Evaluation of transition-state mimics in a superior BACE1 cleavage sequence as peptide-mimetic BACE1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.07.023

A superior substrate sequence for BACE1 containing transition-state mimics at the scissile site was evaluated as a protease inhibitor. Hydroxymethylcarbonyl (HMC) and hydroxyethylamine (HEA) isosteres were selected as the transition state mimics, and inco

Full text of DOI:10.1016/j.bmc.2015.07.023

Accepted Manuscript
Evaluation of transition-state mimics in a superior BACE1 cleavage sequence
as peptide-mimetic BACE1 inhibitors
Yasunao Hattori, Kazuya Kobayashi, Ayaka Deguchi, Yukie Nohara, Tomomi
Akiyama, Kenta Teruya, Akira Sanjoh, Atsushi Nakagawa, Eiki Yamashita,
Kenichi Akaji
PII:
S0968-0896(15)00606-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.07.023
BMC 12452
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 June 2015
10 July 2015
11 July 2015
Please cite this article as: Hattori, Y., Kobayashi, K., Deguchi, A., Nohara, Y., Akiyama, T., Teruya, K., Sanjoh,
A., Nakagawa, A., Yamashita, E., Akaji, K., Evaluation of transition-state mimics in a superior BACE1 cleavage
sequence as peptide-mimetic BACE1 inhibitors, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/
10.1016/j.bmc.2015.07.023
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Evaluation of transition-state mimics in a superior BACE1 cleavage
sequence as peptide-mimetic BACE1 inhibitors
Yasunao Hattoria, Kazuya Kobayashia, Ayaka Deguchia, Yukie Noharaa, Tomomi
Akiyamaa, Kenta Teruyab, Akira Sanjohc, Atsushi Nakagawad, Eiki Yamashitad, and
Kenichi Akaji*a
a Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Yamashina-ku,
Kyoto 607-8412, Japan
b
Department of Neurochemistry, Tohoku University Graduate School of Medicine,
Sendai, 980-8575 Japan
c R&D Center, Protein Wave Co. Nara 631-0006, Japan
d Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan
KEY WORDS
BACE 1, inhibitor, transition state mimic, hydroxyethylamine, hydroxymethylcarbonyl
*Corresponding author;
Kenichi Akaji, Ph.D.
Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Kyoto 607-8412,
Japan
Tel/Fax: +81-75-595-4635, E-mail: akaji@mb.kyoto-phu.ac.jp
1

Abstract
A superior substrate sequence for BACE1 containing transition-state mimics at the
scissile site was evaluated as a protease inhibitor. Hydroxymethylcarbonyl (HMC) and
hydroxyethylamine (HEA) isosteres were selected as the transition state mimics, and
incorporated into the scissile site of the superior sequence covering the P4 to P1’ sites
(Glu-Ile-Thi-Thi*Nva; *denotes the cleavage site). Isosteres having different absolute
configurations of the hydroxyl group were synthesized separately, and the effect of the
configuration was evaluated. Configuration of the hydroxyl group of each isostere
showed a marked effect on the inhibitory activity; anti-configuration to the scissile site
substituent had potent inhibitory activity in an HMC-type inhibitor, whereas
anti-configuration of HEA-type inhibitors showed no inhibitory activity. Structural
evaluations based on X-ray crystallographic analyses of recombinant BACE1 in
complex with each inhibitor provided insights into the protein-ligand interactions,
especially at the prime sites.
2

1. Introduction
Alzheimer’s disease (AD) is the most common cause of senile dementia and is
thought to be caused by the increased production and accumulation of amyloid peptide
(A) in the brain1. Most As consist of 40 or 42 amino acid residues and derive from a
type I transmembrane protein APP (amyloid precursor protein) by cleavage with two
aspartic proteases, - and -secretase. -Secretase, also called BACE1 (-site APP
cleaving enzyme 1), cleaves the luminal domain of APP at the N-terminus of A, which
is followed by -secretase cleavage at the C-terminus of A located in the membrane.
Thus, an inhibitor of BACE1 would prevent A production at the initial processing step,
and could be a promising anti-AD drug2-8. Inhibition of BACE1 is expected to be
clinically feasible, since BACE1 null mice proved viable with few phenotypical
abnormalities.
BACE1 is a transmembrane protease active at a mildly acidic pH of about 5.5,
which suggests that it would act in endosomes. The extracellular catalytic domain of
mature BACE1 takes a flap open conformation, whereas the flap is closed when the
substrate or transition-state mimic inhibitor is bound9. The flap-open conformation is
energetically stable and destabilization of this conformation is required for
substrate/inhibitor binding. This destabilization is compensated to some extent by
interactions with the substrate/inhibitor with the flap region covering Tyr71 to Lys75 of
BACE1.
In our previous paper10, we reported an efficient procedure for the preparation of a
large amount of recombinant BACE1 (rBACE1) corresponding to positions 47-454 of
an extracellular domain. The purified 45-kDa rBACE1 had kinetic parameters of
Km=5.5 M and kcat=1719 s-1. Using substrate peptide libraries to be digested with
rBACE1, it was found that a unique peptide containing unnatural amino acids,
Ile-Ser-Glu-Ile-Thi-Thi*Nva-Ala-Glu-Phe-Arg-His-NH2 (*denotes the cleavage site)
could be cleaved 10 times faster than a Swedish mutant substrate. Taking advantage of
rBACE1 and the superior cleavage sequence, we incorporated transition-state mimics at
the cleavage site of this superior sequence, and evaluated the inhibitory activity for
rBACE1. Two types of the most widely used isosters, hydroxymethylcarbony (HMC)11
and hydroxyethylamine (HEA)12, were selected as the transition-state mimics.
Inhibitory activities of synthesized inhibitors varied significantly depending on the
stereo-structures of transition-state mimics. To evaluate the interactions of these
inhibitors with rBACE1, structure-based analyses of rBACE1 in complex with each
inhibitor were conducted by X-ray crystal analyses.
3

2. Results and Discussions
2.1. Chemistry
Structures of synthesized HMC- and HEA-type inhibitors are summarized in Figure
1. In both type inhibitors, each isostere with a specific conformation of the key hydroxyl
group was separately prepared and incorporated in the superior substrate sequence
covering the P4 to P1’ sites, (R)- or (S)-1 and (R)- or (S)-2. In addition, several analogs
containing different P1 site substituents were also synthesized to evaluate additional
effects of side chain structures at the scissile site.
Figure 1
In HMC-type inhibitors, (R)- and (S)-hydroxyl inhibitors with linear or cyclic
substituents at the P1 site were synthesized, respectively. Thus, four different amino
acids, Thi (R=2-thienyl), Leu (R=isopropyl), Cha (R=cyclohexyl), and Phe (R=phenyl),
were selected as the starting amino acids. Key intermediate, -hydroxyl carboxylic acid,
was synthesized according to the route shown in Scheme 1. Starting from commercially
available Fmoc amino acids, a sulfur ylide derivative 8 was prepared by
14
HATU-mediated coupling13 with sulfonium salt 7. Oxidation of 8 with Oxone®
yielded 9 which was then reduced with NaBH4 to give the desired -hydroxyl
carboxylic acid as a diastereomer mixture. Each diastereomer was easily separated by
preparative HPLC to give (R)- or (S)-hydroxyl derivatives 10 to 13 (Figure S1).
1
Absolute configuration of the purified diasteromer was determined by H NMR using a
chiral anisotropic reagent, phenylglycine methyl ester (PGME), as reported by Kusumi
et al15. Each purified diastereomer was coupled with (S)- and (R)-phenyl glycine methyl
1
ether and H NMR was recorded. Signals of -proton and P1 site side chain protons
4

were shifted by the effects of a benzene ring of (S)- or (R)-PGME. The positive or
negative shifts (∆=S−R) were clearly observed as expected from the literature to
determine each absolute configuration (Figure S2).
Scheme 1
Using the (R)-hydroxymethyl carboxylic acid thus obtained, HMC-type inhibitors
were synthesized by conventional Fmoc-based solid phase peptide synthesis (Scheme 2).
Fmoc-Nva-OH was coupled with 2-chlorotrityl chloride resin and the Fmoc group was
removed by 20% piperidine. Fmoc-protected (R)-hydroxyl carboxylic acid R-10, 11, 12,
or 13 was then condensed by an EDCI/HOBt-mediated reaction. Fmoc-Thi-OH and
Fmoc-Ile-OH were then condensed successively by the combination of
piperidine-mediated Fmoc-deprotection and DIC/HOBt-mediated coupling. N-terminal
Boc-Glu(Ot-Bu)-OH was similarly incorporated and the resulting resin was treated with
TFA/H2O/thioanisole. The crude product was purified by preparative HPLC to give
R-14, 15, 16, or 17, each having a single major peak on analytical HPLC (Figure S3).
An HMC-type inhibitor having (S)-hydroxyl configuration containing 2-thienyl S-14,
isopropyl S-15, cyclohexyl S-16, or phenyl S-17 group at the P1 site was similarly
synthesized and purified.
5

Scheme 2
In the synthesis of HEA-type inhibitors, the isostere hydroxyl group was introduced
stereo-selectively, since the above PGME method could not be applied to determine the
hydroxyl configuration in the HEA scaffold. The HEA backbone was constructed by
reductiveamination of -hydroxyl -amino aldehyde with a norvaline derivative
(Scheme 3). The -hydroxyl group in the -hydroxyl -amino aldehyde was
stereo-selectively prepared according to the published procedure. The (S)-hydroxyl
group of an olefinic 1, 2-amino alcohol derivative was constructed by stereo-selective
vinylation16 of amino aldehydes, and the Evans17 aldol reaction was employed to
construct the (R)-hydroxyl group.
Scheme 3
An HEA inhibitor containing an (S)-hydroxyl group was synthesized according to
the route shown in Scheme 4. A Z-protected amino aldehyde derivative 19 was prepared
6

from Z-protected amino acid 18 through the corresponding Weinreb-amide. The
aldehyde was reacted with 2-trimethylsilylethyliden triphenylphosphorane according to
the published procedure16 followed by protection as an acetonide to give 20 as a single
diastereomer through Cram chelation control. In preparation of the corresponding
2-thienyl derivative 21, however, the selectivity and isolation yield were insufficient to
yield diastereomixtures containing the desired product as a major one. Thus, a simple
Grignard reaction using vinylmagnesium chloride was employed for the synthesisis of
21. Undesired diastereomers could be epimerized to the desired ones after conversion to
23 by treatment with K2CO3.18 Dihydroxylation of the olefin bond of 20 or 21 and
NaIO4 mediated oxidation yielded aldehyde 22 or 23. Reductive amination in the
presence of Lewis acid followed by catalytic hydrogenation gave the desired HEA
isostere 24 or 25. Fmoc-Thi-OH, Fmoc-Ile-OH, and N-terminal Boc-Glu(Ot-Bu)-OH
were then condensed successively by the combination of EDCI-mediated coupling and
Et2NH-mediated Fmoc-deprotection. The product was finally treated with
TFA/H2O/thioanisole, and the crude product was purified by preparative HPLC to give
a homogeneous product 26 or 27 (Figure S3).
Scheme 4
An HEA inhibitor containing a (R)-hydroxyl group was synthesized using the Evans
aldol reaction as shown in Scheme 5. Evans asymmetric auxiliary,
(S)-4-isopropyl-2-oxazolidinone, was introduced to 3-substituted carboxylic acid 28 or
29 through acid anhydride to yield 30 or 3119. 2-Propenal was reacted with Z-enolate of
30 or 31 to yield the desired (R)-hydroxyl allyl alcohol derivative 32 or 33 as expected.
Removal of the Evans asymmetric auxiliary and subsequent hydrolysis yielded
7

carboxylic acid 34 or 35. The key intermediate, olefinic 1, 2-amino alcohol 36 or 37 was
obtained by Curtius rearrangement followed by protection with the Z group of the
resulting amino group. A (R)-hydroxyl HEA isostere intermediate was prepared by
acetonide formation and following reductive amination with a norvaline derivative as
above. Peptide chain elongation to the isoster intermediate and deprotection gave the
desired (R)-HEA inhibitor R-26 or R-27 without difficulty.
Scheme 5
In the synthesis of a substituted HEA-type inhibitor having methyl group at the
-position of the isostere hydroxyl group, coupling with H-Nva-Ot-Bu with an
intermediate ketone by reductiveamination was used (Scheme 6). Previously prepared
-(S)-hydroxyl aldehyde 22 was first reacted with MeMgCl followed by oxidation to
yield methyl ketone 38. Imination of 38 with H-Nva-Ot-Bu proceeded in the presence
of 5 equivalent of Ti(Oi-Pr)4 and following reduction with NaBH3CN to give the desired
product 39 with 74% yield. No imination reaction was observed in the absence of the
Lewis acid, and excess or less to 5 equivalent of the Lewis acid lowered the yield. The
product was an inseparable diastereomixtures of 3:1. Using this mixture, a substituted
HEA-type inhibitor S-40 was synthesized by successive coupling of the corresponding
amino acid derivatives and following deprotection as above. The two diastereomers of
inhibitor S-40 derived from the substituted methyl group could be separated on HPLC
(Figure S4) at this stage, and each product was used for evaluation of the inhibitory
activity without determining the absolute configuration. Starting from -(R)-hydroxyl
aldehyde, an epimer of 22, another substituted HEA-type inhibitor R-40 having the
8

opposite configuration of isostere hydroxyl group was similarly prepared.
Diastereomers of the synhtesized inhibitor having an -(R)-hydroxyl group could not be
separated on HPLC (Figure S4), and used for the evaluation of the inhibitory activity as
a diasteromixture.
Scheme 6
An N-Methylated HEA-type inhibitor was synthesized according to the route shown
in Scheme 7. Reductiveaminated intermediate synthesized above was methylated by
reaction with paraformaldehyde and NaBH3CN to yield N-methylated derivative 41
without difficulties. The product was then used as the starting compound for the
N-methylated HEA-type inhibitor S-42. The desired product was purified by preparative
HPLC as above. Another N-methylated inhibitor R-42 containing an opposite hydroxyl
configuration was similarly synthesized.
Scheme 7
2.2. BACE1 inhibitory activity
BACE1 inhibitory activities of the synthesized inhibitors were evaluated using
rBACE110 with the kinetic parameters of Km=5.5 M and kcat=1719 sec-1. The protease
9

was incubated with an eicosa peptide amide (IKTEEISEVNL*DAEFRHDSG-NH2; *
shows the site of cleavage by BACE1) corresponding to the initial cleavage site of
Swedish (SW) mutant APP in the presence of each inhibitor. The mixture was subjected
to an analytical HPLC and IC50 value calculated from a decrease in the substrate at
various inhibitor concentrations was used to evaluate the inhibitory activity (Table 1). A
typical sigmoidal curve used to estimate the IC50 value is shown in Figure S5.
Table 1
In HMC-type inhibitors, (R)-isomer, an anti-configuration of the isostere hydroxyl
group to the scissile site side-chain substituent, showed inhibitory activity (R-14, R-15,
R-16, and R-17) whereas the (S)-isomer showed no (S-15; IC50>1 mM) or weak
10

inhibitory activity (S-14, S-16, and S-17). As the P1 site structure, the aromatic structure
(R-14 and R-17) was better than aliphatic structure (R-15 and R-16), and an inhibitor
containing the superior sequence including P1 2-thienyl (R-14) was the most potent
inhibitor as expected (IC50=26 M). (S)-isomers having ring structure at the P1 site
(S-14, S-16, and S-17) showed weak, but distinct, inhibitory activities (IC50=480~870
M), which suggest that the P1 site structure may bring some conformational change
taking the (S)-hydroxyl group to an accessible distance from the active site Asp residue
of BACE1.
In contrast, (R)-isomer of HEA-type inhibitors, a syn-configuration of the isostere
hydroxyl group to the scissile site side-chain substituent, showed inhibitory activity
(R-27 and R-26), and no inhibitory activity (IC50>1 mM) was observed for the
(S)-isomer (S-27 and S-26). An inhibitor containing the superior sequence including P1
2-thienyl showed 8 times higher inhibitory activity (R-27, IC50=9.4 M) than the P1
Leu-type inhibitor (R-26, IC50=72 M). HEA-type inhibitor R-27 containing the
superior sequence had higher activity than the corresponding HMC-type inhibitor 14,
which suggests that the HEA isostere is more suitable than the HMC isostere to
integrate into the superior substrate sequence. Introduction of a methyl group at the
-position of the isostere hydroxyl group abolished the inhibitory activity (R- and S-40),
which was restored to some extent by the introduction at the -position of the isostere
hydroxyl group (R-42, IC50=680 M). The result suggests that interactions of HEA-type
inhibitors with BACE1 may be significantly influenced by the prime-site substituent.
2.3. Evaluation of the interactions
The results obtained above strongly suggest that the necessary conformation of the
hydroxyl group for potent inhibition depends on the isoster backbone, as well as the
prime-site substituent. To clarify the differences by evaluating each interaction with
BACE1, the structure of rBACE1 in complex with the inhibitor was revealed by X-ray
crystallography. Thus, co-crystals of inhibitor R-14, R-27, and R-26 with rBACE1 were
prepared and analyzed. Structures of rBACE1 in complex with inhibitors R-14, R-27,
and R-26 were refined to a resolutions of 2.80 Å, 3.20 Å, and 2.80 Å, respectively (PDB
code 4TRY, 4TRZ, and 4TRW). The data obtained were summarized in Table 2.
11

Table 2
The X-ray crystallography revealed that the overall structures were similarly folded
on these inhibitors (Figure 2-a). Each inhibitor was located in the active site cleft taking
a linear form. The P1 and P3 site substituents directed to rBACE1, whereas the P2 and P4
site substituents directed toward outside (Figure S6). The isostere hydroxyl group and
its -positon carbonyl group of the HMC isostere R-14 (IC50=26 M), and the
-position nitrogen of HEA R-27 (IC50=9.4 M) and R-26 (IC50=72 M) were in a
hydrogen bond distance from the active center Asp32 and/or Asp228 of rBACE1
(Figure 2-b and S6). The results indicate that the superior substrate sequence contributes
to underlying interactions of rBACE1 with the isostere functional groups. Based on
these X-ray crystallographic analyses, the interactions of less potent (S)-hydroxyl
HMC-type inhibitors, containing an opposite configuration hydroxyl group, were
estimated using a package for molecular structure analyses, MOE (Molecular Operating
Environment). The simulation gave two stable conformation of S-14 (IC50=870 M);
one of which could interact with Asp32 and 228 like the potent inhibitor R-14 (IC50=26
M), but the other could only interact with Asp32 (Figure S7-a). The predicted low
12

contribution of the hydroxyl group could explain the lower inhibitory activity of
(S)-HMC-type inhibitors. The same MOE simulation was also conducted with an
(S)-HEA-type inhibitor S-26 which showed no inhibitory activity. No predicted stable
structure for S-26 could interact with both active center Asp32 and 228 at their hydroxyl
or -site nitrogen atoms like as the active HEA-type inhibitor R-26 (IC50=72 M)
(Figure S7-b). These simulation studies support the essential role of the hydroxyl group
of the HEA isosteres incorporated into the superior sequence.
Figure 2
Among the substituents directed toward outside of rBACE1, side-chain carboxylic
acid and -carbonyl of P4 site Glu in both HMC- and HEA-type inhibitors form
hydrogen bonds with side-chain amide of Asn233 and Gln73 of rBACE1, respectively
(Figure 3-a). Thus, the P4 site Glu residue in the superior sequence functions as a kind
of hydrogen bond mediator to locate the inhibitor in the flap-closed structure of BACE1.
These interactions make it possible to effectively locate the neighboring inside-directed
P3 site substituents in a hydrophobic S3 pocket formed by Ile110 and a turn structure
formed by Ser10 to Gln12 of rBACE1 (Figure 3-b).
13

Figure 3
The P1 site substituents are also directed toward a hydrophobic S1 pocket formed by
Tyr71, Phe108, and Ile118. Although the conformations are nearly the same in R-14,
R-27, and R-26 (Figure 4-a), subtle differences are observed between aliphatic
isopropyl and aromatic 2-thienyl substituents. The planer 2-thienyl group of the
HEA-type inhibitor R-27 (IC50=9.4 M) was slightly twisted compared with inhibitors
containing the aliphatic isoproyl substituent R-26 (IC50=72 M). This slight change was
transferred to the conformations of neighboring sites, which were forced to add several
interactions of inhibitor R-27 with BACE1. Thus, distances between the prime-site
carboxyl of Nva and side-cahin hydroxyl group of Thr231 (3.024 Å) and that between
the nonprime-site P2-Thi sulfur atom and side-chain hydroxyl group of Thr72 at the flap
region (3.896 Å), as well as the N-terminus carboxyl of R-27 and side-cahin amide of
Asn233 (2.959 Å) fell into the range of the hydrogen-bond distance (Figure 4-b). These
slight changes, as well as the hydrophobic interactions at P1 and P3 sites make the
HEA-type inhibitor R-27 a suitable isostere to keep rBACE1 in the flap-closed
conformation20. The important contribution at the prime site interactions was also
supported by the results that introduction of a methyl group at the prime site abolished
the inhibitory activity (compound R-26 vs. R-40) probably due to the interception of
interactions at these sites, and the activity was recovered to some extent by shifting the
substitution position by one-carbon far from the essential hydroxyl group (compound
R-40 vs. R-42).
14

Figure 4
3. Conclusion
Incorporation of transition state mimics for an aspartic protease such as
hydroxymethylcarbonyl (HMC)- or hydroxyethylamine (HEA)-isostere into a superior
substrate sequence for BACE1 was found to be effective as an initial step for the
development of a BACE1 inhibitor. Opposite absolute configurations at the essential
hydroxyl group in these isosteres are necessary to bring the isosters in hydrogen bond
distances of active center Asp32 and Asp228 of rBACE1. Incorporation of the HEA
isostere gave a more potent inhibitor than the incorporation of the HMC isostere. X-ray
crystallographic analyses revealed that both inhibitors had similar modes of interaction
with rBACE1 at non-prime sites, especially at P4 and P3 sites. By P4 site interactions,
both inhibitors are forced to locate at the active center cleft of rBACE1, taking a
flap-closed conformation. Hydrophobic side-chains at P3 and P1 sites in HMC- and
HEA-type inhibitors interacted similarly with the corresponding S3 and S1 pockets.
Subtle changes in interactions at the P1 site between the aliphatic isopropyl and aromatic
substituents makes the inhibitor R-27 take a twisted conformation at the prime site,
resulting in improved interactions with rBACE1. In addition, introduction of a methyl
group at the -position of the HEA-type inhibitors completely abolished the inhibitory
activity. Taken together, it was strongly suggested that the inhibitory activity of the
HEA-type inhibitors is significantly affected by interactions at the prime site.
Replacement of the Nva residues with small aromatic substituents to enhance the
interactions at the prime site is now underway.
15

4. Experimental Section
4.1. General
Melting point was determined with a Yanaco apparatus and was uncorrected.
Analytical TLC was performed on silica gel (Silica gel 70 F254 TLC Plate-Wako).
Column chromatography was carried out on Wakogel® C-200E (particle size, 75-150
m) or Wakogel® C-300E (particle size, 45-75 m). 1H NMR spectra (400 or 300 MHz,
tetramethylsilane at  = 0.00 ppm, CH3COCH3  = 2.04 ppm, or CH3OH  = 3.35 ppm
13
as an internal standard) and C NMR spectra (100 or 75 MHz, CDCl3 at  = 77.0 ppm,
CD3COCD3  = 29.3 ppm, or CD3OD  = 49.3 ppm as an internal standard) were
measured with an Agilent UNITY INOVA 400 NB or Bruker AV-300 FT-NMR
spectrometers. The coupling constants were given in Hz. Mass spectra was obtained on
JEOL JMS-SX 102A (FAB) or JEOL JMS-GCmate II (EI and CI) mass spectrometers.
Optical rotations were determined with a HORIBA SEPA-300 polarimeter.
Preparative HPLC was performed using a COSMOSIL 5C18-ARII column (20 ×
250 mm) with a linear gradient of CH3CN in 0.1% aqueous TFA at a flow rate of 5.0
mL/min on a HITACHI ELITE LaChrom system (OD, 220 nm). For analytical HPLC, a
COSMOSIL 5C18-ARII column (4.6 × 150 mm) was employed with a linear gradient
of CH3CN in 0.1% aqueous TFA at a flow rate of 0.9 mL/min on a HITACHI ELITE
LaChrom system (OD, 230 nm).
4.1.1.
(3S)-3-([{(9H-Fluoren-9-yl)methoxy}carbonyl]amino)-2-hydroxy-4-(thiophen-2-yl)
butanoic acid R- and S-10
To a solution of bromide salt 7 (344 mg, 1.65 mmol) was added HATU (531 mg,
1.40 mmol), DIPEA (0.66 mL, 3.8 mmol), and Fmoc-Thi-OH (500 mg, 1.27 mmol) in
o
DMF (4 mL) at 0 C. After stirring for 14 h at room temperature, the mixture was
o
poured into saturated aqueous NH4Cl at 0 C and the whole was extracted with EtOAc.
The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated.
The residue was roughly purified by silica gel column chromatography (Ether/Acetone
= 2:1). This compound was immediately used for the next step without further
purification. The resultant residue was dissolved in DMF/H2O (1:1, 10 mL). Oxone®
(2.13 g, 3.46 mmol) was added to the mixture. After stirring for 30 min at room
temperature, the reaction was quenched with 1 mol/L HCl, and the whole was extracted
with EtOAc. The organic layer was washed with brine, dried over Na2SO4, filtered, and
concentrated. This compound was immediately used for the next step without
purification. The residue was dissolved in MeOH (5 mL) and NaBH4 (378 mg, 10.0
16

o
mmol) was added at 0 C. The mixture was stirred for 1.5 h at room temperature. The
reaction was quenched with 1 mol/L HCl, and the whole was extracted with EtOAc. The
organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The
residue was purified by HPLC to give R-10 (91 mg, 23%) and S-10 (79 mg, 20%).
R-10: []28 – 62 (c 1.0, MeOH); 1H NMR (400 MHz, CD3COCD3):  = 7.81 (d, J
D
= 7.6 Hz, 2H), 7.64-7.61 (m, 2H), 7.40 (t, J = 7.6 Hz, 2H), 7.33-7.26 (m, 3H), 6.97-6.92
(m, 2H), 6.44 (d, J = 9.2 Hz, 1H), 4.37-4.31 (m, 1H), 4.27 (d, J = 2.4 Hz, 2H), 4.24-4.15
(m, 2H), 3.27 (dd, J = 14.6, 7.4 Hz, 1H), 3.13 (dd, J = 14.6, 7.6 Hz, 1H), 2.02-1.99 (m,
2H); 13C NMR (100 MHz, CD3COCD3):  = 173.6, 156.2, 144.54, 144.49, 141.5, 140.8,
128.0, 127.44, 127.40, 127.2, 126.6, 125.7, 124.5, 120.2, 70.8, 66.6, 55.8, 47.4, 32.1;
HRMS (FAB) Calcd. For C23H22NO5S [M+H]+: 424.1219. Found: 424.1212.
S-10: []28D 13 (c 1.0 , MeOH); 1H NMR (400 MHz, CD3COCD3):  = 7.80 (d, J =
7.6 Hz, 2H), 7.62-7.61 (m, 2H), 7.35 (t, J = 7.4 Hz, 2H), 7.26 (t, J = 7.4 Hz, 2H), 7.19
(d, J = 4.4 Hz, 1H), 6.90-6.85 (m, 2H), 6.64 (brd, J = 9.2 Hz, 1H), 4.33 (d, J = 4.0 Hz,
1H), 4.26 (dd, J = 8.4, 5.6 Hz, 2H), 4.18-4.11 (m, 2H), 3.16 (dd, J = 15.2, 10.4 Hz, 1H),
3.04 (dd, J = 15.0, 3.4 Hz, 1H), 2.03-1.99 (m, 2H); 13C NMR (100 MHz, CD3COCD3):
 = 173.6, 156.3, 144.51, 144.50, 141.9, 141.1, 127.9, 127.4, 127.0, 126.4, 125.7, 125.6,
124.3, 120.2, 72.8, 66.5, 56.1, 56.0, 47.4; HRMS (FAB) Calcd. For C23H22NO5S
[M+H]+: 424.1219. Found: 424.1214.
4.1.2.
(3S)-3-([{(9H-Fluoren-9-yl)methoxy}carbonyl]amino)-2-hydroxy-5-methylhexanoic
acid R- and S-11
Title compounds were similarly prepared starting from Fmoc-Leu-OH as
above.
R-11: []28D 24 (c 0.10, MeOH); 1H NMR (400 MHz, CD3COCD3):  = 7.84 (d, J
= 7.6 Hz, 2H), 7.70-7.65 (m, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.32-7.29 (m, 2H), 4.28 (d, J
= 6.8 Hz, 2H), 4.20-4.17 (m, 3H), 2.06 (dd, J = 6.8, 4.4 Hz, 1H), 1.69-1.60 (m, 2H),
13
1.47-1.31 (m, 2H), 0.94 (d, J = 6.4 Hz, 6H); C NMR (100 MHz, CDCl3):  = 175.0,
156.9, 143.8, 143.4, 141.3, 141.2, 127.7, 127.0, 125.03, 124.99, 119.9, 72.0, 67.2, 51.6,
47.0, 41.1, 24.6, 23.0, 21.9; HRMS (FAB) Calcd. For C22H25NNaO5 [M+Na]+:
406.1630. Found: 406.1636.
S-11: []28 14 (c 0.10, MeOH); H NMR (400 MHz, CD3OD):  = 7.81 (d, J =
1
D
7.2 Hz, 2H), 7.70-7.69 (m, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.35-7.31 (m, 2H), 4.43 (dd, J
= 10.8, 7.2 Hz, 1H), 4.35 (dd, J = 10.8, 6.8 Hz, 1H), 4.25-4.22 (m, 2H), 4.11-4.08 (m,
1H), 1.66-1.61 (m, 2H), 1.17-1.11 (m, 1H), 0.96 (d, J = 6.4 Hz, 3H), 0.92 (d, J = 6.4 Hz,
17

3H); 13C NMR (100 MHz, CD3OD):  = 176.0, 158.8, 145.7, 145.5, 142.9, 129.0,
128.42, 128.40, 126.5, 121.2, 74.7, 68.0, 53.5, 48.8, 39.0, 26.0, 24.4, 22.0; HRMS
(FAB) Calcd. For C22H25NNaO5 [M+Na]+: 406.1630. Found: 406.1636.
4.1.3.
(3S)-3-([{(9H-Fluoren-9-yl)methoxy}carbonyl]amino)-4-cyclohexyl-2-hydroxybuta
noic acid R- and S-12
Title compounds were similarly prepared starting from Fmoc-Cha-OH as
above.
1
R-12: []28 26.6 (c 1.01, CHCl3); H NMR (400 MHz, CDCl3):  = 7.71 (d, J =
D
7.6 Hz, 2H), 7.61 (m, 1H), 7.50 (d, J = 6.8 Hz, 2H), 7.35 (t, J = 7.4 Hz, 2H), 7.28-7.25
(m, 2H), 6.47 (brs, 1H), 5.23 (d, J = 9.2 Hz, 1H), 4.33 (d, J = 6.0 Hz, 2H), 4.23-4.14 (m,
4H), 1.79 (d, J = 12.0 Hz, 1H), 1.65-1.45 (m, 5H), 1.26-1.13 (m, 4H), 0.96-0.83 (m,
2H); 13C NMR (100 MHz, CDCl3):  = 175.3, 156.9, 143.5, 141.25, 141.20, 127.7,
127.0, 125.1, 125.0, 119.9, 72.1, 67.3, 51.0, 47.0, 39.6, 34.1, 33.6, 32.6, 26.4, 26.2,
26.0; HRMS (FAB) Calcd. For C25H29NNaO5 [M+Na]+: 446.1943. Found: 446.1940.
1
S-12: []28 9.88 (c 1.03, CHCl3); H NMR (400 MHz, CDCl3):  = 7.75 (d, J =
D
7.6 Hz, 2H), 7.55 (d, J = 6.4 Hz, 2H), 7.39 (t, J = 7.2 Hz, 2H), 7.29 (t, J = 7.2 Hz, 2H),
5.12 (m, 1H), 4.41 (m, 2H), 4.32 (brs, 1H), 4.20-4.19 (m, 1H), 4.10 (m, 1H), 1.78-1.75
(d, J = 12.0 Hz, 1H), 1.64-1.55 (m, 5H), 1.25-1.13 (m, 5H), 0.93-0.77 (m, 2H); 13C
NMR (100 MHz, CDCl3):  = 175.6, 157.3, 143.6, 141.3, 127.8, 127.1, 127.0, 125.0,
124.9, 120.0, 74.1, 67.1, 51.8, 47.1, 36.7, 34,0, 32.1, 26.4, 26.2, 26.0; HRMS (FAB)
Calcd. For C25H29NNaO5 [M+Na]+: 446.1943. Found: 446.1946.
4.1.4.
(3S)-3-([{(9H-Fluoren-9-yl)methoxy}carbonyl]amino)-2-hydroxy-4-phenylbutanoic
acid R-and S-13
Title compounds were similarly prepared starting from Fmoc-Phe-OH as
above.
R-13: []28D 51.3 (c 0.785, CHCl3); 1H NMR (400 MHz, CD3COCD3):  = 7.80 (d,
J = 7.6 Hz, 2H), 7.61 (t, J = 7.0 Hz, 2H), 7.37-7.14 (m, 9H), 6.38 (brd, J = 9.2 Hz, 1H),
4.31-4.29 (m, 1H), 4.21-4.10 (m, 4H), 2.99 (dd, J = 13.2, 7.6 Hz, 1H), 2.89 (dd, J = 13.2,
13
7.6 Hz, 1H), 2.03-1.99 (m, 1H); C NMR (100 MHz, CD3COCD3):  = 174.0, 156.1,
144.53, 144.47, 141.5, 138.9, 129.7, 128.7, 127.9, 127.42, 127.38, 126.6, 125.7, 120.2,
70.8, 66.6, 55.8, 47.4, 38.1; HRMS (FAB) Calcd. For C25H24NO5 [M+H]+: 418.1654.
Found: 418.1649.
18

S-13: []28D 168 (c 0.565, CHCl3); 1H NMR (400 MHz, CD3COCD3):  = 7.79 (d,
J = 7.6 Hz, 2H), 7.57 (d, J = 7.6 Hz, 2H), 7.35 (t, J = 7.4 Hz, 2H), 7.29-7.19 (m, 6H),
7.13-7.10 (m, 1H), 6.58 (brd, J = 8.8 Hz, 1H), 4.34-4.29 (m, 2H), 4.17-4.08 (m, 3H),
13
2.92-2.81 (m, 2H), 2.03-2.00 (m, 1H); C NMR (100 MHz, CD3COCD3):  = 173.6,
156.2, 144.53, 144.47, 141.5, 139.2, 129.6, 127.9, 127.37, 127.35, 126.5, 125.7, 125.6,
120.2, 73.1, 66.5, 56.0, 47.4, 35.3; HRMS (FAB) Calcd. For C25H24NO5 [M+H]+:
418.1654. Found: 418.1651.
4.1.5. Glu-Ile-Thi-(R)HMC(2-thienyl)-Nva R-14
After agitating 2-chlorotrityl resin (500 mg) in DMF for 20 min, Fmoc-Nva-OH
(407 mg, 1.20 mmol) and DIPEA (0.21 mL, 1.20 mmol) was added with DMF. The
mixture was agitated for 2 h. After the resin was washed with DMF, MeOH and DIPEA
(0.41 mL, 2.40 mmol) in DMF was added to the resin. After agitating for 30 min, the
resin was washed with DMF, CHCl3, and DMF to give protected Nva on resin (575 mg).
After washed with DMF, piperidine (20%) in DMF was added to the resin. After
agitating for 20 min, the deprotected resin was washed with DMF. After agitating in
DMF, CHCl3, and DMF for 20 min, R-10 (34.9 mg, 0.0827 mmol), HOBt•H2O (25.3
mg, 0.165 mmol), EDCI•HCl (31.7 mg, 0.165 mmol), and DIPEA (28 L, 0.165 mmol)
in DMF were added to this resin (114 mg, 0.0532 mmol). The mixture was agitated for
18 h. After the resin was washed with DMF, piperidine (20%) in DMF was added to the
resin. After agitating for 20 min, the deprotected resin was washed with DMF. To this
resin were added Fmoc-AA-OH (0.0827 mmol), HOBt•H2O (25.2 mg, 0.165 mmol),
DIC (26 L, 0.165 mmol), and DIPEA (28 L, 0.099 mmol) in DMF. The mixture was
agitated for 2 h. After the resin was washed with DMF, piperidine (20%) in DMF was
added to the resin. After agitating for 20 min, the deprotected resin was washed with
DMF. Completion of each coupling reaction was ascertained using the Kaiser ninhydrin
test. Finally, coupling of Boc-Glu(Ot-Bu)-OH (25.1 mg, 0.0827 mmol) was carried out
with HOBt•H2O (25.2 mg, 0.165 mmol), DIC (26 L, 0.165 mmol), and DIPEA (28 L,
0.165 mmol) in DMF. The mixture was agitated for 2 h, and the resin was washed with
DMF. The resin was dried under reduced pressure. TFA/H2O/thioanisole (95:2.5:2.5)
was added to the resin and the mixture was agitated for 2 h. The mixture was filtered,
and solvents of the filtrate were removed under reduced pressure. Ether was added to
the residue to form the precipitate. The precipitate was washed with ether and purified
by preparative HPLC to yield R-14 (28.0 mg, 76%). 1H NMR (400 MHz, CD3OD):  =
7.24 (dd, J = 5.0, 1.4 Hz, 1H), 7.20 (dd, J = 5.0, 1.4 Hz, 1H), 6.97-6.90 (m, 4H), 4.72
(dd, J = 9.6, 4.4 Hz, 1H), 4.50 (dd, J = 7.5, 2.1 Hz, 1H), 4.42 (d, J = 7.8, 5.0 Hz, 1H),
19

4.33 (d, J = 7.6 Hz, 1H), 4.13 (d, J = 2.4 Hz, 1H), 3.97 (d, J = 6.4 Hz, 1H), 3.31 (d, J =
4.0 Hz, 1H), 3.21 (dd, J = 14.8, 7.6 Hz, 1H), 3.12 (dd, J = 15.2, 9.6 Hz, 1H), 3.05 (dd, J
= 14.6, 7.4 Hz, 1H), 2.50-2.40 (m, 2H), 2.10 (d, J = 3.2 Hz, 1H), 2.06 (d, J = 8.4 Hz,
1H), 1.91-1.71 (m, 3H), 1.58-1.52 (m, 1H), 1.45-1.37 (m, 2H), 1.20-1.13 (m, 1H),
0.99-0.91 (m, 9H); HRMS (FAB) Calcd. For C31H46N5O9S2 [M+H]+: 696.2737. Found:
696.2740.
4.1.6. Glu-Ile-Thi-(R)HMC(isopropyl)-Nva R-15
1H NMR (400 MHz, CD3OD):  = 7.76 (brd, J = 9.2 Hz, 1H), 7.20 (dd, J = 5.0, 1.0
Hz, 1H), 6.95 (m, 1H), 6.92 (dd, J = 5.2, 3.6 Hz, 1H), 4.69 (dd, J = 9.6, 4.4 Hz, 1H),
4.43-4.40 (m, 2H), 4.35 (d, J = 7.6 Hz, 1H), 4.06 (d, J = 2.4 Hz, 1H), 3.97 (t, J = 6.4 Hz,
1H), 3.31 (d, J = 4.4 Hz, 1H), 3.13 (dd, J = 15.0, 9.6 Hz, 1H), 2.45-2.44 (m, 2H), 2.08
(q, J = 7.1 Hz, 2H), 1.96-1.74 (m, 3H), 1.63-1.53 (m, 3H), 1.46-1.39 (m, 3H), 1.21-1.14
(m, 1H), 1.00-0.91 (m, 15H); HRMS (FAB) Calcd. For C30H50N5O9S [M+H]+: 656.3329.
Found: 656.3326.
4.1.7. Glu-Ile-Thi-(R)HMC(cyclohexyl)-Nva R-16
1H NMR (400 MHz, CD3OD):  = 7.20 (dd, J = 5.0, 1.4 Hz, 1H), 6.95-6.94 (m, 1H),
6.91 (dd, J = 5.0, 3.4 Hz, 1H), 4.71 (dd, J = 9.6, 4.4 Hz, 1H), 4.44-4.40 (m, 2H), 4.36 (d,
J = 8.0 Hz, 1H), 4.05 (d, J = 2.4 Hz, 1H), 3.99 (t, J = 6.4 Hz, 1H), 3.30 (d, J = 4.4 Hz,
1H), 3.12 (dd, J = 15.2, 10.0 Hz, 1H), 2.44 (dd, J = 13.4, 7.8 Hz, 2H), 2.10 (dd, J = 14.4,
7.6 Hz, 2H), 1.92-1.71 (m, 9H), 1.60-1.38 (m, 6H), 1.33-1.14 (m, 5H), 1.02-0.92 (m,
9H); HRMS (FAB) Calcd. For C33H54N5O9S [M+H]+: 696.3642. Found: 696.3637.
4.1.8. Glu-Ile-Thi-(R)HMC(phenyl)-Nva R-17
1H NMR (400 MHz, CD3OD):  = 7.32-7.28 (m, 4H), 7.23-7.21 (m, 1H), 7.19 (dd, J
= 4.6, 1.8 Hz, 1H), 6.91-6.89 (m, 2H), 4.72 (dd, J = 9.6, 4.4 Hz, 1H), 4.51 (td, J = 7.6,
1.9 Hz, 1H), 4.41-4.37 (m, 1H), 4.34 (d, J = 8.0 Hz, 1H), 4.05 (d, J = 2.0 Hz, 1H),
4.01-3.98 (m, 1H), 3.30 (dd, J = 15.0, 4.2 Hz, 1H), 3.10 (dd, J = 15.2, 9.6 Hz, 1H), 2.96
(dd, J = 13.4, 7.8 Hz, 1H), 2.86 (dd, J = 13.4, 7.4 Hz, 1H), 2.47-2.42 (m, 2H), 2.09-2.06
(m, 2H), 1.90-1.70 (m, 3H), 1.56-1.51 (m, 1H), 1.50-1.33 (m, 2H), 1.17-1.11 (m, 1H),
0.96-0.86 (m, 9H); HRMS (FAB) Calcd. For C33H48N5O9S [M+H]+: 690.3173. Found:
690.3176.
4.1.9. Glu-Ile-Thi-(S)HMC(2-thienyl)-Nva S-14
1H NMR (400 MHz, CD3OD):  = 7.24 (dd, J = 5.0, 1.4 Hz, 1H), 7.20 (dd, J = 5.0,
20

1.4 Hz, 1H), 6.97-6.90 (m, 4H), 4.72 (dd, J = 9.6, 4.4 Hz, 1H), 4.50 (dd, J = 7.5, 2.1 Hz,
1H), 4.42 (d, J = 7.8, 5.0 Hz, 1H), 4.33 (d, J = 7.6 Hz, 1H), 4.13 (d, J = 2.4 Hz, 1H),
3.97 (d, J = 6.4 Hz, 1H), 3.34 (d, J = 1.6 Hz, 1H), 3.21 (dd, J = 14.8, 7.6 Hz, 1H), 3.12
(dd, J = 15.2, 9.6 Hz, 1H), 3.05 (dd, J = 14.6, 7.4 Hz, 1H), 2.50-2.40 (m, 2H), 2.10 (d, J
= 7.2 Hz, 1H), 2.06 (d, J = 8.4 Hz, 1H), 1.93-1.71 (m, 3H), 1.58-1.52 (m, 1H), 1.46-1.33
(m, 2H), 1.21-1.13 (m, 1H), 0.99-0.91 (m, 9H); HRMS (FAB) Calcd. For C31H46N5O9S2
[M+H]+: 696.2737. Found: 696.2732.
4.1.10. Glu-Ile-Thi-(S)HMC(isopropyl)-Nva S-15
1H NMR (300 MHz, CD3OD):  = 7.99 (brd, J = 8.7 Hz, 1H), 7.20 (dd, J = 4.8, 1.8
Hz, 1H), 6.91-6.89 (m, 2H), 4.64 (dd, J = 8.7, 5.7 Hz, 1H), 4.43 (dd, J = 8.3, 5.0 Hz,
1H), 4.35-4.30 (m, 2H), 4.05 (d, J = 2.4 Hz, 1H), 3.94 (t, J = 6.3 Hz, 1H), 3.15 (dd, J =
14.9, 8.6 Hz, 1H), 2.46-2.41 (m, 2H), 2.06 (q, J = 7.0 Hz, 2H), 1.89-1.71 (m, 3H),
1.64-1.49 (m, 3H), 1.46-1.31 (m, 2H), 1.21-1.11 (m, 1H), 1.07-1.00 (m, 1H), 0.97-0.86
(m, 12H), 0.82 (d, J = 6.6 Hz, 3H); HRMS (FAB) Calcd. For C30H50N5O9S [M+H]+:
656.3329. Found: 656.3333.
4.1.11. Glu-Ile-Thi-(S)HMC(cyclohexyl)-Nva S-16
1H NMR (400 MHz, CD3OD):  = 7.24 (dd, J = 5.0, 1.4 Hz, 1H), 6.97-6.97 (m, 1H),
6.95 (dd, J = 5.0, 3.4 Hz, 1H), 4.69 (dd, J = 8.8, 5.6 Hz, 1H), 4.47 (dd, J = 8.2, 5.0 Hz,
1H), 4.39-4.37 (m, 1H), 4.35 (d, J = 8.0 Hz, 1H), 4.09 (d, J = 3.2 Hz, 1H), 3.98 (t, J =
6.2 Hz, 1H), 3.37 (dd, J = 7.6, 6.0 Hz, 1H), 3.21 (dd, J = 14.8, 8.4 Hz, 1H), 2.47 (td, J =
7.6, 2.7 Hz, 2H), 2.11 (dd, J = 14.0, 7.6 Hz, 2H), 1.89-1.77 (m, 4H), 1.67-1.56 (m, 6H),
1.47-1.41 (m, 2H), 1.33-1.16 (m, 8H), 1.01-0.93 (m, 9H); HRMS (FAB) Calcd. For
C33H54N5O9S [M+H]+: 696.3642. Found: 696.3635.
4.1.12. Glu-Ile-Thi-(S)HMC(phenyl)-Nva S-17
1H NMR (400 MHz, CD3OD):  = 7.28-7.21 (m, 4H), 7.18-7.15 (m, 1H), 6.94-6.92
(m, 2H), 4.61 (dd, J = 8.8, 5.2 Hz, 1H), 4.52-4.46 (m, 2H), 4.24 (d, J = 8.0 Hz, 1H),
4.19 (d, J = 3.2 Hz, 1H), 3.97 (t, J = 6.4 Hz, 1H), 3.27 (dd, J = 13.6, 8.4 Hz, 1H), 3.13
(dd, J = 15.0, 9.0 Hz, 1H), 2.81 (d, J = 7.2 Hz, 1H), 2.45 (td, J = 7.6, 3.2 Hz, 1H), 2.09
(q, J = 6.9 Hz, 2H), 1.92-1.64 (m, 4H), 1.51-1.42 (m, 2H), 1.37-1.33 (m 3H), 1.14-1.11
(m, 1H), 0.99 (t, J = 7.0 Hz, 3H), 0.90 (t, J = 7.2 Hz, 3H), 0.76 (d, J = 6.8 Hz, 3H);
HRMS (FAB) Calcd. For C33H48N5O9S [M+H]+: 690.3173. Found: 690.3173.
4.1.13. (4S,5S)-Benzyl 4-isobutyl-2,2-dimethyl-5-vinyloxazolidine-3-carboxylate 20.
21

To a suspension of LiAlH4 (36 mg, 0.96 mmol) in THF (mL) was added
o
Z-Ile-N(OMe)Me (380 mg, 0.96 mmol) in THF (5 mL) at 0 C, stirred for 15 min at
same temperature. The reaction was quenched with saturated aqueous H2O, filtered, and
concentrated. The residue was roughly purified by silica gel column chromatography
(hexane/AcOEt = 4:1) to give Z-Leu-al 19, which was immediately used for the next
step without further purification. To a solution of methyltriphenylphosphonium bromide
(686 mg, 1.92 mmol) in THF (5 mL) was added n-BuLi (1.3 mL, 2.1 mmol, 1.6 mol/L
o
in hexane) at 0 C. The mixture was stirred for 1 h at room temperature, and then
o
methyltrimethylsillyl iodide (286 L, 1.92 mmol) was added at 0 C. The resultant
mixture was stirred for 1 h at room temperature. n-BuLi (1.3 mL, 2.1 mmol, 1.6 mol/L
in hexane) was added to the mixture at 78 oC. The reaction mixture was stirred for 1 h
at room temperature, and then Z-Leu-al 19 in ether (3 mL) was added. The resultant
mixture was stirred for 1 h at room temperature. The reaction was quenched with
saturated aqueous NH4Cl, and the whole was extracted with ether. The organic layer
was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was
dissolved in THF (3 mL) and TBAF (0.96 mL, 0.96 mmol, 1.0 mol/L in THF) was
o
added at 0 C and stirred for 1 h at room temperature. The reaction was quenched with
saturated aqueous NH4Cl, and the whole was extracted with ether. The organic layer
was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was
roughly purified by silica gel column chromatography (hexane/EtOAc = 3:1). The
resultant residue was dissolved in acetone (8 mL) and 2,2-dimethoxypropane (2.5 mL)
and BF3•Et2O (25 L, 0.20 mmol) was added. The mixture was stirred for 5 h at room
o
temperature. The reaction was quenched with Et3N at 0 C, filtered, and concentrated.
The residue was purified by silica gel column chromatography (hexane/EtOAc = 6:1) to
give 20 as a single diastereomer (663 mg, 18%, 3 steps). []25D +15 (c 0.49, CHCl3); 1H
NMR (300 MHz, CDCl3):  = 7.37-7.31 (m, 5H), 5.95 (ddd, J = 17.2, 10.1, 7.1 Hz, 1H),
5.32 (brd, J = 17.1 Hz, 1H), 5.21 (d, J = 10.2 Hz, 1H), 5.18 (d, J = 12.0 Hz, 1H), 5.09
(brd, J = 12.3 Hz, 1H), 4.30 (dd, J = 7.2, 3.6 Hz, 1H), 3.84 (brs, 1H), 1.65-1.47 (m, 9H),
0.97-0.80 (m, 6H); 13C NMR (CDCl3, 75 MHz):  = 152.2, 137.8, 136.4, 128.5, 128.10,
128.05, 117.6, 94.6, 82.0, 66.8, 60.3, 43.4, 27.4, 25.4, 23.8, 21.3; HRMS (EI) Calcd. For
C19H27NO3 [M]+: 317.1991. Found: 317.1987.
4.1.14. Z-Thi-N(OMe)Me
To a solution of N,O-dimethylhydroxyamine hydrochloride (0.68 g, 7.0 mmol) was
added Z-Thi-OH (1.8 g, 5.8 mmol), BOP (2.8 g, 6.4 mmol), and NMM (1.3 g, 12 mmol)
o
in CH2Cl2 (12 mL) at 0 C. The mixture was stirred for 48 h at room temperature. The
22

reaction was quenched with water and the whole was extracted with EtOAc. The
organic layer was washed with 1 mol/L HCl, saturated aqueous NaHCO3, brine, dried
over Na2SO4, filtered, and concentrated. The residue was purified by silica gel column
chromatography (hexane/EtOAc = 1:1) to give Z-Thi-N(OMe)Me (2.0 g, 97%). []29
D
+17.6 (c 1.18, CHCl3); 1H NMR (400 MHz, CDCl3):  = 7.37-7.29 (m, 5H), 7.15 (dd, J
= 5.2, 0.8 Hz, 1H), 6.91 (dd, J = 5.2, 3.6 Hz, 1H), 6.81 (d, J = 3.2 Hz, 1H), 5.59 (d, J =
8.8 Hz, 1H), 5.12 (d, J = 12.4 Hz, 1H), 5.07 (d, J = 12.4 Hz, 1H), 4.99-4.97 (m, 1H),
3.72 (s, 3H), 3.30 (dd, J = 15.0, 5.4 Hz, 1H), 3.22-3.16 (m, 1H), 3.20 (s, 3H); 13C NMR
(100 MHz, CDCl3):  = 171.1, 155.7, 137.9, 136.3, 128.5, 128.1, 128.0, 126.9, 126.6,
124.6, 66.8, 61.6, 52.2, 32.6, 32.2; HRMS (EI) Calcd. For C17H20N2O4S [M]+: 348.1144.
Found: 348.1140.
4.1.15. Benzyl {(2S,3S)-3-hydroxy-1-(thiophen-2-yl)pent-4-en-2-yl}carbamate 21
To a solution of Z-Thi-N(OMe)Me (0.63 g, 1.8 mmol) in CH2Cl2 (10 mL) was
added DIBALH (3.6 mL, 3.6 mmol, 1.0 mol/L in hexane) at 78 oC, stirred for 15 min
at the same temperature under an argon gas atomosphere. The reaction was quenched
with MeOH and the temperature was gradually raise upto room temperature and then
filtered through silica gel and celite pad and concentrated. The residue was roughly
purified by silica gel column chromatography (hexane/EtOAc = 3:1). The resulting
Z-Thi-al was immediately used for the next step without further purification. To a
solution of vinylmagnesium chloride (12 mL, 18 mmol, 1.45 mol/L in THF) was added
Z-Thi-al in THF (3 mL) under an argon gas atmosphere at 0 oC. The mixture was stirred
for 12 h at the same temperature. The reaction was quenched with saturated aqueous
NH4Cl, and the whole was extracted with EtOAc. The organic layer was washed with
brine, dried over Na2SO4, filtered, and concentrated. The residue was roughly purified
by silica gel column chromatography (hexane/EtOAc = 3:1). To a solution of the residue
and 2,2-dimethoxypropane (0.54 mL, 4.4 mmol) in acetone (4 mL) was added BF3•Et2O
(1 drop from syringe). The mixture was stirred for 4 h at room temperature. The reaction
was quenched with Et3N and concentrated. The residue was purified by silica gel
1
column chromatography (hexane/EtOAc = 10:1) to give 21 (0.22 g, 33%, 3 steps). H
NMR (400 MHz, CDCl3, 2:1 diasteromer mixture, major isomer):  = 7.39-7.30 (m, 5H),
7.15 (d, J = 4.8 Hz, 1H), 6.92 (dd, J = 4.4, 3.6 Hz, 1H), 6.73 (brs, 1H), 5.86-5.76 (m,
1H), 5.25-5.10 (m, 2H), 5.20 (brs, 2H), 4.40 (t, J = 6.2 Hz, 1H), 3.88 (td, J = 6.8, 2.0 Hz,
1H), 3.16-3.10 (m, 2H), 1.68 (s, 1.5H), 1.65 (brs, 1.5H), 1.60 (s, 1.5H), 1.54 (brs, 1.5H);
13C NMR (100 MHz, CDCl3):  = 152.2, 140.9, 136.3, 131.8, 128.5, 128.2, 128.1, 126.8,
126.1, 124.5, 118.9, 93.5, 79.8, 77.7, 67.0, 62.2, 31.7, 31.1, 27.1, 23.7; HRMS (EI)
23

Calcd. For C20H23NO3S [M]+: 357.1399. Found: 357.1401.
4.1.16.
(4S,5R)-Benzyl
5-formyl-2,2-dimethyl-4-(thiophen-2-ylmethyl)oxazolidine-3-carboxylate 23
To a suspension of K2OsO2(OH)4 (4.4 mg, 0.012 mmol), K3Fe(CN)6 (0.60 g, 1.8 mmol)
K2CO3 (0.25 g, 1.8 mmol) and MeSO2NH2 (57 g, 0.60 mmol) in t-BuOH/H2O (1:1, 3
mL) was added 21 (0.22 g, 6.0 mmol) in t-BuOH/H2O (1:1, 3 mL) at 0 oC. After stirring
for 20 h at the same temperature, the reaction was quenched with aqueous Na2SO3, and
the whole was extracted with EtOAc. The organic layer was washed with brine, dried
over Na2SO4, filtered, and concentrated. The residue was roughly purified by silica gel
column chromatography (hexane/EtOAc = 3:1). To the product in Et2O/H2O (1:1, 3 mL)
was added NaIO4 (0.25 g, 1.2 mmol) at 0 oC. After stirring for 20 min, the reaction was
diluted with water, and the whole was extracted with EtOAc. The organic layer was
washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was
roughly purified by silica gel column chromatography (CHCl3/MeOH = 20:1). The
1
product 23 was immediately used without further purification. H NMR (400 MHz,
CDCl3, 2:1 diasteromer mixture):  = 9.72 (m, 0.67H), 9.47 (m, 0.33H), 7.39-7.32 (m,
5H), 7.17 (dd, J = 5.2, 1.2 Hz, 1H), 6.94-6.92 (m, 0.67H), 6.90-6.88 (m, 0.33H), 6.77
(brs, 0.67H), 6.70 (brs, 0.33H), 5.23-5.10 (m, 2H), 4.94 (d, J = 12.0 Hz, 0.125H), 4.71
(m, 0.25H), 4.62 (m, 0.125H), 4.55 (d, J = 5.6 Hz, 0.25H), 4.48 (td, J = 9.2, 2.9 Hz,
0.5H), 4.31 (brs, 0.5H), 4.14 (m, 0.25H), 3.41-3.05 (m, 2H), 1.71-1.55 (m, 6H).
4.1.17. Z-(S)HEA(2-thienyl)-Nva-Ot-Bu 25
Compound 23 in MeOH (3 mL) was added K2CO3 (80 mg, 0.58 mmol). After
stirring for 2 h, the mixture was filtered and washed bith brine, dried over Na2SO4,
filtered, and concentrated. The residue was roughly purified by silica gel column
chromatography (CHCl3/MeOH = 100:1). This compound was immediately used for the
1
next step without further purification. H NMR (400 MHz, CDCl3):  = 9.72 (m, 1H),
7.39-7.34 (m, 5H), 7.18-7.13 (m, 1H), 6.94-6.89 (m, 1H), 6.78-6.74 (m, 1H), 5.20-5.07
(m, 2H), 4.73-4.72 (m, 0.25H), 4.50-4.47 (m, 0.5H), 4.31 (brs, 0.5H), 4.13-4.11 (m,
0.25H), 4.04-4.02 (m, 0.5H), 3.49-3.07 (m, 2H), 1.57 (brs, 6H).
To a mixture of the resultant residue and HCl•H2N-Nva-Ot-Bu (96 mg, 0.46 mmol)
was added Ti(Oi-Pr)4 (0.68 mL, 2.3 mmol). After stirring for 3 h, the mixture was
diluted with EtOH (2 mL) and then, NaBH3CN (58 mg, 0.92 mmol) was added at 0 oC.
The resulting mixture was stirred for 5 min at room temperature. The reaction was
quenched with H2O and the whole was extracted with EtOAc. The organic layer was
24

washed with 1 mol/L HCl, saturated aqueous NaHCO3, brine, dried over Na2SO4,
filtered, and concentrated. The residue was purified by silica gel column
chromatography (hexane/EtOAc = 6:1) to give 25 (83 mg, 27%, 4 steps from 21). []28
D
3.8 (c 0.98, CHCl3); 1H NMR (400 MHz, CDCl3):  = 7.42-7.31 (m, 5H), 7.14 (dd, J =
5.2, 1.2 Hz, 1H), 6.91 (dd, J = 5.0, 3.4 Hz, 1H), 6.81-6.75 (m, 1H), 5.20-5.13 (m, 2H),
4.11 (quint., J = 4.3 Hz, 1H), 3.90 (brs, 1H), 3.36-3.09 (m, 2H), 3.00 (brt, J = 6.6 Hz,
1H), 2.71 (dd, J = 11.8, 8.6 Hz, 1H), 2.32 (dd, J = 11.6, 4.4 Hz, 1H), 1.72 (brs, 3H),
1.65 (brs, 3H), 1.54-1.46 (m, 2H), 1.44 (s, 9H), 1.38-1.26 (m, 3H), 0.89 (t, J = 7.4 Hz,
3H); 13C NMR (CDCl3, 100 MHz):  = 174.6, 152.2, 136.3, 128.6, 128.13, 128.08,
126.9, 126.4, 124.4, 95.2, 81.9, 79.3, 66.9, 62.1, 61.3, 51.4, 35.7, 33.3, 28.1, 27.5, 26.6,
19.0, 13.9; HRMS (EI) Calcd. For C28H40N2O5S [M]+: 516.2658. Found: 516.2664.
4.1.18. Z-(S)HEA(isopropyl)-Nva-Ot-Bu 24
To a suspension of K2OsO2(OH)4 (3.1 mg, 8.36 mol), K3Fe(CN)6 (2.06 g, 6.27
mmol) K2CO3 (867 mg, 6.27 mmol) and MeSO2NH2 (199 mg, 2.09 mmol) in
t-BuOH/H2O (1:1, 5 mL) was added 20 (663 mg, 2.09 mmol) in t-BuOH/H2O (1:1, 5
o
mL) at 0 C. After stirring for 15 h at the same temperature, the reaction was quenched
with aqueous Na2SO3, and the whole was extracted with EtOAc. The organic layer was
washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was
roughly purified by silica gel column chromatography (hexane/EtOAc = 3:1) to give the
corresponding diol. To a solution of the product diol in Et2O/H2O (1:1, 3 mL) was
o
added NaIO4 (0.25 g, 1.2 mmol) at 0 C. After being stirred for 1 h, the reaction was
diluted with water, and the whole was extracted with EtOAc. The organic layer was
washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was
roughly purified by silica gel column chromatography (hexane/EtOAc = 3:1) to give 22.
The product was immediately used for the next step without further purification. To a
mixture of the residue and HCl•H2N-Nva-Ot-Bu (438 mg, 2.09 mmol) was added
Ti(Oi-Pr)4 (3.1 mL, 10.5 mmol). The mixture was diluted with EtOH (5 mL) and then,
o
NaBH3CN (263 mg, 4.18 mmol) was added at 0 C. The resulting mixture was stirred
for 10 min at room temperature. The reaction was quenched with H2O, and the whole
was extracted with EtOAc. The organic layer was washed with saturated aqueous
NH4Cl, saturated aqueous NaHCO3, brine, dried over Na2SO4, filtered, and concentrated.
The residue was purified by silica gel column chromatography (hexane/EtOAc = 3:1) to
1
give 24 (798 mg, 80%). []22 +5.3 (c 0.79, CHCl3); H NMR (300 MHz, CDCl3, 9:1
D
amide rotamer, major isomer):  = 7.36-7.31 (m, 5H), 5.17 (d, J = 12.3 Hz, 1H), 5.07
(brd, J = 12.3 Hz, 1H), 3.98 (m, 1H), 3.80 (m, 1H), 3.07 (t, J = 6.6 Hz, 1H), 2.80 (dd, J
25

= 11.4, 8.7 Hz, 1H), 2.44 (dd, J = 11.4, 5.6 Hz, 1H), 1.76 (brs, 3H), 1.65-1.50 (m, 8H),
1.45 (s, 9H), 1.41-1.25 (m, 3H), 0.96-0.86 (m, 6H), 0.90 (t, J = 7.2 Hz, 3H); 13C NMR
(CDCl3, 75 MHz):  = 174.7, 152.2, 136.4, 128.4, 128.0, 94.6, 80.9, 66.7, 62.2, 59.1,
52.3, 43.4, 35.9, 29.7, 29.1, 28.1, 27.6, 25.6, 23.8, 21.1, 19.0, 13.9; HRMS (EI) Calcd.
For C27H44N2O5 [M]+: 476.3250. Found: 476.3244.
4.1.19. Glu-Ile-Thi-(S)HEA(2-thienyl)-Nva S-27
The mixture of 25 (83 mg, 0.16 mmol) and Pd-black (83 mg) in MeOH/Et3N (3:1, 1
mL) was stirred under hydrogen atmosphere. After stirring for 48 h, the reaction
mixture was filtered and concentrated to afford deprotected product. To a solution of the
deprotected product in DMF was added Fmoc-Thi-OH (83 mg, 0.21 mmol), HOBt•H2O
(37 mg, 0.24 mmol), EDCI•HCl (46 mg, 0.24 mmol), and NMM (88 L, 0.80 mmol).
After being stirred for 4 h, the reaction mixture was quenched with water and the whole
was extracted with ether. The organic layer was washed with saturated aqueous NH4Cl,
saturated aqueous NaHCO3, brine, dried over Na2SO4, filtered, and concentrated. The
residue was dissolved in Et2NH/MeCN (1:1, 1 mL). After stirring for 1 h, solvents were
removed under reduced pressure, and the residue was dissolved in DMF. To the solution
were added Fmoc-Ile-OH (100 mg, 0.29 mmol), HOBt•H2O (37 mg, 0.24 mmol),
EDCI•HCl (46 mg, 0.24 mmol), and NMM (88 L, 0.80 mmol). After stirring for 4 h,
the reaction mixture was diluted with water and the whole was extracted with ether. The
organic layer was washed with brine and dried over Na2SO4, filtered, and concentrated.
The residue was dissolved in Et2NH/MeCN (1:1, 1 mL). After being stirred for 1 h,
solvents were removed under reduced pressure. The residue was dissolved in DMF and
Boc-Glu(Ot-Bu)-OH (88 mg, 0.29 mmol), HOBt•H2O (37 mg, 0.24 mmol), EDCI•HCl
(46 mg, 0.24 mmol), and NMM (88 L, 0.80 mmol) were added. After stirring for 4 h,
the reaction mixture was diluted with water and the whole was extracted with ether. The
organic layer was washed with brine and dried over Na2SO4, filtered, and concentrated.
Finally, TFA/H2O/TIS (95:2.5:2.5, 6 mL) was added to the resin and the mixture was
agitated for 3 h. The mixture was concentrated. Ether was added to the residue to form
the precipitate. The precipitate was washed with ether and purified by preparative
HPLC to yield 27 (11 mg, 10%, 7 steps). 1H NMR (400 MHz, CD3OD):  = 7.24 (dd, J
= 4.6, 1.8 Hz, 1H), 7.21 (dd, J = 7.0, 3.8 Hz, 1H), 6.93-6.90 (m, 4H), 4.63 (dd, J = 8.4,
6.4 Hz, 1H), 4.49 (d, J = 5.6 Hz, 0.5H), 4.36 (d, J = 5.6 Hz, 0.5H), 4.14 (d, J = 7.2 Hz,
1H), 4.07-4.04 (m, 1H), 4.00-3.97 (m, 2H), 3.76 (brt, J = 6.0 Hz, 1H), 3.19-3.13 (m,
2H), 3.06 (dd, J = 14.8, 8.0 Hz, 1H), 2.88 (dd, J = 12.4, 4.0 Hz, 1H), 2.77 (dd, J = 12.2,
9.6 Hz, 1H), 2.58-2.51 (m, 2H), 2.20-2.15 (m, 2H), 1.89-1.84 (m, 2H), 1.78-1.76 (m,
26

1H), 1.52-1.35 (m, 3H), 1.16-1.09 (m, 1H), 0.97 (t, J = 7.2 Hz, 3H), 0.96-0.81 (m, 6H);
HRMS (FAB) Calcd. For C31H48N5O8S2 [M+H]+: 682.2944. Found: 682.2951.
4.1.20. Glu-Ile-Thi-(S)HEA(isopropyl)-Nva S-26
Compound S-26 was similarly synthesized using 24 (113 mg, 0.237 mmol) as above.
1H NMR (300 MHz, CDCl3):  = 7.94 (d, J = 9.9 Hz, 1H), 7.28 (dd, J = 4.1, 2.6 Hz, 1H),
6.98-6.95 (m, 2H), 4.54 (t, J = 7.7 Hz, 1H), 4.27 (d, J = 7.8 Hz, 1H), 4.07 (t, J = 6.3 Hz,
1H), 3.90 (brd, J = 9.9 Hz, 2H), 3.79 (d, J = 5.4 Hz, 1H), 3.75 (d, J = 3.9 Hz, 1H), 3.26
(dd, J = 9.8, 8.0 Hz, 1H), 3.12 (dd, J = 12.6, 3.3 Hz, 1H), 2.97 (dd, J = 12.3, 9.6 Hz,
1H), 2.54 (t, J = 7.7 Hz, 2H), 2.20-2.08 (m, 2H), 1.96-1.88 (m, 3H), 1.61-1.44 (m, 4H),
1.28-1.18 (m, 3H), 1.03 (t, J = 7.4 Hz, 3H), 1.02-0.94 (m, 6H), 0.88 (d, J = 6.0 Hz, 3H),
0.83 (d, J = 6.0 Hz, 3H); HRMS (FAB) Calcd. For C30H52N5O8S [M+H]+: 642.3537.
Found: 642.3541.
4.1.21. (S)-4-Isopropyl-3-{3-(thiophen-2-yl)propanoyl}oxazolidin-2-one 30
To 2-thiophene-propionic acid 28 (5.23 g, 33.5 mmol) in THF (60 mL) were added
Et3N (5.6 mL, 40 mmol) and isobutylchloroformate (5.0 mL, 36.8 mmol) at 0 °C. After
stirring
for
30
min,
the
mixture
was
added
to
the
lithio-(4S)-4-isopropyl-2-oxazolidinone, prepared by dropwise addition of n-BuLi (14.2
mL, 36.8 mmol, 2.6 mol/L in hexane) to (4S)-4-isopropyl-2-oxazolidinone (4.76g, 36.8
mmol) in THF (60 mL) at −78 °C, and the mixture was stirred for 1 h. The reaction was
quenched with saturated aqueous NaHCO3 and the whole was extracted with EtOAc.
The organic layer was washed with water and brine, dried over Na2SO4, filtered, and
concentrated. The residue was purified by silica gel column chromatography
1
(hexane/EtOAc = 5:1) to yield 30 (3.68 g, 97%). []28 +78 (c 1.0, CHCl3); H NMR
D
(400 MHz, CDCl3):  7.12 (dd, J = 5.2, 1.2 Hz, 1H), 6.91 (dd, J = 5.2, 3.2 Hz, 1H),
6.86-6.85 (m, 1H), 4.44 (td, J = 8.1, 3.6 Hz, 1H), 4.26 (dd, J = 9.2, 8.0 Hz, 1H), 4.21
(dd, J = 9.2, 3.2 Hz, 1H), 3.41-3.25 (m, 2H), 3.24-3.19 (m, 2H), 2.41-2.33 (m, 1H), 0.91
13
(d, J = 6.8 Hz, 3H), 0.85 (d, J = 7.2 Hz, 3H); C NMR (75 MHz, CDCl3):  171.7,
153.9, 143.0, 126.7, 124.8, 123.4, 63.4, 58.3, 37.2, 28.2, 24.3, 17.8, 14.5; HRMS (EI)
Calcd. For C13H17NO3S [M]+: 267.0929. Found: 267.0933.
4.1.22.
(S)-3-{(2S,3R)-3-Hydroxy-2-(thiophen-2-ylmethyl)pent-4-enoyl}-4-isopropyloxazoli
din-2-one 32
To a solution of 30 (8.68 g, 32.5 mmol) in CH2Cl2 (60 mL) were added n-Bu2BOTf
27

o
(5.70 g, 20.8 mmol) and DIEA (5.80 mL, 32.5 mmol) at 0 C. After the mixture had
been stirred for 40 min, acrolein (0.87 mL, 13 mmol) in CH2Cl2 (10 mL) was added.
After stirring for 2 h, the temperature was gradually raised up to room temperature. The
reaction was quenched with saturated aqueous NH4Cl and 30% H2O2. The mixture was
extracted with EtOAc and the whole was washed with saturated aqueous NaHCO3,
water, and brine. The combined organic layer was dried over MgSO4, filtered, and
concentrated. The residue was purified by silica gel column chromatography
(hexane/EtOAc = 4:1) to yield 32 (6.63 g, 63%). []28 +9.1 (c 0.77, CHCl3); 1H NMR
D
(400 MHz, CDCl3):  7.09 (dd, J = 5.2, 1.2 Hz, 1H), 6.86 (dd, J = 5.0, 3.4 Hz, 1H),
6.84-6.83 (m, 1H), 5.93 (ddd, J = 17.0, 10.6, 6.4 Hz, 1H), 5.35 (td, J = 17.2, 1.5 Hz, 1H),
5.25 (td, J = 10.4, 1.4 Hz, 1H), 4.67 (td, J = 11.1, 4.6 Hz, 1H), 4.45 (m, 1H), 4.43 (td, J
= 8.4, 3.5 Hz, 1H), 4.21 (t, J = 8.8 Hz, 1H), 4.12 (dd, J = 9.2, 3.2 Hz, 1H), 3.34 (dd, J =
14.4, 11.0 Hz, 1H), 3.12 (dd, J = 14.8, 4.2 Hz, 1H), 2.44 (m, 1H), 2.17-2.10 (m, 1H),
0.80 (d, J = 6.8 Hz, 3H), 0.50 (d, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3):  173.3,
154.1, 140.9, 136.9, 126.7, 125.7, 123.8, 117.1, 73.6, 62.9, 58.5, 49.9, 28.0, 27.9, 17.8,
13.9; HRMS (EI) Calcd. For C16H21NO4S [M]+: 323.1191. Found: 323.1189.
4.1.23.
(S)-3-{(2S,3R)-3-Hydroxy-2-isobutylpent-4-enoyl}-4-isopropyloxazolidin-2-one 33
Compound 33 was similarly prepared as above starting from 2-isopropyl-propionic
acid 29. []27D +63 (c 1.3, CHCl3); 1H NMR (300 MHz, CDCl3):  5.88 (ddd, J = 17.1,
10.5, 6.0 Hz, 1H), 5.28 (td, J = 17.2, 1.4 Hz, 1H), 5.20 (td, J = 10.5, 1.4 Hz, 1H), 4.50
(td, J = 8.1, 3.5 Hz, 1H), 4.39-4.30 (m, 2H), 4.25 (d, J = 8.1 Hz, 1H), 4.20 (dd, J = 9.3,
3.3 Hz, 1H), 2.39-2.33 (m, 2H), 1.80 (ddd, J = 13.3, 10.3, 4.7 Hz, 1H), 1.53-1.45 (m,
1H), 1.36 (ddd, J = 13.0, 9.1, 3.7 Hz, 1H), 0.92 (d, J = 6.9 Hz, 3H), 0.914 (d, J = 6.6 Hz,
3H), 0.910 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3): 
174.9, 154.3, 137.2, 116.7, 74.4, 63.0, 58.7, 45.8, 37.0, 28.3, 26.3, 23.4, 21.9, 18.0,
14.5; HRMS (EI) Calcd. For C15H25NO4 [M]+: 283.1784. Found: 283.1788.
4.1.24. (2S,3R)-Methyl 3-hydroxy-2-(thiophen-2-ylmethyl)pent-4-enoate
To a solution of the 32 (6.63 g, 20.5 mmol) in MeOH (40 mL) was added NaOMe
o
(3.32 g, 61.5 mmol) at 15 C and stirred for 20 min. The reaction was quenched with
saturated aqueous NH4Cl. The mixture was extracted with EtOAc and the whole was
washed with 1 mol/L HCl, saturated aqueous NaHCO3, water, and brine. The combined
organic layer was dried over Na2SO4, filtered, and concentrated. The residue was
purified by silica gel column chromatography (hexane/EtOAc = 3:1) to afford title
28

1
compound (3.34 g, 72%). []28 28 (c 0.71, CHCl3); H NMR (400 MHz, CDCl3): 
D
7.13 (dd, J = 5.2, 1.2 Hz, 1H), 6.90 (dd, J = 5.2, 3.6 Hz, 1H), 6.80 (dd, J = 3.4, 1.0 Hz,
1H), 5.90 (ddd, J = 16.9, 10.7, 6.1 Hz, 1H), 5.36 (td, J = 17.1, 1.4 Hz, 1H), 5.25 (td, J =
10.4, 1.4 Hz, 1H), 4.39 (brt, J = 5.4 Hz, 1H), 3.65 (s, 3H), 3.26 (dd, J = 15.0, 9.8 Hz,
1H), 3.14 (dd, J = 15.0, 5.0 Hz, 1H), 2.91 (td, J = 9.9, 5.0 Hz, 1H); 13C NMR (100 MHz,
CDCl3):  173.8, 141.3, 137.1, 126.8, 125.5, 123.9, 117.1, 72.8, 53.4, 51.9, 27.6; HRMS
(EI) Calcd. For C11H14O3S [M]+: 226.0664. Found: 226.0667.
4.1.25. (2S,3R)-Methyl 3-hydroxy-2-isobutylpent-4-enoate
1
[]28 6.6 (c 0.71, CHCl3); H NMR (300 MHz, CDCl3):  5.85 (ddd, J = 17.1,
D
10.5, 6.0 Hz, 1H), 5.30 (td, J = 17.1, 1.5 Hz, 1H), 5.19 (td, J = 10.4, 1.4 Hz, 1H),
4.30-4.29 (m, 1H), 3.70 (s, 3H), 2.65 (ddd, J = 10.4, 5.0, 4.1 Hz, 1H), 2.55 (brd, J = 3.9
Hz, 1H), 1.67 (ddd, J = 13.2, 10.5, 5.0 Hz, 1H), 1.58-1.50 (m, 1H), 1.37 (ddd, J = 13.2,
9.0, 2.1 Hz, 1H), 0.91 (d, J = 6.6 Hz, 3H), 0.89 (d, J = 6.6 Hz, 3H); 13C NMR (75 MHz,
CDCl3):  175.3, 137.5, 116.5, 73.5, 51.7, 49.1, 36.2, 26.2, 23.2, 21.7; HRMS (CI)
Calcd. For C10H19O3 [M+H]+: 187.1334. Found: 187.1326.
4.1.26. (2S,3R)-3-Hydroxy-2-(thiophen-2-ylmethyl)pent-4-enoic acid 34
Methyl ester of 34 obtained above (3.34 g, 14.8 mmol) was dissolved in THF/H2O
(1:1, 15 mL) and 1 mol/L NaOH (15 mL) was added. After stirring for 2 h, the reaction
was quenched with 1 mol/L HCl. The mixture was extracted with EtOAc, dried over
Na2SO4, filtered, and concentrated. The residue was purified by silica gel column
chromatography (hexane/EtOAc = 2:1) to yield 34 (3.04 g, 97%). []28 30 (c 0.93,
D
CHCl3) 1H NMR (400 MHz, CDCl3):  7.14 (dd, J = 5.0, 1.0 Hz, 1H), 6.91 (dd, J = 5.0,
3.4 Hz, 1H), 6.83-6.82 (m, 1H), 5.91 (ddd, J = 17.0, 10.6, 6.2 Hz, 1H), 5.37 (d, J = 16.8
Hz, 1H), 5.28 (d, J = 10.4 Hz, 1H), 4.43 (brt, J = 5.4 Hz, 1H), 3.27 (dd, J = 15.0, 9.0 Hz,
13
1H), 3.09 (dd, J = 15.0, 5.2 Hz, 1H), 2.95 (dd, J = 9.6, 4.7 Hz, 1H); C NMR (100
MHz, CDCl3):  178.3, 141.0, 136.4, 126.9, 125.7, 124.0, 117.8, 72.7, 53.0, 27.2;
HRMS (EI) Calcd. For C10H12O3S [M]+: 212.0507. Found: 212.0504.
4.1.27. (2S,3R)-3-Hydroxy-2-isobutylpent-4-enoic acid 35
[]D28 +81 (CHCl3, c 1.5); 1H NMR (300 MHz, CDCl3):  7.00 (brs, 1H), 5.88 (ddd,
J = 17.0, 10.7, 6.2 Hz, 1H), 5.32 (td, J = 17.2, 1.4 Hz, 1H), 5.22 (td, J = 10.5, 1.4 Hz,
1H), 4.36 (dd, J = 6.0, 5.1 Hz, 1H), 2.69-2.63 (m, 1H), 1.73-1.56 (m, 2H), 1.40-1.30 (m,
1H), 0.93 (d, J = 6.6 Hz, 3H), 0.90 (d, J = 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3): 
180.0, 136.9, 117.0, 73.5, 48.9, 35.8, 26.1, 23.2, 21.6; HRMS (EI) Calcd. For C9H16O3
29