Atmospheric pressure chemical ionization multi-stage mass spectrometry in the characterization of stereoisomeric synthons of cyclopropane amino acids

Article abstract of DOI:10.1002/(SICI)1096-9888(200002)35:2<199::AID-JMS930>3.0.CO;2-6

The mass spectrometric behaviour of (1S,2R)-, (1R,2R)-, (1R,2S)- and (1S,2S)-2-[(S)-2,2-dimethyl-1,3-dioxolan-4-yl]-1-spiro-{4'[2'-phenyl-5'(4'H)-oxazolone]} cyclopropane (2) and (1S,2R)-, (1R,2R)-, (1R,2S)- and (1S,2S)-methyl-1-benzamido-2-[(S)-2,2-dimethyl-1,3-dioxolan-4-yl]cyclopropanecarboxylate (3) was studied under atmospheric pression ionization conditions and by multi-stage mass spectrometric (MS(n)) experiments performed with an ion trap. Interestingly, by using methanol as solvent, compounds 2 lead to [M + H + CH₃OH]⁺ ions which, as proved by collisional experiments, exhibit the same structure of the corresponding compound 3. MS/MS of [MH]⁺ ions allows a clear characterization of the different stereoisomers, which give rise to specific fragmentation pathways, rationalized with respect to the structure of the neutral molecules. Copyright (C) 2000 John Wiley and Sons, Ltd.

Full text of DOI:10.1002/(SICI)1096-9888(200002)35:2<199::AID-JMS930>3.0.CO;2-6

JOURNAL OF MASS SPECTROMETRY
J. Mass Spectrom. 35, 199–209 (2000)
Atmospheric pressure chemical ionization
multi-stage mass spectrometry in the
characterization of stereoisomeric synthons of
cyclopropane amino acids
Simone Cristoni,1 Carlos Cativiela,2 Ana I. Jime´nez2 and Pietro Traldi3*
1
CNR, Area della Ricerca, Corso Stati Uniti 4, I-35100 Padova, Italy
Organic Chemistry Department, Zaragoza University–CSIC, 50009 Zaragoza, Spain
CNR, Centro di Studio sulla Stabilita` e Reattivita` dei Composti di Coordinazione, Via Marzolo 1, I-35100 Padova, Italy
2
3
The mass spectrometric behaviour of (1S,2R)-, (1R,2R)-, (1R,2S)- and (1S,2S)-2-[(S)-2,2-dimethyl-1,3-dioxolan-
4-yl]-1-spiro-f40[20-phenyl-50(40H)-oxazolone]g cyclopropane (2) and (1S,2R)-, (1R,2R)-, (1R,2S)- and (1S,2S)-
methyl-1-benzamido-2-[(S)-2,2-dimethyl-1,3-dioxolan-4-yl]cyclopropanecarboxylate (3) was studied under atmo-
spheric pression ionization conditions and by multi-stage mass spectrometric (MSn ) experiments performed with
an ion trap. Interestingly, by using methanol as solvent, compounds 2 lead to [M Y H Y CH3OH]Y ions which,
as proved by collisional experiments, exhibit the same structure of the corresponding compound 3. MS/MS of
[MH]Y ions allows a clear characterization of the different stereoisomers, which give rise to specific fragmen-
tation pathways, rationalized with respect to the structure of the neutral molecules. Copyright  2000 John
Wiley & Sons, Ltd.
KEYWORDS: atmospheric pressure chemical ionization; multi-stage mass spectrometry; ion trap; structural
characterization; stereoisomeric compounds
protein amino acids with their cyclopropane surrogates
INTRODUCTION
generally enhances resistance to proteolytic cleavage.3
Besides this effect, the tensioned three-membered ring
introduces severe constraints in the proximal backbone
torsion angles, which may cause profound changes in
the peptide conformation, thus influencing its biological
properties.3,4 Moreover, owing to the rigidity of the cyclo-
propane, 2-substituents are fixed in space with reference
to the amino and carboxyl functions and, consequently,
cyclopropane residues are particularly useful for evalu-
ating the effect of the side-chain orientation on peptide
secondary structures.5
In recent years, cyclopropane amino acids have attracted
increasing attention owing to their biological activity
and peculiar structural features. The simplest member of
the family, the naturally occurring 1-aminocyclopropane-
carboxylic acid (ACC), has been identified as the imme-
diate biosynthetic precursor of ethylene,1 a plant hormone
that regulates many physiological processes, including
germination, ripening of fruits and senescence. Some 2-
alkyl derivatives of ACC have been shown to inhibit
ethylene production,2 and might therefore have an appli-
cation in agriculture as plant growth and fruit ripening
regulators.
The importance of cyclopropane amino acids has stim-
ulated much effort in the development of synthetic routes
to this peculiar class of constrained residues.6 In this con-
text, a methodology involving the selective cyclopropana-
tion of the easily available chiral (Z)-azlactone derived
from 1,2-O-isopropylidene-D-glyceraldehyde (1) using
diazomethane7 on sulfoxonium methylides8 (Scheme 1)
has been described. The selectivity of the process
depended both on the cyclopropanating agent and on the
experimental conditions. Once the four different stereoiso-
mers 2 had been separated by chromatography, each of
them was transformed into the corresponding ester 3 by
methanolysis. The absolute configuration of compounds
1
The interest in ACC and its substituted derivatives also
stems from their potential use in the design of confor-
mationally constrained peptide analogues. Substitution of
3 and their precursors 2 was assigned by H NMR NOE
difference experiments7 and x-ray diffraction analysis on
single crystals of (1S,2R)-3a7 and (1S,2S)-3d.8 The major
stereoisomer (1S,2R)-3a was further elaborated to obtain
derivative 4,9 bearing a formyl substituent which can
be easily transformed into different groups (Scheme 2).
The value of this chiral synthon was evidenced by the
* Correspondence to: P. Traldi, CNR, Centro di Studio sulla Stabilita`
e Reattivita` dei Composti di Composti di Coordinazione, via Marzolo
1, I-35100 Padova, Italy.
Copyright 2000 John Wiley & Sons, Ltd.
Received 27 July 1999
Accepted 12 October 1999

200
S. CRISTONI ET AL.
Scheme 1
Scheme 2
Figure 1. (a) ESI mass spectrum of 2b; (b) MS/MS of species at m/z 661.
Copyright 2000 John Wiley & Sons, Ltd.
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MS OF STEREOISOMERIC AMINO ACIDS
201
preparation of several cyclopropane amino acids in enan-
EXPERIMENTAL
tiomerically pure form.9,10
Mass spectrometry has been often successfully applied
to the characterization of stereoisomeric compounds.11–15
Generally, the best results have been achieved by the use
of soft ionization techniques linked with collisional exper-
iments, thus avoiding the possible overcoming of isomer-
ization barriers. In this paper, the behaviour, obtained
by atmospheric pressure chemical ionization (APC1),16
of diastereoisomers 2a–d and 3a–d, and MSn, easily
performed with an ion trap,17 are reported and discussed,
showing that this approach is highly effective for the char-
acterization of stereoisomeric compounds.
Compounds 2a–d and 3a–d were analytically pure sam-
ples, synthesized according to the literature.8,9
APCI mass spectra were obtained with an LCQ instru-
ment (Finnigan, Palo Alto, CA, USA). The temperature
of the source capillary was 450 °C and the voltage applied
on the needle was 5 kV. Methanol solutions (10ꢀ5 M) of
2a–d and 3a–d were introduced by direct infusion at a
flow-rate of 30 µl minꢀ1. Both sample concentration and
flow-rate were kept rigorously constant for all the exam-
ined samples. The entrance capillary temperature was
Figure 2. Mass spectra, obtained by APCI analysis using CH3OH as solvent, of diastereoisomers 2a d.
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202
S. CRISTONI ET AL.
180 °C. MSn experiments were performed by selection
of the ions of interest and by their resonance excitation
by a supplementary r.f. voltage in the range 20–25%
of its maximum value (5 V peak-to-peak) for 200 ms;
with these values good reproducibility of the multi-stage
mass spectra was achieved, with variations in the relative
abundance of product ions always <5%. The He pres-
sure inside the trap was kept constant at 2 ð 10ꢀ5 Torr
(1 Torr D 133.3 Pa).
stereoisomeric compounds: the mild conditions and the
relatively simple ionization mechanism typical of the
method would lead to a low energy deposition on the
neutral molecule.
However, in the present case ESI leads to spectra
showing the occurrence of extensive clustering phenom-
ena. As an example, the ESI spectrum of 2b is reported
in Fig. 1(a). Protonated molecules are completely unde-
tectable; a weak signal is present at m/z 310, due to
[M C Na]C species. The most abundant peaks are due to
[M C Na C CH3OH]C (m/z 342) and [2.M C CH3OH/ C
Na]C (m/z 661). MS/MS of the species at m/z 661
[Fig. 1(b)] shows only the production of [M C Na C
CH3OH]C cation. Analogous results were obtained also
for 2a, 2c and 2d. In the case of compounds 3, [M C Na]C,
RESULTS AND DISCUSSION
In principle, electrospray ionization (ESI) would be the
best approach to employ for the characterization of
Figure 3. Mass spectra, obtained by APCI analysis using CH3OH as solvent, of diastereoisomers 3a d.
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MS OF STEREOISOMERIC AMINO ACIDS
203
[2M C Na]C, [3M C Na]C ions are always detected with
increasing abundance, together with other less abundant
ionic species e.g. [M C Na ꢀ CH3OH]C, [.M ꢀ CH3/ C
M C Na]C. These clustering reactions are present even
for different sample concentrations and different sheath
gas pressures.
In order to obtain more abundant, possibly protonated,
molecular species, further experiments were undertaken
under APCI conditions.
The APCI mass spectra of 2a–d are reported in Fig. 2
and those of 3a–d in Fig. 3. It is interesting to observe that
under these conditions 2a–d exhibit a readily detectable
[MH]C species at m/z 288, together with ions at a
higher mass value (m/z 320) (the corresponding species,
cationized by NaC, were observed under ESI conditions
[ions at m/z 342 of Fig. 1(a)]). They could originate
through two different processes: the addition to the neu-
tral molecules of protonated methanol or, alternatively, the
addition of neutral methanol to the protonated molecules.
Considering that the experimental conditions (sample con-
centration, injection flow-rate, instrumental set-up) were
kept rigorously constant, the spectra shown in Fig. 2 indi-
cate that the yield of this addition product is strongly
dependent on the configuration of the original molecule:
in the case of 2a and 2c the peaks corresponding to this
adduct are the most abundant (100%) while their abun-
dance shows a clear decrease on passing to the trans
compounds 2d (32%) and 2b (5%).
Figure 4. MS/MS of (a) the ions at m/z 320 observed in the APCI spectrum of 2c, (b) [MH]C ions of 3c, (c) the ions at m/z 320 observed
in the APCI spectrum of 2a and (d) [MH]C ions of 3a.
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S. CRISTONI ET AL.
Thus, by APCI of 2a–d using methanol as solvent, a
achievement of the above-described data. In this case, only
[MH]C ions are detected and for all the compounds they
lead to the base peak of the spectra, whereas ions at m/z
320 completely disappear.
reaction analogous to that occurring in condensed phase,
leading to compounds 3a–d, seems to take place. In
order to verify this hypothesis, the tandem mass spec-
tra of the ions at m/z 320 observed for 2a and 2b were
compared with those of [MH]C of 3a and 3b, respec-
tively. The spectra are practically superimposable (see,
for example, the data pertaining to 2a–3a and 2c–3c
reported in Fig. 4), proving that the above hypothesis
is correct. Between the possible mechanisms described
above for [M C H C CH3OH]C formation, in our opinion
the latter seems the most plausible: the possible proto-
nation occurring for 2a–d on the nitrogen atom (surely
the most basic site of the molecule) would enhance the
electrophilicity of the carbonyl group, which represents
the reactive centre against methanol. In order to inves-
tigate this aspect, further experiments were performed
using CH3CN as solvent, maintaining all the other exper-
imental conditions identical with those employed for the
In the APCI spectra of 2a–d, obtained by analysis of
their methanol solutions, fragments are observed at m/z
262 and 154, common for all compounds (see Fig. 1). The
latter presumably is formed by cleavages of the oxazolone
and cyclopropane rings followed by further cyclization,
leading to the cation shown in Scheme 3. The former
cannot be considered to originate from the MHC ion: first,
using CH3CN as solvent it completely disappears. Further,
it would correspond to the loss of a species of 26 u from
[MH]C, difficult to explain from the original structures of
2a–d. It is remarkable that this fragmentation product is
completely absent in the collision spectra of [MH]C and
is particularly abundant in the APCI spectra of 3a–d (see
Fig. 3). Consequently, it is reasonable to assume that it is
formed from the [M C H C CH3OH]C adducts at m/z 320,
Scheme 3
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MS OF STEREOISOMERIC AMINO ACIDS
205
through the loss of C2H2O2. As will described later, this
hypothesis is fully confirmed by the tandem mass spectra
reported in Fig. 4.
fragmentation processes can be easily identified. The
detection of different collisionally generated fragmenta-
tion products may be due to their close activation energies
or, alternatively, to the further decomposition of a primary
fragment. The latter case can be easily confirmed by MS3
experiments.
Thus, 2a shows the occurrence of only one collision-
ally induced decomposition route, leading to the ion at m/z
230, which can be explained by an acetone loss, originat-
ing from the cleavage of the 2,2-dimethyl-1,3-dioxolane
ring. The same process is highly favoured for the other
diastereoisomers, but for them other fragmentation chan-
nels are activated. For 2c and 2d a further fragment ion is
detected at m/z 105, due to C6H5COC, originating from
cleavage of the oxazolone ring; as can be seen by the
MS3 results reported in Fig. 5, it originates from the ions
at m/z 230. However, their formation in the MS/MS of
2c and 2d only suggests that in these cases the related
activation energy is closed to that of the process lead-
ing to the ions at m/z 230. Looking at the structures of
Interestingly, the tandem mass spectra of either [MH]C
or [M C H C CH3OH]C ions of 2a–d do not show the
formation of the ion at m/z 154 discussed above. Their
presence in the APCI spectra could indicate that they may
originate not by an APCI-induced decomposition process
of [MH]C but, probably, by thermal decomposition occur-
ring in the heated capillary .450 °C/ of the APCI source.
The collisional behaviour of [MH]C of 2a–d is strongly
dependent on the configuration of the neutral precursor,
as shown by the data reported in Fig. 5.
It should be emphasized that the collision phenomenon
present in MS/MS experiments performed by ion trap
is different from that present in the classical MS/MS
approach as with an ion trap the energy deposition takes
place step by step and consequently the decomposition
pathways at lower activation energy are privileged.17
Therefore, by this method, the more energetically favoured
Figure 5. MSn (n D 1 3) of 2a d.
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S. CRISTONI ET AL.
Figure 6. Structures of 2a d.
Scheme 4
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MS OF STEREOISOMERIC AMINO ACIDS
207
2a–d, shown in Fig. 6, one could hypothesize that the
observed behaviour could be explained by a possible com-
plexation of the proton between the oxazolone and the
dioxolane ring, lowering the activation energy required
for C6H5COC formation.
For 2c, two other ions are detected at m/z 212 and
202. Both are presumably formed by fast decomposition
processes of the ion at m/z 230 described, above through
losses of H2O and CO, respectively. In the case of 2b
the former process is inhibited. Ions at m/z 230 and 202
are still present and further four fragments are detected,
whose possible origin is described in Scheme 4.
The selection and collision of the collisionally gener-
ated ion at m/z 230 still lead to different results. Water
loss is detected for all the compounds except 2b, while
CO loss is particularly favoured for 2b and is absent for
2c. An ion at m/z 184 is present for 2b only.
lead to the base peak in the APCI spectra of 3a, 3c and
3d, whereas in the case of 3b the fragment at m/z 262
represents the most abundant species. As expected, ions
at m/z 262, already observed among the species detected
for 2a–d but originating from the [M C H C CH3OH]C
adducts, are present for all the compounds 3a–d. Inter-
estingly, for all the compounds the retrosynthetic process
is observed: the methanol loss leads to readily detectable
ions at m/z 288. The ions at m/z 154 detected in the APCI
spectra of 3a and 3b are, as described above, typical
of structures 2 (present in 2a–d; not present in 3c and
3d). Also in this case, looking at the structures of 3a–d
(Fig. 8), a possible proton complexation, analogous to that
suggested for compounds 2, could be hypothesized for 3c
and 3d only, which would inhibit the occurrence of the
fragmentation processes leading to the ion at m/z 154.
It should be noted that for 3c and 3d a readily detectable
ion is present at m/z 220, corresponding to the loss of the
2,2-dimethyl-1,3-dioxolane ring with H rearrangement.
Also this behaviour could be related to the more crouching
structure of these two isomers.
In summary, the above collisional data strongly depend
on the configuration of neutral compounds. In particular,
2a and 2d lead to protonated molecules that are more
stable than those obtained from 2b and 2c.
The mass spectra and MSn data of compounds 3a–d
are summarized in Figs 3 and 7. The protonated molecules
Compounds 3a and 3b show, in MS/MS conditions, a
primary water loss (Fig. 7), completely absent for 3c and
Figure 7. MSn (n D 1 3/ of 3a d.
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S. CRISTONI ET AL.
Figure 8. Structures of 3a d.
3d, suggesting that it must be somehow related to the
configuration of the molecule. The same process becomes
particularly relevant among the collisionally activated
decompositions of ions at m/z 262, leading to the species
at m/z 244 (see Fig. 6). A further ion at m/z 140 is detected
in MS3, corresponding to the cyclopropanedimethyldiox-
olane moiety.
configuration of the compounds under study. Interest-
ingly, compounds 2 show under APCI conditions an easy
methanol addition leading, as proved by MS/MS experi-
ments, to the protonated molecules of the corresponding
compounds 3. This process is highly thermodynamically
favoured, as confirmed by the behaviour of compounds
3 which show, under APCI conditions, the occurrence
of the retrosynthetic process leading to the protonated
molecules of 2.
In conclusion, both APCI and MSn reveal the existence
of decomposition pathways strongly related to the original
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