Shifting Native Chemical Ligation into Reverse through N!S Acyl Transfer are phosphorylation and glycosylation. Phosphorylation has been shown to be particularly important in the regu- lation of signal transduction pathways,[5c,33] while glycosy- lation plays a key role in diverse biological processes such as protein folding, cell–cell recognition and adhesion, immune defense, and improving the circulatory lifetime of secreted proteins.[34] Phosphorylation is commonly O- linked through Ser, Thr, or Tyr, and complex carbohy- drates (glycans) are most commonly N-linked through Asn, or O-linked through Ser and Thr. The site of modifi- cation is not under direct genetic control and can be pres- ent at one or more residues.[35] For example, erythropoie- tin (EPO), a glycoprotein hormone, contains three N-gly- cosylation sites (Asn24, Asn38, and Asn83) and a single O- glycosylation site (Ser126).[36] Asparagine residues are gly- cosylated in response to a required Asn–Xaa–Ser/Thr motif[36c] whereas the sites of O-glycosylation and phos- phorylation are dictated more by sequence context.[37] Due to substrate/acceptor competition between the proc- essing enzymes that make up the structure of the glycans, heterogeneously glycosylated structures (glycoforms) are observed.[38] Despite the development of cell culture strat- egies that can achieve human-like glycosylation, the pro- teins are isolated as heterogeneous mixtures with respect to glycan structure and glycosylation site occupancy.[39] Phosphoproteins can often be hyperphosphorylated in substoichiometric fashion, which greatly complicates anal- yses. It is essential to have homogenous forms of these proteins to ensure reliable studies of structure–activity re- lationships. Consequently, much interest has evolved around the synthesis of homogeneous glycoproteins and phosphoproteins. Because solid phase peptide synthesis is limited to pep- tides with up to approximately 50 amino acid residues,[40] NCL is the tool that has been most successful in accessing homogeneous glycoproteins[6b,41] and phosphopro- teins.[5c,42] Unlike in traditional bioconjugation strategies for protein modification,[43] the native glycopeptide struc- tures can be maintained using NCL or using the thioester method developed by Aimoto.[2a,44] However, phospho- peptide and glycopeptide thioesters can be more difficult to prepare due to the increased chemical complexity. During peptide synthesis, the appropriate PTM is usually introduced as a suitably protected amino acid building block, and when this is located in the N-cysteinyl peptide fragment, synthesis can be extremely straightforward. Often, the real challenge encountered is the synthesis of the PTM containing thioester. A major factor contributing to the challenge of post- translationally modified peptide thioester synthesis is the fragile nature of these moieties. They are generally in- compatible with traditional Boc-based chemistry during which they are repeatedly exposed to TFA and then HF.[45] In an attempt to circumvent this problem, an Fmoc-based strategy is most frequently employed. Most PTM-containing thioesters have been prepared using the acylsulfonamide “safety-catch” linker (Scheme 1). How- ever, manipulations with this linker, including loading and cleavage, can be difficult to monitor in the absence of a double linker strategy,[5f,46] and “off target” alkylation can occur.[42] Regardless of this, the Fmoc strategy is still generally preferred for glycopeptides and phosphopepti- des, and the mildly acidic to neutral conditions employed within N!S acyl transfer methods are highly comple- mentary to the acyl sulfonamide approach. 4.2. Phosphopeptide Thioester Synthesis Muir and co-workers previously used the sulfonamide linker in the semisynthesis of the hyperphosphorylated transmembrane kinase TbR-1.[5c,42] The phosphorylated GS region of the protein H– TTLKDLIYDM*pTTpSGpSGpSGLPL–SBn (residues 175–195, 13) were synthesized using an optimized proce- dure in a yield of 4.6 % (Scheme 10). In order to be suc- cessful, the methionine residue was substituted for nor- leucine to preclude a previously observed side reaction between methionine and iodoacetonitrile during resin ac- tivation. The thioester was subsequently ligated to the re- mainder of the TbR-1 cytoplasmic domain, obtained by recombinant methods. Macmillan and co-workers also attempted to use the safety-catch linker but experienced low yields of ~ 2 % in the synthesis of a short thioester (H–LETVSpTQELY– SBn, 14).[47] Upon use of N!S acyl shift, the thioester 15 (H–LETVSpTQELG–SCH2CH2SO3H), which included a Y27G substitution to facilitate thioester formation, was synthesized in an unoptimized overall yield of 13 %.[48] The phosphopeptide thioester was ligated to a recombi- nant Chk2 kinase ForkHead Associated (FHA) domain to produce a stoichiometrically phosphorylated protein that formed a characteristic phosphate-dependant homo- dimer. 4.3. Glycopeptide Thioester Synthesis Using the sulfonamide linker to produce a thioester and NCL, Shin et al.[5b] were able to synthesize an 82-residue antibacterial glycopeptide containing two O-linked GalNAc residues at Thr10 and Thr54. The thioester frag- ment 16 (diptericin residues 1–24) was isolated in 21 % yield (Scheme 11). A similar approach also allowed Mac- millan and Bertozzi to prepare homogeneous semisyn- thetic glycoforms of mouse glycosylation-dependant cell adhesion molecule 1 (GlyCAM-1) containing O-linked sugars.[13] Later, Unverzagt and co-workers were able to synthe- size a peptide thioester containing an N-linked biantenna- ry heptasaccharide to make a fragment of RNase B. The safety-catch linker was activated with trimethysilyldiazo- methane and the glycopeptide thioester was cleaved from the resin with ethyl 3-mercaptopropionate and sodium Isr. J. Chem. 2011, 51, 885 – 899 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.ijc.wiley-vch.de 895