Synthesis of 13C labeled sulfur and nitrogen mustard metabolites as mass spectrometry standards for monitoring and detecting chemical warfare agents

Article abstract of DOI:10.1002/jlcr.2929

¹³C labeled (>M + 4) metabolites of nitrogen and sulfur-based chemical warfare agent metabolites were prepared from readily available and ¹³C labeled commercial starting materials. The new chemical routes are efficient in the number

Full text of DOI:10.1002/jlcr.2929

Research Article
Received 12 April 2012,
Accepted 13 April 2012
Published online 14 May 2012 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.2929
Synthesis of 13C labeled sulfur and nitrogen
mustard metabolites as mass spectrometry
standards for monitoring and detecting
chemical warfare agents
a
a
b
c
*
Ruilian Wu, Siegfried N. Lodwig, Jurgen G. Schmidt, Robert F. Williams,
and Louis A. "Pete" Silksa
13C labeled (>M + 4) metabolites of nitrogen and sulfur-based chemical warfare agent metabolites were prepared from
readily available and 13C labeled commercial starting materials. The new chemical routes are efficient in the number of
chemical steps, can be scaled to afford gram quantities, and occur in good yields on the basis of the 13C label. These labeled
compounds are useful as internal standards in mass spectrometry for monitoring chemical warfare agents and their
metabolites.
Keywords: carbon-13 labeled; sulfur and nitrogen mustard metabolites
In addition, all of the 13C labeled metabolites constructed and
Introduction
reported herein have not been reported to the best of our
knowledge.7
The detection of chemical warfare agents, their precursors and
by-products of synthesis, metabolism, and degradation products
is important in connection with the threat of terrorism, ongoing
The synthesis of the labeled metabolites thiodiglycol (13C4) 2
and thiodiglycol sulfone (13C4) 3 derived from hydrolysis and
military operations, and verification of compliance with the
oxidation processes in biological and environmental systems,
Chemical Weapons Convention.1 Many modern analytical methods
are useful for immediate and accurate detection, such as chromato-
graphy, mass spectrometry, capillary electrophoresis, and on-site
real-time monitoring devices.2–6
and bismethylsulfanylethylsulfone (13C4) 5, bismethylsulfinylsul-
fone (13C4) 6 from glutathione conjugation, and b-lyase pathway
for sulfur mustard (13C4) HD metabolite is showed in Scheme 1.
We modified our previous synthesis (Scheme 2) to avoid the
intermediate sulfur mustard due to its associated hazards.
[1,1′,2,2′-13C4]diethyl thiodiglycolate (13C4) 1 was synthesized
by the reaction of ethyl bromoacetate (13C2) and sodium sulfide
in acetone at room temperature in 74% yield. Diethyl thiodigly-
colate (13C4) 1 was then reduced to thiodiglycol (13C4) 2 in 96%
yield with lithium borohydride in tetrahydrofuran. Thiodiglycol
(13C4) 2 was then oxidized to diglycol sulfone (13C4) 3 in 87%
yield by hydrogen peroxide in dilute phosphoric acid at reflux
temperature for 3 days (or in quantitative yield using Oxone in
1:3 EtOH/EtOAc at room temperature for 2 h). Diglycol sulfone
Labeled compounds are critically important serving as internal
standards for mass spectrometry, which confirm and quantify com-
pounds of interest. They also facilitate the study on the chemical
warfare agent (CWA) degradation processes and allow their
products to be tracked in biological and environmental systems.6
A recent publication on the synthesis of deuterated dialkyl-
aminoethyls as possible standards for the mass spectrometric
monitoring of CWAs6 prompted us to report our own synthetic
efforts in this area. This report is devoted to the design and
development of reliable synthetic methods for the synthesis of
13C labeled metabolites of sulfur and nitrogen vesicants. To the
best of our knowledge, this is the first comprehensive study of
the synthesis of derivatives of sulfur and nitrogen mustards both
in unlabeled and labeled fashion. These compounds have been
used as internal standards for the monitoring of CWAs at the
Centers for Disease Control and Prevention.5
aBioscience Division, Bioenergy and Environmental Science, the National Stable
Isotope Resource, Group B8, MS E529, Los Alamos National Laboratory, Los
Alamos, NM 87545, USA
bChemistry Division, Los Alamos National Laboratory, Los Alamos, NM 87545,
USA
Results and discussion
cBioscience Division, Biosecurity and Public Health Group, Los Alamos National
Previous syntheses of sulfur and nitrogen-based metabolites are Laboratory, Los Alamos, NM 87545, USA
known; however, the synthetic routes used were not amenable
*Correspondence to: Ruilian Wu, Bioscience Division, Bioenergy and Environmen-
tal Science, the National Stable Isotope Resource, Group B8, MS E529, Los Alamos
National Laboratory, Los Alamos, NM 87545, USA.
for 13C labeling. Several begin with compounds that are inconve-
nient to label. All of our routes begin with readily available or
simple to construct materials, such as ethyl bromoacetate. E-mail: Ruilian@lanl.gov
Published 2012. This article is a US Government work
and is in the public domain in the USA.
J. Label Compd. Radiopharm 2012, 55 211–222

R. Wu et al.
Scheme 1. Synthesis of the bismethylsulfinylsulfone (13C4) 6. (i) Na2S, acetone, 0ꢀ-rt, 18 h; (ii) LiBH4, THF, 0ꢀC-rt, 18 h; (iii) H2O2, 2% H3PO4, reflux, 3 days or Oxone,
EtOAc-EtOH, rt, 2 h; (iv) MsCl, 2,6-lutidine, EtOAc-ACN, rt, 2 days; (v) NaSMe, EtOH, 0ꢀC, 0.5 h; (vi) H2O2, H2O, rt, 18 h.
Scheme 2. Synthesis of bismethylsulfinylsulfone (13C4) 6 and monomethyl-sulfinylsulfone (13C4) 8. (i) TsCl, pyridine, CH2Cl2; (ii) Oxone, CH2Cl2-H2O; (iii) NaSMe, EtOH, 0ꢀC-rt;
(iv) 1 eq. H2O2, H2O, rt.
(13C4) 3 was converted to its mesylate derivative (13C4) 4 in 78% oxy-mustards bis(2-chloroethylthiomethyl)ether is shown in
yield with mesyl chloride followed by reaction with sodium Scheme 3.
thiomethoxide to afford bismethylsulfanylethylsulfone (13C4) 5
Ethyl bromoacetate (13C2) reacted with potassium thioacetate
in 66% yield, which was subsequently oxidized to bismethylsulfi- in acetone at room temperature to afford the thioester (13C2) 9 in
nylsulfone (13C4) 6 in 84% yield with hydrogen peroxide in water. 93% yield, which after acetate cleavage with sodium ethoxide
Using one equivalent of hydrogen peroxide gave rise to a mixture underwent nucleophilic displacement with bis(chloromethyl)
of bismethylsulfinylsulfone (13C4) 6, monomethylsulfinylsulfone ether to afford the thioether 10 (13C4) in 100% yield. Reduction
(13C4) 8 and unreacted starting material 5 (Scheme 2).
with lithium borohydride provided the desired bis(2-hydro-
Besides the commonly known bis(2-chloroethyl)sulfide (HD), xyethylthiomethyl)ether 11 (13C4) in 72% yield.
five sesqui-mustard and two oxy-mustard analogs are included
The synthesis of nitrogen mustard metabolites started with
in the Chemical Weapons Convention schedule of chemicals.8 2-bromoethanol (13C2), which was converted to 2-bromoethyl
These compounds have been shown to be two to five t-butyldimethylsilyl ether (13C2) 12 as shown in Scheme 4.
times more potent than HD.7a The synthesis of bis
The preparation of disubstituted metabolites of HN1 and HN2
(2-hydroxyethylthiomethyl)ether (13C4) 11, metabolite of blistering agents is illustrated in Scheme 5. Methyl or ethyl
Scheme 3. Synthesis of bis(2-hydroxyethylthiomethyl)ether (13C4) 11. (i) CH3COSK, acetone, rt, 18 h; (ii) (1) NaOEt, EtOH, 0ꢀC, 5 min; (2) ClCH2OCH2Cl, 0ꢀC-rt, 18 h; (iii) LiBH4,
THF, 0ꢀC-rt, 18h.
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J. Label Compd. Radiopharm 2012, 55 211–222

R. Wu et al.
Scheme 4. 2-Bromoethyl t-butyldimethylsilyl ether (13C2) 12.
Scheme 5. Synthesis of bis-(2-chloroethyl)methyl (or ethyl)amine hydrochloride (13C4) 17. (i) 12, K2CO3, ACN, reflux, 2 days; (ii) Pd(OH)2/C, EtOH, H2 (1 atm), rt, 18h; (iii) 12,
K2CO3, ACN, reflux, 18 h; (iv) THF, 1 M HCl, 0ꢀC-rt, 2 h; (v) SOCl2, CHCl3, reflux, 2.5 h.
benzylamine was reacted with 2-bromoethyl t-butyldimethylsilyl Reaction with t-butyl N-acetyl-(L)-cysteinate in the presence
ether (13C2) 12 to form tertiary amine 13 (13C2) in 92–94% yields, of potassium carbonate afforded the t-butyl N-acetyl-(L)-cysteinate
removal of benzyl group with palladium hydroxide/carbon 19 (13C4) in 63–75% yields. The latter was then reacted with trifluor-
afforded 2-(methyl or ethylamino)ethylsilyl ether 14 (13C2) in oacetic acid/triisopropylsilane to remove the t-butyl protecting
95–100% yields. The secondary amine was then reacted with groups giving rise to the desired product 20 (13C4) as trifluoroacetic
2-bromoethyl t-butyldimethylsilyl ether (13C2) 12 to form acid salt in 97–100% yields (Scheme 6).
2-(methyl or ethylamino)diethylsilyl ether 15 (13C4) in 87–91%
The synthesis of monosubstituted metabolites of HN1 and HN2
yields. These products were then subjected to diluted hydrochlo- is illustrated in Scheme 7. Intermediate 14 (13C2) from Scheme 5
ric acid in THF to afford 2-(methyl or ethylamino)-diethanol was treated with 1 M hydrochloric acid to form N-ethanolamine
hydrochloride (13C4) 16 in 92–95% yields. Subsequently, 16 was hydrochloride 21 (13C2) in 100% yields. Additional reaction with
converted to the corresponding bis-(2-chloroethyl)methyl (or 2-bromoethyl t-butyldimethylsilyl ether 12 (13C2) formed its ter-
ethyl)amine hydrochloride (13C4) 17 in 99–100% yields.
tiary amine 22 (13C4) in 85–89% yields. This was then converted
Reaction of 17 with sodium thiomethoxide smoothly gave to its chloride 23 (13C4) by the treatment of mesyl chloride in
rise to methylsulfanyl metabolites 18 (13C4) in 80–90% yields. 66–68% yields. With 23 (13C4) in hand, the t-butyl N-acetyl-(L)-
Scheme 6. Synthesis of to methylsulfanyl metabolites (13C4) 18 and N-acetyl-(L)-cysteine metabolites (13C4) 20. (i) t-Butyl N-acetyl-(L)-cysteinate, K2CO3, ACN, reflux, 18 h;
(ii) TFA, iPr3SiH, CH2Cl2, 0ꢀC-rt, 18 h; (c) NaSMe, EtOH, 1.5 h.
J. Label Compd. Radiopharm 2012, 55 211–222
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R. Wu et al.
Scheme 7. Synthesis of methyl or ethylethanolamine N-acetyl-(L)-cysteine derivative (13C4) 25. (i) THF, 1 M HCl, 0ꢀC-rt, 2 h; (ii) 12, K2CO3, ACN, reflux, 2 days; (iii) MsCl,
iPr2NEt, CH2Cl2, 0ꢀC-rt, 18 h; (iv) t-Butyl N-acetyl-(L)-cysteinate, K2CO3, ACN, reflux, 2 h; (v) (1) TFA, iPr3SiH, CH2Cl2, 0ꢀC-rt, 18 h; (2) 1 M HCl, THF, 0ꢀC-rt, 18 h.
cysteinate derivative 24 (13C4) could be easily obtained in 96–97% tris(2-t-butyl N-acetyl-(L)-cysteinate ethylamine) (13C6) 32, and
yields. Deprotection of the t-butyl group then gave rise to the tris(2-N-acetyl-(L)-cysteinate ethylamine) (13C6) 33 were pre-
methyl or ethylethanolamine N-acetyl-(L)-cysteinate derivative pared by straightforward substitution of the chlorides in 95%,
25 (13C4) as hydrochloric acid salt in 100% yields.
83%, and 100% yields, respectively (Scheme 9).
The synthesis of trisubstituted metabolites of HN3 is illustrated in
The synthesis of disubstituted metabolites of HN3 was achieved
Scheme 8. Benzylamine was reacted with two equivalents of 2-bro- as illustrated in Scheme 10. The intermediate 27 (13C4) from
moethyl t-butyldimethylsilyl ether 12 (13C2) to form the tertiary Scheme 8 was treated with 1 M hydrochloric acid to form diethano-
amine 26 (13C4) in 92% yield. The benzyl group was then removed lamine hydrochloride 34 (13C4) in 100% yield. Reaction of
with palladium hydroxide/carbon in 100% yield, the resulting sec- 34 with 2-bromoethyl t-butyldimethylsilyl ether 12 (13C2) gave rise
ondary amine 27 (13C4) reacted again with 2-bromoethyl t-butyl to the tertiary amine 35 (13C6) in 98% yield. Treatment of 35 with
dimethylsilyl ether 12 (13C2) to form triethanolamine silyl ether mesyl chloride afforded the chloride 36 (13C6) in 76% yield. With
28 (13C6) in 86% yield. Desilylation took place using 1 M hydrochloric 36 (13C6) in hand, the dimethylthio derivative 37 (13C6) and t-butyl
acid in THF to form triethanolamine hydrochloride 29 (13C6) in N-acetyl-(L)-cysteinate derivative 39 (13C6) could be easily obtained
98% yield. Reaction of 29 with thionyl chloride smoothly con- in 91% and 76% yields, respectively. The protecting group of disub-
verted the triol to the tris(2-chloroethyl)amine hydrochloride stituted methylthio 37 (13C6) was removed with hydrochloric acid
30 (13C6) in 99% yield. Tris(2-methylthioethyl)amine 31 (13C6), giving rise to 38 in 100% yield. The N-acetyl-(L)-cysteine 40 (13C6)
Scheme 8. Synthesis of tris(2-chloroethyl)amine hydrochloride (13C6) 30. (i) 12, K2CO3, ACN, reflux, 3 days; (ii) Pd(OH)2/C, EtOH, H2 (1 atm), rt, 18 h; (iii) 12, K2CO3, ACN,
reflux, 2 days; (iv) THF, 1 M HCl, 0ꢀC-rt, 18 h; (v) SOCl2, reflux, 2.5 h.
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R. Wu et al.
Scheme 9. Synthesis of tris(2-methylthioethyl)amine 31 (13C6) and tris(2-N-acetyl-(L)- cysteine ethylamine) (13C6) 33. (i) t-Butyl N-acetyl-(L)-cysteinate, K2CO3, ACN, reflux,
18 h; (ii) TFA, iPr3SiH, CH2Cl2, 0ꢀC-rt, 18h; (iii) NaSMe, EtOH, reflux, 1.5 h.
Scheme 10. Synthesis of disubstituted methylthio 38 (13C6) and N-acetyl-(L)-cysteine 40 (13C6). (i) THF, 1 M HCl, 0ꢀC-rt, 2 h; (ii) 12, K2CO3, ACN, reflux, 3 days; (iii) MsCl,
iPr2NEt, CH2Cl2, 0ꢀC-rt, 18 h; (iv) NaSMe, EtOH, reflux, 1.5 h; (v) THF, 1 M HCl, 0ꢀC-rt, 18 h; (vi) t-Butyl N-acetyl-(L)-cysteinate, K2CO3, ACN, reflux, 18 h; (vii) (1) TFA, iPr3SiH,
0ꢀC-rt, 18 h; (2) THF, 1 M HCl, 0ꢀC-rt, 18 h.
was obtained by the sequential deprotection of the t-butyl and form tertiary amine 41 (13C6) in 73% yield. Subsequently, this was
silyl groups using trifluoroacetic acid/triisopropyl silane cocktail converted to its chloride 42 (13C6) by the treatment of mesyl
followed by hydrochloric acid in 80% yield.
The synthesis of monosubstituted N-acetyl-(L)-cysteine metabo- 44 (13C6) was obtained by standard procedures in 42% overall yield.
lite of HN3 is shown in Scheme 11. The intermediate 27 (13C4) was
In summary, we have developed efficient routes to a wide
heated to reflux with one equivalent of 2-bromoethanol (13C2) to variety of CWA metabolites that have not previously been
chloride in 72% yield. The mono N-acetyl-(L)-cysteine metabolite
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R. Wu et al.
Scheme 11. Synthesis of mono N-acetyl-(L)-cysteine metabolite 44 (13C6). (i) Br13CH123CH2OH, K2CO3, ACN, reflux, 2 days; (ii) MsCl, iPr2NEt, CH2Cl2, 0ꢀC-rt 3.5 h; (iii) t-Butyl N-
acetyl-(L)-cysteinate, K2CO3, ACN, reflux,18 h; (iv) (1) TFA, iPr3SiH, 0ꢀC-rt, 18 h; (2) THF, 1 M HCl, 0ꢀC-rt, 18 h.
13C-labeled. These compounds are currently being used by the
Centers for Disease Control and Prevention and other national
agencies for the detection, quantitation, and monitoring of
exposures to CW agents.
pressure to provide 1.187 g (87%) of 3 as a colorless liquid. Alternatively,
3 can be made as follows: thiodiglycol (13C4) 2 (3g, 23.8mmol) was
dissolved in 200 ml ethyl acetate/100 ml ethanol/10 ml water. Oxone
™(KHSO5 Á 0.5 KHSO4 Á 0.5 K2SO4, 17.5g, 57 mmol) was added in five
portions over a period of 10 min with vigorous stirring. After 2h, all starting
material was consumed and converted to the sulfone 3. The reaction mix-
ture was evaporated to dryness on a rotary evaporator, the residual salt
Experimental
was suspended in 100-ml ethyl acetate and filtered. The solid was extracted
1H and 13C NMR spectra were obtained with a Bruker DPX-300 or a Bruker
twice with 100 ml dichloromethane. The organic solvents were removed on
Avance 300 MHz spectrometer (Bruker BioSpin Corporation. Billerica, MA,
a rotary evaporator to provide 3.7g (98%) of 3 as a colorless liquid. 1H NMR
USA). Chemical shifts are reported in parts per million and referenced
(acetone-d6) d 3.99 (complex d, J = 143.4Hz), 3.31 (complex d, J = 133.8 Hz).
against tetramethylsilane. The coupling constants J are reported in Hz.
13C NMR (CDCl3) d 57.5, (t, J = 37.0 Hz), 57.0 (t, J= 36.9 Hz).
Mass spectra (MS) were recorded on a ThermoFisher LCQ DECAXPplus
liquid chromatography–mass spectrometry (LC-MS) or Polaris Q gas chro-
Thiodiglycol sulfone dimesylate (13C4) 4
To a solution of thiodiglycol sulfone (13C4) 3 (1.87 g, 7.50 mmol) in
matography–mass spectrometry (GC-MS) spectrometer (Thermo Fisher
Scientific Corporation, Waltham, MA, USA). Reagents were purchased
acetonitrile-ethyl acetate (3:2, 20 ml) was added 2,6-lutidine (1.91 ml,
from commercial sources and used as received. Chromatography was
16.5 mmol) and methanesulfonyl chloride (1.28 ml, 16.5 mmol) all in
one portion. The mixture was stirred at room temperature for 2 days.
The solvents were removed under reduced pressure, and the residue
was suspended in water. The solid was filtered and washed with water
performed on silica gel (230–400 mesh) with the indicated solvent sys-
tem. Thin layer chromatography (TLC) was used to monitor the reaction
progress and for product purifications.
to provide 1.845 g (78%) of 4 as a white solid. 1H NMR (acetone-d6)
Diethyl thiodiglycolate (13C4) 1
d 4.68 (ddt, J = 154.4, 5.6, 2.6 Hz), 3.69 (complex d, J = 139.4 Hz), 3.20 (s).
To a solution of ethyl bromoacetate (13C2) (4.246 g, 25.13 mmol) in
acetone (17.0 ml) was added sodium sulfide (1.318 g, 16.89 mmol) in one
portion at 0ꢀC. The mixture was allowed to warm to room temperature and
stirred overnight. The solvent was removed under reduced pressure, and
the residue was loaded on silical gel and eluted with 10% EtOAc/hexanes
to provide 1.962 g (74%) of 1 as a colorless liquid. 1H NMR (CDCl3) d 4.23
(dq, J= 7.2, 3.0 Hz), 3.41 (dt, J= 141.4, 4.9 Hz), 1.32 (t, J=7.2Hz). 13C NMR
(CDCl3) d 170.0, (d, J=62.0Hz), 61.7 (d, J=2.0Hz), 14.3 (d, J=2.0Hz).
13C NMR (acetone-d6) d 64.2 (complex d, J = 38.7 Hz), 54.7 (t, J = 38.8 Hz).
Bismethylsulfanylethylsulfone (13C4) 5
To a suspension of sodium thiomethoxide (0.524 g, 6.73 mmol) in ethanol
(12.0 ml) was added thiodiglycol sulfone dimesylate (13C4) 4 (0.784 g,
2.49 mmol) in one portion at 0ꢀC. The mixture was stirred at 0ꢀC for
30 min or until TLC showed a complete reaction. 1 M HCl (5.0 ml) was
then added and the mixture was allowed to warm to room temperature
and stirred overnight. The solvent was removed under reduced pressure,
and the residue was adsorbed and loaded on silica gel. Elution with 25%
ethyl acetate/hexanes provided 0.358 g (66%) of 5 as a white solid. 1H
Thiodiglycol (13C4) 2
To a solution of diethyl thiodiglycolate (13C4) 1 (1.94 g, 9.23 mmol) in
tetrahydrofuran (54.0 ml) was added lithium borohydride (11.54 ml,
23.08 mmol, 2.0 M in THF) dropwise at 0ꢀC, and the mixture was allowed
to warm to room temperature and stirred overnight. MeOH was added
slowly until the mixture was cleared and the solvents were removed
under reduced pressure. The residue was loaded on silica gel and eluted
with 10% MeOH/CH2Cl2 to provide 1.114 g (96%) of 2 as a colorless liquid.
NMR (CDCl3)
d 3.38 (complex d, J = 140.5 Hz), 2.92 (complex d,
J = 140.5 Hz), 2.21 (s). 13C NMR (CDCl3) d 53.5 (dt, J = 34.9, 3.18 Hz), 26.3
(dt, J = 34.9, 3.18 Hz), 15.9 (s). LC-MS m/z for 13C4C2H14O2S3 [M + Na À H]:
calc 240.3, found 239.7.
Bismethylsulfinylsulfone (13C4) 6
1H NMR (CDCl3) d 3.81 (d, J = 144.0 Hz), 2.80 (d, J = 140.0 Hz), 1.73 (brs). 13
NMR (CDCl3) d61.2, (d, J = 38.0 Hz), 35.2 (d, J = 38.0 Hz).
C
To a suspension of bismethylsulfanylethylsulfone (13C4) 5 (0.264 g,
1.21 mmol) in H2O (50.0 ml) was added 35% hydrogen peroxide
(0.199 ml. 2.42 mmol). The mixture was stirred at room temperature
overnight. The solvent was removed under reduced pressure, and the resi-
Thiodiglycol sulfone (13C4) 3
To a solution of thiodiglycol (13C4) 2 (1.083 g, 8.58 mmol) in 2% phospho- due was adsorbed on silica gel and loaded to a chromatographic column.
ric acid (12.6 ml) was added 35% hydrogen peroxide (2.10 ml), and the
Elution with 10% MeOH/CH2Cl2 provided 0.254g (84%) of 6 as a white
mixture was heated to reflux for 3 days. 13C NMR showed a complete reac- solid. 1H NMR (CDCl3) d 3.77 (complex d, J = 139.8 Hz), 3.47 (complex d,
tion. The solvent and excess reagents were removed under reduced J = 139.8Hz), 3.28 (complex d, J = 139.8Hz), 2.77 (d, J = 4.33 Hz). 13C NMR
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R. Wu et al.
N-methyl-N-2-t-butyldimethylsilyloxyethylamine (13C2) 14a
(CDCl3) d 46.1 (complex d, J= 35.8 Hz), 43.9 (complex d, J= 35.9 Hz), 36.9
(s). LC-MS m/z for 13C4C2H14O4S3 [M+]: calc 250.3, found 250.9.
To a solution of N-benzyl-N-methyl-N-2-t-butyldimethylsilyloxyethyla-
mine (13C2) 13a (0.243 g, 0.86 mmol) in MeOH (4.0 ml) was added Pd
(OH)2/C (0.050 g). A hydrogen balloon was used with an inlet needle
immersed in the suspension. The mixture was stirred overnight or until
TLC showed a complete reaction. The solids were filtered and washed
with MeOH. The solvent was removed under reduced pressure to provide
0.192 g (100%) of 14a as a colorless liquid. 1H NMR (CDCl3) d 3.69 (ddt,
J = 140.9, 4.77, 1.92 Hz), 2.65 (ddt, J = 132.8, 5.43, 2.96 Hz), 2.45 (d,
J = 5.43 Hz), 0.87 (s), 0.04 (s). 13C NMR (CDCl3) d 62.4(d, J = 40.5 Hz), 54.0
(d, J = 40.5 Hz), 36.5 (d, J = 4.17 Hz), 26.1, 18.6, À5.1.
Ethyl 2-acetylthioacetate (13C2) 9
To a solution of ethyl bromoacetate (13C2) (1.956 g, 11.57 mmol) in
acetone (30.0 ml) was added potassium thioacetate (1.95 g, 17.07 mmol)
at room temperature, and the mixture was stirred overnight. The solvent
was removed under reduced pressure, and the mixture was loaded on
silica gel. Elution with 10% ethyl acetate in hexanes provided 1.774 g
(93%) of 9 as a colorless liquid. 1H NMR (CDCl3) d 4.17 (dq, J = 7.13,
3.12 Hz), 3.67 (dd, J = 142.0, 6.19 Hz), 2.37 (s), 1.25 (t, J = 7.13 Hz). 13C
NMR (CDCl3) d 168.3 (d, J = 62.4 Hz), 31.1 (complex d, J = 62.4 Hz).
N-Methyl-N,N-bis(2-t-butyldimethylsilyloxyethyl)amine (13C4) 15a
Bis(2-ethylcarboxymethylthiomethyl)ether (13C4) 10
A mixture of N-methyl-N-2-t-butyldimethylsilyloxyethylamine (13C2) 14a
(0.344 g, 1.80 mmol), 2-bromoethyl t-butyldimethylsilyl ether (13C2) 12
(0.434 g, 1.80 mmol) and potassium carbonate (0.373 g, 2.70 mmol) in
acetonitrile (4.0 ml) was heated to reflux overnight. The mixture was
filtered and the filtrate was concentrated under reduced pressure to pro-
vide 0.577 g (91%) of 15a as a colorless liquid. 1H NMR (CDCl3) d 3.68
(complex d, J = 138.3 Hz), 2.56 (complex d, J = 141.4 Hz), 2.31 (buried d),
0.86 (s), 0.03 (s). 13C NMR (CDCl3) d 61.9(d, J = 43.5 Hz), 60.4 (d,
J = 43.5 Hz), 26.2, À5.1.
To a solution of 21% sodium ethoxide in ethanol (1.994 g, 6.00 mmol) in
ethanol (4.5 ml) was added ethyl 2-acetylthioacetate (13C2) 9 (0.986 g,
6.00 mmol) dropwise via a cannula at 0ꢀC. The mixture was stirred at this
temperature for 5 min. Bis(chloromethyl)ether (0.3 g, 2.61 mmol) was
added via a syringe. The mixture was allowed to warm to room tempera-
ture and stirred overnight. Water was added to the previous mixture and
was extracted with ethyl acetate. The solvents were removed under
reduced pressure, and the residue was purified on silica gel with 20%
ethyl acetate in hexanes to provide 0.817 g (100%) of 10 as a colorless
liquid. 1H NMR (CDCl3) d 4.88 (q, J = 5.21 Hz), 4.17 (dq, J = 7.13, 3.12 Hz),
N-methyldiethanolamine hydrochloride (13C4) 16a
3.28 (dd, J = 141.1, 5.48 Hz), 1.26 (t, J = 7.13 Hz). 13C NMR (CDCl3) d 170.5 To a solution of N-methyl-N,N-bis(2-t-butyldimethylsilyloxyethyl)amine
(d, J = 61.9 Hz), 69.7, 61.7, 32.0 (d, J = 61.9 Hz), 14.3.
(
13C4) 15a (0.572 g, 1.63 mmol) in THF (3.75 ml) was added 1 M HCl
(3.75 ml) at 0ꢀC. The mixture was allowed to warm to room temperature
and stirred overnight. The solvent and excess reagents were removed
under reduced pressure, and the residue was partitioned between ethyl
acetate and water. The aqueous layer was lyophilized to provide 0.241 g
(92%) of 16a as a colorless liquid. 1H NMR (D2O) d 3.91 (complex d,
J = 138.3 Hz), 2.56 (complex d, J = 141.4 Hz), 2.31 (buried d), 0.86 (s),
0.03 (s). 13C NMR (D2O) d 61.9(d, J = 43.5 Hz), 60.4 (d, J = 43.5 Hz), 26.2, À5.1.
Bis(2-hydroxyethylthiomethyl)ether (13C4) 11
To
a solution of bis(2-ethylcaroxymethylthiomethyl)ether (
13C4) 10
(0.814 g, 2.86 mmol) in THF (15.0 ml) was added lithium borohydride
(3.58 ml, 7.15 mmol, 2.0 M in THF) at 0ꢀC.9 The mixture was allowed to
warm to room temperature and stirred overnight. The solvent was
removed under reduced pressure and partitioned between water and
ethyl acetate. The organic extracts were collected and the solvent was
removed under reduced pressure, and the residue was purified on silica
gel with 40% ethyl acetate in hexanes to provide 0.416 g (72%) of 11 as
a colorless liquid. 1H NMR (CDCl3) d 4.81 (q, J = 5.15 Hz), 3.77 (dq,
J = 143.8, 5.70 Hz), 2.81 (ddq, J = 137.8, 5.75, 2.90 Hz). 13C NMR (CDCl3)
Bis-(2-chloroethyl)methylamine hydrochloride (13C4) 17a
To a solution of N-methyldiethanolamine hydrochloride (13C4) 16a
(0.239 g, 1.497 mmol) in chloroform (3.0 ml) was added thionyl chloride
(0.44 ml, 5.988 mmol), and the mixture was heated to reflux for 2.5 h.
The mixture was cooled to room temperature, and the excess thionyl
chloride was decomposed by the addition of MeOH. The solvents were
removed under reduced pressure to provide 0.291 g (99%) of 17a as a
white solid. 1H NMR (CDCl3) d 4.04 (br d, J = 149.4 Hz), 3.58 (br d,
J = 139.6 Hz), 3.42 (br d J = 141.7 Hz), 2.96 (s). 13C NMR (CDCl3) d 57.5
(dd, J = 38.3, 1.69 Hz), 36.7 (dd, J = 38.3, 1.69 Hz).
d
71.2, 62.0 (d, J = 37.1 Hz), 35.7 (d, J = 37.1 Hz). LC-MS m/z for
13C4C2H14O3S2 (M + H À CH2O) calc 173.3, found 172.9.
2-bromoethyl t-butyldimethylsilyl ether (13C2) 12
To a solution of 2-bromoethanol (13C2) (7.618 g, 60.0 mmol) in dichloro-
methane (35.0 ml) was added t-butyldimethylsilyl chloride (10.853 g,
72.0 mmol).10 The mixture was cooled with an ice bath, and N,N-diisopro-
pylethylamine (15.68 ml, 90.0 mmol) was added dropwise via a syringe
pump. The mixture was allowed to warm to room temperature slowly
and stirred overnight. The solvent and excess reagents were removed
with a rotary evaporator, and the residue was purified on silica gel with
2% ethyl acetate in hexanes to provide 14.922 g (99%) of 12 as a colorless
liquid. 1H NMR (CDCl3) d 3.87 (dt, J = 6.58, 2.90 Hz), 3.38 (dt, J = 6.58,
2.90 Hz), 0.88 (s), 0.07 (s). 13C NMR (CDCl3) d 63.7 (d, J = 40.8 Hz), 45.3 (d,
J = 41.0 Hz), 33.6 (d, J = 40.8 Hz), 26.0, À5.1.
N-bis(2-methylthioethyl)-N-methylamine (13C4) 18a
To a solution of bis-(2-chloroethyl)methylamine (13C4) 17a (0.687 g,
3.50 mmol) in EtOH (20.0 ml) was added sodium thiomethoxide
(0.980 g, 13.98 mmol) in one portion at room temperature. The mixture
was heated for 1–2 h. The solvent was removed under reduced pressure,
and the residue was purified on silica gel with 5% MeOH/CH2Cl2 to pro-
vide 0.515 (80%) of 18a as a light yellow liquid. 1H NMR (CDCl3) d 2.59
(complex d, J = 132.6 Hz), 2.27 (t, J = 5.26 Hz), 2.10 (d, J = 4.17 Hz). 13C
NMR (CDCl3) d 57.1 (dd, J = 38.9, 2.72 Hz), 42.2, 31.9 (d, J = 38.8 Hz), 16.0.
GC-MS m/z for 13C4C3H17NS2 (M + H): calc 184.3, found 184.0.
N-benzyl-N-methyl-N-2-t-butyldimethylsilyloxyethylamine (13C2)
13a
t-butyl-N-acetyl-(L)-cysteinate
A mixture of N-methylbenzylamine (1.82 g, 15.0 mmol), 2-bromoethyl
N-acetyl-(L)-cysteine (4.08 g, 25.0 mmol) was suspended in benzene
t-butyldimethylsilyl ether (13C2) 12 (2.41 g, 10.0 mmol) and potassium (25.0 ml) and t-butanol (10.0 ml). The mixture was heated to reflux.
carbonate (2.073 g, 15.0 mmol) in acetonitrile (20.0 ml) was heated to
Dimethylformamide bisneopentyl acetal (10.0 g, 43.2 mmol) was added
reflux for two days. The solvent was removed under reduced pressure through an additional funnel dropwise in 10 min. The mixture was
and the residue was purified on silica gel with 20% ethyl acetate in
heated to reflux for another 30 min. The mixture was cooled to room
hexanes to provide 2.581 g (92%) of 13a as a colorless liquid. 1H temperature and partitioned between water and ethyl acetate. The
NMR (CDCl3) d 7.32–7.19 (m), 3.76 (complex d, J = 138.2 Hz), 3.54 (d, organic extracts were washed with saturated sodium bicarbonate and
J = 3.84 Hz), 2.54 (complex d, J = 131.4 Hz), 2.24 (d, J = 4.66 Hz), 0.86 (s), brine and dried over sodium sulfate. The residue was purified on silica
0.02 (s). 13C NMR (CDCl3)
d 139.4, 129.2, 128.4, 127.1, 62.0 (d, gel with 50% ethyl acetate in hexanes to provide 2.492 g (45%) of
J = 43.0 Hz), 59.4 (d, J = 42.8 Hz), 43.3, 26.2, 18.5, À5.1.
t-butyl-N-acetyl-(L)-cysteinate as colorless syrup. 1H NMR (CDCl3)
J. Label Compd. Radiopharm 2012, 55 211–222
Published 2012. This article is a US Government work
and is in the public domain in the USA.
www.jlcr.org

R. Wu et al.
d 6.33 (br d, J = 4.66 Hz), 4.73 (two sets overlapping t, J = 3.89 Hz), 2.99 reduced pressure, and the residue was purified on silica gel with 20%
(dd, J = 4.06, 2.85 Hz), 2.96 (dd, J = 4.06, 2.85 Hz), 2.04 (s), 1.47 (s). 13C ethyl acetate in hexanes to provide 0.446 g (66%) of 23a as a colorless
NMR (CDCl3) d 170.0, 169.4, 83.3, 54.0, 28.2, 27.3, 23.5.
liquid. 1H NMR (CDCl3) d 3.70 (dt, J = 6.19, 138.1 Hz), 3.53 (ddt, J = 7.23,
3.07, 150.1 Hz), 2.79 (dhept, J = 3.45, 135.1 Hz), 2.59 (complex d,
J = 131.1 Hz), 2.33 (t, J = 4.88 Hz), 0.87 (s), 0.04 (s). 13C NMR (CDCl3)
d 61.8 (dd, J = 2.49, 42.5 Hz), 59.69 (dd, J = 2.59, 40.1 Hz), 59.66 (dd,
J = 2.59, 42.5 Hz), 43.4, 41.9 (dd, J = 2.59, 40.9 Hz), 26.2, 18.5, À5.1.
N-bis(2-N-acetyl-(L)-cysteine t-butyl ester ethyl)-N-methylamine
(13C4) 19a
To a solution of bis-(2-chloroethyl)-methylamine (13C4) 17a (0.098 g,
0.50 mmol) and t-butyl-N-acetyl-(L)-cysteinate (0.329 g, 1.50 mmol) in
acetonitrile (5.0 ml) was added potassium carbonate (0.276 g, 2.0 mmol),
and the mixture was heated to reflux overnight.11 The solvent was
removed under reduced pressure, and the residue was purified on silica
gel with 10% MeOH/CH2Cl2 to provide 0.165 g (63%) of 19a as a colorless
liquid. 1H NMR (CDCl3) d 6.47 (br d, J = 7.13 Hz), 4.69 (t, J = 4.99 Hz), 4.67 (t,
J = 4.99 Hz), 3.10–2.88 (m), 2.60 (complex d, J = 134.6 Hz), 2.23 (t,
J = 4.99 Hz), 2.02 (s), 1.46 (s). 13C NMR (CDCl3) d 170.0, 169.4, 83.3, 54.0,
28.2, 27.3, 23.5.
N-methyl-N-2-t-butyldimethylsilyloxyethyl-N-2-N-acetyl-(L)-cysteine
t-butyl ester ethylamine (13C4) 24a
To a solution of N-methyl-N-2-t-butyldimethylsilyloxyethyl-N-2-chloro-
ethylamine (13C4) 23a (0.181 g, 0.707 mmol) and t-butyl N-acetyl-(L)-
cysteinate (0.184 g, 0.84 mmol) in acetonitrile (3.50 ml) was added pota-
ssium carbonate (0.307 g, 2.22 mmol). The mixture was heated to reflux
for 1–2 h until TLC showed the reaction was complete. The solvent was
removed under reduced pressure, and the residue was purified on silica
gel with 10% MeOH/CH2Cl2 to provide 0.299 g (97%) of 24a as a colorless
liquid. 1H NMR (CDCl3) d 3.70 (dt, J = 6.19, 138.1 Hz), 3.53 (ddt, J = 7.23,
3.07, 150.1 Hz), 2.79 (dhept, J = 3.45, 135.1 Hz), 2.59 (complex d,
J = 131.1 Hz), 2.33 (t, J = 4.88 Hz), 0.87 (s), 0.04 (s). 13C NMR (CDCl3)
d 61.8 (dd, J = 2.49, 42.5 Hz), 59.69 (dd, J = 2.59, 40.1 Hz), 59.66 (dd,
J = 2.59, 42.5 Hz), 43.4, 41.9 (dd, J = 2.59, 40.9 Hz), 26.2, 18.5, À5.1.
N-bis(2-N-acetyl-(L)-cysteine ethyl)-N-methylamine (13C4) trifluoro-
acetic acid salt 20a
To a solution of N-bis(2-N-acetyl-(L)-cysteine t-butyl ester ethyl)-N-methy-
lamine (13C4) 19a (0.162 g, 0.309 mmol) in dichloromethane (2.54 ml) was
added triisopropylsilane (0.62 ml). The mixture was cooled with an ice
bath and trifluoroacetic acid (0.62 ml) was added. The mixture was then
allowed to warm to room temperature and stirred overnight. The solvent
and excess reagents were removed under reduced pressure, and the resi-
due was partitioned between ethyl acetate and water. The aqueous layer
was washed again with ethyl acetate and decolorized with active char-
coal. The mixture was filtered, and the filtrate was lyophilized to provide
0.218 g (97%) of 20a as a white solid. 1H NMR (D2O) d 4.62 (d, J = 4.77 Hz),
4.59 (d, J = 4.77 Hz), 3.65 (br d, J = 18.6 Hz), 3.27–3.07 (m), 3.0–2.85 (m),
2.85 (s), 2.78–2.66 (br s), 2.03 (s). 13C NMR (D2O) d 54.0 (d, J = 36.6 Hz),
25.7 (d, J = 36.6 Hz). GC-MS m/z for 13C4C11H27N3O6S2 (M À H): calc
412.5, found 412.2.
N-methyl-N-2-hydroxyethyl-N-2-N-acetyl cysteine ethylamine (13C4)
hydrochloric acid salt 25a
To a solution of N-methyl-N-2-t-butyldimethylsilyloxyethyl-N-2-N-acetyl-
(L)-cysteine t-butyl ester ethylamine (13C4) 24a (0.296 g, 0.674 mmol)
and triisopropylsilane (0.67 ml) in dichloromethane (2.8 ml) was added
dropwise trifluoroacetic acid (0.67 ml) at 0ꢀC. The mixture was allowed
to warm to room temperature and stirred overnight. The solvent and
excess reagents were removed under reduced pressure, and tetrahydro-
furan (0.8 ml) was added to the residue. The mixture was cooled to 0ꢀC,
and 1 M HCl (0.8 ml) was added dropwise, and the mixture was allowed
to warm to room temperature and stirred overnight. The solvent was
removed under reduced pressure, and the residue was partitioned
between ethyl acetate and water. The aqueous layer was lyophilized to
provide 0.218 g (100%) of 25a as a colorless hygroscopic solid. 1H NMR
(D2O) d 4.63–4.55 (m), 4.17–4.05 (m), 3.79–3.40 (m), 3.28–2.99 (m),
2.99–2.84 (m), 2.75–2.64(m), 2.02 (s). 13C NMR (D2O) d 174.4, 173.8, 57.2
(d, J = 38.0 Hz), 55.1 (d, J = 37.9 Hz), 54.9 (d, J = 36.7 Hz), 52.4, 40.3, 32.5,
25.5 (d, J = 36.9 Hz), 21.7. GC-MS m/z for 13C4C6H20N2O4S (M+ H): calc
269.3, found 269.0.
N-methylethanolamine hydrochloride (13C2) 21a
To a solution of N-methyl-N-2-t-butyldimethylsilyloxyethylamine (13C2)
14a (0.574 g, 3.0 mmol) in tetrahydrofuran (3.45 ml) was added 1 M
hydrochloric acid (3.45 ml) at 0ꢀC. The mixture was allowed to warm to
room temperature and stirred overnight. The solvent and excess
reagents were removed under reduced pressure, and the residue was
partitioned between ethyl acetate and water. The aqueous layer was con-
centrated and lyophilized to provide 0.347 g (100%) of 21a as a colorless
liquid. 1H NMR (D2O) d 4.62 (d, J = 4.77 Hz), 4.59 (d, J = 4.77 Hz), 3.65 (br d,
J = 18.6 Hz), 3.27–3.07 (m), 3.0–2.85 (m), 2.85 (s), 2.78–2.66 (br s), 2.03 (s).
13C NMR (D2O) d 54.0 (d, J = 36.6 Hz), 25.7 (d, J = 36.6 Hz).
N-benzyl-N-ethyl-N-2-t-butyldimethylsilyloxyethylamine (13C2) 13b
A mixture of N-ethylbenzylamine (2.03 g, 15.0 mmol), 2-bromoethyl
t-butyldimethylsilyl ether (13C2) 12 (2.41 g, 10.0 mmol) and potassium
carbonate (2.073 g, 15.0 mmol) in acetonitrile (20.0 ml) was heated to
reflux for 2 days. The solvent was removed under reduced pressure,
N-methyl-N-2-t-butyldimethylsilyloxyethyl-N-2-hydroxyethylamine
(13C4) 22a
To a solution of N-methylethanolamine hydrochloride (13C2) 21a (0.347 g, and the residue was purified on silica gel with 20% ethyl acetate in
3.0 mmol) and 2-bromoethyl t-butyldimethylsilyl ether (13C2) 12 (0.724 g, hexanes to provide 2.765 g (94%) of 13b as a colorless liquid. 1H NMR
3.0 mmol) in acetonitrile (8.24 ml) was added potassium carbonate
(CDCl3) d 7.34–7.17 (m), 3.67 (ddt, J = 6.36, 140.4, 2.14 Hz), 3.63 (d,
(1.244 g, 9.0 mmol), and the mixture was heated to reflux for 2 days. J = 4.11 Hz), 2.61 (ddt, J = 3.78, 6.91, 131.9 Hz), 2.57 (dq, J = 4.44,
The mixture was filtered, and the filtrate was concentrated to provide 7.07 Hz), 1.04 (d, J = 7.07 Hz), 0.87 (s), 0.02 (s). 13C NMR (CDCl3) d 140.3,
0.632 g (89%) of 22a as a colorless liquid. 1H NMR (CDCl3) d 3.68 (dt, 129.0, 128.3, 126.9, 62.2 (d, J = 43.0 Hz), 59.1, 55.5 (d, J = 43.1 Hz),
J = 5.97, 138.1 Hz), 3.54 (apparent dq, J = 5.49, 140.0 Hz), 2.65 (complex 48.5, 26.2, 18.5, 12.2, -5.1.
d, J = 128.9 Hz), 2.30 (t, J = 4.66 Hz), 0.87 (s), 0.04 (s). 13C NMR (CDCl3)
N-ethyl-N-2-t-butyldimethylsilyloxyethylamine (13C2) 14b
d
61.6 (dd, J = 2.01, 42.5 Hz), 59.4 (dd, J = 1.44, 42.7 Hz), 59.1 (d,
J = 2.16 Hz), 58.7 (d, J = 1.29 Hz), 42.7, 26.1, 18.5, À5.14.
To a solution of N-benzyl-N-ethyl-N-2-t-butyldimethylsilyloxyethylamine
13C2) 13b (0.137 g, 0.464 mmol) in MeOH (4.0ml) was added Pd(OH)2/C
(0.021 g). A hydrogen balloon was used with an inlet needle immersed in
the suspension. The mixture was stirred overnight or until TLC showed a
(
N-methyl-N-2-t-butyldimethylsilyloxyethyl-N-2-chloroethylamine
(13C4) 23a
To a solution of N-methyl-N-2-t-butyldimethylsilyloxyethyl-N-2-hydro- complete reaction. The solids were filtered and washed with MeOH. The
xyethylamine
(
13C4) 22a (0.628 g, 2.29 mmol) in dichloromethane filtrate was concentrated under reduced pressure to provide 0.091g
(6.0 ml) was added N,N-diisopropylethylamine (0.599 ml, 3.44 mmol). (95%) of 14b as a colorless semi-solid. 1H NMR (CDCl3) d 3.71 (ddt,
The mixture was cooled to 0ꢀC, and methanesulfonyl chloride J = 140.6, 5.00, 1.92 Hz), 2.69 (complex d, J = 133.6Hz), 2.64 (dq, J = 6.85,
(0.213 ml, 2.75 mmol) was added. It was then allowed to warm to room 3.23 Hz), 1.13 (t, J = 7.13Hz), 0.88 (s), 0.05 (s). 13C NMR (CDCl3) d 62.5 (d,
temperature and stirred overnight. The solvent was removed under J = 40.2Hz), 51.7 (d, J = 40.2 Hz), 44.1 (d, J = 3.74 Hz), 26.1, 15.5, À5.1.
www.jlcr.org
Published 2012. This article is a US Government work
and is in the public domain in the USA.
J. Label Compd. Radiopharm 2012, 55 211–222

R. Wu et al.
N-ethyl-N,N-bis(2-t-butyldimethylsilyloxyethyl)amine (13C4) 15b
A mixture of N-ethyl-N-2-t-butyl-dimethylsilyloxyethylamine (13C2) 14b
residue was partitioned between ethyl acetate and water. The aqueous
layer was washed again with ethyl acetate and decolorized with active
charcoal. The mixture was filtered, and the filtrate was lyophilized to
provide 0.215 g (100%) of 20b as a white solid. 1H NMR (D2O) d 4.61
(dd, J = 8.11, 4.77), 3.38 (br d, J = 143.5 Hz), 3.28 (m), 3.11 (t, J = 4.44 Hz),
3.0–12.89 (m), 2.95 (m), 2.02 (s), 1.28 (J= 7.23 Hz). 13C NMR (D2O) d 174.0,
173.8, 52.4, 51.4 (d, J = 36.9 Hz), 32.7, 25.6 (d, J = 36.7 Hz), 21.7, 7.96.
GC-MS m/z for 13C4C12H29N3O6S2 (M + H): calc 428.5, found 428.9.
(0.370 g, 1.80 mmol), 2-bromoethylt-butyldimethylsilyl ether
(
13C2)
12 (0.434 g, 1.80 mmol) and potassium carbonate (0.373 g, 2.70 mmol)
in acetonitrile (4.0 ml) was heated to reflux overnight. The mixture
was filtered and the filtrate was concentrated under reduced pressure
to provide 0.570 g (87%) of 15b as a colorless liquid. 1H NMR (CDCl3)
d 3.63 (ddt, J = 140.6, 6.74, 2.47 Hz), 2.60 (complex d, J = 131.5 Hz), 2.58
(m), 1.00 (t, J = 7.29 Hz), 0.87 (s), 0.03 (s). 13C NMR (CDCl3) d 62.3 (d,
J = 43.3 Hz), 56.8 (d, J = 43.3 Hz), 26.2, À5.15.
N-ethylethanolamine hydrochloride (13C2) 21b
To a solution of N-ethyl-N-2-t-butyldimethylsilyloxyethylamine (13C2) 14b
(0.616 g, 3.0mmol) in tetrahydrofuran (3.45 ml) was added 1M hydrochloric
acid (3.45 ml) at 0ꢀC.13 The mixture was allowed to warm to room
temperature and stirred for 2 h. The solvent was removed under
reduced pressure, and the residue was partitioned between ethyl
acetate and water. The aqueous layer was concentrated and lyophilized
to provide 0.382 g (100%) of 21b as a light yellow solid. 1H NMR (D2O) d
3.80 (dt, J = 144.6, 4.88 Hz), 3.09 (br d, J = 143.4 Hz), 3.09 (q, J = 7.29 Hz),
N-ethyldiethanolamine hydrochloride (13C4) 16b
To
(
a solution of N-ethyl-N,N-bis(2-t-butyldimethylsilyloxyethyl)amine
13C4) 15b (0.566 g, 1.55 mmol) in THF (3.60 ml) was added 1 M HCl
(3.60 ml) at 0ꢀC. The mixture was allowed to warm to room temperature
and stirred for 2 h. The solvent was removed under reduced pressure,
and the residue was partitioned between ethyl acetate and water. The
aqueous layer was lyophilized to provide 0.255 g (95%) of 16b as a yellow
solid. 1H NMR (D2O) d 3.91 (complex d, J = 143.6 Hz), 3.34 (complex d,
J = 143.1 Hz), 3.34 (m), 1.30 (t, J = 7.29 Hz). 13C NMR (D2O) d 55.3
(d, J = 38.2 Hz), 54.1 (d, J = 38.2 Hz).
1.18 (t, J = 7.40 Hz). 13C NMR (D2O)
(d, J = 37.8 Hz), 42.3, 9.83.
d 56.2 (d, J = 37.8 Hz), 48.1
Bis-(2-chloroethyl)ethylamine hydrochloride (13C4) 17b
N-ethyl-N-2-t-butyldimethylsilyloxy ethyl-N-2-hydroxyethylamine
(13C4) 22b
To a solution of N-ethyldiethanolamine hydrochloride (13C4) 16b (0.250 g,
1.44 mmol) in chloroform (3.0 ml) was added thionyl chloride (0.42 ml,
5.75 mmol), and the mixture was heated to reflux for 2.5 h. The mixture
was cooled to room temperature, and the excess thionyl chloride was
decomposed by MeOH. The solvents were removed under reduced pres-
sure to provide 0.302 g (100%) of 17b as a white solid. 1H NMR (CDCl3) d
4.05 (br d, J = 152.4 Hz), 3.48 (br d, J = 147.5 Hz), 3.33 (br s), 1.47 (t,
J = 7.29 Hz). 13C NMR (CDCl3) d 57.5 (dd, J = 38.3, 1.69 Hz), 36.7 (dd,
J = 38.3, 1.69 Hz).
To a solution of N-ethylethanolamine hydrochloride (13C2) 21b (0.383 g,
3.0 mmol) and 2-bromoethyl-t-butyldimethylsilyl ether
(
13C2) 12
(0.724 g, 3.0 mmol) in acetonitrile (8.24 ml) was added potassium carbo-
nate (1.244 g, 9.0 mmol), and the mixture was heated to reflux for
2 days.13 The mixture was filtered, and the filtrate was concentrated to
provide 0.642 g (85%) of 22b as a colorless liquid. 1H NMR (CDCl3) d
3.65 (ddt, J = 140.7, 5.59, 1.81 Hz), 3.53 (apparent dq, J = 140.7, 5.26 Hz),
2.71–2.59 (m), 2.64 (complex d, J = 131.7 Hz), 1.02 (t, J = 7.13 Hz), 0.87 (s),
0.04 (s). 13C NMR (CDCl3) d 61.8 (d, J = 42.8 Hz), 58.9 (d, J = 38.5 Hz), 55.7
(d, J = 38.4 Hz), 55.4 (d, J = 42.8 Hz), 48.6, 26.1, 18.5, À5.14.
N-bis(2-methylthioethyl)-N-ethyl amine (13C4) 18b
To
a solution of bis-(2-chloroethyl)ethylamine (
13C4) 17b (0.191 g,
0.90 mmol) in EtOH (7.0 ml) was added sodium thiomethoxide (0.252 g,
3.6 mmol) in one portion at room temperature. The mixture was heated
to reflux for 1–2 h. The solvent was removed under reduced pressure,
and the residue was purified on silica gel with 5% MeOH/CH2Cl2 to pro-
vide 0.160 g (90%) of 18b as a light yellow liquid. 1H NMR (CDCl3) d 2.68
(complex d, J = 132.8 Hz), 2.57 (complex d, J = 136.6 Hz), 2.57 (m), 1.02 (t,
J = 7.13 Hz). 13C NMR (CDCl3) d 53.3 (d, J = 38.7 Hz), 47.8, 32.1 (d,
J = 38.7 Hz), 16.1, 12.1. GC-MS m/z for 13C4C4H19NS2 (M + H): calc 198.3,
found 198.0.
N-ethyl-N-2-t-butyldimethylsilyloxy ethyl-N-2-chloroethylamine
(13C4) 23b
To
a
solution of N-ethyl-N-2-t-butyldimethylsilyloxyethyl-N-2-hydro-
13C4) 22b (0.636 g, 2.21 mmol) in dichloromethane
xyethylamine
(
(6.0 ml) was added N,N-diisopropylethylamine (0.577 ml, 3.32 mmol).
The mixture was cooled to 0ꢀC, and methanesulfonyl chloride
(0.205 ml, 2.65 mmol) was added. It was then allowed to warm to room
temperature and stirred overnight. The solvent was removed under
reduced pressure, and the residue was purified on silica gel with 10%
ethyl acetate in hexanes to provide 0.466 g (68%) of 23b as a colorless
liquid. 1H NMR (CDCl3) d 3.65 (ddt, J = 140.5, 5.70, 1.59 Hz), 3.49 (ddt,
J = 150.2, 7.23, 3.12 Hz), 3.17–3.07 (m), 2.62–2.79 (complex d,
J = 131.8 Hz), 2.72–2.60 (m), 1.01 (t, J = 7.13 Hz), 0.87 (s), 0.04 (s). 13C
N-bis(2-N-acetyl-(L)-cysteine t-butyl ester ethyl)-N-ethylamine
(13C4) 19b
To a solution of bis-(2-chloroethyl)-ethylamine (13C4) 17b (0.105 g,
0.50 mmol) and t-butyl-N-acetyl-(L)-cysteinate (0.329 g, 1.50 mmol) in
acetonitrile (5.0 ml) was added potassium carbonate (0.276 g, 2.0 mmol),
and the mixture was heated to reflux overnight. The solvent was
removed under reduced pressure, and the residue was purified on silica
gel with 10% MeOH/CH2Cl2 to provide 0.203 g (75%) of 19b as a colorless
liquid. 1H NMR (CDCl3) d 6.46 (br d, J = 7.18 Hz), 4.68 (t, J = 4.93 Hz), 4.67 (t,
J = 4.93 Hz), 3.06–2.89 (m), 2.84 (br s), 2.56–2.46 (m), 2.39 (br s), 2.02 (s),
1.46 (s), 0.99 (t, J = 7.07 Hz). 13C NMR (CDCl3) d 170.2, 170.0, 83.0, 53.5
(d, J = 38.8 Hz), 47.8, 37.4, 36.8, 35.0, 31.1 (d, J = 38.8 Hz), 28.2, 23.4, 12.1.
NMR (CDCl3)
d 62.3 (d, J = 42.8 Hz), 56.7 (d, J = 14.9 Hz), 56.1 (d,
J = 17.4 Hz), 49.2, 42.3 (d, J = 40.4 Hz), 26.2, 18.5, 12.5, À5.12.
N-ethyl-N-2-t-butyldimethylsilyloxyethyl-N-2-N-acetyl-(L)-cysteine
t-butyl ester ethylamine (13C4) 24b
To a solution of N-ethyl-N-2-t-butyl dimethylsilyloxyethyl-N-2-chloroethyl
amine (13C4) 23b (0.189 g, 0.70 mmol) and t-butyl N-acetyl-(L)-cysteinate
(0.184 g, 0.84 mmol) in acetonitrile (3.50 ml) was added potassium carbo-
nate (0.307 g, 2.22 mmol). The mixture was heated to reflux for 1–2 h until
TLC showed a complete reaction. The solvent was removed under
reduced pressure, and the residue was purified on silica gel with 10%
N-bis(2-N-acetyl-(L)-cysteine ethyl)-N-ethyl-amine (13C4) trifluor-
oacetic acid salt 20b
To a solution of N-bis(2-N-acetyl cysteine t-butyl ester ethyl)-N-ethyl- MeOH/CH2Cl2 to provide 0.305 g (96%) of 24b as a colorless liquid. 1H
amine (13C4) 19b (0.198 g, 0.366 mmol) in dichloromethane (3.0 ml) was NMR (CDCl3) d 6.29 (br d, J = 7.13 Hz), 4.67 (dt, J = 7.34, 4.88 Hz), 3.65
added triisopropylsilane (0.73 ml). The mixture was cooled with an ice (dt, J = 140.8, 6.36 Hz), 3.05–2.75 (m), 2.64–2.31 (m), 2.01 (s), 1.46 (s),
bath, and trifluoroacetic acid (0.73 ml) was added. The mixture was then
allowed to warm to room temperature and stirred overnight. The solvent (d, J = 43.0 Hz), 55.8 (d, J = 43.0 Hz), 54.4 (d, J = 38.5 Hz), 52.8, 48.6, 34.9,
and excess reagents were removed under reduced pressure, and the
31.1 (d, J = 38.5 Hz), 28.2, 26.2, 23.5, 18.5, 12.3, À5.09.
1.00 (t, J = 7.13 Hz), 0.87, 0.03. 13C NMR (CDCl3) d 170.1, 169.9, 83.0, 62.1
J. Label Compd. Radiopharm 2012, 55 211–222
Published 2012. This article is a US Government work
and is in the public domain in the USA.
www.jlcr.org

R. Wu et al.
N-ethyl-N-2-hydroxyethyl-N-2-N-acetyl-(L)-cysteine ethylamine (13C4) Tris(2-chloroethyl)amine hydrochloride (13C6) 30
hydrochloric acid salt 25b
Triethanolamine hydrochloride (13C6) 29 (0.544 g, 2.84 mmol) was sus-
pended in thionyl chloride (4.13 ml, 56.76 mmol), and the mixture was
heated to reflux for 2–3 h. The solvent was removed under reduced pres-
To a solution of N-ethyl-N-2-t-butyldimethylsilyloxyethyl-N-2-N-acetyl-(L)-
cysteine t-butyl ester ethylamine (13C4) 24b (0.304 g, 0.671 mmol) in
dichloromethane (2.8 ml) was added dropwise triisopropylsilane
(0.67 ml). The mixture was cooled with an ice bath, and trifluoroacetic
acid (0.67 ml) was added at 0ꢀC. The mixture was allowed to warm to
room temperature and stirred overnight. The solvent and excess
reagents were removed under reduced pressure, and tetrahydrofuran
(0.8 ml) was added to the residue. The mixture was cooled to 0ꢀC, and
1 M HCl (0.8 ml) was added dropwise, and the mixture was allowed to
warm to room temperature and stirred overnight. The solvent was
removed under reduced pressure, and the residue was partitioned
between ethyl acetate and water. The aqueous layer was lyophilized to
provide 0.240 g (100%) of 25b as a colorless hygroscopic solid. 1H NMR
(D2O) d 4.59 (dd, J = 8.06, 4.88 Hz), 4.12–4.00 (m), 3.68–3.42 (m), 3.35–
2.80 (m), 2.70–2.56 (m), 2.01 (s), 1.27 (t, J = 7.29 Hz). 13C NMR (D2O)
d 174.4, 173.8, 55.3 (d, J = 37.8 Hz), 54.0 (d, J = 37.6 Hz), 52.4, 51.6 (d,
J = 37.0 Hz), 48.8, 32.6, 25.4 (d, J = 36.7 Hz), 21.7, 7.98. GC-MS m/z for
13C4C7H22N2O4S (M+ + H): calc 283.3, found 283.1.
1
sure to provide 0.696 g (99%) of 30 as a colorless solid. H NMR (CDCl3)
d 4.10 (complex d, J = 140.3 Hz), 3.65 (complex d, J = 140.3 Hz). 13C NMR
(CDCl3) d 55.4 (d, J = 38.5 Hz), 37.3 (d, J = 38.1 Hz).
Tris(2-methylthioethyl)amine (13C6) 31
To a solution of tris(2-chloroethyl)amine hydrochloride (13C6) 30 (0.696 g,
2.82 mmol) in ethanol (20.0 ml) was added sodium thiomethoxide
(0.987 g, 14.09 mmol) in one portion at room temperature. The mixture
was heated to reflux for 1–2 h. The solvent was removed under reduced
pressure, and the residue was purified on silica gel with 5% MeOH/CH2Cl2
to provide 0.659 g (95%) of 31 as a light yellow liquid. 1H NMR (CDCl3)
d 2.74 (complex d, J = 134.1 Hz), 2.56 (complex d, J = 137.2 Hz), 2.13 (d,
J = 4.17 Hz). 13C NMR (CDCl3) d 53.9 (d, J = 38.7 Hz), 32.3 (d, J = 38.7 Hz),
16.1. GC-MS m/z for 13C6C3H21NS3 (M+ + H): calc 246.4, found 246.1.
Tris(2-N-acetyl-(L)-cysteine t-butyl ester ethylamine) (13C6) 32
A mixture of tris(2-methylthioethyl)amine (13C6) 31 (0.123 g, 0.5 mmol),
t-butyl-N-acetyl-(L)-cysteinate (0.374 g, 1.705 mmol), and potassium
carbonate (0.276 g, 2.0 mmol) in acetonitrile (5.0 ml) was heated to reflux
overnight. The solvent was removed under reduced pressure, and the
residue was purified on silica gel using 10% MeOH/CH2Cl2 to provide
0.314 g (83%) of 32 as a colorless liquid. 1H NMR (CDCl3) d 6.62
(d, J = 7.67 Hz), 4.71 (dt, J = 7.56, 4.93 Hz), 3.00 (m), 2.65 (complex d,
J = 134.3 Hz), 2.05 (s), 1.49 (s). 13C NMR (CDCl3) d 170.2, 170.1, 83.0, 54.1
(d, J = 38.4 Hz), 53.1, 35.0 31.1 (d, J = 38.0 Hz), 28.2, 23.4.
N-benzyl-N,N-bis(2-t-butyldimethyl silyloxyethyl)amine (13C4) 26
A mixture of benzylamine (0.256 g, 2.76 mmol), 2-bromoethyl t-butyldi-
methyl silyl ether (13C2) 12 (1.211 g, 5.02 mmol) and potassium carbonate
(1.144 g, 8.28 mmol) in acetonitrile (6.0 ml) was heated to reflux for
3 days. The solvent was removed under reduced pressure, and the
residue was purified on silica gel with 10% ethyl acetate in hexanes to
provide 0.985 g (92%) of 26 as a colorless liquid. 1H NMR (CDCl3)
d 7.37–7.18 (m), 3.71 (t, J = 4.17 Hz), 3.66 (ddt, J = 140.5, 7.02, 2.30 Hz),
2.68 (complex d, J = 132.4 Hz), 0.87 (s), 0.02 (s). 13C NMR (CDCl3) d 140.3,
128.9, 128.3, 127.0, 62.2 (dd, J = 43.0, 1.58 Hz), 60.3, 57.0 (dd, J = 43.0,
1.58 Hz), 26.2, À5.13.
Tris(2-N-acetyl-(L)-cysteine ethylamine) (13C6) trifluoroacetic acid
salt 33
To a solution of tris(2-N-acetyl-(L)-cysteine t-butyl ester ethylamine) (13C6)
32 (0.310 g, 0.408 mmol) in dichloromethane (5.0 ml) was added triiso-
propylsilane (1.23 ml), and the mixture was cooled with an ice bath. Tri-
fluoroacetic acid (1.23 ml) was then added dropwise, and the mixture
was stirred overnight. The solvent and excess reagents were removed
under reduced pressure, and the residue was partitioned between ethyl
acetate and water. The aqueous layer was lyophilized to provide 0.321 g
(100%) of 33 as a white hygroscopic solid. 1H NMR (D2O) d 4.61 (m), 3.73
(br s), 3.22 (br s), 3.23–2.88 (m), 2.74 (br s), 2.03. 13C NMR (D2O) d 174.3,
173.8, 52.5 (d, J = 36.5 Hz), 32.8, 25.7 (d, J = 36.3 Hz), 21.8. GC-MS m/z for
13C6C15H36N4O9S3 (M + H): calc 591.7, found 592.2.
N,N-bis(2-t-butyldimethylsilyl-oxyethyl)amine (13C4) 27
To a solution of N-benzyl-N,N-bis(2-t-butyldimethylsilyloxyethyl) amine
(
13C4) 26 (1.357 g, 3.17 mmol) in MeOH (15.0 ml) was added Pd(OH)2/C
(0.196 g).12 A hydrogen balloon was used with an inlet needle immersed
in the suspension. The mixture was stirred overnight or until TLC showed
a complete reaction. The solids were filtered and washed with MeOH.
The filtrate was concentrated under reduced pressure to provide
1.100 g (100%) of 27 as a colorless semi-liquid. 1H NMR (CDCl3) d 3.74
(ddt, J = 140.8, 5.15, 2.08 Hz), 2.74 (complex d, J = 132.5 Hz), 1.91 (br s),
0.90 (s), 0.06 (s). 13C NMR (CDCl3) d 62.7 (dd, J = 40.5, 4.02 Hz), 52.0 (dd,
J = 40.5, 4.17 Hz), 26.2, 18.5, À5.11.
Diethanolamine hydrochloride (13C4) 34
N,N,N-Tris(2-t-butyldimethylsilyloxyethyl)amine (13C6) 28
To a solution of N,N-bis(2-t-butyldimethylsilyloxyethyl)amine (13C4) 27
(0.603 g, 1.79 mmol) in THF (4.0 ml) was added 1 M HCl (4.0 ml) at 0ꢀC.
The mixture was allowed to warm to room temperature and stirred for
2 h. The solvent was removed under reduced pressure, and the residue
was partitioned between ethyl acetate and water. The aqueous layer
was lyophilized to provide 0.262 g (100%) of 34 as a white solid. 1H
NMR (D2O) d 3.71 (ddt, J = 140.6, 5.00, 1.92 Hz), 2.69 (complex d,
J = 133.6Hz), 2.64 (dq, J = 6.85, 3.23 Hz), 1.13 (t, J= 7.13 Hz), 0.88 (s), 0.05
(s). 13C NMR (D2O) d 62.5 (d, J = 40.2 Hz), 51.7 (d, J = 40.2Hz), 44.1 (d,
J = 3.74 Hz), 26.1, 15.5, À5.11.
A mixture of N,N-bis(2-t-butyldimethylsilyloxyethyl) amine (13C4) 27
(1.356 g, 4.02 mmol), 2-bromoethyl t-butyldimethylsilyl ether (13C2) 12
(0.969 g, 4.02 mmol) and potassium carbonate (1.111 g, 8.04 mmol) in
acetonitrile (16.0 ml) was heated to reflux for 2 days. The solvent was
removed under reduced pressure, and the residue was purified on silica
gel with 50% ethyl acetate in hexanes to provide 1.731 g (86%) of 28 as a
colorless liquid. 1H NMR (CDCl3) d 3.64 (ddt, J = 140.3, 6.36, 2.30 Hz), 2.69
(complex d, J = 132.0 Hz), 0.89 (s), 0.05 (s). 13C NMR (CDCl3) d 62.4 (d,
J = 43.0 Hz), 58.1 (d, J = 43.0 Hz), 26.2, 18.5, À5.09.
Triethanolamine hydrochloride (13C6) 29
To a solution of N,N,N-tris(2-t-butyldimethylsilyloxyethyl)amine (13C6) 28
N-(2-t-butyldimethylsilyloxyethyl)-N, N-bis(2-hydroxyethyl)amine
(13C6) 35
(1.136 g, 2.28 mmol) in THF (8.0 ml) was added 1 M HCl (8.0 ml) dropwise
A mixture of diethanolamine hydrochloride (
13C4) 34 (0.378 g,
at 0ꢀC. The mixture was allowed to warm to room temperature and stir- 2.60 mmol), 2-bromoethyl t-butyldimethyl silyl ether (13C2) 12 (0.626 g,
red for 2 h. The solvent was removed under reduced pressure, and the
2.60 mmol) and potassium carbonate (1.078 g, 7.80 mmol) in acetonitrile
residue was partitioned between ethyl acetate and water. The aqueous (9.0 ml) was heated to reflux for 3 days. The reaction was cooled to room
1
layer was lyophilized to provide 0.428 g (98%) of 29 as a white solid. H temperature and then filtered, and the filtrate was concentrated to pro-
NMR (D2O)
d
3.93 (complex d, J = 142.5 Hz), 3.47 (complex d, vide 0.685 g (98%) of 35 as a colorless liquid. 1H NMR (CDCl3) d 3.60
J = 142.5 Hz). 13C NMR (D2O) d 55.23, 55.16.
(dt, J = 140.3, 5.10 Hz), 2.75 (complex d, J = 130.3 Hz), 0.91 (s), 0.10 (s).
www.jlcr.org
Published 2012. This article is a US Government work
and is in the public domain in the USA.
J. Label Compd. Radiopharm 2012, 55 211–222

R. Wu et al.
13C NMR (CDCl3) d 60.1 (d, J = 39.1 Hz), 57.1 (d, J = 39.1 Hz), 56.4 (d,
J = 42.2 Hz), 26.1, 18.5, À5.25.
pressure, and the residue was dissolved in THF (1.0 ml), and 1 M HCl
(1.0 ml) was added dropwise at 0ꢀC. The mixture was allowed to warm
to room temperature and stirred overnight. The solvent was removed
under reduced pressure, and the residue was partitioned between ethyl
acetate and water. The aqueous layer was collected and decolorized with
active carbon and lyophilized to provide 0.219 g (80%) of 40 as a
colorless hygroscopic solid. 1H NMR (D2O) d 4.61 (dd, J = 8.00, 4.71 Hz),
4.18–4.02 (m), 3.28–3.54 (m), 3.35–3.05 (m), 3.05–2.83 (m), 2.82–2.66
(m), 2.02 (s). 13C NMR (D2O) d 174.3, 174.0, 55.1, 52.5 (d, J = 36.5 Hz),
48.6 (s), 32.7 (s), 25.6 (d, J = 36.5 Hz), 21.8.
N-(2-t-butyldimethylsilyloxyethyl)-N,N-bis(2-chloroethyl)amine
(13C6) 36
To a solution of N-(2-t-butyldimethylsilyloxyethyl)-N,N-bis(2-hydroxyethyl)
amine (13C6) 35 (0.685 g, 2.54 mmol) in dichloromethane (11.0 ml) was
added N,N-diisopropylethylamine (0.788 g, 6.10 mmol), and the mixture
was cooled with an ice bath. Methanesulfonyl chloride (0.641 g,
5.59 mmol) was added dropwise, and the mixture was allowed to warm
to room temperature and stirred overnight. The solvent was removed
under reduced pressure, and the residue was purified on silica gel with
10% ethyl acetate in hexanes to provide 0.595 g (76%) of 36 as a colorless
liquid. 1H NMR (CDCl3) d 3.67 (ddt, J = 140.4, 5.97, 2.03 Hz), 2.96 (ddt,
J = 150.2, 6.96, 3.07 Hz), 2.96 (complex d, J = 140.3 Hz), 2.75 (complex d,
J = 132.3 Hz), 0.89 (s), 0.06 (s). 13C NMR (CDCl3) d 62.4 (d, J = 42.2 Hz), 57.5
(d, J = 40.1 Hz), 57.0 (d, J = 42.2 Hz), 42.4 (d, J = 40.1 Hz), 26.1, 18.4, À5.18.
N,N-bis(2-t-butyldimethylsilyloxyethyl)-N-(2-hydroxyethyl)amine
(13C6) 41
A
mixture of N,N-bis(2-t-butyldimethylsilyloxyethyl)amine (
13C4) 27
(1.065 g, 3.15 mmol), 2-bromoethanol (13C2) (0.400 g, 3.15 mmol), and
potassium carbonate (0.871 g, 6.30 mmol) in acetonitrile (16.0 ml) was
heated to reflux for 2 days. The solvent was removed under reduced
pressure, and the residue was purified on silica gel with 100% ethyl acet-
ate. Two compounds were isolated, the product 41 was 0.491 g, and the
starting material 27 was 0.474 g, providing 73% of 41 on the basis of con-
sumed starting material. 1H NMR (CDCl3) d 3.70 (ddt, J = 140.3, 5.97,
1.92 Hz), 2.75 (complex d, J = 131.2 Hz), 0.90 (s), 0.06 (s). 13C NMR (CDCl3)
d 62.1 (d, J = 42.5 Hz), 59.6 (d, J = 38.4 Hz), 57.1 (d, J = 42.5 Hz), 57.0
(d, J = 38.4 Hz), 26.2, 18.5, À5.16.
N-(2-t-butyldimethylsilyloxyethyl)-N,N-bis(2-methylthioethyl)amine
(13C6) 37
To a solution of N-(2-t-butyldimethylsilyloxyethyl)-N,N-bis(2-chloroethyl)
amine (13C6) 36 (0.230 g, 0.75 mmol) in EtOH (6.0 ml) was added sodium
thiomethoxide (0.158 g, 2.25 mmol), and the mixture was heated to reflux
for 1–2 h. The solvent was removed under reduced pressure, and the resi-
due was purified on silica gel with 5% MeOH/CH2Cl2 to provide 0.226 g
(91%) of 37 as a colorless liquid. 1H NMR (CDCl3) d 3.68 (ddt, J = 140.3,
6.52, 2.30 Hz), 2.78 (complex d, J = 134.6 Hz), 2.67 (complex d,
J = 132.1 Hz), 2.60 (complex d, J = 139.1 Hz), 2.12 (d, J = 4.17 Hz), 0.89 (s),
0.06 (s). 13C NMR (CDCl3) d 62.2 (d, J = 42.7 Hz), 56.4 (d, J = 42.7 Hz), 54.8
(d, J = 38.5 Hz), 32.4 (d, J = 38.5 Hz), 26.2, 18.5, 16.1, À5.09.
N,N-bis(2-t-butyldimethylsilyloxyethyl)-N-(2-chloroethyl)amine
(13C6) 42
To a solution N,N-bis(2-t-butyldimethylsilyloxyethyl)-N-(2-hydroxyethyl)
amine (13C6) 41 (0.352 g, 0.917 mmol) in dichloromethane (2.40 ml) was
added N,N-diisopropylethylamine (0.24 ml, 1.378 mmol). The mixture
was cooled with an ice bath, and methanesulfonyl chloride (0.085 ml,
1.101 mmol) was added dropwise. The mixture was allowed to warm to
room temperature and stirred for 3–4 h until TLC showed a complete
reaction. The solvent was removed under reduced pressure, and the resi-
due was purified on silica gel with 10% ethyl acetate in hexanes to pro-
vide 0.267 g (72%) of 42 as a colorless liquid. 1H NMR (CDCl3) d 3.65
(ddt, J = 140.3, 6.41, 2.19 Hz), 3.50 (complex d, J = 150.6 Hz), 2.95 (complex
d, J = 135.7 Hz), 2.75 (complex d, J = 131.5 Hz), 0.89 (s), 0.05 (s). 13C NMR
(CDCl3) d62.5 (d, J = 42.7 Hz), 57.9 (d, J = 40.2 Hz), 57.6 (d, J = 42.7 Hz),
42.5 (d, J = 40.5 Hz), 26.1, 18.5, À5.12.
N-(2-Hydroxyethyl)-N,N-bis(2-methyl thioethyl)amine hydrochlor-
ide (13C6) 38
To a solution of N-(2-t-butyldimethylsilyloxyethyl)-N,N-bis(2-methylthioethyl)
amine (13C6) 37 (0.378 g, 1.15 mmol) in THF (2.0 ml) was added 1 M HCl
(2.0 ml) at 0ꢀC. The mixture was allowed to warm to room temperature and
stirred overnight. The solvent was removed under reduced pressure, and
the residue was partitioned between ethyl acetate and water. The aqueous
1
layer was lyophilized to provide 0.329 g (100%) of 38 as a yellow liquid. H
NMR (D2O) d 3.92 (complex d, J= 139.1 Hz), 3.50 (complex d, J=146.7Hz),
3.42 (complex d, J= 144.1 Hz), 2.90 (complex d, J= 141.1 Hz), 2.13 (d,
J=4.44Hz). 13C NMR (D2O) d 55.0 (d, J=5.58Hz), 52.0 (d, J= 37.0 Hz), 27.2
(d, J= 36.5 Hz), 14.0. LC-MS m/z for 13C4C4H19NOS2 (M+ + H): calc 216.3, found
216.1.
N,N-bis(2-t-butyldimethylsilyloxy-ethyl)-N-(2-N-acetyl-(L)-cysteine
t-butyl ester ethyl)amine (13C6) 43
A
mixture of N,N-bis(2-t-butyldimethylsilyloxy-ethyl)-N-(2-chloroethyl)
amine (13C6) 42 (0.262 g, 0.651 mmol), t-butyl-N-acetyl-(L)-cysteinate
(0.157 g, 0.717 mmol), and potassium carbonate (0.135 g, 0.977 mmol) in
acetonitrile (3.0 ml) was heated to reflux overnight. The solvent was
removed under reduced pressure, and the residue was purified on silica
gel with 10% MeOH/CH2Cl2 to provide 0.365 g (96%) of 43 as a colorless
liquid. 1H NMR (CDCl3) d 6.27 (br d, J = 7.18 Hz), 4.70 (dt, J = 7.51, 4.82 Hz),
3.64 (ddt, J = 140.3, 6.08, 1.97 Hz), 3.08–2.77 (m), 2.60–2.32 (m), 2.04 (s),
1.49 (s), 0.89 (s), 0.05 (s). 13C NMR (CDCl3) d 170.1, 169.9, 83.0, 62.2 (d,
J = 42.8 Hz), 51.7 (d, J = 42.8 Hz), 55.8 (d, J = 38.4 Hz), 52.5, 35.0, 31.4 (d,
J = 38.4 Hz), 28.2, 26.2, 23.5, 18.5, À5.08.
N-(2-t-Butyldimethylsilyloxyethyl)-N,N-bis(2-N-acetyl-(L)-cysteine
t-butyl ester ethylamine)amine (13C6) 39
A
mixture of N-(2-t-butyldimethylsilyloxyethyl)-N,N-bis(2-chloroethyl)
amine
(
13C6) 36 (0.230 g, 0.75 mmol), t-butyl-N-acetyl-(L)-cysteinate
(0.362 g, 1.65 mmol), and potassium carbonate (0.207 g, 1.5 mmol) in
acetonitrile (8.5 ml) was heated to reflux overnight. The solvent was
removed under reduced pressure, and the residue was purified on silica
gel with 10% MeOH/CH2Cl2 to provide 0.381 g (76%) of 39 as a colorless
liquid. 1H NMR (CDCl3) d 6.50 (br d, J = 7.40 Hz), 4.70 (dt, J = 7.45, 4.99 Hz),
3.63 (complex d, J = 140.6 Hz), 3.09–2.80 (m), 2.55–2.34 (m), 2.05 (s), 1.49
(s), 0.89 (s), 0.05 (s). 13C NMR (CDCl3) d 170.1, 169.9, 82.9, 61.9 (d,
J = 42.6 Hz), 56.2 (d, J = 42.4 Hz), 54.7 (d, J = 38.4 Hz), 52.9, 34.9, 31.3 (d,
J = 38.3 Hz), 28.1, 26.1, 23.3, 18.4, À5.09.
N,N-bis(2-hydroxyethyl)-N-(2-N-acetyl-(L)-cysteine ethyl)amine (13C6)
hydrochloric acid salt 44
To a solution of N,N-bis(2-t-butyl-dimethylsilyloxyethyl)-N-(2-N-acetyl-(L)-
cysteine t-butyl ester ethyl)amine (13C6) 43 (0.361 g, 0.617 mmol) in
dichloromethane (2.5 ml) was added triisopropylsilane (0.62 ml). The mix-
ture was cooled with an ice bath, and trifluoroacetic acid (0.62 ml) was
N-(2-hydroxyethyl)-N,N-bis(2-N-acetyl-(L)-cysteine ethylamine)amine
(
13C6) hydrochloric acid salt 40
To a solution of N-(2-t-butyldimethyl-silyloxyethyl)-N,N-bis(2-N-acetyl-(L)- added dropwise, and the mixture was allowed to warm to room tem-
cysteine t-butyl ester ethylamine)amine (13C6) 39 (0.380 g, 0.565 mmol) perature and stirred overnight. The solvent was removed under reduced
in dichloromethane (5.0 ml) was added triisopropylsilane (1.13 ml), the
pressure, and the residue was dissolved in THF (1.5 ml). The mixture was
mixture was cooled with an ice bath, trifluoroacetic acid (1.13 ml) was cooled with an ice bath, and 1 M HCl (1.5 ml) was added and then allowed
added dropwise, and the mixture was allowed to warm to room tem- to warm to room temperature and stirred overnight. The solvent was
perature and stirred overnight. The solvent was removed under reduced removed under reduced pressure, and the residue was partitioned
J. Label Compd. Radiopharm 2012, 55 211–222
Published 2012. This article is a US Government work
and is in the public domain in the USA.
www.jlcr.org

R. Wu et al.
between ethyl acetate and water. The aqueous layer was lyophilized to
provide 0.174 g (42%) of 44 as a colorless hygroscopic solid. 1H NMR
(D2O) d4.64–4.55 (m), 4.27–4.04 (m), 3.86–3.52 (m), 3.36-3.03 (m),
3.03–2.87 (m), 2.81–2.67 (m), 2.03 (s). 13C NMR (D2O) d55.1, 52.7 (d,
J = 36.7 Hz), 25.3 (d, J = 36.7 Hz). GC-MS m/z for 13C6C5H22N2O5S
(M + H): calc 301.3, found 300.9.
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Acknowledgements
We gratefully acknowledge the support of this work by the
CDC (R-2589-05-0) and the Los Alamos National Laboratory
LDRD program (ER 20100160).
Conflict of Interest
[8] Convention on the Prohibition of the Development, Production,
Stockpiling, and Use of Chemical Weapons and on their Destruction.
The Technical Secretariat of the Organization for the Prohibition of
Chemical Weapons: The Hague, The Netherlands, 1992.
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2910–2913.
[10] See SI section for the following reference, S-H. Kuwabe, K. E. Torraca,
S. L. Buchwald, J. Am. Chem. Soc. 2001, 123, 12202–12206.
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K-I. Miyamoto, Chem. Pharm. Bull. 2007, 55, 328–333.
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The authors did not report any conflict of interest.
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Published 2012. This article is a US Government work
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J. Label Compd. Radiopharm 2012, 55 211–222