Hydrogen-Deuterium Exchange and Selective Labeling of Deprotonated Amino Acids and Peptides in the Gas Phase

Article abstract of DOI:10.1021/ja076599l

Hydrogen-deuterium exchange reactions of deprotonated amino acids and small peptides were studied. Selective labeling can be carried out at the α-amino group of lysine (2 of 4 labile hydrogens undergo exchange with CF₃CH₂OD) and the

Full text of DOI:10.1021/ja076599l

Published on Web 12/08/2007
Hydrogen-Deuterium Exchange and Selective Labeling of Deprotonated
Amino Acids and Peptides in the Gas Phase
Zhixin Tian, Lev Lis, and Steven R. Kass*
Department of Chemistry, UniVersity of Minnesota, Minneapolis, Minnesota 55455
Received August 31, 2007; E-mail: kass@chem.umn.edu
Hydrogen atoms attached to oxygen, nitrogen, and sulfur can
be replaced with deuterium upon reacting protio compounds with
deuterium oxide or deuterated alcohols (ROD). The isotopic
exchange of these labile hydrogens is an important tool for the
determination of protein structures and the study of dynamic
processes such as protein folding.1 Isotopically labeled species also
provide a valuable means for obtaining mechanistic information.
We report herein on the H/D exchange of deprotonated amino acids
and small peptides. To our surprise, site-specific labeling can be
carried out on the M - H ions of arginine, lysine, and a variety of
small peptides with select deuterated reagents. The replacement of
only some of the labile hydrogens should prove useful in probing
Figure 1. Mass spectra for the H/D exchange of deprotonated lysine with
CF3CH2OD (2.4 × 10-7 Torr) at time 0.0 s (inset) and 28.5 s.
reaction mechanisms and the mobile nature of the proton. In
addition, the sequence order is found to effect the extent of
exchange.
The conjugate base of glycine undergoes two H/D exchanges
with CF3CH2OD,2 and molecular modeling indicates that this
process takes place via a four-centered flip-flop process in which
the O-D and N-H bonds interchange via a zwitterionic transition
structure (see Figure S1 in the Supporting Information).3 Given
these results, it is not surprising that the M - H ions of Arg, Asn,
Cys,4 Glu, His, Pro, Thr, Trp, and Tyr undergo exchange of their
R-amino hydrogens with CF3CH2OD. It is less apparent what will
happen to the hydrogens attached to the side-chain heteroatoms,
but they too are replaced with deuterium presumably by a variety
of processes.3-5 Lysine, however, is surprising in that only one of
the two amino groups undergoes H/D exchange (Figure 1).6 Given
that the disappearance rate constant of the d0 ion of Lys is similar
to that of glycine (k ) (2.2 ( 0.7) × 10-11 and (2.6 ( 0.8) ×
10-11 cm3 molecule-1 s-1, respectively),7 this suggests that it is
the hydrogens on the R-amino group which are replaced rather than
those on the side chain.
Figure 2. Mass spectra for the H/D exchange of deprotonated arginine
with C6H5CH2OD (2.7 × 10-7 Torr) at time 0.0 s (inset) and 75 s.
k e 1 × 10-13 cm3 molecule-1 s-1). The conjugate base of arginine
is the sole exception as it undergoes three H/D exchanges with
both of these reagents (k ) (7.1 ( 2.1) × 10-11 and (1.3 ( 0.4) ×
10-12 cm3 molecule-1 s-1, respectively) (Figure 2). These findings
suggest that the deuterium is incorporated into the guanidine side
chain, but to address this issue further, several methylated deriva-
tives and R-desamino arginine (H2NC(dNH)NH(CH2)4CO2-) were
examined. The latter compound and N(R),N(R)-dimethylarginine
(H2NC(dNH)NHCH2CH2CH2CH(NMe2)CO2-) both incorporate up
to three deuterium atoms with C6H5CH2OD (k ) (1.5 ( 0.5) ×
10-10 and (1.0 ( 0.3) × 10-10 cm3 molecule-1 s-1, respectively),
whereas N(γ),N(γ)-dimethylarginine (Me2NC(dNH)NH(CH2)3-
CH(NH2)CO2-) does not react at all. These results confirm that
three of the four hydrogens on the guanidine side chain undergo
exchange, and they indicate that at least one hydrogen on the side-
chain NH2 group is required for the reaction to occur. A pathway
which is consistent with these findings is the six-centered flip-flop
mechanism illustrated in Scheme 1 in which the carboxylate
presumably facilitates the process by hydrogen bonding to one of
the sites on the guanidine side chain. Processes of this sort were
previously proposed by Beauchamp et al.,3 and other viable options
are difficult to envision given the weak acidities of the exchange
reagents that were employed.
To test this inference, the reactivities of N(R)-methyllysine
(H2N(CH2)4CH(NHMe)CO2-) and N(ꢀ)-methyllysine (MeNH-
(CH2)4CH(NH2)CO2-) were explored. The former compound
undergoes one H/D exchange, whereas the latter incorporates two
deuteriums, thereby confirming that it is the R-amino group of lysine
which undergoes exchange. Electrostatic interactions can account
for this observation since the R-amino group is closer to the charged
site. That is, a zwitterionic transition state or intermediate with a
+
NH3 group is expected to be more stable at the R-position than
on the more distant side chain. This difference should diminish
with more acidic exchange reagents since they lead to faster
reactions (measured rate constants are given in Table S1). In accord
with this expectation, all of the labile hydrogens in the M - H ion
of Lys rapidly undergo exchange with d6-phenol (k ) (1.1 ( 0.3)
× 10-9 cm3 molecule-1 s-1), which is 12 kcal mol-1 more acidic
than CF3CH2OH.8
Less acidic exchange reagents react more slowly, and C6H5CH2-
OD and CH3CH2OD were found to be unreactive with deprotonated
glycine and other nonacidic side-chain-containing amino acids (i.e.,
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10.1021/ja076599l CCC: $40.75 © 2008 American Chemical Society

C O M M U N I C A T I O N S
Scheme 1. Proposed Six-Centered Flip-Flop H/D Exchange
Mechanism for the Incorporation of Three Deuteriums into the
Side Chain of Deprotonated Arginine
Several larger peptides have been reacted with CF3CH2OD, as
well, and they too display a wide range of behavior. For example,
all four N-H hydrogens undergo exchange in the M - 1 ion of
Ala-Leu-Gly, but the amide sites react approximately 1 order of
magnitude more rapidly than the N-terminus. In contrast, just three
of the seven labile hydrogens are replaced with deuterium in Trp-
Met-Asp-Phe, and (Gly)6 does not react at all (i.e., k e 1 × 10-13
cm3 molecule-1 s-1).
These results indicate that some small deprotonated peptide ions
can be differentially labeled, which has not been demonstrated
previously.9,10 The extent of H/D exchange depends upon the
primary sequence, both in terms of which amino acids are present
and their order in the peptide chain. These findings raise many
new questions but should be useful in carrying out mechanistic
studies and addressing the mobile proton controversy.11
Scheme 2. A Plausible Relay Mechanism for the H/D Exchange
Reaction of the Gly-Gly M - H Ion with CF3CH2OD
Acknowledgment. Support from the National Science Founda-
tion and the Minnesota Supercomputer Institute is gratefully
acknowledged.
Supporting Information Available: Table S1, Figure S1, the
preparation of N(R),N(R)-dimethylarginine, the purification of R-de-
samino arginine and their NMR spectra along with a brief description
of the gas-phase experiments are provided in conjunction with sample
data (Figures S2 and S3). This material is available free of charge via
the Internet at http://pubs.acs.org.
The above findings are significant in that they indicate some
anions can be differentially labeled in the gas phase even when
this cannot be done in solution. Moreover, these results are not
limited just to deprotonated amino acids. We have examined the
H/D exchange of 24 dipeptide M - H ions with CF3CH2OD (i.e.,
Ala-Met (1), Ala-Phe (1), Ala-Ser (2), Ala-Trp (3), Ala-Val (1),
Arg-Tyr (8), Arg-Val (4), γ-Glu-Gly (4), Gly-Glu (2), Gly-Gly
(1), Gly-His (2), Gly-Phe (1), Gly-Pro (0), His-Gly (2), His-Leu
(4), Leu-Leu (1), Lys-Phe (3), Lys-Ser (4), Met-Trp (1), Phe-Ala
(1), Phe-Tyr (1), Pro-Gly (2), Pro-Leu (1), and Tyr-Arg (6)), where
the number in parentheses indicates the number of exchanges that
were observed. Only 1 deuterium is incorporated in 10 of the
dipeptides even though they have 2-4 labile hydrogens. The amide
hydrogen almost assuredly is the one being replaced in all of these
substrates given that the reaction rates are within a factor of 3 of
each other (i.e., k ) (5.7 ( 1.7) × 10-11 to (1.6 ( 0.5) × 10-10
cm3 molecule-1 s-1), and the one dipeptide that does not have an
amide hydrogen (Gly-Pro) does not react (i.e., k e 1 × 10-13 cm3
molecule-1 s-1). This leads us to suggest a relay process3-5 for
this transformation (Scheme 2) particularly since ∆∆H°acid (CH3-
CONH2 - CH3CO2H) ) 13 ( 3 kcal mol-1 and model calculations
indicate that the difference in acidity between the amide and the
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