Synthesis of deuterium-labeled analogs of the lipid hydroperoxide-derived bifunctional electrophile 4-oxo-2(E)-nonenal

Article abstract of DOI:10.1002/jlcr.1860

Lipid hydroperoxides undergo homolytic decomposition into the bifunctional 4-hydroxy-2(E)-nonenal and 4-oxo-2(E)-nonenal (ONE). These bifunctional electrophiles are highly reactive and can readily modify intracellular molecules including glutathione (GSH), deoxyribonucleic acid (DNA) and proteins. Lipid hydroperoxide-derived bifunctional electrophiles are thought to contribute to the pathogenesis of a number of diseases. ONE is an α,β-unsaturated aldehyde that can react in multiple ways and with glutathione, proteins and DNA. Heavy isotope-labeled analogs of ONE are not readily available for conducting mechanistic studies or for use as internal standards in mass spectrometry (MS)-based assays. An efficient one-step cost-effective method has been developed for the preparation of C-9 deuterium-labeled ONE. In addition, a method for specific deuterium labeling of ONE at C-2, C-3 or both C-2 and C-3 has been developed. This latter method involved the selective reduction of an intermediate alkyne either by lithium aluminum hydride or lithium aluminum deuteride and quenching with water or deuterium oxide. The availability of these heavy isotope analogs will be useful as internal standards for quantitative studies employing MS and for conducting mechanistic studies of complex interactions between ONE and DNA bases as well as between ONE and proximal amino acid residues in peptides and proteins. Copyright

Full text of DOI:10.1002/jlcr.1860

Research Article
Received 21 September 2010,
Revised 26 November 2010,
Accepted 30 November 2010
Published online 17 February 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1860
Synthesis of deuterium-labeled analogs of the
lipid hydroperoxide-derived bifunctional
electrophile 4-oxo-2(E)-nonenal
Ã
Jasbir S. Arora,a Tomoyuki Oe,b and Ian A. Blaira
Lipid hydroperoxides undergo homolytic decomposition into the bifunctional 4-hydroxy-2(E)-nonenal and 4-oxo-2(E)-
nonenal (ONE). These bifunctional electrophiles are highly reactive and can readily modify intracellular molecules including
glutathione (GSH), deoxyribonucleic acid (DNA) and proteins. Lipid hydroperoxide-derived bifunctional electrophiles are
thought to contribute to the pathogenesis of a number of diseases. ONE is an a,b-unsaturated aldehyde that can react in
multiple ways and with glutathione, proteins and DNA. Heavy isotope-labeled analogs of ONE are not readily available for
conducting mechanistic studies or for use as internal standards in mass spectrometry (MS)-based assays. An efficient one-
step cost-effective method has been developed for the preparation of C-9 deuterium-labeled ONE. In addition, a method for
specific deuterium labeling of ONE at C-2, C-3 or both C-2 and C-3 has been developed. This latter method involved the
selective reduction of an intermediate alkyne either by lithium aluminum hydride or lithium aluminum deuteride and
quenching with water or deuterium oxide. The availability of these heavy isotope analogs will be useful as internal
standards for quantitative studies employing MS and for conducting mechanistic studies of complex interactions between
ONE and DNA bases as well as between ONE and proximal amino acid residues in peptides and proteins.
Keywords: 4-oxo-2-nonenal; oxidative stress; lipid peroxidation; deuterium labeling; DNA-adducts; protein adducts; pentyl furan
Introduction
Experimental
Materials and instruments
Reactive oxygen species (ROS) and oxidative stress have received
increasing attention because of their potential role in re-perfusion
injury,1 cancer2 and age-related diseases.3 Oxidative stress results
in ROS-mediated conversion of polyunsaturated lipids into
hydroperoxides. Subsequent homolytic decomposition of the lipid
hydroperoxides leads to the intermediate formation of 4-hydro-
peroxy-2(E)-nonenal which decomposes to the a,b-unsaturated
aldehydes, 4-oxo-2(E)-nonenal (ONE) and 4-hydroxy-2(E)-nonenal
(HNE).4–6 ONE is more neurotoxic and more reactive toward
proteins than HNE.7 It is also a particularly potent cytotoxin and
genotoxin,8,9 which reacts with deoxyribonucleic acid (DNA) bases
such as 20-deoxyguanosine (dGuo) to form heptanone-etheno-
dGuo adducts.10 ONE also reacts with histone proteins leading to
the formation of novel cyclic peptide adducts.11 The ONE adducts
with hemoglobin,12 N-acetyl-cysteine13,14 and GSH15 could be
useful as biomarkers various oxidative stress-related diseases.
The 4-oxo group of ONE inductively enhances the electrophilicity
of C-3 making it more susceptible to nucleophilic attack than HNE.
This enhanced electrophilicity provides an explanation for the
greater cytotoxicity and DNA reactivity of ONE when compared
with HNE. In order to comprehensively study the role of ONE in its
interactions with both small molecules and macromolecule, it is
necessary to have reliable and efficient methods available for the
NBS, sodium thiosulfate, citric acid, magnesium, bromoethane,
propiolaldehyde diethylacetal, hexanal, LAH, MnO2 and citric
acid were purchased from Aldrich. 2-pentyl furan was purchased
from Paulter and Baumann. NMR spectra were determined at
251C using a Varian UNITY 500 instrument with Tetramethylsi-
lane (TMS) as
a reference internal standard. HRMS was
performed using a Micromass Autospec in the Chemistry
department of University of Pennsylvania.
Synthesis section
ONE from 2-pentylfuran (4a)
To a solution of 2-pentyl furan 3a (2.5 g, 18.1 mmol) and pyridine
(2.8 mL, 35.1 mmol) in THF–acetone–H2O (5:4:1, 60 mL) was
added NBS (3.86 g, 21.7 mmol) dissolved in THF–acetone–H2O
aCenter for Cancer Pharmacology, University of Pennsylvania, Philadelphia, PA
19104, USA
bDepartment of Bio-analytical Chemistry, Graduate School of Pharmaceutical
synthesis of specifically heavy isotope-labeled analogs. We have Sciences, Tohoku University, Aoba-ku, Sendai 980-8578, Japan
previously reported a multi-step synthesis of ONE and HNE starting
*Correspondence to: Ian A. Blair, Center for Cancer Pharmacology, University of
from ethyl (triphenylphosphoranylidene) acetate.16 Herein, we
Pennsylvania, School of Medicine, 854 BRB II/III, 421 Curie Boulevard,
Philadelphia, PA 19104-6160, USA.
E-mail: ian@mail.med.upen.edu, ianblair@mail.med.upenn.edu
report the synthesis of the deuterium-labeled version of ONE with
selective labeling at C-9, C-2, C-3 and C-2/C-3 positions.
J. Label Compd. Radiopharm 2011, 54 247–251
Copyright r 2011 John Wiley & Sons, Ltd.

J. S. Arora et al.
(5:4:1, 20 mL) at ꢀ201C. The solution was stirred for 1 h at ꢀ201C (18.24 g, 80%). 1H-NMR (500 MHz, CDCl3)d: 0.89 (t, J = 7 Hz, 3H,
and 6 h at room temperature and then poured into a mixture of CH3), 1.24–1.72 (m, 14H, 4xCH212xCH3), 3.59 (t, J = 7 Hz, 2H,
ethyl acetate and aqueous Na2S2O3. The organic layer was CH2), 3.72 (t, J = 7 Hz, 2H, CH2), 4.43 (s, 1H, CH), 5.30 (s, 1H, CH).
separated and washed with aqueous citric acid solution (pH 3).
The organic layer was dried and evaporated under reduced 4-Hydroxy-non-2-enal-dimethylacetal (7)
pressure. The residue was purified on a silica gel column using
To a solution of 6 (10 g, 43.8 mmol) in anhydrous ether (200 mL)
at ꢀ251C was added lithium aluminum anhydride (3.32 g,
87.5 mmol) slowly, under an atmosphere of argon. The
suspension was stirred for 6 h at ꢀ251C and then carefully
hydrolyzed by adding 10 mL of saturated ammonium chloride
solution. The temperature of the reaction mixture was kept
below ꢀ51C during the addition. The precipitates were filtered.
Washed the mother liquor with water, dried the ether layer with
sodium carbonate and evaporated the solvent under reduced
10% ethyl acetate in hexane to afford the title compound
1
(1.23 g, 44%). H-NMR (500 MHz, CDCl3) d: 0.9 (t, J = 6.9 Hz, 3H,
C(9)-CH3), 1.34–1.38 (m, 4H, C(7,8)-CH2), 1.66 (p, J = 7.5 Hz, 2H,
C(6)-CH2), 2.68 (t, J = 7.4 Hz, 2H, C(5)CH2), 6.76 (dd, J = 6.9,
16.2 Hz, 1H, C(8)-CH), 6.87 (d, J = 16.2 Hz, 1H, C(7)-CH), 9.78
(d, J = 6.9 Hz, 1H, CH). 13C-NMR (CDCl3)d: 13.7, 22.3, 23.2, 31.1,
41.0, 137.1, 144.9, 193.4, 200.0.
9-[2H3]-ONE (4b)
pressure. The purification of the residue by column chromato-
n-Butyl lithium (19.5 mL, 2.5 M in hexane, 48.8 mmol) was added graphy using 15% ethyl acetate in hexanes resulted in pure 7 as
to furan 1 (5.90 mL, 81.21 mmol) in anhydrous THF (75 mL) under yellow oil (6.55 g, 65%). 1H-NMR (500 MHz, CDCl3)d: 0.88–0.89 (m,
argon at 01C. The resulting brown solution was brought to room 3H, CH3), 1.22 (t, J = 7 Hz, 6H, 2xCH3), 1.30–1.32 (m, 6H, 3xCH2),
temperature and stirred for additional 3 h. Bromopentane 2 (2 g, 1.58 (m, 2H, CH2), 3.49–3.52 (m, 2H, CH2), 3.63–3.66 (m, 2H, CH2),
13.1 mmol) was added to the reaction mixture at 01C. The 3.97 (brs, 1H, OH), 4.15 (s, 1H, CH), 4.89–4.90 (m, 1H, CH),
reaction mixture was stirred for additional 4 h at room 5.67–5.76 (m, 1H, CH), 5.85–5.89 (m, 1H, CH). Using similar
temperature and quenched by adding saturated NH4Cl solution. reaction conditions as for 7 and following a combination of
The mixture was extracted with ethyl acetate, dried (sodium lithium aluminum hydride or lithium aluminum deuteride with
sulfate) and evaporated under reduced pressure. Without water or deuterium oxide compounds 9, 11 and 13 were
further purification of deuterated 2-pentyl furan 3b (2.5 g, synthesized.
17.7 mmol), added pyridine (2.8 mL, 35.1 mmol) and a mixture of
THF-acetone-H2O (5:4:1, 60 mL). To the above solution was 2-[2H]-4-hydroxy-non-2-enal-dimethylacetal (9)
slowly added NBS (3.78 g, 21.2 mmol) dissolved in THF–acetone–
Reduction of
6 was conducted using lithium aluminum
H2O (5:4:1, 20 mL) at ꢀ201C. The solution was stirred for 1 h at
ꢀ201C and 6 h at room temperature and then poured into a
mixture of ethyl acetate and aqueous Na2S2O3. The organic layer
was separated and washed with aqueous citric acid solution
(pH 3). The organic layer was dried and evaporated under
reduced pressure. The residue was purified on a silica gel
column using 10% ethyl acetate in hexane to afford the title
compound 4b as yellow oil (1.32 g, 48%). 1H-NMR (500 MHz,
CDCl3)d: 1.30–1.32 (m, 4H, C(7,8)-CH2), 1.66 (p, J = 7.5 Hz, 2H,
C(6)-CH2), 2.68 (t, J = 7.5 Hz, 2H, C(5)-CH2), 6.78 (dd, J = 7.5, 16 Hz,
1H, C(2)-H), 6.87 (d, J = 16 Hz, 1H, C(3)-H), 9.78 (d, J = 7.5 Hz, 1H,
CH). 13C-NMR (CDCl3)d: 14.0, 22.5, 23.5, 31.4, 41.3, 137.4, 144.7 (t),
193.5, 200.3. HRMS (m/z): [M1H]1 calcd for C9[2H3]H11O2,
158.1260; found, 158.1260.
deuteride and the quenching was performed by a saturated
solution of deuterated ammonium chloride in deuterium oxide.
Yield 65%, yellow oil. 1H-NMR (500 MHz, CDCl3)d: 0.89 (t, J = 7 Hz,
3H, CH3), 1.22 (t, J = 7 Hz, 6H, 2xCH3), 1.28–1.32 (m, 6H, 3xCH2),
1.53 (p, J = 7 Hz, 2H, CH2), 3.47–3.53 (m, 2H, CH2), 3.62–3.67 (m,
2H, CH2), 3.96 (brs, 1H, OH), 4.14 (d, J = 5 Hz, 1H, CH), 4.89 (s, 1H,
CH), 5.84 (s, 1H, CH).
3[-2H]-4-hydroxy-non-2-enal-dimethylacetal (11)
Reduction of
6 was conducted using lithium aluminum
deuteride and the quenching was conducted using a saturated
solution of ammonium chloride in water. Yield 65%, yellow oil.
1H-NMR (500 MHz, CDCl3)d: 0.88 (t, J = 7 Hz, 3H, CH3), 1.22
(t, J = 7 Hz, 6H, 2xCH3), 1.30–1.37 (m, 6H, 3xCH2), 1.58–1.54
(m, 2H, CH2), 3.47–3.53 (m, 2H, CH2), 3.61–3.67 (m, 2H, CH2), 4.14
(s, 1H, CH), 4.89 (d, J = 5 Hz, 1H, CH), 5.68 (s, 1H, CH).
4-Hydroxy-non-2-ynal-dimethylacetal (6)
In a three-necked flask containing magnesium (2.43 g, 0.1 mol)
and THF (50 mL) was slowly added a solution of bromoethane
(10.83 g, 0.1 mol) in THF (50 mL) under an atmosphere of argon.
The solution of Grignard was cooled to ꢀ101C while stirring,
resulting in the precipitation of ethyl magnesium bromide. The
suspension was stirred intensely at ꢀ101C and to it was added a
solution of propiolaldehyde diethylacetal 5 (12.81 g, 0.1 mol) in
THF (50 mL). The solution was warmed to 01C and the stirring
was continued for further 2 h. A solution of hexanal (10.01 g,
0.1 mol) in THF (50 mL) was added to the above suspension at
ꢀ151C slowly. The suspension was stirred for further 4 h at
ꢀ101C. After the completion of reaction, saturated ammonium
chloride was added while cooling. The organic phase was
extracted with ether (3 ꢁ 200 mL). Dried the organic layer with
sodium carbonate and evaporated under reduced pressure.
Purification of the residue by column chromatography using
10% ethyl acetate in hexanes as an eluent resulted in pure 6
2,3-[2H2]-4-hydroxy-non-2-enal-dimethylacetal (13)
Reduction of
6 was performed using lithium aluminum
deuteride and the quenching was conducted using a saturated
solution of deuterated ammonium chloride in deuterium oxide.
Yield 65%, yellow oil 1H-NMR (500 MHz, CDCl3)d: 0.88 (t, J = 7 Hz,
3H, CH3), 1.22 (t, J = 7 Hz, 6H, 2xCH3), 1.28–1.32 (m, 6H, 3xCH2),
1.52–1.53 (m, 2H, CH2), 3.47–3.53 (m, 2H, CH2), 3.62–3.67 (m, 2H,
CH2), 3.94 (brs, 1H, OH), 4.14–4.15 (m, 1H, CH), 4.93 (s, 1H, CH).
4-Oxo-non-2-ena-dimethylacetal (8)
To a solution of 7 (6 g, 0.026 mol) in hexanes was added MnO2
(34.02 g, 0.39 mol). The suspension was allowed to stir under
an atmosphere of argon for 24 h. The solid was filtered and
the mother liquor was evaporated under reduced pressure.
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J. S. Arora et al.
Purification of the residue by column chromatography using 5% 2,3-[2H2]-ONE (17)
ethyl acetate in hexanes resulted in pure 8 (3.568 g, 60%).
Yield 60%, yellow oil. 1 H-NMR (500 MHz, CDCl3)d: 0.90 (t, J = 7 Hz,
1H-NMR (500 MHz, CDCl3)d: 0.89 (t, J = 7 Hz, 3H, CH3), 1.21–1.24
(t, J = 7 Hz, 6H, 2xCH3), 1.30–1.33 (m, 4H, 2xCH2), 1.62 (pentet,
J = 7.5 Hz, 2H, CH2), 2.56 (t, J = 7 Hz, 2H, CH2), 3.50–3.56 (m, 2H,
CH2), 3.62–3.68 (m, 2H, CH2), 5.04–5.05 (m, 1H, CH), 6.33 (d,
J = 16.5 Hz, 1H, CH), 6.62 (dd, J = 4.5, 16.25 Hz, 1H, CH). Using the
same procedure, compounds 10, 12 and 14 were synthesized
from 9, 11 and 13, respectively. Yields and NMR spectra are
provided below.
3H, CH3), 1.32–1.33 (m, 4H, 2xCH2), 1.67 (p, J = 7.5 Hz, 2H, CH2),
2.68 (t, J = 7.5 Hz, 2H, CH2), 9.78 (s, 1H, CH). 13C-NMR (CDCl3)d: 14,
22.5, 23.5, 31.4, 41.3, 137.1 (t), 144.6 (t), 193.6, 200.3. HRMS (m/z):
[M1H]1 calculated for C9H12[2H2]O2, 157.1198; found, 157.1202.
Results and discussion
2-Pentyl furan 3a was used to synthesize the unlabeled ONE 4a
by ring opening of the 2-substituted furan using N-bromosucci-
nimide (NBS) in a solvent mixture consisting of acetone, THF and
water containing pyridine as a base, the conditions reported by
Kobayashi et al.17 for the ring opening of 2-substituted furans.
A similar ring opening, for the synthesis of ONE, was reported
recently (24% yield);18 however, in our hands the yields of the
ring opening of the 2-substituted furans with modified
conditions (Figure 1) was higher (44–48%). The analytical data
2-[2H]-4-oxo-non-2-enal-dimethylacetal (10)
Yield, 60%, yellow oil. 1H-NMR (500 MHz, CDCl3)d: 0.89 (t, J = 7 Hz,
3H, CH3), 1.23 (t, J = 7.5 Hz, 6H, 2xCH3), 1.28–1.33 (m, 4H, CH2),
1.62 (p, J = 7 Hz, 2H, CH2), 2.57 (t, J = 7 Hz, 2H, CH2), 3.50–3.56 (m,
2H, CH2), 3.62–3.68 (m, 2H, CH2), 5.04 (s, 1H, CH), 6.33 (s, 1H, CH).
3-[2H]-4-oxo-non-2-enal-dimethylacetal (12)
Yield 60%, yellow oil. 1H-NMR (500 MHz, CDCl3)d: 0.89 (t, J = 7 Hz,
3H, CH3), 1.23 (t, J = 7 Hz, 6H, 2xCH3), 1.33–1.34 (m, 4H, 2xCH2),
1.60 (pentet, J = 7.5 Hz, 2H, CH2), 2.56 (t, J = 7.5 Hz, 2H, CH2),
3.5–3.56 (m, 2H, CH2), 3.6–3.68 (m, 2H, CH2), 5.04 (d, J = 4.5 Hz,
1H, CH), 6.62 (s, 1H, CH).
2,3-[2H2]-4-oxo-non-2-enal-dimethylacetal (14)
Yield 60%, yellow oil. 1H-NMR (500 MHz, CDCl3)d: 0.89 (t, J = 7 Hz,
3H, CH3), 1.23 (t, J = 7 Hz, 6H, 2xCH3), 1.28–1.33 (m, 4H, 2xCH2),
1.60–1.65 (m, 2H, CH2), 2.57 (t, J = 7 Hz, 2H, CH2), 3.50–3.56 (m,
2H, CH2), 3.62–3.68 (m, 2H, CH2), 5.04 (s, 1H, CH).
Synthesis of ONE (4a) from 8
Made a suspension of 8 (3g, 13mmol) in threefold volume (9 mL)
of 1% aqueous citric acid and allowed the reaction to run for
another 24h. After the completion of the reaction, the solution
was extracted with dichloromethane (3ꢁ 25mL). Dried the
organic layer (Na2SO4) and evaporated under reduced pressure.
Purification of the residue using 5% ethyl acetate in hexanes
resulted in 4a as yellow oil (1.2g, 60%). The analytical data of this
compound is similar to the ONE synthesized from 2-pentyl furan.
Using the same procedure, labeled ONE analogs 15, 16 and 17
were synthesized from 10, 12 and 14, respectively. Yields, NMR
spectra and HRMS data are provided below.
Figure 1. Synthesis of deuterium-labeled (C-9) and -unlabeled ONE from furan
derivatives.
2-[2H]-ONE (15)
Yield 60%, yellow oil. 1H-NMR (500 MHz, CDCl3)d: 0.90 (t, J = 7 Hz,
3H, CH3), 1.32–1.33 (m, 4H, 2xCH2), 1.66 (p, J = 7 Hz, 2H, CH2),
2.69–2.70 (t, J = 7 Hz, 2H, CH2), 6.86 (s, 1H, CH), 9.78 (s, 1H, CH).
13C-NMR (CDCl3)d: 13.9, 22.5, 23.4, 31.3, 41.3, 137.0 (t), 144.9,
193.5, 200.3. HRMS (m/z): [M1H]1 calcd for C9H13[2H]O2,
156.1135; found, 156.1132.
3-[2H]-ONE (16)
Yield 60%, yellow oil. 1H-NMR (500 MHz, CDCl3)d: 0.89 (t, J = 7 Hz,
3H, CH3), 1.29–1.33 (m, 4H, 2xCH2), 1.66 (p, J = 7.5 Hz, 2H, CH2),
2.69 (t, J = 7 Hz, 2H, CH2), 6.76–6.77 (m, 1H, CH), 9.79 (d,
J = 7.5 Hz, 1H, CH). 13C-NMR (CDCl3)d: 14.0, 22.5, 23.5, 31.4, 41.3,
137.4, 144.7 (t), 193.5, 200.3. HRMS (m/z): [M1H]1 calculated for
C9H213HO2, 156.1135; found, 156.1143.
Figure 2. Comparison of 1H-NMR spectra between deuterium-labeled and
-unlabeled ONE.
J. Label Compd. Radiopharm 2011, 54 247–251
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J. S. Arora et al.
of 4a was same as has been reported earlier.16 The synthesis of Intermediate 3b was subjected to ring opening using NBS
C-9 labeled ONE started with reaction of the anion of furan 1, and the product was purified using column chromatography.
generated with BuLi, with C-5 deuterium-labeled bromopentane The 1H-NMR spectrum of 4b did not show any C-9 methyl
to provide the labeled 2-pentyl furan analog 3b (Figure 1). protons at 0.9 ppm that were observed for the unlabeled ONE
4a (Figure 2). The 13C-NMR spectrum revealed a very weak
triplet at 13 ppm instead of a singlet in 4a at 13.7 ppm (Figure 3)
confirming the incorporation of three deuterium atoms at C-9
position of ONE. High-resolution (HR) mass spectrometry (MS) of
[M1H]1 further supported the formation of 3b with complete
labeling at C-9.
In a second synthetic strategy (Figure 4), the acetylenic
hydrogen of commercially available propiolaldehyde diethylacetal
5 was abstracted with ethyl magnesium bromide to convert 5 to
propiolaldehyde diethylacetal magnesium bromide in situ. The
addition of hexanal to this intermediate resulted in the formation
of 4-hydroxy-non-2-ynal dimethylacetal 6 in 80% yield. Dimethy-
lacetal 6 was exploited to synthesize ONE and its deuterium-
labeled versions. Addition of lithium aluminum hydride to
dimethylacetal 6 followed by aqueous work-up gave 4-hydroxy-
non-2-enal-dimethylacetal 7, which was oxidized with manganese
dioxide to provide 8 in 60% yield. Deprotection of 8 under acidic
conditions gave ONE 4a. In a slightly modified procedure, the
reduction in dimethylacetal 6 with lithium aluminum hydride and
work-up with deuterium oxide, saturated with deuterated
ammonium chloride, gave intermediate 9, which had deuterium
at C-2 of 4-hydroxy-non-2-enal-dimethylacetal. The 1H-NMR
Figure 3. Comparison of 13C-NMR spectra between deuterium-labeled and
-unlabeled ONE.
spectrum of 9 showed the absence of the multiplet at
5.67ppm as compared with the 1H-NMR spectrum of 7,
Figure 4. Synthesis of C-2, C-3 and C-2/C3-deuterium-labeled ONE and -unlabeled ONE.
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J. Label Compd. Radiopharm 2011, 54 247–251

J. S. Arora et al.
confirming the incorporation of deuterium at C-2. Oxidation of 9 between ONE and DNA bases9 as well as between ONE and
and subsequent deprotection of the deuterium-labeled inter- proximal amino acid residues in peptides19 and proteins.11
mediate 10 gave labeled ONE 15 with deuterium at C-2.
1
The H-NMR spectrum of 15 showed a singlet at 9.78ppm for Acknowledgement
the aldehyde hydrogen instead of the doublet observed in 4a
(Figure 2). The signal at 6.76ppm observed in 4a was also absent
confirming the presence of deuterium at C-2 in 15. The 13C-NMR
spectrum of 15 showed a weak triplet at 137 ppm, whereas the
unlabeled ONE 4a had a strong singlet at 137 ppm (Figure 3).
HRMS confirmed the incorporation of one deuterium into 15.
We acknowledge the support of NIH grants R01CA091016 and
P30ES013508.
For the synthesis of ONE 16 with a deuterium at C-3, the References
dimethylacetal 6 was reduced with lithium aluminum deuteride
and the reaction was quenched by the addition of a saturated
solution of ammonium chloride in water to give intermediate 11
with deuterium incorporated at C-3. Subsequent oxidation with
MnO2 to 12 followed by deprotection resulted in the formation
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from the C-3 proton at 6.87 ppm (Figure 2) and the presence of
week triplet in the 13C-NMR spectrum at 144.7 ppm (Figure 3).
ONE 17 having deuterium atoms at both C-2 and C-3 was
synthesized by reduction of dimethylacetal 6 with lithium
aluminum deuterated with a work-up using deuterium oxide
saturated with deuterium-labeled ammonium chloride. This
yielded intermediate 13 having deuterium labeling at both C-2
and C-3 positions. Subsequent oxidation with MnO2 and
acidic deprotection resulted in the formation of ONE 17. The
1H-NMR spectrum of 17 did not show any signals in the region
6–8 ppm. In addition, the aldehyde proton appeared as singlet
(Figure 2). The 13C-NMR spectrum of 17 showed two weak
triplets at 137.1 and 144.6 due to carbon deuterium coupling
instead of the corresponding singlets seen in unlabeled ONE 4a
at the same positions (Figure 3). HRMS of the labeled compound
further confirmed the incorporation of two deuterium atoms
into ONE 17.
In summary, we have demonstrated that deuterium-labeled
analogs of ONE can be synthesized in good yields starting from
the commercially available propiolaldehyde diethylacetal. The
availability of these heavy isotope analogs will be useful as
internal standards for quantitative studies employing MS and
for conducting mechanistic studies of complex interactions
J. Label Compd. Radiopharm 2011, 54 247–251
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