Glycodendrimersomes from Sequence-Defined Janus Glycodendrimers Reveal High Activity and Sensor Capacity for the Agglutination by Natural Variants of Human Lectins

Article abstract of DOI:10.1021/jacs.5b08844

A library of eight amphiphilic Janus glycodendrimers (Janus-GDs) presenting d-lactose (Lac) and a combination of Lac with up to eight methoxytriethoxy (3EO) units in a sequence-defined arrangement was synthesized via an iterative modular methodology. The length of the linker between Lac and the hydrophobic part of the Janus-GDs was also varied. Self-assembly by injection from THF solution into phosphate-buffered saline led to unilamellar, monodisperse glycodendrimersomes (GDSs) with dimensions predicted by Janus-GD concentration. These GDSs provided a toolbox to measure bioactivity profiles in agglutination assays with sugar-binding proteins (lectins). Three naturally occurring forms of the human adhesion/growth-regulatory lectin galectin-8, Gal-8S and Gal-8L, which differ by the length of linker connecting their two active domains, and a single amino acid mutant (F19Y), were used as probes to study activity and sensor capacity. Unpredictably, the sequence of Lac on the Janus-GDs was demonstrated to determine bioactivity, with the highest level revealed for a Janus-GD with six 3EO groups and one Lac. A further increase in Lac density was invariably accompanied by a substantial decrease in agglutination, whereas a decrease in Lac density resulted in similar or lower bioactivity and sensor capacity. Both changes in topology of Lac presentation of the GDSs and seemingly subtle alterations in protein structure resulted in different levels of bioactivity, demonstrating the presence of regulation on both GDS surface and lectin. These results illustrate the applicability of Janus-GDs to dissect structure-activity relationships between programmable cell surface models and human lectins in a highly sensitive and physiologically relevant manner.

Full text of DOI:10.1021/jacs.5b08844

Subscriber access provided by NEW YORK MED COLL
Article
Glycodendrimersomes from Sequence-Defined Janus
Glycodendrimers Reveal High Activity and Sensor Capacity
for the Agglutination of Natural Variants of Human Lectins
Shaodong Zhang, Qi Xiao, Samuel E Sherman, Adam Muncan, Andrea D. M. Ramos Vicente,
Zhichun Wang, Daniel A Hammer, Dewight Williams, Yingchao Chen, Darrin J Pochan,
Sabine Vértesy, Sabine André, Michael L. Klein, Hans-Joachim Gabius, and Virgil Percec
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b08844 • Publication Date (Web): 30 Sep 2015
Downloaded from http://pubs.acs.org on October 1, 2015
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 free 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 accessible to all
readers and 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.
Journal of the American Chemical Society 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 25
Journal of the American Chemical Society
Glycodendrimersomes from Sequence-Defined Janus Glycodendrimers Reveal High
Activity and Sensor Capacity for the Agglutination of Natural Variants of Human
Lectins
1
2
3
4
5
6
7
8
9
Shaodong Zhang,† Qi Xiao,† Samuel E. Sherman,† Adam Muncan,† Andrea D. M. Ramos Vicente,† Zhichun
Wang,§ Daniel A. Hammer,§ Dewight Williams,⊥ Yingchao Chen,# Darrin J. Pochan,# Sabine Vértesy,|| Sabine
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
André,|| Michael L. Klein,‡ Hans-Joachim Gabius,|| and Virgil Percec*,†
†
Roy & Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia,
Pennsylvania 19104-6323, United States. § Department of Chemical and Biomolecular Engineering, University of
⊥
Pennsylvania, Philadelphia, Pennsylvania 19104-6391, United States.
Electron Microscopy Resource
Laboratory, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6082,
United States. # Department of Materials Science & Engineering, University of Delaware, Newark, Delaware
||
19716, United States. Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-
Maximilians-University, Veterinärstrasse 13, 80539 Munich, Germany. ‡ Institute of Computational Molecular
Science, Temple University, Philadelphia, Pennsylvania 19122, United States
ABSTRACT: A library of eight amphiphilic Janus glycodendrimers (Janus-GDs) presenting D-
lactose (Lac) and a combination of Lac with up to eight methoxytriethoxy (3EO) units in a sequence-
defined arrangement was synthesized via an iterative modular methodology. The length of the linker
between Lac and the hydrophobic part of the Janus-GDs was also varied. Self-assembly by injection
from THF solution into phosphate-buffered saline (PBS) led to unilamellar, monodisperse
glycodendrimersomes (GDSs) with dimensions predicted by Janus-GD concentration. These GDSs
provided a toolbox to measure bioactivity profiles in agglutination assays with sugar binding proteins
(lectins). Three naturally occurring forms of the human adhesion/growth-regulatory lectin galectin-8
(Gal-8S and Gal-8L), which differ by the length of linker connecting their two active domains, and a
single-amino acid mutant (F19Y) were used as probes to study activity and sensor capacity.
Unpredictably, the sequence of Lac on the Janus-GDs was demonstrated to determine bioactivity with
the highest level revealed for a Janus-GD with six 3EO groups and one Lac. A further increase in Lac
density was invariably accompanied by a substantial decrease in agglutination, whereas a decrease in
Lac density resulted in similar or lower bioactivity and sensor capacity. Both changes in topology of Lac
presentation of the GDSs and seemingly subtle alterations in protein structure resulted in different levels
of bioactivity, demonstrating the presence of regulation on both GDS surface and lectin.
1
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 25
These results illustrate the applicability of Janus-GDs to dissect structure-activity relationships between
programmable cell surface models and human lectins in a highly sensitive and physiologically relevant
manner.
1
2
3
4
5
6
7
8
INTRODUCTION
9
One of the key challenges of current biomedical research is to gain understanding of the
molecular basis of cell-cell/matrix interactions. It is known that already at the stage of sperm-zona
pellucida recognition in fertilization, various binding parameters such as the epitope structure of
determinants and their local density and topology of presentation cooperate to generate the required
avidity, selectivity, specificity and contact stability of binding.1 Increasingly gaining attention, loading
and delivery of exosomes and microvesicles, with diameters of 40 to about 150 nm or more, are likewise
of central biological relevance as a means for directed transport and understanding mechanisms of
assembly.2 Prominent among surface interactions is the interplay between glycans and their receptors
(lectins), involved in many physiological and pathological processes.3 However, although the nominal
carbohydrate specificity of lectins is being characterized in detail, the intriguing selectivity of this
recognition mode on cell and vesicle surfaces indicates that topological factors can play major roles. To
address this issue, the design and synthesis of readily programmable model systems is a challenge for
supramolecular chemistry. Access to these models will enable understanding of the contributions of
structure, topology of presentation and particle size on bioactivity towards human lectins.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
So far, two different strategies have been employed to prepare tools for unraveling the complexity of
carbohydrate-lectin interactions, including a covalent approach with synthetic glycopeptides,4
glycopolymers5 and glycodendrimers,6 and a supramolecular approach7 including glycan-presenting
vesicles. For example, the Kiessling laboratory observed in a model study that a higher density of
carbohydrates on glycopolymers5d results in increased activity per polymer but decreased efficiency per
carbohydrate towards lectins, while the Kiick laboratory found increased relative activity to
carbohydrate ligands at lower density of binding epitopes on a glycopolymer.5e The Seeberger laboratory
designed programmable sequences of carbohydrates on monodisperse glycooligomers, and concluded
2
ACS Paragon Plus Environment

Page 3 of 25
Journal of the American Chemical Society
that the increase of the number of sugars from one to three enhanced the relative activity,5i while dilution
with non-cognate sugars on these glycooligomers improved the relative activity.5n Although these
pioneering approaches provided valuable insight into the “multivalency” of glycan ligands,3b,8 they also
demonstrated that the influence of ligand structure on the mechanism of carbohydrate-lectin interaction
process is incompletely understood.5d,e,I,n,l,7a For example the previous mimics of biological membranes
could not closely model their surface with spatial display of glycan ligands containing an optimal
density and defined sequence. With the aim to design such biological mimics, glycodendrimersomes
(GDSs)9 that provide access to density and sequence control were recently introduced. They are
generated by the self-assembly of amphiphilic Janus-glycodendrimers (Janus-GDs) and have potent
bioactivity as docking sites for lectins, establishing a model system for studying their trans-bridging
capacity.9b-d In this study, eight sequence- and density-defined Janus-GDs were synthesized and shown
to self-assemble into unilamellar GDSs of predictable dimensions. Agglutination assays of the GDSs
were performed with the biomedically relevant human lectin Gal-8 to reveal an optimal glycan topology
that unexpectedly occurs for a low density of Lac in a defined sequence to generate the highest
agglutination relative activity and sensor capacity.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
RESULTS AND DISCUSSION
Rational Design and Iterative Modular Synthesis of Amphiphilic Janus Dendrimers with Density-
and Sequence-Defined D-Lactose. A distinctive feature of the presence of glycans on the cell surface is
the natural heterogeneity of density. It can be based on microclusters such as branching of N-glycans and
mucin-type O-glycans. It can also be based on macroclusters referring to local vicinity of individual
glycan chains in glycoproteins such as mucins, or in microdomains with glycoprotein/glycolipid
clusters. It was previously reported that bioinspired GDSs self-assembled from “single-single” (1-Lac),
“twin-twin” (2-Lac) and “twin-mixed” (3-Lac) amphiphilic Janus-GDs (Scheme 1) and their D-mannose
(Man) containing analogues.9b With agglutination assays of GDSs with identical concentration of
carbohydrates, it was discovered that GDSs self-assembled from 3-Lac or its Man-containing analogue
3
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 25
with lower density of glycan ligands exhibited higher relative bioactivity toward their cognitive
lectins.9b-d However, no optimal density and sequence of carbohydrate toward glycan ligands on
biological membranes was determined. This fundamental question prompted the design of new Janus-
GDs from 4-Lac to 6-Lac (Scheme 1) with increasingly reduced density and defined sequences of
epitopes. The general aim was to gain fundamental insight into the multivalency and the impact of
topology of glycan presentation, and also to attempt to find out if an optimal density and sequence of
glycan ligands providing the highest activity can be realized.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Summary of Lac-containing amphiphilic Janus dendrimers used for agglutination assays with Gal-8:
3EO = methoxytriethoxy group; Lac = D-lactose. The hydrophobic segments of these molecules, triazoles, and
aromatic rings are omitted for clarity. The numbers in the parentheses define the position of each hydrophilic tail
that is counted from the left to the right of each molecule.
Inspired by the notation coined by the Seeberger laboratory5m the amphiphilic molecules
designed for the current study are denoted with the self-explanatory notations illustrated in Figure 1.
Taking 3EO(1,2,3)-3EOLac(4) for example, “3EO” denotes methoxytriethoxy group, “3EOLac” refers
to triethoxy-lactoside group, and the numbers within the parentheses define the position of each
hydrophilic tail in the hydrophilic segment. These molecules were also given a code such as 3-Lac.
Together with Scheme 1, Figure 1 demonstrates the rational design of the series of Lac-containing
Janus-GDs. As described previously by our laboratory,9b 1-Lac (3EOLac(1)) contains a single
4
ACS Paragon Plus Environment

Page 5 of 25
Journal of the American Chemical Society
hydrophobic first-generation minidendron and a single carbohydrate headgroup, which simply reduces
its molecular weight by half compared to 2-Lac (3EOLac(1,2)). Of note, these two molecular
frameworks share the same density of Lac. Comparing to 1-Lac and 2-Lac, the density of Lac in 3-Lac
(3EO(1,2,3)-3EOLac(4)) is reduced by introducing three chains of 3EO in the hydrophilic part. From 3-
Lac to 4-Lac (3EO(1,2,3,4,5,6)-3EOLac(7)) and to 5a-Lac (3EO(1,2,3,4,5,6,7)-3EOLac(8)-3EO(9)),
the density of Lac further decreases. 5a-Lac and 5b-Lac (3EO(1,2,3,4,5,6,7,8)-3EOLac(9)) are a pair of
isomers with Lac located in different positions on the hydrophilic segment, and the same is true for 6a-
Lac (3EO(1,2,3,4,5,6,7)-6EOLac(8)-3EO(9)) and 6b-Lac (3EO(1,2,3,4,5,6,7,8)-6EOLac(9)). Compared
to 5a-Lac/5b-Lac, 6a-Lac/6b-Lac are only different in that their Lac-headgroups are attached to a
longer chain of oligo(ethylene glycol) monomethyl ether.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In accordance with the method used for the synthesis of the “twin-mixed” molecule 3-Lac
previously reported,9b 4-, 5a-, 5b-, 6a- and 6b-Lac were synthesized via an accelerated modular strategy
by using the acetonide-protected tris(hydroxymethyl)aminomethane (Tris) (5) to form the Janus
dendrimers with three different groups (A, B, C) as outlined in Figure 2. First, the presence of the amino
group in 5 allowed for the selective addition of an acid or anhydride-containing group A to the amino
group in molecule 5 was realized via amidation. The presence of a single unprotected hydroxyl group on
the resulting molecules (6, 20a, 20b) allowed for the targeted addition of an acid containing the
hydrophilic group B. This esterification yielded the desired hydrophilic portion of the Janus dendrimers
(7, 21a, 21b). Their acetonide groups were then removed via acid catalysis to yield two hydroxyl groups
in the resulting products (8, 22a, 22b). Esterification of the hydroxyl groups in 8, 22a, 22b with an acid-
containing group C was performed to generate the hydrophobic portion of the Janus dendrimers. Finally,
the modular synthesis was completed with the addition of two possible azide-functionalized D-lactose
derivatives (Lac-3EO-N3 or Lac-N3) to the alkyne in group A of the hydrophilic portion of the Janus
dendrimers via copper-catalyzed click chemistry10 to give 4-, 5a-, 5b-, 6a- and 6b-Lac. Detailed
reagents and conditions for each reaction are presented in the Supporting Information (Scheme SS1–7).
5
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 25
1
2
3
4
Scheme 1. Summary of Amphiphilic Janus Dendrimers with Different Density and Sequence-Defined
Arrangement of D-Lactose (Lac) in the Hydrophilic Segmenta
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aCodes of the amphiphilic molecules are indicated in black and their short notations are indicated in red. The
diameter (DDLS, in nm) and polydispersity (in the parentheses) were determined by dynamic light scattering (DLS)
at 0.1 mM of Lac in phosphate buffered saline (PBS 1×).
6
ACS Paragon Plus Environment

Page 7 of 25
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Summary of the accelerated iterative modular synthetic strategy employed in the preparation of the
amphiphilic Janus-GDs 4-Lac, 5a-Lac, 5b-Lac, 6a-Lac and 6b-Lac. “A” represents the structure incorporated in
the dendrimers between the triazole ring and the Tris framework, “B” represents a second-generation hydrophilic
minidendron, “C” represents a second-generation hydrophobic minidendron, and “S” represents the Lac group.
Self-assembly of Lac-Containing Janus-GDs into Monodisperse, Unilamellar GDSs with
Predictable Size. The GDSs were prepared by injection of their THF solution into PBS.9,11 As
determined by cryogenic transmission electron microscopy (cryo-TEM), all Lac-containing Janus-GDs
self-assemble into unilamellar vesicles in PBS. Representative images of 4-, 5a-, 5b-, 6a- and 6b-Lac
GDSs are shown in Figure 3. Images of 1-, 2- and 3-Lac have been reported previously.9b
7
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 25
1
2
3
4
5
6
7
8
9
Figure 3. Selected cryo-TEM images of GDSs self-assembled by amphiphilic molecules (a) 4-Lac
(3EO(1,2,3,4,5,6)-3EOLac(7)), (b) 5a-Lac (3EO(1,2,3,4,5,6,7)-3EOLac(8)-3EO(9)), (c) 5b-Lac
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(3EO(1,2,3,4,5,6,7,8)-3EOLac(9)), (d) 6a-Lac (3EO(1,2,3,4,5,6,7)-6EOLac(8)-3EO(9)), and (e) 6b-Lac
(3EO(1,2,3,4,5,6,7,8)-6EOLac(9)) at 0.1 mM in PBS.
Figure 4. Short notations and the summary of Lac-containing amphiphilic molecules with different topologies
and their corresponding GDSs. The diameter (DDLS, in nm) and polydispersity (in the parentheses) were measured
by DLS (0.1 mM of Lac in PBS). 3D topological vesicular structures are drawn as 2D cross-section models for
better clarify of their surface arrangement and density of glycans. For simplicity Lac-groups are isolated although
most probably they interact with each other during the self-assembly process.
8
ACS Paragon Plus Environment

Page 9 of 25
Journal of the American Chemical Society
Dynamic light scattering (DLS) was used to assess differences in diameter of GDSs prepared
1
2
with the same molar concentration of Lac (0.1 mM) in PBS. As shown in Scheme 1 and Figure 4, the
size and size-distribution of the vesicles self-assembled from 1-Lac and 2-Lac are almost identical.
Considering that 1-Lac shares similar chemical structure with half of 2-Lac, this identical self-assembly
behavior (Figure 4) indicates that the power of two molecules of 1-Lac is equal to that of one molecule
of 2-Lac during the self-assembly process. Intuitively, the volume occupied by an individual molecule
in a GDS should increase with the molecular weight of the Janus-GD, and this agrees with the increasing
size of the vesicles following the order 2-Lac < 3-Lac < 4-Lac (Figure 4). On the other hand, the size of
4-Lac is almost identical with that of 5a-Lac and 5b-Lac, even though their molecular weights are not
identical. More surprisingly, the GDSs formed by 6a-Lac and 6b-Lac shrink significantly. The
complexity of the chemical structure of these Janus-GDs rendered the mechanism of self-assembly
rather complex, and acquisition of more understanding of this self-assembly process by computer
simulations is currently in progress. In addition, it is important to notice that testing with DLS gave
polydispersities (PDI) between 0.14 and 0.30. These values indicate monodisperse vesicles.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. Concentration dependence of the (a) diameter (DDLS, in nm) and (b) square of diameter (DDLS2) of GDSs
self-assembled by Lac-containing Janus-GDs in PBS. R2 = coefficient of determination.
Based on the results previously reported by our laboratory, the sizes of “twin-twin”11b or “single-
single”11c Janus dendrimersomes were predictable by their concentration. The sizes (diameters) could be
calculated from the thickness of the bilayer and the concentration of dendrimer solution. Furthermore,
9
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 25
the square of the dendrimersome diameter exhibits a linear correlation with the concentration. In the
current study all the Janus-GDs self-assembled into unilamellar vesicles with predictable dimension in
PBS in a suitable range of concentration as indicated in Figure 5. It should be noted that since the sizes
of the isomeric pairs 5a-/5b-Lac and 6a-/6b-Lac were identical, 5b-Lac 3EO(1,2,3,4,5,6,7,8)-
3EOLac(9) and 6a-Lac 3EO(1,2,3,4,5,6,7)-6EOLac(8)-3EO(9) were chosen as representative cases for
the isomers. Figure 5a shows the experimental diameter values obtained from DLS experiments. When
converting into square of diameter as shown in Figure 5b, the mass concentration of each Janus-GD is
proportional to the square of diameter of the corresponding vesicle. This physically significant
correlation was consistent with previous conclusions.11b,c The curves in Figure 5a can therefore be used
as a convenient calibration tool11b to predict the size of the vesicles in the concentration range of 0 to 1
mg·mL–1. The reliability of this prediction was validated by the results summarized in Table 1. The
measured sizes of the GDSs agree with the predicted values at the final Lac concentration of 0.1 mM.
Table 1. Comparison of the Size of Various Lac-Containing Glycodendrimersomes Predicted by Diameter-
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Concentration Correlation and Size Determined by DLS at Lac = 0.1 mM in PBS
b
Glycodendrimersomes
3EOLac(1)
a
Concentration
mg·mL–1
0.109
Dpredicted
nm
DDLS
nm
39
38
51
151
145
100
Errorc
(D2 = aC)a
11426
12360
10963
53910
47130
22565
35
9.1%
3.5%
1.8%
0.3%
1.0%
2.5%
3EOLac(1,2)
0.108
37
3EO(1,2,3)-3EOLac(4)
0.229
52
3EO(1,2,3,4,5,6)-3EOLac(7)
3EO(1,2,3,4,5,6,7,8)-3EOLac(9)
3EO(1,2,3,4,5,6,7)-6EOLac(8)-3EO(9)
0.418
0.452
0.466
150
146
103
aequations are derived from the linear correlation between square of diameter (D2) and mass concentration (C) in
Figure 5, and a is the constant of the equation. bDpredicted is calculated according to the equation D2 = aC. cError is
the absolute value calculated according to the equation Error = [(Dpredicted–DDLS)/Dpredicted] × 100%.
Supramolecular Models of Biological Membranes Containing Multivalent Glycan Ligands with
Programmable Density, Sequence and Topology of Presentation. These GDSs provide a valuable
toolbox to dissect the contribution of diverse parameters of spatial display on the bioactivity toward
lectins. To add physiological relevance a human lectin, the tandem-repeat-type galectin-8 (Gal-8), was
examined. Gal-8 has recently been discovered to be a potent sensor for endosome/lysosome integrity12
via its glycan binding and switching on of the autophagy machinery. It is also a matricellular modulator
10
ACS Paragon Plus Environment

Page 11 of 25
Journal of the American Chemical Society
of adhesion and migration, with further effector capacity on cell growth.13 Its occurrence in two isoforms
is caused by alternative splicing so that the two carbohydrate recognition domains (CRD) are connected
by a peptide linker composed of 33 or 75 amino acids (Gal-8S/8L) (Figure SF2). The functional
relevance of the length of linker is so far unknown. The agglutination assays of all GDSs with Gal-8
were monitored by UV-vis spectroscopy for 1000 s so that the optical density (OD) value of all GDSs
could reach a plateau (Figure 6). Before the study of the influence of spatial display of glycan ligands on
the bioactivity of GDSs, non-selective binding was rigorously excluded by control experiments testing a
suspension of GDSs with non-cognate sugar head group, i.e. D-mannose for Gal-8 (Figure 6) and with a
non-specific lectin, i.e. GDS from 3-Lac with ConA (Figure SF3). Additional control experiments were
reported in references 9a (Figure SF 26), 9b (Figure SF 5), 9c (Figure SF 2) and 9d (Figures 4, 5 and 6).
The absence of secondary interactions during the agglutination process were demonstrated by saturating
Gal-8S with 100 mM D-lactose and perform the agglutination with GDSs from 3-Lac (Figure SF4). No
agglutination was observed in this experiment. However the control experiment of Gal-8S containing
100 mM D-fructose provided the expected agglutination of GDSs from 3-Lac (Figure SF4). Finally, the
addition of 100 mM D-lactose 100 s after the agglutination of the GDSs from 3-Lac with Gal-8S
provided quantitative dissociation and therefore demonstrated the absence of secondary interactions
during the agglutination process (Figure SF5).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6. Agglutination assays between different Lac-containing GDSs at identical concentration of Lac and two
11
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 12 of 25
Gal-8 natural variants. Lac-containing GDSs (0.1 mM of Lac in 900 µL of PBS) were incubated with (a) Gal-8S
or (b) Gal-8L (2 mg·mL–1 in 100 µL of PBS). The molar attenuation coefficient ε = A/(cl), adapted from Beer–
Lambert law, where A = plateau OD-value, c = molar concentration of Lac, and l = semi-micro cuvette path
length (0.23 cm). Control experiments were carried out by incubating 3-Man9b (3EO(1,2,3)-3EOMan(4), 0.1 mM
of Man in 900 µL of PBS, its chemical structure was described in Scheme SS8 with Gal-8S/8L (2 mg·mL–1 in 100
µL of PBS).
1
2
3
4
5
6
7
8
9
In the first set of agglutination experiments, the molar concentration of Lac in all vesicles was
maintained at 0.1 mM before incubation with Gal-8. In order to better quantify the relative agglutination
activity of each type of Lac-containing vesicle, the molar attenuation coefficient per Lac (ε) was
calculated according to the Beer–Lambert law (A = εcl), where A, c and l respectively stand for the
plateau value of OD, molar concentration of Lac, and the path length of light that is equal to the width of
the cuvette. This is valid under the assumption that there is no lysis of GDSs during the course of the
agglutination assay. This assumption was validated previously by cryo-TEM9a,c experiments, that in
agreement with the mechanical properties of GDSs,9a,11a,b supported their expected shape integrity
during the agglutination process. The increase of OD-value from Figure 6 is therefore the result of
crosslinking of intact and undeformed GDSs. Therefore the results of GDSs integrity during
agglutination and the absence of secondary interactions is in agreement with isothermal titration
calorimetry (ITC) and hemaglutination experiments reported for Gal-8.13c
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
As an internal control, the series of 1-Lac, 2-Lac and 3-Lac headgroup display was first tested
with Gal-8S. Confirming previous experience,9d a grading of activity was observed with the molar
attenuation coefficient ε increasing from 8.3 for 1-Lac to 10 for 2-Lac and 13 ×103 M–1·cm–1 for 3-Lac
(Figure 6a). Indeed, the binding experiments in Figure 6 reveal a different range of responses for all
GDSs, although their contact sites for Gal-8, i.e. the disaccharide Lac, are identical. This therefore
underscores the influence of spatial factors. The already significant reactivity of 3-Lac was raised by up
to 2-fold by altering the mode of presentation to 4-Lac, despite the reduced density of Lac. Of note, the
compounds 4-Lac vs. 5-Lac/6-Lac also likely differ in degree of lateral flexibility when self-
assembling. This second factor concerns the possibility of a physiological elongation. It is realized either
within the glycan chain, most prominently by adding N-acetyllactosamine repeats especially to the β1,6-
12
ACS Paragon Plus Environment

Page 13 of 25
Journal of the American Chemical Society
branch of complex-type N-glycans and the core 2/4 of mucin-type O-glycans, or the scaffold by
incorporation of long-chain fatty acids into the ceramide backbone of glycosphingolipids.14 Intuitively,
such an extension should make the sugar headgroup especially accessible for lectin binding, as
delineated for sulfatides with a C24-long anchor within apical transport processes in enterocyte-like cells
by a human galectin.15 In contrast to the intuitive expectations, an extension of the linker (in 6a/b-Lac)
did not lead to OD-increase, since 5a/b-Lac with identical structure but shorter linker length were more
conducive to yield aggregates (Figure 6). The comparison between 5a/b-Lac and 6a/b-Lac implies that
an extended arm may allow backfolding to the surface of vesicles, which restricts headgroup
accessibility or compromises stability of the trans-interaction of lectins. In summary, the highest signal
was seen with 4-Lac as building block, and this indicates there could be an optimal sequence and
density of glycan ligands to ensure the high level of their bioactivity.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
We also tried to study the impact of length variation of the natural linker in Gal-8, if there is any.
Instead of the rigid positioning of the four binding sites in Concanavalin A (ConA),16 the linker between
the two carbohydrate-binding domains of Gal-8 may furnish spatial adaptability, both rotationally and
laterally. Whether the length extension of natural linker of Gal-8 will affect capacity for agglutination
and sensitivity to surface design is answered in Figure 6. Fine-tuning by structural changes, for example
between 6a-Lac and 6b-Lac, is in this case achievable, making Gal-8L more sensitive (21 vs. 17 ×103
M–1·cm–1 in Figure 6b) to changes in Lac display than Gal-8S (23 vs. 23 ×103 M–1·cm–1 in Figure 6a).
This difference nourishes the idea for higher spatial adaptability by longer linker length for this
adhesion/growth-regulatory tissue effector.
13
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 14 of 25
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 7. Rate of change in turbidity, k, of GDSs with Gal-8S (blue) and Gal-8L (red) calculated from the data in
Figure 6 at t0.5, where t0.5 is the time at which the observed absorbance is equal to half of the plateau absorbance.
Binding was too fast (t0.5 ≈ 5–20 s) and the initial rate could not be determined. The initial rate is higher than the
rate at t0.5 and hence the calculated values of k presented here represent an underestimate of the true initial rate.
It is instructive to compare the agglutination data from Figure 6 with the rate change in turbidity,
k, of the same GDSs with Gal-8S (in blue) and Gal-8L (in red) from Figure 7. Since the agglutination
process is over in about 100 s the initial rate was determined at t0.5 ≈ 5–20 s in order to have a fair
comparison of the rates from different processes and therefore these values are underestimated.
Nevertheless they demonstrate the same trend as the data from Figure 6.
GDSs present the carbohydrates both on the interior and exterior surface of the supramolecular
assemblies (Figure 4). It is not yet known at this time if any of the exterior surface carbohydrates may be
or not quantitatively available for binding and to what extent they can be hidden within the GDS
structure. Nevertheless indication that most of the carbohydrates from the outer surface of the GDSs are
available for binding was provided by previous co-assembly experiments performed with amphiphilic
Janus glycodendrimers containing binding and nonbinding carbohydrates. These mixed binding-
nonbinding GDSs demonstrated an increase in agglutination parallel with the increase in the
concentration of the binding sugar.9d
However, Figure 6 does not take into consideration that constant concentration of Lac at 0.1 mM
results in a range of diameters for the various GDSs. When the concentration of a specific Janus-GD is
kept constant, it is expected that the bioactivity of the GDSs is size-dependent.9a Thus, experiments to
investigate the relationship between vesicle size and agglutination were performed. The impact of this
14
ACS Paragon Plus Environment

Page 15 of 25
Journal of the American Chemical Society
parameter was considered with 2-Lac (3EOLac(1,2)) (Figure 8a and 8b) and 3-Lac (3EO(1,2,3)-
3EOLac(4)) (Figure 8c and 8d) as two representative examples. These GDSs with identical
concentration but different dimension were prepared by a successive dilution method (Figure SF1).
Immediately after Gal-8L was incubated with the GDSs, the evolution of size of GDS-lectin aggregates
was monitored by DLS (Figures 8a and 8c) and UV-vis spectroscopy over a period of 600 s (Figure 8b
and 8d). As determined by DLS, the fastest agglutination was provided by the smallest GDSs self-
assembled by 2-Lac (Figure 8a). The plateau value of OD shows that the smallest size led to the highest
bioactivity of the GDSs (Figure 8b). On the contrary, in the investigated range of dimensions both the
rate of binding and the bioactivity of the GDSs self-assembled by 3-Lac increased with increasing size
(Figure 8c and 8d). The opposite tendency implies that the impact of size on bioactivity is specific rather
than general for GDSs formed by Janus-GDs having different structural frameworks, with possible
ramifications for exosome/microvesicle recognition in situ. Thus, the conclusions made from Figure 6
had to be verified with additional experimentation that corrects for vesicle size.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
15
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 16 of 25
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 8. Agglutination of 2-Lac (3EOLac(1,2), 900 L, 0.0625 mg·mL–1) and 3-Lac (3EO(1,2,3)-3EOLac(4)
vesicles, 900 L, 0.0625 mg·mL–1) GDSs of different sizes in the presence of Gal-8L (100 L, 0.5 or 1.0 mg·mL–
1) in PBS. The evolution of sizes of GDS-lectin aggregates was monitored by DLS (a and c) and UV-vis
spectroscopy (b and d).
16
ACS Paragon Plus Environment

Page 17 of 25
Journal of the American Chemical Society
Figure 9. Agglutination assays between different GDSs of identical sizes. (a) Gal-8S (2 mg·mL–1 in 100 µL of
PBS) or (b) Gal-8L (2 mg·mL–1 in 100 µL of PBS) was incubated with Lac-containing GDSs (DDLS = 63 ± 3 nm,
in 900 µL of PBS). The molar attenuation coefficient ε = A/(cl), adapted from Beer–Lambert law, where A =
plateau OD-value, c = molar concentration of Lac, and l = semi-micro cuvette pathlength (0.23 cm). The boxes
divide the GDSs into two groups: small Janus-GDs (top) and large Janus-GDs (bottom). The blue boxes indicate
relatively high sensitivity and the red boxes indicate comparatively low sensitivity of lectins towards different
glycan topologies.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
By applying the prediction method illustrated in the previous section, all GDSs of identical size
(DDLS = 63 ± 3 nm) were prepared from the corresponding Janus-GDs with different concentration.
Their agglutination assays were again carried out with Gal-8. Inevitably, the concentration of all Janus-
GDs cannot be kept constant in the case of identical size, but the impact of concentration can easily be
excluded by calculation of the molar attenuation coefficient per Lac (ε) (Figure 9), yielding a set of data
that controls for both size and concentration of the various GDSs. The ε-value can evaluate their relative
bioactivities due to the identical size with similar curvature and ratio of carbohydrates outside and inside
the GDSs membranes. This series of binding experiments showed similar tendency in both cases of Gal-
8S and Gal-8L as presented in Figure 6, and this further confirms that the spatial display of glycan
ligands significantly affects the relative bioactivity of the GDSs.
Figure 10. Agglutination assays between different GDSs with identical sizes. (a) Gal-8S (2 mg·mL–1 in 100 µL of
PBS) or (b) Gal-8S F19Y (2 mg·mL–1 in 100 µL of PBS) was incubated with Lac-containing GDSs (DDLS = 73 ±
5 nm, in 900 µL of PBS). The molar attenuation coefficient ε = A/(cl), adapted from Beer–Lambert law, where A
= plateau OD-value, c = molar concentration of Lac, and l = semi-micro cuvette path length (0.23 cm). The boxes
divide the GDs into two groups: small Janus-GDs (top) and large Janus-GDs (bottom). The blue boxes indicate
high sensitivity and the red boxes indicate low sensitivity of lectins towards different glycan topologies.
17
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 18 of 25
As reported previously in our laboratory, a single-site mutation of the peptide linker of wild-type
(WT) Gal-8S can impair significantly its cross-linking activity with GDSs self-assembled by 3-Lac.9d
Extending from 3-Lac to all Janus-GDs with different structural pattern in the current study, we
compared the bioactivity of WT Gal-8S with F19Y (the mutated form) (Figure SF2) with all Lac-
containing GDSs with identical size (DDLS = 73 ± 5 nm) (Figure 10). In line with previous results,9d
significant drop in agglutination level from WT Gal-8S to F19Y was observed for all GDSs. Of note,
different GDSs showed different sensitivity toward the impaired function of Gal-8. For example, 30%,
85%, and 25% of the original bioactivity was retained for 3-Lac, 4-Lac and 6b-Lac when they were
incubated with F19Y, respectively. As can be judged from the value of the molar attenuation coefficient
(ε), Gal-8S always exhibited the highest affinity with 4-Lac, regardless of its WT or mutated form
(Figure 9).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
After excluding both the factors of dimension of GDSs and concentration of Lac, the coverage of
Lac on GDS surfaces decreased from 100% for 1-Lac and 2-Lac to 25% for 3-Lac (containing a 3/1
ratio of 3EO/Lac), 14% for 4-Lac (containing a 6/1 ratio of 3EO/Lac) and 11% for 5a-, 5b-, 6a-, 6b-Lac
(all containing a 8/1 ratio of 3EO/Lac). For the series 1-, 2-, 3-, 4-Lac, the tendency of relative
bioactivity towards Gal-8 variants always increased significantly, reaching a maximum ε-value for 4-
Lac (6/1 of 3EO/Lac). Compared to 4-Lac, the series 5a-, 5b-, 6a-, 6b-Lac demonstrated a decrease in
relative bioactivity, but they showed greater values than 1-, 2- and 3-Lac. It is possible that the
decreased Lac coverage on 4-Lac compared to 1-, 2- and 3-Lac could have better accessibility, resulting
in the most efficient protein-sugar interactions. However, further Lac dilution, as in the cases of 5a-, 5b-,
6a- and 6b-Lac, may reduce the necessary quantity of ligand epitopes for binding, offsetting any added
steric benefit of greater dilution. Thus, 4-Lac provides an optimal balance between Lac density and
accessibility, even with a dilution factor of 1/7 but still an increased relative bioactivity factor of 6 (Gal-
8S), 7 (Gal-8L) and 12 (Gal-8S F19Y), compared with 1-Lac.
It is also worth noting that, regarding the behavior of binding of all GDSs, they can be
categorized into two groups: the group of small Janus-GDs including 1-, 2-, and 3-Lac, and the group of
18
ACS Paragon Plus Environment

Page 19 of 25
Journal of the American Chemical Society
large Janus-GDs including 4-, 5a-, 5b-, 6a-, and 6b-Lac. The relative bioactivity of Gal-8S is clearly
distinguishable for the group of small Janus-GDs with their ε values being 4.8, 6.5, 8.9 ×103 M–1·cm–1
for 1-, 2- and 3-Lac (blue box in Figure 9a) respectively, while the GDSs in the group of large Janus-
GDs showed similar affinity toward the same lectin with ε = 24~27 ×103 M–1·cm–1 (red box in Figure
9a). On the other hand, this tendency is opposite in the case of Gal-8L (Figure 9b) and 8S F19Y (Figure
10b). Taking Gal-8L for instance, the relative bioactivity of small Janus-GDs 1-, 2-, and 3-Lac was
fairly similar with ε = 4.7~6.2 ×103 M–1·cm–1 (red box in Figure 9b), whereas the large Janus-GDs
showed significantly different relative activities with ε = 31, 27, 21, 19 and 16 ×103 M–1·cm–1 for 4-, 5a-
, 5b-, 6a-, and 6b-Lac (blue box in Figure 9b). This discrimination ability toward different lectins could
be amplified by “GDS array patterning” and used as the principle for their sensing and identification.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CONCLUSIONS
The current study employed a rational chemical design strategy involving sequence- and density-defined
parameters to create a library of amphiphilic Janus-GDs that self-assemble into GDSs with
programmable glycan topology on their surface. These GDSs can evidently be designed to mimic the
spatial properties of biological membranes and therefore they provide a versatile tool for research in
glycobiology. Agglutination assays with the human lectin Gal-8, unraveled the impact of density,
sequence and topology of the glycans on the bioactivity of the GDS. Various modes of sugar
presentation on the GDS surface led to conspicuously different extents of stable trans-interactions that
can be used to to study structure-activity correlations with relevance for understanding how glycan
display on biological membranes and lectin design team up to their intriguing functions. Since the
influence of ligand structure on binding processes of biological membranes is incompletely
understood5d,k and contradictory results were reported with different models,5d,k,7a the up to twelve times
increased relative agglutination activity at seven times lower Lac concentration observed for 4-Lac with
Gal-8S F19Y was unpredictable and is a remarkable conclusion of these investigations. Thus, this
supramolecular platform offers not only the highest activity but also sensor capability and versatility for
19
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 20 of 25
establishing and exploiting structure-activity relationships of far-reaching biomedical relevance. The
detailed presentation of Lac on the surface of these GDSs requires additional experiments. Nevertheless,
we believe that these results will impact the design of more efficient glycopolymers, glycopeptides,
glycodendrimers and of any other multivalent glycoconjugates by decreasing the density of the
carbohydrate while employing a proper and well defined-sequence. Last but not least, our laboratory’s
approach to discovery and prediction by screening rationally designed libraries of building blocks17 has
been shown to be extremely efficient when applied to amphiphilic Janus dendrimers, their
dendrimersomes,12 amphiphilic Janus-GDs and their GDSs.9 These investigations provided substantial
progress in the field of synthetic vesicles and liposomes.18 The generality of the sequence- and density-
defined presentation of ligands on the concept of multivalency8 reported here is currently being
expanded to additional GDSs libraries with more complex structure, different glycan ligands as well as
to other classes of ligands.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ASSOCIATED CONTENT
Supporting Information: Synthetic procedures with complete structural and self-assembly analysis,
sample preparation, and experimental protocol. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: percec@sas.upenn.edu
Notes: The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Financial support from the National Science Foundation (grants DMR-1066116 and DMR-1120901), the
P. Roy Vagelos Chair at the University of Pennsylvania, and the Humboldt Foundation (all to VP),
National Science Foundation (grant DMR-1120901 to MLK) and the EC Seventh Framework
20
ACS Paragon Plus Environment

Page 21 of 25
Journal of the American Chemical Society
Programme (GLYCOPHARM) are gratefully acknowledged. The authors also thank Dr. H.-J. Sun for
performing one cryo-TEM experiment.
1
2
3
4
5
6
7
REFERENCES
8
9
(1)
(a) Thaler, C. D.; Cardullo, R. A. J. Biol. Chem. 1996, 271, 23289–23297. (b) Clark, G. F.; Dell,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A. J. Biol. Chem. 2006, 281, 13853–13856.
(2)
(a) Fevrier, B.; Raposo, G. Curr. Opin. Cell. Biol. 2004, 16, 415–421. (b) Valadi, H.; Ekström,
K.; Bossios, A.; Sjöstrand, M.; Lee, J. J.; Lötvall, J. O. Nat. Cell. Biol. 2007, 9, 654–659. (c) EL
Andaloussi, S.; Mäger, I.; Breakefield, X. O.; Wood, M. J. A. Nat. Rev. Drug Discov. 2013, 12, 347–
357. (4) Raposo, G.; Stoorvogel, W. J. Cell. Biol. 2013, 200, 373–383.
(3)
(a) Sharon, N.; Lis, H. Science 1972, 177, 949–959. (b) Lee, Y. C.; Lee, R. T. Acc. Chem. Res.
1995, 28, 321–327. (c) Lis, H.; Sharon, N. Chem. Rev. 1998, 98, 637–674. (d) Gabius, H.-J.; André, S.;
Jiménez-Barbero, J.; Romero, A.; Solís, D. Trends Biochem. Sci. 2011, 36, 298–313. (e) Lepenies, B.;
Lee, J.; Sonkaria, S. Adv. Drug Deliv. Rev. 2013, 65, 1271–1281. (f) André, S.; Kaltner, H.; Manning, J.
C.; Murphy, P. V.; Gabius, H.-J. Molecules 2015, 20, 1788–1823.
(4)
(a) Aoi, K.; Tsutsumiuchi, K.; Okada, M. Macromolecules 1994, 27, 875–877. (b) Allen, J. R.;
Harris, C. R.; Danishefsky, S. J. J. Am. Chem. Soc. 2001, 123, 1890–1897. (c) Kramer, J. R.; Deming, T.
J. J. Am. Chem. Soc. 2010, 132, 15068–15071. (d) Kramer, J. R.; Deming, T. J. Polym. Chem. 2014, 5,
671–682. (e) Krannig, K.-S.; Sun, J.; Schlaad, H. Biomacromolecules 2014, 15, 978–984.
(5)
(a) Yamada, K.; Yamaoka, K.; Minoda, M.; Miyamoto, T. J. Polym. Sci. Part A: Polym. Chem.
1997, 35, 255–261. (b) Horan, N.; Yan, L.; Isobe, H.; Whitesides, G. M.; Kahne, D. Proc. Natl. Acad.
Sci. U.S.A. 1999, 96, 11782–11786. (c) Matsuura, K.; Hibino, M.; Yamada, Y.; Kobayashi, K. J. Am.
Chem. Soc. 2001, 123, 357–358. (d) Cairo, C. W.; Gestwicki, J. E.; Kanai, M.; Kiessling, L. L. J. Am.
Chem. Soc. 2002, 124, 1615-1619. (e) Polizzotti, B. D.; Kiick, K. L. Biomacromolecules 2006, 7, 483–
490. (f) Geng, J.; Mantovani, G.; Tao, L.; Nicolas, J.; Chen, G.; Wallis, R.; Mitchell, D. A.; Johnson, B.
R. G.; Evans, S. D.; Haddleton, D. M. J. Am. Chem. Soc. 2007, 129, 15156–15163. (g) Godula, K.;
Bertozzi, C. R. J. Am. Chem. Soc. 2010, 132, 9963–9965. (h) Ruff, Y.; Buhler, E.; Candau, S.-J.;
Kesselman, E.; Talmon, Y.; Lehn, J.-M. J. Am. Chem. Soc. 2010, 132, 2573–2584. (i) Ponader, D.;
Wojcik, F.; Beceren-Braun, F.; Dernedde, J.; Hartmann, L. Biomacromolecules 2012, 13, 1845–1852. (j)
Miura, Y. Polym J 2012, 44, 679–689. (k) Kiessling, L. L.; Grim, J. C. Chem. Soc. Rev. 2013, 42,
4476–4491. (l) Zhang, Q.; Haddleton, D. M. Adv. Polym. Sci. 2013, 262, 39–60. (m) Jimenez Blanco, J.
L.; Ortiz Mellet, C.; Garcia Fernandez, J. M. Chem. Soc. Rev. 2013, 42, 4518–4531. (n) Ponader, D.;
21
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 22 of 25
Maffre, P.; Aretz, J.; Pussak, D.; Ninnemann, N. M.; Schmidt, S.; Seeberger, P. H.; Rademacher, C.;
1
2
3
Nienhaus, G. U.; Hartmann, L. J. Am. Chem. Soc. 2014, 136, 2008–2016.
4
5
(6)
(a) Roy, R.; Zanini, D.; Meunier, S. J.; Romanowska, A. J. Chem. Soc., Chem. Commun. 1993,
6
7
8
9
1869–1993. (b) Turnbull, W. B.; Kalovidouris, S. A.; Stoddart, J. F. Chem. Eur. J. 2002, 8, 2988–3000.
(c) Woller, E. K.; Walter, E. D.; Morgan, J. R.; Singel, D. J.; Cloninger, M. J. J. Am. Chem. Soc. 2003,
125, 8820–8826. (d) Wang, S.-K.; Liang, P.-H.; Astronomo, R. D.; Hsu, T.-L.; Hsieh, S.-L.; Burton, D.
R.; Wong, C.-H. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 3690–3695. (e) Thomas, B.; Berthet, N.;
Garcia, J.; Dumya, P.; Renaudet, O. Chem. Commun. 2013, 49, 10796–10798. (f) Munoz, E. M.; Correa,
J.; Riguera, R.; Fernandez-Megia, E. J. Am. Chem. Soc. 2013, 135, 5966–5969. (g) Chabre, Y. M.; Roy,
R. Chem. Soc. Rev. 2013, 42, 4657–4708. (h) Appelhans, D.; Klajnert-Maculewicz, B.; Janaszewska, A.;
Lazniewska, J.; Voit, B. Chem. Soc. Rev. 2015, 44, 3968–3996. (i) Roy, R.; Shiao, T. C. Chem. Soc.
Rev. 2015, 44, 3924–3941.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(7)
(a) Grant, C. W. M.; Peters, M. W. Biochim. Biophys. Acta 1984, 779, 403–422. (b) DeFrees, S.
A.; Phillips, L.; Guo, L.; Zalipsky, S. J. Am. Chem. Soc. 1996, 118, 6101–6104. (c) Voskuhl, J.; Stuart,
M. C. A.; Ravoo, B. J. Chem. Eur. J. 2010, 16, 2790–2796. (d) Kramer, J. R.; Rodriguez, A. R.; Choe,
U.-J.; Kamei, D. T.; Deming, T. J. Soft Matter 2013, 9, 3389–3395. (e) Jayaraman, N.; Maiti, K.;
Naresh, K. Chem. Soc. Rev. 2013, 42, 4640–4656. (f) Chmielewski, M. J.; Buhler, E.; Candau, J.; Lehn,
J.-M. Chem. Eur. J. 2014, 20, 6960–6977.
(8)
(a) Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem. Int. Ed. 1998, 37, 2754–2794.
(b) Lundquist, J. J.; Toone, E. J. Chem. Rev. 2002, 102, 555–578. (c) Fasting, C.; Schalley, C. A.;
Weber, M.; Seitz, O.; Hecht, S.; Koksch, B.; Dernedde, J.; Graf, C.; Knapp, E.-W.; Haag, R., Angew.
Chem. Int. Ed. 2012, 51, 10472–10498.
,(9)
(a) Percec, V.; Leowanawat, P.; Sun, H.-J.; Kulikov, O.; Nusbaum, C. D.; Tran, T. M.; Bertin,
A.; Wilson, D. A.; Peterca, M.; Zhang, S.; Kamat, N. P.; Vargo, K.; Moock, D.; Johnston, E. D.;
Hammer, D. A.; Pochan, D. J.; Chen, Y.; Chabre, Y. M.; Shiao, T. C.; Bergeron-Brlek, M.; André, S.;
Roy, R.; Gabius, H.-J.; Heiney, P. A. J. Am. Chem. Soc. 2013, 135, 9055–9077. (b) Zhang, S.;
Moussodia, R.-O.; Sun, H.-J.; Leowanawat, P.; Muncan, A.; Nusbaum, C. D.; Chelling, K. M.; Heiney,
P. A.; Klein, M. L.; André, S.; Roy, R.; Gabius, H.-J.; Percec, V. Angew. Chem. Int. Ed. 2014, 53,
10899–10903. (c) Zhang, S.; Moussodia, R.-O.; Murzeau, C.; Sun, H.-J.; Klein, M. L.; Vértesy, S.;
André, S.; Roy, R.; Gabius, H.-J.; Percec, V. Angew. Chem. Int. Ed. 2015, 54, 4036–4040. (d) Zhang, S.;
Moussodia, R.-O.; Vértesy, S.; André, S.; Klein, M. L.; Gabius, H.-J.; Percec, V. Proc. Natl. Acad. Sci.
U.S.A. 2015, 112, 5585–5590.
22
ACS Paragon Plus Environment

Page 23 of 25
Journal of the American Chemical Society
(10) (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int. Ed. 2001, 40, 2004–2021. (b)
1
2
3
Rostovtsev, V. V.; Green, L. G.; Fokin, V.V.; Sharpless, K. B. Angew. Chem. Int. Ed. 2002, 41, 2596–
2599.
4
5
6
7
8
9
(11) (a) Percec, V.; Wilson, D. A.; Leowanawat, P.; Wilson, C. J.; Hughes, A. D.; Kaucher, M. S.;
Hammer, D. A.; Levine, D. H.; Kim, A. J.; Bates, F. S.; Davis, K. P.; Lodge, T. P.; Klein, M. L.;
DeVane, R. H.; Aqad, E.; Rosen, B. M.; Argintaru, A. O.; Sienkowska, M. J.; Rissanen, K.; Nummelin,
S.; Ropponen, J. Science 2010, 328, 1009–1014. (b) Peterca, M.; Percec, V.; Leowanawat, P.; Bertin, A.,
J. Am. Chem. Soc. 2011, 133, 20507–20520. (c) Zhang, S.; Sun, H.-J.; Hughes, A. D.; Draghici, B.;
Lejnieks, J.; Leowanawat, P.; Bertin, A.; Otero De Leon, L.; Kulikov, O. V.; Chen, Y.; Pochan, D. J.;
Heiney, P. A.; Percec, V. ACS Nano 2014, 8, 1554–1565. (d) Zhang, S.; Sun, H.-J.; Hughes, A. D.;
Moussodia, R.-O.; Bertin, A.; Chen, Y.; Pochan, D. J.; Heiney, P. A.; Klein, M. L.; Percec, V. Proc.
Natl. Acad. Sci. U.S.A. 2014, 111, 9058–9063.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(12) (a) Thurston, T. L. M.; Wandel, M. P.; von Muhlinen, N.; Foeglein, Á.; Randow, F. Nature 2012,
482, 414–418. (b) Meunier, E.; Dick, M. S.; Dreier, R. F.; Schürmann, N.; Broz, D. K.; Warming, S.;
Roose-Girma, M.; Bumann, D.; Kayagaki, N.; Takeda, K.; Yamamoto, M.; Broz, P. Nature 2014, 509,
366–370.
(13) (a) Zick, Y.; Eisenstein, M.; Goren, R. A.; Hadari, Y. R.; Levy, Y.; Ronen, D. Glycoconj. J.
2004, 19, 517–526. (b) Eshkar Sebban, L.; Ronen, D.; Levartovsky, D.; Elkayam, O.; Caspi, D.; Aamar,
S.; Amital, H.; Rubinow, A.; Golan, I.; Naor, D.; Zick, Y.; Golan, I. J. Immunol. 2007, 179, 1225–1235.
(c) Ruiz, F. M.; Scholz, B. A.; Buzamet, E.; Kopitz, J.; André, S.; Menéndez, M.; Romero, A.; Solís, D.;
Gabius, H.-J. FEBS J 2014, 281, 1446–1464.
(14) (a) Sandhoff, R. FEBS Lett. 2010, 584, 1907–1913. (b) Togayachi, A.; Narimatsu, H. Trends
Glycosci. Glycotechnol. 2012, 24, 95–111.
(15) Delacour, D.; Gouyer, V.; Zanetta, J.-P.; Drobecq, H.; Leteurtre, E.; Grard, G.; Moreau-
Hannedouche, O.; Maes, E.; Pons, A.; André, S.; Le Bivic, A.; Gabius, H. J.; Manninen, A.; Simons, K.;
Huet, G. J. Cell Biol. 2005, 169, 491–501.
(16) Goldstein, I. J.; Poretz, R. D. In The Lectins Properties, Functions and Applications in Biology
and Medicine; Goldstein, I. J.;Liener, I. E.; Sharon, N., Eds.; Academic, San Diego, 1986, pp 233–247.
(17) (a) Percec, V.; Cho, W.-D.; Ungar, G.; Yeardley, D. J. P. J. Am. Chem. Soc. 2001, 123, 1302–
1315. (b) Percec, V.; Mitchell, C. M.; Cho, W.-D.; Uchida, S.; Glodde, M.; Ungar, G.; Zeng, X.; Liu, Y.;
Balagurusamy, V. S. K.; Heiney, P. A. J. Am. Chem. Soc. 2004, 126, 6078–6094. (c) Percec, V.;
Holerca, M. N.; Nummelin, S.; Morrison, J. J.; Glodde, M.; Smidrkal, J.; Peterca, M.; Rosen, B. M.;
23
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 24 of 25
Uchida, S.; Balagurusamy, V. S. K.; Sienkowska, M. J.; Heiney, P. A. Chem. Eur. J. 2006, 12, 6216–
6241. (d) Percec, V.; Peterca, M.; Sienkowska, M. J.; Ilies, M. A.; Aqad, E.; Smidrkal, J.; Heiney, P. A.
J. Am. Chem. Soc. 2006, 128, 3324–3334. (e) Percec, V.; Won, B. C.; Peterca, M.; Heiney, P. A. J. Am.
Chem. Soc. 2007, 129, 11265–11278. (f) Rosen, B. M.; Wilson, D. A.; Wilson, C. J.; Peterca, M.; Won,
B. C.; Huang, C.; Lipski, L. R.; Zeng, X.; Ungar, G.; Heiney, P. A.; Percec, V. J. Am. Chem. Soc. 2009,
131, 17500–17521.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(18) (a) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem. Int. Ed. Engl. 1988, 27, 113–158. (b)
Kunitake, T. Angew. Chem. Int. Ed. Engl. 1992, 31, 709–726. (c) Thomas, J. L.; Tirrell, D. A. Acc.
Chem. Res. 1992, 25, 336–342. (d) Guo, X.; Szoka, F. C. Acc. Chem. Res. 2003, 36, 335–341. (e)
Torchilin, V. P. Nat. Rev. Drug. Discov. 2014, 13, 813–827. (f) Allen, T. M.; Cullis, P. R. Science 2004,
303, 1818–1822.
24
ACS Paragon Plus Environment

Page 25 of 25
Journal of the American Chemical Society
TOC Graphic
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
25
ACS Paragon Plus Environment