The concept of selective and reversible labelling

The first example of purification by selective labelling was described in 1968 for a peptide that was s)mthesized in solution (2). In 1976, Merrifield and co-workers (3) applied this concept to SPPS and demonstrated that peptides bearing an intentionally introduced Cys-Met N-terminal dipeptide can be purified by absorption onto an organometallic immobilized matrix. The native sequence lacking Cys-Met was recovered through a CNBr-mediated Met-X specific cleavage. 

Following these pioneering studies, several chromatographic probes have been proposed as a result of intentional design or modification of other procedures (for a review on the subject see refs 1 and 4). 

The Fmoc group is ideeiUy suited as a template molecule to develop chromatographic probes, because its urethane link with the amino acid N is stable to the harsh, acidic conditions used to cleave peptides from the resin support, but it is labile under mild basic conditions that do not harm the polypeptide chain (5). Furthermore, because the probe is added to the peptide chain at the end of synthesis, Fmoc-probes can aid the purification of peptides synthesized 

The first Fmoc-based chromatographic probe was described in 1978 by Merrifield (6) and contained negative charges provided by a sulpho group at position 2 of the fluorenyl moiety. The strongly acidic peptides thus derivatized were separated from unlabelled peptides by ion-exchange chromatography. 

Another Fmoc-based probe, recently developed, contains benzo-fused analogues of the Fmoc molecule. It can be used to purify peptides by either porous graphitized carbon chromatography (7) or RP-HPLC (8). 

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