The CLIP/CLAP-HSQC: Pure absorptive spectra for the measurement of one-bond couplings Andreas Enthart a , J. Christoph Freudenberger a,b , Julien Furrer a,c , Horst Kessler a , Burkhard Luy a, * a Munich Center for Integrated Protein Science, Department Chemie, Organische Chemie II, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany b Currenta GmbH & Co. OHG, SER-ANT Spektroskopie, Geb. Q18-2, 51368 Leverkusen, Germany c Institut de Chimie, Université de Neuchâtel, Avenue de Belleveaux 51, CH-2009 Neuchâtel, Switzerland article info Article history: Received 6 December 2007 Revised 28 February 2008 Available online 21 March 2008 Keywords: RDCs Partial alignment HSQC Pure absorption IPAP abstract Heteronuclear residual dipolar one-bond couplings of organic molecules at natural abundance are most easily measured using t 2 coupled HSQC spectra. However, inevitably mismatched transfer delays result in phase distortions due to residual dispersive antiphase coherences in such experiments. In this article, slightly modified t 2 coupled HSQC experiments with clean inphase (CLIP) multiplets are introduced which also reduce the intensities of undesired long-range cross peaks. With the corresponding antiphase (CLAP) experiment, situations where a and b components overlap can be resolved for all multiplicities in an IPAP manner. A comparison of the experiments using hard pulses and shaped broadband excitation and inversion pulses on the heteronucleus is given and potential spectral artefacts are discussed in detail. Ó 2008 Elsevier Inc. All rights reserved. 1. Introduction Since the introduction of residual dipolar couplings (RDCs) in biomolecular NMR many experiments have been developed for the measurement of heteronuclear one-bond couplings [1–5]. Most of these experiments are designed for IS spin systems, where mea- surement in the indirectly acquired dimension is easily possible without loss of information and the smaller linewidths of the het- eronucleus give the advantage of better defined splittings. For small- to medium-sized organic molecules, however, methylene and methyl groups must be taken into account and therefore t 2 coupled HSQC or HMQC experiments are usually used for coupling extraction in such molecules [6–9]. This kind of experiment is eas- ily applied to isotropic samples, as the small range of one-bond heteronuclear 1 J CH coupling constants allows the setting of close to optimal transfer delays. In partially aligned samples, neverthe- less, the wide distribution of ( 1 J CH + D CH ) couplings does not allow a correct match of the INEPT-type delays for all groups so that sig- nificant phase distortions from residual antiphase magnetization reduce the quality of the spectra (see also Fig. 1 for demonstration). Here, several versions of HSQC experiments with purely absorp- tive multiplet patterns are studied that allow the direct measure- ment of one-bond heteronuclear couplings without phase distortions. In addition to the so-called clean inphase (clean-IP, or simply CLIP) HSQC, a second experiment resulting in absorptive antiphase (CLAP) spectra is introduced that allows the quantitative extraction of coupling constants in an a/b or IPAP-type approach. All HSQC-type experiments were implemented using either hard pulses or recently introduced BEBOP and BIBOP pulses [10,11] for excitation, inversion, and refocussing on carbon nuclei with clear advantages for the soft pulse versions. 2. Pure absorption spectra The introduction of orienting media with sufficiently low align- ment [12–14] started a surge of experimental techniques for the measurement of one-bond heteronuclear couplings, usually ( 1 J NH + D NH ) couplings of protein amide groups. Compared to the measurement of 1 H, 15 N couplings in isotope labeled biomolecules, the measurement of 1 H, 13 C RDCs in organic molecules at natural abundance that contain CH, CH 2 , and CH 3 spin systems requires pulse sequences with different abilities. A widely used approach is the acquisition of already established HSQC or HMQC experi- ments without heteronuclear decoupling [6–9,15]. In these exper- iments, couplings can be directly read from the heteronuclear splitting in the directly detected dimension. Unfortunately, the wide distribution of couplings due to the orientationally depen- dent D CH RDCs makes the exact match of the INEPT transfer delay to 1/(2 1 J CH +2D CH ) impossible. Ingenious approaches like the use of adiabatic inversion pulses for compensation of chemical shift dependent spin–spin coupling effects [16] do not help, since RDCs are structurally dependent and do not scale with the chemical shift. As a result, cross peaks are significantly phase distorted as 1090-7807/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jmr.2008.03.009 * Corresponding author. Fax: +49 89 289 13210. E-mail address: Burkhard.Luy@ch.tum.de (B. Luy). Journal of Magnetic Resonance 192 (2008) 314–322 Contents lists available at ScienceDirect Journal of Magnetic Resonance journal homepage: www.elsevier.com/locate/jmr