Stereocontrolled Synthesis of a Potential Transition-State Inhibitor of the Salicylate Synthase MbtI from Mycobacterium tuberculosis

Article abstract of DOI:10.1021/acs.joc.5b00455

Mycobactins are small-molecule iron chelators (siderophores) produced by Mycobacterium tuberculosis (Mtb) for iron mobilization. The bifunctional salicylate synthase MbtI catalyzes the first step of mycobactin biosynthesis through the conversion of the primary metabolite chorismate into salicylic acid via isochorismate. We report the design, synthesis, and biochemical evaluation of an inhibitor based on the putative transition state (TS) for the isochorismatase partial reaction of MbtI. The inhibitor mimics the hypothesized charge buildup at C-4 of chorismate in the TS as well as C-O bond formation at C-6. Another important design element of the inhibitor is replacement of the labile pyruvate side chain in chorismate with a stable C-linked propionate isostere. We developed a stereocontrolled synthesis of the highly functionalized cyclohexene inhibitor that features an asymmetric aldol reaction using a titanium enolate, diastereoselective Grignard addition to a tert-butanesulfinyl aldimine, and ring closing olefin metathesis as key steps.

Full text of DOI:10.1021/acs.joc.5b00455

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Article
Stereocontrolled Synthesis of a Potential Transition-State Inhibitor
of the Salicylate Synthase MbtI from Mycobacterium tuberculosis
Zheng Liu, Feng Liu, and Courtney C. Aldrich
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 02 Jun 2015
Downloaded from http://pubs.acs.org on June 5, 2015
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Stereocontrolled Synthesis of a Potential
Transition-State Inhibitor of the Salicylate
Synthase MbtI from Mycobacterium tuberculosis
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Zheng Liu,1,2 Feng Liu, 1 and Courtney C. Aldrich1 *
1Department of Medicinal Chemistry, University of Minnesota, 308 Harvard St. SE,
Minneapolis, MN 55455 (USA).
2Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, College of Chemistry,
Central China Normal University, 152 Luoyu Road, Wuhan, Hubei 430079 (China)
TBSO
Mg2+
O
HO
O
O
O
RCM
1
OH
2
3
OH
O
6
OH
5
4
MgCl
CO2
NH2
O
OH
OH
OH
Putative transition state
Transitionꢀstate analogue
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ABSTRACT: Mycobactins are smallꢀmolecule iron chelators (siderophores) produced by
Mycobacterium tuberculosis (Mtb) for iron mobilization. The bifunctional salicylate synthase
MbtI catalyzes the first step of mycobactin biosynthesis through the conversion of the primary
metabolite chorismate into salicylic acid via isochorismate. We report the design, synthesis and
biochemical evaluation of an inhibitor based on the putative transitionꢀstate (TS) for the
isochorismatase partial reaction of MbtI. The inhibitor mimics the hypothesized charge buildꢀup
at Cꢀ4 of chorismate in the TS as well as CꢀO bondꢀformation at Cꢀ6. Another important design
element of the inhibitor is replacement of the labile pyruvate sideꢀchain in chorismate with a
stable Cꢀlinked propionate isostere. We developed a stereocontrolled synthesis of the highly
functionalized cyclohexene inhibitor that features an asymmetric aldol reaction using a titanium
enolate, diastereoselective Grignard addition to a tertꢀbutanesulfinyl aldimine, and ring closing
olefin metathesis as key steps.
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INTRODUCTION
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Tuberculosis (TB), the leading cause of infectious disease mortality after HIVꢀAIDS, is due
to members of the Mycobacterium tuberculosis (Mtb) complex that includes seven closely related
species.1 The emergence of multiꢀdrug and extensivelyꢀdrug resistant strains of Mtb has renewed
focus on the development of antiꢀtubercular agents with novel modes of action.2 With the
exception of pyrazinamide, whose mechanism remains an enigma,3 the antibiotics presently used
in TB chemotherapy target a limited set of biochemical pathways in macromolecular and cofactor
biosynthesis.4 Iron is a required cofactor for more than 40 enzymes in Mtb, but is highly
restricted in a vertebrate host where the concentration of free iron (Fe2+) is estimated as 10ꢀ24 M.5
In order to obtain iron Mtb deploys two complementary strategies: synthesis of the mycobactin
siderophores,6 which are smallꢀmolecule iron chelators that scavenge iron from host tissues and
uptake of heme through a specialized heme receptor followed by heme degradation to release the
iron.7 The relative contribution of each mechanism for iron mobilization in vivo is unknown and
both may play a role during different stages of infection. Mtb mutants unable to produce
mycobactins cannot replicate under ironꢀrestricted conditions and are cleared in vivo, thereby
confirming the importance of mycobactinꢀmediated iron acquisition in Mtb.8
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The bifunctional salicylate synthase MbtI is responsible for the first committed step in
mycobactin biosynthesis through the conversion of chorismate into salicylic acid, which is
accomplished in two partial reactions at the same active site.9 In the first reaction, MbtI catalyzes
the interconversion of chorismate to isochorismate that has been hypothesized to proceed through
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a concerted SN2′′ reaction mechanism via transition state TS1 (Figure 1).9c,
The
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isochorismatase activity of MbtI requires Lys205, which nucleophilically activates a water
molecule for attack on chorismate at Cꢀ6 and Glu252 that polarizes the Cꢀ4 hydroxylꢀleaving
group. In the second reaction, pyruvate is eliminated through an intramolecular [3,3]ꢀ
sigmatropic rearrangement, formerly a retroꢀEne reaction, to afford salicylic acid via bicyclic
transition state TS2 (Figure 1).11
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MbtI represents an appealing target for development of inhibitors of mycobactin biosynthesis
since it is structurally and biochemically characterized, has no human orthologs, and is
conditionally essential under ironꢀdeficient conditions. Following the foundational studies of
Abell et al. on inhibitors of chorismateꢀutilizing enzymes,12aꢀe Payne and coworkers were the first
to disclose inhibitors of MbtI and their most potent compound is the rationally designed substrate
mimic 1 (Figure 2A) containing a 2,3ꢀdihydroxybenzoate scaffold.12fꢀg This compound is a
competitive inhibitor and possesses an inhibition constant (Ki) of approximately 10 µM. In a
complementary approach employing highꢀthroughput screening, Vasan and coꢀworkers reported
benzimidazoleꢀ2ꢀthione 2 as a noncompetitive inhibitor with similar potency to 1 (Figure 2A).13
Based on the modest potency of the described MbtI inhibitors, we chose to investigate potential
transitionꢀstate (TS) inhibitors of MbtI. TS inhibitors exploit the affinity of an enzyme for the TS
relative to the Michaelis complex.14 Capturing even a small percentage of the TS binding energy
with an inhibitor, in theory, allows the design of exceptionally potent inhibitors. TS strategies
are less successful when the nonꢀcatalyzed rate is high since the amount of potential TS
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stabilization will be smaller. In the case of MbtI, the first transition state TS1 is more desirable
to mimic than the second bicyclic transition state TS2 due to ease of the nonꢀcatalyzed
sigmatropic rearrangment.11, 15
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Inspired by the pioneering work of Kozlowski, Bartlett et al., who reported on the synthesis
of 3 as a TS inhibitor for several chorismateꢀutilizing enzymes,16 we report the synthesis and
characterization of cyclohexene 4 that mimics putative TS1 through incorporation of an amino
group at Cꢀ4 to resemble the charged leaving group of TS1 (Figure 2B). Another key design
element is replacement of the sigmatropically labile pyruvate sideꢀchain at Cꢀ5 with a stable Cꢀ
linked propionate fragment because MbtI possesses pyruvate lyase activity, unlike the
monofunctional chorismateꢀutilizing enzymes investigated by Kozlowski et al.. Interestingly,
inhibitor 4 bears a striking resemblance to Oseltamivir (trade name Tamiflu) 5 (Figure 2C), an
antiviral drug developed at Gilead that is a TS inhibitor of viral neuraminidase.17
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H2N Lys205
2+
Mg
Mg2+
Mg2+
O
H
O
O
O
O
HO
O
O
H
1
2
3
H
O
OH
O
6
5
O
CO2
4
CO2
CO2
OH
OH2
isochorismate
chorismate
HH
TS1
H
δ
O
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O
Glu252
Mg2+
Mg2+
O
O
O
HO
O
O
O
+
Me
H
O
OH
O
CO2
pyruvate
H
salicylate
TS2
O
N
R
OH
O
O
OH
H
OH
N
O
N
N
O
N
H
O
Me
O
mycobactins
Figure 1. Conversion of chorismate to salicylate via isochorismate catalyzed by MbtI.
A
C
HO
O
EtO
O
H
N
OH
O
S
N
O
NH2
NHAc
O
HO2C
1, Ki = 11 ± 1 ꢁM
2, Ki = 9.2 ± 0.9 ꢁM
5, Ki >100 ꢁM
B
HO
O
HO
O
HO
O
OH
OH
O
OH
O
δ
OH
OH
CO2
NH3
O
OH2
NH3
O
δ
TS1
(±)-3
4
Figure 2. (A) Reported MbtI inhibitors; (B) Rationale for TS inhibitor design of 4; (C)
Oseltamivir (Tamiflu) a neuraminidase TS inhibitor. Ki values are with respect to MbtI.
RESULTS AND DISCUSSION
Retrosynthetic Plan. The challenge of the target molecule 4 resides in the construction of
three contiguous stereocenters in the highly functionalized cyclohexene scaffold. Our first
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retrosynthetic plan involved disconnection of the Cꢀ5 propionate sideꢀchain to thionocarbonate 6
through a radical coupling with tertꢀbutyl acrylate. This would allow us to dovetail into the
elegant racemic synthesis of 7 reported by Kozlowski and coꢀworkers (Figure 3A).16b We also
noted the structural similarity between 4 and Oseltamivir 5 (Figure 2B and 2C), and were
inspired by the efficient ringꢀclosing metathesis (RCM) reaction reported by Yao and
coworkers18 for construction of the cyclohexene core of Oseltamivir 5. Accordingly, as shown in
Figure 3B, RCM disconnection of 8 leads to acyclic diene 9 that can be further simplified to syn
aldol adduct 10 via an asymmetric Grignard addition employing Ellman's Nꢀsulfinamide
auxiliary.19 Intermediate 10 can be readily assembled through synꢀaldol reaction between
oxazolidinone 11 and aldehyde 12. An advantage of this second approach is the ability to control
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stereochemistry on an acyclic precursor using contemporary methods in asymmetric synthesis.
A
HO
O
EtO
O
EtO
O
OH
OR1
S
OTBS
OH
O
O
NHR2
OPh
NH2
OH
NHBoc
Barton radical
coupling
(±)ꢀ4
(±)ꢀ6
(±)ꢀ7
B
R1O
RCM
R1O
asymmetric
allyllation
HO
O
OH
OR3
OR3
O
NH2
OH
NHBoc
OR2
tꢀBu
NH
S (S)
OR2
4
8
9
O
asymmetric
aldol
OR3
O
O
O
N
OR1
10
H
OR1
O
+
O
OR2
11
12
Bn
OR2
Figure 3. Retrosynthetic analysis.
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Radical coupling strategy. Silyloxybutadiene 13 was prepared from crotonaldehyde in 78%
yield through modification of the reported procedure16b using TBSOTf. The key DielsꢀAlder
reaction between 13 and ethyl propiolate afforded cyclohexadiene in variable yields from 0–61%
(from more than a dozen attempts) with the aromatized benzoate derivative as a major byproduct
even though the reaction was rigorously degassed and antioxidants were employed.
Notwithstanding the inconsistent yields several grams of 14 were secured and elaborated to
cyclohexene derivative (±)ꢀ7 as described16b in four steps with an overall yield of 24% (average
of 70% per step). Conversion to the thionocarbonate (±)ꢀ6 mediated by Nꢀmethylimidazole
(NMI) proceeded in 90% yield at room temperature, whereas 4ꢀdimethylaminopyridine (DMAP),
a more conventional acylation catalyst resulted in only low conversion (<10%).20 Several
conditions were explored to effect a radical coupling with tertꢀbutyl acrylate and other radical
traps21; however, despite considerable effort, only the deoxygenated product (±)ꢀ16 was isolated
in yields up to 63% yield (84% brsm) likely due to the sterically demanding environment at Cꢀ5.
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Scheme 1. Attempted introduction of propionate sideꢀchaina
EtO
O
(24%, 4 steps)
(lit. 35%)
OTBS
b) ꢀ
CO2Et
OTBS
a) TBSOTf
CHO
13
14, 0–61%
EtO
O
EtO
O
EtO
O
S
OTBS
S
OTBS
OTBS
d) AIBN
c)
Cl
OPh
CO2tBu
O
OPh
OH
NHBoc
NHBoc
NHBoc
(±)ꢀ7
(±)ꢀ15 (90%)
(±)ꢀ16 (63%)
aReagents and conditions: (a) TBSOTf, Et3N, CH2Cl2, ꢀ20 °C→rt, (b) 2,6ꢀdiꢀtertꢀbutylꢀ4ꢀ
methylphenol (BHT) or methylene blue (0.05 equiv), PhCH3, 65–80 °C, 6–8 d; (c) Nꢀ
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methylimidazole (3.5 equiv), CH2Cl2, rt, 24 h; (d) representative conditions: (Me3Si)3Si (1.05
equiv), tertꢀbutyl acrylate (10 equiv), AIBN (0.8 equiv), PhCH3, reflux, 2 h.
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RCM approach. As a result of the capricious yield of the cyclohexadiene 14 coupled with
inability to advance intermediate 15, we next focused attention on the complementary RCM
route as described below since the propionate sideꢀchain is installed early in the synthesis.
Synthesis began with acylation of Evans' (R)ꢀphenylalanineꢀderived oxazolidinone with the
known carboxylic acid 1722 under mixed anhydride conditions to afford Nꢀacyloxazolidinone 18
in 93% yield. The required aldehyde 19 coupling partner was prepared with a single purification
step through BaylisꢀHillman reaction23 of tertꢀbutyl acrylate and formaldehyde in aqueous
tetrahydrofuran (THF) followed by tertꢀbutyldimethylsilyl (TBS) protection and
diisobutylaluminum hydride (DIBALꢀH) reduction (Scheme 2, bottom insert). Initially,
asymmetric boronꢀmediated aldol reaction24 of 18 with aldehyde 19 using dibutylboron triflate as
a Lewis acid in Et2O provided the syn aldol adduct 20 in high diastereoselectivity (dr > 20:1) but
only 36% yield. As an alternative, the titanium enolate of 18 was investigated according to
methodology developed by Crimmins and coworkers.25 Treatment of 18 with TiCl4 and 2.5
equiv of (–)ꢀsparteine in CH2Cl2 at ꢀ78 °C followed by the addition of aldehyde 19 afforded 20
(dr > 20:1) in an improved 48% yield. Protection as a methoxymethyl (MOM) ether employing
CH3OCH2Cl and catalytic tetrabutylammonium iodide (TBAI) furnished 21. The chiral auxiliary
was then reductively removed with sodium borohydride26 and DessꢀMartin oxidation27 of the
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resultant alcohol 22 provided aldehyde 23.
Condensation with Ellman's (S)ꢀ(–)ꢀtertꢀ
butanesulfinamide auxiliary yielded tertꢀbutanesulfinyl aldimine 24. As first reported by Xu and
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coꢀworkers the combination of pyridinium pꢀtoluenesulfonate (PPTS) and anhydrous CuSO4
dramatically promoted imine formation, which was necessary for the sterically congested
aldehyde 23.28 The stereocontrolled installation of the allyl group in 25 was achieved in 87%
yield and excellent diastereoselectivity (dr > 20:1) by the addition of allyl magnesium bromide to
24 in CH2Cl2 at ꢀ78 °C.19, 29 Ringꢀclosing metathesis of 25 employing the Grubbs secondꢀ
generation catalyst30 gave cyclohexene derivative 26 in 95% yield.
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Scheme 2. Synthesis of cyclohexene 26a
O
O
O
a) PivCl
HO
OPMB
OPMB
O
N
O
b)
17
Bn
18 (93%, two steps)
O
NH
(R)
Bn
O
O
OMOM
c) TiCl4
O
O
OH OTBS
d) MOMCl
sparteine
O
N
OTBS
O
N
O
OTBS
Bn
Bn
H19
OPMB
OPMB
OTBS
20 (48%)
21 (82%)
OMOM
OMOM
e) NaBH4
f) DMP
HO
OTBS
O
OPMB
22 (67%)
OPMB
23 (90%)
tBu
tꢀBu
O
g)
h)
S
(S)
S
(S)
S
NH
O
OMOM
MgBr
tBu
NH2
O
N
OMOM
(S)
OTBS
OTBS
24 (62%)
(75% brsm)
25 (87%)
OPMB
OPMB
TBSO
10
1
TBSO
O
O
j) HCHO
DABCO
OMOM
i) Grubbsꢀ2
2
3
H
6
OtBu
8
k) TBSCl
OPMB
5
19
4
l) DIBALꢀH
9
7
tBu
NH
(36%, three steps)
S (S)
O
26
(69%, 79% brsm)
aReagents and conditions: (a) PivCl, Et3N, THF, 0 °C, 6 h; (b) (R)ꢀ4ꢀ(benzyl)oxazolidinꢀ2ꢀone,
nꢀBuLi, ꢀ78 °C, 5 min, then added to the mixed anhydride, ꢀ78 °C for 30 min, 0 °C for 30 min; (c)
TiCl4, (ꢀ)ꢀsparteine, CH2Cl2, ꢀ78 °C, 1.5 h, then 19 was added, ꢀ40 °C for 3 h, ꢀ20 °C overnight;
(d) CH3OCH2Cl, iꢀPr2NEt, TBAI, toluene, 90 °C, 7 h; (e) NaBH4, THF/H2O (3:1), 0 °C to rt,
overnight; (f) DessꢀMartin periodinane, NaHCO3, CH2Cl2, rt, 30 min; (g) (S)ꢀ(–)ꢀ2ꢀmethylꢀ2ꢀ
propanesulfinamide, anhydrous CuSO4, PPTS, CH2Cl2, rt, 24 h; (h) allylmagnesium bromide,
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CH2Cl2, ꢀ20 °C, 2.5 h; (i) 14 mol% Grubbs secondꢀgeneration catalyst, CH2Cl2, reflux, 14 h, then
DMSO was added, rt, 16 h; (j) HCHO, DABCO, THF–H2O (1:1), reflux, 16 h; (k) TBSCl,
imidazole, CH2Cl2, rt, 16 h; (l) DIBALꢀH, CH2Cl2, ꢀ78 °C, 1 h.
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The remaining synthetic operations involve oxidation of the primary alcohols at Cꢀ9 and Cꢀ
10, removal of the tertꢀbutanesulfinamide auxiliary, and deprotection of the MOM group at Cꢀ6
of cyclohexene 26. We initially sought to adjust the oxidation level at Cꢀ9 and Cꢀ10 and
maintain the tertꢀbutanesulfinamide as a nitrogen protecting group. However we discovered the
tertꢀbutanesulfinamide moiety is readily oxidized under mild oxidation conditions to a
sulfonamide,19 which undergoes facile elimination due to its axial conformation combined with
the enhanced acidity of the vicinal allylic proton. Thus, the sulfinamide auxiliary was first
removed along with the TBS group by treatment with 4 M HCl in dioxane–MeOH. The resultant
amino alcohol was Bocꢀprotected to afford 27 in 89% yield over two steps. Subsequent PMB
deprotection with 2,3ꢀdichloroꢀ5,6ꢀdicyanoꢀ1,4ꢀbenzoquinone (DDQ) gave diol 28. Direct
oxidation to the dicarboxylic acid 29 was very low yielding, so we employed a twoꢀstage
procedure with tetrapropylammonium perruthenate (TPAP)31 to provide an intermediate
dialdehyde that was converted to dicarboxylic acid 29 through PinnickꢀLindgren oxidation.32
Deprotection of 29 with trimethylsilyl bromide33 in CH2Cl2 at ꢀ78 °C afforded enantiopure target
compound 4 as the acetate salt that was purified by silica gel chromatography employing a
quaternary solvent system consisting of CHCl3–MeOH–H2O–AcOH.
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Scheme 3. Synthesis of 5a
5
HO
HO
6
7
8
9
OMOM
OMOM
OH
c) DDQ
a) HCl
26
OPMB
b) Boc2O
NHBoc
NHBoc
28 (89%)
27 (89%, two steps)
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HO
O
HO
O
OMOM
OH
f) TMSBr
d) TPAP
e) NaClO2
OH
OH
NHBoc
O
NH
O
3 OAc
29 (46%, two steps)
4 (50%)
aReagents and conditions: (a) 4 M HCl in 1,4ꢀdioxane, MeOH, 0 °C, 1 h; (b) Boc2O, Et3N, 1,4ꢀ
dioxane/H2O (2:1), rt, 1 h; (c) DDQ, CH2Cl2–H2O (20:1), rt, 2 h; (d) tetrapropylammonium
perruthenate, NMO, 4 Å molecular sieves, CH2Cl2, rt, 15 min; (e) NaClO2, 2ꢀmethylꢀ2ꢀbutene, tꢀ
BuOH–THF–H2O (5:1:1), rt, 2 h; (f) TMSBr, CH2Cl2, ꢀ78 °C, 1 h.
The relative configurations of three contiguous stereocenters in 5 were confirmed by the 2D
NOESY studies of cyclohexene 28. As shown in Figure 4, NOE correlation of Hꢀ4 with Hꢀ7 and
Hꢀ8 indicates an anti relationship between Hꢀ4 and Hꢀ5. Due to NOE correlation of Hꢀ6 with Hꢀ
7 and Hꢀ8 as well as the correlation of Hꢀ5 with the CH2 of the MOM group at Cꢀ6, we conclude
that Hꢀ5 and Hꢀ6 also have an anti relationship.
OH
0.35
0.90
9
8
7
2
3
OH
5
0.76
4
6
1.00
NH
O
Boc
0.66
OMe
Figure 4. Key NOE correlations of 28. The integral of the Hꢀ6/Hꢀ9 crossꢀpeak was used as the
internal calibrant (its integral was set to 1.00).
Biological Evaluation. The putative transitionꢀstate inhibitor 4 was evaluated for enzyme
inhibition against recombinant MbtI under initialꢀvelocity conditions as described,13 but showed
less than 10% inhibition at 100 µM. The modest potency of 4 clearly indicates it is a poor TS
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mimic. To rationalize the observed activity, we docked 4 into MbtI using the recently reported
coꢀcrystal structure of MbtI with a chorismate analog.34 Introduction of a CH2 moiety in place of
the Cꢀ5 oxygen atom of chorismate, led to loss of key hydrogen bond with Arg405 while the
protonated Cꢀ4 amino group made a potentially repulsive electrostatic interaction with Arg405
(Figure S1).
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CONCLUSION
We designed and synthesized an inhibitor based on the hypothetical transition state of the
isochorismate partial reaction catalyzed by MbtI wherein the Cꢀ4 hydroxyl group of chorismate is
protonated by Glu252 resulting in bond cleavage and concomitant CꢀO bond formation at Cꢀ6
due to nucleophilic activation of a water molecule by Lys205. MbtI is a bifunctional enzyme and
also catalyzes pyruvate elimination via an intramolecular [3,3]ꢀsigmatropic reaction. In order to
prevent this potential reaction from occurring in our inhibitor, the pyruvate sideꢀchain was
replaced with a stable propionate isostere. Two complementary synthetic routes were explored to
the target inhibitor 4. The initial route capitalized on the beautiful chemistry developed by
Bartlett and Kozlowski for the preparation of a cyclohexene intermediate (±)ꢀ7. Frustrated by
our inability to install the propionate sideꢀchain through a radicalꢀmediated process and the fickle
yield in the key DielsꢀAlder reaction, we undertook a novel synthetic route to enantiopure 4.
This second approach featured an asymmetric aldol reaction of a titanium enolate, a
diastereoselective Grignard addition to a tertꢀbutylsulfinyl aldimine, and ring closing olefin
metathesis as key steps. Enzyme inhibition studies revealed 4 is a poor TS mimic and potentially
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suggests an alternate TS that does not involve charge buildꢀup at Cꢀ4 and/or a more prominent
role of the Cꢀ5 oxygen atom in chorismate for binding. These studies provide a synthetic and
mechanistic foundation for future efforts to develop TSꢀbased inhibitors of MbtI.
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EXPERIMENTAL SECTION
General Method. All reactions were carried out under a dry Ar atmosphere using ovenꢀdried
glassware and magnetic stirring. The solvents were dried before use as follows: THF and Et2O
were heated at reflux over sodium benzophenone ketyl; toluene was heated at reflux over sodium;
CH2Cl2 was dried over CaH2. Anhydrous N,Nꢀdiisopropylethylamine, triethylamine were used
directly as purchased. Commercially available reagents were used without further purification
unless otherwise noted. Aluminum TLC sheets (silica gel 60 F254) of 0.2ꢀmm thickness were
used to monitor the reactions. The spots were visualized with short wavelength UV light or by
charring after spraying with a solution prepared from one of the following solutions:
phosphomolybdic acid (5.0 g) in 95% EtOH (100 mL); pꢀanisaldehyde solution (2.5 mL of pꢀ
anisaldehyde, 2 mL of AcOH, and 3.5 mL of conc. H2SO4 in 100 mL of 95% EtOH); or
ninhydrin solution (0.3g ninhydrin in 100 ml of nꢀbutanol; add 3 ml AcOH). Flash
chromatography was carried out with silica gel 60 (230ꢀ400 ASTM mesh). NMR spectra were
obtained on a 400ꢀMHz or 600ꢀMHz spectrometer. Proton chemical data are reported as follows:
chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br =
broad), coupling constant, and integration. Chemical shifts were referenced on residual solvent
peaks: CDCl3 (δ = 7.26 ppm for 1H NMR and 77.00 ppm for C NMR), CD2Cl2 (δ = 5.32 ppm
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for 1H NMR and 53.84 ppm for 13C NMR), CD3OD (δ = 3.31 ppm for H NMR and 49.00 ppm
for 13C NMR), D2O (δ = 4.79 ppm for 1H NMR). Optical rotations were measured at rt in a 1.0ꢀ
dm cell. Highꢀresolution mass spectrometry was performed on an linear trap quadrupole (LTQ)
mass spectrometer with electrospray ionization and a resolution of 60,000 (at m/z=400).
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(E)-1-(tert-Butyldimethylsilyloxy)-1,3-butadiene (13). To a solution of freshly distilled
crotonaldehyde (5.00 mL, 60.3 mmol, 1.00 equiv) in CH2Cl2 (70 mL) at ꢀ10 °C was added Et3N
(12.0 mL, 36.6 mmol, 1.40 equiv) and the reaction stirred 5 min. Next, TBSOTf (15.0 mL, 65.3
mmol, 1.10 equiv) was slowly added and the reaction warmed to 23 °C over 2 h, then stirred an
additional 16 h at 23 °C. The reaction was partitioned between CH2Cl2 (75 mL) and saturated
aqueous NaHCO3 (75 mL), dried (MgSO4) and concentrated under reduced pressure. The title
compound was purified by distillation over a Vigreux column (bp 60–62°C/11 mm Hg) to afford
the title compound (8.70 g, 78%) as a colorless liquid. The analytical data (1H, C NMR and
13
HRMS) matched the reported data for this compounds prepared by an alternate procedure
[reported yield 61% using crotonaldehyde (1.0 equiv) TBSCl (1.2 equiv), Et3N (1.2 equiv), ZnCl2
(0.013 equiv), hydroquinone (0.02 equiv), benzene, 80 °C, 24 h].16b
(±)-(4R,5S,6S) Ethyl 4-(tert-butoxycarbonylamino)-6-(tert-butyldimethylsilyloxy)-5-
(phenoxythiocarbonyloxy)cyclohex-1-enecarboxylate (15). To a solution of alcohol (±)ꢀ716b
(76.0 mg, 0.180 mmol, 1.00 equiv) in CH2Cl2 (5 mL) at 23 °C was added phenyl
thionochloroformate (50.0 ꢁL, 0.36 mmol, 2.00 equiv) followed by Nꢀmethylimidazole (50.0 ꢁL,
0.65 mmol, 3.60 equiv). The reaction mixture was stirred for 16 h at 23 °C then quenched with
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H2O (30 mL) and extracted with CH2Cl2 (3 × 30 mL). The combined organic extracts were
washed with saturated aqueous NaCl (30 mL), dried (Na2SO4) and concentrated in vacuo.
Purification by flash chromatography (5:1 hexane–EtOAc) afforded the title compound (89 mg,
90%) as white solid: 1H NMR (400 MHz, CDCl3) δ 0.16 (s, 3H), 0.30 (s, 3H), 0.90 (s, 9H), 1.33
(t, J = 7.1 Hz, 3H), 1.42 (s, 9H), 2.44–2.69 (m, 2H), 4.12–4.25 (m, 1H), 4.25–4.38 (m, 2H), 4.83
(s, 1H), 5.49–5.57 (m, 1H), 6.24 (d, J = 9.1 Hz, 1H), 7.04–7.11 (m, 3H), 7.27–7.32 (m, 1H),
7.37–7.44 (m, 2H); 13C NMR (100 MHz, CDCl3) δ –5.1, –4.5, 14.3, 18.0, 25.8, 28.3, 29.6, 43.9,
60.9, 63.7, 79.0, 79.5, 121.7, 126.7, 129.55, 129.64, 138.8, 153.3, 154.9, 165.9, 193.6; HRMS
(ESI+) calcd for C27H41NNaO7SSi+ [M + Na]+ 574.2265, found 574.2266 (error 0.2 ppm).
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(±)-(4R,6R) Ethyl 4-(tert-butoxycarbonylamino)-6-(tert-butyldimethylsilyloxy)cyclohex-
1-enecarboxylate (16). To a solution of ester (±)ꢀ15 (44.0 mg, 0.080 mmol, 1.00 equiv) in
refluxing toluene (4 mL) was added dropwise a solution of (Me3Si)3SiH (40 ꢁL, 0.12 mmol, 1.55
equiv), AIBN (7 mg, 0.04 mmol, 0.50 equiv) and tertꢀbutyl acrylate (20 ꢁL, 0.12 mmol, 1.55
equiv) in toluene (2 mL). The reaction mixture was heated at reflux for 2 h. After cooling to
room temperature, the solvent was removed in vacuo. Purification by flash chromatography (5:1
1
hexane–EtOAc) afforded the title compound (20 mg, 63%) as white solid: H NMR (400 MHz,
CDCl3) δ 0.10 (s, 3H), 0.18 (s, 3H), 0.88 (s, 9H), 1.31 (t, J = 7.1 Hz, 3H), 1.40 (s, 9H), 1.73 (dt, J
= 14.3, 3.8 Hz, 1H), 2.01–2.09 (m, 1H), 2.27–2.39 (m, 1H), 2.55 (dd, J = 20.0, 5.1 Hz, 1H), 4.07
(q, J = 4.4 Hz, 1H), 4.10–4.20 (m, 1H), 4.21–4.32 (m, 1H), 4.75–4.81 (m, 1H), 6.55 (d, J = 8.1
Hz,
1H),
6.91–6.98
(m,
1H);
13C
NMR
(100
MHz,
CDCl3)
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δ −5.0, −4.8, 14.3, 18.0, 25.8, 28.4, 32.6, 33.9, 42.0, 60.6, 62.7, 78.6, 132.0, 138.8, 155.3, 166.3;
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HRMS (ESI+) calcd for C20H37NNaO5Si+ [M + Na]+ 422.2333, found 422.2337 (error 0.9 ppm).
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(R)-3-[5-(4-Methoxybenzyloxy)pentanoyl]-4-benzyloxazolidin-2-one (18). To a solution
of acid 1722 (10.66 g, 44.7 mmol, 1.15 equiv) in THF (500 mL) at ꢀ78 °C was added Et3N (10.8
mL, 77.2 mmol, 2.00 equiv) followed by pivaloyl chloride (6.00 mL, 49.2 mmol, 1.15 equiv) and
the reaction was warmed to 0 °C. The resulting thick white precipitate was stirred for 6 h at 0 °C
and then reꢀcooled to ꢀ78 °C. In a separate flask, nꢀBuLi (2.5 M in hexane, 17.0 mL, 42.5 mmol,
1.10 equiv) was added by syringe over 5 min to a solution of (R)ꢀ4ꢀbenzylꢀ2ꢀoxazolidinone (6.84
g, 38.6 mmol, 1.00 equiv) in THF (100 mL) at ꢀ78 °C. The oxazolidinone solution was
transferred by cannula to the flask containing the mixed anhydride. The mixture was stirred for
30 min at ꢀ78 °C and 30 min at 0 °C, then quenched with saturated aqueous NH4CI (200 mL).
THF was removed under reduced pressure and the aqueous layer was extracted with CH2Cl2 (3 ×
200 mL). The combined organic layers were washed with 10% aqueous NaOH solution, dried
(MgSO4), and concentrated. The crude residue was purified by flash chromatography
(3:1→2:1→3:2 hexanes–EtOAc) to afford the title compound (14.2 g, 93%) as a colorless oil:
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[α]23 –37.8 (c 3.20, CHCl3); H NMR (400 MHz, CDCl3) δ 1.67–1.75 (m, 2H), 1.76–1.85 (m,
D
2H), 2.75 (dd, J = 13.4, 9.6 Hz, 1H), 2.89–3.05 (m, 2H), 3.29 (dd, J = 13.3, 3.2 Hz, 1H), 3.51 (t,
J = 6.2 Hz, 2H), 3.80 (s, 3H), 4.14–4.19 (m, 2H), 4.45 (s, 2H), 4.66 (dddd, J = 13.3, 10.1, 7.1, 3.5
Hz, 1H), 6.87–6.92 (m, 2H), 7.19–7.23 (m, 2H), 7.25–7.37 (m, 5H); 13C NMR (100 MHz, CDCl3)
δ 21.0, 29.0, 35.1, 37.8, 55.0, 55.2, 66.1, 69.6, 72.5, 113.7, 127.2, 128.9, 129.2, 129.3, 130.6,
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135.3, 153.4, 159.0, 173.0; HRMS (ESI+) calcd for C23H27NNaO5 [M + Na]+ 420.1781, found
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420.1786 (error 1.2 ppm).
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2-(tert-Butyldimethylsilyloxymethyl)acrolein (19). To a solution of tertꢀbutyl 2ꢀ
(hydroxymethyl)acrylate prepared according to the reported procedure23, 35 (10.0 g, 63.2 mmol,
1.00 equiv) in CH2Cl2 (500 mL) at 0 °C was added imidazole (11.1 g, 165 mmol, 2.61 equiv)
followed by tertꢀbutyldimethylsilyl chloride (19.1 g, 126 mmol, 2.00 equiv). The reaction
mixture was stirred at 23 °C overnight. After 16 h, the solvent was removed in vacuo to provide
a colorless oil, which was dissolved in 10:1 hexane–EtOAc (220 mL). The solution was passed
through a short pad of silica gel, which was washed with hexane–EtOAc (10:1). The filtrate was
concentrated in vacuo and dried under high vacuum to afford a colorless oil, which was then used
directly in the next step without further purification.
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To the solution of the crude tertꢀbutyl 2ꢀ(tertꢀbutyldimethylsilyloxymethyl)acrylate prepared
above in CH2Cl2 (300 mL) at ꢀ78 °C was added diisobutylaluminum hydride (100 mL, 1.0 M in
hexane, 100 mmol) dropwise. The reaction mixture was stirred for 1 h at ꢀ78 °C, then diluted
with Et2O (300 mL), and allowed to slowly warm to 23 °C. While the reaction mixture slowly
warmed to room temperature, the reaction mixture was quenched by the sequential dropwise
addition of H2O (4 mL), 15% aqueous NaOH solution (4 mL), and H2O (10 mL). After warming
to 23 °C, the reaction mixture was vigorously stirred for 30 min, then treated with anhydrous
MgSO4, and stirred for an additional 15 min. The mixture was filtered through a pad of Celite
and the resulting filtrate was concentrated in vacuo to afford the title compound (6.00 g, 50%,
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two steps) as a colorless oil, whose H and 13C NMR agreed with the reported data for 19
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prepared by an alternate synthetic route.36
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(R)-4-Benzyl-3-[(2R,3R)-4-(tert-butyldimethylsilyloxy)methyl-2-{3-[(4-
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methoxybenzyl)oxy]propyl}-3-(methoxymethoxy)pent-4-enoyl]oxazolidin-2-one (21). To a
solution of Nꢀacyloxazolidinone 18 (3.00 g, 7.50 mmol, 1.00 equiv) in CH2Cl2 (100 mL) at ꢀ
78 °C was added a 1.0 M solution of TiCl4 in CH2Cl2 (8.50 mL, 8.50 mmol, 1.13 equiv)
dropwise and the solution was stirred for 10 min. (–)ꢀSparteine (4.30 mL, 18.8 mmol, 2.50 equiv)
was added dropwise to the mixture and the solution stirred at ꢀ78 °C for 1.5 h. Freshly distilled
aldehyde 19 (3.00 g, 15.0 mmol, 2.00 equiv) was then added dropwise. The reaction was stirred
for 1.5 h at ꢀ78 °C, 3 h at ꢀ40 °C, and 12 h at ꢀ20 °C. The reaction was quenched at ꢀ40 °C by
addition of halfꢀsaturated aqueous NH4Cl (100 mL) and quickly transferred to a separatory funnel.
The organic layer was separated and the aqueous layer extracted with CH2Cl2 (2 × 100 mL). The
combined organic layers were dried (MgSO4), filtered and concentrated. Purification of the
crude residue by flash chromatography (10:1→8:1→7:1→6:1 PhMe–EtOAc) provided aldol
adduct 20 (2.13 g, 48%) as a colorless oil, which was directly carried onto the next step.
Chloromethyl methyl ether (1.69 g, 21.0 mmol, 5.90 equiv) was added to a solution of aldol
adduct 20 (2.13 g, 3.56 mmol, 1.0 equiv), iꢀPr2NEt (3.66 mL, 21.0 mmol, 5.90 equiv), and Bu4NI
(131 mg, 0.36 mmol, 0.10 equiv) in PhMe (50 mL) and the mixture stirred at 90 °C for 7 h. The
reaction mixture was cooled to rt and treated with saturated aqueous NaHCO3 (30 mL). The
layers were separated and the aqueous layer was extracted with EtOAc (3 × 50 mL). The
combined organic extracts were washed with saturated aqueous NaCl (30 mL), dried (Na2SO4)
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and concentrated. Purification by flash chromatography (4:1→3:1 hexane–EtOAc) afforded the
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title compound (1.88 g, 82%) as colorless oil: [α]23 +13.5 (c 1.10, CHCl3); H NMR (400 MHz,
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D
8
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CDCl3) δ 0.00 (s, 6H), 0.84 (s, 9H), 1.57–1.67 (m, 2H), 1.82–1.92 (m, 2H), 2.55 (dd, J = 13.2,
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10.2 Hz, 1H), 3.22 (dd, J = 13.2, 3.0 Hz, 1H), 3.27 (s, 3H), 3.37–3.42 (m, 2H), 3.70 (s, 3H),
3.97–4.13 (m, 4H), 4.16–4.23 (m, 1H), 4.26 (d, J = 7.9 Hz, 1H), 4.35 (s, 2H), 4.41 (d, J = 6.8 Hz,
1H), 4.47 (dddd, J = 12.9, 9.8, 6.4, 3.0 Hz, 1H), 4.55 (d, J = 6.8 Hz, 1H), 5.09 (s, 1H), 5.29 (s,
1H), 6.76–6.81 (m, 2H), 7.10–7.15 (m, 2H), 7.15ꢀ7.27 (m, 5H); 13C NMR (100 MHz, CDCl3) δ ꢀ
5.6, 18.2, 25.0, 25.7, 27.0, 37.7, 46.4, 55.0, 55.7, 55.8, 62.1, 65.8, 69.7, 72.3, 77.9, 94.2, 112.8,
113.5, 127.1, 128.8, 129.1, 129.2, 130.5, 135.3, 145.8, 152.9, 158.9, 173.3; HRMS (ESI+) calcd
for C35H51NNaO8Si+ [M + Na]+ 664.3276, found 664.3283 (error 1.05 ppm).
(2S,3R)-4-{[(tert-Butyldimethylsilyl)oxy]methyl}-2-{3-[(4-methoxybenzyl)oxy]propyl}-3-
(methoxymethoxy)pent-4-en-1-ol (22). Sodium borohydride (450 mg, 11.7 mmol, 4.00 equiv)
was added in one portion to a solution of oxazolidinone 21 (1.88 g, 2.93 mmol, 1.00 equiv) in
3:1 THF–H2O (60 mL) at 0 °C. The reaction mixture was allowed to warm to 23 °C and stirred
overnight (~16 h). The reaction was quenched by the slow addition of saturated aqueous NH4Cl
(60 mL) and extracted with diethyl ether (3 × 50 mL). The combined organic extracts were
washed with saturated aqueous NaCl, dried (MgSO4), and concentrated under reduced pressure.
Purification by flash chromatography (3:1→2:1→3:2 hexane–EtOAc) afforded the title
1
compound (920 mg, 67%) as a colorless oil: [α]23 +60.7 (c 0.400, CHCl3); H NMR (400 MHz,
D
CDCl3) δ 0.00 (s, 6H), 0.84 (s, 9H), 1.22–1.34 (m, 1H), 1.43–1.59 (m, 2H), 1.59–1.71 (m, 2H),
2.67 (br s, 1H), 3.35 (s, 3H), 3.41 (t, J = 6.3 Hz, 2H), 3.58–3.63 (m, 2H), 3.76 (s, 3H), 4.09 (s,
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2H), 4.19 (d, J = 6.1 Hz, 1H), 4.39 (s, 2H), 4.48 (d, J = 6.5 Hz, 1H), 4.58 (d, J = 6.5 Hz, 1H),
5.12 (s, 1H), 5.32 (s, 1H), 6.83 (d, J = 8.5 Hz, 2H), 7.22 (d, J = 8.5 Hz, 2H); 13C NMR (100 MHz,
CDCl3) δ ꢀ5.58, ꢀ5.56, 18.2, 22.7, 25.8, 27.3, 43.1, 55.0, 55.8, 62.4, 63.2, 70.2, 72.4, 78.5, 94.6,
112.9, 113.6, 129.0, 130.5, 146.1, 159.0; HRMS (ESI+) calcd for C25H44NaO6Si+ [M + Na]+
491.2799, found 491.2806 (error 1.4 ppm).
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(2S,3R)-4-{[(tert-Butyldimethylsilyl)oxy]methyl}-2-{3-[(4-methoxybenzyl)oxy]propyl}-3-
(methoxymethoxy)pent-4-enal (23). To a solution of alcohol 22 (920 mg, 1.96 mmol, 1.00
equiv) in CH2Cl2 (20 mL) at 23 °C was added solid NaHCO3 (1.65 g, 19.6 mmol, 10.0 equiv)
followed by Dess–Martin periodinane (1.25 g, 2.94 mmol, 1.50 equiv). The reaction was stirred
for 30 min at 23 °C then quenched by 1:1 10% aqueous Na2S2O3–saturated aqueous NaHCO3 (20
mL). The layers were separated and the aqueous layer was extracted with CH2Cl2 (3 × 20 ml).
The combined organic layers were dried (MgSO4) and concentrated under reduced pressure. The
resulting residue was dissolved in a minimum amount of 3:1 hexane–EtOAc and filtered through
a short pad of silica gel, which was rinsed with 3:1 hexane–EtOAc (200 mL). The filtrate was
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concentrated to afford the title compound (821 mg, 90%) as colorless oil: H NMR (400 MHz,
CDCl3) δ 0.00 (s, 6H), 0.84 (s, 9H), 1.43–1.54 (m, 1H), 1.57–1.79 (m, 3H), 2.50 (dddd, J = 11.5,
8.9, 5.6, 2.7 Hz, 1H), 3.26 (s, 3H), 3.36 (t, J = 6.0 Hz, 2H), 3.73 (s, 3H), 4.05 (s, 2H), 4.34 (s,
2H), 4.41 (d, J = 6.7 Hz, 1H), 4.44 (d, J = 5.9 Hz, 1H), 4.56 (d, J = 6.8 Hz, 1H), 5.08 (s, 1H),
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5.27 (s, 1H), 6.80 (d, J = 8.6 Hz, 2H), 7.19 (d, J = 8.6 Hz, 2H), 9.61 (d, J = 2.2 Hz, 1H); C
NMR (100 MHz, CDCl3) δ ꢀ5.54, ꢀ5.52, 18.2, 21.2, 25.8, 27.6, 54.6, 55.1, 55.9, 63.2, 69.7, 72.4,
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75.8, 94.1, 113.6, 114.0, 129.1, 130.5, 144.8, 159.0, 203.1; HRMS (ESI+) calcd for
C25H42NaO6Si+ [M + Na]+ 489.2643, found 489.2647 (error 0.8 ppm).
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(S,E)-N-[(2S,3R)-4-{[(tert-Butyldimethylsilyl)oxy]methyl}-2-{3-[(4-
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methoxybenzyl)oxy]propyl}-3-(methoxymethoxy)pent-4-en-1-ylidene]-tert-butylsulfinamide
(24). To a solution of aldehyde 23 (820 mg, 1.76 mmol, 1.00 equiv) in CH2Cl2 (10 mL) was
added (S)ꢀ(–)ꢀtertꢀbutylsulfinamide (235 mg, 1.94 mmol, 1.10 equiv), anhydrous CuSO4 (562 mg,
3.52 mmol, 2.00 equiv) and pyridinium pꢀtoluenesulfonate (442 mg, 1.76 mmol, 1.00 equiv).
The mixture was stirred at 23 °C for 24 h, then filtered through a pad of Celite, and the filter cake
was washed with CH2Cl2. The combined organic filtrate was washed with saturated aqueous
NaCl, dried (MgSO4), and concentrated. Purification by flash chromatography (3:1→2:1
hexane–EtOAc) afforded the title compound (616 mg, 62%, 75% brsm) as a colorless oil: [α]D23
1
+165 (c 0.700, CHCl3); H NMR (400 MHz, CDCl3) δ 0.00 (s, 6H), 0.84 (s, 9H), 1.13 (s, 9H),
1.42–1.69 (m, 3H), 1.80–1.91 (m, 1H), 2.73–2.83 (m, 1H), 3.30 (s, 3H), 3.36 (t, J = 6.1 Hz, 2H),
3.72 (s, 3H), 4.06 (s, 2H), 4.24 (d, J = 7.1 Hz, 1H), 4.34 (s, 2H), 4.42 (d, J = 6.7 Hz, 1H), 4.58 (d,
J = 6.7 Hz, 1H), 5.06 (s, 1H), 5.25 (s, 1H), 6.80 (d, J = 8.3 Hz, 2H), 7.17 (d, J = 8.3 Hz, 2H),
7.87 (d, J = 6.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ ꢀ5.71, ꢀ5.69, 18.0, 22.2, 24.2, 25.7, 27.2,
48.7, 54.9, 55.8, 56.6, 62.3, 69.4, 72.2, 77.8, 93.7, 113.5, 114.1, 128.9, 130.4, 144.6, 158.9, 169.8;
HRMS (ESI+) calcd for C29H51NNaO6SSi+ [M + Na]+ 592.3099, found 592.3105 (error 1.0 ppm).
(S)-N-[(4R,5S,6R)-7-{[(tert-Butyldimethylsilyl)oxy]methyl}-5-{3-[(4-
methoxybenzyl)oxy]propyl}-6-(methoxymethoxy)octa-1,7-dien-4-yl]-tert-butylsulfinamide
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(25). To a solution of tertꢀbutanesulfinyl imine 24 (600 mg, 1.05 mmol, 1.00 equiv) in CH2Cl2
(20 mL) was added allylmagnesium bromide (1.0 M in Et2O, 3.15 mL, 3.15 mmol, 3.00 equiv) at
ꢀ78 °C. The reaction was warmed to ꢀ20 °C and stirred for 2.5 h at this temperature then
quenched with saturated aqueous NH4Cl (20 mL). The layers were separated and the aqueous
layer was extracted with CH2Cl2 (3 × 20 ml). The combined organic layers were dried (MgSO4),
filtered, and concentrated. The resulting residue was dissolved in a minimum amount of Et2O
and filtered through a short pad of silica gel, which was rinsed with Et2O. The filtrate was
concentrated to provide the title compound (560 mg, 87%) as colorless oil: [α]D23 +80.1 (c 0.600,
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CHCl3); H NMR (400 MHz, CDCl3) δ 0.02 (s, 6H), 0.87 (s, 9H), 1.16 (s, 9H), 1.37–1.51 (m,
2H), 1.55–1.79 (m, 3H), 2.35–2.45 (m, 1H), 2.49–2.59 (m, 1H), 3.22 (d, J = 8.3 Hz, 1H), 3.28 (s,
3H), 3.35–3.38 (m, 3H), 3.74 (s, 3H), 3.99 (d, J = 15.0 Hz, 1H), 4.05 (d, J = 14.6 Hz, 1H), 4.16
(d, J = 7.3 Hz, 1H), 4.36 (s, 2H), 4.39 (d, J = 6.7 Hz, 1H), 4.56 (d, J = 6.7 Hz, 1H), 5.05–5.10 (m,
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3H), 5.34 (s, 1H), 5.66–5.80 (m, 1H), 6.81 (d, J = 8.2 Hz, 1H), 7.19 (d, J = 8.2 Hz, 1H); C
NMR (100 MHz, CDCl3) δ ꢀ5.55, ꢀ5.52, 18.2, 22.6, 22.8, 25.7, 29.5, 38.5, 42.7, 55.0, 55.9, 56.0,
56.5, 62.1, 70.0, 72.3, 79.0, 93.9, 113.5, 113.9, 118.6, 129.0, 130.5, 134.5, 145.3, 158.9; HRMS
(ESI+) calcd for C32H58NO6SSi+ [M + H]+ 612.3749, found 612.3754 (error 0.8 ppm).
(S)-N-[(1R,5R,6S)-4-{[(tert-Butyldimethylsilyl)oxy]methyl}-6-{3-[(4-
methoxybenzyl)oxy]propyl}-5-(methoxymethoxy)cyclohex-3-en-1-yl]-tert-butylsulfinamide
(26).
[1,3ꢀbisꢀ(2,4,6ꢀTrimethylphenyl)ꢀ2ꢀ
imidazolidinylidene]dichloro(phenylmethylene)(tricyclohexylphosphine)ruthenium (13.8 mg,
0.016 mmol, 0.14 equiv) was added to a solution of diene 25 (70.0 mg, 0.114 mmol, 1.0 equiv)
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in CH2Cl2 (20 mL) and the reaction was heated at reflux for 14 h. The reaction was cooled to
23 °C, then treated with DMSO (1.5 mL),37 and stirred 16 h at 23 °C. The reaction mixture was
washed with saturated aqueous NaCl (50 mL), and the organic layer was dried (MgSO4), filtered
and concentrated. Purification by flash chromatography (2:1→3:2→1:1 hexane–EtOAc)
afforded the title compound (63 mg, 95%) as a light brown oil: 1H NMR (400 MHz, CDCl3) δ ꢀ
0.01 (s, 3H), 0.00 (s, 3H), 0.85 (s, 9H), 1.11 (s, 9H), 1.16–1.29 (m, 2H), 1.54–1.70 (m, 2H),
1.99–2.07 (m, 1H), 2.23–2.34 (m, 1H), 2.48–2.59 (m, 1H), 3.26–3.46 (m, 6H), 3.73 (s, 3H), 3.92
(s, 1H), 4.02 (d, J = 12.8 Hz, 1H), 4.14 (d, J = 12.8 Hz, 1H), 4.35 (s, 2H), 4.54 (d, J = 6.6 Hz,
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1H), 4.60–4.69 (m, 2H), 5.65 (s, 1H), 6.80 (d, J = 8.2 Hz, 2H), 7.17 (d, J = 8.2 Hz, 2H); C
NMR (100 MHz, CDCl3) δ ꢀ5.6, ꢀ5.4, 18.0, 22.5, 25.7, 26.5, 27.5, 29.2, 40.9, 53.5, 55.0, 55.5,
55.6, 64.3, 69.5, 71.6, 72.4, 95.3, 113.5, 121.5, 129.0, 130.3, 135.0, 158.9; HRMS (ESI+) calcd
for C30H53NNaO6SSi+ [M + Na]+ 606.3255, found 606.3252 (error 0.5 ppm).
tert-Butyl
[(1R,5R,6S)-4-(hydroxymethyl)-6-{3-[(4-methoxybenzyl)oxy]propyl}-5-
(methoxymethoxy)cyclohex-3-en-1-yl]carbamate (27). To a solution of 26 (55 mg, 0.094
mmol, 1.0 equiv) in MeOH (2.0 mL) was added dropwise 4 M HCl in 1,4ꢀdioxane (190 µL, 0.75
mmol, 8.0 equiv) at 0 °C. The resulting mixture was stirred at 0 °C for 1 h then quenched with
saturated aqueous NaHCO3 (3 mL). Methanol was removed under reduced pressure and the
aqueous layer was treated with CH2Cl2 (2 mL) and 6 M aqueous NaOH (2 mL) at 0 °C. After
stirring for 10 min at 0 °C, the layers were separated. The aqueous layer was extracted with
CH2Cl2 (3 × 3 mL) and the combined organic layers were dried (K2CO3) and concentrated under
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reduced pressure to afford the crude amino alcohol as a viscous yellow oil, which was used
directly in the next step without further purification.
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To the crude amino alcohol in 1:2 H2O–1,4ꢀdioxane (3 mL) at 23 °C was added Et3N (26 µL,
0.19 mmol, 2.0 equiv) followed by diꢀtertꢀbutyl dicarbonate (28 mg, 0.13 mmol, 1.4 equiv) in
1,4ꢀdioxane (0.5 mL). The reaction mixture was stirred for 1 h at 23 °C, then partitioned
between H2O (10 mL) and CH2Cl2 (10 mL). The aqueous layer was extracted with CH2Cl2 (3 × 3
mL) and the combined organic layers were dried (MgSO4), filtered, and concentrated.
Purification by flash chromatography (1:1 hexane–EtOAc) afforded the title compound (39 mg,
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89%) as a colorless oil: H NMR (400 MHz, CDCl3) δ 1.21–1.31 (m, 2H), 1.42 (s, 9H), 1.63–
1.74 (m, 2H), 1.96–2.11 (m, 2H), 2.19–2.26 (m, 2H), 3.37–3.46 (m, 5H), 3.80 (s, 3H), 3.82–3.89
(m, 1H), 3.96 (s, 1H), 4.09 (d, J = 12.0 Hz, 1H), 4.18 (d, J = 12.7 Hz, 1H), 4.40 (s, 2H), 4.64 (d,
J = 6.7 Hz, 1H), 4.74 (d, J = 6.7 Hz, 1H), 5.76 (br s, 1H), 5.93 (d, J = 8.1 Hz, 1H), 6.87 (d, J =
13
8.5 Hz, 2H), 7.23 (d, J = 8.5 Hz, 2H); C NMR (100 MHz, CDCl3) δ 27.0, 27.68, 27.72, 28.4,
40.0, 46.3, 55.2, 56.1, 65.4, 69.7, 72.5, 74.0, 78.8, 96.2, 113.8, 124.6, 129.2, 130.5, 134.8, 155.4,
159.1; HRMS (ESI+) calcd for C25H39NNaO7 [M + Na]+ 488.2619, found 488.2621 (error 0.4
+
ppm).
tert-Butyl
[(1R,5R,6S)-4-(hydroxymethyl)-6-(3-hydroxypropyl)-5-
(methoxymethoxy)cyclohex-3-en-1-yl]carbamate (28). To a solution of 27 (40 mg, 0.086
mmol, 1.0 equiv) in 20:1 CH2Cl2–H2O (10 mL) was added DDQ (29 mg, 0.13 mmol, 1.5 equiv)
at 0 °C. The reaction was stirred for 2 h at 23 °C, then partitioned between saturated aqueous
NaHCO3 (10 mL) and CH2Cl2 (10 mL). The aqueous layer was extracted with CH2Cl2 (2 × 10
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mL) and the combined organic layers were dried (Na2SO4), filtered, and concentrated.
Purification by flash chromatography (Et2O→EtOAc) afforded the title compound (23 mg, 89%)
as a colorless oil: 1H NMR (400 MHz, CDCl3) δ 1.21–1.31 (m, 2H), 1.42 (s, 9H), 1.58–1.72 (m,
2H), 2.04–2.12 (m, 1H), 2.20–2.26 (m, 2H), 2.30–2.40 (m, 2H), 3.43 (s, 3H), 3.55–3.68 (m, 2H),
3.80–3.89 (m, 1H), 3.97–4.01 (m, 1H), 4.07 (d, J = 12.4 Hz, 1H), 4.18 (d, J = 12.6 Hz, 1H), 4.67
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(d, J = 6.8 Hz, 1H), 4.77 (d, J = 6.8 Hz, 1H), 5.72–5.79 (m, 1H), 5.93 (d, J = 7.5 Hz, 1H); C
NMR (100 MHz, CDCl3) δ 26.6, 27.8, 28.4, 30.4, 39.9, 46.4, 56.1, 62.4, 65.1, 73.7, 78.9, 96.2,
124.7, 134.9, 155.5; HRMS (ESI+) calcd for C17H31NNaO6 [M + Na]+ 368.2044, found
+
368.2046 (error 0.5 ppm).
(4R,5S,6R)-4-tert-(Butylcarbonyl)amino-5-(2-carboxyethyl)-6-
(methoxymethoxy)cyclohex-1-enecarboxylic acid (29). To a solution of diol 28 (23 mg, 0.076
mmol, 1.0 equiv), NꢀmethylmorpholineꢀNꢀoxide (53 mg, 0.45 mmol, 6.0 equiv), and powdered 4
Å molecular sieves (35 mg) in CH2Cl2 (1 mL) at 23 °C was added tetrapropylammonium
perruthenate (2.0 mg, 0.0056 mmol, 0.075 equiv). The mixture was stirred for 15 min at 23 °C,
then filtered through a short pad of silica gel, eluting with EtOAc. The filtrate was concentrated
under reduced pressure to afford the crude dialdehyde, which was directly used in the next step
without further purification.
To a solution of freshly prepared dialdehyde and 2ꢀmethylꢀ2ꢀbutene (0.20 mL, 1.9 mmol, 25
equiv) in 5:1:1 tertꢀbutyl alcohol–THF–H2O (1 mL) at 0 °C was slowly added a solution of
sodium chlorite (80% w/w technical grade, 52 mg, 0.46 mmol, 6.0 equiv) and sodium phosphate
monobasic monohydrate (63 mg, 0.46 mmol, 6.0 equiv) in H2O (0.3 mL). The resulting
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suspension was stirred at 23 °C for 2 h. The reaction was quenched with saturated aqueous
NaHSO3 (3 mL) at 0 °C and the aqueous layer was extracted with CH2Cl2 (3 × 5 mL). The
combined organic layers were dried (Na2SO4), filtered, and concentrated under reduced pressure.
Purification by flash chromatography on silica gel38 (210:25:4→180:25:4 CHCl3–MeOH–AcOH)
afforded the title compound (13 mg, 46% for two steps) as a colorless oil that was ~90% pure: 1H
NMR (400 MHz, CDCl3) δ 1.41 (s, 9H), 1.44–1.54 (m, 2H), 2.18–2.25 (m, 1H), 2.39–2.60 (m,
4H), 3.40 (s, 3H), 3.88–3.95 (m, 1H), 4.31–4.34 (m, 1H), 4.72 (d, J = 6.9 Hz, 1H), 4.86 (d, J =
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6.9 Hz, 1H), 6.29 (d, J = 8.1 Hz, 1H), 7.14–7.18 (m, 1H); C NMR (100 MHz, CDCl3) δ 25.0,
28.4, 29.0, 31.8, 40.1, 45.4, 56.1, 71.8, 79.3, 97.5, 128.0, 142.4, 155.5, 171.1, 178.2; HRMS
(ESI–) calcd for C17H26NO8 [M – H]− 372.1664, found 372.1668 (error 1.1 ppm).
−
(4R,5S,6R)-4-Amino-5-(2-carboxyethyl)-6-hydroxycyclohex-1-enecarboxylic
acid
acetate salt (4). To a solution of dicarboxylic acid 29 (13 mg, 0.035 mmol, 1.0 equiv) in CH2Cl2
(1 mL) was added TMSBr (40 µL, 0.30 mmol, 8.6 equiv) at ꢀ78 °C. The reaction mixture was
stirred for 1 h at ꢀ78 ° then concentrated in vacuo during which time the flask warmed to 23 °C.
The residue was washed with Et2O, and purified by flash chromatography on silica gel38
(65:25:4:2→100:56:8:8 CHCl3–MeOH–H2O–AcOH) to afford the acetate salt of the title
compound (5.0 mg, 50%) as a white solid: [α]23 +6.2 (c 0.40, H2O); 1H NMR (600 MHz, D2O) δ
D
1.57–1.64 (m, 2H), 2.22–2.29 (m, 1H), 2.46 (t, J = 7.4 Hz, 2H), 2.56 (d, J = 13.5 Hz, 1H), 2.78
13
(d, J = 13.5 Hz, 1H), 3.68–3.74 (m, 1H), 4.55–4.59 (m, 1H), 6.84–6.88 (m, 1H); C NMR (150
MHz, D2O) δ 21.1 (AcOH), 24.0, 65.0, 32.7, 40.7, 46.9, 65.1, 131.8*, 134.3, 172.4*, 177.4*
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(AcOH), 179.6* (Chemical shifts denoted by * are derived from HMBC); HRMS (ESI–) calcd
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−
for C10H14NO5 [M – H]− 228.0877, found 228.0883 (error 2.6 ppm).
8
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MbtI Assay. Reactions were performed under initial velocity conditions in a total volume of
50 µL at 37 °C for 30 min and the production of salicylic acid was monitored continuously by
following changes in fluorescence at 420 nm with excitation at 305 nm on a microplate reader.
Assays were set up in duplicate and contained 0.5 µM MbtI in reaction buffer (100 mM Trisꢀ
HCl, pH 8.0, 1 mM MgCl2, 50 µM chorismate, and 0.0025% Igepal CAꢀ630). A threeꢀfold serial
dilution of inhibitor in H2O was added to black 384 well plates coated with a nonꢀbinding surface
(Greiner). A positive control (H2O only) and negative control (10 mM EDTA) were also
included. The IC50 values were calculated from the Hill equation (eq 1). In this equation the
fractional activity (vi/v0) versus inhibitor concentration was fit by nonꢀlinear regression analysis
using GraphPad prism version 6.0 where vi is the reaction velocity at a given [I] and v0 is the
reaction velocity of the DMSO control, and h is the Hill slope.
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vi
1
(1)
=
v0 1+ ([I]/IC50 )h
ASSOCIATED CONTENT
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Supporting Information: copies of H NMR and C NMR spectra of all new compounds
and Figure illustrating a docked pose of 4 in MbtI. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
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The Journal of Organic Chemistry
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Corresponding Author
*Eꢀmail: aldri015@umn.edu.
Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by a grant from the National Institutes of Health Grant (RO1 AI070219
to CCA) and the National Natural Science Foundation of China (21402058 to ZL). We thank Dr.
Youlin Xia from NMR center of University of Minnesota for NMR spectroscopic analysis. Mass
spectrometric analysis was carried out in the Analytical Biochemistry Shared Resource of the
Masonic Cancer Center, University of Minnesota, funded in part by Cancer Center Support Grant
CAꢀ77598.
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