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.

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.

Tetrahydroisoquinoline-3-carboxylate based matrix-metalloproteinase inhibitors: design, synthesis and structure-activity relationship.

The design, synthesis and structure-activity relationship (SAR) of a series of nonpeptidic 2-arylsulfonyl-1,2,3,4-tetrahydro-isoquinoline-3-carboxylates and-hydroxamates as inhibitors of the matrix metalloproteinase human neutrophil collagenase (MMP-8) is described here. Based on available X-ray structures of MMP-8/inhibitor complexes, our structure-based design strategy was directed to complement major protein-ligand interaction regions mainly in the S1' hydrophobic specificity pocket close to the catalytic zinc ion. Here, the rigid 1,2,3,4-tetrahydroisoquinoline scaffold (Tic) provides ideal geometry to combine hydroxamates and carboxylates as typical zinc complexing functionalities, with a broad variety of S1' directed mono- and biaryl substituents consisting of aromatic rings perfectly accommodated within this more hydrophobic region of the MMP-8 inhibitor binding site. The effect of different S1' directed substituents, zinc-complexing groups, chirality and variations of the tetrahydroisoquinoline ring-system is investigated by systematic studies. X-ray structure analyses in combination with 3D-QSAR studies provided an additional understanding of key determinants for MMP-8 affinity in this series. The hypothetical binding mode for a typical molecule as basis for our inhibitor design was found in good agreement with a 1.7 A X-ray structure of this candidate in complex with the catalytic domain of human MMP-8. After analysis of all systematic variations, 3D-QSAR and X-ray structure analysis, novel S1' directed substituents were designed and synthesized and biologically evaluated. This finally results in inhibitors, which do not only show high biological affinity for MMP-8, but also exhibit good oral bioavailability in several animal species.