CN111850061B - Preparation method of valienamine A ester - Google Patents
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Abstract
The invention discloses a preparation method of valienamine A ester shown in formula (I), wherein R is C 1 ‑C 21 Saturated aliphatic radical of (2), C 1 ‑C 21 Unsaturated aliphatic radical of (A) or (C) 1 ‑C 21 The halogenated saturated aliphatic group of (a), the process being: the valienamine A and fatty acid RCOOH react in tertiary butanol under the action of a biocatalyst to prepare an esterified product of the valiolamine A shown in a formula (I); the biocatalyst is one of Lipozyme RMIM, lipase CALB, porcine pancreatic Lipase, candida Lipase and Candida rugosa Lipase. The method can accurately control the reaction sites and obtain the validamine A esterified substance with high purity.
Description
Technical Field
The invention relates to a method for preparing valienamine A esterified substance by enzyme catalysis.
Background
Validamycin (Validamycin) is a pseudo trisaccharide compound, which is separated from a secondary metabolite of streptomyces hygroscopicus var. In 1973, streptomyces hygroscopicus validus variant (s. Hygroscopicus var. Kingganensis) strains capable of inhibiting rice sheath blight disease (Rhizoctonia solani) were found and screened in soil of jinggangshan mountain and hangzhou, zhejiang, where pesticide research was carried out in Shanghai city, and through research, the chemical structure of active compounds extracted from the strains is consistent with that of validamycin, and the active ingredients are named as validamycin (jinggangmycin). The validamycin can effectively inhibit rice sheath blight, has a certain sterilization effect on vegetable seedling blight, and has the advantages of long-term efficacy, low toxicity, low residue, high safety and small environmental pollution, so that the validamycin is a pollution-free biological pesticide with the largest popularization and use area and the lowest mu cost in China.
Validamycin A is the most important and main component in validamycin compounds, and the molecular structure of the validamycin A comprises validamycin A and D-type glucopyranosyl, so that the validamycin A is a pseudo trisaccharide compound.
The chemical structure of validamine A is similar to that of trehalose (shown in the specification), the validamine A is considered to be a natural trehalase competitive inhibitor with high activity, the validamine A is novel in structure, good in-vitro sterilization effect and strong in selectivity, and the inhibition effect on trehalase can reach 10 -8 -10 -9 And in addition, the product has high safety to mammals and higher plants, and has good application prospect.
Validamine A has strong in-vitro trehalase inhibition activity, but is difficult to enter into organisms, so that the validamine A needs to be modified in structure to increase the capability of entering into the organisms, so that the problem that the insecticidal and bactericidal activity can be exerted by 'injection' is solved, and ester synthesis becomes a preferred scheme. However, the number of hydroxyl groups on validoxylamine a is large, and the esterification sites are difficult to accurately control by the conventional chemical synthesis method, but the reaction sites can be accurately controlled by the lipase catalytic synthesis method adopted by the invention, and the high-purity derivatives can be finally obtained. The biological activity of the compound is further researched, and the research shows that the compound has good biological activity.
Disclosure of Invention
The invention aims to provide a method for preparing valienamine A esterified substance by enzyme catalysis, which can accurately control reaction sites and obtain high-purity valienamine A esterified substance.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a preparation method of valienamine A ester shown in formula (I) comprises the following steps: the valienamine A and fatty acid RCOOH react in tertiary butanol under the action of a biocatalyst to prepare an esterified product of the valiolamine A shown in a formula (I); the biocatalyst is one of Lipozyme RMIM, lipase CALB, porcine pancreatic Lipase, candida Lipase and Candida rugosa Lipase;
r is C 1 -C 21 Saturated aliphatic radical of (2), C 1 -C 21 Unsaturated aliphatic radical of (C) 1 -C 21 A halogenated saturated aliphatic group of (2).
Preferably, R = C n H 2n+1 Wherein n =1-21. Further preferably n =8 to 18, still further preferably n =10 to 18, and still further preferably 10 to 17.
Preferably, R = C n H 2n-1 Wherein n =1-21. Further preferably n =3-18, still further preferably n =5-18, particularly preferably n =7-18, such as R = CH 2 ＝CHCH 2 CH 2 CH 2 CH 2 CH 2 。
Preferably, R = C n H 2n+1-m X m Wherein n =1-21, m is less than or equal to 2n +1, X = F, cl, br or I. Further preferably, n =3 to 18, still more preferably n =5 to 18, and particularly preferably n =7 to 18. Further preferably X = Cl. Particularly preferably, R = CCl 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 。
Preferably, the reaction system is further added with an activated molecular sieve, wherein the amount of the molecular sieve is 0.4-2g/mmoL, more preferably 1.2-2.0g/mmoL, and most preferably 1.2g/mmoL based on the mole number of the validoxylamine A. As a further preference, the molecular sieve is a 4A ° molecular sieve. As a further preference, the molecular sieve is activated prior to use. The activation method is recommended as follows: activating at 400 ℃ for 2h.
Preferably, the biocatalyst is Lipozyme RMIM, where the conversion of validamine A is highest.
Preferably, the feeding mode is as follows: the validamine A is refluxed and stirred in tertiary butanol at 90-110 ℃ for 4-6h to prepare validamine A saturated solution, and then the validamine A saturated solution and fatty acid RCOOH are mixed in a reaction vessel. The feeding mode can obviously improve the solubility of valiolamine A in tertiary butanol, thereby effectively improving the reaction efficiency.
As a further preference, the preparation process is carried out specifically as follows: fully mixing a valienamine A-tert-butanol saturated solution and fatty acid RCOOH in a reaction vessel at 25-50 ℃, adding a biocatalyst to carry out oscillation reaction at 25-50 ℃, and after full reaction, separating and purifying reaction liquid to obtain an esterified product of valienamine A; the feeding molar ratio of the valienamine A to the fatty acid RCOOH is 4.
As a further preference, the reaction system is further added with a molecular sieve, and the dosage of the molecular sieve is 0.4-2g/mmoL, more preferably 1.2-2.0g/mmoL, and most preferably 1.2g/mmoL based on the mole number of the valiohydroxylamine A.
As a further preference, the biocatalyst is Lipozyme RMIM, where the conversion of validoxylamine A is highest.
As a further preference, the biocatalyst is Lipozyme RMIM, and the amount of the biocatalyst is 160-240g/moL, most preferably 160g/moL based on the number of moles of validamine A.
As a further preference, the biocatalyst is Lipozyme RMIM and equilibrium is reached at a reaction time of 48h.
As a further preference, the biocatalyst is Lipozyme RMIM, the reaction temperature is 45-50 ℃, and the optimal temperature is 45 ℃.
As a further preferable example, the biocatalyst is Lipozyme RMIM, and the feeding molar ratio of the valiolamine a to the fatty acid RCOOH is 1.
The most preferred reaction conditions for the present invention when the biocatalyst is Lipozyme RMIM are: the molar ratio of validamine A to fatty acid RCOOH is 1, the enzyme addition amount is 160g/moL of validamine A, the molecular sieve amount is 1.2 g/moL of validamine A, the reaction temperature is 45 ℃, and the reaction time is 48 hours.
As a further preference, the specific steps of the separation and purification are as follows: centrifuging to remove the biocatalyst (and molecular sieve), evaporating to remove the solvent, adding water, adjusting pH to neutral with sodium hydroxide, separating with macroporous adsorbent resin column, gradient eluting with 0-55% ethanol water solution as eluting reagent, collecting target solution, concentrating, and drying.
The validamine A esterified substance shown in the formula (I) has a good inhibition effect on rice sheath blight disease.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention provides a method for improving the solubility of validamine A, which can improve the solubility of validamine A in tertiary butanol.
(2) The invention provides a method for preparing valienamine A esterified substance by enzyme catalysis, which can accurately control reaction sites and obtain high-purity valienamine A esterified substance.
(3) The invention can obviously improve the reaction efficiency of validoxylamine A and fatty acid by adding the molecular sieve.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.
Example 1: fermentation of lipase producing strains
The invention applies acinetobacter junii zjutfet-1 (preserved in China center for type culture collection, with preservation date of 2015, 9 and 6 days, preservation number of CCTCC NO.M2015511, preservation address of Wuhan university, wuhan, china, zip code 430072) to the synthesis of the compound. The culture method of the strain is as follows:
inoculating acinetobacter junii zjutfet-1 to a slant culture medium, and culturing for 20 hours at 35 ℃ to obtain a slant colony; the final concentration of the slant culture medium is as follows: LB medium +2% agar powder.
Inoculating the slant colony to a seed culture medium, and culturing at 30 ℃ for 6h to obtain a seed solution, wherein the final concentration of the seed culture medium comprises 10g/L of peptone, 5g/L of yeast extract and 10g/L of NaCl, the solvent is distilled water, and the pH value is 7.0;
inoculating the seed solution into a fermentation culture medium at the volume concentration of 0.2%, culturing for 48h at 30 ℃ and 180rpm, centrifuging the fermentation liquor, removing the supernatant to obtain wet thalli, and freeze-drying to obtain the thalli freeze-dried powder described below. The fermentation medium comprises 10g/L of peptone, 5g/L of sucrose, 10g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate and 50g/L of olive oil, the solvent is distilled water, and the pH value is 7.0.
Example 2: preparation of validoxylamine A acetate (Compound 1) (R) 1 ＝CH 3 CO-，R 2 -R 8 ＝H)：
Validoxylamine A (1.34g, 4.0 mmol) and acetic acid (0.120g, 2.0 mmol) were sequentially added to a 50mL conical flask, and cyclohexane and t-butanol were added each 10mL. Shaking table at 45 deg.C for 30min, adding lyophilized thallus powder (1 g) after the reaction system is balanced sufficiently, and shaking table at 45 deg.C for 48h. After the reaction is finished, 20mL of trichloromethane is added to stop the reaction, the reaction solution is filtered and evaporated in a rotary manner, 20mL of deionized water is added, then 1M of sodium hydroxide solution is added until the pH is =7, then the mixture is separated by a macroporous adsorption resin column (HZ-801, shanghai Huasha science and technology Co., ltd.), gradient elution is carried out on the eluent which is 0% -55% of ethanol water solution, the target solution is collected, and the target solution is concentrated and dried to obtain 0.38g of validamine A acetate with the purity of 98.8%. And detecting the concentrated solution by TLC, wherein a developing solvent is n-propanol: water: acetic acid =4 (volume ratio) 1, developer is iodine vapor and ninhydrin solution.
1 H NMR(600MHz,DMSO)δ5.58(dd,J＝6.2,0.6Hz,1H),4.74(d,J＝0.9Hz,2H),4.06(s,1H),3.72(dd,J＝2.5,1.5Hz,1H),3.57–3.54(m,1H),3.52(dd,J＝12.2,8.0Hz,1H),3.35(t,J＝8.3Hz,1H),3.27(dd,J＝12.4,8.2Hz,1H),3.17(dd,J＝7.9,1.8Hz,1H),3.13–3.11(m,1H),3.09(dd,J＝9.2,6.3Hz,1H),2.08(d,J＝1.0Hz,1H),2.07(dd,J＝6.5,4.1Hz,1H),2.01(s,3H),1.76(s,1H),1.33–1.31(m,1H),1.07–1.05(m,1H).ESI-MS:[M+H]:378.1719.
Example 3: preparation of validoxylamine A propionate (Compound 2) (R) 1 ＝CH 3 CH 2 CO-，R 2 -R 8 ＝H)：
Validoxylamine A (1.34g, 4.0mmol) and propionic acid (0.148g, 2.0mmol) were sequentially added to a 50mL Erlenmeyer flask, and 10mL of each of cyclohexane and t-butanol was added. Shaking table at 45 deg.C for 30min, adding lyophilized thallus powder (1 g) after the reaction system is balanced sufficiently, and shaking table at 45 deg.C for 48h. And after the reaction is finished, adding 20mL of trichloromethane to stop the reaction, filtering the reaction solution, performing rotary evaporation, adding 20mL of deionized water, then adding 1M sodium hydroxide solution to achieve pH =7, separating by using a macroporous adsorption resin column, performing gradient elution on eluent which is 0-55% ethanol water solution with gradient concentration, collecting target solution, concentrating and drying to obtain 0.42g of validamine A propionate with the purity of 98.5%. And detecting the concentrated solution by TLC, wherein a developing solvent is n-propanol: water: acetic acid = 4.
1 H NMR(600MHz,DMSO)δ5.58(dd,J＝6.2,0.6Hz,1H),4.72(d,J＝0.6Hz,2H),4.06(dd,J＝4.2,0.7Hz,1H),3.85(dt,J＝5.5,3.7Hz,1H),3.70(dd,J＝9.4,4.3Hz,1H),3.60(dd,J＝9.3,4.2Hz,1H),3.52(dd,J＝12.4,7.6Hz,1H),3.27(dd,J＝12.4,7.6Hz,1H),3.19–3.14(m,2H),3.09(dd,J＝6.2,4.4Hz,1H),2.88–2.81(m,1H),2.48(dd,J＝7.5,2.9Hz,1H),2.46–2.42(m,2H),1.68(s,1H),1.37–1.26(m,1H),1.20(t,J＝6.8Hz,3H),1.09(dd,J＝8.2,3.6Hz,1H).
ESI-MS:[M+H] + :392.1867.
Example 4: preparation of validoxylamine A butyrate (Compound 3) (R) 1 ＝CH 3 CH 2 CH 2 CO-，R 2 -R 8 ＝H)：
Validoxylamine A (1.34g, 4.0 mmol) and butyric acid (0.176g, 2.0 mmol) were sequentially added to a 50mL conical flask, and cyclohexane and t-butanol were added each 10mL. Shaking table at 45 deg.C for 30min, adding lyophilized thallus powder (1 g) after the reaction system is balanced sufficiently, and shaking table at 45 deg.C for 48h. And after the reaction is finished, adding 20mL of trichloromethane to stop the reaction, filtering the reaction solution, performing rotary evaporation, adding 20mL of deionized water, adding 1M of sodium hydroxide solution to achieve pH =7, separating by using a macroporous adsorption resin column, performing gradient elution on eluent which is 0-55% of ethanol water solution with gradient concentration, collecting target solution, concentrating and drying to obtain 0.45g of validamine A butyrate with the purity of 98.2%. And detecting the concentrated solution by TLC, wherein a developing solvent is n-propanol: water: acetic acid = 4.
1 H NMR(600MHz,DMSO)δ4.73(d,J＝13.9Hz,2H),4.56(s,1H),4.41(dd,J＝9.5,5.8Hz,1H),4.31(s,1H),3.77(t,J＝8.4Hz,1H),3.53(dd,J＝10.4,3.8Hz,1H),3.49–3.41(m,1H),3.41–3.23(m,6H),3.09(t,J＝8.7Hz,1H),2.13(dt,J＝11.2,6.9Hz,1H),1.89(s,5H),1.87–1.80(m,1H),1.53(ddd,J＝14.9,11.7,5.8Hz,1H).
ESI-MS[M+H] + 406.2067,[M+Na] + 428.1890
Example 5: preparation of validamine a valerate (compound 4) (R) 1 ＝CH 3 CH 2 CH 2 CH 2 CO-，R 2 -R 8 ＝H)：
Validoxylamine A (1.34g, 4.0mmol) and valeric acid (0.204g, 2.0mmol) were sequentially added to a 50mL Erlenmeyer flask, and 10mL of each of cyclohexane and t-butanol was added. Shaking table at 45 deg.C for 30min, adding lyophilized thallus powder (1 g) after the reaction system is balanced sufficiently, and shaking table at 45 deg.C for 48h. And after the reaction is finished, adding 20mL of trichloromethane to stop the reaction, filtering the reaction solution, performing rotary evaporation, adding 20mL of deionized water, then adding 1M sodium hydroxide solution to achieve pH =7, separating by using a macroporous adsorption resin column, performing gradient elution on eluent which is 0-55% ethanol water solution, collecting target solution, concentrating and drying to obtain 0.46g of validamine A valerate, wherein the purity is 99.1%. And detecting the concentrated solution by TLC, wherein a developing solvent is n-propanol: water: acetic acid = 4.
1 H NMR(600MHz,DMSO)δ5.76(s,1H),5.44(s,1H),4.65(s,5H),4.17(dd,J＝10.6,2.8Hz,1H),4.09–3.86(m,3H),3.70(d,J＝4.7Hz,1H),2.28(t,J＝7.3Hz,2H),2.03–1.93(m,1H),1.77(d,J＝14.1Hz,1H),1.51(dt,J＝15.0,7.4Hz,2H),1.38–1.03(m,4H),0.86(t,J＝7.3Hz,3H).
ESI-MS[M+H] + 420.2227,[M+Na] + 442.2040
Example 6: preparation of validoxylamine A caproate (Compound 5)
(R 1 ＝CH 3 CH 2 CH 2 CH 2 CH 2 CO-，R 2 -R 8 ＝H)：
Validoxylamine A (1.34g, 4.0mmol) and hexanoic acid (0.232g, 2.0mmol) were sequentially added to a 50mL Erlenmeyer flask, and 10mL of each of cyclohexane and t-butanol was added. Shaking table at 45 deg.C for 30min, adding lyophilized thallus powder (1 g) after the reaction system is balanced sufficiently, and shaking table at 45 deg.C for 48h. And after the reaction is finished, adding 20mL of trichloromethane to stop the reaction, filtering the reaction solution, performing rotary evaporation, adding 20mL of deionized water, then adding 1M sodium hydroxide solution to achieve pH =7, separating by using a macroporous adsorption resin column, performing gradient elution on eluent which is 0-55% ethanol water solution with gradient concentration, collecting target solution, concentrating and drying to obtain 0.45g of valienamine A caproate with purity of 98.4%. And detecting the concentrated solution by TLC, wherein a developing solvent is n-propanol: water: acetic acid = 4.
1 H NMR(600MHz,DMSO)δ5.81(dd,J＝37.3,3.0Hz,1H),4.68(d,J＝13.1Hz,2H),4.35(d,J＝13.2Hz,1H),3.96(d,J＝17.8Hz,1H),3.69(dd,J＝11.3,5.7Hz,1H),3.49(d,J＝3.8Hz,31H),3.46–3.17(m,6H),3.11–3.02(m,1H),2.31(dt,J＝22.2,7.3Hz,1H),2.15(t,J＝7.4Hz,1H),1.74(dd,J＝14.1,3.2Hz,1H),1.56–1.36(m,2H),1.36–1.03(m,6H),,0.89(t,J＝6.9Hz,3H).
ESI-MS[M+H] + 434.2381,[M+Na] + 456.2189
Example 7: preparation of validoxylamine A heptanoate (Compound 6)
(R 1 ＝CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CO-，R 2 -R 8 ＝H)：
Valkanehydroxylamine A (1.34g, 4.0 mmol) and heptanoic acid (0.260g, 2.0 mmol) were sequentially added to a 50mL Erlenmeyer flask, and 10mL each of cyclohexane and t-butanol was added. Shaking table at 45 deg.C for 30min, adding thallus lyophilized powder (1 g) after the reaction system is balanced sufficiently, and shaking table at 45 deg.C for 48h. And after the reaction is finished, adding 20mL of trichloromethane to stop the reaction, filtering the reaction solution, performing rotary evaporation, adding 20mL of deionized water, adding 1M sodium hydroxide solution to achieve pH =7, separating by using a macroporous adsorption resin column, performing gradient elution on eluent which is 0-55% ethanol water solution, collecting target solution, concentrating and drying to obtain 0.48g of validamine A heptanoate with the purity of 98.5%. And detecting the concentrated solution by TLC, wherein a developing solvent is n-propanol: water: acetic acid = 4.
1 H NMR(600MHz,DMSO)δ5.79(dd,J＝37.3,3.0Hz,1H),4.62(d,J＝13.1Hz,2H),4.33(d,J＝13.2Hz,1H),3.94(d,J＝17.8Hz,1H),3.71(dd,J＝11.3,5.7Hz,1H),3.45(d,J＝3.8Hz,31H),3.43–3.17(m,6H),3.11–3.01(m,1H),2.31(dt,J＝22.2,7.3Hz,1H),2.18(t,J＝7.4Hz,1H),1.71(dd,J＝14.1,3.2Hz,1H),1.56–1.38(m,2H),1.36–1.03(m,8H),0.91(t,J＝6.9Hz,3H).
ESI-MS[M+H] + 448.2532,[M+Na] + 470.2341
Example 8: preparation of validoxylamine A octanoate (Compound 7)
(R 1 ＝CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CO-，R 2 -R 8 ＝H)：
Validoxylamine A (1.34g, 4.0 mmol) and octanoic acid (0.288g, 2.0 mmol) were added sequentially to a 50mL conical flask, and 10mL each of cyclohexane and t-butanol were added. Shaking table at 45 deg.C for 30min, adding lyophilized thallus powder (1 g) after the reaction system is balanced sufficiently, and shaking table at 45 deg.C for 48h. And after the reaction is finished, adding 20mL of trichloromethane to stop the reaction, filtering the reaction solution, performing rotary evaporation, adding 20mL of deionized water, adding 1M sodium hydroxide solution to achieve pH =7, separating by using a macroporous adsorption resin column, performing gradient elution on an eluent which is a 0-55% ethanol aqueous solution, collecting a target solution, concentrating and drying to obtain 0.51g of validamine A octanoate with a purity of 98.3%. And detecting the concentrated solution by TLC, wherein a developing solvent is n-propanol: water: acetic acid = 4.
1 H NMR(600MHz,DMSO)δ5.81(dd,J＝37.3,3.0Hz,1H),4.65(d,J＝13.1Hz,2H),4.31(d,J＝13.2Hz,1H),3.94(d,J＝17.8Hz,1H),3.71(dd,J＝11.3,5.7Hz,1H),3.45(d,J＝3.8Hz,31H),3.40–3.17(m,6H),3.11–3.01(m,1H),2.30(dt,J＝22.2,7.3Hz,1H),2.18(t,J＝7.4Hz,1H),1.74(dd,J＝14.1,3.2Hz,1H),1.56–1.40(m,2H),1.36–1.01(m,10H),0.91(t,J＝6.9Hz,3H).
ESI-MS[M+H] + 462.2686,[M+Na] + 484.2492
Example 9: preparation of validoxylamine nonanoate A (Compound 8)
(R 1 ＝CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CO-，R 2 -R 8 ＝H)：
Validoxylamine A (1.34g, 4.0 mmol) and nonanoic acid (0.316g, 2.0 mmol) were added sequentially to a 50mL conical flask, and 10mL each of cyclohexane and t-butanol were added. Shaking table at 45 deg.C for 30min, adding thallus lyophilized powder (1 g) after the reaction system is balanced sufficiently, and shaking table at 45 deg.C for 48h. And after the reaction is finished, adding 20mL of trichloromethane to stop the reaction, filtering the reaction solution, performing rotary evaporation, adding 20mL of deionized water, then adding 1M sodium hydroxide solution to achieve pH =7, separating by using a macroporous adsorption resin column, performing gradient elution on eluent which is 0% -55% of ethanol water solution with gradient concentration, collecting target solution, concentrating and drying to obtain 0.49g of validamine pelargonate A ester with the purity of 98.9%. And detecting the concentrated solution by TLC, wherein a developing solvent is n-propanol: water: acetic acid = 4.
1 H NMR(600MHz,DMSO)δ5.79(dd,J＝37.3,3.0Hz,1H),4.66(d,J＝13.1Hz,2H),4.45(d,J＝13.2Hz,1H),3.97(d,J＝17.8Hz,1H),3.71(dd,J＝11.3,5.7Hz,1H),3.48(d,J＝3.8Hz,31H),3.45–3.18(m,6H),3.13–3.04(m,1H),2.29(dt,J＝22.2,7.3Hz,1H),2.17(t,J＝7.4Hz,1H),1.76(dd,J＝14.1,3.2Hz,1H),1.58–1.40(m,2H),1.24(s,10H),1.16–1.00(m,2H),0.86(t,J＝6.9Hz,3H).
ESI-MS[M+H] + 476.2856,[M+Na] + 498.2616
Example 10: preparation of validoxylamine A decanoate (Compound 9)
(R 1 ＝CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CO-，R 2 -R 8 ＝H)：
Validoxylamine A (1.34g, 4.0 mmol) and decanoic acid (0.344g, 2.0 mmol) were added sequentially to a 50mL Erlenmeyer flask, and 10mL each of cyclohexane and t-butanol were added. Shaking table at 45 deg.C for 30min, adding lyophilized thallus powder (1 g) after the reaction system is balanced sufficiently, and shaking table at 45 deg.C for 48h. And after the reaction is finished, adding 20mL of trichloromethane to stop the reaction, filtering the reaction solution, performing rotary evaporation, adding 20mL of deionized water, then adding 1M sodium hydroxide solution to achieve pH =7, separating by using a macroporous adsorption resin column, performing gradient elution on eluent which is 0-55% ethanol water solution with gradient concentration, collecting target solution, concentrating and drying to obtain 0.55g of validamine A caprate ester with purity of 98.4%. And detecting the concentrated solution by TLC, wherein a developing solvent is n-propanol: water: acetic acid = 4.
1 H NMR(600MHz,DMSO)δ5.86(dd,J＝37.6,3.5Hz,1H),4.62(d,J＝13.0Hz,1H),4.48(d,J＝13.2Hz,1H),3.92(d,J＝18.1Hz,1H),3.65(dd,J＝11.0,5.7Hz,1H),3.43(d,J＝3.6Hz,1H),3.40–3.13(m,6H),3.11–3.04(m,1H),2.29(dt,J＝22.2,7.1Hz,1H),2.17(t,J＝7.7Hz,1H),1.81(dd,J＝14.5,3.6Hz,1H),1.58–1.42(m,2H),1.24(s,12H),1.16–0.98(m,2H),0.86(t,J＝6.5Hz,3H).
m/z(ESI-MS):[M+H] + 490.3008,[M+Na] + 512.2746.
Example 11: preparation of validoxylamine A n-undecanoate (Compound 10)
(R 1 ＝CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CO-，R 2 -R 8 ＝H)：
Validoxylamine A (1.34g, 4.0 mmol) and n-undecanoic acid (0.372g, 2.0 mmol) were sequentially added to a 50mL Erlenmeyer flask, and 10mL each of cyclohexane and t-butanol was added. Shaking table at 45 deg.C for 30min, adding lyophilized thallus powder (1 g) after the reaction system is balanced sufficiently, and shaking table at 45 deg.C for 48h. And after the reaction is finished, adding 20mL of trichloromethane to stop the reaction, filtering the reaction solution, performing rotary evaporation, adding 20mL of deionized water, then adding 1M of sodium hydroxide solution to achieve pH =7, separating by using a macroporous adsorption resin column, performing gradient elution on eluent which is 0-55% of ethanol water solution with gradient concentration, collecting target solution, concentrating and drying to obtain 0.52g of validamine n A-undecanoate with purity of 98.1%. And detecting the concentrated solution by TLC, wherein a developing solvent is n-propanol: water: acetic acid = 4.
1 H NMR(600MHz,DMSO)δ5.92(dd,J＝37.8,3.7Hz,1H),4.71(d,J＝13.5Hz,1H),4.48(d,J＝13.7Hz,1H),3.98(d,J＝17.2Hz,1H),3.71(dd,J＝11.8,5.4Hz,1H),3.46(d,J＝3.4Hz,1H),3.42–3.18(m,6H),3.13–3.06(m,1H),2.30(dt,J＝22.2,7.2Hz,1H),2.21(t,J＝7.4Hz,1H),1.85(dd,J＝14.0,3.2Hz,1H),1.56–1.42(m,2H),1.20(s,14H),1.16–0.98(m,2H),0.86(t,J＝6.3Hz,3H).
m/z(ESI-MS):[M+H] + 504.3161,[M+Na] + 527.1816.
Example 12: preparation of validoxylamine A dodecanoate (Compound 11):
(R 1 ＝CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CO-，R 2 -R 8 ＝H)
validoxylamine A (1.34g, 4.0 mmol) and n-dodecanoic acid (0.400g, 2.0 mmol) were sequentially added to a 50mL Erlenmeyer flask, and 10mL each of cyclohexane and t-butanol were added. Shaking table at 45 deg.C for 30min, adding lyophilized thallus powder (1 g) after the reaction system is balanced sufficiently, and shaking table at 45 deg.C for 48h. And after the reaction is finished, adding 20mL of trichloromethane to stop the reaction, filtering the reaction solution, performing rotary evaporation, adding 20mL of deionized water, then adding 1M of sodium hydroxide solution to achieve pH =7, separating by using a macroporous adsorption resin column, performing gradient elution on eluent which is 0% -55% of ethanol water solution with gradient concentration, collecting target solution, concentrating and drying to obtain 0.54g of validamine laurate-A ester with purity of 98.2%. And detecting the concentrated solution by TLC, wherein a developing solvent is n-propanol: water: acetic acid = 4.
1 H NMR(600MHz,DMSO)δ5.95(dd,J＝36.5,4.1Hz,1H),4.77(d,J＝12.8Hz,1H),4.50(d,J＝12.8Hz,1H),3.41(d,J＝17.8Hz,1H),3.768(dd,J＝11.3,5.7Hz,1H),3.43(d,J＝3.8Hz,1H),3.40–3.18(m,6H),3.13–3.06(m,1H),2.32(dt,J＝22.2,7.3Hz,1H),2.25(t,J＝7.4Hz,1H),1.86(dd,J＝14.1,3.2Hz,1H),1.56–1.42(m,2H),1.23(s,12H),1.16–0.92(m,4H),0.81(t,J＝6.8Hz,3H).
m/z(ESI-MS):[M+H] + 518.3310,[M+Na] + 540.2876.
Example 13: preparation of validoxylamine-a n-tetradecanoate (compound 12):
(R 1 ＝CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CO-，R 2 -R 8 ＝H)
validoxylamine A (1.34g, 4.0 mmol) and n-tridecanoic acid (0.428g, 2.0 mmol) were sequentially added to a 50mL Erlenmeyer flask, and 10mL each of cyclohexane and t-butanol was added. Shaking table at 45 deg.C for 30min, adding lyophilized thallus powder (1 g) after the reaction system is balanced sufficiently, and shaking table at 45 deg.C for 48h. And after the reaction is finished, adding 20mL of trichloromethane to stop the reaction, filtering the reaction solution, performing rotary evaporation, adding 20mL of deionized water, then adding 1M sodium hydroxide solution to achieve pH =7, separating by using a macroporous adsorption resin column, performing gradient elution on eluent which is 0-55% ethanol water solution in a gradient concentration, collecting target solution, concentrating and drying to obtain 0.49g of validamine n A tetradecanoate with the purity of 98.6%. And detecting the concentrated solution by TLC, wherein a developing solvent is n-propanol: water: acetic acid = 4.
1 H NMR(600MHz,DMSO)δ5.93(dd,J＝37.3,3.0Hz,1H),4.70(d,J＝13.1Hz,1H),4.50(d,J＝13.2Hz,1H),3.94(d,J＝17.4Hz,1H),3.72(dd,J＝11.3,5.7Hz,1H),3.48(d,J＝4.2Hz,1H),3.44–3.17(m,6H),3.13–3.02(m,1H),2.31(dt,J＝22.2,7.3Hz,1H),2.24(t,J＝7.4Hz,1H),1.87(dd,J＝14.1,3.2Hz,1H),1.65–1.40(m,2H),1.18(s,12H),1.15–0.81(m,6H),0.74(t,J＝6.4Hz,3H).
m/z(ESI-MS):[M+H] + 546.3624,[M+Na] + 568.3201
Example 14: preparation of validoxylamine A n-pentadecanoic acid (compound 13)
(R 1 ＝CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CO-，R 2 -R 8 ＝H)：
Validoxylamine A (1.34g, 4.0 mmol) and n-pentadecanoic acid (0.484g, 2.0 mmol) were added sequentially to a 50mL conical flask, and 10mL each of cyclohexane and t-butanol were added. Shaking table at 45 deg.C for 30min, adding lyophilized thallus powder (1 g) after the reaction system is balanced sufficiently, and shaking table at 45 deg.C for 48h. After the reaction is finished, 20mL of trichloromethane is added to stop the reaction, the reaction solution is filtered and steamed in a rotary mode, 20mL of deionized water is added, then 1M sodium hydroxide solution is added until the pH is =7, the separated sodium aliphatate is removed through filtration, then separation is carried out through a macroporous adsorption resin column, the eluent is 0% -55% of ethanol water solution with gradient concentration for gradient elution, the target solution is collected, and the target solution is concentrated and dried to obtain 0.58g of validamine A pentadecanoate with the purity of 98.4%. And detecting the concentrated solution by TLC, wherein a developing solvent is n-propanol: water: acetic acid = 4.
1 H NMR(600MHz,DMSO)δ5.58(dd,J＝6.2,0.6Hz,1H),4.76(d,J＝0.9Hz,2H),4.06(dd,J＝7.3,0.6Hz,1H),3.75(dd,J＝9.2,7.4Hz,1H),3.70(t,J＝8.8Hz,1H),3.52(dd,J＝12.4,7.1Hz,1H),3.27(dd,J＝12.4,7.1Hz,1H),3.19(t,J＝8.9Hz,1H),3.13–3.06(m,2H),2.83(dd,J＝10.5,8.7Hz,1H),2.61(dd,J＝16.9,7.8Hz,1H),2.36(t,J＝8.0Hz,2H),2.02(t,J＝7.9Hz,1H),1.97(s,1H),1.69(p,J＝7.8Hz,2H),1.40–1.29(m,23H),1.21–1.16(m,1H),0.99(t,J＝6.4Hz,3H).
m/z(ESI-MS):[M+H] + 560.3833,[M+Na] + 582.3432
Example 15: preparation of validoxylamine A n-hexadecanoic acid (compound 14)
(R 1 ＝CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CO-，R 2 -R 8 ＝H)：
Validoxylamine A (1.34g, 4.0 mmol) and n-hexadecanoic acid (0.512g, 2.0 mmol) were sequentially added to a 50mL Erlenmeyer flask, and 10mL each of cyclohexane and t-butanol was added. Shaking table at 45 deg.C for 30min, adding thallus lyophilized powder (1 g) after the reaction system is balanced sufficiently, and shaking table at 45 deg.C for 48h. After the reaction is finished, 20mL of trichloromethane is added to stop the reaction, the reaction solution is filtered and steamed in a rotary mode, 20mL of deionized water is added, then 1M sodium hydroxide solution is added until the pH is =7, the separated sodium aliphatate is removed by filtration, then separation is carried out through a macroporous adsorption resin column, the eluent is 0% -55% of ethanol water solution with gradient concentration for gradient elution, the target solution is collected, and the target solution is concentrated and dried to obtain 0.56g of validamine A-hexadecanoic acid ester with the purity of 98.4%. And detecting the concentrated solution by TLC, wherein a developing solvent is n-propanol: water: acetic acid = 4.
1 H NMR(600MHz,DMSO)δ5.58(dd,J＝6.2,0.6Hz,1H),4.74(d,J＝0.6Hz,2H),4.06(dd,J＝4.4,0.9Hz,1H),3.75(dd,J＝9.2,4.4Hz,1H),3.55–3.49(m,2H),3.27(dd,J＝12.4,7.0Hz,1H),3.22–3.17(m,1H),3.14–3.07(m,2H),2.83(dd,J＝10.3,8.1Hz,1H),2.49(dt,J＝9.3,7.6Hz,1H),2.32(t,J＝8.2Hz,2H),2.19(t,J＝8.0Hz,1H),1.71(p,J＝8.0Hz,2H),1.51(s,1H),1.41–1.26(m,25H),1.18(t,J＝7.9Hz,1H),0.99(t,J＝6.4Hz,3H).
m/z(ESI-MS):[M+H] + 574.4101,[M+Na] + 596.3614
Example 16: preparation of validoxylamine A n-octadecanoic acid (compound 15)
(R 1 ＝CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CO-，R 2 -R 8 ＝H)：
Validoxylamine A (1.34g, 4.0 mmol) and n-octadecanoic acid (0.568g, 2.0 mmol) were sequentially charged into a 50mL conical flask, and 10mL each of cyclohexane and t-butanol was added. Shaking table at 45 deg.C for 30min, adding lyophilized thallus powder (1 g) after the reaction system is balanced sufficiently, and shaking table at 45 deg.C for 48h. After the reaction is finished, 20mL of trichloromethane is added to stop the reaction, the reaction solution is filtered and steamed in a rotating mode, 20mL of deionized water is added, then 1M sodium hydroxide solution is added until the pH value is =7, the separated sodium aliphatate is removed through filtration, then separation is carried out through a macroporous adsorption resin column, gradient elution is carried out on eluent which is 0% -55% of ethanol water solution with gradient concentration, target solution is collected, concentration and drying are carried out, and 0.54g of validamine A octadecanoic acid ester with the purity of 98.6% is obtained. And detecting the concentrated solution by TLC, wherein a developing solvent is n-propanol: water: acetic acid = 4.
1 H NMR(500MHz,Chloroform)δ5.58(dd,J＝6.2,0.6Hz,1H),4.74(d,J＝0.9Hz,2H),4.06(dd,J＝4.2,0.6Hz,1H),3.75(dd,J＝8.9,4.3Hz,1H),3.52(dd,J＝12.4,7.6Hz,1H),3.50–3.46(m,1H),3.27(dd,J＝12.4,7.6Hz,1H),3.18–3.13(m,1H),3.12–3.07(m,2H),2.83(dd,J＝10.4,8.1Hz,1H),2.59(dt,J＝9.2,7.7Hz,1H),2.26(dt,J＝11.2,8.1Hz,3H),1.70(p,J＝8.0Hz,2H),1.43–1.30(m,29H),1.17(s,1H),1.11(t,J＝8.0Hz,1H),0.99(t,J＝6.5Hz,3H).
m/z(ESI-MS):[M+H] + 602.4508,[M+Na] + 624.5136
Example 17: preparation of 7, 8-Octenoic acid validoxylamine A ester (Compound 16) (R) 1 ＝CH 2 ＝CHCH 2 CH 2 CH 2 CH 2 CH 2 CO-，R 2 -R 8 ＝H)：
Validoxylamine A (1.34g, 4.0 mmol) and 7, 8-octenoic acid (0.28g, 2.0 mmol) were sequentially added to a 50mL conical flask, and 10mL each of cyclohexane and t-butanol were added. Shaking table at 45 deg.C for 30min, adding lyophilized thallus powder (1 g) after the reaction system is balanced sufficiently, and shaking table at 45 deg.C for 72h. And after the reaction is finished, adding 20mL of trichloromethane to stop the reaction, filtering the reaction solution, performing rotary evaporation, adding 20mL of deionized water, then adding 1M sodium hydroxide solution to achieve pH =7, separating by using a macroporous adsorption resin column, performing gradient elution on eluent which is 0% -55% of ethanol water solution with gradient concentration, collecting target solution, concentrating and drying to obtain 0.62g of 7, 8-octenoic acid validoxylamine A ester with purity of 97.9%. And detecting the concentrated solution by TLC, wherein a developing solvent is n-propanol: water: acetic acid = 4.
1 H NMR(600MHz,DMSO)δ5.65(ddt,J＝16.3,10.0,6.1Hz,1H),5.58(dd,J＝6.2,0.6Hz,1H),5.02–4.97(m,1H),4.97–4.93(m,1H),4.77(d,J＝0.9Hz,2H),4.06(d,J＝1.3Hz,1H),3.77–3.72(m,2H),3.52(dd,J＝12.5,7.5Hz,1H),3.35(s,1H),3.27(dd,J＝12.5,7.5Hz,1H),3.22–3.17(m,1H),3.14–3.11(m,1H),3.09(t,J＝4.3Hz,1H),2.36(dt,J＝9.8,7.9Hz,3H),2.08(t,J＝8.0Hz,1H),2.02(dd,J＝14.2,7.8Hz,2H),1.74–1.66(m,3H),1.38–1.34(m,4H),1.34–1.30(m,1H),1.15(t,J＝7.9Hz,1H).
m/z(ESI-MS):[M+H] + :460.2506
Example 18: preparation of validoxylamine A Trichlorooctanoate (Compound 17) (R) 1 ＝CCl 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CO-，R 2 -R 8 ＝H)：
Validoxylamine A (1.34g, 4.0 mmol) and trichlorooctanoic acid (0.50g, 2.0 mmol) were sequentially added to a 50mL Erlenmeyer flask, and 10mL each of cyclohexane and t-butanol were added. Shaking table at 45 deg.C for 30min, adding thallus lyophilized powder (1 g) after the reaction system is balanced sufficiently, and shaking table at 45 deg.C for 72h. And after the reaction is finished, adding 20mL of trichloromethane to stop the reaction, filtering the reaction solution, performing rotary evaporation, adding 20mL of deionized water, then adding 1M sodium hydroxide solution to achieve pH =7, separating by using a macroporous adsorption resin column, performing gradient elution on eluent which is 0-55% ethanol water solution with gradient concentration, collecting target solution, concentrating and drying to obtain 0.49g of validoxylamine A trichlorooctanoate with purity of 98.1%. And detecting the concentrated solution by TLC, wherein a developing solvent is n-propanol: water: acetic acid = 4.
1 H NMR(500MHz,Chloroform)δ5.58(dd,J＝6.2,0.6Hz,1H),4.74(d,J＝0.6Hz,2H),4.06(dd,J＝2.6,0.6Hz,1H),4.00(dd,J＝7.0,3.3Hz,1H),3.74(t,J＝3.1Hz,1H),3.52(dd,J＝12.4,7.1Hz,1H),3.35(t,J＝8.3Hz,1H),3.27(dd,J＝12.4,7.1Hz,1H),3.21–3.15(m,1H),3.11–3.06(m,1H),2.82(dd,J＝10.3,8.3Hz,1H),2.43(dt,J＝9.2,7.7Hz,1H),2.37–2.28(m,4H),1.90(t,J＝7.9Hz,1H),1.73(p,J＝8.0Hz,2H),1.51(s,1H),1.42–1.32(m,6H),1.31(d,J＝4.1Hz,1H),1.24(t,J＝7.9Hz,1H).
m/z(ESI-MS):[M+H] + :565.1462
Example 19: determination of antibacterial Activity
The measurement method adopted is a hyphal growth rate method. The method comprises the following specific operations: taking an activated R.solani bacterial dish from the edge of a potato culture medium by using a puncher with the diameter of 6.0mm, respectively inoculating the bacterial dish with the hypha facing downwards to the middles of a drug-containing agar culture medium plate and a control group culture medium plate, culturing in a constant-temperature incubator at 25 ℃, measuring by using a vernier caliper when the diameter of a control bacterial colony is between 50mm and 80mm (36 h is generally needed by R.solani), calculating the diameter of the bacterial colony from the average value of the data, and finally calculating the inhibition rate. The hypha growth inhibition rate calculation formula 4-1 (length unit: mm) is as follows:
in the experimental process, the inhibition rate obtained by diluting a plurality of test agents to the concentration of 1000 mu g/mL is taken as a reference, if the inhibition rate is 0, the test agents are abandoned, and if the inhibition rate is more than 0, the test agents are used for carrying out the determination of the antibacterial activity by the concentration gradients of 500 mu g/mL, 250 mu g/mL, 125 mu g/mL, 62.5 mu g/mL, 31.25 mu g/mL and the like.
Regression equation of toxicity of medicine and effective intermediate concentration EC 50 And EC 5 The calculation of (2): the virulence of the drug on r.solani is expressed as the hyphal growth inhibition. Converting the inhibition rate of the hyphal growth into an inhibition rate value (y), converting the medicament concentration into a concentration logarithm (x), calculating a regression equation determination coefficient R of the inhibition rate value of the medicament to R.solani according to a least square method, and respectively calculating the inhibition concentration EC of the medicament to the hyphal growth according to a virulence regression equation 5 And EC 50 The value is obtained.
Table 1 virulence equation of different drugs on r
As seen from the table above, the compounds have good biological activity, wherein the inhibition effect of part of the compounds on rice sheath blight is higher than that of validamycin A, and even the lowest action effect is 2-3 orders of magnitude lower than that of validamycin A or validamycin A. Therefore, the compound has huge application prospect and commercial value, and is expected to be developed into a novel efficient and green bactericide for replacing validamycin.
Example 20: preparation of valienamine A-tert-butanol saturated solution
In an oil bath kettle at 90 ℃, valienamine A (5 g) is refluxed and stirred in tertiary butanol (300 mL) for 5h to prepare a valienamine A-tertiary butanol saturated solution.
In an oil bath kettle at 90 ℃, valienamine A (5 g) is stirred in tert-butyl alcohol (300 mL) for 30 hours without reflux to prepare valiolamine A-tert-butyl alcohol saturated solution.
The solubility of the two solutions is shown in table 2:
TABLE 2
Example 21:
in an oil bath kettle at 90 ℃, valienamine A (5 g) is refluxed and stirred in tertiary butanol (300 mL) for 5 hours to prepare valienamine A-tertiary butanol saturated solution. Fully mixing a valienamine A-tert-butanol saturated solution (21mL, 0.5mmoL) and undecanoic acid (1.0mmoL, 186.33mg) in a reaction vessel at 40 ℃, adding lipase Lipozyme RMIM (100 mg) and a 4A-degree molecular sieve (1 g) which is activated at 400 ℃ for 2 hours, oscillating and reacting at 40 ℃, sampling after reacting for 30 hours, adding water for diluting, passing through a membrane, and analyzing by a high performance liquid chromatography; wherein the mobile phase is a mixture of a PBS solution with pH =7.0 and methanol (wherein the volume fraction of methanol is 3%). And (3) analyzing results by a high performance liquid chromatograph: the validoxylamine a conversion rates were 26.87%, respectively.
Centrifuging the reaction solution to remove molecular sieve and catalyst, evaporating to remove solvent, adding water, adjusting pH to neutral with sodium hydroxide, separating with macroporous adsorbent resin column (HZ-801, shanghai Huasha science and technology Co., ltd.), gradient eluting with 0-55% ethanol water solution as eluting reagent, collecting target solution, concentrating, drying to obtain product 1 Characterization by H NMR (600MHz, DMSO) and m/z (ESI-MS) showed that it was the same as compound 10 above, validoxylamine A undecanoate in 98% purity.
Example 22: synthesis of validamine undecanoate by screening optimal lipase
The lipase was changed, and other reaction steps were the same as in example 21, and the obtained products were validoxylamine undecanoate, wherein the hplc analysis results are shown in the following table:
table 3: different lipases a Catalytic validoxylamine A conversion rate
a The lipases listed in the table were all purchased from Xiamen Kogya Biotech, inc.
The results show that the Lipozyme RMIM has the highest catalytic efficiency, and the validamine A conversion rate reaches 26.87%.
Example 23: optimization of conditions for synthesizing validamine A undecanoate by Lipozyme RMIM catalysis-reaction time
Fully mixing a valienamine A-tert-butanol saturated solution (21mL, 0.5mmoL) and undecanoic acid (1.0mmoL, 186.33mg) in a reaction vessel at 40 ℃, adding Lipozyme RMIM (100 mg) and a 4A-degree molecular sieve (1 g) which is activated at 400 ℃ for 2 hours, oscillating and reacting at 40 ℃, sampling in the reaction process, adding water for dilution, passing through a membrane, and analyzing by a high performance liquid chromatograph; wherein the mobile phase is a mixture of a PBS solution with pH =7.0 and 3% methanol. The hplc analysis results are shown in table 4:
TABLE 4 conversion of validoxylamine A at different reaction times
The results show that the optimum catalysis time of Lipozyme RMIM is 48 hours, and the conversion rate of valixylamine A reaches 26.54%.
Example 24: condition optimization-temperature for synthesizing validamine A undecanoate by Lipozyme RMIM catalysis
Mixing valienamine A-tert-butanol saturated solution (21 mL,0.5 mmoL) and undecanoic acid (1.0 mmoL, 186.33mg) in a reaction vessel (at 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C), adding lipase (100 mg) and 4A molecular sieve (1 g) activated at 400 deg.C for 2 hr, shaking at 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, reacting for 48 hr, sampling, diluting with water, sieving, and analyzing with high performance liquid chromatography; wherein, the mobile phase is a mixed solution of PBS solution with pH =7.0 and 3% methanol. The hplc analysis results are shown in table 5:
table 5: validoxylamine A conversion at different temperatures
The results showed that the optimum catalytic temperature of Lipozyme RMIM was 45 ℃.
Example 25: condition optimization-molecular sieve addition for synthesizing validamine undecanoate A under catalysis of Lipozyme RMIM
Mixing valienamine A-tert-butanol saturated solution (21 mL,0.5 mmoL) and undecanoic acid (1.0 mmoL, 186.33mg) in a reaction vessel at 45 deg.C, adding lipase (100 mg) and 4A ° molecular sieve (0 g, 0.2g, 0.4g, 0.6g, 0.8g, 1.0 g) activated at 400 deg.C for 2 hr, shaking at 45 deg.C for reaction for 48 hr, sampling, diluting with water, passing through membrane, and analyzing with HPLC; wherein, the mobile phase is a mixed solution of PBS solution with pH =7.0 and 3% methanol. The hplc analysis results are shown in table 6:
TABLE 6 conversion of validoxylamine A at different molecular sieve addition
The results showed that the optimum molecular sieve amount was 1.2g/mmoL.
Example 26: optimization of conditions for synthesizing validamine undecanoate A under catalysis of Lipozyme RMIM-substrate molar ratio
Mixing valienamine A-tert-butanol saturated solution (21 mL,0.5 mmoL) and undecanoic acid (0.125mmoL, 0.167mmoL,0.25mmoL,0.5mmoL,1.0mmoL,1.5mmoL,2.0 mmoL) in a reaction vessel at 45 deg.C, adding lipase (100 mg) and 4A ° molecular sieve (0.6 g) activated at 400 deg.C for 2 hr, oscillating at 45 deg.C, reacting for 48 hr, sampling, diluting with water, sieving, and analyzing with high performance liquid chromatography; wherein the mobile phase is a mixture of a PBS solution with pH =7.0 and 3% methanol. The hplc analysis results are shown in table 7:
TABLE 7 conversion of validamine A at different substrate molar ratios
The results show that the optimum molar ratio for the Lipozyme RMIM catalyzed reaction is 1.
The reaction solution obtained in different substrate molar ratios was worked up in the same manner as in example 21, and all the products were validoxylamine A undecanoate.
Example 27: optimization of conditions for synthesizing validamine undecanoate A under catalysis of Lipozyme RMIM-enzyme addition
Fully mixing a valienamine A-tert-butanol saturated solution (21 mL,0.5 mmoL) and undecanoic acid (1.5 mmoL,279.495 mg) in a reaction vessel at 45 ℃, then adding lipase (20mg, 40mg,60mg,80mg,100mg, 120mg) and a 4A-degree molecular sieve (0.6 g) which is activated at 400 ℃ for 2 hours to perform oscillation reaction at 45 ℃, sampling after the reaction is performed for 48 hours, adding water for dilution, performing film passing, and analyzing by a high performance liquid chromatography; wherein the mobile phase is a mixture of a PBS solution with pH =7.0 and 3% methanol. The hplc analysis results are shown in table 8:
TABLE 8 conversion of validoxylamine A at different enzyme dosages
As a result, it was found that the optimum amount of the enzyme was 80mg, that is, 160g/moL, in the case of 21mL of the reaction system.
In conclusion, in the validamine A-tert-butyl alcohol saturated solution, when Lipozyme RMIM is used as a catalyst, the molar ratio of the validamine A to the undecanoic acid is 1.
Claims (13)
1. A preparation method of valienamine A ester shown in formula (I) comprises the following steps: reacting validamine A with undecanoic acid in tert-butyl alcohol under the action of a biocatalyst to prepare an esterified product of validamine A shown in a formula (I); the biocatalyst is Lipozyme RMIM;
R=C n H 2n+1 wherein n =10.
2. The method of claim 1, wherein: the feeding mode of the raw materials is as follows: refluxing and stirring validamine A in tertiary butanol at 90-110 ℃ for 4-6h to prepare validamine A saturated solution, and mixing the validamine A saturated solution and undecanoic acid in a reaction container.
3. The method of claim 2, wherein: the preparation method is implemented as follows: fully mixing a valienamine A-tert-butanol saturated solution and undecanoic acid in a reaction vessel at 25-50 ℃, then adding a biocatalyst for oscillation reaction at 25-50 ℃, and after full reaction, separating and purifying the reaction liquid to obtain an esterified product of valienamine A; the feeding molar ratio of the valienamine A to the undecanoic acid is 4-1.
4. The method of claim 3, wherein: and adding an activated molecular sieve into the reaction system, wherein the dosage of the molecular sieve is 0.4-2g/mmoL in terms of the mole number of the validoxylamine A.
5. The method of claim 4, wherein: the dosage of the molecular sieve is 1.2-2.0g/mmoL calculated by the mole number of the validoxylamine A.
6. The method of claim 5, wherein: the dosage of the molecular sieve is 1.2g/mmoL calculated by the mole number of the valienamine A.
7. The method of claim 3, wherein: the feeding molar ratio of the valienamine A to the undecanoic acid is 1; the dosage of the biocatalyst is 160-240g/moL based on the mole number of the validoxylamine A.
8. The method of claim 7, wherein: the feeding molar ratio of the valienamine A to the undecanoic acid is 1.
9. The method of claim 7, wherein: the amount of the biocatalyst used was 160g/moL based on the number of moles of validamine a.
10. The method of claim 3, wherein: the reaction temperature is 45-50 ℃; the reaction time was 48h.
11. The method of claim 10, wherein: the reaction temperature was 45 ℃.
12. The method of claim 3, wherein the reaction conditions are: the molar ratio of validamine A to undecanoic acid is 1.
13. The method of claim 4, wherein: the separation and purification steps are specifically as follows: centrifuging to remove the biocatalyst and the molecular sieve, evaporating to remove the solvent, adding water, adjusting the pH value to be neutral by using sodium hydroxide, separating by using a macroporous adsorption resin column, performing gradient elution by using an ethanol water solution with the volume concentration of 0-55% as an elution reagent, collecting a target solution, concentrating and drying.
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