Summary and Outlook 64

Transcription

1 Summary and utlook 64 7 Summary and utlook 71 Summary The importance of enantiomeric pure substances is steadily growing in the pharmaceutical and chemical industries Research groups worldwide struggle to find new [37, ] and efficient ways for their production Chiral epoxides are particularly valuable as versatile building blocks in organic synthesis as they offer easy access to numerous functionalities The catalytic epoxidations developed by SHARPLESS and JACBSEN are the most relevant amidst the reported methods [6-8] However, limited substrate scope and low turnover number are recognized as major drawbacks Biocatalysts then emerged as a promising candidate for the production of chiral epoxides and vicinal diols [82] Enzymes are masterpieces of Mother Nature However, features required for industrial applications, such as non-natural substrate acceptance, organic solvent tolerance or high thermostability, are not inherent in enzymes In the 1990s, molecular evolution flourished and became a powerful tool to genetically modify the desired protein properties ( Figure 55) [29] Target gene Mutagenesis X X X X Gene with mutations Transformation Bacteria Repeat the whole process Expression of the target protein Biocatalysis Identification of improved variants Enzyme variants Figure 55 Genetic optimization of an enzyme by means of molecular evolution In this thesis, the Aspergillus niger epoxide hydrolase was chosen as the target enzyme and glycidyl phenyl ether 1 as the model substrate This is an important intermediate in the synthesis of β-blockers which are normally used in the treatment H H NHR [37, 38] of hypertension and angina pectoris They share in common an aryl oxypropanolamine structure ( Figure 56) R Figure 56 Common structure of β-blockers The A niger epoxide hydrolase exhibits only relatively poor enantioselectivity

2 Summary and utlook 65 towards glycidyl phenyl ether 1 The enzyme was therefore subjected to a molecular optimization process with the aim to improve its enantioselectivity (Figure 57) A niger epoxide hydrolase ph = 72, T = 30 o C E = Figure 57 Model reaction of A niger epoxide hydrolase using rac-1 as model substrate H H ne of the major challenges in a directed evolution process is the evaluation of large numbers of samples As most mutations are damaging or neutral, the percentage of inactive enzyme variants is generally high An efficient assay to identify active clones is therefore necessary A biologically inherent function of epoxide hydrolases is detoxification [22] Epoxides are well known carcinogens due to their high lability towards different biomolecules, such as DNA, RNA and proteins [23, 51] This cytotoxic attribute allows the design of a genetic selection assay [42] A toxicity-based selection system was successfully developed and offers a material-, cost- and manpower-efficient method to assay large numbers of the enzyme variants created ( Figure 58) Its use was also extended to high-throughput applications that significantly accelerated the evolution process (Figure 59) [54] Figure 58 Toxicity-based selection Figure 59 High-throughput version of the selection assay in 96-format Quick determination of the enantioselectivity is still a difficult task Conventional techniques such as chiral HPLC or GC require relatively long analysis time and are therefore scarcely suitable for high-throughput screening Different methods have been reported previously by several research groups to overcome this bottleneck [57, 64-66] Mass spectrometry was previously introduced by our group for high-throughput

3 Summary and utlook 66 ee screening using quasienantiomers [59] A multiplexed electrospray system was further incorporated to improve the assay efficiency by shortening the analysis time (Figure 60) Figure 60 MUX interface (left) and the schematic representation (right) Several problems were identified Direct determination of conversion and calculation of E-value was hampered by the undetectable signal of the substrate To circumvent this difficulty the internal standard method was employed However, with the previously used ametryne as the reference substance, the reproducibility of the measurements was low due to the ion suppression phenomenon A new internal standard was introduced and satisfactory precision was thus achieved (Figure 61) Moreover, the accuracy of the MUX-ESI-MS system was confirmed by comparing the evaluation of different ee-values using HPLC ( Figure 62) I diol / I new IS ee p (HPLC) c diol / c new IS ee p (MUX-ESI-MS) Figure 61 Calibration line using new IS Figure 62 Accuracy test of the MUX-ESI-MS In conclusion, MUX-ESI-MS was optimized and shown to be feasible for a highthroughput analysis of the enantioselectivity However, scale-up of the bacterial culture and of the biocatalytic reaction and chiral HPLC analysis were required

4 Summary and utlook 67 Not only is the application of an efficient screening assay crucial to the identification of improved enzyme variants, a comprehensible and simple representation of the huge amounts of data produced by the screening also plays an extremely important role A new data processing criterion was thus adapted and proved to be very effective in the recognition of variants with enhanced properties ( Figure 63) ee p LW080 LW081 LW037 LW LW Conversion Figure 63 Data procession criteria for high-throughput screening for enantioselectivity In summary, an efficient, material- as well as time-saving procedure for the identification of the improved enzyme variants was achieved by the integration of activity-selection assay, enantioselectivity-ms evaluation and data processing For a successful genetic evolution, not only is an efficient screening systems essential, but also the choice of an optimal mutagenesis strategy Different approaches have been reported [87] Based on the available structural information of the A niger epoxide hydrolase [95] and the crucial influence of the residues situated in the active site, [96] an approach with focused mutations emerged as the best candidate After thorough inspection of the 3D structure of the target enzyme, six different libraries were selected and subjected to the combinatorial active site saturation (CAST) [97] These libraries covered the whole tunnel-like substrate binding site ( Figure 64) In order to mimic the Darwinian evolution, the concept of CAST was further extended in this thesis by performing the process iteratively The variants with the most improved selectivity in each round became therefore the parent gene of the next generation The accumulation of positive mutations and cooperative effects between the residues were thereof achieved

5 Summary and utlook 68 Figure 64 Top view of tunnel-like binding pocket showing sites A-E (blue) and the catalytic active Asp192 (red) After an evolution process of six generations, the highly enantioselective variant LW202 (E > 100) was identified ( Figure 65) This improvement is equivalent to a 25- fold increase in selectivity in comparison to the wild-type enzyme Additionally, several enzyme variants that exhibited a higher substrate conversion rate were also found 120 E = 115 LW E Selectivity factor (E) E = 49 LW126 A or F 40 E = 35 LW144 E no substantial improvements E = 14 LW081 E = 21 LW086 C F D E = 24 LW wild-typ B A or C no substantial improvements Figure 65 Iterative CASTing in the evolution of the A niger epoxide hydrolase

6 Summary and utlook 69 Variants with improved enzyme properties offer a unique possibility to gain a deeper understanding of the protein structure and function, which is one of the major goals in protein engineering Detailed kinetic studies and screening of the substrate scope were therefore carried out Furthermore, enzyme variants with single, double and triple mutations were created A reverse engineering experiment was also performed to identify the origin of the observed high enantioselectivity Examination of the substrate scope showed that LW202 retains its high enantioselectivity towards most of the chosen substances ( Figure 66 and Figure 67) However, the wild-type enzyme exhibited surprisingly higher activity and enantioselectivity than the identified variant towards the substrates with nitro and methoxy substituents at the ortho position A niger epoxide hydrolase H H H H R Na-phosphate buffer R R rac-7 to 18 (S)-7 to 18 (R)-7 to 18 Figure 66 Schematic representation of the substrate mapping for A niger epoxide hydrolase LW202 wild-type 50 E-value ,2-Me 1,2-Me 1,2-Cl 1,2-N2 1,3-Me 1,3-Me 1,3-Cl 1,4-t-butyl 1-naphtyl 2-naphtyl R-Substituent Figure 67 Graphical representation of the substrate screening results As the mutation introduced at position 350 led to only slight changes in the enantioselectivity, it was of interest to explore the necessity of this mutation to achieve high selectivity Reverse engineering was performed by restoring the original amino acid at this position A sharp decrease in the E-value indicated that mutation at this position is of significant influence The 3D structure of the LW202 was also constructed with the aid of a computer programme This revealed evident narrowing of the tunnel-shaped substrate binding

7 Summary and utlook 70 site ( Figure 68) This may affect the affinity of both enantiomers to different extents [ ] Drastic changes in the K M value of the improved variants estimated in the kinetic studies corroborate with this observation Figure 68 Comparison of the tunnel-shaped substrate binding site between the wild-type enzyme and the variant LW202 In addition, an in-depth inspection of the active site revealed a large number of residues with aromatic side chains Four out nine mutations of LW202 also possess phenyl ring functionality (Figure 69) This may allow π-stacking interactions to occur which could destabilize the (R)-enantiomer or stabilize the (S)-1 that lead to high enantioselectivity [95, 119, 120] The hypothesis is supported by results from the analysis of the kinetic parameters k cat and K M Figure 69 Closer look of the active site of LW202 In conclusion, integration of the region-focused iterative CAST-mutagenesis strategy with the developed toxicity-based selection system and the optimized MUX-ESI-MS screening method led to rapid genetic optimization in the enantioselectivity of the

8 Summary and utlook 71 A niger epoxide hydrolase The E-value of the best protein variant was greater than 100 This is a 25-fold increase in selectivity in comparison to that of the wild-type enzyme Detailed studies were additionally performed to gain greater insights into the origin of this remarkable improvement 72 utlook The present work provides an optimized platform for further investigations in different facets of the A niger epoxide hydrolase Evolution of the substrate acceptance appears to be a promising objective since CASTing has been applied to the Pseudomonas aeruginosa lipase with great success [97] As a mutagenesis strategy, iterative cycles of CASTing proved to be an effective tool However, it is hard to identify the best evolutionary route amongst the numerous combinations Further studies on different paths combining with computational methods, such as genetic algorithms, may therefore offer a predictive basis for a more time- and cost-efficient optimization process The improved variant LW202 catalyzes the resolution of rac-glycidyl phenyl ether 1 with high enantioselectivity (E > 100) Results from preliminary experiments suggested that it may have potential application in other substrates Preparative implementtations of LW202 are currently in progress in collaboration with the FURSTSS group (Marseilles, France) Substrate mapping experiments [110] may allow access to valuable information on the differences between the binding pocket of the wild-type enzyme and the variant LW202 Furthermore, thorough theoretical studies using molecular modelling and quantum mechanics, combining with experimental data, may reveal the origin of great improvements in enantioselectivity The drastic drop observed of the E-value as a result of reverse engineering indicates the importance of position 350 in achieving high enantioselectivity An extended study by deleting single mutations from LW202 is needed to gain a greater understanding of their influences and the cooperativity between them Resolution of the crystal structure of LW202 is crucial in gaining greater insights into this enzyme variant and its remarkably enhanced enantioselectivity Furthermore, detailed theoretical studies may help to formulate a general enzyme design principle with which to improve the enantioselectivity [102] As such, crystallization experiments were initiated in collaboration with the MWBRAY group from the University of Uppsala