Thiazolidine Formation as a General and Site-Specific Conjugation Method for Synthetic Peptides and Proteins

Zhang, Lianshan; Tam, James P.

doi:10.1006/abio.1996.0011

Cited by 88 publications

(87 citation statements)

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“…The feasibility of this approach has been demonstrated in experiments to prevent the inducible nuclear import of transcription factors in human monocytic, endothelial, and T lymphocyte cell lines (23,25). We have also implemented a recently developed approach of modular peptide synthesis, which involves separate synthesis of the MPS and functional peptides with subsequent chemical ligation of the two peptides under mild aqueous conditions (24). The biological activity of the resulting modular peptides has been extensively documented by comparing modular peptides with those synthesized conventionally (23,25,48,49).…”

Section: Discussionmentioning

confidence: 99%

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Dissecting G Protein-coupled Receptor Signaling Pathways with Membrane-permeable Blocking Peptides

Chang¹,

Zhang²,

Tam³

et al. 2000

Journal of Biological Chemistry

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To determine the intracellular signaling mechanism of the 5-HT 2C receptor endogenously expressed in choroid plexus epithelial cells, we implemented a strategy of targeted disruption of protein-protein interactions. This strategy entails the delivery of conjugated membrane-permeable peptides that disrupt domain interaction at specific steps in the signaling cascade. As proof of concept, two peptides targeted against receptor-G protein interaction domains were examined. Only G q CT, which targets the receptor-G q protein interacting domain, disrupted 5-HT 2C receptor-mediated phosphatidylinositide hydrolysis. G s CT, targeting the receptor-G s protein, disrupted ␤2 adrenergic receptor-mediated activation of cAMP but not 5-HT 2C receptor-mediated phosphatidylinositide hydrolysis. The peptide MPS-PLC␤1M, mimicking the domain of phospholipase C␤1 (PLC␤1) interacting with active G␣ q , also blocked 5-HT 2C receptor activation. In contrast, peptides PLC␤2M and Phos that bind to and sequester free G␤␥ subunits were ineffective at blocking 5-HT 2C receptormediated phosphoinositol turnover. However, both peptides disrupted G␤␥-mediated ␣ 2A adrenergic receptor activation of mitogen-activated protein kinase. These results provide the first direct demonstration that active G␣ q subunits mediate endogenous 5-HT 2C receptor activation of PLC␤ and that G␤␥ subunits released from G␣ q heterotrimeric proteins are not involved. Comparable results were obtained with metabotropic glutamate receptor 5 expressed in astrocytes. Thus, conjugated, membrane-permeable peptides are effective tools for the dissection of intracellular signals.The 5-HT 2 receptor family consists of three members, 5-HT 2A , 5-HT 2B , and 5-HT 2C . All three receptors belong to the G protein-coupled serpentine receptor superfamily. Their pharmacological profiles are very similar, leading to difficulty in defining their functional roles. 5-HT 2 receptors have been implicated in behaviors such as sleep, feeding, aggression, pain, and anxiety and are thought to play a role in a number of central nervous system disorders including affective disease, schizophrenia, and epilepsy (1). In addition, 5-HT 2 receptors may play a major role in mediating the actions of hallucinogenic drugs (2) as well as antipsychotic drugs (3, 4). Mice expressing nonfunctional 5-HT 2C receptors exhibit epileptic and obese phenotypes (5, 6), suggesting that these receptors play a crucial role in moderating central nervous system function.Expression of the 5-HT 2C receptor is exceptionally high in the choroid plexus (7,8), where it plays a role in the regulation of production and composition of cerebrospinal fluid (9 -11). Initial studies of 5-HT 2C receptor signaling showed that these receptors activate the downstream intracellular effector, phospholipase C␤ (PLC␤) 1 resulting in the hydrolysis of phosphatidylinositol-4,5-bisphosphate into inositol-1,4,5-triphosphate and diacylglycerol (12). In addition, activation of the 5-HT 2C receptor has been observed to release arachidonic acid (13...

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Section: Discussionmentioning

confidence: 99%

“…The membrane-permeable sequence (MPS) peptide was based on a hydrophobic membrane-permeable sequence described previously (23)(24)(25). Lysine and serine residues, as a pseudo-dipeptide, were added at the carboxyl terminus to serve as a linker and a masked aldehyde to facilitate conjugation.…”

Section: Methodsmentioning

confidence: 99%

Dissecting G Protein-coupled Receptor Signaling Pathways with Membrane-permeable Blocking Peptides

Chang¹,

Zhang²,

Tam³

et al. 2000

Journal of Biological Chemistry

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“…Thiazolidine formation fulfills these requirements. It requires a 1,2-amino thiol which is derived from cysteine on one peptide and an aldehyde derived from mild periodate oxidation of serine or threonine on the other (7,20). In our hands, there appears to be no discernible difference which moiety (the aldehyde or cysteine) is placed on which peptide module (hydrophobic or functional).…”

Section: Discussionmentioning

confidence: 99%

“…We have previously described a general and mild method for sitespecific biotinylation of peptides and glycoproteins that is based on the chemoselective ligation of an aldehyde on one reactant with a 1,2-amino thiol moiety on a second reactant (7,8). Ligation of the aldehyde with the 1,2-amino thiol is rapid and site specific, forming a stable thiazolidine ring.…”

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Preparation of functionally active cell-permeable peptides by single-step ligation of two peptide modules

Zhang

Torgerson

Liu

et al. 1998

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

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Noninvasive cellular import of synthetic peptides can be accomplished by incorporating a hydrophobic, membrane-permeable sequence (MPS). Herein, we describe a facile method that expedites synthesis of biologically active, cell-permeable peptides by site-specific ligation of two free peptide modules: one bearing a functional sequence and the second bearing a MPS. A nonpeptide thiazolidino linkage between the two modules is produced by ligation of the COOH-terminal aldehyde on the MPS and the NH 2 -terminal 1,2-amino thiol moiety on the functional sequence. This thiazolidine ligation approach is performed with stoichiometric amounts of fully unprotected MPS and functional peptide in an aqueous buffered solution, eliminating the need for additional chemical manipulation and purification prior to use in bioassays. Two different MPSs were interchangeably combined with two different functional sequences to generate two sets of hybrid peptides. One set of hybrid peptides, carrying the cytoplasmic cell adhesion regulatory domain of the human integrin ␤ 3 , inhibited adhesion of human erythroleukemia cells to fibrinogen-coated surfaces. A second set of hybrid peptides, carrying the nuclear localization sequence of the transcription factor NF-B, inhibited nuclear import of transcription factors NF-B, activator protein 1, and nuclear factor of activated T cells in agonist-stimulated Jurkat T lymphocytes. In each assay, these nonamide bond hybrids were found to be functionally comparable to peptides prepared by the conventional method. Cumulatively, this new ligation approach provides an easy and rapid method for engineering of functional, cell-permeable peptides and demonstrates the potential for synthesis of cell-permeable peptide libraries designed to block intracellular protein-protein interactions.

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“…[12][13][14][15] As an alternative to the oxime bond, thiazolidine ring formation could be used as a site-specific ligation method via reaction of a 1,2-thiol-amine function with a carbonyl group -including masked carbonyls of carbohydrates and hemiacetals -in mildly acidic or basic conditions (pH 4 to 8). [16][17][18] Importantly, the thiazolidine ring is generally stable in a wide pH range (from 4 to 10), thus representing an attractive linkage option. In order to further investigate the efficiency of [ 18 F]FDR as a radiolabelling agent and expand the library of prospective PET tracers for hypoxia imaging, we designed a novel class of candidate tracers [ 18 F]1 ( Figure 1) taking advantage of the last-step formation of a thiazolidine ring linkage between [ 18 F]FDR 2 and terminal 2-amino-thiols 3 carrying a hypoxia-reactive 2-nitroimidazole group.…”

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The first prospective fluorinated PET tracers for imaging hypoxia obtained via thiazolidine-ligation are reported. Three 1,2-thiolamine linkers were combined with four different 2-nitroimidazole spacers via amide or urea bond formation. The resulting compounds were submitted to thiazolidine-ring-forming ligation reaction with the fluorinated carbohydrate L-5-fluoro-5-deoxy-ribose (FDR), affording the desired candidate PET tracers in variable yields. The same ligation reactions performed on L-ribose -a by-product of [ 18 F]FDR radiosynthesisunder conditions mimicking a radiochemical production showed that the fluorinated adducts can be efficiently purified and isolated by HPLC. Finally, one of the prospective hypoxia tracers was successfully produced in radiolabelled form in 29.2% radiochemical yield from [ 18 F]FDR.

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Thiazolidine Formation as a General and Site-Specific Conjugation Method for Synthetic Peptides and Proteins

Cited by 88 publications

References 4 publications

Dissecting G Protein-coupled Receptor Signaling Pathways with Membrane-permeable Blocking Peptides

Dissecting G Protein-coupled Receptor Signaling Pathways with Membrane-permeable Blocking Peptides

Preparation of functionally active cell-permeable peptides by single-step ligation of two peptide modules

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