Combined Approach of Backbone Amide Linking and On-Resin N-Methylation for the Synthesis of Bioactive and Metabolically Stable Peptides

Article abstract of DOI:10.1021/acs.jmedchem.7b01809

Rhabdopeptides are a large class of nonribosomal peptides from the bacteria Xenorhabdus and Photorhabdus with low micromolar activity against different protozoa, which are the causative agents of several tropical diseases. The development of a facile and flexible synthesis combining backbone amide linking with on-resin peralkylation for the synthesis of permethylated rhabdopeptides is described. This strategy allows the fast generation of permethylated naturally occurring and artificial rhabdopeptides for a structure-activity study. Furthermore, in vitro experiments revealed their superior properties regarding their stability and passive membrane diffusion.

Full text of DOI:10.1021/acs.jmedchem.7b01809

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Combined Approach of Backbone Amide Linking and On-Resin N-
Methylation for the Synthesis of Bioactive and Metabolically Stable Peptides
Frank Wesche, Helene Adihou, Astrid Kaiser, Mario Wurglics,
Manfred Schubert-Zsilavecz, Marcel Kaiser, and Helge B. Bode
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01809 • Publication Date (Web): 16 Apr 2018
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Combined Approach of Backbone Amide Linking and On-Resin N-Methylation
for the Synthesis of Bioactive and Metabolically Stable Peptides
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Frank Wesche1, Hélène Adihou1, Astrid Kaiser2, Mario Wurglics2, Manfred Schubertꢀ
Zsilavecz2, Marcel Kaiser3, 4, Helge B. Bode1, 5,*
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1
Molekulare Biotechnologie, Fachbereich Biowissenschaften, Goethe University
Frankfurt, MaxꢀvonꢀLaueꢀStrasse 9, Dꢀ60438 Frankfurt am Main, Germany.
2 Institute of Pharmaceutical Chemistry, GoetheꢀUniversity Frankfurt, MaxꢀvonꢀLaueꢀ
Strasse 9, Dꢀ60438 Frankfurt am Main, Germany.
3 Swiss Tropical and Public Health Institute, Parasite Chemotherapy, Socinstrasse
57, CHꢀ4051 Basel, Switzerland.
4 University of Basel, Petersplatz 1, CHꢀ4003 Basel, Switzerland.
5 Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University
Frankfurt, MaxꢀvonꢀLaueꢀStrasse 15, Dꢀ60438 Frankfurt am Main, Germany.
* Corresponding author: h.bode@bio.uniꢀfrankfurt.de
Abstract
Rhabdopeptides are a large class of nonꢀribosomal peptides from the bacteria
Xenorhabdus and Photorhabdus with low micromolar activity against different
protozoa, which are the causative agents of several tropical diseases. The
development of a facile and flexible synthesis combining backbone amide linking
with onꢀresin peralkylation for the synthesis of permethylated rhabdopeptides is
described. This strategy allows the fast generation of permethylated naturally
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occurring and artificial rhabdopeptides for a structure activity study. Furthermore, in
vitro experiments revealed their superior properties regarding their stability and
passive membrane diffusion.
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Introduction
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To provide and maintain the symbiosis with their host nematodes, the
entomopathogenic bacteria Xenorhabdus and Photorhabdus produce a broad range
of natural products, including small molecular weight molecules and nonꢀribosomal
peptides.1,2 One compound class are the bioactive and chemically diverse
rhabdopeptides (RXPs), which were originally identified in X. nematophila.3
However, RXPs or closely related compounds, like xenortides4 or
mevalagmapeptide,5 occur in many Xenorhabdus and Photorhabdus strains but are
exclusively found in these two genera. Due to their interesting biosynthesis using for
example a combination of iterative and flexible use of monomodular nonꢀribosomal
peptide synthetases, a huge chemical diversity is created in a single bacterial strain.6
RXPs are linear peptides consisting of two to eight amino acids, including valine (V),
leucine (L) and phenylalanine (F), where valine is the most abundant amino acid,
followed by leucine. Furthermore, RXPs always bear a Cꢀterminal amine like
phenylethylamine (PEA), tryptamine (TRA), tyramine (TYA) and agmatine (AGM)
(figure 1). In addition different classes of RXPs can be assigned differing by their
methylation patterns of the amide NH ranging from partially over highly methylated
up to permethylated peptide backbone. Approximately over one quarter of all
identified RXPs contain a permethylated peptide backbone. Therefore, a facile and
flexible chemical synthesis was developed to obtain synthetic and artificial nonꢀ
methylated and permethylated RXPs combining an approach of backbone amide
linking and onꢀresin alkylation, respectively. Herein the synthetic and experimental
details are discussed and the synthesized RXPs were used for bioactivity testing
comprising the analysis of their structureꢀactivity relationship and further in vitro
experiments regarding their pharmacokinetic properties.
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Results and Discussion
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The goal of our work was to develop a fast and reproducible procedure for the
synthesis of highly methylated RXPs. Many methods are described for the
successful incorporation of Nꢀmethylamino acids into peptide backbones.7 Yet, the
synthesis of peptides containing clustered Nꢀmethylamino acids as present in RXPs
is still challenging. Using Nꢀmethylamino acid building blocks or applying Nꢀ
methylation of selective position during the elongation of the peptide chain on solid
phase8 or in solution Nꢀmethyl peptides could be synthesized.9 However, coupling of
an Nꢀmethylated acid component and a Nꢀmethylated terminal amine is challenging
and while for example triphosgene has been described for such coupling reactions10–
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it was not considered for the RXP synthesis due to its handling and safety
problems. Instead we decided to perform a postꢀsynthetic alkylation step onꢀresin
resulting in a permethylated peptide backbone.
Permethylation of peptide backbone on-resin
As previously described for the permethylation of linear and cyclic peptides onꢀresin,
lithiumꢀtertꢀbutoxide (LiOtBu) in THF is the base of choice followed by the treatment
of an alkylating reagent in DMSO14–16 while for complete alkylation still the treatment
has to be repeated several times.14 Therefore, our aim was to optimize these
reaction conditions using different amounts of base (LiOtBu) and alkylation reagent
in single alkylation treatment. As we expected an influence of steric hindrance during
alkylation by the size of the amino acid side chain, we investigated the conditions of
permethylation for a sequence containing four isoleucyl residues (Ile, I), though
isoleucine is not an amino acid naturally occurring in RXPs. A single batch of 400
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ꢁmol of BocꢀIleꢀIleꢀIleꢀIleꢀ2ꢀchlorotrityl chloride polystyrene resin (2CTCꢀPS, 1) was
synthesized under standard Fmocꢀconditions in a peptide synthesizer using Oꢀ(1Hꢀ6ꢀ
chlorobenzotriazoleꢀ1ꢀyl)ꢀ1,1,3,3ꢀtetramethyluronium hexafluorophosphate (HCTU)
and the batch was split in smaller portions of 10 ꢁmol each, which were used for the
following experiments. The tertꢀbutoxycarbonylꢀprotected (Boc) amino acid (Bocꢀ
isoleucine) at the Nꢀterminus was used to avoid higher methylation or the formation
of quaternary ammonia at the primary amine. Then the influence of the amount of
base as well as the amount of alkylation reagent were studied, and the time for
deprotonation and methylation was varied. The amount of base and methylation
reagent was set to 5 or 20 equivalents per amide bond, respectively, and the
reaction times for deprotonation and methylation was set to 5 and 60 min,
respectively. The resins were treated under these different conditions and the
conversion to permethylated peptide was analysed by a test cleavage by HPLCꢀ
ESI/MS (scheme 1, table 1, and Supplementary table S1). In a first reaction with
only five equivalents of base and methyl iodide (MeI) only 0.9% of permethylated
product (mIꢀmIꢀmIꢀmI, 2 (Nꢀmethylꢀisoleucine, mI)) could be observed. Higher
methylation rates could only be observed when the amount of base was increased. If
both reagents (MeI and LiOtBu) were increased up to 20 equivalents the conversion
raised up to 73.7% in only a single treatment of each step for 5 min. Furthermore, if
the reaction time is increased up to 60 min, for deprotonation and methylation, the
conversion is > 90% in just a single treatment.14,16 Additionally, it became clear that
DMSO has to be added as it is described as an efficient solvent for methylation.17,18
In total 32 experiments were performed and interpreted in a design of experiment
(DOE). A DOE provides information about the interaction of different factors and thus
how the total system, here onꢀresin alkylation, works. The response graphs clearly
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show that the addition of DMSO and the amount of LiOtBu and MeI are important
factors (Supplementary table S2 and S3), compared to the reaction times for
deprotonation and methylation that were less significant. In summary, it was possible
to obtain very high conversion rates (~95%) of permethylated peptide backbone in
single treatment and short reaction times.
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Combined approach of backbone amide linking and on-resin permethylation
Instead of coupling the appropriate amine in solution phase, we decided to
incorporate the Cꢀterminal amine moiety on a backbone amide linker (BAL) resin
with subsequent solidꢀphase elongation of peptide and onꢀresin peralkylation thereby
avoiding further steps. In addition, in this approach the relevant amide bond is
protected by the backbone linker during methylation thus yielding the desired natural
products. Therefore, the commercially available 2ꢀ(3,5ꢀdimethoxyꢀ4ꢀformylphenoxy)
ethyl (DFPE) resin was first treated with phenylethylamine using NaBH3CN in
DMF/MeOH/AcOH (80:19:1) (v/v%) at 60°C overnight as described previously19
(scheme 2). The first NꢀFmocꢀprotected amino acid (Fmocꢀvaline) was successfully
introduced by activation with Nꢀ[(dimethylamino)ꢀ1Hꢀ1,2,3ꢀtriazolo[4,5ꢀb] pyridinoꢀ1ꢀ
ylmethylene]ꢀNꢀmethylmethanaminium hexafluorophosphate (HATU) and 1ꢀhydroxyꢀ
7ꢀazabenzotriazole (HOAt) overnight. After Fmocꢀquantification,20 which gave a
loading of 80% of initial loading of DFPE resin, automated SPPS of 25 ꢁmol was
performed using HCTU. As described above, Bocꢀprotected valine was incorporated
at the Nꢀterminus. With this resin (5) in hand, an alkylation reaction was performed
with half of the resin (12.5 ꢁmol) under the optimized permethylation conditions
described above. After a test cleavage with trifluoroacetic acid (TFA) from the BAL
resin and analysis by HPLCꢀESI/MS, a single product peak of mVꢀmVꢀmVꢀPEA (8,
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Nꢀmethylꢀvaline, mV) could be observed in the chromatogram (conversion >99%,
Supplementary table S5). This method also enables the synthesis of nonꢀ
methylated RXPs, for example VꢀVꢀVꢀVꢀPEA (7) and is an ideal set up for a fast
synthesis of a large number of different naturally occurring and artificial RXPs.
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Flexible Synthesis Enables Easy Access to Structure Activity Study for RXPs
Initially, we synthesised naturally occurring permethylated RXPs, and their nonꢀ
methylated derivatives containing up to seven valyl, leucyl or phenylalanyl residues
with Cꢀterminal PEA and single or double valine substituted derivatives (7ꢀ54, Nꢀ
methylꢀleucine, mL, Nꢀmethylꢀphenylalanine, mF). To obtain TRA containing RXPs
(55ꢀ62) a N1ꢀBocꢀprotected tryptamine derivative (S4) was synthesized21 and
coupled by reductive amination to the BAL resin. Particularly, the permethylated
RXPs 8, 10, 12, 14, 32, 38, 44, 54, 56 and 60 are reported as natural products from
Xenorhabdus and Photorhabdus strains.4,6 Furthermore and in addition to the
already published data from our group were the substrate flexibility of the terminal
condensation domains was shown, several new amines were added to E. coli
expressing the RXP biosynthetic genes kj12B and kj12C from Xenorhabdus KJ12.1
but with Kj12B carrying the adenylationꢀmethylation didomain (AꢀMT) from X.
vietnamensis (VietC).6 Interestingly, all supplemented amines were accepted and
incorporated into RXPs, resulting in over 40 new derivatives and demonstrating once
again the substrate flexibility of this enzyme system (Supplementary table S4). In
particular, the four amino acid long and permethylated derivatives were always found
as major derivatives, rendering their efficient synthesis as a key objective and also
allowed a comparison of heterologous produced and synthetic RXP for structure
confirmation (exemplarily shown in Supplementary figure S1).
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Furthermore, it is possible to investigate the characteristic features of natural RPXs
and compare them with its synthetic nonꢀmethyl derivatives. Since the synthesis is
not limited to naturally occurring amino acids it was possible to expand the approach
to aliphatic amino acids not appearing in RXPs, like (Nꢀmethylꢀ) alanine (A, mA, 63ꢀ
70), (Nꢀmethylꢀ) aminobutanoic acid (Abu, mAbu 81 and 82), (Nꢀmethylꢀ) norvaline
(Nva, mNva, 83 and 84) and (Nꢀmethylꢀ) isoleucine (85 and 86) as well as amino
acids that contain tertꢀbutylesters and ethers (for example aspartate (D, mD, 91 and
92), glutamate (E, mE, 93 and 94) and tyrosine (Y, mY, 103 and 104)). With the
exception of tryptophan (W, mW, 101 and 102), amino acids that carry a side chain
with a protected amine (for example lysine) or amide (for example asparagine and
glutamine) were not used in this study, as it would lead to Nꢀmethylation of the side
chain. Additionally, it was possible to incorporate the fed amines, besides other
amines, for example diverse PEA moieties like TYA (106), 4ꢀfluorophenylethylamine
(116), 4ꢀbromophenylethylamine (118) and 4ꢀphenylbutylamine (124) or
naphthylethylamines (112 and 114), as well as simple alkyl moieties (for example
methyl (128), ethyl (130), amyl (136)). The synthesis of these RXPs was possible
when different amino acids and amines were used as building blocks and thus gave
an easy access to various RXPs (scheme 3 and supplementary table S5).
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Bioactivity against protozoa and structure activity study
As already mentioned, Xenorhabdus and Photorhabdus produce a broad range of
natural products to provide and support the symbiosis with the entomopathogenic
host nematodes. It is suggested that all these produced natural products have a
specialized function, such as defending the prey for exclusive exploration against
other competitors like bacteria, fungi, and soilꢀliving protozoa.2 In order to address
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this, all synthesized permethylated and several nonꢀmethylated derivatives were
tested against different protozoa and against rat skeletal muscle cell line (L6) for
cytotoxicity assessment. The tested protozoa are causative agents of tropical
diseases like sleeping sickness (Trypanosoma brucei rhodesiense), leishmaniasis
(Leishmania donovani) and malaria (Plasmodium falciparum) (supplementary table
S6). Previously investigated natural products from Xenorhabdus and Photorhabdus
also showed antiparasitic activities like szentiamide (IC50 = 0.995 ꢂg/mL against P.
falciparum)22 and xenobactin (IC50 of 31.64 ꢁg/mL against Trypanosoma brucei
rhodesiense).23
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As some of the synthesized RXPs were active down to the sub ꢂg/mL range (for
example 26 (IC50 = 0.2 ꢁg/mL), 36 (IC50 = 0.1 ꢁg/mL), 52 (IC50 = 0.14 ꢁg/mL) and
108 (IC50 = 0.60 ꢁg/mL) against P. falciparum) the data clearly shows that
methylation is advantageous for bioactivity (figure 2) and thus may explain the high
abundance of highly and permethylated RXPs in Xenorhabdus and Photorhabdus
strains. Longer peptide chains also increase activity for Nꢀmethylvaline derivatives
(8, 10, 12, and 14), while Nꢀmethylleucine (16, 18, 20, and 22) and Nꢀ
methylphenylalanine derivatives (24, 26, and 28) in general have an increased
activity for example against T. b. rhodesiense, but do not show a simple chain length
dependency. An increased bioactivity and higher selectivity could be observed for
most derivatives by substitution of one or more Nꢀmethylvalines by Nꢀmethylleucine
or Nꢀmethylphenylalanine in four, five or six amino acid long RXPs against T. b.
rhodesiense and P. falciparum (supplementary figure S2). Since the bioactivity of
RXPs against protozoa show a correlation to cytotoxicity against mammalian L6 cells
this may indicate nonꢀspecific cytotoxicity. However, in all cases higher doses are
required for cytotoxic effects.
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Furthermore, by increasing the size of the amino acid side chains in a permethylated
RXP from Abu and Nva up to isoleucine or leucine an increased bioactivity could be
observed in T. b. rhodesisense, P. falciparum, and L6 cells (supplementary figure
S3). All these changes add to the hydrophobicity of these peptides and thus might
enhance their permeation.
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To further investigate the importance of specific residues, an alanine and Dꢀamino
acid scan of mLꢀmLꢀmLꢀmLꢀPEA (16) was devised resulting in derivatives 64ꢀ80.24,25
The alanine substituted permethylated RXPs (64ꢀ70) showed an activity against T. b.
rhodesiense and P. falciparum that correlated with the distance to the Cꢀterminus
(figure 3). Best activity was observed for the Ala present at the Nꢀterminus (70, IC50
= 1.85 ꢁg/mL against P. falciparum) and essentially no activity for the Cꢀterminal Ala
derivative 64 (IC50 = 31 ꢁg/mL against P. falciparum). Therefore, the Cꢀterminal
position was chosen for further modification and incorporation of the above
mentioned amino acids resulting in RXPs 87ꢀ104. In the
Dꢀamino acid scan four
derivatives (16, 72, and 76ꢀ80, Nꢀmethylꢀ ꢀleucine, ml) showed comparable activity
D
for example against T. b. rhodesiense (IC50 1.4 ꢀ 2.1 ꢁg/mL), only mLꢀmLꢀmlꢀmLꢀ
PEA (74, IC50 = 17.5 ꢁg/mL against T. b. rhodesiense) showed a decreased activity.
Although these results are slightly contradictory to the observed data from the
alanine scan, they might imply that the substitution of the
Dꢀamino acid on the
biological activity is dependent to the recognition of a chiral target.25 Nonetheless,
the results suggest that the two Cꢀterminal amino acids had the most impact on the
bioactivity suggesting one or several specific biological and chiral targets in the
different protozoa and mammalian L6 cells that awaits their identification in the
future.24,26
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Incidentally, since all six stereoisomers from the
Dꢀamino acid scan (16, 72ꢀ80) could
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be separated on analytical HPLC it could be assumed that indeed neither
epimerization nor racemization took place during deprotonation under strong basic
conditions or is below detection limit (supplementary figure S5).
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Motivated by the results of the alanine scan, further single substituted artificial
permethylated RXPs were synthesized in which the Cꢀterminal amino acid was
exchanged against other methylated amino acids similar to RXPs 8, 30 and 38. As
mentioned earlier, only amino acids were chosen where no methylation of side chain
could occur during the alkylation step. Whereas the incorporation of serine,
threonine, aspartate, and glutamate showed a decrease in activity, for tryptophan,
tyrosine and cysteine (mLꢀmLꢀmLꢀmCꢀPEA, 90, IC50 = 3.0 against T. b. rhodesiense)
an increase in bioactivity could be observed (supplementary figure S3). Moreover,
the terminal amine has a strong impact on bioactivity (supplementary figure S4)
with the naphthylethylamine derivative (114, IC50 = 1.0 ꢁg/mL against T. b.
rhodesiense) being the most active derivative and a clear trend of increasing
bioactivity by increasing chain length of the terminal amine Me<Et<Pr<Bu.
Considering all obtained data, RXPs 140 (IC50 = 5.6 ꢁg/mL against T. b.
rhodesiense) and 142 (IC50 = 7.5 ꢁg/mL against T. b. rhodesiense) were synthesized
combining three leucyl residues, with cysteine at the Cꢀterminal position and PEA
and 1ꢀnaphthylethylamine as terminal amines but no increased activity could be
observed for both derivatives compared with its parent derivatives 16, 90 and 114,
respectively.
When selected RXPs (8, 12, 16, 20, 26, 48, 50 and 106) were tested against
different bacterial pathogens like Staphylococcus aureus (ATCC29213), S. aureus
(MRSA, ATCC43300), Enterococcus faecalis (ATCC29212), Micrococcus luteus
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(ATCC9341), Escherichia coli (ATCC25922) and Pseudomonas aeruginosa
(ATCC27853), no growth inhibition was observed up to a concentration of 128
ꢁg/mL, although at least an activity for 26 was expected against S. aureus as already
shown for permethylated polyphenyalanines.16 However, these findings suggest that
the target of RXPs is maybe parasiteꢀspecific.
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For further characterization of RXPs we tested the chemically synthesized peptides
for their passive membrane diffusion as well as for their proteolytic and metabolic
stability.
Passive membrane diffusion of RXPs
The passive diffusion rates of nonꢀmethylated RXP 29, 37 and their permethylated
derivatives 30 and 38 were tested in a parallel artificial membrane permeability
assay (PAMPA).27 PAMPA can be used to measure passive membrane diffusion
behaviour in the absence of transporters or efflux systems28 and it is normally used
to screen drug candidates for their oralꢀabsorption potential in the discovery and
development phase.29 As expected, the two nonꢀmethylated RXPs 29 and 37
showed essentially no permeability through an artificial hexadecane membrane
(HDMꢀPAMPA)30, whereas the PAMPA permeability of the permethylated derivatives
30 and 38 were comparable to the orally available drug verapamil (figure 4). The
methylation significantly increases the permeability and thus might contribute to the
observed high activity of the permethylated RXPs against protozoa or might promote
their uptake inside the eukaryotic cells.15,31–33
In vitro pharmacokinetics studies
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A set of nonꢀ and permethylated RXPs were tested for their in vitro stability against
insect hemolymph plasma (from the greater waxmoth Galleria mellonella) and rat
liver microsomes. These assays are indicative of the compounds stability against
proteolytic and cytochrome P450 enzymes, respectively. Nonꢀmethylated RXPs 7
and 29 revealed a short halfꢀlife of approximately 30 min in insect hemolymph
plasma, whereas permethylated RXPs 8 and 30 showed a much longer halfꢀlife (t1/2
> 4 h) (figure 5 and supplementary figure S6) as expected for the stabilizing effect
of Nꢀmethylation of peptide bonds.15,31
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Contrarily, in the microsomal stability assay permethylated 8 was less stable then the
nonꢀmethylated 7 (supplementary figure S7 and S8). Detailed analysis of MS2ꢀ
fragmentation and HRꢀHPLCꢀMS revealed the products of metabolic degradation of
7 and 8 as single hydroxylated products of the starting material. The oxidation by
cytochrome enzymes occurred at different valines as well as on the phenyl, resulting
in βꢀhydroxy valine and tyramine derivatives, respectively (supplementary figure S9
and S10). The tyramine structure could be confirmed by synthesis of the appropriate
derivatives (supplementary figure S11). Multiple oxidized products could not be
observed or are below the detection limit. As cytochrome P450 enzymes are also
encoded in insect34 as well as in genome of Xenorhabdus and Photorhabdus a
possibly metabolic derivatization of RXPs could also occur during the complex life
cycle of insect, nematode and bacteria. Additionally, the TYA derivative 106 (IC50
=
30.6 ꢁg/mL) is less cytotoxic then the PEA derivative 16 (IC50 = 16.7 ꢁg/mL) and
might represent a detoxifying mechanism similar to insecticide resistance.35
However, such TYA derivatives have only been found as natural products in X.
vietnamensis.6
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Conclusion
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After optimization of the alkylation step onꢀresin on a sterically hindered model
peptide, a high conversion of permethylated peptides could be achieved in single
treatment. The combination of backbone amide linking of the Cꢀterminal amine
followed by the optimized alkylation step led to the successful synthesis of
permethylated RXPs. As this facile and flexible approach allows the synthesis of
diverse and artificial nonꢀ and permethylated RXPs, a structure activity study
revealed important characteristics of RXPs regarding their bioactivity against
protozoa. This approach expands the toolꢀbox for solidꢀphase peptide chemists and
might permit facile synthesis routes for new derivatives of highly and permethylated
peptide natural products, for example almiramides36 and dragonamides37 or it can be
used for the synthesis of permethylated core structures of other peptides, for
example nematotoxic omphalotin38. Additionally, the passive membrane diffusion of
permethylated RXPs is superior to their nonꢀmethylated derivatives and might
contribute to their better bioactivity. In two in vitro pharmacokinetics studies RXPs
are further described and illustrate the improved stability of permethylated RXPs at
least in insect hemolymph plasma.
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Experimental Section
Chemistry
General Procedures. All reactions were carried out under an atmosphere of nitrogen
and with dry solvents, unless otherwise stated. Dry solvents and chemicals were
purchased from SigmaꢀAldrich (Germany) or Acros Organics (Belgium) in highest
commercial available purity and were used without further purification. Protected
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amino acids and resins were supplied from Carbolution (Germany), Bachem
(Switzerland), Iris Biotech (Germany) and Merck Millipore (Germany).
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HPLC-ESI/MS
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5 ꢁL of the sample (dissolved crude or purified peptides) were injected and analysed
via HPLCꢀESI/MS by a Dionex UltiMate 3000 system coupled to a Bruker AmaZon X
mass spectrometer with a ACQUITY UHPLC™ BEH C18 column (130 Å, 2.1 mm ×
100 mm, 1.7 ꢁm particle size, Waters GmbH) at a flow rate of 0.4 mL/min for 16 min,
using ACN and water supplemented with 0.1% formic acid (v/v) in a gradient ranging
from 5 to 95% ACN. For detection, positive mode with a scanning range from 100ꢀ
1200 m/z was used. HRꢀESIꢀMS analysis was carried out by using an internal
calibrant (10 mM sodium formate solution) and the same linear gradient from 5 to
95% ACN (0.1% formic acid (FA)). HRꢀmasses were detected by Bruker Impact II
QTOF.
Fermentation of RXP producer and culture extraction
For RXP production, the E. coli containing the heterologous expressed RXP
biosynthetic gens kj12B and kj12C from Xenorhabdus KJ12.1 with AꢀMT replaced by
AꢀMT from X. vietamiensis (vietC)6 was inoculated into liquid Lysogeny broth (LB, pH
7.0) medium and grown at 30 °C with shaking at 200 rpm overnight. An 100 ꢁL
aliquot of overnight culture was transferred into 20 mL fresh LB medium containing
2% (v/v) Amberlite XADꢀ16 resin (Sigma Aldrich), 35 ꢁg/mL chloramphenicol, 1 mM
amine (TRA, MTRA, 1Nphth, 2Nphth, 4FPEA and 4BrPEA) and 0.1% Lꢀarabinose.
The cultures were grown at 30 °C for 24 h at 200 rpm. After the growth the cultures
were centrifuged (15 min @ 4000 rpm) and the pellet (XAD and bacteria) extracted
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with 10 mL methanol for 30 min. Afterwards, XAD and bacteria were removed by
filtration and the filtrate evaporated to dryness. The resulting residue was redissolved
in 1 mL methanol and analysed by HPLCꢀESI/MS.
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Purification
Peptides were purified by semiꢀpreparative reverse phase HPLC using UV detection
at 228 nm or 254 nm on an Agilent 1260 Infinity system with a ZORBRAX
StableBond 300 C18 (9.4 mm × 250 mm, 5.0ꢂm, Agilent) using an different aqueous
ACN gradients with 0.1% FA or manually with StrataꢀC18E columns (Phenomex).
Therefore, columns were conditioned according to manufacture and equilibrated with
aqueous 0.1% FA. Crude peptides were dissolved in 30% ACN/ 0.1% FA and loaded
onto the cartridge. The peptides were eluted with stepwise increasing ACN
concentration. All collected fraction were analysed by HPLCꢀMS and only fractions
containing a single peak of correct mass were combined and used in further
experiments. Afterwards purity was confirmed ≥95%, as determined by HPLCꢀMS or
HPLCꢀUV at a wavelength of 228 nm.
Synthesis of RXPs
Reductive Amination
For reductive amination a mixture of amine or amine hydrochloride (1.0 mmol,
10 eq.) in 1.4 mL DMF/MeOH/AcOH (80:19:1) (v/v%), NaBH3CN (62.8 mg, 1.0
mmol, 10 eq.) and DFPE resin (95.2 mg, 0.1 mmol, 1 eq.) were placed in a 2 mL
Eppendorf tube and incubated in a thermoshaker at 60 °C overnight. The resin was
collected by filtration in a plastic reactor vessel equipped with a Teflon frit and
subsequently washed with DMF (5 ×) and DCM (5 ×). For acylation of first amino
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acid, the appropriate FmocꢀaaꢀOH (1.0 mmol, 10 eq.) was dissolved with HATU
(381 mg, 1.0 mmol, 10 eq.), HOAt (136 mg, 1.0 mmol, 10 eq.) and DIPEA (0.34 mL,
2.0 mmol, 20 eq.) in 2.0 mL DMF and added to resin. The resulting mixture was
incubated at room temperature overnight. The resin was washed with DMF (5 ×) and
DCM (5 ×) and treated with 20% piperidine in NMP (3 × 5 min) to remove the Fmoc
protecting group. The combined filtrates were used to determine the actual loading
(ε301 nm = 7800 mol Lꢀ1 cmꢀ1)20 of the resin and subsequently washed with DCM (5 ×).
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Solid Phase Peptide Synthesis
The linear sequence was synthesized on the preloaded resin on a 25 ꢁmol scale
with a Syro Wave peptide synthesizer (Biotage, Sweden) by using standard Fmoc/tꢀ
Bu chemistry. Therefore, the resin was placed in a plastic reactor vessel with a
Teflon frit and an amount of 6 eq. of amino acid derivatives (c = 0.2 M) was activated
in situ at room temperature with 6 eq. of HCTU (c = 0.6 M) in DMF in the presence of
12 eq. DIPEA (c = 2.4 M) in NMP for 50 min. Fmocꢀprotecting groups were removed
with a solution of 40% piperidine in NMP (v/v%) for 5 min and the deprotection step
was repeated for another 10 min with 20% piperidine in NMP (v/v%). After each
coupling and deprotection step, the resin was washed with NMP (4 ×). After the
addition of the final residue as Bocꢀamino acid, the resin was washed with NMP (5
×), DMF (5 ×) and DCM (5 ×).
Permethylation of Peptide Backbone On-Resin
Onꢀresin permethylation of peptide backbone was performed with half of the
synthesised peptidyl resin (12.5 ꢁmol) from above. Therefore, the resin was placed
in a plastic reactor vessel equipped with a Teflon frit and a Teflon valve. The reactor
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vessel was flushed three times with nitrogen and the resin was washed three times
with dry THF. Under an atmosphere of nitrogen, a mixture of 1 M LiOtBu (1.0 mL,
20 eq/amide bond) in THF and dry DMSO (0.5 mL, 125 ꢁL/amide bond) was
prepared in a separate round bottom flask and added to the resin. The reaction
mixture was agitated for 30 min at room temperature. Afterwards the LiOtBu
containing solution was poured into a round bottom flask and MeI (100 ꢁL,
20 eq/amide bond) was added to it. The mixture was added again to the resin and
agitated for another 30 min at room temperature. The resin was filtered and washed
with MeOH (5 ×), DCM (5 ×) and dried. The peptide was cleaved from the resin by
adding a total of 0.5 mL TFA/TIS/water (95:2.5:2.5) to the permethylated or nonꢀ
methylated peptidyl resin (12.5 ꢁmol) and the mixture was agitated for at least 1 h at
room temperature. The resin was removed by filtration and washed twice with TFA.
The solution was evaporated to dryness. Purity was confirmed by HPLCꢀMS.
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Bioactivity Testing
For determination of bioactivity, RXPs were tested against different protozoan
parasites T. brucei rhodesiense STIB900 (trypomastigotes), L. donovani
(amastigotes), P. falciparum NF54 (stage IEF) and against rat skeletal myoblasts (Lꢀ
6 cells) for cytotoxicity assessment as previously described.39
In Vitro Characterization of RXPs
Parallel Artificial Membrane Permeability Assay (PAMPA) to Determine Passive
Membrane Diffusion of Non-Methylated and Permethylated RXPs
For the assay a multiscreen permeability filter plate assembly form Merck Millipore®
(Germany, MPC4NTR10, consists of lid, donor plate (96ꢀwell filter plate), acceptor
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plate (96ꢀwell receiver plate) and single well tray) was used and the assay was
performed as described in the user manual. Briefly, stock solutions of investigated
compounds (verapamil, metoprolol, RXPs) were prepared (50 mM in DMSO), and
subsequently diluted to the final concentration. Therefore, 10 ꢁL of stock solution
were added to 990 mL PBS buffer (pH 6.5, 50 mM) to obtain transport samples
(500 ꢁM). Artificial hexadecane membrane was created by applying 15 ꢁL of 5%
hexadecane in hexane to the centre of each well in the 96ꢀwell donor plate and
allowed to dry for one hour in the hood. Afterwards 200 ꢁL buffer were added to the
acceptor plate wells and 125 ꢁL of transport samples to the donor well plates. Each
compound was tested in four individual wells. The donor plate was carefully stacked
on top of the acceptor plate and covered with the supplied lid to minimize
evaporation during incubation. At the end of the incubation time (5 h at room
temperature) donor plate was removed from the acceptor plate and 50 ꢁL of samples
were taken from each well. Samples were then dried in an Eppendorf/concentrator
plus (VꢀAQ mode, 60 °C, 1 h) and resuspended in 500 ꢁL methanol and analysed by
HPLCꢀMS. Concentrations of samples in donor and acceptor wells were calculated
form serial dilutions of each compound. Assay was repeated with 16 h incubation
time with comparable results (data not shown).
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In vitro Pharmacokinetics Studies – Insect Hemolymph Stability of Non-
Methylated and Permethylated RXPs
To obtain insect hemolymph plasma, Galleria mellonella were chilled for at least
20 min at ꢀ20 °C. Afterwards, the frozen larvae were thawed on ice and cut between
the second proleg and tail, the resigned hemolymph was collected in a chilled 1.5 mL
Eppendorf tube on ice. The pooled hemolymph was diluted in a 3:1 ratio (v/v) with
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PBS buffer (pH 6.5, 50 mM) and centrifuged at 4,000 g for 10 min at 4 °C to obtain
insect hemolymph plasma. Immediately 20 ꢁL hemolymph plasma were mixed with
475 ꢁL BPS buffer (pH 6.5, 50 mM) and preꢀwarmed for 5 min at 37 °C in a 1.5 mL
Eppendorf tube. Subsequently 5 ꢁL stock solution of RXP were added (final
concentration 0.5 mM) and incubated on a thermoshaker at 37 °C. Periodically (0ꢀ
240 min), aliquots (50 ꢁL) of the incubation mixture were added to 200 ꢁL methanol.
Samples were dried in an Eppendorf/concentrator plus (VꢀAL and VꢀAQ mode, 45
and 60 °C, respectively, each 30 min) and resuspended in 500 ꢁL methanol and
analysed by HPLCꢀMS. For every compound three independent assays were
performed in parallel. Furthermore, the assay was performed under different
conditions (pH, length, concentrations of compound and hemolymph) resulting in
similar results (data not shown).
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In Vitro Drug Metabolism Using Liver Microsomes
A solution of RXP (final concentration 1 mM) was prepared in 100% DMSO. 432 ꢁL
phosphate buffer (0.1 M, pH 7.4) together with 50 ꢁl NADPHꢀregenerating system
(30 mM glucoseꢀ6ꢀphosphate, 4 U/mL glucoseꢀ6ꢀphosphate dehydrogenase, 10 mM
NADP, 30 mM MgCl2) and 5 ꢁL of the corresponding RXPs were preꢀincubated at 37
°C. After 5 min the reaction was started by the addition of 13 ꢁL microsome mix from
the liver of SpragueꢀDawley rats (Invitrogen, Germany; 20 mg protein/mL in 0.1 M
phosphate buffer). The incubation was performed in a shaking water bath at 37 °C.
The reaction was stopped by the addition of 500 ꢁL iceꢀcold methanol at 0, 15, 30
and 60 min. The samples were centrifuged at 10,000 g for 5 min at 4 °C. Afterwards,
samples were dried in an Eppendorf/concentrator plus (VꢀAl, 45 °C, 30 min) and
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resuspended in 500 ꢁL methanol and analysed by HPLCꢀMS. No further replications
were performed with compounds.
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Additional supplementary data
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Experimental setup for synthesis of amines S1ꢀS4 and Design of Experiment;
Formula for calculation of permeability log Pe; supplementary tables showing results
of DOE, HPLCꢀESI/MS analysis of synthesized RXPs, summarized bioactivity of all
tested RXPs against protozoa and cytotoxicity against L6 cell line; supplementary
figures of further structure activity study of synthesized RXPs, HPLCꢀMS analysis of
RXPs for Dꢀamino acid scan, analysis of proteolytic and metabolic stability.
Molecular formula strings for all synthesized RXPs
Corresponding author: *Email: h.bode@bio.uniꢀfrankfurt.de. T 0049ꢀ69ꢀ798ꢀ29557.
Abbreviations Used:
2CTC, 2ꢀchlorotrityl chloride polystyrene resin;
4BrꢀPEA, 4ꢀbromophenylethylamine;
4FꢀPEA, 4ꢀfluorophenylethylamine;
Abu, aminobutanoic acid;
ACN, acetonitrile;
AcOH, acetic acid;
AGM, agmatine;
Ala, alanine;
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AꢀMT, adenylationꢀmethylation domain;
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BAL, backbone amide linking;
DFPE, 2ꢀ(3,5ꢀdimethoxyꢀ4ꢀformylphenoxy) ethyl resin;
DIPEA, N,Nꢀdiisopropylethylamine;
DOE, design of experiment;
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F, phenylalanine;
FA, formic acid;
HOAt, 1ꢀhydroxyꢀ7ꢀazabenzotriazole;
HATU, Nꢀ[(dimethylamino)ꢀ1Hꢀ1,2,3ꢀtriazolo[4,5ꢀb] pyridinoꢀ1ꢀylmethylene]ꢀNꢀ
methylmethanaminium hexafluorophosphate;
HCTU, Oꢀ(1Hꢀ6ꢀchlorobenzotriazoleꢀ1ꢀyl)ꢀ1,1,3,3ꢀtetramethyluronium
hexafluorophosphate;
HDMꢀPAMPA, parallel artificial membrane permeability assay through hexadecane
membrane;
Ile/I, isoleucine;
L, leucine;
LB, Lysogeny broth;
LiOtBu, lithiumꢀtertꢀbutoxide;
MeI, methyl iodide;
MeOH, methanole;
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mA, Nꢀmethyꢀalanine;
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mC, Nꢀmethylcysteine,
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mD, Nꢀmethyꢀasapartic acid;
mE, Nꢀmethylglutamic acid;
mF, Nꢀmethylphenylalaine;
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ml, NꢀmethylꢀDꢀleucine;
mL, Nꢀmethylleucine;
mM, Nꢀmethylmethionine;
mNva, Nꢀmethylnorvaline;
mS, Nꢀmethylserine;
mT, Nꢀmethylthreonine;
mV, Nꢀmethylvaline;
mW, Nꢀmethyltrypotophane;
mY, Nꢀmethyltyrosine;
NaBH3CN, sodium cyanoborohydride;
Nva, norvaline;
PEA, phenylethylamine;
pip, piperidine;
PS, polystyrene
RXP, rhabdopeptide/xenortide peptide;
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TIS, triisopropylsilane;
TRA, tryptamine;
TYA, tyramine;
V, valine;
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Acknowledgment
The authors are grateful to Andreas Lück for the cultivation and supply of Galleria
mellonella, as well as to former interns Tien Duy Vo and Sina Roth for helping with
the synthesis of RXPs. Further thanks goes to Xiaofeng Cai for providing the RXP
producer E. coli strain. The authors are grateful to Prof. Thomas A. Wichelhaus and
Denia Frank (Institute of Medical Microbiology and Infection Control, J.W. Goethe
University) for antimicrobial bioactivity testing of RXPs. This work was supported by
an ERC starting grant to H.B.B. (grant agreement no. 311477) and the LOEWE
program of the state of Hesse as part of the MegaSyn research cluster. H.B.B.
acknowledges the Deutsche Forschungsgemeinschaft for funding of the Impact II
qTof mass spectrometer (INST 161/810ꢀ1).
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