Differential Large-Amplitude Breathing Motions in the Interface of

Article pubs.acs.org/biochemistry

Differential Large-Amplitude Breathing Motions in the Interface of FKBP12−Drug Complexes† Chun-Jiun Yang,‡,∥ Mitsuhiro Takeda,§,⊥ Tsutomu Terauchi,‡ JunGoo Jee,‡,@ and Masatsune Kainosho*,‡,§ ‡

Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 minami-ohsawa, Hachioji, Tokyo 192-0397, Japan § Structural Biology Research Center, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan S Supporting Information *

ABSTRACT: The tight complexes FKBP12 forms with immunosuppressive drugs, such as FK506 and rapamycin, are frequently used as models for developing approaches to structure-based drug design. Although the interfaces between FKBP12 and these ligands are well-defined structurally and are almost identical in the X-ray crystallographic structures of various complexes, our nuclear magnetic resonance studies have revealed the existence of substantial large-amplitude motions in the FKBP12−ligand interfaces that depend on the nature of the ligand. We have monitored these motions by measuring the rates of Tyr and Phe aromatic ring flips, and hydroxyl proton exchange for residues clustered within the FKBP12− ligand interface. The results show that the rates of hydroxyl proton exchange and ring flipping for Tyr26 are much slower in the FK506 complex than in the rapamycin complex, whereas the rates of ring flipping for Phe48 and Phe99 are significantly faster in the FK506 complex than in the rapamycin complex. The apparent rate differences observed for the interfacial aromatic residues in the two complexes confirm that these dynamic processes occur without ligand dissociation. We tentatively attribute the differential interface dynamics for these complexes to a single hydrogen bond between the ζ-hydrogen of Phe46 and the C32 carbonyl oxygen of rapamycin, which is not present in the KF506 complex. This newly identified Phe46 ζ-hydrogen bond in the rapamycin complex imposes motional restriction on the surrounding hydrophobic cluster and subsequently regulates the dynamics within the protein−ligand interface. Such information concerning large-amplitude dynamics at drug−target interfaces has the potential to provide novel clues for drug design.

P

were studied for FKBP12−ligand complexes, on the basis of nuclear magnetic resonance (NMR) relaxation experiments focused on the backbone 15N and side-chain 13C atoms.11−13 Brath and Akke concluded from their relaxation dispersion experiments that the backbone of the FK506-bound FKBP12 is confined to a single conformation, while conformational exchange prevails for various side-chain methyls.12 In a 13C NMR analysis of [8,7-13C2]FK506, Rosen et al. detected two conformers about the C8−N7 amide bond in chloroform, at a ratio of ∼2:1 (cis:trans), but a single conformer was observed for the FKBP12-bound state in an aqueous solution.14 Very interestingly, Sapienza et al. found widespread microsecond to millisecond backbone dynamics for the rapamycin−FKBP12 complex and postulated that these slow motions represent the coupled dynamics of FKBP12 and cis−trans isomerization for the C8−N7 amide.14 They concluded that the marked motional differences between these two structurally very similar complexes indicate that “relatively subtle changes in drug structure can translate into substantial motional differences in

roteins undergo internal motions with different amplitudes and frequencies.1,2 Such features of protein dynamics are commonly not detected in three-dimensional structures determined by X-ray crystallography. In the hierarchy of protein dynamics, large-amplitude, slow breathing motions (LASBM) have long been considered to be potentially relevant to their biological functions.3,4 These interface motions between protein−protein and protein−ligand complexes are especially interesting, because their rates provide unprecedented opportunities for the analysis of interface dynamics. This avenue of research has remained virtually unexplored, mainly because of the lack of versatile and robust experimental methodologies for their study. FK506 binding protein 12 (FKBP12)5 tightly binds the immunosuppressive drugs FK506 and rapamycin.6 FK506 and rapamycin are cyclic macrolides that contain on one side a common FKBP binding motif and on the other side an effector binding region for calcineurin and mTOR, respectively.7−10 Comparison of the crystal structures of the FKBP12−FK506 and FKBP12−rapamycin complexes reveals that the atomic coordinates within the binding regions are almost identical, although a few different interactions outside the common binding region are present.8 Dynamics on various time scales © 2015 American Chemical Society

Received: July 22, 2015 Revised: November 10, 2015 Published: November 11, 2015 6983

DOI: 10.1021/acs.biochem.5b00820 Biochemistry 2015, 54, 6983−6995

Article

Biochemistry FKBP12”.13 From the viewpoint of protein−ligand interactions, the dynamic aspects of protein−ligand interfaces with and without bound ligands are particularly interesting.15−17 In this context, quantitative evaluations of the large-amplitude motions for the bulky side-chain groups of aromatic amino acid residues belonging to the ligand binding interface of FKBP12 in the free and bound states are most crucial. Conceivably, the aromatic rings on the ligand binding surface of FKBP12 are highly dynamic in the free state, whereas their motions are severely restricted in the bound states. Note that the ring-flipping motion results in the identical conformation; therefore, the exact flipping rates can only be directly manifested by the flipping rate-dependent NMR line shapes for symmetry-related aromatic ring signals.18,19 In this report, we have focused on the aromatic amino acid residues clustered within the ligand binding surface of FKBP12, i.e., Tyr26, Tyr80, Tyr82, Phe36, Phe46, Phe48, Phe99, and Trp59. The side-chain rings of the Tyr and Phe residues in the ligand binding region (Tyr26, Tyr82, Phe36, Phe46, Phe48, and Phe99) are exceptionally useful for this study, because measurement of their ring-flipping rates would allow us to quantitatively evaluate large-amplitude dynamics within the ligand binding interface. It should be emphasized that aromatic ring-flipping rates can be studied by only NMR spectroscopy; because the structures of the aromatic rings are identical before and after flipping about the χ 2 axis, the states are indistinguishable by other methods. The slow aromatic ringflipping motions of the Phe and Tyr residues embedded in the interior of proteins were observed many years ago.18,19 The ring-flipping motions were subsequently subjected to various physical chemical studies.20−27 These analyses primarily used one-dimensional (1D) 1H NMR or two-dimensional (2D) 1H−13C correlation spectroscopy for conventionally [U-13C,15N]-labeled proteins, and thus, the quantitative flipping rate measurements for these proteins were severely hampered by the spectral complexities. To avoid these problems, a number of biosynthetic labeling methods have been developed to incorporate isolated 13C nuclei into proteins, using various selectively 13C-labeled or 13C- and 2Hlabeled precursors.28−30 Because the methods generally employ relatively inexpensive 13C-labeled precursors, they have been widely used for studying aromatic ring dynamics on various time scales.31−35 Alternate ways to simplify the aromatic ring spin systems employ selectively isotope-labeled aromatic amino acids or precursors just preceding the synthesis of the aromatic amino acids of interest.36,37 In principle, these alternate strategies allow the preparation of proteins with any type of labeling pattern for each aromatic amino acid, to obtain the desired structural and dynamic information with the highest sensitivity. They are particularly useful for proteins containing many aromatic amino acids, which often give spectra with excessively overlapped aromatic ring signals, because one can select both the amino acid type and labeling pattern to simplify the spectra. In this study, we focused on the large-amplitude dynamics as revealed by the aromatic ring-flipping motions and hydroxyl proton exchange rates of the five Phe and three Tyr aromatic rings, which are clustered in the ligand binding interface of FKBP12. Therefore, we extensively used a variety of selectively isotope-labeled amino acids, including the stereoarray isotope-labeled (SAIL) amino acids,38−41 and successfully obtained a complete set of NMR data for all of the aromatic ring atoms.

In addition to the information available from the aromatic ring signals, the hydroxyl groups of Tyr residues in the FKBP12−ligand interface also provide useful details. The proton exchange rates of the hydroxyl groups of proteins in aqueous solutions have been studied for many years,42,43 but accurate hydrogen exchange rate measurements of the Tyr hydroxyl groups in the interior of proteins had been very difficult prior to a recent strategy employing selective labeling and monitoring of the H/D isotope effect.44,45 We report here the application of these two new methodologies to measurements of Phe and Tyr aromatic ring-flipping rates and Tyr hydroxyl proton exchange rates in FKBP12− FK506 and FKBP12−rapamycin protein−ligand interfaces.

■

MATERIALS AND METHODS Sample Preparation. Three types of SAIL tyrosine ([β 2 ,ε 1 , 2 - 2 H 3 ;0,α,β,δ 1 , 2 - 1 3 C 5 ; 1 5 N]Tyr, “δ-SAIL Tyr”, [β2 ,δ 1,2 - 2 H3 ;0,α,β,γ,ε 1, 2- 13 C 6 ;15N]Tyr, “ε-SAIL Tyr”, and [β2,ε1,2-2H3;0,α,β,ζ-13C4;15N]Tyr, “ζ-SAIL Tyr”) and three types of SAIL phenylalanine ([β2,ε1,2,ζ-2H4;0,α,β,δ1,2-13C5;15N]Phe, “δ-SAIL Phe”, [β2,δ1,2,ζ-2H4;0,α,β,γ,ε1,2-13C6;15N]Phe, “εSAIL Phe”, and [β2,δ1,2,ε1,2-2H5;0,α,β,γ,ζ-13C5;15N]Phe, “ζ-SAIL Phe”) (Figure S1)39,40,44 were synthesized and obtained from SAIL Technologies, Inc. (http://www.sail-technologies.com). These labeled residues were incorporated into FKBP12 using Escherichia coli auxotrophic strain AB2826(DE3), which is defective in the synthetic pathways for aromatic amino acids.46 The cells were initially grown at 37 °C in M9 medium containing unlabeled NH4Cl, D-glucose, and 20 amino acids (25 mM each) to an OD600 of 0.6−0.7. Subsequently, the cells were collected by centrifugation, washed with M9 medium, and then suspended in M9 medium containing 19 amino acids, except for Tyr or Phe, depending on the residue type of the labeling target. After a 15 min incubation at 37 °C, SAIL Tyr or Phe was added to the culture (3 mg/L), and protein expression was induced by adding isopropyl β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Even with this small amount of SAIL Tyr or Phe within the medium, the labeling percentage of SAIL Tyr was found to be more than 90%. The expressed FKBP12 proteins were purified as described previously.47 Unless noted otherwise, the NMR samples contained 0.3−0.4 mM FKBP12, in 25 mM KPO4 (pH 7.0), 1 mM dithiothreitol, and 0.1 mM NaN3 with 10% D2O. To obtain NMR data for FKBP12 bound to either rapamycin or FK506, a 1.2-fold molar excess of each ligand was added to the protein solutions, to ensure the presence of fully bound states. NMR Experiments. The 1H−13C HSQC spectra of the aromatic region of the δ-/ε-SAIL Tyr-labeled and δ-/ε-/ζ-SAIL Phe-labeled FKBP12 proteins were acquired at 30 °C, on an Avance600 spectrometer (Bruker BioSpin, 600.03 MHz for 1H) equipped with a TXI cryogenic probe. The data size and the spectral width were 192 (t1) × 2048 (t2) points and 900 Hz (ω1, 13C) × 7200 Hz (ω2, 1H), respectively. Signals from the Tyr and Phe Hδ and Hε protons were assigned on the basis of intraresidue nuclear Overhauser effects (NOEs) with Hβ,44 HBCB(CG)HE,39 and then the inter-residue NOEs (vide inf ra). HBCB(CGCZ)HZ experiments led to assignments of the Hζ proton of Phe residues.39 The unambiguous assignments of NMR signals from the δ1/δ2 and ε1/ε2 CH atoms of Tyr and Phe residues exhibiting slow flipping motions are described in the Supporting Information. The 2D 1H NMR EXSY experiments were conducted on the ε-SAIL Tyr-labeled or ε-SAIL Phe-labeled FKBP12, in complex 6984

DOI: 10.1021/acs.biochem.5b00820 Biochemistry 2015, 54, 6983−6995

Article

Biochemistry

RD represent the longitudinal relaxation rates of the 13Cζ atoms at the two isotopomers, and kex is the exchange rate. Because the proton/deuterium fractionation factor of the OH groups is almost unity, the site populations for the two isotopomers were set to be equal (i.e., pH = pD = 0.5) in the 1:1 H2O:D2O solution, and the decay rates were also set to be equal (i.e., RH = RD = R). The peak fitting was performed as a function of the mixing times by using the MATLAB software (MathWorks, Inc., Natick, MA). Five hundred Monte Carlo simulation cycles were performed for error analysis (standard deviation). In the case of Tyr26 in the FK506 complex, the exchange rates were too slow, and the exchange peaks were undetectable even at a mixing time of 600 ms. The upper limits of kex were estimated by using the equation for the two-state exchange model,53 with the assumption that the intensities of the cross peaks at a 600 ms mixing time are less than 10% of those of the auto peaks. The ring-flipping rate was evaluated by fitting the intensities of the auto and cross peaks as a function of the mixing times to the following equation:

with either FK506 or rapamycin, using an Avance800 spectrometer (Bruker BioSpin, 800.33 MHz for 1H) equipped with a TXI cryogenic probe. In the pulse scheme for the 2D EXSY experiments, HSQC elements were employed without the carbon chemical shift evolution before and after the NOESY mixing period, to eliminate the NOE peaks involving protons attached to 12C.48 The data points and spectral widths were 128 (t1) × 1024 (t2) points and 4800 Hz (ω1, 1H) × 9600 Hz (ω2, 1H), respectively. The number of transients (FIDs) was 128. The mixing times were set to 10, 20, 30, 50, 70, 100, 150, and 200 ms. Spectra were processed with NMRPipe49 and analyzed with SPARKY.50 The high-sensitivity 1D 13C NMR spectra of ζ-SAIL Tyrlabeled FKBP12 at different H2O/D2O ratios were obtained at 30 °C, using an Avance500 spectrometer (Bruker BioSpin, 500.2 MHz for 1H, 125.8 MHz for 13C) equipped with a 13C direct DCH cryogenic probe. During the free induction decay, 1 H decoupling was applied with the WALTZ16 decoupling scheme.51 The carrier frequency of the carbon was 156 ppm, and the sweep width was 2500 Hz. The repetition time was 2 s. For samples in 100% H2O, a 4.1 mm outside diameter Shigemi inner tube containing the FKBP12 solution was inserted into a 5 mm outside diameter outer tube containing D2O, for the lock signal. The long-range 1H−13C HSQC experiments through 3JCH scalar coupling between Cζ and Hδ were recorded on the ζSAIL Tyr-labeled FKBP12 protein at 30 °C, using the Avance500 spectrometer. The data size and the spectral width were 256 (t1) × 1024 (t2) points and 500 Hz (ω1, 13 C) × 6700 Hz (ω2, 1H), respectively. The carrier frequencies were 156 ppm for 13C and 4.7 ppm for 1H. In the INEPT elements composed of [(π/2)x(1H)−τ−π(1H,13C)−τ−(π/ 2)y(1H)], the delay τ was set to 12.5 ms. The 13C NMR EXSY experiments were performed at 30 °C, by using 0.8 mM ζ-SAIL Tyr-labeled FKBP12 in complexes with either FK506 or rapamycin, in a solution containing a 1:1 H2O:D2O ratio. The pulse scheme for the EXSY experiment was reported previously.44 Proton decoupling was achieved by the WALTZ16 scheme.51 The carrier frequency of carbon was set to 156 ppm. The data points and the spectral widths were 80 (t1) × 4096 (t2) points and 200 Hz (ω1, 13C) × 10000 Hz (ω2, 13C), respectively. The number of transients (FIDs) was 320. The mixing times were 50, 100, 200, 400, and 600 ms, and the repetition time was 2 s. Samples for 13C NMR EXSY experiments for evaluating H/D exchange rates at different pH values were adjusted by adding hydrochloric acid or sodium hydroxide in H2O buffer and subsequently lyophilized. The protein samples were then dissolved in a solution containing a 1:1 H2O:D2O ratio to perform the exchange rate studies. Data Analysis. The proton/deuterium exchange rate constant (kex = kHD + kDH) of the Tyr OH group was evaluated by fitting the peak intensity as a function of the mixing times in the 13C NMR EXSY experiments. The change in peak intensities is given by the Bloch−McConnell equation,52,53 as follows: ⎛ ⎞⎛ HH ⎞ kexpH d ⎛ I HH ⎞ ⎜−RH − kexpD ⎟⎜⎜ I ⎟⎟ ⎜⎜ ⎟⎟ = ⎜ HD dt ⎝ I ⎠ ⎝ −RD − kexpH ⎟⎠⎝ I HD ⎠ kexpD

⎛ k flip ⎞⎛ I auto ⎞ d ⎛ I auto ⎞ ⎜−R − k flip ⎟⎜ ⎟ ⎜ cross ⎟ = ⎜ dt ⎝ I ⎠ ⎝ k flip −R − k flip ⎟⎠⎝ I cross ⎠ auto

(2)

cross

where I and I are the peak intensities of the diagonal and cross peaks, respectively, R is the longitudinal relaxation rate, and kflip is the ring-flipping rate of the phenyl ring. Equation 2 is almost identical to eq 1, the difference being that the forward and reverse reactions are equal, and thus, kex is replaced by 2kflip (i.e., kex = 2kflip). The temperature dependence of kflip was analyzed by the Eyring equation,54 to obtain the free energy of activation (ΔG⧧): ΔG⧧ ΔH ⧧ = −R ln(k fliph/kBT ) = − ΔS ⧧ T T

(3)

where T is the temperature and h, kB, and R are the Planck, Boltzmann, and gas constants, respectively. The enthalpy (ΔH⧧) and entropy (ΔS⧧) of activation were determined by least-squares fitting for ΔG⧧/T, as a function of temperature.

■

RESULTS The ligand binding pocket of FKBP12 in the rapamycin and FK506 complexes consists of the aromatic side chains of Tyr26, Tyr80, Tyr82, Phe36, Phe46, Phe48, Phe99, and Trp59 and the aliphatic side chains of Val55 and Ile56, as illustrated in panels D and E of Figure 1. The side chains of these residues form an extremely hydrophobic concave surface, which specifically recognizes the FKBP12 binding regions of rapamycin and FK506. The almost identical C1−C14 segments in rapamycin and FK506 were identified by X-ray analyses to be the FKBP12 binding regions (colored red in Figure 1A,B). In the FKBP12− ligand complexes, the FKBP12 binding regions of the ligands are deeply buried within the hydrophobic pocket and wrapped by the surrounding aromatic rings (Figure 1C,D). Therefore, the effects of ligand binding on the dynamics of the FKBP12− ligand interfaces should be clearly manifested by the aromatic ring NMR signals, although in practice such approaches have been hampered by the complex NMR spectra of aromatic rings. However, by taking advantage of the SAIL method, we could investigate the effects of ligand binding on the structures and dynamics of the hydrophobic cluster in the ligand binding region of FKBP12 proteins, selectively labeled with a series of

(1)

where IHH and IHD are the peak intensities of the diagonal and cross peaks, respectively, and pH and pD are the site populations of the protonated and deuterated species, respectively. RH and 6985

DOI: 10.1021/acs.biochem.5b00820 Biochemistry 2015, 54, 6983−6995

Article

Biochemistry

Figure 2. Aromatic ring 1H−13C signals in 600 MHz HSQC spectra, obtained at different temperatures in the range of 1040 °C, for FKBP12 selectively labeled with either δ- or ε-SAIL Tyr, at pH 7.0, in the rapamycin-bound state (A and B) and in the FK506-bound state (C and D). The stereospecific assignments for δ1/δ2- and ε1/ε2-CH were unambiguously established by virtue of the stereospecific deuteration of the β-methylene protons (see the Supporting Information and Figure S4).

Figure 1. Ligand binding interface of FKBP12. Chemical structures of the cyclic macrolides rapamycin (A) and FK506 (B), which contain a common FKBP binding motif (C1−C14) on one side (red) but different effector binding sites for mTOR and calcineurin, respectively, on the opposite side (blue). (C) Ribbon model of rapamycin (colored) bound to FKBP12 (gray), showing the overall arrangement of FKBP12 and the ligand. (D and E) Stick models of the protein− ligand interfaces, circled in panel C, for the FKBP12−rapamycin (PDB entry 2DG3)65 and FKBP12−FK506 (PDB entry 1FKJ) complexes, respectively.66 Hydrogen bonds between FKBP12 and the ligands identified in the crystalline structures are shown by dotted lines, and the additional hydrogen bond between the ζ-hydrogen of Phe46 and the C32 carbonyl oxygen, identified in this work for the rapamycin complex, is circled (D). The numbering of the ligand atoms was adopted from the literature.7

signals of Tyr26 were split into two discrete signals even at 40 °C, indicating that the ring-flipping rates of this residue became much slower upon FK506 binding. At temperatures below 20 °C, the Tyr26 signals in the rapamycin-bound state started to appear, indicating a slower rate of ring flipping. At 10 °C, the δand ε-CH in Tyr26 each gave a pair of sharp signals in the rapamycin-bound state. We also noticed substantial line broadenings along the 13C axis for the ε-CH of Tyr82, for both the rapamycin and FK506 complexes below 20 °C (Figure 2B,D). All of the chemical shifts determined for the Tyr δ- and ε-CH in the free and bound states are listed in Table 1. Comparison of the chemical shift differences between the free and bound states revealed that the chemical shifts of Tyr80 were minimally influenced by ligand binding. Although the backbone of Tyr80 sits at the periphery of the binding pocket, its side chain is located on the opposite side of the ligand binding region (Figure 1D,E). Both Tyr26 and Tyr82 participate directly in ligand recognition through hydrogen bonds, including their hydroxyl groups, and they exhibit large chemical shift and line-shape differences between the free and bound states. Further information about the effect of ligand binding on the structures and dynamics of the FKBP12 ligand binding region can be obtained from the Phe residues (Phe36, Phe46, Phe48, and Phe99) that are clustered in the hydrophobic ligand binding pocket. Therefore, we examined the 1H−13C HSQC spectra of FKBP12 proteins selectively labeled with either δ-, ε-, or ζ-SAIL Phe, in the free and rapamycin- or FK506-bound states. In the free state, the aromatic ring 1H−13C signals for all five of the Phe residues of FKBP12, including Phe15 located outside of the ligand binding region, gave well-separated HSQC signals for the δ-, ε-, and ζ-CH at 30 °C (Figure S3A). These results indicated that the aromatic rings of all five of the Phe residues in FKBP12 flip rapidly in the free state. However, appreciable chemical shift and line-shape changes were induced

SAIL aromatic amino acids, such as δ- and ε-SAIL Tyr and δ-, ε-, and ζ-SAIL Phe (Figure S1). Aromatic Ring CH Signals for Tyr and Phe Residues of FKBP12 in the Free and Bound States. In the free state, a single cross peak for the δ- and ε-CH was observed for each of the three Tyr residues, i.e., Tyr26, Tyr80, and Tyr82, of the FKBP12 proteins selectively labeled with δ- or ε-SAIL Tyr (Figure S2A). The results indicated that all three of the Tyr residues located on the ligand binding surface of FKBP12 are rapidly flipping in the free state. However, once binding to rapamycin or FK506 has occurred, significant changes were observed in both the chemical shifts and line shapes of the εand δ-CH signals of Tyr26 and Tyr82 (Figure S2B,C). To investigate the structural perturbations caused by ligand binding, we acquired 1H−13C HSQC spectra of FKBP12 proteins selectively labeled with δ- or ε-SAIL Tyr, in the rapamycin- and FK506-bound states, at 10−40 °C (Figure 2). In the case of the rapamycin-bound state, the δ-CH of Tyr26 appeared at 40 °C as a broad single peak whereas the ε-CH signal of Tyr26 was already broadened beyond detection (Figure 2A,B). Similar but more notable effects were observed for the Tyr26 signals in the FK506-bound state, as shown in panels C and D of Figure 2. Namely, the ε1/ε2- and δ1/δ2-CH 6986

DOI: 10.1021/acs.biochem.5b00820 Biochemistry 2015, 54, 6983−6995

Article

Biochemistry

Table 1. Aromatic Ring 1H and 13C Chemical Shifts (parts per million) of the Tyr Residues in the δ-, ε-, and ζ-SAIL Tyr-Labeled FKBP12, in Their Free, Rapamycin-Bound, and FK506-Bound States, Measured at 30 °Ca 13

Cδ/1Hδ

free FKBP12

rapamycin-bound FKBP12

Tyr26 Tyr80 Tyr82 Tyr26b

FK506-bound FKBP12

Tyr80 Tyr82 Tyr26b Tyr80 Tyr82

132.70/7.00 130.74/6.72 132.85/7.11 132.06/7.05 132.40/7.12 130.63/6.71 132.72/7.14 132.40/7.06 132.23/7.16 130.64/6.72 133.13/7.25

13

Cε/1Hε

117.87/6.61 118.77/6.85 118.03/6.58 117.13/6.83 118.76/6.31 118.80/6.83 117.15/6.38 117.59/6.89 118.29/6.31 118.83/6.85 117.10/6.58

(δ1) (δ2)

(δ1) (δ2)

Hη

kex (s−1)c

158.64 158.27 157.55 158.68

− − − 8.68

− − − 5.26 ± 0.61

158.39 158.56 159.02

− 7.44 9.75

− 5.98 ± 0.86