Measurement of long-range interatomic distances by solid-state tritium-NMR spectroscopy

Article abstract of DOI:10.1021/ja908915v

(Chemical Equation Presented) For the structural determination of a ligand bound to an amorphous macromolecular system, solid-state NMR can be used to provide interatomic distances. It is shown here that selective labeling in discrete locations with tritium enables accurate measurement of long-range distances owing to the high gyromagnetic ratio of this nucleus, without structural modification of the molecule. This approach gives access to the largest NMR distance ever measured between two nuclei (14.4 A). ³H MAS NMR appears to be a promising tool for structural applications in the biological and material sciences. Copyright

Full text of DOI:10.1021/ja908915v

Published on Web 01/21/2010
Measurement of Long-Range Interatomic Distances by Solid-State
Tritium-NMR Spectroscopy
Alexander K. L. Yuen,† Olivier Lafon,§ Thibault Charpentier,*,§ Myriam Roy,† Francine Brunet,§
Patrick Berthault,§ Dimitrios Sakellariou,§ Bruno Robert,‡ Sylvie Rimsky,| Florence Pillon,†
Jean-Christophe Cintrat,† and Bernard Rousseau*,†
CEA, iBiTecS, SerVice de Chimie Bioorganique et de Marquage, F-91191 Gif sur YVette, France, CEA, iBiTecS,
SerVice de Bioe´nerge´tique, Biologie Structurale et Me´canismes, F-91191 Gif sur YVette, France, CEA, IRAMIS,
SIS2M, Laboratoire Structure et Dynamique par Re´sonance Magne´tique, F-91191 Gif sur YVette, France, and
LBPA, CNRS, ENS de Cachan, 61, aVenue du Pre´sident Wilson, F-94235 Cachan, France
Received January 29, 2009; Revised Manuscript Received October 19, 2009; E-mail: thibault.charpentier@cea.fr;
bernard.rousseau@cea.fr
Solid-state NMR has clearly proven to be valuable for probing
molecular structure, owing to its ability to measure accurate
interatomic distances.1 In the absence of high-resolution X-ray data,
such measurements are mandatory for determining the conformation
of a small molecule bound to an insoluble or membrane protein.
Knowledge of this conformation is of key importance in drug
design.2 Long-range distances can be determined using either
beacons such as 19F (distances up to 8 Å)3 or paramagnetic spin
labels (up to 20 Å).4 In both of these cases, however, modification
of the ligand structure is an unwanted consequence. Isotopic labeling
using 13C-13C or 13C-15N does not lead to structural modifications,
but only short distances can be measured (5-6 Å).1 Most of the
conformational changes when a ligand binds its receptor involve
variation of long-range interatomic distances. For instance, the
bioactive conformation of paclitaxel requires the determination of
a key distance greater than 10 Å.5 To gain access to such distances
without structural modifications, we speculated that tritium would
mixture of 50 mCi (∼ 0.15 to 0.3 mg) of the tritiated compound,
40 mg of its unlabeled form, and 0.2 wt % of paramagnetic salt
Cu(NO3)2 was prepared (the latter being added to shorten relaxation
times).
Figure 1. Model compounds for distance measurement.
The 3H spectra of compound 1 obtained in 8 scans are displayed
in Figure 2. They prove that a high signal-to-noise ratio can be
obtained with small quantities of tritiated material. The use of
1H-3H cross-polarization significantly increases the sensitivity,
mostly as a consequence of the shorter proton relaxation time. For
compounds 1-4 the proton T1 was determined to be approximately
10 times shorter than that of tritium (e.g., T1 > 360 s for 3H and T1
1
be an attractive label owing to the fact that it has a spin of /2 and
the highest gyromagnetic ratio of any nucleus (1.07 times that of
proton). As the natural abundance of tritium is negligible (3 ×
10-16%), no background signal would be observed. Furthermore,
modern synthetic protocols can furnish a variety of selectively
labeled materials with tritium atoms at precise locations.6 Tritiated
compounds can be purchased from commercial companies at a price
in the same range as that for a 13C- or 15N-labeled compound. Their
safe handling is well-known, and countless biochemistry and
biology laboratories routinely use them. In addition, tritium has
already proven useful in liquid-state NMR where it has helped
elucidate stereochemical and mechanistic aspects of small-molecule
chemistry over the past few decades.7
1
) 26 s for H in compound 1).
3
In this Communication, we show that solid-state H NMR is a
powerful tool for accurately determining long-range interatomic
distances without modification of molecular structure. The largest
NMR distance ever measured between two nuclei was obtained
using a model compound.
To assess the potential of solid-state tritium-NMR, we designed
small molecules rigid enough to obtain nonfluctuating 3H-3H
distances. Compounds 1-4 (Figure 1) incorporating two tritium
atoms separated by 4.31, 6.0, 9.4, and 13.8 Å, respectively, were
synthesized by catalytic hydrogenolysis of their brominated precur-
sors using tritium gas and Pd/C.8 For each NMR analysis, a powder
Figure 2. Comparison between 3H spectra of compound 1 obtained by
direct acquisition (top) and 1H-3H cross-polarization (bottom) in eight scans.
Total experiment times are indicated on the right; MAS ) magic angle
spinning; CP ) cross-polarization.
For interatomic distance measurements, numerous NMR recou-
pling techniques have been reported in the literature and their
improvement is still the subject of intensive research. Among the
several common pulse sequences we investigated, the best results
† CEA, iBiTecS, Service de Chimie Bioorganique et de Marquage.
§ CEA, IRAMIS.
‡ CEA, iBiTecS, Service de Bioe´nerge´tique, Biologie Structurale et Me´canismes.
| LBPA, CNRS.
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1734 J. AM. CHEM. SOC. 2010, 132, 1734–1735
10.1021/ja908915v  2010 American Chemical Society

C O M M U N I C A T I O N S
were obtained with HORROR.9 This sequence offered the advantage
of requiring only a moderate 3H rf-field amplitude (matched at half
the spinning frequency) so that a high 1H-decoupling rf-field (here
80 kHz) could be applied, keeping the total power reasonable on
two channels very close in frequency.
As shown in Figure 3, remarkably long dipolar oscillations are
observed for compound 1, allowing precise 3H-3H distance
measurement. From these oscillations, the interatomic distance was
extracted, accounting for the damped signal arising from the
simultaneous response of the 3H-3H spin pair and that of isolated
3H spins. For 1, the discrepancy between the measured and the
expected values is less than 1% (Table 1). Even for longer distances
such as those found in compounds 2 and 3, the accuracy remained
excellent, with a discrepancy in the range of 2%. To the best of
our knowledge, this constitutes unprecedented accuracy for such
long-range interatomic distances.
decoupling, which is more efficient than 1H-1H decoupling,
contributes to the precision of the method.12 Finally, this method
relies upon direct measurement of the dipolar oscillation, which is
more precise than using diffusion measurements.
3
Table 1. Predicted vs Measured H-3H Distances in Å for
Compounds 1-4
compound
1
2
3
4
predicted distancea 4.31
6.0
9.4
13.8
measured distance
4.35 ( 0.02 5.9 ( 0.1 9.3 ( 0.2 14.4 ( 2.2
a Distances predicted for molecules 1-4 using DFT relaxed
molecular structures with the PWSCF package.13
In summary, we have described a simple, sensitive, and accurate
approach for the determination of short- to long-range interatomic
distances using standard probe electronics and sample holders and
have successfully applied it to model compounds. The measured
distance of 14.4 ( 2.2 Å reported here is the highest ever obtained
using NMR. To generalize this approach to samples with a larger
3H chemical shift distribution or shorter T1F values, modifications
of the experimental conditions such as use of higher spinning
frequency, sample volume reduction, or design of new pulse
sequences would be beneficial.
This novel method will allow the study of drugs at their binding
sites in insoluble or membrane proteins. Application of this approach
to the study of biological questions such as the bioactive conforma-
tion of microtubule-bound paclitaxel is underway in our laboratories.
Acknowledgment. This work was supported by an ANR grant
from the French Ministry of Research (Program MASTRIT NT05-
2_42116). Nathalie Thromat is warmly acknowledged for her help
in addressing safety issues.
Supporting Information Available: Experimental details, spectro-
scopic data, and safety procedures. This material is available free of
charge via the Internet at http://pubs.acs.org.
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Figure 3. Variation of the H-signal intensity S(t) (in arbitrary units) for
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The case of compound 4 revealed that, even in the absence of
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