Arginine binding motifs: Design and synthesis of galactose-derived arginine tweezers as galectin-3 inhibitors

Article abstract of DOI:10.1021/jm701266y

Anionic O2 derivatives of methyl 3-deoxy-3-(4-methylbenzamido)-1-thio- β-D-galactopyranoside have been synthesized as inhibitors against galectin-3. The sulfate, H-phosphonate, and benzyl phosphate derivatives showed an increased affinity as compared to t

Full text of DOI:10.1021/jm701266y

J. Med. Chem. 2008, 51, 2297–2301
2297
Brief Articles
Arginine Binding Motifs: Design and Synthesis of Galactose-Derived Arginine Tweezers as
Galectin-3 Inhibitors
Christopher T. Öberg,† Hakon Leffler,‡ and Ulf J. Nilsson*,†
Organic Chemistry, Lund UniVersity, PO Box 124, SE-221 00 Lund, Sweden, and Section MIG, Department of Laboratory Medicine, Lund
UniVersity, SölVegatan 23, SE-223 62 Lund, Sweden
ReceiVed December 6, 2007
Anionic O2 derivatives of methyl 3-deoxy-3-(4-methylbenzamido)-1-thio-ꢀ-D-galactopyranoside have been
synthesized as inhibitors against galectin-3. The sulfate, H-phosphonate, and benzyl phosphate derivatives
showed an increased affinity as compared to the parent unsubstituted galactopyranoside. Modeling revealed
arginine-144 being pinched by the C3 benzamide and O2 anionic substituents in that the benzamide stacked
face-to-face and the anionic O2 substituent ion-paired with the guanidinium moiety.
Introduction
1a). In the presence of a LacNAca C3-benzamido substituent,
the arginine moves 3.5 Å to interact with the benzamido
substituent. As a consequence of this arginine move, it ends up
in close proximity to the O2 of the galactose moiety (Figure
1b). The usefulness of utilizing an additional O2 substituent to
pinch the arginine was corroborated with computer simulations.
First, GRID simulations indicated favorable polar interactions
around the arginine guandinium hydrogens, pointing toward the
potential of anionic O2 substituents, such as sulfates and
phosphates. Second, computer docking of proposed inhibitor
structure 2-O-sulfate 10 to galectin-3 using MacroModel showed
reasonable distances for polar interactions between the guan-
dinium hydrogens and the sulfate substituent. Here we disclose
the synthesis and evaluation of the O2 derivatives 12-17
(sulfate, H-phosphonate, phosphate, benzyl phosphate, acetate,
and benzoate) of methyl 3-deoxy-3-(4-methylbenzamido)-thio-
ꢀ-D-galactopyranoside and present an explanatory model where
the aromatic amide forms a face-to-face interaction with the
arginine π-electrons, while an electron-rich (e.g., anionic) added
substituent is placed in close proximity to the guanidinium
protons leading to potent galectin-3 inhibitors (Figure 1).
The galectins, a family of ꢀ-galactoside-binding proteins, have
been implicated in numerous biological activities,1–11 including
regulation of apoptosis,12,13 intracellular trafficking,14 cells
signaling,15–19 and cell adhesion.20 These activities in turn play
important roles in inflammation,3,15,21–25 immunity,26 and cancer
progression.4,5,8,27–29 Recently, insight into their molecular
mechanisms of actions has deepened, as exemplified by the
T-cell cell surface protein glycosylation patterns controlling
sensitivity to galectin-1-induced apoptosis,16 galectin-8 ligand
specificities regulating its cell sorting,30,31 and by the discovery
that galectin-3-induced lattice formation with branched N-
glycans on cell surfaces to regulate cell surface receptor
trafficking.17–19 Several of these discoveries point to galectins
being potential targets for novel anticancer and anti-inflamma-
tory compounds. Hence, the development of high-affinity
inhibitors showing selectivity for individual members of the
galectin family of proteins has emerged as an important task.32
In a series of publications,33–35 we have described the
development of inhibitors with down to low nanomolar affinity
for galectin-3,35 one member of the galectin family. These made
use of arginine residues that line the carbohydrate recognition
domain by forming π-cation interactions with an aromatic
substituent of the inhibitors. Herein, we report on taking this
design concept one step further by adding a substituent located
close to the edge of the arginine guanidine group that, together
with a face-to-face stacking aromatic substituent, “pinches” an
arginine residue. Such interactions with arginines are found in
protein structures and have also been suggested in protein–ligand
complexes, for example, binding of Adenophostin A mimics
by the Adenophostin-Ins(1,4,5)P3 receptor, where a purine
mimic together with a phosphate group synergistically enhance
binding to an arginine side chain,36,37 as well as in synthetic
arginine receptors.38–40 In an extended galectin-3 binding pocket
close to O3 of bound galactose is an arginine (Arg144; Figure
Chemistry
Starting from the known galactoside 1, the key intermediate
5 was obtained through deacetylation (f2), benzylidenation
(f3), subsequent reduction with polymer-supported triphe-
nylphosphine (f4), and finally N-acylation (Scheme 1). With
this, we were in position to create a small library of galectin-3
“tweezers” by derivatizing the O2 position. Sulfation of 5 using
sulfur trioxide was straightforward to obtain 6, as was acylations
with acetyl and benzoyl chloride to obtain 8 and 9. The
introduction of H-phosphonate41 using phosphorus trichloride
required care in maintaining dry conditions to obtain good yields
of 7. Sequential treatment of 7 with trimethylsilyl chloride,
iodine, and water/pyridine resulted in 10.42 With benzylidene-
protected compounds 6-10 in hand, all that remained was
deprotection to obtain the final compounds 12-14, 16, and 17.
* To whom correspondence should be addressed. Tel.: +46 46 2228218.
Fax: +46 46 2228209. E-mail: ulf.nilsson@organic.lu.se.
a Abbreviations: LacNAc, N-acetyllactosamine; TEA, triethylamine; Bn,
benzyl; Piv, pivaloyl; DCM, dichloromethane; pTsOH, 4-toluenesulfonic
acid.
†
Organic Chemistry, Lund University.
‡
Department of Laboratory Medicine, Lund University.
10.1021/jm701266y CCC: $40.75
2008 American Chemical Society
Published on Web 03/05/2008

2298 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Brief Articles
While deprotection of 8 and 9 using acidic Dowex 50 × 8–400
resin proved uneventful, the anionic substituents of 6,7, and 10
(also 11, vide infra) were more labile and required milder
conditions. For instance, Dowex 50 × 8–400 caused partial
desulfation of 6 and hydrogenation over Pd(OH)2/C is incom-
patible with 7. Fortunately, debenzylation could be successfully
achieved of 6 with hydrogenation over Pd(OH)2/C and by
treatment of 7 and 10 with aqueous acetic acid. Synthesis of
the benzylphosphate 11 from 7 by pivaloyl chloride-mediated
coupling of benzyl alcohol followed by iodine oxidization and
water/pyridine hydrolysis41 was accompanied by concomitant
debenzylidenation to give the deprotected 15 together with 11.
Immediate treatment of 11 with aqueous acetic acid completed
the debenzylidenation to provide 15.
To evaluate the effect on binding affinity toward galectin-3,
reference compounds 18, 19, and 20 were needed. With the
amide 18 available from 5 via acetal hydrolysis, and the parent
unsubstituted galactoside 19 commercially available, only the
2-O-sulfate 20 remained to be synthesized. Hence, compound
20 was obtained in a short three-step synthesis from 19 via acetal
protection,43 sulfation, and deprotection (Scheme 2).
Biology
Compounds 12-17, together with the three reference com-
pounds 18-20, were evaluated in vitro against galectin-3 using
a fluorescence polarization assay44 (Table 1). The sulfate 12,
H-phosphonate 13, and benzyl phosphate 15 derivates show
increased affinity relative to the parent compound 18, with the
sulfate 12 showing a 2.5-fold increase (Table 1). The O2
phosphate 14 and the O2 acetate 16 have similar affinities as
that of 18, while the O2 benzoate 17 shows weaker, but not
abolished, binding. Of interest is the relative and combined effect
of each arm of the “tweezers”, that is, the effect of the 2- and
3-substituent alone and taken together. The O2-sulfate 20
increases the affinity 2-fold, while the 3-benzamide 18 increases
the affinity 27-fold. Thus, taken together (i.e., 68-fold enhance-
ment by 12), the effects of the arene and the anion were roughly
additive. The 2-fold affinity enhancement due to the sulfate
moiety at first appear less than expected, because arginine salt
bridges have in many cases been demonstrated to confer much
larger affinity enhancements.45 However, large affinity enhance-
ments due to salt bridges are typically observed for buried45
ion pairs in more hydrophobic local environments, while the
salt bridge of the galectin-3:20 complex is exposed to surround-
ing water. In addition, the conformational flexibility of galectin-3
Arg144 is presumably restricted by ion pairing with a sulfate
group, which would result in a larger entropy loss upon binding
to sulfated 20 compared to the nonsulfated 18. This hypothesis
is supported by dynamic simulations, where galectin-3 Arg144
experience larger flexibility in complex with 18 than in complex
with 20.
Figure 1. (a) CRD-section of galectin-3 cocrystallized with LacNAc.34,48
Emphasized in the transparent surface is an arginine (Arg144) in the
extended binding pocket close to galactose O3. Arg144 is adopting a
position close to the remainder of the protein surface. (b) CRD-section
of galectin-3 cocrystallized with a 3-benzamido-LacNAc derivative.34,49
Arg144 has moved 3 Å to adopt a conformer that allows for a π-cation
interaction with the benzamido substituent. This unexpected change in
preferred Arg144 side-chain conformation potentially allows for further
interactions with galactose derivatives, as the arginine now is suitably
placed for interactions with O2 substituents. (c) GRID simulations
suggested polar interactions to be favorable around the edges of the
guanidine of the arginine. As shown here, MacroModel simulations
further support this model and place the O2 substituent sulfate of
galactose derivative 12 at reasonable distances to the arginine for polar
interactions.
Conclusions
Substitution at the O2 position offers a new affinity-enhancing
venue of derivatizing methyl galactosides as galectin-3 inhibi-
tors. The enhancements are significant for the sulfate 12,
H-phosphonate 13, and benzyl phosphate 15. For the phosphate
14, acetate 16, and benzoate 17, the substitution was neutral or
slightly disadvantageous. In no case did the substituent cause
abolished binding. Computer simulations corroborated our
model of arginine pinching in that a compound that shows
favorable binding in the simulations does in fact show increased
affinity in vitro. Hence, the combined use of suitably positioned
aromatic and of electron-rich (anionic) structural elements in

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2299
Scheme 1a
a (a) NaOMe, MeOH, 91%; (b) PMB(OMe)2, CSA, MeCN, 91%; (c) PPh3-PS, THF/H2O, 86%; (d) pMeBzCl, DCM, pyr, 98%; (e) SO3-NMe3, DMF,
50 °C, quant; (f) (i) PCl3, imidazole, DCM/MeCN, Et3N, (ii) pyr/H2O, 79%; (g) pyr, AcCl, 94%; (h) pyr, BzCl, 77%; (i) Pd(OH)2, MeOH, 52%; (j) HOAc
(aq 30%), 90-95%; (k) (i) pyr, TMSCl, (ii) I2, (iii) H2O; (l) (i) BnOH, pyr, PivCl, (ii) DCM, MeCN, I2, (iii) pyr/H2O, crude 11 together with 44% of 15;
(m) HOAc (aq 30%), 52% from 5; (n) HOAc (aq 46%), 84% in total yield from 7; (o) Dowex 50 × 8, MeOH, 88%; (p) Dowex 50 × 8, MeOH/DCM, 62%;
(q) Dowex 50 × 8, MeOH/H2O/DCM, 60% (PMP ) 4-methoxyphenyl).
Scheme 2a
Table 1. Kd Values against Galectin-3 as Measured by a Fluorescence
Polarization Assay44
a (a) DMP, TsOH; (b) SO3-NMe3, DMF, TEA; (c) HOAc (aq 30%),
26% from 19.
an inhibitor can indeed pinch arginine side chains and result in
beneficial interactions where both the aromatic and the anionic
group contribute to affinity enhancements. As arginine side
chains are common in protein binding sites, we believe that the
present findings are of importance for the development of
protein–ligands/inhibitors in general.
Materials and Methods
General Methods. NMR spectra were recorded with Bruker
DRX 400 MHz and Avance II 400 MHz spectrometers at ambient
temperature. 1H NMR spectra were assigned using two-dimensional
methods (COSY). Chemical shifts are given in ppm downfield from
a Average and standard deviation of repeated single point measurements.
the signal for Me4Si, with reference to residual CHCl3, DMSO,
HDO, or CD2HOD. HRMS (ESI) was recorded on a Micromass
Q-TOF micro spectrometer. Reactions were monitored by TLC
using aluminum-backed silica gel plates (Merck 60F254) and
visualized using UV light and by charring with ethanolic H2SO4
(7%). Preparative chromatography was performed using silica gel
(Amicon Matrex 35–70 µm, 60 Å) columns. Preparative TLC was
performed using glass-backed silica gel plates (200 × 200 × 1
mm, 60F254). DMF was distilled and stored over 4 Å molecular
sieves. THF was dried by passing through activated alumina. Other
solvents were dried by storing over activated 4 Å molecular sieves.
Reagents were supplied by Sigma-Aldrich and used as is. The
anionic compounds were typically stored as their TEA salts,
prepared by concentrating a solution of the anion with excess TEA
(after flash chromatography etc). This is reflected in the NMR
spectra.
Methyl 3-Deoxy-3-(4-methylbenzamido)-2-O-sulfo-1-thio-ꢀ-
D-galactopyranoside 12. Compound 6 (14 mg, 22.3 µmol) and
Pd(OH)2 (30 mg) was stirred in MeOH (1 mL) under H2 (ca. 60
bar) at rt for 48 h (more Pd(OH)2 (30 mg) was added after 20 h).

2300 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Brief Articles
The Pd(OH)2 was filtered off before the reaction was concentrated
with excess TEA and purified by column chromatography (SiO2,
DCM/MeOH/H2O 135:35:5) to give 12 in 52% yield. 1H NMR
(CD3OD, 400 MHz) δ 7.83 (d, J ) 8.2 Hz, 2H), 7.25 (d, J ) 8.3
Hz, 2H), 4.66 (app. t, J ) 9.6 Hz, 1H), 4.53 (d, J ) 9.5 Hz, 1H),
4.41 (d, 2.6 Hz, 1H), 4.04 (dd, J ) 2.7, 10.5 Hz, 1H), 3.80–3.65
(m, 3H), 3.11 (q, J ) 7.3 Hz, 1.3H), 2.37 (s, 3H), 2.22 (s, 3H),
1.26 (t, J ) 7.3 Hz, 2.3H). ESIMS m/z calcd for [C15H20NO8S2]-,
406.0630; found, 406.0612.
3.9 Hz, 1H), 2.38 (s, 3H), 2.21 (s, 3H), 2.03 (s, 3H). ESIMS m/z
calcd for [C17H23NO6S+Na]+, 392.1144; found, 392.1135.
Methyl 2-O-Benzoyl-3-deoxy-3-(4-methylbenzamido)-1-thio-
ꢀ-D-galactopyranoside 17. Dowex 50 × 8–400 (∼20 mg) was
added to a suspension of 9 (15 mg, 27 µmol) in MeOH (4 mL).
DCM was added until the solution cleared and the reaction was
stirred at rt for 17 h. The reaction was concentrated and purified
by preparative TLC (SiO2, DCM/MeOH 100:5) to give 17 in 62%
1
yield. H NMR (CDCl3, 400 MHz) δ 8.00 (bd, J ) 7.1 Hz, 2H),
Methyl 3-Deoxy-3-(4-methylbenzamido)-2-O-phosphono-1-
thio-ꢀ-D-galactopyranoside 13. HOAc (300 µL) was added to a
stirred solution of 7 (30 mg, 49 µmol) in H2O (700 µL) at rt. After
3 h, the reaction was concentrated and purified by column
chromatography (SiO2, DCM/MeOH 100:5 f 100:10 f 100:15
gradient) to give 13 in 99% yield of 90–95% purity. 1H NMR
(CD3OD, 400 MHz) δ 7.89 (d, J ) 8.2 Hz, 2H), 7.25 (d, J ) 8.0
Hz, 2H), 6.94 (d, J ) 636 Hz, 1H), 4.50 (d, J ) 9.5 Hz, 1H), 4.42
(app. q, J ) 10 Hz, 1H), 4.28 (d, J ) 2.7 Hz, 1H), 4.05 (dd, J )
2.8, 10.1 Hz, 1H), 3.80–3.62 (m, 3H), 3.16 (q, J ) 7.3 Hz, 3H),
2.38 (s, 3H), 2.33 (s, 3H), 1.28 (t, J ) 7.3 Hz, 5.5H). ESIMS m/z
calcd for [C15H21NO7SP]-, 390.0776; found, 390.0759.
Methyl 3-Deoxy-3-(4-methylbenzamido)-2-O-phospho-1-thio-
ꢀ-D-galactopyranoside 14. Compound 10 (6.0 mg, 9.6 µmol) was
dissolved in HOAc (aq, 30%, 1 mL). After 2.5 h, the reaction
mixture was concentrated and purified by column chromatography
(SiO2, DCM/MeOH/H2O 130:35:5 f 65:35:5 gradient) to give 14
in 88% yield. 1H NMR (CD3OD, 400 MHz) δ 7.92 (d, J ) 8.2 Hz,
2H), 7.25 (d, J ) 8.0 Hz, 2H), 4.53–4.44 (m, 2H), 4.42 (d, J ) 2.5
Hz, 1H), 3.93 (dd, J ) 2.6, 9.9 Hz, 1H), 3.80–3.65 (m, 3H), 3.13
(q, J ) 7.3 Hz, 13H), 2.37 (s, 3H), 2.23 (s, 3H), 1.27 (t, J ) 7.3
Hz, 22H). ESIMS m/z calcd for [C15H21NO8SP]-, 406.0726; found,
406.0715.
7.57–7.48 (m, 3H), 7.39 (app. t, J ) 7.9 Hz, 2H), 7.13 (d, J ) 8.0
Hz, 2H), 6.92 (d, J ) 8.7 Hz, 1H), 5.54 (app. t, J ) 10.1 Hz, 1H),
4.71 (d, J ) 9.8 Hz, 1H), 4.59 (ddd, J ) 10.6, 8.7, 2.8 Hz, 1H),
4.31 (d, J ) 2.5 Hz, 1H), 4.04 (dd, J ) 4.2, 12.1 Hz, 1H), 3.97
(dd, J ) 4.0, 12.1 Hz, 1H), 3.77 (app. t, J ) 3.9 Hz, 1H), 2.33 (s,
3H), 2.24 (s, 3H). ESIMS m/z calcd for [C22H25NO6S+Na]+,
454.1300; found, 454.1285.
Methyl 3-Deoxy-3-(4-methylbenzamido)-1-thio-ꢀ-D-galacto-
pyranoside 18. Compound 5 (18 mg, 40.4 µmol) and acidic resin
Dowex 50 × 8–400 (18 mg) were suspended in MeOH/H2O
(95:5, 2 mL) and DCM (∼2 mL) was added until the solution
cleared. The reaction was stirred at rt for 20 h before being
concentrated and unreacted 5 removed by column chromatography
(SiO2, DCM/MeOH 100:5 f 100:10 gradient) to give 18 in 60%
1
yield. H NMR (CD3OD, 400 MHz) δ 7.77 (d, J ) 8.2 Hz, 2H),
7.27 (d, J ) 7.9 Hz, 2H), 4.38 (d, J ) 9.5 Hz, 1H), 4.11 (dd, J )
3.1, 10.3 Hz, 1H), 4.02 (d, J ) 3.0 Hz, 1H), 3.81–3.63 (m, 4H),
2.39 (s, 3H), 2.22 (s, 3H).
Methyl 2-O-Sulfo-1-thio-ꢀ-D-galactopyranoside 20. Methyl
ꢀ-D-thiogalactopyranoside 19 (100 mg, 0.48 µmol) and pTsOH (9.0
mg, 47.6 µmol) were suspended in 2,2-dimethoxypropane (9.6 mL)
rt for 21 h. The reaction was quenched by addition of TEA (100
µL), concentrated, and purified by column chromatography (SiO2,
DCM/TEA 100:1) to give methyl 3,4-O-isopropylidene-6-O-(2-
methoxy-2-propyl-1-thio-ꢀ-D-galactopyranoside (143 mg). TEA
(200 µL) and SO3-NMe3 (280 mg, 2.0 mmol) were added to a
solution of this compound (130 mg) in DMF (4 mL). The reaction
was stirred at 70 °C for 48 h to give a mixture containing methyl
3,4-O-isopropylidene-6-O-(2-methoxy-2-propyl-2-O-sulfo-1-thio-
ꢀ-D-galactopyranoside. Half of the volume was removed before
HOAc (aq, 30%, 25 mL) was added and the reaction was stirred at
rt. After about 24 h, the reaction was concentrated and purified by
column chromatography twice (DCM/MeOH/H2O 130:35:5 f
65:35:5) to give 20 in 26% yield starting from 19. 1H NMR (CDCl3,
400 MHz) δ 4.53 (d, J ) 9.8 Hz, 1H), 4.37 (app. t, J ) 9.4 Hz,
1H), 4.05 (d, J ) 3.3 Hz, 1H), 3.88 (dd, J ) 3.0, 9.3 Hz, 1H),
3.81–3.70 (m, 3H), 3.21 (q, J ) 7.3 Hz, 5.5H), 2.23 (s, 3H), 1.29
(t, J ) 7.3 Hz, 8.3H). ESIMS m/z calcd for [C7H13O8S2]-, 496.1195;
found, 496.1180.
Methyl 3-Deoxy-3-(4-methylbenzamido)-2-O-(O-benzyl-phos-
pho)-1-thio-ꢀ-D-galactopyranoside 15. Compound 7 was rendered
anhydrous by repeated evaporation with dry pyridine. BnOH (17
µL, 164 µmol) followed by PivCl (30 µL, 245 µmol) was added to
a stirred solution of 7 (50 mg, 81.8 µmol) in pyridine (2 mL) at rt
under a nitrogen atmosphere. Additional BnOH and PivCl was
added until all of 5 was consumed, at which point iodine (41.5
mg, 164 µmol) in pyridine/H2O (2 mL) was added. Excess Na2S2O5
was added to scavenge unreacted iodine and the reaction was
concentrated. The reaction was purified by column chromatography
(SiO2, DCM/MeOH 100:0 f 100:5 f 100:10 f 100:15 with 1%
TEA gradient) to yield the pure debenzylidenated 15 in 44%
together with methyl 3-deoxy-4,6-O-(4-methoxybenzylidene)-3-(4-
methylbenzamido)-2-O-(O-benzyl-phospho)-1-thio-ꢀ-D-galactopy-
ranoside 11. Compound 11 was debenzylidenated without further
purification by dissolving in HOAc (1.2 mL) and H2O (2.8 mL),
and the reaction was stirred at rt for 1.5 h before more HOAc (1.2
mL) was added. After another 3 h, the reaction was concentrated
cold and purified by column chromatography (SiO2, DCM/MeOH/
TEA 100:5:1 f 100:10:1 f DCM/MeOH/H2O 65:35:5 gradient)
to provide additional portion of pure 15. The two portions of 15
Fluorescence-Polarization Experiments and Kd Determina-
tions. The Kd values were determined as previously described44,46
with human galectin-3 at 1 µM, the fluorescent probe 2-(fluorescein-
5/6-yl-carbonylamino)-ethyl 3-(4-methoxybenzyl)-ꢀ-D-galactopy-
ranosyl-(1 f 4)-2-acetamido-2-deoxy-ꢀ-D-glucopyranoside47 at 0.1
µM, and inhibitors at either 5000, 1000, 200, 40, 8, or 1.6 µM.
The average values and standard deviations are from 4 to 12
measurements.
1
were pooled to provide a total yield of 84%. H NMR (CD3OD,
400 MHz) δ 9.23 (d, J ) 5.0 Hz, 1H), 7.91 (d, J ) 8.2 Hz, 2H),
7.35–7.18 (m, 7H), 5.07 (dd, J ) 5.5, 12.1 Hz, 1H), 4.93 (dd, J )
5.7, 12.1 Hz, 1H), 4.61–4.50 (m, 2H), 4.35 (d, J ) 2.6 Hz, 1H),
4.07–4.00 (m, 1H), 3.79–3.65 (m, 3H), 3.11 (q, J ) 7.3 Hz, 7H),
2.37 (s, 3H), 2.17 (s, 3H), 1.24 (t, J ) 7.3 Hz, 10H). ESIMS m/z
calcd for [C22H27NO8SP]-, 496.1195; found, 496.1180.
Acknowledgment. We thank Lund University, the Swedish
Research Council, the programs “Glycoconjugates in Biological
Systems” (GLIBS) and “Chemistry for Life Sciences” sponsored
by the Swedish Strategic Research Foundation for financial
support. We thank Barbro Kahl-Knutsson for performing the
fluorescence polarization assay and Molecular Discovery Ltd.
for providing an academic licence of the GRID program suite.
C.T.Ö. thanks Dr. Anne Imberty for laboratory time and space.
Methyl 2-O-Acetyl-3-deoxy-3-(4-methylbenzamido)-1-thio-ꢀ-
D-galactopyranoside 16. Dowex 50 × 8–400 (∼10 mg) was added
to 8 (12 mg, 24.6 µmol) in MeOH (1.0 mL). More Dowex 50 ×
8–400 was added after 2 h and the reaction was stirred at rt for
22 h. The reaction was filtered, concentrated, and purified by
preparative TLC (SiO2, DCM/MeOH 100:5) to give 16 in 88%
1
yield. H NMR (CDCl3, 400 MHz) δ 7.62 (d, J ) 8.2 Hz, 2H),
7.21 (d, J ) 8.0 Hz, 2H), 6.80 (d, J ) 8.9 Hz, 1H), 5.27 (app. t,
J ) 10.1 Hz, 1H), 4.54 (d, J ) 9.8 Hz, 1H), 4.42 (ddd, J ) 2.9,
9.1, 10.3 Hz, 1H), 4.21 (d, J ) 2.6 Hz, 1H), 4.00 (dd, J ) 4.3,
12.1 Hz, 1H), 3.93 (dd, J ) 4.1, 12.1 Hz, 1H), 3.68 (app. t, J )
Supporting Information Available: Experimental procedures
and data for compounds 2–11 and 1H NMR spectra of compounds
2-18 and 20. This material is available free of charge via the
Internet at http://pubs.acs.org.

Brief Articles
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(48) PDB id code 1kjl.
(49) PDB id code 1kjr.
JM701266Y