15N-labeled ionic probe attachment mass spectrometry of carbon clusters

Article abstract of DOI:10.1039/c0ob00887g

An ionization method that uses metal-complex-based ionization probes, malonic acid 3-[2,6-bis(4,4-dimethyloxazolin-2-yl)pyridin-4-yloxy]propyl ethyl ester (EM-TMpybox) and potassium N-{3-[2,6-bis(4,4-dimethyloxazolin-2-yl) pyridine-4-yloxy]propyl} aminoac

Full text of DOI:10.1039/c0ob00887g

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PAPER
15N-labeled ionic probe attachment mass spectrometry of carbon clusters†
Fumihiro Ito and Kentaro Yamaguchi*
Received 1st December 2010, Accepted 3rd February 2011
DOI: 10.1039/c0ob00887g
An ionization method that uses metal-complex-based ionization probes, malonic acid
3-[2,6-bis(4,4-dimethyloxazolin-2-yl)pyridin-4-yloxy]propyl ethyl ester (EM-TMpybox) and potassium
N-{3-[2,6-bis(4,4-dimethyloxazolin-2-yl)pyridine-4-yloxy]propyl} aminoacetate (Sar-TMpybox), was
developed for isotope ratio analysis and the effective ionization of unsubstituted carbon clusters. The
preparation of Sar-TMpybox and EM-TMpybox and their applications in cold-spray ionization mass
spectrometry are reported. A probe applicable to a substituted fullerene is also demonstrated.
MS measurement in carbon cluster chemistry. By comparing
Introduction
the isotopic shifts of 15N-labeled and non-labeled fullerenes, the
Mass spectrometry (MS) is an essential tool for characterizing
precise elucidation of the complex structure will be realized. This
is the first example of the observation of 15N labeling isotopic
shift and ionization using an MS probe for carbon clusters. Our
probe is comprised of three functional parts: a charged site, a
linker, and an anchoring site (Fig. 1), as reported previously.10 We
attempted to modify fullerenes by means of the Prato reaction11
and the Bingel–Hirsch reaction.12 In the Prato reaction, an
unstable azomethine ylide obtained from sarcosine and aldehyde
reacts with the carbon clusters. In the Bingel–Hirsch reaction, the
enolate of diethyl bromomalonate reacts with the carbon clusters.
Therefore, we designed two new probes that have diethyl malonate
(EM-TMpybox 1) and sarcosinate (Sar-TMpybox 2) as anchoring
sites (Fig. 1). In this paper, we describe the synthesis of the new
probes and their application to carbon clusters and the analysis
of their isotopic shifts using 14N– and 15N–3 in soft ionization
conditions by means of cold-spray ionization mass spectrometry
(CSI-MS).13
various compounds. Not only is MS used for the analysis of
molecular weights, it is also one of the most sensitive detection
methods for biological, chemical, pharmaceutical, and environ-
mental substances. With regard to recent advances in ionization
technology for MS, soft ionization, such as electrospray ionization
(ESI),1 matrix-assisted laser desorption/ionization (MALDI),2
and chemically modified ionization using ammonium salts, the
so-called “MS probe” or “tag” has been investigated. The MS
probe is basically used to increase the relative abundance of ions
formed from a compound3 and for isotope labeling4 in the analysis
of higher-order structures of biomolecules. Previously, we have
reported MS probes that can donate plural charges contained
in their metal charged sites to the target compound in order to
analyze biomolecules. Furthermore, the isotopic shift was detected
easily by the probes using 14N- and 15N-pybox–La complexes
3 (14N–3 and 15N–3).5 Recently, the synthesis and analysis of
complex fullerene derivatives have been widely conducted.6 MS
measurement is often troublesome because large and stable carbon
clusters are usually difficult to ionize by soft ionization techniques.
As regards previous observations of the ionization of carbon
clusters by MS, limited reports, such as the electron ionization
(EI) MS of a C60/C70 mixture by Luffer and Schram7 and the
high-energy collision-induced dissociation (CID) of singly and
multiply charged C60 and C70 by Young et al.8 and Doyle and
Ross,9 are known. Therefore, we planned to analyze carbon
clusters with our ionic probe, which would make it possible to
adopt soft ionization in order to establish facile and effective
Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima
Bunri University, 1314-1 Shido, Sanuki, Kagawa, 769-2193, Japan. E-mail:
h-itoh@kph.bunri-u.ac.jp, yamaguchi@kph.bunri-u.ac.jp; Fax: +81 87 894
0181; Tel: +81 87 894 5111
† Electronic supplementary information (ESI) available: Multiply charged
ionization of fullerenes C60 and C70 using Sar-TMpybox 2, CSI mass
spectra of fullerenes having some probes 2 and 1H and 13C NMR spectra
of 1, 2, 6–10, and 12. See DOI: 10.1039/c0ob00887g
Fig. 1 The construction of the ionic probe.
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Scheme 1 The synthesis of EM-TMpybox 1 and Sar-TMpybox 2.
of probe 1, benzaldehyde,14 and fullerene in toluene was refluxed
for 24 h. After the reaction mixture was filtered, toluene was
removed in vacuo. The obtained residue was washed with Et2O.
The mixture of fullerene derivative and 14N– or 15N–3 was stirred in
MeCN. This solution diluted with MeCN was then applied to CSI-
MS measurement.15 Using the excess probe 2 in the modification
reaction, all of the fullerenes should react.
Probe 2 was reacted with C60 and singly charged ions of C60
having 1–6 probes 2 attached were detected (see ESI†).16 Multiply
charged ions [C60 + (14N–3 + 2) + acac-]2+ (m/z 812), [C60 + 2(14N–
3 + 2) + 2acac-]4+ (m/z 632), and [C60 + 3(14N–3 + 2) + 3acac-]6+
(m/z 572) were detected by the formation of charged sites coupled
with 14N–3 including La3+ (Fig. 3a). One of the three ions at the
charged site was cancelled out by an acac-. In the case of 15N–3, C60,
to which the 15N-labeled probes were attached, this was detected
as multiply charged ions [C60 + (15N–3 + 2) + acac-]2+ (m/z 813),
[C60 + 2(15N–3 + 2) + 2acac-]4+ (m/z 633), and [C60 + 3(14N–3 + 2) +
3acac-]6+ (m/z 573), as observed in the non-labeled ones with a
definite isotopic shift of m/z 1 (Fig. 3b). Compared with the other
Results and discussion
MS probes 1 and 2 were synthesized as follows. EM-TMpybox
was prepared by nucleophilic aromatic substitution of 4-Cl-pybox
4 with 1,3-propanediol and conversion of the hydroxyl group on 6
into ethyl malonate with ethyl 3-chloro-3-oxo-propionate. Alkyla-
tion of 4-hydroxypybox 5 with 1,3-dibromopropane afforded the
bromide 7 in 89% yield. Amine 9 was obtained by conversion of the
bromide into benzylamine 8 with BnNH2, alkylation into 9, and
removal of the benzyl group. Finally, the desired Sar-TMpybox 2
was obtained by hydrolysis of methyl ester 10 (Scheme 1). At first,
EM-TMpybox 1 was attached to fullerene C60 in the presence
of DBU and CBr4 at room temperature. Probe 1 was reacted
with C60 and singly charged ions of C60 having 1–4 probes 1
attached were detected (Fig. 2). However, the excess amount of
DBU prevented the formation of the La complex (3 + 1) with
the desired charges and it was difficult to remove the DBU salt.
Next, Sar-TMpybox 2 was attached to fullerenes C60 and C70. To
promote the reaction of the probe with the fullerenes, a suspension
Fig. 2 The CSI mass spectrum of 1 binding fullerene C60.
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Fig. 3 CSI mass spectra of a) (14N–3 + 2)–C60 and b) (15N–3 + 2)–C60.
multiply charged ions, [C60 + 3(14N–3 + 2) + 3acac-]6+ was weak
because the relative amount of the fullerenes is definitely small in
the case of the three probes that are attached. Although the C60
derivatives with more than three probes 2 were observed, multiply
charged ions were not detected for these species because of the
difficulty in forming the charged site due to the steric hinderance
and/or the electronic repulsion of the pybox–La complexes.
In the case of C70, ions of C70 with 1–5 probes 2 attached were
(m/z 593) with a definite isotopic shift of m/z 1 compared to the
non-labeled ones (Fig. 4b).
Finally, this method was adopted for (1,2-methano-fullerene
C60)-61 carboxylic acid17 as a substituted C60. As complex fullerene
derivatives possessing various functional groups have been pre-
pared only quite recently,6 we examined the ionic probe attachment
ionization of commercially available fullerene derivatives. In the
case of fullerenes with a carboxyl group as the functional group,
BrAc-TMpybox 11 was attached to the carboxyl group by the
method previously reported by us.10 The probe (14N–3 + 11)-bound
C60 derivative was detected as a doubly charged ion [M + acac-]2+
(m/z 817) (Fig. 5a). In the case of the (15N–3 + 11)-C60 derivative,
a doubly charged ion [M + acac-]2+ (m/z 818) was observed as well
(Fig. 5b). The isotopic shift of m/z 1 was also observed in these
spectra.
detected (see ESI†). Similar to C60, multiply charged ions [C70
+
(14N–3 + 2) + acac-]2+ (m/z 872), [C70 + 2(14N–3 + 2) + 2acac-]4+
(m/z 662), and [C70 + 3(14N–3 + 2) + 3acac-]6+ (m/z 592) were
detected by the formation of the charged site coupled with 14N–3
including La3+ (Fig. 4a). In the case of 15N–3, (15N–3 + 2)–C70 was
detected as [C70 + (15N–3 + 2) + acac-]2+ (m/z 873), [C70 + 2(15N–
3+2) + 2acac-]4+ (m/z 663), and [C70 + 3(15N–3 + 2) + 3acac-]6+
Fig. 4 CSI mass spectra of a) (14N–3 + 2)–C70 and b) (15N–3 + 2)–C70.
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350 mesh, No. HU80502) was used. All anhydrous solvents were
purchased from Wako Pure Chemical. Fullerenes C60, C70, and
(1,2-methano-fullerene C60)-61-carboxylic acid were purchased
from Aldrich.
Synthesis of 4-(3-bromo-prop-1-yloxy)-2,6-bis(4,4-
dimethyloxazolin-2-yl)pyridine (7)
Under N2, after a suspension of 5 (550 mg, 1.90 mM) and
powdered KOH (345 mg, 6.16 mM) in DMF (5 mL) was stirred
for 10 min at room temperature, 1,3-dibromopropane (1.2 mL,
11.8 mM) was added. After this was stirred at room temperature
for 2 h, the mixture was poured into H2O. The mixture was
extracted with ethyl acetate and the organic layer was washed
with H2O and brine, dried over Na2SO4, and evaporated in vacuo.
The residue was purified by column chromatography (n-hexane–
ethyl acetate = 1 : 1) to give 7 (692 mg, 89%) as colorless prisms. mp
132–133 ◦C. IR (ATR, cm-1): 1641. 1H NMR (400 MHz) d: 1.39
(12H, s), 2.34 (2H, quin., J = 6.0), 3.58 (2H, t, J = 6.0), 4.21 (4H,
s), 4.28 (2H, t, J = 6.0 Hz), 7.71 (2H, s). 13C NMR (100 MHz) d:
28.5, 29.3, 31.9, 66.0, 68.0, 79.9, 112.2, 148.7, 148.7, 161.0, 165.7.
HRCSIMS m/z 432.0932 (calcd for C18H2479BrN3NaO3: 432.0899)
and 434.0880 (calcd for C18H2481BrN3NaO3: 434.0878). Anal. calcd
for C18H24BrN3O3: C, 52.69; H, 5.90; N, 10.24. Found: C, 52.69;
H, 5.99; N, 10.20.
Fig. 5 CSI mass spectra of a) the (14N–3 + 11)-C60 derivative and b) the
(15N-3 + 11)-C60 derivative.
Synthesis of N-benzyl-3-[2,6-bis(4,4-dimethyloxazolin-2-
yl)pyridine-4-yloxy]propyl-1-amine (8)
Conclusions
The preparation of a new ionic probe, Sar-TMpybox 2, and the
development of an effective and reliable ionization method using
this probe, together with the isotopic labeling of unsubstituted
fullerenes C60 and C70, were reported. By using the probe BrAc-
TMpybox 11, 14N– and 15N–3 isotopic shifts and multiply charged
ions were clearly observed in CSI-MS, in the case of the substituted
fullerene, (1,2-methano-fullerene C60)-61 carboxylic acid. This
method was proven to be effective for 15N-labeled multiply charged
ionization of stable fullerenes. We found that halving the number
of the observed charge state indicates the number of the attached
probe. The doubly charged ion is preferably observed in the pybox
complex formed from the probe–complex 3 species by taking a
single counter anion. Further application of the charged probes
and the ionization of more complex large carbon clusters, such as
carbon nanotubes, are under consideration.
Under N2, a suspension of 7 (1.16 g, 2.82 mM), K2CO3 (2.24
g, 16.2 mM), and BnNH2 (0.80 mL, 7.32 mM) in DMF (5 mL)
was stirred at room temperature for 24 h. This reaction mixture
was poured into H2O and extracted with ethyl acetate. The
organic layer was washed with H2O and brine, dried over Na2SO4,
and evaporated in vacuo. The residue was purified by column
chromatography (n-hexane : acetone = 6 : 1) to give 8 (1.00 g, 82%)
1
as a colorless oil. IR (ATR, cm-1): 3446, 1642. H NMR (400
MHz) d: 1.39 (12H, s), 1.99 (2H, quin., J = 6.5 Hz), 2.80 (2H, t,
J = 6.5 Hz), 4.20 (4H, s), 4.21 (2H, t, J = 6.5 Hz), 7.22–7.32 (5 H,
m, overlapped with CHCl3), 7.69 (2H, s). 13C NMR (100 MHz) d:
28.5, 29.5, 45.8, 54.1, 67.1, 68.0, 79.8, 112.3, 127.1, 128.2, 128.5,
140.4, 148.6, 161.1, 166.0. HRCSIMS m/z 437.2543 (calcd for
C25H33N4O3: 437.2553).
Synthesis of methyl N-benzyl-N-{3-[2,6-bis(4,4-dimethyloxazolin-
2-yl)pyridine-4-yloxy]prop-1-yl}aminoacetate (9)
Experimental
Under N2, a suspension of 8 (392 mg, 0.90 mM), K2CO3 (623 mg,
4.50 mM), and methyl a-bromoacetate (0.17 mL, 1.80 mM) in
DMF (3 mL) was stirred at room temperature for 12 h. This
reaction mixture was poured into H2O and extracted with ethyl
acetate. The organic layer was washed with H2O and brine, dried
over Na2SO4, and evaporated in vacuo. The residue was purified
by column chromatography (n-hexane–ethyl acetate = 1 : 3) to give
9 (419 mg, 92%) as a colorless oil. IR (ATR, cm-1): 1737, 1640.
1H NMR (400 MHz) d: 1.40 (12H, s), 1.93 (2H, quin., J = 6.4
Hz), 2.81 (2H, t, J = 6.4 Hz), 3.34 (2H, s), 3.69 (3H, s), 3.78 (2H,
s), 4.15 (2H, t, J = 6.4 Hz), 7.17–7.30 (5 H, m, overlapped with
CHCl3), 7.63 (2H, s). 13C NMR (100 MHz) d: 27.3, 28.5, 49.9,
51.4, 54.4, 58.5, 66.5, 68.0, 79.8, 112.3, 127.3, 128.4, 129.0, 138.8,
General
All melting points were measured on a Yanaco MP-500D and
are uncorrected. Infrared spectra were recorded on a JASCO
FT/IR 6300 spectrophotometer with an ATR (attenuated total
reflectance) system. H and 13C NMR spectra were recorded in
CDCl3 unless otherwise stated, on a JEOL JNM-ECP 400 and a
Bruker 400 MHz with tetramethylsilane (TMS) as the internal
reference. CSI mass spectra were recorded on a JEOL JMS-
T100LC mass spectrometer equipped with a cold-spray ion source.
NH plates (Fuji Silysia Chemical, Ltd., No. TO80817) were used
for TLC. For column chromatography, NH silica gel (Fuji Silysia
Chemical Ltd., particle size 100–200 mesh, No. IO61280 and 200–
1
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148.5, 161.1, 166.0, 171.9. HRCSIMS m/z 509.2733 (calcd for
C28H37N4O5: 509.2764).
was added. After this was stirred under reflux for 2 h, the mixture
was poured into H2O. The mixture was extracted with ethyl acetate
and the organic layer was washed with H2O and brine, dried over
Na2SO4, and evaporated in vacuo. The residue was purified by
column chromatography (n-hexane–ethyl ace◦tate = 1 : 1) to give 1
(330 mg, 50%) as colorless prisms. mp 96–97 C. IR (ATR, cm-1):
1746, 1727, 1632. 1H NMR (400 MHz) d: 1.24 (3H, t, J = 7.3 Hz),
1.36 (12H, s), 2.14 (2H, quin., J = 6.2 Hz), 3.37 (2H, s), 4.18 (4H,
s), 4.18 (2H, t, J = 6.2 Hz), 4.18 (2H, q, J = 7.3 Hz), 4.30 (2H, t, J =
6.2 Hz), 7.67 (2H, s). 13C NMR (100 MHz) d: 14.2, 28.2, 28.5, 41.6,
61.7, 64.9, 68.0, 79.8, 112.2, 148.6, 161.0, 165.7, 166.5. HRCSIMS
m/z 484.2084 (calcd for C23H31N3NaO7: 484.2060). Anal. calcd
for C23H31N3O7: C, 59.91; H, 6.87; N, 9.11. Found: C, 59.86; H,
6.77; N, 9.10.
Synthesis of methyl N-{3-[2,6-bis(4,4-dimethyloxazolin-
2-yl)pyridine-4-yloxy]prop-1-yl}aminoacetate (10)
A solution of 9 (1.09 g, 2.14 mM) in MeOH (10 mL) was hydro-
genated over 10% Pd/C (519 mg) under a hydrogen atmosphere
at room temperature for 8 h. After removal of the catalyst by
filtration through a Celite pad, the filtrate was evaporated in
vacuo. The residue was purified by column chromatography (n-
hexane : acetone = 4 : 1) to give 10 (751 mg, 84%) as colorless oil.
1
IR (ATR, cm-1): 3437, 1738, 1641. H NMR (400 MHz) d: 1.39
(12H, s), 1.98 (2H, quin., J = 6.5 Hz), 2.79 (2H, t, J = 6.5 Hz),
3.42 (2H, s), 3.74 (3H, s), 4.20 (4H, s), 4.23 (2H, t, J = 6.5 Hz),
7.70 (2H, s). 13C NMR (100 MHz) d: 28.5, 29.4, 46.0, 50.8, 51.9,
66.8, 68.0, 79.8, 112.2, 148.5, 161.0, 166.0, 172.9. HRCSIMS m/z
419.2335 (calcd for C21H31N4O5: 419.2294).
Synthesis of 5-[2,6-bis-(4,4-dimethyl-4,5-dihydro-oxazol-2-yl)-
pyridin-4-yloxy]-pentan-2-one (12)
A
solution of 4-benzyloxy-2,6-bis-(4,4-dimethyl-4,5-dihydro-
oxazol-2-yl)-pyridine5 (1.0 g, 2.65 mM) in degassed dry MeOH
(10 mL) was hydrogenated over 10% Pd/C (326 mg) under a
H2 atmosphere at room temperature for 4 h. After removal of
the catalyst by filtration through a Celite pad, the filtrate was
evaporated in vacuo. The residue was washed with Et2O to give
crude 5. Under N2, 5-chloro-2-pentanone (0.6 mL, 5.26 mM) was
added to a mixture of crude 5 and K2CO3 (1.1 g, 8.07 mM) in DMF
(5 mL). The whole was stirred at room temperature for 24 h. The
reaction was quenched with H2O and extracted with CH2Cl2. The
organic layer was washed with H2O and brine, dried over Na2SO4,
and evaporated in vacuo. The residue was purified by NH column
chromatography (200–350 mesh, n-hexane–CHCl3 = 5 : 1–2 : 1) to
Synthesis of potassium N-{3-[2,6-bis(4,4-dimethyloxazolin-2-
yl)pyridine-4-yloxy]prop-1-yl}aminoacetate (2)
Powdered KOH was continuously added to a solution of 10
(751 mg, 1.79 mM) in MeOH (10 mL) with stirring until 10
disappeared. Then, the mixture was purified directly by NH
column chromatography (200–350 mesh, EtOH–MeOH = 10 : 1)
to give 2 (1.10 g, quant.) as a colorless powder. mp >300 ◦C. IR
1
(ATR, cm-1): 3378, 1646. H NMR (400 MHz, CD3OD) d: 1.36
(12H, s), 2.04 (2H, quin, J = 6.5 Hz), 2.79 (2H, t, J = 6.5 Hz),
3.16 (2H, s), 4.24 (4H, s), 4.25 (2H, t, J = 6.5 Hz), 7.63 (2H, s).
13C NMR (100 MHz, CD3OD) d: 27.0, 28.4, 45.8, 52.6, 67.1, 67.7,
79.6, 112.1, 148.0, 161.4, 166.7, 177.0. HRCSIMS m/z 443.1741
(calcd for C20H28KN4O5: 443.1697).
1
give colorless oil 12 (20 mg, 2.0%). H NMR (400 MHz) d: 1.37
(12H, s), 2.06 (2H, quin, J = 6.6 Hz), 2.16 (3H, s), 2.62 (2H, t,
J = 6.6 Hz), 4.12 (2H, t, J = 6.6 Hz), 4.18 (4H, s), 7.64 (2H, s).
13C NMR (100 MHz) d: 22.9, 28.5, 30.1, 39.5, 67.7, 68.0, 79.8,
112.2, 148.5, 161.0, 165.9, 207.7. HRCSIMS m/z 374.2072 (calcd
for C20H28N3O4: 374.2080).
Synthesis of 3-[2,6-bis-(4,4-dimethyl-4,5-dihydro-oxazol-2-yl)-
pyridin-4-yloxy]-propan-1-ol (6)
Under N2, after a suspension of 4 (1.0 g, 3.3 mM) and powdered
KOH (947 mg, 17 mM) in DMF (20 mL) was stirred for 30 min
at 70 ◦C, 1,3-propanediol (0.3 mL, 4.2 mM) was added. After this
was stirred at 70 ◦C for 2 h, the mixture was poured into H2O.
The mixture was extracted with CH2Cl2 and the organic layer was
washed with H2O and brine, dried over Na2SO4, and evaporated
in vacuo. The residue was purified by NH column chromatography
(n-hexane–ethyl acetate = 1 :◦1 to 0 : 1) to give 6 (661 mg, 58%) as
Acknowledgements
This work was supported by JST, Grant-in-Aid for Scientific
Research on Regional Innovation Areas “Development of a
Nitrogen-15 Separation System Using Non-ferrous Smelting Flow
& Utilization Technology”(20R7006), and the Japanese Ministry
of Education, Culture, Sports, Science and Technology (MEXT) in
the form of a Grant-in-Aid for Scientific Research on Innovation
Areas “Emergence in Chemistry” (20111007).
1
colorless prisms. mp 93–94 C. IR (ATR, cm-1): 3226, 1673. H
NMR (400 MHz) d: 1.40 (12H, s), 2.06 (2H, quin., J = 6.0 Hz),
3.84 (2H, t, J = 6.0 Hz), 4.21 (4H, s), 4.28 (2H, t, J = 6.0 Hz),
7.70 (2H, s). 13C NMR (100 MHz) d: 28.4, 31.6, 59.2, 65.9, 67.9,
79.8, 112.2, 148.4, 161.0, 165.9. HRCSIMS m/z 348.1929 (calcd
for C18H26N3O4: 348.1923). Anal. calcd for C18H25N3O4+H2O: C,
59.16; H, 7.45; N, 11.50. Found: C, 58.89; H, 7.39; N, 11.41.
Notes and references
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