FMP Publications

Our publications are recorded in a searchable database since 2010, updates will be added regularly.

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References
Soluble peptidyl phosphoranes for metal-free, stereoselective ligations in organic and aqueous solution
Ahsanullah, R.J., Al-Gharabli, S. I.; Rademann, J.
Org Lett, 14:14-17
(2012)

Tags: Medicinal Chemistry (Rademann)

Abstract: Protocols for solid-phase syntheses of soluble peptidyl phosphoranes are presented. Various supported phosphoranylidene acetates were prepared on Rink amide or via alkylation of trialkyl- and triarylphosphines with bromoacetyl Wang ester. C-Acylation was conducted racemization-free with activated Fmoc-amino acids, followed by SPPS (solid-phase peptide synthesis). Acidic conditions released decarboxylated peptidyl phosphoranes into solution. The protocol allowed for the electronic variation of peptidyl phosphoranes which were investigated in ligation reactions with azides in organic and aqueous solvents.

High-Temperature Dynamic Nuclear Polarization Enhanced Magic-Angle-Spinning NMR
Akbey, Ü., Linden, A. H.; Oschkinat, H.
Appl Magn Reson, 43:81-90
(2012)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Dynamic nuclear polarization (DNP) transfers electron spin-polarization to nuclear spins in close proximity, increasing sensitivity by two-to-three orders of magnitude. This enables nuclear magnetic resonance (NMR) experiments on samples with low concentrations of analyte. The requirement of using cryogenic temperatures in DNP-enhanced solid-state NMR (ssNMR) experiments may impair the resolution and hence limit its broad application to biological systems. In this work, we introduce a "High-Temperature DNP" approach, which aims at increasing spectral resolution by performing experiments at temperatures of around 180 K instead of similar to 100 K. By utilizing the extraordinary enhancements obtained on deuterated proteins, still sufficiently large DNP enhancements of 11-18 are obtained for proton and carbon, respectively. We recorded high sensitivity 2D C-13-C-13 spectra in similar to 9 min with higher resolution than at 100 K, which has similar resolution to the one obtained at room temperature for some favorable residues.

Practical aspects of high-sensitivity multidimensional (1)(3)C MAS NMR spectroscopy of perdeuterated proteins
Akbey, Ü., Rossum, B. J.; Oschkinat, H.
J Magn Reson, 217:77-85
(2012)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: The double nucleus enhanced recoupling (DONER) experiment employs simultaneous irradiation of protons and deuterons to promote spin diffusion processes in a perdeuterated protein. This results in 4-5 times higher sensitivity in 2D (13)C-(13)C correlation experiments as compared to PDSD [1]. Here, a quantitative comparison of PDSD, (1)H-DARR, (2)H-DARR, and (1)H+(2)H DONER has been performed to analyze the influence of spin diffusion on polarization transfer processes. Cross peak buildup curves were analyzed to obtain guidelines for choosing the best experimental parameters. The largest cross peak intensities were observed for the DONER experiments. The fastest build-up rate was observed in the (2)H-DARR experiment within a buildup range of approximately 18-45 ms, whereas values between 24 and 69 ms are observed for the DONER experiment. Furthermore, the effects of direct excitation and cross polarization (CP) are compared. A comparison between DONER and RFDR experiments reveal approximately 50% more intense cross peaks in the C(alpha)-CO and C(alpha)-C(alip) regions of the 2D (13)C-(13)C DONER spectrum applying proton CP ((1)H-(13)C). As a parameter determining the S/N in (13)C-(13)C correlation experiments, proton CP efficiency is investigated using deuterated samples with proton/deuterium ratios at 20%, 40%, and 100% H(2)O. Sufficiently strong (13)C CPMAS signal intensity is observed for such proteins even with very low proton concentration. The effect of proton and/or deuterium decoupling is analyzed at various MAS spinning frequencies. Deuterium decoupling was found most crucial for obtaining high resolution. Long range correlations are readily observed representing distances up to approximately 6 A by using DONER approach.

Rapid solid-state NMR of deuterated proteins by interleaved cross-polarization from H-1 and H-2 nuclei
Bjerring(*), M., Paaske(*), B., Oschkinat, H., Akbey, Ü.; Nielsen(*), N. C.
Journal of Magnetic Resonance, 214:324-328
(2012)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: We present a novel sampling strategy, interleaving acquisition of multiple NMR spectra by exploiting initial polarization subsequently from H-1 and H-2 spins, taking advantage of their different T-1 relaxation times. Different H-1- and H-2-polarization based spectra are in this way simultaneously recorded improving either information content or sensitivity by adding spectra. The so-called Relaxation-optimized Acquisition of Proton Interleaved with Deuterium (RAPID) H-1 -> C-13/H-2 -> C-13 CP/MAS multiple-acquisition method is demonstrated by 1D and 2D experiments using a uniformly H-2, N-15, C-13-labeled alpha-spectrin SH3 domain sample with all or 30% back-exchanged labile H-2 to H-1. It is demonstrated how 1D C-13 CP/MAS or 2D C-13-C-13 correlation spectra initialized with polarization from either H-1 or H-2 may be recorded simultaneously with flexibility to be added or used individually for spectral editing. It is also shown how 2D C-13-C-13 correlation spectra may be recorded interleaved with H-2-C-13 correlation spectra to obtain C-13-C-13 correlations along with information about dynamics from H-2 sideband patterns. (C) 2011 Elsevier Inc. All rights reserved.

The effect of biradical concentration on the performance of DNP-MAS-NMR
Lange, S., Linden, A. H., Akbey, Ü., Franks, W. T., Loening(*), N. M., van Rossum, B. J.; Oschkinat, H.
J Magn Reson, 216:209-212
(2012)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: With the technique of dynamic nuclear polarization (DNP) signal intensity in solid-state MAS-NMR experiments can be enhanced by 2-3 orders of magnitude. DNP relies on the transfer of electron spin polarization from unpaired electrons to nuclear spins. For this reason, stable organic biradicals such as TOTAPOL are commonly added to samples used in DNP experiments. We investigated the effects of biradical concentration on the relaxation, enhancement, and intensity of NMR signals, employing a series of samples with various TOTAPOL concentrations and uniformly (13)C, (15)N labeled proline. A considerable decrease of the NMR relaxation times (T(1), T(2)( *), and T(1)(rho)) is observed with increasing amounts of biradical due to paramagnetic relaxation enhancement (PRE). For nuclei in close proximity to the radical, decreasing T(1)(rho) reduces cross-polarization efficiency and decreases in T(2)( *) broaden the signal. Additionally, paramagnetic shifts of (1)H signals can cause further line broadening by impairing decoupling. On average, the combination of these paramagnetic effects (PE; relaxation enhancement, paramagnetic shifts) quenches NMR-signals from nuclei closer than 10A to the biradical centers. On the other hand, shorter T(1) times allow the repetition rate of the experiment to be increased, which can partially compensate for intensity loss. Therefore, it is desirable to optimize the radical concentration to prevent additional line broadening and to maximize the signal-to-noise observed per unit time for the signals of interest.

Fast passage dynamic nuclear polarization on rotating solids
Mentink-Vigier(*), F., Akbey, Ü., Hovav(*), Y., Vega(*), S., Oschkinat, H.; Feintuch(*), A.
Journal of Magnetic Resonance, 224:13-21
(2012)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Magic Angle Spinning (MAS) Dynamic Nuclear Polarization (DNP) has proven to be a very powerful way to improve the signal to noise ratio of NMR experiments on solids. The experiments have in general been interpreted considering the Solid-Effect (SE) and Cross-Effect (CE) DNP mechanisms while ignoring the influence of sample spinning. In this paper, we show experimental data of MAS-DNP enhancements of H-1 and C-13 in proline and SH3 protein in glass forming water/glycerol solvent containing TOTAPOL. We also introduce a theoretical model that aims at explaining how the nuclear polarization is built in MAS-DNP experiments. By using Liouville space based simulations to include relaxation on two simple spin models, (electron-nucleus) and (electron-electron-nucleus), we explain how the basic MAS-SE-DNP and MAS-CE-DNP processes work. The importance of fast energy passages and short level anti-crossing is emphasized and the differences between static DNP and MAS-DNP is explained. During a single rotor cycle the enhancement in the (electron-electron-nucleus) system arises from MAS-CE-DNP involving at least three kinds of two-level fast passages: an electron-electron dipolar anti-crossing, a single quantum electron MW encounter and an anti-crossing at the CE condition inducing nuclear polarization in- or decrements. Numerical, powder-averaged, simulations were performed in order to check the influence of the experimental parameters on the enhancement efficiencies. In particular we show that the spinning frequency dependence of the theoretical MAS-CE-DNP enhancement compares favorably with the experimental and 13C MAS-DNP enhancements of proline and SH3. (C) 2012 Elsevier Inc. All rights reserved.

Surfactant-assisted formation of organophilic CeO2 nanoparticles
Tunusoglu(*), O., Munoz-Espi(*), R., Akbey, Ü.; Demir(*), M. M.
Colloid Surface A, 395:10-17
(2012)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: We report a simple one-pot method to prepare organically functionalized CeO2 nanoparticles by controlled chemical precipitation. The particles were nucleated by mixing aqueous solutions of Ce(NO3)(3)center dot 6H(2)O and ammonia at room temperature. Different small organic molecules were chosen as capping agents and injected into the reaction medium at the beginning of the synthesis: 3-(mercaptopropyl) trimethoxy silane (MPS), hexadecyltrimethyl ammonium bromide (CTAB), 3-mercapto propionic acid (3-MPA), and thioglycolic acid (TGA). The resulting nanocrystals were quasi-spherical and had a narrow mean size distribution with an average size smaller than 10 nm. Dynamic nuclear polarization enhanced NMR (DNP-NMR) and FTIR measurements suggested a chemical grafting of the surfactant and a homogeneous surface modification. The colloidal stabilities were characterized by dynamic light scattering and zeta potential measurements. The stabilization by aliphatic groups was tested with a frequently used hydrophobic monomer, methyl methacrylate. According to the results. CTAB is the most effective of the used stabilizing surfactant. The mechanism of formation of the organophilic CeO2 nanoparticles is discussed. (C) 2011 Elsevier B.V. All rights reserved.

Assignment strategies for aliphatic protons in the solid-state in randomly protonated proteins
Asami, S.; Reif, B.
J. Biomol. NMR, 52:31-39
(2012)

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: Biological solid-state nuclear magnetic resonance spectroscopy developed rapidly in the past two decades and emerged as an important tool for structural biology. Resonance assignment is an essential prerequisite for structure determination and the characterization of motional properties of a molecule. Experiments, which rely on carbon or nitrogen detection, suffer, however, from low sensitivity. Recently, we introduced the RAP (Reduced Adjoining Protonation) labeling scheme, which allows to detect backbone and sidechain protons with high sensitivity and resolution. We present here a H-1-detected 3D (H)CCH experiment for assignment of backbone and sidechain proton resonances. Resolution is significantly improved by employing simultaneous (CO)-C-13 and C-13 beta J-decoupling during evolution of the C-13 alpha chemical shift. In total, similar to 90% of the H-1 alpha-C-13 alpha backbone resonances of chicken alpha-spectrin SH3 could be assigned.

Optimal degree of protonation for (1)H detection of aliphatic sites in randomly deuterated proteins as a function of the MAS frequency
Asami, S., Szekely(*), K., Schanda(*), P., Meier(*), B. H.; Reif, B.
J Biomol NMR, 54:155-168
(2012)

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: The (1)H dipolar network, which is the major obstacle for applying proton detection in the solid-state, can be reduced by deuteration, employing the RAP (Reduced Adjoining Protonation) labeling scheme, which yields random protonation at non-exchangeable sites. We present here a systematic study on the optimal degree of random sidechain protonation in RAP samples as a function of the MAS (magic angle spinning) frequency. In particular, we compare (1)H sensitivity and linewidth of a microcrystalline protein, the SH3 domain of chicken alpha-spectrin, for samples, prepared with 5-25 % H(2)O in the E. coli growth medium, in the MAS frequency range of 20-60 kHz. At an external field of 19.96 T (850 MHz), we find that using a proton concentration between 15 and 25 % in the M9 medium yields the best compromise in terms of sensitivity and resolution, with an achievable average (1)H linewidth on the order of 40-50 Hz. Comparing sensitivities at a MAS frequency of 60 versus 20 kHz, a gain in sensitivity by a factor of 4-4.5 is observed in INEPT-based (1)H detected 1D (1)H,(13)C correlation experiments. In total, we find that spectra recorded with a 1.3 mm rotor at 60 kHz have almost the same sensitivity as spectra recorded with a fully packed 3.2 mm rotor at 20 kHz, even though ~20x less material is employed. The improved sensitivity is attributed to (1)H line narrowing due to fast MAS and to the increased efficiency of the 1.3 mm coil.

Optimal degree of protonation for H-1 detection of aliphatic sites in randomly deuterated proteins as a function of the MAS frequency
Asami, S., Szekely(*), K., Schanda(*), P., Meier(*), B. H.; Reif, B.
J. Biomol. NMR, 54:155-168
(2012)

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: The H-1 dipolar network, which is the major obstacle for applying proton detection in the solid-state, can be reduced by deuteration, employing the RAP (Reduced Adjoining Protonation) labeling scheme, which yields random protonation at non-exchangeable sites. We present here a systematic study on the optimal degree of random sidechain protonation in RAP samples as a function of the MAS (magic angle spinning) frequency. In particular, we compare H-1 sensitivity and linewidth of a microcrystalline protein, the SH3 domain of chicken alpha-spectrin, for samples, prepared with 5-25 % H2O in the E. coli growth medium, in the MAS frequency range of 20-60 kHz. At an external field of 19.96 T (850 MHz), we find that using a proton concentration between 15 and 25 % in the M9 medium yields the best compromise in terms of sensitivity and resolution, with an achievable average H-1 linewidth on the order of 40-50 Hz. Comparing sensitivities at a MAS frequency of 60 versus 20 kHz, a gain in sensitivity by a factor of 4-4.5 is observed in INEPT-based H-1 detected 1D H-1,C-13 correlation experiments. In total, we find that spectra recorded with a 1.3 mm rotor at 60 kHz have almost the same sensitivity as spectra recorded with a fully packed 3.2 mm rotor at 20 kHz, even though similar to 20x less material is employed. The improved sensitivity is attributed to H-1 line narrowing due to fast MAS and to the increased efficiency of the 1.3 mm coil.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
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13125 Berlin, Germany
+4930 94793 - 100 
+4930 94793 - 109 (Fax)
info(at)fmp-berlin.de

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