Synthesis of β-(S-methyl)thioaspartic acid and derivatives

Article abstract of DOI:10.1016/j.bmc.2008.04.069

β-(S-Methyl)thioaspartic acid occurs as a posttranslational modification at position 88 in Escherichia coli ribosomal protein S12, a position that is a mutational hotspot resulting in both antibiotic-resistant and antibiotic-sensitive phenotypes. Critical

Full text of DOI:10.1016/j.bmc.2008.04.069

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5908–5913
Synthesis of b-(S-methyl)thioaspartic acid and derivatives
Jorge Heredia-Moya and Kenneth L. Kirk*
Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, DHHS, Bethesda, MD 20892, USA
Received 21 February 2008; revised 24 April 2008; accepted 24 April 2008
Available online 29 April 2008
Abstract—b-(S-Methyl)thioaspartic acid occurs as a posttranslational modification at position 88 in Escherichia coli ribosomal pro-
tein S12, a position that is a mutational hotspot resulting in both antibiotic-resistant and antibiotic-sensitive phenotypes. Critical to
research designed to determine the biological function of b-(S-methyl)thioaspartic acid will be the availability of synthetic
b-(S-methyl)thioaspartic acid as well as derivatives designed for peptide incorporation. We report here the synthesis of b-(S-
methyl)thioaspartic acid and derivatives. The installation of the b-methylthio moiety into the aspartic acid structure was accom-
plished by electrophilic sulfenylation of N-protected-L-aspartic acid derivatives with 2,4-dinitrophenyl methyl disulfide. Following
this key transformation, we were able to prepare protected b-(S-methyl)thioaspartic acid derivative suitable for peptide coupling.
Published by Elsevier Ltd.
1. Introduction
b-methylthiolation to form the b-(S-methyl)thioaspartic
acid (1).7 This modified residue is located at position
D88, near the streptomycin binding site and in the midst
of residues altered in streptomycin-resistant mutants.
This modification has also been found to occur in the
phototropic bacterium Rhodopseudomonas palustris,8
and in the extremely thermophilic bacterium Thermus
thermophilus in the same location.9 Mutations in the
immediate vicinity of residue 88 have functional
consequences. For example, the mutation that confers
streptomycin resistance corresponds to a single amino
acid replacement of Lys ! Arg at residue 87,10 and
these mutant ribosomes are capable of performing
spontaneous nonenzymatic translocation in vitro.11
However, recently it was reported that b-methylthiola-
tion is not a determinant of the streptomycin phenotype
in T. thermophilus.12
The ribosome is the universal macromolecular machine
involved in translating mRNA transcript into polypep-
tides. Several antibiotics act by targeting protein biosyn-
thesis, interacting with ribosomal structural proteins,
rRNAs, and ribosomal-associated proteins.1 These anti-
biotics are useful mechanistic tools since studies on anti-
biotic–ribosomal proteins interactions can be especially
informative about the structure of the ribosome.2 For
example, mutations in ribosomal proteins L22, S5, and
S12 have been observed to confer resistance to erythro-
mycin,3 spectinomycin,4 and streptomycin,5 respectively,
in Escherichia coli. The role of ribosomal protein S12 in
maintenance of translational accuracy has been well
established.6
Genetic and biochemical analyses of ribosomes from
streptomycin-resistant mutants implicated ribosomal
protein S12 as the determinant of the various streptomy-
cin phenotypes, including resistance, dependence, and
pseudodependence. The aspartic acid in the E. coli ribo-
somal protein S12 is posttranslationally modified via a
In bacteria, S12 binds to 16S rRNA in regions associ-
ated with the fidelity of codon recognition. When cou-
pled with the parsimonious nature of bacterial
genetics, it is likely that b-(S-methyl)thioaspartic acid
is both structurally and functionally important. How-
ever, the synthesis of this amino acid was not reported
yet and its role in ribosome function has not been
delineated.
Keywords: Unnatural amino acids; Ribosomal protein; Posttransla-
tional modification; Sufenylationl; Selective deprotection.
*
We report herein the synthesis of b-(S-methyl)thioaspar-
tic acid (1) and selectively protected derivatives that are
Corresponding author. Tel.: +1 301 496 2619; fax: +1 301 402 4182;
e-mail: kennethk@bdg8.niddk.nih.gov
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.04.069

J. Heredia-Moya, K. L. Kirk / Bioorg. Med. Chem. 16 (2008) 5908–5913
5909
designed for incorporation into peptides. These peptides
in turn will be used to elucidate the enzymology of post-
transcriptional modification as part of a study to deter-
mine the biological function of this modification.
28% of enamine and no a-aminonitrile was formed. At-
tempted 1,4 addition of TMSCN to the isolated enam-
ine15 under a variety of conditions also failed as did
aminocyanation of 2 catalyzed by lithium perchlorate/
diethylether (LPDE).16
2. Results and discussion
Due to these failures to elaborate the amino acid side
chain from 2, we decided to install the methylthio moi-
ety onto an appropriate b-amino ester enolate using
electrophilic sulfenylation.17 The necessary 2,4-dinitro-
phenyl methyl disulfide (5) was synthesized, on a mul-
ti-gram-scale, in 77% yield by microwave irradiation
For the synthesis of this amino acid 1, we initially con-
sidered a Strecker synthesis shown retrosynthetically in
Scheme 1. The aldehyde 2 was synthesized in 67% yield
from methyl (methylthio)-acetate (3) by a published pro-
cedure.13 However, the Strecker reaction of 2 with
NaCN and NH4Cl was unsatisfactory. Although, there
was some 1H NMR evidence of product formation,
none was isolated. This poor result may be due to low-
ered reactivity of the aldehyde due to enolization as well
as to isomerization of the intermediate imine to an en-
amine that is less reactive toward cyanide. The 1H
NMR shows that 2 is in equilibrium with the 3-hydro-
xyl-acrylates 2a and 2b in a ratio 7:53:40, respectively
(Scheme 2).
(130 °C) of
a
series of solutions of 2,4-dini-
trobenzenesulfenyl chloride (4) in acetonitrile with
1 equiv of DMSO (Scheme 3).18 The best result was ob-
tained by irradiating several solutions of 500 mg of 4,
(0.43 M in acetonitrile) because irradiation on a gram-
scale (2 g) resulted in lower yields (53%) of 5.
With 5 in hand, we next prepared substrates for electro-
philic sulfenylation. The remaining carboxylic acid of Z-
Asp(Ot-Bu)OH 6a was esterified with TMSCHN2 in
15 min at room temperature19 to afford 7a in 87% yield.
The reaction of 7a with LDA, 5 and subsequent acidic
hydrolysis of 8a without isolation afforded the target
amino acid 1. After purification using ion exchange
chromatography, 3-(S-methyl)thioaspartic acid 1 was
obtained in 33% yield as a mixture (1:1) of both diaste-
Attempts to increase the yield by functionalization of
the aldehyde using less direct methods also failed. The
reaction of 2 with tert-butyl carbamate14 in the presence
of CuSO4 to form the tert-butylvinyl carbamate fol-
lowed by in situ treatment with TMSCN produced only
O
S
S
O
H2N
HO
Cl
S
a
O
OH
S
OMe
S
O2N
NO2
O2N
NO2
OMe
S
4
5
CHO
O
1
2
3
Scheme 3. Reagents and conditions: (a) DMSO 1 equiv, CH3CN,
MW, 130 ° C, 10 min.
Scheme 1. Retrosynthesis of amino acid 1.
O
OMe
O
O
H
S
S
H
S
OMe
OMe
H
H
2a
H
O
O
O
2
2a
2b
12.0
11.0
10.0
9.0
8.0
ppm (f1)
Scheme 2. Portion of 1H NMR of 2 showing the equilibrium with the 3-hydroxyl-acrylates 2a and 2b.

5910
J. Heredia-Moya, K. L. Kirk / Bioorg. Med. Chem. 16 (2008) 5908–5913
Table 1. Synthesis of N-protected-3-methylsulfanyl-L-aspartic acid
derivatives
reomers, contaminated with 17% of aspartic acid com-
ing from unreactive 6a. All attempts to isolate separate
diastereomers were unsuccessful.
Compound R1
Yield 7 Base
(%)
Yield 8 Yield 9
(%)
(%)
For biological studies involving peptide synthesis we
required N-protected-3-methylsulfanyl-L-aspartic acid-
4-esters. Because the selective hydrolysis of the methyl
ester of 8a while leaving the tert-butyl ester intact is
problematic, we explored other protective groups more
amenable to selective deprotection using the commercial
available L-aspartic acid derivatives 6a, d, and e as start-
ing materials (Scheme 4).
7a
7b
7c
7d
7e
Me
87
79
—
—
—
—
TBDMS
Bn
Bn
LDA
LDA
LDA
NR
16
93
NR
86
100
85
16
Bn
LiHMDS 26
87
options for deprotection and, instead, explored other
protecting groups.
Another promising starting material for this reaction is
the amino acid 6d. To avoid thermolytic cleavage of
the tert-butyl carbamate,21 the benzylation was carried
out at 60 °C with the cooling system in the microwave
oven activated. Under those conditions, 7d was obtained
in almost quantitative yield. In the sulfenylation reac-
tion, the monosulfenylated amino acid 8d was isolated
in 16% yield and 1% of disulfenylated product was ob-
served by NMR. The benzyl ester was easily cleaved
by transfer hydrogenation, using palladium-black with
1,4-cyclohexadiene as hydrogen donor.22 The presence
of the sulfur moiety did not poison the catalyst and
the yield of 9d was 86%.
Initially, we selected the TMDBS group as a good op-
tion for the protection of 6a. Using TBDMSCl and
imidazole the protected amino acid 7b was obtained in
79% yield. However no evidence of 8b was observed
after the sulfenylation reaction.
The benzylation of 6a with BnBr and Cs2CO3 using
microwave irradiation at 130 °C for 10 min afforded 7c
in 93% yield. The sulfenylation of 7c produced a com-
plex mixture that complicated purification. However,
we were able to isolate the product 8c in 16% yield (Ta-
ble 1). We have not investigated this transformation in
detail and have no ready explanation for the low yields
of methylsulfenylation product compared to higher re-
ported yields of arylsulfenylation.17 The electrophilic
sulfenylation proceeded via an anti addition,20 and it
has been reported that the product obtained with aspar-
tic acid derivative has the (2R,3R) configuration.17a
Additionally, the configuration of the newly stereogenic
center when (2,4-dimethoxybenzylthio)-4-methylphenyl
sulfonate was used as sulfenylating agent has been con-
firmed by X-ray analysis.17b Comparisons of the Ha–Hb
coupling constant of 8c (4.9 Hz) and the products re-
ported (4.5–5.4 Hz) suggested the same configuration.
Unfortunately, deprotection of the benzyl ester under
mildly basic conditions (0.1 N K2CO3, H2O, and
THF) failed. The benzyl group can be removed under
harsher basic condition, but this would make retention
of the tert-butyl ester difficult. Other conditions that
would also remove the benzyl carbamate were not appli-
cable. Due to the low yield obtained in the synthesis of
8c, we abandoned this intermediate before exploring all
We also explored the reaction using 6e as a starting
material. Using the same procedure, described above,
the protected amino acid 7e was obtained in 85% yield.
Using LiHMDS as base, 8e was obtained in 26% after
the sulfenylation reaction. The cleavage under the same
conditions affords the amino acid 9e in 87% yield. This
provides the Fmoc protected mono-tert-butyl ester of
(2R,3R)-1 as a single diastereomer, an orthogonally pro-
tected derivative suitable for peptide incorporation. The
product appeared as a single peak in two HPLC systems
(Chirobiotic R and C18 columns) supporting likely dia-
stereomeric homogeneity.
3. Conclusion
The electrophilic sulfenylation of N-protected-L-aspartic
acid-1-benzyl-4-esters (7d–7e) with 2,4-dinitrophenyl
O
O
O
O
O
H
H
H
N
N
N
R1
R2
R1
R2
H2N
S
R3
OH
O
R3
O
O
R3
O
O
OH
OH
a
b
d
R2
S
c
O
O
O
6a-e
7a-e
8a-e
1
R1
R2
O
O
R3
Cbz
Cbz
Cbz
Boc
H
N
a
Me
t-Bu
R3
OH
O
b
c
d
e
TBDMS t-Bu
Bn
Bn
Bn
t-Bu
Me
t-Bu
S
R2
Fmoc
9d-e
Scheme 4. Reagents and conditions: (a) compound 7a: TMSCHN2 2.4 equiv, toluene/MeOH 3:1, rt, 15 min; compound 7b: TBDMSCl 1.1 equiv,
imidazole 2.2 equiv, DMF, 0 °C, 5 min, rt, 3 days; compound 7c–7e: BnBr 1.2 equiv, Cs2CO3 1.2 equiv, CH3CN, MW, 60 °C or 130 °C, 10 min. (b)
LiHMDS or LDA 2.2 equiv, compound 5 1.4 equiv, ꢀ78 °C. (c) 1,4-cyclohexadiene 10 equiv, black Pd (equal weight), 25 °C, 3 h; (d) compound 8a:
6 N HCl, reflux, 5 h.

J. Heredia-Moya, K. L. Kirk / Bioorg. Med. Chem. 16 (2008) 5908–5913
5911
methyl disulfide (5) affords (2R,3R)-N-protected-3-
methylsulfanyl-L-aspartic acid-1-benzyl-4-esters 8d and
8e in 16% and 26% yields, respectively, as a single diaste-
reomer. The benzyl ester was cleaved by catalytic trans-
fer hydrogenation to afford the N-protected-3-
methylsulfanyl-L-aspartic acid-4-esters 9d and 9e in high
yield. These protected derivatives of amino acid 1 are
suitable for peptide incorporation.
4.3.1. N-Benzyloxycarbonyl-L-aspartic acid-1-benzyl ester-
4-tert-butyl ester (7c). Oil; 93% yield; 1H NMR
(300 MHz, CDCl3) d ppm 1.38 (s, 9H), 2.75 (dd,
J = 16.9 Hz, J = 4.5 Hz, 1H), 2.96 (dd, J = 17.0 Hz,
J = 4.6 Hz, 1H), 4.63 (ddd, J = 8.6 Hz, J = 4.6 Hz,
J = 4.5 Hz, 1H), 5.12 (s, 2H), 5.15 (d, J = 12.4 Hz, 1H),
5.22 (d, J = 12.4 Hz, 1H), 5.77 (d, J = 8.9 Hz, 1H),
7.40–7.29 (m, 10H); 13C NMR (75 MHz, CDCl3) d
ppm 27.88, 37.66, 50.56, 66.97, 67.30, 81.71, 127.98,
128.08, 128.12, 128.29, 128.43, 128.48, 135.21, 136.14,
155.93, 169.79, 170.70; HRMS (TOF ES+) m/z
calcd for C23H27NO6Na (M+Na)+: 436.1736; found:
436.1729.
4. Experimental
4.1. General
All solvents and reagents were from Aldrich and used
without further purification. NMR spectra were run in
CDCl3 on a Varian Gemini 300 MHz or Mercury
300 MHz spectrometer. Chemical shifts are expressed
in ppm with TMS as internal reference. Mass spectra
were determined using a Hewlett–Packard 1100 MSD
instrument. Reactions were monitored by TLC on sil-
ica gel using 10% methanol in dichloromethane as sol-
vent and compounds visualized by a UV lamp. Flash
chromatography was carried out in a Biotage SP4TM
Purification System. The microwave assisted reactions
were run in a Biotage InitiatorTM microwave oven.
Optical rotations were determined with a JASCO P-
2000 polarimeter. The reported yields are for purified
material and are not optimized.
4.3.2. N-tert-Butoxycarbonyl-L-aspartic acid-1-benzyl
ester-4-methyl ester (7d). White needles; quantitative;
mp 62–63 °C; 1H NMR (300 MHz, CDCl3) d ppm
1.43 (s, 9H), 2.82 (dd, J = 17.0 Hz, J = 4.7 Hz, 1H),
3.02 (dd, J = 17.0 Hz, J = 4.5 Hz, 1H), 3.62 (s, 3H),
4.62 (ddd, J = 8.4 Hz, J = 4.7 Hz, J = 4.5 Hz, 1H), 5.15
(d, J = 12.3 Hz, 1H), 5.21 (d, J = 12.3 Hz, 1H), 5.50
(d, J = 8.4 Hz, 1H), 7.30–7.37 (m, 5H); 13C NMR
(75 MHz, CDCl3) d ppm 28.26, 36.64, 50.05, 51.93,
67.41, 80.14, 128.22, 128.37, 128.53, 135.26, 155.36,
170.88, 171.28; HRMS (TOF ES+) m/z calcd for
C17H23NO6Na: (M+Na)+ 360.1423; found: 360.1427.
4.3.3. N-9H-Fluoren-9-ylmethoxycarbonyl-L-aspartic
acid-1-benzyl ester-4-tert-butyl ester (7e). White solid;
1
85% yield; mp 56–58 °C; H NMR (300 MHz, CDCl3) d
4.2. Synthesis of 2,4-dinitrophenyl methyl disulfide (3)
ppm 1.41 (s, 9H), 2.78 (dd, J = 16.9 Hz, J = 4.4 Hz, 1H),
2.97 (dd, J = 16.9 Hz, J = 4.7 Hz, 1H), 4.23 (dd,
J = 7.3 Hz, J = 7.1 Hz, 1H), 4.33 (dd, J = 10.4 Hz,
J = 7.3 Hz, 1H), 4.42 (dd, J = 10.4 Hz, J = 7.1 Hz, 1H),
4.65 (ddd, J = 8.7 Hz, J = 4.7 Hz, J = 4.4 Hz, 1H), 5.17
(d, J = 12.2 Hz, 1H), 5.24 (d, J = 12.2 Hz, 1H), 5.84 (d,
J = 8.7 Hz, 1H), 7.44–7.26 (m, 9H), 7.59 (d, J = 7.6 Hz,
2H), 7.76 (d, J = 7.6 Hz, 2H); 13C NMR (75 MHz, CDCl3)
d ppm 27.98, 37.72, 47.08, 50.63, 67.27, 67.45, 81.87,
119.95, 125.11, 125.16, 127.05, 127.69, 128.23, 128.40,
128.56, 135.23, 141.24, 141.27, 143.69, 143.89, 155.96,
169.93, 170.77; HRMS (TOF ES+) m/z calcd for
C30H31NO6Na: (M+Na)+ 524.2049; found: 524.2043.
To a solution of 500.0 mg of 2,4-dinitrobenzene-sulfe-
nyl chloride in 5 mL of acetonitrile was added
1.15 mL of DMSO, and the mixture was irradiated at
130 °C for 10 min (absorption level = normal). The
undissolved material was removed by filtration and
the solution was concentrated under reduced pressure.
The crude product was purified by column chromatog-
raphy (KP-Sil column, 0–20% of EtOAc in hexane, UV
235–245 nm) to afford 389.7 mg of product. Yellow
needles; 77% yield; mp 96–97 °C; 1H NMR
(300 MHz, CDCl3) d ppm 2.48 (s, 3H), 8.50 (d,
J = 1.4 Hz, 2H), 9.13 (t, J = 1.4 Hz, 1H); 13C NMR
(75 MHz, CDCl3)
d
ppm 21.77, 121.64, 127.46,
4.4. General procedure for the 3-sulfenylation of
N-protected-L-aspartic acid-1-benzyl ester-4-esters
128.23, 144.91, 145.43, 145.91; HRMS (TOF AP+)
m/z calcd for C7H7N2O4S2 (M+H)+: 246.9847; found:
246.9846.
To a solution of 1 mmol of ester in 4 mL of anhydrous
THF at ꢀ78 °C and under N2 was added 2.2 mmol of
LDA or LiHMDS over a period of 10 min. The solution
was stirred for 30 min at this temperature and 1.2 mmol
of 2,4-dinitrophenyl methyl disulfide was added in one
portion as a solid. The mixture was stirred for 60 min
at the same temperature and then allowed to reach
ꢀ40 °C in 30 min. Then 3 mL of 2 N HCl was added
and the product was extracted with EtOAc. The organic
layer was washed with brine, dried over Na2SO4, and
evaporated to dryness in vacuum. The orange crude
product was dissolved in hot cyclohexane and the undis-
solved material was removed by filtration. The solvent
was removed under reduced pressure and the product
purified by flash chromatography (KP-SIL column,
5–30% of EtOAc/CH2Cl2 (1:1) in cyclohexane, UV
4.3. General procedure for the benzylation of N-protected-
L-aspartic acid 4 esters
To a solution of 6 mmol of N-protected-L-aspartic
acid b-esters in 10 mL of CH3CN under N2 were
added 7.2 mmol of anhydrous Cs2CO3 and 7.2 mmol
of BnBr, and the mixture was irradiated 10 min at
130 °C (normal absorption and cooling OFF) or
60 °C (normal absorption and cooling ON). The solid
was removed by filtration and the solvent removed
under reduced pressure. The crude product was puri-
fied by column chromatography (KP-SIL column, 7–
60% of EtOAc in hexane, UV 235–245 nm) to afford
the pure product.

5912
J. Heredia-Moya, K. L. Kirk / Bioorg. Med. Chem. 16 (2008) 5908–5913
235–245 nm). Recrystallization (EtOH/hexane) afforded
the pure product.
5.76 (d, J = 9.4 Hz, 1H), 7.80 (s, 1H); 13C NMR
(75 MHz, CDCl3) d ppm 16.05, 28.24, 49.21, 52.17,
52.89, 54.67, 80.76, 156.08, 171.96, 173.76; HRMS
(TOF ESꢀ) m/z calcd for C11H18NO6S: (Mꢀ1)
292.0855; found: 292.0872.
4.4.1. (2R,3R)-N-Benzyloxycarbonyl-3-methylsulfanyl-
aspartic acid-1-benzyl-ester-4-tert-butyl ester (8c). Oil;
1
16% yield; H NMR (300 MHz, CDCl3) d ppm 1.40 (s,
9H), 2.25 (s, 3H), 3.78 (d, J = 4.9 Hz, 1H), 4.87 (dd,
J = 10.1 Hz, J = 5.0 Hz, 1H), 5.24–5.08 (m, 4H), 5.92
(d, J = 10.1 Hz, 1H), 7.29–740 (m, 10H); 13C NMR
(75 MHz, CDCl3) d ppm 15.84, 27.83, 50.27, 55.28,
67.18, 67.53, 82.88, 127.95, 128.07, 128.23, 128.40,
128.45, 128.55, 135.06, 136.21, 156.48, 169.73, 169.89;
HRMS (TOF ES+) m/z calcd for C24H30NO6S:
(M+H)+ 460.1794; found: 460.1818.
4.5.2. (2R,3R)-N-9H-Fluoren-9-ylmethoxycarbonyl-3-
methylsulfanyl-aspartic acid-4-tert-butyl ester (9e). Yellow
27
D
solid; 87% yield; mp 55–57 °C; ½aꢁ +13.95° (c 0.52,
1
MeOH); H NMR (300 MHz, CDCl3) d ppm 1.50 (s,
9H), 2.28 (s, 3H), 3.81 (d, J = 4.4 Hz, 1H), 4.25 (dd,
J = 7.7 Hz, J= 7.1 Hz, 1H), 4.34–4.45 (m, 2H), 4.84
(dd, J = 9.3 Hz, J = 4.4 Hz, 1H), 6.09 (d, J = 9.3 Hz,
1H), 7.29 (dt, J = 7.5 Hz, J = 0.6 Hz, 2H), 7.39 (t,
J = 7.2 Hz, 2H), 7.61 (d, J = 7.3 Hz, 2H), 7.75 (d,
J = 7.5 Hz, 2H); 13C NMR (75 MHz, CDCl3) d ppm:
16.08, 27.93, 47.04, 50.13, 55.15, 67.56, 83.58, 119.93,
125.18, 127.07, 127.68, 141.24, 143.72, 156.59, 171.10,
173.33; HRMS (TOF ES+) m/z calcd for C24H27NO6
NaS: (M+Na)+ 480.1457; found: 480.1442.
4.4.2. (2R,3R)-N-tert-Butoxycarbonyl-3-methylsulfanyl-
aspartic acid-1-benzyl ester-4-methyl ester (8d). Yellow
27
D
needles; 16% yield; mp 83–85 °C; ½aꢁ +7.89° (c 0.76,
1
MeOH); H NMR (300 MHz, CDCl3) d ppm 1.44 (s,
9H), 2.26 (s, 3H), 3.64 (s, 3H), 3.87 (d, J = 4.8 Hz,
1H), 4.87 (dd, J = 10.1 Hz, J = 4.8 Hz, 1H), 5.11 (d,
J = 12.2 Hz, 1H), 5.22 (d, J = 12.2 Hz, 1H), 5.61 (d,
J = 10.1 Hz, 1H), 7.30–7.38 (m, 5H); 13C NMR
(75 MHz, CDCl3) d ppm 15.88, 28.23, 49.68, 52.56,
54.75, 67.51, 80.27, 128.32, 128.43, 128.53, 135.08,
155.73, 169.86, 171.22; HRMS (TOF AP+) m/z
calcd for C18H26NO6S: (M+H)+ 384.1481; found:
384.1466.
Acknowledgments
This work was supported by the intramural research
program of NIDDK, NIH. We are grateful to NIMH,
NIH colleagues Dr. Sanford Markey, Dr. Jeffrey A.
Kowalak, and Dr. Michael B. Strader for helpful discus-
sions concerning the biology of b-(S-methyl)thioaspartic
acid and Mr. Anthony J. Makusky for chiral HPLC
analyses.
4.4.3. (2R,3R)-N-9H-Fluoren-9-ylmethoxycarbonyl-3-
methylsulfanyl-aspartic acid-1-benzyl ester-4-tert-butyl
ester (8e). White solid; 26% yield; mp 109.5–112.5 °C;
27
½aꢁ +0.95° (c 1.47, MeOH); 1H NMR (300 MHz,
D
CDCl3) d ppm 1.44 (s, 9H), 2.27 (s, 3H), 3.81 (d,
J = 4.8 Hz, 1H), 4.25 (dd, J = 7.7 Hz, J = 7.0 Hz, 1H),
4.36 (dd, J = 10.4 Hz, J = 7.0 Hz, 1H), 4.42 (dd,
J = 10.4 Hz, J = 7.7 Hz, 1H), 4.89 (dd, J = 10.2 Hz,
J = 4.8 Hz, 1H), 5.19 (s, 2H), 5.99 (d, J = 10.2 Hz,
1H), 7.44–7.27 (m, 9H), 7.61 (d, J = 7.4 Hz, 2H), 7.76
(d, J = 7.5 Hz, 2H); 13C NMR (75 MHz, CDCl3) d
ppm: 15.91, 27.89, 47.08, 50.30, 55.27, 67.52, 67.61,
82.97, 119.92, 125.23, 127.06, 127.65, 128.27, 128.45,
128.58, 135.04, 141.24, 143.79, 143.87, 156.48, 169.75,
170.05; HRMS (TOF ES+) m/z calcd for C30H33NO6-
NaS: (M+Na)+ 570.1926; found: 570.1916.
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27
D
aspartic acid-4-methyl ester (9d). Oil; 86% yield; ½aꢁ
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J = 4.5 Hz, 1H), 4.83 (dd, J = 9.5 Hz, J = 4.4 Hz, 1H),

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