Notes on Synthesis of perdeutero-5-13 C , 5 , 5 , 5-trifluoroisoleucine VI 56

The CF3 group is a promising label for heteronuclear ( F,C) 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. CF3 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-C versions and of 2-Ctrifluoroacetate and its utility as a precursor for introduction of the CF3 group into proteins.


Introduction
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 C ) HMQC are predicted to depend on terms which are derived from relaxation via reorientation of either the CF or FF intramolecular bonds i.e., transverse rates proportional to 6  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  ammonium acetate (4.0 g, 0.05 mol) (or their perdeutero counterparts) in dry benzene (100 mL) were refluxed with a Dean-Stark trap. Reflux was performed until one equivalent of water (or D 2 O) was collected in the trap. Due to the cost of perdeuteroammonium acetate, a minimal amount of this catalyst was used. The quantity 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  The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/140681 doi: bioRxiv preprint first posted online May. 22, 2017; condensation product was 99.4% isotopically pure. This isotopic purity is consistent with that of the reagents used. See the 2 H NMR spectrum in Figure 1. below.

Electrochemical trifluoromethylation of methallylcyanide-d 7
Muller 33 chose to perform electrochemical trifluoromethylation of methallylcyanide in aqueous 90% methanol between platinum electrodes whereas we chose a later system 37 of Because both methallylcyanide-d 7 and 2-13 C-trifluoracetate are very expensive these should be used in 1:1 molar ratio and with 50% excess current. Both authors achieved mild basic condition with 10% Na.
We used a Sargent Slomin S-29460 electrolytic analyzer, for radical trifluoromethylation.
Both rotating and stationary electrodes were platinum. The rotating platinum electrode was chosen to be the anode. At the anode, reactions CF 3 CO 2 Muller's workup 33  The mixture was poured into 700 mL of water, the dense oil collected, and the aqueous layer extracted with two 40 mL portions of dichloromethane. The combined organic layers from three identical runs were distilled to remove the solvent and then steam distilled. The non-aqueous layer was a mixture of 3-methyl-5,5,5-trifluoropentanonitrile, several isomeric 3-methyl-5,5,5-trifluoropentenonitriles, methallyl cyanide, and unidentified by-products. It was diluted with methanol and hydrogenated at low pressure over 5% Pd/C. Distillation afforded 66 g of nearly pure 3-methyl-5,5,5- For the perdeutero route, once the various isomeric perdeutero-3-methyl-5,5,5trifluoropentenonitriles are reduced by D 2 , exchange is no longer possible. Subsequent steps closely follow the protocol outlined by Muller as does this exposition. Quantities were adjusted proportionately.
The distillation product, 3-methyl-5,5,5-trifluoropentanonitrile was stirred with sufficient concentrated aqueous hydrochloric acid to bring most of the organic material into solution, for several days, then diluted with water and refluxed overnight. The separated organic layer was isolated, dried over Na 2 SO 4 , and distilled at 6 torr, giving 58. 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 40,41,42. The use of a C 8 F 17 BOC derivative 42 proved to be problematic because the only rational method for its hydrolysis was the use of TFA The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/140681 doi: bioRxiv preprint first posted online May. 22, 2017; was purified by solid phase extraction on fluorous silica 40 . This derivative was subjected to atmospheric pressure hydrogenolysis over 5%Pd/C and the subsequent product mixture subjected to fluorous SPE. Lyophilization yielded a soluble product that was white. Incorporation of this product in calmodulin (Takeda & Kainosho, 2012) 55  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
. CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/140681 doi: bioRxiv preprint first posted online May. 22, 2017; 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 . CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/140681 doi: bioRxiv preprint first posted online May. 22, 2017; 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: 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.

Conversion of 2-13 C-bromal hydrate to 2-13 C-tribromoacetic acid
This reaction is conducted in dichloromethane. This three-step reaction is exceedingly slow. For practical synthesis, it is necessary to conduct this reaction in a sealed pressure reaction vessel at above 75 °C. The completion of this reaction can be determined by 13  SiO 2 reacts with BF 3 . 2-13 C-trifluoroacetate is extracted into aqueous ammonia and excess removed under vacuum. Ammonium 2-13 C-trifluoroacetate was converted into a fine powder by lyophilization, to aid in sublimation. Ammonium 2-13 C-trifluoroacetate 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 F and 13 C NMR. Whereas high vacuum sublimation has only one theoretical plate, the greater load capacity compared to chromatography affords it an advantage for the first stages of purification. Repeated stages of high vacuum sublimation yielded a product that gave three spots on silica gel TLC. Neutral alumina TLC gave streaks. One spot showed an R f =0.78 on silica gel TLC that was identical to the R f of natural abundance ammonium trifluoroacetate, eluted with 10% aqueous ammonia in methanol. Based on this observation, the ammonium 13 C haloacetate mixture was subjected to preparative TLC under the same conditions. 19 F and 13 C NMR of the product from the preparative TLC . CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/140681 doi: bioRxiv preprint first posted online May. 22, 2017; target band showed that the mixture consisted of ammonium salts of 2-13 Ctrifluoroacetate, 2-13 C-bromodifluoroacetate and 2-13 C-dibromofluoroacetate (See Table   I).
It is interesting to note that CF 3 I has been enriched to 86% in 13 C by selective multiphoton dissociation of 12 CF 3 I at pressures less than 1.0 torr 48 . A more practical method would rely on enrichment in the condensed phase. An isotope effect is often observed on melting points 49 . In recent work, boron has been enriched to 93.21% in 10 B and 99.01% in 11 B by zone refining 50 . It is known that isotope effects on heat capacity and crystal transition temperature can be detected by differential scanning calorimetry 51 .
Zone refining of low melting ammonium trifluoroacetate (M.P. 123 °C) would be more energy efficient than that of high melting boron (M.P. 2079 °C). In future, we plan to develop an analytical HPLC method and to use preparative HPLC for the purification of ammonium 2-13 C-trifluoroacetate. Infrared difference spectroscopy may be able to resolve the carbon isotope effect for ammonium 2-13 C-trifluoroacetate in the condensed phase. Differential scanning calorimetry of ammonium 2-13 C-trifluoroacetate will provide the thermodynamic information needed to plan and develop a zone refining method for the extraction of ammonium 2-13 C-trifluoroacetate from an inexpensive natural abundance melt.
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.
Whereas incorporation of monofluorinated aromatic amino acids in proteins using in vivo or in vitro expression systems is now routine, incorporation of trifluoromethyl analogs of branched chain amino acids is at present a challenging technology. A biochemical hurdle is the specificity of an aminoacyl tRNA synthetase. Wang et al. 24 determined that the specificity constant, k cat /K M of 5,5,5-trifluoroisoleucine is only 1/134 that of isoleucine for E. coli isoleucyl tRNA synthetase. Considerations of enzyme kinetics and competitive inhibition dictate that the background concentration of isoleucine needs to be effectively zero for tRNA Ile to be charged with 5,5,5-trifluoroisoleucine by isoleucyl tRNA synthetase. In addition to the catalytic domain, where the amino acid and tRNA aa are specifically ligated, aminoacyl tRNA synthetase also has an editing domain for the hydrolysis of mischarged tRNA. In the case of isoleucyl tRNA synthetase, the editing domain has been evolved most specifically for the hydrolysis of val· tRNA Ile . The successful incorporation of 5,5,5-trifluoroisoleucine in mDHFR, mIL II (24) and in calmodulin 55 (M. Kainosho: private communication) implies that 5TFI· tRNA Ile is too large for the editing site. Practical questions remain. Is an expression system based upon E. coli more advantageous than one based upon a eukaryotic organism? Is a cell free lysate system preferable to in vivo expression system? 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 . CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/140681 doi: bioRxiv preprint first posted online May. 22, 2017; 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 .
. CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/140681 doi: bioRxiv preprint first posted online May. 22, 2017;   The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/140681 doi: bioRxiv preprint first posted online May. 22, 2017;