Mannose/glucose-functionalized dendrimers to investigate the predictable tunability of multivalent interactions

Article abstract of DOI:10.1021/ja053008n

G4-, G5-, and G6-PAMAM dendrimers were functionalized with mixtures of mannose and glucose in varying ratios, and the relative affinities of these compounds for Concanavalin A (Con A) were evaluated using the hemagglutination assay. As the ratio of mannose to glucose increases, the relative activity in the hemagglutination assay (on a per sugar basis) increases linearly. Methyl mannose binds to Con A with an affinity 4-fold higher than that of methyl glucose; multivalency amplifies this trend. The mannose/glucose-functionalized dendrimer results reported here suggest that the affinity of multivalent associations can be attenuated in predictable, reliable ways based on monovalent affinities of the ligands. Copyright

Full text of DOI:10.1021/ja053008n

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Mannose/Glucose-Functionalized Dendrimers To Investigate the Predictable
Tunability of Multivalent Interactions
Mark L. Wolfenden and Mary J. Cloninger*
Department of Chemistry and Biochemistry and Center for Bioinspired Nanomaterials, Montana State UniVersity,
108 Gaines Hall, Bozeman, Montana 59717
Received May 9, 2005; E-mail: mcloninger@chemistry.montana.edu
Scheme 1 a
Multivalent binding between proteins and carbohydrates mediates
many biological events.1 Carbohydrate-protein interactions often
occur with low monomeric binding affinities (Kd ) ∼10-3 M);2 a
multivalent presentation of ligands is generally required to achieve
physiologically relevant associations.3 A variety of scaffolds have
been used as frameworks on which to support the carbohydrate
residues.4
Synthetic multivalent molecules are generally empirically opti-
mized for a particular application. However, two noteworthy studies
to quantify the effects one would expect from multivalent presenta-
tion have been reported. Lees and co-workers describe a binding
enhancement value for divalent, pentameric, and linear polymer
systems,5 while Reinhoudt and co-workers relate the monovalent
association constant to the multivalent association constant using
an effective concentration value and a scaling factor.6 Both of these
methods progress the discussion of how monovalent association
constants effect multivalent interactions, but both suffer from the
difficulty of determining the appropriate value for the effective
concentration.
Whitesides et al. suggested that polyvalent interactions should
equal monovalent interactions raised to the Nth power (eq 1), where
a (a) Synthesis of isothiocyanato carbohydrates. Mannose is shown;
N is the number of receptor-ligand interactions. A cooperativity
glucose (1b and 2b) and galactose (1c and 2c) syntheses are analogous. (b)
Synthesis of mannose/glucose-functionalized dendrimers (galactose addition
factor, R, is also in eq 1.3
does not occur; see text for details). Amounts of x and y are provided in
Table 1, and amounts of a, b, and c are provided in Table S1 of the
Supporting Information.
KNpoly ) (Kmono RN
)
(1)
The beauty of Whitesides’ model as a predictive tool lies in its
simplicity. If monovalent association constants can be measured,
then one should be able to predict multivalent values in a
straightforward fashion, regardless of the nature of the framework
or the type of multivalent interaction involved. The goal of the
research reported here was to determine whether monovalent
differences in affinity affect multivalent association constants in
predictable ways (as described by eq 1).
To investigate Whitesides’ prediction regarding the relationship
between monovalent and multivalent associations, we functionalized
PAMAM dendrimers7 with mannose and glucose. We evaluated
the relative affinities of the dendrimers with Concanavalin A (Con
A), a plant lectin that binds methyl D-mannopyrannoside 4 times
better than it binds methyl D-glucopyrannoside.2 Con A exists as a
tetramer at neutral pH, with four carbohydrate binding sites located
6.5 nm apart.8 Previously, we showed that mannose/hydroxyl-
functionalized G(4)- to G(6)-PAMAM dendrimers with 50%
mannose incorporation showed the highest activity in hemagglu-
tination assays with Con A.9 The G(4), G(5), and G(6) dendrimers
are large enough to bind divalently to Con A, so these generations
were chosen to study the tunability of affinity for this report.
Because of the roughly spherical shape of dendrimers, the den-
drimer/Con A associations that we observe are almost certainly
divalent. Trivalent and tetravalent associations are not possible due
to the shape and size of PAMAMs. We describe here the synthesis
of mannose/glucose dendrimers and the results of hemagglutination
assays with these dendrimers and Con A.
To synthesize the carbohydrate tethers, allylation of the anomeric
hydroxyl of R sugar starting material,10 peracetylation of the 2,3,4-
and 6-hydroxyls11 and thiol radical addition of Boc-protected
aminoethanethiol12 afforded intermediate 1 (Scheme 1a). Removal
of the Boc group and addition of thiophosgene afforded the requisite
mannose, glucose, and galactose isothiocyanates 2a-c.
Isothiocyanato carbohydrates 2a-c were sequentially added to
PAMAM dendrimers. MALDI-TOF analyses were performed 24
h after each addition to determine the degree of functionalization.
The acetyl protecting groups were removed under Zemplen condi-
tions.
We functionalized 50% of the dendrimers’ amino endgroups with
mannose and glucose since our previous work indicated that 50%
mannose functionalization caused the highest activity in the
hemagglutination assay.9 After mixtures of mannose and glucose
were added to the dendrimers in varying ratios to total 50% (Table
1), galactose (which does not bind to Con A) was added. Although
galactose additions of about 50% were intended, galactose addition
was negligible. Warming the galactose additions to 40 °C with both
9
12168
J. AM. CHEM. SOC. 2005, 127, 12168-12169
10.1021/ja053008n CCC: $30.25 © 2005 American Chemical Society

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C O M M U N I C A T I O N S
Table 1. Hemagglutination Assay Results
between Con A and the dendrimer, which can significantly change
the association motif.14 The linearity of the fit shown in Figure 1
suggests that man/glc-functionalized dendrimers bearing varying
ratios of mannose to glucose also exhibit the affinity changes
predicted by eq 1.
The results reported here with two ligands (mannose and glucose)
that vary by a factor of 4 in the strength of their monovalent
associations to Con A but vary by almost a factor of 16 in their
divalent dendrimer/Con A associations indicate that multivalency
can be influenced in predictable, and therefore, tunable ways.
Monovalent differences are amplified by multivalent associations,
and mixtures of low and high affinity ligands can be used to
attenuate multivalent affinities.
No. mannose
residuesa
No. glucose
residuesa
relative activity
per active sugarb
4a
4b
4c
4d
4e
5a
5b
5c
5d
6a
6b
6c
6d
30
24
18
10
0
44
38
16
0
53
34
16
0
0
7
13
26
29
0
13
40
45
0
35
50
77
3820 ( 1650
2660 ( 0
2260 ( 780
1090 ( 380
260 ( 110
4830 ( 2090
3040 ( 0
1270 ( 440
310 ( 130
5350 ( 0
3510 ( 1220
2150 ( 0
470 ( 0
In summary, hemagglutination assays with Con A and mannose/
glucose-functionalized dendrimers 4-6 indicate that multivalent
affinities can be predicted based on monovalent association
constants. The glucose and mannose monomers differ in binding
strength only by a factor of 4; multivalent association amplifies
this difference. Transposition of the observed relationship between
monovalent and multivalent association constants into more com-
plex systems (for example, polyvalent rather than divalent com-
plexes and nondendritic frameworks) should reasonably follow and
is currently being explored. Further evaluation of mannose/glucose
dendrimer-Con A complexes using the hemagglutination and
precipitation assays is also underway. That multivalent affinity can
be attenuated by mixing ligands of varying binding strengths
provides a new element of control and predictability to the design
of synthetic multivalent molecules for biological applications.
a No. of sugar residues was determined using MALDI-TOF MS data
after deacetylation (MW ) 168 g/mol for 4 Ac) and after addition of tethered
sugar (MW ) 507 g/mol per tethered sugar). See the Supporting Information
for details. b Active sugar ) man + glc. Standard deviation values are large
because of serial 2-fold dilutions. For standard deviation ) 0, all inhibitory
concentrations were equal. All values represent at least three trials. Relative
activity of methyl mannose ) 1.
Acknowledgment. Support of this research by NIH RO1
GM62444 is gratefully acknowledged.
Figure 1. Percent mannose of the glucose/mannose mixture versus relative
activity (per glucose + mannose).
Supporting Information Available: Experimental procedures and
characterization data for 1-6, hemagglutination assay procedure. This
material is available free of charge via the Internet at http://pubs.acs.org.
acetylated and deacetylated mannose/glucose dendrimers and adding
isothiocyanatoethoxyethanol 39 failed to cause significant galactose
loadings.
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