One-pot biocatalytic amine transaminase/acyl transferase cascade for aqueous formation of amides from aldehydes or ketones

Enzyme cloning, expression and purification The cloning of the gene of Silicibacter pomeroyi amine transaminase (Sp-ATA) has been described elsewhere.1 The codon-optimized gene of Mycobacterium smegmatis acyl transferase (MsAcT) was purchased cloned into pET-28a(+) from GenScript (Piscataway, U.S.A.). Expression of Sp-ATA was performed by inoculating 20 mL LB-medium (100 μg/mL ampicillin) with Escherichia coli (E. coli) BL21 (DE3) cells containing the Sp-ATA encoding plasmid. The culture was incubated at 37 °C and 210 rpm overnight. 180 mL LB-medium (100 μg/mL ampicillin and 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG)) was then inoculated with the overnight culture and incubated at 25 °C and 140 rpm for 24 h. After expression, the cells were harvested by centrifugation (8000 rpm for 15 min). The cell pellet was dissolved in Immobilized Metal ion Affinity Chromatography (IMAC) binding buffer (sodium phosphate buffer (20 mM, pH 7.4) and NaCl (0.5 M)) and cells were disrupted by sonication. Cell debris was removed by centrifugation (20000 rpm form 15 min) and the supernatant was collected and filtered (0.45 μm). The enzyme was then purified by IMAC according to the manufacturers’ protocol. Pyridoxal-5 ́-phosphate (PLP) was added in an excess (1 mM) and the solution was incubated in room temperature for 30 minutes. Finally, a buffer change to CHES buffer (0.2 M, pH 10) was performed by running the solution through a PD-10 column according to the manufacturers’ protocol. The enzyme solution was stored at 4 °C. Expression of MsAcT was performed by inoculating 10 mL LB-medium (50 μg/mL kanamycin) with E. coli BL21 (DE3) cells containing the MsAcT encoding plasmid. The culture was incubated at 37 °C and 210 rpm overnight. 1 L LB-medium (50 μg/mL kanamycin) was inoculated with the overnight culture and incubated at 37 °C and 210 rpm until an optical density (OD600) of 0.6-0.8 was reached. 1 mM IPTG was added and the culture was incubated at 30 °C and 140 rpm for 18h. Harvesting, disruption and IMAC purification was performed as with Sp-ATA. After purification, the buffer was changed into potassium phosphate buffer (20 mM, pH 7.2) using a PD-10 column. The enzyme solution was stored at -20 °C.

Biocatalytic acylation/amidation is well-known and has been applied for decades 1 but it has often been limited to the use of organic solvents to avoid hydrolysis.The acyl transferase from Mycobacterium smegmatis (MsAcT) is an enzyme that can perform trans-acylations in aqueous solution. 2,3Only a few hydrolases can catalyze trans-acylation in water [4][5][6][7][8] and MsAcT also has the ability to act as a perhydrolase and catalyze the formation of peracids from esters. 3 Since MsAcT can catalyze acylation for the formation of esters in aqueous solution we hypothesized that it could also utilize amines for amidation under the same conditions.Amidation should also in theory be able to reach higher conversions due to the higher nucleophilicity of the amine compared to the alcohol and the more stable amide product which would drive the equilibrium towards product formation.
Amine transaminases have been extensively studied during the last decades due to their ability to perform asymmetric synthesis of both chiral and achiral primary amines from the corresponding pro-chiral ketones or aldehydes with excellent yield and enantiomeric excess (ee). 9,102][13][14][15][16][17] There are also recent reports of synthetic cascades where amine transaminases are involved in longer reactions sequences. 18There have however been fewer reports of amine transaminase cascades where the reductive amination is followed by amidation in a later stage of the cascade 19 and it has never been performed in a one-pot one-step fashion where one-step means that the reaction is carried out in only one operational step even though the cascade contains several reaction steps working simultaneously.The amide is a common functional group in a wide variety of important compounds such as pharmaceuticals 1,20,21 and polymers 22 and an aqueous onepot biocatalytic synthesis route starting from inexpensive aldehydes and ketones would be a cheap and green alternative to existing procedures.The irreversible nature of amide formation in these mild conditions and in the presence of the previously mentioned enzymes 23 would also serve as efficient equilibrium displacement of the thermodynamically challenging reductive amination.
One theoretical drawback of a one-pot one-step amine transaminase/acyl transferase cascade is that the amine donor of the transamination will compete with the amine product in the second step of the cascade.This can however be circumvented when MsAcT is used since it does not accept amino acids as substrates for amidation (unpublished results).This opens up the possibility of using alanine as amine donor for the transamination step of the cascade since it is accepted by most amine transaminases.L-alanine (used in the case of an (S)-selective amine transaminase) is cheap and readily available.It also has a high solubility in water and can therefore be used in large excess to displace the equilibrium towards product formation.
Since there is a plethora of available amine transaminases that catalyze the desired reaction under a wide range of pHvalues, the optimization of the cascade conditions was focused on MsAcT.The pH-optimum was investigated using a model reaction with benzylamine as acyl acceptor/nucleophile and methyl acetate as acyl donor forming the product N-benzylacetamide (Scheme 1, MsAcT catalyzed step).The optimal pH was found to be around pH 11 (Fig. S1, ESI ‡).The reason for the high pH-optimum is believed to be due to the protonation state of benzylamine at higher pH-values (pK a = 9.34). 24There is however no known amine transaminase operating well at pH-values above 10 and therefore pH 10 was chosen for the continuation of the cascade optimization.One amine transaminase that is active at this alkaline pH is the amine transaminase from Silicibacter pomeroyi (Sp-ATA).It has a wide substrate scope and a pH-optimum of approximately 9.5 25 and was therefore chosen as the catalyst for the first step of the cascade.
With an established pH of the cascade, reactions could now be run and followed to see how far the reaction could proceed.The above mentioned model reaction (Scheme 1, MsAcT catalyzed step) and the same reaction using methyl methoxyacetate as acyl donor were followed over time (Fig. 1).7][28] The reason for this is that the possibility to form an internal hydrogen bond between the methoxy oxygen and the nitrogen of the formed amide makes the amine a better competitor against water and less hydrolysis will therefore theoretically occur. 29As can be seen in Fig. 1, the reaction utilizing methyl acetate as acyl donor reached a maximum conversion of 86% after 90 minutes while methyl methoxyacetate gave a slower reaction rate but a higher conversion of 93% after 270 minutes.
The cascade was optimized by varying the enzyme concentrations.It was hypothesized that Sp-ATA needed to be ap-plied in large excess over MsAcT to avoid too much hydrolysis of the acyl donor before enough amounts of amine could be formed to compete with water.The optimization with respect to enzyme concentration was performed with benzaldehyde as starting substrate.Sp-ATA uses the amine donor L-alanine to perform the transamination of benzaldehyde to benzylamine.MsAcT then amidates the formed benzylamine using either methyl acetate or methyl methoxyacetate to form the final corresponding amide (Scheme 1).The enzymes were applied at three different concentrations each and as can be seen in Fig. 2, the highest conversion of 29% was achieved with 0.16 U mL −1 MsAcT and 3 U mL −1 Sp-ATA using methyl acetate as acyl donor.However, in the case of methyl methoxyacetate, the highest conversions were reached when more MsAcT was added.The highest conversions of 74% and 71% (Fig. 2) were achieved with 1.6 U mL −1 MsAcT and 0.3 U mL −1 or 3 U mL −1 Sp-ATA respectively.This is most likely due to the above mentioned ability of methyl methoxyacetate to make the amine a better competitor against water.
The cascade was further optimized by continuing with one of the conditions that gave the highest conversions (3 U mL −1 Sp-ATA, 1.6 U mL −1 MsAcT and 1% v/v methyl methoxyacetate) (Table 1, entry 1).The concentration of methyl methoxyacetate was increased to 2% in order to achieve a higher conversion of the cascade.A higher concentration of methyl methoxyacetate was not tested due to the resulting low activity of Sp-ATA (Fig. S2, ESI ‡).The decreased activity of Sp-ATA when the concentration of methyl methoxyacetate is increased was the reason for the choice of continuing with the higher Sp-ATA concentration (3 U mL −1 ).Since the acyl  donor concentration was increased, the buffer concentration also needed to be increased (CHES, 0.4 M, pH 10).By applying a higher acyl donor concentration the conversion of the cascade to form N-benzyl-2-methoxyacetamide could be improved from 84% (Table 1, entry 2) when 1% v/v methyl methoxyacetate was used to 97% (Table 1, entry 3) when 2% v/v was used.The higher conversion of 84% compared to 71% in the previous optimization step when the same substrate and catalyst concentrations were used can be explained by the higher buffer concentration which allows the enzymes to operate under optimal pH for a longer time.The substrate scope of the amine transaminase/acyl transferase cascade using the now optimized conditions was investigated by applying the cascade in the synthesis of N-phenylethyl-2methoxyacetamide (Table 1, entry 5), N-butyl-2methoxyacetamide (Table 1, entry 6) and N-heptyl-2methoxyacetamide (Table 1, entry 7).The conversions were lower compared to the synthesis of N-benzyl-2methoxyacetamide (Table 1) but this can be explained by the different activities of the enzymes towards the substrates and conversions can probably be increased by optimizing the amine transaminase/acyl transferase ratio for each substrate.
The ability of MsAcT to displace the equilibrium of the transamination step was investigated by running the first step of the cascade without the addition of MsAcT and methyl methoxyacetate in the same conditions as the complete cascade with benzaldehyde as a substrate.Sp-ATA was able to synthesize benzylamine from benzaldehyde at a conversion of 40% which clearly shows that MsAcT contributes to the equilibrium displacement of the transamination.
To further increase the applications of the amine transaminase/acyl transferase cascade it was evaluated for the synthesis of enantiomerically pure chiral amides.The chiral substrate analog of benzylamine, 1-phenylethylamine, was initially investigated for amidation catalyzed by MsAcT.No activity was however detected and focus was shifted to the aliphatic amine 2-aminohexane as the aliphatic secondary alco-hol 2-octanol previously has been shown to be accepted by MsAcT. 3 The ability of MsAcT to amidate both enantiomers of 2-aminohexane was investigated in a reaction with methyl methoxyacetate (2% v/v).The results show that the activity for 2-aminohexane is substantially lower than for benzylamine.MsAcT also has a poor enantioselectivity as it was able to synthesize both (S)-and (R)-N-(hexan-2-yl)-2methoxyacetamide at conversions of 18% and 5% respectively (Fig. 3).When the racemic 2-aminohexane was applied the reaction reached a conversion of 13% with an ee of 56% (S).However, the low enantioselectivity of MsAcT means that both product enantiomers can be synthesized if enantioselectivity is introduced in the first step of the cascade.Amine transaminases are highly enantioselective and enzymes with both (S)-and (R)-selectivity are available.
The amine transaminase/acyl transferase cascade was employed in the synthesis of (S)-N-(hexan-2-yl)-2methoxyacetamide (MsAcT activity against the (R)-enantiomer was deemed too low for a successful cascade) starting from 2-hexanone.However, no conversion to product was detected and the reason for this is probably due to low activity of Sp-ATA against 2-hexanone. 25ble 1 Results of the continued cascade optimization after optimal enzyme concentrations and choice of acyl donor had been established.Common conditions of all entries are 20 mM aldehyde, 0.5 M L-alanine, 3 U mL −1 Sp-ATA and 1.6 U mL a CHES, pH 10. b Result from previous optimization (Fig. 2) that was chosen for continuation.c Preparative synthesis.50 times increased reaction volume from entry 3. d 5% DMSO was added due to solubility issues.Finally, to demonstrate the applicability of the developed cascade, a scaled up preparative synthesis of N-benzyl-2methoxyacetamide was performed at a 100 mg scale.The optimal cascade conditions (2% v/v methyl methoxyacetate, 0.5 M L-alanine, 3 U mL −1 Sp-ATA and 1.6 U mL −1 MsAcT) were used and after 24 h the reaction had reached 92% conversion (Table 1, entry 4) with a 75% isolated yield.
In conclusion, a novel and efficient one-pot one-step amine transaminase/acyl transferase cascade for the formation of amides from aldehydes in aqueous solution has been developed.The cascade conditions were optimized to give a conversion of 97% to N-benzyl-2-methoxyacetamide from the corresponding aldehyde benzaldehyde (20 mM) using 0.5 M L-alanine and 2% v/v methyl methoxyacetate as amine-and acyl donor, respectively.A preparative synthesis of N-benzyl-2methoxyacetamide was also performed utilizing the developed transaminase/acyl transferase cascade.134 mg of N-benzyl-2-methoxyacetamide was synthesized from benzaldehyde with 92% conversion and 75% isolated yield.The cascade was also evaluated for the synthesis of chiral amides starting from the corresponding ketones.MsAcT was shown to catalyze the amidation of 2-aminohexane but the overall activity of the system was too low for a successful cascade.In order to develop a cascade for the synthesis of chiral amides, MsAcT needs to be engineered for improved amidation of chiral primary amines.

Fig. 2 Scheme 1
Fig.2Results from cascade optimization with varying enzyme concentrations using (A) methyl acetate or (B) methyl methoxyacetate as acyl donor.Conversion was analyzed after 18 h.