JP6332600B2 - Method for producing polyamine conjugate - Google Patents

Description

  The present invention relates to a novel method for producing a polyamine conjugate, and more specifically to a method for producing a polyamine conjugate produced from cinnamic acid and polyamine in a simple and high yield.

  Polyamine is a general term for aliphatic hydrocarbons having two or more primary amino groups, and is a natural product that exists universally in all living organisms. Exemplary polyamines include putrescine, spermidine and spermine. The main physiological functions of polyamines are as follows: (1) Nucleic acid stabilization and structural change by interaction with nucleic acid, (2) Various nucleic acid synthesis system promoting action, (3) Protein synthesis system activation, (4 It is known to stabilize cell membranes, enhance membrane permeability of substances, (5) eliminate active oxygen, and (6) promote cell growth.

  Polyamines exist by interacting with acidic substances in cells, and most bacteria and animal cells exist by binding to RNA. Therefore, it is considered that the physiological action of polyamine is mainly caused by interaction with RNA (see Non-Patent Document 1).

  On the other hand, in plant cells, various phenylpropanoids such as cinnamic acid and caffeic acid as well as free polyamines and compounds in which other acids are amide-bonded to polyamines, that is, polyamine conjugates have been isolated. These polyamine conjugates are present in plants of various families, and are thought to regulate intracellular polyamine concentrations, and physiological functions such as promotion of flower bud growth and suppression of infection with pathogenic bacteria have been reported (non- (See Patent Documents 2 and 3).

  Although the physiological function of such a polyamine conjugate has many unclear points, its usefulness is expected as a material having the functions of cinnamic acid and polyamine. However, polyamine conjugates have been conventionally obtained by isolation from plants or chemical synthesis, and are difficult to mass-produce at an industrial level (see Non-Patent Documents 4 and 5). Accordingly, there is a need to develop a simple and high-yield production method for polyamine conjugates.

Kazue Igarashi “Mysterious Life Material-Polyamine”, Kyoritsu Shuppan, 1993 Martin-Tanguy, J. et al. et al. (1978) Phytochemistry, 17, 1927 Martin-Tanguy, J. et al. (1985) Plant Growth Regulation, 3,381 Bagni, N .; and Tassoni, A.M. (2001) Amino Acids, 20, 301 Fixon-Owoo, S .; et al. (2003) Phytochemistry, 63, 315

  The present invention has been made in view of the current state of the prior art, and an object of the present invention is to provide a simple and high-yield production method for polyamine conjugates.

  In order to achieve the above object, the present inventors have intensively studied the amide synthesis exchange reaction catalyzed by an enzyme. As a result, in the amide synthesis reaction between cinnamic acids and polyamines, cinnamic acids and polyamines were used as substrates. The inventors have found that a polyamine conjugate can be synthesized easily and in a high yield by a one-step reaction by performing a catalytic reaction with an enzyme in an organic solvent, and the present invention has been completed.

That is, the present invention has the following configurations (1) to ( 3 ).
(1) In the method for producing a polyamine conjugate by an amide synthesis reaction of cinnamic acid and polyamine, using cinnamic acid and polyamine as a substrate, performing a catalytic reaction with an enzyme in an organic solvent , Cinnamic acid, caffeic acid, hydroxycinnamic acid, ferulic acid, hesperic acid, 3,4-obtained by transesterification of a carboxy group in cinnamic acid to an alkyl group or vinyl group having 1 to 4 carbon atoms Selected from the group consisting of alkyl esters or vinyl esters of dihydroxyphenylpropionic acid, 3-phenylpropionic acid and sinapinic acid, and the organic solvent is acetone, chloroform, tetrahydrofuran, t-butanol, 1,4-dioxane, isoamyl Ether, n-hexane, 2-methyl-2- It is an organic solvent or a combination thereof selected from the group consisting of ethanol and diisopropyl ether, and wherein said enzyme is characterized in that it is a lipase.
( 2 ) The method according to ( 1), wherein the polyamine is selected from the group consisting of putrescine, spermidine and spermine.
( 3 ) The method according to (1) or (2), wherein the organic solvent is an organic solvent selected from the group consisting of tetrahydrofuran, t-butanol and 2-methyl-2-butanol or a combination thereof. .

  Unlike the conventional chemical synthesis method, the production method of the present invention utilizes a catalytic reaction by an enzyme, so that a polyamine conjugate can be produced simply and with high efficiency by a one-step reaction. Therefore, the production method of the present invention is expected to contribute to the production of polyamine conjugates expected to be useful as materials having the physiological functions of cinnamic acids and polyamines at the industrial level.

An HPLC chromatogram of an enzyme reaction solution obtained by reacting ethyl cinnamate and putrescine as substrates and adding lipase in 2M2B for 24 hours. ESI-MS spectrum of purified sample 1. ESI-MS / MS spectrum of purified sample 1. The cleavage pattern of the purified sample 1 estimated from the fragment ion peak of MS / MS analysis. The result of the NMR analysis which identified the refinement | purification sample 1 as N-cinnamate putrescine. The HPLC chromatogram of the enzyme reaction liquid which made ethyl cinnamate and spermidine a substrate, added lipase in 2M2B, and was made to react for 24 hours. ESI-MS spectrum of purified sample 2. ESI-MS / MS spectrum of purified sample 2 The cleavage pattern of the purified sample 2 estimated from the fragment ion peak of MS / MS analysis. The HPLC chromatogram of the enzyme reaction liquid which made ethyl cinnamate and spermine a substrate and added lipase in 2M2B and made it react for 24 hours. ESI-MS spectrum of purified sample 3 ESI-MS / MS spectrum of purified sample 3 The cleavage pattern of the purified sample 3 estimated from the fragment ion peak of MS / MS analysis. Results of NMR analysis in which purified sample 3 was identified as N, N'-di-cinnamic acid spermine. The HPLC chromatogram of the enzyme-reaction liquid which made p-methylcoumarate and putrescine a substrate, added lipase in 2M2B, and was made to react for 24 hours. ESI-MS spectrum of purified sample 4 ESI-MS / MS spectrum of purified sample 4 The cleavage pattern of the purified sample 4 estimated from the fragment ion peak of MS / MS analysis. The result of the NMR analysis in which the purified sample 4 was identified as Np-putrescine coumarate. The HPLC chromatogram of the enzyme-reaction liquid which added lipase in 2M2B and made it react for 24 hours, using methyl p-coumarate and spermidine as a substrate. ESI-MS spectrum of purified sample 5. ESI-MS / MS spectrum of purified sample 5 The cleavage pattern of the purified sample 5 estimated from the fragment ion peak of MS / MS analysis. The result of the NMR analysis in which the purified sample 5 was identified as N1-p-spermidine coumarate. ESI-MS spectrum of purified sample 6 ESI-MS / MS spectrum of purified sample 6 The cleavage pattern of the purified sample 6 estimated from the fragment ion peak of MS / MS analysis. Results of NMR analysis in which purified sample 6 was identified as N1, N10-di-p-coumarate spermidine. The HPLC chromatogram of the enzyme-reaction liquid which made p-methyl coumarate and spermine a substrate and added lipase in 2M2B and made it react for 24 hours. ESI-MS spectrum of the purified sample 7 ESI-MS / MS spectrum of the purified sample 7 The cleavage pattern of the purified sample 7 estimated from the fragment ion peak of MS / MS analysis. The result of the NMR analysis which identified the refinement | purification sample 7 as Np-coumarate spermine. ESI-MS spectrum of purified sample 8. ESI-MS / MS spectrum of purified sample 8 The cleavage pattern of the purified sample 8 estimated from the fragment ion peak of MS / MS analysis. Results of NMR analysis where the purified sample 8 was identified as spermine N, N'-di-p-coumarate. Production amount of putrescine Np-coumarate in an enzyme reaction solution in which lipase was added in various solvents and reacted for 24 hours using methyl p-coumarate and putrescine as substrates.

  The method for producing a polyamine conjugate of the present invention is characterized in that, in an amide synthesis reaction between cinnamic acids and polyamines, cinnamic acids and polyamines are used as substrates, and an enzyme catalytic reaction is performed in an organic solvent.

  Cinnamic acids used in the present invention include cinnamic acid, caffeic acid, hydroxycinnamic acid, ferulic acid, hesperic acid, 3,4-dihydroxyphenylpropionic acid, 3-phenylpropionic acid, sinapinic acid and the like. These cinnamic acids are preferably alkyl esters or vinyl esters in which a carboxy group is transesterified to an alkyl group having 1 to 4 carbon atoms or a vinyl group as a form in an amide synthesis reaction with a polyamine.

  The polyamine used in the present invention is a general term for aliphatic hydrocarbons having two or more primary amino groups, and is a natural product that exists universally in the living body. For example, 1,3-diaminopropane, putrescine, cadaverine, cardine, spermidine, homospermidine, aminopropyl cadaverine, theremin, spermine, thermospermine, canabalmin, aminopentylnorspermidine, N, N-bis (aminopropyl) cadaverine, homo Examples include spermine, cardopentamine, homocardopentamine, cardohexamine, and homocardohexamine. Typical polyamines include putrescine, spermidine and spermine.

  The polyamine conjugate used in the present invention is a general term for amide compounds in which the above cinnamic acids and polyamine are produced by an amide synthesis reaction, and is sometimes called a polyamine amide or a polyamine-based alkaloid. In this case, since an amide is formed according to the number of amino groups of the polyamine, at least one polyamine conjugate in which one molecule or more of cinnamic acid is bonded to one molecule of polyamine is synthesized. In the production method of the present invention, two or more cinnamic acids may be used simultaneously. Accordingly, a polyamine conjugate in which two or more different cinnamic acids are bonded to a polyamine is also included in the product obtained by the production method of the present invention.

  The organic solvent used in the present invention is not particularly limited, and examples thereof include acetone, chloroform, tetrahydrofuran, t-butanol, 1,4-dioxane, isoamyl ether, n-hexane, 2-methyl-2-butanol and diisopropyl ether. It is done. These organic solvents may be used alone or in combination of two or more.

  The enzyme used in the present invention is not particularly limited as long as it is an enzyme capable of performing a catalytic reaction in an organic solvent using cinnamic acid and polyamine as substrates in the amide synthesis reaction, but lipase is preferable.

  The reaction of enzyme synthesis in the production method of the present invention is carried out by allowing the above-described enzyme to act on cinnamic acid derivative and polyamine in an organic solvent. The concentration of the cinnamic acid derivative as a substrate is usually 10 mM to 200 mM, preferably 50 to 150 mM. The density | concentration of the polyamine which is another substrate is 10 mM-200 mM normally, Preferably it is 25-75 mM. The molar ratio of cinnamic acid to polyamine is preferably 6: 1 to 1: 1, and particularly preferably 3: 1 to 2: 1.

  The amount of enzyme to be used varies depending on the degree of activity of the biocatalyst for the amide synthesis reaction, but is usually preferably 10 to 200 mg / mL, particularly preferably 60 to 100 mg / mL.

  The reaction temperature is usually preferably 30 to 60 ° C. If there is no adverse effect on enzyme deactivation, life reduction, or reaction rate, the reaction can be performed at a temperature of 60 ° C. or higher or 30 ° C. or lower. The reaction can be carried out under reduced pressure or under a pressure of 0.1 MPa or more, but it is usually preferred to carry out at normal pressure.

  The reaction time may be appropriately adjusted, but can usually be about 1 hour or more and 96 hours or less. Usually, 24 hours is preferable, although it depends on the degree of activity of the biocatalyst for the amide synthesis reaction.

  The polyamine conjugate produced by the amide synthesis reaction can be easily separated by extraction with, for example, acetic acid or methanol. The separated polyamine conjugate can be purified with high purity by using a cation exchange column, a HPLC reverse phase column, or the like. In this case, as a form of purification, not only column chromatography but also a batch system can be applied using an appropriate resin.

  In addition, since the reaction rate decreases when a large amount of water is present in the reaction system, it is preferable to remove the water from the reaction system as much as possible in order to efficiently perform the amide synthesis reaction of the present invention. For example, it is desirable to take a water removal measure such as using a molecular sieve, and to carry out the reaction under conditions where water is not substantially present (water content: 0.5% by weight or less).

  According to the above-described method, it becomes possible to produce a polyamine conjugate simply and in high yield from a cinnamic acid derivative and a polyamine. In addition, separation and recovery of the product after the reaction are easy, and it is suitable for implementation on an industrial level.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited by these Examples.

(Example 1) Enzymatic synthesis of putrescine N-cinnamate 2.5 mL of a reaction solution containing 200 mg of 150 mM ethyl cinnamate, 50 mM putrescine, and molecular seaves 3 mg in 2-methyl-2-butanol (2M2B) was heated to 60 ° C. After preheating for 10 minutes, 100 mg of immobilized lipase (Novozym 435) was added and stirred at 60 ° C. and 350 rpm for 24 hours. The reaction solution 24 hours after the start of the reaction was subjected to analysis by high performance liquid chromatography (HPLC; High Performance Liquid Chromatography) and thin layer chromatography (TLC; Thin-Layer Chromatography).

  Cosmosil ODS 5C18-PAQ (4.6 mm ID x 250 mm, Nacalai Tesque) was used as the HPLC analytical column, and 0.2% (v / v) acetic acid water (A) and methanol (B) were used as the mobile phase. In linear gradient mode (0% → 80% B / 50 min), the flow rate was 1 ml / min at 25 ° C., and the absorbance at 254 nm was monitored to detect the reaction product. FIG. 1 shows an example of HPLC results obtained by analyzing a reaction solution 24 hours after the start of the reaction. The peak detected after 15 to 16 minutes of elution time is the peak of the reaction product detected 24 hours after the start of the reaction after adding the enzyme.

  For TLC, SILICA GEL 60 RP-18 F254S aluminum plate (MERCK) was used. After developing with methanol: water: acetic acid (7: 3: 0.2), UV irradiation (254 nm) and ninhydrin staining were performed in the reaction solution. Were used as indicators for detecting cinnamic acids, polyamines and polyamine conjugates. As a result of subjecting the reaction solution 24 hours after the start of the above reaction to TLC, new spots showing UV absorption and ninhydrin positivity were observed (results not shown).

  In order to obtain a reaction product, the reaction solution was dried under reduced pressure, dissolved in 2.5 mL of 0.2% (v / v) acetic acid water, and subjected to the following purification. Cosmosil ODS 5C18-PAQ (10 mm ID x 250 mm, Nacalai Tesque) was used as a preparative column for purifying the reaction product. After injecting 500 μL of the reaction solution sample, the mobile phase was set to gradient mode (0% B). After 10 ml, 10% → 30% B (60 ml) was allowed to flow at 25 ° C. at a flow rate of 5 ml / min, the absorbance at 254 nm was monitored, and the fraction eluted was collected by a fraction collector. The obtained elution fraction was detected as a single peak after elution time of 15 to 16 minutes in the analysis by HPLC described above. By this purification, 9.6 mg of purified sample 1 was obtained.

  As a result of ESI-MS of the purified sample 1, a molecular ion peak was detected at m / z 219 (FIG. 2). When MS / MS analysis was performed on the peak at m / z 219, fragment ion peaks were detected at m / z 202, 131, and 103 (FIG. 3). From these results, purified sample 1 was assigned to the structure of N-cinnamoyl putrescine (FIG. 4).

  Furthermore, as a result of analyzing the purified sample 1 by NMR, it was assigned to the structure of N-cinnamoyl putrescine (FIG. 5). From the above MS and NMR analysis results, the purified sample 1 was identified as N-cinnamoyl putrescine.

(Example 2) Enzymatic synthesis of spermidine N1-cinnamate After preheating a 2.5 mL reaction solution containing 200 mg of 150 mM ethyl cinnamate, 50 mM spermidine and molecular seaves 3Å in 2M2B at 60 ° C. for 10 minutes, Novozym 435 100 mg was added and stirred at 60 ° C. and 350 rpm for 24 hours. The reaction solution 24 hours after the start of the reaction was subjected to analysis by HPLC and TLC.

  FIG. 6 is an example of the HPLC result obtained by analyzing the reaction product in the same manner as in Example 1. The peak detected after 3 minutes of elution time is the peak of the reaction product detected 24 hours after the start of the reaction after adding the enzyme. Furthermore, as a result of subjecting the reaction solution after 24 hours to TLC in the same manner as in Example 1, new spots showing UV absorption and ninhydrin positivity were observed (results not shown).

  In order to obtain a reaction product, the reaction solution was dried under reduced pressure, dissolved in 2.5 mL of 0.2% (v / v) acetic acid water, and subjected to the following purification. In the same manner as in Example 1, using an HPLC preparative column, 500 μL of the reaction solution sample was injected, and the mobile phase was changed to gradient mode (50 ml at 0% B, then 20 ml at 0% → 100% B). ) At 25 ° C. at a flow rate of 3 ml / min, the absorbance at 254 nm was monitored, and the fraction eluted was collected by a fraction collector. The obtained elution fraction was detected as a plurality of peaks after elution time 3 to 5 minutes in the analysis by HPLC described above. By this purification, 1.9 mg of purified sample 2 was obtained.

  As a result of ESI-MS of the purified sample 2, a molecular ion peak was detected at m / z 276 (FIG. 7). When MS / MS analysis was performed on the peak at m / z 276, a fragment ion peak was detected at m / z 259, 188, 131 (FIG. 8). In spermidine, aminopropyl group and aminobutyl group having different carbon numbers are bonded to NH at the 5-position. When one molecule of cinnamic acid forms an amide bond with spermidine, two types of spermidine cinnamate with cinnamic acid bonded to either the 1-position or the 10-position amino group may be synthesized. It is considered that these two types of spermidine cinnamate are contained in a plurality of peaks detected after 3 to 5 minutes of elution time on HPLC. From the above results, the purified preparation 2 was assigned to the structure of N1-cinnamoyl spermidine (FIG. 9).

(Example 3) Enzymatic synthesis of N, N'-di-cinnamate spermine 2.5 mL of a reaction solution containing 150 mg of ethyl cinnamate, 50 mM spermine and 200 mg of molecular saves in 2M2B was preliminarily prepared at 60 ° C for 10 minutes. After warming, 100 mg of Novozym 435 was added and stirred at 60 ° C. and 350 rpm for 24 hours. The reaction solution 24 hours after the start of the reaction was subjected to analysis by HPLC and TLC.

  FIG. 10 is an example of the HPLC result obtained by analyzing the reaction product in the same manner as in Example 1. The peak detected after 20 to 21 minutes of elution time is the peak of the reaction product detected 24 hours after the start of the reaction after adding the enzyme. Furthermore, as a result of subjecting the reaction solution 24 hours after the start of the reaction to TLC in the same manner as in Example 1, new spots showing UV absorption and ninhydrin positivity were observed (results not shown).

  In order to obtain a reaction product, the reaction solution was dried under reduced pressure, dissolved in 2.5 mL of 0.2% (v / v) acetic acid water, and subjected to the following purification. In the same manner as in Example 1, using a preparative HPLC column, 500 μL of the reaction solution sample was injected, and the mobile phase was changed to gradient mode (40 ml at 10% B, then 160 ml at 10% → 70% B). ) At 25 ° C. at a flow rate of 3 ml / min, the absorbance at 254 nm was monitored, and the fraction eluted was collected by a fraction collector. The elution fraction corresponding to the largest peak among a plurality of peaks is again in gradient mode (40 ml at 0% B, then 120 ml at 10% → 40% B) at a flow rate of 3 ml / min at 25 ° C. The absorbance at 254 nm was monitored, and the eluted fraction was collected by a fraction collector. The obtained elution fraction was detected as a single peak after an elution time of 20 minutes in the analysis by HPLC described above. By this purification, 11.7 mg of purified sample 3 was obtained.

  As a result of ESI-MS of the purified sample 3, a molecular ion peak was detected at m / z 463 (FIG. 11). When MS / MS analysis was performed on the peak at m / z 276, a fragment ion peak was detected at m / z 259, 188, 131 (FIG. 12). From these results, the purified sample 3 was assigned to the structure of N, N'-dicinnamoyl spermine (FIG. 13).

  Furthermore, as a result of analyzing the purified sample 3 by NMR, it was assigned to the structure of N, N′-dicinnamylylspermine (FIG. 14). From the above MS and NMR analysis results, the purified sample 3 was identified as N, N'-dicinnamylylspermine.

(Example 4) Enzymatic synthesis of N-p- putresuccinate putrescine 25 mL of a reaction solution containing 2Om2B of 150mM methyl p-coumarate, 50mM putrescine and 3 ~ 2000mg molecular saves was pre-warmed at 60 ° C for 10 minutes. Then, Novozym 435 1000 mg was added, and it stirred at 280 rpm for 24 hours at 60 degreeC. The reaction solution 24 hours after the start of the reaction was subjected to analysis by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC).

  FIG. 15 is an example of the HPLC results obtained by analyzing the reaction product in the same manner as in Example 1. The peak detected after 13 minutes of elution time is the peak of the reaction product detected 24 hours after the start of the reaction after adding the enzyme. Furthermore, as a result of subjecting the reaction solution after 24 hours to TLC in the same manner as in Example 1, new spots showing UV absorption and ninhydrin positivity were observed (results not shown).

  In order to obtain a reaction product, the reaction solution was dried under reduced pressure, dissolved in 20 mL of 0.2% (v / v) acetic acid water, and subjected to the following purification. The reaction solution sample was passed through a CM Sepharose Fast Flow column (φ1.6 × 11 cm, 20 mL, GE Healthcare) equilibrated with 0.2% (v / v) acetic acid water, and the non-adsorbed fraction was removed with distilled water. The adsorbed component was eluted with 100 mL of 3M acetic acid. The fraction eluted with 3M acetic acid was dried under reduced pressure, dissolved in 5 mL of 0.2% (v / v) acetic acid water, and subjected to purification by HPLC. In the same manner as in Example 1, using a preparative column, after injecting 500 μL of sample, the mobile phase was set to 25 ° C. in stepwise mode (20 ml at 10% B, then 40 ml at 100% B). The flow rate was 5 ml / min, the absorbance at 254 nm was monitored, and the eluted fraction was collected by a fraction collector. The elution fraction obtained as the maximum peak in the latter half was detected as a single peak after 13 minutes elution time in the analysis by HPLC described above. By this purification, 48 mg of purified sample 4 was obtained.

  As a result of ESI-MS of the purified sample 4, a molecular ion peak was detected at m / z 235 (FIG. 16). When MS / MS analysis was performed on the peak at m / z 235, fragment ion peaks were detected at m / z 218, 147, and 119 (FIG. 17). From these results, the purified sample 4 was assigned to the structure of Np-coumaroyl putrescine (FIG. 18).

  Furthermore, as a result of analyzing the purified sample 4 by NMR, it was attributed to the structure of Np-comumaroyl putrescine (FIG. 19). From the MS and NMR analysis results described above, the purified sample 4 was identified as Np-cumaroyl putrescine.

(Example 5) Enzymatic synthesis of spermidine N1-p-coumarate 25 mL of a reaction solution containing 100 mM methyl p-coumarate, 75 mM spermidine and 2000 mg of molecular sieves 3 mg in 2M2B was pre-warmed at 60 ° C. for 10 minutes. Then, Novozym 435 1000 mg was added, and it stirred at 280 rpm for 24 hours at 60 degreeC.

  FIG. 20 is an example of HPLC results obtained by analyzing the reaction solution after 24 hours of reaction in the same operation as in Example 1. The peaks detected at 4 minutes and 24 minutes after the elution time are peaks of the reaction product detected 24 hours after the start of the reaction after addition of the enzyme. Furthermore, as a result of subjecting the reaction solution 24 hours after the start of the reaction to TLC in the same manner as in Example 1, new spots showing UV absorption and ninhydrin positivity were observed (results not shown).

  In order to obtain these reaction products, the reaction solution was dried under reduced pressure, dissolved in 20 mL of 0.2% (v / v) acetic acid water, and subjected to the following purification. The reaction solution sample was passed through a CM Sepharose Fast Flow column (φ1.6 × 11 cm, 20 mL, GE Healthcare) equilibrated with 0.2% (v / v) acetic acid water, and the non-adsorbed fraction was removed with distilled water. 100 mL of the adsorbed component was eluted with 0.2 M, 0.5 M, 1 M, 2 M and 3 M acetic acid, respectively. As a result of subjecting the obtained elution fraction to TLC, spots showing UV absorption and ninhydrin positivity were observed in the elution fractions of 0.2 M and 0.5 M acetic acid (results not shown).

  The fraction eluted with 0.2 M acetic acid was dried under reduced pressure, dissolved in 5 mL of 0.2% (v / v) acetic acid water, and 500 μL was injected using a preparative column in the same manner as in Example 1. Then, the mobile phase was flowed at a flow rate of 5 ml / min at 25 ° C. in stepwise mode (20 ml at 10% B, then 60 ml at 100% B), and the absorbance at 254 nm was monitored. Minutes were collected. The elution fraction obtained as the maximum peak in the latter half was detected as a single peak after elution time of 4 to 5 minutes in the analysis by HPLC described above. By this purification, 86 mg of purified sample 5 was obtained.

  As a result of ESI-MS of the purified sample 5, a molecular ion peak was detected at m / z 292 (FIG. 21). When the peak of m / z 292 was analyzed by MS / MS, a fragment ion peak was detected at m / z 275, 204, 147 (FIG. 22). From these results, the purified sample 5 was assigned to the structure of N1-p-cumaroyl spermidine (FIG. 23).

  Furthermore, as a result of analyzing the purified sample 5 by NMR, it was attributed to the structure of N1-p-cumaroyl spermidine (FIG. 24). From the MS and NMR analysis results described above, the purified sample 5 was identified as N1-p-cumaroyl spermidine.

(Example 6) Enzymatic synthesis of N1, N10-di-p-coumarate spermidine In the purification by CM Sepharose Fast Flow column in Example 5, the fraction eluted with 0.5 M acetic acid was dried under reduced pressure, 0.2 % (V / v) dissolved in 5 mL of acetic acid water, and 500 μL was injected into the preparative column in the same manner as in Example 1, and then the mobile phase was changed to stepwise mode (20 ml at 10% B, then 100% B was flowed at 25 ° C. at a flow rate of 5 ml / min, the absorbance at 254 nm was monitored, and the eluted fraction was collected by a fraction collector. The elution fraction obtained as the maximum peak in the latter half was detected as a single peak after the elution time of 24 to 25 minutes in the analysis by HPLC described above. By this purification, 77 mg of purified sample 6 was obtained.

  As a result of ESI-MS of the purified sample 6, a molecular ion peak was detected at m / z 439 (FIG. 25). When the peak at m / z 439 was analyzed by MS / MS, a fragment ion peak was detected at m / z 292, 204, and 147 (FIG. 26). Although m / z 439 is 1 larger than the molecular ion peak m / z 438 of N1, N10-di-p-comaroyl spermidine, the substance showing m / z 439 is N1, N10-di-p from the analysis of the fragment ion peak. -Probably a substance in which a proton is added to NH at the 5-position of coumeric ylmidine. From these results, the purified sample 6 was assigned to the structure of spermidine N1, N10-di-p-coumaroyl spermidine (FIG. 27).

  Furthermore, as a result of analyzing the purified sample 6 by NMR, it was assigned to the structure of N1, N10-di-p-comaroylylmidine (FIG. 28). From the above MS and NMR analysis results, the purified sample 6 was identified as spermidine N1, N10-di-p-coumaroyl spermidine.

(Example 7) Enzymatic synthesis of Np-spermic acid spermine 25 mL of a reaction solution containing 150 mg of methyl p-coumarate, 75 mM spermine and 2000 mg of molecular saves 3 mg in 2M2B was pre-warmed at 60 ° C. for 10 minutes. Then, Novozym 435 1000 mg was added, and it stirred at 280 rpm for 24 hours at 60 degreeC.

  FIG. 29 is an example of HPLC results obtained by analyzing the reaction solution after a 24-hour reaction in the same manner as in Example 1. The peaks detected after 2 to 3 minutes and 15 to 16 minutes after the elution time are peaks of reaction products detected 24 hours after the start of the reaction after addition of the enzyme. Furthermore, as a result of subjecting the reaction solution 24 hours after the start of the reaction to TLC in the same manner as in Example 1, new spots showing UV absorption and ninhydrin positivity were observed (results not shown).

  In order to obtain these reaction products, the reaction solution was dried under reduced pressure, dissolved in 20 mL of 0.2% (v / v) acetic acid water, and subjected to the following purification. The reaction solution sample was passed through a CM Sepharose Fast Flow column (φ1.6 × 11 cm, 20 mL, GE Healthcare) equilibrated with 0.2% (v / v) acetic acid water, and the non-adsorbed fraction was washed with distilled water. 100 mL of adsorbed components were eluted with 0.2 M, 0.5 M, 1 M and 3 M acetic acid, respectively. As a result of subjecting the eluted fraction to TLC, spots showing UV absorption and ninhydrin positivity were observed in the eluted fractions of 0.2 M and 0.5 M acetic acid (results not shown).

  The fraction eluted with 0.2 M acetic acid was dried under reduced pressure, dissolved in 5 mL of 0.2% (v / v) acetic acid water, and 500 μL was injected into the preparative column in the same manner as in Example 1. Run the mobile phase in stepwise mode (20 ml at 10% B, then 60 ml at 100% B) at 25 ° C. with a flow rate of 5 ml / min, monitor the absorbance at 254 nm, and extract the eluted fraction with a fraction collector. It was collected. The elution fraction obtained as the maximum peak in the latter half was detected as a single peak after elution time of 4 to 5 minutes in the analysis by HPLC described above. By this purification, 60 mg of purified sample 7 was obtained.

  As a result of ESI-MS of the purified sample 7, a molecular ion peak was detected at m / z 349 (FIG. 30). When MS / MS analysis was performed on the peak at m / z 292, a fragment ion peak was detected at m / z 275, 204, 129 (FIG. 31). From these results, the purified sample 7 was assigned to the structure of Np-coumaroyl spermine (FIG. 32).

  Furthermore, as a result of analyzing the purified sample 7 by NMR, it was attributed to the structure of Np-comuaroyl spermine (FIG. 33). From the MS and NMR analysis results described above, the purified sample 7 was identified as Np-cumaroyl spermine.

(Example 8) Enzymatic synthesis of N, N'-di-p-coumarate spermine In the purification using the CM Sepharose Fast Flow column of Example 7, the fraction eluted with 0.5 M acetic acid was dried under reduced pressure. Dissolve in 5 mL of 2% (v / v) acetic acid water, and inject the 500 μL using the preparative column in the same manner as in Example 1. , 60 ml at 100% B) at 25 ° C. with a flow rate of 5 ml / min, the absorbance at 254 nm was monitored, and the eluted fraction was collected by a fraction collector. The elution fraction obtained as the maximum peak in the latter half was detected as a single peak after the elution time of 24 to 25 minutes in the analysis by HPLC described above. By this purification, 92 mg of purified preparation 8 was obtained.

  As a result of ESI-MS of the purified sample 8, a molecular ion peak was detected at m / z 495 (FIG. 34). When the peak at m / z 439 was analyzed by MS / MS, a fragment ion peak was detected at m / z 275, 204, 147 (FIG. 35). From these results, the purified sample 8 was assigned to the structure of N, N′-di-p-comauroyl spermine (FIG. 36).

  Furthermore, as a result of analyzing the purified sample 8 by NMR, it was attributed to the structure of N, N′-di-p-comaroyl spermine (FIG. 37). From the above MS and NMR analysis results, the purified sample 8 was identified as N, N′-di-p-coumaroyl spermine.

(Example 9) Enzymatic synthesis of Np-putrescine couprate in various solvents Using 150 mM methyl p-coumarate and 50 mM putrescine as substrates, 10 mg of Novozym 435 was added in various solvents as shown in FIG. Reaction was performed and the production amount of putrescine Np-coumarate was examined (FIG. 38). Tert-butanol, 1,4-dioxane, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 3-methyl-1-butanol (3M1B) and isoamyl ether having a boiling point of 70 ° C. or higher are 60 ° C. N-hexane, chloroform, tetrahydrofuran (THF), acetone and diisopropyl ether having a boiling point of 70 ° C. or lower were reacted at 45 ° C., and 2-methyl-2-butanol (2M2B) was reacted at both temperatures. As a result, a particularly high production amount of putrescine Np-coumarate was obtained in tert-butanol, THF and 2M2B.

  According to the method of the present invention, a polyamine conjugate can be produced simply and with high yield. Further, the present invention is suitable for industrial production of polyamine conjugates that are expected to be useful as functional materials having the physiological functions of cinnamic acids and polyamines. Furthermore, the polyamine conjugate produced by the method of the present invention can be used in applications such as pharmaceuticals, chemical products, cosmetics, and foods.

Claims (3)

In a method for producing a polyamine conjugate by an amide synthesis reaction between cinnamic acids and a polyamine, an enzyme-catalyzed reaction is carried out in an organic solvent using cinnamic acids and polyamine as a substrate, and the cinnamic acids are contained in cinnamic acids. Cinnamic acid, caffeic acid, hydroxycinnamic acid, ferulic acid, hesperic acid, 3,4-dihydroxyphenylpropion obtained by transesterification of the carboxy group of 1 to 4 with an alkyl group or vinyl group having 1 to 4 carbon atoms Selected from the group consisting of acids, alkyl esters or vinyl esters of 3-phenylpropionic acid and sinapinic acid, and the organic solvent is acetone, chloroform, tetrahydrofuran, t-butanol, 1,4-dioxane, isoamyl ether, n -Hexane, 2-methyl-2-butano How is an organic solvent or a combination thereof selected from the group consisting of Le and diisopropyl ether, and the enzyme, characterized in that it is a lipase. The method of claim 1, wherein the polyamine is selected from the group consisting of putrescine, spermidine and spermine. The method according to claim 1 or 2, wherein the organic solvent is an organic solvent selected from the group consisting of tetrahydrofuran, t-butanol and 2-methyl-2-butanol or a combination thereof.