The crystallographic study of left-handed Z-DNA d(CGCGCG)2 and thermine complexes crystallized at various temperatures and at various concentration of cations.

In crystals of complexes of thermine and d(CGCGCG)2 molecules grown at 4, 10, and 20 degrees C, the numbers of thermine molecules connected to the DNA molecule were dependent on the temperature of the crystallization. Two molecules of thermine and one Mg2+ ion were connected to DNA molecule when thermine and d(CGCGCG)2 were co-crystallized at 4 and at 20 degrees C. When an increased concentration of magnesium and thermine molecules were co-crystallized with d(CGCGCG)2 molecules at 10 degrees C, three Mg2+ ions and only one thermine molecule were bound with a d(CGCGCG)2 molecule. The number of polyamines and of Mg2+ ions connected to DNA was dependent on the atomic values of the polyamine and of the metal ion. The binding of more Mg2+ ions occurred when the atomic value of Mg2+ exceeded that of the corresponding mono- or polyamine, and when the Mg2+ ion concentration was elevated. Furthermore, this study is the first documentation of a naturally occurring polyamine bound to the minor groove of DNA in a crystal structure.

Polyamines stabilize left-handed Z-DNA: using X-ray crystallographic analysis, we have found a new type of polyamine (PA) that stabilizes left-handed Z-DNA.

There are many great reports of polyamine stabilization of the Z-DNA by bridge conformation between neighboring, symmetry-related Z-DNA in the packing of crystals. However, polyamine binding to the minor groove of Z-DNA and stabilizing the Z-DNA structure has been rarely reported. We proved that the synthesized polyamines bind to the minor groove of Z-DNA and stabilize the conformation under various conditions, by X-ray crystallographic study. These polyamines consist of a polyamine nano wire structure. The modes of the polyamine interaction were changed under different conditions. It is the first example that the crystals consisted of metal free structure. This finding provides a basis for clarifying B-Z transition mechanics.

Self-assembled octameric cage constructed by the potassium salt of p-tert-butylcalix[6]arene p-bromophenylalanine derivative in the solid state.

Potassium salts of p-tert-butylcalix[6]arene p-bromophenylalanine derivative formed sizable octameric cages in the solid state that were revealed by X-ray crystallographic analysis.

The crystal structure of N(1)-[2-(2-amino-ethylamino)-ethyl]-ethane-1,2-diamine (polyamines) binding to the minor groove of d(CGCGCG)(2), hexamer at room temperature.

The crystal structure of a left-handed Z-DNA hexamer, d(CG)(3) in complex with a synthetic polyamine, N(1)-[2-(2-amino-ethylamino)-ethyl]-ethane-1,2-diamine, NH(3)(+)-(CH(2))(2)-NH(2)(+)-(CH(2))(2)-NH(2)(+)-(CH(2))(2)-NH(3)(+) [PA(222)], has been determined by the X-ray diffraction method at 1.0 A resolution. In an orthorhombic crystal, the d(CG)(3) duplex binds two PA(222) molecules, and this synthetic polyamine exhibits dual conformational properties. One of the two PA(222) molecules resides on the floor of the minor groove of a Z-DNA duplex and imino groups bridge the two phosphate chains across a double helix, while the terminal amino groups link the oxygen atoms O2 of four cytosine bases. This PA(222) molecule makes a U-turn like a fishhook at one of its ends to provide a micro-environmental network previously unseen in complexes of DNA with polyamines. The width of the minor groove does not become considerably greater with the looped end of the polyamine, indicating conformational rigidity of the Z-DNA backbone imposed by the high stacking energy of the GC base pairs. While polyamine binding to the minor groove has been postulated by theoretical studies for stabilizing the Z-DNA double helical conformation, the finding in the crystal of the looped polyamine chain binding the minor groove of Z-DNA is observed for the first time from the data collected at 10 degrees C (so-called room temperature data). Another PA(222) molecule binds on the convex outer surface of the major groove of the Z-DNA duplex and links three d(CG)(3) duplexes which are symmetrically related to each other. The structure of this PA(222) presents the previously reported zig-zag type conformation [Egli et al., Biochemistry 30 (1991) 11388-11402]. Comparison of this structure with other polyamine-DNA cocrystals reveals structural themes and differences that may relate to the length of the polyamine.

Concerted interaction between conjugated double bond CHs and multiple OHs in polyene macrolide antibiotic chainin: weak =C-H...O interactions responsible for intrinsic molecular assembly.

The concerted interactions observed between five conjugated double bond CHs and four hydroxy Os in the crystal of chainin, a polyene macrolide antibiotic, clarified the existence of unprecedented, weak =C-H...O interactions, which is important for forming its intrinsic molecular assembly.

Efficient and beta-Stereoselective Synthesis of 4(5)-(beta-D-Ribofuranosyl)- and 4(5)-(2-Deoxyribofuranosyl)imidazoles(1).

A synthetic route to 4(5)-(beta-D-ribofuranosyl)imidazole (1), starting from 2,3,5-tri-O-benzyl-D-ribose (5), was developed via a Mitsunobu cyclization. Reaction of 5 with the lithium salt of bis-protected imidazole afforded the corresponding 5-ribosylimidazole 7RS. Hydrolysis of 7RS gave a 1:1 mixture of diol isomers 8R and 8S having an unsubstituted imidazole. Mitsunobu cyclization of the mixture 8RS using N,N,N',N'-tetramethylazodicarboxamide and Bu(3)P exclusively afforded benzylated beta-ribofuranosyl imidazole 9beta in 92% yield, accompanied by alpha-anomer 9alpha, in a ratio of 26.3:1. The configuration of 9beta was established by X-ray crystallography of ethoxycarbonyl derivative 10beta. Reductive debenzylation of 9beta over Pd/C was carried out, and the synthesis of 1 was attained from starting 5 in four steps and 87% overall yield. This synthetic methodology was extended to the synthesis of 4(5)-(2-deoxy-beta-D-ribofuranosyl)imidazole (2). Mitsunobu cyclization of a 1:1 mixture of the corresponding diol isomers 14RS produced 15beta and 15alpha in a ratio of 5.4:1. The synthesis of 2 was attained in a 59% overall yield from the starting 3,5-di-O-benzyl-2-deoxy-D-ribose (12). beta-Stereoselective glycosylation in the key step is discussed and explained by intramolecular hydrogen bonding between an NH in the imidazole and the oxygen functional group in the sugar moiety.

Inclusion effect and structural basis of cyclodextrins for increased extraction of medicinal alkaloids from natural medicines.

The enhancing effects of alpha-, beta-, and gamma-cyclodextrins (CyDs) on the aqueous extraction of ephedrine and berberine from the natural medicines were investigated in HPLC analysis, and the greatest effect was observed for beta- and gamma-CyDs. To clarify the structural basis of such an increased extraction effect with beta-CyD, possible interaction modes of (1R,2S)-ephedrine with alpha-, beta-, and gamma-CyDs were investigated using molecular dynamic simulations in an aqueous solution system. It was shown that the wrapping model of ephedrine by beta-CyD is the most compact and thus increases the solubility most effectively, compared with those by other CyDs. The same mode could be possible for the greatest effect of gamma-CyD on the extraction of berberine from natural medicines. This clearly shows that CyDs are useful additives for the effective extraction of bioactive alkaloids from natural medicines.

The rare crystallographic structure of d(CGCGCG)(2): the natural spermidine molecule bound to the minor groove of left-handed Z-DNA d(CGCGCG)(2) at 10 degrees C.

Several crystal structure analyses of complexes of synthetic polyamine compounds, including N(1)-(2-(2-aminoethylamino))ethyl)ethane-1,2-diamine PA(222) and N(1)-(2-(2-(2-aminoethylamino)ethylamino)ethyl)ethane-1,2-diamine PA(2222), and left-handed Z-DNA d(CGCGCG)(2) have been reported. However, until now, there have been no examples of naturally occurring polyamines bound to the minor groove of the left-handed Z-DNA of d(CGCGCG)(2) molecule. We have found that spermidine, a natural polyamine, is connected to the minor groove of left-handed Z-DNA of d(CGCGCG)(2) molecule in a crystalline complex grown at 10 degrees C. The electron density of the DNA molecule was clear enough to determine that the spermidine was connected in the minor groove of two symmetry related molecules of left-handed Z-DNA d(CGCGCG)(2). This is the first example that a spermidine molecule can form a bridge conformation between two symmetry related molecules of left-handed Z-DNA d(CGCGCG)(2) in the minor groove.

Variation in cytostatic constituents of a sponge-derived Gymnascella dankaliensis by manipulating the carbon source.

The Halichondria sponge-derived fungus, Gymnacella dankaliensis, was cultured in two different media conditions. A modified malt extract medium containing soluble starch instead of glucose resulted in two extremely unusual steroids, dankasterones A (2) and B (3), while four additional unusual steroids, gymnasterones A (4), B (5), C (6), and D (7), were isolated from the original malt extract medium. Their stereostructures have been established on the basis of spectroscopic analyses along with X-ray crystal structure analyses, modified Mosher's method, CD exciton method, and a chemical transformation. All the steroids except for 4 exhibited significant growth inhibition against the murine P388 cancer cell line. Dankasterone A (2) also exhibited potent growth inhibition against human cancer cell lines.

Structure determination of inonotsuoxides A and B and in vivo anti-tumor promoting activity of inotodiol from the sclerotia of Inonotus obliquus.

Two new lanostane-type triterpenoids, inonotsuoxides A (1) and B (2) along with three known lanostane-type triterpenoids, inotodiol (3), trametenolic acid (4), and lanosterol (5), were isolated from the sclerotia of Inonotus obliquus (Pers.: Fr.) (Japanese name: Kabanoanakake) (Russian name: Chaga). Their structures were determined to be 22R,25-epoxylanost-8-ene-3beta,24S-diol (1) and 22S,25-epoxylanost-8-ene-3beta,24S-diol (2) on the basis of spectral data including single crystal X-ray analysis. These compounds except for 2 were tested for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), as a test for potential cancer chemopreventive agents. The most abundant triterpene, inotodiol (3), was investigated for the inhibitory effect in a two-stage carcinogenesis test on mouse skin using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA as a promoter. Compound 3 was found to exhibit the potent anti-tumor promoting activity in the in vivo carcinogenesis test.

A crystal structure study when crystallized only in d(CG)3 + methylamine and a d(CG)3 + metal ion.

There is not an example of the crystallization only of d(CG)(3) and a metallic cation without putting the amine in addition in there is no example of the crystallization d(CG)(3) with methylamine that is the mono-amine so far either. We were able to obtain a very beautiful crystal with all sides of 0.3mm by the sitting drop method that had done the crystallization of d(CG)(3)+ methylamine and d(CG)(3)+ Mg(2 +). The crystal was diffracted up to 1.0 A by using the synchrotron radiation. As a result of the X-ray crystal structure analysis, methylamine was not seen while d(CG)(3)+ methylamine was crystallizing mysteriously. d(CG)(3) had stabilized with five Mg(2+) ions. d(CG)(3) had stabilized similarly with five Mg(2+) ions in the crystal of d(CG)(3)+ Mg(2+). The position of these five Mg(2+) ions understood that I agreed on superimpose in a crystal of d(CG)(3) + methylamine, a crystal of d(CG)(3)+Mg(2+), a crystal of d(CG)(3) + spermidine, and crystals of d(CG)(3) + PA (24) which was already analyzed so as to cut it and stabilized d(CG)(3) at an about the same position.

Amine free crystal structure: the crystal structure of d(CGCGCG)2 and methylamine complex crystal.

We succeeded in the crystallization of d(CGCGCG)2 and methylamine Complex. The crystal was clear and of sufficient size to collect the X-ray crystallographic data up to 1.0 A resolution using synchrotron radiation. As a result of X-ray crystallographic analysis of 2Fo-Fc map was much clear and easily traced. It is the first time monoamine co-crystallizes with d(CGCGCG)2. However, methylamine was not found from the complex crystal of d(CGCGCG)2 and methylamine. Five Mg ions were found around d(CGCGCG)2 molecules. These Mg ions neutralized the anion of 10 values of the phosphate group of DNA with five Mg2+. DNA stabilized only by a metallic ion and there is no example of analyzing the X-ray crystal structure like this. Mg ion stabilizes the conformation of Z-DNA. To use monoamine for crystallization of DNA, we found that we can get only d(CGCGCG)2 and Mg cation crystal. Only Mg cation can stabilize the conformation of Z-DNA. The method of using the monoamine for the crystallization of DNA can be applied to the crystallization of DNA of long chain of length in the future like this.

Synthesis and properties of 2'-O,4'-C-methyleneoxymethylene bridged nucleic acid.

A novel bridged nucleic acid (BNA) analogue, 2'-O,4'-C-methyleneoxymethylene bridged nucleic acid (2',4'-BNA(COC)), was synthesized and incorporated into oligonucleotides. The 2',4'-BNA(COC) modified oligonucleotides showed high binding affinity with an RNA complement and significant enzymatic stability against snake venom phosphodiesterase.

Antineoplastic agents. 551. Isolation and structures of bauhiniastatins 1-4 from Bauhinia purpurea.

Bioassay-guided (P388 lymphocytic leukemia cell line) separation of extracts prepared from the leaves, stems, and pods of Bauhinia purpurea, and, in parallel, its roots, led to the isolation of four new dibenz[b,f]oxepins (2a, 3-5) named bauhiniastatins 1-4, as well as the known and related pacharin (1) as cancer cell growth inhibitors. The occurrence of oxepin derivatives in nature is quite rare. Bauhiniastatins 1-4 were found to exhibit significant growth inhibition against a minipanel of human cancer cell lines, and bauhiniastatin 1 (2a) was also found to inhibit the P388 cancer cell line. Structures for these new cancer cell growth inhibitors were established by spectroscopic techniques that included HRMS and 2D NMR.

Measurement of circular dichroism and structural chemical research of d(CG)6 and d(TA)6.

It was known that d(CG)(6) became left-handed Z-DNA by a theoretical calculation. We carried out a CD measurement of d(CG)(6) at room temperature. B-Z transition was occurred when I raised the concentration of Mg salt. In addition, I confirmed that the Mg salt concentration caused B-Z transition by the lower concentration under the existence of cobalt hexamine. Structure of d(TA)(6) is not known. We performed the CD measurement of d(TA)(6) at low temperature. Structure changed from B type when I raised the Mg salt concentration in an existence of cobalt hexamine. I am examining structure now.

Circular dichroism spectra of d(CGCGCGCGCGCG): evidence for intermediate models in the B-to-Z transition.

We analyze CD spectra of d(CGCGCGCGCGCG) to study the potential intermediate states produced during transition between both types of secondary structures of B- and Z-forms as ionic strength of PBS buffer increases. Decomposition of the CD spectra by linear superposition provides evidence for three intermediate states that distinct from B and Z-DNA. Intermediate states of B-form and Z-form found from CD spectra analysis are discussed in terms of early proposed by others the models for B- to Z-DNA transition and W-DNA model refined by molecular modelling program.

The development of crystallization and X-ray crystallography method of long stem DNA.

We clarified stabilization mechanism of left-handed Z-DNA by X-ray crystal structure analysis. I was able to achieve it by burying big minor groove of Z-DNA because metal ion and polyamine connected it to minor groove of Z-DNA. On that occasion I understood that an electric charge and length of polyamine gave big influence to a combination brain very much. I succeeded in crystallization of Z-DNA of a long chain, X-ray crystal structure analysis by examining these combination ability.

Stabilization of the left-handed Z-DNA with monoamines and polyamines.

It is known that the crystal structure of d(CG)3 become left-handed Z-DNA under high salt concentration and various polyamines stabilize the Z-DNA structure. We have structurally investigated how polyamines stabilize the Z-DNA by the X-ray crystallographic analysis of d(CG)3-polyamine cocrystals. In this study, we determined the Z-DNA structures with di- to pentavalent polyamines at high resolution. Comparison with the structures revealed that the valence of polyamine determined number of the polyamine(s) and metal ion(s) chelating to the Z-DNA. Furthermore, we succeeded in crystallizing d(CG)3-monoamine (methylamine and ethylamine) complexes.

The X-ray crystallographic study of the conformation of Z-DNA which bound to polyamine for the minor groove at room temperature and low temperature.

We succeeded in x-ray crystal analysis of d(CG)3 with N1-{2-[(2-amino-ethylamino)-ethylamino]-ethyl}-ethane-1,2-diamine complex at three conditions (1: room temperature 2: low temperature 3: highly salt condition). At room temperature two polyamines took extended form and bound to minor groove of d(CG)3, at low temperature two polyamines took U-shape form and lied in minor groove of d(CG)3, at highly salt condition two polyamines took fishhook form and one polyamine lied in upper side of minor groove and the other polyamine lied in down side of minor groove of d(CG)3.

Function of AGCA tetrads in (CAG)n repeats.

We investigate the structural properties of d(CAG)n repeats in d[CG(CAG)4CG)]*d[CG(CTG)4CG]. The crystals diffract up to 3.5 A and completeness of data is only 60%. We also measure Tm-UV and CD spectra and gel electrophoresis of this oligomer.