Synthesis of N -acetyl glucosamine analogs as inhibitors for hyaluronan biosynthesis

Article abstract of DOI:10.1080/07328303.2013.815196

Elevated hyaluronan expression is a hallmark of many types of cancer. Therefore, inhibition of hyaluronan biosynthesis can potentially slow the growth of tumor cells. Herein, we explore a chain termination strategy to reduce hyaluronan synthesis by tumor cells. Several analogs of glucosamine were prepared, which contained modifications at the C-3 positions. These analogs can possibly cap the nonreducing end of a growing hyaluronan chain, thus lowering the amount of hyaluronan synthesized. Upon incubation with pancreatic cancer cells, a fluorine-containing glucosamine analog was found to exhibit significant inhibitory activities of hyaluronan synthesis. Furthermore, it drastically reduced the proliferation of cancer cells. Supplemental materials are available for this article. Go to the publisher's online edition of Journal of Carbohydrate Chemistry to view the supplemental file.

Full text of DOI:10.1080/07328303.2013.815196

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Synthesis of N-acetyl Glucosamine
Analogs as Inhibitors for Hyaluronan
Biosynthesis
Gilbert Wasonga a , Yota Tatara b , Ikuko Kakizaki b & Xuefei Huang a
a Department of Chemistry , Michigan State University , East
Lansing , MI , 48824 , USA
b Department of Glycotechnology, Center for Advanced Medical
Research , Hirosaki University Graduate School of Medicine ,
Hirosaki , 036-8224 , Japan
Published online: 02 Sep 2013.
To cite this article: Gilbert Wasonga , Yota Tatara , Ikuko Kakizaki & Xuefei Huang (2013) Synthesis
of N-acetyl Glucosamine Analogs as Inhibitors for Hyaluronan Biosynthesis, Journal of Carbohydrate
Chemistry, 32:5-6, 392-409
To link to this article: http://dx.doi.org/10.1080/07328303.2013.815196
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Journal of Carbohydrate Chemistry, 32:392–409, 2013
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Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2013.815196
Synthesis of N-acetyl
Glucosamine Analogs as
Inhibitors for Hyaluronan
Biosynthesis
Gilbert Wasonga,1 Yota Tatara,2 Ikuko Kakizaki,2
and Xuefei Huang1
1Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA
2Department of Glycotechnology, Center for Advanced Medical Research, Hirosaki
University Graduate School of Medicine, Hirosaki 036-8224, Japan
Elevated hyaluronan expression is a hallmark of many types of cancer. Therefore, in-
hibition of hyaluronan biosynthesis can potentially slow the growth of tumor cells.
Herein, we explore a chain termination strategy to reduce hyaluronan synthesis by
tumor cells. Several analogs of glucosamine were prepared, which contained modifica-
tions at the C-3 positions. These analogs can possibly cap the nonreducing end of a
growing hyaluronan chain, thus lowering the amount of hyaluronan synthesized. Upon
incubation with pancreatic cancer cells, a fluorine-containing glucosamine analog was
found to exhibit significant inhibitory activities of hyaluronan synthesis. Furthermore,
it drastically reduced the proliferation of cancer cells.
Supplemental materials are available for this article. Go to the publisher’s online
edition of Journal of Carbohydrate Chemistry to view the supplemental file.
Keywords Biosynthesis; Chain terminator; Hyaluronan; Inhibitor; Salvage pathway
INTRODUCTION
Hyaluronan (HA), a member of the glycosaminoglycan family, is composed
of repeating disaccharides of β-1,3-N-acetyl-glucosamine-β-1,4-glucuronic
acid.[1] HA plays important roles in tumor development, metastasis, and drug
resistance.[2–4] HA expression is elevated in a variety of tumors,[5] with the
high levels of HA shown to have prognostic values for various cancers, such
Received April 23, 2013; accepted June 11, 2013.
Address correspondence to Xuefei Huang, Department of Chemistry, Chemistry Build-
ing, Room 426, 578 S. Shaw Lane, Michigan State University, East Lansing, Michigan,
48824 USA. E-mail: xuefei@chemistry.msu.edu
392

Synthesis of N-acetyl Glucosamine Analogs
393
as non–small cell lung, bladder, gastric, and prostate cancer.[6–9] Therefore,
inhibition of HA biosynthesis can be an interesting approach toward cancer
treatment.
HA biosynthesis is mediated by HA synthases,[10] which use UDP-α-N-
acetyl-D-glucosamine (UDP-GlcNAc) and UDP-α-D-glucuronate (UDP-GlcA) as
substrates to sequentially transfer GlcNAc and GlcA to the nonreducing end
of a growing HA chain. 4-Methylumbelliferone (4-MU) was found to be an HA
synthesis inhibitor,[11] which functions by suppressing the expression of HA
synthases.[12] 4-MU can also serve as an acceptor for UDP-GlcA, thus compet-
ing for the limited intracellular pool of UDP-GlcA and further reducing the
amount of HA produced.[13,14] 4-MU can reduce proliferation, motility, inva-
sion, and metastatic properties of various types of cancer including prostate
cancer and breast cancer.[12,15–18] These observations have demonstrated the
utility of HA biosynthesis inhibitors as potential antitumor agents.
Herein, we report an alternative approach to inhibit HA biosynthesis
through potential early chain termination. We envision that if a modified Glc-
NAc analog such as 1–3 is incorporated into an HA chain, as the C-3 position
does not bear a free hydroxyl moiety, HA synthesis will be terminated, lead-
ing to a reduction of the amount of HA synthesized. The key to the success
of this strategy is the incorporation of the potential chain terminators (e.g.,
1–3) into the growing HA chain. The GlcNAc donor (i.e., UDP-GlcNAc) is syn-
thesized inside the cells primarily from glucose through a series of enzymatic
reactions.[19] However, there exists a salvage pathway, which utilizes exoge-
nous GlcNAc as the source for UDP-GlcNAc.[20] The salvage pathway has a
broad substrate scope and is well tolerant of GlcNAc structural modifications,
which can allow the transformation of a variety of GlcNAc analogs into UDP
glycosyl donors.[21] The HA synthase can potentially utilize the UDP-GlcNAc
analog as a glycosyl donor and incorporate the modified GlcNAc into the HA
chain. The chain termination strategy has been applied to inhibition of biosyn-
thesis of a variety of glycans[22] including sialyl Lewis X, β-glucan, heparan
sulfate, and chitin.[23–28] Herein, we report the chemical synthesis of GlcNAc
analogs and investigate their abilities to inhibit HA synthesis by cancer cells.
A fluorine-containing GlcNAc analog showed high inhibitory activities toward
HA synthesis and cancer cellproliferation.
Figure 1: Structures of compounds 1–3 (color figure available online).

G. Wasonga et al.
394
RESULTS AND DISCUSSION
Our synthetic targets are the 3-OMe, 3-oxo, and 3-F[29] analogs of GlcNAc 4–6.
The hydroxyl groups of these analogs are protected as acetates to reduce their
hydrophilicity and increase their potential to cross cell membranes during
cellular incubation. Acetate groups have been shown to significantly enhance
intracellular concentrations of exogenous carbohydrates added to mammalian
cells, which can then be deacetylated in situ by cytosolic esterases.[30]
Figure 2: Structures of compounds 4–6 (color figure available online).
In our initial route for the synthesis of these GlcNAc derivatives, we opted
to use furanose oxazoline 8 as a key intermediate since it contains a free 3-
OH moiety. Compound 8 was conveniently prepared in large quantities from
commercially available GlcNAc 7 using anhydrous FeCl3 as a catalyst in ace-
tone.[31] With oxazoline 8 in hand, methylation of its free 3-OH group by MeI
and NaH gave compound 9 in quantitative yield. Subsequent acid-catalyzed
hydrolysis of compound 9 with p-toluene sulfonic acid (PTSA) furnished the de-
sired 3-O-methyl-N-acetyl-D-glucosamine 1, which was acetylated using acetic
anhydride in pyridine to give the product 4 in 50% overall yield over the two
steps (Sch. 1).
Scheme 1: Reagents and Conditions: i) Acetone/2 eq FeCl3, reflux, 60◦C, 65%; ii) 2 eq NaH,
1.5 eq Mel, DMF, 0◦C to rt, 96%; iii) 0.4 eq PTSA, THF/H2O 2:1; iv) Ac2O/Pyridine, rt, 50%.
Next we explored the synthesis of the 3-oxo GlcNAc derivative 5. Swern
oxidation of 8 produced the ketone 10 in 92% yield (Sch. 2a). However, acid-
catalyzed deprotection of 10 in a similar manner, as in the synthesis of com-
pound 4, failed to provide the desired product 2. One possible explanation was
that compound 2 was unstable under the acidic reaction condition. Based on
this consideration, we designed an alternative route to avoid the acidic condi-
tion.
GlcNAc 7 was selectively benzylated at the anomeric position using con-
centrated HCl leading to compound 11,[28] which was followed by benzylidena-
tion of the 4,6-hydroxyl groups generating compound 12[32] in an excellent 95%

Synthesis of N-acetyl Glucosamine Analogs
395
Scheme 2: Reagents and Conditions: i) 1.4 eq COCl2, 2.5 eq DMSO, 5 eq Et3N, −75◦C to rt,
92%; ii) 0.4 eq PTSA, THF/H2O 2:1; iii) BnOH, HCl, 90◦C, 71%, iv) 5 eq PhCH(OMe)2, 0.6 eq
camphorsulfonic acid, DMF, rt, 95%, v) 1.4 eq COCl2, 2.5 eq DMSO, 5 eq Et3N, −75◦C to rt,
90%; vi) H2, Pd/C, MeOH.
yield (Sch. 2b). Subsequent Swern oxidation of 12 produced 3-uloside deriva-
tive 13 in 90% yield. However, when subjected to the mild catalytic hydro-
genation condition to remove the benzyl and benzylidene groups, compound 13
was transformed to multiple species. One of the major side products isolated
was ketone 14, which was presumably formed due to a retro-Claisen reaction
through nucleophilic attack of methanol on 2 (Sch. 2c). Therefore, compound 2
would most likely be unstable in a nucleophilic solvent such as water and was
not pursued further.
We shifted our attention to the preparation of the 3F-GlcNAc derivative 6.
The first route explored was the substitution of the free 3-OH in oxazoline 8 by
a fluorine atom. However, treatment of 8 with diethylaminosulfur trifluoride
(DAST) gave a complex product mixture. Exchanging 8 in this reaction with
compound 12 or the corresponding epimers containing axial 3-OH did not yield
any desired fluorine substitution product. This was consistent with a literature
report where the yield for fluorine substitution onto C3 of a GlcNAc analog was
only 10%.[29]
Kajihara and coworkers described that 6-OH of a glucosamine analog could
be substituted with fluorine with a modest yield (∼38%) when phthalimide
(Phth) was used as the protective group for the nitrogen.[33] To test the effect
of Phth on our fluorination reaction, we started from the Phth-containing glu-
cosamine tetraacetate 15,[34,35] which was efficiently converted to β-benzyl gly-
coside 16[36] using BF3.Et2O in CH2Cl2 (Sch. 3). Subsequent acetate removal
with 0.3 M NaOMe in a mixture of MeOH-CH2Cl2 at −10◦C to 0◦C produced

G. Wasonga et al.
396
Scheme 3: Reagents and Conditions: i) 2 eq BnOH, 3 eq BF3OEt2, CH2Cl2, 79%; ii) 0.3 M
NaOMe, MeOH/CH2Cl2, −10◦C; iii) PhCH(OMe)2, 0.08 eq camphorsulfonic acid, CH3CN,
77%; iv) 2 eq Tf2O, 5 eq pyridine, −20◦C to 10◦C; 6 eq tetrabutylammonium acetate,
toluene, 60◦C%; v) 6 eq DAST, CH2Cl2, −5◦C to rt; vi) 6 eq AcCl, MeOH/CH2Cl2, 28% yield
from 18; vii) H2N(CH2)2NH2, nBuOH, 90◦C; acetic anhydride,pyridine rt, 80%; viii) H2, Pd/C,
MeOH; acetic anhydride, pyridine, rt, 60%.
triol 17 in 77% yield. The low reaction temperature was important to ensure
the phthalimide ring was not opened by NaOMe. The O-4 and O-6 free hydroxyl
groups in 17 were protected as a benzylidene acetal by treatment with ben-
zaldehyde dimethyl acetal in the presence of a catalytic amount of camphorsul-
fonic acid to yield the benzylidene glycoside 18.[36] To install the fluorine atom
in the equatorial orientation, it was necessary to invert the configuration of
the 3-OH group. This was accomplished through a four-step double-inversion
protocol. Compound 18 was treated with Tf2O in CH2Cl2 in the presence of
pyridine to convert the free hydroxyl group into a good leaving group (i.e., tri-
flate). Displacement of the triflate by acetate via an SN2 manner yielded the
3-OAc glycoside, which was subsequently deprotected with NaOMe at −10◦C to
produce the corresponding allosamine glycoside 19. Fluorination of compound
19 was accomplished using DAST, resulting in the inversion of configuration
at C-3 to access the fluorinated analog mixed with a side product. Upon re-
moval of the benzylidene-protecting group, we were able to purify the desired
product 20 and identify the side product as 21; 21 is presumably formed due
to elimination of hydrogen fluoride followed by tautomerization and opening
of the phthalimide ring. The overall yield of 20 from benzylidene glycoside 18
was 28%.
To remove the Phth group in 20, it was initially treated with methylamine,
but this attempt resulted in multiple degradation products. In contrast, heat-
ing 20 with excess ethylene diamine in n-butanol at 90◦C allowed the access
to the free amine derivative, which on subsequent acetylation provided the ac-
etamide 22 in 80% yield (Sch. 3). Pd-catalyzed hydrogenolysis of 22 followed

Synthesis of N-acetyl Glucosamine Analogs
397
Table 1: Amount of HA secreted into culture medium and on surface of human
pancreatic cancer cell KP1-NL upon incubation with various compounds
Culture medium
Cell surface
Amount of HA
% of control
Amount of HA
% of control
(μg/106 cells)
(μg/106 cells)
DMSO
4-MU
3.02 0.03
3.00 0.05
1.66 0.04
2.95 0.02
2.65 0.01
2.40 0.02
1.89 0.06
100
99.2
54.9
97.5
87.7
79.6
62.7
0.17 0.02
0.17 0.01
0.12 0.01
0.15 0.02
0.14 0.02
0.14 0.01
0.11 0.01
100
100.2
68.4
89.1
81.8
82.9
64.9
10 μM
100 μM
10 μM
4
6
100 μM
10 μM
100 μM
by acetylation led to the desired product 6 with the α-anomer as the major
product in 60% yield.
With compounds 4 and 6 in hand, we evaluated their abilities to inhibit
HA biosynthesis in human pancreatic cancer cell line KP1-NL cells. 4-MU was
used as a positive control. The amount of HA secreted into the culture medium
as well as that residing on the cell surface was quantified by an ELISA-like
assay using a Hyaluronan Assay Kit and reported on a per cell basis (Table 1).
While compound 4 was not very effective in inhibiting HA biosynthesis, com-
pound 6 significantly reduced the amount of HA formed, with an inhibition
potency comparable to that of 4-MU.
As HA present in the pericellular matrix can create a barrier to the cell
membrane, its presence can be visualized by the steric exclusion of fixed
red blood cells to form a clear, halo-like area surrounding the KP1-NL cells
(Fig. 3 and Fig. S1 in supporting information). Treatment of the cells with
hyaluronidase, an enzyme capable of HA degradation, reduced the clear area
around the cells to 48.2% (Fig. 3b; for quantification of the size of clear area
see Fig. 3f). Compound 4 was much less potent, only leading to a small re-
duction to 93.1% of the vehicle control (Fig. 3d,f). In comparison, compound 6
(100 μM) reduced the area to 40.9% (Fig. 3e,f), which was consistent with the
ELISA analysis results supporting the idea that 6 could significantly inhibit
HA synthesis. 4-MU (100 μM) generated the largest change in the area under
the same assay condition (Fig. 3c,f).
Next we analyzed the effects of compounds 4 and 6 on cell proliferation
using an Alamar blue assay (Fig. 4). At the concentration of 100 μM, 4-MU did
not show much inhibitory activities against the pancreatic cancer cell line KP1-
NL cells. 3-Methoxy analog 4 (100 μM) reduced the proliferation of the cancer
cells to 60.5% of the DMSO control, while the 3-fluorine-containing analog 6
exhibited the highest antiproliferative activities, leading to a 91% reduction in
cell numbers at 100 μM. The IC50 value of 6 was determined to be 30 μM.

G. Wasonga et al.
398
Figure 3: Effect of various compounds on the formation of a pericellular HA matrix by KP1-NL
cells as assessed by a particle exclusion assay. Representative pictures of KP1-NL cells
following incubation with (a) DMSO control; (b) Streptomyces hyaluronidase (1.0 U/mL); (c)
4-MU (100 μM); (d) GlcNAc analog 4 (100 μM); and (e) GlcNAc analog 6 (100 μM). Fixed
sheep erythrocytes (the small cells in the pictures) were then added to the cells to help
visualize the pericellular matrix around the cancer cells. The pictures were taken at identical
magnification (each picture represents an area of 100 μm in width). (f) Quantification of the
relative sizes of areas in particle exclusion assay assessed using Image J.
In conclusion, we have developed synthetic routes to GlcNAc analogs bear-
ing structural modifications at C-3 positions as potential inhibitors of HA
biosynthesis. While the 3-OMe compound 4 was readily prepared, the inter-
mediate to 3-oxo analog was found to be unstable. To synthesize the 3-F-
containing analog 6, fluorination of the C-3 turned out to be highly challenging.
The Phth group used for protection of the nitrogen was crucial, which gave bet-
ter yield for the fluorination step compared to the previous report.[29] Upon in-
cubation of human pancreatic cell line with compounds 4 and 6, the 3-F analog
6 significantly reduced the amount of HA both in secreted form and on the can-
cer cell surface. The inhibitory activity of 6 was further confirmed by a particle
exclusion assay and 6 exhibited significant antiproliferative activities against

Synthesis of N-acetyl Glucosamine Analogs
399
Figure 4: Number of KP1-NL cells upon incubation with 4-MU, compound 4, and compound
6 respectively at 100 μM for 72 hours as determined by Alamar blue assay. The number of
cells treated with DMSO was set as 100% (color figure available online).
pancreatic cancer cells. It is possible that besides the potential for serving as
a chain terminator, 3-F GlcNAc analog 6 can reduce HA biosynthesis by inter-
fering with other cellular processes. The exact mechanism will require further
investigation.[37]
EXPERIMENTAL SECTION
General Experimental Procedures
All reactions were carried out under nitrogen with anhydrous solvents in
flame-dried glassware, unless otherwise noted. Chemicals used were reagent
grade as supplied except where noted. Compounds were visualized by UV light
(254 nm) and by staining with a yellow solution containing Ce(NH4)2(NO3)6
(0.5 g) and (NH4)6Mo7O24 4H2O (24.0 g) in 6% H2SO4 (500 mL). Flash column
chromatography was performed on silica gel 60 (230–400 mesh). NMR spec-
1
tra were referenced using residual CHCl3 (δ H NMR 7.26 ppm) and CDC13
(δ 13C NMR 77.0 ppm). Peak and coupling constant assignments are based on
1
1
1
1H NMR, H-1H gCOSY, and/or H-13C gHMQC and H-13C gHMBC experi-
ments.
2-Methyl-(1,2-dideoxy-5,6-O-isopropylidene-α-D-glucofurano)-
[2,1-d]-2-oxazoline (8)[38]
Anhydrous FeCl3 (15 g, 0.092 mol) was added to a suspension of
2-acetamido-2-deoxy-D-glucopyranose 7 (10 g, 0.068 mol) in dry acetone

G. Wasonga et al.
400
(200 mL), and the mixture was stirred and heated to reflux for 20 min with
exclusion of moisture. The solution was cooled to 0◦C, followed by addition of
triethylamine (35 mL), acetone (135 mL), and a solution of sodium bicarbonate
(21.3 g) in water (135 mL) with continuous stirring. Acetone, triethylamine,
and some water were then removed in vacuo at <30◦C. The mixture was ex-
tracted with diethyl ether (5 × 200 mL) and the combined extracts dried over
MgSO4 and concentrated at rt to give compound 8 as brownish syrup (7.15 g,
1
65%). The H NMR spectrum is identical to what is reported in the litera-
ture.[38] 1H NMR (600 MHz, CDCl3) δ 1.33 (s, 3H, CH3), 1.39 (s, 3H, CH3), 2.00
(s, 3H, N = COCH3), 2.92 (s, 1H, OH-3), 3.72 (dd, 1H, J3 = 3.6, 9.6 Hz, H-
4), 3.97 (dd, 1H, J3 = 4.8, 8.4 Hz, H-6), 4.11 (dd, 1H, J3 = 5.4, 7.8 Hz, H-6’),
4.28–4.32 (m, 1H, H-5), 4.39 (d, 1H, J3 = 3 Hz, H-3), 4.44 (dd, 1H, J3 = 1.2,
4.8 Hz, H-2), 6.14 (d, J3 = 4.8 Hz, H-1).
2-Methyl-(1,2-di-deoxy-3-methoxy-5,6-O-isopropylidene-α-D-
glucofurano)-[2,1-d]-2-oxazoline (9)
Compound 8 (0.4 g, 1.64 mmol) was dissolved in DMF (15 mL) and cooled
to 0◦C. NaH (0.08 g, 3.29 mmol) was added in three portions with vigorous
stirring followed by dropwise addition of MeI (0.15 mL, 2.47 mmol). The reac-
tion mixture was allowed to warm up to rt. After 3 h, DMF was evaporated in
vacuo and the residue coevaporated twice with toluene. The resultant residue
was dissolved in CH2Cl2 (20 mL) and washed with ice cold water and the aque-
ous phase extracted twice with CH2Cl2 (2 × 20 mL). The organic phases were
combined, washed with brine (30 mL), and dried with Na2SO4. Upon filtration
and concentration in vacuo, compound 9 was obtained in 96% yield (0.4 g). [α]D20
−15.5 (c = 1, CH2Cl2); 1H NMR (600 MHz, CDCl3) δ 1.27 (s, 3H, CH3), 1.33 (s,
3
ˆ
3H, CH3), 1.94 (s, 3H, N = CCH3), 3.40 (s, 3H, OCH3), 3.71 (dd, 1H, J = 3.6,
9 Hz, H-4), 3.77 (d, 1H, J3 = 4.2 Hz, H-3), 3.91–3.94 (m, 1H, H-6), 3.97–3.99
(m, 1H, H-6ꢁ), 4.21 (m, 1H, H-5), 4.42 (dd, 1H, J3 = 1.8, 6 Hz, H-4), 6.01 (d, 1H,
J3 = 6.6 Hz, H-1); 13C NMR (150 Hz, CDCl3) δ 14.3. 25.5, 26.99, 57.98, 67.0,
72.7, 74.8, 81.5, 83.7, 107.1, 109.2, 167.2. HRMS C12H20NO5 [M + H+] calc.
258.1341 found 258.1333.
1,4,6-Tri-O-acetyl-2-acetamido-2-deoxy-3-methoxy-β-D-
glucopyranoside (4)
Compound 9 (0.204 g, 0.793 mmol) was dissolved in 1:2 H2O/THF mix-
ture (10 mL:20 mL) and p-toluenesulfonic acid (0.06 g, 0.264 mmol) was added
to the reaction mixture then stirred overnight. The reaction was quenched
with Et3N to a neutral pH and the solvents were evaporated in vacuo. The
residue was washed several times with CH2Cl2 and the resultant white pow-
der dried under vacuum. The residue was then dissolved in pyridine (8 mL).

Synthesis of N-acetyl Glucosamine Analogs
401
Ac2O (0.5 mL) was added and the reaction mixture was stirred at rt overnight.
The reaction mixture was concentrated. The residue was dissolved in CH2Cl2,
washed with H2O and brine, dried over Na2SO4, and purified by column chro-
matography (CH2Cl2/MeOH, 8:1) to give compound 4 in 50% (0.143 g) yield
over two steps. [α]2D0 + 10.5 (c = 1, CH2Cl2); 1H NMR (600 MHz, CDCl3) δ 1.99
(s, 3H, NHCOCH3), 2.07 (s, 3H, COCH3), 2.10 (s, 3H, COCH3), 2.11 (s, 3H,
COCH3), 3.40 (s, 3H, OCH3), 3.75–3.84 (m, 3H, H-2, H-4, H-5), 4.09 (dd, 1H,
J3 = 2.4, 10.8 Hz, H-6), 4.23 (dd, 1H, J3 = 4.5, 12Hz, H-6’), 5.02 (t, 1H, J3 =
3
8.4 Hz, H-3), 5.70 (d, 1H, J3 = 7.2 Hz, H-1), 5.89 (d, 1H, J = 6.6 Hz, CONH);
13C NMR (125 Hz, CDCl3) 20.8, 20.84, 20.9, 23.4, 53.8, 58.0, 62.0, 68.5, 72.9,
79.2, 91.97, 169.4, 169.41, 170.2, 170.7. HRMS C15H23NO9 Na[M + Na+] calc.
384.1271 found 384.1259.
Benzyl-2-acetamido-2-deoxy-α-D-glucopyranoside (11)
N-Acetyl glucosamine 7 (6.00 g, 27.1 mmol) was dissolved in benzyl alcohol
(50 mL) and concentrated HCl (2.9 mL) was added. The mixture was heated
to 90◦C for 3 h, cooled to rt, and then poured onto 500 mL Et2O and stored
overnight at −20◦C. The resulting precipitate was recovered by filtration and
rinsed with Et2O and hexanes to yield 17.64 g of crude material, which was
purified by silica gel chromatography (8% to 15% MeOH/CH2Cl2) to provide
11 (5.98 g, 71%) as white foam. Comparison of 1H NMR with literature values
confirmed the identity of compound 11.[28] 1H NMR (500 MHz, CD3OD), δ 1.95
(s, 3H, COCH3), 3.37–3.39 (m, 1H), 3.69–3.73 (m, 3H), 3.82 (d, 1H, J3 = 9.5 Hz),
3.89 (dd, 1H, J3 = 3.5, 11 Hz), 4.49 (d, 1H, J3 = 12 Hz), 4.74 (d, 1H, J3 =
11.5 Hz), 7.28–7.40 (m, 5H, aromatic).
Benzyl-2-acetamido-2-deoxy-4,6-O-benzylidene-α-D-
glucopyranoside (12)
Compound 11 (0.412 g, 1.32 mmol) was dissolved in DMF (20 mL) followed
by the addition of benzaldehyde dimethyl acetal (0.6 mL, 3.97 mmol) and a
catalytic amount of p-toluenesulfonic acid (0.123 g, 0.53 mmol). The reaction
mixture was stirred for 3 h. DMF was removed in vacuo and the resultant
white residue was suspended in saturated sodium bicarbonate solution. Upon
filtration, the residue was washed several times with hexanes/EtOAc/CH2Cl2
(4/1/0.5) solvent system then dried under vacuum to afford compound 12 in
95% yield (0.5 g). Comparison of 1H NMR with reported literature values con-
firmed the identity of compound 12.[32] 1H NMR (600 MHz, CDCl3) δ 1.82 (s, 3H,
CH3), 3.59 (t, 1H, J3 = 9 Hz, H-6), 3.75 (t, 1H, J3 = 10.2 Hz, H-6’), 3.84–3.91
(m, 1H, H-5), 3.92–3.95 (m, 1H, H-4), 4.23–4.26 (m, 2H, H-2, H-3), 4.93 (d, 1H,
J3 = 12 Hz, CH2Ph), 4.74 (d, 1H, J3 = 12 Hz, CH2Ph), 4.93 (d, 1H, J3 = 3.5 Hz,
H-1), 5.57 (s, 1H, CHPh), 5.82 (d, 1H, J3 = 8.4 Hz, CONH), 7.33–7.51 (m, 10H,
aromatic).

G. Wasonga et al.
402
Benzyl-2-acetamido-2-deoxy-4,6-O-benzylidene-3-oxo-α-D-
glucopyranoside (13)
To a solution of COCl2 (0.11 mL, 1.25 mmol) and DMSO (0.16 mL,
2.23 mmol) in CH2Cl2 (30 mL) cooled to −78◦C and stirred for 20 min was
added compound 12 (0.36 g, 0.894 mmol) dissolved in CH2Cl2 (20 mL). After
20 min, Et3N (0.63 mL, 4.47 mmol) was added and the reaction mixture al-
lowed to warm to rt then quenched with H2O (100 mL). The aqueous layer
was extracted with CH2Cl2 (50 mL). The organic layers were combined and
washed with brine and dried over MgSO4. Compound 13 was obtained in 90%
yield (0.32 g) by recrystallization from hexanes/EtOAc/CH2Cl2 (5/1/0.5). [α]D20
+ 38.8 (c = 0.1, CH2Cl2); 1H NMR (500 MHz, CDCl3) δ 2.00 (s, 3H, CH3), 3.90
(t, 1H, J3 = 10.5 Hz, H-6), 4.11–4.16 (m, 1H), 4.29 (dd, 1H, J3 = 5, 10.5 Hz,
H-6’), 4.37 (d, 1H, J3 = 10.5 Hz, H-4), 4.50 (d, 1H, J3 = 12 Hz, CH2Ph), 4.69
(d, 1H, J3 = 12 Hz, CH2Ph), 4.95–4.97 (m, 1H, H-2), 5.41 (d, 1H, J3 = 4.5 Hz,
H-1), 5.56 (s, 1H, CHPh), 6.22 (d, 1H, J3 = 8.0 Hz, CONH), 7.25–7.49 (m, 10H,
aromatic); 13C NMR (125 Hz, CDCl3) δ 23.2, 59.1, 66.6, 69.6, 70.8, 77.0, 77.2,
77.5, 82.9, 100.8, 102.2, 126.6, 128.2, 128.6, 128.9, 129.6, 136.5, 170.1, 195.2.
HRMS C22H24NO6 [M + H+] calc. 398.1580 found 398.1577.
Benzyl-3,4,6-tri-O-acetyl-2-deoxy 2-phthalamido-β-
D-glucopyranoside (16)
Compound 15 (2.2 g, 4.61 mmol) was dissolved in CH2Cl2 (30 mL), followed
by addition of BnOH (1.1 mL, 9.22 mmol). BF3OEt2 (1.75 mL, 11.4 mmol) was
added dropwise and the reaction mixture was stirred at rt for 24 h. The mix-
ture was diluted with CH2Cl2 (20 mL) and washed with saturated NaHCO3 (3
× 20 mL). The aqueous layer was extracted with CH2Cl2 (60 mL) and the com-
bined organic phases were dried over Na2SO4, filtered, concentrated, and puri-
fied by column purification (hexanes/EtOAc 3:2) to yield compound 16 (1.92 g,
1
79%). Comparison of H NMR with reported literature values confirmed the
identity of compound 16.[36] 1H NMR (600 MHz, CDCl3) δ 1.86 (s, 3H, COCH3),
2.02 (s, 3H, COCH3), 2.13 (s, 3H, COCH3), 3.85–3.88 (m, 1H, H-5), 4.19 (dd, 1H,
J3 = 2.4, 12 Hz, H-6), 4.33–4.39 (m, 2H, H-2, H-6’), 4.53 (d, 1H, J3 = 12 Hz,
CHPh), 4.84 (d, 1H, J3 = 12.6 Hz, CHPh), 5.17 (t, 1H, J3 = 9.6 Hz, H-4) 5.37
(d, 1H, J3 = 8.4 Hz, H-1), 5.77 (dd, 1H, J3 = 9.0, 10.2 Hz, H-3), 7.08–7.79 (m,
9H, aromatic).
Benzyl-4,6-O-benzylidene-2-deoxy-2-phthalamido-β-D-
glucopyranoside (18)
Compound 16 (1.88 g, 3.79 mmol) was dissolved in MeOH/CH2Cl2 solvent
mixture (3:2, 20 mL) and cooled to −10◦C followed by dropwise addition of 0.3M
NaOMe in MeOH (6 mL). Upon completion of the reaction as established by

Synthesis of N-acetyl Glucosamine Analogs
403
TLC in 2 h, the reaction mixture was neutralized with Amberlite ion exchange
resin to pH 7. The crude triol 17 was concentrated and the residue was coevap-
orated with toluene twice followed by drying under vacuum overnight. The
residue was then suspended in CH3CN (15 mL) and PhCH(OCH3)2 (0.7 mL,
4.55 mmol) was added, followed by addition of camphorsulfonic acid (0.2 g,
0.87 mmol). The mixture was stirred for 3 h then quenched with Et3N upon
completion of reaction as confirmed by TLC. Solvents were evaporated and
the residue was purified by column chromatography (hexanes/EtOAc 3:1) to
yield compound 18 (1.42 g, 77%). The identity of compound 18 was confirmed
by comparison with reported literature values.[36] 1H NMR (600 MHz, CDCl3)
δ 3.62–3.67 (m, 2H, H-4, H-6), 3.85–3.88 (m, 1H, H-6ꢁ), 4.29–4.32 (m, 1H, H-
2), 4.42 (dd, 1 H, J3 = 4.8, 10.8 Hz, H-5), 4.52 (d, 1H, J3 = 12.6 Hz, CHPh),
3
4.63–4.67 (m, 1H, H-3), 4.84 (d, 1H, J = 12 Hz, CHPh), 5.28 (d, 1H, J3 =
8.4 Hz, H-1), 5.58 (s, 1H CHPh) 7.03–7.79 (m, 14H, aromatic).
Benzyl-2-deoxy-3-fluoro-2-phthalamido-β-D-
glucopyranoside (20)
To a solution of compound 18 (0.833 g, 1.71 mmol) dissolved in CH2Cl2
(30 mL), pyridine (0.7 mL, 8.54 mmol) was added and the mixture was cooled to
−20◦C. Tf2O (0.6 mL, 3.42 mmol) dissolved in CH2Cl2 (1 mL) was added drop-
wise and the reaction mixture was allowed to warm up to rt within 1.5 h. The
reaction mixture was diluted with CH2Cl2 (20 mL) and then quenched with
saturated NaCHO3, washed with water and brine, dried over Na2SO4, and
concentrated under reduced pressure at ambient temperature (25–30◦C). The
resultant yellow syrup was dissolved in toluene, TBAOAc (3.09 g, 10.26 mmol)
added, and the mixture stirred at 65◦C overnight. The mixture was allowed
to cool to rt then diluted with EtOAc. Solvents were evaporated and the
resultant residue was purified by column chromatography (hexanes/EtOAc
3.5:1) to yield benzyl-3-O-acetyl-2-deoxy-4,6-O-benzylidene-2-phthalamido-β-
D-allopyranoside. [α]2D0 −47.0 (c = 0.5, CH2Cl2); H NMR (600 MHz, CDCl3)
1
δ 2.07 (s, 3H, COCH3), 3.86–3.93 (m, 2H, H-4, H-6), 4.18–4.22 (m, 1H, H-
6ꢁ), 4.44–4.49 (m, 2H, H-2, H-4), 4.64 (d, 1H, J3 = 10.8 Hz, CHPh), 4.93 (d,
CHPh J3 = 12 Hz, CHPh), 5.61 (s, 1H, CHPh), 5.77–5.78 (m, 1H, H-3), 6.15
(d, 1H, J3 = 8.4, H-1), 7.21–7.84 (m, 14H, aromatic). HRMS C30H27NO8Na
[M + Na+] calc. 552.1634 found 552.1595. Benzyl-3-O-acetyl-2-deoxy-4,6-O-
benzylidene-2-phthalamido-β-D-allopyranoside (0.6 g, 1.13 mmol) was dis-
solved in MeOH/CH2Cl2 (3:2, 10 mL), cooled to −10◦C, and treated with 0.3M
NaOMe solution in methanol (3 mL). After 1 h, the reaction was complete as
confirmed by TLC and subsequently quenched with Amberlite ion exchange
resin (IR 120) to pH 7. Solvents were evaporated and the resultant white solid
was coevaporated with toluene (3 × 10 mL) then dried under vacuum for 2 h.
The allosamine analog 19 was dissolved in CH2Cl2 (10 mL) in a 50-mL fal-
con tube and cooled to −5◦C. This was followed by dropwise addition of DAST

G. Wasonga et al.
404
(0.27 mL, 6.78 mmol) and the reaction mixture was allowed to warm to rt
over 3 h. The reaction mixture was cooled to −5◦C followed by dropwise addi-
tion of MeOH to destroy the excess DAST, concentrated, and passed through
a short silica column. The resultant mixture of compound was dissolved in
MeOH/CH2Cl2 (3:2, 10 mL) followed by addition of AcCl (0.16 mL, 2.26 mmol),
then stirred overnight at rt. The reaction mixture was quenched with Et3N to
pH 7. Solvents were evaporated in vacuo and the resultant residue was puri-
fied by column chromatography (hexanes/EtOAc/MeOH 3:1:0.1) to acquire the
desired compound 20 (0.17 g, 28% yield from 18) and elimination product 21
(0.07 g, 14.5%). 20: [α]2D0 −64.0 (c = 0.5, CH2Cl2); H NMR (600 MHz, CDCl3)
1
δ 2.29 (s, 1H, OH), 3.11 (s, 1H, OH), 3.52–3.54 (m, 1H, H-5), 3.89–3.99 (m,
3H, H-4, H-6, H-6ꢁ), 4.34–4.39 (m, 1H, H-2), 4.53 (d, 1H, J3 = 12 Hz, CHPh),
4.79 (d, 1H, J3 = 12 Hz, CHPh), 5.13–5.25 (m, 3H), 7.05–7.26 (m, 5H, aro-
matic), 7.22–7.80 (m, 4H, aromatic); 19F NMR (282 MHz, CDCl3) −194.44 (dt,
1F, J3 = 51.9, 13.8 Hz). HRMS C21H20NaO6NF [M + Na+] calc. 424.1167 found
424.1172.
Benzyl-3-deoxy-2-(methylcarbamoyl)benzoate-6-methoxy-4-oxo-
β-D-glucopyranoside (21)
[α]2D0 −51.4 (c = 0.5, CH2Cl2); 1H NMR (600 MHz, CDCl3) 2.24 (dd, 1H, J3
= 4.2, 13.2 Hz, H-3), 2.48 (t, 1H, J3 = 13.2 Hz, H-3), 3.32 (s, 3H, OCH3), 3.36
(s, 3H, OCH3), 3.73–3.74 (m, 1H, H-5), 3.88–3.96 (m, 2H, H-6, H-6ꢁ), 4.43–4.48
(m, 1H, H-2), 4.58 (d, 1H, J3 = 12 Hz, CHPh), 4.79 (d, 1H, J3 = 12 Hz, CHPh),
5.33 (d, 1H, J3 = 8.4 Hz, H-1), 7.08–7.16 (m, 5H, aromatic), 7.70–7.84 (m, 4H,
aromatic); 13C NMR (150 Hz, CDCl3) 32.6, 49.4, 49.95, 50.6, 60.9, 71.2, 80.1,
98.7, 99.4, 123.3, 123.6, 127.6, 127.8, 127.9, 128.1, 128.3, 128.5, 131.4, 131.5,
131.7, 134.0, 167.6, 167.8, 204.4, 204.7. HRMS C23H29N2O7 [M + NH4+] calc.
445.1975 found 445.1994.
Benzyl-4,6-tri-O-acetyl-2-acetamido-2-deoxy-3-fluoro-β-D-
glucopyranoside (22)
Compound 20 (0.34 g, 0.847 mmol) was dissolved in nbutanol (10 mL) and
treated with ethylene diamine (6 mL). The reaction mixture was heated at
90◦C for 23 h. Solvents were then removed under reduced pressure and the
resultant residue was coevaporated with toluene (3 × 10 mL), which was
followed by drying in vacuo for 3 h. The intermediate free amine was dis-
solved in pyridine (10 mL), followed by addition of Ac2O (6 mL), and was
stirred overnight at rt. The mixture was treated with EtOH to react with ex-
cess Ac2O, concentrated, dissolved in CH2Cl2, washed with water and brine,
and dried over Na2SO4, followed by purification by column chromatography
(hexanes/EtOAc/CH2Cl2/MeOH, 2:1:1:0.2) to afford compound 22 (0.27 g, 80%).

Synthesis of N-acetyl Glucosamine Analogs
405
1H NMR (600 MHz, CDCl3) δ 1.94 (s, 3H, NHCOCH3), 2.09 (s, 3H, COCH3),
2.10 (s, 3H, COCH3), 3.33–3.38 (m, 1H, H-2), 3.66–3.67 (m, 1H, H-5), 4.14–4.17
(m, 1H, H-6), 4.27 (dd, 1H, J3 = 5, 12.5 Hz, H-6’) 4.57 (d, 1H, J3 = 12.5 Hz,
CHPh), 4.88 (d, 1H, J3 = 12 Hz, CHPh), 5.10–5.24 (m, 3H, H-1, H-3, H-4), 5.78
3
(d, 1H, J3 = 7 Hz, CONH); 19F NMR (282.2 MHz, CDCl3) −193.26 (dt, 1F, J
= 53.3, 12.4 Hz). HRMS C19H25FNO7 [M + H+] calc. 398.1615 found 398.1595.
1,4,6-Tri-O-acetyl-2-acetamido-2-deoxy-3-fluoro-α-D-
glucopyranoside (6)
Compound 22 (20 mg, 0.05 mmol) was dissolved in MeOH (6 mL) and
treated with Pd/C (30 mg). The reaction mixture was the stirred under hy-
drogen for 36 h and the reaction confirmed as complete by TLC. The mixture
was filtered and concentrated and the residue coevaporated with toluene (3
× 10 mL) followed by drying under vacuum. The resultant residue was dis-
solved in pyridine (3 mL), treated with Ac2O (0.5 mL), and stirred overnight at
rt. The mixture was concentrated; washed with 1M HCl, saturated NaHCO3,
and brine; and dried over Na2SO4. Column purification (hexanes/EtOAc, 1.5:1)
afforded compound 6 (15 mg, 76% yield over two steps). [α]2D0 +10.1 (c = 1,
1
CH2Cl2); H NMR (600 MHz, CDCl3) δ 2.03 (s, 3H, NHCOCH3), 2.09 (s, 3H,
COCH3), 2.11 (s, 3H, COCH3), 2.16 (s, 3H, COCH3), 3.93–3.96 (m, 1H, H-5),
4.06–4.09 (m, 1H, H-6), 4.22 (d, 1H, J3 = 4.2, 12.6 Hz, H-6ꢁ), 4.58–4.69 (m, 2H,
H-2, H-3), 5.25–5.30 (m, 1H, H-4), 5.52 (d, 1H, J3 = 7.8 Hz, NH), 6.20 (t, 1H,
J3 = 3 Hz, H-1); 13C NMR (150 Hz, CDCl3) 20.6, 20.7, 20.8, 23.2, 50.9, 50.96,
61.3, 68.1, 68.2, 69.5, 69.6, 88.97, 90.2, 90.9, 91.0, 168.4, 169.0, 170.0, 170.7;
19F NMR (282.2 MHz, CDCl3) −194.24 (dt, 1F, J3 = 50.8, 13.5 Hz,). HRMS
C14H24N2O8F [M + NH4+] calc. 367.1517 found 367.1512.
Determination of HA Concentration by an ELISA-Like Assay
KP1-NL cells (passage 3) were seeded in 35-mm culture dishes (6 ×
105 cells/dish) and cultured for 24 h. Cells were further cultured in fresh
medium containing each compound for 48 h. The culture supernatants were
collected and the cells were lysed by 1% Nonidet P-40, 140 mM NaCl, 10 mM
EDTA, and 20 mM Tris-HCl (pH 7.4). HA released into the culture medium
and accumulated in cell layers was quantified by an ELISA-like assay using
HA-binding protein (HABP) according to the manufacturer’s instructions for
the Hyaluronan Assay Kit (Seikagaku Co., Tokyo, Japan). The absorbance
at 490 nm (control wavelength, 630 nm) of each well was measured by a
microplate spectrophotometer xMark (Bio-Rad, Tokyo, Japan). The quantity
of HA was expressed per live cell number. The viability of cells was assessed
by trypan blue staining.

G. Wasonga et al.
406
Particle Exclusion Assay
KP1-NL cells (passage 5) plated at 2.5 × 104 cells in a 35-mm dish were
cultured for 24 h and then further cultured for 48 h with or without 100 μM of
the compound of interest in fresh medium containing 0.1% DMSO. An aliquot
of glutaraldehyde-stabilized sheep erythrocytes (5 × 108) in 0.75 mL of PBS
was then added to the culture medium. After 15 min, the culture was observed
using an inverted phase-contrast microscope (Olympus IMT-2).[39] The sizes of
the clear areas were quantified using ImageJ and the standard deviations of
at least five areas in each image were reported.
Estimation of Cell Proliferation
Cell numbers were estimated using the Alamar Blue assay, which is based
on the oxidation–reduction reaction of cells.[40] Briefly, KP1-NL cells (passage
3, 625 cells/well) were cultured overnight in 90 μL of medium in 96-well flat-
bottomed microplates (Iwaki Glass Co., Chiba, Japan) in triplicate and were
further cultured for 72 h with or without 0.1 mM of the test compound in
freshly served medium containing 0.1% DMSO. Alamar Blue working solution
(10 μL, BioSource, Camarillo, CA) was then added to each well and the plate
was further incubated for 3 h at 37◦C in a CO2 incubator. The fluorescence
of each well was measured at ex 544 nm and em 590 nm using a Fluoroskan
II (Japan Flow Laboratories, Tokyo, Japan). Reported values represented the
means of replicate wells and the standard deviations for each are reported. The
reaction was linear in the range of 300–3500 fluorescence units, corresponding
to 300–20,000 KP1-NL cells/well.
ACKNOWLEDGMENTS
We thank the National Institute of General Medical Sciences (R01 GM72667),
NIH, for financial support of our work. This work was also partially supported
by the Regional Innovation Strategy Support Program (City Area Type) of the
Ministry of Education, Culture, Sports, Science and Technology of Japan.
Supporting Information Available: Selected NMR spectra and images
of the particle exclusion assay at low magnification.
REFERENCES
1. Lapcik, L., Jr.; Lapcik, L.; De Smedt, S.; Demeester, J.; Chabrecek, P. Hyaluronan:
preparation, structure, properties, and applications. Chem. Rev. 1998, 98, 2663–2684.
2. Misra, S.; Ghatak, S.; Zoltan-Jones, A.; Toole, B.P. Regulation of multidrug resis-
tance in cancer cells by hyaluronan. J. Biol. Chem. 2003, 278, 25285–25288.

Synthesis of N-acetyl Glucosamine Analogs
407
3. Delpech, B.; Girard, N.; Bertrand, P.; Courel, N.M.; Chauzy, C.; Delpech, A.
Hyaluronan: fundamental principles and applications in cancer. J. Intern. Med. 1997,
242, 41–48.
4. Knudson, W. Tumor associated hyaluronan: providing an extracellular matrix that
facilitates invasion. Am. J. Pathol. 1996, 148, 1721–1726.
5. Franzmann, E.J.; Schroeder, G.L.; Goodwin, W.J.; Weed, D.T.; Fisher, P.; Lokesh-
war, V.B. Expression of tumor markers hyaluronic acid and hyaluronidase (HYAL1) in
head and neck tumors. Int. J. Cancer 2003, 106, 438–445.
6. Posey, J.T.; Soloway, M.S.; Ekici, S.; Sofer, M.; Civantos, F.; Duncan, R.C.; Lokesh-
war, V.B. Evaluation of the prognostic potential of hyaluronic acid and hyaluronidase
(HYAL1) for prostate cancer. Cancer Res. 2003, 63, 2638–2644.
7. Pirinen, R.; Tammi, R.; Tammi, M.; Hirvikoski, P.; Parkkinen, J. J.; Johansson, R.;
Bo¨hm, J.; Hollme´n, S.; Kosma, V.M. Prognostic value of hyaluronan expression in non-
small-cell lung cancer: increased stromal expression indicates unfavorable outcome in
patients with adenocarcinoma. Int. J. Cancer 2001, 95, 12–17.
˚
8. Seta¨la¨, L.P.; Tammi, M.I.; Tammi, R.H.; Eskelinen, M.J.; Lipponen, P.K.; Agren,
U.M.; Parkkinen, J.; Alhava, E.M.; Kosma, V.-M. Hyaluronan expression in gastric can-
cer cells is associated with local and nodal spread and reduced survival rate. Br. J.
Cancer 1999, 79, 1133–1138.
¨
9. Lokeshwar, V.B.; Obek, C.; Soloway, M.S.; Block, N.L. Tumor-associated
hyaluronic acid: a new sensitive and specific urine marker for bladder cancer. Cancer
Res. 1997, 57, 773–777.
10. Weigel, P.H.; Hascall, V.C.; Tammi, M. Hyaluronan synthases. J. Biol. Chem. 1997,
272, 13997–14000.
11. Nakamura, T.; Takagaki, K.; Shibata, S.; Tanaka, K.; Higuchi, T.; Endo,
M. Hyaluronic-acid-deficient extracellular matrix induced by addition of 4-
methylumbelliferone to the medium of cultured human skin fibroblasts. Biochem. Bio-
phys. Res. Commun. 1995, 208, 470–475.
12. Nakazawa, H.; Yoshihara, S.; Kudo, D.; Morohashi, H.; Kakizaki, I.; Kon, A.; Tak-
agaki, K.; Sasaki, M. 4-Methylumbelliferone, a hyaluronan synthase suppressor, en-
hances the anticancer activity of gemcitabine in human pancreatic cancer cells. Cancer
Chemother. Pharmacol. 2006, 57, 165–170 and references cited therein.
13. Kakizaki, I.; Kojima, K.; Takagaki, K.; Endo, M.; Kannagi, R.; Ito, M.; Maruo,
Y.; Sato, H.; Yasuda, T.; Mita, S.; Kimata, K.; Itano, N. A novel mechanism for the
inhibition of hyaluronan biosynthesis by 4-methylumbelliferone. J. Biol. Chem. 2004,
279, 33281–33289.
14. Kultti, A.; Pasonen-Seppa¨nen, S.; Jauhiainen, M.; Rilla, K. J.; Ka¨rna¨, R.; Pyo¨ria¨,
E.; Tammi, R.H.; Tammi, M.I. 4-Methylumbelliferone inhibits hyaluronan synthesis by
depletion of cellular UDP-glucuronic acid and downregulation of hyaluronan synthase
2 and 3. Exp. Cell Res. 2009, 315, 1914–1923.
15. Kudo, D.; Kon, A.; Yoshihara, S.; Kakizaki, I.; Sasaki, M.; Endo, M.; Takagaki,
K. Effect of a hyaluronan synthase suppressor, 4-methylumbelliferone, on B16F-10
melanoma cell adhesion and locomotion. Biochem. Biophys. Res. Commun. 2004, 321,
783–787.
16. Piccioni, F.; Malvicini, M.; Garcia, M.G.; Rodriguez, A.; Atorrasagasti, C.; Kippes,
N.; Buena, I.T.P.; Rizzo, M.M.; Bayo, J.; Aquino, J.; Viola, M.; Passi, A.; Alaniz, L.; Maz-
zolini, G. Antitumor effects of hyaluronic acid inhibitor 4-methylumbelliferone in an
orthotopic hepatocellular carcinoma model in mice. Glycobiology 2012, 22, 400–410.

G. Wasonga et al.
408
17. Urakawa, H.; Nishida, Y.; Wasa, J.; Arai, E.; Zhuo, L.; Kimata, K.; Kozawa, E.;
Futamura, N.; Ishiguro, N. Inhibition of hyaluronan synthesis in breast cancer cells
by 4-methylumbelliferone suppresses tumorigenicity in vitro and metastatic lesions of
bone in vivo. Int. J. Cancer 2012, 130, 454–466.
18. Lokeshwar, V.B.; Lopez, L.E.; Munoz, D.; Chi, A.; Shirodkar, S.P.; Lokeshwar,
S.D.; Escudero, D.O.; Dhir, N.; Altman, N. Antitumor activity of hyaluronic acid syn-
thesis inhibitor 4-methylumbelliferone in prostate cancer cells. Cancer Res. 2010, 70,
2613–2623.
19. Ghosh, S.; Blumenthal, H.J.; Davidson, E.; Roseman, S. Glucosamine metabolism.
V. Enzymatic synthesis of glucosamine 6-phosphate. J. Biol. Chem. 1960, 235,
1265–1273.
20. Boehmelt, G.; Wakeham, A.; Elia, A.; Sasaki, T.; Plyte, S.; Potter, J.; Yang, Y.J.;
Tsang, E.; Ruland, J.; Iscove, N.N.; Dennis, J.W.; Mak, T.W. Decreased UDP-GlcNAc
levels abrogate proliferation control in EMeg32-deficient cells. EMBO J. 2000, 19,
5092–5104.
21. Vocadlo, D.J.; Hang, H.C.; Kim, E.-J.; Hanover, J.A.; Bertozzi, C.R. A chemical
approach for identifying O-GlcNAc modified proteins in cells. Proc. Natl. Acad. Sci.
USA 2003, 100, 9116–9121.
22. Gloster, T.M.; Vocadlo, D.J. Developing inhibitors of glycan processing enzymes
as tools for enabling glycobiology. Nature Chem. Biol. 2012, 8, 683–694 and references
cited therein.
23. Dimitroff, C.J.; Kupper, T.S.; Sackstein, R. Prevention of leukocyte migration to
inflamed skin with a novel fluorosugar modifier of cutaneous lymphocyte-associated
antigen. J. Clin. Invest. 2003, 112, 1008–1018.
24. Gold, H.; Munneke, S.; Dinkelaar, J.; Overkleeft, H.S.; Aerts, J.M.G.; Code´e, J.D.C.;
van der Marel, G.A. A practical synthesis of capped 4-methylumbelliferyl hyaluronan
disaccharides and tetrasaccharides as potential hyaluronidase substrates. Carbohydr.
Res. 2011, 346, 1467–1478.
25. Danac, R.; Ball, L.; Gurr, S.J.; Fairbanks, A.J. Synthesis of UDP-glucose deriva-
tives modified at the 3-OH as potential chain terminators of β-glucan biosynthesis. Car-
bohydr. Res. 2008, 343, 1012–1022 and references cited therein.
26. Muller, T.; Danac, R.; Ball, L.; Gurr, S.J.; Fairbanks, A.J. Synthesis of UDP-GlcNAc
derivatives modified at OH-4 as potential chain-terminators of chitin biosynthesis.
Tetrahedron Assym. 2007, 18, 1299–1307.
27. Kisilevsky, R.; Szarek, W.A.; Ancsin, J.B.; Elimova, E.; Marone, S.; Bhat, S.;
Berkin, A. Inhibition of amyloid A amyloidogenesis in vivo and in tissue culture by
4-deoxy analogues of peracetylated 2-acetamido-2-deoxy-α- and β-D-glucose. Am. J.
Pathol. 2004, 164, 2127–2137 and references cited therein.
28. Yeager, A.R.; Finney, N.S. Synthesis of fluorescently labeled UDP-GlcNAc ana-
logues and their evaluation as chitin synthase substrates. J. Org. Chem. 2005, 70,
1269–1275.
29. Sharma, M.; Bernacki, R.J.; Hillman, M.J.; Korytnyk, W. Fluorinated carbohy-
drates as potential plasma membrane modifiers. Synthesis of 3-deoxy-3-fluoro deriva-
tives of 2-acetamido-2-deoxy-D-hexopyranoses. Carbohydr. Res. 1993, 240, 85–93.
30. Dube, D.H.; Bertozzi, C.R. Metabolic oligosaccharide engineering as a tool for gly-
cobiology. Curr. Opin. Chem. Biol. 2003, 7, 616–625.
31. Cai, Y.; Ling, C.-C.; Bundle, D.R. Facile approach to 2-acetamido-2-deoxy-β-D-
glucopyranosides via a furanosyl oxazoline. Org. Lett. 2005, 7, 4021–4024.

Synthesis of N-acetyl Glucosamine Analogs
409
32. Horton, D.; Hughes, J.B.; Jewell, J.S.; Philips, K.D.; Turner, W.N. Anomeric equi-
libria in derivatives of amino sugars. Nuclear magnetic resonance studies on acetylated
amino sugars and specifically deuterated analogs. J. Org. Chem. 1967, 32, 1073–1080.
33. Kajihara, Y.; Kodama, H.; Endo, T.; Hashimoto, H. Novel features of acceptor
recognition by β-(1–4)-galactosyltransferase. Carbohydr. Res. 1998, 306, 361–378.
34. Wang, Z.; Zhou, L.; El-Boubbou, K.; Ye, X.-S.; Huang, X. Multi-component one-pot
synthesis of the tumor-associated carbohydrate antigen Globo-H based on preactivation
of thioglycosyl donors. J. Org. Chem. 2007, 72, 6409–6420.
35. Hernandez-Torres, J.M.; Liew, S.-T.; Achkar, J.; Wei, A. Optimized synthesis of an
orthogonally protected glucosamine. Synthesis 2002, 487–490.
36. Ogawa, T.; Nakabayashi, S. Synthetic studies on cell surface glycans. Part
XI. Synthesis of 3,6-di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6-tetra-O-acetyl-β-D-
galactopyranosyl)-β-D-glucopyranosyl chloride. Carbohydr. Res. 1981, 97, 81–86.
37. Barthel, S.R.; Antonopoulos, A.; Cedeno-Laurent, F.; Schaffer, L.; Hernandez, G.;
Patil, S.A.; North, S.J.; Dell, A.; Matta, K.L.; Neelamegham, S.; Haslam, S.M.; Dimitroff,
C.J. Peracetylated 4-fluoro-glucosamine reduces the content and repertoire of N- and O-
glycans without direct incorporation. J. Biol. Chem. 2011, 286, 21717–21731.
38. Mack, H.; Basabe, J.V.; Brossmer, R. 2-Acetamido-2-deoxy-D-gluco- and -D-manno-
furanose. A simple preparation of 2-acetamido-2-deoxy-D-mannose. Carbohydr. Res.
1988, 175, 311–316.
39. Knudson, W.; Knudson, C.B. Assembly of a chondrocyte-like pericellular matrix on
non-chondrogenic cells. Role of the cell surface hyaluronan receptors in the assembly of
a pericellular matrix. J. Cell Sci. 1991, 99, 227–235.
40. Ahmed, S.A.; Gogal, R.M.J.; Walsh, J.E. A new rapid and simple non-radioactive
assay to monitor and determine the proliferation of lymphocytes: an alternative to
[3H]thymidine incorporation assay. J. Immunol. Methods 1994, 170, 211–224.