AMACE1: Versatile aminoacetamide electrophore reagent

Article abstract of DOI:10.1021/ac990384f

Acetamide, 2-amino-N-[[3,5-bis(trifluoromethyl)phenyl]methyl]-N-methyl-, monohydrochloride, which we have named AMACE1, was synthesized in three steps starting from N-tritylglycine. AMACE1 was coupled via its primary amine group (pK(a) 8.2) under aqueous conditions to four model analytes for oxidative sugar damage to DNA: glycolate, 3-hydroxy-2-butanone, 3-phenylbutyraldehyde, and α-hydroxy-γ-butyrolactone, relying on cyanoborohydride for coupling to a keto function and a water-soluble carbodiimide for coupling to a carboxyl function. Further reaction with butyric anhydride led to products that could be detected by gas chromatography/electron capture mass spectrometry when 1 μL of ethyl acetate containing essentially 20 amol of each product was injected, on the basis of selected ion monitoring of the analyte characteristic anion fragment from dissociative loss of the 3,5- bis(trifluoromethyl)phenylmethyl moiety: m/z 215, 289, 299, and 329, respectively. Since many small, organic analytes contain a keto or carboxylic acid group, AMACE1 should be useful in general in the area of trace organic analysis.

Full text of DOI:10.1021/ac990384f

Anal. Chem. 2000, 72, 1798-1801
AMACE1: Versatile Aminoacetamide Electrophore
Reagent
Rong J. Lu and Roger W. Giese*
Department of Pharmaceutical Sciences in the Bouve´ College of Pharmacy and Health Professions, Barnett Institute, and
Chemistry Department, Northeastern University, Boston, Massachusetts 02115
Acetamide, 2 -amino-N-[[3 ,5 -bis(trifluoromethyl)phenyl]-
methyl]-N-methyl-, monohydrochloride, which we have
named AMACE1 , was synthesized in three steps starting
from N-tritylglycine. AMACE1 was coupled via its primary
amine group (pKa 8 .2 ) under aqueous conditions to four
model analytes for oxidative sugar damage to DNA:
glycolate, 3 -hydroxy-2 -butanone, 3 -phenylbutyraldehyde,
and r-hydroxy-γ-butyrolactone, relying on cyanoborohy-
dride for coupling to a keto function and a water-soluble
carbodiimide for coupling to a carboxyl function. Further
reaction with butyric anhydride led to products that could
be detected by gas chromatography/ electron capture mass
spectrometry when 1 µL of ethyl acetate containing
essentially 2 0 amol of each product was injected, on the
basis of selected ion monitoring of the analyte character-
istic anion fragment from dissociative loss of the 3 ,5 -bis-
(trifluoromethyl)phenylmethyl moiety: m / z 2 1 5 , 2 8 9 ,
2 9 9 , and 3 2 9 , respectively. Since many small, organic
analytes contain a keto or carboxylic acid group, AMACE1
should be useful in general in the area of trace organic
analysis.
Figure 1. Synthesis of AMACE1 (12).
Electrophores are substances that have a high electron affinity
number of diverse, water-soluble products also makes their
in the gas phase. This usually makes them very detectable with
analysis challenging.8 We decided to tackle this detection problem
an electron capture detector1,2 or an electron capture mass
by using electrophore derivatization-GC/ EC-MS.
spectrometer,2,4 traditionally after gas chromatography (GC-ECD
The presence of a keto, carboxylic acid, lactone, or aldehyde
and GC/ EC-MS). Commonly an electrophore is a polyhalogenated
moiety on many of the DNA sugar oxidation products motivated
organic compound, and small analytes that are not electrophores
us to develop an electrophore reagent containing an amino group
inherently often can become so by derivatization. For example,
with a relatively low pKa so that all of these functional groups
the detection of DNA adducts by electrophore derivatization-GC/
could be derivatized conveniently and efficiently under aqueous
EC-MS is of interest.5,6
conditions. Further, we wanted to generate a reagent possessing
There is a need to improve the detection of oxidative damage
an appropriately oriented, internal dissociative electron capture
to the sugars of DNA, a class of DNA adducts.7 In part more
site in order to provide a sensitive, universal, analyte-characteristic
sensitive detection is needed so that small biological samples can
response. This latter concept previously was introduced by others.9
be analyzed. This could help, for example, to elucidate the
Here we report success in this overall endeavor by the preparation
metabolic pathways that repair these lesions in humans. The large
and testing of an aminoacetamide electrophore reagent that we
have named “AMACE1” (a-maj′-sej-1).
(1) Zlatkis, A., Poole, C. F., Eds.; Electron Capture; Elsevier: Amsterdam, 1981.
(2) Corkill, J. A.; Joppich, M.; Kuttab, S. H.; Giese, R. W. Anal. Chem. 1 9 8 2 ,
54, 481-485.
EXPERIMENTAL SECTION
(3) Knighton, W. B.; Sears, L. J.; Grimsrud, E. P. Mass Spectrom. Rev. 1 9 9 6 ,
Reagents. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hy-
14, 327-343.
drochloride (EDAC) and 2-(N-morpholino)ethanesulfonic acid
(4) Wang, P.; Murugaiah, V.; Yeung, B.; Vouros, P.; Giese, R. W. Anal. Chem.
1 9 9 6 , 721, 289-296.
(5) Giese, R. W. Chem. Res. Toxicol. 1 9 9 7 , 10, 255-270.
(6) Giese, R. W.; Saha, M.; Abdel-Baky, S.; Allam, K. Methods. Enzymol. 1 9 9 6 ,
271, 504-522.
(8) von Sonntag, C. The Chemical Basis of Radiation Biology; Taylor & Francis:
New York, 1987.
(9) Caesar, J. P., Jr.; Sheeley, D. M.; Reinhold: V. N. Anal. Biochem. 1 9 9 0 ,
191, 247-252.
(7) Demple, B.; Harrison, L. Annu. Rev. Biochem. 1 9 9 4 , 63, 915-948.
1798 Analytical Chemistry, Vol. 72, No. 8, April 15, 2000
10.1021/ac990384f CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/17/2000

Figure 2. Synthesis of electrophoric derivatives of compounds 1-4.
(MES) were from Sigma (St. Louis, MO). N-Hydroxysuccinimide-
3-sulfonate (NHS-SO3) was from Pierce Chemical Co. (Rockford,
IL). All solvents were from Fisher Scientific (Pittsburgh, PA), and
all reagents were from Aldrich (Milwaukee, WI) unless indicated
otherwise. Anhydrous ethanol was prepared by distillation of
anhydrous ethanol (Aldrich) from Mg/ catalytic I2, and converted
to 1 N HCl/ ethanol by HCl bubbling with weight monitoring. The
pyridine (Py) was distilled and then stored over KOH. Immu-
nopure epoxy-activated Agarose was from Pierce Chemical Co.
(Rockford, IL). AMACE1 will soon be available from Fluka (Buchs,
Switzerland) as product number 29245.
P rocedures. Flash column chromatography was performed
with silica gel 60 (170-400 mesh) from Fisher, and the bed was
19 mm × 45.7 cm (d × h) unless indicated otherwise. 1H and 13C
NMR were obtained on a Varian ZL300 NMR. GC/ EC-MS spectra
were recorded on a Hewlett-Packard 5973 by injecting 1µL of
sample in ethyl acetate (Ultra from Fisher). All evaporations or
concentrations were done on a rotary evaporator with water
aspirator vacuum, and all extractive-washing steps were 3× with
0.1 volume, unless indicated otherwise.
2 -Tritylam ino-N-[(3 ,5 -bis(triflu orom ethyl)benzyl]acet-
amide (1 0 ). To a solution of N-tritylglycine (0.635 g, 2 mmol),
3,5-bis[(trifluoromethyl)benzyl]amine (0.535 g, 2.2 mmol), and
1-hydroxybenzotriazole hydrate (HOBt, 0.554 g, 4.1 mmol) in 4
mL of dimethylformamide (DMF) was added triethylamine (710
µL, 5.1 mmol) followed by EDAC (0.595 g, 3.1 mmol). The mixture
was stirred overnight at room temperature, then quenched with
10 mL of water, extracted with ethyl acetate, and washed with
ice-cold 0.5 N HCl, water, ice-cold 0.5 N NaOH, and brine. After
neutral pH was confirmed with pH paper, the solution was dried
over MgSO4 and the solvent removed by evaporation. The
resulting crude product was dissolved in 1 mL of ethyl acetate
and purified by flash chromatography using 1:4 acetone/ hexanes,
monitoring 2-mL fractions by TLC-spotting UV quench, to give
an oil (0.943 g, 77%): 1H NMR (CDCl3, 300 MHz) δ 7.80-7.60 (d,
3H), 7.55-7.20 (m, 15H), 5.12 (s, 2H), 3.24 (s, 2H), 1.5 (b, 1H).
2 -Tritylamino-N-[(3 ,5 -bis(trifluoromethyl)benzyl]-N-me-
thylacetamide (1 1 ). Under N2, to 0.814 g (1.5 mmol) of 1 0 in
15 mL of anhydrous (distilled from Na) tetrahydrofuran (THF)
was added 0.060 g (1.5 mmol) of NaH (as a 60% dispersion in
mineral oil; used as received) at -5 °C. The resulting mixture
was stirred for 30 min and then 0.277 g (1.95 mmol) of MeI in 5
mL of THF was added dropwise over 30 min. The mixture was
stirred for 4 h and then quenched by pouring into 30 mL of cold
water saturated with NH4Cl. The solution was extracted with ethyl
acetate, and the combined organic layers were washed with brine,
dried, and concentrated to give an oil that was purified by flash
chromatography on silica gel with 1:4 acetone/ hexanes to give
an oil (0.78 g, 93%): 1H NMR (CDCl3, 300 MHz) δ 7.82-7.60 (d,
3H), 7.52-7.16 (m, 15H), 4.09 (s, 2H), 3.25 (s, 2H), 2.65 (s, 3H).
Acetamide, 2-Amino-N-[[3,5-bis(trifluoromethyl)phenyl]-
methyl]-N-methyl-, Monohydrochloride (12) (AMACE1: Ami-
noacetamide Electrophore 1 ). To 0.556 g (1 mmol) of 1 1 was
added 2 mL of 1 N HCl/ EtOH (see Reagents), and the solution
was hand-shaken in a 60 °C bath for 5 min. Cooling to room
temperature gave an off-white flocculent precipitate which was
scintered-glass filtered and washed with 5 mL of cold diethyl ether
to give white cubes (0.344 g, 98%), mp 227-228 °C: 1H NMR
(DMSO, 300 MHz) δ 8.15 (b, 2H), 8.05-7.95 (d, 3H), 4.75 (s,
2H), 4.00 (s, 2H), 3.00 (s, 3H); 13C NMR (DMSO, 300 MHz)
168.31, 142.10, 132.15, 131.80, 131.36, 129.66, 126.42, 122.73,
122.55, 51.09, 35.54. Calcd mass for M, 315.24; obsd, 314.9. pKa
) 8.2 by pH titration in 0 or 8% methanol.
Coupling of AMACE1 to Glycolic Acid, Yielding 1 3 . A
solution of 0.040 g (0.3 mmol) of glycolic acid, 0.148 g of EDAC
(0.775 mmol), and 0.217 g of NHS-SO3 (1 mmol) in 4 mL of 0.1
M MES buffer (pH 6.0, adjusted with NaOH) was stirred at room
temperature for 15 min under N2, followed by the addition of 0.175
Analytical Chemistry, Vol. 72, No. 8, April 15, 2000 1799

Figure 3. (A) Structures and fragmentation by electron capture of electrophoric derivatives 5-8 prepared by separately reacting compounds
1-4, respectively, with AMACE1 followed by butyric anhydride, and mass chromatogram obtained by injecting 1 µL of ethyl acetate containing
10 fg each of these derivatives into a GC/EC-MS. (B) Blank chromatogram obtained by injecting ethyl acetate. GC/EC-MS conditions: model
6890 GC interfaced to a 5973 MS (Hewlett-Packard), Ultra 1 (cross-linked methyl siloxane) column, L ) 50 m, i.d. ) 0.2 mm, 0.11-µm film
thickness; inject with column at 100 °C, then immediately ramp at 20 °C/min up to 300 °C, and hold for 10 min; multiple selected ion monitor
m/z 215, 289, 299, and 329.
g (0.5 mmol) of 1 2 . After 6 h of further stirring, the reaction
mixture was quenched with 15 mL of water and extracted with
ethyl acetate. The extract was washed with ice-cold 0.5 N HCl,
water, ice-cold 0.5 N NaOH, and brine and dried over MgSO4.
Evaporation then flash chromatography with 2:1 acetone/ hexanes
gave white needles (0.151 g, 81%), mp 138-139.5 °C: 1H NMR
(CDCl3, 300 MHz) δ 7.91-7.82 (d, 3H), 7.25 (b, 1H), 4.85 (s, 2H),
4.24-4.21 (d, 2H), 4.06-4.02 (d, 2H), 3.15 (s, 3H).
way as 1 3 except pH 7.0 MES buffer was utilized in place of pH
6.0 MES buffer, giving a colorless oil or white plates (0.166 g,
80%), mp 121-123 °C: 1H NMR(CDCl3, 300 MHz) δ 8.21-7.96
(d, 3H), 7.78-7.74 (d, 1H), 7.59-7.54 (m, 1H), 7.49-7.25 (m, 1H),
4.83 (s, 2H), 4.09-4.07 (d, 2H), 3.79-3.68 (m, 1H), 3.20 (s, 2H),
2.09 (s, 3H), 2.08-2.03 (m, 2H); 13C NMR (CDCl3, 300 MHz) δ
174.94, 169.18, 139.26, 132.38, 131.93, 128.06, 124.94, 121.81,
121.33, 71.40, 60.21, 50.93, 40.92, 36.07, 34.28.
Butyrylation of 1 3 , Yielding 5 . To a mixture of 0.149 g of
1 3 (0.4 mmol), 0.146 g of 4-(dimethylamino)pyridine (DMAP)
(1.2 mmol), and 1 mL of pyridine in 5 mL of anhydrous acetonitrile
(distilled from P2O5) was added 196 µL (1.2 mmol) of butyric
anhydride and the mixture was stirred at 45-50 °C for 12 h, when
TLC with 1:1 ethyl acetate/ hexanes showed reaction completion.
The reaction was quenched by pouring into 10 mL of ice water
and extracted with ethyl acetate. The combined organic layers
were washed with ice-cold 0.5 N NaOH, water, and ice-cold
saturated aqueous NH4Cl and dried over Na2SO4. Concentration
gave a crude yellow oil which was purified by flash chromatog-
raphy with 1:1 ethyl acetate/ hexanes to give a colorless oil (0.127
g, 72%): 1H NMR (CDCl3, 300 MHz) δ 7.87-7.71 (d, 3H), 4.74
(s, 2H), 4.66 (s, 2H), 4.21-4.20 (d, 2H), 3.01 (s, 3H), 2.47-2.44
(t, 2H), 1.78-1.76 (m, 2H), 1.04-0.99 (t, 3H).
Butyrylation of 1 4 , Yielding 8 . Compound 8 was prepared
in the same way as 5 except that the molar amounts of pyridine,
butyric anhydride, and DMAP relative to starting material were
doubled, yielding a colorless light oil (0.162 g, 73%): 1H NMR
(CDCl3, 300 MHz): δ 7.84-7.71 (d, 3H), 7.22 (b, 1H), 5.39-5.37
(t, 1H), 4.73 (s, 2H), 4.73-4.12 (m, 6H), 3.00 (s, 3H), 2.48-2.43
(m, 2H), 2.66-2.32 (m, 2H), 2.65-2.18 (m, 2H), 1.79-1.64 (m,
4H), 1.02-0.95 (m, 6H).
Attachment of AMACE1 to 3 -P henylbutyraldehyde (3 ) by
Reductive Amination, Yielding 1 5 . To a solution of 0.525 g (1.5
mmol) of AMACE1 in 4 mL of 0.1 M MES buffer (pH 6.0) was
added 74 mg (0.5 mmol) of 3-phenylbutyraldehyde, followed by
33 mg (0.5 mmol) of NaCNBH3. The mixture was stirred at room
temperature under N2 overnight, quenched into 15 mL of ice-cold,
saturated, aqueous sodium bicarbonate, and extracted with ethyl
acetate. The combined organic layers were washed with brine,
dried over Na2SO4, and concentrated to give a yellow oil, which
Coupling of AMACE1 to r-Hydroxy-γ-Butyrolactone
(4 a, b), Yielding 1 4 . Compound 1 4 was prepared in the same
1800 Analytical Chemistry, Vol. 72, No. 8, April 15, 2000

was purified by flash chromatography with 3:5 ethyl acetate/
hexanes to give a colorless light oil (0.156 g, 70%): 1H NMR-
(CDCl3, 300 MHz) δ 7.82, 7.70 (d, 3H), 7.34-7.16 (m, 5H), 4.70
(s, 2H), 3.48 (s, 2H), 2.94 (s, 3H), 2.89-2.79 (m, 1H), 2.62-2.48
(m, 2H), 1.9-1.81 (m, 2H), 1.29-1.25 (d, 3H).
electron capture conditions (see below). Fifth, the electrophoric
3,5-bis(trifluoromethyl)benzyl moiety makes our reagent easy to
handle (UV-active and nonvolatile at room temperature) and is
itself chemically inert.
As shown in Figure 2, true or model analytes for oxidative
sugar damage, glycolate (1 ), 3-hydroxy-2-butanone (2 ), 3-phenyl-
butyraldehyde (3), and R-hydroxy-γ-butyrolactone (4a) were each
tagged with AMACE1 by a direct nucleophilic (4 ), Schiff base/
NaCNBH3 (2 , 3 ), or carbodiimide-induced (1 , 4 b) reaction,
followed by reaction with butyric anhydride at residual active
hydrogen sites, to form electrophores 5 -8 . The reactions were
conducted at the milligram level, and TLC showed essentially
complete conversion of starting material to product in each case;
isolated yields for the products were g60%.
Shown in Figure 3 is a GC/ EC-MS chromatogram obtained
by injecting a mixture containing 10 fg each of the products
(ranging from 18 to 23 amol) as diluted standards. To obtain this
multiple selected ion chromatogram, four ions were monitored,
one for each of the analytes, on the basis of the intended,
dissociative electron capture. In part, high sensitivity is achieved
since the electron capture behavior of each derivative (full-scan
data not shown) is dominated by the characteristic ion that is
monitored in Figure 3.
Butyrylation of 1 5 , Yielding 7 . Compound 7 was prepared
from 1 5 in the same way that 8 was prepared from 1 4 , yielding
product as a colorless light oil (0.136 g, 66%): 1H NMR (CDCl3,
300 MHz) δ 7.83-7.70 (d, 3H), 7.32-7.19 (m, 5H), 4.67 (s, 2H),
4.14-3.98 (m, 2H), 3.36-3.25 (m, 2H), 3.62-3.60 (d, 3H), 2.72-
2.66 (m, 1H), 2.36-2.31 (t, 2H), 2.10-2.05 (m, 2H), 1.89-1.81
(m, 2H), 1.72-1.57 (m, 2H), 1.32-1.30 (d, 3H), 1.01-0.98 (t, 3H).
Attachment of AMACE1 to 3 -Hydroxy-2 -butanone (2 ) by
Reductive Amination, Yielding 1 6 . Compound 1 6 was pre-
pared from 2 in the same way that 1 5 was prepared from 3 ,
yielding a colorless light oil (0.115 g, 62%): 1H NMR (CDCl3, 300
MHz) δ 7.85-7.72 (d, 3H), 4.74 (s, 2H), 4.15-4.10 (m, 1H), 3.66
(s, 2H), 3.00 (s, 3H), 3.09-2.99 (m, 1H), 1.58-1.55 (d, 3H), 1.46-
1.40 (d, 3H).
Butyrylation of 1 6 , Yielding 6 . Compound 6 was prepared
from 1 6 in the same way that 8 was prepared from 1 4 except
the reaction temperature was raised to 60-65 °C, yielding a
colorless light oil (0.183 g, 61%): 1H NMR (CDCl3, 300 MHz) δ
7.85-7.70 (d, 3H), 4.96-4.91 (m, 1H), 4.82-4.59 (q, 2H), 4.32-
3.70 (q, 2H), 4.11-4.03 (m, 1H), 3.08-3.05 (d, 3H), 2.99 (s, 3H),
2.46-2.11 (m, 3H), 1.78-1.63 (m, 4H), 1.31-1.28 (d, 4H), 1.03-
0.90 (m, 6H).
Since the AMACE1 labeling reactions are all conducted in
water under mild, similar conditions, potentially they can be
conducted sequentially in the same vial. Work on this is in
progress, along with effort to perform the overall analysis on
oxidized sugar analytes at the trace level starting with a biological
sample.
RESULTS AND DISCUSSION
Prior to selecting butyrylation as a way to remove residual
active hydrogens (NH and OH) in the intermediate products
derived from the labeling reaction with AMACE1, we explored
pivaloylation and acetylation. The former reaction gave high yields
of product from compounds 1 3 -1 5 , but the yield from 1 6 was
very low, apparently for steric reasons. Acetylation gave good
yields in all cases, but the acetyl products tailed more than the
pivaloyl products by GC/ MS. Butyrylation provided high yields
throughout, and the peak shapes of the products were similar to
those of the pivaloyl derivatives.
Other sugar and sugar-related analytes contain one or more
of the functional groups that can be labeled by AMACE1, and so
do other kinds of small analytes. AMACE1 should be useful in
general for trace organic analysis, given the above-cited advan-
tages including the aqueous coupling conditions.
AMACE1 (1 2 ) was prepared by the scheme shown in Figure
1. As seen, N-tritylglycine was coupled to 3′,5′-bis(trifluoromethyl)-
benzylamine using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride, triethylamine, and 1-hydroxybenzotriazole in dim-
ethylformamide (87% yield), followed by methylation with methyl
iodide in NaH/ tetrahydrofuran (93%) and deprotection using 1 N
HCl anhydrous ethanol (98%).
AMACE1 was designed to have some favorable properties for
our purposes, all of which have been realized. First, it possesses
a primary amine as a functional group with a low pKa of 8.2. This
enhances the ease of amino coupling AMACE1 onto electrophilic
sites (e.g., aldehydes, ketones, activated carboxyls) under aqueous
conditions, as about to be demonstrated. Second, aldehyde-
substituted scavenger supports are available to remove residual,
primary amine reagents.10,11 Third, this functional amino group
exists as an R-aminoketone, which sets up another way to remove
residual reagent: immobilized metal ion affinity chromatogra-
phy.12,13 Fourth, AMACE1 incorporates a novel electrophoric
structure which efficiently yields an N-methylamide anion under
ACKNOWLEDGMENT
This work was supported by NIH Grant CA 71993 received as
a subcontract from Harvard Medical School. The authors thank
Gang Shao for valuable comments concerning the manuscript.
Contribution No. 769 from the Barnett Institute.
(10) Mort, A. J.; Zhan, D.; Rodriguez, V. Electrophoresis 1 9 9 8 , 19, 2129-2132.
(11) Kaldor, S. W.; Siegel, M. G.; Fritz, J. E.; Dressman, B. A.; Hahn, P. J.
Tetrahedron Lett. 1 9 9 6 , 37, 7193-7196.
(12) Hansen, P.; Lindeberg, G.; Andersson, L. J. Chromatogr. 1 9 9 2 , 627, 125-
135.
Received for review April 13, 1999. Accepted June 25,
1999.
(13) Shen, X.; Giese, R. W. J. Chromatogr., A 1 9 9 7 , 777, 261-265.
AC990384F
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