or via energetics in the MS16,17), followed by detection. Sequenc-
ing by MS also can involve the detection of “mass tag labels” such
as sulfur isotopes18 or metal ions19 attached to DNA.
Chemstation computer for data acquisition in the single ion
monitoring mode. Dwell time was set to be 50 ms with live screen
display off when four ions were monitored. An Ultra-2 HP capillary
column (25 m × 0.32 mm i.d., 0.17 µm film thickness) from
Hewlett Packard was used. With helium as carrier gas, the
column head pressure was set to 20 psi. The source pressure
(methane) was set to 2.0 Torr at 250 °C. Injection was on-column
at 120 °C, hold for 2 min, ramp column oven to 165 °C at 10 °C/
min, then ramp to 290 °C at 70 °C/ min, and hold for 3 min.
GC-ECD was performed with a Varian 3500 instrument (Walnut
Creek, CA) fitted with a splitless glass insert (1/ 4 in. o.d. × 2 mm
i.d. × 3 in. long) in a direct injector at 300 °C, and an Ultra-1
capillary column (10 m × 0.2 mm, 0.11 µm film thickness, HP).
Detector temperature was 350 °C.
Synthesis. (a) 4′-[(P entafluorobenzyl)oxy]acetophenone,
1 a′, and 2 a′, 3 a, and 4 a. 4′-Hydroxyacetophenone (2.72 g, 20
mmol) and sodium hydroxide (0.90 g, 22.5 mmol) were dissolved
in acetonitrile (50 mL) and water (30 mL). To this solution,
R-bromo-2,3,4,5,6-pentafluorotoluene (3 mL, 19.9 mmol) was
added. The reaction mixture was heated to reflux for 5 h and
then cooled to room temperature. To this solution, a 5% sodium
carbonate solution (150 mL) was added. The precipitate was
collected by suction filtration, washed with water (200 mL × 2),
and then recrystallized with methanol/ water. Yield, 5.98 g (95%).
The same method was used for the preparation of 2 a′, 3 a, and
4 a. For 3 a and 4 a, an equimolar amount of 3,5-bis(trifluoro-
methyl)benzyl bromide was used in place of R-bromo-2,3,4,5,6-
pentafluorotoluene. The yield was 93% for 2 a′, 88% for 3 a, and
95% for 4 a.
(b) 4′-[(p-Methoxytetrafluorobenzyl)oxy]acetophenone, 1a,
and 2 a. 1 a′ (5.98 g, 18 mmol) was dissolved in methanol (30
mL). To this solution, sodium methoxide (1.08 g, 20 mmol) was
added. The reaction mixture was heated to reflux for 2 h and
then evaporated to dryness. The resulting solid was washed with
water (100 mL × 2) and air-dried. Yield, 6.1 g (99%). The same
method was used for the preparation of 2 a. The yield was 89%.
(c) Ethyl N-[(Diethylphosphono)acetyl]isonipecotate (5 ).
In a 50 mL round-bottom flask were placed 1 g (5.1 mmol) of
diethylphosphonoacetic acid, 0.7 g (6 mmol) of N-hydroxysuccin-
imide, and 10 mL of dioxane that had been distilled from LiAlH4.
After cooling in an ice bath under nitrogen with stirring, 1.25 g
(6.0 mmol) of N,N-dicyclohexylcarbodiimide in 5 mL of dioxane
were added slowly. After stirring at room temperature overnight,
1.18 g (7.5 mmol) of ethyl isonipecotate in 5 mL of dioxane was
added, followed by stirring at room temperature overnight. The
white precipitate of dicyclohexylurea was filtered off, and the
filtrate was concentrated on a rotary evaporator. The product
could be purified by silica flash chromatography with ethyl acetate,
giving 1.2 g (70%) of product based on diethylphosphonoacetic
acid. In practice, the entire evaporated reaction mixture (a viscous
oil) was used directly in the next step, when the amount of starting
diethylphosphonoacetic acid was 1.15 mL (7.1 mmol).
Here we present our initial progress toward a goal of sequenc-
ing DNA based on electrophore mass tag labels. An electrophore
is a compound that readily ionizes in the gas phase by an event
called “electron capture”. Typically this takes place in an electron
capture detector for gas chromatography (GC-ECD) or in the ion
source of an electron capture mass spectrometer (typically GC/
EC-MS). Several features of electrophore mass tags make them
attractive for DNA sequencing. Electrophores can be easy to
prepare, stable both chemically and physically, detectable in trace
amounts, and available as numerous analogs for high multiplexing.
In principle, the detection of electrophores by time-of-flight (TOF)
EC-MS could be very sensitive and fast, helping to make it
possible, with use of highly multiplexed electrophores, to se-
quence DNA quite rapidly. Some of this potential has been
pointed out previously.20-29
EXPERIMENTAL SECTION
Materials. Organic solvents, such as THF, acetonitrile,
methanol, ethyl ether, methylene chloride, and ethyl acetate were
purchased from J. T. Baker (Phillipsburg, NJ). Sodium bicarbon-
ate, sodium sulfate, sodium carbonate, sodium bisulfite, prepara-
tive TLC plates, and regular silica TLC plates were also purchased
from J. T. Baker. PCR buffer was Sequitherm (Epicenter,
Madison, WI), pH 9.3/ 50 mM Tris/ 2.5 mM MgCl2. All other
chemicals were purchased from Aldrich Chemicals Co., Inc.
(Milwaukee, WI). All compounds (except the electrophore-labeled
DNA oligomers) synthesized in our laboratories were confirmed
1
by H NMR and 13C NMR. In some cases 19F NMR also was
used.
Equipment. A Zorbax RX-C18 (4.6 mm × 15 cm) reversed-
phase column (Mac-Mod Analytical, Chadds Ford, PA) was
employed for HPLC.
Model thermal release studies were conducted on a Varian
3300 gas chromatograph fitted with a thermal conductivity
detector. An aluminum column (3/ 16 in. i.d., 4 ft long) was packed
with 10% SP-2100 (Supelco, Bellefonte, PA) on Chromosorb W
(Alltech, Deerfield, IL).
GC/ EC-MS was performed using a Hewlett-Packard (HP)
5988A mass spectrometer coupled to a HP 5890 gas chromato-
graph. The instrument was controlled by a HP 59970C MS
(16) Nordhoff, E.; Karas, M.; Cramer, R.; Hahner, S.; Hillenkamp, F.; Kirpekar,
F.; Lezius, A.; Muth, J.; Meier, C.; Engels, J. W. J. Mass Spectrom. 1 9 9 5 ,
30, 99-112.
(17) Ni, J.; Pomerantz, S. C.; Rozenski, J.; Zhang, Y.; McCloskey, J. A. Anal.
Chem. 1 9 9 6 , 68, 1989-1999.
(18) Brennen, T.; Chakel, J.; Bente, P.; Field, M. In Biological Mass Spectroscopy;
Burlingame, A. L., McCloskey, J. A., Eds.; Elsevier Science Publishing:
Amsterdam, The Netherlands, 1990; pp 219-27.
(19) Jacobson, K. B.; Arlinghaus, H. F. Anal. Chem. 1 9 9 2 , 64, 315A-328A.
(20) Joppich-Kuhn, R.; Joppich, M.; Giese, R. W. Clin. Chem. 1 9 8 2 , 28, 1844-
1847.
(21) Giese, R. W. Trends Anal. Chem. 1 9 8 3 , 2, 166-168.
(22) Abdel-Baky, S.; Klempier, N.; Giese, R. W. Tetrahedron 1 9 9 0 , 17, 5859-
5880.
(23) Xu, L.; Giese, R. W. J. Fluorine Chem. 1 9 9 4 , 67, 47-51.
(24) Giese, R. W. U.S. Patent 4,650,750, 1987.
(25) Giese, R. W. U.S. Patent 4,709,016, 1987.
(26) Giese, R. W. U.S. Patent 5,360,819, 1994.
(27) Giese, R. W.; Abdel-Baky, S.; Xu, L. U.S. Patent 5,516,931, 1996.
(28) Abdel-Baky, S.; Giese, R. W. Anal. Chem. 1 9 9 3 , 65, 498-499.
(29) Xu, L.; Magiera, D.; Abushamaa, A.; Kugabalasooriar, S.; Giese, R. W. J.
Chromatogr., A 1 9 9 7 , 764, 95-102.
(d) N-[3 -[4 ′-[(p -Methoxytetrafluorobenzyl)oxy]phenyl]-
crotonyl]isonipecotic Acid, 1 b, and 2 b-4 b. The preceding
viscous oil was dissolved in THF (50 mL). Sodium hydride (0.87
g, 21.75 mmol) was added followed by 1 a (1.61 g, 4.9 mmol).
The reaction mixture was refluxed for 10 h, cooled to room
temperature, and filtered. To the filtrate, a solution of KOH (1 g,
18 mmol) in water (20 mL) and acetonitrile (10 mL) was added.
The reaction was stirred at room temperature for 24 h followed
3596 Analytical Chemistry, Vol. 69, No. 17, September 1, 1997