Trifluoroselenomethionine: A New Unnatural Amino Acid

Article abstract of DOI:10.1002/cbic.201600266

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.

Full text of DOI:10.1002/cbic.201600266

Accepted Article
Title: Trifluoroselenomethionine - a New Non-Natural Amino Acid
Authors: Eric Block; Squire Booker; Sonia Flores-Penalba; Graham George; Sivaji
Gundala; Brad Landgraf; Jun Liu; Stephene Lodge; Jake Pushie; Sharon Ro-
zovsky; Abith Vattekkatte; Rama Yaghi; Huawei Zeng
This manuscript has been accepted after peer review and the authors have elected
to post their Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by using the Digital
Object Identifier (DOI) given below. The VoR will be published online in Early View as
soon as possible and may be different to this Accepted Article as a result of editing.
Readers should obtain the VoR from the journal website shown below when it is pu-
blished to ensure accuracy of information. The authors are responsible for the content
of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.201600266
Link to VoR: http://dx.doi.org/10.1002/cbic.201600266
A Journal of
www.chembiochem.org

ChemBioChem
10.1002/cbic.201600266
Trifluoroselenomethionine - a New Non-Natural
Amino Acid
*
,[a]
[b],[c]
[a]
[d]
Eric Block,
Squire J. Booker,
Sonia Flores-Penalba, Graham George,
Sivaji
[a]
[b]
[e]
[a]
[d],[f]
Gundala, Bradley J. Landgraf, Jun Liu, Stephene N. Lodge, M. Jake Pushie,
Sharon
Rozovsky,* Abith Vattekkatte,[a], Rama Yaghi,
,
[e]
[a],#
and Huawei Zeng
[g]
[
a]
Department of Chemistry, University at Albany, State University of New York, Albany, New
York 12222, United States;
[
b]
[c]
Department of Chemistry and Department of
Biochemistry and Molecular Biology, Penn
State University, University Park, Pennsylvania 16802, United States;
[d]
Department of Geological Sciences, University of Saskatchewan, Saskatoon, Saskatchewan
S7N 5E2, Canada;
[
e]
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware
1
9716, United States;
[
f]
College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5E5,
Canada;
[
g]
United States Department of Agriculture, Agricultural Research Service, Grand Forks Human
Nutrition Research Center, Grand Forks, North Dakota 58203, United States
1

ChemBioChem
10.1002/cbic.201600266
ABSTRACT
Trifluoroselenomethionine (TFSeM), a novel non-natural amino acid, was synthesized in seven
steps from N-(tert-butoxycarbonyl)-L-aspartic acid tert-butyl ester. TFSeM shows enhanced
methioninase-induced cytotoxicity toward human colon cancer derived HCT-116 cells compared
to selenomethionine (SeM). 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.
None-the-less, when two different variants of the protein GB1 were expressed in an E. coli
methionine auxotroph cell line using TFSeM and methionine (Met) in a 9:1 molar ratio, it was
found that, surprisingly, 85% of the proteins were composed of SeM, even though no SeM had
been added, implying loss of the trifluoromethyl group from TFSeM. The recycling of TFSeM to
SeM is enzymatically catalyzed by E. coli extracts. However, TFSeM is not a substrate of E. coli
methionine adenosyltransferase.
Keywords: trifluoroselenomethionine • selenomethionine • methionine γ-lyase • Fluorinated
amino acids • Se-adenosyltrifluoromethylselenomethionine
Introduction
Trifluoromethionine (3; TFM; Scheme 1) and selenomethionine (5; SeM) are of interest as
prodrugs, affording cytotoxic trifluoromethanethiol (4) and methaneselenol (6), respectively,
when cleaved by methioninase (MGL; L-methionine -lyase; EC 4.4.1.11), analogous to
[
1]
cleavage of methionine (1; Met) to methanethiol (2). In the presence of methioninase, 3 is
[
1a]
effective against anaerobic pathogens, while 5 inhibits cell growth and induces apoptosis in
[
1c, 1d]
colon cancer derived HCT-116 cells.
The K values for 3 and 5 toward methioninase are
m
2

ChemBioChem
10.1002/cbic.201600266
[1a, 1b,
significantly smaller than that for 1, consistent with higher acidity of 4 and 6 compared to 2.
1
f]
Very different mechanisms have been proposed for the cytotoxicity of 4 and 6: 4 is unstable
under physiological conditions, breaking down to the amine cross-linker S=CF (9; thiocarbonyl
2
[
1f]
fluoride), while 6 generates superoxide, inducing oxidative stress as it oxidizes glutathione and
[2]
other cellular thiols. In other work, 3, incorporated into recombinant proteins by E. coli,
1
9
provided a F NMR spectroscopic probe. It also showed diminished activity at the sulfur atom
[3]
of 3, compared to 1, toward oxidation and reaction with electrophilic reagents.
Would trifluoroselenomethionine (7, TFSeM; Se-trifluoromethyl-L-homoselenocysteine), com-
bining characteristics of both and 5, show similar biochemistry, affording
3
trifluoromethaneselenol (8)? If so, how would the cytotoxicity of 8 compare to that of 4 and 6?
Would 7 be incorporated into recombinant proteins in E. coli, paralleling the case of 3, but with
enhanced resistance to oxidation compared to 5? We describe here the first synthesis of 7
(Scheme 2), its reactivity toward methioninase, comparative computational studies of one-
electron reduction of oxygen by 2, 6, and 8, as well as comparative computational and
experimental studies of loss of fluoride ion from 4 and 8 to give thiocarbonyl fluoride, S=CF (9)
2
and selenocarbonyl fluoride, Se=CF (10), respectively. We also report physical, spectroscopic
2
and calculated structural properties of 7 relative to those of 1, 3 and 5, as well the outcome of
efforts to incorporate 7 into recombinant proteins in E. coli.
Results and Discussion
Synthesis of Trifluoroselenomethionine
A short and convenient synthesis of trifluoroselenomethionine (7) was developed from the
known, chiral starting material (S)-tert-butyl 2-(tert-butoxycarbonylamino)-4-hydroxybutanoate
(12, Scheme 2) which, by successive mesylation (13) and treatment with NaBr (14) followed by
3

ChemBioChem
10.1002/cbic.201600266
KSeCN gave the crystalline (S)-tert-butyl 4-selenocyanato-2-(tert-butoxycarbonylamino)-
butanoate (15; ~80% overall yield), which could be characterized by x-ray crystallography
(
Figure 1). The highly lipophilic CF Se group was conveniently introduced giving 16 through
3
reaction of commercially available trifluoromethyl trimethylsilane (CF SiMe ) with
3
3
selenocyanate 15 with slow (syringe pump) addition of catalytic (0.2 eq) TBAF
[
4]
(
tetrabutylammonium fluoride) at 0 °C. The selenocyanate route is preferred since all of the
selenium group is consumed. The synthesis was completed by removal of the protecting groups
1
9
in 16 with trifluoroacetic acid giving trifluoroacetate salt 7a as a colorless solid, showing F-
7
7
19
NMR  –35.12 and –76.07 ppm and Se-NMR  400 ppm. The F NMR signal for alkyl-
substituted CF Se groups generally appears at –35 ppm (relative to CFCl ) compared to –41 ppm
3
3
[
4]
for CF S; trifluoroacetic acid appears at –76.55. Treatment of 7a with the chloride form of
3
1
9
AG® 1-X8 Resin gave 7b, the hydrochloride salt of 7, as a colorless solid, showing F-NMR 
7
7
–
35.35 ppm and Se-NMR  400. Salt 7b was used in all biological studies.
Generation and Decomposition of Trifluoromethaneselenol (8)
In order to evaluate the consequences of enzymatic generation of 8 in the presence of human
colon cancer derived HCT-116 cells, we sought to generate 8. While 8 has been previously
[
5]
[5a]
characterized, it is said to be unstable in water and to decompose to HF and F C=Se upon
2
[
5b]
heating. We sought to generate 8, or its sodium salt, by base-catalyzed decomposition of 3-
trifluoromethylselanyl)propionitrile (18), which in turn we proposed to prepare by treatment of
-selenocyanatopropanenitrile (17) with CF SiMe (Scheme 3). In the event, 18 was smoothly
(
3
3
3
prepared from 17, which in turn was easily prepared from 3-chloropropionitrile. When 18 was
1
9
19
treated with sodium methoxide and the reaction monitored by F NMR, the only F signal seen
corresponded to fluoride ion, as confirmed by NMR analysis of a standard. When 18 was treated
4

ChemBioChem
10.1002/cbic.201600266
with sodium methoxide in the presence of excess cyclopentadiene at low temperature and then
[
6]
slowly warmed, the known
cyclopentadiene adduct of F C=Se, 3,3-difluoro-2-
2
selenabicyclo[2.2.1]hept-5-ene (19), was detected by GC-MS. Hence decomposition of 8 in
[
5b]
solution follows the path seen upon heating.
Spectroscopic and Computational Comparison of Structures of Selenomethionine (5) and
Trifluoroselenomethionine (7)
A combined spectroscopic and computational approach was used to determine the physical and
electronic structural changes that occur on replacing the methyl group of 5 with the
trifluoromethyl group in 7. In particular, extended X-ray absorption fine structure (EXAFS)
measurements of 5 and 7 (as 7b) were conducted on beamline 7-3 of the Stanford Synchrotron
Radiation Lightsource (SSRL). The data obtained was analyzed using density functional theory
(DFT) calculations and other methods. Analysis of the Se K near-edge spectrum for both 5 and 7,
shown in Figure 2, yielded strikingly similar near edge spectra, with no discernible shift in the
near edge between the two. The first derivatives for both near edge spectra (inset in Figure 2) are
also very similar, such that it would not be possible to distinguish the two chemical species from
one another using the Se K near edge spectrum alone.
Like the near-edge spectra, the EXAFS spectra and associated Fourier transforms (Figure 3),
for 5 and 7 were very similar. The theoretical resolution of bond lengths that can be obtained
from the EXAFS data (calculated as /(2k), where k is the extent of the fitted EXAFS data)
-1
-1
for 5 is 0.13 Å (k-range 1-12.5 Å ) and 0.12 Å (k-range 1-14 Å ) for 7. Given the bond length
resolution of the EXAFS data, both of the Se–C bond lengths in each of the analogs are
sufficiently similar that they must be modeled as a single shell of two Se–C scattering
interactions, resulting in an average fit distance (R) for the two interactions. The EXAFS curve
5

ChemBioChem
10.1002/cbic.201600266
fitting analysis, using single scattering paths for the two Se–C backscattering interactions yielded
very similar bond length results between 5 and 7, with 2 Se–C at 1.957(2) Å for 5 and 2 Se–C at
1
.955(4) Å for 7 (Table 1).
A search of the Cambridge Structural Database (CSD) for Se–C bond lengths from 5 gives
an average distance of 1.944 ± 0.006 Å for SeCH and 1.954 ± 0.008 Å for SeCH , slightly
[7]
3
2
below the average obtained from EXAFS data fitting (averages from the CSD search were taken
from three small molecule crystal structures, all of which contained Met within a peptide chain:
o
o
MAJTAT C–Se–C = 97.23 , CH –Se = 1.950, CH –Se = 1.947; DEYFOD C–Se–C = 98.68 ,
3
2
o
CH –Se = 1.939, CH –Se = 1.963; AVIKOG C–Se–C = 97.94 , CH –Se = 1.942, CH –Se =
3
2
3
2
1
.952). The average interatomic distance for Se–C obtained by EXAFS is still within the
experimental error obtained from the CSD search for the Se–CH bond and only 0.002 Å longer
2
than the maximum value for Se–CH found in the CSD search.
3
DFT Structure Calculations
Gas-phase geometry optimizations were carried out for each of the isostructural charge neutral
amino acid models: methionine (1) and selenomethionine (5), as well as each of their CF3-
bearing derivatives, 3 and 7, respectively. The calculated Se–CH bond length, 1.96(2) Å,
3
presented the largest deviation, ~0.03(8) Å from the EXAFS value of 1.92(8) Å. Other calculated
heteroatom carbon bond lengths, summarized in Table 2, were within 0.02 Å of the EXAFS
values.
The DFT calculations indicate that when the terminal methyl group in the methionine model is
changed to CF the sulfur bond length shortens, with concomitant elongation of the S–CH bond.
3
2
Similarly, for the Se-containing analogs the Se–CH bond also elongated in the CF bearing
2
3
derivative, relative to 5, while the Se–CH and Se–CF bond lengths remained largely
3
3
6

ChemBioChem
10.1002/cbic.201600266
unchanged. Molecular volume calculations, using the united atom topological model, were
performed on each of the optimized structures (Figure 4). Each of the investigated modifications
to the methionine side chain results in an increase in side chain volume.
The molecular volume of amino acid 5 is 104% larger than 1 (111% increase considering only
the hetero atom and terminal methyl group moieties). Modification of the methyl group of 1 to
CF represents a 106% increase in amino acid volume (123% increase for the sulfur and terminal
3
groups), while modification of the methyl group of 5 to CF represents a 107% increase in
3
molecular volume (120% increase for Se–CX ). Modification of 1 to 7 represents the most
3
significant increase, at 111% relative to the molecular volume of methionine, and a 133%
increase for Se–CF , relative to S–CH .
3
3
Relative to the sulfur heteroatom in 1, the Se-atom in 5 has low-lying d-orbitals, which are
more energetically-accessible than those in the sulfur-containing analog. The result is that the
LUMO of 5 is at a lower energy than in the sulfur-containing analog and a greater fraction of the
orbital density resides on the Se-atom in the LUMO (Figure 4, LUMO density at the sulfur atom
not apparent at the contour value used). Modification of the terminal methyl group in 3
introduces an electron withdrawing moiety to the heteroatom, with the result that in the sulfur-
containing methionine analog the LUMO contains some SC * character. These trends are
doubly enhanced in the CF -bearing selenomethionine analog, where the decreased electron
3
density on the Se-atom results in a significant change to the character of the LUMO, such that a
significant fraction of the overall density residues on the heteroatom, despite there being no
discernable change in the core 1s excitation energy in the Se K near-edge spectrum (Figure 2).
The observed C–X–C bond angle is also closer to 90° (see Figure 4).
7

ChemBioChem
10.1002/cbic.201600266
The HOMO-LUMO gaps for 5, 3, and 7, relative to the calculated HOMO-LUMO gap for 1,
-1
are -26.5, +100.9, and +32.5 kJ mol . Relative to the methyl-bearing methionine residue, the
presence of the electron withdrawing CF moiety serves to widen the HOMO/LUMO gap, while
3
the change from sulfur-containing 1 to selenium-containing 5 results in a smaller HOMO-LUMO
gap. The HOMO-LUMO gap for 7 supports the trend of the CF modification observed in the
3
sulfur-containing models, where the HOMO-LUMO gap is larger than in the methyl-bearing
analogs.
-1
Comparing the HOMO-LUMO gap for 7 with 5 gives a relative change of +59.0 kJ mol ,
again mirroring the 1 vs. 3 trends. These data are also summarized in Table S2 in the
Supplementary Material. The character of the HOMO of the various analogs changes relatively
little between 3, 5 and 7. Comparing the LUMO of the two selenomethionine analogs, both
possess some character on the Se-atom; the LUMO of 5 is spread across the amino acid, with
some contribution from SeC * as well as the CC(O) * orbital; however, in the CF analog
3
the SeC * contributes significantly to the character of the LUMO, with relatively little *
contribution on the amino acid backbone region. Interestingly, although energetically different,
the HOMO and LUMO for 3 and 7 are qualitatively similar, although the HOMO/LUMO gap in
-1
3
is calculated to be ~70 kJ mol greater (see Table S2 in Supporting Material), and unlike 7, the
LUMO of 3 has comparatively more * character on the backbone atoms, similar to 5 (Figure 4).
Leroux previously determined that the past assertion that the CF moiety is at least as large as
3
[
8]
isopropyl is incorrect, and in fact the volume for CF is considerably smaller than the volume
3
[
9]
of isopropyl. We also performed model calculations comparing isopropyl and CF , and our
3
findings support Leroux’s previous conclusion that the CF substituents are indeed larger than
3
CH , but certainly of smaller volume than models incorporating isopropyl (data not shown).
3
8

ChemBioChem
10.1002/cbic.201600266
Furthermore, we have compared the molecular cavities for the side chain fragments of the series
S–CH , S–CF , Se–CH and Se–CF . The cavities of the molecular fragments calculated during
3
3
3
3
the solvation procedure included an additional margin for a solvent probe molecule (based on
3
3
water). Relative to S–CH (47.39 Å ) the volume of the S–CF moiety is 123% larger (58.43 Å ),
3
3
3
3
the Se–CH moiety is 111% larger (52.60 Å ), and the Se–CF is 133% larger (63.22 Å ).
3
3
Methioninase Studies
Figure 5 shows that the cell growth rate was inhibited by 16.7%, 26.1%, and 44.6% in
HCT116 cells treated by incubating Trichomonas vaginalis methioninase (40 U/L) with 1.25, 2.5
and 5 μmol/L 5, and by 49.4%, 60.0% and 76.3% in the cells treated with 1.25, 2.5 and 5 μ
mol/L 7b, respectively. That is, 7b is substantially more effective in reducing the concentration
of cells than 5 at all concentrations tested. There are three possible explanations for the enhanced
cytotoxicity of 7b compared to 5, if it is assumed that 8 is released from 7b, and 6 from 5,
respectively, by the methioninase: 1) CF SeH 8 is more effective in generating reactive oxygen
3
species than methaneselenol (6) (Figure 6); 2) CF SeH (8) is assumed to break down under
3
physiological conditions, as it does in our model studies, to Se=CF (10), which should act as a
2
potent cross-linker of primary amine groups, as proposed for the cytotoxicity of 4 from 3,
through formation of S=CF (9) (Figure 7); 3) some combination of the first and second
2
postulated processes occurs.
Figure 8 illustrates the proposed redox cycling of selenolate, the anionic form of selenols such
as 6 and 8. Figure 6 compares the ease of reduction of dioxygen by methanethiol (2) with
selenols 6, 6a, 6b and 8, as calculated by DFT methods. These calculations show that the
selenols are superior reducing agents to thiols and that 6 and 6a are comparable (within the error-
limits of the method), and slightly superior to 6b. These latter results are in agreement with the
9

ChemBioChem
10.1002/cbic.201600266
[
2]
findings of the reported relative cytotoxicity 6>6a>6b>2. The fact that the relative observed
cytotoxicity of 8 is not correctly predicted by the DFT calculations of Figure 6 suggests that
additional, unique properties of 8 need to be considered, e.g., loss of fluoride ion to give
[
1a]
selenocarbonyl fluoride 10 (as calculated in Figure 7, and previously reported for 4 ), as we
have confirmed experimentally for 8. A third possibility is that the cytotoxicity associated with 8
is due to its enhanced acidity (as calculated in Figure 7) or to redox properties of its
[
10]
corresponding anion. The reported pKa values of 2, 4, 6, and 8, are 10.09 ± 0.10, 2.77 ± 0.10,
.50 ± 0.70, and -1.29 ± 0.70, respectively.
6
Incorporation of TFSeM into proteins in E. coli
Incorporation of fluorinated amino acids into proteins provides a structural probe as well as
[
11]
potentially introduces useful functional changes. The methionyltRNA synthetase (MetRS) is
[
12]
among the more promiscuous aminoacyl-tRNAsynthetases, though it still exercises selectivity
[
13]
based on the length and geometry of the side chain.
Given that difluoromethionine and
[
3, 14]
trifluoromethionine (3) have been successfully incorporated into proteins,
it was of interest
to determine if TFSeM (7) could be similarly incorporated. The standard procedure to substitute
a given amino acid with its fluorinated analog, involves expressing the target recombinant
protein in an E. coli auxotroph that is unable to synthesize the conventional amino acid to be
substituted. The fluorinated amino acid is added to a defined media, taken up by the cell, and
[15]
subsequently incorporated into the protein.
The successful substitution relies on both the
ability of the respective aminoacyltRNA synthetase to utilize the fluorinated analog and the
capacity of the nascent chain to fold into a stable three-dimensional structure when the analog is
inserted. The incorporation ratio using this procedure tends to be low due to challenges in
[
16]
loading the fluorinated amino acid to the respective aminoacyltRNA synthetase.
Previous
1
0

ChemBioChem
10.1002/cbic.201600266
[
14a]
publications reported 10-15% successful incorporation of 3 into green fluorescent protein
and
[
14c]
3
1-70% incorporation into different positions in bacteriophage lambda lysozyme.
These
studies suggest that the incorporation of fluorinated amino acids would be most efficient for
solvent-exposed positions where the larger volume and the change in hydrophobicity may be
better tolerated.
To assess the incorporation of 7, we chose as a target the compact, 56 amino acid protein im-
munoglobulin-binding B1 domain of streptococcal protein G (GB1). GB1 possesses a
remarkably stable fold and is often employed in studies of protein folding and thermal
[
17]
stability.
We have constructed variants of GB1 which, following purification, have a single
Met present in a unique location in the protein: a Val39Met variant in which the Met is located in
a loop and a Leu5Met variant in which the Met is located in the hydrophobic core of the protein
[
17-18]
(Figure 9A). Similar substitutions have been shown to be stable and well-folded.
Comparing
the expression level of the two different variants allows for a test of whether incorporation of 7
was position dependent.
We followed the expression procedure reported for incorporation of 3 and other methionine
[
13-14, 14c, 19]
analogs
with the following modifications. First, we utilized a C43(DE3)-based E. coli
[
20]
amino acid auxotrophic host strain named RF11 in which metA was deleted. Second, Met was
included in the growth media since a previous study had shown that including 10% Met in the
[
21]
growth media reduced the toxicity of SeM while allowing for a high SeM incorporation ratio.
In order to reduce the toxicity of 7, in the form of its hydrochloride salt 7b, the Met auxotroph
RF11 was grown in LB media and switched into a defined media at the mid-logarithmic phase of
growth. The defined media was supplemented with either Met, SeM and Met at a 9:1 molar ratio,
or TFSeM (as 7b) and Met at a 9:1 molar ratio. The expression levels were compared using
1
1

ChemBioChem
10.1002/cbic.201600266
SDS–PAGE analysis (Figure 9B and 9C). The expression level of GB1 grown in the presence of
TFSeM (as 7b) was lower than that detected in the presence of Met alone or SeM and Met,
indicating that TFSeM was more cytotoxic than SeM, in agreement with previous reports about
fluorinated amino acids toxicity.
To determine the incorporation rate of TFSeM (as 7b), the two proteins were purified by
IMAC chromatography as detailed in the experimental section. The mass spectra of the purified
GB1 Leu5Met and GB1 Val39Met grown with 9:1 molar ratio of TFSeM (as 7b):Met contained
no clear evidence for TFSeM insertion (Figure 10B and 10D). Instead, the molecular mass of the
protein suggested that SeM was incorporated. When cells were grown in the presence of a 9:1
molar ratio of SeM:Met, GB1 Leu5Met and GB1 Val39Met contained primarily SeM reflecting
its abundance in the defined media compared to that of Met (Figure 10A and 10C).
Since neither SeM nor any other source of selenium was included in the growth medium of the
Met auxotroph, if SeM is indeed incorporated into GB1 then the observed SeM inserted in GB1
must have originated from TFSeM. To confirm the identity of the protein species detected by
ESI-MS the preparation of GB1 Val39Met in RF11 was scaled up. The resulting samples were
analyzed by both intact protein ESI-MS and sequencing of trypsin-derived peptides by tandem
MS. GB1 Val39Met was grown with solely 22 M Met (Figure 11A), 22 M Met and 200 M
TFMet (as 7b) (Figure 11B), and solely 200 M TFMet (as 7b) (Figure 11C). The predominant
protein form observed in this experiment was GB1 Val39Met (calculated molecular mass is 6179
Da and observed is 6180 Da). Also present were lower molecular weight forms that are likely to
arise from a loss of methyl (6166 Da) and water (6161 Da) from the protein. These modifications
may be attributed to the stress of the Met auxotroph growing with low Met concentrations. The
protein forms at 6220/1 Da are most likely due to acetylation in vivo or interactions with
1
2

ChemBioChem
10.1002/cbic.201600266
acetonitrile used for LC. Only when GB1 Val39Met was grown with 22 M Met and 200 M
TFMet was it possible to detect the 6227 Da peak suspected as containing SeM. The sample
shown in Figure 11B was trypsin digested and sequenced by tandem MS. As Figure 11D shows,
the peptide QYANDNG(SeM )GDEWTYDDATK was identified based on its molecular weight
3
9
and the distinct isotope pattern of selenium. It was not possible to fully sequence the peptide due
to its low abundance in the sample. Interestingly, even though this sample also contains a species
with 6280 Da, matching the calculated MW of GB1 Val39TFSeM, the corresponding peptide
QYANDNG(TFSeM )GDEWTYDDATK was not detected. Hence, we could not confirm
3
9
experimentally that TFSeM was incorporated into GB1 under these specific growth conditions.
Since as mentioned neither SeM nor any other source of selenium was included in the growth
medium, and proteins with SeM were not observed when GB1 Val39Met was grown without
TFSeM, we reasoned that the fraction of TFSeM recycled to SeM in vivo. The incorporation of
SeM into GB1 Val39Met and Leu5Met during its expression in E. coli cultured in the presence
of TFSeM might be envisaged to take place via intermediate formation of TFSeSAM. In such a
pathway, the trifluoromethyl group could be removed by one or more of the S-
adenosylmethionine (SAM)-dependent methyltransferases (MTases) that normally transfer the
[
22]
methyl moiety of SAM to a target acceptor (Scheme 4).
adenosylhomoselenocysteine (SeAH), would then
The resulting molecule, Se-
acted upon by S-
be
adenosylhomocysteine/methylthioadenosine nucleosidase to yield adenine and Se-
ribosylhomoselenocysteine, the latter of which would be converted to homoselenocysteine and
(4S)-4,5-dihydroxypentan-2,3-dione by ribosylhomocysteine lyase. Homoselenocysteine would
then be methylated by either the cobalamin-dependent or the cobalamin-independent methionine
synthase to afford selenomethionine. Methylation of homoselenocysteine could also take place
1
3

ChemBioChem
10.1002/cbic.201600266
by lesser appreciated mechanisms, such as homocysteine S-methyltransferase, the product of the
mmuM gene in E. coli, which uses L-S-methylmethionine or the S(+) isomer of SAM as the
[
23]
methyl donor.
To determine whether TFSeSAM is a key intermediate in the formation of SeM from TFSeM,
the ability of E. coli methionine adenosyltransferase (MAT) to convert TFSeM into TFSeSAM
was assessed. MAT catalyzes the condensation of L-methionine and ATP to SAM, and also
yields pyrophosphate and inorganic phosphate as byproducts. Shown in 16A is an HPLC elution
profile of the products of a MAT reaction incubated for 30 or 60 min at 25 °C under turnover
conditions in the presence of TFSeM (as 7b). A trace containing authentic standards of related
adenine-containing compounds (dashed line) and tryptophan, used as an internal standard,
indicates that SAM and SeSAM elute between 11 and 13 min. In both the 30 and 60 min time
points, no peaks are present in the region corresponding to SAM or SeSAM in the reaction
containing TFSeM, and observable peaks throughout the chromatogram are present before
initiation of the reaction (0 min) and do not increase with time (blue, yellow, and red lines). An
HPLC elution profile of a control reaction, conducted under identical conditions, but containing
SeM rather than TFSeM, shows robust and time-dependent formation of SeSAM (Figure 12B,
red, yellow, green, and blue traces), indicating that the MAT preparation is active. The unknown
peak eluting just after SAH is also present in the TFSeM reaction, and is most likely a
contaminant in the MAT preparation. The observation of SeSAH suggests that the MAT
preparation is also contaminated with one or more methyltransferases.
Similar reactions were also analyzed by LC-MS to assess whether the concentration of TFSeM
(as 7b) changes as a function of time. Shown in Figure S1A is a total ion chromatogram of a
MAT reaction at various extents of incubation in the presence of SeM. Peaks at 1.14, 1.39, 1.54,
1
4

ChemBioChem
10.1002/cbic.201600266
and 4.46 min undergo time-dependent changes. The peak at 1.39 min (m/z 198.1) corresponds to
SeM, and decreases as a function of time, while peaks at 1.14 (m/z 447.1), 1.54 (m/z 433.1) and
4
.46 (m/z 346.1) correspond to SeSAM, SeAH, and methylselenoadenosine (MSeA),
respectively, and increase as a function of time. The formation of SeSAH and MSeA is not
unexpected, given the methyltransferase contamination in the MAT preparation and the
[
24]
propensity of SeSAM to form MSeA and homoserine lactone nonenzymatically. By contrast,
the total ion chromatogram of a MAT reaction at various extents of incubation in the presence of
TFSeM shows that the concentration of TFSeM does not change significantly during 18 h of
incubation (Figure S1B).
Given that E. coli MAT does not appear to use TFSeM as a substrate, experiments were
conducted to assess whether other proteins in E. coli can act on it. In these experiments, 1 mM
TFSeM (as 7b) was incubated for various extents of time with crude lysate from E. coli
methionine auxotroph strain B834(DE3)pLysS with a metE deletion, and the reactions were
analyzed by LC-MS. As shown in Figure 13A and 13B, both TFSeM and SeM are consumed as
a function of time. When similar reactions were conducted with TFSeM using crude lysate that
had been boiled for 10 min or the filtrate from crude lysate that had been subjected to
centrifugation in a spin concentrator containing a membrane with a 3 kDa molecular weight
cutoff, the concentration of both SeM and TFSeM stayed constant throughout the 18 h
incubation (Figure S2A-D). This behavior strongly suggests that TFSeM is acted upon by an
unidentified protein in E. coli. Together, these results indicate that although TFSeM is not
converted into TFSeSAM, it is a target of another protein in E. coli that most likely converts it
into homoselenocysteine or a precursor of this metabolite.
Conclusion
1
5

ChemBioChem
10.1002/cbic.201600266
Trifluoroselenomethionine (7), a novel non-natural amino acid, was synthesized in seven steps
from N-(tert-butoxycarbonyl)-L-aspartic acid tert-butyl ester. Trifluoroselenomethionine shows
enhanced methioninase-induced cytotoxicity toward human colon cancer derived HCT-116 cells
compared to selenomethionine (5). Mechanistic explanations for this enhanced activity were
computationally and experimentally examined. Comparison of 5 and 7 by selenium EXAFS and
DFT calculations showed them to be spectroscopically and structurally very similar. However,
when two different mutants of the protein GB1 were expressed in the E. coli methionine
auxotroph cell line RF11 using 7 (as 7b) and methionine in a 9:1 ratio, it was found that,
surprisingly, over 80% of the proteins were composed of 5, even though no 5 had been added,
implying loss of the trifluoromethyl group from 7. A yet unidentified enzyme catalyzes the
process. It is of interest to determine which enzyme is responsible for the loss of trifluoromethyl
from 7, as this chemical group is present in many drugs and thus is of inherent medicinal interest.
Experimental Section
Materials and methods: L-Tryptophan, 2-mercaptoethanol, S-adenosyl-L-homocysteine (SAH),
adenine, 5'-methylthioadenosine (MTA), and phenylmethanesulfonyl fluoride (PMSF) were
purchased from Sigma Corp (St. Louis, MO). Tris(hydroxymethyl)aminomethane (Tris-HCl) and
magnesium chloride hexahydrate were purchased from Avantor Performance Materials (Center
Valley, PA). Selenomethionine (>98.0%) was purchased from TCI (Tokyo, Japan), ATP
(
disodium salt) from CalBioChem (San Diego, CA), potassium chloride from EMD Chemicals
Gibbstown, NJ), dithiothreitol (DTT) from Gold Biotechnology (St. Louis, MO), and
(
ammonium acetate (ACS grade) from Affymetrix (Cleveland, OH). All other chemicals were of
the highest grade available. S-adenosyl-L-methionine (SAM), and Se-adenosyl-L-
selenomethionine (SeSAM) were synthesized and purified as described previously, as was
1
6

ChemBioChem
10.1002/cbic.201600266
[
13]
methionine adenosyltransferase (MAT).
HPLC methods 1 and 2 were previously described
[
13]
and used without any modifications.
Synthetic procedure
(S)-tert-Butyl 2-(tert-butoxycarbonylamino)-4-hydroxybutanoate (12): Ethyl chloroformate
(1.4 mL, 14.6 mmol) was added at -10 °C to a stirred solution of N-(tert-butoxycarbonyl)-L-
aspartic acid 1-tert-butyl ester (11) (4 g, 13.8 mmol) and Et N (2.0 mL, 14.2 mmol, 1.03 eq) in
3
THF (60 mL) and the mixture was stirred at -10 °C for 30 min. The precipitated mass was
filtered and the filtrate was added over a period of 30 min to a solution of NaBH (1.6 g, 42.5
4
mmol, 3.1 eq) in H O-THF (3 mL:12 mL) at 10-15 °C. The reaction was stirred for 4 h and
2
acidified with HCl, and the THF was removed in vacuo. The aqueous solution was extracted
twice with EtOAc (25 mL each time), washed with 10% NaOH (25 mL), H O (25 mL) and brine
2
(
25 mL). The organic solution was dried (Na SO ) and concentrated to give a clear oil. The crude
2 4
oil product was purified by flash chromatography on silica (elution with EtOAc and toluene
[
25]
1
(
1:4)) to give the known title compound as a clear, viscous oil (5.93 g, 85%); H-NMR (400
MHz, CDCl ) =1.45 (s, 9 H), 1.47 (s, 9 H), 2.00–2.22 (m, 2 H), 3.54–3.58 (m, 2 H), 4.20 (m, 1
3
1
3
H), 5.41 ppm (s, 1 H); C-NMR (CDCl , 100 MHz) =172.5, 152.9, 82.2, 79.9, 53.2, 36.5, 29.7,
3
2
8.4 ppm.
S)-tert-Butyl 4-bromo-2-(tert-butoxycarbonylamino)butanoate (14): To a solution containing
2 (1.39 g, 5.04 mmol) in anhydrous CH Cl (60 mL) at 0 °C was added Et N (1.05 mL, 7.56
(
1
2
2
3
mmol, 1.5 eq) followed by CH SO Cl (0.39, mL, 2.3 mmol, 2.2 eq). The reaction mixture was
3
2
stirred at 0 °C under argon atmosphere for 1 h, diluted with brine (100 mL) and the organic layer
was separated. The aqueous layer was then back-extracted twice with diethyl ether (50 mL each
time) and the combined organic layer was dried (Na SO ) and concentrated in vacuo to give the
2
4
1
7

ChemBioChem
10.1002/cbic.201600266
[
26]
known mesylate 13 as a white solid. Sodium bromide (2.07 g, 20.16 mmol, 4 eq) was added to
a solution containing the crude mesylate 13 in dry acetone (100 mL). The solution was heated to
reflux for 24 h. The cooled reaction mixture was filtered and then concentrated in vacuo. The
residue was purified by flash chromatography using EtOAc and hexane (1:4) to give the
[
25, 27]
1
known
title compound as a colorless solid (1.5 g, 88%); H-NMR (CDCl , 400 MHz)
3
=1.45 (s, 9 H), 1.46 (s, 9 H), 2.21–2.41 (m, 2 H), 3.43–3.58 (m, 2 H), 4.43 (m, 1 H), 5.13 (s, 1
H) ppm.
(S)-tert-Butyl 4-selenocyanato-2-(tert-butoxycarbonylamino)butanoate (15): To a solution of
(S)-tert-butyl 4-bromo-2-(tert-butoxycarbonylamino)butanoate (1.0 g, 2.96 mmol) in THF (45
mL), KSeCN (0.64 g, 4.5 mmol) and tetra-n-butylammonium bromide (0.096 g, 0.29 mmol)
were added, and the mixture was refluxed for 3 h. After completion of the reaction, the solvent
was evaporated under reduced pressure. The residue was purified by flash chromatography (1:4
EtOAc:hexane) giving the title compound as a colorless crystalline solid, m.p. 78-80 °C (0.65 g,
1
6
3
1
5 %); H-NMR (CDCl , 400 MHz) =1.44 (s. 9 H), 1.46 (s, 9 H), 2.13–2.42 (m, 2 H), 2.96–
3
1
3
.14 (m, 2 H), 4.31 (m, 1 H), 5.23 ppm (m, 1 H); C-NMR (CDCl , 100 MHz) =170.4, 155.7,
3
7
7
02.3, 83.0, 80.3, 53.1, 35.0, 28.2, 27.9, 26.0 ppm; Se-NMR (CDCl , 95.3 MHz) =216 ppm;
3
-1
+
IR: max 3387, 2980, 2516, 2159, 1707, 1688, 1503, 1190 cm ; DART-HR-TOF-MS (M + H )
8
0
C H O N Se: found for Se 365.0979, calcd 365.0980. The structure crystallizes in the non-
14
25
4
2
centrosymmetric P212121 space group. Having a heavy Se atom in the light-atoms molecule
allowed us to use Mo radiation to determine the absolute configuration by utilizing the
anomalous dispersion effects in the x-ray diffraction measurements on the crystal. Flack
parameter (refined to essentially zero with low e.s.d -0.010(12)) confirms the correct assignment
of the absolute configuration.
1
8

ChemBioChem
10.1002/cbic.201600266
(S)-tert-Butyl 4-trifluoromethylselanyl-2-(tert-butoxycarbonylamino)butanoate (16): To an
ice-cooled solution of selenocyanate 15 (500 mg, 1.37 mmol) and CF SiMe (0.44 mL, 2.7
3
3
mmol) in anhydrous THF (5 mL) under argon, tetra-n-butylammonium fluoride (TBAF) (0.3
mL, 0.3 mmol) was added dropwise with a syringe pump (1 mL/h). The reaction was monitored
1
9
by F-NMR (CDCl , CFCl ). After stirring for 1 h at room temperature, the crude mixture was
3
3
purified by flash chromatography (petroleum ether/ether) giving the title compound as a clear oil
1
(
0.32 g, 35%); H-NMR (CDCl , 400 MHz) =1.44 (s. 9 H), 1.47 (s, 9 H), 1.99–2.24 (m, 2 H),
3
1
3
2
.82–2.94 (m, 2 H), 4.24-4.26 (m, 1 H), 5.11 ppm (bd, J = 6.8, 1 H); C-NMR (CDCl , 100
3
19
MHz) =170.7, 155.3, 122.6 (q, J=328), 82.5, 79.9, 53.6, 34.3, 28.1, 27.8, 21.1 ppm; F-NMR
7
7
(
CFCl , 376.5 MHz) =–35.1 ppm; Se-NMR (CDCl , 95.3 MHz) =400 ppm; DART-HR-
3
3
+
80
76
TOF-MS (M + H ) C H O NF Se: for Se found 408.0900, calcd 408.0901; for Se, found
1
4
25
4
3
4
04.0931, calcd 404.0928.
(S)-1-Carboxy-3-((trifluoromethyl)selanyl)propan-1-aminium 2,2,2-trifluoroacetate (7a):
(S)-tert-Butyl 4-trifluoromethylselanyl-2-(tert-butoxycarbonylamino)butanoate (16; 0.2 g, 0.5
mmol) was dissolved in a 10% solution of freshly distilled TFA in dry CH Cl (45 mL) and the
2
2
mixture was stirred for 3 h. When TLC indicated complete deprotection, the mixture was
extracted twice with water (15 mL each time) and the collected aqueous layers were evaporated
under reduced pressure to give the title compound as a colorless solid (110 mg; 70%), m.p. 224-
1
2
26 °C (decomp.); H-NMR (D O, 400 MHz) =2.32–2.46 (m, 2 H), 3.08-3.20 (m, 2 H), 3.85
2
1
3
ppm (t, J=6.8, 1 H); C-NMR (D O, 100 MHz) =173.5, 162.7 (q, J=38), 122.5 (q, overlap with
2
1
9
CF of acetate), 115.5 (q, overlap with -SeCF3), 54.3, 31.8, 20.5 ppm; F-NMR (D O, 376.5
3
2
77
MHz) =–35.1, –76.0 ppm; Se-NMR (D O, 95.3 MHz) =400 ppm; DART-HR-TOF-MS (M +
2
1
9

ChemBioChem
10.1002/cbic.201600266
+
80
76
H ) C H O NF Se for Se: found 251.9748, calcd 251.9751; for Se: found 247.9748, calcd
5
9
2
3
2
47.9778. By absolute qNMR the purity of 7a was determined to be 98% (see SI).
(S)-1-Carboxy-3-((trifluoromethyl)selanyl)propan-1-aminium chloride (7b): To 100 mg
(0.28 mmol) of 7a and AG® 1-X8 Resin (500 mg, chloride form, 50-100 mesh) in a 25 mL
round bottomed flask was added 10 mL of water and stirred for 24 h. The heterogeneous mixture
was filtered through cotton and aqueous solution was evaporated under reduced pressure to give
1
the title compound as a colorless solid (52 mg; 63%), m.p. 228-231 °C (decomp.); H-NMR
13
(
D O, 400 MHz) =2.24–2.31 (m, 2 H), 2.96-3.08 (m, 2 H), 3.74 ppm (t, J=6.8, 1 H); C-NMR
2
19
(
D O, 100 MHz) =173.5, 122.7(q, J=328), 54.3, 31.8, 20.5 ppm; F-NMR (D O, 376.5 MHz)
2
2
7
7
+

=–34.56 ppm; Se-NMR (D O, 95.3 MHz) =400 ppm; DART-HR-TOF-MS (M + H )
2
80
76
C H O NF Se for Se: found 251.9745, calcd 251.9751; for Se: found 247.9750, calcd
5
9
2
3
2
47.9778. By absolute qNMR the purity of 7b was determined to be 98% (see SI).
3
-Selenocyanatopropanenitrile (17): To a solution of 3-chloropropionitrile (2.0 g, 22.0 mol) in
acetone (50 mL) was added potassium selenocyanate (3.2 g, 0.22 mol). The reaction mixture was
refluxed overnight, filtered, and evaporated in vacuo. The crude product was purified by flash
chromatography (n-pentane/EtOAc) giving the title compound 17 as a yellow oil (2.3 g, 65%);
1
13
H NMR (CDCl , 400 MHz) δ=3.19-3.24 (m, 2 H), 3.00-3.04 ppm (m, 2 H); C NMR (CDCl ,
3
3
+
80
1
01 MHz) δ=117.1, 99.9, 22.8, 20.0 ppm; DART-HR-TOF-MS (M+H ) C H N Se: for Se
4 5 2
7
6
found 160.9605, calcd 160.9618; for Se, found 156.9630, calcd 156.9645.
-(Trifluoromethylselanyl)propionitrile (18): To an ice-cooled solution of selenocyanate 17
800 mg, 5.0 mmol) and CF SiMe (1.42 g, 10.0 mmol) in anhydrous THF (10 mL) under argon,
3
(
3
3
tetra-n-butylammonium fluoride (TBAF) (2.0 mL, 2.0 mmol) was added dropwise with a syringe
1
9
pump (1 mL/h). The reaction was monitored by F-NMR (CDCl , CFCl ). After stirring for 1 h
3
3
2
0

ChemBioChem
10.1002/cbic.201600266
at room temperature, the crude mixture was purified by flash chromatography (n-
1
pentane/EtOAc) giving the title compound as a yellow oil (300 mg, 30%); H NMR (CDCl
3
, 400
1
3
MHz) δ=3.14-3.17 (m, 2 H), 2.85-2.89 ppm (m, 2 H); C NMR (CDCl , 101 MHz) δ=120.5 (q,
3
1
9
J=330, CF ), 117.7, 19.6, 19.5 ppm; F NMR (CDCl
3
, 376 MHz) δ=–34.89 ppm. DART-HR-
3
+
80
76
TOF-MS (M+H ) C H F NSe: found for Se 203.9538, calcd 203.9539; for Se, found
4
5 3
1
99.9564, calcd 199.9566.
Generation and decomposition of trifluoromethaneselenol (8); trapping of 3,3-difluoro-2-
selenabicyclo[2.2.1]hept-5-ene: Under argon, NaOMe (20 mg, 0.37 mmol) in methanol (2 mL)
was added dropwise to 3-(trifluoromethylselanyl)propanenitrile (18) (75 mg, 0.37 mmol), and
the mixture was stirred at room temperature for 30 min. Methanol was then removed in vacuo,
1
9
CD CN was added under argon, and the solution was directly examined by F NMR (376 MHz).
3
1
9
The F NMR spectrum showed a singlet at δ=–82.71, which can be compared to a signal at δ=–
8
4.48 for a standard solution of KF in CD CN. In a second experiment, a mixture of
3
cyclopentadiene (2.0 eq) and 18 (75 mg, 0.37 mmol) in a 10 mL flask was frozen using liquid
nitrogen and the flask was filled with argon. A solution of NaOMe (0.37 mmol) in methanol (2
mL) was added and the reaction was warmed to room temperature. Analysis of the product by
GC-MS showed a molecular ion at m/z 196 and fragment ions at m/z 115 and 95, corresponding
to the mass spectrum reported for the F C=Se (10) cyclopentadiene Diels-Alder reaction product
2
[
6]
3
,3-difluoro-2-selenabicyclo[2.2.1]hept-5-ene (19) (Scheme 3).
Cell cultures: HCT116 colorectal carcinoma cells were obtained from American Type Culture
Collection and maintained in DMEM (GIBCO Invitrogen, Carlsbad, CA). Stock cells were
passaged twice weekly at 80% confluency [0.25% trypsin (GIBCO), 1 mmol/L EDTA, in Ca–
Mg–free Hanks’ balanced salt (Sigma Chemical Co., St. Louis, MO); viability was determined
2
1

ChemBioChem
10.1002/cbic.201600266
by trypan blue exclusion using a hemocytometer] and incubated in a humidified chamber at
3
1
6.5 °C, 5% CO . HCT116 cells with passages 22∼40 and all media were supplemented with
2
0% fetal bovine serum (FBS) (Sigma Chemical Co., St. Louis, MO).
Methaneselenol and trifluoromethaneselenol generation: Purified recombinant Trichomonas
vaginalis methioninase produced in E. coli was purchased from Wako Pure Chemical Industries
(Japan). For cell treatments, the enzyme substrate 5 or 7 (1.25 to 5 μmol/L) was added to the
culture medium and was immediately followed by the addition of methioninase (40 U/L) for 16
[
1c, 1d]
h. The method of methaneselenol generation has been previously described.
Statistical analysis: Results are given as means ±SEM. Data were analyzed using mixed model
analyses of variance (ANOVA), blocking on group and allowing heterogeneous variances (JMP,
version 9.0, SAS Institute, Inc., Cary, NC). If the overall ANOVA was statistically significant,
then Tukey’s test for multiple comparisons was used to analyze treatment means. Differences
with a p value < 0.05 were considered statistically significant.
X-ray absorption spectroscopy: X-ray absorption measurements of the synthesized samples
were carried out at the Stanford Synchrotron Radiation Lightsource (SSRL Stanford, CA, USA)
on beamline 7-3 using a Si(220) double-crystal monochromator and Rh-coated silicon mirrors
for focusing and harmonic rejection, with the storage ring SPEAR containing 200 mA at 3.0
GeV. During data acquisition samples were maintained at a temperature of approximately 10 K
using an Oxford Instruments CF1204 helium cryostat. The incident and transmitted X-ray
intensities were measured using nitrogen-filled gas ionization chambers. The X-ray absorption
was measured as the Se Kα fluorescence excitation spectrum using an array of 30 Ge detectors
[
28]
(
Canberra Industries), with 3 absorption units of As filters. The spectrum of a hexagonal Se
foil was recorded simultaneously with each scan. Energy calibration was by reference to the
2
2

ChemBioChem
10.1002/cbic.201600266
lowest-energy inflection point in the Se K-edge absorption spectrum from the Se foil, assumed to
be 12,658 eV.
XAS data analysis: The EXAFS oscillations χ(k) were quantitatively analyzed by curve-fitting
[
29]
using the EXAFSPAK suite of computer programs,
using ab initio theoretical phase and
[
30]
amplitude functions calculated with the program FEFF, version 8.25. The threshold energy (k
-1
=
0 Å ) of the EXAFS oscillations was assumed to be 12675.0 eV. Only background-subtracted
EXAFS data was analyzed, with no Fourier filtering or smoothing applied.
Density functional structure calculations: Density functional calculations were carried out
[
31]
with the Gaussian09, revision C.01, suite of software.
Density functional theory (DFT)
provides good estimates of energetic trends, particularly between related molecular structures,
and one can generally anticipate bond-length accuracies of better than 0.05 Å. Spin restricted
calculations were optimized without geometry or symmetry constraints using the B3LYP hybrid
functional method. The 6-311+G(2df,2p) basis set was employed for geometry optimizations, as
well as harmonic frequency calculations, which were used to confirm that structures were at a
stationary point on the potential energy surface. Structures were considered optimized when the
change in energy between subsequent optimization steps fell below 0.2 J/mol. Aqueous solvation
[
32]
calculations were performed using the polarizable continuum method IEFPCM,
with the
molecular volume defined using united atom radii. Partial atomic charges for each of the
[
33]
isostructural models were calculated with the grid-based CHelpG charge derivation method at
the same level of theory as the structure calculations. In order to obtain more accurate geometries
for non-bonded complexes involving selenium and the -electron systems of aromatic rings, we
[
34]
employed Mφller-Plesset perturbation theory at the MP2/6-311+G(2df,2p)-level for geometry
optimizations.
2
3

ChemBioChem
10.1002/cbic.201600266
Expression and purification of GB1 variants: The gene of the immunoglobulin-binding B1
domain of streptococcal protein G (GB1) was codon optimized for expression in E. coli and the
gene synthesized by DNA2.0 (Menlo Park, CA). The synthetic gene of N-terminal hexahistidine-
tag tagged GB1 was cloned into the bacterial expression vector pJexpress414 (DNA2.0). The
hexahistidine-tag included the tobacco etch virus (TEV) protease cleavage sequence ENLYFQG
to allow for removal of the hexahistidine-tag. Following cleavage with TEV protease the
resulting GB1 has Gly at position 1 instead of Met, which allowed us to insert a sole Met at
specific locations. In addition, the resulting protein also contained a substitution of Gln for Thr at
position 2 since the majority of studies on GB1 utilize this variant protein to improve sample
[
35]
homogeneity.
This vector was subsequently used as a template to introduce Met at either
position Val39Met or position Leu5Met using PCR-based mutagenesis.
For protein expression, the expression plasmid of the appropriate variant was transformed into
[
20]
E. coli methionine auxotroph RF11 cells, in which the gene metA was deleted (Addgene). To
enrich the protein with TFSeM, cells were grown in LB media (10 g/L tryptone, 10 g/L NaCl, 5
g/L yeast extract), supplemented with 100 μg/mL ampicillin, with good aeration at 37 C. At the
mid exponential phase of cell growth (when the OD at 600 nm reached 0.6-0.7), the cells were
harvested by centrifugation (5000 g), and resuspended in an equal volume of a defined media
[
21, 36]
adapted from Studier’s MDAG media.
The media contained 25 mM Na HPO , 25 mM
2 4
KH PO , 50 mM NH Cl, 5 mM Na SO , 2 mM MgSO , 0.2 metals, 0.5% glucose (27.8 mM),
2
4
4
2
4
4
0
.25 mM aspartate (18.8 mM) and 200 g/ml each of 17 canonical amino acids (not Cys, Met or
Tyr). A 1 concentration of trace metals solutions contained 50 M FeCl , 20 M CaCl , 10 M
3
2
MnCl , 10 M ZnSO , 2 M CoCl , 2 M CuCl , 2 M NiCl , 2 M Na MoO , and 2 M
2
4
2
2
2
2
4
[
21]
H BO . The media also contained vitamins as described by Studier.
3
3
2
4

ChemBioChem
10.1002/cbic.201600266
The resuspended cell solution was divided into three equal aliquots (10 mL each) and
supplemented with either 200 M Met, 200 M SeM + 22 M Met, or 200 M TFSeM (as 7b) +
2
2 M Met. Protein expression was induced with 0.5 mM isopropyl-1-thio--D-
galactopyranoside (IPTG) at 18 C. The cell paste, following an 18 h expression period, was
resuspended in 400 μL B-PER™ Bacterial Protein Extraction Reagent (Thermo Scientific
Pierce) supplemented with 1 mM PMSF and 0.5 mM benzamidine. The resuspended cell paste
was then lysed using a syringe needle. The cell debris was removed following a 15 min
incubation at 25 C by centrifugation at 15000 g for 30 min. The supernatant was mixed with
4
00 μL 50 mM phosphate buffer (pH 7.5), 200 mM NaCl, and 10 mM imidazole (IMAC buffer).
The mixture was incubated with 150 μL IMAC beads (Gold Biotechnology) for 1 h while
shaking. The target protein was eluted using IMAC buffer with 500 mM imidiazole. Following
elution, the volume was concentrated to 100 μL using a spin concentrator (Amicon ultra-0.5
mL). The buffer was exchanged into 25 mM phosphate buffer (pH 7.5), 50 mM NaCl, and 1 mM
EDTA by dialysis and the hexahistidine-tag was cleaved off with TEV protease for 18 h at 4
[37]

C.
A 200 mL scale expression and purification of GB1 Val39Met was carried out as specified
above. The growth medium was supplemented with either 22 M Met, 200 M TFSeM (as 7b)
and 22 M Met, or 200 M TFSeM (as 7b).
Mass spectrometry: Mass spectra of intact proteins were obtained using a QTOF Ultima
(Waters) mass spectrometer, operating under positive electrospray ionization (+ESI) mode,
connected to an LC-20AD (Shimadzu) liquid chromatography unit. Protein samples were
separated from small molecules by reverse phase chromatography on a Waters Xbridge BEH C4
column (300 Å, 3.5 μm, 2.1 mm  50 mm), using an acetonitrile gradient from 30-71.4%, with
2
5

ChemBioChem
10.1002/cbic.201600266
0
.1% formic acid. Each analysis was 25 min under constant flow rate of 0.2 mL/min at RT. Data
were acquired from m/z 350 to 2500, at a rate of 1 sec/scan. Alternatively, spectra were acquired
by Xevo G2-S QTOF on a Waters ACQUITY UPLC Protein BEH C4 reverse-phase column
(300 Å, 1.7 μm, 2.1 mm  150 mm). An acetonitrile gradient from 5%-95% was used with 0.1%
formic acid, over a run time of 5 min and constant flow rate of 0.5 mL/min at RT. Spectrum were
acquired from m/z 350 to 2000, at a rate of 1 sec/scan. The spectra were deconvoluted using
maximum entropy in MassLynx.
Analysis and sequencing of peptides was carried out using a Q Exactive Orbitrap interfaced
with Ultimate 3000 LC system. Data acquisition by Q Exactive Orbitrap was as follows: 10 μl of
trypsin-digested protein was loaded on an Ace UltraCore super C18 reverse-phase column (300
Å, 2.5 μm, 75 mm  2.1 mm) via an autosampler. An acetonitrile gradient from 5%-95% was
used with 0.1% formic acid, over a run time of 45 min and constant flow rate of 0.2 mL/min at
RT. MS data were acquired using a data-dependent top10 method dynamically choosing the most
abundant precursor ions from the survey scan for HCD fragmentation using a stepped
normalized collision energy of 28, 30, and 35 eV. Survey scans were acquired at a resolution of
7
0,000 at m/z 200 on the Q Exactive. Theoretical patterns of isotopic patterns of peptides were
9
[38]
calculated using UCSF MS-ISOTOPE or enviPat Web 2.1.
Time-dependent activity assays of methionine adenosyltransferase with SeM and TFSeM as
substrates: The time-dependent formation of SeSAM, Se-adenosyl-
trifluoromethylselenomethionine (TFSeSAM), Se-adenosylhomoselenocysteine (SeAH), and
methylselenoadenosine (MSeA) was determined from the same reaction mixtures, which
contained the following in a volume of 400 µL when analyzed by HPLC: 0.168 mg/mL MAT, 10
mM SeM or TFSeM (as 7b), 100 mM Tris-HCl, pH 8.0, 15 mM ATP, 40 mM MgCl , 50 mM
2
2
6

ChemBioChem
10.1002/cbic.201600266
KCl, 8% v/v 2-mercaptoethanol, and 1 mM tryptophan, used as an internal standard (IS).
Reaction mixtures analyzed by LC-MS were identical except that the total reaction volume was
2
50 µL and the final concentration of SeM or TFSeM (as 7b) was 1 mM. All components except
MAT were incubated at 25 °C for 3 min before initiating the reaction with MAT. Aliquots of the
reaction mixture (50 µL for HPLC, 20 µL for LC-MS) were withdrawn at various times from 0-
1
8 h and added to 10 µL of 2 M H SO (HPLC) or 10 µL of 1 M H SO (LC-MS) to quench the
2 4 2 4
reaction. Precipitated protein was removed by centrifugation at 18,000 g for 15 min, and a 20
µL aliquot of the resulting supernatant was subjected to analysis by HPLC using method 2 or by
+
ESI LC-MS in scan mode (m/z 150 - 600) using the method described below. Standards were
generated for each analyte and analyzed under identical conditions to determine the identity of
the products generated in assays. HPLC elution times are as follows: ATP, 3.2 min; SAM, 11.0
min; SeSAM 12.3 min; adenine, 18.4 min; S-adenosylhomocysteine, (SAH), 21.8 min;
tryptophan (IS) 25.1 min; methylthioadenosine (MTA), 29.4 min. The LC-MS buffer system
consisted of solvent A (40 mM ammonium acetate and 5% v/v methanol titrated to pH 6.2 with
acetic acid) and solvent B (100 % acetonitrile). The column was equilibrated in 98% solvent A,
-1
2
% solvent B at a flow rate of 0.5 mL min . After sample injection (2 µL), a gradient was
applied from 2% solvent B to 45% solvent B over 5 min and then 45% to 5% over 3 min. The
observed ions (m/z) and retention times (min), respectively, were 447.1 and 1.1 (SeSAM), 198.0
and 1.3 (SeM), 433.1 and 1.5 (SeAH), 188.0 and 3.0 (tryptophan), 252.0 and 3.1 (TFSeM), and
3
46.1 and 4.5 (MSeA). Data were analyzed using the Agilent Technologies MassHunter
qualitative and quantitative analysis software.
Time-dependent assays of crude lysate, boiled crude lysate, and lysate filtrate with SeM and
[
21]
TFSeM. E. coli B834(DE3)pLysS cells (B834 is a methionine auxotroph ) were grown
2
7

ChemBioChem
10.1002/cbic.201600266
overnight in M9 minimal media containing 1 µg/mL chloramphenicol and 80 mg/L methionine.
The cells were isolated by centrifugation at 10,000 g at 4 ˚C for 15 min and then frozen in
liquid nitrogen. The frozen cell paste was thawed in lysis buffer containing 50 mM Tris-HCl, pH
8
.0, 200 mM KCl, and 10 mM 2-mercaptoethanol. Lysozyme and PMSF were added to final
concentrations of 1 mg/mL and 2 mg/mL, respectively, and the cells were lysed by sonication
using a Fisher sonic dismembrator (Fisher Scientific). The lysate was transferred to sterile
centrifuge tubes and subjected to centrifugation at 50,000 g for 1 h at 4 ˚C. The supernatant was
kept on ice until use that day. An aliquot of the supernatant was boiled for 10 min and then
subjected to centrifugation at 18,000 g at 4 ˚C for 30 min. An additional aliquot of the
supernatant was transferred to a spin concentrator containing a 3 kDa molecular weight cutoff
membrane and subjected to the same centrifugation as the boiled lysate to produce a protein-free
filtrate. Aliquots (200 µL) of crude lysate, boiled crude lysate, or filtrate were added to reaction
mixtures at 25 ˚C containing 1 mM SeM or 1 mM TFSeM (as 7b) and 100 mM Tris-HCl, pH
8
.0. Aliquots (20 µL) were withdrawn at various times from 0-18 h, quenched in 10 µL of 1 M
H SO , and analyzed by LC-MS as described above.
2
4
2
8

ChemBioChem
10.1002/cbic.201600266
ASSOCIATED CONTENT
Supporting Information. X-ray Crystal Data and Structure Refinement, supporting information
for DFT calculation, MAT activity assays and information about synthesis and compounds
purity. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*
*
E-mail: eblock@albany.edu. Phone 518-442-4459
E-mail: rozovsky@udel.edu. Phone 302-831-7028
Present Addresses

Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Jena, 07745
Germany
#
Atlanta Metropolitan State College, Atlanta, Georgia 30310, United States
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Funding Sources
Work at the University of Albany was supported by grants from the National Science
Foundation (CHE-0744578; CHE-1265679; CHE-1337594 [MRI]; CHE-1429329 [MRI]) and
the University at Albany (E.B.). Work at Grand Forks Human Nutrition Research Center was
supported by USDA-ARS, CRIS project (5450-51000-045-00D) (H.Z.). Work at the University
2
9