Synthesis and cellular penetration properties of new phosphonium based cationic amphiphilic peptides

Article abstract of DOI:10.1039/c8md00113h

A new category of phosphonium based cationic amphiphilic peptides has been developed and evaluated as potential antimicrobial peptides and cell penetrating peptides. The required building blocks were conveniently accessible from cysteine and could be applied in a solid phase peptide synthesis protocol for incorporation into peptide sequences. Evaluation of the antimicrobial properties and cellular toxicity of these phosphonium based peptides showed that these "soft" cationic side-chain containing peptides have poor antimicrobial properties and most of them were virtually non toxic (on HEK cells tested at 256 and 512 μM) and non-haemolytic (on horse erythrocytes tested at 512 μM), hinting at an interesting potential application as cell penetrating peptides. This possibility was evaluated using fluorescent peptide derivatives and showed that these phosphonium based peptide derivatives were capable of entering HEK cells and depending on the sequence confined to specific cellular areas.

Full text of DOI:10.1039/c8md00113h

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T. Postma, M. Hezwani, A. Pirvan, S. Gannon, C. Smith, M. O. Riehle and R. Liskamp, Med. Chem.
Commun., 2018, DOI: 10.1039/C8MD00113H.
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DOI: 10.1039/C8MD00113H
Synthesis and Cellular Penetration Properties of new Phosphonium
based Cationic Amphiphilic Peptides
a
a
a
a
Ezequiel Silva Nigenda , Tobias M. Postma , Mohammed Hezwani , Alin Pirvan ,
a
b
b
a
Susan Gannon , CarolꢀAnne Smith , Mathis Riehle , Rob M.J. Liskamp*
a
School of Chemistry, University of Glasgow, Joseph Black Building, University Avenue, Glasgow G12 8QQ,
United Kingdom
b
Centre for Cell Engineering, Institute of Molecular, Cell and Systems Biology, Joseph Black Building, University
Avenue, Glasgow G12 8QQ, United Kingdom
A new category of phosphonium based cationic amphiphilic peptides has been developed and evaluated as
potential antimicrobial peptides and cell penetrating peptides. The required building blocks were conveniently
accessible from cysteine and could be applied in a solid phase peptide synthesis protocol for incorporation into
peptide sequences. Evaluation of the antimicrobial properties and cellular toxicity of these phosphonium based
peptides showed that these "soft" cationic sideꢀchain containing peptides have poor antimicrobial properties and
most of them were virtually non toxic (on HEK cells tested at 256 and 512 ꢁM) and nonꢀhaemolytic (on horse
erythrocytes tested at 512 ꢁM), hinting at an interesting potential application as cell penetrating peptides. This
possibility was evaluated using fluorescent peptide derivatives and showed that these phosphonium based
peptide derivatives were capable of entering HEK cells and depending on the sequence confined to specific
cellular areas.
Introduction
Cationic Amphiphilic Peptides (CAPs) are a class of compounds that are attracting extensive attention in
medicinal chemistry because of their wide range of biological activities. The broad spectrum antimicrobial
1
properties of Antimicrobial Peptides (AMPs) are of great interest for the development of potential new antibiotics,
since as compared to the widely used small molecule antibiotics, virtually no resistance has been observed so
2
3
far . Also, the membrane permeation capabilities of Cell Penetrating Peptides (CPPs) and their low toxicity or
side effects, will become increasingly important for the intraꢀcellular delivery of small molecule drugs and
4,5,6
biologics
.
All these activities are a consequence of the amphipathic nature of CAPs conferred by the presence of both
hydrophobic amino acid residues (Trp, Phe, Leu) and basic amino acid residues (Lys, Arg, His) leading to a net
7
positive charge of CAPs . The role of positively charged amino acids in CAPs is especially noteworthy, since they
form the anchors for interactions with the negatively charged phospholipids. Bacterial membranes are net
negatively charged because of the presence of anionic phospholipids such as phosphatidylglycerol and
2
8
phosphatidylserine in gramꢀnegative bacteria , or techoic and lipoteichoic acids in gramꢀpositive bacteria . In
contrast, eukaryotic cell membranes are composed of more neutral zwitterionic lipids like phosphatidyl choline
and cholesterol. In these cells, the anionic lipids are located in the inner part of the plasma membrane.
The cytotoxic effect on both prokaryotic and eukaryotic cells is closely related to the structure of the CAPs and
the nature of their interaction with the membranes. The presence in CAPs of amino acid residues like Tryptophan
tends to lead to stronger interactions with membranes causing lysis, whereas Leucine tends to increase the
9
lipophilicity of CAPs facilitating crossing membranes. Also, the replacement of very hydrophobic residues in the
apolar face of CAPs, by less hydrophobic amino acid residues reduces interaction with red blood cell membranes
10
leading to diminished haemolysis.
1

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This duality in the behaviour of the CAPs is fascinating and poses a great challenge for tuning their activity. In
many Cationic Antimicrobial Peptides, this can be achieved by altering the balance between the charged and
11
hydrophobic residues . Therefore, in this research, we wished to look at the charge character of the positively
charged amino acids by changing the naturally occurring positively charged nitrogen amino acids to positively
charged phosphorus containing amino acids. To our knowledge, the influence on the bioꢀactivity of CAPs
containing other positively charged atoms has not been studied so far. By introducing phosphonium amino acids,
we can study the triangular relation between antimicrobial activity (AM), cell penetrating ability (CP) and cellular
toxicity (CT) of a new class of CAPs. Determination of the relative AM, CP and CT parameters, should steer us
ultimately towards cationic amphiphilic peptides with optimal activity and selectivity. It might be even possible
that, by judicious modification of the sequences, we will be able to tune the AM, CP and CT properties.
As a suitable starting point for our studies we chose HHC10, a wellꢀknown antimicrobial peptide developed in the
5
group of Hancock, (Figure 1) which is an optimized AMP with low cytotoxicity properties as was evidenced from
its low haemolysis properties. HHC10 has potent broadꢀspectrum antimicrobial activity against multidrug
resistant superbugs in vitro but the presence of Lys and Arg residues, makes HHC10 also susceptible to
1
2
proteolytic digestion by trypsinꢀlike proteases . An attractive possibility to reduce this proteolytic degradation is
the incorporation of Dꢀamino acids. For example, we have recently shown that synthesis of the Dꢀisomer of the
13,14
HHC10 peptide led to an increase of its stability in serum and subsequent incorporation into a coating
led to a
serumꢀstable antimicrobial coating. Thus, introduction of other unnatural amino acids such as analogues of the
positively charged lysine and arginine residues with a different positively charged atom, that is not recognised by
proteolytic enzymes, may favourably affect the bioactivity or stability.
5
Figure 1. HHC10 Cationic Amphiphilic Peptide developed by Hancock et al.
In this work we describe the development of these new positively charged amino acids, containing a
phosphonium moiety, used in the synthesis of new CAPs with possible medicinal chemistry applications. The
antimicrobial and cytotoxic activities of these PhosphoniumꢀHHC10 analogues were studied, which led to the
development of fluorescent versions to investigate their behaviour in mammalian cells and study of potential cell
penetrating properties.
Results and discussion
Cysteine is perhaps the most versatile amino acid for postꢀsynthesis modification by alkylation either in a peptide
15
or a protein due to the excellent nucleophilic character of the thiol group . Work developed by Shokat et. al,
1
6
describes the functionalisation of cysteine to produce a series of methylated lysine analogues . By using suitable
alkylating reagents containing a phosphonium group, it was possible to synthesise the phosphonium isosteric
P'
amino acid of (trimethylamine)lysine 6 ('K ) and one as a more distant analogue of the positive charge present in
P'
the side chain of arginine 7 ('R ).
2

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The required bromo phosphonium alkylating reagents 4 and 5 were prepared by addition of trimethyl phosphine
to 1,2ꢀdibromo ethane and 1,3ꢀdibromo propane, respectively. Using 4, it was possible to prepare the pure
phosphonium cysteine derivative 6 directly from FmocꢀCysꢀOH 2. However, to facilitate purification of
phosphonium derivative 7, synthesis was more conveniently carried out starting from FmocꢀCysꢀOMe 3, followed
by ester hydrolysis under the acidic conditions of the work up. (Scheme 1).
Scheme 1. Synthesis of the phosphonium containing amino acid derivatives.
In principle, it is possible to introduce the desired phosphonium cysteine derivatives postꢀassembly of the
peptides in which the positively charged chains must be incorporated. This can be achieved by alkylation of
deprotected cysteine derivatives using alkylating agents 4 or 5. However, it was found that completion of the
alkylation reactions was difficult to realize in peptides containing more than one cysteine residue. In addition,
peptides (for example CAP 9) containing two different phosphonium cysteine derivatives cannot be synthesized
postꢀassembly. Hence, it was decided to employ the modified amino acid building block approach and use Fmoc
17
protected derivatives 6 and 7 in a manual solid phase synthesis approach on Rink amide resin . To synthesise
the three different HHC10 phosphonium analogues 8 ꢀ 10 (Figure 2), DIC/Oxymaꢀpure was used as a coupling
reagent after failed attempts of using HCTU/DIPEA. After cleavage and deprotection, crude peptides were
purified by reverse phase HPLC and obtained with purities >97%
3

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Figure 2. HHC10 derivatives with the phosphonium charged amino acids
For the evaluation of the bioꢀactivity of phosphonium CAPs 8 ꢀ 10 their antimicrobial properties, (eukaryotic)
cellular toxicity and haemolytic activity were determined (Table 1)
Determination of the antimicrobial activity using the modified MIC method for Cationic Amphiphilic Peptides
1
8
developed by Hancock and coꢀworkers showed that peptides CAPs 8 ꢀ 10 were virtually inactive towards E. coli
compared to HHC10. This inactivity towards bacteria was paralleled by a lower cellular toxicity towards HEK 293
1
9
cells as compared to HHC10 using the Alamar blue assay . The haemolytic activity of CAPs 8 ꢀ 10 was also
considerably lower than that of HHC10, which is known to possess relatively low haemolytic properties.
Haemolysis of
horse erythrocytes
at 512 ꢁM (%)
MIC on
E. Coli(ꢁM)
Cell viability of HEK 293
cells at 256 ꢁM (%)
CAP
8
9
64
64
85 ± 6
87 ± 4
103 ± 3
2.0 ± 1.5
3.4 ± 0.1
3.3 ± 0.1
3.2 ± 0.1
14.3 ± 3.6
10
128
2
0
HHC10
1.5
Table 1. Antimicrobial and cytotoxicity evaluation of the phosphonium based HHC10 analogues and
comparison with HHC10: Lower antimicrobial activities of the phosphonium peptides are translated into a
diminished toxicity profile as compared to HHC10. All experiments were carried out in triplicate and cell viability
and haemolytic effect are presented as the mean ± SEM.
The considerable antimicrobial activity of HHC10 is probably related to the high interaction properties of Arg/Trp
21
rich peptides with bacterial membranes . Similarly, HHC10 can interact with membranes of mammalian cells,
which led to a decrease of cell viability and increase of the haemolytic activity. However, the increased size of the
4

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phosphorus atom as well as the presence of methyl groups likely led to a more diffuse positive charge of the side
chains of the phosphonium CAPs and therefore less strong interaction with the negatively charged bacterial
membrane, thereby decreasing the antimicrobial effect. Concomitantly, this decrease of the positive charge
character might be responsible for the diminished eukaryotic toxicity and haemolysis of CAPs 8ꢀ10.
This relatively low toxicity of CAPs 8ꢀ10 prompted us to investigate two additional phosphonium containing
peptides that might have better antimicrobial properties. To achieve this, Trp amino acid residues were changed
P
P
P
P
P
to Pheꢀresidues in the repetitive sequence X FFX FFX FFX ('X ' represents an incorporated phosphonium
amino acid) leading to 11 and 12, which were synthesized by solid phase peptide synthesis (Figure 3). This
2
2
sequence is based on the cationic bactericidal peptide KFFKFFKFFK previously used by our group which was
23
reported to have a direct activity against E. coli and low eukaryotic toxicity and haemolysis.
Figure 3. Phosphonium Analogues of the bactericidal peptide KFFKFFKFFK
Interestingly, the antimicrobial activity of 11 and 12 increased slightly (MIC 32 and 64 ꢁM, respectively), but was
24
still considerably less than that of HHC10 (1.5 ꢁM). According to Eriksson et al. , the KFFKFFKFFK sequence
acts as a cellꢀpermeabilising peptide for the delivery of Peptide Nucleic Acids, thus hinting at possible favourable
cell penetrating properties of 11 and 12.
To investigate the potential cell penetrating properties induced by the phosphonium containing amino acid
P
P
P
P
residues, a small collection of peptides in which the Pheꢀresidues in the X FFX FFX FFX sequence of peptides
1 and 12 were changed by other common hydrophobic residues (Trp, Leu, Ile), was synthesised thereby
1
affording six additional phosphoniumꢀCAPs (13ꢀ18) (Figure 5). The cytotoxicity of these compounds was tested
and compared to compounds 11 and 12 trying to understand the relation of the hydrophobic residues on the
activity of the phosphoniumꢀCAPs.
Haemolysis of horse
erythrocytes at 512 ꢁM
%)
1.5 ± 0.1
Cell viability of HEK 293
cells at 512 ꢁM (%)
CAP
Sequence
(
P
P
P
ꢀ
11
H
ꢀ
K FFK FFK FFK NH
2
46 ± 1
5

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DOI: 10.1039/C8MD00113H
P
P
P
Pꢀ
1
1
1
1
1
1
1
2
3
4
5
6
7
8
H
H
H
H
H
H
H
H
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
R FFR FFR FFR NH
2
83 ± 1
14 ± 5
44 ± 1
110 ± 21
93 ± 3
120 ± 23
83 ± 29
0.0
3.1 ± 0.1
P
P
P
Pꢀ
K WWK WWK WWK NH
2
11.0 ± 1.6
P
P
P
Pꢀ
R WWR WWR WWR NH
2
5.8 ± 1.0
P
P
P
Pꢀ
K LLK LLK LLK NH
2
2.1 ± 0.5
0.6 ± 0.2
2.3 ± 1.5
3.9 ± 0.7
14.3 ± 3.6
P
P
P
Pꢀ
R LLR LLR LLR NH
2
P
P
P
Pꢀ
K IIK IIK IIK NH
2
P
P
P
Pꢀ
R IIR IIR IIR NH
2
ꢀ
HHC10
KRWWKWIRW NH
2
P'
Table 2. Cytotoxicity evaluation of the phosphoniumꢀCAPs: 'K represents the phosphonium isostere of
P'
(
trimethyl)lysine and 'R with a phosphonium side chain length comparable to that of arginine. All peptides are
TFA salts with Cꢀterminal amide and free Nꢀterminus. All experiments were carried out in triplicate and cell
viability and haemolytic effect are presented as the mean ± SEM.
Replacement of the phenylalanine residues by tryptophan residues in the peptide increased the cytotoxicity of the
phosphoniumꢀCAPs as was evidenced by compound 13 which led to a very low cell viability and the highest
haemolytic effect of all synthesised peptides. In addition, interaction of Trp rich sequences with membrane
components and disruption of red blood cells was increased as comparison of the haemolytic effect of
compounds with similar cell viability like compounds 11 and 14 showed. The latter had a higher haemolytic
activity. Peptides 15ꢀ18 showed a lower toxicity on red blood cells.
Nevertheless, the generally low toxicity of these phosphonium CAPs is remarkable. We think that this is due to
the character of these added positively charged amino acids as compared to the positively charged amino acid
residues (Arg, Lys, His), which are normally found in antimicrobial peptides and cytotoxic peptides. The
phosphonium charge is much more diffuse and therefore interaction with the negative charges of the lipids are
weaker, possibly sufficient for cell penetrating properties but less strong for a display of toxicity effects or
antimicrobial activity.
To evaluate possible cell penetrating properties of phosphonium CAPs, fluorescent derivatives of the
P
P
P
P
phosphonium HHC10 analogues (8ꢀ10) and X FFX FFX FFX peptide sequences (11,12) were prepared. These
peptides were selected because of their similarly low antimicrobial and low cytotoxic activities, thereby rendering
them potentially interesting to serve as cell penetrating peptide carriers for future cargos.
For visualization by fluorescence microscopy the BODIPYꢀFl label, which was prepared according to a modified
25
literature procedure , was coupled to the N terminus of the peptide mimics using EDCI—HCl and 6ꢀCl HOBt in
DMF leading to fluorescent Bodipyꢀ CAPs 19ꢀ23 (Figure 4).
6

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P
+
P
+
P
+
P
+
F
NH
O
NH
F
NH
O
NH
S
S
S
S
N
B
ꢀ
F
+
O
O
O
O
S
N
B
ꢀ
F
O
O
O
O
H
H
N
H
H
N
H
H
N
H
H
N
N
N
N
NH
2
N
+
N
N
NH
2
N
H
N
N
N
N
H
N
H
N
N
N
N
H
H
H
H
H
H
H
O
P
O
O
O
NH
O
O
O
O
O
NH
O
S
S
S
NH
NH
+
P
+
P
+
P
+
P
P
P
P
P
P
P
P
1
9: BodipyꢀK K WWK WIK WꢀNH
2
20: BodipyꢀK R WWK WIR WꢀNH
2
P
+
P
+
P
+
P
+
F
NH
O
NH
F
Bꢀ
N+
S
S
S
S
O
O
O
S
N
B
N+
ꢀ
F
O
O
O
O
S
N
F
O
O
O
H
H
H
H
N
H
H
H
H
H
N
N
N
N
N
N
NH
2
N
N
N
N
N
H
NH
2
N
H
N
N
H
N
N
H
N
H
N
H
H
H
H
O
P
H
O
O
O
O
O
O
O
NH
O
O
S
S
NH
P
+
+
P
+
P
+
P
P
P
P
P
P
P
P
2
1: BodipyꢀR R WWR WIR WꢀNH
2
22: BodipyꢀK FFK FFK FFK ꢀNH
2
P
+
P
+
F
S
N
B
ꢀ
F
O
S
O
O
O
O
N+
H
H
N
H
H
O
S
N
H
N
H
N
N
N
N
N
N
H
H
N
NH
2
O
O
H
H
O
O
S
O
P
+
P
+
P
P
P
P
2
3: BodipyꢀR FFR FFR FFR ꢀNH
2
Figure 4. Green fluorescent phosphonium peptides.
MCF7 cells were incubated with solutions (20 ꢂM) of the fluorescent peptides according to a slightly modified
26
literature protocol (Figure 5). These cells were used because of their wellꢀknown morphology and controlled
grow rate. The cells were observed with an Axiovert Fluorescent Microscope. Controls using the fluorescent
BODIPY ligand were carried out to confirm that the green fluorescence was due to the presences of the
fluorescent peptides (See electronic supporting information).
Rhodamine
Phalloidin-
Bodipy-CAP-
cellular uptake
CAP
Phase-contrast
DAPI-stain
Merged images
stain
1
2
2
2
9
0
1
2
7

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2
3
Figure 5. Fluorescence micrographs of MCF7 cells with 20 ꢁM of BODIPYꢀCAPs 19, 20, 21, 22 and 23. Blue
fluorescence of DAPI (4',6ꢀDiAmidinoPhenylIndole) stained nuclei and red fluorescent components of the
cytoskeleton were obtained by marking with Rhodamine Phalloidin. Green (BODIPY) fluorescent Cationic
Amphiphilic Peptides showed their cellular uptake and localisation. Pictures were merged to visualise the
localisation of the fluorescent peptides inside the cell thus confirming their cell penetrating properties. The white
scale bar in the right figure of each frame is 10 ꢂm.
The observed association of the fluorescent CAPs with membranes was not merely due to the CAPs associating
with just any lipid containing organelle, since neither Golgi, ER nor mitochondria were stained. The staining was
diffuse throughout the cells but stayed in place. There was no real entry into the nucleus. In general, and perhaps
not entirely unexpectedly, association of all fluorescent phosphonium CAPs seemed to be similar. Subtle
differences in the staining may indicate the importance of structural variations of phosphonium CAPs. For
P
example, tryptophan/K containing CAP 19 seemed to be uniformly distributed over membrane containing
P
P
structures, whereas tryptophan/R /K containing CAP 20 showed staining of well localised areas in cell periphery
with diffuse staining in the cytoplasm but not the nucleus. The bright actin patches were reflected in bright
P
patches of the CAP. Tryptophan/R containing CAP 21 showed a more diffuse staining throughout the cytoplasm
as no dark nuclear area was visible looking at the green fluorescence of CAP 21. Here also the bright actin
P
patches were reflected in bright patches of the CAP. Phenylalanine/K containing CAP 22 showed a granular
pattern and perinuclear staining with possible association with areas inside or around (Golgiꢀarea) the nucleus.
P
Finally, phenylalanine/R containing CAP 23 showed a less granular pattern than 22 with diffuse association with
areas inside the nucleus. There was a very clear perinuclear staining and here too the actin bright patches were
also bright in the bright patches of this CAP.
Conclusions
A new class of positively charged nonꢀnatural amino acids derived from cysteine was synthesised. These were
used to prepare a new category of Cationic Amphiphilic Peptides that are less toxic than normal Lysine or
Arginine based CAPs, possibly due to presence of a more diffused charge of the positive phosphonium moiety.
Their Antimicrobial activity is lower than the wellꢀknown AMP HHC10. However, their low toxicity and ability to
cross membranes without causing damage was further investigated to evaluate possible cell penetrating
properties. This study showed that introduction of phosphonium amino acids diminished the toxicity compared to
of positively charged nitrogen containing peptides such as HHC10. Further modification of the sequence of the
peptides by introduction of other lipophilic amino acid residues favoured their cell penetrating properties. The
data showed that this class of phosphonium based CAPs may well comprise a new category of cell penetrating
peptides with low cytotoxicity.
8

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Conflict of Interest
The authors declare no competing interest.
Acknowledgements
E.S.N thanks the National Council of Science and Technology of Mexico (CONACyT) for financial support.
Assistance by Dr Alex Hoose with MSꢀinterpretation is gratefully acknowledged.
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MedChemComm
P
N
N+
P
B-
High MIC values
F
F
S
S
O
Low cytotoxicity
Non- haemolytic
Cell penetrating
properties as shown
by fluorescent
S
S
microscopy
P
P