Self-interrupted synthesis of sterically hindered aliphatic polyamide dendrimers

Article abstract of DOI:10.1073/pnas.1700922114

2,2-Bis(azidomethyl)propionic acid was prepared in four steps and 85% yield from the commercially available 2,2-bis(hydroxymethyl) propionic acid and used as the starting building block for the divergent, convergent, and double-stage convergent-divergent iterative methods for the synthesis of dendrimers and dendrons containing ethylenediamine (EDA), piperazine (PPZ), and methyl 2,2- bis(aminomethyl)propionate (COOMe) cores. These cores have the same multiplicity but different conformations. A diversity of synthetic methods were used for the synthesis of dendrimers and dendrons. Regardless of the method used, a self-interruption of the synthesis was observed at generation 4 for the dendrimer with an EDA core and at generation 5 for the one with a PPZ core, whereas for the COOMe core, self-interruption was observed at generation 6 dendron, which is equivalent to generation 5 dendrimer. Molecular modeling and molecular-dynamics simulations demonstrated that the observed self-interruption is determined by the backfolding of the azide groups at the periphery of the dendrimer. The latter conformation inhibits completely the heterogeneous hydrogenation of the azide groups catalyzed by 10% Pd/carbon as well as homogeneous hydrogenation by the Staudinger method. These self-terminated polyamide dendrimers are enzymatically and hydrolytically stable and also exhibit antimicrobial activity. Thus, these nanoscale constructs open avenues for biomedical applications.

Full text of DOI:10.1073/pnas.1700922114

Self-interrupted synthesis of sterically hindered
aliphatic polyamide dendrimers
a,1
b,1
a,1
a
a
Davit Jishkariani , Christopher M. MacDermaid , Yam N. Timsina , Silvia Grama , Syeda S. Gillani ,
a
c
a
a
Masoumeh Divar , Srujana S. Yadavalli , Ralph-Olivier Moussodia , Pawaret Leowanawat , Angely M. Berrios
a
d,2
c
b,3
a,3
Camacho , Ricardo Walter , Mark Goulian , Michael L. Klein , and Virgil Percec
a
b
Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323; Institute of Computational
c
Molecular Science, Temple University, Philadelphia, PA 19122; Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6313
and Department of Preventive and Restorative Sciences, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104-6030
d
Contributed by Michael L. Klein, February 7, 2017 (sent for review January 18, 2017; reviewed by Ezequiel Perez-Inestrosa and Donald A. Tomalia)
2
8
,2-Bis(azidomethyl)propionic acid was prepared in four steps and
5% yield from the commercially available 2,2-bis(hydroxymethyl)
tional groups on the periphery of the dendrimer would provide a
critical packing state, above which a sterically inhibited reaction
rate would be observed (32). Above this critical packing state,
the lower reactivity of the functional groups on the periphery
would provide globular dendrimers with incompletely reacted
functional groups, thus generating an imperfect dendrimer struc-
ture (1, 2). Indeed, the most investigated, and best-elucidated,
aliphatic polyamide [polyamidoamine (PAMAM)] dendrimer
discovered and commercialized by Tomalia (Fig. 1) has been
shown to exhibit an inhibited reaction rate of its functional groups
at generations 4–7 as well as display a significant decrease in re-
action rate at generations 7–8, although it could be synthesized
with an imperfect structure up to generation 12 (2). Moreover, all
other aliphatic polyamide dendrimers have been reported for only
relatively low generations, without any explanation (Fig. 1).
This decrease in functional group reactivity on the surface of
dendrimers was also explained by backfolding of peripheral
groups (33, 34). Regardless of the mechanism that controls re-
activity, the efficacy of a biomedical application is likely corre-
lated with the reactivity of the peripheral functional groups of
the dendrimer because incomplete interactions or reactions
would generate, by analogy with iterative synthesis of higher
dendrimer generations, an imperfect biologically active conju-
gated product. Herein, we report the divergent, convergent, and
double-stage convergent–divergent iterative synthesis of a steri-
cally hindered aliphatic polyamide dendrimer (12) by a diversity
propionic acid and used as the starting building block for the
divergent, convergent, and double-stage convergent–divergent iter-
ative methods for the synthesis of dendrimers and dendrons con-
taining ethylenediamine (EDA), piperazine (PPZ), and methyl 2,2-
bis(aminomethyl)propionate (COOMe) cores. These cores have the
same multiplicity but different conformations. A diversity of syn-
thetic methods were used for the synthesis of dendrimers and den-
drons. Regardless of the method used, a self-interruption of the
synthesis was observed at generation 4 for the dendrimer with an
EDA core and at generation 5 for the one with a PPZ core, whereas
for the COOMe core, self-interruption was observed at generation 6
dendron, which is equivalent to generation 5 dendrimer. Molecular
modeling and molecular-dynamics simulations demonstrated that
the observed self-interruption is determined by the backfolding
of the azide groups at the periphery of the dendrimer. The latter
conformation inhibits completely the heterogeneous hydrogena-
tion of the azide groups catalyzed by 10% Pd/carbon as well as
homogeneous hydrogenation by the Staudinger method. These
self-terminated polyamide dendrimers are enzymatically and
hydrolytically stable and also exhibit antimicrobial activity.
Thus, these nanoscale constructs open avenues for biomedical
applications.
dendrons
| ethylenediamine core | piperazine core | antimicrobials
|
nanomedicine
Significance
liphatic polyamide (1–14) and peptide (15) dendrimers are
Hydrolytically and enzymatically stable nanoscale synthetic
constructs, with well-defined structures that exhibit antimi-
crobial activity, offer exciting possibilities for diverse applica-
tions in the emerging field of nanomedicine. Herein, we
demonstrate that it is the core conformation, rather than pe-
riodicity, that ultimately controls the synthesis of sterically
hindered aliphatic polyamide dendrimers. The latter self-
interrupt at a predictable low generation number due to back-
folding of their peripheral groups, which in turn leads to well-
defined nanoarchitectures.
A
complex monodisperse, functional compounds (16) synthesized
by iterative divergent (1), convergent (17), and double-stage con-
vergent–divergent (18) methods. Their nanoarchitecture emerges by
increasing their generation number from ellipsoidal or disk-like 2D
to 3D globular constructs (16, 19–25). Their core and branch mul-
tiplicities, as well as branch cell segment length (l), determine the
generation number at which dendrimers become globular (19–21).
Dendrimers synthesized by divergent iterative methods are expected
to contain their functional groups, which increase in number with
core and branch multiplicities as well as with their generation
number, at the periphery of their 2D or 3D architectures (16, 19,
Author contributions: D.J., C.M.M., Y.N.T., S.S.Y., R.W., M.G., M.L.K., and V.P. designed
research; D.J., C.M.M., Y.N.T., S.G., S.S.G., M.D., S.S.Y., R.-O.M., P.L., and A.M.B.C. per-
formed research; D.J., C.M.M., Y.N.T., S.G., S.S.Y., R.-O.M., P.L., M.G., M.L.K., and V.P.
contributed new reagents/analytic tools; D.J., C.M.M., Y.N.T., S.G., S.S.G., M.D., S.S.Y.,
R.-O.M., P.L., M.G., M.L.K., and V.P. analyzed data; and D.J., C.M.M., Y.N.T., S.G., S.S.Y.,
R.W., M.G., M.L.K., and V.P. wrote the paper.
2
1). Dendrons and dendrimers produced by convergent strategies
contain one functional group at their apex, whereas the rest of them
are at their periphery (16, 17, 20, 23–25). Ultimately at a certain
generation, dendrons also become globular dendrimers with
their functional group from the apex becoming encapsulated at
the core of the globular object (22–25). The large concentration
of functional groups at the periphery of the globular object in-
spired a diversity of functions of biomedical interest that are not
accessible with their linear homologs (26–31). Biomedical utility
has propelled the field of aliphatic polyamide dendrimers to an
unprecedentedly high level of expectations (26–31). Simultaneous
with the interest in biomedical applications, it was predicted that a
dense surface packing state generated by the presence of all func-
Reviewers: E.P.-I., University of Malaga; and D.A.T., NanoSynthons LLC.
The authors declare no conflict of interest.
1
D.J., C.M.M., and Y.N.T. contributed equally to this work.
2
Present address: Department of Operative Dentistry, University of North Carolina,
Chapel Hill, NC 27599-7450.
3
To whom correspondence may be addressed. Email: percec@sas.upenn.edu or mlklein@
temple.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1700922114/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1700922114
PNAS Early Edition
|
1 of 10

Fig. 1. (A–L) Name of the laboratory, synthetic method used, maximum generation number synthesized (n) of Gn, and the numbers of atoms in the linear
part of their repeat unit or branch cell segment length (l, in-between parentheses) of aliphatic polyamide and peptide dendrimers reported in the literature.
of synthetic methods. Unexpectedly, self-interruption of the syn- Modification of this synthesis led to a more efficient four steps
thesis of this dendrimer was discovered at a generation that is procedure involving 1, 4, 5, and 6 to produce 7 in 85% yield while
determined by the conformation rather than the multiplicity of reducing the overall reaction time from 92 to 38 h (Fig. 2) and
its core.
performing all steps under milder reaction conditions.
Results and Discussion
Divergent Synthesis of Four Dendrimer Generations with an
Ethylenediamine Core. The first divergent synthesis investigated
used a modified literature procedure that involved the use of an
ethylenediamine (EDA) core and the acid chloride-activated form
of 7 to produce three generations of polyamide dendrimers (12).
The main modification involved the replacement of the amidation
step consisting of the addition of 8 to a water/NaOH mixture
containing EDA and a higher-generation dendrimer with the ad-
Selecting the Aliphatic Poly(amide) Dendrimer and the Synthesis of
Its Building Block. The most sterically congested dendrimer
reported to date (Fig. 1) (12) was selected to evaluate the re-
activity of its functional groups as a function of generation
number and core structure and compare it with nonsterically
hindered aliphatic polyamide dendrimers.
This sterically hindered aliphatic polyamide dendrimer is expec-
ted to provide an amplification effect of the reactivity of similar
functional groups available in less sterically hindered dendrimers.
This sterically hindered aliphatic polyamide is also synthetically at-
tractive because it was originally prepared from the inexpensive and
dition of 8 to a CH Cl /Et N solution of EDA at 0–23 °C (Fig. 2).
2 2 3
This modification improved the yield of the amidation step at
all generations with a remarkable increase at G3 from 27 to 59%.
We expect that, due to steric reasons, the rate of amidation of
the acid chloride competes with its rate of hydrolysis, and
commercially available 2,2-bis(hydroxymethyl)propionic acid AB
2
building block (1 in Fig. 2) (12), which was previously used in the
divergent iterative synthesis of five generations of aliphatic polyester
dendrimers (35, 36) that are commercially available (37), as well as
in the synthesis of amphiphilic Janus dendrimers that self-assemble
in biomedically interesting dendrimersomes (38, 39).
2 2
therefore the transition from amidation in basic water to CH Cl
improved its yield (Fig. 2). The maximum generation of the
previously reported synthesis of this dendrimer was G3 (12). Under
the modified conditions used here, the maximum generation that
could be synthesized with the EDA core was G4 (Fig. 2). All at-
tempts to perform the heterogeneous reduction of the azide groups
of G4 to amine with 10% Pd/C and up to stoichiometric amount
The starting material for the iterative synthesis of the current ali-
phatic polyamide dendrimer is the AB 2,2-bis(azidomethyl)propionic
2
acid (compound 7 in Fig. 2) that was previously obtained in
6% yield via the three reactions steps involving 1, 2, and 3 (Fig. 2)
12). Attempts to improve the low yield of this synthesis failed.
of catalyst under 120 psi H pressure as well as Staudinger ho-
2
6
(
mogeneous hydrogenation have failed. An amidation method
inspired from the synthesis of peptides (40–42) was elaborated.
2
of 10
|
www.pnas.org/cgi/doi/10.1073/pnas.1700922114
Jishkariani et al.

Fig. 2. Synthesis of 2,2-bis(azidomethyl)propionic acid, its carboxylic acid-activated forms, and the divergent synthesis of four generations of aliphatic
polyamide dendrimers containing ethylenediamine (EDA) core. Reagents and conditions: (i) SOCl , dimethylformamide (DMF) (23 °C, 18 h, reflux, 24 h); (ii)
O, 7 M NaOH (0 °C, 2 h); (iii) NaN , DMF:H O (9:1) (80 °C, 48 h); (iv) MeOH, Amberlyst-15 (reflux, 5 h); (v) methanesulfonyl chloride (MsCl), triethylamine
Et N), CH Cl (0–23 °C, 7 h); (vi) NaN , DMF (90 °C, 24 h); (vii) NaOH, MeOH/THF/H O [1/1/1 (vol/vol/vol)] (50 °C, 2 h); (viii) SOCl , CH Cl (3 h, reflux); (ix)
-hydroxybenzotriazole (HOBt), Et N, CH Cl (0 °C, 0.5 h, then 23 °C, 12 h); (x) HOBt, N,N’-dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP),
(23 °C, 1 h, then 35 °C, 2 h); (xi) 8, Et N, CH Cl (–78 °C, 0.5 h, then 23 °C, 12 h); (xii) 9, Et N, CH Cl (23 °C, 24 h); (xiii) 10% Pd/C [10% (wt/wt)], H
(120 psi) (23 °C, 3 h); and (xv) 10% Pd/C [80% (wt/wt)], H (120 psi) (23 °C, 3 h).
2
H
2
3
2
(
3
2
2
3
2
2
2
2
1
3
2
2
CH
2
Cl
2
3
2
2
3
2
2
2
(
120 psi) (23 °C, 2 h); (xiv) 10% Pd/C [30% (wt/wt)], H
2
2
The carboxylic group of 7 was activated as an ester of
the EDA core that allowed the synthesis of only four generations,
1
-hydroxybenzotriazole (HOBt) either by the direct esterification
the PPZ generated five generations of dendrimers. G5(PPZ)N was
3
of 7 with HOBt in the presence of N,N′-dicyclohexylcarbodii-
obtained in 44–38% yield with both methods. Attempts to hydro-
mide (DCC) and a catalytic amount of 4-dimethylaminopyridine
genate G5(PPZ)N to G5(PPZ)NH failed.
3
2
(
DMAP) to produce 9 in 92% yield or by the esterification of 8 in
the presence of Et N to generate 9 in only 69% yield. Amidation of
Divergent Synthesis of Six Dendron Generations. The focal point of
the polyamide dendrons was generated by the reduction of the
3
EDA followed by iterative reduction/amidation steps produced
consistently four generations of dendrimers in up to 20% higher
yields than the acid chloride methods (Fig. 2). However, the re-
azide groups of G1(COOMe)N
NH to generate G1(COOMe)NH
dation of G1(COOMe)NH with 9 (Fig. 2) or with 8 (Fig. 2)
produced G2(COOMe)N in 92% and 84% yield, respectively
3
(compound 6 from Fig. 2) to
2
2
in 98% yield (Fig. 4). Ami-
duction of G4(EDA)N generated by the HOBt-activated method
2
3
could not be accomplished.
3
(
Fig. 4). Hydrolysis of the methyl ester of G2(COOMe)N pro-
3
Divergent Synthesis of Five Dendrimer Generations with a Piperazine
Core. Disubstituted EDA and piperazine (PPZ) exhibit the same
multiplicity but different conformations (43–45). The same core
multiplicity with different conformations was not previously in-
vestigated in the synthesis of dendrimers (1, 2, 16, 46). The di-
vergent iterative methods based on the acid chloride 8 and the
HOBt ester 9 of 7 were both used in the synthesis of polyamide
duced G2(COOH)N in 93% yield (Fig. 4). Activation of the
3
COOH group of G2(COOH)N as HOBt ester or as acid chlo-
3
ride followed by amidation with G1(COOMe)N3 produced
G3(COOMe)N (85% and 55% yields), which was hydrolyzed
3
to G3(COOH)N (91% yield) (Fig. 4).
3
Reduction of the azide groups of G2(COOMe)N
3
to
G2(COOMe)NH
2
(94% yield) followed by its amidation with 9
(60% yield), which was reduced to
G3(COOMe)NH (95% yield) and amidated with 9 to generate
dendrimers. High yields (63–92%) were obtained for the first four produced G3(COOMe)N
generations with both methods (Fig. 3). However, by contrast with
3
2
Jishkariani et al.
PNAS Early Edition
|
3 of 10

Fig. 3. Divergent synthesis of five generations of aliphatic polyamide dendrimers containing piperazine (PPZ) core. Reagents and conditions: (i) 9, Et
CH Cl (23 °C, 24 h); (ii) 8, Et N, CH Cl (–78 °C, 0.5 h, then 23 °C, 12 h); (iii) 10% Pd/C [15% (wt/wt)], H (120 psi) (23 °C, 3 h); (iv) 10% Pd/C [20% (wt/wt)], H
120 psi) (23 °C, 3 h); (v) 9, Et N, CH Cl (23 °C, 48 h); (vi) 10% Pd/C [50% (wt/wt)], H (120 psi) (23 °C, 8 h); (vii) 9, Et N, CH Cl (23 °C, 72 h); (viii) 8, Et N, CH Cl
–78 °C, 1 h, then 23 °C, 24 h); and (ix) 10% Pd/C [80% (wt/wt)], H (120 psi) (23 °C, 8 h).
3
N,
2
2
3
2
2
2
2
2
(
(
3
2
2
2
3
2
2
3
2
2
G4(COOMe)N (49% yield) that was reduced to G4(COOMe)NH
without purification). These activated compounds were reacted
3
2
in 92% yield. Repeating this amidation/reduction iteration on
with EDA and PPZ to produce G2(EDA)N (78% yield),
3
G4(COOMe)NH
2
produced G5(COOMe)NH
2
in 40% yield.
in 38% yield.
All attempts to reduce the azide groups of G6(COOMe)N to
G6(COOMe)NH failed. This is not surprising because the
G2(PPZ)N (65% yield), G3(EDA)N (78% yield), and G3(PPZ)N
3
3
3
Amidation of G5 with 9 produced G6(COOMe)N
3
(65% yield) (Fig. 5). Amidation of G2(PPZ)NH (Fig. 3) with
2
3
G2(COBt)N and G2(COCl)N generated G4(PPZ)N in 16%
3
3
3
2
and 42% yields (Fig. 5).
G6(COOMe)N
3
dendron is equivalent to a G5 dendrimer that
Finally, G3(COBt)N
3
was reacted with G2(PPZ)NH
2
to produce
contains at the focal point the methyl ester of 2,2-bis(aminomethyl)
propionic acid (Fig. 4).
3
G5(PPZ)N in 18% yield. Repeated attempts to reduce the azide
groups of G5(PPZ)N to generate G5(PPZ)NH did not succeed.
3
2
This focal point has the same multiplicity as EDA (Fig. 2) and
PPZ (Fig. 3) except that it contains an additional tetrahedral
carbon isolating the methylenic units from EDA or PPZ (Fig. 4).
Structural Analysis of Dendrimers and Dendrons. Structural analysis
of all dendrimers and dendrons prepared by divergent and
accelerated double-stage convergent–divergent methods by a
1
Accelerated Double-Stage Convergent–Divergent Synthesis of Three
EDA Generations and Five PPZ Generations of Dendrimers. An
accelerated double-stage convergent-divergent synthesis was also
used for the synthesis of dendrimers containing EDA and PPZ at
combination of 500-MHz H-NMR (SI Appendix, Figs. S1 and
1
3
S2), 126-MHz C-NMR spectroscopy (SI Appendix, Figs. S3 and
S4), gel permeation chromatography (GPC) (Fig. 6A), and ma-
trix-assisted laser desorption/ionization time of flight (MALDI-
TOF) (Fig. 6B) demonstrated perfectly monodisperse com-
pounds with molar masses equal to their theoretical values. This
demonstrates that, although the reactivity of the azide and amine
groups from their periphery may decrease while the generation
their core (Fig. 5). G2(COOH)N (Fig. 4) was activated both as
3
HOBt ester and as acid chloride (both in 99% conversion and
used without purification) (Fig. 5), whereas G3(COOH)N
3
(Fig.
4
) was activated only as HOBt ester (99% conversion and used
4
of 10
|
www.pnas.org/cgi/doi/10.1073/pnas.1700922114
Jishkariani et al.

Fig. 4. Synthesis of the methyl ester of 2,2-bis(aminomethyl)propionic acid, the convergent synthesis of three generations, and the divergent synthesis of six
generations of aliphatic polyamide dendrons. Reagents and conditions: (i) 10% Pd/C [10% (wt/wt)], H (120 psi), MeOH (23 °C, 3 h); (ii) 9, Et N, CH Cl (23 °C,
O [1/1/1 (vol/vol/vol)] (50 °C, 2 h); (v) DCC, HOBt, CH Cl (23 °C, 6 h, then
(reflux, 4 h), then G1(COOMe)NH , Et N, CH Cl (–78 °C, 0.5 h, then 23 °C, 15 h);
Cl (23 °C, 24 h); (ix) 9, Et N, CH Cl (23 °C, 48 h); (x) 10% Pd/C [20% (wt/wt)], H
(23 °C, 48 h); (xii) 10% Pd/C [30% (wt/wt)], H (120 psi), MeOH (23 °C, 2 h); (xiii) 9, Et N, CH Cl (23 °C, 48 h); and
(120 psi), MeOH (23 °C, 2 h).
2
3
2
2
2
3
4 h); (iii) 8, Et
5 °C, 2 h), then G1(COOMe)NH
3
N, CH
2
Cl
2
(–78 °C, 0.5 h, then 23 °C, 12 h); (iv) NaOH, THF/MeOH/H
, Et N, CH Cl , (23 °C, 24 h); (vi) SOCl , CH Cl
(120 psi), MeOH (23 °C, 2 h); (viii) 9, Et N, CH
120 psi), MeOH (23 °C, 2 h); (xi) 9, Et N, CH Cl
xiv) 10% Pd/C [80% (wt/wt)], H
2
2
2
2
3
2
2
2
2
2
2
3
2
2
(
(
(
vii) 10% Pd/C [10% (wt/wt)], H
2
3
2
2
3
2
2
2
3
2
2
2
3
2
2
2
number increases, there is a generation number at which at least
the azide groups become completely inactive toward heteroge-
neous hydrogenation catalyzed with 10% Pd/C as well as ho-
mogeneous hydrogenation by the Staudinger reaction. This lack
of reactivity produces a self-interruption of the dendrimer syn-
thesis process. Notably, this generation number is controlled by
the structure of the core even when the multiplicity of the core is
identical. This demonstrates that the conformation of the core is
either as important or even more important than its multiplicity.
demonstrate that the observed decrease in reactivity of the den-
drimer’s peripheral groups is commensurate with increasing se-
questration of these groups and an overall compactness of the
dendrimer. Calculated atom number densities and radial distribu-
tion functions (Figs. 7 and 8) indicate that an increasing number of
terminal groups are backfolded into the dendrimer interior or are
trapped in invaginations created on the dendrimers surface.
The calculated radius of gyration of the peripheral groups
g 3
R (N ), demarcated by the solid white line in the density con-
tours of Fig. 7, is only slightly greater than the radius of gyration
of the dendrimer (inner gray line), but always much less than that
of its calculated radius of solvent accessible surface area (rSASA,
outer gray line), indicating a tightly compact structure.
Molecular Modeling and Molecular-Dynamics Simulations of Azido-
Terminated Dendrimers. Long-timescale fully atomistic molecu-
lar-dynamics simulations, based on state-of-the-art force fields,
Jishkariani et al.
PNAS Early Edition
|
5 of 10

Fig. 5. Accelerated double-stage convergent–divergent
synthesis of three EDA generations and five PPZ gener-
ations of aliphatic polyamide dendrimers. (i) HOBt, DCC,
CH
reflux, 4 h); (iii) EDA, Et
Et N, CH Cl (23 °C, 24 h); (v) G2(PPZ)NH
23 °C, 12 h); (vi) G2(PPZ)NH , Et N, CH Cl
then 23 °C, 12 h); (vii) 10% Pd/C [50% (wt/wt)], H
2
Cl
2
(23 °C, 1 h, then 35 °C, 5 h); (ii) SOCl
N, CH Cl (23 °C, 24 h); (iv) PPZ,
, Et N, CH Cl
(–78 °C, 0.5 h,
2 2 2
, CH Cl
(
3
2
2
3
2
2
2
3
2
2
(
2
3
2
2
2
2
(
(
120 psi) (23 °C, 8 h); (viii) 10% Pd/C [80% (wt/wt)], H
120 psi) (23 °C, 8 h).
This ratio, R
g
(N
3
)/rSASA is always less than 1 and decreases as
surface area of a sphere is linearly proportional to the probe
radius, any computed deviations from linearity between these
values indicate penetration by the probe into the dendrimer in-
terior. Only a smaller probe can penetrate into surface invagina-
tions, leading to an apparent increase in surface area. Deviations
are indeed observed with probe radii less than ∼3 Å (Fig. 9) be-
ginning with the fourth-generation dendrimer and are more pro-
nounced at smaller probe radii and larger dendrimer generations.
This suggests that, in order for reactants to penetrate the
surface of the dendrimer, they must have an apparent radius less than
∼3 Å, or about the average distance between two oxygen atoms in
bulk water. Couple this with the likelihood for the reactant molecule
to at least partially desolvate to penetrate, and the observed decrease
in reactivity of the terminal groups becomes quite clear.
the dendrimer generation increases, indicating a propensity of
the outer groups to compact into the dendrimer structure, rather
than expand into the bulk solvent. This presumably leads to the
observed decrease and eventual cessation of their reactivity.
Furthermore, and most importantly, this ratio is also dependent
on the chosen core type, with the R (N )/r decrease per
SASA
g
3
generation being most pronounced for the EDA core, followed
by that of PPZ and finally COOMe. Thus, the computations fully
support the observation that the chosen core type influences the
reactivity of the peripheral groups, with the most rigid core
having the most pronounced effect. Last, the calculated radial
distribution functions, ρ(r) show a nonzero probability of finding
terminal groups (Fig. 8, solid teal line) backfolded into the
dendrimer. Calculated 3D atom number densities (Fig. 7, yellow
hotspots) indicate buried terminal groups as well.
Antimicrobial Properties of Dendrimers and Susceptibility to Enzymatic
and Chemical Hydrolysis. Naturally occurring peptides as well as
synthetic mimics including peptoids are known to exhibit antimi-
crobial activities (47–53). To test for potential antimicrobial ac-
tivity of the present aliphatic polyamide dendrimers terminated
with amine, cell viability assays using resazurin were performed.
Given the observed compactness of the dendrimer’s structure,
we wondered whether reactants could penetrate into the den-
dritic interior to react with the buried terminal groups. Using a
spherical probe with various radii, the surface areas of the model
dendrimers were calculated. Because the square root of the
Fig. 6. GPC traces (A) of Gn(EDA)N
Gn(EDA)NH (n = 1–3) and Gn(PPZ)NH
observed in the presence of sodium ions [M+Na] .
3
(n = 1–4) and Gn(PPZ)N
3 3
(n = 1–5) dendrimers and Gn(COOMe)N (n = 1–6) dendrons and MALDI-TOF spectra (B) of
2
2
(n = 1–4) dendrimers and Gn(COOMe)NH
2
(n = 2–5) dendrons. Calc. and Obs. are the molecular weights calculated and
+
6
of 10
|
www.pnas.org/cgi/doi/10.1073/pnas.1700922114
Jishkariani et al.

Fig. 7. Two-dimensional slices of the 3D average atom number densities (in atoms per cubic angstrom) of the terminal N
3
groups, and molecular renders for
Gn(EDA)N (n = 3–6), Gn(PPZ)N (n = 3–6) dendrimers, and Gn(COOMe)N (n = 3–6) dendrons. Continuous red line marks the maximum generation number
3
3
3
synthesized for the azide dendrimers with EDA, PPZ, and COOMe cores. Dotted red line marks the maximum generation number of the azide dendrimers that
could be hydrogenated to the corresponding amines. In the molecular renders, carbon atoms are colored gray; nitrogen, blue; oxygen, red; and hydrogen,
white. The number density contours are centered on the dendrimer’s center of mass (x = 0, z = 0) and look along the x axis. Dashed gray lines demarcate radial
g g 3
separations of 5 Å. The solid lines denote calculated properties: the radius of gyration (R ) of the dendrimer (inner gray line), the R of the terminal N groups
(thick white line), and the radius of the solvent-accessible surface area (rSASA) (outer gray line).
Resazurin is a redox indicator dye, which provides a readout expectedly, no hydrolysis was observed under any of these con-
for bacterial cell growth and viability (54). The minimal inhibitory ditions (Methods and SI Appendix). The enzymatic and hydrolytic
concentration (MIC) is measured as described in Methods.
G4(PPZ)NH
stability of these aliphatic polyamide dendrimers make them
2
was found to be active against all Gram-positive likely candidates for biomedical applications.
bacteria that were tested (Bacillus subtilis, Bacillus cereus, Micro-
coccus luteus, and Staphylococcus aureus—see SI Appendix, Table
Conclusions
S2, for MIC estimates). G2(PPZ)NH
2
did not display antibacterial Sterically hindered aliphatic polyamide dendrimers are expected
activity at concentrations up to 1,024 μg/mL.
to amplify the dependence of the reactivity of their peripheral
functional groups on their core structure and generation number.
Understanding this reactivity–core structure–generation de-
pendence is important to elucidate the synthesis of such con-
structs for potential applications in nanomedicine. Toward this
goal, a study of the divergent, convergent, and double-stage
convergent–divergent iterative synthesis of the most sterically
To test susceptibility to enzymatic hydrolysis, polyamide den-
2 2 3
drimers G2(PPZ)NH , G4(PPZ)NH , and G4(PPZ)N were
treated with a broad-spectrum protease, proteinase K (EC
3
.4.21.64) (55), which is a serine protease with a broad specificity
1
toward peptide substrates, and analyzed by H NMR and
MALDI-TOF (Methods). All three dendrimers were found to be
stable against enzymatic degradation (Methods and SI Appendix). hindered polyamide dendrimer known to date (12) was per-
The hydrolytic stability of these dendrimers was tested for 24 h at
formed in gram quantities (Fig. 10) with three different cores
(EDA, PPZ, COOMe) having the same multiplicity but different
conformations.
2
3 °C and 80 °C under base-catalyzed conditions for G4(PPZ)N3
and for G3(EDA)NH2 under acid-catalyzed conditions. Un-
Jishkariani et al.
PNAS Early Edition
|
7 of 10

3 3 3
Fig. 8. Radial distribution functions ρ(r) of (A) Gn(EDA)N (n = 3–6), (B) Gn(PPZ)N (n = 3–6) dendrimers, and (C) Gn(COOMe)N (n = 3–6) dendrons. The
calculations were done using the dendrimer and dendron center of mass as a reference (r = 0 Å). The unit value for ρ(r) is expressed in atoms per cubic
2
angstrom and quantifies the number of atoms in the volume element given by 4πr . The abbreviations in the legends stand for the following: ALL, Gn(EDA/
3
PPZ/COOMe)N dendrimer or dendron; AmMPA, 2,2-bis(aminomethyl)propionic acid repetitive units; AzMPA, 2,2-bis(azidomethyl)propionic acid unit; CORE,
EDA/ PPZ/COOMe. Note that, regardless of core type, there exists a nonzero terminal group density within the dendrimer (teal line) indicating backfolding
and sequestering of the terminal azido groups away from the dendrimer surface.
A four-step synthesis in high yield, short reaction time, and
under mild reaction conditions of the starting compound [2,2-
bis(azidomethyl)propionic acid obtained from the commercial
make these dendrimer nanoarchitectures interesting candidates
for nanomedicine.
2
,2-bis(hydroxymethyl)propionic acid] was elaborated to facili-
Methods
tate this study. Unexpectedly, the conformation rather than the
multiplicity of the core provided self-interruption of the iterative
synthesis at a predictable generation number. These results ex-
pand on previous work that predicted and demonstrated a
change in the reactivity of the functional groups for less sterically
hindered polyamide dendrimers such as PAMAM (1, 2, 32).
Molecular modeling and molecular-dynamics simulations dem-
onstrated that this self-interruption is likely due to the back-
folding of the functional groups on the dendrimer periphery. The
steric hindrance of these aliphatic polyamide dendrimers makes
them behave like peptoids (53) rather than as peptides of
β-amino acids, and consequently they are both enzymatically and
hydrolytically stable. While demonstrating self-interruption syn-
thesis of sterically hindered aliphatic polyamide dendrimers,
these experiments also provided quantitative mechanistic in-
sights into the De Gennes self-limiting growth and dense packing
parameters (32). This combination of function together with
their gram quantity synthesis, and their antimicrobial properties,
Synthesis of Dendrimers and Dendrons. Dendrimers and dendrons were syn-
thesized by divergent, convergent, and double-stage convergent–divergent
iterative methods starting from the commercially available 2,2-bis(hydroxy-
methyl)propionic acid. Details of their synthesis and structural analysis
methods and data are available in SI Appendix.
In Silico Modeling and Molecular-Dynamics Simulations. The monomers used in
constructing models of the dendrimer macromolecules were taken from ref.
12. The molecules were described by the Amber GAFF forcefield (56) except
for the terminal azido group for which parameters were used from previous
work (57). Fully atomistic molecular-dynamics simulations were performed
using NAMD 2.11 (58). The NPT ensemble was applied with a temperature of
300 K and a pressure of 1.0 atm. Methanol was used as a solvent and was
described using the Amber force field. A time step of 1 fs was used. Tra-
jectories of each of the 18 systems (3 cores, 6 generations each) were col-
lected for a minimum of 100 ns, of which the last 20 ns was used for analysis.
Dendrimer rmsd and autocorrelation functions of the square of the radius of
gyration were used to establish equilibrium and relevant decorrelation
8
of 10
|
www.pnas.org/cgi/doi/10.1073/pnas.1700922114
Jishkariani et al.

3 3
Fig. 9. Square root of solvent-accessible surface area (SASA) as a function of probe radius p for (A) Gn(EDA)N (n = 1–6) and (B) Gn(PPZ)N (n = 1–6)
dendrimers, and (C) Gn(COOMe)N (n = 1–6) dendrons. Color code: blue (n = 1), green (n = 2), gray (n = 3), red (n = 4), orange (n = 5), and yellow (n = 6).
3
Deviations from linearity indicate probe sizes necessary to penetrate the dendrimer surface and explore small cavities and surface invaginations. For gen-
erations n = 4–6, probe sizes less than r ∼ 3 Å are required for penetration, suggesting that reactants must be at least this small to interact with the se-
questered 2,2-bis(azidomethyl)propionic acid (AzMPA) groups.
times. The VMD software package (59) was used to perform the following
analyses: the radial distribution function, autocorrelation function, radius of
gyration, solvent-accessible surface area, fractal dimension, and aspect ra-
tios. The MD analysis package (60) was used to calculate the 3D atomic
densities. Thorough accounts of the modeling and simulation details are
available in SI Appendix.
OD600 were diluted 1:100 fold and 50 μL of cells were added to each well.
The plate was placed on a shaker for 15 min, followed by stationary in-
cubation at 37 °C for 16–18 h. For M. luteus, the incubation was carried out
for ∼48 h as the strain grows slowly at 37 °C. Resazurin turns from blue to
pink color as a function of cell growth. A MIC value was determined by the
lowest concentration of dendrimer at which there was no color change. All
estimates were derived from at least three independent trials.
Cell Viability Assays Using Resazurin. Cell viability assays to determine MICs
were performed using resazurin based on established protocols (61). Satu-
rated cultures of the strains to be tested were grown in Mueller Hinton
broth (MHB) (Difco) at 37 °C with aeration for 16–18 h. The cultures were
then diluted 1:100 in fresh MHB and allowed to grow to an optical density at
Proteinase K Digestion. Proteinase K digestions of the dendrimers terminated
with an amine group were performed as follows: 5 mg of the dendrimer
2 2
G2(PPZ)NH or G4(PPZ)NH was added to 5 mg of proteinase K in a micro-
centrifuge tube and resuspended in 200 μL of 30 mM Tris·HCl, pH 8. A
control for proteinase K without addition of dendrimer was included. The
tubes were incubated at 37 °C overnight. Following ∼16 h at 37 °C, the
above reactions were dehydrated at room temperature in a Speedvac
to facilitate analysis of samples by NMR and MALDI-TOF. All of the
6
00 nm (OD600) of ∼0.2. A 2× dilution scheme of the test dendrimer was
prepared in MHB in a 96-well clear microtiter plate such that the final
concentrations were 0, 1, 2, 4, 8, 16, 32, 64, 128, 256, 512, and 1,024 μg·mL−
in a 50-μL volume. Five microliters of 0.675% resazurin stock solution was
added to each well containing the dendrimer solution. Cultures grown to
1
2
Fig. 10. Gram-scale synthesis of G4(PPZ)NH using the 1H-benzo[d][1,2,3]triazol-1-yl-2,2-bis(azidomethyl)propanoate, 9.
Jishkariani et al.
PNAS Early Edition
|
9 of 10

dendrimers were recovered, and their structures were confirmed by NMR
and MALDI-TOF.
search from the School of Dental Medicine at the University of Pennsylvania
(to V.P. and R.W.), National Science Foundation Grant DMR-1120901 (to M.L.K.
and M.G.), and National Institutes of Health Grant R01-GM080279 (to M.G.).
Computer time was provided through National Science Foundation Grant ACI-
1614804, the Petascale Computing Resource Allocations allocation baeg, and
generous support from the National Center for Supercomputer Applications
(C.M.M. and M.L.K.).
ACKNOWLEDGMENTS. This work was supported by National Science Foun-
dation Grants DMR-1066116 and DMR-1120901 (to V.P.), the P. Roy Vagelos
Chair at the University of Pennsylvania (to V.P.), the Humboldt Foundation
(
V.P.), the Joseph and Josephine Rabinowitz Award for Excellence in Re-
1
2
. Tomalia DA, et al. (1985) A new class of polymers: Starburst-dendritic macromole-
cules. Polym J 17(1):117–132.
. Tomalia DA, Naylor AM, Goddard WA (1990) Starburst dendrimers: Molecular-level
control of size, shape, surface chemistry, topology and flexibility from atoms to
macroscopic matter. Angew Chem Int Ed Engl 29(2):138–175.
32. De Gennes PG, Hervet H (1983) Statistics of “Starburst” polymers. J Phys Lett 44(9):
L-351–L-360.
33. Lescanec RL, Muthukumar M (1990) Configurational characteristics and scaling be-
havior of starburst molecules:
A computational study. Macromolecules 23(8):
2280–2288.
3
4
5
. Newkome GR, Yao Z, Baker GR, Gupta VK (1985) Cascade molecules: A new approach
to micelles. A [27]-Arborol. J Org Chem 50(11):2003–2004.
. Pittelkow M, Christensen JB (2005) Convergent synthesis of internally branched
PAMAM dendrimers. Org Lett 7(7):1295–1298.
. Liu XX, et al. (2009) PAMAM dendrimers mediate siRNA delivery to target Hsp27 and
produce potent antiproliferative effects on prostate cancer cells. ChemMedChem 4(8):
34. Mourey TH, et al. (1992) Unique behavior of dendritic macromolecules: Intrinsic vis-
cosity of polyether dendrimers. Macromolecules 25(9):2401–2406.
35. Ihre H, Hult A, Söderlind E (1996) Synthesis, characterization, and H NMR self-dif-
1
fusion studies of dendritic aliphatic polyesters based on 2,2-bis(hydroxymethyl)pro-
pionic acid and 1,1,1-tris(hydroxyphenyl)ethane. J Am Chem Soc 118(27):6388–6395.
36. Ihre H, Padilla De Jesús OL, Fréchet JMJ (2001) Fast and convenient divergent syn-
thesis of aliphatic ester dendrimers by anhydride coupling. J Am Chem Soc 123(25):
5908–5917.
1
302–1310.
6
. Liu X, et al. (2011) Structurally flexible triethanolamine core PAMAM dendrimers are
effective nanovectors for DNA transfection in vitro and in vivo to the mouse thymus.
Bioconjug Chem 22(12):2461–2473.
. Yoon K, Goyal P, Weck M (2007) Monofunctionalization of dendrimers with use of
microwave-assisted 1,3-dipolar cycloadditions. Org Lett 9(11):2051–2054.
. Ornelas C, Broichhagen J, Weck M (2010) Strain-promoted alkyne azide cycloaddition
for the functionalization of poly(amide)-based dendrons and dendrimers. J Am Chem
Soc 132(11):3923–3931.
37. García-Gallego S, Nyström AM, Malkoch
M (2015) Chemistry of multifunctional
polymers based on bis-MPA and their cutting-edge applications. Prog Polym Sci 48:
85–110.
7
38. Percec V, et al. (2010) Self-assembly of Janus dendrimers into uniform den-
drimersomes and other complex architectures. Science 328(5981):1009–1014.
39. Peterca M, Percec V, Leowanawat P, Bertin A (2011) Predicting the size and properties
of dendrimersomes from the lamellar structure of their amphiphilic Janus den-
drimers. J Am Chem Soc 133(50):20507–20520.
40. König W, Geiger R (1970) [A new method for synthesis of peptides: Activation of the
carboxyl group with dicyclohexylcarbodiimide using 1-hydroxybenzotriazoles as ad-
ditives]. Chem Ber 103(3):788–798. German.
8
9
. Ornelas C, Pennell R, Liebes LF, Weck M (2011) Construction of a well-defined mul-
tifunctional dendrimer for theranostics. Org Lett 13(5):976–979.
1
1
1
1
1
0. Kim C, et al. (2001) Supramolecular assembly of amide dendrons. J Am Chem Soc
23(23):5586–5587.
1
1. Rannard S, Davis N, McFarland H (2000) Synthesis of dendritic polyamides using novel
selective chemistry. Polym Int 49(9):1002–1006.
2. Ruiz-Sanchez AJ, et al. (2015) Synthesis of all-aliphatic polyamide dendrimers based
on a 3,3′-diaminopivalic acid scaffold. Polym Chem 6(16):3031–3038.
3. Mesa-Antunez P, et al. (2016) Fluorescent BAPAD dendrimeric antigens are efficiently
internalized by human dendritic cells. Polymers (Basel) 8(4):111.
4. Newkome RG, Shreiner DC (2007) Poly(amidoamine), polypropylenimine, and related
dendrimers and dendrons possessing different 1 → 2 branching motifs: An overview
of the divergent procedures. Polymer (Guildf) 49(1):1–173.
5. Denkewalter RG, Kolc JF, Lukasavage WJ (1983) Macromolecular highly branched
homogeneous compound. US Patent 4,410,688.
6. Rosen BM, et al. (2009) Dendron-mediated self-assembly, disassembly, and self-
organization of complex systems. Chem Rev 109(11):6275–6540.
7. Hawker CJ, Fréchet JMJ (1990) Preparation of polymers with controlled molecular
architecture. A new convergent approach to dendritic macromolecules. J Am Chem
Soc 112(21):7638–7647.
41. Batz H-G, Franzmann G, Ringsdorf H (1972) Model reactions for synthesis of phar-
macologically active polymers by way of monomeric and polymeric reactive esters.
Angew Chem Int Ed Engl 11(12):1103–1104.
42. El-Faham A, Albericio F (2011) Peptide coupling reagents, more than a letter soup.
Chem Rev 111(11):6557–6602.
43. Jhon MS, Cho U-L, Kier LB, Eyring H (1972) Molecular orbital studies of ethylenedi-
amine conformations. Proc Natl Acad Sci USA 69(1):121–123.
44. Boiocchi M, et al. (2004) The influence of the boat-to-chair conversion on the de-
metallation of the nickel(II) complex of an open-chain tetramine containing a pi-
perazine fragment. Dalton Trans 2004(4):653–658.
1
1
1
45. Paital AR, et al. (2009) Structure and dimensionality of coordination complexes cor-
II
II
related to piperazine conformation: From discrete [Cu
μ
2
] and [Cu
3 2 n
1,3-N bridged [Cu ] chain. Dalton Trans 2009(8):1352–1362.
4
] complexes to a
−
II
46. Tomalia DA, Khanna SN (2016) A systematic framework and nanoperiodic concept for
unifying nanoscience: Hard/soft nanoelements, superatoms, meta-atoms, new
emerging properties, periodic property patterns, and predictive Mendeleev-like
nanoperiodic tables. Chem Rev 116(4):2705–2774.
47. Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415(6870):
389–395.
48. Fernandez-Lopez S, et al. (2001) Antibacterial agents based on the cyclic D,L-α-peptide
architecture. Nature 412(6845):452–455.
49. Porter EA, Wang X, Lee HS, Weisblum B, Gellman SH (2000) Non-haemolytic β-amino-
acid oligomers. Nature 404(6778):565.
50. Hamuro Y, Schneider JP, DeGrado WF (1999) De novo design of antibacterial β-pep-
tides. J Am Chem Soc 121(51):12200–12201.
51. Tew GN, et al. (2002) De novo design of biomimetic antimicrobial polymers. Proc Natl
Acad Sci USA 99(8):5110–5114.
52. Kuroda K, DeGrado WF (2005) Amphiphilic polymethacrylate derivatives as antimi-
crobial agents. J Am Chem Soc 127(12):4128–4129.
1
1
2
8. Wooley KL, Hawker CJ, Fréchet JMJ (1991) Hyperbranched macromolecules via a
novel double-stage convergent growth approach. J Am Chem Soc 113(11):4252–4261.
9. Naylor AM, Goddard WA, III, Kiefer GE, Tomalia DA (1989) Starburst dendrimers. 5.
Molecular shape control. J Am Chem Soc 111(6):2339–2341.
0. Percec V, Cho W-D, Mosier PE, Ungar G, Yeardley DJP (1998) Structural analysis of
cylindrical and spherical supramolecular dendrimers quantifies the concept of mon-
odendron shape control by generation number. J Am Chem Soc 120(43):11061–11070.
1. Maiti PK, Cagin T, Wang G, Goddard WA, III (2004) Structure of PAMAM dendrimers:
Generations 1 through 11. Macromolecules 37(16):6236–6254.
2
2
2
2. Percec V, et al. (2000) Design and structural analysis of the first spherical mono-
dendron self-organizable in a cubic lattice. J Am Chem Soc 122(17):4249–4250.
3. Rosen BM, et al. (2009) Predicting the structure of supramolecular dendrimers via the
3 2
analysis of libraries of AB and constitutional isomeric AB biphenylpropyl ether self-
assembling dendrons. J Am Chem Soc 131(47):17500–17521.
4. Percec V, et al. (2006) Synthesis and retrostructural analysis of libraries of AB and
3
53. Chongsiriwatana NP, et al. (2008) Peptoids that mimic the structure, function, and
mechanism of helical antimicrobial peptides. Proc Natl Acad Sci USA 105(8):
2794–2799.
54. Fields RD, Lancaster MV (1993) Dual-attribute continuous monitoring of cell pro-
liferation/cytotoxicity. Am Biotechnol Lab 11(4):48–50.
2
2
2
constitutional isomeric AB
J Am Chem Soc 128(10):3324–3334.
5. Percec V, Cho W-D, Ungar G, Yeardley DJP (2001) Synthesis and structural analysis of
two constitutional isomeric libraries of AB -based monodendrons and supramolecular
2
phenylpropyl ether-based supramolecular dendrimers.
2
55. Ebeling W, et al. (1974) Proteinase K from Tritirachium album Limber. Eur J Biochem
47(1):91–97.
dendrimers. J Am Chem Soc 123(7):1302–1315.
6. Esfand R, Tomalia DA (2001) Poly(amidoamine) (PAMAM) dendrimers: From bio-
mimicry to drug delivery and biomedical applications. Drug Discov Today 6(8):
56. Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA (2004) Development and testing
of a general amber force field. J Comput Chem 25(9):1157–1174.
57. Carvalho ATP, Fernandes PA, Ramos MJ (2007) Parameterization of AZT—a widely
used nucleoside inhibitor of HIV-1 reverse transcriptase. Int J Quantum Chem 107(2):
292–298.
58. Phillips JC, et al. (2005) Scalable molecular dynamics with NAMD. J Comput Chem
26(16):1781–1802.
59. Humphrey W, Dalke A, Schulten K (1996) VMD: Visual molecular dynamics. J Mol
Graph 14(1):33–38, 27–28.
4
27–436.
7. Duncan R, Izzo L (2005) Dendrimer biocompatibility and toxicity. Adv Drug Deliv Rev
7(15):2215–2237.
2
2
2
5
8. Lee CC, MacKay JA, Fréchet JMJ, Szoka FC (2005) Designing dendrimers for biological
applications. Nat Biotechnol 23(12):1517–1526.
9. Kannan RM, Nance E, Kannan S, Tomalia DA (2014) Emerging concepts in dendrimer-
based nanomedicine: From design principles to clinical applications. J Intern Med
2
76(6):579–617.
60. Michaud-Agrawal N, Denning EJ, Woolf TB, Beckstein O (2011) MDAnalysis: A toolkit
for the analysis of molecular dynamics simulations. J Comput Chem 32(10):2319–2327.
61. Sarker SD, Nahar L, Kumarasamy Y (2007) Microtitre plate-based antibacterial assay
incorporating resazurin as an indicator of cell growth, and its application in the in
vitro antibacterial screening of phytochemicals. Methods 42(4):321–324.
3
3
0. Svenson S, Tomalia DA (2005) Dendrimers in biomedical applications—reflections on
the field. Adv Drug Deliv Rev 57(15):2106–2129.
1. Svenson S (2015) The dendrimer paradox—high medical expectations but poor clinical
translation. Chem Soc Rev 44(12):4131–4144.
1
0 of 10
|
www.pnas.org/cgi/doi/10.1073/pnas.1700922114
Jishkariani et al.