CN114478280A - Improved process for the preparation of polyaspartic acid esters from 1,5-pentanediamine salts - Google Patents
Improved process for the preparation of polyaspartic acid esters from 1,5-pentanediamine salts Download PDFInfo
- Publication number
- CN114478280A CN114478280A CN202111386011.4A CN202111386011A CN114478280A CN 114478280 A CN114478280 A CN 114478280A CN 202111386011 A CN202111386011 A CN 202111386011A CN 114478280 A CN114478280 A CN 114478280A
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- China
- Prior art keywords
- water
- pentanediamine
- pda
- hydrochloride
- free
- Prior art date
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- 238000002360 preparation method Methods 0.000 title claims description 14
- 150000002148 esters Chemical class 0.000 title description 12
- 108010064470 polyaspartate Proteins 0.000 title description 12
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- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
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Abstract
The application provides a method for preparing free PDA by desalting and purifying PDA hydrochloride obtained by biological fermentation, which comprises the following steps: converting lysine hydrochloride to PDA hydrochloride using lysine decarboxylase; preparing the PDA hydrochloride into an aqueous solution, and adding an alkali metal hydroxide into the aqueous solution; evaporating most of water under reduced pressure; adding a certain amount of lower aliphatic alcohol; filtering the generated chloride; evaporating most of the solvent under reduced pressure; optionally, the alcohol addition is repeated and the chloride filtered offAnd evaporating the solvent at least once under reduced pressure; and free PDA containing a small amount of the above alcohol and a very small amount of water was obtained. The present application also provides a process for preparing an asparagus resin of formula (I) from PDA hydrochloride obtained from a biological fermentation, the process comprising: reacting diethyl fumarate with the free PDA under certain conditions; and carrying out post-treatment on the obtained reaction liquid to obtain the product, namely the asparagus resin shown in the formula (I).
Description
Technical Field
The present application relates to the production of Polyaspartic Acid Esters (PAE). More particularly, the present application describes an improved process for the production of polyaspartic acid esters from 1,5-pentanediamine hydrochloride using a solvent-free process that can produce Polyaspartic Acid Esters (PAE) from free 1,5-pentanediamine at a lower cost, in simpler equipment, and in higher yields, with very low levels of piperidine impurities.
Background
Polyaspartic Acid Esters (PAEs), also known as Polyaspartic acid resins or aspartic acid resins, are a special class of sterically hindered secondary amines that can be obtained by polymerizing aspartate. The PAE resin contains amino and carboxyl groups, has strong chelation, adsorption and other effects, is widely applied to the fields of bioengineering, pharmaceutical chemistry, agriculture and forestry planting and the like, and an aspartic acid polymerization product with excellent biodegradation performance is bound to be widely applied along with the increasing severe problem of environmental pollution.
In addition, aspartic polyurea resins prepared from the reaction of polyaspartic esters and isocyanates have attracted increasing attention in applications other than coatings due to their good mechanical properties, and are referred to as third-generation polyureas, and their use in the market has been increasing. The use of polyaspartic acid esters for the preparation of polyurea coatings has been known for nearly a decade. The polyurea coating has excellent ageing resistance, ultraviolet light resistance and excellent wear resistance and waterproof performance, and is mainly applied to industrial terraces, outdoor road pavement and waterproof surface coating of swimming pools and water orchards in the early stage. In recent years, polyaspartic ester polyurea coatings have also begun to be used in the field of industrial coatings, on the one hand, because such polyurea coatings can be rapidly cured at low temperatures, even at room temperature, and because the surface properties, mechanical properties and chemical resistance of the paint film all exceed or are equivalent to those of conventional high-temperature baking paints, they are high-performance coatings that can be baked free; on the other hand, as a solvent-based coating, the construction solid content of the polyurea coating is usually more than 65%, which is far higher than 35-45% of the construction solid content of the traditional high-temperature baking paint. For the two reasons, the polyurea industrial coating taking the polyaspartic acid ester as the raw material is a type of energy-saving and environment-friendly coating.
An asparagus resin is N, N-pentamethylene-bis (diethyl aspartate) with the following structure (I):
the aspartic resin of formula (I) is formed by the Michael addition reaction of a primary amine and a double bond between 1,5-pentanediamine and diethyl fumarate. The specific reaction is shown in scheme 1 below.
Scheme 1
One of the raw materials for synthesizing the asparagus resin of the formula (I) is 1,5-pentanediamine (1,5-pentanediamine, PDA), also known as cadaverine (cadaverine), 1,5-diaminopentane (1, 5-diaminopentane). PDA can not be extracted from petroleum, only a biological fermentation method can carry out large-scale production at present, and many reports on the biological preparation of the pentanediamine are reported, and a commonly used method is a microbial fermentation method, namely, a whole cell catalysis method takes lysine as a substrate and utilizes lysine decarboxylase in somatic cells to catalyze and produce the pentanediamine. The biofermentation method first obtains PDA hydrochloride, and in order to carry out the reaction of the above scheme 1, it is first necessary to carry out a process of liberating PDA hydrochloride to obtain free PDA. For the dissociation process, at present, the most economical and convenient method is to perform dissociation by reacting an equivalent amount of aqueous solution of sodium hydroxide with hydrochloric acid (HCl) contained in PDA hydrochloride to generate sodium chloride, and the dissociated aqueous solution is separated and purified by a rectifying tower. The method can ensure that the purity of the free PDA reaches more than 99.5 percent, and does not contain solvents such as ethanol and the like. However, this method requires a rectification step, and the direct consequence of rectification is that the production and equipment costs are greatly increased; in addition, the PDA rectification purification process needs higher temperature (boiling point of 150-.
For example, when the ring-closed by-product is piperidine, it is reacted with diethyl fumarate in scheme 2 below to give 2-piperidinyl-diethyl succinate (ZZA for short). The presence of such cyclic impurities in the aspartic resin of formula (I) results in a decrease in the yield of the resin and can severely affect the quality of the resin and downstream products.
Scheme 2
Accordingly, the present invention seeks to overcome the above-mentioned technical challenges encountered in the production of aspartic resins of formula (I), and thereby provide a process which can produce PDA and subsequent aspartic resins of formula (I) at higher purity at lower cost, simpler equipment and at relatively lower temperatures.
Disclosure of Invention
In a first aspect, the present application provides a method for desalting and purifying PDA hydrochloride obtained by biological fermentation to obtain free PDA, the method comprising:
(1) converting lysine hydrochloride to PDA hydrochloride using lysine decarboxylase;
(2) preparing the PDA hydrochloride into an aqueous solution, and adding an alkali metal hydroxide into the aqueous solution;
(3) evaporating most of water under reduced pressure;
(4) adding a certain amount of lower aliphatic alcohol;
(5) filtering the generated chloride;
(6) evaporating most of the solvent under reduced pressure;
(7) optionally, repeating steps (4) - (6) at least once; and
(8) free PDA is obtained which contains small amounts of the above-mentioned alcohol and very little water.
In a second aspect, the present application provides a process for preparing N, N-pentamethylenebis (diethyl aspartate) (i.e., an aspartic resin of formula (I)) from PDA hydrochloride obtained from a biological fermentation, the process comprising:
(1) converting lysine hydrochloride to PDA hydrochloride using lysine decarboxylase;
(2) preparing the PDA hydrochloride into an aqueous solution, and adding an alkali metal hydroxide into the aqueous solution;
(3) evaporating most of water under reduced pressure;
(4) adding a certain amount of lower aliphatic alcohol;
(5) filtering the generated chloride;
(6) evaporating most of the solvent under reduced pressure;
(7) optionally, repeating steps (4) - (6) at least once; and
(8) obtaining free PDA containing a small amount of the above alcohol and a very small amount of water;
(9) providing diethyl fumarate;
(10) reacting diethyl fumarate with the free PDA in the step (8) under certain conditions; and
(11) and (4) carrying out post-treatment on the reaction liquid in the step (10) to obtain a product N, N-pentamethylene di (diethyl aspartate).
Drawings
In the drawings:
FIG. 1 shows the preparation of Polyaspartic Acid Esters (PAE)1H NMR spectrum.
Detailed Description
Definition of
Headings and other identifiers, such as (1), (2), (a), (B), etc., are given for ease of reading the specification and claims only. The use of headings or other identifiers in the specification or claims does not necessarily require that the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.
The use of the words "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one", but is also consistent with the meaning of "one or more", "at least one", and "one or more than one".
The term "about" is used to indicate that a numerical value includes the standard deviation of error for the device or method being used in order to determine the value. Generally, the term "about" is intended to mean up to 10% of the possible variations. Thus, variations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10% of a value are included in the term "about". Unless otherwise indicated, the term "about" used in connection with a range also applies to both endpoints of the range.
The terms "comprising" (and any form of comprising), "having" (and any form of having), "including" (and any form of including), or "containing" (and any form of containing) as used herein are inclusive or open-ended and do not exclude additional unrecited elements or process/method steps.
Sequence listing
The present application contains a sequence listing in computer readable form created on day 28/9/2020, which is approximately 12kb in size. This computer readable form is incorporated by reference into this application.
In a first aspect, the present application provides a method for purifying biologically fermented PDA hydrochloride to produce free PDA, the method comprising:
(1) converting lysine hydrochloride to PDA hydrochloride using lysine decarboxylase;
(2) preparing the PDA hydrochloride into an aqueous solution, and adding an alkali metal hydroxide into the aqueous solution;
(3) evaporating most of water under reduced pressure;
(4) adding a certain amount of lower aliphatic alcohol;
(5) filtering the generated chloride;
(6) evaporating most of the solvent under reduced pressure;
(7) optionally, repeating steps (4) - (6) at least once; and
(8) free PDA with little or no piperidine content was obtained.
Preferably in the process of the first aspect of the invention, the lysine decarboxylase in step (1) is a highly active lysine decarboxylase having an amino acid sequence as set forth in SEQ ID NO:2, which can be added at a significantly lower lysine decarboxylase addition rate as compared to the prior art, wherein the lysine decarboxylase addition rate is defined as the ratio of the added weight of lysine decarboxylase on a lysine decarboxylase cell dry basis to the weight of lysine in the lysine fermentation broth on a lysine salt molecular weight basis.
Preferably in the process of the first aspect of the present invention, the alkali metal hydroxide in step (2) is selected from: sodium hydroxide, potassium hydroxide, lithium hydroxide. Among them, it is preferable that the alkali metal hydroxide is sodium hydroxide.
Preferably in the process of the first aspect of the present invention, in step (3), 70% or more of the water is distilled off. More preferably, in step (3), more than 75% of the water is distilled off. Still preferably, in step (3), 80% or more of water is distilled off. Further preferably, in step (3), 84% or more of water is distilled off.
Preferably in the process of the first aspect of the present invention, the lower aliphatic alcohol in step (4) is selected from: methanol, ethanol, n-propanol, isopropanol. More preferably, the lower aliphatic alcohol in step (4) is ethanol.
Preferably in the process of the first aspect of the present invention, the chloride in step (5) is sodium chloride.
Preferably in the method of the first aspect of the present invention, steps (4) to (6) are repeated once. Alternatively, steps (4) - (6) are not repeated. Alternatively, steps (4) - (6) are repeated twice. Alternatively, steps (4) - (6) are repeated more than twice.
Preferably in the process of the first aspect of the invention the obtained free PDA has a piperidine content of less than 0.01%, more preferably less than 0.001%, more preferably less than 0.0001%, even more preferably the obtained free PDA is completely free of piperidine.
In a second aspect, the present application provides a process for preparing N, N-pentamethylenebis (diethyl aspartate) (i.e., an aspartic resin of formula (I)) from PDA hydrochloride obtained from a biological fermentation, the process comprising:
(1) converting lysine hydrochloride to PDA hydrochloride using lysine decarboxylase;
(2) preparing the PDA hydrochloride into an aqueous solution, and adding an alkali metal hydroxide into the aqueous solution;
(3) evaporating most of water under reduced pressure;
(4) adding a certain amount of lower aliphatic alcohol;
(5) filtering the generated chloride;
(6) evaporating most of the solvent under reduced pressure;
(7) repeating steps (4) - (6) at least once; and
(8) obtaining free PDA containing a small amount of the above alcohol and a very small amount of water;
(9) providing diethyl fumarate;
(10) reacting diethyl fumarate with the free PDA in the step (8) under certain conditions; and
(11) and (4) carrying out post-treatment on the reaction liquid in the step (10) to obtain a product N, N-pentamethylene di (diethyl aspartate).
Preferably, in the method of the second aspect of the present invention, the lysine decarboxylase in step (1) is a high-activity lysine decarboxylase having an amino acid sequence as set forth in SEQ ID NO:2, which can be added at a significantly lower lysine decarboxylase addition rate as compared to the prior art, wherein the lysine decarboxylase addition rate is defined as the ratio of the added weight of lysine decarboxylase on a lysine decarboxylase cell dry basis to the weight of lysine in the lysine fermentation broth on a lysine salt molecular weight basis.
Preferably in the process of the second aspect of the present invention, the alkali metal hydroxide in step (2) is selected from: sodium hydroxide, potassium hydroxide, lithium hydroxide. Among them, it is preferable that the alkali metal hydroxide is sodium hydroxide.
Preferably, in the method of the second aspect of the present invention, in the step (3), 70% or more of water is distilled off. More preferably, in step (3), more than 75% of the water is distilled off. Still preferably, in step (3), 80% or more of water is distilled off. Further preferably, in step (3), 84% or more of water is distilled off.
Preferably in the process of the second aspect of the present invention, the lower aliphatic alcohol in step (4) is selected from: methanol, ethanol, n-propanol, isopropanol. More preferably, the lower aliphatic alcohol in step (4) is ethanol.
Preferably in the process of the second aspect of the present invention, the chloride in step (5) is sodium chloride.
Preferably in the method of the second aspect of the present invention, steps (4) to (6) are repeated once. Alternatively, steps (4) - (6) are not repeated. Alternatively, steps (4) - (6) are repeated twice. Alternatively, steps (4) - (6) are repeated more than twice.
Preferably in the method of the second aspect of the invention the obtained free PDA has a piperidine content of less than 0.01%, more preferably less than 0.001%, more preferably less than 0.0001%, even more preferably the obtained free PDA is completely free of piperidine.
Preferably, in the method of the second aspect of the present invention, the certain conditions in step (10) are that diethyl fumarate is added at low temperature under nitrogen protection and reacted at room temperature. More preferably, the low temperature is 0 ℃.
Preferably, in the process of the second aspect of the present invention, the post-treatment of step (11) is a low boiler removal treatment. More preferably, the low boiler removal treatment is carried out at a pressure of about 1-2mmHg and a temperature of about 120 ℃.
In addition, the highly active lysine decarboxylase described herein can be used in significantly reduced amounts during fermentation due to its higher activity. The highly active lysine decarboxylase described herein is a lysine decarboxylase treated by a process comprising the steps of: (1) a polypeptide comprising a sequence represented by SEQ ID NO:1, transforming the plasmid of the lysine decarboxylase kdc gene into an escherichia coli cell; (2) selecting the transformed positive single colony, inoculating an LB (10g/L peptone, 5g/L yeast extract powder, 10g/L sodium chloride) test tube culture medium, and culturing at 30 ℃ and 180RPM overnight; (3) a shake flask containing TB medium (12g/L peptone, 24g/L yeast extract, 4g/L glycerol) was inoculated with 5% inoculum from the overnight culture; (4) putting the shake flask into a shaking table for culturing under the following culture conditions: culturing at 30 deg.C and 250RPM for about 2 hr; (5) adding isopropyl beta-D-1-thiogalactopyranoside (IPTG) to induce and express protein, wherein the induction conditions are as follows: inducing at 30 deg.C and 250RPM for about 4 hr; (6) the fermentation broth was centrifuged to collect the cells and the centrifuged wet cells were stored at-80 ℃.
Examples
Example A: versatile materials and methods
The following reference materials were used in the examples: recombinant DNA manipulations generally follow the methods described in the following documents: sambrook et al, 2001. Restriction enzyme, T4 DNA ligase, Rapid DNA ligation kit, SanPrep Column DNA gel extraction kit, Plasmid Mini-Prep kit, and agarose were purchased from Sangon Biotech (Shanghai, China). The TE buffer contained 10mM Tris-HCl (pH 8.0) and 1mM Na2EDTA (pH 8.0). The TAE buffer contained 40mM Tris-acetate (pH 8.0) and 2mM Na2EDTA。
In example B, restriction enzyme digestion was performed in buffer provided by Sangon Biotech.
A typical restriction enzyme digest comprises: 0.8 μ g DNA in 8 μ L TE, 2 μ L restriction enzyme buffer (10 Xconcentration), 1 μ L bovine serum albumin (0.1mg/mL), 1 μ L restriction enzyme, and 8 μ LTE. The reaction was incubated at 37 ℃ for 1 hour and analyzed by agarose gel electrophoresis. The DNA used for the cloning experiments was digested, the reaction was terminated by heating at 70 ℃ for 15 minutes, and then the DNA was extracted using the SanPrep Column DNA gel extraction kit. The concentration of DNA in the sample was determined as follows. Aliquots of DNA (10. mu.L) were diluted to 1mL in TE and the absorbance at 260nm relative to the absorbance of TE was measured. The DNA concentration was calculated based on the fact that the absorbance at 260nm of 50. mu.g/mL of the double-stranded DNA was 1.0.
Agarose gels typically comprise: 0.7% agarose (w/v) in TAE buffer. Ethidium bromide (0.5. mu.g/ml) was added to the agarose to allow visualization of the DNA fragments under UV light. Agarose gels were run in TAE buffer. Two sets of 1kb Plus DNA Ladder from Sangon Biotech were used to determine the size of the DNA fragments.
NCO-terminated polyurethane prepolymer: reaction product of IPDI with LM2000 polyether diol, NCO% ═ 7.5%.
Example B: cloning, expression and Activity testing of lysine decarboxylase expressed in E.coli
The E.coli lysine decarboxylase kdc (2-keto-acid decarboxylase) gene was synthesized and cloned into pET21a (Millipore Sigma, formerly Novagen). The wild-type kdc nucleic acid sequence from E.coli strain BW25113(E.C.4.1.1.18) is represented by SEQ ID NO:1 and the amino acid sequence is represented by SEQ ID NO:2, annotated as lysine decarboxylase.
The plasmid containing the kdc gene was transformed into BL21(DE3) E.coli cells. The empty plasmid pET21a was also transformed into a negative control. For enzyme expression and characterization experiments, flasks containing 40mL of TB were inoculated at 5% from overnight culture and shaken. The flask was incubated at 30 ℃ for 2 hours with shaking at 250rpm, then induced to produce protein with 0.2mM isopropyl β -D-1-thiogalactopyranoside (IPTG), and incubated at 30 ℃ for another 4 hours with shaking. Cells were collected by centrifugation and aggregates were stored at-80 ℃.
KDC enzyme activity was assessed using a pH-based in vitro assay. Enzyme activity was tested using commercial lysine-HCl salt. Unless otherwise indicated, all chemicals were purchased from Sigma-Aldrich Chemical Company (St. Louis, Mo.). First, cells were lysed using a bench-top sonicator according to the manufacturer's instructions. The cell lysate was partially clarified by centrifugation (14,000g for 5 minutes). The protein concentration of the resulting clarified lysate was measured by the Bradford protein assay kit (Sangon Biotech) according to the manufacturer's instructions. Lysates were normalized by protein concentration by dilution in 10mM Tris buffer. The normalized lysates were then diluted 1:5 in 10mM Tris buffer. 20 μ L of lysate was added to each well for multi-well plate analysis. Each condition was repeated three times.
The reaction mixture contained 15% lysine-HCl, 0.04% pyridoxal-5' -phosphate (PLP). By adding 1M H2SO4And 1N NaOH adjusted the pH of each reaction mixture to about pH 6.5. The lysate was then added to the reaction mixture simultaneously with the addition of 1M H2SO4The pH was kept constant at 6.5. Record H2SO4The amount is used to calculate the activity of the enzyme. When the addition of H is no longer necessary2SO4The assay reaction was complete while maintaining the pH at 6.5.
Example C: fermentation of transformed E.coli overexpressing KDC
For the present examples, growth media were prepared as follows: all solutions were prepared in distilled deionized water. LB medium (1L) contained: bactoTMtryptone (i.e., enzymatic digest of casein) (10g), BactoTMYeast extract (i.e., autolysed water-soluble fraction of yeast extract) (5g), and NaCl (10 g). The LB-glucose medium comprises 1L of LB medium: glucose (10g), MgSO4(0.12g), and thiamine hydrochloride (0.001 g). LB-freezing buffer contained in 1L of LB medium: k2HPO4(6.3g)，KH2PO4(1.8g)，MgSO4(1.0g)，(NH4)2SO4(0.9g), sodium citrate dihydrate (0.5g), and glycerol (44 mL). The M9 salt (1L) comprises: na (Na)2HPO4(6g)，KH2PO4(3g)，NH4Cl (1g), and NaCl (0.5 g). M9 minimal medium comprises in 1L M9 salts: d-glucose (10g), MgSO4(0.12g), and thiamine hydrochloride (0.001 g). Antibiotics were added to the following final concentrations when appropriate: ampicillinPenicillin (Ap), 50. mu.g/mL; chloramphenicol (Cm), 20. mu.g/mL; kanamycin (Kan), 50. mu.g/mL; tetracycline (Tc), 12.5. mu.g/mL. Antibiotic stock solutions were prepared in water, except for two: chloramphenicol was prepared in 95% ethanol, and tetracycline was prepared in 50% aqueous ethanol. Aqueous stock solutions of IPTG were prepared at various concentrations.
The standard fermentation medium (1L) contained: k2HPO4(7.5g), ferric ammonium citrate (III) (0.3g), citric acid monohydrate (2.1g), and concentrated H2SO4(1.2 mL). By adding concentrated NH prior to autoclaving4OH adjusted the pH of the fermentation medium to 7.0. The following supplements were added just before the start of fermentation: d-glucose; MgSO (MgSO)4(0.24 g); potassium; and trace minerals including (NH)4)6(Mo7O24)·4H2O(0.0037g)，ZnSO4·7H2O(0.0029g)，H3BO3(0.0247g)，CuSO4·5H2O (0.0025g), and MnCl2·4H2O (0.0158 g). IPTG stock solutions were added as necessary (e.g. when the optical density at 600nm was between 15-20) to the final concentrations indicated. Glucose feed solution and MgSO4(1M) autoclaving of the solution. By mixing 300g of glucose with 280mL of H2O mixing to prepare a glucose feed solution (650 g/L). The solution of trace minerals and IPTG was sterilized through a 0.22- μm membrane. Antifoam agent (Sigma 204) was added to the fermentation broth as required. Typical wet E.coli cell densities reach 120 g/L.
Example 1: conversion of lysine hydrochloride to PDA hydrochloride Using lysine decarboxylase
To produce Pentamethylenediamine (PDA) -HCl, 2g of wet engineered E.coli containing lysine decarboxylase was added to 1L of a 200g/L lysine hydrochloride solution containing 0.1g/L PLP. The pH was maintained at 6.5 with HCl. The temperature of the solution was raised to 37 ℃. The reaction was then started and continued for 10 hours while maintaining the pH at 6.5.
The reaction mixture was passed through a 0.2 micron microfiltration membrane (to remove large particles such as cells, bacterial debris and aggregates) and a 10kDa ultrafiltration membrane (to remove proteins and other soluble macromolecules in the culture medium). Recrystallization was carried out by adding ethanol at 15 ℃ to obtain white solid PDA hydrochloride (PDA-HCl).
Example 2A: desalting PDA hydrochloride and purifying with ethanol once with water
PDA hydrochloride (175g, 1mol) obtained in example 1 was dissolved in 300g of water, and sodium hydroxide (80g, 2mol) was added thereto, followed by stirring at room temperature for 1 hour. Then dehydrated under reduced pressure of 40mmHg at 60 deg.C. After about 250g of water had evaporated, 100g of ethanol was added to the remaining reaction solution, and the mixture was stirred for 1 hour. The precipitated sodium chloride is filtered off. The filtrate was subjected to reduced pressure evaporation at 40 ℃ under 40mmHg to remove the solvent, to obtain 100.58g of a colorless transparent solution, i.e., free PDA, in a yield of 98.6%, containing 0.7% of ethanol residue and having a water content of 0.52%.
Example 2B: PDA hydrochloride was desalted and twice water-borne purification with ethanol
PDA hydrochloride (175g, 1mol) obtained in example 1 was dissolved in 300g of water, and sodium hydroxide (80g, 2mol) was added thereto, followed by stirring at room temperature for 1 hour. Then dehydrating under reduced pressure of 40mmHg at 60 deg.C. After about 250g of water had evaporated, 100g of ethanol was added to the remaining reaction solution, and the mixture was stirred for 1 hour. The precipitated sodium chloride was filtered off. The filtrate was evaporated to remove the solvent under reduced pressure at 40 ℃ and 40 mmHg. After most of the solvent was distilled off, 100g of ethanol was added to the remaining reaction solution, and the mixture was stirred for 1 hour. The precipitated sodium chloride is filtered off. The filtrate was subjected to reduced pressure evaporation at 40 ℃ under 40mmHg to remove the solvent, to obtain 99.55g of a colorless transparent solution, i.e., free PDA, in a yield of 97.6%, with a sample containing 0.8% of residual ethanol and a residual water content of 0.04%.
Example 2B-1: desalting PDA hydrochloride, twice purifying with water by using ethanol and further removing solvent
The solvent of the free PDA obtained in example 2B was further distilled off under reduced pressure at 60 ℃ and 20mmHg to obtain 72.83g of a colorless transparent liquid, and the total yield was found to be 71.4%, and the sample contained less than 0.01% of ethanol residue, less than 0.01% of water residue, and no piperidine and ZZA were detected.
Example 2C: desalting PDA hydrochloride and purifying with ethanol for three times with water
PDA hydrochloride (175g, 1mol) obtained in example 1 was dissolved in 300g of water, and sodium hydroxide (80g, 2mol) was added thereto, followed by stirring at room temperature for 1 hour. Then dehydrating under reduced pressure of 40mmHg at 60 deg.C. After about 250g of water had evaporated, 100g of ethanol was added to the remaining reaction solution, and the mixture was stirred for 1 hour. The precipitated sodium chloride was filtered off. The filtrate was subjected to reduced pressure distillation at 40 ℃ under 40mmHg to remove the solvent. After most of the solvent was distilled off, 100g of ethanol was added to the remaining reaction solution, and the mixture was stirred for 1 hour. The filtration was continued, and the solvent was distilled off from the filtrate under reduced pressure of 40mmHg at 40 ℃. After most of the solvent was distilled off, 100g of ethanol was added to the remaining reaction solution, and the mixture was stirred for 1 hour. The precipitated sodium chloride is filtered off. The filtrate was subjected to reduced pressure evaporation at 40 ℃ under 40mmHg to remove the solvent, to obtain 97.82g of a colorless transparent solution, i.e., free PDA, in a yield of 95.9%, containing 0.8% of ethanol residue and having a water content of 0.02%.
Example 2D: PDA hydrochloride was desalted and purified by atmospheric distillation
PDA hydrochloride (175g, 1mol) obtained in example 1 was dissolved in 300g of water, and sodium hydroxide (80g, 2mol) was added thereto, followed by stirring at room temperature for 1 hour. The obtained aqueous solution was put into a rectifying column and subjected to separation and purification at normal pressure and a temperature of about 150 ℃ to 160 ℃ to obtain 99.35g of free PDA, with a yield of 97.4%, a sample containing no ethanol, a moisture-remaining amount of 0.02%, and a piperidine content of 1.12%.
Example 2E: PDA hydrochloride was desalted and purified by distillation under reduced pressure
PDA hydrochloride (175g, 1mol) obtained in example 1 was dissolved in 300g of water, and sodium hydroxide (80g, 2mol) was added thereto, followed by stirring at room temperature for 1 hour. The obtained aqueous solution was subjected to separation and purification in a vacuum distillation column at a pressure of 30mmHg and a temperature of 100 ℃ and 110 ℃ to obtain 93.23g of free PDA, the yield was 91.4%, the sample contained no ethanol, the moisture content was 0.02%, and the piperidine content was 0.75%.
Example 3: preparation of N, N-Pentamethylene bis (diethyl aspartate) (PAE) from PDA
PDA (102g, 1mol) obtained in example 2A was placed in a 1L four-necked flask and dissolved in175Putting diethyl fumarate (516g, 3mol) into a 1L dropping funnel in ethanol, opening the dropping funnel under the protection of nitrogen, slowly dropping diethyl fumarate into a four-mouth bottle, stirring for 16 hours, and removing low-boiling-point substances from the obtained reaction solution at the pressure of 1-2mmHg and the temperature of 120 ℃. After 6 hours, 417.2g of a clear yellowish liquid, i.e. PAE, were obtained in a yield of 93.5%1The purity by HNMR was 97.9%.
Example 3': preparation of N, N-pentamethylenebis (aspartic acid diethyl ester) (PAE) from PDA
PDA (102g, 1mol) obtained in example 2A was placed in a 1L four-necked flask and cooled to 0 ℃ with an ice-water bath. Diethyl fumarate (516g, 3mol) was placed in a 1L dropping funnel. The whole system was protected with nitrogen. The dropping funnel is opened, diethyl fumarate is slowly dropped into the four-mouth bottle, the temperature of the system in the reaction bottle is controlled not to exceed 30 ℃, and the dropping is finished in about 1 hour. The ice-water bath was removed and the reaction was allowed to warm to room temperature (25-30 ℃) and stirred for 16 hours to give a colorless slightly viscous liquid. The reaction solution is subjected to low boiling point substance removal treatment under the pressure of 1-2mmHg and the temperature of 120 ℃ while stirring. After 6 hours 415.8g of a clear, slightly yellow liquid, i.e. PAE, were obtained in a yield of 93.2%1The purity by HNMR was 97.2%.
Example 4: preparation of N, N-pentamethylenebis (aspartic acid diethyl ester) (PAE) from PDA
PDA (102g, 1mol) obtained in example 2B was placed in a 1L four-necked flask and dissolved in300Putting diethyl fumarate (516g, 3mol) into a 1L dropping funnel in ethanol, opening the dropping funnel under the protection of nitrogen, slowly dropping diethyl fumarate into a four-mouth bottle, stirring for 16 hours, and removing low-boiling-point substances from the obtained reaction solution at the pressure of 1-2mmHg and the temperature of 120 ℃. At 6 hoursAfter this time, 430.1g of a clear, slightly yellow liquid, i.e. PAE, was obtained in a yield of 96.4%1The purity by HNMR was 99.2%.
Example 4': preparation of N, N-pentamethylenebis (aspartic acid diethyl ester) (PAE) from PDA
PDA (102g, 1mol) obtained in example 2B was placed in a 1L four-necked flask and cooled to 0 ℃ with an ice-water bath. Diethyl fumarate (516g, 3mol) was placed in a 1L dropping funnel. The whole system was protected with nitrogen. The dropping funnel is opened, diethyl fumarate is slowly dropped into the four-mouth bottle, the temperature of the system in the reaction bottle is controlled not to exceed 30 ℃, and the dropping is finished in about 1 hour. The ice-water bath was removed and the reaction was allowed to warm to room temperature (25-30 ℃) and stirred for 16 hours to give a colorless slightly viscous liquid. The reaction solution is subjected to low boiling point substance removal treatment under the pressure of 1-2mmHg and the temperature of 120 ℃ while stirring. After 6 hours 433.7g of a clear, slightly yellow liquid, i.e. PAE, were obtained in 97.2% yield1Purity by HNMR was greater than 99.6%.
Example 5: preparation of N, N-pentamethylenebis (aspartic acid diethyl ester) (PAE) from PDA
PDA (102g, 1mol) obtained in example 2D was placed in a 1L four-necked flask and cooled to 0 ℃ with an ice-water bath. Diethyl fumarate (516g, 3mol) was placed in a 1L dropping funnel. The whole system was protected with nitrogen. The dropping funnel is opened, diethyl fumarate is slowly dropped into the four-mouth bottle, the temperature of the system in the reaction bottle is controlled not to exceed 30 ℃, and the dropping is finished in about 1 hour. The ice-water bath was removed and the reaction was allowed to warm to room temperature (25-30 ℃) and stirred for 16 hours to give a colorless slightly viscous liquid. The reaction solution is subjected to low boiling point substance removal treatment under the pressure of 1-2mmHg and the temperature of 120 ℃ while stirring. After 6 hours 428.3g of a clear, slightly yellow liquid, i.e. PAE, were obtained in a yield of 96.0%1The purity by HNMR was 97.3%.
Example 6: preparation of N, N-pentamethylenebis (aspartic acid diethyl ester) (PAE) from PDA
PDA (102g, 1mol) obtained in example 2E was placed in a 1L four-necked flaskThe ice water bath cooled it to 0 ℃. Diethyl fumarate (516g, 3mol) was placed in a 1L dropping funnel. The whole system was protected with nitrogen. The dropping funnel is opened, diethyl fumarate is slowly dropped into the four-mouth bottle, the temperature of the system in the reaction bottle is controlled not to exceed 30 ℃, and the dropping is finished in about 1 hour. The ice-water bath was removed and the reaction was allowed to warm to room temperature (25-30 ℃) and stirred for 16 hours to give a colorless slightly viscous liquid. The reaction solution is subjected to low boiling point substance removal treatment under the pressure of 1-2mmHg and the temperature of 120 ℃ while stirring. After 6 hours 431.5g of a clear, slightly yellow liquid, i.e. PAE, were obtained in a yield of 96.7%1The purity by HNMR was 98.4%.
TABLE 1
TABLE 2
Examples 2A-2E relate to the desalination purification of biologically fermented PDA hydrochloride to free PDA in different ways, wherein examples 2A-2C use the process according to the invention and examples 2D-2E use the usual rectification method. As can be seen from the results in Table 1 above, examples 2A-2C, although having relatively high residual amounts of ethanol and water, were completely free of any piperidine type by-product; whereas example 2D-2E was completely free of ethanol and had relatively low residual moisture levels, but had significantly higher amounts of piperidine-based by-products. It can also be seen that the yields of free PDA obtained by the process of the invention and by the atmospheric distillation process are substantially comparable.
The method utilizes the characteristic that sodium chloride is insoluble in ethanol, removes the sodium chloride in the system by a method of adding ethanol after distilling most of water, and simultaneously utilizes the boiling point of the ethanol to carry out azeotropy to distill off both water and the ethanol. In this way, the method of the present invention avoids the use of relatively expensive rectification columns for the rectification step and thus the high temperatures involved in the desalination process (e.g. about 150-.
Examples 3, 3 ', 4', 5 and 6 relate to the preparation of PAE with different starting materials free PDA and in the presence or absence of solvent. Examples 3 and 3 'both used the starting material free PDA, obtained by a single aqueous purification of ethanol, to perform the michael addition reaction with diethyl fumarate, except that example 3 was performed using an ethanol solvent, whereas example 3' was performed without using an ethanol solvent, and the product PAE was obtained in a yield of 93% or more, a purity of 97% or more, and completely free of any piperidine-derived impurity ZZA. Examples 4 and 4 'both used ethanol, twice purified with water, to give the starting material free PDA, for the michael addition reaction with diethyl fumarate, except that example 4 used an ethanol solvent for the reaction, whereas example 4' reacted without an ethanol solvent, with a product PAE yield of 96% or more, a purity of 99% or more, and completely free of any piperidine-derived impurity ZZA; in particular, example 4' gave a yield of 97.2% and a purity as high as 99.6%. Examples 4 'and 4 "both performed michael addition reactions using ethanol as the solvent, except that example 4' used the starting material free PDA of example 2B (residual amount of ethanol was 0.8%), and example 4" used the starting material free PDA of example 2B-1 (residual amount of ethanol was 0.01%). It can be seen that examples 4' and 4 "have essentially the same yield and purity of the product PAE and do not contain the piperidine-derived impurity ZZA. This indicates that ethanol, whether present in trace amounts or in large amounts as a solvent, does not substantially affect the final yield and purity of PAE during the preparation of PAE from PDA. However, adding a step of removing the ethanol solvent in the previous PDA production process greatly reduced the PDA yield (71.4% compared to 97.6% for example 2B), resulting in a cost-ineffective overall process. Examples 5 and 6 the raw material free PDA obtained by atmospheric distillation and vacuum distillation, respectively, was subjected to michael addition reaction with diethyl fumarate without using an ethanol solvent, and although the product PAE was obtained in yields of 96% or more (96.0% and 96.7%, respectively) as compared with examples 4 and 4 ', the purity was only in the range of 97% to 98%, which was comparable to examples 3 and 3'; however, the most serious drawback of the product is that it contains a significantly higher amount of the piperidine-derived impurity ZZA (1.4% and 1.0%), which seriously affects the quality of the product PAE.
Example 7: performance determination of polyaspartic acid ester polyurea waterproofing coatings made from PAE
The PAE obtained in example 4' was labeled PAE4#, and the PAE obtained in example 5 was labeled PAE5 #. The polyaspartate polyureas were prepared by reacting PAE4# and PAE5# with NCO-terminated polyurethane prepolymer (100g, 0.05% excess in terms of NCO to PAE equivalents), respectively. The formulation and properties of the resulting polyurea coatings are shown in Table 3 below, where the test standards for spray polyurea waterproofing coatings were performed in accordance with GB/T23446-2009.
TABLE 3
NCO-terminated polyurethane prepolymers are the reaction products of IPDI and LM2000 polyether diols, where the% NCO is tested according to HG/T2409-
As can be seen from the data in Table 3, the polyurea coating 1# prepared by using PAE4# has high purity, no impurities, long gel time, flat coating and mechanical properties meeting the national standard II type standard. In contrast, the polyurea coating 2# prepared from PAE5# contains tertiary amine impurities, and the tertiary amine impurities promote the reaction of NCO and water, so that the film has defects after being sprayed and formed into a film under the same condition, the problems of poor surface smoothness of an orange peel coating and the like are easy to occur, and the field painting effect and the waterproof capability are influenced; particularly, the coating effect is influenced under the construction condition of high humidity (more than 60 percent of relative humidity), micro bubbles are easily generated on the surface, and the product quality is influenced; the gel speed is too high, and the requirement on spraying equipment is high; and insufficient tensile properties due to coating film defects.
Therefore, the PAE prepared by the method of the invention does not contain cyclic tertiary amine impurities, has high purity and can prepare high-quality waterproof paint.
Claims (40)
1. A method for desalting and purifying 1,5-pentanediamine hydrochloride obtained by biological fermentation to obtain free 1,5-pentanediamine, wherein the method comprises the following steps:
(1) converting lysine hydrochloride to 1,5-pentanediamine hydrochloride using lysine decarboxylase;
(2) preparing the obtained 1,5-pentanediamine hydrochloride into an aqueous solution, and adding an alkali metal hydroxide into the aqueous solution;
(3) evaporating most of water under reduced pressure;
(4) adding a certain amount of lower aliphatic alcohol;
(5) filtering the generated chloride;
(6) evaporating most of the solvent under reduced pressure;
(7) optionally, repeating steps (4) - (6) at least once; and
(8) free 1,5-pentanediamine containing a small amount of the above alcohol and a very small amount of water is obtained.
2. The method according to claim 1, wherein the amino acid sequence of the lysine decarboxylase in step (1) is as set forth in SEQ ID NO:2, which is added at a low lysine decarboxylase addition rate.
3. The process according to claim 1 or 2, wherein the alkali metal hydroxide in step (2) is selected from the group consisting of: sodium hydroxide, potassium hydroxide, lithium hydroxide.
4. A process according to any one of claims 1 to 3, wherein the alkali metal hydroxide in step (2) is sodium hydroxide and the chloride produced in step (5) is sodium chloride.
5. The process according to any one of claims 1 to 4, wherein in step (3), 70% or more of water is distilled off.
6. The process according to any one of claims 1 to 5, wherein in step (3), more than 75% of the water is distilled off.
7. The process of any one of claims 1 to 6, wherein in step (3), more than 80% of the water is distilled off.
8. The process of any one of claims 1 to 7, wherein in step (3), 84% or more of the water is distilled off.
9. The process according to any one of claims 1 to 8, wherein the lower aliphatic alcohol in step (4) is selected from the group consisting of: methanol, ethanol, n-propanol, isopropanol.
10. The process according to any one of claims 1 to 9, wherein the lower aliphatic alcohol in step (4) is ethanol.
11. The method of any one of claims 1 to 10, wherein steps (4) - (6) are repeated once.
12. The method of any one of claims 1 to 11, wherein steps (4) - (6) are not repeated.
13. The method of any one of claims 1 to 12, wherein steps (4) - (6) are repeated twice.
14. The method of any one of claims 1 to 13, wherein steps (4) - (6) are repeated more than twice.
15. The process according to any one of claims 1 to 14, wherein the piperidine content of the resulting free 1,5-pentanediamine is less than 0.01%.
16. The method of claim 15, wherein the piperidine content is less than 0.001%.
17. The method of claim 15, wherein the piperidine content is less than 0.0001%.
18. The process of claim 15, wherein the resulting free 1,5-pentanediamine is completely free of piperidine.
19. A process for the preparation of an asparagus resin of formula (I) from 1,5-pentanediamine hydrochloride that is biologically fermented, said process comprising:
(1) converting lysine hydrochloride to 1,5-pentanediamine hydrochloride using lysine decarboxylase;
(2) preparing the obtained 1,5-pentanediamine hydrochloride into an aqueous solution, and adding an alkali metal hydroxide into the aqueous solution;
(3) evaporating most of water under reduced pressure;
(4) adding a certain amount of lower aliphatic alcohol;
(5) filtering the generated chloride;
(6) evaporating most of the solvent under reduced pressure;
(7) optionally, repeating steps (4) - (6) at least once; and
(8) obtaining free 1,5-pentanediamine containing a small amount of the above alcohol and a very small amount of water;
(9) providing diethyl fumarate;
(10) reacting diethyl fumarate with the free 1,5-pentanediamine in the step (8) under certain conditions; and
(11) and (4) carrying out post-treatment on the reaction liquid in the step (10) to obtain the asparagus resin in the formula (I).
20. The method according to claim 19, wherein the amino acid sequence of the lysine decarboxylase in step (1) is as set forth in SEQ ID NO:2, which is added at a low lysine decarboxylase addition rate.
21. The process according to claim 19 or 20, wherein the alkali metal hydroxide in step (2) is selected from the group consisting of: sodium hydroxide, potassium hydroxide, lithium hydroxide.
22. The process according to any one of claims 19 to 21, wherein the alkali metal hydroxide in step (2) is sodium hydroxide and the chloride produced in step (5) is sodium chloride.
23. The process of any one of claims 19 to 22, wherein in step (3) more than 70% of the water is distilled off.
24. A process according to any one of claims 19 to 23, wherein in step (3) more than 75% of the water is distilled off.
25. The process of any one of claims 19 to 24, wherein in step (3) more than 80% of the water is distilled off.
26. The process of any one of claims 19 to 25, wherein in step (3) more than 84% of the water is distilled off.
27. The process according to any one of claims 19 to 26, wherein the lower aliphatic alcohol in step (4) is selected from the group consisting of: methanol, ethanol, n-propanol, isopropanol.
28. The process according to any one of claims 19 to 27, wherein the lower aliphatic alcohol in step (4) is ethanol.
29. The method of any one of claims 19 to 28, wherein steps (4) - (6) are repeated once.
30. The method of any one of claims 19 to 29, wherein steps (4) - (6) are not repeated.
31. The method of any one of claims 19 to 30, wherein steps (4) - (6) are repeated twice.
32. The method of any one of claims 19-31, wherein steps (4) - (6) are repeated more than twice.
33. A process according to any one of claims 19 to 32, wherein the piperidine content of the resulting free 1,5-pentanediamine is less than 0.01%.
34. The method of claim 33, wherein the piperidine content is less than 0.001%.
35. The method of claim 33, wherein the piperidine content is less than 0.0001%.
36. The process of claim 33, wherein the resulting free 1,5-pentanediamine is completely free of piperidine.
37. The method according to any one of claims 19 to 36, wherein the certain conditions in step (10) are that diethyl fumarate is added at low temperature under nitrogen protection and reacted at room temperature.
38. The method of claim 37, wherein the low temperature is 0 ℃.
39. The process according to any one of claims 19 to 38, wherein the work-up of step (11) is a low boiler removal process.
40. The process of claim 39 wherein the low boiler removal treatment is carried out at a pressure of about 1-2mmHg and a temperature of about 120 ℃.
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