Correction: A benzylic linker promotes methyltransferase catalyzed norbornene transfer for rapid bioorthogonal tetrazine ligation.

[This corrects the article DOI: 10.1039/C7SC03631K.].

A benzylic linker promotes methyltransferase catalyzed norbornene transfer for rapid bioorthogonal tetrazine ligation.

Site-specific alkylation of complex biomolecules is critical for late-stage product diversification as well as post-synthetic labeling and manipulation of proteins and nucleic acids. Promiscuous methyltransferases in combination with analogs of S-adenosyl-l-methionine (AdoMet) can functionalize all major classes of biomolecules. We show that benzylic moieties are transferred by Ecm1 with higher catalytic efficiency than the natural AdoMet. A relative specificity of up to 80% is achieved when a norbornene moiety is placed in para-position, enabling for the first time enzymatic norbornene transfer to specific positions in DNA and RNA- even in cell lysate. Subsequent tetrazine ligation of the stable norbornene moiety is fast, efficient, biocompatible and - in combination with an appropriate tetrazine - fluorogenic.

13C and 15N NMR study of the hydration response of T4 lysozyme and alphaB-crystallin internal dynamics.

The response to hydration of the internal protein dynamics was studied by the means of solid state NMR relaxation and magic angle spinning exchange techniques. Two proteins, lysozyme from bacteriophage T4 and human alphaB-crystallin were used as exemplars. The relaxation rates R1 and R1rho of 13C and 15N nuclei were measured as a function of a hydration level of the proteins in the range 0-0.6 g of water/g of protein. Both proteins were totally 15N-enriched with natural 13C abundance. The relaxation rates were measured for different spectral bands (peaks) that enabled the characterization of the dynamics separately for the backbone, side chains, and CH3 and NH3+ groups. The data obtained allowed a comparative analysis of the hydration response of the protein dynamics in different frequency ranges and different sites in the protein for two different proteins and two magnetic nuclei. The most important result is a demonstration of a qualitatively different response to hydration of the internal dynamics in different frequency ranges. The amplitude of the fast (nanosecond time scale) motion gradually increases with increasing hydration, whereas that of the slow (microsecond time scale) motion increases only until the hydration level 0.2-0.3 g of water/g of protein and then shows almost no hydration dependence. The reason for such a difference is discussed in terms of the different physical natures of these two dynamic processes. Backbone and side chain nuclei show the same features of the response of dynamics with hydration despite the fact that the backbone motional amplitudes are much smaller than those of side chains. Although T4 lysozyme and alphaB-crystallin possess rather different structural and biochemical properties, both proteins show qualitatively very similar hydration responses. In addition to the internal motions, exchange NMR data enabled the identification of one more type of motion in the millisecond to second time scale that appears only at high hydration levels. This motion was attributed to the restricted librations of the protein as a whole.

Mechanisms of potentiation of the mammalian GABAA receptor by the marine cembranoid eupalmerin acetate.

BACKGROUND AND PURPOSE: Eupalmerin acetate (EPA) is a marine diterpene compound isolated from the gorgonian octocorals Eunicea succinea and Eunicea mammosa. The compound has been previously shown to modulate muscle-type and neuronal nicotinic acetylcholine receptors, which are inhibited in the presence of low micromolar concentrations of EPA. In this study, we examined the effect of EPA on another transmitter-gated ion channel, the GABA(A) receptor. EXPERIMENTAL APPROACH: Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing rat wild-type and mutant alpha1beta2gamma2L GABA(A) receptors. KEY RESULTS: Our findings demonstrate that, at micromolar concentrations, EPA potentiates the rat alpha1beta2gamma2L GABA(A) receptor. The analysis of single-channel currents recorded in the presence of EPA showed that the kinetic mode of action of EPA is similar to that of neuroactive steroids. Mutations to residues alpha1Q241 and alpha1N407/Y410, previously shown to affect receptor modulation by neurosteroids, also diminished potentiation by EPA. Exposure to a steroid antagonist, (3alpha,5alpha)-17-phenylandrost-16-en-3-ol, reduced potentiation by EPA. Additionally, exposure to EPA led to potentiation of GABA(A) receptors activated by very high concentrations (1-10 microM) of allopregnanolone. In tadpole behavioural assays, EPA caused loss of righting reflex and loss of swimming reflex. CONCLUSIONS AND IMPLICATIONS: We conclude that EPA either interacts with the putative neurosteroid binding site on the GABA(A) receptor or shares with neurosteroids the key transduction elements involved in channel potentiation by steroids. The results indicate that cembranoids represent a novel class of GABA(A) receptor modulators.

Bond-shift rearrangement in solid Li(3)P(7)(monoglyme)(3): a (31)P MAS NMR study.

The (31)P MAS NMR spectrum of solid Li(3)P(7)(monoglyme)(3) has been reinvestigated over a wide temperature range (-70 to +77 degrees C) and under conditions of better resolution (Larmor frequency of 162 MHz and spinning rate of approximately 30 kHz) than previously measured (121 MHz and 13 kHz). At low temperatures three spinning sideband (ssb) manifolds are observed: a singlet (centered at -45 ppm relative to 85% H(3)PO(4)) due to the apical atom (A) of the P(7)-cage trianion; a 1 : 1 : 1 triplet (at -110, -117, and -124.5 ppm) due to the negatively charged equatorial (E) atoms, and a one to two doublet (at -161 and -168.5 ppm) due to the basal (B) atoms. These results are consistent with the P(7) cage having nearly, but not perfect, C(3v) symmetry. The compound appears to be well ordered in the solid state with very little structural dispersity. On heating, the NMR lines broaden and eventually coalesce into a single ssb manifold. This behavior is ascribed to bond-shift rearrangement similar to the Cope rearrangement in bullvalene. A MAS 2D exchange experiment and a quantitative analysis of the 1D NMR lineshapes indicate that, unlike in solution where the rearrangement involves a single bond shift at a time, in the solid the process involves a succession of two bond shifts: The first leads to an intermediate species in which the rearranged P(7) cage is inverted, while in the subsequent step a second bond shift takes place that also restores the original orientation of the cage in the lattice. The overall effect of the double bond shift is equivalent to cyclic permutation of the phosphorus atoms within the five member rings of the P(7)-cage. The quantitative analysis of the dynamic lineshapes shows that this cyclic permutation proceeds at a different rate in one ring (k(d)(1)) than in the other two (k(d)(2,3)). The kinetic parameters for these processes are E(a)(1)=18.7 kJ/mol, E(a)(2,3)=58.0 kJ/mol, k(d)(1)(17 degrees C)=k(d)(2,3)(17 degrees C)=10(4) s(-1). No indications for independent threefold molecular jumps of the P(7) cage were found.

Molecular modeling of bifunctional chelate peptide conjugates. 1. Copper and indium parameters for the AMBER force field.

In this work we describe the development of parameters for In(III) and Cu(II) for the AMBER force field as found in the modeling package MacroModel. These parameters were developed using automated procedures from a combination of crystallographic structures and ab initio calculations. The new parameters were added in the form of AMBER substructures containing specific metal-ligand parameters to the existing force field. These new parameters have produced results in good agreement with experiment without requiring additional changes to the existing AMBER parameters. These parameters were then utilized to examine the conformational effects caused by the conjugation of InDTPA (DTPA = diethylenetriaminepentaacetic acid) and CuDOTA (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to the cyclic octapeptide octreotide.

Slow-down of 13C spin diffusion in organic solids by fast MAS: a CODEX NMR Study.

One- and two-dimensional 13C exchange nuclear magnetic resonance experiments under magic-angle spinning (MAS) can provide detailed information on slow segmental reorientations and chemical exchange in organic solids, including polymers and proteins. However, observations of dynamics on the time scale of seconds or longer are hampered by the competing process of dipolar 13C spin exchange (spin diffusion). In this Communication, we show that fast MAS can significantly slow down the dipolar spin exchange effect for unprotonated carbon sites. The exchange is measured quantitatively using the centerband-only detection of exchange technique, which enables the detection of exchange at any spinning speed, even in the absence of changes of isotropic chemical shifts. For chemically equivalent unprotonated 13C sites, the dipolar spin exchange rate is found to decrease slightly less than proportionally with the sample-rotation frequency, between 8 and 28 kHz. In the same range, the dipolar spin exchange rate for a glassy polymer with an inhomogeneously broadened MAS line decreases by a factor of 10. For methylene groups, no or only a minor slow-down of the exchange rate is found.

PATROS-A new MAS exchange method using sideband separation: application to Poly(n-butylmethacrylate)

The tr-ODESSA method (Reichert et al., J. Magn. Reson. 125, 245 (1997)), which is a 1D MAS experiment designed to monitor spin exchange involving both equivalent and inequivalent sites, is extended to situations where the spectrum consists of several spinning side band (ssb) manifolds with small chemical shift anisotropies. To increase the spectral resolution in such situations, the tr-ODESSA sequence is combined with that of PASS to a single experiment, which we term PATROS. In this hybrid experiment, magnetization transfer is monitored by the tr-ODESSA part, while the increase in resolution is provided by the separation of the ssb according to their order, during the PASS part. We demonstrate the feasibility of the method on a standard solid dimethylsulfone (DMS) sample and then apply it to monitor separately the ultraslow motions of the main- and side-chains in the polymer poly(n-butylmethacrylate). Theoretical expressions for the ssb intensities in PATROS experiments are derived and the merits and limitations of the method are discussed. Copyright 2000 Academic Press.

Labeling and in vivo evaluation of novel copper(II) dioxotetraazamacrocyclic complexes.

64Cu shows promise as both a positron emission tomography imaging and radiotherapeutic radionuclide due to its half-life (T(1/2) = 12. 7 h), decay characteristics (beta(+) [19%]; beta(-) [40%]), and the capability to produce it on a large-scale with high specific activity on a biomedical cyclotron. Macrocyclic chelators are generally used as bifunctional chelators to attach Cu(II) to antibodies and peptides due to their relatively high in vitro stability. To investigate neutral Cu(II) complexes, we performed labeling experiments with six tetraazamacrocyclic ligands with different chelate ring sizes. 1,4,8,11-Tetraazacyclotetradecane-3, 9-dione (1), 1,4,8,11-tetraazacyclotetradecane-5,7-dione (2), 1,4,7, 10-tetraazacyclotridecane-11,13-dione (3), 1,4,7, 10-tetraazacyclotridecane-2,9-dione (4), 1,4,7, 10-tetraazacyclododecane-2,9-dione (5), and 1,4,7, 10-tetraazacyclotridecane-3,8-dione (6) were radiolabeled with (64)Cu. Only (64)Cu-labeled 1 readily formed a complex in high purity, and therefore was evaluated in vivo. The rapid blood, liver, and kidney clearance of (64)Cu-labeled 1 suggest that ligand 1 may be useful as a macrocyclic structure to design new bifunctional chelators for copper radionuclides in diagnostic or radiotherapeutic studies and is a potential alternative to currently used macrocyclic bifunctional chelators.

Initial conditions for carbon-13 MAS NMR 1D exchange involving chemically equivalent and inequivalent nuclei

A major problem in dynamic 1D (13)C MAS NMR concerns the exchange between magnetically inequivalent, but chemically equivalent sites, whose signals are not resolved in the regular 1D spectrum. This difficulty may be overcome by properly preparing the initial nonequilibrium state of the spin system in the exchange experiments. In the present paper we discuss the advantages and limitations of several such experiments already in use and propose a new sequence, which we term SELDOM-ODESSA. Unlike the other 1D-exchange methods, this experiment yields pure absorption spectra that can more readily be analyzed quantitatively. The experiment is a hybrid comprising a SELDOM sequence, for selective excitation of one of the spinning sideband manifolds in the spectrum, followed by the ODESSA sequence, which induces alternate polarization in the excited sideband manifold. The evolution of the spectrum following this sequence provides information on both the exchange between congruent sites belonging to the same group of equivalent nuclei, and the exchange between inequivalent sites. Results are presented for a tropolone sample specifically enriched in carbon-13 at the carbonyl and hydroxyl sites. The dominant exchange mechanism in this sample involves spin diffusion. The various spin exchange processes in this sample, in the presence and absence of proton decoupling during the mixing time, are measured and discussed. Copyright 2000 Academic Press.

Dynamic carbon-13 MAS NMR: application to benzene ring flips in polyaryl ethers.

Carbon-13 dynamic MAS NMR is used to determine the pi-flip rates of the phenyl rings in the low-molecular-weight members of the polyaryl ethers series (phenyl-O(-phenylene-O)n-phenyl). The first member in the series (diphenyl ether, n = 0) does not undergo measurable dynamic processes up to its melting point (28 degrees C). The second and third members (n = 1 and 2) exhibit, above room temperature, line broadening effects due to fast pi-flips of the terminal rings, while the spectra of the n = 1 homologue also exhibit line broadening for the inner phenylene ring. Kinetic parameters for the various pi-flip processes were derived by a detailed lineshape analysis of the MAS spectra. The measurements were extended to lower temperatures by time-reverse ODESSA experiments. The kinetic parameters derived from these experiments are, k(t)(300 K) = 31 s(-1), E(t) = 84 kJ/mol, and k(i)(300 K) = 1.3 s(-1), Ei = 77 kJ/mol for the n = 1 homologue and k(t)(300 K) = 3.2 s(-1), E(i) = 78 kJ/mol, for the n = 2 homologue, where the subscripts t and i refer to the terminal and inner benzene rings, respectively. For the simulation of the dynamic MAS spectra the Floquet expansion method was used. In an introductory chapter the Floquet method is reviewed with emphasis on the practical aspects of the computation procedure, on the sensitivity of the results to the isotropic and anisotropic chemical shift parameters, and on the form of the results in the limiting fast and slow exchange regimes.

Synthesis and characterization of racemic mixture and meso isomers of bis(trans-2-aminocyclohexyl)aminepentaacetic acid and the stabilities of their Gd(III) complexes.

The synthesis and characterization of two multidentate ligands, the racemic mixture and meso isomers of bis-(trans-2-aminocyclohexyl)aminepentaacetic acid, are described. Equilibrium constants for their Gd(III) complexes were determined by direct potentiometry. The formation constants (KML = [ML]/[M][L]) of Gd(III)-Cycyracemic in 0.10 M KCl at 25.0 degrees C is 10(20.71; that for the meso isomer is 10(20.42). The crystal structure of (1S,2S,1'S,2'S)-bis(trans-2-aminocyclohexyl)amine-N,N,N',N",N"-pentaacetic acid, penta-tert-butyl ester (C42H75N3O10), is reported. This compound crystallizes in the triclinic system with space group P1 with cell parameters a = 10.805(2) A, b = 11.382(2) A, c = 20.999(4) A, alpha = 91.41(3) degrees, beta = 98.23(3) degrees, gamma = 113.88(3) degrees, V = 2327.8(8) A3, Z = 2, Dx = 1.116 g mL-1. The results are compared to the crystal structures of the gadolinium complexes and the predictions derived from molecular mechanics.

Narrowband excitation of (2)H powder pattern and its application to (2)H 1D exchange sample-turning NMR.

Frequency-selective narrowband excitation of the (2)H powder pattern was examined. Selection of a single spectral band with a linewidth of ca. 15 kHz was achieved by a narrowband (1)H --> (2)H cross polarization by using the time-averaged precession frequency method. Further narrowing with a ca. 5 kHz linewidth is achieved by DANTE irradiation. The narrowband excitation was applied to transform a recently developed 2D spin-exchange method for obtaining structural information (Chem. Phys. Lett. 260, 159, (1996)) into its 1D analogue. The determination of the D-C-D bond angle was demonstrated for alpha-glycine-[2,2-d(2)]. Further, the intermolecular polarization transfer between two deuterons separated by 0.299 nm was detected with the mixing time of 500 ms.

Solid-state photodimerization of 4-aryl-1,4-dihydropyridines studied by 13C CPMAS NMR spectroscopy.

13C CPMAS NMR spectroscopy has been applied to monitor the solid-state reaction of two different photodimerizing 4-phenyl-1,4-dihydropyridines yielding a cage dimer in one case and an anti-dimer in the other case. The spectra of the reacting monomers exhibit a magnetical inequivalence of chemically equivalent CO and C2/4 carbon atoms caused by a rotation of the pseudoaxially oriented 4-phenyl substituent out off the plane through N1, C3, C8 which could be determined by X-ray crystal structure analyses of the centrosymmetrically arranged monomers. The 13C CPMAS NMR monitoring of the cage dimer formation proves that the reaction takes place in two steps via a syn-dimer for which a non-symmetrical structure was derived from the spectrum. The non-symmetrical structure was confirmed by X-ray crystal structure analysis of one structurally related derivative. A centrosymmetric structure for both the finally formed cage dimer and the anti-dimer of the other monitored photoreaction was proved by their spectra with one set of signals for each half of the dimers. respectively. Thus, conformational properties of the molecules as well as the symmetry of the products can be directly derived from the 13C CPMAS NMR spectra.

Evaluation of gallium-68 tris(2-mercaptobenzyl)amine: a complex with brain and myocardial uptake.

Previous research into development of a gallium-radiolabeled agent that crosses the blood-brain barrier has met with limited success. In this study, we focused our attention on a Ga(III) complex of a 4-coordinate amine trithiolate tripod ligand, tris(2-mercaptobenzyl) amine (S3N). The Ga(III) S3N complex is small, neutral, and lipophilic, meeting the requirements for a potential brain imaging agent. The Ga-68 complex was easily formed with a radiochemical purity of >95%. In vitro stability of the Ga-S3N complex, determined in rat serum incubated at 37 degrees C, was greater than 95% intact at 2 h by silica gel and reversed-phase radio-thin layer chromatography. Biodistribution studies conducted in female Sprague-Dawley rats showed the complex cleared rapidly from the blood with initial high liver uptake followed by rapid washout. Significant uptake was observed in the brain, with brain:blood ratios increasing from 0.11 at 2 min postinjection to 3.8 at 60 min postinjection. Uptake was also observed in the heart going from a heart:blood ratio of 2.3 at 2 min postinjection to 11 at 60 min postinjection. Molecular mechanics were used to determine the coordination number, and demonstrated that the Ga(III) complex prefers to be 4-coordinate. Imaging studies with 68Ga-S3N in a Nemestrina macaque showed significant brain uptake, similar to other lipophilic agents. The extraction of 68Ga-S3N into the brains of both rodents and primates, higher than any 68Ga agent reported in the literature, suggests that this compound may have potential as a brain imaging agent for positron emission tomography.

Superslow backbone protein dynamics as studied by 1D solid-state MAS exchange NMR spectroscopy.

Superslow backbone dynamics of the protein barstar and the polypeptide polyglycine was studied by means of a solid-state MAS 1D exchange NMR method (time-reverse ODESSA) that can detect reorientation of nuclei carrying anisotropic chemical shift tensors. Experiments were performed on carbonyl 13C in polyglycine (natural abundance) and backbone 15N nuclei in uniformly 15N-enriched barstar within a wide range of temperatures in dry and wet powders for both samples. Two exchange processes were observed in the experiments: molecular reorientation and spin diffusion. Experimental conditions that are necessary to separate these two processes are discussed on a quantitative level. It was revealed that the wet protein undergoes molecular motion in the millisecond range of correlation times, whereas in dry protein and polyglycine molecular reorientations could not be detected. The correlation time of the motion in the wet barstar at room temperature is 50-100 ms; the activation energy is about 80 kJ/mol. Previously, protein motions with such a long correlation time could be observed only by methods detecting chemical exchange in solution (e.g., hydrogen exchange). The application of solid-state MAS exchange spectroscopy provides new opportunities in studying slow biomolecular dynamics that is important for the biological function of proteins.