Michinori Waki

Supramolecular Complex Formation by β-Cyclodextrin and Ferrocenylnaphthalene Diimide-intercalated Double Stranded DNA and Improved Electrochemical Gene Detection

Ferrocenylnaphthalene diimide 1 can bind to double stranded DNA (dsDNA) by the threading intercalation mode and the resulting complex was stabilized further by β-cyclodextrin (CD) by forming a supramolecular complex. These complex formation processes were studied by spectroscopic, viscometric, and electrochemical means in the absence or presence of β-CD. Quantitative analysis by quartz crystal microbalance (QCM) and electrochemical experiments strongly suggested a 2:1 binding stoichiometry for β-CD to 1 threading-intercalated to the dsDNA-immobilized electrode. Owing to this supramolecular complex formation, electrochemical DNA detection based on 1 was improved considerably.

[4,4′-(Z)-Dehydrophenylalanine]gramicidin S with stabilized bioactive conformation and strong antimicrobial activity

Dehydrophenylalanine (ΔPhe) was incorporated into an antibiotic peptide gramicidin S (GS) in place of D‐Phe4,4′ to prepare an unsaturated analog. Conformational analysis with 1H‐NMR indicated that the unsaturated analog has much the same backbone conformation as that of natural gramicidin S as shown by NOE experiments. Studies on temperature dependences and on the chemical shift differences showed that the hydrogen bonds between Val‐NH and Leu‐CO in the unsaturated analog are strengthened by the incorporation of ΔPhe4,4′. This resulted in the reinforcement of the β‐sheet structure which is the most important structural element for GS bioactivity. [ΔPhe4,4′]gramicidin S exhibited indeed very strong antimicrobial activities against Gram‐positive bacteria as well as the natural peptide.

Facile synthesis of Gramicidin S via cyclication of a linear pentapeptide

Abstract Azide and N -hydroxysuccinimide ester of five pentapeptides related to gramicidin S (cyclic decapeptide) were cyclized to determine the ratio of dimer to monomer in the cyclization product, the pentapeptide derivative with D-Phe as C -terminus giving the dimer in good yield.

Enzyme inhibition by dipeptides containing 2,3‐methanophenylalanine, a sterically constrained amino acid

Both isomers of (E)‐2,3‐methanophenylalanine (▽EPhe), a sterically restricted amino acid, were incorporated into peptides in order to examine their possible enzyme inhibitory activity. Both (2R,3S)‐ and (2S,3R)‐▽EPhe‐Phe(or Leu)‐OMe were found to inhibit effectively the hydrolysis of Ac‐Tyr‐OEt by chymotrypsin in a competitive manner. The ester groups of these dipeptides were quite resistant to chymotrypsin hydrolysis, and the ▽EPhe‐Phe peptide bond was also entirely stable. The inhibition constant (K i) of the most potent dipeptide of H‐(2R,3S)‐▽EPhe‐Phe‐OMe was 0.16 mM at 25°C. The inhibitory action of ▽Phe‐containing peptides was found to depend on the configuration of the ▽Phe residue. The electrophilic nature of the cyclopropane ring which is conjugated with both the phenyl ring and the ester carbonyl group appears to be relevant to the inhibitory activity. Fully irreversible inactivation of chymotrypsin was achieved by its incubation with H‐(2R,3S)‐▽EPhe‐Leu‐OMe. An enzyme carboxylate group is thought to be responsible for nucleophilic attack on the cyclopropane ring leading to irreversible inactivation.

Environment-dependent conformation and antimicrobial activity of a gramicidin S analog containing leucine and lysine residues

An analog of gramicidin S, cyclo(‐L‐Leu‐L‐Lys‐L‐Leu‐D‐Leu‐L‐Leu‐)2, in which four out of five amino acid components of gramicidin S were substituted, has been synthesized. This analog assumes a conformation similar to that of gramicidin S in acidic liposomes and a random conformation in neutral liposomes. The antimicrobial activity of this analog corresponded to one‐fourth of that of gramicidin S. A possible mechanism for conformational changes in acidic liposomes is discussed.

Anti-chymotrypsin and anti-elastase activities of a synthetic bicyclic fragment containing a chymotrypsin-reactive site of soybean Bowman-Birk inhibitor

A bicyclic hexadecapeptide, which corresponds to the sequence 36-51 and contains the chymotrypsin-reactive Leu-43-Ser-44 bond of soybean Bowman-Birk inhibitor, has been synthesized. This peptide consists of two loops formed by disulfide bridges between Cys-36 and Cys-51 and between Cys-41 and Cys-49. The bicyclic peptide showed a strong anti-chymotryptic activity with a Ki of 7.1.10(-7) M. Comparison of inhibitory activity and digestive stability against chymotrypsin with other hexadecapeptides having the same sequence but lacking one or both disulfide bridges suggested that the compact bicyclic structure increases the activity and protects the Leu-Ser bond from chymotryptic digestion. Interestingly, the bicyclic peptide was found to inhibit porcine pancreatic elastase with a Ki of 4.3.10(-5) M, indicating the broad specificity of this ring system.

Dimerization of neurokin A and B COOH-terminal heptapeptide fragments enhanced the selectivity for tachykinin receptor subtypes

We have synthesized dimeric analogues of neurokinin A and B COOH-terminal heptapeptide fragments and evaluated their biological activities to contract isolated smooth muscle preparations of the guinea-pig ileum and rat vas deferens. The dimers were fairly active and showed two-fold increased selectivity for receptors in the guinea-pig ileum as compared with monomers. Extremely slow dissociation indicated a possible bivalent interaction between dimer and receptor.

[4,4′-D-Diaminopropionic acid]gramicidin S: a synthetic gramicidin S analog with antimicrobial activity against Gram-negative bacteria

Gramicidin S is especially active against Gram‐positive bacteria; e.g., Staphylococcus aureus. An analog, [4,4′‐D‐diaminopropionic acid]gramicidin S, which contains D‐diaminopropionic acid residues instead of D‐phenylalanine residues, has been synthesized. This analog is active against some of the Gram‐negative bacteria; e.g., Escherichia coli and Salmonella typhosa. Activities of several related analogs are discussed.

Regional distribution of DNA and RNA in rat brain: A sensitive determination using high-performance liquid chromatography with electrochemical detection

DNA and RNA contents in 20 brain regions or nuclei of the rat were determined by a highly sensitive method using high-performance liquid chromatography with electrochemical detection. The high DNA and RNA contents were found in the hypothalamic nuclei, especially the median eminence-arcuate nucleus. These results may be available for the preparation of nucleic acids as the regional control.

Dimeric substance P analogue shows a highly potent activity of the in vivo salivary secretion in the rat.

We have synthesized a dimeric analogue of substance P (SP) COOH-terminal nonapeptide fragment (D-SP-(3-11] and examined the in vivo and in vitro biological activities in the submaxillary gland of the rat. The dimer elicited an enhanced biological response as compared with receptor binding, showing 2.4-fold more potent receptor affinity than its monomer and 75-fold more potent in vivo salivary secretion activity.