Evaluation of the antibacterial and antibiofilm activities of novel CRAMP-vancomycin conjugates with diverse linkers

Article abstract of DOI:10.1039/c5ob00830a

We report the design, synthesis and antibacterial activity analysis of conjugates of vancomycin and cathelicidin-related antimicrobial peptides (CRAMP). Vancomycin inhibits the nascent peptidoglycan synthesis and is highly active against Gram-positive bacteria, whereas Gram-negative bacteria are generally insensitive due to a protective outer membrane. CRAMP is known to translocate across the Gram-negative outer membrane by a self-promoted uptake mechanism. Vancomycin-CRAMP conjugates were synthesized using click chemistry with diverse hydrophilic and hydrophobic linkers, with CRAMP functioning as a carrier peptide for the transfer of vancomycin through the outer membrane. Small hydrophobic linkers with an aromatic group result in the most active conjugates against planktonic Gram-negative bacteria, while maintaining the high activity of vancomycin against Gram-positive bacteria. These conjugates thus show a broad-spectrum activity, which is absent in CRAMP or vancomycin alone, and which is strongly improved compared to an equimolar mixture of CRAMP and vancomycin. In addition, these conjugates also show a strong inhibitory activity against S. Typhimurium biofilm formation.

Full text of DOI:10.1039/c5ob00830a

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Evaluation of the antibacterial and antibiofilm activity of novel
CRAMP-vancomycin conjugates with diverse linkers†
Nigam M. Mishra,a Yves Briers,b Chris Lamberigts,b Hans Steenackers,c Stijn Robijns,c Bart
Landuyt,d Jos Vanderleyden,c Liliane Schoofs,d Rob Lavigne,b Walter Luytene and Erik V. Van der
Eycken*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
We report the design, synthesis and antibacterial activity analysis of conjugates of vancomycin and cathelicidin-related
antimicrobial peptides (CRAMP). Vancomycin inhibits the nascent peptidoglycan synthesis and is highly active against
Gram-positive bacteria, whereas Gram-negative bacteria are generally insensitive due to a protective outer membrane.
CRAMP is known to translocate across the Gram-negative outer membrane by a self-promoted uptake mechanism.
Vancomycin-CRAMP conjugates were synthesized using click chemistry with diverse hydrophilic and hydrophobic linkers,
with CRAMP functioning as a carrier peptide for the transfer of vancomycin through the outer membrane. Small
hydrophobic linkers with an aromatic group result in the most active conjugates against planktonic Gram-negative
bacteria, while maintaining the high activity of vancomycin against Gram-positive bacteria. These conjugates thus show a
broad-spectrum activity, which is absent in CRAMP or vancomycin alone, and which is strongly improved compared to an
equimolar mixture of CRAMP and vancomycin. In addition, these conjugates also show a strong inhibitory activity against
Salmonella Typhimurium biofilm formation.
drug discovery. However, the diversity of natural products that
is nowadays explored is much larger than ever. Antimicrobial
peptides (AMPs) represent such a new source of potent,
Introduction
The increase and spread of antibiotic resistance is a major
challenge for the healthcare at a global scale. The massive use
of antibiotics, often in an inadequate and inappropriate way,
has led to an increasing incidence of multidrug-resistant
bacteria both in hospitals and the community. Simultaneously,
the discovery of new classes of antibiotics drastically
diminished after the so-called golden age of antibiotic
discovery during the 1960s. Since 1987, no novel classes of
antibiotics have been reported.1 To meet the societal need for
new antibiotics, there is a global call to policymakers to install
a new regulatory framework with incentives to reinvigorate
antibiotic development in an economically viable way.2
broad-spectrum antibacterials. They are isolated from
throughout the animal and plant kingdom. Animals and plants
protect themselves against pathogenic microorganisms such as
bacteria, fungi, viruses and protozoa to which they are
continuously exposed. They are generally protected from
infections by these microbes by a variety of responses
produced by their innate immune system, which includes the
production and release of AMPs.3 During the past decades, a
large number of naturally occurring AMPs (also called host
defense peptides) have been isolated. Their abundance, tissue
distribution and in vitro activity confirm an important role in
innate immunity and host defense.4-6 The peptides do not only
possess the ability to directly kill invaders, but also stimulate
effector molecules of the host immune system.7
Natural products have been a pivotal source for many
successful antibiotics and are currently revisited in antibiotic
Cathelicidins are an important class of AMPs. They have been
identified in several species ranging from fish, amphibians,
reptiles to mammals.7-9 Members of the cathelicidin family are
distinguished by a highly conserved N-terminal cathelicidin
a.Laboratory for Organic & Microwave-Assisted Chemistry (LOMAC), Department of
Chemistry, KU Leuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium. E-mail:
Erik.VanderEycken@chem.kuleuven.be
b.Laboratory of Gene Technology, Department of Biosystems, KU Leuven,
Kasteelpark Arenberg 21 box 2462, B-3001 Leuven, Belgium.
c.Centre of Microbial and Plant Genetics (CMPG), Department of Microbial and
Molecular Systems, KU Leuven, Kasteelpark Arenberg 20, Box 2460, B-3001
Leuven, Belgium.
domain and
a
more variable C-terminal cathelicidin
antimicrobial peptide domain.7,8,10,11 In humans and mice only
one cathelicidin is expressed, hCAP18/LL-37 and cathelicidin-
related antimicrobial peptide (CRAMP), respectively. They act
rapidly on Gram-positive and Gram-negative bacteria and thus
d.Department of Biology, Animal Physiology and Neurobiology Section, KU Leuven,
Naamsestraat 59 - box 2465, 3000 Leuven, Belgium.
e.Department of Pharmaceutical and Pharmacological Sciences, KU Leuven,
Naamsestraat 59 - box 2469, KU Leuven, 3000, Leuven, Belgium.
†Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
contribute to the protection against
a wide variety of
pathogens. In Gram-negative bacteria, the positively charged
cathelicidins interact with the lipopolysaccharide (LPS) cation
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binding sites with high affinity, leading to the competitive of antibacterial and antibiofilm activity. We demonstrate that
displacement of the stabilizing divalent cations. The outer specific CRAMP-vancomycin conjugates using short
membrane is disrupted and cathelicidins can pass the outer hydrophobic linkers with an aromatic group have an enhanced
membrane in
a so-called self-promoted uptake process. antibacterial activity against Gram-negative bacteria compared
Interaction with the negatively charged cytoplasmic to CRAMP or vancomycin alone, or a 1:1 mixture of them. In
membrane leads to a transition of the cathelicidins from an addition, they show good antibacterial activity against Gram-
unstructured to a structured form. They aggregate in clusters positive species and prevent S. Typhimurium biofilm
that perforate the cytoplasmic membrane. The membrane formation.
integrity is disrupted, resulting in
a
rapid cell death.12
Generally, AMPs that target the cytoplasmic membrane are
less prone to resistance development given the relatively
immutable nature of the membrane.13
In this study, we covalently link mouse CRAMP to vancomycin.
Vancomycin is a glycopeptide and it is used for the treatment
of Gram-positive bacterial infections, including methicillin-
resistant Staphylococcus aureus (MRSA), multiple resistant
enterococci and Clostridium difficile infections.14 Vancomycin
binds to the C-terminal D-Ala-D-Ala of the pentapeptide of
lipid II and the nascent peptidoglycan, inhibiting both
Figure 1: CRAMP vancomycin conjugate covalently linked via various linkers
Results and discussion
transpeptidation
and
transglycosylation
during
the
A mixture of CRAMP and vancomycin does not inhibit
bacterial growth better than its separate compounds. A set of
four Gram-positive (Bacillus cereus, Bacillus subtilis,
Micrococcus luteus, Staphylococcus aureus) and four Gram-
negative bacterial species (Escherichia coli, Pseudomonas
putida, Salmonella Typhimurium, Yersinia enterocolitica) were
selected to screen for inhibition of bacterial growth. We
initially analyzed the minimum inhibitory concentration (MIC)
for CRAMP, vancomycin and an equimolar (1:1) mixture of
CRAMP and vancomycin (Table 3). Generally, we observed that
the MIC of the mixture was equal to the MIC of vancomycin for
Gram-positive species and equal to the MIC of CRAMP for
Gram-negative species. This was expected given the
ineffectiveness of vancomycin against Gram-negative bacteria
and the higher sensitivity of Gram-positive bacteria for
vancomycin than for CRAMP. Only for S. Typhimurium and Y.
enterocolitica, a four-fold lower MIC was observed for the
mixture in comparison to CRAMP alone, which might indicate a
potential synergy when both molecules are added as a
mixture.
peptidoglycan synthesis. Vancomycin is only active against
Gram-positive bacteria as it cannot penetrate the Gram-
negative outer membrane given its large size.15 We reasoned
that a direct coupling of CRAMP and vancomycin may improve
antibacterial activity against Gram-negative species if the
CRAMP moiety could act as a carrier molecule for the
transport of vancomycin through the outer membrane. After
passage across the outer membrane, both the vancomycin and
CRAMP moiety may exert their specific antibacterial action.
AMPs often have only a moderate affinity for the target
membrane. Since vancomycin has a high affinity for lipid II,
which is embedded in the cytoplasmic membrane, vancomycin
is hypothesized to act as a high-affinity targeting molecule for
CRAMP towards the cytoplasmic membrane, again resulting in
an increased antibacterial effect against Gram-negative
bacteria. Similarly, the targeting effect of vancomycin could
also contribute to an improved activity against Gram-positive
species. The targeting principle has been shown for conjugates
of Magainin-2 and its truncated version with vancomycin. The
conjugates showed enhanced membrane permeabilizing
activity against large unilamellar vesicles with embedded lipid
II molecules. Also the antibacterial activity of conjugates
against Gram-positive vancomycin-resistant enterococci was
improved.16 In another report, the antimicrobial peptides
anoplin and temporin L were conjugated via click chemistry to
vancomycin. The vancomycin targeting function led to an
enhanced membrane disruption of large unilamellar vesicles
(LUVs) for anoplin but not for temporin L.13 An additional
argument for our approach is that the combination of two
active molecules in a single molecule is always preferred over
mixtures, both from the clinical and regulatory perspective.
Specifically, CRAMP (H-KIGEKLKKIGQKIKNFFQKLVPQPEQ-NH2)
was conjugated with vancomycin using various aliphatic and
aromatic linkers of different length with minimal steric bulk of
the linker group, and without affecting the binding affinity of
CRAMP and vancomycin (Figure 1 and Table 2). In total, 23
CRAMP-vancomycin conjugates were synthesized for analysis
A short hydrophobic linker to conjugate vancomycin and
CRAMP shows most promise. In our approach, CRAMP was
initially linked to vancomycin using a diamino 4,7,10-Trioxa-
1,13-tridecanediamine derivative based linker 10a (Table 2).
This linker was selected to enhance the solubility of the final
compound. Synthesis of CRAMP was carried out by applying
the strategy of solid phase peptide synthesis (SPPS) using rink
amide resin. Subsequently, the conjugate was synthesized
using the highly efficient and widely used CuAAC click
chemistry, which is a copper-mediated coupling between an
18
azide and an alkyne.17, We firstly coupled 4-azidobutanoic
acid
3 on the N-terminus of CRAMP which was on the solid
support (Scheme 1). Simultaneously, the C-terminally modified
vancomycin-linker-alkyne derivative 11a (Scheme 1, Table 2)
was prepared by standard solution-phase synthesis using
EDC/HOAt as coupling reagents and linker 10a and was
purified using preparative HPLC. To couple this vancomycin-
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TEG linker 11a with CRAMP-butyl azide derivative
5 via click (Table 3). It was found that 12a showed inhibitory activity
reaction, different conditions were tested (Table 1). Firstly, against all species tested, however, 12a was comparatively less
conjugation was evaluated by dissolving the functionalized active than vancomycin in case of Gram-positive species and
precursors in an aqueous solution (DMF/H2O; 1:9) with less or equally active than CRAMP in case of Gram-negative
addition of sodium ascorbate and CuSO4·5H2O and under bacteria (Table 3). Therefore it was hypothesized that the
microwave irradiation. This approach turned out to be rather length and/or the nature of the TEG linker was not optimal for
sluggish and isolation of the product was not possible due to proper interaction of vancomycin and CRAMP to their
very small amount of product formation (entry 1, Table 1). In respective targets. Therefore, shorter linkers (10b and 10c
addition, it was noted that the use of tris [(1-benzyl-1H-1,2,3- entry 2 & 3; Table 2) and a longer linker 10d (Table 2, entry 4;
triazol-4-yl)methyl]amine (TBTA) ligand along with for synthesis refer to the SI, Scheme 3) and the corresponding
,
CuSO4·5H2O/sodium ascorbate did not significantly improve CRAMP-linker-vancomycin conjugates 12b-d were synthesized
the outcome of the reaction (entry 2, Table 1). Several following the same strategy as for CRAMP-vancomycin
conditions were evaluated unsuccessfully, employing various conjugate 12a. However, this resulted again in two major
copper sources like Cu(OAc)2 (entry 3, Table 1), CuI (entry 4, peaks with similar mass in the LC-MS spectrum. We could
Table 1) and Cu(ACN)4PF6 (entry 5, Table 1). Cu(OAc)2 and isolate these two major peaks (annotated as 12b-d and 12b’-
Cu(ACN)4PF6 made the reaction mixture sluggish with d’, respectively) in all three cases by preparative HPLC. These
unreacted starting materials and a very small amount of isolated products 12b-d and 12b’-d’ were tested against the
desired product, along with other unidentified compounds. same bacterial species. Compound 12b’ showed the highest
Ruthenium catalyzed click condition was also tested but inhibitory activity, which was improved compared to 12a
,
resulted in the decomposition of starting materials (Entry 7, specifically for B. subtilis (four-fold), M. luteus (eight-fold), E.
Table 1). Using CuI, iodination of vancomycin linker was coli (four-fold) and Y. enterocolitica (four-fold). For Gram-
observed, giving rise to the formation of iodinated CRAMP- positive bacteria, 12b’ showed comparable activity as
vancomycin conjugate along with desired product 12a in a very vancomycin (except for B. cereus with a four-fold higher MIC
small amount contaminated with impurities although exact for 12b’); for Gram-negative bacteria, 12b’ performs equally or
position of iodination could not be defined. Finally, optimal better (Y. enterocolitica) than CRAMP alone. Compounds with
conditions were obtained with CuSO4·5H2O and sodium linker 10c and especially 10d showed less inhibitory activity
ascorbate at 40 oC in DMF/H2O (1:9) for 20h (entry 6, Table 1). compared to the compound with linker 10a (Table 3). From
However, LC-MS and HPLC analysis surprisingly showed two these results, it was concluded that short and hydrophobic
major peaks of the same mass, which could not be separated.
The obtained CRAMP-vancomycin conjugate 12a was tested
against the Gram-positive and Gram negative bacterial species
linkers such as 10b may have the highest potential.
Scheme 1. General scheme for the synthesis of the target molecule (the carboxyl group attached to vancomycin is part of vancomycin itself i.e. the C-terminus of its heptapeptide
backbone)
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Table 1. Optimization of CuAAC reaction
Entry
Catalyst (ligand)
CuSO4·5H2O
CuSO4·5H2O, (TBTA)
Cu(OAc)2
Base
Sodium ascorbate
Sodium ascorbate
Sodium ascorbate
DIPEA
Solvent
DMF/H2O (1:9)
DMF/H2O (1:9)
H2O/t-BuOH (1:1)
DMF
Temp
70oC (MW)
70oC (MW)
40oC
Time
0.5h
0.5h
30h
24h
20h
20h
24h
LC-MS Conversion 12a (%)
1
2
3
4
5
6
7
5
5
10
10
40
63
0
CuI
40oC
Cu(ACN)4PF6
CuSO4·5H2O
CpRu(PPh3)2Cl2
--
MeOH
rt
Sodium ascorbate
--
DMF/H2O (1:9)
DMF
40oC
40oC
Table 2. Structure of linkers used for the synthesis of CRAMP-vancomycin conjugates
Entry
1
Product
Vancomycin linker
Final product
10a
11a
12a
2
3
10b
10c
11b
12b & 12b′
11c
11d
12c & 12c′
12d & 12d′
4
5
10d
10e
O
11e
11f
11g
11h
11i
12e & 12e′
12f & 12f′
12g & 12g′
12h & 12h′
12i & 12i′
HN
N
H
6
7
8
9
10f
10g
10h
10i
O
HN
N
H
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10
10j
11j
12j & 12j′
11
10k
11k
12k & 12k′
12l& 12l′
12
10l
11l
13
14
10m
10n
11m
11n
12m & 12m′
12n & 12n′
15
16
10o
10p
11o
11p
12o & 12o′
12p & 12p′
17
10q
11q
12q & 12q′
18
19
10r
10s
11r
11s
12r & 12r′
12s & 12s′
20
21
10t
11t
12t & 12t′
12u & 12u′
10u
11u
HN
O
HN
22
23
10v
11v
12v & 12v′
10w
11w
12w & 12w′
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Table 3. Antibacterial activity of CRAMP-vancomycin conjugates
MIC (µM)
E coli XL-1
Gram-positive species
Gram-negative species
B. cereus
B. subtilis
M. luteus
S. aureus
ATCC6538
> 100
≤ 0.75
≤ 0.75
nd
P. putida
G1
S. Typhimurium
LT2
Y. enterocolitica
Entry
Product
LMG 9610
PSB3
25
ATCC4689
50
1.5
1.5
12.5
1.5
1.5
25
12.5
50
50
1.5
3
Blue MRF’
ATCC9610
50
1
CRAMP
V
25
1.5
1.5
6
3
100
3
12.5
100
12.5
12.5
25
100
2
1.5
1.5
6
100
100
12.5
50
3
C+V
12a
12b
12b’
12c
25
4
12.5
6
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
100
5
3
6
nd
25
6
6
1.5
25
nd
3
12.5
25
12.5
25
7
3
nd
12.5
6
8
12c’
12d
12d’
12e
12e’
12f
3
12.5
50
nd
25
12.5
100
50
9
6
nd
25
25
nd
6
100
50
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
25
6
25
nd
6
nd
>100
25
>100
12.5
100
6
1.5
6
12.5
6
nd
1.5
3
nd
nd
6
50
12f’
12g
12g’
12h
12h’
12i
1.5
6
12.5
6
nd
12.5
100
12.5
>100
12.5
>100
12.5
100
25
1.5
3
nd
nd
6
>100
100
100
6
3
6
nd
12.5
1.5
6
12.5
6
3
nd
nd
6
>100
>100
>100
>100
>100
>100
>100
50
>100
6
3
nd
6
1.5
3
nd
nd
3
100
6
12i’
12j
1.5
6
6
nd
3
1.5
3
nd
nd
6
100
6
12j’
12k
12k’
12l
1.5
6
6
nd
3
1.5
3
nd
nd
3
>100
12.5
>100
12.5
>100
12.5
>100
6
>100
6
1.5
6
3
nd
3
3
nd
nd
6
100
>100
12.5
>100
12.5
100
3
12l’
12m
12m’
12n
12n’
12o
12o’
12p
12p’
12q
12q’
12r
3
12.5
3
3
nd
100
6
3
nd
nd
6
>100
100
3
12.5
3
6
nd
6
3
nd
nd
3
>100
100
1.5
3
6
3
nd
3
1.5
3
nd
nd
6
100
12.5
>100
>100
100
>100
>100
25
>100
100
50
3
6
nd
6
6
3
3
nd
nd
6
>100
>100
>100
>100
>100
>100
>100
>100
1.5
>100
6
1.5
6
12.5
3
3
nd
3
nd
nd
6
100
12.5
100
12.5
>100
12.5
1.5
6
6
12.5
6
3
nd
6
1.5
3
nd
nd
6
12r’
12s
3
12.5
3
nd
6
3
nd
nd
12.5
3
>100
>100
3
12s’
12t
6
25
6
nd
1.5
3
≤ 0.75
1.5
1.5
≤ 0.75
≤ 0.75
1.5
≤ 0.75
≤ 0.75
nd
nd
nd
nd
nd
nd
nd
nd
6
12t’
12u
12u’
12v
12v’
12w
12w’
12.5
25
6
6
6
6
6
6
3
6
3
3
6
12.5
6
6
6
3
6
3
3
3
3
6
6
6
1.5
6
1.5
1.5
3
1.5
3
6
≤ 0.75
1.5
3
6
3
3
MIC (µM): lowest concentration of an antibacterial agent that completely inhibits growth of the microorganism as detected by the unaided eye; (nd): bioactivity not
determined; V: vancomycin; C+V: mixture of CRAMP and vancomycin in a 1:1 ratio.; in case of the equimolar mixture (C+V), the concentration represents the
concentration of the individual compounds
Conjugates with short linkers comprising an aromatic group Gram-positive species
.
Based on these findings, we
show broad inhibitory activity against Gram-negative and synthesized nine shorter hydrophobic linkers 10e-m, and five
6 | J. Name., 2012, 00, 1-3
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Biofilm inhibition (IC50 in µM)
aromatic linkers; 10n and 10t-w (Table 2), according to the
reported procedures18-20 using shorter commercially available
building blocks (refer to the SI: Table 2 & 3; Scheme 2, 5 & 6)
and utilized five commercially available shorter linkers 10o-s
(Table 4, refer to the SI). Subsequently, these linkers were
coupled to vancomycin via its C-terminus to obtain
vancomycin-linker conjugates 11e-w (Table 2), resulting in
CRAMP-vancomycin conjugates 12e-w and 12e’-w’ via CuAAC.
This new series of CRAMP-vancomycin conjugates was
analyzed for inhibitory activity on the panel of species. From
Entry
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
Product
12d’
12e
12e’
12f
12f’
12g
12g’
12h
12h’
12i
12i’
12j
12j’
12k
12k’
12l
12l’
12m
12m’
12n
12n’
12o
12o’
12p
12p’
12q
12q’
12r
12r’
12s
12s’
12t
12t’
12u
12u’
12v
12v’
12w
12w’
IC50
3.38
0.33
3.46
1.29
1.38
0.76
1.79
2.18
1.10
1.01
1.13
0.62
2.00
2.34
1.43
0.71
0.99
~1.56
2.18
1.27
1.42
0.35
0.88
1.71
2.53
0.41
1.57
1.10
1.41
0.38
~6.7
1.56
5.00
8.64
10.77
1.88
7.27
0.91
1.60
95% confidence interval
2.44 to 4.68
0.19 to 0.59
2.14 to 5.59
0.87 to 1.90
1.07 to 1.78
0.28 to 2.10
1.24 to 2.59
0.79 to 6.03
0.62 to 1.96
0.69 to 1.48
0.77 to 1.66
0.45 to 0.86
1.57 to 2.46
1.55 to 3.53
1.16 to 1.76
0.43 to 1.20
0.57 to 1.71
nd
the entire series conjugates with aromatic linkers (12t-w
/12t’-
w’ except 12n/12n’) showed the strongest inhibitory activity
against Gram-negative strains, which was stronger than the
mixture of individual components. This improvement was most
prominent for S. Typhimurium (up to 32-fold for 12w in
comparison to the mixture and 128-fold compared to
vancomycin or CRAMP alone), followed by Y. enterocolitica (up
to 8-fold for 12t in comparison to the mixture, and 16-32-fold
compared to CRAMP and vancomycin alone, respectively). In
case of Gram-positive species, the aromatic conjugates
outperformed vancomycin against B. subtilis, but there was no
conjugate with improved activity against all Gram-positive
species tested compared to vancomycin. Nevertheless, the
aromatic conjugates represent a new class of antibacterial
compounds that have broad activity against Gram-negative
and Gram-positive species (MIC < 10 µM), which is neither the
case for CRAMP or vancomycin alone nor for the equimolar
mixture of both individual molecules.
0.76 to 6.24
0.83 to 1.96
1.02 to 1.97
0.24 to 0.53
0.56 to 1.40
0.72 to 4.05
1.33 to 4.81
0.30 to 0.56
0.90 to 2.70
0.58 to 2.07
0.40 to 5.00
0.18 to 0.79
nd
1.30 to 1.85
3.43 to 7.28
5.97 to 12.50
5.97 to 12.50
1.25 to 2.84
4.47 to 11.84
0.58 to 1.45
1.34 to 1.92
CRAMP-vancomycin conjugation effectively prevents S.
Typhimurium biofilm formation. A large majority of bacterial
infections is biofilm-related.21 Therefore we evaluated the
prevention of S. Typhimurium biofilm formation for the
complete set of CRAMP-vancomycin conjugates. Inhibition is
expressed as IC50, which corresponds to the concentration of
the compound that is needed to inhibit biofilm formation by
50%. In addition, a 95% confidence interval is calculated (Table
4). CRAMP or vancomycin alone have high IC50 values, however
a mixture of CRAMP and vancomycin appears to be even more
efficient (IC50 = 0.36 µM) in biofilm prevention. This result is
consistent with lower synergy at the 1:1 ratio of CRAMP and
IC50 in (µM): concentration of inhibitor needed to inhibit biofilm formation by
50%; (nd): not determined; V: vancomycin; C+V: mixture of CRAMP and
vancomycin in 1:1 ratio; ~: approximately.
Structural differences between 12 and 12’ compounds
related to isomerization of CRAMP affect inhibitory activity
against Gram-negative species
.
When comparing the
vancomycin, since the Interaction Index
I < 1 (I =
inhibitory activity of the two compounds 12 and 12’ against
planktonic bacteria, a high variability among the species tested
is observed. For Gram-negative species, 12’ is often more
active (up to 8-16-fold) than 12, whereas for Gram-positive
species 12’ is mostly slightly less active against B. subtilis and
M. luteus. We tried to identify the structural differences
between the two compounds 12 and 12’ obtained after CuAAC
reaction. As this reaction was performed using Cu, which is
known to give rise to the 1,4 triazole product selectively, there
was a negligible chance for the formation of the 1,5-
IC50C+V/IC50CRAMP + IC50C+V /IC50V = 0.57).22 Although most
conjugates of CRAMP and vancomycin have slightly higher IC50
values than the mixture, the results indicate that they still
show potent biofilm inhibitory activity.23
Table 4. Influence of CRAMP-vancomycin conjugates 12a-w and 12a’-w’ on the
prevention of biofilm formation of Salmonella Typhimurium (expressed as IC50 in µM)
Biofilm inhibition (IC50 in µM)
Entry
Product
CRAMP
V
C+V
12a
12b
12b’
12c
12c’
12d
IC50
95% confidence interval
0.75 to 1.06
1
2
3
4
5
6
7
8
9
0.88
2.29
0.36
1.41
1.86
0.64
1.86
1.97
1.32
1
cycloadduct. It was indeed confirmed by H NMR that the 1,4-
1.87 to 2.80
0.32 to 0.40
1.06 to 1.87
1.23 to 2.78
0.25 to 1.65
1.52 to 2.25
1.33 to 2.93
0.95 to 1.83
triazole was formed in both cases. Therefore it was considered
that some isomerisation took place under CuAAC reaction
conditions. To verify if isomerisation is occurring in the peptide
or in vancomycin part, they were treated separately with
CuSO4·5H2O (3.0 equiv) and sodium ascorbate (6.0 equiv) in
H2O/DMF (4.5:0.5) upon stirring for 20h at 40°C. The reactions
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were monitored by mass and LC-MS. The mass spectrum did in the ɑ-C position followed by gas chromatographic
not show any changes, while in the LC-MS chromatogram of separation of the enantiomers of the peptide via GC-MS (Table
the reaction with CRAMP-butyl azide
5 (Figure 2 and 3) a new 7, refer to the SI). No enantiomeric formation was observed in
peak was observed with a retention time of 28.18 min with amino acids, indicating that the observation of the two peaks
similar intensity. The reaction with the vancomycin-linker 11 could not be explained in this way.
did not show any change in the LC-MS chromatogram. Hence,
it was clear that some changes were occurring in CRAMP-butyl Isomerization of CRAMP is caused by exposure to sulphate
azide
5
. The same observation was made when only CRAMP
2
anions. Trying to avoid the problem, the amount of copper
was treated. Apparently only CRAMP was interacting with sulphate was reduced. This led to incomplete conversion. To
CuSO4·5H2O and/or sodium ascorbate, leading to the determine the amount of copper sulphate needed for the
formation of isomerised compound with the same mass.
generation of the side compound, a set of reactions was
carried out starting with 0.1 to 2.0 equiv of CuSO4·5H2O. It was
observed that even 0.1 equiv of CuSO4·5H2O was sufficient for
the generation of the second peak in the chromatogram
although to a lesser extent. To investigate the role of the
oxidation state of copper, CRAMP was treated with
CuSO4·5H2O in the absence of sodium ascorbate. However, in
this case the side compound was also generated, indicating
that the oxidation state of the copper ion does not play any
role in this transformation of the peptide. To further confirm
this, CuI was used as a copper source instead of CuSO4·5H2O.
Surprisingly, in this case there was no generation of second
peak-compound in the LC-MS chromatogram of CRAMP. Also,
increasing the amount of CuI from 2.0 to 4.0 equiv did not
cause a second peak. Similar results were obtained with CuCl
and CuCl2. Hence, it was proven that the oxidation state of
copper does not play any role in the generation of the second
peak-compound. It was also confirmed that not all copper
sources lead to the generation of the double peak compound
as chloride and iodide salts did not cause any change.
Apparently, only CuSO4·5H2O was causing this problem.
According to the literature, anions (especially divalent anions)
might interact with peptides.24 They could bind to the
positively charged residue of the peptide and cause either
Figure 2. LC-MS chromatogram of CRAMP-butyl azide 5 before reaction with
CuSO4·5H2O and sodium ascorbate (retention time 26.19 min, method 0-60% ACN/H2O
in 40 min)
Figure 3. LC-MS chromatogram of CRAMP-butyl azide 5 after reaction with CuSO4·5H2O
and sodium ascorbate (a new peak was observed at a retention time of 28.18 min,
method 0-60% ACN/H2O in 40 min)
Isomerisation is not due to helicity distortion or racemization.
To our knowledge, this is the first report of the generation of
two isomers of a peptide under CuAAC reaction conditions.
One obvious explanation could be distortion of the helicity of
the peptide due to the interaction with CuSO4·5H2O. To
confirm this, both compounds corresponding with the two
peaks in the LC-MS chromatogram of CRAMP were isolated by
preparative HPLC, and a circular dichroism (CD) spectrum was
recorded along with this of pure CRAMP (Figure 3, SI).
According to the CD spectrum, both compounds were found to
be α-helical in TFE (trifluoroethanol) and random coiled in pure
water hence excluding the helicity distortion hypothesis
(Figure 3 & 4, SI). An alternative explanation could be
attributed to the racemization of the amino acids of the
peptide to some extent, resulting in the formation of
diastereoisomers. To confirm this, the enantiomeric purity of
the each amino acid in both the isolated peptides
(corresponding with the two peaks in the LC-MS
2-
aggregation or nanostructure formation.24 The mass of SO4
was observed by LC-MS in one of the isolated peaks of CRAMP
after reaction with CuSO4·5H2O. X-ray fluorescence (XRF)
2-
analysis suggested that SO4 is trapped in the isolated
compounds of CRAMP (probably at the positively charged
lysine residues) after treatment with CuSO4·5H2O (Table 5).
However, the exact position of the binding site could not be
defined. CRAMP was also treated with another sulphate
source, i.e. Na2SO4, and as expected the two peaks appeared
again in the LC-MS chromatogram. Notably, when these two
compounds were isolated by preparative HPLC and treated
again with CuSO4·5H2O to analyze if further conversions into
two peaks occur, this was indeed observed, although, the rate
of conversion was now rather slow. To investigate the
possibility of nanofibre formation or aggregation of CRAMP in
the presence of sulphate anions, atomic force microscopy
(AFM) measurements were performed (Figure 4). Although
there were some differences in both images, nanofibre
formation or aggregation of the peptide could not be
confirmed unambiguously. Therefore, at this stage, we have
come to the conclusion that the sulphate anion binds to the
positively charged amino acid residue(s), changing the
conformation of the peptide.
chromatogram
of
CRAMP
upon
was
treatment
analyzed
with
by
CuSO4·5H2O/sodium
ascorbate)
determination of the optical purity of each amino acid of the
sequence by hydrolyzing in a 6N HCl solution. This hydrolysis
caused the suitable derivatisation of the free amino acids
whereby racemisation is accomplished by deuterium exchange
8 | J. Name., 2012, 00, 1-3
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ARTICLE
the Promotion of Innovation through Science and Technology
in Flanders under grant IWT-SBO 120050 (NEMOA) and
through a PhD fellowship to S.R., the European Commission's
Seventh Framework Programme (FP7/2007(2013) under the
grant agreement COATIM (Project no. 278425), the KU Leuven
(IDO/11/008) and the F.W.O. (Fund for Scientific Research –
Flanders (Belgium)) through a postdoctoral fellowship to H.S.
The authors thank Wout Frederickx and Prof. Steven De Feyter
for AFM measurements, Koen Nuyts for helping in CD analysis,
Dr. Claudia Cavalluzzo for scientific discussions and theoretical
inputs, Prometa, KU leuven for MALDI-TOF measurements and
Dirk Henot for XRF analysis.
Figure 4a
Figure 4b
Figure 4. AFM-image of CRAMP in the absence of CuSO4·5H2O (Figure 4a) and in the
presence of CuSO4·5H2O (Figure 4b)
Notes and references
Table 5: Percentage of Cu and S present in CRAMP samples
Electronic Supplementary Information (ESI) is available, including
detailed experimental procedure and characterization of
compounds etc.
Samples
Amount of Cu
0.006%
Amount of
S
CRAMP pure (before treatment with
CuSO4·5H2O)
0.110%
1
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0.010%
0.009%
0.250%
0.267%
CRAMP peak-2 compound
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We aimed to synthesize a compound with broad-spectrum
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