Fluorine Label Research Articles

Selective patterning of covalent molecular grafting on doped amorphous silicon templates

AbstractLinear alkene molecules have been covalently immobilized on amorphous silicon surfaces. The larger thermal reactivity of n+‐doped vs. nominally undoped a‐Si:H surfaces towards perfluorodecene (PFD) molecules in the gas phase has been exploited to investigate a selective patterning of molecular grafting on a previously defined template of doped / undoped regions. PFD has been chosen to obtain hydrophobic monomolecular layers with fluorine labelling, useful for photoelectron spectroscopy (XPS) and NanoSIMS imaging.The template was obtained by growing undoped a‐Si:H on top of n+ a‐Si‐H, and defining 40 μm‐edge patterns of undoped a‐Si:H by reactive ion etching in a (CF4, O2) mixture. After native oxide removal, the patterned surface was exposed to PFD at 230 °C. NanoSIMS images, taken at increasing Cs+ irradiation doses, show the expected 19F and 28Si contrast on as‐grafted surfaces; this contrast disappears after ∼340 seconds Cs+ irradiation, corresponding to the desorption of the PFD monomolecular layer. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Fluoroglycoproteins: ready chemical site-selective incorporation of fluorosugars into proteins

A tag-and-modify strategy allows the practical synthesis of homogenous fluorinated glyco-amino acids, peptides and proteins carrying a fluorine label in the sugar and allows access to first examples of directly radiolabelled ([(18)F]-glyco)proteins.

19F‐MAS NMR on Proteorhodopsin: Enhanced Protocol for Site‐Specific Labeling for General Application to Membrane Proteins†

Proteorhodopsin (PR) is a light-driven proton pump found in near-surface marine gamma-proteobacteria. The green absorbing variant has three cysteines at positions 107, 156 and 175. We probed the accessibility of these residues by (19)F-MAS NMR. For this purpose, an efficient but simple protocol for chemical fluorine labeling of accessible cysteines in membrane proteins was established. This one-step reaction was applied to detergent-solubilized PR before reconstitution into phospholipids. All three cysteines could be labeled and showed distinct (19)F chemical shifts with different integral intensities. The accessibility of these cysteines is discussed in the context of a homology model. With the chemical cysteine labeling procedure shown here, an attractive option for site-directed solid-state NMR studies on other membrane proteins is offered due to the high intrinsic sensitivity of (19)F-MAS NMR.

Preparation of N β-Fmoc-Protected Aza-β³-Amino Acids with Nonproteino­genic Hydrophobic Side Chains for Solid-Phase Syntheses of Pseudopeptides

The preparation of twelve new N b -Fmoc-protected aza- b 3 -amino acids (aza-b 3 aa) with nonproteinogenic hydrophobic side Due to proteolytic degradation, peptides are not ideal can- didates for pharmaceutical development. For this reason, numerous research groups try to develop non-natural pep- tidic analogues in order to enhance metabolic stability, bioavailability, and biological absorption. 1-4 In this class of peptidomimetics, pseudopeptides either consisting ex- clusively or including aza-b 3 -amino acids have emerged as a promising new class of compounds that favor hydro- gen bond formation and can enhance biological activi- ties. 5-7 N b -Fmoc-protected aza-b 3 -amino acids with both nonfunctionalized and functionalized side chains that are analogues of natural proteinogenic amino acids have been already published, 8-10 and we have demonstrated that pseudopeptides including aza-b 3 -amino acids can be more active than the natural parent peptides. 6 In light of the in- stability of aza-b 3 -Tyr and aza-b 3 -Trp analogues in a strongly acidic medium, and in the context of our ongoing projects on the synthesis of antimicrobial pseudopeptides, we needed to develop N b -Fmoc-protected aza-b 3 -amino acid building blocks with non-proteinogenic side chains that are more hydrophobic than natural side chains and more stable. We already know that Trp residues can be re- placed by naphthylalanine, pyridylalanine, biphenylala- nine residues and many other compounds. 11-13 Furthermore, lipophilic long alkyl chains are very useful for enhancing or modifying antimicrobial activities and cytotoxicity. 14-17 Additionally, the incorporation of fluor- inated amino acids have generated great interest since such modifications can enhance the activity of antimicro- bial peptides. Such modifications can also allow structural NMR studies since fluorine labels are very suitable for an- alyzing molecular orientations. 18-20 Thus, we describe here a detailed preparation of twelve new aza-b 3 -amino acid derivatives (Table 1), with the side chains of 9-anthracenylalanine (9-Ath), 4,4¢-biphenylala-

Synthesis and Biological Evaluation of Pyridazinone Analogues as Potential Cardiac Positron Emission Tomography Tracers

A series of fluorinated pyridazinone derivatives with IC50 values ranging from 8 to 4000 nM for the mitochondrial complex 1 (MC1) have been prepared. Structure-activity relationship (SAR) assessment indicated preference of the fluorine label to be incorporated on an alkyl side chain rather than directly on the pyridazinone moiety. Tissue distribution studies of a series of analogues ([18F] 22-28) in Sprague-Dawley (SD) rats identified [18F]27 as the most promising radiotracer with high uptake in cardiac tissue (3.41%ID/g; 30 min post injection) in addition to favorable heart to nontarget organ distribution ratios. MicroPET images of SD rats and nonhuman primates after [18F]27 administration allowed easy assessment of the myocardium through 60 min with minimal lung or liver interference.

Combined NMR and computational study for azide binding to human manganese superoxide dismutase

Human manganese superoxide dismutase (MnSOD) labeled with 3-fluorotyrosine (Tyf) was complexed with the 15N-labeled inhibitor azide ([ 15N 3 −]). The sample was characterized by solid-state NMR (SSNMR) spectroscopy ( 19F-MAS and 15N-CPMAS). Employing 19F- 15N-REDOR spectroscopy, we determined the distances between the fluorine label in Tyrosine-34 and the three 15N-nuclei of the azide and the relative orientation of the azide in the binding pocket of the MnSOD. A distance of R 1=4.85 Å between the 19F-label of Tyf34 and the nearest 15N of the azide and an azide–fluorotyrosine Tyf34 angle of 90° were determined. These geometry data are employed as input for molecular modeling of the location of the inhibitor in the active site of the enzyme. In the computations, several possible binding geometries of the azide near the Mn-complex were assumed. Only when the azide replaces the water ligand at the Mn-complex we obtained a geometry of the azide–Mn-complex, which is consistent with the present NMR data. This indicates that the water molecule ligating to the Mn-complex is removed and the azide is placed at this position. As a consequence the azide forms an H bond with Gln143 instead with Tyf34, in contrast to non- 19F-labeled MnSOD, where the azide is hydrogen bonded to the hydroxy group of Tyr34.

Synthesis of 7α-(Fluoromethyl)dihydrotestosterone and 7α-(Fluoromethyl)nortestosterone, Structurally Paired Androgens Designed To Probe the Role of Sex Hormone Binding Globulin in Imaging Androgen Receptors in Prostate Tumors by Positron Emission Tomography

Although prostate cancer growth is regulated by androgens through the androgen receptor (AR), in vitro assays of AR levels in prostate tumors have limited prognostic value. This might be improved by direct measurement of tumor AR in vivo using positron emission tomography (PET) imaging with fluorine-18-labeled androgens. Most AR PET imaging agents have been designed to limit steroid binding to serum proteins, but there is evidence that binding to sex hormone binding globulin (SHBG) might enhance tumor uptake. To probe the role of SHBG in prostate tumor uptake of PET imaging agents, we have synthesized two fluoro steroids, 7alpha-(fluoromethyl)dihydrotestosterone (7alpha-FM-DHT) and 7alpha-(fluoromethyl)nortestosterone (7alpha-FM-norT), by a route amenable to their labeling with [18F]fluoride ion. Both compounds have high affinity for AR, but 7alpha-FM-norT has much lower affinity for SHBG. Thus, these two fluoro steroids are well matched in terms of their site of fluorine labeling, similarity of structure, and equivalent AR binding affinity-but contrasting SHBG binding-and therefore can be used as agents for evaluating the role of SHBG binding in the target tissue uptake of AR PET imaging agents in humans.

Site-Specific Incorporation of a 19F-Amino Acid into Proteins as an NMR Probe for Characterizing Protein Structure and Reactivity

19F NMR is a powerful tool for monitoring protein conformational changes and interactions; however, the inability to site-specifically introduce fluorine labels into proteins of biological interest severely limits its applicability. Using methods for genetically directing incorporation of unnatural amino acids, we have inserted trifluoromethyl-l-phenylalanine (tfm-Phe) into proteins in vivo at TAG nonsense codons with high translational efficiency and fidelity. The binding of substrates, inhibitors, and cofactors, as well as reactions in enzymes, were studied by selective introduction of tfm-Phe and subsequent monitoring of the 19F NMR chemical shifts. Subtle protein conformational changes were detected near the active site and at long distances (25 Angstrom). 19F signal sensitivity and resolution was also sufficient to differentiate protein environments in vivo. Since there has been interest in using 19F-labeled proteins in solid-state membrane protein studies, folding studies, and in vivo studies, this general method for genetically incorporating a 19F-label into proteins of any size in Escherichia coli should have broad application beyond that of monitoring protein conformational changes.

19F NMR studies of solvent exposure and peptide binding to an SH3 domain

19F NMR was used to study topological features of the SH3 domain of Fyn tyrosine kinase for both the free protein and a complex formed with a binding peptide. Metafluorinated tyrosine was biosynthetically incorporated into each of 5 residues of the G48M mutant of the SH3 domain (i.e. residues 8, 10, 49 and 54 in addition to a single residue in the linker region to the C-terminal polyhistidine tag). Distinct 19F NMR resonances were observed and subsequently assigned after separately introducing single phenylalanine mutations. 19F NMR chemical shifts were dependent on protein concentration above 0.6 mM, suggestive of dimerization via the binding site in the vicinity of the tyrosine side chains. 19F NMR spectra of Fyn SH3 were also obtained as a function of concentration of a small peptide (2-hydroxynicotinic-NH)–Arg–Ala–Leu–Pro–Pro–Leu–Pro-diaminopropionic acid –NH 2, known to interact with the canonical polyproline II (PPII) helix binding site of the SH3 domain. Based on the 19F chemical shifts of Tyr8, Tyr49, and Tyr54, as a function of peptide concentration, an equilibrium dissociation constant of 18 ± 4 μM was obtained. Analysis of the line widths suggested an average exchange rate, k ex, associated with the peptide–protein two-site exchange, of 5200 ± 600 s − 1 at a peptide concentration where 96% of the FynSH3 protein was assumed to be bound. The extent of solvent exposure of the fluorine labels was studied by a combination of solvent isotope shifts and paramagnetic effects from dissolved oxygen. Tyr54, Tyr49, Tyr10, and Tyr8, in addition to the Tyr on the C-terminal tag, appear to be fully exposed to the solvent at the metafluoro position in the absence of binding peptide. Tyr54 and, to some extent, Tyr10 become protected from the solvent in the peptide bound state, consistent with known structural data on SH3–domain peptide complexes. These results show the potential utility of 19F-metafluorotyrosine to probe protein–protein interactions in conjunction with paramagnetic contrast agents.

Structures of Staphylococcus aureus Cell-Wall Complexes with Vancomycin, Eremomycin, and Chloroeremomycin Derivatives by 13C{19F} and 15N{19F} Rotational-Echo Double Resonance

Solid-state NMR has been used to examine isolated cell walls and intact whole cells of Staphylococcus aureus complexed to five different vancomycin, eremomycin, and chloroeremomycin derivatives. The cell walls and whole cells were specifically labeled with d-[1-(13)C]alanine, or a combination of [1-(13)C]glycine and [epsilon-(15)N]lysine. Each of the bound glycopeptides had a (19)F-labeled substituent at either its C-terminus or its disaccharide position. The (13)C{(19)F} rotational-echo double-resonance (REDOR) dephasing for the cell-wall (13)C-labeled bridging pentaglycyl segment connecting a glycopeptide-complexed peptidoglycan stem with its neighboring stem indicates that the fluorine labels for all bound glycopeptides are positioned at one or the other end of the bridge. An exception is N'-(p-trifluoromethoxybenzyl)chloroeremomycin, whose hydrophobic substituent differs in length by one phenyl group compared to that of oritavancin, N'-4-[(4-chlorophenyl)benzyl)]chloroeremomycin. For this drug, the fluorine label is near the middle of the pentaglycyl segment. (15)N{(19)F} REDOR dephasing shows the proximity of the fluorine to the bridge-link site of the pentaglycyl bridge for C-terminus-substituted moieties and the cross-link site for disaccharide-substituted moieties. Full-echo REDOR spectra of cell-wall complexes from cells labeled by d-[1-(13)C]alanine (in the presence of an alanine racemase inhibitor) reveal three different carbonyl carbon chemical-shift environments, arising from the d-Ala-d-Ala binding site and the d-Ala-Gly-1 cross-link site. The REDOR results indicate a single fluorine dephasing center in each peptidoglycan complex. Molecular models of the mature cell-wall complexes that are consistent with internuclear distances obtained from (13)C{(19)F} and (15)N{(19)F} REDOR dephasing allow a correlation of structure and antimicrobial activity of the glycopeptides.

Solvent-Free Vapor-Phase Photografting of Acrylamide onto Poly(ethylene terephthalate)

Poly(ethylene terphthalate) (PET) films were photografted under reduced pressure in a solvent-free vapor of acrylamide and a co-initiator, benzophenone. Characterization of grafted samples by ESCA and contact angles showed that the grafting increased with grafting time and temperature. The amide groups obtained by the acrylamide grafting were converted into amine groups by the Hofmann rearrangement to be used in coupling reactions. The amine groups were confirmed by reaction with pentafluorobenzoyl chloride, which provides a fluorine label for ESCA. Surface grafting of polymeric substrates in the vapor phase induced by plasma or high energy and UV irradiation is reviewed.

Orientation of the antimicrobial peptide PGLa in lipid membranes determined from 19F-NMR dipolar couplings of 4-CF 3-phenylglycine labels

A highly sensitive solid state 19F-NMR strategy is described to determine the orientation and dynamics of membrane-associated peptides from specific fluorine labels. Several analogues of the antimicrobial peptide PGLa were synthesized with the non-natural amino acid 4-trifluoromethyl-phenylglycine (CF 3-Phg) at different positions throughout the α-helical peptide chain. A simple 1-pulse 19F experiment allows the simultaneous measurement of both the anisotropic chemical shift and the homonuclear dipolar coupling within the rotating CF 3-group in a macroscopically oriented membrane sample. The value and sign of the dipolar splitting determines the tilt of the CF 3-rotational axis, which is rigidly attached to the peptide backbone, with respect to the external magnetic field direction. Using four CF 3-labeled peptide analogues (with l-CF 3-Phg at Ile9, Ala10, Ile13, and Ala14) we confirmed that PGLa is aligned at the surface of lipid membranes with its helix axis perpendicular to the bilayer normal at a peptide:lipid ratio of 1:200. We also determined the azimuthal rotation angle of the helix, which agrees well with the orientation expected from its amphiphilic character. Peptide analogues with a d-CF 3-Phg label resulting from racemization of the amino acid during synthesis were separately collected by HPLC. Their spectra provide additional information about the PGLa structure and orientation but allow only to discriminate qualitatively between multiple solutions. The structural and functional characterization of the individual CF 3-labeled peptides by circular dichroism and antimicrobial assays showed only small effects for our four substitutions on the hydrophobic face of the helix, but a significant disturbance was observed in a fifth analogue where Ala8 on the hydrophilic face had been replaced. Even though the hydrophobic CF 3-Phg side chain cannot be utilized in all positions, it allows highly sensitive NMR measurements over a wide range of experimental conditions and dynamic regimes of the peptide.

Bioorganic chemistry of plant lipid desaturation

The desaturation of long-chain fatty acids is a ubiquitous biotransformation which plays a critical role in the biosynthesis of plant lipids. Species-specific variations lead to unusual fatty acid signatures. Of particular interest is the unique ability of desaturases to oxidize unactivated hydrocarbon chains in such a chemo-, regio- and stereoselective manner. As part of ongoing research into the structure/activity relationships of this large class of enzymes, useful mechanistic tools have been developed to probe the active site. Recently a combination of stereo- and regiospecific deuterium, sulfur and fluorine labelling has been used to study the mechanism of a soluble plant Δ9 desaturase. The study of several membrane-bound desaturases has led to the conclusion that the dehydrogenation (desaturation) process is initiated by a kinetically important hydrogen activation step at the carbon of the incipient double bond which is closest to the acyl terminus of the fatty acid chain. A detailed analysis of a related 1,4-desaturation process has also been carried out.

Imaging TOF-SIMS for the surface analysis of silver halide microcrystals

Photographic materials are interesting to gain insight in the general applicability of TOF-SIMS imaging. On the one hand this study was concentrated on the distribution of elements (in particular halides) and small molecular fragments such as SCN. Using a Ga + liquid metal ion gun the distribution of these compounds could be studied with high lateral resolution, up to 70 nm. Moreover by applying higher primary ion doses, the in-depth distributions of these compounds could be studied for thin conversion layers. On the other hand, the capability for imaging larger organic molecules (organic dyes) was studied. The achievable lateral resolution for this type of compounds is limited due to lower secondary ion yields. In order to overcome this problem specific elements such as fluorine can be introduced into the molecules, this allows direct imaging of the compound at the surface of silver halide microcrystals (using the fluorine label). Analyzing the samples with polyatomic primary ions can improve molecular identification, but at this moment imaging capabilities remain limited.

Role of entropy in protein thermostability: folding kinetics of a hyperthermophilic cold shock protein at high temperatures using 19F NMR.

We used (19)F NMR to extend the temperature range accessible to detailed kinetic and equilibrium studies of a hyperthermophilic protein. Employing an optimized incorporation strategy, the small cold shock protein from the bacterium Thermotoga maritima (TmCsp) was labeled with 5-fluorotryptophan. Although chaotropically induced unfolding transitions revealed a significant decrease in the stabilization free energy upon fluorine labeling, the protein's kinetic folding mechanism is conserved. Temperature- and guanidinium chloride-dependent equilibrium unfolding transitions monitored by (19)F NMR agree well with the results from optical spectroscopy, and provide a stringent test of the two-state folding character of TmCsp. Folding and unfolding rate constants at high temperatures were determined from the (19)F NMR spectra close to the midpoint of thermal unfolding by global line shape analysis. In combination with results from stopped-flow experiments at lower temperatures, they show that the folding rate constant of TmCsp and its temperature dependence closely resemble those of its mesophilic homologue from Bacillus subtilis, BsCspB. However, the unfolding rate constant of TmCsp is two orders of magnitude lower over the entire temperature range that was investigated. Consequently, the difference in conformational stability between the two proteins is solely due to the unfolding rate constant over a wide temperature range. A thermodynamic analysis points to an important role of entropic factors in the stabilization of TmCsp relative to its mesophilic homologues.

Fluorine as an NMR probe for structural studies of chemical and biological systems

Applications of fluorine NMR to probe long-range interactions in rigid organic molecules, namely cubanes, and to study structures and dynamics of proteins and other biological systems, are presented. 19F NMR parameters, especially long-range coupling constants are shown to reveal intrinsic features of the molecules. Correlations between long-range (more than three-bond) couplings involving fluorine and other chemical and physico-chemical properties of the compounds are demonstrated, and examples of unusual couplings which cannot be explained by existing theories of ‘through-bond’ and ‘through-space’ mechanisms are discussed. Recent applications of fluorine labeling of aromatic and aliphatic amino acids in investigating protein structures and dynamics are highlighted. The potential of using 19F NMR parameters, such as large spin–spin coupling constants observed for organic compounds, for structural analysis of biological macromolecules is outlined. Copyright © 2000 John Wiley & Sons, Ltd.

Synthesis of Fluorinated Macrocyclic Bis(indolyl)maleimides as Potential19F NMR Probes for Protein Kinase C

Six macrocyclic bis(indolyl)maleimides 1−6 bearing a fluorine label on the aliphatic portion of the macrocycle have been prepared as potential fluorine NMR probes for the catalytic domain of protein kinase C. The macrocyclic bis(indolyl)maleimides such as LY333531 are reversible, ATP competitive, and isoform-selective inhibitors of protein kinase C and may thus serve to probe for subtle differences between protein kinase catalytic domains. The key stereochemical elements were put in place by a Welch aldol condensation between ethyl fluoroacetate and (R)-cyclohexylidene glyceraldehyde, which was followed by allylation of the secondary alcohol, elaboration of the alkene and ester to alcohols, and mesylation. The macrocycle was formed by slow addition of a mixture of the fluorine-labeled aliphatic dimesylate and N-methyl 2,3-bis[1H-indol-3-yl]maleimide to a suspension of cesium carbonate. Adjusting the functionality led to the six fluorine-labeled macrocyclic bis(indolyl)maleimides. These compounds retain th...

Structural Parameters from19F Homonuclear Dipolar Couplings, Obtained by Multipulse Solid-State NMR on Static and Oriented Systems

Local macromolecular structure can be determined by solid-state NMR measurements of weak dipolar couplings between selectively labeled groups. The nonperturbing use of2H,13C, or15N in biological systems, however, faces drawbacks in terms of a low sensitivity and a comparatively short distance range relative to1H. To extend these limitations, we illustrate the use of19F as an alternative NMR probe. The Carr–Purcell–Meiboom–Gill (CPMG) multipulse sequence was adapted here to measure homonuclear dipolar couplings between two fluorine labels in static samples at 470 MHz. Two lipids (4,4-DMPC-F2, and a difluorinated sterol), which are arranged in liquid crystalline bilayers, serve as models to assess the scope of the technique. In these19F-background-free biological samples, weak couplings down to 100 Hz could be resolved directly from the splitting of the pure dipolar powder lineshape, and1H-decoupling was not required. Order parameters were determined for the anisotropic motion of the lipids, consistent with their expected behavior in the membrane. Besides measuring the distance-dependent term of the dipolar coupling in powder samples, we have also used oriented membranes to extract additional angular information from the dipolar anisotropy. The strategy presented here thus has the potential to obtain not only the internuclear distance between two labels, but also their angular orientation in the sample, provided the molecules are aligned as a membrane or a fiber.

NMR Investigation of the Interactions between 4‘-Fluoro-1‘-acetonaphthone and the Suwannee River Fulvic Acid

The association of 4‘-fluoro-1‘-acetonaphthone (F-acetonaphthone) with the International Humic Substances Society (I.H.S.S.) Suwannee River fulvic acid (SRFA) standard in 55% CH3OD/45% D2O was investigated as a function of SRFA concentration (4.5−13.5 mg/mL) as well as solution pαH* (3.88−7.56). 19F NMR measurements of T1 and T2 relaxation times were used in conjunction with chemical shift and diffusion coefficient measurements to study the interaction, and the results were interpreted using the current association models reported in the literature (1). Analysis of diffusion coefficients measured as a function of SRFA concentration suggests that a small amount of the F-acetonaphthone is associated with SRFA. Unusual behavior of the T1 and T2 relaxation time constants, correlation times calculated from these parameters, and diffusion coefficients were observed for F-acetonaphthone in solutions of SRFA in 55% CH3OD/45% D2O at pαH* 6.10. Analysis of the pαH* dependence of the diffusion coefficients suggests two competing mechanisms of association between F-acetonaphthone and SRFA, hydrophobic interactions, and hydrogen bonding. The contribution of the fluorine label to the interaction of F-acetonaphthone with SRFA was investigated using 19F NOE measurements as well as measurements of competitive binding by unfluorinated acetonaphthone and fluorobenzene.

Conformational isomers of a class II MHC-peptide complex in solution

A number of kinetic measurements of peptide dissociation from class II MHC-peptide complexes provide compelling evidence for the existence of conformational isomers in solution. There is evidence that T-lymphocytes can distinguish such isomers. However, virtually nothing is known about the structure of these isomers. Accordingly, we have investigated a water-soluble version of the murine class II MHC molecule I-E k complexed with an antigenic peptide derived from pigeon cytochrome c residues 89-104 (PCC) by 19F-NMR. Two fluorine labels were placed on the PCC peptide; one fluorine label was placed at a MHC contact site, the other at a position involved in T-cell receptor (TCR) recognition. Introduction of these labels did not alter the observed kinetics of the PCC/I-E k complex. The NMR data show two conformational isomers of this immunogenic complex. The presence of conformational isomers at a TCR contact site suggests that these structures may be recognized differently by the TCR. The agreement between the dissociation kinetics and the 19F-NMR data demonstrate that kinetic heterogeneity is correlated with structural counterparts observed by NMR. Dissociations in the presence of dimethyl sulfoxide were used to show that the rate of interconversion of these conformational isomers at pH 7.0 is low, with a lifetime on the order of hours or more. Modification of a peptide residue of PCC occupying the minor MHC binding pocket P6 alters the 19F-NMR spectra of both labels. This demonstrates that distant changes of amino acid residues can influence the conformation of the whole antigenic peptide inside the MHC binding cleft.