Programming Bioactive Architectures with Cyclic Peptide Amphiphiles

Article abstract of DOI:10.1002/cplu.201500218

We present a versatile approach for the synthesis of cyclic peptide amphiphiles of the hormone somatostatin (SST) with tunable lipophilic tails to program bioactive nanoarchitectures. A novel bis-alkylation reagent is synthesized that facilitates the functionalization of SST with a thiol anchor. Different hydrophobic moieties are introduced inspired by a biomimetic palmitoylation approach which opens access to cyclic peptide amphiphiles that display rich self-organization and cell membrane interactions. Made to order: Cyclic peptide amphiphiles prepared by a bioinspired approach are employed to program various bioactive nanoarchitectures that display rich self-organization and cell membrane interactions.

Full text of DOI:10.1002/cplu.201500218

DOI: 10.1002/cplu.201500218
Full Papers
Programming Bioactive Architectures with Cyclic Peptide
Amphiphiles
Seah Ling Kuan,* Tao Wang, Marco Raabe, Weina Liu, Markus Lamla, and Tanja Weil*[a]
We present a versatile approach for the synthesis of cyclic pep-
tide amphiphiles of the hormone somatostatin (SST) with tuna-
ble lipophilic tails to program bioactive nanoarchitectures. A
novel bis-alkylation reagent is synthesized that facilitates the
functionalization of SST with a thiol anchor. Different hydro-
phobic moieties are introduced inspired by a biomimetic pal-
mitoylation approach which opens access to cyclic peptide
amphiphiles that display rich self-organization and cell mem-
brane interactions.
Introduction
The synthesis and design of peptide amphiphiles (PAs) has at-
tracted considerable attention owing to various applications in
biotechnology and biomedicine including drug delivery and re-
generative medicine.[1–5] Peptide derivatives with amphiphilic
properties due to alternating sequences of hydrophobic and
hydrophilic (charged) residues that assemble into higher order
nanostructures have been reported.[6–8] Additionally, peptides
that are modified through the attachment of hydrophobic
lipid chains have displayed unique self-assembly properties
and have also been adopted as noncovalent anchors for inser-
tion into nanocarriers such as liposomes.[9,10] In particular, the
design of PAs with bioactive peptides as targeting ligands is
highly attractive for the directed delivery of drugs or bioimag-
ing agents.[10] Although the library of synthetic linear PAs is
abundant in the literature, examples of synthetic cyclic PAs still
remain scarce.[11–13]
active cyclic peptides that introduces new features for control-
ling bioactivity or self-assembly[13,16–20] could be valuable for
many therapeutic applications.
Still, the site-directed attachment of hydrophobic moieties
such as lipids to cyclic peptides and subsequent purification
are challenging.[21,22] One of the few examples is the well-
known cyclic RGDFK PA; it contains a dialkyl lipid chain at-
tached to the cyclic peptide through the lysine e-amino
group.[23] This elegant approach requires the presence of only
a single lysine residue that is not involved in the bioactivity of
the peptide. Therefore, larger cyclic peptides with multiple ly-
sines located in or close to the protein binding site cannot be
modified by this reaction. As a result, there is a considerable
interest in developing a synthetic PA platform by a more versa-
tile chemical approach.
In the cellular machinery, posttranslation processes allow for
the modification of proteins with lipids and one of the pre-
dominant modifications occurs at the free cysteine residue.[24]
In this respect, the physiological palmitoylation machinery
could serve as a good synthetic model for deriving amphiphilic
cyclic peptides in a site-directed manner. Evaluation of protein
databases shows that most therapeutically relevant polypep-
tides and proteins offer at least one disulfide bond close to the
surface that is directed away from the active sites.[25] Many
cyclic peptides found in nature such as somatostatin and oxy-
tocin possess a single disulfide bridge that imparts stability
and is often crucial for generating the correct bioactive three-
dimensional architecture.[26,27] However, the presence of disul-
fide bridges in cyclic peptides often limits the possibilities to
introduce additional free cysteine residues due to disulfide
scrambling, which leads to wrongly folded nonactive peptides
and thus reduces the yields during peptide expression and
solid-phase synthesis.[28,29]
Cyclic peptides play important roles in many cellular pro-
cesses including cellular signaling. Moreover, they are known
to be more stable and usually exhibit higher target-binding af-
finity than the corresponding linear counterparts due to their
rigid scaffolds.[14] Cyclic lipopeptides comprising a cyclic pep-
tide headgroup with a lipid tail are expressed by bacteria and
display a rich pharmacology with antibacterial, antifungal, and
antiviral activities.[15] Consequently, the functionalization of bio-
[a] Dr. S. L. Kuan,+ Dr. T. Wang,+ M. Raabe, W. Liu, Dr. M. Lamla,
Prof. Dr. T. Weil
Institute of Organic Chemistry III–Macromolecular Chemistry & Biomaterials
Ulm University, Albert-Einstein-Allee 11
89081 Ulm (Germany)
E-mail: seah-ling.kuan@uni-ulm.de
tanja.weil@uni-ulm.de
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cplu.201500218. It contains experimental de-
tails and syntheses of compounds 1, 3–5.
Herein, we report the synthesis of three cyclic PAs of the
peptide hormone somatostatin (SST) whose amphiphilic prop-
erties are tuned in a versatile manner to tailor different biona-
noarchitectures. SST is a neuropeptide that is efficiently taken
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up into cells and translocated into the cytosol. This is mediated
by five G protein coupled membrane-bound receptors (SSTR1–
5) that are overexpressed in several tumors including breast,
lung, prostate, and neuroendocrine cancers.[30–32] SST deriva-
tives can serve both as targeting ligands as well as antiprolifer-
ative drugs.[33,34] Due to their bioactivities, SST conjugates are
of immense clinical interest for radiotherapy, tumor imaging,
and chemotherapy.[35,36]
Results and Discussion
The functionalization of SST relies on the structurally novel bis-
alkylation reagent 2-((tert-butoxycarbonyl)thio)ethyl 2-(tosylme-
thyl)acrylate (3), which introduces a single thiol anchor group
by converting the chemically labile disulfide bond into two
SCH001 more stable bis-sulfides (Scheme 1). Bis-alkylation reagents
have been used for the functionalization of peptide hor-
mones,[37–39] antibodies,[25,40] oligonucleotides,[41] and therapeu-
tic proteins.[40] In contrast to conventional bis-sulfone reagents
containing three phenyl rings,[37] 3 offers improved solubility
and the thiol anchor can be attached under aqueous condi-
tions; various modifications with hydrophobic substituents can
be introduced subsequently (Scheme 1a).[42]
Scheme 1. Synthesis of SST-SH 4. i) K2CO3, Boc2O, ACN, 24 h, 85%; ii) Et3N,
methacryloyl chloride, anhydrous DCM, overnight, 83%; iii) I2, sodium p-tolu-
ene sulfinate, DCM, 3d, then 3 equiv Et3N RT overnight and reflux overnight
in EA, 59%; iv) SST, TCEP, 50 mm PB, pH 7.8, 24 h, then TFA/MiliQ H2O (2:1),
1 h, 41%. ACN=acetonitrile, DCM=dichloromethane, EA=ethyl acetate,
TCEP=(tris(2-carboxyethyl)phosphine), TFA =trifluoroacetic acid.
ics, atom efficiency, catalyst-free conditions, and the improved
stability of the thioether addition product.[43,44] Thus, it is at-
tractive to apply thiol–maleimide chemistry to program the
amphilicity of cyclic peptides through a thiol anchor. Hydro-
phobic maleimide derivatives 6–8 were prepared by the reac-
tion of N-(2-aminoethyl) maleimide trifluoroacetate salt (5)
with either the NHS esters (Py-NHS, Pal-NHS) or the acid chlo-
ride (Cho-Cl) as depicted in Scheme 2a in moderate to high SCH002
yield (63–81%). The hydrophobic groups were selected for
their different functions and lipophilicities, as determined from
the calculated values of the octanol/water partition coefficients
(logP(o/w)); hydrophobic character of the groups increased in
the following order: pyrene<palmitoyl<cholesteryl (Table 1). TAB001
The bis-alkylation reagent 3 was synthesized by a multistep
reaction sequence (Scheme 1b, Supporting Information (SI)).
O-(tert-butyl) S-(2-hydroxyethyl) carbonothioate (1) was first
synthesized in 85% yield to protect the thiol group for subse-
quent reactions. Thereafter, 1 reacted with methacryloyl chlo-
ride to afford 2-((tert-butoxycarbonyl)thio)ethyl methacrylate
(2) in 83% yield. Finally, 3 was obtained in 59% yield from the
oxidation of 2 and sodium p-toluene sulfinate. Compound 3
contains a monosulfone group as the reactive species and can
react with SST via two consecutive Michael additions in aque-
ous buffer.[42]
SST was first reduced at the disulfide by treatment
with tris(2-carboxyethyl)phosphine hydrochloride
(TCEP) in 50 mm phosphate buffer (PB) at pH 7.8 and
then incubated with 3 overnight at room tempera-
ture. We postulated that 3 reacted with a free thiol
group of the reduced disulfide, followed by the sub-
sequent elimination of the p-toluenesulfinic acid
group to form a second Michael system. In the pres-
ence of another thiol group in close vicinity, the
second Michael addition occurred with the concomi-
tant formation of a C3-rebridged bis-sulfide.[40] The
product was purified by HPLC and the Boc protect-
ing group was removed by treatment with TFA/H2O
(2:1) for 1 h at room temperature to afford the thiol-
functionalized SST, SST-SH (4) in 41% yield over two
steps. Application of this method rather than the
strategy of using conventional bis-sulfone reagent
that we have reported previously[37] leads to an
almost twofold increase in the yield of SST-SH due
to the improved solubility of the bis-alkylation re-
agent in aqueous medium.
Scheme 2. a) Synthesis of hydrophobic maleimide derivatives 6–8: i) pyrene-N-hydroxy-
succinimide ester (Py-NHS), DIEA, DMF, RT, overnight, 81%; ii) cholesteryl chloroformate
(Cho-Cl), DIEA, DMF/CHCl3, RT, overnight, 63%; iii) palmitic acid N-hydroxysuccinimide
ester (Pal-NHS), DIEA, DMF/CHCl3, RT, overnight, 70%. b) Synthesis of somatostatin am-
phiphiles 9a–c by a biomimetic approach: iv) maleimides 6–8, DMF/CHCl3, 48 h (56%,
23%, 69%, respectively). DIEA=N,N-diisopropylethylamine, DMF=dimethylformamide.
Thiol–maleimide chemistry is often employed in
bioconjugation reactions due to fast reaction kinet-
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).The calculated logP(o/w) values (Table 1) corroborate the
Table 1. logP(o/w) values obtained for SST and compounds 4–8.
SP1
HPLC retention times of the cyclic PAs and indicate that the
hydrophobic character of the cyclic amphiphiles is strongly in-
fluenced by the hydrophobic units, increasing in the order:
pyrene<palmitoyl<cholesteryl.
SST
6
7
8
À3.01
4.29
6.97
5.23
SST-SH (4)
À3.24
9a
9b
9c
It had been demonstrated that SST conjugates prepared
using a similar bis-alkylation scheme act as potent SSTR2 ago-
nists and that they are internalized into MCF-7 cells which
overexpress the SST receptors.[38] Thus, formation of a nano-
structure with the SST PAs on the exterior could be important
for therapeutic applications given the epitope’s known bioac-
tivity and was therefore further investigated. TEM images of
9a at 0.5 wt% and 1 wt% showed that the spherical assem-
blies formed have more homogenous size distribution at
1 wt% with an average diameter of 265Æ40 nm (Figure 1a–b, FIG001
SI, Figure S10). The pyrene residue in 9a is expected to localize
within the hydrophobic compartment of the spherical assem-
bly and interact with aromatic hydrophobic dyes such as Nile
Red (NR). Following a literature protocol for encapsulation
studies,[47] 9a was then mixed with NR in a 1:1 mole ratio to
form 9aꢀNR. The NR fluorescence lifetime has been reported
to decrease significantly with an increase in the hydrogen-
bonding capability of the solvent.[45,48] At a 9a concentration
of 0.25 wt%, there was a sharp increase in fluorescence intensi-
ty upon the formation of the spherical assemblies which indi-
cates the presence of NR within the hydrophobic compart-
ments of the amphiphilic nanostructures (Figure 1c). In addi-
tion, a concomitant blue shift of the fluorescence emission
(from 660 nm to 650 nm) and a nonradiative energy transfer
(NRET) from the pyrene excimer to NR (Figure 1d) at lex =
346 nm were also observed, indicating the close proximity of
9a and NR which is a result of the encapsulation of NR in the
hydrophobic compartments of the spherical assemblies.
0.43
(18.7)[a]
3.11
(20.1)[a]
1.36
(19.7)[a]
[a] HPLC retention time in min.
Pyrene has interesting photophysical properties due to the
long lifetime of its monomers and the efficient formation of
excimers;[45] peptides with this modification might provide
a fluorescence probe for additional characterization and may
also interact with hydrophobic drugs through p–p interactions
for host–guest encapsulation. The two lipids cholesterol and
palmitic acid are essential components in mammalian cell
membranes and facilitate the insertion of bioactive peptide
amphiphiles into nanocarriers such as liposomes and carrier
proteins in a noncovalent fashion.[9,10] The resultant bioactive
amphiphiles had been exploited as targeting ligands to modu-
late transport across biological barriers and the localization of
the carrier inside tumor cells.[46]
The reaction of 4 with the respective hydrophobic malei-
mides (6–8) was carried out under argon atmosphere in dime-
thylformamide (DMF)/chloroform (CHCl3) over 48 h to afford
amphiphilic somatostatin analogues 9a–c as shown in
Scheme 2b. Cyclic peptide 9a was obtained in 56% yield after
purification and MALDI-TOF MS is in agreement with mass sig-
nals at 2194.92 [M+H]+ (SI, Figure S4). Successful conjugation
of pyrene in 9a was further corroborated by the characteristic
absorption of pyrene at 346 nm and its fluorescence emission
spectrum (SI, Figure S5). Excimer formation of 9a was observed
at 0.25 wt% (2.5 mgmLÀ1). Peptides 9b and 9c were obtained
in about 23% and 69% yield, respectively, and were identified
by MALDI-TOF MS (9b: 2337.14 [M+H]+; 9c: 2163.04 [M+H]+
The lipophilic tails of 9b and 9c can be employed as nonco-
valent anchors to arrange SST on the exterior of defined three-
dimensional architectures of nanocarriers in order to direct and
modulate their localization in cells. We have chosen human
Figure 1. a) Schematic representation of the formation of spherical assemblies of 9a and encapsulation of Nile Red (NR) dye. i) 1 wt% 9a in H2O from
a 5 wt% solution of 9a in DMSO and ii) 0.5 wt% 9a and NR. b) TEM image of 1 wt% 9a. Sample is stained with 2% sodium phosphotungstate adjusted to
pH 7. c) Emission spectra of NR incubated with varying concentrations of 9a showing incorporation of the hydrophobic dye at 0.25 wt%. d) Emission spectra
of NR incubated 9a. Inset shows NRET from excimer of 9a to NR at higher concentration.
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serum albumin (HSA) as the carrier core for the proposed
system based on its biocompatibility. HSA is a natural trans-
porter for various molecules and one of the most exploited
proteins used as a drug delivery vehicle in therapeutics and
clinical biochemistry.[49–53] It is known to be involved in trans-
port and storage in biological processes and possesses hydro-
phobic binding pockets for both exogenous and endogenous
ligands.[50,54] It has seven binding sites for fatty acids (Fig-
FIG002 ure 2a)[55] and cholesterol has been shown to bind to HSA with
the somatostatin amphiphiles. An AFM study of 9c-HSA shows
single macromolecules with a height of 1.5Æ0.4 nm which is
comparable to HSA with a height of 1.4Æ0.4 nm (Figure S13).
To investigate the bioactivity of the supramolecular SST to-
wards SSTR-positive cell lines after modification, a cellular
uptake assay was accomplished in the human breast cancer
cell line MCF-7 which expresses the SSTR2 receptor.[33] After 2 h
of incubation with 9a the fluorescence emission of lysed MCF-
7 was obtained as an indicator for successful membrane trans-
location. A concentration-dependent uptake of 9a
(Figure 3a) was observed and the translocation of FIG003
9aꢀNR across cellular membranes was also substan-
tiated by confocal microscopy (Figure 3b).
Next, we investigated the translocation of 9b-HSA
and 9c-HSA across cell membranes in comparison to
native HSA, which does not permeate membranes of
non-inflamed tissue.[57,58] It was observed that the
cyclic PAs 9b and 9c facilitate the transport of fluo-
rescein-labeled HSA into cells (Figure 3d). Interest-
ingly, 9b-HSA was localized in the cell membranes
while 9c-HSA was internalized into the cells. The dif-
ference in the localization is presumably because
the cholesterol moiety in 9b-HSA directs the anch-
ored HSA to the cholesterol-enriched domains,
which are known to be highly expressed in tumor
cells.[59,60] Cell uptake studies of 9a, 9b-HSA, and 9c-
HSA carried out at 48C and 378C were compared
Figure 2. a) Human serum albumin and its binding sites for fatty acids. b) Binding 9b
and 9c (10 mol equiv) to HSA (1 mol equiv), 3 h, RT. c) Absorbance spectra of HSA, 9b-
HSA, and 9c-HSA showing successful binding. Inset depicts the expansion for absorb-
ance at 280 nm. HSA used is fluorescein-labeled.
a Kb value on the order of 105.[54] This unique feature of HSA to
bind hydrophobic molecules has been exploited clinically:
ABRAXANE, an albumin-bound form of the antitumor drug pa-
clitaxel, is currently available on the market as a drug for
breast cancer treatment.[56] The combination of HSA with
tumor-targeting groups and drugs is therefore highly promis-
ing for designing an efficient translational drug transporter.[49]
Compounds 9b and 9c were applied as noncovalent an-
chors that assembled SST at the periphery of HSA via the inter-
action of the lipid or cholesterol substituents with the protein’s
binding pockets (Figure 2b). An excess of 9b or 9c was added
to fluorescein-labeled HSA and the mixture was incubated for
3 h. Thereafter, any unbound 9b and 9c was removed through
ultrafiltration (molecular weight cut-off=30 kDa) to afford the
SST-loaded HSA analogues 9b-HSA and 9c-HSA. Figure 2c
shows the absorbance spectra of 9b-HSA, 9c-HSA, and HSA
with their concentrations normalized using the absorbance
signal of fluorescein at 480 nm. The absorbance values at
280 nm for 9b-HSA and 9c-HSA are much higher than that of
native HSA due to the additional contribution of aromatic
amino acid residues from somatostatin. This supports the suc-
cessful binding of 9b and 9c to HSA. This was further substan-
tiated by agarose gel electrophoresis where a new band ap-
peared for 9b-HSA with lower mobility (Figure S12b) due to
the increase in molecular weight and decrease in negative
charge upon successful binding of the electropositive 9b to
HSA. The sizes of 9b-HSA and 9c-HSA as determined by dy-
namic light scattering (DLS) are 7.9Æ1.1 nm and 6.4Æ0.2 nm
respectively (Table S1). The slight increase in size compared to
native HSA (6.3Æ1.0 nm) is presumably due to the binding of
since receptor-mediated uptake is an energy-dependent pro-
cess.[61] There is a significant inhibition of the cell uptake of 9a,
9b-HSA, and 9c-HSA (Figure 3a,c) at 48C of 57% (average
over all concentrations), 53% and 67%, respectively (Tables S2
and S3), thereby clearly indicating that 9a, 9b-HSA, and 9c-
HSA enter cells via receptor-mediated cell uptake rather than
via passive diffusion.
Conclusion
Drawing inspiration from the cellular palmitoylation process,
we have presented herein a two-step approach that allows the
site-directed attachment of hydrophobic substituents in a con-
venient fashion in order to derive bioactive cyclic PAs
1) through the introduction of a thiol anchor to cyclic polypep-
tides and 2) capitalizing on the atom-efficient thiol–maleimide
chemistry to achieve different lipid modifications. The peptide
hormone SST was successfully functionalized with a thiol, and
thus three different SST amphiphiles (9a–9c) were obtained
from the SST-SH building block which complement the limited
number of cyclic PAs in the literature. The SST amphiphiles
were used to direct the assembly of different supramolecular
bionanoarchitectures which could be used for drug delivery by
encapsulating hydrophobic drugs with bioactive epitopes on
the exterior as shown with 9a. Or they could be employed to
decorate the periphery of membrane impermeable proteins to
facilitate their transport across cell membranes as shown with
9b and 9c. Notably, the SST amphiphiles 9a and 9c retain
their SST bioactivity while the interaction of 9b with cell mem-
branes is seemingly dictated by the cholesterol group. Further
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Figure 3. a) Uptake of 9a into MCF-7 cells, 2 h incubation, 378C. Comparison of uptake of 9a into MCF-7 cells (10 mm) showed 75% inhibition at 48C. b) Con-
focal images of NR and 9aꢀNR in MCF-7 cells, 4 h incubation. c) Comparison of uptake of 9b-HSA and 9c-HSA (1 mm) into MCF-7 cells at 48C and 378C, 2 h
incubation, with 53% and 65% inhibition, respectively. d) Confocal images of NR and 9b-HSA, 9c-HSA, and HSA in MCF-7 cells, 4 h incubation.
ed for 2 min with a flow rate of 4 mLminÀ1. The absorbance was
investigation should be undertaken to evaluate the influence
monitored at 280 nm and 220 nm. The product was dried by lyo-
philization and redissolved in 3 mL of TFA/MilliQ H2O 2:1. The solu-
tion was stirred for 1 h at RT and 18 mg of the SST-SH 4 was ob-
of the lipid residue on the subcellular localization of nanocarri-
ers. Based on these studies, new strategies for the design of bi-
onanomaterials that can be directed to various subcellular lo-
calizations for targeted drug transport could be evolved. One
can envision that the chemical amphiphile approach presented
herein represents a versatile platform with significant potential
to chemically program various cyclic peptides through self-or-
ganization to direct drug molecules or imaging reagents to
specific cellular compartments in order to achieve efficient tar-
geting at the subcellular level, which is of great interest in bio-
medicine and bioimaging.
tained after lyophilization in 41% yield and characterized by
MALDI-TOF-MS (m/z=1783.7583 [M+H]+). C82H114N18O21S3. Calc.
1783.75915 [M+H]+
.
Synthesis of somatostatin amphiphiles (9a–c)
All manipulation and reactions were carried out under argon. To
a solution of SST-SH (4) dissolved in anhydrous DMF at a concentra-
tion of about 1 mgmLÀ1 was added 10 mol equiv of the respective
maleimides (6–8) dissolved in anhydrous DMF (6) or CHCl3 (7, 8) at
a concentration of ca. 5 mgmLÀ1 and the reaction mixture was left
to stir for 48 h at RT. The solvent was removed under reduced pres-
sure and the solid was washed with 3”2 mL DCM to remove un-
reacted 6–8 to yield 9a–c. The resultant residue was then purified
by HPLC for the SST amphiphiles 9a,b. All the products were char-
acterized by MALDI-MS or LC-MS.
Experimental Section
Synthesis of SST-SH (4)
Somatostatin (SST, 40 mg, 24.4 mmol) was dissolved in 35 mL of
phosphate buffer (PB, 50 mm, pH 7.8) and TCEP (14 mg, 48.8 mmol)
in 1 mL PB (50 mm, pH 7.8) was added. To this solution, com-
pound 3 (19 mg, 48.8 mmol) in 24 mL of acetonitrile (ACN) was
added slowly. The resulting reaction mixture was incubated at RT
for 24 h. The mixture was then concentrated and purified by Agi-
lent 1260 HPLC using a LiChrospher 100, RP-18, 10 mm, LiChroCART
250–10 column with the mobile phase starting at 95% solvent A
(0.1% TFA in water) and 5% solvent B (0.1% TFA in ACN), increas-
ing to 30% B at 5 min, further increasing to 80% B at 10 min,
reaching 95% B at 22 min, maintained at 95% B for 3 min, and fi-
nally returning to 5% B at 30 min; the column was then equilibrat-
9a: MALDI-TOF MS: 2194.92010 [M+H]+; ESI-MS: 1098 [M+2H]2+
;
731 [M]3+
.
Chemical formula: C108H136N20O24S3, exact mass:
2194.93009 [M+H]+.
9b: MALDI-TOF MS: 2337.14319 [M+H]+; ESI-MS: 1169 [M+2H]2+
,
779 [M]3+
.
Chemical formula: C116H166N20O25S3, exact mass:
2337.15979 [M+H]+
.
9c: ESI-MS: 1082 [M+2H]2+; 721 [M]3+; MALDI-TOF MS: 2163.0415
[M+H]+. Chemical formula: C116H166N20O25S3, exact mass:
2163.0052 [M+H]+. HPLC purity: 81%.
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scription in the Supporting Information. Imaging was then per-
formed using a LSM 710 laser scanning confocal microscope
system (Zeiss, Germany) coupled to an XL-LSM 710 S incubator
and equipped with a 63” oil immersion objective. The acquired
images were processed with Zen software developed by Carl Zeiss.
Transmission electron microscopy (TEM)
Holey carbon coated TEM grids were treated with 2 mL of 1 wt% or
0.5 wt% 9a, washed with water to remove excess material, and
negatively stained with 2 wt% aqueous sodium phosphotungstate
acid adjusted to pH 7. The TEM samples were air-dried overnight
prior to imaging.
Acknowledgements
Encapsulation of Nile Red (NR) with 9a
We are grateful for financial support from the ERC Synergy
(grant 319130-BioQ) and the Volkswagen Foundation (grant
number Akz 86 366). We thank Stephan Fischer for technical sup-
port and Andrew Norris and Adewola Osunsade for proofreading.
In this experiment 1 mL of Nile Red (22.8 mm in CHCl3/MeOH, 4:1)
was mixed with 1 mL of 9a (5 wt% in DMSO) and the volatile sol-
vent was allowed to evaporate. Thereafter, water was added to
reach a total volume of 10 mL (0.5 wt% 9a) and the mixture was in-
cubated for 30 min at RT. The solution was diluted to 20 mL
(0.25 wt% 9a) for absorbance and fluorescent measurements. Six
serial dilutions of 9a were also made from 5 wt% to show the dif-
ference in encapsulation of NR. Fluorescence spectra were ob-
tained at lex =346 nm and 550 nm.
Keywords: peptide
amphiphiles
·
supramolecular
bioconjugates · protein nanoarchitecture · somatostatin
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Binding of 9b or 9c to human serum albumin
In this experiment 10 mol equiv of 9b or 9c was added to 0.5 mg
of FITC-labeled HSA in water and the mixture was incubated for
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Published online on July 14, 2015
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim