Characterization of Heterogeneously Functionalized Dendrimers by

Oct 20, 2005 - Eric D. Walter, Karl B. Sebby, Robert J. Usselman, David J. Singel,* and Mary J. Cloninger*. Department of Chemistry and Biochemistry, ...
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J. Phys. Chem. B 2005, 109, 21532-21538

Characterization of Heterogeneously Functionalized Dendrimers by Mass Spectrometry and EPR Spectroscopy Eric D. Walter, Karl B. Sebby, Robert J. Usselman, David J. Singel,* and Mary J. Cloninger* Department of Chemistry and Biochemistry, OpTeC, and Center for Bioinspired Nanomaterials, 108 Gaines Hall, Montana State UniVersity, Bozeman, Montana 59717 ReceiVed: March 28, 2005; In Final Form: August 25, 2005

Starburst dendrimers are receiving considerable attention as templates for the assembly of structured arrays of molecular components. This research motivates the development of improved methods for dendrimer characterizationsspecifically, for determining the numbers, distributions of numbers, and spatial distribution of molecular species synthetically attached to macromolecular templates. Such information provides the basis for advancing strategies aimed at controlling dendrimer functionalization, and thus represents enabling technology for tailoring the composition and structure of molecular arrays fashioned on dendrimer templates. Moreover, this information is vital to the proper interpretation of ongoing experiments in which dendrimers sparsely functionalized with reporter groups are used as probes. In this article, we report MALDI-TOF mass spectrometry and EPR spectroscopy of heterogeneously functionalized G(4)-PAMAM dendrimers bearing nitroxide spin-labels.

Introduction Dendrimers provide an attractive template for the chemical assembly of complex arrays of molecular components. The commercial availability of dendrimer frameworks in a variety of sizes with numerous functionalization sites has advanced the practical utility of these templates; modification of these frameworks has emerged as an area of vigorous research activity.1,2 For example, heterogeneously surface-functionalized dendrimers have been reported for diverse applications such as the synthesis of combinatorial libraries,3 the development of drug delivery systems,4 and the creation of dendrimer redox systems.5,6 Newkome et al. have reported the reaction of mixtures of branched isocyanates with DAB-Am-34 and 12cascade acid dendrimers and have shown that different ratios of functional groups can be added to the dendrimer surface.7,8 However, little attention has been given to determining the relative spatial locations of the different endgroups in partially and heterogeneously functionalized dendrimers. Such information is of fundamental importance, particularly in the use of dendrimers sparsely functionalized with molecular reporter groups to probe intermolecular and interfacial contacts of macromolecules.9-11 We are developing experimental methods to characterize this property of products formed from the heterogeneous surface functionalization of dendrimers. Our objective includes three specific goals, namely the determination of (a) the overall ratio of functional groups attached in heterogeneously or partially functionalized dendrimers, i.e., the aVerage loading of the dendrimer by each substituent; (b) the loading distribution over a population of dendrimers, i.e., the loading homogeneity; and (c) the relative position of the various functional groups that are attached to the dendrimer template, i.e., the spatial distribution of the attached groups. * Corresponding authors. E-mail: [email protected] (D.J.S.); [email protected] (M.J.C.).

Frequently, NMR spectroscopy is used to determine the average degree of dendrimer functionalization.7,8 We have also found that MALDI-TOF MS (matrix assisted laser desorption ionization time-of-flight mass spectrometry) is useful in this determination.12 For spin-labeled substituents, quantitative EPR (electron paramagnetic resonance) spectroscopy, as reported here, provides another useful route to determine the average loading. In this article, we report an approach for the use of MALDI-TOF MS to illuminate the loading distribution. We also introduce a novel spin-label EPR approach to provide information on the spatial distribution of functional groups attached to a dendrimer template. Detailed analysis of the EPR line broadening changes with progressive loading of the spin-label provides a direct means to test models of the spatial distribution of spin-labels and also indirectly provides complementary information on the spatial distribution of other groups attached in heterogeneously functionalized systems. Here, we specifically describe the application of MALDI-TOF MS and EPR spectroscopy to the characterization of G(4)-PAMAM dendrimers13 heterogeneously functionalized via isothiocyanate coupling methods.9,14 This approach is related to spin-label methods for solid-state EPR methods for determining long-range distance constraints in spin-labeled proteins.15,16 A novel feature of the work reported here is the treatment of systems with a plethora of coupled spins. Experimental Results The general strategy for heterogeneous dendrimer functionalization is shown in Scheme 1. We use 4-isothiocyanato2,2,6,6-tetramethylpiperidine-N-oxide (NCS-TEMPO, 1) as one reagent and a primary, secondary, tertiary, or aromatic isothiocyanate 2-5 to incorporate a second surface residue. The synthesis is carried out with a stock solution of G(4)-PAMAM17 in DMSO (dimethyl sulfoxide; 200 µL, 25 mM in endgroups). Aliquots of a solution of NCS-TEMPO (1, 25 mM), and of the additional isothiocyanate (2-5, 25 mM) in DMSO were added

10.1021/jp0515683 CCC: $30.25 © 2005 American Chemical Society Published on Web 10/20/2005

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SCHEME 1: Synthesis of Spin-Labeled Dendrimers 6-10

Figure 2. CW-EPR spectra of G(4)-PAMAM dendrimers variously loaded with TEMPO. 10a (black), 10b (red), 10c (orange), 10d (green), 10e (light blue), 10g (dark blue), and 10h (violet), exhibiting a strong dependence of EPR line width on loading. Samples of ∼300 µL contained in 3 mm i.d. fused silica EPR tubes were immersed in boiling nitrogen (76 K) during the scans. The field was scanned over 200 G in a scan of 2 min. The field was modulated at 100 kHz with an amplitude of 1 G. The detection time constant was 250 ms. Microwave irradiation was applied at ∼9.2 GHz at a power of 2 nW.

Figure 1. MALDI-TOF MS of TEMPO-functionalized G(4)-PAMAM dendrimers. G(4)-PAMAM (gray), 10a (black), 10b (red), 10c (orange), 10d (green), 10e (light blue), 10f (dark blue), and 10h (violet).

to the dendrimer solution either sequentially or simultaneously in a specific ratio (total added volume ) 200 µL). For sequential reactions, the second reactant was added 48 h after the first reactant and was allowed to react for an additional 48 h before EPR spectra were acquired. The time interval was selected to ensure completion of the reaction; control EPR spectra suggest that samples are not altered after a year of storage. Partially spin-labeled dendrimers 10a-g were synthesized by reaction of dendrimer with a limiting amount of 1, and fully spin-labeled dendrimer 10h was synthesized by reaction of the dendrimer with 55 equivalents of 1. Overall loadings were primarily determined by MADLI-TOF MS, as reported previously.9,12,14 MALDI-TOF MS spectra of partially functionalized intermediates (after addition of one isothiocyanate compound) and of fully functionalized dendrimers (after addition of the second isothiocyanate) were obtained. Comparison of values of MW for 6-10, for unfunctionalized PAMAMs, and for synthetic intermediates allowed us to determine the number and ratio of surface groups per dendrimer.9 EPR spectroscopy was also used to compute TEMPO loadings through comparison of the double integrated EPR signal amplitude against calibrated, standard TEMPO solutions. Representative MALDI-TOF mass spectra are shown in Figure 1. A noteworthy feature of the MS data is that the peaks are very broad and appear at lower mass values than would be expected for the idealized compounds. Inasmuch as the unfunctionalized dendrimer sustains this broadening, it must reflect an intrinsic heterogeneity in chemical structure of the template that presumably derives from errors in the dendrimer synthesis. Absence of expected terminal -CH2CH2CONHCH2CH2NH2 arms is documented; similarly, intramolecular bis-amide formation, in which 1 equivalent of ethylenediamine is effectively

lost, can occur instead of the intermolecular addition of 2 equivalents.12a,18 The depicted spectra also clearly show a progressive increase in the breadth of the MALDI peaks with increasing m/z. Analysis of this trend is used to assess the loading homogeneity, as discussed in the Analysis and Discussion section. CW (continuous wave) EPR spectra were recorded at ∼9.2 GHz for TEMPO-labeled dendrimers 6-10 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.19 The computer interface of the systems provides for sweep control and data acquisition in a LabView environment. Exemplary spectra are exhibited in Figure 2. 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. Similarly, serial dilutions were performed to establish conditions under which line broadening from interdendrimer spin-spin interactions was eliminated. Analysis and Discussion Average Loading. We have found the synthetic strategy depicted in Scheme 1 to be generally applicable for heterogeneous dendrimer functionalization.9,20 For isothiocyanates of comparable reactivity (e.g., 1 and 2), the ratio of the two functionalities on the dendrimer surface was the same as the reactant mole ratio and was independent of the sequence of addition. For isothiocyanates with differing reactivities (e.g., 1 and 3 or 4), the less hindered isothiocyanate reacted more rapidly. We found that sequential additions gave surface functionalization ratios that most faithfully reflect the reactant mole ratios. Reactions with 4 had to be performed with reagents at 3-fold lower concentrations than in the case of the other reactions to avoid product dendrimer aggregation and/or precipitation, especially for high loadings of 4. In the case of very reactive isothiocyanates such as 5, it was necessary to stir reactions rapidly to avoid bolus effects. Overall, the MALDITOF MS (and EPR) data indicate that coupling reactions go to completion for all of the reactions studied, independent of reactant and order of addition. When reagents of comparable reactivity are added as a mixture to the dendrimer, the relative

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loadings simply reflect the composition of the reagent mixture. Loading Distributions. We examined the trend in the widths of the MS peaks (full width at half-maximum height, fwhm) with loading to obtain information about the loading distribution. This trend in peak width involves two effects. The primary broadening effect has a linear dependence on loading. The broad mass distribution of unreacted dendrimers implies, as noted above, a corresponding mass-dependent distribution in the number of reactive sites. As molecules are attached at these sites, each point in the MS shifts to an extent that is determined by the product of the mass of the attached group and the number of sites occupied. Provided that the loading is not correlated to the number of sites, the latter factor is just the loading times number of sites on dendrimers belonging to that point in the MS. Thus not only does the peak position shift with loading, but line width linearly increases with mass.21 To clarify this point, consider the fwhm, which is determined by the positions of the half-maximum points on the high- and low-mass sides of the peak in the MALDI traces. When reacted, for example to 100% loading, the high-mass point increases in mass by an amount determined by the number of endgroups on the dendrimer and by the mass of the functional group. The same holds true for the low-mass point, but since it belongs to a dendrimer with less mass, it has (as noted above) fewer endgroups. Accordingly, the lower mass point sustains a smaller increase in mass. Overall, the difference between the low- and high-mass points, the fwhm, increases proportionally to the mass of the reactants. The secondary effect gives rise to a broadening that is nonlinear in loading and from which we gain insight into the loading distribution. Even in the idealized case of an error-free dendrimer with no variance over the sample in the number of reactive sites, partial loading of a dendrimer is likely to occur with some inherent heterogeneity. For example, a half-loaded dendrimer is likely to include molecules in which the number of sites that are functionalized is somewhat more and somewhat less than 50%. To the extent that the number of reactive sites scales with mass, this loading-dependent broadening involves a convolution of the MALDI-TOF MS peak with the product of the loading distribution and the mass of the attached group. The contributions to the line broadening from these two effects are readily separated. Since the loadings of the unreacted and fully loaded dendrimers are necessarily homogeneous, they sustain no secondary broadening. Thus, on a plot of width versus loading, a straight line between data points at 0 and 100% loading indicates the linear effect.22 If the loadings were similarly homogeneous for all intermediate loadings, then the trend in the peak width would adhere to this straight line. As illustrated in Figure 3, however, there is some evidence of a departure of the MALDI-TOF MS peak widths from a straight line. The evident departure of the observed peak widths from a straight line implies some heterogeneity in the loading. We suspected that the loading distribution might follow a normal distribution law; that is

Xg ) {LNg(1 - L)(N-Ng)N!}/{Ng!(N - Ng)!}

(1)

in which Xg is the fraction of molecules with Ng attached groups on a dendrimer of N reactive sites, and L is the loading Ng/N. At large N, this distribution function, tends toward Gaussian shapes with widths of [4NL(1 - L)]1/2; the maximal width, namely N1/2, occurs for the half-loaded dendrimer. By numerical simulations, we ascertained that the convolution of the observed

Figure 3. Analysis of MALDI-TOF MS line widths. (a) Full width at half-maximum height of the MS peaks, as shown in Figure 1, observed for different loadings of the dendrimer with TEMPO. Circles are experimental data. The primary effect of loading on the MS line width, as discussed in the text, is the linear trend (solid line). Broadening from the loading distribution (as illustrated in (b)) convolved with the linear trend leads to the curved line (dashed line) in the graph. (b) Random loading distributions on a template with N ) 55 sites, for average loadings L of 0.05 (black), 0.1 (red), 0.25 (orange), 0.5 (green), 0.75 (light blue), 0.9 (dark blue), and 0.95 (violet).

MS peaks with a Gaussian function enlarges their widths according to

W ) (WMS3/2 + Wg3/2)2/3

(2)

in which W is the resultant width of a peak, after convolution of a MS peak of width WMS with a Gaussian function of width Wg. To test this idea of a random loading distribution, we computed the loading-dependent width as a function of L. We employed eq 1 with N ) 55 to determine loading dependent distribution functions.2a The widths of these functions were combined, via eq 2, with values read from the straight line in Figure 3 to obtain the resultant values plotted as the curved line in Figure 3. The agreement between the curved line and the experimental data is very satisfactory: notwithstanding the limitations of poor spectral resolution in the MALDI-TOF MS of this system, a simple analysis makes a strong case for a normal distribution of the loading. Most importantly, the data clearly rule out any broad distributions in which there are substantial numbers of dendrimers having Ng/N far removed from the overall loading value. Such distributions would lead to a pattern of broadening that is inconsistent with the experimental observations summarized in Figure 3. Spatial Distribution. With the average loading and loading homogeneity established, EPR spectroscopy was used to evalu-

Heterogeneously Functionalized Dendrimers

Figure 4. TEMPO EPR spectral A/B peak height ratio vs loading for 6-10, 5-95% TEMPO.

ate the presentation of endgroups. The EPR method that we present here is an extension of known methodologies for longrange (7-25 Å) distance determination in proteins and other complex systems.15 In protein studies, site-directed spin-labeling methods16 are used to attach pairs of labels. The distance between the labels is determined from the dipolar line broadening of the EPR spectra of the isolated spin-labels. A novel feature of the work reported here is the use of dipolar interactions among a large network of spins to characterize their overall spatial distribution. The core problem is reminiscent of van Vleck’s classic treatment of dipolar line widths of spins in crystal lattices.23 We have found that the ratio of the EPR spectral amplitudes at the positions marked “A” and “B” in Figure 2 provides a useful measure, analogous to that introduced by Kokorin,24 of the interspin broadening. Comparison of the double integral signal intensity values for 6-10 gives results analogous to those obtained from comparison of A/B peak height ratios. In Figure 4, we plot the loading-dependent trends of the A/B peak height ratio for the products of all of the reactions carried out in this study. The trends in A/B ratios depicted in Figure 4 are remarkably similar for the entire array of compounds studied. When 1 is added alone or before 2-5, the A/B ratio varies from ∼0.7 to ∼1.4 in a nearly linear fashion as the amount of spinlabel is increased. This behavior is also observed when TEMPO and 2 are simultaneously added to the reaction. When 3-5 are added prior to 1, congruent behavior is observed for reactant ratios of less than 0.5; for reactant ratios above 0.8, a slight diminution of A/B is observed. This deviation is attributable the presence of a small amount of unreacted 1 (which is evidenced by the presence of a sharp feature in the spectrum between points A and B).25 Decomposition of the spectra into sharp and broad components indicate that the free TEMPO represents less than 3% of the total in all compounds surveyed. We also find that double integration of the spectra, and comparison to free TEMPO standards, accounts for greater than 94% of the spin-label introduced in the reaction. The common trend in the A/B ratio for the EPR spectra indicates that the dipolar broadening, as a function of TEMPO loading, is very similar in all of the studied compounds. The simplest explanation for this similarity is that the spatial distribution of the spins is the same and is evidently dictated by the dendrimer scaffold itself. This result by itself strongly suggests that the groups attach in a spatially random fashion; were they either to cluster or avoid each other in the reaction, then distinct, complementary trends would likely be observed when the order of sequential reactions was reversed.

J. Phys. Chem. B, Vol. 109, No. 46, 2005 21535 To investigate the spatial distribution in more detail, we have undertaken numerical computations of the dipolar line broadening functions for assumed structural models; we used the results of these computations to simulate trends in A/B ratio by convolving the dipolar line broadening functions with the EPR spectrum of an isolated TEMPO spin-label. These computed trends are compared to the data in Figure 4 to assess the compatibility of the model with experimental results. We model the spin-labeled dendrimer sphere of radius R that possesses Nspins spin-labels distributed among Nsites reactive sites for a loading, L, of Nspins/Nsites. As a starting point, we neglect heterogeneity in Nsites and in L. For the G-4 dendrimer preparation used in these experiments, Nsites is taken to be 55 (because this is the routine maximum loading that we can achieve). We assume the existence of a minimum (cutoff) interspin distance Rmin. As limiting cases, we probe three models of spatial cooperativity; we assume that the labels are distributed on the dendrimer surface such that they alternatively (a) occupy remote positions (maximizing the interspin distances), (b) form in clusters (minimizing the interspin distances), or (c) occupy positions at random. We surveyed R from 15 to 45 Å in 5 Å steps and Rmin from 0 to 16 Å in 2 Å steps. By a Monte Carlo method, we generated ∼10-20 specific structures for each set of model parameters. Given each realization of each structural model, the calculation of the dipolar line broadening function largely follows directly. For computational expediency, however, we employ a simplification that follows from the classical treatment of vanVleck.23 For each spin, i (on each structure), which sees Nspins - 1 other spins, j, on that structure, we directly compute 〈∑rij-6〉, where rij is the distance between spins i and j. We then assume that the dipolar line broadening sustained by this ith spin is modeled as a Gaussian function with the full width at half-maximum height, B, which is given by

∑rij-6〉1/2

B ) (1.95 × 104)〈

(3)

The numerical factor in expression 3 involves physical constants and numerical factors that relate the second moment and line width, for B in gauss and for rij in angstroms.26 The dipolar line-broadening function associated with a given set of model parameters is taken as the normalized sum of the individual Gaussians calculated for each of the Nspins spins, averaged over the 10-20 realizations of each structural model. A more elaborate direct calculation gives essentially identical results.27 To compute dipolar broadened EPR spectra, these dipolar spectra are convolved with the spectrum of a dilute (2 mM) frozen solution of 4-amino TEMPO. A/B values are determined by inspection of the resultant, broadened EPR spectra. To determine the structure parameters R and Rmin, we took advantage of the fact that the three loading models give identical results at L ) 1. Good agreement between calculation and experiment at L ) 1 is obtained for Rmax ) 34 ( 2 Å and Rmin ) 7 ( 1 Å. The curves in Figure 5 represent trends in A/B peak height ratios computed for these values according to the three loading models. Comparison to the experimental data (Figure 4) clearly rules out the remote loading model. Moreover, the sharp initial rise that characterizes the cluster model is absent from the experimental trend. Overall, the data are most consistent with the random loading model. This result is of particular importance in understanding the interaction of partially functionalized dendrimers with target macromolecules and molecular assemblies. Interestingly, the experimental trend for

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Figure 5. Calculated trends in A/B ratio with loading. The depicted curves represent computations with Rmax ) 34 Å and Rmin ) 7 Å, and with one of the three limiting spatial distribution models of the loading: random (solid line), clustered (dotted line), and remote (dashed line).

a random distribution of spins does show noticeable differences from the computed trend. This subtle disagreement presumably has its origin in one of the several idealizations built into the model. The effect of loading heterogeneity could be reintroduced by smoothing the A/B trends with a moving average, in which the averaging is weighted by the loading distribution function. As seen from the MALDI-TOF MS experiments, however, the loading distribution attaches substantial weight only within a narrow range of the nominal L. We have verified that such a smoothing does not improve the agreement appreciably. In fact this smoothing actually increases the curvature of the computed line and thus decreases the agreement with experiment. This result underscores the facts that the underlying dipolar spectral broadening functions are not simple, constant line shapes whose widths are simply additive in determining the width of a composite spectrum: the fwhm is primarily determined by the sharper features, while the broader components extend the wings.28 Assumption of a spherical shape may seem particularly severe, but it is actually not necessary. Any smooth surface that affords the same pair correlation function would give the same results. Previous work29 has established a strictly linear relation between A/B and concentration in a normal, three-dimensional solution. In Figure 6, we show experimental results that verify this relation for frozen solutions of 4-amino TEMPO in 3:1 DMSO:glycerol. It is worth noting that, while the broadening trends of the spectra in this solution are grossly similar to those of the dendrimer depicted in Figure 3, there are noticeable qualitative differences, which are especially evident in the troughs between peaks and in the wings of the spectrum. By performing additional calculations following the method described above for random lattices of three dimensions, we further verified that our computational approach reproduces the result for 4-amino TEMPO. More generally, we find, for random lattices of various dimensionalities d, that A/B varies approximately with the respective volume, surface, or linear density of spins raised to the power 4 - d. The relationship is accurately reflected in the width of the line-broadening function. On the basis of these results, we suspected that the calculated trend in line broadening would reproduce the experiments better if we allow for a “rough” surface, which introduces some threedimensional character to the spatial distribution. We thus elaborated our calculation, for the random model, to allow the spins to be distributed within surface shells of various thicknesses. In Figure 7, we show the progression in the line width (fwhm) of the dipolar spectra thus calculated. Included in the figure are dipolar line widths obtained from the experimental EPR spectra by deconvolution15 against the spectrum of a dilute

Figure 6. (a) EPR spectra of 4-amino TEMPO in 3:1 DMSO:glycerol at concentrations of 2 mM (black), 30 mM (red), 60 mM (green), 100 mM (light blue), and 160 mM (dark blue). (b) Concentration-dependent trends in A/B ratio for 4-amino TEMPO. EPR conditions are as in Figure 2. In this system, A/B can be calculated from the TEMPO concentration as follows: A/B ) [TEMPO concentration (mM) × 0.00379 ((0.00006)] + 0.703 ((0.005).

Figure 7. Calculated trends in dipolar line broadening with loading. Circles are experimental points for 10a-h. The depicted curves represent computations with the random loading model elaborated to allow for occupancy within shells of various thicknesses: 0 Å (long dash), 10 Å (dash), 20 Å (short dash), and 16 Å (line). In all computations, Rmin ) 7 Å and Rmax is varied between 34 and 35 Å to achieve agreement with experiment.

frozen solution of 4-amino TEMPO. We found that a minimum shell thickness of 16 Å is required to obtain a computed line width trend that matches the experimental results. This three-dimensional character might originate from several causes. Defects in synthesis that result in lost branches and shortened arms are linked to heterogeneity in N, the extent of which is clearly apparent in the MALDI-TOF mass spectra, and must create dendrimer shape imperfections. The elaboration of our model to include a shell thickness provides a simple route to account for the heterogeneity in N, but cannot fully account for the 16 Å shell required to accommodate our results. The volume shell may derive from the variability in the position of

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the dendrimer endgroups, which have been suggested alternatively to be folded back into the dendrimer or extended into the solvent.28 The variation in end-group conformation could cause the observed shell thickness. A final issue that remains to be addressed is the possible impact of dendrimer dynamics on the randomness of the spatial distribution. It has been suggested that the dendrimer framework is flexible enough to allow extensive movements of the endgroups.1,13 Such dynamics could give rise to a random distribution of attached groups by scrambling any order imparted during synthesis. The results reported here do not answer this question, but nevertheless provide a methodological framework for addressing this and analogous problems in large-scale structural dynamics. If a macromolecule can be multiply labeled in some nonrandom fashion, then the structural rearrangements that lead to a random distribution of the labels could, in principle, be monitored by following the temporal trend in dipolar broadening. Work is in progress in our laboratories to extend the methods reported here to investigate structures and structural dynamics of other multiply functionalized macromolecular templates. While described in the context of dendrimer studies, the EPR method reported here could be enormously useful for studies of other systems such as large, multisubunit protein assemblies.

distribution of attached groups is achieved only in a statistical sense. As such, sparsely loaded dendrimers provide for an inherent library of structures, in which intergroup distances are distributed over an appreciable range, the upper limit of which is controllable by selection of the size of the template. Molecular recognition studies that involve multivalent interactions can take advantage of this diverse library for rapid assessment of the fundamental structural features of multivalent receptor sites.9 Characterization information such as the work described here is imperative for applications such as the use of dendrimers in targeted drug delivery, where a recognition element, a prodrug, a solubilizing group, and an imaging group might all be present on the same dendrimer.1f,g,4 While the EPR methods reported here were developed in the context of dendrimer templates, they are immediately applicable to the exploration of structural organization and structural transitions of other macromolecular assemblies such as multisubunit protein assemblies that are highly loaded with nitroxide spin-labels.

Conclusions

Supporting Information Available: Procedures for the synthesis of 1, 2, 3, and 5, general procedure for addition of isothiocyanates to dendrimers, and EPR Stackplots for 6-10. This material is available free of charge via the Internet at http:// pubs.acs.org.

MS and EPR data were used to quantify loadings in heterogeneously functionalized dendrimers. Under the conditions employed, the coupling reactions were shown to go to completion for all of the reactions studied, independent of the sequence of reactions. Relative loadings in heterogeneously functionalized systems are thus controllable by adjustment of the initial reagent mixture. Despite the modest resolution of the MALDI-TOF MS spectra, the spectral peak widths suggest good sample homogeneity for a given overall loading. Loading-dependent trends in the dipolar broadening of the EPR spectra were employed to test models of the spatial distribution of the attached groups. The broadening trends were found to be sensitive to the overall size and effective dimensionality of the template, but relatively insensitive to the shape. They are also sensitive to the manner in which the attached groups are distributed in space. For the G(4)-PAMAM dendrimer, a random spatial distribution of attached groups located in a surface layer of thickness at least 16 Å, on a sphere with a 35 Å radius, gives an excellent account of the experimental trends. The three-dimensional character of this distribution appears to require some heterogeneity in positioning of the endgroups. The degree to which these properties are influenced by the dynamics of the structure is under continuing investigation. The randomness of the spatial distribution has important consequences for experiments conducted with sparsely functionalized templates. Spin-labeling of dendrimers with TEMPO derivatives has been previously reported. The adsorption of spinlabeled dendrimers onto porous surfaces, DNA, and vesicles has been probed by EPR spectroscopy.10 Intermolecular interactions have also been investigated with TEMPO-labeled dendrimers.11 Interpretation of the results of some of these experiments was limited by the lack of knowledge of the manner in which the spin-labels are spatially distributed on the dendrimer template. In addition to their use as probes, TEMPOlabeled dendrimers have been used for “living” radical polymerizations30 and for reoxidation of small molecule nitroxides for EPR imaging.31 In such systems, control over the spatial

Acknowledgment. The authors thank NIH (RO1 GM6244) and DOE (DE-FG02-01ER45869) and NSF (DMR-0210915) for funding of this research. K.B.S. thanks the NSF REU program and NSF EPSCoR for support.

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