Efficient Access to Elusive 1D 13C NMR Spectra through Highly Resolved 1H, 13C-Long-Range Correlation Spectroscopy.

A method for obtaining 1D 13C NMR spectra from natural products or metabolites using proton detection is described. The approach delivers singlets for every 13C signal without conducting any broadband 1H decoupling (CPD) and is based on calculating 13C projections from constant-time HMBC and conventional HSQC experiments, recorded at high digital resolution and processed to pure phases. Paramount to the proposed method is the implication of nonuniform sampling and echo processing. The echo processing produces phase-sensitive 2D CT-HMBC spectra with narrow 13C signal line shapes. Two simple HMBC pulse sequences are utilized with the suppression of homo- and heteronuclear couplings. Due to the removal of the 1H multiplet structure in F1 (no tilt at higher digital resolution), 13C singlets arise. An overall increase in 13C signal-to-noise (SINO) for all types of carbon multiplicities is observed, making the proposed technique superior compared to direct 13C excitation. For otherwise difficult-to-measure quaternary carbon atoms, a SINO enhancement of up to 6 and 12 depending on F1 resolution (3 and 6 Hz/point) is reported. Echo/anti-Echo signal detection cleans up the spectrum. Nonuniform sampling (NUS) lays the groundwork to significantly reduce the total acquisition time. Final 1D 13C projections are obtained by combining the 13C projection from CT HMBC and conventional HSQC. This orthogonal concept of combining the 13C projections from different spectra inherently minimizes the risk of missing 13C cross-peaks by inappropriate setting of long-range nJHC coupling delays and the shortcoming of T2 relaxations. The advantages and some limitations of the concept are discussed.

Synthetic Peptide Antibodies as TNF-α Inhibitors: Molecularly Imprinted Polymer Nanogels Neutralize the Inflammatory Activity of TNF-α in THP-1 Derived Macrophages.

Tumor Necrosis Factor-α (TNF-α) is a cytokine that is normally produced by immune cells when fighting an infection. But, when too much TNF-α is produced as in autoimmune diseases, this leads to unwanted and persistent inflammation. Anti-TNF-α monoclonal antibodies have revolutionized the therapy of these disorders by blocking TNF-α and preventing its binding to TNF-α receptors, thus suppressing the inflammation. Herein, we propose an alternative in the form of molecularly imprinted polymer nanogels (MIP-NGs). MIP-NGs are synthetic antibodies obtained by nanomoulding the 3-dimensional shape and chemical functionalities of a desired target in a synthetic polymer. Using an in-house developed in silico rational approach, epitope peptides of TNF-α were generated and 'synthetic peptide antibodies' were prepared. The resultant MIP-NGs bind the template peptide and recombinant TNF-α with high affinity and selectivity, and can block the binding of TNF-α to its receptor. Consequently they were applied to neutralize pro-inflammatory TNF-α in the supernatant of human THP-1 macrophages, leading to a downregulation of the secretion of pro-inflammatory cytokines. Our results suggest that MIP-NGs, which are thermally and biochemically more stable and easier to manufacture than antibodies, and cost-effective, are very promising as next generation TNF-α inhibitors for the treatment of inflammatory diseases.

Boosting the resolution of multidimensional NMR spectra by complete removal of proton spin multiplicities.

Over decades multidimensional NMR spectroscopy has become an indispensable tool for structure elucidation of natural products, peptides and medium sized to large proteins. Heteronuclear single quantum coherence (HSQC) spectroscopy is one of the work horses in that field often used to map structural connectivity between protons and carbons or other hetero nuclei. In overcrowded HSQC spectra, proton multiplet structures of cross peaks set a limit to the power of resolution and make a straightforward assignment difficult. In this work, we provide a solution to improve these penalties by completely removing the proton spin multiplet structure of HSQC cross peaks. Previously reported sideband artefacts are diminished leading to HSQC spectra with singlet responses for all types of proton multiplicities. For sideband suppression, the idea of restricted random delay (RRD) in chunk interrupted data acquisition is introduced and exemplified. The problem of irreducible residual doublet splitting of diastereotopic CH2 groups is simply solved by using a phase sensitive JRES approach in conjunction with echo processing and real time broadband homodecoupling (BBHD) HSQC, applied as a 3D experiment. Advantages and limitations of the method is presented and discussed.

Molecularly Imprinted Polymer Nanogels for Protein Recognition: Direct Proof of Specific Binding Sites by Solution STD and WaterLOGSY NMR Spectroscopies.

Molecularly imprinted polymers (MIPs) are tailor-made synthetic antibodies possessing specific binding cavities designed for a target molecule. Currently, MIPs for protein targets are synthesized by imprinting a short surface-exposed fragment of the protein, called epitope or antigenic determinant. However, finding the epitope par excellence that will yield a peptide "synthetic antibody" cross-reacting exclusively with the protein from which it is derived, is not easy. We propose a computer-based rational approach to unambiguously identify the "best" epitope candidate. Then, using Saturation Transfer Difference (STD) and WaterLOGSY NMR spectroscopies, we prove the existence of specific binding sites created by the imprinting of this peptide epitope in the MIP nanogel. The optimized MIP nanogel could bind the epitope and cognate protein with a high affinity and selectivity. The study was performed on Hepatitis A Virus Cell Receptor-1 protein, also known as KIM-1 and TIM-1, for its ubiquitous implication in numerous pathologies.

Mirror symmetric broadband homodecoupled perfect echo spectroscopy.

A new experiment for recording phase sensitive ω1-broadband homodecoupled TOCSY spectra is presented. The method is an extension of the already existing perfect echo (PE) filter, proposed to sample t1 chemical shift under sustained homodecoupling. The modification is made by attaching a time reversed perfect echo filter to a regular perfect echo scheme. Thus it becomes possible to acquire for longer t1 acquisition times without compromising the quality of homodecoupling. The mirror symmetric double perfect echo is implemented into the evolution period of a TOCSY experiment. A spin lock pulse purges undesired dispersive antiphase components at the end of the central t1 evolution period. Pure absorptive lineshapes with reduced proton spin multiplicities are obtained. The approach can be used in conjunction with real or constant time chemical shift evolution. In case of compounds with reduced T2 relaxation time, the real time approach is advisable, where the echo delays are an extension of the t1 evolution period. In this way, an unnecessary loss due to T2 relaxation is avoided. Using the pulse sequence in constant time mode at high t1max values gives ω1-homodecoupled TOCSY spectra without a significant dependence of the transfer amplitude on J. All experiments were carried out using non uniform sampling to decrease the measurement time. Experimental setup, advantages and limitations are discussed.

Small angle double quantum spectroscopy (SAQS NMR).

A new experiment for recording double quantum spectra is introduced. The 2D DQ NMR experiment yields phase sensitive spectra with double quantum frequencies in F1. The appearance of remote peaks is vastly suppressed by using a small flip angle double quantum excitation and reconversion. Pulse sequences and phase sensitive processing are discussed. The complexity of the SQ antiphase magnetization given in larger proton spin networks could be reduced by using the option of band selective decoupling during the preparation period. In addition, an ACCORDION element is applied by incrementing the J evolution delay in concert with the t1 period. With this the excitation of double quantum coherence over a wider range of J values is achieved. A broadband homodecoupled version of the DQ experiment is proposed, where correlation peaks with singlet response at F2 chemical shifts and double quantum frequencies in F1 are obtained. We call this experiment Small Angle double Quantum Spectroscopy SAQS NMR.

Svetamycins A-G, Unusual Piperazic Acid-Containing Peptides from Streptomyces sp.

Seven new halogenated peptides termed svetamycins A-G (1-7) have been isolated from laboratory cultures of a Streptomyces sp. Svetamycins A-D, F, and G are cyclic depsipeptides, whereas svetamycin E is a linear analogue of svetamycin C. Their structures were determined using extensive spectroscopic analysis, and their stereochemical configuration was established by a combination of NMR data, quantum mechanical calculations, and chemical derivatizations. Svetamycins are characterized by the presence of a hydroxyl acetic acid and five amino acids including a rare 4,5-dihydroxy-2,3,4,5-tetrahydropyridazine-3-carboxylic acid, a γ-halogenated piperazic acid, and a novel δ-methylated piperazic acid in svetamycins B-C, E, and G. Moreover, isotope-labeled substrate feeding experiments demonstrated ornithine as the precursor of piperazic acid and that methylation at the δ position of the piperazyl scaffold is S-adenosyl-l-methionine (SAM)-dependent. Svetamycin G, the most potent antimicrobial of this suite of compounds, inhibited the growth of Mycobacterium smegmatis with an MIC80 value of 2 µg/mL.

Faster and cleaner real-time pure shift NMR experiments.

Real-time pure shift experiments provide highly resolved proton NMR spectra which do not require any special processing. Although being more sensitive than their pseudo 2D counterparts, their signal intensities per unit time are still far below regular NMR spectra. In addition, scalar coupling evolution during the individual data chunks produces decoupling sidebands. Here we show that faster and cleaner real-time pure shift spectra can be obtained through the implementation of two parameter alterations. Variation of the FID chunk lengths between individual transients significantly suppresses decoupling sidebands for any kind of real-time pure shift spectra and thus allows for example the analysis of minor components in compound mixtures. Shifting the excitation frequency between individual scans of real-time slice-selective pure shift spectra increases their sensitivity obtainable in unit time by allowing faster repetitions of acquisitions.

A different approach to multiplicity-edited heteronuclear single quantum correlation spectroscopy.

A new experiment for recording multiplicity-edited HSQC spectra is presented. In standard multiplicity-edited HSQC experiments, the amplitude of CH2 signals is negative compared to those of CH and CH3 groups. We propose to reverse the sign of (13)C frequencies of CH2 groups in t1 as criteria for editing. Basically, a modified [BIRD](r,x) element (Bilinear Rotation Pulses and Delays) is inserted in a standard HSQC pulse sequence with States-TPPI frequency detection in t1 for this purpose. The modified BIRD element was designed in such a way as to pass or stop the evolution of the heteronuclear (1)JHC coupling. This is achieved by adding a 180° proton RF pulse in each of the 1/2J periods. Depending on their position the evolution is switched on or off. Usually, the BIRD- element is applied on real and imaginary increments of a HSQC experiment to achieve the editing between multiplicities. Here, we restrict the application of the modified BIRD element to either real or imaginary increments of the HSQC. With this new scheme for editing, changing the frequency and/or amplitude of the CH2 signals becomes available. Reversing the chemical shift axis for CH2 signals simplifies overcrowded frequency regions and thus avoids accidental signal cancellation in conventional edited HSQC experiments. The practical implementation is demonstrated on the protein Lysozyme. Advantages and limitations of the idea are discussed.

Improving the sensitivity of conventional spin echo spectra by preservation of initial signal-to-noise ratio.

A simple processing strategy is introduced to enhance the spectral quality and signal-to-noise ratio in conventional J resolved spectra. The idea of pseudo echo filtering is extended to conserve the primary signal-to-noise, predominating at the beginning of the FID in the indirect dimension. This is achieved by matching the maximum amplitude of the FID with that of the sine window function. Practically, the FID is right shifted by the number of acquired points in the indirect dimension; missing data points are backward predicted and finally multiplied with the unshifted sine window function. Standard processing tools are employed for this purpose. The results of data processing using different window functions with and without right shifts and back predictions are discussed. The signal-to-noise ratio of the J resolved spectrum is increased by a factor of 6 compared to standard data processing using pseudo echo filtering alone.

Broadband homodecoupled NMR spectroscopy with enhanced sensitivity.

A new type of broadband homodecoupling technique is described, which is based on the original version of the Zangger-Sterk experiment, but results in a spectrum with higher sensitivity. The homodecoupling is performed by a combination of selective and non-selective 180° RF pulses in the presence of weak rectangular pulsed field gradients in a pseudo 2D experiment. The proposed experiment uses a fast pulsing approach to increase the signal-to-noise ratio per unit time. The recycle delay is significantly shortened typically to about 100 ms. After each scan, the offset of the selective shaped pulse is changed to access fresh magnetisation from adjacent frequency/spatial regions. The physical acquisition time was limited to 40 ms to keep the total length of the pulse sequence as short as possible. Broadband inversion BIP pulses are used instead of 180° hard pulses. They are used pairwise to cancel out unwanted phase shifts over the bandwidth. Reconstruction of the homodecoupled spectrum was done by concatenating the first 10 ms of the FID from each single increment to obtain the final homodecoupled proton FID followed by Fourier transformation. The new method can either be used to acquire broadband homodecoupled spectra in a shorter time or to increase the signal-to-noise ratio compared to the original Zangger-Sterk experiment. Using eight different frequencies can thus lead to a signal to noise gain of a factor √8 or a factor of eight in time.

Monitoring fast reactions by spatially-selective and frequency-shifted continuous NMR spectroscopy: application to rapid-injection protein unfolding.

The repetition rate of an NMR experiment is usually limited by the longitudinal relaxation times of the investigated molecule. Here we show that continuous excitation and data acquisition, without any interscan delay, is possible by a spatially resolved experiment where different nuclei are excited in consecutive scans.

Broadband homodecoupled heteronuclear multiple bond correlation spectroscopy.

A general concept for removing proton-proton scalar J couplings in 2D NMR spectroscopy is proposed. The idea is based on introducing an additional J resolved dimension into the pulse sequence of a conventional 2D experiment to design a pseudo 3D NMR experiment. The practical demonstration is exemplified on the widely used gradient coherence selected heteronuclear long-range correlation spectroscopy (HMBC). We refer to this type of pulse sequence as tilt HMBC experiment. For every (13)C chemical shift evolution increment, a homonuclear J resolved experiment is recorded. The long-range defocusing delay of the HMBC pulse sequence is exploited to implement this building block. The J resolved evolution period is incremented in a way very similar to ACCORDION spectroscopy to accommodate the buildup of heteronuclear long-range antiphase magnetisation as well. After Fourier transformation in all dimensions the spectra are tilted in the J resolved dimension. Finally, a projection along the J resolved dimension is calculated leading to almost disappearance of proton-proton spin multiplicities in the 2D tilt HMBC spectrum. The tilt HMBC experiment combines sensitivity with simple experimental setup and can be recorded with short recycle delays, when combined with Ernst angle excitation. The recorded spectra display singlet proton signals for long-range correlation peaks making an unambiguous signal assignment much easier. In addition to the new experiment a simple processing technique is applied to efficiently suppress the noise originating from forward linear prediction in the indirect evolution dimensions. In case of issues with fast repetition times, probe heating and RF power handling most of the RF pulses can be replaced by broadband, frequency swept pulses operating at much lower power.

Experimental access to HSQC spectra decoupled in all frequency dimensions.

A new operator called RESET "Reducing nuclEar Spin multiplicitiEs to singuleTs" is presented to acquire broadband proton decoupled proton spectra in one and two dimensions. Basically, the homonuclear decoupling is achieved through the application of bilinear rotation pulses and delays. A [BIRD](r,x) pulse building block is used to selectively invert all proton magnetization remotely attached to (13)C isotopes, which is equivalent to a scalar J decoupling of the protons directly attached to (13)C from all other protons in the spin system. In conjunction with an appropriate data processing technique pure shift proton spectra are obtained. For this purpose, the concept of constant time acquisition in the observe dimension is exploited. Both ideas were merged together producing superior HSQC based pseudo 3D pulse sequences. The resulting HSQC spectra show cross peaks with collapsed multiplet structures and singlet responses for the proton chemical shift frequencies. An unambiguous assignment of signals from overcrowded spectra becomes much easier. Finally, the recently introduced SHARC technique is exploited to enhance the capability of the scalar J decoupling method. A significant reduction of the total measurement time is achieved. The time is saved by reducing the number of (13)C chemical shift evolution increments and working with superimposed narrow spectral bandwidths in the (13)C indirect domain.

An alternative approach for recording of multidimensional NMR data based on frequency dependent folding mechanism.

A new scheme for obtaining HSQC spectra with improved resolution or in a shorter time called SHARC (Shaped Arrayed data aCquisition protocol) is proposed, which uses region selective RF pulses and allows the sweep width to be adjusted individually for each region. It thus bypasses the problems with the Nyquist theorem associated with other method suggested for this purpose. Assignment of the cross-peaks to their respective region is achieved by manipulating the phases of the RF pulses and/or their frequencies. SHARC NMR can be applied without any previous knowledge of the chemical shift distribution, but can be further optimized on the basis of a quick overview spectrum.

Convenient synthesis of multifunctional EDTA-based chiral metal chelates substituted with an S-mesylcysteine.

We describe the synthetic route to ethylenediaminetetraacetic acid (EDTA) derivatives that can be attached to surface-exposed thiol functional groups of cysteine residues in proteins, via a methylthiosulfonate moiety that is connected in a stereochemically unique way to the C-1 carbon atom of EDTA. Such compounds can be used to align proteins in solution without the need to add liquid crystalline media, and are, therefore, of great interest for the NMR spectroscopic analysis of biomolecules. The binding constant for the paramagnetic tag to lanthanide ions was determined by measuring luminescence. For the Tb(+3)-ligand complex, a K(b) value of 6.5 x 10(17) M(-1) was obtained. This value is in excellent agreement with literature values for the related EDTA compound. In addition, it could be shown that there is no significant reduction in the luminescence intensity upon addition of a 10(4) excess of Ca2+ ions, indicating that this paramagnetic tag is compatible with buffers containing high concentrations of divalent alkaline earth ions.

Novel techniques for weak alignment of proteins in solution using chemical tags coordinating lanthanide ions.

A molecule with an anisotropic magnetic susceptibility is spontaneously aligned in a static magnetic field. Alignment of such a molecule yields residual dipolar couplings and pseudocontact shifts. Lanthanide ions have recently been successfully used to provide an anisotropic magnetic susceptibility in target molecules either by replacing a calcium ion with a lanthanide ion in calcium-binding proteins or by attaching an EDTA derivative to a cysteine residue via a disulfide bond. Here we describe a novel enantiomerically pure EDTA derived tag that aligns stronger due to its shorter linker and does not suffer from stereochemical diversity upon lanthanide complexation. We observed residual (15)N,(1)H-dipolar couplings of up to 8 Hz at 800 MHz induced by a single alignment tensor from this tag.

Measurement of long range H,C couplings in natural products in orienting media: a tool for structure elucidation of natural products.

In this paper we show that water insoluble compounds dissolved in poly-gamma-benzyl-glutamate are amenable to the measurement of a number of homo- and heteronuclear dipolar couplings. The sensitivity and experimental precision of dipolar couplings are sufficient to obtain a good match with the structure. In order to achieve the necessary precision for H,C dipolar couplings between protons and carbons that are not directly bound a new method for the measurement of heteronuclear long range couplings is introduced that allows a one-parameter fit to a HSQC-based experiment as reference experiment. The methodology is applied to menthol (1R, 3S, 4R).