C. Griesinger

A general enhancement scheme in heteronuclear multidimensional NMR employing pulsed field gradients.

SummaryGeneral pulse sequence elements that achieve sensitivity-enhanced coherence transfer from a heteronucleus to protons of arbitrary multiplicity are introduced. The building blocks are derived from the sensitivity-enhancement scheme introduced by Cavanagh et al. ((1991) J. Magn. Reson., 91, 429–436), which was used in conjunction with gradient coherence selection by Kay et al. ((1992) J. Am. Chem. Soc., 114, 10663–10665), as well as from a multiple-pulse sequence effecting a heteronuclear planar coupling Hamiltonian. The building blocks are incorporated into heteronuclear correlation experiments, in conjunction with coherence selection by the formation of a heteronuclear gradient echo. This allows for efficient water suppression without the need for water presaturation. The methods are demonstrated in HSQC-type experiments on a sample of a decapeptide in H2O. The novel pulse sequence elements can be incorporated into multidimensional experiments.

Transformation of homonuclear two-dimensional NMR techniques into one-dimensional techniques using Gaussian pulses

Abstract A general procedure is proposed, which allows the transtormation of most of the homonuclear two-dimensional NMR techniques into one-dimensional sequences by using semiselective Gaussian pulses. These techniques are particularly advantageous in cases where a limited amount of information is required for solving a chemical problem, e.g., a few connectivities, coupling constants, or NOE values. Applications of 1D COSY, 1D NOESY, 1D homonuclear relayed techniques, and 1D TOCSY are demonstrated. Furthermore, a variant of the ID COSY, the refocused ID COSY with z filter is suggested, which in combination with 1 D COSY allows the application of DISCO for the determination of coupling constants from multiplets in crowded regions. The 1 D NOESY sequence allows the measurement of NOES for very short mixing times and thus provides reliable values for buildup rates of transient NOEs. Selective excitation of a certain resonance may be impossible by conventional techniques, because it lies in a crowded region. In such cases by exploiting the J dependence of transfer functions (directed magnetization transfer) magnetization can be transferred selectively from a coupled nucleus to the one of interest. Its NMR parameters (J coupling, NOE) are then accessible via relayed COSY or relayed NOESY.

A simultaneous 15N,1H- and 13C,1H-HSQC with sensitivity enhancement and a heteronuclear gradient echo

SummaryNew pulse sequences are introduced and discussed that allow for simultaneous acquisition of 15N,1H-and 13C,1H-HSQC correlations for fully 13C,15N-labeled biomacromolecules in combination with hetero-nuclear gradient echoes and sensitivity enhancement. The pulse sequence experimentally found to be optimal can be used as a building block, especially in time-consuming multidimensional NMR experiments. Due to the excellent solvent suppression obtained by employing heteronuclear gradient echoes, which allows detection of resonances under the water resonance, it would be possible to record two sensitivity-enhanced 4D experiments simultaneously on one sample dissolved in H2O, e.g. a 4D 13C,1H-HSQC-NOESY-15N, 1H/13C,1H-HSQC.

Differentiation of HMBC two- and three-bond correlations: A method to simplify the structure determination of natural products

Abstract The structure elucidation of natural products, today, relies heavily on the application of proton-detected heteronuclear NMR experiments. Perhaps the most useful of these methods is the HMBC experiment, which provides correlations between protons and carbons over two and three bonds. The application of the HMBC method for the direct translation of H,C correlations to yield bonding information is limited, however, by the fact that it does not distinguish between 2 J CH and 3 J CH correlations. Reported here is an application of the recently described 1,1-ADEQUATE experiment that yields only two bond H,C connectivities in HCC moieties and therefore allows the differentiation of HMBC two- and three-bond correlations. The method is demonstrated on a 14 mg sample of a new marine natural product, 5,6-dihydro lamellarin H ( 1 ). An NMR methodology is presented which allows the differentiation of two- and three-bond correlation in HMBC spectra. The ω 1 -refocused 1,1-ADEQUATE is used to obtain exclusively two-bond correlations which thereby facilitates the assignment of the constitution of complex structures. The method is demonstrated on a new polycyclic marine natural product, 5,6-dihydro lamellarin H.

Novel strategies for sensitivity enhancement in heteronuclear multi—dimensional NMR experiments employing pulsed field gradients

SummaryNovel strategies for sensitivity enhancement in heteronuclear multidimensional spectra are introduced and evaluated theoretically and experimentally. It is shown that in 3D sequences employing several Coherence Order Selective Coherence Transfer (COS-CT) steps, enhancement factors of up to 2 can be achieved. This sensitivity enhancement is compatible with the use of heteronuclear gradient echoes, yielding spectra with excellent water suppression. HNCO and HCCH-TOCSY pulse sequences are proposed and experimentally tested. These experiments employ recently developed coherence order selective pulse sequence elements, e.g., COS-INEPT and planar TOCSY for antiphase to in-phase transfers 2F-S2↔S- or in-phaseaCOS-CT for in-phase transfer F-↔S-, and the well-known isotropic TOCSY mixing sequences for homo- and heteronuclear in-phase transfer.

Determination of a complete set of coupling constants in 13C-labeled oligonucleotides.

SummaryThree experiments are introduced to determine a complete set of coupling constants in RNA oligomers. In the HCCH-E.COSY experiment, the vicinal proton-proton coupling constants can be measured with high accuracy. In the P-FIDS-CT-HSQC experiment, vicinal proton-phosphorus and carbon-phosphorus couplings are measured that depend on the phosphodiester backbone torsion angles β and ε. In the refocussed HMBC experiment, vicinal carbon-proton couplings are measured that depend on the glycosidic torsion angle χ.

Chemical synthesis of 13C-labelled monomers for the solid-phase and template controlled enzymatic synthesis of DNA and RNA oligomers

The preparation of 13C-labelled ribonucleosides starting from [13C6]-glucose 1 and the corresponding nucleobases 5a-e or 6a-e (N6-benzoyl-adenine, N2-acetyl-guanine, N4-benzoyl-cytosine, uracil and thymine) in 47 – 66% overall yield is described. Their subsequent transformation into 5′-O-dimethoxytrityl protected DNA-phosphoramidites and 5′-O-dimethoxytrityl-2′-O-trialkylsilyl protected RNA-phosphor-amidites for the solid phase synthesis of DNA- and RNA-oligomers and to 5′-O-ribo- and deoxyribo-nucleosidetriphosphates for template controlled enzymatic synthesis (polymerase- or reverse transcriptase reaction) has been carried out.

Determination of HN,H? and HN,C? coupling constants in 13C, 15N-labeled proteins

SummarySensitive three-dimensional NMR experiments, based on the E.COSY principle, are presented for the measurement of the 3J(HN,Hα) and 3J(HN,C′) coupling constants in uniformly 13C- and 15N-labeled proteins. They employ gradient coherence selection in combination with the sensitivity enhancement method in HSQC-type spectra (Cavanagh et al., 1991; Palmer et al., 1991). In most cases, the two measured coupling constants unambiguously define the ϕ-angle for protein structure determination. The method is applied to uniformly 13C, 15N-labeled ribonuclease T1.

The measurement of heteronuclear transverse relaxation times in ax3 spin systems via polarization-transfer techniques

Abstract Pulse schemes for the measurement of 13C transverse relaxation times in AX33 spin systems are described which make use of the sensitive 1H spin for detection. The experiments are based on reverse-DEPT and reverse-INEPT polarization-transfer sequences. It is shown that relaxation rates obtained from 13C-direct-observe and from polarization-transfer experiments are identical only if magnetization from each of the 13C transitions is transferred equally to the detected 1H spins. This requires judicious choices in pulse angles and delays in reverse-DEPT and reverse-INEPT experiments. For application to macromolecules, experimental and theoretical results suggest that polarization-transfer schemes based on reverse INEPT are superior to reverse-DEPT-based sequences and give results which are in good agreement with values measured via 13C-observe methods.

PLUSH TACSY: Homonuclear planar TACSY with two-band selective shaped pulses applied to Cα,C′ transfer and Cβ,Caromatic correlations

SummaryA new homonuclear Hartmann-Hahn-type mixing scheme is introduced that effects coherence transfer between resonances in two separated frequency bands. The mixing scheme relies on the irradiation of two-band selective shaped pulses that are expanded in an MLEV-16 supercycle. Similar to heteronuclear Hartmann-Hahn experiments, a planar effective coupling tensor is created. This novel mixing scheme is applied to Cα,C′ transfer and to the transfer between Cβ and aromatic carbon spins.