Improved synthesis of (S)-N-Boc-5-oxaproline for protein synthesis with the α-ketoacid-hydroxylamine (KAHA) ligation

Article abstract of DOI:10.1016/j.bmc.2017.06.019

We describe a new route for the synthesis of (S)-N-Boc-5-oxaproline. This building block is a key element for the chemical synthesis of proteins with the α-ketoacid-hydroxylamine (KAHA) ligation. The new synthetic pathway to the enantiopure oxaproline is

Full text of DOI:10.1016/j.bmc.2017.06.019

Accepted Manuscript
Improved synthesis of (S)-N-Boc-5-Oxaproline for protein synthesis with the
α-ketoacid-hydroxylamine (KAHA) ligation
Claudia E. Murar, Thibault J. Harmand, Jeffrey W. Bode
PII:
S0968-0896(17)30774-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.06.019
BMC 13802
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
9 April 2017
6 June 2017
13 June 2017
Please cite this article as: Murar, C.E., Harmand, T.J., Bode, J.W., Improved synthesis of (S)-N-Boc-5-Oxaproline
for protein synthesis with the α-ketoacid-hydroxylamine (KAHA) ligation, Bioorganic & Medicinal Chemistry
(2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.06.019
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Improved Synthesis of (S)-N-Boc-5-Oxaproline
for Protein Synthesis with the
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α-Ketoacid-Hydroxylamine (KAHA) Ligation
Claudia E. Murar a, Thibault J. Harmanda, Jeffrey W. Bodea,b
a Laboratorium für Organische Chemie, Department of Chemistry and Applied Biosciences, ETH–Zürich,
CH-8093 Zürich, Switzerland
b Institute of Transformative bio-Molecules (WPI–ITbM), Nagoya University, Chikusa,
Nagoya 464-8602, Japan

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Improved synthesis of (S)-N-Boc-5-Oxaproline for protein synthesis with the
α-ketoacid-hydroxylamine (KAHA) ligation
Claudia E. Murar a, Thibault J. Harmand a and Jeffrey W. Bode a,b
∗
aLaboratorium für Organische Chemie, Department of Chemistry and Applied Biosciences, ETH–Zürich, CH-8093 Zürich, Switzerland
bInstitute of Transformative bio-Molecules (WPI–ITbM), Nagoya University, Chikusa, Nagoya 464-8602, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
We describe a new route for the synthesis of (S)-N-Boc-5-oxaproline. This building block is a
key element for the chemical synthesis of proteins with the α-ketoacid-hydroxylamine (KAHA)
ligation. The new synthetic pathway to the enantiopure oxaproline is based on a chiral amine
mediated enantioselective conjugate addition of a hydroxylamine to trans-4-oxo-2-butenoate.
This route is practical, scalable and economical and provides decagram amounts of material for
protein synthesis and conversion to other protected forms of (S)-oxaproline.
Received in revised form
Accepted
Available online
Keywords:
2009 Elsevier Ltd. All rights reserved.
KAHA ligation
(S)-N-Boc-5-Oxaproline
Chemical ligation
Chemical protein synthesis
1. Introduction
to the corresponding backbone amide in basic buffers by an O-N
shift, generating a homoserine residue at the ligation site
(Scheme 1).2 The C-terminal peptide α-ketoacids are readily
prepared using a variety of solid supported linkers and we have
reported KAHA ligations with Leu, Phe, Val, Ile, Glu, Arg, Tyr,
and Glu-derived α-ketoacids. The use of hydroxylamines derived
from amino acids, however, is plagued by poor stability of the
unprotected forms.3,4
As part of our effort to establish a new platform for the
chemical synthesis of proteins using Fmoc-SPPS, we reported in
2012 the α-ketoacid–hydroxylamine ligation (KAHA ligation)
with (S)-N-Boc-5-oxaproline ((S)-N-5-Opr).1 The KAHA ligation
enables coupling of an unprotected C-terminal peptide α-
ketoacid with a segment bearing an N-terminal 5-oxaproline (5-
Opr). The initially formed depsipetide product readily rearranges
Scheme 1. Coupling of (S)-N-Boc-5-oxaproline ((S)-N-Boc-5-Opr) onto a protected peptide segment and acidic cleavage from the resin,
followed by KAHA ligation
———
∗ Corresponding author. Tel.: +41 44 633 21 03; e-mail: bode@org.chem.ethz.ch

Scheme 2. a) Prior route to the synthesis of (S)-N-Boc-5-oxaproline. b) New route for the preparation of (S)-N-Boc-5-oxaproline.
To address this, we identified
a
number of cyclic
recovery.1 Above all, the requirement of a pressurized reactor in
this procedure is a major impediment for preparing this building
block in a practical way.
hydroxylamine derivatives as being both chemically stable to
standard Fmoc-SPPS and excellent performance in the KAHA
ligation (Scheme 1). (S)-N-Boc-5-oxaproline has been utilized in
the synthesis of SUMO2, SUMO3,5 irisin,6 betatrophin,7 AS48,8
and NP4.9 This building block can be readily converted to
orthogonally protected (S)-N-protected-5-oxaprolines, which are
used for sequential KAHA ligations in the synthesis of small and
medium-size proteins.
In this report we document our development of a new
synthetic pathway for the synthesis of (S)-N-Boc-5-Opr based on
an enantioselective organomediated reaction (Scheme 2b). This
procedure can be used to easily prepare >5 g of (S)-N-Boc-5-Opr
in a research lab and has been employed by a research partner for
the production of >1 kg of this building block.
Our efforts to expand the use of KAHA ligation for the
chemical synthesis of peptides and proteins required a scalable
and practical synthesis for the key monomer (S)-N-Boc-5-Opr.
Our original approach to the synthesis of (S)-N-Boc-5-Opr was
based on a modified procedure of Vasella et al.10 and utilized a
[3+2] cycloaddition with ethylene in a pressurized reactor
(Scheme 2a). However, this protocol required an expensive chiral
auxiliary that afforded a 6:4 diastereomeric ratio after the
cycloaddition reaction. After several recrystallizations the two
diastereoisomers can be separated but with relatively poor
2. Enantioselective organomediated route for the synthesis of
(S)-N-Boc-5-Oxaproline
2.1. Route 1: from 5-hydroxyisoxazolidine
Inspired by the work of Cordova on asymmetric tandem reaction
between N-protected hydroxylamines and α β-unsaturated
aldehydes catalyzed by diarylprolinols. We envisaged a route for
the synthesis of (S)-N-Boc-5-Opr from the corresponding 5-
hydroxyisoxazolidine in three steps (Scheme 3).11
Scheme 3. a) Cordova’s work to substituted 5-hydroxyisoxazolidines. b) Our envisaged route for the preparation of (S)-N-Boc-5-Opr
inspired from the work of Cordova.

Scheme 4. Conjugate addition of silyl protected hydroxylamines to α,β-unsaturated aldehydes.
Substituted 5-hydroxyisoxazolidine 11 was readily prepared
from N-Boc-protected hydroxylamine and ethyl trans-4-oxo-2-
butenoate catalyzed by TMS protected diphenylprolinol in a 3:1
diastereomeric ratio. The synthesis of the TMS protected
diphenylprolinol catalyst was straightforward starting from the
documented the asymmetric addition of silyl protected
hydroxylamines to α β-unsaturated aldehydes in the presence of
a chiral catalyst and we turned our attention to a modified
synthetic protocol (Scheme 4).19,20
We proceeded with the TMS catalyst 7 that gave the addition
product 8 in a 96:4–98:2 er.21 Evaluation of the conditions for the
conjugate addition revealed that using 50 mol% of the
organocatalyst improved the addition yield to 48%.
Unfortunately, increasing the quantity of the TMS protected
diphenylprolinol to 75% or even an equimolar ratio did not show
any yield improvement. In all cases, the reaction did not go to
completion.
commercially
available,
affordable
intermediate
(S)-
diphenyl(pyrrolidin-2-yl)methanol. Otherwise, this catalyst is
commercially available from a great number of vendors. The key
step in this route was the mild reduction of the cyclic
hydroxylamine 11. We therefore probed conditions for this step.
Mild activation of the cyclic hydroxylamine 11 with Shoda’s
catalyst12–14 followed by reduction with sodium borohydride led
to decomposition of the activated adduct as indicated by 1H
NMR. Treatment with methanesulfonyl chloride and acetic
anhydride under basic conditions or with triphenylphosphine
under basic conditions failed to give the activated intermediate
validating the low stability of this intermediate. We consequently
turned our attention to the use of Lewis acids in the presence of
reducing agents but both InCl3 and TiCl4 entailed significant
Further reduction of the β-amino aldehyde 8 with sodium
borohydride provided alcohol 12 in good yield and without the
need for further purification (Scheme 5a). We attempted to
cyclize this alcohol in
a
one-pot fashion using
nonafluorobutanesulfonyl fluoride (NfF) and DBU. After
treatement of alcohol with NfF (1.1 equiv) and DBU (3.2 equiv)
in a mixture of CH2Cl2:pentane 1:2 at 0 ºC for 1 h, we were able
to observe partial conversion to the desired cyclic product.
However, due to the high basicity of DBU the alcohol 12
underwent transesterification and the desired product 4 was
obtained together with the byproduct 13 (1:1 ratio). We evaluated
the use of pyridine as base or lowering the temperature to –15 ºC,
but the outcome of the reaction was not favorable and the isolated
yield was never higher than 38%.
.
degradation of starting material. Silane reduction using BF3 Et2O
as Lewis acid or in acidic conditions with TFA were also
unfruitful. Ultimately, attempts of reduction with borohydrides
were deemed unproductive.
Although the conditions described above are well established
for cyclic hemiacetals,15–17 these were unsuccessful for our cyclic
hydroxylamine-hemiacetal. Therefore we turned towards the
preparation of Boc-Opr using an asymmetric addition of silyl-
protected hydroxylamines to α β-unsaturated aldehydes,
followed by cyclization.
We next examined the stepwise preparation of cyclic ester 4.
Mesylation of alcohol 12 smoothly afforded compound 9 in
complete conversion. Treatment of the crude mesylated
compound 9 with TBAF (1.5 equiv, 1 M in THF) at low substrate
concentration (0.05 M) at 4 ºC for 2 h furnished cyclic ester 4 as
a single product (Scheme 5b). Hydrolysis of the ethyl ester
afforded the (S)-N-Boc-5-Opr building block 1 in 96:4–98:2 er
and good overall yield (60% over four steps). The (S)-N-Boc-5-
Opr 1 was derivatized with p-bromoaniline, and the er of the
2.2. Route 2: from β-amino aldehydes
In the next strategy we first examined literature examples
based on the extensive work of Cordova and MacMillan in the
field of conjugate addition reactions involving N-centered
nucleophiles and chiral amine catalysts.11,18 Both groups
Scheme 5. a) Design of a new strategy towards the synthesis of (S)-N-Boc-5-oxaproline. b) Synthesis of (S)-N-Boc-5-oxaproline from
alcohol 12.

newly formed product was investigated (see experimental part).
With a practical method to prepare (S)-N-Boc-5-Opr 1 in hand,
we set out to explore the scalability of this route and we
established a new route for the preparation of more than 5 g of
(S)-N-Boc-5-Opr with a good overall yield. Ultimately, a
research partner prepared 1 kg of desired oxaproline using our
new route.
under reduced pressure to afford 5 (18.5 g, 75.0 mmol, quant) as
a low melting solid that was used in the next step without further
purification. 1H NMR (400 MHz, CDCl3) δ 6.67 (s, 1H), 1.48 (s,
9H), 0.95 (s, 9H), 0.162 (s, 6H) ppm. 13C NMR (101 MHz,
CDCl3) δ –5.1, 18.7, 26.5, 28.8, 82.2, 158.5 ppm. HR-MS (ESI):
calculated for (C11H25NO3Si) [M+Na]+: 270.1496, found
270.1497. IR (thin film): υ = 3292, 2960, 1752, 1693, 1473,
1463, 1392, 1367, 1251, 1171 cm-1.
The (S)-N-Boc-5-Opr could also serve as a starting point for a
variety of different protected building blocks for sequential
KAHA ligations.22,23
4.1.3. Ethyl trans-4-oxo-2-butenoate (6)
Ethyl trans-4-oxo-2-butenoate 6 was obtained from ABCR-
Chemicals (96%) and purified by distillation. Ethyl trans-4-oxo-
2-butenoate (20 mL) was placed in a 100 mL flask with a
magnetic stirbar. The flask was equipped with a Vigreux
containing a thermometer on the top and a water condenser on
the side to collect the distillate. The pure fractions distilled at 35
°C at 8.10-2 mbars and the product was obtained with an isolated
yield of 85%.
3. Conclusions
We have developed a scalable and practical synthesis of the
enantioenriched (S)-5-oxaproline building block using an
improved enantioselective organomediated reaction. The key
aldehyde intermediate 8 is also a valuable material for the
preparation of novel monomers for KAHA ligation.
4.2. Synthesis of (S)-N-Boc-5-oxaproline
4. Experimental
1H an13C NMR spectra were recorded on Bruker DRX400 and
Bruker AVIII400 spectrometers using CDCl3 as solvent.
Chemical shift (δ) are reported in ppm with the solvent resonance
as the internal standard relative to chloroform (δ 7.26 ppm for 1H
and 77.0 ppm for 13C).
4.2.1.
butyldimethylsilyl)oxy)amino)-4-oxobutanoate (8)
(S)-Ethyl
2-((tert-Butoxycarbonyl)((tert-
To a solution of (S)-(–)-α,α-Diphenyl-2-pyrrolidinemethanol
trimethylsilyl ether 7 (19.0 g, 58.6 mmol, 0.50 equiv) in CHCl3
(130 mL) was added dropwise ethyl (2E)-4-oxo-2-butenoate 6
(14.1 mL, 117 mmol, 1.00 equiv) at 4 ºC. tert-Butyl (tert-
butyldimethylsilyl)oxycarbamate 5 (34.8 g, 141 mmol, 1.20
equiv) was dissolved in CHCl3 (70 mL) and added dropwise over
1 h. The reaction was stirred for 16 h at rt. TLC was taken in
25% EtOAc in hexanes using KMnO4 as stain (product 8 has an
Rf = 0.6 the starting material 5 has an Rf = 0.7 and the starting
material aldehyde 6 has an Rf = 0.5). The reaction mixture was
concentrated under reduced pressure. The crude compound was
purified by column chromatography (gradient of 5–15% EtOAc
in hexanes) to give 8 (21.2 g, 97:3 er, 56.1 mmol, 48% yield) as a
pale yellow oil. HPLC: Chiralcel IA, hexanes:isopropyl alcohol
97:3, 20 °C, 30 µL, 0.8 mL/min, 210 nm, tR = 6.2 min (major), tR
= 6.8 min (minor). 1H NMR (400 MHz, CDCl3) δ 9.83 (t, J = 1.1
Hz, 1H), 4.91 (dd, J = 8.1, 5.3 Hz, 1H), 4.32 – 4.09 (m, 2H), 3.24
(ddd, J = 17.5, 8.1, 1.3 Hz, 1H), 2.80 (ddd, J = 17.6, 5.3, 0.9 Hz,
1H), 1.49 (s, 9H), 1.36 – 1.19 (m, 3H), 0.93 (s, 9H), 0.17 (d, J =
7.0 Hz, 6H) ppm. 13C NMR (101 MHz, CDCl3) δ 198.70, 169.05,
157.90, 82.56, 61.65, 60.52, 42.76, 28.17, 28.14, 25.88, 25.86,
17.94, 14.16, -4.71, -4.79 ppm. HR-MS (ESI): calculated for
HRMS data were recorded by the mass spectrometry service
of the laboratory of Organic Chemistry at ETH Zurich either with
a Bruker maXis instrument equipped with an ESI source and a
Qq-TOF detector.
TLC analysis was performed using pre-coated glass plate
(Merck, silica gel 60 F254) and visualized by ultraviolet at 254
nm, by staining with potassium permanganate or cerium
ammonium molybdate. Products were purified via column
chromatography using silica gel type F60 (230–400 mesh) using
a forced flow air at 0.5–1.0 bar.
4.1. Synthesis of starting materials
4.1.1. (S)-2-(Diphenyl((trimethylsilyl)oxy)methyl)pyrrolidine (7)
To (S)-diphenylprolinol (4.00 g, 15.8 mmol, 1.00 equiv) in
CH2Cl2 (40 mL) was added imidazole (3.22 g, 47.4 mmol, 3.00
equiv) at 0 ºC. TMSCl (5.00 mL, 39.5 mmol, 2.50 equiv) was
added dropwise and the reaction was stirred for 12 h at rt. MTBE
(100 mL) was added to the reaction and the mixture was filtered.
The organic phase was washed with H2O (50 mL) and saturated
aqueous NaCl (2 x 50 mL), dried over MgSO4, filtered and
concentrated under reduced pressure to a colorless oil 11 (5.00 g,
15.3 mmol, 97%). 1H NMR (400 MHz, CDCl3) δ 7.54 – 7.46 (m,
2H), 7.42 – 7.36 (m, 2H), 7.35 – 7.20 (m, 6H), 4.07 (t, J = 7.4
Hz, 1H), 2.98 – 2.75 (m, 2H), 1.84 – 1.72 (m, 1H), 1.68 – 1.55
(m, 3H), 1.48 – 1.37 (m, 1H), -0.06 (s, 9H) ppm. 13C NMR (101
MHz, CDCl3) δ 146.83, 145.78, 128.44, 127.61, 127.57, 127.53,
126.90, 126.73, 83.17, 65.42, 47.16, 27.51, 25.06, 2.20 ppm. HR-
MS (ESI): calculated for (C20H28NOSi) [M+H]+: 326.1935,
found: 326.1937.
(C17H33NNaO6Si) [M+Na]+: 398.1969, found: 398.1968. [α ]D
= – 23.23 (c = 2.25, CH2Cl2). IR (thin film): υ = 3381, 2979,
2931, 2858, 1729, 1462, 1369, 1302, 1164, 1042, 1014, 835, 785
cm-1.
26.4
4.2.2.
butyldimethylsilyl)oxy)-L-homoserinate (12)
Ethyl
N-(tert-Butoxycarbonyl)-N-((tert-
The aldehyde 8 (15.0 g, 40.0 mmol, 1.0 equiv, 97:3 er) was
dissolved in MeOH (200 mL, concentration of substrate 0.20 M)
and sodium borohydride (3.02 g, 79.8 mmol, 2.0 equiv) was
added in ten portions (~300 mg every 3 min) at –20 ºC. After
complete addition, the reaction was stirred for 45 min in the
CH3CN-dry ice bath at -20 ºC, after which the reaction was
allowed to warm to 0 ºC. The reaction was monitored by TLC in
25% EtOAc in hexanes using ninhydrin for stain (product 12 has
Rf = 0.33 and stains violet and starting material 8 has Rf = 0.65
and stains yellow). Ice-water was added to the solution with
vigorous stirring and the solution was stirred for 10 min at 0 ºC.
To this mixture EtOAc and H2O were added and the aqueous
solution was poured into a 2-L separatory funnel. The aqueous
layer was extracted with EtOAc (3 x 150 mL). The combined
4.1.2. tert-Butyl (tert-butyldimethylsilyl)oxycarbamate (5)
A 500 mL round-bottom flask charged with tert-butyl
hydroxycarbamate (10 g, 75 mmol, 1.0 equiv) in CH2Cl2 (150
mL) and Et3N (11.1 mL, 82.5 mmol, 1.10 equiv) was cooled to 0
ºC, and TBSCl (11.2 g, 75.0 mmol, 1.00 equiv) in CH2Cl2 (100
mL) was added. The reaction was allowed to warm to ambient
temperature and stirred for 12 h. Upon completion, the reaction
was diluted with H2O, the organic layer washed with H2O,
saturated aqueous NaCl, dried over MgSO4 and concentrated

organic layers were washed with saturated aqueous NH4Cl (100
mL) and saturated aqueous NaCl (100 mL). The organic layer
was dried over Na2SO4, filtered and concentrated under reduced
pressure to a pale yellow oil 12 (14.8 g, 39.2 mmol, 98.2%) that
was used without further purification. 1H NMR (400 MHz,
CDCl3): δ 4.47 (dd, J = 8.8, 5.8 Hz, 1H), 4.22 (dd, J = 7.1, 5.1
Hz, 2H), 3.77 (t, J = 5.7 Hz, 2H), 2.50–2.00 (m, 2H), 1.50 (s,
9H), 1.29 (d, J = 7.2 Hz, 3H), 0.95 (s, 9H), 0.21 (d, J = 3.1 Hz,
6H) ppm. 13C NMR (101 MHz, CDCl3): δ 172.77, 157.40, 83.09,
65.67, 60.86, 60.39, 50.88, 28.20, 28.13, 26.92, 25.90, 24.95,
21.05, 17.98, 14.20, -4.87, -4.90 ppm.
4.2.5. (S)-2-(tert-Butoxycarbonyl)isoxazolidine-3-carboxylic acid
(1)
The oil 4 (6.34 g, 25.8 mmol, 1.0 equiv) was diluted with THF
(105 mL) and was added a chilled solution of aqueous 1 M LiOH
(103 mL, 103 mmol, 4.0 equiv) at 4 ºC. The reaction mixture was
stirred for 1.5 h at rt. The reaction was monitored by TLC in 5%
MeOH in CH2Cl2 using ninhydrin for staining. CHCl3 (250 mL)
was added and the solution was acidified to pH 3 with an
aqueous solution of 2 M KHSO4 (100 mL). The mixture was
poured into a 1-L separatory funnel. The aqueous layer was
separated and washed with CHCl3 (5 x 100 mL). The combined
organic layers were dried over Na2SO4, filtered and concentrated
under reduced pressure to a clear yellow oil 1 that solidified upon
standing in the refrigerator (5.39 g, 24.8 mmol, 96.0% yield,
4.2.3.
butyldimethylsilyl)oxy)-O-(methylsulfonyl)-L-homoserinate (9)
Ethyl
N-(tert-Butoxycarbonyl)-N-((tert-
The oil 12 (14.8 g, 39.2 mmol, 1.0 equiv) was diluted with
CH2Cl2 (195 mL) and Et3N (16.6 mL, 119 mmol, 3.0 equiv) was
added afterwards at 4 ºC. Methanesulfonyl chloride (7.40 mL,
75.7 mmol, 1.93 equiv) was added dropwise. The reaction
mixture was stirred for 15 min at 4 ºC after which the ice-water
bath was removed and the reaction was stirred at rt for 1 h. The
reaction was monitored by TLC in 40% EtOAc in hexanes using
ninhydrin stain (product 9 has Rf = 0.65 and starting material 12
has Rf = 0.57). Saturated aqueous NH4Cl (150 mL) was added to
the reaction and the mixture was poured into a 2-L separatory
funnel. The combined organic layers were washed with saturated
aqueous NH4Cl (200 mL), saturated aqueous NaHCO3 (200 mL)
and saturated aqueous NaCl (200 mL). The organic layer was
dried over Na2SO4, filtered and concentrated under reduced
pressure. The resulting dark-orange oil containing ethyl N-(tert-
butoxycarbonyl)-N-((tert-butyldimethylsilyl)oxy)-O-
1
62.4% overall yield, 97:3 er). H NMR (400 MHz, CDCl3): δ
7.43 (s, 1H), 4.75–4.70 (m, 1H), 4.73 (dd, J = 5.2 Hz, 9.3 Hz,
1H), 3.85 (ddd, J = 8.0 Hz, 8.0 Hz, 8.0 Hz, 1H), 2.72–2.50 (m,
2H), 1.50 (s, 9H) ppm. 13C NMR (101 MHz, CDCl3): δ 174.5,
156.0, 83.2, 68.4, 59.4, 32.7, 28.0 ppm.
4.3. Determination of the enantiomeric ratio of (S)-N-Boc-5-
oxaproline
4.3.1. tert-Butyl (S)-3-((4-bromophenyl)carbamoyl)isoxazolidine-
2-carboxylate (S1)
To 1 (1.0 mg, 4.6 µmol, 1.0 equiv) in DMF (19 µL), N,N’-
diisopropylcarbodiimide (1.4 µL, 9.2 µmol, 2.0 equiv),
hydroxybenzotriazole (1.2 mg, 9.2 µmol, 2.0 equiv) were added
and the mixture stirred for 10 min at rt. To this, p-bromoaniline
(1.6 mg, 9.2 µmol, 2.0 equiv) was added and the reaction stirred
at rt for 3 h. The mixture was diluted with CH2Cl2 (3 mL) and
washed with H2O (3 mL), saturated aqueous NaCl (3 mL), dried
over Na2SO4, filtered and concentrated under reduced pressure.
The residue was diluted with a mixture of 9:1 hexanes:isopropyl
alcohol (250 µL) and separated by preparative achiral HPLC
(normal phase, 9:1 hexanes:isopropyl alcohol) on an Alltima
column to give amide derivate S1 (1.1 mg, 2.9 µmol, 97:3 er,
64% yield). HPLC: Chiralcel IA, hexanes/isopropyl alcohol 8:2,
20 °C, 30 µL, 1.0 mL/min, 210 nm, tR = 8.3 min (minor), tR
(methylsulfonyl)-L-homoserinate 9 was used in the next step
with no further purification (17.6 g, 38.6 mmol, 98.1% crude
1
yield, 83% purity by NMR). H NMR (400 MHz, CDCl3): δ 4.75
(dd, J = 8.8, 5.8 Hz, 1H), 4.58 (dd, J = 7.1, 5.1 Hz, 2H), 4.40 (m,
2H), 3.21 (s, 3H), 2.63–2.46 (m, 2H), 1.68 (s, 9H), 1.48 (d, J =
7.2 Hz, 3H), 1.21 (s, 9H), 0.38 (d, J = 3.1 Hz, 6H) ppm. 13C
NMR (101 MHz, CDCl3): δ 177.71, 162.29, 87.95, 70.59, 65.78,
33.02, 30.80, 29.85, 22.87, 0.03, 0.00 ppm.
4.2.4. (S)-2-(tert-Butyl)
dicarboxylate (4)
3-ethyl
(S)-isoxazolidine-2,3-
1
=15.95 min (major). H NMR (600 MHz, CDCl3) δ 8.51 (s, 1H),
7.62 – 7.40 (m, 3H), 4.93 – 4.74 (m, 1H), 4.14 (dddd, J = 8.4,
7.4, 4.4, 0.8 Hz, 1H), 3.93 – 3.67 (m, 1H), 2.66 (ddd, J = 8.8, 4.3,
3.4 Hz, 1H), 1.54 (s, 9H) ppm. 13C NMR (151 MHz, CDCl3) δ
168.43, 157.63, 136.31, 131.95, 121.41, 117.22, 84.05, 69.38,
62.71, 32.45, 28.08 ppm.
The compound 9 (17.6 g, 38.6 mmol, 1.0 equiv) was diluted
with THF (775 mL) tetrabutylammonium fluoride (1 M in THF,
58.0 mL, 58.0 mmol, 1.50 equiv) was added drpwise at 4 ºC. The
reaction mixture was stirred for 1 h at 4 ºC. The reaction was
monitored by TLC in 40% EtOAc in hexanes using ninhydrin as
stain (product 4 has Rf = 0.45 and stains yellow and starting
material 9 has Rf = 0.65 and stains brown). Saturated aqueous
NaHCO3 (120 mL) was added to the reaction and stirred for 10
min. The aqueous solution was diluted with Et2O (300 mL) and
the mixture was poured into a 2-L separatory funnel. The
aqueous layer was separated and washed with Et2O (3 x 150 mL).
The combined organic layers were washed with saturated
aqueous NaHCO3 (150 mL) and saturated aqueous NaCl (150
mL). The organic layer was dried over Na2SO4, filtered and
concentrated under reduced pressure. The resulting brown oil
Acknowledgments
This work was supported by the ETH Zürich and the Swiss
National Science Foundation (200020_150073). We thank the
LOC MS Service for analyses.
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