Goutam Mukherjee

Hyperthermophilic asparaginase mutants with enhanced substrate affinity and antineoplastic activity: structural insights on their mechanism of action

Thermophilic l‐asparaginases display high stability and activity at elevated temperatures. However, they are of limited use in leukemia therapy because of their low substrate affinity and reduced activity under physiological conditions. In an attempt to combine stability with activity at physiological conditions, 3 active‐site mutants of Pyrococcus furiosus l‐asparaginase (PfA) were developed. The mutants, specifically K274E, showed improved enzymatic properties at physiological conditions as compared to the wild type. All variants were thermodynamically stable and resistant to proteolytic digestion. None of the enzymes displayed glutaminase activity, a highly desirable therapeutic property. All variants showed higher and significant killing of human cell lines HL60, MCF7, and K562 as compared to the Escherichia coli l‐asparaginase. Our study revealed that increased substrate accessibility through the active site loop plays a major role in determining activity. A new mechanistic insight has been proposed based on molecular dynamics simulated structures, where dynamic flipping of a critical Tyr residue is responsible for the activity of thermophilic l‐asparaginases. Our study not only resulted in development of PfA mutants with combination of desirable properties but also gave a mechanistic insight about their activity.—Bansal, S., Srivastava, A., Mukherjee, G., Pandey, R., Verma, A. K., Mishra, P., Kundu, B. Hyperthermophilic asparaginase mutants with enhanced substrate affinity and antineoplastic activity: structural insights on their mechanism of action. FASEB J. 26, 1161‐1171 (2012). www.fasebj.org

Aminotroponiminatogermaacid halides with a Ge(E)X moiety (E = S, Se; X = F, Cl).

Fluorination of aminotroponiminate (ATI) ligand-stabilized germylene monochloride [(t-Bu)(2)ATI]GeCl (1) with CsF gave the aminotroponiminatogermylene monofluoride [(t-Bu)(2)ATI]GeF (2). Oxidative addition reaction of compound 2 with elemental sulfur and selenium led to isolation of the corresponding germathioacid fluoride [(t-Bu)(2)ATI]Ge(S)F (3) and germaselenoacid fluoride [(t-Bu)(2)ATI]Ge(Se)F (4), respectively. Similarly, reaction of aminotroponiminatogermylene monochloride [(i-Bu)(2)ATI]GeCl (9) with elemental sulfur and selenium gave the aminotroponiminatogermathioacid chloride [(i-Bu)(2)ATI]Ge(S)Cl (11) and aminotroponiminatogermaselenoacid chloride [(i-Bu)(2)ATI]Ge(Se)Cl (12), respectively. Compound 9 has been prepared through a multistep synthetic route starting from 2-(tosyloxy)tropone 5. All compounds (2-4 and 6-12) were characterized through the multinuclear NMR spectroscopy, and single-crystal X-ray diffraction studies were performed on compounds 2, 4, and 8-12. The germaselenoacid halide complexes 4 and 12 showed doublet (-142.37 ppm) and singlet (-213.13 ppm) resonances in their (77)Se NMR spectra, respectively. Germylene monohalide complexes 2 and 9 have a germanium center in distorted trigonal pyramidal geometry, whereas a distorted tetrahedral geometry is seen around the germanium center in germaacid halide complexes 4, 11, and 12. The length of the Ge═E bond in germathioacid chloride (11) and germaselenoacid halide (4 and 12) complexes is 2.065(1) and 2.194(av) Å, respectively. Theoretical studies (based on the DFT methods) on complexes 4, 11, and 12 reveal the nature of the Ge═E multiple bond in these germaacid halide complexes with computed Wiberg bond indices (WBI) being 1.480, 1.508, and 1.541, respectively.

Selenadiazolopyridine: a synthon for supramolecular assembly and complexes with metallophilic interactions.

The synthesis and characterization of the complexes of Cu(I), Ag(I), Cu(II), and Co(II) ions with 1,2,5-selenadiazolopyridine (psd) is reported. The following complexes have been prepared: [Cu(2)(psd)(3)(CH(3)CN)(2)](2+)2(PF(6)(-)); [(CuCl)(2)(psd)(3)]; [Cu(2)(psd)(6)](2+)2(ClO(4))(-); [Ag(2)(psd)(2)](2+)2(NO(3))(-); [Ag(2)(psd)(2)](2+)2(CF(3)COO)(-); [Cu(psd)(2)(H(2)O)(3)](2+)2(ClO(4))(-)·(psd)(2); [Cu(psd)(4)(H(2)O)](2+)2(ClO(4))(-)·(CHCl(3)); [Cu(psd)(2)(H(2)O)(3)](2+)2(NO(3))(-)·(H(2)O)·(psd)(2), and [Co(psd)(2)(H(2)O)(4)](2+)2(ClO(4))(-)·(psd)(2). The electronic structure of ligand psd, in particular the bond order of Se-N bonds, has been probed by X-ray diffraction, (77)Se NMR, and computational studies. A detailed analysis of the crystal structures of the ligand and the complexes revealed interesting supramolecular assembly. The assembly was further facilitated by the presence of neutral ligands for some complexes (Cu(II) and Co(II)). The molecular structure of the ligand showed that it was present as a dimer in the solid state where the monomers were linked by strong secondary bonding Se···N interactions. The crystal structures of Cu(I) and Ag(I) complexes revealed the dinuclear nature with characteristic metallophilic interactions [M···M] (M = Cu, Ag), while the Cu(II) and Co(II) complexes were mononuclear. The presence of M···M interactions has been further probed by Atoms in Molecules (AIM) calculations. The paramagnetic Cu(II) and Co(II) complexes have been characterized by UV-vis, ESI spectroscopy, and room temperature magnetic measurements.

Are Ligand-Stabilized Carboxylic Acid Derivatives with Ge═Te Bonds Isolable?

The stability of ligand-stabilized carboxylic acid derivatives (such as esters, amides, anhydrides, and acid halides) with terminal Ge═Te bonds is highly questionable as there is no report on such compounds. Nevertheless, we are able to isolate germatelluroester [LGe(Te)Ot-Bu] (4), germatelluroamide [LGe(Te)N(SiMe3)2] (5), and germatelluroacid anhydride [LGe(Te)OGe(Te)L] (6) complexes (L = aminotroponiminate (ATI)) as stable species. Consequently, the synthetic details, structural characterization, and UV-vis spectroscopic and theoretical studies on them are reported for the first time.

Use of thio and seleno germanones as ligands: silver(I) halide complexes with Ge═E→Ag-I (E = S, Se) moieties and chalcogen-dependent argentophilic interaction.

The potential of thio and seleno germanones [LPhGe═E] (L = aminotroponiminate (ATI) ligand, E = S 3, Se 4) to function as ligands has been demonstrated through the isolation of their silver(I) iodide complexes [{(t-Bu)2ATIGe(E)Ph}2(Ag2I2)] (E = S 5, Se 6) with a planar and discrete Ag2I2 core. Compounds 5 and 6 possess the hitherto unknown Ge═E→Ag-I moieties and the crystallographic data reveals the presence of a strong argentophilic interaction (2.950(1) Å) in complex 6, but is inconclusive in complex 5 (3.470(1) Å). Using theoretical studies, proof for the presence and absence of argentophilic interactions in complexes 6 and 5 was obtained, respectively. Further, it is disclosed that the donor ability of the chalcogen atoms in the Ge═E→Ag-I moieties dictate the Ag···Ag interaction in these complexes.

Selective interception of gelsolin amyloidogenic stretch results in conformationally distinct aggregates with reduced toxicity.

The pathogenesis of protein misfolding diseases is attributed to the cytotoxicity caused by amyloidogenic prefibrillar aggregates, rather than mature fibrils. The presence of one or more amyloidogenic stretches in different proteins has been proven critical for initiating fibril formation. In the present study, we show that two natural compounds, curcumin and emetine, bind tightly (Kd < 1.6 μM) to the core amyloidogenic stretch (182-192) of gelsolin (AGel). Binding happens in different structural orientations, distinctly modulating the amyloidogenic pathway of AGel. While AGel alone undergoes sigmoidal transition to thioflavin T (ThT)-responsive fibrillar aggregates with clear lag phase, the presence of curcumin or emetine abolishes the lag phase and produces starkly different, noncytotoxic end products. Atomic force microscopy revealed that while curcumin augments fibril formation, emetine arrests it at an intermediate aggregated stage with no fibrillar morphology. FTIR spectroscopy, dynamic light scattering, and ANS fluorescence experiments also suggest that these two species are distinct. Curcumin and emetine also differentially affect the preformed amyloids with the former thickening the fibrils and the latter releasing reclusive oligomers. MD simulations further provided mechanistic insights of differential interaction by the two compounds modulating amyloid formation. The results were also confirmed on the disease-associated amyloidogenic fragment of gelsolin (fAGel). Thus, our findings suggest that targeting amyloidogenic stretches in proteins could be useful in designing novel molecules against protein misfolding diseases.

Digermylene oxide complexes: facile synthesis and reactivity.

A simple heating of aminotroponiminate (ATI) ligand stabilized germylene monochlorides [(R)2ATIGeCl] (R = t-Bu 1, i-Bu 2) with an excess of potassium hydroxide in toluene resulted in the first ATI ligand stabilized digermylene oxides [{(R)2ATIGe}2O] (R = t-Bu 3, i-Bu 4), respectively. Reaction of compound 3 with elemental sulfur and selenium gave the first germaacid anhydride complexes [{(t-Bu)2ATIGe(E)}2O] (E = S 5, Se 6) with (S)Ge-O-Ge(S) and (Se)Ge-O-Ge(Se) moieties, respectively. The digermylene oxide complexes 3 and 4 and germaacid anhydride complexes 5 and 6 were characterized by multinuclear NMR spectroscopy and single-crystal X-ray diffraction analysis. In its (77)Se NMR spectrum, compound 6 showed a resonance at -78.9 ppm. The Ge-O-Ge bond angles in compounds 5 and 6 are 178.66(2)° and 179.81(2)°, respectively. To understand further the bonding features, DFT calculations followed by MO, AIM, and NBO analysis were carried out on compounds 3, 5, and 6. The computed Wiberg bond indices of Ge-O bonds are slightly less than 0.5 in all the aforementioned compounds, and the same for the Ge═E bonds in compounds 5 and 6 are close to 1.4.

The Preparation of Complexes of Germanone from a Germanium μ‐Oxo Dimer

Complexes of germanone containing formal Ge=O→M bonds (M=Zn, B, Ge, Sn) were isolated and characterized. The compounds were prepared through a novel synthetic route using a germanium μ-oxo dimer 3 as the starting material. This method circumvents the need to employ germanones to prepare complexes of germanones.

Pseudohalogenogermylenes versus Halogenogermylenes: Difference in their Complexation Behavior towards Group 6 Metal Carbonyls

Pseudohalogenogermylenes [(iBu)2 ATI]GeY (Y=NCO 4, NCS 5) show different coordination behavior towards group 6 metal carbonyls in comparison to the corresponding halogenogermylenes [(iBu)2 ATI]GeX (X=F 1, Cl 2, Br 3) (ATI=aminotroponiminate). The reactions of compounds 4-5 and 1-3 with cis-[M(CO)4 (COD)] (M=Mo, W, COD=cyclooctadiene) gave trans-germylene metal complexes {[(iBu)2 ATI]GeY}2 M(CO)4 (Y=NCO, M=Mo 6, W 11; Y=NCS, M=Mo 7) and cis-germylene metal complexes {[(iBu)2 ATI]GeX}2 M(CO)4 (M=Mo, X=F 8, Cl 9, Br 10; M=W, X=Cl 12), respectively. Theoretical studies on compounds 7 and 9 reveal that donor-acceptor interactions from Mo to Ge atoms are better stabilized in the observed trans and cis geometries than in the hypothetical cis and trans structures, respectively.