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

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Universidad de Oviedo Facultad de Qum ica Departam ento de Qum ica Orgnica e I norgnica 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

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SLIDE 1

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)

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SLIDE 2

Introduction: Quinic and Shikimic Acids

CO2H OH OH HO OH OH HO OH HO2C

1 2 3 4 5 6 2 3 4 5 6 1

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
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SLIDE 3

Background: Enzymatic Acylation of Quinic and Shikimic Acid Derivatives 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

CAL-A

  • J. Org. Chem. 2 0 0 2 , 67, 4978-4981
  • J. Org. Chem. 2 0 0 3 , 68, 5784-5787

CO2Me OH OH HO

3 4 5

OH OH HO OH MeO2C

3 4 5

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SLIDE 4

Objectives: Monoacyl Derivatives and Molecular Basis for Enzyme Selectivity

OH OH HO CO2Me Methyl 3-epi-Shikimate OH OH HO CO2Me Methyl 4-epi-Shikimate OH OH HO CO2Me Methyl 5-epi-Shikimate

  • 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

OH OH HO Methyl 3-epi-Quinate OH MeO2C OH OH HO Methyl 4-epi-Quinate OH MeO2C OH OH HO Methyl 5-epi-Quinate OH MeO2C

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SLIDE 5

Results: Monoacyl 3-epi-Quinic and 3-epi-Shikimic Acid Derivatives OH OAc HO CO2Me OH OAc HO OH MeO2C

3 5 4 3 5 4

Conditions: CAL-A, vinyl acetate, molecular sieves 4Å OH OH HO 7 OH MeO2C OH OAc HO 8 OH MeO2C O OAc O OH MeO2C OMe MeO 17 b a

(a) CAL-A, CH2= CHOAc, sieves 4Å, 40 °C, 4.5 h (b) butane-2,3-dione, CH(OMe) 3, (±)-CSA, MeOH, 65 °C, 2 h

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SLIDE 6

Results: Monoacyl 3-epi-Quinic and 3-epi-Shikimic Acid Derivatives

OH OH HO 7 OH MeO2C O OH O (±)-18 OH MeO2C OMe MeO O OAc O (±)-17 OH MeO2C OMe MeO a b

(a) butane-2,3-dione, CH(OMe) 3, (±)-CSA, MeOH, 65 °C, 2 h; (b) Ac2O, DMAP, Py, CH2Cl2, 0 °C, 4 h

OH OH HO 3 OH MeO2C O OH O (+)-18 OH MeO2C OMe MeO O OAc O (+)-17 OH MeO2C OMe MeO ref. b

Tetrahedron Lett. 2 0 0 0 , 41, 8759-8762

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SLIDE 7

Results: Monoacyl 3-epi-Quinic Acid Derivative: Relative Configuration

Figure 1 . HPLC chromatograms of corresponding: A, racemic ester ( )-1 7 ; B, ester 1 7 from enzymatic reaction; C, enantiomerically pure ester (+ )-1 7 (1R,3S,4S,5R)-(+ )-1 7 60% ee racemic

A B

pure

C

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SLIDE 8

Results: Monoacyl 4-epi-Quinic and 4-epi-Shikimic Acid Derivatives

OAc OH HO CO2Me

3 5 4 Transesterification in 1.5 h Total selectivity 80% isolated yield

O O O H5 C OH H4 H H O H3

Change conditions:

  • no molecular sieves 4Å
  • temperature 20 °C (40 °C)

Exclusively 4-O-acetyl derivative in 24 h Identical conditions:

  • lactone detected
  • mixture of acyl compounds

OAc OH HO OH MeO2C

3 5 4

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SLIDE 9

HO OH H5 OH CO2Me H4 OH H3 OH HO H4 H3 HO H5 CO2Me OH

MeOH-d4 CDCl3

OH HO H4 H3 HO H5 CO2Me HO CO2Me OH H5 H4 H3 HO

MeOH-d4 CDCl3

In both cases:

  • Different chair conformation
  • No changes in selectivity with hydrogen and non-hydrogen bonding solvents

In 4.5 h at 40 °C, 100% conversion Exclusively C-5 product

OAc OH HO OH MeO2C

3 5 4

Results: Monoacyl 5-epi-Quinic and 5-epi-Shikimic Acid Derivatives OH OH AcO CO2Me

3 5 4

In 0.3 h at 20 °C, 100% conversion Exclusively C-5 product

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SLIDE 10

Background: Conformational Analysis To explain selectivity of CAL-A, w e study the role of intram olecular H-bonds w ithin ligand

  • IR and 1H NMR spectroscopy are common methods to determine

intra- and intermolecular H-bonding

  • IR not suitable since our molecules posses several OH groups
  • 1H NMR concentration dependent hydroxyl protons exchange is not

adequate (poor solubility)

  • 1H 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
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SLIDE 11

Determining structures by homo- and heteronuclear 2D NMR experiments Hydroxyl exchange indicates an acidity order of OH-5 > OH-3 > OH-4

Results: Conformational Analysis. Methyl Shikimate

O O CO2Me H H O CO2Me H O H H4 H4 H3 H3 H5 H5 O H O H 0.05 mM CF3CO2H

First Second

  • H-bonds:

OH-4/ OH-5; OH-4/ OH-3

  • OH-3> OH-4, then OH-3 is

H-acceptor

  • Equatorial OH functionalyzed

before than axial

  • enzymatic acylation on OH-4
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SLIDE 12

Hydroxyl exchange indicates an acidity order of OH-5 > OH-4 > OH-3 Since acidity does no indicate the direction of H-bonds, vicinal 3JCH,OH were used

Results: Conformational Analysis. Methyl Quinate

0.05 mM CF3CO2H O O O H H H O O O H O H OH CO2Me CO2Me H H3 H4 H5 H4 H5 H3 H

First Second

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SLIDE 13

Results: Conformational Analysis. Methyl 3-epi-Shikimate and 3-epi-Quinate

  • Same half-chair:

MeOH-d4, acetone-d6 or CDCl3

  • Acidity order of OH-4 ≈OH-5 > OH-3
  • OH-3 lesser acidic, then H-donor

indicates the direction of the H-bonds

  • Enzymatic acylation led to 3-OAc

derivative

  • More efficient H-bond between OH-3

(pe) and OH-4

O O CO2Me H H H5 H4 H3 O H

  • Chair from coupling constants analysis
  • Three secondary OHs weak H-bonds
  • Hydroxyl exchange is similar (OH-3 or

5 seems to be more stable)

  • direction of the H-bonds, both are

equivalents (plane of symmetry)

  • Enzymatic acylation led to 3-OAc (or

5-OAc) derivative

O O H H OH CO2Me H5 H3 H4 O H

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SLIDE 14

Results: Conformational Analysis. Methyl 4-epi-Shikimate and 4-epi-Quinate

H5 H4 O H3 O H H O CO2Me H

  • Half-chair (MeOH-d4, acetone-d6 or

CDCl3)

  • Acidity order of OH-5 > OH-3 ≈OH-4
  • JCH,OH plus acidity indicate the

direction of the H-bonds. OH-5 the most acidic then H-acceptor

  • Enzymatic acylation led to 4-OAc

derivative, as predicted

O H3 O CO2Me H O H4 H5 O H H H

  • Hydroxyl exchange study unsuccessful (low

solubility in CDCl3)

  • In all solvents the same chair
  • From JCH,OH values, OH-4 acts as H-acceptor
  • According to that, acylation on OH-3 (a)
  • Actually, enzymatic acylation is on OH-4
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SLIDE 15

O O O H5 C OH H4 H H O H3

Results: Conformational Analysis. Methyl 4-epi-Shikimate and 4-epi-Quinate

O O O H5 C OH O OMe H H4 H H H3

  • 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

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SLIDE 16

Results: Conformational Analysis. Methyl 5-epi-Shikimate and 5-epi-Quinate

O CO2Me O H H4 H3 O H H H5

  • Hydroxyl exchange provide an acidity
  • rder of OH-5 > OH-4 > OH-3
  • OH-5 should be the acceptor of OH-3
  • JCH,OH-3 indicates an angle close to 180º
  • Also, JCH,OH-4 and JCH,OH-5 indicate the

direction of the H-bonds

  • Enzymatic acylation should be on OH-4

(e); However, 5-OAc derivative was

  • btained (acetyl migrations)

O O H O H H H4 H3 O CO2Me H H5

  • Hydroxyl exchange did not provide

information

  • Chair conformation in CDCl3
  • All JCH,OH indicate angles close to 180º

(1,3-diaxial H-bonds)

  • Similar directions of H-bonds (meso)
  • OH equatorial more reactive. Enzymatic

acylation was on OH-4, as predicted

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SLIDE 17

N N H His Asp CO2- O- R O Ser O R O Ser O H N N H His Asp CO2- O H O O H H + δ+ δ+ δ-

Additional interactions in TI-2:

  • Transition state H-bond plays an

important role in enzyme selectivity

  • Two of the essentials H-bonds in the

active site are: His-Ser and His-OR1

  • In Q and S acid derivatives, positive

charge can be delocalized through another H-bonds network within ligand

Results: Tetrahedral Intermediate 2 (TI-2) in Serine Mechanism

N N H His Asp CO2- R1O O- R O Ser O R O Ser O H R1 N N H His Asp CO2- H + TI-2

General mechanism of lipases:

  • Catalytic triad: Ser-His-Asp
  • TI-2 is the key to enzymatic selectivity

In view of these results, we propose a cooperative effect of H-bond between E-S, which lead to selectivity

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SLIDE 18

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

  • 1H NMR is used to determine the strength of intramolecular H-bond
  • Acid exchange of OH and JCH,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

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SLIDE 19

Acknowledgements:

  • Prof. Vicente Gotor
  • Dr. Susana Fernández
  • Dr. Nuria Armesto

Financial support by MEC (Spain) Project CTQ-2004-04185