Syntheses of D-labelled oxidative metabolites of acrylamide and acrylonitrile for the quantification of their toxicities in humans

Article abstract of DOI:10.1002/ejoc.200800291

Syntheses of the labelled oxidative metabolites of acrylamide and acrylonitrile - reference compounds for the evaluation of human exposure to important toxicants - are reported. For that, L-cystine tert-butyl ester was acetylated and the product reductively cleaved to L-cysteine tert-butyl ester, which reacted with carbamoyl[D₃]oxirane (obtained from [D ₃]acrylonitrile and 30% aq. H₂O₂ at pH = 7.0-7.5) and afforded a separable mixture of tert-butyl N-acetyl-S-(2-hydroxy-2- carbamoyl[ D₃]ethyl)cysteinate and tert-butyl N-acetyl-S-(1- carbamoyl-2-hydroxy[D₃]ethyl)cysteinate (ca. 9:1). Removal of the tert-butyl group in these intermediates with aq. HCl gave the final deuterated internal standards with carbamoyl residues. Protection of the secondary hydroxy group in the major intermediate with tBuMe₂SiCl/imidazole in DMF followed by dehydration of the carbamoyl group (trifluoroacetic anhydride/pyridine in CH₂Cl₂) and stepwise removal of the tert-butyl and tBuMe₂Si protecting groups (TFA, Et₃SiH, CH₂Cl₂; aq. HF in MeCN) yielded N-acetyl-S-(2-cyano-2- hydroxy[D₃]ethyl)cysteine. Monoprotection of [D₄]ethylene glycol with tBuMe₂SiCl and NaH in THF, oxidation to tBuMe ₂SiOCD₂CDO, conversion to tBuMe₂SiOCD ₂CD(OH)CN and tBuMe₂SiOCD₂CD(OTs)CN followed by nucleophilic substitution of the tosyloxy group with N-acetyl-L-cysteine (MeOD, Et₃N) and deprotection with 4 M HCl in dioxane resulted in N-acetyl-S-(1-cyano-2-hydroxy[D₃]ethyl)cysteine. All transformations (except the last but one) gave the respective products in good yields. Wiley-VCH Verlag GmbH & Co. KGaA, 2008.

Full text of DOI:10.1002/ejoc.200800291

FULL PAPER
DOI: 10.1002/ejoc.200800291
Syntheses of D-Labelled Oxidative Metabolites of Acrylamide and
Acrylonitrile for the Quantification of Their Toxicities in Humans
Vladimir N. Belov,[a,b] Sergei M. Korneev,[a] Jürgen Angerer,[c] and Armin de Meijere*[a,b]
Keywords: Isotopic labelling / Acrylonitrile / Acrylamide / Metabolism / Toxicology
Syntheses of the labelled oxidative metabolites of acrylamide
and acrylonitrile – reference compounds for the evaluation of
human exposure to important toxicants – are reported. For
dride/pyridine in CH2Cl2) and stepwise removal of the tert-
butyl and tBuMe2Si protecting groups (TFA, Et3SiH,
CH2Cl2; aq. HF in MeCN) yielded N-acetyl-S-(2-cyano-2-
hydroxy[D3]ethyl)cysteine. Monoprotection of [D4]ethylene
glycol with tBuMe2SiCl and NaH in THF, oxidation to tBu-
Me2SiOCD2CDO, conversion to tBuMe2SiOCD2CD(OH)CN
and tBuMe2SiOCD2CD(OTs)CN followed by nucleophilic
that,
L-cystine tert-butyl ester was acetylated and the product
reductively cleaved to
L-cysteine tert-butyl ester, which re-
acted with carbamoyl[D3]oxirane (obtained from [D3]acrylo-
nitrile and 30% aq. H2O2 at pH = 7.0–7.5) and afforded a
separable mixture of tert-butyl N-acetyl-S-(2-hydroxy-2-car- substitution of the tosyloxy group with N-acetyl-
L-cysteine
bamoyl[D3]ethyl)cysteinate and tert-butyl N-acetyl-S-(1-car- (MeOD, Et3N) and deprotection with 4 HCl in dioxane re-
M
bamoyl-2-hydroxy[D3]ethyl)cysteinate (ca. 9:1). Removal of
the tert-butyl group in these intermediates with aq. HCl gave
the final deuterated internal standards with carbamoyl resi-
dues. Protection of the secondary hydroxy group in the major
intermediate with tBuMe2SiCl/imidazole in DMF followed by
dehydration of the carbamoyl group (trifluoroacetic anhy-
sulted in N-acetyl-S-(1-cyano-2-hydroxy[D3]ethyl)cysteine.
All transformations (except the last but one) gave the respec-
tive products in good yields.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
readily react with glutathione (GSH), furnishing the
corresponding mercapturic acids [-AcNHCH(COOH)-
CH2SCH2CH2E, E = CONH2 or CN]. Another type of
metabolism – epoxidation to glycidamide (GA) and glyci-
donitrile (GN) – is catalyzed by the Cytochrome P450 2E1
enzyme.[8] These poisonous intermediates are believed to be
responsible for the carcinogenic activity of the parent com-
pounds, and therefore the quantitative determination of
these oxiranes is very important. In vivo, oxiranes can alkyl-
ate such nucleophiles as the N-terminus of haemoglobin,
DNA, cysteine-containing enzymes or GSH.[9] Conjugation
of GA and GN with nucleophilic sites is accomplished by
the opening of the oxirane ring activated with carbamoyl
or cyano electron-acceptor groups. A priori, it is not easy
to predict the regioselectivity of this reaction. Electronic
and steric effects in the starting oxiranes oppose each other.
In vitro, a similar reaction of MeSH with oxiranes obtained
from CH2=CHR (R = COMe, COOMe, CONH2, COONa
and Me) always gives mixtures of two products. The con-
tent of the regioisomer derived from the α-attack varies in
this sequence from nearly 100 to 0%.[10]
Introduction
Acrylonitrile (AN) and acrylamide (AA) are industrially
produced worldwide in amounts of 6 million and 100 thou-
sand tons per year, and approximately 1% of these amounts
is lost polluting the environment. Acrylamide is also formed
from asparagine and carbohydrates[1a–1g] or from alanine-
containing peptides (without carbonyl compounds)[1h,1i]
upon production and heating of food, such as French fries,
potato chips, popcorn, coffee, bread, cereals, etc. AA and
AN are components of cigarette smoke.[2] AA is neurotoxic
for humans,[3] carcinogenic[4,5] and has genotoxic, neuro-
toxic and reproductive hazardous effects.[5] AN was found
to be carcinogenic in rats[6] and mutagenic in E. coli.[7] It is
also toxic in cardiovascular (blood), development, gastroin-
testinal (liver), immuno, neuro, reproductive and respira-
tory systems, and acts as a skin or sense organ toxicant.
Due to their high reactivities, AA and AN show complex
metabolisms. As they are good Michael acceptors, they
[a] Institut für Organische und Biomolekulare Chemie der Georg-
August Universität Göttingen,
Tammannstrasse 2, 37077 Göttingen, Germany
Fax: +49-551-399475
GSH conjugates of AA and AN are excreted in urine as
the corresponding mercapturic acids (S-alkylated N-acetyl-
-cysteines), after enzymatic cleavage of the glutamine and
glycine residues from glutathione and acetylation of the
amino group of the cysteine moiety. In humans, only one
oxidative metabolite of AA has been found, which arises
E-mail: ameijer1@gwdg.de
[b] KAdemCustomChemGmbH,
Brombeereweg 13, 37077 Göttingen, Germany
[c] Institut und Poliklinik für Arbeits-, Sozial und Umweltsmedi-
zin, Universität Erlangen-Nürnberg,
Schillerstrasse 25/29, 91054 Erlangen, Germany
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V. N. Belov, S. M. Korneev, J. Angerer, A. de Meijere
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from a β-attack of the SH-group on the unsubstituted car- internal standards are used as additives to urine, and the
bon atom of the oxirane ring in GA (compound 4-H).[11] metabolites are not chemically transformed during the sam-
The other regioisomer was not detected in this study,[11] as ple preparation and analysis, the mass-spectrometric frag-
well as in the simultaneously published report, in which the mentation pattern diagnostic of the nature of the observed
labelled compound 4-D was used as an internal standard charged species determines the required positions of the iso-
for the determination of GA in human urine.[12] Yet, no topic labels in the additives. LC-MS/MS spectra of the oxi-
experimental details of the preparation of the labelled oxi- dative metabolites of AA and AN are simpler in the nega-
dative metabolites of AA have been reported. In another tive ionization mode, and – at least in the case of the main
study, in which [13C3]-AA (alone or along with [13C3]-AN) oxidative metabolite of acrylamide[12] – the main peak cor-
was administered to mice and rats, and the composition of responds to the negatively charged fragment SCH2CH-
a complex mixture of metabolites was analyzed by 13C (OH)CONH2. Therefore, it was necessary to synthesize the
NMR spectroscopy, the same regioisomer ([13C3]-4-H) was compounds with stable isotopes in their acrylamide and
detected, and, additionally, some 13C NMR signals (C*) acrylonitrile fragments, respectively.
were attributed to the “minor” metabolite ([13C3]-5-H).[13]
Epoxidation of acrylonitrile and the commercially avail-
These results indicate, that for the quantification of the oxi- able [D3]acrylonitrile with 30% aq. H2O2 at pH = 7.0–7.5
dative metabolites of AA, it is necessary to develop a practi- according to a published method furnishes the glycidamide
cal synthesis of both isotopically labelled mercapturic acids and its deuterated analogue 2-D in 65–70% yield.[18] Reac-
4 and 5.[14] Isotopic labelling allows one to use these deuter- tion of 2-D with N-acetylcysteine (3) under basic conditions
ated analyte analogues as “ideal” internal standards when gave a mixture of the two regioisomeric mercapturic acids
employing the method of isotopic dilution, in which the 4 and 5 in a ratio of ca. 9:1, which could not be separated
quantitative results do not depend on the conditions of by simple chromatography on silica gel (Scheme 1), but by
sample preparation.
preparative reversed-phase (RP) HPLC. Each of the com-
Similar metabolism studies of acrylonitrile and [13C3]- pounds 4 and 5 was thus isolated as a mixture of two epi-
AN were conducted only with mice or rats (not with hu- mers from the complex crude product, which also contained
mans).[8,15] In 1988 Linhart et al. reported that the conjuga- 2,3-dihydroxypropanamide, glyceric acid, traces of the
tion of GN with GSH in vivo (rats) does not proceed re- starting material, hydrogen peroxide, which oxidized N-ace-
gioselectively, and both α- as well as β-carbon atoms of GN tylcysteine to N-acetylcystine, and other unidentified sub-
are attacked by the nucleophilic thiol group.[15] However, stances. However, starting with tert-butyl N-acetylcysteinate
the primary product of the β-attack was not detected, but (8), which can easily be prepared from commercial tert-
its stable secondary metabolite [-AcNHCH(COOH)- butyl cystinate (6) by N-acetylation with acetic anhydride
CH2SCH2CH2OH] was identified. Later this result was and subsequent reductive cleavage of the disulfide bond
confirmed by another group. It was found that the metabo- with sodium borohydride (other reducing agents such as
lism of [13C3]-AN via the epoxide in mice and rats leads to Zn in AcOH or Ph3P in refluxing aq. EtOH gave inferior
the stable product of an α-attack ([13C3]-13-H) which is results), led to a crude product from which the tert-butyl
formed as a mixture of two epimers, as well as in several esters of the regioisomeric mercapturic acids 9 and 10 could
other substances which presumably arise from the unstable be isolated by ordinary column chromatography on silica
primary product of a β-attack.[8] It is not clear, why the gel.
primary product of the β-attack is unstable in vivo. In vitro,
The tert-butyl ester groups in the hydroxyamides 9 and
the reaction of methyl N-acetylcysteinate with GN (under 10 could easily be cleaved by treatment with 10% hydro-
basic conditions) gives predominantly the product of the β- chloric acid at 0 °C to furnish the free acids 4 and 5,
attack (methyl ester of 12-H).[15] Quantitative data have not respectively. The hydroxyamide residues in compounds 4
been reported, and earlier the same group stated that this and 5 are stable enough under the reaction conditions
regioisomer had been formed exclusively.[16] To clarify the and during workup, but for prolonged storage, the final
product distribution in the oxidative metabolism of AN, it mercapturic acids 4 and 5 should be purified by ordinary
was necessary to synthesize both regioisomeric ring-open- chromatography or RP-HPLC in order to remove any
ing products from glycidonitrile and N-acetylcysteine along traces of a strong acid. When performing the addition of
routes, which do not leave any doubt about their structures 8 to the epoxide 2-R at pH ≈ 9 in a mixture of acetonitrile
and allow one to prepare them in isotopically labelled forms and aq. Na2CO3/NaHCO3, the ratio of the regioisomeric
as internal standards for determination and quantification adducts 4/5 was found to be ca. 9:1. In the case of MeSH
of the corresponding urinary metabolite(s) in rodents and at pD = 9.8, the products of the α-/β-attack on glycid-
humans.[17]
amide 2-H were formed in a ratio of ca. 22:78,[10] i.e. the
α-adduct was obtained to a smaller extent than the β-
adduct in this study. At pH Ն 8, the thiolate ion of
cysteine (pKa = 8.2) is in fact the reactive nucleophile. At
physiological pH values (7.8–8.3) the selectivity of gluta-
Results and Discussion
In modern analytical procedures, the metabolites are sep- thione towards GA may even be higher. This may explain
arated by HPLC and detected by ESI tandem mass spec- why the minor oxidative metabolite of GA has not yet
trometry in positive or negative ionization mode. If labelled been detected in vivo.[11,12]
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D-Labelled Oxidative Metabolites of Acrylamide and Acrylonitrile
Scheme 2. Preparation of the oxidative metabolites of acrylonitrile
12-H, 13-H and the internal standard 12-D from cyanooxirane and
the deuterated hydroxyamide 9-D, respectively: (a) aq. Na2CO3,
MeCN, room temp.; (b) tBuMe2SiCl, DMF, imidazole, 5 °C, 4 d;
(c) (CF3CO)2O, pyridine (2 equiv.), CH2Cl2, room temp., 16 h; (d)
Et3SiH, TFA, CH2Cl2, 5 °C, 2 d; room temp. 1 d; (e) 51% aq. HF,
MeCN, 2 h, room temp.
Scheme 1. Preparation of the oxidative metabolites of acrylamide
(4-H, 5-H) and internal standards (4-D, 5-D) from acrylonitrile and
[D3]acrylonitrile, respectively: (a) 30% aq. H2O2, aq. NaOH, pH =
7.3–7.8; (b) aq. Na2CO3, MeCN, pH = 8–9; (c) Ac2O, pyridine,
0 °C; (d) NaBH4, EtOH; (e) aq. HCl, THF, 0 °C.
(TFAA) and 90% aq. H2O2 in a phosphate buffer. Even
when carrying out this reaction in refluxing CH2Cl2, no
product could be detected, and only the starting material 1-
D was recovered. An attempted dehydration of the crude
Labelled oxidative metabolites of acrylonitrile would be glycidamide 2-D with TFAA/pyridine was also unsuccess-
accessible analogously by the nucleophilic addition of N- ful.
acetylcysteine (3) to glycidonitrile, provided this oxirane
Therefore, an alternative approach to the deuterated hy-
would be available in its deuterated form. The undeuterated droxynitriles 12-D and 13-D had to be developed. The syn-
glycidonitrile (GN) 11-H reacted with N-acetylcysteine (3) thesis of the labelled major oxidative metabolite of acryloni-
to produce the regioisomeric mercapturic acids 12-H/13-H trile turned out to be straightforward (Scheme 2). Towards
in a ratio of ca. 9:1 (Scheme 2).
that, the hydroxy group in the amide 9-D was first protected
Unfortunately, the only practical synthesis of the highly as a tert-butyldimethylsilyl ether, and then the amide was
reactive cyanooxirane 11 requires a very large excess of dehydrated to a nitrile by treatment with TFAA/pyridine in
acrylonitrile (1-H): from 200 mL only ca. 5 g of 11 is ob- CH2Cl2. After some screening of conditions, the tert-butyl
tained in its reaction with an aqueous solution of sodium ester and the silyl ether were cleaved in two steps. The tert-
hypochlorite,[19] and the excess of 1-H is distilled off in butyl group was removed by treatment with TFA and
vacuo through a long Vigreux column. Thus, from 1 g of Et3SiH as a cation scavenger in anhydrous CH2Cl2. The
[D3]-AN only 50 mg of [D3]-GN may be prepared at best, dimethylsilyl group was cleaved off by the addition of aq.
and it is rather difficult to completely remove unreacted 1- HF to an acetonitrile solution of the concentrated reaction
D from the few drops of the product [D3]-GN. An attempt mixture from the previous step. Finally, the hydroxynitrile
to use a larger amount of sodium hypochlorite on [D3]- 12-D was purified by preparative HPLC.
AN did not help much, and after careful evaporation of the
A reliable synthesis of the minor oxidative metabolite of
starting material 1-D and addition of the estimated equi- AN started from ethylene glycol 17-H (Scheme 3), which is
molar amount of N-acetylcysteine (3) under basic condi- also commercially available in the tetradeuterated form 17-
tions, only a trace of the major of the two regioisomeric D. The diol was protected on one hydroxy group as a tert-
products in its deuterated form 12-D could be detected by butyldimethylsilyl ether, and the alcohol 18-R[20] was oxid-
HPLC, along with the unreacted N-acetylcysteine. Because ized to the aldehyde 19-R,[21] which was converted into the
the secondary kinetic isotopic effect of the deuterium atoms cyanohydrine 20-R. The latter was tosylated, and then the
may somewhat reduce the epoxidation rate, the epoxidation tosyloxy group in 21-R was replaced by the thiol group of
of 1-D was also attempted with trifluoroperacetic acid, N-acetylcysteine (3) in the presence of an excess of Et3N in
which was prepared in situ from trifluoroacetic anhydride methanol. The yields in all steps, except the last one, were
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V. N. Belov, S. M. Korneev, J. Angerer, A. de Meijere
FULL PAPER
good. The low yield (25–30%) of the desired product 22 conventional silica gel chromatography or RP-HPLC. With
may be due to the bis(nucleophilic) character of the reagent both labelled oxidative metabolites 12-D and 23c of acrylo-
3 and the bis(electrophilic) nature of the substrate 21-R, nitrile at hand, it will be easy not only to prove the results
which may lead to substitution of both the tosyloxy and of Sumner et al., who claimed that, after administration of
the cyano leaving groups with formation of a six-membered [1,2,3-13C3]-AN to rats and mice, only one oxidative metab-
lactone ring. The use of the aprotic solvent DMF did not olite, namely [1,2,3-13C3]-23, had been excreted with ur-
improve the yield. However, the simplicity of the procedure ine,[8,13] but also to quantitatively determine its or their con-
and the isolation of the product make this protocol more tent in urine. The structure of the latter metabolite was as-
convenient than the two-step variant employing tert-butyl signed on the basis of the proton multiplicities of 13C NMR
N-acetylcysteinate 8 for the substitution and subsequent re- signals, which were determined by heteronuclear 2D J-re-
moval of the tert-butyl protecting group.[22] After workup, solved spectroscopy (HET2DJ). The carbon–carbon con-
the crude product mixture was distributed between diethyl nectivities were established with the INADEQUATE tech-
ether and aqueous sodium hydrogen carbonate solution. nique. Resonances were assigned on the basis of chemical
The latter, which contained the carboxylic acid 22 and the shifts, proton multiplicities, values of the 13C-13C coupling
excess N-acetylcysteine (3), was carefully acidified with cit- constants and calculated values of the chemical shifts.[8b]
ric acid. Subsequent extraction with diethyl ether left N- For a rigorous proof, a model (unlabelled) compound was
acetylcysteine in the aqueous phase; yet some of the prod- synthesized according to the method of Linhart et al.[15,17]
uct 22 may be lost during acidification, because the silyl For comparison, the diagnostically important 13C NMR
ether may be cleaved to a certain extent even at pH = 2–3. chemical shifts (in [D6]DMSO) for the major diastereomer
The deuterium label adjacent to the cyano group (in 22) is 12 are δ = 36.3 and 61.2/61.3 ppm for the SCH2 and
prone to deuterium/hydrogen exchange in the presence of a CH(OH)CN groups, respectively (in the two epimers). For
base in a protic solvent, as the neighboring sulfur atom also the minor diastereomer 23 the CH(CN)S and CH2OH
stabilizes an anionic center adjacent to it. Therefore, under groups have the chemical shifts of δ = 35.4/35.5 and 61.8/
the employed conditions, the product 22b with only two 61.9 ppm, respectively. Sumner et al. were aware of this
deuterium labels was obtained. When MeOD was used as similarity,[8b] but stated that the carbon–carbon couplings
a solvent instead of MeOH, and Na2CO3 in D2O for the and proton multiplicities are consistent with the S-(1-
workup, as well as D2SO4 in D2O for the acidification, the cyano-2-hydroxyethyl)cysteine derivative [1,2,3-13C3]-23.
1
deuterated product 22c was indeed obtained.
Therefore, it is crucial to compare the H NMR chemical
shifts of Linhart’s compound (presumably 23a),[15,17] which
were measured in [D6]acetone, with the values obtained in
this study. Esterification of both mercapturic acids 12-H
and 23a with diazomethane and subsequent measurement
1
of their H NMR spectra unequivocally confirmed the re-
sults of Linhart et al. in that only the minor regioisomer
23a gave a methyl ester with the same spectrum as the re-
ported one.[23]
The major regioisomer resulting from a β-attack of N-
acetylcysteine on cyanooxirane prepared in this study (com-
pound 12-H/D), which previously had not been detected in
vivo and thought to be unstable in rodents,[8,13b,15] may be
used in the unlabelled and labelled form to clarify, if any
stable secondary metabolites are going to be formed from
it in vivo. Towards that, new urine metabolites have to be
found by means of LC-MS, after administration of the hy-
droxynitrile 12-H/D to rodents.
Conclusion and Outlook
Practical syntheses of all four oxidative metabolites of
acrylamide and acrylonitrile in their unlabelled and deute-
rium-labelled forms, which can be reproduced in all toxico-
logical and analytical laboratories, make it possible to fur-
ther study the metabolism of acrylamide and acrylonitrile
and quantify human exposure to these toxic electrophiles.
An approach to compounds with the rare structural frag-
Scheme 3. Total synthesis of the oxidative metabolites of acryloni-
trile 1-H(D) in the unlabelled (13-H) and labelled form (23b–c): (a)
NaH, THF; tBuMe2SiCl; (b) (COCl)2, DMSO, –78 °C; Et3N, room
temp.; (c) Na2S2O5, KCN, aq. MeOH; (d) p-MeC6H4SO2Cl (TsCl),
pyridine; (e) MeOH/(f) [D]MeOH, Et3N, 40 °C 36 h; (g) 4  HCl
in dioxane.
Finally, the tert-butyldimethylsilyl protecting group in ment R1OCH2CH(SR2)CN [R1 = protecting group or H,
22a–c was removed by treatment with 4  HCl in dioxane, R2 = (functionally substituted) alkyl or aryl group] has been
and the mercapturic acids 23a–c were purified either by developed.
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D-Labelled Oxidative Metabolites of Acrylamide and Acrylonitrile
idine (25 mL), cooled to 0 °C. The reaction flask was shaken for
several minutes, and the solid dissolved while an exothermic reac-
tion took place. The reaction mixture was left at +4 °C for 2 d,
then ice (100 g) was added, followed by concd. aq. HCl (25 mL),
which was introduced in small portions with stirring, so that the
temperature did not exceed 20 °C and the pH value remained above
4–5. The reaction mixture was extracted with diethyl ether
(4ϫ100 mL); the combined organic solutions were washed with
cold 0.5  aq. H2SO4, until the pH value of the aqueous layer
reached 2–3, and subsequently with satd. aq. NaHCO3. After dry-
ing and evaporation of the solvent, the residue was dissolved in hot
CHCl3 (20 mL), and hexane was added, until the solution became
turbid. The mixture was left at +4 °C for 16 h, the colorless precipi-
tate was collected by filtration to give 2.16 g (85%) of 7 with m.p.
Experimental Section
General: NMR spectra were recorded with Varian MERCURY 300
and Bruker AM 250 spectrometers at 300 (1H) and 75.5 MHz (13C
and APT), as well as at 250 (1H) and 62.9 MHz (13C and DEPT),
respectively. All spectra are referenced to tetramethylsilane as an
internal standard (δ = 0 ppm) by using the signals of the residual
protons of CHCl3 (δ =7.26 ppm) in CDCl3, CHD2OD (δ
=3.31 ppm) in CD3OD, HOD (δ =4.79 ppm) in D2O or [D5]DMSO
(δ =2.50 ppm) in [D6]DMSO. Multiplicities of signals are described
as follows: br. = broad, s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet, mc = centrosymmetrical multiplet. Coup-
ling constants (J) are given in Hz. Multiplicities in the 13C NMR
spectra were determined by DEPT or APT (Attached Proton Test)
measurements. Isotopic purity of all deuterated compounds was
found to be Ͼ95% by 13C NMR spectroscopy and mass spectrome-
try. Low-resolution mass spectra (electrospray ionization, ESI)
were obtained with an LCQ spectrometer. High-resolution mass
spectra (ESI-HRMS) were obtained with an APEX IV spectrome-
ter. HPLC: 20- and 100-µL injection loops for the analytical and
preparative columns, respectively; analytical column: Eurospher-
100 C18, 5 µm, 250ϫ4 mm, 1 mL/min; preparative column: Euro-
sphere-100 C18, 5 µm, 250ϫ8 mm, 4 mL/min; 25 °C, solvent A:
1
80–81 °C (diethyl ether). H NMR (250 MHz, CDCl3): δ = 1.43 (s,
9 H), 2.00 (s, 3 H), 3.14 (d, J = 5.1 Hz, 2 H), 4.71 (mc, 1 H), 6.69
(br.d, J = 7.3 Hz, 1 H, NH) ppm. 13C NMR (62.9 MHz, CDCl3):
δ = 23.0 (Me), 27.9 (Me ϫ3), 41.4 (CH2), 52.5 (CH), 82.9 (C-O),
169.4 and 170.0 (CO) ppm. MS (ES): m/z (positive mode, %) = 895
(100) [2 M + Na]+, 459 (69) [M + Na]+; m/z (negative mode, %) =
481 (100) [M + HCOO–]. C18H32N2O6S2 (436.6): calcd. C 49.52, H
7.39, N 6.41; found C 49.63, H 7.39, N 6.41.
tert-Butyl N-Acetylcysteinate (8): NaBH4 (1.78 g, 49.5 mmol) was
added in small portions to a solution of compound 7 (2.16 g,
4.95 mmol) in anhydrous EtOH (100 mL) with cooling at 20 °C.
The cooling bath was removed, and the mixture was left to warm
up to ca. 30 °C. An exothermic reaction occurred, and the EtOH
eventually started to boil. After it ceased, an amorphous solid sepa-
rated; CHCl3 (200 mL) and 5% aq. HCl (50 mL) were carefully
added to the reaction mixture with cooling by ice/water. The or-
ganic layer was separated, washed with water (100 mL), brine
(50 mL), dried and concentrated to yield 8 (1.95 g, 90%) as an oil,
which slowly crystallized at +4 °C to a colorless low-melting solid.
It was readily oxidized by air oxygen (to compound 7) and was
water
+ 0.1% v/v trifluoroacetic acid (TFA); solvent B:
MeCN+0.1% v/v TFA; detection at 220 nm. Elemental analyses
were carried out in the Mikroanalytisches Laboratorium des Insti-
tuts für Organische und Biomolekulare Chemie der Universität
Göttingen. Analytical TLC was performed on Merck ready-to-use
plates with silica gel 60 (F254), detection under UV light at 254 nm
and development with MOPS reagent (10% molybdophosphoric
acid in ethanol). Column chromatography: Merck silica gel, grade
60, 0.04–0.063 mm. Organic solutions were dried with anhydrous
Na2SO4. All reactions were carried out with magnetic stirring.
Reaction of N-Acetylcysteine (3) with Glycidamide (2-H). N-Acetyl-
S-[(R/S)-2-carbamoyl-2-hydroxy]ethylcysteine (4-H) and N-Acetyl-
S-[(R/S)-1-carbamoyl-2-hydroxyethyl]cysteine (5-H): A suspension
of N-acetylcysteine (3, 16.3 mg, 0.10 mmol) in water (0.3 mL) was
flushed with argon, cooled to 4 °C, then 20% aq. Na2CO3 (50 µL)
and subsequently the glycidamide 2-H (17 mg, 0.20 mmol) were
added. The reaction mixture was warmed up to room temperature
with rapid stirring and, after 4 h, acidified with 1  aq. HCl to pH
= 2. The compounds 4-H and 5-H were detected by HPLC (column
4ϫ250 mm; A/B, 99:1; isocratic mode) in the complex reaction
mixture with the help of reference samples, which were prepared
and characterized as described below. Compound 5-H: tR = 4.5
and 4.7 min (2:3, 2 epimers of the minor regioisomer); compound
4-H: tR = 6.0 min (for both epimers of the major regioisomer).
1
therefore kept under an inert gas. H NMR (250 MHz, CDCl3): δ
= 1.28 (t, J = 8.9 Hz, 1 H, SH), 1.42 (s, 9 H), 1.99 (s, 3 H), 2.92
(mc, 2 H), 4.71 (dt, J = 3.5 and 4 Hz, 1 H), 6.69 (br. d, J = 7 Hz,
1 H, NH) ppm. 13C NMR (62.9 MHz, CDCl3): δ = 23.0 (Me), 26.9
(CH2), 27.8 (Me, ϫ3), 53.7 (CH), 82.7 (C-O), 169.1 and 169.8 (CO)
ppm. MS (ES): m/z (positive mode, %) = 461 (19) [2 M + Na]+,
242 (100) [M + Na]+; m/z (negative mode, %) = 218 (41)
[M – H]–.
tert-Butyl N-Acetyl-S-[(R/S)-2-carbamoyl-2-hydroxyethyl]cysteinate
(9-H) and tert-Butyl N-Acetyl-S-[(R/S)-1-carbamoyl-2-hydroxyeth-
yl]cysteinate (10-H): Glycidamide 2-H (200 mg, 2.3 mmol, as a
waxy solid),[18] then water (10 mL) and 20% aq. Na2CO3 (0.5 mL)
were added to a solution of compound 8 (450 mg, 2.05 mmol) in
MeCN (20 mL), kept under argon. After 1 h, TLC (CH2Cl2/
MeOH, 10:1) disclosed that the starting thiol had been fully con-
sumed, and two new compounds with Rf ≈ 0.30 (major) and 0.27
(minor) appeared. The reaction mixture was diluted with EtOAc
(100 mL), the organic layer was separated, washed with brine and
concentrated in vacuo to give an oily residue (1.3 g). The latter was
subjected to chromatography on SiO2 (120 g), eluting with CH2Cl2/
MeOH (12:1) to give two regioisomeric products; 400 mg (64%) of
9-H as an oil, 40 mg (6.4%) of 10-H as an oil. 9-H: 1H NMR
(250 MHz, CDCl3, 2 diastereomers): δ = 1.40 (s, 9 H), 1.98 (s, 3
H), 2.72–3.10 (m, 4 H, CH2S), 4.20 (mc, 1 H, CHOH), 4.62 (mc, 1
H, CHNH), ca. 5.4 (br. s, 1 H, OH), 6.70 (br. s, 1 H, CONH2),
7.10 (br. s, 1 H, CONH2), 7.21 (d, J = 7 Hz, 1 H, NHAc) ppm.
13C NMR (62.9 MHz, CDCl3, 2 diastereomers): δ = 22.8 (Ac), 27.7
(Me, ϫ3), 35.2 (CH2S), 37.7 (CH2S), 52.9/53.0 (CHNH), 70.8/71.1
(CHOH), 82.77/82.83 (C-O), 169.8, 170.9 and 175.97/176.00 (CO)
[D3]Glycidamide (2-D): Compound 2-D was obtained from freshly
distilled [D3]acrylonitrile (1.0 g, 18 mmol) in H2O (7.0 mL) and
30% aq. H2O2 (1.9 g, 17 mmol) by adding 1  aq. NaOH (ca.
0.5 mL) at such a rate that the pH value was maintained at 7.3–7.8
(pH meter!).[18] Unreacted H2O2 was destroyed by adding 10% Pd/
C (ca. 50 mg) to the reaction mixture, when the consumption of
NaOH ceased, and the pH value did not return to 7.3 anymore.
After standing at +4 °C overnight, the reaction mixture was centri-
fuged, and the supernatant solution was used for the reaction with
N-acetylcysteine (3) (see: preparation and HPLC isolation of the
compounds 4-D and 5-D) and for the synthesis of preparatively
useful amounts of the esters 9 and 10.
Di-tert-butyl N-Acetylcystinate (7): Acetic anhydride (2.7 mL,
29 mmol) was added in one portion to a suspension of di-tert-butyl
cystinate dihydrochloride (6) (2.48 g, 5.83 mmol) in anhydrous pyr-
Eur. J. Org. Chem. 2008, 4417–4425
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4421

V. N. Belov, S. M. Korneev, J. Angerer, A. de Meijere
FULL PAPER
ppm. MS (ES): m/z (positive mode, %) = 635 (32) [2 M + Na]+,
HCl, 0 °C, 2 h). Yield: 158 mg (70%) as a colorless foam. 1H NMR
329 (100) [M + Na]+. 10-H: 1H NMR (250 MHz, CDCl3, 2 dia- (300 MHz, [D6]DMSO, 2 diastereomers): δ = 1.84 (s, 3 H) ppm;
stereomers): δ = 1.42 (s, 9 H), 2.05/2.07 (s, Σ 3 H), 2.82–3.15 (m, 2
H, CH2S), 3.50 (m, 1 H, CHS), 3.88 (m, 2 H, CH2O), 4.69 (m, 1
H, CHNH), ca. 4.8 (br. s, 1 H, OH), 6.49/6.62 (br. s, Σ 1 H,
CONH2), 6.96/7.05 (d, J = 7 Hz, Σ 1 H, NHAc), 7.11/7.27 (br. s,
Σ 1 H, CONH2) ppm. 13C NMR (62.9 MHz, CDCl3, 2 dia-
ABX system: δA = 2.775/2.784, δB = 2.94/2.95, δX = 4.35 (JAB =
13.6, JAX = 8.4, JBX = 5.1) ppm; δ = 7.27 (br. s, 4 H, OH, COOH,
NH2), 8.20 (d, J = 8.1 Hz, 1 H, NH) ppm. 13C NMR (75.5 MHz,
[D6 ]DMSO, 2 diastereomers): δ = 22.4 (Ac), 33.8/33.9
(CH2CHNH), ca. 36 (br., CD2CHOH), 52.30/52.35 (CHNH), ca.
stereomers): δ = 22.97/23.05 (Ac), 27.9 (Me, ϫ3), 33.9/34.4 (CH2S), 71 (br., CDHOH), 169.5, 172.2, 174.83/174.88 (CO) ppm. MS (ES):
49.2/50.2 (CHS), 52.4/52.9 (CHNH), 62.6/62.8 (CH2OH), 83.2/83.3
m/z (negative mode, %) = 505 (100) [2 M – H]–, 252 (39) [M – H]–.
(C-O), 169.65/169.71, 170.9/171.1 and 174.05/174.11 (CO) ppm.
N-Acetyl-S-[(R/S)-1-carbamoyl-2-hydroxyethyl]cysteine (5-H) and
N-Acetyl-S-{(R/S)-1-carbamoyl-2-hydroxy[1,2,2-D3]ethyl}cysteine
(5-D): Compounds 5-H and 5-D were prepared from the esters 10-
H (32 mg, 0.10 mmol) and 10-D (100 mg, 0.33 mmol) in water (0.5
and 1 mL, respectively) with concd. aq. HCl (0.2 and 0.3 mL,
respectively), as described above for the major regioisomer 4-H.
The products were isolated by preparative HPLC as glassy oils; tR
= 4.5/4.7 min (2:3, 2 diastereomers); 11 mg (42%) of 5-H, 36 mg
(44 %) of 5-D. 5-H: 1H NMR (300 MHz, D2O, major dia-
stereomer): δ = 2.05 (s, 3 H) ppm; ABX system (SCH2CHNH): δA
= 3.02, δB = 3.19, δX = 4.63 (JAB = 14, JAX = 8.5, JBX = 4.8) ppm;
tert-Butyl N-Acetyl-S-{(R/S)-2-carbamoyl-2-hydroxy[1,1,2-D3]ethyl}-
cysteinate (9-D) and tert-Butyl N-Acetyl-S-{(R/S)-1-carbamoyl-2-
hydroxy[1,2,2-D3]ethyl}cysteinate (10-D): The tert-butyl ester 8
(1.8 g, 8.2 mmol) was added under argon to an aqueous solution
(ca. 5 mL) of [D3]glycidamide (2-D, ca. 700 mg, 7.8 mmol), fol-
lowed by MeCN (20 mL) and 20% aq. Na2CO3 (0.5 mL). The mix-
ture was stirred rapidly under argon for 4 h, and the title com-
pounds were isolated as described above for the unlabelled ana-
logues; 0.90 g (37%) of 9-D as an oil, 0.13 g (5.4%) of 10-D as an
oil. 9-D: 1H NMR (250 MHz, CDCl3, 2 diastereomers): δ = 1.46
(s, 9 H), 2.03 (s, 3 H), 2.84–3.08 (mc, 2 H, CH2S), 4.69 (mc, 1 H, ABX system (OHCH2CHS): δA = 3.80, δB = 3.84, δX = 3.57 (JAB
CHNH), 4.98/5.08 (s, Σ 1 H, OH), 6.33 (br. s, 1 H, CONH2), 6.90 = 12, JAX = 5.9, JBX = 7.7) ppm. 13C NMR (75.5 MHz, D2O, 2:1
(“t”, J = 7 Hz, 1 H, NHAc), 7.03 (br. s, 1 H, CONH2) ppm. 13C mixture of 2 diastereomers): δ = 24.85 (Ac, assigned chemical
NMR (62.9 MHz, CDCl3, 2 diastereomers): δ = 23.1 (Ac), 27.9
shift), 35.4/35.5 (CH2S), 53.4/53.6 (CHS), 55.6/55.8 (CHN), 64.9
(Me ϫ3), 35.5 (CH2S), 53.0/53.4 (CHNH), 83.1/83.2 (C-O), 169.8, (CH2O), 176.6, 177.46/177.43, 178.7/178.8 (CO) ppm. HRMS
1
170.9 and 175.5 (CO) ppm. 10-D: H NMR (250 MHz, CDCl3, 2 (C8H14N2O5S): m/z (ESI, positive mode; %) = 273.05169 (100) [M
diastereomers): δ = 1.43 (s, 9 H), 2.02/2.04 (s, Σ 3 H), 2.88–3.13 + Na] {273.05156 (calcd.)}, 251.06977 (20) [M + H] {251.06962
(m, 2 H, CH2S), 4.3/4.4 (br. s, Σ 1 H, OH), 4.68 (mc, 1 H, CHNH),
6.53/6.67 (br. s, Σ 1 H, CONH2), 7.09 (br. m, 1 H, NHAc), 7.17/
(calcd.)}. 5-D: 1H NMR (300 MHz, D2O, 2:1 mixture of dia-
stereomers): δ = 2.04 (s, 3 H)ppm; ABX system: δA = 3.00/3.03, δB
7.27 (br. s, Σ 1 H, CONH2) ppm. 13C NMR (62.9 MHz, CDCl3, 2 = 3.17, δX = 4.62 (JAB = 14, JAX = 8.3, JBX = 4.7) ppm. 13C NMR
diastereomers): δ = 22.9/23.0 (Ac), 27.9 (Me, ϫ3), 33.6/34.1
(CH2S), 52.5/52.9 (CHNH), 83.1/83.2 (C-O), 169.66/169.73, 170.9/ signed chemical shift), 35.4/35.5 (CH2S), ca. 53.2 (br., CD), 55.6/
171.0 and 174.0 (CO) ppm. MS (ES): m/z (positive mode, %) = 641 55.7 (CHN), ca. 64.1 (br., CD2), 176.6, 177.42/177.38, 178.66/
(75.5 MHz, D2O, 2:1 mixture of diastereomers): δ = 24.85 (Ac, as-
(100) [2 M + Na]+, 332 (82) [M + Na]+; m/z (negative mode, %) = 178.75 (CO) ppm. HRMS (C8H11D3N2O5S): m/z (ESI, positive
354 (100) [M + HCOO–].
mode; %) = 276.07033 (100) [M + Na] {273.07040 (calcd.)},
254.08839 (75) [M + H] {254.08845 (calcd.)}.
N-Acetyl-S-[(R/S)-2-carbamoyl-2-hydroxyethyl]cysteine
(4-H):
Concd. aq. HCl (1.5 mL) was added dropwise with stirring to an
ice-cold solution of the tert-butyl ester 9-H (179 mg, 0.585 mmol)
N-Acetyl-S-[(R/S)-2-cyano-2-hydroxyethyl]cysteine (12-H) and N-
Acetyl-S-[(R/S)-1-cyano-2-hydroxyethyl]cysteine (13-H): A suspen-
in water (3 mL). The reaction mixture was kept at +4 °C for 2 h, sion of N-acetylcysteine (3) (163 mg, 1.00 mmol) in water (3 mL)
the aq. HCl was evaporated in vacuo (1–2 mbar), and the residue
was purified by chromatography on SiO2 (50 g) eluting with a
PrOH/water/AcOH (9:1:1) mixture; Rf ≈ 0.5, developed with aq.
was flushed with argon, cooled to 4 °C, then 20% aq. Na2CO3
(0.5 mL) was added, and subsequently cyanooxirane 11 (140 mg,
2.03 mmol). The reaction mixture was warmed up to room tem-
perature with rapid stirring, and after 1 h, it was acidified with 1 
aq. HCl to pH = 2. Then it was concentrated in vacuo to ca. 1 mL,
and the title compounds were isolated by preparative RP-HPLC
KMnO4. Yield: 112 mg (76%) of a colorless glassy solid; tR
=
6.0 min; A/B, 99:1; isocratic mode. 1H NMR (300 MHz, [D6]-
DMSO, 2 diastereomers): δ = 1.85 (s, 3 H) ppm; ABX system
(SCH2CHOH): δA = 2.67/2.69, δB = 2.81/2.83, δX = 3.98 (JAB
13.6, JAX = 7.3, JBX = 4) ppm; ABX system (SCH2CHNH): δA
2.77/2.78, δB = 2.95/2.96, δX = 4.36 (JAB = 13.6, JAX = 8.3, JBX
=
=
=
(column 8ϫ250 mm; A/B, 96:4; 10 injections, 0.1 mL each): tR =
6.6 min (for both epimers of the minor regioisomer 13-H), yield
9 mg (4%) of a glassy solid; 8.2 min (for both epimers of the major
5.1) ppm; δ = ca. 5.8 (br. s, OH), 7.2 (br. s, 2 H, CONH2), 8.20 (d, regioisomer 12-H), yield 85 mg (37%) of a glass-like foam. 12-H:
J = 6 Hz, 1 H, NH) ppm. 13C NMR (75.5 MHz, [D6]DMSO, 2 1H NMR (300 MHz, D2O): δ = 2.08 (s, 3 H) ppm; ABX system
diastereomers): δ = 22.4 (Ac), 33.9/34.0 (CH2CHNH), 36.75/36.83 (SCH2CHOH): δA/δB = 3.03–3.07, δX = 4.84 ppm; ABX system
(CH2CHOH), 52.2/52.3 (CHNH), 71.3/71.4 (CHOH), 169.4, 172.2,
174.7/174.8 (CO) ppm. MS (ES): m/z (positive mode, %) = 790 (58)
(SCH2CHNH): δA = 3.08, δB = 3.24, δX = 4.65 (JAB = 14, JAX
=
8, JBX = 5) ppm. 13C NMR (75.5 MHz, D2O): δ = 22.4 (Ac), 34.2
[3 M + NH3 + Na]+, 523 (67) [2 M + Na]+, 273 (56) [M + Na]+, (CH2CHNH), 36.7 (CH2CHOH), 53. 3 (CHNH), 61.7 (CHOH),
251 (100) [M + H]+; m/z (negative mode, %) = 499 (100) [2 M – 120.6 (CN), 174.2, 174.9 (CO) ppm. MS (ES): m/z (negative mode,
H] –, 249 (67) [M – H]–. HRMS (C8H14N2O5S): m/z = 251.06981
[M + H] {251.06962 (calcd.)}, 273.05177 [M + Na] {273.05156
(calcd.)}.
%) = 463 (100) [2 M – H]– , 231 (24) [M – H]– . HRMS
(C8H12N2O4S): m/z = 233.05922 [M + H] {233.05905 (calcd.)},
255.04126 [M + Na] {255.04100 (calcd.)}.
N-Acetyl-S-{(R/S)-2-carbamoyl-2-hydroxy[1,2,2-D3]ethyl}cysteine
(4-D): Compound 4-D was obtained from the tert-butyl ester 9-D
(276 mg, 0.89 mmol) in water (3 mL) as described above for the
corresponding unlabelled compound 4-H (1.5 mL of concd. aq.
tert-Butyl N-Acetyl-S-[(R/S)-2-(tert-butyldimethylsilyl)oxy-2-car-
bamoyl[1,1,2-D3]ethylcysteinate (14): Imidazole (122 mg,
1.82 mmol) and then tert-butyldimethylsilyl chloride (271 mg,
1.80 mmol) were added to a solution of hydroxyamide 9-D
4422
www.eurjoc.org
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 4417–4425

D-Labelled Oxidative Metabolites of Acrylamide and Acrylonitrile
(473 mg, 1.53 mmol) in anhydrous DMF (2 mL). The reaction mix-
ture was left at +5 °C for 4 d and was then taken up in an EtOAc/
water mixture (10:1, 100 mL). The organic layer was separated,
washed with water (20 mL), brine (20 mL) and dried. After evapo-
ration of the solvent, the residual oil (652 mg, 100%) was used in
the next step without further purification; Rf = 0.3 (EtOAc). 1H
NMR (300 MHz, CDCl3, 1:1 mixture of 2 diastereomers): δ = 0.09/
0.10/0.13/0.14 (s ϫ4, Me2Si, Σ 6 H), 0.91 (s, 9 H, tBuSi), 1.44/1.45
(s, 9 H, tBuO), 2.00/2.03 (s, 3 H, ac), 2.89–3.10 (m, 2 H,
SCH2CHNH), 4.65–4.77 (m, 1 H, CHNH), 5.72/5.77 (br. s, 1 H,
(5 mL) kept at 0 °C in the presence of methanol (5 mL) appeared
a colorless paste, and after 20 min, a solution of KCN (0.585 g,
9.0 mmol) in water (1.5 mL) was added dropwise at 0 °C. CAU-
TION: The reaction should be carried out in a well-ventilated hood,
and the remaining KCN in all aqueous solutions after the workup
should be oxidized with KMnO4 before disposal! The reaction mix-
ture was left to warm to room temperature and stirred for 2 h.
Water (20 mL) and subsequently diethyl ether (50 mL) were added.
The organic layer was separated, washed with brine, dried and con-
centrated in vacuo. The residue (0.75 g, corresponding to 81% yield
CONHH), 6.34/6.73 (d ϫ2, J = 7.8/7.9 Hz, Σ 1 H, NHCH), 6.59 of the title compound) was used in the next step without further
1
(br. s, 1 H, CONHH) ppm. MS (ES): m/z (positive mode; %) = 869
(100) [2 M + Na]+, 446 (13) [M + Na]+; m/z (negative mode) = 468
(100) [M + HCOO–].
purification. H NMR (300 MHz, CDCl3): δ = 0.11/0.12 (s ϫ2, 6
H, Me2Si), 0.91 (s, 9 H, tBu), 3.05 (d, J = 8.6 Hz, 1 H, OH), 3.84
(d, J = 3.8 Hz, CH2O), 4.46 (mc, 1 H, CHCN) ppm. 13C NMR
(75.5 MHz, CDCl3): δ = –5.5 (Me2Si), 18.2 (C), 25.7 (Me3C), 62.2
(CHCN), 64.2 (CH2), 118.3 (CN) ppm. MS (CI, NH3): m/z = 219
[M + NH4+].
tert-Butyl N-Acetyl-S-{(R/S)-2-(tert-butyldimethylsilyl)oxy-2-
cyano[1,1,2-D3]ethyl}cysteinate (15): Pyridine (0.29 mL, 3.6 mmol)
was added to a solution of amide 14 (652 mg, 1.54 mmol) in anhy-
drous CH2Cl2 (5 mL) at 0 °C, and subsequently trifluoroacetic an-
hydride (0.21 mL, 1.8 mmol) was added dropwise with stirring. The
reaction mixture was stirred at room temperature overnight, diluted
with CH2Cl2 (50 mL), washed with water (20 mL), 0.5  aq. H2SO4
(20 mL), water (20 mL) and brine (20 mL). After drying and evapo-
ration of the solvent, the residue was subjected to column
chromatography on SiO2 (100 mL) eluting with a hexane/EtOAc
(1:1) mixture, to yield the title compound as a colorless oil (415 mg,
3-(tert-Butyldimethylsilyloxy)-2-hydroxy[2,3,3-D3]propanenitrile
(20-D): Compound 20-D was synthesized from [D4]ethylene glycol
(OH,OD) (5 g) which was monoprotected with tert-butyldimethyl-
silyl chloride as described for the unlabelled compound,[20] and the
alcohol 18-D was then oxidized to the aldehyde 19-D according to
the procedure described previously for the unlabelled compound.[21]
The reaction of 19-D with aq. KCN in the presence of Na2S2O5
1
was carried out as described above for compound 20-H. H NMR
1
(300 MHz, CDCl3): δ = 0.10/0.11 (s ϫ2, 6 H, Me2Si), 0.90 (s, 9 H,
tBu), 3.20 (br. s, 1 H, OH) ppm. 13C NMR (75.5 MHz, CDCl3): δ
= –5.5 (Me2Si), 18.3 (C), 25.7 (Me3C), ca. 62 (t, CDCN), ca. 64
(m, CD2), 118.5 (CN) ppm.
66%); Rf = 0.8 (EtOAc), Rf = 0.3 (hexane/EtOAc, 1:1). H NMR
(300 MHz, CDCl3, 1:1 mixture of 2 diastereomers): δ = 0.13/0.14/
0.17/0.18 (s ϫ4, Me2Si, Σ 6 H), 0.879/0.885 (s, 9 H, tBuSi), 1.46 (s,
9 H, tBuO), 2.01/2.02 (s, 3 H, ac), 2.97–3.14 (m, 2 H,
SCH2CHNH), 4.66 (mc, 1 H, CHNH), 6.32 (d, J = 7.3 Hz, 1 H,
NHCH) ppm. 13C NMR (75.5 MHz, CDCl3, mixture of 2 dia-
stereomers): δ = –5.31/–5.25 (Me2Si), 18.0 (C), 23.1 (Ac), 25.4 (Me),
27.9 (Me), 35.4/35.5 (CH2S), ca. 37.5 (CD2), 52.5/52.6 (CHNH), ca.
62.3 (CD), 83.20/83.23 (CO), 118.98/119.00 (CN), 169.50/169.52,
169.79/169.83 (CO) ppm. MS (ES): m/z (positive mode; %) = 833
(49) [2 M + Na]+, 428 (100) [M + Na]+; m/z (negative mode; %) =
450 (100) [M + HCOO–], 464 (62) [M + AcO–].
3-(tert-Butyldimethylsilyloxy)-2-(tosyloxy)propanenitrile (21-H): To
a solution of the crude cyanohydrine 20-H (4.2 g, 21 mmol) in pyr-
idine (12 mL) was added tosyl chloride (4.2 g, 22 mmol) at 0 °C.
The mixture was left at +5 °C overnight, poured into a cold mixture
of concd. HCl (12 mL) and ice (50 g), and then CHCl3 (50 mL)
was added. The mixture was shaken, the organic layer was sepa-
rated, and the aqueous layer was extracted with CHCl3
(2 ϫ 30 mL). The combined organic layers were washed with a
phosphate buffer solution (pH = 7, 100 mL), dried and concen-
trated in vacuo. The title compound 21-H (6.5 g, 83%) was isolated
as a clear colorless oil by column chromatography on SiO2 (100 g)
eluting with a hexane/ether mixture (5:1). 1H NMR (300 MHz,
CDCl3): δ = 0.06 (s, 6 H, Me2Si), 0.85 (s, 9 H, tBu), 2.45 (s, 3 H,
Me), 3.91 (d, J = 5.8 Hz, 1 H, CH2O), 5.02 (t, J = 5.8 Hz, CHCN),
7.37 (d, J = 8.2 Hz, 2 H, Har), 7.82 (d, J = 8.4 Hz, 2 H, Har) ppm.
13C NMR (75.5 MHz, CDCl3): δ = –5.6/–5.6 (Me2Si), 18.1 (C),
21.7 (Me), 25.5 (Me3C), 63.0 (CH2O), 67.0 (CHCN), 114.0 (CN),
128.2 (CH), 130.2 (CH), 131.9 (C), 146.2 (C) ppm. MS (CI, NH3):
m/z (%) = 728 (96) [2 M + NH4+], 373 (100) [M + NH4+].
N-Acetyl-S-{(R/S)-2-cyano-2-hydroxy[1,1,2-D3]ethyl}cysteine (12-
D): Et3SiH (0.15 mL, 0.11 g, 0.94 mmol) was added to a solution
of the nitrile 15-D (61 mg, 0.15 mmol) in CH2Cl2 (0.3 mL) at 0 °C,
and subsequently TFA (0.18 mL, 2.34 mmol) was added. The
reaction mixture was kept at +5 °C for 2 d and at room tempera-
ture for 1 d, concentrated in vacuo and taken up with CHCl3
(2ϫ10 mL), then concentrated again. The residue was dissolved in
MeCN (0.15 mL), and aq. HF (51–56 %, 0.1 mL) was added at
0 °C. After stirring at room temperature for 2 h, oily drops of tBu-
Me2SiF appeared in the reaction mixture. It was concentrated in
vacuo, taken up with water (2ϫ1 mL), concentrated again and the
residue subjected to preparative HPLC (A/B, 94:6) to yield the title
compound (tR = 8.2 min) as a viscous oil (14 mg, 40 %). 1H
NMR (300 MHz, D2O): δ = 2.13 (s, 3 H) ppm; ABX system
3-(tert-Butyldimethylsilyloxy)-2-tosyloxy[2,3,3-D3]propanenitrile-
(21-D): Compound 21-D was obtained from 20-D (1.33 g,
6.5 mmol) in pyridine (5 mL) with p-toluenesulfonyl chloride
(1.33 g, 7.0 mmol) in 92% yield (2.15 g) as described above for
(SCH2CHNH): δA = 3.14, δB = 3.30, δX = 4.69 (JAB = 14, JAX
=
8, JBX = 5) ppm. 13C NMR (75.5 MHz, D2O): δ = 22.7 (Ac), 34.4
(CH2CHNH), ca. 36.5 (CD2CHOH), 53.7 (CHNH), ca. 62
(CDOH), 120.9 (CN), 174.7, 175.3 (CO) ppm. MS (ES): m/z (nega-
tive mode; %) = 469 (100) [2 M – H]–, 234 (23) [M – H]–. HRMS
(C8H9D3N2O4S): m/z = 236.07797 [M + H] {236.07788 (calcd.)},
258.05996 [M + Na] {258.05982 (calcd.)}, 234.06339 [M – H]
{234.06333 (calcd.)}.
1
compound 21-H. H NMR (300 MHz, CDCl3): δ = 0.06 (s, 6 H,
Me2Si), 0.85 (s, 9 H, tBu), 2.45 (s, 3 H, Me), 7.37 (d, J = 8.2 Hz,
2 H, Har), 7.82 (d, J = 8.4 Hz, 2 H, Har) ppm. 13C NMR
(75.5 MHz, CDCl3): δ = –5.6/–5.6 (Me2Si), 18.1 (C), 21.7 (Me),
25.7 (Me3C), 62.5 (m, CD2O), 66.8 (t, CDCN), 114.0 (CN), 128.2
(CH), 130.2 (CH), 132.1 (C), 146.1 (C) ppm. MS (CI, NH3): m/z
(%) = 376 (100) [M + NH4+].
3-(tert-Butyldimethylsilyloxy)-2-hydroxypropanenitrile (20-H): In a
suspension of the aldehyde 19-H[21] (806 mg, 4.62 mmol) and
Na2S2O5 (977 mg, 5.14 mmol) in water (10 mL) and methanol
N-Acetyl-S-[(R/S)-1-cyano-2-hydroxyethyl]cysteine (13-H ϵ 23a):
Et3N (1 mL) was added to a solution of tosylate 21-H (0.33 g,
0.92 mmol) and N-acetylcystein (0.21 g, 1.3 mmol) in MeOH
Eur. J. Org. Chem. 2008, 4417–4425
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4423

V. N. Belov, S. M. Korneev, J. Angerer, A. de Meijere
FULL PAPER
(5 mL) under Ar, and the mixture was stirred at 40 °C for 36 h.
The solvent and excess of base were evaporated in vacuo, and satd.
aq. NaHCO3 (10 mL) was added to the residue. The mixture was
extracted with diethyl ether (3ϫ20 mL), and the organic layers
were discarded. The aqueous layer was acidified with 5% aq. citric
acid (pH ≈ 3) and then extracted with diethyl ether (3ϫ20 mL).
The combined organic solutions were dried and the solvents evapo-
rated in vacuo. The residue (ca. 0.1 g of the crude compound 22a)
was dissolved in THF (2 mL), and a cold mixture of concd. HCl
(0.5 mL) and water (0.2 mL) was added carefully at 0 °C. After
having kept the mixture at 0 °C for 2 h, it was concentrated in
vacuo (ca. 1 mbar) at 0 °C, the residue was dissolved in water
(1 mL), and the title compound was isolated by preparative RP-
HPLC (column 8ϫ250 mm; A/B, 96:4; 10 injections, 0.1 mL each):
tR = 6.6 min (for both epimers of the regioisomer 13-H), yield
30 mg (14%) of a glassy solid (after 3 co-evaporations with water
to remove traces of TFA from the eluent and lyophilization). 1H
NMR (300 MHz, D2O, mixture of 2 diastereomers): δ = 2.14 (s, 3
H) ppm; ABX system (cysteine residue): δA = 3.25/3.27, δB = 3.42/
3.44, δX = 4.79 (JAB = 14.2, JAX = 7.7/8.3, JBX = 4.8/5.3) ppm;
ABX system [SCH(CN)CH2OH]: δA = 3.94, δB = 4.02, δX = 4.13
(t) (JAB = 11.4, JAX = 6.2, JBX = 5.2) ppm. 13C NMR (75.5 MHz,
D2O): δ = 22.39/22.42 (Ac), 33.28/33.40 (CH2S), 36.35/36.46
(CHCN), 52.96/53.12 (CHNH), 62.15/62.12 (CH2OH), 119.69/
119.72 (CN), 173.88/173.91, 174.88/174.94 (CO) ppm. MS (ESI):
m/z (negative mode, %) = 463 (100) [2 M – H]–, 231 (24) [M –
H]–. HRMS (C8H12N2O4S): m/z (%) = 233.05908 (60) [M + H]
{233.05905 (calcd.)}, 255.04105 (100) [M + Na] {255.04100
(calcd.)}, 271.00606 (37) [M + K].
Acknowledgments
The authors are grateful to R. Machinek and Dr. H. Frauendorf
et al. (Georg-August-Universität Göttingen) for measuring NMR
and mass spectra.
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N-Acetyl-S-{(R/S)-1-cyano-2-hydroxy[2,2-D2]ethyl}cysteine (23b):
Compound 23b was obtained from the tosylate 21-D as described
1
above for compound 23a. H NMR (300 MHz, D2O, mixture of 2
diastereomers): δ = 2.14 (s, 3 H) ppm; ABX system (cysteine resi-
due): δA = 3.22/3.26, δB = 3.39/3.43, δX = 4.76/4.78 (JAB = 14, JAX
= 7.8/8.3, JBX = 4.65/5.05) ppm; 4.10 [s, 1 H, SCH(CN)CD2OH]
ppm. 13C NMR (75.5 MHz, D2O): δ = 21.8 (Ac), 32.66/32.78
(CH2S), 35.57/35.69 (CHCN), 52.37/52.532 (CHNH), ca. 61.5
(CD2), 119.08 (CN), 173.31/173.34, 174.30/174.36 (CO) ppm.
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{235.07160 (calcd.)}, 257.05370 (49) [M
(calcd.)}.
+ Na] {257.05355
N-Acetyl-S-{(R/S)-1-cyano-2-hydroxy[1,2,2-D3]ethyl}cysteine (23c):
Compound 23c was obtained from the tosylate 21-D (0.36 g,
1.0 mmol) and N-acetylcystein (0.28 g, 1.7 mmol) in MeOD
(10 mL) with Et3N as described above for compound 23a (ϵ 13-
H). The residue after evaporation of the solvent and the excess of
Et3N, was treated with D2O (6 mL) and Na2CO3, until the pH
value of the aqueous phase reached 9–10. The mixture was ex-
tracted with diethyl ether (3ϫ5 mL), and the aqueous layer was
carefully acidified at 0 °C with D2SO4 diluted with D2O (1:4), until
the pH value of the solution reached 3. Further workup was per-
formed as described above for compound 23a, and finally 32 mg
(14%) of the title compound 23c was isolated. 1H NMR (300 MHz,
D2O, mixture of 2 diastereomers): δ = 2.13 (s, 3 H) ppm; ABX
system (cysteine residue): δA = 3.23/3.27, δB = 3.38/3.42, δX = 4.77/
4.80 (JAB = 14.1, JAX = 7.6/8.2, JBX = 4.6/5.1) ppm. 13C NMR
(75.5 MHz, D2O): δ = 22.35/22.39 (Ac), 33.2/33.3 (CH2S), ca. 36
(m, CDCN), 52.9/53.1 (CHNH), ca. 62 (m, CD2), 119.7 (CN),
173.82/173.85, 174.89/174.95 (CO) ppm. MS (ESI): m/z (negative
mode; %) = 469 (100) [2 M – H]–, 234 (16) [M – H]–. HRMS
(C8H9D3N2O4S): m/z (%) = 236.07792 (100) [M + H] {236.07788
(calcd.)}, 258.05992 (63) [M + Na] {258.06038 (calcd.)}.
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4424
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 4417–4425

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[23] The use of [D6]acetone allows one to identify the signals of the
CH2OH group: in our case the OH proton gave a broad signal
at δ ≈ 5.0 ppm, and the CH2 group was detected as a broad
multiplet at δ = 3.83 ppm; Linhart found the former signal for
the two diastereomers at δ = 4.84 and 4.87 ppm as two triplets
with J = 6.2 Hz and the latter at δ = 3.85 ppm as a multiplet.
The chemical shifts of the CHS(CN) fragment signals were
found to be the same as reported in ref.[15]: δ = 4.01 and
4.05 ppm (J = 5.4 and 6.9 Hz) for 2 diastereomers.
Received: March 19, 2008
[14] Because an amide group may easily be converted into a cyano
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ˇ
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ˇ
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presence of Et3N to give the methyl ester of 23a. We have found
that under similar conditions, the nucleophilic substitution of
the tosyl group in 21-H proceeds rather slowly (see Results and
Published Online: June 9, 2008
Eur. J. Org. Chem. 2008, 4417–4425
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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