Synthesis,biodistribution and antitumor activity of hematoporphyrin-platinum(II) conjugates

A new series of platinum(II) complexes of pegylated hematoporphyrin derivatives with controlled hydrophobic/hydrophilic balance were synthesized by introducing different kinds of poly(ethylene glycol) and amine ligands to the porphyrin ring. The antitumor activity of the porphyrin-platinum(II) conjugates was assayed in vitro and in vivo against leukemia L1210 cell line and various human tumor cell lines. The present complexes exhibited high antitumor activity and improved water solubility as well as considerable lipophilicity. In particular, complex 16 showed not only higher in vivo activity (T/C%=258) than cisplatin (T/C%=184) and carboplatin (T/C%=168), but also excellent solubility in water and organic solvent. The antitumor activity of complex 20 was superior to that of carboplatin against all human tumor cell lines tested. Moreover, some amphiphilic complexes (7 and 12) exhibited elevated tumor-localizing effect (tumor/muscle ratio>2).     

Crystal structure of the inclusion complex of the antibacterial agent triclosan with cyclomaltoheptaose and NMR study of its molecular encapsulation in positively and negatively charged cyclomaltoheptaose derivatives

The inclusion complexes of triclosan with native cyclomaltoheptaose (beta-cyclodextrin, betaCD) as well as with negatively and positively charged derivatives are studied. The structure of the inclusion complex betaCD/triclosan in the crystalline state [P1, a=15.189(5), b=15.230(6), c=16.293(6), alpha=91.07(4), beta=91.05(3) gamma=100.71(3)] comprises two crystallographically independent host macrocycles A and B. The packing results in betaCD dimers that align head-to-head and form infinite channels along the c-axis. Only one guest molecule statistically disordered over two positions, (the dichlorophenyl ring in the cavities of either A or B) corresponds to each dimer (a 2:1 host/guest complex). The enclosed dichlorophenyl ring enters the dimer through the primary side, whereas the hydrophilic chlorophenol ring extends in the space between dimers. Water molecules in five positions are also enclosed in the intradimer region, arranged on a plane perpendicular to the sevenfold axis of betaCD. The NMR spectroscopic studies in aqueous solution show the presence of both 1:1 and 2:1 betaCD/triclosan complexes. In the first case, two different 1:1 complexes are simultaneously present, each with either ring entering the narrow primary side of one betaCD molecule. In the 2:1 complex both rings of triclosan are included in two independent betaCD hosts, a precursor to the supramolecular arrangement found in the crystalline form. In the case of the negatively charged sodium heptakis[6-deoxy-6-(3-thiopropionate)]-betaCD, the NMR studies at pH 7.9 show a complete inclusion of triclosan inside the host in two orientations, one for the non-ionized (phenol) and reverse for the ionized (phenolate) form. Finally, for the positively charged heptakis(6-aminoethylamino-6-deoxy)-betaCD, inclusion of triclosan is possible only when the pH is raised to 10 and it is concluded that both aromatic rings are alternatively inside the cavity. However in that case also, inclusion of the entire guest in the elongated cavity is suggested.     

Synthesis and evaluation of isatin analogs as caspase-3 inhibitors: introduction of a hydrophilic group increases potency in a whole cell assay

A series of isatin analogs containing a hydrophilic group, including a pyridine ring, ethylene glycol group, and a triazole ring, have been synthesized, and their inhibition potency for caspase-3 was measured both in vitro (i.e., recombinant enzyme) and in whole cells (HeLa cells). The analogs having a hydrophilic group, including 12, 13, 16, 38, and 40, have dramatically increased activity in vitro and in HeLa cells compared to the corresponding unsubstituted N-phenyl isatin analogs.     

Photopatterning of antibodies on biosensors

The immobilization of biomolecules on surfaces in defined micropatterns has become increasingly important for the development of new diagnostic devices and high-throughput genetic and drug screening protocols. We describe the synthesis and testing of thiol-reactive, photoactivatable linkers that will permit laser micropatterning or photolithographic patterning of surfaces. In these linkers, a benzophenone photophore is tethered through a variable-length poly(ethylene glycol) hydrophilic spacer to a maleimide group. Spacers containing one to five ethylene glycol units were examined. Antibodies were photoimmobilized on polystyrene waveguides and the resulting biosensors were used for fluorescence immunoassays. The spacer with five ethylene glycol units optimally decreased the steric interactions among large molecules (antibodies and antigens) and increased binding capacity and response rate of the biosensor. Two different sandwich assay protocols were examined. In the first, the antigen and fluorescently labeled second antibody were added sequentially to the biosensor ("stepwise"). In the second, the antigen and antibody were premixed before injection into the biosensor ("premixed"). The stepwise protocol gave a significantly higher response than that of the premixed protocol. Although the premixed protocol is more convenient, the stepwise protocol provides enhanced sensitivity.     

Synthesis and biological activity of novel platinum(II) complexes of glutamate tethered to hydrophilic hematoporphyrin derivatives

A new series of hematoporphyrin-platinum(II) conjugates was prepared by platination of the glutamate ligand tethered to hydrophilic hematoporphyrin derivatives, in which different numbers of ethylene oxide unit were introduced to modulate the hydrophobic/hydrophilic balance of the conjugates. The antitumor activity of the hematoporphyrin-platinum(II) conjugates was assayed in vitro and in vivo against the leukemia L1210 cell line. Among the complexes, compound 11 exhibited not only higher in vivo activity (T/C% = 192) than cisplatin (T/C% = 184) and carboplatin (T/C% = 168), but also elevated tumor-localizing effect (tumor/muscle ratio > 3).     

Crystallization and preliminary crystallographic data of chicken gizzard G-actin . DNase I complex and Physarum G-actin . DNase I complex.

Smooth muscle G-actin from chicken gizzard and Physarum plasmodium G-actin both interact with DNase I and form 1 : 1 complexes. These complexes were crystallized by using polyethylene glycol 6000 as a precipitant. Both crystals belong to the same orthorhombic space group P2(1)2(1)2(1). The cell dimensions of chicken gizzard G-actin.DNase I complex are a=42.00 +/- 0.07 A, b=225.3 +/- 0.4 A, and c=77.4 +/- 0.1 A, while those of Physarum G-actin.DNase I complex are a=42 A, b=221 A, and c=77 A.     

Multinuclear NMR study of the interaction of vanadate with mononucleotides, ADP, and ATP

The interaction of vanadate with 5'-mononucleotides, ADP, ATP, and various molecules containing some of their chemical moieties was studied in aqueous solution in the pH region of 5-9 using proton, 13C, 31P, and 51V nuclear magnetic resonance (NMR) spectroscopy. All the compounds studied formed noncyclic vanadate esters through interaction of monovanadate or divanadate with the hydroxyl groups of the ribose ring. Noncyclic anhydrides were also formed with the phosphate groups of ribose 5-phosphate, the mononucleotides, ADP, ATP, phosphate, pyrophosphate, and tripolyphosphate. In particular, ADP and ATP analogs resulted from AMP (AMPV and AMPV2) and from ADP (ADPV). Cyclic esters of trigonal bipyramidal geometry resulted from the interaction of vanadate with two ribose ring cis hydroxyl groups. AMP, CMP, and UMP formed two such complexes of 1:1 and 1:2 stoichiometries, similar to what has been observed for uridine and other nucleosides. However, 2'-deoxy-AMP does not yield this type of complexes. ADP and ATP also form similar cyclic ester complexes with vanadate, which does not chelate their pyrophosphate and tripolyphosphate moieties. Nevertheless, the separate pyrophosphate (PP) and tripolyphosphate (PPP) ligands form cyclic anhydrides of octahedral geometry with vanadate. However, their binding to vanadate is weaker than that of the ribose ring of nucleotides. Competition experiments between ethylene glycol and phosphate (P), pyrophosphate (PP), or tripolyphosphate (PPP) show that the relative strength of the interaction of these ligands with vanadate is PP greater than ethylene glycol greater than PPP greater than P.     

A fluorometric assay to determine antioxidant activity of both hydrophilic and lipophilic components in plant foods

This study aimed to develop a fluorometric method to determine total antioxidant activity of plant foods. The antioxidant activities in plant foods were determined after extracting (1) hydrophilic components with acidified methanol (methanol:glacial acetate acid:water=50:3.7:46.3), (2) lipophilic components with methanol followed by tetrahydrofuran (THF), or (3) both hydrophilic and lipophilic components using sequential extraction of acidified methanol and THF together. Both the hydrophilic assay [using the hydrophilic radical initiator 2,2'-azobis-(2-amidinopropane)dihydrochloride (10 mmol/L) and hydrophilic probe 2,7-dichlorodihydrofluorescein (DCFH)] and the lipophilic assay [using the lipophilic radical initiator [2,2'-azobis (4-methoxiy-2,4-dimethylvaleronitrile), 2 mmol/L], and the lipophilic probe 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY 581/591) (BODIPY: 2 micromol/L)] were used to measure antioxidant activity. The inhibition of BODIPY oxidation was significantly increased (P<.01) when both the hydrophilic and lipophilic components were extracted using acidified methanol and organic solvent as compared to those extracted by organic solvent alone. In addition, the rate of DCFH oxidation was significantly delayed (P<.05) when both components coexisted compared to DCFH oxidation of the hydrophilic component alone. The combination of lipophilic and hydrophilic components in these plant foods showed significantly greater antioxidant activity than that of either hydrophilic or lipophilic component alone. Thus, both hydrophilic and lipophilic components in plant foods and their interactions should be considered when determining their antioxidant activity.     

Crystallization of the Fv fragment of mouse myeloma protein M315.

The Fv fragment of mouse myeloma protein M313 was crystallized from poly(ethylene glycol) solution in the form of monoclinic crystals, space group C2 and unit cell dimensions a = 5.96 nm (59.6 A), b = 5.66 nm (56.6 A), c = 13.79 nm (13.9 A) and beta = 99.7 degrees. Some unusual effects of poly(ethylene glycol)on protein crystals were noted and are discussed.     

Preparation, characterization, and antitumor activity of new cisplatin analogs with homopiperazines: crystal structure of [PtII(1-methylhomopiperazine)(methylmalonato)].2H2O

A series of new platinum(II) and (IV) complexes with homopiperazine have been synthesized and characterized by elemental analysis, infrared, and 195Pt nuclear magnetic resonance spectroscopic techniques. The complexes are of two types: [PtIILX] (where L = homopiperazine (hpip), 1-methylhomopiperazine (mhpip), or 1,4-dimethylhomopiperazine (dmhpip), and X = 1,1-cyclobutanedicarboxylato (CBDCA), or methylmalonato ligand) and [PtIV(L-)trans-(Y)2Cl2] (where Y = hydroxo, acetato, or chloro ligand). Among the complexes synthesized, the crystal structure of [PtII(mhpip)(methylmalonato)].2H2O was determined by the single crystal X-ray diffraction method. The crystallographic parameters were orthorhombic, P2(1)2(1)2(1) (no. 19), a = 7.2014(14), b = 7.3348(15), c = 26.971(5) A, and Z = 4. The structure refinements converged to R1 = 0.0641 and wR2 = 0.1847. In this complex, platinum has a slightly distorted square planar geometry with the two adjacent corners being occupied by two nitrogens of the mhpip ligand, whereas the remaining cis positions are coordinated with two oxygen atoms of the methylmalonato group. The mhpip ligand is in a boat conformation and forms five and six membered chelating rings with platinum. The intricate network of intermolecular hydrogen bonds holds the crystal lattice together. Some of these synthesized cisplatin analogs have good in vitro cytotoxic activity against the cisplatin-sensitive human ovarian A2780 (IC50 = 0.083-17.8 microM) and the isogenic cisplatin-resistant 2780CP (IC50 = 20.1-118.1 microM) cell lines.     

Metal ion complexes of macrocyclic polyamines enhance both the phosphate hydrolysis and imidazole ring opening of RNA 5'-cap structure

The cleavage of P1-(7-methylguanosyl-5') P3-(guanosyl-5') triphosphate, a RNA 5'-cap model, by 2-hydroxyethyl- (6a-6c) and 2-aminoethyl- (7a-7c) substituted macrocycles in the presence and absence of Zn2+ and Cu2+ ions has been studied at pH 7.2 and 60 degrees. In the presence of the metal ions, hydrolysis of the phosphate group is enhanced. The mono- and dinuclear Zn2+ complexes promote solely the phosphate hydrolysis, whereas the corresponding Cu2+ complexes accelerate both the phosphate hydrolysis and the imidazole ring opening of the 7-methylguanine base. In the absence of the metal ions, the macrocycles mainly promote breakdown of the 7-methylguanine base, most probably by enhancing the nucleophilic attack of hydroxide ion on the C(8)-atom by shielding the repulsive negative charge on the phosphate moiety. The 2-hydroxyethyl and 2-aminoethyl side arms exhibit a two- to three-fold rate acceleration. Opening of the imidazole ring eventually results in cleavage of the triphosphate bridge.     

Preliminary crystallographic studies on duck ovotransferrin

Crystals of duck ovotransferrin and duck apo-ovotransferrin have been grown from polyethylene glycol solutions. For both crystals, the space group is P2(1)2(1)2(1), the unit cell dimensions for the ovotransferrin are a = 49.6 A, b = 85.6 A, c = 178.7 A and for the apo-ovotransferrin a = 77.6 A, b = 98.8 A, c = 127.0 A, giving four molecules in the unit cell.     

Crystallization and preliminary x-ray crystallographic study of NADH-cytochrome b5 reductase from pig liver microsomes

Crystals of the hydrophilic, catalytic domain (30 kDa) of pig liver NADH-cytochrome b5 reductase solubilized by the protease (cathepsin D) have been grown in a solution of polyethylene glycol by the vapor-diffusion procedure. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1) with unit-cell dimensions of a = 87.1, b = 73.2, and c = 49.0 A. The asymmetric unit contains one molecule of the enzyme. The x-ray diffraction patterns extend to 2.0-A resolution. On the other hand, the intact enzyme (35 kDa) containing the hydrophobic membrane-binding domain solubilized by the detergent (Triton N-101) has been crystallized also from the polyethylene glycol solution. The crystals are needle-shaped and still too small for x-ray diffraction study.     

Synthesis and comparative molecular field analysis (CoMFA) of argentatin B derivatives as growth inhibitors of human cancer cell lines

Synthesis, characterization, anticancer activity, and comparative molecular field analysis (CoMFA) of 14 argentatin B (1) analogs are described. The effect of argentatin B derivatives on the growth of K562 (leukemia), PC-3 (prostate), U251 (CNS), and HCT-15 (colon) human cancer cell lines was determined using the sulforhodamine B test. The most active compound in this series, 2-formyl-(16beta,24R)-16,24-epoxy-25-hydroxycycloart-1-en-3-one (12), was about 35-50 times more potent than argentatin B (1). Structures were built using the X-ray crystallography of six derivatives for 3D modeling with Sybyl6.9. CoMFA of Log (1/IC50) in K562 cell line gave q2 = 0.507, r2 = 0.907, and three components. The standard deviation CoMFA contours indicate that increased activity is associated with a bulky group at C-2, a C1-C2 double bond, and low electronic density at C-25. Experimental Log P values for argentatin B and one derivative were 1-2 Log units more hydrophilic than the calculated CLog P values.     

Preliminary X-ray crystallographic study of adenylosuccinate synthetase from Escherichia coli

Adenylosuccinate synthetase, a dimeric enzyme of 96,000 Mr, catalyzes the first committed step toward the de novo biosynthesis of AMP. Large, single crystals of adenylosuccinate synthetase from Escherichia coli grow from solutions of polyethylene glycol and ammonium sulfate. Crystals from ammonium sulfate belong to the orthorhombic space group P212121 with unit cell parameters a = 79.0 A, b = 70.2 A and c = 152.6 A. Crystals from polyethylene glycol belong to the space group P21, having unit cell parameters of a = 71.16 A, b = 71.99 A, c = 82.95 A, and beta = 71.52 degrees. The asymmetric units of both crystal forms probably contain the entire dimeric enzyme.     

Combined biotin-terpyridine systems: a new versatile bridge between biology, polymer science and metallo-supramolecular chemistry

Biotin, a well-known binding unit for the proteins avidin and streptavidin, was combined with the chelating ligand terpyridine via polymeric and nonpolymeric spacers. An omega-amino-functionalized terpyridyl-poly(ethylene glycol) was prepared and utilized for complex formation with iron(II), nickel(II), and ruthenium(II) ions. The biocompatibility of the complex formation was investigated in aqueous media. Moreover, biotin was functionalized with a methoxy-poly(ethylene glycol) as a model system. The compounds were characterized by UV/vis and NMR spectroscopy as well as MALDI-TOF mass spectrometry. The systems represent a new combination of strong noncovalent binding units from both biology and synthetic supramolecular chemistry.     

Effect of PEG and mPEG-anthracene on tRNA aggregation and particle formation

Poly(ethylene glycol) (PEG) and its derivatives are synthetic polymers with major applications in gene and drug delivery systems. Synthetic polymers are also used to transport miRNA and siRNA in vitro. We studied the interaction of tRNA with several PEGs of different compositions, such as PEG 3350, PEG 6000, and mPEG-anthracene under physiological conditions. FTIR, UV-visible, CD, and fluorescence spectroscopic methods as well as atomic force microscopy (AFM) were used to analyze the PEG binding mode, the binding constant, and the effects of polymer complexation on tRNA stability, aggregation, and particle formation. Structural analysis showed that PEG-tRNA interaction occurs via RNA bases and the backbone phosphate group with both hydrophilic and hydrophobic contacts. The overall binding constants of K(PEG?3350-tRNA)= 1.9 (±0.5) × 10(4) M(-1), K(PEG?6000-tRNA) = 8.9 (±1) × 10(4) M(-1), and K(mPEG-anthracene)= 1.2 (±0.40) × 10(3) M(-1) show stronger polymer-RNA complexation by PEG 6000 and by PEG 3350 than the mPEG-anthracene. AFM imaging showed that PEG complexes contain on average one tRNA with PEG 3350, five tRNA with PEG 6000, and ten tRNA molecules with mPEG-anthracene. tRNA aggregation and particle formation occurred at high polymer concentrations, whereas it remains in A-family structure.     

Survival of vitrified sheep embryos in vitro and in vivo

The effects of the composition of vitrification media, the duration of exposure to the media and the stage of development were examined on the survival of vitrified Day-6 sheep embryos. Vitrification media that contained two cryoprotectants in equal molar concentrations were used. In Experiment 1, the effects of the types (glycerol + propylene glycol or glycerol + ethylene glycol) and concentrations (3.5 + 3.5 or 4.5 + 4.5 M) of cryoprotectants and the level of BSA supplementation (0.4 or 20%) were investigated in a 2 x 2 x 2 design. The embryos were exposed to vitrification media for 30 sec at 18 to 24 degrees C before vitrification. The in vitro survival rate was not affected by the level of BSA supplementation, but there was an interaction between the types and concentrations of cryoprotectants used (P<0.01). Embryos cryopreserved in mixtures of glycerol + propylene glycol survived better when the concentration of cryoprotectants was 3.5 M while the survival of embryos cryopreserved in mixtures of glycerol + ethylene glycol was higher at 4.5 M cryoprotectant concentration. In Experiments 2 and 3, the effect of the duration of exposure (15, 30, 60 or 120 sec) to vitrification media at 4 to 12 degrees C was investigated on the survival rate in vivo. Vitrification media contained 3.5 M glycerol + 3.5 M propylene glycol or 4.5 M glycerol + 4.5 M ethylene glycol in Experiments 2 and 3, respectively. The survival rate in vivo, increased when the duration of exposure to vitrification media was increased from 15 to 30 sec, but the viability declined when the duration of exposure was further increased to 60 (Experiment 3) or to 120 sec (Experiment 2). The effect of the stage of development was significant only in Experiment 1 (P = 0.032), but in all three experiments the rate of survival increased with advancing stages of development from late morulae to late blastocysts. The best result was achieved in Experiment 2, when embryos were exposed to a mixture of 3.5 M glycerol + 3.5 M propylene glycol for 30 or 60 sec. Under these conditions 52% (22 42 ) of rapidly cryopreserved sheep embryos developed into lambs. This result shows that a simple rapid procedure for the cryopreservation of sheep embryos can produce a survival rate comparable to that obtained using more complex traditional procedures.     

Effect of ethylene glycol on the interaction of different myosin subfragment 1.nucleotide complexes with actin

To elucidate the structure of the cross-bridge intermediates in the actomyosin ATPase cycle, several laboratories have added both ethylene glycol and AMP-PNP to muscle fibers. These studies suggested that ethylene glycol shifts the structure of myosin.AMP-PNP toward the weak-binding conformation, i.e., toward the structure of myosin.ATP. Since only the weak-binding conformation of myosin subfragment 1 (S-1) binds with no apparent cooperativity to the troponin-tropomyosin-actin complex (regulated actin), we used this as a probe to examine the conformation of various S-1.nucleotide complexes in ethylene glycol. Our results show that ethylene glycol markedly weakens the binding strength of S-1, S-1.ADP, and S-1.AMP-PNP to actin but has almost no effect on the binding strength of S-1.ATP. As in muscle fibers, at 40% ethylene glycol, the binding strength of S-1.AMP-PNP to actin becomes very similar to the binding strength of S-1.ATP. In the presence of troponin-tropomyosin, the binding of S-1.AMP-PNP to actin shows no apparent cooperativity in 40% ethylene glycol. Therefore, our results confirm that ethylene glycol shifts the structure of the myosin.AMP-PNP toward the weak-binding conformation. However, our results also suggest that ethylene glycol has a direct effect on the regulated actin complex. This is shown by the fact that ethylene glycol markedly increases the cooperative binding of S-1.ADP to regulated actin both in the presence and in the absence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)