Hydrogen/deuterium exchange of cross-linkable α-amino acid derivatives in deuterated triflic acid

Article abstract of DOI:10.1080/09168451.2014.917267

In this paper we report here a hydrogen/deuterium exchange (H/D exchange) of cross-linkable α-amino acid derivatives with deuterated trifluoromethanesulfonic acid (TfOD). H/D exchange with TfOD was easily applied to o-catechol containing phenylalanine (DOPA) within an hour. A partial H/D exchange was observed for trifluoromethyldiazirinyl (TFMD) phenylalanine derivatives. N-Acetyl-protected natural aromatic α-amino acids (Tyr and Trp) were more effective in H/D exchange than unprotected ones. The N-acetylated TFMD phenylalanine derivative afforded slightly higher H/D exchange than unprotected derivatives. An effective post-deuteration method for cross-linkable α-amino acid derivatives will be useful for the analysis of biological functions of bioactive peptides and proteins by mass spectrometry.

Full text of DOI:10.1080/09168451.2014.917267

This article was downloaded by: [University of West Florida]
On: 05 October 2014, At: 05:04
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer
House, 37-41 Mortimer Street, London W1T 3JH, UK
Bioscience, Biotechnology, and Biochemistry
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/tbbb20
Hydrogen/deuterium exchange of cross-linkable α-
amino acid derivatives in deuterated triflic acid
a
a
a
a
b
Lei Wang , Yuta Murai , Takuma Yoshida , Masashi Okamoto , Katsuyoshi Masuda , Yasuko
a
a
c
c
Sakihama , Yasuyuki Hashidoko , Yasumaru Hatanaka & Makoto Hashimoto
a
Division of Applied Science, Graduate School of Agriculture, Hokkaido University,
Sapporo, Japan
b
Suntory Institute for Bioorganic Research, Osaka, Japan
c
Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama,
Toyama, Japan
Published online: 17 Jun 2014.
To cite this article: Lei Wang, Yuta Murai, Takuma Yoshida, Masashi Okamoto, Katsuyoshi Masuda, Yasuko Sakihama,
Yasuyuki Hashidoko, Yasumaru Hatanaka & Makoto Hashimoto (2014) Hydrogen/deuterium exchange of cross-linkable
α-amino acid derivatives in deuterated triflic acid, Bioscience, Biotechnology, and Biochemistry, 78:7, 1129-1134, DOI:
10.1080/09168451.2014.917267
To link to this article: http://dx.doi.org/10.1080/09168451.2014.917267
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of
the Content. Any opinions and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied
upon and should be independently verified with primary sources of information. Taylor and Francis shall
not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other
liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or
arising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions

Bioscience, Biotechnology, and Biochemistry, 2014
Vol. 78, No. 7, 1129–1134
Hydrogen/deuterium exchange of cross-linkable α-amino acid derivatives in
deuterated triflic acid
1
1,a
1
1
2
Lei Wang , Yuta Murai , Takuma Yoshida , Masashi Okamoto , Katsuyoshi Masuda ,
1
1
3
1,
Yasuko Sakihama , Yasuyuki Hashidoko , Yasumaru Hatanaka and Makoto Hashimoto *
1
2
Division of Applied Science, Graduate School of Agriculture, Hokkaido University, Sapporo, Japan; Suntory
3
Institute for Bioorganic Research, Osaka, Japan; Graduate School of Medicine and Pharmaceutical Sciences,
University of Toyama, Toyama, Japan
Received January 28, 2014; accepted February 27, 2014
http://dx.doi.org/10.1080/09168451.2014.917267
6
)
7)
In this paper we report here a hydrogen/deute-
rium exchange (H/D exchange) of cross-linkable
α-amino acid derivatives with deuterated trifluoro-
methanesulfonic acid (TfOD). H/D exchange with
TfOD was easily applied to o-catechol containing
phenylalanine (DOPA) within an hour. A partial
H/D exchange was observed for trifluoromethyldiaz-
irinyl (TFMD) phenylalanine derivatives. N-Acetyl-
protected natural aromatic α-amino acids (Tyr and
Trp) were more effective in H/D exchange than
unprotected ones. The N-acetylated TFMD phenylal-
anine derivative afforded slightly higher H/D
exchange than unprotected derivatives. An effective
post-deuteration method for cross-linkable α-amino
acid derivatives will be useful for the analysis of bio-
logical functions of bioactive peptides and proteins
by mass spectrometry.
acidic condition and/or metal catalysis on aromatics
have been reported in the literature. Although aro-
matic α-amino acids were considered to be the sub-
strate for H/D exchange, the α-amino acid skeleton
sometimes inhibited to apply previous H/D exchange
methods due to their solubility in many organic
8
,9)
solvent.
Recently we developed a method for effective H/D
exchange for natural aromatic α-amino acids and their
containing peptides with trifluoromethanesulfonic acid-
d (TfOD) at a temperature lower than room temperature
without a side effect on stereochemistry. The exchange
can be monitored regularly and controlled by adjusting
the reaction temperature and reagent ratio. We report
in this paper the post-H/D exchange for cross-linkable
aromatic α-amino acid derivatives with TfOD under
mild conditions.
1
0)
Key words: Hydrogen/deuterium exchange; aromatic
α-amino acid; cross-link; photophore; tri-
flic acid
Results and discussion
3,4-Dihydroxy-L-phanylalanine (L-DOPA, 1) has cat-
echol moiety on the benzene ring, which can be uti-
lized as a cross-linkable functional group in the
11–13)
presence of periodate.
The hydroxyl groups in
Introduction
the molecule might enhance the H/D exchange due to
their electron-donating properties. H/D exchange of
about 90% takes place at three exchangeable sites on
the benzene ring at 0 °C or room temperature within
Cross-linking reagents are valuable tools for investi-
gating ligand–biomolecule interactions. Based on the
progress of the mass analysis, stable isotope-labeled
bioactive ligands are attractive tools in the field.
Deuterium is one of the simplest stable isotopes and
can be utilized for post-introductions to bioactive
compounds. Many attempts for deuterium introduction
in the aromatic portions of compounds have been
reported.
1
)
1
20 min in TfOD as see in the H-NMR measurement
2
)
(Fig. 1). The degree of exchange did not change with
longer incubation time. ESI-TOF mass analysis also
indicated that three hydrogen atoms exchanged with
deuterium (m/z 201 and 198 with and without deute-
rium exchange, respectively). It has been reported that
the H/D exchange of 1 in 37%DCl/D
ducted at 50 °C for 24 h.
O was con-
Our H/D exchange with
TfOD proceeded very swiftly even though it was
2
3
–5)
14)
Hydrogen/deuterium exchange (H/D exchange)
at a specific position and universal H/D exchange in
*
Corresponding author. Email: hasimoto@abs.agr.hokudai.ac.jp
a
Present address: Faculty of Advanced Life Science, Frontier Research Center for Post-Genome Science and Technology, Hokkaido University, Kita
1, Nishi 11, Kita-ku, Sapporo 001-0021, Japan.
2
©
2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry

1130
L. Wang et al.
1
Fig. 1. Time-course H/D exchange for L-DOPA (1) treated with TfOD at 0 °C by H-NMR spectrum in D
2
O.
1
Notes: H-NMR of the reaction mixture at 0 min, 20 min, 1 h and 3 h are (A), (B), (C) and (D), respectively.
conducted at low temperature. The H/D exchange
ratio for 1 was enhanced significantly than that for
L-tyrosine, which favored H/D exchange at the 3-posi-
tion. All C–H bonds on the aromatic moiety of 1
were placed at o- or p- positions against two hydro-
xyl groups. These characters promoted H/D exchange
for DOPA swiftly even though the reaction was con-
ducted at 0 °C.
in no significant detectable differences on the mass
spectrum compared to untreated samples. These results
indicated that the carbonyl group acted as the electron
withdrawing group to the benzene ring and hampered
the H/D exchange reaction at room temperature
(Fig. 2(B)).
L-Phe(4-TFMD) (4) was subjected to H/D
exchange with TfOD. It was observed that the diazir-
inyl ring was decomposed within 1 h at room tem-
perature. The result was consistent with the previous
reports of instability of the diazirinyl ring in strong
Photoaffinity labeling is the most attractive method
for creating
biomolecule interactions.
α-amino acid derivatives have been used to investi-
a
cross-link between ligand and
1
5,16)
Photoreactive aromatic
2
2,23)
acidic conditions over room temperature.
No
1
7–20)
gate bioactive peptides interactions.
Three com-
decomposition of the diazirine ring was observed
when the reaction was conducted at 0 °C for 5 h.
The H/D exchange proceeded in a time-dependent
manner and the H/D exchange ratio was observed up
to 35% (35 and 10% for the 2- and 3- position,
mon photophores, arylazide, benzophenone, and
diazirine, were utilized for the photoaffinity labeling.
The H/D or tritium exchange for photoreactive aro-
matic α-amino acid derivatives has not yet been
reported.
1
respectively) in H-NMR measurement (Fig. 2(C)).
L-Phe(4-azido) (2) was treated with TfOD at room
temperature and the reaction mixture afforded a compli-
cated mixture within a half hour. It has been reported
Further reaction at the same temperature caused
decomposition of diazirinyl ring.
To improve the H/D exchange ratio for compound
4, we used N-acetyl-protected aromatic α-amino acid
derivatives in the H/D exchange. N-Ac-L-Tyr (6) was
subjected to treatment with TfOD at room tempera-
ture. H/D exchange at both, the 2- and 3- position of
6 was more rapid than that of unprotected L-Tyr (5).
It was observed that 90% H/D exchange occurred
within 3 h at the 3-position and over 24 h at the
2
1)
that the azide group easily reacts in TfOH
and no
improvement was observed in the exchange reaction
conducted at 0 °C (Fig. 2(A)).
L-Phe(4-benzoyl) (3) was stable in the treatment with
TfOD at room temperature for a day. However, low incor-
poration (less than 8% per aromatic proton) was observed
1
in H-NMR measurement. The low incorporation resulted

Hydrogen-deuterium exchange of cross-linkable α-amino acids
1131
Fig. 2. H/D exchange of photophore containing aromatic α-amino acid.
Notes: (A) L-Phe(4-azido) (2), (B) L-Phe(4-benzoyl) (3), and (C) L-Phe(4-TFMD) (4) at 0 °C.
Time-course H/D exchange ratio for 2- and 3- position of compound 4 was presented by open square and circle, respectively.
2
-position for 5 at room temperature (Fig. 3(A)). The
exchange per aromatic proton) (Fig. 4(B)). The results
indicated that the electron-withdrawing properties of
the carbonyl group were the main factor for a lower H/
D exchange ratio.
exchange rate was drastically improved using 6 for
over 90% exchange, within 1 and 6 h at the 3- and
2
- position, respectively (Fig. 3(B)). The same tenden-
cies were observed for L-Trp derivatives. The H/D
exchange for aromatic protons of Trp was observed
to be up to 80% at room temperature. Although
unprotected, L-Trp (7) took over 8 h and N-Ac-L-Trp
For trifluoromethyldiazirinyl (TFMD) derivative 11,
H/D exchange at room temperature promoted decom-
position of the diazirinyl ring within 1 h, which was
consistent with previous results. On the other hand,
H/D exchange conducted at 0 °C promoted higher H/
D exchange at both the 2- (up to 35%) and 3- (up to
70%) position for 5 h than unprotected L-Phe(4-
TFMD) (4) (Fig. 4(C)). N-Protection improved H/D
exchange with TfOD for the unfavored substrate due
to the prevention of neutralization of TfOD by amino
groups.
(
8) took less than 3 h (Fig. 3(C)). These results indi-
cate that the exchange rate for N-acetyl-protected aro-
matic α-amino acids is faster than that for
unprotected compounds.
Based on the above observations, N-Ac-L-DOPA (9),
N-Ac-L-Phe(4-benzoyl) (10), and N-Ac-L-Phe(4-TFMD)
(
11) were subjected to H/D exchange in TfOD. For
DOPA derivative 9, the N-protection did not cause
improvement in H/D exchange drastically because
unprotected 1 is a good substrate for our H/D
exchange. Compound 9 was easily deuterated by over
We described post-deuteration (H/D exchange) of
cross-linkable aromatic α-amino acid derivatives in
TfOD at low temperature. The exchange conditions
were beneficial due to the introduction of deuterium at
the final steps, reaction at low temperature, and the
detectable progress of the reaction. These reactions
were not easy to perform using previously used
9
0% at 0 °C within 20 min (Fig. 4(A)). No improve-
ment was observed for the treatment of 10 at room
1
temperature with H-NMR analysis (less than 8%

1132
L. Wang et al.
Fig. 3. Time-course H/D exchange of L-Tyr (5), N-Ac-L-Tyr (6), L-Trp (7), and N-Ac-L-Trp (8).
Notes: ((A), (B)) Time-course H/D exchange ratio for 2- and 3- position of compound 5 or 6 was presented by open square and circle, respec-
tively. (C) Time-course H/D exchange ratio for indole protons of compound 7 or 8 was presented by open triangle and diamond, respectively.
methods. The advanced synthetic methods will be uti-
lized to elucidate ligand–biomolecule interaction with
mass spectrometry.
General procedures for H/D exchange with TfOD.
Cross-linkable aromatic α-amino acid derivatives
(0.25 mM) were dissolved in TfOD (0.9 mL, 10 mM) at
a specific temperature. The reaction mixture was
1
diluted with H O (0.6 mL) followed by H-NMR and
2
ESI-TOF mass analysis.
Experimental
General methods.
1
H-NMR spectra were measured
General procedures for acetylation of aromatic
α-amino acid derivatives.
derivatives (0.4 mM) were dissolved in methanol (2 mL)
and acetic anhydride (0.8 mM) was added dropwise at
by a Jeol EX 270 spectrometer for determining the H/
D exchange ratio. MS data were obtained with a
Waters LCT Premier XE instrument. ESI-TOF-MS data
were obtained with a Waters UPLC ESI-TOF mass
spectrometer. TfOD (98 atom % D) was purchased
from Sigma–Aldrich. L-DOPA was purchased from
Wako chemicals. Photoreactive L-phenylalanine deriva-
tives were synthesized by methods described in a previ-
Aromatic α-amino acid
4
0 °C. The reaction mixture was stirred at same temper-
ature for overnight and concentrated. The residue was
subjected to silica column chromatography (CH Cl :
2
2
CH OH = 4:1, then ethyl acetate:CH OH = 1:1) to afford
3
3
2
4)
pure N-acetyl compounds in 60–75% yield.
ous study.

Hydrogen-deuterium exchange of cross-linkable α-amino acids
1133
[
2] Arsene CG, Ohlendorf R, Burkitt W, Pritchard C, Henrion A,
O’Connor G, Bunk DM, Güttler B. Protein quantification by
isotope dilution mass spectrometry of proteolytic fragments:
cleavage rate and accuracy. Anal. Chem. 2008;80:4154–4160.
3] Dormán G, Olszewski JD, Prestwich GD, Hong Y, Ahern DG.
Synthesis of highly tritiated 4-benzoyl-L-phenylalanine, a pho-
toactivatable amino acid. J. Org. Chem. 1996;60:2292–2297.
4] Ambroise Y, Mioskowski C, Djéga-Mariadassou G, Rousseau B.
Consequences of affinity in heterogeneous catalytic reactions:
highly chemoselective hydrogenolysis of iodoarenes. J. Org.
Chem. 2000;65:7183–7186.
[
[
[
5] Faucher N, Ambroise Y, Cintrat JC, Doris E, Pillon F, Rousseau B.
Highly chemoselective hydrogenolysis of iodoarenes. J. Org. Chem.
2002;67:932–934.
[
6] Atzrodt J, Derdau V, Fey T, Zimmermann J. The renaissance of
H/D exchange. Angew. Chem. Int. Ed. 2007;46:7744–7765.
7] Ito N, Watahiki T, Maesawa T, Maegawa T, Sajiki H. Synergistic
effect of a palladium-on-carbon/platinum-on-carbon mixed cata-
lyst in hydrogen/deuterium exchange reactions of alkyl-substituted
aromatic compounds. Adv. Synth. Catal. 2006;348:1025–1028.
8] Benfenati E, Icardi G, Chen S, Fanelli R. Syntheses of deuterated
leu-enkephalins and their use as internal standards for the quantifi-
cation of leu-enkephalin by fast atom bombardment mass
spectrometry. J. Labelled Compd. Radiopharm. 1990;28:411–419.
9] Faull KF, Barchas JD, Murray S, Halpern B. Deuterated
peptides: platinum catalyzed exchange labeling of leucine
enkephalin and related peptides. Biomed. Mass Spectrom. 1983;
[
[
[
1
0:463–470.
[
10] Murai Y, Wang L, Masuda K, Sakihama Y, Hashidoko Y,
Hatanaka Y, Hashimoto M. Rapid and controllable hydrogen-
deuterium exchange on aromatic rings of α-amino acids and
peptides. Eur. J. Org. Chem. 2013;5111–5116.
[
[
[
[
11] Umanah GKE, Son C, Ding FX, Naider F, Becker JM.
Cross-linking of a dopa-containing peptide ligand into its g
protein-coupled receptor. Biochemistry. 2009;48:2033–2044.
12] Burdine L, Gillette TG, Lin HJ, Kodadek T. Periodate-triggered
cross-linking of dopa-containing peptide−protein complexes. J.
Am. Chem. Soc. 2004;126:11442–11443.
13] Liu B, Burdine L, Kodadek T. Chemistry of periodate-mediated
cross-linking of 3,4-dihydroxylphenylalanine-containing mole-
cules to proteins. J. Am. Chem. Soc. 2006;128:15228–15235.
14] Pająk M, Kańska M. Enzymatic synthesis of dopamine ring
labeled with hydrogen isotopes. J. Radioanal. Nucl. Chem.
2
009;279:455–458.
[
[
15] Brunner J. New photolabeling and crosslinking methods. Annu.
Rev. Biochem. 1993;62:483–514.
16] Kotzyba-Hibert F, Kapfer I, Goeldner M. Recent trends in
photoaffinity labeling. Angew. Chem. Int. Ed. Engl. 1995;34:
1
296–1312.
[
[
[
17] Nassal M. 4′-(1-Azi-2,2,2-trifluoroethyl)phenylalanine, a photola-
bile carbene-generating analog of phenylalanine. J. Am. Chem.
Soc. 1984;106:7540–7545.
18] Shih LB, Bayley H. A carbene-yielding amino acid for incorpo-
ration into peptide photoaffinity reagents. Anal. Biochem.
Fig. 4. H/D exchange of photophore containing N-acetyl aromatic
α-amino acid.
Notes: (A) N-Ac-L-DOPA (9), N-Ac-L-Phe(4-benzoyl) (10), and (B)
N-Ac-L-Phe(4-TFMD) (11) at 0 °C.
(
A) Time-course H/D exchange ratio for 2-, 5-, and 6- position of
1
985;144:132–141.
compound 9 was presented by open square, triangle, and circle, respec-
tively. (C) Time-course H/D exchange ratio for 2- and 3- position of
compound 11 was presented by open square and circle, respectively.
19] Masuda K, Koizumi A, Misaka T, Hatanaka Y, Abe K, Tanaka
T, Ishiguro M, Hashimoto M. Photoactive ligands probing the
sweet taste receptor. Design and synthesis of highly potent diaz-
irinyl D-phenylalanine derivatives. Bioorg. Med. Chem. Lett.
2
010;20:1081–1083.
Acknowledgments
[
20] Murai Y, Masuda K, Sakihama Y, Hashidoko Y, Hatanaka Y,
Hashimoto M. Comprehensive synthesis of photoreactive (3-tri-
fluoromethyl)diazirinyl indole derivatives from 5- and 6- triflu-
oroacetylindoles for photoaffinity labeling. J. Org. Chem. 2012;
77:8581–8587.
[21] Olah GA, Ramaiah P, Wang Q, Prakash GKS. Synthetic meth-
ods and reactions. 188. Triflic acid catalyzed phenylamination of
aromatics with phenyl azide. J. Org. Chem. 1993;58:6900–6901.
This research was partially supported by the Fugaku
Foundation, Noastec Foundation and the Cooperative
Research Program of “Network Joint Research Center
for Materials and Devices” for financial support.
[
22] Moss RA, Fedé JM, Yan S. SbF
5
-mediated reactions of oxafluo-
References
rodiazirines. Org. Lett. 2001;3:2305–2308.
[
1] Paramelle D, Miralles G, Subra G, Martinez J. Chemical cross-
linkers for protein structure studies by mass spectrometry. Pro-
teomics. 2013;13:438–456.
[23] Murai Y, Masuda K, Ogasawara Y, Wang L, Hashidoko Y, Hata-
naka Y, Iwata S, Kobayashi T, Hashimoto M. Synthesis of photo-
reactive 2-phenethylamine derivatives. –Synthesis of adenosine

1
134
L. Wang et al.
derivatives enabling functional analysis of adenosine receptors via
photoaffinity labeling–. Eur. J. Org. Chem. 2013;2428–2433.
24] Wang L, Hisano W, Murai Y, Sakurai M, Muto Y, Ikemoto H,
Okamoto M, Murotani T, Isoda R, Kim D, Sakihama Y, Sitepu
IR, Hashidoko Y, Hatanaka Y, Hashimoto M. Distinct metabo-
lites for photoreactive L-phenylalanine derivatives in Klebsiella
sp. CK6 isolated from rhizosphere of a wild dipterocarp sapling.
Molecules. 2013;18:8393–8401.
[