We describe an efficient regiospecific method for cyclization of unprotected peptide segments based on intramolecular transthioesterification of unprotected cysteinyl peptide thioesters under the control of ring−chain tautomeric equilibrium in aqueous buffered solutions at pH ranging from 5 to 7.5. The initial cyclization to form an intramolecular thioester under the ring−chain tautomeric equilibrium is reversible and could be performed in relatively high concentrations without observable oligomerization. This method overcomes the limitation of conventional cyclization methods that require high dilutions. The reaction becomes irreversible by a subsequent, spontaneous proximity-driven S- to N-acyl transfer to the adjacent N α-amine of cysteine to form an end-to-end cyclic peptide. The cyclization is regioselective. No side reactions were observed with side-chain functionalities such as the N ε-amine of lysine, thiol of internal cysteine, or imidazole of histidine. Since a free thiol group was introduced to the product after cyclization, these cyclic peptides were exploited as building blocks for synthesizing peptides with unusual architectures such as bicyclic peptides containing end-to-end backbones and disulfide bridges as well as cascade branched peptide dendrimers.

An orthogonal cyclization strategy has been developed to prepare cyclic peptides individually or as a mixture in aqueous solutions. In this strategy, we propose a dual activation by entropy and enthalpy through Ag + ion coordination of the reactive ends of an unprotected peptide thioester to permit long-range acyl migration. As a result, both lactamization and lactonization of linear peptides can be realized simultaneously or selectively by controlling the reaction conditions. At pH 4, lactonization is favored due to the strong protonation of the amino moieties. However, at pH 5-5.7 lactamization of R-amine of the N-terminal amino acid is favored, particularly with DMSO as a cosolvent. At pH > 6, lactamization of the -amine of lysine becomes significant. Furthermore, the Ag + ion complexation with the peptide thioester precursor in aqueous buffered solutions may facilitate entropy-favored ring-chain tautomerization, which in turn promotes formation of monomeric cyclic products and thereby reduces unwanted oligomerization. Evidence supporting Ag + ion-directed ringchain tautomerization includes product distribution profiles, the concentration-independent nature of the cyclization, and lack of competition by intermolecular coupling with other unprotected peptide segments. The N-terminal amino acid has been found to exert a profound effect in conferring high specificity toward endto-end cyclization. Examination of 23 linear peptide precursors containing different N R -amino acids showed that the probability of lactamization is almost 20-fold greater in R-amines than -amines with N-terminal Gly, Asn, Asp, and Ser. Unprotected linear peptides ranging from 5 to 16 amino acid residues were cyclized individually or as a mixture with good to excellent yields. More importantly, this strategy paves the way for a new approach to synthesizing cyclic peptide libraries in which unprotected peptides are cyclized and released from the resin at the same time. The obtained libraries can, therefore, be used directly for bioassays without further chemical manipulations. This method is also useful for the synthesis of bicyclic peptides containing both sulfur-sulfur and lactam linkages.

No abstract

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...

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.