Measuring enzyme (enantio)selectivity

Transcription

1 Measuring enzyme (enantio)selectivity Types of selectivity - review stereoisomers Stereoselective synthesis (create) vs. resolutions (separate) Enantioselectivity & enantiomeric purity Ways to measure enantiomeric purity

2 Synthesis vs. separation asymmetric synthesis, or stereoselective synthesis, introduces one or more new and desired elements of chirality; e.g., convert sitigliptin ketone to (R)-enantiomer using transaminase kinetic resolution is a separation of enantiomers by their different reaction rates. The product contains more of the more reactive enantiomer, while the starting material contains more of the less reactive enantiomer. This excess goes through a maximum and disappears on full completion of the reaction.

3 Enantioselective synthesis to make drug intermediates R lipase Ac H S SiMe 2 t-bu H SiMe 2 t-bu achiral >99% ee Wirz et al. Tetrahedron: Asymmetry 2000, 11, Meso compound - a compound that has at least two stereocenters but is achiral. anti-psoriasis Vitamin D derivative

4 Enantiomeric purity and enantioselectivity Enantiomeric excess - purity of one enantiomer in a sample, e.g. 98% ee is a 99:1 mix of enantiomers Enantioselectivity (or enantiomeric ratio) - selectivity of an enzyme or reagent for one enantiomer over the other. e.e. = F major F minor F major + F minor E = rate of formation of fast enantiomer rate of formation of slow enantiomer

5 Asymmetric synthesis: ee constant throughout reaction Enantioselectivity is defined from the relative amount of the enantiomers formed. It is more convenient to rewrite the equation in terms of ee because that is how product purity is characterized. E = F major F minor e.e. = F major F minor F major + F minor Using the two definitions above, derive the two more convenient equations below. E = 1+ e.e. 1 e.e. or e.e. = E 1 E + 1

6 Kinetic resolution ee varies with conversion; must use E, not ee, to compare kinetic resolutions To determine E, you must measure two things: ees and conv, eep and conv or ees and eep.

7 A perfect kinetic resolution product C 2 CH 3 C 2 CH 3 slow enzyme fast C 2 H C 2 H enantiomeric excess,% remaining starting material conversion, % ee p & ees change as reaction proceeds Stop at 50% conversion (Perfect resolution will stop by itself.)

8 An imperfect resolution C 2 CH 3 slow pig liver esterase C 2 H E = 9.7 C 2 CH 3 fast C 2 H Product is never above 80% ee, but starting material reaches >95%ee

9 Measure eep & conv to determine E ( ) ( ) ( )( 1 ee S ) ( )( 1+ ee S ) E = ln 1 c 1+ ee P ln 1 c 1 ee P E = ln 1 c ln 1 c Conversion and E calculated using : c = ee S ee S + ee P and E = 1 ee ln S 1+ ee S ee P 1 + ee ln S 1+ ee S ee P ( ) ( ) Chen, C. S., Fujimoto, Y., Girdaukas, G., Sih, C. J. (1982) Quantitative analyses of biochemical kinetic resolutions of enantiomers, J. Am. Chem. Soc. 104,

10 Diastereomeric derivatives R, S (sample) + R' (reagent) R R' + S R' (diastereomers) t-bu Cl CF 3 Me Ph t-bu CF 3 Me Ph t-bu Mosher's acid chloride t-bu Me CF 3 Ph Both reagent and sample stable to racemization under reaction conditions. Sample must react completely; otherwise one enantiomer may react faster. Avoid purification methods (e.g., crystallization) that could fractionate diastereomers. Requires a method to distinguish diastereomers.

11 Chiral Stationary Phases for Gas Chromatography -C 5 ChiralDex G-TA: non-bonded 2,6- di--pentyl-3-trifluoroacetyl derivative of γ-cyclodextrin (7 Glc) CP-Chirasil-DEX CB: β- cyclodextrin (6 Glc) directly bonded to dimethylpolysiloxane Chirasil-Val: valine derivative bonded to siloxane polymer (can racemize at high T) C 5 - C 5 - -C 5 C 5 - F 3 -Ac -C 5 -C Ac-F 3 F 3 -Ac 5 C 5 - Ac-F 3 F 3 -Ac -C 5 Ac-F 3 F 3 -Ac C 5 - -C 5 C 5 - Me C 5 - -C 5 Me Me Me Si Si Me Me Me Me Me Me Me Me Me Me Me Me H N N H Si Si Me

12 Chiral Stationary Phases for HPLC

13 Examples chiral polymers ligand exchange chiral crown ether

14 Quiz In the transaminase-catalyzed synthesis of sitagliptin, the product had 99.5% ee. What was the enantioselectivity of the reaction? Draw a picture of what an HPLC analysis of enantiomeric purity might look like. A protease shows a k cat/km of 2 x 10 4 M -1 s -1 toward peptide A and 3 x 10 5 M -1 s -1 toward peptide B. What is the selectivity of this protease for the two peptides? Explain (qualitatively) how you could use the protease to make a sample of 99 mol% peptide A.