Synthesis of aromatic C-xylosides by position inversion of glucose

Article abstract of DOI:10.1016/j.bmc.2006.05.068

Two formally C-xylosylated analogs to 2-naphthyl β-d-xylopyranoside, which is known to initiate priming of glucosaminoglycan chains, were synthesized by a position inversion of glucose (i.e., position 1 becomes position 5). The d-C-xyloside showed priming, while the l-C-xyloside did not initiate priming.

Full text of DOI:10.1016/j.bmc.2006.05.068

Bioorganic & Medicinal Chemistry 14 (2006) 6659–6665
Synthesis of aromatic C-xylosides by position inversion of glucose
a
b
a
a
a,
*
´
´
Jesper Malmberg, Katrin Mani, Elin Sawen, Anders Wiren and Ulf Ellervik
¨
aOrganic Chemistry, Lund University, PO Box 124, SE-221 00 Lund, Sweden
bDepartment of Experimental Medical Science, Division of Neuroscience, Lund University,
Biomedical Center C13, SE-221 84 Lund, Sweden
Received 29 March 2006; revised 22 May 2006; accepted 31 May 2006
Available online 16 June 2006
Abstract—Two formally C-xylosylated analogs to 2-naphthyl b-D-xylopyranoside, which is known to initiate priming of glucosami-
noglycan chains, were synthesized by a position inversion of glucose (i.e., position 1 becomes position 5). The D-C-xyloside showed
priming, while the L-C-xyloside did not initiate priming.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
primer depends on the structure of the aglycon, which
may reflect selective partitioning of primers into differ-
ent intracellular compartments or into different branch-
es of biosynthetic pathways. In most cases, priming of
CS dominates and synthesis of free HS chains is low
or undetectable. Increased yields of HS can be obtained
when the aglycon of the xylosides comprises aromatic,
polycyclic structures, such as naphthol-derivatives. The
xyloside-primed GAG chains can be retained inside
the cells but are usually mainly secreted into the medium
and possibly also re-internalized. b-D-Xyloside-primed
HS chains have interesting biological properties, such
as activation of basic fibroblast growth factor,2 anti-
thrombotic effects,3 and growth inhibition of trans-
formed cells.4–6
Xylose is an unusual structural component in mammali-
an cells. So far, it has only been found in one unique po-
sition, that is, as the linker between protein and
carbohydrate in proteoglycans. Proteoglycans are com-
posed of glucosaminoglycan (GAG) chains covalently
attached to a core protein (Fig. 1). The first step in
GAG assembly is xylosylation of a serine residue. A spe-
cific
linker
tetrasaccharide,
GlcA(b1–3)Gal(b1–
3)Gal(b1–4)Xylb, is assembled and serves as an acceptor
for elongation of GAG chains. It is still unclear what
determines whether heparan sulfate (HS), with a
HexA–GlcNAc repeating motif, or chondroitin sulfate/
dermatan sulfate (CS/DS), with a HexA–GalNAc
repeating motif, is attached to the core protein but
repetitive Ser-Gly sequences and a high proportion of
Phe, Tyr, or Trp promote the formation of HS.1 The
resultant HS precursor polymers are subsequently mod-
ified through N-deacetylation/N-sulfation, epimeriza-
tion, and O-sulfation.
We have previously reported that the HS-priming glyco-
side 2-(6-hydroxynaphthyl)-b-D-xylopyranoside selec-
tively inhibits growth of transformed or tumor-derived
cells in vitro as well as in vivo.5 Treatment with this
xyloside at a pharmacologically relevant dose reduced
the average tumor load by 70–97% in SCID mice. At-
tempts to determine the mechanism for the selective
growth-inhibition suggest that the effect on transformed
cells is not caused by the xyloside itself but by products
derived from priming of HS.5,7 Furthermore, the bioac-
tivity is dependent on the hydroxyl substitution pattern
in the naphthalene rings of the xyloside and on nuclear
targeting of the xyloside-derived products. However, the
knowledge of structure recognition of the proteoglycan-
synthesizing enzymes is still scarce.
Biosynthesis of GAG chains can also take place inde-
pendently of core protein synthesis by using xylosides
as primers. Xylosides with hydrophobic aglycon can
penetrate cell membranes and initiate GAG synthesis
by serving as acceptors in the first galactosylation step.
The composition of the GAG assembled on the xyloside
Keywords: C-Glycosides; Carbohydrates; Structure–activity relation-
ships; Xylose.
*
To further investigate these effects, we decided to
synthesize the C-xyloside 2 as well as the enantiomeric
Corresponding author. Tel.: +46 46 2228220; fax: +46 46 2228209;
e-mail: ulf.ellervik@organic.lu.se
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.05.068

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J. Malmberg et al. / Bioorg. Med. Chem. 14 (2006) 6659–6665
HN
HN
OH
OH
OH
HO
O
HO
O
HO
O
COOH
O
COOH
O
O
O
O
O
O
O
O
NH
O
HO
O
HO
HO
OH
AcHN
OH
OH
OH
OH
O
n
core protein
linker tetrasaccharide
Figure 1. Glucosaminoglycan chains consist of a linker tetrasaccharide unit (GlcA(b1–3)Gal(b1–3)Gal(b1–4)Xylb) coupled to serine residues of the
protein chain.
tion of bromo-naphthalene with n-BuLi gave good re-
O
HO
HO
sults. The 6R- and 6S-diastereomers were formed in
equal amounts. Reduction of the benzylic hydroxyl
group proved to be troublesome, but Et3SiH in combi-
nation with BF3OEt2 gave acceptable results. The opti-
mized conditions turned out to be high concentration
of BF3OEt2 and short reaction times (0.4 mM, 10 min,
rt) which gave 71% yield. Deprotection of the benzyl
groups using hydrogenation resulted in an inseparable
mix of 2 and the analogous tetrahydronaphthalene
derivative. Instead the benzyl groups were exchanged
for acetyl groups,11 which were subsequently removed
O
OH
1
2
O
HO
HO
OH
O
HO
OH
HO
3
Figure 2. Xylosides used in this study.
´
under Zemplen conditions. The lower yields in some of
the reactions starting from 4a, compared to 4b, are
due to the smaller scale.
C-xyloside 3 (Fig. 2) and compare these with 2-naphthyl
b-D-xylopyranoside (1) which is known to prime GAG
synthesis.8
There are several observations that the biological effects
of xylosides are dependent on the polarity (i.e., logP) of
the compounds.7,12 The gradient HPLC retention times,
in contrary to isocratic retention times, can be treated as
linear free-energy-related parameters,13 that is, gradient
HPLC retention times can be used to substitute logP
values in biological evaluations. The gradient HPLC
retention times for the naphthoxylosides were measured
using a C-18 column and a mobile phase of water (0.1%
trifluoroacetic acid) with a gradient of acetonitrile from
one minute increasing by 1.2% per minute up to 30 min.
The retention times were measured for three separate
runs per compound and the calculated mean retention
times are presented in Table 1. The C-xylosides 2 and
3 are slightly less polar compared to the O-xyloside 1.
2. Results and discussion
C-Glycosides are generally difficult to synthesize in good
yields and defined stereochemistry. Instead for a C-gly-
cosylation strategy we decided to synthesize formal C-
xylosides by cleavage of the anomeric position of glu-
cose and subsequent coupling of the naphthalene residue
to position 6. This position inversion strategy (i.e., posi-
tion 1 becomes position 5) was first introduced by Guo
et al. for simple compounds (i.e., C-(b-pentopyranosyl)-
methanols).9
The commercially available peracetylated D- or L-glu-
cose was converted into the anomeric bromides that
were subsequently reduced using Bu3SnH. The addition
of the radical initiator AIBN gave stable yields (97%
over two steps). The acetyl-protective groups were then
exchanged for benzyl ethers and the primary benzyl
group was selectively deprotected using DIBAL-H by
To test the xyloside’s ability to prime GAG synthesis,
mouse 3T3 fibroblasts were incubated with 0.1 mM
xyloside and [35S]sulfate followed by isolation and size
separation of free GAG chains. All cells secreted alka-
li-sensitive proteoglycans (Fig. 3, Pool I). However,
treatment with some xylosides also initiated synthesis
of free GAG chains (Fig. 3, Pool II). The total amount
of GAG-priming was determined by integration of frac-
tions in Pool II (Table 1).
the method recently introduced by Sinay and co-work-
¨
ers.10 The conversion of the primary alcohol to an alde-
hyde was first carried out using Dess–Martin
periodinane which gave 70% and several byproducts.
Unfortunately, purification on SiO2 resulted in epimer-
ization. However, Swern oxidation gave clean quantita-
tive conversion and the product could be used in the
next step without further purification.
Table 1. Data for compounds 1–3
Compound
Retention time (min)
Priming (dpm)
1
2
3
19.0 0.2
20.9 0.3
20.9 0.3
8749
735
10
We tried several reagent combinations for the coupling
of the naphthalene unit to the aldehyde but only reac-

J. Malmberg et al. / Bioorg. Med. Chem. 14 (2006) 6659–6665
6661
3. Conclusions
We have synthesized two C-xylosides (2 and 3) by a po-
sition inversion-strategy starting with glucose. This
strategy opens up for the synthesis of other xylosides,
as well as lyxose, arabinose, and ribose derivatives (see
Scheme 1).
Our results show that the ability of GAG priming is
much lower for the C-xyloside 2 compared to the O-xy-
loside 1 despite the lower polarity of 2. However, the
lower GAG-priming ability of 2 is in accordance with
an earlier study where the corresponding phenyl glyco-
sides were tested.14 The L-C-xyloside 3 was not able to
initiate any GAG priming, which correlates well with
earlier results on L-O-xylosides.4 All together these re-
sults demonstrate the importance of the sugar structure
(D-xylose) as well as the glycosidic bond (O vs C) for
GAG priming.
4. Experimental
4.1. General synthetic methods
NMR spectra were recorded at 300, 400 or 500 MHz. 1H
NMR spectra were assigned using 2D-methods (COSY,
HETCOR, and long range HETCOR). Chemical shifts
are given in parts per million downfield from the signal
for Me4Si, with reference to residual undeuterated sol-
vents. Reactions were monitored by TLC using alumina
plates coated with silica gel and visualized using either
UV light or by charring with p-anisaldehyde (dip solu-
tion). Preparative chromatography was performed with
15
silica gel (35–70 lm, 60 A). Known (4a, 5a,16 5b,17
˚
7a,18 7b,17 8b,9 and 9b10) and commercially available
compounds (4b and 6b) were in agreement with previ-
ously published data.
Figure 3. Priming of GAG-chains in mouse 3T3 fibroblasts incubated
with 1 (a), 2 (b) or 3 (c). Pool II contains xyloside-primed GAG chains.
Dashed lines show the result for untreated cells.
AcO
O
O
O
AcO
AcO
AcO
c
a
b
AcO
AcO
AcO
OAc
OAc
OAc
OAc
OAc
OAc
Br
5a
(5b)
6a, 96%
(6b, 97%)
4a
4b
O
O
O
HO
HO
BnO
BnO
BnO
BnO
d
e
f
OH
OH
OBn
OH
OBn
OBn
7a, 95%
(7b, 98%)
8a, 87%
(8b, 83%)
9a, 87%
(9b, 90%)
OH
O
O
O
g
BnO
BnO
BnO
BnO
BnO
CHO
BnO
OBn
OBn
OBn
10a, quant.
(10b, quant.)
11a, 54%
(11b, 76%)
12a, 53%
(12b, 71%)
O
O
AcO
HO
HO
j
AcO
OAc
13a, 82%
(13b, 89%)
OH
2, 92%
(3, 94%)
Scheme 1. Reagents and conditions: (a) Ac2O, HBr/AcOH, 90 min; (b) Bu3SnH, AIBN, KF, 20 min; (c) NaOMe, 45 min; (d) NaH, BnBr, DMF, 16 h;
(e) DIBAL-H, toluene, 50 ꢁC, 2 h; (f) (COCl)2, DMSO, CH2Cl2, Et3N, ꢀ61 ꢁC, 45 min; (g) 2-bromonaphthalene, n-BuLi, THF, ꢀ78 ꢁC, 2.5 h; (h)
Et3SiH, BF3OEt2, CH2Cl2, 10 min; (i) Ac2O, BF3OEt2, 0 ꢁC, 17 h; (j) NaOMe, 1 h. Parenthesized yields are for the synthesis starting with D-Glc (4b).

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J. Malmberg et al. / Bioorg. Med. Chem. 14 (2006) 6659–6665
4.2. Tetra-O-acetyl-a-L-glucopyranosyl bromide (5a)
4.8. 1,5-Anhydro-2,3,4,6-tetra-O-benzyl-L-glucitol (8a)
Compound 4a (1.03 g, 2.64 mmol) was dissolved in
AcOH (3.5 mL). Ac2O (0.5 mL) was added followed
by fresh HBr/AcOH 33% (3.5 mL) and stirred for
90 min at rt. Argon was flushed through the solution
for 15 min. The solution was then lyophilized to give
crude material that was used in the next step without
further purification.
Compound 7a (374 mg, 2.28 mmol) was dissolved in
freshly distilled DMF (9.5 mL) and NaH (1.2 g) was
added under Ar. The mixture was cooled to 10 ꢁC
and BnBr (2.0 mL, 17 mmol) was added dropwise.
The mixture was stirred at rt for 16 h. MeOH
(10 mL) was slowly added, followed by H2O
(11 mL). HCl (4 M) was added until the solution be-
comes neutral. The crude product was extracted with
CH2Cl2 and the organic phase was washed with
H2O, dried (MgSO4), and concentrated. The residue
4.3. Tetra-O-acetyl-a-D-glucopyranosyl bromide (5b)
Synthesized as compound 5a.
was chromatographed (SiO2, 3:1 heptane/EtOAc) to
21
give 8a (1.05 g, 87%) as a colorless syrup. ½aꢁ ꢀ9.8
D
4.4. 2,3,4,6-Tetra-O-acetyl-1,5-anhydro-L-glucitol (6a)
(c 1.0 in CDCl3). 1H NMR (CDCl3): d 7.19–7.32
(m, 18H; ArH), 7.07–7.11 (m, 2H; ArH), 4.80, 4.93
(ABq, 1H each, J = 11.0 Hz; OBn), 4.45, 4.78 (ABq,
1H each, J = 10.7 Hz; OBn), 4.59, 4.67 (ABq, 1H
each, J = 11.6 Hz; OBn), 4.46, 4.54 (ABq, 1H each,
J = 12.2 Hz; OBn), 3.99 (dd, 1H, J1 = 11.1 Hz,
J2 = 4.7 Hz; H-1e), 3.48–3.66 (m, 5H; H-2, H-3, H-
6 · 2, H-4), 3.33 (ddd, 1H, J1 = 9.5 Hz, J2 = 4.3 Hz,
J3 = 2.1 Hz ; H-5), 3.12–3.21 (m, 1H; H-1a); 13C
NMR data (CDCl3): d 139.0, 138.38, 138.36, 138.1,
128.7, 128.6, 128.2, 128.15, 128.11, 128.08, 127.93,
127.91, 127.8, 86.6, 79.5, 78.7, 78.0, 75.8, 75.3, 73.8,
73.5, 69.2, 68.4; HRMS calcd for C34H37O5 (M+H):
525.2641, found: 525.2639.
The crude compound 5a was dissolved in dry Et2O
(4.6 mL) under Ar. Bu3SnH (0.780 mL, 2.90 mmol)
was added to the solution followed by a catalytic
amount of AIBN. The solution was stirred in rt for
30 min. KF (0.520 g) dissolved in H2O (2 mL) was add-
ed and the mixture was stirred rapidly for 20 min. The
mixture was filtered through Celite and the filter cake
was washed with Et2O. The H2O phase was extracted
with Et2O. The organic phases were washed with
H2O, concentrated, and recrystallized from Et2O and
pentane to give 6a (0.844 g, 96%) as white crystals;
21
1
mp 70–71 ꢁC; ½aꢁ ꢀ40.0 (c 0.7 in CDCl3); H NMR
D
(CDCl3): d 5.21 (t, 1H, J = 9.5 Hz; H-3), 4.99–5.06
(m, 2H, H-2, H-4), 4.12–4.24 (m, 3H; H-1e, 2· H-6),
3.58–3.63 (m, 1H; H-5), 3.31 (t, 1H, J = 11.0 Hz; H-
1a), 2.10, 2.04, 2.04, 2.02, (s, 3H each; OAc); 13C
NMR data (CDCl3): d 170.9, 170.6, 170.0, 169.7,
76.7, 73.9, 69.2, 68.6, 67.1, 62.4, 21.0, 20.95, 20.92,
20.8; HRMS calcd for C14H21O9 (M+H): 333.1186,
found: 333.1193.
4.9. 1,5-Anhydro-2,3,4,6-tetra-O-benzyl-D-glucitol (8b)
Synthesized as compound 8a (6.90 g, 83%). ½aꢁ21 +10.6 (c
D
21
D
+27 (c 0.5 in CHCl3)].
20
D
0.9 in CHCl3), ½aꢁ +27.1 (c 0.8 in CH2Cl2); [lit.9 ½aꢁ
4.10. 1,5-Anhydro-2,3,4-tri-O-benzyl-L-glucitol (9a)
4.5. 2,3,4,6-Tetra-O-acetyl-1,5-anhydro-D-glucitol (6b)
Compound 8a (1.02 g, 1.95 mmol) was dissolved in
distilled toluene (27 mL). DIBAL (20 mL, 1 M in
CH2Cl2), was added dropwise under Ar. The tempera-
ture was increased to 50 ꢁC and the mixture was stir-
red for 2 h. The solution was poured on ice, HCl
(26 mL, 1 M) was added and the mixture was stirred
rapidly for 10 min. The mixture was diluted with
EtOAc (100 mL) and the H2O phase was extracted
with EtOAc. The combined organic phases were
washed with NaCl (satd aq), dried (MgSO4), and
concentrated. The residue was chromatographed
21
Synthesized as compound 6a (3.30 g, 97%). ½aꢁ +42.3 (c
D
1.0 in CHCl3); [lit.19 [a]D +42.6 (c 1.4 in CHCl3)].
4.6. 1,5-Anhydro-L-glucitol (7a)
Compound 6a (0.813 g, 2.45 mmol) was dissolved in
NaOMe/MeOH (20 mL, 0.05 M) and stirred for
45 min at rt. Amberlite IR-120 H+ was added and stir-
red until the solution turned neutral. The Amberlite
was filtered off and washed with MeOH. The organic
(SiO2, 1:1 heptane/EtOAc) to give 9a (733 mg, 87%)
20
phase was concentrated to give 7a (382 mg, 95%) as a
as a white amorphous solid. ½aꢁ ꢀ17.0 (c 0.6 in
D
21
white amorphous solid; ½aꢁ ꢀ39.0 (c 0.5 in MeOD);
CHCl3) 1H NMR (CDCl3): d 7.28–7.38 (m, 15H;
ArH), 4.88, 5.00 (ABq, 1H each, J = 11.0 Hz; OBn),
4.65, 4.90 (ABq, 1H each, J = 11.0 Hz; OBn), 4.65,
4.74 (ABq, 1H each, J = 11.6 Hz; OBn), 4.01 (dd,
1H, J1 = 11.0 Hz, J2 = 4.9 Hz; H-1e), 3.81–3.87 (m,
1H; H-1e), 3.58–3.70 (m, 3H; H-2, H-3, H-6), 3.50 (t,
1H, J = 9.1 Hz; H-4), 3.27–3.32 (m, 2H; H-5, H-6),
3.24 (t, 1H, J = 10.7 Hz; H-1a), 1.76 (t, 1H, J =
6.45 Hz; OH); 13C NMR data (CDCl3): d 138.9,
138.34, 138.27, 128.7, 128.6, 128.3, 128.12, 128.06,
127.9, 86.4, 79.9, 78.8, 77.8, 75.8, 75.4, 73.6, 68.2,
62.5; HRMS calcd for C27H31O5 (M+H): 435.2171,
found: 435.2182.
D
1H NMR (MeOD): d 3.89 (dd, 1H, J1 = 11.0 Hz,
J2 = 5.4 Hz, H-1), 3.83 (dd, 1H, J1 = 11.8 Hz,
J2 = 2.1 Hz; H-6), 3.60 (dd, 1H, J1 = 11.8 Hz,
J2 = 5.8 Hz, H-6), 3.19–3.49 (m, 5H; H-1, H-2, H-3,
H-4, H-5); 13C NMR data (MeOD): d 82.6, 80.1, 72.0,
71.6, 71.1, 63.2; HRMS calcd for C6H12O5Na
(M+Na): 187.0582, found: 187.0578.
4.7. 1,5-Anhydro-D-glucitol (7b)
21
Synthesized as compound 7a (1.6 g, 98%). ½aꢁ +41.9 (c
D
25
D
0.7 in MeOH); [lit.9 ½aꢁ +42 (c 0.5 in H2O)].

J. Malmberg et al. / Bioorg. Med. Chem. 14 (2006) 6659–6665
6663
4.11. 1,5-Anhydro-2,3,4-tri-O-benzyl-D-glucitol (9b)
ArH), 5.02, 4.87 (ABq, 1H each, J = 10.9 Hz; OBn),
5.00, 4.70 (ABq, 1H each, J = 11.0 Hz; OBn), 4.71,
4.63 (ABq, 1H each, J = 11.7 Hz; OBn), 3.94 (dd, 1H,
J1 = 11.2 Hz, J2 = 4.9 Hz; H-5), 3.60–3.72 (m, 2H; H-3,
H-4), 3.54 (dt, 1H, J1 = 9.3 Hz, J2 = 2.3 Hz; H-1),
3.29–3.38 (m, 2H; H-2, H-2, Nap-CH2), 3.09 (t, 1H,
J = 10.7 Hz; H-5), 2.77 (dd, 1H, J1 = 14.3 Hz,
J2 = 9.1 Hz; Nap-CH2); 13C NMR data (CDCl3): d
138.9, 138.5, 138.4, 136.4, 133.7, 132.4, 128.72, 128.68,
128.6, 128.1, 128.05, 128.03, 127.98, 127.89, 127.89,
127.87, 127.8, 127.7, 126.0, 125.5, 86.7, 81.8, 80.9,
79.1, 75.8, 75.5, 73.4, 68,2, 38.7 ; HRMS calcd for
C37H36O4 (M+H): 545.2692, found: 545.2714.
20
Synthesized as compound 9a (170 mg, 90%). ½aꢁ +16.5
D
20
D
(c 0.9 in CHCl3); [lit.10 ½aꢁ +18 (c 1.0 in CHCl3)].
4.12. 2,6-Anhydro-3,4,5-tri-O-benzyl-D-glucose (10a)
Oxalyl chloride (0.015 mL, 0.175 mmol) was diluted
with dry CH2Cl2 (0.28 mL) and cooled to ꢀ61 ꢁC.
DMSO (0.022 mL, 0.310 mmol) in dry CH2Cl2
(0.140 mL) was added and the mixture was stirred for
5 min. Compound 9a (50 mg, 0.115 mmol) in dry
CH2Cl2 (0.140 mL) was added dropwise and stirred
for 30 min at ꢀ61 ꢁC. Et3N (0.081 mL) was added and
the stirring continued for 15 min at ꢀ61ꢁC. The mixture
was allowed to attain rt and H2O (1 mL) was added. The
mixture was diluted with CH2Cl2 and the organic phase
was washed with H2O, concentrated, and dried under
vacuum overnight to give crude 10a as a yellow syrup
that was used in the next step without further
purification.
4.17. 2-((2,3,4-Tri-O-benzyl-b-L-xylopyranosyl)methyl)-
naphthalene (12b)
22
D
Synthesized as compound 12a (27.6 mg, 71%). ½aꢁ
ꢀ10.3ꢁ (c 1.0 in CDCl3). HRMS calcd for C37H37O4
(M+H): 545.2692, found: 545.2700.
4.18. 2-((2,3,4-Tri-O-acetyl-b-D-xylopyranosyl)methyl)-
naphthalene (13a)
4.13. 2,6-Anhydro-3,4,5-tri-O-benzyl-L-glucose (10b)
Synthesized as compound 10a.
Compound 12a (17.0 mg, 0.0312 mmol) was dissolved in
Ac2O (3.2 mL) and cooled to 0 ꢁC. BF3ÆOEt (0.050 mL)
was added and the mixture was stirred for 17 h at 0 ꢁC.
The mixture was diluted with EtOAc and washed with
NaHCO3 (satd aq) and H2O. The organic phase was
dried (Na2SO4), and concentrated. The residue was
4.14. (6R and 6S)-1,5-Anhydro-2,3,4-tri-O-benzyl-6-C-
naphthyl-L-glucitol (11a)
2-Bromonaphthalene (95.3 mg, 0.460 mmol) was dis-
solved in distilled THF (1.0 mL) and cooled to
ꢀ78 ꢁC. n-BuLi (1.6 M in hexane, 0.287 mL,
0.460 mmol) was added dropwise and stirred at
ꢀ78 ꢁC for 45 min. Compound 10a was dissolved in dis-
tilled THF (0.5 mL) and added dropwise (10 min) and
stirred for 2.5 h at ꢀ78 ꢁC. The mixture was allowed
to attain rt and NH4Cl (2 mL, satd aq) was added.
The mixture was diluted with Et2O and the organic
phase was washed with H2O, concentrated, and chro-
matographed (SiO2, 2:1 heptane/EtOAc) to give a 6R/
6S-mixture of 11a (35.0 mg, 54%) as a white amorphous
solid used in the next step without separation of
diastereomers.
chromatographed (SiO2, 2:1 heptane/EtOAc) to give
21
D
13a (10.3 mg, 82%) as a white amorphous solid. ½aꢁ
ꢀ17.4 (c 0.8 in CDCl3). 1H NMR (CDCl3): d 7.75–
7.83 (m, 3H; ArH), 7.63 (s, 1H; ArH), 7.49–7.40 (m,
2H; ArH), 7.33 (dd, 1H, J1 = 8.4 Hz, J2 = 1.6 Hz;
ArH), 5.19 (t, 1H, J = 9.4 Hz; H-3), 5.03–4.92 (m, 2H;
H-2, H-4), 4.06 (dd, 1H , J1 = 11.2 Hz, J2 = 5.6 Hz; H-
5), 3.66–3.73 (m, 1H; H-1), 3.18 (t, 1H, J = 11.0 Hz;
H-5), 3.00, 2.92 (ABq, 1H, J = 14.3 Hz; Nap-CH2),
3.00, 2.90 (ABq, 1H, J = 14.4 Hz; Nap-CH2); 13C
NMR data (CDCl3): d 170.7, 170.0, 134.9, 133.6,
132.5, 128.1, 127.9, 127.83, 127.78, 126.3, 125.7, 79.2,
74.2, 72.7, 69.5, 67.0 38.6, 21.0, 20.94, 20.92; HRMS
calcd for C22H24O7Na (M+Na): 423.1420, found:
423.1431.
4.15. (6S and 6R)-1,5-Anhydro-2,3,4-tri-O-benzyl-6-C-
naphthyl-D-glucitol (11b)
4.19. 2-((2,3,4-Tri-O-acetyl-b-L-xylopyranosyl)methyl)-
naphthalene (13b)
Synthesized as compound 11a (195 mg, 76%).
22
Synthesized as compound 13a (18 mg, 89%). ½aꢁ +18.5
4.16. 2-((2,3,4-Tri-O-benzyl-b-D-xylopyranosyl) methyl)-
naphthalene (12a)
D
(c 1.2 in CDCl3); HRMS calcd for C22H24O7Na
(M+Na): 423.1420, found: 423.1416.
Compound 11a (35.0 mg, 0.0624 mmol) was dissolved in
dry CH2Cl2 (3.5 mL) and Et3SiH (0.140 mL) was added
under Ar. BF3ÆOEt (0.170 mL) was added and the mix-
ture was stirred for 10 min. NaHCO3(4 mL, satd aq)
was added, the H2O phase was extracted with CH2Cl2,
and the organic phases were washed with H2O, dried
(MgSO4) and concentrated. The residue was chromato-
graphed (SiO2, 4:1 heptane/EtOAc) to give 12a (18.0 mg,
4.20. 2-((b-D-Xylopyranosyl)methyl)-naphthalene (2)
Compound 13a (10.3 mg, 0.0257 mmol) was dissolved in
NaOMe/MeOH (25 mL, 0.05 M) and stirred for 60 min
at rt. Amberlite IR-120 H+ was added and stirred until
the solution turned neutral. The Amberlite was filtered
off and washed with MeOH. The organic phase was con-
21
1
53%) as a clear syrup. ½aꢁ +11.0 (c 1.0 in CDCl3). H
centrated to give 2 (6.5 mg, 92%) as a white amorphous
D
21
solid. ½aꢁ ꢀ21.1 (c 0.2 in MeOD). 1H NMR (MeOD): d
NMR (CDCl3): d 7.75–7.82 (m, 3H; ArH), 7.65 (s, 1H;
ArH), 7.41–7.46 (m, 2H; ArH), 7.28–7.38 (m, 16H;
D
7.67–7.82 (m, 4H; ArH), 7.37–7.45 (m, 3H; ArH), 3.81

6664
J. Malmberg et al. / Bioorg. Med. Chem. 14 (2006) 6659–6665
(dd, 1H, J1 = 11.1 Hz, J2 = 5.4 Hz; H-5e), 3.27–3.47 (m,
4H; H-4, H-1, Nap-CH2, H-3), 3.13 (t, 1H, J = 9.1 Hz;
H-2), 3.05 (t, 1H, J = 10.9 Hz; H-5a), 2.80 (dd, 1H,
J1 = 14.2 Hz, J2 = 8.6 Hz; Nap-CH2), 13C NMR data
(MeOD): d 136.6, 133.8, 132.5, 128.1, 127.7, 127.3,
127.2, 125.6, 125.0, 81.5, 78.8, 73.9, 70.4, 69.8, 38.0;
HRMS calcd for C16H19O4 (M+H): 275.1283, found:
275.1290.
ovalbumin containing 10 mM N-ethylmaleimide, and
1 mM diisopropylphosphoro-fluoridate on a slow shaker
at 4 ꢁC for 10 min.
4.25. Isolation of xyloside-primed radiolabeled GAG
The procedures have been described in detail previous-
ly.4 [35S]Sulfate-labeled polyanionic macromolecules
were isolated from the culture medium by ion ex-
change-chromatography on DEAE–cellulose at 4 ꢁC.
Samples were mixed with 1.3 vol of urea (7 M), Tris
(10 mM), pH 7.5, and Triton X-100 (0.1%), and passed
over a 1 mL-column of DE-53 equilibrated with urea
(6 M), NaOAc (0.5 M), pH 5.8, ovalbumin (5 lg/mL),
and Triton X-100 (0.1%). After sample application, the
columns were washed successively with 10 mL-portions
of (a) equilibration buffer, (b) urea (6 M), Tris
(10 mM), pH 8.0, ovalbumin (5 lg/mL), and Triton X-
100 (0.1%), and (c) 50 mM Tris, pH 7.5. Bound material
was eluted with 5· 1 mL guanidine–HCl (4 M), NaOAc
(50 mM), pH 5.8, and ovalbumin (5 lg/mL). Radioac-
tive fractions were pooled, precipitated with ethanol
(95%, 5 vol) overnight at ꢀ20 ꢁC using 100 lg of dextran
as carrier. After centrifugation in a Beckman JS-7.5 at
4000 rpm and 4 ꢁC for 45 min, the material was dis-
solved in guanidine–HCl (4 M), NaOAc (50 mM), pH
5.8, and free xyloside-primed GAG chains were separat-
ed from PG by hydrophobic interaction chromatogra-
phy on octyl-Sepharose followed by gel permeation
FPLC on Superose 6. Radioactivity was determined in
a b-counter.
4.21. 2-((b-L-Xylopyranosyl)methyl)-naphthalene (3)
22
Synthesized as compound 2 (18 mg, 94%). ½aꢁ +22.3 (c
D
0.2 in MeOD). HRMS calcd for C16H19O4Na (M+Na):
297.1103, found: 297.1111.
4.22. HPLC of naphthoxylosides
High-performance liquid chromatography was run on a
Hewlett Packard Series II 1090 Liquid chromatograph
and a SupelcoTM LC-18-DB column (15 cm · 4.6 mm,
5 lm). The system was controlled by the Hewlett Packard
ChemStation for LC software suite. Mobile phase
consisted of H2O + 0.1% trifluoroacetic acid (TFA) with
a gradient of acetonitrile from one minute increasing
by 1.2% per minute until 30 min. The mean retention
times were calculated from three separate runs per
compound.
4.23. General biological methods
The normal mouse 3T3 fibroblasts were obtained from
ATCC, Rockville, MD. Regular cell culture media, L-
glutamine, penicillin–streptomycin, trypsin, and donor
calf serum were obtained from Life Technologies. Min-
imal essential medium (MEM) was purchased from Sig-
ma. Na235SO4 (1310 Ci/mmol) and D-[1-3H]galactose
(20 Ci/mmol) were obtained from Amersham Interna-
tional, UK. The prepacked Superdex Peptide HR 10/
30, Dextran T-500, and octyl-Sepharose CL-4B were
from Pharmacia-LKB, Sweden, and DE-53 DEAE–cel-
lulose was from Whatman. Water for HPLC-analysis
was from a Millipore Milli-Q system.
Acknowledgments
The Swedish Research Council, Association for Interna-
tional Cancer Research, the Cancer Fund, the Swedish
Cancer Society, the American Cancer Society, the
Crafoord Foundation, the Thelma Zoegas Foundation,
and the Bergvall Foundation supported this work.
Supplementary data
4.24. Cell culture, radiolabeling, and extraction
procedures
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2006.05.068.
Cells were cultured as monolayers in MEM supplement-
ed with fetal bovine serum (10%, v/v), L-glutamine
(2 mM), penicillin (100 U/mL), and streptomycin
(100 lg/mL) in an incubator with humidified atmo-
sphere and 5% CO2 at 37 ꢁC. Confluent cells were incu-
bated in low-sulfate, MgCl2-labeling medium
supplemented with glutamine (2 mM), [35S]sulfate
(50 mCi/mL), and different xylosides. Dilutions were
made from 20 mM stock solutions in DMSO/H2O
(1:1, v/v). Incorporation of [3H]galactose was performed
in MEM containing 20 lCi/mL [3H]galactose. After the
incubation period, culture medium was collected
and pooled with two washings of ice-cold PBS
(0.137 M NaCl, 3 mM KCl, 8 mM Na2HPO4, 2 mM
KH2PO4, pH 7.5). Cells were extracted with 0.1–0.2 mL/
cm2 dish of a solution of 0.15 M NaCl, 10 mM EDTA, 2%
(v/v)Triton X-100, 10 mM KH2PO4, pH 7.5, and 5 lg/mL
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