Insights into the Finer Issues of Native Chemical Ligation: An Approach to Cascade Ligations

Tan, Zhongping; Shang, Shiying; Danishefsky, Samuel J.

doi:10.1002/ange.201005513

Cited by 36 publications

(25 citation statements)

References 41 publications

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“…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

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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.

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Section: Introductionmentioning

confidence: 99%

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

et al. 2011

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“…4). To engage the third and fourth fragments in a kinetic leucine ligation (39), it was necessary to protect the N terminus of F3 as a thioleucine thiazolidine. In the event, fragment 6 and fragment 7 with an N-terminal thioproline failed to undergo kinetic ligation to form 8 with the predominant products arising from hydrolysis of the thioester, 9 (40), and macrocyclization of fragment 7 resulting in 10.…”

Section: Resultsmentioning

confidence: 99%

Chemical synthesis of the ATAD2 bromodomain

Creech

Paresi

2014

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

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Due to the emerging importance of the bromodomain binding region in the study of epigenetic effectors and the vast implications for a wide variety of human disease, the bromodomain region of human ATPase family AAA+ (ATPases associated with diverse cellular activities) domain-containing protein 2 (ATAD2) was targeted for chemical synthesis. The ATAD2 bromodomain (130 aa) was divided into five strategic fragments to be assembled using native chemical ligation with a focus on maximal convergency and efficiency. The fragments were assembled with one cysteine and three thioleucine ligations, unveiling the native alanine and leucine amino acids at the ligation points following metal-free dethiylation. Synthetic highlights of the study are a photolabile dimethoxynitrobenzyl-protected glutamic acid side chain used to impede hydrolysis of the C-terminal Glu-thioester, a thiazolidine-protected thioleucine, and an efficient assembly of three fragments in a single reaction vessel with dual-mode kineticstandard chemical ligation. With a focus on material throughput and convergency, the five peptide fragments were assembled into the native ATAD2 bromodomain region with a total of three HPLC events in 8% overall yield from the fragments.solid-phase peptide synthesis | desulfurization | peptide retrosynthesis

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“…The fully protected peptides to be ligated were synthesized by Fmoc chemistry on a 0.05 mmol scale. The preleucine and prevaline surrogates were attached to the N termini of the fully protected peptides by 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate (HATU) coupling (17). The peptide fragments, bearing C-terminal thioesters, were prepared from the fully protected peptides using the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI)-mediated amide formation reaction under the nonepimerizing conditions developed by Sakakibara et al (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

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confidence: 94%

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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.

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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...

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Insights into the Finer Issues of Native Chemical Ligation: An Approach to Cascade Ligations

Cited by 36 publications

References 41 publications

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

Chemical synthesis of the ATAD2 bromodomain

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

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