Studies towards enzymatic kinetic resolutions of 1,3-diol peptidomimetics obtained via the Ugi reaction

Enzymatic methods in combination with the multicomponent Ugi condensation make a very efficient method for simple synthesis of non-racemic peptidomimetics. The aim of the studies was to develop an enzymatic kinetic resolution of 1,3-diol peptidomimetics providing non-racemic compounds. Among many applications, 1,3-diols can serve as intermediates in the synthesis of anticancer agents - β -acyloxymethacrylic amides. Stereoselective enzymatic acylation and hydrololysis of Ugi products were investigated. The enantiomeric or diastereomeric excesses were determined in both cases. As a result, an efficient enzymatic method for the synthesis chiral, non-racemic 1,3-diol peptidomimetics was developed.


Introduction
Enzymes have become common catalysts for kinetic resolution of racemic mixtures.2][3] Interesting combinations of enzymatic procedures and multicomponent reactions, such as the Passerini 4 or Ugi reactions, [5][6] have been reported.][9][10] The Ugi reaction is a four-component condensation of an aldehyde, amine, carboxylic acid and an isocyanide that provide α-acylaminoamides. 7Although recently, the assymetric, catalytic versions of Ugi threecomponent reactions were described, [11][12][13] the stereocontrol of the classical, four-component Ugi reaction still remains very limited.We reported previously that enzymatic desymmetrization of cyclic anhydrides and subsequent Ugi reaction can be used for the synthesis of non-racemic small peptide analogues in a one-pot procedure. 5Another example of the combination of the biocatalytic method and the Ugi reaction is the enzymatic oxidation of a substituted pyrrolidine to an optically active 1-pyrroline, which was subsequently used in an asymmetric Ugi reaction. 6n this paper we present our studies on the enzymatic desymmetrization of 1,3-diol peptidomimetics I obtained via the Ugi reaction ( Figure 1).5][16] We also have recently described 1,3-diol I as an intermediate for the synthesis of peptidomimetics possessing various electrophilic fragments including a new class of -acyloxymethacrylatespotent anticancer agents. 17Peptidomimetics I were previously obtained via the classic Ugi reaction as a racemic mixture.The proper method of the synthesis non-racemic diol I can be applied for further synthesis of optically active, anticancer β-acyloxymethacrylates.

Results and Discussion
The synthetic strategies, presented in Scheme 1, are based on the enzymatic kinetic resolution of enantiomers of compounds I. Considering the structure of I, two approaches have been proposed.The first method is based on enzymatic acylation of compound 1 to alcohol 4. The second approach is based on the enzymatic hydrolysis reaction of the ester group to an acid 5.For the first approach, diol 1 as its benzyl ester was chosen, since it was judged to be less likely to undergo side reactions involving the ester moiety, than an ethyl ester.For the enzymatic hydrolysis, diol 2 was chosen ( Scheme 1).The synthesis of substrates for enzymatic reaction.The synthesis of diol 1 was described previously. 17For the synthesis of analogue 2, similar conditions were applied (Scheme 2).Compound 7 was obtained in the Ugi reaction with acid 3, ethyl isocyanoacetate, 2,4dimethoxybenzylamine and isovaleric aldehyde in 49% yield (Scheme 2).Then the acetal protecting group was cleaved in 80%aq acetic acid providing diol 2 in 77% yield.Diasteroselective approach.Since compound 1 contains two diastereotopic hydroxyl groups, the monoacylation reaction can lead to a mixture of stereoisomers.As can be seen in Scheme 3, the acylation reaction of 1 should be catalyzed diastereoselectively and enantioselectively, in order to provide efficient desymmetrisation.Initially, the diastereoselectivity of enzymatic acylation with vinyl acetate was studied as described before. 18n our previous studies, it was found that the proper choice of solvent is crucial for stereoselectivity of an enzyme. 19Thus different solvents were examined, such as: toluene, vinyl acetate, 1,4-dioxane, tert-butyl methyl ether (TBME).After enzymatic screening of different reaction conditions, seven enzymes were found to catalyze the reaction.(For the full list of enzymes tested, see the Experimental Section, 4.7) The results are presented in Table 1.Pseudomonas cepaciae 20 a Determined by RP-HPLC (Method A)

HO
As summarized in the Table 1, diol 1 can be acylated in a chemoselective way (only monoacylation product was observed in the reaction mixture).The Novozym 435 and the lipases from: Pseudomonas fluorescens, Pseudomonas cepaciae, Aspergillus oryzae, Mucor javanicus, Penniclium roqueforti and Candida antarctica were able to catalyze the reaction.The highest diastereoselectivity was observed when lipase from Aspergillus oryzae was used (entry 4).These results were still not satisfactory, therefore the enantioselectivity was not further studied.
Enantioselective approach.Since the enzymatic acylation of diol 1 was not very selective, the second approach was based on enzymatic enantioselective hydrolysis.In this approach the number of possible products is reduced, which simplifies stereochemical course of the reaction.Formation of only the carboxylic acid 5 and unreacted substrate 2 is observed.Scheme 4. Enzymatic hydrolysis of 1,3-diol 2. Reagents and conditions: see Table 2.
For the initial screening, the typical conditions were applied.In order to increase solubility of substrate, acetone was used as a co-solvent.Among all the tested enzymes, six of them catalyzed the reaction.The results are summarized in Table 2.The best results were obtained when porcine lipase acetone powder, porcine pancreas lipase type II, papaine, pektynase, CALB and lipase from Rhisopus arrhisus were used as biocatalysts.The enatiomeric excess of the substrate was determined by HPLC.The acetone powder required a very long reaction time and despite moderate conversion, the enantioselectivity was very poor.Interestingly, papain exhibited lipase activity and catalyzed the reaction.The best results were obtained in the case of Rhisopus arrhisus lipase and Candida antarctica lipase which catalyzed the reaction with excellent enantioselectivity (Table 2, entries 5 and 6).

Conclusions
Herein we have described the results of our studies on enzymatic kinetic resolution of 1,3-diol peptidomimetics using two different approaches.The enzymatic diasteroselective acylation of hydroxyl groups was not efficient and therefore not optimized.On the other hand, the enantioselective enzymatic hydrolysis of the ester group was found to be very efficient and the respective carboxylic acid 5 was obtained with excellent enantioselectivity.The proposed methodology is simple, efficient and could be expanded to other type of substrates.In future studies this methodology will be applied for the synthesis of non-racemic β-acyloxyamides.
General method for enzymatic acylation.Compound 1 was dissolved in solvent (Table 1, c = 10 mg/mL) and enzyme was added (Table 1, 2 mg for each 10 mg of substrate).Vinyl acetate (3 eq) was added.The reaction was carried out at 40 o C. Solvent was evaporated and the product was analyzed by HPLC (Method A).
General method for enzymatic hydrolysis.Compound 2 was dissolved in buffered 7.0/acetone (8:2, v:v) and the enzyme was added (Table 2, 10%g).The reaction was carried out at room temperature (25 o C).Acetone was then evaporated and aqueous phase was extracted with ethyl acetate.The organic layers was combined and washed with 1N NaHCO3 (3x).The ethyl acetate was evaporated.The product was analyzed by HPLC (Method B).

Scheme 1 .
Scheme 1. Synthetic strategy for kinetic resolution of 1,3 diol peptidomimetics I.The synthesis of substrates for enzymatic reaction.The synthesis of diol 1 was described previously.17For the synthesis of analogue 2, similar conditions were applied (Scheme 2).Compound 7 was obtained in the Ugi reaction with acid 3, ethyl isocyanoacetate, 2,4dimethoxybenzylamine and isovaleric aldehyde in 49% yield (Scheme 2).Then the acetal protecting group was cleaved in 80%aq acetic acid providing diol 2 in 77% yield.

Table 1 .
Initial screening of acylation reaction of 1

Table 2 .
Enzymatic hydrolysis of 2 a Yields determined by RP-HPLC (Method C). b ees determined by HPLC (Method B). c Enantioselectivity (E) was calculated according to equation: