Combinatorial approach toward synthesis of small molecule libraries as bacterial transglycosylase inhibitors

Article abstract of DOI:10.1039/c000622j

The development of iminocyclitol-based small molecule libraries against a bacterial TGase is described. An iminocyclitol was conjugated with a pyrophosphate mimic using either a 1,3-dipolar cycloaddition or reductive amination reaction, which was then condensed with a variety of lipophilic carboxylic acids in an amide bond coupling to generate a desired molecular library. With assistance of microtiter plate-based combinatorial chemistry and in situ screening, a potential inhibitor, the first potent iminocyclitol-based inhibitor against bacterial TGases was efficiently developed. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/c000622j

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Combinatorial approach toward synthesis of small molecule libraries as
bacterial transglycosylase inhibitors†
Hao-Wei Shih, Kuo-Ting Chen, Shao-Kang Chen, Chia-Ying Huang, Ting-Jen R Cheng, Che Ma,
Chi-Huey Wong* and Wei-Chieh Cheng*
Received 15th January 2010, Accepted 19th February 2010
First published as an Advance Article on the web 29th March 2010
DOI: 10.1039/c000622j
The development of iminocyclitol-based small molecule libraries against a bacterial TGase is described.
An iminocyclitol was conjugated with a pyrophosphate mimic using either a 1,3-dipolar cycloaddition
or reductive amination reaction, which was then condensed with a variety of lipophilic carboxylic acids
in an amide bond coupling to generate a desired molecular library. With assistance of microtiter
plate-based combinatorial chemistry and in situ screening, a potential inhibitor, the first potent
iminocyclitol-based inhibitor against bacterial TGases was efficiently developed.
to the progress of TGase inhibitor discovery are the lack of
Introduction
both a convenient high throughput screening (HTS) assay, and
The crisis of multi-drug resistance requires the urgent discovery
an efficient synthetic protocol for the small molecule library
of new antibacterial drugs and targets.1 Inhibition of bacterial
preparation.
cell wall biosynthesis receives much attention as the lack of a
Recently, we have developed a HTS TGase assay called Flu-
eukaryotic counterpart reduces the potential for side effects.2
orescence Anisotropy (FA)-based Moenomycin A-binding assay
Among the several enzymes involved in cell-wall biosynthesis,
(also called fluorescence polarization binding assay (FP-binding
assay)).6 This assay can be used as a primary assay to quickly
identify potential hits from compound libraries, the activity
of which would be confirmed using the HPLC-based lipid II
polymerizing activity analysis (lipid II-based functional assay).7
The design and preparation of a TGase inhibitor library is
therefore the present challenge. To the best of our knowledge,
few TGase inhibitors have been reported, all of which are based
on the chemical structures of 1 or 2.8
We posited that a molecule comprising an iminocyclitol (also
called an azasugar), a pyrophosphate mimic and a lipophilic
moiety would have the potential to impart inhibitory activity, since
azasugars are thought to mimic the shape and charge distribution
of the oxonium ion transition state during the sugar transfer
process;9 the lipophilic part would interact with TGases or cell
membrane; and the pyrophosphate-like moiety would mimic the
pyrophosphate group or be a tether (Fig. 2).
transglycosylase (TGase), a class of glycosyltransferases (GTases)
(EC 2.4), catalyzes the transfer of the sugar moiety from the
activated polymeric peptidoglycan (a glycosyl donor) to the
specific hydroxyl group (4-OH) of Lipid II (1, a glycosyl acceptor),
with concomitant release of a C55 undecaprenyl pyrophosphate
carrier (Fig. 1).3 This bacterial enzyme (TGase) is considered
a promising antibiotic target, due to its essential function and
ready accessibility, it is located on the external surface of bacterial
membrane, within easy reach of inhibitors.4
As neither the undecaprenyl moiety (C55) of 1 nor moenocinol
(C25) moiety of 2 are easily accessible starting materials, we
adopted a combinatorial approach to identify a new, simple
lipophilic moiety. To simplify the library preparation, it was
decided to conjugate an adduct (iminocyclitol-pyrophosphate
mimic) with a lipophilic part by means of an amide bond
(temporary linker Z) rather than the native ether bond (linker
Y) because the amide bond is much easier than the ether bond
to form (Fig. 2). These simplified libraries are called ‘primary
libraries’. Each member of a given primary library was screened in
the FP-binding assay, and the bioactivity of the hits was confirmed
by using the lipid II-based functional assay. The structures of hits
were turned by, for example, synthesizing analogues containing the
native ether linker (Fig. 3). This technique allows rapid screening
of lipid moieties, as well as optimization of the orientation and
identities of the other fragments. Herein, a new TGase inhibitor
discovered using this strategy is disclosed.
Fig. 1 Chemical structures of lipid II (1) and moenomycin A (2) and
general function of transglycosylase (TGase).
To date, only moenomycin complexes such as moenomycins A
(2), A12, C1, C3, and C4, are known to inhibit TGases. However,
due to their poor bioavailability, moenomycins are currently used
only as growth promoters in animal feeds.5 The main impediments
Genomics Research Center, Academia Sinica, No. 128, Academia Road Sec.
2, Nankang District, Taipei, 11529, Taiwan
† Electronic supplementary information (ESI) available: Experimental
details and NMR spectra. See DOI: 10.1039/c000622j
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Fig. 4 Proposed TGase inhibitor library and retro-synthetic analysis.
Fig. 2 General structures and principles for a primary library design.
Scheme 1 Preparation of unsaturated phosphonate diester 4. Reagents
and conditions: (a) (COCl)2, CH2Cl2; (b) benzyl 3-hydroxypropylcarba-
mate, Et3N, CH2Cl2.
Fig. 3 The relationship between library synthesis and bio-evaluation.
Results and discussion
Preparation of the primary library A
Inspired by uridine diphosphono-b-Galf mimics,10 our initial
desired structure consists of an iminocyclitol, a lipophilic part, and
a 2-hydroxypropyl tether as shown in Fig. 4. In our retrosynthetic
analysis (Fig. 4), 3 is conjugated with 4 via a 1,3-dipolar
cycloaddition to give an adduct, which is linked to members of
a diverse acid library 5 in an amide-bond forming reaction to
generate our first primary library.
Cyclic nitrone 3 was prepared in four steps from tri-O-benzyl D-
arabinose according to our previous report.11 For the preparation
of 4, dibenzyl allylphosphonate (6)12 was treated with oxalyl
chloride to give phosphonochloridate 7, which was reacted with
benzyl 3-hydroxypropylcarbamate in the presence of triethylamine
to obtain 4 in overall 61% yield for two steps (Scheme 1).
As illustrated in Scheme 2, the cycloaddition of 3 and 4
gave 8 as a single product in 42% yield with high region- and
stereoselectivity. The configuration of the newly established chiral
Scheme 2 Preparation of the primary library A (10). Reagents and
conditions: (a) 4, TCE, 80 ◦C; (b) H2, Pd(OH)2, MeOH; (c) acid library 5,
HBTU, HOBt, DIEA, DMF.
centers in 8 was determined by the NMR analysis and similar
literature reports.10
Hydrogenation of 8 gave amine 9, which was coupled with
a randomly selected 63-membered acid library 5 (Table 1) by
prior activation of the carboxylic acids in the presence of HBTU
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Table 1 Diverse carboxylic acid library 5 for amide bond formation
(1.5 equiv), HOBt (1.5 equiv) and DIEA (3 equiv) in DMF. After
24 h, the reactions were analyzed by LC-MS. It was found that
amine 9 had been completely consumed and significant amount
of 10 formed (estimate conversion). The 63 members (10 mM) in
the primary library A (10) were directly evaluated in the HTS FP-
binding assay without further purification.13 Unfortunately, at an
inhibitor concentration of 1 mM, no hits were identified.
Scheme 3 Preparation of unsaturated phosphono methylserine 14.
Reagents and conditions: (a) 7, Et3N, CH2Cl2.
library 5 to generate the primary library B (17) in a microtiter
plate (Scheme 4). After the FP-binding assay screening, 17 hits
were found at 1 mM, none of which was potent in the further
functional assay screening at the same final concentration (1 mM).
This disappointing result suggested that a fundamental re-design
of the inhibitor was required.
Preparation of the primary library B
Our re-design was inspired from the phosphoglycerate moiety
in moenomycin A (2). Initial attempts to conjugate alcohol 11
with phosphonochloridate 7 were not successful, perhaps due to
decomposition of 13 via b-elimination.14 In contrast, the stable,
unsaturated phosphonomethylserine 14 could be prepared from
1215 in a reasonable yield (55%) (Scheme 3).
In a similar procedure to that depicted in Scheme 2, amine
16, prepared from 3 via 1,3-dipolar cycloaddition with 14, N–O
bond reduction, and debenzylation, was coupled with the acid
Preparation of the primary library C
After detailed studies of the chemical structures of the proposed
transition state, we designed a new iminocyclitol-based structure
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Table 2 N-Acetylation of 18 under various conditions
Entry conditions
time
T/◦C 19 (%)a 20 (%)a 18 (%)a
1
2
3
4
Ac2O
Ac2O/py.
AcOH/EDCI
10 min 4 to rt 18
42
33
47
8
33
42
33
8
2 h
8 h
4 to rt 13
rt
rt
8
73
Ac2O/py./2N HCl 3 h
a Isolated yield after column chromatography.
Dihydroxylation of alkene 14 using OsO4 and NMO followed
by oxidative cleavage with NaIO4 gave 21. Since aldehyde 21
was not stable to chromatography or even when left to stand at
room temperature, it was freshly prepared and used immediately
without purification. Condensation of 21 with 19 in the presence
of NaCNBH3, followed by hydrogenolysis gave 22 in 54% overall
yield over the four steps from 14 (Scheme 5). After rapid library
generation and in situ screening, 15 hits were found out at
1 mM. These were submitted for the lipid II-based functional
assay (activity assay) and two compounds 24 and 25 were found
as inhibitors (~60% inhibition) (Fig. 6). Our screening data
indicated the structure of the lipid moiety dramatically influences
the inhibition: lipophilic moieties in both active molecules were
branched not linear, biphenyl, or cholesterol-related.
Scheme 4 Preparation of the primary library B (17). Reagents and
conditions: (a) 14, TCE, 80 ◦C; (b) H2, Pd(OH)2, MeOH; (c) acid library
5, HBTU, HOBt, DIEA, DMF.
bearing an N-acetyl methyl group at the C-2 position and linking
with other fragments at the nitrogen on the ring system via
reductive amination (Fig. 5).
Fig. 5 Proposed transition state and desired iminocyclitol fragment 19.
According to our previous report,11 18 (ADMDP) was conve-
niently prepared from cyclic nitrone 3 via a four-step procedure
in 77% overall yield. Unfortunately, selective mono-acetylation of
18 on a multi-gram scale was not successful.16 Various conditions
were screened (Table 2), and it was found that the use of acetic
anhydride (1.5 equiv) in a buffer solution (pyridine and aqueous
HCl, pH ~ 4) could obtain 19 in satisfactory yield (73%; see
entry 4).
Scheme 5 Preparation of the primary library C (23). Reagents and
conditions: (a) (i) OsO4, NMO, acetone; (ii) NaIO4, THF; (b) (i) 19,
NaCNBH3, MeOH; (ii) H2, Pd(OH)2, MeOH; (c) acid library 5, HBTU,
HOBt, DIEA, DMF.
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Fig. 6 Chemical structures of 24 and 25.
Table 3 TGase inhibition study
Compound
Inhibition% a(1 mM)
Inhibition% (100 mM)
b
22
—
—
11
15
>80
< 10
< 10
< 10
—
—
13
24
69
73
100
38
71
82
—
—
41
77
100
25
31
32
Scheme 6 Preparation of 31 bearing the ether linkage. Reagents and
conditions: (a) (i) NaH, 3,7,11-trimethyldodecane bromide, DMF; (ii)
CSA, MeOH; (b) (i) BAIB, TEMPO, acetone; (ii) DCC, MeOH; (iii) BCl3,
CH2Cl2; (c) 7, Et3N, CH2Cl2; (d) (i) OsO4, NMO, acetone; (ii) NaIO4,
THF; (e) (i) 19, NaCNBH3, MeOH; (ii) H2, Pd(OH)2, MeOH.
33
34
35
36
37
38
42
100
Moe A (2)c
a 1. Inhibition % calculated by comparing the reaction rate in the presence
of the compound to the rate in the absence of any inhibitory compound.
2. E. Coli. PBP1b test system. b — refers to no inhibition. c IC50 of
moenomycin A (2) was around 40 nM in our HPLC activity assay system.
results showed the lipophilic moiety to be essential for inhibition of
TGase activity. For example, compounds 24 and 25 exhibited the
inhibitory potency but 22, the truncated form without a lipophilic
part, did not. Similar observations have been previously reported
in studies of Moenomycin A (2), when the lipophilic moiety
(moenocinol) was removed from 2, the inhibitory activity was
completely lost.20 Our results also indicated that the branched
lipid-linked 31 was a more potent inhibitor (80% inhibition at
100 mM) than the linear lipid-linked 32 (almost no inhibition
at 100 mM). The linkage between the lipophilic moiety and
the pyrophosphate-mimic moiety was also found to affect the
inhibitory activity, 31 (with an ether linkage) was five times more
potent than 25 (with an amide linkage) (Table 3).
In this study, 31 exhibited the best inhibitory potency against
E. coli transglycosylase PBP1b. When presented at concentrations
of 1 mM and 100 mM, the inhibition activities were 100% and
80%, respectively, two and six times more potent than the lipid
II-based inhibitor 38 or lipid I (37), respectively. In contrast,
35 and 36, with no iminocyclitol moiety, exhibited no inhibitory
activity. Presumably, 31 is a transition-state mimic inhibitor and
its lipophilic part can interact both with the enzyme and also the
cell membrane, increasing its local concentration.21 In addition,
the N-acetyl methyl moiety at the C2 position on the pyrrolidine
ring played a crucial role for inhibition (Table 3; 31 v.s. 33).
Compound 31 was also tested against other TGases including C.
difficile, S. aureus and H. pylori, and found to exhibit an inhibitory
effect against all three (>80% inhibition at 100 mM). Although
the MIC value of 31 against S. aureus was lower (125 mM), it
still constituents a useful lead in the search for more potent and
selective inhibitors.
After re-synthesis of 24 and 25, followed by purification, the
inhibitory activities were re-examined (Table 3). It can be seen
that they are slightly higher than that of the unpurified samples,
synthesized in the microtiter plate-based parallel synthesis.
Next, the amide linkage of 24 was replaced by the original
ether linkage and the inhibitory potency was investigated again
(Scheme 6). Alkylation of 2617 with 3,6,9-trimethyldodecanyl
bromide, followed by acid medicated hydrolysis gave 27 (60%).
Alcohol 27 was converted to the corresponding 28 in 47%
overall yield for three steps: oxidation (bis-acetoxyiodobenzene
(BAIB), 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMO)),18
esterification, and debenzylation. Notably, debenzylation in acid
mediated conditions (BCl3/CH2Cl2) was found to be more efficient
and practical than catalytic hydrogenolysis, even after a prolonged
reaction time (3 days) under 70 psi of hydrogen.
Following the similar transformations (14 → 22) in Scheme 5,
compound 31, containing an ether linkage instead of amide
group, was generated in 38% overall yield for four steps from 29.
Similarly, compound 32 was prepared from 26 using dodecanyl
bromide as an alkylation reagent. For comparison purposes,
several interesting molecules, shown in Fig. 7, were prepared such
as two iminocyclitol-based compounds 33 and 34, lipid I (37), and
lipid II analogue 38.19
All of the compounds described above were tested for inhibitory
activity against the E. coli transglycosylase PBP1b (Table 3). The
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(1H at 600 MHz; 13C at 150 MHz) were recorded on a spectrometer
in d-solvent at ambient temperature. Mass spectra were obtained
by BRUKER Daltonics Bio-TOF III. CC refers to column
chromatography.
19. Ac2O (0.12 mL, 1.2 mmol) in water (1 mL) was added
dropwise to a solution of diamine 18 (0.2 g, 1.2 mmol) in a buffer
solution of 2 N HCl (0.6 mL, 1.2 mmol) and pyridine (0.2 mL,
2.6 mmol) in water (5 mL). The mixture was stirred for 3 h. After
the reaction was completed (monitored by TLC), Dowex 550A
resin was added to adjust the pH value to 8. The resin was removed
by filtration and filtrate was evaporated to dryness. The residue
was purified by CC (n-propanol : 2 N NH4OH = 7 : 3) to give oil
1
compound in 73% yield. H NMR (600 MHz, CDCl3) d 1.87 (s,
3H), 3.40–3.60 (m, 4H), 3.79 (dd, 1H, J = 6.1 and 12.4 Hz), 3.87
(dd, 1H, J = 3.7 and 12.4 Hz), 3.97 (t, 1H, J = 6.6 Hz), 4.01 (t, 1H,
J = 7.2 Hz); 13C NMR (150 MHz, CDCl3) d 170.7, 76.2, 74.7, 62.4,
60.9, 58.6, 39.1, 21.6; HRMS calcd for [C8H16N2O4+H]+ 205.1188,
found 205.1198.
22. To a solution of 14 (1.5 g, 3.5 mmol) in acetone (30 mL) and
water (5 mL) was added N-methylmorpholine N-oxide (NMO)
(1 g, 7.7 mmol) and catalytic amount of osmium tetraoxide (OsO4).
The solution was stirred overnight. The reaction was quenched
by Na2SO3 (2 g) and stirred for another 1 h. The solution was
evaporated to dryness and the residue was washed with EtOAc
then filtrated. The organic layer was dried with MgSO4 and
concentrated. The residue was purified by CC (10% MeOH in
EA) to give unseparable diastereomers as a yellow oil in 77%
yield. 1H NMR (600 MHz, CDCl3) d 1.43 (s, 3H), 2.13–2.28 (m,
2H), 3.44 (m, 1H), 3.86–4.44 (m, 5H), 4.94–5.07 (m, 4H), 7.36–
7.44 (m, 10H); 13C NMR (150 MHz, CDCl3) d 169.9, 136.3, 134.9,
128.6 (¥ 2), 128.5 (¥ 2), 128.2 (¥ 2), 127.2 (¥ 4), 71.6, 69.1, 67.6,
67.4, 67.1, 60.1, 37.7, 20.1; HRMS calcd for [C21H26N3O7P+H]+
464.1587, found 464.1593.
The diol (1 g, 2 mmol) in THF (5 mL) and water (5 mL) was
added by sodium periodate (NaIO4) (0.64 g, 3 mmol) and stired
1 h at RT. The precipitate was filtrated and washed with THF.
The organic layer was evaporated to dryness and the aldehyde was
used directly without further purification. The amine 19 (0.27 g,
1.3 mmol) in MeOH (10 mL) was added to the aldehyde and stirred
10 min at RT, then NaCNBH3 (0.2 g, 2.6 mmol) was added in one
portion and the mixture was stirred overnight. After the reaction
was complete, the solution was evaporated to dryness. The residue
was purified by CC (10% MeOH in DCM) to give unseparable
diastereomers as an oil in 72% yield. 1H NMR (600 MHz, CDCl3)
d 1.36 (s, 3H), 1.98 (s, 3H), 2.06–2.18 (m, 2H), 2.88–3.11 (m, 5H),
3.66–3.72 (m, 6H), 3.91–3.93 (m, 1H), 4.96–5.25 (m, 4H), 7.31–
7.41 (m, 10H); 13C NMR (150 MHz, CDCl3) d 170.1, 136.3, 134.9,
128.6 (¥ 2), 128.5 (¥ 2), 128.2 (¥ 2), 127.2 (¥ 4), 79.1, 69.3, 68.2,
67.9, 67.7, 67.6, 67.5, 66.8, 65.9, 65.1, 59.4, 39.5, 37.1, 21.3, 18.3;
HRMS calcd for [C28H38N5O9P+H]+ 620.2485, found 620.2491.
Asolutionofazide(0.2g, 0.5mmol), Pd(OH)2 and1dropAcOH
in MeOH (10 mL) was stirred 24 h under hydrogen atmosphere.
After the reaction was complete, the solution was filtrated and
evaporated to dryness to give the title compound as unseparable
diastereomers in 89% yield. 1H NMR (600 MHz, CDCl3) d 1.45 (s,
3H), 1.82–2.01 (m, 5H), 2.95–3.01 (m, 2H), 3.11 (br, 1H), 3.15 (br,
1H), 3.40–3.47 (m, 1H), 3.55 (dd, 1H, J = 4.2 and 14.4 Hz), 3.78
(d, 2H, J = 4.2 Hz), 3.86 (t, 1H J = 3 Hz), 3.93–3.95 (m, 1H), 3.99
(br, 1H), 4.11 (dd, 1H, J = 4.2 and 10.8 Hz); 13C NMR (150 MHz,
Fig. 7 Chemical structures of 32–38.
Conclusion
We have successfully developed a strategy to efficiently assemble
an iminocyclitol, a pyrophosphate mimic, and a lipophilic moiety
for the discovery of bacterial TGase inhibitors, and used it
to discover a novel, broad spectrum TGase inhibitor. Several
primary libraries were rapidly synthesized and evaluated, and
the optimized moieties and their orientations were disclosed.
The phosphono methylserine moiety in 22 can be considered
a preliminary surrogate of the pyrophosphate mimic, and the
amino group is the diversity point, by virtue of its capacity to
be easily, quickly, and directly conjugated with various acids. We
are currently using this approach to synthesize and identify other
potent glycosyltransferase inhibitors and also explore more potent
bacterial TGase inhibitors.
Experimental section
General information. All the solvents and reagents were obtained
commercially and used without further purification. NMR spectra
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CDCl3) d 174.5, 174.4, 78.6, 78.0, 67.7, 66.8, 66.7, 66.7, 66.2, 63.4,
61.2, 61.1, 58.4, 58.3, 41.7, 37.1, 37.0, 24.7, 24.4, 23.8, 21.8, 18.4;
HRMS calcd for [C14H28N3O9P+H]+ 414.1641, found 414.1649.
24. A solution of acid (0.08 g, 0.54 mmol), HBTU (0.2 g,
0.54 mmol), HOBT (0.07 g, 0.54 mmol) and DIEA (0.18 mL,
1.1 mmol) in dried DMF was stirred 15 min, then amine 22 (0.15 g,
0.36 mmol) was added. The reaction was stirred at RT overnight.
The solution was evaporated to dryness and residue was purified
by CC (n-propanol : 2 N NH4OH = 7 : 3) to give title compound
as unseparable diastereomers in 68% yield. 1H NMR (600 MHz,
CDCl3) d 0.84 (d, 3H, J = 6.6 Hz), 0.96 (br, 1H), 1.09–1.28 (m,
2H), 1.40 (s, 3H), 1.52 (s, 3H), 1.59 (s, 3H), 1.67 (br, 1H), 1.78–1.82
(m, 1H), 1.88–2.07 (m, 10H), 2.19 (dd, 1H, J = 6.0 and 14.4 Hz),
3.31–3.37 (m, 2H), 3.52–3.62 (m, 5H), 3.88–3.91 (m, 3H), 3.99
(br, 1H), 4.06–4.14 (m, 2H), 5.04 (t, 1H, J = 6.6 Hz); 13C NMR
(150 MHz, CDCl3) d180.9, 176.2, 176.2, 175.6, 175.5, 133.3, 125.8,
78.1, 18.0, 77.9, 77.8, 71.7, 71.5, 70.1, 68.7, 62.7, 62.7, 62.6, 58.9,
58.8, 46.2, 46.1, 44.6, 37.9, 31.5, 26.5, 26.3, 23.2, 20.3, 19.9, 18.1;
HRMS calcd for [C23H42N3O10P+H]+ 552.2686, found 552.2695.
25. The procedure used for the synthesis of 25 was similar to
24. Compound 25 was obtained as unseparable diastereomers.1H
NMR (600 MHz, CDCl3) d 0.77–0.90 (m, 12H), 1.01–1.33 (m,
12H), 1.44 (s, 3H), 1.88–1.85 (m, 1H), 1.98–2.23 (m, 1H), 3.28–
3.35 (m, 2H), 3.45–3.55 (m, 3H), 3.61–3.66 (m, 1H), 3.86–3.97 (m,
4H), 4.06–4.08 (m, 1H), 4.15–4.19 (m, 1H); 13C NMR (150 MHz,
CDCl3) d 178.6, 174.2, 174.1, 174.0, 173.9, 77.3, 77.2, 77.0, 70.0,
69.8, 68.4, 67.3, 65.7, 61.3, 61.2, 57.8, 57.6, 48.3, 48.1, 44.1, 43.1,
43.0, 39.3, 39.1, 37.3, 37.2, 37.1, 37.0, 37.0, 36.9, 36.6, 32.7,
32.4, 30.4, 30.1, 27.8, 27.7, 24.7, 24.4, 24.3; HRMS calcd for
[C29H56N3O10P+H]+ 638.3782, found 638.3788.
1.50–1.61 (m, 4H), 3.51–3.56 (m, 4H), 3.60–3.71 (m, 3H), 5.08
(d, 2H, J = 2.4 Hz), , 7.27–7.32 (m, 5H); 13C NMR (150 MHz,
CDCl3) d 137.9, 128.4 (¥ 2), 127.7, 127.6 (¥ 2), 78.4, 73.5, 69.9,
68.6, 62.7, 39.3, 37.4, 37.3, 37.2, 37.1, 37.0, 32.7, 29.8, 27.9, 24.8,
24.3, 22.6, 19.6, 19.5; HRMS calcd for [C25H44O3+H]+ 393.3369,
found 393.3363
28. To a solution of alcohol 27 (2.6 g, 6.3 mmol) in ace-
tone (30 mL) and water (5 mL) at 0 ◦C was added (bis-
acetoxyiodobenzene) (BAIB) (5.4 g, 16 mmol) and TEMPO (0.2 g,
1.3 mmol). The reaction was slowly warmed to RT and stirred
for 3 h. The mixture was poured into water, diluted with EtOAc
and extracted with water. The aqueous layer was extracted with
EtOAc twice and the combined EtOAc was further extracted with
Na2S2O3 and water. The organic layer was dried with MgSO4
and concentrated. The residue was used directly without further
purification. To a solution of acid in DCM (20 mL) was added
DCC (2.7 g, 13 mmol), MeOH (5 mL) and catalytic amount of
DMAP. The mixture was stirred at RT overnight. The reaction
was concentrated, washed with hexane and filtrated. The filtrate
was concentrated and used directly without further purification.
A solution of benzyl protected glycerate (1.3 g, 2.9 mmol) in DCM
◦
(50 mL) was cooled to -50 C and BCl3 (1M in hexane, 4.5 mL,
4.5 mmol) was added dropwisely. After the reaction was complete
(~ 1h), the solution was quenched with MeOH (10 mL) and TEA
(10 mL) and evaporated to dryness. The residual was purified
by CC (20% EtOAc in hexane) to give unseparable enantiomers
Yield : 88%. 1H NMR (600 MHz, CDCl3) d 0.81–1.58 (m, 29H),
3.40–3.46 (m, 1H), 3.70–3.86 (m, 5H), 3.96 (dd, 1H, J = 3.0 and
6.0 Hz); 13C NMR (150 MHz, CDCl3) d 171.4, 79.5, 69.7, 63.4,
52.0, 39.3, 37.7 (¥ 4), 32.8, 29.7, 27.9, 24.7, 24.3, 22.6 (¥ 2), 19.6
(¥ 2); HRMS calcd for [C19H38O4+H]+ 331.2848, found 331.2855.
29. The procedure used for the synthesis of 29 was similar to 4
26. To a solution of diol (5.7 g, 31 mmol) and tr◦iphenylmethyl
chloride (8.7 g, 31 mmol) in DCM (100 mL) at 0 C was slowly
added TEA (6.5 mL, 47 mmol). The reaction was slowly warmed to
RT and stirred for 2 h. The mixture was poured into water, diluted
1
(see supporting information). Yield : 71%. H NMR (600 MHz,
CDCl3) d 0.81–1.32 (m, 27H), 1.50 (br, 1H), 1.63 (br, 1H), 2.62
(m, 2H), 3.44 (m, 1H), 3.66 (m, 1H), 3.71 (m, 3H), 4.04 (br,
1H), 4.17–4.33 (m, 2H), 5.04 (m, 2H), 5.17 (m, 2H), 5.74 (m,
1H), 7.30–7.35 (m, 5H); 13C NMR (150 MHz, CDCl3) d 170.2,
136.2, 128.6 (¥ 2), 128.5, 127.8, 126.9 (¥ 2), 120.3, 78.2, 69.7, 67.2,
65.8, 52.1, 39.3, 37.7 (¥ 4), 37.3, 32.8, 29.7, 27.9, 24.7, 24.3, 22.6
(¥ 2), 19.6 (¥ 2); HRMS calcd for [C29H49O6P+H]+ 525.3345, found
525.3351.
31. The procedure used for the synthesis of 31 was similar to
22. Yield : 92%. 1H NMR (600 MHz, MeOH-d4) d 0.85–0.90 (m,
12H), 1.07–1.68 (m, 17H), 2.01 (s, 3H), 2.08 (m, 2H), 3.45–3.76
(m, 8H), 3.92–3.97 (m, 2H), 4.05 (t, 1H, J = 2.8 Hz), 4.09–4.16 (m,
4H); 13C NMR (150 MHz, MeOH-d4) d 176.6, 172.4, 81.7, 81.5,
79.3, 78.5, 69.6, 69.1, 68.9, 68.4, 68.2, 66.1, 64.8, 63.4, 58.5, 58.4,
48.5, 40.7, 39.1, 37.4 (¥ 4), 37.3, 37.2, 37.0, 36.4, 36.3, 32.6, 29.8,
29.7, 27.7, 24.5, 24.1, 24.0, 21.8 (¥ 2), 19.6 (¥ 2); HRMS calcd for
[C28H55N2O10P+H]+ 611.3673, found 611.3682.
with DCM and extracted with 1 N HCl, water and NaHCO3(aq)
.
The organic layer was dried with MgSO4 and concentrated.
The residue was purified by CC (DCM with 1% TEA) to give
1
unseparable enantiomers as yellow oil in 87% yield. H NMR
(600 MHz, CDCl3) d 3.21 (dd, 2H, J = 5.4 and 9.6 Hz), 3.57 (dd,
2H, J = 6.0 and 9.6 Hz), 3.98 (m, 1H), 4.53 (s, 2H), 7.18–7.42 (m,
20H); 13C NMR (150 MHz, CDCl3) d 144.1 (¥ 3), 138.3, 128.7
(¥ 6), 128.4 (¥ 2), 128.3 (¥ 2), 127.7 (¥ 6), 127.5, 126.9 (¥ 3), 86.5,
78.3, 73.2, 70.7, 70.5; HRMS calcd for [C29H28O3+H]+ 425.2116,
found 425.2122.
27. To a solution of alcohol 26 (5.1 g, 12 mmol) and 3,7,11-
trimethyldodecan-1-ol (4.5 mL, 18 mmol) in dried DMF (150 mL)
at 0 ◦C was added NaH (0.58 g, 14 mmol) in one portion. The
reaction was slowly warmed to RT and stirred for 5 h. The mixture
was poured into water, diluted with ether and extracted with water
and NaHCO3(aq). The organic layer was dried with MgSO4 and
concentrated. The residue was used without further purification.
A solution of alkane (5.1 g, 7.6 mmol) and camphorsulfonic acid
(CSA) (0.53 g, 2.2 mmol) in MeOH (100 mL) was reflux for
1 h. The reaction was quenched with TEA and evaporated to
32. The procedure used for the synthesis of 32 was similar to
1
22. Yield : 88%. H NMR (600 MHz, MeOH-d4) d 0.88 (t, 3H,
J = 7.0 Hz), 1.27 (br, 18H), 1.60 (br, 2H), 2.02 (s, 3H), 2.12 (br,
2H), 3.44 (br, 1H), 3.51–3.79 (m, 7H), 3.94 (br, 1H), 3.97 (t, 1H,
J = 3.8 Hz), 4.08 (br, 1H), 4.12 (br, 3H); 13C NMR (150 MHz,
MeOH-d4) d 173.8, 173.3, 78.9, 77.0, 76.9, 71.0, 69.9 (¥ 2), 65.1,
63.8, 57.8, 48.8, 32.0, 29.7 (¥ 4), 29.6, 29.4, 26.1, 24.2, 22.7, 21.7,
13.4 (¥ 2); HRMS calcd for [C25H49N2O10P+H]+ 569.3203, found
569.3211.
dryness. The mixture was extracted with water and NaHCO3(aq)
.
The organic layer was dried with MgSO4 and concentrated. The
residue was purified by CC (30% EtOAc in hexane) to give
1
unseparable enantiomers as yellow oil. Yield : 82%. H NMR
(600 MHz, CDCl3) d 0.81–0.87 (m, 12H), 1.03–1.48 (m, 13H),
2592 | Org. Biomol. Chem., 2010, 8, 2586–2593
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Acknowledgements
This work was supported by National Science Council and
Academic Sinica.
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