Enzyme Mimics Principles Cyclodextrins as Mimics Corands as Mimics Metallobiosites 1
Enzyme Mimics Biochemical systems: Binding is a trigger to events: Binding induces a conformational change in the receptor that turns on a process Hormones chemical dephosphorylation reactions eurotransmitters nerve impulses (membrane depolarization) 2
Enzyme Mimics Enzymes: living selectivity stabilize every point along the reaction pathway 3
Enzyme Mimics Characteristic features of an enzyme model: Induce fast reaction rates under mild conditions Show a high degree of structural and chiral recognition of the substrates High turnover number Subject to competitive inhibition Artificial systems: Molecular weight less than 2000 Da, stability to handling at high temperatures, solubility in a range of solvents, few chiral centers 4
Enzyme Mimics Basic requirements for an enzyme model: Provides a hydrophobic binding site for the substrate Affords hydrogen bonding and/or electrostatic binding sites complementary to the substrate Appropriate catalytic group should be attached to the model The structure of the model should be rigid The model should be water soluble and catalytically active under physiological conditions 5
Enzyme Mimics Two main approaches: The functional model : a system that carries out the desired reaction without the binding characteristics of the enzyme The structural model : a system that shows efficient binding characteristics of the substrate or of an analogue of the transition state 6
Enzyme Mimics Mechanism of enzymatic catalysis: Enzymes are molecule complementary to the transition states of the reaction they catalyze. It should result in strain at the scissile bond (-73.2 kj/mol for the transition state of the deamination of ATP) k 1 E S kcat ES P E k -1 k 1 /k -1 = K 11 7
Enzyme Mimics Mechanism of enzymatic catalysis: Further factors: Desolvation of the bound substrate, the reactants are hold together in their first solvation sphere (spatiotemporal theory) Increase of the effective molarity (Intramolecular reactions are faster than intermolecular one, ES can be considered as one molecule). - unimolecular reactions result only in a loss of internal rotational entropy, they may be favored by 60 kj/mol on entropic grounds. - the entropic price is paid in advance by the substrate binding. 8
Enzyme Mimics Effective Molarity (EM) a) H 2 H H - k 1 - Phenyl succinate reacts 100000 times faster than phenyl acetate X X EM = k 1 /k 2 = 100000 M b) - 2 H - k 2 X X Intramolecular reactions (a) are much faster than bimolecular equivalents (b) 9
Cyclodextrins as Enzyme Mimics Main features: Water solubility at physiological ph Reversible, non-covalent binding with the release slower than the binding Well-defined structures Wide range of accessible derivatives Juxtaposition of hydroxy groups which may act as catalysts and a hydrophobic cavity for guest binding 10
Cyclodextrins as Enzyme Mimics Most simple enzyme model, but not catalytic since the cyclodextrin remains acylated Applied for selective ester cleavage Rate enhancement : 100 11
Cyclodextrins as Enzyme Mimics Rate acceleration for 4a: 5 900 000 12
Cyclodextrins as Esterase Mimics - P P P - - - H A H β -CD, Pr 3 A - P H (9.4) γ -CD, Ce 4 - P - A H H H 2 A = Adenylate cyclase/phosphodiesterase activity by cyclodextrin complexes (after Sumaoka et al.1994) Rate enhancement for the cleavage: 10 13 13
Corands as ATPase Mimics H P P - P - - - H 2 H 2 H 2 H 2 A H 2 A P - H 2 H P P H - - - H 2 H 2 H 2 A H 2 Catalytic cycle driven by the replacement of ADP by ATP (more strongly bound) P - - (4.9)-5H ATP P P A - - D E H 2 B - P P A - - acceleration rate: 100 at ph 4 9.7 is isolable ADP H H 2 H 2 H 2 C - P - H 2 H 2 H 2 H 2 H 2 H 2 H 2 H 2 (4.9)-5H (9.7) Mechanism of ATP hydrolysis by (4.9)-5H (A = adenosine sugar) 14
Corands as ATPase Mimics 15
Corands as ATPase Mimics with 10 eq acetyl phosphate (AcP) and Mg 2 /Ca 2, reversibility of all the steps ATP synthase 16
Chiral Corands as Transacylase Mimics Transition state is more stable for the S-L pair Ratio L/D = 1 for alanine, 6-10 for branched amino acids 17
A Structure Mimic of Chymotrypsin Active site of chymotrypsin, a protease used as a transacylase planned model 18
A Structure Mimic of Chymotrypsin Me H Me Me Me Me p- 2 C 6 H 4 Bound into hemispherand binding site H Me H H H H Me Me H H H Me Me Me Me 10 11 time faster than reaction with phenylbenzylalcohol (9.12) (9.13) Partial model transacylase activity in a chymotrypsin mimic. (9.14) a 30-step-synthesis competitive inhibition by a slow transfer to the hydroxy group 19
Metallobiosites Coordination compound models for the metal-containing site in biological models (structural models) speculative model if the structure is unknown corroborative model : direct imitation of the structure macrocycles as ligand are useful to coordinate two metal ions at a defined-distance 20
Haemocyanin Models A metalloprotein involved in oxygen transport in molluscs Knowledge basis for a speculative model: two copper centers, each coordinated by three histidines an endogeneously bridging donor atom (e.g. H from tyrosine) strong antiferromagnetic coupling tetragonal Cu coordination environment from UV-spectroscopy Cu Cu = 364 pm Raman v(--) 750 cm -1 21
Haemocyanin Models 22
Haemocyanin Models binds oxygen irreversibly 23
Zinc-containing Enzyme Zn 2 a non redox active Lewis acid, present in more than 100 enzymes i.e Carbonic anhydrase: - C 2 fixation - a tetrahedral Zn 2 center coordinated by 3 histidines and a water - a pka of 7 due to the hydrophobic cavity and the high positive charge Base(B) BH (His) 3 Zn 2 H 2 (His) 3 Zn H HC 3 - Key steps in the action of carbonic anhydrase C 2 H 2 (His) 3 Zn C 2 H 24
Zinc-containing Enzyme o hydrophobic cavity only slightly acidic 25
Haem Analogues Models of oxygen uptake and transport oxygen is a weak but very reactive ligand Use of oxidisible metals: Iron (II) complexes of porphyrin ligands or Co(II) have a vacant axial coordination site available for 2 the main issue is to generate a five-coordinate Fe(II) 3 different approaches 26
Haem Analogues Use of 2-methylimidazole as the axial ligand 27
Haem Analogues Covalent attachment of a single axial ligand (at high concentrations, dimerization can still occur) 28
Haem Analogues fit one side of the porphyrin with a hydrophobic molecular cage (picket fences porphyrins) should prevent dimerization and also protonation (formation of inactive Fe(III) porphyrin and highly reactive hydroxyradical H 2. 29
Picket fences porphyrins interconvertable at 80 C and separable by chromatography H CH 2 H 2 H 2 H 2 α,α,α,α H 2 1.Condensation 2.Reduction H 2 H 2 α,α,α,β H 2 H 2 H 2 H 2 H H H 2 H 2 (9.24) H 2 H 2 H 2 H 2 H 2 H 2 α,α,β,β (9.24) H 2 α,β,α,β H 2 H 2 H H H H H H Synthesis of "picket fence" porphyrins (9.25) 30
Picket fences porphyrins stable for several hours in solution at RT and crystallizable 31
Picket fences porphyrins pocket and picnic basket porphyrins can reduce the C binding affinity due to the capping of the binding site (Fe-C= is linear) 32
Cytochrome P-450 Models an oxygen-binding haem unit with an axial cysteine thiolate residue instead of histidine used for the catabolism of xenobiotics RH 2 RH H 2 difficult to construct since the thiolate is a weak ligand and should not be oxidized 33
Cytochrome P-450 Models RH H 2 Fe 3 CySH RH H 2 (RH) Fe 5 CyS H 2 (RH) Fe 3 CyS e- 2 H (RH) Fe 3 CyS 2- (RH) Fe 2 CyS 2 - Analog with a tethered axial thiolate e - (RH) Fe 3 - CyS 34 Catalytic reactivity cycle for cytochrome P-450
An abiotic molecular device R R Me Mn 3 R Me Me Hydrophilic group Hydrophobic portion A membrane bound metalloporphyrin with an oxidase activity carry out the epoxidation of styrene and dihydrofuran use methylene blue as electron carrier use a vesicle filled with a Ptsuspension (by reduction of Pt(II) salts with H 2 ) as electron source low turn-over (1-8) Vesicle membrane wall Pt H 2 2 H R = C 16 H 33 (9.30) MB ox MB red Polymer wall of vesicle Mn II Mn III 2 2 H Mn porphyrin complex Cytochrome P-450-mimic microreactor based on a Pt-filled vesicle.(mbox/red = methylene blue electron carrier,oxidised and reduced forms,respectively.) R (9.31) Me C - Hydrophilic group 35 H 2