Selective Modification of Ribosomally Synthesized and Post-Translationally Modified Peptides (RiPPs) through Diels–Alder Cycloadditions on Dehydroalanine Residues

Article abstract of DOI:10.1002/chem.201902907

We report the late-stage chemical modification of ribosomally synthesized and post-translationally modified peptides (RIPPs) by Diels–Alder cycloadditions to naturally occurring dehydroalanines. The tail region of the thiopeptide thiostrepton could be modified selectively and efficiently under microwave heating and transition-metal-free conditions. The Diels–Alder adducts were isolated and the different site- and endo/exo isomers were identified by 1D/2D ¹H NMR. Via efficient modification of the thiopeptide nosiheptide and the lanthipeptide nisin Z the generality of the method was established. Minimum inhibitory concentration (MIC) assays of the purified thiostrepton Diels–Alder products against thiostrepton-susceptible strains displayed high activities comparable to that of native thiostrepton. These Diels–Alder products were also subjected successfully to inverse-electron-demand Diels–Alder reactions with a variety of functionalized tetrazines, demonstrating the utility of this method for labeling of RiPPs.

Full text of DOI:10.1002/chem.201902907

A Journal of
Accepted Article
Title: Selective Modification of RiPPs via Diels-Alder Cycloadditions on
Dehydroalanine Residues
Authors: Reinder de Vries, Jakob Viel, Ruben Oudshoorn, Oscar
Kuipers, and Gerard Roelfes
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To be cited as: Chem. Eur. J. 10.1002/chem.201902907
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Selective Modification of Ribosomally Synthesized and Post-
translationally Modified Peptides (RiPPs) via Diels-Alder
Cycloadditions on Dehydroalanine Residues
Reinder H. de Vries,[a] Jakob H. Viel,[b] Ruben Oudshoorn,[a] Oscar P. Kuipers[b] and Gerard Roelfes*[a]
Abstract: We report the late stage chemical modification of
ribosomally synthesized and posttranslationally modified peptides
(RIPPs) by Diels-Alder cycloadditions to naturally occurring
dehydroalanines. The tail region of the thiopeptide thiostrepton could
be modified selectively and efficiently under microwave heating and
transition metal free conditions. The Diels-Alder adducts were isolated
and the different site- and endo/exo isomers were identified by 1D/2D
1H NMR. Via efficient modification of the thiopeptide nosiheptide and
the lanthipeptide nisin Z the generality of the method was established.
MIC assays of the purified thiostrepton Diels-Alder products against
thiostrepton-susceptible strains displayed high activities comparable
to that of native thiostrepton. These Diels-Alder products were also
subjected successfully to Inverse-electron-demand Diels-Alder
reactions with a variety of functionalized tetrazines, demonstrating the
utility of this method for labeling of RiPPs.
hydrogenations,[25] cross-coupling reactions,[26,27] photoredox
catalysis,[28] cyclopropanations[29]
and 1,3-dipolar
cycloadditions.[30] These studies have highlighted the potential of
dehydroamino acid modification in RiPPs, but also illustrate the
challenge of achieving selectivity due to the high structural
complexity of RiPPs and the difficulties of discriminating between
the various dehydroamino acids present.
Here we now report the Diels-Alder reaction with
cyclopentadiene as a mild and selective modification reaction for
of dehydroalanine residues in antimicrobial RiPPs (Scheme 1).
Furthermore, the unactivated, strained alkene in the formed
norbornene product could be employed in Inverse Electron
Demand Diels-Alder (IEDDA, “click”) reactions with tetrazines
(Scheme 1), a popular labeling tool in chemical biology.[31]
Ribosomally synthesized and post-translationally modified
peptides (RiPPs),[1] such as thiopeptides[2–5] and lanthipeptides[1,6]
have attracted attention as potential alternatives to small-
molecule antibiotics because of their high activity against a broad
range of bacteria and low level of resistance development.[7,8] Yet
chemical editing of these peptides is necessary in order to
mitigate their poor pharmacological properties and to make them
suitable for clinical application and to synthesize analogues and
derivatives for the study of their mechanism of action. Over the
years, progress has been made towards late-stage chemical
modification of antimicrobial peptides isolated from producing
strains, although achieving (site) selective derivatization of these
structurally diverse and complex natural products often poses a
major synthetic challenge.[9]
Scheme 1. 2-step labeling of dehydroalanines in RiPPs via a Diels-Alder and
IEDDA sequence.
As a starting point, the Diels-Alder reaction between
cyclopentadiene and a protected dehydroalanine substrate (1)
was studied (SI-7). In previous studies only anhydrous conditions
and also high temperatures had been reported for this reaction.[32]
The Diels-Alder reaction is known to be significantly accelerated
in water.[33] Indeed, appreciable conversion was observed in
water at room temperature after 48 hours, whereas no product
was observed when using dichloromethane as solvent (SI-7).
Next, different co-solvents that are tolerated by peptides
were tested in order to help solubilize the cyclopentadiene and
thereby increase the conversion. It was found that 2,2,2-
trifluoroethanol (TFE) gave the best results, likely due to its mild
Brønsted acidity, which can give rise to activation of the
dienophile.[34] Using 20 mol% Sc(OTf)3 to activate the dienophile
improved the conversion further, up to 88 % after 48 hours with
10 eq. cyclopentadiene.
The endo/exo ratio was ~ 40:60 in all cases, which is in
agreement with previous reports about the secondary orbital
interactions between this particular Dha substrate (1) and
cyclopentadiene.[32] 1,3-cyclohexadiene, 1,3-dimethylbutadiene
and furan were also evaluated as dienes, but did not give any
conversion at room temperature (SI-7).
The conditions established with the protected Dha
substrate appeared suitable for modification of the thiopeptide
thiostrepton (Figure 1A), given its high solubility in TFE. During
initial screening and subsequent LC-MS analysis, it was found
that addition of Sc(OTf)3 did not give rise to increased conversions
compared to reactions performed without the scandium salt.
Many thiopeptides and lanthipeptides contain one or
more uniquely reactive dehydroamino acids such as
dehydroalanine (Dha) and dehydrobutyrine (Dhb), which are the
result of post-translational enzymatic dehydration of Ser and Thr
residues, respectively.[10] The electrophilic nature of
dehydroamino acids has made them attractive functionalities for
biorthogonal reactions.[11–20] In recent years, these dehydroamino
acids have emerged as interesting targets for the late-stage
modification
of
RiPPs,
via
Michael
additions,[21–24]
[a]
[b]
R.H. de Vries, R. Oudshoorn, Prof. Dr. G. Roelfes
Stratingh Institute for Chemistry
University of Groningen
Nijenborgh 4, 9747 AG Groningen, The Netherlands
E-mail: j.g.roelfes@rug.nl
J.H. Viel, Prof. Dr. O.P. Kuipers
Department of Molecular Genetics
Groningen Biomolecular Sciences and Biotechnology Institute
University of Groningen
Nijenborgh 7, 9747 AG Groningen, The Netherlands
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.201902907
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Figure 1. A) Scheme depicting the Diels-Alder reaction between thiostrepton and cyclopentadiene to give the corresponding products 2a-c. Conditions: 1 mM
thiostrepton and 0.6 M freshly distilled cyclopentadiene in 1 mL H2O/TFE 1:1, microwave-assisted heating at 50 °C for 16 hours B) Zoom in of LC-MS chromatogram
of the crude product showing products 2a-c (* = single modification, ** = double modification). C) Full LC-MS chromatograms of purified products 2a-c. D) Stacked
1H NMR spectra of thiostrepton (top) and product 2b (bottom), showing the region between 5.0 ppm and 7.0 ppm.
On the contrary, the transition metal free conditions gave rise to
the cleanest transformations to mainly single- and double
modified thiostrepton (Figure 1B). After 7 days of reaction time
(while adding freshly distilled cyclopentadiene daily) 64 %
conversion to single- and double modified thiostrepton was
obtained as based on peak integration of the starting material and
the products in analytical HPLC.
reaction has taken place in the tail region of thiostrepton.
Moreover, the appearance of two doublets of doublets (red) is
characteristic for the formation of the alkene of norbornene. The
NMR spectra of 2a and 2c showed similar changes in signals (SI-
11, 33-37).
1
Using H-1H TOCSY NMR, products 2a and 2b were both
identified as Dha16-modified thiostrepton (see SI-11 for a detailed
explanation). By comparing the methylene signals of Dha17 in
products 2a and 2b, thereby taking into account the shielding
effect of the newly formed carbon-carbon double bond in the
norbornene, it was established that product 2a is Dha16-endo and
product 2b is Dha16-exo (see SI-11). In a similar manner, using
1H NMR and 1H-1H TOCSY NMR techniques, product 2c could be
identified as Dha17-modified thiostrepton (SI-12).
Performing the reaction at 50 °C in a microwave reactor
greatly improved the conversion to 72 % after only 16 hours of
reaction time, compared to 28 % conversion after 16 hours at
room temperature and 50 % conversion when heating the reaction
at 50 °C in an oil bath. A mixture of single- and double modified
products was obtained and the starting material and the products
proved to be stable under the microwave conditions. Even
hydrolytic cleavage of the Dha-tail, which is a common side
reaction in thiostrepton modification,[23] was not observed.
The reaction was performed on a 25 mg scale, after which
the three major single modified products (2a-c) were isolated
using preparative HPLC (Figure 1C). Products 2a-c, obtained as
mixtures of diastereomers that could not be separated were
analyzed by NMR. When comparing the 1H NMR spectra of
unmodified thiostrepton and the products, with particular focus on
the region between 5.00 ppm and 7.00 ppm (Figure 1D, only
showing product 2b for this example, see SI-10-12, 33-37 for all
spectra) it can be seen that the methylene signals of Dha3
(purple) and Dhb8 (yellow) are conserved in product 2b. From the
two sets of signals originating from the methylenes in the tail, i.e.
Dha16 (blue) and Dha17 (green), one set of signals has
disappeared and the other has shifted upfield, indicating that the
Scheme 2. Synthesis and Diels-Alder reaction of truncated thiostrepton (3).
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To further demonstrate the selectivity for the tail region, a
truncated variant of thiostrepton (3) was synthesized via selective
base-mediated cleavage of Dha17 from the tail of thiostrepton
using Et2NH, leaving only Dha16 as a reactive site (Scheme 2,
SI-6).[23] When 3 was subjected to the optimized reaction
conditions, only two major single modified products (4a and 4b,
Scheme 2) were observed. Using analytical HPLC a 41 % total
conversion was observed (SI-13). Both products were isolated as
mixtures of diastereomers and identified (SI-16-17) as endo- (4a)
and exo (4b) isomers of Dha16-modified 3 (SI-13) using NMR
analysis analogous to the identification of products 2a-c.
Collectively, these results show that the reaction is highly
selective for the tail region of thiostrepton. Also, the LC-MS UV
signal areas of products 2a and 2b compared to product 2c
(Figure 1B) indicate a significant preference for modification at
Dha16, which can be explained by the fact that this residue is the
most electron poor site due to the neighbouring thiazole15 and
Dha17, both electron withdrawing moieties.
The scope of the reaction was evaluated by performing the
reaction on different RiPPs. The Diels-Alder reaction of
cyclopentadiene and the thiopeptide nosiheptide was performed
under the optimized conditions and after microwave-assisted
heating at 50 °C for 32 hours a conversion of 75 % to single
modified nosiheptide was observed (Scheme 3A, SI-18). The
commercial nosiheptide starting material contained a small
amount of nosiheptide that lacks the terminal Dha, having a
terminal amide instead. The product of the reaction of this impurity
with cyclopentadiene was not observed in the LC-MS analysis,
confirming that the reaction is selective for the terminal Dha over
the internal Dhb residue, which is consistent with the results
obtained using thiostrepton and 3.
The reaction between cyclopentadiene and the
lanthipeptide nisin Z was investigated next (Scheme 3B). In this
case, the same conditions as for the thiopeptides were used,
except for the substitution of ddH2O for 0.1% AcOH (aq.) due to
solubility- and stability issues of nisin at pH>5. In addition to the
inevitable, but well-documented addition of water to Dha in
nisin,[35] a 52 % conversion to single Diels-Alder modified product
was observed after 16 hours of microwave irradiation at 50 °C (SI-
19-20). For nisin Z, which bears one Dhb and two Dha residues,
the site selectivity could not be determined due to poor separation
of isomers on LC-MS and HPLC. However, good stabilities under
microwave irradiation were observed for both nosiheptide and
nisin Z, demonstrating the general applicability of our approach
for the modification of Dha-containing RiPPs.
Previous studies have shown that modification of the tail
region of thiostrepton does not severely impact its activity.[23,26] To
confirm that this is also true for the norbornene modifications,
thiostrepton and purified derivatives 2a-c, 3 and 4a-b were tested
against S. aureus (ATCC29213) and E. faecalis (ATCC29212)
strains in a MIC-assay (SI-21). The results (Table 2) show that all
derivatives have excellent antimicrobial activity, with a MIC value
that is within one order of magnitude compared to native
thiostrepton for both strains. Moreover, variations in activity
towards both strains and between the different site- and endo/exo
isomers remained limited to a factor of 4. The activity of 3 also
very closely resembles that of thiostrepton, showing that even
removing part of the tail region has little effect on its activity.
A)
HO
HO
NH
O
N
S
NH
O
N
S
S
S
O
O
HN
HN
NH
N
NH
N
O
O
O
O
O
O
N
O
N
O
OH
OH
OH
OH
S
S
H2O/TFE 1:1
microwave (50 °C)
O
O
NH
N
N
NH
N
N
N
N
S
S
S
HN
S
HN
Table 2. MIC-assay results of Diels-Alder analogues of thiostrepton against S.
aureus and E. faecalis
S
S
NH2
NH2
N
N
O
O
H
O
O
H
Nosiheptide
Antibiotic
MIC (μg/mL)
against S. aureus
MIC (μg/mL)
against E. faecalis
O
H
N
O
B)
O
HN
O
H2N
O
HN
O
NH
S
Vancomycin
1
0.5
2
4
0.5
2
N
O
H
S
NH
O
NH
S
O
O
O
H
N
O
HN
O
N
H
O
N
H
Thiostrepton
O
HN
O
H
N
O
NH
NH
NH
O
0.1% AcOH (aq.)/
TFE 1:1
microwave (50 °C)
H2N
O
O
NH
O
S
N
H
O
S
NH
S
H
2a
2b
2c
3
O
NH
NH
O
N
HN
NH2
O
O
N
O
NH2
H
N
HN
HN
O
S
H
N
H2
N
O
O
O
2
2
OH
NH2
NH
O
O
Nisin Z
NH
NH
HN
HN
O
O
2
1
OH
O
H
O
N
O
O
HN
HN
O
N
H2N
O
HN
0.5
4
1
O
NH
S
N
H
O
S
NH
O
O
NH
S
O
O
H
N
4a
4b
2
O
HN
N
H
O
N
H
O
O
HN
O
H
N
O
NH
NH
H2N
O
O
NH
S
2
2
N
H
O
S
NH
S
NH
H
O
NH
NH
O
N
HN
O
NH2
O
O
N
O
NH2
H
N
HN
HN
O
S
H
H2N
N
O
O
O
O
OH
The selective incorporation of the norbornene functionality
in the tail of thiostrepton while leaving the inherent activity intact
enables further derivatization via IEDDA click reactions with
tetrazines.[31] Purified 2a was treated with di-2-pyridyl tetrazine (5)
in H2O/ACN 1:1 at room temperature (Figure 2A) and after
NH
NH2
O
O
N
H
NH
HN
HN
O
O
OH
O
HN
N
Scheme 3. Diels-Alder reactions of cyclopentadiene with A) the thiopeptide
nosiheptide and B) the lanthipeptide nisin Z. For nisin Z only one of the possible
products is shown.
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fluorescence turn-on effect could even be visualized by shining
UV light (365 nm) on the undiluted samples (Figure 3B), which
shows the potential for using this 2-step labeling method in the
detection of new Dha-containing peptides.
Figure 3. A) Scheme depicting fluorescence turn-on of BODIPY-tetrazine upon
click reaction with the norbornene-modified peptide. B) Image showing
fluorescence under UV light (365 nm) for DMSO control (left) and click reaction
with 2a (right).
We have established the Diels-Alder reaction as a powerful
tool for efficient and selective late-stage chemical editing of
peptide antibiotics. This approach, which only requires
cyclopentadiene as a reagent and microwave-assisted heating,
allows for straightforward and transition metal free installation of
the norbornene functionality on these complex natural products
by reacting with the naturally occurring Dha residues under mild
conditions. Especially attractive is the possibility of employing the
norbornene product in Inverse Electron Demand Diels-Alder
reactions with tetrazines, which gives access to a variety of new
semisynthetic derivatives. Additionally, the norbornene moiety
could potentially be used in other labeling reactions.[38,39] These
results demonstrate the potential of this methodology for the
tailoring of RiPPs.
Figure 2. A) IEDDA reaction of norbornene-modified thiostrepton with di-2-
pyridyl tetrazine (5). B) MALDI-TOF MS spectra of IEDDA reaction of di-2-
pyridyl tetrazine with 2a and control reaction with unmodified thiostrepton (inset).
C) Structures of fluorescein-tetrazine (9) and biotin-tetrazine (10).
overnight reaction full conversion to singly labeled
dihydropyridazine (m/z = 1938) and pyridazine (m/z = 1936)
products was observed by MALDI-TOF MS of the crude reaction
mixture (Figure 2B). As a control, unmodified thiostrepton was
subjected to the same conditions, after which only starting
material (m/z = 1664, Figure 2B inset) was observed, illustrating
the high chemoselectivity for the norbornene moiety over the
other unsaturated motifs in thiostrepton.
Acknowledgements
The authors thank Dowine de Bruijn for useful advice and
discussions. This project was supported by the Netherlands
Organisation for Scientific Research (NWO) (Vici grant
724.013.003 and ALWOP.214). G.R. acknowledges support from
the Ministry of Education Culture and Science (Gravitation
programme no. 024.001.035).
Next, the IEDDA reaction with a range of different
functionalized tetrazines was investigated. An amine-
functionalized tetrazine building block (8)[36] was derivatized with
a fluorescein- (9) or biotin (10) moiety (Figure 2C). MALDI-TOF
MS showed efficient labeling of 2b with both tetrazines using the
same conditions as described above (SI-22).
Keywords: RiPPs • late stage chemical modification •
thiopeptides • bio-orthogonal chemistry • Diels-Alder
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