Application of the logic of cysteine-free native chemical ligation to the synthesis of Human Parathyroid Hormone (hPTH)

Abstract

The power of chemical synthesis of large cysteine-free polypeptides has been significantly enhanced through the use of nonproteogenic constructs which bear strategically placed thiol groups, enabling native chemical ligation. Central to these much expanded capabilities is the specific, radical-induced, metal-free dethiolation, which can be accomplished in aqueous medium. Anticipating this type of problem, our laboratory (9, 11, 14, 17) and others (5-7, 10) have developed strategies by which to accomplish ligation at a range of noncysteine amino acid residues. The logic of our approach is outlined in Fig. 1. The N-terminal peptide, D, to be ligated is equipped with an N-terminal sulfurbearing amino acid surrogate, A, itself prepared by synthesis and coupled, by a suitable method, to the peptide B. In this way, N-terminal ligation candidate D is ready for coupling with C-terminal acyl donating peptide C (usually a thioester). Ligation product E can be maintained as such, thereby affording a specifically placed thiol group in a nonnatural context. Theoretically, de-thiolation of E will produce F wherein the "R" group had been governed by the synthetically derived A. In principle, the strategy shown in Fig. 1 is of universal scope.Studies using a variety of model peptides have demonstrated the fragment-coupling capability of the cysteine-free strategy. Here, in this paper, we seek to examine the general applicability of this strategy to the total synthesis of cysteine-poor proteins. Human Parathyroid Hormone (hPTH) is chosen to serve as a model molecule because of its representativeness in terms of amino acid composition and its therapeutic value. hPTH is a biological messenger that is secreted by the parathyroid glands as a peptide containing 84 amino acids (18,19). Upon binding to its receptor, hPTH can enhance the concentration of calcium (Ca 2þ ) in the blood (20). Because of their important physiological roles, hPTH and one of its fragments, hPTH (1-34), are now given (by subcutaneous injection) for the treatment of hypoparathyroidism and osteoporosis in men, as well as for postmenopausal women at high risk of fracture (21-23). Not unlike most other hormone drugs, the recombinant hPTH therapeutics have very short half-lives in the human body and need to be administered at least once a day (24, 25). The need for continuous daily subcutaneous injection is an obvious disadvantage which serves to compromise use of the hormone. Clearly, the production of more stable forms of hPTH, where "pharmacolability" is attenuated without undercutting biological activity, would be of great interest (26). It is also of interest to interrogate the consequences of employing nonproteogenic inserts (27). While this goal can be accomplished by cleverly designed recombinant methods, chemical synthesis could well be more convenient for servicing the initial production of probe structures for such structure-activity relationship (SAR) evaluations (28,29). Previously, the chemical synthesis of hPTH required either the solid phase synthes...

Synthetic Amino Acids for Applications in Peptide Ligation–Desulfurization Chemistry

“…Spontaneous folding of the synthetic protein was confirmed using circular dichroism, and the chemically prepared hPTH was also shown to be of higher purity than the recombinantly produced hPTH used as a reference sample. [45] These results further validate the utility of chemical synthesis, particularly ligation-desulfurization chemistry, for the preparation of well defined protein samples for use in biological assays.…”

“…Just as the pioneering synthetic work of Sir John Cornforth enabled the careful investigation of the mechanisms of enzyme catalysis, the programmed construction of homogeneous peptide and protein targets using the tools afforded by chemical synthesis will no doubt prove an invaluable resource for understanding the intricate relationship between protein structure and function. Madanin-1 (30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46) Madanin-1 (49-60)…”

“…[44] Another powerful example of the disconnection of proteins using a combination of ligation-desulfurization at Cys and thiol-derived amino acid building blocks was provided by Danishefsky and co-workers for the preparation of the 84-amino acid, Cys-free human parathyroid hormone (hPTH) 115 (Scheme 19). [45] By employing a convergent ligation-global desulfurization protocol, the authors of this study accessed the target protein 115 through ligation at Cys, b-thiol Leu, and g-thiol Val from peptide fragments 116-119. First, thiophenyl thioester 116 was utilised in a kinetically controlled ligation reaction [7] with bifunctional peptide 117, containing an N-terminal b-thiol Leu residue and a C-terminal alkyl thioester to generate hPTH(1-38) 120.…”

Modern Extensions of Native Chemical Ligation for Chemical Protein Synthesis

“…As discussed, these building blocks are used in combination with ligation at Cys or ligation-desulfurization at Ala for the construction of complex proteins. A powerful example of the scope of ligation using a combination of these building blocks was provided by Danishefsky and coworkers in 2011 with the construction of human parathyroid hormone (hPTH) 131 using a convergent ligation-global desulfurization strategy employing Cys, β-thiol Leu, and γ-thiol Val ligation disconnections (Scheme 33) [133]. It was envisaged that the target hPTH protein could be synthesized from peptide fragments 132-135 by combining iterative ligation technologies for the construction of proteins in both the N-to-C and C-to-N directions (Scheme 34).…”

Protein chemical synthesis by serine and threonine ligation

“…In an effort to expand the scope of NCL, a number of research groups have developed variants, wherein β-or γ-thiol containing amino acids are temporarily installed on the peptide N terminus, thus promoting NCL-like ligation. Subsequent desulfurization serves to restore the natural amino acid at the site of ligation (23)(24)(25)(26). Whereas this NCLdesulfurization strategy has been widely used in protein synthesis, a menu of complementary thiol-independent ligation approaches (27-34) may offer new opportunities for convergent protein synthesis.…”