Measurement of inclusion complex formation between cyclophane and biological relevant amino acids using electrospray ionization, cold-spray ionization and fast atom bombardment mass spectrometry

Article abstract of DOI:10.1248/cpb.53.1029

The investigation of the host-guest complex formations between cyclophane (TGDMAP) (1) as a host and L-acidic amino acids such as L-glutamic acid (Glu) and L-aspartic acid (Asp) as guests was carried out using fast atom bombardment (FAB), electrospray ion

Full text of DOI:10.1248/cpb.53.1029

August 2005
Notes
Chem. Pharm. Bull. 53(8) 1029—1033 (2005)
1029
Measurement of Inclusion Complex Formation between Cyclophane and
Biological Relevant Amino Acids Using Electrospray Ionization, Cold-
Spray Ionization and Fast Atom Bombardment Mass Spectrometry
Koichi METORI,a Yoshihisa SEI,b Yumiko KIMURA,a Tomoyuki OZAWA,c Kentaro YAMAGUCHI,b and
Muneharu MIYAKE*,a
a College of Pharmacy, Nihon University; 7–7–1 Narashinodai, Funabashi, Chiba 274–8555, Japan: b Faculty of
Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University; 1314–1 Shido, Sanuki, Kagawa 769–2193,
c
Japan: and Chemical Research Laboratories, Nissan Chemical Industries, LTD.; 722–1 Tsuboi-cho, Funabashi, Chiba
274–8507, Japan. Received February 14, 2005; accepted May 18, 2005
The investigation of the host–guest complex formations between cyclophane (TGDMAP) (1) as a host and L-
acidic amino acids such as L-glutamic acid (Glu) and L-aspartic acid (Asp) as guests was carried out using fast
atom bombardment (FAB), electrospray ionization (ESI) and cold-spray ionization (CSI) mass spectrometry
(MS). The stability constant (Ks) values obtained by the three different MS methods almost agreed. However, the
complex ion peaks of a novel cyclophane (CPCn) (2) with Glu and Asp were not observed in FAB-MS. Then,
these host–guest complex formations by use of CSI-MS and ESI-MS was examined, as the results, these complex
ion peaks were observed clearly and the measurement values by the two MS methods are mostly in agreement. It
was concluded that ESI-MS and CSI-MS are available for the determination of Ks value as well as FAB-MS.
Key words cyclophane; glutamic acid; aspartic acid; electrospray ionization (ESI)-MS; cold-spray ionization (CSI)-MS; FAB-
MS
1H-NMR measurement is widely used for determination of
the brain was made by FAB-MS, cold-spray ionization mass
spectrometry (CSI-MS), electrospray ionization mass spec-
trometry (ESI-MS). The Ks values of 1·Glu and 1·Asp were
obtained by the three different MS methods almost agreed.
On the other hand, a novel water-soluble cyclophane (2) as a
host bearing 1,4,7,10-tetraazacyclododecane group on the
alkyl bridge as blanches of cyclophane having diphenyl-
methane skeleton in order to bind to the carboxyl group and
also lead to remedy the solubility of cyclophane was de-
signed. The synthesis of the novel cyclophane (2) is shown in
Chart 1. Examination on the complex formation of 2 with
host–guest complex formation, however, the method cannot
be used for measuring confirmation of host–guest complex
formation in case either the peaks of guest and host overlap
each other or these signals show broad. Recently, it reported
that mass spectrometry can be used to detect noncovalent as-
sociation complexes.1—4)
1
We reported that FAB-MS is useful method as well as H-
NMR method not only for confirmation of TGDMAP (1) and
biological relevant phosphates such as nucleotide complex
geometry but also for determination of stability constant (Ks)
values of the complexes because the Ks value calculated by
FAB-MS (100 Mꢀ1 for UMP) was most similar to the Ks value
calculated by 1H-NMR spectrometry (110 Mꢀ1 for UMP).5—7)
These results show that the binding stability in aqueous solu-
tion is preserved in the gas phase.
1
Glu was performed by H-NMR spectrometry in 0.1 M phos-
1
phate buffer solution (pD 6.8). However, the H-NMR spec-
trum of Glu was complicated and the chemical shift changes
of Glu in the presence of 2 were negligible. Therefore, it was
impossible to calculate the Ks values between 2 and Glu and
also the complex ion peaks of 2 with Glu and Asp were not
observed in FAB-MS. For the purpose to solve the problem,
we investigated the measurement of the Ks values by use of
ESI-MS and CSI-MS. Especially ionization procedure of
CSI-MS8,9) which is milder than that of ESI-MS is expected
to provide a powerful means to study noncovalent host–guest
complexes such as hydrogen bond formation. Our primary
purpose in this report is to judge if ESI-MS and CSI-MS as
well as FAB-MS are useful for determination of the complex
formations of 2·L-acidic amino acids such as Glu and Asp
when FAB-MS cannot be used for measuring confirmation of
host–guest complex formation as described above. In fact,
the complex ion peaks were observed clearly by using ESI-
MS and CSI-MS instead of FAB-MS, and also the Ks values
calculated by the two different mass spectrometry methods
almost agreed.
In this report, investigation on the complex formation of 1
with L-glutamic acid (Glu) and L-aspartic acid (Asp) as
guests which play an important role in receptor-signaling in
Results and Discussion
In order to determine the complex formation and Ks value
of 1 with Glu or Asp, three ionizing methods (FAB-MS, ESI-
Fig. 1. Structure of Cyclophane Host
∗ To whom correspondence should be addressed. e-mail: miyake@pha.nihon-u.ac.jp
© 2005 Pharmaceutical Society of Japan

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Vol. 53, No. 8
a) BrCH2CO2Me, K2CO3, DMF, b) 5 N KOH/MeOH, c) pentafluorophenol, N,Nꢃ-dicyclohexylcarbodiimide, CH2Cl2, d) 25% NH4OH, THF, e) BH3, THF, f) 6, TEA, CH2Cl2,
g) 1-carboxymethil-4,7,10-tris(tert-butoxylcarbonyl)-1,4,7,10-tetraazacyclodecane, 1-ethil-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, CH2Cl2, h) conc. HCl, THF.
Chart 1
MS and CSI-MS) were investigated. FAB-MS spectrum for a
1 : 1 mixture of a equivalent amount (2.5 mmol) of 1 and Glu
in 50% glycerol contained in 20 mmol of triethylammonium
acetate (TEAA) solution (pH 7.0) showed the peak of the
1 : 1 complex at m/z 1054.7 [(1ꢀ2Cl)ꢁ(Glu)ꢀH]ꢁ and mol-
ecular ion peaks of higher mass could not be detected (Fig.
2). Figure 3 shows ESI and CSI mass spectra obtained for an
equivalent amount (0.1 mmol) of 1 and Glu in 2 mmol of
TEAA solution (pH 7.0). These data suggest that 1 forms the
1 : 1 complex with Glu. The 1 : 1 stoichiometry for the com-
plex between 1 and Glu was also confirmed by using Job’s
method10,11) of continuous variations using FAB-MS. The
Fig. 2. FAB Mass Spectrum for the Inclusion Complex Formation be-
tween TGDMAP and Glu in a Glycerol Matrix
Job’s method gave a maximum at 0.5, indicative of a 1 : 1 sto-
ichiometry (Fig. 4). The Ks value of the complex was deter-
mined using the absolute intensity of the complex on the
basis of double reciprocal plots according to the previous re-
port.7) The plots gave excellent linearity with a correlation
coefficient (rꢂ0.997) (Fig. 5). In case of using Asp as a
guest, the similar results was obtained and the 1 : 1 complex
ion peak [(1ꢀ2Cl)ꢁ(Asp)ꢀH]ꢁ of 1 with Asp was shown at
m/z 1040.7 in FAB-MS and at m/z 1040.8 in ESI-MS and
CSI-MS, respectively (data not shown). The Ks values ob-
tained by FAB-MS (70 Mꢀ1 for Glu and 60 Mꢀ1 for Asp) mea-
surement method were mostly identical to the results of CSI-
ꢀ1
ꢀ1
MS (105 M for Glu and 80 M for Asp) and ESI-MS
ꢀ1
ꢀ1
(120 M for Glu and 75 M for Asp) measurement methods
in spite of using different ionization, temperature and sol-
vent. The binding constants (Ks) are summarized in Table 1.
However, the complex ion peaks of between a novel cyclo-
phane (CPCn) (2) and Glu and Asp were not observed in
FAB-MS measurement method. Then, the measurement of
these host–guest complex formations by use of CSI-MS and
ESI-MS was examined. An equivalent amount (0.2 mmol) of
2, Glu and Asp were dissolved in 2 mmol of TEAA solution
(pH 7.0) and then were mixed at a rate of 1 : 1. The ion peaks
corresponding to [2ꢁGluꢁH]ꢁ was observed at m/z 1110.9
in positive mode ESI-MS and at m/z 1110.7 in CSI-MS (Fig.
6) and [2ꢁAspꢁH]ꢁ was observed at m/z 1096.8 (data not
Fig. 3. Comparison of (a) ESI and (b) CSI Mass Spectra for the Inclusion
Complex Formation between TGDMAP and Glu in a TEAA Solution
shown). The ESI-MS measurement of host–guest complex The Job’s methods gave a maximum at 0.5, indicative of a
ion intensity advantaged over the CSI-MS, therefore the ex- 1 : 1 stoichiometry monitored the absolute intensity at m/z
amination of the Ks value of the 2·Glu and 2·Asp com- 1110.9 for Glu and at m/z 1096.8 for Asp. The good correla-
plexes, and the Job’s plots were performed using ESI-MS. tion coefficients were obtained in the double reciprocal plots

August 2005
1031
Fig. 4. Job’s Plots for the Inclusion Complex Formation between
TGDMAP and Glu
The absolute intensity was measured at m/z 1054.7 (host–guest complex ion). The
total concentration (TGDMAPꢁGlu) was 5 mmol.
Fig. 6. Comparison of (a) ESI and (b) CSI Mass Spectra for the Inclusion
Complex Formation between CPCn and Glu
weak noncovalent inclusion complexes in host–guest chem-
istry.
Fig. 5. The Double Reciprocal Plots for the Inclusion Complex Formation
between TGDMAP and Glu
Experimental
Apparatus Melting points were determined using a Yanagimoto Melt-
ing point Apparatus Yanaco MP and were uncorrected. 1H-NMR was
recorded on a JEOL JNM-LA400 spectrometer containing tetramethylsilane
as standard. FAB-MS measurement was performed using a JEOL JMS-
GC-mate instrument equipped with double-focusing mass analyzer. ESI-MS
and CSI-MS measurements were performed using a JEOL JMS-T100CS in-
strument equipped with time-of-flight mass analyzer. Elemental analyses
were performed on a Perkin Elmer 2400 II CHN analyzer.
Reagents The following reagents were commercially available and used
without further purification: Borane–methyl sulfide complex (BH3·DMS),
Sigma-Aldrich Chemical; 4,4ꢃ-dihydroxydiphenylmethane, 1-ethyl-3-(3-di-
methylaminopropyl)carbodiimide hydrochloride, N,Nꢃ-dicyclohexylcarbo-
diimide, pentafluorophenol, guest compounds (L-glutamic acid and L-aspar-
tic acid potassium salt) triethylammonium acetate (TEAA) solution (pH
7.0); Tokyo Kasei Kogyo. All organic solvents were purchased from Wako
Pure Chemical.
The absolute intensity was measured at m/z 1054.7 (host–guest complex ion). The
final concentration of TGDMAP was 2.5 mmol and Glu concentration ranges from
0.83 mmol to 7.5 mmol.
Table 1. The Ks (Mꢀ1) Values of Guests in the Presence of TGDMAP or
CPCn by FAB, ESI and CSI-MS
TGDMAP
CPCn
Method
Glu
Asp
Glu
Asp
FAB-MS
ESI-MS
CSI-MS
70
120
105
60
75
80
N.D.
340
N.T.
N.D
115
N.T.
N.D.: not detected. N.T.: not tested.
4,4ꢀ-Bis(methoxycarbonylmethoxy)diphenylmethane (4)
A mixture
of 4,4ꢃ-dihydroxydiphenylmethane (3) (1.0 g, 5 mmol), methyl bromoacetate
(1.53 g, 10 mmol) and K2CO3 (1.38 g, 10 mmol) in DMF (20 ml) was stirred
at room temperature. After 24 h, the reaction mixture was filtered. The fil-
trate was extracted with EtOAc (50 mlꢄ3), washed with brine and dried over
MgSO4. After removal of the solvent under reduced pressure, the residue
was purified by column chromatography on silica gel with EtOAc : CHCl3
(1 : 9) as an eluent to give 1.6 g, 93% as a colorless solid. An analytical sam-
ple was obtained by recrystallizing this material from EtOAc–hexane, color-
less needles. mp 51—52 °C. 1H-NMR (CDCl3) dꢂ3.80 (6H, s), 3.85 (2H, s),
4.60 (4H, s), 6.82 (4H, d, Jꢂ8.8 Hz), 7.08 (4H, d, Jꢂ8.8 Hz). EI-MS m/z:
344 [Mꢁ]. HR-EI-MS m/z: 344.1256 (Calcd for C19H20O6: 344.1259). Anal.
for the inclusion complex formation for Ks measurements of
2·Glu and 2·Asp monitored the absolute intensity of the
host–guest complex ion. As shown in Table 1, the Ks value of
ꢀ1
Glu and Asp were obtained 340 M and 115 Mꢀ1, respec-
tively. In the case of using 1, the Ks values of the 2·Glu and
2·Asp in ESI-MS were obtained 120 Mꢀ1 and 75 Mꢀ1, respec-
tively.
In conclusion, a novel cyclophane (2) works as host that
form complexes with Glu and Asp as guests and the ability Calcd for C19H20O6: C, 66.27; H, 5.85. Found: C, 66.54; H, 5.91.
4,4ꢀ-Bis(carboxymethoxy)diphenylmethane (5)
A
mixture of
4
of inclusion complex formation of 2 has stronger than 1. The
ESI-MS and CSI-MS methods were found to be a convenient
way to evaluate the stability constant of host–guest com-
plexes because the ionizing methods of ESI-MS and CSI-
MS, which are milder than that of FAB-MS, are useful for
confirmation of host–guest complex which cannot be de-
tected by FAB-MS. In this experiments, although the deter-
mined binding constants did not differ significantly between
CSI-MS and ESI-MS, CSI-MS is milder ionization than that
of ESI-MS and FAB-MS, could apply for determination of a
(1.19 g, 3.46 mmol) and 5 N KOH/MeOH (4 ml) in MeOH (40 ml) was re-
fluxed 2 h. After removal of the solvent under reduced pressure, the residue
was dissolved in 100 ml of H2O. The solution was extracted with EtOAc
(100 ml). The aqueous solution was acidified to pHꢂ1 with 10% HCl and
extracted with EtOAc (300 ml). The EtOAc layer was washed with brine,
dried over MgSO4, and concentrated under reduced pressure to give a color-
less powder (1.09 g, 100%). An analytical sample was obtained by recrystal-
lizing this material from EtOAc–hexane, colorless needles. mp 199—
200 °C. 1H-NMR (DMSO-d6) dꢂ3.79 (2H, s), 4.59 (4H, s), 6.80 (4H, d,
Jꢂ8.8 Hz), 7.10 (4H, d, Jꢂ8.8 Hz), 12.90 (2H, s). EI-MS m/z: 316 [Mꢁ].
HR-EI-MS m/z: 316.0944 (Calcd for C17H16O6: 316.0946). Anal. Calcd for

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Vol. 53, No. 8
C17H16O6: C, 64.55; H, 5.10. Found: C, 64.60; H, 5.11.
mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (45
mg, 0.23 mmol) in CH2Cl2 (5 ml) was stirred at r.t. for 18 h under N2 atmos-
phere. The reaction mixture was diluted with CH2Cl2 (10 ml), washed with
2 N NaOH and dried over Na2SO4. The solvent was concentrated under re-
duced pressure. The residue was purified by column chromatography on sil-
ica gel with EtOAc : hexane (3 : 1) and EtOAc as eluent to give a colorless
4,4ꢀ-Bis(pentafluorophenoxycarbonylmethoxy)diphenylmethane (6)
A mixture of 5 (2.95 g, 9.3 mmol), pentafluorophenol (3.46 g, 18.8 mmol)
and N,Nꢃ-dicyclohexylcarbodiimide (3.88 g, 18.8 mmol) in THF (100 ml)
was stirred at room temperature for 24 h. The mixture was filtered, and the
filtrate was evaporated under reduced pressure. The residue was purified by
column chromatography on silica gel with CH2Cl2 as an eluent to give amorphous powder (115 mg, 74%). mp 151—153 °C. 1H-NMR (CDCl3)
5.56 g, 92% as a colorless solid. An analytical sample was obtained by re-
crystallizing this material from EtOAc–hexane, colorless needles. mp 135—
dꢂ1.43 (36H, s), 1.46 (18H, s), 3.04 (8H, br), 3.22—3.42 (16H, br), 3.45—
3.62 (8H, br), 3.72—3.82 (16H, m), 4.05—4.12 (4H, m), 4.17—4.22 (4H,
136 °C. 1H-NMR (CDCl3) dꢂ3.90 (2H, s), 4.97 (4H, s), 6.89 (4H, d, m), 6.62 (2H, d, Jꢂ8.5 Hz), 6.66 (2H, d, Jꢂ8.5 Hz), 6.70 (2H, d, Jꢂ8.5 Hz),
Jꢂ8.4 Hz), 7.13 (4H, d, Jꢂ8.4 Hz). FAB-MS m/z: 648 [Mꢁ]. FAB-HR-MS
6.73 (2H, d, Jꢂ8.5 Hz), 6.94—7.00 (8H, m). FAB-MS m/z: 1564 [MꢁH]ꢁ.
m/z: 648.0628 (Calcd for C29H14F10O6: 648.0630). Anal. Calcd for FAB-HR-MS m/z: 1563.9335 (Calcd for C84H127N10O18: 1563.9329). Anal.
C29H14F10O6: C, 53.72; H, 2.18. Found: C, 53.73; H, 2.14.
Calcd for C84H126N10O18: C, 64.51; H, 8.12; N, 8.96. Found: C, 64.40; H,
8.29; N, 8.81.
N,Nꢀ-Bis(1,4,7,10-tetraazacyclododecane-1-ylacetyl)-10,26-diaza-
7,13,23,29-tetraoxa[7.1.7.1]paracyclophane Octahydrochrolide (2) Cy-
4,4ꢀ-Bis(carbamoylmethoxy)diphenylmethane (7) To a solution of 6
(4.0 g, 6.17 mmol) in THF (30 ml) was added 25% NH4OH (12 ml) at room
temperature. After stirring for 12 h, sat. NaHCO3 (200 ml) was added to the
reaction mixture. The precipitate was collected by filtration, washed with clophane (11) (100 mg, 0.064 mmol) was dissolved in THF (1 ml), to which
H2O, EtOH and Et2O and dried under vacuum at 60 °C to give 1.9 g, 98% as
a colorless powder which was used in the next step without further purifica-
tion. mp 233—234 °C. H-NMR (DMSO-d6) dꢂ3.80 (2H, s), 4.36 (4H, s),
conc. HCl (0.2 ml) was added. After the reaction mixture was stirred at r.t.
for 12 h. The reaction mixture was diluted with CH2Cl2 (10 ml), washed with
2 N NaOH and dried over Na2SO4. The solvent was diluted with THF (10 ml)
and the precipitate was collected by filtration, washed with THF, and dried to
1
6.85 (4H, d, Jꢂ8.8 Hz), 7.11 (4H, d, Jꢂ8.8 Hz), 7.32 (2H, s), 7.43 (2H, s).
FAB-MS m/z: 315 [MꢁH]ꢁ. HR-FAB-MS m/z: 315.1346 (Calcd for
C17H19N2O4: 315.1344). Anal. Calcd for C17H18N2O4: C, 64.96; H, 5.77; N,
8.91. Found: C, 64.74; H, 5.67; N, 8.69.
4,4ꢀ-Bis(2-aminoethoxy)diphenylmethane (8) A mixture of 7 (314 mg,
1 mmol) and BH3·DMS (1.16 ml, 12 mmol) in THF (12 ml) was stirred for
24 h at 80 °C under N2 atmosphere, then was cooled to room temperature.
Six milliliters of 0.7 M hydrogen chloride–MeOH solution was added, and
the mixture was refluxed for 0.5 h, and evaporated under reduced pressure.
The residue was basified with 25% NH4OH. The mixture was extracted with
1
give a white powder (80 mg, 100%). mp 280—282 °C (decomp). H-NMR
(D2O 0.1 M phosphate buffer (pD 6.8)) dꢂ2.76—2.89 (32H, m), 3.41—3.53
(16H, m), 3.04 (8H, br), 3.76 (4H, br), 3.84—3.88 (4H, m), 6.18 (2H, d,
Jꢂ8.3 Hz), 6.31 (2H, d, Jꢂ8.3 Hz), 6.36 (2H, d, Jꢂ8.3 Hz), 6.40 (2H, d,
Jꢂ8.3 Hz), 6.63—6.69 (8H, m). FAB-MS 963 [Mꢀ8HClꢁH]ꢁ. FAB-HR-
MS m/z: 963.6206 (Calcd for C54H79N10O6: 963.6183). Anal. Calcd for
C54H86Cl8N10O6: C, 51.68; H, 6.91; N, 11.16. Found: C, 51.75; H, 7.08; N,
11.35.
FAB-MS Measurements for the Complex Formation Measurement
CH2Cl2, washed with brine and dried over Na2SO4. Removal of the solvent conditions were as follows: equimolar solutions (5 mmol) of host and guest
under reduced pressure afforded a pale yellow oil, which was purified by were prepared in 20 mmol of triethylammonium acetate (TEAA) solution
column chromatography on silica gel with CHCl3 : MeOH : 25% NH4OH containing 50% glycerol (pH 7.0). An equal volume (5 ml) of the host and
(100 : 40 : 4) as an eluent to give 243 mg, 85% as a colorless amorphous
powder which was used in the next step without further purification. 1H- employed as a fast atom bombardment gas. Scanning was performed from
m/z 100 to 2000 in 10 s and several scans were summed to obtain the final
guest solution was mixed and then 1 ml of the mixture was loaded. Xe was
NMR (CD3OD) dꢂ2.99 (4H, t, Jꢂ5.6 Hz), 3.82 (2H, s), 3.98 (4H, t,
Jꢂ5.6 Hz), 6.84 (4H, d, Jꢂ8.4 Hz), 7.07 (4H, d, Jꢂ8.4 Hz). FAB-MS m/z: spectrum. All of FAB mass spectra of the mixture were employed positive-
287 [MꢁH]ꢁ. FAB-HR-MS m/z: 287.1757 (Calcd for C17H23N2O2: ion mode.
287.1759). Anal. Calcd for C17H22N2O2: C, 71.30; H, 7.74; N, 9.78. Found:
ESI-MS Measurements In the case of using of TGDMAP (1) as host,
C, 71.51; H, 7.78; N, 9.52.
ESI-MS measurement conditions and sample preparation procedures were as
9,27-Dioxo-10,26-diaza-7,13,23,29-tetraoxa[7.1.7.1]paracyclophane (9) follows: the probe heater temperature was set at 250 °C and needle, ring and
A mixture of 6 (973 mg, 1.5 mmol), 8 (430 mg, 1.5 mmol) and TEA (2.1 ml, orifice voltages were held at 2000, 20 and 130, in positive-ion mode, respec-
15 mmol) in CH2Cl2 (300 ml) was stirred at 60 °C under N2 atmosphere. tively. On the other hand, in the case of using of CPCn (2) as host, the probe
After 24 h, the reaction mixture was concentrated under reduced pressure.
The residue was purified by column chromatography on silica gel with
EtOAc : MeOH (9 : 1) as an eluent to give 590 mg, 69% as a colorless solid.
heater temperature was set at 250 °C and needle, ring and orifice voltages
were held at 1800, 22 and 33, in positive-ion mode, respectively. Equimolar
solutions (0.2 mmol) of host and guest were prepared in 2 mmol of TEAA
solution (pH 7.0). An equal volume of the host and guest solution was mixed
An analytical sample was obtained by recrystallizing this material from
1
EtOAc–hexane, colorless fine needles. mp 195—196 °C. H-NMR (CDCl3) and then the sample solution was introduced into the spectrometer at a flow
dꢂ3.53 (2H, s), 3.70—3.734 (4H, m), 3.80 (2H, s), 3.92 (4H, t, Jꢂ5.2 Hz),
6.63 (4H, d, Jꢂ8.8 Hz), 6.71 (4H, d, Jꢂ8.8 Hz), 6.87 (4H, d, Jꢂ8.8 Hz),
rate of 1.5 ml/h using a syringe pump.
CSI-MS Measurements An orthogonal cold-spray apparatus equipped
6.94 (2H, t, Jꢂ5.2 Hz), 7.05 (4H, d, Jꢂ8.8 Hz). FAB-MS m/z: 567 [MꢁH]ꢁ. with a spray temperature control system using liquid N2 was used and the
FAB-HR-MS m/z: 567.2496 (Calcd for C34H35N2O6: 567.2495). Anal. Calcd spray temperature was set at 4 °C. In the case of using of TGDMAP (1) as
for C34H34N2O6·1/3 H2O: C, 71.30; H, 6.10; N, 4.90. Found: C, 71.36; H, host, needle, ring and orifice voltages were held at 1800, 18 and 140, in pos-
6.11; N, 4.83.
itive-ion mode, respectively. In the case of using of CPCn (2) as host, nee-
10,26-Diaza-7,13,23,29-tetraoxa[7.1.7.1]paracyclophane (10) A mix- dle, ring and orifice voltages were held at 2000, 18 and 33, in positive-ion
ture of 9 (610 mg, 1.07 mmol) in THF (13 ml) was stirred under N2 atmo-
sphere. BH3·DMS (1.3 ml, 1.34 mmol) was added dropwise. The reaction
mixture was stirred for 24 h at 80 °C, and then 0.7 ml of 0.7 M hydrogen
chloride–MeOH solution was added. After 0.5 h, the reaction mixture was
evaporated under reduced press. The residue was basified with excess 25%
NH4OH. The mixture was extracted with CH2Cl2 (100 ml), washed with
brine and dried over Na2SO4. Removal of the solvent, the residue was puri-
fied by column chromatography on silica gel with CHCl3 : MeOH : 25%
NH4OH (100 : 10 : 1) as an eluent to give 480 mg, 83% as a colorless pow-
mode, respectively. Sample preparation procedures and flow rate followed in
ESI-MS measurement.
Determination of Ks Values of the Complexes by FAB-MS, ESI-MS
and CSI-MS The Ks values of the host–guest complexes were determined
using the absolute intensities of host–guest complexes on the basis of the
double reciprocal plots. For FAB-MS measurement, the stock solutions of
the host and all of the guests were prepared in 20 mmol of TEAA solution
(pH 7.0) containing 50% glycerol. The concentration of the host was
2.5 mmol, while those of the guest ranges from 0.83 mmol to 7.5 mmol (6
points). For ESI-MS and CSI-MS measurements, the stock solutions of the
der. An analytical sample was obtained by recrystallizing this material from
1
CH2Cl2–hexane, colorless fine needles. mp 135—136 °C. H-NMR (CDCl3) host and all of the guests were prepared in 2 mmol of TEAA solution (pH
dꢂ3.00 (8H, t, Jꢂ4.8 Hz), 3.80 (4H, s), 4.06 (8H, t, Jꢂ4.8 Hz), 6.75 (8H, d, 7.0). The concentration of the host was 0.1 mmol, while guest ranges from
Jꢂ8.4 Hz), 7.00 (8H, d, Jꢂ8.4 Hz). FAB-MS m/z: 539 [MꢁH]ꢁ. FAB-HR- 0.05 mmol to 0.3 mmol (5 points). The double reciprocal plots (1/absolute
MS m/z: 539.2908 (Calcd for C34H39N2O4: 539.2909). Anal. Calcd for intensity (I) vs. 1/[G]) gave excellent linearity with a correlation coefficient
C34H38N2O4: C, 75.81; H, 7.11; N, 5.20. Found: C, 75.82; H, 7.39; N, 5.20.
rꢅ0.997 (in FAB-MS measurement) and 0.989 (in ESI-MS and CSI-MS
N,Nꢀ-Bis[tris(4,7,10-tert-butoxycarbonyl)-1,4,7,10-tetraazacyclodode- measurements).
cane-1-ylacetyl]-10,26-diaza-7,13,23,29-tetraoxa[7.1.7.1]-paracyclo-
phane (11) A mixture of 10 (54 mg, 0.1 mmol), 1-carboxymethyl-4,7,10-
Acknowledgments This work was supported by a grant from “Acade-
tris(tert-butoxycarbonyl)-1,4,7,10-tetraazacyclododecane12) (106 mg, 0.2 mic Frontier” Project for Private Universities: matching fund subsidy from

August 2005
1033
MEXT (Ministry of Education, Culture, Sports, Science and Technology)
2002—2006.
6) Metori K., Kimura Y., Miyake M., J. Mass Spectrom. Soc. Jpn., 50,
301—303 (2002).
7) Metori K., Kimura Y., Miyake M., J. Mass Spectrom. Soc. Jpn., 51,
566—569 (2003).
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