Synthesis of chiral hydrazine reagents and their application for liquid chromatographic separation of carbonyl compounds via diastereomer formation.
Five new chiral derivatizing reagents 5-hydrazino-2,4-dinitrophenyl-l-alaninamide (HDNP-l-Ala-NH2), 5-hydrazino-2,4-dinitrophenyl-l-phenylalaninamide (HDNP-l-Phe-NH2), 5-hydrazino-2,4-dinitrophenyl-l-valinamide (HDNP-l-Val-NH2), 5-hydrazino-2,4-dinitrophenyl-l-leucinamide (HDNP-l-Leu-NH2) and 5-hydrazino-2,4-dinitrophenyl-l-phenylglycinamide (HDNP-d-Phg-NH2) were synthesized by straightforward two-step synthesis starting from 1,5-difluoro-2,4-dinitrobenzene. Nucleophilic substitution of one fluorine atom in DFDNB with different amino acid amides yielded Marfey's reagent (5-fluoro-2,4-dinitrophenyl-l-alaninamide) and its structural variants (5-fluoro-2,4-dinitrophenyl-l-phenylalaninamide, 5-fluoro-2,4-dinitrophenyl-l-valinamide, 5-fluoro-2,4-dinitrophenyl-l-leucinamide and 5-fluoro-2,4-dinitrophenyl-d-phenylglycinamide). Chiral hydrazine reagents were prepared by nucleophilic substitution of remaining fluorine atom in Marfey's reagent and its variants with hydrazine under basic conditions. These reagents react quantitatively with chiral carbonyl compounds under mild conditions (30 degrees C, 30 min) to form hydrazone diastereomers. The labeling reaction occurs only in the presence of acid which has a catalytic action and diastereomers have strong absorbance around 348 nm. The separation of diastereomers was tried on a reversed-phase C18 HPLC column using different binary solvent combinations. Excellent separation was achieved in case of cyclic ketones having substitution at alpha-position. Optimization for derivatization yield, limit of detection, limit of quantification, linearity, accuracy and precision was carried out with respect to HDNP-l-Val-NH2. Studies related to effects of structural modification in reagents and analytes on chromatographic behavior of diastereomers were also analyzed.
Bhushan
Ravi
R
Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247667, India. rbushfcy@iitr.ernet.in <rbushfcy@iitr.ernet.in>
Kumar
Virender
V
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006834:Hydrazines / Q000138:chemical synthesis* / Q000737:chemistry; D007202:Indicators and Reagents / Q000138:chemical synthesis / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013055:Spectrophotometry, Infrared; D013237:Stereoisomerism
D006834:Hydrazines; D007202:Indicators and Reagents; C029424:hydrazine