Nobuki Kato

Facile synthesis of peptide–porphyrin conjugates: Towards artificial catalase

A facile synthetic method for peptide-porphyrin conjugates containing four peptide units on one porphyrin was developed using chemoselective reactions. The key building blocks, 5,10,15,20-tetrakis(3-azidophenyl)porphyrin 1 and 5,10,15,20-tetrakis(5-azido-3-pyridyl)porphyrin 2, were efficiently synthesized and used as substrates for two well-known chemoselective reactions, traceless Staudinger ligation and copper-catalyzed azide alkyne cycloaddition (so-called click chemistry). Both reactions gave the desired compounds, and click chemistry was superior for our purpose. To confirm the value of the established methodology, nine peptide-porphyrin conjugates were synthesized, and their catalase- and peroxidase-like activity in water was evaluated. Our synthetic strategy is expected to be valuable for the preparation of artificial heme protein models.

Manganese Salen Complexes with Acid–Base Catalytic Auxiliary: Functional Mimetics of Catalase

Antioxidant therapies have been considered for a wide variety of disorders associated with oxidative stress, and synthetic catalytic scavengers of reactive oxygen species would be clinically superior to stoichiometric ones. Among them, salen-manganese complexes (Mn(Salen)) seem promising, because they exhibit dual functions, i.e. superoxide dismutase- and catalase-mimetic activities. We have been developing enzyme-mimetic Mn(Salen) complexes bearing a functional group that enhances their catalytic activity. Here, we describe the design and synthesis of novel Mn(Salen) complexes with general acid-base catalytic functionality, inspired by the reaction mechanism of catalase. As expected, these Mn(Salen) complexes showed superior catalase-like activity and selectivity, while retaining moderate SOD-like activity. An unsubstituted pyridyl group worked well as a functionality to promote catalase-like activity. The introduced functionality did not alter the redox potential suggesting that the auxiliary-modified complex acted as an acid-base catalyst analogous to catalase. We believe that our approach provides a new design principle for sophisticated catalyst design. Further, the compounds described here appear to be good candidates for use in antioxidant therapy.

Synthesis of the carbon framework of scholarisine A by intramolecular oxidative coupling.

Scholarisine A, isolated from the leaves of Alstonia scholaris, is a monoterpene indole alkaloid with an unprecedented cage-like structure. In this paper, preparation of the distinctive cage-like core skeleton of scholarisine A is described. The key feature of this synthetic strategy is an intramolecular oxidative coupling reaction at the late stage to construct a 10-oxa-tricyclo[5.3.1.0(3, 8)]undecan-9-one structure fused with indolenine. Intramolecular oxidative coupling by using N-iodosuccinimide gave the carbon framework of scholarisine A in moderate yield, which is the first example of intramolecular oxidative-coupling reaction between non-activated enolate and indole. This study lays the foundation for continued investigations towards the total synthesis of scholarisine A.

Effect of helical conformation and side chain structure on γ-secretase inhibition by β-peptide foldamers: insight into substrate recognition.

Substrate-selective inhibition or modulation of the activity of γ-secretase, which is responsible for the generation of amyloid-β peptides, might be an effective strategy for prevention and treatment of Alzheimers disease. We have shown that helical β-peptide foldamers are potent and specific inhibitors of γ-secretase. Here we report identification of target site of the foldamers by using a photoaffinity probe. The photoprobe directly and specifically labeled the N-terminal fragment of presenilin 1, in which the initial substrate docking site is predicted to be located. We also optimized the foldamer structure by preparing a variety of derivatives and obtained two highly potent foldamers by incorporation of a hydrophilic and neutral functional group into the parent structure. The class of side chain functional group and the position of incorporation were both important for γ-secretase-inhibitory activity. The substrate selectivity of the inhibitory activity was also quite sensitive to the class of side chain group incorporated.

Array-based fluorescence assay for serine/threonine kinases using specific chemical reaction

We report herein the development of an efficient fluorescence assay for serine/threonine kinases using a peptide array. Our approach is based on chemical reactions specific to phosphoserine and phosphothreonine residues, that is, base-mediated beta-elimination of the phosphate group and subsequent Michael addition of a thiol-containing fluorescent reagent. This procedure enables the covalent introduction of a fluorescent moiety into the phosphorylated peptide. Novel fluorescent reagents were designed for this purpose and synthesized. With these reagents, protein kinase A (PKA) and Akt-1 activities were readily detected. Our method can also be used to measure the activity of kinase inhibitors. This assay is expected to be widely applicable in kinase research.

Photocontrol of Peptide Function: Backbone Cyclization Strategy with Photocleavable Amino Acid

Biologically active compounds with light-responsive function offer experimental possibilities that are otherwise difficult to achieve. Temporal and spatial regulation of the functions of proteins is central to biological processes. Hence, the ability to artificially trigger molecular events in a biologically relevant context is expected to be useful for the study of living organisms. Caged compounds, namely photoactivatable bioagents, are a representative class of such compounds. Such caged compounds are biologically inactive and stable under physiological conditions. The classical technology to develop caged compounds is the modification of a key functional group with a photoremovable protective moiety, such as the o-nitrobenzyl or coumarin-4-ylmethyl group. Upon irradiation with light, the caged molecule is irreversibly activated by the removal of the photolabile group. Since caged peptides are powerful tools for controlling cellular signaling, many have been reported to date. Typical caged peptides have been synthesized by means of standard solid-phase methods by using side-chain caged amino acids, such as Ser, Thr, Tyr, Asp, Glu, Lys, Arg and phosphorylated derivatives of Ser, Thr, and Tyr. d] This strategy is popular and effective, but a caging group can only be introduced into certain amino acids; some amino acids (e.g. , Phe and Leu) cannot be caged in this way. Also, identification of a single critical residue is difficult or sometimes impossible; multiple incorporation of photolabile groups is sometimes necessary. To overcome these limitations, backbone-caged peptides have been reported, in which a photolabile group is introduced on the amide backbone. However, the position of the photolabile group is limited; for example, incorporation of a photolabile group close to a bulky side-chain is difficult. As another approach, a number of successful examples of the control of peptide conformation with light have been reported. The most successful approach seems to be the use of a photoisomerizable group, such as azobenzene or stilbene. The conformation of a cyclic peptide was photocontrolled by the introduction of azobenzene into the backbone of peptides. b] In the case of a-helical peptides, the photoresponsive group was incorporated through side-chain cross-linking. e, 8a, b, d] For b-sheet peptides, an artificial b-hairpin has been used as the photoresponsive unit, f, 8c] and, in some cases, the sheet structure was cyclized. 8c] The affinity toward biological targets, such as DNA and proteins (Bcl-XL, PDZ domain, coiled-coil protein) has been successfully photocontrolled. These results suggest that the photocontrol of peptide conformation is a promising approach. The potential reversibility of photoisomerization is an attractive feature, although the active conformation and mode of action of the peptides must be identified for efficient peptide design. Also, thermal cis–trans isomerization of photoisomerizable groups is a concern. Head-to-tail peptide cyclization is a classical and general strategy for making peptides more resistant to enzymatic degradation by exopeptidases, amino and carboxy peptidases. In addition, cyclization reduces the flexibility of peptide conformation and might result in high receptor binding affinity. We envisioned that cyclization would also restrict favorable peptide conformation and reduce the activity compared with the corresponding linear peptide. The structural change of cyclicto-linear by using light could lead to the photocontrol of peptide conformation and activity. Recently, Takahashi et al. reported the photomodulation of protein–ligand interaction (phosphatidylinositol 3-kinase SH3 domain with a peptide ligand); the conformation of a proline-rich peptide was successfully photocontrolled by side-chain-to-side-chain cross-linking with a photocleavable nitrobenzene-derived linker. The binding affinity of the cyclic peptide to the SH3 domain increased modestly (fourfold) upon photoirradiation. This pioneering work elegantly proved the potential of the combination of peptide cyclization and a photocleavable moiety, although the degree of photocontrol remained modest. Here, we present a proof-of-principle study for photocontrol of peptide function, a backbone cyclization strategy with photocleavable amino acid. Our approach is summarized in Figure 1. The peptide conformation is restricted by head-to-tail main-chain cyclization, so that an active conformation cannot be formed; that is, the cyclic peptide is supposed to be inactive. A photocleavable amino acid is incorporated into the cyclic peptide sequence to obtain photoresponsiveness. Upon photoirradiation, the photocleavable amino acid is cleaved to form a linear peptide, which can adopt the active conformation, and is, therefore, biologically active. Our head-to-tail cyclization strategy is based on the occurrence of a large structural change after photoirradiation, and structural information about the peptide active conformation is not needed. To test our idea, we chose matrix metalloproteinase-3 (MMP3) inhibitor peptide as a target; the active conformation of MMP-3 inhibitor peptide (MMPI), to our knowledge, has not [a] Dr. N. Umezawa, Y. Noro, K. Ukai, Dr. N. Kato, Prof. Dr. T. Higuchi Graduate School of Pharmaceutical Sciences, Nagoya City University 3–1 Tanabe-dori, Mizuho-ku, Nagoya (Japan) E-mail : umezawa@phar.nagoya-cu.ac.jp higuchi@phar.nagoya-cu.ac.jp Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201100212.

Turn-on fluorescent probe with visible light excitation for labeling of hexahistidine tagged protein

We report here the development of a novel fluorescein-based probe which shows selective fluorescence enhancement on binding to a hexahistidine-tagged protein. No fluorescence change was observed with untagged protein. This probe is excitable with visible light and is considered to be suitable for use in biological applications.

Structurally Diverse Polyamines: Solid-Phase Synthesis and Interaction with DNA.

A versatile solid‐phase approach based on peptide chemistry was used to construct four classes of structurally diverse polyamines with modified backbones: linear, partially constrained, branched, and cyclic. Their effects on DNA duplex stability and structure were examined. The polyamines showed distinct activities, thus highlighting the importance of polyamine backbone structure. Interestingly, the rank order of polyamine ability for DNA compaction was different to that for their effects on circular dichroism and melting temperature, thus indicating that these polyamines have distinct effects on secondary and higher‐order structures of DNA.

Synthesis of the Bicyclo[7.3.0]dodecadiyne Core of the Maduropeptin Chromophore

Maduropeptin, an extremely potent antitumor agent, consists of a 1:1 complex of a carrier protein and a chromophore. We report herein a general and efficient route for the synthesis of the highly strained bicyclo[7.3.0]dodecadiyne core of the chromophore. The key feature of the synthetic strategy is the use of two Sonogashira coupling reactions in a stepwise manner to construct the conjugated dienyne substructure of the fused-ring system, including the Z alkene at C4,C13.

Role of Thiolate Ligand in Spin State and Redox Switching in the Cytochrome P450 Catalytic Cycle

The catalytic cycle of cytochrome P450 involves a change from the resting-state, water-bound, six-coordinated form (1, low-spin state) to a five-coordinated form (2, high-spin state) upon binding of a hydrophobic substrate. Here, we used a heme-thiolate model complex (SR complex) with THF as a model of nonionic H2O to address the question of whether or not coordination of nonionic water is sufficient to induce the low-spin state. Measurements of electronic absorption spectra and magnetic properties confirmed that five-coordinated SR complex has a high-spin state, and THF-bound, six-coordinated SR has a low-spin state in dichloromethane at ambient temperature. The redox potential E1/2 (FeII/FeIII) of THF-bound SR was 80-90 mV more negative than that of five-coordinated SR. These properties indicate SR is a good model of P450. Our results suggest that thiolate coordination plays a key role in setting the low energy barrier between the high-spin and low-spin states.