A strategy for the synthesis of sulfated peptides.

The sulfation of tyrosine residues is a posttranslational modification that occurs in all eukaryotes.[1] This ubiquitous modification has been identified on many secretory proteins with diverse biological activities. Tyrosine O-sulfate (Tyr(SO3H)) residues often are important for facilitating specific protein±protein interactions.[2] However, there is little information on the molecular basis for the importance of tyrosine sulfation.[3] Characterization of the molecular interactions of Tyr(SO3H) has been hampered by lack of access to the necessary quantities of sulfated proteins and peptides. We report a method for the efficient production of sulfated peptides using a new protecting-group strategy. Although several strategies for the synthesis of sulfated peptides have been reported,[4] a general, efficient approach is still being sought. The primary obstacle in the synthesis of sulfated peptides is the predilection of sulfotyrosine to undergo desulfation under acidic conditions. Sulfotyrosinecontaining peptides generally decompose rapidly in the presence of trifluoroacetic acid, the standard reagent used for side-chain deprotection and cleavage from the solid supports employed in Fmoc-based (Fmoc1⁄4 (9H-fluoren-9ylmethoxy)carbonyl) solid-phase peptide synthesis (FmocSPPS). The sulfate group counterion has been shown recently to have a dramatic influence on the stability of sulfated peptides toward acidic treatment.[5] Alternatively, acid-sensitive substrates can be released from the 2-chlorotrityl(2-Clt-) derivatized resin without significant decomposition.[6] Indeed, sulfated peptides may be liberated from this support without significant accompanying desulfation.[7] Thus, the most attractive published method for sulfated peptide synthesis involves stepwise incorporation of FmocTyr(SO3Na)OH into the growing peptide.[8] Unfortunately, couplings using this salt and subsequent reactions to elongate the resulting peptide can be sluggish.[9] Moreover, our attempts to synthesize peptides containing multiple sulfotyrosine residues were plagued by poor resin swelling and the need for extended coupling times. Given the lack of general methods for the synthesis of sulfated peptides, we set out to develop a new approach. For the greatest flexibility, we employed a postsynthetic sulfation strategy. We envisioned that selective unmasking and sulfation of the desired tyrosine residues could be conducted on the solid support. Incorporation of orthogonally protected tyrosine derivatives at the peptide-assembly stage would allow the synthesis of multiply sulfated peptides with various sulfation patterns. Additionally, a solid-phase sulfation step would avoid the complicated purification of partially protected, sulfated peptide intermediates and, in the case of polysulfated peptides, mixtures with different sulfation patterns. Excess reagents could also be used to drive the sulfation reaction to completion. With this strategy, both sulfated and unmodified peptides could be produced from the same peptide synthesis. To implement this approach, we required a protecting group for the tyrosine phenol that is stable to the conditions used in Fmoc-SPPS, yet can be unmasked selectively and quantitatively under mild conditions. Strongly acidic or basic conditions for protecting-group removal must therefore be avoided. We also wished to circumvent the laborious process of cleavage and characterization of intermediates to confirm the success of the deprotection reaction. We therefore sought to design a protecting group that could be removed under mild conditions and that possesses a characteristic spectroscopic signal by which to monitor the deprotection step. We identified Loubinoux©s little-used azidomethyl (Azm) phenol protecting group as an excellent candidate for tyrosine protection.[10] The mild conditions required for azide reduction were expected to release the phenol group without cleavage of the Clt-ester bond or modification of oxidation-sensitive amino acid residues such as cysteine and methionine.[11] We therefore pursued the synthesis of FmocTyr(OAzm)OH (Scheme 1). The synthesis of target 1 proceeds from BocTyrOMe (2, Boc1⁄4 tert-butyloxycarbonyl). Attempted alkylation of 2 with dibromomethane or bromochloromethane affords only the dimeric tyrosine methylene acetal, even when high concentrations of the electrophile are employed. Alkylation with an alternate, bifunctional methylene equivalent, chloromethyl methylsulfide, proceeds in good yield (Scheme 2). Initial COMMUNICATIONS