Computer-optimized decoupling scheme for wideband applications and low-level operation

Shaka, A.J.; Barker, Peter B.; Freeman, Ray

doi:10.1016/0022-2364(85)90122-2

Cited by 871 publications

(808 citation statements)

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“…The HSQC pulse sequence included the TANGO block to suppress signals from N14-coupled protons (22) and 15 N-decoupling during proton acquisition, using a 2.1 kHz field strength and the GARP decoupling scheme (23). The HMBC pulse sequence included a low-pass J filter tuned to exclude one-bond couplings of 90 Hz and to include long-range couplings of 4−5 Hz (20); it was acquired without 15 N-decoupling to further suppress one-bond coupling cross peaks.…”

Section: Synthesis Of Model Compoundsmentioning

confidence: 99%

Electronic and Nuclear Magnetic Resonance Spectroscopic Features of the 1‘,4‘-Iminopyrimidine Tautomeric Form of Thiamin Diphosphate, a Novel Intermediate on Enzymes Requiring This Coenzyme

2006

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Appropriate compounds were synthesized to create models for the 1',4'-imino tautomer of the 4'-aminopyrimidine ring of thiamin diphosphate recently found to exist on the pathway of enzymatic reactions requiring this cofactor (Jordan, F., and Nemeria, N.S. (2005) Bioorganic Chemistry, 33, 190−215). The N1-methyl-4-aminopyrimidinium compounds synthesized on treatment with a strong base produce the 1,4-imino tautomer whose UV spectrum indicates a maximum between 300−320 nm, depending on the absence or presence of a methyl group at the 4-amino nitrogen. The λ max found is in the same wavelength range as the positive circular dichroism band observed on several enzymes and showed a very strong dependence on solvent dielectric constant. To help with the 15 N chemical shift assignments, the model compounds were specifically labeled with 15 N at the amino nitrogen atom. The chemical shift of the amino nitrogen was deshielded by N1-methylation, then dramatically further deshielded by more than 100 ppm on formation of the 1,4-iminopyrimidine tautomer. Both the UV spectroscopic values and the 15 N chemical shift for the 1,4-iminopyrimidine tautomer should serve as useful guides to the assignment of enzyme-bound signals.The notion that the 4'-aminopyrimidine group of thiamin diphosphate (ThDP) undergoes tautomerization to the 1',4'-imino form (1',4'-iminoThDP) during the catalytic cycle of enzymes that utilize it has gained wider acceptance since the appearance of X-ray crystal structural data (1). The role and likelihood of tautomerization is suggested by two totally conserved structural features on all ThDP enzymes: (a) the V coenzyme conformation ensuring that the C2 thiazolium atom and the N4' atom of the 4'-aminopyrimidine ring are within less than 3.5Å from each other (2), potentially enabling the 1',4'-imino tautomer to participate in proton transfers; and (b) the presence of a glutamate within hydrogen bonding distance of the N1' atom of the 4-aminopyrimidine ring, as a potential catalyst for the tautomerization (Scheme 1, as exemplified with the reaction of yeast pyruvate decarboxylase, YPDC). As illustrated in the active site structure of YPDC in Figure 1, the residue E51 probably carries out this function (3). Chemical evidence for the importance of the 4'-aminopyrimidine moiety of ThDP in catalysis was obtained from ThDP analogues in which one or another nitrogen atom was replaced by carbon (4), while a model for activation of the ring for catalysis via tautomerization by N1-protonation was suggested from our laboratories (5-7). Notwithstanding the attractive features of this hypothesis, until recently no direct spectroscopic or structural evidence was available on any ThDP enzymes for the presence of the 1',4'-imino tautomer. In rapid-scan stopped flow experiments, mixing slow active-center variants of YPDC with pyruvate, a UV # Supported by National Institutes of Health grants GM050380 and GM062330. In these examples, the keto group of the substrate analog phosphonate forms a covalent adduct with the t...

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Section: Synthesis Of Model Compoundsmentioning

confidence: 99%

Electronic and Nuclear Magnetic Resonance Spectroscopic Features of the 1‘,4‘-Iminopyrimidine Tautomeric Form of Thiamin Diphosphate, a Novel Intermediate on Enzymes Requiring This Coenzyme

2006

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“…CARP decoupling was applied on the "N channel during acquisition (Shaka et al, 1985). Phase cycling: 4 I, 4(x), 4 ( -x ) ; 42, x, "x; 43, 2(x), 2( -x); receiver, x, -x, -x, x.…”

Section: Identification Of the Imidazole 'H And "N Resonances Of His 49mentioning

confidence: 99%

“…CARP decoupling of 15N was performed during acquisition (Shaka et al, 1985). Time-proportional phase incrementation was used to obtain quadrature detection in all indirectly detected dimensions of 2D and 3D experiments.…”

Section: Nmr Spectroscopymentioning

confidence: 99%

Redox‐dependent dynamics of putidaredoxin characterized by amide proton exchange

Lyons¹,

Ratnaswamy²,

Pochapsky³

1996

Protein Science

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Multidimensional NMR methods were used to obtain 'H-I'N correlations and "N resonance assignments for amide and side-chain nitrogens of oxidized and reduced putidaredoxin (Pdx), the Fe2S2 ferredoxin, which acts as the physiological reductant of cytochrome P-450,,, (CYPlOl). A model for the solution structure of oxidized Pdx has been determined recently using NMR methods (Pochapsky TC, Ye XM, Ratnaswamy G, Lyons TA, 1994, Biochemistry 33:6424-6432) and redox-dependent ' H NMR spectral features have been described (Pochapsky TC, Ratnaswamy G, Patera A, 1994, Biochemistry33:6433-6441). "N assignments were made with NOESY-('H/''N) HMQC and TOCSY-('H/''N) HSQC spectra obtained using samples of Pdx uniformly labeled with "N. Local dynamics in both oxidation states of Pdx were then characterized by comparison of residue-specific amide proton exchange rates, which were measured by a combination of saturation transfer and H 2 0 / D 2 0 exchange methods at pH 6.4 and 7.4 (uncorrected for isotope effects). In general, where exchange rates for a given site exhibit significant oxidation-state dependence, the oxidized protein exchanges more rapidly than the reduced protein. The largest dependence of exchange rate upon oxidation state is found for residues near the metal center and in a region of compact structure that includes the loop-turn Val 74-Ser 82 and the C-terminal residues (Pro 102-Trp 106). The significance of these findings is discussed in light of the considerable dependence of the binding interaction between Pdx and CYPlOl upon the oxidation state of Pdx.

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“…In general, once the acquisition and processing parameters have been established for any qHNMR experiment, either a parameter set or a set-up macro may be created on the NMR spectrometer, which can then be used to initiate and control the experiment in a much more routine manner and typically require little or no modification to the acquisition parameters (potential exceptions are the use of unusual solvents and/or relaxation behavior of the analyte). Two instrumental conditions distinguish the proposed qHNMR approach from a "normal" survey proton NMR experiment: (i) the qHNMR data are obtained non-spinning, and (ii) the qHNMR spectra are acquired with inverse gated 13 C decoupling using a GARP (Globally-optimized Alternating-phase Rectangular Pulses) 8 decoupling scheme. All additional key factors that have to be taken into account when establishing a routine qHNMR experimental protocol are discussed in the following.…”

Section: A Cookbook Approach To Qhnmrmentioning

confidence: 99%

A Routine Experimental Protocol for qHNMR Illustrated with Taxol

2007

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Quantitative 1 H NMR (qHNMR) provides a value-added dimension to the standard spectroscopic data set involved in structure analysis, especially when analyzing bioactive molecules and elucidating new natural products. The qHNMR method can be integrated into any routine qualitative workflow without much additional effort by simply establishing quantitative conditions for the standard solution 1 H NMR experiments. Moreover, examination of different chemical lots of taxol and a Taxus brevifolia extract as working examples led to a blueprint for a generic approach to performing a routinely practiced 13 C-decoupled qHNMR experiment, and for recognizing its potential and main limitations. The proposed protocol is based on a newly assembled 13 C GARP broadband decoupled proton acquisition sequence that reduces spectroscopic complexity by removal of carbon satellites. The method is capable of providing qualitative and quantitative NMR data simultaneously and covers various analytes from pure compounds to complex mixtures such as metabolomes. Due to a routinely achievable dynamic range of 300:1 (0.3%) or better, qHNMR qualifies for applications ranging from reference standards to biologically active compounds to metabolome analysis. Providing a "cookbook" approach to qHNMR, acquisition conditions are described that can be adapted for contemporary NMR spectrometers of all major manufacturers. The world's pool of natural products plays an important role as an (in)exhaustible resource for evolutionary-shaped molecules. Natural products are valuable research tools, which in part is due to their biological potency (see comprehensive reviews [1][2][3][4] ). When natural products are used as biomedical agents, and in consistency with the pharmacophore model, it is the combination of their specific chemical structure and/or reactivity that forms their relationship with a biological target and ultimately defines their essential structural features. Consequently, chemical constitution plays a key role in biological activity, and, therefore, all structure related information obtainable from a biologically active agent is by default relevant. Ultimately, any variation of structural parameters has the potential to introduce variations in biological activity. This relationship holds regardless of the magnitude of the biological perturbation, i.e., whether there is slight or a substantial change in potency, or even an alteration in the type of biological response. The well-documented subtleties of mammalian hormonal steroids can serve as a distinguished example in this regard.⊥ Dedicated to Dr. Norman R. Farnsworth on the occasion of his 77 th birthday.* To whom correspondence should be addressed. Tel (312) 355-1949355- . Fax (312)-355-2693. gfp@uic.edu. Supporting Information Available.This material is available free of charge via the Internet at http://pubs.acs.org. Further information on qNMR methodology, instrument parameter files for the NMR major manufacturer's spectrometers, and original FIDs of the taxol qHNMR experime...

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Computer-optimized decoupling scheme for wideband applications and low-level operation

Cited by 871 publications

References 7 publications

Electronic and Nuclear Magnetic Resonance Spectroscopic Features of the 1‘,4‘-Iminopyrimidine Tautomeric Form of Thiamin Diphosphate, a Novel Intermediate on Enzymes Requiring This Coenzyme

Electronic and Nuclear Magnetic Resonance Spectroscopic Features of the 1‘,4‘-Iminopyrimidine Tautomeric Form of Thiamin Diphosphate, a Novel Intermediate on Enzymes Requiring This Coenzyme

Redox‐dependent dynamics of putidaredoxin characterized by amide proton exchange

A Routine Experimental Protocol for qHNMR Illustrated with Taxol

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