Yi-An Lu

Synthesis of large cyclic cystine-knot peptide by orthogonal coupling strategy using unprotected peptide precursor

Abstract A simple and direct method for the synthesis of large end-to-end, disulfide rich cyclic peptide using orthogonal coupling of unprotected N α -cysteinyl thioester peptide is described.

Functional and Structural Comparisons of Linear and Dendritic Arrays of Polypeptides

Bioactive biopolymers with repeating sequences are often found in nature. Polypeptides usually contain an array of linear repeats, whereas both linear and dendritic repeats are found in polysaccharides. To determine the functional and structural consequences of polypeptides in a dendritic array, we have designed dendritic polypeptides [1] and compared them to the linearly repeating polypeptides. Both series of polypeptides contain two to eight copies of tetrapeptides with BHHB motifs (B = basic amino acid, H = hydrophobic amino acid) derived from naturally occurring antimicrobials.

Preparation of Site-Specific Peptide Immunogens Using Multiple Antigen Peptide Approach System

Publisher Summary This chapter discusses the preparation of site-specific peptide immunogens using the multiple antigen peptide (MAP) approach system. The MAP system is designed as a new approach to prepare peptide immunogens to overcome the ambiguity of the conventional approach. The MAP produces carrierless, chemically defined, peptide immunogens with a high degree of homogeneity. As a result, immunogens prepared by the MAP approach elicit high but uniform antibody responses in outbred animals. As such, the MAP approach is particularly suitable for the preparation of site-specific antibodies for biochemical and immunological applications. The MAP system consists of an oligomeric branching lysine core, usually composed of three or seven lysines, and four or eight copies of dendritic arms of peptide antigens. Because each peptide arm may consist of 10–20 amino acids, the overall appearance of the MAP system is of a macromolecule with a high density of surface peptide antigen and a molecular weight exceeding 10,000. Advantages of the MAP system include the aforementioned chemical control and precision of producing peptide antigens, the flexibility of incorporating two or more types of peptide antigens, and the replacement of the protein carrier.

Orthogonal ligation of free peptides

Over past eight years, our laboratory has focused on developing new methodologies for ligating free peptide segments to form complex proteins and biopolymers in aqueous or organic solutions. Recently, we and others have developed a novel segment ligation strategy [l-3] in which an amide bond is formed regiospecifically to the desired Nterminal amine between unprotected peptide segments containing more than one free Nterminal amine. This strategy represents a significant methodological advance. We refer it as “orthogonal ligation strategy” in accordance with other orthogonal concepts in chemistry, including orthogonal protection schemes [4], orthogonal activation [5] and coupling [6] in organic chemistry that distinguish two functional sites based on chemoselectivity. The orthogonal ligation is a cascade consisting of two reactions of capture and activation. The capture step utilizes the principle of chemoselective ligation to form a covalent intermediate between two peptide segments. Then, an amide bond is formed via an intramolecular acyl transfer through entropic activation (fig. 1). The intramolecular acylation rate, which is first order and often spontaneous, minimizes side reactions associated with enthalpic activation methods.

Orthogonal segment ligation

Orthogonal ligation is a convergent, amide-bond condensation strategy for two unprotected peptide segments regiospecific to a particular N-terminal amino acid. Conceptually, it is similar to other orthogonal strategies such as orthogonal protection, activation, and coupling used in chemistry to distinguish one functional group from another based on chemoselectivity. The ability of orthogonal ligation methods to avoid polymerization reactions may provide a tandem ligation scheme for coupling multiple peptide segments to further enhance the efficiency of convergent synthesis. To achieve the tandem ligation scheme using unprotected peptide segments without any protection or deprotection step, regioselectivity is required to distinguish one Nterminal amino acid from another during the sequential ligation steps. Over the past six years, our laboratory has developed a repertoire of orthogonal ligation methods toward this end [1-4]. These methods are based on two types of capture mechanisms: imine [1] and thioester [2,5]. Thiaproline ligation [1] is the first example demonstrating imine capture and the orthogonal ligation concept (Fig. 1). In aqueous conditions, this ligation employs an acyl segment carrying a glycoaldehyde ester 1 to capture an Nterminal (Nt) Cys segment 2a through an imine 3a, which rapidly tautomerizes to a thiazolidine ester 4a. The O-ester then rearranges to a stable amide bond, thiaproline (SPro) product 5a, at the ligation site. The thiaproline ligation is facile under aqueous conditions at pH 4 to 7. Although similar ligation reactions could occur with five other Nt-amino acids, including Nt-Ser 2b, -Thr 2c, -Trp 2d, -His 2e and -Asn 2f, these N-terminal amino acids do not readily undergo imine capture reaction in aqueous solutions and occur only slowly under non-aqueous conditions. This paper describes a new reaction condition for imine ligation with these six different N-terminal amino acids that leads to a thiaproline bond with Nt-Cys, an oxaproline bond with Nt-Ser or Nt-Thr, as well as other imidic bonds with NtTrp, Nt-His, and Nt-Asn (Fig. 1).

Cyclic peptides from unprotected precursors through ring-chain tautomerism

In 1993, our laboratory introduced a novel approach of blockwise ligation through amide bonds for peptide synthesis using unprotected peptide segments under aqueous conditions [I]. This approach uses a pair of mutually reactive groups to achieve selective amide bond formation between a specific α-amine on one peptide and an α-acyl moiety on another peptide with both peptides containing free αand e-amines. Because of its exclusive chemoselectivity, we have referred to this approach as the orthogonal coupling strategy [ 151. By exploiting different pairs of reactive groups, several methods based on this strategy have been developed and applied successfully to the synthesis of proteins [5, 6]. In addition, we have found that the orthogonal coupling strategy is well suited for preparing cyclic peptides [7,8] when two reactive ends are present on a single chain, largely due to unprotected peptide segments undergoing the entropy-favored ring-chain tautomerization. Ring-chain tautomerization is a novel concept and has the attendant advantage of eliminating the requirement for high dilution in the macrocyclization of unprotected peptides. In this paper, we describe the concept of ring-chain tautomerization in combination with the orthogonal ligation strategy for intramolecular cyclization of unprotected peptides to form end-to-end cyclic peptides using two different orthogonal coupling methods.

Antimicrobial and Chemotactic Activities of ω-Conotoxin Cyclic Analogues

The family of ω-conotoxins isolated from paralytic venomous of Conus snails has been known to be an antagonist of presynaptic N-type calcium ion channels. They share certain similarities to the newly discovered family of antimicrobial macrocyclic peptides found in plant [1]. Both are basic peptides with a four-β-loop scaffold constrained by a cystine-knot disulfide motif. We have prepared three cyclic ω-conotoxin analogues with different disulfide constraints to determine whether the open-chain co-conotoxins are microbicidal and whether their activity can be improved by end-to-end cyclic analogues mimicking the plant macrocycles. In addition, we have also determined their hemolytic and chemotactic activities attendant to membranolytic and defense mechanisms of antimicrobials.