Ionic probe attachment ionization mass spectrometry

Article abstract of DOI:10.1016/j.tetlet.2009.09.021

A new ionization method that uses metal-complex-based ionization probes containing the 2,6-bis(oxazolinyl)pyridine (pybox) ligand is presented. This method was proven to effectively ionize large complex molecules, including biomolecules. The preparation o

Full text of DOI:10.1016/j.tetlet.2009.09.021

Tetrahedron Letters 50 (2009) 6252–6255
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Ionic probe attachment ionization mass spectrometry
*
*
Fumihiro Ito , Tomoko Nakamura, Satoko Yorita, Hiroshi Danjo, Kentaro Yamaguchi
Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, 1314-1 Shido, Sanuki, Kagawa 769-2193, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 July 2009
Revised 1 September 2009
Accepted 3 September 2009
Available online 9 September 2009
A new ionization method that uses metal-complex-based ionization probes containing the 2,6-bis(oxaz-
olinyl)pyridine (pybox) ligand is presented. This method was proven to effectively ionize large complex
molecules, including biomolecules. The preparation of the charged probes and their application to the
ionization of biomolecules using cold-spray ionization mass spectrometry are shown.
Ó 2009 Elsevier Ltd. All rights reserved.
The continuous development of the soft ionization technique is
thought to have sparked the advancement of mass spectrometry
(MS). Since electron ionization (EI), the first practical ionization
method used in chemistry, was coined, remarkable progress has
been made in this area. Recent soft ionization technique has made
it possible to analyze such biomacromolecules as proteins.1 The
technique is based on electrospray ionization (ESI)2 and matrix-as-
sisted laser desorption ionization (MALDI).3 During analysis under
soft ionization conditions, the higher order structure of a biomac-
romolecule could be preserved. Therefore, the elucidation of pri-
mary structures as well as higher order structures4 and their
dynamics has become possible, and post-translational modifica-
tion analysis has become a reality.
However, problems that hinder the reliable and reproducible
ionization of complex large biomolecules by these methods still re-
main, because of the often observed ambiguous protonation during
ESI-MS analysis. The protonation seems to be more difficult in the
case of cold spray ionization (CSI), a variant of ESI method operat-
ing under low temperature to ionize labile organic species.5 There-
fore, an effective adjustable-charge-state ionization technique6 is
required for these molecules. We have developed a new method
called ‘ionic probe attachment ionization.’ We have designed ionic
probes that can donate plural charges contained in the metal
charged site of the probe molecule to the target compound. As re-
gards previous studies of instrumental analysis using chemical
probes, the caged lanthanide NMR probe by Ubbink and co-work-
ers7 and the ruthenium(II) bisterpyridine complexes of an electron
spectroscopic probe by Thordarson and co-workers8 are known.
The former is used as a relaxation reagent and/or a pseudo con-
tact-shift reagent to obtain long-distance restraints for solving
solution structures. In the latter, the probe is introduced to cyto-
chrome c to observe photoelectron transfer between the probe do-
nor and the cytochrome c acceptor. Furthermore, ruthenium(II)
bisterpyridine complex was used in various related research
areas.9 In this Letter, we present new metal-complex-based ioniza-
tion probes containing the 2,6-bis(oxazolinyl)pyridine (pybox) li-
gand, which can ionize target molecules by donating plural
charges effectively and reliably. We also describe the application
of the charged probes to the ionization of biomolecules by cold-
spray ionization mass spectrometry (CSI-MS).5
The probe comprises three functional parts: a charged site, a
linker, and an anchoring site (Fig. 1), similar to biotin reagents.10
The pybox ligand is used to enclose the charged metal in the
charged site, although the terpyridine-type ligand also seems to
be a good candidate for this site. Compared with the terpyridine-
type ligand, the pybox ligand has structural diversity that can be
easily controlled by choosing the structure of the amino alcohol
used as the starting material. In addition, complexation with metal
cations takes place with the pybox ligand under mild conditions. A
haloalkane moiety having variable alkyl chain length was used as a
linker.11 Two compounds, succinimide (NHS) and maleimide (Mal),
were used as the anchoring site to bind the probe to the target mol-
ecule. The two probes used in this study, NHS-tetramethylpybox
(NHS-TMpybox) and Mal-tetramethylpybox (Mal-TMpybox), were
prepared as follows.12 4-Chloropybox 5 was prepared from chel-
idamic acid (1) using a previously reported method for pybox syn-
thesis.13 Nucleophilic aromatic substitution of
5 with BnOH
afforded 6 in 94% yield. 4-Hydroxypybox 7 was obtained by remov-
ing the benzyl group on 6 in 68% yield. Ethyl ester 8 was furnished
from 6 in 86% yield by alkylation with ethyl 4-bromobutyrate. Fi-
nally, desired NHS-TMpybox 10 was obtained by hydrolysis of 8
n+
CH2
O
O
n+
O
CH2
OR
O
N
M
X
M
N
m
m
O
linker
O
O
O
charged site
multi-charged ionization probe
* Corresponding authors. Tel.: +81 87 894 5111; fax: +81 87 894 0181.
E-mail addresses: h-itoh@kph.bunri-u.ac.jp (F. Ito), yamaguchi@kph.bunri-
u.ac.jp (K. Yamaguchi).
anchoring site
Figure 1. Constitution of the charged probe.
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.09.021

F. Ito et al. / Tetrahedron Letters 50 (2009) 6252–6255
6253
X
N
Cl
Cl
H2N
OH
OH
SOCl2
DMF
X
X
NaH
H
N
H
N
NEt3
O
O
Cl
Cl
N
N
HO2C
N
CO2H reflux
THF
reflux
93%
CH2Cl2
rt
N
N
O
O
O
O
1
2
3: X = OH
SOCl2, reflux
3 steps 53%
BnOH
NaH
5: X = Cl
6: X = Bn
O
4: X = Cl
THF
rt, 94%
O
O
N
3 CO2X
O
N
N
NHS
EDCI
O
O
Br
CO2Et
3
KH2PO4
O
O
O
O
K2CO3
N
N
N
N
DMF, rt
CH2Cl2, rt
2 steps
76%
OH
2 steps from 6
8: X = Et
H2
KOH
86%
NHS-TMpybox 10
Pd/C
MeOH, rt
9: X = K
O
O
N
MeOH
rt
O
O
O
N
N
N
TsO
7
68%
3
3 N
O
N
O
N
N
O
O
O
O
O
O
K2CO3
O
N
O
O
DMF, rt
96%
toluene
reflux
99%
N
N
N
11
Mal-TMpybox 12
H2N
OH
NEt3
X
X
H
NaH
La(acac)3
X
X
O
N
O
H
N
N
N
O
O
N
CH2Cl2
rt
N
N
THF
rt
THF
rt
N
N
O
O
La3+
N
O
O
SOCl2
13: X = OH
3X
94%
17
15: X = OH
DMF
SOCl2, reflux
3 steps 56%
18: X = acac
reflux
14: X = Cl
NH4PF6
16: X = Cl
2 steps quant.
19: X = acac + 2PF6
Scheme 1. Synthesis of NHS-TMpybox 10 and Mal-TMpybox 12.
and conversion of potassium salt 9 into NHS ester 10 in two steps
in 76% yield. Mal-TMpybox 12 was prepared by coupling 7 with
tosylate14 and subsequent retro Diels–Alder reaction of 11
(Scheme 1). As previously described,13 pybox 17 was prepared
from dipicolinic acid (13). We chose lanthanum(III) ion as the
charged center because of its robustness to oxidation/reduction
and low isotope abundance ratio (138La:139La = 0.09:99.91). Treat-
ment of pybox 17 with La(acac)3 (acac: acetylacetonato) gave
La(III) complex 18. Finally, 19 was obtained by counter anion ex-
trile solution of metal complex 19 was added. The other method
involved the direct mixing of the acetonitrile solution of 19 with
10 or 12-binding crude compound that was freeze-dried without
further purification (Scheme 2, method B). Metal complex 19 was
also added to obtain triply charged metal cation probes. Then,
the synthesized probes were applied to the CSI-MS measurement17
of several amino acids and peptides.
Probe 20-binding lysine prepared by method A exhibited a doubly
charged ion peak [M+acacꢀ]2+ in the CSI mass spectrum (Fig. 3a). The
expected triply charged ion peak was not detected because the
acacꢀ moietycanceledonechargeofthision.IntheCSImassspectrum
of the molecule prepared by method B, [M+acacꢀ+NHSꢀ]2+ ion peak
wasdetectedinadditiontothedoublychargedionpeakthatappeared
in the mass spectrum of the molecule prepared by method A (Fig. 3b).
Again, NHSꢀ canceled one of the three positive charges of this ion.
In the case of charged probe 21-binding cysteine, a doubly
charged ion peak involving acac as the counter anion was detected,
similar to probe 20-binding lysine. The ion intensity in the CSI
mass spectrum of the molecule prepared by method A and B were
both almost identical (Fig. 4). Method B, which is simpler than
method A, is thought to be more favorable for the analysis of com-
plex large biomolecules.
The NHS-TMpybox-charged probe in method A ionized two
pentapeptides, TTKTT and KTTTK. The CSI mass spectra of these
peptides are shown in Figure 5. Two probes were introduced to
KTTTK to give a quadruply charged ion peak, while single probe
attachment to TTKTT yielded a doubly charged ion peak. The
charge of the target molecule could be controlled by the number
of probes attached.
This charged probe method was applied to the ionization of
three bioactive peptides: glutathione (GSH), arginine vasopressin
(AVP), and somatostatin (SS). Probe 21-binding GSH prepared by
method A was analyzed and the CSI mass spectrum of this com-
change of acacꢀ with PF6 (Scheme 1).15 Although we attempted
ꢀ
to replace all the three acacꢀ with PF6ꢀ, only two of them could
be exchanged to give La(III) complex 19. Then, 19 was mixed with
10 and 12 to give respective charged probes 20 and 21 in which the
metal cation was enclosed by two pybox ligands to prevent coordi-
nation with the counter anion, which promote ionic dissociation
(Fig. 2).16 However, as the yield of charged probe 20 was low by
elimination of NHS and purification of 20 and 21 was difficult, their
use as probes remains a formidable task.
Then, two methods to introduce the probe to the target mole-
cule were examined. One involved the addition of a DMSO solution
of previously prepared NHS-TMpybox 10 or Mal-TMpybox 12 to
the phosphate-buffered standard solution of the target molecule,
followed by purification (Scheme 2, method A). Then, the acetoni-
O
O
N
N
N
N
O
N
N
O
3-
N
3-
(2PF6 + acac)
N
N
N
N
(2PF6 + acac)
O
La3+
La3+
O
O
O
O
N
O
N
O
O
O
N
O
O
O
20
21
Figure 2. Two charged probes 20 and 21.

6254
F. Ito et al. / Tetrahedron Letters 50 (2009) 6252–6255
n+
n+
H2N
H2N
Method A
ligand
ligand
purification
H2N
CO2H
pure
CO2H
10 or 12
NHS
H2N
NHS
H2N
CO2H
Method B
freeze-dry
NHS
CO2H
crude
NHS
CO2H
NHS
NHS
Scheme 2. Introduction of multiply charged ion probe.
Figure 3. CSI mass spectra of probe 20-binding lysine prepared by methods A (a)
and B (b).
Figure 5. CSI mass spectra of probe 20—(a) TTKTT and (b) KTTTK prepared by
method A.
Figure 4. CSI mass spectra of probe 21-binding cysteine prepared by methods A.
Figure 6. CSI mass spectra of probe 21-binding GSH prepared by methods A.
pound is shown in Figure 6. A deprotonated doubly charged molec-
ular ion peak was clearly observed, the intensity of which was
three times higher than that of the ion peak observed in the mass
spectrum of unmodified native GSH. Deprotonation occurred in
one of the two carboxylic groups that had a tendency to release
their protons.
In the case of AVP modified by charged probe 21 via method B,
three major ion peaks were observed, as shown in Figure 7. Two
reaction sites, arginine and glycine, of this peptide were available. A
doubly charged ion peak based on NHS-TMpybox-AVP (M), in which
the probe bound to arginine or glycine in AVP, was observed at m/z
947 ([M+acacꢀ]2+). Interestingly, triply charged ion peaks based on
2(NHS-TMpybox)-AVP (M0) at m/z 955 ([M0+2acacꢀ+NHSꢀ+MeCN]3+
)
and m/z 967 ([M0+2acacꢀ+2MeCN]3+) were also observed. The
observed ion intensities were almost identical to those in the mass
spectrum of unmodified native AVP. However, the charge-weight
ratio was clearly reduced by the multiple charge formation.
Figure 7. CSI mass spectrum of probe 21-binding AVP prepared by method B.
Finally, SS was analyzed by attaching charged probe 20. Two
attachment sites were available in SS because this peptide con-
tained two lysines. Doubly charged ion peaks due to Mal-TMpy-
box-SS with some adducts were observed (Fig. 8). The four major

F. Ito et al. / Tetrahedron Letters 50 (2009) 6252–6255
6255
References and notes
1. Siuzdak, G. The Expanding Role of Mass Spectrometry in Biotechnology; MCC
Press: San Diego, 2003.
2. Karas, M.; Bachmann, D.; Hillenkamp, F. Anal. Chem. 1985, 57, 2935.
3. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whithouse, C. M. Science 1989,
246, 64.
4. Kaltashov, I. A.; Abzalimov, R. R. J. Am. Soc. Mass Spectrom. 2008, 19, 1239.
5. Yamaguchi, K. J. Mass Spectrom. 2003, 38, 473.
6. Maki, T.; Ishida, K. J. Org. Chem. 2007, 72, 6427; Che, F.-Y.; Fricker, L. D. J. Mass
Spectrom. 2005, 40, 238; Mirzaei, H.; Regnier, F. Anal. Chem. 2006, 78, 4175.
7. Vlasie, M. D.; Comuzzi, C.; van den Nieuwendijk, A. M. C. H.; Prudêncio, M.;
Overhand, M.; Ubbink, M. Chem. Eur. J. 2007, 13, 1715; Keizers, P. H. J.; Desreux,
J. F.; Overhand, M.; Ubbink, M. J. Am. Chem. Soc. 2007, 129, 9292.
8. Peterson, J. R.; Smith, T. A.; Thordarson, P. Chem. Commun. 2007, 1899.
9. Kaneko, M.; Masui, R.; Ake, K.; Kousumi, Y.; Kuramitsu, S.; Yamaguchi, M.;
Kuyama, H.; Ando, E.; Norioka, S.; Nakazawa, T.; Okamura, T.; Yamamoto, H.;
Ueyama, N. J. Proteome Res. 2004, 3, 983; Ito, A.; Okamura, T.; Yamamoto, H.;
Ueyama, N.; Yamaguchi, M.; Kuyama, H.; Ando, E.; Tsunasawa, S.; Ake, K.;
Masui, R.; Kuramitsu, S.; Nakazawa, T.; Norioka, S. Rapid Commun. Mass
Spectrom. 2007, 21, 2647.
10. Lin, P.-C.; Ueng, S.-H.; Yu, S.-C.; Jan, M.-D.; Adak, A.-K.; Yu, C.-C.; Lin, C.-C. Org.
Lett. 2007, 9, 2131.
11. Three different lengths of the alkyl chain –[–CH2–]n– were prepared. Length
n = 3 was used in this experiment because it exhibited the highest yield (56%)
compared to the other lengths (n = 4: 39% and n = 5: 25%).
12. An increase in solubility due to dimethyl substitution into the dihydrooxazole
ring of both pybox probes in organic solvent was noted compared to
unsubstituted or monomethyl substituted compounds.
13. Vermonden, T.; Branowska, D.; Marcelis, A. T. M.; Sudhölter, E. J. R. Tetrahedron
2003, 59, 5039.
14. Tang, F. H. Y.; Wang, M. Y. S.; Zhu, Y. L. Adv. Funct. Mater. 2007, 17, 996.
15. Although other counter ions including BF4 were tested, PF6 seems to be
favorable for the CSI process based on solvation, because the low coordination
ability of this anion may promote dissociation. Sakamoto, S.; Fujita, M.; Kim, K.;
Yamaguchi, K. Tetrahedron 2000, 56, 955.
16. Unpublished data. Though a doubly charged ion was detected in the case of 1:2
(cation to pybox ligand ratio) complex, single charged ions containing solvents
and counter ions were detected in the case of 1:1 complex.
Figure 8. CSI mass spectrum of probe 20-binding SS prepared by method B.
ion peaks were assigned as [M+acacꢀ]2+ (m/z 1225), [M+acacꢀ+
H2O]2+ (m/z 1234), [M+acacꢀ+MeCN+H2O]2+ (m/z 1253), and
[M+acacꢀ+MeCN+2H2O]2+ (m/z 1262). These results confirm that
the newly developed charged probes can ionize biomolecules.18
In the ionization of pentapeptide by means of charged probe
attachment, quadruply charged ions produced by the attached
two probes were observed. This method reduced the charge-
weight ratio by donating a charge using the probe.
In conclusion, an effective and reliable ionization method called
ionic probe attachment ionization was presented. Two charged
probes, Mal-TMpybox 10 and NHS-TMpybox 12, were prepared
and biomolecules including peptides were ionized using these
probe attachments. This method was proven to be effective for ion-
izing various large complex molecules, including biomolecules.
Using this method, multiply charged biomolecules were clearly ob-
served by deducing the charge-weight ratio from the CSI mass
spectrum. Further application of the charged probes is in progress.
17. CSI mass spectra were recorded on a JEOL JMS-T100LC mass spectrometer
equipped with
a cold-spray ion source. This setup makes it possible to
conduct measurements at low temperatures. Measurement conditions were
as follows. sprayer temperature: ꢀ20 to 20 °C; needle voltage: +3030 V;
ring lens voltage: +15 V; orifice 1 voltage: +150 V; orifice 2 voltage: +15 V;
concd ca. 1 mM.
Supplementary data
Supplementary data (synthetic procedures for 3 to 19, charac-
terization data of 3 to 17 and modification method A and B)
associated with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2009.09.021.
18. This method was also applied to the ionization of large peptides. Multiply
charged molecular ion peaks of high charge states were clearly observed in the
case of nocicptine. The results will be published elsewhere.