Akira Odani

Metal–amino acid chemistry. Weak interactions and related functions of side chain groups

α-Amino acids are highly functional small molecules whose side chain groups are like prototypes of metal coordination and weak interactions in proteins. This perspective focuses on non-covalent or weak interactions of the side chain groups of amino acids, such as the charged groups of arginine and aspartic acid and the aromatic rings of tyrosine and tryptophan, in metal complexes in solution and in the solid state. The structure and function of small complexes exhibiting weak interactions and their biological relevance are described.

Simultaneous determination of salivary testosterone and dehydroepiandrosterone using LC–MS/MS: Method development and evaluation of applicability for diagnosis and medication for late-onset hypogonadism

Late-onset hypogonadism (LOH) is a male-specific disorder caused by the age-related decline in androgens, such as testosterone (T). A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method for the simultaneous quantification of T and its precursor, dehydroepiandrosterone (DHEA), in human saliva has been developed and validated. The saliva was deprotenized with acetonitrile, purified using a Strata-X cartridge, derivatized with 2-hydrazino-1-methylpyridine, and subjected to LC-MS/MS. The recovery rates of the steroids during the pretreatment were about 90%. Quantification was based on selected reaction monitoring using characteristic transitions, and deuterated T and DHEA were used as internal standards. This method allowed the reproducible (inter- and intra-assay precisions, <2.9%) and accurate (accuracy, 98.5-101.8%) quantification of the salivary androgens using a 500-microl sample and the limits of quantification for both androgens were 10 pg/ml. As preliminary steps in the practical application of the developed method in diagnosis and medication for LOH, the diurnal rhythms, inter-day alternations and age differences in the salivary T and DHEA were examined; the method found that the salivary T and DHEA show specific diurnal rhythms, significant alternations in early morning and pronouncedly decline with age. The method also enabled the determination of the changes in the individual T and DHEA levels after the DHEA supplementation, which is expected to be a new and easy medication for LOH. Thus, the developed method has satisfactory applicability in the diagnosis and medication for LOH.

Structural evidence for the intramolecular charge-transfer interaction involving an indole ring in ternary copper(II) complexes with L-tryptophan and aromatic diamines

Abstract With a view to understanding the precise binding mode and strength of the stacking interaction in the ternary copper(II) complexes comprising an aromatic diamine such as 2,2′-bipyridine (bpy) and 1,10-phenanthroline (phen) and an aromatic amino acid such as L -phenylalanine, L -tyrosine and L -tryptophan ( L -trp), the crystal structure of [Cu(bpy)( L -trp)]ClO 4 and the circular dichroism (CD) and absorption spectra of [Cu(bpy)( L -trp)]ClO 4 and [Cu(phen)( L -trp)]ClO 4 have been investigated. The complex [Cu(bpy)( L -trp)]ClO 4 crystallizes in the monoclinic space group, P 2 1 , with two molecules in a unit cell of dimensions a =13.022(1), b =7.753(1), c =10.533(1) A, and β=91.18(1)°. The Cu(II) ion is five-coordinate square-pyramidal, with the two nitrogen atoms of bpy and the nitrogen and oxygen atoms of the amino acid coordinated at the equatorial positions in a slightly distorted square-planar form and the carboxylate oxygen atom of the neighboring molecule at the axial position. The most interesting structural feature of the complex is the existence of the intramolecular stacking interaction between the aromatic rings of L -trp and bpy with the average spacing of 3.67 A from the vacant axial position. The CD spectra in the d-d region for [Cu(bpy)( L -trp)ClO 4 and [Cu(phen)( L -trp)]ClO 4 in aqueous solution showed a large negative peak at 587 and 598 nm, respectively, and the magnitudes were greatly reduced in dioxane-water, which indicates that the aromatic ring stacking interaction is weakened in a hydrophobic environment. The absorption bands due to the charge transfer (CT) interaction between the indole ring and the aromatic diamine have been observed in the difference spectra in the near ultraviolet region. The strength of the stacking interactions has been demonstrated by the CT band intensity and the distance between the stacked rings to be in the order [Cu(phen)( L -trp)]ClO 4 >[Cu(bpy)( L -trp)]ClO 4 both in solution and in the solid stare.

The phosphate clamp: a small and independent motif for nucleic acid backbone recognition

The 1.7 Å X-ray crystal structure of the B-DNA dodecamer, [d(CGCGAATTCGCG)]2 (DDD)-bound non-covalently to a platinum(II) complex, [{Pt(NH3)3}2-µ-{trans-Pt(NH3)2(NH2(CH2)6NH2)2}](NO3)6 (1, TriplatinNC-A,) shows the trinuclear cation extended along the phosphate backbone and bridging the minor groove. The square planar tetra-am(m)ine Pt(II) units form bidentate N-O-N complexes with OP atoms, in a Phosphate Clamp motif. The geometry is conserved and the interaction prefers O2P over O1P atoms (frequency of interaction is O2P > O1P, base and sugar oxygens > N). The binding mode is very similar to that reported for the DDD and [{trans-Pt(NH3)2(NH2(CH2)6(NH3+)}2-µ-{trans-Pt(NH3)2(NH2(CH2)6NH2)2}](NO3)8 (3, TriplatinNC), which exhibits in vivo anti-tumour activity. In the present case, only three sets of Phosphate Clamps were found because one of the three Pt(II) coordination spheres was not clearly observed and was characterized as a bare Pt2+ ion. Based on the electron density, the relative occupancy of DDD and the sum of three Pt(II) atoms in the DDD-1 complex was 1:1.69, whereas the ratio for DDD-2 was 1:2.85, almost the mixing ratio in the crystallization drop. The high repetition and geometric regularity of the motif suggests that it can be developed as a modular nucleic acid binding device with general utility.

Preferential formation of ternary copper(II) complexes involving substituted ethylenediamines and amino acids with an aromatic side chain

Abstract The stability constants have been determined potentiometrically for the ternary copper(II)-amine-amino acid systems, where amine refers to ethylenediamine(en), N,N′-dimethylethylenediamine(dmen), N,N,N′,N′-tetramethylethylenediamine(tmen), or N,N′-dibenzylethylenediamine(dben), and amino acid to L-alanine(ala), L-valine(val), L-phenylalanine(phe), L-tyrosine(tyr), O-methyl-L-tyrosine(mtyr), L-tryptophan(trp), or 5-hydroxy-L-tryptophan(htrp). The tendency of the ternary complex formation (Δlog K) decreased in the order, dben > tmen > dmen > en for the amines and htrp ⋍ trp >tyr ⋍ mtyr ⋍ phe > val ⋍ ala for the amino acids. The preferred ternary complex formation of the systems containing dben or htrp was comparable with that observed for phenanthroline. The stability enhancement was found to be additive and proportional to the aromatic ring size. These observations suggest that the proximity of an aromatic ring around the copper(II) ion increases the electronegativity of copper(II) by both the copper(II)-aromatic ring interaction and decreased hydration.

Weak interactions in metal complexes of amino acids with a phosphorylated side chain. Conversion of aromatic ring stacking to electrostatic bonding by tyrosine phosphorylation

Abstract Ternary Cu(II) complexes with the phosphoesters of amino acids (A=O-phosphoserine (Pser) or I=0.1 and 1.0 M(KNO3) and the stability enhancement due to the ligand-ligand interactions was evaluated by calculating the equilibrium constant K for the following hypothetical equilibrium from the overall stability constant of each ternary species: , where A′ refers to serine or tyrosine (Tyr) and B′ to alanine and the interactions are possible only in CU(L-A)(L-B). The ionic strength dependence of the log K values (> 0) indicated that Cu(L-A)(L-B) is stabilized by electrostatic interactions. X-ray crystal structure analysis of [Cu(bpy)(L-Tyr)ClO4]·2H2O (bpy=22′-bipyridine) revealed that the non-phosphorylated complex involves aromatic ring stacking between the Tyr phenol and coordinated bpy rings with the average spacing of 3.35 A in a tetrahedrally distorted square-pyramidal structure with the two nitrogen atoms of bpy and the nitrogen and oxygen atoms of L-Tyr at the equatorial positions and an oxygen atom of the perchlorate ion at the axial position. The results show that the stacked Tyr phenol ring is phosphorylated to be involved in the electrostatic interactions with the positively charged side chain of Arg or Lys which may be a possible mechanism of the conformational change due to Tyr phosphorylation in proteins.

Stability enhancement and circular dichroism spectral anomaly as an indication of non-covalent interactions in histidine- and tyrosine-containing ternary copper(II)—amino acid systems

Visible absorption and CD spectral and potentiometric studies on the His- and Tyr-containing ternary copper(II) complexes Cu(A)(L-B), where A refers to L-His, D-His, or L-Tyr and B to Lys, Tyr, Trp, Phe, Ala, Val, Arg, Glu, Asn, Gln, Ser, or Thr, were made to study ligand-ligand interactions in the complexes. While the CD spectral magnitudes in the d—d region are additive in the absence of side chain interactions and can be estimated from the magnitudes for the ternary systems involving DL-A or DL-B, deviation from the additivity was observed for Cu(L-His)(L-B) (B = LysH, Tyr, Trp, or Phe) and Cu(L-Tyr)(L-Trp). From the stability constants determined at 25 °C and I = 0.1 M (KNO3), the equilibrium constants, K, for the following hypothetical equilibria were calculated to be large (0.14–0.60) for formation of Cu(L-/D-His)(L-B)(B = Tyr or Trp) and Cu(D-His)(L-Phe) with Cu(en)(L-Ala) as standard: Cu(A)(L−Ala)+Cu(en)(L−b)⇌KCu(A)(L−B)+Cu(en)(L−Ala) The positive values indicate the stabilization due to the stacking between the imidazole ring of His and the aromatic side chain of L-B. Solvent dependence of the CD spectra for Cu(L-His)(L-LysH) and Cu(L-His) L-Trp) further supported the existence of the intramolecular electrostatic and hydrophobic interactions.

Development of Novel Radiogallium-Labeled Bone Imaging Agents Using Oligo-Aspartic Acid Peptides as Carriers

68Ga (T 1/2 = 68 min, a generator-produced nuclide) has great potential as a radionuclide for clinical positron emission tomography (PET). Because poly-glutamic and poly-aspartic acids have high affinity for hydroxyapatite, to develop new bone targeting 68Ga-labeled bone imaging agents for PET, we used 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as a chelating site and conjugated aspartic acid peptides of varying lengths. Subsequently, we compared Ga complexes, Ga-DOTA-(Asp)n (n = 2, 5, 8, 11, or 14) with easy-to-handle 67Ga, with the previously described 67Ga-DOTA complex conjugated bisphosphonate, 67Ga-DOTA-Bn-SCN-HBP. After synthesizing DOTA-(Asp)n by a Fmoc-based solid-phase method, complexes were formed with 67Ga, resulting in 67Ga-DOTA-(Asp)n with a radiochemical purity of over 95% after HPLC purification. In hydroxyapatite binding assays, the binding rate of 67Ga-DOTA-(Asp)n increased with the increase in the length of the conjugated aspartate peptide. Moreover, in biodistribution experiments, 67Ga-DOTA-(Asp)8, 67Ga-DOTA-(Asp)11, and 67Ga-DOTA-(Asp)14 showed high accumulation in bone (10.5±1.5, 15.1±2.6, and 12.8±1.7% ID/g, respectively) but were barely observed in other tissues at 60 min after injection. Although bone accumulation of 67Ga-DOTA-(Asp)n was lower than that of 67Ga-DOTA-Bn-SCN-HBP, blood clearance of 67Ga-DOTA-(Asp)n was more rapid. Accordingly, the bone/blood ratios of 67Ga-DOTA-(Asp)11 and 67Ga-DOTA-(Asp)14 were comparable with those of 67Ga-DOTA-Bn-SCN-HBP. In conclusion, these data provide useful insights into the drug design of 68Ga-PET tracers for the diagnosis of bone disorders, such as bone metastases.

Preparation and evaluation of a radiogallium complex-conjugated bisphosphonate as a bone scintigraphy agent

INTRODUCTION (68)Ga is a radionuclide of great interest as a positron emitter for positron emission tomography (PET). To develop a new bone-imaging agent with radiogallium, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was chosen as a chelating site and Ga-DOTA complex-conjugated bisphosphonate, which has a high affinity for bone, was prepared and evaluated. Although we are interested in developing (68)Ga-labeled bone imaging agents for PET, in these initial studies (67)Ga was used because of its longer half-life. METHODS DOTA-conjugated bisphosphonate (DOTA-Bn-SCN-HBP) was synthesized by conjugation of 2-(4-isothiocyanatebenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid to 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (alendronate). (67)Ga-DOTA-Bn-SCN-HBP was prepared by coordination with (67)Ga, and its in vitro and in vivo evaluations were performed. RESULTS (67)Ga-DOTA-Bn-SCN-HBP was prepared with a radiochemical purity of over 95% without purification. (67)Ga-DOTA-Bn-SCN-HBP had great affinity for hydroxyapatite in binding assay. In biodistribution experiments, (67)Ga-DOTA-Bn-SCN-HBP accumulated in bone rapidly but was hardly observed in tissues other than bone. Pretreatment of an excess amount of alendronate inhibited the bone accumulation of (67)Ga-DOTA-Bn-SCN-HBP. CONCLUSIONS (67)Ga-DOTA-Bn-SCN-HBP showed ideal biodistribution characteristics as a bone-imaging agent. These findings should provide useful information on the drug design of bone imaging agents for PET with (68)Ga.

Synthesis of antitumor azolato-bridged dinuclear platinum(II) complexes with in vivo antitumor efficacy and unique in vitro cytotoxicity profiles

We synthesised four tetrazolato-bridged dinuclear Pt(ii) complexes, [{cis-Pt(NH3)2}2(μ-OH)(μ-5-R-tetrazolato-N2,N3)](n+), where R is CH3 (1), C6H5 (2), CH2COOC2H5 (3), or CH2COO(-) (4) and n = 2 (1-3) or 1 (4). Their structures were characterised by (1)H, (13)C, and (195)Pt NMR spectroscopy, mass spectrometry, and elemental analysis, and the crystal structure of 1 was determined by X-ray crystallography. The cytotoxicities of the complexes to human non-small-cell lung cancer (NSCLC) cell lines sensitive and resistant to cisplatin were assayed. Complex 1 was more cytotoxic than cisplatin in both PC-9 and PC-14 NSCLC cell lines, and cross-resistance to 1 in the cisplatin-resistant cells was largely circumvented. Complex 3 was moderately cytotoxic, whereas 2 and 4 were only marginally cytotoxic. We also determined the growth inhibitory activities of 1 and 3, as well as prototype azolato-bridged complexes [{cis-Pt(NH3)2}2(μ-OH)(μ-pyrazolato)](2+) (AMPZ), [{cis-Pt(NH3)2}2(μ-OH)(μ-1,2,3-triazolato-N1,N2)](2+) (AMTA), [{cis-Pt(NH3)2}2(μ-OH)(μ-tetrazolato-N1,N2)](2+) (5-H-X), and [{cis-Pt(NH3)2}2(μ-OH)(μ-tetrazolato-N2,N3)](2+) (5-H-Y), against a panel of 39 human cancer cell lines (JFCR39). The average 50% growth inhibition concentrations of the complexes against the JFCR39 cell lines ranged from 0.933 to 23.4 μM. The cytotoxicity fingerprints of the complexes based on the JFCR39 cytotoxicity data were similar to one another but completely different from the fingerprints of clinical platinum-based anticancer drugs. Complex 3 exhibited marked antitumor efficiency when tested in vivo on xenografts of PANC-1 pancreatic cancer in nude mice. The high potency of 3 confirmed that the tetrazolato-bridged structure exhibits high in vivo antitumor efficacy.