Synthesis and antitumor activity of platinum complexes of protonated diamines

Complexes of the formula cis-[Pt(H$\text{N}\text{+}$^～N)(L)Cl₂], where (H$\text{N}\text{+}$^～N) are the protonated diamines including 3-aminoquinuclidine, N-aminopiperidine, piperazine, N-methylpiperazine, 1,1,4-trimethylpiperazine, and N-methyl-1,4-diazabicyclo [2,2,2] octane (N-methyl-dabco) and L = SCN^?, NO₂^?, Br^?, and F^?, were synthesized from the protonated diamine complexes, [Pt(H$\text{N}\text{+}$^～N)Cl₃]. The antitumor activities of the complexes were evaluated in vitro against L1210 murine leukemia cells, and ID₅₀ values for the L-substituted complexes were compared to values of the parent complexes. In each case it was found that replacement of a chloride ion by SCN^?, NO₂^?, Br^?, or F^?, either reduced or completely eliminated antitumor activity. This effect is explained in terms of the trans-directing ability of the ligand, L, compared to chloride. The NO₂-substituted complex of 3- aminoquinuclidine was tested in vivo and found to exhibit little or no antitumor activity.     

Chloride and tropolonato mixed complexes of uranium(IV) and thorium(IV)

The synthesis of some new chloride and tropolonato mixed complexes of the general composition MCl_4?nTrop_n·mL (M = U^IV, Th^IV; n = 1, 2, 3; m = $\text{1}\text{2}$, $\text{2}\text{3}$, 1, 2 and 3; L = DME, THF, LiCl and 18-crown-6) by the reaction of uranium and thorium tetrachlorides with M′Trop (M′ = Tl and Li) is described. The residual chloride atoms in UTropCl₃·THF undergo further substitution reactions involving TlCp, while UTrop₂Cl₂·THF shows a different behaviour toward TlCp, since Cp₃UCl as one of the main reaction products is obtained. Protolysis of Cp₂U(NEt₂)₂ by HTrop affords a mixture of CpUTrop₃ and Cp₂UTrop₂. Finally both UTropCl₃·THF and UCl₄ react directly with HTrop, the nature of the resulting products depending on the reaction solvent.     

Electron paramagnetic resonance crystallography of spin-labeled hemoglobin-protein fine structures.

Various hemoglobin derivatives have been labeled at the Cys-β93 residue with a bulky and “strongly immobilized” nitroxide maleimide (I) and a smaller, more flexible and “weakly immobilized” nitroxide iodoacetamide (II) and crystallized. The angular dependence of the paramagnetic resonance of the spin-label was measured for the ab, $\text{ac}{}^1$ and $\text{bc}{}^1$ planes at 298 K and 77 K for spin-labeled crystals of Oxyhemoglobin, methemoglobin fluoride, and methemoglobin azide. In the case of the methemoglobin crystals, the angular variation of the heme resonance was also monitored at 77 K. From the hyperfine splitting data, the spin-label I was found to assume specific orientations at both temperatures with some motional narrowing at 298 K. Spin-label II is specifically oriented only at room temperature but is frozen at 77 K in random orientations. Oxyhemoglobin labeled with I (I-HbO₂²) has the most prominent spin-label orientation (z∥b, x∥a) and the less abundant spin-labels with (z∥b ± 15 °) (Ohnishi et al., 1966). The corresponding spin-label orientations for I-Hb⁺ F^? are ($\text{z∥a, x∥c}{}^1$) and ($\text{z∥c}{}^1\text{, x∥a}$). Crystals of I-Hb⁺ N^?₃ have spin-labels oriented along angular directions similar, but not identical to those of I-Hb⁺F^?. Therefore, there are probably significant peptide segmental displacements when HbO₂ is oxidized to methemoglobins. At 25 °C II-Hb⁺ N^?₃ has spin-label orientations not too different from those in I-Hb⁺ N^?₃, whereas in HbO₂ the two spin-labels show significant differences in their orientations.     

Acylated des-(Ala1-Gly2)-somatostatin analogs: prolonged inhibition of growth hormone secretion

The following eight analogs of somatostatin were synthesized by solid phase: des-[Ala¹-Gly²]-somatostatin (I); des-[Ala¹-Gly²]-H₂somatostatin (II); $\text{N}$-acetyl-Cys³-somatostatin (III); $\text{N}$-acetyl-Cys³-H₂somatostatin (IV); $\text{N}$-pyvalyl-Cys³-H₂somatostatin (V); $\text{N}$-acrylyl-Cys³-H₂somatostatin (VI); $\text{N}$-benzoyl-Cys³-H₂somatostatin (VII); $\text{N}$-hexanoyl-Cys³-H₂somatostatin (VIII). Deletion of the N-terminal dipeptide Ala¹-Gly² is compatible with high biological activity. A single s.c. injection of these analogs as a microsuspension in saline inhibits for 24–72 hours (depending on the compound) the secretion of growth hormone normally stimulated in rats by pentobarbital.     

Analysis of metallonucleoside hydrolysis by high-pressure liquid chromatography

A number of ammine complexes of transition metals in the platinum group exhibit antitumor and mutagenic activities, which probably result from in vivo metal ion coordination to nucleic acids. Coordination at N-7 purine sites on nucleic acids may induce depurination through a general acid-catalyzed cleavage of the sugar-purine bond. This possibility was tested by observing the hydrolysis of [dGuo)(NH₃)₅Ru]³⁺ under physiological conditions. Since multiple products result from this reaction, a high-pressure liquid chromatography technique was developed for separating various ammineruthenium(III) complexes with purine, pyrimidine, and nucleoside ligands. Using this technique it was possible to identify and follow the relative concentrations of several of the hydrolysis products. These data were used to develop a preliminary reaction scheme for the decomposition of (dGuo)(NH₃)₅Ru(III). The net rate constant (1.8 × 10^?6 s^?1) for the disappearance of this complex yields an upper limit for the rate of metal-induced sugar hydrolysis. While the half-life ($\text{5}\text{days}\text{< t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{< 28}\text{days}$) for the sugar hydrolysis reaction is substantially shorter than that for the free nucleoside under the same conditions, it cannot be concluded that this represents a major contribution to the mutagenicity of Ru(III) complexes.     

Kinetic studies on the reaction of ethylenediaminetetraacetatochromium(III) complex with hydrogen peroxide

The rate of reaction of [Cr(III)Y]_aq (Y is EDTA anion) with hydrogen peroxide was studied in aqueous nitrate media [μ = 0.10 M (KNO₃)] at various temperatures. The general rate equation, Rate = $\text{k}{}_1\text{+}\text{k}{}_2\text{K}{}_1\text{[H}{}^{\mathrm{+}}\text{]}{}^{\mathrm{?1}}\text{1 +}\text{K}{}_1\text{[H}{}^{\mathrm{+}}\text{]}{}^{\mathrm{?1}}$ [Cr(III)Y]_aq[H₂O₂] holds over the pH range 5–9. The decomposition reaction of H₂O₂ is believed to proceed via two pathways where both the aquo and hydroxo-quinquedentate EDTA complexes are acting as the catalyst centres. Substitution-controlled mechanisms are suggested and the values of the second-order rate constants k₁ and k₂ were found to be 1.75 × 10^?2 M^?1 s^?1 and 0.174 M^?1 s^?1 at 303 K respectively, where k₂ is the rate constant for the aquo species and k₂ is that for the hydroxo complex. The respective activation enthalpies (ΔH^*₁ = 58.9 and ΔH^*₂ = 66.5 KJ mol^?1) and activation entropies (ΔS^*₁ = ?85 and ΔS^*₂ = ?40 J mol^?1 deg^?1) were calculated from a least-squares fit to the Eyring plot. The ionisation constant pK₁, was inferred from the kinetic data at 303 K to be 7.22. Beyond pH 9, the reaction is markedly retarded and ceases completely at pH ? 11. This inhibition was attributed in part to the continuous loss of the catalyst as a result of the simultaneous oxidation of Cr(III) to Cr(VI).     

Specific platinum chelation by the guanines of the deoxyhexanucleotide d(T-G-G-C-C-A) upon reaction with cis-[Pt(NH3)2(H2O)2](NO3)2

The stoichiometric reaction between d-TpGpGpCpCpA (d(T-G-G-C-C-A)) and $\text{cis}$-[Pt(NH₃)₂(H₂O)₂](NO₃)₂ (8.4 × 10^?6 to 1.3 × 10^?4M in water at pH 5.5–6) gives a single complex. High pressure gel permeation chromatography and pH-dependent ¹H NMR analyses of the nonexchangeable base protons, show that it is a platinum chelate with the $\text{cis}$-Pt^II(NH₃)₂ moiety bound to the two N7 atoms of the adjacent guanines. A 3 × 10^?3M reaction gives the same platinum chelate, via the formation of intermediate complexes, together with unsoluble adducts.     

Analysis of the kinetics of electron transfer reactions of hemoglobin and myoglobin with inorganic complexes.

The kinetics of methemoglobin reduction by Fe(EDTA)^2? have been studied and found to follow a second order rate law with k = 29.0 $\text{M}$^?1 s^?1 [25°C, μ = 0.2 $\text{M}$, pH 7.0 (phosphate)], $\text{ΔH}{}^3\text{= 5.5 ± 0.7 kcal/mol}$, and $\text{ΔS}{}^2\text{= ?33 ± 2 e.u.}$. The electrostatics-corrected self-exchange rate constant (k₁₁^corr) for hemoglobin based on the Fe(EDTA)^2? cross-reaction is 2.79×10^?3$\text{M}$^?1 s^?1. This rate constant is compared with others reported for a water-soluble iron porphyrin and calculated from published data for the reactions of myoglobin and hemoglobin with Fe(EDTA)^2? and Fe(CDTA)^2?/?. The k₁₁^corr values for these systems range over ten orders of magnitude with heme ? myoglobin > hemoglobin.     

Polar group interaction and molecular packing of membrane lipids: The crystal structure of lysophosphatidylethanolamine

The conformation and molecular packing of 3-palmitoyl-dl-glycerol-1-phosphoryl-ethanolamine has been determined by a single crystal analysis (R = 0.115); it crystallizes in the monoclinic space group $\text{P2}{}_1\text{a}$ with a unit cell of $\text{a = 7.66}\text{A}\text{?}\text{, b = 9.08}\text{A}\text{?}\text{, c = 37.08}\text{A}\text{?}\text{and}\text{β = 90.2 °}$, with four molecules per unit cell. The molecules exist as configurational and conformational enantiomers and pack in a bilayer arrangement. The phosphorylethanolamine groups have an orientation parallel to the layer surface. The hydrocarbon chains are arranged according to the T∥ chain packing mode and adopt an extreme tilt of 57.5 ° with respect to the layer normal. The free glycerol hydroxyl group forms an intramolecular hydrogen bond with, a phosphate oxygen and thus affects the conformation and orientation of the head group. The phosphorylethanolamine dipoles are oriented parallel to each other in double rows, while they are antiparallel and form a continuous network in dilauroylphosphatidylethanolamine (Elder et al., 1977). The area per molecule in 3-palmitoyl-dl-glycerol-1-phosphorylethanolamine (34.8 Ų) is less than in diacylphosphatidylethanolamine (38.6 Ų), indicating that in the latter the hydrocarbon chains determine the molecular cross-section. The significance of the interaction and space requirement of the phosphorylethanolamine group for the phase behaviour of phosphatidylethanolamine is discussed.     

Protein synthesis in rabbit reticulocytes: control of protein synthesis initiation by a met-tRNA Met f deacylase and peptide chain initiation factors

The 0.5M KCl wash of rabbit reticulocyte ribosomes (I fraction) catalyzes the deacylation of Met-tRNA_f^Met. Upon DEAE-cellulose column chromatography, the deacylase activity elutes with the 0.1M KCl wash of the column (f1) and is well-resolved from the peptide chain initiation factors (1–3). The deacylase activity is specific for Met-tRNA_f^Met (retic., $\text{E.}$$\text{coli}$). Other aminoacyl tRNAs tested including fMet-tRNA_f^Met (retic., $\text{E.}$$\text{coli}$), Phe-tRNA ($\text{E.}$$\text{coli}$), Val-tRNA (retic.), and Arg-tRNA (retic.) are completely resistant to the action of the deacylase. In the presence of the peptide chain initiation factor (IF1) and GTP, retic. Met-tRNA_f^Met forms the initiation complex Met-tRNA_f^Met:IF1:GTP (2), and in this ternary complex Met-tRNA_f^Met is not degraded by the deacylase. $\text{E.}$$\text{coli}$ Met-tRNA_f^Met binds to IF1 independent of GTP, and in this complex, this Met-tRNA_f^Met is degraded by the deacylase.Prior incubation of f1 with Met-tRNA_f^Met (retic.) strongly inhibited protein synthesis initiation, presumably due to deacylation of the initiator tRNA. This inhibition by f1 was completely prevented when Met-tRNA_f^Met (retic.) was pre-incubated with peptide chain initiation factors.     

A Cl−/HCO3−-ATPase in the gils of Carassius Auratus. Its inhibition by thiocyanate

An ATPase is demonstrated in plasma membrane fractions of goldfish gills. This enzyme is stimulated by Cl^? and HCO₃^?, inhibited by SCN^?.Biochemical characterization shows that HCO₃^? stimulation ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 2.5}\text{mequiv./l}$) is specifically inhibited in a competitive fashion by SCN^? ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.25}\text{mequiv./l}$). The residual Mg²⁺-dependent activity is weakly is weakly affected by SCN^?.In the microsomal fraction chloride stimulation of the enzyme occurs in the presence of HCO₃^? ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{for chloride = 1 mequiv./l}$); no stimulation is observed in the absence of HCO₃^?. Thiocyanate exhibits a mixed type of inhibition ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.06}\text{mequiv./l}$) towards the Cl^? stimulation of the enzyme.Bicarbonate-dependent ATPase from the mitochondrial fraction is stimulated by Cl^?, but this enzyme has a relatively weak affinity for this substrate ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 14}\text{mequiv./l}$).     

The crystal structure of galactaric acid (mucic acid) at −147°: An unusually dense,hydrogen-bonded structure

Galactaric acid, C₆H₁₀O₈, (CAS Reg. No. 526-99-8), is triclinic, P$\text{1}$, with cell dimensions at ?147° [and 20°], a = 4.900(1) [4.918(1)], b = 5.728(1) [5.816(1)], c = 6.784(1) [6.849(1)] Å, α = 92.32(2) [92.31(2)], β = 93.74(2) [94.16(2)], γ = 93.08(2) [93.49(2)]°, V = 189.5 ų, Z = 1, D_x = 1.831 [1.800], D_m = [1.790] g.cm^?3, molecular symmetry $\text{I}$. The structure was solved by the direct method, MULTAN, and refined to R = 0.034, R_w = 0.039 for 787 reflections with F_Obs > 3σ(F_obs). The crystal structure has a system of strong, intermolecular hydrogen-bonds, which accounts for the high crystal density and low solubility in water.     

Behavior of rat hypothalamic and extrahypothalamic immuno-reactive TRF in thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC)

[³H] quinuclidinyl benzilate (QNB), a specific muscarinic antagonist, was utilized to identify muscarinic cholinergic receptors on dispersed anterior pituitary cells. Scatchard analysis of [³H] QNB binding to receptors departs from linearity with upward concavity. A high affinity binding site having a dissociation constant (K_d) of 1.5 nM was observed when the [³H] QNB concentration was varied from 0.15 to 20 nM. A low affinity binding site (K_d 20 nM) was observed when [³H] QNB concentration was above 20 nM. Using 10 nM [³H] QNB for binding, the second order association rate constant (k₁) of 0.064 nM^?1 min^?1 and first order dissociation rate constant (k₂) of 0.078 min^?1$\text{(}\text{T}\text{1}\text{2}\text{8}\text{min}\text{)}$ were observed. k₂/k₁ = K_d of 1.22 nM is in good agreement with K_d = 1.5 nM from equilibrium data. Muscarinic cholinergic receptor antagonists, atropine and scopolamine, and agonist oxtoremorine potently competed with [³H] QNB binding. A nicotinic cholinergic receptor agonist was 50 times less potent as a competitor of [³H] QNB binding than the muscarinic agonist.     

The use of bacitracin as an inhibitor of the degradation of thyrotropin releasing factor and luteinizing hormone releasing factor

Bacitracin was found to be an effective inhibitor of the $\text{in}\text{vitro}$ degradation of both thyrotropin releasing factor¹ (TRF) and luteinizing hormone releasing factor (LRF) by guinea pig hypothalamic and whole brain homegenates and rat hypothalamic homogenates and subcellular fractions. Bacitracin was effective in inhibiting the degradation of TRF and LRF, as determined by radioimmunoassay, where it exhibited no interference with the assays. Kinetic studies of the degradation of exogenous synthetic [³H]-TRF demonstrated non-competitive inhibition by bacitracin with K_i = 1.9 × 10^?5 M, while studies on the degradation of [³H] LRF indicated competitive inhibition with K_i = 1.7 × 10^?5 M. Electrophoretic and amino acid analysis revealed that bacitracin itself was not degraded during the course of the $\text{in}\text{vitro}$ incubation.     

Ligand displacement reactions of oxyhemocyanin: comparison of reactivities of arthropods and molluscs.

The ligand displacement reactions of oxyhemocyanin have been compared over a series of arthropods and molluscs. The arthropods (with the exception of $\text{Limulus}$) are found to be more reactive than the molluscs, (k_cancer = .04 hr^?1, k_busycon = .002 hr^?1, k_limulus ? 10^?4hr^?1 N₃^? reactions). Correlation of the spectral properties of the oxy sites require these to be extremely similar with small differences being associated with shifts in the dd transition energies. The met produced by ligand displacement contains variable amounts of EPR detectable, group 2 exogenous ligand damaged sites (arthropods 25–35%, molluscs 3–9%, $\text{Limulus}$ <1%). A parallel (arthropod > mollusc ～ $\text{Limulus}$ ～ 0) group 2 ligand damaged site is also reported for the half met derivatives.     

Purification and structural properties of the precursors of the globin messenger RNAs

X-ray diffraction data have been obtained from sodium and calcium salts of a proteoglycan rich in chondroitin 4-sulfate isolated from the Swarm rat chondrosarcoma. When sodium is the only countercation associated with the proteoglycan, the oriented polysaccharide chains adopt a 3-fold helical conformation in the solid state and pack in a trigonal unit cell with dimensions a = b = 1.45 nm and c = 2.88 nm. Addition of small amounts of calcium or full conversion of the polyanion from a sodium to a calcium salt form results in a conformational transition to a somewhat more extended 2-fold structure.For the calcium salt X-ray intensity data were used to refine the polysaccharide conformation and packing arrangement in the unit cell. Two antiparallel chains were found to crystallize in an orthorhombic unit cell with space group P22₁2₁ and dimensions a = 0.745 nm, b = 1.781 nm and c = 1.964 nm. The individual helix axes intersect the base plane of the unit cell at (x_f = 0, y_f = 0) and ($\text{x}{}_{\mathrm{f}}\text{= 0, y}{}_{\mathrm{f}}\text{=}\text{1}\text{2}$), and the polyanions are crystallographically equivalent, being related by the symmetry of the space group.The conformation of chondroitin 4-sulfate is stabilized intramolecularly by O.3 … O.5 hydrogen bonds across the β(1 → 4) linkage as well as by OSO^?₃ … Ca²⁺ … ^?OOC co-ordination across the β(1 → 3) linkage. Within the lattice adjacent parallel chains interact through COO^? … Ca²⁺ … ^?OOC bridges, and each calcium co-ordination shell is completed with an additional five water molecules to form a distorted, square antiprism. These water molecules are hydrogen-bonded to neighboring polyanions, and all intermolecular interactions involve water bridges or calcium ion co-ordination.On the basis of the refined packing model and the known structural features of the proteoglycan, models are considered for proteoglycan organization in connective tissue. Consideration of the conformational directing influence and relative abundance of calcium in the intercellular matrix suggest that the secondary structure of chondroitin 4-sulfate in vivo is likely to be similar to the conformation described in this study.     

Two temporal phases for the control of histone gene activity in cleaving sea urchin embryos (S. purpuratus)

This report presents an analysis of histone gene expression in the cleaving embryo of the sea urchin, Strongylocentrotus purpuratus, with emphasis on whether the regulatory site(s) in the pathway of gene expression change as development proceeds. The analysis focuses on the equation, $\text{dP1}\text{dt}\text{= M·f·n·}\text{A}\text{t}$, where $\text{dP1}\text{dt}$ = the absolute rate of histone synthesis; M = the mole quantity of histone messenger RNA; f = the fraction of histone mRNA in polysomes; n = the polysome size; and $\text{A}\text{t}$ = the rate of elongation of nascent histone polypeptide chains. The embryo solves this rate equation differently at different times. Measurements were made (at 15°C) of absolute rates of histone synthesis ($\text{dP1}\text{dt}$). The rate of histone synthesis increases at least 48-fold during the first 6 hr after fertilization from less than 0.5 to 24 pg embryo^?1 hr^?1; in the period from 6 to 12 hr, this rate rises to 182 pg embryo^?1 hr^?1, an additional 7.7-fold rise, resulting in an overall increase of 370-fold between the 1-cell and 200-cell stage. The fraction of newly synthesized (zygotic) histone messenger RNA that partitions into polysomes (f^zygotic) has also been measured during the first 12 hr of development. This fraction increases from 0.2 in the 2-hr embryo to 0.8 in the 6-hr embryo (16-cell stage), increasing slowly thereafter to near unity by 12 hr. The size of histone-synthesizing polysomes (n) does not change substantially over the 12-hr interval, remaining constant at a weighted mean of 5 ribosomes per polysome (range 3 to 7). Utilizing the data on f^zygotic and $\text{dP1}\text{dt}$, the rate of elongation of nascent histone polypeptide chains ($\text{A}\text{t}$) during the first 6 hr of development was estimated; $\text{A}\text{t}$ remains constant at 1.11 codons per second. This calculated value is in fair agreement with a direct measurement of histone peptide elongation rate in the 12-hr embryo. It is proposed that histone gene expression in cleaving sea urchin embryos be divided into two phases, distinguished on the basis of their pivotal translational parameters: Phase I (0–6 hr), during which f is rate determining, and Phase II (6 hr on), during which M is the rate-determining parameter.