Synthesis of Cyclic Peptides and Cyclic Proteins via Ligation of Peptide Hydrazides

Zheng, Ji‐Min; Tang, Shan; Guo, Ye; Chang, Haonan; Liu, Lei

doi:10.1002/cbic.201100580

Cited by 95 publications

(76 citation statements)

References 82 publications

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“…2A). Our initial version is designed to work with thioesters prepared using the hydrazide method 66–69 , so segments containing “incompatible” C-terminal residues are not included in the segment list (Asp, Glu, Asn, Gln, and Pro). Specifically, Asp/Glu are excluded because of their potential for thioester migration to the side chain 70 , although recent work has suggested that this is a pH-dependent reaction more prevalent in Asp thioesters 71 .…”

Section: Discussionmentioning

confidence: 99%

“…Aligator is centered on NCL using the Fmoc-compatible hydrazide method for generating peptide thioesters 66–69 , but should be generally applicable to other chemoselective ligation reactions (e.g., KAHA 92 and Ser/Thr ligation 93 ). The programs described here are freely available for non-commercial use on Github: https://github.com/kay-lab.…”

Section: Discussionmentioning

confidence: 99%

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Aligator: A computational tool for optimizing total chemical synthesis of large proteins

Jacobsen

Erickson

Kay

2017

Bioorganic & Medicinal Chemistry

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The scope of chemical protein synthesis (CPS) continues to expand, driven primarily by advances in chemical ligation tools (e.g., reversible solubilizing groups and novel ligation chemistries). However, the design of an optimal synthesis route can be an arduous and fickle task due to the large number of theoretically possible, and in many cases problematic, synthetic strategies. In this perspective, we highlight recent CPS tool advances and then introduce a new and easy-to-use program, Aligator (Automated Ligator), for analyzing and designing the most efficient strategies for constructing large targets using CPS. As a model set, we selected the E. coli ribosomal proteins and associated factors for computational analysis. Aligator systematically scores and ranks all feasible synthetic strategies for a particular CPS target. The Aligator script methodically evaluates potential peptide segments for a target using a scoring function that includes solubility, ligation site quality, segment lengths, and number of ligations to provide a ranked list of potential synthetic strategies. We demonstrate the utility of Aligator by analyzing three recent CPS projects from our lab: TNFα (157 aa), GroES (97 aa), and DapA (312 aa). As the limits of CPS are extended, we expect that computational tools will play an increasingly important role in the efficient execution of ambitious CPS projects such as production of a mirror-image ribosome.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Aligator: A computational tool for optimizing total chemical synthesis of large proteins

Jacobsen

Erickson

Kay

2017

Bioorganic & Medicinal Chemistry

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“…在我们近期关于蛋白质化学合成的研究中 [17] , 发现使用含有 N 端半胱氨酸的多肽酰肼作为原料, 可以高效率地合成含 5～42 个氨基酸的环肽(图 2a) [18] . 该方法的原理是 C 端酰肼在 NaNO 2 的活化条件下原位生成硫酯, 通过分子内硫酯交换生成大环内硫酯, 再经过自发的 S-to-N 酰基迁移得到环化产物.…”

Section: Lactobacillus Helveticus 中分离得到的环(L-脯-l-酪-l-脯 -L-异亮 ) 四肽具 unclassified

“…以环(丙-酪-精-苯丙) [14] 为模型化合物, 我们首先合成四肽酰肼 H-Cys-Tyr-Arg-Phe-NHNH 2 (1a), 再利用已优化出的条件进行环化测试 [18] (图 3). 实验表明, 浓度为 1.0 mmol/L 的未经纯化的四肽酰肼 1a (ESI found .…”

Section: 四肽酰肼环化反应的发现unclassified

Synthesis of Cyclic Tetrapeptides via Ligation of Peptide Hydrazides

Tang¹,

Zheng²,

Yang³

et al. 2012

Acta Chim. Sinica

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Naturally occurring or man-made cyclic tetrapeptides have unique rigid skeletons and therefore, represent an interesting class of candidate bioactive molecules in drug discovery. However, efficient chemical synthesis of cyclic tetrapeptides often presents a difficult problem due to the large strain involved in this category of target compounds. To overcome this problem we describe in the present study the use of peptide hydrazides for the preparation of highly strained 12-membered all-L cyclic tetrapeptides. The new synthetic route starts with the easy Fmoc solid phase synthesis of a linear N-Cys-tetrapeptide hydrazide precursor. Upon quick activation by NaNO 2 at pH 3 and -10 ℃ for 20 min followed by treatment with a pH 7 buffer containing an external thiol (4-mercaptophenylacetic acid, 40 equiv.) at room temperature, the N-Cys-tetrapeptide hydrazide precursor is converted in situ to an N-Cys-tetrapeptide thioester. This thioester undergoes a fast intramolecular thioester exchange reaction to generate a 13-membered thiolactone intermediate. Then an S-to-N acyl shift is expected to take place to create an amide bond, which affords the desired cyclic tetrapeptide in a modest overall yield (ca. 40%). Finally, desulfurization of the cyclic peptide product can be carried out to produce the target cyclic tetrapeptide that does not contain any Cys residue. Through detailed 1 H, 13 C, and TOSCY NMR and HPLC analyses, it is found that the new method for tetrapeptide synthesis can be carried out at relatively high substrate concentrations (0.8-1.0 mmol/L) without causing the formation of much cyclic octapeptide byproduct. Our test also showed that the reaction can generate the desired cyclic tetrapeptide in an epimerization free manner. By using the new method we have successfully prepared several cyclic tetrapeptides including cyclo(Ala-Leu-Ala-Leu), cyclo(Ala-His-Gly-Trp), and cyclo(Ala-Val-Gly-Ile) in 18%-25% overall yields. We expect that our method of cyclic tetrapeptide synthesis may find applications in the development of cyclic peptide libraries for bioactivity screening.

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“…For synthesizing DapA, we used a recently developed method to join peptide segments via native peptide bonds formed between a peptide with a C-terminal hydrazide (for selective conversion to a thioester) and a peptide with an N-terminal Cys (40)(41)(42)(43). We selected this chemistry because of the convenient route to peptide hydrazides via Fmoc SPPS (less hazardous and more compatible with acid-sensitive modifications than Boc chemistry), the robustness of the native chemical ligation reaction (30,44), and the ease of carrying out convergent protein assembly (vs. linear C-to N-assembly).…”

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confidence: 99%

Synthesis and folding of a mirror-image enzyme reveals ambidextrous chaperone activity

Weinstock

Jacobsen

Kay

2014

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

126

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Mirror-image proteins (composed of D-amino acids) are promising therapeutic agents and drug discovery tools, but as synthesis of larger D-proteins becomes feasible, a major anticipated challenge is the folding of these proteins into their active conformations. In vivo, many large and/or complex proteins require chaperones like GroEL/ES to prevent misfolding and produce functional protein.The ability of chaperones to fold D-proteins is unknown. Here we examine the ability of GroEL/ES to fold a synthetic D-protein. We report the total chemical synthesis of a 312-residue GroEL/ESdependent protein, DapA, in both L-and D-chiralities, the longest fully synthetic proteins yet reported. Impressively, GroEL/ES folds both L-and D-DapA. This work extends the limits of chemical protein synthesis, reveals ambidextrous GroEL/ES folding activity, and provides a valuable tool to fold D-proteins for drug development and mirror-image synthetic biology applications.

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Synthesis of Cyclic Peptides and Cyclic Proteins via Ligation of Peptide Hydrazides

Cited by 95 publications

References 82 publications

Aligator: A computational tool for optimizing total chemical synthesis of large proteins

Aligator: A computational tool for optimizing total chemical synthesis of large proteins

Synthesis of Cyclic Tetrapeptides via Ligation of Peptide Hydrazides

Synthesis and folding of a mirror-image enzyme reveals ambidextrous chaperone activity

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