Chapter 6
A Pilot Study towards the Preparation of
Antimicrobial Coated Surfaces
Abstract. The synthesis of a lipidated Gramicidin S analogue via copper(I)-mediated [3+2] azide-alkyne
cycloaddition is described and the ability of this amphiphilic molecule to form stable monolayers at the air-water
interface was investigated. Furthermore, information about the organization of the monolayers of the lipidated
and non lipidated Gramicidin S analogues was provided by Grazing Incidence X-ray Diffraction (GIXD)
measurements performed at the air-water interface. Finally, monolayers of the lipidated Gramicidin S analogue
were transferred onto microscope glass slides and the efficiency of the coating was evaluated by contact angle
measurements and X-ray Photoelectron Spectroscopy (XPS). The results described in this Chapter constitute a
pilot study towards the preparation of antimicrobial coated surfaces.
Chapter 6
6.1 Introduction
Gramicidin S (GS), cyclo-(DPhe-Pro-Val-Orn-Leu)2, is a potent cationic antimicrobial
peptide (CAP) isolated from Bacillus Brevis with a β-sheet structure.1 Studies of the
interaction of GS and its analogues with bacterial cells showed that the destruction of the
integrity of the lipid bilayer of the membrane is the primary mode of antimicrobial action of
this peptide.2 In support of this hypothesis, GS has been found to interact strongly with
phospholipid model membranes, perturbing their organization and increasing their
permeability.3
In this Chapter, an analogue of GS, the 2S,4S-azido-proline (2S,4S-Azp) GS
derivative 1 (Chart 6.1),4 was studied for its ability to form monolayers at the air-water
interface. Being partially soluble in water, GS analogue 1 was not able to form stable
monolayers. Only when NaCl was present in the subphase stable monolayers were obtained.
O
N
H O
H2N
N
O
O
N
H
H
N
O
N
H
O
H
N
O
N
H
H
N
O
O
NH2
N
H
N
O
N3
1
Chart 6.1. Chemical structure of 2S,4S-azido-proline (2S,4S-Azp) GS analogue 1.
To increase the amphiphilicity of GS analogue 1, a lipid tail was introduced affording
GS analogue 3 (Scheme 6.1). Moreover, compound 3 was prepared with the ultimate goal to
make antimicrobial coated surfaces.
6.2 Synthesis, Langmuir Monolayer Studies and Structural Insight by Grazing
Incident X-ray Diffraction (GIXD) at the Air-Water Interface
The 2S,4S-azido-proline (2S,4S-Azp) GS analogue (1) was conjugated via
copper(I)-mediated [3+2] azide-alkyne cycloaddition to a terminal alkyne derivative of
1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE), compound 2.5 GS analogue (1)
was reacted with lipid 2 to form the copper(I)-mediated cycloaddition product 3. Cu(I) was
136
A Pilot Study towards the Preparation of Antimicrobial Coated Surfaces
generated in situ by reduction of Cu(II) with sodium ascorbate. The reaction was allowed to
proceed for 10 hrs at RT in a 1/1 (v/v) H2O/tBuOH solvent system6 and product 3 was
purified by dialysis and HPLC (Scheme 6.1).
O
N
H O
H2N
N
O
O
N
H
H
N
O
N
H
H
N
O
H
N
O
O
O
NH2
N
H
N
N
H
O
N3
1
O
O
O
O
O
OPO
OH
O
N
H
a
2
O
N
H O
H2N
N
O
O
N
H
H
N
O
N
H
O
H
N
O
N
H
H
N
O
O
NH2
N
H
N
O
N
N
N
O
O
H
N
3
OH
O P O
O
O
O
O
Scheme 6.1. Reagents and conditions. (a) 1 mol% CuSO4·5H2O and 5 mol% sodium ascorbate for 16 hrs at RT
in 1/1 (v/v) H2O/tBuOH.
The secondary structure of GS has been extensively investigated and the molecule was
found to adopt a C2-symmetry with an internal β-sheet like structure, which is stabilized by
four interstrand hydrogen bonds between the Leu and the Val residues.7 Furthermore, the
D
Phe-Pro dipeptide motif occupies the i+1 and i+2 position in the two type II’ β-turns
stabilizing further the β-pleated cyclic architecture. In this conformation, the hydrophobic Val
and Leu and the hydrophilic Orn residues are positioned on opposite sides of the cyclic
structure in an antiparallel fashion, resulting in a hydrophobic and a hydrophilic face of the
molecule. A single-crystal structure of a hydrated gramicidin S-urea complex was solved by
Dodson and co-workers in 1978.8 In the crystal structure, a slight twist in the β-sheet was
observed, while its C2-symmetry was retained. Unexpectedly, the side-chains of the Orn
residues where found to be taking part in the formation of hydrogen bonds with the carbonyl
oxygen atom of the DPhe residue. A more recent refined structure reported by the same group
showed the formation of channels.9 Six equivalents of GS molecules assembled into a
left-handed double spiral, exposing the hydrophobic side-chains at the outside surface,
whereas the hydrophilic side-chains occupied the inner surface. In additional studies, crystal
137
Chapter 6
structures of several derivatives of GS have been resolved. As an example, needle-shaped
crystals of an analogue of GS, in which one of the DPhe-Pro dipeptide sequence was replaced
by a furanoid sugar amino acid, were examined by X-ray diffraction analysis.10 The
crystallographic data revealed that the compound adopted a similar structure to the one
reported for GS but with a larger right-handed twist of the β-sheet. However, inspection of the
molecular packing of this GS analogue showed that a β-barrel-like arrangement was achieved
arising from the stacking of six individual β-sheets, which profoundly affected the oligomeric
assembled architecture. It is likely, therefore, that the substitution of one of the two Pro
residues with the 2S,4S-Azp should not affect the β-pleated structure itself. However, this
modification could influence the overall assembly into larger structures.
Surface pressure-surface area (π-A) isotherms studies and grazing incidence X-ray
diffraction (GIXD) experiments were performed to gain a better understanding of the
organization of the peptide domain in the monolayers of GS analogue 1 and its lipidated
derivative 3.
35
π (mN/m)
30
25
20
15
10
5
0
0
50
100
150
200
Mma (Ų/molecule)
Figure 6.1. Surface area - surface pressure (π-A) isotherms of 2S,4S-Azp GS analogue 1 (−−−) on milli-Q
water and (⋅⋅⋅⋅⋅) on 0.14 mM NaCl. π-A isotherms of lipidated GS analogue 3 (⎯) on milli-Q water.
Preliminary Langmuir monolayer studies11 showed that GS analogue 1 did not form a
stable monolayer at the air-water interface (Figure 6.1). The isotherm on milli-Q water was
characterized by a low collapsing surface pressure (πc = 10 mN/m). However, when a
0.14 mM NaCl solution was used as subphase, the isotherm of the cyclodecapeptide 1 reached
π = 25 mN/m before collapsing. Interestingly, a transition at π = 11 mN/m was observed. In
138
A Pilot Study towards the Preparation of Antimicrobial Coated Surfaces
contrast amphiphile 3, prepared by the conjugation of the lipid tail to GS analogue 1 resulted
in the formation of a stable monolayer at the air-water interface (πc = 35 mN/m).
GIXD measurements of GS derivatives were performed to elucidate the organization
of the monolayers. A solution of 2S,4S-Azp GS analogue 1 was spread on an aqueous
subphase containing 0.14 mM NaCl and seven Bragg’s peaks were found at A = 155
Å2/molecules and π = 24 mN/m. These diffraction data (see Table 2 in Appendix 1)
corresponded to a crystalline phase of Gramicidin S already reported,12 which is not consistent
to the structure reported by Dobson and revealed that compound 1 has a “pseudo-rectangular”
unit cell with a = 14.90 Å, b = 17.78 Å and γ = 90.09°, which is almost a rectangular unit cell.
The 17.78 Å distance corresponds to the expected value of 5 x 3.5 = 17.5 Å (along the peptide
backbone direction).13 In contrast, the 14.90 Å distance does not match a multiplication of
4.75 Å, suggesting that the molecules are tilted relative to the surface. If no tilt was present,
the unit cell axis would be 4 x 4.7 = 19 Å. Therefore, a tilt of 19/14.9 = ~ 38˚ is expected.
Qxy
QZ
Figure 6.2. Two contour plots of the peaks at (0,1) and (1,0) related to the main Bragg’s peak at qxy = 1.35.
The relatively large peak at qxy = 1.35 (Figure 6.2) is probably due to an organization,
which is independent of the lattice that gives rise to the other peaks. It could come from
molecules lying flat at the interface, which do not organize in the b direction. On the contrary,
lipidated GS 3 did not show any diffraction peaks. Thus, in this case the conjugation of the
lipid tail (DOPE) to the cyclic β-sheet peptide interfere with the crystalline structure of its
monolayer, in contrast with what was observed for the linear hexa- and octa-ALP monolayers
(Chapter 2).
139
Chapter 6
6.3 Transferred Monolayers towards the Preparation of Antimicrobial Coated
Surfaces
Monolayers of compound 3 were transferred by the Langmuir-Schaefer14 technique
(horizontal dipping) on glass microscope slides, therefore, exhibiting the peptide part on the
surface. As a control, Langmuir-Blodgett14 (vertical dipping) transfer was performed. In this
case the lipid tails are expected to “stick out” from the solid surface, shielding the peptide
domain.
Table 6.1. (A) Contact angle measurements on transferred monolayers of 3. Contact angles were measured on
glass slides. (B) Contact angle values on a clean glass surface with water θW = 0, formaminde θF = 11,
methyleniodide θM = 49 and α-bromo-naphthalene θα-Br = 22 degrees, respectively, which were used for
comparison.
A
advancing
SD
(n=3)
receding
SD
(n=3)
a
32.3
4
14
4
b
30
4.6
14.3
0.6
c
40.3
7.4
4.7
2.1
d
37.7
5.8
10.3
0.6
B
θW
SD
(n=3)
θF
θM
SD
(n=3)
θα-Br
SD
(n=3)
a
29
1.3
28
28
3
28
3
b
27.5
4.8
20.7
20.7
2.5
20.7
2.5
c
26.8
4.5
32.5
32.5
3.3
32.5
3.3
d
23.2
4.5
23.2
23.2
4.3
23.2
4.3
a = vertical dipping, b = vertical dipping after immersion in water, c = horizontal dipping and d = horizontal
dipping after immersion in water. All contact angles are in degrees.
140
A Pilot Study towards the Preparation of Antimicrobial Coated Surfaces
Contact angle measurements demonstrated that the monolayers were successfully
transferred onto glass slides and that the material was still present also after immersion of the
coated surfaces in aqueous solution (Table 6.1).15
The transferred monolayers were further investigated using X-ray Photoelectron
Spectroscopy (XPS). The XPS analysis showed that the horizontal dipping was more effective
than the vertical transfer, as can be observed from the data relative to %N (Table 6.2). Indeed,
the successful preparation of solids exposing the antimicrobial peptide on their surface was
achieved. However, after immersion into water most of the material dissolved, leaving hardly
any monolayer on the substrate. Due to the problems encountered in the preparation of stable
coated surfaces after immersion in water, bacterial adhesion on these surfaces was not studied,
as these tests require a continuous flow of bacterial solution.
Table 6.2. X-ray Photoelectron Spectroscopy (XPS) on transferred monolayers of 3. Elemental surface
compositions were expressed as a ratio of the percentage element over the percentage carbon.
%C
%O
% Si
%N
% Na
% Ca
others
a
31,7
40,5
16,4
0,0
9,2
1,2
Cu, Zn
b
55,5
26,9
8,2
2,2
6,0
1,2
c
33,2
41,8
16,2
1,1
6,3
1,5
d
38,7
34,3
9,0
4,2
13,1
0,5
Zn, K
e
34,6
40,0
15,9
1,1
6,5
1,3
Zn
a = clean glass slide (control), b = vertical dipping, c = vertical dipping after immersion in water, d = horizontal
dipping and e = horizontal dipping after immersion in water.
6.4. Conclusions and Future Prospects
In summary, lipidation of the GS derivative 1 resulted in a more stable monolayer
compared to the GS analogue 1 itself. However, in the case of the monolayer of lipidated GS
3, no well-ordered crystalline assembly could be detected by GIXD. In contrast, GS derivative
1 formed crystalline structures at the air-water interface, when NaCl was present in the
subphase. Indeed, monolayers of 3 were successfully transferred onto glass slides as
demonstrated by contact angles and XPS measurements. Unfortunately, the coated surfaces
were not stable after immersion in water. Improvements for a more efficient coating are
141
Chapter 6
currently under investigation and take into consideration either the modification of the lipid
tail (e.g. chains bearing crosslinkable units) or the use of a different substrate (i.e.
“hydrophobic” glass or slides covered by a thin Teflon layer). This results constitute a pilot
study towards the preparation of antimicrobial coated surfaces.
6.5 Experimental Section
General materials and methods. All reagents and solvents were commercial products purchased
from Sigma-Aldrich B.V. or Biosolve B.V. and used as received. Lipids were purchased from Lipoid and Avanti
Polar Lipids, Inc. Milli-Q water with a resistance of more than 18.2 MΩ/cm was provided by a Millipore Milli-Q
filtering system with filtration trough a 0.22 µm Millipak filte.
1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-propyl-2’-amide-1,4-triazole-cycloD
( Phe-2S,4S-Pro-Val-Orn-Leu-DPhe-Pro-Val-Orn-Leu) 3. GS analogue 14 (30 mg, 25 µmol) and lipid
derivative 25 (20 mg, 25 µmol) were dissolved in 240 µl of a mixture of 1/1 (v/v) H2O/tBuOH. To this mixture,
2.5 µl of 0.1 M of CuSO4⋅5H2O in water and 25 µl of 0.1 M sodium ascorbate in water were added. After 16 hrs,
TLC analysis (2/8 v/v MeOH/CHCl3) revealed the formation of the product. Dialysis against water was followed
by lyophilization and 11 mg (6 µmol, 22%) of 3 was collected, purified by RP-HPLC and characterized by LCMS (Rt = 16.6 min, m/z = 1979.6 [M+H]+; m/z = 990.0 [M+H]2+; m/z = 604,16 a linear gradient of 40 → 90% B
was applied in 5 CV)17 and stored at -20 °C. IR (thin film from CH3OH, cm-1): 3271 (br, NH stretch, Amide A
band), 2957 (-CH3 antisymmetric stretch), 2924 (CH2 antisymmetric stretch), 2853 (CH2 symmetric stretch),
1677, 1637 (br, C=O stretch, Amide I band), 1536 (C-N stretch, Amide II band).
Langmuir monolayer studies. Surface pressure - surface area (π-A) isotherms of the monolayer
films were measured as described in the experimental section of Chapter 2. Glass surfaces were cleaned first by
spotting with 2% Extran MA02 (Merck KgaA, Darmstadt, Germany), followed by sonication for 10 min in 0.5%
RBS25 (Fluka Chemie AG, Buchs, Switzerland) and thorough rinsing with tap water, methanol, tap water and
finally milli-Q water to obtain a fully water wettable, hydrophilic surface (i.e. a zero degrees water contact angle
glass surface). Monolayers were transferred onto clean glass microscope slides (76 x 26 mm) by LangmuirSchaefer deposition, using a vacuum system to hold the glass slides or by upstroke Langmuir-Blodgett transfer
with a speed of 5 mm/min. Both dipping experiments were performed at a constant surface pressure of 20 mN/m.
Grazing incidence X-ray diffraction (GIXD). GIXD measurements were performed in
collaboration with Hanna Rapaport (Ben Gurion University of the Negev, Beer-Sheva, Israel) at HASYLAB
(DESY, Hamburg, Germany). The experiments were curried out as described in the experimental section of
Chapter 2.
X-ray Photoelectron Spectroscopy (XPS) and contact angle measurements. XPS and
contact angle measurements were performed in the group of Prof. dr. ir. Henk J. Busscher (Department of
Biomedical Engineering, University Medical Center Groningen and University of Groningen, The Netherlands).
XPS was performed using an S-Probe spectrometer (Surface Science Instruments, Mountain View, CA, USA),
equipped with an aluminium anode (10 kV, 22 mA) and a quartz monochromator, as previously described.18
Elemental surface compositions were expressed as a ratio of the percentage element over the percentage carbon.
Contact angles were measured on glass slides by the sessile drop technique. The advancing and receding contact
142
A Pilot Study towards the Preparation of Antimicrobial Coated Surfaces
angles were obtained by placing the needle in the water droplet (1-1.5 µl) and carefully moving the sample until
the advancing angle appeared to be maximal. All contact angles were calculated from droplet profiles determined
with a home-made contour monitor. Each contact angle is the mean of three drops.
143
Chapter 6
6.6 References and Notes
1
a) Izumiya, N.; Kato, T.; Aoyagi, H.; Waki, M.; Kondo, M. Synthetic aspects of biologically active cyclic
peptides-gramicidin S and tyrocidines; Halstead (Wiley), New York, 1979. b) Prenner, E. J.; Lewis, R. N. A. H.;
McElhaney, R. N. Biochim. Biophys. Acta 1999, 1462, 201-221.
2
Prenner, E. J.; Lewis, R. N. A. H.; McElhaney, R. N. Biochim. Biophys. Acta 1999, 1462, 201–221.
3
Kiricsi, M.; Prenner, E. J.; Jelokhani-Niaraki, M.; Lewis, R. N. A. H.; Hodges, R. S.; McElhaney, R. N. Eur. J.
Biochem. 2002, 269, 5911-5920.
4
Azidoproline analogue of GS (1) was kindly provided by Gijsbert M. Grotenbreg (Biosyn group, Leiden
University, The Netherlands). For the synthetic approach see Grotenbreg, G. M.; Christina, A. E.; Buizet, A. E.
M.; van der Marel, G. A.; Overkleeft, H. S.; Overhand, M. Org. Biomol. Chem. 2005, 3, 233-238.
5
For the synthesis of compound 2 see Chapter 5.
6
Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41, 2596-2599.
7
a) Schmidt, G. M. J.; Hodgkin, D. C.; Oughton, B. M. Biochem. J. 1957, 65, 744-756. b) De Santis, P.; Liquori,
A. M. Biopolymers 1971, 10, 699-710.
8
Hull, S. E.; Karlsson, R.; Main, P.; Woolfson, M. M.; Dodson, E. J. Nature, 1978, 275, 206-207.
9
Tishchenko, G. N.; Andrianov, V. I.; Vainstein, B. K.; Woolfson, M. M.; Dodson, E. Acta Crystallog. 1997,
D53, 151-159.
10
Grotenbreg, G. M.; Timmer, M. S. M.; Llamas-Saiz, A. L.; Verdoes, M.; van der Marel, G. A.; van Raaij, M.
J.; Overkleeft, H. S.; Overhand, M. J. Am. Chem. Soc. 2004, 126, 3444-3446.
11
Preliminary Langmuir monolayer studies were performed in order to carry out GIXD experiments. However,
due to a low number of repeated experiments, the value of limiting molecular area could be affected by error.
Therefore, surface pressure - surface area (π-A) isotherms were described only taking into account π values.
12
Klostermeyer, H. Chem. Ber. 1968, 101, 2823-2831.
13
In crystalline β-sheet structures, the dimensions of the unit cell have been estimated, taking into consideration
repeat distances of 4.7 and 6.9 Å and the number of the amino acid residues. See Rapaport, H.; Kjaer, K.; Jensen,
T. R.; Leiserowitz, L.; Tirrell, D. A. J. Am. Chem. Soc. 2000, 122, 12523-12529.
14
For details about the Langmuir-Schaefer and Langmuir-Blodgett transfers see Chapter 3 (experimental
section).
15
The bacterial adhesion test needs to be performed under a continuous flow of bacterial solution. Therefore,
stability of the transferred monolayer under these conditions is needed for a correct evaluation of the results.
16
Characteristic peak of DOPE, attributed to the loss of the peptide-N-succinyl-phosphoethanolamine part. See
a) Gross, M. L. Mass spectrometry in the biological sciences: a tutorial 1992, Kluwer Academic Publishers,
Dordrecht, p.430; b) Wood, G. W.; Tremblay, P. A.; Kates, M. Biomed. Mass Spectrom. 1980, 7, 11-12.
17
For the conditions used for HPLC and LC-MS see general material and methods in Charter 2.
18
Everaert, E. P.; van der Mei, H. C.; de Vries, J.; Busscher, H. J. J. Adhes. Sci. Technol. 1995, 9, 1263-1278.
144