Enantioselective ester hydrolysis by an achiral catalyst co-embedded with chiral amphiphiles into a vesicle membrane

Co-embedding of an amphiphilic non-chiral hydrolysis catalyst with amphiphilic chiral additives into the membrane of a phospholipid vesicle induces different rates of ester hydrolysis for enantiomeric amino acid esters.

The origin of chirality in nature, 1 why natural molecules are homochiral, and how such a rapid amplication of single enantiomers occurred, are still unanswered questions. 2,3ynthetic chemistry uses chiral catalyst or ligands to convert achiral substrates enantio-selective. 4The reactant and the source of chirality require typical close contact.The simple addition of chiral additives to the reaction mixture or using enantiopure chiral solvents 5,6 provide none or minute enantioselectivity.][15] We report here the hydrolysis of enantiomeric amino acid esters on the modied surface of phospholipid vesicles (Fig. 1).A chiral, catalytically inactive membrane additive is coembedded with a catalytically active achiral metal complex Zn 2 Cy into the phospholipid membrane. 12,16The membrane serves as two-dimensional platform with higher concentration of the amphiphilic membrane additives compared to the bulk solution. 17This proximity of the chiral additive to the achiral metal complex affects its selectivity in ester hydrolysis and induces thereby different reaction rates for both enantiomers.
The bis-zinc-cyclen complex Zn 2 Cy is anchored into the surface of the vesicle by its lipophilic alkyl chain and promotes the hydrolysis of activated carboxylic esters as previously reported (Fig. 2). 18The Lewis acidic zinc ions coordinate one water molecule and the ester functionality.Enantiopure amphiphilic derivatives of L-proline l-Pro, (À)-sparteine (À)-Spa, L-glucose l-Glu, L-tartaric acid l-Tar and L-histidine l-His-COOH and the corresponding alcohol l-His-OH were used as chiral membrane additives.Enantiometrically pure 4nitrophenol esters of phenylalanine, PN-Phe, serve as substrates for the catalysed hydrolysis.Upon cleavage of the ester bond, the coloured 4-nitrophenolate anion is released, which enables a facile determination of the reaction progress.However, derivatives PN-Phe with free amine group show fast spontaneous hydrolysis under the reaction conditions.Therefore compounds PN-C12-Phe and PN-C2-Phe with a protected amino-group were prepared, which are stable in the absence of the hydrolysis catalyst.

Results and discussion
All measurements were done in buffered solutions (HEPES buffer, 25 mM, 7.4 pH), at room temperature.Samples were prepared by sonication according to a previously reported procedure to form micellar solutions or 100 nm unilamellar functionalised vesicles. 18The rate of substrate hydrolysis was determined colorimetrically as an increase in absorbance intensity at 400 nm (absorption maximum of 4-nitrophenol at pH 7.4; no other species absorb at this wavelength).The relative error of the measurement was estimated to be below 10% (Fig. 3).
Pseudo rst order rate constants were calculated using the initial slope method.Every membrane additive was examined as a sole micellar solution (without lipid or Zn 2 Cy), co-micellar solution (without lipid in the presence of Zn 2 Cy), and in vesicular membranes with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) lipids (5 mol% Zn 2 Cy, 10 mol% membrane additive, 85 mol% lipid).Lipids differ in their transition temperatures providing uid (DOPC) and rigid (DSPC) membranes at room temperature.We measured the hydrolytic rate constants separately for the two enantiomers of the substrate PN-C12-Phe.This substrate is equipped with a long alkyl chain, which increases its lipophilicity and the adsorption to the surface of the membrane.The initial hydrolysis rates for different functionalized vesicles and co-micelles are summarised in Table 1.

Kinetic effects
Hydrolysis of the ester PN-C12-Phe is favoured in micellar solutions (Table 1).Conrming previous results, 18 membrane additives can affect the hydrolysis activity in vesicular and comicellar solutions signicant.
In DOPC membranes the hydrolytic rates are affected by two types of membrane additives: tartrate l-Tar decreases and histidine derivatives l-His-COOH and l-His-OH increase the initial rate of ester hydrolysis (Table 1).These observations are in accordance to effects on the previously studied hydrolysis of uorescein diacetate by Zn 2 Cy. 18

Enantiodiscrimination in ester hydrolysis
The rates of ester hydrolysis were determined for both enantiomers as previously reported for micellar solutions. 10The largest relative difference in hydrolysis rates for the enantiomeric esters is observed in DOPC vesicles with addition of amphiphilic tartrate l-Tar or histidine acid l-His-COOH as membrane additives (Fig. 4).
Both compounds contain free carboxy group.When the carboxyl group of histidine is reduced the effect in vesicles is lost.This indicates that the free acid might have a crucial role in this cooperative action.In buffered solution the acid is deprotonated and might interact with the Lewis acidic zinc complex.This interaction is weak in bulk solution, but may signicantly increase due to the close proximity of the binding  Table 1 Pseudo first order kinetic rate constants for the hydrolysis of the L and D enantiomer of P-C12-Phe using different systems.All kinetic values are given in 10 À3 s À1

Comicelles
No cyclen, no lipid a No effect on hydrolysis observed.
Fig. 4 Effect of membrane additives on the relative ratio of ester hydrolysis rates of enantiomeric amino acid esters represented by the fraction of pseudo first order kinetic constants.
partners at the vesicular surface.In uid DOPC membranes, embedded components diffuse and may thereby arrange optimal for the catalysis.0][21] This was also previously observed by us. 18Other membrane additives than tartrate or histidine do not show considerable effects on the relative hydrolysis rate of the enantiomeric esters.Substrates PN-C2-Phe and Pn-Phe exhibit similar enantio-selective enhancement (Table 2).
In order to conrm our observation, we prepared the enantiomeric amphiphilic histidine d-His-COOH.This compound was investigated under identical conditions as l-His-COOH and induced a similar hydrolysis rate enhancement of d-PN-C12-Phe (Fig. 5), but with opposite enantioselectivity.
In addition, the effect of the membrane additive loading on the enantioselectivity of the hydrolysis was investigated for d-His-COOH (Fig. 6).Addition of 10 mol% provided the highest relative difference between the hydrolysis rate constants.

Conclusions
The relative catalytic hydrolysis rates of enantiomeric amino acid esters with a non-chiral catalyst become signicantly different, if the catalyst is co-embedded into the surface of DOPC vesicles with chiral amphiphiles.The rates for enantiomeric phenylalanine nitrophenyl esters differ by a factor of two with co-embedded amphiphiles prepared from tartaric acid or histidine.This resembles an ee of approx.up to 40%, which may be expected for reactions performed from racemate.The uidity of the membrane is essential to achieve the catalytic enantio-discrimination and therefore only observed in DOPC vesicles.Although the measured rate differences may not be useful for practical applications, the results prove that the intermolecular interaction between a Lewis acidic metal complex, chiral amphiphiles and activated amino acid esters co-embedded into a uid membrane without covalent connection can affect reaction rates enantioselective.

Fig. 1
Fig. 1 Proposed concept of additive-induced enantioselectivity in a hydrolytic reaction.

Fig. 2
Fig. 2 Molecular structures of the achiral catalyst for hydrolysis, racemic substrates and chiral membrane additives.

Fig. 6
Fig. 6 Pseudo first order rate constants of hydrolysis for both enantiomers by a vesicular solution of Zn 2 Cy (5 mol%) and DOPC with different loadings of d-His-COOH.