A program for ligation at threonine sites: application to the controlled total synthesis of glycopeptides

Chen, Jin; Wang, Ping; Zhu, Jianglong; Wan, Qian; Danishefsky, Samuel J.

doi:10.1016/j.tet.2010.01.067

Cited by 131 publications

(83 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…One such strategy is the use of ligation junctions that contain thiolmodified residues that can be desulfurized later to give the native structure. This NCL-desulfurization method, since its invention for an Ala junction, [21] has captured the interest of several laboratories, including ours, and enabled performing NCL at Phe, [22] Val, [23,24] Leu, [25,26] Lys, [27,28] and Thr [29] ligation junctions. Moreover, this concept has been extended to include sugar-assisted ligation [30,31] for glycopeptide synthesis, and lysine-mediated peptide and protein ubiquitination.…”

Section: Introductionmentioning

confidence: 99%

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

et al. 2011

View full text Add to dashboard Cite

The HIV‐1 Rev protein is responsible for shuttling partially spliced and unspliced viral mRNA out of the nucleus. This is a crucial step in the HIV‐1 lifecycle, thus making Rev an attractive target for the design of anti‐HIV drugs. Despite its importance, there is a lack of structural, biophysical, and quantitative information about Rev. This is mainly because of its tendency to undergo self‐assembly and aggregation; this makes it very difficult to express and handle. To address this knowledge gap, we have developed two new highly efficient and reproducible methods to prepare Rev in large quantities for biochemical and structural studies: 1) Chemical synthesis by using native chemical ligation coupled with desulfurization. Notably, we have optimized our synthesis to allow for a one‐pot approach for the ligation and desulfurization steps; this reduced the number of purification steps and enabled the obtaining of desired protein in excellent yield. Several challenges emerged during the design of this Rev synthesis, such as racemization, reduced solubility, formylation during thioester synthesis, and the necessity for using orthogonal protection during desulfurization; solutions to these problems were found. 2) A new method for expression and purification by using a vector that contained an HLT tag, followed by purification with a Ni column, a cation exchange column, and gel filtration. Both methods yielded highly pure and folded Rev. The CD spectra of the synthetic and recombinant Rev proteins were identical, and consistent with a predominantly helical structure. These advances should facilitate future studies that aim at a better understanding of the structure and function of the protein.

show abstract

Section: Introductionmentioning

confidence: 99%

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

et al. 2011

View full text Add to dashboard Cite

show abstract

“…This step is typically followed by several protecting group manipulations, including hydrolysis of the thioacetate moiety, to enable protection of the thiol handle as an asymmetric disulfide or the corresponding S-trityl (Trt) derivative, both of which are compatible with standard conditions employed in Fmoc-strategy SPPS. [19] This overall synthetic strategy has been employed for the synthesis of several thiol-derived amino acids, including b-thiol phenylalanine (Phe), [20] g-thiol Val, [21] g-thiol lysine (Lys), [22] d-thiol Lys, [23] g-thiol threonine (Thr), [24] b-thiol leucine (Leu), [25] g-thiol Pro, [26] g-thiol glutamine (Gln), [27] and b-thiol arginine (Arg). [8d] In 2007, Crich and Banerjee prepared b-thiol Phe building block 3 [20a] for use in ligation-desulfurization chemistry in a 9-step synthesis from L-Phe methyl ester 4 (Scheme 3).…”

Section: Synthesis Of Thiol-derived Amino Acids Through Nucleophilic mentioning

confidence: 99%

“…[22b] In 2010, Danishefsky and co-workers reported the construction of a g-thiol Thr derivative for use in ligation-desulfurization chemistry. [24] This synthetic strategy employed a slight variation on the activation-displacement pathway for the installation of the crucial thiol moiety (Scheme 8). Beginning with protected vinylglycine derivative 42, treatment with meta-chloroperbenzoic acid (m-CPBA) first afforded epoxide 43 as a 5 : 1 mixture of syn/anti diastereomers, which were separated by chromatography.…”

Section: Synthesis Of Thiol-derived Amino Acids Through Nucleophilic mentioning

confidence: 99%

Synthetic Amino Acids for Applications in Peptide Ligation–Desulfurization Chemistry

Malins

Payne

2015

Aust. J. Chem.

View full text Add to dashboard Cite

Native chemical ligation is a powerful tool for the convergent assembly of homogeneous peptide and protein targets from unprotected peptide fragments. The method involves the chemoselective coupling of a peptide thioester with a peptide bearing an N-terminal cysteine (Cys) residue and is mediated by the nucleophilic Cys thiol functionality. A widely adopted extension of the technique for the disconnection of protein targets at alanine (Ala) ligation junctions has been the application of post-ligation desulfurization protocols for the mild removal of the Cys thiol moiety. Recently, attention has turned to the construction of synthetic amino acid building blocks bearing suitably positioned b-, g-, or d-thiol ligation auxiliaries with a view to expanding the scope of the ligation-desulfurization manifold. To date, several thiol-derived amino acids have been prepared, greatly increasing the generality and flexibility of chemoselective ligation technologies for the chemical synthesis of diverse protein targets. This review will highlight the current synthetic approaches to these important amino acid building blocks.

show abstract

“…Such methods are not ideal for the generation of analogs (30)(31)(32)(33). It was recognized that enhancement of the power of chemical synthesis for such objectives, including that of hPTH itself, could be accomplished by extending the reach of the underlying elegant concept of NCL (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). To this end, we report a pleasing and encouraging example wherein these recent findings have been pooled such that the molecule hPTH can be conveniently assembled from small synthetic peptide fragments.…”

mentioning

confidence: 94%

“…(2,3). However, given the relative scarcity of cysteine residues in nature, a clear impetus arises for the realization of new NCL capabilities (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17).…”

mentioning

confidence: 99%

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

Shang

Tan

Danishefsky

2011

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

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...

show abstract

A program for ligation at threonine sites: application to the controlled total synthesis of glycopeptides

Abstract: A method by which to accomplish formal threonine ligation has been developed. The method accomplishes ligations of two peptide domains. We have also demonstrated the ability to successfully ligate two independent glycopeptide domains.

Cited by 131 publications

References 55 publications

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

Synthetic Amino Acids for Applications in Peptide Ligation–Desulfurization Chemistry

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

Contact Info

Product

Resources

About