Solid state 19F NMR parameters of fluorine-labeled amino acids. Part II: Aliphatic substituents

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Abstract

A representative set of amino acids with aliphatic 19F-labels has been characterized here, following up our previous compilation of NMR parameters for single 19F-substituents on aromatic side chains. Their isotropic chemical shifts, chemical shift tensor parameters, intra-molecular 19F dipole–dipole couplings and temperature-dependent T1 and T2 relaxation times were determined by solid state NMR on twelve polycrystalline amino acid samples, and the corresponding isotropic 19F chemical shifts and scalar couplings were obtained in solution. Of particular interest are amino acids carrying a trifluoromethyl-group, because not only the 19F chemical shift but also the intra-CF3 homonuclear dipolar coupling can be used for structural studies of 19F-labeled peptides and proteins. The CF3-groups are further compared with CH2F-, CD2F-, and CD3-groups, using both 19F and 2H NMR to describe their motional behavior and to examine the respective linebroadening effects of the protonated and deuterated neighbors. We have also characterized two unnatural amino acids in which a CF3-label is rigidly connected to the backbone by a phenyl or bicyclopentyl moiety, and which are particularly well suited for structure analysis of membrane-bound polypeptides. The 19F NMR parameters of the polycrystalline amino acids are compared with data from the correspondingly labeled side chains in synthetic peptides.

Introduction

For analyzing a solid compound by NMR it is usually essential to know the fundamental parameters of the nucleus at the site of interest, such as its chemical shift anisotropy and dipolar couplings in the static limit. In the preceding contribution we had outlined the advantages of 19F-labelling for studying peptides and proteins, and had compiled the NMR parameters for aromatic amino acids with a single 19F-substituent on the ring [1]. These aromatic groups can be readily incorporated into proteins biosynthetically, but their use in structural investigations may be complicated by the flexibility of the side chain and the lack of an axially symmetric electronic environment. Certain aliphatic 19F-labels, on the other hand, can overcome this limitation, especially when carrying a CF3-group. The advantage of this reporter-group over a single 19F-substituent lies in its fast rotational averaging, which creates an environment that is axially symmetric to a first approximation, in contrast to most other 19F CSA tensors. Furthermore, the CF3-group provides structural information not only via its 19F chemical shift anisotropy, but also via its intra-CF3 homonuclear dipolar coupling [2], [3], [4], [5], [6], [7]. The present compilation of 19F NMR parameters is thus focused on aliphatic 19F-segments, as illustrated in Fig. 1, with special attention to CF3-labels in aliphatic and aromatic amino acids. The study includes two unnatural CF3-labeled amino acids that have been specifically developed to describe the conformation, alignment and mobility of membrane-bound peptides, namely CF3-labeled phenylglycine (Phg) and bicyclopentylglycine (Bpg) [4], [5], [6], [7], [8].

Section snippets

Amino acids

All amino acids and their abbreviations are summarized in Fig. 1 (for stereoisomers see Table 1). The polycrystalline 19F-labeled amino acids were used as supplied by the following commercial sources: 3F-Ala was purchased from Bachem (Bubendorf, Switzerland), 3,3,3F3-Ala from Chempur (Karlsruhe, Germany), 3F-Val and 5,5,5F3-Leu from Lancaster (now Alfa Aesar, Karlsruhe, Germany), 4CF3-Phe and 4CF3-Phg from ABCR (Karlsruhe, Germany). The leucine derivatives 5F-(2S,4S)-leucine (5F-Leu(2S,4S)),

Liquid state NMR

As a first step in characterizing the isotropic chemical shift values (δiso), J-coupling constants, and the types of multiplets of the various 19F-labeled amino acids, we examined their 19F NMR spectra in aqueous solution. The results are summarized in Table 1. As expected from the large dispersion known for this nucleus, the isotropic chemical shifts cover a wide range from −60 to −230 ppm. Clear differences are noted between the mono-fluorinated analogues of alanine and leucine (shifts between

Conclusions

To extend the compilation of 19F NMR parameters for the most relevant amino acids, we have here examined aliphatic 19F-labels, especially CF3-groups, in aliphatic and aromatic side chains. Compared to the aromatic ring-substituted 19F-labels characterized in Part I [1], the most pronounced differences were found in the relaxation behavior. Whereas the aromatic 19F-substituents have long T1 times in the range of several minutes, the labeled methyl groups never exceed a few seconds, which is

Acknowledgments

The authors thank Prof. D. Young and Dr. J.-D. Charrier (formerly at the Department of Chemistry of the University of Sussex) for the generous gift of the 19F-labeled leucine samples, and Prof. T. Asakura (Tokyo University) for the gift of 3CF3-Phe. The Deutsche Forschungsgemeinschaft is gratefully acknowledged for financial support of SFB 197 (TP B13) and the Center for Functional Nanostructures (E1.2).

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