Synthesis, structural elucidation, and biochemical analysis of immunoactive glucuronosyl diacylglycerides of mycobacteria and corynebacteria

Article abstract of DOI:10.1021/jo302508e

Glucuronosyl diacylglycerides (GlcAGroAc₂) are functionally important glycolipids and membrane anchors for cell wall lipoglycans in the Corynebacteria. Here we describe the complete synthesis of distinct acyl-isoforms of GlcAGroAc₂ bearing both acylation patterns of (R)-tuberculostearic acid (C_19:0) and palmitic acid (C _16:0) and their mass spectral characterization. Collision-induced fragmentation mass spectrometry identified characteristic fragment ions that were used to develop rules allowing the assignment of the acylation pattern as C_19:0 (sn-1), C_16:0 (sn-2) in the natural product from Mycobacterium smegmatis, and the structural assignment of related C_18:1 (sn-1), C_16:0 (sn-2) GlcAGroAc₂ glycolipids from M. smegmatis and Corynebacterium glutamicum. A synthetic hydrophobic octyl glucuronoside was used to characterize the GDP-mannose-dependent mannosyltransferase MgtA from C. glutamicum that extends GlcAGroAc₂. This enzyme is an Mg²⁺/Mn²⁺- dependent metalloenzyme that undergoes dramatic activation upon reduction with dithiothreitol.

Full text of DOI:10.1021/jo302508e

Featured Article
pubs.acs.org/joc
Synthesis, Structural Elucidation, And Biochemical Analysis of
Immunoactive Glucuronosyl Diacylglycerides of Mycobacteria and
Corynebacteria
Benjamin Cao,† Xingqiang Chen,† Yoshiki Yamaryo-Botte,‡ Mark B. Richardson,† Kirstee L. Martin,‡
George N. Khairallah,† Thusita W.T. Rupasinghe,§ Roisin M. O’Flaherty,† Richard A.J. O’Hair,†
Julie E. Ralton,‡ Paul K. Crellin,∥ Ross L. Coppel,∥ Malcolm J. McConville,*,‡ and Spencer J. Williams*,†
‡
§
†School of Chemistry, Department of Biochemistry and Molecular Biology, and Metabolomics Australia, Bio21 Molecular Science
and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia
∥Department of Microbiology, Monash University, Victoria 3800, Australia
S
* Supporting Information
ABSTRACT: Glucuronosyl diacylglycerides (GlcAGroAc2) are func-
tionally important glycolipids and membrane anchors for cell wall
lipoglycans in the Corynebacteria. Here we describe the complete
synthesis of distinct acyl-isoforms of GlcAGroAc2 bearing both acylation
patterns of (R)-tuberculostearic acid (C19:0) and palmitic acid (C16:0
)
and their mass spectral characterization. Collision-induced fragmentation
mass spectrometry identified characteristic fragment ions that were used
to develop “rules” allowing the assignment of the acylation pattern as
C19:0 (sn-1), C16:0 (sn-2) in the natural product from Mycobacterium
smegmatis, and the structural assignment of related C18:1 (sn-1), C16:0 (sn-
2) GlcAGroAc2 glycolipids from M. smegmatis and Corynebacterium
glutamicum. A synthetic hydrophobic octyl glucuronoside was used to
characterize the GDP-mannose-dependent mannosyltransferase MgtA
from C. glutamicum that extends GlcAGroAc2. This enzyme is an Mg2+/
Mn2+-dependent metalloenzyme that undergoes dramatic activation upon reduction with dithiothreitol.
(PIMs), lipomannan (LM), and lipoarabinomannan (LAM).6−9
INTRODUCTION
■
Recently, there has been increased interest in a range of
glycosylglycerolipids that contain glucuronic acid linked to
diacylglycerol (Figure 1b).10,11 These immunoreactive α-
glucuronosyl diacylglycerols were initially isolated from M.
smegmatis MNC strain 13 by Brennan and colleagues12 and
recently shown to function as alternative membrane anchors for
LM in Corynebacterium glutamicum.13−15 Although depicted as
nominal structures 1a and 2a, respectively, the precise arrange-
ment of the ester-linked (R)-tuberculostearic acid 4 or oleic acid
and palmitic acid on the glycerol backbone was not determined,
and structures 1b and 2b are viable alternatives (Figure 2a,b). We
have recently shown that structure 1a, and only this natural
arrangement, yielded a CD1d-presented antigen that could
activate murine-derived NKT cells through Vα10-Jα50 T cell
receptors,16 highlighting the importance of acyl regiochemistry.
Compound 1a produced a Th2-biased response, characterized
by the release of immunosuppressive cytokines, suggesting that
mycobacterial-derived α-glucuronosyl diacylglycerols may have
roles in modulating host−immune responses.16 Besra and co-
workers have also isolated the structurally related α-glucuronosyl
The suborder Corynebacterineae includes a range of agricultur-
ally, medically, and biotechnologically important bacteria.
Corynebacterium glutamicum is used extensively in the bio-
technology industry for the production of the amino acids L-
glutamate and L-lysine and is now a focus for intensive metabolic
engineering.1 Many mycobacteria are responsible for human and
animal diseases, especially Mycobacterium tuberculosis, the
causative agent of tuberculosis. The fast-growing bacterium
Mycobacterium smegmatis is generally considered to be non-
pathogenic2,3 and provides a readily handled fast-growing model
for the basic biochemistry and genetics of M. tuberculosis.4 All
mycobacteria and corynebacteria contain an atypical cell wall in
which the peptidoglycan layer is overlaid with layers of covalently
linked arabinogalactan and long-chain mycolic acids. The
mycolic acids effectively form the inner leaflet of an asymmetric
bilayer with noncovalently associated lipids, glycolipids, and
glycophospholipids forming the outer layer (Figure 1a).5 These
noncovalently associated lipids contribute to the bulk membrane
lipids and can also act as membrane anchors for abundant cell
wall lipoglycans that influence host immune interactions.5
The structure, biosynthesis, and function of several
corynebacterium/mycobacterium glycolipids have been inten-
sively studied, including the phosphatidylinositol mannosides
Received: November 15, 2012
Published: January 23, 2013
© 2013 American Chemical Society
2175
dx.doi.org/10.1021/jo302508e | J. Org. Chem. 2013, 78, 2175−2190

The Journal of Organic Chemistry
Featured Article
diacylglycerols (termed Gl-A) bearing oleic acid and palmitic
acid from C. glutamicum. Gl-A was depicted as having the
nominal structure 2b (viz. the isomer of 2a), although evidence
for this acylation pattern was not provided.17 In C. glutamicum it
was reported that nominal 2b undergoes mannosylation by the
action of the glycosyltransferase MgtA (NCgl0452) to form
nominal 3b (termed Gl-X) (Figure 2a); however, the acylation
pattern of this compound will be defined by its biosynthetic
precursor Gl-A, and the structure 3a represents a viable
alternative.17 Despite evidence that the M. tuberculosis ortholog
(Rv0557) can complement a C. glutamicum deletion mutant of
MgtA17,18 and the existence of an M. smegmatis MgtA ortholog,
there is no evidence that M. smegmatis can elongate its
endogenous GlcAGroAc2 species nor of the presence of any
GlcAGroAc2 species in M. tuberculosis. Indeed, knockout studies
of MgtA in M. tuberculosis H37Rv failed to abolish production of
any cell wall mannosylated glycans, although the rate of bacterial-
induced cell death was increased.19
Methods for the synthesis of glucuronosyl diacylglycerides
have not yet been reported. In this work, we have developed a
synthetic approach to the putative M. smegmatis glycolipid
structures 1a and 1b containing (R)-tuberculostearic acid at the
sn-1 or sn-2 position. These authentic reference materials are
used to establish rules governing the fragmentation of
glucuronosyl diacylglycerides using negative-mode collision-
induced fragmentation electrospray ionization mass spectrom-
etry, allowing the unambiguous assignment of the natural
material as possessing structure 1a. We extend our analysis to the
related oleic acid containing glycolipids from M. smegmatis and C.
glutamicum, demonstrating that both possess structure 2a. Next,
we use these reference materials to guide a search for
corresponding structures in M. smegmatis mc2155 and M.
tuberculosis H37Rv. Finally, we develop simplified structural
analogues that can act as substrates for C. glutamicum MgtA,
enabling its biochemical characterization.
Figure 1. (a) Schematic of major structural elements of the
mycobacterial and corynebacterial cell wall. (b) Outline of involvement
of GlcAGroAc2 in the biosynthesis of C. glutamicum Gl-LM and
comparison with PI-LM/LAM biosynthesis in Corynebacterinae.
Figure 2. (a) Possible structures of glucuronosyl diacylglycerides isolated from C. glutamicum and M. smegmatis. (b) Structures of fatty acids. (c)
Retrosynthesis for GlcAGroAc2 (1a).
2176
dx.doi.org/10.1021/jo302508e | J. Org. Chem. 2013, 78, 2175−2190

The Journal of Organic Chemistry
Featured Article
a
Scheme 1. (a) Synthesis of (R)-Tuberculostearic Acid and (b) Synthesis of Homochiral Glycerol Fragment 11
a
Reagents and conditions for part a: (i) NaHDMS, THF, MeI, 86%; (ii) NaBH4, THF/H2O, 93%; (iii) refs 22 and 23. Reagents and conditions for
part b: (iv) aq HCl, hexane/MeOH, 39%; (v) TPSCl, imidazole, DMF (94%); (vi) (1) NaIO4, THF/H2O, (2) NaBH4, EtOH/H2O, 91%, two steps.
a
Scheme 2. Synthesis of GlcAGroAc2 Isomers
a
Reagents/conditions (i) 11, Bu4NI, TTBP, CH2Cl2, 6 d, 63%; (ii) NaOMe, MeOH, CH2Cl2, 75%; (iii) cat. TEMPO, PhI(OAc)2, CH2Cl2/H2O,
87%; (iv) BnOH, HBTU, DIPEA, cat. DMAP, CH2Cl2, 84%; (v) HF·pyr, THF, 94%; (vi) stearic acid, COMU, DIPEA, cat. DMAP, DMF, 50° C, 24
h, 72%; (vii) (R)-tuberculostearic acid, COMU, DIPEA, cat. DMAP, DMF, 50 °C, 24 h, 63%; (viii) two portions of palmitic acid, COMU, DIPEA,
cat. DMAP, DMF, 50 °C, 24 h, 87%; (ix) CAN, MeCN/H2O; (x) palmitic acid, COMU, DIPEA, DMAP, DMF, rt, 30 h, 60% over two steps; (xi)
palmitic acid, COMU, DIPEA, DMAP, DMF, rt, 2 d, 67% over 2 steps; (xii) two portions of (R)-tuberculostearic acid, COMU, DIPEA, DMAP,
DMF, rt, 2 d, 75% over two steps; (xiii) H2, Pd(OH)2, MeOH/THF/AcOH; 60% for 21, 85% for 1a, 90% for 1b.
difficult than using glucosyl donors owing to the strongly
electron-withdrawing nature of the carboxyl of C6. For this
reason, we elected to use a glucosyl donor that could be oxidized
at the 6-position of the sugar after forming the challenging 1,2-cis
α-glycosidic linkage. Several syntheses of enantiopure (R)-
RESULTS AND DISCUSSION
■
Total Synthesis of GlcAGroAc2 (C19:0/C16:0) and GlcA-
GroAc2 (C16:0/C19:0).20 Our strategy toward the synthesis of
compound 1a and isomer 1b is summarized in Figure 2c.
Glycosylations using glucuronosyl donors are known to be more
2177
dx.doi.org/10.1021/jo302508e | J. Org. Chem. 2013, 78, 2175−2190

The Journal of Organic Chemistry
Featured Article
tuberculostearic acid (4) have been reported that utilize
resolution approaches,21 chiral pool starting materials,22−25 and
catalytic enantioselective reactions.26 The approach used here
employs an Evans’ chiral auxiliary to set the 10-(R)-methyl
stereocenter27 and then merges with the approaches of
Seeberger22 and Painter23 to conclude the synthesis (Scheme
1a). Diastereoselective methylation of N-decanoyloxazolidinone
(5)28 via the chelated Z-enolate was achieved by deprotonation
with NaHDMS followed by addition of iodomethane and yielded
6 as a single stereoisomer as determined by 1H NMR
spectroscopy, in 86% yield.28 Reductive cleavage of the chiral
auxiliary of 6 proceeded smoothly using NaBH4 in THF
affording the alcohol 7 in 93% yield with recovery of 76% of
the chiral auxiliary. Also obtained was 3% of the N-
acylphenylalaninol resulting from hydride attack at the
endocyclic carbonyl, which was easily removed by chromatog-
raphy. The optical rotation of 7 ([α]D = +10.4) was in excellent
agreement with that reported previously ([α]D = +11.0).22 The
synthesis of (R)-tuberculostearic acid 4 was concluded following
the route outlined by Seeberger22 involving tosylation of 7,
Cu(I)-catalyzed sp3−sp3 cross-coupling with a THP-protected
C8 fragment, and deprotection and finally oxidation according to
the method of Painter.23
Given the need to install different acyl groups at the sn-1 and
sn-2 positions of the glyceride following glycosylation, we elected
to prepare an orthogonally protected glycerol fragment 11
bearing a p-methoxybenzyl group at sn-2 and a tert-
butyldiphenylsilyl group at the sn-1 position. The dibenzylidene
diether 8 was prepared in two steps from D-mannitol29,30 and was
debenzylidenated using dilute HCl in a mixture of hexane and
MeOH providing the tetraol 9 in a moderate 39% yield (Scheme
1b). Regioselective silylation of the primary alcohols using
TPSCl (TPS: tert-butyldiphenylsilyl) and imidazole in DMF
afforded disilyl ether 10 in 94% yield. The vicinal diol was
oxidatively cleaved by exposure to NaIO4 in THF/H2O, and the
crude glyceraldehyde reduced with NaBH4 in EtOH/H2O to
afford the selectively protected sn-glycerol 11 in 91% yield over
two steps.
Unlike 1,2-trans glycosidic linkages, which can be readily
constructed by virtue of neighboring group participation,
stereoselective formation of 1,2-cis-glycosidic linkages can be
problematic. We chose to explore the glycosyl iodide method,
owing to the outstanding α-stereoselectivities observed in
glycosylations of a range of primary alcohols.31−33 Attempted
glycosylation of 11 with freshly prepared 1234 under promotion
by Ph3PO in CH2Cl2, according to Kobashi’s protocol,35 resulted
in the formation of significant amounts of a 1,3-disilylated
glycerol, arising from intermolecular silyl group transfer between
molecules of the acceptor. Speculating that silyl transfer was
catalyzed by the release of HI in the glycosylation reaction, the
hindered base tris-tert-butylpyrimidine (TTBP)36 was added, but
resulted in a poorer yield. Changing from Ph3PO to a more
powerful promoter, Bu4NI, in the presence of TTBP, allowed
isolation of the α-glucoside 13 in 63% yield with no evidence of
the formation of the β-anomer (Scheme 2).
With the glucoside in hand, our attention turned to oxidation
of the sugar to the glucuronoside. Deprotection of glycoside 13
with NaOMe in MeOH/CH2Cl2 afforded the primary alcohol 14
in 91% yield (Scheme 2). Oxidation of 14 using TEMPO
(2,2,6,6-tetramethylpiperidin-1-oxyl) and BAIB (bis(acetoxy)-
iodobenzene)37 in a biphasic solvent system consisting of
CH2Cl2/H2O gave the D-glucuronic acid 15 in 87% yield.
Treatment of 15 with benzyl alcohol and HBTU in the presence
of N-methylmorpholine (NMM) and DMAP in CH2Cl2 afforded
the D-glucuronate 16 in 84% yield.
The closing stages of the synthesis required the selective
installation of the acyl chains. We chose to first install the primary
acyl group as 1,2-acyl migration favors the primary ester, and as
the conditions for subsequent PMB removal were perceived to
be milder than that required for TPS deprotection. Cleavage of
the silyl ether of 16 using HF.pyridine in THF gave the alcohol
17 in 94% yield (Scheme 2). Our initial exploratory efforts
targeted the C18:0/C16:0 isomer 21 as a model. However, initial
attempts to esterify the primary alcohol of 17 with stearic acid
and DIC or HBTU resulted in sluggish reactions with variable
yields. Therefore we investigated the third generation uronium-
type reagent COMU, based on Oxyma (ethyl 2-cyano-2-
(hydroxyimino)acetate).38−40 Superior reactivity has been
ascribed to COMU over classical peptide coupling reagents
owing to the exclusive formation of O-acyl Oxyma intermediates
compared to the less reactive predominantly N-acyl species
present using HOBt-based reagents such as HBTU.38 Alcohol 17
was treated with stearic acid, COMU, DIPEA, and catalytic
DMAP in DMF at 50 °C for 24 h. After purification with flash
chromatography the monoacylated species 18 was isolated in
72% yield. The PMB group of 18 was cleaved under oxidative
conditions using CAN in MeCN/H2O. Attempts to purify the
secondary alcohol 19 by silica gel chromatography led to acyl
group migration; in practice, best results were obtained when the
crude secondary alcohol was directly acylated with palmitic acid
using COMU, DIPEA, and DMAP, affording the diacylglyceride
20 in a modest 60% yield over two steps. The benzyl ethers and
benzyl ester of 20 were cleaved simultaneously using H2/
Pd(OH)2, delivering 21 in 60% yield.
The synthesis of the two targets 1a and 1b was achieved from
17 using a similar approach (Scheme 2). Thus, esterification of
the primary hydroxyl with (R)-tuberculostearic acid 4 (17 →
22), oxidative removal of the PMB group and esterification of the
secondary hydroxyl with palmitic acid (22 → 23 → 24) and
finally deprotection afforded GlcAGroAc2 (C19:0/C16:0) 1a. In
the case of 1b, some minor procedural improvements were
implemented. Addition of COMU in two portions, omission of
DIPEA, and use of excess DMAP gave improved yields. Thus, the
alcohol 17 was treated with palmitic acid, COMU and DMAP in
DMF for 24 h before addition of a second portion of palmitic
acid, COMU, and DMAP. The reaction was stirred for a further
24 h and the resultant monoglyceride 25 was isolated in 87%
yield. Removal of the PMB group followed by esterification of the
liberated alcohol 26 with 4 under the modified COMU/DMAP
reaction conditions gave the diglyceride 27 in 75% yield over two
steps. Finally, hydrogenolysis of 27 (H2/Pd(OH)2) afforded
GlcAGroAc2 (C16:0/C19:0) 1b in 90% yield.
Structural Elucidation of Natural GlcAGroAc2 Com-
pounds from C. glutamicum and M. smegmatis. With
authentic synthetic targets 1a and 1b in hand, the collision-
induced dissociation (CID) mass spectra of the [M − H]− of
both compounds were compared with that of the natural
product. ESI CID-MS/MS of the deprotonated isomers 1a and
1b (m/z 785) revealed the formation of fragment ions with
identical m/z values but with clear differences in relative intensity
(Figure 3a,b). For example, fragmentation of the [M − H]− of
both isomers via loss of two different ketenes derived from the
palmitic acyl and tuberculostearic acyl chains gives fragment ions
at m/z 547 and 505, respectively. For 1a, the ion at m/z 547 is
formed in greater abundance than the ion at m/z 505 (ratio of
3:1), indicating that loss of the ketene from the palmitate at the
2178
dx.doi.org/10.1021/jo302508e | J. Org. Chem. 2013, 78, 2175−2190

The Journal of Organic Chemistry
Featured Article
The structure of 1 isolated from M. smegmatis MNC strain 13
was originally assigned as 1a (C19:0/C16:0). However, no
definitive evidence was provided to verify this structure and
this assignment appears to have been arbitrary.12 In the original
report of the FAB-MS/MS spectrum of deprotonated native 1,
distinct fragment ions at m/z 547 and 505 are observed,
corresponding to loss of ketenes via cleavage of the palmitic acyl
and tuberculostearic acyl groups respectively.12 The former ion
(m/z 547) is more abundant than the latter (m/z 505) (approx
3:1), identical to the fragment ratios observed with synthetic 1a
and clearly different from the ratios observed with 1b. In fact, the
MS2 spectrum of 1a (Figure 3a) appears identical to the native
glycolipid from M. smegmatis in all respects including the relative
intensity of the peaks at m/z 255 and 297 and m/z 487 and 539.12
This therefore provides strong evidence that the natural
glycolipid from M. smegmatis MNC strain 13 is 1a, confirming
the original assignment of Brennan and co-workers.12 As well,
structure 1a is consistent with related C19:0/C16:0 phosphogly-
ceride-containing mycobacterial natural products, suggesting a
common biogenesis of the diacylglyceride fragment.23,26,41−43
In the case of the deprotonated glucuronosyl diacylglycerides,
the characteristic peaks observed at m/z 547 and 505 correspond
to the secondary and primary alcohols 28 and 29, respectively,
and are generated through loss of ketenes derived from the acyl
residues (Figure 4a). Similarly, cleavage of palmityl and
tuberculostearyl groups can also occur through neutral loss as
the carboxylic acids by elimination, to give alkene fragments 30
and 31 observed at m/z 529 and 487, respectively (Figure 4a).
These ions are formed in a 1:1 ratio and unlike the phospholipids
these peaks are not useful for assignment of fatty acid
distribution. The carboxylate anions of the fatty acids at m/z
297 and 255 are also less suitable for assigning the fatty acid
positions as they exhibit only minor intensity differences. Thus,
the fragments ions generated from loss of the ketenes from the
Figure 3. Multistage mass spectrometry (MSn) experiments involving
collision-induced dissociation of negative ions formed via ESI. (a) MS2
spectrum of deprotonated 1a; (b) MS2 spectrum of deprotonated 1b;
(c) MS2 spectrum for the ion at m/z = 769.8 from C. glutamicum,
assigned as 2a; (d) MS3 spectrum of collision-induced dissociation of
m/z 751.4, derived from the ion m/z = 931.6, assigned as 3a. All
experiments were performed on a Finnigan hybrid LTQ-FT-MS.
sn-2 position is the preferred fragmentation pathway. In contrast,
the CID MS/MS spectrum of 1b has a reciprocal intensity ratio
with the peaks at m/z 547 and 505 in a 1:3 ratio, consistent with
preferential loss of the ketene derived from the sn-2 tuber-
culostearate. This data confirms that cleavage of the fatty acyl
residue at the sn-2 position yields a more intense product ion
than for cleavage at the sn-1 position.
Figure 4. Proposed fragment ions formed in the CID spectra of deprotonated GlcAGroAc2 and ManGlcAGroAc2 shown in Figure 5. Fragment ions of
[M − H]− from: (a) 1a, (b) 2a, (c) 3a, (d) 34.
2179
dx.doi.org/10.1021/jo302508e | J. Org. Chem. 2013, 78, 2175−2190

The Journal of Organic Chemistry
Featured Article
acyl residues are the most diagnostic for assignment of the
acylation pattern of glucuronosyl diacylglycerides.
simple solvent partitioning of products from radiolabeled sugar
donors in enzymic assays. As well, the detergent-like behavior of
octyl glycosides ensures excellent solubility and distribution
within aqueous and micellar phases within the cell free systems to
be used for biochemical assays.
With the establishment of “rules” for the assignment of the sn-1
and sn-2 acyl chains based on the CID spectra of deprotonated 1a
and 1b, we next applied these findings to the structures of other
GlcAGroAc2 compounds. Brennan and co-workers also reported
FAB-MS/MS spectra for the fragmentation of [M − H]− of
natural 2 isolated from M. smegmatis MNC strain 13, which
revealed ion fragments at m/z 531 (assigned as 32) and m/z 505
(assigned as 33), corresponding to cleavage of palmitic and oleic
acids, respectively (Figure 3b).12 The former ion (m/z 531) is
approximately 3-fold more abundant than the latter (m/z 505),
which supports the oleyl group being attached at the sn-1
position and the palmityl group at sn-2, thus confirming the
structure 2a as the structure of the M. smegmatis natural product.
Besra and co-workers did not report fragmentation spectra for 2
isolated from C. glutamicum.17 We therefore studied a crude lipid
extract from C. glutamicum by CID ESI-MS/MS containing 2.
Fragmentation of the parent ion (m/z 769) yielded ion
fragments at m/z 531 and 505, corresponding to cleavage of
palmitic and oleic acids, respectively (Figure 3c). The fact that
the former ion (m/z 531) is approximately 3-fold more abundant
than the latter (m/z 505) supports structure 2a, which is the
reverse acylation pattern of that originally assigned by Besra and
co-workers17 but identical to the material from M. smegmatis. We
also studied 3, which is present within the same C. glutamicum
lipid extract. Fragmentation of the parent ion (m/z 931.6)
produces one major ion (m/z 751.4) assigned as 34, presumably
derived from elimination of the mannopyranosyloxy group
(Figure 4c). Isolation and subsequent fragmentation of this ion
resulted in the MS3 spectrum shown in Figure 3d, which displays
the characteristic ion fragment ions at m/z 513 (assigned as 35)
and m/z 487 (assigned as 36), corresponding to loss of palmitic
and oleic acids, respectively (Figure 4d). The 3:1 ratio of 35/36 is
consistent with the presence of oleic acid at the sn-1 position and
structure 3a, which is the same acylation pattern as established
for its biosynthetic precursor 2a, and is the reverse of that
assigned by Besra and co-workers.17
We next screened total lipid extracts of log-phase M. smegmatis
mc2155 and M. tuberculosis H37Rv for the presence of 1 or 2
using Finnigan LTQ and Agilent LCMS triple-quadrupole and
QToF instruments. Using the authentic reference materials 1a
and 1b, a limit of detection was established defining the
sensitivity limit of the analysis. Based on these results, we
conclude that within these strains grown under the specified
conditions, 1 and 2 are not present at levels of more than 100 ng
per mg of dried lipid extract.
Synthesis of Hydrophobic Glycosides for Study of
ManGlcAGroAc2 biosynthesis. While the GDP-mannose-
dependent mannosyltransferase MgtA responsible for the
biosynthesis of ManGlcAGroAc2 from GlcAGroAc2 has been
cloned,17 relatively little is known about the biochemical
properties of this enzyme. Preliminary experiments revealed
that the synthetic glycolipids 1a, 1b, or 21 do not act as substrates
for MgtA from a C. glutamicum cell free extract, possibly because
they do not have the correct acylation pattern for C. glutamicum,
or more probably owing to the poor physicochemical properties
of these glycolipids which results in poor solubility in the cell free
system, even with inclusion of Triton X-100. This disappointing
result provided an incentive to investigate simplified substrate
analogues 37, 38, 39 and 40. We elected to prepare these
compounds as octyl glycosides as such hydrophobic derivatives
are readily purified by reverse-phase chromatography, and allow
A key requirement for our approach was the ability to generate
large amounts of octyl α-D-glucopyranoside 37. Adasch and co-
workers reported that treatment of D-glucose with 10 equiv of
octanol in dioxane containing 7% H2SO4 gave 37 in 26% yield,
with reduced equivalents of octanol leading to oligomerization of
D-glucose.44 However, their procedure involves a complex
product isolation procedure requiring distillation of excess
octanol and chromatography prior to recrystallization. In this
work, we investigated the use of DMF as a cosolvent. After
considerable optimization, we found that use of 12 equiv of
octanol in DMF, AcCl as a source of HCl, and a reaction
temperature of 60 °C for 6 h provided the cleanest conversion to
octyl D-glucopyranoside as a mixture of anomers (Scheme 3a). As
a general rule, the Krafft points of alkyl α-D-glucosides are higher
than the corresponding β-anomers, allowing separation of the
pure α-anomers by crystallization at temperatures between their
Krafft points.44 The mixture of anomers were readily separated
by careful recrystallization above the Krafft point of the β-
anomer, affording 37 in 30% yield. The chemoselective oxidation
of 37 was achieved using TEMPO in aqueous Ca(OCl)2 and
KBr45 affording 38 in 90% yield.
Treatment of 37 with benzaldehyde dimethyl acetal and
catalytic p-toluenesulfonic acid afforded the benzylidene acetal
41 in 83% yield (Scheme 3b). The benzylidene acetal 41 was
acetylated by treatment with acetic anhydride and pyridine to
afford the acetylated benzylidene acetal 42 in 91% yield.
Regioselective reduction of the acetylated benzylidene acetal
42 with trifluoroacetic acid and triethylsilane in dichloro-
methane46 afforded the alcohol 43 in 83% yield. TMSOTf
activation of a mixture of the trichloroacetimidate 4447 and
alcohol 43 with 0.06 equiv of TMSOTf at −60 °C for 2 h and
then at room temperature for another 2 h afforded the
disaccharide 45 directly in 90% yield. Debenzylation of the
disaccharide 45 was achieved by treatment with H2 and catalytic
Pd(OH)2, affording the primary alcohol 46 in 84% yield. This
primary alcohol was treated with NaOMe in MeOH to afford 39
in 72% yield. Alternatively, oxidation of the primary alcohol 46
with TEMPO and BAIB in CH2Cl2/water, afforded the
carboxylic acid 47 in 90% yield. In this case the choice of an
organic soluble co-oxidant was influenced by the lipophilic
nature of the alcohol, which prevents its efficient partitioning into
the aqueous phase, as is required for biphasic TEMPO oxidations
using Ca(OCl)2. Finally, the carboxylic acid 47 was treated with
2180
dx.doi.org/10.1021/jo302508e | J. Org. Chem. 2013, 78, 2175−2190

The Journal of Organic Chemistry
Featured Article
Scheme 3. (a) Synthesis of Octyl Monosaccharides 37 and 38
a
and (b) Synthesis of Octyl Disaccharides 39 and 40
Figure 5. Biochemical characterization of C. glutamicum MgtA. (a) Left:
Orcinol-stained HPTLC of synthetic 37−40. Right: Fluorogram of
HPTLC of octyl α-D-glucoside 37 (1, 10 mM) and octyl α-D-
glucuronoside 38 (1, 10 mM) incubated with GDP-[3H]mannose in
centrifuged lysate of midlog C. glutamicum. (b) Left: C. glutamicum
cytosolic fraction was incubated with amphomycin, prior to addition of
GDP-[3H]mannose. Right: Cytosolic fraction was oxidized using 50
mM CuCl2, prior to addition of 10 mM DTT. (c) Midlog culture of C.
glutamicum was lysed by sonication (total), and unbroken cells were
removed by centrifugation (lysate). The lysate was fractioned to afford
cytosolic and membrane fractions. Fractions were incubated in the
presence of 5 mM octyl α-D-glucuronoside 38 and GDP-[3H]mannose
for 2 h, and the lipid was extracted and separated on HPTLC. (d) Wild-
type and ΔPimB′ C. glutamicum-derived cytosolic fractions were
incubated with 38 (5 mM).
a
Reagents and conditions for part a: (i) AcCl, octanol, DMF, 60 °C,
30%; (ii) TEMPO, KBr, Ca(OCl)2, CH2Cl2, H2O, 0 °C, 90%.
Reagents and conditions for part b: (iii) PhCH(OMe)2, p-
toluenesulfonic acid, CHCl3, 83%; (iv) Ac2O, pyridine, 91%; (v)
Et3SiH, TFA, CH2Cl2, 83%; (vi) TMSOTf, CH2Cl2, 85%; (vii) H2,
Pd(OH)2, EtOAc/EtOH, 84%; (viii) NaOMe, MeOH, 72%; (ix)
TEMPO, BAIB, H2O, CH2Cl2, 90%; (x) NaOMe, MeOH, 65%.
NaOMe in MeOH to cleave all the acetate esters, affording 40 in
65% yield.
Evaluation of Hydrophobic Glycosides in a C.
glutamicum Cell-Free System: Characterization of
MgtA. The synthetic analogues 37−40 were examined for
their ability to act as substrates for C. glutamicum mannosyl-
transferases in a cell-free system obtained by nitrogen cavitation
of log phase cells. The in vitro biosynthesis of PIMs and
ManGlcAGroAc2 can be reconstituted in this system following
addition of GDP-[2-3H]mannose as a mannosyl donor and the
lipophilic products isolated by biphasic partitioning of the
suspension into 1-butanol and separation of radiolabeled
products by HPTLC. In the absence of an exogenous acceptor,
the major products labeled are ManGlcAGroAc2 (Gl-A) 3a,
polyprenylmannose phosphate (PPM), and acylphosphatidyl-
myo-inositol dimannoside (AcPIM2),48 which are derived largely
from existing endogenous pools of direct intermediates rather
than de novo synthesis from PI (Figure 5a). Upon addition of
synthetic glucuronoside 38, a new lipid is synthesized. The
formation of this product requires the 6-position to be oxidized
as octyl glucoside 37 did not yield a new product. The new
product formed from 38 coeluted with synthetic 40 and was
sensitive to digestion by jack bean α-mannosidase but was
resistant to snail β-mannosidase demonstrating the presence of
an α-linked mannosyl group (Figure S1, Supporting Informa-
tion). Negative-ion LC−MS analysis of the total lipid extract
from the in vitro assay revealed a new species (m/z = 467.1), with
a fragmentation pattern that matches synthetically prepared 40
(Figure S2, Supporting Information). We conclude that the
product formed in the in vitro assay is 40. The synthesis of 40 was
not inhibited by amphomycin, a selective inhibitor of
polyprenolphosphomannose synthesis, indicating that the
mannosyltransferase involved in the conversion 38 → 40, is
most likely the GDP-mannose dependent mannosyltransferase,
MgtA (Figure 5b).
The synthetic substrate 38 facilitated the further character-
ization of MgtA. Incubation of 38 with C. glutamicum total lysate,
a cleared lysate, and fractionated cytosol and membrane/cell wall
fractions revealed that the majority of MgtA activity was localized
to the cytosol, which is consistent with MgtA being a peripheral
membrane protein that under the lysis conditions can be released
into the cytosol (Figure 5c). Interestingly, in the presence of 10
mM DTT, the enzyme activity was dramatically enhanced
(Figure 5b). This is an unusual observation for a glycosyl-
transferase and suggests that the activity of the enzyme can be
regulated by the redox status of the bacterium. A screen for metal
ion dependence revealed that the activity of MgtA was stimulated
by Mg2+ and Mn2+ in preference to various other metal ions
(Ca2+, Cu2+, Fe3+, and Zn2+) (Figure S1, Supporting
Information).
Incubation of 38 with C. glutamicum ΔPimB′ resulted in
similar levels of mannosylation, providing evidence that the
2181
dx.doi.org/10.1021/jo302508e | J. Org. Chem. 2013, 78, 2175−2190

The Journal of Organic Chemistry
Featured Article
enzymatic activity monitored by the use of 38 arises from MgtA
(Figure 5d). Incubation of 38 with a cell-free extract derived from
M. smegmatis mc2155 provided no evidence for the formation of
40, suggesting that under conditions of exponential growth this
strain does not express MgtA (Figure S1, Supporting
Information).
GlcAGroAc2 to form ManGlcAGroAc2.17 The genes responsible
for the synthesis of PIM2 were subsequently identified as
Rv2188c, MSMEG4253, and NCgl2106 in M. tuberculosis, M.
smegmatis, and C. glutamicum, respectively, and annotated
PimB′.13,56 Complementation studies with Rv2188c and
Rv0557 in a ΔMgtA/ΔPimB′ C. glutamicum background have
confirmed these roles for the M. tuberculosis orthologs.18
However, in vitro studies using C. glutamicum ΔMgtA and
ΔPimB′ mutants showed that cell-free extracts could each
catalyze the synthesis of both AcPIM2 and ManGlcAGroAc2,
revealing a relaxed specificity for the C. glutamicum PimB′ and
MgtA transferases within a single mutant background.18 In
addition, in vitro studies of M. tuberculosis MgtA expressed within
a C. glutamicum ΔMgtA/ΔPimB′ double knockout revealed that
this enzyme was equally able to mannosylate AcPIM1 or
GlcAGroAc2, whereas PimB′ expressed within the same double
mutant exhibited a preference for mannosylation of AcPIM1.18
In order to study the GlcAGroAc2 mannosylating mannosyl-
transferase MgtA, we synthesized the artificial substrate octyl α-
D-glucuronoside 38. Our data, including studies with ΔPimB′ C.
glutamicum, demonstrate that this compound is exclusively a
substrate for MgtA. This substrate was used to undertake the
initial biochemical characterization of MgtA and confirms that
this enzyme is a GDP-mannose-dependent mannosyltransferase.
Further, using 38 we show that MgtA is a metalloenzyme with a
preference for Mg2+/Mn2+, and also demonstrate that MgtA
undergoes a dramatic enhancement of activity under reducing
conditions, an atypical feature for a glycosyltransferase. Finally,
using the artificial substrate 38, we demonstrate the absence of
MgtA activity within M. smegmatis mc2155.
CONCLUSIONS
■
Glucuronosyl diacylglycerols of varying lipid composition have
been isolated from C. glutamicum and M. smegmatis. However,
the precise arrangement of the acyl chain on the glycerol
backbone has not been defined. To assist in resolving their
structure, we chemically synthesized two candidate structures 1a
and 1b to use as reference materials. Using collision-induced
dissociation and multistage mass spectrometry, we were able to
establish that these synthetic materials yielded characteristic
fragmentation spectra. The fragmentation profiles established for
glucuronosyl diacylglycerides is consistent with related observa-
tions using electrospray ionization multistage mass spectrometry
for glycosyldiglycerides,49 glycosylphosphatidic acids,50 phos-
phatidylinositol phosphates,51 phosphatidylethanol-
amines,26,52,53 and phosphatidylinositol mannosides.23,41,42
That the fragmentation patterns of the glucuronosyl diacylgly-
cerides should match those for these other classes of molecules is
not immediately obvious. Previous mechanistic studies of
phospholipids has shown that charge-driven fragmentation
determines the production of diagnostic fragment ions.52 In
the case of neutral glycosylglycerides, analysis is typically
performed on positive ions, which fragment through charge-
remote mechanisms.49 In this case, fragmentation of the negative
ions also occurs through charge-remote mechanisms, yielding
larger peaks derived from loss of the acyl group on the secondary
(sn-2) position relative to the primary (sn-1) position. This work
has allowed (re)assignment of structures 1a, 2a, and 3a for the
glucuronosyl diacylglycerides from M. smegmatis and C.
glutamicum and notably demonstrates the identity of the
GlcAGroAc2 (C18:0/C16:0) structure 2a from these two
organisms.
This work describes the first comprehensive study of the
chemical synthesis and structural analysis of glucuronosyl
diacylglycerides, as well as the processing mannosyltransferase
MgtA. Synthetic approaches to these glycolipids are of
importance as isolating these glycolipids in sufficient quantities
and purity from their natural sources is impractical. Under-
standing the structure−activity relationships of these glycolipids
and their impact on the immunopathology of mycobacteria
represent important topics for future study.
We next investigated whether compounds 1a, 2a, and 3a could
be detected in the common laboratory strains M. smegmatis
mc2155 and M. tuberculosis H37Rv. Using Q-trap and LCMS-
QToF and LCMS-triple quadrupole mass spectrometers, we
were unable to find any evidence for these compounds within the
limit of detection. Compounds 1a and 1b were originally isolated
from M. smegmatis MNC strain 13, and the absence of this
compound from M. smegmatis mc2155 provides a note of caution
for future studies of the biosynthesis of these compounds within
this laboratory strain. M. smegmatis strain mc2155 ultimately
derives from the heterogeneous American type culture collection
(ATCC) 60754 and is a mutant selected on the basis of an
efficient plasmid transformation phenotype,2 and its relationship
to MNC strain 13 is unclear. The absence of any detectable
GlcAGroAc2 species from M. tuberculosis H37Rv to within the
limit of detection is consistent with the absence of a clear
phenotype observed upon deletion of Rv2188c (PimB′) in this
organism, and there are no literature reports on this compound
in any strains of M. tuberculosis.18
EXPERIMENTAL SECTION
■
General Experimental Methods. Pyridine was distilled over KOH
before use. Dichloromethane and THF were dried over alumina
according to the method of Pangborn et al.57 Reactions were monitored
using thin-layer chromatography (TLC), performed with silica gel 60
F254. Detection was effected by charring in a mixture of 5% sulfuric acid
in methanol, vanillin stain (6% vanillin, 1% H2SO4 in EtOH), 10%
phosphomolybdic acid in EtOH, and/or visualizing with UV light. Flash
chromatography was performed according to the method of Still et al.58
using silica gel 60. Melting points were obtained using a hot-stage
microscope (corrected). [α]D values are given in deg 10−1 cm2 g−1.
(4R)-3-((2′R)-2′-Methyldecanoyl)-4-benzyl-2-oxazolidinone
(6). NaHMDS (60 mL, 60 mmol, 1.0 M in THF) was added dropwise to
a stirred solution of (4R)-3-decanoyl-4-benzyl-2-oxazolidinone (5)28
(7.50 g, 22.6 mmol) in dry THF (80 mL) at −78 °C under N2 and the
resultant mixture stirred for 1 h. MeI (7.0 mL, 112 mmol) was added via
syringe, and the mixture was stirred for 2 h at −78 °C at which time TLC
indicated consumption of starting material and formation of a less polar
product. The reaction was quenched with satd aq NH4Cl (40 mL), and
the volatile components were removed by rotary evaporation. The
residue was extracted with CH2Cl2 (3 × 50 mL), and the combined
organic phase was washed with 1 M sodium sulfite (50 mL), dried
(MgSO4), and concentrated under reduced pressure. The residue was
purified by flash chromatography (10:90 EtOAc/petroleum spirits) to
give 6 (6.73 g, 86%) as a colorless oil: [α]D −64.4 (c 1.0 in CHCl3) (lit.28
MgtA (Rv0557 in M. tuberculosis; MSMEG1113 in M.
smegmatis) was originally annotated as PimB, a GDP-mannose-
dependent α-mannosyltransferase that catalyzes the conversion
of (Ac)PIM1 to (Ac)PIM2 in mycobacteria.55 However, deletion
of the corresponding ortholog (NCgl0452) from C. glutamicum
revealed that MgtA catalyzes the transfer of mannose to
2182
dx.doi.org/10.1021/jo302508e | J. Org. Chem. 2013, 78, 2175−2190

The Journal of Organic Chemistry
Featured Article
[α]D −73.5 (c 1.0, CH2Cl2)); 1H NMR (500 MHz, CDCl3) δ 0.88 (3 H,
t, J = 7.0 Hz, CH2CH3), 1.22 (3 H, d, J = 7.0 Hz, CHCH3), 1.26−1.35
(12 H, m, (CH2)6CH2), 1.40−1.42 (1 H, m, NCOCHCH2), 1.71−1.75
(1 H, m, NCOCHCH2), 2.77 (1 H, m, dd, J = 9.5, 13.5 Hz, CH2Ph), 3.27
(1 H, dd, J = 3.0, 13.5 Hz, CH2Ph), 3.68−3.74 (1 H, m, CHCO), 4.15−
4.22 (2 H, m, CH2O), 4.66−4.70 (1 H, m, CHN), 7.21−7.35 (5 H, m,
Ph); 13C NMR (125 MHz, CDCl3) δ 14.2 (1 C, CH2CH3), 17.5 (1 C,
CHCH3), 22.8, 27.4, 29.4, 29.6, 29.8, 32.0, 33.6, 37.8 (8 C,
CH(CH3)(CH2)7CH3), 38.1, 55.5, 66.1 (3 C, OCH2CH(CH2Ph)NH),
127.4, 129.0, 129.6, 135.5 (6 C, Ph), 153.2, 177.5 (2 C,
OCONHCOCH); HRMS (ESI+) calcd for C21H31NNaO3 [M + Na]+
m/z 368.2196, found 368.2196.
(R)-2-Methyldecan-1-ol (7). A freshly prepared solution of NaBH4
(947 mg, 29.6 mmol) in water (3.5 mL) was added to a stirred mixture of
6 (1.28 g, 3.70 mmol) in THF (11 mL) at 0 °C. The mixture was stirred
overnight at rt and then neutralized with 1 M HCl with cooling (ice/
water). The aqueous phase was extracted with EtOAc (3 × 20 mL), and
the combined organic phase was washed with water (30 mL) and brine
(30 mL), dried (MgSO4), and concentrated under reduced pressure.
The residue was purified by flash chromatography (15:85 EtOAc/
petroleum spirits) to give 7 (600 mg, 94%) as a colorless oil: [α]D +10.4
(c 3.2 in CHCl3) (lit.22 [α]D +11.0 (c 2.3 in CHCl3)); 1H NMR (400
MHz, CDCl3) δ 0.88 (3 H, t, J = 6.8 Hz, CH2CH3), 0.91 (3 H, d, J = 6.7
Hz, CHCH3), 1.07−1.64 (15 H, m, CH(CH2)7CH3), 3.41 (1 H, dd, J =
6.6, 10.5 Hz, HOCH2CH), 3.50 (1 H, dd, J = 5.7, 10.5 Hz, HOCH2CH);
13C NMR (100 MHz, CDCl3) δ 14.2 (1 C, CH2CH3), 16.7 (1 C,
CHCH3), 22.8, 27.1, 29.5, 29.8, 30.1, 32.0, 33.3 (7 C, CH(CH2)7CH3),
35.9 (1 C, CHCH3), 68.6 (HOCH2). Next to elute was (2R)-N-((1R)-2-
hydroxyethyl-1-benzyl)-2-methyldecanamide (36 mg, 3%) as a colorless
solid, 1H NMR (400 MHz, CDCl3) δ 0.87 (3 H, t, J = 6.8 Hz, CH2CH3),
1.03 (3 H, d, J = 6.9 Hz, CHCH3), 1.22−1.59 (14 H, m, (CH2)7CH3),
2.07−2.16 (1 H, m, CH(CH3)CH2), 2.83 (1 H, dd, J = 7.7, 13.8 Hz,
CH2Ph), 2.92 (1 H, dd, J = 7.1, 13.8 Hz, CH2Ph), 3.14 (1 H, br s, OH),
3.60 (1 H, dd, J = 5.1, 11.0 Hz, HOCH2), 3.68 (1 H, dd, J = 3.5, 11.0 Hz,
HOCH2), 4.17 (1 H, m, CONHCH), 5.76 (1 H, d, J = 7.2 Hz, CONH),
7.21−7.31 (5 H, m, Ph); 13C NMR (100 MHz, CDCl3) δ 14.2, 18.0 (2
C, 2 × CH3), 22.8, 27.5, 29.4, 29.6, 29.8, 33.0, 34.4, 37.1 (8 C,
(CH2)7CH3, CH2Ph), 41.8 (1 C, COCH), 52.9 (1 C, CONHCH), 64.7
(1 C, HOCH2), 126.8, 128.8, 129.3, 137.8 (6 C, Ph), 177.6 (1 C,
NHCOCH); HRMS (ESI+) calcd for C20H33NO2H [M + H]+ m/z
320.2584, found 320.2584. Last to elute was (4R)-4-benzyl-2-
oxazolidinone (490 mg, 75% recovered).
2,5-Di-O-(4-methoxybenzyl)-D-mannitol (9). HCl (12 M, 0.5
mL) was added to 829,30 (2.78 g, 4.64 mmol) in a mixture of MeOH (100
mL) and hexane (65 mL) at rt with vigorous stirring. The mixture was
stirred for 5 h, water (10 mL) was added, and the resulting mixture was
stirred for an additional 1 h. The resultant two layers were separated, and
the bottom layer was treated with satd aq Na2CO3 (4 mL). Additional
Na2CO3 powder was added to adjust the pH to between 8 and 9. The
mixture was filtered and the filtrate concentrated to give a residue, which
was taken up in boiling EtOAc (200 mL). The hot mixture was filtered,
the filtrate concentrated, and the residue recrystallized (EtOAc/hexane)
to give 9 (0.74 g, 38%) as colorless needles: mp 99−101 °C (lit.59 mp
98.5−100 °C); [α]D −1.0 (c 1.0 in MeOH) (lit.60 [α]D −9.0 (c 1.9 in
MeOH)); 1H NMR (500 MHz, CDCl3) δ 2.00 (4 H, br s, 4 × OH), 3.58
(2 H, ddd, J = 5.0, 6.4, 9.1 Hz, H2,5), 3.75−3.84 (4 H, m,
H1(a,b),6(a,b)), 3.80 (6 H, 2 × OCH3), 3.93 (2 H, d, J = 6.4 Hz,
H3,4), 4.52, 4.59 (4 H, 2d, J = 11.3 Hz, 2 × CH2Ph), 6.87−7.26 (8 H,
AA′XX′, Ar); 13C NMR (125 MHz, CDCl3) δ 55.4 (2 C, OCH3), 61.4,
70.0, 72.4, 79.6 (8 C, C1,2,3,4,5,6 and 2 × CH2Ph), 114.1, 129.8, 130.0,
159.6 (12 C, Ar).
NMR (500 MHz, CDCl3) δ 1.05 (18 H, s, 2 × tert-butyl), 3.10 (2 H, d, J
= 5.5 Hz, 2 × OH), 3.69 (2 H, ddd, J = 4.5, 5.0, 5.5 Hz, H2,5), 3.78 (6 H,
s, 2 × OCH3), 3.83 (2 H, dd, J = 5.0, 11.0 Hz), 3.75 (2 H, dd, J = 5.0, 11.0
Hz, H1b,6b), 3.90 (2 H, dd, J = 4.5, 11.0 Hz, H1a,6a), 4.00 (2 H, t, J = 5.5
Hz, H3,4), 4.47, 4.61 (4 H, 2 × d, J = 11.0 Hz, 2 × CH2Ar), 6.80−7.69
(28 H, m, Ar,Ph); 13C NMR (125 MHz, CDCl3) δ 19.3, 27.0 (8 C,
C(CH3)3), 55.4 (2 C, 2 × OCH3), 64.3, 70.0, 73.0, 80.5 (8 C,
C1,2,3,4,5,6 and 2 × CH2Ph), 113.9, 127.9, 129.6, 129.9, 130.6, 133.3,
133.4, 135.8, 135.8, 159.3 (36 C, Ar,Ph); HRMS (ESI+) calcd for
C54H66NaO8Si2 [M + Na]+ m/z 921.4188, found 921.4192.
1-O-(tert-Butyldiphenylsilyl)-2-O-(4-methoxybenzyl)-sn-
glycerol (11). NaIO4 (1.96 g, 9.18 mmol) was added to a stirred
solution of 10 (1.50 g, 1.67 mmol) in a mixture of THF (25 mL) and
water (5 mL) at rt. The mixture was stirred for 3 h and then diluted with
EtOAc (100 mL). The organic layer was separated, washed with water
(2 × 40 mL) and brine (2 × 30 mL), dried (MgSO4), and concentrated
under reduced pressure to give the crude aldehyde. The residue was
dissolved in a mixture of ethanol (27 mL) and water (3 mL), cooled to 0
°C, and treated with NaBH4 (758 mg, 20.0 mmol). The mixture was
stirred for 2 h and then quenched with the careful addition of AcOH to
adjust to pH 8. The mixture was extracted with EtOAc (3 × 30 mL), and
the combined organic phases were dried (MgSO4) and concentrated.
The residue was purified by flash chromatography (20:80 EtOAc/
petroleum spirits) to give 11 (1.36 g, 91% over two steps) as a pale
yellow oil: [α]D −27.5 (c 1.0 in CHCl3); 1H NMR (500 MHz, CDCl3) δ
1.06 (9 H, s, tert-butyl), 2.03 (1 H, dd, J = 5.0, 6.0 Hz, OH), 3.58−3.62 (1
H, m, H2), 3.67 (1 H, dd, J = 6.0, 11.5 Hz, H3a), 3.77−3.80 (1 H,
obscured m, H3b), 3.72 (1 H, dd, J = 6.0 Hz, 10.5 Hz, H1a), 3.78 (1 H,
dd, J = 5.0, 10.5 Hz, H1b), 3.80 (3 H, s, OCH3), 4.44, 4.56 (2 H, 2d, J =
11.5 Hz, CH2Ar), 6.84−7.68 (14 H, m, Ar,Ph); 13C NMR (125 MHz,
CDCl3) δ 19.3, 27.0 (4 C, C(CH3)3), 55.4 (1 C, OCH3), 63.0, 63.7, 72.0
(3 C, C1,2,3), 79.4 (1 C, CH2Ar), 114.0, 127.9, 129.5, 129.9, 130.6,
133.3, 133.4, 135.7, 135.8, 159.4 (18 C, Ar,Ph); HRMS (ESI+) calcd for
C27H34O4SiNH4 [M + NH4]+ m/z 468.2565, found 468.2565.
1-O-(tert-Butyldiphenylsilyl)-2-O-(4-methoxybenzyl)-sn-
glyceryl 2,3,4-Tri-O-benzyl-6-O-acetyl-α-D-glucopyranoside
(13). TMSI (1.22 mL, 8.99 mmol) was added to a solution of 1,6-di-
O-acetyl-2,3,4-tri-O-benzyl-D-glucopyranose61 (4.00 g, 7.49 mmol) in
dry CH2Cl2 (10 mL) at 0 °C under N2. The resultant mixture was stirred
for 1 h at 0 °C, at which point TLC indicated consumption of all starting
material. The mixture was azeotroped with dry toluene (3 × 10 mL) and
then placed under high vacuum for 1 h. Crude 12 was dissolved in dry
CH2Cl2 (50 mL) and cannulated into a stirred mixture of 11 (1.34 g,
2.99 mmol), TBAI (3.86 g, 10.5 mmol), 2,4,6-tri-tert-butylpyrimidine
(2.05 g, 8.24 mmol), and freshly activated 4 Å molecular sieves in dry
CH2Cl2 (50 mL). The reaction was stirred for 6 d at rt under N2 at which
point TLC indicated consumption of the glycerol. The mixture was
diluted with EtOAc, quenched with 1 M Na2S2O3, and stirred for 1 h.
The mixture was then filtered through a layer of Celite and the organic
phase washed with water (50 mL), brine (50 mL), dried (MgSO4), and
concentrated under reduced pressure. The residue was diluted with
Et2O and the excess TBAI filtered. The filtrate was concentrated and the
residue purified by flash chromatography (20:80 then 25:75 then 30:70
EtOAc/petroleum spirits) to give 13 (1.73 g, 63%) as a pale yellow oil:
[α]D +29.6 (c 1.24 in CHCl3); 1H NMR (500 MHz, CDCl3) δ 1.10 (9 H,
s, tert-butyl), 2.02 (3 H, s, COCH3), 3.54 (1 H, dd, J3,4 = 9.0, J4,5 = 9.9 Hz,
H4), 3.57−3.60 (1 H, m, H2′), 3.55 (1 H, dd, J1,2 = 3.5, J2,3 = 9.6 Hz,
H2), 3.77−3.81 (3 H, m, H1′(a,b),3′a), 3.79 (3 H, s, OCH3), 3.89 (1 H,
ddd, J4,5 = 10.1, J5,6 = 2.1, J5,6 = 4.1 Hz, H5), 3.93 (1 H, dd, J = 3.5, 10.5
Hz, H3′b), 4.04 (1 H, t, J2,3 = J3,4 = 9.2 Hz, H3), 4.20 (1 H, dd, J5,6 = 2.1,
J6,6 = 12.0 Hz, H6a), 4.30 (1 H, dd, J5,6 = 4.2, J6,6 = 12.0 Hz, H6), 4.60,
4.63 (2 H, 2 × d, J = 11.5 Hz, CH2Ph), 4.60, 4.92 (2 H, 2 × d, J = 11.0 Hz,
CH2Ph), 4.70, 4.74 (2 H, 2 × d, J = 12.0 Hz, CH2Ph), 4.86, 5.05 (2 H, 2
× d, J = 10.8 Hz, CH2Ph), 4.92 (1 H, d, J1,2 = 3.2 Hz, H1), 6.80, 7.26 (4
H, 2 × d, J = 8.7 Hz), 7.29−7.72 (25 H, m, Ar,Ph); 13C NMR (125 MHz,
CDCl3) δ 19.4 (1 C, C(CH3)3), 21.0 (1 C, COCH3), 27.0 (3 C,
C(CH3)3), 55.4 (1 C, OCH3), 63.2, 63.7 (2 C, C6,1′), 68.8, 68.9 (2 C,
C2,5), 72.0, 72.9, 75.2, 75.8 (4 C, 4 × CH2Ph), 77.4, 78.4 (2 C, C2′,3′),
80.2 (1 C, C4), 82.0 (1 C, C3), 97.5 (1 C, C1), 113.8, 127.8−128.6,
129.3, 129.8, 130.9, 133.4, 133.5, 135.7, 135.8, 138.1, 138.4, 138.8, 159.2
1,6-Di-O-(tert-butyldiphenylsilyl)-2,5-di-O-(4-methoxyben-
zyl)-D-mannitol (10). TBDPSCl (200 μL, 0.78 mmol) was added to a
stirred mixture of 9 (150 mg, 0.36 mmol) and imidazole (97.0 mg, 1.42
mmol) in DMF (1 mL) at 0 °C under N2. The mixture was warmed to rt
and stirred for 4 h. The mixture was diluted with EtOAc (20 mL),
washed with water (3 × 10 mL) and brine (10 mL), dried (MgSO4), and
concentrated under reduced pressure. The residue was purified by flash
chromatography (15:85 then 20:80 EtOAc/petroleum spirits) to give
10 (300 mg, 94%) as a pale yellow oil: [α]D −12.5 (c 1.0 in CHCl3); 1H
2183
dx.doi.org/10.1021/jo302508e | J. Org. Chem. 2013, 78, 2175−2190

The Journal of Organic Chemistry
Featured Article
(30 C, Ar,Ph), 170.7 (1 C, CO); HRMS (ESI+) calcd for
and 5 × CH2Ar), 98.2 (1 C, C1), 127.7, 127.75, 127.83, 127.85, 127.88,
127.90, 128.1, 128.4, 128.50, 128.53, 128.57, 128.65, 129.3, 129.8, 130.9,
133.4, 133.5, 135.2, 137.7, 135.8, 138.1, 138.3, 138.8, 159.1 (42 C,
Ar,Ph), 169.9 (1 C, C6); HRMS (ESI+) calcd for C61H66NaO10Si [M +
Na]+ m/z 1009.4318, found 1009.4318.
C56H64SiNaO10 [M + Na]+ m/z 947.4161, found 947.4157.
1-O-(tert-Butyldiphenylsilyl)-2-O-(4-methoxybenzyl)-sn-
glyceryl 2,3,4-Tri-O-benzyl-α-D-glucopyranoside (14). NaOMe in
MeOH (0.05 M, 1 mL) was added to a stirred solution of 13 (174 mg,
0.188 mmol) in CH2Cl2 (3 mL). The mixture was stirred overnight and
then neutralized with Amberlite 120R (H+ form) resin. The mixture was
filtered and the filtrate concentrated to give a residue, which was purified
by flash chromatography (30:70 EtOAc/petroleum spirits) affording 14
Benzyl (2-O-(4-Methoxybenzyl)-sn-glyceryl 2,3,4-Tri-O-ben-
zyl-α-D-glucopyranosid)uronate (17). HF·pyridine (70%, 290 μL)
was added to a mixture of 16 (140 mg, 0.142 mmol) in dry THF (4.5
mL). The mixture was stirred under N2 overnight, diluted with EtOAc
(30 mL), and poured into satd aq NaHCO3. The organic layer was
separated and then washed with satd aq NaHCO3 (5 × 10 mL), dried
(MgSO4), and concentrated under reduced pressure. The residue was
purified by flash chromatography (40:60 to 45:55 EtOAc/petroleum
spirits) to give 17 (100 mg, 94%) as a colorless oil: [α]D +28.9 (c 1.13 in
CHCl3); 1H NMR (500 MHz, CDCl3) δ 2.22 (1 H, br s, OH), 3.58 (1 H,
dd, J = 5.4, 10.5 Hz, H3′a), 3.66 (1 H, dd, J1,2 = 3.5, J2,3 = 9.6 Hz, H2),
3.67 (1 H, dd, J = 5.5, 11.4 Hz, H1′a), 3.72 (1 H, m, H2′), 3.76−3.80 (2
H, m, H4,1′b), 3.80 (3 H, s, OCH3), 3.86 (1 H, dd, J = 4.7, 10.5 Hz,
H3′b), 4.01 (1 H, dd, J2,3 = J3,4 = 9.3 Hz, H3), 4.33 (1 H, d, J4,5 = 10.0 Hz,
H5), 4.49, 4.77 (2 H, 2 × d, J = 10.8 Hz, CH2Ph), 4.59, 4.80 (2 H, 2 × d, J
= 11.4 Hz, CH2Ph), 4.66, 4.68 (2 H, 2 × d, J = 11.0 Hz, CH2Ph), 4.81 (1
H, d, J = 3.2 Hz, H1), 4.83, 4.96 (2 H, 2 × d, J = 10.9 Hz, CH2Ph), 5.17,
1
(151 mg, 91%) as a colorless oil: [α]D +27.3 (c 0.99 in CHCl3); H
NMR (500 MHz, CDCl3) δ 1.13 (9 H, s, tert-butyl), 1.77 (1 H, br s,
OH), 3.59 (1 H, dd, J1,2 = 3.5, J2,3 = 9.7 Hz, H2), 3.61−3.66 (2 H, m,
H4,2′), 3.73−3.85 (6 H, m, H1′(a,b),3′a,5,6(a,b)), 3.81 (3 H, s, OCH3),
3.96 (1 H, dd, J = 3.2, 10.5 Hz, H3′b), 4.09 (1 H, t, J2,3 = J3,4 = 9.2 Hz,
H3), 4.65 (2 H, s, CH2Ph), 4.73, 4.78 (2 H, 2 × d, J = 11.5 Hz, CH2Ph),
4.73, 4.97 (2 H, 2 × d, J = 11.0 Hz, CH2Ph), 4.91, 5.07 (2 H, 2 × d, J =
11.0 Hz, CH2Ph), 4.93 (1 H, d, J1,2 = 3.5 Hz, H1), 6.86, 7.29 (4 H, 2 × d, J
= 8.5 Hz), 7.32−7.75 (25 H, m, Ar,Ph); 13C NMR (125 MHz, CDCl3) δ
19.3, 27.0 (4 C, C(CH3)3), 55.3 (1 C, OCH3), 61.8, 63.6, 68.6, 71.0,
72.0, 72.9, 75.1, 75.7, 77.5, 78.4, 80.3, 81.9 (12 C, C2,3,4,5,6,1′,2′,3′ and
4 × CH2Ph), 97.5 (1 C, C1), 113.8, 127.6, 127.8, 127.8, 127.8, 127.9,
128.0, 128.1, 128.5, 128.5, 129.3, 129.8, 130.9, 133.4, 133.5, 135.7, 135.7,
138.4, 138.4, 138.9, 159.1 (36 C, Ar,Ph); HRMS (ESI+) calcd for
C54H62NaO9Si [M + Na]+ m/z 905.4055, found 905.4064.
1-O-(tert-Butyldiphenylsilyl-2-O-(4-methoxybenzyl)-sn-glyc-
eryl 2,3,4-Tri-O-benzyl-α-D-glucopyranosiduronic Acid (15).
Compound 14 (180 mg, 0.204 mmol), TEMPO (19.0 mg, 0.122
mmol), and BAIB (394 mg, 1.22 mmol) were stirred in a mixture of
CH2Cl2/H2O (2:1, 6 mL) at rt for 3 h and then quenched with 0.25 M
Na2S2O3. The aqueous phase was extracted with EtOAc (3 × 5 mL). The
organic layer was washed with water, dried (MgSO4), and concentrated
under reduced pressure. The residue was purified by flash chromatog-
raphy (30:70 then 40:60 EtOAc/petroleum spirits with 2% AcOH) to
give 15 (160 mg, 87%) as a pale yellow oil: [α]D +15.3 (c 1.09 in
CHCl3); 1H NMR (500 MHz, CDCl3) δ 1.05 (9 H, s, tert-butyl), 3.58 (1
H, dd, J1,2 = 3.5, J2,3 = 9.6 Hz, H2), 3.62 (1 H, dd, J = 6.0, 10.5 Hz, H3′a),
3.75 (3 H, s, OCH3), 3.60−3.77 (4 H, m, H4,1′(a,b),2′), 3.90 (1 H, dd, J
= 2.5, 10.5 Hz, H3′b), 4.02 (1 H, t, J2,3 = J3,4 = 9.3 Hz, H3), 4.30 (1 H, d,
J4,5 = 10.0 Hz, H5), 4.55 (2 H, s, CH2Ph), 4.64, 4.71 (2 H, 2 × d, J = 12.0
Hz, CH2Ph), 4.66, 4.84 (2 H, 2 × d, J = 10.6 Hz, CH2Ph), 4.82, 4.98 (2
H, 2 × d, J = 10.9 Hz, CH2Ph), 4.91 (1 H, d, J = 3.5 Hz, H1), 6.79, 7.21 (4
H, 2 × d, J = 8.5 Hz), 7.23−7.68 (25 H, m, Ar,Ph); 13C NMR (125 MHz,
CDCl3) δ 19.3, 27.0 (4 C, C(CH3)3), 55.3 (1 C, OCH3), 63.4, 69.3, 69.9,
71.9, 73.0, 75.3, 75.9, 78.3, 79.3, 79.5, 81.3 (11 C, C2,3,4,5,1′,2′,3′ and 4
× CH2Ph), 98.0 (1 C, C1), 113.8, 127.8, 127.8, 127.8, 127.9, 128.0,
128.0, 128.0, 128.2, 128.48, 128.50, 128.53, 129.3, 129.8, 130.7, 133.37,
133.43, 135.69, 135.73, 137.7, 138.1, 138.6, 159.1 (36 C, Ar,Ph), 173.7
(1 C, C6); HRMS (ESI+) calcd for C54H60NaO10Si [M + Na]+ m/z
919.3848, found 919.3851.
Benzyl (1-O-tert-Butyldiphenylsilyl-2-O-(4-methoxybenzyl)-
sn-glyceryl 2,3,4-tri-O-benzyl-α-D-glucopyranosid)uronate
(16). Benzyl alcohol (27 μL, 0.264 mmol) was added to a stirred
mixture of 15 (158 mg, 0.176 mmol), HBTU (133 mg, 0.352 mmol),
DIPEA (77 μL, 0.44 mmol), and DMAP (8.6 mg, 0.07 mmol) in CH2Cl2
(4 mL). The mixture was stirred at rt for 2 h, quenched with water, and
diluted with EtOAc and the organic phase separated. The organic layer
was concentrated under reduced pressure to give a residue, which was
purified by flash chromatography (10:90 to 15:85 EtOAc/petroleum
spirits) to give 16 (146 mg, 84%) as a colorless oil: [α]D +9.47 (c 0.97 in
CHCl3); 1H NMR (500 MHz, CDCl3) δ 1.05 (9 H, s, tert-butyl), 3.59 (1
H, m, H3′a), 3.61 (1 H, dd, J1,2 = 3.5, J2,3 = 9.5 Hz, H2), 3.71−3.79 (4 H,
H4,1′(a,b),2′), 3.76 (3 H, s, OCH3), 3.94 (1 H, dd, J = 2.0, 10.8 Hz,
H3′b), 4.01 (1 H, t, J2,3 = J3,4 = 9.3 Hz, H3), 4.33 (1 H, d, J4,5 = 10.0 Hz,
H5), 4.46, 4.76 (2 H, 2 × d, J = 10.8 Hz, CH2Ph), 4.53, 4.57 (2 H, 2 × d, J
= 11.5 Hz, CH2Ph), 4.65, 4.71 (2 H, 2 × d, J = 12.1 Hz, CH2Ph), 4.81,
4.96 (2 H, 2 × d, J = 10.9 Hz, CH2Ph), 4.93 (1 H, d, J = 3.5 Hz, H1), 5.13,
5.16 (2 H, 2 × d, J = 12.2 Hz, CH2Ph), 6.78, 7.20 (4 H, 2 × d, J = 8.5 Hz),
7.12−7.15 (2 H, m), 7.25−7.68 (28 H, Ar,Ph); 13C NMR (125 MHz,
CDCl3) δ 19.3, 27.0 (4 C, C(CH3)3), 55.4 (1 C, OCH3), 63.5, 67.6, 69.3,
70.8, 72.0, 73.0, 75.2, 75.9, 78.3, 79.6, 79.9, 81.3 (12 C, C2,3,4,5,1′,2′,3′
5.20 (2 H, 2 × d, J = 12.3 Hz, CH2Ph), 6.85−7.34 (24 H, m, Ar,Ph); 13
C
NMR (125 MHz, CDCl3) δ 55.4 (1 C, OCH3), 62.7, 67.4, 68.9, 70.8,
71.9, 73.6, 75.2, 75.9, 77.1, 79.6, 79.7, 81.3 (12 C, C2,3,4,5,1′,2′,3′ and 5
× CH2Ar), 98.2 (1 C, C1), 114.0, 127.8, 127.9, 128.06, 128.09, 128.11,
128.4, 128.5, 128.55, 128.57, 128.62, 128.7, 129.6, 130.4, 135.2, 138.0,
138.1, 138.6, 159.4 (30 C, Ar,Ph), 169.6 (1 C, C6); HRMS (ESI+) calcd
for C45H48NaO10 [M + Na]+ m/z 771.3140, found 771.3147.
Benzyl (1-O-Stearyl-2-O-(4-methoxybenzyl)-sn-glyceryl
2,3,4-tri-O-benzyl-α-D-glucopyranosid)uronate (18). DIPEA (34
μL, 0.197 mmol) was added to a mixture of 17 (37 mg, 0.049 mmol),
stearic acid (28 mg, 0.099 mmol), and COMU (42 mg, 0.099 mmol) in
DMF (1.5 mL). The mixture was stirred under N2 for 20 min at which
point the solution turned from pale yellow to dark orange. DMAP (2.4
mg, 0.02 mmol) was added, and the reaction was heated to 50 °C and
stirred overnight. The mixture was diluted with EtOAc, washed with satd
aq NaHCO3, water, and brine, dried (MgSO4), and concentrated. The
residue was purified by flash chromatography (10:60:30 EtOAc/
toluene/petroleum spirits) to give 18 (36 mg, 72%) as a colorless oil:
[α]D +22.2 (c 1.2 in CHCl3); 1H NMR (500 MHz, CDCl3) δ 0.91 (1 H,
t, J = 7.0 Hz, CH2CH3), 1.28−1.62 (30 H, m, OCOCH2(CH2)14-
CH2CH3), 2.30 (2 H, t, J = 7.7 Hz, OCOCH2CH2), 3.54 (1 H, m, H3′a),
3.61 (1 H, dd, J1,2 = 3.5, J2,3 = 9.6 Hz, H2), 3.77 (1 H, dd, J3,4 = 9.1, J4,5
=
9.9 Hz, H4), 3.78 (3 H, s, OCH3), 3.80−3.84 (3 H, m, H2′,3′b), 4.00 (1
H, dd, J2,3 = J3,4 = 9.3 Hz, H3), 4.16 (1 H, dd, J = 5.0, 11.8 Hz, H1′a), 4.31
(1 H, d, J4,5 = 9.9 Hz, H5), 4.32 (1 H, dd, J = 4.1, 11.8 Hz, H1′b), 4.47,
4.76 (2 H, 2 × d, J = 10.8 Hz, CH2Ph), 4.61 (2 H, s, CH2Ph), 4.65, 4.76
(2 H, 2 × d, J = 12.0 Hz, CH2Ph), 4.82, 4.96 (2 H, 2 × d, J = 10.9 Hz,
CH2Ph), 4.86 (1 H, d, J1,2 = 3.5 Hz, H1), 5.15, 5.18 (2 H, 2 × d, J = 12.3
Hz, CH2Ph), 6.82−7.34 (24 H, m, Ar,Ph); 13C NMR (125 MHz,
CDCl3) δ 14.3 (1 C, CH3), 22.8, 25.0, 29.3, 29.46, 29.50, 29.6, 29.79,
29.80, 29.82, 29.84, 32.1, 34.3 (16 C, OCO(CH2)16CH3), 55.4 (1 C,
OCH3), 63.2 (1 C, C1′), 67.4 (1 C, C3′), 70.8 (1 C, C5), 68.4, 71.8,
73.3, 75.0, 75.2, 75.9 (6 C, C2′ and 5 × CH2Ar), 79.6, 79.8 (2 C, C2,4),
81.3 (1 C, C3), 98.1 (1 C, C1), 113.9, 127.76, 127.77, 127.9, 128.0,
128.1, 128.4, 128.5, 128.55, 128.56, 128.58, 128.7, 129.5, 130.3, 135.2,
138.1, 138.2, 138.7, 159.4 (30 C, Ar,Ph), 169.7 (1 C, C6), 173.7 (1 C,
OCOCH2); HRMS (ESI+) calcd for C63H82NaO11 [M + Na]+ m/z
1037.5759, found 1037.5758.
Benzyl (1-O-Stearyl-2-O-palmityl-sn-glyceryl 2,3,4-tri-O-ben-
zyl-α-D-glucopyranosid)uronate (20). Cerium ammonium nitrate
(60.0 mg, 109 μmol) was added to a stirred solution of 18 (35.0 mg, 34.5
μmol) in MeCN/H2O (9:1, 3 mL) at rt. The mixture was stirred at rt for
2 h, diluted with EtOAc (10 mL) and H2O (5 mL), and stirred for 10
min. The organic layer was separated and the aqueous layer extracted
with EtOAc (2 × 5 mL). The combined organic extract was washed with
water (2 × 5 mL) and brine (5 mL), dried (MgSO4), and concentrated
under reduced pressure. The crude alcohol 19 was placed on high
vacuum for 2 h and then used directly in the next step. DIPEA (25 μL,
2184
dx.doi.org/10.1021/jo302508e | J. Org. Chem. 2013, 78, 2175−2190

The Journal of Organic Chemistry
Featured Article
140 μmol) was added to a stirred mixture of crude 19 (36 mg), palmitic
acid (36 mg, 142 μmol), and COMU (61 mg, 142 μmol) in DMF (2
mL). The mixture was stirred for 10 min under N2, and then DMAP (1.7
mg, 14 μmol) was added. The reaction was stirred for 30 h at rt and then
diluted with EtOAc, washed sequentially with 1 M HCl (5 mL), 0.5 M
K2CO3 (3 × 5 mL), water (5 mL), and brine (10 mL), dried (MgSO4),
and concentrated. The residue was purified by flash chromatography
(10:90 then 15:85 EtOAc/petroleum spirits) to give 20 (24 mg, 60%
over two steps) as a colorless oil: [α]D +19.1 (c 0.5 in CHCl3); 1H NMR
(400 MHz, CDCl3) δ 0.91, 0.91 (6 H, 2 × t, J = 7.1 Hz, 2 × CH3), 1.26−
1.61 (56 H, m, stearyl and palmityl CH2), 2.25, 2.31 (4 H, m, 2 ×
OCOCH2CH2), 3.56 (1 H, dd, J = 5.1, 10.7 Hz, H3′a), 3.59 (1 H, dd, J1,2
= 3.5, J2,3 = 9.6 Hz, H2), 3.75 (1 H, dd, J3,4 = 9.0, J4,5 = 9.9 Hz, H4), 3.82
(1 H, dd, J = 5.2, 10.7 Hz, H3′b), 3.97 (1 H, dd, J2,3 = J3,4 = 9.3 Hz, H3),
4.22 (1 H, dd, J = 6.0, 11.9 Hz, H1′a), 4.28 (1 H, d, J4,5 = 9.9 Hz, H5),
4.44 (1 H, dd, J = 3.8, 11.8 Hz, H1′b), 4.45, 4.75 (2 H, 2 × d, J = 10.8 Hz,
3.5 Hz, H1), 5.12, 5.15 (2 H, 2 × d, J = 12.2 Hz, CH2Ph), 6.79−7.30 (24
H, m, Ar,Ph); 13C NMR (125 MHz, CDCl3) δ 14.2 (1 C, CH2CH3),
19.8 (1 C, CHCH3), 22.8, 25.0, 27.2, 29.3, 29.47, 29.49, 29.7, 29.8, 30.1,
30.2, 32.0, 32.9, 34.3, 37.2 (16 C, (CH2)8CH(CH3)(CH2)7CH3), 55.4
(1 C, OCH3), 63.2 (1 C, C1′), 68.3 (1 C, C3′), 70.8 (1 C, C5), 67.4,
71.8, 73.3, 75.0, 75.1, 75.9 (6 C, C2′ and 5 × CH2Ar), 79.6 (1 C, C2),
79.72 (1 C, C4), 81.2 (1 C, C3), 98.1 (1 C, C1), 113.9, 127.8, 127.9,
128.0, 128.1, 128.39, 128.43, 128.5, 128.55, 128.57, 128.7, 129.5, 130.3,
136.2, 138.0, 138.2, 138.6, 159.3 (30 C, Ar,Ph), 169.6 (1 C, C6), 173.7
(1 C, OCOCH2); HRMS (ESI+) calcd for C64H84NaO11 [M + Na]+ m/z
1051.5906, found 1051.5909.
Benzyl (1-O-((R)-10-Tuberculostearyl)-2-O-palmityl-sn-glyc-
eryl 2,3,4-Tri-O-benzyl-α-D-glucopyranosid)uronate (24). Ce-
rium ammonium nitrate (129 mg, 0.236 mmol) was added to a stirred
solution of 22 (81 mg, 0.079 μmol) in MeCN/H2O (11:1, 6 mL) at rt.
The mixture was stirred at rt for 2 h, diluted with EtOAc (10 mL) and
H2O (5 mL), and stirred for 10 min. The organic layer was separated and
the aqueous layer extracted with EtOAc (2 × 5 mL). The combined
organic extract was washed with water (2 × 5 mL) and brine (5 mL),
dried (MgSO4), and concentrated under reduced pressure. The residue
was placed on high vacuum for 2 h and then used directly in the next
step. DIPEA (27 μL, 0.157 μmol) and palmitic acid (40.4 mg, 0.157
μmol) were added to a stirred mixture of the crude alcohol 23 (75 mg)
and COMU (67.4 mg, 0.157 μmol) in DMF (2 mL). The mixture was
stirred for 10 min under N2, and then DMAP (0.2 mg, 16 μmol) was
added. The reaction was stirred overnight, a second portion (as above)
of DIPEA, palmitic acid, COMU, and DMAP was added, and the
mixture was stirred for a further 24 h. The mixture was diluted with
EtOAc (20 mL), washed sequentially with 1 M HCl (10 mL), 0.5 M
K2CO3 (3 × 10 mL), water (10 mL), and brine (10 mL), dried
(MgSO4), and concentrated. The residue was purified by flash
chromatography (5:65:30 then 10:60:30 EtOAc/toluene/petroleum
spirits) to give 24 (60.4 mg, 67% over two steps) as a colorless oil: [α]D
+19.1 (c 1.16 in CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.85 (3 H, d, J
= 6.5 Hz, CHCH3), 0.90, 0.90 (6 H, 2 × t, J = 7.0 Hz, 2 × CH2CH3),
1.08−1.60 (55, m, palmityl and tuberculostearyl CH + CH2), 2.25−2.31
(4 H, m, 2 × OCOCH2), 3.56 (1 H, dd, J = 5.1, 10.7 Hz, H3′a), 3.59 (1
H, dd, J1,2 = 3.5, J2,3 = 9.6 Hz, H2), 3.75 (1 H, dd, J3,4 = 9.1, J4,5 = 9.9 Hz,
H4), 3.81 (1 H, dd, J = 5.2, 10.7 Hz, H3′b), 3.96 (1 H, dd, J2,3 = J3,4 = 9.3
Hz, H3), 4.22 (1 H, dd, J = 6.0, 11.9 Hz, H1′a), 4.27 (1 H, d, J4,5 = 9.9 Hz,
H5), 4.43 (1 H, dd, J = 3.7, 11.9 Hz, H1′b), 4.45, 4.75 (2 H, 2 × d, J =
10.8 Hz, CH2Ph), 4.62, 4.77 (2 H, 2 × d, J = 12.0 Hz, CH2Ph), 4.76 (1 H,
d, J1,2 = 3.5 Hz, H1), 4.80, 4.94 (2 H, 2 × d, J = 10.8 Hz, CH2Ph), 5.16 (2
H, s, CH2Ph), 5.24 (1 H, m, H2′), 7.12−7.14 (2 H, m), 7.25−7.35 (18
H, m, Ph); 13C NMR (125 MHz, CDCl3) δ 14.3 (2 × CH2CH3), 19.8 (1
C, CHCH3), 22.8, 25.0, 27.2, 29.2, 29.3, 29.46, 29.49, 29.52, 29.67,
29.71, 29.8, 29.9, 30.16, 30.20, 32.1, 32.9, 34.2, 34.3, 37.3 (30 C, palmityl
and tuberculostearyl CH + CH2), 62.5 (1 C, C1′), 66.9 (1 C, C3′), 69.8
(1 C, C2′), 70.9 (1 C, C5), 67.4, 73.5, 75.2, 76.0 (4 C, 4 × CH2Ph), 79.7
(2 C, C2,4), 81.2 (1 C, C3), 98.2 (1 C, C1), 127.8, 127.9, 128.0, 128.1,
128.4, 128.5, 128.57, 128.64, 128.7, 135.1, 138.0, 138.2, 138.6 (24 C,
Ph), 169.6 (1 C, C6), 173.1, 173.5 (2 C, 2 × OCOCH2); HRMS (ESI+)
calcd for C72H106NaO11 [M + Na]+ m/z 1169.7627, found 1169.7628.
1-O-((R)-10-Tuberculostearyl)-2-O-palmityl-sn-glyceryl α-D-
glucopyranosiduronic Acid (1a). Pd(OH)2−C (20%, 20 mg) was
added to a degassed solution of 24 (25 mg, 21.8 μmol) in MeOH/THF
(3:2, 2.5 mL) containing AcOH (50 μL). The mixture was stirred under
H2 atmosphere for 2 h. The mixture was filtered through a layer of
Celite, rinsed with MeOH/THF (3:2), and then concentrated under
reduced pressure. The residue was dissolved in minimal AcOH/CHCl3
and purified by silica gel chromatography (99:1 CHCl3/AcOH then
7:2:0.1:0.1 CHCl3/MeOH/H2O/AcOH) to give 1a (14.5 mg, 85%) as a
CH2Ph), 4.63, 4.75 (2 H, 2 × d, J = 11.7 Hz, CH2Ph), 4.76 (1 H, d, J1,2
=
3.5 Hz, H1), 4.80, 4.92 (2 H, 2 × d, J = 10.8 Hz, CH2Ph), 5.17 (2 H, s,
CH2Ph), 5.25 (1 H, m, H2′), 7.13−7.35 (20 H, m, Ph); 13C NMR (125
MHz, CDCl3) δ 14.3 (2 C, 2 × CH3), 22.8, 25.0, 29.25, 29.29, 29.5, 29.7,
29.8, 29.9, 32.1, 34.2, 34.3 (30 C, stearyl and palmityl CH2), 62.5 (1 C,
C1′), 66.9 (1 C, C3′), 69.7 (1 C, C2′), 70.9 (1 C, C5), 67.4, 73.5, 75.2,
76.0 (4 C, 4 × CH2Ph), 79.7 (2 C, C2,4), 81.2 (1 C, C3), 98.2 (1 C, C1),
127.8, 127.9, 128.0, 128.1, 128.4, 128.5, 128.6, 128.66, 128.71, 135.2,
138.0, 138.2, 138.7 (24 C, Ar), 169.6 (1 C, C6), 173.2, 173.5 (2 C, 2 ×
OCOCH2); HRMS (ESI+) calcd for C71H104NaO11 [M + Na]+ m/z
1155.7471, found 1155.7472.
2-O-Palmityl-1-O-stearyl-sn-glyceryl-α-D-glucopyranosidur-
onic Acid (21). Pd(OH)2−C (20%, 37 mg) was added to a degassed
solution of 20 (22 mg, 19.4 μmol) in MeOH/THF (3:2, 2.5 mL)
containing AcOH (50 μL). The mixture was stirred under H2
atmosphere for 2 h. The mixture was filtered through a layer of Celite,
rinsed with MeOH/THF (3:2), and then concentrated under reduced
pressure. Flash chromatography (CHCl3 with 1% AcOH then
7:2:0.1:0.1 CHCl3/MeOH/H2O/AcOH) gave 21 (9 mg, 60%) as a
colorless powder: [α]D +29.3 (c 0.25 in CHCl3); 1H NMR (500 MHz,
DMSO-d6) δ 0.85, 0.85 (6 H, 2 × t, J = 7.2 Hz, 2 × CH3), 1.23−1.50 (56
H, m, stearyl and palmityl CH2), 2.24−2.30 (4 H, m, 2 × OCOCH2),
3.21−3.23 (1 H, m, H2), 3.36−3.40 (2 H, m, H3,4), 3.55 (1 H, dd, J =
5.0, 10.9 Hz, H3′a), 3.69 (1 H, dd, J = 5.6, 10.9 Hz, H3′b), 3.78 (1 H, d,
J4,5 = 9.8 Hz, H5), 4.16 (1 H, dd, J = 6.9, 12.0 Hz, H1′a), 4.32 (1 H, dd, J
= 2.6, 12.0 Hz, H1′b), 4.69 (1 H, d, J1,2 = 3.5 Hz, H1), 4.80, 4.91 (2 H, 2
× br s, 2 × OH), 5.11 (1 H, m, H2′); 13C NMR (100 MHz, DMSO-d6) δ
13.9 (2 × CH3), 22.1, 24.4, 24.4, 28.4, 28.70, 28.74, 28.9, 29.1, 32.3, 33.4,
33.5 (30 C, stearyl and palmityl CH2), 62.2 (1 C, C1′), 65.5 (1 C, C3′),
69.6, 71.4, 71.7, 71.8, 72.6 (5 C, C2,3,4,5,2′), 99.4 (1 C, C1), 171.0 (1 C,
C6), 172.3, 172.5 (2 C, 2 × OCOCH2); HRMS (ESI+) m/z 795.5596
(C43H80NaO11 [M + Na]+ requires 795.5593).
Benzyl (1-O-((R)-10-Tuberculostearyl)-2-O-(4-methoxyben-
zyl)-sn-glyceryl 2,3,4-Tri-O-benzyl-α-D-glucopyranosid)uronate
(22). DIPEA (111 μL, 0.742 mmol) was added to a stirred mixture of 17
(95 mg, 0.127 mmol), (R)-tuberculostearic acid (4) (61 mg, 0.204
mmol), and COMU (174 mg, 0.406 mmol) in DMF (4 mL). The
mixture was stirred under N2 for 20 min, at which point the solution
turned from pale yellow to dark orange. DMAP (3.1 mg, 0.025 mmol)
was added, and the reaction was heated to 50 °C and stirred overnight.
The mixture was diluted with EtOAc, washed with satd aq NaHCO3,
water, and brine, dried (MgSO4), and concentrated. The residue was
purified by flash chromatography (10:60:30 EtOAc/toluene/petroleum
spirits) to give 22 (83 mg, 63%) as a colorless oil: [α]D +20.6 (c 1.3 in
1
CHCl3); H NMR (500 MHz, CDCl3) δ 0.86 (3 H, d, J = 6.6 Hz,
CHCH3), 0.90 (3 H, t, J = 7.1 Hz, CH2CH3), 1.08−1.57 (30 H, m,
tuberculostearyl CH + CH2), 2.26 (3 H, t, J = 7.7 Hz, OCOCH2), 3.50
(1 H, dd, J = 7.5, 12.0 Hz, H3′a), 3.58 (1 H, dd, J1,2 = 3.5, J2,3 = 9.6 Hz,
H2), 3.74 (1 H, dd, J3,4 = 9.1, J4,5 = 10.0 Hz, H4), 3.74 (3 H, s, OCH3),
3.76−3.81 (2 H, m, H2′,3′b), 3.97 (1 H, dd, J2,3 = J3,4 = 9.3 Hz, H3), 4.13
(1 H, dd, J = 5.0, 11.8 Hz, H1′a), 4.27 (1 H, d, J4,5 = 9.8 Hz, H5), 4.29 (1
H, dd, J = 4.1, 11.7 Hz, H1′b), 4.43, 4.73 (2 H, 2 × d, J = 10.8 Hz,
CH2Ph), 4.58 (2 H, s, CH2Ph), 4.62, 4.72 (2 H, 2 × d, J = 12.0 Hz,
1
colorless glass: [α]D +39.2 (c 0.25 in DMSO); H NMR (500 MHz,
DMSO-d6) δ 0.81 (3 H, d, J = 6.5 Hz, CHCH3), 0.85, 0.85 (6 H, 2 × t, J =
7.0 Hz, 2 × CH2CH3), 1.03−1.23 (51 H, m), 1.50 (4 H, m, palmityl and
tuberculostearyl CH + CH2), 2.24−2.31 (4 H, m, 2 × OCOCH2), 3.22
(1 H, dd, J1,2 = 3.7, J2,3 = 9.4 Hz, H2), 3.55 (1 H, dd, J = 5.3, 10.9 Hz,
H3′a), 3.69 (1 H, dd, J = 5.2, 10.7 Hz, H3′b), 3.77 (1 H, d, J4,5 = 9.9 Hz,
H5), 4.16 (1 H, dd, J = 6.8, 12.0 Hz, H1′a), 4.32 (1 H, dd, J = 2.7, 12.0
Hz, H1′b), 4.69 (1 H, d, J1,2 = 3.3 Hz, H1), 5.11 (1 H, m, H2′); 13C
CH2Ph), 4.78, 4.93 (2 H, 2 × d, J = 10.9 Hz, CH2Ph), 4.82 (1 H, d, J1,2
=
2185
dx.doi.org/10.1021/jo302508e | J. Org. Chem. 2013, 78, 2175−2190

The Journal of Organic Chemistry
Featured Article
NMR (125 MHz; DMSO-d6) δ 13.9 (1 C, 2 × CH2CH3), 19.6 (1 C,
CHCH3), 22.1, 24.4, 24.4, 26.40, 26.44, 28.43, 28.45, 28.69, 28.71, 28.8,
29.0, 29.0, 29.1, 29.36, 29.37, 31.3, 32.1, 33.4, 33.5, 36.42, 36.44 (30 C,
palmityl and tuberculostearyl CH + CH2), 62.2 (1 C, C1′), 65.5 (1 C,
C3′), 69.6 (1 C, C2′), 71.4, 71.8, 72.6 (4 C, C2,3,4,5), 99.4 (1 C, C1),
171.1 (1 C, C6), 172.2, 172.5 (2 C, 2 × CO2CH2); HRMS (ESI+) calcd
for C44H82NaO11 [M + Na]+ m/z 809.5749, found 809.5739.
Benzyl (1-O-Palmityl-2-O-(4-methoxybenzyl)-sn-glyceryl
2,3,4-Tri-O-benzyl-α-D-glucopyranosid)uronate (25). Palmitic
acid (45.3 mg, 0.177 mmol), COMU (75.8 mg, 0.177 mmol), and
DMAP (21.6 mg, 0.177 mmol) was added to a stirred solution of 17
(87.6 mg, 0.117 mmol) in DMF (2 mL). The mixture was stirred under
N2 for 24 h, a second portion of palmitic acid, COMU, and DMAP (as
above) was added, and the mixture was stirred for a further 24 h. The
mixture was diluted with EtOAc, washed with satd aq NaHCO3, water,
and brine, dried (MgSO4), and concentrated. The residue was purified
by flash chromatography (10:60:30 EtOAc/toluene/petroleum spirits)
to give 25 (100 mg, 87%) as a colorless oil: [α]D +22.8 (c 1.0 in CHCl3);
1H NMR (500 MHz, CDCl3) δ 0.90 (3 H, t, J = 7.0 Hz, CH3), 1.27−1.33
(24 H, m), 1.57−1.61 (2 H, m, (CH2)13), 2.28 (2 H, t, J = 7.5 Hz,
OCOCH2), 3.53 (1 H, t, J = 7.5, 12.0 Hz, H3′a), 3.61 (1 H, dd, J1,2 = 3.5,
J2,3 = 9.6 Hz, H2), 3.76 (1 H, dd, J3,4 = 9.1, J4,5 = 10.0 Hz, H4), 3.78 (3 H,
s, OCH3), 3.79−3.83 (2 H, m, H2′,3′b), 4.00 (1 H, dd, J2,3 = J3,4 = 9.3 Hz,
H3), 4.16 (1 H, dd, J = 5.0, 11.8 Hz, H1′a), 4.30 (1 H, d, J4,5 = 9.7 Hz,
H5), 4.32 (1 H, dd, J = 4.1, 11.7 Hz, H1′b), 4.46, 4.75 (2 H, 2 × d, J =
10.8 Hz, CH2Ph), 4.60 (2 H, s, CH2Ph), 4.65, 4.75 (2 H, 2 × d, J = 12.0
Hz, CH2Ph), 4.81, 4.96 (2 H, 2 × d, J = 10.9 Hz, CH2Ph), 4.85 (1 H, d,
J1,2 = 3.5 Hz, H1), 5.15, 5.18 (2 H, 2 × d, J = 12.2 Hz, CH2Ph), 6.82−
6.84 (2 H, m), 7.13−7.15 (2 H, m), 7.24−7.33 (20 H, m, Ar,Ph); 13C
NMR (125 MHz, CDCl3) δ 14.2 (1 C, CH3), 22.8, 25.0, 29.3, 29.4, 29.5,
29.6, 29.77, 29.79, 29.80, 29.83, 32.1, 34.3 (14 C, (CH2)14), 55.4 (1 C,
OCH3), 63.2 (1 C, C1′), 68.4 (1 C, C3′), 70.8 (1 C, C5), 67.4, 71.8,
73.3, 75.0, 75.1, 75.9 (6 C, C2′ and 5 × CH2Ar), 79.6 (1 C, C2), 79.7 (1
C, C4), 81.3 (1 C, C3), 98.1 (1 C, C1), 113.9, 127.75, 127.76, 127.9,
128.0, 128.1, 128.4, 128.49, 128.54, 128.55, 128.57, 128.7, 129.5, 130.3,
135.2, 138.1, 138.2, 138.7, 159.4 (30 C, Ar,Ph), 169.6 (1 C, C6), 173.7
(1 C, CO2CH2); HRMS (ESI+) calcd for C61H78O11 [M + Na]+ m/z
1009.5436, found 1009.5441.
Benzyl (1-O-Palmityl-2-O-(R)-tuberculostearyl-sn-glyceryl
2,3,4-tri-O-benzyl-α-D-glucopyranosid)uronate (27). Cerium am-
monium nitrate (127 mg, 0.231 mmol) was added to a stirred solution of
25 (76 mg, 77 μmol) in MeCN/H2O (11:1, 6 mL) at rt. The mixture
was stirred at rt for 2 h, diluted with EtOAc (20 mL) and H2O (5 mL),
and stirred for 10 min. The organic layer was separated, washed with
water (2 × 5 mL) and brine (5 mL), dried (MgSO4), and concentrated
under reduced pressure. The residue was placed on high vacuum for 2 h
and then used directly in the next step. (R)-Tuberculostearic acid (4)
(23 mg, 77 μmol in 0.5 mL DMF), COMU (36 mg, 85 μmol), and
DMAP (14 mg, 116 μmol) were added to a stirred solution of the crude
alcohol 26 in DMF (1 mL). The mixture was stirred under N2 for 24 h, a
second equal portion of (R)-tuberculostearic acid, COMU, and DMAP
(as above) was added, and the mixture was stirred for a further 24 h. The
mixture was diluted with EtOAc, washed with satd aq NaHCO3, water,
and brine, dried (MgSO4), and concentrated. The residue was purified
by flash chromatography (5:60:35 EtOAc/toluene/petroleum spirits)
to give 27 (67 mg, 76%) as a colorless oil: [α]D +19.5 (c 1.35 in CHCl3);
1H NMR (400 MHz, CDCl3) δ 0.85 (3 H, d, J = 6.5 Hz, CHCH3), 0.90,
tuberculostearyl CH + CH2), 62.5 (1 C, C1′), 66.9 (1 C, C3′), 69.7 (1 C,
C2′), 70.9 (1 C, C5), 67.4, 73.5, 75.2, 76.0 (4 C, 4 × CH2Ph), 79.7 (2 C,
C2,4), 81.2 (1 C, C3), 98.2 (1 C, C1), 127.8, 127.9, 128.0, 128.1, 128.4,
128.5, 128.57, 128.65, 128.7, 135.2, 138.0, 138.2, 138.6 (24 C, Ph), 169.6
(1 C, C6), 173.1, 173.5 (2 C, OCOCH2); HRMS (ESI+) calcd for
C72H106NaO11 [M + Na]+ m/z 1169.7627, found 1169.7628.
1-O-Palmityl-2-O-((R)-10-tuberculostearyl)-sn-glyceryl α-D-
glucopyranosiduronic Acid (1b). Pd(OH)2−C (20%, 30 mg) was
added to a degassed solution of 27 (37 mg, 32 μmol) in MeOH/THF
(3:2, 5 mL) containing AcOH (100 μL). The mixture was stirred under
a H2 atmosphere for 2 h. The mixture was filtered through a layer of
Celite, rinsed with MeOH/THF (3:2), and then concentrated under
reduced pressure. The residue was dissolved in minimal CHCl3/AcOH
and purified by silica gel chromatography (99:1 CHCl3/AcOH then
7:2:0.1:0.1 CHCl3/MeOH/H2O/AcOH) to give 1b (23 mg, 90%) as a
colorless powder: [α]D +33.5 (c 0.48 in DMSO); 1H NMR (500 MHz,
DMSO-d6) δ 0.80 (3 H, d, J = 6.5 Hz, CHCH3), 0.84, 0.84 (6 H, 2 × t, J =
7.0 Hz, 2 × CH2CH3), 1.04−1.49 (55 H, 3 × m, palmityl and
tuberculostearyl CH + CH2), 2.25−2.29 (4 H, m, 2 × OCOCH2), 3.22
(1 H, dd, J1,2 = 3.5, J3,4 = 9.6 Hz, H2), 3.54 (1 H, dd, J = 5.6, 11.0 Hz,
H3′a), 3.69 (1 H, dd, J = 4.9, 11.0 Hz, H3′b), 3.76 (1 H, d, J4,5 = 10.1 Hz,
H5), 4.16 (1 H, dd, J = 6.6, 12.0 Hz, H1′a), 4.32 (1 H, dd, J = 2.1, 12.0
Hz, H1′b), 4.69 (1 H, d, J1,2 = 3.5 Hz, H1), 5.11 (1 H, m, H2′); 13C
NMR (100 MHz, DMSO-d6) δ 13.9 (1 C, 2 × CH2CH3), 19.6 (1 C,
CHCH3), 22.1, 24.42, 24.44, 26.4, 26.5, 28.4, 28.7, 28.76, 28.79, 28.95,
29.02, 29.1, 29.36, 29.39, 31.3, 32.1, 33.4, 33.5, 36.43, 36.46 (30 C,
palmityl and tuberculostearyl CH + CH2), 62.2 (1 C, C1′), 65.5 (1 C,
C3′), 69.6 (1 C, C2′), 71.4, 71.7, 71.8, 72.6 (4 C, C2,3,4,5), 99.4 (1 C,
C1), 171.0 (1 C, C6), 172.3, 172.5 (2 C, CO2CH2); HRMS (ESI+) calcd
for C44H82NaO11 [M + Na]+ m/z 809.5749, found 809.5743.
Octyl α-D-Glucopyranoside (37). Acetyl chloride (6.59 g, 84.0
mmol) was slowly added to a mixture of D-glucose (6.29 g, 34.9 mmol)
and octanol (54.6 g, 419 mmol) in DMF (25 mL) at 60 °C, resulting in
the complete dissolution of D-glucose. The mixture was heated at 60 °C
for 6 h, and then NaHCO3 (7.1 g) was added to quench the reaction.
The mixture was filtered, and the filtrate was purified by flash
chromatography (100% petroleum spirits then 9:1 EtOAc/EtOH).
The mixture was recrystallized from water, twice, to yield 37 as white
needles (3.06 g, 30%): mp 71−72 °C (lit.44 mp 73−74.4 °C); [α]25
D
+141 (c 1.05 in MeOH) (lit.44 [α]25D +120.0); 1H NMR (499.7 MHz,
CD3OD) δ 0.92−0.95 (3 H, m, CH3), 1.34−1.45 (10 H, br m, 5 × CH2),
1.62−1.72 (2 H, m, CH2), 3.30−3.34 (1 H, m, H4), 3.40−3.43 (1 H, dd,
J1,2 3.5, J2,3 9.5 Hz, H2), 3.45−3.49 (1 H, m, OCH2), 3.58−3.62 (1 H, m,
H5), 3.65−3.78 (3 H, m, H3,6,OCH2), 3.81−3.84 (1 H, dd, J5,6′ 2.5, J6,6′
12.5 Hz, H6′), 4.79−4.80 (1 H, d, J1,2 4.0 Hz, H1); 13C NMR (125.6
MHz, CD3OD) δ 14.4, 23.7, 27.3, 30.4, 30.56, 30.61, 33.0 (7 C, octyl),
62.7, 69.1, 71.8, 73.57, 73.59, 75.2 (6 C, C2,3,4,5,6,C1′-octyl), 100.0
(C1).
Octyl α-D-Glucopyranosiduronic Acid (38). A solution of 37
(640 mg, 2.20 mmol), TEMPO (68 mg, 0.4 mmol), and KBr (260 mg,
2.2 mmol) was dissolved in NaHCO3/Na2CO3 solution (10 mL, pH
9.5). The mixture was cooled to 0 °C, and Ca(OCl)2 (629 mg, 4.4
mmol) was added. The mixture was stirred under N2 for 40 min, and
then Na2S2O3 was added to quench the reaction. The pH was adjusted
to pH 2 by dropwise addition of 2 M HCl. The mixture was extracted
with 1-butanol (5 × 10 mL), and the organic layers was combined and
evaporated under reduced pressure. The residue was purified by flash
chromatography (7:2:1 EtOAc/MeOH/H2O + 0.5% AcOH) to give 38
as a colorless paste (607 mg, 90%): mp 71−72 °C (lit.62 mp >250 °C);
0.90 (6 H, 2 × t, J = 7.0 Hz, 2 × CH2CH3), 1.07−1.60 (55 H, 3 × m,
palmityl and tuberculostearyl CH + CH2), 2.25−2.29 (4 H, m, 2 ×
OCOCH2CH2), 3.56 (1 H, dd, J = 5.1, 10.8 Hz, H3′a), 3.59 (1 H, dd, J1,2
= 3.5, J2,3 = 9.6 Hz, H2), 3.75 (1 H, dd, J3,4 = 9.1, J4,5 = 9.8 Hz, H4), 3.81
(1 H, dd, J = 5.2, 10.8 Hz, H3′b), 3.96 (1 H, t, J2,3 = J3,4 = 9.2 Hz, H3),
4.22 (1 H, dd, J = 6.0, 11.9 Hz, H1′a), 4.27 (1 H, d, J4,5 = 10.0 Hz, H5),
4.43 (1 H, dd, J = 3.7, 11.9 Hz, H1′b), 4.45, 4.74 (2 H, 2 × d, J = 10.8 Hz,
25
1
[α]D +120 (c 1.02 in MeOH); H NMR (499.7 MHz, CD3OD) δ
0.92−0.95 (3 H, m, CH3), 1.34−1.45 (10 H, br m, 5 × CH2), 1.62−1.72
(2 H, m, CH2), 3.54−3.81 (5 H, m, H2,3,4,OCH2), 4.06 (1 H, d, J4,5 9.5
Hz, H5), 4.97 (1 H, d, J1,2 3.5 Hz, H1); 13C NMR (125.6 MHz, CD3OD
+ CF3CO2H) δ 14.4, 23.7, 27.3, 30.4, 30.5, 33.0 (7 C, octyl), 69.8, 72.9,
73.1, 73.4, 74.6 (6 C, C2,3,4,5,C1′-octyl), 100.6 (C1), 173.4 (CO).
Octyl 4,6-O-Benzylidene-α-D-glucopyranoside (41). A mixture
of 37 (3.38 g, 12.0 mmol), benzaldehyde dimethyl acetal (4.40 g, 16.0
mmol), and p-toluenesulfonic acid (100 mg) in CHCl3 (50 mL) was
refluxed under Dean−Stark conditions for 20 min. The mixture was
neutralized by addition of K2CO3 (5 g) and was stirred for 1 h. The
CH2Ph), 4.62, 4.77 (2 H, 2 × d, J = 12.0 Hz, CH2Ph), 4.76 (1 H, d, J1,2
=
3.5 Hz, H1), 4.80, 4.94 (2 H, 2 × d, J = 10.8 Hz, CH2Ph), 5.16 (2 H, s,
CH2Ph), 5.24 (1 H, m, H2′), 7.12−7.14 (2 H, m), 7.25−7.35 (18 H, m,
Ph); 13C NMR (100 MHz, CDCl3) δ 14.3 (2 C, CH2CH3), 19.8 (1 C,
CHCH3), 22.8, 25.0, 27.2, 29.26, 29.29, 29.47, 29.49, 29.52, 29.66, 29.72,
29.81, 29.85, 30.17, 30.20, 32.1, 32.9, 34.2, 34.4, 37.3 (30 C, palmityl and
2186
dx.doi.org/10.1021/jo302508e | J. Org. Chem. 2013, 78, 2175−2190

The Journal of Organic Chemistry
Featured Article
mixture was filtered, and the filtrate was concentrated under reduced
pressure to give a yellow oil. The oil was purified by flash
chromatography (1:1 EtOAc/petroleum spirits) to give 41 as a white
solid (3.79 g, 83%): mp 101−102 °C (lit.63 mp 74 °C); [α]D25 +120 (c
1.05 in CHCl3); 1H NMR (499.7 MHz, CDCl3) δ 0.88−0.91 (3 H, m,
CH3), 1.28−1.37 (10 H, br m, 5 × CH2), 1.60−1.66 (2 H, m, CH2), 2.37
(1 H, br s, OH), 3.00 (1 H, br s, OH), 3.43−3.49 (2 H, m, H4,OCH2),
3.59−3.60 (1 H, m, H2), 3.70−3.75 (2 H, m, H6,OCH2), 3.78−3.83 (1
H, m, H5), 3.91 (1 H, t, J 9.5 Hz, H3), 4.28 (1 H, dd, J5,6′ 4.5, J6,6′ 10.0 Hz,
H6′), 4.85−4.86 (1 H, d, J1,2 4.0 Hz, H1), 5.52 (1 H, s, PhCH), 7.35−
7.51 (5 H, m, Ar−H); 13C NMR (125.6 MHz, CDCl3) δ 14.4, 22.8, 26.2,
29.3, 29.5, 29.6, 31.9 (7 C, octyl), 62.7, 69.0, 69.2, 72.1, 73.2, 81.2 (6 C,
C2,3,4,5,6,C1′-octyl), 98.9, 102.1 (C1, PhCH), 126.5, 128.5, 129.4,
137.3 (Ar).
Octyl 2,3-O-Acetyl-4,6-O-benzylidene-α-D-glucopyranoside
(42). A mixture of 41 (3.58 g, 6.78 mmol) and acetic anhydride (18
mL, 190 mmol) in pyridine (35 mL) was stirred for 3 h and then was
diluted with ice−water (200 mL) and the organic phase extracted with
EtOAc (3 × 150 mL). The extract was washed with 2 M HCl (2 × 100
mL), satd NaHCO3 (3 × 150 mL), and water (3 × 100 mL). The extract
was dried (MgSO4) and the solvent evaporated under reduced pressure
to afford 42 as a white solid (2.87 g, 91%): mp 86−87 °C; [α]D25 +104 (c
1.03 in CHCl3); 1H NMR (499.7 MHz, CDCl3) δ 0.87−0.90 (3 H, m,
CH3), 1.28−1.36 (10 H, br m, 5 × CH2), 1.57−1.63 (2 H, m, CH2), 2.06
(1 H, s, Ac), 2.07 (1 H, s, Ac), 3.37−3.42 (1 H, m, OCH2), 3.63 (1 H, t, J
10.0 Hz, H4), 3.59 (1 H, m, H2), 3.67−3.72 (1 H, m, OCH2), 3.75 (1 H,
t, J 10.0 Hz, H6), 3.93−3.98 (1 H, m, H5), 4.29 (1 H, dd, J5,6′ 5, J6,6′ 10.5
Hz, H6′), 4.86 (1 H, d, J1,2 5.0, J2,3 10.0 Hz, H2), 5.05 (1 H, d, J1,2 4.0 Hz,
H1), 5.50 (1 H, s, PhCH), 5.60 (1 H, t, J 9.5 Hz, H3), 7.33−7.46 (5 H,
m, Ar-H); 13C NMR (125.6 MHz, CDCl3) δ 14.0, 20.6, 20.8, 22.6, 26.0,
29.18, 29.24, 29.3, 31.8 (9 C, octyl, CH3CO × 2), 62.3, 68.6, 68.8, 69.0,
71.7, 79.3 (6 C, C2,3,4,5,6,C1′-octyl), 96.5, 101.4 (C1, PhCH), 126.1,
128.1, 130.0, 136.9 (Ar), 169.7, 170.4 (CO × 2). Anal. Calcd for
C25H36O8: C, 64.64; H, 7.81. Found C, 64.69; H, 7.90.
Octyl 2,3-O-Acetyl-6-O-benzyl-α-D-glucopyranoside (43). Tri-
fluoroacetic acid (3.29 g, 28.5 mmol) was added dropwise to a solution
of 42 (2.64 g, 5.68 mmol), triethylsilane (3.31 g, 28.5 mmol), and 4 Å
molecular sieves (5 g) in CH2Cl2 (30 mL) at 0 °C. When the addition
was complete (5 min), the mixture was warmed to room temperature
and stirred overnight. The mixture was filtered through Celite and
poured into ice-cold satd NaHCO3 (100 mL). The organic phase was
extracted with CH2Cl2 (3 × 30 mL), and the combined extract was dried
(MgSO4) to give 43 as a pale yellow oil (2.21 g, 83%): [α]25D +81 (c 0.97
in CHCl3); 1H NMR (499.7 MHz, CDCl3) δ 0.88 (3 H, t, J 6.8, CH3),
1.23−1.33 (10 H, br m, 5 × CH2), 1.54−1.60 (2 H, m, CH2), 2.04 (1 H,
s, Ac), 2.08 (1 H, s, Ac), 2.87 (1 H, br s, OH), 3.36−3.41 (1 H, m,
OCH2), 3.66−3.86 (5 H, m, H4,5,6,6′,OCH2), 4.57 4.62 (2 H, 2 d, J
12.9, PhCH2), 4.82 (1 H, dd, J1,2 3.5, J2,3 10.0 Hz, H2), 5.01 (1 H, d, J1,2
3.5 Hz, H1), 5.32 (1 H, dd, J3,4 9.0, J2,3 10.0, H3), 7.25−7.35 (5 H, m,
Ar−H); 13C NMR (125.6 MHz, CDCl3) δ 14.0, 20.7, 20.9, 26.0, 29.2,
29.2, 31.8 (9 C, octyl, CH3CO × 2), 68.4, 69.5, 70.0, 70.7, 70.9, 73.4,
73.6 (7 C, C2,3,4,5,6,PhCH2,C1′-octyl), 95.6 (C1), 127.6, 127.7, 128.4,
137.8 (4 C, Ar), 170.3, 171.6 (2 C, CO). Anal. Calcd for C25H38O8: C,
64.36; H, 8.21. Found: C, 64.39; H, 8.20.
Octyl 2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyl-(1→4)-2,3-
di-O-acetyl-6-O-benzyl-α-D-glucopyranoside (45). A suspension
of the trichloroacetimidate 4447 (2.03 g, 4.12 mmol), acceptor 43 (1.28
g, 2.74 mmol), and 4 Å molecular sieves (3 g) in dry CH2Cl2 (20 mL)
was stirred at room temperature for 30 min and then cooled to −40 °C.
TMSOTf (98 μL, 0.12 g, 0.55 mmol) was slowly added, and the mixture
was stirred at −40 °C for 2 h. The mixture was warmed to room
temperature, stirred for a further 2 h, neutralized with Et3N, and filtered
through Celite. The filtrate was reduced to dryness and purified by flash
chromatography (3:7 EtOAc/petroleum spirits) to give 45 as a pale
yellow oil (1.97 g, 90%): [α]25D +89 (c 1.07 in CHCl3); 1H NMR (499.7
MHz, CDCl3) δ 0.86−0.89 (3 H, m, CH3), 1.24−1.35 (10 H, br m, 5 ×
CH2), 1.57−1.63 (2 H, m, CH2), 1.97, 2.02, 2.03, 2.04, 2.08, 2.12 (18H,
6 × s, Ac), 3.34−42 (1 H, m, OCH2), 3.65−3.94 (7 H, m,
H4,5,5′,6,6,6′,OCH2), 4.11 (1 H, dd, J5′,6′ 4.5, J6′,6′ 12.0 Hz, H6′),
4.53 (1 H, dd, J 11.2 Hz, PhCH2), 4.59 (1 H, dd, J 11.2 Hz, PhCH2), 4.70
(1 H, dd, J1,2 3.5, J2,3 10.5 Hz, H2), 4.95−4.96 (2 H, m, H1,1′), 5.03−
5.04 (1 H, m, H2′), 5.18−5.23 (2 H, m, H3′,4′), 5.49−5.53 (1 H, m,
H3), 7.25−7.35 (5 H, m, Ar−H); 13C NMR (125.6 MHz, CDCl3) δ
13.9, 20.37, 20.44. 20.5, 20.57, 20.65, 22.4, 25.8, 29.0, 29.1, 31.6 (13 C,
octyl, CH3 × 6), 62.2, 65.8, 68.3, 68.4, 69.3, 69.5, 69.6, 71.37, 71.40, 73.2,
77.1 (12 C, C2,2′,3,3′,4,4′,5,5′,6,6′,PhCH2O,C1″-octyl), 95.43, 99.19
(C1,1′), 127.3, 127.4, 128.2, 137.8 (4 C, Ar), 169.3, 169.4, 169.6, 169.8,
170.1, 170.2 (6 C, CO). Anal. Calcd for C39H56O17: C, 58.78; H, 7.08.
Found: C, 58.72; H, 7.11. Evidence for the exclusive formation of the α-
anomer in this glycosylation was obtained through examination of the
1JC,H coupling constants for the anomeric carbons of the disaccharide.
Each constant was >170 Hz (175 and 171 Hz), thereby showing that all
O-glycosidic linkages formed in this work are α.64
Octyl 2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyl-(1→4)-2,3-
di-O-acetyl-α-D-glucopyranoside (46). A mixture of 45 (2.31 g, 2.90
mmol) and Pd(OH)2 (10%, 200 mg) in EtOAc/EtOH (2:1, 30 mL) was
stirred under H2 overnight. The mixture was filtered through Celite and
the solvent evaporated to give a residue, which was purified by flash
chromatography (1:1 EtOAc/petroleum spirits) to afford 46 as a pale
yellow oil (1.87 g, 91%): [α]25D +85 (c 1.02 in CHCl3); 1H NMR (499.7
MHz, CDCl3) δ 0.86−0.89 (3 H, m, CH3), 1.24−1.34 (10 H, m, 5 ×
CH2), 1.56−1.61 (2 H, m, CH2), 1.98, 2.03, 2.03, 2.06, 2.08, 2.12 (18H,
6 × s, Ac), 3.36−3.40 (1 H, m, OCH2), 3.63−3.67 (1 H, m, OCH2),
3.77−3.86 (3 H, m, H5,6,6), 3.92 (1 H, t, J3,4 9.5, J4,5 9.5 Hz, H4), 4.08 (1
H, ddd, J4,5 9.5, J5,6 2.4, 6.9 Hz, H5), 4.12−4.15 (1 H, dd, J5′,6′ 2.5, J6′,6′
12.0 Hz, H6′), 4.20−4.23 (1 H, dd, J5′,6′ 6.3, J6′,6′ 12.0 Hz, H6′), 4.72 (1
H, dd, J1,2 3.5, J2,3 10.0 Hz, H2), 5.01 (1 H, d, J1,2 3.5 Hz, H1), 5.04−5.06
(2 H, m, H1′,2′), 5.22 (1 H, t, J 10.0 Hz, H4′), 5.29 (1 H, dd, J1′,2′ 2.5,
J
2′,3′ 10.0 Hz, H3′), 5.58 (1 H, dd, J3,4 9.3, J2,3 10.0 Hz, H3); 13C NMR
(125.6 MHz, CDCl3) δ 13.9, 20.4, 20.46, 20.50, 20.51, 20.6, 20.7, 22.5,
25.8, 29.07, 29.11, 29.13, 31.7 (13 C, octyl, CH3CO × 6), 61.0, 62.7,
66.2, 68.2, 68.5, 69.6, 69.8, 70.0, 71.47, 71.54, 76.3 (11 C,
C2,2′,3,3′,4,4′,5,5′,6,6′,C1″-octyl), 95.5, 99.2 (C1,1′), 169.4, 169.5,
169.6, 169.8, 170.3, 170.4 (6 C, CO); HRMS (ESI+) calcd for
C32H50NaO17 [M + Na]+ m/z 729.2940, found 729.2933.
Octyl α-D-Mannopyranosyl-(1→4)-α-D-glucopyranoside (39).
A mixture of 46 (380 mg, 0.54 mmol) and 0.5 M NaOMe in MeOH (15
mL) was stirred for 4 h. The mixture was reduced to dryness and purified
by flash chromatography (17:2:1 EtOAc/MeOH/H2O). Further
purification by reversed-phase chromatography (water/MeOH)
afforded 39 as a white powder (177 mg, 72%): [α]25 +98 (c 0.99 in
D
MeOH); 1H NMR (499.7 MHz, CD3OD) δ 0.92−0.95 (3 H, m, CH3),
1.33−1.41 (10 H, m, 5 × CH2), 1.63−1.68 (2 H, m, CH2), 3.39 (1 H, J1,2
3.4, J2,3 9.3 Hz, H2), 3.42−3.47 (2 H, m, OCH2), 3.51−3.83 (10 H, m,
H2,3,4,5,6,6,3′,4′,5′,6′,OCH2), 3.86 (1 H, dd, J5′,6′ 1.5, J6′,6′ 11.5 Hz,
H6′), 3.95 (1 H, dd, J1′,2′ 1.5, J2′,3′ 3.0 Hz, H6′), 4.77 (1 H, d, J1,2 3.5 Hz,
H1), 5.35 (1 H, d, J1′,2′ 1.5 Hz, H1′); 13C NMR (125.6 MHz, CD3OD) δ
14.4, 23.7, 27.3, 30.4, 30.5, 30.6, 33.0 (7 C, octyl), 62.3, 63.0, 68.6, 69.2,
7 2 . 2 , 7 2 . 3 , 7 2 . 5 , 7 3 . 7 , 7 5 . 5 , 7 5 . 6 , 7 7 . 3 ( 1 1 C ,
C2,2′,3,3′,4,4′,5,5′,6,6′,C1″-octyl), 99.9, 102.8 (C1,1′); HRMS (ESI+)
calcd for C20H38NaO11 [M + Na]+ m/z 477.2306, found 477.2304.
Octyl 2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyl-(1→4)-2,3-
di-O-acetyl-α-D-glucopyranosiduronic Acid (47). A mixture of 46
(466 mg, 0.66 mmol), TEMPO (62 mg, 0.40 mmol), and BAIB
(bisacetoxyiodobenzene) (1.28 g, 3.96 mmol) in CH2Cl2−H2O (2:1, 15
mL) was stirred at room temperature for 3 h. The reaction was
quenched with 10% aq Na2S2O3 (40 mL), and the mixture was extracted
with CH2Cl2 (3 × 20 mL). The organic phase was washed with water
and dried (MgSO4). After evaporation of the solvent, the residue was
purified by flash chromatography (1:1 EtOAc−petroleum spirits then
7:1:0.1 EtOAc−petroleum spirits−AcOH) to give 47 as a colorless paste
(428 mg, 90%): [α]25D +89 (c 1.07 in CHCl3); 1H NMR (499.7 MHz,
CDCl3) δ 0.84−0.87 (3 H, m, CH3), 1.25−1.34 (10 H, m, 5 × CH2),
1.54−1.65 (2 H, m, CH2), 1.97, 2.01, 2.02, 2.07, 2.10, 2.12 (18H, 6 × s,
Ac), 3.38−3.43 (1 H, m, OCH2), 3.69−3.74 (1 H, m, OCH2), 3.99−
4.02 (2 H, m, H4,5′), 4.10−4.13 (2 H, m, H6′,6′), 4.28 (1 H, d, J4,5 10.0
Hz, H5), 4.78 (1 H, dd, J1,2 3.5, J2,3 10.0 Hz, H2), 5.07 (1 H, d, J1,2 3.5 Hz,
H1), 4.99−5.01 (2 H, m, H1,2′), 5.24−5.33 (2 H, m, H3′,4′), 5.51−5.55
(1 H, dd, J3,4 9.5, J2,3 10.0 Hz, H3); 13C NMR (125.6 MHz, CDCl3) δ
14.0, 20.55, 20.58, 20.7, 20.9, 22.6. 25.8, 29.13, 29.14, 29.2, 31.7 (13 C,
2187
dx.doi.org/10.1021/jo302508e | J. Org. Chem. 2013, 78, 2175−2190

The Journal of Organic Chemistry
Featured Article
octyl, CH3 × 6), 62.7, 66.2, 68.3, 68.7, 69.1, 69.8, 69.9, 70.91, 70.94, 78.4
(10 C, C2,2′,3,3′,4,4′,5,5′,6′,C1″-octyl), 96.0, 99.1 (C1,1′), 169.6,
169.7, 169.76, 169.84, 170.0, 170.3, 172.8 (7 C, CO) HRMS (ESI+)
calcd for C32H48NaO18 [M + Na]+ m/z 743.2733, found 743.2733.
Octyl α-D-Mannopyranosyl-(1→4)-α-D-glucopyranosiduronic
Acid (40). A mixture of 47 (273 mg, 0.38 mmol) and NaOMe in MeOH
(0.5 M, 10 mL) was stirred for 4 h. The solution was acidified with
trifluoroacetic acid, and then reduced to dryness, and the residue was
purified by flash chromatography (17:2:1 EtOAc−MeOH−H2O) to
give the product as a colorless paste. Further purification by reversed-
performed. The second supernatant was pooled with the first and the
total extract dried under a stream of N2. The dried material was
partitioned in a biphasic system of 1-butanol and water (2:1, v/v). After
centifugation (16000g for 2 min), the lower aqueous phase was washed
once with water-saturated 1-butanol and the combined butanol phases
back-extracted with I volume of water before being dried in a rotational
vacuum concentrator (RVC). The lipid residue was dissolved in 10 μL of
water-saturated 1-butanol and analyzed by high-performance thin layer
chromatography (HPTLC) using aluminum-backed silica gel 60 sheets.
HPTLC sheets were developed in chloroform/methanol/1 M
phase chromatography (water/MeOH) gave 40 as a colorless paste (115
ammonium acetate/13
M ammonium solution/water
1
mg, 65%): [α]25 +89 (c 1.07 in MeOH); H NMR (499.7 MHz,
(180:140:9:9:23, v/v) and then sprayed with En3Hance and exposed
to high sensitivity X-ray film (BiomaxMR film) at −80 °C to detect 3H-
labeled lipids.
D
CD3OD) δ 0.92−0.95 (3 H, m, CH3), 1.34−1.43 (10 H, m, 5 × CH2),
1.65−1.72 (2 H, m, CH2), 3.47−3.58 (3 H, m, H2,5′,OCH2), 3.68−3.83
(7 H, m, H3,4,3′,4′,6′,6′,OCH2), 3.91 (1 H, dd, J1,2 1.7, J2,3 3.2 Hz, H2′),
4.10−4.12 (1 H, m, H5), 4.83 (1 H, d, J1,2 4.0 Hz, H1), 5.39 (1 H, d, J1′,2′
1.0 Hz, H1′); 13C NMR (125.6 MHz, CD3OD) δ 14.4, 23.7, 27.3, 30.4,
30.5, 30.6, 33.0 (7 C, octyl), 62.4, 68.0, 69.8, 72.3, 72.4, 73.3, 74.6, 75.2,
78.4 (10 C, C2,2′,3,3′,4,4′,5,5′,6′,C1″-octyl), 100.6, 102.2 (C1,1′),
173.1 (CO); HRMS (ESI+) calcd for C20H36NaO12 [M + Na]+ m/z
491.2099, found 491.2091.
For Studying the Effect of Metal Ions. EGTA (to a final
concentration of 1 mM) and one each of CaCl2, CuCl2, FeCl3, MgCl2,
MnCl3, or ZnCl2 (to a final concentration of 15 mM) were added to the
assay mixtures.
For Studying the Effect of Amphomycin. MgCl2 (7.5 mM), 5 μg of
amphomycin, and 1 mM DTT were added to the assay mixture for 10
min prior to the addition of GDP-[3H]mannose.
Mannosidase Treatment of Glycolipids. 3H-mannose-labeled
total lipids were dried and resuspended in 0.1 M sodium acetate buffer
(pH 5.0) and treated with jack bean α-mannosidase (4 units) or 0.1 M
citric acid-phosphate buffer (pH 4.5) and treated with snail β-
mannosidase (0.5 units) in both cases in the presence of 0.1%
taurodeoxycholate. The mixtures were incubated at 37 °C for 48 h. The
reaction was stopped by the addition of 3.75 volumes of
chloroform:methanol (1:2, v/v, and insoluble material removed by
centrifugation (16000g, 5 min). The supernatant was dried under a
stream of nitrogen, and the lipids were recovered by 1-butanol, water
(2:1, v/v) biphasic partitioning as described above. The lipids were
separated by HPTLC and detected on X-ray film using fluorography as
described previously.
Preparation of Crude Cellular Fractions. Coyrnebacterium
glutamicum ATCC 13032 was grown in BHI medium (brain heart
infusion, Oxoid) until mid log phase (OD600 = 3−8) and then harvested.
The pellet was washed in 25 mM Hepes, pH 7.4 and resuspended in ice-
cold sonication buffer (25 mM Hepes, pH7.4, protease inhibitor
cocktail, “Complete, EDTA-free”, 1 mM DTT) to 0.25 g wet weight/mL
prior to sonication at 20 μm 4 × 45 s, on ice with 1 min interval
(Soniprep 150, MSE, UK). The lysate was centrifuged at 4000g for 10
min at 4 °C, and the supernatant was used for assay as “lysate”. The
lysate was further centrifuged at 100000g for 1 h at 4 °C. The resulting
supernatant was used as the “cytosol” fraction. The pellet was
resupended in the sonication buffer to the same volume as cytosol
fraction and used as the “membrane” fraction.
Preparation of Lipid Extracts. Total lipid extract of M. tuberculosis
H37Rv was obtained from the Colarado State University TB Vaccine
Testing and Research Materials Center (funded by NIH, NIAD N01-AI-
40091).
Coyrnebacterium glutamicum ATCC 13032 was grown in 1 liter BHI
(brain heart medium) at 30 °C to midlog phase (OD = 5.9) and
harvested. The bacteria pellet was washed in PBS once and its wet
weight measured (0.45 g).
Multistage Mass Spectrometric Analysis of GlcAGroAc2 (C19:0
/
C16:0) (1a), GlcAGroAc2 (C16:0/C19:0) (1b), and C. glutamicum, M.
smegmatis, and M. tuberculosis Extracts. Negative-ion multistage
tandem mass spectrometry experiments were conducted using a
modified Finnigan LTQ quadrupole ion-trap mass spectrometer
equipped with a Finnigan electrospray ionization source. Compounds
1a and 1b were dissolved in CHCl3 containing 1% AcOH to give 1 mg/
mL stock solutions. Mass spectrometry samples were prepared by
diluting the stock solutions to 0.01 mg/mL in methanol. Samples were
directly injected into the mass spectrometer via the ESI probe at a rate of
5 μL/min. The sheath gas, spray voltage and capillary temperature were
adjusted to approximately 5−20 au, 3−4.5 kV, and 330 °C, respectively.
The tube lens and capillary voltages were adjusted to maximize the yields
of the desired ions. Collision induced dissociation (CID) experiments
were performed by mass selecting the desired precursor ion, with a
selection window of 1.0−8 m/z (depending on the desired isotope
envelope), and then subjecting it to an activation potential of 20−40%
and an activation Q of 0.25−0.4 for a period of 30 ms. Crude glycolipid
extracts were at 0.1 mg/mL in 1:1 CHCl3/MeOH.
M. smegmatis mc2155 was grown in 10 mL of M7H10 medium
supplemented with glycerol (0.2%), DC enrichment (0.2% glucose,
0.085% NaCl, final), at 37 °C for 2 days for a seed culture. The seed
culture was diluted to 1/100 and cultured in the same medium as above
for 15 h to midlog phase at 37 °C and harvested. The bacteria pellet was
washed once using PBS and its wet weight measured (0.25 g).
Both bacterial pellets were treated with 15 mL of chloroform/
methanol (2:1 (v/v)) for 2 h, 6 mL of chloroform/methanol (2:1 (v/v))
for 2 h, and 7.8 mL of chloroform/methanol/water (1:2:0.8 (v/v))
overnight. The first two chloroform/methanol extracts were transferred
to another tube and dried under a stream of N2. The third extract was
transferred into the same tube, and then 6 mL of chloroform and 2.4 mL
of water were added to make a final composition of chloroform/
methanol/water 8:4:3 (v/v). The lower organic phase was transferred to
a new glass tube and washed twice with chloroform/methanol/water
3:48:47(v/v). The organic phase was dried under a stream of N2. This
yielded the following crude lipid extracts: C. glutamicum, 31.5 mg, and
M. smegmatis, 2.2 mg.
Mannosyltransferase Assays. A 90 μL aliquot of each fraction was
mixed with MgCl2 to 7.5 mM, 100 mM substrate (37−40) to 5 mM final
concentration (or as indicated in the figure) or water for a total volume
of 100 μL. After preincubation at 37 °C for 5 min, 2 μL of GDP-
[2-3H]mannose (6.3 Ci/mmol, final concentration, 170 nCi/reaction)
or 100 mM cold GDP-mannose was added to start the reaction. After 2
h, the reaction was stopped by the addition of 375 μL of 1:2 (v/v)
chloroform/methanol and vortexing. After the centrifugation at 4000g
for 10 min, the supernatant was removed, an aliquot of chloroform/
methanol/water (1:2:0.8, v/v) was added, and another extraction was
Mass Spectrometric Analysis of 40. The total lipid obtained after
incubation of octyl α-D-glucopyranosiduronic acid (38) was 10 times
diluted with a solution of 10 mM ammonium formate in methanol. The
solution was flow injected into an Agilent 6520 Accurate-Mass Q-TOF
LC/MS at the rate of 0.2 mL/min in H2O/methanol/THF (14:20:66,
v/v/v). The capillary voltage was set at 4000 V, and the gas temperature
and flow rate were set to 250 °C and 7 L/min, respectively. The
nebulizer was set at 40 psi. For MS2 total ion scanning, the fragmentator
was set to 135 V in negative mode. MS2 experiments were conducted
using an Agilent 6410 triple quadrupole mass spectrometer in negative
mode with a fragmentator voltage of 135 V and collision energy of 20 eV.
ASSOCIATED CONTENT
■
S
* Supporting Information
Figures S1 (additional characterization of MgtA) and S2 (mass
spectrometric characterization of octyl ManGlcA 40). Copies of
2188
dx.doi.org/10.1021/jo302508e | J. Org. Chem. 2013, 78, 2175−2190

The Journal of Organic Chemistry
Featured Article
the 1H and 13C NMR spectra of new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org
(23) Dyer, B. S.; Jones, J. D.; Ainge, G. D.; Denis, M.; Larsen, D. S.;
Painter, G. F. J. Org. Chem. 2007, 72, 3282.
(24) Roberts, I. O.; Baird, M. S. Chem. Phys. Lipids 2006, 142, 111.
(25) Linstead, R. P.; Lunt, J. C.; Weedon, B. C. L. J. Chem. Soc. 1951,
1130.
(26) ter Horst, B.; Seshadri, C.; Sweet, L.; Young, D. C.; Feringa, B. L.;
Moody, D. B.; Minnaard, A. J. J. Lipid Res. 2010, 51, 1017.
(27) Evans, D. A.; Ennis, M. D.; Mathre, D. J. J. Am. Chem. Soc. 1982,
104, 1737.
(28) Darley, D. J.; Butler, D. S.; Prideaux, S. J.; Thornton, T. W.;
Wilson, A. D.; Woodman, T. J.; Threadgill, M. D.; Lloyd, M. D. Org.
Biomol. Chem. 2009, 7, 543.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: sjwill@unimelb.edu.au, malcolmm@unimelb.edu.au.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(29) Kałuza, Z.; Furman, B.; Krajewski, P.; Chmielewski, M.
̇
We thank Prof. Mark Rizzacasa for helpful advice. Financial
support from the Australian Research Council and Australian
National Health and Medical Research Council is gratefully
acknowledged. M.J.M. is an NHMRC Principal Research Fellow.
Tetrahedron 2000, 56, 5553.
(30) Baggett, N.; Stribblehill, P. J. Chem. Soc., Perkin Trans. 1 1977,
1123.
(31) Meloncelli, P. J.; Martin, A. D.; Lowary, T. L. Carbohydr. Res.
2009, 344, 1110.
(32) Lam, S. N.; Gervay-Hague, J. Org. Lett. 2002, 4, 2039.
(33) Hadd, M. J.; Gervay, J. Carbohydr. Res. 1999, 320, 61.
(34) Thiem, J.; Meyer, B. Chem. Ber./Recl. 1980, 113, 3075.
(35) Kobashi, Y.; Mukaiyama, T. Chem. Lett. 2004, 33, 874.
(36) Crich, D.; Smith, M.; Yao, Q. J.; Picione, J. Synthesis 2001, 323.
(37) Epp, J. B.; Widlanski, T. S. J. Org. Chem. 1998, 64, 293.
(38) El-Faham, A.; Albericio, F. J. Peptide Sci. 2010, 16, 6.
(39) El-Faham, A.; Funosas, R. S.; Prohens, R.; Albericio, F. Chem.
Eur. J. 2009, 15, 9404.
REFERENCES
■
(1) Wendisch, V. F.; Bott, M.; Eikmanns, B. J. Curr. Opin. Microbiol.
2006, 9, 268.
(2) Snapper, S. B.; Melton, R. E.; Mustafa, S.; Kieser, T.; Jacobs, W. R.,
Jr. Mol. Microbiol. 1990, 4, 1911.
(3) Katoch, V. M. Indian J. Med. Res. 2004, 120, 290.
(4) Shiloh, M. U.; DiGiuseppe Champion, P. A. Curr. Opin. Microbiol.
2010, 13, 86.
(5) Brennan, P. J.; Nikaido, H. Annu. Rev. Biochem. 1995, 64, 29.
(6) Guerin, M. E.; Kordulakova, J.; Alzari, P. M.; Brennan, P. J.;
Jackson, M. J. Biol. Chem. 2010, 285, 33577.
(40) Twibanire, J. D. K.; Grindley, T. B. Org. Lett. 2011, 13, 2988.
(41) Hsu, F.-F.; Turk, J.; Owens, R. M.; Rhoades, E. R.; Russell, D. G. J.
Am. Soc. Mass Spectrom. 2007, 18, 466.
(7) Mishra, A. K.; Driessen, N. N.; Appelmelk, B. J.; Besra, G. S. FEMS
Microbiol. Rev. 2011, 35, 1126.
(42) Hsu, F.-F.; Turk, J.; Owens, R. M.; Rhoades, E. R.; Russell, D. G. J.
(8) Umesiri, F. E.; Sanki, A. K.; Boucau, J.; Ronning, D. R.; Sucheck, S.
J. Med. Res. Rev. 2010, 30, 290.
(9) Cao, B.; Williams, S. J. Nat. Prod. Rep. 2010, 27, 919.
(10) Hunter, S. W.; McNeil, M. R.; Brennan, P. J. J. Bacteriol. 1986, 168,
917.
(11) Schultz, J. C.; Elbein, A. D. J. Bacteriol. 1974, 117, 107.
(12) Wolucka, B. A.; McNeil, M. R.; Kalbe, L.; Cocito, C.; Brennan, P.
J. Biochim. Biophys. Acta 1993, 1170, 131.
(13) Lea-Smith, D. J.; Martin, K. L.; Pyke, J. S.; Tull, D.; McConville,
M. J.; Coppel, R. L.; Crellin, P. K. J. Biol. Chem. 2008, 283, 6773.
(14) Mishra, A. K.; Klein, C.; Gurcha, S. S.; Alderwick, L. J.; Babu, P.;
Hitchen, P. G.; Morris, H. R.; Dell, A.; Besra, G. S.; Eggeling, L. Antonie
van Leeuwenhoek 2008, 94, 277.
(15) Mishra, A. K.; Krumbach, K.; Rittmann, D.; Appelmelk, B.;
Pathak, V.; Pathak, A. K.; Nigou, J.; Geurtsen, J.; Eggeling, L.; Besra, G.
S. Mol. Microbiol. 2011, 80, 1241.
(16) Uldrich, A. P.; Patel, O.; Cameron, G.; Pellicci, D. G.; Day, E. B.;
Sullivan, L. C.; Kyparissoudis, K.; Kjer-Nielsen, L.; Vivian, J. P.; Cao, B.;
Brooks, A. G.; Williams, S. J.; Illarionov, P.; Besra, G. S.; Turner, S. J.;
Porcelli, S. A.; McCluskey, J.; Smyth, M. J.; Rossjohn, J.; Godfrey, D. I.
Nat. Immunol. 2011, 12, 616.
(17) Tatituri, R. V. V; Illarionov, P. A.; Dover, L. G.; Nigou, J.; Gilleron,
M.; Hitchen, P.; Krumbach, K.; Morris, H. R.; Spencer, N.; Dell, A.;
Eggeling, L.; Besra, G. S. J. Biol. Chem. 2007, 282, 4561.
(18) Mishra, A. K.; Batt, S.; Krumbach, K.; Eggeling, L.; Besra, G. S. J.
Bacteriol. 2009, 191, 4465.
(19) Torrelles, J. B.; DesJardin, L. E.; MacNeil, J.; Kaufman, T. M.;
Kutzbach, B.; Knaup, R.; McCarthy, T. R.; Gurcha, S. S.; Besra, G. S.;
Clegg, S.; Schlesinger, L. S. Glycobiology 2009, 19, 743.
(20) Cn:x/Cm:y refers to the glycerol bearing an acyl group with n
carbons and x degree(s) of unsaturation at the sn-1 position and an acyl
group with m carbons and y degree(s) of unsaturation at the sn-2
position of glycerol, respectively. C16:0, C18:1, and C19:0 represent
palmityl, oleyl, and (R)-tuberculostearyl residues, respectively.
(21) Prout, F. S.; Cason, J.; Ingersoll, A. W. J. Am. Chem. Soc. 1947, 69,
1233.
Am. Soc. Mass Spectrom. 2007, 18, 479.
(43) Rankin, G. M.; Compton, B. J.; Johnston, K. A.; Hayman, C. M.;
Painter, G. F.; Larsen, D. S. J. Org. Chem. 2012, 77, 6743.
(44) Adasch, V.; Hoffmann, B.; Milius, W.; Platz, G.; Voss, G.
Carbohydr. Res. 1998, 314, 177.
(45) Lin, F.; Peng, W.; Xu, W.; Han, X.; Yu, B. Carbohydr. Res. 2004,
339, 1219.
(46) DeNinno, M. P.; Etienne, J. B.; Duplantier, K. C. Tetrahedron Lett.
1995, 36, 669.
(47) Matsuo, I.; Isomura, M.; Miyazaki, T.; Sakakibara, T.; Ajisaka, K.
Carbohydr. Res. 1997, 305, 401.
(48) In AcxPIMY, X refers to the number of acyl groups attached not
including those attached to the glycerol, and Y refers to the number of
mannose residues.
(49) Guella, G.; Frassanito, R.; Mancini, I. Rapid Commun. Mass
Spectrom. 2003, 17, 1982.
(50) Hsu, F.-F.; Turk, J. J. Am. Soc. Mass Spectrom. 2000, 11, 797.
(51) Hsu, F.-F.; Turk, J. J. Am. Soc. Mass Spectrom. 2000, 11, 986.
(52) Hsu, F.-F.; Turk, J. J. Am. Soc. Mass Spectrom. 2000, 11, 892.
(53) Smith, P. B. W.; Snyder, A. P.; Harden, C. S. Anal. Chem. 1995, 67,
1824.
(54) Jacobs, W. R.; Tuckman, M.; Bloom, B. R. Nature 1987, 327, 532.
(55) Schaeffer, M. L.; Khoo, K. H.; Besra, G. S.; Chatterjee, D.;
Brennan, P. J.; Belisle, J. T.; Inamine, J. M. J. Biol. Chem. 1999, 274,
31625.
(56) Guerin, M. E.; Kaur, D.; Somashekar, B. S.; Gibbs, S.; Gest, P.;
Chatterjee, D.; Brennan, P. J.; Jackson, M. J. Biol. Chem. 2009, 284,
25687.
(57) Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.;
Timmers, F. J. Organometallics 1996, 15, 1518.
(58) Still, W. C.; Kahn, M.; Mitra, A. M. J. Org. Chem. 1978, 43, 2923.
(59) Keck, G. E.; Yu, T.; McLaws, M. D. J. Org. Chem. 2005, 70, 2543.
(60) Kaluza, Z.; Furman, B.; Krajewski, P.; Chmielewski, M.
Tetrahedron 2000, 56, 5553.
(61) Yang, G.; Ding, X.; Kong, F. Tetrahedron Lett. 1997, 38, 6725.
(62) Ferlin, N.; Grassi, D.; Ojeda, C.; Castro, M. J. L.; Grand, E.;
(22) Liu, X.; Stocker, B. L.; Seeberger, P. H. J. Am. Chem. Soc. 2006,
128, 3638.
Fernan
́
dez Cirelli, A.; Kovensky, J. Carbohydr. Res. 2008, 343, 839.
2189
dx.doi.org/10.1021/jo302508e | J. Org. Chem. 2013, 78, 2175−2190

The Journal of Organic Chemistry
Featured Article
(63) Mereyala, H. B.; Bhavani, S.; Rudradas, A. P. Synth. Commun.
2004, 34, 1057.
(64) Bock, K.; Pedersen, C. J. Chem. Soc., Perkin Trans. 2 1974, 293.
2190
dx.doi.org/10.1021/jo302508e | J. Org. Chem. 2013, 78, 2175−2190