Preparation of cysteine adducts by regioselective ring-opening reactions of phenyloxirane

Article abstract of DOI:10.1515/hc-2015-0042

Regioselective ring-opening reactions of phenyloxirane by protected cysteine are described, which enable the synthesis of pure regioisomeric cysteine adducts required for bioanalytical studies on adducts of protein with styrene. The reaction catalyzed by tris(pentafluorophenyl)borane proceeds regioselectively and stereospecifically to give protected S-(2-hydroxy-1-phenylethyl)cysteine (α-adduct) with the inversion of configuration at the α-carbon. By contrast, when the same reaction is catalyzed by tetraalkylammonium fluorides, S-(2-hydroxy-2-phenylethyl)cysteine (β-adduct) is formed predominantly, and a compete racemization is observed.

Full text of DOI:10.1515/hc-2015-0042

Heterocycl. Commun. 2015; 21(2): 61–65
Preliminary Communication
Alena Moulisová and Igor Linhart*
Preparation of cysteine adducts by regioselective
ring-opening reactions of phenyloxirane
Abstract: Regioselective ring-opening reactions of pheny- ongoing research on protein adducts, we needed to
loxirane by protected cysteine are described, which ena- prepare cysteine adducts of phenyloxirane (styrene oxide,
ble the synthesis of pure regioisomeric cysteine adducts 1), a mutagenic and carcinogenic metabolite of styrene.
requiredforbioanalyticalstudiesonadductsofproteinwith The direct reaction of ꢀ-cysteine (2) with 1 in strongly
styrene. The reaction catalyzed by tris(pentafluorophenyl) alkaline solution (EtOH/EtONa) afforded a 1:2 mixture of
borane proceeds regioselectively and stereospecifically S-(2-hydroxy-1-phenylethyl)cysteine (3) and S-(2-hydroxy-
to give protected S-(2-hydroxy-1-phenylethyl)cysteine 2-phenylethyl)cysteine (4) as reported by Cundari et al.
(
α-adduct) with the inversion of configuration at the [6] (Scheme 1). These authors also attempted to achieve
α-carbon. By contrast, when the same reaction is catalyzed the regioselectivity of this reaction by using unprotected
by tetraalkylammonium fluorides, S-(2-hydroxy-2-phenyle- 2-bromo-2-phenylethanol and 2-bromo-1-phenylethanol
thyl)cysteine (β-adduct) is formed predominantly, and a as synthetic equivalents of 1. However, under the alka-
compete racemization is observed.
line reaction conditions, these bromohydrines under-
went dehydrobromination to give 1 so that mixtures 3
Keywords: cysteine adducts; oxirane ring opening; protein and 4 were always formed. This mixture was converted to
adducts; styrene; styrene oxide.
(t-butoxycarbonyl)amino (N-BOC) derivatives 3a and 4a
6]. Small samples of 3 and 4 were previously obtained by
[
the direct reaction of 1 with cysteine. This reaction gave
a 2:1 mixture of 3 and 4, each of them consisting of two
diastereomers. Individual isomers were then separated
by high-performance liquid chromatography. However, it
was not possible to scale up this procedure to obtain suf-
ficient quantities of the individual products [7]. As a rule,
a direct attack by nucleophiles proceeds on the less sub-
stituted (β) carbon of the oxirane ring. By contrast, in an
acidic environment, where the oxirane oxygen is either
protonated or coordinated to a Lewis acid, the nuclephilic
attack is redirected to the more substituted (α) carbon
DOI 10.1515/hc-2015-0042
Received October 21, 2014; accepted February 26, 2015; previously
published online March 26, 2015
Adducts with blood proteins, mainly albumin and globin,
are formed by reactions of numerous xenobiotics enter-
ing human body and/or their electrophilic metabolites.
They found applications in biological monitoring of
cumulative exposure to environmental and occupational
mutagens and carcinogens [1, 2]. The main site of attack
in both globin and albumin is the highly nucleophilic SH
group of cysteine [3]. For bioanalytical studies on protein
adducts, S-substituted cysteines are needed as analytical
standards as well as other research tools. The nucleophilic
cysteine SH group of glutathione also plays a fundamen-
tal role to prevent cell damage by various electrophiles
as well as by free radicals [4, 5]. In connection with our
[
8]. However, 1 shows a rather anomalous regioselectiv-
ity of ring-opening reactions. In reactions of 1 with thiols
in slightly alkaline aqueous solutions [9, 10] or in ionic
liquids [11, 12] mainly but not exclusively, the products
of α-attack were observed. However, with a wide range of
substituted oxiranes, the same reactions gave exclusively
products of β-attack. This may be due to a combination of
the resonance stabilization of α-carbocation by adjacent
aromatic ring and of the electron withdrawing effect of
the phenyl group. The resonance stabilization of the car-
bocation at α-position is expected to play a crucial role if
*
Corresponding author: Igor Linhart, Institute of Chemical
Technology, Faculty of Chemical Technology, Department of Organic
Chemistry, Technická 1905, Prague CZ-166 28, Czech Republic,
e-mail: linharti@vscht.cz
the reaction proceeds by S 1 mechanism, which should
Alena Moulisová: Institute of Chemical Technology, Faculty of
Chemical Technology, Department of Organic Chemistry, Technická
N
be preferred in acidic solutions. However, when 1 was
allowed to react with 2 in acidic aqueous solutions at pH
1905, Prague CZ-166 28, Czech Republic
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2ꢀꢀꢀꢀꢁꢀA. Moulisová and I. Linhart: Cysteine adducts by ring-opening reactions of phenyloxirane
O
Table 2:ꢀReactions of racemic 1 with protected cysteines 2a and 2b.
COOH
NH2
pH 3–7
HS
Cysteine ꢁ
derivative
Reagent ꢁ
Conditionsꢁ
Yield of
ꢁ
Ratio of
protected
ꢃ:ꢄb
1
2
protected
a
ꢃ+ꢄ
OH
OH
COOH
NH2
COOH
NH2
S
ꢂa
ꢂb
ꢂa
ꢂb
ꢂa
ꢂa
ꢂb
ꢂa
ꢂa
ꢂa
ꢂb
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
NBS/Et Nꢄ
CH CN/rt
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢊꢅ%
ꢈꢅ%
ꢈꢅ%
ꢇꢅ%
traces
ꢉꢅ%
ꢉꢋ%
ꢉꢊ%
ꢈꢅ%
ꢄ<ꢄꢋ%
ꢄ<ꢄꢊꢅ%
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢃa only
ꢃb only
ꢃ
ꢃ
S
NBS/Et Nꢄ
CH CN/rt
ꢃ
ꢃ
c
3
4
B(C
ꢉ
F
ꢋ
)
ꢃ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
CH
ꢈ
Cl
ꢈ
/rt
ꢃa only
c
B(C F )
CH Cl /rt
ꢃb only
ꢉ
ꢋ
ꢃ
ꢈ
ꢈ
Scheme 1ꢀꢂ
KF
CH CN/rt
–
ꢃ
TBAF
TBAF
CH CN/rt
ꢊ:ꢊꢅc
ꢊ:ꢊꢅc
ꢊ:ꢆ
ꢃ
CH CN/rt
ꢃ
TMAF
CH CN/rt
ranging from 3 to 6, we observed the formation of both
adducts 3 and 4 in the ratio ranging from 2:1 to 3:4. Less
α-adduct was formed with decreasing pH but the ratio did
not differ very much from that observed at pH 7. Appar-
ently, at this pH range, the reaction does not proceed by
ꢃ
Et N
CH CN/rt
ꢊ:ꢃ
ꢃa only
ꢃb only
ꢃ
ꢃ
ZnClꢈ
NBS
CH CN/rt
ꢃ
CH CN/rt
ꢃ
a
ꢃa+ꢄa or ꢃb+ꢄb.
b
ꢃa:ꢄa or ꢃb:ꢄb.
c
SN1 mechanism. The reaction is shown in Scheme 1.
This reaction was performed with racemic ꢅ as well as with its pure
enantiomers.
Observed minor changes in regioselectivity may
-
be attributed to decreasing ratio of S /SH in cysteine
(
Table 1). Ring-opening reactions of 1 were further tested
with two protected cysteine derivatives, namely, N-t-bu- reaction with 2a and 2b, which afforded the correspond-
toxycarbonyl- and N-benzyloxycarbonylcysteine methyl ing protected isomer 3 exclusively (Scheme 2). However,
esters, 2a and 2b, respectively. Ring opening proceeding the reaction proceeded slowly, and conversion was still
in the presence of N-bromosuccinimide (5 mol%), which incomplete after 3 days at room temperature (Table 2).
for various benzenethiols and alkanethiols was reported Increasing the reaction temperature did not lead to any
to give mainly products of α-attack [13], gave low yields of improvement of the yield but resulted in lower product
expected products with the protected cysteines 2a and 2b. purity. Reaction with 2b afforded a better yield than that
These products were accompanied by the corresponding
cysteine derivatives (Table 2).
OH
OH
In an attempt to improve regioselectivity of the reac-
COOMe
COOMe
NH-PG
tion and/or yields of the products, we further tested
several combinations of the activating agent and solvent,
such as Et N, LiClO , or ZnCl in dichloromethane or ace-
Ph
S
Ph
S
NH-PG
S,R - 3a-b
R,R - 3a-b
3
4
2
tonitrile. Products of the α-attack always prevailed, and
the yields were always low due to both low conversion
and by-product formation. Efficient and regioselective
cleavage of 1 via α-attack by benzenethiol, aniline, allyl
alcohol, and propargyl alcohol was reported in the pres-
B(C6F5)3/CH2Cl2
H
COOMe
Ph
H
O
HS
O
ence of tris(pentafluorophenyl)borane as a Lewis acid Ph
NH-PG
2a-b
catalyst [14]. This process proved to be applicable for the
R - 1
S - 1
TBAF
MeCN-THF
TBAF
MeCN-THF
rac - 1
Table 1:ꢀConversion of 2 and product ratio (3:4) after 3 h of reaction
with 1 in neutral and acidic aqueous solutions as determined by LC/MS.
TBAF
MeCN-THF
Acidityꢁ
Conversion of ꢂ (%)ꢁ
Product ratio ꢃ:ꢄ
HO
COOMe
NH-PG
pH ꢃ
pH ꢇ
pH ꢋ
pH ꢉ
pH 7a
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢅ.ꢆꢄ
ꢊ.ꢅꢄ
ꢋ.ꢊꢄ
ꢇꢊꢄ
7ꢇ:ꢈꢉ
7ꢃ:ꢈ7
ꢉ7:ꢃꢃ
ꢉꢉ:ꢃꢇ
7ꢅ:ꢃꢅ
S
Ph
4a-b
2
a, 3a, 4a: PG = tert-butyloxycarbonyl (BOC)
2b, 3b, 4b: PG = benzyloxycarbonyl
ꢊꢅꢅꢄ
aReaction at pH ꢌ was performed with both R- and S-enantiomers of 1.
Scheme 2ꢀꢂ
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A. Moulisová and I. Linhart: Cysteine adducts by ring-opening reactions of phenyloxiraneꢂꢁꢀꢀꢀꢂ63
with 2a (Table 2). Acid-catalyzed ring-opening reactions expected, acid-catalyzed racemization was observed after
of substituted oxiranes generally proceed by S 1 mecha- the deprotection of pure diastereomers of 3a. Attempts to
N
nism, resulting in the racemization of the products [8]. remove the Z-protecting group of 3b and 4b by Pd-cata-
Nevertheless, we observed stereospecific ring opening lyzed hydrogenolysis were unsuccessful probably because
with the inversion of configuration at the α-carbon, so of catalyst poisoning by sulfur-containing substrates. This
that the reaction of pure (R)-1 gave exclusively S,R- 3a or protecting group can be removed by reaction with tri-
S,R-3b from 2a and 2b, respectively. In a similar way, pure methylsilyl iodide (TMSI) [18]. With 3b, this reaction fol-
substrate (S)-1 was transformed into the diastereomer R,R- lowed by alkaline ester hydrolysis afforded a mixture of 3
3
a or R,R-3b (Scheme 2). Absolute configuration of the and 4. Pure diastereomers R,R- and S,R-3b gave predomi-
13
products was confirmed by the comparison of C NMR nantly but not exclusively R,R- and S,R-diastereomers,
shifts with those of the analogous N-acetyl derivatives [15]. respectively, accompanied with a mixture of R,R- and S,R-
The inversion of configuration is characteristic for diastereomer of 3, as determined by LC/MS analysis com-
SN2 reactions. Therefore, it can be concluded that, at least paring retention times and mass spectra with those of the
under the conditions used, oxirane ring opening cata- products obtained by the reaction of pure enantiomers of
lyzed by B(C F ) proceeds by S 2 mechanism, without for- 1 with 2. Although this reaction was not a useful prepara-
6
5
3
N
mation of an intermediary carbocation but probably with tive route to pure 3, it helped us to confirm the absolute
a significant weakening of the C -O bond of 1 in the transi- configuration of R,R-3 and S,R-3. The deprotection of 4b by
α
tion state due to the binding interaction of the Lewis acid TMSI also yielded a product mixture.
with the oxirane oxygen.
In conclusion, simple regioselective ring-opening
Eventually, experiments with tetrabutylammonium methods were developed, enabling the preparation of
fluoride (TBAF) as a base afforded predominantly products pure regioisomeric adducts 3 and 4 of phenyloxirane with
of β-attack. Thus, the reaction of 1 with 2a in the presence cysteine. These products are useful tools in the research on
of 10 mol% of TBAF led to a 10:1 mixture of protected prod- proteinadductsandinthebiologicalmonitoringoflong-term
ucts 4a and 3a. After chromatographic separation, pure cumulative exposure to styrene. Surprisingly, the oxirane
protected isomer 4a was obtained in a satisfactory yield ring opening of 1 catalyzed by tris(pentafluorophenyl)
(
Table 2). In fact, the SH group of cysteine can be easily borane proceeds by S 2 mechanism with full inversion of
N
deprotonated (pK ꢁ=ꢁ8.2). Hence, the observed exceptional configuration. By contrast, the ring opening catalyzed by
a
regioselectivity indicates that TBAF does not serve solely TBAF, which was expected to proceed stereospecifically,
as a base because other bases direct the SH-attack mainly always led to the mixture of diastereomers because of full
to the α-carbon. A commercially available solution of TBAF racemization of 1 enantiomers in the presence of TBAF.
in THF used in these experiments contained approxi-
mately 5% of water so that the nucleophilic attack could
be redirected to the β-carbon of 1 by the formation of com-
plexes held together by hydrogen bonding. ‘Anhydrous’ Experimental details
TBAF was reported to contain about 0.3 equivalents of
Tris(pentafluorophenyl)borane, 97% pure, was obtained from Alfa
water [16]. Attempts to remove this residual water results in
Aesar. TBAF, 1 ꢂ solution in THF, TMAF, TMSI, racemic styrene oxide
the degradation of TBAF due to Hofmann elimination [17].
Therefore, we also used tetramethylammonium fluoride
(
1), and (R)- and (S)-enantiomers of 1 and ꢀ-cysteine were purchased
from Sigma-Aldrich. All other chemicals were of analytical or syn-
thetic grade and were used as received. SPE columns Strata X (1 g/12
(
TMAF) as a catalyst that, unlike TBAF, can be obtained in
an anhydrous form. The result, however, was very similar mL) were purchased from Chromservis, precoated plates of silica gel
6
0 F254 were from Merck and silica gel 60, and 230–400 mesh for
to that obtained with TBAF. Unlike the reaction catalyzed
by Lewis acids, base-catalyzed reaction should be stere-
ospecific so that a single diastereomer of 4a and 4b should
be obtained from each enantiomer of 1. However, we
column chromatography was from Sigma-Aldrich.
NMR spectra were recorded on a Varian 400 spectrometer oper-
1
13
ating at 400 MHz for H and 100 MHz for C. LC/MS analyses were
conducted on a Thermo Scientific LXQ linear trap mass spectrometer
observed the formation of 1:1 diastereomer mixtures of 4a in tandem with a Janeiro LC system consisting of two Rheos 2200
and 4b in the reaction with pure enantiomers (R)-1 and (S)- pumps and CTC PAL autosampler. Electrospray ionization in the pos-
itive ion mode was used. The collision gas was helium, the capillary
1
as well as with racemic 1. Control experiments showed
temperature was set at 300°C, and the capillary voltage was 4.6 kV.
High-resolution mass spectra (HRMS) were measured on a Thermo
Fisher Scientific LTQ Orbitrap mass spectrometer using electrospray
ionization in the positive mode. Specific optical rotation was meas-
that in the presence of enantiomeric 1, the products rapidly
racemized in the presence of TBAF. The deprotection of 3a
and 4a was conducted by treatment of trifluoroacetic acid
(
TFA) in dichloromethane for 4 h at room temperature. As ured on a polarimeter Perkin-Elmer 241.
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4ꢀꢀꢀꢀꢁꢀA. Moulisová and I. Linhart: Cysteine adducts by ring-opening reactions of phenyloxirane
1
Reaction of styrene oxide (1) with protected cysteines in
the presence of Lewis acid
yield 139 mg (38%); R ꢁ=ꢁ0.25 (chloroform/ethyl acetate, 10/1); H NMR
f
(
CDCl ): δ 2.85 (m, 2H), 3.69 (s, 3H), 3.82 (m, 2H), 3.99 (m, 1H), 4.60 (m,
3
1
3
1
H), 5.13 (s, 2H), 5.73 (d, 1H, J ꢁ=ꢁ 8.2 Hz), 7.32 (m, 10H); C NMR (CDCl3)
δ 34.0, 52.7, 53.6, 53.8, 65.9, 67.2, 127.9, 128.0, 128.1, 128.3, 128.6, 128.8,
To a stirred solution of protected cysteine, 2a [19] or 2b [20]
+
1
36.1, 139.1, 156.0, 171.0; HRMS: Found m/z 412.1185 [M+Na] , calcd
(
0.94 mmol) and 1 (0.12 g, 0.95 mmol) in dry dichloromethane B(C F )
+
20
6
5
3
m/z 412.1189; ESI-MS: m/z 390 [M+H] ; [ α]D -243.5° (cꢁ=ꢁ0.6, CHCl ).
3
(
25 mg, 5 mol%) was added, and stirring was continued for 3 days
at room temperature. The mixture was concentrated, and the crude
residue was purified by column chromatography using cyclohexane/
acetone 5:1 as an eluent.
Reaction of styrene oxide (1) with protected cysteines
in the presence of TBAF
N-(tert-Butoxycarbonyl)-S-(2-hydroxy-1-phenylethyl)-ꢀ-cysteine
methyl ester (3a)ꢃColorless oil; 1:1 diastereomeric mixture; yield A solution of protected cysteine 2a or 2b (0.68 mmol) and 1 (0.082 g,
1
6
7 mg (21%); R ꢁ=ꢁ0.22 (chloroform/ethyl acetate, 10/1); H NMR 0.68 mmol) in dry CH CN was treated with 1 ꢂ TBAF solution in
f
3
(
CDCl ): δ 1.43 and 1.45 (s, 9H), 2.24 (br s, 1H), 2.85 (m, 2H), 3.69 and THF (0.068 mL, 10 mol%). The solution was stirred until the reac-
3
3
.73 (s, 3H), 3.85 (m, 2H), 3.98 (m, 1H), 4.50 (m, 1H), 5.24 and 5.36 (br tion was completed as judged by thin layer chromatographic (TLC)
13
s, 1H), 7.32 (m, 5H); C NMR (CDCl ): δ 28.5, 33.9 and 34.5, 52.8, 53.6, analysis (approximately 5 h). The solvent was removed in vacuo, and
3
5
3.7, 65.9 and 66.1, 80.5, 128.1, 128.3, 129.0, 139.3 and 139.5, 155.3 and the product was then isolated by column chromatography using
+
1
55.6, 171.6; HRMS: Found m/z 378.1352 [M+Na] , calcd m/z 378.1346; cyclohexane/acetone, 5:1, as an eluent.
+
ESI-MS: m/z 356 [M+H] .
N-(tert-Butoxycarbonyl)-S-(2-hydroxy-2-phenylethyl)-ꢀ-cysteine
(
1S)-N-(tert-Butoxycarbonyl)-S-(2-hydroxy-1-phenylethyl)-ꢀ- methyl ester (4a)ꢃColorless oil; 1:1 diastereomeric mixture, yield
1
cysteine methyl ester (S,R-3a)ꢃColorless oil; one diastereomer; 125 mg, (52%), R ꢁ=ꢁ0.32 (chloroform/ethyl acetate, 10/1); H NMR
f
1
yield 65 mg (20%); R ꢁ=ꢁ0.22 (chloroform/ethyl acetate, 10/1); H NMR (CDCl ): δ 1.44 (s, 9H), 2.76 (m, 1H), 2.97 (m, 3H), 3.75 and 3.76 (s, 3H),
f
3
(
CDCl ): δ 1.43 (s, 9H), 2.88 (m, 2H), 3.74 (s, 3H), 3.86 (m, 2H), 3.97 (m, 4.57 (m, 1H), 4.76 (m, 1H), 5.43 and 5.55 (d, 1H, J ꢁ=ꢁ 7.6 and 7.6 Hz), 7.31
3
13
(m, 5H); 13C NMR (CDCl ): δ 28.3, 35.2 and 35.3, 52.6, 53.6, 53.7, 72.3
1
H), 4.50 (m, 1H), 5.23 (d, 1H, J ꢁ=ꢁ 7.4 Hz), 7.32 (m, 5H); C NMR (CDCl ):
3
3
δ 28.3, 33.7, 52.6, 53.4, 53.5, 65.6, 80.3, 127.9, 128.0, 128.8, 139.0, 155.1, and 72.6, 80.3, 125.7, 127.9, 128.5, 142.4, 155.3, 171.4; HRMS: Found m/z
+
+
+
378.1352 [M+Na] , calcd m/z 378.1346; ESI-MS: m/z 356 [M+H] .
1
71.3; HRMS: Found m/z 378.1349 [M+Na] , calcd m/z 378.1346; ESI-
+
20
MS: m/z 356 [M+H] ; [ α]D +100.5° (cꢁ=ꢁ1, CHCl ).
3
N-(Benzyloxycarbonyl)-S-(2-hydroxy-2-phenylethyl)-ꢀ-cysteine
methyl ester (4b)ꢃColorless oil; 1:1 diastereomeric mixture; yield
(
1R)-N-(tert-Butoxycarbonyl)-S-(2-hydroxy-1-phenylethyl)-ꢀ-
1
1
28 mg; (48%), R ꢁ=ꢁ0.30 (chloroform/ethyl acetate, 10/1); H NMR
cysteine methyl ester (R,R-3a)ꢃColorless oil; one diastereomer;
f
1
(CDCl ) δ 2.90 (m, 4H), 3.75 and 3.76 (s, 3H), 4.63 (m, 1H), 4.75 (m, 1H),
yield 57 mg (17%); R ꢁ=ꢁ0.22 (chloroform/ethyl acetate, 10/1); H NMR
3
f
1
3
5
(
.12 (s, 2H), 5.79 and 5.93 (br s, 1H, J ꢁ=ꢁ 7.3 Hz), 7.32 (m, 10H); C NMR
(
CDCl ): δ 1.46 (s, 9H), 2.51 (br s, 1H), 2.84 (m, 2H), 3.69 (s, 3H), 3.84
3
CDCl ) δ 35.1, 42.3 and 42.5, 52.8, 53.9 and 54.1, 67.2, 72.5 and 72.7,
(
m, 2H), 4.01 (m, 1H), 4.54 (m, 1H), 5.36 (d, 1H, J ꢁ=ꢁ 7.4 Hz), 7.32 (m, 5H);
3
13
125.7, 127.9, 128.1, 128.3, 128.6, 136.1, 142.4, 155.9, 171.1; HRMS: Found
C NMR (CDCl ): δ 28.3, 34.3, 52.6, 53.2, 53.5, 65.9, 80.3, 127.8, 128.0,
3
+
+
+
m/z 412.1198 [M+Na] , calcd m/z 412.1189; ESI-MS: m/z 390 [M+H] .
1
28.8, 139.2, 155.4, 171.3; HRMS: Found m/z 378.1349 [M+Na] , calcd
+
20
m/z 378.1346; ESI-MS: m/z 356 [M+H] ; [ α]D -103.5° (cꢁ=ꢁ1, CHCl ).
3
N-(Benzyloxycarbonyl)-S-(2-hydroxy-1-phenylethyl)-ꢀ-cysteine Deprotection of Z-protected cysteine methyl ester
methyl ester (3b)ꢃColorless oil; 1:1 diastereomeric mixture; yield
adducts 3b and 4b
1
1
45 mg (40%); R ꢁ=ꢁ0.25 (chloroform/ethyl acetate, 10/1); H NMR
f
(
CDCl ): δ 2.89 (m, 2H), 3.69 and 3.74 (s, 3H), 3.84 (m, 2H), 3.96 (m,
3
A solution of protected cysteine 3b or 4b (30 mg, 0.08 mmol) in
dichloromethane was treated at 0°C with three equivalents of
TMSI, and the mixture was stirred overnight. Then, the mixture was
quenched with water, concentrated, extracted with ethyl acetate,
1
H), 4.59 (m, 1H), 5.09 and 5.13 (s, 2H), 5.55 and 5.70 (d, 1H, J ꢁ=ꢁ 7.3 and
3
8
.0 Hz), 7.32 (m, 10H); 1 C NMR (CDCl ): δ 33.6 and 34.1, 52.8, 53.5 and
3
5
1
3.6, 53.8, 65.7 and 65.9, 67.1 and 67.2, 127.9, 128.0, 128.1, 128.2, 128.5,
28.8, 136.0, 138.9 and 139.1, 155.7 and 155.9, 171.0; HRMS: Found m/z
and dried over MgSO . The solvent was evaporated, and the resulting
+
+
4
4
12.1194 [M+Na] , calcd m/z 412.1189; ESI-MS: m/z 390 [M+H] .
N-deprotected methyl ester was hydrolyzed with aqueous NaOH (1 ꢂ,
0
.1 mL) diluted with 1 mL of methanol. The solvent was evaporated,
(
1S)-N-(Benzyloxycarbonyl)-S-(2-hydroxy-1-phenylethyl)-ꢀ-
the residue was diluted with water, and the pH level of the solution
was adjusted to 5–6 before analysis by LC/MS.
cysteine methyl ester (S,R-3b)ꢃColorless oil; one diastereomer;
1
yield 128 mg (35%); R ꢁ=ꢁ0.25 (chloroform/ethyl acetate, 10/1); H NMR
f
(
CDCl ): δ 2.91 (m, 2H), 3.73 (s, 3H), 3.83 (m, 2H), 3.94 (m, 1H), 4.55 (m,
3
13
1
H), 5.09 (s, 2H), 5.60 (d, 1H, J ꢁ=ꢁ 7.8 Hz), 7.32 (m, 10H); C NMR (CDCl ):
3
Deprotection of Boc-protected cysteine methyl ester
adducts 3a and 4a
δ 33.5, 52.7, 53.4, 53.8, 65.8, 67.1, 127.9, 128.0, 128.1, 128.2, 128.5, 128.8,
+
1
36.1, 139.0, 155.7, 171.0; HRMS: Found m/z 412.1187 [M+Na] , calcd
+
20
m/z 412.1189; ESI-MS: m/z 390 [M+H] ; [ α]D +137.1° (cꢁ=ꢁ0.6, CHCl ).
3
Protected compound 3a or 4a (30 mg, 0.09 mmol) was dissolved in a
(
1R)-N-(Benzyloxycarbonyl)-S-(2-hydroxy-1-phenylethyl)-ꢀ- 1:1 mixture of TFA and dichloromethane. The solution was stirred at
cysteine methyl ester (R,R-3b)ꢃColorless oil; one diastereomer; room temperature for 4 h then concentrated on a rotary evaporator.
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A. Moulisová and I. Linhart: Cysteine adducts by ring-opening reactions of phenyloxiraneꢂꢁꢀꢀꢀꢂ65
The residue was dissolved in water (5 mL), and pH of the solution was
aspects of their formation, analysis and applications.
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adjusted to 5–6. The solution was poured on a Strata X SPE column,
which was then washed with 10 mL of water and eluted with 10 mL
of pure methanol.
[4] Chasseaud, L. F. Nature and distribution of enzymes catalyzing
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the very useful building blocks N-Boc-S-alkylated cysteines.
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[ꢌ] Jágr, M.; Mráz, J.; Linhart, I.; Stránský, V.; Pospíšil, M.
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S-(2-Hydroxy-1-phenylethyl)-ꢀ-cysteine [7]ꢃThis compound was
obtained as white solid; yield 10 mg (50%); R ꢁ=ꢁ0.20 (chloroform/
f
1
methanol/acetic acid, 7/2/1); H NMR (D 0): δ 2.87 (m, 2H), 3.56 and
2
1
3
3
.71 (m, 1H), 3.81 (m, 2H), 4.01 (t, 1H, J ꢁ=ꢁ 6.7 Hz), 7.28 (m, 5H); C NMR
(
D O): δ 31.2 and 31.4, 50.8 and 51.6, 53.6, 64.4 and 64.5, 128.1, 128.2,
2
+
1
[
28.9, 138.7 and 139.3, 172.4; ESI-MS: m/z 242 [M+H] ; MS/MS: m/z 225
+
+
M+H – NH ] , 121 [PhCHCH OH] .
3
2
S-(2-Hydroxy-2-phenylethyl)-ꢀ-cysteine [7]ꢃThis compound was
obtained as white solid; yield 32 mg (80%): R ꢁ=ꢁ0.20 (chloroform/
f
1
methanol/acetic acid, 7/2/1); H NMR (D O): δ 2.92 (m, 4H), 3.82 (m,
2
13
1
3
H), 4.79 (t, 1H, J ꢁ=ꢁ 6.6 Hz), 7.32 (m, 5H); C NMR (D O): δ 32.4 and
2
2.5, 39.5 and 39.6, 53.3, 72.4 and 72.5, 126.1, 128.2, 128.7, 141.9, 172.0;
+
+
ESI-MS: m/z 242 [M+H] ; MS/MS: m/z 242 [M+H – H O] .
2
[
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4
Racemization of styrene oxide enantiomers
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2
0
Two solutions were used, one with (R)-1 ( [ α] +5.3° (cꢁ=ꢁ2.1, CH CN))
D
3
and 10 mol% TBAF in acetonitrile and the other without TBAF. In a [11] Khosropour, A. R.; Khodaei, M. M.; Ghozati, K. BiCl catalyzed
3
few hours, the complete racemization of styrene oxide in the pres-
ence of TBAF was observed. However, there was no change in specific
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[
[
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4
6
4ꢌ1–64ꢌ4.
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under mild reaction conditions. Afr. J. Chem. 2008, 61,
Aliquot samples (10 μL) of the reaction mixture of 1 and 2 at various
pH were injected onto a 150ꢁ×ꢁ2 mm Phenomenex Aqua C18 column,
particle size 5μm, and eluted with 4% acetonitrile in 0.1% aqueous
formic acid at a flow rate of 150 μL/min. The mass analyzer was set to
monitor full-scan MS in the range of m/z 100–400 and collisionally
1
15–118.
[
14] Chandrasekhar, S.; Reddy, Ch. R.; Babu, B. N.; Chandrasekhar,
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6
5 3
+
activated MS/MS taken at m/z 242 for (M+H) ion of 3 and 4. These
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[
Acknowledgments: Financial support from the Ministry of [16] Cox, D. P.; Terpinski, J.; Lawrynowicz, W. ‘Anhydrous’ tetrabu-
tylammonium fluoride: a mild but highly efficient source of
Health of the Czech Republic (grant no. NT13401-4/2012) is
nucleophilic fluoride ion. J. Org. Chem. 1984, 49, 3216–3219.
gratefully acknowledged.
[
[
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Total synthesis of hydrocinchonidine and hydrocinchonine
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