Hydrogen/Deuterium Exchange on Aromatic Rings
Experimental Section
General Remarks: NMR spectra were measured with a JEOL EX-
280 spectrometer. ESI-TOF-MS data were obtained with a Waters
UPLC ESI-TOF mass spectrometer. Deuterated trifluoromethane-
sulfonic acid (98 atom-% D) and maculosin were purchased from
Sigma–Aldrich. Chymotrypsin was purchased from Wako chemi-
cals. Leucine-enkephakine and Delta-sleeping inducing peptide
(
DSIP) were purchased from the Peptide Institute.
Friedel–Crafts Reaction of Toluene (1) and N-Trifluoroacetyl-L-
Asp(Cl)-OMe (2) in TfOD: Compound 2[ (31.0 mg, 0.113 mmol)
and toluene (1; 12 μL, 0.113 mmol, 1 equiv.) were dissolved in
TfOD (0.5 mL) at 0 °C, and the reaction mixture was stirred for
6b]
1
h at room temperature and poured into ice-cold H
and EtOAc (15 mL). The organic layer was washed with saturated
aqueous NaCl and dried with MgSO , filtered, and then concen-
trated. The residue was purified by silica gel chromatography
EtOAc/n-hexane, 1:4) to yield 3 (34.8 mg, 0.105 mmol, 94%). [α]
+24.0 (c = 2.0, CHCl ). The chirality was also verified by chiral
HPLC (Chirobiotic T2; EtOH/H O, 10%). Deuterium incorpora-
2
O (15 mL)
4
(
=
D
3
2
1
tion was calculated to be 68–75% on the basis of H NMR spectro-
scopic analysis (see the Supporting Information, Figure SI-1).
General Procedures for Hydrogen/Deuterium Exchange with TfOD
For Aromatic α-Amino Acids: Aromatic α-amino acid (0.25 mmol)
was dissolved in TfOD (0.9 mL, 10 mmol, 40 equiv.) at the indi-
2
cated temperature. The reaction mixture was diluted with H O
1
(
0.6 mL), and the solution was analyzed by H NMR spectroscopy
and ESI-TOF MS.
For Peptides: Peptide (9 μmol) was dissolved in TfOD (32 μL,
.36 mmol for one aromatic α-amino acid) at the indicated tem-
perature. The reaction mixture was diluted with H O (0.5 mL), and
0
2
1
Figure 5. Extent of deuteration of the aromatic residue of maculo-
sin in hydrogen/deuterium exchange with TfOD.
the solution was analyzed by H NMR spectroscopy and ESI-TOF
MS. The hydrogen/deuterium exchange samples were analyzed by
ESI-TOF MS several times. No change in the analytical data for
the sample was observed when measurements were repeated after
storing the sample in a refrigerator for 1 month.
Conclusions
Chymotrypsin Digestion for Analysis of Hydrogen/Deuterium-Ex-
changed Leucine-Enkephaline: The hydrogen/deuterium-exchanged
leucine-enkephalin was made neutral by addition of 1 m NaOH. To
the solution, chymotrypsin (1/20 against mol-equiv.) was added.
The mixture was incubated at 37 °C for 24 h, then subjected to
ESI-TOF MS.
This first detailed analysis of hydrogen/deuterium ex-
change at aromatic α-amino acids with TfOD at low tem-
perature (from 0 °C to room temperature) was examined.
The reaction time for exchange was extremely fast com-
pared with previous methods, and this encouraged us to
examine which factors determine the deuterium content in
α-amino acids and peptides. It was found that deuterium
incorporation can be controlled by the amount of reagent,
the reaction time, and the temperature. To the best of our
Supporting Information (see footnote on the first page of this arti-
cle): MS and MSMS data of hydrogen/deuterium exchanged pept-
ides.
knowledge, there has been no comprehensive study of hy- Acknowledgments
drogen/deuterium exchange for aromatic rings in biolo-
This research was partially supported by a Ministry of Education,
Science, Sports and Culture Grant-in-Aid for Scientific Research
C), Japan (19510210, 21510219.) M. H. also thanks the Fugaku
gically active peptides. This new method is easier to apply
to small amounts of peptides than current standard ap-
proaches and does not require special equipment. Hydro-
gen/deuterium exchange for aromatic rings of α-amino ac-
ids and peptides should encourage researchers to use IDMS
techniques for the quantitative analysis of biologically
(
Foundation for financial support. Y. M. thanks the Clark Memo-
rial Foundation and Sapporo Agriculture Organization for finan-
cial support. Part of this work was performed under the Coopera-
tive Research Program of “Network Joint Research Center for Ma-
active peptides. Further application of this hydrogen/deuter- terials and Devices”.
ium exchange with TfOD for other aromatic compounds
is underway. Our findings show that aromatic C–H bond
activation can occur at low temperature, which could con-
tribute to the establishment of new development processes.
[
1] a) C. G. Arsene, R. Ohlendorf, W. Burkitt, C. Pritchard, A.
Henrion, G. O’Connor, D. M. Bunk, B. Güttler, Anal. Chem.
2008, 80, 4154–4160.
Eur. J. Org. Chem. 2013, 5111–5116
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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