A new 2-(9-anthrylmethylamino)ethyl-appended cyclen, L(3) (1-(2-(9-anthrylmethylamino)ethyl)-1,4,7,10-tetraazacyclododecane) (cyclen = 1,4,7,10-tetraazacyclododecane), was synthesized and characterized for a new Zn(2+) chelation-enhanced fluorophore, in comparison with previously reported 9-anthrylmethylcyclen L(1) (1-(9-anthrylmethyl)-1,4,7,10-tetraazacyclododecane) and dansylamide cyclen L(2). L(3) showed protonation constants log K(a)(i)() of 10.57 +/- 0.02, 9.10 +/- 0.02, 7.15 +/- 0.02, <2, and <2. The log K(a3) value of 7.15 was assigned to the pendant 2-(9-anthrylmethylamino)ethyl on the basis of the pH-dependent (1)H NMR and fluorescence spectroscopic measurements. The potentiometric pH titration study indicated extremely stable 1:1 Zn(2+)-L(3) complexation with a stability constant log K(s)(ZnL(3)) (where K(s)(ZnL(3)) = [ZnL(3)]/[Zn(2+)][L(3)] (M(-)(1))) of 17.6 at 25 degrees C with I = 0.1 (NaNO(3)), which is translated into the much smaller apparent dissociation constant K(d) (=[Zn(2+)](free)[L(3)](free)/[ZnL(3)]) of 2 x 10(-)(11) M with respect to 5 x 10(-)(8) M for L(1) at pH 7.4. The quantum yield (Phi = 0.14) in the fluorescent emission of L(3) increased to Phi = 0.44 upon complexation with zinc(II) ion at pH 7.4 (excitation at 368 nm). The fluorescence of 5 microM L(3) at pH 7.4 linearly increased with a 0.1-5 microM concentration of zinc(II). By comparison, the fluorescent emission of the free ligand L(1) decreased upon binding to Zn(2+) (from Phi = 0.27 to Phi = 0.19) at pH 7.4 (excitation at 368 nm). The Zn(2+) complexation with L(3) occurred more rapidly (the second-order rate constant k(2) is 4.6 x 10(2) M(-)(1) s(-)(1)) at pH 7.4 than that with L(1) (k(2) = 5.6 x 10 M(-)(1) s(-)(1)) and L(2) (k(2) = 1.4 x 10(2) M(-)(1) s(-)(1)). With an additionally inserted ethylamine in the pendant group, the macrocyclic ligand L(3) is a more effective and practical zinc(II) fluorophore than L(1).

A new bis-zinc@) receptor (Zn,L), which has two macrocyclic 12-

A new ditopic host molecule (31, composed of the macromonocyclic 1,4,7,10,13,16-hexa-azacyclo-octadecane (1) and the macrocyclic polyether benzo-15-crown-5 (2) covalently linked, forms stable 1 : 1 complexes with zwitterionic molecules such as amino acids, peptides, and dopamine in aqueous solution at neutral pH.

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1,1'-(m-Xylenediyl)-bis(1,4,7,10-tetraazacyclododecane)-Z n2II complex (m-xylenediyl-bicyclin-Zn2II), a potent inhibitor of human immunodeficiency virus (HIV), was obtained from cyclin by a combination of dimerization and metal complexation. The ratio of median cytotoxic concentration against host cells (CC50) and median effective concentration against HIV cytopathogenicity (EC50), referred to as the selectivity index (SI), was regarded to be a measure of anti-HIV activity. These two chemical modifications contributed to a potent, in vitro anti-HIV activity of m-xylenediyl-bicyclin-Zn2II by respectively increasing the CC50 and decreasing the EC50 in comparison with those of cyclin.

Therapy for glaucoma is generally individualized for each glaucoma patient, with several factors, including intraocular pressure (IOP) and the degree of optic nerve damage, given consideration. In the treatment of glaucoma, several types of agents such as beta-blockers, prostaglandin analogues and carbonic anhydrase (CA) inhibitors are clinically used at present in pharmacotherapy.1-2) Beta-blockers or prostaglandin F2-alpha analogues have been shown to be effective ocular hypotensive agents when used alone in ocular hypertensive or open-angle glaucoma patients.3,4) These two types of agents are currently recognized as the first-line medications for treating glaucoma.5) However, mono-therapy with timolol, a beta-blocker, or latanoprost, a prostaglandin analogue, is often insufficient to maintain a desirable IOP over the long term in many patients. 6) In these cases, CA inhibitors are often used as an adjunctive therapy to timolol or to latanoprost in order to achieve a desirable IOP. 7) Although it has been shown that monotherapy with timolol results in a smaller IOP reduction than monotherapy with latanoprost, the combination therapies of either timolol or latanoprost with CA inhibitors show comparable IOP reduction activity in glaucoma patients. 8) These clinical observations suggest that there exist different interactions between CA inhibitors and latanoprost or timolol that affect IOP reduction.In our previous paper, 9) latanoprost was shown in an in vitro study to be a weak noncompetitive inhibitor of human carbonic anhydrase (HCA) I and II and to bind to the zinc ion, which plays an important role in the expression of enzyme activity according to the AutoDocking simulation method.In the present study, to elucidate the interaction mechanism between timolol and CA inhibitors, the reaction mechanism and binding mode of timolol with respect to CA were investigated by enzyme-kinetic studies and an AutoDocking simulation method. MATERIALS AND METHODS MaterialsHuman CA I and II (HCA I and HCA II) were purchased from Sigma Co., Ltd. Since the purities of HCA I and II were shown to be more than 95% by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), these enzymes were used without further purification. Timolol hemihydrate (formal name:,5-thiadiazol-3-yl}oxy]-2-propanol 1/2H 2 O) was provided by Santen Pharmaceutical Co. Ltd. All other reagents used in this experiment were of analytical grade and were purchased from Wako Pure Chemical Co., Ltd. (Tokyo, Japan).Determination of the Concentration of CO 2 The CO 2 concentration of pure water saturated with CO 2 was measured using a CO 2 electrode (Orion 9502BN) connected to an Orion Ion-meter 720A. The standard solutions of CO 2 were prepared using 0.5-5 ml of 0.1 M NaHCO 3 solutions mixed with 5 ml of 1 M citric acid buffer (pH 4.5) and filled up to 50 ml with pure water.Determination of the Enzyme Activity of Carbonic Anhydrase Determination of the enzyme activity was based on the hydration of CO 2 catalyzed by HCA I and II. The technique used for...