Review D. Macmillan et al. Scheme 10. Fmoc phosphopeptide thioester synthesis; reagents and conditions i) a) Fmoc SPPS; b) ICH2CN, DIPEA; c) BnSH, DIPEA; d) TFA, scavengers. ii) a) Fmoc SPPS; b) ICH2CN, DIPEA (twice); c) BnSH, PhSH; d) TFA, scavengers. iii) 10 % w/v MESNa, 0.1 m Na phosphate pH 5.8, 55 8C, 72 h. thiophenolate. The side-chain protecting groups were then removed under acidic conditions with TFA and pu- rification of the thioester yielded 46 %.[5f] However, since TFA cleavage conditions have been found to be incom- patible with certain glycosidic linkages (for example, fu- cosidic linkages in EPO), Danishefsky et al.[49] explored the possibility of incorporating a glycan after peptide syn- thesis via Lansbury aspartylation.[50] An unsymmetrical aryl–alkyl disulfide was coupled to a peptide to form 17, which was subsequently glycosylated with a chitobiose- derived glycosylamine to give 18. After reduction under ligation conditions, it was proposed that 18 underwent in- tramolecular O!S acyl shift to generate thioester 19 in situ, which was then able to participate in NCL. Ozawa et al. were able to use an N!S acyl shift to generate glycopeptide thioesters.[51] After installation of N-alkyl cysteine (NAC) on CLEAR amide resin, the rest of the peptide chain was elongated using standard Fmoc protocols. The carbohydrate moieties were introduced using the appropriate glycoaminoacid building block. After complete assembly, the product 20 was cleaved from the solid support and dissolved in 5 % MPA solu- tion, facilitating conversion to the thioester 21 in 2 days. This proceeded without significant decomposition of car- bohydrate appendages, with an overall yield of 20 % (cal- culated from initial resin loading). Masania et al. were also able to apply N!S acyl trans- fer in the context of glycopeptides. Glycosylation-depend- ant cell adhesion molecule 1 (GlyCAM-1) residues 75–82 H–GSSQLEETSGC–OH (22) were assembled as a model O-linked glycopeptide incorporating Fmoc– GalNAc(OAc)3-Thr–OH at position 82.[48] An analogue of cell surface glycopeptide CD52 (24) was also prepared, substituting Asn3 with azidohomoalanine (Aha). This ena- bled the attachment of N-acetyl glucosamine through an unnatural triazole linkage formed through CuI-mediated cycloaddition.[52] After being cleaved from the solid sup- port, both glycopeptides were subjected to thioesterifica- tion conditions (10 % w/v MESNa, 0.1 m Na phosphate; pH 5.8, 55 8C for 72 h) to generate thioesters 23 (10 %) and 25 (18 %), respectively. More recently we investigated N!S acyl shift in the preparation of N-linked glycopeptides.[27] EPO residues 22–28 H–AEN(GlcNAc(OAc)3)ITTGC (26) were assem- bled on NovaSynTGT resin and subjected to thioesterifi- cation (10 % w/v MESNa, 0.5 % w/v TCEP, 0.1 m Na Phosphate; pH 5.8, 55 8C for 48 h) to give thioester 27 with a good overall yield of 21 %. Conducting the reac- tion at pH 5.8 was important, since at lower pH the acetyl esters were cleaved, giving rise to a complex mixture of partially deacetylated compounds. It would have been ex- tremely convenient if quantitative deacetylation had ac- companied thioester formation, but deacetylation pro- ceeded slowly (at pH 2) and longer reaction times result- ed in increased thioester hydrolysis. Although only a single monosaccharide was introduced, glycopeptide/pro- tein remodelling strategies can elaborate these simple gly- copeptides into more complex structures.[53] Overall, use of N!S acyl transfer in the formation of post-translationally modified peptide thioesters has dem- onstrated versatility in application to phosphoproteins, glycoproteins, and their analogues. We have also shown the tolerance of a variety of non-peptidic linkages to this reaction. Moreover the efficiency of this process is re- flected in good yields without the requirement for special- ized resins, linkers, and unnatural building blocks. 896 www.ijc.wiley-vch.de 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Isr. J. Chem. 2011, 51, 885 – 899