A new amine protecting group which can be used orthogonally with both Fmoc and Boc protection is reported; by employing lysine protected appropriately as the branching motif, a 34 residue di-epitopic peptide has been constructed by continuous flow solid phase peptide synthesis.Currently solid phase peptide synthesis (SPPS) of multiple antigen peptide systems' (MAPs) containing different peptide sequences, e.g. both B and T cell epitopes, is only feasible using Merrifield methodology.2t In the preceding communication3 we described a novel amino protecting group, Dcm 1, and its application in continuous flow SPPS. We now report that the methyl derivative 2, unlike its precursor, is stable to the deprotection conditions for the Fmoc group and can therefore be used as orthogonal protection to Fmoc in SPPS of branched peptides, in particular MAPs.Fmoc-Lys-OH reacts readily with 2-acetyldimedone to afford the NE-1-(4,4-dime thyl-2,6-dioxocyclohexylidene)e thy1 (Dde) derivative 3.$ The lability of the Dde group to 2% v/v hydrazine in DMF under continuous flow conditions was demonstrated with the model peptide Dde-Ala-Ala-Pepsyn KA( 100). Deprotection which results in the formation of 3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazole 4 (Scheme 1) and can be monitored by the absorption at 300 nm was complete after 3 min. Exposure of Fmoc-Lys(Dde)-OH to neat trifluoroacetic acid (TFA) followed by periodic monitoring of the amine concentration using the picrylsulfonic acid amine test5 modified for quantitative analysis resulted in only ca. 0.1% loss of Dde after 24 h. Stability of Dde to 20% piperidine in DMF under continuous flow conditions was investigated using the model peptide Boc-Ala-Lys(Dde)-AlaTyr(But)-Gly-Gly-Phe-Leu-Polyhipe SU 500 amide resin. A loss of ca. 3.0 and 6.5% was observed after 2 and 4 h respectively as monitored by HPLC analysis of the cleaved peptide. A contact time of 2 h with 20% piperidine represents 17 x 7 min Fmoc deprotection cycles, allowing the synthesis of medium size peptide chains without significant loss of the Dde group. 9 '0 1; R' = H, R2 = CH(X)C02H, a-amino acid 2; R' = Me, F? = CH(X)C02H, a-amino acid 3; R' = Me, R2 = [CH2],CH(Fmoc-NH)C02H

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