Supporting Information

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1 Supporting Information Amide Synthesis via Aminolysis of Ester or Acid with an Intracellular Lipase Shichao Zeng, a Ji Liu, a Sampson Anankanbil, b Ming Chen, a Joseph P. Adams, c Radka Snajdrova c and Zhi Li *a a. Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore b. Department of Engineering, Faculty of Science and Technology, Aarhus University, 8000 Aarhus, Denmark c. Chemical Sciences, GSK R&D Medicines Research Centre, Gunnelswood Road, Stevenage, SG1 2NY, UK The authors contributed equally to this work. * Corresponding author. chelz@nus.edu.sg S1

2 Table of Contents 1. Chemicals... S3 1.1 Chemicals for analysis... S3 1.2 Chemicals for the preparation of product standards and biotransformation... S3 1.3 Chemicals for strain screening... S4 1.4 Chemicals for protein purification... S4 2. Strains, plasmids and biochemicals... S4 3. HPLC analysis... S5 4. SDS-PAGE analysis... S6 5. Molecular weight determination of purified his-tagged SpL... S6 6. Preparation of product standards... S7 6.1 Preparation of N-benzylhexanamide 3a... S7 6.2 Preparation of N-benzylcyclohexanecarboxamide 3b... S8 6.3 Preparation of N-benzylbenzenepropanamide 3d... S8 6.4 Preparation of N-phenylbenzenepropanamide 3e... S8 6.5 Preparation of N-(2-phenylethyl)benzenepropanamide 3f... S9 6.6 Preparation of N-pentylbenzenepropanamide 3g... S9 6.7 Preparation of (rac)-α-hydroxy-n-(phenylmethyl)benzeneacetamide 3h... S9 6.8 Preparation of (rac)-n-(1-cyclohexylethyl)benzenepropanamide 3i... S Preparation of (rac)-n-(1-phenylethyl)benzenepropanamide 3j... S10 7. Determination of the activity of E. coli (SpL) cells for the hydrolysis of PNPB... S11 8. CALB-catalyzed aminolysis of ester in the presence of water... S12 9. General procedure for aminolysis of esters to give amides 3a-g and (R)-3h-j with free CALB... S NMR spectra... S HRMS spectra... S HPLC chromatograms... S26 S2

3 1. Chemicals All the following chemicals were obtained from commercial suppliers and used without further purification. 1.1 Chemicals for analysis 4-nitrophenyl butyrate (>98%), dipotassium phosphate (>98%), monopotassium phosphate (>99.0%), tert-butyl methyl ether (99.8%) and benzyl alcohol (>99.0%) were purchased from Sigma Aldrich. Acetonitrile (HPLC grade) and n-hexane (HPLC grade) were purchased from Tedia. Isopropanol (HPLC grade) and Chloroform (HPLC) were purchased from Fisher. 1.2 Chemicals for the preparation of product standards and biotransformation Methyl hexanoate 1a (>99%), methyl cyclohexanecarboxylate 1b (>98%), methyl benzoate 1c (99%), hexanoic acid 4b (>99.5%), octanoic acid 4c (>98%), decanoic acid 4d (>98%), oleic acid 4e (>99%), cyclopentanenoic acid 4f (>99%), cyclohexanecarboxylic acid 4g (>98%),, mandelic acid 4h (99%), (rac)-methyl mandelate 1e (97%), methyl (S)-(+)- mandelate (S)-1e (>99%), benzylamine 2a (99%), aniline 2b (>99.5%), phenethylamine 2c (>99%), amylamine 2d (99%), (R)-(-)-1-cyclohexylethylamine (R)-2e (98%), (S)-(+)-1- cyclohexylethylamine (S)-2e (98%), α-methylbenzylamine 2f (99%), (R)-(+)-αmethylbenzylamine (R)-2f (98%), N-benzylbenzamide 3c (98%), molecular sieve 4A (powder, 325 mesh), sodium sulfate (>99%), N-(3-Dimethylaminopropyl)-N - ethylcarbodiimide hydrochloride (EDC, >99%) and dichloromethane (>99.8%) were purchased from Sigma Aldrich. Methyl 3-phenylpropionate 1d (>98%) and 3- phenylpropionic acid 4a (>98.0%) were purchased from TCI. Ethyl acetate (HPLC grade) was purchased from Fisher. Novozyme 435 is a gift from Novozymes (Bagsvaerd, Denmark). S3

4 1.3 Chemicals for strain screening ctane (>99%), sodium phosphate dibasic heptahydrate (>99.99%), sodium chloride (>99.5%), ammonium chloride (>99.5%), magnesium sulfate (>99.5%) and calcium chloride dihydrate (>99%) were purchased from Sigma Aldrich. 1.4 Chemicals for protein purification Imidazole (>99%) and sodium phosphate monobasic monohydrate (>98%) were purchased from Sigma Aldrich. 2. Strains, plasmids and bio-reagents Escherichia coli T7 expression strain, restriction enzymes (NdeI, Bgl II, BamHI and XhoI) and T4 DNA quick ligase were purchased from New England Biolabs. Expression vector prsfduet-1 was acquired from Novagen. ligoes (primers), IPTG (>99%), agarose (molecular biology grade), and 10 TAE buffer (ultrapure grade) were purchased from 1st BASE, Singapore. Kanamycin (>99%) and glycerol (>99%) were purchased from Sigma Aldrich. Phusion DNA polymerase, plasmid miniprep kit, gel extraction kit and PCR purification kit were purchased from Thermo Scientific. DNeasy blood & tissue kit was bought from Qiagen. LB broth, LB agar, tryptone and yeast extract were purchased from Biomed Diagnostics. Free Candida antarctica lipase B (CALB) was bought from SPRIN technologies. 4% 12% NuPAGE Bis-Tris precast gel (in MES-SDS Running Buffer) was acquired from life technologies. DNA sequencing was carried out by 1st BASE, Singapore. S4

5 3. HPLC analysis N-pentylhexanamide was analyzed on an Agilent 7890A gas chromatography equipped with a HP-5 capillary column (30 m 322 µm 0.25 µm) with inlet temperature of 280 o C and detector temperature of 300 o C. The temperature program used was: 40 o C keep for 1 min, then increase to 120 o C at a rate of 5 o C/ minute, then increase to 250 o C at a rate of 10 o C/minute. The retention time for N-pentylhexanamide is min. 3a-3j were analyzed on a Shimadzu prominence HPLC system (LC-20AD, normal phase) equipped with a Diacel Chiralpak IA-3 column ( mm, 3 µm). The UV detection wavelength was set at 210 nm. ven temperature was set at 25 o C. The mobile phase consisted of a mixture of n-hexane and isopropanol at a flow rate of 1 ml/min. 3k-n were analyzed on a Shimadzu prominence HPLC system (LC-20AD, normal phase) equipped with an Agilent RX-SIL column ( mm). The UV detection wavelength was set at 210 nm. ven temperature was set at 25 o C. The mobile phase consisted of a mixture of n-hexane (with 0.1% triacetic acid) and isopropanol at a flow rate of 1 ml/min. Table S1. HPLC Analysis Conditions and the Retention Time for 3a-3j Product RT n-hexane: isopropanol 3a 7.9 min 90: 10 3b 9.1 min 90: 10 3c 16.1 min 90: 10 3d 11.2 min 90: 10 3e 11.3 min 90: 10 3f 9.8 min 90: 10 3g 7.0 min 90: 10 (R)-3h 13.8 min (S)-3h 20.4 min 90: 10 (R)-3i 11.1 min (S)-3i 13.5 min 95: 5 (R)-3j 8.4 min (S)-3j 10.6 min 90: 10 3a 3.1 min 90: 10 3k 2.9 min 90: 10 3l 2.8 min 90: 10 3m 4.0 min 95: 5 3n 3.1 min 90: 10 S5

6 4. SDS-PAGE analysis SDS-PAGE was used to analyze the protein expression level of E.coli (his-tagged SpL). The freshly harvested cells were suspended in DI water to an D 600 of 10. The cells were passed through a homogenizer (Stansted fluid power LTD) twice, followed by centrifugation to give the cell-free extract (CFE). The CFE was mixed with 2 SDS loading buffer at a volume ratio of 1:1, heated at 95 o C for 5 min and subjected to SDS-PAGE analysis according to the standard method. 5. Molecular weight determination of purified his-tagged SpL The molecular weight of the purified his-tagged SpL was determined by MALDI-TF-MS. The results are given below: Calculated mass (Da) for 6 his-tagged SpL is , based on the amino acid sequence: MGSSHHHHHHSQDPMTDSTTHYTRPDVAAFLAFLNAQEGPKMEEMPPAGAREMM RVMGQLADVPRGEIAKVEDRMIPGPDGDIPIRLYDNRPDREAGPVMVFYHGGGWVI GDLETHDPYCAEAARILPVIAIDYRLAPEHPFPAAPIDCEAATRWVADNIACTGLVLS GDSAGGNLTIVTALALRDEPAAKPVIAIHPIYPAVTTHNDWQSYRDFGEGHLLTEGS MTWFGNHYAADPADRRAAPIDFPADGLPTLITASLDPLRDQGRAYAAKLIEAGVPTT YREAKGTIHGYICLAQGIPSAKDDIRGALTVLKAIVAEATGAA Measured mass (Da) for 6 his-tagged SpL is The decrease of Da suggested the N-terminal des-met protein. S6

7 Figure S1. Mass spectrum of 6 his-tagged SpL 6. Preparation of product standards 6.1 Preparation of N-benzylhexanamide 3a Hexanoic acid 4b (239.3 mg, 2.06 mmol), benzylamine 2a (214.3 mg, 2 mmol), EDC (479.3 mg, 2.5 mmol) and 10 ml dichloromethane were added into a 50 ml round bottom flask. The reaction was conducted at room temperature for 12 h. Afterwards, the solvent was evaporated and the crude product was washed with 20% ethanol and water and then, dried in vacuum oven for overnight to give N-benzylhexanamide 3a as white solid at 60% isolated yield. 1 H NMR (400 MHz, CDCl 3 ) δ (m, 5H), (s, br, 1H), (d, 2H, J = 5.6 Hz), (t, 2H, J = 7.6 Hz), (m, 2H), (m, 4H), (t, 3H, J = 6.8 Hz); 13 C NMR: (100 MHz, CDCl 3 ) δ , , , , , 43.52, 36.70, 31.43, 25.40, 22.33, S7

8 6.2 Preparation of N-benzylcyclohexanecarboxamide 3b Cyclohexanecarboxylic acid 4g (264 mg, 2.06 mmol), benzylamine 2a (214.3 mg, 2 mmol), EDC (479.3 mg, 2.5 mmol) and 10 ml dichloromethane were added into a 50 ml round bottom flask. The reaction was conducted at room temperature for 12 h. After reaction the solvent was evaporated. The crude product was washed with 20% ethanol and water and then, dried in vacuum oven for overnight to give N-benzylcyclohexanecarboxamide 3b as white solid at 71% isolated yield. 1 H NMR (400 MHz, CDCl 3 ) δ (m, 5H), (s, br, 1H), (d, 2H, J = 5.6 Hz), (tt, 1H, J=12.0, 3.6 Hz), (m, 4H), (m, 1H), (m, 2H), (m, 3H); 13 C NMR: (100 MHz, CDCl 3 ) δ , , , , , , , , Preparation of N-benzylbenzenepropanamide 3d 3-Phenylpropionic acid 4a (309.4 mg, 2.06 mmol), benzylamine 2a (214.3 mg, 2 mmol), EDC (479.3 mg, 2.5 mmol) and 10 ml dichloromethane were added into a 50 ml round bottom flask. The reaction was conducted at room temperature for 12 h. Afterwards, the solvent was evaporated and the crude product was washed with 20% ethanol and water and then, dried in vacuum oven for overnight to give N-benzylbenzenepropanamide 3d as white solid at 56% isolated yield. 1 H NMR (400 MHz, CDCl 3 ): δ (m, 10H), (s, br, 1H), (d, 2H, J = 5.6 Hz), (t, 2H, J=7.6 Hz), (t, 2H, J = 7.6 Hz); 13 C NMR (100 MHz, CDCl 3 ): δ , , , , , , , , , 43.54, 38.44, Preparation of N-phenylbenzenepropanamide 3e The reaction mixture contained 30 ml n-hexane, 2.4 ml water, 150 mg lyophilized SpL enzyme, methyl 3-phenylpropionate 1d (328 mg, 2 mmol) and aniline 2b (279 mg, 3 mmol). The mixture was incubated at 30 o C and stirred at 500 rpm for 2 h. The product was extracted using 50 ml dichloromethane. The resulting organic phase was taken and solvent was evaporated. The resulting content was purified using flash chromatography with n- S8

9 hexane:ethyl acetate (10:1) eluent to give N-phenylbenzenepropanamide 3e as white solid with 61% isolated yield. 1 H NMR (400 MHz, CDCl 3 ): δ (m, 11H), (t, 2H, J = 7.6 Hz), (t, 2H, J = 7.6 Hz); 13 C NMR (100 MHz, CDCl 3 ): δ , , , , , , , , , , IR (KBr): 3321, 1654 cm -1 ; ESI-MS m/z ([M+Na] + ); HRMS (ESI) for C 15 H 15 NNa +, calculated , found Preparation of N-(2-phenylethyl)benzenepropanamide 3f 3-Phenylpropionic acid 4a (309.4 mg, 2.06 mmol), phenethylamine 2c (243.6 mg, 2 mmol), EDC (479.3 mg, 2.5 mmol) and 10 ml dichloromethane were added into a 50 ml round bottom flask. The reaction was conducted at room temperature for 12 h. Afterwards the solvent was evaporated and the crude product was washed with 20% ethanol and water and then, dried in vacuum oven for overnight to give N-(2-phenylethyl)benzenepropanamide 3f as white solid at 77% isolated yield. 1 H NMR (400 MHz, CDCl 3 ): δ (m, 10H), (s, br, 1H), (t, 2H, J=6.8 Hz), (t, 2H, J = 7.6 Hz), (t, 2H, J = 6.8 Hz), (t, 2H, J = 7.6 Hz); 13 C NMR (100 MHz, CDCl 3 ): δ , , , , , , , , , 40.51, 38.47, 35.62, Preparation of N-pentylbenzenepropanamide 3g 3-Phenylpropionic acid 4a (309.4 mg, 2.06 mmol), amylamine 2d (174.3 mg, 2 mmol), EDC (479.3 mg, 2.5 mmol) and 10 ml dichloromethane were added into a 50 ml round bottom flask. The reaction was conducted at room temperature for 12 h. Afterwards, the solvent was evaporated and the crude product was washed with 20% ethanol and water and then, dried in vacuum oven for overnight to give N-pentylbenzenepropanamide 3g as white solid at 62% isolated yield. 1 H NMR (400 MHz, CDCl 3 ): δ (m, 5H), (s, br, 1H), (t, 2H, J=7.2 Hz), (t, 2H, J = 7.6 Hz), (t, 2H, J = 7.6 Hz), (m, 2H), (m, 4H), (t, 3H, J = 7.2 Hz). 13 C NMR (100 MHz, CDCl 3 ): δ , , , , , 39.46, 38.53, 31.76, 29.20, 28.93, 22.27, Preparation of (rac)-α-hydroxy-n-(phenylmethyl)benzeneacetamide 3h S9

10 Mandelic acid 4h (313.4 mg, 2.06 mmol), benzylamine 2a (214.3 mg, 2 mmol), EDC (479.3 mg, 2.5 mmol) and 10 ml dichloromethane were added into a 50 ml round bottom flask. The reaction was conducted at room temperature for 12 h. Afterwards the solvent was evaporated and the crude product was purified using flash chromatography (n-hexane:ethyl acetate=2:1) to give (rac)-α-hydroxy-n- (phenylmethyl)benzeneacetamide 3h as white solid at 37% isolated yield. 1 H NMR (400 MHz, CDCl 3 ): δ (m, 10H), (s, br, 1H), (s, 1H), (m, 2H); 13 C NMR (100 MHz, CDCl 3 ): δ , , , , , , , , , , 74.16, Preparation of (rac)-n-(1-cyclohexylethyl)benzenepropanamide 3i 3-phenylpropionic acid 4a (309.4 mg, 2.06 mmol), (rac)-1-cyclohexylethylamine 2e (254.5 mg, 2 mmol), EDC (479.3 mg, 2.5 mmol) and 10 ml dichloromethane were added into a 50 ml round bottom flask. The reaction was conducted at room temperature for 12 h. Afterwards the solvent was evaporated and the crude product was purified using flash chromatography (n-hexane:ethyl acetate=5:1) to give (rac)-n-(1-cyclohexylethyl) benzenepropanamide 3i as white solid at 11% isolated yield. 1 H NMR (400 MHz, CDCl 3 ): δ (m, 5H), (s, br, 1H), (m, 1H), (t, 2H, J = 7.2 Hz), (t, 2H, J=6.8 Hz), (m, 5H), (m, 4H), (d, 3H, J = 6.8 Hz), (m, 2H). 13 C NMR (100 MHz, CDCl 3 ): δ , , , , , 49.19, 42.91, 38.73, 31.81, 28.87, 28.82, 26.31, 26.11, 26.10, 17.79; IR (KBr): 3309, 1639 cm -1 ; ESI-MS m/z ([M+Na] + ); HRMS (ESI) for C 17 H 25 NNa +, calculated , found Preparation of (rac)-n-(1-phenylethyl)benzenepropanamide 3j 3-Phenylpropionic acid 4a (309.4 mg, 2.06 mmol), (rac)-α-methylbenzylamine 2f (242.4 mg, 2 mmol), EDC (479.3 mg, 2.5 mmol) and 10 ml dichloromethane were added into a 50 ml round bottom flask. The reaction was conducted at room temperature for 12 h. Afterwards the S10

11 solvent was evaporated and the crude product was purified using flash chromatography (nhexane:ethyl acetate=2:1) to give (rac)-n-(1-phenylethyl)benzenepropanamide 3j as white solid at 23% isolated yield. 1 H NMR (400 MHz, CDCl 3 ): δ (m, 10H), (s, br, 1H), (m, 1H), (t, 2H, J = 7.6 Hz), (t, 2H, J=8 Hz), (d, 3H, J=7.2 Hz); 13 C NMR (100 MHz, CDCl 3 ): δ , , , , , , , , , 48.57, 38.54, 31.69, Determination of the activity of E. coli (SpL) cells for the hydrolysis of PNPB Freshly prepared E. coli (SpL) cells were suspended in KP buffer (100 mm, ph 7.5) to 0.5 g cdw/l. The cell suspension was then passed through a cell homogenizer (Stansted fluid power LTD) twice, and cell-free extract (CFE) was obtained by removal of cell debris via centrifugation. The CFE was diluted with KP buffer (100 mm, ph 7.5) properly to a fixed protein concentration and 10 µl of the diluted cell-free extract was added to 990 µl of KP buffer (100 mm, ph 7.5). 10 µl of 50 mm PNPB acetonitrile solution was added and the absorption at 400 nm was monitored by a photometer. The specific activity was calculated based on the UV absorption, extinction coefficient of p-nitrophenol, pass length, reaction time, and protein concentration. df = Dilution factor = Micromolar extinction coefficient of p-nitrophenol at 400 nm 0.01 = Volume (in ml) of enzyme used S11

12 8. CALB-catalyzed aminolysis of ester in the presence of water A mixture consisting of 10 mm methyl hexanoate 1a, 20 mm benzylamine 2a, and 10 mg free CALB in 5 ml n-hexane containing 0-8% (v/v) water was shaken at 250 rpm and 30 ºC. For the case of 0% (v/v) water, 200 mg of activated 4A molecular sieve was added to ensure totally anhydrous condition. Specific activity was measured at 30 min, and amide yield was determined at 20 h. 120 Specific activity (U/g protein) Final amide yield (%) % 0.50% 1% 2% 4% 8% Water content (%) Figure S2. Effect of water content on aminolysis of methyl hexanoate 1a with benzylamine 2a catalyzed by free CALB in n-hexane. : Specific activity (U/g protein); : Final amide yield (%). 9. General procedure for aminolysis of esters to give amides 3a-g and (R)- 3h-j with free CALB Free CALB was prepared by removing the glycerol and salts inside the commercially available CALB solution from SPRIN by washing the enzyme solution with deionized water in an Amicon Ultra-15 Centrifugal Filter Units (20 kd). The dry enzyme powder was acquired by lyophilizing the enzyme solution for at least 24 h. 10 mg free CALB was added to a 10 ml shaking flask containing 5 ml n-hexane, 200 mg activated 4A molecular sieve powder and 20 mm amine 2a-f. 10 mm ester 1a-e was then S12

13 added to initiate the reaction and the mixture was shaken at 30 C, and 250 rpm. Specific activity was measured at 30 min. Amide yield was determined at 20 h. Table S2. Synthesis of Amide 3a-g via Aminolysis of Ester 1a-d with Amine 2a-d by Using CALB. R 1 1 1a: R 1 = n-pentyl 1b: R 1 = Cy 1c: R 1 = Ph 1d: R 1 = PhCH 2 CH 2 1d 1d 1d + R 2 NH 2 Enzyme R 2 R 1 NH 2 3 2a: R 2 = PhCH 2 2a 2a 2a 2b: R 2 = Ph 2c: R 2 = PhCH 2 CH 2 2d: R 2 = n-pentyl + CH 3 H 3a: R 1 = n-pentyl; R 2 = PhCH 2 3b: R 1 = Cy; R 2 = PhCH 2 3c: R 1 = Ph; R 2 = PhCH 2 3d: R 1 = PhCH 2 CH 2 ; R 2 = PhCH 2 3e: R 1 = PhCH 2 CH 2 ; R 2 = Ph 3f: R 1 = PhCH 2 CH 2 ; R 2 = PhCH 2 CH 2 3g: R 1 = PhCH 2 CH 2 ; R 2 = n-pentyl CALB a Activity (U/g) b Yield (%) c 21.5 > a The reaction was performed with 10 mm of ester 1a-d and 20 mm of amine 2a-d in 5 ml anhydrous n-hexane containing 10 mg of free enzyme. b Specific activity (U/g enzyme) was determined after 30 min. c Yield (%) of the amide was determined after 20 h. Table S3. Enantioselective Aminolysis of Ester with Amine to Synthesize Chiral Amides by Using Free CALB. Entry a Enzyme Ester Amine Product Time (h) Activity (U/g enzyme) b ee p (%) c Yield (%) c E e 1 CALB 1e 2a (R)-3h CALB 1d 2e (R)-3i d CALB 1d 2f (R)-3j d a The reaction was performed with 10 mm of ester and 20 mm of amine in 5 ml anhydrous n-hexane with 10 mg of pure enzyme. b Specific activity (U/g enzyme) was determined after 30 min. c ee p (%) and yield (%) of the amide were determined after the time stated in the table d Specific activity was determined after 20 h. The specific activity at 30 min was too low to be detectable. e E was calculated according to the equation E=ln [1-c (1+ee p )]/ln [1-c (1-ee p )], c is the conversion. S13

14 10. NMR spectra a) N H 3a b) Figure S3. a) 1 H NMR spectrum of 3a. b) 13 C NMR spectrum of 3a. 3a was prepared via aminolysis of ester 3a with amine 2a by using SpL. S14

15 a) b) Figure S4. a) 1 H NMR spectrum of 3b. b) 13 C NMR spectrum of 3b. 3b was prepared via aminolysis of ester 3b with amine 2a by using SpL. S15

16 a) b) Figure S5. a) 1 H NMR spectrum of 3d. b) 13 C NMR spectrum of 3d. 3d was prepared via aminolysis of ester 3d with amine 2a by using SpL. S16

17 Figure S6. a) 1 H NMR spectrum of 3a. b) 13 C NMR spectrum of 3a. 3a was prepared via aminolysis of hexanoic acid 4b with benzylamine 2a by using E. coli (SpL). S17

18 a) b) Figure S7. a) 1 H NMR spectrum of 3k. b) 13 C NMR spectrum of 3k. 3k was prepared via aminolysis of octanoic acid 4c with benzylamine 2a by using E. coli (SpL). S18

19 a) NH 3l b) Figure S8. a) 1 H NMR spectrum of 3l. b) 13 C NMR spectrum of 3l. 3l was prepared via aminolysis of decanoic acid 4d with benzylamine 2a by using E. coli (SpL). S19

20 a) b) Figure S9. a) 1 H NMR spectrum of 3m. b) 13 C NMR spectrum of 3m. 3m was prepared via aminolysis of oleic acid 4e with benzylamine 2a by using E. coli (SpL). S20

21 a) b) Figure S10. a) 1 H NMR spectrum of 3n. b) 13 C NMR spectrum of 3n. 3n was prepared via aminolysis of cyclopentanecarboxylic acid 4f with benzylamine 2a by using E. coli (SpL). S21

22 11. HRMS spectra [M+Na] + [M+Na+ACN]+ [M+H] + Figure S11. HRMS spectrum of 3a. 3a was prepared via aminolysis of ester with SpL. [M+H] + [M+Na] + + [M+Na+ACN] Figure S12. HRMS spectrum of 3b. 3b was prepared via aminolysis of ester with SpL. [M+Na] + [M+H] + Figure S13. HRMS spectrum of 3d. 3d was prepared via aminolysis of ester with SpL. S22

23 Figure S14. HRMS spectrum of 3a. 3a was prepared via aminolysis of hexanoic acid 4b with benzylamine 2a catalyzed by E.coli (SpL). [M+H] + S23

24 Figure S15. HRMS spectrum of 3k. 3k was prepared via aminolysis of octanoic acid 4c with benzylamine 2a catalyzed by E.coli (SpL). [M+H] + Figure S16. HRMS spectrum of 3l. 3l was prepared via aminolysis of decanoic acid 4d with benzylamine 2a catalyzed by E.coli (SpL). [M+H] + Figure S17. HRMS spectrum of 3m. 3m was prepared via aminolysis of oleic acid 4e with benzylamine 2a catalyzed by E.coli (SpL). S24

25 [M+H] + Figure S18. HRMS spectrum of 3n. 3n was prepared via aminolysis of cyclopentanenoic acid 4f with benzylamine 2a catalyzed by E.coli (SpL). S25

26 12. HPLC chromatograms + NH 2 SpL N H 1a 2a 3a a) 3a b) 3a Figure S19. a) Normal phase HPLC chromatogram of 3a prepared by condensation of hexanoic acid 4c and benzylamine 2a. b) Normal phase HPLC chromatogram of the reaction mixture of SpLcatalyzed aminolysis of 1a with 2a at 30 min. S26

27 + NH 2 SpL N H 1b 2a 3b a) 3b b) 3b Figure S20. a) Normal phase HPLC chromatogram of 3b prepared by condensation of cyclohexanecarboxylic acid 4b and benzylamine 2a. b) Normal phase HPLC chromatogram of the reaction mixture of SpL-catalyzed aminolysis of 1b with 2a at 30 min. S27

28 + NH 2 SpL N H 1c 2a 3c a) 3c b) 1c 3c Figure S21. a) Normal phase HPLC chromatogram of 3c (Sigma-Aldrich). b) Normal phase HPLC chromatogram of the reaction mixture from SpL-catalyzed aminolysis of 1c with 2a at 30 min. S28

29 + NH 2 SpL N H 1d 2a 3d a) 3d b) 3d 1d Figure S22. a) Normal phase HPLC chromatogram of 3d prepared by condensation of 3- phenylpropionic acid 4a and benzylamine 2a. b) Normal phase HPLC chromatogram of the reaction mixture of SpL-catalyzed aminolysis of 1d with 2a at 30 min. S29

30 + NH 2 SpL N H 1d 2b 3e a) 3e b) 1d 2b 3e Figure S23. a) Normal phase HPLC chromatogram of 3e prepared by SpL-catalyzed aminolysis of 1d with 2b. b) Normal phase HPLC chromatogram of the reaction mixture of SpL-catalyzed aminolysis of 1d with 2b at 30 min. S30

31 + NH 2 SpL N H 1d 2c 3f a) 3f b) 3f 1d Figure S24. a) Normal phase HPLC chromatogram of 3f prepared by condensation of 3- phenylpropionic acid 4a and phenethylamine 2c. b) Normal phase HPLC chromatogram of the reaction mixture of SpL-catalyzed aminolysis of 1d with 2c at 30 min. S31

32 + NH 2 SpL N H 1d 2d 3g a) 3g b) 1d 3g Figure S25. a) Normal phase HPLC chromatogram of 3g prepared by condensation of 3- phenylpropionic acid 4a and amylamine 2d. b) Normal phase HPLC chromatogram of the reaction mixture of SpL-catalyzed aminolysis of 1d with 2d at 30 min. S32

33 H + NH 2 SpL H NH 1e 2a 3h a) (R)-3h (S)-3h b) (S)-3h (R)-3h c) (R)-3h (S)-3h Figure S 26. a) Normal phase HPLC chromatogram of racemic 3h prepared by condensation of mandelic acid 4d and benzylamine 2a. b) Normal phase HPLC chromatogram of the reaction mixture of Novozyme 435-catalyzed aminolysis of (S)-1e with 2a. c) Normal phase HPLC chromatogram of the reaction mixture of SpL-catalyzed aminolysis of racemic 1e with 2a at 30 min. S33

34 + H 2 N SpL NH 1d 2e 3i a) (R)-3i (S)-3i b) (R)-3i c) (R)-3i (S)-3i Figure S27. a) Normal phase HPLC chromatogram of racemic 3i prepared by condensation of 3- phenylpropionic acid 4a and 1-cyclohexylethylamine 2e. b) Normal phase HPLC chromatogram of the reaction mixture of Novozyme 435-catalyzed aminolysis of 1d with (R)-2e. c) Normal phase HPLC chromatogram of the reaction mixture of SpL-catalyzed aminolysis of 1d with racemic 2e at 30 min. S34

35 + H 2 N SpL NH 1d 2f 3j a) (R)-3j (S)-3j b) (R)-3j c) 1d (R)-3j (S)-3j Figure S28. a) Normal phase HPLC chromatogram of racemic 3j prepared by condensation of 3- phenylpropionic acid 4a and α-methylbenzylamine 2f. b) Normal phase HPLC chromatogram of the reaction mixture of Novozyme 435-catalyzed aminolysis of 1d with (R)-2f. c) Normal phase HPLC chromatogram of the reaction mixture of SpL-catalyzed aminolysis of 1d with racemic 2f at 30 min. S35

36 CH + NH 2 SpL NH 4b 2a 3a 3a Figure S29. Normal phase HPLC chromatogram of 3a prepared from the aminolysis of hexanoic acid 4b with benzylamine 2a catalyzed by E. coli (SpL). S36

37 CH + NH 2 SpL NH 4c 2a 3k 3k Figure S30. Normal phase HPLC chromatogram of 3k prepared from the aminolysis of octanoic acid 4c with benzylamine 2a catalyzed by E. coli (SpL). S37

38 CH + NH 2 SpL NH 4d 2a 3l 3l Figure S31. Normal phase HPLC chromatogram of racemic 3l prepared from the aminolysis of decanoic acid 4d with benzylamine 2a catalyzed by E. coli (SpL). S38

39 H + NH 2 SpL N H 4e 2a 3m 3m Figure S32. Normal phase HPLC chromatogram of racemic 3m prepared from the aminolysis of oleic acid 4e with benzylamine 2a catalyzed by E. coli (SpL). S39

40 CH + NH 2 SpL NH 4f 2a 3n 3n Figure S33. Normal phase HPLC chromatogram of racemic 3n prepared from the aminolysis of cyclopentanecarboxylic acid 4f with benzylamine 2a catalyzed by E. coli (SpL). S40