Rapid chemo-enzymatic synthesis of peracetylated glcnacβ3galβ- aglycones

Article abstract of DOI:10.1080/07328303.2011.617023

The inhibition of sialyl Lewis X can prevent unwanted cellular extravasation that is associated with inflammatory diseases and tumor metastasis. Described is an efficient methodology to prepare peracetylated GlcNAcβ3Gal-aglycone disaccharides subsequently

Full text of DOI:10.1080/07328303.2011.617023

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Rapid Chemo-Enzymatic Synthesis of
Peracetylated GlcNAcβ3Galβ-Aglycones
N. Merbouh a b , J. R. Brown c , F. K. Wallner a , M. Morton d , J. D.
Esko c & P. H. Seeberger a e f
a The Sanford Burnham Institute for Medical Research, La Jolla, CA,
USA
b Chemistry Department, Simon Fraser University, Burnaby, B.C.,
Canada
c Department of Cellular and Molecular Medicine, Glycobiology
Research and Training Center, University of California, San Diego, La
Jolla, CA, USA
d University of Connecticut, Storrs, CT, USA
e Max-Planck Institute for Colloids and Interfaces, Potsdam,
Germany
f Freie Universität Berlin, Berlin, Germany
Version of record first published: 09 Nov 2011.
To cite this article: N. Merbouh , J. R. Brown , F. K. Wallner , M. Morton , J. D. Esko & P. H. Seeberger
(2011): Rapid Chemo-Enzymatic Synthesis of Peracetylated GlcNAcβ3Galβ-Aglycones, Journal of
Carbohydrate Chemistry, 30:4-6, 373-390
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Journal of Carbohydrate Chemistry, 30:373–390, 2011
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Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2011.617023
Rapid Chemo-Enzymatic
Synthesis of Peracetylated
GlcNAcβ3Galβ-Aglycones
N. Merbouh,1,2 J. R. Brown,3 F. K. Wallner,1 M. Morton,4
J. D. Esko,3 and P. H. Seeberger1,5
1The Sanford Burnham Institute for Medical Research, La Jolla, CA, USA
2Chemistry Department, Simon Fraser University, Burnaby, B.C., Canada
3Department of Cellular and Molecular Medicine, Glycobiology Research and Training
Center, University of California, San Diego, La Jolla, CA, USA
4University of Connecticut, Storrs, CT, USA
5Max-Planck Institute for Colloids and Interfaces, Potsdam, Germany;
and Freie Universita¨t Berlin, Berlin, Germany
The inhibition of sialyl Lewis X can prevent unwanted cellular extravasation that
is associated with inflammatory diseases and tumor metastasis. Described is an effi-
cient methodology to prepare peracetylated GlcNAcβ3Gal-aglycone disaccharides sub-
sequently used as metabolic decoys to inhibit sialyl Lewis X expression. Four glycosides
were synthesized from one single parent disaccharide that in turn was prepared by
large-scale enzymatic synthesis. The procedure avoids lengthy and inefficient synthetic
steps to afford the desired disaccharides quickly with good overall yields.
Keywords Disaccharides; Decoy; Sialyl Lewis X; Glycosyltransferase
INTRODUCTION
Both the inflammatory response and circulating tumor cells are known to
use a similar mode of action at the molecular level by binding cell surface
glycoproteins containing sialyl Lewis X (sLex) to selectin receptors present
on the surface of leukocytes and the blood vessel.[1] During an in-
flammatory response, vascular leukocytes are recruited by interactions
between sialyl Lewis X (sLex)-containing glycoproteins on the leukocyte sur-
face and selectin receptors on endothelial cells. Similarly, metastasizing tu-
mor cells use comparable ligand-receptor interactions to bind to platelets as
Received May 29, 2011; accepted August 20, 2011.
Address correspondence to J. D. Esko, Department of Cellular and Molecular Medicine,
Glycobiology Research and Training Center, University of California, San Diego, La
Jolla, CA, USA. E-mail: jesko@ucsd.edu
373

N. Merbouh et al.
374
well as endothelial cells.[2,3] Great efforts have been directed at the design of
therapeutics that will inhibit metastasis and inflammation by interfering with
selectin-dependent adhesion using carbohydrate-based competitive ligands,[4]
or inhibit/decrease expression of sialyl Lewis X on the cell surface by blocking
the assembly of specific oligosaccharides. Exogenous decoy acceptors such as
peracetylated sugars were shown to efficiently cross the hydrophobic barriers
of the cell and Golgi membranes and to down-regulate the expression of sLex
ligands, resulting in suppression of intercellular adhesion in HL-60 leukemia
cells.[5–9] The decoys act as a substrate for the fucosyltransferase, thereby in-
terrupting the formation of the natural ligand.
Benzyl-N-acetylgalactosamine glycoside (GalNAcα-O-Bn) was used to de-
crease cell surface expression of Lewis blood group antigens, thus success-
fully inhibiting adhesion of the treated cells to activated endothelial cells
(Fig. 1).[10] This simple benzyl glycoside interfered with a very early step of
glycoprotein synthesis and consequently affected all cellular O-linked oligosac-
charide biosynthesis.[5,10,11] GalNAcα-O-Bn was found to prime the production
of O-linked oligosaccharides, similar to those typically found on mucins, inter-
rupting the incorporation of endogenous acceptors necessary for the synthe-
sis of the natural ligand. Interestingly, the nature of the aglycone was found
to greatly influence the amount and the structure of the oligosaccharide pro-
duced.[12–14]
In addition to monosaccharides, disaccharides can also act as primers for
O-linked oligosaccharides, since they closely resemble natural glycan inter-
mediates and can better target specific enzymes in biosynthetic pathways.[7]
However, due to their hydrophilic nature, disaccharides and larger oligosac-
charides are often poorly taken up by cells. The use of short-chain acyl
or acetoxymethyl esters to block the hydroxyl groups in a biologically re-
versible manner is often necessary to overcome the delivery problem.[15,16]
Intracellular carboxylesterases provide access to the deblocked nonacetylated
Figure 1: Examples of exogenous primers for O-linked Oligosaccharides production.

Synthesis of Peracetylated GlcNAcβ3Galβ-Aglycones
375
molecules, which in turn are used in the biosynthetic Golgi apparatus, enabling
peracetylated disaccharides such as Galβ1-4GlcNAcβ-O-naphthalenemethanol
(Galβ1-4GlcNAcβ-O-NM), GlcNAcβ1-3Galβ-O-NM, and Galβ1-3GalNAcα-O-
NM (Fig. 1) to prime oligosaccharide synthesis and to generate mucin-like
molecules.[8,17] As a result, these metabolic decoys, much like GalNAcα-O-Bn,
divert the assembly of the O-linked chains from endogenous glycoproteins and
inhibit the expression of the cell surface Lewis determinants, which are rec-
ognized by selectins.[7,17–20] A much lower dose of disaccharides (∼25 µM) can
be used to effect the same degree of Lewis X inhibition compared with the
monosaccharide primers (1 to 2 mM).
Despite the promise of this strategy, a full exploration of its therapeu-
tic potential is impeded by the serious hurdle of synthesizing a large col-
lection of decoys. Access to disaccharides such as Galβ1-4GlcNAcβ-O-NM or
GlcNAcβ1-3Galβ-O-NM requires several lengthy steps. Synthetic schemes that
are neither modular nor flexible leave room for improvement. Here we describe
a shorter and more flexible method to generate GlcNAcβ1-3Galβ-O-aglycone
derivatives. The new synthetic pathway will facilitate the synthesis of more
diverse collections of decoys.
RESULTS AND DISCUSSION
Generally, disaccharides such as Galβ1-4GlcNAcβ-O-NM or GlcNAcβ1-3Galβ-
O-NM are chemically synthesized[7,17,20] using protocols requiring multiple pro-
tecting group manipulations. Chemo-enzymatic syntheses, utilizing native or
engineered glycosyltransferases, represent an attractive alternative. Nowa-
days, purified bacterial glycosyltransferases are a reliable source of stable and
catalytically efficient enzymes that serve for the facile preparation of syn-
thetic derivatives.[21–23] Enzymatic glycosylations are highly stereo- and re-
gioselective[24] and provide the opportunity to generate saccharides coupled
to different aglycones when combined with the use of a traceless linker (3-
mercaptopropanol).[25] Here, we describe a combined enzymatic and chemical
approach to synthesize peracetylated GlcNAcβ1,3Galβ-aglycone decoys. The
method relies on a three-step protocol: (a) selection of an appropriate linker
group that is compatible with the enzymatic coupling step and allows for (b)
enzymatic coupling to produce 250 to 500 mg of a parent GlcNAcβ1,3Galβ-
linker disaccharide and (c) rapid chemical coupling of the parent disaccharide
with different aglycones.
Enzymatic Activity/Evaluation and Synthesis
Initially, several galactose derivatives were evaluated to determine
whether they were suitable for enzymatic glycosylation. Using aglycones 1–8
(Sch. 1), a variety of galactose derivatives carrying a 3-mercaptopropanol

N. Merbouh et al.
376
linker (12–20) were photochemically synthesized starting from tetraacetyl-β-1-
thio-galactopyranose (Sch. 2).[25] All the aglycones were chosen for their strong
UV or fluorescence activity to facilitate the subsequent reactions and purifi-
cations. The photochemical reaction was followed by the removal of the acetyl
groups using catalytic sodium methoxide in methanol, and the subsequent con-
tinuous extraction of the resulting sugar using dichloromethane from a water/
methanol mixture. The resulting sugars were purified by chromatography us-
ing silica gel and lyophilized as a precaution against sulfur oxidation, prior to
their use.
The resulting galactose derivatives (12–20) were subsequently screened
as acceptor substrates using bacterial β3-N-acetylglucosaminyltransferase
(β3GlcNAcT) to select the best candidate for a large-scale enzymatic
synthesis. β3GlcNAcT utilizes UDP-GlcNAc to transfer GlcNAc to galactose-
containing substrates. A crude bacterial lysate from Escherichia coli express-
ing the fusion protein was used since the recombinant form of the enzyme
Scheme 1: (i) 1–8, MeOH, hν; (ii) MeONa/MeOH.

Synthesis of Peracetylated GlcNAcβ3Galβ-Aglycones
377
Scheme 2: (i) β3GlcNAcT, UDP-[3H]GlcNAc, H2O, 37◦C.
is not available.[26] A single colony from an agar plate containing lgtA β3-N-
acetylglucosaminyltransferase (pWc:lgtA/AD202)[31] was inoculated into cul-
tures containing Y2T medium and the cell lysate was prepared as described by
Blixt et al.[21] The amount of glycosylated product formed with each galactose
acceptor was compared to the amount of product formed when either lactose
9 (10.97 nmol product), Galβ-p-nitrophenol 10 (7.86 nmol product), or Gal-β-
naphthalenemethanol 11 (4.33 nmol product) was used since these sugars are
all known acceptors for this enzyme.[26] Importantly, the crude cell lysate suf-
ficed for efficient production of these glycosides, thus obviating the need to
utilize the purified enzyme. These assays revealed that galactose derivative 19
(9.27 nmol product) acted as an excellent acceptor (Fig. 2). Compound 19 was
an ideal acceptor with good affinity for the enzyme and with good solubility in
aqueous and organic media, and it carried a coumarin chromophore, to aid the
separation and purification from the aqueous reaction mixture.
In order to produce a large amount of starting material 21, a large-scale
enzymatic reaction was required. The large-scale enzymatic glycosylation reac-
tion with 19 to produce GlcNAcβ3Galβ-S-coumarin 21 relied on the fusion pro-
tein. Disaccharide product 21 was readily purified from the enzyme mixture by

N. Merbouh et al.
378
Figure 2: Screening aglycone/linkers using a radioactive enzyme assay with β3GlcNAcT and
UDP-[3H]GlcNAc. The amount of radiolabeled disaccharide product formed
[3H]GlcNAcβ3Gal-O-R was measured (color figure available online).
centrifugation followed by C18 Sep Pak chromatography. Subsequently, disac-
charide 21 was chemically peracetylated using acetic anhydride in pyridine to
produce peracetylated disaccharide 22 in 80% yield after purification.[27] The
configuration of the C3-C1ꢁ newly formed bond was assessed using COSY and
HETCOR 2D-NMR to confirm the β-configuration with a coupling constant
ꢁ
ꢁ
JH1 -H2 of 7.9 Hz (Fig. 3).
Glycosylations
Compound 22 served as a glycosylating agent (parent molecule) to produce
four GlcNAcβ3Gal-aglycone disaccharides. Activation of the peractetylated
thioglycoside 22 using N-iodosuccinimide and silver triflate allowed for cou-
pling to benzyl alcohol, 1-naphthalenemethanol, 2-naphthaleneethanol, and
2-naphthalenemethanol, yielding compounds 23, 24, 25, and 26, respectively
(Sch. 3). Compound 26 was identical to a sample prepared according to the lit-
erature procedure and was used to prove the efficiency of the method for the
rapid synthesis of the decoys. [7]

Synthesis of Peracetylated GlcNAcβ3Galβ-Aglycones
379
Figure 3: Partial HETCOR of compound 22 used to determine the stereochemistry of the
newly formed glycosidic bond.
CONCLUSION
In conclusion, we have devised a method for the rapid production of peracety-
lated GlcNAcβ1,3Galβ-aglycone derivatives. A variety of derivatives can be
synthesized from one single parent molecule with good overall yields. The syn-
thesis combined a large-scale enzymatic glycosylation of a monosaccharide car-
rying a traceless linker, followed by acetylation of the product. Glycosylation of
the desired aglycone afforded the “decoy” ready for use. This method uses just a
single acetyl-protecting group in an overall sequence that requires three steps
and three purifications to afford the final compounds in short time and rather
high yield starting from readily available tetra-acetyl-β-1-thio-galactopyranose
and allyloxycoumarin.
EXPERIMENTAL
All solvents and reagents used were reagent grade or better and were used
as received. Analytical thin-layer chromatography was performed on Merck
silica gel 60 F254 plates (glass backed, 0.25 mm). Compounds were visualized
by dipping the plates in a cerium sulfate-ammonium molybdate solution or

N. Merbouh et al.
380
Scheme 3: (i) β3GlcNAcT, UDP-GlcNAc, H2O, 37◦C; (ii) Ac2O, pyridine, rt; (iii) alcohol, NIS,
AgOTf, DCM, 4 Å sieves, 0◦C.
10% H2SO4 in MeOH, followed by heating. Liquid column chromatography
was performed using forced nitrogen flow (unless stated otherwise) on silica
1
gel (standard grade, 60 A, 32–63 µm) provided by Fluka. H NMR and 13C
˚
NMR were recorded on a Varian Inova 300, a Bruker AC300F, or a Bruker
Avance 500, and were referenced to residual solvent peaks. High-resolution
FAB mass spectra were provided by the Mass Spectrometry Facility, Depart-
ment of Chemistry and Biochemistry, University of Notre Dame. The ESI/MS

Synthesis of Peracetylated GlcNAcβ3Galβ-Aglycones
381
spectra were recorded on a Waters Micromass ZQ. The microscale photochem-
ical reactor used was purchased from Ace Glass and equipped with a quartz
well. The mercury lamp (254 nm) with its power supply was purchased from
UVP.
Compounds 1, 9, and 10 were commercially available, while compounds
4,[25] 5,[25] 7,[28] 8,[29] 11,[7] and 12[30] were prepared according to pub-
lished procedures. 2-Naphthylmethyl 2-acetamido-3,4-6-tri-O-acetyl-2-deoxy-
β-D-glucopyranosyl-(1,3)-2,4,6-tri-O-acetyl-β-D-galactopyranoside 26 was used
as the control disaccharide in all biological assays and was identical to a sam-
ple prepared according to literature procedures.[7]
Allyl Ethers and Allyl Esters Preparation
1-(Allyloxymethyl)naphthalene (2)
To a solution of 1-naphthalenemethanol (1.58 g, 10 mmol) in DMF (30 mL)
was added NaH (0.5 g, 60% dispersion in paraffin, washed with hexane) at
rt, over 30 min. To the mixture, allylbromide (1.30 g, 10.7 mmol) was added,
and the mixture further stirred for 1 h. When TLC showed almost quanti-
tative conversion of the alcohol to the corresponding allyl ether, the reaction
was quenched by addition of water in small portions, and the solvent was re-
moved under vacuum. The resulting oil was partitioned between water (50 mL)
and DCM (50 mL), and the aqueous phase was further extracted with DCM
(2 × 50 mL). The organic phase was dried (Na2SO4) and the solvent removed
under vacuum. The resulting allylether was isolated by chromatography on
silica gel (hexanes/EtOAc, 4/1 as eluent) as an oil in about 60% yield. 1H
NMR (300 MHz, CDCl3) δ: 8.12 (dd, 1H, J = 1.5, 8.55 Hz), 7.90–7.79 (m, 2H),
7.56–7.40 (m, 4H), 6.05–5.90 (m, 1H), 5.33 (dq, 1H, J = 1.2, 10.3 Hz), 5.22 (dq,
1H, J = 1.5, 17.2 Hz), 4.96 (s, 2H), 4.10 (dt, 2H, J = 1.5, 5.7 Hz) ppm. 13C NMR
(75 MHz, CDCl3) δ: 135.0, 134.0, 132.0, 128.8, 128.7, 126.6, 126.4, 126.0, 125.4,
124.4, 117.5, 71.5, 70.8, 30.0 ppm. HR-MS (FAB+ of M+H, PEG): m/z calcd for
C14H14O: 198.1045, found: 198.1044.
1-(2-(Allyloxy)ethyl)naphthalene (3)
To a solution of 1-naphthaleneethanol (1.72 g, 10 mmol) in DMF (30 mL)
was added NaH (0.5 g, 60% dispersion in paraffin, previously washed with hex-
ane) at rt, over 30 min. To the mixture, allylbromide (1.30 g, 10.7 mmol) was
added, and the mixture further stirred for 1 h. After 1 h, TLC showed almost
quantitative conversion of the alcohol to the corresponding allyl ether. The re-
action was quenched by addition of water in small portion, and the solvent was
removed under vacuum. The resulting oil was partitioned between water (50
mL) and DCM (50 mL), and the aqueous phase further extracted with DCM

N. Merbouh et al.
382
(2 × 50 mL). The organic phase was dried over Na2SO4 and the solvent re-
moved under vacuum. The resulting allylether was isolated by chromatogra-
phy on silica gel (hexanes/EtOAc, 4/1 as eluent) as an oil with a 55% yield.
1H NMR (300 MHz, CDCl3) δ: 8.07 (d, 1H, J = 8.1 Hz), 7.87–7.71 (m, 2H),
7.55–7.35 (m, 4H), 6.00–5.86 (m, 1H), 5.33 (dq, 1H, J = 1.8, 17.1 Hz), 5.22
(dq, 1H, J = 1.8, 10.2 Hz), 4.01 (dt, 2H, J = 1.5, 5.7 Hz), 3.78 (t, 2H, J =
7.2 Hz), 3.39 (t, 2H, J = 7.3 Hz) ppm. 13C NMR (75 MHz, CDCl3) δ: 135.0, 134.0,
132.3, 129.0, 127.3, 127.0, 126.1, 125.8, 125.7, 124.0, 117.2, 72.2, 70.9, 33.6,
29.9 ppm. HR-MS (FAB+ of M+H, PEG): m/z calcd for C15H16O: 212.1201,
found: 212.1201.
2-(Allyloxy)ethyl 1-naphthoate (6)
To a solution of allyloxyethanol (2.04 g, 20 mmol) and pyridine (1.6 mL)
in DCM (50 mL), a solution of 1-naphthoyl chloride (1.90 g, 10 mmol) in DCM
(50 mL) was added dropwise, at rt, over 30 min. The mixture was stirred for
an additional hour until the TLC showed almost quantitative conversion of the
acyl chloride to the corresponding allyl ester. The reaction was quenched by ad-
dition of water in small portions, and the solvent was removed under vacuum.
The resulting oil was partitioned between water (50 mL) and DCM (50 mL),
and the aqueous phase further extracted with DCM (2 × 50 mL). The organic
phase was dried (Na2SO4) and the solvent removed under vacuum. The result-
ing allyl ester was isolated by chromatography on silica gel (hexanes/EtOAc,
3/2 as eluent) as an oil with a yield of 45%. 1H NMR (300 MHz, CDCl3) δ: 8.92
(dd, 1H, J = 1.2, 8.7 Hz), 8.21 (dd, 1H, J = 1.3, 8.2 Hz), 7.99 (d, 1H, J = 8.4
Hz), 7.86 (dd, 1H, J = 1.2, 8.4 Hz), 7.63–7.44 (m, 3H), 6.00–5.86 (m, 1H), 5.32
(dq, 1H, J = 1.5, 17.1 Hz), 5.20 (dq, 1H, J = 1.2, 10.6 Hz), 4.71 (m, 2H), 4.08
(dt, 2H, J = 1.5, 5.4 Hz), 3.82 (m, 2H) ppm. 13C NMR (75 MHz, CDCl3) δ: 167.8,
134.7, 134.0, 133.6, 131.6, 130.6, 128.7, 128.0, 127.3, 126.4, 126.0, 124.7, 117.5,
72.3, 68.2, 64.4 ppm. HR-MS (FAB+ of M+H, PEG): m/z calcd for C16H17O3:
257.1178, found: 257.1187.
Preparation of Galactose Derivatives 13–20
To a solution of tetra-acetyl-β-1-thio-galactopyranose (0.5 g, 1.38 mmol) in
MeOH (15 mL) was added the allylester or allylether derivative (1.2–2 equiv.),
and the solution was transferred into a photochemical cell. The solution was
flushed with N2 for 15 min and exposed to UV light (mercury lamp 254 nm)
through a quartz well. The solution was irradiated until the TLC (silica gel,
hexanes/EtOAc: 3/2) showed no remaining sugar; most reactions were com-
plete after 4 to 6 h, and longer irradiations only resulted in small yield changes.
The solution was then transferred into an Erlenmeyer flask (50 mL) and a
freshly prepared solution of sodium methoxide in MeOH was added to reach pH
11. The solution was stirred until the TLC showed complete deprotection of the

Synthesis of Peracetylated GlcNAcβ3Galβ-Aglycones
383
acetyl groups: only one spot on TLC (silica gel, hexanes/EtOAc: 3/2), with an
Rf of zero (for the sugar part); the deprotected compound can be visualized by
TLC (silica gel, EtOAc/MeOH: 8/1). The methanolic solution was then filtered
through a short plug of silica gel, and the solvent removed under vacuum. The
resulting solid/oil was dissolved in a minimum amount of water (8 mL max.
containing 1 to 2 mL of methanol) and charged onto a heavy solvent extractor,
and continuously extracted with DCM overnight. The DCM (containing at this
point only the desired sugar and the excess ether/ester) was subsequently re-
moved under vacuum and the residue purified by chromatography (silica gel,
EtOAc/MeOH: 8/1) to yield the desired compound as either a solid or oil. The
compounds were found to be stable, and were freeze-dried as a precaution.
All reported yields are based on the tetra-acetyl-β-1-thio-D-galactopyranose
amount used in the photochemical reaction, with only the pure fractions used
for the enzymatic assays.
3-(Naphthalen-1-yloxy)propyl-1-thio-β-D-galactopyranoside (13)
1
Isolated in 42% yield, H NMR (300 MHz, DMSO d6) δ: 8.19 (d, 1H, J =
6.9 Hz) 7.87 (d, 1H, J = 2.1 Hz) 7.63 (m, 4H) 6.97 (d, 1H, J = 7.3 Hz) 4.97
(d, 1H, J = 5.6 Hz) 4.79 (d, 1H, J = 5.5 Hz) 4.57 (t, 1H, J = 5.5 Hz) 4.41
(d, 1H, J = 4.4 Hz) 4.26 (m, 3H) 3.69 (s, 1H) 3.41 (m, 5H) 2.89 (m, 2H) 2.16
(t, 2H, J = 6.5 Hz) ppm. 13C NMR (75 MHz, DMSO d6) δ: 154.46, 134.48,
127.89, 126.88, 126.71, 125.74, 125.42, 122.03, 120.31, 105.62, 86.21, 79.61,
75.18, 70.26, 68.83, 66.92, 60.98, 29.89, 26.37 ppm. HR-MS (FAB+ of M+H,
PEG): m/z calcd for C19H24O6S: 380.1294, found: 380.1311.
3-(Naphthalen-1-ylmethoxy)propyl-1-thio-β-D-galactopyranoside (14)
1
Isolated in 47% yield, H NMR (300 MHz, DMSO d6) δ: 8.07 (d, 1H, J =
8.5 Hz), 7.93 (d, 1H, J = 8.0 Hz), 7.87 (d, 1H, J = 8.5 Hz), 7.56–7.44 (m, 4H),
4.93 (d, 1H, J = 6.0 Hz), 4.898 (s, 2H), 4.81 (d, 1H, J = 5.5 Hz), 4.60 (t, 1H, J =
5.5 Hz), 4.42 (d, 1H, J = 4.5 Hz), 4.19 (d, 1H, J = 9.5 Hz), 3.68 (d, 1H, J =
3.5 Hz), 3.59 (t, 2H, J = 6.0 Hz), 3.51–3.43 (m, 2H), 3.36–3.33 (m. 2H),
3.28–3.25 (m, 2H), 2.76–2.59 (m, 2H), 1.83 (quintet, 2H, J = 7.0 Hz) ppm. 13C
NMR (75 MHz, D2O) δ: 140.1, 139.5, 138.0, 135.6, 135.1, 134.3, 133.2, 132.8,
132.1, 130.5, 92.6, 85.2, 80.2, 77.3, 76.1, 75.2, 75.1, 67.4, 36.1, 33.4 ppm. HR-
MS (FAB+ of M+H, PEG): m/z calcd for C20H27O6S: 395.1528, found: 395.1526.
3-(2-(Naphthalen-1-yl)ethoxy)propyl-1-thio-β-D-galactopyranoside (15)
1
Isolated in 39% yield, H NMR (300 MHz, DMSO d6) δ: 8.02 (d, 1H, J =
8.5 Hz), 7.90 (d, 1H, J = 7.5 Hz), 7.77 (d, 1H, J = 7.5 Hz), 7.55–7.48 (m, 2H),
7.44–7.39 (m, 2H), 4.93 (d, 1H, J = 6.0 Hz), 4.79 (d, 1H, J = 5.5 Hz), 4.59 (t,
1H, J = 5.5 Hz), 4.40 (d, 1H, J = 4.5 Hz), 4.17 (d, 1H, J = 9.5 Hz), 3.67 (t, 2H, J
= 7.0 Hz), 3.49–3.35 (m, 6H, under solvent peak), 3.34–3.36 (m. 4H), 2.66–2.54
(m, 2H), 1.74 (quintet, 2H, J = 7.0 Hz) ppm. 13C NMR (75 MHz, D2O) δ: 135.0,

N. Merbouh et al.
384
133.6, 131.6, 128.7, 127.2, 127.0, 126.2, 125.8 (2C), 123.8, 86.5, 78.7, 73.9, 70.4,
69.7, 68.8, 68.7, 60.9, 32.3, 29.5, 27.1 ppm. HR-MS (FAB+ of M+H, PEG): m/z
calcd for C21H29O6S: 409.1685, found: 409.1663.
3-(2-(2-(Naphthalen-1-yl)ethoxy)ethoxy)propyl-1-thio-β-D-
galactopyranoside (16)
1
Isolated in 45% yield, H NMR (300 MHz, DMSO d6) δ: 7.89 (m, 4H), 7.50
(m, 3H), 4.92 (d, 1H, J = 5.4 Hz), 4.77 (d, 1H, J = 5.4 Hz), 4.67 (s, 2H), 4.55
(t, 1H, J = 5.5 Hz), 4.39 (d, 1H, J = 4.3 Hz), 4.20 (d, 1H, J = 9.2 Hz), 3.47
(m, 12H), 2.66 (m, 2H), 1.79 (m, 2H) ppm. 13C NMR (75 MHz, DMSO d6) δ:
136.63, 133.27, 132.88, 128.29, 128.18, 128.02, 126.63, 126.29, 86.26, 79.59,
75.17, 72.55, 70.25, 69.96, 69.64, 69.48, 68.83, 60.99, 40.83, 39.16, 30.26, 26.38
ppm. HR-MS (FAB+ of M+H, PEG): m/z calcd for C22H31O7S: 439.1791, found:
439.1768.
3-Propyl-1-naphtoate-1-thio-β-D-galactopyranoside (17)
1
Isolated in 50% yield, H NMR (300 MHz, DMSO d6) δ: 8.76 (d, 1H, J =
8.4 Hz), 8.18 (t, 2H, J = 8.5 Hz), 8.04 (d, 1H, J = 7.9 Hz), 7.64 (m, 3H), 4.99
(d, 1H, J = 5.5 Hz), 4.82 (d, 1H, J = 5.3 Hz), 4.57 (t, 1H, J = 5.5 Hz), 4.45 (dd,
3H, J = 12.2, 6.2 Hz), 4.28 (d, 1H, J = 9.3 Hz), 3.71 (t, 1H, J = 3.6 Hz), 3.42
(m, 5H), 2.82 (m, 2H), 2.08 (t, 2H, J = 6.7 Hz) ppm. 13C NMR (75 MHz, DMSO
d6) δ: 166.85, 133.43, 133.30, 129.96, 128.73, 127.88, 126.83, 126.39, 125.09,
124.97, 85.73, 79.17, 74.72, 69.78, 68.39, 63.78, 60.54, 28.82, 25.82 ppm. HR-
MS (FAB+ of M+H, PEG): m/z calcd for C20H25O7S: 409.1321, found: 409.1332.
3-(2-Propoxyethyl)-1-naphthoate-1-thio-β-D-galactopyranoside (18)
1
Isolated in 40% yield, H NMR (300 MHz, DMSO d6) δ: 8.73 (d, 1H, J =
8.5 Hz), 8.18 (d, 1H, J = 8.5 Hz), 8.12 (d, 1H, J = 6.0 Hz), 8.03 (d, 1H, J =
8.0 Hz), 7.67–7.58 (m, 2H), 4.93 (d, 1H, J = 5.5 Hz), 4.80 (d, 1H, J = 5.5 Hz),
4.54 (t, 1H, J = 5.5 Hz), 4.48 (m, 2H), 4.39 (d, 1H, J = 4.5 Hz), 4.18 (d, 1H,
J = 9.5 Hz), 3.75 (m, 2H), 3.66 (t, 1H, J = 3.5 Hz) 3.56 (t. 2H, J = 6.5 Hz),
3.50–3.40 (m, 2H), 3.38–3.314 (m, 2H), 3.27–3.23 (m, 1H), 2.72–2.59 (m, 2H),
1.81 (quintet, 2H, J = 6.5 Hz) ppm. 13C NMR (75 MHz, D2O) δ: 169.8, 134.9
(2C), 132.1, 131.8, 130.0, 129.3, 128.0, 127.7, 126.7, 126.1, 87.8, 80.2, 75.6, 71.3,
70.9, 70.2, 69.7, 65.7, 62.4, 31.0, 28.5 ppm. HR-MS (FAB+ of M+H, PEG): m/z
calcd for C22H29O8S: 453.1583, found: 453.1568.
3-(7-Propoxy)-2H-chromen-2-one-1-thio-β-D-galactopyranoside (19)
1
Isolated in 40% yield, H NMR (300 MHz, DMSO d6) δ: 7.99 (d, 1H, J =
9.5 Hz), 7.63 (d, 1H, J = 8.4 Hz), 6.97 (m, 2H), 6.29 (d, 1H, J = 9.5 Hz), 4.96
(d, 2H, J = 5.5 Hz), 4.79 (d, 1H, J = 5.4 Hz), 4.56 (t, 1H, J = 5.5 Hz), 4.40 (d,
1H, J = 4.4 Hz), 4.23 (d, 1H, J = 9.2 Hz), 4.17 (t, 2H, J = 6.2 Hz), 3.68 (m, 1H),
3.46 (m, 1H), 3.29 (m, 3H), 2.77 (m, 2H), 2.03 (m, 2H) ppm. 13C NMR (75 MHz,

Synthesis of Peracetylated GlcNAcβ3Galβ-Aglycones
385
DMSO d6) δ: 162.22, 160.78, 155.87, 144.82, 129.98, 113.15, 112.92, 112.81,
101.71, 86.22, 79.64, 75.15, 70.21, 68.84, 67.46, 61.00, 29.56, 26.05 ppm. HR-
MS (FAB+ of M+H, PEG): m/z calcd for C18H23O8S: 399.1114, found: 399.1111.
3-(2-(3-Oxo-6-propoxy-3H-xanthen-9-yl)benzoic acid-1-thio-β-D-
galactopyranoside (20)
Isolated in 22% yield, 1H NMR (300 MHz, DMSO d6) δ: 10.13 (broad s, 1H),
8.01 (d, 1H, J = 7.5 Hz), 7.76 (m, 2H), 7.28 (d, 1H, J = 7.8 Hz), 6.93 (d, 1H, J =
2.4 Hz), 6.66 (m, 4H), 4.95 (d, 1H, J = 5.7 Hz), 4.79 (d, 1H, J = 5.4 Hz), 4.56
(t, 1H, J = 5.4 Hz), 4.39 (d, 1H, J = 4.5 Hz), 4.22 (d, 1H, J = 9.3 Hz), 4.13 (t,
2H, J = 6.0 Hz), 3.67 (m, 1H), 3.47 (m, 1H), 3.32 (m, 5H), 2.74 (m, 2H), 2.03
(t, 2H, J = 6.6 Hz) ppm. 13C NMR (75 MHz, DMSO d6) δ: 168.6, 160.3, 152.5,
151.8, 151.7, 135.6, 130.1, 129.1, 128.9, 126.0, 124.6, 124.0, 112.2, 110.9, 101.3,
85.7, 79.1, 74.6, 69.7, 68.3, 66.7, 60.5, 29.1, 25.6 ppm. HR-MS (FAB+ of M+H,
PEG): m/z calcd for C29H29O10S: 569.1481, found: 569.1469.
Radioactive N-Acetylglucosaminyltransferase Assays to Screen
the Galactose Derivatives
The assays were conducted in a final volume of 100 µL at 37◦C for 1 h,
containing the different acceptor substrates 9–20 (10 mM) and donor sub-
strate UDP[6-3H]GlcNAc (2 mM, 1086 cpm/nmol, NEN Life Science Prod-
ucts) in sodium cacodylate (50 mM, pH 7.5), MnCl2 (10 mM), and bovine
serum albumin (1%). Reactions were initiated by adding crude lysate (10 µL,
∼0.5 mU) diluted in buffer containing bovine serum albumin (1%). Trans-
fer of GlcNAc to the various galactose acceptor substrates using N-
acetylglucosaminyltranferase is expressed in nmol of disaccharide product
formed. Each reaction product was diluted with 0.5 mL of 0.5M NaCl and ap-
plied to a Sep-Pak C18 column (100 mg, Waters). After washing with 25 mL
of water, the disaccharide product is eluted with 50% methanol, dried, and
counted by scintillation counting.
Preparation of Compound 21
Scale-up of the enzymatic reaction was carried out in several steps and
any solubility problems were resolved by adding 10% ethanol. The enzyme re-
actions were performed first on a small scale (∼10 mg) and then on a larger
scale (∼1 g). Reactions contained the acceptor substrate (19) (20 mmol, 40 mM)
and UDP-GlcNAc (20 mmol, 40 mM) in sodium cacodylate buffer (125 mL, 100
mM, pH 7.5) with MnCl2 (20 mM) and bovine serum albumin (0.1%). The re-
actions were initiated by adding the crude enzyme lysate (10 U, based on the
N-acetylglucosaminyltranferase activity). The reaction was stirred at rt for 3
to 5 days. The reactions were monitored by TLC using EtOAC/MeOH: 8/1 as

N. Merbouh et al.
386
eluent, as well as by mass spectrometry. At the end of the reaction, the disac-
charide was centrifuged, the supernatant was applied to a C18 Sep Pak (20g,
prepacked), the column was washed with several portions of water, and the
desired disaccharide was eluted using pure methanol. The elution of the com-
pound can readily be followed by the use of a UV lamp (long wave), as the
coumarin moiety tends to fluoresce. This enzymatic step typically gave an over-
all yield of ≥45% (based on the monosaccharide starting material). Although
on large-scale reactions it is known that UDP inhibits the enzyme-catalyzed
reactions,[26] it was difficult to circumvent. Finally, the disaccharide was per-
acetylated and purified to afford compound 22 in 82% yield, 1H NMR (300 MHz,
CDCl3) δ: 7.64 (d, 1H, J = 9.1 Hz), 7.37 (d, 1H, J = 7.8 Hz), 6.84 (s, 1H), 6.83
(d, 1H, J = 7.8), 6.26 (d, 1H, J = 9.1 Hz), 5.54 (d, 1H, J = 4.6 Hz), 5.41 (d,
1H, J = 3.2 Hz), 5.21 (t, 1H, J = 9.3 Hz), 5.11 (d, 1H, J = 7.9 Hz), 5.04 (t,
1H, J = 9.5 Hz), 4.39 (dd, 2H, J = 17.2, 10.2 Hz), 4.34 (dd, 1H, J = 12.0, 2.3
Hz), 4.13–4.01 (m, 7H), 3.86 (dd, 1H, J = 9.3, 3.2 Hz), 3.83 (t, 1H, J = 6.5 Hz),
3.68 (dt, 1H, J = 9.3, 3.2 Hz), 3.31 (dt, 1H, J = 0.2, 8.3 Hz), 2.93 (m, 1H), 2.80
(m, 1H), 2.15 (s, 3H), 2.10 (s, 6H), 2.05 (s, 3H), 2.02 (s, 6H), 1.9 (s, 3H) ppm.
13C NMR (75 MHz, CDCl3) δ: 170.8, 170.6, 170.5, 169.8, 162.1, 161.2, 155.9,
143.5, 128.8, 113.2, 112.8, 112.6, 101.5, 99.7, 83.9, 75.2, 71.6, 71.1, 69.4, 68.8,
68.7, 66.7, 62.2, 61.3, 56.3, 29.2, 26.4, 23.3, 21.1, 20.8, 20.8, 20.7 ppm. HR-MS
(FAB+ of M+H, PEG): m/z calcd for C38H48O19NS: 854.2541, found: 854.2546.
General Procedure for the Glycosylation of Compound 22
The glycosylation agent and the nucleophile were dried by coevaporation
with toluene (3 × 5 mL) and then kept under vacuum for 2 h. A solution of al-
˚
cohol (1 equiv.), thioglycoside (1.2 equiv.), and powdered molecular sieves (4 A,
50 to 100 mg) in anhydrous DCM (5 to 10 mL) was cooled to 0◦C and then
N-iodosuccinimide (3 equiv.) and silver triflate (0.3 equiv.) were added. After
stirring for 15 to 20 min at 0◦C, Et3N (0.05 mL) was added and the reaction
mixture was diluted with DCM (10 mL) and filtered through Celite. The filtrate
was washed successively with a saturated aqueous solution of Na2S2O3, wa-
ter, and brine. After drying (Na2SO4), the organic phase was filtered and con-
centrated and the residue was purified by chromatography (hexanes/EtOAc,
1:1→1:2).
Benzyl 3-(3,4,6-O-acetyl-2-acetamide-2-deoxy-β-glucosyl)-2,4,6-O-acetyl-β-
galactoside (23)
Compound 14 (45.1 mg, 52.8 mmol) was dried under vacuum for 3 h. Acti-
˚
vated molecular sieves (MS, 4A, powdered, 44 mg) were added, followed by
CH2Cl2 (5 mL) and benzyl alcohol (4.6 mL, 45 mmol). The resulting mix-
ture was cooled to 0◦C and N-iodosuccinimide (NIS, 31.6 mg, 0.14 mmol) and
silver(I) trifluoromethanesulphonate (AgOTf, 5.1 mg, 20 mmol) were added.

Synthesis of Peracetylated GlcNAcβ3Galβ-Aglycones
387
Within 20 min, the mixture turned red. The mixture was stirred at 0◦C for
1 h and the reaction was quenched with triethylamine (50 mL). The mix-
ture was diluted with CH2Cl2 and filtered through celite, which was subse-
quently washed with CH2Cl2. The combined filtrates were washed with sat.
Na2S2O3, water, and brine (20 mL each); dried; and concentrated to give 41
mg crude product. Flash chromatography (hexanes/EtOAc 1:3) yielded com-
pound 15 (7.3 mg, 23%). 1H NMR (500 MHz, CDCl3) δ: 7.87–7.28 (m, 5H), 5.52
(dd, 1H, J = 10.4, 9.0 Hz), 5.46 (d, 1H, J = 7.7 Hz), 5.39 (d, 1H, J = 3.5 Hz),
5.21 (dd, 1H, J = 9.8, 8.0 Hz), 5.07–5.02 (2H, m), 4.90 (d, 1H, J = 12.4 Hz),
4.64 (d, 1H, J = 12.4 Hz), 4.42 (d, 1H, J = 8.0 Hz), 4.32 (dd, 1H, J = 12.2,
2.5 Hz), 4.20–4.08 (m, 3H), 3.84–3.79 (m, 2H), 3.66 (dt, 1H, J = 10.0, 3.3 Hz),
3.32 (dt, 1H, J = 10.5, 7.9 Hz), 2.15 (s, 3H), 2.12 (s, 6H), 2.07 (s, 3H), 2.03
(s, 3H), 1.93 (s, 3H) ppm. 13C NMR (125 MHz, CDCl3) δ: 170.9, 170.8, 170.7,
170.6, 170.0, 169.8, 169.7, 137.0, 128.6, 128.2, 128.0, 99.8, 99.6, 76.3, 71.8, 71.5,
71.3, 71.0, 70.6, 69.4, 69.0, 62.3, 61.5, 56.4, 23.5, 21.2, 21.0, 20.9, 20.9 ppm.
HR-MS (FAB+ of M+H, PEG): m/z calcd for C33H44O17N: 726.2609, found:
726.2592.
1-Naphtalenemethyl 3-(3,4,6-O-acetyl-2-acetamide-2-deoxy-β-glucosyl)-
2,4,6-O-acetyl-β-galactoside (24)
Compound 14 (51.5 mg, 60.3 mmol) and 1-naphtalenemethanol (19.1 mg,
121 mmol) were evaporated from toluene thrice, NIS (44.7 mg, 0.2 mmol)
was added, and the residue was protected from light, dried under vacuum
˚
overnight, and dissolved in CH2Cl2 (5 mL). MS (4A, powdered) was added and
the mixture was cooled to 0◦C. AgOTf (36 mL, 0.51 M in toluene, 18 mmol) was
added and the reaction was stirred at 0◦C to 12◦C for 1.5 h. Triethylamine (50
mL) was added and the mixture was diluted with CH2Cl2 (10 mL) and filtered
through celite, which was subsequently washed with CH2Cl2 (10 mL). The com-
bined filtrates were washed with sat. Na2S2O3, water, and brine (25 mL each);
dried; and concentrated to give 76 mg crude product. Flash chromatography
1
(hexanes/EtOAc 1:3 to 0:1) yielded the title compound 16 (17 mg, 36%). H
NMR (500 MHz, CDCl3) δ: 8.07 (dd, 1H, J = 9.0, 1.9 Hz), 7.86 (m, 2H), 7.51 (m,
2H), 7.42 (dd, 1H, J = 7.8, 4.1 Hz), 7.41 (s, 1H), 5.45–5.33 (m, 3H), 5.14 (dd,
1H, J = 10.1, 8.1 Hz), 4.98 (m, 1H), 4.92 (d, 1H, J = 8.1 Hz), 4.34 (d, 1H, J =
8.0 Hz), 4.26 (dd, 1H, J = 12.2, 2.6 Hz), 4.18, (m, 2H), 4.03 (dd, 1H, J = 12.2,
3.9 Hz), 3.75 (t, 1H, J = 6.4 Hz), 3.70 (dd, 1H, J = 9.9, 3.5 Hz), 3.57 (dt, 1H, J
= 6.4, 2.9 Hz), 3.26 (dt, 1H, J = 10.4, 7.9 Hz), 2.13–1.95 (m, 13H), 1.84 (m, 2H)
ppm. 13C NMR (125 MHz, CDCl3) δ: 170.9, 170.8, 170.6, 170.0, 169.7, 169.6,
134.0, 132.1, 132.1, 129.5, 128.8, 127.8, 126.6, 126.2, 125.2, 124.4, 99.8, 98.6,
76.2, 71.8, 71.5, 71.3, 70.9, 69.5, 62.2, 61.5, 56.3, 21.0, 20.9, 20.9, 20.8 ppm.
HR-MS (FAB+ of M+H, PEG): m/z calcd for C37H46O17N: 776.2766, found:
776.2781.

N. Merbouh et al.
388
2-Naphtaleneethyl 3-(3,4,6-O-acetyl-2-acetamide-2-deoxy-β-glucosyl)-
2,4,6-O-acetyl-β-galactoside (25)
Compound 14 (51.3 mg, 60.1 mmol) and 2-naphthaleneethanol (20.9 mg,
121 mmol) were evaporated from toluene thrice. NIS (43.9 mg, 0.2 mmol) was
added and the residue was protected from light, dried under vacuum overnight,
˚
and dissolved in anhydrous CH2Cl2 (5 mL). Powdered molecular sieves (4 A)
were added and the mixture was cooled to 0◦C. AgOTf (36 mL, 0.51 M in
toluene, 18 mmol) was added and the reaction was stirred at 0◦C to 12◦C for
1.5 h. Triethylamine (50 mL) was added and the mixture was diluted with
CH2Cl2 (10 mL) and filtered through celite, and was subsequently washed with
CH2Cl2 (10 mL). The combined filtrates were washed with sat. Na2S2O3, water,
and brine (25 mL each); dried; and concentrated to give 79 mg crude product.
Flash chromatography (hexanes/EtOAc 1:3) yielded 17 (23 mg, 48%). 1H NMR
(500 MHz, CDCl3) δ: 7.81 (t, 2H, J = 7.4 Hz), 7.78 (d, 1H, J = 8.5 Hz), 7.65 (s,
1H), 7.45 (ddd, 2H, J = 8.0, 6.5, 1.9 Hz), 5.48 (dd, 1H, J = 10.5, 9.3 Hz), 5.44 (d,
1H, J = 7.8 Hz), 5.38 (d, 1H, J = 3.3 Hz), 5.15 (dd, 1H, J = 10.0, 8.1 Hz), 5.04 (t,
1H, J = 9.7 Hz), 5.02 (d, 1H, J = 8.1 Hz), 4.38 (d, 1H, J = 8.1 Hz), 4.32 (dd, 1H,
J = 12.2, 2.5 Hz), 4.24 (dt, 1H, J = 9.7, 5.3 Hz), 4.18–4.08 (m, 3H), 3.83–3.79
(m, 2H), 3.75 (dd, 1H, J = 9.3, 7.3 Hz), 3.66 (dt, 1H, J = 9.2, 3.6 Hz), 3.32
(dt, 1H, J = 10.5, 7.7 Hz), 3.12–3.02 (m, 2H), 2.14 (s, 3H), 2.11 (s, 3H), 2.09 (s,
3H), 2.02 (s, 6H), 1.90 (s, 3H), 1.73 (s, 3H) ppm. 13C NMR (125 MHz, CDCl3) δ:
170.9, 170.8, 170.6, 170.6, 170.0, 169.7, 169.7, 136.3, 133.7, 132.3, 128.0, 127.7,
127.7, 127.7, 127.5, 126.2, 125.6, 101.3, 99.8, 76.3, 71.8, 71.4, 71.4, 70.9, 70.6,
69.4, 69.0, 62.3, 61.5, 56.3, 36.3, 23.4, 21.0, 20.9, 20.8 ppm. HR-MS (FAB+ of
M+H, PEG): m/z calcd for C38H48O17N: 790.2922, found: 790.2949.
ACKNOWLEDGEMENTS
P.H.S. gratefully acknowledges financial support from NIH (5 R01 HL64799-
06 and 5 P01 CA071932-10). J.D.E. gratefully acknowledges financial support
from NIH (CA112278). We thank Dr. V. Mountain for critically editing this
manuscript. We would like to thank Dr. Warren Wakarchuk and Dr James
Paulson for the kind gift of lgtA β-3-N-acetylglucosaminyltransferase.
N. Merbouh and J. R. Brown contributed equally to the work and should
be considered as first coauthors.
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