RP-HPLC using lipophilic chromatographic probes

In a comparative study, several hydrophobic probes differing in the type of substitution at position 4 of 1 were prepared (12). Parameters evaluated included stability of the probe during coupling and acid deprotection, RP-HPLC separation between derivatized and underivatized molecules, and time required for probe removal. Probe 2 offered the best compromise between all these parameters it is easily converted to an activated form (e.g. 2a) by using the same chemistry as for activation of the Fmoc molecule. If the reaction between 2a and the peptidyl-resin is carried out immediately after completion of chain assembly, the base-catalysed reaction requires 2-3 h. Longer reaction times might be necessary if derivatization is not carried out on freshly prepared polypeptides. In this case it is advisable to store the peptidyl-resin in an 

The probe and the probe-peptide link are stable to acids and in the presence of the most widely used scavengers. However, both scavengers and acids must be of high quality if side reactions at the Fmoc group and the peptide chain are to be avoided. In general, the solubility profile of large polypeptides is not significantly altered by the presence of lipophilic probe 2. 

The homogeneity and yields of the final preparation depend strongly on the length of the polypeptide chain and on the conditions used for both synthesis and cleavage from the resin support. Under optimized conditions. 

In a comparative study we found that yields of a 100 amino acid-long protein purified with probe 2 are 5-15% and about 10-times those from a conventional multistep purification scheme (13). Under optimized conditions, the entire process of derivatization, cleavage, semi-preparative purification, fraction analysis, probe removal, and further semi-preparative chromatography requires about one week using standard laboratory equipment. 

Attachment of lipophilic probe 2 to the N-terminus of the peptidyl-resin 

---