Modeling and monitoring the effects of three partly conserved Ile residues in the dimerization domain of a Mip-like virulence factor from Escherichia coli.

FKBP22, an Escherichia coli-made peptidyl-prolyl cis-trans isomerase, has shown considerable homology with Mip-like virulence factors. While the C-terminal domain of this enzyme is used for executing catalytic function and binding inhibitor, the N-terminal domain is employed for its dimerization. To precisely determine the underlying factors of FKBP22 dimerization, its structural model, developed using a suitable template, was carefully inspected. The data show that the dimeric FKBP22, like dimeric Mip proteins, has a V-like shape. Further, it dimerizes using 40 amino acid residues including Ile 9, Ile 17, Ile 42, and Ile 65. All of the above Ile residues except Ile 9 are partly conserved in the Mip-like proteins. To confirm the roles of the partly conserved Ile residues, three FKBP22 mutants, constructed by substituting them with an Ala residue, were studied as well. The results together indicate that Ile 65 has little role in maintaining the dimeric state or enzymatic activity of FKBP22. Conversely, both Ile 17 and Ile 42 are essential for preserving the structure, enzymatic activity, and dimerization ability of FKBP22. Ile 42 in particular looks more essential to FKBP22. However, none of these two Ile residues is required for binding the cognate inhibitor. Additional computational studies also indicated the change of V-shape and the dimeric state of FKBP22 due to the Ala substitution at position 42. The ways Ile 17 and Ile 42 protect the structure, function, and dimerization of FKBP22 have been discussed at length.Communicated by Ramaswamy H. Sarma.

Cp2TiCl-Promoted Endo-Trig Radical Cyclization to Six- and Seven-Membered Carbocycles: Synthesis of (±)-Isoclavukerin A.

A systematic study is undertaken to investigate the less explored endo-trig radical cyclization in activated olefin-appended epoxides using Cp2TiCl. The radical generated by the Ti(III)-promoted reductive opening of the epoxy ring promptly underwent endo-trig cyclization, giving access to differently 1,3-disubstituted six- and seven-membered carbocycles in good yields and diastereoselectivity. This protocol was successfully employed in the construction of 5,7- and 6,7-fused bicyclic frameworks entailing a de novo synthesis of (±)-isoclavukerin A belonging to tri-nor-guaiane class of sesquiterpene natural products in eight simple steps from commercially available starting materials. Besides the Ti(III)-mediated reaction serving as a key step in the synthesis, a sequential [2,3]-sigmatropic rearrangement/syn-elimination of an allyl sulfenate intermediate successfully rendered the highly constrained diene moiety in the hydroazulene core of the target molecule.

An attempt to construct an indole-fused azabicyclo[3.3.1]nonane framework via radical cyclization.

The structural motif of an indole-fused azabicyclo[3.3.1]nonane is common in many biologically significant indole-based natural products. Because of its structural complexity, this N-bridged scaffold has become an enticing target for organic chemists. Many efficient strategies have been developed to access this ring system synthetically, but a radical approach remains unexplored. Herein, we report a radical-based strategy to construct an indole-fused azabicyclo[3.3.1]nonane structural framework. Although our initial attempt to use a Cp2TiCl-mediated radical cyclization method was found to be unsuccessful, an alternative approach using a SmI2-mediated radical cyclization protocol was effective for enabling the desired ring closure, leading to the target indole-fused azabicyclo[3.3.1]nonane ring system. The modular approach developed here can be extended with appropriate functionalities on this indole-fused N-bridged ring system to synthesize many alkaloids.

One-Pot Tandem Aldol-Cycloetherification Protocol in the Enantioselective Synthesis of Davanoids.

Total synthesis of cis and trans diastereomers of prenylated davanoids like davanone, nordavanone, and davana acid ethyl ester was achieved in an enantioselective strategy. Various other davanoids could also be synthesized using standard procedures from the Weinreb amides derived from davana acids. Enantioselectivity in our synthesis was achieved employing a Crimmins' non-Evans syn aldol reaction that fixed the stereochemistry of the C3-hydroxyl group, while the C2-methyl group was epimerized in a late stage of the synthesis. A Lewis acid-mediated cycloetherification reaction was used to establish the tetrahydrofuran core of these molecules. Interestingly, a slight alteration of the Crimmins' non-Evans syn aldol protocol led to the complete conversion of the aldol adduct to the core tetrahydrofuran ring of davanoids, thus essentially dovetailing two important steps in the synthesis. The resulting one-pot tandem aldol-cycloetherification strategy enabled the enantioselective synthesis of trans davana acid ethyl esters and 2-epi-davanone/nordavanone in just three steps in excellent overall yields. The modularity of the approach will enable the synthesis of various other isomers in stereochemically pure forms for further biological profiling of this important class of molecules.

The cofactors and domains of a staphylococcal capsule-producing enzyme preserve its structure, stability, shape and dimerization ability.

CapF, a staphylococcal capsule-producing enzyme, binds Zn2+ ion and NADPH using its C-terminal domain (CTD) and N-terminal domain (NTD), respectively. To elucidate the roles of cofactors and domains, we have systematically investigated the related recombinant proteins, rCapF, rCTD, recombinant NTD (rNTD) and the Zn2+-free rCapF/rCTD, Apo-rCapF/Apo-rCTD. The results show that the secondary structure, tertiary structure, shape and surface hydrophobicity of Apo-rCapF and Apo-rCTD are different from those of rCapF and rCTD. The removal of Zn2+ made rCapF thermo-sensitive, whereas both rCTD and Apo-rCTD are thermo-resistant proteins. Further, Apo-rCapF and rCapF existed as the dimers, whereas rCTD and Apo-rCTD formed a mixture of dimers and tetramers in the aqueous solution. Zn2+ maintained the structure of NTD as well. The NADPH binding activity and Cys accessibility of rNTD, rCapF and Apo-rCapF were significantly different from each other. The binding of NADPH to the above three proteins freely occurred, liberated heat at 25°C and increased their diameters. In addition, the structure, stability, shape and oligomerization ability of rNTD, rCTD and rCapF little resembled each other. Collectively, the domains and cofactors of CapF contribute to preserving its conformation, stability, shape and dimerization ability.

Serine 106 preserves the tertiary structure, function, and stability of a cyclophilin from Staphylococcus aureus.

SaCyp, a staphylococcal cyclophilin involved in both protein folding and pathogenesis, has a Ser residue at position 106 and a Trp residue at position 136. While Ser 106 of SaCyp aligned with a cyclosporin A (CsA) binding Ala residue, its Trp 136 aligned with a Trp or a Phe residue of most other cyclophilins. To demonstrate the exact roles of Ser 106 and Trp 136 in SaCyp, we have elaborately studied rCyp[S106A] and rCyp[W136A], two-point mutants of a recombinant SaCyp (rCyp) harboring an Ala substitution at positions 106 and 136, respectively. Of the mutants, rCyp[W136A] showed the rCyp-like CsA binding affinity and peptidyl-prolyl cis-trans isomerase (PPIase) activity. Conversely, the PPIase activity, CsA binding affinity, stability, tertiary structure, surface hydrophobicity, and Trp accessibility of rCyp[S106A] notably differed from those of rCyp. The computational experiments also reveal that the structure, dimension, and fluctuation of SaCyp are not identical to those of SaCyp[S106A]. Furthermore, Ser at position 106 of SaCyp, compared to Ala at the same position, formed a higher number of non-covalent bonds with CsA. Collectively, Ser 106 is an indispensable residue for SaCyp that keeps its tertiary structure, function, and stability intact.Communicated by Ramaswamy H. Sarma.

A conserved arginine residue in a staphylococcal anti-sigma factor is required to preserve its kinase activity, structure, and stability.

RsbW, σB, and RsbV, encoded by Staphylococcus aureus and related bacteria, act as an anti-sigma factor, an sigma factor, and an anti-anti-sigma factor, respectively. The interaction between RsbW and σB blocks the transcription initiation activity of the latter protein. RsbW also functions as a serine kinase and phosphorylates RsbV in the presence of ATP. Our modeling study indicates that the RsbW-RsbV complex is stabilized by twenty-four intermolecular non-covalent bonds. Of the bond-forming RsbW residues, Arg 23, and Glu 49 are conserved residues. To understand the roles of Arg 23 in RsbW, rRsbW[R23A], a recombinant S. aureus RsbW (rRsbW) harboring Arg to Ala change at position 23, was investigated using various probes. The results reveal that rRsbW[R23A], like rRsbW, exists as the dimers in the aqueous solution. However, rRsbW[R23A], unlike rRsbW, neither interacted with a chimeric RsbV (rRsbV) nor formed the phosphorylated rRsbV in the presence of ATP. Furthermore, the tertiary structure and hydrophobic surface area of rRsbW[R23A] matched little with those of rRsbW. Conversely, both rRsbW[R23A] and rRsbW showed interaction with a recombinant σB (rσB). rRsbW and rRsbW[R23A] were also unfolded via the formation of at least one intermediate in the presence of urea. However, the thermodynamic stability of rRsbW significantly differed from that of rRsbW[R23A]. Our molecular dynamics (MD) simulation study also reveals the substantial change of structure, dimension, and stability of RsbW due to the above mutation. The ways side chain of critical Arg 23 contributes to maintaining the tertiary structure, and stability of RsbW was elaborately discussed.Communicated by Ramaswamy H. Sarma.

Structurally distinct unfolding intermediates formed from a staphylococcal capsule-producing enzyme retained NADPH binding activity.

CapF, a capsule-producing enzyme expressed by Staphylococcus aureus, binds NADPH and exists as a dimer in the aqueous solution. Many other capsule-producing virulent bacteria also express CapF orthologs. To understand the folding-unfolding mechanism of S. aureus CapF, herein a recombinant CapF (rCapF) was individually investigated using urea and guanidine hydrochloride (GdnCl). Unfolding of rCapF by both the denaturants was reversible but proceeded via the synthesis of a different number of intermediates. While two dimeric intermediates (rCapF4 and rCapF5) were formed at 0.5 M and 1.5 M GdnCl, three dimeric intermediates (rCapF1, rCapF2, and rCapF3) were produced at 1 M, 2 M, and 3 M urea, respectively. rCapF5 showed 3.6 fold less NADPH binding activity, whereas other intermediates retained full NADPH binding activity. Compared to rCapF, all of the intermediates (except rCapF3) had a compressed shape. Conversely, rCapF3 possessed a native protein-like shape. The maximum shape loss was in rCapF4 though its secondary structure remained unperturbed. Additionally, the tertiary structure and hydrophobic surface area of the intermediates neither matched with each other nor with those of the native rCapF. Of the four Trp residues in rCapF, one or more Trp residues in the intermediates may have higher solvent accessibility. Using sequence alignment and a tertiary structural model of CapF, we have demonstrated that the region around Trp 137 of CapF may be most sensitive to unfolding, whereas the NADPH binding motif carrying region at the N-terminal end of this protein may be resistant to unfolding, particularly at the low denaturant concentrations.Communicated by Ramaswamy H. Sarma.

Cp2TiCl-Mediated Reductive Cyclization: Total Synthesis of Pestalotiolactone A, Myrotheciumone A, and Scabrol A.

The first stereoselective total syntheses of fungal secondary metabolites monoterpenoid (+)-pestalotiolactone A, meroterpenoid (-)-myrotheciumone A, and iridoid lactone (+)-scabrol A have been accomplished in an expedient unified approach starting from d-(+)-malic acid employing an epoxide opening-radical cyclization protocol initiated by Cp2Ti(III)Cl as a key step to assemble the core bicyclic lactone moieties of these molecules with complete diastereoselective control. Finally, the deoxygenation and methylation delivered the target natural products.

Environment-Sensitive Fluorescence of 7-Nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-Labeled Ligands for Serotonin Receptors.

Serotonin is a neurotransmitter that plays a crucial role in the regulation of several behavioral and cognitive functions by binding to a number of different serotonin receptors present on the cell surface. We report here the synthesis and characterization of several novel fluorescent analogs of serotonin in which the fluorescent NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group is covalently attached to serotonin. The fluorescent ligands compete with the serotonin1A receptor specific radiolabeled agonist for binding to the receptor. Interestingly, these fluorescent ligands display a high environmental sensitivity of their fluorescence. Importantly, the human serotonin1A receptor stably expressed in CHO-K1 cells could be specifically labeled with one of the fluorescent ligands with minimal nonspecific labeling. Interestingly, we show by spectral imaging that the NBD-labeled ligand exhibits a red edge excitation shift (REES) of 29 nm when bound to the receptor, implying that it is localized in a restricted microenvironment. Taken together, our results show that NBD-labeled serotonin analogs offer an attractive fluorescent approach for elucidating the molecular environment of the serotonin binding site in serotonin receptors. In view of the multiple roles played by the serotonergic systems in the central and peripheral nervous systems, these fluorescent ligands would be useful in future studies involving serotonin receptors.

Total Synthesis of Panaginsene.

The total synthesis of panaginsene has been accomplished in 11 linear steps starting from methyl 3,3-dimethyl-5-oxocyclopent-1-ene-1-carboxylate. The key steps are a Sharpless asymmetric epoxidation and Ti(III)-mediated reductive epoxide opening-radical cyclization to construct the chiral quaternary carbon stereocenter followed by a very challenging HWE olefination reaction on an 1,3-keto aldehyde and a late stage McMurry olefination using low valent titanium to construct the highly constrained angular tetrasubstituted olefin in a five-membered ring.

Alternative Sigma Factor of Staphylococcus aureus Interacts with the Cognate Antisigma Factor Primarily Using Its Domain 3.

σB, an alternative sigma factor, is usually employed to tackle the general stress response in Staphylococcus aureus and other Gram-positive bacteria. This protein, involved in S. aureus-mediated pathogenesis, is typically blocked by RsbW, an antisigma factor having serine kinase activity. σB, a σ70-like sigma factor, harbors three conserved domains designated σB2, σB3, and σB4. To better understand the interaction between RsbW and σB or its domains, we have studied their recombinant forms, rRsbW, rσB, rσB2, rσB3, and rσB4, using different probes. The results show that none of the rσB domains, unlike rσB, showed binding to a cognate DNA in the presence of a core RNA polymerase. However, both rσB2 and rσB3, like rσB, interacted with rRsbW, and the order of their rRsbW binding affinity looks like rσB > rσB3 > rσB2. Furthermore, the reaction between rRsbW and rσB or rσB3 was exothermic and occurred spontaneously. rRsbW and rσB3 also associate with each other at a stoichiometry of 2:1, and different types of noncovalent bonds might be responsible for their interaction. A structural model of the RsbW-σB3 complex that has supported our experimental results indicated the binding of rσB3 at the putative dimeric interface of RsbW. A genetic study shows that the tentative dimer-forming region of RsbW is crucial for preserving its rσB binding ability, serine kinase activity, and dimerization ability. Additionally, a urea-induced equilibrium unfolding study indicated a notable thermodynamic stabilization of σB3 in the presence of RsbW. Possible implications of the stabilization data in drug discovery were discussed at length.

Understanding the structure, stability, and anti-sigma factor-binding thermodynamics of an anti-anti-sigma factor from Staphylococcus aureus.

Staphylococcus aureus and many related bacteria encode both anti-sigma factor RsbW and anti-anti-sigma factor RsbV to control stress response by σB, an alternative sigma factor. Our structural and thermodynamic studies of a recombinant S. aureus RsbV (rRsbV) show that the monomeric protein contains five α-helices and a mostly parallel but mixed ß-sheet composed of five ß-strands, and interacts with a chimeric S. aureus RsbW (rRsbW) in vitro. In addition, rRsbV binds rRsbW with a Kd of 0.058 µM using spectroscopy and 0.008 µM using calorimetry at 25 °C. From a gel-shift assay, the affinity of rRsbV to rRsbW was found to be higher than its affinity with a recombinant S. aureus σB (rσB). Moreover, the heat generated from the spontaneous rRsbV - rRsbW interaction changed in a compensatory manner with entropy in the 20°-35 °C range. The association between rRsbV and rRsbW yielded a negative heat capacity change, suggesting that both hydrogen bonds and hydrophobic interactions participate in the formation of the rRsbV-rRsbW complex. Computational analyses of a homology-based RsbV-RsbW model has mostly supported the formation of a 2: 2 complex verified by gel filtration chromatography, the experimental ΔG and the existence of these non-covalent bonds. Our unfolding experiments show that the thermodynamic stability of rRsbV is significantly increased in the presence of rRsbW. Thus, these studies have provided valuable insights into the structure, stability, and the anti-sigma-binding thermodynamics of an anti-anti-sigma factor.Communicated by Ramaswamy H. Sarma.

Application of Cp2TiCl-Promoted Radical-Induced Cyclization: An Expeditious Access to [a]-Annelated Indoles.

An efficient and novel route for assembling pyrrolo/piperido[1,2-a]indoles is portrayed involving a radical-mediated reductive epoxide opening reaction of N-tethered epoxy-indoles that trigger facile intramolecular cyclization followed by an oxidative quenching step. Capitalizing on the operational simplicity of the method involving just two steps and use of an efficient C-C bond-forming reaction, this radical-based protocol enables the modular assembly of an important class of N-fused indole derivatives with versatile functional and structural diversity.

Synthesis and Biological Studies of Dodecameric Cationic Antimicrobial Peptides Containing Tetrahydrofuran Amino Acids.

We report here a concise route to synthesize various stereoisomers of tetrahydrofuran amino acids (TAAs) and the synthesis of TAA-containing linear cationic dodecapeptides. Some of these linear peptides show slightly better antimicrobial activities than their tetra- and octameric congeners, but no activity against Mycobacterium tuberculosis, for which octapeptides exhibited by far the best results; this implies that antibacterial activity is dependent on the length of these linear peptides. All the dodecapeptides described here were found to be toxic in nature against Vero cells. The study helps to delineate the optimal length of this series of linear peptides and select potential leads in the development of novel cationic peptide-based antibiotics.

Development of Minimal Diguanosinyl Motif toward RNA G-Quadruplex-Like Structures in Solution.

Among the non-canonical structures of B-DNA, the G-quadruplex is of particular interest because of its well-defined conformation, high stability, and versatility. Herein we report our studies on the development of an amide-linked minimal diguanosinyl motif that forms a G-quadruplex-like structure in solution in the presence of potassium cations; various linear guanosine amino acid dimers were synthesized with linkers of different chain lengths to investigate the optimum flexibility required to form such structures.

Removal of an atypical region from a staphylococcal cyclophilin affects its structure, function, stability, and shape.

SaCyp, a cyclophilin having 197 amino acid residues, acts both as a protein-folding catalyst and a virulence factor in Staphylococcus aureus. Interestingly, a region, homologous to the SaCyp region carrying 121-148 amino acid residues, is present in many putative cyclophilins but absent in well-studied cyclophilins. To determine the exact roles of this unusual region in SaCyp and related proteins, we have investigated a deletion mutant (rCypΔ) of a recombinant SaCyp (rCyp) using various probes. The data reveal that rCypΔ has significantly less catalytic activity and possesses altered structure and hydrophobic surface compared to rCyp. Conversely, the deletion substantially increased inhibitor binding affinity and altered the shape of rCyp. However, both proteins were unfolded by a non-two-state mechanism in the presence of urea. Additionally, the stability of rCyp was significantly reduced due to the deletion of the residues 121-148. Our MD simulation study also indicated the considerable alteration in structure, shape, and fluctuations of SaCyp due to the removal of the region carrying 121-148 residues. Hence, the atypical region located in SaCyp might be vital for maintaining its unique structure, function, stability, and shape.

Titanocene(III)-Mediated 5-exo-trig Radical Cyclization: En Route to Spirooxindole-Based Tetrahydrofuran and Bicyclic Lactone.

The isatin core system is of immense importance due to the highly reactive prochiral C-3 position, which paves an easy way to construct large arrays of spirooxindole heterocyclic motifs. Herein, we depict an isatin-derived and 3,3'-disubstituted oxindole-appended epoxy-acrylate undergoing Cp2Ti(III)Cl-mediated reductive oxirane-ring opening with concomitant intramolecular 5-exo-trig radical cyclization leading to tetrahydrofuran-based oxa-spirooxindole systems. The fused spirooxindole structural feature is embedded in many natural products and tends to exhibit a wide spectrum of biological activities. The presence of more than one quaternary center and the availability of multiple functional groups like hydroxyl, ester, or lactone in the resultant products expand the scope of synthetic applications of the newly acquired oxa-spirooxindole molecules.

Studies on sugar puckering and glycosidic stabilities of 3'-amino-5'-carboxymethyl-3',5'-dideoxy nucleoside mimics.

The synthesis of nucleoside amino acid monomers and dimers has been carried out to evaluate and characterize the impact of the neutral amide backbone on key attributes like puckering of the sugar rings and glycosidic bond strengths of these analogs. The conformational analysis suggests that amide-linked nucleotides have a high predilection towards N-type conformers. The glycosidic bond strength was found to be slightly weaker compared to ribonucleosides under acidic conditions at high temperatures. The results will be helpful to explore in future the development of fully amide-linked oligonucleotides for therapeutic purposes.

Application of Cp2TiCl-Promoted Radical Cyclization: A Unified Strategy for the Syntheses of Iridoid Monoterpenes.

An expedient approach toward the unified total syntheses of (+)-iridomyrmecin, (-)-isoiridomyrmecin, (+)-7- epi-boschnialactone, (+)-teucriumlactone, and (-)-dolichodial in chirally pure forms starting from readily available (+)-ß-citronellene is delineated combining step economy and simplicity. Highlights include a Ti(III)-mediated reductive epoxide opening-cyclization for the construction of the core cyclopenta[ c]pyran skeleton of the iridoid lactones with complete diastereoselectivity for the newly created bridgehead stereogenic centers. Subsequent transformations facilitate a short access to (+)-teucriumlactone and (-)-dolichodial and formal access to potentially other iridoids.