The function of proteins and enzymes is much related to the tertiary structure formed with the folding of secondary structures, e.g., the a-helix, b-sheet, or b-turn. Although bsheet or b-turn structures are found in peptides and proteins, their biological functions are poorly understood yet compared with the a-helix. In an effort to clarify the chemical properties, functions, and stereochemistry of b-turn (or bhairpin) and antiparallel b-sheet structures, the design and synthesis of peptides have been reported by a number of research groups. [1][2][3][4][5][6] Model compounds which mimic b-sheet and b-turn structures have been shown to adopt b-hairpin structures in an aqueous solution, and hydrogen bonding can stabilize the b-turn structure. Recently, we have reported the synthesis and chemical properties of mini parallel doublestranded peptides, (L-AA 3 -L-AA 2 -L-AA 1 ) 2 -spacer(S) (AAϭ amino acids; Sϭethano-and dodecano-, etc.) conjugated two peptide strands to a mini loop (ϭspacer).-diamine mediated b-sheet-like conformation were described in the previous paper because the sequences like -Phe-Phe-, -Leu-Phe-Phe-, and -Val-Phe-Phe-which are little existence in natural peptides and proteins. Moreover, the -Val-Phe-Phe-, interestingly, is contained in Alzheimer's disease related b-amyloid peptide (Ab).The aims of the present study are the synthesis of doublestranded peptides containing chiral -x-Phe-y-Phe-sequences and elucidation of the structure-activity relationship between antibacterial activity and chirality of the -Phe-Phesequence. We synthesized four chiral types Showa Phamaceutical University; 3-3165 Higashi-tamagawagakuen, Machida, Tokyo 194-8543, Japan. Received September 8, 2003; accepted November 7, 2003 The synthesis of a series of mini double-stranded peptides containing chiral-x-Phe-y-Phe-peptide residues and the diastereomeric selective effects of these compounds on Escherichia coli NIHJ JC-2 and Staphylococcus aureus FDA 209P

Signal transduction factors that mediate cell division and growth such as protein tyrosine and serine kinases play important roles in many proliferative diseases. Binding of ligands to specific cell-surface receptors leads to aberrant signals, such as gene deletion and abnormal protein function, which lead to abnormal proliferation. 1,2) Better understanding these pathways can potentially aid in designing drugs that inhibit malignant neoplastic proliferation. Tyrosine (Tyr), with its phenol side chain, provides a phosphorylation site for tyrosine kinases, which play an important role in signal transduction (such as SH2 and SH3 domains).3) From a pharmacological viewpoint, drugs which contain Tyr residues in their structure compete for tyrosine kinase phosphorylation and may affect the signal transduction of living cells.Recently, we reported the relationship between optical isomers and growth inhibition activity by using a set of parallel, double-stranded peptides, bis(y-Phe-x-Phe) 2 -spacer(S) (7) (symbols x and yϭL-or D-configurations, AAϭamino acid, and Sϭ(CH 2 ) 12 , with two Phe residues conjugated to a polyamine spacer as a model of neoplastic targeting. [4][5][6] In order to elucidate the relationship between chirality and effect of Tyr on cell growth inhibition by the double-stranded peptides, we synthesized the bis(y-Tyr-x-Phe) 2 -(CH 2 ) 12 (6) replacing y-Phe-x-Phe with y-Tyr-x-Phe sequence. To elucidate the mechanism of action of the compound, we assayed (i) the change in the level of tyrosine phosphorylation of total proteins of src ts NRK cells, and (ii) the inhibition of epidermal growth factor receptor protein tyrosine kinase domain (EGFRKD) autophosphorylation 7,8) following treatment with 6 or 7. The proliferation of these cells is regulated by tyrosine phosphorylation and dephosphorylation, by tyrosine kinases and tyrosine phosphatases, respectively.The results showed that bis(L-Tyr-L-Phe)-N,N-dodecane-1,12-diamine (6a) and bis(L-Tyr-L-Ile)-N,N-dodecane-1,12-diamine (6e), containing the L-Tyr-L-Phe and L-Tyr-L-Ile sequences, markedly increased the tyrosine phosphorylation of 32-34 kDa proteins in src ts NRK cells. However, the profiles of the total phosphotyrosine of proteins from cells treated with 7a and 7b, containing L-Phe-L-Phe and D-Phe-LPhe sequences, are strikingly different from the profiles of those treated with 6a and 6e. The action of 6a on the tyrosine phosphorylation of proteins in src ts NRK cells is very similar to that of the tyrosine kinase inhibitor, erbstatin.9) The EGF-R protein tyrosine kinase, a transmembrane glycoprotein, is one target for the treatment of proliferative diseases. 8,10) Our results show that compounds 6a and 6b are inhibitors of EGF-R tyrosine phosphorylation.Thus, we believe that the double-stranded peptides, 6a, b and 6c, d, can be used to control the inhibitory effect on proliferation shown by both diastereomers, (D-, L-) and (L-, D-), and we describe new insights which could explain the different inhibitory effects on proliferation of Phe and Tyr, (...

In a previously published study, we designed and synthesized parallel double-stranded peptides (1), bis(z-Phey-Phe-x-Val) 2 -spacer(S) and bis(y-Phe-x-Phe) 2 -spacer(S) (symbols x, y and zϭL-or D-configurations, AAϭamino acid, Sϭspacer, and Pheϭphenylalanine), conjugated with two -Val-Phe-Phe-and -Phe-Phe-peptide residues, respectively, to a polyamine spacer.1,2) The -Val-Phe-Phe-and -Phe-Phe-sequences are p-electron rich and in hydrophobic sequence, and the appearance rate is low in the natural proteins. For example, the appearance rates of the -Phe-Pheand -Val-Phe-Phe-sequences in a human progesterone receptor form B (hPR; 933AA (AAϭamino acid) 3) ) were 0%. However, these sequences are also contained in the positions from 21st to 23rd in b-amyloid (Ab) peptide in Alzheimer's disease. 4,5) Thus, the Phe-Phe sequence as a hydrophobic amino acid may be playing an important role in cell viability, but the biological role and effects are not known at all. To elucidate the chemical and biological functions, we are very interested in designing functional double-stranded peptides improving on cell growth inhibition, penetration, and viability by conjugation of Phe and Phe-Phe. The introduction of p-electron rich and a hydrophobic side chain in the doublestranded peptides can have a role of molecular inclusion and recognition by interaction between the double-stranded peptide and the target. (L-, D-), and (D-, D-), to bis(y-Phe-x-Phe) 2 -spacer(S) are possible in the synthesis and we synthesized them.From a pharmacological standpoint, we are interested in what difference four optical isomers, enantiomer and diastereomer, have on the viability of living cells. Especially, the anti-proliferative effects on tumor cells of the diastereomers are poorly understood when compared with that of the enantiomer. Here, our aim in this study is to understand better the structure-activity relationships between cell anti-proliferative effects and four optical isomers of the classes of bis(y-Phe-x-Phe) 2 -spacer(S). We found that the bis(y-Phex-Phe) 2 -spacer(S)s are very valuable for investigating the anti-proliferative effect of tumor cells since the evidence for the configurational pattern of the compound-target cell interaction on cell death is lacking.In stimulation of N 1 ,N 12 -bis(y-Phe-x-Phe)dodecanediamine (18a-d) to src ts NRK cells, 6,7) for instance, we first found that the growth inhibitory decreases in the following order:-bis(L-Phe-L-Phe)dodecanediamine (18a). Similar trends were also observed for other cells, such the A431 and A549 cells. 8,9) These results indicate that the diastereomers, (D-, L-) and (L-, D-) are more potent than the (L-, L-) configuration. The death of src ts NRK and A431 cells is controlled in the chilarity of 18a-d. The geometric control by the diastereomers of the cell growth inhibition (IC 50 ) may present good advantages for the uptake of the drug. Because the double-stranded peptides interact with the cell membrane and may play an important role in the transportation of polyamines, 10) Na ϩ ,...