A concise stereoselective synthesis of orthogonally protected lanthionine and β-methyllanthionine

Article abstract of DOI:10.1039/b618178c

Lantibiotics such as nisin are active against most Gram-positive bacteria and constitute an important class of antibacterial agents. These ribosomally synthesized peptides contain either one or both of the unusual amino acids meso-lanthionine (m-Lan) or β

Full text of DOI:10.1039/b618178c

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
A concise stereoselective synthesis of orthogonally protected lanthionine and
b-methyllanthionine†
Steven L. Cobb and John C. Vederas*
Received 13th December 2006, Accepted 26th January 2007
First published as an Advance Article on the web 7th February 2007
DOI: 10.1039/b618178c
Lantibiotics such as nisin are active against most Gram-positive bacteria and constitute an important
class of antibacterial agents. These ribosomally synthesized peptides contain either one or both of the
unusual amino acids meso-lanthionine (m-Lan) or b-methyllanthionine (b-MeLan). Nucleophilic ring
opening of sulfamidates allows facile preparation of stereochemically pure derivatives of m-Lan and
b-MeLan with orthogonal protection for solid phase synthesis of lantibiotic analogues.
these m-Lan derivatives have been successfully applied to the
solid phase synthesis of fragments of natural lantibiotics, such
Introduction
as Tabor’s elegant synthesis of the C ring of nisin.8a However,
the additional stereocentre present at the C-3 position makes
the stereoselective synthesis of orthogonally protected derivatives
of b-MeLan more challenging. Consequently, to the best of our
knowledge, there have been only two previously reported syntheses
of linear orthogonally protected derivatives of b-MeLan.9 The
best procedure requires about seven synthetic steps, and further
use in lantibiotic synthesis would necessitate removal of a benzyl
carbamate (Cbz) and a benzyl ester (Bn) in the presence of a sulfur
atom. As orthogonally protected derivatives of amino acids 1 and
2 would be key components in solid phase peptide synthesis of
lantibiotics, it seemed useful to develop an alternative synthetic
route to these compounds. A key objective was to develop an
efficient methodology that utilizes protecting groups that could be
selectively removed under mild conditions.
Lantibiotics are ribosomally synthesized antimicrobial peptides
that have extremely potent activity against a broad spectrum
of Gram-positive bacteria, including food pathogens (e.g. Lis-
teria monocytogenes) and organisms that exhibit resistance to
conventional antibiotics (e.g. MRSA and VRE).1 Consequently,
lantibiotics have emerged as a promising new class of antibacterial
agents. Lantibiotics such as nisin,2 lacticin 31473 and gallidermin4
are characterized by the presence of the unusual amino acids meso-
lanthionine (m-Lan) (1) and b-methyllanthionine ((2S,3S,6R)-b-
MeLan) (2) (Fig. 1). m-Lan and b-MeLan arise due to the action
of a series of post-translational modification enzymes.
Fig.
1 meso-Lanthionine (m-Lan) (1) and b-methyllanthionine
Results and discussion
((2S,3S,6R)-b-MeLan) (2).
Sulfamidates are useful synthetic building blocks that can be
opened stereo- and regioselectively by a variety of nucleophiles.10
Hence, it would appear that a suitably protected cysteine 5 could
attack a cyclic sulfamidate (6 or 7) derived from a protected
serine 8 or threonine 9 to give orthogonally protected derivatives
of of m-Lan or b-MeLan (Scheme 1). However, attack of
sulfur nucleophiles on sulfamidates can be plagued by undesired
elimination reactions11 unless the a-position is blocked.10c A fully
unprotected threonine-derived sulfamidate has been successfully
opened in water with a thio-sugar,12 but attack of cysteine-derived
nucleophiles on protected serine and threonine sulfamidates is
relatively unexplored.10c In the present study, we investigate the
In particular, dehydration of serine or threonine residues in
the original linear peptide sequence introduces dehydroalanine
or dehydrobutyrine residues respectively. A cyclase enzyme then
mediates the stereoselective Michael addition of a nearby cysteine
residue onto the dehydro residue, thereby leading to the formation
of m-Lan or b-MeLan bridges within the lantibiotic peptide.5 In an
attempt to further enhance the bioactivity and stability of natural
lantibiotics such as nisin, we are currently pursuing the solid phase
synthesis of analogues of these peptides. In order to achieve this
goal, facile access to orthogonally protected derivatives of both
m-Lan and b-MeLan is necessary.
To date, nisin A is the only lantibiotic that has been prepared
by total synthesis.6 However, since this monumental synthesis
was reported by Shiba and co-workers, several stereoselective
methods for the preparation of orthogonally protected derivatives
of m-Lan have been developed and reported.7,8 Furthermore,
Department of Chemistry, University of Alberta, Edmonton, Alberta,
Canada. E-mail: john.vederas@ualberta.ca; Fax: +1 (780) 492-2134;
Tel: +1 (780) 492-5475
† Electronic supplementary information (ESI) available: 1H and 13C NMR
spectra. See DOI: 10.1039/b618178c
Scheme 1
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use of cyclic sulfamidates to prepare orthogonally protected
derivatives of lanthionine 3 and b-methyllanthionine 4 (Scheme 1).
Although N-alloc and allyl ester protecting groups on m-Lan
have previously been shown to allow sequential construction of
lanthionine rings during standard peptide synthesis,8 they are
incompatible with the oxidation conditions required to prepare
the cyclic sulfamidates from their corresponding sulfamidites.13
The alkene moieties in these groups are also prone to add aliphatic
thiols in Markovnikov fashion. Hence, a p-methoxybenzyl (PMB)
group was chosen for the nitrogen protection, as it can be
removed using mild reaction conditions and has been successfully
used in the preparation of D-allo-threonine- and L-serine-derived
cyclic sulfamidates.12 Similarly, methyl ester protection of the acid
functionality of an amino acid has been shown to be compatible
with the preparation of cyclic sulfamidates.11,13 In addition, the
required amino acid methyl ester starting materials, 10a–e, are
commercially available, and D-allo-Thr-OMe·HCl 10e can be
prepared in quantitative yield using acetyl chloride/MeOH.
The PMB protecting group was introduced to compounds 10a–
e using a reductive amination procedure to afford the bis-protected
amino acids 11a–e in good to excellent yields (65–89%) (Fig. 2).
The conversion of compounds 11a–e to their corresponding cyclic
sulfamidates 12a–e was achieved in good yields (74–78%) by using
a slight modification of the procedure detailed by Cohen and
Halcomb12 (Fig. 2).
The ring opening of a cyclic sulfamidate with a thiol nucleophile
requires the use of a base. However, the presence of an acidic a-
proton whose removal can initiate elimination reactions prevents
the use of previously employed bases such as DBU.10c Therefore, we
first examined the Cs2CO3-mediated nucleophilic ring opening of
the L-Thr-derived sulfamidate 12a with trityl thiol 13. After acidic
hydrolysis (1 M NaH2PO4 buffer, pH 5.4)10b,11 of the intermediate
sulfamic acid, the orthogonally protected b-methyl-L-cysteine
((2R,3S)-b-MeCys), 14 was obtained in good yield (67%; 45%
overall yield from 10a). The ring opening reaction of 12a with
Fmoc-Cys-OtBu 158 and commercially available Boc-Cys-OMe
16 was examined next. Fmoc-Cys-OtBu 15 has been successfully
used as a thiol nucleophile under basic conditions (Cs2CO3/DMF)
in other reactions.14 However, in our case all attempts to use 15 as a
nucleophile with sulfamidates were unsuccessful and the reactions
yielded only a complex mixture of products. We assume that the
problems that we encountered were due to instability of the Fmoc
protecting group under the basic reactions conditions utilised.
Similar stability issues were reported by Smith and Goodman,
who were unable to use Fmoc-Cys-OMe in the Cs2CO3-meditated
ring opening of an a-methyl-D-serine-b-lactone.15 However, the
same reaction conditions could be successfully used in the ring
opening of 12a with Boc-Cys-OMe 16 to afford the b-MeLan
derivate 17, albeit in low yield (40%). No unreacted sulfamidate
12a was recovered from the reaction mixture, but it was possible to
confirm by electrospray mass spectrometry (ESMS) the presence
of the open sulfamic acid intermediate. This result indicated a
problem with the final hydrolysis stage that should liberate the N-
PMB amino group. Fortunately, the conditions developed by Kim
and So (nPrSH/BF3·Et2O, CH2Cl2)16 could be used to hydrolyze
the intermediate sulfamic acid even in the presence of the Boc
protecting group. When this system was employed in place of the
phosphate buffer, the yield of 17 could be increased considerably
from 40% to 79% (Fig. 3).
With the reaction conditions refined, attention refocused on
the initial goal of preparing orthogonally protected derivatives of
Lan and b-MeLan. In order to achieve this, a cysteine derivative
with acid labile protecting groups was required. To this end,
Boc-Cys-OtBu 18 was prepared in a good yield (63% over two
steps) from commercially available (Boc-Cys-OH)2.17 The ring
openings of the L- and D-serine-derived cyclic sulfamidates 12b
and 12c with 18 were done in DMF using Cs2CO3 as a base.
nPrSH/BF3·Et2O hydrolyses of the intermediate sulfamic acids
then furnished the orthogonally protected Lan 19a and m-Lan
19b in excellent yields (85% and 89%) (Fig. 4). The diastereomeric
purity of both 19a and 19b were shown by 1H NMR spectroscopy
to be >20 : 1 (other isomers undetectable, see ESI†). Similarly,
the nucleophilic ring opening of the D-threonine- and D-allo-
threonine-derived sulfamidates 12d and 12e with 18 using the
aforementioned reaction conditions afforded the orthogonally
protected (2S,3R,6R)-b-MeLan 20a (72%) and (2S,3S,6R)-b-
MeLan 20b (70%), respectively (Fig. 4). The diastereomeric purity
of both the b-MeLan derivatives 20a and 20b was again determined
by 1H NMR spectroscopy to be >20 : 1.
Fig. 2 Preparation of cyclic sulfamidates. Reagents and conditions: a)
NaCNBH3, p-anisaldehyde, acetic acid, MeOH, 0 ◦C to rt, 16 h; b)
(i) SOCl2, pyridine, CH2Cl2 −78 ◦C to rt, (ii) RuCl3·3H2O, NaIO4,
H2O–MeCN (1 : 1), 0 ◦C to rt.
Conclusions
In conclusion, we have developed a facile synthetic route to
stereochemically pure and orthogonally protected lanthionines
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to be readily prepared from easily available starting materials.
Studies to incorporate m-Lan 19b and b-MeLan 20b into natural
lantibiotics and their synthetic analogues are ongoing.
Experimental
General
All reactions involving air- or moisture-sensitive reagents were
performed in oven-dried glassware under an atmosphere of dry
argon. Solvents were reagent grade and were used as supplied
unless otherwise stated. For anhydrous reactions, solvents were
dried according to the procedures detailed in Perrin and Armarego
(D. D. Perrin and W. L. F. Armarego, Purification of Laboratory
Chemicals, 3rd edn, Pergamon Press). All reactions were mon-
itored by thin layer chromatography (TLC) using glass plates
with UV fluorescent indicator (normal SiO2, Merck 60 F254).
Nuclear magnetic resonance (NMR) spectra were obtained on
Inova Varian 300, 400 or 500 MHz spectrometers. 1H NMR
chemical shifts are reported in parts per million (ppm) relative
1
to CDCl3 (d 7.27). H NMR data are reported in the following
order multiplicity (s, singlet; d, doublet; t, triplet; q, quartet and
m, multiplet), number of protons, coupling constants (J) in Hertz
and assignment. 13C NMR chemical shifts are reported relative
to CDCl3 d 77.0. Optical rotations were measured on a Perkin–
Elmer 241 polarimeter with a microcell (10 cm, 1 mL) at ambient
temperature and are reported in units of 10−1 deg cm2 g−1. Infrared
spectra (IR) were recorded on a Nicolet Magna 750 or a 20SX
FT-IR spectrometer. High-resolution mass spectra (HRMS) were
obtained on a Kratos AEIMS-50 high resolution instrument.
Fig. 3 Initial sulfamidate ring opening reactions. Regents and conditions:
a) (i) Cs2CO3, DMF, rt, (ii) 1 M NaH2PO4 buffer pH 5.4, rt; b) (i)
Cs2CO3, DMF, rt, (ii) nPrSH/BF3·Et2O, CH2Cl2, rt, then NH4OH, rt.
* No formation of the desired product was detected.
D-allo-Threonine methyl ester (10e)
Acetyl chloride (6.00 mL, 83.9 mmol) was slowly added to a
solution of the amino acid (1.00 g, 8.39 mmol) in MeOH (40 mL)
at 0 ◦C. With the addition complete, the ice bath was removed and
the reaction mixture was heated to reflux and left for 16 h. Upon
cooling, the reaction solution was concentrated under reduced
pressure to afford 10e as a white solid, which was used without
further purification (1H NMR indicated a purity of >95%);
dH(500 MHz; D2O) 1.22 (3 H, d, J 6.6, CH3), 3.78 (3 H, s, OCH3),
4.14 (1 H, d, J 3.6, H-a), 4.27 (1 H, dq, J 6.6 and 3.6, H-b);
dC(100 MHz; D2O) 18.4 (CH3), 54.3 (OCH3), 58.7 (C-a), 66.3 (C-
b), 169.0 (CO); m/z (ES+) calcd for C5H12NO3 134.0812, found
134.0812 [MH+].
N-(p-Methoxybenzyl)-L-threonine methyl ester (11a)
NaCNBH3 (556 mg, 8.85 mmol) and p-anisaldehyde (0.79 mL,
6.49 mmol) were added to a cooled (0–5 ◦C) solution of 10a
(1.00 g, 5.90 mmol) in MeOH (50 mL) and acetic acid (0.68 mL,
11.80 mmol). The reaction solution was stirred at 0–5 ◦C for
1 h and then at rt for a further 16 h. Solid NaHCO3 (1.50 g,
17.86 mmol) was added and the solvent was removed under
reduced pressure. The resulting white residue was partitioned
between CH2Cl2 (75 mL) and H2O (30 mL) and the aqueous layer
was re-extracted with CH2Cl2 (75 mL). The organic layers were
combined and the solvent removed under vacuum. After column
purification (SiO2, 2 : 1 Hex–EtOAc to 100% EtOAc), 11a was
obtained as a clear oil (1.330 g, 89%). TLC (SiO2, 1 : 1 Hex–EtOAc)
Fig. 4 Preparation of orthogonally protected Lan 19a and 19b and
b-MeLan 20a and 20b. Reagents and conditions: a) (i) Cs2CO3, DMF,
rt, (ii) nPrSH/BF3·Et2O, CH2Cl2, rt, then NH4OH, rt.
19a and 19b as well as b-methyllanthionines 20a and 20b.
The methodology described is versatile enough to allow other
orthogonally protected stereoisomers of both Lan and b-MeLan
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Rf 0.36; [a]2D6 −49.41 (c 1.23, CHCl3); mmax(microscope)/cm−1 3450
(br), 2953, 2837, 1735, 1612, 1514, 1462, 1373, 1249, 1199, 1108,
1035; dH(400 MHz; CDCl3) 1.17 (3 H, d, J 6.0, CH3), 3.02 (1 H, d,
J 7.2, H-a), 3.62 (1 H, d, J 12.4, PMB-CH2), 3.67 (1 H, dq, J 7.2
and 6.0, H-b), 3.76 (1 H, d, J 12.4, PMB-CH2), 3.70 (3 H, s, OCH3),
3.77 (3 H, s, OCH3), 6.84-6.82 (2 H, m, Ar-H), 7.29-7.19 (2 H, m,
Ar-H); dC(100 MHz; CDCl3) 19.2 (CH3), 51.8 (PMB-CH2), 51.9
(OCH3), 55.9 (OCH3), 66.9 (C-a), 67.8 (C-b), 113.7 (Ar-C), 129.4
(Ar-C), 131.1 (Ar-C), 158.8 (Ar-C), 174.0 (CO), m/z (ES+) Calcd
for C13H20NO4 254.1387, found 254.1385 [MH+].
N-(p-Methoxybenzyl)-D-threonine methyl ester (11d)
NaCNBH3 (3.80 g, 60.4 mmol) and p-anisaldehyde (6.40 mL,
52.4 mmol) were added to a cooled (0–5 ◦C) solution of 10d
(6.83 g, 40.3 mmol) in MeOH (180 mL) and acetic acid (4.60 mL,
80.4 mmol). The reaction solution was stirred at 0–5 ◦C for
1 h and then at rt for a further 16 h. Solid NaHCO3 (10.20 g,
120.9 mmol) was added and the solvent was removed under
reduced pressure. The resulting white residue was partitioned
between CH2Cl2 (200 mL) and H2O (120 mL) and the aqueous
layer was re-extracted with CH2Cl2 (150 mL). The organic layers
were combined and the solvent removed under vacuum. After
column purification (SiO2, 2 : 1 Hex–EtOAc to 100% EtOAc)
11d was obtained as a clear oil (6.81 g, 71%). TLC (SiO2, 1 :
1 Hex–EtOAc) Rf 0.35; [a]2D6 +49.71 (c 0.44, CHCl3); mmax(CHCl3
cast)/cm−1 3445 (br), 2952, 2837, 1734, 1612, 1513, 1462, 1248,
1175; dH(400 MHz; CDCl3) 1.18 (3 H, d, J 6.4, CH3), 3.02 (1 H, d,
J 7.6, H-a), 3.62 (1 H, d, J 12.8, PMB-CH2), 3.68 (1 H, m, H-b),
3.71 (3 H, s, OCH3), 3.77 (1 H, d, J 12.8, PMB-CH2), 3.79 (3 H, s,
OMe), 6.87 (2 H, m, Ar-H), 7.23-7.19 (2 H, m, Ar-H); dC(100 MHz;
CDCl3) 19.3 (CH3), 51.9 (PMB-CH2), 52.0 (OCH3), 55.2 (CH3),
66.8 (C-a), 67.9 (C-b), 113.8 (Ar-C), 129.5 (Ar-C), 131.1 (Ar-C),
158.9 (Ar-C), 174.1 (CO); m/z (ES+) Calcd for C13H19NO4Na
276.1206, found 276.1207 [MNa+].
N-(p-Methoxybenzyl)-L-serine methyl ester (11b)
NaCNBH3 (606 mg, 9.65 mmol) and p-anisaldehyde (0.85 mL,
7.07 mmol) were added to a cooled (0–5 ◦C) solution of 10b
(1.00 g, 6.43 mmol) in MeOH (50 mL) and acetic acid (0.74 mL,
12.86 mmol). The reaction solution was stirred at 0–5 ◦C for
1 h and then at rt for a further 16 h. Solid NaHCO3 (1.62 g,
19.29 mmol) was added and the solvent was removed under
reduced pressure. The resulting white residue was partitioned
between CH2Cl2 (75 mL) and H2O (30 mL) and the aqueous layer
was re-extracted with CH2Cl2 (75 mL). The organic layers were
combined and the solvent removed under vacuum. After column
purification (SiO2, 1 : 1 Hex–EtOAc to 100% EtOAc) 11b was
obtained as a white solid (1.123 g, 73%). TLC (SiO2, EtOAc) Rf
0.30; [a]2D6 −41.65 (c 1.04, CHCl3); mmax(microscope)/cm−1 3320
(br), 2998, 2952, 2836, 1736, 1611, 1513, 1462, 1248; dH(400 MHz;
CDCl3) 3.40 (1 H, dd, J 6.0 and 4.4, H-a), 3.60 (1 H, dd, J 10.8
and 6.0, Ser-CH2), 3.62 (1 H, d, J 12.8, PMB-CH2), 3.73 (3 H, s,
OCH3), 3.75 (1 H, dd, J 10.8 and 4.4, Ser-CH2), 3.78 (3 H, s,
OCH3), 3.78 (1 H, d, J 12.8, PMB-CH2), 6.87-6.83 (2 H, m, Ar-H),
7.24-7.20 (2 H, m, Ar-H); dC(100 MHz; CDCl3) 51.5 (PMB-CH2),
52.1 (OCH3), 55.2 (OCH3), 61.7 (C-a), 62.4 (C-b), 113.9 (Ar-C),
129.4 (Ar-C), 131.3 (Ar-C), 158.9 (Ar-C), 173.5 (CO); m/z (ES+)
Calcd for C12H17NO4Na 262.1050, found 262.1052 [MNa+].
N-(p-Methoxybenzyl)-D-allo-threonine methyl ester (11e)
NaCNBH3 (0.79 g, 12.59 mmol) and p-anisaldehyde (1.12 mL,
9.23 mmol) were added to a cooled (0–5 ◦C) solution of 10e
(1.42 g, 8.39 mmol) in MeOH (40 mL) and acetic acid (1.36 mL,
16.78 mmol). The reaction solution was stirred at 0–5 ◦C for
30 min and then at rt for a further 16 h. Solid NaHCO3 (2.10 g,
25.17 mmol) was added and the solvent was removed under
reduced pressure. The resulting white residue was partitioned
between CH2Cl2 (75 mL) and H2O (30 mL) and the aqueous layer
was re-extracted with CH2Cl2 (50 mL). The organic layers were
combined and the solvent removed under vacuum, giving 11e as a
clear oil (1.28 g, 65%). TLC (SiO2, 1 : 1 Hex–EtOAc) Rf 0.21; [a]D26
+42.88 (c 0.52, CHCl3); mmax(microscope)/cm−1 3440 (br), 2952,
2837 1734, 1612, 1514, 1248 cm−1; dH(400 MHz; CDCl3) 1.02
(3 H, d, J 6.4, CH3), 3.40 (1 H d, J 4.8, H-a), 3.60 (1 H, d, J 12.8,
PMB-CH2), 3.75 (3 H, s, OCH3), 3.80 (3 H, s, OCH3), 3.83 (1 H,
d, J 12.8, PMB-CH2), 4.00 (1 H, dq, J 6.4 and 4.8, H-b), 6.89-6.85
(2 H, m, Ar-H), 7.27-7.23 (2 H, m, Ar-H); dC(400 MHz; CDCl3)
18.8 (CH3), 51.9 (PMB-CH2), 52.1 (OCH3), 55.2 (OCH3), 65.3
(C-a), 67.2 (C-b), 113.8 (Ar-C), 129.5 (Ar-C), 131.5 (Ar-C), 158.9
(Ar-C), 173.6 (CO); m/z (ES+) Calcd for C13H20NO4 254.1387,
found 254.1388 [MH+].
N-(p-Methoxybenzyl)-D-serine methyl ester (11c)
NaCNBH3 (606 mg, 9.65 mmol) and p-anisaldehyde (0.85 mL,
7.07 mmol) were added to a cooled (0–5 ◦C) solution of 10c
(1.00 g, 6.43 mmol) in MeOH (50 mL) and acetic acid (0.74 mL,
12.86 mmol). The reaction solution was stirred at 0–5 ◦C for
1 h and then at rt for a further 16 h. Solid NaHCO3 (1.62 g,
19.29 mmol) was added and the solvent was removed under
reduced pressure. The resulting white residue was partitioned
between CH2Cl2 (75 mL) and H2O (30 mL) and the aqueous layer
was re-extracted with CH2Cl2 (75 mL). The organic layers were
combined and the solvent removed under vacuum. After column
purification (SiO2, 2 : 1 Hex–EtOAc to 100% EtOAc) 11c was
obtained as a white solid (1.046 g, 68%). TLC (SiO2, EtOAc)
Rf 0.30; [a]2D6 +40.78 (c 1.39, CHCl3); mmax(DCM)/cm−1 3327 (br),
2999, 2952, 2837, 1736, 1612, 1513, 1248, 1177, 1035; dH(400 MHz;
CDCl3) 3.40 (1 H, dd, J 6.0 and 4.4, H-a), 3.60 (1 H, dd, J 11.2
and 6.0, Ser-CH2), 3.63 (1 H, d, J 12.0, PMB-CH2), 3.79-3.70 (8H,
m, 2 × OCH3, PMB-CH2 and Ser-CH2), 6.86-6.82 (2 H, m, Ar-H),
7.23-7.20 (2 H, m, Ar-H); 13C (100 MHz; CDCl3) 51.3 (PMB-CH2),
52.0 (OCH3), 55.1 (OCH3), 61.6 (C-a), 62.4 (C-b), 113.7 (Ar-C),
129.4 (Ar-C), 131.2 (Ar-C), 158.8 (Ar-C), 173.4 (CO); m/z (ES+)
Calcd for C12H17NO4Na 262.1050, found, 262.1048 [MNa+].
(4S,5R)-N-(p-Methoxybenzyl)-2,2-dioxo-1,2,3-oxathiazolidinone-
5-methyl-4-carboxylic acid methyl ester (12a)
Pyridine (5.67 mL, 70.1 mmol) was added to a solution of the di-
protected amino acid 11a (3.55 g, 14.02 mmol) in CH2Cl2 (40 mL)
and the reaction solution was then cooled to −78 ◦C. SOCl2
(1.23 mL, 16.8 mmol) was added dropwise over 5 min, and the
solution was left to stir at −78 ◦C for 5 min and allowed to
warm to rt over 1 h. The reaction mixture was quenched by the
addition of 1% HCl (25 mL). The aqueous layer was extracted
with CH2Cl2 (100 mL and 50 mL), and the combined organic
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layers were washed with saturated NaHCO3 (20 mL), dried over
Na2SO4, filtered and concentrated under reduced pressure to give
a yellow residue. The residue was dissolved in MeCN (50 mL)
and the solution was cooled to 0–5 ◦C. RuCl3·3H2O (220 mg,
0.85 mmol), NaIO4 (3.30 g, 15.42 mmol) and H2O (50 mL) were
then added sequentially, and the reaction mixture was left to stir for
10 min at 0–5 ◦C and a further 10 min at rt. The reaction solution
was then partioned between CH2Cl2 (200 mL) and saturated
NaHCO3 (50 mL). The aqueous layer was extracted with CH2Cl2
(2 × 50 mL) and the combined organic layers were washed with
brine (50 mL), dried over Na2SO4, filtered and concentrated under
reduced pressure. Column purification over silica gel (4 : 1 to 3 :
1 Hex–EtOAc) afforded 12a as a clear oil (3.37 g, 76%). TLC
(SiO2, 4 : 1 Hex–EtOAc) Rf 0.16; [a]2D6 −32.32 (c 0.54, CHCl3);
mmax(microscope)/cm−1 3000 (br), 2957, 2840, 1754, 1613, 1515,
1441, 1350, 1252, 1187; dH(400 MHz; CDCl3) 1.49 (3 H, d, J 6.4,
CH3), 3.68 (1 H, d, J 6.4, H-a), 3.65 (3 H, s, OCH3), 3.76 (3 H, s,
OCH3), 4.36 (2 H, s, PMB-CH2), 4.84 (1 H, m, H-b), 6.86-6.82 (2
H, m, Ar-H), 7.28-7.24 (2 H, m, Ar-H); dC(100 MHz; CDCl3) 19.3
(CH3), 50.1 (PMB-CH2), 53.0 (OCH3), 55.2 (OCH3), 64.8 (C-a),
77.5 (C-b), 114.1 (Ar-C), 125.2 (Ar-C), 130.7 (Ar-C), 159.8 (Ar-C),
167.9 (CO); m/z (ES+) Calcd for C13H17NO6SNa 338.0669, found
338.0670 MNa+.
(4R)-N-(p-Methoxybenzyl)-2,2-dioxo-1,2,3-oxathiazolidinone-
4-carboxylic acid methyl ester (12c)
Pyridine (1.24 mL, 15.36 mmol) was added to a solution of the di-
protected amino acid 11c (735 mg, 3.07 mmol) in CH2Cl2 (12 mL)
and the reaction solution was then cooled to −78 ◦C. SOCl2
(0.27 mL, 3.68 mmol) was added dropwise over 5 min, and the
solution was left to stir at −78 ◦C for 5 min and allowed to
warm to rt over 1 h. The reaction mixture was quenched by the
addition of 1% HCl (25 mL). The aqueous layer was extracted
with CH2Cl2 (100 mL) and the combined organic layers were
washed with saturated NaHCO3 (20 mL), dried over Na2SO4,
filtered and concentrated under reduced pressure to give a yellow
residue. The residue was dissolved in MeCN (50 mL) and the
solution was cooled to 0–5 ◦C. RuCl3·3H2O (48 mg, 0.18 mmol),
NaIO4 (787 mg, 3.68 mmol) and H2O (50 mL) were then added
sequentially, and the reaction mixture was left to stir for 10 min
◦
at 0–5 C and a further 10 min at rt. The reaction solution was
then partioned between CH2Cl2 (200 mL) and saturated NaHCO3
(50 mL). The aqueous layer was extracted with CH2Cl2 (2 ×
50 mL) and the combined organic layers were washed with brine
(50 mL), dried over Na2SO4, filtered and concentrated under
reduced pressure. Column purification over silica gel (2 : 1 to 1
: 1 Hex–EtOAc) afforded 12c as a clear oil (718 mg, 76%); TLC
(SiO2, 1 : 1 Hex–EtOAc) Rf 0.33; [a]2D6 +49.30 (c 0.29, CHCl3);
mmax(microscope)/cm−1 3005, 2958, 2840, 1750, 1612, 1515, 1441,
1350, 1251, 1185 cm−1; dH(400 MHz, CDCl3) 3.74 (3 H, s, OCH3),
3.80 (3 H, s, OCH3), 4.06 (1 H, dd, J 7.6 and 4.8, H-a), 4.40 (1
H, d, J 14.0, PMB-CH2), 4.47 (1 H, d, J 14.0, PMB-CH2), 4.58 (1
H, dd, J 8.8 and 7.6, Ser-CH2), 4.65 (1 H, dd, J 8.8 and 4.8, Ser-
CH2), 6.90-6.86 (2 H, m, Ar-H), 7.34 (2 H, m, Ar-H); dC(100 MHz,
CDCl3) 49.8 (PMB-CH2), 53.0 (OCH3), 55.2 (OCH3), 57.8 (C-a),
67.3 (C-b), 114.1 (Ar-C), 125.3 (Ar-C), 130.6 (Ar-C), 159.8 (Ar-C),
168.3 (CO); m/z (ES+) Calcd for C12H15NO6SNa 324.0512, found
324.0509 [MNa+].
(4S)-N-(p-Methoxybenzyl)-2,2-dioxo-1,2,3-oxathiazolidinone-4-
carboxylic acid methyl ester (12b)
Pyridine (1.18 mL, 14.63 mmol) was added to a solution of the di-
protected amino acid 11b (700 mg, 2.93 mmol) in CH2Cl2 (10 mL)
and the reaction solution was then cooled to −78 ◦C. SOCl2
(0.26 mL, 3.56 mmol) was added dropwise over 5 min, and the
solution was left to stir at −78 ◦C for 5 min and allowed to warm to
rt over 1 h. The reaction mixture was quenched by the addition of
HCl (1% sol. 15 mL). The aqueous layer was extracted with CH2Cl2
(75 mL), and the combined organic layers were washed with
saturated NaHCO3 (25 mL), brine (40 mL), dried over Na2SO4,
filtered and concentrated under reduced pressure to give a yellow
residue. The residue was dissolved in MeCN (20 mL) and the
solution was cooled to 0–5 ◦C. RuCl3·3H2O (46 mg, 0.18 mmol),
NaIO4 (752 mg, 3.52 mmol) and H2O (20 mL) were then added
sequentially, and the reaction mixture was left to stir for 10 min at
0–5 ◦C and to warm to rt over 45 min. The reaction solution
was then partioned between CH2Cl2 (100 mL) and saturated
NaHCO3 (50 mL). The aqueous layer was extracted with CH2Cl2
(2 × 50 mL) and the combined organic layers were washed with
brine (50 mL), dried over Na2SO4, filtered and concentrated under
reduced pressure. Column purification over silica gel (2 : 1 to 1 :
1 Hex–EtOAc) afforded 12b as a clear oil (683 mg, 74%); TLC
(SiO2, 1 : 1 Hex–EtOAc) Rf 0.33; [a]2D6 −49.76 (c 1.54, CHCl3);
mmax(cast)/cm−1 3004, 2957, 2840, 1754 (br), 1613, 1586, 1515,
1440, 1352 (br); dH(400 MHz, CDCl3), 3.74 (3 H, s, OCH3), 3.80
(3 H, s, OCH3), 4.06 (1 H, dd, J 7.6 and 4.8, H-a), 4.39 (1 H, d, J
14.0, PMB-CH2), 4.47 (1 H, d, J 14.0, PMB-CH2), 4.58 (1 H, dd,
J 8.8 and 7.6, Ser-CH2), 4.64 (1 H, dd, J 8.8 and 4.8, Ser-CH2),
6.90-6.86 (2 H, m, Ar-H), 7.34-7.30 (2 H, m, Ar-H), dC(100 MHz,
CDCl3) 49.8 (PMB-CH2), 53.0 (OCH3), 55.2 (OCH3), 57.9 (C-a),
67.3 (C-b), 114.1 (Ar-C), 125.3 (Ar-C), 130.6 (Ar-C), 159.8 (Ar-C),
168.3 (CO); m/z (ES+) Calcd for C12H15NO6SNa 324.0512, found
324.0512 [MNa+].
(4R,5S)-N-(p-Methoxybenzyl)-2,2-dioxo-1,2,3-oxathiazolidinone-
5-methyl-4-carboxylic acid methyl ester (12d)
Pyridine (1.60 mL, 19.75 mmol) was added to a solution of the di-
protected amino acid 11d (1.00 g, 3.95 mmol) in CH2Cl2 (20 mL)
and the reaction solution was then cooled to −78 ◦C. SOCl2
(0.35 mL, 4.74 mmol) was added dropwise over 10 min, and the
solution was left to stir at −78 ◦C for 10 min and allowed to
warm to rt over 1 h. The reaction mixture was quenched by the
addition of 1% HCl (25 mL). The aqueous layer was extracted with
CH2Cl2 (80 mL), and the combined organic layers were washed
with saturated NaHCO3 (20 mL), dried over Na2SO4, filtered and
concentrated under reduced pressure to give a yellow residue. The
residue was dissolved in MeCN (20 mL) and the solution was
cooled to 0–5 ◦C. RuCl3·3H2O (62 mg, 0.24 mmol), NaIO4 (0.93 g,
4.35 mmol) and H2O (20 mL) were then added sequent◦ially, and
the reaction mixture was left to stir for 10 min at 0–5 C and a
further 10 min at rt. The reaction solution was then partitioned
between CH2Cl2 (80 mL) and saturated NaHCO3 (40 mL). The
aqueous layer was extracted with CH2Cl2 (2 × 50 mL), and the
combined organic layers were washed with brine (100 mL), dried
over Na2SO4, filtered and concentrated under reduced pressure.
Column purification over silica gel (2 : 1 to 1 : 1 Hex–EtOAc)
This journal is
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afforded 12d as a colourless oil (0.97 g, 78%). TLC (SiO2, 2 :
1 Hex–EtOAc) Rf 0.19; [a]2D6 +29.97 (c 0.87, CHCl3); mmax(DCM,
microscope)/cm−1 3000, 2957, 2840, 1754, 1613, 1515, 1441, 1351,
1252, 1187; dH(400 MHz, CDCl3) 1.53 (3 H, d, J 6.4, CH3), 3.69
(3 H, s, OCH3), 3.70 (1 H, d, J 6.4, H-a), 3.80 (3 H, s, OCH3), 4.41
(2 H, s, PMB-CH2), 4.88 (1 H, app. p, J 6.4, H-b), 6.90-6.86 (2 H,
m, Ar-H), 7.32-7.28 (2 H, m, Ar-H); dC(100 MHz; CDCl3) 19.3
(CH3); 50.0 (PMB-CH2), 53.0 (OCH3), 55.2 (OCH3), 64.8 (C-a),
77.5 (C-b), 114.1 (Ar-C), 130.7 (Ar-C), 159.8 (Ar-C), 167.8 (CO),
m/z (ES+) Calcd for C13H17NO6SNa 338.0669, found 338.0671
[MNa+].
purification over silica gel (2 : 1 Hex–EtOAc) afforded 14 as a
clear oil (106 mg, 67%). TLC (SiO2, 2 : 1 Hex–EtOAc) Rf 0.35;
[a]D26 −14.90 (c 0.30, CHCl3), mmax(CHCl3, microscope)/cm−1 3339,
3057, 2951, 2835, 1733, 1611, 1512, 1445, 1248; dH(400 MHz,
CDCl3) 0.96 (3 H, d, J 7.2, CH3), 2.78 (1 H, dq, J 7.2 and 3.3,
H-b), 2.88 (1 H, d, J 3.3, H-a), 3.53 (1 H, d, J 12.8, PMB-CH2),
3.57 (3 H, s, OCH3), 3.72 (1 H, d, J 12.8, PMB-CH2), 3.81 (3
H, s, OCH3), 6.89-6.86 (2 H, m, Ar-H), 7.34-7.19 (11 H, m, Ar-
H), 7.55-7.52 (6 H, m, Ar-H), dC(100 MHz, CDCl3) 16.7 (CH3),
43.2 (C-b), 51.3 (OCH3), 52.4 (PMB-CH2), 55.2 (OCH3), 64.3 (C-
a), 67.5 (C(Ar)3), 113.6 (Ar-C), 126.4 (Ar-C), 127.8 (Ar-C), 129.6
(Ar-C), 132.0 (Ar-C), 145.0 (Ar-C), 158.7 (Ar-C), 173.5 (CO), m/z
(ES+) Calcd for C32H33NO3SNa 534.2073, found 534.2076 MNa+.
(4R,5R)-N-(p-Methoxybenzyl)-2,2-dioxo-1,2,3-oxathiazolidinone-
5-methyl-4-carboxylic acid methyl ester (12e)
3-(S)-[(R)-2-tert-Butoxycarbonyl-2-(tert-butoxycarbonylamino)-
ethylsulfanyl]-(R)-(p-methoxybenzylamino)butanoic acid methyl
ester (17)
Pyridine (0.57 mL, 7.07 mmol) was added to a solution of the di-
protected amino acid 11e (358 mg, 1.41 mmol) in CH2Cl2 (8 mL)
and the reaction solution was then cooled to −78 ◦C. SOCl2
(0.13 mL, 1.69 mmol) was added dropwise over 5 min, and the
solution was left to stir at −78 ◦C for 5 min and allowed to
warm to rt over 1 h. The reaction mixture was quenched by the
addition of 1% HCl (10 mL). The aqueous layer was extracted
with CH2Cl2 (50 mL), and the combined organic layers were
washed with saturated NaHCO3 (20 mL), dried over Na2SO4,
filtered and concentrated under reduced pressure to give a yellow
residue. The residue was dissolved in MeCN (10 mL) and the
solution was cooled to 0–5 ◦C. RuCl3·3H2O (22 mg, 0.08 mmol),
NaIO4 (787 mg, 3.68 mmol) and H2O (10 mL) were then added
sequentially, and the reaction mixture was left to stir for 15 min
Procedure 1. Boc-Cys-OMe 16 (152 mg, 0.65 mmol) was added
to a solution of 12a (136 mg, 0.43 mmol) in DMF (4 mL) at rt.
Cs2CO3 (210 mg, 0.65 mmol) was added and the solution was left
to stir for 16 h. The reaction was added to 1 M NaH2PO4 buffer
(20 mL), and the solution was left to stir for 24 h at rt. EtOAc
(25 mL) and the layers separated. The aqueous layer was extracted
with EtOAc (2 × 25 mL), and the combined organic extracts
were dried, filtered and concentrated under reduced pressure.
Column purification over silica (2 : 1 Hex–EtOAc) afforded 17
as a colourless oil (81 mg, 40%). TLC (SiO2, 2 : 1 Hex–EtOAc)
Rf 0.31; [a]2D6 −6.71 (c 1.08, CHCl3), mmax(microscope)/cm−1 33.65
(br), 2975, 2837, 1715, 1612, 1512, 1454, 1367, 1248, 1169, 1034;
dH (400 MHz, CDCl3) 1.27 (3 H, d, J 7.2, CH3), 1.44 (9 H, s,
C(CH3)3), 2.88 (1 H, dd, J 13.2 and 6.0, CH2), 2.96 (1 H, dd,
J 13.2 and 5.0, CH2), 3.04 (1 H, dq, J 7.2 and 5.2, H-3), 3.33
(1 H, d, J 5.2, H-2), 3.60 (1 H, d, J 12.8, PMB-CH2), 3.73 (3 H, s,
OCH3), 3.74 (3 H, s, OCH3), 3.80 (3 H, s, OCH3), 3.80 (1 H, d, J
12.8, PMB-CH2), 4.51 (1 H, m, H-6), 5.43 (1 H, br d, J 6.8, NH),
6.87-6.83 (m, 2H, Ar-H), 7.26-7.23 (m, 2H, Ar-H); dC(100 MHz,
CDCl3) 17.6 (CH3), 21.3 (C(CH3)3), 33.6 (C-5), 43.8 (C-3), 51.8
(PMB-CH2), 51.8 (OCH3), 52.5 (OCH3), 53.3 (C-6), 55.2 (OCH3),
64.5 (C-2), 80.1 (C(CH3)3), 113.7 (Ar-C), 129.5 (Ar-C), 131.5 (Ar-
C), 155.1 (CO), 158.8 (Ar-C), 171.4 (CO), 173.7 (CO); m/z (ES+)
Calcd for C22H32N2O7SNa 493.1979, found 493.1982 [MNa+].
◦
at 0–5 C and a further 30 min at rt. The reaction solution was
then partioned between CH2Cl2 (50 mL) and saturated NaHCO3
(15 mL). The aqueous layer was extracted with CH2Cl2 (2 ×
40 mL) and the combined organic layers were washed with brine
(30 mL), dried over Na2SO4, filtered and concentrated under
reduced pressure. Column purification over silica gel (2 : 1 to
1 : 1 Hex–EtOAc) afforded 12e as a white solid (333 mg, 75%).
TLC (SiO2, 2 : 1 Hex–EtOAc) Rf 0.21; [a]2D6 +62.59 (c 0.91, CHCl3);
mmax(CHCl3, microscope)/cm−1 2956, 2841, 1755, 1613, 1515, 1443,
1346, 1252, 1188, 1029; dH(400 MHz; CDCl3) 1.41 (3 H, d, J 6.4,
CH3), 3.70 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 3.97 (1 H, d,
J 6.4, H-a), 4.21 (1 H, m, J 14.0, PMB-CH2), 4.38 (1 H, d, J
14.0, PMB-CH2), 4.99 (1 H, app. p, J 6.4, H-b), 6.86-6.83 (2 H,
m, Ar-H), 7.26-7.22 (2 H, m, Ar-H), dC(100 MHz; CDCl3) 15.8
(CH3), 48.8 (PMB-CH2), 52.6 (OCH3), 55.3 (OCH3), 63.4 (C-a),
76.4 (C-b), 114.2 (Ar-C), 125.4 (Ar-C), 130.6 (Ar-C), 158.9 (Ar-C),
167.3 (CO); m/z (ES+) Calcd for C13H17NO6SNa 338.0669, found
338.0672 [MNa+].
Procedure 2. Boc-Cys-OMe 16 (94 mg, 0.40 mmol) was added
to a solution of 12a (105 mg, 0.33 mmol) in DMF (1.5 mL) at
rt. Cs2CO3 (130 mg, 0.40 mmol) was added, and the solution was
left to stir for 18 h. The solvent was removed under vacuum to
give a thick resid◦ue. This residue was dissolved in CH2Cl2 (2 mL)
and cooled to 0 C. BF3·Et2O (0.08 mL, 0.60 mmol) was added
and the reaction solution was left to stir for 30 min at 0 ◦C.
nPrSH (0.05 mL, 0.60 mmol) was then added and the reaction
was left to stir for a further 18 h at rt. NH4OH solution (30%
NH3, 1 ml) was added and the resulting solution was left to
stir for 30 min before CH2Cl2 (10 ml) and MgSO4 (excess) were
added. The reaction solution was filtered and the solid washed
with CH2Cl2. The organic washes were combined, and the solvent
was removed under reduced pressure to give a pale yellow oil.
Column chromatography (SiO2, 2 : 1 Hex–EtOAc) yielded 17 as a
(2R,3S)-N-(p-Methoxylbenzyl)-3-tritylsulfanyl-
3-methylcarboxylic acid methyl ester (14)
Trityl-SH 13 (132 mg, 0.48 mmol) was added to a solution of
12a (100 mg, 0.32 mmol) in DMF (2 mL) at rt. Cs2CO3 (155 mg,
0.48 mmol) was then added and the solution was left to stir for
16 h. The reaction was added to 1 M NaH2PO4 buffer (20 mL),
and the solution left to stir for 1 h at rt. EtOAc (25 mL) was
added, and the layers separated. The aqueous layer was extracted
with EtOAc (2 × 25 mL), and the combined organic extracts were
dried, filtered and concentrated under reduced pressure. Column
1036 | Org. Biomol. Chem., 2007, 5, 1031–1038
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©

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colourless oil (124 mg, 79%). The physical and spectral properties
for 17 obtained marched those reported for Procedure 1.
H-3), 2.95 (2 H, m, H-5), 3.46 (1 H, t, J 6.0, H-2), 3.66 (1 H, d,
J 12.8, PMB-CH2), 3.72 (3 H, s, CO2CH3), 3.78 (1 H, d, J 12.8,
PMB-CH2), 3.79 (3 H, s, PMB-OCH3), 4.39 (1 H, m, H-6), 5.66 (1
H, br s, NH), 6.86-6.83 (2 H, m, Ar-H), 7.26-7.23 (2 H, m, Ar-H);
dC(100 MHz, CDCl3) 27.9 (C(CH3)3), 28.3 (C(CH3)3), 35.5 (C-3),
36.0 (C-5), 51.2 (PMB-CH2), 51.9 (PMB-OCH3), 54.1 (C-6), 55.2
(CO2CH3), 60.1 (C-2), 79.8 (C(CH3)3), 82.4 (C(CH3)3), 113.8 (Ar-
C), 129.4 (Ar-C), 131.4 (Ar-C), 155.2 (CO), 158.8 (Ar-C), 169.8
(C-7), 173.8 (C-1); m/z (ES+) Calcd for C24H39N2O7S 499.2473,
found 499.2475 [MH+].
3-[(R)-2-tert-Butoxycarbonyl-2-(tert-butoxycarbonylamino)-
ethylsulfanyl]-(R)-(p-methoxybenzylamino)propionic acid
methyl ester (19a)
Boc-Cys-OtBu 18 (213 mg, 0.77 mmol) and Cs2CO3 (251 mg,
0.77 mmol) were added to a solution of 12b (193 mg, 0.64 mmol)
in DMF (2 mL). The reaction solution was left to stir at rt for
20 h. The solvent was removed under vacuum to give a thick
residue. This residue was dissolved in CH2Cl2 (4 mL) and cooled
to 0 ◦C. BF3·Et2O (0.12 mL, 0.96 mmol) was added, and the
reaction solution was left to stir for 30 min. nPrSH (0.09 mL,
0.99 mmol) was then added, and the reaction was left to stir for
a further 16 h. NH4OH (30% NH3, 1.5 mL) was added, and the
resulting solution was left to stir 30 min before MgSO4 (excess)
and CH2Cl2 (10 ml) were added. The reaction solution was filtered
and the solid washed with CH2Cl2. The solvent was removed
under reduced pressure, and the resulting oil was purified by
column chromatography (SiO2, 2 : 1 Hex–EtOAc) to give 19a as a
colourless oil (257 mg, 85%). TLC (SiO2, 2 : 1 Hex–EtOAc) Rf 0.28;
[a]D26 −9.18 (c 1.25, CHCl3); mmax(cast)/cm−1 3363 (br), 2977, 2933,
2836, 1737, 1713, 1611, 1513, 1392, 1367, 1155, 1035; dH(400 MHz,
CDCl3) 1.44 (9 H, s, C(CH3)3), 1.46 (9 H, s, C(CH3)3), 2.85 (2 H,
m, H-3), 2.95 (2 H, m, H-5), 3.45 (1 H, t, J 6.4, H-2), 3.65 (1 H,
d, J 12.8, PMB-CH2), 3.73 (3 H, s, CO2CH3), 3.77 (1 H, d, J 12.8,
PMB-CH2), 3.79 (3 H, s, PMB-OCH3), 4.39 (1 H, m, H-6), 5.56 (1
H, br d, NH), 6.87-6.83 (2 H, m, Ar-H), 7.26-7.23 (2 H, m, Ar-H);
dC(100 MHz, CDCl3) 27.9 (C(CH3)3), 28.3 (C(CH3)3), 35.5 (C-3),
36.0 (C-5), 51.3 (PMB-CH2), 52.0 (PMB-OCH3), 54.2 (C-6), 55.2
(CO2CH3), 60.3 (C-2), 79.8 (C(CH3)3), 82.4 (C(CH3)3), 113.8 (Ar-
C), 129.5 (Ar-C), 131.4 (Ar-C), 155.2 (CO), 158.8 (Ar-C), 169.8
(C-7), 173.8 (C-1); m/z (ES+) Calcd for C24H39N2O7S 499.2473,
found 499.2471 [MH+].
3-(R)-[(R)-2-tert-Butoxycarbonyl-2-(tert-butoxycarbonylamino)-
ethylsulfanyl]-(S)-(p-methoxybenzylamino)butanoic acid methyl
ester (20a)
Boc-Cys-OtBu 18 (322 mg, 1.16 mmol) and Cs2CO3 (377 mg,
1.16 mmol) were added to a solution of 12d (304 mg, 0.96 mmol)
in DMF (5 mL). The reaction solution was left to stir at rt for
20 h. The solvent was removed under vacuum to give a thick
residue. The residue was dissolved in CH2Cl2 (6 mL), and cooled
to 0 ◦C. BF3·Et2O (0.18 mL, 1.44 mmol) was added, and the
reaction solution was left to stir for 30 min. nPrSH (0.13 mL,
1.44 mmol) was then added, and the reaction was left to stir for
a further 16 h. NH4OH (30% NH3, 2.0 mL) was added, and the
resulting solution was left to stir 30 min before MgSO4 (excess)
and CH2Cl2 (15 mL) were added. The reaction solution was filtered
and the solid washed with CH2Cl2. The solvent was removed under
reduced pressure, and the resulting oil was purified by column
chromatography (SiO2, 4 : 1 Hex–EtOAc), to give 20a as an oil
(354 mg, 72%). TLC (SiO2, 4 : 1 Hex–EtOAc) Rf 0.15; [a]2D6 +15.19
(c 0.65, CHCl3); mmax(CHCl3, microscope)/cm−1 3363 (br), 2978,
2933, 1735, 1716, 1513, 1368, 1248, 1155, 1035; dH(400 MHz,
CDCl3) 1.28 (3 H, d, J 7.2, CH3); 1.44 (9 H, s, C(CH3)3), 1.47
(9 H, s, C(CH3)3), 2.89 (1 H, dd, J 13.8 and 4.8, H-5), 2.97 (1 H, dd,
J 13.8 and 4.8, H-5), 3.07 (1 H, m, H-3), 3.33 (1 H, d, J 5.6, H-2),
3.62 (1 H, d, J 12.8, PMB-CH2), 3.72 (3 H, s, PMB-OCH3), 3.79
(3 H, s, CO2CH3), 3.81 (1 H, d, J 12.8, PMB-CH2), 4.42 (1 H,
m, CH), 5.69 (1 H, d, J 8.4, NH), 6.87-6.83 (2 H, m, Ar-H),
7.27-7.23 (2 H, m, Ar-H); dC(100 MHz, CDCl3) 18.3 (CH3), 27.9
(C(CH3)3), 28.3 (C(CH3)3), 34.0 (C-5), 44.4 (C-3), 51.7 (PMB-
CH2), 51.7 (PMB-OCH3), 54.3 (C-6), 55.2 (CO2CH3), 64.5 (C-2),
79.7 (C(CH3)3), 82.4 (C(CH3)3), 113.7 (Ar-C), 129.5 (Ar-C), 131.5
(Ar-C), 155.3 (CO), 158.8 (Ar-C), 169.8 (CO), 173.7 (CO); m/z
(ES+) Calcd for C25H41N2O7S 513.2629, found 513.2628 [MH+].
3-[(S)-2-tert-Butoxycarbonyl-2-(tert-butoxycarbonylamino)-
ethylsulfanyl]-(R)-(p-methoxybenzylamino)propionic acid
methyl ester (19b)
Boc-Cys-OtBu 18 (201 mg, 0.73 mmol) and Cs2CO3 (236 mg,
0.73 mmol) were added to a solution of 12c (182 mg, 0.60 mmol)
in DMF (2 mL). The reaction solution was left to stir at rt for
20 h. The solvent was removed under vacuum to give a thick
residue. The residue was dissolved in CH2Cl2 (4 mL) and cooled
to 0 ◦C. BF3·Et2O (0.11 mL, 0.91 mmol) was added, and the
reaction solution was left to stir for 30 min. nPrSH (0.08 ml,
0.91 mmol) was then added, and the reaction was left to stir for
a further 16 h. NH4OH (30% NH3, 1.5 mL) was added, and the
resulting solution was left to stir 30 min before MgSO4 (excess)
and CH2Cl2 (10 mL) were added. The reaction solution was filtered
and the solid washed with CH2Cl2. The solvent was removed under
reduced pressure, and the resulting oil was purified by column
chromatography (SiO2, 4 : 1 to 2 : 1 Hex–EtOAc) to give 19b
as an oil (252 mg, 89%). TLC (SiO2, 2 : 1 Hex–EtOAc) Rf 0.29;
[a]2D6 +7.47 (c 2.16, CHCl3); mmax(CHCl3, microscope)/cm−1 3360
(br), 2978, 2934, 1737, 1715, 1513, 1393, 1368, 1248, 1156, 1035;
dH(400 MHz, CDCl3) 1.44 (9 H, s, C(CH3)3), 1.46 (9 H, s, C(CH3)3),
2.84 (1 H, dd, J 13.2 and 6.0, H-3), 2.89 (1 H, dd, J 13.2 and 6.0,
3-(S)-[(R)-2-tert-Butoxycarbonyl-2-(tert-butoxycarbonylamino)-
ethylsulfanyl]-(S)-(p-methoxybenzylamino)butanoic acid methyl
ester (20b)
Boc-Cys-OtBu 18 (178 mg, 0.64 mmol) and Cs2CO3 (210 mg,
0.64 mmol) were added to a solution of 12e (169 mg, 0.54 mmol)
in DMF (3 mL). The reaction solution was left to stir at rt for
20 h. The solvent was removed under vacuum to give a thick
residue. The residue was dissolved in CH2Cl2 (4 mL) and cooled to
0 ◦C. BF3·Et2O (0.11 mL, 0.80 mmol) was added, and the reaction
solution was left to stir for 30 min. nPrSH (0.06 ml, 0.80 mmol)
was then added, and the reaction was left to stir for a further 16 h.
NH4OH (30% NH3, 1.5 mL) was added, and the resulting solution
was left to stir 30 min before MgSO4 (excess) was added. The
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reaction solution was filtered and the solid washed with CH2Cl2.
The solvent was removed under reduced pressure and the resulting
oil was purified by column chromatography (SiO2, 4 : 1 to 2 : 1
Hex–EtOAc) to give 20b as an oil (194 mg, 70%). TLC (SiO2, 4 :
1 Hex–EtOAc) Rf 0.13; [a]2D6 +11.02 (c 2.04, CHCl3); mmax(CHCl3,
microscope)/cm−1 3372 (br), 2978, 2934, 1737, 1715, 1513, 1456,
1513, 1368, 1248 1155; dH(400 MHz; CDCl3) 1.31 (3 H, d, J 6.8,
CH3), 1.45 (9 H, s, C(CH3)3), 1.46 (9 H, s, C(CH3)3), 2.91 (1 H, dd,
J 13.6 and 5.2, H-5), 2.96 (1 H, dd, J 13.6 and 4.8, H-5), 3.13 (1 H,
m, H-3), 3.25 (1 H, d, J 5.6, H-2), 3.60 (d, 1H, J 13.2, PMB-CH2),
3.74 (3 H, s, PMB-OCH3), 3.79 (3 H, s, CO2CH3), 3.83 (1 H, d,
J 13.2, PMB-CH2), 4.37 (1 H, m, H-6), 5.49 (1 H, d, J 7.2, NH),
6.86-6.83 (2 H, m, Ar-H), 7.27-7.23 (2 H, m, Ar-H), dC(100 MHz;
CDCl3) 19.3 (CH3), 28.0 (C(CH3)3), 28.3 (C(CH3)3), 33.6 (C-5),
44.0 (C-3), 51.7 (PMB-CH2), 51.8 (PMB-OCH3), 53.8 (C-6), 55.2
(CO2CH3), 64.9 (C-2), 79.8 (C(CH3)3), 82.5 (C(CH3)3), 113.7 (Ar-
C), 129.7 (Ar-C), 131.7 (Ar-C), 155.2 (CO), 158.7 (Ar-C), 169.8
(CO), 173.9 (CO); m/z (ES+) Calcd for C25H41N2O7S 513.2629,
found 513.2627 [MH+].
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Acknowledgements
Financial support was provided by the Natural Sciences and
Engineering Research Council of Canada (NSERC), the Canada
Research Chair in Bioorganic and Medicinal Chemistry, and the
Alberta Heritage Foundation for Medical Research (AHFMR)
(postdoctoral fellowship to SLC).
14 M. F. Mohd Mustapa, R. Harris, N. Bulic-Subanovic, S. L. Elliot,
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1038 | Org. Biomol. Chem., 2007, 5, 1031–1038
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The Royal Society of Chemistry 2007
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