Synthesis of Asparagusic Acid Modified Lysine and its Application in Solid-Phase Synthesis of Peptides with Enhanced Cellular Uptake

Article abstract of DOI:10.1055/s-0036-1591847

Cyclic disulfides, such as asparagusic acid, enhance the uptake of a variety of cargoes into live cells. Here, we report a robust and scalable synthesis of an asparagusic acid modified lysine. This amino acid can be used in solid-phase peptide synthesis. We confirmed that incorporation of this building block into the sequence of a peptide increases its cellular uptake substantially.

Full text of DOI:10.1055/s-0036-1591847

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2017, 28, A–D
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A. Tirla et al.
Letter
Synthesis of Asparagusic Acid Modified Lysine and its
Application in Solid-Phase Synthesis of Peptides with Enhanced
Cellular Uptake
Alina Tirla
Moritz Hansen
Pablo Rivera-Fuentes*
Laboratorium für Organische Chemie, ETH Zürich,
HCI G329, Vladimir-Prelog-Weg 3, 8093 Zürich,
Switzerland
pablo.rivera-fuentes@org.chem.ethz.ch
Received: 02.10.2017
Accepted after revision: 07.11.2017
Published online: 08.12.2017
DOI: 10.1055/s-0036-1591847; Art ID: st-2017-b0728-l
Abstract Cyclic disulfides, such as asparagusic acid, enhance the up-
take of a variety of cargoes into live cells. Here, we report a robust and
scalable synthesis of an asparagusic acid modified lysine. This amino
acid can be used in solid-phase peptide synthesis. We confirmed that
incorporation of this building block into the sequence of a peptide in-
creases its cellular uptake substantially.
Alina Tirla (left) obtained her M.Sc. in medicinal chemistry from the
Key words asparagusic acid, modified amino acid, cellular uptake,
University of Glasgow in 2015. Since 2015 she has been Ph.D. student
solid-phase peptide synthesis, cell-penetrating peptides
at ETH Zurich. Her research interests include the synthesis and imple-
mentation of chemical probes to study redox biology.
Moritz Hansen (center) is currently studying towards his M.Sc. degree
at ETH Zurich. In the summer of 2017, he worked in the lab of Pablo
Rivera-Fuentes developing new cell-penetrating peptides.
The cellular membrane has a highly regulated mecha-
nism for controlling the uptake of compounds, which gen-
erally impedes the internalization of large or highly polar
molecules, such as peptides, oligonucleotides, and nanopar-
Pablo Rivera-Fuentes (right) received his Ph.D. from ETH Zurich in
2012. After postdoctoral studies at MIT (2012–2014) and the Universi-
ty of Oxford (2014–2015), he joined the faculty at ETH Zurich in Octo-
ber 2015 as assistant professor of organic chemistry. His research is
focused on the development of chemical tools to observe and perturb
biological function with subcellular precision.
1
ticles. This barrier must be overcome to use biomolecules
2
as therapeutic candidates, and several strategies have been
3
4
5
developed employing liposomes, viral vectors, polymers,
6
and cell-penetrating peptides (CPPs) as delivery vectors.
CPPs are short cationic sequences that have been used
to deliver small molecules, proteins, nucleic acids, and
change between exofacial thiols and the thiols attached to
the cargo, such as fluorescent probes, peptides, CPPs, pro-
teins, or oligonucleotides.9 Matile and coworkers have
shown that cellular uptake can be further improved by at-
6,7
quantum dots to the intracellular space. CPPs can be ob-
tained from natural sources or be synthetically designed,
and the most common peptides used for translocation
across the plasma membrane are Tat, polyarginine, pene-
10
taching either cell-penetrating poly(disulfide)s (siCPDs)
7
tratin, and transportan. The cellular uptake usually takes
or ring-strained disulfides such as asparagusic acid (AspA)
9
place via an endocytotic mechanism, which can be a draw-
back of CPPs if they fail to escape the vesicles (endosomes),
thus not delivering the cargo to the correct intracellular lo-
to the cargo. Employing these strategies, small fluorescent
9
molecules as well as larger compounds, such as 20-mer
1
1
12
peptides, liposomes and polymersomes, and quantum
6,8
13
cation.
dots were successfully brought into the cell.
Recently, a new method of intracellular delivery has
emerged, which uses the endogenous thiol groups present
on the cell surface (exofacial thiols) to enhance cellular up-
AspA has a very small molecular weight (134 Da) and a
simple structure, thus making it an attractive target for fur-
ther studies (Figure 1, a). Specifically, we were interested in
its ability to improve the uptake of any peptides, indepen-
1
take. The method of action is believed to be disulfide ex-
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A. Tirla et al.
Letter
dent of their net charge. For convenience, we envisioned
that the AspA fragment could be attached to the side chain
of an Fmoc-protected lysine. This building block (Fmoc-
Lys(AspA)-OH, Figure 1, b) could be used like any other ami-
no acid in solid-phase peptide synthesis (SPPS), streamlin-
ing the generation of peptides with AspA tags in defined
positions within the amino acid sequence, even in the pres-
ence of other nucleophilic side chains.
It is noteworthy to mention that pure AspA is prone to
polymerization under aerobic atmosphere, even when
stored as a solid at –20 °C. Once polymerized, the solid is
insoluble in most organic solvents. The polymer can be re-
versed back to dithiol 4 by reaction with Zn dust in a 2 M
ammonium hydroxide suspension for 1 h at room tempera-
ture.15
To modify commercially available Fmoc-Lys-OH, AspA
(
1) was converted into the N-hydroxysuccinimide (NHS) es-
HO
S
O
ter intermediate 5 by reacting 1 with NHS in presence of N-
N′-dicyclohexylcarbodiimide (DCC, Scheme 2).16 After fil-
tering the resulting urea and solvent removal, Fmoc-Lys-OH
was added to the reaction mixture. The starting amino acid
is relatively insoluble, so the reaction needs to be carried
out in a mixture of 1,4-dioxane and phosphate-buffered sa-
line (PBS, 0.05 M), 3:1.
H
S
N
NHFmoc
O
S
O
S
HO
a
b
Figure 1 a) AspA; b) Fmoc-Lys(AspA)-OH
Even though AspA clearly enhances the uptake of pep-
tides into cells, there are few reports concerning the syn-
thesis and stability of this molecule and its derivatives.
Herein, we report a robust synthesis of AspA, an AspA-
tagged lysine building block and its application in SPPS.
The synthesis starts following a modified procedure
O
HO
NHS, DCC
MeCN
N
O
O
O
O
25 °C, 1.5 h
S
S
S
14
S
from Venditti et al. Commercially available 3-bromo-2-
bromomethyl)propanoic acid (2) was dissolved in 1 M
(
1
5
NaOH and reacted with potassium thioacetate (KSAc) at
room temperature for 21 h (Scheme 1). The progress of the
reaction can be followed both by thin-layer chromatogra-
phy (TLC) and liquid chromatography–mass spectrometry
Fmoc-Lys-OH
dioxane/PBS 3:1
25 °C, 4 h
30% (over 2 steps)
S
S
(LC–MS, in negative ion mode), and it is crucial that the re-
H
N
NHFmoc
action is allowed to reach completion.
O
HO
O
O
KSAc
O
6
1
M NaOH
Br
OH
AcS
OH
Scheme 2 Synthesis of Fmoc-Lys(AspA)-OH (6)
25 °C, 21 h
Br
SAc
2
3
We found that extraction with CH Cl is an efficient way
2
2
1
M NaOH
5 °C, 16 h
to obtain the product from the reaction mixture. Although a
stable emulsion is formed under these conditions, it can be
easily separated by centrifugation at 1750 × G (see Support-
ing Information). Compound 6 (Fmoc-Lys(AspA)-OH) was
obtained by evaporation of the organic phase and tritura-
tion of the solid with pentane. The obtained solid can be
purified further by flash column chromatography if needed
(see Supporting Information). This synthesis is reproduc-
ible and has been performed on a gram scale.
2
O
HO
O
DMSO
HS
OH
7
5 °C, 15 h
SH
S
S
82% (over 3 steps)
4
1
Scheme 1 Synthesis of AspA (1)
Next, we tested the suitability of the building block for
SPPS. Using Wang resin, we synthesized the peptide Lys(As-
pA)-Gly-Gly-Lys-Asp-Glu-Leu (K(AspA)GGKDEL, see Sup-
porting Information), and the deprotected N-terminus was
capped with 5-carboxyrhodamine B. Cleavage and global
deprotection resulted in peptide 7 (Figure 2). HPLC purifi-
cation and ESI-MS analysis confirmed the purity and iden-
tity of the peptide (see Supporting Information). This result
confirms that the modified amino acid can be incorporated
into a peptide using SPPS. Moreover, this peptide contains
Next, crude thioacetate 3 was hydrolyzed with NaOH to
obtain dithiol 4, which was then oxidized in DMSO, at 75 °C
under aerobic conditions, to obtain the desired AspA (1).15
When the reaction reaches full conversion, the thiol triplet
peak at around δ = 1.53 ppm disappears from the H NMR
spectrum and the obtained product is pure enough for use
in further modifications.
1
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A. Tirla et al.
Letter
two lysine residues, but only one bears an AspA tag. The se-
lective synthesis of this peptide is greatly simplified by the
availability of amino acid 6.
cells were washed, the medium was exchanged, and the
cells were imaged after 2 h using a confocal fluorescence
microscope (Figure 3).
To investigate the ability of AspA to improve cellular up-
take, we also synthesized peptide 8, which has the same
amino acid sequence and fluorophore as 7, but no AspA tag
As it can be seen in Figure 3 (C and F), the uptake of pep-
tide 7 is superior to that of 8. By comparing fluorescence in-
tensity of cells treated with the two peptides, we can con-
clude that the AspA moiety improves the cellular uptake by
at least a factor of four (Figure 4).
(Figure 2)
GGKDEL-OH
NH
O
O
O
HOOC
Et2N
N
H
N
S
H
S
O
+NEt2
7
Figure 4 Quantification of intracellular fluorescence intensity of cells
treated with peptides 7 or 8
H
O
N
O
GGKDEL-OH
NH3
HOOC
Et2N
N
H
In summary, we report the synthesis of both AspA and
an AspA-tagged lysine. This amino acid can be obtained in
five steps, with only one flash column chromatography re-
quired in the last step of the synthesis. Furthermore, the
modified amino acid is stable under standard SPPS condi-
tions and its incorporation into a peptide sequence increas-
es the cellular uptake of the attached cargo.
O
+NEt2
8
Figure 2 Structures of peptides 7 and 8
The availability of this building block will enable the
rapid generation of libraries of peptides with AspA tags in
defined positions. We envision that this modified amino
acid will facilitate the discovery of peptides with enhanced
cellular uptake and potential applications in chemical biol-
ogy and medicinal chemistry.
The peptides were tested for uptake in live human cer-
vical cancer (HeLa) cells. The cells were incubated for 2 h at
7 °C with a 10 μΜ solution of peptide in Dulbecco’s Modi-
3
fied Eagle’s Medium (DMEM), in the absence of serum. The
Funding Information
This work was supported by the Swiss National Science Foundation
(grant 200021_165551).
)(
Acknowledgment
Confocal microscopy was carried out at the Scientific Center for Opti-
cal and Electron Microscopy (ScopeM) of ETH Zurich.
Supporting Information
Supporting information for this article is available online at
https://doi.org/10.1055/s-0036-1591847.
S
u
p
p
o
nrtogI
i
f
rm oaitn
S
u
p
p
ortioIgnfmr oaitn
Figure 3 Live HeLa cell imaging. A) Brightfield image; B) same cells as
in (A), blue channel image of nuclei labeled with dye Hoechst 33342; C)
same cells as in (A), red channel image of cells incubated with 10 μM 7;
D) Brightfield image; E) same cells as in (D), blue channel image of nu-
clei labeled with dye Hoechst 33342; F) same cells as in (D), red channel
image of cells incubated with 10 μM 8. Scale bar = 10 μm.
References and Notes
(1) Torres, A. G.; Gait, M. J. Trends Biotechnol. 2012, 30, 185.
(2) Stewart, M. P.; Sharei, A.; Ding, X.; Sahay, G.; Langer, R.; Jensen,
K. F. Nature 2016, 538, 183.
(3) Allen, T. M.; Cullis, P. R. Adv. Drug Delivery Rev. 2013, 65, 36.
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–D

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A. Tirla et al.
Letter
(
4) Ma, Y.; Nolte, R. J. M.; Cornelissen, J. J. L. M. Adv. Drug Delivery
dioxane-PBS, 3:1 (28 mL) at room temperature (25 °C) and
Fmoc-Lys-OH (538 mg, 1.46 mmol, 1.1 equiv) was added in one
portion. After 4 h, the solution was acidified by slow addition of
1 M HCl (ca. 2 mL) and CH Cl (ca. 25 mL) until phase separation
Rev. 2012, 64, 811.
(5) Haag, R.; Kratz, F. Angew. Chem. Int. Ed. 2006, 45, 1198.
(6) Bechara, C.; Sagan, S. FEBS Lett. 2013, 587, 1693.
(7) Fonseca, S. B.; Pereira, M. P.; Kelley, S. O. Adv. Drug Delivery Rev.
2
2
occurred. The aqueous phase was extracted again with CH Cl2
2
2
009, 61, 953.
(2 × 25 mL). A white emulsion formed, which could be sepa-
rated by centrifugation (3 min at 1750 × G). The combined
(
(
8) Bruce, V. J.; McNaughton, B. R. Cell Chem. Biol. 2017, 24, 924.
9) Gasparini, G.; Sargsyan, G.; Bang, E. K.; Sakai, N.; Matile, S.
Angew. Chem. Int. Ed. 2015, 54, 7328.
organic phases were dried over MgSO , filtered, and concen-
4
trated under reduced pressure to give a very viscous light-
yellow oil. The product was precipitated from pentane. The
obtained solid was purified by flash column chromatography
(SiO ; CH Cl –MeOH, 100 to 90:10) to give a white solid (198
(
10) Gasparini, G.; Bang, E.-K.; Molinard, G.; Tulumello, D. V.; Ward,
S.; Kelley, S. O.; Roux, A.; Sakai, N.; Matile, S. J. Am. Chem. Soc.
2014, 136, 6069.
2
2
2
1
(
11) Abegg, D.; Gasparini, G.; Hoch, D. G.; Shuster, A.; Bartolami, E.;
Matile, S.; Adibekian, A. J. Am. Chem. Soc. 2017, 139, 231.
12) Chuard, N.; Gasparini, G.; Moreau, D.; Lörcher, S.; Palivan, C.;
Meier, W.; Sakai, N.; Matile, S. Angew. Chem. Int. Ed. 2017, 56,
mg, 30% yield). H NMR (400 MHz, DMSO-d ): δ = 1.24–1.46 (m,
6
4 H, H-5 and H-6), 1.54–1.76 (m, 2 H, H-7), 2.98–3.22 (m, 5 H,
H-4, H-2, two of H-1), 3.37 (m, 2 H, two of H-1), 3.86–3.95 (m, 1
H, H-8), 4.19–4.26 (m, 1 H, H-11), 4.25–4.30 (m, 2 H, H-10), 7.33
(td, J = 7.4, 1.1 Hz, 2 H, H-13), 7.42 (td, J = 7.5, 1.6 Hz, 2 H, H-14),
7.59 (d, J = 8.0 Hz, 1 H, H-9), 7.73 (d, J = 7.5 Hz, 2 H, H-12), 7.89
(
2947.
(
(
(
(
13) Derivery, E.; Bartolami, E.; Matile, S.; Gonzalez-Gaitan, M. J . Am.
Chem. Soc. 2017, 139, 10172.
(d, J = 7.5 Hz, 2 H, H-15), 8.11 (t, J = 5.6 Hz, 1 H, H-3) ppm. 13
C
14) Venditti, A.; Mandrone, M.; Serrilli, A. M.; Bianco, A.; Iannello,
C.; Poli, F.; Antognoni, F. J. Agric. Food Chem. 2013, 61, 6848.
15) Yanagawa, H.; Kato, T.; Sagami, H.; Kitahara, Y. Synthesis 1973,
NMR (101 MHz, DMSO-d ): δ = 23.5, 28.9, 30.8, 39.0, 42.5, 42.6,
6
47.1, 52.0, 54.1, 66.0, 120.5, 120.6, 125.7, 125.8, 127.5 (2 C),
128.1 (2 C), 141.1, 141.2, 144.2, 144.3, 156.6, 170.6, 174.4 ppm.
+
6
07.
ESI-HRMS: m/z calcd for [C25H28N O S Na] : 523.1332; found:
2
5 2
2
6
16) N -{[(9H-Fluoren-9-yl)methoxy]carbonyl}-N -(1,2-dithio-
lane-4-carbonyl)-L-lysine (Fmoc-Lys(AspA)-OH, 6)
523.1340.
Crude NHS ester 5 (328 mg, 1.32 mmol) was dissolved in
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–D