Methylenediphosphonotetrathioate: Synthesis, Characterization, and Chemical Properties

Metal chelators are potential therapeutic agents for treating diseases such as Wilson’s and Alzheimer’s where the pathology involves an excess of metal-ions (Cu­(II) and Zn­(II)/Cu­(II)/Fe­(II/III), respectively). In addition to the high affinity of the metal-ion to the chelators, metal selectivity of the chelators is essential to achieve the therapeutic goal, that is, the successful removal of excess of harmful metal-ions in a physiological extracellular medium rich in alkali and alkali earth metal-ions. For this purpose, we synthesized a novel chelator, methylenediphosphonotetrathioate (MDPT) which is the tetrathio analogue of methylenediphosphonic acid (MDP). MDPT was synthesized from bis-methylene­(phosphonicdichloride) in a 3-step synthesis and a 31% overall yield. MDPT formed a stable complex with Zn­(II) (log <i>K</i> = 10.84), which is 10⁷ times more stable than the corresponding Ca­(II) complex. Moreover, the MDPT-Zn­(II) complex was 50-fold more stable than the MDP-Zn­(II) complex. In addition, MDPT was found to inhibit the Cu­(I)-catalyzed Fenton reaction (IC₅₀ 26 μM) 2.5 times more potently than a Fe­(II)-catalyzed Fenton reaction, and 2.5 times more potently than EDTA (IC₅₀ 64 μM) in the Cu­(I)/H₂O₂ system, as monitored by electron spin resonance (ESR). Furthermore, MDPT was found to be relatively stable in both acidic (pD 1.9, <i>t</i>_¹/₂ = 71.5 h) and basic media (pD 12.4, <i>t</i>_¹/₂ = 81 h) as monitored by ³¹P/¹H NMR. However, MDPT was not stable in air because of intramolecular oxidation and disulfide formation (33% oxidation after 27 h). In conclusion, MDPT was found to be a water-soluble chelator showing a clear preference to soft/borderline metal-ions and a remarkable selectivity to those metal-ions vs Ca­(II) ions. The relative sensitivity of MDPT to oxidation may limit its use; however, the application of MDPT in acidic or basic media will increase its lifetime

Nucleoside2′,3′/3′,5′-Bis(thio)phosphate Analogues Are Promising Antioxidants Acting Mainly via Cu⁺/Fe²⁺ Ion Chelation

We synthesized a series of adenine/guanine 2′,3′- or 3′,5′-bisphosphate and -bisphosphorothioate analogues, <b>1</b>–<b>6</b>, as potential Cu⁺/Fe²⁺ chelators, with a view to apply them as biocompatible and water-soluble antioxidants. We found that electron paramagnetic resonance (EPR)-monitored inhibition of OH radicals production from H₂O₂, in an Fe²⁺-H₂O₂ system, by bisphosphate derivatives <b>1</b>, <b>3</b>, and <b>5</b> (IC₅₀ = 36, 24, and 40 μM, respectively), was more effective than it was by ethylenediaminetetraacetic acid (EDTA), by a factor of 1.5, 2, and 1.4, respectively. Moreover, 2′-deoxyadenosine-3′,5′-bisphosphate, <b>1</b>, was 1.8- and 4.7-times more potent than adenosine 5′-monophosphate (AMP) and adenosine 5′-diphosphate (ADP), respectively. The bisphosphorothioate derivatives <b>2</b>, <b>4</b>, and <b>6</b> (IC₅₀ = 92, 50, and 80 μM, respectively), exhibited a dual antioxidant activity, acting as both metal-ion chelators and radical scavengers [2,2′-azino-bis­(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay data indicates IC₅₀ = 50, 70, and 108 μM vs 27 μM for Trolox]. Only 2′-deoxyadenosine-3′,5′-bisphosphorothioate, <b>2</b>, exhibited good inhibition of Cu⁺-induced H₂O₂ decomposition (IC₅₀ = 78 vs 224 μM for EDTA). Nucleoside–bisphosphorothioate analogues (<b>2</b>, <b>4</b>, and <b>6</b>) were weaker inhibitors than the corresponding bisphosphate analogues (<b>1</b>, <b>3</b>, and <b>5</b>), due to intramolecular oxidation under Fenton reaction conditions. ¹H- and ³¹P NMR monitored Cu⁺ titration of <b>2</b>, showed that Cu⁺ was coordinated by both 3′,5′-bisphosphorothioate groups, as well as N7-nitrogen atom, while adenosine-2′,3′-bisphosphorothioate, <b>6</b>, coordinated Cu⁺ only by 2′,3′-bisphosphorothioate groups. In conclusion, an additional terminal phosphate group on AMP/guanosine 5′-monophosphate (GMP) resulted in Fe²⁺-selective chelators highly potent as Fenton reaction inhibitors

Characterization of Complexes of Nucleoside-5′-Phosphorothioate Analogues with Zinc Ions

On the basis of the high affinity of Zn²⁺ to sulfur and imidazole, we targeted nucleotides such as GDP-β-S, ADP-β-S, and AP₃(β-S)­A, as potential biocompatible Zn²⁺-chelators. The thiophosphate moiety enhanced the stability of the Zn²⁺-nucleotide complex by about 0.7 log units. ATP-α,β-CH₂-γ-S formed the most stable Zn²⁺-complex studied here, log <i>K</i> 6.50, being ∼0.8 and ∼1.1 log units more stable than ATP-γ-S-Zn²⁺ and ATP-Zn²⁺ complexes, and was the major species, 84%, under physiological pH. Guanine nucleotides Zn²⁺ complexes were more stable by 0.3–0.4 log units than the corresponding adenine nucleotide complexes. Likewise, AP₃(β-S)­A-zinc complex was ∼0.5 log units more stable than AP₃A complex. ¹H- and ³¹P NMR monitored Zn²⁺ titration showed that Zn²⁺ coordinates with the purine nucleotide N7-nitrogen atom, the terminal phosphate, and the adjacent phosphate. In conclusion, replacement of a terminal phosphate by a thiophosphate group resulted in decrease of the acidity of the phosphate moiety by approximately one log unit, and increase of stability of Zn²⁺-complexes of the latter analogues by up to 0.7 log units. A terminal phosphorothioate contributed more to the stability of nucleotide-Zn²⁺ complexes than a bridging phosphorothioate