Synthesis of an enlarged library of dynamic DNA activators with oxime, disulfide and hydrazone bridges

Article abstract of DOI:10.1002/chem.201200550

Dynamic amphiphiles have a bridge between their charged head and their hydrophobic tails. The presence of dynamic covalent bonds is of interest for differential and biosensing applications as well as for rapid access to the libraries needed to screen for gene delivery or cellular uptake of siRNA. However, efforts to develop libraries have so far concentrated on hydrazone bridges to monocationic heads. Here, we report synthesis efforts to enlarge this focused library with oxime and disulfide bridges and dynamic amphiphiles with more than one positive charge. Evaluation in fluorogenic vesicles reveals best activation of DNA as ion transporters by dynamic amphiphiles with dendritic scaffolds, doubly charged heads and four tails. Moreover, oximes, contrary to hydrazones, remain active under acidic conditions. Linear elongation of dendritic head-groups seems to cause increasing detergent effects and should therefore be avoided. Head on: Design, synthesis and evaluation of dynamic amphiphiles with singly or doubly charged peptide dendrons as head-groups; hydrazones, oximes and disulfides as bridges; and with fragrant tails are reported. Amphiphiles with two charges and four tails (see figure) are identified as the most powerful activators of DNA as ion transporters and thus as the most promising family to screen for gene and siRNA delivery. Copyright

Full text of DOI:10.1002/chem.201200550

FULL PAPER
DOI: 10.1002/chem.201200550
Synthesis of an Enlarged Library of Dynamic DNA Activators with Oxime,
Disulfide and Hydrazone Bridges
Javier Montenegro,[a, b] Eun-Kyoung Bang,[a] Naomi Sakai,[a] and Stefan Matile*[a]
Abstract: Dynamic amphiphiles have a
“bridge” between their charged head
and their hydrophobic tails. The pres-
ence of dynamic covalent bonds is of
interest for differential and biosensing
applications as well as for rapid access
to the libraries needed to screen for
gene delivery or cellular uptake of
siRNA. However, efforts to develop li-
braries have so far concentrated on hy-
drazone bridges to monocationic heads.
Here, we report synthesis efforts to en-
large this focused library with oxime
and disulfide bridges and dynamic am-
phiphiles with more than one positive
charge. Evaluation in fluorogenic vesi-
cles reveals best activation of DNA as
ion transporters by dynamic amphi-
philes with dendritic scaffolds, doubly
charged heads and four tails. More-
over, oximes, contrary to hydrazones,
remain active under acidic conditions.
Linear elongation of dendritic head-
groups seems to cause increasing deter-
gent effects and should therefore be
avoided.
Keywords: amphiphiles · disulfides ·
DNA · hydrazones · ion transport-
ers
Introduction
Dynamic amphiphiles have a charged head, a hydrophobic
tail, and a dynamic connector or “bridge” (Figure 1).[1–4] The
dynamic covalent bonds of bridges, for example, hydrazones,
disulfides or oximes, are weaker than covalent bonds but
stronger than noncovalent interactions, such as hydrogen
bonds.[5,6] Because of the dynamic nature of their bridges,
dynamic amphiphiles can be formed, modified and de-
stroyed, in situ, and they can transform during function, de-
pending on conditions. The unique characteristics of dynam-
ic amphiphiles are of interest in the context of polyion–
counterion transport systems.[1–4,7–10] In general, the move-
ment of polyions across lipid bilayer membranes is strongly
influenced by their counterions. Hydrophilic counterions are
referred to as inactivators because they produce hydrophilic
polyion–counterion complexes that do not interact with the
membrane. Amphiphilic counterions are activators because
they give hydrophobic polyion–counterion complexes that
can move across bulk and bilayer membranes and act as car-
Figure 1. Dynamic amphiphiles are composed of charged heads and hy-
drophobic tails that are linked together by dynamic “bridges” that can
form, change and break in situ in response to changing conditions. This
report adds oximes and disulfides to the previously reported collection of
hydrazone bridges.
riers of hydrophilic counterions. Dynamic amphiphiles with
positively charged heads can thus activate DNA or RNA as
cation transporters in lipid bilayer membranes.[8,9] Negative-
ly charged dynamic amphiphiles can activate cell-penetrat-
ing peptides (CPPs, or protein transduction domains,
PTDs)[7,10] as anion transporters.[11]
In this context, dynamic amphiphiles offer several advan-
tages. They were introduced originally to sense hydrophobic
analytes with membrane-based sensing systems.[1,2] Early ap-
plications toward cholesterol biosensors focused on signal
generation with cholesterol oxidase, covalent capture of the
cholestenone product with an anionic hydrazide, and activa-
tion of CPPs with the obtained dynamic amphiphile.[2] How-
ever, attention soon shifted to the general challenge of dif-
ferential sensing with membrane-based sensing systems.[1]
Covalent capture of analytes with several different cationic
[a] Dr. J. Montenegro, Dr. E.-K. Bang, Dr. N. Sakai, Prof. S. Matile
Department of Organic Chemistry
University of Geneva, Geneva (Switzerland)
Fax : (+41)22-379-3215
E-mail: stefan.matile@unige.ch
Homepage: www.unige.ch/sciences/chiorg/matile/
[b] Dr. J. Montenegro
Current address: Departamento de Quꢀmica Orgꢁnica
y Centro Singular de Investigaciꢂn en Quꢀmica Biolꢂgica
y Materiales Moleculares (CIQUS)
University of Santiago de Compostela
Santiago de Compostela (Spain)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201200550.
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Figure 2. Library design for dynamic amphiphiles based on established heads (black box) and selected tails T with emphasis on oxime bridges, doubly
bridged amphiphiles with disulfide and hydrazone bridges, and with doubly charged heads with as many or as few hydrazone bridges as possible.
hydrazides A1H2, G1H2, A1H3 and G1H3 gave the dynam-
ic amphiphiles that activated DNA differently and thus
could be used to generate patterns. Analysis of the obtained
patterns by routine methods was found to discriminate odor-
ants, including enantiomers, cis–trans isomers and single-
atom homologues, as well as perfumes (Figure 2).[1]
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Dynamic DNA Activators
FULL PAPER
For gene delivery or cellular uptake of siRNA,[4,9] dynam-
ic amphiphiles are attractive because they provide facile
access to libraries.[3] With rational design and predictions
from model systems remaining problematic, the screening of
large libraries appears to be a most promising approach to
achieve progress in this field. So far, we have reported ten
peptidic head-groups with one ammonium or guanidinium
cation and one to six hydrazone tails (Figure 2).[1,3] Com-
bined with 39 hydrophobic tails, this small collection has al-
ready given access to 390 dynamic amphiphiles without
much effort spent on their synthesis. This focused library is
currently being screened for siRNA uptake.[12] Here, we de-
scribe the expansion of this library toward the inclusion of
oximes, disulfides as well as multiply charged head-groups.
We report high activity for doubly charged amphiphiles and
high stability of oximes under acidic conditions, whereas the
linear elongation of dendritic heads needed to install oximes
and disulfides leads to increasing interference from nonspe-
cific detergent effects.
creasing the number of charges in the head-group should be
most effective with four tails, in which case singly charged
G1H4 starts to lose activity because of being too hydropho-
bic. Guanidinium (G2H4) and ammonium cations (A2H4)
were selected to modulate the proximity effects originating
from intramolecular charge repulsion.[7] Single tail, multiply
charged amphiphiles at the other end of the established
structural space, that is, G2H1 and higher homologues, will
be of interest to handle unusual tails of exceptional hydro-
phobicity. G2H1 and the GnH1 family will be described
elsewhere once all matching tails are made and studied.[12]
Synthesis: G1O1 was readily synthesized from monoprotect-
ed ethylenediamine (1) and the protected alkoxyamine of
glycolic acid (2; Scheme 1; see the Supporting Information).
Standard peptide coupling conditions with TBTU as activat-
ing agent gave amide 3, which was deprotected chemoselec-
tively by hydrogenolysis. Guanidinylation of amine 4 with
Results and Discussion
Design: The head-groups so far available for the construc-
tion of libraries of cationic amphiphiles contain an ammoni-
um (A) or a guanidinium (G) cation and one (A1H1,
G1H1) to six (A1H6, G1H6, not shown) hydrazone
bridges.[1,3] DNA activation was always better with guanidi-
nium than with ammonium cations. This known difference is
attributed to the high pKa of guanidinium cations (i.e., per-
manent charges),[7] the in-plane directionality of their hydro-
gen bonds, and the delocalization of their positive charge to
possibly support arene-templated ion pairing. Activities in-
creased with the number of tails until G1H3.[1] With G1H4
and particularly G1H6, satisfactory to high activities were
observed with shorter tails, whereas longer tails did not
function well, presumably because the polyion–counterion
complexes obtained with calf-thymus (ct) DNA became too
hydrophobic.[3]
To expand our library based on these trends, four possibil-
ities were considered. Substitution of the hydrazone bridges
with the more stable, acid resistant oxime bridges[6] should
reveal the impact of different stability of dynamic amphi-
philes on DNA activation, sensing and cellular uptake. For
these reasons, we decided to develop the oxime series with
one guanidinium head and up to four tails, that is, from
G1O1 to G1O4 (Figure 2). Dynamic amphiphiles with disul-
fide and hydrazone bridges should respond to both acid and
reduction.[5] We decided to explore these doubly bridged
amphiphiles first with the most informative combination of
one guanidinium head and two (G1S2H2) to three tails
(G1S3H3).
Scheme 1. Synthesis of G1O1 and G1O2: a) TBTU, DIEA, DCM, room
temperature, 54%; b) Pd/C, H2, MeOH, 1m HCl in Et2O, room tempera-
ture, 87%; c) N,N’-di-Boc-1H-pyrazole-1-carboxamidine, DIEA, MeOH,
room temperature, 67%; d) 1m HCl in H2O, MeOH, 708C, 5 min, 71%;
e) TBTU, DIEA, DCM, room temperature, 66%; f) Et3SiH, TFA/DCM
1:1, room temperature, quantitative; g) TBTU, DIEA, DMF, room tem-
perature, 52%; h) Pd/C, 1m HCl in Et2O, H2, MeOH, room temperature,
89%; i) N,N’-di-Boc-1H-pyrazole-1-carboxamidine, DIEA, MeOH, room
temperature, 65%; j) 1m HCl in H2O, MeOH, 708C, 5 min, 72%.
Multiply charged head-groups are very attractive because,
driven by their need to minimize intramolecular charge re-
pulsion, their ability to form inert intermolecular ion pairs
should be exceptional (as long as their acidity is weak
enough to avoid charge reduction by deprotonation).[7,13] In-
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N,N’-di-Boc-1H-pyrazole-1-carboxamidine gave the protect-
ed target molecule 5, which was converted into G1O1 under
acidic conditions.
Multiaminoxy head-groups were all synthesized from glu-
tamate. The synthesis of the simplest member of this family,
G1O2, was carried out from amine-protected glutamate 6
(Scheme 1). Amide formation with partially Boc-protected
ethylenediamine 7 gave diamide 8. Boc removal with acid
gave diamine 9, which was treated with the aminoxy glyco-
late 2. Tetraamide 10 was chemoselectively deprotected, and
the resulting amine 11 was guanidinylated. Deprotection of
the obtained product 12 gave the desired G1O2.
The minimalist peptide dendron[14] G1O3 was prepared
from glutamate derivatives 13 and 14 (Scheme 2). Selective
deprotection of the coupling product 15 gave triacid 16,
which was treated with amine 7 to give triamide 17. Boc re-
moval prepared for the attachment of the aminoxy glycolate
2 to amine 18. Selective deprotection of 19 by hydrogenoly-
sis gave amine 20 in quantitative yield. Guanidinylation af-
forded 21, which was deprotected to give the target mole-
cule G1O3.
G1O4, the most complex peptide dendron of the series,
could be prepared from intermediate 11. Coupling to gluta-
mate 6 afforded 22 with the desired four-tail scaffold. Selec-
tive deprotection followed by guanidinylation of 23 and
final deprotection of 24 gave the desired target molecule.
The less demanding disulfide hydrazide G1S2H2 was syn-
thesized from glutamate 25 (Scheme 3). Coupling with mo-
noprotected ethylenediamine 1 gave diamide 26, which was
deprotected selectively under acidic conditions. The ob-
tained amine 27 was guanidinylated. Selective deprotection
of diamide 28 by hydrogenolysis gave diamine 29. To intro-
duce the disulfide bridges, diacid 30 was partially coupled
with the Boc-protected hydrazine 31. The resulting acid 32
was treated with diamine 29, and product 33 was fully de-
protected to give the target molecule, G1S2H2.
The higher homologue G1S3H3 was synthesized analo-
gously (Scheme 3). The glutamates 14 and 34 were coupled
to give the branched minidendron 35. Deprotection and
“umpolung” of triacid 36 with monoprotected ethylenedi-
Scheme 2. Synthesis of G1O3 and G1O4: a) TBTU, DIEA, DCM, room
temperature, 79%; b) Et3SiH, TFA/DCM 1:1, room temperature, 79%;
c) TBTU, DIEA, DMF, room temperature, 61%; d) Et3SiH, TFA/DCM
1:1, room temperature, 88%; e) TBTU, DIEA, DMF, room temperature,
59%; f) Pd/C, 1m HCl in Et2O, H2, MeOH, room temperature, quantita-
tive; g) N,N’-di-Boc-1H-pyrazole-1-carboxamidine, DIEA, MeOH, room
temperature, 66%; h) 1m HCl in H2O, MeOH, 708C, 5 min, 63%;
i) TBTU, DIEA, DMF, room temperature, 49%; j) Pd/C, H2, MeOH, 1m
HCl in Et2O, room temperature, 93%; k) N,N’-di-Boc-1H-pyrazole-1-car-
boxamidine, DIEA, MeOH, room temperature, 46%; l) 1m HCl in H2O,
MeOH, 708C, 5 min, 63%.
ACHTUNGTRENNUNGamine (1) followed by deprotection and guanidinylation
gave dendron 37. To introduce disulfides and hydrazides to-
gether, the three amines were liberated chemoselectively for
the amide coupling of 38 with acid 32. Deprotection of the
obtained product 39 gave the desired trihydrazide, G1S3H3.
The dicationic G2H4 was readily accessible from inter-
mediate 40 of the previously reported syntheses of A1H4
(Scheme 4). Attachment of lysine 41 and liberation of the
Cbz-protected amines in 42 gave diamine 43. Guanidinyla-
tion and complete deprotection of product 44 gave the
doubly charged target molecule G2H4. The diamine ana-
logue A2H4 was obtained from the same starting material
(see the Supporting Information). The synthesis of G2H1
will be described in the context of efforts to introduce more
unusual tails from the materials sciences, including fuller-
enes, mesogens or fluorophiles, with multiply charged head-
groups.
Evaluation: The new head-groups were evaluated with
linear alkyl tails of different length from six carbons in T6
to eighteen carbons in T18 plus the branched 2-methyl un-
9
decyl tail T11M and the kinked oleoyl tail T18D (Figure 2).
Hydrazones and oximes were produced in situ by incubation
for 1 h in DMSO at 608C. Product formation was confirmed
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Dynamic DNA Activators
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Scheme 4. Synthesis of G2H4: a) TBTU, DIEA, DCM, room tempera-
ture, 4 h, 80%; b) Pd(OH)2/C, H2, MeOH, room temperature, 3 h, quanti-
tative; c) N,N’-di-Boc-1H-pyrazole-1-carboxamidine, DIEA, CH3CN/H2O
10:1, 558C, 12 h, 52%; d) 1m HCl in Et2O, DCM, 708C, 24 h, 61%.
Scheme 3. Synthesis of G1S2H2 and G1S3H3: a) TBTU, DIEA, DCM,
room temperature, 72%; b) Et3SiH, TFA/DCM 1:1, room temperature,
81%; c) N,N’-di-Boc-1H-pyrazole-1-carboxamidine, DIEA, MeOH, room
temperature, 47%; d) Pd/C, H2, MeOH, 1m HCl in Et2O, room tempera-
ture, quantitative; e) EDC, DIEA, DCM, room temperature, 15%;
f) TBTU, DIEA, DMF, room temperature, 64%; g) 1m HCl in H2O,
MeOH, 708C, 5 min, 85%; h) TBTU, DIEA, DCM, room temperature,
70%; i) Et3SiH, TFA/DCM 1:1, room temperature, 86%; j) 1) TBTU,
DIEA, 1, DMF, room temperature, 2) DMF/DMSO/piperidine 8:2:3,
room temperature, 3) N,N’-di-Boc-1H-pyrazole-1-carboxamidine, DIEA,
MeOH, room temperature, 30% (3 steps); k) Pd/C, 1m HCl in Et2O, H2,
MeOH, room temperature, quantitative; l) TBTU, DIEA, 32, DCM,
room temperature, 61%; m) 1m HCl in H2O, MeOH, 708C, 5 min, 67%.
ternatively, the dynamic amphiphiles can form membrane-
active polyion–counterion complexes with DNA. Export of
the DPX quenchers[8] or HPTS fluorophores by these poly-
ion–counterion complexes is then reported as fluorescence
recovery (Figure 3b).
In a typical experiment, the dynamic amphiphile was
added first to the EYPC-LUVsꢀHPTS/DPX. Fluorescence
recovery at this point demonstrates that the dynamic amphi-
phile is active also in the absence of DNA, and presumably
acts as a detergent to destroy the lipid bilayer. In the ab-
sence of detergent effects, variation of the incubation time
before addition of the DNA can be of use to determine the
stability of the dynamic amphiphile under experimental con-
ditions (see below). ctDNA was then added as representa-
tive polyanion transporter, and the increase in activity with
increasing amphiphile concentration was recorded in dose-
response curves (ctDNA without counterion activator is in-
active, Figure S51a in the Supporting Information).[16] Dose-
in all cases by ESI MS (Tables S1 in the Supporting Infor-
mation).
The activation of DNA as cation transporters by the new
dynamic amphiphiles was evaluated under routine condi-
tions.[1,3,8,13,15] In brief, egg yolk phosphatidylcholine large
unilamellar vesicles (EYPC-LUVs) are loaded with the
anionic 8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS) and the
cationic p-xylene-bis-pyridinium bromide (DPX, Figure 3b).
The addition of dynamic amphiphiles can then destroy the
vesicles, converting the bilayer membranes into mixed mi-
celles. In the HPTS/DPX assay, such detergent effects can
be monitored as fluorescence recovery in the absence of
DNA because release of the entrapped probes increases the
average distance between fluorophores and quenchers. Al-
response curves were subjected to Hill analysis, and YMAX
,
which is the maximal accessible fractional activity under
these conditions, the EC50, which is the effective activator
concentration needed to reach 50% of YMAX, and the Hill
coefficient n were obtained (Figure S51b in the Supporting
Information).
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Table 1. DNA activation data for dynamic amphiphiles with T11M
tails.[a]
Cpd[b]
m/z[c]
YMAX [%][d]
EC50 [mm][e]
n[f]
G1O1
G1O2
343
753
55.4Æ1.6
D
16.6Æ0.2
D
14.1Æ2.0
D
G1O3
G1O4
G1S2H2
G1S3H3
G2H4
1163
1574
963
1478
1339
1226
550
509
860
818
46.5Æ5.2
22.5Æ1.0
D
3.1Æ0.7
1.4Æ0.1
D
2.5Æ1.5
4.6Æ2.1
D
D
D
D
84.5Æ9.4
39.0Æ5.3
59.9Æ2.4
66.2Æ2.4
26.8Æ0.3
18.9Æ1.4
1.4Æ0.1
2.1Æ0.2
19.6Æ0.9
46.6Æ1.1
10.4Æ0.7
35.7Æ2.4
3.4Æ0.8
3.7Æ1.5
3.2Æ0.3
3.3Æ0.2
6.2Æ0.2
3.9Æ0.2
A2H4
G1H2[g]
A1H2[g]
G1H3[g]
A1H3[g]
[a] From transport experiments in fluorogenic vesicles; see Figure S52 in
the Supporting Information; D=detergent effects. Complete data for all
tails can be found in the Supporting Information; [b] compounds, see
Figure 2; [c] [M+H]+ peak in the mass spectra of hydrazones and
oximes formed with T11M; [d] maximal fractional activity of DNA as ion
transporters at saturation with counterion activators; from Hill analysis;
[e] effective counterion concentration needed to reach 50% of YMAX
;
from Hill analysis; [f] Hill coefficient; [g] from ref. [1].
nations can be found in the Supporting Information (Fig-
ure S52 and Table S2). The best results were obtained for
G2H4 (Table 1, entry 7). The YMAX =84.5
G
ceptional for this series. This high YMAX indicates that trans-
port occurs in nearly all vesicles, excluding significant inter-
ference from inactivating processes, such as precipitation, ir-
reversible partitioning, disappearance in the membrane
core, amphiphile hydrolysis, and so on. At the same time,
the EC50 =1.4
G
EC50 values ever seen for polyion–counterion complexes
with satisfactory YMAX >50%. Compared to G1H2 with the
same head/tail ratio,[1] G2H4 was 14-times more active
(Table 1, entries 7 and 9). This significant improvement was
not achieved at the cost of reduced YMAX. Quite the contra-
ry, the maximal fractional activities also increased from
Y
MAX =59.9
G2H4. High YMAX with four T11M tails was also remarkable
considering the strong decrease to unsatisfactory YMAX
26.8
(Æ0.3)% reported for the three-tail G1H3 (Table 1,
entry 11).
The analogue A2H4 with two ammonium heads and four
T11M tails gave similarly outstanding EC50 =2.1
(Æ2.4)% for G1H2 to YMAX =84.5
N
AHCTUNGTRENNUNG
Figure 3. a) Dependence of the fractional activity Y of DNA as ion trans-
*
porters in fluorogenic vesicles in the presence of G1H1T18 ( ) and
G1O1T18 (&) on the time of incubation of the amphiphiles at pH 5.5
prior to DNA addition. At pH 7.4, both amphiphiles act independently
of incubation time. b) Design of vesicle experiments: i) addition of dy-
namic amphiphiles as counterion activators to vesicles with internal re-
porter ions (e.g., DPX, HPTS) possibly causes membrane destruction
(detergent effects: D in Table 1; data not shown), or ii) the formation of
membrane-active polyion–counterion complexes with DNA for iii) the
export of the DPX quenchers and fluorescence recovery of the internal
HPTS fluorophores.
(Table 1, entry 8). However, this 22-fold improvement in ef-
fective concentration compared to A1H2 came at the cost
of a decreasing maximal fractional activity from YMAX
66.2 MAX =39.0
=
G
Y
(Æ5.3)%
(Table 1, entries 8 and 10). This important decrease is con-
trary to the increase found with two guanidinium head-
groups. It suggested that the proximity effects to strengthen
ion pairing in weakly acidic guanidinium but not in more
acidic ammonium cations are essential to reach perfect ac-
tivity of dynamic amphiphiles, that is, minimal EC50 at maxi-
The results for T11M, 2-methylundecanal, an ambergris
odorant from kumquat peel oil used in perfumes, soaps and
detergents, are summarized in Table 1 because they illus-
trate the identified trends very well. All dose-response
curves and complete transport data for all head–tail combi-
mal YMAX
.
In the oxime series, decreasing EC50 with increasing
number of tails coincided with decreasing YMAX (Table 1, en-
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Dynamic DNA Activators
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tries 1–4). Whereas the EC50 =1.4
outstanding as that of G2H4, the YMAX =22.1ACTHUNGTRENNNUG
G
trations (EC50) were reached without losses in maximal ac-
tivity, YMAX, at saturation. Less satisfactory performances
were obtained with diammonium analogues A2H4. They
confirmed that proximity effects to minimize intramolecular
charge repulsion by maximized intermolecular ion pairing
account for the excellent activity of G2H4.[13]
Oxime (O) bridges were introduced in singly charged am-
phiphiles with one to four tails, and disulfide (S) bridges in
singly charged amphiphiles with two or three tails. Com-
pared to hydrazone bridges, oxime bridges provided the ex-
pected insensitivity to acidic conditions. However, the in-
creasingly long linear elongations that had to be introduced
between the dendritic heads and the bridges resulted in in-
creasing detergent effects, particularly for the doubly bridg-
ed G1S2H2 and G1S3H3. These detergent effects render
the dynamic amphiphiles less useful for sensing applications
in fluorogenic vesicles but do not exclude activity with
regard to cellular uptake.
With eight new members added to the ten original head-
groups and 39 tails tested so far, the expanded library pro-
vides formal access to 702 dynamic amphiphiles, and in-
creasing the number of meaningful tails beyond 39 is abso-
lutely unproblematic. Current efforts focus on the import of
more unusual tails such as fullerenes, mesogenes or fluoro-
philes from the materials sciences to increase the probability
to discover unexpected hits, and the growing library is being
screened for gene and siRNA delivery. Preliminary results
are encouraging, and confirm the construction and screening
of expanded libraries of dynamic amphiphiles as a meaning-
ful approach, because the current hits are: 1) competitive
with the current delivery agents on the market, 2) unpredict-
able from model studies in vesicles, and 3) unpredictable
from the literature.[12a] These results will be reported in due
course.
insufficient and indicated marginal activity that is overshad-
owed by presumably much precipitation or irreversible dis-
appearance in the bilayer core of the dynamic amphiphiles
G1O4T11M as well as their DNA complexes (Table 1,
entry 4).
The difference in stability of oxime and hydrazone bridges
under acidic conditions was explored with G1O1T18 and
G1H1T18. With use of the same assay in fluorogenic vesi-
cles at pH 5.5 instead of the usually maintained pH 7.4, the
incubation time before addition of DNA and detection of
activity was varied. With increasing incubation time, the
ability of G1H1T18 to activate DNA as ion transporters de-
*
creased rapidly (Figure 3a, ). Exponential curve fitting
suggested that the half-life of the hydrazone in G1H1T18 is
about 3 min at pH 5.5. The ability of G1O1T18 to activate
DNA as ion transporters did not decrease significantly
during incubation at pH 5.5 (Figure 3a, &). The clear con-
trast between the stable G1O1T18 and the unstable
G1H1T18 was in agreement with the expected lifetimes of
oximes and hydrazones under acidic conditions.[6] Prolonged
incubation of both amphiphiles at pH 7.4 did not reduce
their activity significantly.
G1O2T11M and all other amphiphiles obtained from
G1O2, except for the shorter G1O2T8 and G1O2T10, were
detergents, and showed membrane activity already in the
absence of DNA (Table 1 and Table S2 in the Supporting In-
formation). Nearly identical detergent effects were observed
with the doubly bridged G1S2H2, whereas the original
G1H2 was not affected (Table 1 and Table S2 in the Sup-
porting Information). Detergent effects were even worse
with G1S3H3, whereas the homologous G1O3 and G1H3
acted reliably as activators of DNA as ion transporters
(Table 1 and Table S2 in the Supporting Information). These
trends suggested that not the nature of the bridges but the
linear elongations of the original dendrons account for de-
tergent effects.
Acknowledgements
We thank A. Fin, S. Solemanpour and D.-H. Tran for contributions to
synthesis, D. Jeannerat, A. Pinto and S. Grass for NMR spectroscopy
measurements, the Sciences Mass Spectrometry (SMS) platform for mass
spectrometry services, and the University of Geneva, the European Re-
search Council (ERC Advanced Investigator), the National Centre of
Competence in Research (NCCR) Chemical Biology and the Swiss NSF
for financial support.
Conclusion
The objective of this study was to expand a collection of dy-
namic amphiphiles based on observed trends and innovative
chemistry. For this purpose, the existing hydrazone (H)
bridges were complemented by oxime (O) and disulfide (S)
bridges. Secondly, head-groups with two rather than one
positive charge and amphiphiles with two rather than one
bridge connecting head and tails were designed, synthesized
and evaluated.
Lessons learned with singly charged hydrazone amphi-
philes suggested the preparation of doubly charged amphi-
philes with four tails. The obtained G2H4 peptide dendrons
with two guanidinium cations and four reactive hydrazides
gave, when coupled in situ with hydrophobic aldehyde tails,
the best dynamic activators of DNA as ion transporter in
fluorogenic vesicles known so far, and low effective concen-
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Received: February 19, 2012
Published online: && &&, 0000
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ꢃ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!

Dynamic DNA Activators
FULL PAPER
Amphiphiles
J. Montenegro, E.-K. Bang, N. Sakai,
S. Matile* ...................... &&&& — &&&&
Synthesis of an Enlarged Library of
Dynamic DNA Activators with
Oxime, Disulfide and Hydrazone
Bridges
Head on: Design, synthesis and evalu-
ation of dynamic amphiphiles with
singly or doubly charged peptide den-
drons as head-groups, hydrazones,
oximes and disulfides as bridges, and
with fragrant tails are reported.
Amphiphiles with two charges and
four tails (see figure) are identified as
the most powerful activators of DNA
as ion transporters and thus as the
most promising family to screen for
gene and siRNA delivery.
Chem. Eur. J. 2012, 00, 0 – 0
ꢃ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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These are not the final page numbers!