The unexpected racemization and hydrogen-deuterium exchange of the hydrogen at the α-carbon of proline analogs containing the 5-azoniaspiro[4.4]nonyl-group

Article abstract of DOI:10.1039/c7ob02926h

Recently, we developed a novel non-fragmenting quaternary ammonium ionization tag for the mass spectrometric sensitive sequencing of peptides, based on the N-spiro proline residue (5-azoniaspiro[4.4.]nonyl-carbonyl). Herein, we present an unexpected racemization and the hydrogen-deuterium exchange (HDX) at the α-C atom of the proline derivative under basic aqueous conditions (1% water solution of triethylamine). The deuterium atom, substituted for the α-C atom, does not undergo back-exchange under acidic aqueous conditions which makes the deuterated isotopologue a promising stabile isotope-coded internal standard for quantitative analysis by mass spectrometry. The applicability of the prepared isotopologues of the quaternary ammonium salt labeled peptides for quantification experiments using the isotopic dilution method was also examined.

Full text of DOI:10.1039/c7ob02926h

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Unexpected racemization and the hydrogen-deuterium exchange
of the hydrogen at the α-carbon of proline analogs containing the
5-azoniaspiro[4.4]nonyl-group †
Received 00th January 20xx,
Accepted 00th January 20xx
B. Setner,a M. Wierzbicka,a L. Jerzykiewicz,a M. Lisowski,a and Z. Szewczuk a
DOI: 10.1039/x0xx00000x
Recently, we developed a novel non-fragmenting quaternary ammonium ionization tag, for mass spectrometric sensitive
sequencing of peptides, based on the N-spiro proline residue (5-azoniaspiro[4.4.]nonyl-carbonyl). Herein we present an
unexpected racemization and the hydrogen-deuterium exchange (HDX) at the α-C atom of the proline derivative at basic
aqueous conditions (1% water solution of triethylamine). The deuterium atom, substituted for the α-C atom, does not
undergo back exchange at acidic aqueous conditions which makes the deuterated isotopologue a promising stabile
isotope-coded internal standard for quantitative analysis by mass spectrometry. The applicability of the prepared
isotopologues of the quaternary ammonium salts labeled peptides for quantification experiments using isotopic dilution
method was also examined.
www.rsc.org/
5-azoniaspiro[4.4]nonyl, which can be used for quantitative
experiments in mass spectrometry (isotopic dilution method8),
is an important goal of modern analytical methods.
Introduction
The introduction of a fixed positive charge by means of a
quaternary ammonium salt (QAS) to the analyte molecule
increases the ionization efficiency of peptides in ESI-MS
analysis as well as lowers the detection limit.2 Therefore,
betaine derivatives allow for the efficient analysis of trace
amounts of analyte by MS.3 Furthermore, it has been
previously found that the fixed charged tags influence the
fragmentation patterns in MS/MS experiments and the
methodology that combines a positive charge derivatization
and tandem MS spectra will greatly facilitate the de novo
peptide sequencing and therefore discovery of novel
biomarkers.4,5 However, during collision-induced dissociation
(CID) experiments, both linear and bicyclic QAS undergo the
unwanted side reactions – the Hofmann elimination and/or
the tertiary amine loss.6 Thus, a properly designed QAS tag was
required to facilitate peptide sequencing by tandem MS.
O
O
O
H
H
H
1,4-dibromobutane
DIPEA
α,α’-dibromo-o-xylene
DIPEA
NH
NH
NH
N+
NH
N+
Figure 1. Synthesis of 5-azoniaspiro[4.4]nonyl-carbonyl scaffolds from the L-proline
residue. DIPEA – N,N-diisopropylethylamine.
Labile hydrogens in the backbone as well as in the side-chain
functional groups of peptides and proteins undergo exchange
with deuterons of the solvent (e.g. D2O) within few minutes.9
On the other hand, hydrogens bound to carbon atoms are
generally considered as not exchangeable. However, under
specific conditions it is possible to replace the protons
attached to carbon atoms by deuterons.10 Recently, Pieters et
Recently, we developed
a highly stabile QAS tag – 5-
al. described
activation/deuteration
nanoparticles dispersed in a polyvinylpyrrolidone matrix11
a
mild and enantiospecific C(sp3)-H
azoniaspiro[4.4]nonyl for improved MS peptide analysis.7 We
chosed the rigid 5-azoniaspiro[4.4]nonyl scaffold based on the
L-proline residue, in which all bonds susceptible to cleavage via
Hofmann elimination are protected by cyclization (Fig. 1). The
synthesis of isotopologues of QAS tags such as
reaction utilizing ruthenium
,
whereas the other group proposed Ni(II) complexes for the
efficient preparation of α-D-α-amino acids12. Although the
proposed reactions offer high yields, utilization of heavy
metals requires prolonged reaction times (up to 36 h) at
elevated temperatures for completion. Therefore, more robust
and straightforward HDX reactions are desired.
We found previously that hydrogens at the α-C atom in
betaine (N,N,N-trialkylglycine) can be exchanged by
deuterons13 (Fig. 2a). The process is base-catalyzed by
triethylamine (TEA) [1% solution], and the exchange rate is
negligible at acidic conditions. Thus, introduced deuterium
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atoms are stabile in acidic aqueous solution and are not back- fragments of ovalbumin SWVE-NH2 26 as well as the N-terminal
27
exchanged during LC-MS analysis which makes this reaction fragment of ubiquitin MQIFVKT were synthesized. The base-
useful in preparation of the deuterium-labeled analogs for catalyzed HDX provides us with a deuterated model peptide
quantitative analysis of trace amounts of peptides. This with the isotopically labeled quaternary ammonium ionization
strategy was successfully applied for a facile synthesis and tag. We also analyzed the fragmentation pathways of QAS-
quantification experiments on deuterium-labeled denatonium peptides with the aid of deuterium-labeled analogs.
cation14 as well as quaternary cryptando-peptidic conjugates15
.
Furthermore, we synthesized a benzo-5-azoniaspiro[4.4]nonyl-
However, the introduced deuterons may affect the retention carbonyl dipeptide as well as a benzo-5-azoniaspiro[4.4]nonyl-
parameters of the isotopologues.16 Thus, it is important for carbonyl derivative to investigate the possibility of
quantification analysis that the deuterated standards have racemization at the α-C atom.
similar retention times during LC-MS analysis as their non-
deuterated analogues. This could be achieved by placing them
Results and discussion
next to a hydrophilic group (such as a quaternary ammonium
center) that has little to no affinity to the stationary phase in
RP-HPLC.17,18
Bąchor et al. have noticed that the unmodified proline residue
is not susceptible to the HDX at the α-C atom.22 Based on the
proposed mechanism in which HDX is initiated by the
abstraction of a hydrogen atom from the α-C atom and
incorporation of the deuterium atom to the planar
intermediate, racemization is expected. However, a proline
residue does not undergo racemization even in its azlactone
form, hence HDX should not occur.24 Thus, the aim of this work
was to investigate the base-catalyzed HDX in a peptide
containing the L-proline derivative – 5-azoniaspiro[4.4]nonyl-
carbonyl group attached to the model peptides N-terminus. To
verify the influence of different QAS groups on the exchange
reaction, we synthesized on a solid support a series of model
peptides containing the 5-azoniaspiro[4.4]nonyl-carbonyl as
1% TEA/D2
[HDX]
O
N
O
O
O
N
O
+
+
N
NH
NH
N
(a)
NH
NH
NH
2
2 min
NH
H
D
H
O
O
D
[Rudowska et al. 2012]
1% TEA/D2
[HDX]
O
N
O
O
O
N
O
N+
H
NH
NH
+
NH
2
N
NH
(b)
(c)
N
NH
NH
N
48 h
H
D
D
H
O
O
O
D
O
[Bąchor et al. 2014]
O
D
O
O
O
H
1% TEA/D2
[HDX]
O
NH
NH
NH
NH
NH
NH2
N+
N+
O
O
60 min
Presented in this work
Figure 2. Schemes of the HDX reactions at the α-C of QAS-derivatized model synthetic
peptides.
well
as
benzo-5-azoniaspiro[4.4]nonyl-carbonyl
group
(molecular masses and sequences of all the synthesized QAS-
peptides are presented in Table 1S in the Supplementary
Data).
Previous analysis of the DHX (deuterium-hydrogen exchange)
reaction at the C2 atom of the histidine imidazole ring in
peptides containing
a phosphorylated histidine residue
We examined the HDX reaction of all the synthesized peptide
conjugates with the azoniaspiro group by ESI-MS technique (to
avoid a possibility of back-exchange caused by ambient water
within mass spectrometer, the instrument was saturated with
D2O for 1 h prior to MS measurements). The representative
ESI-MS spectra of ASN+-CO-DVYT-NH2 are presented in Fig. 3.
First, the sample of 0.1 mg was dissolved in 1 μL of the
water/acetonitrile 1:1 mixture containing 0.1% formic acid and
its ESI-MS spectrum was recorded which showed a peak at m/z
647.3, corresponding to the QAS-peptide (Fig. 3a). Next, the
sample was lyophilized and dissolved in D2O (pD=7.4) to
exchange all labile hydrogen atoms. The ESI-MS spectrum
recorded in D2O after 20 minutes showed that the peak was
shifted to m/z 656.4 (+9 Da), due to the exchange of nine labile
hydrogens by deuterons (Fig. 3b). After addition of 1% of TEA
[v/v] (pD=12.3), the m/z value started to shift by another 1 Da
and after 60 minutes a nearly complete exchange was
observed. This suggests that one additional hydrogen atom in
the QAS-peptide was exchanged by deuteron (Fig. 3g). After
lyophilization and redissolving the sample in the
water/acetonitrile 1:1 mixture containing 0.1% formic acid, the
peak corresponding to the analyte was shifted to m/z 648.3
(Fig. 3h). The obtained m/z value indicates that one deuterium
atom did not undergo the back-exchange by a proton. Finally,
we examined the back-exchange of the α-C deuterium atom.
The sample was lyophilized and redissolved in 1% solution of
demonstrated that phosphorylation dramatically slows the
rate of the DHX reaction.19 An unusual HDX at the α-C of the
sarcosine residue has been also noticed in peptides incubated
in 1% solution of TEA in D2O.20,21 The ESI-MS analysis unveils
the complete HDX for the oligosarcosine peptide after 10 days
of incubation at the room temperature. However, in the case
of N-termini of QAS derivatized oligosarcosine peptides the
HDX occurs more easily (up to five times) as compared to
unmodified oligosarcosine peptides.22 This phenomenon could
be explained by the effect of the quaternary ammonium (QA)
group on the acidity of the α-C hydrogens.23 Therefore, it can
be concluded that the QA group may catalyze the HDX at the
α-C in sarcosine and other N-methylated amino acid residues
(Fig. 2b).
HDX reaction at the α-carbon (α-C) of amino acid residues
proceeds with racemization. However, the proline residue is
known as a non-racemizable amino acid residue24, thus it is
resistant to racemization and HDX at the α-C atom. In this
article, we investigate the HDX at the α-C of the
5-azoniaspiro[4.4]nonyl group after incubation in 1% solution
of TEA in D2O (Fig. 2c) by means of MS, NMR, polarimetry, and
X-ray studies. To analyze the influence of various amino acid
residues on the HDX reaction, the immunosuppressive
fragment of the HLA-DQ protein containing the DVYT-NH2
sequence was used as a model peptide.25 Furthermore, the
2 | J. Name., 2012, 00, 1-3
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1+
TEA in H2O (pH=12) which results in return of the peak
corresponding to the deuterated QAS-peptide to the initial m/z
value, due to the back-exchange of a deuterium atom by a
hydrogen one (Fig. 3i). A nearly complete DHX reaction took
place in 180 minutes of incubation. This phenomenon could be
explained by the racemization reaction at the α-C atom of the
5-azoniaspiro[4.4]nonyl-carbonyl group, which could be
analyzed by optical activity measurements.
(a)
100
80
60
40
20
0
647.3
H2O/CH3CN
HCOOH
OH
O
O
O
OH
NH
O
O
H
640 645 650 655 660 m/z →
NH
1+
NH
NH
NH2
(b)
100
80
60
40
20
0
656.4
N+
O
OH
D2O
Spectrum (a) m/zcalc 647.3
D2O
640 645 650 655 660 m/z →
Similar results on the HDX reaction were obtained for other
investigated azoniaspiro-peptides (Supplementary Data, Figs.
26S. and 28S.), which indicates that the peptide sequence does
not affect the isotope exchange reaction. On the other hand,
the results indicate that a more hydrophobic quaternary
ammonium group (in benzoazoniaspiro-peptides versus
azoniaspiro-peptides) undergoes the HDX reaction significantly
faster (up to four-times) regardless of the peptide sequence,
as judged from the ESI-MS spectra (Supplementary Data, Figs.
25S. and 27S.).
1+
(c)
100
80
60
40
20
0
OD
O
656.4
O
D2O/Et3N
2 min
O
OD
O
H
D
N
N
D
N
D
N
D
ND2
D
640 645 650 655 660 m/z →
N+
O
O
1+
OD
(d)
100
80
60
40
20
0
656.4
Spectrum (b) m/zcalc 656.4
D2O/Et3N
5 min
D2O/Et3N
60 min
OD
O
640 645 650 655 660 m/z →
O
1+
(e)
100
80
60
40
20
0
657.4
O
OD
O
D
The obtained ESI-MS/MS spectra of a representative QAS-
peptide are presented in Fig. 4 (for other MS/MS spectra, see
Supplementary Data, Figs. 31S-36S.). Comparing the MS/MS
spectra of unlabeled ASN+-CO-DVYT-NH2 (Fig. 4a) and
deuterium labeled ASN+{d1}-CO-DVYT-NH2 (Fig. 4b) shows that
the molecular peak shifted by 1 Da (from m/z 647.340 to m/z
648.341), due to the HDX reaction. Three most intense signals
in the MS/MS spectrum of ASN+-CO-DVYT-NH2 are of ions: b2*
(m/z 267.135), a2* (m/z 239.140), and a2*-CO2 (m/z 195.152),
the loss of carbon dioxide coming from the aspartic acid
residue side chain (asterisk is added to denote the presence of
D2O/Et3N
15 min
D
N
N
D
N
D
N
D
O
ND2
D
N+
O
OD
640 645 650 655 660 m/z →
Spectrum (g) m/zcalc 657.4
1+
(f)
100
80
60
40
20
0
657.4
H2O/MeCN
HCOOH
D2O/Et3N
30 min
OH
O
O
640 645 650 655 660 m/z →
O
OH
NH
O
D
1+
(g)
100
80
60
40
20
0
657.4
NH
NH
NH
NH2
D2O/Et3N
60 min
N+
O
O
OH
Spectrum (h) m/zcalc 648.3
the derivative moiety that represents
a charged (non-
640 645 650 655 660 m/z →
1+
(h)
H2O/Et3N
180 min
100
80
60
40
20
0
648.3
protonated) fragment). The same ion fragments were
observed in the MS/MS spectrum of ASN-{d1}-CO-DVYT-NH2.
All peaks are shifted by 1 Da: b2* (m/z 268.135), a2* (m/z
240.141), and a2*-CO2 (m/z 196.153). These results revealed
that the base-catalyzed HDX in D2O occurs at the N-terminus
where the QA group is located. To unambiguously localize the
deuterium atom placement in the QAS-peptide, 1H NMR
spectroscopy was used (Supplementary Data, Figs. 37S.; for
other NMR spectra, see Figs. 38S-40S.). The obtained results
unveiled that the deuterium atom is located at the α-C atom of
the 5-azoniaspiro[4.4]nonyl-carbonyl group which is evidenced
by the disappearance of the of the α-hydrogen signal at 4.34
ppm
H2O/CH3CN
HCOOH
OH
O
O
O
OH
NH
O
H
640 645 650 655 660 m/z →
NH
1+
NH
NH
NH2
(i)
100
80
60
40
20
0
647.3
N+
O
O
H2O/Et3N
180 min
OH
Spectrum (i) m/zcalc 647.3
640 645 650 655 660 m/z →
Figure 3. ESI-MS spectra of ASN+-CO-DVYT-NH2 dissolved in various solvents: (a)
H2O/CH3CN/HCOOH (50:50:0.1); (b) D2O; (c-g) D2O/Et3N (1% solution); (h)
H2O/CH3CN/HCOOH (50:50:0.1) after lyophilization from D2O/Et3N (1% solution); (g)
H2O/Et3N (1% solution) after lyophilization from H2O/CH3CN/HCOOH (50:50:0.1).
To verify the applicability of the investigated deuterium
labeled QAS-peptides in quantitative analysis, we prepared the
1:1 mixture of ASN+-CO-DVYT-NH2 and ASN+{d1}-CO-DVYT-NH2.
However, due to the overlap of the first monoisotopic signal of
the unlabeled QAS-peptide (C3013CH47N6O9, m/z 648.343) with
that of the α-d1 isotopomer (C31H46DN9O6, m/z 648.346) (Fig.
4c, grey inset), the quantification information cannot be
obtained directly from a regular MS experiment. One solution
to that problem has been presented by Mirgorodskaya et al.
who proposed the mathematical algorithm which uses the
isotopic patterns of the substance, the internal standard, and
the substance/internal standard mixture for accurate
quantitation of the substance.28 The second solution is MS/MS
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experiment which provides a peptide sequence together with analysis of ASN+-CO-DVYT-NH2 and its isotopologue with the
a clean signals ratio. The obtained intensity ratios of the deuterium at the α-C atom, using standard RP-HPLC-MS
monoisotopic signals corresponding to the fragment ions are conditions (for details, see the Supplementary Data).
presented in Table 1. The calculated ratios of isotopic signals Comparison of isotopic profiles of signals corresponding to the
before and after HDX are similar and correspond to the QAS-peptide and its deuterated isotopologue obtained during
expected values. Similar results were also observed for different LC-MS analysis (Fig. 5) shows that the signals isotopic ratios are
ratios of ASN+-CO-DVYT-NH2 and ASN+{d1}-CO-DVYT-NH2. (4:1, 2:1, similar in both experiments. The obtained results reveal the
1:2, and 1:4) (Figures 46S - 49S). The correlation between the co-elution of non-deuterated and deuterated QAS-peptides.
theoretical and the obtained ratios of non-deuterated and This could be explained based on the solvophobic theory. In
deuterated a2* and b2* ion fragments was very good (Figure 50S) this theory, the probability of deuterium atom interactions
Thus, the obtained results may indicate that the proposed with a stationary phase during a chromatographic separation is
procedure based on the base-catalyzed HDX reaction of QAS reduced by placing it next to a hydrophilic group (such as a
labeled peptides may be a useful, less expensive alternative to quaternary ammonium one).17 To confirm the observed lack of
15
the AQUA technique.29 It should be noted that at this stage the isotopic effect, the ‘peak purity’ analysis of the signal at
this method is not suitable for the quantification of real 12.7 min of the total ion chromatogram was performed (see
biological
samples.
However,
the
deuterated
5- Fig. 5). A high similarity of the isotopic distribution for signals
azoniaspiro[4.4]nonyl-carbonyl group could be used in the recorded at different points within the peak demonstrates the
future as a part of a new isotopically labeled derivatization unchanged intensities of the isotopomers and, therefore, their
agent able to react with amines present in biological samples.
co-elution. The obtained results indicate a high stability of the
α-C-bound deuterium atom during LC-MS experiments, which
allows for the accurate and sensitive analysis of a labeled
peptide using the LC-MS technique.
1+
647.340
100
(a)
*
a2*-CO2
a2
1+
195.152
75
*
1+
239.140 b2
Intens.
50
25
1+
a
5
x10
267.135
b
0
1+
648.341
100
(b)
(c)
3
2
1
0
75
50
25
c
1+
240.141
1+
268.135
1+
196.153
0
1+
648.335
100
75
50
25
0
1+
239.133
1+
267.127
1+
195.146
100
1+
195.146
200
300
400
500
600
m/z
1+
1+
1+
1+
1+
196.152
239.133
1+
648.335
267.127 268.133
1+
647.332
240.139
11.0
11.5
12.0
12.5
13.0
13.5
14.0
Time [min]
+MS, 12.7min #755
+MS, 12.9min #770
+MS, 13.1min #783
1+
260 264 268 272 m/z
→
186 190 194 198 m/z
→
230 234 238 242 m/z→
640 644 648 652 m/z
→
1+
648.362
(a)
Figure 4. ESI-MS/MS spectra of ASN+-CO-DVYT-NH2: (a) before HDX reaction, (parent
ion m/z 647.340, collision energy 25 V) and (b) after HDX reaction, (parent ion m/z
648.335, collision energy 25 V); (c) ESI-MS/MS spectrum of the 1:1 mixture of ASN+-CO-
DVYT-NH2 and its deuterium labeled isotopologue (collision energy 25 V). * – asterisks
denote QAS, non-protonated ion fragments.
647.356
100
80
60
40
20
0
1+
649.363
1+
650.366
1+
647.356
1+
648.361
Table
1 ESI-MS/MS analysis of the mixture of non-deuterated and deuterated
(b)
(c)
100
80
60
40
20
0
isotopologue of the peptide with the 5-azoniaspiro[4.4]nonyl ionization tag.
Fragment
ion
m/z
m/z
m/z
m/z
Theoretical Obtained
1+
649.363
intensity
ratio
intensity
ratio
calculated
found
calculated
for α-C
found for
α-C
1+
650.363
exchanged
exchanged
QAS-
QAS-
1+
647.353
peptide
268.1
240.1
196.2
peptide
268.1
240.1
196.2
1+
100
80
60
40
20
0
648.357
*
b2
267.3
239.1
195.1
267.1
239.1
195.1
1:1
1:1
1:1
1:0.97
1:0.93
1:0.89
*
a2
a2*-CO2
1+
649.362
1+
650.362
In the HDX reaction, a considerable back-exchange of labile
deuterons during MS and LC-MS analysis is
a known
645
646
647
648
649
650
651
652
653
654 m/z
drawback.30 To verify whether the stability of the deuterium
labeling in QAS-peptides is high, we performed the LC-MS
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-
Figure 5. The LC-MS analysis of the mixture of ASN+-CO-DVYT-NH2 and its deuterated
isotopologue ASN+{d1}-CO-DVYT-NH2. Total ion chromatogram – peak at Rt 12.7 min
was selected for peak purity analysis; a-c) ESI-MS spectra recorded for points a-c of the
total ion chromatogram.
O
O
D
O
H
NH
NH
NH
1. D2O
TEA
N+
N+
N+
2. H2O
The HDX at the α-C atom in a chiral proline-based QA group
should result in formation of the racemic mixture. Hence, the
effect was studied for two compounds containing the benzo-5-
azoniaspiro[4.4]nonyl-carbonyl group. Both compounds were
synthesized in solution using the mixed anhydride method (Fig
Scheme 1. Proposed mechanism of HDX at the α-C atom in a proline derivative - 5-
azoniaspiro[4.4]nonyl-carbonyl group. Based on the Richard et al. proposition.31
It has been previously reported that the pKa value of the α-C
hydrogen atom in a proline residue (zwitterion) is 29±1.32
Richard et al. showed that the esterification of proline
effectively lowers the pKa value to 21±1 for N-protonated
proline methyl ester.32 Moreover, introduction of the positive
charge at the α-amine group further decreases the pKa value
of α-C hydrogen atom(s).23 Thus, combining two effects –
amidation of proline as well as introduction of a positive
charge at the α-amine group enables a facile preparation of
deuterated isotopologues of proline derivatives presented in
this article.
6.).31
Figure 6. (a) Scheme of the synthesis of BASN+-CO-γAbu-OEt∙BPh4; (b) X-ray crystal
Experimental
structure of BASN+-CO-γAbu-OEt∙BPh4. NMM
–
N-methylmorpholine; THF
–
–
Materials and reagents
tetrahydrofuran; DMF
dichloromethane.
–
dimethylformamide; TFA
–
trifluoroacetic acid; DCM
All reagents for peptide synthesis were purchased from Iris
Biotech GmbH (Marktredwitz, Germany), Novabiochem
(Darmstadt, Germany) or Fluorochem (Hadfield, Derbyshire,
United Kingdom). All other reagents, including deuterium
oxide (D2O, 99.9% purity) and solvents, were purchased from
Sigma Aldrich (St. Louis, MO, USA). All reagents were used
without further purification.
‡
Colorless crystals of BASN+-CO-γAbu-OEt∙BPh4 were sufficient
for
a single crystal X-ray diffraction experiment (for
monocrystal structure analysis, see the Supplementary Data).
Analysis of the obtained structure confirmed stereochemistry
at the chiral α-C atom connected to the spiro nitrogen one. To
provide evidence that the HDX observed in solution occurs at
α-C of the QA group, the ESI-MS (Supplementary Data, Figs. Model QAS-peptides synthesis
1
29S. and 30S.) as well as H NMR (Supplementary Data, Figs.
The peptides were synthesized manually according to a
41S. and 42S.) analyses were performed. Polarimetry was used
to provide another independent evidence for the HDX at the
α-C atom. The obtained specific rotations of the synthesized
compounds, [α]D25°C, are presented in Table 2. They are
significantly decreased after HDX which shows that the
exchange is accompanied by racemization.
standard Fmoc (9-fluorenylmethoxycarbonyl) solid phase
synthesis procedure.34 Peptides were modified with the 5-
azoniaspiro[4.4]nonylcarbonyl
(ASN+)
or
benzo-5-
azoniaspiro[4.4]nonylcarbonyl group (BASN+) according to the
procedure described by us previously.7 After removal of the
Fmoc group from the N-terminal proline residue amino group,
the peptidyl-resin was coupled with
2 Polarimetric analysis of compounds with the benzo-5-azoniaspiro[4.4]nonyl
5
equiv. of
Table
1,4-dibromobutane or α,α’-dibromo-ortho-xylene for 24 h in
the presence of 5 equiv. of N,N-diisopropylethylamine (DIPEA)
to obtain ASN+ or BASN+ groups, respectively. The derivatized
peptides were cleaved from the resin simultaneously with the
side chain deprotection using a solution of trifluoroacetic
acid/water/triisopropylsilane [TFA/H2O/TIS] (95:2.5:2.5; v/v/v)
at room temperature for 2 h and then transferred to the cold
diethyl ether. Precipitated QAS-peptides were decanted,
dissolved in water, and lyophilized. All quaternary ammonium
groups were stable at these conditions according to RP-HPLC
analysis. The QAS-peptides were purified by the semi-
preparative RP-HPLC.
ionization tag before and after HDX.
Compound
[α]D25°C before
HDX
[α]D25°C after
HDX
BASN+-CO-ethylenediamine-N-Boc
1.76°
0.70°
BASN+-CO-γAbu-OEt
–13.93°
1.20°
There are sufficient evidences that racemization at the α-C
atom in proline proceeds through planar enolate
intermediate.32,33 We assume the same mechanism through an
enolate which can undergo the deuterium transfer from D2O
to give the racemic 5-azoniaspiro[4.4]nonyl QA group
containing deuterium at the α-C atom (Scheme 1).
a
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DOI: 10.1039/C7OB02926H
ARTICLE
Journal Name
Model compounds synthesis
selected gradient elution (A = 0.1% formic acid in water; B =
0.1% formic acid in acetonitrile), flow rate 0.1 mL/min, at room
temperature.
For the synthetic details of BASN+-CO-γAbu-OEt∙BPh4 and
BASN+-CO-ethylenediamine-N-Boc∙BPh4,
see
the
Supplementary Data.
Nuclear magnetic resonance
Isotopic exchange
Nuclear magnetic resonance (NMR) spectra were recorded on
a high-field spectrometer Bruker Avance III 500 MHz, equipped
with a broadband inverse gradient probe-head. Spectra were
referenced to the residual solvent signal ([D]acetonitrile: 1.94
ppm and [13C]acetonitrile: 1.32 ppm; [D]chloroform: 7.26 ppm;
[13C]chloroform: 77.16 ppm).
HDX was initiated by dissolving 0.1 mg of a QAS-peptide
derivative in 200 μL of D2O at room temperature. After 20
minutes, all labile protons (connected to heteroatoms) in the
amide bonds and amino acid side chains were exchanged by
deuterons, as judged from ESI-MS analysis. Then, 2 μL of TEA
were added and the mixture was analyzed by ESI-MS. The
acidic α-C hydrogen atom was substituted completely by the
deuterium one after 60 and 15 minutes in ASN+-CO-peptides
and BASN+-CO-peptides, respectively (see the Results section
for details).
Polarimetry
The specific rotation values were measured on a JASCO DIP–
1000
(JASCO,
Japan)
polarimeter.
Benzo-5-
azoniaspiro[4.4]nonyl-carbonyl derivatives were dissolved at
concentrations of approximately 1% in acetonitrile. The
measurements were run at 25°C. For each compound, the
measurement was performed three times. Obtained values
were averaged by a built-in software.
Back-exchange of amide protons
The sample was lyophilized and redissolved in 200 μL of the
50:50 acetonitrile/water mixture containing 0.1% of formic
acid which caused the back-exchange of labile deuterons for
protons after 20 minutes of incubation at room temperature.
X-ray crystallography
X-ray quality crystals were grown by dissolving BASN+-CO-
γAbu-OEt∙BPh4 in methanol. The crystals were mounted on
Back-exchange of protons in the 5-azoniaspiro[4.4]nonyl-carbonyl
residue
glass fibers and then flash-frozen to 100(2)
K (Oxford
Cryosystem-Cryostream Cooler). Preliminary examination and
intensities data collections were carried out on an Xcalibur PX
The sample was lyophilized again and dissolved in 200 μL of
H2O containing 2 μL of TEA. The acidic deuterium atom in
peptides derivatized with the 5-azoniaspiro[4.4]nonyl-carbonyl
and benzo-5-azoniaspiro[4.4]nonyl-carbonyl group was
substituted by a hydrogen atom from the solvent after 180
minutes at room temperature.
with Sapphire2 CCD diffractometer with
a
graphite-
monochromated MoKα radiation. Data reduction and analysis
were carried out with a CrysAlisPro program. The structure
was solved by a direct method with SHELXS-97 and refined
with full-matrix least-squares techniques on F2 with SHELXL-
2014/7. The C-bonded hydrogen atoms were included in
calculated positions and refined in the riding mode using
SHELXTL default parameters. The N-bonded hydrogen atom
was located from difference maps and refined without any
restraints. CCDC 1578235 contains the supplementary
crystallographic data for this paper. These data can be
obtained free of charge from the Cambridge Crystallographic
Data Centre at www.ccdc.cam.ac.uk/data_request/cif.
Mass spectrometry
Mass spectrometry experiments were performed on a Bruker
micrOTOF-Q mass spectrometer (Bruker Daltonics, Bremen,
Germany) and on an FT-ICR (Fourier transform-ion cyclotron
resonance) Apex-Qe Ultra 7 T instrument (Bruker Daltonics,
Bremen, Germany), equipped with an ESI source. ESI–MS
analyses were performed by infusion of peptide solutions
directly to the electrospray ionization chamber at a flow rate
of 3 μL/min. The instrument was operated in the positive ion
mode and calibrated before each analysis with the Tunemix™
mixture (Agilent Technologies, Santa Clara, USA) by the
quadratic method. In the MS/MS experiments, the singly
charged [M]+ precursor ions or doubly charged protonated
[M+H]2+ ions were selected. The collision voltage was
optimized for the best fragmentation. For MS as well as
MS/MS analysis, a Bruker Compass DataAnalysis 4.0 software
was used.
Conclusions
It was found that the proline-derived 5-azoniaspiro[4.4]nonyl-
carbonyl residue undergoes the unexpected HDX reaction at
basic conditions at room temperature. The obtained results
indicate that it proceeds at a much slower rate as compared to
the 'classical' proton exchange reactions in amines or amides.
However, as opposed to these processes, HDX in the
5-azoniaspiro[4.4]nonyl-carbonyl residue is dramatically slow
at neutral or acidic conditions. Optical activities of the
synthesized model compounds were investigated to confirm
the racemization at the α-C atom of proline-based QAS.
Analysis of the obtained results confirms that the presented
ionization tag is the first known example of a proline derivative
that undergoes racemization in the presence of 1% solution of
TEA.
LC-MS
Agilent 1200 Series binary LC gradient system connected to a
Bruker micrOTOF-Q mass spectrometer (Bruker Daltonics,
Bremen, Germany) was used for LC-MS experiment.
Separation of QAS-peptides isotopologues was performed on
an Aeris Peptide XB-C18 column (50×2.10 mm, 3.6 μm), with a
6 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C7OB02926H
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ARTICLE
21 R. Bąchor, D. Dębowski, A. Łęgowska, P. Stefanowicz, K.
Rolka and Z. Szewczuk, J. Pept. Sci., 2015, 21, 819.
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Observed co-elution of the model QAS-peptides isotopologues
under LC-MS conditions confirms the applicability of the
presented HDX reaction as an inexpensive stabile isotope
labeling of the 5-azoniaspiro[4.4]nonyl-carbonyl group for the
peptide analysis.
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Conflicts of interest
There are no conflicts to declare.
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‡ For crystallographic data see CCDC 1578235 (Data can be
obtained free of charge from the Cambridge Crystallographic
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