Development of carbapenem-based fluorogenic probes for the clinical screening of carbapenemase-producing bacteria

Article abstract of DOI:10.1016/j.bioorg.2019.103405

This report describes the synthesis of a library of fluorogenic carbapenemase substrates consisting of carbapenem derivatives, fluorescence dyes, and active cleavable linkers and their evaluation for specifically detecting carbapenemase-producing organism

Full text of DOI:10.1016/j.bioorg.2019.103405

Journal Pre-proofs
Development of carbapenem-based fluorogenic probes for the clinical screen-
ing of carbapenemase-producing bacteria
Juhyeon Kim, Yihoon Kim, Ahmed Z. Abdelazem, Hoon Seok Kim, Jung Ok
Kim, Yeon-Joon Park, Sun-Joon Min
PII:
S0045-2068(19)30957-5
DOI:
https://doi.org/10.1016/j.bioorg.2019.103405
YBIOO 103405
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
18 June 2019
18 September 2019
26 October 2019
Please cite this article as: J. Kim, Y. Kim, A.Z. Abdelazem, H. Seok Kim, J. Ok Kim, Y-J. Park, S-J. Min,
Development of carbapenem-based fluorogenic probes for the clinical screening of carbapenemase-producing
bacteria, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103405
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Development of carbapenem-based fluorogenic probes for the clinical screening of
carbapenemase-producing bacteria
a,b
c
d
e
e
Juhyeon Kim , Yihoon Kim , Ahmed Z. Abdelazem , Hoon Seok Kim , Jung Ok Kim , Yeon-
e
c,f,
Joon Park , Sun-Joon Min *
aDepartment of Chemistry, Korea University, Seoul, 02841, Republic of Korea
bCenter for Neuro-Medicine, Korea Institute of Science and Technology (KIST), Seoul, 02792,
Republic of Korea
cDepartment of Applied Chemistry, Hanyang University, Ansan, Gyeonggi-do, 15588,
Republic of Korea
dBiotechnology and Life Sciences Department, Faculty of Postgraduate Studies for Advanced
Sciences, Beni-Suef University, Egypt
eDepartment of Laboratory Medicine, Seoul St. Mary’s Hospital, College of Medicine, The
Catholic University of Korea, Seoul, 06591, Republic of Korea
fDepartment of Chemical & Molecular Engineering, Hanyang University, Ansan, Gyeonggi-
do, 15588, Republic of Korea
email: sjmin@hanynag.ac.kr
Abstract
This report describes the synthesis of a library of fluorogenic carbapenemase substrates
consisting of carbapenem derivatives, fluorescence dyes, and active cleavable linkers and their
evaluation for specifically detecting carbapenemase-producing organisms (CPOs). We
synthesized a series of compounds having three different types of linkers such as benzyl ether,
carbamate, and amine using hydroxymethyl carbapenem 7a and hydroxyally carbapenem 7b
1

as key intermediates. Probe 1b exhibited high stability and a prompt turn-on fluorescence
signal upon hydrolysis by carbapenemases. In particular, the screening of clinical samples
indicated that the probe 1b exhibited excellent selectivity to the CPOs over other β-lactamases
or non-carbapenemase producing bacteria, which may be of clinical use for the rapid and
accurate detection of CPOs for timely diagnosis and treatment.
Keywords: carbapenems; carbapenemase-producing bacteria; linker; fluorescence; probes
2

1
. Introduction
Carbapenems are an important class of last-resort antibiotics [1,2] used for the treatment of
severe and life-threatening bacterial infections due to their broad spectrum of activity and
exceptional stability to β-lactamases, including extended spectrum β-lactamases (ESBLs) (Fig.
1
a) [3]. However, the carbapenem resistance is frequently occuring as a result of the overuse
and misuse of this antibiotic and rapid transfer of carbapenemase gene through plasmids in
pathogenic strains. The major mechanism of pathogenic resistance to carbapenem is related to
the acquisition and expression of carbapenemases [4]. Since the first carbapenemase in
Enterobacteriaceae was identified in 1993 [5], the number of carbapenemase-producing
bacteria has increased worldwide. Currently, the spread of carbapenemase-producing species
is the most important clinical issue in antibiotic resistance to pathogenic bacteria, and it must
be prevented or mitigated.
Three major classes of β-lactamases are involved in carbapenem resistance: Ambler class A,
B, and D [6]. On the basis of their hydrolysis mechanisms, class A and D carbapenemases are
serine-hydrolyzing enzymes possessing a serine residue in the active site for hydrolysis. Class
B carbapenemases belong to metallo-β-lactamases in which zinc ions in the active site play a
crucial role in hydrolytic activity [7,8]. Most carbapenemases expressed from
Enterobacteriaceae deactivate almost all β-lactam antibiotics, including carbapenems, because
they have a very broad substrate profile. The bacterial infectionrelated mortality is significantly
increased and the problem is intensified by the lack of efficient early-stage detection methods
and insufficient new antibiotic development. Therefore, recent research has focused on solving
these issues.
Current detection methods of carbapenem-resistant bacteria are mainly classified into
phenotypic and genotypic methods [9,10]. The Clinical Laboratory Standards Institute (CLSI)
guideline recommends two phenotypic methods, the CarbaNP test and the mCIM (modified
3

carbapenem inactivation method) test. However, the CarbaNP test has several limitations; the
special reagents are required and can be stored in a short period time, invalid results occur with
some isolates, and OXA-48 type carbapenemases are not consistently detected. The mCIM test
is readily performed in most laboratories, but it requires overnight incubation [11]. Currently,
colorimetric based assay kits such as RAPIDEC CARBA NP (bioMérieux) and β-CARBA test
(Bio-Rad) which requires only 2 h and 30 min, respectively, are also commercially available.
In addition, a new MALDI-TOF MS (Matrix-assisted laser desorption ionization-time of flight
mass spectrometry) based carbapenem hydrolysis assay has been developed, but it is not yet
used as routine protocol to detect carbapenemases from clinical samples.[12] Genotypic
methods, such as PCR-based molecular assays, enable rapid detection with high sensitivity and
specificity. In spite of their high accuracy, genotypic methods are less useful because they
require special equipment and expertise to perform, and cannot detect unknown carbapenemase
genes [13,14].
4

(
a) Structures of carbapenems
H
N
O
R
HO
HO
O
H
H
N
H
H
N
CO H
NH
NH
2
S
S
O
CO2H
CO H
2
meropenem (R = CONMe2)
ertapenem
doripenem (R = CH NHSO NH )
2
2
2
(b) Previous carbapenemase substrates (Xie group)
O
Ph
O
HO
O
HO
O
H
H
N
H
H
O
N
N
B
N
F
F
CO2H
CO2H
CPC-1 (1a)
CB-1
(c) This work
O
HO
H
O
H
N
Y
X
R'
O
X = O, NH
CO2H
R' = H, CH , CF3
3
carbapenem (Cp) active linker coumarin (dye)
dye
O
dye
Cp
O
Cp
Cp
O
n
n
1
2
a (CPC-1, n = 0)
a (n = 1)
1b (n = 0)
2b (n = 1)
O
dye
dye
Cp
Cp
O
O
N
N
n
H
H
dye
O
1c
1d (n = 0, R' = Me)
3
1
2
e (n = 0, R' =CF3)
c (n = 1, R' =CF3)
Fig. 1. Design of a chemical library of carbapenem-based fluorogenic probes.
Fluorescence-based assays have garnered considerable attention recently because of the ease
of the procedure, high sensitivity, cost effectiveness, and rapid detection time. Since the use of
a fluorescent chemical probe for detecting β-lactamases was first reported by the Tsien group
[
15], the development of β-lactamase fluorogenic probes has been studied by many researchers
16-18]. However, most reported fluorescence-based probes in detection of β-lactam resistance
[
are based on a core structure of cephalosporin antibiotics, which are easily hydrolyzed by most
β-lactamases. Thus, cephalosporin-based substrates are not suitable for selective detection of
5

carbapenemase-producing bacteria.
Recently, Xie and colleagues reported carbapenem-based substrates CB-1 and CPC-1 as
fluorogenic probes for detecting carbapenemase-producing bacteria (Fig. 1b) [19,20]. Both
probes demonstrated that fluorogenic substrates derived from a carbapenem core structure are
well-recognized by carbapenemases. Although they exhibited high specificity to metallo-
carbapenemases (VIM-27, NDM-1, and IMP-1) over other β-lactamases such as TEM-1 and
CTX-M-9, it is still necessary to develop a method to detect a broad range of carbapenemases
including KPC or OXA-type carbapenemases and explore its clinical applications.
Based on these chemical probes and our studies on β-lactamase probes [21-23], we designed a
chemical library of fluorogenic probes, consisting of a carbapenem core structure, an active
linker, and a fluorophore to identify new fluorogenic probes specific to all types of
carbapenemases (Fig. 1c). In this approach, we envisaged that the different types of the active
linker would operate as a functional spacer between the enzyme recognition site and fluorescent
dye, which may facilitate the enzymatic acceptance of our probes as substrates, so that a wide
range of carbapenemases would be detectable. Additionally, we expect that their
physicochemical properties, such as molecular stability in an aqueous solution, would improve.
Herein, we describe a chemical library of carbapenem-based fluorogenic probes, which are
clinically evaluated for the specific detection of carbapenemase-producing bacteria.
6

2
. Results and discussion
The synthesis of carbapenem-based fluorogenic probes is illustrated in Scheme 1. Following
the procedure reported in previous literature [24,25], we first synthesized hydroxymethyl
carbapenem 7a and hydroxyallyl carbapenem 7b as key versatile intermediates for preparation
of the library. Thus, hydroxy diazo β-lactam 4 was transformed to the corresponding triflate 5
in good yield through the following three steps: TBS protection, intramolecular insertion, and
treatment of trifilic anhydride. Then, triflate 5 was coupled with hydroxymethyl stannane 6a
[
26] through the Stille coupling reaction in the presence of Pd (dba) ·CHCl afforded
2 3 3
hydroxymethyl carbapenem 7a as a whited solid. The allyl alcohol derivative 7b was also
prepared in a higher yield using (E)-3-(tributylstannyl)prop-2-en-1-ol 6b [27] as a coupling
partner under the modified reaction condition. With alcohols 7a and 7b in hand, we initially
attempted to synthesize probes 1a and 2a containing ether moiety as a linker. Although the
synthesis of CPC-1 has already been reported, we synthesized this compound as well in order
to use it as a control substrate. Based on the reported procedure [28,29], we performed the
Mitsunobu reaction of 7a with umbelliferone and subsequent removal of TBS group and PNB
group to yield compound 1a, which was purified by semi-prep HPLC, dried by lyophilization,
and stored at a low temperature. Using the same reaction sequence, we were able to obtain the
desired carbapenem derivative 2a without difficulty.
7

HO
N2
TBSO
O
TBSO
OH
H
H
H
H
N
H
H
N
a-c
d
CO2PNB
OTf
n
NH
O
O
OH
O
CO2PNB
CO2PNB
4
5
Bu3Sn
n
7a (n = 0)
7b (n = 1)
6
6
a (n = 0)
b (n = 1)
O
O
O
O
TBSO
O
HO
O
O
e
H
H
N
f
H
H
n
n
N
O
CO2PNB
CO2R
8
9
a (n = 0)
a (n = 1)
8a', R= PNB (n = 0)
9a', R= PNB (n = 1)
g
1
2
a (CPC-1), R = H (n = 0)
a, R = H (n = 1)
Scheme 1. Synthesis of the carbapenemase-based fluorogenic probes 1a and 2a. Reagents and
conditions: a) TBSCl, imidazole, DMF, rt; b) Rh (OAc) , ZnCl , CH Cl , 60 °C (in pressure
2
4
2
2
2
tubes); c) TMP, DIPEA, Tf O, −50 °C, 92% (three steps from 4); d) 6a or 6b, Pd (dba) ·CHCl ,
2
2
3
3
P(2-furyl) , ZnCl , HMPA or NMP, 70 °C, 52% (7a) and 74% (7b); e) umbelliferone, DIAD,
3
2
PPh , toluene, 0 °C, 90% (8a) and 91% (9a); f) NH HF , NMP/DMF, rt, 82% (8a') and 75%
3
4
2
(
9a'); g) Rh/C, H , 1 M NaHCO , THF/H O, rt, 73% (1a) and 44% (2a).
2 3 2
Next, we expanded these optimized procedures to synthesize a series of carbapenemase
substrates with different types of active linkers as described in Scheme 2. The synthesis of
chemical probes 1b, 2b, and 1c having benzyl ether as an active linker was achieved by the
Mitsunobu reaction of 7a or 7b with coumarin-attached phenols 10a and 10b (See Scheme S1
in ESI for the synthesis of 10a and 10b) followed by the deprotection of both protecting groups.
The reaction of 7a or 7b with 12a or 12b isocyanate, generated in-situ from the treatment of
1
1a or 11b with triphosgene [30] afforded the corresponding carbamate intermediates, which
underwent protecting group eliminations to provide 1d, 1e, and 2c. Finally, the carbapenem-
based probe 3 having an amine linker was prepared from alcohol 7a via Dess-Martin oxidation,
reductive amination [31], and selective deprotection reaction sequences. Therefore, we
effectively constructed a chemical library of nine different carbapenem-based probes, including
CPC-1 (1a) [32].
8

Benzyl ether linker
O
HO
O
a-c
H H
N
O
7a or 7b
O
n
O
CO2H
1
2
b (n = 0)
b (n = 1)
HO
O
a-c
H H
N
O
O
O
CO2H
O
1c
Carbamate linker
O
R
O
R
R
HN
O
HO
d
b,c
H
H
O
n
H2N
O
O
OCN
O
O
N
O
CO2H
1
1
1a (R= Me)
1b (R= CF3)
12a (R= Me)
12b (R= CF3)
1d (n=0, R= Me)
1e (n=0, R= CF3)
2c (n=1, R= CF3)
Amine linker
O
O
TBSO
O
1
R O
H
H
e
H
H
f
O
HN
7a
Me
N
N
O
CO2PNB
CO2R2
13
14 (R1,R2 = TBS, PNB)
b,c
1
2
3
(R ,R = H, H)
Synthesis of 10a and 10b
OH
OH
or
OH
OH
g,h(or i)
R1
R2
HO
1
2
1
1
6a (R , R = OTBS, H)
15a
15b
1
2
6b (R , R = H, OTBS)
j,k
O
O
O
or
O
O
O
HO
OH
10a
10b
Scheme 2. Synthesis of the carbapenem-based fluorogenic probes with different linkers such
as benzyl ether, carbamates, and amine. Reagents and conditions: a) 10a or 10b, DIAD, PPh3,
toluene, 0 °C, 1 h, 50-84%; b) NH HF , NMP/DMF, rt, 3 d, 48-72%; c) Rh/C, H , 1M NaHCO ,
4
2
2
3
THF/H O, rt, 2 h, 24-60%; d) i) triphosgene, TEA, DCE, 80 °C, 2 h; ii) 7a or 7b, TEA, THF,
2
rt, 12 h, 33-78%; e) Dess-Martin periodinane, DCM, rt, 64%; f) 11a, AcOH, NaBH(OAc) , rt,
3
1
2 h, 48%; g) TBSCl, Imidazole, DMF, rt, 12 h, 99% (15a') and 76% (15b'); h) NIS, MeOH,
rt, 2 h, 88% (for 16a); i) CeCl ·7H O, MeCN, 90 °C, 12 h, 72% (for 16b); j) umbelliferone,
3
2
DIAD, PPh , toluene, rt, 1 h, 98% (16a') and 77% (16b'); k) AcOH, TBAF, THF, rt, 2 h, 91%
3
(10a) and 98% (10b').
9

Having synthesized new fluorescent probes, we then examined their intrinsic stability in
aqueous solution by measuring their fluorescence intensity in phosphate buffered saline (PBS,
pH 7.4) for 12 h (Fig. 2). Unlike the control 1a having good stability, allyl ether probe 2a
slowly decomposed up to 27% in PBS solution. We found that probes 1b and 1c containing
benzyl ether linkers exhibit excellent stability with fluorescence increases lower than 15%,
whereas carbamate-linked probes 1d, 1e, and 2c were easily hydrolyzed to generate high
fluorescent signals within a short period of time. The low stability of these probes is
presumably due to the higher susceptibility of the carbamate moiety to nucleophilic attack [33].
We also confirmed that probe 3 is more stable when we observed its remaining amount by the
analysis of HPLC, but it cannot be used as a turn-on fluorogenic probe because there was no
difference in fluorescence intensity between probe 3 and aminocoumarin 11a.
1
0

Fig. 2. Intrinsic stability of fluorescence probes 1a-1e, 2a-2c and 3 in PBS. The enhanced
fluorescence intensity of probes (10 μM) was measured for 12 hours in PBS (pH 7.4). λ =
ex
3
65 nm (1a-1c) and 375 nm (1d-3).
Next, we investigated the absorbance and fluorescence of 1b and 1c before and after treatment
by carbapenemase (Fig. 3). Probe 1b displayed a strong absorption peak at 320 nm. After the
treatment of NDM-1, the maximum absorption wavelength shifted to 360 nm, which is
identical to the characteristic absorption band of deprotonated umbelliferone (Fig. 2a). A
similar absorbance shift was observed upon incubation of probe 1c with NDM-1 (not shown).
When probes 1b and 1c were treated with NDM-1, they showed over 150-fold fluorescence
signal enhancement at 460 nm (Fig. 3b and 3c).
1
1

Fig. 3. Absorbance and fluorescence of 1b and 1c. (a) Changes in absorption spectra for probe
b before (red) and after (black) incubation with NDM-1 (100 nM) at 25 °C. (b) The
fluorescence emission profile of 1b (10 μM) upon incubation with NDM-1 (100 nM) at 25 °C.
c) The fluorescence emission profile of 1c (10 μM) upon incubation with NDM-1 (100 nM)
1
(
at 25 °C.
To verify the possibility of using the newly synthesized compounds as fluorogenic probes to
detect carbapenemases, we conducted the enzyme assay of probes 1a (CPC-1), 1b and 1c (Fig.
4
). Four of the most clinically widespread carbapenemases, including two metallo-β-lactamses
(IMP-1, NDM-1), one class A β-lactamase (KPC-3), and one class D β-lactamase (OXA-48),
were prepared. We observed a significant increase in fluorescence signals when all
carbapenemases were treated with probe 1b for one hour (Fig. 4a). However, treatment with
probe 1c under the same condition showed a slower signal induction, which may be due to the
high stability of 1c (Fig. 4b). In particular, probe 1b exhibited higher detection efficiency
against NDM-1, IMP-1, and KPC-3 than with compound 1a (Fig. 4c). Additionally, TEM-1, a
serine β-lactamase (Class A) most commonly found in gram-negative bacteria, was utilized as
a negative control. As expected, no fluorescence enhancement was observed in TEM-1 (Fig.
4
d). We further confirmed the detection of TEM-1 as the fluorogenic probe reported in the
literature [18] was used as a positive control. (Fig. S1). These results indicate that probe 1b is
useful for the specific detection of carbapenemases.
1
2

Fig. 4. Fluorescence response of probes 1a (CPC-1), 1b and 1c to carbapenemases. (a) and (b)
Time dependence of the fluorescence of 1b and 1c (10 μM in PBS, pH 7.4) upon incubation
with the indicated carbapenemase. (c) Fluorescent enhancement of 1a (CPC-1) and 1b (10 μM
in PBS, pH 7.4) upon incubation with a series of carbapenemases for 60 min. (d) Time
dependence of the fluorescence of 1b in the presence of NDM-1 and TEM-1 for 30 min.
Finally, we tested the two probes (1a and 1b) for detecting carbapenemase-producing
Enterobacteriaceae (CPE) isolated from clinical specimens. A total of 42 previously
characterized isolates of Enterobacteriaceae were used: 24 CPEs (9 KPC, 9 NDM, and 6 OXA)
and 18 carbapenem-susceptible Enterobacteriaceae (CSE) isolates. The isolates were collected
from various clinical samples from multiple Korean hospitals. The identification of bacterial
isolates was performed using MALDI-TOF MS analysis via Vitek MS (bioMérieux, Marcy
L'Etoile, France). They were previously characterized by PCR amplification and molecular
1
3

sequencing as described in the literatures [34,35]. The detailed characteristics of isolates
including minimum inhibitory concentrations (MIC) of carbapenems are shown in Table S1
(
Supplementary Material). In addition, the manual Carba NP test was conducted as described
in the CLSI guidelines [11]. As a result, probe 1b exhibited higher sensitivity (91.7%) than 1a
CPC-1) (58.3%) for detecting CPE (Table 1). The probe 1a (CPC-1) exhibited poor
sensitivity (44.4%) in detecting KPC enzyme, which reflects the findings of a previous report
18], whereas probe 1b showed high sensitivity (100%) for detecting KPC enzyme. In addition,
(
[
probe 1b exhibited higher sensitivity (66.7%) for detection of OXA-48 like carbapenemases
compared to probe 1a (CPC-1) (16.7%) and the manual Carba NP test (16.7%), which is known
to exhibit low sensitivity for detection of OXA-48 type carbapenemases [36]. The fluorescence
signals of the probes according to the carbapenemase types are presented in Fig. 5.
Table 1. Results of the fluorogenic assay using 1a (CPC-1) and probe 1b, and the Carba NP
test in 24 CPE and 18 CSE isolates.
No. of
1a
Carba
NP
Classification Gene
Pos.%
1b Pos.%
Pos.%
isolates (CPC-1)
Class A
Class B
Class D
KPC
9
9
6
4 44.4
9 100
1 16.7
9
9
100
100
9
9
100
100
NDM
OXA
4 66.7
1 16.7
Total
Total
CPE
CSE
24
18
14 58.3
22 91.7
19 79.2
0
0
0
0
0
0
1
4

Fig. 5. Fluorescence signals after treatment of each gene (or group) with probes 1a (CPC-1)
and 1b.
3
. Conclusion
In summary, we have described a library of carbapenem-based fluorogenic probes with
different active linkers for the detection of carbapenemase-producing bacteria. Among the
synthesized fluorescent probes, compound 1b with a benzyl ether linker generated an enhanced
fluorescence signal upon hydrolysis by different types of carbapenemases, which proved that
the active linker plays a crucial role in the stability of the probes and their ability to potentiate
the activity of carbapenemases. Finally, we confirmed that probe 1b could detect pathogenic
bacteria expressing carbapenemase with excellent sensitivity and specificity. Therefore, probe
1
b can be clinically used as a potential substrate for diagnosing CPO-induced diseases.
1
5

4
. Experimental Section
4
.1 Chemistry
General: All reactions were conducted using oven-dried glassware under an atmosphere of
argon (Ar). All commercially available reagents and anhydrous solvents were obtained from
Sigma Aldrich, TCI, Alfa, Junsei, Samchun, DaeJung Chemical and were used without further
purification. Solvents CH Cl was dried and distilled following usual protocols. Organic
2
2
solvents were evaporated with reduced pressure using a rotary evaporator. Reactions were
followed by TLC analysis using silica gel 60 F254 with fluorescent indicator using UV lamp
and ninhydrin solution with heat as visualizing agents. Flash chromatography was carried out
using Merck silica gel 60 (0.063-0.200 mm) and KANTO silica gel 60N (spherical, neutral).
1
13
The H NMR spectra and C NMR spectra were measured with Bruker AVANCE III HD 400.
1
H NMR chemical shifts are expressed in parts per million (δ) downfield to CHCl (δ = 7.26),
3
13C NMR chemical shifts are expressed in parts per million (δ) relative to the central CDCl3
1
resonance (δ = 77.0). Coupling constants in H NMR are in Hz. The following abbreviations
were used to designate multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, dd =
doublet of doublets, m = multiplet. CDCl was used as NMR solvent and standard material
3
TMS (tetramethylsilane) wasn’t contained. LC/MS analyses were performed on Agilent 6125
SQ LCMS system.
4
.1.1. 4-Nitrobenzyl (R)-4-((2R,3S)-3-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-4-
oxoazetidin-2-yl)-2-diazo-3-oxopentanoate (4′) [25]
To a solution of alcohol 4 (5.00 g, 12.8 mmol) in anhydrous DMF (43.0 mL) cooled at 0 °C
was added imidazole (4.42 g, 64.8 mmol) and tert-butyldimethylsilyl chloride (7.72 g, 51.2
mmol). The mixture was allowed to warm up to room temperature over 12 h. After completion
of the reaction (monitored by TLC), it was quenched with saturated aqueous NH Cl, extracted
4
1
6

with ethyl acetate and washed with brine. The organic layers were dried over anhydrous MgSO4
and concentrated in vacuo. The resulting residue was purified by flash column chromatography
on silica gel (EtOAc:n-hexane = 1:2) to afford target compound 4' (6.16 g, 95%) as a white
1
solid. H-NMR (400 MHz, CDCl ) δ 8.26 (d, J = 8.6 Hz, 2H), 7.54 (d, J = 8.6 Hz, 2H), 5.89
3
(s, 1H), 5.37 (d, J = 13.2 Hz, 1H), 5.33 (d, J = 13.2 Hz, 1H), 4.21-4.16 (m, 1H), 3.90-3.89 (m,
2
0
1
4
H), 2.96 (d, J = 2.9 Hz, 1H), 1.19 (d, J = 6.0 Hz, 3H), 1.17 (d, J = 5.9 Hz, 3H), 0.85 (s, 9H),
1
3
.06 (s, 3H), 0.05 (s, 3H); C-NMR (100 MHz, CDCl ) δ 194.29, 168.15, 160.72, 148.09,
3
41.90, 128.77, 124.08, 65.60, 65.08, 61.16, 51.48, 43.30, 25.75, 22.61, 17.94, 12.28, -4.28, -
.98.
4
3
.1.2. 4-Nitrobenzyl (4R,5R,6S)-6-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-4-methyl-7-oxo-
-(((trifluoromethyl)sulfonyl)oxy)-1-azabicyclo[3.2.0]heptane-2-carboxylate (5) [25]
A sealed tube was charged with diazo carbonyl compound 4' (100 mg, 0.198 mmol) in
dichloromethane (0.600 mL). Rhodium acetate dimer (1 mg, 0.002 mmol) and zinc chloride (1
mg, 0.006 mmol) was added to the mixture. The reaction mixture was allowed to stir at 60 °C
for 1 h. After completion of the reaction (monitored by TLC), 2,2,6,6-tetramethyl piperidine
(40 μL, 0.24 mmol) and N,N-diisopropylethylamine (17 μL, 0.10 mmol) was added to the
mixture at -78 °C and trifluoromethanesulfonic anhydride (37 μL, 1.68 mmol) was dropwise
for 5 min. After 1 h, the resulting residue was purified by flash column chromatography on
neutral silica gel (dichloromethane only) to afford triflate 5 (117 mg, 97%) as a white solid.
1
H-NMR (400 MHz, CDCl ) δ 8.21(d, J = 8.7 Hz, 2H), 7.62 (d, J = 8.7Hz, 2H), 5.42 (d, J =
3
1
3
0
3.6 Hz, 1H), 5.35 (d, J = 13.6 Hz, 1H), 4.35 (dd, J = 3.4, 11.0 Hz, 1H), 4.30-4.24 (m, 1H),
.41-3.33 (m, 2H), 1.29 (d, J = 7.3 Hz, 3H), 1.22 (d, J = 6.2 Hz, 3H), 0.86 (s, 9H), 0.08 (s, 3H),
.07 (s, 3H).
1
7

4
.1.3. 4-Nitrobenzyl (4S,5R,6S)-6-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-3-
hydroxymethyl)-4-methyl-7-oxo-1-azabicyclo[3.2.0]heptane-2-carboxylate (7a) [25]
To a solution of tri(2-furyl)phosphine (11.4 mg, 0.049 mmol), Pd (dba) -CHCl (25.4 mg,
(
2
3
3
0
.025 mmol), zinc chloride (22.4 mg, 0.164 mmol), in anhydrous HMPA (0.5 mL) was slowly
added triflate 5 (100 mg, 0.164 mmol) in HMPA (0.1 mL) and hydroxymethyl stannane 6a
211 mg, 0.656 mmol) in HMPA (0.1 mL) at 70 °C. After 2 h, it was quenched with saturated
(
aqueous NH Cl at 5 °C, extracted with ethyl acetate and washed with brine. The organic layers
4
were dried over anhydrous MgSO and concentrated in vacuo. The resulting residue was
4
purified by flash column chromatography on silica gel (EtOAc:n-hexane = 1:8 to 1:2) to afford
1
target compound 7a (42.0 mg, 52%) as a white solid. H-NMR (400 MHz, CDCl ) δ 8.22 (d, J
3
=
8.7 Hz, 2H), 7.66 (d, J = 8.7 Hz, 2H), 5.46 (d, J = 13.9 Hz, 1H), 5.27 (d, J = 13.9 Hz, 1H),
4
3
.55 (d, J = 15.0 Hz, 1H), 4.39 (d, J = 14.6 Hz, 1H), 4.27-4.22 (m, 2H), 3.30-3.23 (m, 2H),
.14 (s, 1H), 1.24 (d, J = 6.2 Hz, 3H), 1.21 (d, J = 7.4 Hz, 3H), 0.86 (s, 9H), 0.08 (s, 3H), 0.07
1
3
(
s, 3H); C-NMR (100 MHz, CDCl ) δ 175.14, 161.88, 153.43, 147.67, 142.48, 128.06, 127.44,
3
1
23.79, 65.73, 65.70, 60.34, 57.68, 55.73, 41.65, 25.68, 22.35, 17.96, 15.37, -4.20, -5.02.
4
.1.4. 4-Nitrobenzyl (4S,5R,6S)-6-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-3-((E)-3-
hydroxyprop-1-en-1-yl)-4-methyl-7-oxo-1-azabicyclo[3.2.0]heptane-2-carboxylate (7b)
To a solution of tri(2-furyl)phosphine (19.1 mg, 0.082 mmol), Pd (dba) -CHCl (42.5 mg,
2
3
3
0
.041 mmol), zinc chloride (10.9 mg, 0.182 mmol), in anhydrous N-Methylpyrrolidine (10.3
mL) was added triflate 5 (500 mg, 0.821 mmol) and (E)-3-(tributylstannyl)prop-2-en-1-ol 6b
428 mg, 1.232 mmol) at room temperature. After 12 h, it was quenched with saturated aqueous
(
NH Cl, extracted with ethyl acetate and washed with brine. The organic layers were dried over
4
anhydrous MgSO and concentrated in vacuo. The resulting residue was purified by flash
4
column chromatography on silica gel (EtOAc:n-hexane = 1:4 to 1:2) to afford target compound
1
8

1
7
7
1
1
b (315 mg, 74%) as a white solid. H-NMR (400 MHz, CDCl ) δ 8.22 (d, J = 8.8 Hz, 2H),
3
.67 (d, J = 8.8 Hz, 2H), 7.31 (d, J = 16.3 Hz, 1H), 6.20 (dt, J = 5.5, 16.2 Hz, 1H), 5.44 (d, J =
3.9 Hz, 1H), 5.27 (d, J = 13.9 Hz, 1H), 5.27 (d, J = 13.9 Hz, 1H), 4.32 (bs, 2H), 4.29-4.23 (m,
H), 4.20 (dd, J = 2.7, 9.4 Hz, 1H), 3.41-3.33 (m, 1H), 3.23 (dd, J = 2.7, 5.6 Hz, 1H), 1.27 (d,
1
3
J = 6.2 Hz, 3H), 1.22 (d, J = 7.3 Hz, 3H), 0.86 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H); C-NMR
(
100 MHz, CDCl ) δ 175.17, 174.90, 160.88, 148.60, 147.58, 142.97, 137.57, 128.09,
3
1
4
25.22,123.71, 122.40, 65.96, 65.21, 63.25, 59.12, 56.19, 39.41, 30.68, 29.59, 25.67, 17.64, -
.22, -4.99.
4
3
.1.5. 4-Nitrobenzyl (4S,5R,6S)-6-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-4-methyl-7-oxo-
-(((2-oxo-2H-chromen-7-yl)oxy)methyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (8a)
[19]
To a solution of alcohol 7a (15.5 mg, 0.032 mmol) in toluene (0.6 mL) was added
umbelliferone (6.2 mg, 0.038 mmol) and triphenylphosphine (10.1 mg, 0.038 mmol) at 0 °C.
After 2 min, diisopropyl azodicarboxylate (8 μL, 0.047 mmol) was added to the mixture. The
mixture was warm up to room temperature over 30 min. After completion of the reaction
(
monitored by TLC), it was quenched with saturated aqueous NH Cl, extracted with ethyl
4
acetate and washed with brine. The organic layers were dried over anhydrous MgSO and
4
concentrated in vacuo. The resulting residue was purified by flash column chromatography on
silica gel (EtOAc:n-hexane = 1:2) to afford target compound 8a (18.2 mg, 90%) as a white
1
solid. H-NMR (400 MHz, CDCl ) δ 8.21 (d, J = 8.6 Hz, 2H), 7.67 (d, J = 8.6 Hz, 2H), 7.63
3
(
d, J = 9.6 Hz, 1H), 7.38 (d, J = 8.3 Hz, 1H), 6.86 (d, J = 2.1 Hz, 1H), 6.83 (s, 1H), 6.27 (d, J
9.5 Hz, 1H), 5.54 (d, J = 14.4 Hz, 1H), 5.48 (d, J = 13.8 Hz, 1H), 5.29 (d, J = 13.8 Hz, 1H),
.78 (d, J = 14.4 Hz, 1H), 4.28-4.13 (m, 2H), 3.44-3.35 (m, 1H), 3.31-3.23 (m, 1H), 1.25 (d, J
=
4
1
3
=
6.2 Hz, 3H), 1.22 (d, J = 6.6 Hz, 3H), 0.85 (s, 9H), 0.07 (s, 3H), 0.06 (s, 3H); C-NMR (100
1
9

MHz, CDCl ) δ 175.18, 161.25, 161.04, 160.75, 155.78, 147.66, 146.91, 143.31, 142.51,
3
1
4
29.05, 128.13, 127.87, 123.78, 113.58, 113.10, 112.53, 101.82, 65.65, 65.5, 63.0, 60.8, 55.4,
0.3, 25.7, 22.3, 17.9, 15.5, -4.2, -5.0.
4
.1.6. 4-Nitrobenzyl (4S,5R,6S)-6-((R)-1-hydroxyethyl)-4-methyl-7-oxo-3-(((2-oxo-2H-
chromen-7-yl)oxy)methyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (8a′) [19]
To a solution of silyl ether 8a (65.4 mg, 0.103 mmol) in NMP:DMF =1:3 (2.1 mL) was added
ammonium hydrogen fluoride (23.5 mg, 0.412 mmol). The reaction mixture was allowed to
stir at the same temperature for 30 h. After completion of the reaction (monitored by TLC), the
mixture was diluted with saturated aqueous NaHCO , extracted with EtOAc. The organic
3
layers were dried over anhydrous MgSO and concentrated in vacuo. The resulting residue was
4
purified by flash column chromatography on silica gel (EtOAc:n-hexane = 3:1) to afford
1
alcohol 8a' (44.0 mg, 82%) as a white solid. H-NMR (400 MHz, CDCl ) δ 8.24 (d, J = 8.7
3
Hz, 2H), 7.68 (d, J = 8.6 Hz, 2H), 7.64 (d, J = 9.4 Hz, 1H), 7.38 (d, J = 8.2 Hz, 1H), 6.85 (d, J
=
2.4 Hz, 1H), 6.82 (s, 1H), 6.28 (d, J = 9.4 Hz, 1H), 5.54 (d, J = 14.7 Hz, 1H), 5.29 (d, J =
1
1
1
1
1
3.5 Hz, 1H), 4.80 (d, J = 14.4 Hz, 1H), 4.29-4.25 (m, 2H), 3.45-3.38 (m, 1H), 3.36-3.33 (m,
1
3
H), 1.35 (d, J = 6.2 Hz, 3H), 1.27 (d, J = 7.4 Hz, 3H); C-NMR (100 MHz, CDCl ) δ 175.18,
3
61.25, 161.04, 160.75, 155.78, 147.66, 146.91, 143.31, 142.51, 129.05, 128.13, 127.87,
23.78, 113.58, 113.10, 112.53, 101.82, 65.65, 65.50, 63.00, 60.75, 55.43, 40.33, 25.70, 22.28,
7.96, 15.49, -4.24, -4.99.
4
.1.7. (4S,5R,6S)-6-((R)-1-Hydroxyethyl)-4-methyl-7-oxo-3-(((2-oxo-2H-chromen-7-
yl)oxy)methyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid (1a, CPC-1) [19]
A mixture of compound 8a′ (30.0 mg, 0.057 mmol) and 5% Rh/C (3.6 mg) in a mixed solution
of THF/H O (1.9 mL, 2:1) under hydrogen atmosphere (1 atm) were stirred at room
2
2
0

temperature for 2 h. The mixture were passed through a PTFE syringe filter to remove
precipitate and the filtrate was washed with ethyl acetate. The water phase was purified with
preparative RP-HPLC with CH CN-H O as mobile phase. Lyophilization afforded compound
3
2
1
1
a (CPC-1, 72.7 mg, 73%) as a white solid. H-NMR (400 MHz, CD OD) δ 7.86 (d, J = 9.5
3
Hz, 1H), 7.52 (d, J = 8.6 Hz, 1H), 6.98 (dd, J = 2.4, 8.6 Hz, 1H), 6.95 (d, J = 2.1 Hz, 1H), 6.22
d, J = 9.5 Hz, 1H), 5.64 (d, J = 13.5 Hz, 1H), 4.79 (d, J = 13.5 Hz, 1H), 4.08-4.04 (m, 2H),
(
3
3
.24-3.20 (m, 1H), 3.19 (dd, J = 2.9, 7.3 Hz, 1H), 1.26 (d, J = 6.3 Hz, 3H), 1.18 (d, J = 7.3 Hz,
-
H); HRMS (ESI) m/z calcd for C H N O (M-C H N) 486.2366, found 384.1096.
2
6
34
2
7
6
16
4
3
.1.8. 4-Nitrobenzyl (4S,5R,6S)-6-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-4-methyl-7-oxo-
-((E)-3-((2-oxo-2H-chromen-7-yl)oxy)prop-1-en-1-yl)-1-azabicyclo[3.2.0]hept-2-ene-2-
carboxylate (9a)
Following the same procedure as that used for the synthesis of 8a, the reaction of allyl alcohol
7
b (100 mg, 0.194 mmol), umbelliferone (34.6 mg, 0.213 mmol), triphenylphosphine (68.0
mg, 0.233 mmol) and diisopropyl azodicarboxylate (49 μL, 0.290 mmol) in toluene (3.9 mL)
gave target compound 9a (119 mg, 91%) as a white solid after purification by column
1
chromatography on silica gel (EtOAc:n-hexane = 1:2). H-NMR (400 MHz, CDCl ) δ 8.22 (d,
3
J = 8.6 Hz, 2H), 7.67 (d, J = 8.7 Hz, 2H), 7.64 (d, J = 9.6 Hz, 1H), 7.47 (d, J = 8.3 Hz, 1H),
7
2
.38 (d, J = 8.6 Hz, 1H), 6.85 (dd, J = 2.3, 8.6 Hz, 1H), 6.81 (d, J = 7.0 Hz, 1H), 6.28-6.17 (m,
H), 5.45 (d, J = 13.8 Hz, 1H), 5.28 (d, J = 14.0 Hz, 1H), 4.74 (d, J = 5.8 Hz, 2H), 4.27-4.21
(m, 2H), 3.42-3.37 (m, 1H), 3.25 (dd, J = 2.6, 5.4 Hz, 1H), 1.26 (d, J = 6.2 Hz, 6H), 0.85 (s,
1
3
9
1
1
1
H), 0.08 (s, 3H), 0.07 (s, 3H); C-NMR (100 MHz, CDCl ) δ 172.70, 161.42, 161.13, 160.74,
3
55.78, 147.62, 147.29, 143.35, 142.80, 131.20, 128.90, 128.11, 126.40, 125.57, 123.73,
13.41, 113.00, 112.89, 101.78, 68.80, 65.85, 65.35, 59.26, 56.17, 39.28, 25.66, 22.41, 17.62,
6.80, -4.22, -5.01.
2
1

4
.1.9. 4-Nitrobenzyl (4S,5R,6S)-6-((R)-1-hydroxyethyl)-4-methyl-7-oxo-3-((E)-3-((2-oxo-2H-
chromen-7-yl)oxy)prop-1-en-1-yl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (9a′)
Following the same procedure as that used for the synthesis of 8a', the reaction of silyl ether
9
a (40.1 mg, 0.059 mmol) and ammonium hydrogen fluoride (13.6 mg, 0.238 mmol) in a mixed
solution of NMP/DMF (1.2 mL, 1:3) gave target compound 9a' (24.0 mg, 75%) as a white
solid after purification by column chromatography on silica gel (EtOAc:n-hexane = 3:1).
1
H-NMR (400 MHz, CDCl ) δ 8.21 (d, J = 8.7 Hz, 2H), 7.66 (d, J = 8.7 Hz, 2H), 7.63 (d, J =
3
9
6
1
.6 Hz, 1H), 7.45 (d, J = 16.3 Hz, 1H), 7.37 (d, J = 8.6 Hz, 1H), 6.83 (dd, J = 2.4, 8.6 Hz, 1H),
.80 (d, J = 2.3 Hz, 1H), 6.28 (d, J = 4.8 Hz, 1H), 6.20 (dt, J = 5.7, 16.3 Hz, 1H), 5.49 (d, J =
3.8 Hz, 1H), 5.25 (d, J = 13.8 Hz, 1H), 4.73 (d, J = 5.8 Hz, 2H), 4.26-4.23 (m, 2H), 3.47-3.43
(m, 1H), 3.28 (dd, J = 2.6, 6.9 Hz, 1H), 1.37 (d, J = 6.2 Hz, 3H), 1.25 (d, J = 7.2 Hz, 3H).
4
.1.10. (4S,5R,6S)-6-((R)-1-Hydroxyethyl)-4-methyl-7-oxo-3-((E)-3-((2-oxo-2H-chromen-7-
yl)oxy)prop-1-en-1-yl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid (2a)
Following the same procedure as that used for the synthesis of 1a, the reaction of compound
9
a′ (36.0 mg, 0.066 mmol) and 5% Rh/C (4.2 mg) in a mixed solution of THF/H O (2.1 mL,
2
2
:1) under hydrogen atmosphere (1 atm) gave target compound 2a (12.0 mg, 44%) as a white
solid after purification by preparative RP-HPLC with CH CN-H O as mobile phase and
3
2
1
lyophilization. H-NMR (400 MHz, CD OD) δ 7.87 (d, J = 9.5 Hz, 1H), 7.52 (d, J = 8.6 Hz,
3
1
H), 7.46 (d, J = 16.3 Hz, 1H), 6.97 (dd, J = 2.4, 8.6 Hz, 1H), 6.93 (d, J = 2.3 Hz, 1H), 6.22
(d, J = 9.5 Hz, 1H), 6.03 (dt, J = 6.1, 16.2 Hz, 1H), 4.74 (d, J = 6.0 Hz, 2H), 4.10-4.05 (m, 2H),
3
3
.37-3.26 (m, 1H), 3.15 (dd, J = 2.4, 7.5 Hz, 1H), 1.28 (d, J = 6.3 Hz, 3H), 1.27 (d, J = 7.2 Hz,
+
H); HRMS (ESI) m/z calcd for C H NO (M+Na) 434.1210, found 434.1212.
2
2
21
7
2
2

4
.1.11. tert-Butyl((4-((tert-butyldimethylsilyl)oxy)benzyl)oxy)dimethylsilane (15a')
To a solution of para-hydroxybenzyl alcohol 15a (500 mg, 4.03 mmol) in anhydrous DMF
20.2 mL) was added imidazole (1.10 g, 16.1 mmol) and tert-butyldimethylsilyl chloride (2.42
(
g, 16.1 mmol) at 0 °C. The mixture was allowed to warm up to room temperature over 12 h.
After completion of the reaction (monitored by TLC), it was quenched with saturated aqueous
NH Cl, extracted with ethyl acetate and washed with brine. The organic layers were dried over
4
anhydrous MgSO and concentrated in vacuo. The resulting residue was purified by flash
4
column chromatography on silica gel (EtOAc:n-hexane = 1:15) to afford target compound 15a'
1
(
1.40 g, 99%) as a colorless oil. H-NMR (400 MHz, CDCl ) δ 7.17 (d, J = 8.4 Hz, 2H), 6.79
3
1
3
(
d, J = 8.5 Hz, 2H), 4.67 (s, 2H), 0.98 (s, 9H), 0.93 (s, 9H), 0.18 (s, 6H), 0.08 (s, 6H); C-
NMR (100 MHz, CDCl ) δ 154.60, 134.17, 127.42, 119.83, 64.80, 26.00, 25.72, 18.46, 18.23,
3
-4.41, -5.17.
4
.1.12. (4-((tert-butyldimethylsilyl)oxy)phenyl)methanol (16a)
To a solution of bis-silyl ether 15a' (35.3 mg, 0.10 mmol) in MeOH (0.3 mL) was added N-
iodosuccinimide (1.1 mg, 0.005 mmol) at room temperature. After 12 h, it was quenched with
saturated aqueous NH Cl, extracted with ethyl acetate and washed with brine. The organic
4
layers were dried over anhydrous MgSO and concentrated in vacuo. The resulting residue was
4
purified by flash column chromatography on silica gel (EtOAc:n-hexane = 1:8 to 1:4) to afford
1
target compound 16a (21.0 mg, 88%) as a colorless oil. H-NMR (400 MHz, CDCl ) δ 7.22 (d,
3
J = 8.5 Hz, 2H), 6.82 (dt, J = 2.3, 9.0 Hz, 2H), 4.60 (s, 2H), 0.98 (s, 9H), 0.19 (s, 6H).
1
3
C-NMR (100 MHz, CDCl ) δ 155.28, 133.74, 128.58, 120.17, 65.05, 25.71, 18.24, -4.41.
3
4
.1.13. 7-((4-((tert-Butyldimethylsilyl)oxy)benzyl)oxy)-2H-chromen-2-one (16a')
To a solution of benzyl alcohol 16a (1.50 g, 5.34 mmol) in toluene (108 mL) was added
2
3

umbelliferone (961 mg, 5.93 mmol) and triphenylphosphine (1.89 mg, 6.47 mmol) at 0 °C.
After 2 min, diisopropyl azodicarboxylate (1.37 mL, 8.08 mmol) was added to the mixture.
The mixture was warm up to room temperature over 30 min. After completion of the reaction
(
monitored by TLC), it was quenched with saturated aqueous NH Cl, extracted with ethyl
4
acetate and washed with brine. The organic layers were dried over anhydrous MgSO and
4
concentrated in vacuo. The resulting residue was purified by flash column chromatography on
silica gel (EtOAc:n-hexane = 1:8) to afford target compound 16a (2.02 g, 98%) as a white
solid.
1
H-NMR (400 MHz, CDCl ) δ 7.63 (d, J = 9.5 Hz, 1H), 7.37 (d, J = 8.4 Hz, 1H), 7.29 (d, J =
3
8
.4 Hz, 2H), 6.91 (d, J = 2.3 Hz, 1H), 6.88 (d, J = 8.7 Hz, 2H), 6.86 (d, J = 8.4 Hz, 1H), 6.25
(d, J = 9.4 Hz, 1H), 5.03 (s, 2H), 0.98 (s, 9H), 0.20 (s, 6H).
1
3
C-NMR (100 MHz, CDCl ) δ 161.99, 161.20, 155.91, 155.82, 143.44, 129.26, 128.79, 128.38,
3
1
20.32, 113.25, 113.12, 112.65, 101.85, 70.39, 25.68, 18.21, -4.39.
4
.1.14. 7-((4-Hydroxybenzyl)oxy)-2H-chromen-2-one (10a)
Under Ar atmosphere, to a solution of 16a' (1.00 g, 2.61 mmol) in THF (anhydrous, 26.1 mL)
at 0°C were added slowly AcOH (1.05 mL, 18.3 mmol) and TBAF (1M solution in THF, 13
mL, 13.0 mmol). The reaction mixture were stirred at room temperature for 3 h, and then
concentrated under reduced pressure. The residues were purified by chromatography on silica
gel column (Acetone:n-hexane = 1:2) to afford the title compound 10a (635 mg, 91%) as a
1
white solid. H-NMR (400 MHz, Acetone) δ 7.89 (d, J = 9.5 Hz, 1H), 7.68 (d, J = 9.3 Hz, 1H),
7
5
1
.35 (d, J = 8.5 Hz, 2H), 6.98-6.96 (m, 2H), 6.87 (d, J = 8.5 Hz, 2H), 6.21 (d, J = 9.5 Hz, 1H),
1
3
.13 (s, 2H); C-NMR (100 MHz, Acetone) δ 162.16, 160.07, 157.54, 155.98, 143.67, 129.79,
29.24, 127.23, 115.29, 112.95, 112.75, 112.63, 101.51, 70.24.
2
4

4
.1.15. tert-Butyl((2-((tert-butyldimethylsilyl)oxy)benzyl)oxy)dimethylsilane (15b')
Following the same procedure as that used for the synthesis of 15a', the reaction of ortho-
hydroxybenzyl alcohol 15b (500 mg, 4.03 mmol), imidazole (686 mg, 10.1 mmol) and tert-
butyldimethylsilyl chloride (1.52 g, 10.1 mmol) in anhydrous DMF (20.2 mL) gave target
compound 15b' (1.08 g, 76%) as a colorless oil after purification by column chromatography
1
on silica gel (EtOAc:n-hexane = 1:15). H-NMR (400 MHz, CDCl ) δ 7.46 (dt, J = 1.0, 9.4 Hz,
3
1
1
H), 7.11 (td, J = 2.0, 9.6 Hz, 1H), 6.97 (td, J = 1.2, 9.3 Hz, 1H), 6.74 (dd, J = 1.2, 10.0 Hz,
1
3
H), 4.76 (s, 2H), 1.01 (s, 9H), 0.95 (s, 9H), 0.22 (s, 6H), 0.10 (s, 6H); C-NMR (100 MHz,
CDCl ) δ 151.93, 132.23, 127.23, 126.97, 121.09, 117.81, 60.58, 26.01, 25.75, 18.49, 18.25, -
3
4
.19, -5.33.
4
.1.16. (2-((tert-Butyldimethylsilyl)oxy)phenyl)methanol (16b)
To a solution of bis-silyl ether 15b' (636 mg, 1.80 mmol) in acetonitrile (25.8 mL) was added
cerium chloride heptahydrate (1.34 g, 3.61 mmol) at 90 °C. After 12 h, it was quenched with
saturated aqueous NH Cl, extracted with ethyl acetate and washed with brine. The organic
4
layers were dried over anhydrous MgSO and concentrated in vacuo. The resulting residue was
4
purified by flash column chromatography on silica gel (EtOAc:n-hexane = 1:8 to 1:4) to afford
1
target compound 16b (49.0 mg, 72%) as a colorless oil. H-NMR (400 MHz, CDCl ) δ 7.31
3
(dd, J = 2.0, 9.3 Hz, 1H), 7.18 (td, J = 2.1, 9.7 Hz, 1H), 6.96 (dt, J = 1.2, 9.3 Hz, 1H), 6.82 (d,
1
3
J = 10.1 Hz, 1H), 4.68 (s, 2H), 1.02 (s, 9H), 0.26 (s, 6H); C-NMR (100 MHz, CDCl ) δ
3
1
153.50, 131.49, 128.85, 128.63, 121.36, 118.41, 61.96, 25.76, 18.19, -4.15.
4
.1.17. 7-((2-((tert-Butyldimethylsilyl)oxy)benzyl)oxy)-2H-chromen-2-one (16b')
Following the same procedure as that used for the synthesis of 16a', the reaction of benzyl
alcohol 16b (68.0 mg, 0.285 mmol), umbelliferone (50.8 mg, 0.314 mmol), triphenylphosphine
2
5

(100 mg, 0.342 mmol) and diisopropyl azodicarboxylate (0.072 mL, 0.428 mmol) in toluene
(5.80 mL) gave target compound 16b' (84.0 mg, 77%) as a white solid after purification by
1
column chromatography on silica gel (EtOAc:n-hexane = 1:8). H-NMR (400 MHz, CDCl ) δ
3
7
.62 (d, J = 11.8 Hz, 1H), 7.39 (dd, J = 1.9, 9.5 Hz, 1H), 7.36 (d, J = 10.5 Hz, 1H), 7.22 (td, J
2.0, 9.7 Hz, 1H), 6.97 (td, J = 1.2, 9.4 Hz, 1H), 6.91-6.85 (m, 3H), 6.24 (d, J = 11.8 Hz, 1H),
=
1
3
5
1
2
.12 (s, 2H), 0.97 (s, 9H), 0.25 (s, 6H); C-NMR (100 MHz, CDCl ) δ 162.15, 161.30, 155.91,
3
53.52, 143.45, 129.47, 128.75, 126.31, 121.32, 118.67, 113.16, 113.08, 112.57, 101.69, 66.12,
5.71, 21.97, 18.22, -4.16.
4
.1.18. 7-((2-Hydroxybenzyl)oxy)-2H-chromen-2-one (10b)
Following the same procedure as that used for the synthesis of 10a, the reaction of 16b' (427
mg, 1.12 mmol), AcOH (0.447 mL, 7.81 mmol) and TBAF (1M solution in THF, 5.6 mL, 5.58
mmol) in THF (anhydrous, 11.2 mL) gave compound 10b (292 mg, 98%) as a white solid after
1
purification by column chromatography on silica gel (acetone:n-hexane = 1:2). H-NMR (400
MHz, CDCl ) δ 7.89 (d, J = 11.8 Hz, 1H), 7.58 (d, J = 10.4 Hz, 1H), 7.41 (dd, J = 1.8, 9.4 Hz,
3
1
1
1
1
H), 7.20 (td, J = 1.9, 9.6 Hz, 1H), 7.01-6.98 (m, 2H), 6.94 (d, J = 9.4 Hz, 1H), 6.88 (td, J =
1
3
.1, 9.3 Hz, 1H), 6.21 (d, J = 11.9 Hz, 1H), 5.25 (s, 2H); C-NMR (100 MHz, acetone-D ) δ
6
62.22, 161.13, 160.11, 156.00, 155.17, 143.84, 129.75, 129.25, 122.62, 119.68, 115.34,
12.87, 112.72, 112.06, 101.39, 65.45.
4
3
2
.1.19. 4-Nitrobenzyl (4S,5R,6S)-6-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-4-methyl-7-oxo-
-((4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)phenoxy)methyl)-1-azabicyclo[3.2.0]hept-2-ene-
-carboxylate (8b)
Following the same procedure as that used for the synthesis of 8a, the reaction of alcohol 7a
2
6

(94 mg, 0.192 mmol), phenol compound 10a (56.7 mg, 0.211 mmol), triphenylphosphine (67.3
mg, 0.230 mmol) and diisopropyl azodicarboxylate (49 μL, 0.288 mmol) in toluene (3.8 mL)
gave target compound 8b (120 mg, 84%) as a white solid after purification by column
1
chromatography on silica gel (EtOAc:n-hexane:CHCl = 2:5:4). H-NMR (400 MHz, CDCl )
3
3
δ 8.21 (d, J = 8.7 Hz, 2H), 7.66 (d, J = 9.0 Hz, 2H), 7.63 (d, J = 9.8 Hz, 1H), 7.37 (d, J = 8.5
Hz, 1H), 7.35 (d, J = 8.6 Hz, 2H), 6.93 (d, J = 8.7 Hz, 2H), 6.91-6.87 (m, 2H), 6.25 (d, J = 9.4
Hz, 1H), 5.47 (d, J = 14.6 Hz, 1H), 5.47 (d, J = 13.8 Hz, 1H), 5.27 (d, J = 13.8 Hz, 1H), 5.05
(s, 2H), 4.72 (d, J = 15.1 Hz, 1H), 4.29-4.25 (m, 2H), 3.50-3.42 (m, 1H), 3.30 (dd, J = 3.2, 5.2
1
3
Hz, 1H), 1.24 (d, J = 6.2 Hz, 6H), 0.86 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H); C-NMR (100 MHz,
CDCl ) δ 175.34, 161.87, 161.23, 160.82, 158.37, 155.82, 148.49, 147.67, 143.43, 142.61,
3
1
5
29.47, 128.82, 128.10, 123.79, 114.74, 113.30, 101.85, 70.21, 65.64, 65.53, 62.58, 60.66,
5.54, 40.40, 25.70, 22.32, 21.97, 17.98, 15.50, -4.23, -4.97.
4
.1.20. 4-Nitrobenzyl (4S,5R,6S)-6-((R)-1-hydroxyethyl)-4-methyl-7-oxo-3-((4-(((2-oxo-2H-
chromen-7-yl)oxy)methyl)phenoxy)methyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (8b′)
Following the same procedure as that used for the synthesis of 8a', the reaction of silyl ether
8
b (95.0 mg, 0.128 mmol) and ammonium hydrogen fluoride (29.3 mg, 0.513 mmol) in a mixed
solution of NMP/DMF (2.4 mL, 1:3) gave target compound 8b' (58.0 mg, 72%) as a white
1
solid after purification by column chromatography on silica gel (EtOAc:n-hexane = 3:1). H-
NMR (400 MHz, CDCl ) δ 8.22 (d, J = 8.8 Hz, 2H), 7.67-7.62 (m, 3H), 7.38-7.33 (m, 3H),
3
6
5
4
1
1
.92-6.89 (m, 3H), 6.86 (d, J = 2.2 Hz, 1H), 6.25 (d, J = 9.4 Hz, 1H), 5.52 (d, J = 13.6 Hz, 1H),
.47 (d, J = 14.8 Hz, 1H), 5.26 (d, J = 13.8 Hz, 1H), 5.06 (s, 2H), 4.74 (d, J = 14.8 Hz, 1H),
.29-4.24 (m, 2H), 3.52-3.44 (m, 1H), 3.33 (dd, J = 3.0, 6.3 Hz, 1H), 1.35 (d, J = 6.2 Hz, 3H),
1
3
.25 (d, J = 7.2 Hz, 3H). C-NMR (100 MHz, CDCl ) δ 174.72. 161.86. 161.28. 160.70.
3
58.21. 155.80. 149.47. 147.73. 143.47. 142.56. 129.39. 128.82. 128.63, 128.24.127.05.
2
7

1
2
23.85. 114.75. 113.39. 113.22. 112.71. 101.88. 70.19. 65.65. 62.57. 59.87. 55.90. 40.57. 22.04.
1.79. 15.44.
4
.1.21. (4S,5R,6S)-6-((R)-1-Hydroxyethyl)-4-methyl-7-oxo-3-((4-(((2-oxo-2H-chromen-7-
yl)oxy)methyl)phenoxy)methyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid (1b)
Following the same procedure as that used for the synthesis of 1a, the reaction of compound
8
b′ (26.0 mg, 0.041 mmol) and 5% Rh/C (2.6 mg) in a mixed solution of THF/H O (1.4 mL,
2
2
:1) under hydrogen atmosphere (1 atm) gave target compound 1b (10.0 mg, 50%) as a white
solid after purification by preparative RP-HPLC with CH CN-H O as mobile phase and
3
2
1
lyophilization. H-NMR (400 MHz, CD OD) δ 7.87 (d, J = 9.6 Hz, 1H), 7.52 (d, J = 9.3 Hz,
3
1
H), 7.35 (d, J = 8.5 Hz, 2H), 6.98-6.96 (m,4 H), 6.23 (d, J = 9.5 Hz, 1H), 5.54 (d, J = 13.5
Hz, 1H), 5.08 (s, 2H), 4.68 (d, J = 13.4 Hz, 1H), 4.08-4.02 (m, 2H), 3.47-3.46 (m, 1H), 3.17
(dd, J = 2.8, 7,5 Hz, 1H), 3.12-3.11 (m, 1H), 1.25 (d, J = 6.3 Hz, 3H), 1.17 (d, J = 7.1 Hz, 3H);
1
3
C-NMR (100 MHz, CD OD) δ 175.50, 161.15, 162.38, 16199, 158.72, 155.69, 144.39,
3
1
6
5
38.33, 134.92, 129.19, 129.02, 128.40, 114.42, 113.13, 112.70, 111.97, 101.34, 70.03, 65.51,
+
2.64, 59.03, 56.15, 39.38, 20.34, 14.36; HRMS (ESI) m/z calcd for C H NO (M+Na)
2
7
25
8
14.1472, found 514.1474.
4
3
.1.22. 4-Nitrobenzyl (4S,5R,6S)-6-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-4-methyl-7-oxo-
-((E)-3-(4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)phenoxy)prop-1-en-1-yl)-1-
azabicyclo[3.2.0]hept-2-ene-2-carboxylate (9b)
Following the same procedure as that used for the synthesis of 8a, the reaction of allyl alcohol
7
b (20 mg, 0.039 mmol), phenol 10a (11.5 mg, 0.043 mmol), triphenylphosphine (13.7 mg,
.047 mmol) and diisopropyl azodicarboxylate (9.8 μL, 0.058 mmol) in toluene (0.8 mL) gave
0
target compound 9b (22 mg, 74%) as a white solid after purification by column
2
8

1
chromatography on silica gel (EtOAc:n-hexane:CHCl = 2:5:4). H-NMR (400 MHz, CDCl )
3
3
δ 8.21 (d, J = 8.7 Hz, 2H), 7.66 (d, J = 8.7 Hz, 2H), 7.63 (d, J = 9.5 Hz, 1H), 7.43 (d, J =16.3
Hz, 1H), 7.38-7.34 (m, 3H), 6.93-6.84 (m, 4H), 6.25 (d, J = 9.5 Hz, 1H), 6.24-6.19 (m, 1H),
5
4
1
.44 (d, J = 13.9 Hz, 1H), 5.27 (d, J = 13.9 Hz, 1H), 5.05 (s, 2H), 4.69 (d, J = 5.7 Hz, 2H),
.27-4.24 (m, 1H), 4.20 (dd, J = 2.6, 9.4 Hz, 1H), 3.43-3.35 (m, 1H), 3.24 (dd, J = 2.6, 5.6 Hz,
H), 1.26 (d, J = 6.2 Hz, 3H), 1.23 (d, J = 7.3 Hz, 3H), 0.86 (s, 9H), 0.08 (s, 3H), 0.06 (s, 3H).
4
.1.23. 4-Nitrobenzyl (4S,5R,6S)-6-((R)-1-hydroxyethyl)-4-methyl-7-oxo-3-((E)-3-(4-(((2-
oxo-2H-chromen-7-yl)oxy)methyl)phenoxy)prop-1-en-1-yl)-1-azabicyclo[3.2.0]hept-2-ene-2-
carboxylate (9b′)
Following the same procedure as that used for the synthesis of 8a', the reaction of silyl ether
9
b (44.0 mg, 0.057 mmol) and ammonium hydrogen fluoride (13.1 mg, 0.229 mmol) in a mixed
solution of NMP/DMF (1.2 mL, 1:3) gave target compound 9b' (17.0 mg, 51%) as a white
1
solid after purification by column chromatography on silica gel (EtOAc:n-hexane = 3:1). H-
NMR (400 MHz, CDCl ) δ 8.21 (d, J = 8.6 Hz, 2H), 7.66 (d, J = 8.8 Hz, 2H), 7.63 (d, J = 9.7
3
Hz, 1H), 7.43 (d, J =16.5 Hz, 1H), 7.38-7.33 (m, 3H), 6.92-6.85 (m, 4H), 6.26-6.19 (m, 2H),
5
4
1
.48 (d, J = 13.8 Hz, 1H), 5.25 (d, J = 13.8 Hz, 1H), 5.05 (s, 2H), 4.69 (d, J = 5.4 Hz, 2H),
.28-4.21 (m, 2H), 3.48-3.41 (m, 1H), 3.28 (dd, J = 2.4, 6.7 Hz, 1H), 1.38 (d, J = 6.2 Hz, 3H),
.24 (d, J = 7.5 Hz, 3H).
4
7
.1.24. (4S,5R,6S)-6-((R)-1-Hydroxyethyl)-4-methyl-7-oxo-3-((E)-3-(4-(((2-oxo-2H-chromen-
-yl)oxy)methyl)phenoxy)prop-1-en-1-yl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid
(
2b)
Following the same procedure as that used for the synthesis of 1a, the reaction of compound
b′ (40.0 mg, 0.061 mmol) and 5% Rh/C (3.6 mg) in a mixed solution of THF/H O (2.0 mL,
9
2
2
9