[Development of New Synthetic Methods for Carbazole Compounds Aimed at Drug Discovery and Exploratory Research on Pharmaceutical Materials].

We are developing the synthesis of biologically interesting carbazole compounds, including natural products by tandem cyclic reactions. In this report, we describe the new synthesis of carbazole-1,4-quinones as follows; 1) the synthesis of carbazole-1,4-quinones using a tandem ring closing metathesis (RCM) -dehydrogenation reaction, 2) a novel one-pot synthesis of carbazole-1,4-quinone by consecutive Pd-catalyzed cyclocarbonylation, desilylation, and oxidation reactions. Two new synthetic strategies were applied to the synthesis of carbazole-1,4-quinone alkaloids and ellipticine quinones, and then the antiproliferative activity against HCT-116 and HL-60 cells of the synthesized compounds were evaluated.

A Facile Procedure for One-Pot Stable Conjugation of Two Proglucagon Cysteine-Containing Peptide Analogs.

Optimization of peptides for therapeutic purposes often includes chemical conjugation or modification with substituents that serve to broaden pharmacology or improve pharmacokinetics. We report a convenient and rapid procedure for one-pot, site-specific conjugation of two cysteine-containing peptides that utilizes a bivalent linker comprising maleimide and iodoacetyl functional groups. Following maleimide-mediated peptide conjugation the linker was converted from an unstable thiosuccinimide to a stable thioether bond suitable for biological study by mild aqueous hydrolysis. The procedure is exemplified by peptide-peptide, peptide-small molecule, and peptide-fatty acid conjugations. The method provides a facile approach to search for enhanced biological outcomes through additive and sustained peptide pharmacology unencumbered by the prospect of chemical rearrangement in the course of biological study.

Recent Advances and Trends in Chemical CPP-Drug Conjugation Techniques.

This review summarizes recent developments in conjugation techniques for the synthesis of cell-penetrating peptide (CPP)-drug conjugates targeting cancer cells. We will focus on small organic molecules as well as metal complexes that were used as cytostatic payloads. Moreover, two principle ways of coupling chemistry will be discussed direct conjugation as well as the use of bifunctional linkers. While direct conjugation of the drug to the CPP is still popular, the use of bifunctional linkers seems to gain increasing attention as it offers more advantages related to the linker chemistry. Thus, three main categories of linkers will be highlighted, forming either disulfide acid-sensitive or stimuli-sensitive bonds. All techniques will be thoroughly discussed by their pros and cons with the aim to help the reader in the choice of the optimal conjugation technique that might be used for the synthesis of a given CPP-drug conjugate.

The Activation of Procarcinogens by CYP1A1/1B1 and Related Chemo-Preventive Agents: A Review.

CYP1A1 and CYP1B1 are extrahepatic P450 family members involved in the metabolism of procarcinogens, such as PAHs, heterocyclic amines and halogen-containing organic compounds. CYP1A1/1B1 also participate in the metabolism of endogenous 17-ß-estradiol, producing estradiol hydroquinones, which are the intermediates of carcinogenic semiquinones and quinones. CYP1A1 and CYP1B1 proteins share approximately half amino acid sequence identity but differ in crystal structures. As a result, CYP1A1 and CYP1B1 have different substrate specificity to chemical procarcinogens. This review will introduce the general molecular biology knowledge of CYP1A1/1B1 and the metabolic processes of procarcinogens regulated by these two enzymes. Over the last four decades, a variety of natural products and synthetic compounds which interact with CYP1A1/1B1 have been identified as effective chemo-preventive agents against chemical carcinogenesis. These compounds are mainly classified as indirect or direct CYP1A1/1B1 inhibitors based on their distinct mechanisms. Indirect CYP1A1/1B1 inhibitors generally impede the transcription and translation of CYP1A1/1B1 genes or interfere with the translocation of aryl hydrocarbon receptor (AHR) from the cytosolic domain to the nucleus. On the other hand, direct inhibitors inhibit the catalytic activities of CYP1A1/1B1. Based on the structural features, the indirect inhibitors can be categorized into the following groups: flavonoids, alkaloids and synthetic aromatics, whereas the direct inhibitors can be categorized into flavonoids, coumarins, stilbenes, sulfur containing isothiocyanates and synthetic aromatics. This review will summarize the in vitro and in vivo activities of these chemo-preventive agents, their working mechanisms, and related SARs. This will provide a better understanding of the molecular mechanism of CYP1 mediated carcinogenesis and will also give great implications for the discovery of novel chemo-preventive agents in the near future.

Finding a resveratrol analogue as potential anticancer agent with apoptosis and cycle arrest.

Resveratrol has been extensively studied as the anti-cancer agent. A variety of resveratrol analogues have been developed with structural modification to improve its bioactivity. In this work, resveratrol analogues, compound 1-4, were designed and synthesized with the Stille-Heck reaction. These results showed compound 1-4 had better anticancer effect than that of parent resveratrol. Especially compound 1 ((E)-4,4'-(ethene-1,2-diyl)bis(3-methylphenol)) displayed the excellent cytotoxicity and high selectivity. The mechanism research indicated compound 1 inhibited cell proliferation by binary paths of cell cycle arrest in S phase regulated by cyclin A1/A2 and apoptosis induction mediated by Bax/Bcl2 in a prooxidant manner.

[Medicinal Chemical Studies Based on the Theoretical Design of Bioactive Compounds].

I have engaged in medicinal chemical studies based on the theoretical design of bioactive compounds. First, I present a three-dimensional structural diversity-oriented conformational restriction strategy for developing bioactive compounds based on the characteristic steric and stereoelectronic features of cyclopropane. Using this strategy, various biologically active small molecule compounds, such as receptor agonists/antagonists and enzyme inhibitors, were effectively developed. The strategy was also applied to develop versatile peptidomimetics and membrane-permeable cyclic peptides. Next, studies on Ca2+-mobilizing second messengers, cyclic ADP-ribose (cADPR) and myo-inositol trisphosphates (IP3), are described. In these studies, stable equivalents of cADPR were developed, since biological studies of cADPR have been limited due to its instability. Various potent IP3 receptor ligands, which were designed using the d-glucose structure as a bioisostere of the myo-inositol moiety of IP3, have been identified. Organic chemistry studies have also been extensively performed, because excellent organic chemistry is essential for promoting high-level medicinal chemical studies. For examples, new methods for the synthesis of chiral cyclopropanes, new radical reactions with silicon tethers, and kinetic anomeric effect-dependent stereoselective glycosidations have been developed.

Cysteine-specific protein multi-functionalization and disulfide bridging using 3-bromo-5-methylene pyrrolones.

Many reagents have been developed for cysteine-specific protein modification. However, few of them allow for multi-functionalization of a single Cys residue and disulfide bridging bioconjugation. Herein, we report 3-bromo-5-methylene pyrrolones (3Br-5MPs) as a simple, robust, and versatile class of reagents for cysteine-specific protein modification. These compounds can be facilely synthesized via a one-pot mild reaction and they show comparable tagging efficiency but higher cysteine specificity than the maleimide counterparts. The addition of cysteine to 3Br-5MPs generates conjugates that are amenable to secondary addition by another thiol or cysteine, making 3Br-5MPs valuable for multi-functionalization of a single cysteine and disulfide bridging bioconjugation. The labeling reaction and subsequent treatments are mild enough to produce stable and active protein conjugates for biological applications.

An unusual double radical homolysis mechanism for the unexpected activation of the aldoxime nerve-agent antidotes by polyhalogenated quinoid carcinogens under normal physiological conditions.

We have recently shown that the pyridinium aldoximes, best-known as therapeutic antidotes for chemical warfare nerve-agents, could markedly detoxify the carcinogenic tetrachloro-1,4-benzoquinone (TCBQ) via an unusual double Beckmann fragmentation mechanism. However, it is still not clear why pralidoxime (2-PAM) cannot provide full protection against TCBQ-induced biological damages even when 2-PAM was in excess. Here we show, unexpectedly, that TCBQ can also activate pralidoxime to generate a reactive iminyl radical intermediate in two-consecutive steps, which was detected and unequivocally characterized by the complementary application of ESR spin-trapping, HPLC/MS and nitrogen-15 isotope-labeling studies. The same iminyl radical was observed when TCBQ was substituted by other halogenated quinones. The end product of iminyl radical was isolated and identified as its corresponding reactive and toxic aldehyde. Based on these data, we proposed that the reaction of 2-PAM and TCBQ might be through the following two competing pathways: a nucleophilic attack of 2-PAM on TCBQ forms an unstable transient intermediate, which can decompose not only heterolytically to form 2-CMP via double Beckmann fragmentation, but also homolytically leading to the formation of a reactive iminyl radical in double-steps, which then via H abstraction and further hydrolyzation to form its corresponding more toxic aldehyde. Analogous radical homolysis mechanism was observed with other halogenated quinones and pyridinium aldoximes. This study represents the first detection and identification of reactive iminyl radical intermediates produced under normal physiological conditions, which provides direct experimental evidence to explain only the partial protection by 2-PAM against TCBQ-induced biological damages, and also the potential side-toxic effects induced by 2-PAM and other pyridinium aldoxime nerve-agent antidotes.

Organometallic Gold(III) Reagents for Cysteine Arylation.

An efficient method for chemoselective cysteine arylation of unprotected peptides and proteins using Au(III) organometallic complexes is reported. The bioconjugation reactions proceed rapidly (<5 min) at ambient temperature in various buffers and within a wide pH range (0.5-14). This approach provides access to a diverse array of S-aryl bioconjugates including fluorescent dye, complex drug molecule, affinity label, poly(ethylene glycol) tags, and a stapled peptide. A library of Au(III) arylation reagents can be prepared as air-stable, crystalline solids in one step from commercial reagents. The selective and efficient arylation procedures presented in this work broaden the synthetic scope of cysteine bioconjugation and serve as promising routes for the modification of complex biomolecules.

Phosphine Induced Dimerization of Propargyl Alcohols Leading to Allyl Propargyl Ethers.

A facile method for synthesizing allyl propargyl ethers (APEs) was developed based on the dimerization of propargyl alcohols. The reaction proceeded via an oxaphosphetane intermediate, which was generated without the use of a strong base, thus making this process a pseudo-Wittig reaction under mild reaction conditions. A wide variety of functional groups, including formyl and pyridyl groups were tolerated, thus yielding the corresponding functionalized APEs, which are otherwise not readily prepared via conventional methods. Moreover, a cross-reaction was found to occur when the reaction was conducted in the presence of alcohols that were more acidic than propargyl alcohol, which suggests that the proton transfer from the intermediately formed betaine to the second alcohol is crucial for undergoing the dimerization.

Facile design of organic-inorganic hybrid gels for molecular recognition of nucleoside triphosphates.

We report the molecular recognition for nucleoside triphosphates inside the ligand-modified water-soluble organic-inorganic hybrid gels composed of polyhedral oligomeric silsesquioxane (POSS). The series of ligands were designed to make hydrogen bonds with each nucleobase and introduced into the hybrid gels. From the titration experiments, the binding constants between the ligand inside the gels and nucleosides were evaluated. Accordingly, it was found that the ligands inside the gels can form a stable complex only with the target nucleoside triphosphate which has the complementary pattern of the hydrogen bonds (Ka=G-gel/cytidine triphosphate (CTP), 3.5×10(8)M(-1); U-gel/adenosine triphosphate (ATP), 1.6×10(3)M(-1); A-gel/uridine triphosphate (UTP), 1.9×10(7), respectively). With other nucleoside derivatives which have different numbers of phosphate units or different types of nucleobases, the much weaker interaction was detected. These data indicate that the complex formation only with nucleoside triphosphates should occur inside the hybrid gels, and selective recognition of each nucleoside triphosphate can be accomplished.

Enzymatic production of biodiesel from microalgal oil using ethyl acetate as an acyl acceptor.

Microalgae have become an important source of biomass for biodiesel production. In enzymatic transesterification reaction, the enzyme activity is decreased in presence of alcohols. The use of different acyl acceptors such as methyl/ethyl acetate is suggested as an alternative and effective way to overcome this problem. In this study, ethyl acetate was used for the first time in the enzymatic production of biodiesel by using microalga, Chlorella vulgaris, as a triglyceride source. Enzymatic conversion of such fatty acids to biodiesel was catalyzed by Novozym 435 as an efficient immobilized lipase which is extensively used in biodiesel production. The best conversion yield of 66.71% was obtained at the ethyl acetate to oil molar ratio of 13:1 and Novozym 435 concentration of 40%, based on the amount of oil, and a time period of 72 h at 40℃. The results showed that ethyl acetate have no adverse effect on lipase activity and the biodiesel amount was not decreased even after seven transesterification cycles, so ethyl acetate has a great potential to be substituted for short-chain alcohols in transesterification reaction.

Reaction parameters for the synthesis of N,N-dimethyl fatty hydrazides from oil.

Hydrazide derivatives have been synthesized from methyl esters, hydrazones and vegetable oils. They are important due to their diverse applications in pharmaceutical products, detergents as well as in oil and gas industries. The chemical synthesis of fatty hydrazides is well-established; however, only a few publications described the synthesis of fatty hydrazide derivatives, particularly, when produced from refined, bleached and deodorized palm olein. Here, the synthesis and characterization of N,N-dimethyl fatty hydrazides are reported. The N,N-dimethyl fatty hydrazides was successfully synthesized from fatty hydrazides and dimethyl sulfate in the presence of potassium hydroxide with the molar ratio of 1:1:1, 6 hours reaction time and 80℃ reaction temperature in ethanol. The product yield and purity were 22% and 89%, respectively. The fatty hydrazides used were synthesized from refined, bleached and deodorized palm olein with hydrazine monohydrate at pH 12 by enzymatic route. Fourier transform infrared, gas chromatography and nuclear magnetic resonance (NMR) spectroscopy techniques were used to determine the chemical composition of N,N-dimethyl fatty hydrazides. Proton NMR confirmed the product obtained were N,N-dimethyl fatty hydrazides.

Nucleophilic substitution as a mechanism of atrazine sequestration in soil.

Formation of nonextractable residue was widely observed as a sink of atrazine (ATZ) in soil. However, the mechanisms by which ATZ binds to soil organic matter remain unclear. In this study, we demonstrated that neucleophilic substitution could serve an important pathway causing ATZ sequestration. The carbon bonded to the chlorine in ATZ molecule is partially positively charged due to the strong electronegativity of chlorine and is susceptible to the attack of nucleophiles such as aniline. Since aromatic amines are relatively rare in natural soils, amino acids/peptides were hypothesized to act as the main nucleophiles in real environment. However, substantially ATZ transformation was only observed in the presence of those species containing thiol functionality. Thus, we speculated that it was the thiol group in amino acids/peptides acting as the nucleophile. Nitrogen in amino acids was in fact not an active nucleophile toward ATZ. In addition to the sulfur-containing amino acids, other thiol compounds, and sulfide were also proved to be reactive to ATZ. Thus, the sequestration potential of ATZ probably correlates to the availability of thiol compounds in soil.

Synthesis and characterization of polyurethanes made from copolymers of epoxidized natural oil and tetrahydrofuran.

Polyols were synthesized from epoxidized natural oils and tetrahydrofuran through ring opening copolymerization catalyzed by Lewis acid. The properties of these polyols depend on the type of natural oils as well as the reaction conditions (monomer concentration, catalyst amount, reaction temperature and reaction time). These polyols were evaluated as a raw material for making polyurethane (PU) in order to understand the structure-property relationship between a natural oil and PU made from it. The tensile test analysis shows that the incorporation of natural oil moiety into the PU polymer network improves the elasticity of these PU samples when compared to a benchmark PU sample. In addition, the PU samples made from palm oil and soybean oil based polyols exhibit better tensile strength than benchmark PU. These polyols samples are suitable for making elastomeric PU, where high flexibility (high elongation at break) of PU is a common requirement.

Organocatalytic, diastereo- and enantioselective synthesis of nonsymmetric cis-stilbene diamines: a platform for the preparation of single-enantiomer cis-imidazolines for protein-protein inhibition.

The finding by scientists at Hoffmann-La Roche that cis-imidazolines could disrupt the protein-protein interaction between p53 and MDM2, thereby inducing apoptosis in cancer cells, raised considerable interest in this scaffold over the past decade. Initial routes to these small molecules (i.e., Nutlin-3) provided only the racemic form, with enantiomers being enriched by chromatographic separation using high-pressure liquid chromatography (HPLC) and a chiral stationary phase. Reported here is the first application of an enantioselective aza-Henry approach to nonsymmetric cis-stilbene diamines and cis-imidazolines. Two novel mono(amidine) organocatalysts (MAM) were discovered to provide high levels of enantioselection (>95% ee) across a broad range of substrate combinations. Furthermore, the versatility of the aza-Henry strategy for preparing nonsymmetric cis-imidazolines is illustrated by a comparison of the roles of aryl nitromethane and aryl aldimine in the key step, which revealed unique substrate electronic effects providing direction for aza-Henry substrate-catalyst matching. This method was used to prepare highly substituted cis-4,5-diaryl imidazolines that project unique aromatic rings, and these were evaluated for MDM2-p53 inhibition in a fluorescence polarization assay. The diversification of access to cis-stilbene diamine-derived imidazolines provided by this platform should streamline their further development as chemical tools for disrupting protein-protein interactions.

Use of a novel 5'-regioselective phosphitylating reagent for one-pot synthesis of nucleoside 5'-triphosphates from unprotected nucleosides.

5'-Triphosphates are building blocks for enzymatic synthesis of DNA and RNA. This unit presents a protocol for convenient synthesis of 2'-deoxyribo- and ribonucleoside 5'-triphosphates (dNTPs and NTPs) from any natural or modified base. This one-pot synthesis can also be employed to prepare triphosphate analogs with a sulfur or selenium atom in place of a non-bridging oxygen atom of the α-phosphate. These S- or Se-modified dNTPs and NTPs can be used to prepare diastereomerically pure phosphorothioate or phosphoroselenoate nucleic acids. Even without extensive purification, the dNTPs or NTPs synthesized by this method are of high quality and can be used directly in DNA polymerization or RNA transcription. Synthesis and purification of the 5'-triphosphates, as well as analysis and confirmation of natural and sulfur- or selenium-modified nucleic acids, are described in this protocol unit.

Synthesis of radiolabeled protein disulfide isomerase (PDI) inhibitors as new potential PET agents for imaging of the enzyme PDI in neurological disorders and cancer.

Carbon-11-labeled substituted ß-tetrahydrocarbolines were prepared from their corresponding phenolic hydroxyl precursors with [(11)C]CH3OTf through O-[(11)C]methylation and isolated by simplified SPE in 50-60% decay corrected radiochemical yields at EOB with 185-370GBq/µmol specific activity at EOS. A fluorine-18-labeled substituted ß-tetrahydrocarboline was prepared from its corresponding halo-precursors (X=Cl, Br, I) with K[(18)F]F/Kryptofix 2.2.2 via the nucleophilic substitution and isolated by HPLC combined with SPE in 25-40% decay corrected radiochemical yield with 37-222GBq/µmol specific activity at EOB.

Synthesis and morphogenesis of organic and inorganic polymers by means of biominerals and biomimetic materials.

We have studied the simultaneous synthesis and morphogenesis of polymer materials with hierarchical structures from nanoscopic to macroscopic scales. The morphologies of the original materials can be replicated to the polymer materials. In general, it is not easy to achieve the simultaneous synthesis and morphogenesis of polymer material even using host materials. In the present work, four biominerals and three biomimetic mesocrystal structures are used as the host materials or templates and polypyrrole, poly(3-hexylthiopehene), and silica were used as the precursors for the simultaneous syntheses and morphogenesis of polymer materials. The host materials with the hierarchical structure possess the nanospace for the incorporation of the monomers. After the incorporation of the monomers, the polymerization reaction proceeds in the nanospace with addition of the initiator agents. Then, the dissolution of the host materials leads to the formation and morphogenesis of the polymer materials. The scheme of the replication can be classified into the three types based on the structures of the host materials (types I-III). The type I template facilitates the hierarchical replication of the whole host material, type II mediates the hierarchical surface replication, and type III induces the formation of the two-dimensional nanosheets. Based on these results, the approach for the coupled synthesis and morphogenesis can be applied to a variety of combinations of the templates and polymer materials.

Carbohydrate synthesis and biosynthesis technologies for cracking of the glycan code: recent advances.

The glycan code of glycoproteins can be conceptually defined at molecular level by the sequence of well characterized glycans attached to evolutionarily predetermined amino acids along the polypeptide chain. Functional consequences of protein glycosylation are numerous, and include a hierarchy of properties from general physicochemical characteristics such as solubility, stability and protection of the polypeptide from the environment up to specific glycan interactions. Definition of the glycan code for glycoproteins has been so far hampered by the lack of chemically defined glycoprotein glycoforms that proved to be extremely difficult to purify from natural sources, and the total chemical synthesis of which has been hitherto possible only for very small molecular species. This review summarizes the recent progress in chemical and chemoenzymatic synthesis of complex glycans and their protein conjugates. Progress in our understanding of the ways in which a particular glycoprotein glycoform gives rise to a unique set of functional properties is now having far reaching implications for the biotechnology of important glycodrugs such as therapeutical monoclonal antibodies, glycoprotein hormones, carbohydrate conjugates used for vaccination and other practically important protein-carbohydrate conjugates.