Synthesis and antimicrobial activity of the symmetric dimeric form of temporin A based on 3-N,N-di(3-aminopropyl)amino propanoic acid as the branching unit

Article abstract of DOI:10.1016/S0968-0896(01)00047-5

Dimeric derivative of antimicrobial peptide amide Temporin A (TA) was synthesized by using a new branching unit 3-N,N-di(3-aminopropyl) amino propanoic acid (DAPPA), which allows building of the parallelly symmetric α-helical structures. Antimicrobial effect of the original peptide amide, its monomeric carboxy (TAc) and novel dimeric (TAd) analogues were tested against Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative). Both TA and TAd completely inhibited the growth of S. aureus at the concentrations of 5 and 10μM, respectively, whereas TAc did not show any inhibitory activity. The activities of TAc TA and TAd correlate directly with the net charges of the molecules, +1, +2 and +4, respectively. Interestingly, TAd displayed antibacterial effect against E. coli at a concentration of 10μM, whereas as monomeric TA did not show any activity at concentration as high as 20μM. The results indicate that the novel structural modification improves the antibacterial properties of Temporin A especially towards Gram-negative bacteria. Copyright

Full text of DOI:10.1016/S0968-0896(01)00047-5

Bioorganic & Medicinal Chemistry 9 (2001) 1601–1607
Synthesis and Antimicrobial Activity of the Symmetric Dimeric
Form of Temporin A Based on 3-N,N-di(3-aminopropyl)amino
Propanoic Acid as the Branching Unit
Helena Hujakka,a,* Jari Ratilainen,b Timo Korjamo,a Hilkka Lankinen,c
Pentti Kuusela,d,e Harri Santa,a Reino Laatikainena and Ale Narvanena
aUniversity of Kuopio, Department of Chemistry, PO Box 1627, FIN-70211 Kuopio, Finland
bOrion Corporation, Orion Pharma, Department of Synthetic Chemistry, PO Box 65, FIN-02101 Espoo, Finland
cHaartman Institute, Peptide and Protein Laboratory, Department of Virology, PO Box 21, FIN-00014 Helsinki University,
Helsinki, Finland
dHaartman Institute, Department of Bacteriology and Immunology, FIN-00014 Helsinki University, Helsinki, Finland
eHUCH Laboratory Diagnostics, Division of Clinical Microbiology, Helsinki University Central Hospital,
PO Box 400, FIN-00025 University Central Hospital, Helsinki, Finland
Received 9 November 2000; revised 8 February 2001; accepted 13 February 2001
Abstract—Dimeric derivative of antimicrobial peptide amide Temporin A (TA) was synthesized by using a new branching unit
3-N,N-di(3-aminopropyl)amino propanoic acid (DAPPA), which allows building of the parallelly symmetric a-helical structures.
Antimicrobial effect of the original peptide amide, its monomeric carboxy (TAc) and novel dimeric (TAd) analogues were tested
against Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative). Both TA and TAd completely inhibited the
growth of S. aureus at the concentrations of 5 and 10 mM, respectively, whereas TAc did not show any inhibitory activity. The
activities of TAc, TA and TAd correlate directly with the net charges of the molecules, +1, +2and +4, respectively. Interestingly,
TAd displayed antibacterial effect against E. coli at a concentration of 10 mM, where as monomeric TA did not show any activity at
concentration as high as 20 mM. The results indicate that the novel structural modification improves the antibacterial properties of
Temporin A especially towards Gram-negative bacteria. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
They have antimicrobial activity against a broad spec-
trum of bacteria including clinically important methi-
cillin resistant Staphylococcus aureus and vancomysin
resistant Enterococcus faecium.6 However, their lethal
concentrations toward Gram-negative bacteria are
poor.4,7
Antimicrobial peptides play a very important role in
innate immunity. They have been isolated from plants,
insects, amphibians and vertebrates, including
humans.1,2 Amphibian skin has proven to be an espe-
cially rich source of antibacterial peptides. For example
bradykinin, brevinins, esculentins, magainin, caerulein,
xenopsin and laevitide have been isolated from the skin
extracts of several frog species.3À5 Temporins, a group
of antimicrobial peptides, were isolated from the skin of
European red frog Rana temporaria. These peptides
(temporins A–H, K and L) form a group of short (10–
13 amino acid residues), alpha helical, amphipathic,
hydrophobic peptides with amidated C-terminus and
free N-terminus. Temporins are reported to have anti-
bacterial activity without hemolysis to human red cells.4
Antibacterial peptides are thought to kill cells by caus-
ing leaking of water, ions, and even small macro-
molecules through the cell membrane. There are
currently two alternative hypotheses concerning the
mechanisms of action by which amphipathic, a-helical,
lytic peptides, such as Temporin A, permeate the cell
membrane. These models, ‘barrel stave’ and ‘carpet-
like’, share a conceptual difference. In the former, the
peptides form transmembrane pores by inserting into
the hydrophobic core of the membrane. In the ‘carpet-
like’ model the peptides do not insert into the membrane
core but the charged hydrophilic regions of the peptide
interact with anionic heads of bacterial phospholipids
and the hydrophobic regions with the hydrocarbon
*Corresponding author. Tel.:+358-17-163245; fax: +358-17-163259;
e-mail: helena.hujakka@uku.fi
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00047-5

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H. Hujakka et al. / Bioorg. Med. Chem. 9 (2001) 1601–1607
tails.8À10 It has been proposed, too, that there is not
necessarily direct association between the ability to per-
meabilize the cytoplasmic membrane and peptide anti-
microbial activity, but some peptides may act also
intracellular by binding to DNA or altering enzyme
activities.11,12 Recently Temporin A all-D enantiomer
has been shown to have the same antibacterial activity
as Temporin A,13 indicating that the antimicrobial
mechanism of Temporin A is not chiral, but is based on
simple hydrophobic interactions.
Both carboxy and amidated C-terminals of helical anti-
mirobial peptides have been reported. In some cases
replacing the amide group with free acid abolished their
activity,16 in the other cases it did not affect activity.17
In this paper, we describe the synthesis and the anti-
microbial activity of symmetrical dimeric Temporin A
analogue. We also report a comparison of the activities of
Temporin A, its carboxy analogue (TAc) and the dimeric
analogue (TAd) (Fig. 4 A–C) against Gram-positive and
Gram-negative bacteria and show that the TAd owns an
improved activity toward Gram-negative bacteria.
A substitution of a single amino acid can enhance anti-
bacterial activity, if it increases amphiphilic or amphi-
pathic nature of the peptide and enhances the formation
of secondary structure, as shown, for example with
Temporin A13 and ESF1.14 Modifications, which dis-
turb the secondary structure or amphipathic nature of
antimicrobial peptides, usually reduce their ability to
permeabilize the zwitterionic or slightly anionic mem-
branes, e.g., blood cells. Nevertheless, if a peptide is
sufficiently cationic, it can still permeate highly anionic
bacterial cell membranes. Other crucial parameters that
affect biological activity of antimicrobial peptides are
the hydrophobicity and the hydrophobic moment.15
Results
The bacterial assays were performed in Bioscreen C
(Labsystems, Finland). The initial titres of both S. aur-
eus and E. coli were 1Â104 cfu/mL and the total growth
time was 21 h. The growth of the bacteria was followed
turbidometrically every 10 min after a vigorous shaking
for 10 s. After 21 h incubation, the numbers of bacteria
in the control wells were about 1Â108 for S. aureus and
1Â109 E. coli.
Figure 1. The effect of Temporin A, TA, (A) and its dimeric derivative, TAd, (B) on growth of S. aureus. Peptide concentrations were 0 (-&-), 2.5
(-^-), 5 (-~-) and 10 (-*-) mM. The initial bacterial concentration was 1Â104 cfu/mL.

H. Hujakka et al. / Bioorg. Med. Chem. 9 (2001) 1601–1607
1603
The monomeric carboxy analogue of Temporin A
(TAc) was totally inactive against both test bacteria at
concentrations up to 20 mM. The growth of Gram-posi-
tive bacterium S. aureus was inhibited totally with TA
and TAd at 5 and 10 mM concentration, respectively
(Fig. 1). For TAd, some inhibitory effect was seen
already at 5 mM. The situation was different with Gram-
negative E. coli. TA was totally inactive at concentra-
tions up to 20 mM, whereas TAd inhibited the growth
totally at 10 mM concentration and the inhibitory effect
was already seen at 2.5 mM (Fig. 2).
which enables the building up of symmetrical dimeric
peptides. Commonly, branching of multiple peptide
chains is based on lysine, which instead allows nonsym-
metric18 and pseudosymmetric19 structures. In this work
we show that DAPPA increases the positive charge of
the Temporin A peptide in structurally optimal site for
its antibiotic activity.
DAPPA was modified for conventional Fmoc peptide
synthesis strategy. The possibility to add the molecule
directly to automated peptide synthesizer, without any
manual preparations, makes its use convenient. There
are some advantages in employment of branched pep-
tide dimers for pharmaceutical purposes. The pre-
organizing of the chains reduces the binding entropy of
the effector peptide and the increase of molecular
weight, although generally considered as a disadvantage
Discussion
We synthesized a novel, symmetrical backbone, 3-N,N-
di(3-aminopropyl)amino propanoic acid (DAPPA),
Figure 2. The effect of Temporin A, TA, (A) and its dimeric derivative, TAd, (B) on growth of E. coli. Peptide concentrations were 0 (-&-), 2.5
(-^-), 5 (-~-), 10 (-*-) and 20 (-&-) mM. The initial bacterial concentration was 1Â104 cfu/mL.

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H. Hujakka et al. / Bioorg. Med. Chem. 9 (2001) 1601–1607
for a drug, would decrease the diffusion of a compound
in local use.
Conclusion
The two Temporin A derivatives (TA and TAd) have
similar antimicrobial effects against S. aureus, the
Gram-positive test strain. However, only the dimeric
derivative of Temporin A (TAd) has antimicrobial effect
against E. coli, the Gram-negative test strain. The per-
meability of TAd through the outer membrane of E. coli
can be explained by higher positive net charge of TAd.
It is also possible that the positive amino group of the
DAPPA unit enhances the activity by interactions
between the cell membrane. However, the activity of the
TAd is only half that of TA against Gram-positive
strains if calculated per one TA unit. This suggests that
the present structures are not completely optimized, yet,
but provide a good starting point for a further work.
The antibiotic peptides, Temporins, contain 10 to 13
amino acids, and according to the CD studies, they
adapt an a-helical structure in hydrophobic environ-
ment.7 It has been suggested that Temporins form bar-
rel-stave structures20 and due to the amphipathic nature
of the peptide may form pores where the hydrophobic
side of the helix is adjacent to the hydrophobic tails of
fatty acids.21 From the mode of action point of view it is
required that the antibiotic peptides penetrate deep
enough into the cell membrane in order to lyse the cell.
The length of the helical Temporin A is about 24 A,
which is only about half of the thickness of the lipid
bilayer. While the mechanism by which Temporins lyse
bacteria is not known, we argued that by addition of a
spacer tripeptide GGA between DAPPA and the Tem-
porin part would provide the Temporin more freedom
for its proposed alignment to the bacterial membrane.
The spacer allows also the structure to penetrate deeper
into the membrane and its flexibility allows 10–20 A
distance between the C-terminals of the Temporin parts.
Experimental
Synthesis
propanoic acid (DAPPA)
of
the
3-N,N-di(3-aminopropyl)amino
According to the synthesis protocol, N-(3-aminopro-
pyl)-1,3-propanediamine (1) was converted to the cor-
The positive charge at the amino terminus is not neces-
sary for the lytic activity of short helical antimicrobial
peptides and can be replaced with hydrophobic groups
like Fmoc. Unfortunately, such a modification also
increases the undesirable lytic activity of Temporin A
against eucaryotic cells (erythrocytes).22 On the con-
trary, the total positive charge generally improves the
antibiotic peptide activity, obviously due to the fact that
the surface of the bacteria is negatively charged. This
appears to be true also for TA as exemplified by our
modifications. The formal total net charges of TA, TAc
and TAd are +2, +1 and +4, respectively. It is notable
that DAPPA contains one positively charged amino
group in its branching point, and this leads to the total
net charge +4, instead of +3, of TAd. Also the loca-
tions of the charges are of essential importance. It is
notable that TA contains a neutral amide group, TAc a
negatively charged carboxy group, and TAd a positively
charged amino group in that part of the molecule which
can be assumed to locate close to the surface of the cell
membrane after the peptide is embedded in it for lysis.
responding symmetrical backbone 4.
A selective
protection of the primary amino-groups of 1 with
phthalic anhydride in acetic acid,24 followed by utiliza-
tion of methyl acrylate (Michael’s addition), afforded
pure 3 in a fair yield. Deprotection of the amino and
acid functions with concentrated HCl to compound 4
and its Fmoc-protection resulted in the desired Fmoc
protected symmetrical molecule 5, which has a free acid
function readily available for the automated solid phase
peptide synthesis (Fig. 3).
Procedure for the synthesis of Fmoc-DAPPA (5)
Norspermidine (8.96 g, 68.3 mmol) was dissolved in gla-
cial acetic acid (180 mL). Phthalic anhydride (20.23 g,
136.6 mmol) was added neat and the reaction mixture
was refluxed for 1 h. The solvent was distilled in vacuo
to give quantitative yield of 2 with the following spectral
properties: 1H NMR (400 MHz, CDCl3) d 7.84 (m, 4H),
7.73 (m, 4H), 3.79 (t, J=6.7 Hz, 4H), 2.97 (t, J=7.1 Hz,
4H), 2.06 (m, J=6.9 Hz, 4H); 13C NMR (100 MHz,
CDCl3) d 168.42, 134.13, 131.97, 123.43, 45.60, 34.92,
25.86. Phthalic anhydride protected 2 was dissolved into
methyl acrylate (80 mL) and the mixture was stirred at
room temperature for 6 h and then refluxed for 4 h. The
solvent was distilled in vacuo and the residue was mixed
with methanol (50 mL). The precipitate was filtered and
washed with cold methanol (20 mL). The yield 26.0 g (80
The interaction between the negatively charged bacterial
inner membrane and the C-terminus of TAc is unfavor-
able and could render TAc totally inactive against S.
aureus. The negative charge of TAd at the end of the
arm of DAPPA seems to be of less importance for its
activity against the Gram-positive bacterium. TA is not
able to penetrate the outer membrane (OM) of Gram-
negative E. coli at the concentrations used. However,
the positive charge of TAd makes it active also against
E.coli, suggesting that it has structural properties that
facilitate its OM-penetration. It is known that anions
are able to brake OMs of Gram-negative bacteria and
that their net total charge is a very essential property for
the permeabilization.23 Our results suggest that the net
charge of TA (+2) is not enough, but the net charge
of +4 enables total penetration through the outer
membrane.
1
%) of 3: H NMR (400 MHz, CDCl3) d 7.82(m, 4H),
7.70 (m, 4H), 3.71 (t, J=7.3 Hz, 4H) 3.66 (s, 3H), 2.79
(t, J=7.1 Hz, 2H), 2.51 (t, J=7.0 Hz, 4H), 2.45 (t,
J=7.1 Hz, 2H), 1.81 (m, J=7.2Hz, 4H); 13C NMR
(100 MHz, CDCl3) d 173.10, 168.38, 133.82, 132.21,
123.17, 51.61, 51.25, 49.12, 36.30, 32.18, 26.23.
Compound 3 (4.0 g, 8.4 mmol) was mixed with con-
centrated HCL (30 mL) and refluxed for 20 h. The mix-
ture was allowed to cool to room temperature and the
precipitate was filtered. The aqueous phase was

H. Hujakka et al. / Bioorg. Med. Chem. 9 (2001) 1601–1607
1605
extracted with diethyl ether (5Â20 mL) and evaporated
to give 2.62 g (99%) of DAPPA (4) as a colourless thick
oil: 1H NMR (400 MHz, D2O) d 3.57 (t, J=6.8 Hz, 2H),
3.36 (m, 4H), 3.11 (t, J=7.6 Hz, 4H), 2.94 (t, J=6.8 Hz,
2H), 2.19 (m, 4H); 13C NMR (100 MHz, D2O) d 176.65,
53.41, 51.60, 39.41, 31.38, 24.53. DAPPA (1.54 g,
4.9 mmol) was dissolved in water (15 mL) and neu-
tralized with 10 % NaOH solution (2.4 mL). Ten percent
Na2CO3 (60 mL) and dioxan (10 mL) was added to the
mixture and cooled in an ice bath during the slowly
addition of Fmoc-Cl (3.2g, 10.8 mmol) in dioxan
(37 mL). The resulting mixture was stirred for 2h in an
ice bath and 46 h at room temperature. Water (225 mL)
was added and the aqueous phase was extracted with
diethyl ether (4Â50 mL). The pH was set to 6 with con-
centrated HCl and the aqueous phase was extracted
with ethyl acetate (6Â60 mL). The organic phase was
dried over MgSO4 and evaporated. Yield 2.52 g (79%)
of Fmoc-DAPPA (5) as a white solid. 1H NMR
(400 MHz, CDCl3) d 7.71 (bd, J=6.6 Hz, 4H), 7.55 (bd,
J=6.8 Hz, 4H), 7.34 (bt, J=6.7 Hz, 4H), 7.24 (bt,
J=6.7 Hz, 4H), 4.31 (bd, J=6.1 Hz, 4H), 4.12(bt,
J=6.1 Hz, 2H), 3.18 (bs, 4H), 2.94 (bs, 2H), 2.78 (bs,
The distance between carboxy carbon of Leu(13) and
the central nitrogen of the DAPPA unit was estimated to
be ca. 15 A in extented conformation and ca. 17 A to
the carboxy group of DAPPA. The Leu(13) carboxy
carbons in the two branches are separated by ca. 27 A.
Peptide synthesis
The monomeric Temporin A peptide amide (NH2-
FLPLIGRVLSGIL-CONH2), the monomeric carboxy
(NH2-FLPLIGRVLSGIL-COOH) and the dimeric
derivative [2Â(NH2-FLPLIGRVLSGILAGG)-dappa-
G-COOH] (Fig. 4) were synthesized using PerSeptive
9050 Plus automated peptide synthesizer, with Fmoc
strategy and TBTU/DIPEA as the coupling reagent.
For the synthesis of the peptide amide NovaSyn TGR
resin with a modified Rink linker and for the peptide
acids
NovaSyn
TGA
with
4-hydroxymethyl-
phenoxyacetic acid linker were used (Novabiochem,
Laufelfingen, Switzerland). The side-chain protecting
groups used in synthesis were trityl (Trt) for Asn and
His, O-tert-Butyl (OtBu) for Glu, t-Butyloxycarbonyl
(t-Boc) for Lys and tert-Butyl (tBu) for Ser. The pep-
tides were purified by HPLC (Shimadzu, Japan) with
C18 reverse phase column and acetonitrile (ACN) as
eluent (0.1%TFA in H2O/0–60% ACN gradient for 60
min) and verified with MALDI-TOF mass spectrometer
(Bruker, Germany). The concentrations of the peptides
were confirmed by using amino acid analysis (Protein
analysis center, Karolinska Institutet, Sweden).
4H), 2.53 (bs, 2H), 1.80 (bs, 4H); MALDI-TOF MS m/z
+
648.2(M
)
Peptide design
The branched peptide was designed based on the pro-
posed amphipatic a-helical structure of Temporin A.
The size of the helix structure is proposed to be 24 A in
length and about 12A diameter. To facilitate the cor-
rect orientation of parallel amphipatic helices, an addi-
tional AGG was added between Leu(13) and DAPPA.
Microorganisms and growth conditions
The organisms used in these experiments were Staphy-
lococcus aureus (ATCC 25923) and Escherichia coli
Figure 3. The synthesis strategy of Fmoc-DAPPA (5).

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H. Hujakka et al. / Bioorg. Med. Chem. 9 (2001) 1601–1607
Figure 4. . Schematic representation of original Temporin A, TA, (A), its monomeric carboxy, TAc, (B), and the novel dimeric, TAd, (C) analogues.
Acknowledgements
(ATCC 25922). The bacteria were stored on blood agar
plates at +4 ꢀC and subcultured every second week.
The authors thank Ms. Kirsi Lehikoinen for her
technical assistance.
To obtain a suspension culture, a bacterial inoculum
was suspended into 5 mL of tryptic soy broth (TSB)
medium (Difco, USA), and the suspensions were incu-
bated at +37 ꢀC for 20 h. After the incubation, bacterial
cells were centrifuged at 1680Â g for 10 min (Br-i,
Jouan, France), resuspended into 50 mL of fresh TSB-
medium and stored at +4 ꢀC. Part of the suspension
was diluted from 10À1 to 10À9 in buffered peptone water
(OXOID, UK) and 100 ml of each dilution was spread
on two parallel plate count agar (PC agar) plates (LAB
M, UK). The plates were incubated at +37 ꢀC for 24 h,
and the number of colony forming units (cfu) were
counted. For the antimicrobial assays the stored sus-
pension was diluted with TSB to obtain the desired
bacterial concentrations.
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