The Effect of Global Compaction on the Local Secondary Structure of Folded Dendrimers

Article abstract of DOI:10.1021/ja037895a

The effect of nonlocal interactions on the local structural propensities of folded dendrimers was evaluated in this work by comparing, under identical conditions, the conformational properties of isomeric dendrimers differing in their global packing effic

Full text of DOI:10.1021/ja037895a

Published on Web 10/31/2003
The Effect of Global Compaction on the Local Secondary
Structure of Folded Dendrimers
Baohua Huang, Matthew A. Prantil, Terry L. Gustafson, and Jon R. Parquette*
Contribution from the Department of Chemistry, The Ohio State UniVersity,
Columbus, Ohio 43210
Received August 12, 2003; E-mail: parquett@chemistry.ohio-state.edu
Abstract: The effect of nonlocal interactions on the local structural propensities of folded dendrimers was
evaluated in this work by comparing, under identical conditions, the conformational properties of isomeric
dendrimers differing in their global packing efficiency. Accordingly, a modular synthesis of two series of
dendrimers up to the third generation was developed to provide efficient access to isomeric dendrimers
displaying different levels of overall compaction. Dendrimer compaction levels were adjusted by connecting
the folded dendrons to 1,3,5-benzenetricarbonyl chloride, as the central core, via either a 2- or a
4-aminobenzamide linkage to induce relatively “compacted” or “expanded” conformations, respectively.
The hydrodynamic volumes of the dendrimers were measured by time-resolved fluorescence anisotropy
(TRFA) measurements as a function of the dendrimer series, generation level, and solvent. Packing
efficiencies (compaction levels) were estimated by the ratio (Vh/Vvw) of the experimental hydrodynamic
volume (Vh) to the calculated van der Waals volume (Vvw). The extent and stability of local helical bias was
measured using circular dichroism and correlated with the packing efficiency (Vh/Vvw). These studies
suggested that compaction plays an extremely important role in determining the secondary structural
preferences of the dendrimers; however, the nature of compaction was more important than the extent of
compaction.
Introduction
the long-term goal of this work is directed at establishing control
of the hierarchical folding process of a synthetic macromolecule
Many cellular functions such as catalysis, molecular recogni-
tion, and information storage are mediated by biomacromol-
ecules that reversibly fold into compact, three-dimensional
conformations.1 The motivation for developing nonnatural
oligomers capable of reversibly folding into unique structures
is predicated on the notion that a capacity to control the long-
range conformational equilibria of nonbiological materials will
permit functions that lie outside the biological realm to be
realized. Although many new folding oligomers, called “fol-
damers,”2 have been reported that fold into compact secondary
structures, functional biomolecules exhibit structures displaying
much higher structural organization (tertiary and quaternary
structure) that are likely to be necessary for function.1 Recent
progress toward the design of folding oligomers has produced
an understanding of how primary structure can be programmed
to afford stable secondary structural order through local non-
covalent interactions. However, there is little information on
how the long-range interaction of multiple elements of secondary
structure affects the conformational properties of the molecule.
An understanding of the nature of the local T global confor-
mational interplay would significantly assist the design of
nonnatural molecules having tertiary structural order. Therefore,
by understanding how various elements of structural order
cooperate to create a compact, three-dimensional structure.
Global compaction of a denatured protein into a condensed
conformational state occurs early in the folding process of a
protein into its native conformational state and is thought to be
an important step in the folding reaction.3 The cooperative
interplay between large-scale organization and short-range local
conformational propensities ultimately directs the folding
process and determines the stability of the native state.4 The
effect of such long-range tertiary interactions on the formation
and stability of short-range secondary structural order in natural
protein macromolecules suggests that compacting a dendrimer
structure into a condensed state will have a dramatic effect on
the local conformational propensities of the dendrimer. However,
the degree of cooperativity between the collapse of a protein
into a compact shape and the development of secondary structure
is not well understood. For example, early lattice model studies
suggested that the nonlocal interactions that drive protein
collapse are strongly coupled to the formation of secondary
structure and that compaction alone is sufficient to create
secondary structure in proteins.5,6 Several subsequent theoretical7
and experimental8 studies concluded that the degree of local T
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Effect of Global Compaction on Folded Dendrimers
A R T I C L E S
global cooperativity was low when the collapse transition was
driven only by nonspecific, long-range interactions. Neverthe-
less, the importance of both short- and long-range interactions
in stabilizing the folded state of a protein can be inferred from
the observation that isolated R-helices are rarely stable in water9
and that protein fragments often exhibit different conformational
properties in the absence of the tertiary structure of the intact
protein.10 There also is a good correlation between the relative
decrease in hydrodynamic volume and the increase in secondary
structure content among proteins in the literature.3b,11 Similarly,
the hydrophobic interiors of proteins, in their native state,
maintain an extremely high packing density of side chains
similar to the packing found in organic solids.12 This efficient
filling of internal space in a protein appears to be an important
determinant of protein stability13 and is thought to be a crucial
factor responsible for the remarkable thermal stability of
thermophilic proteins.14,15 Consequently, the organization of
elements of protein secondary structure on a template has
become an effective tool for inducing and stabilizing secondary
and tertiary structures of a variety of folding motifs.16
macromolecules in which multiple elements of secondary
structure can interact intramolecularly. Based on the studies
described above, the induction of secondary structural order in
dendrimeric macromolecules should be enhanced by the compact
structure of higher generations. However, in many dendrimer
systems, significant backfolding of the termini occurs, indicating
a certain lack of local conformational order in the dendrimer
structure.20 This internal flexibility appears to be responsible
for the difficulties experienced in efforts to develop three-
dimensional organization in these systems,21,22 and recent efforts
in our laboratory to compact polyaryl ether dendrimers in
aqueous media indicated that compaction alone was not a
sufficient criterion to induce secondary structure in flexible
dendrimers.24
Design Considerations
Recently, we described a series of dendrons whose local
conformational properties were restricted through the interven-
tion of intramolecular hydrogen-bonding and electrostatic
interactions present in the AB2 building block (Figure 1,
bottom).25 These dendrons adopt a specific, chiral helical
secondary structure that occurs throughout the internal and
peripheral regions and depends critically on the development
of intramolecular packing interactions at higher dendron genera-
tion (Figure 1, top). These nonbonded packing interactions
couple the motions and conformational preferences of each pair
of terminal groups, or helical fold, causing the highly dynamic
equilibrium interconverting the M and P helical conformations
shown below to shift toward a single helical sense.26
The highly branched and regularly repeating connectivity of
dendritic macromolecules creates a three-dimensional structure
that adopts an increasingly compact, globular shape at higher
generations,17,18 potentially mimicking the morphology of
globular proteins.19 Consequently, dendrimers would seem to
present an opportunity to approach the design and synthesis of
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(26) Although the barrier could not be measured for these dendrons, we have
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diamide system using NMR line-shape analysis. This barrier most likely
represents an upper limit for the dendrons and dendrimers described in
this manuscript. Preston, A. J.; Fraenkel, G.; Chow, A.; Gallucci, J. C.;
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A R T I C L E S
Huang et al.
Figure 1. Structure and conformational equilibria of helically folded dendrons.
volumes. Specifically, the ortho linkage provided dendrimers
with a more compact structure (smaller hydrodynamic volume)
than did the isomeric para linkage.
Dendron Synthesis: First-Generation Strategy
We previously reported a synthetic strategy toward dendrons
having a 2-aminobenzamide linkage at each generational shell
that employed 4-chloropyridine-2,6-dicarbonyl chloride as the
branching monomer.25d Convergent generational growth was
accomplished using amide bond-forming reactions, and focal
point activation occurred by NaN3 displacement of the focal
chloro group followed by hydrogenation (Scheme 1). Acylation
of the focal amino group of a dendron with 2-nitrobenzoyl
chloride followed by hydrogenation over Pd/C installed a
2-aminobenzamide moiety that became the connecting unit
between generational levels upon reaction with 4-chloropyridine-
2,6-dicarbonyl chloride. Although this strategy provided den-
drons up to the third generation, there were three issues that
needed to be addressed to efficiently construct larger dendrimers
from these dendron precursors. First, attaching the 2-aminobenz-
amide connector at each generational level by this process was
a strictly linear strategy and required five transformations for
each generational growth step. Second, the focal amino group
at each generation was severely deactivated by the electron-
deficient pyridine ring, causing the reaction with 2-nitrobenzoyl
chloride to be slow and capricious, particularly on a large scale.
Finally, hydrogenation of the nitro group proceeded much more
slowly (2-3 d) than was expected for a nitro function
presumably due to poisoning of the catalyst by the dendron
structure.28 Therefore, a more convergent strategy that circum-
vented these limitations was developed to improve the efficiency
of the dendrimer synthesis.
Figure 2. Notional depiction of structural compaction of a folded
dendrimer.
Correlating this local helical conformational equilibria with
the nonlocal interactions experienced between adjacent dendrons
requires a capability to compare the conformational properties
of isomeric dendrimers differing only in their global packing
efficiency.23 However, previous studies addressing the effect
of structure on the hydrodynamic properties of dendrimers have
reported that relatively minor structural modifications, such as
those observed in the cis/trans photoisomerization of azobenzene
dendron/core linkages, induce comparatively small volume
changes in flexible dendrimers.27 Fortuitously, relatively minor
modifications of the dendron/core linkage in the structure of
these folded dendrimers afforded large variations in hydrody-
namic volumes, thereby allowing the effect of compaction on
local secondary structure to be investigated in isomeric systems
having minimal constitutional differences (Figure 2). Accord-
ingly, attaching the dendrons to the central core via either
4-aminobenzamide (para) or 2-aminobenzamide (ortho) linkage
produced structures with significantly different hydrodynamic
(27) (a) Junge, D. M.; McGrath, D. V. J. Am. Chem. Soc. 1999, 121, 4912. (b)
Li, S.; McGrath, D. V. J. Am. Chem. Soc. 2000, 122, 6795. (c) Liao, L.-
X.; Junge, D. M.; McGrath, D. V. Macromolecules 2002, 35, 319.
9
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Effect of Global Compaction on Folded Dendrimers
A R T I C L E S
Scheme 1. First-Generation Dendron Synthesisa
Scheme 2. Branching Unit Synthesisa
a (a) 2-NO2C6H4COCl, pyr., (b) H2, Pd-C, EtOAc-CH3OH, (c) 4-
chloropyridine-2,6-dicarbonyl chloride, pyr., (d) NaN3, DMF, 50 °C.
a (a) (i) POCl3, 128 °C, 12 h, (ii) t-C4H9OH, DMAP (cat.), CH2Cl2/pyr.,
12 h, (b) NaN3, DMF, 50 °C, (c) H2, Pd-C, EtOAc-CH3OH, (d)
2-nitrobenzoyl chloride, DMAP (cat.), CH2Cl2/pyr., 4 h (93%), (e) 4-ni-
trobenzoyl chloride, DMAP (cat.), CH2Cl2/pyr., 6 h (87%), (f) H2, Pd-C,
EtOAc-CH3OH (95% for 5-ortho, 85% for 5-para), (g) allyl chloroformate,
DMAP (cat.), CH2Cl2/pyr., (82% for 5-ortho, 91% for 5-para), (h)
CF3CO2H/CH2Cl2 (1:1), 2.5 h, 92% (for 5-ortho and 5-para), (i) 5-ortho
or 5-para, (COCl)2, CH2Cl2, DMF (cat.).
Second-Generation Strategy: Synthesis of
Functionalized Branching Monomers
We envisioned that employing a branching moiety function-
alized at the 4-position with a suitably protected 2- or 4-ami-
nobenzamide connector would address all three limitations of
the initial synthetic strategy. Directly incorporating the turn unit
in the branching unit would decrease each generational growth
step by three transformations, and the problematic amine
acylation and subsequent nitro reduction steps would only need
to be addressed once in a relatively simple molecule rather than
at every generational step. This strategy would also provide the
flexibility to construct dendrons with a 4-aminobenzamide
linkage at the focal point by employing an appropriately
functionalized branching moiety just prior to attachment to a
dendrimeric central core. Accordingly, exposure of chelidamic
acid to phosphorus oxychloride at 128 °C followed by reaction
of the crude acid chloride with tert-butyl alcohol provides tert-
butyl-4-chloropyridine-2,6-dicarboxylate 4a in 68% yield (Scheme
2). The use of tert-butyl esters was necessary to avoid
intramolecular cleavage of the benzamido linkage by the
carbamate linkage that occurred when the analogous propyl
dicarboxylate of 5-ortho was exposed to basic hydrolysis
conditions. Extension of the focal function was achieved by
sodium azide displacement followed by hydrogenation affording
amine 4c. Reaction of 4c with either 2-nitro- or 4-nitrobenzoyl
chloride followed by hydrogenation, acylation with allyl chlo-
roformate, and deprotection of the tert-butyl esters with CF3-
CO2H/CH2Cl2 (1:1) afforded, after conversion to the corre-
sponding acid chlorides with oxalyl chloride, branching units
5-ortho and 5-para.
Dendron Synthesis
Construction of the focal point derivatized dendrons pro-
ceeded in a convergent manner by treating 5-ortho with 2 equiv
of amine 6,25b thereby providing first-generation dendron 7a in
79% yield (Scheme 3). Deprotection of the N-allyloxylcarbonyl
group of the anthranilamide connector using a catalyst prepared
from 2.5 mol % Pd2(dba)3-CHCl3/25 mol % PPh3 and
tributylammonium formate as a nucleophile provided the free
amine (7b, 46%) along with equal amounts of the corresponding
N-allyl derivative (46%), which was difficult to separate
chromatographically. This side product was formed by nucleo-
philic attack on the palladium π-allyl complex by the amino
group of the product, generated by the deprotection, and
occurred in competition with capture by ammonium formate.
Employing dimedone as a more nucleophilic acceptor29 provided
the free amine in 90% yield; however, this material contained
approximately 10% of the N-allyl compound as an inseparable
impurity. When N,N′-dimethylbarbituric acid was utilized as
allyl acceptor,30 deprotection reproducibly provided 98% yield
of amine 7b after 30 min at ambient temperature without
contamination by the N-allyl byproduct. Subsequent reaction
of amine 7b with 5-ortho followed by deprotection with the
Pd(0) catalyst afforded second-generation dendron 9b in good
yield and high purity. Repetition of the generational growth and
deprotection steps afforded third-generation dendron 11b.
(28) We conclude that the decreased hydrogenation rate is due to catalyst
poisoning rather than steric encumbrance for two reasons. (1) Hydrogenation
is relatively slow even for first-generation dendron 1b. (2) These dendrons
are known to efficiently coordinate to transition metals, see: Rauckhorst,
M. R.; Wilson, P. J.; Hatcher, S. A.; Hadad, C. M.; Parquette, J. R.
Tetrahedron 2003, 59, 3917.
(29) Kunz, H.; Unverzagt, C. Angew. Chem. 1984, 96, 426.
(30) Kunz, H.; Maerz, J. Angew. Chem. 1988, 100, 1424.
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Huang et al.
Scheme 3. Second-Generation Dendrimer Synthesisa
a (a) (i) 5-ortho and 6, 7b, 8b, 9b, or 10b, CH2Cl2/pyr., DMAP (cat.), (b) (i) 5-para and 6, 7b, 8b, 9b, or 10b, CH2Cl2/pyr., DMAP (cat.), (c) Pd2(dba)3‚CHCl3/
P(C6H5)3, N,N-dimethylbarbituric acid, THF, (d) 1,3,5-benzenetricarbonyl trichloride, CH2Cl2/pyr., DMAP (cat.).
Dendrimer Synthesis
been used extensively as fluorescent probes because of the high
quantum yield of fluorescence, thereby enabling the dendrimers
to be studied by fluorescence depolarization.31 Accordingly,
upon excitation at 300 nm, all of the dendrons and dendrimers
exhibited a broad emission band in the range of 350-590 nm
with apparent maxima at approximately 425 and 460 nm.
Significantly lower fluorescence quantum yields were observed
for the dendrons/dendrimers relative to simple 2-acylaminoben-
zamides. The lower quantum yields may be a consequence of
the deviation of the amides in the dendritic structures from
periplanarity inducing nonradiative processes to contribute to
the deactivation of the excited state.31b At higher generations,
the intensity of the emission band at 425 nm increased relative
to that of the 460 nm band. This behavior may have been due
to a variation in the absorption characteristics of the dendrimers
that would result in different chromophores being excited at
300 nm as a function of dendrimer structure and generation.
To examine this possibility, the relative emission intensity at
425 and 460 nm was monitored as the excitation wavelength
was scanned from 250 to 400 nm for dendron 3 and dendrimer
7c. However, the relative intensity of the two emission bands
did not vary over that excitation range, thus ruling out this
possibility. The fluorescence spectra similarly did not depend
on concentration (within the range of 10-6-10-8 M) or solvent
quality (THF, CH2Cl2, 10-50% EtOH/CH2Cl2, MeCN, 10-
50% MeCN/EtOH), indicating that intermolecular aggregation
or solvent-induced structural collapse was not responsible (see
Table 1 in Supporting Information and Figure 6 for the effect
of solvent on compaction). Lifetime data were then collected
for 3 and 7c at 10-20 nm increments across the fluorescence
Dendrimers were prepared by linking the dendrons to 1,3,5-
benzene tricarbonyl chloride through either a 2-aminobenzamide
or a 4-aminobenzamide connecting unit to create isomeric
dendrimers differing only in their hydrodynamic volumes. The
appropriate connector was introduced by reacting with either
5-ortho or 5-para in the generational growth step just preceding
reaction with the core to form a particular dendrimer generation.
For example, dendrimers containing a 4-aminobenzamide link-
age to the central core were prepared by reacting amines 6, 7b,
or 9b with 5-para, affording dendrons having a 4-aminoben-
zamide group (para-linked) at the focal point (8b, 10b, and 12b)
following deprotection (Scheme 3). These para-linked dendrons
were subsequently reacted with 1,3,5-benzenetricarbonyl chlo-
ride, giving the first (p-link[G1]-dend, 8c)-, second (p-link[G2]-
dend, 10c)-, or third (p-link[G3]-dend, 12c)-generation den-
drimers (Figure 4). Similarly, to prepare the ortho-linked
dendrimers, amines 6, 7b, or 9b were treated with 5-ortho,
affording, after deprotection, the corresponding ortho-linked
dendrons 7b, 9b, and 11b, respectively. Reaction with 1,3,5-
benzenetricarbonyl chloride in pyridine-CH2Cl2 afforded the
first (o-link[G1]-dend, 7c)-, second (o-link[G2]-dend, 9c)- or
third (o-link[G3]-dend, 11c)-generation dendrimers (Figure 3).
All dendrons and dendrimers displayed 1H and 13C NMR
spectra, along with mass spectra and elemental analyses,
consistent with the expected structures.
Steady-State Absorption and Emission Properties
Ultraviolet-visible absorption (UV-vis) and fluorescence
spectra of dendrons 1a-2a, 3, and dendrimers 7c-12c are
shown in Figure 5. The transition in the UV-vis spectrum that
occurs at 316 nm corresponds to a π f π* excitation of the
2-acylaminobenzamide chromophores present throughout the
dendrimer structures.25 2-Aminobenzamide chromophores have
(31) (a) Ferro, V.; Weiler, L.; Withers, S. G. Carbohydr. Res. 1998, 306, 531.
(b) Ito, A. S.; Turchiello, R. d. F.; Hirata, I. Y.; Cezari, M. H. S.; Meldal,
M.; Juliano, L. Biospectroscopy 1998, 4, 395. (c) Turchiello, R. F.; Lamy-
Freund, M. T.; Hirata, I. Y.; Juliano, L.; Ito, A. S. Biophys. Chem. 1998,
73, 217.
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Effect of Global Compaction on Folded Dendrimers
A R T I C L E S
Figure 3. Structures of “compact” dendrimers constructed with an ortho-aminobenzamide linkage.
region (380-500 nm), and all of the spectra yielded four distinct
lifetimes upon fitting the data with discrete exponentials (Figure
1, Supporting Information).32 These lifetime values, both relative
population and lifetime, only varied slightly across the spectral
region, indicating that the two apparent maxima in the fluores-
cence were not a consequence of unique intramolecular excited-
9
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A R T I C L E S
Huang et al.
Figure 4. Structure of “expanded” dendrimers constructed from a para-aminobenzamide linkage.
state dimers (excimers or exciplexes) or of the localized emission
of multiple, different chromophores.33 Rather, the appearance
and variation in the spectra likely reflect conformational
differences and the emission of spatially delocalized excitons.34
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14524 J. AM. CHEM. SOC. VOL. 125, NO. 47, 2003

Effect of Global Compaction on Folded Dendrimers
A R T I C L E S
Figure 5. Absorption and emission spectra of dendrons 1a-2a, 3, and dendrimers 7c-12c. Spectra normalized for comparison. Quantum yields:35 1a
(0.004), 2a (0.002), 3 (0.004), 8c (0.011), 10c (0.009), 12c (0.011), 7c (0.007), 9c (0.011), 11c (0.011).
Figure 6. Packing efficiencies (Vh/Vvw) of dendrimers 7c-10c as a function of solvent.
Time-Resolved Fluorescence Anisotropy Studies
to determine the molecular dimensions of the dendrimers in
solvents of varying quality at micromolar concentrations that
minimized aggregation and were comparable to the concentra-
tions required for the circular dichroism measurements. The high
sensitivity of fluorescence measurements ideally suited time-
resolved fluorescence anisotropy (TRFA)39 as a method to
estimate the degree of compaction imparted to the dendrimer
structure as a function of solvent, the nature of the connector,
and dendrimer generation at low concentrations. Accordingly,
the hydrodynamic properties of the dendrimers were measured
by TRFA measurements40 using the time-correlated single-
photon counting (TCSPC) method41 as a function of the
dendrimer series, generation level, and solvent. Fluorescence
anisotropy decays showed a biexponential profile consisting of
a fast (10-30 ps) and a slow depolarization component (190-
2000 ps). The fast decay component likely represents an
intramolecular energy transfer depolarization process,38 consis-
tent with the presence of a delocalized excited state, whereas
the slow component corresponds to the global rotation of the
molecule. The hydrodynamic volumes (Vh) were calculated from
these values of the rotational correlation times (Θ2) via the
The molecular dimensions of dendrimers have been previ-
ously estimated using intrinsic viscosity measurements36 or by
diffusion-ordered nuclear magnetic resonance.37 However, these
techniques generally require relatively high dendrimer concen-
trations and, therefore, must be performed in good solvents. To
measure the secondary structural propensities of the dendrimers
by circular dichroism as a function of hydrodynamic volume, a
property greatly affected by solvent quality,38 it was essential
(32) Demas, J. N. Excited-State Lifetime Measurements; Academic Press: New
York, 1983.
(33) (a) Maus, M.; Mitra, S.; Lor, M.; Hofkens, J.; Weil, T.; Herrmann, A.;
Muellen, K.; De Schryver, F. C. J. Phys. Chem. A 2001, 105, 3961. (b)
Maus, M.; De, R.; Lor, M.; Weil, T.; Mitra, S.; Wiesler, U.-M.; Herrmann,
A.; Hofkens, J.; Vosch, T.; Muellen, K.; De Schryver, F. C. J. Am. Chem.
Soc. 2001, 123, 7668. (c) Schweitzer, G.; Gronheid, R.; Jordens, S.; Lor,
M.; De Belder, G.; Weil, T.; Reuther, E.; Muellen, K.; De Schryver, F. C.
J. Phys. Chem. A 2003, 107, 3199.
(34) (a) Shortreed, M. R.; Swallen, S. F.; Shi, Z.-Y.; Tan, W.; Xu, Z.; Devadoss,
C.; Moore, J. S.; Kopelman, R. J. Phys. Chem. B 1997, 101, 6318. (b)
Swallen, S. F.; Kopelman, R.; Moore, J. S.; Devadoss, C. J. Mol. Struct.
1999, 485-486, 585. (c) Swallen, S. F.; Shi, Z.-Y.; Tan, W.; Xu, Z.; Moore,
J. S.; Kopelman, R. J. Lumin. 1998, 76-77, 193. (d) Swallen, S. F.; Zhu,
Z.; Moore, J. S.; Kopelman, R. J. Phys. Chem. B 2000, 104, 3988.
(35) Olmsted, J. J. Phys. Chem. 1979, 83, 2581.
(36) Mourey, T. H.; Turner, S. R.; Rubinstein, M.; Frechet, J. M. J.; Hawker,
C. J.; Wooley, K. L. Macromolecules 1992, 25, 2401.
(37) Young, J. K.; Baker, G. R.; Newkome, G. R.; Morris, K. F.; Johnson, C.
S. J. Macromolecules 1994, 27, 3464.
(38) (a) De Backer, S.; Prinzie, Y.; Verheijen, W.; Smet, M.; Desmedt, K.;
Dehaen, W.; De Schryver, F. C. J. Phys. Chem. A 1998, 102, 5451. (b)
Hofkens, J.; Latterini, L.; De Belder, G.; Gensch, T.; Maus, M.; Vosch,
T.; Karni, Y.; Schweitzer, G.; De Schryver, F. C.; Hermann, A.; Mullen,
K. Chem. Phys. Lett. 1999, 304, 1. (c) Tande, B. M.; Wagner, N. J.; Mackay,
M. E.; Hawker, C. J.; Jeong, M. Macromolecules 2001, 34, 8580.
(39) For examples of time-resolved fluorescence depolarization studies on
dendrimers, see refs 38a,b and: Matos, M. S.; Hofkens, J.; Verheijen, W.;
De Schryver, F. C.; Hecht, S.; Pollak, K. W.; Frechet, J. M. J.; Forier, B.;
Dehaen, W. Macromolecules 2000, 33, 2967.
(40) Fleming, G. R. Chemical Applications of Ultrafast Spectroscopy; Oxford
University Press: New York, 1986.
(41) Buterbaugh, J. S.; Toscano, J. P.; Weaver, W. L.; Gord, J. R.; Hadad, C.
M.; Gustafson, T. L.; Platz, M. S. J. Am. Chem. Soc. 1997, 119, 3580.
9
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A R T I C L E S
Huang et al.
Debye-Stokes-Einstein relation (DSE):
VhηCf
Θ )
kT
where η is the viscosity of the solvent, k is the Boltzmann
constant, and T is the absolute temperature. The shape factor f
corrects for the shape of the rotating molecule if the molecule
is not spherical (f ) 1 for spherical molecules).40 C is a measure
of the coupling of the rotating solute and solvent molecules and
has a value of 1 when the solute is much larger than the
solvent.42 Because the lowest molecular weight dendrons in this
study are at least 10-100 times larger than the volume of the
solvent and the rotating rotors are approximately spherical, a
value of 1 is used for f and C (see Table 1 in Supporting
Information for rotational correlation times (φ), hydrodynamic
volume (Vh), and free volume (Vfree) for dendrons and dendrim-
ers as a function of solvent).
Comparing the measured hydrodynamic volume (Vh) to the
calculated van der Waals volume (Vvw), calculated from Edwards
increments43 (Vh/Vvw), provides an estimate of the packing
efficiency wherein a lower ratio indicates tighter packing
(proteins typically have Vh/Vvw ratios of 1.2-1.444). Inspection
of the Vh/Vvw ratios listed in Table 1 (Supporting Information)
and Figure 6 revealed several noteworthy trends: (1) All ortho-
linked dendrimers exhibited significantly higher packing ef-
ficiencies (lower Vh/Vvw), ranging from 0.81 to 1.68, than their
para-linked counterparts, which maintained Vh/Vvw ratios ranging
from 1.55 to 6.3. (2) The hydrodynamic volumes of the para-
linked dendrimers displayed higher solvent sensitivity than those
of the corresponding ortho-linked dendrimers. This increased
solvent sensitivity evolved from the relatively expanded con-
formations of the para-linked dendrimer, which support greater
free volumes (Vh - Vvw) that collapse in poor solvents. (3) In
both series, going from the first to second generation resulted
in a more tightly packed conformation. (4) Generally, solvents
expanded the dendrimer structures in the order THF > CH2Cl2
≈ MeCN. Interestingly, whereas the addition of 50% ethanol
to CH2Cl2 or MeCN dramatically collapsed the structures, the
addition of 10% ethanol to CH2Cl2 expanded the dendrimer
structures.
Figure 7. Helical interconversion of anthranilamide chromophores.
containing C3 and C6 as shown in Figure 7. Further, using the
direction of the electric transition dipole moment, the excitonic
couplet occurring at 316 nm in the CD spectra could be
correlated with the helical relationship (M or P) between two
adjacent anthranilamide chromophores (Figure 7). Thus, the bias
of the dynamic equilibrium interconverting two diastereomeric
helical conformations (M and P helices) relating a pair of
anthranilamide termini could be determined.
First-Generation Dendrimers
Comparison of the volumes determined by TRFA of o-link-
[G1]-dend 7c and p-link-[G1]-dend 8c revealed three charac-
teristic trends (Table 1 (Supporting Information), Figure 6). (1)
o-Link-[G1]-dend 7c maintained a significantly more compact
global structure than did p-link-[G1]-dend 8c in all solvents (e.g.,
Vh/Vvw(MeCN) ) 1.68 (7c); 4.86 (8c)). (2) The hydrodynamic
volume (Vh) of o-link-[G1]-dend 7c exhibited very little solvent
sensitivity relative to that of p-link-[G1]-dend 8c, indicating the
presence of a highly compact structure not having significant
free volume (Vfree). (3) In contrast, p-link-[G1]-dend 8c exhibited
a maximum Vh in THF, a minimum Vh in 50% ethanol/MeCN,
and expanded slightly upon the addition of 10% ethanol to a
CH2Cl2 solution of the dendrimer. Interestingly, although
dendrons 7b and 8b exhibited simple Cotton effects at 316 nm,
indicating an unbiased helical equilibrium, dendrimers 7c and
8c exhibited strong, negative excitonic couplets centered at 316
nm in MeCN at -20 °C (Figure 8). The negative chirality of
the couplet is consistent with a shift in the conformational
equilibrium toward the M helical conformation. This excitonic
couplet was relatively insensitive to temperature up to 60 °C in
THF and MeCN for 7c, whereas the couplet for 8c was highly
sensitive to increasing temperature in MeCN and the couplet
was extremely weak at any temperature in THF. This chiroptical
behavior correlates well with the packing efficiency displayed
by the dendrimers. For example, the Vh/Vvw ratio was similarly
low for 7c in THF and MeCN, indicating that the packing
efficiency was an intrinsic property of the ortho-linked den-
drimer structure and relatively insensitive to solvent quality. In
contrast, 8c exhibited an expanded conformation in MeCN
(Vh/Vvw ) 4.86) that expanded further in THF (Vh/Vvw ) 6.3).
Accordingly, the conformational behavior of 7c was similarly
temperature-insensitive in THF and MeCN, whereas 8c did not
show a helical bias in THF and exhibited a highly temperature-
dependent helical bias in MeCN. The correlation of low values
of Vh/Vvw with an increase in the level and stability of chiral
secondary structure suggests that compacting the dendrimer
structure is an important factor in reinforcing the secondary
structural propensity of the dendrimers.
Conformational Studies
The TRFA studies established that linking the dendrons to
the central core through a 2-aminobenzamide (ortho-linked)
produces a more efficiently packed structure than linking
through a 4-aminobenzamide (para-linked). Accordingly, cir-
cular dichroism studies could be used to establish the effect of
the differential compaction levels, under a specific set of solvent
and temperature conditions, on the degree and stability of chiral
secondary structure expressed by the dendrons linked through
the central core. During previous conformational studies on
dendrons 1a-3,25b,d we found that the transition that occurs in
the CD spectra in the region of 300-340 nm is exclusively
due to a π f π* transition of the anthranilamide chromophores.
Time-dependent density functional calculations determined that
this transition, centered at 316 nm, is polarized along the axis
(42) Hartman, R. S.; Konitsky, W. M.; Waldeck, D. H.; Chang, Y. J.; Castner,
E. W. J. J. Chem. Phys. 1997, 106, 7920.
(43) Edward, J. T. J. Chem. Educ. 1970, 47, 261.
(44) Chalikian, T. V.; Totrov, M.; Abagyan, R.; Breslauer, K. J. J. Mol. Biol.
1996, 260, 588.
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Effect of Global Compaction on Folded Dendrimers
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Figure 8. Circular dichroism spectra (normalized for concentration and number of terminal groups) of first-generation dendron and dendrimers as a function
of solvent, temperature, and linkage: (A) o-link-[G1]-dend 7c and dendron 7b in MeCN as function of temperature. (B) o-link-[G1]-dend 7c as a function
of solvent at -20 °C. (C) p-link[G1]-dend 8c as a function of temperature. (D) p-link[G1]-dend 8c as a function of solvent at -20 °C.
The addition of 50% ethanol (a poor dendrimer solvent) to
MeCN resulted in the most compact structures for both 7c (Vh/
Vvw ) 1.17) and 8c (Vh/Vvw ) 1.73). Unexpectedly, in this
solvent system, neither dendrimer exhibited a significant helical
bias. We have previously observed that the addition of 10%
ethanol reduces or destroys the helical bias of chiral dendrons.25b
This observation was attributed to the partial disruption of the
intramolecular hydrogen-bonding interactions in the presence
of small amounts of ethanol. Therefore, it is noteworthy that
both 7c and 8c slightly expand when 10% ethanol is added to
CH2Cl2 solutions of the dendrimers whereas at 50% ethanol, a
dramatic structural collapse occurs. Consequently, this behavior
can be attributed to a partial disruption of the intramolecular
hydrogen-bonding interactions at low concentrations of ethanol
that reduces the conformational order in a manner that slightly
increases the hydrodynamic volume of the dendrimers. Because
ethanol is a poor solvent for the dendrimers (i.e., the dendrimers
are insoluble in pure ethanol), at higher concentrations the
partially denatured structure experiences a nonspecific collapse
resulting in a compact structure lacking a helical bias. Therefore,
we conclude, in this case, that an increase in secondary structural
order will occur only when the dendrimer collapses in a manner
that results in specific nonlocal interactions between elements
of local secondary structure in contrast to a nonspecific structural
collapse.
dendron and to the greater overall packing efficiency of the
folded state. The highly compact structure of 2a was confirmed
by TRFA studies that revealed Vh/Vvw ratios ranging from 1.06
in MeCN to 2.0 in THF. Moreover, o-link-[G2]-dend 9c
maintained an even more compact morphology than 2a in all
of the solvents studied. Accordingly, CD studies for o-link-
[G2]-dend 9c indicated a greater bias for the M helical
conformation, relative to dendron 2a, as evidenced by the
increased magnitude of the excitonic couplet centered at 316
nm. Surprisingly, the CD spectra of 9c, in contrast to the isolated
dendron 2a, were extremely solvent and temperature dependent
(Figure 9). For example, the couplet was present in MeCN;
however, in THF or 50% ethanol/CH2Cl2, a significantly
decreased couplet intensity was observed. These solvent effects
correlate well with the associated solvent-induced changes in
the Vh/Vvw ratios for THF and MeCN. For example, o-link-[G2]-
dend 9c exhibited a larger volume in THF (Vh/Vvw ) 1.5) than
in MeCN (Vh/Vvw ) 0.94). However, similar to p-link-[G1]-
dend 8c, 9c expanded slightly upon the addition of 10% ethanol
to CH2Cl2 and dramatically collapsed in 50% ethanol/CH2Cl2,
exhibiting no helical bias in the compact state. Additionally,
heating the sample to 60 °C in MeCN resulted in a lack of any
helical bias, in contrast to the thermally stable bias present in
dendron 2a. Similar to the first-generation dendrimers (7c and
8c), o-link-[G2]-dend 9c exhibited significantly lower Vh/Vvw
ratios than the isomeric para-linked dendrimer (p-link-[G2]-
dend, 10c). The hydrodynamic volumes of both dendrimers
exhibited a similar solvent sensitivity profile (Figure 6). This
behavior is in qualitative agreement with dendrimers 7c and
8c; however, o-link-[G2]-dend 9c and p-link-[G2]-dend 10c
were more compact than their first-generation counterparts, 7c
and 8c, and exhibited qualitatively similar, but greater overall,
sensitivity to solvent. p-Link-[G2]-dend 10c also exhibited a
Second-Generation Dendrimers
We previously reported that the second-generation dendron
2a exhibited a highly thermally stable (up to 110 °C) and
solvent-insensitive helical conformational order as evidenced
by circular dichroism and NOESY NMR spectroscopy.25d This
unusual thermal stability was attributed to the synchronization
of internal and peripheral conformational equilibria within the
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A R T I C L E S
Huang et al.
Figure 9. Circular dichroism spectra (normalized for concentration and number of terminal groups) of second-generation dendron and dendrimers as a
function of solvent, temperature, and linkage: (A) o-link-[G2]-dend 9c and dendron 2a in MeCN as function of temperature. (B) o-link-[G2]-dend 9c as a
function of solvent at -20 °C. (C) p-link[G2]-dend 10c as a function of temperature. (D) p-link[G2]-dend 10c as a function of solvent at -20 °C.
Discussion
couplet in the CD spectra that was maximal in MeCN and
minimal in THF, similar to o-link-[G2]-dend 9c; however, the
intensity was greatly reduced relative to 9c.
The degree of structural compaction correlates closely with
the extent and stability of secondary structural bias when
comparing the ortho-linked dendrimers (7c, 9c, 11c), having
highly compact structures, to the para-linked counterparts (8c,
10c, 12c) exhibiting relatively expanded conformations. This
correlation is most apparent for the first-generation dendrimers
o-link-[G1]-dend 7c and p-link-[G1]-dend 8c. For example,
although the parent dendron, 1a, exhibits no helical bias,
assembling the dendrons into o-link-[G1]-dend 7c produces a
highly compact structure with a strong M helical bias that is
thermally stable and insensitive to solvent quality. In contrast,
the expanded structure of para-linked isomer 8c displays a
relatively unstable secondary structure. Moreover, the helical
bias is lost going from MeCN to the more expanded conforma-
tion in THF. This correlation also holds when comparing the
o-link-[G2]-dend 9c and p-link-[G2]-dend 10c. Accordingly,
o-link-[G2]-dend 9c, which is significantly more compact than
p-link-[G2]-dend 10c, exhibits far greater secondary structure
than 10c, and both 9c and 10c lose secondary order in THF,
which expands the dendrimers relative to MeCN. However, there
are several observations that do not directly correlate the
secondary order with the extent of dendrimer compaction. For
example, although the dendrimers collapse to their most compact
forms in 50% ethanol/MeCN, a minimal helical bias is observed
for all dendrimers in this solvent system. Similarly, with the
exception of the first-generation dendrimers, 7c and 8c, the
dendrons exhibit a greater and more stable secondary structural
bias than the corresponding dendrimers, which are uniformly
more compact than the associated dendrons. Thus, second-
generation dendron 2a maintains a high helical bias even at 110
Third-Generation Dendrimers
Unfortunately, at the third generation, determination of the
hydrodynamic volumes was not possible because the fluores-
cence depolarization curve was dominated by the fast component
of the process, which became increasingly dominant going from
the first- to the third-generation dendrimers. Nevertheless, it is
likely that the trend of increasing compactness going from the
first to second generation continues upon progressing to the
third-generation dendrimers. Surprisingly, in MeCN, the CD
spectra of the o-link-[G3]-dend, 11c, exhibited a positive
excitonic couplet, indicating that a shift in equilibrium toward
the P helical conformation occurred, in contrast to the M-helical
bias present in all of the other dendrons and dendrimers (Figure
10). Interestingly, increasing the temperature or changing solvent
to THF or 50% ethanol/MeCN reverts the couplet to negative
chirality, indicating a return to an M helical bias, similar in
magnitude to the third-generation dendron 3. Although this
unique conformational preference cannot be fully rationalized,
it is consistent with the general trend observed beginning at
the second generation for the nonlocal interactions to oppose
the secondary structural preference of the isolated dendrons. In
conrast, p-link-[G3]-dend 12c exhibits an M helical bias only
slightly lower in magnitude to dendron 3. Changing solvent to
THF or 50% EtOH/MeCN decreases the amplitude of the
couplet similar to the effect of these solvents on the first- and
second-generation dendrimers of both series.
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Effect of Global Compaction on Folded Dendrimers
A R T I C L E S
Figure 10. Circular dichroism spectra (normalized for concentration and number of terminal groups) of second-generation dendron and dendrimers as a
function of solvent, temperature, and linkage: (A) o-link-[G3]-dend 11c and dendron 3 in MeCN as function of temperature. (B) o-link-[G3]-dend 11c as
a function of solvent at -20 °C. (C) p-link[G3]-dend 12c as a function of temperature. (D) p-link[G3]-dend 12c as a function of solvent at -20 °C.
°C, whereas the corresponding dendrimer 9c experiences a
complete loss of bias at 60 °C and the para-linked isomer 10c
shows little bias at all temperatures. Finally, in contrast to the
M helical preference of all of the other dendrons and dendrimers,
ortho-linked third-generation dendrimer 11c switches to a P
helical preference in MeCN at low temperature, reverting to an
M bias at higher temperatures or in a structure-expanding solvent
such as THF. These apparent anomalies indicate that, although
compaction can enhance secondary structural propensities, the
nature of compaction is more important than the extent of
compaction in determining the amount and stability of local
secondary structure. Specifically, these anomalies suggest that
local secondary structure in the dendrimers is enhanced when
compaction packs the subunits in a mutually complementary
fashion that synchronizes conformational equilibria. Alterna-
tively, the nonspecfic collapse of a structure may induce a
perturbation of local secondary order in a manner that maximizes
packing but opposes the intrinsic local order, resulting in a
decrease or a change in helical bias. For example, the loss of
secondary structure upon the addition of ethanol to the solvent
can be understood by the observation that adding a small amount
(10%) expands the structures. The protic nature of ethanol
partially disrupts the intramolecular interactions that stabilize
the local helical conformation, leading to a disordering of the
local helical conformation and an expansion of the structure,
whereas at higher concentrations (50%), ethanol behaves as a
poor dendrimer solvent, inducing a nonspecific collapse of this
disordered structure. Similarly, model-building studies suggest
that the 2-aminobenzamide linkage present in o-link-[G1]-dend
7c arranges the dendrons around the central core in a propeller-
like arrangement that packs mutually complementary faces of
the dendrons together. This complementary packing synchro-
nizes conformational equilibria in the dendrons in a manner that
amplifies the small M helical bias of the parent dendron. Linking
these dendrons through a 4-aminobenzamide function orients
the dendrons in a radially expanded conformation, causing the
dendrons to potentially interact via several different orientations.
This decreases the complementarity and efficiency of packing,
resulting in a lowered helical bias. Why is the helical bias less
pronounced at higher generations even though compaction has
increased? We hypothesize that the nonspecific, intramolecular
interactions between adjacent dendrons increase at higher
generations as a consequence of the steric congestion that
normally develops among the branches and at the surface of
dendrimers. In o-link-[G2]-dend 9c, these nonspecific interac-
tions compete with the tendency of the 2-aminobenzamide
linkage to pack the dendrons in a complementary fashion,
resulting in a thermally unstable helical bias, relative to dendron
2a. The 4-aminobenzamide linkage reduces the packing comple-
mentarity further so that the nonspecific interactions dominate,
leading to a loss of secondary order. These nonspecific effects
are enhanced at o-link-[G3]-dend 11c to the extent that a shift
in the local helical equilibria toward a P helical bias occurs.
Decreasing the nonspecific packing with the 4-aminobenzamide
linkage shifts the equilibria back to an M bias, albeit slightly
lower in extent than parent dendron 3. It is noteworthy that a
shift from a P to M bias also occurs in o-link-[G3]-dend 11c
upon going from MeCN to THF, which expands the structures
of all of the other dendrimers, and is consistent with the
hypothesis that nonspecific packing of the dendrimer structure
is responsible for the shift to a P helical bias.
Conclusion
In contrast to dendrimers constructed from flexible subunits,
relatively minor modifications in the structure of folded den-
drimers, composed of dendrons exhibiting rigid, folded con-
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A R T I C L E S
Huang et al.
formations, afford large variations in hydrodynamic volumes.
Further, the concomitant changes in overall compaction greatly
affect the intrinsic local secondary structural preferences of the
constituent dendrons. It is noteworthy that the most rapidly
folding biopolymers in nature exhibit a near perfect balance
between local and nonlocal interactions, producing intermediates
along the folding pathway that maintain an overall topology
similar to that of the native state to minimize the creation of
nonnative interactions that inhibit the folding process.45 Similar
to protein folding, dendrimer compaction must occur in a
manner that preserves the topology of the folded dendron
structure. A reinforcement of the local conformational prefer-
ences of the dendrons occurs when compaction packs the
dendrons in a mutually complementary fashion. Packing mutu-
ally complementary surfaces together reinforces secondary
structural preferences, resulting in greater or more stable
secondary structure with increasing compaction. In contrast,
nonspecific compaction increases the nonlocal interactions
between adjacent dendrons but not necessarily in a manner that
preserves the dendron topology and may oppose local secondary
preferences.
Acknowledgment. This work was supported by the National
Science Foundation (CHE-0239871).
Supporting Information Available: Experimental procedures,
complete analytical data for compounds 1-12, lifetime data for
3 and 7c, a table of rotational correlation times (φ), hydrody-
namic volumes (Vh) and free volume (Vfree) for dendrons and
dendrimers as a function of solvent; experimental setup details
for TCSPC and fluorescence studies; and copies of the 1H NMR
and mass spectral data for dendrimers 7c-12c (PDF). This
material is available free of charge via the Internet at
http://pubs.acs.org.
(45) Millet, I. S.; Townsley, L. E.; Chiti, F.; Doniach, S.; Plaxco, K. W.
Biochemistry 2002, 41, 321.
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