Efficient access to enantiomerically pure cyclic α-amino esters through a lipase-catalyzed kinetic resolution

Abstract

A series of α-amino acid derivatives containing the 2,3-dihydroindole or octahydroindole core have been chemoenzymatically synthesized in good overall yields and high enantiomeric purity under mild reaction conditions using lipases for the introduction of chirality. Candida antarctica lipase type A has shown excellent activity and high enantiodiscrimination ability towards the two cyclic amino esters used as substrates. The selectivity of the process proved to be greatly dependent on the alkoxycarbonylating agent. Thus, the enzymatic kinetic resolution of methyl indoline-2-carboxylate has been successfully achieved using 3-methoxyphenyl allyl carbonate, whereas (2R,3aR,7aR)-benzyl octahydroindole-2-carboxylate required the less reactive diallyl carbonate.

1. Introduction

The preparation of optically active compounds is a great challenge for organic chemists, and enantiomerically pure amines and amino acid derivatives represent one of the most important targets.¹ Nowadays, enzyme-mediated processes are becoming usual techniques for enantioselective transformations and, among the biocatalysts of synthetic interest, lipases have gained much attention over the last two decades due to their ease of manipulation, low cost and wide substrate tolerance.² Traditionally, most lipase-catalyzed enantioselective reactions have corresponded to the acylation of alcohols or hydrolysis of esters, whereas the preparation of chiral amides or carbamates have been much less investigated in spite of the fact that mild reaction conditions are required for the formation of these nitrogenated compounds.³ Examples of enzymatic kinetic or dynamic kinetic resolutions of cyclic secondary amines described in the literature are rare,⁴ even though they are present in a large number of biologically relevant systems and are useful building blocks for the preparation of many valuable products.

Among cyclic secondary amines, the indoline (2,3-dihydroindole) subunit is found in a wide range of natural products and biologically active compounds.⁵ Only a few catalytic methods for the production of optically active indolines, either through non-enzymatic⁶ or enzymatic4b,4g,4j methodologies, have been reported. Kurokawa and Sugai described the enzymatic kinetic resolution of methyl N-Boc-indoline-2-carboxylate through a Candida antarctica lipase B (CAL-B) enantioselective hydrolysis, which yielded both the substrate and the product in enantiopure form.^4g In our ongoing research on the synthesis of optically active secondary amines, we observed^4j a different reactivity of Candida antarctica lipase A (CAL-A) and CAL-B towards the N-protection of indolines. Thus, whereas CAL-A exhibited a high enantiopreference towards 2-substituted derivatives, CAL-B showed remarkable stereoselectivity towards 3-methyl indoline. This fact suggests that CAL-A prefers to act at sterically hindered positions,⁷ while the approach of indolines to the active site of CAL-B demands sterically non-hindered situations.⁸

Octahydroindole-2-carboxylic acid (Oic), the completely hydrogenated analogue of indoline-2-carboxylic acid, is the core structure of numerous compounds with applications in medicinal chemistry. For example, it is present in marine compounds with antithrombotic properties;⁹ in the dipeptide perindropil (a potent antihypertensive drug used in the prevention of cardiovascular disorders such as heart failure);¹⁰ and in the prolyl oligopeptidase inhibitor S 17092, a compound with antiamnesic and cognitive-enhancing properties.¹¹ Recently, some of us reported¹² the synthesis of enantiomerically pure (2S,3aS,7aS)- and (2R,3aR,7aR)-Oic derivatives using preparative chiral HPLC,^12a and that of the (2R,3aS,7aS) isomer by selective formation of a trichloromethyloxazolidinone.^12b The only enzymatic process for producing optically active Oic derivatives was described by Hirata¹³ and is based on the enzymatic hydrolysis of N-protected octahydroindole-2-carboxylates.

Our interest in indoline-2-carboxylic acid and octahydroindole-2-carboxylic acid to be used as proline analogues together with our experience in the enzymatic resolution of secondary amines, prompted us to apply procedures to the isolation of these compounds in enantiomerically pure form. Herein, we report the application of a straightforward and highly selective lipase-catalyzed methodology for the alkoxycarbonylation of the secondary amino group in these compounds.

2. Results and Discussion

Initially, we focused on the enzymatic resolution of methyl indoline-2-carboxylate rac-2. This compound was obtained in excellent yield by treatment of commercially available indoline-2-carboxylic acid rac-1 with thionyl chloride in refluxing methanol (Scheme 1). Prior to the enzymatic resolution assays, compound rac-2 was transformed into carbamate rac-3 by reaction with allyl chloroformate, in order to establish the appropriate analytical conditions for HPLC separation of the two enantiomers.

Scheme 1.

Chemical synthesis and enzymatic kinetic resolution of methyl indoline-2-carboxylate

The enzymatic kinetic resolution of rac-2 was then attempted using Candida antarctica lipase type A (CAL-A) from different sources and Candida antarctica lipase type B (CAL-B) as biocatalysts. The data are summarized in Table 1. Based on previous results with 2-substituted indoline derivatives,^4j 3-methoxyphenyl allyl carbonate¹⁴ 4a was initially selected as the alkoxycarbonylating reagent for enzyme activity screening, which was carried out at 30 °C using dry tert-butyl methyl ether (TBME) as the solvent and a 1:2.5 amino ester to carbonate ratio (entries 1–4). For all the tested CAL-A preparations, a complete enantioselectivity was observed in the alkoxycarbonylation process. Lipase from Biocatalytics showed a lower reaction rate than the ones from Roche or Codexis, although all of them reached 50% conversion (entries 1–3). An excellent selectivity was also observed for CAL-B, but low conversions were reached in longer reaction times (entry 4). Finally, the reaction was carried out using CAL-A from Codexis and commercially available diallyl carbonate 4b (entries 5 and 6). The use of this reagent avoids the need for the purification step to separate the 3-methoxyphenol released during the enzymatic process from the reaction crude. However, this change resulted in lower kinetics (entry 5), while higher temperatures led to a decrease in the enantioselectivity value (entry 6) probably due to the inactivation of the enzyme.

Table 1.

Lipase-catalyzed kinetic resolution of amino ester rac-2 using carbonates 4a–b (ratio 1:2.5) in TBME

Entry	Enzyme	R	T (°C)	t (h)	eeP (%)a	eeS (%)a	c (%)b	Ec
1	CAL-A Biocatalytics	3-MeO-C6H4	30	20	>99	98	50	>200
2	CAL-A Roche	3-MeO-C6H4	30	8	>99	98	50	>200
3	CAL-A Codexis	3-MeO-C6H4	30	4	>99 (92)d	>99 (86)d	50	>200
4	CAL-B	3-MeO-C6H4	30	60	99	85	46	>200
5	CAL-A Codexis	CH2=CHCH2	30	74	92	16	15	29
6	CAL-A Codexis	CH2=CHCH2	45	49	94	8	8	35

^aCalculated by HPLC

^bc = ee_S/(ee_S+ee_p)

^cE = ln[(1−c)×(1−ee_P)]/ln[(1−c)×(1+ee_P)]

^dIsolated yields in brackets.

Thus, under the optimal conditions, we were able to isolate one enantiomer of both the substrate and the product in enantiomerically pure form and good yield (Table 1, entry 4). The absolute configuration of the amino ester 2 remaining from the enzymatic kinetic resolution was determined by comparing its HPLC retention time¹⁵ with that of a sample of enantiopure (S)-2 obtained by esterification of commercially available (S)-indoline-2-carboxylic acid, (S)-1. This allowed us to assign an (R)-configuration to the unreacted amine 2 and an (S)-stereochemistry to the isolated carbamate 3 (Scheme 1).

We next undertook the development of adequate chemoenzymatic procedures to obtain Oic derivatives in enantiopure form (Scheme 2). Hydrogenation of racemic indoline-2-carboxylic acid rac-1 following our previously described methodology,^12a afforded rac-5, which was transformed into the corresponding methyl rac-6a and benzyl rac-6b esters using standard procedures. Reaction with allyl chloroformate led to carbamates rac-7a and rac-7b, respectively, which were analyzed by HPLC to find adequate conditions for the separation of their enantiomers.

Scheme 2.

Chemical synthesis and enzymatic kinetic resolution of Oic derivatives

For the enzymatic kinetic resolution of rac-6a, CAL-A from Codexis was initially chosen as the biocatalyst, since it gave the best results in the resolution of 2, and different carbonates were tested. No reaction was observed when using dibenzyl carbonate, even when the temperature was raised to 45 °C. In contrast, the more reactive methoxyphenyl allyl carbonate 4a led to the formation of racemic carbamate 7a (Table 2, entry 1). The use of diallyl carbonate 4b, which is known to exhibit intermediate reactivity, allowed the formation of 7a with excellent selectivity, although only 20% conversion was reached after 23 h at 30 °C (entry 2). An increase in the temperature (entry 3) did not result in a higher conversion but rather in a loss of enzyme activity, as observed before for 2.

Table 2.

Lipase-catalyzed kinetic resolution of amino esters rac-6a–b using carbonates 4a–b (ratio 1:2.5) in TBME

Entry	Enzyme	R1	R2	T (°C)	t (h)	eeP (%)a	eeS (%)a	c (%)b	Ec
1	CAL-A Codexis	Me	3-MeO-C6H4	30	23.5	----	----	100	----
2	CAL-A Codexis	Me	CH2=CHCH2	30	23	>99	25	20	>200
3	CAL-A Codexis	Me	CH2=CHCH2	45	48	>99	13	12	>200
4	CAL-A Codexis	Bn	CH2=CHCH2	30	57	88	>99	53	121
5	CAL-A Biocatalytics	Bn	CH2=CHCH2	30	72	98 (91)d	>99 (90)d	50	>200

^aCalculated by HPLC

^bc = ee_S/(ee_S+ee_p)

^cE = ln[(1−c)×(1−ee_P)]/ln[(1−c)×(1+ee_P)]

^dIsolated yields in brackets.

Our attention was then focused on the resolution of amino ester rac-6b that, with it being more sterically hindered than rac-6a, could be a better substrate for the active site of CAL-A.⁷ Indeed, the enzymatic kinetic resolution of rac-6b using CAL-A from Codexis was found to proceed with very high enantiopreference, thus allowing recovery of the remaining starting material in enantiopure form (entry 4). Alternatively, when CAL-A from Biocatalytics was used, a 50% conversion was reached, with both the product and the starting material being obtained in almost enantiomerically pure form and excellent isolated yields (entry 5). The absolute configuration of both compounds was assigned after transformation of the remaining substrate into a derivative of known stereochemistry. Thus, the unreacted enantiomerically pure amino ester 6b was subjected to N-tert-butoxycarbonyl protection, as previously described for the racemic compound,^12a to afford 8b (Scheme 2); the HPLC retention time of the latter compound was found to be identical¹⁶ to that of a sample of (2R,3aR,7aR)-8b prepared in our previous work.^12a Thereafter, we concluded that the unreacted amino ester 6b has a (2R,3aR,7aR)-configuration and the carbamate formed in the enzymatic process 7b is (2S,3aS,7aS).

3. Conclusions

In conclusion, the development of a practical synthetic route for the preparation of optically active indoline-2-carboxylic acid and octahydroindole-2-carboxylic acid derivatives has been successfully achieved through enzymatic kinetic resolution using lipases in organic solvents. The different lipases tested exhibited distinct behaviors in the alkoxycarbonylation process, depending on the structure of the starting materials. In all cases, CALA has been found to be the most efficient biocatalyst. The choice of the appropriate allyl carbonate also proved crucial to reach a good selectivity and conversion. Under the best conditions, all substrates and products were isolated in good yields and enantiomerically pure form (98% ee in one case). This methodology represents an excellent alternative to those previously described for the preparation of these biologically significant compounds.

4. Experimental

4.1. General

Candida antarctica lipase type B (CAL-B, Novozyme 435, 7300 PLU/g) was a gift from Novo Nordisk Co. Candida antarctica lipase type A was acquired from different commercial sources:¹⁷ CAL-A Chirazyme L-5, c–f, lyophilized, 1000 U/g using tributyrin from Roche, CAL-A immobilized NZL-101, 5.0 U/g using 1-butanol and ethyl laurate from Codexis; CAL-A IMB-104, 2.6 U/mg using tributyrin from Biocatalytics). All other reagents were purchased from Aldrich and used without further purification. Solvents were distilled over an adequate desiccant under nitrogen. Flash chromatography was performed using silica gel 60 (230–240 mesh). High performance liquid chromatography (HPLC) analyses were carried out at 20 °C in a Hewlett Packard 1100 chromatograph under the conditions specified for each substrate and with UV monitoring at 210 nm. IR spectra were recorded on using NaCl plates or KBr pellets in a Perkin-Elmer 1720-X FT. ¹H, ¹³C NMR, DEPT, and ¹H-¹³C heteronuclear experiments were obtained using AC-200 (¹H, 200.13 MHz and ¹³C, 50.3 MHz), AC-300 (¹H, 300.13 MHz and ¹³C, 75.5 MHz), DPX-300 (¹H, 300.13 MHz and ¹³C, 75.5 MHz) or AV-400 (¹H, 400.13 MHz and ¹³C, 100.6 MHz) Bruker spectrometers. The chemical shifts are given in delta (δ) values and the coupling constants (J) in Hertz (Hz).¹⁸ HP1100 chromatograph mass detector was used to record mass spectra experiments (MS) through APCI⁺ or ESI⁺ experiments. Measurement of the optical rotation was done in a Perkin-Elmer 241 polarimeter.

4.2. Synthesis of methyl indoline-2-carboxylate rac-2

To a solution of compound rac-1 (1.0 g; 6.13 mmol) in methanol (100 mL) under a nitrogen atmosphere, thionyl chloride (671 µL, 9.19 mmol) was added dropwise and the reaction mixture was heated at reflux for 2 h. The solvent was removed and the resulting residue was suspended in saturated aqueous NaHCO₃ and extracted with CH₂Cl₂ (3 × 30mL). The organic fractions were combined, dried and evaporated to dryness. The crude product was purified by flash chromatography (30% EtOAc/hexane) to afford 1.01 g of rac-2 as an oil (97%). R_f (40% EtOAc/hexane): 0.44; IR (NaCl): ν 3335, 2950, 2356, 1732, 1684, 1652, 1609, 1531, 1487, 1436, 1313, 1257, 1207, 1151, 992, 773, 748 cm⁻¹; ¹H-NMR (CDCl₃, 300.13 MHz): δ 7.02-7.11 (m, 2H, H₄+H₆), 6.71-6.79 (m, 2H, H₅+H₇), 4.39 (dd, J= 9.0, 6.0 Hz, 1H, H₂), 3.80 (s, 3H, H₁₀), 3.28-3.45 (m, 2H, 2H₃); ¹³C-NMR (CDCl₃, 75.5 MHz): δ 174.5 (C₈), 149.9 (C_7a), 127.6 (C₅), 126.5 (C_3a), 124.4 (C₄), 119.4 (C₆), 110.0 (C₇), 59.7 (C₂), 52.4 (C₁₀), 33.6 (C₃). MS (APCI⁺, m/z): 200 [(M+Na)⁺, 100%], 178 [(M+H)⁺, 12%]. HPLC elution times: (S)-2 15.7 min, (R)-2 17.1 min; conditions: 25 × 0.46 cm ID Chiralcel OD column, eluent n-hexane/ethanol (97:3) at 0.8 mL/min flow rate.

4.3. Synthesis of methyl N-(allyloxycarbonyl)indoline-2-carboxylate rac-3

Pyridine (14.5 µL, 0.18 mmol) was added to a solution of amino ester rac-2 (30 mg, 0.17 mmol) in dry CH₂Cl₂ (1.13 mL) under a nitrogen atmosphere and the mixture was cooled in an ice bath. Allyl chloroformate (15.3 µL, 0.18 mmol) was added dropwise and the system was allowed to warm up to room temperature. After stirring for 4 h, no starting material was detected by TLC analysis. The solvent was evaporated under reduced pressure and the residue obtained was purified by flash chromatography (30% EtOAc/hexane) to afford 40 mg of carbamate rac-3 as a pale yellow oil (90%). R_f (40% EtOAc/hexane): 0.51; IR (NaCl): ν 3336, 2954, 2365, 2342, 1756, 1701, 1648, 1604, 1530, 1487, 1400, 1268, 1205, 826, 749 cm⁻¹; ¹H-NMR (CDCl₃, 400.13 MHz): δ 7.70 (t, J= 8.0 Hz, 1H, H₇), 7.43 (d, J= 8.3 Hz, 1H, H₄), 7.33 (t, J= 15.1 Hz, 1H, H₅), 7.16 (t, J= 15.1 Hz, 1H, H₆), 6.00-6.25 (m, 1H, H₁₄), 5.25-5.60 (m, 2H, H₁₅), 5.05-5.15 (m, 1H, H₂), 4.70-5.00 (m, 2H, H₁₃), 3.90 (s, 3H, H₁₀), 3.69 (t, J = 12.0 Hz, 1H, H₃), 3.30 (d, J = 15.0 Hz, 1H, H₃); ¹³C-NMR (CDCl₃, 100.6 MHz): δ 172 (C₈), 152 (C₁₁), 142.1 (C_7a), 132.1 (2C, C₁₄ and C_3a), 127.9 (C₅), 124.3 (C₄), 122.9 (C₆), 117.7 (C₁₅), 114.7 (C₇), 66.0 (C₁₃), 59.9 (C₂), 52.9 (C₁₀), 32.8 (C₃); MS (APCI⁺, m/z): 300 [(M+K)⁺, 34%], 284 [(M+Na)⁺, 100%], 262 [(M+H)⁺, 51%]. HPLC elution times: (S)-3 12.3 min, (R)-3 14.9 min; conditions: 25 × 0.46 cm ID Chiralcel OD column, eluent n-hexane/ethanol (97:3) at 0.8 mL/min flow rate.

4.4. Typical procedure for the enzymatic kinetic resolution of methyl indoline-2-carboxylate rac-2

To a suspension of compound rac-2 (50 mg, 0.28 mmol) and lipase (100 mg) in TBME (1.9 mL) kept under a nitrogen atmosphere was added the corresponding carbonate 4a–b (0.70 mmol) and the system was shaken at 30 °C and 250 rpm. The course of the reaction was followed by HPLC till conversions were around 50% (see Table 1). The mixture was then filtered and the filtrate was concentrated under reduced pressure. The crude product was purified by flash chromatography (eluent gradient 5–15% EtOAc/hexane) to give (R)-2 {[α]_D ²⁰ = −47.3 (c 0.28, CH₂Cl₂) for >99% ee} and (S)-3 {[α]_D²⁰ = −106.5 (c 1.2, CH₂Cl₂) for >99% ee}.

4.5. Synthesis of (2S*,3aS*,7aS*)-methyl octahydroindole-2-carboxylate rac-6a

Thionyl chloride (1.18 mL, 16.18 mmol) was added dropwise to a solution of rac-5 (1.32 g, 7.81 mmol) in dry methanol (20 mL) at 0° C. The resulting solution was stirred at room temperature for 24 h. The solvent was evaporated to dryness and the resulting residue was solved in water and lyophilized. The white solid obtained was partitioned between saturated aqueous NaHCO₃ (25 mL) and CH₂Cl₂ (50 mL). The organic layer was separated and the aqueous phase was further extracted with CH₂Cl₂ (2 × 50 mL). The combined organic layers were dried over anhydrous MgSO₄, filtered and evaporated to afford rac-6a as an oil (1.20 g; 6.56 mmol; 84%). R_f (5% MeOH/CH₂Cl₂): 0.24; IR (NaCl): ν 3319, 3033, 2927, 2856, 1733, 1454, 1191, 750 cm⁻¹; ¹HNMR (CDCl₃, 400.13 MHz): δ 3.81 (dd, J = 10.0, 6.0 Hz, 1H, H₂), 3.74 (s, 3H, 3H₁₀), 3.09 (m, 1H, H_7a), 2.38 (bs, 1H, NH), 2.17 (ddd, J= 12.8, 10.0, 6.8 Hz, 1H, H₃), 2.03 (m, 1H, H_3a), 1.74-1.60 (m, 3H, H₃+2H₇), 1.52-1.30 (m, 3H, H₄+H₅+H₆), 1.30-1.15 (m, 3H, H₆+H₄+H₅); ¹³C-NMR (CDCl₃, 100.6 MHz): δ 176.2 (C₈); 58.4 (C₂); 58.0 (C_7a); 52.0 (C₁₀); 37.9 (C_3a); 35.6 (C₃); 27.9 (C₇); 27.0 (C₄); 23.4 (C₅); 21.5 (C₆); MS (APCI⁺, m/z): 184 [(M+H)⁺, 100%], 124 [(M-CO₂Me)⁺, 32%]. HPLC elution times: first enantiomer 8.7 min, second enantiomer 10.7 min; conditions: 25 × 0.46 cm ID Chiralcel OD column, eluent n-hexane/2-propanol (95:5) at 0.8 mL/min flow rate.

4.6. Synthesis of (2S*,3aS*,7aS*)-benzyl octahydroindole-2-carboxylate rac-6b

To a solution of rac-5 (1.00 g, 5.92 mmol) in toluene (21 mL) p-toluenesulphonic acid monohydrate (1.63 g, 8.59 mmol) and benzyl alcohol (2.30 mL, 22.21 mmol) were added. The resulting system was heated at reflux for 4 h using a Dean-Stark trap. After evaporation to dryness, the residue was triturated with diisopropyl ether and the white solid formed was collected by filtration. It was then partitioned between saturated aqueous NaHCO₃ (25 mL) and CH₂Cl₂ (50 mL). The organic layer was separated and the aqueous phase further extracted with CH₂Cl₂ (2 × 50 mL). The combined organic extracts were dried over anhydrous MgSO₄, filtered and evaporated to afford rac-6b as an oil (1.380 g; 5.33 mmol; 90%). R_f (5% MeOH/CH₂Cl₂): 0.41; IR (NaCl): ν 3319, 3033, 2927, 2856, 1733, 1454, 1191, 750 cm⁻¹; ¹H-NMR (CDCl₃, 400.13 MHz): δ 7.37-7.28 (m, 5H, Ph); 5.20 (s, 2H, H₁₀); 3.88 (dd, J = 10.1, 5.9 Hz, 1H, H₂); 3.12 (m, 1H, H_7a); 2.52 (bs, 1H, NH); 2.20 (ddd, J = 12.8, 10.1, 6.9 Hz, 1H, H₃); 2.04 (m, 1H, H_3a); 1.77-1.65 (m, 3H, H₃+2H₇); 1.48 (m, 3H, H₄+H₅+H₆); 1.39 (m, 1H, H₆); 1.26 (m, 2H, H₄+H₅); ¹³C-NMR (CDCl₃, 100.6 MHz): δ 175.7 (C₈); 135.7 (C_ipsoPh), 128.5, 128.2, 128.1 (5C, Ph), 66.7 (C₁₀); 58.6 (C₂); 58.1 (C_7a); 37.9 (C_3a); 35.8 (C₃); 27.9 (C₇); 27.0 (C₄); 23.5 (C₅); 21.5 (C₆). MS (APCI⁺, m/z): 260 [(M+H)⁺, 100]. HPLC elution times: (2S,3aS,7aS)-6b 8.2 min, (2R,3aR,7aR)-6b 9.6 min; conditions: 25 × 0.46 cm ID Chiralcel OD column, eluent n-hexane/2-propanol (90:10) at 0.8 mL/min flow rate.

4.7. Synthesis of carbamates rac-7a–b

Allylchloroformate (18.0 µL, 0.17 mmol) was added dropwise to a solution of the amino ester rac-6a–b (0.15 mmol) in dry CH₂Cl₂ (1.03 mL) kept under a nitrogen atmosphere at 0 °C and the system was allowed to warm up to room temperature. After stirring for 4 h, no starting material was detected by TLC analysis. The solvent was evaporated under reduced pressure and the residue obtained was purified by flash chromatography (20% EtOAc/hexane) to afford the corresponding carbamate rac-7a–b as an oil (40–49%).

4.7.1. (2S*,3aS*,7aS*)-Methyl N-(allyloxycarbonyl)-octahydroindole-2-carboxylate rac-7a

R_f (20% EtOAc/hexane): 0.25; IR (NaCl): ν 2929, 2857, 1753, 1705, 1407, 1345, 1303, 1262, 1176, 1123, 1010 cm⁻¹; ¹H-NMR (CDCl₃, 300.13 MHz): δ 5.98-5.77 (m, 1H, H₁₁), 5.31-5.13 (m, 2H, 2H₁₅), 4.58-4.44 (m, 2H, H₁₃), 4.33-4.25 (m, 1H, H₂), 3.93-3.78 (m, 1H, H_7a), 3.73 (d, J = 9.8 Hz, 3H, H₁₀), 2.34-2.29 (m, 1H, H_3a), 2.14-1.92 (m, 3H, 2H₃+H₇), 1.73-1.58 (m, 3H, H₅+2H₄), 1.50-1.36 (m, 2H, H₇+H₆), 1.34-1.08 (m, 2H, H₅, H₆); ¹³C-NMR (CDCl₃, 75.5 MHz): δ (duplicate signals are observed for all carbon atoms) 173.6, 173,4 (C₈), 154.4, 153.7 (C₁₁), 133.0, 132.7 (C₁₄), 117.1, 116.8 (C₁₅), 65.7, 65.5 (C₁₃), 59.0, 58.9 (C₂), 57.7, 57.3 (C_7a), 52.1, 52.0 (C₁₀), 37.0, 36.4 (C_3a), 32.5, 31.5 (C₃), 27.8, 27.2 (C₇), 25.7, 25.6 (C₄), 23.6, 23.5 (C₅), 20.4, 20.3 (C₆); MS (APCI⁺, m/z): 268 [(M+H)⁺, 100%], 210 [(M-CO₂Me+2H)⁺, 10%], 182 [(M-CO₂CH₂CHCH₂)⁺, 31%], 124 [(M-CO₂CH₂CHCH₂-CO₂Me+H)⁺, 5%]. HPLC elution times: first enantiomer 9.6 min, second enantiomer 15.6 min; conditions: 25 × 0.46 cm ID Chiralcel OD column, eluent n-hexane/2-propanol (95:5) at 0.8 mL/min flow rate.

4.7.2. (2S*,3aS*,7aS*)-Benzyl N-(allyloxycarbonyl)-octahydroindole-2-carboxylate rac-7b

R_f (20% EtOAc/hexane): 0.29; IR (NaCl): ν 2929, 2857, 1750, 1703, 1407, 1347, 1303, 1262, 1172, 1124, 989 cm⁻¹; ¹H-NMR (CDCl₃, 300.13 MHz): δ 7.43-7.28 (m, 5H, Ph), 5.98-5.68 (m, 1H, H₁₄), 5.32-5.08 (m, 4H, 2H₁₀+2H₁₃), 4.67-4.50 (m, 1H, H₁₅), 4.47-4.44 (m, 1H, H₁₅), 4.40-4.30 (m, 1H, H₂), 3.95-3.80 (m, 1H, H_7a), 2.32-2.23 (m, 1H, H_3a), 2.21-2.10 (m, 2H, H₃), 2.06-1.96 (m, 1H, H₇), 1.73-1.58 (m, 3H, H₅+2H₄), 1.50-1.38 (m, 2H, H₇+H₆), 1.32-1.14 (m, 2H, H₅+H₆); ¹³C-NMR (CDCl₃, 75.5 MHz): δ (duplicate signals are observed for most carbon atoms) 173.0, 172.8 (C₈), 154.4, 153.7 (C₁₁), 135.8, 135.6 (C_ipso Ph), 133.0, 132.7 (C₁₄), 128.5, 128.4, 128.2, 128.0, 127.9 (5C, Ph), 117.1, 116.9 (C₁₅), 66.6 (C₁₄), 65.7, 65.6 (C₁₃), 59.1, 59.0 (C₂), 57.7, 57.3 (C_7a), 37.0, 36.4 (C_3a), 32.5, 31.4 (C₃), 27.8, 27.2 (C₇), 25.7, 25.6 (C₄), 23.6, 23.5 (C₅), 20.4, 20.3 (C₆). MS (APCI⁺, m/z): 366 [(M+Na+H)⁺, 70%], 344 [(M+2H)⁺, 100%]. HPLC elution times: (2S,3aS,7aS)-7b 9.1 min, (2R,3aR,7aR)-7b 12.1 min; conditions: 25 × 0.46 cm ID Chiralcel OD column, eluent n-hexane/2-propanol (90:10) at 0.8 mL/min flow rate.

4.8. Typical procedure for the enzymatic kinetic resolution of (2S*,3aS*,7aS*)-methyl and benzyl octahydroindole-2-carboxylate rac-6a–b

To a suspension of the amino ester rac-6a–b (0.12 mmol) and lipase (1:2 in weight with respect to the amino ester) in TBME (1.8 mL) kept under a nitrogen atmosphere was added the corresponding carbonate 4a–b (0.30 mmol) and the system was shaken at the required temperature and 250 rpm. The course of the reaction was followed by HPLC (see Table 2). After filtration, the solution was concentrated under reduced pressure and the crude was purified by flash chromatography (eluent gradient 10–100% EtOAc/hexane) to afford 6a–b {(2R,3aR,7aR)-6b: [α]_D²⁰ = +23.1 (c 1.0, CHCl₃) for >99% ee} and 7a–b {one enantiomer of 7a: [α]_D²⁰ = +30.4 (c 0.76, CHCl₃) for >99% ee; (2S,3aS,7aS)-7b: [α]_D²⁰ = −42.8 (c 1.0, CHCl₃) for 98% ee}.