INTRODUCTION

The AKR/Gross murine leukemia viruses （MuLV） are C-type retroviruses that cause the development of leukemia in AKR （H-2k） mice.1 Type-specific antiretroviral cytotoxic T-lymphocyte （CTL） responses have been reported that allow differentiation between infection by the AKR/Gross MuLV and other MuLV.1 A 10-mer derived from the C-terminal portion of the AKR/Gross MuLV CTL epitope has been used as a model “difficult” peptide for this study because it cannot be synthesized under standard conditions.2,3 UV monitoring of Fmoc deprotection allows the optimization of difficult syntheses. Typically, Fmoc removal is accomplished with piperidine, although other reagents such as piperazine may also be used. Piperidine reacts with the Fmoc-protected amino acid to remove the Fmoc group （which is converted to dibenzofulvene） yielding the free amine following decarboxylation （Figure 1）. The dibenzofulvene can then react with piperidine to form the dibenzofulvene-piperidine adduct （DBF-pip）.

Figure 1. Fmoc removal by piperidine.

The DBF-pip molecule shows a distinct UV absorbance at 301 nm that is easily discernible from the background absorbance of the other reagents present in the deprotection reaction.Thus, by monitoring the UV absorbance at 301 nm, the relative efficiency of a given deprotection reaction can be measured. When the UV spectrometer is connected to the Liberty/Liberty1, all liquid waste from the deprotection step flows through the UV flow cell, and the absorbance is measured. If the absorbance detected after the second （3 min） deprotection is above the set threshold, the deprotection is considered failed, and additional 3 minute deprotections are performed until either a passing value is obtained or a maximum number of deprotections has been reached. In theory, a difficult deprotection will correspond to a difficult coupling. When using the UV Monitoring Option, the coupling conditions can be modified （double coupling, extended coupling time, and/or capping） on failure to compensate for this difficulty.

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