Highly Efficient Lipase Catalyzed Monoaminolysis Reaction of Diesters with Benzylamine

Abstract

Preparation of amides by biocatalyzed aminolysis reactions has greatly increased because these processes play an important role in the preparation of some pharmaceutical products and their intermediates. A highly efficient synthesis of malonic (5), succinic (6), and malic (8) monoamide esters by desymmetrization transformations (mono aminolysis reactions) of their diesters is presented. The immobilized lipase from Candida antarctica (CaL-B, Novozym 435) catalyzed the mono aminolysis of bifunctional compounds: diesters such as diethyl malonate (1), diethyl succinate (2), and (R,S)-malate (3), leading to their corresponding monoamides (5), (6), and (8), respectively, with high conversions after a 24 hour reaction in the presence of an organic solvent. Increasing the solvent polarity from toluene, methyl t-butyl ether to dioxane led to an improved conversion. According to qualitative and quantitative GC-MS analysis, conversions of diesters (1), (2), and (3) into their mono amide esters were in the range 65%-97%. CaL-B was the best biocatalyst of the commercial lipases used, including those from Candida rugosa, Rhizomucor miehei, Carica papaya, and Pseudomonas cepacia. Likewise, a yield of 91% regioisomeric, but virtually racemic (R,S)-mono amide (8) was obtained as the single product in dioxane.

Dicarboxylic acid esters and their derivatives possess a myriad of applications in different academic and industrial sectors; they are important starting materials in the manufacture of polyamides and of di- and polyesters. Dicarboxylic acid monoamides are interesting synthons and intermediates for the preparation of biologically active amino acids, lactams, and other fine and pharmaceutical compounds by ammonolysis and aminolysis reactions. ¹ In addition, the selective transformation of a unique functional group in a molecule presenting more complex functionality and avoiding side reactions is one of the most important tasks in the preparation of many compounds. ²

The development of sustainable and efficient methods for reaching chiral building blocks has become imperative for contemporary chemistry over the past decade. Biocatalysis and biotransformations are the most attractive routes to obtain a range of bioactive molecules using clean, easy, and eco-friendly procedures, and are the most targeted by the fine-chemical industry. ³

Enzymes can catalyze chemo-, regio-, and stereoselective reactions of building blocks and achieve the desymmetrization of diacids, diesters, and other symmetric compounds by modification of one functional group that eliminates one or more elements of symmetry of the substrate. ⁴ Particularly, the aminolysis of diesters is a process of great utility for the synthesis of nitrogenated organic compounds; in some cases, this process is useful for the preparation of mono amide esters by means of the desymmetrization of diesters, which are difficult to obtain by chemical procedures under benign conditions (Figure 1). In this context, the purpose of this work was to study the chemo-/regioselectivity of the lipase aminolysis desymmetrization of the diesters diethyl malonate (1), diethyl succinate (2), and diethyl malate (3) with benzyl amine (4). The benzyl group is commonly used as a protecting group in different synthetic procedures and is typically cleaved using reductive methods to obtain amino or amide groups, as in ethyl 3-amino-3-oxopropanoate (7). ⁵

Figure 1

Synthesis of monoamides (5) and (6) by desymmetrization lipase catalyzed aminolysis of diesters diethyl malonate (1) and diethyl succinate (2) with amine (4) in an organic solvent.

Initially, the enzymatic aminolysis of diethyl malonate (1) with amine (4) was tested. All reactions were conducted at several temperatures (30°C-60°C), in an organic solvent, using near equimolecular amounts of reagents, and the reactions were stopped when no further progress was observed by thin layer chromatography (TLC). The aminolysis reaction was analyzed by gas chromatography/mass spectrometry (GC-MS). Candida antarctica lipase (CaL-B), the most commonly used hydrolase for biocatalysis, was selected as the biocatalyst to test the further development of diester desymmetrization. Other lipases from Candida rugosa, Rhizomucor miehei, Carica papaya, and Pseudomonas cepacia were also tested in these experiments, but showed low catalytic activity. In contrast, CaL-B was very efficient in these processes; mono amide ester (5) was obtained as the principal product with a high conversion, and no reaction took place in the absence of the enzyme under similar conditions (Figure 1). Subsequently, the diester (1)/ amine (4) ratio was adjusted to 1:1.2 mmol, so that the enzymatic synthesis of monoamide ester (5) was achieved using 2 mL of either toluene, methyl-t-butyl ether (MTBE) or dioxane as the solvent (Table 1, entries 1-9). The results observed indicated a good CaL-B catalyzed monoaminolysis reaction between ester (1) and benzyl amine (4) at 30°C to 50°C for 24 hours, but one highly efficient process at 60°C. Next, the same CaL-B catalyzed aminolysis reaction between diester (2) and amine (4) was studied according to the aforementioned and similar protocols (see Experimental section). These results were similar to those reported by Astorga et al, ⁶ who reported an efficient process between (2) and several diamines to obtain similar amido esters as the sole products with high yields at room temperature. In that study the optimal yield for amido esters was obtained with the use of a 1:2 molar ratio of diamine: diester (2) with dioxane as the solvent. ⁶

Table 1

Lipase Catalyzed Synthesis of Monoamides From Diethyl Malonate (1) and Diethyl Succinate (2) with Benzyl Amine (4) in an Organic Solvent at 30°C to 60°C.

Entry	Diester	Amine	T °C	Solvent	Conversion (%) a b
1	1	4	30	toluene	75 ± 1
2	1	4	40	toluene	85 ± 1
3	1	4	60	toluene	95 ± 1
4	1	4	30	MTBE	67 ± 1
5	1	4	40	MTBE	83 ± 1
6	1	4	60	MTBE	90 ± 1
7	1	4	30	dioxane	84 ± 1
8	1	4	40	dioxane	94 ± 1
9	1	4	60	dioxane	96 ± 1
10	2	5	30	toluene	78 ± 1
11	2	5	40	toluene	88 ± 1
12	2	5	60	toluene	95 ± 1
13	2	5	30	MTBE	72 ± 1
14	2	5	40	MTBE	88 ± 1
15	2	5	60	MTBE	96 ± 1
16	2	5	30	dioxane	87 ± 1
17	2	5	40	dioxane	95 ± 1
18	2	5	60	dioxane	97 ± 1

^aConversion degree determined by GC-MS analysis (see Experimental).

^bMean of triplicates (mean values with standard deviations).

As can be seen in Table 1, the mono amides (6) and (7) were chemoselectively obtained with good-tohigh conversions (65%-97%) in the range of conditions established (Table 1); their identity was confirmed by gas chromatography/mass spectrometry (GC-MS), Fourier-transform infrared spectroscopy (FT-IR), and nuclear magnetic resonance (¹H NMR) spectroscopy.

Besides being an environmentally friendly and an economical alternative to traditional chemical synthesis, CaL-B as a biocatalyst has many important advantages and added benefits: mild reaction conditions, high chemo- and regio-specificity, and also catalyzes of enantioselective transformations of racemic mixtures of acids, esters, and amines yielding optically active amidoesters with excellent enantiomeric excess. In a previous study, ⁷ enantioselectivity was not observed when several racemic 2-hydroxy acid ester aminolysis reactions were tested by analysis of the corresponding Mosher’s derivatives; virtually only racemic 2-hydroxy amides were obtained. These results were in sharp contrast to the high enantioselectivity reported for a similar aminolysis process of regioisomeric racemic 3-hydroxy esters. ⁸ Because of these different results displayed by CaL-B, with a great differencehz. in the enantioselectivity of amidation between a racemic (R,S)-β-hydroxy ester and a racemic (R,S)-α-hydroxy ester as substrates leading to the corresponding amides, the study of the aminolysis reaction of the racemic (R/S) hydroxysuccinic acid methyl ester (or (R/S)-malic acid methyl ester) (3) was decided on. The reaction of the compound (3), which contains the α- and β-hydroxyl groups, with respect to each one of the ester groups in (3), was followed in order to know what type of their corresponding amido esters, (8) and (9), respectively, could be obtained (Figure 2). Therefore CaL-B was selected as the biocatalyst to test the further analysis of the enantiodifferentiation of (R or S)-(3) to produce one of the two expected monoamidoesters, either (R or S)-(8) or (R or S)-(9). With different reaction parameters such as temperature, ester/amine concentration ratio, and enzyme amount, the reaction of diester (R/S)-(3) and amine (4) was conducted. ⁹ A yield of between 65%and 91% of a single product was obtained after a 24 hour reaction time at 30°C to 60°C, whichever solvent was employed, the reaction being followed by TLC and GC-MS (Table 2). The chemical identity of the regioisomeric, but virtually racemic (R,S)-mono amide (8), over its not observed regioisomer mono amide (9) was confirmed by ¹H NMR spectroscopy. In addition, optical rotation measurements indicated no optical activity, showing no enantiodifferentiation of (R/S)-(3) by CaL-B.

Figure 2

Monoamide (8) synthesis by regioselective CaL-B catalyzed aminolysis of diester dimethyl malonate (3) with amine (4) in an organic solvent.

Table 2

Regioselective Monoamide Synthesis by CaL-B Catalyzed Aminolysis of Diesters (R,S)-Diethyl Malonate (3) with Amine (4) in an Organic Solvent.

Entry	Diéster	Amine	T °C	Solvent	Conversion (%) a b
1	3	4	30	Toluene	65 ± 1
2	3	4	40	Toluene	66 ± 1
3	3	4	60	Toluene	85 ± 1
4	3	4	30	MTBE	67 ± 1
5	3	4	40	MTBE	73 ± 1
6	3	4	60	MTBE	88 ± 1
7	3	4	30	Dioxane	80 ± 1
8	3	4	40	Dioxane	84 ± 1
9	3	4	60	Dioxane	91 ± 1

^aConversion degree determined by GC-MS analysis (see Experimental).

^bMean of triplicates (mean values with standard deviations).

After the aminolysis reaction of diester dimethyl malonate (3), two peaks were particularly compared in the ¹HNMR spectrum of substrate (3): one singlet for the methyl group CH₃-O-(C = O)-CH(OH)CH₂-COOCH₃ at δ 3.8, whose chemical shift is at lower field and deshielded due to the electron-withdrawing effect of the –OH group with respect to the other methyl group CH₃-O-(C = O)-CH(OH)CH₂-COOCH₃, whose signal at δ 3.7 is explained because it is most distant from the deshielding effect of the same hydroxyl group. This signal remained in the ¹H NMR spectrum of the only isolated compound, suggesting mono amido ester (8) as the aminolysis reaction product. Finally, the retention time and mass spectra were constantly the same in all the observed products in the CAL-B catalyzed aminolysis experiments of dimethyl malonate (3) with amine (4) in an organic solvent. Increasing the solvent polarity from toluene to methyl t-butyl ether and to dioxane led to improved conversion.

In conclusion, in this study C. antarctica B lipase catalyzed the aminolysis reaction of diesters (1 - 3), with benzyl amine (4) as the nucleophile reagent in the presence of an organic solvent, yielding mono amido esters (5 , 6) and (8) with high conversions. Of the different parameters considered, increasing the solvent polarity from toluene to dioxane led to the best and most significant improvement in the general processes studied.

Experimental

Lipases and Chemicals

Chemical reagents, solvents, recombinant CAL-B lipase (Novozyme SP-435, 7000 propyl laureate units/g solid enzyme), lipases from Candida rugosa, Rhizomucor miehei, Carica papaya, and Pseudomonas cepacia were all purchased from Sigma-Aldrich (Sigma-Aldrich Química S.A. de C.V., Mexico). Analytical grade organic solvents were dried by refluxing with 3 Å molecular sieves, distilled, and stored over 3 Å molecular sieves. Flash chromatography was performed using silica gel 60 (230-240 mesh).

CAL-B Catalyzed Mono Amide Ester 5, 6, and 8 Synthesis

A suspension was prepared, under nitrogen atmosphere, of the corresponding diesters (1 - 3), (1 mmol), benzyl amine (4) (1.2 mmol), 4 Å molecular sieves (50 mg), and CaL-B (50 mg), in a dry solvent. The reaction mixture was shaken (250 rpm) at 30°C, 40°C, or 60°C. Reactions were followed until no further progress was observed by TLC, after which the reaction was stopped, the enzyme and molecular sieves were removed by filtration and the solvent was then evaporated under reduced pressure. The crude product was purified by flash chromatography. The product was weighed and stored at 10°C until its analysis by ¹H NMR.

Gas Chromatography/Mass Spectrometry Analysis of Mono Amide Esters

Biocatalyzed reactions were monitored by gas chromatography-mass spectrometry on a Clarus 580 S GC coupled to a Clarus SQ 8 S MS (Perkin Elmer, Mexico). Samples were analyzed in an Elite 5 ms fused-silica capillary column (poly(dimethylsiloxane), 30 m × 0.32 mm i.d. × 0.25 µm film thickness (Perkin Elmer,USA). Carrier gas was helium, 1.5 mL/min constant flow; 250°C injector temperature; 1:30 split ratio; at several temperature gradients; 230°C ion source temperature; 250°C transfer line temperature; 70 eV ionization energy. Electron ionization mass spectra were acquired over the mass range of 35-400 amu.

¹H NMR Analysis

¹H NMR spectra, recorded in CDCl₃ on an Agilent-Varian, Mercury Plus spectrometer at 300 MHz are reported as parts per million (ppm) downfield from tetramethylsilane internal standard.

Ethyl 3-(Benzylamino)-3-Oxopropanoate (5)

IRν_max/cm^-1: 3447 (NH₂), 1731 (C = O ester), 1651 (C = O amide).

¹H NMR (CDCl₃) δH: 1.24 (t, 3 H, CH₃), 3.64 (s, 2 H, CH₂), 4.21 (q, 2 H, CH₂), 4.32 (s, 2 H, CH₂), 6.63 (br s, 1 H, NH), 7.27-7.39 (m, 5 H, Ar).

Ethyl 4-(Benzylamino)-4-Oxobutanoate (6)

IRν_max/cm^-1:3441 (NH₂), 1733 (C = O ester), 1650 (C = O amide).

¹H NMR (CDCl₃) δH: 1.10 (t, 3 H, CH₃), 2.51 (t, 2 H, CH₂), 2.62 (t, 2 H, CH₂), 4.10 (t, 3 H, CH₃), 6.63 (br s, 1 H, NH), 7.24-7.32 (m, 5 H, Ar).

(R,S)-Methyl 4-(Benzylamino)-3-Hydroxy-4-Oxobutanoate (8)

IRν_max/cm^-1: 3351 (NH₂), 1730 (C = O ester), 1650 (C = O amide). (CDCl₃) δH: 2.7 (q, 1 H, CH), 3.0 (q, 1 H, CH), 3.7 (s, 3 H, CH₃), 4.07 (br s, 1 H, NH), 4.50 (m, 1 H, CH), 7.20-7.45 (m, 5 H, Ar)