Enzymatic Approaches to Attaining Enantiomerically Pure Amino Acids

Transcription

1 Enzymatic Approaches to Attaining Enantiomerically Pure Amino Acids Undergraduate Literature Seminar Adam Reed Advisor: Seth Elsheimer, PhD 1

2 Outline Importance Current Methods Chiral Specificity of Enzymes Enzymes Separation D-Threonine (2R,3S) Phosphoproline (L-Pro-Pro P ) L-(5-phenylfuran-2-yl)alanine Conclusions Acknowledgments L-Pro-Pro P D-Threonine (2R,3S) L-(5-Phenyl-2-yl)alanine X H Br Cl H R H H H Cl 2

3 Thalidomide (+)(R) enantiomer is a sedative and antiemetic. (-)(S) enantiomer is teratogenic Banned in 1961 (-)(S)-thalidomide Enantiomeric excess (ee%) ee% = 100*(X-Y) / (X+Y) (+)(R)-thalidomide Enantiomer excess (ee) is the ratio of enantiomers in a solution. A racemic mixture has an ee of 0% whereas a solution of only one enantiomer has an ee of 100% Stephens, T; Brynner, R. Dark Remedy. 1 st ed.; Perseus Publishing: Cambridge, Otis Historical Archives: National Museum of Health and Medicine, Flickr account. 3

4 Additional Examples Compound R Enantiomer S Enantiomer General Structure Ethambutol Causes blindness Treats Tuberculosis * Naproxen Liver poisoning Treats arthritis pain * Carvedilol Adrenoceptor blocker 100x more potent Ibuprofen Negligible effect Pain relief * * = chiral center 4

5 Amino Acids are Chiral Compounds Non-superimposable mirror images. (Often, incorrectly, thought of as a center with four different ligands.) Research Objective: Resolution methods of racemic mixtures of amino acids that have both high enantiomeric excess (ee) and can be replicated at an industrial level. 5

6 Amino Acids in Novel Drug Design Amino acids can be used in the rational design of building compounds to treat a multitude of medical conditions, such as: Anti-cancer compounds Viral inhibitors Diabetes treatment HIV treatment Migraine treatment Serine Protease inhibitor Peripheral opioid side effects Analgesic applications Need ee% > 99% for use in medicine 6

7 Techniques for Enantiomer Resolution Liquid-liquid extraction Asymmetric biotransformation Membranes Sensors Enantioseparation Techniques Crystallization (Spontaneous resolution) Capillary electrophoresis Chromatography Carvalho, P.O. et al. B. J. Chem. Eng. 2006, 23.3,

8 Diastereomeric Salts 2-hydroxypropionic acid Enantiomers have identical physical properties Advantages: High optical purity (>95%) Disadvantages: Time consuming, expensive, requires skilled labor, need acid/base functionality, poor reproducibility, not generally applicable. Bernard, H.; Riechers, H. Method for racemate splitting of 2-hydroxypropionic acids. US B1, May 6,

9 High Performance Liquid Chromatography: Chiral Columns Advantages: Baseline resolution (high ee% ) Disadvantages: Costly, maximum capacity, specialized column Wallworth, D.; Cramer, H. Accurate Biological Testing for Amphetamine and Methamphetamine Abuse using chiral HPLC and MS Detection. Sigma Aldrich. 9

10 Active site of enzyme is chiral specific Often times, enzymes will only react with one enantiomer of the substrate, or will react weakly with one and strongly with the other. Typically work well. Better than non-biological catalysts. 10

11 Preparation of Enzymes for Experimental Use Bacteria Mutated (by design) Protein producing bacteria Grown for X hours Whole cells with protein Large amount of desired protein in cell 1. Break up cell 2. Purify Asymmetric Biotransformation Purified protein 11

12 Experimental Confirmation of Product: Circular Dichroism Picture: 12

13 D-Threonine Production D- Threonine (2R,3S) L- Threonine (2S,3R) L-threonine is easily mass produced Previous works have established methods for synthesis of DL-Threonine with negligible levels of impurities. Enzymes used: Threonine Deaminase (TD) (EC ) ω-transaminase (ω-ta) (R/S selective versions) Han, S.-W.; Shin, J.-S. Journal of Molecular Catalysis B: Enzymatic. 2015, 122,

14 Synthetic Scheme TD Enzymes Threonine Deaminase: TD ω-transaminase: ω-ta Cation exchange chromatography >99% ee 90.9% yield ω-ta >99% ee 90.7% yield Han, S.W. et al. Journal of Molecular Catalysis B: Enzymatic. 2015, 122,

15 Phosphoproline (L-Pro-Pro P ) Synthetic Scheme- Work in Progress 1. CAL-A Candida antarctica Lipase Type A: CAL-A R= 3-OMe-C 6 H 4 -G 2. TsCl, Et 3 N, THF, rt 3. Chromatography separation %ee verified by HPLC Remember: need ee > 99% for medical use. Future work needed 94% ee 82% ee Arizpe, A. et al Tetrahedron: Asymmetry. 2015, 26, T = Tosylate G = carbamate 15

16 L-(5-phenylfuran-2-yl)alanine Applications of derivatives Anti-cancer compounds Viral inhibitors Hypertension Diabetes HIV Migraine X H Br Cl H R H H H Cl W for synthetic scheme Bencze, L. C. et al.tetrahedron: Asymmetry. 2015, 26,

17 L-(5-phenylfuran-2-yl)alanine Synthetic Scheme 3. PLE mediated hydrolysis 1.DCC, CH 2 Cl 2 2. EtOH, CAL-B, Toluene 4. Acylase I, ph 7-8 Candida antarctica lipase Type B: CAL-B Pig Liver Esterase: PLE ee>99% Bencze, L. C. et al.tetrahedron: Asymmetry. 2015, 26,

18 Conclusions Enatiopurity is critical for advancement of medicinal chemistry Enzymes are: Highly stereoselective Work well at moderate reaction conditions Inexpensive to produce once expressed in bacteria. Rational design: potential for modification 18

19 Acknowledgments Dr. Heider Dr. Koculi Haley Nolen 19

20 References Bencze, L. C.; Komjáti, B.; Pop, L.-A.; Paizs, C.; Irimie, F.-D.; Nagy, J.; Poppe, L.; Toşa, M. I. Synthesis Of Enantiopure l-(5-phenylfuran-2- Yl)Alanines by a Sequential Multienzyme Process. Tetrahedron: Asymmetry. 2015, 26, Arizpe, A.; Rodríguez-Mata, M.; Sayago, F. J.; Pueyo, M. J.; Gotor, V.; Jiménez, A. I.; Gotor-Fernández, V.; Cativiela, C. Enzymatic And Chromatographic Resolution Procedures Applied to the Synthesis of the Phosphoproline Enantiomers. Tetrahedron: Asymmetry. 2015, 26, Han, S.-W.; Shin, J.-S. Preparation Of D-Threonine by Biocatalytic Kinetic Resolution. Journal of Molecular Catalysis B: Enzymatic. 2015, 122,