A novel rearrangement in electrospray ionization multistage tandem mass spectrometry of amino acid ester cyclohexyl phosphoramidates of AZT

Article abstract of DOI:10.1002/jms.834

Several amino acid ester cyclohexyl phosphoramidates of AZT as anti-HIV prodrugs were synthesized and investigated by electrospray ionization tandem mass spectrometry (ESI-MSⁿ). A novel methoxy group migration from the carbonyl group to the pho

Full text of DOI:10.1002/jms.834

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JOURNAL OF MASS SPECTROMETRY
J. Mass Spectrom. 2005; 40: 636–641
Published online 15 February 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.834
A novel rearrangement in electrospray ionization
multistage tandem mass spectrometry of amino acid
ester cyclohexyl phosphoramidates of AZT
Yi Chen,1,2 Yingwu Yin,2 Xiaobin Sun,2 Xiaohong Liu,2 Haiyan Wang2 and Yufen Zhao1,2∗
1
Key Laboratory of Chemical Biology, Chemistry Department, Zhengzhou University, Zhengzhou 450052, China
Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Ministry of Education, Department of Chemistry, Tsinghua University,
2
Beijing 100084, China
Received 21 May 2004; Accepted 30 December 2004
Several amino acid ester cyclohexyl phosphoramidates of AZT as anti-HIV prodrugs were synthesized
and investigated by electrospray ionization tandem mass spectrometry (ESI-MSn). A novel methoxy group
migration from the carbonyl group to the phosphoryl group was observed in ESI-MS2. This migration is
believed to be a general pathway for ions with a methyl ester moiety at the g-position to a phosphoric acid
moiety, which is assisted with metal ions such as Li+, Na+ and K+. Coordination between metal ions with
both the carbonyl oxygen and phosphoryl oxygen might be a key factor responsible for this migration.
Copyright  2005 John Wiley & Sons, Ltd.
KEYWORDS: rearrangement; methoxy migration; electrospray ionization; multistage tandem mass spectrometry; amino acid
ester cyclohexyl phosphoramidates of AZT
thiophosphoramidates of nucleosides and 30,50-dithymidine
INTRODUCTION
phosphoramidates. Novel rearrangement reactions, e.g.
30-Azido-20,30-deoxythymidine (AZT) is one of the most
carbonyl oxygen migration,7 benzyl migration,8 P—N to
effective inhibitors of HIV reverse transcriptase and has
P—O rearrangement9 and formamide extrusion10 have been
been approved for AIDS treatment.1 AZT itself is a cell
observed. In this paper, we report our investigations on
activation-dependent nucleoside analog. It does not show
several amino acid ester cyclohexyl phosphoramidates of
antiviral activity2 to terminate the viral DNA clone until it
AZT (1–5 in Scheme 1) by ESI-MS combined with tandem
is phosphorylated to the corresponding monophosphates,
techniques, in which a novel rearrangement of methoxy
diphosphates and triphosphates by cellular kinases. The
migration was discovered.
dependence on phosphorylation for activation became an
issue in cells where the nucleoside kinase activity is known
to be low or even lacking.3 In order to enhance membrane
penetration and to overcome this dependence on nucleoside
EXPERIMENTAL
Chemicals syntheses
kinase activation, a series of amino acid phosphoramidate
di- and triesters of nucleotides have been synthesized as
potential anti-HIV prodrugs.2
Electrospray ionization tandem mass spectrometry (ESI-
MSn) is a very powerful tool for structural determination
and has been widely used in chemistry, biochemistry and
pharmaceutical research.4–6 In our previous work, we used
this technique to study the organophosphorus derivatives
of nucleosides including d4T H-phosphonates, amino acid
General
All compounds synthesized were identified by 31P NMR, 1H
NMR, 13C NMR and ESI-MS.
Cyclohexyl 30-azido-30-deoxythymidine-50-yl
H-phosphonate (AZTPH)11
To a solution of PCl3 (10 mmol) in dichloromethane (10 ml)
was added AZT (1 mmol) (J&K Chemical, Beijing, China)
°
at ꢀ30 C under a nitrogen atmosphere and the solu-
tion was stirred for 1 h at this temperature and then
for 6 h at room temperature. The solvent and excess of
PCl3 were removed under reduced pressure. The residue
was dissolved in dichloromethane (10 ml). Cyclohexyl alco-
hol (2.5 mmol) in dichloromethane (5 ml) was then added
ŁCorrespondence to: Yufen Zhao, Key Laboratory of Bioorganic
Phosphorus Chemistry and Chemical Biology, Ministry of
Education, Department of Chemistry, Tsinghua University, Beijing
100084, China. E-mail: yinyw@tsinghua.edu.cn
Contract/grant sponsor: Chinese National Natural Science
Foundation; Contract/grant numbers: 20175026; 29632004;
39870415.
Contract/grant sponsor: Major State Basic Research Development
Program; Contract/grant number: 2003CB514126.
Contract/grant sponsor: Tsinghua University.
°
dropwise to the solution at 0 C, followed by triethy-
lamine (2 mmol). After 10 min, the solvent was removed
by rotary evaporation and the product was purified by
chromatography on silica gel using CH2Cl2 –MeOH (20 : 1)
Contract/grant sponsor: Zhengzhou University.
Copyright  2005 John Wiley & Sons, Ltd.

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Rearrangement of AZT cyclohexyl phosphoramidates in ESI-MS 637
Scheme 1. Structures of compounds AZT and 1–5.
as eluent. The final product AZTPH was obtained in 58%
yield.
RESULTS AND DISCUSSION
Compounds 1–5 displayed similar fragmentation patterns.
As a representative, the ESI-MSn positive ion mass spec-
tral fragmentation pattern of cyclohexyl methoxyisoleucinyl
phosphoramidate of AZT (cyclo-AZT-IleOMe) (3) is dis-
cussed in detail. Its spectra obtained by ESI-MS, ESI-MS2,
ESI-MS3 and ESI-MS4 are shown in Fig. 1.
Amino acid ester cyclohexyl phosphoramidates of AZT
To a solution of amino acid ester (0.4 mmol) and triethy-
lamine (0.5 ml) was added dropwise a solution of AZTPH
(0.3 mmol) in CH2Cl2 (10 ml), followed by chlorodiisopropy-
lamine cooled with an ice-bath. The solution was then stirred
°
for 10 h at 0 C. The solvent was removed by rotary evapo-
ration and the residue was purified by chromatography on
silica gel using CH2Cl2 –MeOH (20 : 1) as eluent.
×105
AZOM0025.d: +MS
[M+H]+
A
557.4
[M+Na]+
6
4
2
0
Mass spectrometry
Mass spectra were acquired using a Bruker ESQUIRE-LC
ion-trap mass spectrometer equipped with a gas nebulizer
probe, capable of analyzing ions up to m/z 6000. Nitrogen
was used as the drying gas at a flow-rate of 4 l minꢀ1. The
nebulizer pressure was 7 psi and the electrospray needle was
typically held at 4 kV. The heated capillary temperature was
702.4
274.5
475.4
414.6
×105
AZOM0026.d: +MS2(579.0)
1.0
B
497.2
0.8
0.6
0.4
0.2
°
300 C. The samples dissolved in methanol were ionized by
324.2
202.2
202.0
580.2
ESI and continuously infused into the ESI chamber at a flow-
rate of 0.16–0.25 ml hꢀ1 by a Cole-Parmer 74 900 syringe
pump. The scan range was generally from m/z 50 to 800.
Five scans were averaged for each spectrum. The selected
[M C H]C, [M C Li]C, [M C Na]C and [M C K]C ions were
analyzed by multistage tandem mass spectrometry (MSn).
The selected ion was first isolated and then fragmented
through collisions with helium to yield MSn spectra. The
fragmentation amplitude values were 0.5–1.0 V and the
fragmentation time was 40 ms.
The high-resolution mass spectral data for 3 were
recorded on a Bruker APEX II Fourier transform ion cyclotron
resonance (FTICR) MS instrument with an external ion source
and analyzed using liquid secondary ion (LSI) MS with a Cs
ion gun in the positive ion mode. The accelerating voltage
was 10 kV and the full scan was from m/z 50 to 700. The
sample was analyzed as a solution in glycerine mixed with
NaCl.
0.0
×104
AZOM0031.d: +MS3(579.0->248.0)
C
2.5
2.0
1.5
168.1
1.0
248.0
0.5
0.0
AZOM0033.d: +MS4(579.0->248.0->202.0)
1500
1250
1000
750
500
250
0
202.0
D
108.1
100
200
300
400
m/z
500
600
700
Figure 1. MS of cyclo-AZT-IleOMe (3). (A) Full MS; (B) MS2 of
[M C Na]C at m/z 579; (C) MS3 of the ion at m/z 248; (D) MS4 of
the ion at m/z 202.
Copyright  2005 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2005; 40: 636–641

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638
Y. Chen et al.
Scheme 2. Positive ESI-MS2 fragmentation pathway of [M C Na]C of cyclo-AZT-IleOMe (3).
Table 1. High-resolution mass spectral data for the main ions of 3a
Ion species
Theoretical mass (m/z)
Measured mass (m/z)
Relative error (ppm)
[M C Na]C
579.2303
497.1520
352.0417
324.0356
248.0658
202.0603
579.2310
497.1539
352.0423
324.0362
248.0657
202.0606
1.2
3.8
1.7
1.8
0.4
1.4
[M ꢀ cyclohexene C Na]C
[M ꢀ cyclohexene ꢀ IleOMe C Na]C
[M ꢀ cyclohexene ꢀ IleOMe ꢀ N2 C Na]C
[M ꢀ cyclohexene ꢀ 249 C Na]C
Rearrangement ion
a Obtained with LSI-MS in the positive ion mode.
In ESI-MS, [M C H]C is predominant, followed by
[M C Na]C. A possible fragmentation pathway of [M C Na]C
is shown in Scheme 2. The fragment ion at m/z 497
was produced by loss of a molecule of cyclohexene, and
that at m/z 469 with an additional loss of N2, a similar
fragmentation pattern to that observed previously for other
azido-containing compounds.12 The ions at m/z 352, 324, 309
and 198 arose from the [M C Na]C ion by loss of a molecule
of amino acid ester and cyclohexene. The other ions were
also as expected and were further identified by ESI-MS3.
However, there was an unexpected ion at m/z 202, which
was found not only in ESI-MS2 (Fig. 1(B)), but also in ESI-MS3
(Fig. 1(C)). According to ESI-MSn, this ion at m/z 202 comes
from the ion at m/z 248, and can be further fragmented to
the ion at m/z 108, the NaC adduct ion of isopentalyimine
(Fig. 1(D)). High-resolution FTICR-MS of 3 indicated that the
exact mass of the ion at m/z 202 was 202.0606, corresponding
to the formula C6H14O3NPNa (calculated 202.0603, relative
error 1.4 ppm) (Table 1).
A possible structure of the ion at m/z 202 is proposed in
Scheme 3 (VI or VII). The formation of this ion could possibly
involve a sodium ion-mediated rearrangement pathway
(Scheme 3). Starting from the NaC adduct ion I at m/z 248, the
sodiumionmightbecloselycoordinatedwiththephosphoryl
Copyright  2005 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2005; 40: 636–641

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Rearrangement of AZT cyclohexyl phosphoramidates in ESI-MS 639
Scheme 3. Possible rearrangement mechanism for the ion at m/z 202.
Scheme 4. Structures of compounds 6–9.
oxygen and carbonyl oxygen to form an intermediate II
relationship made it possible to form the five-membered
cyclic anhydride intermediate/transition state III. Hence
we would predict that any phosphorus compound that
can produce an ion with such a general structural feature
would be subjected to a similar methoxy migration; other
structures with different relative positions of the ester
moiety to phosphorus would have less opportunity for
methoxy migration. In order to test this hypothesis, four N-
diisopropyloxyphosphoryl derivatives of amino acids and
peptides (6–9, Scheme 4) were synthesized and analyzed
by ESI-MS2. Just as we predicted, methoxy migration was
observed for 6 and 7, which have ester moieties at the ꢀ-
position to phosphorus, but was not observed for 8 and 9,
whose ester moiety was located three atoms away from the
ꢀ-position (Table 2).
with a seven-membered ring. Then the oxygen in the
nucleophilic methoxy group could attack the phosphorus via
a five-membered cyclic phosphoric–carboxylic anhydride
intermediate/transition state III13 to form IV, followed by
a carbon–carbon bond cleavage to expel a molecule of
CO to give V. Loss of a molecule of H2O provided the
observed rearrangement ion at m/z 202. In this process,
a methoxy group migrates from the ester moiety on to the
phosphorus. Although the migration of a methoxy group has
been observed before, for example in the chemical ionization
mass spectra of olefinic methyl esters,14 it seems that they
follow different rearrangement pathways.
According to the proposed mechanism in Scheme 3,
the key structural feature of the NaC adduct ion prone
to methoxy migration is an ester moiety (methyl ester in
our case) at the ꢀ-position to phosphorus. Such a structural
It is very interesting that this rearrangement occurs only
for the NaC adduct ion [M C Na]C and not for the protonated
Copyright  2005 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2005; 40: 636–641