in 10 ml of water.

the pH of the solution was removed by acidic side to pH of 3.9, then the solution tank warmed up to the 35oC.

Created electric field applying current to the plate electrodes, the field strength E 0,1 B/m

A constant magnetic field B0=30 T and AC magnetic field Bn=25 NT. The direction vectors

Electrical signal, generates an alternating magnetic field, contained harmonic frequencies 3, 11 and 2,33 Hz, corresponding eigenfrequenciesCcyclic movement of the ionized molecules of the amino acids Glu-and Try-+calculated by the earlier formula.

The solution was subjected to simultaneous action of electric and magnetic field for 10 minutes, then for 10 min reduced the temperature of the solution from 35 to 10oC and the final product of the synthesis was subjected to chemical analysis.

Provodili, S/N 7UEON494 using eluents A and B: eluent A 2.0 mm TEA-phosph, eluent B Acetonitril gradient feeder 10% B + 1% min.

The column was put in 50 ml of the final product, then carried out the registration of the optical density of eluent using ultrasonic detector Uvicord SD-2158 at 220 nm.

As a benchmark for comparison used the peptide timogen production company "Peptos Russia".

The results showed that the synthesized dipeptide-timogen, and the output of thymogen amounted to 60% of the dry matter of the peptide relative to the initial amount of dry matter components in the original solution. The total duration of 20 minutes

Example 2. Obtaining peptide NH2aspartic acid - asparagine

For the synthesis of peptide as the original product used equimolar solution of asparagine (ASP-) and aspartic acid (Asn+) in distilled water: 2,27 M dry matter of each component of the solution in 10 ml of water.

the pH of the solution was shifted to 3.2, then the solution was heated to 30oC.

Created an electric field E 0.5 V/m

A constant magnetic field B030 T AC and the magnet is Arial two harmonic frequencies 3,50 3,53 and Hz, the corresponding eigenfrequenciesCcycloidal movement of ionized molecules Asp-and Asp-and Asn+.

Subjected to the solution of the simultaneous action of electric and magnetic field for 15 min, then decreased for 8 minutes the temperature in the tank 1 from 30 to 10oC and the product of the synthesis was subjected to chemical analysis.

Spent the separation of the synthesized peptides similar to that described in example 1 at column Nucleosyl C18, 3 m 100x4,6 mm eluent QA composition of H2O 5% + Acetonitril + 0,1% triperoxonane acid (TFA) and eluent B composition Acetonitril + 0,1% TFA with a gradient feeder 5% B + 2,83/min. for a period of 7.5 min Suecia passed without a gradient, then to 37.5 min with gradient B.

The column was put in 50 ml of concentrated after gel chromatography in 10 times desalted fraction of the original component. Check the optical density of eluent was carried out similarly as described in example 1.

As a benchmark for comparison used the peptide aspartic acid-asparagine obtained previously described by chemical synthesis. The results showed that the synthesized peptide aspartic acid-asparagine, the yield of the final product was the 23 minutes

Example 3. Getting Tripeptide NH2-glutamic acid-arginine-tryptophan

For the synthesis of Tripeptide as the original product used 10 ml of an equimolar solution containing 210-5M glutamic acid (Glu), arginine (Arg) and tryptophan (Try) in distilled water. For ionization of Glu and Arg pH was shifted to 6.0. Created an electric field E 0.5 B/m is a constant field with induction B042,1 T and AC magnetic field Bn25 NT. Electrical signal, generates an alternating magnetic field, contained two harmonic frequency 3,39 and was 4.42 Hz, corresponding eigenfrequencies Cions Glu-and Arg+.

Subjected to the solution of the simultaneous action of electric and magnetic fields for 15 min, then changed the pH of the solution to 4.0 for translation in the ionized state of the molecules Try and continued exposure to an alternating magnetic field.

Into an electrical signal, generates an alternating magnetic field, added harmonic frequency of 3.14 Hz, corresponding to the natural frequencyCcycloidal movement of ionized molecules Try.

The influence of electric and magnetic field was continued for another 15 min, after CLASS="ptx2">

The separation of the synthesized peptides was carried out similarly as described in example 1 using eluents A and B, where A H2O+5% Acetonitril+0,1% TFA; B Acetonitril + 0,1% TFA with a gradient of filing of 99.9% Acetonitril + 2,83/min

500 l of the final product was evaporated to 50 l and applied to the column. Check the optical density of eluent was carried out similarly as described in example 1. As a benchmark for comparison used the Tripeptide Glu-Arg-Try by the well-known chemical methods. The results showed that the synthesized Tripeptide, corresponding to the accepted standard of the Tripeptide. The yield of the final product amounted to 60% of dry matter of the original components, the total duration of 38 minutes

Example 4. Getting tetrapeptide arginine-lysine-glutamic acid - tryptophan-Arg-Lys-Glu-Try

For the synthesis of peptide as the original product used two peptide Arg-Lys and Glu-Try in equimolar concentrations of 310-3/l (in 10 ml of water at 310-5M of each peptide). the pH of the solution was shifted to 7.0, the solution was heated to 30oC.

Created an electric field of E=2 B/m, constant magnetic field B042,1 mT and an alternating magnetic field with induction B

Subjected to the solution of the simultaneous action of electric and magnetic fields within 15 minutes

Then the solution was cooled to 10oC for 8 min and the product of the synthesis was subjected to chemical analysis, similar to that described in example 1. The results showed that the synthesized tetrapeptide Arg-Lys-Glu-Try. The yield of the final product was 61% of the dry matter of the original components, the duration of 23 minutes

The above-described embodiments of the invention are best for their industrial implementation, however, any well-versed in this field specialist clear that they can be made various improvements and modifications within the scope of the claims.

Example 5. Obtaining peptide NH2-Cys-Ser

For the synthesis of peptide as the original product used axiallary solution of amino acids Cys and Ser in phosphate buffer: 410-5M dry matter of each amino acid in 10 ml of buffer.

the pH of the solution to 5.5, then the solution was heated to 30oC. Created an electric field E 2 B/m, a constant magnetic field B030 T and an alternating magnetic field with indukti is 28 Hz, the corresponding natural frequencies VCcycloidal movement of ionized molecules ys-and Ser+.

Subjected to the solution of the simultaneous action of electric and magnetic field for 40 min, then decreased for 10 minutes the temperature in the tank 1 with 30 to 6oC and the product of the synthesis was subjected to chemical analysis.

Spent the separation of the synthesized peptides on the installation HPLC firm LKB-Pharmacia (Sweden) column Nucleosyl 5 M, 100x4,6 mm using eluents A and B: eluent A H2O + 5% Acetonitril + 0,1% TFA; B Acetonitril + 0,1% TFA with a gradient feeder 5% B + 2,83/min

As a benchmark for comparison used the peptide Cys-Ser, obtained by known chemical methods.

The results showed that the synthesized dipeptide Cys-Ser. The yield of the final product amounted to 24% of the dry matter of the original components.

Example 6. The example was carried out at the boundary and the mean values of the intervals of values of the effective factors in the synthesis of peptide timogen described in example 1:

when the value of electric field intensity E=10 V/m, the magnitude of the magnetic field B010 T and AC field Bn10 NT; as initial components used in the tion of each amino acid in 10 ml of water, the pH of 3.9; design of amino acids, pH and temperature conditions identical to those described in example 1;

when the value E=10 B/m; B0100 T and Bn200 HT output thymogen amounted to 4%

when the value E=5 B/m; B050 T and Bn100 HT output thymogen was 52%

1. The method of producing peptides by synthesis from a solution containing as components of amino acids, wherein the pre at least part of the components is transferred to the ionized state, and then the solution is subjected to the simultaneous impact of cross between an electric field and a magnetic field having a unidirectional constant and variable magnetic components, the frequency of the variable component corresponds to the natural frequency cycloidal motion components of the solution.

2. The method according to p. 1, characterized in that the translation components in the ionized state is performed by changing the pH of the solution.

3. The method according to PP. 1 and 2, characterized in that the magnitude of the electric field strength is chosen in the range of 0.1 to 10.0 V/m, the magnitude of induction of a constant magnetic field is chosen in the range 10 to 100 MKT, and the magnitude of induction of an alternating magnetic field in the range of 10 to 200 what redstem for submission and selection of a solution, characterized in that it is equipped with a regulator of pH and source of the magnetic field formed by the two covering the container and placed coaxially one within the other magnetic coils, one of which is connected to a source of a constant and the other with a source of alternating current, and installed inside the tank connected to the constant current source electrodes of biologically inert material, arranged so that the generated electric field and magnetic field are crossed among themselves, with the capacity performed thermally.

 

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