Probing the carbohydrate recognition domain of E-selectin: The importance of the acid orientation in sLex mimetics

Article abstract of DOI:10.1016/j.bmc.2009.11.024

The selectin-leukocyte interaction is the initial event in the early inflammatory cascade. This interplay proceeds via the terminal tetrasaccharide sialyl Lewis^x (sLe^x), present on physiological selectin ligands and E- and P-selectins located on the endothelial surface. Blocking this process is regarded as a promising therapeutic approach for inflammatory diseases where excessive leukocyte efflux is responsible for tissue damage. Selectin antagonists are generally based on sLe^x as lead structure, containing the essential pharmacophores pre-oriented in the bioactive conformation. In this work, we describe a set of competitive sLe^x mimetics possessing the carboxylic acid pharmacophore equipped with additional hydrophobic substituents as neuraminic acid (Neu5Ac) replacements. This small library of antagonists derived from Huisgen-1,3-dipolar cycloadditions allows to further probe the carbohydrate recognition domain of E-selectin.

Full text of DOI:10.1016/j.bmc.2009.11.024

Bioorganic & Medicinal Chemistry 18 (2010) 19–27
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Probing the carbohydrate recognition domain of E-selectin:
The importance of the acid orientation in sLex mimetics
Alexander Titz a, John Patton b, Martin Smiesko a, Zorana Radic a, Oliver Schwardt a,
John L. Magnani b, Beat Ernst a,
*
a Institute of Molecular Pharmacy, University of Basel, CH-4056 Basel, Switzerland
b GlycoMimetics Inc., Gaithersburg, MD 20878, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The selectin–leukocyte interaction is the initial event in the early inflammatory cascade. This interplay
proceeds via the terminal tetrasaccharide sialyl Lewisx (sLex), present on physiological selectin ligands
and E- and P-selectins located on the endothelial surface. Blocking this process is regarded as a promising
therapeutic approach for inflammatory diseases where excessive leukocyte efflux is responsible for tissue
damage. Selectin antagonists are generally based on sLex as lead structure, containing the essential phar-
macophores pre-oriented in the bioactive conformation. In this work, we describe a set of competitive
sLex mimetics possessing the carboxylic acid pharmacophore equipped with additional hydrophobic sub-
stituents as neuraminic acid (Neu5Ac) replacements. This small library of antagonists derived from Huis-
gen-1,3-dipolar cycloadditions allows to further probe the carbohydrate recognition domain of E-selectin.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 25 August 2009
Accepted 7 November 2009
Available online 13 November 2009
Keywords:
E-selectin
Sialyl Lewisx
Pre-organization
Antagonist
Neu5Ac replacement
1. Introduction
ments11,12 and subsequently confirmed by X-ray crystallography.13
To improve the low affinity of the sLex/E-selectin interaction (IC50 ap-
Selectins, a family of cell adhesion molecules, play a key role in
the early inflammatory response, an essential mechanism of the
immune defense.1 In case of an inflammation, interactions of selec-
tins with leukocytes lead to the characteristic tethering and rolling
of the white blood cells on the vascular endothelium. In a second
step, firm adhesion and migration through the endothelial mono-
layer, a process mediated by protein–protein interactions as for
example of integrins with members of the IgG superfamily,2 lead
to the extravasation of leukocytes to the site of inflammation.
However, in case of excessive extravasation of leukocytes, this
mechanism originally intended as a defensive strategy can gener-
ate severe acute or chronic reactions as, for example, in reperfusion
injury, stroke or rheumatoid arthritis.3,4 Therefore, the antagonism
of the selectins is considered to be a valuable approach for the
treatment of inflammatory diseases.5
prox. 1 mM),8 pre-organization of the pharmacophores in their bioac-
tive orientation resulting in a reduction of entropy costs upon binding
has been successfully explored.14–16
Whereas numerous modifications of the Lex core have been
studied,8,9 only a few reports examined replacements of the
Neu5Ac moiety (reviewed in 17), for example by anionic substitu-
ents (sulphate18–21 or phosphate20), lactic acid derivatives (e.g.,
CGP69669A, Fig. 1C)14 or (S)-isoserine derivatives (Fig. 1D).22 For
the latter, the examined substituents R1 and R2 cover a broad range
including aryl sulfonamides, fatty acid amides or polyhydroxylated
alkyl chains, but do not include heterocyclic fragments. The
reported relative affinities (rIC50 with sLex having a rIC50 of 1)
range from 0.6 to 2.0 for 2° amines and amides, 0.2–0.6 for N-alkyl-
ated amides and 3° amines and 0.12–0.26 for sulfonamides (R1 = H,
R2 = S(O2)R3). Overall, none of the analyzed isoserine derivatives
showed an improved affinity compared to the (S)-cyclohexyllac-
tate derivative CGP69669A14,23 (rIC50 = 0.08, Fig. 1C).
In the search for selectin antagonists, the terminal tetrasaccharide
epitope sialylLewisx (1, sLex, Fig. 1A),6,7 present inphysiologicalselec-
tin ligands, served as lead structure.8–10 The pharmacophores essen-
tial for binding to E-selectin are four hydroxy groups (3- and 4-OH
In this communication, we report the synthesis and affinity data
of a library of E-selectin antagonists 2 where Neu5Ac is replaced by
(S)-isoserine modified by 1,3-dipolar cycloaddition (Fig. 1E).
of L-fucose and 4- and 6-OH of D-galactose) and the carboxylate of
N-acetylneuraminic acid (Neu5Ac).8 The bioactive conformation
(Fig. 1B) of sLex and thus the spatial arrangement of the pharmaco-
phores in the bound state, was determined by trNOE NMR experi-
2. Results and discussion
Molecular modeling studies with 2a (Fig. 1E, R = H) containing an
unsubstituted triazole predicted a limited pre-organization of the
carboxylate in the bioactive conformation11–13 (see acid orientation
* Corresponding author. Tel.: +41 61 267 1550; fax: +41 61 267 1552.
E-mail address: beat.ernst@unibas.ch (B. Ernst).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.024

20
A. Titz et al. / Bioorg. Med. Chem. 18 (2010) 19–27
Figure 1. (A) The minimal E-selectin binding epitope: sialyl Lewisx (1, sLex, essential pharmacophores in bold face); (B) the bioactive conformation of sLex as determined by
trNOE NMR experiments;11,12 (C) replacement of Neu5Ac by (S)-cyclohexyllactate led to CGP69669A14,23 with a 12-fold improved affinity compared to 1; (D) potential
interactions of isoserine derivatives with the E-selectin binding site; sc: interactions with amino acid side chains; bb: interactions with the backbone of E-selectin; (E) E-
selectin antagonists where Neu5Ac is replaced by (S)-isoserine modified by 1,3-dipolar cycloaddition.
in Fig. 2). Furthermore, a hydrophobic pocket formed by Tyr44, Pro46
and Tyr48 offers additional contacts for substituents of anti-substi-
tuted triazoles. Therefore, in a click chemistry approach24,25 based
on the Huisgen 1,3-dipolar cycloaddition,26 the azido functionality
in isoserine derivative 16 was reacted with an array of commercially
available alkynes to obtain the desired triazoles 2 (Scheme 1).
ForthesynthesisoftheNeu5Acreplacingmoiety, theelectrophile
12 is derived from (R)-isoserine (9), which was obtained from (R)-
malic acid (3, Scheme 1). Acetal protection of the hydroxy group
and the 1-carboxylate of 3 led to cyclohexyl-dioxolane 4 in 98%
yield,27 presenting the second carboxylic acid for a selective trans-
formation into the acid chloride 5 and the corresponding acyl azide
6, the precursor for a Curtius rearrangement. Under acidic condi-
tions, the rearrangement product, the isocyanate 7, cyclized to car-
12. Deprotection by hydrogenolysis (?15) followed by reinstalla-
tion of the azido group with a CH2Cl2-free diazo transfer28 yielded
16. Finally, a small library of E-selectin antagonists (2a–f, Table 1)
was obtained by click chemistry with the corresponding alkynes.
Compound 2a was obtained in low yield by cyloaddition with
TMS–acetylene and subsequent fluoride mediated de-silylation.
The tetrahydro-benzotriazolyl compound 2g was obtained by
inverting the reaction sequence, that is, the fully protected azide
14 was first reacted with benzotriazolylpropyne to give 17, fol-
lowed by hydrogenolysis of the benzyl groups and partial hydroge-
nation of the benzotriazol moiety (Scheme 2).
The obtained sLex mimetics were tested in a static cell-free
in vitro assay for their binding to E-selectin.29 All compounds with
the exception of 2e are competitive antagonists (Table 1), with
affinities in the range of sLex (1). Interestingly, azide 16 and the
unsubstituted triazole 2a show the best affinities in this series with
rIC50s of 0.29 and 0.48, respectively.
bamate
8
and subsequently hydrolyzed to (R)-isoserine
hydrochloride (9). The overall yield for the four steps from 4 to 9
was 51%. Carbamate 8, formed as an intermediate, could clearly be
verified by 1H NMR (dd for H-2 at 5.00 ppm, corresponding to a
downfield shift of 0.8 ppm compared to 9). For the protection of
the primary amine, 9 was transformed into an azide by a CH2Cl2-free
diazo transfer (?10).28 After the formation of a benzyl ester (?11),
the triflate 12 was obtained by treatment with triflic anhydride.
The synthesis of 14 was achieved by a regioselective, Bu2SnO-
mediated alkylation of the core trisaccharide 1316 with triflate
3. Conclusions
A small library of E-selectin antagonists was synthesized and
biologically evaluated. The isoserine modifications were selected
based on molecular modeling considerations, that is, the degree
of pre-organization of the carboxylate in the bioactive conforma-

A. Titz et al. / Bioorg. Med. Chem. 18 (2010) 19–27
21
Figure 2. Histograms of the preferred core conformations/acid orientations for sLex (1, Fig. 2A) and triazole 2a (Fig. 2B) as observed during 12 ns MD simulations using
periodic boundary conditions and explicit solvent (TIP3P water, sampling frequency 1.2 ps). Area color coding: gray—unpopulated, white—low, yellow to orange—medium,
red to black—high population. Pink frame indicates a bioactive region as established by transfer-NOE NMR experiment11,12, blue frame indicates corresponding core
conformation/acid orientation as observed in the crystal structure of sLex (1) complexed with E-selectin13 (PDB-ID: 1G1T); for both compounds, the core conformation is
approx. ꢀ40° and therewith in the range of the bioactive value; the acid orientation of the bound conformation of sLex (1) is approx. 110°, although it is populated by sLex (1)
and triazole 2a, some flexibility is predicted leading to entropy costs upon binding.
Table 1
only in the range of sLex (1). Only the unsubstituted triazole 2a
Relative IC50s (rIC50) of the E-selectin antagonists 16 and 2a–g
and the azido compound 16 showed slightly improved affinities.
Entry
Compound
R =
rIC50
However, when substituents were added to the triazoles (2b–g),
the observed inhibitory potency dropped. Overall, our results as
well as the data published by Kolb22 might allow the conclusion
that the substituents of the triazole attached in close proximity
to the essential carboxylate indeed establish additional interac-
tions with E-selectin, but for the price of an alteration of the acid
orientation. As a result, a weakening of the essential salt bridge,
which was previously shown to be a prerequisite for binding,8,13,30
and consequently a decrease in affinity is observed. Since this
interpretation is based on a computational model, experimental
structural data by X-ray crystallography or NMR spectroscopy of
such a mimetic in complex with E-selectin is needed for a final
proof of our hypothesis.
1
2
16
2a
—
H
0.29
0.48
3
4
2b
2c
1.03
0.88
S
5
6
2d
2e
3.13
n.a.
N
F3C
N
N
7
8
2f
1.18
1.22
N
N
4. Experimental
4.1. General Methods
N
N
2g
Nuclear magnetic resonance spectroscopy was performed on a
Bruker Avance 500 UltraShield spectrometer at 500.13 MHz (1H)
or 125.76 MHz (13C). Chemical shifts are expressed in ppm using
residual solvent peaks31,32 or tetramethylsilane (TMS) as refer-
ences. Assignment of 1H and 13C NMR spectra was achieved using
2D DEPT-135, 1H,1H-COSY/TOCSY and 1H,13C-HSQC/HMBC experi-
ments. For complex molecules, the following prefixes for substruc-
tures are used: Cy (cyclohexyl), Fuc (fucose), Gal (galactose), Lac
(lactate), and Bt (benzotriazol). Ci indicates the ipso substituted
carbons of aromatic systems. IR spectra were recorded on a Per-
kin–Elmer Spectrum One as KBr pellets or as films on NaCl plates.
Optical rotations were measured on a Perkin–Elmer 341 polarime-
ter in the indicated solvents in p.a. quality. ESI mass spectra were
recorded on a Waters micromass ZQ instrument. High resolution
mass spectra were obtained on a ESI Bruker Daltonics micrOTOF
spectrometer equipped with a TOF hexapole detector. TLC was per-
formed using silica gel 60 coated glass plates containing fluores-
cence indicator (Merck KGaA, Darmstadt, Germany) and
visualized by using UV light (254 nm) and/or by charring either
in aqueous KMnO4 solution or in a molybdate solution (a 0.02 M
Relative IC50s (rIC50s) were measured using CGP69669A (rIC50 = 0.08) as reference
compound and are scaled on sLex (rIC50 = 1); n.a. = no competitive inhibition of
CGP69669A binding observed.
tion and the possibility of additional contacts with a hydrophobic
pocket formed by Tyr44, Pro46 and Tyr48. It was earlier reported22
that a related set of antagonists did not substantially improve
binding affinity, probably due to the zwitterionic character of an
amine series and conformational restrictions of an amide series.
Since triazole 2a fulfilled the criteria of pre-organization of the car-
boxylate in the bioactive conformation, a library of triazoles was
synthesized. Docking studies clearly showed that for anti-substi-
tuted triazoles, which are easily available by copper-catalyzed click
chemistry, the above-mentioned hydrophobic pocket appeared
accessible.
The library of the triazoles 2a–g was tested in an in vitro assay.
The affinities towards E-selectin, with the exception of 2e, were

22
A. Titz et al. / Bioorg. Med. Chem. 18 (2010) 19–27
solution of ammonium cerium sulfate dihydrate and ammonium
molybdate tetrahydrate in aqueous 10% H2SO4) with heating to
140 °C for 5 min. Column chromatography was performed using
silica gel 60 (0.040–0.063 mm, Fluka). Hydrogenation reactions
were performed in a shaking apparatus (Parr Instruments Com-
pany, Moline, Illinois, USA) in 250 mL or 500 mL bottles with
4 bar H2 pressure. Solvents were purchased from Fluka and dried
prior to use. CH2Cl2 was dried by filtration through basic aluminum
oxide (Fluka). Dioxane, DME, Et2O and PhMe were dried by distil-
lation from sodium/benzophenone. DMF was dried over activated
molecular sieves and MeOH by distillation from sodium methox-
ide. Molecular sieves (4 Å) were activated in vacuo at 500 °C for
2 h immediately before use.
(125.8 MHz, DMSO-d6): d 172.6 (C-1), 67.0 (C-2), 41.7 (C-3); IR
(film): 3422 (s), 1735 (s), 1623 (m), 1234 (m), 1150 (m), 1079
(m) cmꢀ1; ESI-MS Calcd for C3H8NO5 [MꢀCl]+: 106.1; found: 106.0.
4.1.3. (R)-3-Azido-2-hydroxy propanoic acid (10)
To a solution of 9 (363 mg, 2.56 mmol), NaHCO3 (893 mg,
10.6 mmol) and CuSO4ꢁ5H2O (24 mg, 0.10 mmol) in water (2 mL)
were added TfN3 in PhMe (1 M, 3.1 mL) and MeOH (12 mL).28 After
stirring for 21 h, the organic solvents were removed, and the residue
was taken up in water (20 mL). The olive green mixture (pH 8) was
washed with CH2Cl2 (20 mL) and acidified with 6 N HCl. The result-
ing light red mixture was extracted with EtOAc (3 ꢂ 80 mL). The
combined ester phases were dried over Na2SO4, filtered and the sol-
vent was removed in vacuo to give 635 mg of a 1:1.1 mixture of 10
(83%) and TfNH2. The material was used in the next step without fur-
ther purification. The 1H NMR data were identical to those found in
literature.34
Biological data were obtained using the published ELISA proce-
dure with CGP69669A as reference compound.29
1H NMR (500.1 MHz, DMSO-d6): d 4.22 (X of ABX, J = 3.6, 5.8 Hz,
1H, H-2), 3.47 (A of ABX, J = 3.5 12.8 Hz, 1H, H-3a), 3.40 (B of ABX,
J = 5.9, 12.8 Hz, 1H, H-3b); 13C NMR (125.8 MHz, DMSO-d6): d
173.2 (C-1), 69.8 (C-2), 53.5 (C-3); IR (KBr): 2117 (s, N3), 1733 (s,
CO) cmꢀ1; ESI-MS Calcd for C3H4N3O3 [MꢀH]ꢀ: 130.0; found: 129.9.
4.1.1. (5R)-(2,2-Cyclohexylidene-4-oxo-1,3-dioxolan-5-yl)-
acetic acid (4)
Compound 4 was prepared in analogy to Hanessian et al.27 To a
suspension of (R)-malic acid (3) (4.00 g, 29.8 mmol) in dry Et2O
(100 mL) was added cyclohexanone (3.09 mL, 29.8 mmol) at 0 °C
followed by BF3ꢁEt2O (5.62 mL, 44.8 mmol) via syringe. The mix-
ture was stirred at 0 °C for 1 h and turned into a light red solution.
After stirring for another 18 h at rt, Et2O (100 mL) was added, and
the organic phase was washed with aqueous sodium acetate
(3 ꢂ 10 mL, 10% w/v). The combined aqueous phases were ex-
tracted with Et2O (2 ꢂ 50 mL). The combined organic layers were
dried over Na2SO4 and the solvent evaporated in vacuo affording
4 (6.25 g; 98%) as slightly red colored crystals. The analytical data
were identical to those found in literature.33
4.1.4. Benzyl (R)-3-azido-2-hydroxy propanoate (11)
To a solution of crude 10 (3.97 g) in DMF (30 mL) was added
benzyl bromide (13 mL, 110 mmol) followed by dropwise addition
of NEt3 (19 mL, 137 mmol) at rt. Upon addition of the base, the
exothermic reaction needed to be cooled with a water bath at rt.
After stirring for 1 d, the mixture was concentrated, diluted with
water (100 mL) and extracted with CH2Cl2 (3 ꢂ 100 mL). The com-
bined organic phases were dried over Na2SO4, filtered and the sol-
vents removed in vacuo. The crude product was purified by column
chromatography (petrol ether/EtOAc, 4:1) to give 11 (1.92 g, 63%)
as white solid. The 1H NMR data were identical to those found in
literature.22
[a
]
D ꢀ2.9 (c 1.0, CHCl3); 1H NMR (500.1 MHz, CDCl3): d 10.60 (br
s, RCO2H), 4.72 (X of ABX, J = 3.9, 6.5 Hz, 1H, H-2), 3.00 (A of ABX,
J = 3.9, 17.2 Hz, 1H, H-3a), 2.85 (B of ABX, J = 6.5, 17.2 Hz, 1H, H-
3b), 1.90–1.81 (m, 2H, C5H10), 1.80–1.60 (m, 6H, C5H10), 1.55–
1.35 (m, 2H, C5H10); 13C NMR (125.8 MHz, CDCl3): d 175.1, 172.0
(2 CO), 112.2 (C-ketal), 70.0 (C-2), 36.2 (2C, C5H10), 35.3 (C-3),
24.4 (C5H10), 23.4 (2C, C5H10); IR (KBr): 3208 (m), 2939 (m), 1802
(s), 1733 (s), 1375 (m), 1291 (m), 1197 (s), 932 (m) cmꢀ1; ESI-MS
Calcd for C10H13O5 [MꢀH]ꢀ: 213.1; found: 213.0.
[a]
D +82.9 (c 1.04, CHCl3); 1H NMR (500.1 MHz, CDCl3): d 7.41–
7.39 (m, 5H, Ar–H), 5.26 (s, 2H, PhCH2), 4.40 (m, 1H, H-2), 3.64 (A
of ABX, J = 3.3, 12.8 Hz, 1H, H-3a), 3.50 (B of ABX, J = 4.3, 12.8 Hz,
1H, H-3b); 13C NMR (125.8 MHz, CDCl3): d 172.3 (C-1), 134.9 (Ar–
Ci), 129.02, 128.96, 128.67 (5C, Ar–C), 70.4 (C-2), 68.3 (PhCH2),
54.0 (C-3); IR (KBr): 3392 (m), 2110 (s, N3), 1739 (s, CO), 1199 (s)
cmꢀ1; ESI-MS Calcd for C10H11N3O3Na [M+Na]+: 244.1; found: 243.9.
4.1.2. (R)-3-Amino-2-hydroxy-propionic acid hydrochloride (9)
Compound 4 (6.25 g, 29.2 mmol) was dissolved in SOCl2 (48 mL)
with strong gas evolution. The solution was heated to reflux and the
gas evolution ceased after 15 min. After refluxing the dark solution
for 1 h, the solvent was evaporated and the residue dried under high
vacuum over night. Compound 5 was obtained as a brownish oil, dis-
solved in acetone (56 mL) and cooled to ꢀ20 °C. A solution of sodium
azide (2.56 g, 39.4 mmol) in water (16 mL) was added dropwise to
the solution via syringe and the mixture was stirred for 1.5 h. After
removal of the acetone in vacuo at 0 °C, water (150 mL) was added
and the aqueous phase was extracted with PhMe (3 ꢂ 200 mL).
The combined organic layers containing 6 were dried (Na2SO4) and
concentrated to a final volume of ca. 75 mL. The remaining solution
was slowly heated to reflux for 1 h to yield 7 after removal of the sol-
vent. After addition of aqueous HCl (6 N, 35 mL) to crude 7, the mix-
ture was refluxed for 7 h, then the solvent removed in vacuo, the
residue taken up in water (100 mL) and washed with CH2Cl2
(1 ꢂ 100 mL, 2 ꢂ 50 mL). The combined organic phases were ex-
tracted with aqueous HCl (3 N, 2 ꢂ 50 mL). The aqueousphases were
combined and the water removed by evaporation to give a brownish
oil. Precipitation from PhMe/EtOAc/MeOH gave 9 as off-white solid
(2.10 g, 51%).
4.1.5. Benzyl (R)-3-azido-2-O-trifluoromethanesulfonyl-
propanoate (12)
To a solution of 11 (1.00 g, 4.52 mmol) in dry CH2Cl2 (30 mL) un-
der argon was added 2,6-di-tert-butylpyridine (1.73 mL, 7.69 mmol)
at ꢀ20 °C. After dropwise addition of Tf2O (1.29 mL, 7.69 mmol), the
reaction was stirred for 3 h at ꢀ20 °C. Then, the reaction was allowed
to warm to 0 °C. After stirring for 1 h, additional 2,6-di-tert-butyl-
pyridine (1.73 mL, 7.69 mmol) and Tf2O (1.29 mL, 7.69 mmol) were
added at 0 °C. After stirring for 3 h, the reaction mixture was diluted
with CH2Cl2 (100 mL), washed with ice-cold aqueous KH2PO4 (1 M,
50 mL) and the aqueous phase was extracted with CH2Cl2
(2 ꢂ 100 mL). The combined organic layers were dried over Na2SO4
and the solvent was evaporated in vacuo. Purification by column
chromatography (petrol ether/EtOAc, 10:1) yielded 12 (1.40 g,
88%) as colorless oil. The 1H and 13C NMR data were identical to those
found in literature.22
1H NMR (500.1 MHz, CDCl3): d 7.35–7.41 (m, 5H, ArH), 5.31,
5.28 (A, B of AB, J = 12.0 Hz, 2H, PhCH2), 5.23 (X of A0B0X, J = 3.6,
6.0 Hz, 1H, H-2), 3.84 (A0 of A0B0X, J = 3.6, 13.9 Hz, 1H, H-3a), 3.81
(B0 of A0B0X, J = 6.0, 13.9 Hz, 1H, H-3b); 13C NMR (125.8 MHz,
CDCl3): d 164.4 (C-1), 133.9 (Ar–Ci), 129.1, 128.8, 128.6 (5C, Ar–
C), 118.4 (q, J = 320 Hz, CF3), 81.0 (C-2), 69.0 (PhCH2), 51.5 (C-3);
ESI-MS Calcd for C11H10F3N3O5SNa [M+Na]+: 376.0; found: 376.0.
[a]
+9.9 (c 0.48, H2O); 1H NMR (500.1 MHz, D2O): d 4.52 (X of
D
ABX, J = 4.1, 8.4 Hz, 1H, H-2), 3.45 (A of ABX, J = 4.0, 13.3 Hz, 1H, H-
3a), 3.23 (B of ABX, J = 8.4, 13.3 Hz, 1H, H-3b); 13C NMR

A. Titz et al. / Bioorg. Med. Chem. 18 (2010) 19–27
23
O
O
O
O
C
N
OH
OH
R
(iv)
(i)
(ii)
O
O
O
O
OH
O
O
O
HO
O
O
O
5, R = Cl
6, R = N3
3
4
7
(iii)
NH2*HCl
N3
N3
NH
(viii)
(v)
(vi)
(vii)
O
O
O
O
O
O
OH
OH
OH
OH
HO
HO
BnO
8
9
10
11
N3
H2N
OH
O
OH
O
O
N3
O
O
O
O
(ix)-(x)
(xi)
O
O
O
O
OBn
OH
HO
HO
OH
O
OTf
OBn
OH
HO
BnO
O
O
O
O
O
OBn
OH
HO
OBn
OBn
OH
12
BnO
HO
OBn
OBn
BnO
14
15
1316
N
N
N
N3
R
OH
O
OH
O
O
O
O
O
(xii)
(xiii)-(xiv)
O
O
O
OH
O
ONa
HO
HO
OH
O
OH
OH
O
OH
OH
HO
OH
HO
16
2a-f, see Table I
Scheme 1. Reagents and conditions: (i) Cyclohexanone, BF3ꢁEt2O, Et2O, 0 °C to rt, 1 d (98%); (ii) SOCl2, reflux, 30 min (?5); (iii) NaN3, H2O, acetone, ꢀ20 °C, 1 h (?6); (iv)
PhMe, reflux, 30 min (?7); (v) 6 M HCl, reflux, 6 h (?9, 51% over four steps); (vi) TfN3, CuSO4, NaHCO3, PhMe/H2O/MeOH, rt, 24 h; (vii) BnBr, NEt3, DMF, rt, 1 d (63% over two
steps); (viii) Tf2O, 2,6-tBu2py, CH2Cl2, ꢀ20 °C, 5 h (88%); (ix) 1316, Bu2SnO, MeOH, mol. sieves 3 Å, reflux, 15 h; (x) 12, CsF, DME, rt, 6 d (39% over two steps); (xi) Pd(OH)2/C, H2,
THF, H2O, 4 bar, rt, 72 h (95%); (xii) TfN3, CuSO4, NaHCO3, PhMe/H2O/MeOH, rt, 22 h (36%); for 2a: (xiii) TMS–acetylene, DIPEA, CuCl, MeCN/H2O, 9 d, rt; (xiv) (a) TBAF, HOAc/
THF, rt, 30 min; (b) 1 equiv NaOH, (19% over two steps); for 2b–f; (xiii) (a) alkyne, Cu, CuSO4, EtOH/H2O, 24 h, rt; (b) 1 equiv NaOH; yields for 2b (97%), 2c (97%), 2d (42%), 2e
(94%), 2f (91%).
N
N
N
N
N
N
N
N
N
N
N
N
(i)
(ii)
OH
O
OH
O
O
O
14
O
O
O
O
O
O
O
O
OBn
ONa
HO
HO
OBn
OH
OBn
OH
OBn
OH
BnO
HO
17
2g
Scheme 2. Reagents and conditions: (i) 1-Benzotriazolyl-prop-2-yne, Cu, MeOH/H2O, 48 h, rt (86%); (ii) (a) Pd(OH)2/C, H2, dioxane/H2O, 4 bar, rt, 24 h; (b) 1 equiv NaOH
(39%).

24
A. Titz et al. / Bioorg. Med. Chem. 18 (2010) 19–27
4.1.6. Benzyl (2S)-3-azido-2-O-{1-O-[(1R,2R)-2-O-(2,3,4-tri-O-
benzyl- -fucopyranosyl)-cyclohexyl]-6-O-benzyl-b-
galactopyranos-3-yl}-propionate (14)
82.9 (Gal-C3), 79.0 (Cy–CH), 77.6 (2C, Lac-C2, Cy–CH), 74.7, 72.4,
70.8 (Fuc-C4, Gal-C2, -C5), 69.9 (Fuc-C3), 68.2 (Fuc-C2), 66.9
(Gal-C4), 66.8 (Fuc-C5), 61.8 (Gal-C6), 42.2 (Lac-C3), 29.9, 29.6,
29.5, 23.5 (4 Cy–CH2), 15.6 (Fuc-C6); HR-MS Calcd for
C21H36NO13 [MꢀH]ꢀ: 510.2192; found: 510.2197.
a-
L
D-
Compound 1316 (667 mg, 0.85 mmol) and Bu2SnO (235 mg,
0.94 mmol) were dried in high vacuum for 1 h and subsequently
dissolved in dry MeOH (50 mL) under argon with warming. Acti-
vated molecular sieves (3 Å, 2.00 g) were added to the solution,
which was then refluxed under argon for 15 h. Filtration of the sus-
pension over Celite, evaporation of the solvent, co-evaporation
with dry PhMe and drying for 8 h in high vacuum gave a yellow
oily substance (880 mg). This residue was dissolved in dry DME
(10 mL) under argon and a solution of dry 12 (384 mg, 1.09 mmol)
in dry DME (4 mL) was added. When extensively dried CsF
(168 mg, 1.11 mmol) was added, the solution turned slightly tur-
bid. After stirring for 6 d at rt, TLC indicated a ratio of acceptor to
product of ca. 1:1, but no triflate. A solution of KF (10%) in aqueous
KH2PO4 (1 M, 50 mL) was added and after stirring for 1 h, the reac-
tion was extracted with CH2Cl2 (3 ꢂ 50 mL). The combined organic
phases were dried over Na2SO4 and the solvents were removed in
vacuo. Purification of the residue by column chromatography
(CH2Cl2/iPrOH, gradient 0–4%) yielded 14 (329 mg, 39%) as a white
solid.
4.1.8. Sodium (2S)-3-azido-2-O-{1-O-[(1R,2R)-2-O-(-
L-fucopyra
nosyl)-cyclohexyl]-b- -galactopyranos-3-yl}-propionate (16)
D
To a solution of crude 15 (126 mg) in MeOH/water (3 mL, 7.5:1)
28
was added a solution of TfN3 in PhMe (0.91 mL, 0.36 M). After
stirring for 22 h at rt, the reaction mixture was concentrated and
purified by column chromatography (CH2Cl2/MeOH/water,
10:4:0.8) to give 16 (79 mg, contaminated with silica gel). This
product was used for the 1,3-dipolar cycloaddition reactions with-
out further purification. For analysis and biological testing, a small
sample (6.6 mg) was purified by preparative HPLC to give pure 16
(4.7 mg, 35%, two steps) as free acid. The sodium salt was obtained
by addition of an equimolar amount of aqueous NaOH (1 M, 87
to an aqueous solution of the acid.
lL)
Analytical data for the free acid: [
a]
D ꢀ112.1 (c 0.24, MeOH); 1H
NMR (500.1 MHz, CD3OD): d 4.85 (d, J = 3.8 Hz, 1H, Fuc-H1), 4.62
(q, J = 6.5 Hz, 1H, Fuc-H5), 4.51 (dd, J = 3.5, 5.4 Hz, 1H, Lac-H2),
4.30 (d, J = 7.7 Hz, 1H, Gal-H1), 4.00 (d, J = 2.7 Hz, 1H, Gal-H4),
3.87 (dd, J = 3.4, 9.9 Hz, 1H, Fuc-H3), 3.78–3.74 (m, 2H, Lac-H3a,
Gal-H6a), 3.73–3.63 (m, 5H, Fuc-H2, -H4, Gal-H2, -H6b, 1H of
Cy–CH), 3.57–3.51 (m, 2H, 1H of Cy–CH, Lac-H3b), 3.45–3.41 (m,
2H, Gal-H3, -H5), 2.05–2.03 (m, 2H, Cy–CH2), 1.71 (br s, 2H, Cy–
CH2), 1.41–1.22 (m, 4H, Cy–CH2), 1.18 (d, J = 6.4 Hz, 3H, Fuc-H6);
13C NMR (125.8 MHz, CD3OD): d 175.1 (Lac-C1), 102.8 (Gal-C1),
97.5 (Fuc-C1), 84.8 (Gal-C3), 80.5 (Lac-C2), 79.4 (CH), 77.5 (Cy–
CH), 76.2 (Gal-C5), 74.0 (CH), 72.4 (Gal-C2), 71.7 (Fuc-C3), 70.2
(CH), 68.6 (Gal-C4), 67.6 (Fuc-C5), 62.9 (Gal-C6), 54.3 (Lac-C3),
31.1, 30.2, 24.6 (4C, Cy–CH2), 16.7 (Fuc-C6); IR (KBr, Na-form of
5): 3412 (s), 2111 (s, N3), 1606 (s, CO), 1447 (m) cmꢀ1; HR-MS
Calcd for C21H34N3O13 [MꢀH]ꢀ: 536.2097; found: 536.2091.
1H NMR (500.1 MHz, CDCl3): d 7.37–7.23 (m, 25 H, Ar–H), 5.30
(m, 1H, Lac-H2), 5.23, 5.15 (A, B of AB, J = 12.1 Hz, 2H, PhCH2OCO),
4.96–4.94 (m, 2H, Fuc-H1, 1H of PhCH2), 4.80 (A00 of A00B00,
J = 11.6 Hz, 1H, PhCH2), 4.74 (A000 of A000B000, J = 11.9 Hz, 1H, PhCH2),
4.68 (B00 of A00B00, B000 of A000B000, J = 11.7 Hz, 2H, PhCH2), 4.60 (B0 of
A0B0, J = 11.0 Hz, 1H, PhCH2), 4.57 (M of ABM, J = 2.2, 5.2 Hz, 1H,
Lac-H2), 4.52, 4.48 (A0000, B0000 of A0000B0000, J = 12.0 Hz, 1H, PhCH2),
4.38 (q, J = 7.8 Hz, 1H, Fuc-H5), 4.30 (d, J = 7.7 Hz, 1H, Gal-H1),
4.02–3.97 (m, 3H, Fuc-H2, -H3, Gal-H4), 3.80 (t, J = 8.3 Hz, 1H,
Gal-H2), 3.76–3.74 (m, 2H, 1H of Cy–CH, Gal-H6a), 3.67 (m, 1H,
Fuc-H4), 3.65–3.50 (m, 5H, 1H of Cy–CH, Gal-H5, -H6b, Lac-H3),
3.43 (dd, J = 2.1, 9.3 Hz 1H, Gal-H3), 2.02–1.81 (m, 2H, Cy–CH2),
1.67–1.60 (m, 2H, Cy–CH2), 1.39–1.12 (m, 4H, Cy–CH2), 1.10 (d,
J = 6.3 Hz, 3H, Fuc-H6); 13C NMR (125.8 MHz, CDCl3): d 170.5
(Lac-C1), 139.2, 138.9, 138.8, 138.0, 134.8 (5C, Ar–Ci), 128.8–
127.2 (25C, Ar–CH), 100.2 (Gal-C1), 94.6 (Fuc-C1), 82.8 (Gal-C3),
79.7 (CH), 79.0 (Lac-C2), 78.2 (Fuc-C4), 77.2 (Gal-C2), 76.3 (Cy–
CH), 76.1 (CH), 74.9, 73.7 (2 PhCH2), 73.2 (CH), 73.01, 72.97 (2
PhCH2), 71.1 (CH), 68.9 (Gal-C6), 67.6 (Ph–CH2–OCO), 67.3 (CH),
66.3 (Fuc-C5), 52.8 (Lac-C3), 30.1, 29.7, 29.2, 23.3 (4C, Cy–CH2),
16.6 (Fuc-C6); IR (KBr): 2923 (s), 2106 (s, N3), 1742 (s, CO), 1453
(m) cmꢀ1; ESI-MS Calcd for C56H65N3O13Na [M+Na]+: 1010.4;
found: 1010.5.
4.1.9. Sodium (2S)-3-N-(1,2,3-triazol-1-yl)-2-O-{1-O-[(1R,2R)-2-
O-(-
propionate (2a)
A solution of crude 16 (14 mg) and DIPEA (13.7
MeCN/water (1.5 mL, 2:1) was thoroughly degassed by bubbling
through argon. Subsequently, TMS–acetylene (7.4 L, 54 mol)
and CuCl (5.3 mg, 80 mol) were added and the mixture was stir-
red under argon. After 4 d, additional CuCl (5.3 mg, 80 mol) and
TMS–acetylene (15 L, 110 mol) were added and the reaction
was stirred for another 5 d. The solvents were removed in vacuo,
the residue was taken up in MeOH, filtered (0.2 m) and evapo-
L-fucopyranosyl)-cyclohexyl]-b-D-galactopyranos-3-yl}-
lL, 80 lmol) in
l
l
l
l
l
l
4.1.7. (2S)-3-Amino-2-O-{1-O-[(1R,2R)-2-O-(-
cyclohexyl]-b- -galactopyranos-3-yl}-propanoic acid (15)
To a solution of 14 (293 mg, 297 mol) in THF/water (5 mL, 4:1)
L-fucopyranosyl)-
l
D
rated. After drying for 2 h in high vacuum, the residue was taken
up in TBAF in THF (1 M, 1 mL) and HOAc was added (0.2 mL).
The reaction was stirred for 30 min at rt. Evaporation of the solvent
and purification of the residue by preparative HPLC, followed by
ion-exchange chromatography (Dowex-50, Na-form) and a second
preparative HPLC gave 2.0 mg (19%, two steps) of pure 2a as the
free acid. The sodium salt was obtained by addition of equimolar
amounts of aqueous NaOH to an aqueous solution of the acid.
Analytical data for the free acid: 1H NMR (500.1 MHz, D2O): d
8.08 (s, 1H, triazol-H), 7.78 (s, 1H, triazol-H), 4.95 (d, J = 3.5 Hz,
1H, Fuc-H1), 4.90 (dd, J = 3.4, 14.1 Hz 1H, Lac-H3a), 4.76 (m, 1H,
Lac-H3b), 4.63–4.57 (m, 2H, Fuc-H5, Lac-H2), 4.41 (d, J = 7.0 Hz,
1H, Gal-H1), 4.01 (d, J = 1.7 Hz, 1H, Gal-H4), 3.87 (dd, J = 3.4,
10.4 Hz, 1H, Fuc-H3), 3.77–3.65 (m, 5H, Fuc-H2, -H4, Gal-H6, 1H
of Cy–CH), 3.54 (dd, J = 4.2, 7.4 Hz, 1H, Gal-H5), 3.50–3.43 (m,
3H, Gal-H2, -H3, 1H of Cy–CH), 2.09–2.02 (m, 2H, Cy–CH2), 1.69–
1.63 (m, 2H, Cy–CH2), 1.27–1.17 (m, 4H, Cy–CH2), 1.15 (d,
J = 6.9 Hz, 3H, Fuc-H6); 13C NMR (125.8 MHz, D2O): d 174.9 (Lac-
l
was added Pd(OH)2/C (50 mg) and the reaction mixture was hydro-
genated at 4 bar for 72 h at rt. Additional Pd(OH)2/C (100 mg) was
added and the mixture hydrogenated at 4 bar for another 48 h.
After dilution with MeOH/water (1:1, 20 mL), the mixture was fil-
tered and the solvents were removed to give crude 15 (144 mg),
which was used without further purification.
1H NMR (500.1 MHz, D2O): d 4.99 (d, J = 3.9 Hz, 1H, Fuc-H1),
4.63 (q, J = 6.6 Hz, 1H, Fuc-H5), 4.52 (d, J = 7.8 Hz, 1H, Gal-H1),
4.29 (dd, J = 3.9, 8.2 Hz, 1H, Lac-H2), 4.04 (d, J = 2.8 Hz, 1H, Gal-
H4), 3.91 (dd, J = 6.6, 10.4 Hz, 1H, Fuc-H3), 3.79 (dd, J = 3.0,
9.3 Hz, 1H, Fuc-H2), 3.77–3.71 (m, 4H, Fuc-H4, Gal-H5, -H6a, 1H
of Cy–CH), 3.65–3.60 (m, 2H, Gal-H2, -H6b), 3.55 (dd, J = 3.2,
9.6 Hz, 1H, Gal-H3), 3.51 (m, 1H, 1H of Cy–CH), 3.41 (dd, J = 3.9,
13.8 Hz, 1H, Lac-H3a), 3.22 (dd, J = 8.3, 13.4 Hz, 1H, Lac-H3b),
2.14–2.04 (m, 2H, Cy–CH2), 1.69 (br s, 2H, Cy–CH2), 1.33–1.21
(m, 4H, Cy–CH2), 1.19 (d, J = 6.8 Hz, 3H, Fuc-H6); 13C NMR
(125.8 MHz, D2O): d 171.0 (Lac-C1), 100.1 (Gal-C1), 95.9 (Fuc-C1),

A. Titz et al. / Bioorg. Med. Chem. 18 (2010) 19–27
25
C1), 99.7 (Gal-C1), 95.5 (Fuc-C1), 82.1 (Gal-C3), 78.3 (CH), 78.1
4.1.12. Sodium (2S)-3-N-[4-(3-pyridyl)-1,2,3-triazolyl]-2-O-
{1-O-[(1R,2R)-2-O-( -fucopyranosyl)-cyclohexyl]-b-
galactopyranos-3-yl}-propionate (2d)
In analogy to 2b, compound 2d was prepared from crude 16
(7.1 mg) and 3-ethynyl-pyridine (15 mg, 145 mol). After HPLC,
(CH), 77.2 (Lac-C2), 74.3 (CH), 72.0 (Gal-C5), 70.2 (Fuc-C3), 69.5
(Fuc-C2), 67.9 (Gal-C4), 67.6 (Fuc-C5), 66.4 (2C, 2 CH), 61.3 (Gal-
C6), 52.0 (Lac-C3), 29.6, 29.1, 23.1 (4C, 4 Cy–CH2), 15.2 (Fuc-C6);
HR-MS Calcd for C23H37N3O13Na [M+H]+: 586.2224; found:
586.2214.
a-
L
D-
l
2.2 mg (42%) of the free acid were obtained, which was transferred
into the sodium salt as described above.
Analytical data for the free acid: [
a]
D
ꢀ44.7 (c 0.11, MeOH); 1H
4.1.10. Sodium (2S)-3-N-[4-benzyl-1,2,3-triazol-1-yl]-2-O-{1-O-
NMR (500.1 MHz, CD3OD): d 9.04 (br s, 1H, Ar–H), 8.78 (s, 1H, tria-
zol-H), 8.52 (br s, 1H, Ar–H), 8.30 (d, J = 7.6 Hz, 1H, Ar–H), 7.53 (m,
1H, Ar–H), 5.02 (dd, J = 2.8, 14.5 Hz, 1H, Lac-H3a), 4.83 (d,
J = 3.5 Hz, 1H, Fuc-H1), 4.79 (dd, J = 7.1, 14.2 Hz, 1H, Lac-H3b),
4.72 (dd, J = 2.7, 7.0 Hz, 1H, Lac-H2), 4.59 (q, J = 6.7 Hz, 1H, Fuc-
H5), 4.27 (d, J = 7.2 Hz, 1H, Gal-H1), 3.98 (d, J = 2.3 Hz, 1H, Gal-
H4), 3.85 (dd, J = 3.1, 9.8 Hz, 1H, Fuc-H3), 3.75 (dd, J = 7.2,
11.7 Hz, 1H, Gal-H6a), 3.71–3.65 (m, 4H, Fuc-H2, -H4, Gal-H6b,
1H of Cy–CH), 3.62 (t, J = 8.0 Hz, 1H, Gal-H2), 3.52 (dt, J = 4.4,
9.4 Hz, 1H, 1H of Cy–CH), 3.44–3.38 (m, 2H, Gal-H3, -H5), 2.07–
2.00 (m, 2H, Cy–CH2), 1.72–1.65 (m, 2H, Cy–CH2), 1.41–1.19 (m,
4H, Cy–CH2), 1.13 (d, J = 6.5 Hz, 3H, Fuc-H6); 13C NMR
(125.8 MHz, CD3OD): d 147.2, 135.1, 126.1, 124.8 (4C, Ar–CH),
102.3 (Gal-C1), 97.0 (Fuc-C1), 84.3 (Gal-C3), 78.6 (Lac-C2), 77.0
(CH), 75.9 (CH), 73.1 (CH), 71.9 (CH), 71.3 (Fuc-C3), 71.2 (CH),
67.5 (Gal-C4), 66.7 (Fuc-C5), 62.8 (Gal-C6), 54.1 (Lac-C3), 31.1,
[(1R,2R)-2-O-(-
galactopyranos-3-yl}-propionate (2b)
Crude 16 (7.1 mg) was dissolved in a solution of 1-phenyl-prop-
2-yne (16.4 L, 132 mol) in EtOH/water (1 mL, 3:2). Copper pow-
der (1.0 mg, 15.7 mol) were
L-fucopyranosyl)-cyclohexyl]-b-D-
l
l
l
mol) and CuSO4ꢁ5H2O (3.5 mg, 14
l
added, and the reaction was vigorously stirred for 24 h at rt. Filtra-
tion through cotton, evaporation of the solvents, and microfiltra-
tion (0.2 lm) of a solution of the residue in MeOH/water (1:1)
gave the crude product, which was purified by preparative HPLC.
Pure 2b was obtained as free acid (5.4 mg, 97%) and was subse-
quently transferred into the sodium salt by addition of equimolar
amounts of NaOH.
Analytical data for the free acid: [
a]
ꢀ67.7 (c 0.27, MeOH); 1H
D
NMR (500.1 MHz, CD3OD): d 8.00 (s, 1H, triazol-H), 7.30–7.22 (m,
4H, Ar–H), 7.19 (m, 1H, p-Ar–H), 4.88–4.84 (m, 2H, Fuc-H1, Lac-
H3a), 4.71 (m, 1H, Lac-H3b), 4.62–4.56 (m, 2H, Fuc-H5, Lac-H2),
4.26 (d, J = 7.7 Hz, 1H, Gal-H1), 4.03 (s, 2H, PhCH2), 3.93 (m, 1H,
Gal-H4), 3.86 (dd, J = 3.3, 10.0 Hz, 1H, Fuc-H3), 3.77–3.64 (m, 5H,
Fuc-H2, -H4, Gal-H6, 1H of Cy–CH), 3.61–3.52 (m, 2H, Gal-H2, 1H
of Cy–CH), 3.41 (m, 1H, Gal-H5), 3.33 (m, 1H, Gal-H3), 2.07–2.01
(m, 2H, Cy–CH2), 1.73–1.67 (m, 2H, Cy–CH2), 1.40–1.22 (m, 4H,
Cy–CH2), 1.18 (d, J = 6.5 Hz, 3H, Fuc-H6); 13C NMR (125.8 MHz,
CD3OD): d 174.5 (Lac-C1), 140.7 (triazol-Ci), 131.4 (Ar–Ci), 129.8,
129.7 (4 Ar–CH), 127.6 (2C, triazol-CH, Ar–CH), 102.8 (Gal-C1),
97.4 (Fuc-C1), 84.9 (Gal-C3), 79.5 (Lac-C2), 77.5 (Cy–CH), 76.1
(Cy–CH), 74.0 (Gal-C5), 72.1 (Fuc-C4), 71.7 (Gal-C2), 70.5 (Fuc-
C3), 68.0 (Fuc-C2), 67.6 (2C, Fuc-C5, Gal-C4), 63.0 (Gal-C6), 53.7
(Lac-C3), 32.7 (PhCH2), 31.1, 30.9, 24.6 (4C, 4 Cy–CH2), 16.8 (Fuc-
C6); HR-MS Calcd for C30H43N3O13Na [M+H]+: 676.2694; found:
676.2696.
30.2, 24.6 (4C,
4 Cy–CH2), 16.8 (Fuc-C6); HR-MS Calcd for
C28H40N4O13Na [M+H]+: 663.2490; found: 663.2490.
4.1.13. Sodium (2S)-3-N-[4-(3-trifluoromethyl-phenyl)-1,2,3-
triazol-1-yl]-2-O-{1-O-[(1R,2R)-2-O-(
cyclohexyl]-b- -galactopyranos-3-yl}-propionate (2e)
In analogy to 2b, compound 2e was prepared from crude 16
(7.1 mg) and 3-ethynyl-trifluoromethylbenzene (18.7 L,
130 mol). After HPLC, 5.9 mg (94%) of the free acid were obtained,
a-L-fucopyranosyl)-
D
l
l
which was transferred into the sodium salt as described above.
Analytical data for the free acid: [
a]
D
ꢀ52.9 (c 0.27, MeOH); 1H
NMR (500.1 MHz, CD3OD): d 8.75 (s, 1H, triazol-H), 8.16 (s, 1H, Ar–
H), 8.10 (m, 1H, Ar–H), 7.65–7.61 (m, 2H, Ar–H), 5.01 (dd, J = 2.3,
13.7 Hz, 1H, Lac-H3a), 4.83 (d, J = 3.6 Hz, 1H, Fuc-H1), 4.82–4.76
(m, 2H, Lac-H2, -H3b), 4.59 (q, J = 6.7 Hz, 1H, Fuc-H5), 4.27 (d,
J = 7.6 Hz, 1H, Gal-H1), 3.99 (d, J = 2.5 Hz, 1H, Gal-H4), 3.85 (dd,
J = 3.3, 10.1 Hz, 1H, Fuc-H3), 3.75 (dd, J = 6.9, 11.4 Hz, 1H, Gal-
H6a), 3.72–3.64 (m, 4H, Fuc-H2, -H4, Gal-H6b, 1H of Cy–CH),
3.63 (m, 1H, Gal-H2), 3.52 (dt, J = 4.3, 9.4 Hz, 1H, 1H of Cy–CH),
3.41–3.31 (m, 2H, Gal-H3, -H5), 2.04–1.99 (m, 2H, Cy–CH2), 1.68
(br s, 2H, Cy–CH2), 1.41–1.21 (m, 4H, Cy–CH2), 1.13 (d, J = 6.6 Hz,
3H, Fuc-H6); 13C NMR (125.8 MHz, CD3OD): d 174.5 (Lac-C1),
147.4 (triazol-Ci), 133.2 (Ar–Ci), 131.0, 130.4 (2 Ar–CH), 125.8 (tria-
zol-CH), 125.0, 123.4 (2 Ar–C), 102.8 (Gal-C1), 97.5 (Fuc-C1), 84.7
(Gal-C3), 79.6 (Lac-C2), 79.0 (Cy–CH), 77.6 (Cy–CH), 76.1 (Gal-
C5), 74.0 (Fuc-C4), 72.2 (Gal-C2), 71.7 (Fuc-C3), 70.2 (Fuc-C2),
68.2 (Gal-C4), 67.5 (Fuc-C5), 62.9 (Gal-C6), 53.8 (Lac-C3), 31.1,
4.1.11. Sodium (2S)-3-N-[4-(3-thiophenyl)-1,2,3-triazol-1-yl]-2-
O-{1-O-[(1R,2R)-2-O-(
galactopyranos-3-yl}-propionate (2c)
In analogy to 2b, compound 2c was prepared from crude 16
(7.1 mg) and 3-ethynyl-thiophene (12.7 L, 129 mol). After HPLC,
a-L-fucopyranosyl)-cyclohexyl]-b-D-
l
l
5.3 mg (97%) of the free acid were obtained, which was transferred
into the sodium salt as described above.
Analytical data for the free acid: [
a]
D
ꢀ60.8 (c 0.27, MeOH); 1H
NMR (500.1 MHz, CD3OD): d 8.54 (s, 1H, triazol-H), 7.75 (m, 1H,
Ar–H), 7.50–7.48 (m, 2H, Ar–H), 4.98 (dd, J = 2.9, 14.3 Hz, 1H,
Lac-H3a), 4.84 (d, J = 3.9 Hz, 1H, Fuc-H1), 4.77 (dd, J = 6.8,
14.2 Hz, 1H, Lac-H3b), 4.64 (dd, J = 2.9, 6.7 Hz, 1H, Lac-H2), 4.58
(q, J = 6.6 Hz, 1H, Fuc-H5), 4.28 (d, J = 7.7 Hz, 1H, Gal-H1), 3.97 (d,
J = 2.7 Hz, 1H, Gal-H4), 3.86 (dd, J = 3.4, 10.1 Hz, 1H, Fuc-H3),
3.75 (dd, J = 6.9, 11.4 Hz, 1H, Gal-H6a), 3.72–3.62 (m, 5H, Fuc-H2,
-H4, Gal-H2, Gal-H6b, 1H of Cy–CH), 3.54 (dt, J = 4.5, 9.4 Hz, 1H,
1H of Cy–CH), 3.44–3.38 (m, 2H, Gal-H3, -H5), 2.07–2.01 (m, 2H,
Cy–CH2), 1.74–1.66 (m, 2H, Cy–CH2), 1.43–1.22 (m, 4H, Cy–CH2),
1.15 (d, J = 6.7 Hz, 3H, Fuc-H6); 13C NMR (125.8 MHz, CD3OD): d
145.1 (triazol-Ci), 133.2 (Ar–Ci), 127.6, 127.0 (2C, Ar–C), 124.2 (tria-
zol-CH), 122.2 (Ar–C), 102.7 (Gal-C1), 97.4 (Fuc-C1), 85.0 (Gal-C3),
79.5 (CH), 77.5 (Cy–CH), 76.0 (Gal-C5), 74.0 (CH), 72.0 (Gal-C2),
71.7 (Fuc-C3), 70.2 (CH), 67.9 (Gal-C4), 67.6 (Fuc-C5), 63.0 (Gal-
C6), 54.1 (Lac-C3), 31.1, 30.2, 24.6 (4C, 4 Cy–CH2), 16.8 (Fuc-C6);
HR-MS Calcd for C27H39N3O13SNa [M+H]+: 668.2101; found:
668.2100.
30.3, 24.6 (4C,
4 Cy–CH2), 16.7 (Fuc-C6); HR-MS Calcd for
C30H40F3N3O13Na [M+H]+: 730.2411; found: 730.2419.
4.1.14. Sodium (2S)-3-N-[4-(benzotriazolylmethyl)-1,2,3-
triazol-1-yl]-2-O-{1-O-[(1R,2R)-2-O-(
cyclohexyl]-b- -galactopyranos-3-yl}-propionate (2f)
In analogy to 2b, compound 2f was prepared from crude 16
(7.1 mg) and 1-N-benzotriazolyl-prop-2-yne (20.7 mg, 132 mol).
a-L-fucopyranosyl)-
D
l
After HPLC, 5.4 mg (91%) of the free acid were obtained, which
was transferred into the sodium salt as described above.
Analytical data for the free acid: [
a
]
ꢀ53.7 (c 0.27, MeOH); 1H
D
NMR (500.1 MHz, CD3OD): d 8.37 (br s, 1H, triazol-H), 7.98 (A of
ABCD, J = 7.5 Hz, 1H, Bt–H), 7.81 (D of ABCD, J = 7.5 Hz, 1H, Bt–H),
7.55 (C of ABCD, J = 7.5 Hz, 1H, Bt–H), 7.43 (B of ABCD, J = 7.5 Hz,
1H, Bt–H), 6.03 (br s, 2H, Bt–CH2), 4.92–4.86 (m, 2H, Fuc-H1, Lac-

26
A. Titz et al. / Bioorg. Med. Chem. 18 (2010) 19–27
H3a), 4.74 (dd, J = 6.5, 13.5 Hz, 1H, Lac-H3b), 4.62 (m, 1H, Lac-H2),
4.59 (q, J = 6.4 Hz, 1H, Fuc-H5), 4.24 (d, J = 7.8 Hz, 1H, Gal-H1), 3.93
(m, 1H, Gal-H4), 3.87 (dd, J = 3.5, 10.0 Hz, 1H, Fuc-H3), 3.77–3.65
(m, 5H, Fuc-H2, -H4, Gal-H6, 1H of Cy–CH), 3.61–3.53 (m, 2H,
Gal-H2, 1H of Cy–CH), 3.40 (m, 1H, Gal-H5), 3.32 (m, 1H, Gal-
H3), 2.08–2.01 (m, 2H, Cy–CH2), 1.71 (br s, 2H, Cy–CH2), 1.42–
1.22 (m, 4H, Cy–CH2), 1.18 (d, J = 6.7 Hz, 3H, Fuc-H6); 13C NMR
(125.8 MHz, CD3OD): d 176.8 (Lac-C1), 147.2 (Bt–Ci), 142.9 (tria-
zol-Ci), 134.4 (Bt–Ci), 139.1 (Bt–CH), 127.4 (triazol-CH), 125.9
(Bt–CH), 120.1 (Bt–CH), 112.0 (Bt–CH), 102.7 (Gal-C1), 97.4 (Fuc-
C1), 85.1 (Gal-C3), 79.5 (Cy–CH), 78.7 (Lac-C2), 77.4 (Cy–CH),
76.0 (Gal-C5), 74.0 (Fuc-C4), 72.8 (Gal-C2), 71.7 (Fuc-C3), 70.2
(Fuc-C2), 67.9 (Gal-C4), 67.6 (Fuc-C5), 63.0 (Gal-C6), 53.8 (Lac-
C3), 44.6 (Bt–CH2), 30.8, 24.4 (4C, 4 Cy–CH2), 16.8 (Fuc-C6); HR-
MS Calcd for C30H42N6O13Na [M+Na]+: 717.2708; found: 717.2705.
Analytical data for the free acid: [
a
]
ꢀ40.8 (c 0.12, MeOH); 1H
D
NMR (500.1 MHz, CD3OD): d 8.27 (br s, 1H, triazol-H), 5.57 (br s,
2H, 4H-Bt–CH2), 4.93–4.84 (m, 2H, Fuc-H1, Lac-H3a), 4.75 (dd,
J = 5.9, 14.6 Hz, 1H, Lac-H3b), 4.62 (m, 1H, Lac-H2), 4.59 (q,
J = 6.8 Hz, 1H, Fuc-H5), 4.27 (d, J = 8.0 Hz, 1H, Gal-H1), 3.94 (m,
1H, Gal-H4), 3.86 (dd, J = 3.6, 9.9 Hz, 1H, Fuc-H3), 3.77–3.65 (m,
5H, Fuc-H2, -H4, Gal-H6, 1H of Cy–CH), 3.61–3.52 (m, 2H, Gal-
H2, 1H of Cy–CH), 3.41 (t, J = 5.6 Hz, 1H, Gal-H5), 3.35 (m, 1H,
Gal-H3), 2.70–2.64 (m, 4H, 4H-Bt–H), 2.07–2.02 (m, 2H, Cy–CH2),
1.87–1.77 (m, 4H, 4H–Bt–H), 1.73–1.68 (m, 2H, Cy–CH2), 1.41–
1.22 (m, 4H, Cy–CH2), 1.18 (d, J = 6.6 Hz, 3H, Fuc-H6); HR-MS Calcd
for C30H46N6O13Na [M+Na]+: 721.3021; found: 721.3021.
4.2. Molecular modeling
The three-dimensional structures of all compounds were gener-
ated using MacroModel.35 Next, a conformational search was per-
formed to identify the global minimum conformation by sampling
a total of 10,000 structures (MacroModel, mixed torsional/low-
mode sampling method, extended torsional sampling, OPLS 2005
force-field,36 implicit water solvent model). A periodic boundary
system was created by placing the global minimum in a box of pre-
organized TIP3P water molecules. The system charge was neutral-
ized and sodium and chloride ions were added to reach
physiological electrolyte concentration of 0.15 M. Special attention
was paid to building proper solvation shell around the solute: First,
the solvent environment was minimized using a gradient criterion
of 0.1 kcal/mol followed by a 24 ps molecular dynamics (MD) sim-
ulation, so that the water molecules could reorganize around the
solute (geometry of the solute was kept fixed). The whole system
was then completely minimized using a gradient criterion of
0.05 kcal/mol. A 12 ns MD simulation was performed using NPT
ensemble and standard conditions (T = 300 K, p = 101.325 kPa)
with frames sampled every 1.2 ps. All MD simulations were done
using Desmond.37–39 The statistical analysis of the structural data
was performed from the 9950 frames collected during the MD
run (first 50 frames were skipped due to equilibration of the
system).
4.1.15. Benzyl (2S)-3-N-[4-(benzotriazolylmethyl)-1,2,3-triazol-
1-yl]-2-O-{1-O-[(1R,2R)-2-O-(2,3,4-tri-O-benzyl-a-L-
fucopyranosyl)-cyclohexyl] 6-O-benzyl-b-D-galactopyranos-3-yl}-
propionate (17)
3-Benzotriazolyl-propyne (95 mg, 60.7
lmol) was dissolved in
MeOH/water (1 mL, 9:1) and copper powder (1 mg) was suspended
in this solution. A solution of 14 (10 mg, 10.1 mol) in MeOH/
l
water (1 mL, 9:1) was added and the reaction mixture was stirred
for 24 h at rt. Additional copper powder (5 mg) was added and stir-
ring continued for further 24 h before the solvent was removed in
vacuo. Purification by column chromatography (PhMe/EtOAc 1:1)
yielded 17 (10 mg, 86%) as a white solid.
1H NMR (500.1 MHz, CDCl3): d 7.97–7.92 (m, 1H, Bt–H), 7.85 (br
s, 1H, triazol-H), 7.59 (m, 1H, Bt–H), 7.38 (m, 1H, Bt–H), 7.32–7.13
(m, 26H, 25 Ar–H, 1 Bt–H), 5.80 (br s, 2H, Bt–CH2), 5.13, 5.06 (A, B
of AB, J = 11.9 Hz, 2H, PhCH2OCO), 4.88–4.86 (m, 2H, Fuc-H1, 1H of
PhCH2), 4.74–4.70 (m, 2H, 1H of PhCH2, Lac-H3a), 4.66 (A000 of
A000B000, J = 12.0 Hz, 1H, PhCH2), 4.61 (B00 of A00B00, B000 of A000B000,
J = 11.7 Hz, 2H, PhCH2), 4.53 (B0 of A0B0, J = 11.5 Hz, 1H, PhCH2),
4.50–4.46 (m, 2H, Lac-H2, -H3b), 4.42, 4.38 (A000, B0000 of A0000B0000
,
J = 12.0 Hz, 2H, PhCH2), 4.31 (q, J = 6.3 Hz, 1H, Fuc-H5), 4.07 (d,
J = 7.7 Hz, 1H, Gal-H1), 3.94 (dd, J = 3.5, 10.1 Hz, 1H, Fuc-H2),
3.89 (dd, J = 2.7, 10.1 Hz, 1H, Fuc-H3), 3.77 (d, J = 2.5 Hz, 1H, Gal-
H4), 3.66–3.60 (m, 3H, Fuc-H4, Gal-H6a, 1H of Cy–CH), 3.57–3.51
(m, 3H, Gal-H2, -H6b, 1H of Cy–CH), 3.31 (t, J = 6.0 Hz, 1H, Gal-
H5), 3.39 (dd, J = 2.7, 9.3 Hz, 1H, Gal-H3), 1.97–1.87 (m, 2H, Cy–
CH2), 1.66–1.56 (m, 2H, Cy–CH2), 1.35–1.13 (m, 4H, Cy–CH2),
1.01 (d, J = 6.5 Hz, 3H, Fuc-H6); 13C NMR (125.8 MHz, CDCl3): d
170.2 (Lac-C1), 139.4, 139.1, 139.0, 138.2, 134.5 (5C, Ar–Ci),
129.1–127.5 (Ar–C), 124.4, 120.0, 110.4 (3C, Bt–C), 100.7 (Gal-
C1), 95.0 (Fuc-C1), 83.6 (Gal-C3), 79.9 (Fuc-C3), 78.4 (Fuc-C4),
77.9 (Cy–CH), 77.4 (Lac-C2), 76.6 (Fuc-C2), 76.3 (Cy–CH), 75.1,
73.8, 73.2 (4C, 4 PhCH2), 73.0 (Gal-C5), 70.6 (Gal-C2), 69.0 (Gal-
C6), 68.4 (PhCH2–O–CO), 66.7 (Gal-C4), 66.4 (Fuc-C5), 52.3 (Lac-
C3), 44.0 (Bt–CH2), 30.2, 29.9, 29.4, 23.4 (4C, 4 Cy–CH2), 16.9
(Fuc-C6); ESI-MS Calcd for C65H72N6O13Na [M+Na]+: 1167.5;
found: 1167.7.
Acknowledgments
We are grateful to the Swiss National Science Foundation and
GlycoMimetics Inc., Gaithersburg, MD for financial support. The
authors thank Bea Wagner for technical support and Tobias Mohn
for the acquisition of the HR-MS spectra.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.11.024.
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