Four macrocyclic cystine-knot peptides of 29-31 residues, kalata, circulin A and B (CirA and CirB), and cyclopsychotride, have been isolated from coffee plants but have undetermined physiological functions. These macrocycles and 10 of their analogs prepared by chemical synthesis were tested against nine strains of microbes. Kalata and CirA were specific for the Gram-positive Staphylococcus aureus with a minimum inhibition concentration of Ϸ0.2 M. They were relatively ineffective against Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa. However, CirB and cyclopsychotride were active against both Gram-positive and Gram-negative bacteria. In particular, CirB showed potent activity against E. coli with a minimum inhibitory concentration of 0.41 M. All four cyclic peptides were moderately active against two strains of fungi, Candida kefyr and Candida tropicalis, but were inactive against Candida albicans. These macrocycles are cytotoxic and lysed human red blood cell with a lethal dose 50% of 400 M. Modifying the Arg residue in kalata with a keto aldehyde significantly reduced its activity against S. aureus whereas blocking the arg in CirA produced no significant effect. The two-disulfide variants and their scrambled disulfide isomers exhibited antimicrobial profiles and potency similar to their native peptides. However, in high-salt assays (100 mM NaCl), few of these macrocyclic peptides, natives or analogs, retained antimicrobial activity. These results show that the macrocyclic peptides possess specific and potent antimicrobial activity that is saltdependent and that their initial interactions with the microbial surfaces may be electrostatic, an effect commonly found in defensin antimicrobial peptides. Furthermore, their endto-end cyclic structure with a cystine-knot motif represents a molecular structure of antimicrobials and may provide a useful template for the design of novel peptide antibiotics.Although cyclic peptides are produced naturally in microbes and plants, they are usually restricted to small and medium size peptides of Ͻ15 amino acids. Thus, it is interesting to note the recent discovery of four large end-to-end cyclic peptides of 29-31 amino acids from plants of the Rubiacease family. These peptides contain six cysteines that can be aligned and share Ϸ45% of sequence homology (Fig.

Dendrimeric peptides selective for microbial surfaces have been developed to achieve broad antimicrobial activity and low hemolytic activity to human erythrocytes. The dendrimeric core is an asymmetric lysine branching tethered with two to eight copies of a tetrapeptide (R4) or an octapeptide (R8). The R4 tetrapeptide (RLYR) contains a putative microbial surface recognition BHHB motif (B ¼ basic, H ¼ hydrophobic amino acid) found in protegrins and tachyplesins whereas the octapeptide R8 (RLYRKVYG) consists of an R4 and a degenerated R4 repeat. Antimicrobial assays against 10 organisms in high-and low-salt conditions showed that the R4 and R8 monomers as well as their divalent dendrimers contain no to low activity. In contrast, the tetra-and octavalent R4 and R8 dendrimers are broadly active under either conditions, exhibiting relatively similar potency with minimal inhibition concentrations < 1 lM against both bacteria and fungi. Based on their size and charge similarities, the potency and activity spectrum of the tetravalent R4 dendrimer are comparable to protegrins and tachyplesins, a family of potent antimicrobials containing 17-19 residues. Compared with a series of linearly repeating R4 peptides, the R4 dendrimers show comparable antimicrobial potency, but are more aqueous soluble, more stable to proteolysis, less toxic to human cells and more easily synthesized chemically. These results suggest repeating peptides that cluster the charge and hydrophobic residues may represent a primitive form of microbial pattern-recognition. Incorporating such knowledge in a dendrimeric design therefore presents an attractive approach for developing novel peptide antibiotics.

We describe an approach to the synthesis of peptides from segments bearing no protecting groups through an orthogonal coupling method to capture the acyl segment as a thioester that then undergoes an intramolecular acyl transfer to the amine component with formation of a peptide bond. Two orthogonal coupling methods to give the covalent ester intermediate were achieved by either a thiol-thioester exchange mediated by a trialkylphosphine and an alkylthiol or a thioesterification by C"-thiocarboxylic acid reacting with a f8-bromo amino acid. With this approach, unprotected segments ranging from 4 to 37 residues were coupled in aqueous solution to give free peptides up to 54 residues long with high efficiency.Recent advances in the design of artificial proteins with unusual architectures (1) and the ready availability of large peptides and protein domains from both solid-phase synthesis (2, 3) and recombinant DNA have led to the need for the development of convergent strategies in peptide synthesis with these large unprotected peptide segments as building blocks. A requirement of such a strategy is the exceptionally high regiospecificity in the amide bond formation between the a-amine and Ca-acyl moiety of the two segments. Recently, we have proposed (4-7) an orthogonal coupling method to achieve this goal.An orthogonal coupling method to form an amide bond is conceptually different from an orthogonal protecting group strategy (8). It is site-specific and allows only a single specific coupling reaction between the C' moiety of one peptide segment and the Na-amine of another peptide segment, in the presence of other reactive amino moieties. In such a method, a capture step (9) brings the respective N and C termini into close proximity and the amide bond formation is effected by an intramolecular acyl transfer at high effective concentration. So far, all orthogonal couplings have exploited the unique reactivity of the 1,2-aminothiol moiety of an N-terminal cysteine on the amine segment to achieve selectivity to form thiazolidine, thioester, and disulfide with acyl segments bearing glycolaldehyde ester (4, 5), thioester (6, 10), and mixed disulfide (6, 7). In orthogonal couplings involving Ca-thioester and Na-cysteine, the capture step occurs when the covalent thioester is formed between these two segments leading to a spontaneous S-to N-acyl transfer to form a cysteinyl peptide bond (Fig. 1) using fully unprotected peptide segments. The capture is effected by a thiol-thioester exchange in method A and thioesterification in method B. Both will give a covalent thioester leading to a spontaneous intramolecular acyl transfer to give a cysteinyl peptide bond. P-I (shaded oval), peptide-1; P-2, peptide-2 (open square); R, CH2CH2CO2H.N-terminal ,3-bromoalanine (BrAla) and forms a similar covalent thioester that will rearrange rapidly via an intramolecular S-to-N acyl transfer to give the peptide bond (Fig. 1 Table 3), were synthesized by the Fmoc/t-Bu strategy on Wang resin (11) by using the benzotriazol-1-yl-oxy-t...

This paper describes a simple biomimetic strategy to prepare small cyclic proteins containing multiple disulfide bonds. Our strategy involves intramolecular acyl transfer reactions to assist both the synthesis and fragmentation of these highly constrained cyclic structures in aqueous solution. To illustrate our strategy, we synthesized the naturally occurring circulin B and cyclopsychotride (CPT), both consisting of 3 1 amino acid residues tightly packed in a cystine-knot motif with three disulfide bonds and an end-to-end cyclic form. The synthesis of these small cyclic proteins can be achieved by orthogonal ligation of free peptide thioester via the thia zip reaction, which involves a series of reversible thiolthiolactone exchanges to arrive at an a-amino thiolactone, which then undergoes an irreversible, spontaneous ring contraction through an S,N-acyl migration to form the cyclic protein. A two-step disulfide formation strategy is employed for obtaining the desired disulfide-paired products. Partial acid hydrolysis through intramolecular acyl transfer of X-Ser, X-Thr, Asp-X, and Glu-X sequences is used to obtain the assignment of the circulins disulfide bond connectives. Both synthetic circulin B and CPT are identical to the natural products and, thus, the total synthesis confirms the disulfide connectivity of circulin B and CPT contain a cystine-knot motif of 1-4,2-5, and 3-6. In general, our strategy, based on the convergence of chemical proteolysis and aminolysis of peptide bonds through acyl transfer, is biomimetic and provides a useful approach for the synthesis and characterization of large end-to-end cyclic peptides and small proteins.

This paper describes the mechanism and application of an efficient thia zip cyclization that involves a series of intramolecular rearrangements in a cysteine-rich peptide for the synthesis of large end-to-end cyclic peptides. Key functional groups required in this reaction include an N α-cysteine, a thioester, and at least one internal free thiol in a peptide. The zip reaction is initiated by intramolecular transthioesterification through an internal thiol with the thioester. A thiolactone is formed under ring−chain tautomeric equilibrium that favors ring formation in aqueous buffered solution at pH > 7. Successive ring expansions through thiol−thiolactone exchanges in the direction of the amino terminus lead finally to a large Nα-amino thiolactone which then undergoes a spontaneous and irreversible ring contraction through a sequence-independent S to N acyl isomerization to form an end-to-end lactam. The reversible thiolactone exchanges are sequence-dependent, and the rate-determining steps are shown by rate studies on model peptides. The assistance of internal thiols in reducing the ring sizes and hence the entropy of the thiolactone exchanges correlates well with cyclization rates. Zip-assisted end-to-end cyclizations forming 93- and 99-atom rings through a series of small thiolactone intermediates were 60−200-fold faster under strongly denaturing conditions such as 8 M urea than the corresponding unassisted lactamization. The thia zip reaction has been applied successfully to the synthesis of a 31-amino acid cyclic peptide, the naturally occurring cyclopsychotride that shows the antimicrobial activity. In addition, the thia zip reaction also enables the synthesis of an engineered cyclic 33-amino acid animal defensin by replacing the end-to-end disulfide with a lactam, which retains the antimicrobial activities of the native open-chain form.