Cell-Penetrating Peptides Containing Fluorescent d-Cysteines

Article abstract of DOI:10.1002/chem.201800603

A series of fluorescent d-cysteines (Cys) has been synthesized and their optical properties were studied. The key synthetic step is the highly diastereoselective 1,4-conjugate addition of aryl thiols to a chiral bicyclic dehydroalanine recently developed by our group. This reaction is fast at room temperature and proceeds with total chemo- and stereoselectivity. The Michael adducts were easily transformed into the corresponding amino acids to study their optical properties and, in some selected cases, into the corresponding N-Fmoc-d-cysteine derivatives to be used in solid-phase peptide synthesis (SPPS). To further demonstrate the utility of these non-natural Cys-derived fluorescent amino acids, the coumaryl and dansyl derivatives were incorporated into cell-penetrating peptide sequences through standard SPPS and their optical properties were studied in different cell lines. The internalization of these fluorescent peptides was monitored by fluorescence microscopy.

Full text of DOI:10.1002/chem.201800603

A Journal of
Accepted Article
Title: Cell Penetrating Peptides containing Fluorescent D-Cysteines
Authors: Claudio D. Navo, Alicia Asín, Eva Gómez-Orte, Marta I.
Gutiérrez-Jiménez, Ismael Compañón, Begoña Ezcurra,
Alberto Avenoza, Jesús H. Busto, Francisco Corzana, María
M. Zurbano, Gonzalo Jiménez-Osés, Juan Cabello, and
Jesús Peregrina
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To be cited as: Chem. Eur. J. 10.1002/chem.201800603
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Cell Penetrating Peptides containing Fluorescent D-Cysteines
Claudio D. Navo,[a] ,† Alicia Asín,[a],† Eva Gómez-Orte,[b],† Marta I. Gutiérrez-Jiménez,[a],† Ismael
Compañón,[a] Begoña Ezcurra,[b] Alberto Avenoza,[a] Jesús H. Busto,[a] Francisco Corzana,[a] María M.
Zurbano,[a] Gonzalo Jiménez-Osés,[a] Juan Cabello*[b] and Jesús M. Peregrina*[a]
Abstract: A series of fluorescent D-cysteines (Cys) has been
synthesized and their optical properties were studied. The key
synthetic step is the highly diastereoselective 1,4-conjugate addition
of aryl thiols to a chiral bicyclic dehydroalanine recently developed by
our group. This reaction is fast at room temperature and proceeds with
total chemo- and stereoselectivity. The Michael adducts were easily
transformed into the corresponding amino acids to study their optical
properties and, in some selected cases, into the corresponding N-
Fmoc-D-cysteine derivatives to be used in solid phase peptide
synthesis (SPPS). To further demonstrate the utility of these non-
natural Cys-derived fluorescent amino acids, the coumaryl and dansyl
derivatives were incorporated into cell penetrating peptide sequences
through standard SPPS and their optical properties studied in different
cell lines. The internalization of these fluorescent peptides was
monitored by fluorescence microscopy.
shown that fluorescent D-amino acids allow quick detection of cell
wall biosynthesis in live cells and in numerous bacterial species.6
This method has also been used to study the synthesis of
peptidoglycans in some bacterial species.7
Functionalized amino acids such as lysine, cysteine or tyrosine
have been found to be ideal candidates for the development of
fluorescent labels through conjugation of the corresponding
electrophilic dye. Thus, the aim of this work is to introduce
different commercially available fluorophores into the D-Cys
scaffold for potential diagnostic or imaging purposes when the
resulting amino acids are incorporated into peptides.
L-Cys-derived fluorescent amino acids are normally accessed
through nucleophilic attack of the sulfhydryl group of L-Cys to a
fluorescent building block equipped with a suitable leaving group.
(Scheme 1).
LG
HS
Introduction
H
dye containing
Deprotection
R1
a leaving group
(R)
N
CO2R3
Nowadays, fluorescence spectroscopy is routinely used to
investigate complex biological processes as well as a diagnostic
and research tool in many fields of biomedical sciences.1
Although natural amino acids such as tryptophan, tyrosine and
phenylalanine have been used as intrinsic fluorescent probes to
monitor different biological events, their use is restricted due to
their poor optical properties.2 Therefore, the design and synthesis
of fluorescent non-natural amino acids and their incorporation into
peptides or proteins has emerged as a powerful tool to study
biological processes using fluorescence spectroscopy.3-4 Given
the continuous development of imaging techniques and synthetic
biology methods, there is an urgent need for expanding the
toolbox of available amino acids incorporating a variety of
fluorescent moieties with different spectroscopic properties.3-4
Although several synthetic protocols have been developed for the
synthesis of fluorescent non-natural L-amino acids,1,3-4 very few
are described for D-amino acids.5 In this context, it has been
R2
Nucleophilic
Substitution
protected
L
-Cys
S
H
(R)
SH
H2N
CO2H
L
-Cys-derived
fluorescent
amino acids
dye containing
a thiol group
R1
N
R2
CO2R3
Deprotection
protected
dehydroamino acid
S-Michael
additon
Scheme 1. Retrosynthetic analysis of Cys-derived fluorescent amino acids.
However, the formation of C-S bonds involving the opposite
approach –the nucleophilic reaction of dyes incorporating a thiol
group with electrophilic amino acid derivatives- has received less
attention. In this work, we report a new methodology using thiol-
containing dyes as nucleophiles which undergo stereoselective S-
Michael addition to a chiral dehydroamino acid (Scheme 1).
Dehydroalanine (Dha)8 is an a,b-unsaturated amino acid of
biological and synthetic interest9 which acts as a chemical
[a]
C. D. Navo, A. Asín, M. I. Gutiérrez-Jiménez, I. Compañón, Prof. A.
Avenoza, Dr. J. H. Busto, Dr. F. Corzana, Dr. M. M. Zurbano, Dr. G.
Jiménez-Osés and Prof. J. M. Peregrina
Dpto. de Química. Centro de Investigación en Síntesis Química,
Universidad de La Rioja
C/ Madre de Dios, 53, 26006. Logroño, La Rioja, Spain
jesusmanuel.peregrina@unirioja.es
Dr. J. Cabello, Dr. B. Ezcurra, Dr. Gómez-Orte
Center for Biomedical Research of La Rioja (CIBIR)
C/ Piqueras, 98, 26006, Logroño, La Rioja, Spain
precursor of
a
range of site-selective post-translational
modifications. However, in general, these reactions are poorly
stereoselective. We recently reported the synthesis of a new
cyclic chiral Dha-derivative ent-1 derived from D-serine. This
compound behaves as a versatile substrate for stereoselective
S-Michael additions, generally providing high yields and
diastereoselectivity (Scheme 2).10 More recently, we developed a
rigidified variant of this Dha11 through a lactonization reaction to
[b]
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.201800603
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give the bicyclic chiral Dha ent-2. The reduced conformational
flexibility of this bicyclic scaffold confers higher diastereoinducing
properties even at room temperature (Scheme 2).
Results and Discussion
Starting from the chiral bicyclic Dha 2,11 we assayed the S-
Michael addition reaction of different SH-containing fluorophores
(3a-f) as nucleophiles, applying the same procedure11 developed
for its enantiomer ent-2. Such protocol involves dropwise addition
of equimolecular amounts of aromatic thiols in the presence of
triethylamine as a base and THF or CH2Cl2 as a solvent at room
temperature (Table 1).
Previous work
RS
RSH
base,THF
H
O
O
(R)
CO2Me
N(R)
(S)
CO2Me
N(R)
(S)
O
OH
O
S-Michael
OH
MeO
ent-1
Cyclic chiral Dha
MeO
Michael adduct
(J. Am. Chem. Soc. 2014, 136, 789)
Lactonization
Table 1. Stereoslective S-Michael addition of thiols 3a-f to the chiral Dha 2.
RS
H
O
RSH
base,THF
RS
O
O
(R)
N
(S)
H
O
O
(R)O
Et3N, solvent, r.t., t
(S)
O
N
O
O
(R)O
N
(R)
(S)
O
O
O
(S)O
+
RSH
N
S-Michael
(S)O
MeO
(R)
O
MeO
ent-2
MeO
MeO
Michael adduct
(Org. Lett. 2016, 18, 2796)
Bicyclic chiral Dha
2
3a-f
4a-f
RSH
This work
SH
SH
SH
SH
SH
S
O
N
H
O
N
(R)
S
H
O
(S)
base,THF
S-Michael
HCl
O
(S)O
3a
3b
3c
3d
H
O
N
(S)O
Hydrolisis
(R)
O
(S)
H2N
CO2H
MeO
N
SH
MeO
Michael adduct
O
S
2
D-Cys-derived
fluorescent non-natural
amino acids
Bicyclic chiral Dha
NH
O
O
O
3e
SH
3f
Scheme 2. Synthesis of fluorescent D-Cys from a bicyclic chiral dehydroalanine
(Dha 2) through S-Michael additions.
Entry
RSH
Solvent/time (min)
THF/5
Product
Yield[a]
89
1
3a
4a
This new scaffold (ent-2) also behaved as an excellent S-Michael
acceptor when several thiols, including tri-O-acetyl-2-acetamido-
2-deoxy-1-thio-α-D-galactopyranose, were used as nucleophiles.
The advantage of this synthetic approach over the previously
published work10a is an increased yield in the 1,4-conjugate
addition and the ability to hydrolize the Michael adduct in a
cleaner way.10b
2
3
4
5
6
3b
3c
3d
3e
3f
THF/60
THF/60
THF/60
THF/5
4b
4c
4d
4e
4f
96
95
60
93
92
From this previous, it was apparent that D-Cys-derived amino
acids with enhanced fluorescence properties could be readily
accessible using the same methodology. We envisioned the S-
Michael addition of different commercially available fluorescent
aryl thiols to chiral Dha 2, (the enantiomer of ent-2) as the key
step to obtain a family of S-functionalized D-Cys-derived amino
acids after hydrolysis of the corresponding Michael adducts
(Scheme 2). To date, enantiopure fluorescent non-natural amino
acids have been synthesized in poor global yields, using
stereoselective procedures that need for multiple steps.1 Herein,
we report an efficient and versatile two-step synthesis of
fluorescent non-natural amino acids. Our synthetic protocol
includes general guides for researchers to synthesize their own
custom fluorescent side chain-functionalized D-Cys derivatives,
with different user-defined features from an appropriately SH-
activated fluorescent dye. We focused on coumaryl and dansyl
tags because they show strong fluorescence and comprise a
wide range of commercially available fluorescent tags. Moreover,
they are photostable and present good solubility in many
solvents.1
CH2Cl2/180
[a] Yield after a column chromatography. Diastereoselectivity was higher
than 95:5 in all cases, since only one product was detected by 1H NMR
spectra of the reaction mixtures before purification.
In all cases, the Michael adducts were cleanly formed with
excellent yields (89 to 96%), with the exception of thiol 3d,
probably due to its bidentate (S and N nucleophile) character.
Adducts 4a and 4e were formed in five minutes (entries 1 and 5,
Table 1), while adducts 4b, 4c and 4d needed one hour to
achieve complete conversion, probably due to steric hindrance
(entries 2-4, Table 1). When thiol 3f were used (entry 6, Table 1),
the reaction was carried out in dichloromethane as a solvent
because of reduced solubility in THF, and 3 h were required to
complete the reaction. This thiol is not commercially available and
was prepared following a reported procedure,12
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RS
Interestingly, all S-Michael reactions proceeded with complete
diastereoselectivity, since only a unique diastereoisomer was
observed in the reaction mixture. Therefore, the results reported
herein indicate a robust stereoinduction mechanism for the
protonation of the enolate adduct upon conjugate addition. In
general, the short time needed, together with the mild conditions
used (room temperature), the high yield, and total chemo- and
stereoselectivity achieved, make these 1,4-conjugate additions
true click reactions.
H
RS
O
(S)
4 M HCl, 16 h, 60 o
C
H
O
N
(S)O
(R)
O
(S)
H2N
CO2H
MeO
5a-c and 5e,f
4a-f
Fluorescent amino acids
The high diastereoselectivity obtained can be explained attending
to the pyramidalization of the enolate intermediate, as we
previously demonstrated by theoretical calculations11 in the case
of its enantiomer Dha ent-2. The absolute configuration of the
new stereocenter created in the Michael addition (C3) was
S
S
H
S
H
H
(S)
(S)
(S)
H2N
CO2H
H2N
CO2H
H2N
CO2H
5c (96%)
5a (97%)
5b (97%)
N
determined
by NOESY-NMR experiments (Supporting
O
Information). For instance, the observation of a NOE cross-peak
between the hydrogens of the –CH2S– substituent at C3 and the
CH3 group at C7a of the 2,5-dioxotetrahydro-5H-oxazolo[4,3-
b]oxazol rigid bicyclic system confirmed that C3 displays an S-
configuration in compound 4e (Figure 1).
O
S
O
S
NH
O
S
H
H
(S)
(S)
CO2H
H2N
CO2H
H2N
5e (88%)
5f (96% at 40 oC)
Scheme 3. Synthesis of D-Cys derived fluorescent non-natural amino acids 5a-
c and 5e,f from acid hydrolysis of the corresponding Michael adducts 4a-c and
5e,f.
All these amino acids are stable solids at room temperature and
can be stored without the requirement of any special precautions.
Absorption and fluorescence emission were measured for each
compound and are presented in Table 2. The optical properties of
the aromatic natural amino acids L-tyrosine, L-tryptophan and L-
phenylalanine were also included for comparison purposes.13
All non-natural amino acids showed absorption maxima
wavelengths (lmax) in the ultraviolet region ranging from 302–350
nm, being compounds 5e and 5f the ones with absorption peaks
at lower energies (337 and 350 nm, respectively). In all cases,
single absorption bands at lower energies were observed (Table
2 and Supporting Information).
Figure 1. 2D-NOESY-NMR experiment for compound 4e performed in a 400
MHz equipment using CDCl3 as a solvent at 20 oC.
Upon photoexcitation of all non-natural amino acids 5a-c and 5e-
f at wavelengths corresponding to their maximum absorptions, all
these compounds showed a strong emission in the ultraviolet
(compounds 5a-c and 5e) or in the visible regions (compound 5f).
Spectra emission corresponding to normalized fluorescence
intensity are given in Figure 2. Coumaryl and dansyl derivatives,
5e (396 nm, blue region) and 5f (519 nm, green region), are the
more suitable candidates to be used as fluorescent dyes. These
two amino acids looked more whitish when visualized under UV
lamp (Figure 2).
These bicyclic adducts 4a-f can be visualized as protected Cys-
derivatives, in which the amino moiety is protected as a cyclic
carbamate and the carboxylic acid as a lactone. Therefore, the
next step involved a simple acid hydrolysis with aqueous 4 M HCl
at 60 oC for 16 h, giving the corresponding D-Cys derived
fluorescent non-natural amino acids 5a-c and 5e-f in good yields
(88-97%, Scheme 3). To avoid hydrolysis of the sulfonamide
group in compound 4f, the temperature of the reaction was fixed
at 40 oC, leading to amino acid 5f in 96% yield.
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with the amine or thiol nucleophilic groups of the peptides. This
strategy can be challenging with peptides bearing more than one
amine or thiol group, since an non-homogenous mixture of
labeled species can be generated. An alternative strategy to
circumvent this issue involves incorporation in the peptidic
sequence of commercially available amino acids labelled at the
N terminus with a fluorescent dye. However, this methodology
presents the handicap that the fluorescent amino acid only can be
incorporated at the N-terminus of the sequence. Therefore, the
use of adequately protected (Fmoc) fluorescent non-natural
amino acids, such as 5e and 5f, incorporating a dye in the side
chain, allows the direct and site-specific incorporation of the
fluorescent dye as an integrated part of the SPPS methodology
(Scheme 4).
Initially, we protected amino acids 5e,f as Fmoc derivatives 6e,f,
which are ready-to-use in solid phase peptide synthesis (SPPS,
Scheme 4 and Supporting Information). To evaluate the utility of
these amino acids in cell imaging, they were incorporated into the
well-known cell penetrating peptides14 Val-Pro-Ala-Leu-Lys
(VPALK) and Val-Pro-Ala-Leu-Arg (VPALR). We selected these
pentapeptides, also named Bax-Inhibiting Peptides (BIP),
because they belong to the subclass of linear hydrophobic
peptides based on natural amino acids containing the minimal
cell-penetrating sequence able to be internalized. Additionally,
these short sequences composed of five natural amino acids are
non-cytotoxic and it has been demonstrated that they could be
potentially utilized as tools to deliver therapeutic molecules into
cells.14a,15
Table 2. Optical properties of fluorescent D-Cys-derived amino acids 5a-
c,e,f and natural amino acids L-Trp, L-Tyr and L-Phe.
Entry
1
Amino
acid
Concentration[a]
(M)
lmax Abs.[b]
(nm)
lmax Em.[b]
(nm)
5a
1.1·10-5
302
334
2
3
4
5
6
7
8
5b
8.3·10-5
307
285
337
350
287
254
254
370
375
396
519
352
303
282
5c
7.3·10-5
5e
7.2·10-5
5f
5.0·10-5
L-Trp
L-Tyr
L-Phe
-
-
-
[a] Determined in DMSO at room temperature for 5a-c and 5e,f and in water
at pH 7 for L-Trp, L-Tyr and L-Phe.13 [b] Maximum wavelength of the
absorption UV-Vis spectra (lmax Abs.) and maximum wavelength of the
emission (lmax Em.)
Using a standard SPPS protocol involving the use of a 2-
chlorotrityl chloride resin and 1-(bis(dimethylamino)methylene)-
1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate
(HATU), as a coupling agent, along with the Hünig's base (N,N-
diisopropylethylamine, DIEA) and DMF as a solvent, we manually
synthesized the following peptides: Ac-D-Cys(Coum)-L-Val-L-Pro-
L-Ala-L-Leu-L-Lys-OH, 7e, (where D-Cys(Coum) = 5e) and Ac-D-
Cys(Dans)-L-Val-L-Pro-L-Ala-L-Leu-L-Lys-OH, 7f, (where D-
Cys(Dans) = 5f).
Furthermore, and using an automatic microwave assisted solid
phase peptide synthesizer (MW-SPPS) and, a Rink amide MBHA
resin and Oxyma pure/DIC (N,N’-diisopropylcarbodiimide) as a
coupling cocktail, we promptly synthesized with higher yields the
following peptides: Ac-D-Cys(Dans)-L-Val-L-Pro-L-Ala-L-Leu-L-
Lys-NH2, 8f, and Ac-D-Cys(Dans)-L-Val-L-Pro-L-Ala-L-Leu-L-Arg-
NH2, 9f (Scheme 4).
Figure 2. Fluorescence emission spectra of amino acids 5a-c,e,f (left to right)
when they were irradiated at the corresponding maximum absorption. Vials
show the fluorescence of amino acids 5e and 5f after irradiation at 254 nm (see
Supporting Information for details).
In principle, only those derivatives displaying absorbance maxima
at wavelengths longer than 320 nm1d are adequate to be
efficiently incorporated into peptides and/or proteins, thus
avoiding any interference from the endogen aromatic residues.
In our case, non-natural amino acids 5e,f satisfy this requirement
(entries 5 and 6, Table 2) and therefore, were subsequently used
to synthesize well-known cell-penetrating peptides that can be
used in cell imaging.
The common method to obtain fluorescently labeled peptides
involves the bioconjugation of
a fluorescent dye to the
corresponding synthesized peptide.1d,13 Because of this,
commercial fluorescent dyes often contain leaving groups to react
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N
O
O
O
S
NH
O
S
H
S
H
(S)
(S)
CO2H
RHN
CO2H
RHN
R = H: 5f
R = Fmoc: 6f (48%)
R = H: 5e
FmocOSu, NaHCO3
H2O/MeCN (1:2)
r.t., 24 h
R = Fmoc: 6e (47%)
SPPS protocol
Peptide 7e
Peptides 7f, 8f and 9f
Peptide 7e
O
NH
NH2
O
D
-Cys*
S
HN
O
O
O
O
H
N
OH
N
O
N
H
L
-Val
N
H
O
O
L
-Pro
L
-Lys
L
-Ala
L
-Leu
Peptide 7f (R = OH)
Peptide 8f (R = NH2)
O
NH
Figure 3. Absorption and emission spectra of coumaryl (up) and dansyl (down)
peptides 7e and 7f in DMSO.
NH2
O
N
D
-Cys*
S
HN
O
O
O
O
N
H
N
S
As a result, the fluorescence observed with peptide 7e was similar
to that obtained in the control experiment. This fact is probably
due to the fact that the intrinsic fluorescence of cells is
comparable to those emitted by this peptide. Thus, we decided to
focus on the peptides incorporating the dansyl Cys (7f, 8f and 9f).
Fluorescence observed with peptides 7f and 8f was very similar
and different from the control experiment, both molecules being
adequate candidates for this type of assay. However, and in
agreement with the above-described difference of internalization
capacity of sequences VPALK and VPALR,14a the best result was
obtained for peptide 9f, thus validating the use of these amino
acids as intrinsic fluorescent labels.
N
H
R
N
H
L
-Val
N
H
O
O
O
-Ala
L
-Pro
L
-Lys
L
L
-Leu
Peptide 9f
O
NH
NH2
HN
O
D
-Cys*
NH
S
HN
O
H
O
S
O
O
N
H
N
NH2
N
N
L
-Val
N
N
O
H
H
O
O
L
-Arg
L
-Pro
L
-Ala
L
-Leu
Scheme 4. SPPS protocol to achieve peptides 7e and 7-9f.
After purification by semi-preparative reverse-phase high
performance liquid chromatography (RP-HPLC) and lyophilization,
these peptides 7e,f, 8f and 9f were further characterized by UV-
Vis and fluorescence spectroscopy, the excitation and emission
spectra being shown in figure 3. To evaluate both their cell
permeability and fluorescent properties, all peptides were
incubated for 3 h with a human epithelial cell line (HeLa) and
internalization was visualized using fluorescence microscopy
(Figure 4 and Supporting Information).
Figure 4. Control (left) an internalization of peptide 9f by HeLa (right) cells
visualized by a fluorescence microscope. Differences between control cells and
any of the treated cells are significant at p<0.01 (Mann-Whitney U Test, see
Supporting Information).
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with magnetic stirring under inert atmosphere unless noted otherwise.
Analytical thin layer chromatography (TLC) was performed on glass plates
precoated with a 0.25 mm thickness of silica gel. TLC plates were
visualized with UV light and by staining with phosphomolibdic acid (4.5 g)
in ethanol (100 mL) or potassium permanganate (1.5 g KMnO4, 10 g
K2CO3 and 1.25 mL 10% NaOH in 100 mL H2O) solutions Column
chromatography was performed on silicagel (230-400 mesh). Optical
rotations (OR) were measured with a Perkin-Elmer 341 polarimeter at a
concentration (c) expressed in g/100 mL in 1.0 dm cells of capacity 1.0 or
0.35 mL. Melting points were determined on a Büchi B-545 melting-point
apparatus and are uncorrected. 1H and 13C NMR spectra were measured
with a Bruker Avance-400 and Bruker ARX-300 spectrometer with TMS as
the internal standard. Multiplicities are quoted as singlet (s), broad singlet
(br s), doublet (d), doublet of doublets (dd), triplet (t), quartet (q), or
multiplet (m). Spectra were assigned using COSY and HSQC. All NMR
chemical shifts (d) were recorded in ppm, and coupling constants (J) were
reported in Hz. The results of these experiments were processed with
MestreNova software. High-resolution electrospray mass (ESI) spectra
Although the use of D-amino acids is a common strategy used for
developing therapeutic peptides with improving properties, control
experiments with peptides containing L-cysteine are always
convenient. Hence, we also synthesized the peptide Ac-L-
Cys(Dans)-L-Val-L-Pro-L-Ala-L-Leu-L-Arg-NH2,
9f‘,
which
incorporates the fluorescent L-Cys ent-5f. This amino acid ent-5f
is the enantiomer of amino acid 5f and was obtained through
acidic hydrolysis from the Michael adduct ent-4f. This
intermediate was synthesized starting from the enantiomer of
chiral bicyclic Dha 2 (ent-2) and dansyl fluorescent thiol 3f, in the
same conditions described for the S-Michael addition reaction
between Dha 2 and thiol 3f. The cell permeability and fluorescent
properties of this new peptide 9f’ were also evaluated and
resulted to be very similar to those of peptide 9f (Supporting
Information).
The stability of both fluorescent peptides 9f and 9f’ was studied
in human plasma. Both peptides are stable for at least 4 h, which
is the standard time required for the cell permeability assays with
HeLa cells (Supporting Information).
Cell toxicity assays were carried out with fluorescent peptides 9f
and 9f’ using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
hydrolysis method to determine the cell viability as an indicator of
HeLa cells sensitivity to the peptides. As a result, none of the
studied peptides resulted to be toxic in the range of 7.8 μM to 200
μM (Supporting Information).
were recorded on
a microTOF-Q-BRUKER spectrometer; accurate
masseasurements were achieved by using sodium formate as an external
reference. UV-Vis spectra were recorded in approximately 10-5 M solutions
in water (or DMSO) using an OceanOptics USB4000UV-Vis spectrometer
and quartz cells (1 cm path length). Luminescence measurements were
recorded with same solutions using a Jobin-Yvon Horiba Fluorolog 3-22
Tau-3 spectrofluorimeter.
NMR experiments. NMR experiments were performed on a 400 MHz
spectrometer at 298 K. Magnitude-mode ge-2D COSY spectra were
acquired with gradients by using the cosygpqf pulse program with a pulse
width of 90º. Phase-sensitive ge-2D HSQC spectra were acquired by using
z-filter and selection before t1 removing the decoupling during acquisition
by use of the invigpndph pulse program with CNST2 (JHC)=145. Phase-
sensitive ge-2D NOESY experiments were performed. NOE intensities
were normalized with respect to the diagonal peak at zero mixing time.
Conclusions
There is a need for developing non-natural, stereochemically
defined fluorescent amino acids labeled at their side chains to
expand the toolkit of commercially available N-tagged amino
acids. We have developed a direct, simple, cost effective and
efficient synthesis of enantiopure, fluorescent non-natural amino
acids, derived from D-cysteine. The key synthetic step is a S-
Michael addition of thiol-containing dyes, as nucleophiles, on a
chiral bicyclic dehydroalanine, as a Michael acceptor, which can
be easily obtained from the inexpensive natural amino acid L-
serine. This 1,4-conjugate addition proceeds with total chemo-
and stereoselectivity, fast and at room temperature, making it a
true click reaction. The Michael adducts were easily transformed
into the corresponding amino acids to study their optical
properties. In view of the optical properties of the coumaryl and
dansyl D-cysteine derivatives, these two free amino acids were N-
Fmoc-protected to be used in solid phase peptide synthesis
(SPPS) and incorporated into cell penetrating pentapeptides
through standard SPPS. The resulting fluorescent peptides were
treated with different HeLa cell lines, monitoring their
internalization by fluorescence microscopy.
General procedure for the S-Michael addition reaction. To a solution
of chiral α,ß-dehydroamino acid (Dha) derivative 2 (or ent-2) as a Michel
acceptor and triethylamine as a base in dry THF, a solution of the
corresponding SH-containing dye (3a-f) as a nucleophile in dry THF was
added dropwise (0.2 mL/min) at room temperature. After the completed
addition, the reaction was followed by TLC chromatography until total
conversion of the Dha derivative 2 and an aqueous saturated NH4Cl
solution (same volume as THF) was added. The aqueous phase was
extracted twice with ethyl ether and the combined organic phases were
dried with anhydrous Na2SO4, filtered and concentrated. The conversion
and the diastereomeric excess were determined by 1H-NMR experiments.
In order to optimize the Michael additions different conditions were tested.
In the case of compound 3f, dichloromethane was used instead THF due
to problems of solubility in THF.
(3S,7R,7aS)-7-Methoxy-7,7a-dimethyl-3-
((phenylthio)methyl)dihydro-5H-oxazolo[4,3-b]oxazole-2,5(3H)-dione
(4a). Following the general procedure for the Michael additions, Dha
derivative 2 (20.0 mg, 0.094 mmol), triethylamine (14.4 µL, 0.103 mmol),
and thiophenol 3a (11.4 mg, 0.103 mmol) were reacted affording
compound 4a as a colourless oil (27 mg, 0.084 mmol, 89%) after
purification by silica gel column chromatography (hexane/ethyl acetate
25
8:2). [a]D (1.00, CHCl3) = -4.2. HRMS (ESI) m/z: 346.0725 [M+Na]+;
calculated C15H17NO5SNa+ 346.0720. 1H NMR (300 MHz, CDCl3) δ (ppm):
1.62 (s, 3H, Me7a), 1.63 (s, 3H, Me7), 3.14 (dd, 1H, J = 14.2, 10.5 Hz,
CH2S), 3.49 (m, 4H, CH2S, OMe), 4.49 (dd, 1H, J = 10.5, 4.3 Hz, H3), 7.35
(m, 3H, arom), 7.57 (m, 2H, arom). 13C NMR (100 MHz, CDCl3) δ (ppm):
16.6 (Me7a), 22.1 (Me7), 36.6 (CH2S), 51.6 (OMe), 60.4 (C3), 101.6 (C7a),
Experimental Section
Reagents and general procedures. Commercial reagents were used
without further purification. Solvents were dried and redistilled prior to use
in the usual way. All reactions were performed in oven dried glassware
This article is protected by copyright. All rights reserved.

10.1002/chem.201800603
Chemistry - A European Journal
FULL PAPER
108.2 (C7), 127.9, 129.3, 132.2, 133.4 (5xCarom), 158.8 (C5=O), 170.4
(C2=O).
Hz, Me4c), 3.17-3.27 (m, 1H, Hb), 3.48 (s, 3H, OMe7), 3.54-3.62 (m, 1H,
Hb), 4.50 (dd, 1H, J = 10.0, 4.4 Hz, Ha), 6.25 (d, 1H, J = 1.2 Hz, H3c), 7.30-
7.36 (m, 2H, H6c, H8c), 7.50-7.56 (m, 1H, H5c). 13C NMR (75 MHz, CDCl3)
d (ppm): 16.8 (Me7a), 18.7 (Me4c), 22.4 (Me7), 34.9 (CH2S), 51.9 (OMe7),
60.1 (C3), 101.7 (C7a), 108.4 (C7), 115.0 (C3c), 117.2 (C6c), 118.8 (Cqc),
125.3, 125.2 (C8c, C5c), 139.4, 152.0, 153.9 (3xCqc), 158.8 (C5=O), 160.4
(Cc=O), 170.1 (C2=O).
(3S,7R,7aS)-7-Methoxy-7,7a-dimethyl-3-((naphthalen-1-
ylthio)methyl)dihydro-5H-oxazolo[4,3-b]oxazole-2,5(3H)-dione (4b).
Following the general procedure for the Michael additions, Dha derivative
2 (25.0 mg, 0.117 mmol), triethylamine (18.0 µL, 0.129 mmol) and 1-
naphtalenethiol 3b (16.0 µL, 0,129 mmol) were reacted affording
compound 4b as a yellow foam (42 mg, 0.113 mmol, 96%) after purification
5-(Dimethylamino)-N-(2-((((3S,7R,7aS)-7-methoxy-7,7a-dimethyl-2,5-
dioxotetrahydro-2H-oxazolo[4,3-b]oxazol-3-
25
by silica gel column chromatography (CH2Cl2/MeOH 95:5). [a]D (1.00,
CHCl3): -3.3. HRMS (ESI+) (m/z): 396.0879 [M+Na]+; calculated
C19H19NO5SNa+: 396.0876. 1H NMR (400 MHz, CDCl3) d (ppm): 1.48 (s,
3H, Me7a), 1.57 (s, 3H, Me7), 3.15 (dd, 1H, J = 14.2, 10.5 Hz, Hb), 3.11-
4.46 (m, 4H, Hb, OMe), 4.50 (dd, 1H, J = 10.9, 4.2 Hz, Ha), 7.42-7.61 (m,
3H, Harom), 7.83-7.94 (m, 3H, Harom), 8.50 (d, 1H, J = 8.5 Hz, Harom). 13C
NMR (100 MHz, CDCl3) d (ppm): 16.7 (Me7a), 21.9 (Me7), 36.4 (Cb), 51.5
(OMe), 60.6 (Ca), 101.6 (C7a), 107.8 (C7), 125.3, 125.6, 126.4, 127.1,
128.8, 129.6, 130.2, 133.2, 133.6, 134.3 (10xCarom), 158.8 (C5=O), 170.3
(C2=O).
yl)methyl)thio)ethyl)naphthalene-1-sulfonamide (4f). Following the
general procedure for the Michael additions, acrylate 2 (103.0 mg, 0.484
mmol), triethylamine (67 µL, 0.491 mmol) and compound 3f (149 mg,
0,481) were reacted for 3 hours affording compound 4f as a light green-
yellowish viscous solid (231 mg, 0.441 mmol, 92%). [α]D25 (1.00, CHCl3): -
+
36.2. HRMS (ESI+) (m/z): 524.1515 [M+H]+; calculated C23H30N3O7S2
:
524.1520. 1H NMR (400 MHz, CDCl3) d (ppm): 1.58 (s, 3H, Me7a), 1.63 (s,
3H, Me7), 2.75 (td, 2H, J = 6.0, 1.9 Hz, CH2-S), 2.85 (dd, 2H, J = 14.6, 4.4
Hz, Hb), 3.09-3.17 (m, 2H, CH2-NH), 2.91 (s, 6H, Me), 3.53 (s, 3H, OMe7),
4.39 (dd, 1H, J = 10.2, 4.4 Hz, Ha), 7.22 (d, 1H, J = 7.6 Hz, H6d), 7.52-7.65
(m, 2H, H7d, H3d), 8.3- 8.57 (H2d, H4d, H8d). 13C NMR (100 MHz, CDCl3) d
(ppm): 16.7 (Me7), 22.3 (Me7a), 32.3 (CH2-S), 32.6 (Cb), 42.0 (CH2-NH),
45.5 (Med), 51.8 (OMe7), 61.3 (Ca), 101.5 (C7a), 108.5 (C7), 115.4 (C6d),
118.8, 130.0, 130.7 (C2d, C4d, C8d). 123.3, 128.6 (C7d, C3d), 129.6, 129.7,
134.7, 152.1 (C1d, C4ad, C5d, C8ad), 159.0 (C2=O), 170.2 (C5=O).
(3S,7R,7aS)-7-Methoxy-7,7a-dimethyl-3-((naphthalen-2-
ylthio)methyl)dihydro-5H-oxazolo[4,3-b]oxazole-2,5(3H)-dione (4c).
Following the general procedure for the Michael additions, Dha derivate 2
(25.0 mg, 0.117 mmol), triethylamine (18.0 µL, 0.129 mmol) and 2-
naphtalenethiol 3c (18.8 mg, 0,129 mmol) were reacted affording
compound 4c as a yellow foam (42 mg, 0.113 mmol, 95%) after purification
25
by silica gel column chromatography (CH2Cl2/MeOH 95:5). [a]D (1.00,
5-(Dimethylamino)-N-(2-((((3R,7S,7aR)-7-methoxy-7,7a-dimethyl-2,5-
dioxotetrahydro-2H-oxazolo[4,3-b]oxazol-3-
CHCl3): -53.7. HRMS (ESI+) (m/z): 396.0872 [M+Na]+; calculated
C19H19NO5SNa+: 396.0876. 1H NMR (400 MHz, CDCl3) d (ppm): 1.58 (s,
3H, Me7a), 1.61 (s, 3H, Me7), 3.20 (dd, 1H, J = 14.2, 10.4 Hz, Hb), 3,43 (s,
3H, OMe), 3.53-4.53 (m, 1H, Hb), 4.51 (dd, 1H, J = 10.4, 4.2 Hz, Ha), 7.47-
7.50 (m, 2H, Harom), 7.57 (dd, 1H, J = 8.6, 1.8 Hz, Harom), 7.80-7.82 (m, 3H,
Harom), 8.04 (d, 1H, J = 1.5 Hz, Harom). 13C NMR (100 MHz, CDCl3) d (ppm):
16.7 (Me7a), 22.1 (Me7), 36.3 (Cb), 51.6 (OMe), 60.3 (Ca), 101.6 (C7a),
108.0 (C7), 126.5, 126.8, 127.6, 127.7, 129.0, 130.6, 131.1, 132.6, 133.7
(10xCarom), 158.8 (C5=O), 170.4 (C2=O).
yl)methyl)thio)ethyl)naphthalene-1-sulfonamide (ent-4f). Following the
general procedure for the S-Michael addition reactions, acrylate ent-2
(290.0 mg, 1.36 mmol), triethylamine (190 µL, 1.36 mmol) and compound
3f (425 mg, 1.36 mmol) were reacted for 1.5 hours affording compound
25
ent-f as a light green-yellowish syrup (665 mg, 1.27 mmol, 93%). [α]D
(1.00, CHCl3): +40.6. HRMS (ESI+) (m/z): 524.1518 [M+H]+; calculated
C23H30N3O7S2+: 524.1520. NMR data agree with those above reported for
its enantiomer 4f.
(3S,7R,7aS)-3-(((1H-Indol-3-yl)thio)methyl)-7-methoxy-7,7a-
dimethyldihydro-5H-oxazolo[4,3-b]oxazole-2,5(3H)-dione
Following general procedure for the Michael additions, Dha derivate 2
(25.0 mg, 0.117 mmol), triethylamine (18.0 µL, 0.129 mmol) and 1H-
indole-3-thiol 3d (17.50 mg, 0.129 mmol) were reacted affording
compound 4d as a yellow foam (25 mg, 0.069 mmol, 60%) after purification
General procedure for the hydrolysis reaction. A 4 M HCl aqueous
solution was added to the corresponding Michael adduct 4a-c or 4e-f or
ent-4f and the mixture was stirred at 60 oC for 16 h. The solvent was
removed under vacuum and the crude was redissolved in water and
washed with ethyl acetate. The aqueous phase was evaporated affording
the corresponding free amino acid 5a-c, 5e-f and ent-5f. In the case of
Michael adduct 4f, the mixture was stirred at 40 oC.
(4d).
25
by silica gel column chromatography (CH2Cl2/MeOH 95:5). [α]D (1.00,
CHCl3): +174.3. HRMS (ESI+) (m/z): 385.0831 [M+Na]+; calculated
C17H18N2O5SNa+: 385.0829. 1H NMR (400 MHz, CDCl3) d (ppm): 1.63 (s,
3H, Me7a), 1.63 (s, 3H, Me7), 2.82 (dd, 1H, J = 14.1, 11.6 Hz, Hb), 3.26 (dd,
1H, J = 14.2, 3.8 Hz, Hb), 3,51 (s, 3H, OMe), 4.43 (dd, 1H, J = 11.5, 3.8
Hz, Ha), 7.25-7.29 (m, 2H, Harom), 7.42-7.45 (m, 1H, Harom), 7.73-7.74 (m,
1H, Harom), 7.82 (dd, 1H, J = 7.5, 1.5 Hz, Harom), 8.51 (m, 1H, NH). 13C NMR
(100 MHz, CDCl3) d (ppm): 16.7 (Me7a), 22.0 (Me7), 36.7 (Cb), 51.7 (OMe),
60.2 (Ca), 101.7 (C7a), 108.0 (C7), 111.9, 119.0, 121.0, 123.0, 129.0, 132.5,
136.6 (7xCarom), 159.5 (C5=O), 171.0 (C2=O).
S-Phenyl-D-cysteine (5a). Following the general methodology for acid
hydrolysis, starting from 4a (27 mg, 0.084 mmol) and adding a 4 M HCl
aqueous solution (5 mL), compound 5a was obtained as a yellow foam
25
after purification (16 mg, 0.081 mmol, 97%). [α]D (1.00, DMSO): -2.5.
HRMS (ESI+) (m/z): 198.0588 [M+H]+; calculated C9H12NO2S+: 198.0583.
1H NMR (400 MHz, D2O) d (ppm): 3.36-3.41 (m, 1H, Hb), 3.48-3.53 (m, 1H,
Hb), 4.09 (dd, 1H, J = 7.1, 4.4 Hz, Ha), 7.27-7.29 (m, 3H, Harom), 7.42-7.44
(m, 2H, Harom). 13C NMR (100 MHz, D2O) d (ppm): 34.2 (Cb), 52.1
(Ca),128.1, 129.5, 131.4, 132.1 (5xCarom), 170.2 (COOH).
(3S,7R,7aS)-7-Methoxy-7,7a-dimethyl-3-(((4-methyl-2-oxo-2H-
chromen-7-yl)thio)methyl)dihydro-2H-oxazolo[4,3-b]oxazole-2,5(3H)-
dione (4e). Following the general procedure for the Michael additions, Dha
derivative 2 (53.0 mg, 0.249 mmol), triethylamine (38.2 µL, 0.274 mmol)
and 7-mercapto-4-methylcoumarin 3e (52 mg, 0,270) were reacted
affording compound 4e as a yellow solid (96 mg, 0.237 mmol, 93%) after
purification by silica gel column chromatography (CH2Cl2/MeOH 95:5).
[α]D25 (1.00, CHCl3): -51.9. Mp: 128-130 °C. HRMS (ESI+) (m/z): 428.0763
[M+Na]+; calculated C19H19NO7SNa+: 428.0774. 1H NMR (400 MHz,
CDCl3) d (ppm): 1.60 (s, 3H, Me7a), 1.66 (s, 3H, Me7), 2.41 (d, 3H, J = 1.2
S-(Naphthalen-1-yl)-D-cysteine (5b). Following the general methodology
for acid hydrolysis, starting from Michael adduct 4b (42 mg, 0.112 mmol)
and adding a 4 M HCl aqueous solution (5 mL), compound 5b was
obtained as a yellow foam after purification (27 mg, 0.109 mmol, 97%).
25
[α]D (1.00, DMSO): +25.1. HRMS (ESI+) (m/z): 248.0738 [M+H]+;
calculated C13H14NO2S+: 248.0740. 1H NMR (400 MHz, D2O) d (ppm):
3.36-3.41 (m, 1H, Hb), 3.48-3.53 (m, 1H, Hb), 4.09 (dd, 1H, J = 7.1, 4.4 Hz,
Ha), 7.27-7.29 (m, 3H, Harom), 7.42-7.44 (m, 2H, Harom). 13C NMR (100 MHz,
This article is protected by copyright. All rights reserved.

10.1002/chem.201800603
Chemistry - A European Journal
FULL PAPER
DMSO) d (ppm): 42.1 (Cb), 54.2 (Ca), 124.3, 125.8, 126.7, 127.2, 128.9,
129,7, 130.1, 131.9, 132.1, 133.8 (10xCarom), 169.1 (COOH).
7.82 (m, 11H, Harom). 13C NMR (75 MHz, CD3OD) d (ppm): 18.4 (Me4c),
35.1 (Hb), 48.4 (CHFmoc), 54.8 (Ha), 68.1 (CH2Fmoc), 114.5 (C3c), 116.2 (C6c),
120.8, 120.9, 125.2, 126.2, 126.3, 126.5, 128.2, 128.8, 128.8, 129.2
c
(CHarom), 119.0, 142.5, 142.5, 143.6, 145.1, 145.2, 154.9, 155.0 (Cq
CqFmoc), 158.3 (Cc=O), 162.7 (COFmoc), 174.9 (COOH).
,
S-(Naphthalen-2-yl)-D-cysteine (5c). Following the general methodology
for acid hydrolysis, starting from Michael adduct 4c (51 mg, 0.133 mmol)
and adding a 4 M HCl aqueous solution (5 mL), compound 5c was
obtained as a yellow solid after purification (31 mg, 0.127 mmol, 96%).
[α]D25 (1.00, DMSO): -5.7. Mp: 128-130 °C. HRMS (ESI+) (m/z): 248.0744
[M+H]+; calculated C13H14NO2S+: 248.0740. 1H NMR (300 MHz, DMSO) d
(ppm): 3.48-3.64 (m, 2H, Hb), 4.03 (t, 1H, J = 5.8 Hz, Ha), 7.46-7.58 (m,
3H, Harom), 7.84-7.93 (m, 3H, Harom), 7.97-8.02 (m, 1H, Harom). 13C NMR (75
MHz, DMSO) d (ppm): 33.4 (Cb), 51.6 (Ca),126.1, 126.8, 127.1, 127.2,
127.2, 127.7, 128.7, 131.6 131.7, 133.3 (10xCarom), 169.2 (COOH).
N-(((9H-Fluoren-9-yl)methoxy)carbonyl)-S-(2-((5-
(dimethylamino)naphthalene)-1-sulfonamido)ethyl)-D-cysteine (6f).
Derivative 5f (207 mg, 0.521 mmol), FmocOSu (263 mg, 0.133 mmol) and
NaHCO3 (196 mg, 2.33 mmol) were dissolved in a mixture of H2O/CH3CN
(1:2) (15 mL). The solution was stirred at room temperature for 24 h.
CH3CN was removed in vacuo and the aqueous phase was acidified with
2 M HCl (pH 2-3). The solvent was then removed and the crude was
purified by silica gel column chromatography (ethyl acetate/MeOH 95:5)
affording compound 6f as a brownish viscous solid (145 mg, 0.234 mmol,
S-(4-Methyl-2-oxo-2H-chromen-7-yl)-D-cysteine hydrochloride (5e).
Following the general methodology for acid hydrolysis, starting from
Michael adduct 4e (176 mg, 0.435 mmol) and adding a 6 M HCl aqueous
solution (20 mL), compound 5e was obtained as yellow oil (120 mg, 0.381
48%). [α]D (1.00, DMSO): 3.7. HRMS (ESI+) (m/z): 620.1884 [M+H]+;
25
calculated C32H33N3O6S2H+: 620.1884. 1H NMR (400 MHz, DMSO) d
(ppm): 2.58-2.60 (m, 1H, CH2-S), 2.64 (dd, 1H, J = 13.6, 9.8 Hz, Hb), 2.73-
2.76 (m, 1H, CH2-S), 2.77-2.80 (m, 1H, Hb), 2.81 (s, 6H, Me), 2.92-2.99 (m,
2H, CH2-NH), 3.99-4.06 (m, 1H, Ha), 4.17-4.33 (m, 3H, CHFmoc, CH2Fmoc),
7.231-8.46 (m, 16H, Harom, NHFmoc, NHSO2).13C NMR (100 MHz, DMSO)
25
mmol, 88%). [α]D (1.00, H2O): -27.7. HRMS (ESI+) (m/z): 302.0455
[M+Na]+; calculated C13H13NO4SNa+: 302.0457. 1H NMR (400 MHz, D2O)
d (ppm): 2.20 (d, J = 1.1 Hz, 3H, Me4c), 3.44-3.64 (m, 2H, Hb), 4.23 (dd,
1H, J = 7.2, 4.4 Hz, Ha), 6.05 (d, 1H, J = 1.2 Hz, H3c), 7.04 (d, 1H, J = 1.8
Hz, H9c), 7.17 (dd, 1H, J = 8.4, 1.9 Hz, H7c), 7.41 (d, 1H, J = 8.4 Hz, H6c).
13C NMR (100 MHz, D2O) d (ppm):17.8 (Me4c), 32.3 (CH2S), 51.8 (CHNH2),
112.9 (C3c), 115.7 (C6c), 118.1 (Cqc), 125.0, 125.6 (C8c C5c), 138.8, 152.3,
155.5 (3xCqc), 163.3 (Cc=O), 169.7 (COOH).
d (ppm): 25.2 (CH2-S), 32.6 (Cb), 42.5 (CH2-NH), 45.1 (Me), 46.6 (CHFmoc
)
53.9 (Ca), 65.7 (CH2Fmoc), 115.1, 119.1, 119.9, 120.0, 121.4, 123.6, 125.3,
127.1, 127.3, 127.6, 127.9, 128.2, 128.9, 129.5, 136.0, 137.4, 139.4, 140.7,
142.6, 143.8, 151.4, 156.0 (Carom), 172.2, 173.8 (COOH, COFmoc).
N-(((9H-Fluoren-9-yl)methoxy)carbonyl)-S-(2-((5-
(dimethylamino)naphthalene)-1-sulfonamido)ethyl)-L-cysteine (ent-
6f). Derivative ent-5f (500 mg, 1.15 mmol), FmocOSu (583 mg, 1.73
mmol) and NaHCO3 (437 mg, 5.18 mmol) were dissolved in a mixture of
H2O/CH3CN (1:2) (45 mL). The solution was stirred at room temperature
for 24 h. CH3CN was removed in vacuo and the aqueous phase was
acidified with 2 M HCl (pH 2-3). The solvent was then removed and the
crude purified by silica gel column chromatography (ethyl acetate/MeOH
95:5) affording compound ent-6f as a brownish syrup (365 mg, 0.590
S-(2-((5-(Dimethylamino)naphthalene)-1-sulfonamido)ethyl)-D-
cysteine hydrochloride (5f). Following the general methodology for acid
hydrolysis, starting from Michael adduct 4f (231mg, 0.441 mmol) and
adding a 4 M HCl aqueous solution (20 mL), compound 5e was obtained
25
as green-yellowish foamy oil (168 mg, 0.422 mmol, 96%). [α]D (1.00,
D2O): +4.8. HRMS (ESI+) (m/z): 398.1218 [M+H]+; calculated
C17H23N3O4S2NH+: 398.1203. 1H NMR (400 MHz, D2O) d (ppm): 2.41-2.49
(m, 2H, CH2-S), 2.60-2.81 (m, 2H, Hb), 3.01-3.08 (m, 2H, CH2-NH), 3.42
(s, 6H, Me), 3.99 (dd, 1H, J = 7.3, 4.4 Hz, Ha), 7.70-7.84 (m, 2H, H7d, H3d),
7.98 (t, 1H, J = 8.6 Hz, H6d), 8.21-8.61 (m, 3H, H2d,H4d, H8d). 13C NMR (100
MHz, D2O) d (ppm): 30.9 (Cb), 31.3 (CH2-S), 41.7 (CH2-NH), 46.7 (Me),
46.8 (Me) 52.3 (Ca), 119.6, 125.7, 126.6, 130.5 (C2, C4, C6, C8), 125.5,
127.0 (C4a, C8a) 128.1, 128.5 (C7, C3), 135.1 (C1), 138.3 (C5), 170.6
(COOH).
25
mmol, 51%). [α]D (1.00, DMSO): -4.8. HRMS (ESI+) (m/z): 620.1887
[M+H]+; calculated C32H33N3O6S2H+: 620.1884. NMR data agree with
those above reported for its enantiomer 6f.
General methodology for C-terminal acid peptides. 2-Chlorotrityl
chloride-PS resin (0.05 mmol) was swelled in CH2Cl2 for 15 min and
reacted with the first Fmoc-protected amino acid (0.1 mmol) and
diisopropylethylamine (DIPEA) (0.25 mmol) in CH2Cl2 (3 mL) for 2 h at
room temperature. A mixture of CH2Cl2/MeOH/DIPEA (80:15:5, 5 mL) was
added to the resin and the mixture was shaken for 10 min at room
temperature and repeated once with fresh solution. Solid phase peptide
synthesis (SPPS) was performed manually on the resulting resin using 1-
(bis(dimethylamino)methylene)-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide
hexafluorophosphate) (HATU) and DIPEA for coupling and 20% (v/v)
solution of piperidine in DMF for Fmoc deprotection. Fmoc-protected
fluorescent cysteine (0.15 mmol) was coupled using HATU (0.145 mmol)
and DIPEA (1.8 mmol) for 1 hour, as judged by Keiser test.16 Acetic
anhydride and pyridine (1:2) was used for N-terminus acetylation and
trifluoroacetic acid (TFA), thioanisole, anisole and 1,2-ethanedithiol
(90:5:2:3) for the final cleavage and removal of the side chain protecting
groups. The peptide was precipitated with cold diethyl ether, isolated by
centrifugation, washed with cold diethyl ether, dissolved in
acetonitrile/water containing 0.1% TFA (1:1, v/v), and lyophilized to afford
the crude peptide. Purification by reverse phase high-performance liquid
chromatography (RP-HPLC) afforded the corresponding peptide.
S-(2-((5-(Dimethylamino)naphthalene)-1-sulfonamido)ethyl)-L-
cysteine hydrochloride (ent-5f). Following the general methodology for
acid hydrolysis, starting from Michael adduct ent-4f (665 mg. 1.27 mmol)
and adding a 4 M HCl aqueous solution (20 mL), compound ent-5f was
25
obtained as green-yellowish foamy oil (540 mg, 1.24 mmol, 98%). [α]D
(1.00, D2O): -3.2. HRMS (ESI+) (m/z): 398.1209 [M+H]+; calculated
C17H23N3O4S2NH+: 398.1203. NMR data agree with those above reported
for its enantiomer 5f.
N-(((9H-Fluoren-9-yl)methoxy)carbonyl)-S-(4-methyl-2-oxo-2H-
chromen-7-yl)-D-cysteine (6e). Derivative 5e (207 mg, 0.521 mmol),
FmocOSu (263 mg, 0.780 mmol) and NaHCO3 (196 mg, 2.33 mmol) were
dissolved in a mixture of H2O/CH3CN (1:2) (15 mL). The solution was
stirred at room temperature for 24 h. CH3CN was removed in vacuo and
the aqueous phase was acidified with 2 M HCl (pH 2-3). The solvent was
then removed and the crude was purified by silica gel column
chromatography (ethyl acetate/MeOH 95:5) affording compound 6e as
yellow foamy oil (145 mg, 0.234 mmol, 47%). [α]D25 (1.00, CH3OH): 13.1.
HRMS (ESI+) (m/z): 524.1094 [M+Na]+; calculated C28H23NO6SNa+:
524.1100. 1H NMR (300 MHz, CD3OD) d (ppm): 2.33 (s, Hz, 3H, Me4c),
3.37-3.47 (m, 1H, Hb), 3.67-3.75 (m, 1H, Hb), 4.04-4.10 (m, 1H, CHFmoc),
4.19-4.37 (m, 2H, CH2Fmoc), 4.43-4.50 (m, 1H, Ha), 6.17 (s, 1H, H3c), 7.23-
General methodology for C-terminal amide peptides. Microwave
assisted solid phase peptide synthesis (MW-SPPS) was performed
automatically on Rink Amide MBHA resin (0.05 mmol) using an automated
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10.1002/chem.201800603
Chemistry - A European Journal
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synthesizer Liberty Blue (CEM). DIC/Oxyma pure were used for coupling
and 20% (v/v) solution of piperidine in DMF for Fmoc deprotection. Fmoc-
protected fluorescent cysteine (0.15 mmol) was coupled manually using
HATU (0.145 mmol) and DIPEA (1.8 mmol) for 1 hour, as judged by Keiser
test.16 Acetic anhydride and pyridine (1:2) was used for N-terminus
acetylation and TFA, thioanisole, anisole and 1,2-ethanedithiol (90:5:2:3)
for the final cleavage and removal of the side chain protecting groups. The
peptide was precipitated with cold diethyl ether, isolated by centrifugation,
washed with cold diethyl ether, dissolved in acetonitrile/water containing
0.1% TFA (1:1, v/v), and lyophilized to afford the crude peptide. Purification
by RP-HPLC afforded the corresponding peptide.
HaL, HaP, HaR, HaV ), 7.79-7.87 (m, 3H, H3d, H6d, H7d), 8.31-8.62 (m, 3H ,
H2d, H4d, H8d).
Ac-L-Cys(Dans)-L-Val-L-Pro-L-Ala-L-Leu-L-Arg-NH2 (9f’). Peptide 9f’
was synthesized following the general methodology for C-terminal amide
peptides using ent-6f as fluorescent cysteine. Acetylation reaction was
performed by dissolving the resulting peptide in Ac2O and pyridine (1:2).
After stirring for 2 h at room temperature, the solvent was removed in
vacuo and the crude purified by RP-HPLC. HRMS (ESI+) (m/z): 975.4889
[M+H]+; calculated C44H70N12O9S2H+: 975.4903. 1H NMR (400 MHz, D2O)
d (ppm): 0.83-0.97 (m, 12H, MeV, MeL), 1.38 (d, 3 H, J = 7.2 Hz, MeA),
1.55-1.71 (m, 5H, HbL, CHL, CH2-CH2-NH2R), 1.73-1.93 (m, 4H, HbR, CH2P),
2.00 (s, 3H, NHAc), 2.02-2.08 (m, 2 H, HbV, CH2P), 2.23-2.32 (m, 1H, CH2P),
2.46-2.69 (m, 4H , CH2-Slinker, HbC*), 3.00 (s, 6H, MeC*), 3.13-3.24 (m, 4 H,
CH2-NHlinker, CH2-NH2R), 3.63-3.73 (m, 1H, CH2P), 3.80-3.89 (m, 1H, CH2P),
Ac-D-Cys(Coum)-L-Val-L-Pro-L-Ala-L-Leu-L-Lys-OH (7e). Peptide 7e
was synthesized following the general methodology for C-terminal acid
peptides using 6e as fluorescent cysteine. HRMS (ESI+) (m/z): 830.4041
[M+H]+; calculated C40H59N7O10SH+: 830.4044. 1H NMR (400 MHz, D2O)
d (ppm): 0.81 (dd, 12 H, J = 22.6, 6.3 Hz, MeV, MeL), 1.27 (d, 3 H, J = 7.2
C*
4.24-4.37 (m, 6H, Ha HaA, HaL, HaP, HaR, HaV ), 7.56 (d, 1H, H6d), 7.71-
7.80 (m, 2H, H3d, H7d), 8.29-8.55 (m, 3H , H2d, H4d, H8d).
Hz, MeA), 1.31-1.41 (m, 2H, CH2-CH2-CH2-NH2K), 1.45-1.62 (m, 5H, CH2-
K
CH2-NH2K, CHL, HbL), 1.65-1.75 (m, 2H, CH2P), 1.75-1.88 (m, 4H, Hb
,
Cell line stocks. Cell lines were obtained from European Collection of
Animal Cell Cultures (ECACC, United Kingdom) and they were maintained
in liquid nitrogen for long-term storage. The freezing process was done
slowly to avoid cell death due to possible crystal formation. Cells were
trypsinized and spun down (1000 rpm, 5 min) at room temperature.
Afterwards, cells were resuspended in culture medium with 10% FBS. In
addition, dimethyl sulfoxide (DMSO, Sigma), a cryoprotectant, was added
to a 10% final concentration. Cells were frozen in cryotubes at -80 ºC in
isopropanol containers for a week. Afterwards, they were preserved
permanently in liquid nitrogen. In contrast, thawing and recovery of cells
from liquid nitrogen was done quickly. Cryotubes were removed from liquid
nitrogen and maintained in dry ice until they thawed, at which point they
were immediately placed in a 37 ºC water bath. Cells were washed with
culture medium Dulbecco’s Modified Eagle’s Medium (DMEM)
supplemented with 10% v/v fetal calf serum to eliminate DMSO. Finally,
cells were resuspended in fresh medium, transferred to a flask for cell
culture.
CH2P), 1.89 (s, 3H, NHAc), 1.92 (d, 1H, J = 7 Hz, HbV), 2.10-2.21 (m, 2H,
CH2P), 2.32 (s, 3H, Me4c), 2.90 (t, 2H, J = 7.5 Hz, CH2-NH2K), 3.46-3.62 (m,
P
2H, HbC*), 4.00 (d, 1H, J = 7.7 Hz, HaV), 4.14-4.31 (m, 4H, HaA, HaL, Ha
,
HaK), 4.53 (t, 1H, J = 6.3 Hz, HaC*), 6.20 (s, 1H, H3c), 7.13 (s, 1H, H9c),
7.22 (d, 1H, J = 8.2 Hz, H7c), 7.53 (d, 1H, J = 8.4 Hz, H6c).
Ac-D-Cys(Dans)-L-Val-L-Pro-L-Ala-L-Leu-L-Lys-OH (7f). Peptide 7f was
synthesized following the general methodology for C-terminal acid
peptides using 6f as fluorescent cysteine. HRMS (ESI+) (m/z): 948.4673
[M+H]+; calculated C44H69N9O10S2H+: 948.4682. 1H NMR (400 MHz,
H2O/D2O) d (ppm): 0.75-0.99 (m, 12 H, MeV, MeL), 1.39 (d, 3H, J = 7.2 Hz,
K
MeA), 1.54-1.76 (m, 7H, CHL, HbL, CH2-CH2-NH2K, CH2-CH2-CH2-NH2 ),
1.99 (s, 3H, NHAc), 2.03-2.06 (m, 1 H, HbV), 2.22-2.37 (m, 2H, CH2P), 2.41-
2.70 (m, 6H , HbC*, CH2P, CH2-Slinker), 2.91 (s, 6H, MeC*), 2.96-3.20 (m, 4H,
CH2-NHlinker, CH2-NH2K), 3.63-3.86 (m, 4H, HbK, CH2P), 4.14-4.21 (m, 1H,
HaC*), 4.26-4.40 (m, 5H, HaA, HaL, HaP, HaK, HaV), 7.34-8.87 (m, 12H , Harom
,
CONH).
Cell culture. Briefly, Hela cells were cultured in Dulbecco’s Modified
Eagle’s Medium (DMEM) supplemented with 10% v/v fetal calf serum, 2
mM L-glutamine and penicillin–streptomycin (100 units/mL and 100 mg/mL,
respectively). Cells were maintained in an incubator at 37 °C in an
atmosphere of 5% CO2. Hela cells were seeded in 24-well plates at a
density of 50000 cells/well and were cultured overnight. Prior to starting
the experiment, medium was removed from the plates, and fresh medium
containing 10 μM from the stock solution (10 mM in DMSO) of the required
peptide was added to the cells. Cells were cultured for 2 h before
microscopy visualization. After that, peptide media was removed and cells
were rinsed to take the images.
Ac-D-Cys(Dans)-L-Val-L-Pro-L-Ala-L-Leu-L-Lys-NH2 (8f). Peptide 8f was
synthesized following the general methodology for C-terminal amide
peptides using 6f as fluorescent cysteine. HRMS (ESI+) (m/z): 970.4836
[M+H]+; calculated C44H70N10O9S2H+: 947.4841. 1H NMR (400 MHz, D2O)
d (ppm): 0.86-0.98 (m, 12 H, MeV, MeL), 1.38 (d, 3H, J = 7.2 Hz, MeA),
P
1.51-1.97 (m, 11H, CHL, HbL, CH2-CH2-NH2K, CH2-CH2-CH2-NH2K, CH2
,
HbK), 2.01 (s, 3H, NHAc), 2.02-2.11 (m, 1H, HbV), 2.22-2.37 (m, 2H, CH2P),
2.49-2.77 (m, 4H, HbC*, CH2-Slinker), 3.01 (t, 2H, J = 7.5 Hz, CH2-NH2 ),
K
3.20 (t, 2H, J = 6.4 Hz, CH2-NHlinker), 3.45 (s, 6H, MeC*), 3.65-3.74 (m, 1H,
V
CH2P), 3.83-3.91 (m, 1H, CH2P), 4.26-4.41 (m, 6H, HaA, HaL, HaP, HaK, Ha
HaC*), 7.91 (t, 2H, J=8.2 Hz, H3d, H7d), 8.00-8.06 (m, 1H, H6d), 8.40-8.79
(m, 3H , H2d, H4d, H8d).
Fluorescence microscopy. Data were obtained with a Leica microscope
(DM6000B) equipped with a Hamamatsu Orca-ER C10600 and an A4
Leica filter.
Ac-D-Cys(Dans)-L-Val-L-Pro-L-Ala-L-Leu-L-Arg-NH2 (9f). Peptide 9f was
synthesized following the general methodology for C-terminal amide
peptides using 6f as fluorescent cysteine. Acetylation reaction was
performed by dissolving the resulting peptide in Ac2O and pyridine (1:2).
After stirring for 2 h at room temperature, the solvent was removed in
vacuo and the crude was purified by RP-HPLC. HRMS (ESI+) (m/z):
975.4941 [M+H]+; calculated C44H70N12O9S2H+: 975.4903. 1H NMR (300
Degradation studies with human plasma: Resistance to proteases was
evaluated for both peptides 9f and 9f’. Enzimatic degradation in human
plasma was analysed using commercially available serum from human
male AB plasma. Peptides 9f and 9f’ were suspended in a solution of 20%
human serum in water at 37 oC and the degradation of the peptides was
determined at 0.5, 1,5 and 4 h (time in which these peptides are incubated
with HeLa cells), by using UPLC-micrOTOF-Q (column: ACQUITY UPLC
BEH C18 1.7; diameter: 2.1 mm and length: 100 mm), eluting a mixture of
5 mL of the peptide-serum solution in 795 mL of water and employing an
isocratic method (70% H2O, 30% acetonitrile, temperature: 40 oC).
MHz, D2O) d (ppm): 0.81-1.00 (m, 12H, MeV, MeL), 1.38 (d, 3H, J = 7.2 Hz,
R
MeA), 1.53-1.72 (m, 5H, HbL, CHL, CH2-CH2-NH2R), 1.71-1.96 (m, 4H, Hb
,
CH2P), 1.99 (s, 3H, NHAc), 2.03 (s, 1 H, HbV), 2.04-2.08 (m, 1H, CH2P),
2.22-2.33 (m, 1H, CH2P), 2.48-2.56 (m, 2H , CH2-Slinker), 2.57-2.75 (m, 2H ,
HbC*), 3.11-3.22 (m, 4 H, CH2-NHlinker, CH2-NH2R), 3.24 (s, 6H, MeC*), 3.63-
A
3.73 (m, 1H, CH2P), 3.80-3.91 (m, 1H, CH2P), 4.24-4.39 (m, 6H, HaC* Ha
,
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Cytotoxicity
assay. The
MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-
hydrolysis
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microplate in growing media. 24 h later cells were incubated with the
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We are grateful to MINECO (projects CTQ2015-67727-R and
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(EFA038_Momeneu) and the Rioja Salud Foundation.
Keywords: dehydroalanine • Michael addition • cysteine •
fluorescence • cell penetrating peptides
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C. D. Navo, A. Asín, E. Gómez-Orte, M.
I. Gutiérrez-Jiménez, I. Compañón, B.
Ezcurra, A. Avenoza, J. H. Busto, F.
Corzana, M. M. Zurbano, G. Jiménez-
Osés, J. Cabello* and J. M. Peregrina*
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This work describes the efficient synthesis of enantiopure and fluorescent D-
cysteines, using highly chemo- and stereoselective S-Michael additions of thiol-
containing dyes on a chiral bicyclic dehydroalanine. Their use as fluorescent labels
was validated by incorporation into cell penetrating pentapeptides, which were
treated with different HeLa cell lines, monitoring their internalization by fluorescence
microscopy.
Cell Penetrating Peptides containing
Fluorescent D-Cysteines
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