Chemical Protein Synthesis Using a Second-Generation …
We have developed approaches to synthesize unnatural proline analogues in a manner to allow control of structure and the introduction of novel functional groups within proteins, including groups for spectroscopic probes and for bioorthogonal chemistry.
Chemical Protein Synthesis Using ..
Chemical synthesis is the most powerful approach for constructing proteins of novel design and structure, allowing for variation of covalent structure without limitations.
The ability to generate small molecule mimics of protein recognition elements permits their use as chemical probes of protein-protein interactions.
Numerous diseases, including Alzheimer's disease, Parkinson's disease and spongiform encephalopathies (i.e.
Chemical protein synthesis is a field ..
This Account documents both the state of the KAHA ligation and the challenges in identifying, inventing, and optimizing new reactions and building blocks needed to interface KAHA ligation with Fmoc solid-phase peptide chemistry. With these challenges largely addressed, peptide segments ready for ligation are formed directly upon resin cleavage, facilitating rapid assembly of four to five segments into proteins. This work sets the stage for applications of the KAHA ligation to chemical biology and protein therapeutics.
Chemical synthesis of proteins - [PDF Document]
Thus far, most research on chemically synthesized protein molecules () has been focused on relatively small proteins in the size range of 50 to ≈150 aa (i.e., made from two or three peptide segments). This size limitation is the result of two phenomena: (i) practical constraints on the size of the unprotected peptide segment building blocks () and (ii) technical challenges in the chemical ligation of more than two or three peptide segments. Even highly optimized stepwise solid-phase peptide synthetic procedures are limited to ≈50 amino acid residues for the practical preparation of high-purity unprotected peptides (). Thus, the chemical synthesis of a protein of typical size (≈300 aa) () would require the use of at least six synthetic peptide segments as building blocks.
Chemical Synthesis of Proteins | Annual Review of …
The broad utility of native chemical ligation (NCL) in protein synthesis has fostered a search for methods that enable the efficient synthesis of Cterminal peptide-thioesters, key intermediates in NCL. We have developed an acylurea (Nbz) approach for the synthesis of thioester peptide precursors that efficiently undergo thiol exchange yielding thioester peptides and subsequently NCL reaction. However, the synthesis of some glycine-rich sequences revealed limitations, such as diacylated products that can not be converted into -acylurea peptides. Here, we introduce a new acylurea linker bearing an amino(methyl)aniline (MeDbz) moiety that enables in a more robust peptide chain assembly. The generality of the approach is illustrated by the synthesis of a pentaglycine sequence under different coupling conditions including microwave heating at coupling temperatures up to 90 C, affording the unique and desired acyl-′-methylacylurea (MeNbz) product. Further extension of the method allowed the synthesis of all 20 natural amino acids and their NCL reactions. The kinetic analysis of the ligations using model peptides shows the MeNbz peptide rapidly converts to arylthioesters that are efficient at NCL. Finally, we show that the new MeDbz linker can be applied to the synthesis of cysteine-rich proteins such the cyclotides Kalata B1 and MCoTI-II through a one cyclization/folding step in the ligation/folding buffer.
Chemical synthesis of proteins using hydrazide intermediates
The most effective way of covalently joining unprotected peptide segments to form a protein molecule is native chemical ligation (, ). Native chemical ligation involves the reaction of a peptide-αthioester with a Cys-peptide; reversible thioesterthiol exchange with the N-terminal Cys residue gives a thioester-linked intermediate that undergoes an irreversible intramolecular rearrangement to give a near-quantitative yield of a single product linked by a native peptide bond at the ligation site. The native chemical ligation reaction is both practical and highly effective. Each ligation product can be purified by reverse-phase HPLC and characterized with great precision by electrospray MS. In principle, the ability to purify and characterize intermediate products at each successive stage of construction of a protein molecule is one of the major advantages of the chemical ligation approach to total protein synthesis; it ensures accurate construction of high-purity protein molecules. However, in practice, such purification and characterization are arduous: the consecutive chemical ligation of several peptide segments involves multiple laborious purifications carried out by reverse-phase HPLC (see ). The repetitive HPLC purifications result in significant handling losses. Moreover, each of these purification steps entails time-consuming lyophilization of the product to enable solvent exchange for subsequent reactions.