Erythropoietin is a signaling glycoprotein that controls the fundamental process of erythropoiesis, orchestrating the production and maintenance of red blood cells. As administrated clinically, erythropoietin has a polypeptide backbone with complex dishomogeneity in its carbohydrate domains. Here we describe the total synthesis of homogeneous erythropoietin with consensus carbohydrate domains incorporated at all of the native glycosylation sites. The oligosaccharide sectors were built by total synthesis and attached stereospecifically to peptidyl fragments of the wild-type primary sequence, themselves obtained by solid-phase peptide synthesis. The glycopeptidyl constructs were joined by chemical ligation, followed by metal-free dethiylation, and subsequently folded. This homogeneous erythropoietin glycosylated at the three wild-type aspartates with N-linked high-mannose sialic acid–containing oligosaccharides and O-linked glycophorin exhibits Procrit-level in vivo activity in mice.

Application of native chemical ligation logic to the case of an N-terminal proline is described. Two approaches were studied. One involved incorporation of a 3R-substituted thiyl-proline derivative. Improved results were obtained from a 3R-substituted selenol function, incorporated in the context of an oxidized dimer.

Native chemical ligation (NCL) is widely applicable for building proteins in the laboratory. Since the discovery of this method, many strategies have been developed to enhance its capability and efficiency. Because of the poor reactivity of proline thioesters, ligation at a C-terminal proline site is not readily accomplished. Here, we demonstrate that ligation at an N-terminal protein is feasible using the combined logic of NCL and metal free dethiylation (MFD).

The bicyclo[2.2.2]octenone skeleton is found in a number of natural products (Figure 1) including homodimers 1 2 and 2 (aquaticol), 3 and the hetero adduct chamaecypanone C (3). 4 While Diels-Alder cycloaddition of 2,4-cyclohexadienones and ortho-quinols with activated alkenes has frequently been used for the synthesis of bicyclo[2.2.2]octenones, 2,4-cyclohexadienones also have a high propensity to undergo spontaneous [4+2] We first investigated oxidation of the 2,5-disubstituted phenol carvacrol (4) using conditions previously reported for 2,4-dihydroxybenzaldehyde substrates enroute to the azaphilones 8 (Table 1, entry 1). In the event, reaction of 4 with a [(−)-sparteine] 2 Cu 2 O 2 (PF 6 ) 2 complex and N,N-diisopropylethylamine (DIEA) in CH 2 Cl 2 at −78 °C (16 h) afforded a mixture of the [4 +2] dimer (3S,10S)-1 in 25% isolated yield (99% ee by chiral HPLC analysis) and biaryl coupling product 5 (23%). 9 The backbone structure and absolute configuration of (3S, 10S)-1 were determined by comparison to NMR and CD spectral data reported for natural product (3R,10R)-1. 2, 10Further optimization studies revealed that use of LiHMDS to generate the phenolate in THF as solvent, 11 followed by oxidative dearomatization, cleanly afforded dimer 1 in 58% isolated yield (> 99% ee) with a trace amount of biaryl formation (Table 1, entry 2). Use of DIEA as base in THF (entry 3) also led to preferential formation of dimer 1. This result, along with reactions in propionitrile (entry 4) and acetone (entry 5), revealed a strong solvent effect for the reaction. Solvent and ligand effects reported in the literature 12 have generally been attributed to the equilibrium of binuclear copper-peroxo (P, μ-η 2 :η 2 -peroxodicopper(II)) and copper-oxo (O, bis(μ-oxo)dicopper(III)) complex forms. 13, 14 In the case at hand, the solvent effects may be rationalized by greater levels of the corresponding radical abstracting 15 [(−)-sparteine] 2 bis(μ-oxo)dicopper(III) (O) complex in CH 2 Cl 2 and the electrophilic μ-η 2 :η 2 -peroxodicopper(II) (P) complex in THF. Although evaluation of alternative counterions 16 (e.g. BF 4 − , OTf − , Cl − ) to favor formation of the corresponding P complex did not show substantial improvement over PF 6 −,10 we found that pre-formation of the phenolate with LiOH E-mail: porco@bu.edu. To evaluate the scope and limitations of this methodology, a number of phenol substrates were transformed into lithium phenolates and subsequently subjected to copper-mediated oxidative dearomatization ( Table 2). Use of 2,5-dimethyl and 2-methyl-5-tert-butyl substituted phenols 6 (entry 1) and 7 (entry 2), led to the production of [4+2] dimers 8 and 9 in high enantioselectivity, with a noticeable lower conversion observed for substrate 6. Substrate 10 (entry 3) bearing an electrondonating methoxy group at C5 was also successfully converted into dimer 11 after thermolysis of the crude monomer. 10 Attempted oxidation of 2,5-disubstituted phenols with electron-withdrawing groups at C5 gave poor conversion.10 Phenol 12 bearing...

Prolyl thioesters have shown significantly lower reactivities in native chemical ligation (NCL) in comparison to that of the alanyl thioester. This report describes a mild and efficient internal activation protocol of peptidyl prolyl thioesters in NCL without using any thiol-based additives, where the introduction of a 4-mercaptan substituent on the C-terminal proline significantly improves the reactivity of prolyl thioesters via the formation of a bicyclic thiolactone intermediate. The kinetic data indicate that the reaction rate is comparable to that of the reported data of alanyl thioesters, and the mechanistic studies suggest that the ligation of two peptide segments proceeds through an NCL-like pathway instead of a direct aminolysis, which ensures the chemoselectivity and compatibility of various amino acid side chains. This 4-mercaptoprolyl thioester-based protocol also allows an efficient one-pot ligation-desulfurization procedure. The utility of this method has been further demonstrated in the synthesis of a proline-rich region of Wilms tumor protein 1.