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Selective radical trifluoromethylation of native residues in proteins Mateusz Imio#ek, Gogulan Karunanithy, Wai-Lung Ng, Andrew J. Baldwin, Veronique Emilie Gouverneur, and Benjamin G. Davis J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b10230 • Publication Date (Web): 05 Jan 2018 Downloaded from http://pubs.acs.org on January 5, 2018
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Journal of the American Chemical Society
Selective radical trifluoromethylation of native residues in proteins Mateusz Imiołek, Gogulan Karunanithy, Wai-Lung Ng, Andrew J. Baldwin, Véronique Gouverneur, Benjamin G. Davis* Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K. Supporting Information Placeholder ABSTRACT: The incorporation of fluorine can not only significantly facilitate the study of proteins but also potentially modulate their function. Whilst some biosynthetic methods allow global residue-replacement methods, posttranslational fluorine incorporation would constitute a fast and efficient alternative. Here, we reveal a mild method for direct protein radical trifluoromethylation at native residues as a strategy for symmetric-multi-fluorine incorporation on mg scales with high recoveries. High selectivity towards tryptophan residues enhanced the utility of this direct trifluoromethylation technique allowing ready study of fluorinated 19 protein constructs using F-NMR.
Although absent from nearly all-natural products, small organofluorine compounds have found widespread applica1 19 18 tions from materials , to clinical diagnostics ( F-MRI / F2,3 4 PET) and medicinal chemistry (where 30% of new small molecule drugs contain F to alter physicochemical proper5 6 ties and/or modulate affinity ). Incorporation of fluorine into biomolecules is more rare but potential applications in 7 structural re-engineering and as reporter tags for ‘zero19 8,9 background’ F-NMR raise striking possibilities. For the latter, the benefits include: high sensitivity, extreme responsiveness to local environment, broad chemical shift range 10 and low reactivity once incorporated. It has helped to eluci11,12 date structural, functional and dynamic interactions in 13 14 receptor function landscapes and enzymic catalysis. Indeed, fluorine’s features can render it superior over other 15 nuclei in, for example, in-cell NMR spectroscopy or protein19 16-19 observed F-NMR (PrOF) protein-ligand binding. 20
Apart from (semi)synthetic ligations, two main strategies are currently used for generating fluorinated proteins: incorporation of fluorinated amino acids using biosynthetic meth21 ods or chemical modification. The former is typically only 22 residue-specific but can be achieved in a site-selective manner through sense or non-sense codon reassignment; (e.g., amber codon suppression for positioning of CF3-L23 phenylalanine ). However, for operational simplicity and to 24 avoid identified issues with the former, the latter modification method has often been preferred as more direct. It has relied largely on reaction of nucleophilic side chains with prosthetic groups bearing fluorine (typically cysteine and 25,26 lysine) often attached via linkers or prosthetics that can significantly perturb structure and create distance impreci-
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sion. A lack of other methods for installing fluorine into biomolecules has therefore been highlighted as a restriction 24,27 due to on the sites that may be studied within proteins; low relative abundance and roles in interiors, aromatic amino acids are attractive targets. Therefore, direct (hetero)aromatic trifluoromethylation could be a powerful tool allowing a) ‘zero/short linker’ fluorine incorporation via b) a 28,29 Here we higher sensitivity multi-equivalent-F system. describe a novel method that exploits the chemoselectivity of radicals in protein chemistry to achieve this aim (Fig. 1).
Figure 1. Direct radical trifluoromethylation of protein substrates. •CF3 radical targeting (hetero)aromatic residues can be generated from sodium trifluoromethanesulfinate (NaTFMS, Langlois’ reagent) under aqueous, oxidative conditions. Direct C–CF3-bond trifluoromethylation presents a striking synthetic challenge in biomolecules. Notably, most strategies for C-H functionalization have been applied only to small molecules using methods or conditions essentially incompat30 31 ible with proteins (elevated temperatures , organic solvents 32 or strong acids ). Among possible strategies, we identified 33 radical-based approaches. Very recently we have demonstrated the benign nature of radicals for C–C-bond formation 34 through use of a designed unnatural amino acid. This illustrated radical efficiency under ambient aqueous conditions and operational simplicity. We therefore considered whether a suitable •CF3 precursor might be generated that could allow natural amino acid modification. 35
Various putative •CF3 sources were tested using model amino acid substrates. Langlois’ reagent (NaTFMS) proved most promising via multiple redox initiation modes (e.g. transi36-38 tion-metal-free, photocatalytic) under mild conditions. Notably, Baran et al have identified that NaTFMS may be used to modify small molecule N-heterocycle caffeine when spiked into cell lysate, which, as they note, has ‘important 39 implications in the area of bioconjugation’. Advantageous19 ly, F-NMR allowed ready reactivity screening of all abundant natural amino acids (SI Fig. S1) and revealed selective 39 reactivity of NaTFMS/TBHP with only aromatic amino ac-
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ids and free cysteine ((hetero)arene-C–H or Cys-S–H trifluoromethylation) and none towards any other natural amino 19 acids. Notably, F-NMR suggested near homogeneous product formation from tryptophan (Trp) and cysteine (Cys); low levels of products from His, Tyr and Phe, appeared as complex mixtures of apparent regioisomers.
Figure 2. Limited-conversion competition assay of natural amino acids (0.03 mmol) revealed chemo- and regio- selectivity towards Trp with minor products (Cys and lower levels 19 of Phe, His, Tyr isomers, determined by F-NMR, LC-MS). See Fig. S3 for parallel reaction in absence of Cys. Next, limited-conversion (~7.5-fold. This could be further increased to krel >~30-fold by lowering the pH to 6 (SI Table T2), potentially due to reduced reactivity of protonated His residues. Notably, the main side reaction was oxidative dimerization of Cys without trifluoromethylation. These observations suggested importantly not only that the cysteine disulfide is itself inert but that in the absence of free Cys, strongly chemo- and regio- Trp-selective trifluoromethylation might be possible in proteins. This was confirmed by a similar competition reaction in the absence of Cys (SI Fig. S3), along with His modification as minor (50%) were obtained at a moderate pH ~6 (Fig. S4), optimal for most biomolecules.
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Figure 3. Reaction of melittin (MWcalc= 2847) with NaTFMS/TBHP. (a) Relative conversions from LC-MS with the optimal reaction time highlighted in blue. (b) Deconvoluted LC-MS spectra of reaction mixtures after 5 min +/- Met. Undesired oxidation (+32) is curtailed by sacrificial reductant giving melittin-TrpCF3 (+68, MWcalc = 2915, MWfound = 2915). Together these promising results prompted investigation in more complex peptidic and protein substrates. First, reaction of N-acetyl-L-tryptophanamide confirmed reactivity and identical regio-selectivity to that seen for Trp. Secondly, as a simple model peptide, we chose melittin – a 26-residue peptide with a single Trp residue at Trp19. Use of excess NaTFMS/TBHP with methionine as an oxidative buffer allowed short reaction times (Fig. 3). The exclusive site of modification at Trp19 was confirmed by LC-MS/MS analysis (SI Fig. S3); Trp19 oxidation and dual Trp19 CF3-ylation was found observed under prolonged conditions. 4-hydroxy TEMPO not only allowed ready termination of the reaction at varied timepoints but also confirmed the radical-mediated nature of the observed direct trifluoromethylation.
Figure 4. Direct trifluoromethylation of proteins (a) Myoglobin (Mb, MWcalc = 16951.5) deconvoluted LC-MS spectrum showing singly- (MWcalc = 17019, MWfound = 17020) and doubly- modified (MWcalc = 17087, MWfound = 17087), (b) Tryptic-LC-MS/MS confirmed site-selectivity at Trp7 and Trp14. As a natural protein substrate, we chose model haemoprotein – horse heart myoglobin (Mb). Mb contains two buried Trp (Trp7, Trp14) in the presence of several other potentially reactive, more accessible residues (SI Fig. S6). Optimization of conditions (pH, temperature, reactants loading, SI Table T4, SI Fig. S7) allowed modulation of reactivity by CF3 copynumber. In this way, at pH 6 (100 mM ammonium acetate), 0˚C, using 200 eq. NaTFMS after 10 min we were able to create myoglobin bearing primarily one or two CF3 groups (Fig. 4a). Notably higher equivalents of Langlois’ reagent allowed further shortening of reaction times, without apparent adverse effects on protein (SI Table T4, entries 5,6). In contrast 39 to literature observations, we observed no significant influ2+ ence of Zn as an additive. Control of pH also minimized concomitant oxidation (SI Fig. S8). Tryptic digest-LCMS/MS revealed that Trp7 and Trp14 were the dominant site of modification (Fig. 3b and SI Fig. S9) despite their buried nature, highlighting the low steric hindrance of •CF3, even in these congested environments. This accessibility to buried sites complements existing nucleophilic, prosthetic-mediated
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Journal of the American Chemical Society methods, which are more applicable to exposed sites, requir41-43 ing partial denaturation to access buried sites. Similarly, accessibility is needed for standard labelling of Cys with fluorinated tags. Importantly, CD spectroscopy revealed that trifluoromethylation had negligible influence on the structure of Mb-TrpCF3 protein (SI Fig. S10). Next, we tested the prospective utility of our developed di19 rect trifluoromethylation as a tool for enabling protein FNMR. Pleasingly, mg-scale reactions allowed straightforward ‘labelling’ of Mb with CF3 with ~65-80% recovery. As a proof19 of-principle, we used F-NMR to directly probe sites of modification (Fig. 5a). The native protein spectra of modified 19 Mb revealed two major F-peaks. To elucidate their origin protein was digested with trypsin (to free residues from their microenvironment and remove non-equivalence); the resonances collapsed to δF -58.8 ppm, in good agreement to that for 2-CF3-Trp in model Trp-CF3 bis-amide (δF -58.76). This 19 was further reinforced by denaturative F-NMR structural 44 analyses : titration with urea induced a gradual change in chemical shift (Fig. 5b) that could be explained by a known progressive loss of secondary structure in Mb under such 45 conditions. Treatment with 10M urea resulted in full coa46,47 lescence to δ-58.74 ppm (Fig. 5a, full spectrum Fig. S11).
19 Figure 5. F-NMR analyses enabled by direct trifluorometh19 ylation. (a) F-NMR of CF3Trp-Mb (700 µM in 100 mM NH4OAc, pH 8, blue), denatured by 10M urea (300 µM, red) and peptide mixture from tryptic digest (from 2 mM protein, green). Dashes indicate chemical shift for small molecule model. Inset at higher field with individual Trp resolved. (b) 19 Urea titration by F-NMR (protein 200–400 µM). (c) Chemical shift differences from ligands in PrOF, protein concentration (100–300 µM). We extended essentially the same protocol to other model proteins with different folds and Trp copy number: pantothenate synthetase (PanC with single Trp306) and more demanding enzyme lysozyme (Ly), which possess six potentially reactive Trp (Trp28, Trp62, Trp63, Trp108, Trp111 and Trp123) and eight Cys engaged within each other as disul19 fides. Both tryptic-LC-MS/MS and F-NMR (SI Fig. S12-17) not only confirmed Trp residues as primary modification sites in both proteins but also revealed no reaction of Cys residues, consistent with our prior observations. Thermal denaturation also gave rise to resonance coalesence, as for Mb (SI Fig. S19). We were also able to determine that trifluoromethylation had negligible influence upon both the structure of Ly (via CD, SI Fig. S20) but also upon its enzymatic
function (SI Fig. 22, SI Table T5), which was essentially unaltered. We were also able to use this fully active CF3-ylated Ly 19 16-19 in quantitative F-NMR PrOF (SI Fig. 21) to directly determine the binding of known alkaloid inhibitor ligand berberine (Kd 20 µM, consistent with values determined by 48 complementary methods ). The potential of our direct trifluoromethylation method was also demonstrated in more complex structural experiments for different protein-ligand 16-19 states studied by PrOF. This allowed observation of displacement of water bound at hemin iron (which we con49 firmed by UV-Vis was retained after CF3-ylation, Fig. S23) 50 by known ligands cyanide, fluoride, imidazole (Im) (Fig. 5c, 16 full spectra Fig. S24) . We also tested effects of common 51 osmo/cryo- protectants (sarcosine – Sar, trehalose - Tre) 52 and putative ligand phenol. CN , Im caused clear upfield 47 shifts, consistent with being known specific ligands. Both Trp residues are located within the same helix; both signals reported changes, yet these were more pronounced for moretightly packed Trp14 (Fig. S26). Interestingly, known ligand F did not trigger a change in chemical shift suggesting that this small ligand has a negligible impact on overall protein conformation. Conversely, Tre and Sar caused globallyobserved changes, consistent with non-specific interactions or changes in solute properties, in agreement with the mode 53 of actions of these compounds. Phenol, which inhibits the 52 dehaloperoxidase activity of myoglobins, showed only small changes implying only non-specific binding, likely not at hemin (Fig. S25). In conclusion, we have demonstrated direct trifluoromethylation of natural residues in proteins. Tuned radical chemistry allows fast (
