Enzym e-Catalyzed Regioselective Acylation of the Quinic and Shikim - PowerPoint PPT Presentation

Universidad de Oviedo Facultad de Quím ica Departam ento de Quím ica Orgánica e I norgánica Oviedo - SPAI N Enzym e-Catalyzed Regioselective Acylation of the Quinic and Shikim ic Acid Derivatives Prof. Miguel Ferrero COST D2 5 3 July 2 0 0 5 Delft ( The Netherlands)

Introduction: Quinic and Shikimic Acids CO 2 H HO 2 C OH 1 6 2 6 2 1 5 3 5 3 HO OH HO OH 4 4 OH OH Shikimic Acid Quinic Acid - Biosynthesis of L-amino acids (Phe, Trp, Tyr), folate coenzymes, and isoprenoid quinones - Shikimate pathway is unique to plants, fungi, and microorganisms - Potential herbicide, antifungal, and antibacterial agents - Increasing effort toward the synthesis of shikimate analogues - Quinic acid is an alternate carbon source in the shikimate pathway - Enzymes offer efficient discrimination among OHs vs. chemical methods

Background: Enzymatic Acylation of Quinic and Shikimic Acid Derivatives CO 2 Me MeO 2 C OH 5 3 5 3 HO OH HO OH 4 4 OH OH CAL-A CAL-A - Exhaustive study of enzymatic processes (Enz., solvent, T, acyl chain, … ) - CAL-A acylates the most hindered C-4 hydroxyl group - Preparation of novel saturated, unsaturated, and aromatic monoesters, including cinnamate analogues - Used to check their therapeutic and antimicrobial properties J. Org. Chem. 2 0 0 2 , 67 , 4978-4981 J. Org. Chem. 2 0 0 3 , 68 , 5784-5787

Objectives: Monoacyl Derivatives and Molecular Basis for Enzyme Selectivity � Although use of enzymes in synthesis is common, the basis of their selectivity is not well understood � Goals: - prepare valuable monoacyl derivatives - identify the molecular basis of CAL-A selectivity as function of the degree of intramolecular H-bond within the ligand CO 2 Me CO 2 Me CO 2 Me HO OH HO OH HO OH OH OH OH Methyl 3- epi -Shikimate Methyl 4- epi -Shikimate Methyl 5- epi -Shikimate MeO 2 C OH MeO 2 C OH MeO 2 C OH HO OH HO OH HO OH OH OH OH Methyl 3- epi -Quinate Methyl 4- epi -Quinate Methyl 5- epi -Quinate

Results: Monoacyl 3- epi -Quinic and 3- epi -Shikimic Acid Derivatives CO 2 Me MeO 2 C OH 3 3 5 5 4 4 HO OAc HO OAc OH OH Conditions: CAL-A, vinyl acetate, molecular sieves 4Å MeO 2 C OH MeO 2 C OH MeO 2 C OH a b HO OAc O OAc HO OH MeO OH O OH OMe 8 7 17 (a) CAL-A , CH 2 = CHOAc, sieves 4Å, 40 °C, 4.5 h (b) butane-2,3-dione, CH(OMe) 3 , (±)-CSA, MeOH, 65 °C, 2 h

Results: Monoacyl 3-epi-Quinic and 3-epi-Shikimic Acid Derivatives MeO 2 C OH MeO 2 C OH MeO 2 C OH a b O OAc O OH HO OH MeO MeO O O OH OMe OMe 7 (±)- 18 (±)- 17 MeO 2 C OH MeO 2 C OH MeO 2 C OH ref. b O OAc O OH HO OH MeO MeO O O OH OMe OMe 3 (+)- 18 (+)- 17 (a) butane-2,3-dione, CH(OMe) 3 , (±)-CSA, MeOH, 65 °C, 2 h; (b) Ac 2 O, DMAP, Py, CH 2 Cl 2 , 0 °C, 4 h Tetrahedron Lett. 2 0 0 0 , 41 , 8759-8762

Results: Monoacyl 3- epi -Quinic Acid Derivative: Relative Configuration A B 60% ee racemic C pure (1 R ,3 S ,4 S ,5 R )-(+ )- 1 7 Figure 1 . HPLC chromatograms of corresponding: A , racemic ester (� )- 1 7 ; B , ester 1 7 from enzymatic reaction; C , enantiomerically pure ester (+ )- 1 7

Results: Monoacyl 4- epi -Quinic and 4- epi -Shikimic Acid Derivatives CO 2 Me MeO 2 C OH 3 3 5 5 4 4 HO OH HO OH OAc OAc Transesterification in 1.5 h Identical conditions: Total selectivity - lactone detected 80% isolated yield - mixture of acyl compounds H 4 H 5 Change conditions: O O - no molecular sieves 4Å OH H - temperature 20 ° C (40 ° C) O H C Exclusively 4- O -acetyl derivative in 24 h H 3 O

Results: Monoacyl 5- epi -Quinic and 5- epi -Shikimic Acid Derivatives CO 2 Me MeO 2 C OH 3 5 3 5 4 4 AcO OH HO OH OH OAc In 0.3 h at 20 ° C, 100% conversion In 4.5 h at 40 ° C, 100% conversion Exclusively C-5 product Exclusively C-5 product HO OH OH OH HO HO CO 2 Me OH OH OH H 4 H 5 CO 2 Me H 5 H 4 OH HO HO HO HO H 5 H 3 H 3 CO 2 Me CO 2 Me H 3 H 5 H 4 H 4 H 3 MeOH- d 4 CDCl 3 MeOH- d 4 CDCl 3 In both cases: - Different chair conformation - No changes in selectivity with hydrogen and non-hydrogen bonding solvents

Background: Conformational Analysis To explain selectivity of CAL-A, w e study the role of intram olecular H-bonds w ithin ligand - IR and 1 H NMR spectroscopy are common methods to determine intra- and intermolecular H-bonding - IR not suitable since our molecules posses several OH groups - 1 H NMR concentration dependent hydroxyl protons exchange is not adequate (poor solubility) - 1 H NMR temperature dependent is not possible since conformational equilibrium is present - We study the proton exchange by acid catalysis - Rigorous removal of acid from: sample, solvent, and NMR tube

Results: Conformational Analysis. Methyl Shikimate - H-bonds: OH-4/ OH-5; OH-4/ OH-3 H 5 - OH-3> OH-4, then OH-3 is H H 5 CO 2 Me CO 2 Me H O H O H H-acceptor O H O - Equatorial OH functionalyzed H O O before than axial H 3 H 3 H 4 H 4 - enzymatic acylation on OH-4 Determining structures by homo- and heteronuclear 2D NMR experiments Second 0.05 mM CF 3 CO 2 H First Hydroxyl exchange indicates an acidity order of OH-5 > OH-3 > OH-4

Results: Conformational Analysis. Methyl Quinate H 5 H H 5 H O OH H H O O H O H CO 2 Me CO 2 Me O H O O H 3 H 3 H 4 H 4 Since acidity does no indicate the direction of H-bonds, vicinal 3 J CH,OH were used 0.05 mM CF 3 CO 2 H Second First Hydroxyl exchange indicates an acidity order of OH-5 > OH-4 > OH-3

Results: Conformational Analysis. Methyl 3- epi -Shikimate and 3- epi -Quinate H 5 H 5 H H OH CO 2 Me H 3 H 3 O O CO 2 Me H O H O H O H O H 4 H 4 - Same half-chair: - Chair from coupling constants analysis MeOH- d 4 , acetone- d 6 or CDCl 3 - Three secondary OHs weak H-bonds - Acidity order of OH-4 ≈ OH-5 > OH-3 - Hydroxyl exchange is similar (OH-3 or - OH-3 lesser acidic, then H-donor 5 seems to be more stable) indicates the direction of the H-bonds - direction of the H-bonds, both are - Enzymatic acylation led to 3-OAc equivalents (plane of symmetry) derivative - Enzymatic acylation led to 3-OAc (or - More efficient H-bond between OH-3 5-OAc) derivative ( p e) and OH-4

Results: Conformational Analysis. Methyl 4- epi -Shikimate and 4- epi -Quinate H 4 H H H 5 O H 5 O O O CO 2 Me O H 4 H CO 2 Me H O H H H 3 H 3 O H - Half-chair (MeOH- d 4 , acetone- d 6 or - Hydroxyl exchange study unsuccessful (low solubility in CDCl 3 ) CDCl 3 ) - Acidity order of OH-5 > OH-3 ≈ OH-4 - In all solvents the same chair - J CH,OH plus acidity indicate the - From J CH,OH values, OH-4 acts as H-acceptor direction of the H-bonds. OH-5 the most acidic then H-acceptor - According to that, acylation on OH-3 (a) - Enzymatic acylation led to 4-OAc - Actually, enzymatic acylation is on OH-4 derivative, as predicted

Results: Conformational Analysis. Methyl 4- epi -Shikimate and 4- epi -Quinate H 4 H 4 H 5 H 5 O O O O OH OH H H H O O H C C OMe H 3 H 3 O H O - The results of enzymatic acylation is in agreement with this chair - Efficient H-bond between OH-5 and carbonyl of ester extent the H-bond network - OH-4 is in equatorial - Also, enzymatic acylation of lactone (restricted chair conformation) gives exclusively 4-OAc derivative

Results: Conformational Analysis. Methyl 5- epi -Shikimate and 5- epi -Quinate H H H O O O CO 2 Me H H H O H O CO 2 Me H 5 H 5 O O H 3 H 3 H 4 H 4 - Hydroxyl exchange provide an acidity - Hydroxyl exchange did not provide information order of OH-5 > OH-4 > OH-3 - OH-5 should be the acceptor of OH-3 - Chair conformation in CDCl 3 - J CH,OH-3 indicates an angle close to 180º - All J CH,OH indicate angles close to 180º (1,3-diaxial H-bonds) - Also, J CH,OH-4 and J CH,OH-5 indicate the - Similar directions of H-bonds (meso) direction of the H-bonds - Enzymatic acylation should be on OH-4 - OH equatorial more reactive. Enzymatic (e); However, 5-OAc derivative was acylation was on OH-4, as predicted obtained (acetyl migrations)

Results: Tetrahedral Intermediate 2 (TI-2) in Serine Mechanism � In view of these results, we propose a cooperative effect of H-bond between E-S, which lead to selectivity � General mechanism of lipases: Asp Asp CO 2- CO 2- His His - Catalytic triad: Ser-His-Asp H H N N Ser + Ser - TI-2 is the key to enzymatic selectivity N N H O H O O O R R 1 R 1 O O - R TI-2 � Additional interactions in TI-2: Asp Asp - Transition state H-bond plays an CO 2- CO 2- important role in enzyme selectivity His His H H N N Ser - Two of the essentials H-bonds in the Ser N N active site are: His-Ser and His-OR 1 O δ + O H δ - H + δ + O H O O - R - In Q and S acid derivatives, positive O H O O R charge can be delocalized through another H-bonds network within ligand

Conclusions: - Selective acylated derivatives of natural, 3-epi, 4-epi, and 5-epi- isomers of quinic and shikimic acid have been efficiently synthesized - Useful as chiral building-blocks for organic synthesis and as inhibitors - Selectivity of CAL-A is related to both inherent receptor selectivity and intramolecular H-bond in the ligand - 1 H NMR is used to determine the strength of intramolecular H-bond - Acid exchange of OH and J CH,OH provides structural information in terms of dihedral angles (H-O-C-H) and direction of H-bond network - Satisfactory correlation between reactivity of hydroxyls and effectiveness of the corresponding H-bond network