Construction of protein analogs by site-specific condensation of unprotected fragments

Gaertner, Hubert; Rose, Keith; Cotton, Ron; Timms, David; Camble, Roger; Offord, Robin E.

doi:10.1021/bc00015a010

Cited by 146 publications

(54 citation statements)

References 32 publications

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“…Furthermore, aniline is soluble in a range of solvents and can be used as a convenient aqueous buffer with acetic acid, and both its solubility and nucleophilicity can be tuned through the introduction of substituents on the aromatic ring. Aniline catalysis will have a major impact on the numerous applications of imine chemistry including bioconjugation, [1][2][3][4][5][6][7][8] cellular labeling, [18,19] surface immobilization, [20] and dynamic combinatorial libraries. [21,22] .…”

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confidence: 99%

Nucleophilic Catalysis of Oxime Ligation

2006

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Imine-based reactions have found wide application in the conjugation of biomolecules as a result of their high chemoselectivity. [1][2][3][4][5][6][7][8] Aldehydes and ketones can be readily introduced into biomolecules and are virtually inert towards reaction with other functional groups in these molecules. Under acidic conditions the carbonyl group reacts with primary amines to form a reversible imine (Scheme 1 a), and the equilibrium favors the free carbonyl. However, when a-effect nitrogens [9] such as aminooxy groups and hydrazides are used, the equilibrium favors the imine. [1][2][3][4][5][6][7][8] Oxime ligations are often called upon to link complex and precious macromolecules. [7,8] The oxime bond is stable under physiological conditions, whereas more dynamic imines, such as hydrazones, are often reduced to obtain a stable linkage. Oxime ligations proceed with modest reaction rates in acidic solution but are poorly reactive at pH 7. which limits their use in many biological applications. To improve the reaction rate, oxime ligations typically require millimolar concentrations of Scheme 1. a) Imine formation and b) transimination under acidic conditions.

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Nucleophilic Catalysis of Oxime Ligation

2006

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“…Although powerful, each of these techniques has associated with it certain practical or synthetic limitations which have to some extent restricted their widespread application. Total chemical synthesis, which provides unparalleled freedom to manipulate protein structure, has been dominated in recent years by the use of chemical ligation techniques (7)(8)(9)(10)(11)(12)(13). Among these, the "native chemical ligation" approach by Kent et al (14) has proven a particularly useful route to synthetic proteins.…”

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Expressed Protein Ligation, a Novel Method for Studying Protein-Protein Interactions in Transcription

Severinov¹,

Muir²

1998

Journal of Biological Chemistry

192

173

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Expressed protein ligation is a novel protein semisynthesis method that permits the in vitro ligation of a chemically synthesized C-terminal segment of a protein to a recombinant N-terminal segment fused through its C terminus to an intein protein splicing element. In principle, the practical convenience of this method, combined with the expanded opportunities in protein engineering that it provides, makes it well suited for probing the molecular basis of complex processes such as transcription. Here we describe the successful application of expressed protein ligation to the ϳ600 amino acid 70 subunit of Escherichia coli RNA polymerase. The resulting semi-synthetic 70 constructs are shown to be fully functional and have been used to map the binding region of the bacteriophage T4 anti-sigma protein, AsiA, to within amino acids 567-600 of 70 . The success of these semi-synthesis studies sets the stage for the future generation of semi-synthetic 70 molecules in which unnatural amino acids and biophysical probes are site-specifically incorporated in the RNA polymerase complex.

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“…However, these approaches do not overcome the problem of the poor coupling efficiency between large peptide segments, because of the intrinsic difficulty of obtaining effective molar concentrations for high molecular weight molecules. Alternative approaches to overcome the difficulty of forming amide bonds use hydrazone (12,13) or thioether and thioester (14) as surrogate bonds. The advantage of these peptide-bond replacement approaches resides in the high and specific reactivity between the two functional groups not found in amino acids to make the conjugation efficient.…”

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Peptide segment ligation strategy without use of protecting groups.

Liu

Tam

1994

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

210

161

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We describe the concept and the verification of a chemical ligation approach to the synthesis of proteins using peptide se-ents with no protecting groups and no activation of the C-tenal a-carboxyl group. This approach of three steps: (i) aldehyde introduction, in which a masked glycolaldehyde ester Is linked to the carboxyl terminus of an unprotected peptide by reverse protedlysis; (ii) ring formation, in which the umaked aldehyde reacts with the N-teinal a-amino group of the second unprotected peptide containing either a cysteine or a threonine residue to form a t ne or olidine ring at an acidic pH; and (iii) rearrangement in which O-acyl ester linkage is transferred to N-acyl amide linkage to form a peptide bond with a pseudoproline stuture at higher pH. The (4), and the semi-synthesis of proteins consisting of components from synthetic peptides with unusual amino acids and proteins obtained through recombinant technology.The conventional segment-synthesis approaches (5) usually require a maximal protection strategy which has the limitations ofpoor solubility, low coupling efficiency, and the possible danger of racemization due to the overactivation of the carboxyl group. Considerable efforts have been made to overcome these limitations through the use of partial or minimal protecting-group strategies and improvements in the activation methods (6-11). However, these approaches do not overcome the problem of the poor coupling efficiency between large peptide segments, because of the intrinsic difficulty of obtaining effective molar concentrations for high molecular weight molecules. Alternative approaches to overcome the difficulty of forming amide bonds use hydrazone (12,13) or thioether and thioester (14) as surrogate bonds. The advantage of these peptide-bond replacement approaches resides in the high and specific reactivity between the two functional groups not found in amino acids to make the conjugation efficient. The disadvantage is that these linkages are substantially different from the peptide bond, which may lead to conformational distortion, and are usually not stable at either acidic (12) or basic (14) pH.We have now designed and developed a chemical ligation method by which two unprotected peptide segments can be ligated together through an amide bond. In our design, we make use of a highly regiospecific and efficient reaction to link covalently two unprotected peptides. The selectivity of this reaction would also obviate the need for any protecting groups. A peptide bond is then formed in a second step through an intramolecular rearrangement between the two closely neighboring carboxyl and amino groups. The efficiency of the first reaction would solve the slow kinetic problem for reactions between large molecules, and the second reaction, designed as a spontaneous process to form amide bonds without activating the carboxyl group, would overcome the racemization problem of the conventional segment condensation method. EXPERIMENTAL PROCEDURESGeneral. 1H NMR spectra were obtained at 300 ...

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Construction of protein analogs by site-specific condensation of unprotected fragments

Cited by 146 publications

References 32 publications

Nucleophilic Catalysis of Oxime Ligation

Nucleophilic Catalysis of Oxime Ligation

Expressed Protein Ligation, a Novel Method for Studying Protein-Protein Interactions in Transcription

Peptide segment ligation strategy without use of protecting groups.

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