Exploring the thermal stability of α-chymotrypsin in protic ionic liquids

Ammonium based ionic liquids (ILs) are biocompatible co-solvents that stabilize the native state of proteins. Experimentally, we have explored the stability of α-chymotrypsin (CT) in the presence of nine ILs, i.e., diethylammonium acetate (DEAA), diethylammonium hydrogen sulfate (DEAS), diethylammonium dihydrogen phosphate (DEAP), triethylammonium acetate (TEAA), triethylammonium hydrogen sulfate (TEAS), triethylammonium dihydrogen phosphate (TEAP), trimethylammonium acetate (TMAA), trimethylammonium hydrogen sulfate (TMAS), trimethylammonium dihydrogen phosphate (TMAP). Thermodynamic folding properties such as transition temperature (T_m), Gibbs free energy change of unfolding (ΔG_U), enthalpy change (ΔH) and heat capacity change (ΔC_p) of CT in ILs are obtained by fluorescence spectra analysis. Fluorescence and circular dichroism (CD) spectroscopy experiments were performed to probe CT stabilization and structural changes in the presence of ILs. Our experimental results suggest that the ILs act as stabilizers for the CT structure and the stability of CT depends on the structural arrangement of the ions of ILs. Our experimental results reveal that ILs (DEAA, DEAS and DEAP) having more hydrophobic ammonium cations [DEA⁺] are weak stabilizers for CT, while trimethyl ammonium cations [TMA⁺] ILs having small alkyl chain length such as TMAA, TMAS and TMAP are strong stabilizers and therefore more biocompatible for the native structure of CT.     

Electrochemical recognition of anions by 1,1'-N,N'-ferrocenoylbisamino acid esters

A series of novel ferrocene-based receptors, 1,1'-N,N'-ferrocenoylbisamino acid methyl esters 2-5 have been prepared and their electrochemical properties determined. The amino acids employed were glycine (2), beta-alanine (3), gamma-aminobutyric acid (4) and l-norleucine (5). These receptors are composed of an electroactive core and two parallel strands of amino acids that can interact with anions via electrostatic interactions in the oxidized state as well as secondary interactions, such as hydrogen bonding and hydrophobic interactions. Furthermore, the semi-rigid molecular clefts between the two strands of amino acids in these receptors are capable of discerning anions of different geometries and sizes. The anion sensing capabilities of receptors 2-5 were studied using cyclic voltammetry (CV). The anions studied were chloride (Cl-), nitrate (NO3-), dihydrogen phosphate (H(2)PO(4)-), hydrogen sulfate (HSO(4)-), acetate (CH(3)COO-) and neurologically important anions such as lactate (CH(3)CH(OH)COO-), pyruvate (CH(3)COCOO-) and glutamate (HOOC-CH(NH2)CH(2)CH(2)COO-). The receptors 2-5 exhibit selectivity towards chloride, dihydrogen phosphate and acetate, over hydrogen sulfate and nitrate ions and generate a redox response in organic media. Also, binding studies of receptor 3 with neurologically important anions show it displays selectivity towards lactate and pyruvate, over glutamate ions and generates a redox response. However the response is ill defined in all cases, with poorly separated "free" and "ion-pair" peaks, which preclude accurate measurement of the response by CV. These results emphasize the considerations required for the design of ferrocene-based receptors and the necessary parameters for efficient electrochemical recognition of small anions by CV.     

Molecular orbital study of complexes of zinc(II) with sulphide, thiomethanolate, thiomethanol, dimethylthioether, thiophenolate, formiate, acetate, carbonate, hydrogen carbonate, iminomethane and imidazole. Relationships with structural and catalytic zinc in some metallo-enzymes.

Geometry optimization and energy calculations have been performed at the density functional B3LYP/LANL2DZ level on hydrogen sulfide (HS-), dihydrogensulfide (H2S), thiomethanolate (CH3S-), thiomethanol (CH3SH), thiophenolate (C6H5S-), methoxyde (CH3O-), methanol (CH3OH), formiate (HCOO-), acetate (CH3COO-), carbonate (CO3(2-)), hydrogen carbonate (HCO3-), iminomethane (NH=CH2), [ZnS], [ZnS2]2-, [Zn(HS)]+, [Zn(H2S)]2+, [Zn(HS)4]2-, [Zn(CH3S)]+, [Zn(CH3S)2], [Zn(CH3S)3]-, [Zn(CH3S)4]2-, [Zn(CH3SH)]2+, [Zn(CH3SCH3)]2+, [Zn(C6H5S)]+, [Zn(C6H5S)2], [Zn(C6H5S)3]-, [Zn(HS)(NH=CH2)2]+, [Zn(HS)2(NH=CH2)2], [Zn(HS)(H2O)]+, [Zn(HS)(HCOO)], [Zn(HS)2(HCOO)]-, [Zn(CH3O)]+, [Zn(CH3O)2], [Zn(CH3O)3]-, [Zn(CH3O)4]2, [Zn(CH3OH)]2+, [Zn(HCOO)]+, [Zn(CH3COO)]+, [Zn(CH3COO)2], [Zn(CH3COO)3]-, [Zn(CO3)], [Zn(HCO3)]+, and [Zn(HCO3)(Imz)]+ (Imz, 1,3-imidazole). The computed Zn-S bond distances are 2.174A for [ZnS], 2.274 for [Zn(HS)]+, 2.283 for [Zn(CH3S)]+, and 2.271 for [Zn(C6H5S)]+, showing that sulfide anion forms stronger bonds than substituted sulfides. The nature of the substituents on sulfur influences only slightly the Zn-S distance. The optimized tetra-coordinate [Zn(HS)2(NH=CH2)2] molecules has computed Zn-S and Zn-N bond distances of 2.392 and 2.154A which compare well with the experimental values at the solid state obtained via X-ray diffraction for a number of complex molecules. The computed Zn-O bond distances for chelating carboxylate derivatives like [Zn(HOCOO)]+ (1.998A), [Zn(HCOO)]+ (2.021), and [Zn(CH3COO)]+ (2.001) shows that the strength of the bond is not much influenced by the substituent on carboxylic carbon atom and that CH3- and HO- groups have very similar effects. The DFT analysis shows also that the carboxylate Ligand has a preference for the bidentate mode instead of the monodentate one, at least when the coordination number is small.     

Catalytic conversion of cellulose to 5-hydroxymethyl furfural using acidic ionic liquids and co-catalyst

Efficient catalytic conversion of microcrystalline cellulose (MCC) to 5-hydroxymethyl furfural (HMF), is achieved using acidic ionic liquids (ILs) as the catalysts and metal salts as co-catalysts in the solvent of 1-ethyl-3-methylimidazo-lium acetate ([emim][Ac]). A series of acidic ILs has been synthesized and tested in conversion of MCC to HMF. The effect of reaction conditions, such as reaction time, temperature, catalyst dosage, metal salts, water dosage, Cu(2+) concentration and various acidic ILs are investigated in detail. The results show that CuCl(2) in 1-(4-sulfonic acid) butyl-3-methylimidazolium methyl sulfate ([C(4)SO(3)Hmim][CH(3)SO(3)]), is found to be an efficient catalyst for catalytic conversion of MCC to HMF, and 69.7% yield of HMF is obtained. A mechanism to explain the high activity of CuCl(2) in [C(4)SO(3)Hmim][CH(3)SO(3)] is proposed. To the best of our knowledge, this report first proposes that the Cu(2+) and [C(4)SO(3)Hmim][CH(3)SO(3)] show better catalytic performance in catalytic conversion of MCC to HMF.     

The non-enzymatic hydrolysis of oligoribonucleotides VI. The role of biogenic polyamines.

Single-stranded oligoribonucleotides containing UA and CA phosphodiester bonds can be hydrolyzed specifically under non-enzymatic conditions in the presence of spermidine, a biogenic amine found in a wide variety of organisms. In the present study, the rate of oligonucleotide and tRNA(i)(Met)hydrolysis was measured in the presence of spermidine and other biogenic amines. It was found that spermine [H(3)N(+)(CH(2))(3)(+)NH(2)(CH(2))(4)(+)NH(2)(CH(2))(3)(+)NH(3)] and putrescine [H(3)N(+)(CH(2))(4)(+)NH(3)] can replace spermidine [H(3)N(+)-(CH(2))(4)(+)NH(2)(CH(2))(3)(+)NH(3)] to induce the hydrolysis. For all three polyamines, a bell-shaped cleavage rate versus concentration relationship was observed. The maximum rate of hydrolysis was achieved at 0.1, 1.0 and 10 mM spermine, spermidine and putrescine, respectively. Moreover, we found that the hydrolysis requires at least two linked amino groups since two aminoalcohols, 2-aminoethanol and 3-aminopropanol, were not able to induce the cleavage of the phospho-diester bond. The optimal cleavage rate of the oligo-ribonucleotides was observed when amino groups were separated by tri- or tetramethylene linkers. The methylation of the amino groups reduced the ability of diamines to induce oligoribonucleotide hydrolysis. Non-enzymatic cleavage of tRNA(i)(Met)from Lupinus luteus and tRNA(i)(Met)from Escherichia coli demonstrate that both RNAs hydrolyze as expected from principles derived from oligoribonucleotide models.     

Synthetic analogs for oxovanadium(IV/V)-glutathione interaction: an NMR, EPR, synthetic and structural study of oxovanadium(IV/V) compounds with sulfhydryl-containing pseudopeptides and dipeptides

The reaction of [VO(CH3COO)2(phen)] (phen = 1,10-phenanthroline) with the sulfhydryl-containing pseudopeptides (scp), N-(2-mercaptopropionyl)glycine (H3mpg), N-(2-mercaptopropionyl)cysteine (H4m2pc), N-(3-mercaptopropionyl)cysteine (H4m3pc) and the dipeptides glycylglycine (H2glygly) and glycyl-L-alanine (H2glyala), in the presence of triethylamine, results in the formation of the compounds Et3NH[VO(mpg)(phen)] (1), (Et3NH)2[VO(m2pc)] (4), [(Et3NH)2[VO(m3pc) (5), [VO(glygly)(phen)] x 2CH3OH (2 x 2CH3OH) and [VO(glyala)(phen)] x CH3OH (3 x CH3OH). Evidence for the molecular connectivity in 2 x CH3OH was established by X-ray crystallography, showing the vanadium(IV) atom ligated to a tridentate glygly2- ligand at the N(amine), N(peptide) and O(carboxylato) atoms. Combination of the correlation plot of the EPR parameters gz versus Az, together with the additivity relationship supported the prediction of the equatorial donor atom sets of the V(IV)O2+ center at various pH values for the V(IV)O2+-glutathione system considered in this study. Model NMR studies (interaction of vanadium(V) with the scp H3mpg) showed that there is a possibility of vanadium(V) ligation to glutathione.     

Synthesis,structural characterization and in vitro cytotoxicity of organotin(IV) derivatives of heterocyclic thioamides, 2-mercaptobenzothiazole, 5-chloro-2-mercaptobenzothiazole, 3-methyl-2-mercaptobenzothiazole and 2-mercaptonicotinic acid

Five new organotin(IV) molecules with the heterocyclic thioamides; 2-mercaptobenzothiazole (Hmbzt), 5-chloro-2-mercaptobenzothiazole (Hcmbzt), 3-methyl-2-mercaptobenzothiazole (mmbzt) and 2-mercaptonicotinic acid (H(2)mna) of formulae [(n-C(4)H(9))(2)Sn(mbzt)(2)] (1), [(C(6)H(5))(2)Sn(mbzt)(2)] (2), [(CH(3))(2)Sn(cmbzt)(2)].1.7(H(2)O)] (3), [(n-C(4)H(9))(2)SnCl(2)(mmbzt)(2).(CH(2)Cl(2))] (4) and [[(C(6)H(5))(3)Sn](2)(mna).[(CH(3))(2)CO]] (5) have been synthesized and characterized by elemental analysis, 1H-, 13C-NMR, FT-IR and M?ssbauer spectroscopic techniques. Crystal structures of molecules 1, 3 and 5 have been determined by X-ray diffraction at 173(1) K (1 and 5) and 293(2) K (3). Compound 1 C(22)H(26)N(2)S(4)Sn, is monoclinic, space group C2/c, a=44.018(2), b=8.8864(5), c=12.8633(7) A, beta=104.195(5) degrees, Z=8. Compound 3 is also monoclinic, space group P2(1)/c and a=17.128(2) A, b=17.919(2) A, c=7.3580(10) A, beta=98.290(10) degrees, Z=4. In both molecules 1 and 3, two carbon atoms from aryl groups, two sulfur and two nitrogen atoms from thione ligands form a distorted octahedral geometry around tin(IV) with trans-C(2), cis-N(2), cis-S(2) configurations. Compound 5 C(45)H(39)NO(3)SSn(2) is monoclinic, space group P2(1)/n, a=9.1148(2) A, b=29.2819(6), c=15.5556(4) A, beta=106.2851(9) degrees, Z=4. Complex 5 contains two [(C(6)H(5))(3)Sn(IV)] moieties linked by a double deprotonated 2-mercaptonicotinic acid (H(2)mna). Both tin(IV) ions are five coordinated. This complex is the an example of a pentacoordinated Ph(3)SnXY system with an axial-equatorial arrangement of the phenyl groups at Sn(1) atom. Compounds 1, 3 and 5 were tested for in vitro cytotoxicity against the cancer cell line of sarcoma cells (mesenchymal tissue) from the Wistar rat, polycyclic aromatic hydrocarbons (benzo[a]pyrene) carcinogenesis. Compound 5 exhibits strong cytotoxic activity, while complexes 1 and 3 show less cytotoxic activity.     

Effects of geometric isomerism in dinuclear platinum antitumor complexes on aquation reactions in the presence of perchlorate, acetate and phosphate

The aquation and subsequent reactions of the dinuclear Pt antitumor complexes [{trans-PtCl(NH(3))(2)}(2)(mu-NH(2)(CH(2))(6)NH(2))](2+) (1,1/t,t) and [{cis-PtCl(NH(3))(2)}(2)(mu-NH(2)(CH(2))(6)NH(2))](2+) (1,1/c,c) in 15 mM perchlorate, acetate or phosphate solutions were followed at 298 K by [(1)H,(15)N] HSQC 2D NMR spectroscopy. Rate and equilibrium constants for the initial reversible aquation and the subsequent reversible reaction with phosphate or acetate are reported. The rate constant for the first aquation step is two-fold lower for 1,1/c,c than 1,1/t,t but the anation rate constants are similar so that the equilibrium lies further towards the chloro form for the 1,1/c,c compound. A pK (a) value of 6.01+/-0.03 was determined for the diaquated species [{cis-Pt(NH(3))(2)(H(2)O)}(2)(mu-NH(2)(CH(2))(6)NH(2))](4+) (1,1/c,c-3) which is 0.4 units higher than that of the 1,1/t,t compound. The rate constants for the binding of acetate and phosphate to 1,1/t,t are similar, but the rate constant for the reverse reaction is close to ten-fold higher in the case of phosphate so that equilibrium conditions are attained more rapidly (12 h compared with 64 h). On the other hand, for 1,1/c,c the rate constants for the forward and reverse reactions with acetate and phosphate are quite similar so that equilibrium conditions are reached very slowly (80-100 h) and a greater proportion of phosphate-bound species are present. The reduced lability of the bound phosphate for 1,1/c,c is attributed to the formation of a macrochelate phosphate-bridged species which was characterized by (31)P NMR and ESI-MS. The speciation profiles of 1,1/t,t and 1,1/c,c under physiological conditions are explored.     

Pyrazolyl derivatives as bifunctional chelators for labeling tumor-seeking peptides with the fac-[M(CO)3]+ moiety (M = 99mTc, Re): synthesis, characterization, and biological behavior

Radiolabeling of biologically active molecules with the [(99m)Tc(CO)(3)](+) unit has been of primary interest in recent years. With this in mind, we herein report symmetric (L(1)) and asymmetric (L(2)-L(5)) pyrazolyl-containing chelators that have been evaluated in radiochemical reactions with the synthon [(99m)Tc(H(2)O)(3)(CO)(3)](+) (1a). These reactions yielded the radioactive building blocks [(99m)Tc(CO)(3)(k(3)-L)](+) (L = L(1)-L(5), 2a-6a), which were identified by RP-HPLC. The corresponding Re surrogates (2-6) allowed for macroscopic identification of the radiochemical conjugates. Complexes 2a-6a, with log P(o/w) values ranging from -2.35 to 0.87, were obtained in yields of > or =90% using ligand concentrations in the 10(-5-)10(-4) M range. Challenge studies with cysteine and histidine revealed high stability for all of these radioactive complexes, and biodistribution studies in mice indicated a fast rate of blood clearance and high rate of total radioactivity excretion, occurring primarily through the renal-urinary pathway. Based on the framework of the asymmetric chelators, the novel bifunctional ligands 3,5-Me(2)-pz(CH(2))(2)N((CH(2))(3)COOH)(CH(2))(2)NH(2) (L(6)) and pz(CH(2))(2)N((CH(2))(3)COOH)(CH(2))(2)NH(2) (L(7)) have been synthesized and their coordination chemistry toward (NEt(4))(2)[ReBr(3)(CO)(3)] (1) has been explored. The resulting complexes, fac-[Re(CO)(3)(k(3)-L)]Br (L(6)(7), L(7)(8)), contain tridentate ancillary ligands that are coordinated to the metal center through the pyrazolyl and amine nitrogen atoms, as observed for the other related building blocks. L(6) and L(7) were coupled to a glycylglycine ethyl ester dipeptide, and the resulting functionalized ligands were used to prepare the model complexes fac-[Re(CO)(3)(kappa(3)-3,5-Me(2)-pz(CH(2))(2)N(glygly)(CH(2))(2)NH(2))](+) (9/9a) and fac-[Re(CO)(3)(kappa(3)-pz(CH(2))(2)N(CH(2))(3)(glygly)(CH(2))(2)NH(2))](+) (10/10a) (M = Re, (99m)Tc). These small conjugates have been fully characterized and are reported herein. On the basis of the in vitro/in vivo behavior of the model complexes (2a-6a, 9a, 10a), we chose to evaluate the in vitro/in vivo biological behavior of a new tumor-seeking Bombesin pyrazolyl conjugate, [(L(6))-G-G-G-Q-W-A-V-G-H-L-M-NH(2)], that has been labeled with the [(99m)Tc(CO)(3)](+) metal fragment. Stability, in vitro cell binding assays, and pharmacokinetics studies in normal mice are reported herein.     

N,N-bis(2-mercaptoethyl)methylamine: a new coligand for Tc-99m labeling of hydrazinonicotinamide peptides

Hydrazinonicotinamide (HYNIC) forms stable coordination complexes with Tc-99m when reacted with Tc(V)oxo species such as Tc-mannitol or other Tc-polyhydric complexes. However, radio-HPLC of [Tc-For-MLFK-HYNIC] labeled via Tc-polyhydric ligands demonstrated multiple radiochemical species each with unique biodistribution patterns. This is likely due to the fact that Tc can bind to the hydrazino moiety, as well as polyhydric ligands, in a variety of coordination geometries. Tridentate ligands, such as bis(mercaptoethyl)methylamine (NS2), may constrain the possible coordination geometries and improve overall stability. To investigate this, we synthesized NS2, converted the [Tc-mannitol-For-MLFK-HYNIC] to the corresponding NS2-containing complex [Tc-NS2-For-MLFK-HYNIC], and compared its infection imaging and biodistribution properties with [Tc-mannitol-For-MLFK-HYNIC]. Conversion to the NS2 complex was confirmed by HPLC which showed a single unique hydrophobic species with retention time greater than the [Tc-mannitol-For-MLFK-HYNIC] complex. Imaging experiments with both preparations were performed in rabbits with E. coli infections in the left thigh. Tissue radioactivity measurements demonstrated that compared to Tc-mannitol-peptide, accumulation of Tc-NS2-peptide was lower in blood, heart, and normal muscle and higher in spleen, infected muscle, and pus (p < 0.01). These results indicate that the Tc-NS2-peptide complex is chemically more homogeneous and exhibits improved infection localization and biodistribution properties. In an effort to model the interactions of the metal-HYNIC core with NS2 and related ligand types, the reactions of [ReCl3(NNC5H4NH)(NHNC5H4N)] and [99TcCl3(NNC5H4NH)(NHNC5H4N)], effective structural analogues for the [M(NNC5H4NH(x))2] core, with NS2, C5H3N-2,6-(CH2SH)2, O(CH2CH2SH)2, and S(CH2CH2SH)2 were investigated and the compounds [M[CH3N(CH2CH2S)2](NNC5H4N)(NHNC5H4N] (M = 99Tc (5a), Re (5b)), [Re[C5H3N-2,6-(CH2S)2](NNC5H4N)(NHNC5H4N)].CH2Cl2.0.5MeOH (7), [Re[SCH2CH2)2O] (NNC5H4N)(NHNC5H4N)] (8), and [Re[(SCH2CH2)2S](NNC5H4NH)(NHNC5H4N)]Cl (9) were isolated. Similarly, the reaction of [ReCl3(NNC5H4NH)(NHNC5H4N)] with the bidentate ligands pyridine-2-methanethiol and 3-(trimethlysilyl)pyridine-2-thiol led to the isolation of [ReCl(C5H4N-2-CH2S) (NNC5H4N)(NHNC5H4N)] (10) and [Re(2-SC5H3N-3-SiMe3)2 (NNC5H4N)(NHNC5H4N)] (11), respectively, while reaction with N-methylimidazole-2-thiol yielded the binuclear complex [Re(OH)Cl(SC3H2N2CH3)2(NNC5H4N)2 (NHNC5H4N)2] (12). The analogous metal-(HYNIC-OH) precursor, [ReCl3[NNC5H3NH(CO2R)] [NHNC5H3N(CO2R)]] (R = H, 13a; R = CH3, 13b) has been prepared and coupled to lysine to provide [RCl3[NNC5H3NH(CONHCH2CH2CH2CH2CH(NH2)CO2H)] [NHNC5H3NH(CONHCH2CH2CH2CH2CH(NH2)CO2H)]].2HCl (14.2HCl), while the reaction of the methyl ester 13b with 2-mercaptopyridine yields [Re(2-SC5H4N)2[NNC5H3N(CO2Me)][NHNC5H3N(CO2Me)]] (15). While the chemical studies confirm the robustness of the M-HYNIC core (M = Tc, Re) and its persistence in ligand substitution reactions at adjacent coordination sites of the metal, the isolation of oligomeric structures and the insolubility of the peptide conjugates of 13, 14, and 15 underscore the difficulty of characterizing these materials on the macroscopic scale, an observation relevant to the persistent concerns with reagent purity and identity on the tracer level.     

Single diastereomers of unsymmetrical tris-spirocyclic cyclotriphosphazenes based on 1,1'-bi-2-naphthol--synthesis and structures

Diastereoselective synthesis and characterization of chiral unsymmetrical tris-spirocyclic cyclotriphosphazenes based on chiral 1,1'-bi-2-naphthol (BINOL) are reported. Specifically, the chiral compounds (-)N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](O-2,2'C(6)H(4)-C(6)H(4)O)Cl(2) [(-)-4] and (-)N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](OCH(2)CH(2)NMe)(2) [(-)-5] are prepared by starting with the chiral mono-spiro compound (-)N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)]Cl(4) [(-)-3]. Synthesis of four other chiral spirocyclics, N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](OCH(2)CH(2) NMe)(O-2,2'C(6)H(4)-C(6)H(4)O)[(-)-6 and (+)-6], N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](NMe(2))(4) [(-)-7], N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](O-2,2'C(6)H(4)-C(6)H(4)O)(NMeCH(2)CH(2)OH)(2) [(-)-8 and (+)-8], and N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](O-2,2'C(6)H(4)-C(6)H(4)O)[NHCH(2)CH(2)CH(2)Si(OEt)(3)](2) (9) is also reported herein. Compounds 4-6 are obtained in the solid state diastereoselectively and their X-ray structures have been determined and discussed. The diastereoselectivity is also shown by structural characterization of two distinct isomers in the case of 6 [(-)-6 and (+)-6, respectively] by starting with precursor of 3 having (R) or (S)-BINOL residue. The (1)H NMR spectra of 7 and 8 exhibit doublets with virtual coupling for the methyl protons, consistent with the chiral nature of the binaphthoxy residue. The potential of 9, which hydrolyzes readily in CDCl(3) solution, as a useful precursor for chiral polymer applications is highlighted.     

Circular dichroism study of the irreversibility of conformational changes induced by polyamine-linked dinuclear platinum compounds

In this work, the reversibility of both the B-->Z and B-->A conformational change in polymer DNA induced by polynuclear platinum compounds was studied. The compounds examined were: [[trans-PtCl(NH(3))(2)](2)[NH(2) (CH(2))(6)NH(2)]](2+) (BBR3005); [[trans-PtCl(NH(3))(2)](2)[mu-spermine-N1,N12]](4+) (BBR3535); [[trans-PtCl(NH(3))(2)](2)[mu-spermidine-N1,N8]](3+) (BBR3571); [[trans-PtCl(NH(3))(2)](2)[mu-BOC-spermidine]](2+) (BBR3537); and [[trans-PtCl(NH(3))(2)](2)[mu-trans-Pt(NH(3))(2)(H(2)N(CH(2))(6)NH(2))(2)]](4+) (BBR3464). The conformational changes were assessed by circular dichroism and the reversibility of the transitions was tested by subsequent titration with the DNA intercalator ethidium bromide (EtBr). Fluorescent quenching was also used to assess the ability of ethidium bromide to intercalate into A and/or Z-DNA induced by the compounds. The results were compared with those produced by the simple hexamminecobalt cation [Co(NH(3))(6)](3+). The data suggest that while conformational changes induced by electrostatic interactions are confirmed to be reversible, covalent binding induces irreversible changes in both the A and Z conformation. The relevance of these changes to the novel biological action of polynuclear platinum compounds is discussed.     

Chemo-enzymatic synthesis of tetra-, penta-, and hexasaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 14

The chemo-enzymatic synthesis is described of beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->O(CH(2))(6)NH(2) (1), beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->O(CH(2))(6)NH(2) (2), beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->O(CH(2))(6)NH(2) (3), and beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (4), representing fragments of the repeating unit of the Streptococcus pneumoniae serotype 14 capsular polysaccharide. Linear intermediate oligosaccharides 5-8 were synthesized via chemical synthesis, followed by enzymatic galactosylation using bovine milk beta-1,4-galactosyltransferase as a catalyst. The title oligosaccharides form suitable compounds for conjugation with carrier proteins, to be tested as potential vaccines in animal models.     

Enantiomeric and mesomeric mandelate complexes of molybdenum -- on their stereospecific formations and absolute configurations

The stereospecific formation and absolute configuration of R-homocitrate coordinated FeMo-co in nitrogenase was mimicked through the structural analyses of a collection of enantiomeric and mesomeric mandelato molybdenum complexes, i.e., (NH(4))(2)[Mo(Delta)O(2)(R-mand)(2)]x3H(2)O (1a), (NH(4))(2)[Mo(Lambda)O(2)(S-mand)(2)]x3H(2)O (1b), (NH(4))(4)[Mo(Delta)O(2)(RS-mand)(2)][Mo(Lambda)O(2)(RS-mand)(2)]x8H(2)O (2), (NH(4))(2)[W(Delta)O(2)(R-mand)(2)]x2H(2)O (3a), (NH(4))(2)[W(Lambda)O(2)(S-mand)(2)]x2H(2)O (3b) (H(2)mand=mandelic acid, C(8)H(8)O(3)), which have been characterized by elemental analyses, optical rotation, circular dichroism, IR, NMR spectroscopes and X-ray single crystal studies. The R and S chiral mandelic acids induce the formations of the enantiomeric pair of chiral complexes, which are supported by the characterizations of optical rotation and circular dichroism. The configuration of the resulted metal center could be assigned as Delta or Lambda. While the RS racemic reagent yields only mesomeric compound. The Delta(R,R)-complexes 1a and 3a are enantiomers of Lambda(S,S)-1b and 3b, respectively. Of the five complexes, Mo and W atoms are all hexa-coordinated by two cis-oxo groups and two bidentate mandelate ligands through the deprotonated alpha-alkoxyl and alpha-carboxyl groups, forming a stable five-membered chelated rings. The average Mo(VI)-O bond distances with alpha-alkoxyl and alpha-carboxyl are 1.944 and 2.210 A, respectively. Further comparison indicates that bonds of alpha-alkoxyl groups in the hydroxycarboxylato molybdenum complexes are much sensitive to the change in the oxidation state of molybdenum, which support the possible Mo activation model in FeMo-co through the protonation and cleavage of alpha-alkoxyl group in homocitrate ligand.     

Preparation of platinum(IV) complexes with dipeptide and diimine. X-ray crystal structure and 195Pt NMR spectra

We prepared platinum(IV) complexes containing dipeptide and diimine or diamine, the [PtCl(dipeptide-N,N,O)(diimine or diamine)]Cl complex, where -N,N,O means dipeptide coordinated as a tridentate chelate, dipeptide=glycylglycine (NH(2)CH(2)CON(-)CH(2)COO(-), digly, where two protons of dipeptide are detached when the dipeptide coordinates to metal ion as a tridentate chelate), glycyl-L-alanine (NH(2)CH(2)CON(-)CHCH(3)COO(-), gly-L-ala), L-alanylglycine (NH(2)CH CH(3)CON(-)CH(2)COO(-), L-alagly), or L-alanyl-L-alanine (NH(2)CHCH(3)CON(-)CHCH(3)COO(-), dil-ala), and diimine or diamine=bipyridine (bpy), ethylenediamine (en), N-methylethylenediamine (N-Me-en), or N,N'-dimethylethylenediamine (N,N'-diMe-en). In the complexes containing gly-L-ala or dil-ala, two separate peaks of the (195)Pt NMR spectra of the [PtCl(dipeptide-N,N,O)(diimine or diamine)]Cl complexes appeared in, but in the complexes containing digly or L-alagly, one peak which contained two overlapped signals appeared. One of the two complexes containing gly-L-ala and bpy, [PtCl(gly-L-ala-N,N,O)(bpy)]NO(3), crystallized and was analyzed. This complex has the monoclinic space group P2(1)2(1)2(1) with unit cell dimensions of a=9.7906(3)A, b=11.1847(2)A, c=16.6796(2)A, Z=4. The crystal data revealed that this [PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) complex has the near- (Cl, CH(3)) configuration of two possible isomers. Based on elemental analysis, the other complex must have the near- (Cl, CH(3))-[PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) configuration. The (195)Pt NMR chemical shifts of the near- (Cl, CH(3))-[PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) complex and the far- (Cl, CH(3))-[PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) complex are 0 ppm and -19 ppm, respectively (0 ppm for the Na(2)[PtCl(6)] signal). The additive property of the (195)Pt NMR chemical shift is discussed. The (195)Pt NMR chemical shifts of [PtCl(dipeptide-N,N,O)(bpy)]Cl appeared at a higher field when the H attached to the dipeptide carbon atom was replaced with a methyl group. On the other hand, the (195)Pt NMR chemicals shifts of [PtCl(dipeptide-N,N,O)(diamine)] appeared at a lower field when the H attached to the diamine nitrogen atom was replaced with a methyl group, in the order of [PtCl(digly-N,N,O)(en)]Cl, [PtCl(digly-N,N,O)(N-Me-en)]Cl, and [PtCl(digly-N,N,O)(N,N'-diMe-en)]Cl.     

Solubility and stability of cytochrome c in hydrated ionic liquids: effect of oxo acid residues and kosmotropicity

Hydrated ionic liquids (ILs) were prepared by adding appropriate amounts of water to hydrophilic ILs. Some hydrated ILs show excellent solubilizing ability for proteins, keeping the basic properties of ILs. The solubility of cytochrome c (cyt c) depended on the structure of the component ions. When component anions have oxo acid residues, the resulting hydrated ILs solubilize cyt c quite well. In such hydrated ILs, the structure and activity of cyt c is influenced by the kosmotropicity of the component ions. We synthesized ILs from various ions having different kosmotropicity, including dihydrogen phosphate (dhp), dibutylphosphate, acetate, lactate, and methanesulfonate as anions. The activity of the dissolved cyt c depends on the permutations of kosmotropicity of the component ions. cyt c shows no structural change and retains its activity when dissolved in the hydrated choline dhp, which is an excellent combination of chaotropic cation and kosmotropic anion. Furthermore, cyt c dissolved in the hydrated choline dhp remained in a native state and was active after 18 months of storage at room temperature.     

Cooperative effects in long-range 1,4 DNA-DNA interstrand cross-links formed by polynuclear platinum complexes: an unexpected syn orientation of adenine bases outside the binding sites

The novel phase II anticancer drug BBR3464 ([[ trans-PtCl(NH(3))(2)](2)- micro -[ trans-Pt(NH(3))(2)(NH(2)(CH(2))(6)NH(2))(2)]](NO(3))(4)) forms a 1,4-interstrand cross-link adduct with the self-complementary DNA octamer 5'-d(ATG*TACAT)(2)-3', with the two platinum atoms coordinated in the major groove at the N7 positions of guanines that are four base pairs apart on opposite DNA strands. The "central" tetraamine linker [ trans-H(2)N(CH(2))(6)NH(2)Pt(NH(3))(2)NH(2)(CH(2))(6)NH(2)] was located in or close to the minor groove. The adduct was characterized and analyzed by MS, UV and NMR spectroscopy. NMR analysis of the adduct shows strong H8/H1' intraresidue crosspeaks observed for the A1 and A7 resonances, consistent with a syn conformation for these bases which is usually not observed for adenine residues and bases not directly involved in the cross-link in oligonucleotides. The strong intraresidue H8/H1' crosspeak is also observed for G3. Examination of the structure thus reveals unusual cooperative effects unique to this class of anticancer drugs and is the first demonstration of cooperative effects in solution for an anticancer drug. The significant characteristic of the structure is the lack of severe DNA distortion such as a kink, directed bend or significant unwinding of the helices which are characteristic for DNA adducts of mononuclear complexes. This may contribute to the lack of protein recognition of the cross-link by HMG-domain proteins, a biological consequence significantly different from that of mononuclear complexes such as cisplatin. Since DNA is the principal target in vivo for these Pt cross-linking agents, the unique structural perturbations induced by BBR3464 cross-links are likely related to its increased cytotoxicity and antitumor activity as compared to cisplatin ( cis-DDP).     

Nucleoside phosphorylation by phosphate minerals

In the presence of formamide, crystal phosphate minerals may act as phosphate donors to nucleosides, yielding both 5'- and, to a lesser extent, 3'-phosphorylated forms. With the mineral Libethenite the formation of 5'-AMP can be as high as 6% of the adenosine input and last for at least 10(3) h. At high concentrations, soluble non-mineral phosphate donors (KH(2)PO(4) or 5'-CMP) afford 2'- and 2':3'-cyclic AMP in addition to 5'-and 3'-AMP. The phosphate minerals analyzed were Herderite Ca[BePO(4)F], Hureaulite Mn(2+)(5)(PO(3)(OH)(2)(PO(4))(2)(H(2)O)(4), Libethenite Cu(2+)(2)(PO(4))(OH), Pyromorphite Pb(5)(PO(4))(3)Cl, Turquoise Cu(2+)Al(6)(PO(4))(4)(OH)(8)(H(2)O)(4), Fluorapatite Ca(5)(PO(4))(3)F, Hydroxylapatite Ca(5)(PO(4))(3)OH, Vivianite Fe(2+)(3)(PO(4))(2)(H(2)O)(8), Cornetite Cu(2+)(3)(PO(4))(OH)(3), Pseudomalachite Cu(2+)(5)(PO(4))(2)(OH)(4), Reichenbachite Cu(2+)(5)(PO(4))(2)(OH)(4), and Ludjibaite Cu(2+)(5)(PO(4))(2)(OH)(4)). Based on their behavior in the formamide-driven nucleoside phosphorylation reaction, these minerals can be characterized as: 1) inactive, 2) low level phosphorylating agents, or 3) active phosphorylating agents. Instances were detected (Libethenite and Hydroxylapatite) in which phosphorylation occurs on the mineral surface, followed by release of the phosphorylated compounds. Libethenite and Cornetite markedly protect the beta-glycosidic bond. Thus, activated nucleic monomers can form in a liquid non-aqueous environment in conditions compatible with the thermodynamics of polymerization, providing a solution to the standard-state Gibbs free energy change (DeltaG degrees ') problem, the major obstacle for polymerizations in the liquid phase in plausible prebiotic scenarios.     

Condensation of DNA by multivalent cations: experimental studies of condensation kinetics

DNA in viruses and cells exists in highly condensed, tightly packaged states. We have undertaken an in vitro study of the kinetics of DNA condensation by the trivalent cation hexaammine cobalt (III) with the aim of formulating a quantitative, mechanistic model of the condensation process. Experimental approaches included total intensity and dynamic light scattering, electron microscopy, and differential sedimentation. We determined the average degree of condensation, the distribution of condensate sizes, and the fraction of uncondensed DNA as a function of reaction time for a range of [DNA] and [Co(NH(3))(3+)(6)]. We find the following: (1) DNA condensation occurs only above a critical [Co(NH(3))(3+)(6)] for a given DNA and salt concentration. At the onset of condensation, [Co(NH(3))(3+)(6)]/[DNA-phosphate] is close to the average value of 0.54, which reflects the 89-90% charge neutralization criterion for condensation. (2) The equilibrium weight average hydrodynamic radius of the condensates first decreases, then increases with increasing [Co(NH(3))(3+)(6)] as they undergo a transition from intramolecular (monomolecular) to intermolecular (multimolecular) condensation. However, is insensitive to [DNA]. (3) The uncondensed DNA fraction decays approximately exponentially with time. The equilibrium uncondensed DNA fraction and relaxation time decrease with increasing [Co(NH(3))(3+)(6)] but are insensitive to [DNA]. (4) The condensation rate in its early stages is insensitive to [DNA] but proportional to [Co(NH(3))(3+)(6)](xs) = [Co(NH(3))(3+)(6)] - [Co(NH(3))(3+)(6)](crit). (5) Data for low [DNA] and low [Co(NH(3))(3+)(6)] at early stages of condensation are most reliable for kinetic modeling since under these conditions there is minimal clumping and network formation among separate condensates. A mechanism with initial monomolecular nucleation and subsequent bimolecular association and unimolecular dissociation steps with rate constants that depend on the number of DNA molecules in the condensate, accounts reasonably well for these observations.     

Effect of phosphate ions on the fluorescence of tryptophan derivatives. Implications in fluorescence investigation of protein-nucleic acid complexes

In order to test the ability of phosphate groups to quench the fluorescence of tryptophan in protein-nucleic acid complexes we have studied the effect of various phosphate ions on the fluorescence of tryptophan derivatives. Unsubstituted and monoalkyl monoanions (H2PO4- and CH3OPO3H-) quench the fluorescence of all investigated indole derivatives while the dimethyl anion (CH3O)2 PO2- does not. This suggests that quenching of tryptophan fluorescence by phosphate monoanions requires the presence of an acidic OH group and could be due to a proton transfer from the phosphate ion to the indole chromophore. Trianions (PO4 3-4) which are strong proton acceptors quench the fluorescence of all tryptophan derivatives except N(1)methyl tryptophan. This result strongly supports our proposal that quenching of tryptophan fluorescence by phosphate trianions occurs through deprotonation of the NH indole group. Bianions (HPO '4(7), and CH3O PO3 2-3) quench the fluorescence of several indole derivatives including N-acetyl tryptophanamide but have no effect on tryptophan or N(1)-methyl tryptophan. From our results we conclude that phosphate groups of nucleic acids are not able to quench the fluorescence of tryptophyl residues in protein-nucleic acid complexes except if an accessible residue is located near a phosphorylated polynucleotide chain end.