Synthesis of galactofuranose-based acceptor substrates for the study of the carbohydrate polymerase GlfT2

Article abstract of DOI:10.1016/j.bmc.2010.04.068

Despite the prevalence and importance of carbohydrate polymers, the molecular details of their biosynthesis remain elusive. Many enzymes responsible for the synthesis of carbohydrate polymers require a 'primer' or 'initiator' carbohydrate sequence. One example of such an enzyme is the mycobacterial galactofuranosyltransferase GlfT2 (Rv3808c), which generates an essential cell wall building block. We recently demonstrated that recombinant GlfT2 is capable of producing a polymer composed of alternating β-(1,5) and β-(1,6)-linked galactofuranose (Galf) residues. Intriguingly, the length of the polymers produced from a synthetic glycosyl acceptor is consistent with those found in the cell wall. To probe the mechanism by which polymer length is controlled, a collection of initiator substrates has been assembled. The central feature of the synthetic route is a ruthenium-catalyzed cross-metathesis as the penultimate transformation. Access to synthetic substrates has led us to postulate a new mechanism for length control in this template-independent polymerization. Moreover, our investigations indicate that lipids possessing but a single galactofuranose residue can act as substrates for GlfT2.

Full text of DOI:10.1016/j.bmc.2010.04.068

Bioorganic & Medicinal Chemistry 18 (2010) 3753–3759
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of galactofuranose-based acceptor substrates for the study
of the carbohydrate polymerase GlfT2
Rebecca A. Splain a, Laura L. Kiessling a,b,
*
a Department of Chemistry, University of Wisconsin, 1101 University Avenue, Madison, WI 53706, USA
b Department of Biochemistry, University of Wisconsin, 433 Babcock Drive, Madison, WI 53706, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Despite the prevalence and importance of carbohydrate polymers, the molecular details of their biosyn-
thesis remain elusive. Many enzymes responsible for the synthesis of carbohydrate polymers require a
‘primer’ or ‘initiator’ carbohydrate sequence. One example of such an enzyme is the mycobacterial galac-
tofuranosyltransferase GlfT2 (Rv3808c), which generates an essential cell wall building block. We
recently demonstrated that recombinant GlfT2 is capable of producing a polymer composed of alternat-
ing b-(1,5) and b-(1,6)-linked galactofuranose (Galf) residues. Intriguingly, the length of the polymers
produced from a synthetic glycosyl acceptor is consistent with those found in the cell wall. To probe
the mechanism by which polymer length is controlled, a collection of initiator substrates has been assem-
bled. The central feature of the synthetic route is a ruthenium-catalyzed cross-metathesis as the penul-
timate transformation. Access to synthetic substrates has led us to postulate a new mechanism for length
control in this template-independent polymerization. Moreover, our investigations indicate that lipids
possessing but a single galactofuranose residue can act as substrates for GlfT2.
Received 27 March 2010
Revised 20 April 2010
Accepted 21 April 2010
Available online 28 April 2010
Keywords:
Galactan
Carbohydrate polymer
Glycosyl acceptor
Galactofuranose
Glycosyltransferase
Cross-metathesis
Mycobacterium tuberculosis
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
synthesized a collection of analogs of lipid II, which is polymerized
by glycosyltransferases to build bacterial peptidoglycan.4 The pep-
Carbohydrate polymers mediate critical functions in all organ-
isms, including energy storage, cellular structure and protection,
cell differentiation and proliferation, and immune responses. Poly-
saccharides can vary in length from tens of residues to thousands,
and polymer length appears to be important for function. For in-
stance, polysaccharides that are involved in signaling tend to be
shorter, whereas long polysaccharides are components of the
extracellular matrix of eukaryotes and the protective capsules of
bacteria.1,2 Despite the ubiquity of polysaccharides, many of the
details of their biosynthesis and function continue to elude
researchers. For example, cellulose is composed of packed glucose
polymers, which are primarily linked through b-(1,4)-linkages with
a varying percentage of b-(1,6)-linkages. The biosynthesis of cellu-
lose is catalyzed by cellulose synthase and thought to occur via a
highly processive, template-independent mechanism. Interest-
ingly, the factors that determine cellulose polymer length are un-
known,3 highlighting the difficulty of understanding the control
mechanisms that operate in template-independent polymerization
reactions.
tidoglycan glycosyltransferases use lipid-linked saccharides as
both the donor and acceptor for polymerization, and the synthetic
compounds provide the means to differentiate between the lipid
requirement for the donor and acceptor sites. We have employed
chemical synthesis to explore the acceptor requirements and the
mechanisms that govern the assembly of a building block for the
mycobacterial cell wall.
Because of the devastating consequences that can result from
mycobacterial infection, our efforts to illuminate the mechanisms
that govern the biosynthesis of glycan polymers have focused on
the carbohydrate polymerase GlfT2 from Mycobacterium tuberculo-
sis. GlfT2, encoded by the glfT2 (or RV3808c) gene in M. tuberculosis,
is an essential galactofuranosyltransferase.5,6 GlfT2 can catalyze
the synthesis of a linear polymer of 20–40 Galf residues that are
connected by alternating b-(1,5) and b-(1,6) glycosidic bonds
(Fig. 1).7,8 This polymer, termed the galactan, serves as a covalent
bridge between the peptidoglycan and the protective mycolic
acid–arabinan layer.9 We recently demonstrated that recombinant
His6-GlfT2 is capable of producing galactan polymers of endoge-
nous length from synthetic lipid-bearing acceptors.8 Fundamental
to this study was the availability of synthetic acceptors. Herein,
we describe a divergent synthetic route and demonstrate its utility
for assembling a wide range of acceptors. These compounds can
dissect the molecular features relevant for elongation and tem-
plate-independent length control by GlfT2.
Chemical synthesis can provide probes to elucidate the mecha-
nisms that underlie the biosynthesis of carbohydrate and glycan
polymers. For example, Walker, Kahne and co-workers recently
* Corresponding author. Tel.: +1 608 262 0541; fax: +1 608 265 0764.
E-mail address: kiessling@chem.wisc.edu (L.L. Kiessling).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.068

3754
R. A. Splain, L. L. Kiessling / Bioorg. Med. Chem. 18 (2010) 3753–3759
Figure 1. GlfT2 is a polymerizing glycosyltransferase responsible for the synthesis of the mycobacterial galactan polymer.
the common intermediate disaccharide 1. Second, the outstanding
compatibility of functional groups with metathesis reactions al-
lows the installation of lipids with diverse functional groups. Third,
in our experience, direct glycosylation reactions of hydroxyl-termi-
nated lipids, especially with longer lipids, are more difficult to opti-
mize. In contrast, the yields of cross-metathesis reactions are less
affected by the length of the alkenyl substituent. We envisioned
that cross-metathesis could provide compounds that could be used
to explore how GlfT2 functions to control polysaccharide length
and the molecular features19 of compounds that serve as GlfT2
acceptors.
2. Results and discussion
GlfT2 was identified as a critical glycosyltransferase in myco-
bacterial galactan assembly.10–13 Recombinant GlfT2 is a soluble
galactofuranosyl transferase that was first shown to add up to four
new Galf residues to acceptors displaying galactofuranose-termi-
nated oligosaccharides.14 Polymers of lengths comparable to those
observed endogenously were absent. We sought to develop a syn-
thetic route to rapidly generate acceptors that could be converted
into polymers of Galf residues. We postulated that the ability to
vary acceptor structure could illuminate the mechanisms involved
in determining polymer length. Herein, we outline the logic of our
synthetic route to disaccharide acceptors for GlfT2, describe its
application to the synthesis of previously employed acceptors,8
as well as new acceptors, and compare the ability of GlfT2 to elon-
gate these different compounds.
We chose allyl-bearing 1,6-linked Galf disaccharide 1 as the key
substrate for the cross-metathesis reaction. The assembly of this
building block began with the preparation of protected Galf deriv-
ative 2 (Scheme 1a), which was generated from D
-galactose.20,21
Fischer glycosylation with allyl alcohol provided 3, which could
be converted into monosaccharide acceptor 4 by selective removal
of the primary acetate protecting group (Scheme 1a). This reaction
was accompanied by a small amount of benzoyl group migration to
the primary alcohol. The extent of migration varied based on the
number of equivalents of acetyl chloride added. Presumably, a
minimum amount of acid is required for acetate hydrolysis, but in-
creased amounts lead to acid-catalyzed migration. The major prod-
uct (4) could be coupled to monosaccharide donor 522,23 in the
presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) to
afford 1,6-linked Galf disaccharide 1.
2.1. Rationale for acceptor substrate design
The galactan portion of the mycobacterial cell wall consists of
alternating b-(1,5) and b-(1,6)-linked Galf residues. In principle,
synthetic acceptors with either a terminal 1,5- or 1,6-linked sugar
might be effective substrates for GlfT2. Studies with synthetic
disaccharide acceptors suggested that acceptors with a 1,6-Galf
linkage at the non-reducing end were better substrates than 1,5-
terminated acceptors.14 Still, neither gave rise to full-length poly-
mers. Accordingly, we selected a lipid-linked 1,6-Galf disaccharide
as a starting point for our mechanistic investigations.8 In particu-
lar, we were interested in probing the role of the lipid functionality
present in the putative natural acceptor, which we suspected
might be relevant to GlfT2 elongation.4,15 As a result, altering the
lipid became a major focus for our synthetic strategy.
We were attracted by the possibility of a divergent synthesis in
which different lipophilic substituents could be appended late in
the synthetic scheme. To this end, we envisioned using an olefin
cross-metathesis reaction between a sugar substrate equipped
with an anomeric allyl group and an alkenyl-lipid in the penulti-
mate step of the synthesis (Fig. 2).16–18 There are several advanta-
ges of this approach over conventional variation by glycosylation.
First, it provides the means to rapidly diversify acceptors from
A series of phenoxy-terminated alkenyl-lipids were prepared
from the corresponding alkenyl bromides. We reasoned that the
installation of phenoxy groups would add lipophilicity and provide
a means to quantify acceptor substrate concentration in biological
assays. Olefin cross-metathesis of lipids and disaccharide
1
(Scheme 1b), followed by removal of the ester protecting groups
with sodium methoxide, provided a collection of acceptor analogs
(Fig. 3a). The variation of lipophilicity of our acceptor analogs affor-
ded key insights into the mechanism of GlfT2-catalyzed galactan
polymerization.
Acceptor 9 provided the first evidence for the polymerase activ-
ity of GlfT2 (Table 1). MALDI-TOF MS analysis showed enzymatic
reaction products that contained as many Galf residues as are
found in the natural galactan (27 additional Galf residues).8 Be-
Figure 2. Retrosynthetic coupling strategy for glycolipid formation.

R. A. Splain, L. L. Kiessling / Bioorg. Med. Chem. 18 (2010) 3753–3759
3755
Scheme 1. (a) Route to b-1,6-linked Galf disaccharide 1. (b) Ruthenium-catalyzed cross-metathesis of 1 with a collection of lipids can provide protected glycolipids.
Figure 3. Synthetic acceptor substrates for GlfT2. (a) A collection of b-1,6-linked Galf disaccharides was used to reveal that GlfT2 forms galactan polymers and that lipid
length is critical for activity. (b) Compounds 11–14 represent examples of an expanded collection of chemical tools for studying GlfT2.
cause GlfT2 acts on a physiological substrate that can insert into
membranes, it was possible that GlfT2 depends upon the presence
of a hydrophobic environment. We therefore tested the ability of
acceptor 9 to form micelles in dye solubilization assays and found
that it could. This observation led us to question whether the abil-
ity of compound 9 to serve as a substrate depends upon its incor-

3756
R. A. Splain, L. L. Kiessling / Bioorg. Med. Chem. 18 (2010) 3753–3759
Table 1
acceptor for the synthesis of 11 was obtained. Acceptor 11 is a sub-
strate for GlfT2 (Fig. 4; Table 1), and longer polymeric products are
observed. These data provide additional importance of lipid substi-
tution and suggesting bifunctionality may be intrinsic to the recog-
nition event.
The assays for evaluating GlfT2 activity rely on mass spectrom-
etry analysis. Time–course assays and competition studies might
benefit from an isotopically labeled substrate that would provide
distinct peaks in a mass spectrometry trace compared to com-
pound 9. To this end, we used our synthetic route to access com-
pound 12 (Fig. 3b), which contains a d5-phenoxy-terminated lipid
that can be distinguished from acceptor 9 in MALDI-TOF MS anal-
ysis of enzymatic reaction mixtures. We anticipate that this com-
pound will be a valuable of GlfT2 catalysis.
Summary of the ability of synthetic acceptors to act as substrates of GlfT2
Compound
Substrate
Polymerized?
Galf residues added
6
7
8
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
4
12
25
27
48
35
27
46
0
Yes
Yes
Yes
Yes
Yes
Yes
No
9
10
11
12
13
14
poration into micelles. To test this possibility, we used our syn-
thetic route to vary the anomeric substituent and therefore alter
the propensity of compounds to form micelles. We found that
disaccharides 8 and 9 were polymerized by GlfT2 to similar extents
(Table 1), but the former did not form micelles. Thus, the ability of
compounds to act as substrates for polymerization by GlfT2 is not
dictated by their ability to form micelles. Previous studies had
demonstrated GlfT2 can elongate an octyl-linked trisaccharide.14
Therefore, we synthesized glycolipid 6 as a benchmark substrate
for glycosyltransferase activity. When crude enzymatic reaction
mixtures were analyzed by MALDI-TOF MS, disaccharide 6 afforded
products elongated by, at most, four Galf residues (Table 1). Disac-
charide 7 was synthesized to investigate how an increase in lipo-
philicity without an increase in lipid length would affect
polymerization. Similar extents of elongation were observed with
disaccharides 7 and 6 (Table 1), demonstrating that a threshold li-
pid length was required for galactan formation by GlfT2.
These results suggest that the lipid substituent, and not the sac-
charide functionality, is a key determinant of polymer length. We
hypothesize that the acceptor substrate occupies not only the ac-
tive site but also is tethered to a lipid-binding secondary site. If this
prediction is accurate, the longer lipid present in compound 10
should bind with increased affinity to this lipid-binding site as
compared to acceptor 9. Thus, if elongation occurs through biva-
lent substrate binding, the product distribution obtained from
compound 10 versus compound 9 should be shifted to higher-
molecular weight polymers. Our previous results indicate that
compound 10 is elongated to give longer polymer products than
those seen for acceptor 9 (Table 1). Thus, access to acceptors pos-
sessing anomeric substituents with a range of lengths allowed us
to determine how GlfT2 recognizes and polymerizes acceptor
substrates.
We also used our synthetic strategy to generate compounds
that could be used to investigate the role of the sugar substituent.
As mentioned previously, others had shown GlfT2 to be capable of
elongation of both di- and trisaccharides. Still, the production of
full-length galactan formation by GlfT2 depends on the acceptor
substrate attributes. In addition to exploiting our synthetic route
to examine the substrate lipid requirements, we also wanted to
probe the minimum requirements for the acceptor saccharide. To
this end, we assembled monosaccharide Galf acceptor 13
(Fig. 3b) by exploiting the modularity of our synthetic route
(Scheme 2). Fischer glycosylation of allyl alcohol and Galf penta-
acetate (15) gave compound 16. Ruthenium-catalyzed cross-
metathesis provided lipid-linked Galf 17, and sodium methoxide-
catalyzed hydrolysis of the ester yielded 13.
We tested whether GlfT2 could elongate this simple acceptor.
Analysis of crude enzymatic reaction mixtures using MALDI-TOF
MS indicate that monosaccharide Galf acceptor 13 is a substrate
(Fig. 5a). The products obtained possess up to 46 additional Galf
residues, and this length is comparable to those generated from
1,6-linked disaccharide acceptors 8, 9, and 10 (Table 1). This result
is particularly surprising because the putative endogenous sub-
strate for GlfT2 is a tetrasaccharide terminated by a 1,5-linked Galf
disaccharide. While these investigations do not assess how the
number of Galf residues in the acceptor influence the efficiency
of elongation, they do indicate that the disaccharide itself is not
essential for GlfT2-catalyzed polymer formation.
The apparent promiscuity of GlfT2 for simple lipid-linked sac-
charides led us to probe the requirements for GlfT2 elongation fur-
ther. The preceding enzyme in the pathway, GlfT1, is homologous
to GlfT2, and the former acts on a substrate terminated with a
pyranose residue. Thus, GlfT2 might be capable of elongating a
substrate with a pyranose sugar. To test that GlfT2 requires at least
one Galf residue for activity, the galactopyranose (Galp) analog of
acceptor 13 was synthesized in a manner analogous to that de-
scribed for compound 13. As expected, Galp acceptor 14 (Fig. 3b)
is not a substrate for GlfT2, and no elongation was observed
2.2. Expanding the armamentarium of chemical tools
Based on the insight afforded by examining the GlfT2-catalyzed
elongation of 1,6-linked acceptors, we sought to expand the
breadth of our acceptor collection to address additional questions
regarding the mechanism of galactan polymerization. In addition
to the ability of GlfT2 to control polymer length, the enzyme also
was reported to be bifunctional, producing alternating b-(1,5)
and b-(1,6) glycosidic bonds. 1,5-Linked synthetic acceptors with
short hydrophobic anomeric substituents had been shown to serve
as GlfT2 substrates, but only short oligomers were observed.14 Our
results with 1,6-linked acceptors suggest that 1,5-linked acceptors
equipped with a more lipophilic substituent could give rise to long-
er polymers. Thus, acceptor 11 (Fig. 3b) was synthesized using a
route analogous to that described above (Scheme 1). As previously
mentioned, the conditions employed to remove the primary ace-
tate group of 3 were not entirely selective; they afforded regioiso-
meric products that could be separated by column
chromatography. In this way, an appropriate monosaccharide
Figure 4. MALDI-TOF MS spectrum obtained from the reaction of His6-GlfT2, UDP-
Galf, and 1,5-linked disaccharide 11.

R. A. Splain, L. L. Kiessling / Bioorg. Med. Chem. 18 (2010) 3753–3759
3757
Scheme 2. Route for the synthesis of monosaccharide Galf acceptor 13.
required a chemical biology approach to elucidate factors that
influence galactan length. A divergent and modular synthetic strat-
egy allowed for incorporation of lipids to explore the role of the li-
pid and the saccharide of GlfT2 acceptors. Interestingly, a lipid with
a single galactofuranose residue was capable of serving as an
acceptor, suggesting a relaxed specificity for GlfT2. We anticipate
that the synthetic acceptors generated by our cross-metathesis
strategy will continue to provide insight into the mechanism of
GlfT2 and other enzymes that act on glycolipid acceptors.
4. Materials and methods
4.1. General synthetic methods
All chemicals used were reagent-grade from Sigma–Aldrich.
Reactions were carried out under argon in oven-dried glassware.
Methanol (MeOH) was distilled from magnesium and dichloro-
methane (CH2Cl2) was distilled from calcium hydride. Analytical
TLC was carried out on E. Merck TLC plates precoated with Silica
Gel 60 F254 (250 lm layer thickness). Analyte visualization was
accomplished by using a UV lamp and charring with a solution of
p-anisaldehyde (3.5 mL), acetic acid (15 mL), H2SO4 (50 mL), and
ethanol (350 mL). Flash chromatography was performed on Scien-
tific Adsorbents silica gel (32–63 m; 60 Å pore size) by using re-
agent-grade hexanes, ACS-grade ethyl acetate (EtOAc), MeOH or
CH2Cl2. 1H NMR spectra were recorded on Bruker AC-300 spec-
trometers. 1H chemical shifts are reported relative to tetramethyl-
silane (0.00; CDCl3) or CD3OD (3.30; CD3OD). Peak multiplicity is
reported as singlet (s), doublet (d), doublet of doublets (dd), triplet
(t), doublet of triplets (dt), etc. Coupling constants (J) are listed in
Hertz. High-resolution electrospray ionization mass spectrometry
(HRESI MS) was performed on a Micromass LCT instrument. Syn-
thetic procedures and characterization data for compounds 1–10
have been reported previously.8
Figure 5. MALDI-TOF MS spectrum obtained from the reaction of His6-GlfT2, UDP-
Galf, and monosaccharide acceptor substrates. (a) Monosaccharide Galf acceptor
(13) is sufficient for galactan polymerization by GlfT2. Reaction products range
from those resulting from the addition of 2–46 Galf residues. (b) Monosaccharide
Galp acceptor (14) is not a substrate for GlfT2 under identical conditions.
(Fig. 5b; Table 1). Thus, a lipid bearing a terminal galactofuranose
substituent is the minimal requirement for GlfT2-catalyzed poly-
saccharide synthesis. As expected, the galactofuranose residue
serves as a key attribute for GlfT2 acceptors.
That GlfT2 can recognize a single Galf residue suggests there is
variation or flexibility in the endogenous acceptor substrate re-
quired by GlfT2. To probe this possibility requires access to the nat-
ural substrate and analogs. Efforts to generate the requisite
acceptors are ongoing. Because of its ease of synthesis, acceptor
13 is attractive as a substrate for studies that do not require differ-
entiation between 1,5-linked and 1,6-linked acceptor substrates,
including inhibitor screens. Inhibitors of mycobacterial galactan
biosynthesis may be valuable leads for tuberculosis therapies be-
cause the gene encoding GlfT2 is essential for mycobacterial
growth.
4.2. 12-Phenoxy-dodec-2-enyl-b-D-galactofuranosyl-(1,5)-b-D-
galactofuranoside (11)
Allyl 2,3,6-tri-O-benzoyl-5-hydroxy-b-D-galactofuranoside was
obtained as a minor migration side product from the deacylation
of allyl 6-O-acetyl-2,3,5-tri-O-benzoyl-b-D-galactofuranoside de-
scribed previously (in 8% yield).8
1H NMR (CDCl3): d 8.09–8.03 (m, 6H, Ar); 7.59–7.39 (m, 9H, Ar);
5.99–5.86 (m, 1H); 5.66 (dd, J = 4.8, 0.8, 1H); 5.55 (d, J = 1.3, 1H);
5.32 (s, 1H, H-1), 5.36–5.28 (m, 1H); 5.22–5.17 (m, 1H); 4.65–
4.58 (dd, J = 12.8, 8, 1H); 4.52–4.46 (m, 2H); 4.41 (dd, J = 4.8, 2.1,
1H); 4.29–4.22 (m, 1H); 4.12–4.04 (m, 1H); 2.71 (d, J = 8.2, 1H, 5-
OH).
3. Conclusion
The acceptor monosaccharide (71 mg, 0.13 mmol) and ethyl
2,3,5,6-tetra-O-acetyl-1-thio-b-
0.15 mmol) were dissolved in CH2Cl2 (4.7 mL) with activated 4 Å
molecular sieve beads in an ice bath. N-Iodosuccinimide (41 mg,
D
-galactofuranoside24
(57 mg,
Our interest in the mechanism underlying polysaccharide bio-
synthesis led us to study the mycobacterial galactofuranosyltrans-
ferase GlfT2. Interrogation of the mechanism and activity of GlfT2

3758
R. A. Splain, L. L. Kiessling / Bioorg. Med. Chem. 18 (2010) 3753–3759
0.18 mmol) and silver triflate (8 mg, 0.03 mmol) were added to the
stirring solution. The reaction was stirred for 1.5 h in an ice bath.
The mixture was filtered to remove the molecular sieves, and the
organic layer was washed successively with 10% Na2S2O3 (5 mL),
saturated Na2HCO3 (5 mL), water (5 mL), and brine (5 mL). The or-
ganic layer was dried over MgSO4 and concentrated under reduced
pressure. Purification by flash chromatography [40% (vol/vol)
EtOAc in hexanes] provided 62 mg (55%) of allyl 2,3,6-tri-O-ben-
sure. The crude product was purified by flash chromatography [0–
40% (vol/vol) gradient EtOAc/hexanes] to yield 17 mg (24%) of 12-
d5-phenoxy-dodec-2-enyl 2,3,5-tri-O-benzoyl-b-
D-galactofurano-
syl-(1,6)-2,3,5,6-tetra-O-acetyl-b- -galactofuranoside.
D
1H NMR (CDCl3): d 8.08–8.04 (m, 4H, Ar); 7.90–7.87 (m, 2H, Ar);
7.60–7.26 (m, 9H, Ar); 5.85–5.54 (m, 4H); 5.47–5.45 (m, 1H); 5.40–
5.31 (m, 1H); 5.33 (s, 1H, H-1); 5.10 (s, 1H, H-10); 5.00–4.94 (m,
2H); 4.63 (dd, J = 5.1, 3.6, 1H); 4.35–4.03 (m, 6H); 3.96–3.90 (m,
3H); 2.10–1.96 (m, 14H), 1.82–1.71 (m, 2H); 1.49–1.25 (m, 12H).
zoyl-b-
D-galactofuranosyl-(1,5)-2,3,5,6-tetra-O-acetyl-b-D-galacto-
furanoside as a white crystalline solid.
To
the
deuterium-labeled
lipid–disaccharide
(16 mg,
1H NMR (CDCl3): d 8.15–8.03 (m, 6H, Ar); 7.62–7.41 (m, 9H, Ar);
6.01–5.88 (m, 1H); 5.73 (d, J = 5.1, 1H); 5.55 (d, J = 1.1, 1H); 5.51 (s,
1H, H-10); 5.38–5.31 (m, 2H); 5.31 (s, 1H, H-1); 5.23–5.19 (m, 2H);
5.00 (dd, J = 5.8, 2.4, 1H); 4.72–3.80 (m, 9H); 2.04, 2.01, 1.99, 1.78
(4s, 12H, 4 ꢀ CH3CO).
0.015 mmol) in MeOH (0.63 mL) was added sodium methoxide
solution (0.12 mL, 0.5 M in MeOH). The reaction was stirred for
1.5 h at room temperature and neutralized with Amberlite (IR-
120 H+) ion exchange resin, filtered, and concentrated under re-
duced pressure. Purification by flash chromatography [0–20%
(vol/vol) gradient MeOH/CH2Cl2] provided 6 mg (61%) of com-
pound 12 as a white solid.
A
solution of Grubbs first generation catalyst (4.1 mg,
0.005 mmol) in CH2Cl2 (0.2 mL) was added to a stirring solution
of allyl b-(1,5)-disaccharide (62 mg, 0.072 mmol) and 11-phen-
oxy-1-undecene8 (80 mg, 0.32 mmol) in CH2Cl2 (0.52 mL). The
mixture was heated at reflux for 17 h and concentrated under re-
duced pressure. The crude product was purified by flash chroma-
tography [0–40% (vol/vol) gradient EtOAc/hexanes] to yield
60 mg (77%) of 12-phenoxy-dodec-2-enyl 2,3,6-tri-O-benzoyl-b-
1H NMR (CD3OD): d 5.78–5.49 (m, 2H); 4.98 (s, 1H, H-10); 4.95
(d, J = 1.9, 1H, H-1); 4.23–3.50 (m, 16H); 2.10–2.01 (m, 2H);
1.80–1.71 (m, 2H); 1.52–1.28 (m, 12H). HRESI MS m/z calculated
for [M+Na]+ C30H43 D5O12Na: 628.3352. Found: 628.3325.
4.4. Allyl 2,3,5,6-tetra-O-acetyl-b-D-galactofuranoside (16)
D
-galactofuranosyl-(1,5)-2,3,5,6-tetra-O-acetyl-b-
D-
galactofuranoside.
Monosaccharide 1523 (700 mg, 1.8 mmol) and allyl alcohol
(0.25 mL, 3.6 mmol) were dissolved in CH2Cl2 (9 mL). The flask
was placed into an ice bath and stirred for 15 min. BF3ꢁEt2O
(0.67 mL, 5.4 mmol) was added dropwise to the stirring solution.
After 15 min, the ice bath was removed, and the mixture was stir-
red for 1 h at room temperature. The reaction mixture was
quenched with triethylamine and diluted with CH2Cl2 (20 mL).
The organic layer was washed with saturated NaHCO3 solution
(2 ꢀ 15 mL) and brine (15 mL) and dried over MgSO4. The solvent
was removed, and the crude mixture purified by column chroma-
tography [10–40% (vol/vol) gradient EtOAc/hexanes] to give
566 mg of 16 (81%) as a colorless oil.
1H NMR (CDCl3): d 8.13–8.03 (m, 6H, Ar); 7.62–7.41 (m, 9H, Ar);
7.30–7.24 (m, 2H, Ar); 6.95–6.87 (m, 3H, Ar); 5.73–5.51 (m, 4H);
5.51 (s, 1H, H-10); 5.35–5.23 (m, 2H); 5.29 (s, 1H, H-1); 4.99 (dd,
J = 5.8, 2.5, 1H); 4.68–4.50 (m, 4H); 4.43 (dd, J = 5.7, 3.9, 1H);
4.32–4.01 (m, 4H); 3.96–3.91 (m, 2H); 2.03, 2.00, 1.96 (3s, 9H,
3 ꢀ CH3CO); 1.78–1.70 (m, 5H); 1.44–1.23 (m, 12H).
Sodium methoxide solution (0.43 mL, 0.5 M in MeOH) was
added to a stirring solution of the protected lipid–disaccharide
(58 mg, 0.054 mmol) in MeOH (2 mL). The reaction was stirred
for 2 h at room temperature and neutralized with Amberlite (IR-
120 H+) ion exchange resin, filtered, and concentrated under re-
duced pressure. Purification by flash chromatography [100% CH2Cl2
to 20% (vol/vol) gradient MeOH/CH2Cl2] provided 26 mg (80%) of
compound 11 as a colorless oil.
1H NMR (CDCl3): d 5.96–5.83 (m, 1H); 5.42–5.37 (m, 1H); 5.35–
5.28 (m, 1H); 5.23–5.19 (m, 1H); 5.09 (s, 1H, H-1); 5.08 (d, J = 0.9,
1H); 5.02 (dd, J = 1.5, 6, 1H); 4.36 (dd, J = 11.8, 4.5, 1H); 4.29–4.16
(m, 4H); 4.07–3.99 (m, 1H); 2.14, 2.11, 2.09, 2.06 (4s, 12H,
4xCH3CO). HRESI MS m/z calculated for [M+Na]+ C17H24O10Na:
411.1262. Found: 411.1270.
1H NMR (CD3OD): d 7.27–7.20 (m, 2H, Ar); 6.91–6.85 (m, 3H,
Ar); 5.77–5.47 (m, 2H); 5.17 (s, 1H, H-10); 4.86 (d, J = 1.6, 1H, H-
1); 4.21–3.59 (m, 14H); 2.12–2.01 (m, 2H); 1.80–1.71 (m, 2H);
1.52–1.33 (m, 12H). HRESI MS m/z calculated for [M+Na]+
C30H48O12Na: 623.3038. Found: 623.3035.
4.5. 12-Phenoxy-dodec-2-enyl-2,3,5,6-tetra-O-acetyl-b-D-galac-
tofuranoside (17)
4.3. 12-d5-Phenoxy-dodec-2-enyl-b-D-galactofuranosyl-(1,6)-b-
D
-galactofuranoside (12)
To a stirring solution of 16 (33 mg, 0.085 mmol) and 11-phen-
oxy-1-undecene (84 mg, 0.34 mmol) in CH2Cl2 (0.85 mL) was
added Grubbs first generation catalyst (5.0 mg, 0.006 mmol). The
mixture was heated at reflux for 14 h and concentrated under re-
duced pressure. The crude product was purified by flash chroma-
tography [0–30% (vol/vol) gradient EtOAc/hexanes] to yield
39 mg (76%) of 17 as a light brown oil.
11-d5-Phenoxy-1-undecene was prepared according to the pre-
viously published procedure for preparation of unlabeled 11-phen-
oxy-1-undecene.8 Phenol-d5 (1.0 g, 1.0 mmol) was added to a
solution of sodium ethoxide (68 mg, 1.0 mmol) in ethanol
(0.6 mL), and the reaction was stirred for 30 min at room temper-
ature. 11-Bromo-1-undecene (0.22 mL, 1.0 mmol) was added, and
the reaction mixture was stirred at reflux for 14 h. The solution
was cooled to room temperature, diluted with H2O (1.5 mL), and
concentrated under reduced pressure. The crude reaction mixture
was purified by flash chromatography (hexanes) to yield 110 mg
(43%) of 11-d5-phenoxy-1-undecene as a colorless liquid.
1H NMR (CDCl3): d 7.23–7.18 (m, 2H, Ar); 6.88–6.81 (m, 3H, Ar);
5.71–5.61 (m, 1H); 5.49–5.40 (m, 1H); 5.35–5.30 (m, 1H); 5.00,
4.99 (2s, 2H, H-1, H-2); 4.94–4.91 (m, 1H); 4.28 (dd, J = 11.7, 4.5,
1H); 4.21–4.03 (m, 3H); 3.92–3.86 (m, 3H); 2.07, 2.03, 2.02, 1.99
(4s, 12H, 3 ꢀ CH3CO); 2.05–1.94 (m, 2H); 1.75–1.66 (m, 1H);
1.41–1.18 (m, 12H). HRESI MS m/z calculated for [M+Na]+
C32H46O11Na: 629.2933. Found: 629.2921.
1H NMR (CDCl3): d 5.82 (m, 1H); 5.04–4.92 (m, 2H); 3.95 (t,
J = 6.5, 2H); 2.04 (m, 2H); 1.78 (m, 2 H); 1.48–1.30 (m, 12H).
A
solution of Grubbs first generation catalyst (3.7 mg,
4.6. 12-Phenoxy-dodec-2-enyl-b-D-galactofuranoside (13)
0.005 mmol) in CH2Cl2 (0.3 mL) was added to a stirring solution
of allyl disaccharide 1 (59 mg, 0.065 mmol) and 11-d5-phenoxy-
1-undecene (65 mg, 0.26 mmol) in CH2Cl2 (0.35 mL). The mixture
was heated at reflux for 14 h and concentrated under reduced pres-
To compound 17 (39 mg, 0.064 mmol) in MeOH (0.5 mL) was
added sodium methoxide solution (0.3 mL, 0.5 M in MeOH). The
reaction was stirred for 3 h at room temperature and neutralized

R. A. Splain, L. L. Kiessling / Bioorg. Med. Chem. 18 (2010) 3753–3759
3759
with Amberlite (IR-120 H+) ion exchange resin, filtered, and con-
centrated under reduced pressure. Purification by flash chromatog-
raphy [0–10% (vol/vol) gradient MeOH/CH2Cl2] provided 21 mg
(75%) of compound 13 as a white solid.
1250 mM UDP-Galf in 50 mM Hepes, pH 7.0, 25 mM MgCl2, and
100 mM NaCl. Reactions were incubated at room temperature for
18 h, then quenched with 120 mL of a 1:1 mixture of CHCl3/MeOH.
Quenched reaction mixtures were evaporated to dryness under
vacuum in a SpeedVac SC100 (Varian) then resuspended in
50 mL 50% MeCN for MALDI MS analysis. Samples for MALDI MS
1H NMR (CD3OD): d 7.26–7.20 (m, 2H, Ar); 6.90–6.85 (m, 3H,
Ar); 5.76–5.67 (m, 1H); 5.59–5.48 (m, 1H); 4.89 (d, J = 1.8, 1H, H-
1); 4.16–4.10 (m, 1H); 4.01–3.90 (m, 5H); 3.73–3.68 (m, 1H);
3.63–3.60 (m, 2H); 2.08–2.01 (m, 2H); 1.80–1.71 (m, 2H); 1.52–
1.28 (m, 12H). HRESI MS m/z calculated for [M+Na]+ C24H38O7Na:
461.2510. Found: 461.2524.
analysis were spotted as a 1:3 mixture with
a-cyano-4-hydroxy-
cinnamic acid matrix and spectra were recorded in positive linear
mode using a Bruker Ultraflex III mass spectrometer.
Acknowledgments
4.7. 12-Phenoxy-dodec-2-enyl-b-D-galactopyranoside (14)
This paper is dedicated to Peter H. Seeberger on the occasion of
his receipt of the Tetrahedron Young Investigator Award. This re-
search was supported by National Institutes of Health Grant
AI063596. R.A.S. thanks the American Chemical Society Division
of Medicinal Chemistry for a graduate fellowship. We acknowledge
M. R. Levengood and J. F. May for MALDI-TOF MS experiments with
compounds 13 and 14 and C. Brotschi for her preliminary synthetic
studies. MALDI-TOF MS and HRESI MS data were obtained at the
University of Wisconsin—Madison Chemistry Instrument Center
Mass Spectrometry Facility supported by NIH Grant Number NIH
NCRR 1S10RR024601-01 (MALDI-TOF MS) and NSF Grant Number
NSF CHE-9974839 (HRESI MS). The NMR facility is supported in
part by the NSF (CHE-9208463).
Galactopyranose pentaacetate (0.50 g, 1.3 mmol) and allyl alco-
hol (0.20 mL, 2.6 mmol) were dissolved in CH2Cl2 (6.4 mL). The
flask was placed into an ice bath and stirred for 10 min. BF3ꢁEt2O
(0.50 mL, 3.8 mmol) was added dropwise to the stirring solution.
After 20 min, the ice bath was removed, and the mixture was stir-
red for 2 h at room temperature. The reaction mixture was
quenched with triethylamine and diluted with CH2Cl2 (20 mL).
The organic layer was washed with saturated NaHCO3 solution
(2x15 mL) and brine (15 mL) and dried over MgSO4. The solvent
was removed, and the crude mixture purified by column chroma-
tography [10–40% (vol/vol) gradient EtOAc/hexanes] to give
311 mg (63%) of allyl 2,3,5,6-tetra-O-acetyl-b-D-galactopyranoside
as a colorless oil. The characterization of this compound has been
previously reported.25
References and notes
To a stirring solution of allyl 2,3,5,6-tetra-O-acetyl-b-D-galacto-
1. Weigel, P. H.; DeAngelis, P. L. J. Biol. Chem. 2007, 282, 36777.
2. Whitfield, C. Annu. Rev. Biochem. 2006, 75, 39.
3. Stubbe, J.; Tian, J.; He, A.; Sinskey, A. J.; Lawrence, A. G.; Liu, P. Annu. Rev.
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4. Perlstein, D. L.; Wang, T.-S. A.; Doud, E. H.; Kahne, D.; Walker, S. J. Am. Chem.
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5. Pan, F.; Jackson, M.; Ma, Y. F.; McNeil, M. R. J. Bacteriol. 2001, 183, 3991.
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7. Besra, G. S.; Khoo, K. H.; McNeil, M. R.; Dell, A.; Morris, H. R.; Brennan, P. J.
Biochemistry 1995, 34, 4257.
8. May, J. F.; Splain, R. A.; Brotschi, C.; Kiessling, L. L. Proc. Natl. Acad. Sci. U.S.A.
2009, 106, 11851.
9. Brennan, P. J.; Nikaido, H. Annu. Rev. Biochem. 1995, 64, 29.
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P. J. J. Biol. Chem. 2000, 275, 33890.
pyranoside (311 mg, 0.801 mmol) and 11-phenoxy-1-undecene
(767 mg, 3.11 mmol) in CH2Cl2 (8.0 mL) was added Grubbs first
generation catalyst (46 mg, 0.056 mmol). The mixture was heated
at reflux for 15 h and concentrated under reduced pressure. The
crude product was purified by flash chromatography [0–40% (vol/
vol) gradient EtOAc/hexanes] to yield 298 mg (61%) of 12-phen-
oxy-dodec-2-enyl-2,3,5,6-tetra-O-acetyl-b-D-galactopyranoside as
a brown oil.
1H NMR (CDCl3): d 7.30–7.25 (m, 2H, Ar); 6.95–6.88 (m, 3H, Ar);
5.74–5.59 (m, 1H); 5.52–5.43 (m, 1H); 5.38 (d, J = 3.3, 1H); 5.22
(ddd, J = 10.6, 7.9, 2.6, 1H); 5.02 (dd, J = 10.4, 3.5, 1H); 4.51 (dd,
J = 7.8, 1.2, 1H, H-1); 4.31–4.02 (m, 3H); 3.95 (t, J = 6.6, 2H); 3.88
(m, 1H); 2.15 (s, 3H, CH3CO); 2.05–1.98 (m, 11H, 3 ꢀ CH3CO);
1.82–1.73 (m, 2H); 1.48–1.28 (m, 12H).
11. Belanova, M.; Dianiskova, P.; Brennan, P. J.; Completo, G. C.; Rose, N. L.; Lowary,
T. L.; Mikusova, K. J. Bacteriol. 2008, 190, 1141.
12. Mikusova, K.; Belanova, M.; Kordulakova, J.; Honda, K.; McNeil, M. R.;
Mahapatra, S.; Crick, D. C.; Brennan, P. J. J. Bacteriol. 2006, 188, 6592.
13. Kremer, L.; Dover, L. G.; Morehouse, C.; Hitchin, P.; Everett, M.; Morris, H. R.;
Dell, A.; Brennan, P. J.; McNeil, M. R.; Flaherty, C.; Duncan, K.; Besra, G. S. J. Biol.
Chem. 2001, 276, 26430.
14. Rose, N. L.; Completo, G. C.; Lin, S. J.; McNeil, M.; Palcic, M. M.; Lowary, T. L. J.
Am. Chem. Soc. 2006, 128, 6721.
15. Lovering, A. L.; de Castro, L. H.; Lim, D.; Strynadka, N. C. J. Science 2007, 315,
1402.
16. Roy, R.; Dominique, R.; Das, S. K. J. Org. Chem. 1999, 64, 5408.
17. Leeuwenburgh, M. A.; van der Marel, G. A.; Overkleeft, H. S. Curr. Opin. Chem.
Biol. 2003, 7, 757.
18. Plettenburg, O.; Mui, C.; Bodmer-Narkevitch, V.; Wong, C. H. Adv. Synth. Catal.
2002, 344, 622.
19. Adams, J. H.; Cook, R. M.; Hudson, D.; Jammalamadaka, V.; Lyttle, M. H.;
Songster, M. F. J. Org. Chem. 1998, 63, 3706.
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1999, 18, 1121.
Sodium methoxide solution (2.0 mL, 0.5 M in MeOH) was added
to the acetate-protected lipid–galactopyranose (298 mg,
0.491 mmol). The reaction was stirred for 2 h at room temperature
and neutralized with Amberlite (IR-120 H+) ion exchange resin, fil-
tered, and concentrated under reduced pressure. Purification by
flash chromatography [10% (vol/vol) MeOH/CH2Cl2] provided
184 mg (85%) of compound 14 as a colorless oil.
1H NMR (CD3OD): d 7.27–7.20 (m, 2H, Ar); 6.90–6.85 (m, 3H,
Ar); 5.79–5.53 (m, 2H); 4.37–4.27 (m, 1H); 4.24 (d, J = 7.3, 1H, H-
1); 4.08 (ddd, J = 11.9, 6.8, 1, 1H); 3.94 (t, J = 6.4, 2H); 3.82–3.69
(m, 3H); 3.54–3.40 (m, 3H); 2.09–2.01 (m, 2H); 1.80–1.70 (m,
2H); 1.49–1.33 (m, 12H). HRESI MS m/z calculated for [M+Na]+
C24H38O7Na: 461.2510. Found: 461.2510.
22. Pathak, A. K.; Pathak, V.; Seitz, L.; Maddry, J. A.; Gurcha, S. S.; Besra, G. S.;
Suling, W. J.; Reynolds, R. C. Bioorg. Med. Chem. 2001, 9, 3129.
23. Chittenden, G. J. F. Carbohydr. Res. 1972, 25, 35.
4.8. Procedure for MALDI MS analysis of GlfT2 reaction products
24. Gelin, M.; Ferrieres, V.; Plusquellec, D. Eur. J. Org. Chem. 2000, 2000, 1423.
25. Pastore, A.; Adinolfi, M.; Iadonisi, A. Eur. J. Org. Chem. 2008, 2008, 6206.
Reactions consisted of 120 mL total volume containing final
concentrations of 0.2 mM His6-GlfT2, 50–200 mM acceptor, 150–