University of Groningen
Chemical Modification of Peptide Antibiotics
de Vries, Reinder
DOI:
10.33612/diss.171585325
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Publication date:
2021
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de Vries, R. (2021). Chemical Modification of Peptide Antibiotics. University of Groningen.
https://doi.org/10.33612/diss.171585325
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Chapter 2
Selective Modification of Ribosomally Synthesized and
Post-translationally Modified Peptides (RiPPs) via
Diels-Alder Cycloadditions on Dehydroalanine Residues
This chapter describes 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 was 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.
Published as: R. H. de Vries, J. H. Viel, R. Oudshoorn, O. P. Kuipers, G. Roelfes, Chem. Eur. J. 2019,
Chapter 2
32
2.1 Introduction
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]
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 bio-orthogonal reactions.[11,12,21,13–20] In recent years,
these dehydroamino acids have emerged as interesting targets for the late-stage modification of RiPPs, via Michael additions,[22–25] hydrogenations,[26] cross-coupling
reactions,[27,28] photoredox catalysis,[29] cyclopropanations[30] and 1,3-dipolar
cycloadditions.[31] 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 2.1).
Scheme 2.1: 2-step labeling of dehydroalanines in RiPPs via a Diels-Alder and IEDDA
sequence. NN N N Inverse Electron Demand Diels-Alder R N H H N O O N H H N O O N NH R N H H N O O Diels-Alder
Selective Modification of RiPPs via Diels-Alder Cycloadditions on Dehydroalanine Residues
33
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 2.1), a popular labeling tool in chemical biology.[32]
2.2 Results and Discussion
As a starting point, the Diels-Alder reaction between cyclopentadiene and a protected dehydroalanine substrate (1) was studied (Table 2.1). In previous studies only anhydrous conditions and also high temperatures had been reported for this reaction.[33] The Diels-Alder reaction is known to be significantly accelerated in
water.[34] Indeed, appreciable conversion was observed in water at room temperature
after 48 hours, whereas no product was observed when using dichloromethane as solvent (Table 2.1).
Table 2.1: Optimization table of Diels-Alder reaction on 1.
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.[35] 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.[33] 1,3-cyclohexadiene,
1,3-dimethylbutadiene and furan were also evaluated as dienes, but did not give any conversion at room temperature.
Entry Solvent (1:1) Lewis Acid Reaction Time Conversion (%) 1 DCM - 48h - 2 H2O - 51h 36 ± 7 3 H2O/DMF - 51h 41 ± 6 4 H2O/ACN - 51h 27 ± 2 5 H2O/TFE - 51h 61 ± 1
6 H2O/TFE Sc(OTf)3 (10 mol%) 48h 43 ± 3
7 H2O/TFE Sc(OTf)3 (20 mol%) 48h 88 ± 3
N H O O O Conditions NHAc CO2Me CO2Me NHAc exo endo 1 60 : 40
Chapter 2
34
The conditions established with the protected Dha substrate appeared suitable for modification of the thiopeptide thiostrepton (Figure 2.1A), 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. On the contrary, the transition metal free conditions gave rise to the cleanest transformations to mainly single- and double modified thiostrepton (Figure 2.1B). 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.
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,[22] 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 2.1C). 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 product 2b, with particular focus on the region between 5.00 ppm and 7.00 ppm (Figure 2.2A) 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 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.
Selective Modification of RiPPs via Diels-Alder Cycloadditions on Dehydroalanine Residues 35 N H N O S N HN O S N H N HO O HN N S H N O S N OH NH NH O O H N O N H O S N NH H N O O NH2 O N OH OH O HO H H O H2O/TFE 1:1 50 °C, 16h S N NH H N O O NH2 O S N NH H N O O NH2 O Dha16-endo product 2a Dha16-exo product 2b Dha16 Dha17 Dhb8 Dha3 S N NH H N O O NH2 O thiostrepton Dha17 product 2c A) B) C)
Figure 2.1: 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.
Using 1H-1H TOCSY NMR, products 2a and 2b were both identified as
Dha16-modified thiostrepton (Figure 2.2B, example product 2B shown). In the TOCSY NMR of product 2b (Figure 2.2B) the strong correlation between the two shifted methylene signals (6.48 and 5.39 ppm) confirms that they originate from the same Dha residue. Also, a correlation is observed between these methylene signals and the amide N-H (9.19 ppm) that is characteristic for Dha17 (green). This is evidence for Dha17 still being intact but the signal has shifted and, conversely, the reaction has taken place at Dha16.
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
Chapter 2
36
the norbornene, it was established that product 2a is Dha16-endo and product 2b is Dha16-exo (Figure 2.2C). The methylene signals of Dha17 were shifted significantly more upfield in product 2a, indicating they are more shielded by the new double bond in the norbornene and thus residing in the endo-position in 2a and in the exo-position in 2b. Product 2c was identified as Dha17-modified thiostrepton using 1H NMR and 1H-1H TOCSY NMR techniques (Figure 2.2D). As opposed to the Dha16 products,
product 2c shows a correlation between the shifted methylene signals (6.59 and 5.25 ppm) and the characteristic Dha16 amide proton (9.78 ppm), while on the other hand it can be seen that the Dha17 amide signal (appearing at around 9 ppm in thiostrepton) has disappeared in this case.
A) B)
C) D)
Figure 2.2: A)Stacked 1H NMR spectra of thiostrepton (top) and product 2b (bottom), showing the region between 5.0 ppm and 7.0 ppm. B) Zoom of TOCSY NMR of product
2b, showing the key correlations. C) Stacked NMR spectra of product 2b (top) and 2a
(bottom), showing comparison of Dha17 methylene signals. D) Zoom of TOCSY NMR of product 2c, showing the key correlations.
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 (Figure
Selective Modification of RiPPs via Diels-Alder Cycloadditions on Dehydroalanine Residues
37
2.3A).[22] When 3 was subjected to the optimized reaction conditions, only two major
single modified products (4a and 4b) were observed in LC-MS (Figure 2.3B) and 41 % total conversion was observed using analytical HPLC. Both products were isolated as mixtures of diastereomers and identified as endo- (4a) and exo (4b) isomers of Dha16-modified 3 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 2.1B) 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.
Figure 2.3: A) Synthesis and Diels-Alder reaction of truncated thiostrepton (3). B)
LC-MS UV chromatogram (280 nm) of the crude reaction mixture (*single modification).
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 (Figure 2.4). The commercial nosiheptide starting material contained a small amount of nosiheptide that lacks the terminal Dha, having
S N NH H N O O NH2 O Dha16
Dha17 microwave (50 °C), H2O/TFE 1:1 16h S N NH NH2 O O Dha16 CHCl3 Et2NH S N NH NH2 O O
Trunc. Dha16-endo product
4a
thiostrepton Truncated thiostrepton 3
(42 %) S N N
H NH2
O O
Trunc. Dha16-exo product
4b A)
Chapter 2
38
a terminal amide instead. The product of the reaction of this impurity with cyclopentadiene was not observed in the LC-MS analysis (Figure 2.4B), 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.
Figure 2.4: A)
Diels-Alder reaction of nosiheptide. B) LC-MS UV chromatogram (280
nm) of the crude reaction mixture (*single modification).
The reaction between cyclopentadiene and the lanthipeptide nisin Z was investigated next (Figure 2.5A). 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,[36] single Diels-Alder modified
product was observed in the deconvoluted mass spectra after 16 hours of microwave irradiation at 50 °C (Figure 2.5B). The relative areas of the extracted ion chromatograms of the 4 species observed showed a 52 % conversion to Diels-Alder modified nisin Z. 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.
N S N N S N S OH NH S O N H O O S N O OH HN O S N NH O NH HO O HN O O NH2 nosiheptide H2O/TFE 1:1 microwave (50 °C) N S N N S N S OH NH S O N H O O S N O OH HN O S N NH O NH HO O HN O O NH2 A) B)
Selective Modification of RiPPs via Diels-Alder Cycloadditions on Dehydroalanine Residues
39
Figure 2.5: A) Diels-Alder reaction of nisin Z. B) Deconvoluted mass spectrum of the crude reaction mixture (*single modification).
Previous studies have shown that modification of the tail region of thiostrepton does not severely impact its activity.[22,27] 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. The results (Table 2.2) 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. H N O O NH S NH O N H O H N O O NH H N O S N O O H N HN O O NH H2N O HN N H OO NH HN O H N O S O HN O NH O N H S NH O H2N O O HN S O NH NH2 S O HN NH O N H O H N O NH O S O HN OH O NH O NH HN O HN N O N H O HN O OH NH2 NH2 0.1% AcOH (aq.)/ TFE 1:1 microwave (50 °C) O H2N nisin Z A) B)
Chapter 2
40
Table 2.2: MIC-assay results of Diels-Alder analogues of thiostrepton against S. aureus
and E. faecalis.
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.[32] Purified 2a was treated with di-2-pyridyl tetrazine (5) in H2O/ACN 1:1 at room temperature (Figure 2.6A) and after 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 2.6B). As a control, unmodified thiostrepton was subjected to the same conditions, after which only starting material (m/z = 1664, Figure 2.6B inset) was observed, illustrating the high chemoselectivity for the norbornene moiety over the other unsaturated motifs in thiostrepton. Next, the IEDDA reaction with a range of different functionalized tetrazines was investigated. An amine-functionalized tetrazine building block (8)[37] was derivatized with a fluorescein- (9) or biotin (10) moiety
(Figure 2.6C). MALDI-TOF MS showed efficient labeling of 2b with both tetrazines using the same conditions as described above (Figure 2.6C).
Antibiotic MIC (μg/mL) against S.
aureus (MSSA) MIC (μg/mL) against E. faecalis (VSE) Vancomycin 1 4 Thiostrepton 0.5 0.5 2a 2 2 2b 2 2 2c 2 1 3 0.5 1 4a 4 2 4b 2 2
Selective Modification of RiPPs via Diels-Alder Cycloadditions on Dehydroalanine Residues 41 S N NH H N O O NH2 O H2O/ACN 1:1, r.t. -N2 S N NH H N O O NH2 O N N R R 2a O O HO OH O H N S H N N N N N Fluorescein-tetrazine 9 N N N N N N HN NH S O H H H N O N N N N Biotin-tetrazine 10 5 S N NH H N O O NH2 O N NH R R Dihydropyridazine Pyridazine A) B) C)
Figure 2.6: 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) MALDI-TOF MS
Chapter 2
42
A BODIPY-labeled tetrazine (12) with interesting fluorescence turn-on properties was synthesized using a procedure by Carlson et al. with minor modifications (SI-3).[38] The fluorescence of 12 is quenched almost completely by the
tetrazine motif. However this effect is lifted upon reaction of the tetrazine in the IEDDA click reaction (Figure 4A).[38]
NBN F F NN N N Fluorescence Off "Click" N H H N O O N H H N O O N NH N B NF F Fluorescence On A) B) C) 12
Figure 2.7: A) Scheme depicting fluorescence turn-on of BODIPY-tetrazine upon click
reaction with the norbornene-modified peptide (top) and MALDI-TOF MS spectrum of the click product (bottom). B) Fluorimetric measurements of BODIPY-Tz click (red) compared to DMSO control (black). C) Image showing fluorescence under UV light (365 nm) for DMSO control (left) and click reaction with 2a (right).
Selective Modification of RiPPs via Diels-Alder Cycloadditions on Dehydroalanine Residues
43
Upon addition of 2a to a solution of 12, fluorescence measurements indeed showed a rapid increase in fluorescence compared to an identical solution of 12 where only DMSO was added as a control (SI-23). This fluorescence turn-on effect could even be visualized by shining UV light (365 nm) on the undiluted samples (Figure 4B), which shows the potential for using this 2-step labeling method in the detection of new Dha-containing peptides.
2.3 Conclusion
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 employing the norbornene 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 and probing applications.[39,40] These results demonstrate the potential of this methodology for the tailoring of RiPPs.
2.4 Experimental
General remarks
Chemicals were purchased from Sigma-Aldrich, Acros Organics, TCI Europe, Fluorochem and Activate Scientific and used without further purification unless explicitly specified. Thiostrepton , nosiheptide and nisin Z were purchased from CalBioChem, Carbosynth Ltd. and Handary, respectively. Cyclopentadiene was freshly distilled and used immediately. Flash column chromatography was performed on silica gel (Silica gel 60 from Merck, 0.040-0.063 mm, 230-400 mesh). TLC was performed on silica gel (Silica-P flash silica gel from Silicycle, 0.040-0.063 mm , 230-400 mesh). Melting points were recorded on a Büchi B-545 melting point apparatus. 1H-, 13C- and 19F-NMR spectra were recorded on an Agilent 400-MR at 298K
spectrometer operating at 400, 101 and 376 MHz respectively. 1D and 2D 1H NMR on
thiostrepton and its derivatives was performed on a Brüker Ascend 600 operating at 600 MHz. Chemical shifts in 1H and 13C NMR spectra were internally referenced to solvent signals (CDCl
3
at δH = 7.26 ppm, δC = 77.16 ppm; DMSO-d6 at δH = 2.50 ppm, δC = 39.51 ppm). LC-MS analysis
was performed on a Waters Acquity UPLC with TQD mass detector (ESI+). All analysis was performed at 35 °C using a reversed-phase UPLC column (Waters Acquity UPLC BEH C8, 1.7 μm, 2.1 mm x 150 mm). UPLC grade 0.1 % Formic Acid (FA) in H2O (solvent A) and 0.1 % FA in
Chapter 2
44
acetonitrile (solvent B) were used as eluents. Gradient used for thiostrepton and nosiheptide and derivatives: 70 % A to 30 % A over 8 minutes, then to 5 % A over 1 minute (total runtime 15 minutes). All other measurements were done using a gradient of 90 % A to 50 % A over 8 minutes, then to 5 % A over 1 minute (total runtime 15 minutes). High-resolution mass spectrometry was performed on a LTQ Orbitrap XL spectrometer (ESI+). MALDI-TOF MS was performed on an Applied Biosystems 4800 plus TOF/TOF analyzer. Reversed phase HPLC was performed on a Shimadzu HPLC system equipped with LC-20AD solvent chromatographs, a DGU-20A3 degasser unit, a SIL-20A autosampler, a SPD-M20A PDA detector, a CTO-20A column oven, a CBM-20A system controller and a FRC-10A fraction collector. Analysis was performed on a Waters XBridge C8 column (4.6 x 250 mm, particle size 3.5 μm) using a flow of 0.5 mL/min. Preparative HPLC was performed on a Waters XBridge prep C8 column (10 x 150 mm, particle size 5 μm) using a flow of 1.5 mL/min.
Methyl 2-(acetamido)acrylate (1):[41] 5.00 g (84.7 mmol) acetamide, 7 mL (77.5
mmol) methyl pyruvate, a catalytic amount of p-TsOH and a catalytic amount of 4-methoxyphenol were dissolved in 150 mL toluene. The flask was equipped with a Dean-Stark trap and the mixture was heated under reflux for 20 hours. The mixture was then concentrated in vacuo and the residue was taken up in 300 mL DCM. The organic phase was washed with 300 mL NaHCO3 (sat. aq.) and 300 mL H2O. The organic layer was then dried over MgSO4, filtered
and concentrated in vacuo to yield yellow crystals, which were further purified by column chromatography (SiO2, heptane/EtOAc 4:1 -> 1:1). 5.13 g (35.8 mmol, 47 %) of white crystals
were obtained. Melting point: 52.5-54 °C (Lit.: 48 °C). 1H NMR (400 MHz, CDCl
3) δ 7.71 (s, 1H),
6.60 (s, 1H), 5.88 (d, J = 1.5 Hz, 1H), 3.85 (s, 3H), 2.13 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 168.9,
164.8, 131.0, 108.8, 53.1, 24.8.
tert-butyl (4-cyanobenzyl)carbamate (7):[42] To 250 mg (1.48 mmol)
4-(aminomethyl) benzonitrile hydrochloride in 5 mL DCM under N2
atmosphere was added 0.41 mL (2.96 mmol) Et3N, after which a clear solution was obtained.
The mixture was cooled to 0 °C and 0.39 g (1.78 mmol) Boc2O was added. After stirring for 5
minutes at 0 °C the mixture was allowed to warm to r.t. and was stirred overnight under N2
atmosphere. The solvent was evaporated and the residue was redissolved in 10 mL Et2O. The
ethereal layer was washed with 2x5mL 0.5 M HCl (aq.), after which the combined aqueous layers were back-extracted with 10 mL Et2O. The combined organic layers were then washed
with 2x5 mL NaHCO3 (sat. aq.) and 5 mL brine. After drying over MgSO4 the solvent was
evaporated, yielding a white solid, which was further purified by recrystallization from petroleum ether 40-65 (200 mL solvent used). 244 mg (1.05 mmol, 71 %) white crystals were obtained. Melting point: 109-110 °C (Lit.: 106-108 °C). 1H NMR (400 MHz, CDCl
3) δ 7.61 (d, J = 8.2 Hz, 2H), 7.38 (d, J = 8.1 Hz, 2H), 5.01 (s, 1H), 4.36 (d, J = 6.2 Hz, 2H), 1.45 (s, 9H). 13C NMR N H O O O NHBoc NC
Selective Modification of RiPPs via Diels-Alder Cycloadditions on Dehydroalanine Residues
45 (151 MHz, CDCl3) δ 156.0, 144.8, 132.5, 127.9, 118.9, 111.2, 80.2, 44.3, 28.5. LC-MS (ESI+) m/z:
233.2 [M+H]+, 218.1 [M-Me+H]+, 133.1 [M-Boc+H]+.
tert-butyl (4-(1,2,4,5-tetrazin-3-yl)benzyl)carbamate (8):[37] 58 mg (0.25
mmol) tert-butyl (4-cyanobenzyl)carbamate, 46 mg (0.125 mmol) Zn(OTf)2
and 0.26 g (2.5 mmol) formamidine acetate were added to a microwave vial. The vial was sealed, after which 0.2 mL DMF was added, followed by 0.61 mL (12.5 mmol) hydrazine monohydrate. The mixture was left to stir at 40 °C for 72 hours, after which the mixture was allowed to cool to room temperature. Then, 345 mg (5 mmol) NaNO2 in 5 mL H2O was added
slowly to the mixture. 1M HCl (aq.) was then added dropwise until pH ≤ 3 and bubbling ceased. The aqueous layer was then extracted with 5x20 mL EtOAc, the combined organic layers were dried over MgSO4 and the solvent was evaporated. The purple residue was purified by flash
column chromatography (SiO2, n-hexane/EtOAc 7:1) to give 14 mg (0.048 mmol, 19 %) of a
purple solid. 1H NMR (400 MHz, CDCl3) δ 10.21 (s, 1H), 8.60 (d, J = 8.3 Hz, 2H), 7.52 (d, J = 8.1 Hz,
2H), 4.99 (s, 1H), 4.45 (d, J = 5.5 Hz, 2H), 1.48 (s, 9H). 13C NMR (151 MHz, CDCl
3) δ 166.5, 157.9,
156.1, 144.8, 130.7, 128.8, 128.3, 80.1, 44.5, 28.6.
Biotin-NHS (13):[43] 250 mg (1.02 mmol) D-Biotin and 189 mg (1.64
mmol) N-hydroxy succinimide were added to dry DMF under a N2
atmosphere. 255 mg (1.33 mmol) EDC.HCl was added and the mixture was stirred under N2 atmosphere for 24 hours. The mixture was then
poured onto crushed ice and the precipitate was collected by filtration. The resulting white solid was washed with 3x20 mL ice cold H2O and dried in vacuo
overnight. 261 mg (0.77 mmol, 75 %) of a white solid was obtained. Melting point: 200-207 °C (decomp.) (Lit.: 206-207 °C). 1H NMR (400 MHz, DMSO-d6) δ 6.42 (s, 1H), 6.36 (s, 1H), 4.39 – 4.24 (m, 1H), 4.22 – 4.07 (m, 1H), 3.16 – 3.05 (m, 1H), 2.87 – 2.75 (m, 5H), 2.67 (t, J = 7.4 Hz, 2H), 2.58 (d, J = 12.5 Hz, 1H), 1.73 – 1.57 (m, 3H), 1.57 – 1.34 (m, 3H). 13C NMR (101 MHz,
DMSO-d6) δ 170.2, 168.9, 162.7, 61.0, 59.2, 55.2, 30.0, 27.8, 27.6, 25.4, 24.3 (1 signal missing due to overlap).
Fluorescein-H-tetrazine (9): tert-butyl
(4-(1,2,4,5-tetrazin-3-yl)benzyl)carbamate (8) was
deprotected by dissolving 12 mg (42 μmol) in 5 mL DCM/TFA 1:1 and stirring this solution at r.t. for 10 minutes, followed by evaporation of the solvents and flash column chromatography (SiO2, DCM/MeOH 9:1), giving 5 mg (27
μmol, 64 %) of (4-(1,2,4,5-tetrazin-3-yl)phenyl)methanamine TFA salt, which was used without further purification. 2.3 mg (9.3 μmol) (4-(1,2,4,5-tetrazin-3-yl)phenyl)methanamine was then dissolved in 50 μL dry DMF, after which
BocHN N N N N HN NH S O H H O O N O O O O HO OH O H N S H N N N N N
Chapter 2
46
39 μL (0.28 mmol) TEA was added, followed by 4 mg (10.2 μmol) fluorescein isothiocyanate (FITC) in 50 μL dry DMF. The solution was stirred at r.t. under a N2 atmosphere overnight and
then diluted with 100 μL H2O/ACN 1:1, filtered through a microfilter and subjected to prep
RP-HPLC (solvent A: 0.1 % FA in ACN, solvent B: 0.1 % FA in ddH2O, gradient 90 % B to 10 % B
over 40 minutes). After lyophilization of the appropriate fractions trace amounts of product were obtained (≤ 1 mg). HRMS calcd. C30H21N6O5S+ [M+H]+: m/z = 577.1289, found: 577.1290.
Biotin-H-tetrazine (10): The same procedure as for
Fluorescein-H-tetrazine was followed, starting from 2.3 mg (9.3 μmol) (4-(1,2,4,5-tetrazin-3-yl)phenyl)methanamine and 3.5 mg (10.2 μmol) Biotin-NHS (13). After prep-HPLC
purification and lyophilization, trace amounts (≤1 mg) of a pink solid were obtained. HRMS calcd. C19H24N7O2S+ [M+H]+: m/z = 414.1707, found: 414.1713.
m-Cyanophenyl BODIPY (11):[38] 0.50 g (3.8 mmol) 3-formylbenzonitrile was
dissolved in 100 mL DCM. 0.85 mL (8.25 mmol) 2,4-dimethylpyrrole was added, followed by 3 drops of TFA and the mixture was stirred under a N2 atmosphere
for 1 hour until TLC (SiO2, n-hexane/EtOAc 7:1) indicated full consumption of
3-formylbenzonitrile. 0.86 g (3.8 mmol) DDQ in 100 mL DCM was added, after which the solution turned dark purple immediately. 7.8 mL (44.5 mmol) DIPEA was then added, followed by 8 mL (46 %, 29.8 mmol) BF3·OEt2 and the mixture was stirred at r.t. under a N2
atmosphere overnight. 50 mL H2O was then added and the layers were separated. The aqueous
layer was then extracted with 3x150 mL DCM and the combined organic layers were dried over MgSO4 and concentrated in vacuo. The residue was then purified by flash column
chromatography (SiO2, toluene/n-hexane 3:1 -> 9:1 in 3 steps), after which 368 mg (1.06 mmol,
28 %) of a bright orange solid was obtained. Melting point: 233-234 °C. 1H NMR (400 MHz,
CDCl3) δ 7.80 (dt, J = 7.8, 1.4 Hz, 1H), 7.69 – 7.61 (m, 2H), 7.58 (dt, J = 7.8, 1.5 Hz, 1H), 6.01 (s,
2H), 2.56 (s, 6H), 1.35 (s, 6H). 19F NMR (376 MHz, CDCl
3) δ -145.98 – -146.60 (m). 13C NMR (151
MHz, CDCl3) δ 156.8, 142.7, 138.1, 136.7, 133.0, 132.9, 132.0, 131.2, 130.3, 122.0, 118.0, 113.7,
14.9 (1 signal missing due to overlap). LC-MS (ESI+) m/z: 350.2 [M+H]+, 330.2 [M-F-]+.
BODIPY-H-tetrazine (12): 98 mg (0.28 mmol) m-cyanophenyl BODIPY, 51 mg
(0.14 mmol) Zn(OTf)2 and 0.292 g (2.8 mmol) formamidine acetate were added
to a microwave vial. The vial was sealed and 0.35 mL DMF and 0.68 mL (14 mmol) hydrazine monohydrate were added. The mixture was stirred at 60 °C for 24 hours. After allowing the mixture to cool down to r.t., 300 mg (4.35 mmol) NaNO2 in 10 mL H2O was added slowly. To this solution was then added 1M HCl (aq.)
until pH ≤ 3, after which the aqueous solution was extracted with 3x100 mL DCM. The combined organic layers were dried over MgSO4 and the solvent was evaporated. Two times
HN NH S O H H H N O N N N N NBN CN F F NBN F F NN N N
Selective Modification of RiPPs via Diels-Alder Cycloadditions on Dehydroalanine Residues
47 flash column chromatography (SiO2, n-hexane/EtOAc 4:1 to 1:1 in 2 steps and SiO2,
n-hexane/EtOAc 2:1) yielded 3.9 mg (8.4 μmol, 3 %) of a red brittle solid. 1H NMR (400 MHz,
CDCl3) δ 10.26 (s, 1H), 8.76 (d, J = 7.8 Hz, 1H), 8.66 – 8.59 (m, 1H), 7.77 (t, J = 7.8 Hz, 1H), 7.61
(d, J = 7.6 Hz, 1H), 5.99 (s, 2H), 2.61 (s, 6H), 1.42 (s, 6H). HRMS calcd. C21H19BFN6+ [M-F-]+: m/z =
385.1748, found: 385.1747.
Truncated thiostrepton (3): 100 mg (0.06 mmol)
thiostrepton was dissolved in 6 mL CHCl3. The
mixture was cooled to 0 °C and 0.5 mL Et2NH was
added over 5 minutes. The mixture was then allowed to warm up and was stirred at r.t. for 3 hours. TLC (SiO2, CHCl3/MeOH 9:1) indicated
incomplete conversion, so another 0.3 mL Et2NH
was added at once and the mixture was stirred for another 15 minutes. When TLC showed complete conversion, the mixture was co-evaporated with 5 mL toluene. Flash column chromatography (SiO2, CHCl3/MeOH 0-5% MeOH in 5 steps) afforded 40 mg (25 μmol, 42%) of the desired
product that contained a small amount (≤8 %) of thiostrepton truncated at Dha16 (-2Dha), as a colorless brittle solid. 1H NMR (600 MHz, CDCl
3) δ 9.95 (s, 1H), 9.83 (s, 1H), 8.51 (s, 1H), 8.32 (d, J = 9.1 Hz, 1H), 8.27 – 8.23 (m, 2H), 8.11 (s, 1H), 7.80 (s, 1H), 7.58 – 7.55 (m, 2H), 7.54 – 7.52 (m, 1H), 7.46 (s, 1H), 7.30 (s, 1H), 6.91 – 6.86 (m, 2H), 6.85 – 6.80 (m, 1H), 6.74 (d, J = 1.9 Hz, 1H), 6.42 (d, J = 7.5 Hz, 1H), 6.40 – 6.35 (m, 1H), 6.30 (ddd, J = 9.8, 5.5, 1.5 Hz, 1H), 6.19 (q, J = 7.0 Hz, 1H), 5.84 (d, J = 9.1 Hz, 1H), 5.79 – 5.75 (m, 2H), 5.45 – 5.41 (m, 1H), 5.35 – 5.29 (m, 2H), 5.23 – 5.19 (m, 1H), 5.11 (s, 1H), 4.96 (dd, J = 13.4, 8.7 Hz, 1H), 4.80 – 4.73 (m, 1H), 4.68 (d, J = 8.0 Hz, 1H), 4.46 (dd, J = 8.0, 3.3 Hz, 1H), 4.12 – 4.05 (m, 3H), 3.88 – 3.77 (m, 2H), 3.71 (dd, J = 11.4, 8.6 Hz, 1H), 3.63 (dd, J = 5.6, 1.6 Hz, 1H), 3.53 – 3.40 (m, 1H), 3.12 (dd, J = 13.4, 11.4 Hz, 1H), 2.99 (d, J = 6.3 Hz, 1H), 2.98 – 2.85 (m, 1H), 2.26 (td, J = 12.8, 5.8 Hz, 1H), 1.74 (d, J = 6.5 Hz, 3H), 1.62 (d, J = 7.0 Hz, 3H), 1.52 – 1.43 (m, 5H), 1.37 (d, J = 6.4 Hz, 3H), 1.33 (d, J = 6.5 Hz, 3H), 1.20 – 1.16 (m, 8H), 1.10 – 1.05 (m, 1H), 1.00 (d, J = 6.0 Hz, 3H), 0.95 (t, J = 7.4 Hz, 3H), 0.89 (d, J = 6.8 Hz, 3H). HRMS calcd. C69H83N18O17S5+ [M+H]+: m/z = 1595.4782, found: 1595.4761.
Diels-Alder on methyl 2-(acetamido)acrylate (1): 20 mg (0.14 mmol) methyl
2-(acetamido)acrylate (1) was dissolved in 1 mL ddH2O in a 6 mL vial equipped with a stirring
bar. 1 mL of cosolvent and, where appropriate, 0.1 or 0.2 eq. Lewis acid were added. Then, 5 or 10 eq. freshly distilled cyclopentadiene was added and the reaction mixture was stirred at room temperature for 24-50 hours. The reaction mixture was extracted to 5 mL DCM. The organic layer was separated, dried over MgSO4 and concentrated in vacuo. The crude mixture
containing starting material and endo- and exo products was then dissolved 0.6 mL CDCl3. 1H
NMR spectra obtained from the crude product were in accordance with previously reported
N H N O S N HN O S N H N HO O HN N S H N O S N OH NH NH O O H N O N H O S N NH NH2 O O N OH OH O HO H H O
Chapter 2
48
spectra of the endo- and exo products.[33] Conversions and endo/exo ratios were based on
relative integrations of known 1H NMR signals of the starting material and products.
Diels-Alder on thiostrepton and 3: In a typical procedure, 1 μmol thiostrepton or 3 was
dissolved in 0.5 mL TFE. 0.5 mL ddH2O was added, followed by 50 μL (0.6 mmol) freshly distilled
cyclopentadiene. The mixture was stirred overnight in a microwave reactor at 50 °C (50 W power). For LC-MS analysis, samples were prepared by diluting 100 μL of the reaction mixture with 200 μL ddH2O/ACN 1:1 and filtering over a microfilter (0.2 μm). Overall conversion was
determined using analytical RP-HPLC (solvent A: 0.1 % FA in ACN, solvent B: 0.1 % FA in ddH2O,
gradient 60 % B to 10 % B over 40 minutes) of the crude reaction mixture.
Preparative scale and purification: 15 μmol thiostrepton or 3 was dissolved in 5 mL TFE and 5
mL H2O was added. 252 μL (3 mmol) freshly distilled cyclopentadiene was added daily for up to
7 days of total reaction time, during which the mixture was stirred at room temperature. Then, the mixture was transferred to a separatory funnel, and the bottom aqueous layer was separated. The aqueous layer was filtered over a plug of Celite and finally filtered over a microfilter (0.2 μm). The obtained clear filtrate was directly subjected to preparative HPLC. The crude products were purified by preparative HPLC (solvent A: 0.1 % FA in ACN, solvent B: 0.1 % FA in ddH2O, gradient 60 % B to 10 % B over 40 minutes). Analysis of the fractions by
LC-MS, followed by lyophilization of the combined pure fractions resulted in the isolation of the pure products as white brittle solids. The identity of the products was confirmed using HRMS: calcd. C77H92N19O18S5 [M+H]+ (singly modified thiostrepton): 1730.547, found: 1730.536 (2a,
Figure 2.8), 1730.536 (2b), 1730.547 (2c); calcd. C74H89N18O17S5 [M+H]+ (singly modified 3):
Selective Modification of RiPPs via Diels-Alder Cycloadditions on Dehydroalanine Residues
49
Figure 2.8: HRMS spectrum of 2a (top) and theoretical HRMS simulation (bottom).
Figure 2.9: HRMS spectrum of 4a (top) and theoretical HRMS simulation (bottom).
Diels-Alder on nosiheptide: 1.2 mg (1 μmol) nosiheptide was dissolved in 0.5 mL TFE in a
microwave vial. Then, 0.5 mL ddH2O and 50 μL (0.6 mmol) freshly distilled cyclopentadiene
were added and the mixture was heated for 16 hours in a microwave reactor at 50 °C (50 W power). After addition of another portion (50 μL) of freshly distilled cyclopentadiene the mixture was heated again in a microwave reactor at 50 °C for 16 hours. For LC-MS and HPLC
1720 1725 1730 1735 1740 1745 m/z 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 1730.53613 z=1 1731.54033 z=1 1732.54131 z=1 1733.53617 z=1 1734.53729 z=1 1742.55692 z=? 1725.30594 z=? 1728.56745z=? 1739.09191z=1 1721.95401 z=? 1746.04420z=? 1730.54658 1731.54993 1732.55329 1733.54573 1734.54908 1735.55244 1738.55159 1741.54828 1744.54743 1660 1661 1662 1663 1664 1665 1666 1667 m/z 0 20 40 60 80 100 0 20 40 60 80 100 R el at iv e A bundanc e 1661.5343 z=1 1662.5373 z=1 1663.5393 z=1 1664.5334 z=1 1665.5388 z=1 1663.0360 z=2 1666.5341z=1 1662.0383 z=2 1660.5216 z=? 1661.5251 1662.5285 1663.5318 1664.5243 1665.5276 1666.5310
Chapter 2
50
analysis, samples were prepared by diluting 100 μL reaction mixture with 200 μL ddH2O/ACN
1:1 and filtering over a microfilter (0.2 μm). Overall conversion was determined using analytical RP-HPLC (solvent A: 0.1 % FA in ACN, solvent B: 0.1 % FA in ddH2O, gradient 60 % B to 10 % B
over 40 minutes) of the crude reaction mixture, showing a total conversion of 75 % to singly modified nosiheptide.
Diels-Alder on nisin Z: In a typical procedure, 3.3 mg (1 μmol) nisin Z was dissolved in 0.5 mL
ddH2O with 0.1 % AcOH. 0.5 mL TFE was added, followed by 100 μL (1.2 mmol) freshly distilled
cyclopentadiene. The mixture was stirred overnight in a microwave reactor at 50 °C (50 W power). For LC-MS analysis, samples were prepared by diluting 100 μL reaction mixture with 200 μL ddH2O/ACN 1:1 and filtering over a microfilter (0.2 μm). Due to the low UV absorption
coefficient of nisin Z, the conversion could not be determined using UV signal integration, but was instead estimated using ion current integrations, assuming that all nisin derivatives are ionized to a similar extent. The extracted ion chromatogram signals were integrated individually and compared to obtain the relative composition of the mixture in order to give an estimate of the total conversion to Diels-Alder modified nisin Z (Table 2.3). From the composition
percentages a total conversion of 52 % to Diels-Alder modified nisin Z was estimated (including modified nisin Z that also underwent water addition) (Table 2.3).
Table 2.3: Extracted ion chromatograms of modified nisin Z (top) and found masses and relative
compositions (bottom), from which the conversion was estimated.
Species [M+4H]4+
found Mcalc Mdecon Area %
Nisin Z 833.9 3331 3332 24
Nisin Z + H2O 838.0 3349 3348 24
Nisin Z + 1 Mod. 850.5 3397 3397 27
Selective Modification of RiPPs via Diels-Alder Cycloadditions on Dehydroalanine Residues
51
General procedure for IEDDA reaction to Diels-Alder products of thiostrepton: To 44 μL 20 μM
2b in ddH2O/ACN 1:1 was added 2 μL 46 mM tetrazine in DMSO. The mixture was stirred
overnight at room temperature, after which the reaction mixture was analyzed by MALDI-TOF MS (CHCA-matrix) (see Figure 2.6).
Fluorescence turn-on upon IEDDA reaction of BODIPY-Tz with modified thiostrepton: 3 μL
2.16 mM 2a in DMSO and 3 μL 51 μM BODIPY-Tz (12) in H2O/ACN 1:1 (used immediately after
preparative HPLC purification) were added to 50 μL ACN (final concentrations: 116 μM 2a and
2.7 μM BODIPY-Tz (12)). Under UV light (365 nm) a clear fluorescence was observed after
stirring at r.t. for 1 hour. After overnight reaction, the mixture was analyzed by MALDI-TOF MS (CHCA matrix) (see Figure 2.7). For fluorescence measurements, the reaction mixture and
control were both diluted to 2 mL with 1944 μL ACN.
Biological Activity Assays
Preparation of antimicrobial agents: Thiostrepton and its variants were dissolved and diluted in
DMSO to a concentration of 640 µg/mL and stored at -20 °C. Before use, they were diluted 20-fold in Mueller Hinton Broth 2 (CAMHB; cation-adjusted, Sigma-Aldrich). Dilution of the thiostrepton compounds in CAMHB was done in 50 % volume steps to prevent precipitation. Vancomycin for the quality controls was dissolved in MQ to a concentration of 256 mg/mL and stored at -20 °C. The vancomycin stock was diluted in CAMHB to a final concentration of 256 µg/mL before use.
Strains and growth conditions: The MIC values of thiostrepton and its variants were
determined for Staphylococcus aureus LMG 10147 (ATCC29213) and Enterococcus faecalis LMG 08222 (ATCC29212). LMG 10147 and LMG 08222 were cultured from glycerol stocks on LB (Formedium™) and GM17 (Difco™) plates respectively. For the MIC determination tests, LMG 10147 was grown in CAMHB, while LMG 08222 was grown in CAMHB + 3 % v/v lysed horse blood (TCS Biosciences). For all steps, the incubation temperature was 37 °C.
DMSO as a solvent: As the thiostrepton compounds were diluted from stocks in DMSO, residual
amounts of DMSO remained in the MIC test plates (down from 2.5% in the first well). As a control for potential side effects of DMSO, both strains were grown in DMSO concentrations representative of those present in the test plates, without the addition of antimicrobial compounds. No growth inhibition was observed at any of the tested DMSO concentrations.
Broth microdilution: MIC testing and internal controls were performed employing the 96-well
plate broth microdilution method described in Wiegand et al., 2008[44], which is outlined in short here.
Chapter 2
52
First, 50 µL and 100 µL of sterile CAMHB is added to columns 2-11 and 12 of a 96-well plate, respectively. Then, 100 µL of freshly diluted test compound is added to the first well. A serial dilution of the compound is achieved by transferring 50 µL from the first well to the second, mixing, and then continuing these steps until well 10. Finally, 50 µL of bacterial suspension is added to wells 1 to 11, resulting in a final cfu of 5 x 105 mL-1 in each well. Well 11, lacking the
test compound, functions as a growth control and well 12 as a sterility control. Before incubation, several dilutions from a growth control well are plated as a control for the number of cfu’s. To ensure MIC data reliability, MIC values for vancomycin were determined for every series of tests performed, as described by the CLSI standard.[45]
The MIC test plates were placed in an airtight container to prevent evaporation, and incubated for 20 hours before reading. The concentration of compound in the first well of the serial dilution that shows no visible growth of the test strain is considered the MIC value. All compounds were tested three times in triplicates.
Selective Modification of RiPPs via Diels-Alder Cycloadditions on Dehydroalanine Residues
53 Appendix: NMR spectra
Figure S1: 1H NMR spectrum of thiostrepton-Dha16-endo (2a).
Chapter 2
54
Figure S3: 1H NMR spectrum of thiostrepton-Dha16-exo (2b).
Selective Modification of RiPPs via Diels-Alder Cycloadditions on Dehydroalanine Residues
55
Figure S5: 1H NMR spectrum of thiostrepton-Dha17 (2c).
Chapter 2
56
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