Modular Enzymatic Cascade Synthesis of Vitamin B5 and Its Derivatives

Abstract Access to vitamin B5 [(R)‐pantothenic acid] and both diastereoisomers of α‐methyl‐substituted vitamin B5 [(R)‐ and (S)‐3‐((R)‐2,4‐dihydroxy‐3,3‐dimethylbutanamido)‐2‐methylpropanoic acid] was achieved using a modular three‐step biocatalytic cascade involving 3‐methylaspartate ammonia lyase (MAL), aspartate‐α‐decarboxylase (ADC), β‐methylaspartate‐α‐decarboxylase (CrpG) or glutamate decarboxylase (GAD), and pantothenate synthetase (PS) enzymes. Starting from simple non‐chiral dicarboxylic acids (either fumaric acid or mesaconic acid), vitamin B5 and both diastereoisomers of α‐methyl‐substituted vitamin B5, which are valuable precursors for promising antimicrobials against Plasmodium falciparum and multidrug‐resistant Staphylococcus aureus, can be generated in good yields (up to 70 %) and excellent enantiopurity (>99 % ee). This newly developed cascade process may be tailored and used for the biocatalytic production of various vitamin B5 derivatives by modifying the pantoyl or β‐alanine moiety.

Coenzyme A( CoA) is an essential enzyme cofactor in all organisms, and its biosynthetic pathway enzymes have been identified as attractive targets for new antimicrobial drugs. [1,2] An interesting class of new antimicrobials that target CoA biosynthesis is constitutedb yp antothenamides (PanAms), which are secondary or tertiarya mides of pantothenic acid (vitaminB 5 , 5a,F igure 1), the biosynthetic precursor of CoA. Various PanAms have been shown to possess potent antimicrobial activity against several organisms, including the pathogenic bac-terium Staphylococcus aureus [3] as well as the malaria parasite Plasmodium falciparum. [4] However, pantetheinase enzymes that normally hydrolyze pantetheinei nh uman serum also act on the PanAms,thereby reducing their efficacy. [5,6] Interestingly, pantetheinase-mediated hydrolysis of PanAms could be preventedb ym odifying the b-alaninem oiety of the compounds. [7,8] Indeed, aP anAm with an added a-methyl group was shown to have superior antiplasmodial activity compared to its parent molecule. [9] However,s uch modificationsi ntroduce stereochemical complexity to the molecules, recent results of which have indicated the strong relevance to the antimalariala ctivity of PanAm analogues. [10] However,t he particularly challenging chemical synthesis of these compounds poses as ignificant barriert ot he discoveryo ft heir clinical potential. Therefore, it is of high interest to developa na symmetricb iocatalytic synthetic strategy that provides efficient andstep-economical access to pantothenic acid (5a)a nd both diastereoisomers of its a-methyl substituted derivative (5b,F igure 1), avoiding( de-)protecting steps and intermediate purifications.
The desired PanAms can be easily prepared from the correspondingp antothenic acids by transforming the carboxylic acid group to an amide. [11] We envisioned that pantothenic acid (5a)a nd its a-methyl substituted derivative( 5b)c ould be prepared from fumaric acid (1a)a nd mesaconic acid (1b), respectively,t hrough a modulart hree-step enzymaticc ascade (Scheme 1) involving 3methylaspartate ammonia lyase (MAL), an appropriate decarboxylases uch as aspartate-a-decarboxylase (ADC), b-methylaspartate-a-decarboxylase (CrpG) or glutamate decarboxylase (GAD),a nd pantothenate synthetase (PS). The expected chemoselectivity of each biocatalyst could allow for ao ne-pot reaction sequence due to the orthogonal reactivity of each enzyme.I nt his process, the new stereogenic centeri np roduct 5b can be established by either regio-and diastereoselective amination( as catalyzed by MAL), or diastereospecific decarboxylation by one of the decarboxylase enzymes.
MAL of Clostridium tetanomorphum is part of ac atabolic pathway for l-glutamate, in which it catalyzes the conversion of l-threo-3-methylaspartate to ammonia and mesaconate. [12] Using al arge molar excesso fa mmonia, the enzymea lso efficiently catalyzes the amination of mesaconate (1b)t og ive both (2S,3S)-3-methylaspartate (l-threo-2b)a nd (2S,3R)-3-methylaspartate (l-erythro-2b)a sp roducts (Scheme 1). It was found that l-threo-2b forms at am uch faster rate than that of l-erythro-2b,b ut at equilibrium (using a6 5-foldm olare xcesso f ammonia over 1b at pH 9) the molar ratio of these diastereoisomersi sa pproximately 1. [13] In addition, MAL accepts fumarate (1a)a sasubstrate, whichi sc onvertedt ol-aspartate (2a). The mechanism-inspired engineering of aM AL mutant( H194A) with strongly enhanced diastereoselectivity in the amination of 1b,g iving exclusively l-threo-2b,h as been previously reported. [13] Moreover,t he substrate scope of MAL has been expanded by structure-guided site-saturation mutagenesis, allowing for the biocatalytic production of ab road range of valuable 3substituted derivatives of l-aspartic acid. [14] ADC of E. coli is part of the biosynthetic pathway for pantothenate, in which it catalyzest he decarboxylation of 2a to give b-alanine (3a). [15] This enzyme has al imited substrate scope and showed no decarboxylation activity towards 2b. [16] The enzyme b-methylaspartate-a-decarboxylase( CrpG) of Nostoc sp. ATCC 53789 is involved in ab iosynthetic pathway for cryptophycin, in which it catalyzes the decarboxylation of l-erythro-2b to yield (R)-3-amino-2-methylpropanoic acid (3b). [17] CrpG is the only enzyme knownt oc atalyze the decarboxylation of 2b,w ith significant activity towardst he l-erythro isomer only.G AD of the hyperthermophilic archaeon Thermococcusk odakarensis has been reported to function as an ADC and is mostl ikelyr esponsible for the production of b-alanine necessary for pantothenate biosynthesis. [18] In this study,w e demonstrate that this enzyme exhibits decarboxylase activity towards 2b,b ut with the highest activity towards the l-threo isomer.N otably,A DC and CrpG must undergo self-processing leadingt of ormation of the catalytic pyruvoyl group, whereas GAD is aP LP-dependentd ecarboxylase that does not require autocatalytic self-processing.
Pantothenate synthetase (PS) of E. coli is involved in the last step of pantothenateb iosynthesis and catalyzes the adenosine triphosphate (ATP)-dependent condensation of (R)-pantoate (4) and b-alanine (3a)t of orm (R)-pantothenic acid (vitaminB 5 , 5a). PS enzymest ypically accept av ariety of b-alanine analogues in the condensation reaction,a lbeit with reducedc atalytic efficiency compared to that with the natural substrate. [15,19] Initially,w eset out to combine MAL and ADC in one pot to preparep roduct 3a.A ccordingly,s ubstrate 1a and NH 4 Cl were incubated with MAL and ADC, and the reaction was monitored by TLC ( Figure S3, Supporting Information). After 24 h, 1a was completely converted to product 3a,a sc onfirmed by 1 HNMR spectroscopy.T hese initial results showed that the two enzymes MAL and ADC are compatible for cascade synthesis in one pot. To further demonstrate the preparative usefulness of this two-step cascade system, a1 00 mg-scale synthesis was performed. Accordingly,s ubstrate 1a (25 mm)a nd NH 4 Cl (500 mm)w ere incubated with MAL (0.02 mol %) and ADC (0.6 mol %) in one pot (25 mL of buffer,p H8). Under these conditions, excellent conversion (> 99 %a fter 24 h) andg ood isolated yield of product 3a (85 %) were achieved ( Table 1 Unfortunately,A DC showed no decarboxylase activity towards either l-threo-2b or l-erythro-2b.T herefore, we cloned, expressed, and purified the decarboxylase CrpG, and then incubated it with a1 :1 mixture of l-threo-2b and l-erythro-2b. Undert hese conditions, the l-erythro isomer was fully decarboxylated, whereas the l-threo isomer was not converted, not even after prolongedi ncubation for 7d ( Figure S11, Supporting Information). This indicates that CrpG is highly diastereose-Scheme1.Proposed enzymatic cascade synthesis of vitamin B 5 and its derivatives. Table 1. Two-step enzymaticc ascade synthesis of b-alanine (3a)and both enantiomers of 3-amino-2-methylpropanoic acid (3b). [ Having established the preference of CrpG for l-erythro-2b, at wo-step enzymatic cascade reactionw as performeda tt he analytical scale by incubation of 1b and NH 4 Cl with MAL and CrpG in one pot. Interestingly,f ull conversion of startings ubstrate 1b was observed( Figure S4, Supporting Information), yieldings olely the (R)-enantiomer of product 3b,a sc onfirmed by 1 HNMR spectroscopy and chiral HPLC analysis. This is explained by ad ynamic kinetic asymmetric transformation [20,21] of the diastereoisomeric mixture of 2b (Scheme 2). In the first cascade step, MAL produced both l-erythro-a nd l-threo-2b as intermediate products. Subsequently,i nt he second step CrpG only decarboxylated l-erythro-2b to give exclusively( R)-3b. The remaining l-threo-2b was also convertedi nto l-erythro-2b by MAL, leading to full conversion of the startingm aterial (1b) to the desired product (R)-3b.
Several control experiments were also performed. Firstly, when 1b and NH 4 Cl were incubated with MAL alone, an approximately 1:1m ixture of l-threo-2b and l-erythro-2b was obtained ( Figure S12 A, Supporting Information). After removal of MAL from the reactionm ixture by heat inactivation and filtration,C rpG was added. After prolonged incubation (7 d), a mixture of unreacted l-threo-2b and product 3b was obtained (Figure S12 B, Supporting Information).S econdly, incubation of 1b and NH 4 Cl with the diastereospecific mutant of MAL (MAL-H194A)a nd CrpG in one pot resultedi nt he formation of lthreo-2b but did not yield product 3b,w hichi sc onsistent with the inability of CrpG to decarboxylate l-threo-2b (Figure S9, Supporting Information). These results confirm that MAL is responsible for both the synthesis and epimerization of l-threo-a nd l-erythro-2b,a nd that CrpG displays activityt owards l-erythro-2b only,a llowing for the selective synthesis of (R)-3b starting from the simplen on-chirald icarboxylic acid 1b.
CrpG displays activity towards l-erythro-2b,e nabling the enzymatics ynthesis of (R)-3b.T os ynthesize the opposite enantiomer of 3b,adecarboxylase with activity towards l-threo-2b was required. Our attempts to obtain CrpG variants by directed evolution through screening of single-site saturation mutagenesis libraries did not yield any mutants with detectablea ctivity towards l-threo-2b.T herefore, we cloned andp roduced two pyridoxal phosphate (PLP)-dependent GAD enzymes, and tested their ability to decarboxylate l-threo-2b.I nitially,w e workedo nt he GAD from Pyrococcus furiosus (PfGAD), which was reported to accept l-aspartate, l-glutamate and l-tyrosine as substrates. [22] Although many different expression conditions were tested, we weren ot able to produce PfGAD in as oluble form in an E. coli host. In an attempt to produce the soluble protein, different constructs were made as fusions with three solubility enhancers:m altose-binding protein (MBP), small ubiquitin-like modifier protein (SUMO) and Fh8, as mall protein secreted by the parasite Fasciola hepatica. However,i nefficient solubilization of PfGAD limited the effectiveness of this approach.T herefore, we selected the GAD from the hyperthermophilic archaeon Thermococcus kodakarensis (TkGAD), which has 71 %sequence similarityw ith PfGAD ( Figure S2, Supporting Information). TkGAD reportedly catalyzes the decarboxylation of l-glutamate and l-aspartate. [23] The gene encoding TkGAD was cloned and expressed, and the corresponding enzyme purified, yielding the soluble and active protein ( Figure S1, Supporting Information). Initially, TkGAD activity was tested towards l-aspartate (2a)a nd l-erythro-a nd l-threo-2b (Figure S10, Supporting Information). Thee nzyme completely converted 2a to 3a,w hereas the reaction with l-erythro-2b showed < 10 %c onversion. To our delight, l-threo-2b was also accepted as substrate by TkGAD, yieldingt he desired (S)-3b with more than 70 %c onversion.
Considering that TkGAD displays activity towards both diastereoisomers of 2b,adynamic kinetic asymmetric transformation approach, in which MAL and TkGAD are combined in one pot, would not yield the enantiopure (S)-3b product. Hence, for the one-pot,t wo-step, enzymatic cascade synthesis of (S)-3b, TkGAD was used in combination with the diastereospecific MAL-H194A mutant, which produces exclusively l-threo-2b upon aminationo f1b (Scheme 3a nd Figure S5 in the Supporting Information). Accordingly,s ubstrate 1b (10 mm)a nd NH 4 Cl (500 mm)w ere incubated with MAL-H194A (0.02 mol %) Scheme2.One-pot, two-step,enzymatic cascade reaction,involving MAL and CrpG, that fully converts mesaconate (1b)too nly (R)-3b.This is due to CrpG only acting on l-erythro-2b and to the MAL-mediated dynamickinetic asymmetric transformation of the l-threo-2b to the desired diastereomer. Having developed one-pot,t wo-step, enzymatic cascader eactions for the productiono f3a,( R)-3b and (S)-3b,w en ext verifiedw hether the PS enzyme is able to accept these compounds as substrates in condensation with (R)-pantoate (4) using small-scale (1 mL) reactions. We were pleased to find that PS accepted 3a,( R)-3b and (S)-3b as substrates in the condensation reaction, yielding the corresponding pantothenic acid products 5a and 5b,a sc onfirmed by 1 HNMR spectroscopy ( Figure S13, SupportingI nformation).
Having established that PS can be used to set up at hreestep enzymatic cascade reaction, the enzymesM AL, ADC, and PS werec ombined in one pot. Initially,t he reaction was tested by adding the three enzymes simultaneously and using an equimolar ratio of 1a, 4 and ATP. Although full conversion of the startings ubstrate 1a was observed after 24 h, the desired product (R)-5a was obtained in ac rude yield of only 50 % along with accumulationo f3a.T his indicated that furthero ptimization of the reaction conditions was necessary to improve the yield of product (R)-5a.A fter testing different ratios of 4 and ATP, am olar ratio of 2:3w as found to be best, which resulted in excellent conversion of the starting substrate into the desired product 5a (crude yield > 99 %; Figures S6 and S14, Supporting Information). Hence, under these conditions, no significant accumulation of intermediate product 3a was observed.
To assess the performance of this one-potm ulti-enzymatic cascade, a3 2mg-scale synthesis was performed.T he threestep cascade reaction wasperformed by addition of all componentss imultaneously including the three enzymes. After 24 h, the starting material 1a was completely consumed( conversion > 99 %, Figure S15 A, Supporting Information) andt he desired product (R)-5a was obtained in good isolated yield (70 %) and with excellent ee (> 99 %) ( Table 2, Figures S19 and S20, Supporting Information). Importantly,t he modularity of this enzymatic cascadea pproacha lso allows for the facile synthesis of both diastereoisomers of a-methyl-substituted vitamin B 5 ,t hat is (2R,2'R)-5b and (2S,2'R)-5b ( Figures S7 and S8, Supporting Information), with one stereogenic centerb eing set by the selected combination of enzymes and the other by the substrate (R)-pantoate (4). Under suitable reaction conditions (for details, see section 8.2 in the Supporting Information), and using the appropriate combination of enzymes in one-pot,t he desired products (2R,2'R)-5b and (2S,2'R)-5b were obtained with excellent de and ee values (> 99 %) andi n4 6-49 %i solated yield (Table 2; Figures S15, S21-S24, and S26 in the Supporting Information).
In conclusion, we have successfully developed ao ne-pot cascade process for the synthesis of enantiomerically pure vitamin B 5 startingf rom fumarate and utilizing MAL, ADC, and PS enzymes.S tarting from mesaconic acid,t he stereoselective synthesis of both diastereoisomers of a-methyl-substituted vitamin B 5 ,a ni mportanta ntibiotic precursor,w as achieved by using either the CrpG or GAD enzymei nsteado fA DC, with one stereogenic center being set by the selected combination of MAL/CrpG or MAL-H194A/GAD and the other derived from one of the starting substrates. Given the availability of engi-Scheme3.The one-pot, two-step,enzymatic cascade reaction that converts mesaconate (1b)too nly (S)-3b and relies on adiastereospecific mutant MAL (MAL-H194A) and the newly discovered stereoselectivity of TkGAD towards l-threo-2b.   neered MAL mutantsa nd natural PS enzymes with ab road substrate scope, work is in progress to expand the substrate scope of CrpG and GAD by protein engineering. Althought he decarboxylation step is actually stereochemically deconstructive with the loss of one chiral center, the cascade approachs trongly benefits from the use of stereo-divergent decarboxylases. These enzymes not only allow fort he synthesis of both diastereoisomerso fa-methyl-substituted vitamin B 5 ,b ut they also provide as trong drivingf orce to pull the equilibrium of the MAL-catalyzed reaction towards product formation.T he use of an irreversible decarboxylation step, with stereochemical kineticd istinction, is an important strategy in biocatalytic cascade synthesis to overcome thermodynamic limitations and maximize product yield. [24,25] Ap ossible constraint on the use of the developed cascade for large-scale transformations would be the dependence of the PS enzyme on the expensive cofactor ATP. This could be addressed by the incorporation of an auxiliary enzyme-catalyzeds tep fore fficient ATPr ecycling. SeveralA TP-recyclinge nzyme systems are availablea nd af ew have already been successfully implemented in preparative biocatalysis. [26][27][28] However,c onsidering that the starting materials (fumarate and mesaconate) can be efficiently produced in high yields by large-scale fermentation using metabolicallye ngineered E. coli strains, [29,30] we envision to co-express the enzymes employed for the cascade in such a fermentation host to directly obtain vitaminB 5 and its amethyl-substitutedd erivatives from cheap carbon and nitrogen sources. Indeed, such ac ell-baseda pproach would eliminate the need for ATPr ecycling.