Chemoselective Lipase-Catalyzed Synthesis of Amido Derivatives from 5-Hydroxymethylfurfurylamine

The acylations of furfurylamine and 5-hydroxymethylfurfurylamine (HMFA) have been studied finding immobilized Candida antarctica lipase B (CALB) as an ideal biocatalyst. CALB was used immobilized on two different supports (Novozyme 435 and EziG-CALB), with the polymer-coated controlled porosity glass carrier material from EnginZyme being an excellent carrier to yield an active and stable enzymatic preparation for the acylation of the primary amine group. The amount of the acyl donor in the reaction was a key factor to achieve the mono- and chemoselective N-protection of HMFA with large excess of ethyl acetate leading to the formation of the N,O-diacetylated product. Thus, a series of 16 nonactivated esters were used to selectively modify the amine group of HMFA, obtaining 9 hydroxy amides under mild reaction conditions and with quantitative yields through chromatography-free transformations. The influence of substrate concentration was studied, resulting in complete conversions in all cases after 22 h (100–1000 mM). Excellent results were observed at 100 and 200 mM of HMFA, while higher concentrations led to longer reaction times and, to some extent, the formation of the diacetylated product (up to 7% after 22 h at 1 M). After this optimization, a metric analysis was performed to confirm the high sustainability of the presented process (E-factor of 1.1 excluding solvents) upon intensification of the biotransformation to 1 g at 200 mM HMFA concentration. The possibility of obtaining orthogonally protected HMFA-derived amido esters has been achieved through a clean and sequential one-pot process using EziG-CALB, which involved the use of ethyl methoxy acetate as the nonactivated ester for N-acylation and the activated vinyl acetate for O-protection.


Lipase-catalyzed acetylation of amine 4
The lipase-catalyzed acetylation of 2-furfurylamine (4) was developed with different hydrolases (see main text for additional information), prior developing the optimization of the reaction conditions.

General protocol for the lipase screening in the acetylation of amine 4
Amine 4 (20 mg, 0.2 mmol, 100 mM) was dissolved in methyl tert-butyl ether (MTBE, 2 mL) in an Erlenmeyer-flask, then the corresponding hydrolase (20 mg, 1:1 w/w enzyme:4 ratio) and EtOAc (59 µL, 0.6 mmol, 3 equiv) were successively added. The reaction was shaken at 250 rpm for 24 h at 30 °C, and after this time an aliquot was taken and analyzed by GC (see Section 4). The conversion values are depicted in Table S1.

Monitorization of the CALB-catalyzed acetylation of amine 4
Both immobilized CALB forms, the one from Novozymes and the one from EnginZyme were used for the acetylation of amine 4, and a reaction time course study was performed, taking regular aliquots after 0.5, 1 and 2 h ( Figure S1).

Solvent screening for the EziG-CALB-catalyzed acetylation of amine 4
The lipase-catalyzed acetylation of amine 4 was developed following the general protocol described in Section 2.1, but during only 2 h and using different solvents as disclosed in Table S2. Hexane 10 a Reaction conditions: 4 (20 mg, 100 mM), 3 equiv of 5a and EziG-CALB (20 mg, 1:1 w/w enzyme:4 ratio) for 2 h at 30 ºC and 250 rpm. b Product percentage calculated by GC analyses. c Used as solvent and acyl donor.

Lipase screening for the acetylation of HMFA with EtOAc
HMFA (3, 15 mg, 0.12 mmol, 100 mM) was dissolved in 2-MeTHF (1.2 mL) inside an Erlenmeyer-flask, then the corresponding hydrolase (15 mg, 1:1 w/w enzyme:3 ratio) and EtOAc (5a, 15 µL, 0.15 mmol, 1.3 equiv) were successively added. The reaction was shaken at 250 rpm for 24 h at 30 °C, and after that time an aliquot was taken and analyzed by GC (see Section 4). The conversion values are depicted in Table 1 of the manuscript.

Acylation of HMFA with EziG-CALB using different acyl donors 5a-p
Lipase-catalyzed selective N-acylation was performed using EziG-CALB under optimized reaction conditions (Table S3, see experimental section of the manuscript for detailed information). Table S3. Acyl donor study for the acylation of HMFA with EziG-CALB in 2-MeTHF.

Entry
Acyl donor 5a-p a Percentage of products measured by 1 H-NMR analyses of the reaction crude. Quantitative isolated yields were reached after filtration and evaporation of the filtrate for most of the reactions. For ethyl phenylacetate (5e) the reaction crude was dried on the freeze-dryer overnight. For benzyl acetate (5j) and 4-nitrophenyl acetate (5k), the formation of product 7a was observed in complete conversion, but no purification was performed to remove the remaining acyl donor. For ethyl caprate (5o) methyl laurate (5p), to remove the excess of the acyl donor, the reaction crude was washed with cold Et2O (3 x 2 mL).

Study of the influence of the HMFA concentration using EtOAc
Ethyl acetate (1.3 equiv) was added to a mixture of HMFA (3, 30-150 mg, 0.24-1.2 mmol, 200-1000 mM), EziG-CALB (15 mg), and 2-MeTHF (1.2 mL). The corresponding mixture was shaken between 2 and 22 h at 250 rpm and 30 °C, taking aliquots regularly that were analyzed by GC (see Figure S2 and Table S4).  Figure S3. Calculated E-factor for the acetylation of 3 with EtOAc using EziG-CALB at 1-g scale. Figure S4. Contribution of the different components to the E-factor for the acetylation with EtOAc of 3 using EziG-CALB at 1-g scale.
to obtain 1,13 g product (g) to obtain 1 kg Amine (g) to obtain 1 kg Amine (

Analytical methods (Gas chromatography analyses)
GC analyses were performed on an Agilent HP6890 GC chromatograph equipped with a FID detector. In all cases, a HP-1 column (30 m x 0.32 mm x 0.25 m) was used for the determination of conversion values and product percentages (Table S4).