EPR and affinity studies of mannose-TEMPO functionalized PAMAM dendrimers

Article abstract of DOI:10.1039/b411643g

Mannose-TEMPO functionalized G(4)-PAMAM dendrimers with increasing mannose loadings have been synthesized and characterized by MALDI-TOF MS and EPR spectroscopy. Analysis of linebroadening effects in the EPR spectra of these dendrimers allowed us to determine the relative presentation of mannose and TEMPO on the dendrimer surface. Hemagglutination assays and affinity chromatography/EPR experiments to assess the activity of the mannose-TEMPO dendrimers with Concanavalin A are presented.

Full text of DOI:10.1039/b411643g

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A R T I C L E
EPR and affinity studies of mannose–TEMPO functionalized
PAMAM dendrimers†
Lynn E. Samuelson, Karl B. Sebby, Eric D. Walter, David J. Singel and Mary J. Cloninger*
Department of Chemistry and Biochemistry and Center for Bioinspired Nanomaterials,
Montana State University, 108 Gaines Hall, Bozeman, MT 59717, USA.
E-mail: mcloninger@chemistry.montana.edu; Fax: +406 994 5407; Tel: +406 994 3051
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Mannose–TEMPO functionalized G(4)-PAMAM dendrimers with increasing mannose loadings have been
synthesized and characterized by MALDI-TOF MS and EPR spectroscopy. Analysis of linebroadening effects in the
EPR spectra of these dendrimers allowed us to determine the relative presentation of mannose and TEMPO on the
dendrimer surface. Hemagglutination assays and affinity chromatography/EPR experiments to assess the activity of
the mannose–TEMPO dendrimers with Concanavalin A are presented.
Introduction
tional group distribution on the dendrimer. Moreover, although
the work reported here is focused on PAMAM dendrimer func-
tionalization, these studies are widely applicable to characteriza-
tion of macromolecules and nanoparticles.
Although multivalent protein–carbohydrate interactions play a
critical role in many biological recognition events, multivalent
therapeutic agents that utilize protein–carbohydrate interactions
have been difficult to design. This is primarily because the funda-
mental requirements of protein–carbohydrate interactions are
To evaluate the loading distribution of sugars on
heterogeneously functionalized dendrimers, we synthesized
mannose–TEMPO functionalized dendrimers. The synthesis,
characterization, and EPR analysis of these compounds is
reported here. In addition, the use of mannose–TEMPO func-
tionalized dendrimers in hemagglutination assays and affinity
chromatography experiments with Concanavalin A (Con A, a
1
not well understood. Carbohydrates are often covalently linked
to macromolecules as a means of studying protein–carbohydrate
2
3
4
interactions. We and others have used dendrimers as scaffolds
for carbohydrate display. Functionalization of the terminal
amines on the surface of PAMAM dendrimers is readily accom-
plished to form homogeneous mannose functionalized3 and
heterogeneous mannose–hydroxyl functionalized dendrimers.3
Heterogeneous functionalization of dendrimers that incorpo-
rate varying loadings of saccharide residues is attractive because
it allows for the fine-tuning of protein affinities and protein clus-
tering properties of the macromolecules. Not only can the size
of the (dendrimer) framework be changed easily by changing
the generation of dendrimer used, but the display of sugars on
the dendrimer surface can also be readily manipulated through
8
mannose binding lectin) is described.
b,c
a
Results and discussion
In order to study the relative locations of the mannose residues
on heterogeneously functionalized dendrimers, acetyl protected
mannose–TEMPO functionalized dendrimers 3–9 were syn-
thesized as shown in Scheme 1. Mannose derivative 13 and
4-isothiocyanatoTEMPO (2) were added sequentially at room
temperature to G(4)-PAMAM in controlled ratios to obtain het-
erogeneously functionalized dendrimers 3–9. The acetyl groups
were removed with NaOMe–MeOH to form mannose–TEMPO
functionalized dendrimers 10–16.
a
5
heterogeneous functionalization.
Recently, we studied EPR linebroadening effects for
G(4)-PAMAM dendrimers functionalized with a TEMPO
(
2,2,6,6-tetramethylpiperidine N-oxide) spin label and another
functional group. The relative locations of the spin labels were
determined. At all loadings studied (5–95% TEMPO), the spin
label was found to be randomly distributed on the surface of the
dendrimer and therefore the other group (2,2,6,6-tetramethyl-
piperidine, propanol, tert-butane and phenol) must also present
6
a random surface display.
For our work using dendrimers to study protein–carbohydrate
interactions, we use a synthesis strategy where peracetylated sug-
ars are tethered to the dendrimer surface and then deprotected.
Although the peracetylated sugars are almost certainly randomly
distributed on the dendrimer surface, we were concerned that
unmasking of the hydroxyl groups might create hydrogen-bond-
ing networks. Clustering of sugars on the dendrimer surface
comparable to the creation of rafts in lipid membranes could
7
occur. In order to effectively design heterogeneous multivalent
recognition systems using the thiourea functionalization routes
described here, it is imperative that we characterize the func-
†
Electronic supplementary information (ESI) available: Tables of
MALDI-TOF MS and EPR characterization data for compounds 3–16
and 22–24; tables of masses of materials that were isolated in affinity
chromatography experiments; EPR absorbance spectra for 3–16 and
second integral values (11 pages). See http://www.rsc.org/suppdata/ob/
b4/b411643g/
Scheme 1 Synthesis of mannose–TEMPO functionalized G(4)-
PAMAM dendrimers.
T h i s j o u r n a l i s
©
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 0 7 5 – 3 0 7 9
3 0 7 5

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Table 1 MALDI-TOF MS characterization data for 3–9
M
W
after 1
Number of
sugars
M
W
after both 1
Number of
TEMPOs
was added
and 2 were added
3
4
5
6
7
8
9
37740
36310
32060
26100
19220
16090
15590
52
49
40
27
13
6
38390
37570
34940
32490
28130
26470
26590
3
6
14
30
42
49
52
5
Theoretical ratios of mannose to TEMPO are shown in
Scheme 1 (m:n) and include 95%, 90%, 75%, 50%, 25%, 10%,
and 5% mannose functionalization. Actual ratios of mannose to
W
TEMPO were determined from MALDI-TOF MS. The M of
−
1
G(4)-PAMAM starting material was found to be 13170 g mol ,
which suggests that, on average, each dendrimer has fifty-five
9
terminal amino groups. Subtraction of 13170 from the M
W
of
each dendrimer after addition of 1 and division of this value
by 477 (the molecular weight of 1) gives the average number
of sugars on each dendrimer. Subtraction of the intermediate
M
W
from the M
by 213 (the molecular weight of 2) gives the average number
of TEMPO residues per dendrimer. The difference in M for
0–16 and 3–9 divided by 168 (the molecular weight of four
W
obtained for 3–9 and division of this value
W
1
acetyl groups) serves as a check of the number of sugars per
molecule. Table 1 shows the experimentally determined number
of sugars and TEMPO residues for dendrimers 3–9. In all cases,
our calculated number of functionalized endgroups is between
5
4 and 57, which agrees very well with our calculated number of
amino groups on the G(4)-PAMAM (55 endgroups on average)
and suggests that essentially complete reactivity of the primary
amines is observed. Whether primary isothiocyanate 1 or sec-
ondary isothiocyanate 2 is added first has virtually no effect
on the degree of dendrimer functionalization (Table S2 and S3
of the supporting information†). Since we are not using excess
isothiocyanate, it seems reasonable to assume that the isothio-
cyanates are reacting with the primary amines on the dendrimer
rather than the secondary amines that are produced when
incomplete/retro Michael addition occurs. Addition of 1 to
some of the secondary amines (rather than the primary amines
on the dendrimer surface) is a possibility, but the calculated val-
ues of number of endgroups based on the MS data suggest that
the dendrimer’s amino termini react preferentially.
Fig. 1 (a) EPR spectra of 3–9, normalized to peak A. (b) EPR spectra
of 10–16, normalized to peak A. (c) A plot comparing the A/B peak
height ratio versus percent TEMPO loading for dendrimers with and
without acetyl protecting groups.
strongly suggests that all dendrimers 3–16 have a random distri-
bution of surface functional groups.
Hemagglutination assays12 were performed on dendrimers
10–12, and the results were compared with previous results using
mannose-hydroxyl functionalized dendrimers to ensure that the
spin label did not affect lectin binding. The first three entries
in Table 2 summarize our results, which were comparable to
previously reported values for dendrimers lacking the TEMPO
groups. Unfortunately, dendrimers 13–16 were insoluble in
aqueous solutions and could not be studied with lectins.
To increase the water solubility of the spin labeled dendrimers
with low sugar loadings, dendrimers 22–24 were synthesized as
The EPR spectra obtained for 3–9 are shown as a stack
plot in Fig. 1a (normalized to peak A). After removal of the
acetyl groups, EPR spectra were obtained for 10–16 (Fig. 1b).
Dendrimers with high loadings of TEMPO have broader spec-
tra than dendrimers with lower TEMPO loadings (9 and 16 vs.
3
and 10). Qualitatively, the stack plots of 3–9 and 10–16 are
nearly identical. The A/B peak height ratio has shown itself to
be a reasonable measure of linebroadening within the confines
of our system and also has strong precedent in other systems,
so we rely on this value to evaluate linebroadening effects.1
A graph of the A/B peak height ratio versus the percent load-
ing of TEMPO (Fig. 1c) shows the nearly perfect overlay of
the results for acetylated and deprotected dendrimers. The only
differences in the linebroadening effects are at very low and very
high TEMPO loadings, where small concentration measurement
errors or remaining unreacted 2 would cause the most significant
changes in the EPR spectra. Indeed, when placed in the context
of A/B peak height ratio vs. TEMPO loading for a variety of
heterogeneously functionalized dendrimers, differences in line-
broadening effects between 3 and 10 and between 9 and 16 are
negligible (Fig. S1 in the supporting information†).
0,11
3a
shown in Scheme 2. Isothiocyanates 1, 2, and 21 were sequen-
tiallyaddedtoG(4)-PAMAMincontrolledratiosatroomtempe-
rature, and MALDI-TOF MS were obtained after addition of
each component. Removal of the acetyl groups was accom-
plished using NaOMe–MeOH. MALDI-TOF characterization
data is given in the supporting information.† As for 10–16, theo-
retical numbers of functional groups are shown in Scheme 2 and
values determined from MALDI-TOF MS characterization are
reported in Table 2.
The results of the hemagglutination assays on 22 and 23 with
Con A are shown in entries 4 and 5 of Table 2. Good agreement
was observed between values obtained for mannose–TEMPO
dendrimers and previously synthesized mannose–hydroxyl
dendrimers with similar saccharide loadings. The water
solubility of dendrimer 24 was too low to obtain good data
in the hemagglutination assay. Because 10–12 and 22–23 show
We conclude that the surface patterning of peracetylated
mannose–TEMPO dendrimers and of unmasked mannose–
TEMPO dendrimers is the same. Comparison of Fig. 1c to
computer simulations of various dendrimer loading patterns
3
0 7 6
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 0 7 5 – 3 0 7 9

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Table 2 Hemagglutination assay results for spin labeled and non-spin
labeled mannose functionalized dendrimers
Table 3 A/B peak height ratios for mannose–TEMPO dendrimers
before and after affinity chromatography
mL eluteda
Initial A/B ratio
A/B ratio after columnb
Number of
sugars
Relative
activitya
Relative activity without
TEMPOb
1
0
22
22
22
22
22
1–2
0.77
0.79
0.92
0.94
0.99
0.78
0.76
0.74
0.86
0.79
0.93
0.85
1
1
1
2
2
2
0
1
2
2
3
4
52
49
41
26
12
7
410 ± 310
290 ± 120
170 ± 80
200 ± 100
100 ± 50
NAc
180 ± 10
200 ± 10
220 ± 10
170 ± 40
120 ± 40
30 ± 25
11
12
22
23
24
a
b
The fraction in which EPR active material was recovered. The reported
values are the average of three trials.
a
All values are relative to methyl mannose, and concentrations are
b
adjusted to reflect per sugar values. On a per mannose basis and rela-
tive to methyl mannose, the activity of mannose/hydroxyl functionalized
dendrimers with equivalent numbers of sugars. Values are taken from
reference 3a. Because of the low water solubility of 24, this value could
not be determined.
c
one-half to two-thirds of the original EPR-active material was
recovered. Loss of mass probably occurs primarily because of
weighing errors for the small quantities (3 mg per run) of
dendrimer involved.
From the affinity chromatography/EPR experiments, we
conclude that the results observed in the hemagglutination
assay are due to the bulk dendrimer material binding strongly
to Con A. In other words, a small amount of very active den-
drimer (with clustered or disparate mannose residues) is not
responsible for the activity that we observe in the hemaggluti-
nation assay.
Conclusion
In summary, mannose–TEMPO functionalized PAMAM
dendrimers have been synthesized and characterized by
MALDI-TOF MS and EPR spectroscopy. The EPR spectra
of dendrimers 3–16 indicate that both the acetylated mannose
groups and the (deprotected) mannose residues are randomly
distributed on the dendrimer surface. Comparison of the
hemagglutination assay results for mannose–TEMPO and
mannose–hydroxyl functionalized dendrimers suggests that the
presence of a spin label has no affect on binding of the den-
drimer to Con A. Finally, affinity chromatography/EPR experi-
ments indicate that the results observed in the hemagglutination
assay are due to the bulk dendrimer material binding strongly
to Con A and are not caused by the presence of a few idealized
dendrimers.
Scheme 2 Synthesis of tri-functionalized dendrimers.
activities in the hemagglutination assay comparable to those
of mannose–hydroxyl functionalized dendrimers, we conclude
that the presence of the TEMPO spin label does not signifi-
cantly influence protein binding. Of course, dendrimers with
TEMPO only (no mannose) show no affinity toward Con A in
the assay. The spin label and the hydroxyl group both serve in
the assay merely as inactive space-holding groups that separate
the mannose residues.
In a mixture of randomly functionalized dendrimers, it
is certainly possible that a small population of dendrimers
will be responsible for the majority of the activity in the
hemagglutination assay. We wanted to determine whether the
activity that we have observed towards Con A is caused by
the bulk dendrimer mixture (randomly mannose functional-
ized) or some minor component (where the sugars are clus-
tered or are far apart on the dendrimer surface). To address
this point, we performed affinity chromatography with Con
A–sepharose using mannose–TEMPO dendrimers followed by
EPR spectroscopy. In affinity chromatography, molecules with
low affinity towards Con A should elute before those with high
affinity. The linebroadening effects in the EPR spectra of the
eluted dendrimers can then be compared to the linebroadening
effects in the EPR spectrum of the original mannose–TEMPO
dendrimer. Variations in linebroadening effects would be indica-
tive of loading patterns deviating from random.
Solutions of mannose–TEMPO dendrimers 10–12 and 22–24
were run through columns packed with Con A-bound-sepharose.
EPR spectra were obtained of the eluted samples and were com-
pared with those of the starting solutions in order to study any
differences in linebroadening effects. Results are summarized in
Table 3. In all cases except 24, the dendrimer remained on the
column until it was eluted by addition of concentrated methyl
mannose. Dendrimer 24 was eluted off the column in the first
Experimental
General protocols
General reagents were purchased from Aldrich, Sigma, and
Acros. Generation 4 PAMAM dendrimers were purchased from
Dendritech as aqueous solutions. Methylene chloride was puri-
fied on basic alumina. BF
3
2 2
·Et O was distilled from CaH . All
other solvents and reagents were used as supplied. All MALDI-
TOF reference standards were purchased from Sigma. Com-
pound 21 was synthesized as previously reported.3
a
General procedure for the synthesis of peracetylated mannose–
TEMPO dendrimers 3–9
To a stock solution of G(4)-PAMAM in DMSO (500 lL,
25 mM in endgroups), aliquots of solutions of NCS-TEMPO
(2, 25 mM) and of 1 (25 mM) in DMSO were added at room
temperature so that the total added volume was 500 lL (see
Table S1, supporting information†). The second reactant was
added 48 h after the first, when analysis of the MALDI-TOF
spectrum indicated complete reaction had occurred. The mix-
ture was allowed to react for an additional 48 h after the second
reactant was added. Completion was again determined through
the analysis of the MALDI-TOF spectrum. A 100 lL aliquot
was taken for EPR spectral analysis, and the remaining solu-
tion was used to synthesize dendrimers 10–16. Tables S2 and S3
(supporting information†) provide MALDI-TOF MS and EPR
characterization data for 3–9.
1
–2 fractions, which is consistent with the lower relative bind-
ing seen in the hemagglutination assay of comparably loaded
mannose–hydroxyl dendrimers (entry 6, Table 2). In all cases,
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 0 7 5 – 3 0 7 9
3 0 7 7

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General deacetylation procedure to form mannose–TEMPO
dendrimers 10–16
described in reference 6, the peak width at half height is evalu-
ated to determine the overall sample homogeneity. Samples that
deviate significantly from a normal distribution function give
significantly broader peaks than those that are observed for the
compounds described here.
Addition of 0.3 M NaOMe in methanol (1.0–1.3 equiv. per
carbohydrate, see Table S4 in the supporting information†) to
the DMSO solutions of 3–9 and then reaction at room tempera-
ture for 48 h afforded 10–16. Reaction completion was deter-
mined by analysis of MALDI-TOF MS. A 100 lL aliquot was
taken for EPR spectral analysis, and the remaining solution was
purified on either Sephadex G25 gel in DMSO or in Amnicon
Hemagglutination assays
Hemagglutination assays were performed at 22 °C in 0.1 M
Tris–HCl buffer, pH 7.2, containing 0.15 M NaCl, 1 mM CaCl2
W
centrifugation filters (M CO = 5 KDa.) and lyophilized. Table
and 1 mM MnCl . Serial two-fold dilutions of ligands (50 lL)
2
S5 (supporting information†) provides MALDI-TOF MS and
EPR characterization data for 10–16.
were incubated with 1 lM Con A (50 lL) for two hours, after
which 50 lL of 3% suspensions of rabbit erythrocytes was
added. After one hour the inhibiting dose was determined by
visual inspection. At the concentrations used, no precipitation
of Con A was observed.
Synthesis of the tri-functionalized dendrimers 22–24
To a stock solution of G(4)-PAMAM in DMSO (1 mL, 25 mM
in endgroups), aliquots of a solution of 2 (25 mM), 1 (25 mM)
and 21 (25 mM) in DMSO were added sequentially at room tem-
perature (total added volume = 1 mL, see Table S6, supporting
information,† for amounts). The second reactant was added 48 h
after the first, and the third reacted was added 48 h after the
second. The functionalization of each reactant was confirmed
by analysis of the MALDI-TOF spectrum. The mixture was
allowed to react for an additional 48 h after the third reactant
was added, upon which time MALDI-TOF MS indicated that
the reaction had gone to completion. NaOMe in methanol was
added so that there was 1.0 equivalent of NaOMe per mannose
residue. After 48 h at room temperature, when MALDI-TOF
MS indicated reaction completion, water was added to the mix-
ture to quadruple the volume of the solution. The solutions were
EPR
CW-EPR spectra were recorded at 9 GHz for TEMPO-labeled
dendrimers in 3:1 DMSO–glycerol solvent at 76 K in a Varian
E-109 spectrometer modified by the incorporation of an external
field-sweep control unit obtained from the University of Denver.
The computer interface of the systems provides for sweep con-
trol and data acquisition in a LabView environment. For clarity
of display, the spectra have been normalized to exhibit equal
amplitudes at the point marked “A” in the spectra. Recording
conditions were established to avoid artificial broadening of
the EPR spectra. Serial dilutions were performed to establish
conditions under which line-broadening from inter-dendrimer
spin–spin interactions was eliminated.
W
transferred to Amnicon centrifuge filters (M CO = 5 KDa.) and
purified with water and lyophilized to dryness. Table S7 (sup-
porting information†) provides MALDI-TOF MS and EPR
characterization data for 22–24.
Affinity chromatography
A 1 mL disposable syringe with a plug of glass wool was used
as the column, and a second 1 mL syringe was placed on top
of the first and connected with Parafilm™ to hold the buffer.
The column was packed by running 1–2 mL of PBS-buffered
saline through the column and then slowly pipetting the Con A
sepharose gel into the column so that the gel was on the 1 mL
mark of the syringe. The column was wrapped in aluminium foil
because of the light sensitivity of the TEMPO-functionalized
dendrimers and washed with 10 mL of PBS buffer for equilibra-
tion. A 100 lL solution of sample (approximately 30 mg mL−1
in PBS) was loaded onto the column, and PBS buffer (20 mL)
was eluted through the column while 1 or 2 mL fractions were
collected. For the affinity columns performed on 10–12, 1 mL
fractions were collected. For the affinity columns performed
4
-Isothiocyanato-2,2,6,6-tetramethylpiperidine N-oxide (2)
4
5
-Amino-2,2,6,6-tetramethylpiperidine N-oxide (1.0 g,
.8 mmol) was dissolved in 20 mL of aqueous 5% NaOH solu-
tion and cooled in an ice bath. Thiophosgene (600 mg, 400 lL,
.9 equiv.) was added over 15 minutes (syringe pump) with rapid
stirring. Immediate filtration and drying in vacuo over P gave
40 mg (57%) of pale orange product. Comparison of the EPR
0
2
O
5
6
spectra of the product with the nitroxide reagent verified the
preservation of the nitroxide radical. Characterization data
1
was in agreement with previously published results. H NMR
(
300 MHz, CDCl
3
): d 4.0 (br. s, 1 H), 2.2–1.0 (br. m, including
1
3
singlet (4 CH
3
groups) at 1.2).
2
2–24, 2 mL fractions were collected. After 20 mL of PBS was
eluted, 10 mL of 0.1 M methyl mannose in PBS was added to the
column while 1 mL fractions were collected. All fractions were
desalted (and mannose was removed) by either dialysis against
MALDI
Matrix assisted laser desorption ionization (MALDI) mass
spectra were acquired using a Bruker Biflex-III time-of-flight
mass spectrometer. Spectra of dendrimers were obtained using a
trans-3-indoleacrylic acid matrix with a matrix–analyte ratio of
water (M
tubes with water (M
W
CO = 3.5 KDa.) or using Amnicon centrifugation
CO = 5 KDa.). Purified samples were
W
lyophilized. The fractions that contained enough mass to obtain
an EPR spectrum were dissolved in DMSO to make a 12.5 mM
solution of endgroups and scanned using EPR. Up to 4 continu-
ous fractions were combined to make an EPR sample if needed.
Tables S8 and S9 in the supporting information indicate the
masses of material eluted in each fraction.†
−
1
3
W
000:1 or 1000:1. Horse heart myoglobin (M 16952 g mol ),
−
1
bovine serum albumin (M
W
66431 g mol ), Bradykinin
12361 g mol ), and
23982 g mol ) were used as external stan-
−
1
−1
(
M
W
1061 g mol ), Cytochrome C (M
W
−
1
Trypsinogen (M
W
dards. An aliquot corresponding to 12–15 pmol of the analyte
was deposited on the laser target. Positive ion mass spectra were
acquired in linear mode, and the ions were generated by using a
nitrogen laser (337 nm) pulsed at 3 Hz with a pulse width of 3
nanoseconds. Ions were accelerated at 19–20000 volts and ampli-
fied using a discrete dynode multiplier. Spectra (100 to 200) were
summed into a LeCroy LSA1000 high speed signal digitizer. All
data processing was performed using Bruker XMass/XTOF V
Acknowledgements
Support of this research by NIH RO1 GM62444 is gratefully
acknowledged. We thank Drs Scott Busse and Joe Sears for help
with NMR and MS.
5
.0.2. Molecular mass data and polydispersities of the broad
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3
0 7 8
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 0 7 5 – 3 0 7 9

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9 (a) Tomalia et al. have reported that the average molecular weights
of PAMAM dendrimers are smaller than the theoretical weights;
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O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 0 7 5 – 3 0 7 9
3 0 7 9