Cleavage of phosphorus-carbon (P-C) bonds of α-amino phosphonates with intramolecular hydrogen migration in the gas phase using electrospray ionization tandem mass spectrometry

Article abstract of DOI:10.1002/rcm.6984

RATIONALE α-Amino phosphonates with intrinsic biological activities have been used in a wide variety of applications. Because of the widespread existence of natural organophosphorus compounds containing P-C bonds such as the α-amino phosphonates, it is important to investigate the gas-phase chemistry of P-C bonds in order to determine their basic properties, which might provide some insights into their biosynthesis and catalytic cleavage. METHODS Twenty α-amino phosphonates were successfully synthesized and their fragmentation behavior was systematically investigated using in-solution deuterium labeling in combination with high-resolution Fourier transform ion cyclotron resonance (FTICR) electrospray ionization tandem mass spectrometry. RESULTS The fragmentation pathways of twenty α-amino phosphonates with different chemical structures were systematically studied. In general, P-C bonds could be easily cleaved via a novel intramolecular hydrogen atom migration from the amino group to the phosphoryl group through a five-membered-ring intermediate in the gas phase. A possible mechanism of the rearrangement of α-amino phosphonates is proposed. CONCLUSIONS An interesting intramolecular hydrogen atom migration between the amino and phosphoryl groups was observed with cleavage of the P-C bond in the molecule through a five-membered-ring intermediate. This characteristic fragmentation pathway not only provides some insights into the basic chemistry of compounds with P-C bonds, but could also have some applications in the structural determination of the α-amino phosphonate analogues. Copyright

Full text of DOI:10.1002/rcm.6984

Research Article
Received: 28 April 2014
Revised: 8 July 2014
Accepted: 11 July 2014
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 1964–1970
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6984
Cleavage of phosphorus–carbon (P–C) bonds of α-amino
phosphonates with intramolecular hydrogen migration in the gas
phase using electrospray ionization tandem mass spectrometry
Yuzhen Gao1, Jian Xu1, Yaohui He2, Guo Tang1, Zhiwei Lin1, Hongxia Liu3, Xiang Gao2
*
and Yufen Zhao1,2
1Department of Chemistry and The Key Laboratory for Chemical Biology of Fujian Province, College of Chemistry and Chemical
Engineering, Xiamen University, Xiamen, 361005, P. R. China
2School of Pharmaceutical Sciences, Xiamen University, Xiamen, 361102, P. R. China
3The Key Laboratory for Cancer Metabolomics of Shenzhen, Graduate School at Shenzhen, Tsinghua University, Shenzhen,
518055, P. R. China
RATIONALE: α-Amino phosphonates with intrinsic biological activities have been used in a wide variety of applications.
Because of the widespread existence of natural organophosphorus compounds containing P–C bonds such as the
α-amino phosphonates, it is important to investigate the gas-phase chemistry of P–C bonds in order to determine their
basic properties, which might provide some insights into their biosynthesis and catalytic cleavage.
METHODS: Twenty α-amino phosphonates were successfully synthesized and their fragmentation behavior was
systematically investigated using in-solution deuterium labeling in combination with high-resolution Fourier transform
ion cyclotron resonance (FTICR) electrospray ionization tandem mass spectrometry.
RESULTS: The fragmentation pathways of twenty α-amino phosphonates with different chemical structures were
systematically studied. In general, P–C bonds could be easily cleaved via a novel intramolecular hydrogen atom
migration from the amino group to the phosphoryl group through a five-membered-ring intermediate in the gas phase.
A possible mechanism of the rearrangement of α-amino phosphonates is proposed.
CONCLUSIONS: An interesting intramolecular hydrogen atom migration between the amino and phosphoryl groups
was observed with cleavage of the P–C bond in the molecule through a five-membered-ring intermediate. This
characteristic fragmentation pathway not only provides some insights into the basic chemistry of compounds with
P–C bonds, but could also have some applications in the structural determination of the α-amino phosphonate
analogues. Copyright © 2014 John Wiley & Sons, Ltd.
α-Amino phosphonates are bioactive natural products
containing phosphorus–carbon (P–C) bonds. Since the first
natural P–C compound, 2-aminoethylphosphonic acid,
was isolated and identified from rumen Protozoa in 1959,[1]
more such compounds have been synthesized.[2–8] Among
these, α-amino phosphonates have been used in a wide
variety of applications such as anticancer and antivirus
drug development, and as antibiotics, herbicides, and
fungicides,[9–13] and most of their biological activity could
be attributed to the structural properties of their P–C bonds.
Although P–C bonds are chemically stable, it has
been reported that they can be cleaved by enzymes such as
phosphonoacetate hydrolase (PhnA) and phosphonopyruvate
hydrolase (PnPy).[14,15] Considerable efforts have been made
to elucidate the mechanism of formation and cleavage of the
P–C bonds, but the exact details remain unknown.[16,17]
In our previous work, different classes of organophosphorus
compounds with P–C bonds were synthesized and
then investigated by electrospray ionization tandem
mass spectrometry (ESI-MS/MS). It was found that the
fragmentation pathways of P–C bonds were different
from those of phosphates and phosphoramidates containing
P–O and P–N bonds, respectively.[18–21] Several interesting
rearrangement reactions have been observed, such as
hydrogen-atom migration from the hydroxyl to the phosphoryl
group for α-hydroxyl dialkylphosphonic acid esters,[22] neutral
phosphoryl amine loss from phosphonate peptides,[23] and
oxygen-atom migration to phosphoryl groups for
[(4-substituted-benzoylamino)phenylmethyl]phosphonic acid
diisopropyl esters.[24] Furthermore, it was found that P–C
bonds could be easily cleaved in the gas phase under
our experimental conditions. Because of the widespread
existence of organophosphorus compounds such as α-amino
phosphonates, it is valuable to investigate the gas-phase
chemistry of their P–C bonds in order to obtain some insight
into their biosynthesis and cleavage. In the present work, a
series of α-amino phosphonates was synthesized and their
fragmentation behavior was systematically investigated using
* Correspondence to: X. Gao, School of Pharmaceutical
Sciences, Xiamen University, Xiamen, 361102, P. R. China.
E-mail: xgao@xmu.edu.cn
Rapid Commun. Mass Spectrom. 2014, 28, 1964–1970
Copyright © 2014 John Wiley & Sons, Ltd.

Fragmentation of P–C bonds of α-amino phosphonates
ESI-MS/MS in combination with stable isotope labeling.
Simultaneously with the cleavage of P–C bonds, a novel
intramolecular hydrogen-atom migration between the amino
and phosphoryl groups through a five-membered ring was
observed. The proposed mechanism of rearrangement of
α-amino phosphonates was confirmed by deuterium labeling
and high-resolution Fourier transform ion cyclotron resonance
(FTICR) tandem mass spectrometry.
Scheme 1. Chemical structures of α-amino phosphonates 1–20.
Table 1. ESI-MSn data of [M+Na]+ ions of α-amino phosphonates 1–20
Compounds (MW)
Precursor ions (m/z)
Product ions (m/z, relative intensity percentage)
P–H bond formation
1 (361)
384
342 (9)
218 (97)
147 (9)
147 (20)
147 (36)
129 (11)
189 (100)
342
218 (100)
189
129 (100)
Exact massa
HRMSb
2 (333)
384.1704
384.1693
356
342.1235
342.1224
218 (100)
218.0933
218 (100)
218 (100)
263 (18)
248 (100)
252 (100)
236 (56)
210 (100)
190 (100)
160 (100)
210 (20)
292 (70)
184 (9)
218.0946
218.0930
189.0657
189.0646
161 (39)
162.0394
133 (16)
224 (44)
161 (100)
161 (28)
161 (88)
161 (100)
d-2c
357.1444
328
3 (305)
4 (397)
420
5 (378)
401
115 (8)
6 (363)
386
7 (367)
390
115 (5)
115 (6)
8 (351)
374
9 (347)
370
10 (327)
11 (297)
12 (325)
13 (305)
14 (299)
15 (319)
16 (339)
17 (334)
18 (359)
19 (339)
20 (305)
350
148 (5)
320
348
115 (2)
190 (100)
161 (100)
161 (76)
161 (100)
161 (100)
161 (12)
328
115 (7)
322
342
234 (4)
204 (14)
115 (11)
362
224 (100)
219 (100)
244 (100)
224 (100)
357
382
161 (55)
161 (42)
161 (100)
362
328
aThe theoretical mass by calculation.
bThe mass measured by high-resolution ESI-FTICR-MS.
cDeuterium-labeled compound 2.
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Y. Gao et al.
EXPERIMENTAL
0.99 (d, J = 6.1 Hz, 3H). 13C NMR (100 MHz, CDCl3)
δ (ppm) 139.6 (s), 136.1 (d, J = 4.3 Hz), 128.8 (d, J = 6.6 Hz),
128.3 (d, J = 6.6 Hz), 127.7 (d, J = 2.9 Hz), 127.2 (s), 71.3 (d, J
= 7.4 Hz), 60.2 (d, J = 155.1 Hz), 51.4 (d, J = 17.5 Hz), 24.2
(t, J = 3.7 Hz), 23.9 (d, J = 5.3 Hz), 23.4 (d, J = 5.3 Hz).
31P NMR (162 MHz, CDCl3) δ (ppm) 21.87.
Synthesis and identification of α-amino phosphonates
Twenty α-amino phosphonates (compounds 1–20, Scheme 1)
were synthesized according to a novel method developed in
our previous work.[25] All the compounds were characterized
by 1H NMR (400 MHz), 13C NMR (100 MHz), 31P NMR
(162 MHz) and ESI-MS. The general synthetic procedure
was as follows. Amide (0.3 mmol) was treated in anhydrous
tetrahydrofuran (3.0 mL) with Schwartz’s reagent Cp2ZrHCl
(171 mg, 0.66 mmol) under nitrogen and stirred at room
temperature. H-phosphonate (0.36 mmol) was added until
the suspension cleared (about 5–15 min), and the mixture
was stirred at 60 °C for about 12 h in a Schlenk tube. The
reaction was quenched with saturated sodium bicarbonate
(35.0 mL), and extracted with ethyl acetate (3 × 5.0 mL). The
combined organic extracts were washed with brine (10 mL),
dried over Na2SO4, and evaporated under reduced pressure
to leave the crude product, which was subsequently purified
by flash chromatography and eluted using petroleum ether/
ethyl acetate (2:1, v/v) to give the product.
The NMR data for diisopropyl ((benzylamino)(phenyl)
methyl)phosphonate (compound 1) and diethyl ((benzylamino)
(phenyl)methyl)phosphonate (compound 2) were as follows.
Compound 1: 1H NMR (400 MHz, CDCl3): δ (ppm)
7.46-7.45 (m, 2H), 7.39-7.29 (m, 8H), 4.74-4.66 (m, 1H),
4.55-4.48 (m, 1H), 4.00-3.95 (d, J = 20.4 Hz, 1H), 3.84-3.81
(d, J = 13.1 Hz, 1H), 3.58-3.54 (d, J = 13.3 Hz, 1H), 2.32
(s, 1H), 1.31-1.29 (m, 6H), 1.25-1.24 (d, J = 6.1 Hz, 3H), 1.00-
Compound 2: 1H NMR (400 MHz, CDCl3) δ (ppm) 7.44-7.42
(m, 2H), 7.38-7.34 (m, 2H), 7.32-7.28 (m, 3H), 7.26-7.22 (m, 3H),
4.11-3.92 (m, 4H), 3.84-3.78 (m, 2H), 3.56-3.52 (d, J = 13.3 Hz,
1H), 2.31 (s, 1H), 1.27-1.25 (t, J = 7.0 Hz, 3H), 1.14-1.10 (t, J =
7.0 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ (ppm) 139.4 (s),
135.8 (d, J = 3.9 Hz), 128.8 (d, J = 6.5 Hz), 128.6 (d, J = 2.2 Hz),
128.4 (d, J = 2.2 Hz), 128.0 (d, J = 3.6 Hz), 127.2 (s), 63.0 (d, J =
6.8 Hz), 62.9 (d, J = 7.0 Hz), 59.7 (d, J = 153.4 Hz), 51.3 (d, J =
17.5 Hz), 16.5 (d, J = 5.8 Hz), 16.3 (d, J = 5.7 Hz). 31P NMR
(162 MHz, CDCl3) δ (ppm) 23.51.
Mass spectrometric conditions
The ESI mass spectra were acquired in positive ion mode
using a Bruker Esquire 3000plus ion trap mass spectrometer
(Bruker Daltonic Inc., Billerica, MA, USA). Typically,
[M+H]+ and [M+Na]+ ions were observed with high relative
intensities for all twenty α-amino phosphonates studied. The
α-amino phosphonates were dissolved in methanol at a
concentration of about 5–10 ppm and infused continuously
into the ESI chamber at a flow rate of 4 μL min–1 using a
model 74900 syringe pump (Cole-Parmer Instrument Co.,
Vernon Hills, IL, USA). Nitrogen (N2) was used as the drying
gas at a flow rate of 4 L min–1 and the nebulizer gas with a
Figure 1. ESI tandem mass spectra of compound 1 in positive ion mode: (A)
ESI-MS/MS of [M+Na]+ ion at m/z 384; (B) ESI-MS3 of product ion at m/z 342;
and (C) ESI-MS3 of the rearrangement ion at m/z 189.
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Fragmentation of P–C bonds of α-amino phosphonates
pressure of 8 psi. The electrospray capillary was typically
held at 4 kV. The heated capillary temperature was 250 °C.
The tandem mass spectra were obtained by collision-induced
dissociation (CID) with helium after isolation of the
appropriate [M+H]+ and [M+Na]+ precursor ions. Generally,
the scan range was from m/z 50 to 800. The fragmentation
amplitude values ranged from 0.3 to 0.8 eV and the
fragmentation time was 40 ms. Five scans were averaged for
each spectrum.
High-resolution positive ion ESI tandem mass spectra were
acquired on a Bruker APEX-Ultra FTICR mass spectrometer
(7.0 T). Nitrogen (N2) was used as the drying gas and
Scheme 2. (A) Possible fragmentation pathways of [M+Na]+ ion of
compound 1. (B) Proposed rearrangement mechanism for the intramolecular
hydrogen migration.
Figure 2. High-resolution ESI-FTICR tandem mass spectrum of [M+Na]+ ion of
compound 1 at m/z 384.1693.
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Y. Gao et al.
nebulizer gas. The acquisition parameters were as follows: drying
gas temperature, 180 °C; drying gas flow rate, 5.0 L min–1;
nebulizer gas flow rate, 1.0 L min-1; ion accumulation time, 2.0 s.
The capillary entrance voltage was 4.3 kV, the end plate voltage
was 3.8 kV, and the collision energy was 13.5 eV. The precursor
[M+Na]+ ions were selected using an isolation width of 1.0 m/z
unit. The scan range was from m/z 50 to 800. The sample dissolved
in methanol was introduced into the source at a flow rate of 4 mL
min–1. Ten scans were averaged for each spectrum.
representative example, the positive ion mode ESI mass
spectra of compound 1 are shown in Fig. 1, and the
corresponding fragmentation pathways are shown in
Scheme 2(A). In ESI-MS/MS, the precursor [M+Na]+ ion at
m/z 384 fragmented to produce two abundant product ions
at m/z 218 and 189 through cleavage of the P–C bond.
Furthermore, by neutral loss of propene (42 Da), a minor
product ion was observed at m/z 342, which in turn
fragmented to form product ions at m/z 218 and 147 by
cleavage of the P–C bond (Fig. 1(A)). A very low abundance
product ion at m/z 300 was also formed by neutral loss of a
second molecule of propene (Fig. 1(B)). In order to identify
its structure, the product ion at m/z 189 was subsequently
isolated and investigated by ESI-MS3, as shown in Fig. 1(C).
Two product ions at m/z 147 and 129 were generated by loss of
either propene (42 Da) or isopropanol (60 Da), respectively. In the
gas phase, the H-phosphonate ion at m/z 189 and the imine ion at
m/z 218 were formed by P–C bond cleavage of compound 1 under
collision conditions. Interestingly, P–C bonds could be easily
formed through the Pudovik reaction used for the preparation
of α-amino phosphonates by the reaction of imines with H-
phosphonate under basic conditions in the solution phase.[26]
In order to determine the formulae of product ions, high-
resolution ESI-FTICR-MS/MS was performed on the sodium
adduct ion of compound 1 at m/z 384.1693 (theoretical mass
384.1704, relative error 2.8 ppm; Fig. 2 and Table 1). It was
found that the measured masses of the ions at m/z 342, 218,
and 189 (342.1224, 218.0930, and 189.0646) corresponded to
the formulae C17H22NNaO3P (calculated 342.1235, relative
Deuterium-labeling experiment
Hydrogen/deuterium exchange was used to trace and determine
the intramolecular hydrogen migration of α-amino phosphonates.
Briefly, compound 1 (1 mg) was incubated with CD3OD (1 mL) for
24 h at room temperature in order to exchange the labile protons
completely. An aliquot of the reaction mixture (about 10 μL) was
diluted with CD3OD (1 mL) and analyzed by ESI-FTICR-MS/MS
using the above MS conditions. Prior to analysis, CD3OD
(1 mL) was injected into the ESI source in order to remove
active hydrogen atoms in the mass spectrometer system.
RESULTS AND DISCUSSION
ESI-MS and ESI-MSn analysis of [M+Na]+ ions of α-amino
phosphonates
The ESI-MSn data of the sodium adducts [M+Na]+ of
compounds 1–20 are summarized in Table 1. All α-amino
phosphonates shared a general fragmentation pathway. As a
Figure 3. ESI tandem mass spectra of compound 2 in positive ion mode: (A)
ESI-MS/MS of [M+Na]+ ion at m/z 356 and (B) ESI-FT-ICR-MS/MS of
deuterium-labeled [d-M+Na]+ ion at m/z 357.1449.
Figure 4. ESI tandem mass spectrum of [M+Na]+ ion of compound 9 at m/z 370.
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Fragmentation of P–C bonds of α-amino phosphonates
error 3.2 ppm), C14H13NNa (calculated 218.0946, relative
error 7.3 ppm), and C6H15NaO3P (calculated 189.0657,
relative error 5.8 ppm), respectively, in agreement with the
proposed structures shown in Scheme 2(A).
hydrogen migration, which is close to that in the
McLafferty rearrangement with a six-membered ring in
the transition state.[27]
In order to test the proposed hydrogen migration
mechanism of α-amino phosphonates, compound 2 with a
diethyl phosphate group was analyzed by ESI-MS/MS in
combination with deuterium labeling. As shown in Fig. 3(A),
two abundant product ions at m/z 218 and 161 were
produced from the precursor [M+Na]+ ion at m/z 356 by
cleavage of the P–C bond. By comparison with the
fragmentation pathways of compound 1, no alkyl group loss
was observed for compound 2. In addition, compound 2 was
labeled with deuterium via solution-phase hydrogen/
deuterium exchange prior to ESI-FTICR-MS analysis. After
labeling, the [M+Na]+ ion of compound 2 was observed at
m/z 357.1444 (theoretical mass 357.1454, relative error 2.8
ppm), a 1 u increase, which is consistent with the structure
of compound 2 with one labile hydrogen atom. As shown
in Fig. 3(B), the product ion at m/z 162.0394 (theoretical mass
162.0406, relative error 7.4 ppm) was also increased by 1 u
from the product ion at m/z 161 without deuterium labeling
(Fig. 3(A)), indicating that the deuterium atom of the
precursor ion might be transferred to the phosphoryl group.
Meanwhile, an imine ion was observed at m/z 218.0940
(theoretical mass 218.0946, relative error 2.7 ppm), suggesting
that its structure was the same as that of the native product
ion. In summary, cleavage of the P–C bond of the α-amino
phosphonate occurs simultaneously with the intramolecular
hydrogen atom migration.
To study the hydrogen migration pathway further, three
α-amino phosphonates (compounds 9, 10, and 11) without
the active hydrogen atom were synthesized and studied by
ESI-MS/MS (Table 1). As a representative, the ESI-MS/MS
spectrum of the sodium adduct of compound 9 in positive
ion mode is shown in Fig. 4. Only one product ion at
m/z 210, corresponding to an imine ion, was generated from
the precursor ion by a neutral loss of 160 Da through
cleavage of the P–C bond. However, no rearrangement
ion was detected, indicating that the methyl group in the
molecule could not migrate to the phosphoryl group.
Mechanisms of P–C bond cleavage and intramolecular
hydrogen migration
Based on these experimental results, a general intramolecular
rearrangement mechanism is proposed. As shown in
Scheme 2(B), the active hydrogen of the amino group might
be coordinated with the phosphoryl oxygen atom to form
an intermediate ion 1a with
a five-membered ring.
Subsequently, the P–C bond would be cleaved in parallel
with hydrogen-atom transfer to form the trivalent phosphite
ion 1b and imine ion 1c in a coordinated process. Moreover,
1b could exist as three exchangeable isomers, 1d, 1e, and
1f, which might favor its stability, resulting in its high relative
abundance of up to 100%. Based on this proposed mechanism
of rearrangement, the formation of the bi-coordinated
phosphate ion at m/z 129 could also be explained by neutral
loss of propanol from 1b. The five-membered phosphonate
ring proposed is characteristic of the above intramolecular
Table 2. ESI-MSn data of [M+H]+ ions of α-amino
phosphonates 1–20
Imine ions
(m/z, relative
Molecular
weight
Precursor
ions (m/z)
intensity
Compounds
percentage)
1
361
333
305
378
363
367
351
347
327
297
325
299
339
334
359
339
305
362
334
306
379
364
368
352
348
328
298
326
300
340
335
360
340
306
196 (100)
196 (100)
196 (100)
241 (100)
226 (100)
230 (100)
214 (100)
210 (100)
190 (100)
160 (100)
188 (100)
162 (100)
202 (100)
197 (100)
222 (100)
202 (100)
168 (100)
2
3
5
6
7
8
9
10
11
12
14
16
17
18
19
20
ESI-MS/MS analysis of [M+H]+ ions of α-amino phosphonates
In comparison with the fragmentation pathways of [M+Na]+
ions, the ESI-MS/MS spectra of the protonated α-amino
phosphonates were systematically determined and summarized
in Table 2. It was found that the fragmentation pathways of
[M+H]+ ions were simpler than those of the sodium adducts.
As shown in Fig. 5, the precursor [M+H]+ ion at m/z 362
Figure 5. ESI tandem mass spectrum of [M+H]+ ion of compound 1 at m/z 362
in positive ion mode.
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Y. Gao et al.
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fragmented to produce a product ion at m/z 196 by neutral loss
of one molecule of diisopropyl H-phosphite (166 Da) through
cleavage of the P–C bond with an active hydrogen-atom
transfer. However, different from the fragmentation pathways
of sodium adducts, the [M+H]+ ion of the diisopropyl
H-phosphite could not be detected for all the compounds
investigated. It is postulated that the different coordination
affinities of the sodium ion and the proton with the phosphoryl
group in the gas phase might be the key factor responsible
for the appearance of characteristic H-phosphite ions.
CONCLUSIONS
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[18] X. Gao, F. Ni, J. Bao, Y. Liu, Z. Zhang, P. X. Xu, Y. F. Zhao.
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α-Amino phosphonates with different chemical structures were
successfully synthesized and analysed by ESI-MS/MS in
combination with deuterium labeling. An intramolecular
hydrogen atom migration between the amino and phosphoryl
groups was observed, with cleavage of the P–C bond through
a five-membered-ring intermediate. The possible migration
mechanism and structures of key product ions were confirmed
by hydrogen/deuterium exchange and high-resolution FTICR-
MS/MS. In view of these experimental results, selective
cleavage of P–C bonds might be a general process for α-amino
phosphonates under our mass spectrometry conditions. These
characteristic fragmentation pathways might not only provide
some insights into the basic chemistry of P–C bonds, but also
have some potential applications in the structural determination
of α-amino phosphonate analogues.
[19] X. Gao, Z. P. Zeng, P. X. Xu, G. Tang, Y. Liu, Y. F. Zhao.
Comparison of the isomeric α-amino acyl adenylates and
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Acknowledgements
This work was supported by the Chinese National Natural
Science Foundation (21305115, 21375113, and 21173178).
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