Assembly of Divalent Ligands and Their Effect on Divalent Binding to Pseudomonas aeruginosa Lectin LecA

Article abstract of DOI:10.1021/acs.joc.8b02727

Divalent ligands were prepared as inhibitors for the adhesion protein of the problematic Pseudomonas aeruginosa pathogen. Bridging two binding sites enables simultaneous binding of two galactose moieties, which strongly enhances binding. An alternating motif of glucose and triazole and aryl groups was shown to have the right mix of rigidity, solubility, and ease of synthesis. Spacers were varied with respect to the core unit as well as the aglycon portions in an attempt to optimize dynamics and enhance interactions with the protein. Affinities of the divalent ligands were measured by ITC, and K_d's as low as 12 nM were determined, notably for a compounds with either a rigid (phenyl) or flexible (butyl) unit at the core. Introducing a phenyl aglycon moiety next to the galactoside ligands on both termini did indeed lead to a higher enthalpy of binding, which was more than compensated by entropic costs. The results are discussed in terms of thermodynamics and theoretical calculations of the expected and observed multivalency effects.

Full text of DOI:10.1021/acs.joc.8b02727

Subscriber access provided by SWINBURNE UNIV OF TECHNOLOGY
Article
Assembling of divalent ligands and their Effect on
Divalent Binding to Pseudomonas aeruginosa Lectin LecA
Guangyun Yu, Anna Chiara Vicini, and Roland J. Pieters
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02727 • Publication Date (Web): 25 Jan 2019
Downloaded from http://pubs.acs.org on February 3, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 57
The Journal of Organic Chemistry
1
2
3
4
5
6
Assembling of divalent ligands and their Effect on Divalent Binding
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
to Pseudomonas aeruginosa Lectin LecA
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Guangyun Yu, Anna Chiara Vicini, Roland J. Pieters*
Department of Chemical Biology & Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Utrecht University,
P.O.Box 80082, NL-3508 TB Utrecht, The Netherlands
ABSTRACT: Divalent ligands were prepared as inhibitors for the adhesion protein of the problematic
Pseudomonas aeruginosa pathogen. Bridging two binding sites enables simultaneous binding of two galactose
moieties which strongly enhances binding. An alternating motif of glucose and triazole and aryl groups was shown
to have the right mix of rigidity, solubility and ease of synthesis. Spacers were varied with respect to the core unit
as well as the aglycon portions, in an attempt to optimize dynamics and enhance interactions with the protein.
Affinities of the divalent ligands were measured by ITC and K ’s as low as 12 nM were determined, notably for a
d
compounds with either a rigid (phenyl) or flexible (butyl) unit at the core. Introducing a phenyl aglycon moiety
next to the galactoside ligands on both termini did indeed lead to a higher enthalpy of binding which was more than
compensated by entropic costs. The results were discussed in terms of thermodynamics and theoretical calculations
of the expected and observed multivalency effects.
INTRODUCTION
Protein-carbohydrate interactions are involved in many biological processes and diseases.1,2,3,4,5,6 In this context it
is an important goal to find new specific molecular ligands for carbohydrate binding or carbohydrate processing
proteins,7,8,9 to be used as chemical probes or leads for therapeutic application.11,12 One specific aspect of protein-
carbohydrate interactions is the widespread prevalence of multivalency.13,14,15,16,17,18 Numerous carbohydrate
binding proteins of biological or medicinal interest contain more than one binding site either identical or not.
Bridging such binding sites by divalent or higher valency ligands may lead to greatly enhanced binding or inhibitory
potencies.19,20 Increasingly higher potency enhancements are being reported and also larger distances are being
covered by spacers.21 In this context it is likely that rigidified spacers are beneficial with the potential for high
10
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 2 of 57
2
2
potency and specificity. More flexible, often PEG-based spacers, exhibit more shallow affinity optima. Recent
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
calculations
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HO OH
O
OH
HO
O
N
N
N
OH
O
N
N
N
OH
N
N
HO
OH
N
N
HO
HO
O
HO
O
HO
O
N
O
N
OH
OH HO
OH
OH
1
3
4
Figure 1. X-ray structure of 1 bound to LecA (pdb 4YWA ).
involving effective molarity calculations and experiments revealed that PEG-based spacers will have no enhancing
effect for bridging distant weak sites of millimolar binding affinities.21 Nucleic acid based spacers seem to be
preferred for the bridging of long distances,19,23,24,25,26,27 while for shorter distances various structural types have
been reported including e.g. polyproline28 and phenylene-ethynylene.29 Notably, such spacers should have a
persistent linear overall shape, but they should also allow for overall aqueous solubility of the multivalent construct.
Previously, we described a modular spacer based on directly equatorially 1,4-linked glucose and 1,4 linked triazole
moieties.30,31 This system was used for optimization and yielded divalent ligand 1 (Figure 1) of the Pseudomonas
aeruginosa adhesion lectin LecA with two galactose specific binding sites separated by ca. 26 Å, when measured
2
ACS Paragon Plus Environment

Page 3 of 57
The Journal of Organic Chemistry
between the anomeric oxygens of the bound galactosides in pdb entry 1OKO.32,33 The spacer length of 1 was
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
optimized based on inhibition and binding data (K = 28 nM), and it is the most potent reported divalent ligand for
d
LecA. Its divalent binding mode was confirmed by X-ray crystallography, in which the whole spacer was visible,
3
4
a feat not seen before for a synthetic spacer of this length (Figure 1). The structure was also largely predicted by
molecular modeling.31 Interestingly, besides the interactions between the terminal galactoside ligands and the
protein, additional interactions were observed between the protein and the spacer, possibly adding to the binding
affinity. While 1 was clearly a potent LecA inhibitor it was not clear which of its structural features were
contributing significantly to its potency. We here describe the synthesis and detailed thermodynamic evaluation of
a series of variants.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The aim was to first explore the synthetic possibilities of rigid spacers composed of glucose, triazole and phenyl
units and secondly to study the structure activity correlations between the spacer moieties and the binding to LecA.
The syntheses were modular in all cases but different strategies were explored involving building blocks based on
azido-glucose derivatives and 1,4-diethynyl benzene linked together by CuAAC. ITC was used to shed more light
on the effect of various components of the spacers, producing thermodynamic parameters. Multivalent LecA
3
5
36
37,38,39,40
inhibitors have been reported in the literature e.g. based on fullerenes, β-peptoids, peptide dendrimers,
calixarenes,41,42 cyclic carbohydrates,43 perylene,44 tetraphenylethylene,45 on a carbohydrate core46,47,48 and gold
nanoparticles.49 Also, among other potent divalent ligands,50,43 a potent divalent ligand with a K of 82 nM was
d
found by library screening, although less potent than 1, it achieved its potency while being considerably more
flexible.51
The lectin LecA is of medicinal interest as a virulence factor for P. aeruginosa, involved in the adhesion of the
pathogen, biofim formation and causing lung injury.33,52,53 Pseudomonas aeruginosa is a Gram-negative pathogen
involved in diseases such as dermatitis, pancreatitis, urinary tract infection, keratitis and respiratory tract infection.54
It is regarded as a primary cause of death in immuno-compromised patients, and notably those with cystic fibrosis.55
Treating P. aeruginosa infection is becoming more difficult because the increasing spread of drug-resistant
strains56,57 which made it one the highest priorities targets for intervention.58 Another reason for its difficult
5
9
eradication is its tendency to form biofilms. In these biofilms, the bacteria are protected from the host defense
system and the action of antibiotics. It was estimated that within a biofilm, bacterial are upward of 1000-times more
3
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 4 of 57
resistant to conventional antibiotic treatment.60,61,62,63 These issues combined make the search for P. aeruginosa
therapeutics an urgent one. Bacterial adhesion is often a prelude to infection.64,65 For P. aeruginosa, lectin LecA
has been identified to play an important role in the internalization of the pathogen by binding to glycosylated targets
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
displayed on the cell surface. Therefore inhibition of LecA is aimed at affecting adhesion of the bacteria an early
step of the infection process and may provide an alternative to conventional antibiotics.67 This concept68,65 was
supported by the therapeutic effect of a galactose solution against P. aeruginosa pneumonia in mouse models and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
cystic fibrosis patients, through inhibiting the binding of LecA to its glycosylated targets.53,69
methylene series:
HO OH
O
OH
HO
O
N
N
OH
O
N
N
OH
N
N
HO
OH
N
N
HO
O
HO
HO
O
HO
N
O
N
O
N
N
OH
OH HO
OH
1
OH
OH
HO
HO OH
O
OH
O
N
N
N
N
N
N
N
HO
O
N
N
OH
OH
O
HO
O
HO
N
NHO
N
OH
N
O
O
OH
OH
OH
2
3
OH
HO
HO
OH
O
HO OH
O
N
N
N
N
N
N
O
N
HO
O
O
HO
N
N
OH
N
HO
N
OH
OH
OH
phenylene series:
R
HO OH
HO OH
O
OH
O
N
N
O
N
N
N
N
N
OH
N
N
HO
O
O
HO
O
N
HO
N HO
N
O
OH
OH
OH
OH
R
4
a R = H
O
4
b R =
O
HO OH
O
OH
HO OH
O
N
N
N
N
N
N
N
OH
N
HO
OH
O
O
HO
O
N
HO
N
N
N
O
HO
OH
OH
OH
OH
5
HO OH
O
HO
OH
OH
N
N
N
N
N
HO
N
N
O
N
O
O
O
HO
N
O
OH
HO
HO
OH
OH
OH
6
HO OH
O
OH
OH
HO
OH
N
N
N
N
O
N
N
N
HO
O
N
N
O
HO
O
O
O
O
HO
HO
OH
OH
OH
OH
7
HO OH
O
OH
OH
HO OH
O
N
N
N
N
O
N
N
N
HO
O
N
N
O
O
O
HO
O
HO
HO
OH
OH
OH
OH
8
monovalent reference compounds:
HO OH
OH
HO
HO
HO OH
O
N
N
N
N
OH
HO
O
O
O
N
O
HO
OH
N
N3
OH
HO
O
OH
O
OH
OH
9a
9b
10
Figure 2. Structures of mono- and divalent LecA inhibitors used in this study
4
ACS Paragon Plus Environment

Page 5 of 57
The Journal of Organic Chemistry
1
2
3
4
RESULTS AND DISCUSSION
From previous research, we knew that the length of the divalent ligand is a very important factor for the binding
affinity.31 For this reason, ligand 2 and 3 were designed with the same numbers of atoms in the spacer as the
previously optimized 1 (Figure 2). For ligand 2, a phenyl group replaces the central glucose moiety of 1 and
maintains the number of atoms in the spacer (in terms of distance between the two galactosides). Furthermore, the
two remaining glucose units in the spacer of 2 are linked in the opposite direction, i.e. the C(4) is linked to the core
instead of C(1). The molecule is now also symmetrical just like its target protein. The consequences of the
modification is that this synthesis does not require the use of a glucose building block with a C(1) alkyne which is
a more difficult to prepare building block. The strategy for the synthesis of 2 relied on the construction of the di-
azido functionalized spacer 13 (Scheme 1). To this end the two hemiacetals in 12 were converted to two -azides
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
using 2-azido-1,3-dimethylimidazolinium hexafluorophosphate (ADMP).70 CuAAC conjugation of 13 and 14,
followed by Zemplén deprotection yielded 2. Next, a totally unconstrained central unit was introduced in the design
of 3 in order to evaluate the importance of the constraint in 1 and 2. For ligand 3, octa-1,7-diyne was used to
introduce the central unit. For the synthesis, a different strategy was used than
Scheme 1a
OH
OH
O
OH
OH
O
a
N
N
OH
N
N
N
N
N
OH
N
N
N
HO
HO
b
N3
HO
O
O
R
N
N
N3
HO
HO
OH
O
N3
HO OH PF6
3
OH
12
N3
HO
11
H C
CH3
N
13
N
HO OH
a
OH
R
R
R
OH
N
N
OH
N
N
N
N
N
R
N
N
N
O
R
O
R
HO
O
O
N
HO
N
O
O
AcO OAc
O
R
OH
AcO
O
15: R = OAc
OAc
c
14
2
: R = OH
OBz
O
HO
BzO
OBz
O
OAc
OAc
OBz
O
OAc
HO
AcO
O
a, 14
N
N
d
N
AcO
AcO
N
AcO
N3
O
N3
BzO
O
O
BzO
N
N
OAc
OBz
16
OBz
OBz
17
18
R1
R1
a
R2
R1
R1
19: R1 = OAc, R2 = OBz
3: R1 = R2 = OH
R2
N
N
N
N
O
N
N
N
N
R1
R1
R2
O
c
O
O
N
O
N
2
N
O
R1
R2
N
R1
R
R2
a
(
a) CuSO
4
•5H
2
O, Na-ascorbate, DMF/H
2
O 9:1, Microwave, 80°C, 40 min, 65-85%; (b) D
2 3 3
O/CH CN 4/1, Et N, 0°C, 3 days, 50%; (c)
MeONa, MeOH, 40-50% after Prep HPLC; (d) i) Tf
2
O, pyridine, CH Cl , 0°C, 1h; ii) NaN , DMF, 14h, 80% over two steps.
2
2
3
5
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 6 of 57
Scheme 2a
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
R
R
a
OH
OH
O
N
N
N
N
N
OH
N
N
R
13
O
R
HO
N
N
N
N
R
R
O
O
R
N
HO
O
O
AcO OAc
O
AcO
OH
R
O
21: R = OAc
OAc
b
4a: R = OH
20a
HO OBz
O
OAc
OBz
O
OAc
N
AcO
O
a
N
N
AcO
c
N
N
AcO
N3
AcO
16
+
20a
O
O
BzO
N
O
BzO
OAc
OAc
OBz
OBz
22a
23a
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
O
R1 R1
O
R2
O
R1
R1
R2
N
N
N
N
N
N
N
O
R2
N
N
R1
R1
N
O
O
24
O
N
N
O
O
R1
R2
R2
R1
O
R2
a
a
O
O
O
23a
O
5: R1 = OAc, R2 = OBz
2
b
b: R1 = R2 = OH
4
R2
1R1
R1
R1
R2
N
N
N
N
N
R2
N
N
N
R1
R
N
O
O
N
O
N
N
R2
O
O
R1
O
2
R1
R
R2
R1
2
6: R1 = OAc, R2 = OBz
b
: R1 = R2 = OH
5
a
(a) CuSO
4
•5H
2
O, Na-ascorbate, DMF/H
Cl , 0°C, 1h; ii) NaN
2
O 9:1, Microwave, 80°C, 40 min, 65-85%; (b) MeONa, MeOH, 20-40% after prep. HPLC; (c)
i) Tf
2
O, pyridine, CH
2
2
3
, DMF, 14h, 73% over two steps
6
ACS Paragon Plus Environment

Page 7 of 57
The Journal of Organic Chemistry
for 2. Here the galactoside ligand was first coupled to the spacer unit and the resulting compound was linked to the
core structure at the end. The partially benzoylated building block 16 was ‘clicked’ with 14 to yield 17. After
activation as a triflate, the axial hydroxyl at C(4) was displaced by sodium azide leading to equatorial azide 18.
CuAAC conjugation to the central dialkyne, followed by the Zemplén deprotection afforded ligand 3. Overall the
advantage of this strategy was to avoid the relatively low yielding ADMP step. The synthesis is now highly efficient
with only 9 steps from commercial peracetylated sugars and an overall yield of 13%.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The next aim was to introduce a phenyl group as the aglycon part of the terminal galactoside ligands, as this moiety
is known to enhance the LecA binding by a factor of ca. 5-10 fold,71,43,41,72,47,73 benefiting from CH- interactions.74
In the first approach 13 was linked to 20a (Scheme S1) by CuAAC to give 21 and after acetyl removal 4a was
obtained (Scheme 2).
Scheme 3a
AcO OAc
AcO OAc
O
OBz
O
AcO OAc
O
OBz
O
O
a
N
N
N
b
N
N
AcO
O
X
TIPS
AcO
O
X
N
AcO
O
X
OAc
OBz
O
AcO
AcO
OAc
OAc
OAc
OAc
N3
AcO
TIPS
2
2
2
0a: X = -
0b: X = -OCH2-
0c: X = -OCH2CH2-
28a: X = -
29a: X = -
29b: X = -OCH2-
29c: X = -OCH2CH2-
OAc
28b: X = -OCH2-
28c: X = -OCH2CH2-
27
OBz
O
OAc
OBz
O
a
HO
BzO
OAc
N
AcO
c
N
AcO
AcO
AcO
N
N
N
3
BzO
16
+
20a-c
N
X
O
O
X
O
O
OAc
N
OAc
OBz
OBz
2
2
2
2a: X = -
23a: X = -
2b: X = -OCH2-
2c: X = -OCH2CH2-
23b: X = -OCH2-
23c: X = -OCH2CH2-
3
0a-c: R1 = OAc, R2 = OBz
OR1
O
OR2
O
a
1
OR2
OR1
29a
+
+
+
23a
23b
23c
R O
d
1
N
N
N
N
N
N
R O
29b
9c
O
N
N
R1O
O
6: R = R2 = OH, X = -
1
1
R O
O
X
2
X
O
2
R O
2
N
: R1 = R2 = OH, X = -OCH2-
R O
OR1
7
OR1
OR2
OR2
8: R1 = R2 = OH, X = -OCH2CH2-
a
(
a) CuSO
4
•5H
2
O, Na-ascorbate, DMF/H
2
O 9:1, Microwave, 80°C, 40 min, 65-85%; (b) TBAF, Et
3
N, THF, 14 h, 80-90%; (c) i) Tf
2
O,
pyridine, CH
2
2
Cl , 0°C, 1h; ii) NaN
3
, DMF, 14h, 80% over two steps; (d) MeONa, MeOH, 30-40% after prep HPLC.
7
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 8 of 57
Unfortunately, 4a proved to be insoluble in water. For this reason, the central benzene ring was outfitted with two
short PEG units in the synthesis of 4b. As before a C4-bridged version of the molecule (5) was also prepared using
the same synthetic strategy. The sequence started with a ‘click’ coupling between 16 and 20a, resulting in 22a. Its
axial C(4) hydroxyl was converted to an equatorial azide to give 23a. CuAAC coupling of 23a to bis-alkyne 2475
yielded 25 and after deprotection 4b, which exhibited sufficient aqueous solubility. Similarly, 23a was coupled to
octa-1,7-diyne, yielding 26 and after acetyl removal 5 was obtained.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
While the structure of compound 4b was very close to that of compound 2, the modification also introduces an
extra 6 atoms and the spacer is therefore longer. In order to also keep a similar length to 2 while introducing the
phenyl units adjacent to the galactose moiety, compound 6 was designed. In this compound the shortest path
between to two anomeric oxygens of the galactose ligands involves 25 atoms (table 1) , while this number is 26 for
compounds 1-3. To evaluate the sensitivity of the binding to both length and flexibility, compounds 7 and 8 were
added that contain additional flexible units in the spacer.
The syntheses of 6-8 started with alkynes 20 (Schemes S1, S3) that were linked to azide 27 giving 28 (Scheme 3).
For 27 a new route was developed (Scheme S2). Previously we used a galactose moiety where the C(4) OH was
inverted to the glucoside azide.31 As the alkyne introduction chemistry typically works better on glucoside
derivatives, this was used here and a double inversion strategy lead to 27. Removal of the TIPS groups from 28 by
TBAF yielded the free alkynes 29. The other half of the target compounds was prepared similarly, starting again
with alkynes 20, now coupled to azidosugar 16, giving 22. Installing the required equatorial azido groups in 23, set
the stage for the coupling between 29. After the CuAAC coupling and removal of the acetyl and benzoyl groups,
divalent ligands 6-8 were obtained.
The compounds were evaluated for their binding abilities of the LecA lectin using Isothermal Titration Calorimetry
(
ITC) as previously reported by us and others.31,41,76 The bivalent compounds were compared to a number of
monovalent ligands reported in the literature and a few relevant reference compounds, and the numbers are shown
in Table 1. Entry 1 and 2 show similar K ’s for free galactose versus its -OMe derivative at around 70-90 M.
d
Entry 3 shows the effect of an aromatic aglycon part, that enhances the binding to a K of ca. 9 M. The divalent
d
inhibitors (1-8) were divided into two groups. The first, the methylene series (1-3) refers to the methylene group
8
ACS Paragon Plus Environment

Page 9 of 57
The Journal of Organic Chemistry
between the galactose ligand and the spacer (triazole). In the phenylene series (4b-8) this is a phenyl group. Both
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
series showed an n-value of around 0.5, consistent with bivalent binding. In the methylene series, both compounds
2
and 3 that contain a phenyl and an n-butyl unit in the center respectively, were more potent than the glucose
bridged 1. The K ’s of the divalent 2 and 3 were essentially identical and reached unprecedented levels of 12-13
d
nM. Interestingly, the thermodynamic parameters in the methylene series varied widely. In the phenylene series,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
affinities varied but were lower with K ’s between 35 and 94 nM. With the exception of PEG-containing 4b which
d
was measured in the presence of 5% DMSO, thermodynamic parameters of the phenylene group members 5-8 were
relatively close.
One of the notable thermodynamic features of 1 was the low entropic loss associated with its binding event. The
TS component was smaller than that of monovalent ligands. While it is tempting to attribute this to the rigid
spacer, naturally other factors such as solvation also play important roles in the entropic component. A surprising
notion was the observation that the flexible 3 was such a good ligand surpassing 1 in terms of K . Furthermore, this
d
compound’s entropic loss was relatively low and similar to that of the monovalent ligands. Besides this, it also has
a larger favorable enthalpy than 1, but the differences are small. Another notable aspect is the fact that turning the
two glucosides around in 3 versus 1 and removing the central glucose, did not have a deleterious effect. In the X-
31
ray structure of 1 bound to the protein LecA, three, water bridged hydrogen bonds and a direct hydrogen bond
involving the C(3) and C(4) OH’s of the central and adjacent spacer glucoside were observed.
Table 1. LecA binding by ITC.a
Compo Atoms in
K
d
n
H
-TS
G
Rel.pot.
f
und
spacer
(per sugar)
1
2
3
4
5
6
7
D-Galactose71
87,500
1.1
-7.9
2.3
-5.5
-
Gal--OMe43
Gal--OPh71
70,000
0.8
-9.3
3.6
-5.7
8,800
0.9
-11.2
4.8
-6.9
9a31
22,000
0.92
-8.3
2.0
-6.3
0.25 (0.25)
1(1)
9b
6,200 ± 400
8,400 ± 1,700
7,300 ± 800
1.00 ± 0.01
0.94 ± 0.06
0.95 ± 0.01
-11.6 ± 0.9
-8.7 ± 1.0
-9.5 ± 0.6
4.5 ± 0.9
-1.8 ± 0.8
2.5 ± 0.6
-7.1 ± 0.1
-6.9 ± 0.1
-7.0 ± 0.1
10
1 (1)
1(1)d
10
Methylene seriesb
2831
7
1
26
0.55
-11.6
1.3
-10.3
221 (111)
9
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 10 of 57
8
9
2
3
26
26
12 ± 8
13 ± 3
0.41 ± 0.09
0.58 ± 0.06
-22.9 ± 1.9
-14.3 ± 0.2
12 ± 1.8
3.6 ± 0.3
-10.9 ± 0.4
517 (258)
477 (238)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
-10.8 ± 0.14
Phenylene seriesc
61 ± 10
1
1
1
1
0
1
2
3
4b
5
32
32
25
29
31
0.43 ± 0.03
0.43 ±0.03
0.46 ± 0.01
0.43 ± 0.01
0.50 ± 0.01
-10.2 ±0.5
-16.8 ± 0.5
-16.8 ± 0.5
-15.2 ± 0.2
-16.1 ± 0.9
0.4 ± 0.1
7.2 ± 0.5
7.1 ± 0.6
5.6 ± 0.1
5.9 ± 1.0
-9.9 ± 0.2
-9.6 ± 0.2
-9.6 ± 0.1
-9.6 ± 0.1
-10.2 ± 0.3
120 (60)d,e
91 (46)
92 ±15
6
87 ± 17
97 (48)
7
94 ± 13
89 (45)
14
8
35 ± 15
240 (120)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
b
K in nM,  S and  in Kcal/mol, Standard deviations are given over 2 or more experiments; Relative
d
c
d
potency determined vs. 9b; Relative potency determined vs. 10 in buffer; determined in buffer with 5% DMSO;
e
f
Relative potency determined vs. 10 in buffer with 5% DMSO; number of atoms between the two anomeric
e
oxygens of the galactosides of divalent ligands, using the shortest path Relative potency determined vs. 10 in
f
buffer with 5% DMSO; number ofatoms between the two anomeric oxygens of the galactosides of divalent ligands,
using the shortest path.
While the flipped glucose moieties in 3 could similarly make these hydrogen bonds too, the central sugar is
obviously missing in 3. These protein-spacer interactions may have contributed some to the binding energy, but it
seems their contributions were minor as the removal of the central glucose had no negative effect on binding.
Compound 2 bound with essentially the same affinity as compound 3. Interestingly, the binding energy of
compound 2 has a much higher enthalpic contribution (ca. twofold) and a higher entropic loss (ca. 9-fold) than 1.
The origin of this effect is not obvious, but may involve the differential solvation between bound or free state. Even
so, the large and opposite enthalpic and entropic components of the binding of 2 are not extreme and fall between
38
the following two reported divalent ligands: 1) a flexible, peptide-based divalent ligand GalAG1 showed a H of
51
-
29 kcal/mol with a K 83 nM, and 2) and the mentioned flexible structure discovered by library screening showed
d
a H of -18 kcal/mol for a K 82 nM.
d
In the aryl-linked galactoside series (4-8), K differences were relatively moderate, and none of the compounds
d
were as potent as the starting point 1. Compound 4b showed a remarkably low entropic loss (-TS = 0.4 kcal/mol),
even lower than that of 1, and in stark contrast with 2, but may not be directly compared because it was measured
1
0
ACS Paragon Plus Environment

Page 11 of 57
The Journal of Organic Chemistry
in the presence of 5% DMSO. In the phenylene series the enthalpic contribution is typically larger than that of the
methylene linked series (1-3), but the entropic loss is also larger leading to a weaker overall binding.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
In terms of the benefit of the divalent presentation and the usefulness of building a spacer between the two ligands,
a reference monovalent ligand needs to be chosen. This is a delicate issue, as no reference molecule is perfect in
this regard. After previously using 9a we here use 9b for the methylene series as the extended monovalent ligand.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
It is clear that it benefits somewhat from interactions to the protein with a lower K down from 22 M for 9a to 6.2
d
M for 9b. The affinity is now comparable with that of the phenyl aglycon containing 10 and related compounds.
34
Possibly, the observed interactions between the spacer glucose moiety is the cause of this enhancement, while the
phenyl-linked compounds were reported to benefit from the interactions of the phenyl with the nearby CH group
of a histidine.
In our results clearly the methylene series was more potent than the phenylene series. Large relative potencies (and
relative potencies per sugar), were obtained for 2 with a 542-fold (271-fold per sugar) multivalency enhancement.
Note that this number is 1822 when compared to 9a. The 271-fold K -based number is among the highest
d
enhancements in the literature for LecA to the best of our knowledge. For Reymond’s tetravalent peptidic system
the K was 2.5 nM, which calculates to a ca. 300-fold enhancement per sugar when compared to the monovalent
d
ligand (K ca. 3 M). Similarly, for a calixarene based tetramer (K 90 nM) a ca. 300 fold enhancement per sugar
d
d
was calculated, when compared to the reference arm.41
Based on recent mathematical models we calculated whether the observed enhancements were in line with
expectations and possibly whether they were close to optimum or not. The recent reported models for rigid spacers
indicate that rigidity is key for multivalency effects, especially when the binding sites have a weak monovalent
2
1
affinity. In that case a flexible PEG spacer will not induce a measurable multivalency effect, while a rigid spacer
of appropriate length does. The effective molarity is key in these discussions, which is the concentration of the
second ligand around a second binding site after the first one is bound and should be higher than the monovalent
K . For a PEG-based system this concentration was calculated to be in the micromolar range in contrast to the rigid
d
spacer where it was milimolar, thus much more likely to bind with enhanced affinity. In the present system the
binding sites have a relatively high affinity with monovalent ligands binding below 100 M. Even a PEG-based
1
1
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 12 of 57
divalent system in our hands bound with a 30-fold enhancement.30 We calculated the effective concentration
according to the paper using a 30Å distance between the center of the two nearby binding sites in LecA, and
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
subsequently the predicted K ’s as a function of spacer length for rod-like ligands, with a variability of its length
d
2
1
of 4 Å, since no spacer is perfectly rigid. The K ’s were calculated according to eq. 1.
d
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
퐾mono
퐾퐷,bv
=
(1)
∆퐺
푠푝푎푐푒푟
푘푇
exp
[
]
2휋푐푒푓푓
This equation contains the mentioned effective concentration c . The monovalent K is 70M, i.e. K
based on
eff
d
mono
table 1, entry 2, the affinity of the ligand without any parts of the spacer (Gal--OMe). The experimentally
determined interaction with the spacer (Gspacer) is based on entry 5, where the ligand 9b contains a sizeable part of
the spacer which does contribute to the affinity (K = 6.2 M).
d
Figure 3. Calculated dissociation constants of a divalent ligands of various lengths (r is end-to-end distance), for
ete
LecA, according to eq. 1(see SI for details).
1
2
ACS Paragon Plus Environment

Page 13 of 57
The Journal of Organic Chemistry
The graph (Figure 3) shows an optimum K for the ideal 30 Å spacer, the spacing between the binding site centers.
d
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The K was predicted to be ca. 1 nM. Considering that the best compound in this study has a K of 12 nM, our
d
d
compounds are ca. one order of magnitude below their theoretical optimum. It should be pointed out that the
experimental approach has an impact on multivalency effects, e.g. due to protein concentration. This was previously
3
1
noted in the lower inhibition concentrations for 1 (IC =2.7 nM) by ELISA and similar effects were seen for the
50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
multivalent inhibition of cholera toxin.77
The type of modeling described above makes it clear that major advances due to chelation type of bivalent binding
are possible and favorable for rigid spacers of the right length, but would unlikely be able to distinguish between
the subtle structural variations in the present series, that nevertheless show significant binding differences.
Can we look to the thermodynamic parameters for answers? Overall the thermodynamics indicate an enthalpically
7
8
driven binding, that can be associated with an induced fit model. If we take the profile of 2 as exceptional, the
rest behaves in the following way. The addition of the phenyl aglycon does indeed help the binding enthalpy as it
does for the monovalent Gal--OPhe (entry 3), however, unlike the case of the monovalent Gal--OPhe, the gains
are more than balanced by increased entropic losses, resulting in overall weaker binding. Surface burial could be
the source of favorable entropy, often associated with hydrophobic surfaces like the phenyl group. In the present
compounds this factor does not dominate. More likely is the option that conformational rearrangements were
needed in the ligand, and possibly the protein, to accommodate the galactoside ligand with its properly oriented
71
phenyl aglycon to take advantage of the additional CH- interactions. The large difference between 2 and 3 with
respect to thermodynamic parameters, while exhibiting essentially the same K is intriguing. Building a CPK model
d
and handling both compounds reveals the large difference in rigidity. Compound 2 is quite rigid, while 3 has a
central hinge region that allows a range of conformations. The large entropic costs for the binding of 2 suggest
major reorganization of the protein to enable all intermolecular contacts. Ironically, binding the flexible 3 costs far
less entropy, as the single degree of freedom caused by the hinge is not overall very costly, when compared to
rearranging the protein for 2.
CONCLUSIONS
1
3
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 14 of 57
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Rigid, well defined spacers were synthesized that were based on equatorially 1,4-linked glucose moieties or 1,4-
linked phenyl rings alternated with 1,4 triazole moieties. Variations were made in the central unit, and in the part
linked to the galactose ligand with additional flexibility enhancing units. Synthetic strategies varied accordingly
with the most successful synthesis of one the best ligands 3 being only 9 steps from commercial peracetylated
sugars and an overall yield of 13%. This synthesis coupled an azido-galactose moiety to the terminal propargyl
galactoside ligand by CuAAC. After introducing an equatorial azido group at the galactose C(4)-OH, two of these
units were linked to the central bis-alkyne.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Overall a major affinity improvement was obtained by linking either octa-1,7-diyne or 1,4-diethynylbenzene to
1
9a, which yielded divalent ligands 2 and 3 with ca. 500-fold binding enhancements.
Thermodynamic parameters were evaluated in detail and surprisingly large differences were observed, while the
differences in K ’s were relatively minor. The compounds in the phenylene series did generally show more
d
favorable enthalpy as expected for additional CH- interactions, but this advantage was more than erased by
additional entropic costs possibly caused by protein rearrangement. This phenomenon made the methylene series
the more effective ligands. Within this series the large differences in thermodynamic parameters between 2 and 3
were intriguing and tentatively attributed to differences on rigidity and solvation as caused by replacing a phenyl
with a butyl group.
A recent spacer modeling approach was applied and lead to the conclusion that more improvements should
theoretically be possible, but also that the method was too coarse grain to predict the subtle effects that were seen
here. In that sense possibly a full modeling approach may eventually become successful. Bridging ligands is a
common theme in the carbohydrate recognition realm, but also interfaces in general18 or in non-carbohydrate
ligands that were linked together to achieve improved properties.79
EXPERIMENTAL SECTION
General. Unless stated otherwise, chemicals were obtain from commercial sources and were used without further
8
0
2
3
4
purification. Compounds 11 , 14 , 16 , 24 were synthesized following literature procedure. Solvents were
1
4
ACS Paragon Plus Environment

Page 15 of 57
The Journal of Organic Chemistry
purchased from Biosolve (Valkenswaard, The Netherlands). All moisture-sensitive reactions were performed under
nitrogen atmosphere. Anhydrous THF was dried over Na/benzophenone and freshly distilled prior to use. All the
other solvents were dried over molecular sieves 4 Å or 3 Å. TLC was performed on Merck precoated Silica 60
plates. Spots were visualized by UV light, 10% H SO in MeOH and triphenylphosphine in THF followed by
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
4
Ninhydrine (for azides). Microwave reactions were carried out in a Biotage microwave Initiator (Uppsala, Sweden).
The microwave power was limited by temperature control once the desired temperature was reached. Sealed vessels
of 2-5 mL and 10-20 mL were used. Analytical HPLC runs were performed on a Shimadzu automated HPLC
system with a reversed-phase column (phenomenex, C , 250 x 4.60 mm 5u micron 140087-2 for ligand 5, C , 250
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
18
x 2.00 mm 5u micron 132174-4, for ligand 2, 3 4a, 4b, 6, 7, 8 and 9b) that was equipped with an evaporative light
scattering detector (PLELS 1000, Polymer Laboratories, Amherst, MA, USA) and a UV/Vis detector operating at
2
20 nm and 254 nm. Preparative HPLC runs were performed on an Applied Biosystems workstation. Elution was
1
effected by using a linear gradient of 5% MeCN/0.1% TFA in H O to 5% H O/0.1% TFA in MeCN. H NMR
2
2
spectra were recorded at 400 MHz, 500 MHz and 600 MHz and 13C at 101 MHz, 126 MHz and 151 MHz.
Electrospray Mass experiments were performed in a Shimadzu LCMS QP-8000. High resolution mass spectrometry
(HRMS) analysis was performed using an ESI-QTOF II spectrometer (Bruker, Billerica, USA).
Isothermal titration microcalorimetry (ITC)
1
5
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 16 of 57
The lectin LecA was obtained from Sigma-Aldrich and was dissolved in buffer (0.1ꢀM Tris-HCl, 6 mM CaCl , pH
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
.5) and degassed. Protein concentration (between 10 and 40 μM depending on the ligand affinity) was checked by
measurement of optical density by using a theoretical molar extinction coefficient of 28000 units. Carbohydrate
ligands were dissolved directly into the same buffer, degassed, and placed in the injection syringe. ITC was
performed using a MicroCal Auto ITC200 (Malvern, Worcestershire, UK). LecA (0.01-0.04 mM) was placed into
the 200 μL sample cell at 25ꢀ℃. Titration was performed with injections of carbohydrate ligands (5-20 times of
LecA, 2.5 μL) every 120 s. Data were fitted using the “one-site model” using MicroCal Origin 7 software according
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
to standard procedures. Fitted data yielded the stoichiometry (n), the association constant (K ), the enthalpy (ΔH)
a
and the entropy of binding. The K value was calculated as 1/K , and T = 298 K.
d
a
Diglucoside (12).
Compound 11 (63 mg, 500 μmol) and 1,4-diethynylbenzene (256 mg, 1.25 mmol) was dissolved in 0.9 mL of
DMF. Then the aqueous solution of CuSO ‧5H O (18 mg in 25 μL of water, 75 μmol) and Na-ascorbate (30 mg in
4
2
2
5 μL of water, 150 μmol) was added to the resulting mixture. Finally TBTA (40 mg, 75 μmol) was added and the
reaction system was heated by microwave irradiation at 80 ℃ for 40 min. TLC indicated complete conversion of
the reaction. Then Cuprisorb was added and stirred for 30 min and filtered. The filtrate was dried under vacuum
and the residue was purified by column chromatography (MeOH/DCM 1:1) to afford 12 as a colourless syrup (174
1
mg, 348 μmol, 65%). H NMR (400 MHz, D O) δ 8.54 (s, 1H), 8.52 (s, 1H), 7.99 – 7.91 (m, 4H), 5.43 (d, J = 3.7
2
Hz, 1H), 4.92 (d, J = 8.0 Hz, 1H), 4.70 (td, J = 10.4, 3.9 Hz, 2H), 4.58 (ddd, J = 10.6, 4.2, 2.2 Hz, 1H), 4.46 (t, J =
9
.9 Hz, 1H), 4.31 – 4.20 (m, 2H), 3.80 (dd, J = 9.6, 3.7 Hz, 1H), 3.59 (ddd, J = 12.7, 7.5, 2.2 Hz, 2H), 3.54 – 3.47
1
3
1
(
m, 1H), 3.41 – 3.33 (m, 2H). C{ H} NMR (151 MHz, D O) δ 146.8, 129.1, 125.8, 122.3, 122.1, 96.1, 92.3, 74.5,
2
7
5
4.1, 73.4, 71.8, 70.2, 69.7, 62.3, 62.2, 59.9, 59.8. HRMS (ESI, Q-TOF) m/z calcd for C H N O [M + H]+
22 29 6 10
37.1945, found 537.1956.
1
6
ACS Paragon Plus Environment

Page 17 of 57
The Journal of Organic Chemistry
Bis-azide (13).
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
To a D O/CH CN (4:1) solution of 12 (1.3 g, 2.4 mmol), triethylamine (3.4 mL, 24 mmol) was added dropwise to
2
3
the solution and cooled to 0 ℃. Then 2-azido-1, 3-dimethylimidazolinium hexafluorophosphate (ADMP 4 g, 14.2
mmol) was added and the mixture was stirred at 0 ℃ until most of the starting material was converted to the azide
compound. Initially, two new spots formed on the TLC plate (developing eluent n-BuOH/H O/Acetic acid 6:3:1,
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13 highest new spot, mono azide lower new spot). Within 3 days conversion to 13 completed. The solvent was
removed under vacuum and the residue was purified by column chromatography to give compound 13 as a white
1
solid (703 mg, 1.2 mmol, 50%). H NMR (400 MHz, D O) δ 8.44 (s, 2H, H-trizole), 7.92 (s, 4H, ArH), 4.82 (d, J
2
=
8.7 Hz, 2H, H-1), 4.62 (t, J = 10.3 Hz, 2H, H-4), 4.26-4.17 (m, 4H, H-5, H-3), 3.62 (dd, J = 12.7 Hz, 1.8 Hz, 2H,
13
1
H-6a), 3.37 (t, J= 8.8 Hz, 2H, H-2), 3.34 – 3.25 (m, 2H, H-6b). C{ H} NMR (101 MHz, CD OD) δ 147.9, 131.5,
3
+
1
27.2, 123.7, 92.2, 78.0, 75.4, 75.4, 63.3, 61.4. HRMS (ESI, Q-TOF) m/z calcd for C H N O [M+H] 587.2075,
2
2
27 12
8
found 587.2073.
General procedure for the “click reaction”, step a. preparation of compounds 15, 17 and 19
The alkyne-compound, CuSO ‧5H O (0.15 equiv) and sodium ascorbate (0.3 equiv) were added to a solution of the
4
2
azide-compound in DMF containing 10% water. The mixture was heated under microwave irradiation at 80 ˚C for
40 min. After evaporation of the solvent the residue was dissolved in CH Cl . The organic solution was washed
2
2
three times with water, brine and dried over sodium sulfate. The solvent was removed, and the residue was purified
by column chromatography.
General procedure for removal of acetyl and benzoyl protecting groups, step c. Preparation of compounds
2
, 3, 4a, 4b, 5.
1
7
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 18 of 57
The protected substrate was suspended or dissolved in methanol. Sodium methoxide was added to obtain a basic
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
pH (pH ≈ 8). The reaction was stirred at r.t. and it was monitored by HPLC. After disappearance of the substrate,
+
the reaction was neutralized with the DowexH . The mixture was filtered, and the solvent was evaporated under
vacuum, which was subjected to purification by Preparative-HPLC.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Protected divalent ligand (15)
OAc
OH
OAc
O
AcO
AcO
OH
AcO
O
N
N
OH
N
N
N
N
N
N
N
N
N
O
AcO
HO
O
N
O
O
HO
OAc
OH
OAc
1
(
95 mg, 70 μmol, 70%, white solid) H NMR (400 MHz, DMSO-d ) δ 8.72 (s, 2H, H-triazole), 8.37 (s, 2H, H-
6
triazole), 7.98 (s, 4H, ArH), 5.88 (d, J = 9.2 Hz, 2H, H-1), 5.28 (d, J = 3.6 Hz, 2H, H-4’), 5.16 (dd, J = 10.3, 3.6
Hz, 2H, H-3’), 4.94 (t, J = 10.3, 7.9 Hz, 2H, H-2’), 4.89 – 4.78 (m, 4H, H-1’, H-6’a), 4.70 (d, J = 12.4 Hz, 2H, H-
6
’b), 4.61 (t, J = 10.3 Hz, 2H, H-4), 4.35 – 3.99 (m, 12H, H-5, H-3, H-5’, -OCH -, H-2), 3.29 (dd, J = 11.9 Hz, 2H,
2
1
3
1
H-6a), 3.16 – 3.07 (m, 2H, H-6b), 2.00 – 1.85 (24H, 8 x CH COO-). C{ H} NMR (101 MHz, DMSO-d ) δ 206.5
3
6
(Acetone), 170.0, 169.5, 169.2, 145.6, 143.1, 130.2, 125.6, 123.8, 122.1, 99.1, 87.3, 77.3, 74.1, 72.5, 70.3, 70.0,
6
8.6, 67.3, 61.8, 61.7, 61.3, 59.7, 30.7 (Acetone), 20.6, 20.5, 20.4, 20.3. HRMS (ESI, Q-TOF) m/z calcd for
+
+
C H N O [M+H] 1359.4500, found 1359.4471; C H N O Na [M+Na] 1381.4320, found 1381.4301.
5
6
71 12 28
56 70 12 28
Divalent ligand (2).
OH
OH
OH
HO
HO
OH
HO
N
N
OH
N
N
N
N
N
N
N
N
N
O
HO
O
HO
O
N
O
O
O
HO
OH
OH
OH
1
(
10.2 mg, 10 μmol, 40%, white solid) H NMR (400 MHz, D O) δ 8.59 (s, 2H, H-triazole), 8.43 (s, 2H, H-triazole),
2
7
.96 (s, 4H, ArH), 6.08 (d, J = 9.2 Hz, 2H, H-1), 5.09 – 4.97 (m, 6H, 2 x -OCH -, H-4), 4.53 (m, 6H, H-5, H-3,
2
H-1’), 4.31 (t, J = 9.2 Hz, 2H, H-2), 3.95 (d, J = 3.4 Hz, 2H, H-4’), 3.83 – 3.57 (m, 12H, 6’a, 6’b, H-2’, H-3’, H-
1
3
1
6a, H-5’), 3.40 (dd, J = 12.9, 4.3 Hz, 2H, H-6b). C{ H} NMR (101 MHz, D O,extracted from HSQC) δ 147.3,
2
1
44.4, 129.7, 126.5, 125.0, 122.9, 102.34, 87.7, 77.3, 75.5, 73.8, 73.0, 72.9, 70.9, 68.9, 62.1, 61.8, 61.2, 59.9.
+
HRMS (ESI, Q-TOF) m/z calcd for C H N O [M+H] 1023.3650, found 1023.3644.
4
0
55 12 20
1
8
ACS Paragon Plus Environment

Page 19 of 57
The Journal of Organic Chemistry
Galactoside (17)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
OBz
O
OAc
HO
BzO
AcO
O
N
N
AcO
O
N
OAc
OBz
1
(240 mg, 266 μmol, 86%, White solid) H NMR (400 MHz, CDCl ) δ 8.11 (s, 1H, H-triazole), 7.96 (m, 4H, ArH),
3
7
.85 – 7.75 (m, 2H, ArH), 7.56 – 7.23 (m, 9H, ArH), 6.18 – 6.06 (m, 2H, H-1, H-2), 5.58 (dt, J = 7.9, 2.7 Hz, 1H,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H-3), 5.42 (d, J = 3.4 Hz, 1H, H-4’), 5.18 (dd, J = 10.3, 7.9 Hz, 1H, H-2’), 5.05 (dd, J = 10.3, 3.4 Hz, 1H, H-3’),
4.90 – 4.80 (m, 2H, -OCH -), 4.71 (dd, J = 11.8, 5.5 Hz, 1H, H-6a), 4.59 – 4.49 (m, 2H, H-6b, H-4), 4.40 – 4.27
2
(m, 3H, H-1’, H-6’a, H-5), 4.08 (m, 2H, H-6’b, H-5’), 3.67 (d, J = 6.3 Hz, 1H, -OH), 2.15 (s, 3H, CH COO-), 2.13
3
1
3
1
(
s, 3H, CH COO-), 1.96 (s, 3H, CH COO-). C{ H} NMR (101 MHz, CDCl ) δ 171.9, 170.5, 170.2, 169.7, 166.6,
3
3
3
1
8
65.8, 165.4, 143.3, 133.9, 133.8, 133.6, 130.0, 129.9, 129.9, 129.3, 128.8, 128.7, 128.6, 128.6, 128.1, 122.8, 97.8,
6.8, 76.2, 73.8, 71.1, 70.5, 69.0, 68.8, 67.5, 67.4, 63.0, 62.0, 60.7, 20.9, 20.8, 20.7, 20.7. HRMS (ESI, Q-TOF)
+
m/z calcd for C H N O [M+H] 904.2776, found 904.2780.
4
4
46
3
18
Azido-glucoside (18)
OBz
O
OAc
AcO
N
N
AcO
N3
BzO
O
O
N
OAc
OBz
Compound 17 (240 mg, 266 μmol) was first dissolved in DCM/Pyridine (10:1, 11 mL), then triflic anhydride (0.75
g, 2.66 mmol) was dropped at 0 ˚C and reacted for 1 hour at this temperature. The reaction was quenched with 1M
KHSO , then DCM (20 mL) was added to extract the triflate intermediate. The solution was washed with water,
4
brine and dried by sodium sulfate. The solvent was removed under vacuum to afford the crude triflate intermediate
which was used directly for the next step. To the solution of the intermediate in DMF (10 mL), sodium azide (87
mg, 1.33 mmol) was added, the mixture reacted at room temperature overnight. After removal of the solvent, DCM
was added to dilute the product. The organic phase was washed by water, brine and dried with sodium sulfate. The
residue was purified by column chromatography (toluene/ethyl acetate 3:1) to afford the compound as a white solid
1
(217 mg, 234 μmol, 88%). H NMR (400 MHz, CDCl ) δ 8.11 – 8.01 (m, 2H, ArH), 7.98 – 7.90 (m, 2H, ArH),
3
7
.88 (s, 1H, H-triazole), 7.80 – 7.70 (m, 2H, ArH), 7.62 – 7.23 (m, 9H, ArH), 6.13 (d, J = 9.2 Hz, 1H, H-1), 5.90
(t, J = 9.6 Hz, 1H, H-3), 5.80 (t, J = 9.4 Hz, 1H, H-2), 5.37 (dd, J = 3.5, 1.1 Hz, 1H, H-4’), 5.16 (dd, J = 10.4, 7.9
1
9
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 20 of 57
Hz, 1H, H-2’), 4.98 (dd, J = 10.4, 3.4 Hz, 1H, H-3’), 4.88 (d, J = 12.9 Hz, 1H, -OCH -), 4.82 – 4.72 (m, 2H, -
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
OCH -, H-6a), 4.65 (dd, J = 12.5, 4.4 Hz, 1H, H-6b), 4.44 (d, J = 7.9 Hz, 1H, H-1’), 4.26 – 4.02 (m, 4H, H-6’a,
2
H-4, H-6’b, H-5), 3.95 (ddd, J = 7.2, 6.0, 1.2 Hz, 1H, H-5’), 2.12 (s, 3H, CH COO-), 2.04 (s, 3H, CH COO-), 1.94
3
3
1
3
1
(
s, 3H, CH COO-), 1.76 (s, 3H, CH COO-). C{ H} NMR (101 MHz, CDCl ) δ 170.7, 170.4, 170.1, 169.6, 166.0,
3
3
3
1
7
65.5, 165.0, 144.0, 134.0, 134.0, 133.7, 130.0, 129.9, 129.9, 129.4, 128.7, 128.6, 128.4, 127.8, 122.1, 98.9, 86.2,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6.1, 73.7, 71.2, 71.0, 70.6, 68.7, 67.2, 63.0, 61.4, 61.4, 60.6, 20.9, 20.8, 20.7, 20.6. HRMS (ESI, Q-TOF) m/z
+
calcd for C H N O [M+H] 929.2841, found 929.2859.
4
4
45
6
17
Protected divalent ligand (19)
OAc
AcO
OAc
OBz
O
N
N
N
AcO
O
O
N
N
OBz
N
N
N
AcO
AcO
O
BzO
O
N
N
OBz
N
O
N
BzO
OAc
OAc
OBz
(
81 mg, 41 μmol, 87%, White solid) 1H NMR (400 MHz, CDCl ) δ 8.02 – 7.92 (m, 6H, 4 x H-triazole, 2 x ArH),
3
7.72 (m, 8H, ArH), 7.59 – 7.53 (m, 2H, ArH), 7.48 – 7.34 (m, 10H, ArH), 7.22 (m, 8H, ArH), 6.54 – 6.44 (m, 4H,
H-1, H-3), 6.05 (t, J = 9.5 Hz, 2H, H-2), 5.37 (dd, J = 3.5, 1.1 Hz, 2H, H-4’), 5.27 – 5.11 (m, 6H, H-4, H-2’, H-5),
4.99 (dd, J = 10.4, 3.4 Hz, 2H, H-3’), 4.90 (d, J = 12.9 Hz, 2H, -OCH -), 4.79 (d, J = 12.9 Hz, 2H, -OCH -), 4.50
2
2
–
4.44 (m, 4H, H-1’, H-6a), 4.28 – 4.20 (m, 4H, H-6b, H-6’a), 4.07 (dd, J = 11.2, 7.1 Hz, 2H, H-6’b), 3.96 (m, 2H,
H-5’), 2.46 (m, 4H, 2 x -CH -), 2.12 (s, 6H, 2 x CH COO-), 2.05 (s, 6H, 2 x CH COO-), 1.94 (s, 6H, 2 x CH COO-),
2
3
3
3
1
3
1
1
1
1
2
.75 (s, 6H, 2 x CH COO-), 1.36 (m, 4H, 2 x -CH -). C{ H} NMR (101 MHz, CDCl ) δ 170.7, 170.4, 170.1,
3 2 3
69.6, 166.0, 165.0, 164.8, 148.4, 144.1, 133.9, 133.8, 133.7, 130.0, 129.9, 129.7, 129.2, 128.7, 128.6, 128.6, 128.1,
27.8, 122.4, 121.7, 99.0, 86.1, 75.4, 72.9, 71.4, 71.0, 70.6, 68.7, 67.2, 62.7, 61.5, 61.4, 60.4, 28.0, 25.0, 20.9, 20.8,
0.7, 20.6. HRMS (ESI, Q-TOF) m/z calcd for C H N O [M+H]+ 1963.6386, found 1963.6364;
96 99 12 34
+
C H N O Na [M+Na] 1985.6201, found 1985.6166.
96 98 12 34
Divalent ligand (3)
OH
HO
OH
O
OH
OH
N
N
N
O
N
N
OH
N
N
N
HO
HO
O
HO
O
N
N
OH
N
O
O
N
HO
OH
OH
OH
2
0
ACS Paragon Plus Environment

Page 21 of 57
The Journal of Organic Chemistry
(
13 mg, 13 μmol, 42%, White solid) 1H NMR (400 MHz, D O) δ 8.39 (s, 2H, H-triazole), 7.95 (s, 2H, H-triazole),
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
.01 (d, J = 9.2 Hz, 2H, H-1), 5.06 (d, J = 12.7 Hz, 2H, -OCH -), 4.95 (d, J = 12.7 Hz, 2H, -OCH -), 4.84 (m, 2H,
2 2
H-4), 4.52 (d, J = 7.8 Hz, 2H, H-1’), 4.46 – 4.33 (m, 4H, H-3, H-5), 4.23 (t, J = 9.2 Hz, 2H, H-2), 3.93 (d, J = 3.4
Hz, 2H, H-4’), 3.85 – 3.49 (m, 12H, H-5’, H-6’a, H-6’b, H-3’, H-2’, H-6a), 3.27 (dd, J = 13.0, 4.3 Hz, 2H, H-6b),
1
3
1
2
1
.79 (m, 4H, 2 x -CH -), 1.71 (m, 4H, 2 x -CH -). C{ H} NMR (101 MHz, D O) δ 148.5, 143.9, 124.6, 123.4,
2 2 2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
01.9, 87.3, 76.9, 75.1, 73.4, 72.6, 72.5, 70.6, 68.5, 61.7, 61.2, 60.8, 59.5, 27.5, 23.9. HRMS (ESI, Q-TOF) m/z
+
+
calcd for C H N O [M+H] 1003.3968, found 1003.3983; C H N O Na [M+Na] 1025.3788, found
3
8
59 12 20
38 58 12 20
1025.3802.
Galactoside (20a.1)
AcO OAc
O
AcO
O
I
OAc
2
0a.1. To the solution of pentaacetyl β-D-galactopyranoside (200 mg, 513 μmol) in CH Cl (3 mL) with 4Å
2 2
molecular sieves, 4-iodophenol (147 mg, 667 μmol) and BF ·Et O (124 mg, 872 μmol) were added slowly at 0 ̊C .
3
2
Then the mixture was allowed to increase to room temperature and reacted for further 32 hours. The reaction was
quenched with water (0.5 mL) and diluted with ethyl acetate. The organic layer was washed with 1M HCl, saturated
NaHCO , water, brine and dried with sodium sulfate. The solvent was removed and the residue was purified by
3
1
column chromatography (PE/EtOAc 4:1) to obtain the pure β isomer as a white solid (169 mg, 308 μmol, 60%). H
NMR (400 MHz, CDCl ) δ 7.62 – 7.53 (m, 2H, ArH), 6.81 – 6.72 (m, 2H, ArH), 5.50 – 5.41 (m, 2H, H-2, H-4),
3
5.09 (dd, J = 10.4, 3.4 Hz, 1H, H-3), 4.99 (d, J = 7.9 Hz, 1H, H-1), 4.25 – 4.07 (m, 2H, H-6a, H-6b), 4.04 (m, 1H,
13
1
H-5), 2.17 (s, 3H, CH COO-), 2.05 (s, 6H, 2 x CH COO-), 1.98 (s, 3H, CH COO-). C{ H} NMR (101 MHz,
3
3
3
CDCl ) δ 170.4, 170.3, 170.2, 169.4, 156.8, 138.6, 119.3, 99.6, 86.2, 71.2, 70.8, 68.6, 66.9, 61.5, 20.8, 20.8, 20.7.
3
The spectral data are in accordance with literature data.5
Galactoside (20a.2)
AcO OAc
O
AcO
O
Si
OAc
2
1
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 22 of 57
Compound 20a.1 (150 mg, 273 μmol), Pd(PPh ) Cl (5.74 mg, 8.2 μmol), and CuI (1.56 mg, 8.2 μmol) were added
3
2
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
to a round bottomed flask and degassed for 30 min. Then the previously degassed triethylamine (3 mL) was added
to the flask and finally ethynyltrimethylsilane (40.3 mg, 410 μmol) was added via syringe. The resulting system
reacted at r.t. overnight. Triethylamine was removed under vacuum, and CH Cl was added to extract the product.
2
2
The organic layer was washed with water, brine and dried with sodium sulfate. After removal of the solvent, the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
residue was purified by column chromatography (PE/EtOAc 4:1) to obtain 20a.2 as a white solid (114 mg, 218
1
μmol, 80%). H NMR (400 MHz, CDCl ) δ 7.41 – 7.33 (m, 2H, ArH), 6.93 - 6.85 (m, 2H, ArH), 5.50 - 5.39 (m,
3
2H, H-2, H-4), 5.09 (dd, J = 10.4 Hz, 3.4 Hz, 1H, H-3), 5.02 (d, J = 8.0 Hz, 1H, H-1), 4.21 - 4.11 (m, 2H, H-6a, H-
6
b), 4.06 - 4.03 (m, 1H, H-5), 2.15 (s, 3H, CH COO-), 2.03 (s, 6H, 2 x CH COO-), 1.98 (s, 3H, CH COO-), 0.21
3 3 3
13
1
(
s, 9H, Si(CH ) ). C{ H} NMR (101 MHz, CDCl ) δ 170.4, 170.3, 170.2, 169.4, 156.9, 133.6, 118.2, 116.7, 104.5,
3
3
3
9
9.3, 93.7, 71.3, 70.9, 68.7, 67.0, 61.5, 20.8, 20.8, 20.7. HRMS (ESI, Q-TOF) m/z calcd for C H O SiNa
25 32 10
+
[
M+Na] 543.1663, found 543.1659.
Galactoside (20a)
AcO OAc
O
AcO
O
OAc
Compound 20a.2 (108 mg, 219 μmol) was dissolved in THF (10 mL). TBAF·3H O (83 mg, 263 μmol) was added
2
and the mixture was stirred at room temperature for 1 hour. The solvent was removed under vacuum, and the residue
was purified by column chromatography (PE/EtOAc 2:1) to afford 20a as a brown solid (74 mg, 164 μmol, 75%).
1
H NMR (400 MHz, CDCl ) δ 7.46 – 7.38 (m, 2H, ArH), 6.98 – 6.89 (m, 2H, ArH), 5.52 - 5.42 (m, 2H, H-2, H-4),
3
5
.10 (dd, J = 10.4, 3.4 Hz, 1H, H-3), 5.05 (d, J = 8.0 Hz, 1H, H-1), 4.24 - 4.12 (m, 2H, H-6a, H-6b), 4.08 - 4.05 (m,
H, H-5) , 3.03 (s, 1H, CH≡C-), 2.17 (s, 3H, CH COO-), 2.05 (s, 6H, 2 x CH COO-), 2.00 (s, 3H, CH COO-).
1
3
3
3
13
1
C{ H} NMR (101 MHz, CDCl ) δ 170.4, 170.3, 170.2, 169.4, 157.1, 133.7, 117.1, 116.8, 99.3, 83.1, 77.4, 71.3,
3
+
7
4
0.9, 68.6, 66.9, 61.5, 20.8, 20.8, 20.7. HRMS (ESI, Q-TOF) m/z calcd for C H O Na [M+Na] 471.1267, found
2
2
24 10
71.1267.
General procedure for the “click reaction”, step a. preparation of compounds 21, 22a, 25 and 26.
2
2
ACS Paragon Plus Environment

Page 23 of 57
The Journal of Organic Chemistry
The compounds were prepared following the procedure previously described for the synthesis of compound 12
Protected divalent ligand (21)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
AcO OAc
O
OH
O
OAc
OH
N
N
N
N
N
OH
N
N
N
N
N
AcO
O
N
HO
O
AcO
O
N
O
AcO
HO
OAc
OAc
OH
1
(
96 mg, 65 μmol, 65%, White solid) H NMR (400 MHz, DMSO-d ) δ 8.85 (s, 2H, H-trizole), 8.75 (s, 2H, H-
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
trizole), 7.99 (s, 4H, ArH), 7.87 (d, J = 8.4 Hz, 4H, ArH), 7.10 (d, J = 8.5 Hz, 4H, ArH), 5.90 (d, J = 9.2 Hz, 2H,
H-1), 5.53 (d, J = 7.6 Hz, 2H, H-1’), 5.36 (m, 2H, H-3’), 5.33 – 5.19 (m, 4H, H-2’, H-4’), 4.64 (t, J = 10.2 Hz, 2H,
H-4), 4.46 (t, J = 6.5 Hz, 2H, H-5’), 4.41 – 4.33 (m, 2H, H-5), 4.24 (t, J = 9.5 Hz, 2H, H-3), 4.18 – 3.97 (m, 6H,
H-2, H-6’a, H-6’b), 3.32 (m, 2H, H-6a), 3.14 (m, 2H, H-6b), 2.16 (s, 6H, 2 x CH COO-), 2.07 (s, 6H, 2 x CH COO-
3
3
13
1
)
, 2.04 (s, 6H, 2 x CH COO-), 1.96 (s, 6H, 2 x CH COO-). C{ H} NMR (101 MHz, DMSO-d ) δ 206.5 (Acetone),
3
3
6
1
7
70.0, 169.9, 169.6, 169.3, 156.2, 146.1, 145.7, 130.2, 128.7, 126.6, 125.7, 125.4, 122.1, 120.2, 116.9, 97.6, 87.5,
7.3, 74.1, 72.6, 70.4, 70.2, 68.4, 67.3, 61.9, 61.4, 59.7, 30.7 (acetone), 20.5, 20.5, 20.4, 20.4. HRMS (ESI, Q-TOF)
+
m/z calcd for C H N O [M+H] 1483.4813, found 1483.4819.
6
6
75 12 28
Divalent ligand (4a)
HO OH
OH
O
OH
OH
N
N
N
N
N
OH
N
N
N
N
N
HO
N
O
HO
O
HO
O
N
O
O
HO
HO
OH
OH
OH
1
(
11 mg, 9.5 μmol, 20%, White solid) H NMR (500 MHz, DMSO-d ) δ 8.57 (s, 2H, H-triazole), 8.51 (s, 2H, H-
6
triazole), 7.76 (s, 4H, ArH), 7.60 (d, J = 8.6 Hz, 4H, ArH), 6.91 (d, J = 8.3 Hz, 4H, ArH), 5.67 (d, J = 9.2 Hz, 2H,
H-1), 4.66 (d, J = 7.7 Hz, 2H, H-1’), 4.42 (t, J = 10.2 Hz, 2H, H-4), 4.13 (dt, J = 10.7, 3.4 Hz, 2H, H-5), 4.01 (t, J
=
9.5 Hz, 2H, H-3), 3.84 (t, J = 9.0 Hz, 2H, H-2), 3.49 - 3.21 (12H, H-5’, H-2’, H-6’a, H-6’b, H-3’, H-4’), 3.10 (m,
1
3
1
2H, H-6a), 2.92 (m, 2H, H-6b). C{ H} NMR (126 MHz, DMSO-d ) δ 157.6, 146.6, 146.0, 130.3, 126.7, 126.0,
6
124.3, 122.2, 120.2, 116.9, 101.1, 87.7, 77.4, 75.7, 74.1, 73.3, 72.7, 70.4, 68.3, 62.1, 60.6, 59.9. HRMS (ESI, Q-
+
TOF) m/z calcd for C H N O [M+H] 1147.3968, found 1147.3954.
5
0
59 12 20
Galactoside (22a)
2
3
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 24 of 57
HO OBz
O
OAc
AcO
N
N
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
AcO
O
O
BzO
N
OAc
OBz
1
(
365 mg, 378 μmol, 87%, White solid) H NMR (400 MHz, CDCl ) δ 8.08 (s, 1H, H-trizole), 7.93 (d, J = 8.0 Hz,
3
2
6
2
H, ArH), 7.86 (d, J = 8.0 Hz, 2H, ArH), 7.67 (m, 4H, ArH), 7.50 – 7.16 (m, 9H, ArH), 6.98 – 6.90 (m, 2H, ArH),
.22 (t, J = 9.7 Hz, 1H, H-2), 6.08 (d, J = 9.4 Hz, 1H, H-1), 5.51 (dd, J = 10.0, 2.7 Hz, 1H, H-3), 5.48 – 5.34 (m,
H, H-2’, H-4’), 5.07 – 4.94 (m, 2H, H-3’, H-1’), 4.71 (dd, J = 11.7, 6.0 Hz, 1H, H-6a), 4.53 – 4.40 (m, 2H, H-6b,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H-4), 4.30 (t, J = 6.2 Hz, 1H, H-5), 4.17 – 3.95 (m, 3H, H-5’, H-6’a, H-6’b), 3.18 (d, J = 4.0 Hz, 1H, -OH), 2.09
1
3
1
(
s, 3H, CH COO-), 2.02 – 1.90 (s, 9H, 3 x CH COO-). C{ H} NMR (101 MHz, DMSO-d ) δ 170.0, 169.9, 169.6,
3
3
6
1
69.3, 165.6, 165.1, 164.4, 156.4, 146.5, 133.8, 133.7, 133.6, 129.3, 129.3, 129.2, 129.1, 129.0, 128.8, 128.8, 128.7,
128.3, 126.8, 124.9, 119.8, 116.8, 97.6, 84.9, 75.2, 74.3, 70.4, 70.2, 69.2, 68.3, 67.3, 66.2, 63.8, 61.40, 20.5, 20.5,
+
20.4, 20.4. HRMS (ESI, Q-TOF) m/z calcd for C H N O [M+H] 966.2933, found 966.2928; C H N O Na
4
9
48
3
18
49 47
3
18
+
+
[
M+Na] 988.2753, found 988.2742; C H N O K [M+K] 1004.2492, found 1004.2476.
4
9
47
3
18
Azido-galactoside (23a)
OBz
O
OAc
N
N
N
AcO
O
N3
BzO
AcO
O
OAc
OBz
2
4
ACS Paragon Plus Environment

Page 25 of 57
The Journal of Organic Chemistry
The compound was prepared following the procedure previously described for the synthesis of compound 18.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
(
225 mg, 227 μmol, 73%, White solid) H NMR (400 MHz, CDCl ) δ 8.08 (m, 2H, ArH), 8.00 (s, 1H, H-trizole),
3
7
5
.97-7.94 (m, 2H, ArH), 7.76 – 7.27 (m, 13H, ArH), 7.04 – 7.01 (m, 2H, ArH), 6.23 – 6.13 (m, 1H, H-1), 5.97 –
.86 (m, 2H, H-2, H-4), 5.54 – 5.42 (m, 2H, H-2’, H-4’), 5.16 – 5.02 (m, 2H, H-1’, H-3’), 4.79 (d, J = 12.1 Hz, 1H,
H-6a ), 4.67 (dd, J = 12.4, 3.6 Hz, 1H, H-6b), 4.26 – 4.03 (m, 5H, H-3, H-5, H-6’a, H-6’b, H-5’), 2.18 (s, 3H,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
1
CH COO-), 2.11 – 1.99 (s, 9H, 3 x CH COO-). C{ H} NMR (101 MHz, CDCl ) δ 170.5, 170.4, 170.3, 169.5,
3
3
3
166.1, 165.5, 165.1, 157.2, 147.9, 134.0, 133.9, 133.7, 130.0, 129.9, 129.4, 128.8, 128.7, 128.6, 128.4, 127.9, 127.4,
125.4, 117.5, 117.4, 99.7, 86.1, 76.0, 74.0, 71.3, 70.9, 70.8, 68.8, 67.0, 63.1, 61.6, 60.7, 20.9, 20.8, 20.7. HRMS
+
(
ESI, Q-TOF) m/z calcd for C H N O [M+H] 991.2997, found 991.2992.
49 47 6 17
Protected divalent ligand (25)
O
O
N
O
OAc
OAc
AcO
OBz
O
OBz
AcO
O
N
N
N
N
N
N
N
N
N
BzO
AcO
O
O
O
N
N
AcO
O
BzO
OAc
OBz
OAc
OBz
O
O
O
1
(
80 mg, 34 μmol, 85%, White solid) H NMR (400 MHz, DMSO-d ) δ 8.97 (s, 2H, H-triazole), 8.64 (s, 2H, H-
6
triazole), 7.96 (d, J = 7.6 Hz, 4H, ArH), 7.90 – 7.34 (m, 32H, ArH), 7.07 (d, J = 8.4 Hz, 4H, ArH), 6.95 (d, J = 9.1
Hz, 2H, H-1), 6.59 (t, J = 9.8 Hz, 2H, H-3), 6.36 (t, J = 9.2 Hz, 2H, H-2), 5.70 (t, J = 10.2 Hz, 2H, H-4), 5.52 (d, J
= 7.7 Hz, 2H, H-1’), 5.36 – 5.20 (m, 8H, H-5, H-3’, H-2’, H-4’), 4.45 (t, J = 7.0 Hz, 4H, -CH -), 4.26 – 4.06 (m,
2
10H, H-5’, H-6b, H-6’b, -CH -), 3.71 (m, 4H, H-6a, H-6’a), 3.60 (t, J = 4.6 Hz, 4H, -CH -), 3.48 (t, J = 4.3 Hz, 4H,
2
2
1
3
1
-CH -), 3.25 (s, 6H, 2 x -OCH ), 2.20 – 1.90 (24H, 8 x CH COO-). C{ H} NMR (101 MHz, DMSO-d ) δ 170.0,
2
3
3
6
1
1
7
69.8, 169.6, 169.2, 165.2, 164.5, 164.1, 156.5, 149.0, 146.5, 141.8, 134.1, 134.0, 133.6, 129.4, 129.1, 129.1, 128.9,
28.8, 128.8, 128.7, 127.9, 127.8, 126.8, 124.7, 124.4, 120.3, 119.4, 116.9, 111.4, 97.6, 84.3, 73.8, 72.8, 71.4, 70.4,
0.1, 69.5, 68.8, 68.3, 68.2, 67.2, 62.7, 61.4, 59.5, 58.1, 58.1, 20.5, 20.5, 20.4, 20.4. HRMS (ESI, Q-TOF) m/z
+
calcd for C H N O [M+H] 2343.7646, found 2343.7637.
1
18 119 12 40
Divalent ligand (4b)
2
5
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 26 of 57
O
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
OH
O
OH
O
HO
HO
OH
OH
N
N
N
N
OH
N
N
N
N
N
HO
HO
N
O
HO
O
N
N
O
O
HO
OH
OH
OH
O
O
O
(
8.7 mg, 6.3 μmol, 21%, White solid) 1H NMR (500 MHz, DMSO-d ) δ 8.74 (s, 2H, H-triazole), 8.51 (s, 2H, H-
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
triazole), 7.91 (s, 2H, ArH), 7.82 (d, J = 8.2 Hz, 4H, ArH), 7.14 (d, J = 8.2 Hz, 4H, ArH), 5.91 (d, J = 9.0 Hz, 2H,
H-1), 4.88 (d, J = 7.7 Hz, 2H, H-1’), 4.67 (t, J = 10.3 Hz, 2H, H-4), 4.36 (m, 2H, H-5), 4.29 (m, 4H, -OCH -), 4.22
2
(
t, J = 9.5 Hz, 2H, H-3), 4.07 (t, J = 9.0 Hz, 2H, H-2), 3.94 – 3.87 (m, 4H, -OCH -), 3.73 – 3.67 (m, 6H, H-5’, -
2
OCH -), 3.63 – 3.51 (m, 12H, -OCH -, H-2’, H-3’, H-6’a, H-6’b), 3.44 (dd, J = 9.9, 3.2 Hz, 2H, H-4’), 3.29 (m,
2
2
1
3
1
8H, -OCH , H-6a), 3.14 (m, 2H, H-6b). C{ H} NMR (126 MHz, DMSO-d6) δ 157.4, 148.9, 146.3, 141.2, 126.4,
3
124.9, 124.2, 120.0, 119.5, 116.6, 110.6, 100.9, 87.4, 77.3, 75.6, 74.1, 73.3, 72.7, 71.4, 70.3, 69.7, 69.0, 68.2, 68.0,
+
6
1.7, 60.4, 59.8, 58.2. HRMS (ESI, Q-TOF) m/z calcd for C H N O [M+H] 1383.5228, found 1383.5230.
60 79 12 26
Protected divalent ligand (26)
AcO OAc
O
OBz
N
N
OBz
OAc
N
N
OBz
N
N
N
N
N
AcO
O
N
BzO
O
N
AcO
O
N
O
AcO
O
BzO
OAc
OAc
OBz
2
6
ACS Paragon Plus Environment

Page 27 of 57
The Journal of Organic Chemistry
1
(
94 mg, 45 μmol, 90%, White solid) H NMR (400 MHz, DMSO-d ) δ 8.96 (s, 2H, H-triazole), 8.04 (s, 2H, H-
6
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
triazole), 7.99 – 7.91 (m, 4H, ArH), 7.82 – 7.24 (m, 30H, ArH), 7.03 (d, J = 8.6 Hz, 4H, ArH), 6.80 (d, J = 9.1 Hz,
H, H-1), 6.48 (t, J = 9.8 Hz, 2H, H-3), 6.29 (t, J = 9.2 Hz, 2H, H-2), 5.57 – 5.46 (m, 4H, H-4, H-1’), 5.34 – 5.16
m, 8H, H-4’, H2’, H-5, H-3’), 4.43 – 4.30 (m, 4H, H-5’, H-6a), 4.09 (m, 6H, H-6b, H-6’a, H-6’b), 2.32 (m, 4H, 2
x -CH -), 2.16 – 2.07 (6H, 2 x CH COO-), 2.06 – 1.80 (18H, 6 x CH COO-), 1.20 (d, J = 6.4 Hz, 4H, -CH -).
2
(
2
3
3
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
1
C{ H} NMR (101 MHz, DMSO-d ) δ 170.0, 169.8, 169.6, 169.2, 165.2, 164.4, 164.1, 156.4, 147.0, 146.5, 134.1,
6
133.8, 133.6, 129.5, 129.1, 129.1, 128.9, 128.9, 128.8, 128.7, 127.9, 127.8, 126.7, 124.7, 121.9, 120.2, 116.9, 97.6,
84.4, 73.7, 72.8, 71.4, 70.4, 70.1, 68.3, 67.2, 62.5, 61.4, 59.1, 27.6, 24.3, 20.5, 20.5, 20.4, 20.4. HRMS (ESI, Q-
+
TOF) m/z calcd for C H N O [M+H] 2087.6699, found 2087.6693.
1
06 103 12 34
Divalent ligand (5)
HO OH
OH
N
N
OH
O
OH
N
N
N
N
N
N
HO
N
HO
O
N
OH
N
HO
O
O
O
HO
O
HO
N
OH
OH
OH
1
(
10.5 mg, 9.3 μmol, 39%, White solid) H NMR (400 MHz, D O) δ 8.54 (s, 2H, H-triazole), 7.91 (s, 2H, H-triazole),
2
7
.77 (d, J = 8.7 Hz, 4H, ArH), 7.22 (d, J = 8.7 Hz, 4H, ArH), 6.04 (d, J = 9.2 Hz, 2H, H-1), 5.11 (d, J = 7.3 Hz, 2H,
H-1’), 4.86 (t, J = 10.6 Hz, 2H, H-4), 4.43 (m, 4H, H-3, H-5), 4.26 (t, J = 9.2 Hz, 2H, H-2), 4.02 (d, J = 3.2 Hz, 2H,
H-4’), 3.91 – 3.77 (m, 10H, H-5’, H-6’a, H-6’b, H-3’, H-2’), 3.57 (m, 2H, H-6a), 3.31 (dd, J = 13.0, 4.4 Hz, 2H,
1
3
1
H-6b), 2.78 (s, 4H, 2 x -CH -), 1.69 (q, J = 7.6 Hz, 4H, 2 x -CH -). C{ H} NMR (101 MHz, D O, deduced from
2
2
2
HSQC) δ 127.6, 117.3, 100.9, 87.8, 77.3, 75.8, 73.8, 72.9, 72.8, 70.7, 68.7, 61.6, 61.0, 60.0, 59.9, 59.9, 27.9, 24.4.
+
HRMS (ESI, Q-TOF) m/z calcd for C H N O [M+H] 1127.4281, found 1127.4267.
4
8
63 12 20
(3R,4R,5R,6R)-3,4,5-tris(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-2-one (27.1)
OBn
BnO
BnO
O
O
OBn
In a two-necks round bottom flask under nitrogen atmosphere, a solution of 2,3,4,6-tetra-O-benzyl-D-
glucopyranose (7.007 g, 12.96 mmol) in dry CH Cl (65 ml) was treated with Dess-Martin Periodinane (DMP)
2
2
(6.596 g, 15.55 mmol) at r.t. and the reaction mixture was stirred at the same temperature until complete conversion
2
7
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 28 of 57
of the starting material (monitored by TLC, PE/EtOAc 2:1). After 1 h and 45 minutes, the reaction mixture was
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
filtered through a silica pad and rinsed with 1.4 L of CH Cl to afford the lactone 27.1 as a pale yellow oil (6.388
2
2
1
g, 11.86 mmol, 92%) which was used in the next step without any further purification. H NMR (400 MHz, CDCl )
3
δ 7.41 – 7.15 (m, 20H, ArH), 4.99 (d, J = 11.4 Hz, 1H, PhCH), 4.76 – 4.43 (m, 8H, PhCH, H-5), 4.12 (d, J = 6.5
Hz, 1H, H-2), 3.88 – 3.98 (m, 2H, H-3, H-4), 3.73 (dd, J = 11.0, 2.4 Hz, 1H, H-6a), 3.67 (dd, J = 11.0, 3.3 Hz, 1H,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
1
H-6b). C{ H} NMR (101 MHz, CDCl ) δ 169.5, 137.7, 137.7, 137.6, 137.1, 128.6, 128.6, 128.5, 128.3, 128.2,
3
+
128.1, 128.1, 128.0, 81.1, 78.3, 77.6, 76.2, 74.1, 73.9, 73.7, 68.4. MS (ESI) m/z calcd for C H O Na [M+Na]
3
4
34
6
+
561.23, found 561.55, m/z calcd for C H NO [M+NH ] 556.27 found 556.55. Spectroscopic data were in
3
4
38
6
4
accordance with literature data.6
Triisopropyl(((2S,3S,4R,5R,6R)-3,4,5-tris(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-2-
yl)ethynyl)silane (27.2)
OBn
O
TIPS
BnO
BnO
OBn
In a round bottom flask under argon atmosphere, a solution of triisopropylsilyl acetylene (6.5 ml, 29.0 mmol) in
dry THF (35 mL) was treated dropwise at -78˚C with a 2.5 M solution of n-BuLi in hexane (7.0 ml, 17.4 mmol)
and stirred for 15 minutes. Subsequently, a solution of compound 27.1 (6.256 g, 11.61 mmol) in dry THF (12 mL)
was added dropwise within 2 minutes at -78˚C and stirred for an hour at -78˚C (monitored by TLC, PE/EtOAc 2:1).
When the conversion of 27.1 was complete, the reaction was neutralised by the addition of Amberlite IR120 H+
form (checked using pH-paper), allowing at the same time the temperature to increase slowly from -78˚C to r.t. The
resin was filtered, washed with CH Cl , and the solvent was removed in vacuo. The residue, a yellow oil, was
2
2
dissolved in dry CH CN/CH Cl 1:1 (130 mL in total) and transferred to a three-necks round-bottom flask. The
3
2
2
solution was cooled to -15 ˚C by means of an ice and salt bath, and Et SiH (11.0 ml, 68.9 mmol) was added at once,
3
followed by dropwise addition of BF ‧OEt (8.5 ml, 68.9 mmol), while the temperature inside the flask was
3
2
monitored to avoid it exceeding -10°C. The reaction mixture was stirred for 1h at -15°C, and then, as the reaction
was not complete, overnight at -20°C. The reaction was quenched by pouring the mixture into Et O/NaHCO (sat)
2
3
1
: 1 (200 mL in total). Et O (150 mL) and H O (100 mL) were added and the layers were separated. The aqueous
2
2
2
8
ACS Paragon Plus Environment

Page 29 of 57
The Journal of Organic Chemistry
phase was extracted with Et O (3 x 150 mL) and the combined organic layers were dried over Na SO and
2
2
4
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
concentrated in vacuo. Purification by column chromatography (PE 95%, EtOAc 3%, CHCl 2%) gave 6.45 g (9.15
3
1
mmol, 79 % in 2 steps) of 27.2 as white needles. H NMR (400 MHz, CDCl ) δ 7.39 – 7.16 (m, 20H, ArH), 5.11
3
(d, J = 10.7 Hz, 1H, PhCH), 4.90 (d, J = 11.1 Hz, 1H, PhCH), 4.85-4.81 (m, 3H, PhCH), 4.66 – 4.52 (m, 3H,
PhCH), 4.07 – 4.01 (m, 1H, H-2), 3.77 (dd, J = 11.2 Hz, 2.1 Hz, 1H, H-6a), 3.70 (dd, J = 11.2, 4.5 Hz, 1H, H-6b),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
1
3
.68 – 3.59 (m, 3H, H-1, H-3, H-4), 3.47 – 3.41 (m, 1H, H-5), 1.10 (s, 21H, SiCH(CH ) and SiCH(CH ) ). C{ H}
3 2 3 2
NMR (101 MHz, CDCl ) δ 138.7, 138.4, 138.3, 138.2, 128.5, 128.5, 128.5, 128.4, 128.1, 128.0, 128.0, 127.9, 127.8,
3
127.8, 127.7, 104.8, 87.5, 86.3, 82.7, 79.4, 77.9, 75.8, 75.4, 75.2, 73.6, 70.4, 68.8, 18.8, 11.4. HRMS (ESI, Q-TOF)
+
m/z calcd for C H NO Si [M+NH ] 722.4235, found 722.4245.
4
5
60
5
4
2
9
ACS Paragon Plus Environment