Role of Metal Ion in Specific Recognition of Pyrophosphate Ion under Physiological Conditions and Hydrolysis of the Phosphoester Linkage by Alkaline Phosphatase Priyadip Das, † Nellore Bhanu Chandar, † Shishir Chourey, † Hridesh Agarwalla, ‡,# Bishwajit Ganguly,* ,† and Amitava Das* ,‡,# ‡ Organic Chemistry Division, CSIRNational Chemical Laboratory, Pune 411008, Maharashtra, India † CSIR - Central Salt & Marine Chemicals Research Institute, Bhavnagar 364002, Gujarat, India * S Supporting Information ABSTRACT: Complexes synthesized from Zn(II), Cu(II), and Cd(II), using a dipicolyl amine derivative (L), showed unique specificity toward pyrophosphate ion (PPi or P 4 O 7 4- ) among all other common anionic analytes, including different biologically significant phosphate ion (PO 4 3- ,H 2 PO 4 2- ) or phosphate-ion- based nucleotides, such as AMP, ADP, ATP, and CTP. However, the relative affinities of PPi toward these three metal complexes were found to vary and follow the order K a L.Zn-PPi > are given in units of a L.Cu-PPi ≥ K a L.Cd-PPi . Luminescence responses of the receptor L were substantial on binding to Zn 2+ and Cd 2+ , while relatively a much smaller luminescence response was observed in the presence of Cu 2+ . Luminescence responses of L.M-PPi (M is Zn 2+ , Cd 2+ , and Cu 2+ ) were further modified on binding to the PPi ion. This could be utilized for quantitative detection of PPi in physiological condition as well as for developing a real time “turn-on” (for L.Zn and L.Cu) and “turn-off” (for L.Cd) fluorescence assay for evaluating the enzymatic activity of alkaline phosphatase (ALP). Experimental results revealed how the subtle differences in the binding affinities between PPi and M in L.M (M is Zn 2+ , Cd 2+ , and Cu 2+ ), could influence the cleavage of the phosphoester linkage in PPi by ALP. The DFT calculations further revealed that the hydrolytic cleavage of the metal ion coordinated phosphoester bond is kinetically faster than that for free PPi and thus, rationalized the observed difference in the cleavage of the phosphoester bond by an important mammalian enzyme such as ALP in the presence of different metal complexes. ■ INTRODUCTION Recently, research pertaining to fluorescent chemosensors has received considerable significance, with regard to the design of efficient biomarkers, imaging reagents, and developing appropriate enzymatic assay. Such molecular sensors are also being widely used for the analysis of environmental and biological samples as well as for probing biological processes. 1 Simplicity in the detection process, along with the high sensitivity that one can achieve in the analysis of a desired analyte have provided fluorescence-based receptors and associated methodologies an edge over conventional analytical methods that rely on use of sophisticated instrumentation and involve multistep sample preparation. Furthermore, fluorescent marker or imaging reagents offer the advantages of spatial and temporal resolution, along with in vitro or in vivo analysis. 2 Specific recognition and quantitative estimation of various biologically important phosphate ions have received consid- erable attention since these ions play crucial role(s) in various bioenergetic processes. Being the product of ATP hydrolysis under cellular conditions, 3,4 pyrophosphate (PPi) is involved in energy transduction in organisms and in controlling metabolic processes by participation in various enzymatic reactions, e.g., DNA replication. 5 Furthermore, the detection of PPi is important in real-time DNA sequencing method, 6 as well as in cancer research. 7 Patients with chondrocalcinosis, which is a common arthritic condition in which calcium pyrophosphate dehydrate (CPPD) form in articular cartilage have also been shown to have a high level of synovial fluid PPi. 8 Thus, detection and discrimination of PPi is important for evaluating the generation of each of these ions during various biological processes and clarifying its roles in these processes. All these have contributed in the recent surge of interests in developing an improved methodology for the specific recognition and sensitive detection of PPi in physiological condition. However, such an objective (i.e., specific binding and recognition of PPi in aqueous medium) is a challenging one, because of the very high solvation enthalpy (584 kcal mol -1 ) of PPi in aqueous medium. This high hydration enthalpy adversely influences the effective binding of PPi to a receptor with active hydrogen- Received: May 17, 2013 Article pubs.acs.org/IC © XXXX American Chemical Society A dx.doi.org/10.1021/ic401243h | Inorg. Chem. XXXX, XXX, XXX-XXX