Comprehensive screening of octopus amphiphiles as DNA activators in lipid bilayers: Implications on transport, sensing and cellular uptake

Article abstract of DOI:10.1039/c0ob00948b

Dynamic octopus amphiphiles contain one charged "head," here a guanidinium cation, together several hydrophobic "tails" (or "tentacles") that can be attached and exchanged in situ by reversible hydrazone formation. Quite surprisingly, their ability to activate DNA as transporters in lipid bilayer membranes was found to increase with the number of tails (up to four) as well as with their length (up to eight carbons). Both encouraged and puzzled by these results, we decided that a comprehensive screening of octopus amphiphiles with regard to number (from one to six) and length (from three to eighteen carbons) of their tails would be appropriate at this point. For this purpose, we here report the synthesis of cationic hexahydrazide peptide dendrons together with that of aldehydes with long, saturated, unsaturated and branched hydrophobic tails. Comprehensive screening of the completed collection of tails and heads reveals that the ability of octopus amphiphiles to activate DNA transporters shifts with increasing number of tails to decreasing length of the tails. Moreover, cis-alkenyl and branched alkyl tails are more active than their linear analogs, branched aromatic tails are best. These overall very meaningful trends for octopus amphiphiles will be of importance for sensing applications and fragrant cellular uptake.

Full text of DOI:10.1039/c0ob00948b

View Article Online / Journal Homepage / Table of Contents for this issue
Organic &
Biomolecular
Chemistry
Dynamic Article Links
Cite this: Org. Biomol. Chem., 2011, 9, 2641
www.rsc.org/obc
PAPER
Comprehensive screening of octopus amphiphiles as DNA activators in lipid
bilayers: implications on transport, sensing and cellular uptake†
Javier Montenegro, Andrea Fin and Stefan Matile*
Received 28th October 2010, Accepted 12th January 2011
DOI: 10.1039/c0ob00948b
Dynamic octopus amphiphiles contain one charged “head,” here a guanidinium cation, together
several hydrophobic “tails” (or “tentacles”) that can be attached and exchanged in situ by reversible
hydrazone formation. Quite surprisingly, their ability to activate DNA as transporters in lipid bilayer
membranes was found to increase with the number of tails (up to four) as well as with their length (up
to eight carbons). Both encouraged and puzzled by these results, we decided that a comprehensive
screening of octopus amphiphiles with regard to number (from one to six) and length (from three to
eighteen carbons) of their tails would be appropriate at this point. For this purpose, we here report the
synthesis of cationic hexahydrazide peptide dendrons together with that of aldehydes with long,
saturated, unsaturated and branched hydrophobic tails. Comprehensive screening of the completed
collection of tails and heads reveals that the ability of octopus amphiphiles to activate DNA
transporters shifts with increasing number of tails to decreasing length of the tails. Moreover,
cis-alkenyl and branched alkyl tails are more active than their linear analogs, branched aromatic tails
are best. These overall very meaningful trends for octopus amphiphiles will be of importance for
sensing applications and fragrant cellular uptake.
channels,5,9x the operation of membrane-based synthetic sensing
Introduction
systems10 in many variations,1–3,6 and the activity of many catalysts
in organic synthesis.11
The activity of dynamic polyion–counterion complexes in lipid
bilayer membranes is of scientific interest with regard to topics as
diverse as transmembrane transport,1–5 voltage gating,5 sensing1–3,6
and cellular uptake.7–9 In the presence of amphiphilic counterions,
polyions such as anionic DNA and RNA7,8 or the cationic cell-
penetrating peptides (CPPs)9 have been shown to become soluble
in organic solvents (or bulk membranes) such as chloroform.5
Moreover, counterion-activated CPPs or DNA/RNA have been
shown to actually prefer hydrophobic over hydrophilic environ-
ments, that is partition from water into bulk membranes and
lipid bilayer membrane. Moreover, they can carry hydrophilic
counterions as large as fluorescent probes across bulk and intact
lipid bilayer membranes.5
The activity of polyion–counterion complexes originates from
the desire to minimize charge repulsion in weakly acidic or basic
polyions where de-/protonation is impossible and only counterion
scavenging can help.5 By now, it is understood that counterion-
mediated function accounts for the cellular uptake of CPPs
and RNA/DNA,5,7–9 the voltage gating of biological potassium
Recently, polyion–counterion complexes have been used to
create the first differential sensing system10 that works, like
biological olfactory systems, in lipid bilayer membranes.1 For this
purpose, hydrophobic analytes such as octanal T8 are reacted in
situ with cationic hydrazides such as G1H3 to yield amphiphilic
cations such as G1H3T8 (Fig. 1 and 2). These cationic amphiphiles
then can function as activators of polyanionic cation transporters
such as calf-thymus (ct) DNA in fluorogenic vesicles (Fig. 1). For
pattern generation, analytes such as octanal T8 are incubated with
counterions (one to three guanidiniums, ammoniums, carboxy-
lates or phosphonates) carrying one to four reactive hydrazides
(e.g., G1H1–G1H3, Fig. 2).1 The fluorescent response obtained
from polyion activation with the different amphiphiles is then
used to generate n-dimensional patterns that can be recognized
by PCA or HCA.1–3 This differential sensing approach was shown
to discriminate enantiomers, cis–trans isomers and single atom
homologs. Error-free recognition of all tested perfumes confirmed
compatibility of the new sensing system with complex matrices
from the supermarket.1
Covalent capture of hydrophobic “tails” by reactive counterion
“heads” is of interest not only for differential sensing1–3,6,10
but also for new approaches toward cellular uptake7–9 and
catalysis.11 On the one hand, a high number of delivery agents can
be prepared with essentially no effort for comprehensive screening.
Department of Organic Chemistry, NCCR Chemical Biology, University of
Geneva, Geneva, Switzerland. E-mail: stefan.matile@unige.ch; Fax: +41
22 379 3215; Tel: +41 22 379 6523; Web: www.unige.ch/sciences/
chiorg/matile/
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c0ob00948b
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 2641–2647 | 2641
©

View Article Online
The dynamic nature of the non-viral vectors, on the other
hand, can be used to add multifunctionality with regard to
endosomal escape, targeting, fluorescence labeling, and so on.
Toward this end, we found that carboxylates are better than
phosphonates to activate CPPs, and that gemini structures8 are
better than excessive “heads” or “tails”.3 For the activation of
DNA and RNA, guanidinium cations are better than ammonium
cations, and activity increases in the guanidinium series with
increasing number of tails from nearly inactive single-chain
amphiphiles G1H1T to the most active “octopus” amphiphiles
G1H4T with four hydrophobic tails or “tentacles”.2 With one
exception, these findings were all as expected from the literature.5–9
They confirmed, inter alia, the importance of preoganized
hydrogen-bonding as well as weak basicity/acidity to assist oper-
ational ion pairing with powerful proximity effects.5 However, the
increasing activity of cationic activators with increasing number
of tails came as a surprise, culminating with best performances
for octopus amphiphiles of significant structural complexity.2 To
clarify this puzzling octopus effect, we here report a comprehensive
screening of dynamic amphiphiles with regard to number (from
one to six) and length (from three to eighteen carbons) of their tails.
The bottom line is that maximal activity of octopus amphiphiles
shifts with increasing number of tails to shorter tail length, and
that cis-unsaturated and branched alkyl tails are more active.
These findings are of interest for applications toward sensing,6,10
catalysis11 as well as cellular uptake.7–9
Fig. 1 Ion transport by dynamic polyion–counterion complexes in
fluorogenic vesicles. Hydrophobic “tails” (or “tentacles”, e.g., T5) are
covalently captured by hydrophilic cations (e.g., hexahydrazides G1H6) to
give six-tail amphiphiles (e.g., G1H6T5) that can activate polyanions (e.g.,
ctDNA) as transporters in lipid bilayers (here the example for fluorescence
recovery in response to the export of trapped cationic quenchers (green)
but not anionic fluorophores (red) is shown).
Results and discussion
The hexahydrazide peptide dendron12 G1H6 was synthesized in
eight steps in overall 16% yield from the differently protected
Fig. 2 Reactive counterions G1H1–G1H6 composed of one guanidinium cation (G1) and one to six hydrazides (H) for in situ reaction with hydrophobic
tails (T) to yield amphiphilic hydrazones that can activate DNA as transporter in fluorogenic vesicles (see Fig. 1). TAOT for 4-ethyloctanal refers to the
presence of a tail that is similarly branched as in the popular detergent AOT (Aerosol OT, di-2-ethyl-1-hexylsulfosuccinate), TJAS refers to jasminaldehyde
as tail.
2642 | Org. Biomol. Chem., 2011, 9, 2641–2647
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
glutamic acids 1 and 2 (Scheme 1). Their coupling to give
triester 3 was followed by the conversion into trihydrazide 4.1
Hydrazide protection with Boc prepared for the hydrogenolytic Z
removal in 5.1 The two mini-dendrons 6 were then attached to the
glutamic acid core 7 to yield the desired scaffold. Chemoselective
deprotection of 8 yielded amine 9, which was guanidinylated with
the standard reagent 10. Acid-catalyzed removal of the eight Boc
protecting groups in 11 gave the target molecule G1H6.
as fluorogenic vesicles (i.e., egg yolk phosphatidylcholine large
unilamellar vesicles loaded with the anionic fluorophore 8-
hydroxy-1,3,6-pyrenetrisulfonate and the cationic quencher p-
xylene-bis-pyridinium bromide).1,2,5 In this assay, counterion-
activated DNA is thought to export the cationic quencher DPX
from intact vesicles.5b This DPX export is detected as an increase in
emission of the anionic fluorophore HPTS left behind. Although
plausible and consistent with control experiments, we add that
other interpretations for fluorescence recovery in the HPTS/DPX
are always possible.5b
In a typical experiment, the counterion activator is added to
EYPC-LUVs … HPTS/DPX first (Fig. 3). Fluorescence recovery
before the addition of DNA transporters indicates that the
Scheme 1 Reagents and conditions: (a) HBTU, DIEA, CH2Cl2, rt, 85%;1
(b) N2H4 monohydrate, MeOH, reflux, 90%;1 (c) Boc2O, DIEA, MeCN,
H2O, reflux, 90%;1 (d) H2, Pd/C, MeOH, rt, quant; (e) HBTU, DIEA,
CH2Cl2, rt, 62%; (f) H2, Pd/C, MeOH, rt, quant; (g) DIEA, CH3CN,
50 ◦C, 61%; (h) 1 M HCl in Et2O, reflux, 63% (8 steps, 16%).
Tetrahydrazide mini-dendron G1H4, trihydrazide G1H3, dihy-
drazide G1H2 and monohydrazide G1H1 were prepared following
previously reported procedures (Fig. 2).1,2 Almost all tails T were
commercially available. The 9-cis-octadecene-1-aldehyde T18D9
and TAOT , the aldehyde with a 4-ethyl-1-octyl motif reminiscent
of the branched tails in the reversed-micellar surfactant AOT,
were accessible by PCC oxidation in one step following literature
procedures.13
Hydrazides G1H6, G1H4, G1H3, G1H2 and G1H1 were
converted into hydrazones under routine conditions,1–3,14 that is
by incubation for one hour with aldehyde tails T in DMSO at
60 ◦C. In situ hydrazone formation was confirmed by electrospray
mass spectrometry (ESI-MS). Not further activated hydrazones
are hydrolyzed under acidic conditions but are stable in neutral
water, at least for the few minutes of a transport experiment.14
Quantitative kinetics of hydrolysis as a function of pH are currently
under investigation in the context of cellular uptake experiments.
The activity of new hexahydrazide G1H6 with hydrophobic tails
to activate polyanion transporters was explored under routine
conditions in comparison with the previously reported G1H1–
G1H4.1,2 At the same time, the screening of tails was expanded
from octanal T8 or shorter to long, saturated, unsaturated and
branched tails up to T18, T18D9 and TAOT . Calf-thymus (ct) DNA
was used as polyanion transporter, EYPC-LUVs … HPTS/DPX
Fig. 3 Changes in fractional fluorescence intensity IF of HPTS (lex = 413
nm, lem = 510 nm) during addition of (a) G1H6T7 (1.25 (ꢀ), 3.75 ( ), 6.25
᭹
(ꢀ), 10 (+), 12.5 (¥), 25 (᭛), 30 (ꢁ), 40 mM ( ), final concentrations, t ~
᭺
0 s), (b) G1H1T12 (0.375 (ꢀ), 1.25 ( ), 6.25 (ꢀ), 12.5 (+), 20 (¥), 30 (᭛),
᭹
50 (ꢁ), 70 mM ( )) or (c) G1H3TAOT (1.25 (+), 3.25 (¥), 6.25 (᭛), 12.5
᭺
(ꢁ), 25 mM ( )), ctDNA (1.25 mg ml-1 final concentration, t ~ 40 s) and
᭺
Triton X-100 (excess, t ~ 200 s) to EYPC-LUVs … HPTS/DPX. Data for
the highest amphiphile concentrations are in colored in brown and red in
each series to facilitate the comparison of activity before and after DNA
addition (a, red arrow).
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 2641–2647 | 2643
©

View Article Online
cationic amphiphile is a membrane-active surfactant (Fig. 3a,
red arrow). With the current collection of amphiphiles, strong
detergent effects were rare, whereas the onset of detergent activity
at higher concentrations was observed occasionally. With octopus
amphiphile G1H6T7, for example, the onset of detergent activity
was observed around a critical concentration of 30 mM (Fig. 3a,
t = 0–40 s). Interestingly, single-tail amphiphile with G1H1T12,
featuring a classical surfactant motif with a long dodecyl tail, was
inactive without DNA up to 70 mM (Fig. 3b, t = 0–40 s). The
same was true for G1H3TAOT , containing three branched alkyl
tails reminiscent of the classical detergent AOT (Fig. 3c, t = 0–40
s).
Fluorescence recovery in response to the addition of ctDNA
reports on active polyion–counterion complexes (Fig. 3, t = 40–
200 s, ctDNA alone is inactive). At the end of each experiment,
the fluorescence response is calibrated to a fractional activity Y =
1.0 by complete vesicle lysis with an excess of Triton X-100 (Fig.
3, t > 200 s).
For quantitative characterization of polyion–counterion trans-
porters, these experiments were repeated for different activator
concentrations at constant polyion concentration first (Fig. 3).
The fluorescence intensity just before lysis was taken as fractional
activity Y and plotted as a function of activator concentration.
The resulting dose–response curves were subjected to Hill analysis
to yield the YMAX, the maximal accessible activity under these
conditions, and the EC50, the effective activator concentration
needed to reach 50% of YMAX (Fig. S2, Table S1†).15 For a
comprehensive overview of the results for linear alkyl tails, YMAX
and EC50 were then plotted as a function of the number and the
length of the tails (Fig. 4).
In general, the activity of octopus amphiphiles shifted with
increasing number of tails toward decreasing tail length (Fig.
4). Counterions with too few and too short tails were inactive
because they are too hydrophilic to partition into the bilayer
membrane. Counterions with too many and too long tails
were inactive because they are too hydrophobic to reach the
vesicles and precipitate instead. The results from this com-
prehensive mapping thus demonstrated that intermediate hy-
drophobicity is essential for activity, at least within the n-alkyl
series.
Counterion activity was most interesting at the edges. The
shortest functional tail was pentanal T5, addressable with the new
octopus amphiphile G1H6 only (Fig. 4, red). Hexanal T6 already
was active in both six-tail amphiphile G1H6T6 and four-tail
amphiphile G1H4T6. Heptanal T7 was active in six-tail G1H6T7,
four-tail G1H4T7 and three-tail G1H3T7. With octanal T8, four-
tail G1H4T8, three-tail G1H3T8 and two-tail G1H4T8 were
sufficiently hydrophobic to activate DNA transporters, whereas
six-tail G1H6T8 was already too hydrophobic at this point to reach
the membrane. Amphiphiles with fewer tails exhibited weaker
selectivity compared to the six-tail G1H6, losing activity only with
tails such as T12 for four-tail G1H4 and three-tail G1H3, and T16
for two-tail G1H2. Most remarkable was single-tail amphiphile
G1H1, which was inactive in all previous studies with shorter tails.
With T12, G1H1 suddenly became capable of activating DNA
transporters in a significant manner (Fig. 4, dark blue; Fig. 3b).
Together with T13–T16, G1H1 exhibited a not further surprising
detergent activity (not shown in Fig. 4*), whereas DNA activation
reappeared with T18 (Fig. 4, dark blue).
Fig. 4 Dependence of (a) YMAX and (b) EC50 of counterion-activated
DNA transporters on tail number and tail length, measured for varied
counterion concentrations at constant ctDNA concentration (1.25 mg
ml-1). Results cover counterions with heads G1H6 (with six tails, red),
G1H4 (four tails, garnet), G1H3 (three tails, violet), G1H2 (two tails, blue)
and G1H1 (one tail, dark blue) that are coupled with tails T4 (4 carbons)
to T18 (18 carbons). YMAX < 0.2 at 25 mM was considered inactive and put
to YMAX = 0.0 and EC50 = 100 mM for contrast only. Some data points for
G1H1–G1H4 from ref. 1 and 2 are included. * = Missing data points for
membrane-active amphiphiles are bridged with straight lines to emphasize
the trends for the polyion–counterion complexes of interest. All values are
from Hill analysis of dose–response curves as in Fig. 3, compare Table
S1†).15
To probe the validity and reproducibility of the results obtained
for activator screening at constant DNA concentration and varied
activator concentrations, the same activity mapping was made for
varied DNA concentrations at constant counterion concentration
(Fig. 5). The cut was placed at a quite challenging threshold of
6.15 mM activator (compare Fig. 4b). The results from dose–
response curves for DNA (Fig. 5) perfectly complemented the
ones from dose–response curves for counterion activators (Fig.
4). Namely, with increasing number of tails, the ability to activate
DNA shifted to shorter tails. The range covered reached from T12
as best for two-tail G1H2 to T6 for six-tail G1H6 (Fig. 5).
To outline the full potential of the concept of dynamic14
counterion amphiphiles1–3 for applications toward sensing,6,10
catalysis11 and cellular uptake,7–9 the comprehensive analysis of
octopus amphiphiles for linear and saturated alkyl tails T4–T18
2644 | Org. Biomol. Chem., 2011, 9, 2641–2647
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Fig. 5 Dependence of (a) YMAX and (b) EC50 of counterion-activated
DNA transporters on tail number and tail length, measured for varied
ctDNA concentrations at constant counterion concentration (6.15 mM).
Results cover counterions with heads G1H6 (with six tails, red), G1H4
(four tails, garnet), G1H3 (three tails, violet), G1H2 (two tails, blue) and
G1H1 (one tail, dark blue) that are coupled with T4–T18 (YMAX < 0.2
at 1.25 mg ml-1 DNA was considered inactive and put to YMAX = 0.0 and
EC50 = 100 mg ml-1 for contrast only, compare Table S2†).15
Fig. 6 Dependence of (a) YMAX and (b) EC50 of counterion-ctDNA
transporters on the nature of the tails, measured for varied counterion
concentrations at constant DNA concentration (1.25 mg ml-1). Results
cover counterions with heads G1H6 (with six tails, red), G1H4 (four
tails, garnet), G1H3 (three tails, violet), G1H2 (two tails, blue) and G1H1
(one tail, dark blue) that are coupled with T10 (longish, saturated), TAOT
(branched), TJAS (branched, aromatic), T18D9 (very long, unsaturated) and
T18 (very long, saturated. YMAX < 0.2 at 25 mM was considered inactive
and put to YMAX = 0.0 and EC50 = 100 mM to enhance contrast, compare
Table S1†).15
was complemented with a full data set for unsaturated tails with
central cis double bonds (T18D9), branched alkyl tails (TAOT), and
branched alkyl-aryl tails (TJAS , Fig. 6).
Six-tail amphiphiles obtained with G1H6 were inactive with all
examples beyond linear saturated tails (Fig. 6). Confirming the
results from the n-alkyl screening (Fig. 4), this general inactivity
confirmed that the selected tails are all too hydrophobic for a six-
tail dendron G1H6. Four-tail amphiphiles produced with G1H4
functioned as DNA activators when coupled with the branched
G1H1 was inactive with the detergent-like TAOT, where G1H2 and
G1H3 both worked very well.
The only tail that generated activity independent of the number
of tails attached to the head group (excluding the “hypervalent”
G1H6) was the one derived from jasminaldehyde TJAS (Fig. 6). The
EC50 values with TJAS were consistently in the lower micromolar
range, namely EC50 = 10.6 0.7 mM with G1H1, EC50 = 4.1
0.7 mM with G1H2, EC50 = 4.4 0.6 mM with G1H3 and
EC50 = 11.8 1.6 mM with G1H4 (Fig. 6b, Table S1†). The same
T
AOT and jasminaldehyde TJAS , whereas the longer T18D9 failed to
generate activity despite the presence of a solubilizing cis double
bond in the middle (Fig. 6).
Best results were secured with three-tail amphiphiles obtained
from G1H3 and two-tail amphiphiles obtained from G1H2, which
were active with TAOT , TJAS and T18D9 (Fig. 6). Unique again was
the behavior of single-tail amphiphiles obtained from G1H1 (Fig.
6, dark blue). The DNA activation with the unsaturated long-tail
T18D9 was as good as with the saturated analog T18. Here, G1H1
differed from G1H2 and G1H3, which were both active with the
unsaturated T18D9 but inactive with the saturated T18. However,
trend was observed with the YMAX for TJAS , moving from YMAX
=
0.50 0.02 with G1H1 over maximal YMAX = 0.81 0.11 with
G1H2 to YMAX = 0.51 0.05 with G1H3 and YMAX = 0.62 0.06
with G1H4 (Fig. 6a, Table S1†). For comparison, the otherwise
excellent T18D9 was inactive with G1H4, or TAOT , otherwise
outstanding, failed to work with G1H1 (Y < 0.2 at 25 mM, Fig. 6,
Table S1†).
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 2641–2647 | 2645
©

View Article Online
Biology, and the Swiss NSF for financial support. S.M. is an ERC
Advanced Investigator.
Conclusions
New concepts and methods for the identification of counterion
activators of DNA (and the charge-inverted CPP) transporters
in bilayer membranes are of interest because of their importance
for general applications toward sensing,6,10 catalysis11 and cellular
uptake.7–9 The objective of this study was to elaborate a compre-
hensive map connecting the number, the length and the nature
of the hydrophobic tails of single-head guanidinium activators of
DNA transporters.
The three key findings are as follows. Firstly, the concept of
dynamic14 counterion activators,1–3 where different tails can be
characterized with essentially no synthetic effort, is confirmed as
very important and productive from a methodological point of
view. With this approach, it is no problem to rapidly produce and
screen counterion libraries.
The second key finding is that with increasing number of tails,
the maximal activity of counterion activators shifts to decreasing
tail length. This finding highlights the fundamental importance of
a balanced, intermediate amphiphilicity for activity. Hindered ion
pairing of DNA phosphodiesters with guanidinium cations hidden
within multi-tail octopus amphiphiles was of interest as alternative
explanation. However, altered DNA binding explained sharp
drops in activity with single carbons added to or removed from
the tails less convincingly than the need for balanced, intermediate
hydrophobicity of the final active polyion–counterion complex,
where small differences in amphiphile structure are amplified by
multivalency.
The newly introduced octopus amphiphiles with a new record
of six tails added via six hydrazones and a peptide dendron
converging to one cationic guanidinium head turns out to be less
interesting in this context because activity is limited to very short
tails with 5–7 carbons only and often includes some non-specific
leakage. This result suggests that the originally planned synthesis
of “true” octopus amphiphiles with eight tentacles is redundant
because activity is likely to further decrease. The newly introduced
long tails with a new record of up to 18 carbons turn out to be very
interesting because they bring activity to single-tail amphiphiles
that have so far been useless.
References
1 T. Takeuchi, J. Montenegro, A. Hennig and S. Matile, Chem. Sci., 2011,
2, 303–307.
2 J. Montenegro, P. Bonvin, T. Takeuchi and S. Matile, Chem. Eur. J.,
2010, 16, 14159–14166.
3 J. Montenegro and S. Matile, Chem. Asian J., 2011, 2, 281–289.
4 (a) J. T. Davis, O. Okunola and R. Quesada, Chem. Soc. Rev., 2010,
39, 3843–3862; (b) P. A. Gale, Chem. Soc. Rev., 2010, 39, 3746–3771;
(c) G. W. Gokel and N. Barkey, New J. Chem., 2009, 33, 947–96; (d) T.
M. Fyles, Chem. Soc. Rev., 2007, 36, 335–347; (e) A. P. Davis, D. N.
Sheppard and B. D. Smith, Chem. Soc. Rev., 2007, 36, 348–357; (f) A.
L. Sisson, M. R. Shah, S. Bhosale and S. Matile, Chem. Soc. Rev., 2006,
35, 1269–1286; (g) S. Matile, A. Som and N. Sorde´, Tetrahedron, 2004,
60, 6405–6435; (h) I. Park, Y. Yoon, M. Jung, K. Kim, S. Park, S. Shin,
Y. Lim and M. Lee, Chem. Asian J., 2011, 2, 452–458; (i) J. K. W. Chui
and T. M. Fyles, Chem. Commun., 2010, 46, 4169–4171; (j) H. Cho
and Y. Zhao, J. Am. Chem. Soc., 2010, 132, 9890–9899; (k) L. Fischer
and G. Guichard, Org. Biomol. Chem., 2010, 8, 3101–3117; (l) R. J.
Brea, C. Reiriz and J. R. Granja, Chem. Soc. Rev., 2010, 26, 1448–1456;
(m) R. E. Dawson, A. Hennig, D. P. Weimann, D. Emery, S. Gabutti, J.
Montenegro, V. Ravikumar, M. Mayor, J. Mareda, C. A. Schalley and
S. Matile, Nat. Chem., 2010, 2, 533–538; (n) J. T. Davis, P. A. Gale, O. A.
Okunola, P. Prados, J. C. Iglesias-Sanchez, T. Torroba and R. Quesada,
Nat. Chem., 2009, 1, 138–144; (o) X. Li, B. Shen, X. Q. Yao and D.
Yang, J. Am. Chem. Soc., 2009, 131, 13676–13680; (p) B. M. Rosen, C.
J. Wilson, D. A. Wilson, M. Peterca, M. R. Imam and V. Percec, Chem.
Rev., 2009, 109, 6275–6540; (q) C. P. Wilson and S. J. Webb, Chem.
Commun., 2008, 44, 4007–4009; (r) A. Satake, M. Yamamura, M. Oda
and Y. Kobuke, J. Am. Chem. Soc., 2008, 130, 6314–6315; (s) P. V. Jog
and M. S. Gin, Org. Lett., 2008, 10, 3693–3696; (t) M. Jung, H. Kim, K.
Baek and K. Kim, Angew. Chem. Int. Ed., 2008, 47, 5755–5757; (u) P.
L. Boudreault and N. Voyer, Org. Biomol. Chem., 2007, 5, 1459–1465;
(v) S. Bhosale, A. L. Sisson, P. Talukdar, A. Fu¨rstenberg, N. Banerji,
E. Vauthey, G. Bollot, J. Mareda, C. Ro¨ger, F. Wu¨rthner, N. Sakai and
S. Matile, Science, 2006, 313, 84–86.
5 (a) T. Takeuchi, N. Sakai and S. Matile, Faraday Discuss., 2009, 143,
187–203; (b) T. Takeuchi, V. Bagnacani, F. Sansone and S. Matile,
ChemBioChem, 2009, 10, 2793–2799; (c) T. Miyatake, M. Nishihara and
S. Matile, J. Am. Chem. Soc., 2006, 128, 12420–12421; (d) M. Nishihara,
F. Perret, T. Takeuchi, S. Futaki, A. N. Lazar, A. W. Coleman, N. Sakai
and S. Matile, Org. Biomol. Chem., 2005, 3, 1659–1669; (e) N. Sakai,
T. Takeuchi, S. Futaki and S. Matile, ChemBioChem, 2005, 6, 114–122;
(f) N. Sakai and S. Matile, J. Am. Chem. Soc., 2003, 125, 14348–14356.
6 (a) S. M. Butterfield, T. Miyatake and S. Matile, Angew. Chem. Int. Ed.,
2009, 48, 325–328; (b) T. Takeuchi and S. Matile, J. Am. Chem. Soc.,
2009, 131, 18048–18049; (c) N. Sakai, J. Mareda and S. Matile, Acc.
Chem. Res., 2008, 41, 1354–1365; (d) S. Hagihara, H. Tanaka and S.
Matile, J. Am. Chem. Soc., 2008, 130, 5656–5657; (e) S. Litvinchuk, H.
Tanaka, T. Miyatake, D. Pasini, T. Tanaka, G. Bollot, J. Mareda and
S. Matile, Nat. Mater., 2007, 6, 576–580; (f) N. Sakai, J. Mareda and
S. Matile, Acc. Chem. Res., 2005, 38, 79–87; (g) N. Sorde´, G. Das and
S. Matile, Proc. Natl. Acad. Sci. USA, 2003, 100, 11964–11969; (h) G.
Das, P. Talukdar and S. Matile, Science, 2002, 298, 1600–1602; (i) N.
Sakai, B. Baumeister and S. Matile, ChemBioChem, 2000, 1, 123–125.
7 (a) S. C. Semple, A. Akinc, J. Chen, A. P. Sandhu, B. L. Mui, C.
K. Cho, D. W. Y. Sah, D. Stebbing, E. J. Crosley, E. Yaworski, I.
M. Hafez, J. R. Dorkin, J. Qin, K. Lam, K. G. Rajeev, K. F. Wong,
L. B. Jeffs, L. Nechev, M. L. Eisenhardt, M. Jayaraman, M. Kazem,
M. A. Maier, M. Srinivasulu, M. J. Weinstein, Q. Chen, R. Alvarez,
S. A. Barros, S. De, S. K. Klimuk, T. Borland, V. Kosovrasti, W. L.
Cantley, Y. K. Tam, M. Manoharan, M. A. Ciufolini, M. A. Tracy,
A. de Fougerolles, I. MacLachlan, P. R. Cullis, T. D. Madden and
M. J. Hope, Nat. Biotechnol., 2010, 28, 172–176; (b) M. Me´vel, N.
Kamaly, S. Carmona, M. H. Oliver, M. R. Jorgensen, C. Crowther,
F. H. Salazar, P. L. Marion, M. Fujino, Y. Natori, M. Thanou, P.
Arbuthnot, J.-J. Yaouanc, P. A. Jaffre`s and A. D. Miller, J. Controlled
Release, 2010, 143, 222–232; (c) M. E. Davis, J. E. Zuckerman, C. H. J.
Choi, D. Seligson, A. Tolcher, C. A. Alabi, Y. Yen, J. D. Heidel and A.
Ribas, Nature, 2010, 464, 1067–1070; (d) K. A. Whitehead, R. Langer
and D. G. Anderson, Nat. Rev. Drug Discov., 2009, 8, 129–138; (e) V.
Concerning the nature of the tails, we found that long cis-
alkenyl and branched alkyl tails are more active than their linear
analogs. The totally unpredictable identification of the branched
and aromatic tail from jasminaldehyde TJAS as the one that works
well (YMAX ≥ 0.5) with the highest number of different head groups
underscores the power of “high-throughput” counterion screening
with dynamic amphiphiles (Table S1,† Fig. 6). Dodecanal T12 as
closest competitor is poorly active with G1H4 (YMAX = 0.24
0.07, Table S1,† Fig. 4a), all other tails fail with at least two head
groups. Attractive applications of these overall very consistent
lessons learned in this study toward fragrant cellular uptake are
currently under investigation.
Acknowledgements
We thank D. Jeannerat, A. Pinto and S. Grass for NMR mea-
surements, the Sciences Mass Spectrometry (SMS) platform at the
Faculty of Sciences, University of Geneva, for mass spectrometry
services, and the University of Geneva, the NCCR Chemical
2646 | Org. Biomol. Chem., 2011, 9, 2641–2647
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Janout and S. L. Regen, Bioconjugate Chem., 2009, 20, 183–192; (f) J. A.
Boomer, M. M. Qualls, H. D. Inerowicz, R. H. Haynes, V. S. Patri, J. M.
Kim and D. H. Thompson, Bioconjugate Chem., 2009, 20, 47–59; (g) S.
Bhattacharya and A. Bajaj, Chem. Commun., 2009, 45, 4632–4656;
(h) V. Bagnacani, F. Sansone, G. Donofrio, L. Baldini, A. Casnati and
R. Ungaro, Org. Lett., 2008, 10, 3953–3956; (i) L.-A. Tziveleka, A.-M.
G. Psarra, D. Tsiourvas and C. M. Paleos, J. Controlled Release, 2007,
117, 137–146; (j) J. Yang, H. Chen, I. R. Vlahov, J. X. Cheng and P.
S. Low, J. Pharmacol. Exp. Ther., 2007, 321, 462–468; (k) S. Horiuchi
and Y. Aoyama, J. Controlled Release, 2006, 116, 107–114; (l) H. Isobe,
W. Nakanishi, N. Tomita, S. Jinno, H. Okayama and E. Nakamura,
Chem. Asian J., 2006, 1, 167–175; (m) M. Sainlos, M. Hauchecorne,
N. Oudrhiri, S. Zertal-Zidani, A. Aissaoui, J.-P. Vigneron, J.-M. Lehn
and P. Lehn, ChemBioChem, 2005, 6, 1023–1033; (n) C. R. Dass, J.
Mol. Med., 2004, 82, 579–591; (o) S. Zhanga, Y. Xu, B. Wang, W. Qiao,
D. Liu and Z. Li, J. Controlled Release, 2004, 100, 165–180; (p) R.
Haag, Angew. Chem. Int. Ed., 2004, 43, 278–282; (q) M. Liu and J.
M. J. Frechet, Pharm. Sci. Technol. Today, 1999, 2, 393–401; (r) V.
Ciccarone, Y. Chu, K. Schifferli, J. Pichet, P. Hawley-Nelson, K. Evans,
L. Roy and S. Bennett, Focus, 1999, 21, 54–55; (s) D. A. Tomalia, A. M.
Naylor and W. A. Goddard, Angew. Chem. Int. Ed., 1990, 29, 138–175;
(t) P. L. Felgner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M.
Wenz, J. P. Northrop, G. M. Ringold and M. Danielsen, Proc. Natl.
Acad. Sci. USA, 1987, 84, 7413–7417.
S. Gordo, M. Feliz, M. Mene´ndez and E. Giralt, ChemBioChem, 2006,
7, 1105–1113; (t) I. Tsogas, D. Tsiourvas, G. Nounesis and C. M. Paleos,
Langmuir, 2006, 22, 11322–11328; (u) S. M. Fuchs and R. T. Raines,
ACS Chem. Biol., 2007, 2, 167–170; (v) S. Afonin, A. Frey, S. Bayerl, D.
Fischer, P. Wadhwani, S. Weinkauf and A. S. Ulrich, ChemPhysChem,
2006, 7, 2134–2142; (w) T. Takeuchi, M. Kosuge, A. Tadokoro, Y.
Sugiura, M. Nishi, M. Kawata, N. Sakai, S. Matile and S. Futaki,
ACS Chem. Biol., 2006, 1, 299–303; (x) D. Schmidt, Q. X. Jiang and R.
MacKinnon, Nature, 2006, 444, 775–779; (y) F. Perret, M. Nishihara,
T. Takeuchi, S. Futaki, A. N. Lazar, A. W. Coleman, N. Sakai and
S. Matile, J. Am. Chem. Soc., 2005, 127, 1114–1115; (z) R. Fischer,
M. Fotin-Mleczek, H. Hufnagel and R. Brock, ChemBioChem, 2005,
6, 2126–2142; (aa) T. Jiang, E. S. Olson, Q. T. Nguyen, M. Roy, P. A.
Jennings and R. Y. Tsien, Proc. Natl. Acad. Sci. USA, 2004, 101, 17867–
17872.
10 (a) P. Anzenbacher Jr, P. Lubal, P. Bucek, M. A. Palacios and M. E.
Kozelkova, Chem. Soc. Rev., 2010, 39, 3954–3979; (b) L. A. Joyce, S. H.
Shabbir and E. V. Anslyn, Chem. Soc. Rev., 2010, 39, 3621–3632; (c) M.
Florea and W. M. Nau, Org. Biomol. Chem., 2010, 8, 1033–1039; (d) G.
Mirri, S. D. Bull, P. N. Horton, T. D. James, L. Male and J. H. Tucker,
J. Am. Chem. Soc., 2010, 132, 8903–8905; (e) A. Wada, S. Tamaru, M.
Ikeda and I. Hamachi, J. Am. Chem. Soc., 2009, 131, 5321–5330; (f) D.
C. Gonazlez, E. N. Savariar and S. Thayumanavan, J. Am. Chem. Soc.,
2009, 131, 7708–7716; (g) J.-L. Reymond, V. S. Fluxa and N. Maillard,
Chem. Commun., 2009, 45, 34–46; (h) L. Vial and P. Dumy, New J.
Chem., 2009, 33, 939–946; (i) A. Satrijo and T. M. Swager, J. Am.
Chem. Soc., 2007, 129, 16020–16028; (j) A. Buryak, A. Pozdnoukhov
and K. Severin, Chem. Commun., 2007, 2366–2368; (k) R. Jelinek and
S. Kolusheva, Top. Curr. Chem., 2007, 277, 155–180; (l) N. T. Greene
and K. D. Shimizu, J. Am. Chem. Soc., 2005, 127, 5695–5700; (m) J.
J. Lavigne and E. V. Anslyn, Angew. Chem. Int. Ed., 2001, 40, 3118–
3130; (n) N. A. Rakow and K. S. Suslick, Nature, 2000, 406, 710–
703.
11 (a) S. H. Liao and B. List, Angew. Chem., Int. Ed., 2010, 49, 628–631;
(b) R. J. He, C. H. Ding and K. Maruoka, Angew. Chem., Int. Ed.,
2009, 48, 4559–4561; (c) G. L. Hamilton, E. J. Kang, M. Mba and F.
D. Toste, Science, 2007, 317, 496–499; (d) N. Sakai, N. Sorde´ and S.
Matile, J. Am. Chem. Soc., 2003, 125, 7776–7777; (e) J. Lacour and V.
Hebbe-Viton, Chem. Soc. Rev., 2003, 32, 373–382.
12 (a) K. Sadler and J. P. Tam, Rev. Mol. Biotechnol., 2002, 90, 195–229;
(b) T. Darbre and J.-L. Reymond, Acc. Chem. Res., 2006, 39, 925–934.
13 (a) C.-X. Miao, L.-N. He, J.-Q. Wang and J.-L. Wang, Adv. Synth.
Catal., 2009, 351, 2209–2216; (b) M. Uyanik, M. Akakura and K.
Ishihara, J. Am. Chem. Soc., 2009, 131, 251–262; (c) G. Sbardella,
S. Castellano, C. Vicidomini, D. Rotili, A. Nebbioso, M. Miceli, L.
Altucci and A. Mai, Bioorg. Med. Chem. Lett., 2008, 18, 2788–2792;
(d) T. Yoshida, M. Murai, M. Abe, N. Ichimaru, T. Harada, T. Nishioka
and H. Miyoshi, Biochemistry, 2007, 46, 10365–10372; (e) M. J. Schultz,
S. S. Hamilton, D. R. Jensen and M. S. Sigman, J. Org. Chem., 2005,
70, 3343–3352; (f) E. J. Corey and J. W. Suggs, Tetrahedron Lett., 1975,
16, 2647–2650; (g) R. F. Nystrom, J. Am. Chem. Soc., 1959, 81, 610–
612.
8 (a) A. J. Kirby, P. Camilleri, J. B. F. N. Engberts, M. C. Feiters, R. J.
M. Nolte, O. So¨derman, M. Bergsma, P. C. Bell, M. L. Fielden, C. L.
Garc´ıa Rodr´ıguez, P. Gue´dat, A. Kremer, C. McGregor, C. Perrin, G.
Ronsin and M. C. P. van Eijk, Angew. Chem. Int. Ed., 2003, 42, 1448–
1457; (b) F. M. Menger and J. S. Keiper, Angew. Chem. Int. Ed., 2000,
39, 1906–1920.
9 (a) K. Ezzat, S. El Andaloussi, R. Abdo and U. Langel, Curr. Pharm.
Design, 2010, 16, 1167–1178; (b) A. Verma and F. Stellacci, Small,
2010, 6, 12–21; (c) T. I. Rokitskaya, N. V. Sumbatyan, V. N. Tashlitsky,
G. A. Korshunova, Y. N. Antonenko and V. P. Skulachev, Biochim.
Biophys. Acta, 2010, 1798, 1698–1706; (d) A. J. Beevers and A. M.
Dixon, Chem. Soc. Rev., 2010, 39, 2146–2157; (e) A. E. Jablonski, T.
Kawakami, A. Y. Ting and C. K. Payne, J. Phys. Chem. Lett., 2010,
9, 1312–1315; (f) A. A. Stromstedt, M. Pasupuleti, A. Schmidtchen
and M. Malmsten, Biochim. Biophys. Acta, 2010, 1788, 1916–1923;
(g) M. Reuter, C. Schwieger, A. Meister, G. Karlsson and A. Blume,
Biophys. Chem., 2009, 144, 27–37; (h) K. Inomata, A. Ohno, H. Tochio,
S. Isogai, T. Tenno, I. Nakase, T. Takeuchi, S. Futaki, Y. Ito, H. Hiroaki
and M. Shirakawa, Nature, 2009, 458, 106–109; (i) P. A. Wender, W.
C. Galliher, E. A. Goun, L. R. Jones and T. H. Pillow, Adv. Drug
Del. Rev., 2008, 60, 452–472; (j) A. Ziegler, Adv. Drug Del. Rev., 2008,
60, 580–597; (k) K. M. Stewart, K. L. Horton and S. O. Kelley, Org.
Biomol. Chem., 2008, 6, 2242–2255; (l) A. A. Kale and V. P. Torchilin,
Bioconjugate Chem., 2007, 18, 363–370; (m) T. B. Potocky, J. Silvius,
A. K. Menon and S. H. Gellman, ChemBioChem, 2007, 8, 917–926;
(n) E. K. Esbjorner, P. Lincoln and B. Norden, Biochem. Biophys. Acta,
2007, 1768, 1550–1558; (o) T. Shimanouchi, P. Walde, J. Gardiner, Y.
R. Mahajan, D. Seebach, A. Thomae, S. D. Kraemer, M. Voser and R.
Kuboi, Biochem. Biophys. Acta, 2007, 1768, 2726–2736; (p) M. Tang,
A. J. Waring and M. Hong, J. Am. Chem. Soc., 2007, 129, 11438–11446;
(q) J. M. Gump and S. F. Dowdy, Trends Mol. Med., 2007, 13, 443–448;
(r) E. P. Holowka, V. Z. Sun, D. T. Kamei and T. J. Deming, Nat. Mater.,
2007, 6, 52–57; (s) M. Martinell, X. Salvatella, J. Ferna´ndez-Carneado,
14 (a) O. Ramstro¨m and J.-M. Lehn, Nat. Rev. Drug Discovery, 2002, 1,
26–36; (b) P. T. Corbett, J. Leclaire, L. Vial, K. R. West, J. L. Wietor, J.
K. M. Sanders and S. Otto, Chem. Rev., 2006, 106, 3652–3711; (c) A.
Herrmann, Org. Biomol. Chem., 2009, 7, 3195–3204.
15 See Electronic supplementary information†.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 2641–2647 | 2647
©