PREDICTION OF RETENTION TIMES OF PEPTIDE NUCLEIC-ACIDS DURING REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Peptide nucleic acids (PNAs) are synthetic biopolymers consisting of n ucleobase side chains attached to an amino ethyl glycine backbone. At present this family of compounds enjoys a well deserved popularity in biomedical research, due to a number of favorable biological and chemi cal properties of PNAs compared to conventional synthetic oligonucleot ides. PNAs are basically peptides, and are synthesized, purified and a nalyzed by traditional peptide chemistry, chromatography and mass spec trometry techniques. In the current report, we analyzed factors that i nfluence the elution behavior of 29 PNAs on reversed-phase high-perfor mance liquid chromatography using a water-acetonitrile-trifluoroacetic acid gradient elution system on C-18 columns. We found that increasin g the temperature from 25 degrees C to 55 degrees C resulted in improv ed peak shape and resolution. The retention times of the PNA analogs w ere dependent upon the length of the polymers with longer PNAs eluting later. Mixtures of PNAs with varying length, originating from ineffic ient monomer couplings during the polymer assembly, could be separated by single chromatographic runs. The retention time also depended upon the cytosine, thymine, adenine and guanine content of the polymers. T hese differences in the contribution to the retention times could be e xplained by simple hydrophobicity features of the monomer side chains at pH 1.8. Based on all data, a linear equation was generated which pr edicted the retention time of any synthetic PNA based on composition a nd length. Comparison of the predicted and observed retention times sh owed a remarkable reliability of the algorithm, (C) 1998 Elsevier Scie nce B.V. All rights reserved.