Synthesis of a chiral amino acid with bicyclo[1.1.1] pentane moiety and its incorporation into linear and cyclic antimicrobial peptides

Article abstract of DOI:10.1039/b702134h

The synthesis of the lipophilic chiral amino acid 1 bearing the bicyclo[1.1.1]pentane moiety is described. Linear and cyclic hexapeptides of the type Arg-Arg-Xaa-Yaa-Arg-Phe containing 1 instead of one or two tryptophan residues are prepared by solid phase peptide synthesis and the antimicrobial and hemolytic activity of the peptides obtained are discussed. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b702134h

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of a chiral amino acid with bicyclo[1.1.1]pentane moiety and its
incorporation into linear and cyclic antimicrobial peptides†
Stephan Pritz,*a Michael Pa¨tzel,*b Gu¨nter Szeimies,b Margitta Dathea and Michael Bienerta
Received 9th February 2007, Accepted 18th April 2007
First published as an Advance Article on the web 4th May 2007
DOI: 10.1039/b702134h
The synthesis of the lipophilic chiral amino acid 1 bearing the bicyclo[1.1.1]pentane moiety is
described. Linear and cyclic hexapeptides of the type Arg-Arg-Xaa-Yaa-Arg-Phe containing 1 instead
of one or two tryptophan residues are prepared by solid phase peptide synthesis and the antimicrobial
and hemolytic activity of the peptides obtained are discussed.
enhances the antimicrobial activity by inducing a constrained am-
phipathic structure, and additionally increases resistance against
Introduction
proteolysis.7,8 The effect of cyclisation on the enhancement of the
antimicrobial activity is especially pronounced for the tryptophan-
containing sequences. The substitution of tryptophan by the more
lipophilic 2-naphthylalanine (2-Nal) increases the antimicrobial
effect of the linear sequences, and transformation into the cyclic
analogues does not result in further improvements.6,7 Remarkably,
the insertion of the lipophilic, aromatic Nal residues increases also
the lysis of erythrocytes,7 thus making these peptides less suitable
as potential antimicrobial reagents.
In order to study the influence of lipophilic, non-aromatic
amino acid side chains on the antimicrobial and hemolytic activity,
we synthesised the corresponding linear and cyclic peptides,
containing amino acids derived from the bicyclo[1.1.1]pentane
moiety. The chemistry of bicyclo[1.1.1]pentanes has gained con-
siderable attention over the past years and a broad range of
differently substituted derivatives has been synthesised.9 Among
them 2-(3ꢀ-substituted bicyclo[1.1.1]pentyl)glycines are the only
examples of a-amino acids bearing the bicyclo[1.1.1]pentane
moiety.10 These compounds represent selective metabotropic glu-
tamate 1 receptor (mGluR) antagonists, with no activity at other
mGluR subtypes. Recently, the synthesis of the c-amino acid 3-
aminobicyclo[1.1.1]pentane-1-carboxylic acid and its incorpora-
tion into peptides has been reported.11
Antimicrobial peptides are important components of the animal
defence against microbial infections.1 Stimulated by the challenge
of increasing bacterial resistance, considerable efforts are being
made to elucidate the structural basis of peptide selectivity and
their mechanism of action. It is generally accepted that the peptides
exert their activity by permeabilisation of the lipid matrix of
the bacterial membrane. Peptide interaction with the negatively-
charged lipid matrix of prokaryotic cells is the consequence of
several common structural features of the peptides such as cationic
charge and the tendency to adopt an amphipathic conformation
in the membrane-bound state. Because of their activity against
a broad spectrum of pathogens, but low activity against normal
eukaryotic cells and their unique mode of action, the peptides
have been heralded as new antibiotic drug candidates. But, despite
their attractive properties, and many efforts to optimise the peptide
properties with respect to high antibacterial activity and selectivity,
issues regarding toxicity and instability are unresolved and the
high production costs of the large peptides render additional
concerns. It is thus important to develop smaller antibacterial
peptides with adequate metabolic stability. There are only a few
examples of antibacterial peptides of small size in the literature.
A six-residue sequence was identified as the antimicrobial motif
of bovine lactoferricin2 and N-acetylated hexapeptides such as
Ac-RRWWRF-NH2 have been identified by deconvolution of
combinatorial libraries.3 A recent study to define the minimum
requirements of charge and lipophilic properties in antimicro-
bial peptides led to even shorter sequences with a remarkable
antimicrobial effect.4 All these peptides are rich in arginine and
tryptophan residues which play an important role in their activity.
Studies with analogues modified in the position of the aromatic
residue have demonstrated the significance of the size, shape and
character of the side chain for the antimicrobial effect.5–7 Our
recent studies showed that cyclisation of RRWWRF distinctly
Here we report on the synthesis of the novel amino acid 1
bearing the bicyclo[1.1.1]pentane moiety, the incorporation of
1 into peptides of the type Arg-Arg-Xaa-Yaa-Arg-Phe, and the
antimicrobial and hemolytic activity of both the linear and cyclic
analogues.
aLeibniz-Institute of Molecular Pharmacology, Robert-Ro¨ssle-Strasse
10, 13125, Berlin, Germany. E-mail: pritz@fmp-berlin.de; Fax: +49
(0)30 94793 159; Tel: +49 (0)30 94793 362
Results and discussion
bHumboldt-Universita¨t zu Berlin, Institut fu¨r Chemie, Brook-Taylor-Str.
2, 12489, Berlin, Germany, michael.paetzel@rz.hu-berlin.de; Fax: +49
(0)30 2093 6940; Tel: +49 (0)30 2093 7190
The synthesis of target compound 1 is depicted in Scheme 1.
Treatment of the dibromocyclopropane 2 with 2.2 equivalents
of methyllithium generated the [1.1.1]propellane 3.12 Addition of
tert-butylmagnesium chloride to the central bond of 3 afforded a
† Electronic supplementary information (ESI) available: Synthesis and
purification of peptides. See DOI: 10.1039/b702134h
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Scheme 1 Synthesis of amino acid 1 and peptides containing 1.
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3-tert-butylbicyclo[1.1.1]pentyl magnesium chloride, which could
be trapped by carbon dioxide to give acid 4. This compound
was reduced to the alcohol 5 by lithium aluminium hydride
and subsequently converted to the corresponding aldehyde via a
Dess–Martin-oxidation. Condensation of the aldehyde with (R)-
2-phenylglycinol gave imine 6, which was subjected to an asym-
metric Strecker reaction.10,13 The 7/3-mixture of diastereomeric
a-aminonitriles 7 and 8 was separated by flash chromatography.
Finally, the auxiliary was removed oxidatively and the nitrile
hydrolysed to give the amino acid 1.10,13 Treatment of (S)-1 with
base and Fmoc-protection resulted in the formation of compound
9 which is a suitable derivative for solid phase peptide synthesis
possessing good solubility in DMF.
Linear and cyclic analogues of the hexapeptide sequence
RRWWRF (10a) containing 1 instead of one or two tryptophan
residues were synthesised manually by standard Fmoc-SPPS
techniques. Rink amide and 2-chlorotrityl resins were used for
the linear peptides and for the linear precursors of the cyclic
peptides, respectively. The linear peptide amides (10a–12a) were
N-terminally acetylated. Cyclisation was achieved by activation
with HAPyU giving the cyclic analogues 10b–12b. According to
HPLC, purity of linear and cyclic peptides was >95 and >85%,
respectively. Molecular masses of all peptides were confirmed
by MALDI mass spectrometry (see the ESI† for synthesis,
purification and analytical data of the peptides 10a/b–12a/b).
Small, arginine- and tryptophan-rich hexapeptides such as Ac-
RRWWRF-NH2 (10a) have been found to possess antimicrobial
activity.3 Our recent studies showed that cyclisation of RRWWRF
markedly enhances the antimicrobial effect7 by inducing an
amphipathic structure.8 Although the influence of the side chains
upon the structure of the cyclic peptides in a membrane-mimicking
environment was low, substitution of the aromatic side chains by
bulky residues of varying hydrophobicity and size had dramatic
consequences on the biological effect.
Fig. 1 (A) Reciprocal minimal inhibitory concentration (1/MIC) of
linear and cyclic peptides against E. coli and B. subtilis (white and black
bars); (B) peptide-induced dye release from mixed POPG–POPC (1 : 3)
vesicles (striped bars) measured as fluorescence dequenching (F) at cpeptide
2 lM and clipid = 25 lM after t = 5 min.
=
To further elucidate the activity-modifying character of side
chains, linear and cyclic hexapeptides were synthesised with the
novel aliphatic, highly hydrophobic amino acid 1 substituting one
or two of the tryptophan residues, respectively (Table 1).
The linear peptide 10a had little activity against bacteria, as
shown by a minimal inhibitory concentration (MIC) higher than
100 lM (1/MIC < 0.01 lM−1) for E. coli (Gram-negative). B.
subtilis (Gram-positive) were slightly more susceptible (1/MIC =
0.04 lM−1) (Fig. 1A). With introduction of one or two of the
bulky, highly hydrophobic residues 1, the antimicrobial effect of
the linear sequence markedly increased. These results are similar
to effects obtained by introduction of bulky aromatic residues
into lactoferricin14 and confirm the important role of peptide
hydrophobicity for the activity of the hexapeptides.7
The gradual changes in the retention times of the linear peptides
which followed the order 10a < 11a < 12a reflected changes in the
total hydrophobicity and/or amphipathicity as result of amino
acid exchange (Table 1). Comparable retention behaviour of the
corresponding cyclic compounds suggests that conformational
constraints have little influence on the surface behaviour. These
properties are also reflected in the peptide interactions with lipid
bilayers. With introduction of 1, the bilayer-disturbing activity
distinctly increased, but pronounced differences between linear
and cyclic analogues were not observed (Fig. 1B).
In contrast to the pronounced enhancement of the antimicrobial
activity by cyclisation of the parent peptide 10a (Fig. 1A),
the positive effect of cyclisation disappeared for the analogues
Table 1 Sequences and retention times of linear and cyclic peptides
Linear
Cyclic
Name
Sequence
tR/mina
Name
Sequence
tR/mina
10a
11a
12a
Ac-RRWWRF-NH2
Ac-RRW1RF-NH2
Ac-RR11RF-NH2
17.6
20.1
23.0
10b
11b
12b
Cyclo(RRWWRF)
Cyclo(RRW1RF)
Cyclo(RR11RF)
18.5
19.9
23.9
a tR: retention time in RP-HPLC.
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substituted by 1. Whereas 10b exhibited a more than 16-fold
enhanced activity against E. coli and eight times higher activity
against B. subtilis than its linear counterpart, the activities of
the linear and corresponding cyclic peptides containing one
residue 1 (11a/b) were almost the same, while the activity of
12b decreases significantly when compared to 12a. Furthermore,
different to the observations in HPLC (Table 1) and the bilayer
permeabilisation assay (Fig. 1B), the biological activities of all
cyclic peptides were little differentiated. As suggested for the
naphthylalanine-containing hexapeptide, it is likely that the high
global hydrophobicity of the sequences containing 1 eliminated the
activity-enhancing effect of cyclisation-induced amphipathicity.7
Obviously, aromatic residues are not essential. Furthermore, the
different activity patterns on model membranes and bacteria show
that peptide interactions with the biological membrane are highly
complex, and hydrophobic peptide interactions with the lipid
matrix of the cell membrane are not sufficient to explain the
activity towards bacterial cell membranes.
methyl)cyclopropane 2 (40 g, 0.135 mol, 1.00 equiv.) in diethyl
ether (150 ml), which was kept at −30 ◦C under stirring. The mix-
ture was stirred for 1 h at room temperature, until the formation
of a white precipitate was complete. The solvent and the volatile
products were distilled at 14 Torr from a 20 ◦C bath into a Schlenk
flask which was kept at −78 ◦C.12 To the obtained clear solution
was added dropwise tert-butylmagnesium chloride (47 ml of 2.0 M
◦
solution in diethyl ether, 0.094 mol, 0.70 equiv.) at −50 C. The
reaction mixture was stirred for 4 d at room temperature. Then CO2
(from 40 g dry ice, dried over silica gel) was passed into the grey–
green reaction mixture for 2 h at −40 ◦C. The reaction was allowed
to warm to room temperature and then cooled to 0 ◦C. Aqueous
HCl (100 ml, 2 N) was added at this temperature. The organic
phase was separated and the aqueous layer was extracted with
diethyl ether (3 × 25 ml). Drying the combined organic layers over
MgSO4 and evaporation of the solvent afforded carboxylic acid 4
(14.0 g, 61% from 2) as colourless crystals. Mp: 167 ◦C (lit.15: 155–
157 ◦C); 1H NMR (300 MHz, CDCl3) d 0.84 (s, 9 H), 1.86 (s, 6 H),
10.54 (br s, 1 H); 13C NMR (75 MHz, CDCl3) d 25.9, 29.5, 35.5,
48.2, 48.3, 177.4. The NMR data correspond to the literature.15
In summary, our studies showed that substitution of tryptophan
by the highly hydrophobic and bulky residues 1 resulted in
peptides with pronounced antimicrobial activity. Comparable
to the aromatic naphthylalanine, the high hydrophobicity of 1
predominated peptide membrane interactions and the activity-
enhancing effect of cyclisation, observed for the tryptophan-
containing peptide, became negligible.
3-tert-Butylbicyclo[1.1.1]pent-1-ylmethanol (5)
To a suspension of LiAlH4 (11.4 g, 0.3 mol) in THF (250 ml)
carboxylic acid 4 (20.43 g, 0.121 mol), dissolved in THF (110 ml)
was added dropwise over 30 min at 0 ◦C. The mixture was heated
under reflux for 1 h. After cooling to 0 ◦C diethyl ether (300 ml) and
a sat. aq. solution of MgSO4 (40 ml) were added. The suspension
was filtrated through a pad of celite. The filtrate was extracted with
diethyl ether (3 × 50 ml). Drying the combined organic layers over
MgSO4 and evaporation of the solvent afforded the target com-
pound◦5 (15.98 g, 85%) as colourless crystals. Mp: 40–41 ◦C (lit.16:
46–47 C); 1H NMR (300 MHz, CDCl3) d 0.84 (s, 9 H), 1.28 (s, 1 H,
1.48 (s, 6 H), 3.59 (s, 2 H); 13C NMR (75 MHz, CDCl3) d 26.0, 29.7,
37.2, 45.0, 48.4, 63.9. The NMR data correspond to the literature.16
Conclusion
Synthesis of the chiral amino acid 1 was achieved in nine steps from
the tetrahalide 2. Key steps in the synthesis were the Grignard-
addition to [1.1.1]propellane 3 and the asymmetric Strecker-
reaction yielding the two glycinonitriles 7 and 8 in a diastereomeric
ratio of 7 : 3. After Fmoc-protection of 1, several linear and cyclic
peptides containing this amino acid could be synthesised by SPPS
and their antimicrobial activity was determined. Substitution of
tryptophan by 1 in the linear peptide RRWWRF (10a) enhances
both the antimicrobial and bilayer-permeabilising activity. To
the same extent, hydrophobicity increases in these compounds.
Thus, the higher activity of 11a and 12a can be attributed to a
higher membrane-disturbing effect due to higher hydrophobicity.
Cyclisation of 11 and 12 has only minor influence on bioactivity.
On the other hand, the antimicrobial activity of 10b is distinctly
enhanced upon cyclisation, while lysis remains low.
3-tert-Butylbicyclo[1.1.1]pentane-1-carbaldehyde
To a solution of 3-tert-butylbicyclo[1.1.1]pentane-1-methanol 5
(10.08 g, 65.35 mmol) in CH2Cl2 (200 ml) was added Dess–Martin
periodinane17 (27.72 g, 65.35 mmol) at 0 ◦C within 10 min. After
stirring for 2 h a solution of Na2S2O3 × 5 H2O (100 g) in sat. aq.
NaHCO3 (400 ml) and 200 ml diethyl ether were added carefully to
the white suspension formed. After stirring for 1.5 h the layers were
separated and the aqueous phase was extracted with diethyl ether
(3 × 80 ml). Drying the combined organic phases over MgSO4 and
evaporation of the solvents afforded the target aldehyde (9.64 g,
97%) as pale yellow oil. The aldehyde was used in the next step
without further purification. 1H NMR (300 MHz, CDCl3) d 0.85
(s, 9 H), 1.81 (s, 6 H), 9.59 (s, 1 H); 13C NMR (75 MHz, CDCl3) d
25.8, 44.9, 45.9, 46.9, 48.7, 200.1.
Experimental
All experiments were carried out under a nitrogen atmosphere.
Solvents were dried according to standard procedures before being
used. tBuMgCl was purchased from Aldrich Co. 1H and 13C NMR
spectra were recorded at 300 and 75 MHz, respectively, on a Bruker
DPX 300. Mass spectra were obtained on a ThermoFinnigan LCQ
XP. Elemental analyses were performed on a Leco CHNS-932
apparatus. Optical rotations were measured on a Jasco DIP-370
polarimeter at 589 nm and are given in 10−1 deg cm2 g−1.
(2S,1ꢀR)- and (2R,1ꢀR)-2-(3-tert-Butylbicyclo[1.1.1]pent-1-yl)-
N-(2ꢀ-hydroxy-1ꢀ-phenylethyl)glycinonitrile (7 and 8)
To a solution of 3-tert-butylbicyclo[1.1.1]pentane-1-carbaldehyde
(3.08 g, 20.2 mmol) in CH2Cl2 (175 ml) was added (R)-2-
phenylglycinol18 (2.78 g, 20.3 mmol) within 5 min. The solution
was stirred at room temperature for 16 h. The reaction mixture
was cooled to −65 ◦C and trimethylsilylcyanide (5.07 ml, 4.02 g,
3-tert-Butylbicyclo[1.1.1]pentane-1-carboxylic acid (4)
MeLi (225 ml of a 1.2 M solution in diethyl ether, 0.27 mol,
2.00 equiv.) was added dropwise to a solution of 2,2-bis(chloro-
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40.5 mmol) was added via a syringe. The reaction mixture was
10 H2O (0.65 g). In addition, sat. aqueous Na2CO3 solution was
added until pH 9. When required, further Na2CO3 solution was
added to maintain pH 9. To the white suspension was added N-
(9-fluorenylmethoxycarbonyloxy)succinimide (1.44 g, 4.27 mmol).
After stirring for 12 h at room temperature the clear, yellow
solution was diluted with ethyl acetate (20 ml) and carefully
acidified with hydrochloric acid (3 N) to pH 2. The layers were
separated and the aqueous phase extracted with ethyl acetate
(2 × 20 ml). The combined organic layers were washed with
hydrochloric acid (10 ml of a 3 N aq. solution), H2O (5 ml)
and dried over MgSO4. The solvents were removed under reduced
pressure ◦to afford the protected amino acid 8 (1.66 g, 93%). Mp:
194–195 C (CH2Cl2). 1H NMR (300 MHz, DMSO-d6) d 0.81 (s, 9
H), 1.51 (q, J = 9.4 Hz, 6 H), 4.08 (d, J = 8.3 Hz, 1 H), 4.16–4.34
(m, 3 H), 7.33 (t, J = 7.3 Hz, 2 H), 7.42 (t, J = 7.0 Hz, 2 H), 7.60 (d,
J = 8.3 Hz, 1 H), 7.76 (d, J = 7.5 Hz, 2 H), 7.82 (d, J = 7.2 Hz, 2
H), 12.52 (br s, 1 H); 13C NMR (75 MHz, DMSO-d6) d 25.8, 29.2,
35.6, 45.4, 46.7, 46.8, 55.3, 65.8, 120.1, 125.4, 127.1, 127.7, 140.7,
143.9, 156.2, 171.8. Found: C, 74.22; H, 6.76; N, 3.15. C26H29NO4
requires C, 74.44; H, 6.97; N, 3.34%.
allowed to warm up slowly to room temperature. After 20 h the
◦
solution was cooled to 0 C and hydrochloric acid (100 ml of 3
N aq. solution) was added via a dropping funnel and the mixture
was stirred for 1 h. The layers were separated and the organic
phase washed with hydrochloric acid (30 ml of 3 N aq. solution).
The combined aqueous layers were extracted with CH2Cl2. The
combined organic phases were washed with H2O, and dried
over MgSO4. The solvent was removed under reduced pressure
to afford a yellow oil (5.00 g), which was subjected to column
chromatography (pentane–ethyl acetate = 8 : 2) to give the pure
(2S,1ꢀR)-derivative 7 (1.86 g, 30% from the aldehyde, colourless
crystals) and a mixture (1.22 g, 20%) of 7 and diastereomer 8. The
mixture was subjected to a second chromatography with the next
reaction batch. Compound 7: mp: 54–57 ◦C. 1H NMR (300 MHz,
CDCl3) d 0.85 (s, 9 H), 1.54–1.68 (m, 6 H), 2.12 (br s, 2 H), 3.37 (s,
1 H), 3.56 (d × d, J = 9.0 and 10.9 Hz, 1 H), 3.78 (d × d, J = 3.8
and 10.9 Hz, 1 H), 4.08 (d × d, J = 4.1 and 9.0 Hz, 1 H), 7.24–7.41
(m, 5 H); 13C NMR (75 MHz, CDCl3) d 25.9, 29.6, 36.4, 45.1, 48.2,
49.7, 63.1, 67.6, 118.8, 127.7, 128.3, 128.9, 138.5. Found: C, 76.11;
H, 8.98; N, 9.19. C19H26N2O requires C, 76.47; H, 8.78; N, 9.39%.
Compound 8: yellow oil. 1H NMR (300 MHz, CDCl3) d 0.86 (s, 9
H), 1.55–1.68 (m, 6 H), 2.03 (br s, 2 H), 3.64 (d × d, J = 7.2 and
10.9 Hz, 1 H), 3.70 (s, 1 H), 3.75 (d × d, J = 4.5 and 10.9 Hz, 1
Antibacterial studies
H), 3.99 (d × d, J = 4.3 and 7.3 Hz, 1 H), 7.28–7.41 (m, 5 H); 13
C
E. coli (Gram-negative, DH 5a strain) and B. subtilis (Gram-
positive, PY 22 strain) were used to test the antibacterial activity
of the peptides.7 Bacteria were cultivated to the mid-logarithmic
phase of growth (OD600 = 0.5). Aliquots of the cell suspensions
were added to the wells of a microtiter plate containing peptide at
different concentrations. Final concentration of bacteria was 105
CFU per ml. Final concentrations of peptide ranged from 0.05
to 100 lM in 2-fold dilutions. Microtiter plates were incubated
overnight at 37 ◦C and the absorbance was read at 600 nm
(Autoreader EL 311, Bio-Tek Instruments Inc., USA). The
minimum inhibitory concentration (MIC) of bacterial growth is
defined as the lowest concentration of peptide at which there was
no change in optical density.
NMR (75 MHz, CDCl3) d 25.9, 29.6, 36.8, 45.3, 48.2, 50.2, 63.2,
66.3, 118.8, 127.6, 128.4, 129.0, 139.6. Found: C, 76.18; H, 8.93;
N, 9.26. C19H26N2O requires C, 76.47; H, 8.78; N, 9.39%.
(S)-2-(3-tert-Butylbicyclo[1.1.1]pent-1-yl)glycine hydrochloride
[(S)-1]
To a solution of glycinonitrile 7 (1.73 g, 5.80 mmol) in a mixture
of CH2Cl2–MeOH (1 : 1, 20 ml) was added at room temperature
under stirring Pb(OAc)4 (3.08 g, 6.95 mmol) within 2 min. After
stirring for 15 min phosphate buffer (pH 7, 60 ml) was added
and the solution was allowed to stir for further 45 min. The
brown precipitate was filtered over Celite and the solid residue
washed with CH2Cl2 (3 × 40 ml). The layers were separated and
the aqueous phase was extracted with CH2Cl2 (3 × 40 ml). The
organic phases were combined and the solvent was removed under
reduced pressure. To the remaining yellow oil (1.5 g) was added
hydrochloric acid (110 ml of a 6 N aq. solution) and the reaction
mixture was refluxed for 9 h. The hot solution was decanted from
the black residue and the solution was kept in a freezer at −30 ◦C
overnight. The formed white precipitate was filtrated and washed
with CH2Cl2 (2x 15 ml) and dried under high vacuum. The target
compound (S)-1 was obtained as a colourless powder (0.87 g,
64%). Mp: 300 ◦C (decomp.). 1H NMR (300 MHz, TFA-d) d 0.93
(s, 9 H), 1.88 (s, 6 H), 4.49 (s, 1 H); 13C NMR (75 MHz, TFA-d)
d 26.6, 31.4, 36.8, 48.2, 50.5, 57.8, 174.5. Mass spectrum, m/z 198
Peptide-induced dye release from liposomes
To characterise the bilayer permeabilising activity against a
lipid matrix mimicking the lipid composition of a bacterial
membrane, peptide-induced calcein release from vesicles was
determined fluorimetrically, as described.7 Vesicles composed of 1-
palmitoyl-2-oleoylphosphatidylcholine (POPC) and 1-palmitoyl-
2-oleoylphosphatidyl-sn-glycerol (POPG) at a molar ratio of 3 : 1
were prepared by vortexing the dried lipid in dye buffer solution
(70 mM calcein, 10 mM Tris, 0.1 mM EDTA, pH 7.4) and extru-
sion (Lipex Biomembranes Inc., Canada) through polycarbonate
filters (six times through two stacked 0.4 lm pore size filters
followed by eight times through two stacked 0.1 lm pore size
filters). The fluorescence was excited at 490 nm and registered
at 520 nm after 5 min after adding an aliquot of an LUV
suspension to the peptide on a LS 50B spectrofluorimeter (Perkin
Elmer Corp. Germany). The peptide concentration was 2 lM,
the lipid concentration was 25 lM. The fluorescence intensity (F)
corresponding to 100% dye release was determined by addition of
bilayer-disturbing Triton X-100.
(M+–Cl, 100%). [a]D 38.7 (c 0.715 g per 100 ml in 0.1 N HCl).
32
Found: C, 56.21; H, 8.84; N, 5.67, Cl, 14.87. C11H20ClNO2 requires
C, 56.52; H, 8.62; N, 5.99, Cl, 15.17%.
(S)-Fmoc-3-tert-Butylbicyclo[1.1.1]pent-1-yl)glycine (9)
To a suspension of amino acid hydrochloride (S)-1 (1.00 g, 4.27 g)
in a mixture of acetone–H2O (1 : 1, 20 ml) was added Na2CO3 ×
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DMF, N,N-dimethylformamide; Fmoc, 9-fluorenylmethyl-
oxycarbonyl; HAPyU, 1-(1-pyrrolidinyl-1H-1,2,3-triazolo[4,5-b]-
pyridin-1-ylmethylene)pyrrolidinium hexafluorophosphate N-
oxide; MALDI-MS, matrix-assisted laser desorption ionisation-
mass spectrometry; MIC, minimal inhibitory concentration;
POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; POPG, 1-palmi-
toyl-2-oleoylphosphatidyl-sn-glycerol; RP-HPLC, reversed-phase
high performance liquid chromatography; SPPS, solid phase pep-
tide synthesis; TFA, trifluoroacetic acid; TMS-CN, trimethylsilyl-
cyanide.
Acknowledgements
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