Analysis of racemic acid derivitives by chiral high performance liquid chromatography Tara Kuknyo Orech, Knox College

Overview Chiral HPLC Amino and Phosphonic Acids α Hydroxy- phosphonates Results and Future Research

What is HPLC? High Performance Liquid Chromatography P can also stand for “Pressure” Fast! Large number of plates Detected by UV-Vis Nelson, David L., Albert L. Lehninger, and Michael M. Cox. "3.3 Working with Proteins."Lehninger Principles of Biochemistry. New York: W.H. Freeman, Print.

Chirality Chiral molecules differ in “handedness”, or 3-D orientation Common in organic chemistry and biology Very difficult to separate because they have identical physical properties, except for light rotation

Chiral Chromatography Chromatography…with chirality? Affinity Chromatography Separated by three chiral binding site or chiral pocket stationary phases "Online Guide to Chiral HPLC." Mark Earll's Homepage. Web. 20 Aug

Column Used Nucleosil Chiral-3, 4mm diameter, pores 100Å, made for organic solvents Family of columns that uses Nucleosil silica Charge-transfer interactions, hydrogen bonds, dipole-dipole intereactions and steric effects Chiral Selector: N(3,5-dinitrobenzoyl)- L-phenylglycine Nucleosil Silica "NUCLEOSIL CHIRAL." MACHEREY-NAGEL Homepage Web. 13 Apr

Disadvantages to Chiral HPLC Expensive ($1,000-$10,000) Affinity chromatography=specific column for different molecules Interactions between stationary phase and chiral molecule are fairly weak, making it difficult to separate "Online Guide to Chiral HPLC." Mark Earll's Homepage. Web. 20 Aug

Why Chiral Chromatography? Analyzes enantiomeric purity Optical Rotation does not work for new compounds Less sensitive to impurities than opt. rot. Allows separation of enantiomers

α β * * What is a β-amino phosphonic acid? Abrams, Martha Leigh. “Progress towards the synthesis of phosphorus analogs of β-amino acids.” Knox College Honors Thesis, May 2008.

β-Amino Phosphonic Acids Tyrosine and phenylalanine derivatives R and S-Valine first, translate techniques S-PhenylalanineS-Tyrosine S-Valine

FMOC-Derivative Eluent100:18:1 heptane, dioxane and trifluoroacetic acid, very non polar Can detect all amino acids with FMOC at similar wavelengths (~280 nm) Kortenaar, Paul B.W. et al.""Rapid and Efficient Method for the Preparation of F-moc-amino Acids Starting from 9-fluorenylmethanol."" International Journal of Peptide and Protein Research 26.4 (1996): Web. 12 Jan. 2009

Method S and R Valine-FMOC separately Optimize the Peak Run a racemic mixture

First Factor: Wavelength

Optimized Factors Wavelength: 280 nm Flow Rate: 1 mL/min Output Range: 1.0 Amplifier Setting: 0-1V Sample Concentration: 12 mmol Sample Size: 20μL Solvent: Acetone:Acetonitrile, Buffer Eluent: 100:18:1 heptane, dioxane and trifluoroacetic acid

Initial Readings Problem: both at 8 minutes! S-Valine R-Valine

Mixed Example

Separation of the Peaks Dilute from 12 mmol to 1 mmol Slow flow rate to.5 mL/min Alter eluent polarity All methods diluted peaks, even if put them further downfield FMOC-Cl? 8 mins! L-Valine with x5 TFA

Extractions of FMOC-Cl Before extractionsOne extractionTwo extractions

Re-Optimized! Changes: wavelength at 265 nm, sample size 5 µL S-Valine at ~16 mins R-Valine at ~13 mins S-valine

Future Research Tyrosine and Phenylalanine β-amino phosphonic acid derivatives α-hydroxy phosphonates (racemic)

Project 2: Racemic Dimethyl 1-hydroxybenzylphosphonate Pogatchnik, D. M., Weimer, David F. “Enantioselective Synthesis of α-Hydroxy Phosphonates via Oxidation with (Camphor Sulfonyl). University of Iowa Department of Chemistry

Optimal Settings Flow Rate1.0 mL/min Wavelength285 nm Output Range1.0 Amplifier Setting0-200mV Sample Concentration4 mmol Sample Size5 μL SolventHeptane Dioxane TFA 100:20:.1 EluentHeptane Dioxane TFA 100:20:.1

Separation of enantiomers

Results Total Average69.14 : / Average of Batch : / Average of Batch : /-.94

Future Research Optimize further to obtain 50:50 results Nucleosil Chiral-2 Polarimeter to determine on S vs R Complex products from HPLC to form diasteromers, determine S vs R by NMR Another factor…temperature? N(3,5-dinitrobenzoyl)-L- phenylglycine N(3,5-dinitrobenzoyl)- D-phenylglycine

Thank You! Howard Hughes Medical Internship Knox College Dr. Larry Welch Dr. Diana Cermak American Chemical Society Illinois State Academe of Science