Synthesis of Perfluoro-tert-butyl Tyrosine, for Application in ¹⁹F NMR, via a Diazonium Coupling Reaction

Abstract

A practical synthesis of the novel highly fluorinated amino acid Fmoc-perfluoro-tert-butyl tyrosine was developed. The sequence proceeds in 2 steps from commercially available Fmoc-4-NH₂-phenylalanine, via diazotization followed by diazonium coupling reaction with perfluoro-tert-butanol. In peptides, perfluoro-tert-butyl tyrosine was detected in 30 seconds by NMR spectroscopy at 500 nM peptide concentration, due to 9 chemically equivalent fluorines that are a sharp singlet by ¹⁹F NMR. Perfluoro-tert-butyl ether has an estimated σ_p Hammett substituent constant of +0.30.

Graphical Abstract

Sensitivity in NMR spectroscopy is limited by the concentration of the analyte (typically requiring mid-micromolar concentration for rapid analysis) and by challenges in resolution due to spectral overlap (due to competing signals obscuring the signals of interest). These problems are particularly acute for the study of proteins and for investigations in complex solutions. The problem of spectral overlap in proteins can be partially overcome via site-specific isotopic labeling, such as using ¹⁵N-, ¹³C- and/or ²H-labeled amino acids and selective pulse sequences to spectroscopically isolate these amino acids. These approaches have allowed the investigation of protein function in media as complex as living E. coli and human cells and Xenopus oocytes, albeit at a substantial cost in sample preparation, experimental complexity, and instrument time.¹

Alternatively, the introduction of NMR-active nuclei that are not typically present in biological systems provides an approach that results in NMR signals that are only associated with the molecules of interest, eliminating the challenges associated with background signals and spectral assignment. ¹⁹F has been the most widely used non-native NMR-active nucleus applied for interpretation of biological structure and function.² Fluorinated molecules have been used for analysis of events as diverse as protein folding in cells, screening of pharmaceutical inhibitors of protein function, and MRI.^{3 19}F has 100% natural abundance and sensitivity similar to ¹H. Moreover, due to the absence of background signals, including from water, experiments can, compared to ¹H NMR, be typically conducted with increased gain and simpler pulse sequences, further increasing sensitivity. However, despite these substantial advantages, the inherent sensitivity limits of NMR spectroscopy result in an inability to probe many processes at physiologically relevant concentrations.

Increasing the concentration of equivalent signals in a molecule can substantially increase sensitivity and decrease the concentrations accessible for analysis. Thus, highly fluorinated molecules employing multiple chemically equivalent fluorines have found applications of small molecules, polymers, and proteins, in NMR spectroscopy and in MRI.^{3d, 4}

We recently described the synthesis of two conformationally biased amino acids containing perfluoro-tert-butyl groups, 4R- and 4S-perfluoro-tert-butyl hydroxyproline.^{3c, 5} These amino acids were synthesized either in solution phase or within peptides on solid phase via Mitsunobu reaction of perfluoro-tert-butanol with the diastereomeric hydroxyprolines. Peptides containing perfluoro-tert-butyl hydroxyprolines have distinct conformational preferences and, owing to the presence of 9 chemically equivalent fluorines, can be rapidly detected at high nanomolar concentrations (5 minutes at 200 nM peptide concentration).

Given this high sensitivity achievable by ¹⁹F NMR, we sought to extend the applications of perfluoro-tert-butyl groups to applications with aromatic amino acids. In particular, we were inspired by recent work showing that tert-butyl tyrosine (Tby) (Figure 1) can be incorporated in expressed proteins via amber codon suppression using an evolved aminoacyl tRNA synthetase or pyrrolysyl tRNA synthetase.^{4g, 6} Tby was observed by ¹H NMR in expressed proteins as a sharp singlet, due to 9 equivalent hydrogens.⁷ Moreover, this sharp singlet is present even when this amino acid is in proteins as large as 320 kDa: multiple bond rotations of the side chain result in isotropic averaging despite the slow rotational correlation times that typically prevent standard NMR spectroscopy of proteins over 30 kDa. Similarly, isotropic averaging of trifluoromethylphenylalanine allowed the detection of a 98 kDa protein in living E. coli cells.^3d These data suggest significant advantages of tyrosine-tert-butyl ethers in the NMR spectroscopy of large proteins. Therefore, we sought to develop an approach to the synthesis of the amino acid perfluoro-tert-butyl tyrosine, which could combine high signal sensitivity (9 equivalent fluorines) with the potential for future incorporation in expressed proteins and detection in high molecular weight complexes due to isotropic signal averaging.

Figure 1.

tert-Butyl tyrosine (Tby) and its fluorinated analogue. Arrows indicate bond rotations that result in isotropic averaging of the NMR-active ¹H nuclei in Tby, even in large proteins.

There is only one previous synthesis of a molecule with a perfluoro-tert-butyl aryl ether, which was prepared via a diazonium coupling reaction with perfluoro-tert-butanol.⁸ Therefore, we examined the synthesis of Fmoc-perfluoro-tert-butyl tyrosine using the commercially available amino acid Fmoc-4-NH₂-phenylalanine (1) as the starting material (Scheme 1). Diazotization of 1 using sodium nitrite in tetrafluoroboric acid generated the intermediate diazonium 2, which has not previously been described and which was used in the subsequent step without purification. Diazonium coupling was achieved via heating 2 in perfluoro-tert-butanol at reflux, generating Fmoc-perfluoro-tert-butyl tyrosine 3 in good yield in two steps from commercially available starting material, with only a single purification step. This highly practical synthesis, which can be completed in 24 hours, should encourage broad application of this amino acid. In addition, removal of the Fmoc group generated the free amino acid, for potential applications in protein expression and protein engineering. Warning: while tetrafluoroborate salts of diazoniums exhibit greater stability than other diazonium salts, these reactions inherently generate nitrogen gas, yielding potential hazard from explosion. Reactions were conducted in glassware open to air, and the diazonium salt was used immediately after generation, without storage of this intermediate.

Scheme 1.

Synthesis of perfluoro-tert-butyl tyrosine.

Diazonium coupling is a versatile approach to the modification of aromatic rings, via direct coupling or via metal-mediated reactions, including Sandmeyer-type reactions and palladium-catalyzed (Heck, Suzuki, Stille, carbonylation) reactions.⁹ Diazotization of the free amino acid 4-amino-phenylalanine (synthesized from 4-nitro-phenylalanine) has previously been applied to the synthesis of 4-chloro, 4-azido, 4-tetrazole, 4-thiol-, and 4-selenol-phenylalanine derivatives, as well as phenylalanine derivatives containing nitrogen and sulfur mustards.¹⁰ Diazonium salts may also be converted to azo compounds or may be directly reduced to hydrazines. The facile generation of the diazonium of Fmoc-4-NH₂-phenylalanine suggests that this approach may be highly practical and broadly applicable to the synthesis of diverse and functionally useful 4-substituted phenylalanines, for direct application in Fmoc solid-phase peptide synthesis.

To examine the structural and electronic effects of the perfluoro-tert-butyl aryl ether, Fmoc-perfluoro-tert-butyl tyrosine 3 was incorporated via solid-phase peptide synthesis in the model peptide context Ac-TXPN-NH₂, where X = an aromatic amino acid.¹¹ These peptides exhibit cis-trans isomerism about the aromatic-proline amide bond due to a proline/aromatic C–H/π interaction, with the extent of cis amide bond (greater cis amide bond = reduced K_trans/cis) correlating with the electronics of the aromatic π face. This peptide was synthesized via standard solid-phase peptide synthesis, with optimization in yield achieved via PEG-polystyrene graft resin, 24 hour amide coupling with 3, and HATU as a coupling reagent. Subjection of the peptide to TFA cleavage/deprotection conditions using thioanisole and water as scavengers cleanly generated the peptide Ac-TTyr(C₄F₉)PN-NH₂ (Scheme 2a). In contrast, the use of triisopropylsilane (TIS) as a scavenger resulted in product decomposition, generating the peptide with phenylalanine (elimination of the perfluoro-tert-butyl alcohol, Scheme 2b), presumably via an SNAr mechanism with the silyl hydride under acidic conditions. These results were reproduced using the purified peptide Ac-TTyr(C₄F₉)PN-NH₂, indicating the compatibility of perfluoro-tert-butyl tyrosine with TFA cleavage/deprotection conditions as long as hydride sources are avoided. In addition, the purified peptide was exposed to both 20% piperidine in DMF and 8% DIPEA in DMF for 24 hours (Scheme 2cd). No significant decomposition was observed under either condition, confirming the compatibility of perfluoro-tert-butyl tyrosine with extended exposure to reagents employed in peptide synthesis.¹²

Scheme 2.

Stability of Tyr(C₄F₉) in a peptide to standard conditions of (a, b) TFA cleavage/deprotection, (c) Fmoc deprotection, and (d) amide coupling.

An aryl perfluoro-tert-butyl ether has only been described once previously,⁸ and thus no data exist on the aromatic electronic effects of a perfluoro-tert-butyl ether. Peptides with 4-substituted phenylalanines in the Ac-TXPN-NH₂ context (X = 4-Z-Phe) exhibit a Hammett correlation between log K_trans/cis and the Hammett σ constant of the 4-substituent (ρ = 0.295 ± 0.017 across 13 neutral 4-substituents).^{11c, 11e} Therefore, the K_trans/cis of Ac-TTyr(C₄F₉)PN-NH₂ could be employed to provide an estimate of σ for the –OC₄F₉ group. The K_trans/cis = 4.1 measured for this peptide (Figure 2) correlates to a σ of approximately +0.30, indicating a modestly electron-withdrawing effect for this group, despite 9 fluorines. These results are consistent with other fluorinated ethers, which are electron-withdrawing in contrast to the analogous hydrocarbon ethers, but less electron-withdrawing than the equivalent fluorocarbons (Table 1).¹³ Thus, the electronic properties of the perfluoro-tert-butyl aryl ether are affected by a balance of the electron-donating character of the ether and the electron-withdrawing character of the 9 fluorines, whose effect is reduced because they are located 4 atoms away from the aromatic ring.

Figure 2.

(a) cis-trans isomerism of the aromatic-proline amide bond (red) in Ac-TXPN-NH₂ peptides (X = aromatic amino acid) can be used to quantify aromatic electronic effects, via K_trans/cis and via the δ of proline Hα when in the cis conformation (with smaller K_trans/cis and more upfield Pro_cis Hα δ indicating a more electron-rich aromatic and a stronger C–H/π interaction). (b) ¹H NMR spectra (amide-aromatic region, 90% H₂O/10% D₂O, 5 mM phosphate pH 4, 25 mM NaCl) of peptides with X = Tyr (Z = OH) (blue) and X = Tyr(C₄F₉) (Z = OC₄F₉) (red). Pro Hα for X = Tyr, δ_trans = 4.42 ppm, δ_cis = 3.83 ppm; Pro Hα for X = Tyr(C₄F₉), δ_trans = 4.40 ppm, δ_cis = 4.00 ppm.

Table 1.

Hammett σ_p substituent constants for alkyl and alkyl ether groups.^a

substituent	σp	substituent	σp
−CH3	−0.07	−OCH3	−0.27
−CHF2	+0.32	−OCHF2	+0.31
−CF3	+0.54	−OCF3	+0.35
−CF2CF3	+0.52	−OCF2CF3	+0.28
−C(CH3)3	−0.20	−OC(CH3)3	−0.29
−C(CF3)3	+0.55	−OC(CF3)3	+0.30b

^aValues from ref 13, except as indicated.

^bEstimated from results herein.

We also examined the solution-phase synthesis of perfluoro-tert-butyl tyrosine within peptides containing 4-NH₂-phenylalanine (Scheme 3). Diazotization of Ac-TPhe(4-NH₂)PN-NH₂ followed by heating with perfluoro-tert-butanol generated the peptide Ac-TTyr(C₄F₉)PN-NH₂, which was identical by NMR to the peptide synthesized using 3. While this approach will not be compatible with all amino acids (e.g. potential side reactions with Tyr, Cys, Met)¹⁴, it provides an alternative approach to the synthesis of peptides containing Tyr(C₄F₉), and is also suggestive of the general synthesis of peptides containing diverse unnatural substituted phenylalanines via diazonium coupling reactions.

Scheme 3.

Solution-phase diazotization and synthesis of Tyr(C₄F₉) within peptides.

In order to examine potential magnetic resonance applications of perfluoro-tert-butyl tyrosine, Ac-TTyr(C₄F₉)PN-NH₂, was examined by ¹⁹F NMR spectroscopy. Ac-TTyr(C₄F₉)PN-NH₂ exhibited a sharp singlet peak (δ = –69.1 ppm) in water at 298 K (Figure 3a). Interestingly, the ¹⁹F δ of the cis and trans rotamers were identical, consistent with the isolation of the fluorines from the backbone conformation of the peptide. To determine the sensitivity of perfluoro-tert-butyl tyrosine, the ¹⁹F NMR spectrum of this peptide was examined at 500 nM and 200 nM peptide concentrations. Perfluoro-tert-butyl tyrosine was detected in 30 seconds (8 scans) at 500 nM [Ac-TTyr(C₄F₉)PN-NH₂], and in 5 minutes (128 scans) at 200 nM [Ac-TTyr(C₄F₉)PN-NH₂] (Figure 3b, Figures S10-S12), suggesting broad potential applications of this amino acid to sensitively probe peptide and protein structure and function.

Figure 3.

¹⁹F NMR spectra of the peptide Ac-TTyr(C₄F₉)PN-NH₂ in 90% H₂O/10% D₂O, 5 mM phosphate pH 4, 25 mM NaCl. (a) As in Figure 2b. (b) Peptide concentration = 500 nM, signal/noise = 10.1. The experiment was conducted with 8 scans, 2 dummy scans, acquisition time = 0.8 s, relaxation delay = 2.0 s (28 seconds total experiment time).

We have described the highly practical synthesis of Fmoc-perfluoro-tert-butyl tyrosine in two steps from the commercially available amino acid Fmoc-4-NH₂-phenylalanine. The synthesis proceeds via a diazonium coupling reaction. Ready access to this Fmoc-protected phenylalanine diazonium intermediate could provide highly practical access to a wide range of unnatural phenylalanine derivatives for applications in peptide synthesis. Perfluoro-tert-butyl tyrosine was detected at 500 nanomolar peptide concentration in 30 seconds by ¹⁹F NMR, promoting applications of this amino acid in the detection of protein function and protein interactions at physiologically relevant concentrations. Combined with the previously demonstrated ability of tert-butyl tyrosine to be encoded within proteins and to be a useful NMR probe even in extremely large proteins beyond the typical size limit of NMR spectroscopy, these results suggest broad potential future applications of perfluoro-tert-butyl tyrosine in NMR spectroscopy and MRI imaging.