Notes on Synthesis of perdeutero-5-13C,5,5,5-trifluoroisoleucine VI56

The 13CF3 group is a promising label for heteronuclear (19F,13C) NMR studies of proteins. Desirable locations for this NMR spin label include the branched chain amino acid methyl groups. It is known that replacement of CH3 by CF3 at such locations preserves protein structure and function and enhances stability. 13CF3 may be introduced at the δ position of isoleucine and incorporated biosynthetically in highly deuterated proteins. This paper reports our work in synthesis and purification of 5,5,5-trifluoroisoleucine, its perdeutero and 5-13C versions and of 2-13C-trifluoroacetate and its utility as a precursor for introduction of the 13CF3 group into proteins.


Introduction Fluorine NMR and labeling strategies in proteins
Fluorine NMR spectroscopy is a powerful method for the study of both structure and dynamics of proteins and their interactions with other proteins or ligands 1,2,3,4 .
Because of the ability of the 19 F lone-pair electrons to participate in non-bonded interactions with the local environment, 19 F chemical shifts are sensitive to changes in van der Waals contacts, electrostatic fields and hydrogen bonding. As such, 19 F chemical shifts (or changes in shifts) are often indicative of conformational changes 5,6,7,8 , binding 9 , and protein folding or unfolding events 10,11,12 . Fluorinated probes are also frequently used to assess solvent exposure, via chemical shift changes or relaxation effects resulting from: 1) substituting H 2 O for 2 H 2 O 2,14 paramagnetic additives such as Gd 3+ :EDTA 2 heteronuclear nuclear Overhauser effects 13 . In membranous systems, analogous paramagnetic effects are also observed upon addition of nitroxide spin-labels 14 or dissolved oxygen 15,16 , facilitating the study of topology and immersion depth via fluorinated probes. Finally, associated 19 F spin-spin and spin-lattice relaxation rates are useful for studying conformational dynamics over a wide range of timescales, due to the significant chemical shift dispersion, shift anisotropy, and large heteronuclear dipolar relaxation terms 17,18,19,20 .
Fluorine labeling of proteins is achieved in many ways. Via biosynthetic means, monofluorinated versions of tyrosine, phenylalanine, and tryptophan may be substituted for their nonfluorinated equivalents, often with little effect on overall expression yields 2,4 .
Fluorinated versions of methionine 7,21 , proline 22 , leucine 23 , and isoleucine 24 have also been successfully incorporated into proteins. An alternative approach to 19 F labeling of proteins is to make use of a thiol specific fluorinated probe, which frequently consists of a terminal trifluoromethyl group 4 . In this way, a fluorine tag may be placed at virtually any site in the protein, using successive single cysteine mutations of the protein under study. Thus, the majority of 19 F labels used in protein NMR fall under the category of isotopic fluoroaromatic or trifluoromethyl species. For very large proteins, spectral overlap may become problematic for biosynthetically labeled proteins. However, a doubly ( 13 C, 19 F) labeled amino acid should provide greater resolution in twodimensional ( 19 F, 13 C) NMR spectra, with further possibilities of assignment without mutational analysis. Moreover, such two-dimensional NMR schemes should benefit from the relatively large one bond ( 13 C, 19 F) coupling which is between 265 and 285 Hz for both fluoroaromatics and trifluoromethyl groups. Finally, the possibilities for studying dynamics from a 13 C, 19 F pair encompasses a much greater range, since various zero, single, and double quantum coherences in addition to Zeeman and two-spin longitudinal order, may be separately evolved and studied 25,26 . In this paper, we present a method for the preparation of perdeuterated isoleucine, in which the terminal trifluoromethyl group consists of a 13 C-19 F pair. The motivation for this work is to develop a useful doubly labeled species for subsequent nD NMR studies of proteins, whose isoleucine residues have been fluorinated.

Advantages of a trifluoromethyl group
The trifluoromethyl group is expected to be a useful probe of molecular structure and dynamics, particularly in the hydrophobic core of proteins, at the interface between protein complexes, and in the membrane or detergent interior in studies of integral membrane proteins. Expressed within proteins, the CF 3 group offers additional benefits of sensitivity and relatively long transverse relaxation times. However, the inherent slow rotational tumbling associated with large proteins or protein complexes, and membrane proteins, results in line broadening and reduced sensitivity. 19 F spin labels also suffer extensively from dipolar relaxation with nearby proton spins of the protein 2 , which may be largely avoided by extensive deuteration. Furthermore, in situations where 13 C, 19 F two-dimensional NMR schemes are employed, the use of transverse relaxation optimized spectroscopy (TROSY) techniques 27,28 may be considered. The TROSY effect in methyl groups, results from interference between intra-methyl dipolar interactions 28 . As such, the effect is independent of field, to the extent that chemical shift anisotropy does not contribute to relaxation. Since the geometry of the trifluoromethyl group is like that of a CH 3 group, while the gyromagnetic ratio of the 19 F nucleus is 0.83 times that of 1 H, the methyl TROSY effect would be expected to be preserved in appropriate ( 19 F, 13 C) two-dimensional schemes. In particular, in the rigid limit, the maximum peak intensities in the ( 1 H, 13  respectively, the above transverse rates are predicted to be more than three times smaller than those for the CH 3 groups, in the absence of external dipolar relaxation or relaxation due to chemical shift anisotropy.

The trifluoromethyl probe in isoleucine
Considering these anticipated advantages for 1D and 2D NMR, we have developed a protocol for the synthesis and purification of perdeuterated 5,5,5-trifluoroisoleucine, in which the carbon nucleus of the trifluoromethyl group is 13 C enriched. Incorporation, using a cell-free protein expression technique, is reported 55 . We also describe herein a synthesis strategy for 2-13 C-trifluoroacetate and purification of the ammonium salt (or hypothetically CF 3 CO 2 H), to produce perdeutero 5-13 C-5,5,5-trifluoroisoleucine. This report is intended to communicate some of the subtleties involved in these efforts.
Isoleucine has some additional features that make it attractive for ( 19  -helical proteins, no such preference is seen, with these residues equally distributed between the interior and the surface of the protein 32 .

Synthesis outline
The scheme shown below shows the route used to make 5,5,5-trifluoroisoleucine as a racemic mixture of diastereomers 33 . This scheme is modified for perdeuteration by substitution with cyanoacetic acid-d 3 , acetone-d 6 , CF 3 CO 2 D, ammonium-d 4 acetate-d 3 and D 2 /Pd. Modification of this scheme for the 13 CF 3 amino acid requires electrochemical trifluoromethylation using 13 CF 3 CO 2 -.

Experimental
Melting points are uncorrected. 1  was observed not to be critical to yield. One gram of ammonium-d 4 acetate-d 3 will suffice for the synthesis of methallylcyanide-d 7 . The Dean-Stark unit was replaced with a distillation head and the fraction collected between 110 °C and 115 °C.
Compton et al. 35 reports the existence of both 3-methyl-2-butenenitrile and 3-methyl-3butenenitrile in a sample of methallylcyanide. This is consistent with an equilibrium arising from 'active hydrogen' chemistry as evidenced by the 2 H NMR spectrum in Figure 1, below. The predicted boiling points for these isomers are within 2°C, so they cannot be separated by fractional distillation. Accordingly, this product is properly called methallylcyanide 33 rather than 3-methyl-but-3-enenitrile 24 . Appreciation of the active hydrogen nature of this product is required to make methallylcyanide-d 7 .
Synthesis of methallylcyanide-d 7 requires the preparation of cyanoacetic acid-d 3 , first and then its condensation with acetone-d 6   We observed the formation of polymeric materials and so we could not crystallize the product from our reaction mixture. Accordingly, we developed several purification methods discussed in the next section.

Purification of a reaction mixture containing 2-amino-3-methyl-5,5,5trifluoropentanoic acid
Two factors are likely to alter the physical properties of 5,5,5-trifluoroisoleucine relative to native isoleucine. The inductive effect of the CF 3 group 38 will make the amino acid and amino groups more acidic and the greater hydrophobicity of the CF 3 group will enhance the hydrolytic stability of its polymers 39 .
In our hands, reaction of a mixture of diastereomers of 2-bromo-3-methyl-5,5,5trifluoropentanoic acid with aqueous ammonia produced a product mixture that contained a significant quantity of polymeric material. Some of this material was readily soluble in diethyl ether and CDCl 3 . That fraction that was soluble in organic solvent could be hydrolyzed by dissolution in TFA followed by gradual addition of water. We found that the reaction mixture could be stabilized by formation of the TFA salt.
We first chose a method of chemical purification that was appropriate for the partial fluorous character of the amino acid 40 private communication) using a cell free protein expression system proved that the product mixture contained ≤ 25% 5,5,5-trifluoro-L-isoleucine and proved the efficacy of the chemical purification.
During lyophilization, much of the product was lost due to sublimation. Sublimation has recently been revisited as a means for purification of amino acids 43 . We found that our chemically purified product mixture could be sublimed at 150 °C and 6 mm Hg, but that fractional sublimation would require better vacuum and temperature control.
Finally, we exploited ion exchange chromatography using cellulose phosphate to separate monomeric and polymeric fractions. Elution with distilled H 2 O yielded a microcrystalline fraction while elution with dilute aqueous ammonia yielded a waxy fraction identifiable as polymeric material. Refinement of ion exchange column purification should be developed using ion exchange TLC on cellulose phosphate paper.
We explored the use of analytical HPLC-MS first using an acetonitrile gradient in 0.1% aqueous TFA on a C 18 column. 2-amino-3-methyl-5,5,5-trifluoropentanoic acid has a molecular weight of 185. We identified two separated peaks corresponding to [M+] and [M+H+] on an ESI-MS instrument. We conclude that these are the diastereomers and that differential inductive effects due to CF 3 cause differing acid/base properties of the diastereomers. These peaks were followed at longer time by peaks due to polymers. We wished to explore the possibility of preparative scale chromatography where TFA would be counterproductive. An isocratic method was developed using 5% acetonitrile in 0.2% aqueous formic acid on an analytical C 18 column.

Ammonium 2-13 C-trifluoroacetate
To embed the 13 CF 3 group into a synthetic scheme for 5,5,5-trifluoroisoleucine following the synthetic scheme above, a route to 2-13 C-trifluoroacetate is required. Trifluoroacetic acid has been made via electrochemical fluorination 44 . Since this electrolysis would entail use of anhydrous hydrogen fluoride within a customized Teflon reaction vessel, a refrigeration unit, a high current power supply and a process control system, we chose to explore an alternate route involving halogen exchange with 2-13 C-tribromoacetic acid.
We devised a new route to 2-13 C-tribromoacetic acid starting with 2-13 C-ethanol, discussed below.

Halogen exchange reaction design considerations
Preliminary experiments were performed following the halogen exchange reaction originated by 45 . Mass spectral analysis of an early natural abundance test reaction showed that trifluoroacetic acid was formed by reaction of AgBF 4 with CBr 3 CO 2 H in DCM.
However, 13 C NMR multiplet analysis of a test reaction with 13 CBr 3 CO 2 H showed a conversion to 13 CF 3 CO 2 H of only 18% after stirring at for 10 days at room temperature.
A longer reaction time in glassware was found to be counterproductive because of failure of containment. AgBF 4 releases highly aggressive BF 3 during the reaction. The reaction between BF 3 and silica gel 46,47 is useful at the purification stage, however, reaction with ground glass joints may give rise to leakage.

A procedure for making 2-13 C-trifluoroacetic acid
The following procedure was designed to avoid the necessity of handling hydrogen fluoride, either as a solvent, reagent, or product. Synopsis: 2-13 C-ethanol is converted to the tribromoacetaldehyde, 2-13 C-bromal (hydrate) using a molar excess of bromine, Br 2 .
One equivalent of water converts the product mixture to bromal hydrate. Reaction with excess nitric acid at a temperature less than 50 °C converted bromal hydrate to 2-13 Cbromoacetic acid. This product is isolated and converted to 2-13 C-trifluoroacetic acid using AgBF 4 under pressure in dichloromethane. The 2-13 C-trifluoroacetate is extracted into ammonia. Impure ammonium 2-13 C-trifluoroacetate can be enhanced in purity by sublimation at 85 °C and with a vacuum less than 10 microns Hg. The yields were very low.

Conversion of 2-13 C-ethanol to 2-13 C-bromal (hydrate)
The reaction proceeds according to: 2-13 C-ethanol + 4Br 2 → 2-13 C-bromal + 5HBr In a closed system, the above is an equilibrium reaction. To drive the reaction to completion, product HBr gas must be permitted to escape. This loss of mass results in a considerable reduction in the volume of the reaction mixture. We have tried sulfur and I 2 as catalysts. TFA is probably a better catalyst for this reaction. The oxidation potential of Br 2 is not sufficient to carry oxidation beyond the aldehyde. The aldehyde is required for tribromination, because each bromination step proceeds via the enol. To avoid loss of volatiles, 2-13 C-ethanol and excess Br 2 were combined at liquid nitrogen temperature and warmed very slowly to reflux temperature. When the reaction has been driven to completion, 13 C NMR shows the presence of only 2-13 C-bromal (~40 ppm) and its hydrate (~12 ppm). Prior to the next step, it may be desirable to isolate 2-13 C-bromal via distillation, and its hydrate by crystallization but it is not essential. One equivalent of H 2 O is added to convert all to hydrate. 13  was sublimed to improve purity by sublimation at 85°C and less than 10 microns Hg vacuum. Because initial purity was poor, yield was poor. Gram scale quantities of material could be processed in this way.

Manipulation of 13 C haloacetates
The halogen exchange reaction between 2-13 C-tribromoacetic acid and AgBF 4 proceeds in a stepwise fashion and hence yields a mixture of haloacetates. The target compound, 2-13 C-trifluoroacetic acid is too volatile and so the haloacetate mixture is best manipulated as a salt. The ammonium salts have volatilities that were exploited for purification by high vacuum sublimation and the progress of purification was monitored by 19 Table   I).
It is interesting to note that CF 3 I has been enriched to 86% in 13  Another promising route to 13 C enriched TFA that we may explore is that of fluorodeoxygenation 52 , starting with glycine. Recently a new and better synthesis of arylsulfur trifluorides has been reported 53 for reagents that may provide a convenient route to fluorodeoxygenation of carboxylic acids.

Conclusion
We have presented a critical scientific narrative of a promising technology. The advantage of the 13 CF 3 NMR spin label in protein NMR has been explained. Progress in incorporation of this spin label in a perdeuterated amino acid and in an important protein is reported. We comment on some of the synthetic subtleties encountered. This paper covers many areas of synthetic chemistry, organic, fluoro, isotopic, and biochemical. We have identified ammonium 2-13 C-trifluoroacetate as an important synthon for introduction of the 13 CF 3 group into amino acids. Completed synthesis of methallylcyanide-d 7 and conceptual synthesis of 5-13 C-5,5,5-trifluoroisoleucine-d 7 , provide an important building block for the exploitation of 13 CF 3 in protein NMR. 5,5,5-TFI is an unnatural AA, more so than monofluorinated amino acids. Either as uniform, or site specific isotopic labels, deuterium and 13 C amino acids are readily synthesized and incorporated. Fluorine is the 13 th most abundant isotope in the earth's crust, yet even after 3.5b years of biology only about a dozen fluorinated natural products have been evolved, attributed to fluorine's chemistry as a "superhalogen" 54 .
Organofluorine compounds as polymers or as drugs have proven useful in material science and pharmacology. The target spin 13 CF 3 label should prove useful in multidimensional heteronuclear NMR structure dynamics studies of proteins. Synthesis of 5,5,5-TFI has been proven by its incorporation in the calcium binding protein calmodulin. Methallylcyanide-d 7 has been produced with military grade deuterium isotope purity, 99.4%. Trace quantities of 2-13 C-trifluoroacetate have been characterized by 13 C-19 F NMR coupling. This contribution may pave the way to future study.
This work is part of a project in heteronuclear multidimensional NMR 57,58,59 .   Figure 2. These images show conformational changes in calmodulin. On the left is calmodulin without calcium and on the right, is calmodulin with calcium. Sites that bind target proteins are indicated by red stars. These images are from the RCSB Protein Data Bank. http://pdb101.rcsb.org/motm/44, https://meshb.nlm.nih.gov/record/ui?name=Calmodulin