Synthesis of unusual N-acylated aminosugar fragments of Mycobacterium marinum lipooligosaccharide IV

Article abstract of DOI:10.1021/acs.joc.5b00064

A convergent strategy was developed for the stereoselective synthesis of four unusual N-acylated monosaccharides (5-8), which are fragments of lipooligosaccharide IV (LOS-IV) from Mycobacterium marinum. A critical substrate-controlled asymmetric cyclization of an amino acid derived oxazolidine provided a key lactam intermediate 11, which was successfully converted to targets 5-7. The key step in the synthesis of 8 was a one-pot cascade oxidation-cyclization-oxidation reaction of a Boc-protected amino alcohol, prepared from 3-butynol, which led to the formation of lactam 15. The five-membered ring lactam intermediates in these synthetic routes were sensitive to elimination side reactions, but careful manipulation of the reaction sequence allowed for the stereoselective synthesis of the targets. This work represents the first synthesis of these unusual motifs, which have been shown to be essential to the bioactivity of LOS-IV.

Full text of DOI:10.1021/acs.joc.5b00064

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Article
Synthesis of Unusual N-Acylated Aminosugar Fragments
of Mycobacterium Marinum Lipooligosaccharide IV
Lei Wang, Mengyao Dong, and Todd L. Lowary
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 02 Feb 2015
Downloaded from http://pubs.acs.org on February 3, 2015
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Synthesis of Unusual N-Acylated Aminosugar
Fragments of Mycobacterium Marinum Lipooligosaccharide IV
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Lei Wang, Mengyao Dong, and Todd L. Lowary*
Alberta Glycomics Centre and Department of Chemistry, University of Alberta
Gunning–Lemieux Chemistry Centre, Edmonton, AB T6G 2G2, Canada
Email: tlowary@ualberta.ca
Graphical Abstract
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Abstract
A convergent strategy was developed for the stereoselective synthesis of four unusual Nꢀ
acylated monosaccharides (5–8), which are fragments of lipooligosaccharide IV (LOSꢀIV) from
Mycobacterium marinum. A critical substrateꢀcontrolled asymmetric cyclization of an aminoꢀ
acid derived oxazolidine provided a key lactam intermediate 11, which was successfully
converted to targets 5–7. The key step in the synthesis of 8 was a oneꢀpot cascade oxidation–
cyclization–oxidation reaction of a Bocꢀprotected amino alcohol, prepared from 3ꢀbutynol,
which led to the formation of lactam 15. The five member ring lactam intermediates in these
synthetic routes were sensitive to elimination side reactions, but careful manipulation of the
reaction sequence allowed for the stereoselective synthesis of the targets. This work represents
the first synthesis of these unusual motifs, which have been shown to be essential to the
bioactivity of LOSꢀIV.
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Introduction
Lipooligosaccharides (LOSs) are antigenic cell surface glycolipids found in the cell wall
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complex of mycobacteria. They are produced by a range of mycobacteria, including
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4ꢀ6
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Mycobacterium kansasii, M. smegmatis, M. gastri, M. malmoense, M. marinum, and the
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Canetti strain of M. tuberculosis. Recent investigations support a role for LOSs in important
biological events including sliding motility, biofilm formation, and infection of host
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macrophages; however, their precise role in mycobacterial virulence remains unclear.
M. marinum produces four LOSs (LOSꢀI–IV, Figure 1). Similar to all LOSs reported to
date, these molecules contain an acylated trehalose core, which is functionalized with a speciesꢀ
specific glycan. The glycan in M. marinum LOSꢀIV is terminated with a family of Nꢀacylated 4ꢀ
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aminoꢀ4,6ꢀdideoxyꢀgalactopyranose residues. These unusual Nꢀacylated monosaccharides share
a common lactam core, differentiated by substitutions at Cꢀ2 (CO H or H) or Cꢀ4 (MeO or H).
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This heterogeneity generates two acidic (1 and 2, Figure 2) and two neutral (3 and 4) compounds.
Compound 1 and 2, substituted by both carboxy (Cꢀ2) and methoxy (Cꢀ4) groups, account for 95%
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of the total LOSꢀIV derivatives. The absolute stereochemistry of the substituents in the lactam
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ring of 1–3 have been determined using H NMR spectroscopy, in particular through NOE
experiments. However, this was not possible for 4 and it was proposed that its lactam structure
was either that shown in Figure 2, or its enantiomer.
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Figure 1. Structures of M. marinum LOSs.
Figure 2. Structures of unusual Nꢀacylated monosaccharides present in M. marinum LOSꢀIV (1–
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) and synthetic targets (5–8).
Recent studies by Guérardel and coꢀworkers showed that LOSꢀIV induces the expression
of both Intercellular Adhesion Molecule 1(ICAMꢀ1) and CD40 on macrophages. In addition,
LOSꢀIV stimulates Interleukinꢀ8 (ILꢀ8) secretion from THPꢀ1 cells. However, LOSꢀIII did not
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show these activities, thus indicating that these terminal Nꢀacylated monosaccharides confer
important biological functions to LOSꢀIV. The biological activity of these motifs, in conjunction
with their intriguing structure, motivated us to develop a synthetic route that would provide these
compounds in a form that could be used for future investigations of their function. In this paper,
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we describe the first stereoselective synthesis of these unusual Nꢀacylated monosaccharides (5–
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). These molecules were synthesized bearing an aminooctyl aglycone, a group that provides a
convenient handle for conjugation, for example, to proteins for the generation of monoclonal
antibodies.
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Results and discussion
The targets feature a structure consisting of a highly substituted γꢀlactam connected
through an amide bond to a 4ꢀaminoꢀ4,6ꢀdideoxyꢀαꢀDꢀgalactopyranoside moiety. Retrosynthetic
analysis of 5–8 is outlined in Scheme 1. The disconnection of the amide bond in 5–7 affords
protected 4ꢀaminoꢀ4,6ꢀdideoxyꢀgalactose derivative 9 and three γꢀlactams, which could be
accessed from the same key intermediate 10. Compound 10 could be produced from the fused
bicyclic oxazolidine–pyrrolinone 11 through a series of functional group transformations. In turn,
11 could be accessed by stereoselective cyclization of 12, which is accessible from Dꢀserine (13).
With regard to the synthesis of 8, disconnection of the amide bond leads to amine 9 and Nꢀ
methyl lactam 14. Following a series of functional group manipulations, Boc protected lactam 15
could be converted to Nꢀmethyl lactam 14. The key step is the conversion of the highly
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functionalized αꢀamino acid ester 16 to lactam 15.
Scheme 1. Retrosynthetic analysis of 5–8.
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Synthesis of Monosaccharide 9. With a plan in place, the synthesis began with the
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preparation of galactopyranoside derivative 9 (Scheme 2). Glucopyranoside 18 was treated with
tosyl chloride, leading to regioselective tosylation of the Cꢀ6 hydroxyl group to form 19 in 95%
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yield. Subsequent reduction of 19 by LiAlH provided 6ꢀdeoxysugar 20 in 85% yield. Triflation
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of 20 followed by azide substitution at room temperature provided, in 85% overall yield, azide
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1. Azide reduction by the Staudinger reaction and trifluoroacetylation of the resulting amine
gave the corresponding trifluoroacetamide derivative 23 in 79% yield over the two steps. The
anomeric allyl group in 23 was then removed by treatment with a catalytic amount of PdCl in a
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solution of methanol and CH Cl2 to afford the hemiacetal, which upon exposure to
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trichloroacetonitrile, Cs CO and 4 Å molecular sieves in anhydrous CH Cl gave the desired
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trichloroacetimidate donor. Glycosylation of the trichloroacetimidate donor with 8ꢀazidoꢀoctanol
in the presence of TMSOTf was αꢀselective affording 24 in 68% overall yield from 23. Finally,
the trifluroacetyl protecting group was removed under basic conditions to afford 8ꢀazidooctyl 4ꢀ
aminoꢀ4,6ꢀdideoxyꢀgalactopyranoside (9) in 96% yield.
Scheme 2. Synthesis of 8ꢀazidooctyl 4ꢀaminoꢀ4,6ꢀdideoxyꢀαꢀDꢀgalactopyranoside (9).
Synthesis of key lactam precursor 11. After the successful synthesis of 9, we turned our
attention to the preparation of the protected lactam derivative 11. The key issue was to introduce
stereocenters at α and β positions of the lactam. In previous work, Ling and coꢀworkers (Scheme
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) treated αꢀalkylꢀβꢀketo amide 25 with DBU at 110 °C, which led to a cyclization–dehydration
sequence forming α, βꢀunsaturated lactam 27.
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Scheme 3. Reference method for synthesis of α, βꢀunsaturated lactam 27.
Inspired by this reaction, we hypothesized that if αꢀmethoxyꢀβꢀketo amide 12 (Scheme 4)
was reacted with DBU, a product with the hydroxyl group cis to ester would be formed because
of Hꢀbonding with the carbonyl group. In addition, we anticipated that the methoxy group would
be trans to the hydroxy group due to a dipole effect. With this plan in mind, we started our
synthetic work from ꢀserine methylester hydrochloride (13). This αꢀamino ester was condensed
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with pivaldehyde to afford an oxazolidine intermediate that was directly coupled with acetoacetic
acid in the presence of EDC and DMAP giving βꢀketo amide 28 in 77% overall yield.
Iodosobenzenediacetateꢀmediated oxidation of 28 in methanol provided the αꢀmethoxyꢀβꢀketo
amide 12 in 90% yield. DBUꢀpromoted cyclization of 12 was carried out as reported by Ling and
coworkers; however, in our hands, it was necessary to use a lower temperature (60 °C) to avoid
the dehydration following cyclization. After 12 hours, two diastereomeric cyclized products were
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formed (ratio 9:1 from the H NMR spectrum of the crude product), which could be separated by
chromatography. Pleasingly, NOESY experiments (see Supporting Information) showed that the
major product (70% isolated yield) was the desired intermediate 11 and the minor product (8%
isolated yield) was the undesired stereoisomer (29) where the hydroxyl and methoxy groups are
cis. Xꢀray crystallographic analysis of 11 further confirmed the structure of major product (see
Figure S1 and Tables S1 and S2 in the Supporting Information).
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Scheme 4. Synthesis of oxazolidine 11.
The next step was to protect the tertiary alcohol in 11 as a benzyl ether. Mindful of the
anticipated baseꢀsensitivity of 11, initial attempts to benzylate this tertiary alcohol were carried
out under acidic conditions (TMSOTf and benzyl trichloroacetimidate). However, these attempts
were unsuccessful and so we turned out attention to baseꢀpromoted alkylation. Treatment of
alcohol 11 with NaH and benzyl bromide (2 equiv) led to the formation of two benzylated
compounds (ratio 24:71) and a trace amount of elimination byꢀproduct 35 (Table 1).
Disappointingly, NOESY experiments (see Supporting Information) confirmed that the major
product was 34, in which the substituents α and β to carbonyl group were cis. Presumably, upon
deprotonation of alcohol with base, the resulting anion undergoes a reversible retroꢀaldol
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reaction (Scheme 5). Although, upon recyclization both stereoisomers can be formed in this
process, the cis intermediate 32 would be expected to react faster with benzyl bromide given
dipolar effects, leading to a majority of 34. We imagined that using a higher concentration of
benzyl bromide would allow the trapping of alkoxide 30 before ring opening. Gratifyingly, as
outlined in Table 1, increasing the ratio of BnBr to DMF to 1:2, led to a 3:2 ratio of the desired
(33) to undesired (34) stereoisomers. Further increasing the ratio of BnBr to DMF to 3:1 and
decreasing the temperature to –15 °C gave a 9:1 mixture of 33 and 34.
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a
Table 1.Protection of tertiary alcohol in bicyclic intermediate 11
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b
Products distribution (NMR yield )
T
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Entry
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Reaction conditions: 11 (1 equiv), NaH (1.5equiv), nꢀBu NI (1.2 equiv)
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Yields were obtained by integration of CO CH protons of the products observed in the crude H NMR spectrum.
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t-Bu
N
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N
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N
O
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CO2CH3
MeO
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MeO
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MeO
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Scheme 5. Proposed mechanism for the formation of byꢀproduct 34.
Synthesis of target 5. After the successful alkylation of the tertiary alcohol, a series of
functional group manipulations converted 33 into the key intermediate 10 (Scheme 6). Acidic
conditions were used to cleave the oxazolidine ring in 81% yield. The primary alcohol product of
this reaction, 36, was then converted to TBS ether 37 and then Nꢀmethyl amide 38 in 83% yield
over the two steps. Finally, nꢀBu NF deprotection of the TBS ether provided 10 in 89% yield.
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With 10 in hand, we examined a number of different oxidation conditions (Jones, PDC in wet
DMF, RuCl and NaIO , TEMPO and NaClO/NaClO ) in an attempt to oxidize the primary
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alcohol to the corresponding carboxylic acid. Unfortunately, none of these methods afforded the
desired product and therefore a twoꢀstep approach was explored. Ley oxidation (TPAP/NMO)
afforded aldehyde 39 in 84% yield; subsequent Pinnick oxidation easily oxidized 39 to
carboxylic acid 40 in 92% yield. With the acid 40 in hand, amidation with the amine 9 led to 41
in 76% yield. The methyl ester was then hydrolyzed by treatment with LiOH in THF and water
to furnish 42 (96% yield). Global debenzylation and reduction of 42 was achieved in a mixture
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of methanol, water and acetic acid (10:1:0.1) under a H atmosphere with 10% Pd/C as the
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catalyst to give an 89% yield of 5.
Scheme 6. Synthesis of target 5.
When 5, which was homogenous based on HRMS analysis, was dissolved in CD OD, the
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H NMR spectrum showed a mixture of two species in a 4:3 ratio. When the solvent was
changed to DMSOꢀd , the ratio for these two isomers was still 4:3; however, it changed to 10:1
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after two days. Finally, when AcOHꢀd was used as solvent, the ratio changed to 2:3 after one
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day and did not change further (see Supporting Information). Recovery of the samples dissolved
in DMSOꢀd and AcOHꢀd and redissolution in CD OD, resulted in a 4:3 mixture of isomers.
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These experiments suggest that following deprotection, Nꢀacylated monosaccharide 5 exists as a
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mixture of two atropisomers about the Cꢀ4 amide bond, with solventꢀdependent populations.
Attempts to resolve the atropisomers into a single species using elevated temperature (60 °C)
NMR experiments in CD OD did not result in significant changes in the distributions of the two
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species. Interestingly, in the report detailing the structure of these LOSs from M. marinum,
atropoisomerism was not reported. In these synthetic compounds, restricted rotation about this
bond may arise from the presence of the aminooctyl aglyone, which would lead to these
compounds existing at zwitterions at neutral pH. We note, however, that the use of AcOHꢀd4,
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which would be expected to protonate the molecule, did not lead to a single species in the H
NMR spectrum. Unfortunately, direct comparison of the NMR data from 5 with those reported
for the natural product was not possible. The NMR spectra of the natural glycolipids were
measured in D O, a solvent in which 5 (as well as 6–8) is insoluble.
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Synthesis of target 6. To synthesize 6, an inverted order for the oxidation and amidation
sequence was used. The initial plan was to hydrolyze the ester in 38 to afford an acid 43 that
could be coupled to amine 9 (Scheme 7). Unfortunately, presumably due to steric effects arising
from the TBS protecting group, a number of different conditions (e.g., LiOH in THF/H O, LiOH
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in CH OH/H O (r.t. to 50 °C), NaOH in CH OH/H O) only afforded trace amounts of desired
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product. Thus, we decided to hydrolyze the key intermediate 10, which possesses a free hydroxyl
group (Scheme 8). Compound 10 was easily hydrolyzed by treatment with LiOH in THF and
water at room temperature to afford acid 44 in 92% yield. TBTUꢀpromoted amidation of 44 with
amine 9 led to a 67% yield of 45. Lateꢀstage Ley oxidation (TPAP/NMO) and then Pinnick
oxidation straightforwardly afforded acid precursor 47 in 80% yield over two steps. Finally,
global debenzylation and azide reduction provided 6 in 88% yield. Like 5, compound 6
demonstrated atropisomerism in CD OD with a ratio about 5:1 (see Supporting Information).
3
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Scheme 7. Attempts to hydrolyze the ester in 38.
0
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0
Scheme 8. Synthesis of target 6.
Synthesis of target 7. To synthesize 7, dicarboxylic acid monoester 40 (Scheme 9) was
used as the starting material. Decarboxylation of 40 in toluene at reflux afforded ester 48 as a 1:1
mixture of diasteroisomers in 78% yield. These compounds were inseparable using silica gel
column chromatography and were therefore carried forward as a mixture. Hydroylsis of 48 by
treatment with LiOH in THF and water afforded the corresponding carboxylic acids 49 (also as
an inseparable mixture of diastereomers), which were coupled with amine 9. The resulting two
amidation products, 50 and 51, were separated by column chromatography to give major and
minor products in a 4:1 ratio. NOESY experiments (see Supporting Information) showed that the
interaction between the CH (δ1.52 ppm) and Hꢀ2´(δ3.90 ppm) in the major product was stronger
3
than the interaction between the CH (δ1.38 ppm) and Hꢀ2´ (δ4.03 ppm) in the minor product.
3
These results suggest that 50, with a cis relationship between the benzyl ether and amide groups
was formed as the major product (64% yield calculated based on amine 9). Compound 51 was
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formed as minor product in 16% yield. Unreacted 49 was also isolated at the end of the reaction.
These yields presumably arise from differences in reactivities between the two stereoisomeric
acids in the amidation reaction. After deprotection in a mixture of methanol, water and acetic
10
11
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60
acid (10:1:0.1) under a H atmosphere with 10% Pd/C as the catalyst, 7 was obtained in 87%
2
yield. Unlike the 5 and 6, Nꢀacylated monosaccharide 7 did not show any atropisomeric effects in
CD OD, which we attribute to more efficient amide bond rotation due to the lack of an adjacent
3
carboxylic acid group.
O
2'
N
H
O
O
N
O
H
2
, Pd/C,
MeO
BnO
AcOH, CH OH, H O
3
2
MeO
HN
BnO
HO
HN
HO
O
87%
O
BnO
O
N
3
HO
5
0
O
NH
2
LiOH,
2
9, TBTU, DIEA,
6
7
toluene,
reflux
O
O
N
O
6
O
THF, H O
N
DMF
N
64%
CO
2
CH
3
2
CO H
OH
CH
+
CO
2
3
78% MeO
MeO
BnO
MeO
BnO
BnO
2'
O
H
N
O
40
48
49
MeO
BnO
HN
BnO
O
BnO
O
N
3
51
6%
6
1
Scheme 9. Synthesis of target 7.
Synthesis of target 8. The synthesis of 8 (Scheme 10) began with 3ꢀbutynol (17), which
1
4
was converted to αꢀamino acid ester 16 following the route reported by Qin and coꢀworkers.
Treatment of 16 with NaIO and RuCl led to a cascade oxidation–cyclization–oxidation
4
3
sequence, which produced NꢀBoc protected lactam 15 in 69% yield. Protecting the tertiary
alcohol of 15 with an acetyl group and removing the Boc group afforded lactam 55 in 82% yield
over two steps. Because lactam 55 was anticipated to be unstable in basic solution, milder
conditions were chosen for the Nꢀmethylation step. Thus, Nꢀhydroxymethylation of lactam 55
was achieved using paraformaldehyde in acetone in the presence of K CO and water with
2
3
sonication. To reduce the hemiaminal 56 to an Nꢀmethyl group (i.e., compound 57), we initially
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6
explored the use of Pd/C catalyzed hydrogenation at atmospheric pressure in the presence of
trifluoroacetic acid. However, this reaction was very slow and only a trace amount of product
was formed. We found, however, that the hemiaminal 56 could be converted to 57 by treatment
with triethylsilane and trifluoroacetic acid at room temperature. NꢀMethylamide 57 was obtained
in 81% yield over two steps from 55. Hydrolysis of methyl and acetate esters in 57 failed when
LiOH was used in THF and water; the major product formed was elimination of the acetate to
give the α,β unsaturated carboxylic acid. Fortunately, the use of milder conditions (1:2:2 TEA–
H O–CH OH), led to the desired compound 14 in 82% yield. Amidation of the carboxylic acid
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0
2
3
14 with the amine 9 was sluggish and led to 58 in modest 52% yield. Given the ease with which
the amidations leading to 5–7 were carried out, the relatively low yield and sluggishness of the
reaction were surprising. However, attempts to improve the yield of the product by changing the
reaction conditions (e.g., use of EDC and DIEA) were unsuccessful. Debenzylation and
reduction of 58 was achieved in a mixture of methanol, water and acetic acid (10:1:0.1) under a
1
H atmosphere with 10% Pd/C as the catalyst to afford 8. The H NMR spectrum of 8, similar to
2
that obtained for 7, did not show evidence of atropisomerism.
As was the case for 5, comparison of the NMR data for 8 with that reported for the
polysaccharide was prohibited by the insolubility of 8 in D O, the solvent used for NMR studies
2
of the naturallyꢀoccurring glycan. In this context it should be noted that the synthesis of the
enantiomer of 16, which could also be prepared from 3ꢀbutynol using the route described
14
previously, would enable the production of a lactam that could be used to prepare a derivative 8
with the opposable stereochemistry in this sideꢀchain appendage.
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Scheme 10. Synthesis of target 8.
Conclusion
In summary, an efficient convergent strategy was developed for the synthesis of four
unusual Nꢀacylated monosaccharides (5–8) fragments present in LOS IV from M. marinum. The
general approach to the targets involved the formation of a lactam intermediates (40, 44, 49 and
4), which were coupled to aminosugar 9 and then deprotection of the resulting product.
-
1
Monosaccharide 9 was prepared via a conventional route. The lactam moieties required for the
targets were assembled via two approaches. A key feature of the sequence leading to the lactam
precursors needed for the synthesis of 5–7 was the construction of highly substituted
oxazolidine–pyrrolinone bicyclic ring system 11 through a substrate controlled stereoselective
cyclization of αꢀmethoxyꢀβꢀketo amide 12. This reaction installed the two key stereocenters of
the lactam moiety in a single step. A different approach was developed to synthesize the lactam
needed for the preparation of target 8. A cascade oxidation–cyclization–oxidation sequence of
amino acid 16 was used to construct the core lactam 15. Due to the decomposition under strongly
basic conditions, a milder approach (hemiaminal formation with paraformaldehyde and K CO3
2
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6
followed by reaction with triethylsilane and trifluoroacetic acid) was used to furnish the Nꢀ
methyl lactam 14. The routes developed here will be useful in preparation of building blocks
needed for the synthesis of the complete LOS-IV molecule. In addition, 5–8 have been
0
1
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0
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0
synthesized in a form for conjugation to appropriate proteins and/or probes and hence represent
useful biochemical tools.
Experimental section
General experimental methods: All reagents were purchased from commercial sources and
were used without further purification unless noted. All reactions were monitored by TLC on
silica gel 60ꢀF254 (0.25 mm). Visualization of the reaction components was achieved using UV
fluorescence (254 nm) and/or by charring with acidified anisaldehyde solution in ethanol or
KMnO in water or cerium ammonium molybdate stain. Organic solvents were evaporated under
4
reduced pressure and the products were purified by column chromatography on silica gel (230–
400 mesh). Optical rotations were measured in a microcell (1 cm, 1 mL) at ambient temperature
1
and are in units of degree⋅mL/(g⋅dm). H NMR spectra were recorded at 400 MHz, 500 MHz,
600 MHz or 700 Mz and chemical shifts are referenced to residual CHCl (7.26 ppm, CDCl ),
3
3
13
CHD OD (3.30 ppm, CD OD) or DMSOꢀd (2.50 ppm, DMSOꢀd ). C NMR spectra were
2
3
5
6
recorded at 125 MHz and chemical shifts are referenced to CDCl (77.0 ppm) or CD OD (49.3
3
3
ppm). Reported splitting patterns are abbreviated as s = singlet, d = doublet, t = triplet, m =
multiplet, br = broad, app = apparent. Assignments of NMR spectra were based on twoꢀ
1
1
dimensional experiments ( H– H COSY, HSQC and HMBC) and stereochemistry of the
1
anomeric centers of the pyranose rings were confirmed by measuring J
via coupled HSQC
Cꢀ1,Hꢀ1
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experiments. ESI/TOFꢀHRMS spectra were recorded on samples suspended in THF or CH OH
3
and added NaCl.
10
11
12
13
14
15
16
17
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22
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49
50
51
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55
56
57
58
59
60
8
-Azidooctyl
oxopyrrolidine-2´-carboxamido]-4,6-dideoxy-α-
30 mg, 0.037 mmol) in CH OH (5 mL), water (0.5 mL) and acetic acid (0.1 mL) was treated
4-[(2´R,3´S,4´R)-3´-(hydroxy)-4´-methoxy-2´-carboxyl-1´,3´-dimethyl-5´-
D
-galactopyranoside (5) To a solution of 42
(
3
with palladium on charcoal (10%, 10 mg) and subjected to hydrogen atmosphere for 20 h. The
mixture was filtered through Celite and the filtrate was concentrated. The residue was subjected
to flash chromatography (C18 column, gradient 0→50% CH OH–H O) to yield 5 (17 mg, 89%
3
2
yield) as a white amorphous solid. [α] = +28.6 (c 0.3, CH OH); The NMR data showed that
D
3
1
there were two atropisomers in CD OD in a 4:3 ratio. H NMR (500 MHz, CD OD, δ ) 4.80 (d,
3
3
H
1
H, J = 3.5 Hz, Hꢀ1 (major)), 4.78 (d, 1 H, J = 3.5 Hz, Hꢀ1 (minor)), 4.22−4.13 (m, 2H, Hꢀ4 and
Hꢀ5), 3.95 (s, 1 H, CH OCH (major)), 3.96−3.90 (m, 1 H, Hꢀ3), 3.85 (s, 1 H, CH OCH (minor)),
3
3
3
3
2
1
6
.69−3.66 (m, 2 H, octyl OCH and Hꢀ2), 3.66 (s, 3 H, OCH (minor)), 3.55−3.59 (m, 1 H, Hꢀ2),
2
3
.49−3.44 (m, 1 H, octyl OCH ), 2.91−2.88 (m, 2 H, CH NH ), 2.90 (s, 3 H, NCH (major)),
2
2
2
3
.80 (s, 3 H, NCH (minor)), 1.68−1.60 (m, 4 H, CH x 2), 1.55 (s, 3 H, CCH (minor)),
3
2
3
.44−1.32 (m, 8 H, CH x 4), 1.21 (d, 3 H, J = 6.5 Hz, Hꢀ6 (major)), 1.18 (d, 3 H, J = 6.5 Hz, Hꢀ
2
1
3
(minor)), 1.17 (s, 3 H, CCH (major)); C NMR (125 MHz, CD OD, δ ) for major
3
3
C
atropisomer 173.3 (CH NC=O), 167.9 (HNC=O), 99.0 (Cꢀ1), 85.2 (CH OCH), 78.9 (CH COH),
3
3
3
7
2.5 (CCO H), 70.1 (Cꢀ3), 69.1 (Cꢀ2), 68.0 (OCH CH ), 64.5 (Cꢀ5), 58.8 (OCH ), 54.8 (Cꢀ4),
2 2 2 3
13
3
9.3 (CH NH ), 29.2 to 28.5 (NCH and CH x 6), 17.7 (CCH ), 16.3 (Cꢀ6); C NMR (125 MHz,
2
2
3
2
3
CD OD, δ ) for minor atropisomer 174.2 (CH NC=O), 168.6 (HNC=O), 99.0 (Cꢀ1), 83.2
3
C
3
(CH OCH), 75.7 (CH COH), 69.9 (Cꢀ3), 69.2 (Cꢀ2), 68.1 (CCO H), 67.9 (OCH CH ), 64.9 (Cꢀ
3 3 2 2 2
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5
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6
5
), 59.6 (OCH ), 54.7 (Cꢀ4), 39.3 (CH NH ), 29.2 to 28.5 (NCH and CH x 6), 22.0 (CCH ),
3
2
2
3
2
3
16.1 (Cꢀ6); HRMS (ESI) calcd for (M+Na) C H N NaO : 542.2684. Found: 542.2678.
23 41
3
10
0
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8
-Azidooctyl
4-[(2´S,3´S,4´R)-3´-(hydroxy)-4´-methoxy-2´-carboxyl-1´,3´-dimethyl-5´-
-galactopyranoside (6) To a solution of 47
15 mg , 0.018 mmol) in CH OH (5 mL), water (0.5 mL) and acetic acid (0.1 mL) was added
oxopyrrolidine-2´-carboxamido]-4,6-dideoxy-α-
D
(
3
palladium on charcoal (10%, 7 mg) and subjected to hydrogen atmosphere for 20 h. The mixture
was filtered through Celite and the filtrate was concentrated. The residue was subjected to flash
chromatography (C18 column, 0→50% CH OH–H O) to yield 6 (8 mg, 88% yield) as a white
3
2
soild. [α] = +117.8 (c 0.3, CH OH); The NMR data showed that there were two atropisomers in
D
3
1
1
CD OD in a 5:1 ratio. H NMR for major atropisomer: H NMR (500 MHz, CD OD, δ ) 4.77 (d,
3
3
H
1
H, J1,2 = 4.0 Hz, Hꢀ1), 4.26 (dd, 1 H, J3,4 = 4.5 Hz, J4,5 = 1.5 Hz, Hꢀ4), 4.15 (qd, 1 H, J5,6 = 6.5
Hz, J4,5 = 1.5 Hz, Hꢀ5), 3.96 (s, 1 H, CH OCH), 3.84 (dd, 1 H, J = 10.0 Hz, J3,4 = 4.5 Hz, Hꢀ3),
3
2,3
3
.67−3.62 (m, 2 H, octyl OCH and Hꢀ2), 3.56 (s, 3 H, OCH ), 3.46 (dt, 1 H, J = 10.0, 6.5 Hz,
2
3
octyl OCH ), 2.91 (t, J = 8.0 Hz, 2 H, CH NH ), 2.85 (s, 3 H, NCH ), 1.67−1.60 (m, 4 H, CH x
2
2
2
3
2
13
2
), 1.42−1.36 (m, 8 H, CH x 4), 1.31 (s, 3 H, CCH ), 1.07 (d, 3 H, J = 6.5 Hz, Hꢀ6); C NMR
2
3
5,6
1
3
for major atropisomer: C NMR (125 MHz, CD OD, δ ) 173.7 (CH NC=O), 169.1 (HNC=O),
3
C
3
99.0 (Cꢀ1), 84.3 (CH OCH), 79.1 (CH COH), 70.3 (Cꢀ2), 69.8 (Cꢀ3), 67.9 (octyl OCH ), 64.3
3
3
2
(Cꢀ5), 58.9 (OCH ), 55.0 (Cꢀ4), 39.3 (CH NH ), 29.0 (CH ), 28.8 (CH ), 28.7 (CH ), 28.6
3 2 2 2 2 2
(NCH ), 27.2 (CH ), 25.9 (CH ), 25.7 (CH ), 17.8 (CCH ), 15.7 (Cꢀ6); HRMS (ESI) calcd for
3
2
2
2
3
(M+Na) C H N NaO : 542.2684. Found: 542.2677.
23 41 3 10
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-Azidooctyl 4-[(2´S,3´S,4´R)-3´-(hydroxy)-4´-methoxy-1´,3´-dimethyl-5´-oxopyrrolidine-2´-
carboxamido]-4,6-dideoxy-α-D-galactopyranoside (7) A solution of 50 (15.0 mg , 0.019 mmol)
in CH OH (5 mL), water (0.5 mL) and acetic acid (0.1 mL) was treated with palladium on
3
10
11
12
13
14
15
16
17
18
19
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22
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51
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59
60
charcoal (10%, 8 mg) and subjected to hydrogen atmosphere for 20 h. The mixture was filtered
through Celite and the filtrate was concentrated. The residue was triturated with CH Cl to yield
2
2
7
(8.1 mg, 0.016 mmol, 87% yield) as a white amorphous solid. [α] = +118.3 (c 0.3, CH OH);
D 3
1
H NMR (600 MHz, CD OD, , δ ) 4.78 (d, 1 H, J = 4.0 Hz, Hꢀ1), 4.34 (dd, 1 H, J3,4 = 4.5 Hz,
3
H
1,2
J4,5 = 1.5 Hz, Hꢀ4), 4.15 (qd, 1 H, J5,6 = 6.5 Hz, J4,5 = 1.5 Hz, Hꢀ5), 4.06 (s, 1 H, CH OCH), 4.01
3
(s, 1 H, CHC=ONH), 3.87 (dd, 1 H, J2,3 = 10.5 Hz, J3,4 = 4.5 Hz, Hꢀ3), 3.67 (dt, 1 H, J = 9.5, 7.0
Hz, octyl OCH ), 3.62 (dd, 1 H, J2,3 = 10.5 Hz, J1,2 = 4.0 Hz, Hꢀ2), 3.59 (s, 3 H, OCH ), 3.45 (dt,
2
3
1 H, J = 9.5, 6.5 Hz, octyl OCH ), 2.90 (t, 2 H, J = 7.5 Hz, CH NH ), 2.75 (s, 3 H, NCH ),
2
2
2
3
1
.68−1.62 (m, 4 H, CH x 2), 1.43−1.38 (m, 8 H, CH x 4), 1.37 (s, 3 H, CCH ), 1.11 (d, 3 H, J
2
2
3
5,6
13
= 6.5 Hz, Hꢀ6); C NMR (125 MHz, CD OD, δ ) 172.8 (CH NC=O), 170.0 (HNC=O), 98.9 (Cꢀ
3
C
3
1
), 84.1 (CH OCH), 76.1 (CH COH), 70.1 (CHC=ONH), 69.4 (Cꢀ3), 69.2 (Cꢀ2), 67.9 (octyl
3 3
OCH ), 64.1 (Cꢀ5), 58.9 (OCH ), 54.7 (Cꢀ4), 39.2 (CH NH ), 29.0 (CH ), 28.8 (CH ), 28.7
2
3
2
2
2
2
(CH ), 27.8 (NCH ), 27.2 (CH ), 25.9 (CH ), 25.7 (CH ), 20.5 (CCH ), 15.6 (Cꢀ6); HRMS (ESI)
2
3
2
2
2
3
calcd for (M+H) C H N O : 476.2966. Found: 476.2961.
2
2
42
3
8
8
-Azidooctyl 4-[(2´S,3´R)-3´-(hydroxy)-1´,3´-dimethyl-5´-oxopyrrolidine-2´- carboxamido]-
,6-dideoxy-α- -galactopyranoside (8) A solution of 58 (5.1 mg , 0.0077 mmol) in CH OH (5
4
D
3
mL), water (0.5 mL) and acetic acid (0.1 mL) was treated with palladium on charcoal (10%, 5
mg) and subjected to a hydrogen atmosphere for 8 h. The mixture was filtered through Celite and
the filtrate was concentrated. The residue was triturated with CH Cl to afford 8 (2.8 mg, 81%
2
2
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
yield) as a white amorphous solid. [α] = +89.0 (c 0.1, CH OH); H NMR (700 MHz, CD OD,
D
3
3
δH) 8.02 (d, 1 H, J = 10.5 Hz, NH), 4.77 (d, 1 H, J1,2 = 4.0 Hz, Hꢀ1), 4.33−4.35 (m, 1 H, Hꢀ4),
4
.14 (q, 1 H, J5,6 = 6.5 Hz, Hꢀ5), 4.09 (s, 1 H, CHC=ONH), 3.85 (dd, 1 H, J2,3 = 10.5 Hz, J3,4
=
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
.5 Hz, Hꢀ3), 3.66 (dt, 1 H, J = 10.0, 7.0 Hz, octyl OCH ), 3.59 (dd, 1 H, J = 10.5 Hz, J1,2 = 4.0
2
2,3
Hz, Hꢀ2), 3.44 (dt, 1 H, J = 10.0, 6.5 Hz, octyl OCH ), 2.90 (t, 2 H, J = 7.5 Hz, CH NH ), 2.76 (s,
2
2
2
3
H, NCH ), 2.61 (d, 1 H, J = 16.5 Hz, CH C=O), 1.31 (d, 1 H, J = 16.5 Hz, CH C=O),
3 2 2
1.68−1.60 (m, 4 H, CH x 2), 1.49 (s, 3 H, CCH ), 1.44−1.36 (m, 8 H, CH x 4), 1.12 (d, 3 H, J
2
3
2
5,6
1
3
=
6.5 Hz, Hꢀ6); C NMR (125 MHz, CD OD, δ ) 176.4 (CH NC=O), 171.5 (HNC=O), 100.5
3 C 3
(
Cꢀ1), 74.3 (CHC=ONH), 73.2 (CH COH), 70.9 (Cꢀ3 or Cꢀ2), 70.8 (Cꢀ3 or Cꢀ2), 69.4 (octyl
3
OCH ), 65.2 (Cꢀ5), 56.0 (Cꢀ4), 46.2 (CH C=O), 40.8 (CH NH ), 30.5 (CH ), 30.3 (CH ), 30.2
2
2
2
2
2
2
(
CH ), 29.1 (NCH ), 28.9 (CCH ), 28.6 (CH ), 27.4 (CH ), 27.2 (CH ), 17.1 (Cꢀ6); HRMS (ESI)
2
3
3
2
2
2
calcd for (M+H) C H N O : 446.2861. Found: 446.2860.
21 40
3
7
8
-Azidooctyl 4-amino-2,3-di-O-benzyl-4,6-dideoxy-α-
D
-galacatopyranoside (9) To a solution
of 24 (350 mg, 0.59 mmol) in CH OH (16 mL) at room temperature was added NaOH (aq.) (4
3
mL, 1 N, 4 mmol). The mixture was heated at reflux for 4 days, before being cooled, diluted with
water and extracted with CH Cl . The combined organic phases were washed with brine, dried
2
2
over Na SO and concentrated to afford 9 (281 mg, 96% yield) as a colorless oil. R 0.17 (2:3
2
4
f
1
hexane−EtOAc); [α] = +42.3 (c 0.4, CH Cl ); H NMR (500 MHz, CDCl , δ ) δ 7.42−7.30 (m,
D
2
2
3
H
1
0 H, ArH), 4.83 (d, 1 H, J = 12.0 Hz, PhCH , Cꢀ2), 4.79 (d, 1 H, J = 11.5 Hz, PhCH , Cꢀ3),
2 2
4
.77 (d, 1 H, J1,2 = 4.0 Hz, Hꢀ1), 4.72 (d, 1 H, J = 11.5 Hz, PhCH , Cꢀ3), 4.69 (d, 1 H, J = 12.0
2
Hz, PhCH , Cꢀ2), 4.05 (qd, 1 H, J = 6.5 Hz, J4,5 = 1.5 Hz, Hꢀ5), 3.89 (dd, 1 H, J2,3 = 10.0 Hz,
2
5,6
J3,4 = 4.0 Hz, Hꢀ3), 3.78 (dd, 1 H, J2,3 = 10.0 Hz, J1,2 = 4.0 Hz, Hꢀ2), 3.65 (dt, 1 H, J = 10.0, 7.0
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Hz, octyl OCH ), 3.47 (dt, 1 H, J = 10.0, 7.0 Hz, octyl OCH ), 3.29 (t, 2 H, J = 7.0 Hz, CH N ),
2
2
2
3
3.20 (dd, 1 H, J3,4 = 4.0 Hz, J4,5 = 1.5 Hz, Hꢀ4), 1.68−1.60 (m, 4H, CH x 2), 1.48 (br, 2H, NH ),
2
2
13
1
1
7
.42−1.37 (m, 8H, CH x 4), 1.26 (d, 3 H, J = 6.5 Hz, Hꢀ6); C NMR (125 MHz, CDCl , δ ) δ
2
5,6
3
C
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
38.8 (Ar), 128.4 (Ar), 128.3 (Ar), 127.8 (Ar), 127.6 (Ar), 127.63 (Ar), 127.61 (Ar), 97.4 (Cꢀ1),
8.6 (Cꢀ3), 75.5 (Cꢀ2), 73.1 (PhCH , Cꢀ2), 72.3 (PhCH , Cꢀ3), 68.1 (OCH CH ), 65.1 (Cꢀ5), 53.4
2
2
2
2
(
Cꢀ4), 51.5 (CH N ), 29.4 (CH ), 29.3 (CH ), 29.1 (CH ), 28.8 (CH ), 26.7 (CH ), 26.1 (CH ),
2 3 2 2 2 2 2 2
16.8 (Cꢀ6); HRMS (ESI) calcd for (M+H) C H N O : 497.3122. Found: 497.3113.
2
8
41
4
4
(2S,3S,4R)-methyl
3-(benzyloxy)-2-(hydroxymethyl)-4-methoxy-1,3-dimethyl-5-
oxopyrrolidine-2-carboxylate (10) To a solution of 38 (46 mg, 0.10 mmol) in THF (5 mL) was
added nꢀBu NF solution (0.5 mL, 1.0 M, 0.50 mmol) dropwise at room temperature. The mixture
4
was stirred for 3 h. Then water was added and the mixture was extracted with EtOAc. The
organic phases were washed with brine, dried over Na SO and concentrated. The crude residue
2
4
was purified by flash column chromatography (silica gel, gradient 30→50% EtOAc–hexane) to
afford 10 (30 mg, 89% yield) as a white amorphous solid. R 0.32 (2:3 hexane−EtOAc); [α] =
f
D
1
+
82.1 (c 0.1, CH Cl ); H NMR (500 MHz, CDCl , δ ) δ 7.36−7.26 (m, 5 H, ArH), 4.67 (d, 1 H,
2
2
3
H
J = 11.5 Hz, PhCH ), 4.53 (d, 1 H, J = 11.5 Hz, PhCH ), 4.13 (d, 1 H, J = 12.5 Hz, CH OH),
2
2
2
4
.10 (s, 1 H, CH OCH), 4.02 (d, 1 H, J = 12.5 Hz, CH OH), 3.75 (s, 3 H, CO CH ), 3.73 (s, 3 H,
3 2 2 3
1
3
OCH ), 2.92 (s, 3 H, NCH ), 1.48 (s, 3 H, CCH ); C NMR (125 MHz, CDCl , δ ) 172.3
3
3
3
3
C
(NC=O), 170.9 (CO CH ), 138.1 (Ar), 128.3 (Ar), 127.5 (Ar), 126.8 (Ar), 83.4 (PhCH OC), 82.2
2 3 2
(
CH OCH), 74.5 (CCO CH ), 66.0 (PhCH ), 62.5 (CH OH), 59.3 (OCH ), 52.7 (CO CH ), 27.7
3
2
3
2
2
3
2
3
(NCH ), 12.8 (CCH ); HRMS (ESI) calcd for (M+Na) C H NNaO : 360.1418. Found:
3
3
17 23
6
360.1420.
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1
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2
2
2
2
2
2
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3
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4
4
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5
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5
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(
3S,6R,7S,7aS)-methyl
3-(tert-butyl)-7-hydroxy-6-methoxy-7-methyl-5-
oxohexahydropyrrolo[1,2-c]oxazole-7a-carboxylate (11) To a solution of 12（1.50 g, 5.0
mmol）in dry toluene (100 mL) was added 1,8 diazabicyclo[5.4.0]undecꢀ7ꢀene (DBU, 0.38 g,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
.5 mmol) and the mixture was heated at 60 °C for 12 h and then cooled to room temperature.
The mixture was concentrated and the crude product was purified by flash column
chromatography (silica gel, gradient 9→13% EtOAc–hexane) to afford 11 (1.12 g, 70% yield) as
a white amorphous solid. R 0.52 (2:3 hexane−EtOAc); [α] = +35.8 (c 0.2, CH Cl ); a sample
f
D
2
2
1
was recrystallized from hexane and EtOAc, m.p. 136–137 °C; H NMR (500 MHz, CDCl , δ )
3
H
4
.95 (s, 1 H, CH(CH ) ), 4.69 (d, 1 H, J = 9.5 Hz, OCH H C), 4.61 (s, 1 H, CH OCH), 3.95 (d, 1
3 3 R S 3
H, J = 9.5 Hz, OCH H C), 3.85 (s, 3 H, CO CH ), 3.70 (s, 3 H, OCH ), 2.12 (s, 1 H, OH), 1.33
R
S
2
3
3
1
3
(s, 3 H, CCH ), 0.91 (s, 9 H, C(CH ) ); C NMR (125 MHz, CDCl , δ ) 173.0 (NC=O), 171.2
3
3 3
3
C
(CO CH ), 96.1 (CH(CH ) ), 85.6 (CH OCH), 81.2 (HOCCH ), 75.8 (CCO CH ), 69.0
2 3 3 3 3 3 2 3
(OCH H C), 59.8 (OCH ), 52.8 (CO CH ), 36.4 (C(CH ) ), 24.9 (C(CH ) ), 18.6 (CCH );
R
S
3
2
3
3 3
3 3
3
HRMS (ESI) calcd for (M+Na ) C H NNaO : 324.1418. Found: 324.1419.
1
4
23
6
(2S,4R)-methyl 2-(tert-butyl)-3-(2-methoxy-3-oxobutanoyl)oxazolidine-4-carboxylate (12)
To a stirred suspension of PhI(OAc) (1.3 g, 7.2 mmol) in anhydrous CH OH (25 mL) at room
2
3
temperature was added BF •OEt (0.9 mL, 7.2 mmol). After the solution became clear,
3
2
compound 28 (1.5 g, 5.5 mmol) in CH OH (3 mL) was added dropwise and the mixture was
3
stirred at room temperature for 16 h. At that point, half of the solvent was removed and the
BF •OEt was quenched by the addition of a satd aq solution of NaHCO . The mixture was then
3
2
3
extracted with EtOAc. The organic phases were washed with brine, dried over Na SO ,
2
4
concentrated, and the residue was purified by flash column chromatography (silica gel, gradient
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0
→25% EtOAc–hexane) to yield 12 (1.5 g, 90% yield, 96:4 = keto–enol tautomers, 3:1 for two
inseparable diastereoisomeric αꢀmethoxyꢀβꢀketo amides) as a colorless oil; R 0.20 (3:1
f
1
hexane−EtOAc); NMR data for major isomer: H NMR (500 MHz, CDCl , δ ) 5.31 (s, 1 H,
3
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CH(CH ) ), 5.30 (dd, 1 H, J = 7.0, 1.5 Hz, CHCO CH ), 4.73 (s, 1 H, CHOCH ), 4.60 (dd, 1 H,
3
3
2
3
3
J = 8.5, 1.5 Hz, OCH ), 3.98 (dd, 1 H, J = 8.5, 7.0 Hz, OCH ), 3.82 (s, 3 H, CO CH ), 3.51 (s, 3
2
2
2
3
13
H, CHOCH ), 2.34 (s, 3 H, CH C=O), 0.94 (s, 9 H, C(CH ) ); C NMR (125 MHz, CDCl , δ )
3
3
3 3
3
C
207.0 (CH C=O), 170.1 (CO CH ), 167.9 (NC=O), 97.1 (CH(CH ) ), 86.8 (CHOCH ), 67.7
3
2
3
3 3
3
(OCH ), 58.2 (CHCO CH ), 57.5 (OCH ), 52.7 (CO CH ), 37.2 (C(CH ) ), 26.8 (CH C=O), 25.7
2 2 3 3 2 3 3 3 3
1
(
C(CH ) ); NMR data for minor isomer: H NMR (500 MHz, CDCl , δ ) 5.34 (s, 1 H,
3 3
3
H
CH(CH ) ), 4.79 (dd, 1 H, J = 7.0, 2.0 Hz, CHCO CH ), 4.63 (s, 1 H, CHOCH ), 4.46 (dd, 1 H, J
3
3
2
3
3
=
8.5, 2.0 Hz, OCH ), 3.94 (dd, 1 H, J = 8.5, 7.0 Hz, OCH ), 3.81 (s, 3 H, CO CH ), 3.49 (s, 3 H,
2 2 2 3
13
CHOCH ), 2.31 (s, 3 H, CH C=O), 0.99 (s, 9 H, C(CH ) ); C NMR (125 MHz, CDCl , δ )
3
3
3 3
3
C
2
02.5.0 (CH C=O), 170.0 (CO CH ), 168.6 (NC=O), 97.6 (CH(CH ) ), 88.8 (CHOCH ), 68.8
3 2 3 3 3 3
(
OCH ), 59.3 (CHCO CH ), 58.6 (OCH ), 52.5 (CO CH ), 37.1 (C(CH ) ), 27.0 (CH C=O), 26.0
2 2 3 3 2 3 3 3 3
(C(CH ) ); HRMS (ESI) calcd for (M+H ) C H NO : 302.1598. Found: 302.1597.
3
3
14 24
6
(2S,3R)-3-hydroxy-1,3-dimethyl-5-oxopyrrolidine-2-carboxylic acid (14) Compound 57 (80
mg, 0.35 mmol) was dissolved in TEA (5 mL), CH OH (10 mL) and water (10 mL) and the
3
mixture was stirred at room temperature over night. Then the solution was concentrated, and the
resulting residue was subjected to flash column chromatography (latrobeads 6RSꢀ8060, gradient
1
0%→50% CH OH–CH Cl ) to yield 14 (49 mg, 82% yield) as a white amorphous solid; R 0.2
3
2
2
f
1
(
2:1 CH Cl −CH OH); [α] = +11.6 (c 0.3, CH Cl ); H NMR (500 MHz, CD OD, δ ) 3.98 (s, 1
2
2
3
D
2
2
3
H
H, NCH), 2.83 (s, 3 H, NCH ), 2.56 (d, 1 H, J = 17.0 Hz, CH C=O), 2.41 (d, 1 H, J = 17.0 Hz,
3
2
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2
2
2
2
2
2
2
3
3
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3
3
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4
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4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
CH C=O), 1.50 (s, 3 H, CCH ); C NMR (125 MHz, CDCl , δ ) 176.1 (NC=O), 170.0 (CO H),
2
3
3
C
2
7
5.0 (NCH), 73.0 (CH COH), 46.6 (CH C=O), 29.4 (NCH ), 28.1 (CCH ); HRMS (ESI) (MꢀH)
3 2 3 3
calcd for C H NO : 172.0615. Found: 172.0618.
7
10
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
6
7
8
9
0
1
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(2S,3R)-1-tert-butyl 2-methyl 3-hydroxy-3-methyl-5-oxopyrrolidine-1,2-dicarboxylate (15)
14
Compound 16 (0.91g, 3.31 mmol) was dissolved in CCl (15 mL), CH CN (15 mL), and water
4
3
(
18 mL). With vigorous stirring, NaIO (2.12 g, 9.9 mmol) and RuCl ·H O (30 mg) were added
4 3 2
at 0 °C. The mixture was stirred at room temperature for 1 h, and then EtOAc (30 mL) was
added. The mixture was washed with a satd aq solution of NaHCO , brine, dried over Na SO
4
3
2
and concentrated. The residue was purified by flash chromatography (silica gel, gradient
5
0→150% EtOAc–hexane) to give 15 as a colorless liquid (0.61 g, 69% yield). R 0.3 (4:3
f
1
EtOAc−Hexane); [α] = +0.19 (c 0.5, CH Cl ); H NMR (500 MHz, CDCl , δ ) 4.34 (s, 1 H,
D
2
2
3
H
NCH), 3.84 (s, 3 H, OCH ), 2.90 (d, 1 H, J = 17.0 Hz, COCH ), 2.60 (d, 1 H, J = 17.0 Hz,
3
2
13
COCH ), 2.26 (br, 1 H, OH), 1.52 (s, 9 H, C(CH ) ), 1.45 (s, 3 H, CCH ); C NMR (125 MHz,
2
3 3
3
CDCl , δ ) 170.6 (NC=O), 169.1 (CO CH ), 149.0 (CO C(CH ) ), 84.1 (OC(CH ) ), 70.8
3
C
2
3
2
3 3
3 3
(
CH COH), 69.6 (NCH), 52.5 (OCH ), 46.4 (CH ), 28.2 (CCH ), 27.9 (C(CH ) ); HRMS (ESI)
3 3 2 3 3 3
calcd for (M+Na) C H NNaO : 296.1105. Found: 296.1105.
1
2
19
6
Allyl 2,3-di-O-benzyl-6-O-tosyl-α-
D
-glucopyranoside (19) To a solution of allyl 2,3ꢀdiꢀOꢀ
1
5
benzylꢀαꢀ
Dꢀglucopyranoside (18, 1.60 g, 4.07 mmol) in anhydrous pyridine (10 mL) was added
TsCl (1.16 g, 6.10 mmol) at 0 °C. After being stirred for 12 h at room temperature, the mixture
was diluted with EtOAc (30 mL), washed with 1 N HCl, a satd aq solution of NaHCO , brine,
3
dried over Na SO and concentrated. The crude residue was purified by flash column
2
4
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chromatography (silica gel, gradient 20→25% EtOAc–hexane) to afford 19 (2.14 g, 95% yield)
1
as a colorless oil. R 0.45 (2:1 hexane−EtOAc); [α] = +38.1 (c 0.7, CH Cl ); H NMR (500 MHz,
f
D
2
2
CDCl , δ ) 7.80 (d, 2 H, J = 8.5 Hz, ArH), 7.39−7.22 (m, 12 H, ArH), 5.92 (dddd, 1 H, J = 17.0,
3
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
10.0, 6.5, 5.0 Hz, CH =CH), 5.32 (app dq, 1 H, J = 17.0, 1.5 Hz, CH =CH), 5.24 (app dq, 1 H, J
2
2
=
10.0, 1.0 Hz, CH =CH), 5.02 (d, 1 H, J = 12.0 Hz, PhCH , Cꢀ3), 4.79 (d, 1 H, J = 3.5 Hz, Hꢀ
2 2 1,2
1
), 4.74 (d, 1 H, J = 12.0 Hz, PhCH , Cꢀ2), 4.71 (d, 1 H, J = 12.0 Hz, PhCH , Cꢀ3), 4.65 (d, 1 H,
2
2
J = 12.0 Hz, PhCH , Cꢀ2), 4.28−4.22 (m, 2 H, Hꢀ4 and Hꢀ5), 4.13 (app ddt, 1 H, J = 13.0, 5.0,
2
1
.5 Hz, CH =CHCH ), 3.97 (app ddt, 1 H, J = 13.0, 6.5, 1.0 Hz, CH =CHCH ), 3.79 (app t, 1 H,
2 2 2 2
J2,3 = J3,4 = 9.5 Hz, Hꢀ3), 3.79 (dd, 1 H, J6R,6S = 10.0 Hz, J = 2.5 Hz, Hꢀ6), 3.49 (dd, 1 H, J2,3
=
5,6
9.5 Hz, J1,2 = 3.5 Hz, Hꢀ2), 3.45 (dd, 1 H, J6R,6S = 10.0 Hz, J5,6 = 3.0 Hz, Hꢀ6), 2.45 (s, 3 H,
1
3
PhCH ), 2.23 (d, 1 H, J = 3.0 Hz, OH); C NMR (125 MHz, CDCl , δ ) 144.8 (Ar), 138.7 (Ar),
3
3
C
137.9 (Ar), 133.5 (CH=CH ), 133.0 (Ar), 129.8 (Ar), 128.6 (Ar), 128.5 (Ar), 128.1 (Ar), 128.0
2
(Ar), 127.9 (Ar), 118.5 (CH=CH ), 95.6 (Cꢀ1), 81.1 (Cꢀ3), 79.5 (Cꢀ2), 75.4 (PhCH , Cꢀ3), 73.0
2 2
(
PhCH , Cꢀ2), 69.5 (Cꢀ6), 69.2 and 68.9 (Cꢀ4 and Cꢀ5), 68.4 (CH CH=CH ), 21.7 (CH PhSO );
2
2
2
3
2
HRMS (ESI) calcd for (M+Na) C H NaO S: 577.1867. Found: 577.1864.
3
0
34
8
Allyl 2,3-di-O-benzyl-6-deoxy-α-
D
-glucopyranoside (20) Tosylate 19 (2.10 g, 3.79 mmol) was
dissolved in THF (30 mL) and LiAlH (288 mg, 7.58 mmol) was added. The reaction mixture
4
was heated at reflux for 3 h. After completion of the reaction, the LiAlH was quenched by
4
slowly adding the mixture to ice, then the mixture was filtered through Celite. The filter cake
was washed with EtOAc, and the resulting cloudy solution was filtered again through Celite. The
combined organic layers were washed with 1 N HCl, a satd aq solution of NaHCO , brine, dried
3
over Na SO and concentrated. The crude residue was purified by flash column chromatography
2
4
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(
silica gel, gradient 10→15% EtOAc–hexane) to afford 20 (1.25 g, 85% yield) as a colorless oil.
1
R 0.39 (4:1 hexane−EtOAc); [α] = +70.4 (c 0.2, CH Cl ); H NMR (500 MHz, CDCl , δ )
f
D
2
2
3
H
7
.41−7.29 (m, 10 H, ArH), 5.98 (dddd, 1 H, J = 17.0, 10.0, 6.5, 5.0 Hz, CH =CH), 5.37 (app dq,
2
0
1
2
3
4
5
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7
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9
0
1
2
3
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6
7
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9
0
1
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4
5
6
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1 H, J = 17.0, 1.0 Hz, CH =CH), 5.27 (app dq, 1 H, J = 10.0, 1.0 Hz, CH =CH), 5.08 (d, 1 H, J =
2
2
1
1.5 Hz, PhCH , Cꢀ3), 4.81 (d, 1 H, J = 3.5 Hz, Hꢀ1), 4.76 (d, 1 H, J = 12.0 Hz, PhCH , Cꢀ2),
2 1,2 2
4
.73 (d, 1 H, J = 11.5 Hz, PhCH , Cꢀ3), 4.70 (d, 1 H, J = 12.0 Hz, PhCH , Cꢀ2), 4.20 (app ddt, 1
2
2
H, J = 13.0, 5.0, 1.0 Hz, CH =CHCH ), 4.05 (app ddt, 1 H, J = 13.0, 6.5, 1.0 Hz, CH =CHCH ),
2
2
2
2
3
.81 (app t, 1 H, J2,3 = J3,4 = 9.5 Hz, Hꢀ3), 3.75 (dq, 1 H, J4,5 = 9.5 Hz, J5,6 = 6.0 Hz, Hꢀ5), 3.57
(dd, 1 H, J2,3 = 9.5 Hz, J1,2 = 3.5 Hz, Hꢀ2), 3.21 (app td, 1 H, J4,5 = J3,4 = 9.5 Hz, J4,OH = 2.0 Hz,
1
3
Hꢀ4), 2.19 (d, 1 H, J = 2.0 Hz, OH), 1.27 (d, 3 H, J5,6 = 6.0 Hz, Hꢀ6); C NMR (125 MHz,
CDCl , δ ) 138.8 (Ar), 138.1 (Ar), 133.9 (CH=CH ), 128.7 (Ar), 128.5 (Ar), 128.1 (Ar), 128.0
3
C
2
(Ar), 127.92 (Ar), 127.91 (Ar), 118.1 (CH=CH ), 95.5 (Cꢀ1), 81.3 (Cꢀ3), 80.1 (Cꢀ2), 75.4 (Cꢀ4),
2
7
5.3 (PhCH , Cꢀ3), 72.8 (PhCH , Cꢀ2), 68.2 (CH CH=CH ), 67.1 (Cꢀ5), 17.7 (Cꢀ6); HRMS (ESI)
2 2 2 2
calcd for (M+Na) C H NaO : 407.1829. Found: 407.1825.
2
3
28
5
Allyl 4-azido-2,3-di-O-benzyl-4,6-dideoxy-α-
D
-galactopyranoside (21) To a solution of 20
(1.25 g, 3.25 mmol) and pyridine (3 mL) in anhydrous CH Cl at 0 °C was added Tf O (1.37 g,
2
2
2
4
.88 mmol) slowly. After being stirred for 1 h, the mixture was diluted with CH Cl , washed
2 2
with water, a satd aq solution of NaHCO , brine, dried over Na SO and concentrated to obtain
3
2
4
the crude triflate, which was dissolved into DMF (15 mL). Excess NaN (845 mg, 13 mmol) was
3
added and the mixture was stirred overnight at room temperature. After completion of the
reaction, the mixture was filtered through Celite and the residue was washed with EtOAc. The
combined solutions were concentrated and the resulting residue was purified by flash column
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chromatography (silica gel, gradient 4→6% EtOAc–hexane) to afford 21 (1.13 g, 85% yield) as
1
a colorless oil. R 0.61 (4:1 hexane−EtOAc); [α] = +85.1 (c 0.4, CH Cl ); H NMR (500 MHz,
f
D
2
2
CDCl , δ ) 7.44−7.29 (m, 10 H, ArH), 5.95 (dddd, 1 H, J = 17.0, 10.0, 6.0, 5.0 Hz, CH =CH),
3
H
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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35
36
37
38
39
40
41
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44
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5.34 (d, 1 H, J = 17.0 Hz, CH =CH), 5.25 (d, 1 H, J = 10.0 Hz, CH =CH), 4.89 (d, 1 H, J = 12.0
2
2
Hz, PhCH , Cꢀ3), 4.84 (d, 1 H, J = 12.0 Hz, PhCH , Cꢀ2), 4.80 (d, 1 H, J = 3.5 Hz, Hꢀ1), 4.79
2
2
1,2
(
d, 1 H, J = 12.0 Hz, PhCH , Cꢀ3), 4.68 (d, 1 H, J = 12.0 Hz, PhCH , Cꢀ2), 4.12 (dd, 1 H, J =
2
2
12.5, 5.0 Hz, CH =CHCH ), 4.10 (dd, 1 H, J = 9.5 Hz, J3,4 = 3.5 Hz, Hꢀ3), 4.03 (dd, 1 H, J =
2
2
2,3
12.5, 6.0 Hz, CH =CHCH ), 4.01 (q, 1 H, J = 6.5 Hz, Hꢀ5), 3.89 (dd, 1 H, J2,3 = 9.5 Hz, J1,2 =
2 2 5,6
1
3
3
.5 Hz, Hꢀ2), 3.75 (d, 1 H, J3,4 = 3.5 Hz, Hꢀ4), 1.26 (d, 3 H, J5,6 = 6.5 Hz, Hꢀ6); C NMR (125
MHz, CDCl , δ ) 138.4 (Ar), 138.3 (Ar), 133.9 (CH=CH ), 128.5 (Ar), 128.4 (Ar), 128.0 (Ar),
3
C
2
1
27.8 (Ar), 127.7 (Ar), 127.6 (Ar), 118.1 (CH=CH ), 96.3 (Cꢀ1), 78.1 (Cꢀ3), 76.0 (Cꢀ2), 73.6
2
(PhCH , Cꢀ2), 73.2 (PhCH , Cꢀ3), 68.5 (CH CH=CH ), 65.2 (Cꢀ4), 64.5 (Cꢀ5), 17.3 (Cꢀ6);
2
2
2
2
HRMS (ESI) calcd for (M+Na) C H N NaO : 432.1894. Found: 432.1893.
2
3
27
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4
Allyl 4-amino-2,3-di-O-benzyl-4,6-dideoxy-α-D-galactopyranoside (22) To a solution of 21
(1.05 g, 2.56 mmol) in THF (40 mL) at room temperature was added NaOH (1 M, 10 mL). Then
a solution of PMe (10.0 mL, 1M in THF, 10.0 mmol) was added dropwise. The mixture was
3
stirred at room temperature for 10 h. After completion of the reaction, the mixture was diluted
with water, extracted with CH Cl . The combined organic phases were washed with brine, dried
2
2
over Na SO and concentrated. The crude residue was purified by flash column chromatography
2
4
(
silica gel, gradient 0→2% CH OH–EtOAc) to afford 22 (0.91 g, 92% yield) as a colorless oil. R
3
f
1
0
.17 (2:3 hexane−EtOAc); [α] = +98.3 (c 0.5, CH Cl ); H NMR (500 MHz, CDCl , δ )
D
2
2
3
H
7
.42−7.29 (m, 10 H, ArH), 5.97 (dddd, 1 H, J = 17.0, 10.0, 6.5, 5.0 Hz, CH =CH), 5.36 (apt dq,
2
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H, J = 17.0, 1.5 Hz, CH =CH), 5.25 (apt dq, 1 H, J = 10.0, 1.0 Hz, CH =CH), 4.83 (d, 1 H, J
1,2
2
2
=
4.0 Hz, Hꢀ1), 4.81 (d, 1 H, J = 12.0 Hz, PhCH , Cꢀ2), 4.79 (d, 1 H, J = 11.5 Hz, PhCH , Cꢀ3),
2 2
4.72 (d, 1 H, J = 11.5 Hz, PhCH , Cꢀ2), 4.68 (d, 1 H, J = 12.0 Hz, PhCH , Cꢀ3), 4.17 (apt ddt, 1
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0
H, J = 13.0, 5.0, 1.0 Hz, CH =CHCH ), 4.06 (q, 1 H, J = 6.5 Hz, Hꢀ5), 4.05 (apt ddt, J = 13.0,
2
2
5,6
6.5, 1.5 Hz, 1H, CH =CHCH ), 3.92 (dd, 1 H, J = 10.0 Hz, J3,4 = 4.0 Hz, Hꢀ3), 3.78 (dd, 1 H,
2
2
2,3
J2,3 = 10.0 Hz, J1,2 = 4.0 Hz, Hꢀ2), 3.22 (d, 1 H, J3,4 = 4.0 Hz, Hꢀ4), 2.06 (br, 2 H, NH ), 1.26 (d,
2
13
3
H, J5,6 = 6.5 Hz, Hꢀ6); C NMR (125 MHz, CDCl , δ ) 138.7 (Ar), 138.6 (Ar), 134.1
3 C
(CH=CH ), 128.4 (Ar), 128.3 (Ar), 127.9 (Ar), 127.7 (Ar), 127.6 (Ar), 117.9 (CH=CH ), 96.2
2
2
(Cꢀ1), 78.3 (Cꢀ3), 75.3 (Cꢀ2), 73.2 (PhCH , Cꢀ2), 72.4 (PhCH , Cꢀ3), 68.3 (CH CH=CH ), 65.2
2 2 2 2
(Cꢀ5), 53.5 (Cꢀ4), 16.7 (Cꢀ6); HRMS (ESI) calcd for (M+H) C H NO : 384.2169. Found:
23 30 4
384.2165.
Allyl 4-trifluoroacetamido-2,3-di-O-benzyl-4,6-dideoxy-α-D-galactopyranoside (23) To a
solution of 22 (590 mg, 1.5 mmol) in anhydrous pyridine (15 mL) at 0 °C was added
trifluoroacetic anhydride (594 mg, 3.0 mol) dropwise. The mixture was slowly warmed to room
temperature and stirred for 6 h. After completion of the reaction, the mixture was concentrated
under vacuum and diluted with CH Cl . The organic solution was washed with 1 N HCl, brine,
2
2
dried over Na SO and concentrated. The crude residue was purified by flash column
2
4
chromatography (silica gel, gradient 10→15% EtOAc–hexane) to afford 23 (634 mg, 86% yield)
1
as a colorless oil. R 0.48 (4:1 hexane−EtOAc); [α] = +71.1 (c 0.3, CH Cl ); H NMR (500 MHz,
f
D
2
2
CDCl , δ ) 7.40−7.30 (m, 10 H, ArH), 6.32 (d, 1 H, J = 10.0 Hz, NH), 5.96 (dddd, 1 H, J = 17.0,
3
H
1
0.0, 6.5, 5.0 Hz, CH =CH), 5.37 (apt dq, 1 H, J = 17.0, 1.5 Hz, CH =CH), 5.28 (apt dq, 1 H, J =
2
2
10.0, 1.0 Hz, CH =CH), 4.84 (d, 1 H, J = 12.0 Hz, PhCH , Cꢀ2), 4.83 (d, 1 H, J = 4.0 Hz, Hꢀ1),
2
2
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.83 (d, 1 H, J = 11.0 Hz, PhCH , Cꢀ3), 4.68 (d, 1 H, J = 12.0 Hz, PhCH , Cꢀ2), 4.60 (d, 1 H, J =
2
2
11.0 Hz, PhCH , Cꢀ3), 4.57 (dd, 1 H, J
= 10.0 Hz, J = 4.0 Hz, Hꢀ4), 4.23 (qd, 1 H, J = 6.5
3,4 5,6
2
4,NH
Hz, J4,5 = 1.5 Hz, Hꢀ5), 4.19 (apt ddt, 1 H, J = 13.0, 5.0, 1.5 Hz, CH =CHCH ), 4.07 (dd, 1 H,
2
2
10
11
12
13
14
15
16
17
18
19
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21
22
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60
J2,3 = 10.0 Hz, J3,4 = 4.0 Hz, Hꢀ3), 4.06 (apt ddt, 1 H, J = 13.0, 6.5, 1.0 Hz, CH =CHCH ), 3.47
2
2
1
3
(dd, 1 H, J2,3 = 10.0 Hz, J1,2 = 4.0 Hz, Hꢀ2), 1.18 (d, 3 H, J5,6 = 6.5 Hz, Hꢀ6); C NMR (125
2
MHz, CDCl , δ ) 158.0 (q, J = 37.5 Hz, C=O), 138.1 (Ar), 137.9 (Ar), 133.5 (CH=CH2),
3
C
C,F
1
1
28.4 (Ar), 128.3 (Ar), 128.0 (Ar), 127.9 (Ar), 127.7 (Ar), 118.4 (CH=CH ), 115.9 (q, J
=
2
C,F
287.5 Hz, CF ), 96.3 (Cꢀ1), 76.2 (Cꢀ3), 75.1 (Cꢀ2), 73.4 (PhCH , Cꢀ2), 72.1 (PhCH , Cꢀ3), 68.8
3
2
2
(
CH CH=CH ), 63.8 (Cꢀ5), 52.1 (Cꢀ4), 16.4 (Cꢀ6); HRMS (ESI) calcd for (M+Na)
2 2
C H F NNaO : 502.1812. Found: 502.1808.
25 28
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8-Azidooctyl 4-trifluoroacetamido-2,3-di-O-benzyl-4,6-dideoxy-α-D-galactopyranoside (24)
To a solution of 23 (500 mg, 1.04 mmol) in CH OH (10 mL) and CH Cl (10 mL) at room
3
2
2
temperature was added PdCl (18 mg, 0.10 mol, 0.1 equiv). The mixture was stirred at room
2
temperature for 16 h. After completion of the reaction, the mixture was filtered through a plug of
Celite and the filtrate was concentrated to obtain crude 4ꢀtrifluoroacetamidoꢀ2,3ꢀdiꢀOꢀbenzylꢀ
4,6ꢀdideoxyꢀ
Dꢀgalactopyranoside, which was carried forward without further purification. This
crude product was dissolved in anhydrous CH Cl (20 mL) with 4 Å molecular sieves and this
2
2
mixture was treated with trichloroacetonitrile (1.5 g, 10.4 mmol) and Cs CO (676 mg, 2.08
2
3
mmol). The mixture was stirred at room temperature for 6 h and then filtered through Celite. The
filtrate was concentrated to obtain the corresponding glycosyl trichloroacetimidate, which was
dissolved in Et O (5 mL) and added to a mixture of 8ꢀazidooctanol (355 mg, 2.08 mmol) and 4 Å
2
molecular sieves in Et O (5 mL). The mixture was cooled to 0 °C and TMSOTf (15 µL) was
2
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