Quantifying the effect of surface ligands on dendron-DNA interactions: Insights into multivalency through a combined experimental and theoretical approach

Article abstract of DOI:10.1002/chem.200902546

We report the synthesis, DNA binding ability and preliminary gene delivery profiles of dendrons with different amine surface groups, 1,3-diaminopropane (DAP), N,N-di-(3-aminopropyl)-N-(methyl)amine (DAPMA) and spermine (SPM). By using a combination of ethidium bromide displacement, gel electrophoresis and transfection assays, it is shown that the dendrons with SPM groups are the most effective DNA binders, while the DAPMA-functionalised dendrons were the most effective systems for gene delivery (although the gene delivery profiles were still modest). In order to provide deeper insight into the experimental data, we performed a molecular dynamics simulation of the interactions between the dendrons and DNA. The results of these simulations demonstrated that, in general terms, the enthalpic contribution to binding was roughly proportional to the dendron surface charge, but that dendrons with DAP (and DAPMA) surface amines had significant entropie costs of binding to DNA. In the case of DAP, this is a consequence of the fact that the entire dendron structure has to be organised in order for each individual monoamine charge to make effective contact with DNA. For SPM, however, each surface ligand is already a multivalent triamine, therefore, each individual charge has a much lower entropie cost of binding. For DAPMA, we observed that strong binding of the hindered tertiary amine to the DNA double helix led to ligand back-folding and significant geometric distortion of DNA. Although this weakens the overall binding, we suggest that this distortion might be an explanation for the experimentally observed enhanced gene delivery, in which DNA compaction is an important step. Overall, this paper demonstrates how structure-activity relationships can be developed for multivalent dendritic ligands and provides insights into the thermodynamics of multivalent interactions.

Full text of DOI:10.1002/chem.200902546

FULL PAPER
DOI: 10.1002/chem.200902546
Quantifying the Effect of Surface Ligands on Dendron–DNA Interactions:
Insights into Multivalency through a Combined Experimental and
Theoretical Approach
Simon P. Jones,[a] Giovanni M. Pavan,*[b, c] Andrea Danani,[b] Sabrina Pricl,[c] and
David K. Smith*[a]
Abstract: We report the synthesis,
DNA binding ability and preliminary
gene delivery profiles of dendrons with
different amine surface groups, 1,3-dia-
minopropane (DAP), N,N-di-(3-amino-
propyl)-N-(methyl)amine (DAPMA)
and spermine (SPM). By using a com-
bination of ethidium bromide displace-
ment, gel electrophoresis and transfec-
tion assays, it is shown that the den-
drons with SPM groups are the most
effective DNA binders, while the
DAPMA-functionalised dendrons were
the most effective systems for gene de-
livery (although the gene delivery pro-
files were still modest). In order to pro-
vide deeper insight into the experimen-
tal data, we performed a molecular dy-
namics simulation of the interactions
between the dendrons and DNA. The
results of these simulations demonstrat-
ed that, in general terms, the enthalpic
contribution to binding was roughly
proportional to the dendron surface
charge, but that dendrons with DAP
(and DAPMA) surface amines had sig-
nificant entropic costs of binding to
DNA. In the case of DAP, this is a con-
sequence of the fact that the entire
dendron structure has to be organised
in order for each individual mono-
amine charge to make effective contact
with DNA. For SPM, however, each
surface ligand is already a multivalent
triamine, therefore, each individual
charge has a much lower entropic cost
of binding. For DAPMA, we observed
that strong binding of the hindered ter-
tiary amine to the DNA double helix
led to ligand back-folding and signifi-
cant geometric distortion of DNA. Al-
though this weakens the overall bind-
ing, we suggest that this distortion
might be an explanation for the experi-
mentally observed enhanced gene de-
livery, in which DNA compaction is an
important step. Overall, this paper
demonstrates how structure–activity re-
lationships can be developed for multi-
valent dendritic ligands and provides
insights into the thermodynamics of
multivalent interactions.
Keywords: dendrimers
gene technology
dynamics · thermodynamics
·
DNA
·
·
molecular
Introduction
[a] Dr. S. P. Jones, Prof. D. K. Smith
Department of Chemistry, University of York
Heslington, York, YO10 5DD (UK)
Fax : (+44)1904 432516
Multivalent binding of biological targets is a key principle in
enhancing binding strength and hence developing systems
with potential biomedical applications.[1] By employing mul-
tivalent ligand arrays, synthetic systems are able to enhance
their binding to targets with more than one binding site. Ex-
perimental studies and mathematical models have demon-
strated that once the first ligand in a multivalent array has
bound to the target, the binding of a second ligand will be a
cooperative, entropically less disfavoured process, with a
local concentration effect also enhancing binding.[2] Den-
drimers and dendrons are well-defined nanoscale branched
polymers that have repetitive structures and multiple surface
groups, and as such they are of great interest for their partic-
ipation in biologically-relevant multivalent recognition pro-
cesses.[3] There has been considerable interest in the ability
E-mail: dks3@york.ac.uk
[b] G. M. Pavan, Prof. A. Danani
University for Applied Sciences of Southern Switzerland (SUPSI)
Institute of Computer Integrated Manufacturing
for Sustainable Innovation
Centro Galleria 2, 6928 Manno (Switzerland)
Fax : (+41)58 666 6620
E-mail: giovanni.pavan@supsi.ch
[c] G. M. Pavan, Prof. S. Pricl
Molecular Simulations Engineering (MOSE) Laboratory
Department of Chemical Engineering (DICAMP)
University of Trieste
Piazzale Europa 1, 34127 Trieste (Italy)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200902546.
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of dendritic molecules to interact with nucleic acid targets,
such as DNA and RNA.[4] Nucleic acids have multiple po-
tential sites with which ligands can interact. In general
terms, cationic ligands interact with polyanionic DNA/RNA,
and the organisation of multiple charge–charge interactions
can give rise to high-affinity nucleic acid binding.[5] The first
studies of interactions between cationic dendrimers and
DNA were performed by the groups of Tomalia and Szoka,
who employed high generation spherical poly(amidoamine)
(PAMAM) dendrimers.[6] These polycationic, multivalent
dendrimers show high affinity for DNA and can achieve ef-
fective gene delivery into cells.[7] Computer modelling of the
interactions between these dendrimers and single strand
DNA suggested that at high dendritic generations of
growth, single strand DNA could wrap itself around the
large surface of the dendrimer.[8] Since these studies, a wide
range of polycationic multivalent dendrimers and dendrons
have been employed in DNA binding and gene delivery, in-
modify the conformation of DNA, for example, by inducing
bending and aggregation.[16] Although a tetra-amine, the in-
teraction between an individual spemine ligand and DNA is
still relatively weak especially in the competitive high salt
environment of biological systems.[17] We have reported that
dendritic arrays of spermine units can give rise to much en-
hanced ultrahigh affinity DNA binding.[14] We employed an
ethidium bromide (EthBr) displacement assay to gain a
comparative quantitative estimate of the binding strengths,
and discovered that the second generation system (G2-SPM,
Figure 1) with nine surface spermine ligands, displaced 50%
of EthBr from its complex with DNA (ca. 1 mm) at concen-
trations as low as 30 nm. This was a significantly lower con-
centration than that required for monovalent spermine
(high micromolar concentrations). Furthermore, the binding
of G2-SPM to DNA was independent of salt concentration,
and in a recent modelling study we proposed that for the
second generation system under high salt conditions, some
of the spermine surface groups effectively “sacrificed” their
interaction with DNA and acted to screen/optimise the re-
maining spermine ligands that then bound to the DNA
more tightly than would have been expected.[18] In this way,
the dendrimer exhibited a new type of multivalency effect.
We have experimentally explored the effects of dendron
structural variation on DNA binding, in order to develop
structure–activity relationships. We have grafted the dendrit-
ic spermine array onto proteins and demonstrated that the
synthetic nanoscale biohybrids exhibit high DNA affinity,[19]
cluding systems based on dendritic polyACTHNUTRGNEUNG
(l-lysine),[9] poly-
(propyleneimine),[10] and other more specialised dendritic
frameworks.[11] In general, the affinity of dendritic molecules
towards DNA binding increases as they get larger (increas-
ing dendritic generation). This reflects the fact that the pri-
mary interactions between cationic dendrimers and DNA
are electrostatic in nature. Gene delivery profiles also usual-
ly improve at higher dendritic generation, for example,
PAMAM dendrimers exhibit optimal gene delivery at about
the fifth generation of growth (G5).[12] In addition to using
simple monoamine surface
groups on dendritic structures,
there has been some interest in
how the precise structure of the
cationic surface groups modifies
DNA binding and gene deliv-
ery. For example, dendritic sur-
faces have frequently been
functionalised with cationic ar-
ginine (guanidinium) surface
groups; this is a way of signifi-
cantly enhancing gene deliv-
ery.[13]
We have recently developed
a biomimetic approach to mul-
tivalent DNA binding dendrons
and synthesised dendritic arrays
of spermine ligands.[14] Sper-
mine is a naturally occurring
tetra-amine that is used in
nature for DNA binding and
present in millimolar amounts
in eukaryotic cells.[15] Spermine
plays a key role in the compac-
tion of DNA, and there has
been considerable interest in
the mode of DNA binding, and
mechanisms through which oli-
goamines of this type can Figure 1. Chemical structures of the dendrons investigated in this study.
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Effect of Surface Ligands on Dendron–DNA Interactions
FULL PAPER
modified the structures of the dendrons such that cellular
gene delivery can be achieved,[20] and both ourselves and
Kostiainen et al. have developed degradable systems, in
which the multiple spermine ligands are cleaved from the
surface of the dendron, “switching off” the high-affinity
binding.[21] However, we were also interested in the precise
role played by the spermine ligands in binding to the DNA.
In order to probe this in more detail, we decided to synthe-
sise a family of dendrons with different surface groups
(Figure 1). The comparison between these systems and their
ability to bind and deliver DNA is the subject of this study.
We have employed a combined experimental and theoretical
(molecular dynamics modelling) approach in order to gain a
unique insight into the behaviour of these dendrons and the
role played by the surface groups in controlling their multi-
valent interactions with DNA.
scaffolds (G1 and G2) by simple amide coupling methodolo-
gies. The yields for surface functionalisation of the first gen-
eration dendron were acceptable (about 30%) with G1-
DAPACHTNUTRGENNUG(Boc) and G1-DAPMAAHCTUNGTRNE(NUGN Boc) being obtained in high
purity after column chromatography. The yield for ninefold
functionalisation of the second generation dendron with
DAPMAACTHNUGRTNENUG(Boc) to give G2-DAPMAACHTUNGTREN(NUNG Boc) was very low (ca.
5%) but this was sufficient to provide enough material for
biological studies, and we therefore did not pursue the opti-
misation of this procedure further at this point. On the
other hand, pure products could not be obtained when using
DAP
that this latter reaction might have failed due to the greater
steric hindrance of the reactive amine in DAP(Boc) caused
by the proximate Boc protecting group when compared with
the more distant Boc protecting group in DAPMA(Boc).
Consideration of the modelling studies (see below) meant
that we did not pursue the synthesis of G2-DAP(Boc) any
ACHUTGTNRNEUG(N Boc) in an attempt to yield G2-DAPAHCTUNGTRNEN(UGN Boc). We suggest
AHCTUNGTRENNUNG
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
further at this point. Deprotection of the peripheral Boc
groups by using hydrogen chloride in methanol finally yield-
ed the target amine-surfaced dendrons (G1-DAP, G1-
DAPMA and G2-DAPMA) as their hydrochloride salts in
excellent yield.
Results and Discussion
Experimental Study
Synthesis: Compounds G1-SPM and G2-SPM were synthes-
ised using our previously reported methods.[14] We then se-
lected commercially available amines that could be readily
modified using simple syntheses—diaminopropane (DAP)
and N,N-di-(3-aminopropyl)-N-(methyl)amine (DAPMA)—
for attachment to the same Newkome-type dendron scaf-
fold.[22] It is worth noting that each spermine (SPM) ligand
has three protonatable amines once it is attached to the den-
dritic scaffold through an amide linkage, while each
DAPMA group has two, and each DAP group only has one.
Both DAP and DAPMA could be readily monoprotected
with tert-butoxycarbonyl (Boc) groups on one of the two ter-
minal primary amines (Scheme 1).[23] This enabled the incor-
poration of these protected amines onto the periphery of
the previously reported first and second generation dendritic
Ethidium bromide displacement: In order to gain a compa-
rative quantification of the DNA binding abilities of these
dendrons, we employed an ethidium bromide (EthBr) dis-
placement fluorescence assay, which is commonly used to in-
vestigate the binding of polyammonium cations to calf
thymus DNA.[14,21,24] When EthBr is displaced from its com-
plex with DNA, the fluorescence intensity decreases, allow-
ing quantification of the amount of dendron required to ef-
fectively bind DNA. This is most usually expressed as the
“charge excess” of cationic dendron relative to anionic
DNA required for 50% EthBr displacement (CE50, Table 1).
The concentration of dendron required for effective DNA
binding can also be calculated (C50, Table 1). It should be
noted that EthBr displacement is a competitive binding
assay, and therefore only really
appropriate for comparing li-
gands with similar DNA bind-
ing modes: in this case, all of
the dendrons have polyamine
surfaces, and we reasoned that
comparison was valid.
The data in Table 1 indicate
that for each type of dendron,
the G2 systems are significantly
better DNA binders than their
G1 analogues—a clear multiva-
lency effect. Dendrons G1-SPM
and G2-SPM are the most ef-
fective DNA binders in terms
of the CE50 parameter, which
reflects the relative ability of
the cationic charges to bind
Scheme 1. Synthesis of DNA binding dendrons G1-DAPMA, G2-DAPMA and G1-DAP.
anionic DNA. Consideration of
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D. K. Smith, G. M. Pavan et al.
Table 1. DNA binding data extracted from ethidium bromide (EthBr)
displacement assays. Assays were performed by using EthBr (2.54 mm)
and calf thymus DNA (1.00 mm double stranded concentration; the con-
centration of each negatively charged phosphate is therefore 2.00 mm).
[c]
Dendron
CE50 value[a]
C50 value[b]
ACHTUNGTERN[NUNG Amine]50
[mm]
[mm]
G1-DAP
550[d]
32
367
10.7
0.567
0.600
0.056
1100
32
G1-DAPMA
G2-DAPMA
G1-SPM
5.1
2.7
0.76
5.1
1.8
0.50
G2-SPM
[a] CE50 represents the charge excess (N:P ratio) required to decrease
EthBr fluorescence by 50%. [b] C50 represents the concentration of den-
dron required to displace 50% of EthBr. C50 =CE50 ꢁ2.00 mm/No. of pro-
tonatable amines in the dendron. [c] [Amine]50 represents the effective
(normalised) concentration of amine surface ligand required to displace
50% of EthBr; [amine]50 =C50 ꢁNo. of surface ligands on dendron.
[d] This value was estimated by linear extrapolation of the data points.
Figure 2. Gel electrophoresis of DAPMA dendrons: a) Z-G1-DAPMA,
and b) Z-G2-DAPMA (polyamine:DNA, w/w); lane 1: 0:1; lane 2: 0.1:1;
lane 3: 0.2:1; lane 4: 0.3:1; lane 5: 0.4:1; lane 6: 0.5:1; lane 7: 0.6:1; lane 8:
0.7:1; lane 9: 0.8:1; lane 10: 0.9:1; lane 11: 1:1; lane 12: 1.5:1; lane 13: 2:1;
lane 14: 2.5:1; lane 15: 3:1.
the [amine]50 parameter again shows that spermine is the
optimal surface ligand for DNA binding, outperforming
DAPMA by an order of magnitude and DAP by three
orders of magnitude when attached to the same dendron
support. In concentration terms, the performance of G2-
DAPMA with nine surface ligands is similar to that of G1-
SPM, which only has three spermine ligands, even though
the former has 18 surface positive charges and the latter
only has nine. This clearly demonstrates that G1-SPM em-
ploys its ligands for DNA binding more effectively than G2-
DAPMA and shows that it is not just their higher charge
that gives spermine ligands their DNA binding advantage. It
is worth noting that spermine is optimised in nature for
minor groove DNA binding, whereas the synthetic amines
(DAP and DAPMA) are not.
EthBr displacement assay is a competition experiment, and
is only at its most effective when comparing directly equiva-
lent ligands. It should also be noted that gel electrophoresis
depends on effective DNA compaction, and this can differ
between the different classes of surface ligand (see model-
ling study later for further discussion).
Gene transfection: We went on to investigate the ability of
G1-DAPMA and G2-DAPMA to transfect HEK293 cells
using a standard luciferase assay, and the data were normal-
ised against branched poly(ethyleneimine) (bPEI, 20k). As
observed previously for G1-SPM and G2-SPM, these den-
drons, at least in unmodified form, were fairly ineffective in
transfection assays (Figure 3).[20] Similar to G1-SPM, den-
Gel electrophoresis: Gel electrophoresis was also used to
provide complementary insight into DNA binding
(Figure 2); the DNA used was a pGL3 plasmid instead of
calf thymus DNA in order to obtain good electrophero-
grams. We discontinued experimental work with G1-DAP as
the DNA binding was too weak to quantify in a meaningful
way, and focused on the DAPMA-functionalised dendrons,
which had shown a degree of effective DNA binding
(Figure 2). Dendron G1-DAPMA showed relatively weak
DNA binding and condensation by electrophoresis, as would
be expected from the EthBr assay. Dendron G1-DAPMA
showed some retardation of DNA migration from 0.6–1.5
(w/w) ratio (0.59 to 1.76 nmoles) and complete retardation
at higher ratios. On the other hand, G2-DAPMA retarded
DNA migration at 0.7:1 (w/w; 0.23 nmoles); this is in agree-
ment with the hypothesis that multivalent G2-DAPMA is a
more effective DNA binder than G1-DAPMA. The DNA
binding achieved by G2-DAPMA is less effective than that
previously observed by using G2-SPM (retardation at 0.5:1,
w/w, corresponding to 0.12 nmoles).[20a] This difference is
somewhat smaller than what might have been anticipated
from the EthBr assay. It should be noted, however, that the
Figure 3. Transfection data for G1-DAPMA and G2-DAPMA for
HEK293 cells by using a standard luciferase assay. For G1-DAPMA it
was necessary to add chloroquine (100 mm) to observe transfection. Data
for G2-DAPMA are chloroquine-free.
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Effect of Surface Ligands on Dendron–DNA Interactions
FULL PAPER
dron G1-DAPMA required the addition of chloroquine for
effective transfection to be observed, indicative of problems
with endosomal escape. However, like G2-SPM, G2-
DAPMA was a moderate transfection agent even in the ab-
sence of chloroquine. At low loadings, G2-DAPMA gave
similar levels of transfection to G2-SPM even though it has
fewer amine groups and is a significantly weaker DNA
binder (G2-SPM achieved a maximum transfection level of
4% compared with a standard bPEI control; data previously
published).[20] Furthermore, G2-DAPMA exhibited the best
transfection of all dendrons at relatively high loadings (20:1,
w/w). Presumably high N:P ratios are needed because the
DNA binding is weak and sufficient dendron is required to
condense the plasmid and protect it in the extracellular en-
vironment.
tential for further synthetic optimisation as low-toxicity
gene delivery agents. This observation is similar to those
made for the modification of dendritic structures with argi-
nine units;[13] that is, this modification reduces the affinity of
the dendrimers for DNA due to the more charge-diffuse
nature of the guaninidium group compared with a simple
amine, but lowers toxicity and enhances the observed gene
delivery profiles. It is also worth noting that although the
absolute levels of transfection for our structures are low, we
have previously demonstrated with our SPM-surfaced den-
drons that the incorporation of hydrophobic units at the
dendron focal point can dramatically enhance gene deliv-
ery.[20b] We therefore conclude from these experimental stud-
ies that further synthetic modification of the DAPMA-de-
rived dendrons could be worthwhile.
Dendron toxicity: We also monitored the toxicity of the
dendrons using a Cell Titer-Blue cell viability assay. Interest-
ingly, both G1-DAPMA and G2-DAPMA were significantly
less toxic than bPEI (Figure 4). Indeed, both of these den-
Modelling Study
Molecular dynamics methodology: Given the potential of
these dendrons and the interesting experimental DNA bind-
ing effects, we decided to model the effect of surface group
modification on DNA binding using molecular dynamics
(MD) methods in AMBER 9.[25] To achieve this, we em-
ployed a 21 base pair B-DNA double strand containing a
mixture of bases (Figure 5). This choice relied on a compro-
mise between accuracy and computational feasibility, and
was the method previously used for a full modelling study of
compounds G1-SPM and G2-SPM.[18] For the purposes of
modelling, we assumed that in each dendron structure, all
amine groups were protonated. The pKa values of an isolat-
ed spermidine unit are 10.90, 9.71 and 8.25.[26] Spermidine,
with three protonatable amines, separated by three- and
four-carbon spacers is a good analogue for the surface
bound spermine groups, and is, as a consequence of the pKa
values largely protonated at pH 7. Of course, the act of lo-
cating multiple spermidine-like amines on the periphery of a
dendritic scaffold will modify the pKa values making full
Figure 4. Toxicity data for G1-DAPMA and G2-DAPMA compared with
bPEI.
drons showed effectively no toxicity up to relatively high
concentrations of approximately 40 mgmLꢀ1. This is in sharp
contrast to G2-SPM, which exhibited significant toxicity at
concentrations above 20 mgmLꢀ1 (data previously pub-
lished).[20] The relatively lower toxicity of G2-DAPMA per-
haps helps explain why it was still an active transfection
agent at relatively high loadings, whereas under such condi-
tions G2-SPM is too toxic to effectively transfect cells. It is
worth noting that at low loadings, both G1-DAPMA and
G2-DAPMA cause significant cell proliferation; this was not
observed for G1-SPM or G2-SPM, and the reasons for this
are unclear.
Summary of experimental data: In combination, these re-
sults lead us to believe that even though DAPMA surface
groups appear to be significantly less effective than SPM in
terms of absolute affinity for DNA, these dendrons have po-
Figure 5. Structure of the double helical DNA used for modelling in this
study.
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D. K. Smith, G. M. Pavan et al.
protonation more difficult, however, the dendritic scaffold is
relatively flexible, and a preliminary pH titration indicated
that the majority (>90%) of the amines remain protonated
at physiological pH, even for G2-SPM.[27] The force-field pa-
rameters for these residue types were obtained by using the
AM1 semiempirical calculation methods available within
the antechamber module of AMBER 9. We constructed den-
dron models for G1-SPM, G2-SPM (previously reported),[18]
G1-DAPMA, G2-DAPMA, G1-DAP and G2-DAP (mod-
elled but not synthesised; see above). Each of these models
was simulated in a TIP3P water box,[28] with a single DNA
double helix and a single dendron unit; binding affinities
(DGbind) were determined by using molecular dynamics
methods under biologically relevant salt (150 mm) condi-
tions (see the Supporting Information for full details). The
particle mesh Ewald (PME) approach[29] was used to treat
long-range electrostatic effects, and bond lengths involving
bonds to hydrogen atoms were constrained by using the
SHAKE algorithm.[30] The DGbind values were calculated by
using the molecular mechanics Poisson–Boltzmann surface
area (MM-PBSA) method,[31] taking both polar and nonpo-
lar solvation effects into account.[32] Entropic parameters
were estimated by using the normal-mode-analysis ap-
proach.[33] The free energies of binding, as well as the en-
thalpic and entropic contributions, are reported in Table 2 as
an average across a number of snapshots obtained from the
MD trajectories.
groups are SPM (i.e., ca. ꢀ7.0 kcalmolꢀ1 per charge). Those
dendrons with simple DAP surface groups are the least ef-
fective in terms of DNA binding (only ca. ꢀ5.5 kcalmolꢀ1
per charge). Importantly, the DGbind/charge values are in
general agreement with the CE50 values reported in Table 1
and the gel electrophoresis studies; that is, G2-SPM is the
most effective binder on a per charge basis, G2-DAPMA
and G1-SPM have similar, intermediate affinities for DNA,
and the other dendrons (G1-DAPMA and G1-DAP) are sig-
nificantly less effective DNA binders.
The modelling indicates there is an enthalpy–entropy
compensation effect (Table 2). The binding is enthalpically
favourable due to electrostatic attraction, but entropically
unfavourable due to loss of degrees of freedom. However,
closer inspection of the data makes it clear that enthalpy
and entropy vary with charge in very different ways. In gen-
eral terms (with the exception of G1-DAP) each charge con-
tributes approximately ꢀ12.0 kcalmolꢀ1 to the value of
DHbind. This is a consequence of the enthalpy of binding
largely reflecting the simple electrostatics of interaction be-
tween the dendron and the DNA, therefore, the more
highly charged the cationic dendron, the stronger the en-
thalpic interaction with anionic DNA. In contrast to the en-
thalpies, the entropic values are much less predictable in the
way they vary with charge. For example, G2-DAP and G1-
SPM both have the same charge (+9) and similar DHbind
values: (ꢀ109.9 kcalmolꢀ1 vs. ꢀ106.3 kcalmolꢀ1) but the
binding of G2-DAP to DNA is entropically much more dis-
Thermodynamic insights into multivalency: Clearly, each of
the dendrons has a favourable interaction with the DNA
double helix, as indicated by the negative DGbind values. It
would be anticipated that as the charge of the dendron in-
creases, so would the strength of its interaction with DNA.
This is indeed, in general terms, the case; for example, when
comparing G2-SPM (+27) with G1-DAP (+3) it is evident
that, as expected, G2-SPM is a much more effective binder
(>tenfold increase in binding affinity). This is in agreement
with the experimental data. However, Table 2 indicates that
the relationship between charge and binding affinity is not a
straightforward one, for example, dendrons G2-DAP and
G1-SPM both have total charges of +9, but G1-SPM has a
more favourable DGbind value than G2-DAP. This indicates
that the ligand structure plays an important role in organis-
ing the individual charge–charge interactions.
favoured than the binding of G1-SPM (ꢀTDSbind
=
+60.2 kcalmolꢀ1 vs. +45.9 kcalmolꢀ1). This indicates that
more degrees of freedom are lost when G2-DAP binds
DNA. It is this entropic difference that leads to the differ-
ence in DGbind values, with G1-SPM predicted to be a much
more effective DNA binder than G2-DAP. Evidently, the
binding of the higher generation G2 system, which only has
terminal monoamine ligands (G2-DAP), to DNA is not as
efficient on entropic grounds as binding a lower generation
G1 system with multiple amines on each branch of the den-
dron (G1-SPM). This provides a key insight into multivalen-
cy, although both systems contain nine amines, they are dif-
ferently arranged. It is clearly better to have the individual
amines grouped together into three spermine units, than
spread onto the termini of nine separate flexible branches.
Anchoring the nine separate branches of a G2 dendron to
DNA, although enthalpically equivalent to binding three
spermine units in terms of charge–charge interactions, is
clearly entropically more challenging due to the required
It is informative to compare the effective binding energy
per dendron charge (DGbind/charge, Table 2). It is clear that
each charge is most effectively used when the surface
Table 2. Thermodynamic parameters determined by molecular dynamics methods for the binding of dendrons to DNA. Analysis was performed by using
150 mm aqueous NaCl as solvent medium. All data (apart from charge) are in kcalmolꢀ1
.
Dendron
Charge
DHbind
ꢀTDSbind
DGbind
DHbind/charge
ꢀTDSbind/charge
DGbind/charge
G1-DAP
G2-DAP
G1-DAPMA
G2-DAPMA
G1-SPM
+3
+9
+6
+18
+9
+27
ꢀ49.9ꢁ5.3
ꢀ109.9ꢁ13.5
ꢀ68.9ꢁ7.0
+33.2ꢁ12.1
+60.2ꢁ13.8
+35.1ꢁ9.2
ꢀ16.7
ꢀ49.6
ꢀ33.8
ꢀ126.3
ꢀ60.4
ꢀ196.2
ꢀ16.6ꢁ1.8
ꢀ12.2ꢁ1.5
ꢀ11.5ꢁ1.2
ꢀ12.6ꢁ0.5
ꢀ11.8ꢁ1.4
ꢀ11.5ꢁ0.4
+11.1ꢁ4.0
+6.7ꢁ1.5
+5.9ꢁ1.5
+5.6ꢁ0.8
+5.1ꢁ1.9
+4.2ꢁ0.5
ꢀ5.6
ꢀ5.5
ꢀ5.6
ꢀ7.0
ꢀ6.7
ꢀ7.3
ꢀ227.3ꢁ9.5
ꢀ106.3ꢁ12.3
ꢀ310.2ꢁ11.5
+101.0ꢁ14.8
+45.9ꢁ17.0
+114.0ꢁ14.4
G2-SPM
4524
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 4519 – 4532

Effect of Surface Ligands on Dendron–DNA Interactions
FULL PAPER
“immobilisation” of the much larger dendritic structure. The
importance of entropic factors in driving multivalency ef-
fects is often discussed,[2] but this modelling demonstrates an
important example in which entropic factors discriminate
between multivalent binding effects in two enthalpically
equivalent but structurally different dendrons.
In order to probe the binding in more detail, and to
better understand the origin of these enthalpic and entropic
effects on multivalency, we went on to consider the den-
drons as assemblies of residues. Each dendron was consid-
ered to be composed of three different kinds of residue
(Figure 6). We assigned CEN (yellow) as the benzyl carba-
mate protecting group at the focal point of the dendron,
REP (blue) as the repetitive unit of the Newkome-type den-
dron (amide–ether repeat unit, Figure 1) and SPM,
DAPMA or DAP as the surface amine groups (coloured by
atom, Figure 6) in each of the different dendron systems.
This decomposition of molecular mechanics energies al-
lowed us to gain an insight into the interaction between
each individual residue and the DNA double helix, and
hence fully understand the origins of binding.
In Table 3, we report the energetic contributions of each
residue within the dendron structure. These can be defined
in terms of Equation (1), that is, they represent the differ-
ence between the energy of the dendron–DNA complex
(Ecomplex) and the sum of the energies of dendron and DNA
taken separately (Edendron +EDNA). Negative energy values in-
dicate attractive forces and the thermodynamic tendency to
form a complex:
E ¼ EcomplexꢀðEdendron þ EDNA
Þ
ð1Þ
Gas-phase energies (Egas) for each residue are composed
of electrostatic and van der Waals interaction contributions
(Eele and Evdw, respectively) according to Equation (2):
Egas ¼ Eele þ Evdw
ð2Þ
The in-vacuum gas-phase energy for each residue (Egas) is
then corrected for solvent effects by using the mm pbsa.pl
script of AMBER 9 with the generalized Born solvation
method (the Poisson–Boltzmann method is not supported in
residue-based energy decomposition) to obtain the total
energy Etot.[34]
Table 3 presents the contribution of the amine surface
groups SPM, DAPMA and DAP to the binding energy for
each of the G1 dendrons. This allows direct comparison in
binding enthalpy between the different systems. The contri-
butions of the CEN and REP units of the dendron to the
binding energy were found to be minimal, and are therefore
not presented in Table 3 for clarity. The amine groups are
primarily responsible for interacting with the DNA double
helix, and as is clear from Table 3, the interaction is wholly
electrostatic in nature, with no significant van der Waals
contribution. As expected, G1-SPM has the highest affinity
Figure 6. Depiction of G2-DAPMA indicating the three different types of
structural residue: CEN (yellow), REP (blue) and SPM (coloured by
atom: C in silver, N in blue and H atoms in white).
Table 3. Interaction energies determined for individual residues within the G1 dendron structure interacting with DNA. Energies represent the differ-
ence between the complex and the two individual components; negative values represent favourable interactions. The total in-vacuum energy Egas, com-
posed of van der Waals Evdw and electrostatic Eele energies is corrected for solvation giving Etot. All energies are reported in kcalmolꢀ1
.
[a]
[b]
[c]
[d]
Dendron
G1-DAP
Residue[e]
Number
Evdw
Eele
Egas
Mean Egas ꢁSD
ꢀ342.2ꢁ17.0
Etot
Mean Etot ꢁSD
ꢀ6.2ꢁ0.7
DAP
DAP
DAP
1
2
3
0.2
ꢀ351.3
ꢀ356.9
ꢀ318.6
ꢀ1026.8
ꢀ548.9
ꢀ692.4
ꢀ664.0
ꢀ1905.3
ꢀ840.8
ꢀ1145.9
ꢀ940.3
ꢀ2927.0
ꢀ351.1
ꢀ357.1
ꢀ318.5
ꢀ1026.7
ꢀ548.9
ꢀ693.7
ꢀ664.5
ꢀ1907.2
ꢀ841.3
ꢀ1147.9
ꢀ942.8
ꢀ2931.9
ꢀ6.6
ꢀ0.2
ꢀ6.9
0.1
0.1
0.0
ꢀ5.2
sum of surface amines
ꢀ18.7
ꢀ7.1
G1-DAPMA
G1-SPM
DAPMA
DAPMA
DAPMA
1
2
3
ꢀ635.7ꢁ62.5
ꢀ977.3ꢁ127.5
ꢀ10.2ꢁ2.8
ꢀ18.5ꢁ4.9
ꢀ1.4
ꢀ0.5
ꢀ1.9
ꢀ0.5
ꢀ2.1
ꢀ2.4
ꢀ4.9
ꢀ13.9
ꢀ9.6
sum of surface amines
ꢀ30.6
ꢀ11.8
ꢀ23.1
ꢀ20.7
ꢀ55.7
SPM
SPM
SPM
1
2
3
sum of surface amines
[a] Evdw represents the van der Waals interaction energy. [b] Eele represents the electrostatic interaction energy. [c] Egas represents the combination of Evdw
and Eele to yield the overall in vacuo nonbond energy. [d] Etot represents the total energy after correction for solvation.
Chem. Eur. J. 2010, 16, 4519 – 4532
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
4525

D. K. Smith, G. M. Pavan et al.
for DNA, G1-DAPMA the next highest and G1-DAP the
lowest; this is in agreement with the experimental data. In-
terestingly, the overall gas-phase interaction energy between
G1-DAP (charge=3) and DNA is approximately 33% of
the interaction between G1-SPM (charge=9) and DNA.
The overall gas-phase interaction energy between G1-
DAPMA (charge=6) and DNA is approximately 66% of
the interaction between G1-SPM (charge=9) and DNA.
These observations are in agreement with the experimental
data and also with the modelling, which indicated that the
enthalpic term was basically proportional to the number of
charged residues in the dendron.
It is interesting to note the variance in gas-phase interac-
tion energy between the different surface amines. For G1-
DAP, the lowest energy is ꢀ318.5 kcalmolꢀ1, whilst the high-
est is ꢀ357.1 kcalmolꢀ1, with the standard deviation being
about 5.0% of the mean. This indicates that each of the
three surface amine groups binds with similar affinity to the
DNA double helix. For G1-DAPMA, the standard variation
in Egas between the three surface diamine ligands is 9.8% of
the mean, whilst for G1-SPM, the standard deviation in Egas
between the three surface triamine ligands is as high as
13.0% of the mean, with SPM2 contributing considerably
more to the binding than SPM1. Therefore, although the
average gas-phase affinity is proportional to the number of
charges, not every charge is contributing equally, particularly
in the cases in which the surface amine groups are either di-
amines or triamines. This indicates that for G1-SPM in par-
ticular, it is preferable to optimise the interaction of one of
the surface polyamine ligands with DNA, even if this com-
promises somewhat the binding affinity of the other two li-
gands. This is not the case for G1-DAP, which attempts to
optimise the interaction of all three individual point surface
charges with DNA. It is clear that in order to optimise all
three surface groups simultaneously, G1-DAP will suffer
from a much larger relative entropic cost of binding, as the
overall dendron structure will lose many degrees of free-
dom. This observation is in agreement with the data in
Table 2, which demonstrated that the entropic cost per inter-
action (TDSbind/charge) for G1-DAP binding to DNA was
+11.1 kcalmolꢀ1, whilst for G1-SPM it was only +5.1 kcal
molꢀ1. These arguments also hold when considering the Etot
values, which are corrected for solvation.
Table 4 presents the same kind of analysis applied to the
second generation (G2) dendrons. Once again, in agreement
with the experimental data, G2-SPM has the highest affinity
for DNA, G2-DAPMA is intermediate and G2-DAP has the
lowest affinity. However, in this case, the interaction ener-
gies are not proportional to dendron charge. Although the
Egas of G2-DAP binding to DNA is approximately one third
Table 4. Interaction energies determined for individual residues within the G2 dendron structure interacting with DNA. Energies represent the differ-
ence between the complex and the two individual components; negative values represent favourable interactions. All energies are reported in kcalmolꢀ1
.
[a]
[b]
[c]
[d]
Dendron
G2-DAP
Residue[e]
Number
Evdw
Eele
Egas
Mean Egas ꢁSD
ꢀ343.7ꢁ39.5
Etot
Mean Etot ꢁSD
ꢀ5.1ꢁ1.4
DAP
DAP
DAP
DAP
DAP
DAP
DAP
DAP
DAP
14
15
16
17
18
19
20
21
22
0.0
ꢀ333.2
ꢀ388.2
ꢀ350.0
ꢀ334.5
ꢀ363.4
ꢀ390.3
ꢀ305.7
ꢀ259.5
ꢀ361.7
ꢀ3086.6
ꢀ673.8
ꢀ805.5
ꢀ877.1
ꢀ841.3
ꢀ705.1
ꢀ763.2
ꢀ653.0
ꢀ561.1
ꢀ716.6
ꢀ6596.7
ꢀ979.2
ꢀ1141.8
ꢀ1193.1
ꢀ1199.0
ꢀ1029.7
ꢀ786.2
ꢀ953.1
ꢀ763.3
ꢀ994.3
ꢀ9039.8
ꢀ333.3
ꢀ390.3
ꢀ350.3
ꢀ334.8
ꢀ364.7
ꢀ391.9
ꢀ305.9
ꢀ259.5
ꢀ362.2
3092.7
ꢀ674.6
ꢀ811.1
ꢀ879.8
ꢀ845.5
ꢀ705.6
ꢀ765.8
ꢀ653.0
ꢀ561.1
ꢀ716.6
ꢀ6615.6
ꢀ981.3
ꢀ1145.7
ꢀ1195.2
ꢀ1203.9
ꢀ1034.9
ꢀ786.4
ꢀ954.1
ꢀ763.5
ꢀ997.7
ꢀ9062.6
ꢀ6.3
ꢀ2.0
ꢀ0.3
ꢀ0.3
ꢀ1.4
ꢀ1.6
ꢀ0.2
0.0
ꢀ6.2
ꢀ7.3
ꢀ5.6
ꢀ3.8
ꢀ3.1
ꢀ4.0
ꢀ3.5
ꢀ0.5
ꢀ6.2
ꢀ0.8
ꢀ5.6
ꢀ2.7
ꢀ4.2
ꢀ0.6
ꢀ2.6
ꢀ1.4
ꢀ0.1
ꢀ0.9
ꢀ18.9
ꢀ2.1
ꢀ3.9
ꢀ2.1
ꢀ4.9
ꢀ5.1
ꢀ0.2
ꢀ1.0
ꢀ0.1
ꢀ3.4
ꢀ22.8
ꢀ6.3
sum of surface amines
ꢀ46.0
ꢀ11.9
ꢀ17.6
ꢀ19.0
ꢀ26.4
ꢀ19.2
ꢀ16.3
ꢀ15.2
ꢀ7.0
G2-DAPMA
DAPMA
DAPMA
DAPMA
DAPMA
DAPMA
DAPMA
DAPMA
DAPMA
DAPMA
14
15
16
17
18
19
20
21
22
ꢀ734.8ꢁ95.3
ꢀ16.9ꢁ5.1
ꢀ19.9
ꢀ152.4
ꢀ18.3
ꢀ29.4
ꢀ28.9
ꢀ36.1
ꢀ23.6
ꢀ9.5
sum of surface amines
G2-SPM
SPM
SPM
SPM
SPM
SPM
SPM
SPM
SPM
SPM
14
15
16
17
18
19
20
21
22
ꢀ1007.0ꢁ151.2
ꢀ21.3ꢁ8.5
ꢀ18.1
ꢀ9.7
ꢀ18.4
ꢀ191.9
sum of surface amines
[a] Evdw represents the van der Waals interaction energy. [b] Eele represents the electrostatic interaction energy. [c] Egas represents the combination of Evdw
and Eele to yield the overall in vacuum nonbond energy. [d] Etot represents the total energy after correction for solvation.
4526
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 4519 – 4532

Effect of Surface Ligands on Dendron–DNA Interactions
FULL PAPER
of the binding of G2-SPM, the Egas of G2-DAPMA is signifi-
cantly larger than the expected 66.6% of G2-SPM (actually
73.0%). This implies that on a per charge basis, G2-
DAPMA appears to bind more strongly to DNA than might
be predicted/expected. This is even more marked when con-
sidering the Etot values (corrected for solvation). For G2-
DAP the Etot value is only approximately 24.0% of G2-SPM
(i.e., less than expected) whereas for G2-DAPMA it is
79.4% (much more than expected).
Once again, it is interesting to note that for the multiva-
lent surface amines SPM and DAPMA, the interactions be-
tween different surface ligands and the DNA are more
varied in energy. For G2-DAP the standard deviation in Egas
represents 11.5% of the mean, whilst for G2-DAPMA and
G2-SPM this figure rises to 13.0 and 15.0%, respectively.
The same argument applies for the solvated Etot values, for
which the standard deviation rises from 27.5% of the mean
for G2-DAP to 30.1 and 39.9% for G2-DAPMA and G2-
SPM, respectively. Solvation increases the standard devia-
tion, as it amplifies the differences in DNA binding between
different surface ligands on the same dendron because sol-
vent molecules compete for interactions with the surface li-
gands that are not bound to DNA.
As observed for G1-DAP, G2-DAP attempts to optimise
the interaction of as many of the surface point charges as
possible with DNA; clearly this will have a significant en-
tropic cost across the whole dendritic structure (ꢀTDSbind
/
charge=+6.7 kcalmol-1, Table 2). There is a greater range
of binding energies for the surface ligands of G2-DAP than
G1-DAP because it is not actually possible to completely
optimise the binding of nine simple surface monoamines to
the DNA double helix (also reflected in a lower ꢀTDSbind
/
charge value for G2-DAP than G1-DAP). The inability to
organise all of the charge–charge interactions becomes even
more marked for G2-DAPMA and G2-SPM; these den-
drons clearly only optimise the interaction of selected sur-
face ligands with DNA, the other ligands binding less effec-
tively. However, this selective optimisation will have a lower
Figure 7. Root mean square deviation (RMSD) of heavy atoms belonging
to G2+DNA complexes (blue), G2 dendron (green) and DNA (pink) re-
ported for: a) G2-SPM, b) G2-DAPMA, and c) G2-DAP systems as a
function of time (ns) and 1(r). RMSD represents the movements (ꢂ) of
heavy atoms from their starting positions. A horizontal tendency means
that the system reached an equilibrium (i.e., the system is uniformly vi-
brating).
entropic cost per interaction; indeed for G2-SPM ꢀTDSbind
/
charge is as low as +4.2 kcalmolꢀ1 (Table 2) because not all
of the dendritic structure requires such careful organisation.
This entropic difference helps explain why the dendrons
with polyvalent surface amines (SPM and DAPMA) are ex-
perimentally so much more effective than those with indi-
vidual surface amine groups (DAP). This entropically-ampli-
fied difference between dendrons is much larger than what
would be expected purely from the calculation of charge–
charge interactions between the dendrons and DNA.
time for all G2+DNA complexes, the G2 dendron consid-
ered individually within the complex, and the DNA mole-
cule within the complex.
In Equation (3) d is the distance between N pairs of
equivalent heavy atoms (in this study C, N, O and P atoms
belonging to dendron and DNA molecules only, water ex-
cluded). This allows us to represent the way in which each
heavy atom of the structure moves from the starting position
in the simulation. As the value reaches a plateau, it repre-
sents the system having evolved to constant vibration
around an equilibrium position (i.e., the system has reached
equilibrium in the simulation).
Structural insights: In order to understand why the binding
between G2-DAPMA and DNA appeared to be anomalous-
ly large in enthalpic terms (73.0% of G2-SPM in terms of
Egas and 79.4% terms of Etot, when charge–charge interac-
tions alone would have led us to expect 66.6%) we per-
formed a structural study of the dendron–DNA binding pro-
cess. Figure 7 reports the dynamic RMSD (root mean
square deviation, defined by Equation (3)) versus simulation
vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
u
i¼N
u
t
X
1
N
ð3Þ
RMSD ¼
di2
i¼1
Chem. Eur. J. 2010, 16, 4519 – 4532
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
4527

D. K. Smith, G. M. Pavan et al.
It is interesting to compare the behaviour of the curves
for the complex and the two individual components (i.e.,
dendron and DNA) and to consider their superposition
(Figure 7). For G2-SPM the curves are perfectly superim-
posed (Figure 7a), indicating that the complex is uniform,
with the vibration of atoms in the two individual compo-
nents being equivalent to the vibration of the overall com-
plex. However, for G2-DAPMA there is a significant differ-
ence between the curves (Figure 7b) with the dendron
having significantly lower RMSD values than the DNA or
the overall complex. This is due to the dendron conforma-
tion (the different RMSD values mean that the position of
the dendron is shifted from the starting position by about
6 ꢂ, whereas the DNA atoms shift by about 7 ꢂ for DNA).
The DAPMA-functionalised dendrons appear to have a
degree of structural rigidity—a consequence of the different
structure of the DAPMA ligand compared with SPM (we
suggest this might be a consequence of the presence of the
methyl group on the tertiary amine of DAPMA; see later
for further discussion). However, it is worth noting that,
after reaching a convergence in RMSD, the curves proceed
in parallel, meaning that dendron and DNA atoms, after
reaching an equilibrium position, do vibrate uniformly as a
stable complex. For G2-DAP the situation is rather differ-
ent; there is reasonable superposition between the curves,
but at points, the RMSD for the dendron diverges from the
other curves (Figure 7c). This is indicative of a weaker bind-
ing, with the monoamine end groups of the DAP structure
being less attractive to DNA than the diamines and tria-
mines of DAPMA and SPM, and hence the vibrations of
dendron and DNA differ from one another over the time-
course of the simulation.
As is evident from Table 5, the dendron Rg for G2-SPM
(15.4 ꢂ) is larger than that for G2-DAPMA (11.8 ꢂ) or G2-
DAP (10.2 ꢂ) as expected based on the relative sizes of
these dendrons. Considering only the surface ligands of the
dendron leads to larger radii of gyration than the overall
dendron structure, as the surface ligands are the furthest
points, most distant from the centre of mass. However, it is
noteworthy that the difference between surface ligands and
dendron is smallest for G2-DAPMA (only 2.0 ꢂ) not, as
may have been expected G2-DAP (2.6 ꢂ), which actually
has smaller ligand groups. This indicates that the surface li-
gands of G2-DAPMA are not fully extended when they in-
teract with the DNA. Furthermore, the dimensions of the
overall complex are actually the smallest when G2-DAPMA
is used, and the DNA is significantly smaller (2.5 ꢂ) in this
case than when the surface ligand is SPM. This indicates
that the G2-DAPMA dendron deforms the DNA in order to
achieve its interactions, whereas for the binding of G2-SPM
and G2-DAP the DNA remains more extended. There has
been recent literature interest in the different abilities of oli-
goamines to modify the conformational preferences of
DNA, and in some cases it has been suggested that unnatu-
ral amines are more likely to encourage conformational
change.[35]
This DNA deformation/bending can be observed visually
in the molecular mechanics structural models (Figure 8). We
suggest that by bending DNA in this way, G2-DAPMA is
able to achieve larger interaction energies than would have
been predicted on a simple “per charge” basis (helping ex-
plain the stronger than expected energetic data for G2-
DAPMA in Table 4). It also explains why the entropy of
DNA binding is more disfavoured for G2-DAPMA than
G2-SPM, as deforming DNA in this way will lose significant
degrees of freedom.
Table 5 reports the radius of gyrations (Rg) for the G2+
DNA complexes, the G2 dendrons, the DNA molecule, and
also the surface ligands (each considered within the simulat-
In a final analysis to probe why the DAPMA surface li-
gands appear to have this kind of effect, we considered the
detailed radial distribution functions (RDFs) rather than
just the simple averaged radii of gyration values presented
in Table 5. Figure 9a, b, and c represent the overall RDFs.
Since RDF, indicating the presence of atoms in space (i.e.,
as a function of the distance from the centre of mass of the
dendron) is calculated at each frame of simulation, it has a
clear dynamic meaning. Large peaks in these graphs mean
not only high density of atoms in a certain zone, but also
high localisation, and thus low ability of these atoms to
move. The contact point between dendron and DNA corre-
sponds with the peak maximum, close to the centre of mass,
meaning that at this point degrees of freedom are lost be-
cause of the dendron–DNA interactions. Figure 9d, e, and f
represent the distributions of phosphorus (on DNA) and ni-
trogen (on the surface amines). The contact peak between
anionic phosphate and cationic protonated amine is clearly
visible as the peak maximum close to the centre of mass in
each case. Interestingly, however, for G2-DAP the peak for
the protonated amine (Figure 9 f, solid line) is significantly
smaller than for G2-DAPMA or G2-SPM (Figure 9d and e,
solid lines). This lower peak indicates that the terminal
Table 5. Radius of gyration (Rg, as defined by Equation (4)), which rep-
resents the distance of atoms from the centre of mass of the dendron.
The data considered the whole dendron, the surface amine ligands of the
dendron, and the DNA (all within the complex) as well as the overall
complex between dendron and DNA.
Dendron
Rg (den-
Rg (surface
Rg (DNA)/ꢂ
Rg (com-
dron)/ꢂ
ligands)/ꢂ
plex)/ꢂ
G2-DAP
G2-DAPMA
G2-SPM
10.2
11.8
15.4
12.8
13.8
18.1
20.6
19.2
21.7
19.5
18.2
20.4
ed complex). This radius of gyration, as defined by Equa-
tion (4) represents the average distance between each atom
in the structure considered and the centre of mass of the
dendron.
N
X
1
2
R2g ¼
ðrk ꢀ rmean
Þ
ð4Þ
N
k¼1
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Chem. Eur. J. 2010, 16, 4519 – 4532

Effect of Surface Ligands on Dendron–DNA Interactions
FULL PAPER
amines of G2-DAP are more
likely to be oscillating around
the P atoms of DNA; this is
consistent with the relatively
lower binding of this system,
and in agreement with our ar-
guments above about the signif-
icant entropic cost (per charge)
of binding G2-DAP to the
DNA double helix. For G2-
SPM and G2-DAPMA on the
other hand, the multivalent
nature of each individual amine
ligand helps counteract the en-
tropic cost of immobilisation
and lowers the mobility of the
main electrostatic contact point
between dendron and DNA.
Figure 10a and b represent
the radial distribution function
for the ligating nitrogen atoms
of the surface amines in G2-
SPM and G2-DAPMA. This
Figure 8. Snapshots of molecular dynamics simulation of G2 dendrons binding to double helical DNA: a) G2-
DAP, b) G2-DAPMA, and c) G2-SPM. Within the dendron CEN is shown in blue, REP in yellow and the
amine ligands are shown in magenta (DAP), green (DAPMA) and red (SPM). The DNA is portrayed as a
dark grey shadow, water molecules are omitted for clarity, and only those counterions in close proximity to the
complexes are shown.
Figure 9. Radial distribution functions (RDF) of dendron (c), DNA (a) and surface ligands (d) are reported for: a) G2-SPM, b) G2-DAPMA,
and c) G2-DAP. RDF of N atoms of surface ligands (c) and of P atoms of DNA (a) are reported for: d) G2-SPM, e) G2-DAPMA, and f) G2-DAP.
Surface SPM, DAPMA and DAP ligands are illustrated in the bottom of the panel and shaded by atoms (N dark grey, C grey and H white). The most
crucial atoms of these surface ligands are labelled with decreasing index going toward the surface.
Chem. Eur. J. 2010, 16, 4519 – 4532
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4529

D. K. Smith, G. M. Pavan et al.
Figure 10. RDF of selected atoms of surface ligands reported for: a) G2-SPM, and b) G2-DAPM. RDF for N1 is represented in green, for N2 in red and
for N3 and C2 in blue and black, respectively. Superposition of RDF of most internal amines of SPM (N3, blue) and DAPMA (N2, red) represents the
difference in peaks c).
analysis allows a structural decomposition of the role of
each individual protonated amine within the complex with
DNA. Figure 10b also presents the radial distribution func-
tion for the methyl group (identified by C2 atom) attached
to the tertiary amine (N2 position) of DAPMA. For G2-
SPM it is evident that N3 forms the most stable interaction
with DNA (i.e., the highest peak corresponding to the
lowest mobility). This is the amine closest to the dendron
scaffold. The amines N2 and, to an even greater extent N1,
then form more mobile interactions with the DNA (i.e.,
smaller peaks), which are further away from the centre of
mass. For G2-DAPMA, the primary interaction is formed by
N2 (again closest to the dendritic scaffold). This peak is
larger than that observed for N3 of G2-SPM (Figure 10c),
which indicates a lower mobility of this amine in G2-
DAPMA than in G2-SPM. Notably, the attached methyl
group appears coincident with the amine, indicating it is lo-
cated in the same region of space. We argue that this group
hinders the mobility of the N2 group once bound to the
DNA. Terminal atom N1 also forms interactions with DNA,
but these can be either closer to the centre of mass than the
primary interaction between N2 and DNA (surprising) or
further away (as expected and as was observed for G2-
SPM). This indicates the important/primary role played by
the sterically hindered N2 in the binding process, with the
remainder of the DAPMA group then forming the best re-
maining interaction available, which might require either
ligand back-folding or ligand extension. Ligand back-folding
can be seen for some of the DAPMA ligands in Figure 8b
(e.g., 16 and 17) but is not observed for any of the spermine
ligands in Figure 8c. We propose that the steric hindrance of
the N2 group of DAPMA and the strong preference for its
coordination to the DNA double helix provides the stronger
than expected enthalpic interaction with DNA, causes
ligand back-folding, and ultimately leads to the structural
deformation of DNA discussed above. SPM ligands on the
other hand, are more optimised for binding in the minor
groove, and therefore ligand back-folding and DNA defor-
mation are not observed to any major extent.
Summary and Conclusion
By synthesising a family of dendrons with varied surface
amine ligands we have gained a unique insight into DNA
binding, gene delivery and multivalency effects. It was
shown that the dendrons with spermine (SPM) surface
groups are the most effective DNA binders. The dendrons
with DAP and DAPMA surface groups are less effective at
DNA binding. However, the DAPMA-functionalised den-
drons were more effective systems for gene delivery with
low toxicity, performing as well as, if not better than the
SPM-functionalised dendrons (although the gene delivery
profiles were still relatively modest).
In order to provide deeper insight into the experimental
data, we performed a molecular dynamics simulation of the
interactions between the dendrons and DNA. The results of
these simulations demonstrated that in general terms, the
enthalpic contribution to binding was proportional to the
degree of surface charge, but dendrons with DAP and
DAPMA surface amines had significant entropic costs of
binding, which limited their total binding affinity. In the
case of DAP, this is a consequence of the fact that the entire
dendron structure had to be organised in order for all of the
monoamine ligand charges to make effective contact with
DNA. In contrast, for SPM, each surface ligand is already a
multivalent triamine, therefore, each individual charge has a
much lower entropic cost of binding. For DAPMA, we ob-
served that binding of the hindered amine to the DNA was
enthalpically stronger than expected and also caused ligand
back-folding, which led to significant geometric distortion of
the DNA double helix. This weakened the overall binding
from what may have been expected solely on the grounds of
the enthalpic charge–charge interactions. We suggest that
this geometric distortion could help explain the experimen-
tally observed enhanced gene delivery—DNA compaction is
an important step in transfection.
Overall, this study demonstrates how we can begin to de-
velop structure–activity relationships for multivalent den-
dritic ligands using a combined experimental and theoretical
approach. The paper supports other work showing that gene
delivery potential does not necessarily correlate with bind-
4530
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Chem. Eur. J. 2010, 16, 4519 – 4532

Effect of Surface Ligands on Dendron–DNA Interactions
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We thank Nathan P. Gabrielson and Prof. Daniel W. Pack for assistance
with the gene transfection studies, which were carried out in the Depart-
ment of Chemical Engineering at University of Illinois at Urbana–Cham-
paign. The generous financial support to the DRUDE Initiative, project
“Nanovectors for drug delivery in oncology: a combined modelling/ex-
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Ticino to A.D. and S.P., is gratefully acknowledged. D.K.S. also acknowl-
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knowledge the support of COST action TD0802, “Dendrimers in Bio-
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