University of Groningen
Polyamide synthesis by hydrolases
Schwab, Leendert Willem
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Chapter 3 Polycondensation of diesters and diamines catalyzed by lipases A and B from Candida antarctica yeast 3.1
ABSTRACT This chapter discusses the solvent free enzyme catalyzed polycondensation of diesters and diamines. The reactions are monitored with ATR FTIR spectroscopy to determine the rate of the enzymatic polycondensation. The influences of temperature and changing ester and amine monomers are discussed. At three different temperatures the polycondensation of dimethyladipate and diethylene triamine was studied. Below 60 °C the enzymatic and the uncatalyzed reaction proceed with equal rates. At 60 °C however, the uncatalyzed reaction is 5 times as fast as the enzymatic reaction. The diester and diamine components were varied and the reaction was evaluated at 60 °C. The rate of reaction by N435 was not influenced by changing the diester from dimethyladipate to diethyladipate. N435 catalyzed the polycondensation of dimethyladipate with a series of diamines in the order butanediamine > 4,9‐dioxa‐
1,12‐dodecanediamine > diethylene triamine. Experiments with Candida antarctica lipase A showed catalysis at 60 °C but at a lower rate than the N435. Measurements at room temperature showed a negliable catalytic effect of the enzyme. The lack of polymer formation when the enzymatic polycondensation of diesters and diamines is performed in a solvent is due to a preference of N435 for monoamidation of diesters. A condensation of dimethylsuccinate with a two‐fold excess of butylamine showed mainly the mono‐amidated succinate. 3.2
IN TRODUC TIO N The synthesis of amide bonds by enzyme (Candida antarctica lipase B) catalyzed condensation of dicarboxylic esters and diamines is applied mainly for the enantiomeric resolution of secondary amines.1,2 Despite the tendency of carboxylic acids and amines to form salts this reaction is reported by Prasad and co‐workers.3 The immobilized Candida antarctica lipase B (N435) catalyzes the aminolysis of activated esters4,5 and non‐activated esters.6 Other lipases and esterases can be employed in these reactions but are less versatile in substrate tolerance and stability towards organic solvents.7,8,9 Although Gotor et al. used bifunctional reactants in one of their articles6, polymerization was not mentioned. This research aims at synthesizing polyamides by an enzymatic (N435) catalyzed polycondensation of diesters and diamines. Initial experiments in organic solvents (DMSO, NMP, 1,4‐dioxane, toluene) failed to produce polymer. Experiments with dimethylsuccinate and two‐fold excess of butylamine revealed that the N435 synthesized mainly a monoamide see Figure 3‐1. As a consequence we changed to a solvent free system. Figure 3‐1. Monoamidation of dimethylsuccinate by butylamine catalyzed by N435. Enzymatic polymerization in the bulk proved to be accompanied by a significant uncatalyzed reaction especially at elevated temperatures (60 °C). Because this uncatalyzed condensation is present it is unclear how much the N435 contributes to the polymerization. Cheng and Gu published the solvent free polycondensation of diethylene triamine with dialkylesters. They showed that different lipases catalyze the polymerization at temperatures from 50 to 90 °C resulting in polyamides with a Mw of 3,000‐15,000.10,11. In their experiments a high temperature and a N2‐flow assisted the removal of the leaving group (methanol) in favor of the uncatalyzed reaction. To find out what the actual enzymatic contribution to this polycondensation is, we studied the reaction with IR‐spectroscopy. Apart from the diethylene triamine and dimethyladipate we also included 1,4‐butanediamine and 4,9‐dioxa‐1,12‐
dodecanediamine as the diamines and diethyl adipate as a second diester. The reaction scheme is depicted in Figure 3‐2. ‐ 44 ‐ Figure 3‐2. Enzyme catalyzed condensation between diester and diamine discussed in this chapter This chapter discusses first the polycondensation of dimethyladipate and diethylene triamine in a solvent and shows that in the case dimethylsuccinate and butylamine the enzyme produces mainly a monoamide. It continues with describing the solvent free polycondensation in the presence of N435. The main part consisting of ATR FTIR spectroscopy measurements that evaluate the rate of amide bond formation by the enzyme. Since solubility was a main concern variations in the diamine and diester compounds were made. A few experiments were dedicated to the action of Candida antarctica lipase A. 3.3
EXPERIMENTAL 3.3.1 Materials and methods Materials Diethylene triamine, ethylacetate, butanediamine and acetonitril were obtained from Acros. Butanediamine was purified by sublimation before use. 4,9‐dioxa‐1,12‐
dodecanediamine, dimethyladipate, diethyladipate, α‐Cyano‐4‐hydroxycinnamic acid 99+%, bradykin, angiotensin I, and ACTH 18‐39 were obtained from Sigma‐Aldrich. Trifluoroacetic acid was obtained from Merck. N435 was kindly donated by Novozymes, CLEA Cal‐A was kindly donated by Cleatechnologies Delft. Methods H‐NMR spectra were recorded using a 300 MHz and a 400 MHz Varian VXR apparatus. 1
MALDI‐ToF‐MS measurements were performed on a Biosystems Voyager‐DE PRO spectrometer in reflector and linear mode with α‐cyano‐4‐hydroxycinnamic acid as the matrix and acetonitril/H2O (50/50) with 0.5 % TFA as the solvent. Calibration was performed with a mixture consisting of matrix dimer, bradykinin, angiotensin I and ACTH 18‐39. The spots were created by mixing the matrix solution (10 mg mL‐1) with the sample solution (~3 mg mL‐1) in a 1:1 ratio by volume. Sometimes a 10‐fold dilution of the sample is needed to obtain a decent mass spectrum. ‐ 45 ‐ ATR FTIR spectroscopy was performed using a Bruker IFS 88 equipped with a Golden Gate Attenuated Total Reflection apparatus. 3.3.2 Experiments Aminolysis of dimethylsuccinate with n‐butylamine A mixture of dimethylsuccinate (10 mmol), n‐butylamine (40 mmol) and N435 (0.308 g) was stirred in 30 mL of 1,4‐dioxane at 40 °C. After 72 hours of reaction the N435 is removed by filtration. The solvent is removed by evaporation under reduced pressure. A solid product is obtained in 76% yield. H‐NMR (CDCl3): δ = 0.91(CH3) 1.33 (CH2CH3), 1.47(NHCH2CH2), 2.47(NHCOCH2), 2.67(CH2COO), 3.23(NHCH2), 3.68(COOCH3), 5.67(NH) 1
Polycondensation of dimethyladipate with diethylene triamine The reaction was performed according to Gu et al.11 Dimethyladipate (5 mmol) diethylenetriamine (5 mmol), are mixed with N435 (25.6 mg). The mixture is stirred at 7 °C, room temperature and 60 °C. Stirring comes to a halt due to increased viscosity of the mixture reaction times depend on the temperature. At that point methanol is added and the solution is filtrated to remove the N435 beads. The clear filtrate is poured in an excess of ethylacetate and the white precipitate is collected and dried at 46 °C under reduced pressure. Yield 22 wt% H‐NMR (D2O): δ = 1.44(CH2) 2.12(CH2CONH), 2.56(CH2NHCH2), 3.15(CH2NHCO), 3.55(CH3OCO) MALDI‐ToF MS (m/z): Mp = 1191 A(EA)5 1
Polycondensation with other diamines or diester The polycondensation reaction was first repeated using diethyladipate and diethylene triamine. Second the polycondensation was repeated with dimethyladipate in combination with the diamines: butanediamine and 4,9‐dioxa‐1,12‐dodecanediamine. Reactions with these diesters and diamines were performed at 60 °C for the ATR FTIR spectroscopy study and stopped when the increased viscosity made stirring impossible. Polycondensation with Candida antarctica lipase A The aminolysis of dimethyladipate with diethylene triamine was repeated with Candida antarctica lipase A as a cross‐linked enzyme aggregate (CLEA).12,13,14 This catalyst is evaluated against N435. The amount of CLEA is estimated to represent the amount of enzyme on the N435 beads (~20 wt%).15 ‐ 46 ‐ ATR FTIR spectroscopy measurements During the reaction aliquots are taken from the reaction mixture and an IR‐spectrum is recorded immediately. At each interval the area of the ester carbonyl absorption (1733‐1731 cm‐1) and the amide I carbonyl absorption (1689‐1620 cm‐1) is determined from the IR spectrum. By plotting the area of these two absorptions against time the reaction speed is obtained as the slope of a linear fit through the data points. For each slope the r2‐value is determined to validate the fit. The procedure was followed for both the reaction with N435 and the uncatalyzed reaction. All the uncatalyzed reactions produce polyamide. Therefore the actual contribution of the lipase to the amide formation is calculated by subtracting the slope of the uncatalyzed reaction from the slope of the N435 catalyzed reaction. 3.4
RE SUL TS AND DISCUSSIO N 3.4.1 Polycondensation performed in a solvent The aminolysis of dimethyl adipate with diethylene triamine was performed in a 1:1 molar ratio in 1,4‐dioxane, DMSO, NMP and toluene. In none of the solvents polymer was obtained. From the 1H‐NMR results it was suspected that predominantly monosubstition of the diester by the diamine takes place. The aminolysis of dimethylsuccinate with a two‐fold excess of butylamine was performed to find if disubstitution takes place. Although enough amine groups are present to amidate all the ester groups the mono amidated dimethylsuccinate was obtained in 76 % yield after 72 hours of reaction in 1,4‐dioxane at 40 °C. The 1H‐NMR‐spectrum of the product is shown in Figure 3‐3 with the in‐set showing the proton signals originating from the amidated dimethyl succinate. Disubstitution would give a singlet in the region 2.4 ‐ 2.6 ppm. However two triplets are found, at 2.67 ppm (CH2‐CO‐O) and at 2.46 (CH2‐CO‐NH). The singlet at 2.51 ppm can originate from disubstitution. If so, this is a minor fraction in the product. The integrals and numbers of protons for each peak match with a monoamide obtained from butylamine with dimethylsuccinate, see Figure 3‐3 and Table 3‐1. Regioselectivity in amidation of diesters has been reported before by Puertas16 for the amidation of dimethylsuccinate with different aliphatic diamines. Conde17 found monoamidation of D‐glutamic acid diesters yielding a mixture of the two possible monoamides with a preference for the synthesis of γ‐monoamide. For the L‐glutamic acid diester predominantly the α‐monoamide was formed18 Astorga reports the aminolysis of unactivated diesters6 the resulting amides are obtained by an aminolysis reaction on one of the two available ester groups. ‐ 47 ‐ Figure 3‐3. 1H‐NMR spectrum of mono‐amidated dimethylsuccinate with the in‐set showing the splitting up of the CH2 proton signals between the carbonyl groups. This result shows that the N435 has a preference for amidating one of the ester groups when it is used in a solvent. As a consequence the polymerization is continued without a solvent. Table 3‐1. Chemical shifts and integrals of the monoamidated dimethylsuccinate δ (ppm) protons integrals # Hʹs 0,91 CH3 19.13 3.1 1,33 CH2‐CH3 13.25 2.2 1,47 NH‐CH2‐CH2 13.58 2.2 2,47 NH‐CO‐CH2 12.31 2.0 2,67 CH2‐CO‐O 13.54 2.2 3,23 NH‐CH2 12.96 2.1 3,68 CO‐O‐CH3 15.23 2.5 5,67 NH 3.87 0.7 ‐ 48 ‐ 3.4.2 Polycondensation in a solvent free reaction The N435 catalyzed polycondensation of diethylene triamine with dimethyladipate produces a polyamide. After 120 hours of shaking at room temperature the mixture has become viscous and no mixing occurs anymore. The reaction is stopped and after purification the average molecular weight was measured with MALDI‐ToF mass spectrometry. Ions were collected in the range 500‐3000 m/z with a peak maximum molecular weight Mp at a m/z value of 1191. This [M‐Na]+ molecular ion has one diamine A and connected to this are five dimers (EA) giving the final polymer A(EA)5. The molecular weight is limited by the solidification of the products. The uncatalyzed reaction also yields a polyamide but in a lower rate. After an additional 48 hours this uncatalyzed reaction has reached the same viscosity where stirring is no longer possible. 3.4.3 ATR FTIR spectra during the course of polycondensation The reaction between diethylene triamine and dimethyladipate was run at room temperature for 88 hours. By room temperature is meant that the reactions are performed without heating or cooling. During this time samples were taken and measured immediately with ATR FTIR spectroscopy see Figure 3‐4. Figure 3‐4. Enzyme catalyzed condensation of diethylene triamine and dimethyladipate at room
temperature. ATR FTIR spectra taken at regular intervals (grey lines) from 0 ‐ 88 hours. The consumption of ester bonds (1731 cm‐1) and the production of amide bonds (1689‐
1628 cm‐1) is clearly visible. During the reaction the amides are at first soluble in the reaction mixture. Gradually, more amides precipitate and the increased hydrogen bonding between the amides causes broadening and shift of the amide I absorption peak.19 After 24 hours (dotted line) the amide I signal has broadend to a combination of peaks from 1689 cm‐1 to ‐ 49 ‐ 1628 cm‐1 with a maximum at 1662 cm‐1. After 88 hours (dashed line) this has changed to an amide I absorption ranging from 1689 cm‐1 to 1606 cm‐1 with a maximum at 1645 cm‐1. The uncatalyzed reaction also develops the amide bonds but at a much lower rate see Figure 3‐5. Figure 3‐5. Uncatalyzed reaction between diethylene triamine and dimethyladipate performed at room temperature. ATR FTIR spectra taken at regular intervals (grey lines) from 0 ‐ 88 hours. When the reaction is performed at 60 °C solidification starts at a higher extend of reaction. As the result of more solid reaction products the amide I absorption now ranges from 1689 – 1586 cm‐1 with a sharp maximum at 1630 cm‐1 as depicted in Figure 3‐6. Figure 3‐6. Enzyme catalyzed condensation of dimethyladipate and diethylene triamine at 60 °C. ATR FTIR‐spectra taken at regular intervals (grey lines) from 0 – 43 h. ‐ 50 ‐ 3.4.4 Influence of temperature on catalysis by N435 At three different temperatures (7 °C, room temperature, 60 °C) the rate of amide formation by the lipase was evaluated. Below for each temperature the area of the amide I carbonyl absorption is plotted against reaction time. In each graph the catalyzed and the uncatalyzed reaction are depicted. Figure 3‐7. Amide formation at 7 °C in the reaction between diethylene triamine and dimethyladipate catalyzed (squares) and uncatalyzed (circles). The amide bond formation during the polycondensation at 7 °C is plotted in Figure 3‐7. The slope of amide bond formation of the catalyzed (squares) reaction is 0.07 the uncatalyzed reaction (circles) has a slope of 0.03. The enzymatic contribution to the amide formation is therefore (slope catalyzed minus slope uncatalyzed) 0.07 ‐ 0.03 = 0.04. At room temperature the catalyzed reaction (squares) has a slope of 0.13 and the uncatalyzed reaction (circles) has a slope of 0.06. The increasing amide absorptions are plotted in Figure 3‐8. The lipase catalyzed reaction (enzymatic contribution) has a slope of 0.07. ‐ 51 ‐ Figure 3‐8. Amide formation at RT in the reaction between diethylene triamine and dimethyladipate catalyzed (squares) and uncatalyzed (circles). At 60 °C the uncatalyzed reaction has a slope of 0.53 and the catalyzed reaction has a slope of 0.70. The enzymatic contribution to to the amide formation is 0.17. The amide formation at 60 °C is plotted in Figure 3‐9. Figure 3‐9. Amide formation at 60 °C in the reaction between diethylene triamine and dimethyladipate, catalyzed (squares) and uncatalyzed (circles). The enzymatic contribution to the rate of amide bond formation is equal to the uncatalyzed condensation of dimethyladipate with diethylene triamine at 7 °C and at room temperature. By raising the temperature to 60 °C the enzymatic contribution to the reaction catalyzed condensation doubles while the rate of the uncatalyzed reaction increases a ten‐fold. ‐ 52 ‐ Apparently the uncatalyzed reaction passes an activation barrier between RT and 60 °C. In Table 3‐2 the rate of amide formation in the catalyzed and uncatalyzed reaction and the calculated, enzymatic contribution to the amide formation are listed. Table 3‐2. Slope of the amide I absorption against time at different temperatures. Catalyzed reaction /r2 Uncatalyzed reaction /r2 Enzymatic contribution 7 °C 0.07 / 0.98 0.03 / 0.94 0.04 Room temperature 0.13 / 0.99 0.06 / 0.98 0.07 60 °C 0.70 / 1.00 0.53 / 0.99 0.17 In conclusion, adding N435 leads to an increased amide bond formation at all temperatures. The contribution of the lipase relative to the uncatalyzed reaction decreases with temperature. Roughly 50 % of the amide bonds are synthesized by the lipase at 7 °C and RT, while 19 % of the amide bonds are produced by the enzyme at 60 °C. 3.4.5 Influence of the diester compound on catalysis by N435 Diethyladipate and diethylene triamine were polymerized in the bulk at 60 °C. The reaction was carried out for 48 hours. The rate of the enzymatic amide formation was calculated from the absorptions plotted in Figure 3‐10. The catalyzed reaction (squares) has a slope of 0.25 and the uncatalyzed reaction (circles) of 0.08. The contribution of the lipase is therefore 0.17. Figure 3‐10. Amide formation of the condensation reaction between diethyl adipate and diethylene triamine catalyzed (squares) and uncatalyzed (circles). ‐ 53 ‐ The action of the lipase is not affected by changing the leaving group of the diester to ethanol. In the polycondensation of dimethyladipate the lipase contributed a comparable 0.13. The minor effect of the leaving group on the lipase catalysis can be explained by assuming that the stabilization of the tetrahedral intermediate is the driving catalytic mechanism therefore it is of less importance which leaving groups are attached to the target ester carbonyl. Regarding the geometry of the active site there is enough space to accommodate both the tetrahedral intermediate and a nucleophile. An example is the amidation of an ethylester with dodecyl amine or benzylamine published by Quiros et al.4 Figure 3‐11. N435 catalyzed amide formation from diethyl adipate (squares) compared to the aminolysis of dimethyl adipate (circles) at 60 °C. However the uncatalyzed reaction with diethyladipate (slope 0.08) is much slower than the uncatalyzed reaction of dimethyladipate (slope 0.53). The decreased leaving group ability of the ethanol compared to the methanol slows the uncatalyzed reaction down considerably. The enzymatic reaction is not influenced by changing the diester to diethyladipate. In Figure 3‐11 the slopes of both reactions are plotted. The reaction of dimethyladipate (squares) has a slope of 0.70 while the diethyladipate (circles) reaction has a slope of only 0.25. 3.4.6 Influence of diamine compound on catalysis by N435 Since we want to use the aminolysis reaction to synthesize a range of polyamides it is interesting to see if the N435 is able to catalyze the condensation of dimethyladipate with other diamines as well. Two other diamines were chosen an aliphatic linear 1,4‐
butanediane and a diamine with oxygen atoms in the main chain 4,9‐dioxa‐1,12‐
dodecanediamine. ‐ 54 ‐ Figure 3‐12. Amide formation in the condensation reaction of dimethyladipate with 4,9‐dioxa‐1,12‐
dodecanediamine (squares), butanediamine (circles) and diethylene triamine (triangles) as the nucleophile in the presence of N435. The reactions with 1,4‐butanediamine and 4,9‐dioxa‐1,12‐dodecanediamine were run at 60°C for 13 hours. After this period the viscosity made it impossible to take aliquots for ATR FTIR measurements. With diethylene triamine the condensation could proceed for 48 hours as discussed above. In Figure 3‐12 the amide bond formation is plotted for 25 hours of the reaction. The polycondensation proceeds faster with 1,4‐
butanediamine (slope 0.82) as the diamine when compared to diethylene triamine (slope 0.70) and 4,9‐dioxa‐1,12‐dodecanediamine (slope 0.31). Table 3‐3. Slope of amide production at 60 °C from the condensation of dimethyladipate with different diamines. Catalyzed reaction /r2 Uncatalyzed reaction /r2 Enzymatic contribution Diethylenetriamine 0.70 / 1.00 0.53 / 0.99 0.17 Butanediamine 0.82 / 0.95 0.20 / 1.00 0.62 4,9‐dioxa‐1,12‐
dodecanediamine 0.31 / 0.97 0.07 / 0.99 0.24 The enzymatic contribution to the amide formation was calculated for these diamines see Table 3‐3. The slope of amide formation by the enzyme is 0.62 for butanediamine. Enzymatic condensation of 4,9‐dioxa‐1,12‐dodecanediamine has a slope of 0.24. While the enzymatic condensation of diethylene triamine was catalyzed with a slope of 0.13. Of the three diamines used butanediamine is the diamine preferred by the lipase since, Cal‐B produced amide bonds with the highest rate with this nucleophile. ‐ 55 ‐ 3.4.7 Evaluation of Candida antarctica lipase A as a catalyst Aminolysis reactions as described in the experimental section were also carried out with the cross‐linked enzyme aggregate (CLEA) of Cal‐A. In a solvent free system amides were produced and monitored by ATR FTIR spectroscopy. The results are compared with the N435. In Figure 3‐13 the amide production at room temperature is depicted as before. It shows that the condensation (squares) proceeds with a slope of 0.07 while the uncatalyzed reaction (circles) proceeds with a slope of 0.06. These values are close enough to conclude that the Cal‐A does not contribute to the amide formation at this temperature. Figure 3‐13. Amide formation catalyzed by Cal‐A (squares) and uncatalyzed (circles) at room temperature. Below in Figure 3‐14 the amide formation at 60 °C is depicted. The amide formation of the catalyzed reaction is 0.62 and the uncatalyzed reaction has a slope of 0.53. Meaning that the enzymatic contribution at 60 °C is 0.09. ‐ 56 ‐ Figure 3‐14. Amide formation catalyzed by Cal‐A (squares) and uncatalyzed (circles) at 60 °C. In Table 3‐4 the slopes of the CLEA Cal‐A and the N435 catalyzed reaction are compared. The enzymatic contribution of Cal‐A to the polycondensation is 0.01 at room temperature and 0.09 at 60 °C. N435 catalyzes the condensation reaction at all temperatures as outlined above. It can be concluded that Cal‐A does not show activity at room temperature but does so at 60 °C. However, this is negliable compared to the N435 (enzymatic contribution of 0.17). Table 3‐4. Slope of the amide formation by CLEA Cal‐A compared to N435 Slope at RT Catalyzed /r2 Slope at 60 °C Uncatalyzed/r2 Catalyzed/r2 Uncatalyzed/r2 CLEA Cal‐A 0.07 / 0.98 0.06 / 0.98 0.62 / 0.94 0.53 / 0.99 N435 0.06 / 0.98 0.70 / 1.0 0.53 / 0.99 0.13 / 0.99 Suggested improvements for the use of Cal‐A Cal‐A is less effective in catalyzing the condensation of diethylene triamine and dimethyladipate than Cal‐B. Possible explanations can be found in the selectivity of the enzyme, the immobilization technique and/or the reaction medium. The lipase Cal‐A is less tolerant towards substrates than the Cal‐B and only in specific cases good results were reported in literature. Heldt‐Hansen and co‐workers reported a selectivity for long‐chain carboxylic acids in esterification reactions. Esterifications with butyric acid and acetic acid gave no reaction wheras longer chain carboxylic acids are used with success.20 Reactions with activated esters (fluorinated butanoate and hexanoate) were performed by Kanerva et al.21 ‐ 57 ‐ In a review by Dominguez de Maria22 the selectivity for the nucleophile is discussed. Cal‐A has a bigger active site pocket than the lipase B and can perform reactions with sterically hindered (tertiary) alcohols. Furthermore the Cal‐A has a selectivity for N‐
acylation of β‐amino acid esters, transesterification of these compounds is not catalyzed. Other immobilization techniques for Cal‐A exist that might yield better results. The lipase A used in this research is bought as a cross‐linked enzyme aggregate (CLEA)14. Immobilization on celite with sucrose23, silcone particles24, polypropylene beads EP‐
10025 and magnetic particles26 was reported as well. The most used methods of immobilization are the CLEA and the immobilization on Celite in the presence of sucrose. Aminolysis of dimethyladipate was carried out without a solvent and although for Cal‐B this was a good reaction medium this is not necessarily so for Cal‐A. Solvents discussed in the review by Dominguez de Maria are iso‐propylether, acetonitril, diethylether and butylbutanoate (also reactant). None of which are particular good solvents for polyamides.22 It is recommended that future research is directed towards using monomers that resemble substrates that are accepted by the Cal‐A like tertiary alcohols, β‐amino acids and long chain trans fatty acids. The immobilization technique can be changed to Celite in presence of sucrose as this is used sucessfully in the N‐acylation of β‐amino acids.23 Last, it is crucial to find a reaction medium that prevents precipitation of the product amides and leaves the structure and activity of the enzyme intact. 3.5
CO NCLUSIONS This chapter shows that polycondensation of diesters and diamines should not be performed in the solvents DMSO, NMP, 1,4‐dioxane or toluene due to preferred monoamidation as shown for the amidation of dimethylsuccinate with a two‐fold excess of butylamine and illustrated by examples from literature. Polymer can be obtained when the reaction is performed in the bulk. A Polymer with a Mp of 1192 m/z from MALDI‐ToF MS was obtained from a polycondensation of diethylene triamine and dimethyladipate. With IR spectroscopy it was shown that the N435 catalyzes the polycondensation but with a significant uncatalyzed reaction. Especially at 60 °C the uncatalyzed reaction has a higher rate than the enzymatic reaction. Changing the diester to diethyladipate did not affect the enzymatic reaction but the uncatalyzed reaction was much slower than in the case of dimethyladipate caused by reduced leaving group ability. The diamine compound was altered to adress a possible selectivity of the N435 for the nucleophile and decrease the solidification of ‐ 58 ‐ the reaction mixture. The polycondensation was catalyzed in the order butanediamine > 4,9‐dioxa‐1,12‐dodecanediamine > diethylenetriamine Candida antarctica lipase A was used as a catalyst and it is found that this enzyme catalyzes the reaction at 60 °C at a slower rate than the Cal‐B does. At room temperature the Cal‐A hardly contributes to the polyamide formation. It is not recommended to uses Cal‐A as a catalyst in this reaction. In order to use the enzymatic polycondensation as a viable synthesis route to polyamides it is necessary to find a solvent that dissolves the polyamide and leaves the enzyme intact and active. Examples of such solvents are ionic liquids and super critical CO2. Other lipases than the Candida antarctica lipase B might be found active in this reaction. 3.6
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