1H 15N Research Articles

Mapping Site-Specific Changes That Affect Stability of the N-Terminal Domain of Calmodulin

Biophysical tools have been invaluable in formulating therapeutic proteins. These tools characterize protein stability rapidly in a variety of solution conditions, but in general, the techniques lack the ability to discern site-specific information to probe how solution environment acts to stabilize or destabilize the protein. NMR spectroscopy can provide site-specific information about subtle structural changes of a protein under different conditions, enabling one to assess the mechanism of protein stabilization. In this study, NMR was employed to detect structural perturbations at individual residues as a result of altering pH and ionic strength. The N-terminal domain of calmodulin (N-CaM) was used as a model system, and the ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) experiment was used to investigate effects of pH and ionic strength on individual residues. NMR analysis revealed that different solution conditions affect individual residues differently, even when the amino acid sequence and structure are highly similar. This study shows that addition of NMR to the formulation toolbox has the ability to extend understanding of the relationship between site-specific changes and overall protein stability.

Synthesis, spectroscopic characterization, DFT studies and antibacterial assays of a novel silver(I) complex with the anti-inflammatory nimesulide

A novel silver(I) complex with nimesulide (NMS) of composition AgC13H11N2O5S was synthesized and characterized by chemical and spectroscopic measurements, density functional theory (DFT) studies and biological assays. Infrared (IR), ESI-QTOF-mass spectrometric (MS) analyses and 1H, 13C and [1H–15N] nuclear magnetic resonance (NMR) studies indicate that NMS acts as a bidentate ligand, being coordinated to Ag(I) through the nitrogen and one of the oxygen atoms of the sulphonamide group. The proposed structure based on the experimental data was confirmed as a minimum of the potential energy surface (PES) with the calculation of the hessians, showing no imaginary frequencies. Also, the theoretical IR spectra of the free ligand and of the silver(I) complex (Ag–NMS) are in a good agreement with the experimental data. Theoretical Time-Dependent DFT (TD-DFT) studies confirmed that the observed transitions in the UV–Vis spectra of the NMS and Ag–NMS are due to π–π∗ transitions. The antibacterial activity of Ag–NMS was evaluated by an antibiogram assay, using the disk diffusion method. The complex showed an effective antibacterial activity against Staphylococcus aureus (Gram-positive), and Escherichia coli and Pseudomonas aeruginosa (Gram-negative) pathogenic bacterial cells.

Nonionic Homopolymeric Amphipols: Application to Membrane Protein Folding, Cell-Free Synthesis, and Solution Nuclear Magnetic Resonance

Nonionic amphipols (NAPols) synthesized by homotelomerization of an amphiphatic monomer are able to keep membrane proteins (MPs) stable and functional in the absence of detergent. Some of their biochemical and biophysical properties and applications have been examined, with particular attention being paid to their complementarity with the classical polyacrylate-based amphipol A8-35. Bacteriorhodopsin (BR) from Halobacterium salinarum and the cytochrome b(6)f complex from Chlamydomonas reinhardtii were found to be in their native state and highly stable following complexation with NAPols. NAPol-trapped BR was shown to undergo its complete photocycle. Because of the pH insensitivity of NAPols, solution nuclear magnetic resonance (NMR) two-dimensional (1)H-(15)N heteronuclear single-quantum coherence spectra of NAPol-trapped outer MP X from Escherichia coli (OmpX) could be recorded at pH 6.8. They present a resolution similar to that of the spectra of OmpX/A8-35 complexes recorded at pH 8.0 and give access to signals from solvent-exposed rapidy exchanging amide protons. Like A8-35, NAPols can be used to fold MPs to their native state as demonstrated here with BR and with the ghrelin G protein-coupled receptor GHS-R1a, thus extending the range of accessible folding conditions. Following NAPol-assisted folding, GHS-R1a bound four of its specific ligands, recruited arrestin-2, and activated binding of GTPγS by the G(αq) protein. Finally, cell-free synthesis of MPs, which is inhibited by A8-35 and sulfonated amphipols, was found to be very efficient in the presence of NAPols. These results open broad new perspectives on the use of amphipols for MP studies.

A closer look at the nitrogen next door: 1H–15N NMR methods for glycosaminoglycan structural characterization

Recently, experimental conditions were presented for the detection of the N-sulfoglucosamine (GlcNS) NHSO3- or sulfamate 1H and 15N NMR resonances of the pharmaceutically and biologically important glycosaminoglycan (GAG) heparin in aqueous solution. In the present work, we explore further the applicability of nitrogen-bound proton detection to provide structural information for GAGs. Compared to the detection of 15N chemical shifts of aminosugars through long-range couplings using the IMPACT–HNMBC pulse sequence, the more sensitive two-dimensional 1H–15N HSQC–TOCSY experiments provided additional structural data. The IMPACT–HNMBC experiment remains a powerful tool as demonstrated by the spectrum measured for the unsubstituted amine of 3-O-sulfoglucosamine (GlcN(3S)), which cannot be observed with the 1H–15N HSQC–TOCSY experiment due to the fast exchange of the amino group protons with solvent. The 1H–15N HSQC–TOCSY NMR spectrum reported for the mixture of model compounds GlcNS and N-acetylglucosamine (GlcNAc) demonstrate the broad utility of this approach. Measurements for the synthetic pentasaccharide drug Arixtra® (Fondaparinux sodium) in aqueous solution illustrate the power of this NMR pulse sequence for structural characterization of highly similar N-sulfoglucosamine residues in GAG-derived oligosaccharides.

Fragment Based Drug Discovery: Practical Implementation Based on 19F NMR Spectroscopy

Fragment based drug discovery (FBDD) is a widely used tool for discovering novel therapeutics. NMR is a powerful means for implementing FBDD, and several approaches have been proposed utilizing (1)H-(15)N heteronuclear single quantum coherence (HSQC) as well as one-dimensional (1)H and (19)F NMR to screen compound mixtures against a target of interest. While proton-based NMR methods of fragment screening (FBS) have been well documented and are widely used, the use of (19)F detection in FBS has been only recently introduced (Vulpetti et al. J. Am. Chem. Soc.2009, 131 (36), 12949-12959) with the aim of targeting "fluorophilic" sites in proteins. Here, we demonstrate a more general use of (19)F NMR-based fragment screening in several areas: as a key tool for rapid and sensitive detection of fragment hits, as a method for the rapid development of structure-activity relationship (SAR) on the hit-to-lead path using in-house libraries and/or commercially available compounds, and as a quick and efficient means of assessing target druggability.

A new model of binding of rifampicin and its amino analogues as zwitterions to bacterial RNA polymerase

Seven new benzyl (3-9) and four new phenethyl (10-13) amino analogues of ansa-macrolide rifampicin (1) were synthesized using the optimised method of reductive amination. Structures of 3-13 in solution were determined by 1D and 2D NMR and FT-IR methods whereas the energetically most favoured conformation of amino analogues was calculated with the use of the PM5 method. Spectroscopic and semi-empirical studies revealed the presence of zwitterionic forms of all 3-13 analogues in solutions containing water traces. (1)H-(15)N HSQC and (1)H-(15)N HMBC in combination with (1)H-(1)H COSY and (1)H-(13)C HMBC two dimensional spectroscopic methods unambiguously evidenced that the presence of the zwitterionic form of ansa-macrolides was a consequence of proton transfer from the O(8)-H phenolic group to the secondary amine moiety within 3-13 structures. (1)H-(1)H NOESY studies indicated two different orientations of the substituent introduced at the C(3) position for benzyl and phenethyl amino analogues of rifampicin and their similar conformation within the ansa-bridges in solution. FT-IR studies of the deprotonation of molecule and comparison of these data with those for indicated 3-13 C(8)=O double bond character after formation of zwitterions in solution. Results of an antibacterial test against Gram-(-) and Gram-(+) strains were compared with detailed structural information on new analogues of 3-13 to indicate some structure-activity relationships. Molecular recognition studies of 1 and 12 inhibitors at the binding site of bacterial RNA polymerase (RNAP) as zwitterions revealed key intermolecular interactions and led to the proposition of a new model of RNAP inhibition, which explains significant differences in antibacterial properties of rifampicin and its analogues.

Macromolecular ruthenium complexes as anti-cancer agents

Ruthenium complexes have shown promise as anti-cancer therapeutics. Among them, RAPTA-C [RuCl2(p-cymene)(PTA)] with the water-soluble 1,3,5-triaza-7-phosphaadamantane (PTA) ligand holds great promise as an alternative to platinum drugs. This paper explores synthetic pathways by which RAPTA-C can be attached to polymer moieties to create a water-soluble macromolecular drug that can potentially display an enhanced permeation and retention (EPR) effect. A reactive scaffold for RAPTA-C conjugation, poly(2-chloroethyl methacrylate) [PCEMA], was prepared via RAFT polymerisation. To increase the reactivity of the substrate, poly(2-iodoethyl methacrylate) [PIEMA] was prepared from PCEMA via the Finkelstein reaction. Two pathways were employed to conjugate RAPTA-C to the polymer, either by attachment of PTA first followed by reaction with the RuCl2(p-cymene) dimer, or by the direct conjugation of RAPTA-C to the polymer. Prior to the study with the polymer, both routes were tested using n-butyl iodide as a model compound. Several NMR experiments, namely 31P, 1H, [1H–31P] HMBC, [1H–13C] HMBC and [1H–15N] HMBC confirmed that both routes lead to the complete reaction of n-butyl iodide and that only the desired product was obtained. However, the direct reaction of RAPTA-C with the polymer required elevated temperature, which led to the loss of the p-cymene ligand. The two-step process at ambient temperature led to the successful formation of the product, albeit the amount of iodide reacted was now only 50% due to steric hindrance. Subsequently, a copolymer based on 2-hydroxypropyl methacrylamide [HPMA] and IEMA was synthesised. The two-step reaction with PTA and RuCl2(p-cymene) dimer led to a water-soluble polymer with an intense orange colour, indicative of the pendant RAPTA-C complex. The monomodal molecular weight distribution monitored using UV-VIS detection of the macromolecular ruthenium complex confirmed drug attachment and indicates the absence of crosslinking events during drug conjugation. A cytotoxicity profile for the copolymer–RAPTA-C product was measured on the ovarian cancer cell line OVCAR-3 and compared with the profile of the lone RAPTA-C drug.

Molecular structure and acid/base properties of 1,2-dihydro-1,3,5-triazine derivatives

It is shown that guanidine and its N,N-dimethyl-derivative react with substituted carbodiimides, affording hitherto unknown 1,2-dihydro-1,2,3-triazine derivatives. The structures of three novel compounds of this type and their perchlorate salts were elucidated by spectroscopic (IR, 1H and 13C NMR and 15N solid-state NMR) and X-ray diffraction methods. The acid/base properties were also determined experimentally and by using DFT calculations with the B3LYP functional. The most basic compound was found to be dihydrotriazine 3, the basicity of which with the pKa value of 23.3 is of the same order of magnitude as that of tetramethylguanidine. Acidity measurements revealed that all the compounds studied are very weak acids with pKa values in the range of 25.8–30.8 pKa units in acetonitrile.

Mapping the Inositol Hexakisphosphate Binding Sites on the Human Fibroblast Growth Factor

An investigation of the characteristics of the interaction between inositol hexakisphosphate (IP6) and the human acidic fibroblast growth factor (hFGF-1) was performed. Results of the Isothermal titration calorimetry experiments suggest that binds IP6 to hFGF-1 with a high affinity. Interestingly, differential scanning calorimetry data show a significant reduction in the melting temperature (Tm ) suggesting a reduction in the stability of the protein (hFGF-1) upon binding to IP6. This is further confirmed by the increased rate of proteolytic digestion of hFGF-1 in the presence of IP6. Circular dichroism and intrinsic fluorescence spectroscopy data indicate that the ligand (IP6) does not induce major secondary structural changes in the protein. 1H 15N chemical shift data reveal that IP6 binds in the heparin binding pocket in hFGF-1. The significance of binding of IP6 to hFGF-1 and the prospects of designing novel therapeutic principles against hFGF-1 induced pathogenesis will be discussed.

Controlled modification of the inorganic and organic bricks in an Al-based MOF by direct and post-synthetic synthesis routes

Four new porous CAU-1 derivatives CAU-1–NH2 ([Al4(OH)2(OCH3)4(BDC–NH2)3]·xH2O, BDC–NH22− = aminoterephthalate), CAU-1–NH2(OH) ([Al4(OH)6(BDC–NH2)3]·xH2O), CAU-1–NHCH3 ([Al4(OH)2(OCH3)4(BDC–NHCH3)3]·xH2O) and CAU-1–NHCOCH3 ([Al4(OH)2(OCH3)4(BDC–NHCOCH3)3]·xH2O) all containing an octameric [Al8(OH)4+y(OCH3)8−y]12+ cluster, with y = 0–8, have been obtained by MW-assisted synthesis and post-synthetic modification. The inorganic as well as the organic unit can be modified. Heteronuclear 1H–15N, 1H–13C and homonuclear 1H–1H connectivities determined by solid-state NMR spectroscopy prove the methylation of the NH2 groups when conventional heating is used. Varying reaction times and temperatures allow controlling the degree of methylation of the amino groups. Short reaction times lead to non-methylated CAU-1 (CAU-1–NH2), while longer reaction times result in CAU-1–NHCH3. CAU-1–NH2 can be modified chemically by using acetic anhydride, and the acetamide derivative CAU-1–NHCOCH3 is obtained. Thermal treatment permits us to change the composition of the Al-containing unit. Methoxy groups are gradually exchanged by hydroxy groups at 190 °C in air. Solid-state NMR spectra unequivocally demonstrate the presence of the amino groups, as well as the successful post-synthetic modification. Furthermore 1H–1H correlation spectra using homonuclear decoupling allow the orientation of the NHCOCH3 groups within the pores to be unravelled. The influence of time and temperature on the synthesis of CAU-1 was studied by X-ray powder diffraction, elemental analyses, and 1H liquid-state NMR and IR spectroscopy.

Simultaneous acquisition of 13Cα–15N and 1H–15N–15N sequential correlations in proteins: application of dual receivers in 3D HNN

We describe here, adaptation of the HNN pulse sequence for multiple nuclei detection using two independent receivers by utilizing the detectable (13)C(α) transverse magnetization which was otherwise dephased out in the conventional HNN experiment. It enables acquisition of 2D (13)C(α)-(15)N sequential correlations along with the standard 3D (15)N-(15)N-(1)H correlations, which provides directionality to sequential walk in HNN, on one hand, and enhances the speed of backbone assignment, on the other. We foresee that the implementation of dual direct detection opens up new avenues for a wide variety of modifications that would further enhance the value and applications of the experiment, and enable derivation of hitherto impossible information.

Identification of Formation of Initial Native Structure in Onconase from an Unfolded State

In the oxidative folding of onconase, the stabilization of intermediates early in the folding process gives rise to efficient formation of its biologically active form. To identify the residues responsible for the initial formation of structured intermediates, the transition from an ensemble of unstructured three-disulfide species, 3S(U), to a single structured three-disulfide intermediate species, des-[30-75] or 3S(F), at pH 8.0 and 25 °C was examined. This transition was first monitored by far-UV circular dichroism spectroscopy at pH 8.0 and 25 °C, showing that it occurs with the formation of secondary structure, presumably because of native interactions. The time dependence of formation of nativelike structure was then followed by nuclear magnetic resonance spectroscopy after we had arrested the transition at different times by lowering the pH to 3 and then acquiring (1)H-(15)N heteronuclear single-quantum coherence spectra at pH 3 and 16 °C to identify amide hydrogens that become part of nativelike structure. H/D exchange was utilized to reduce the intensity of resonances from backbone amide hydrogens not involved in structure, without allowing exchange of backbone amide hydrogens involved in initial structure. Six hydrogen-bonding residues, namely, Tyr38, Lys49, Ser82, Cys90, Glu91, and Ala94, were identified as being involved in the earliest detectable nativelike structure before complete formation of des-[30-75] and are further stabilized later in the formation of this intermediate through S-S/SH interchange. By observing the stabilization of the structures of these residues by their neighboring residues, we have identified the initial, nativelike structural elements formed in this transition, providing details of the initial events in the oxidative folding of onconase.

A spectrometer designed for 6.7 and 14.1 T DNP-enhanced solid-state MAS NMR using quasi-optical microwave transmission

A Dynamic Nuclear Polarisation (DNP) enhanced solid-state Magic Angle Spinning (MAS) NMR spectrometer operating at 6.7T is described and demonstrated. The 187GHz TE13 fundamental mode of the FU CW VII gyrotron is used as the microwave source for this magnetic field strength and 284MHz 1H DNP-NMR. The spectrometer is designed for use with microwave frequencies up to 395GHz (the TE16 second-harmonic mode of the gyrotron) for DNP at 14.1T (600MHz 1H NMR).The pulsed microwave output from the gyrotron is converted to a quasi-optical Gaussian beam using a Vlasov antenna and transmitted to the NMR probe via an optical bench, with beam splitters for monitoring and adjusting the microwave power, a ferrite rotator to isolate the gyrotron from the reflected power and a Martin–Puplett interferometer for adjusting the polarisation. The Gaussian beam is reflected by curved mirrors inside the DNP-MAS-NMR probe to be incident at the sample along the MAS rotation axis. The beam is focussed to a ∼1mm waist at the top of the rotor and then gradually diverges to give much more efficient coupling throughout the sample than designs using direct waveguide irradiation. The probe can be used in triple channel HXY mode for 600MHz 1H and double channel HX mode for 284MHz 1H, with MAS sample temperatures ⩾85K. Initial data at 6.7T and ∼1W pulsed microwave power are presented with 13C enhancements of 60 for a frozen urea solution (1H–13C CP), 16 for bacteriorhodopsin in purple membrane (1H–13C CP) and 22 for 15N in a frozen glycine solution (1H–15N CP) being obtained. In comparison with designs which irradiate perpendicular to the rotation axis the approach used here provides a highly efficient use of the incident microwave beam and an NMR-optimised coil design.

Preparation, spectrochemical, and computational analysis of L-carnosine (2-[(3-aminopropanoyl)amino]-3-(1H-imidazol-5-yl)propanoic acid) and its ruthenium (II) coordination complexes in aqueous solution.

This study reports the synthesis and characterization of novel ruthenium (II) complexes with the polydentate dipeptide, L-carnosine (2-[(3-aminopropanoyl)amino]-3-(1H-imidazol-5-yl)propanoic acid). Mixed-ligand complexes with the general composition [MLp(Cl)q(H2O)r]·xH2O (M = Ru(II); L = L-carnosine; p = 3 − q; r = 0–1; and x = 1–3) were prepared by refluxing aqueous solutions of the ligand with equimolar amounts of ruthenium chloride (black-alpha form) at 60 °C for 36 h. Physical properties of the complexes were characterized by elemental analysis, DSC/TGA, and cyclic voltammetry. The molecular structures of the complexes were elucidated using UV-Vis, ATR-IR, and heteronuclear NMR spectroscopy, then confirmed by density function theory (DFT) calculations at the B3LYP/LANL2DZ level. Two-dimensional NMR experiments (1H COSY, 13C gHMBC, and 15N gHMBC) were also conducted for the assignment of chemical shifts and calculation of relative coordination-induced shifts (RCIS) by the complex formed. According to our results, the most probable coordination geometries of ruthenium in these compounds involve nitrogen (N1) from the imidazole ring and an oxygen atom from the carboxylic acid group of the ligand as donor atoms. Additional thermogravimetric and electrochemical data suggest that while the tetrahedral-monomer or octahedral-dimer are both possible structures of the formed complexes, the metal in either structure occurs in the (2+) oxidation state. Resulting RCIS values indicate that the amide-carbonyl, and the amino-terminus of the dipeptide are not involved in chelation and these observations correlate well with theoretical shift predictions by DFT.

Ester-linked hen egg white lysozyme shows a compact fold in a molecular dynamics simulation - possible causes and sensitivity of experimentally observable quantities to structural changes maintaining this compact fold

Prediction and understanding of the folding and stability of the 3D structure of proteins is still a challenge. The different atomic interactions, such as non polar contacts and hydrogen bonding, are known but their exact relative weights and roles when contributing to protein folding and stability are not identified. Initiated by a previous molecular dynamics simulation of fully ester-linked hen egg white lysozyme (HEWL), which showed a more compact fold of the ester-linked molecule compared to the native one, three variants of this protein are analyzed in the present study. These are 129-residue native HEWL, partly ester-linked HEWL, in which only 34 peptide linkages that are not involved in the helical or β-strand parts of native HEWL were replaced by ester linkages, and fully (126 residues) ester-linked HEWL. Native and partly ester-linked HEWL showed comparable behaviour, whereas fully ester-linked HEWL could not maintain the native secondary structure of HEWL in the simulation and adopted a more compact fold. The conformational changes were analyzed by comparing simulation averaged values of quantities that can be measured by NMR, such as (1)H-(15)N backbone order parameters, residual dipolar couplings, proton-proton NOE distances and (3)J-couplings with the corresponding values derived from experimental NMR data for native HEWL. The information content of the latter appeared to be insufficient to detect the local conformational rearrangements upon esterification of the loop regions of the protein. For fully ester-linked HEWL, a significantly reduced agreement was observed. Upon esterification, the backbone-side chain and side chain-side chain hydrogen-bonding pattern of HEWL changes to maintain its compactness and thus the structural stability of the ester-linked lysozymes.

Characterizing Surface Acidic Sites in Mesoporous-Silica-Supported Tungsten Oxide Catalysts Using Solid-State NMR and Quantum Chemistry Calculations

The acidic sites in dispersed tungsten oxide supported on SBA-15 mesoporous silica were investigated using a combination of pyridine titration, both fast- and slow-MAS 15N NMR, static 2H NMR, and quantum chemistry calculations. It is found that the bridging acidic −OH groups in surface-adsorbed tungsten dimers or multimers (i.e., W–OH–W) are the Bronsted acid sites. The unusually strong acidity of these Bronsted acid sites is confirmed by quantum chemistry calculations. In contrast, terminal W–OH sites are very stable and only weakly acidic as are terminal Si–OH sites. Furthermore, molecular interactions between pyridine molecules and the Bronsted and terminal W–OH sites for dispersed tungsten oxide species are strong. This results in restricted molecular motion for the interacting pyridine molecules even at room temperature, that is, a reorientation mainly about the molecular C2 symmetry axis. The restricted reorientation results in efficient 1H–15N cross-polarization, making it possible to estimate the ...

Atomic-resolution dynamics on the surface of amyloid-β protofibrils probed by solution NMR

Exchange dynamics between molecules free in solution and bound to the surface of a large supramolecular structure, a polymer, a membrane or solid support are important in many phenomena in biology and material science. Here we present a novel and generally applicable solution NMR technique, known as Dark-state Exchange Saturation Transfer (DEST), to probe such exchange phenomena with atomic resolution. This is illustrated by the exchange reaction between amyloid β (Aβ) monomers and polydisperse, NMR invisible ('dark') protofibrils, a process of significant interest since the accumulation of toxic, aggregated forms of Aβ, from small oligomers to very large assemblies, have been implicated in the etiology of Alzheimer's disease1–6. The 15N-DEST experiment imprints with single residue resolution dynamic information on the protofibril-bound species in the form of 15N transverse relaxation rates (15N-R2) and exchange kinetics between monomers and protofibrils onto the easily observed two-dimensional 1H-15N correlation spectrum of the monomer. The exchanging species on the protofibril surface comprise an ensemble of sparsely-populated states where each residue is either tethered to (via other residues) or in direct contact with the surface. The first eight residues exist predominantly in a mobile tethered state`, while the largely hydrophobic central region and part of the C-terminal hydrophobic region are in direct contact with the protofibril surface for a significant proportion of the time. The C-terminal residues of both Aβ40 and Aβ42 display lower affinity for the protofibril surface indicating that they are likely to be surface exposed rather than buried as in structures of Aβ fibrils7–10, and may therefore comprise the critical nucleus for fibril formation11,12. The values, however, are significantly larger for the C-terminal residues of Aβ42 than Aβ40 which may explain the former’s higher propensity for rapid aggregation and fibril formation13,14.

Analysis of buried heterointerfacial hydrogen in highly lattice-mismatched epitaxy on silicon

We realized the epitaxial growth of a Sr layer on Si(111) with an atomically abrupt heterointerface – in spite of its large lattice mismatch (12%) with Si – by introducing a monoatomic layer of H on Si. In order to identify the buried H, we carried out a combination analysis involving neutron reflectometry and resonant nuclear reaction of 1H(15N,αγ)12C analysis. We found different neutron reflectivity profiles resulting from a contrast variation between the H and D atoms at the buried heterointerface. Furthermore, the depth γ-ray intensity profiles revealed that the H at the heterointerface acts as an effective buffer layer that enables it to manage the highly mismatched epitaxy on Si.

Synthesis of 1,2,3-triazolo-nucleosides via the post-triazole N-alkylation

2-Substituted-2H-1,2,3-triazolo-nucleosides with 3-phosphonopropyl, 2-hydroxyethyl, 2-cyanoethyl, carbamoylmethyl, or 1-deoxy-2,5-anhydro-d-mannitol-1-yl on the triazole N-2 nitrogen atom were obtained via the DBU-promoted N-alkylation of 3-(pivaloyloxymethyl)-1-[(NH-1,2,3-triazol-4-yl)methyl]thymine with diethyl 3-bromopropylphoshonate, 2-bromoethanol, acrylonitrile, methyl bromoacetate, or 3,4,6-tris(O-benzoyl)-2,5-anhydro-d-mannitol 1-tosylate. The N-2/N-1 regioselectivity of the alkylation varied from 57/43 (methyl bromoacetate) to 97/3 (diethyl 3-bromopropylphoshonate). The 1-substituted-1H-1,2,3-triazoles, when formed in the appreciated amount in the alkylation reaction, were converted into the corresponding 1-substituted-1H-1,2,3-triazolo-nucleosides. The substitution pattern of 2-substituted-2H-1,2,3-triazolo-nucleosides was confirmed by 1H–15N HMBC NMR spectra; the triazole nitrogen atoms were identified through their correlations with the triazole exo-cyclic protons.

Expression, purification, and characterization of coiled coil and leucine zipper domains of C-terminal myosin binding subunit of myosin phosphatase for solution NMR studies

Protein–protein interactions between MBS and PKG are mediated by the involvement of C-terminal domain of MBS, MBSCT180 and N-terminal coiled coil (CC) leucine zipper (LZ) domain of PKG-Iα, PKG-Iα1−59. MBSCT180 is comprised of three structurally variant domains of non-CC, CC, and LZ nature. Paucity of three-dimensional structural information of these MBS domains precludes atomic level understanding of MBS–PKG contractile complex structure. Here we present data on cloning, expression, and purification of CC, LZ, and CCLZ domains of MBSCT180 and their biophysical characterization using size exclusion chromatography (SEC), circular dichroism (CD), and two-dimensional 1H–15N HSQC NMR. The methods as detailed resulted in high level protein expression and high milligram quantities of purified isotopically (15N and 13C) enriched polypeptides. SEC, CD, and 1H–15N HSQC NMR experiments demonstrated that recombinantly expressed MBS CC domain is well folded and exists as a dimer within physiologic pH range, which is supported by our previous findings. The dimerization of CC MBS is likely mediated through formation of coiled coil conformation. In contrast, MBS LZ domain was almost unfolded that exists as non-stable low structured monomer within physiologic pH range. Protein folding and stability of MBS LZ was improved as a function of decrease in pH that adopts a folded, stable, and structured conformation at acidified pH 4.5. SEC and NMR analyses of LZ vs. CCLZ MBS domains indicated that inclusion of CC domain partially improves protein folding of LZ domain.