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Rapid 3D NMR using the filter diagonalization method

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Rapid 3D NMR using the filter diagonalization method: application to oligosaccharides derivatized with 13C-labeled acetyl groupsIntroductionTheoryExperimentalResults and discussionAcknowledgementsReferencesRapid 3D NMR using the filter diagonalization method:application to oligosaccharides derivatized with13C-labeledacetyl groupsGeoffrey S. Armstrong,a,1Kristin E. Cano,aVladimir A. Mandelshtam,aA.J. Shaka,aand Brad Bendiakb,*aChemistry Department, University of California, Irvine, CA 92697-2025, USAbDepartment of Cell and Developmental Biology and Biomolecular Structure Program, University of Colorado Health Sciences Center,Box B111, 4200 East Ninth Avenue, Denver, CO 80262, USAReceived 7 April 2004; revised 4 June 2004Available online 20 July 2004AbstractRapid 3D NMR spectroscopy of oligosaccharides having isotopically labeled acetyl ‘‘isotags’’ was made possible with highresolution in the indirect dimensions using the filter diagonalization method (FDM). A pulse sequence was designed for the optimalcorrelation of acetyl methyl protons, methyl carbons, and carbonyl carbons. The multi-dimensional nature of the FDM, coupledwith the advantages of constant-time evolution periods, resulted in marked improvements over Fourier transform (FT) and mirror-image linear prediction (MI-LP) processing methods. The three methods were directly compared using identical data sets. A highlyresolved 3D spectrum was achieved with the FDM using a very short experimental time (28 min).Ó 2004 Elsevier Inc. All rights reserved.Keywords: Filter diagonalization method; Multi-dimensional NMR; Constant-time; C-13 labeling; O-acetylation; Oligosaccharides1. IntroductionIn the quest to provide detailed structural and dy-namic information about macromolecules of ever-largersize and complexity, the greatest single limitation inNMR spectroscopy is the overlap of cross peaks. Atsome upper size limit, the assignment of many signalsand deduction of their associated spin systems simplybecomes impossible. Procedures for diminishing crosspeak area or volume, such as decoupling in indirect ordirect dimensions, tROSY [1], or extracting ‘‘singlet’’spectra using appropriate projections of specific experi-ments [2] do improve the situation significantly, but byfar the most potent improvement in spectral dispersionensues by extendi ng experiments to additional dimen-sions. Such extensions do engender issues of concern.These include time limitations when increasing thenumber of increments in indirect dimensions, lower re-sultant resolution in indirect dimensions using standardprocessing methods, and diminishing returns in signalintensity during multiple concatenated magnetizationtransfers and indirect detection periods.The filter diagonalization method (FDM) [3–7] hasemerged as a powerful tool for the analys is of higherdimensionality experiments. In contrast to the Fouriertransform (FT), which must be applied sequentially toorthogonal dimensions, the FDM is a true multi-di-mensional method. The FDM is able to process thecomplete data set, taking advantage of the large signal‘‘area’’ present in multi-dimensional experiments.However, not all pulse sequences are appropriate for theFDM. Therefore, the conjoint design of new pulse se-quences with FDM processing as a built-in goal must beconsidered for optimizing its potential. In particular, the*Corresponding author. Fax: 1-303-315-4729.E-mail address: [email protected] (B. Bendiak).1Present address: Department of Cell and Developmental Biology,University of Colorado Health Sciences Center, Box B111, 4200 EastNinth Avenue, Denver, CO 80262, USA.1090-7807/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.jmr.2004.06.002Journal of Magnetic Resonance 170 (2004) 156–163www.elsevier.com/locate/jmrFDM has been used in the analysis of a 3D HNCOexperiment that was obtaine d with only a few incre-ments in both indirect dimensions [8]. The successfulapplication of the FDM to proteins is just one illustra-tion of its fundamental advantage s that may be broughtto bear on any group of molecules with aptly arrangednuclei.Investigating the role of complex carbohydrates incellular function is a rapidly growing field of study. It isnow evident that this class of molecules is responsiblefor numerous types of intercellular recognition processes[9]. Disruption of these processes can have far reachingconsequences, and result in various hereditary afflictionsand oncologic events [10]. Unfortunately, elucidation ofthe structure of oligosaccharides is complicated by theneed to define the stereochemistry of very closely relatedmonosaccharide units, and the plethora of configura-tions that arise from different linkages between theseunits. From the perspective of NMR spectroscopy, theyare complicated by a very crowded proton spectrumeven for small carbohydrates and isotopically labeledoligosaccharides are usually not available. Furthermore,the issues that confound structural assignment of car-bohydrates cannot draw on pulse sequences employedfor resolving protein structure. New NMR methodsare necessary, tailored to the unique features of thesemolecules.A method to alleviate these problems has been de-scribed previously [11,12]. It involves the peracetylationof oligosaccharides with doubly13C-labeled acetylgroups (one of a group of derivatives that we havetermed ‘‘isotags’’). This provides several advantages tostructural elucidation of carbohydrates, but the mostimportant are first, that the spectral range of the sugarring protons is increased almost three-fold. Second, the13C-labeled acetyl groups afford a point of entry formagnetization to be transferred into the saccharideunits, allowing for analysis of the coupling networksthrough multi-dimensional correlations to acetyl protonand carbon frequencies. Third, the through-bond cou-pling of the acetyl carbonyl group to the sugar ringproton at the site of acetylation identifies all positionsthat were formerly free hydroxyl groups. By inference,this enables the sit e of a glycosidic linkage to be deter-mined unambiguously, as positions of acetylation andglycosylation are mutually exclusive. However, as withother macromolecules, the upper limit on the number ofspin systems that may be simultaneously elucidated isprimarily restricted by the overlap of crosspeaks.Previously, the correlation of the acetyl proton andcarbon frequencies to sugar ring proton frequencies wascarried


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