Stereoselective preparation of lipidated carboxymethyl- proline/pipecolic acid derivatives via coupling of engineered crotonases with an alkylmalonyl-CoA synthetase†

The trisubstituted enolate- and C-C bond-forming capacities of engineered carboxymethylproline synthases CMPSs are coupled with the malonyl-CoA synthetase MatB to enable stereoselective preparation of 5- and 6-membered N-heterocycles functionalised with alkyl-substituted carboxymethyl side chains, starting from achiral alkyl-substituted malonic acids and L-amino acid semialdehydes. The results illustrate the biocatalytic utility of crotonases in tandem enzyme-catalysed reactions for stereoselective synthesis.


Introduction
Saturated 5-and 6-membered N-heterocycles are important constituents of many natural products and pharmaceuticals (e.g.oxaceprol, captopril and tacrolimus) and are used as catalysts 1 in asymmetric synthesis (e.g.proline derivatives).The development of "asymmetric" biocatalytic routes for the versatile production of functionalised N-heterocycles is thus of interest.
The mechanisms of catalysis of most crotonases, including CarB and ThnE, are proposed to proceed via enolate intermediates, usually generated by decarboxylation of (a derivative of ) 1.The enolate intermediates are stabilised by a conserved oxyanion hole (OAH), which is formed in the case of CarB and ThnE by residues Gly62 CarB /Gly107 ThnE and Met108 CarB / Val153 ThnE (Fig. 1).C-C bond formation can then proceed via reaction of the enolate intermediate with the imine form of 2 to give the t-CMP-CoA thioester, which is then hydrolysed to give 3 and coenzyme A (CoASH) (Fig. 1A). 4,52][13][14] We have reported that (engineered) CMPSs accept L-GHP analogues to give 6-and 7-membered carboxymethyl-N-heterocycles. 15CMPSs also accept methylated derivatives of L-GHP to give 5-carboxymethylproline derivatives functionalised at C-2, C-3, C-4, or C-5 of the proline ring, including products with a quaternary center (at C-2 or C-5) in a stereoselective fashion. 16Active site CMPS variants enabled the stereoselective alkylation of enolates generated from methylmalonyl-CoA 4 and ethylmalonyl-CoA 5. 17 These findings stimulated us to investigate other C-2 alkylmalonyl-CoA derivatives as CMPS substrates, and whether the stereoselectivity of CMPS catalysis could be improved.
We report that CMPS catalysis can be coupled to that of malonyl-CoA synthetase (MatB) or crotonyl-CoA carboxylase reductase (Ccr) for the stereoselective preparation of functionalised prolines and pipecolic acids.The results illustrate the utility of coupling crotonases with other enzymes for stereoselective synthesis.With appropriate optimization, the reactions may be useful in cell-based contexts for the preparation of functionalised heterocycles for use in pharmaceutical research.
Incubation of C-2-alkylated malonic acid derivatives with L-GHPs, MatB and CMPS variants (2R)-4 and (2R)-5 can be produced via reaction of 2-methylmalonic acid 10 and 2-ethylmalonic acid 11, respectively, with CoASH as catalysed by the malonyl-CoA synthetase MatB from Streptomyces coelicolor (Fig. 2C). 20The CarB Trp79 residue forms a part of the hydrophobic face of the CarB active site (Fig. 1B); 17 we envisaged that substituting Trp79 for a less bulky residue e.g.Phe/Ala (Table 1) may enable acceptance of derivatives of 1 with bulky C-2 side chains, also available by MatB catalysis.
We initially tested the MatB/CMPS pair with 10 and 11 (Table 1) as well as 2,2-dimethylmalonic acid 12 (results not shown).In each case, the MatB/wildtype CarB coupled reaction resulted in the production of the anticipated t-CMP derivative, as revealed by LC-MS analyses, in yields comparable to those resulting from direct incubation of the corresponding synthetic derivatives of 1 and 2 with wildtype CarB. 17The observed C-6 epimeric ratio in the case of both 10 and 11 reactions was ≥99 : 1 in favour of (6S)-6/7 (Table 1: entries 1 and 4).The MatB/CarB W79F/A coupled reactions were also selective for production of the (6S)-epimer of 6/7 and the isolated yields were either similar (Table 1: entries 2 and 3) or higher (Table 1: entries 5 and 6) than those for wildtype CarB.
Scale-up and NMR of the LC-MS purified MatB/CarB W79Acatalysed products revealed that a single detectable epimer of 6-alkyl-t-CMP with the (5S,6S)-stereochemistry was formed with isolated yields from 5% to 36% (Table 1: entries 8, 10-20).(There is likely scope for optimization of the smallscale reactions, including in cells) LC-MS analyses of the reaction catalysed by MatB/wildtype CarB pair did not result in observation of detectable quantities of the alkylated products that were observed with the MatB/CarB W79A reactions.In the case of the MatB/CarB W79F reactions, we only observed formation of derivatives with allyl and n-propyl side-chains, at comparable yields to those of the MatB/CarB W79A-catalysed reaction (Table 1: entries 7, 9).with CMPS to give (6R)-6 or -7. 17ote that (6S)-6 or -7 are only observed when the Ccr incubation time is sufficiently long prior to CMPS addition; (C) Coupling of malonyl-CoA synthetase (MatB)catalysed formation of (2R)-alkylmalonyl-CoA 20 with CarB W79F for formation of (6S)-6 or -7.
The MatB/CarB pair catalysed production of 3 from 2, using pantetheine (46) or N-acetyl cysteamine (47), substituting for CoASH, occurs in ∼20% or ∼15%, respectively, of the yields obtained with CoASH (Fig. S6 †).When we tested C-2 alkylated derivatives with 46, in addition to the t-CMP derivative, we observed two previously undetected (by LC-MS, Fig. S5 and S7 †) species, corresponding to the methyl ester-and pantetheinyl-t-CMP derivatives.The methyl ester is presumably produced in the methanol quenching step.In the case of the 2-isoprenyl malonic acid (20)/MatB/CarB-W79A system (Fig. 4), the assignment of the methyl ester (48) was confirmed by NMR (during isolation, the assigned pantetheinyl thioester (49) underwent hydrolysis to give (35)).Thus, in principle it is possible to use MatB/CMPSs for the regioselective production of t-CMP esters.These observations are consistent with the proposed intermediacy of an enzyme-bound t-CMP-CoA thioester (50) in CarB catalysis. 4,5It is possible that the observation (by LC-MS) of the thioester (49)/methyl ester (48) in the case of the C-2 substituted alkyl malonic acid derivatives, reflects steric hindrance in the hydrolysis step, resulting in release of the non-optimal pantetheinylthioester (49).

Conclusions
There is a need to efficiently produce functionalised chiral heterocycles for use in medicinal chemistry and as catalysts.Whilst considerable efforts have been directed towards organic synthesis in this regard, less effort has been directed towards enzyme catalysis, particularly in a coupled sense.However, the use of cell-produced natural products for structure-activity relationship studies can be challenging, as it can be difficult to produce a desirable set of analogues, due to the limitations of working with multi-step processes in cells.An alternative approach is to couple reactions in vitro using purified (engineered) enzymes and substrate analogues, bearing in mind that selected coupled reaction sequences could be introduced into cells at a later stage, when larger scale production is required.Our results in which the stereocontrolled production of C-2 malonyl-CoA derivatives, from "achiral" precursors, is coupled to heterocycle formation using CMPS variants demonstrate how a set of chiral 5-and 6-membered ring heterocycles can be efficiently prepared in two enzyme-catalysed steps (Fig. S9 †).It is notable that near complete control of stereochemistry at C-6/C-7 of t-CMP 5/t-CMPi can be achieved by appropriate choice of the alkylmalonyl-CoA synthesising enzyme (Ccr or MatB, Fig. S9 †) and reaction conditions.Additional diversity might be introduced by the use of (truncated) malonyl-CoA analogues (substituted at C-2) or by using different alcohols to quench the reaction.Thus, we believe that one productive future direction could be the de novo construction of unnatural biosynthetic pathways, for the production of both natural productlike and -unlike structures.a d.r.: diastereomeric ratio of epimers at C-6 or C-7 of 5-or 6-membered ring products, respectively.c HFH: 4,4,5,5,6,6,6-heptafluorohexyl (HFH).2,2-Dimethylmalonic acid (12) was also converted to the corresponding C-6 dimethyl-t-CMP derivative.

Fig. 1
Fig. 1 Engineering carboxymethylproline synthases to accept malonyl-CoA 1 analogues with bulky side chains.(A) CarB and ThnE catalysed synthesis of (2S,5S)carboxymethylproline 3; 7,10 (B) A view from a CarB structure 2 with (2R)-methylmalonyl-CoA 4 and L-pyrroline-5-carboxylate 2 (L-P5C) modeled in the active site.The Trp79 residue, the surface of which is shown in orange, is part of a hydrophobic face in the active site; substitution of Trp79 for Phe-or Ala-residues increases the capacity of CarB to accommodate bulkier substituents at the malonyl-CoA C-2 position (asterisked).
Experimental details and spectroscopic characterisations are given in the ESI.† We thank the Biotechnology and Biological Sciences Research Council, Berrow Foundation (LH) and CONACyT and FIDERH (Mexico, RGC) for funding.The MatB expression plasmid was kindly provided by Dr A. T. Keatinge-Clay.The expression plasmids of the CoA biosynthesis enzymes were kindly provided by Dr M. Tosin.We thank Sven Warhaut for his help during the course of the work.

Fig. 4
Fig.4Products from the incubation of C-2 isoprenylmalonic acid(20) with MatB/CarB W79A using pantetheine as a replacement for coenzyme A.

Table 1 N
-heterocycles resulting from incubations of achiral C-2 alkylmalonic acids and L-GHP (2)/L-AASA (40), in the presence of ATP and coenzyme A, as catalysed by the MatB/CMPS coupled enzyme system