Effect of the lipase on lipase-catalyzed transesterification
To identify the optimal conditions, methyl 1H-pyrrole-2-carboxylate (1a) and benzyl alcohol (2a) were designed as model substrates for the initial investigation. For the first lipase-catalyzed transesterification of 1a with 2a, three available commercially encapsulated lipases, namely Lipozyme TLIM, Novozym 435, and CRL, were used. Other input investigations were 5:1 (1a/2a) reactant molar ratio, lipase load of 6 mg/mL, stirrer speed of 150 r/min, molecular sieve load of 1 g, 40°C reaction temperature, and a 24 h reaction time. According to Fig. 1, Lipozyme TLIM provided the lowest contents of benzyl 1H-pyrrole-2-carboxylate (3a), accounting for less than 3%. Compound 3a has a lower amount of 9% according to CRL. Under the catalysis of Novozym 435, compound 3a was generated in the maximum concentration which showed much higher activity (46% yield) in toluene than Lipozyme TLIM and CRL. It suggested that Novozym 435 was efficient for catalyzing the transesterification of methyl 1H-pyrrole-2-carboxylate with alcohol.
Effect Of The Solvent On Lipase-catalyzed Transesterification
The activity of three lipases is influenced by the various catalytic characteristics [28]. Candida antarctica lipase B (CALB) functioned as the source of Novozym 435. It was formerly believed that Novozym 435 was an all-purpose lipase [31]. The efficiency and stability of enzymes are substantially impacted by the reaction medium [32]. In various solvent media, lipases frequently displayed varying positional selectivity and catalytic activity, which were frequently connected to the solvent characteristic [33]. Here, the lipase Novozym 435 was used to study the effects of solvents, eight kinds of solvent, including toluene, 1,4-Dioxane, CH3CN, DMF, n-Hexane, ethanol, isooctane and DMSO, were used as the reaction medium to carry out this reaction (Table 1). Table 1 demonstrates that the n-Hexane significantly outperformed the competition, yielding the required product 3a in a yield of 61% isolated (Table 1, entry 5). The target compound 3a was produced by the isooctane in a 44% isolated yield (Table 1, entry 8). Unfortunately, 1,4-Dioxane, CH3CN, DMF, ethanol and DMSO cannot be used in this reaction, product 3a was not being found.
Table 1
Effect of the solvent on lipase-catalyzed transesterification of methyl 1H-pyrrole-2-carboxylate with benzyl alcohol in n-Hexane.
Entry | Lipase | Solvent | 3a (%) |
1 | Novozym 435 | Toluene | 46 |
2 | Novozym 435 | 1,4-Dioxane | 0 |
3 | Novozym 435 | CH3CN | 0 |
4 | Novozym 435 | DMF | 0 |
5 | Novozym 435 | n-Hexane | 61 |
6 | Novozym 435 | Ethanol | 0 |
7 | Novozym 435 | DMSO | 0 |
8 | Novozym 435 | Isooctane | 44 |
Reaction conditions: methyl 1H-pyrrole-2-carboxylate 1a (1.0 mmol), benzyl alcohol 2a (0.2 mmol), 60 mg Novozym 435, 10 mL solvent, 1.0 g molecular sieves, 40°C, 150 rpm, 24 h.
Effect Of The Lipase Load On Lipase-catalyzed Transesterification
For an industrial application to be effective, lipase load is a critical component. High lipase load may shorten reaction time and increase the reaction rate. Nevertheless, the cost of lipase increased as its supply increased and was fairly high. So, the impact of Novozym 435 load from 2 mg/mL to 10 mg/mL on the compositiont of compound 3a was evaluated under 5:1 (1a/2a) reactant molar ratio, stirrer speed of 150 r/min, molecular sieves of 1 g, 40°C reaction temperature, and a 24 h reaction time in n-Hexane. It can be seen from the Fig. 2 that the transformation rose from 31–61% when the catalyst amount was raised from 2 mg/mL to 6 mg/mL. If the lipase load was from 8 mg/mL to 10 mg/mL, the amount of compound 3a was slightly reduced. The lipase aggregation at the reaction interface reduced the effective lipase concentration and indeed the region of electrode contact area, may be the origin of this occurrence [34]. The ideal lipase load for Novozym 435 was determined to be 6 mg/mL after taking into account the price of lipase and the presence of component 3a.
Effect Of The Molecular Sieves On Lipase-catalyzed Transesterification
The reaction mixture including alcohol and methyl 1H-pyrrole-2-carboxylate was made using dehydrated n-hexane. Therefore, the effect of molecular sieves from 0 g to 1.5 g on the content of compound 3a was investigated. As shown in Fig. 3, the conversion increased from 22–61% when the molecular sieves were raised from 0 g to 1.0 g. The synthesis of pyrrole ester then gradually decreased when the molecular sieve concentration was raised further. The addition of molecular sieves often increases the conversion of the equilibrium [35]. However, there have also been several reports of unfavorable consequences, such as the breakdown of unstable compounds [36, 37].
Effect Of The Molar Ratio On Lipase-catalyzed Transesterification
The influence of substrate molar ratio was frequently cited as a notable feature in the behavior of enzymes involved in synthesis [38]. Hence, at various molar ratios (1a/2a), the transesterification of methyl 1H-pyrrole-2-carboxylate (1a) with benzyl alcohol (2a) was investigated. Figure 4 shows the impact of molar ratio on the transesterification of 1a with 2a. The yield of compound 3a was 22% at the molar ratio of 2:5. After changing the molar ratio was adjusted from 2:5 to 5:1, the production of compound 3a raised from 22–61%, demonstrating that an increase in 1a might cause the reaction thermodynamic equilibrium to move in favor of 3a pyrrole ester, and enhance the iterate of 1a. The 5:1 molar ratio, which had the greatest conversion rate, was determined as the best molar ratio for further research. But with the subsequent rise in the molar ratio to 10:1 and 15:1, the yield dropped slightly to 47% and 40%, respectively. This is so that when the molar ratio reached a 5:1 ratio, the reactions equilibria would be equivalent. As a result, a rise in 1a may cause the reaction equilibrium to shift in the other way, decreasing conversion [39]. As a consequence, the following tests were optimized using a molar ratio of 5:1.
Effect Of The Reaction Time On Lipase-catalyzed Transesterification
The optimum ester synthesis was time-dependent, as originally described. The yield of ethyl ferulate under Novozym's catalysis steadily rose as the reaction period was extended [39]. In spite of this, the hydrogel-bound lipase of P. aeruginosa MTCC-4713 generated the greatest amount of ester during the 6 h synthesis of methyl acrylate, before steadily declining after that [40]. Therefore, the transesterification of pyrrole ester with benzyl alcohol was studied at different reaction time. It can be seen from the Fig. 5, when the reaction time was raised from 6 h to 24 h, the translation improved from 20–61%. The yield significantly decreased to 60% with the additional increase in reaction time to 48 h. Therefore, the reaction time for the ideal reaction circumstances was determined to be 24 h.
Effect Of The Temperature On Lipase-catalyzed Transesterification
Lipase activity and reaction rate were both impacted by reaction temperature. Although increasing temperature sped up reaction rate and lowered reaction time, but it also decreased lipase activity [28]. As shown in Fig. 6, the translation rate has risen from 61–92% as the temperature rose from 40°C to 50°C. When the temperature rose from 50°C to 60°C, the conversion was dramatically reduced. The yield of compound 3a was 78% at 60°C. This suggested that because of the lengthy reaction time at high temperatures, the enzyme may undergo thermal denaturation. For the progressively advanced, the reaction temperature was maintained at 50°C to prevent thermal inactivation of the enzyme and to achieve high conversion.
Effect Of The Agitation Speed On Lipase-catalyzed Transesterification
Reaction time and rate were altered by appropriate agitation occurring at an ideal pace. Experiments were conducted at agitation rates ranging from 100 to 200 rpm to evaluate the impact of agitation speeds. As shown in Fig. 7, when converting between 100 and 150 rpm, the conversion progressively rises from 55–92%. The layer surrounding the solid lipase particles was reduced with an increase in agitation speed, which reduced the mass transfer resistance. Even yet, the speed was only slightly reduced when it was raised to 200 rpm. Since there was only a slight difference between stirrer speeds of 150 and 200 rpm, 150 rpm was chosen to minimize energy usage.
Optimization Of The Reaction Conditions
With the optimized circumstances, we assessed the scope of the substrate. It can be seen from the Table 2 that different aromatic and primary alcohols (2b-2m) were chosen to react with (1a) with optimal reaction conditions. With moderate to good yields of 75–92%, the reaction of 1a and benzylic alcohols having alkyl and halo substituents at the o-, m-, and p- positions led to the formation of the desired compounds 3a–3f. Importantly, (R)-(+)-1-Phenylethanol reacted smoothly with 1a, 52% of the target product, 3g, was successfully created. Gratifyingly, the reaction could also be applied to a range of cyclic and acyclic aliphatic alcohols with 1a, which gave products 3h-3l with 72–98% isolated yields. In addition, methallyl alcohol was well tolerated, giving 3m in 71% yield. It is interesting that when methyl 1H-indole-2-carboxylate (1b) and benzyl alcohol were combined, the desired product 3n was produced in 81% of the time.
Table 2 Substrate scope of alcohols for transesterification reactions.
Reaction conditions: methyl 1H-pyrrole-2-carboxylate 1a (1.0 mmol), benzyl alcohol 2a (0.2 mmol), Novozym 435 60 mg, 1.0 g molecular sieves, and n-Hexane (10 mL) for 24 h, 50°C.
As shown in Scheme 1, large-scale (10 mmol) experiments between methyl 1H-pyrrole-2-carboxylate 1a (50 mmol, 6.25g) and benzyl alcohol 2a (10 mmol, 1.08g) were conducted to further evaluate the superlative scalability of this established method. These experiments produced the desired 3a with an excellent yield of 88%. (1.77 g).
Table 3
Odor description of compounds.
Entry | Compound | Odor description |
1 | benzhydryl 1H-pyrrole-2-carboxylate (3h) | Sweet, acid |
2 | butyl 1H-pyrrole-2-carboxylate (3i) | Sweet, herbs, acid |
3 | pentyl 1H-pyrrole-2-carboxylate (3j) | Sweet, fruity, acid |
Table 4
Pyrolysis products of 3h, 3i and 3j (TIC area%).
Sample | Time | Compound | Area% |
benzhydryl 1H-pyrrole-2-carboxylate (3h) | 3.18 | cyclopropyl carbinol | 1.97 |
3.74 | pyrrole | 1.55 |
24.16 | 3h | 96.48 |
butyl 1H-pyrrole-2-carboxylate (3i) | 3.74 | pyrrole | 1.87 |
4.23 | 1-butanol | 2.98 |
24.89 | 3i | 95.15 |
pentyl 1H-pyrrole-2-carboxylate (3j) | 3.74 | pyrrole | 1.91 |
4.45 | 1-pentanol | 3.16 |
25.35 | 3j | 94.93 |
The Thermal Behaviors Of Compounds
As shown in Table 3, compounds 3h, 3i and 3j possessed strong characteristic aroma such as sweet, herbs, fruity and acid. Studying the chemicals produced under heated conditions was therefore also crucial. The pyrolysis conditions included an oxidizing atmosphere (91% nitrogen, 9% oxygen) and temperatures between 30 and 900°C. Furthermore, the pyrolysis gas chromatograms obtained under influence of heat are shown in Fig. 8 and the peak position areas of the pyrolysis products are listed in Table 4. As shown in Table 4, the evaporative yield of compound 3h in an oxidizing environment at high temperatures is 96.48%. Compound 3h was also the most stable material tested, releasing just 3.52% of its breakdown at high temperatures in oxidizing environments. When the volatile pyrolysis product of 3h was examined, the main byproducts were cyclopropyl carbinol (1.97%) and pyrrole (1.55%). Under extreme heat in an oxidizing atmosphere, the evaporative yields of compound 3i and compound 3j are 95.15% and 94.93%. Pyrrole (1.87%) and 1-butanol (2.98%) made up the majority of the pyrolysis products of 3i. Pyrole (1.91%) and 1-pentanol (3.16%) made up the majority of the pyrolysis products of 3j. It is important to note that several pyrolysis byproducts of the compound 3i and 3j contained the distinguishable flavour components, including pyrrole, 1-butanol and 1-pentanol. They are all appropriate to use as flavor and aroma mediators.
Figure 9 (a, b) shows the TG, DTG, and DSC profiles of the title compounds with 30 and 400°C at a rate of heating of 10°C min− 1. Compounds 3h, 3i, and 3j decomposed at conditions varying from 70.9 to 250.0, 62.2 to 220.0, and 65.7 to 230.0°C, respectively, as shown in Fig. 9 (a, b). And it shows that 3i and 3j had the greatest rate of disintegration at 124.3 and 136.7°C with starting mass reductions of 41.92% and 38.16%. The samples then showed a consistent and uniform mass loss pattern, with overall weight losses of 98.43% and 99.65%. Most of the thermal mass loss (80.42%) occurred at 219.0°C, with an ultimate total weight loss of 98.41%, based to both the TG and DTG curves of 3h. Due to the chemical structure of 3h includes cyclopropyl while the structures of 3i and 3j contain straight-chain carbon, the majority of thermal mass loss in 3h was higher than in 3i and 3j, which may have been due to distinct stabilizations. These three compounds were found to be steady at ambient temperature by the TG and DTG testing.
Figure 9 (c) shows the DSC individuals for 3h − 3j that were examined in an environment of air. Equipment was used to capture the highest temperature of DSC curves and the change in enthalpy of the samples. The Tonset, Tpeak, and Tend temperatures were similar to those discovered using DTG graphs. With the DSC graph of 3h, it can be seen that there is a clear, significant endothermic peak at 52.8°C. The sharp peak indicates a melting-point of 3h, as there is no weight loss at 52.8°C, on the other hand. Additionally, it demonstrated that the Tpeak of 3h-3j in DSC curves occurred in the major mass loss areas there, suggesting that the samples may have vaporized or broken down during the endothermic phase. Overall, the DSC and TG-DTG results were in agreement with the other and showed that the samples were steady at room temperature. The information regarding the outcomes based on the graphs shown in Table 5.
Table 5
Thermal analysis data of compounds 3h, 3i and 3j.
Compound | DSC | TG-DTG | Mass loss/% |
T melt/°C | T onset/°C | T peak/°C | T end/°C | ∆H/kJ mol− 1 | T p/°C | T range/°C |
3h | 52.8 | 206.9 | 222.5 | 229.2 | 92.6 | 219.0 | 70.9–250.0 | 98.41 |
3i | - | 111.5 | 129.1 | 144.5 | 54.4 | 124.3 | 62.2–220.0 | 98.43 |
3j | - | 124.5 | 137.6 | 146.9 | 25.81 | 136.7 | 65.7–230.0 | 99.65 |