The lectin-binding properties of six generations of mannose-functionalized dendrimers

Article abstract of DOI:10.1021/ol016568+

(matrix presented) First-through sixth-generation PAMAM dendrimers have been functionalized with mannose residues. Characterization with MALDI-TOF MS and ¹H NMR is reported. Different binding enhancements consistent with monovalent interaction,

Full text of DOI:10.1021/ol016568+

ORGANIC
LETTERS
2
002
Vol. 4, No. 1
-10
The Lectin-Binding Properties of Six
Generations of Mannose-Functionalized
Dendrimers
7
Eric K. Woller and Mary J. Cloninger*
Department of Chemistry and Biochemistry, 108 Gaines Hall,
Montana State UniVersity, Bozeman, Montana 59717
mcloninger@chemistry.montana.edu
Received August 13, 2001 (Revised Manuscript Received November 10, 2001)
ABSTRACT
First- through sixth-generation PAMAM dendrimers have been functionalized with mannose residues. Characterization with MALDI-TOF MS
1
and H NMR is reported. Different binding enhancements consistent with monovalent interaction, glycoside clustering, and multivalent binding
are observed for different generations of dendrimers.
Polymeric biomaterials that are designed to investigate and
control specific cell behavior are becoming increasingly
desirable for applications in drug delivery and tissue
engineering. Because protein-carbohydrate interactions
have been implicated in a wide variety of intercellular
reported.5,6 In a recent example, generation one (G(1)-)
through generation five (G(5)-) lactose-functionalized den-
drimers were studied to evaluate how the topology of binding
site presentation and ligand display effect binding selectivity.7
1
Here, we have optimized our model system so that
multivalent binding (the ability of one dendrimer to bind to
multiple lectin binding sites) should be facile for large
saccharide-functionalized dendrimers but unlikely for small
dendrimers. We have chosen concanavalin A (Con A) as
our reference lectin because two mannose binding sites are
located about 65 Å apart on one side of the protein, such
that a large dendrimer should be able to bind simultaneously
recognition events, a clear understanding of the details of
2
the requirements for this interaction is intensely sought.
Under physiologically relevant conditions, it is generally
accepted that adhesion of lectins to saccharides on the surface
3
of a cell involves multipoint attachment. To mimic this
4
motif, a variety of glycopolymers have been developed.
Saccharide-functionalized dendrimers capable of glycoside
clustering have been previously reported, but until recently
studies with dendrimers large enough to span multiple lectin
binding sites (i.e., to bind multivalently) had not been
8
to two binding sites. In this paper, we report a systematic
(
5) For leading references, see: (a) Ashton, P. R.; Boyd, S. E.; Brown,
(
1) Griffith, L. G. Acta Mater. 2000, 48, 263-277.
C. L.; Nepogodiev, S. A.; Meijer, E. W.; Peerlings, H. W. I.; Stoddart, J.
F. Chem. Eur. J. 1997, 3, 974-984. (b) Page, D.; Roy, R. Bioconjugate
Chem. 1997, 8, 714-723. (c) Kieburg, C.; Lindhorst, T. K. Tetrahedron
Lett. 1997, 38, 3885-3888.
(6) For recent reviews of dendrimer chemistry, see: (a) Matthews, O.
A.; Shipway, A. N.; Stoddart, J. F. Prog. Polym. Sci. 1998, 23, 1-56. (b)
Zeng, F.; Zimmerman, S. C. Chem. ReV. 1997, 97, 1681-1712. (c) Bosman,
A. W.; Janssen, H. M.; Meijer, E. W. Chem. ReV. 1999, 99, 1665-1688.
(d) Inoue, K. Prog. Polym. Sci. 2000, 25, 453-571.
(7) Andre, S.; Ortega, P. J. C.; Perez, M. A.; Gabius, H.-J. Glycobiology
1999, 9, 1253-1261.
(
2) (a) Singh, R. S.; Tiwary, A. K.; Kennedy, J. F. Crit. ReV. Biotechnol.
1
6
999, 19, 145-178. (b) Lis, H.; Sharon, N. Chem. ReV. 1998, 98, 637-
74.
(3) (a) Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem.,
Int. Ed. 1998, 37, 2754-2794. (b) Page, M. I.; Jencks, W. P. Proc. Natl.
Acad. Sci. U.S.A. 1971, 68, 1678-1683.
(
4) (a) Bovin, N. V.; Gabius, H.-J. Chem. Soc. ReV. 1995, 24, 413-421.
(
b) Sharon, N.; Lis, H. Sci. Am. 1993, 82-89. (c) Kiessling, L. L. In Recent
Trends in Molecular Recognition; Diederich, F., Kunzer, H., Ed.; 1998;
pp 183-212.
1
0.1021/ol016568+ CCC: $22.00 © 2002 American Chemical Society
Published on Web 12/11/2001

study of mannose-functionalized G(1)- through G(6)-
PAMAM dendrimers with Con A.
Scheme 1. Synthesis of Mannose-Functionalized Dendrimers
We observe significant differences in affinity on the basis
of the size of the polymer, which we suggest is due to
monovalent binding, glycoside clustering, and multivalent
binding motifs. Glycoside clustering has been previously
defined as “affinity enhancement achieved by multivalent
ligands over monovalent ones that is greater than would be
9
expected from a simple effect of concentration increase”.
For the discussion in this paper, we adopt this definition of
glycoside clustering but apply it in a narrower context than
10
it is sometimes used in the carbohydrate literature. Namely,
we define multivalent binding (the ability of one dendrimer
to bind to multiple lectin binding sites) and glycoside
clustering (a ligand concentration effect) as two related but
distinct terms. These definitions are shown pictorially in
Figure 1. To our knowledge, this is the first time that studies
with saccharide-functionalized sixth-generation dendrimers
have been reported.
1
acetyl groups give diagnostic signals in the H NMR spectra
and because we can check the molecular weight (MALDI-
TOF MS) of our products both before and after deacetylation
1
3
(
vide supra).
1
The H NMR (500 MHz) spectra of acetyl-protected
mannose-functionalized dendrimers 4 and 9 (theoretical MWs
,902 and 137,152 g/mol respectively) in d -DMSO are
3
6
shown in Figure 2. For 4-9, the amide protons from the
interior of the dendrimer are the peaks that are farthest
downfield, and the peak at 7.5 ppm is the thiourea NH signal.
The relative integrations of these signals suggest that a high
degree of surface functionalization has been achieved. For
example, in 4 there are 12 amide and 16 thiourea protons,
so a 1:1.33 ratio of peaks is expected. Since the observed
ratio is 1:1.25, this suggests that 96% of the possible
functionalization occurred. In all cases, g90% functional-
ization is indicated by NMR. Unfortunately, the MALDI-
TOF MS results suggest a lower degree of surface loading.
The MALDI-TOF MS of mannose-functionalized den-
drimers 10 and 15 and of the starting G(1)- and G(6)-
PAMAMs are shown in Figure 3. For G(4)- to G(6)-
PAMAMs, the measured molecular weights of the dendrimers
Figure 1. The three likely binding motifs and their expected
relative activities for the interaction of glycodendrimer with Con
A.
Mannose-functionalized dendrimers 10-15 were synthe-
sized as shown in Scheme 1. Peracetylation of D-mannose
followed by selective deprotection and activation at the
11
anomeric position afforded trichloroacetimidate 1. Coupling
12
of 1 with the isothiocyanato alcohol 2 using BF
3 2
‚OEt gave
the mannose monomer 3. Addition of 3 to the dendrimer
followed by global deacetylation gave dendrimers 10-15.
Dialysis (water/cellulose tube, MW cutoff 1000 g/mol)
afforded 10-15 in purified form.
Although the acetyl protecting groups are not required
during thiourea formation, we have chosen to deprotect the
sugars after addition to the dendrimer. This is because the
1
4
were lower than the theoretical values. Subtraction of the
experimentally determined molecular weight of the PAMAM
starting materials from the molecular weight of 4-15 and
division of the remainder by 477 (molecular weight of 3) or
by 309 (molecular weight of deacetylated 3) indicates that
(
8) (a) Bittiger, H.; Schnebli, H. P. ConcanaValin A as a Tool; John
Wiley & Sons: 1976. (b) Derewenda, Z.; Yariv, J.; Helliwell, J. R.; Kalb,
A. J.; Dodson, E. J.; Papiz, M. Z.; Wan, T.; Campbell, J. EMBO J. 1989,
8
, 2189-2193. (c) Lis, H.; Sharon, N. FASEB J. 1990, 4, 3198-3208.
9) Quesenberry, M. S.; Lee, R. T.; Lee, Y. C. Biochemistry 1997, 36,
724-2732.
10) For a broader definition of the glycoside cluster effect, see: Lee,
Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 322-327.
11) (a) Ren, T.; Liu, D. Tetrahedron Lett. 1999, 40, 7621-7625. (b)
Schmidt, R. R.; Michel, J. Angew. Chem., Int. Ed. Engl. 1980, 19, 731-
32. (c) Schmidt, R. R. Angew. Chem., Int. Ed. Engl. 1986, 25, 212-235.
12) Synthesized as described in the Supporting Information from 2-(2-
(13) Our synthesis procedure is similar to our previously reported route
using p-isothiocyanatophenyl-R-D-mannopyranoside, except that the aro-
matic ring in the linkage to the dendrimer was eliminated to improve
solubility in aqueous media: Woller, E. K.; Cloninger, M. J. Biomacro-
molecules 2001, 2, 1052-1054.
(14) Tomalia et al. have reported that the average molecular weights of
the PAMAMs are actually smaller than the theoretical molecular weights;
our MALDI results are consistent with Tomalia’s electrospray MS results:
Tolic, L. P.; Anderson, G. A.; Smith, R. D.; Brothers, H. M.; Spindler, R.;
Tomalia, D. A. Int. J. Mass Spec. Ion Processes 1997, 165/166, 405-418.
(
2
(
(
7
(
aminoethoxy)ethanol with thiophosgene, 70% yield.
8
Org. Lett., Vol. 4, No. 1, 2002

Table 1. MALDI-TOF MS Determination of Mannose
Functionalization of G(1)- to G(6)-PAMAMs. Experimentally
Determined M Values Are Given in Parentheses
W
theor. no.
aminesa
no. sugarsb
(MALDI
of 4-9)
no. sugarsc
(MALDI
ave %
gen. (MALDI MW)
of 10-15)
loading
1
2
3
4
5
6
8 (1430)d
16 (3260)
32 (6910)
64 (13500)
128 (25500)
256 (50800)
8 (5280)d
16 (10960)
30 (21000)
54 (39300)
92 (69600)
8 (3877)d
16 (8250)
29 (15930)
55 (30620)
95 (55000)
100
100
92
84
73
173 (133500) 172 (103800)
67
a
PAMAM starting material. b no. sugars4-9 ) (MW4-9 - MWG(1)-G(6))
d
/
477. c no. sugars10-15 ) (MW10-15 - MWG(1)-G(6))/309. g/mol.
loading is that, because of the high degree of symmetry
present in PAMAMs, NMR does not identify structural
defects as well as MALDI-TOF MS. If we assume loss of
units of 114 (the mass of one missing terminal CH
2 2
CH -
CONHCH CH NH unit) as the predominant defect present
2
2
2
1
2
in the starting materials, then our results indicate that at
least 90% of the amines are functionalized. We suggest that
the NMR and MALDI-TOF results indicate that a high
degree of surface functionalization is occurring, but that
defects in the PAMAMs preclude higher sugar loading.
After demonstrating the feasibility of synthesizing water-
soluble mannose-functionalized G(1)- through G(6)-den-
drimers, we evaluated their relative activities with Con A
by performing hemagglutination assays. Although hemag-
glutination assays do not provide information regarding
Figure 2. 1H NMR spectra (500 MHz) of (a) 4 and (b) 9.
the percent incorporation of mannose residues is 100% for
G(1)- and G(2)-PAMAMs, 92% for G(3)-PAMAM, and
84%, 73%, and 67% for G(4)-, G(5)-, and G(6)-PAMAM,
respectively (Table 1).
The simplest explanation for the discrepancy between the
NMR and the MALDI-TOF MS determinations of percent
15
binding affinity, their use to measure inhibition of protein-
carbohydrate interactions is well documented and gives us
an essential entry-level comparison of our system to other
16
glycopolymers. In the article discussing activity vs affinity
and the use of the hemagglutination assay, Toone and co-
workers note that “in many respects hemagglutination assays
are a far more relevant measure of activity than are assays
designed exclusively to evaluate protein-carbohydrate bind-
1
5
ing”.
Erythrocytes (rabbit) were added to preincubated solutions
of Con A and varying concentrations of dendrimer. The
lowest amount of dendrimer that caused inhibition of
1
7
agglutination was determined. The results are shown in
Table 2. Each value represents at least three assays. The large
standard deviation occurs because, while the overall trends
(
15) Dimick, S. M.; Powell, S. C.; McMahon, S. A.; Moothoo, D. N.;
Naismith, J. H.; Toone, E. J.; J. Am. Chem. Soc. 1999, 121, 10286-10296.
16) For examples, see: (a) Sigal, G. B.; Mammen, M.; Dahmann, G.;
(
Whitesides, G. M. J. Am. Chem. Soc. 1996, 118, 9-3800. (b) Mortell, K.
H.; Weatherman, R. V.; Kiessling, L. L. J. Am. Chem. Soc. 1996, 118, 8,
2297-2298. (c) Roy, R.; Zanini, D.; Meunier, S. J.; Romanowska, A. J.
Chem. Soc., Chem. Commun. 1993, 1869-1872. (d) Mandal, D. K.; Brewer,
C. F. Biochemistry 1993, 32, 5116-5120. (e) Lehmann, J.; Weitzel, U. P.
Carbohydr. Res. 1996, 294, 65-94. (f) Hansen, H. C.; Haataja, S.; Finne,
J.; Magnusson, G. J. Am. Chem. Soc. 1997, 119, 6974-6979.
(17) A detailed experimental protocol is provided in the Supporting
Information. Although precipitation has been observed in previous studies
of Con A binding to mannose-functionalized dendrimers (ref 5b), our assays
were performed at 30-fold lower concentrations, and no precipitation is
observed.
Figure 3. MALDI-TOF MS of (a) G(1)-PAMAM, (b) 10, (c) G(6)-
PAMAM, (d) 15.
Org. Lett., Vol. 4, No. 1, 2002
9

expected approximate area that each endgroup would occupy
on the dendrimer surface. Assuming 13-15 are spherical,6
the area available to the endgroups on 15 is considerably
smaller than the area available to endgroups on 13. We
postulate that, although all the larger dendrimers apparently
bind multivalently, some may be better at glycoside cluster-
Table 2. Hemagglutination Assays for 10-15 with Con A
cmpd
no. sugars
rel activity/sugar
methyl mannose
1
8
1
1
1
1
1
1
1
1
0
1
2
3
4
5
16
29
55
95
172
1.5 ( 0.1
45 ( 25
275 ( 95
510 ( 295
660 ( 230
21
ing than others.
Preliminarily, we suggest that the results with 13-15
indicate that the interplay between glycoside clustering and
multivalent binding (Figure 1) may have important effects
on lectin binding interactions. We are currently synthesizing
dendrimers of the same generation with varying concentra-
tions of sugars to further evaluate glycoside clustering vs
multivalent binding motifs (to be published separately).
In summary, we have synthesized and characterized
mannose-functionalized G(1)- to G(6)-PAMAMs 10-15.
Their relative activities toward Con A were evaluated using
the hemagglutination assay. Depending on the size of the
dendrimer framework, monovalent, glycoside clustering, and
multivalent binding motifs were observed. The variety of
binding motifs available for saccharide-functionalized den-
drimers makes these compounds attractive polymeric bio-
materials for the study of protein-carbohydrate interactions.
are always the same, different blood sources (different rabbits
on different days) give different numbers.
When compared to the control monomer methyl mannose,
dendrimers 10 and 11 did not show any increase in activity
toward Con A. Thus, we surmise that 10 and 11 bind
monovalently. Either clustering or monovalent binding was
expected, since the lower generation dendrimers are too small
to span multiple binding sites (i.e., to bind multivalently).
In addition, our findings indicate that 12 (G(3)-PAMAM
core) binds roughly 1 order of magnitude better than 10, 11,
or methyl mannose. This is suggestive of a glycoside
clustering motif. As with 10 and 11, 12 is too small for
18
multivalent binding to occur. Dendrimers 13-15 all show
increases in activity toward Con A (relative to methyl
mannose) of 2 orders of magnitude, indicating that multi-
valent binding (Figure 1) is occurring.19
Acknowledgment. Partial support of this research by NSF
MONTS and by NIH RO1 GM62444-01A1 is gratefully
acknowledged. We thank Drs. Scott Busse and Joe Sears
for help with NMR and MS.
Molecular mechanics calculations (Macromodel V. 6.5,
MM2*) suggest that reaction of 3 with the dendrimer will
add a maximum of about 13 Å to the radius of the molecule.
Thus, using published radii for the original PAMAMs (G(4)-
PAMAM radius ∼22.5 Å, G(5)-PAMAM radius ∼27 Å,
Supporting Information Available: Experimental pro-
cedures and characterization data for 2-15 and experimental
procedures for MALDI-TOF MS and hemagglutination. This
material is available free of charge via the Internet at
http://pubs.acs.org.
20
G(6)-PAMAM radius ∼ 33.5 Å), we can calculate the
OL016568+
(18) The relative activity results for 12 are same order of magnitude as
those obtained with ELLA assays in ref 5b. Differences in binding activities
for 10-12 (compared to similar compounds in ref 5b) may be caused by
changes in the linker between mannose and the PAMAM.
(20) (a) Uppuluri, S.; Keinath, S. E.; Tomalia, D. A.; Dvornic, P. R.
Macromolecules 1998, 31, 4498-4510. (b) Li, J.; Piehler, D.; Qin, J. R.
B., Jr.; Tomalia, D. A. Langmuir 2000, 16, 5613-5616.
(21) Our dendrimer results (and our analysis) are in good agreement with
a previous study of polymer length on binding affinity: Kanai, M.; Mortell,
K. H.; Kiessling, L. L J. Am. Chem. Soc. 1997, 119, 9931-9932.
(19) It is also possible that the change in shape from circular (G(1)- to
G(3)-PAMAM) to spherical (G(4)- to G(6)-PAMAM) causes the observed
binding enhancement. Studies with heterogeneously functionalized den-
drimers are underway to address this issue.
10
Org. Lett., Vol. 4, No. 1, 2002