Trifluoroselenomethionine: A New Unnatural Amino Acid

Abstract

Trifluoroselenomethionine (TFSeM), a new unnatural amino acid, was synthesized in seven steps from N-(tert-butoxycarbonyl)-l-aspartic acid tert-butyl ester. TFSeM shows enhanced methioninase-induced cytotoxicity, relative to selenomethionine (SeM), toward HCT-116 cells derived from human colon cancer. Mechanistic explanations for this enhanced activity are computationally and experimentally examined. Comparison of TFSeM and SeM by selenium EXAFS and DFT calculations showed them to be spectroscopically and structurally very similar. Nonetheless, when two different variants of the protein GB1 were expressed in an Escherichia coli methionine auxotroph cell line in the presence of TFSeM and methionine (Met) in a 9:1 molar ratio, it was found that, surprisingly, 85 % of the proteins contained SeM residues, even though no SeM had been added, thus implying loss of the trifluoromethyl group from TFSeM. The transformation of TFSeM into SeM is enzymatically catalyzed by E. coli extracts, but TFSeM is not a substrate of E. coli methionine adenosyltransferase.

Keywords: amino acids; fluorine; methionine gamma-lyase; selenomethionine; trifluoroselenomethionine.

Figure 1

Comparison of methioninase cleavage of methionine (1), trifluoromethionine (3), selenomethionine (5) and trifluoroselenomethionine (7) (as 7b).

Figure 2

Synthesis of (S)-trifluoroselenomethionine (7).

Figure 3

Molecular structure of key intermediate 15 drawn with thermal ellipsoids at the 50% probability level.

Figure 4

Formation and decomposition of the sodium salt of trifluoromethaneselenol (8).

Figure 5

Overlay of Se K near edge spectra for 7 (red curve) and 5 (black curve) for reference. The associated derivative spectra are shown in the inset plot.

Figure 6

Se K k³-weighted EXAFS for 7 and 5 (left); associated EXAFS Fourier transforms (right). Experimental data shown as a solid line, the results of EXAFS curve fitting are shown as a dashed line.

Figure 7

Geometry-optimized density functional structures for 1, 3, 5, and 7 calculated at the B3LYP/6-311+G(2df,2p) level. Additional corresponding HOMO (top) and LUMO (bottom) for each analog, rendered using a 0.03 e^–/au³ contour surface.

Figure 8

The comparative effect of methaneselenol (6) and trifluoromethaneselenol (8), generated by incubating 40 U/L methioninase with 0, 1.25, 2.5 or 5 μM SeM (5) or TFSeM (7) (as 7b), respectively, on the cell growth of HCT116 human colon cancer cells for 16 h. Values are means ± SEM, n = 4. The letters represent whether measured values are significantly different (p < 0.05, Tukey’s contrasts) between different conditions. If two bars share at least one letter, than the difference between them is not statistically significant (p>0.05), but if they do not have a letter in common, than the measured difference between them is statistically significant (p<0.05). The vertical scale represents the viable cell number of HCT116 human colon cancer cells per mL after 16 h treatment. Each treatment was done with one flask, individually, containing 5 mL media with equal amount of cells prior to treatment.

Figure 9

Calculated free energies for reactions of 2, 6, 6a, 6b and 8, with O₂.

Figure 10

Calculated free energies for conversion of perfluorinated thiol 4 and selenol 8 to the corresponding thio- and selenocarbonyl compounds.

Figure 11

Proposed redox cycling of RSe^–, where R = CH₃ or CF₃.

Figure 12

Expression levels of GB1 Val39Met and GB1 Leu5Met. (A) Ribbon diagram of GB1 (PDB ID: 2QMT).^[37] In green are the side chains of the residues mutated to Met in the two variants. Val 39 is located on an internal loop between the α-helix and a β-strand while Leu 5 is located on a β-strand and is packed against the hydrophobic core of the protein. (B) Expression levels of GB1 Val39Met and GB1 Leu5Met assessed by a reducing 16% Tris-glycine gel. Lanes 1, 3, 5 are samples of GB1 Val39Met taken before induction, and lanes 2, 4, 6 are samples of GB1 Val39Met taken after induction with 200 μM Met, 200 μM SeM + 22 μM Met, and 200 μM TFSeM (as 7b) + 22 μM Met, respectively. Lanes 7, 9, 11 are samples of GB1 Leu5Met taken before induction, and lanes 8, 10, 12 are samples of GB1 Leu5Met taken after induction with 200 μM Met, 200 μM SeM + 22 μM Met, and 200 μM TFSeM + 22 μM Met, respectively. Induced and uninduced samples were adjusted to have the same optical density to aid the comparison of expression levels. M denotes molecular weight markers. The star denotes the location of expressed GB1 variants. The molecular weight of GB1 Val39Met with the hexahistidine tag is 8823 Da and of GB1 Leu5Met is 8809 Da. Both run with an apparent molecular weight of 12 kDa due to dimerization. (C) Same as in B but the samples’ optical density was not adjusted to the same value.

Figure 13

Evaluation of TFSeM (as 7b) incorporation into GB1 by ESI-MS. While TFSeM incorporation was not observed, SeM’s presence was detected even though it was not included in the defined growth media. (A) Deconvoluted mass spectrum showing GB1 Val39Met grown with a 9:1 ratio of SeM:Met in RF11. The expected and observed molecular weight for GB1 Val39Met with Met is 6181 Da and with SeM is 6228 Da (observed 6229 Da). (B) Mass spectrum showing GB1 Val39Met grown with a 9:1 ratio of TFSeM:Met in RF11. The expected molecular weight GB1 Val39Met with TFSeM is 6282 Da (not observed). (C) Deconvoluted mass spectrum showing GB1 Leu5Met grown with a 9:1 ratio of SeM:Met in RF11. The expected and observed molecular weight for GB1 Leu5Met with Met is 6167 Da and with SeM is 6214 Da. (D) Deconvoluted mass spectrum showing GB1 Leu5Met grown with a 9:1 ratio of TFSeM:Met in RF11. The expected molecular weight GB1 Leu5Met with TFSeM is 6268 Da (not observed).

Figure 14

Analysis of the molecular forms detected for GB1 Val39Met grown in the Met auxotroph RF11. Molecular species: GB1 Val39Met calculated mass is 6181 Da and detected is 6180 Da; GB1 Val39SeM calculated mass is 6228 Da and detected is 6227 Da. Other putative assignments are: GB1 Val39CF3Sem calculated molecular mass is 6282 Da, GB1 Val39Met with a loss of a methyl group is 6166 Da, GB1 Val39Met with a loss of water is 6161 Da; and GB1 Val39Met with acetylation or an acetonitrile adduct is 6221 Da. (A) Deconvoluted mass spectrum showing GB1 Val39Met grown with solely 22 μM Met. (B) Deconvoluted mass spectrum showing GB1 Val39Met grown with 22 μM Met and 200 μM TFSeM. (C) Deconvoluted mass spectrum showing GB1 Val39Met grown with solely 200 μM TFSeM. (D) ESI-MS spectrum of the peptide QYANDNG(SeM)DGEWTYDDATK derived from trypsin digestion of the sample shown in panel B. The observed isotope pattern matches the calculated one (the predicted pattern is not shown).

Figure 15

Proposed metabolic path for recycling TFSeM into SeM.

Figure 16

HPLC elution profiles of MAT time-dependent reactions with (A) TFSeM (as 7b) or (B) SeM as substrates. Authentic standard compounds are shown in the black dotted traces. Elution times in minutes are as follows: 3.7 (ATP); 4.2 (ADP); 7.8 (AMP); 11.5 (SAM); 9.8 and 12.3 (SeSAM); 18.2 (adenine); 21.8 (SAH); 25.2 (tryptophan IS); 29.7 (MTA); 31.3 (SeMTA). TFSeM resulted in no apparent formation of TFSeSAM or SeSAM over the course of 60 min, whereas SeM was converted to SeSAM by MAT as evidenced by the time-dependent formation of SeSAM (elution times of 9.8 and 12.3 min). The two elution times for SeSAM match that of the authentic standard; under Method 1 conditions SeSAM elutes as one sharp, defined peak, indicating the double peak is a result of the chromatographic conditions.

Figure 17

Extracted ion chromatograms for (A) SeM (m/z 198.1) and (B) TFSeM (m/z 252.0) in time-dependent reactions with E. coli B834(DE3)pLysS crude lysate. Both SeM and TFSeM are consumed as a function of time.