A novel hydrogen migration of dialkylphosphonic acid esters using electrospray ionization tandem mass spectrometry

Article abstract of DOI:10.1002/rcm.5215

In this paper, attention is focused on analysis of the fragmentation of α-hydroxy-β-amino phosphonate esters designed as inhibitors of protein kinase A. An interesting proton migration mechanism in the cleavage of the P-C bond is investigated by electrospray ionization tandem mass spectrometry. A possible rearrangement mechanism is proposed and verified by high-resolution mass spectra using isotope deuterium/hydrogen-exchange technology and additionally checked by detailed DFT calculation based on Gaussian software. The result clearly indicates that this mechanism proceeds by a five-membered ring concerted transition state with activation energy 11.3 kcal mol^-1 for the compound 3f. The overall reaction is endothermic with an energy 13.2 kcal mol^-1. The effect of different substituents and different metal ions for rearrangement of these esters is studied by experiment and theory. It is concluded that this rearrangement process is energetically unfavorable and hence only occurs in the mass spectrometer. Copyright

Full text of DOI:10.1002/rcm.5215

Research Article
Received: 22 February 2011
Revised: 7 August 2011
Accepted: 7 August 2011
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2011, 25, 3314–3322
(wileyonlinelibrary.com) DOI: 10.1002/rcm.5215
A novel hydrogen migration of dialkylphosphonic acid esters
using electrospray ionization tandem mass spectrometry
Zhiping Zeng1, Ping Luo1, Yao Jiang1, Yan Liu1, Guo Tang1, Pengxiang Xu1 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
2The Key Laboratory for Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of
Chemistry, School of Sciences, Tsinghua University, Beijing 100084, P.R. China
In this paper, attention is focused on analysis of the fragmentation of a-hydroxy-b-amino phosphonate esters
designed as inhibitors of protein kinase A. An interesting proton migration mechanism in the cleavage of the P-C
bond is investigated by electrospray ionization tandem mass spectrometry. A possible rearrangement mechanism
is proposed and verified by high-resolution mass spectra using isotope deuterium/hydrogen-exchange technology
and additionally checked by detailed DFT calculation based on Gaussian software. The result clearly indicates that
this mechanism proceeds by a five-membered ring concerted transition state with activation energy 11.3 kcal molÀ1
for the compound 3f. The overall reaction is endothermic with an energy 13.2 kcal molÀ1. The effect of different
substituents and different metal ions for rearrangement of these esters is studied by experiment and theory. It is
concluded that this rearrangement process is energetically unfavorable and hence only occurs in the mass spectrometer.
Copyright © 2011 John Wiley & Sons, Ltd.
Organophosphorus compounds containing a P–C bond were
first isolated from living organisms in 1959.[1,2] Soon after-
wards many kinds of related compounds were found in
hundreds of aquatic and terrestrial animals and microorgan-
isms.[3] a-Hydroxyphosphonate esters, considered as an
important class of biologically active compounds, have
attracted attention because of their antibacterial, antiviral,
antibiotic, pesticidal, anticancer, and enzyme inhibitor prop-
erties.[4,5] However, the compounds under investigation here,
a-hydroxy-b-aminophosphonate esters designed as inhibi-
tors of protein kinase A, have not been reported previously.
Hence a study of the fragmentation behavior of these com-
pounds by electrospray ionization tandem mass spectrometry
(ESI-MS/MS) is designed to deepen the understanding of
their fragmentation structures.
Mass spectrometry is a very powerful tool and a unique
physical method for structural determination in chemistry
today. Electrospray ionization multistage tandem mass spec-
trometry (ESI-MSn), coupled with high-performance liquid
chromatography, has been widely developed in biological
research, such as detection of non-covalent complexes,[6] the
sequencing of proteins and polynucleotides,[7–9] pharmakinetics,[10]
and drug metabolism.[11] In this present work, we
synthesized a group of phosphonate esters and investigated
their fragmentation behavior by electrospray ionization
mass spectrometry combined with tandem mass spectrome-
try. A five-membered ring proton transfer mechanism was
observed and checked by Fourier transform mass spectrome-
try (FT-MS) and subsequently this mechanism was eluci-
dated in detail based on a theoretical study.
EXPERIMENTAL
Mass spectrometric conditions
All the experiments were performed in the positive ion mode
using a Bruker Esquire 3000plus ion trap mass spectrometer
(Bruker Daltonic Inc., Billerica, MA, USA) equipped with an
analytic electrospray ionization (ESI) source. Nitrogen was
used as the drying gas at a flow rate of 4 LÁmin–1. The nebu-
lizer pressure was 8 psi and the electrospray capillary was
typically held at 4 kV. The heated capillary temperature was
250 ꢀC. Compounds, dissolved in methanol at a concentration
of ca. 0.01 mgÁmL–1, were ionized by ESI and infused continu-
ously into the ESI chamber at a flow rate of 4 mLÁmin–1 using a
model 74900 syringe pump (Cole-Parmer Instrument Co.,
Vernon Hills, IL, USA). Generally, the scan range was from
m/z 50 to 800. Five scans were averaged for each spectrum.
All of the adducts, obtained from endogenous sources as their
sodium salts, were selected using an isolation width of 1.0 to
1.5 mass-to-charge (m/z) units and analyzed by multistage
tandem mass spectrometry through collision with helium to
obtain the ESI-MSn spectra. The fragmentation amplitude
values ranged from 0.3 to 0.8 V and the fragmentation time
was 40 ms.
* Correspondence to: Y.-F. Zhao, Department 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.
E-mail: yfzhao@xmu.edu.cn
Rapid Commun. Mass Spectrom. 2011, 25, 3314–3322
Copyright © 2011 John Wiley & Sons, Ltd.

Hydrogen migration of dialkylphosphonic acid esters using ESI-MS/MS
The high-resolution tandem mass spectrum of deuterium-
labeled compound 3c was acquired on a Bruker APEX-Ultra
Fourier transform ion cyclotron resonance (FT-ICR) mass
spectrometer equipped with an analytic ESI source in the posi-
tive ion mode. 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 following voltages were used: Capillary entrance
voltage (4.3 kV), end plate (3.8 kV), skimmer 1 (36 V), and
collision energy (13.5 V). The scan range was from m/z 100 to
700. The sample dissolved in CH3OD was introduced into
the source at a flow rate of 4 mLÁmin–1. Ten scans were aver-
aged for each spectrum.
For deuterium-labeling, compound 3c (20 mg) was added
to CH3OD solvent(1 mL) and this solution was incubated
with for 24 h at room temperature for exchange of active pro-
tons. An aliquot of the reaction mixture (10 mL) was diluted
with CH3OD (1 mL) and analyzed by ESI-FT-ICR-MS/MS.
The ESI source was equilibrated before performing this
experiment.
Theoretical calculations
Quantum chemical calculations were carried out on all the
various species occurring in the rearrangement process in
order to understand the atomic details of the main fragmenta-
tion reactions of the rearrangement ion. All calculations were
performed with the GAUSSIAN 03 package.[12] The hybrid
density functional method including Becke’s three-parameter
non-local-exchange functional[13] with the correlation func-
tional of Lee-Yang-Parr[14] (B3LYP) was employed. The basis
set used was the standard all-electron split-valence basis set
6-31 G* including the polarization d-function on non-hydrogen
Scheme 1. Synthetic pathway for formation of compounds
3a–f.
Table 1. ESI-MS/MS of [P + Metal]+ ions of compounds 3a–f [m/z, relative abundance (%)]
Product ions m/z (%)
Precursor
[P + M]+
332(3)
276(12)
232(20)
360(18)
304(2)
[a + M]+
m-56
[b + M]+
m-100
232(100)
232(100)
[c + M]+
m-199
[d + M]+
122
[e + M]+
166
[f + H]+
200
[f + Na]+
222
Compound (MW)
3a
276(28)
133(7)
122(23)
122(10)
122(100)
122(12)
122(13)
122(100)
122(4)
122(<1)
122(21)
122(2)
166(<1)
166(<1)
166(0)
166(1)
166(0)
166(0)
166(<1)
166(0)
166(0)
166(<1)
166(0)
166(0)
166(<1)
166(<1)
166(0)
166(1)
166(0)
166(0)
144(0)
144(0)
150(10)
150(0)
150(0)
166(17)
166(0)
166(100)
200(<1)
200(<1)
200(3)
200(0)
200(0)
200(0)
200(0)
200(1)
200(0)
200(0)
200(2)
200(0)
200(0)
200(0)
200(0)
200(0)
200(0)
200(0)
178(0)
178(0)
184(0)
184(0)
184(0)
200(0)
200(0)
222(3)
222(0)
222(0)
222(2)
222(0)
222(0)
222(1)
222(0)
222(0)
222(<1)
222(0)
222(0)
222(3)
222(0)
222(0)
222(2)
222(0)
222(0)
199(0)
199(0)
206(21)
206(0)
206(0)
222(100)
222(0)
Na
133(3)
133(25)
161(7)
161(5)
161(60)
189(6)
189(<1)
189(30)
189(2)
189(<1)
189(21)
257(28)
257(41)
257(100))
285(45)
285(13)
285(100)
203(3)
3b
Na
304(2)
260(100)
260(100)
260(28)
388(1)
3cSR
Na
332(1)
288(100)
288(100)
332(40)
288(74)
388(22)
332(45)
288(84)
456(11)
400(13)
356(76)
484(1)
3cSS
Na
332(17)
400(100)
428(15)
288(100)
288(100)
122(<1)
122(25)
122(0)
3d
Na
356(60)
356(100)
122(5)
122(11)
122(0)
3e
Na
384(100)
384(100)
428(52)
384(13)
402(12)
302(8)
122(0)
122(2)
3fa
H
346(5)
302(100)
100(0)
203(100)
209(0)
100(49)
106(0)
3fb
Li
408(6)
352(100)
308(96)
308(100)
352(14)
308(21)
424(20)
324(15)
222(14)
166(42)
440(2)
209(2)
106(4)
106(100)
122(4)
209(2)
3f
Na
324(26)
225(0)
225(5)
122(100)
122(13)
122(100)
138(1)
3fc
K
384(0)
340(4)
241(1)
241(43)
182(2)
182(0)
216(0)
216(0)
238(100)
238(0)
340(100)
138(98)
aESI-MS/MS of the [P + H]+ ions of compound 3f.
bESI-MS/MS of [P + Li]+ ions of compound 3f.
cESI-MS/MS of [P + K]+ ions of compound 3f.
Rapid Commun. Mass Spectrom. 2011, 25, 3314–3322
Copyright © 2011 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm

Z.-P. Zeng et al.
atoms.[15,16] We calculated the geometries and vibrational fre-
quencies belonging to each reactant, product, transition state,
and intermediate without any constraint. We confirmed that
all the reactants and intermediates have no imaginary frequen-
cies and each transition state had only one imaginary
frequency. Reported energies are ZPE (zero-point energy)-
corrected, unless otherwise specified.
(0.4 mL) were added dropwise to the resulting solution. The
reaction was monitored by thin-layer chromatography (TLC)
until the reactant disappeared. The solvent was then removed
under reduced pressure to obtain a slurry, purified by silica
gel column chromatography (petroleum ether/ethyl acetate
1:1) to give products 3a–f as colorless amorphous solids or
liquids in yields of 83–90%. Each pair of the compounds 3a–f
was epimeric because the chirality of the connecting carbon
of the hydroxyl group was induced by the L-proline. All of
the structures were characterized by 31P NMR, 1H NMR,
and 13 C NMR and elemental analysis.
Preparation of samples
Compounds 3a–f were synthesized (see Scheme 1) according
to methods described in the literature.[17] General procedures
are shown as follows.
Compound 1 (1.0 mmol yellow oil), as the reactant, was
injected into in a 5 mL flask containing 2 mL dichloromethane,
and then compound 2 (1.2 mmol liquid) and triethylamine
The two diastereoisomers of compound 3c were first purified
by flash column chromatography and then re-chromatographed
to separate them into pure diastereoisomers identified as 3cSS
and 3cSR. The absolute configuration of compound 3cSR was
determined by X-ray diffraction analysis. Analytical data for
these two compounds are as follow: 3cSS: colorless oil,
1H NMR (400 MHz, CDCl3) d 5.40 (s, 1H, OH), 4.88–4.71
(m, 2H, CH(CH3)2), 4.30–4.20 (m, 1H, CH(OH)), 3.71–3.63
(m, 1H, NCHC(OH)), 3.41–3.37 (m, 1H, NCHH), 3.37–3.30
(m, 1H, NCHH), 2.28–2.20 (m, 1H, NCHCHH), 2.05–1.95
(m, 1H, NCHCHH), 1.93–1.81 (m, 2H, NCH2CH2), 1.47 (s, 9H,
C(CH3)3), 1.36 (dd, J = 6.2, 1.8 Hz, 12H, CH(CH3)2).13 C NMR
(101 MHz, CDCl3) d 158.4 (C = O), 80.9 (OC(CH3)3), 73.1 (d,
J = 160.9 Hz, 1 C, CH(OH)), 71.5 (d, J = 7.7 Hz, 1 C, OCH(CH3)
2), 71.0 (d, J = 7.1 Hz, 1 C, OCH(CH3)2), 59.2(d, J = 6.8 Hz, 1 C,
NCH), 47.1 (NCH2), 29.7 (NCHCH2), 28.4(s, 3 C,C(CH3)3 ), 24.2
(d, J = 3.4 Hz, 1 C, NCH2CH2), 24.05 (d, J = 3.7 Hz, 2 C, CH
(CH3)2), 24.00(d, J = 4.0 Hz, 2 C, CH(CH3)2).31P NMR
(162 MHz, CDCl3) d 19.86. 3cSR: white solid, 1H NMR
(400 MHz, CDCl3) d 4.87 (s, 0.7H, OH), 4.80–4.71 (m, 2H, CH
(CH3)2), 4.35–4.09 (m, 2H, CH(OH) and NCHC(OH)), 3.59–3.48
(m, 1H, NCHH), 3.32 (dt, J = 10.6, 7.2 Hz, 1H, NCHH), 2.92 (s,
0.3H, OH), 2.36–2.20 (m, 1H, NCHCHH), 2.10–1.93 (m, 2H,
NCHCHH and NCH2CH2), 1.76–1.66 (m, 1H, NCH2CH2), 1.48
(s, 9H, C(CH3)3), 1.34 (d, J = 6.1 Hz, 12H, CH(CH3)2).13 C NMR
(126 MHz, CDCl3) d 156.4 (C = O), 80.0 (OC(CH3)3), 70.9 (d,
J = 5.1 Hz, 2 C, OCH(CH3)2), 70.8 (d, J = 156.1 Hz, 1 C, CH
(OH)), 60.2 (NCH), 47.8(NCH2), 28.6 (s, 3 C, C(CH3)3), 27.5
(NCHCH2), 24.2 (d, J = 3.2 Hz, 1 C, NCH2CH2), 24.07 (d,
J = 4.1 Hz, 2 C, CH(CH3)2), 23.99 (d, J = 5.3 Hz, 2 C, CH(CH3)
2).31P NMR (162 MHz, CDCl3) d 20.82. ESI-HRMS calcd for
[C16H32NPO6 + Na]+: 388.1859; found: 388.1852.
OH
O
O
H
-99
O
-82
O
O
H
H
Na+
P
P
H
H
N
H
O
H
Na+
O
N
H
Na+
O
m/z 204
O
m/z 105
H
P
3c[d+Na]+
O
H
H
N
H
H
O
m/z 122
-42
3c[d]
OH
P
O
O
-99
O
-124
O
H
Na+
P
O
O
H
H
O
N
H
H
N
H
Na+
Na+
O
O
3c[g+Na]+
m/z 246
H
P
m/z 147
3c[d+Na]+
m/z 122
O
H
N
H
H
O
-42
OH
3c[d]
O
O
-99
O
-166
O
H
Na+
P
O
O
O
P
N
H
N
Na+
Na+
H
H
O
O
3c[b+Na]+
3c[c+Na]+
m/z 189
3c[d+Na]+
H
P
O
N
H
m/z 288
H
O
m/z 122
3c[d]
-44
-44
O C O
O
C
O
O
OH
P
O
Na+
O
P
-143
H
Na+
-166
O
O
O
O
N
H
O
N
O
O
O
H
O
Na+
HO
3c[c+Na]+
m/z 189
H
P
H
3c[a+Na]+
O
N
3c[e+Na]+
m/z 166
O
m/z 332
O
O
H
-56
OH
3c[e]
-56
O
O
O
P
O
N
O
-166
O
O
-199
O
H
Na+
P
N
H
O
Na+
O
O
Na+
O
O
RESULTS AND DISCUSSION
H
P
3c[c+Na]+
m/z 189
O
m/z 388
O
N
[3c+Na]+
H
O
3c[f+Na]+
m/z 222
ESI-MS fragmentation of 3a–f
O
We found that the analysis of compounds 3a–f, (S)-tert-butyl-
2-((S or R)-(dialkyoxyphosphoryl)(hydroxy)methyl)pyrrolidine-
1-carboxylate, by ESI-MS only afforded the corresponding
sodium ions [P + Na]+. ESI-MSn spectra of compounds 3a–f in
positive mode were studied in detail and the data are shown
in Table 1. In addition, mass fragmentation of compound 3c for
a pair of diastereomers, labeled 3cSR and 3cSS, has been studied
and there is no significant difference in their spectra (also see
Supplementary Tables S2 and S3, Supporting Information).
Thus, the fragmentation study of our samples 3a–f with a
mixture diastereomers is valid. Meanwhile, adduct ions of
different cations of compound 3f (H+, Li+, Na+, K+) were studied
O
3c[f]
O
OH
-42
OH
P
P
N
N
Na+
m/z 228
H
Na+
m/z 186
O
H
O
H
-42
H2O
H2O
-18
OH
O
-18
OH
-100
OH
P
H
-42
N
O
O
O
O
O
O
H
O
O
P
O
Na+
H
N
P
O
H
N
H
H
Na+
m/z 204
Na+
O
C O
m/z 346
m/z 246
Scheme 2. Possible fragmentations of sodiated phosphonate
cation from 3c.
wileyonlinelibrary.com/journal/rcm
Copyright © 2011 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2011, 25, 3314–3322

Hydrogen migration of dialkylphosphonic acid esters using ESI-MS/MS
Intens.
20090219-TM.d: +MS
6
x10
2.5
424.1
(A)
2.0
1.5
1.0
0.5
402.1
102.3
440.0
239.2
301.1
0.0
5
20090219-TM(424).d: +MS2(424.0)
x10
8
6
4
2
0
222.0
(B)
(C)
324.0
324.0
424.1
166.0
122.1
122.1
4
x10
20090219-TM(424)324.d: +MS3(424.0->324.0)
2.0
1.5
1.0
0.5
0.0
5
x10
20090219-TM(424)222.d: +MS3(424.0->222.0)
2.5
2.0
1.5
1.0
0.5
0.0
(D)
166.0
222.0
122.1
100
200
300
400
500
m/z
Figure 1. MSn spectra from solidated 3f: (A) MS of [3f + Na]+; (B) MS2 of [3f + Na]+ at m/z 424; (C) MS3 of the ion at m/z 324;
and (D) MS3 of the ion at m/z 222.
0.978
2.199
0.994H1 1.878
H1 2.241
Na
2.182
O3
N
0.973
H1
2.511
O1
O3
N
O3
O2
1.512
P1
1.422
O
O1
1.423
2.149
123.8
O1
Na+
O2
O
O2
106.7
1.870
1.541Na
C1
O
1.441
133.2
1.516
C1
1.867
N
C1
P1
P1
1.892
3f
[3f+Na]+-B
[3f+Na]+-A
Na
Na 2.193
2.197
2.138
O3
1.884
0.987
O1
O3
0.981
H1
O2
1.638
H1
1.289
O1
O
N
1.983
O2
1.248
O
134.9
C1
1.668
N
P1
C1
2.274
P1
2.696
[3f+Na]+-Bts
[3f+Na]+-Ats
[3f+Na]+-Ats
[3f+Na]+-Bts
0.969
H1
Na
O3
O2
Na
O2
2.243
O2
1.500
2.243
H1
1.690
H1
P1
O3
N
O2
Na
O1
0.972
O1
H1 P1
1.350
1.422
P1
O
1.750
O
1.211
C1
N
109.7P1
C
1H
H
3f[f+Na]+
3f[f]
3f[c]-B
3f[c+Na]+-B
3f[c]-A
3f[c+Na]+-A
1
E (kcal.mol )
Scheme 3. Rearrangement mechanism and energy changes (kcal mol–1) among reactants,
transition states, and products of solidated 3f.
Rapid Commun. Mass Spectrom. 2011, 25, 3314–3322
Copyright © 2011 John Wiley & Sons, Ltd.
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Z.-P. Zeng et al.
in depth and the results used to investigate the mechanism of
this metal-assisted fragmentation cleavage.
because this ion is so unstable that loss of carbon dioxide
(44 Da) takes priority to give ion 3c[b + Na]+ (see Scheme 2).
The product ion 3c[c + Na]+ at m/z 189 is the sodium ion of the
phosphonate diisopropyl ester, formed by loss of (S)-tert-butyl
2-formylpyrrolidine-1-carboxylate (199 Da) from the precursor
ion [3c + Na]+. Also, ion 3c[b + Na]+ leads to ion 3c[c + Na]+ in
a similar pathway by loss of (S)-pyrrolidine-2-carbaldehyde
(99 Da). In addition, product ion 3c[f + Na]+ at m/z 222 (only
observed in the ESI-MS1) was detected as the sodium adduct
of (S)-tert-butyl 2-formylpyrrolidine-1-carboxylate (199 Da). It
is noteworthy that the intensity of product ions 3c[f + Na]+ in
the MS spectra are extremely weak apart from compound 3f
(see Fig. 1), where MS2 of [3f + Na]+ at m/z 424 shows product
ion 3f[f + Na]+ is the base peak. By comparison with the other
[f + Na]+ fragmentations, it is found that the phenyl group of
[3f + Na]+ binds directly to phosphorus with the help of the
P1–Cphenyl bond which is more stable than the P1–C1(O1H1)
bond (see Scheme 3) and thus the latter ion takes priority in
cleavage, producing an abundant ion 3f[f + Na]+ at m/z 222.
For the compounds 3a–e, the alkoxy group binds to phosphorus
via the Calkyl–O(P1 = O2) bond, that is much more reactive than
the P1–C1(O1H1) bond so that an alkyl group is firstly removed
from the alkoxy group by the cleavage of the P1O–Calkyl bond
on collision (see Scheme 3). On the other hand, the alkoxy group
exhibits some electronic and stereo effects in inhibiting the inten-
sity of ion [f + Na]+ (m/z 222) through inter- or intramolecular
interaction.
Distinctive fragmentation ions are produced in the MS of the
3c species (see Scheme 2), because of the easier loss of an iso-
propyl group by b-elimination from the precursor ion.[21–24]
All the product ions from 3c lose one or two 2-methylprop-1-
ene species (42 Da), giving ions at m/z 346, 246, and 204. The
b-position of the isopropoxyl group of the phosphonate has
six hydrogen atoms, so this has a great probability to migrate
one hydrogen to the bridging oxygen and give out one or
two neutrals. Though ion [3b + Na]+ has two ethoxyl groups,
it does not show the same b-elimination mechanism, as no
ethylene loss is observed in the MS2 of [3b + Na]+ to give
m/z 360. In addition, water (18 Da) can be eliminated from all
of these ions (m/z 246 and 204). It is possible that a three-
coordinated phosphorus structure (see Scheme 2) is formed
Comparison among the structures of compounds 3a–f,
which only differ in the alkyl or alkoxyl group of the phos-
phonates, reveals that the fragmentation patterns are correlated
with the types of substituents of the phosphonates. Given that
those compounds display similar patterns of fragmentation,
the following discussion will focus on compound 3c as an
example. ESI-MSn of the sodium ion adducts [3c + Na]+ from
3c affords product ions 3c[a + Na]+, 3c[b + Na]+, 3c[c + Na]+,
3c[d + Na]+, 3c[e + Na]+ and 3c[f + Na]+ (see Scheme 2). The
signal at m/z 288 corresponding to the product ion 3c
[b + Na]+, [P + Na-100]+, is observed as the base peak. The frag-
ment ion 3c[a + Na]+, product m/z 332, is produced by loss of
2-methylprop-1-ene (56 Da) from the tert-butoxycarbonyl
group through cleavage of a carbon–oxygen bond, and this is
a common fragmentation pathway for all the compounds.[18,19]
The three hydrogen atoms of the methyl group all locate in the
b-position carbon of the tert-butoxycarbonyl protecting group,
one of which likely migrates from the methyl group to the
oxygen of the carbonyl group. According to the McLafferty
rearrangement, a,b-elimination occurs subsequently by
cleavage of the quaternary carbon–oxygen bond in the tert-
butoxycarbonyl protecting group. One neutral molecule,
2-methylprop-1-ene (56 Da), is released from the precursor ion
[3c + Na]+ and then product ion 3c[a + Na]+ is produced finally.
Next carbon dioxide (44 Da) is lost from the fragment ion 3c
[a + Na]+ to give ion 3c[b + Na]+ at m/z 288.[20] Similarly,
product ions 3c[d + Na]+ and 3c[e + Na]+ were generated by
loss of a molecule of 2-methylprop-1-ene (56 Da) or carbon
dioxide (44 Da) from the precursor ion 3c[f + Na]+ as for the
precursor ion [3c + Na]+. In addition, ion [d + Na]+ is derived
from ion [e + Na]+ as shown for the case of 3f (see Table 1).
These three types of product ion were found in all the MS/MS
spectra of the sodium ion adduct precursors obtained from the
series of compounds (3a–f).
The P–C bonds of the precursor ion [3c + Na]+ and its prod-
uct ion 3c[b + Na]+ are fragmented by collision-induced disso-
ciation (CID) to give ions 3c[c + Na]+, 3c[d + Na]+ and 3c
[f + Na]+. Nevertheless, product ion 3c[a + Na]+ does not show
the same cleavage pattern as for ions [3c + Na]+ and 3c[b + Na]+
Table 2. FTICR-ESI-MS of sodium ion adducts of 3cSR and its product ions
Items
Fragmentation (m/z)
Exp.
Exact by calc.
Formula
[3cSR + Na]+
388
389
332
333
288
289
189
189
190
246
247
388.1852(25)
389.1918(33)
332.1228(4)
333.1290(5)
288.1330(100)
289.1389(100)
189.0659(4)
189.0647(3)
190.0710(3)
246.0867(21)
247.0925(21)
388.1859
389.1922
332.1227
333.1290
288.1329
289.1392
189.0651
189.0651
190.0713
246.0860
247.0922
C16H32NPO6Na
C16H31DNPO6Na
C12H24NPO6Na
C12H23DNPO6Na
C11H24NPO4Na
C11H23DNPO4Na
C6H15PO3Na
[3cSR + Na]+*a
3cSR[a + Na]+b
3cSR[a + Na]+*
3cSR[b + Na]+
3cSR[b + Na]+*
3cSR[c + Na]+
3cSR[c + Na]+*c
C6H15PO3Na
C6H14DPO3Na
C8H18NPO4Na
3cSR[g + Na]+
3cSR[g + Na]+*
C8H DNPO4Na
17
a*: Asterisk marks mean deuterium experiments of [3cSR + Na]+.
b3cSR[a + Na]+ means the fragment ion a of [3cSR + Na]+ in Table 1, similar to other items in column 1 of this table.
c3cSR[c + Na]+* contained two ions m/z 189 and 190 compared to 3cSR[c + Na]+.
wileyonlinelibrary.com/journal/rcm
Copyright © 2011 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2011, 25, 3314–3322

Hydrogen migration of dialkylphosphonic acid esters using ESI-MS/MS
to stabilize the adduct ions (m/z 228 and 186). It is
intriguing that the precursor ion (m/z 346) does not lose water
in this fragmentation, though two hydroxyl groups are adja-
cent at the a- and b-positions of these species. The ion at
m/z 246, formed by loss of a monoalkyl phosphonate, is
observed which implies the cleavage of the P–C bond. This
phenomenon was also observed for the ion at m/z 204, likely
because these phosphonic acids share a common cleavage
mechanism.
Finally, it is noteworthy that formation of product ions
[c + M]+ (m/z 147 and 105) and [f + M]+, [d + M]+, and
[e + M]+ (see Table 1, containing [f + H]+ and [f + M]+) can-
not be directly explained by a simple cleavage, because a
novel proton migration occurs in this process.
Evidence for and mechanism of hydrogen migration
In order to validate the mechanism of formation of product
ions [c + M]+ and [f + M]+, high-resolution ESI-MS/MS was
performed for the sodium ion adducts of 3cSR and its
product ions (see Table 2). Secondly, spectra following deu-
terium exchange of compound 3cSR were recorded by FT-
ESI-MS (see Table 2, data with asterisks). The hydroxyl
group of compound 3cSR was deuterated fully, and its pro-
duct ions 3cSR[a + Na]+* and 3cSR[b + Na]+*, both con-
taining this hydroxyl group, showed an increase of 1 amu.
It is notable that product ions 3cSR[c + Na]+* (190 Da),
the sodium ion adduct of diisopropyl phosphonate from
the precursor on [3cSR + Na]+*, appeared to be incompletely
Figure 2. FTICR-ESI-MS/MS2 of [3cSR + Na]+ (388.18528) and [3cSR + Na]+*.
Rapid Commun. Mass Spectrom. 2011, 25, 3314–3322
Copyright © 2011 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm

Z.-P. Zeng et al.
deuterated. It could be that a deuterium atom from the hydroxyl
group migrates to the phosphoryl group and the product ion
3cSR[c + Na]+* is formed, exchanging with the active proton of
product ion 3cSR[f + Na]+, and so partially converts into product
ion 3cSR[c + Na]+ (189 Da). The ratio of these two ions was
observed to be close to 1:1 (see Fig. 2). This explained the
process in such a reaction, deuterium exchange in mass
spectrometry to achieve a balance rapidly. In our further
theoretical study, this effect also was taken into account to
elucidate the migration mechanism (see Scheme 4).
the transition state for pathway A is 11.3 kcal mol–1, which
is 7.4 kcal mol–1 lower than transition state of pathway B
(18.7 kcal mol–1). Moreover, the energy of the sodium-binding
process of pathway A is exothermic by about À58.0 kcal mol–1,
that is 6.0 kcal mol–1 more than for pathway B. For these two
reasons, pathway A is energetically more favorable to promote
the proton transfer process and produce the fragment ion
3f[f + Na]+, which is in accord with the experimental data.
Overall, both reaction pathways are endothermic with pre-
dicted reaction energies of 7.1 kcal mol–1 and 6.1 kcal mol–1
(see Scheme 3). To determine the favored transition state
for [3f + Na]+ÀAts, the deuterium kinetic isotope effect of
[3cSR + Na]+ was taken into consideration in the theoretical
modeling based on results from the D/H exchange experiment.
The activation energy of the rate-limiting step for the dissocia-
tion of [3cSR + Na]+* (22.4 kcal mol–1) is 0.1 kcal mol–1 higher
than for [3cSR + Na]+ (22.3 kcal mol–1), suggesting that deu-
teration does not change the reaction pathway (see Scheme 4
and Supplementary Fig. 13, see Supporting Information).
In this concerted mechanism of pathway A, one intermole-
cular hydrogen bond of [3f + Na]+–A is formed between the
alpha-hydroxyl group and the phosphoryl oxygen. The bond
length and angle are 1.878 Å and 133.2ꢀ (see Scheme 3), which
means that the hydrogen bond is so strong that it plays a
predominant role in stabilizing this structure. Next, lengthen-
ing of the P1–C1 bond is observed in the transition state of
[3f + Na]+–Ats with its bond length changing from 1.892 Å
to 2.700 Å. At the same time, hydrogen migrates from the
hydroxyl group to the phosphoryl group, resulting in the
formation of a tri-coordinate diisopropyl phosphonate inter-
mediate (3f[c]–A) accompanied by oxidation of the hydroxyl
group to a carbonyl group. This process is endothermic, with
some 7.1 kcal mol–1 energy absorbed in this step (see
Scheme 4). Finally, this tri-coordinate diisopropyl phospho-
nate intermediate is so unstable that it rapidly converts into
a tetra-coordinate phosphonate (3f[c]–B), releasing energy of
about 5.2 kcal mol–1.
It is worth noting that the spectrum of [3f + Na]+ is differ-
ent from the other species. It produced a more intense
3f[f + Na]+ ion and less intense 3f[c + Na]+ ion. In order to
validate the mechanism of formation of these product ions,
a five-membered ring proton transfer mechanism is proposed
based on the experimental data (see Scheme 3). In addition, a
theoretical calculation has been carried out using the density
functional theory (DFT) method. According to the calculation
results, the proton transfer mechanism can adopt a concerted
pathway, involving two five-membered ring transition states
[3f + Na]+–Ats and [3f + Na]+ÀBts, which depend on the
binding position of the sodium ion. The activation energy of
[3cSR+Na]+-Ats
3cSR[c]-A
3cSR[f+Na]+
3cSR[c]-B
3cSR[f+Na]+
Based on this mechanistic analysis, we investigated the
relationship of the nature of the phosphonate alkyl group
to the intensity of the rearrangement ion (see the [f + M]+
column in Table 3). Experimentally, we found that the greater
the steric bulk of the alkyl group, the greater the intensity
of the rearrangement ion. A DFT calculation based upon
Gaussian was introduced to optimize the transition state for
[3cSR+Na]+
Scheme 4. Computed (B3LYP/6-31G*) energy changes for
the transition state and products in the fragmentation of
[3cSR + Na]+ and its corresponding deuterated products.
Table 3. Computed transition state structures and activation barriers in the hydrogen migration process of A to Ats
P1C1
(Å)
P1O2
(Å)
C1O1
(Å)
O1H1
(Å)
O2H1
(Å)
O1M
(Å)
O3M
(Å)
Items
TS
a
[f + M]+a
[3a + Na]+
[3b + Na]+
[3c + Na]+
[3c + Na]+b
[3d + Na]+
[3e + Na]+
[3f + Na]+
[3f + Li]+
20.8
21.6
22.3
22.4
22.8
22.8
11.3
9.5
2.606
2.578
2.700
2.700
2.253
2.919
2.696
2.440
3.046
1.645
1.643
1.652
1.652
1.616
1.664
1.668
1.675
1.669
1.252
1.257
1.249
1.249
1.279
1.239
1.249
1.266
1.235
2.038
2.028
2.108
2.108
1.885
2.194
1.884
2.037
1.962
0.978
0.979
0.977
0.977
0.990
0.974
0.981
0.981
0.976
138.8
137.9
138.8
138.8
132.6
140.7
134.9
114.7
152.1
2.226
2.211
2.219
2.219
2.251
2.237
2.193
1.838
2.633
2.193
2.194
2.196
2.196
2.254
2.195
2.138
1.795
2.595
1.8%
1.6%
0.9%
0.9%
1.6%
1.2%
68.0%
9.3%
92.6%
[3f + K]+
15.2
aThe ratio of the intensity of the [f + M]+ ions to the sum of the intensities of all the products.
bDeuteration of [3c + Na]+.
wileyonlinelibrary.com/journal/rcm
Copyright © 2011 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2011, 25, 3314–3322

Hydrogen migration of dialkylphosphonic acid esters using ESI-MS/MS
(see Scheme 3). The activation energy calculated by B3LYP/
all the compounds in Table 3. The results indicate that a steric
effect controls the activation energy of the transition state,
which is in good accord with experimental data (see the TS col-
umn and [f + M]+ column in Table 3 from entry [3a + Na]+ to
[3f + Na]+). The [3f + Na]+ compounds have the lowest transi-
tion state energy (11.3 kcal mol–1), about 10 kcal mol–1 lower
than for other compounds. Furthermore, the bond lengths of
O1Na (2.193 Å) and O3Na (2.138 Å) in the [3f + Na]+ transition
state are about 0.2–0.3 Å shorter than others and this species
also has the shortest O1H1 bond length (1.884 Å). Evidently,
the transition state of [3f + Na]+ is more product-like. On the
other hand, the sodium ion might have a stronger coordination
interaction with the carbonyl group of [3f + Na]+ to stabilize the
rearrangement product 3f[f] (see Scheme 3 for the structure of
3f[f]), which also pushes the H1 proton much closer to O2 of
the phosphoryl group. This process could reduce the activation
energy significantly (about 10 kcal mol–1) and hence produce
a greater abundance of rearrangement ions. The order of
activation energy {[3f + Na]+ (11.3 kcal mol–1) < [3a + Na]+
(20.8 kcal mol–1) < [3b + Na]+ (21.6 kcal mol–1) < [3c + Na]+
(22.3 kcal mol–1)} is exactly in accord with the experimental
results {[3f + Na]+ (68%) < [3a + Na]+ (1.8%) < [3b + Na]+
(1.6%) < [3c + Na]+ (0.9%)}, which reveals that this steric
effect regulation process is kinetically controlled, but not
thermodynamically.
6-31 G* is 11.3 kcal mol–1, so this fragmentation process is
readily achievable by excitation at the high energies afforded
in the ion trap of a mass spectrometer. Whereas the overall
reaction is endothermic at about 13.2 kcal mol–1, this cleavage
process can be considered as the reverse of the synthetic path-
way used for all of these compounds. Finally, from a study of
fragmentation for different metal cations (H+, Li+, Na+, K+),
we see that the adduct of 3f with Na+ or K+ gives fragment
[f + M]+ with high relative intensity.
SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of this article.
Acknowledgements
We would like to acknowledge financial support from the
Major Program of National Natural Science Foundation of
China (No. 20732004) and the National Natural Science Foun-
dation of China (Nos. 20805037, 20972130 and 21075103). We
thank Prof. G. M. Blackburn for useful discussions and
suggestions.
Because the rearrangement behavior of 3f is assisted by a
sodium ion, the role of different ions such as H+, Li+, Na+, K+
was studied by ESI-MSn. It was found that sodium and
potassium ion adducts have a stronger ability to rearrange.
By contrast, there are no rearrangement ions of product
[3f + H]+ ([f + H]+ = 0) in the absence of metal ion assistance.
The likely reason is that a proton has weak coordination abil-
ity and so leads to a totally different fragmentation pattern
compared with our proposed five-membered ring proton
migration. It is thus clear that this rearrangement only occurs
in the presence of metal ions. We therefore modelled the
process to investigate how metal ions influence the rearrange-
ment process. The results show that all the MS rearrangement
reactions of species 3f occur easily with a low energy of
activation. The order of activation energy is [3f + Li]+
(9.5 kcal mol–1) < [3f + Na]+ (11.3 kcal mol–1) < [3f + K]+
(15.2 kcal mol–1). It is surprising that this sequence is the
oppositeoftheexperimentalresults:[3f +Li]+(9.3%)<[3f+Na]+
(68.0%) < [3f + K]+ (92.6%). It could be that this ion-induced
five-membered ring transition state is not the rate-determining
step in the metal-assisted rearrangement of compound 3f.
Indeed, this process could be controlled thermodynamically.
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Copyright © 2011 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2011, 25, 3314–3322