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 4
Rapid and Selective Chemical Editing of Ribosomally
Synthesized and Post-translationally Modified Peptides
(RiPPs) via Cu(II)-Catalyzed β-Borylation of Dehydroamino
Acids
This chapter describes the fast and selective chemical editing of ribosomally synthesized and post-translationally modified peptides (RiPPs) by β-borylation of dehydroalanine residues. The thiopeptide thiostrepton was modified efficiently using Cu(II)-catalysis under mild conditions and 1D/2D NMR of the purified product showed site-selective borylation of the terminal Dha residues. Using similar conditions, the thiopeptide nosiheptide, lanthipeptide nisin Z and protein SUMO_G98Dha were also modified efficiently. Borylated thiostrepton showed an up to 84-fold increase in water solubility, and MIC assays showed that antimicrobial activity was maintained in thiostrepton and nosiheptide. The introduced boronic acid functionalities were shown to be valuable handles for chemical mutagenesis and in a reversible click reaction with triols for the pH-controlled labeling of RiPPs.
Published as: R.H. de Vries, J.H. Viel, O.P. Kuipers, G. Roelfes, Angew. Chem. 2021, 133, 3992-3996;
Chapter 4
80
4.1 Introduction
Ribosomally synthesized and post-translationally modified peptides (RiPPs) have gained interest as alternative sources of new antibiotics due to their high activity and low development of resistance.[1–5] However, clinical application is hampered by their low water solubility and poor metabolic stability.[6,7] Late-stage chemical editing of
these peptides is a popular approach to make semi-synthetic analogues with improved pharmacological properties.[7] For the chemical fine-tuning of these
properties, achieving derivatization that is both chemo- and site-selective is crucial. However, this is particularly challenging in the case of RiPPs due to their high structural diversity and complexity compared to generic peptides and proteins, which is a result of the post-translational modifications occurring during their biosynthesis.
The dehydroamino acids dehydroalanine (Dha) and dehydrobutyrine (Dhb), which occur naturally in RiPPs[8] and have a distinct reactivity as carbon electrophiles,
have proven to be excellent bio-orthogonal handles for the selective modification of peptides and proteins.[9–17] Michael-type additions,[18–21] radical additions,[22,23]
cross-coupling reactions,[24,25] amidations,[26] cyclopropanations[27] and cycloadditions[28,29] on Dha have been applied successfully in the derivatization of
RiPPs via C-C, C-N and C-S linkages. Recently we reported the formation of C-Si bonds via β-silylation of dehydroalanines.[30] However, methods for forming C-B bonds on
Dha are currently lacking, which leaves the vast utility of such abiological functionalities unexplored in RiPPs.
Boronic acids have been applied in medicinal chemistry to act as targeting groups and to provide increased activity, stability and water solubility,[31,32] which
makes them interesting functional groups for the modification of RiPPs. Additionally, they are an attractive motif for side chain diversification, since they are versatile intermediates for various chemical transformations and can undergo reversible boronate ester formation with alcohols.[33,34] Here we report the straightforward,
rapid and selective Cu(II)-catalyzed β-borylation of dehydroalanine residues in RiPPs.
4.2 Results and Discussion
The β-borylation of α,β-unsaturated carbonyl compounds has been reported with Cu(I)[35–38] or Cu(II) catalysis[39–42] and using tetrahydroxydiboron (B
2(OH)4) or
bis(pinacolato)diboron (B2Pin2) as the borylating agent. In contrast to the Cu(I)
open to the air, which is a promising starting point for peptide modification. To test whether Dha is a good substrate for this reaction, a protected Dha (1) was subjected
to the borylation conditions reported by Santos (Figure 4.1A),[39] followed by oxidation
to the serine derivative 2. After 24 hours complete consumption of starting material 1
accompanied by the formation of the serine product (2) were observed in the LC-MS
of the crude product (Figure 4.1B).
Figure 4.1: A) β-borylation of Dha substrate 1. Conditions: 0.2 M 1, 1.1 eq. B2(OH)4, 1 mol% CuSO4, 5 mol% 4-picoline, then 3 eq. NaBO3.4H2O. B) LC-MS UV chromatogram of the crude product (2).
Encouraged by these results, we studied the β-borylation of Dha residues in the thiopeptide thiostrepton (Figure 4.2A). The conditions were slightly modified, as
2,2,2-trifluoroethanol was used as a co-solvent to solubilize thiostrepton. It was also found that the addition of the base 4-picoline did not have a significant effect on the conversion, but had a negative impact on the stability of the peptide and it was therefore omitted. Full conversion of thiostrepton to one major product (3) was
observed after 1 hour in the LC-MS analysis of the crude mixture (Figure 4.2B). The
mass spectrum of 3 showed an m/z of 1778, which corresponds to the [M+Na]+
adduct of the doubly modified peptide, showing that thiostrepton is modified efficiently under these conditions. Singly and doubly dehydrated adducts (-H2O) were
also observed in LC-MS and HRMS analysis of 3. Loss of water is commonly observed
in the mass spectrometry analysis of borylated peptides and proteins.[43–46] It is unclear whether this is the result of reversible dimerization of the boronic acids or condensation reactions with –OH and –NH residues in the peptide in solution, or a dehydration reaction that occurs under the mass spectrometry conditions. However,
N H O O O CuSO4, 4-picoline NH O O O B(OH)2 1 H2O/THF NH O O O OH NaBO3.4H2O 2 A) B) H2O, r.t. B2(OH)4
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82
this dehydration is fully reversible as is evident from the follow-up derivatization reactions of the borylated products (vide infra).
5 mol% CuSO4, TFE/H2O 1:1, r.t. 1 hour 50 eq. B2(OH)4 thiostrepton (1 mM) 3 A) B) C) 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 Dha16 Dha17 Dhb8 Dha3 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 Dhb8 Dha3 B(OH)2 B(OH)2
Figure 4.2: A) β-borylation of thiostrepton (1 mM). B) LC-MS UV chromatogram of the
crude reaction mixture after 1 hour at room temperature. C) Comparison of 1H NMR spectra of thiostrepton (top) and 3 (bottom), showing the disappearance of the signals
corresponding to Dha16 (blue) and Dha17 (green) and the appearance of new α-proton signals (red box).
Product 3 was isolated as a mixture of diastereomers using preparative
reversed-phase HPLC and characterized by HRMS and 1D and 2D NMR techniques. Using 2D NMR an authentic sample of thiostrepton was studied and the methylene signals of the different dehydrated residues were assigned (Figure 4.2C). The 1H NMR
spectra of thiostrepton and 3 were then compared to show that the methylene signals
of the 2 Dha residues in the tail region of thiostrepton (Dha16 and Dha17) have disappeared (Figure 4.2C). Also, 2 new signals can be observed in the α-proton region
(4.5-4.7 ppm). Together with the HRMS data, these results are consistent with a double borylation of the tail region of thiostrepton, which is known to be the most reactive part of this natural product.[18,29] This signifies that our method is not only rapid, but also displays a high degree of chemo- and site-selectivity.
The thiopeptide nosiheptide was also subjected to the borylation conditions (Figure 4.3A). In this case, the addition of 4-picoline was found to be crucial for
efficient conversion to the borylated peptide. In the LC-MS analysis (Figure 4.3B) of
the crude reaction mixture 94 % conversion to the singly borylated product (4) was
observed based upon integration of the different peaks in the UV chromatogram after 1 hour reaction time.
CuSO4,4-picoline TFE/H2O 1:1, r.t. B2(OH)4 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 A) B) 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 B(OH)2 4
Figure 4.3: A) β-borylation of nosiheptide. Conditions: nosiheptide (1 mM), 50 eq.
B2(OH)4, 1.1 eq. CuSO4, 10 eq. 4-picoline. B) LC-MS UV chromatogram (280 nm) of the crude reaction mixture (*single modification).
Next, it was investigated whether installing the boronic acid functionality on RiPPs can help to increase the water solubility; low solubility has been one of the main limitations for application of the thiopeptide class of RiPPs to which thiostrepton and nosiheptide belong.[6]4 and nosiheptide were purified using preparative HPLC and the
water solubility of thiostrepton, nosiheptide and their borylated variants in ddH2O and
50 mM TEAA (pH=7.9) was measured. A significant (up to 84-fold) increase in water solubility for 3 compared to unmodified thiostrepton was observed (Table 4.1). Due to
its inherently poor water solubility, nosiheptide, as well as borylated variant 4,
demonstrated a solubility that was too low to be detected (Table 4.1). However, the
lower retention times in reversed-phase chromatography qualitatively indicated an increased aqueous solubility also for the borylated variant of nosiheptide (4)
Chapter 4
84
Furthermore, the antimicrobial activity of thiostrepton, 3, nosiheptide and 4
against two Gram-positive strains was evaluated in a MIC assay. The results (Table 4.1) show that although 3 and 4 have a decreased activity compared to the
unmodified peptides, relatively high activities are preserved, especially for nosiheptide. This demonstrates that our borylation strategy can be used to improve the water solubility of RiPPs, while remaining effective against their target strains.
Table 4.1: Results of the water solubility assays in µg/mL and MIC assay results in
µg/mL of vancomycin (quality control), thiostrepton, nosiheptide and the borylated peptides against S. aureus and E. faecalis.
Having established the borylation on thiopeptides, the scope of the reaction was expanded to nisin Z, which belongs to the lanthipeptide family of RiPPs (Figure 4.4A). Its high aqueous solubility enabled performing the reaction in completely
aqueous media without cosolvent. After 1 hour of reaction time at room temperature the mixture was analyzed by LC-MS and the obtained mass spectrum was deconvoluted. Full conversion to triply borylated nisin Z was observed (Figure 4.4B),
as well as the doubly borylated product for nisin Z where the two C-terminal residues were lost (nisin Z – Cterm.), a well-documented hydrolysis side reaction of nisin.[47] For both products the loss of 1-4 H2O was also observed (Figure 4.4B), which is the result
of condensation reactions between the boronic acids and –OH and –NH2 residues that
are known to happen in peptides and proteins.[43–46] The identity of the products was
confirmed by obtaining pure samples using preparative HPLC and subjecting these samples to HRMS. Antibiotic Solubility (µg/mL) in ddH2O Solubility (µg/mL) in 50 mM TEAA (pH=7.9) MIC (µg/mL) against S. aureus MIC (µg/mL) against E. faecalis Vancomycin - - 1 4 Thiostrepton 6.4 ± 0.1 5.0 ± 0.2 0.5 0.25 3 169.7 ± 2.6 (27x) 422.1 ± 8.5 (84x) 64 64 Nosiheptide N.D. N.D. 0.0156 0.0312 4 N.D. N.D. 0.25 0.5
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 NH 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 O H2N B2(OH)4 CuSO4, 4-picoline H2O nisin Z A) B)
Figure 4.4: A) β-borylation of nisin Z. Conditions: nisin Z (1 mM), 100 eq. B2(OH)4, 1.0 eq. CuSO4, 10 eq. 4-picoline. B) Deconvolution result of the crude reaction mixture, showing full conversion to triply borylated nisin Z (3469 Da) and loss of 1-4 H2O (3450, 3433, 3414, 3397 Da). Also, doubly borylated nisin Z – Cterm. – 2H2O was observed (3188 Da).
The scope was expanded further to SUMO_G98Dha, a G98Dha mutant of the 12.5 kDa protein Small Ubiquitin-like Modifier (Figure 4.5A). The reaction was
performed in aqueous buffer and upon analysis of the reaction mixture by HRMS and deconvolution (Figure 4.5B) showed the singly borylated protein. In addition to the
borylated protein, also the loss of water and unmodified protein were observed (Figure 4.5C). SUMO_G98Dha and the borylated product co-eluted from the LC-MS,
indicating that the overall polarity was not affected significantly by the modification. These results show that the reaction can be applied on a wide range of challenging substrates.
Chapter 4 86 SUMO_G98Dha CuSO4, 4-picoline PBS (pH=7.0), r.t. B2(OH)4 B(OH)2 A) B) C)
Figure 4.5: A) β-borylation of SUMO_G98Dha. Conditions: SUMO_G98Dha (0.1 mM),
400 eq. B2(OH)4, 10 eq. CuSO4, 40 eq. 4-picoline. B) Deconvoluted HRMS spectrum of the crude product. C) Zoom of the deconvoluted HRMS spectrum. The borylated
protein – H2O (Mcalc = 12541.25, Mobs = 12541.29) and the borylated protein –2H2O (Mcalc = 12523.24, Mobs = 12523.27, 12525.29 shows slightly higher intensity due to overlap) were observed, as well as unmodified protein (Mcalc = 12513.24, Mobs = 12513.25). The found mass at 12498.31 is an unidentified species.
Boronic acids are known to be useful intermediates for chemical transformations, as well as a variety of reversible reactions. Due to the post-translational dehydration machinery, which leads to the formation of Dha and Dhb motifs, serines and threonines are highly uncommon residues in RiPPs. Since hydroboration-oxidation is known to lead to the anti-Markovnikov hydration of alkenes, we envisioned that chemical mutagenesis via β-borylation of Dha residues followed by mild oxidation could be an attractive approach to re-introduce Ser and Thr in these peptides. Purified 3 was treated with an excess amount of NaBO3 (Figure
4.6A) and full conversion of the borylated residues in 3 to the Ser-Ser motif in the tail
N H O B(OH)2 3 NaBO3 H2O/ACN/THF N H O OH A) B)
Figure 4.6: A) Oxidation of 3 with NaBO3 (conditions: 0.34 mM 3, 10 eq. NaBO3). B) LC-MS UV chromatogram of the crude reaction mixture.
Installation of the boronic acid functionality onto RiPPs also allowed for the exploration of reversible boronate ester formation with alcohols. First, 3 was reacted
with Alizarin Red S (Figure 4.7A), a catecholate-based, water soluble boronic acid
staining agent, which is not fluorescent on its own but becomes highly fluorescent when it forms a boronate ester. When examined under a UV light (365 nm) a clear fluorescence turn-on effect was observed when the reaction was compared to controls containing DMSO and unmodified thiostrepton (Figure 4.7B).
Figure 4.7: A) Fluorescence turn-on labeling of 3 with Alizarin Red S (conditions: 43 µM
peptide, 10 eq. Alizarin Red S). B) Visualization of fluorescence turn-on of Alizarin red S. Left: DMSO control, middle: thiostrepton control, right: reaction with 3.
Boronic acids can also form boronate esters with tetraazaadamantane (TAAD) triols under basic conditions, while the resulting complexes hydrolyze readily under acidic conditions.[34] To explore this reversible labeling on RiPPs, N-Bn-TAAD
was synthesized and reacted with 3 using NaHCO3 as a base (Figure 4.8A). After 24
hours high conversion (>90 %) of 3 and formation of the singly- and doubly labeled
peptide was observed in LC-MS. When an excess amount of aqueous HCl was added, LC-MS showed almost full conversion back to the boronic acid within 3 hours. After the hydrolysis the mixture was basified a second time with excess NaHCO3 and 65 %
A) B) N H O B(OH)2 N H O B HO NaO3S O O O O ACN/H2O 1:1 O O OHOH SO3Na 3
Chapter 4
88
conversion to the boronate-triol complexes was observed in LC-MS, proving that this labeling strategy can be applied in the reversible labeling of RiPPs under pH control. The method is also modular, since the benzyl substituent in N-Bn-TAAD can be
switched for a variety of biologically relevant labels. The boronate-triol coupling was also used to further confirm the presence of boronic acid in borylated SUMO_G98Dha (Figure 4.8B). Additionally, the amount of boronic acid dehydration observed in mass
spectrometry analysis of the triol coupling mixtures was consistent with the extent of boronate ester formation. No dehydration was observed for products where full conversion of all boronic acids to boronate-triol complexes was obtained, suggesting that the dehydration is reversible and showing that it does not hamper further conjugation. A) B) N H O B(OH)2 3 / Borylated SUMO_G98Dha NH O B N NN N O OO Ph NaHCO3, ACN/H2O 1:1, r.t. N NN N HO HOOH Ph Br 6 M HCl (aq.) N-Bn-TAAD
Figure 4.8: A) Reversible formation of boronate-triol complex (conditions: 0.58 mM 3,
25 eq. N-Bn-TAAD, 10 eq. NaHCO3). B) Deconvoluted LC-MS mass spectrum of boronate triol complex formation on SUMO_G98Dha.
Collectively, these results demonstrate that the borylation reaction presented here is a versatile method for the modification of RiPPs and proteins and it compares well with existing Dha modifications in terms of simplicity of execution, efficiency and selectivity.
4.3 Conclusion
In conclusion, the β-borylation of Dha residues is a fast and selective approach for forming unnatural C-B bonds in peptides and proteins under mild conditions, allowing for the exploration of such abiological functionalities in these natural products. Installation of boronic acids on RiPPs leads to variants with improved water solubility, while antimicrobial activities are retained. Moreover, the borylated peptides have proven to be useful intermediates for further chemical transformations and reversible labeling.
4.4 Experimental
General remarksChemicals were purchased from Sigma-Aldrich, Acros Organics and TCI Europe, and used without further purification. Thiostrepton, nosiheptide and nisin Z were purchased from CalBioChem, Carbosynth Ltd. and Handary, respectively. SUMO_G98Dha was prepared following reported literature procedures.[25] 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- and 13C-NMR spectra were
recorded on an Agilent 400-MR at 298K spectrometer operating at 400 and 101 MHz, respectively or on a Brüker Ascend 600 operating at 600 and 151 MHz, respectively. 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 40 °C using a reversed-phase UPLC column (Waters Acquity UPLC BEH C8, 1.7 μm, 2.1 mm x 150 mm), except for SUMO_98Dha, where a Waters Acquity C4 Protein BEH 1.7 μm (2.1 mm x 150 mm) column was used. UPLC grade 0.1 % formic acid (FA) in ddH2O (solvent A) and 0.1 % FA
in acetonitrile (solvent B) were used as eluents. Gradient used for thiostrepton, 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) unless mentioned otherwise. High-resolution mass spectrometry was performed on a LTQ Orbitrap XL spectrometer (ESI+). For nisin Z and SUMO_G98Dha the mass spectra were deconvoluted using the MagTran algorithm (version 1.03).[48] 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 operating at 35 °C, a CBM-20A system controller and a FRC-10A fraction collector. 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. Eluents used were 0.1 % FA in ACN (solvent A) and 0.1 % FA in ddH2O (solvent B), using a gradient of 90
% B to 10 % B over 40 minutes (total runtime 55 minutes).
Methyl 2-(acetamido)acrylate (1):[49] 5.00 g (84.7 mmol) acetamide, 7 mL (77.5
mmol) methyl pyruvate were dissolved in 150 mL toluene and catalytic amounts of p-TsOH and 4-methoxyphenol were added. 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
N H
O O
Chapter 4
90
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, CDCl3) 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) ppm. 13C NMR (101 MHz,
CDCl3) δ 168.9, 164.8, 131.0, 108.8, 53.1, 24.8 ppm.
1,1',1''-nitrilotris(propan-2-one) trioxime (TRISOXH3):[50] 1.00 g (10.8 mmol, 0.9 mL) chloroacetone was dissolved in 20 mL diethyl ether. 1.10 g (15.8 mmol) NH2OH hydrochloride in 10 mL H2O was added and the biphasic
mixture was stirred vigorously at room temperature. 1.1 g (7.96 mmol) K2CO3 was added in small portions, making sure the mixture remained clear. The mixture was
stirred at r.t. for 30 minutes, after which the layers were separated and 0.7 mL 28 % (11.5 mmol) NH3 (aq.) in 5 mL H2O was added to the ethereal layer. The mixture was stirred at r.t. for
1 hour, cooled to 0 °C using an ice bath and stirred at that temperature for another 30 minutes. When left standing at r.t. for 5 minutes a white solid precipitated which was collected by filtration and recrystallized from 5 mL EtOH/H2O 1:1. 172 mg (0.75 mmol, 21 %) white crystals
were obtained. Melting point: 180-183 °C (decomp.) (Lit.: 178 °C (decomp.)). 1H NMR (400 MHz,
DMSO-d6) δ 10.59 (s, 3H), 2.91 (s, 6H), 1.75 (s, 9H) ppm. 13C NMR (101 MHz, DMSO-d6) δ 153.5,
57.1, 12.1 ppm.
1-benzyl-4,6,10-trihydroxy-3,5,7-trimethyl-1,4,6,10-tetraazaadamantan-1-ium bromide (N-Bn-TAAD):[51] 252 mg (1.1 mmol) TRISOXH
3 was dissolved in a
mixture of 4.4 mL MeOH and 1.1 mL H2O. 189 µL (3.3 mmol) AcOH was added,
followed by 196 µL (1.65 mmol) benzyl bromide and the opaque mixture was heated with a heat gun until it became clear. The mixture was then stirred at r.t. for 24 hours, after which the solvent was evaporated and the residual beige solid was washed with 2x4 mL EtOAc/MeOH 3:1 and dried in vacuo. 211 mg (0.53 mmol, 48 %) of a white solid was obtained.
1H NMR (600 MHz, DMSO-d
6) δ 8.86 (br. s, 3H), 7.63 – 7.58 (m, 2H), 7.58 – 7.50 (m, 3H), 4.61 (s,
2H), 4.05 (br. s, 6H), 1.23 (s, 9H) ppm. 13C NMR (151 MHz, DMSO-d
6) δ 133.2, 130.6, 129.1,
126.0, 74.8 (broad), 67.6, 20.6 ppm (1 quaternary carbon signal missing due to low intensity and extensive broadening).
β-borylation of methyl 2-(acetamido)acrylate (1): 29 mg (0.2 mmol) 1 and 20 mg (0.22 mmol) B2(OH)4 were weighed into a 6 mL vial with stirring bar. 0.8 mL ddH2O and 0.2 mL 10 mM (2
µmol) CuSO4 (aq.) were added, followed by 1 µL (10 µmol) 4-picoline. The mixture was stirred at
r.t. overnight, filtered over Celite and the filter cake was washed with 1 mL THF. 92 mg (0.6 mmol) NaBO3 tetrahydrate was added to the combined filtrates and the mixture was stirred at
r.t. for 2 hours. The mixture was extracted with 10 mL EtOAc, the organic phase was dried over MgSO4 and the solvent was evaporated. The residue was analyzed directly by LC-MS (Waters
N N N N HO OH OH N NN N HO HOOH Ph Br
Acquity HSS T3, 1.7 μm, 2.1 mm x 150 mm column). Only product 2 (m/z = 162 [M+H]+) was
observed, indicating full conversion of starting material 1.
β-borylation of thiostrepton: In a typical procedure, 1.7 mg (1 μmol) thiostrepton and 4.5 mg (50 µmol) B2(OH)4 were weighed into a Schlenck tube equipped with a stirring bar. 0.5 mL TFE
was added, followed by 475 µL ddH2O and 25 µL 10 mM (0.25 µmol) CuSO4 (aq.). The mixture
was stirred at r.t. for 1 hour after which the reaction mixture was centrifuged (2 min. at 13400 rpm). 50 µL of the supernatant was diluted with 200 µL H2O/ACN 1:1 and analyzed directly by
LC-MS. Full conversion of thiostrepton was observed, as well as the formation of doubly borylated thiostrepton 3 (m/z = 1778 [M+Na]+) as the only major product.
Preparative scale and purification of borylated thiostrepton: 25 mg (15 μmol) thiostrepton was dissolved in 3.5 mL TFE and 0.6 mg (3.75 µmol) CuSO4 in 3.5 mL H2O was added. 34 mg
(0.38 mmol) B2(OH)4 was added and the mixture was stirred at r.t. for 1 hour. Then, the mixture
was filtered over a plug of Celite, 2 mL H2O/ACN 1:1 was added and finally the solution was
filtered over a microfilter (0.45 μm). The obtained clear filtrate was purified using preparative HPLC. Analysis of the fractions by LC-MS, followed by lyophilization of the combined pure fractions resulted in isolation of pure 3 as a white brittle solid, which was identified using HRMS (calcd. C72H91B2NaN19O22S5 [M+Na]+: 1778.527, found: 1778.525, Figure 4.9). NMR samples
were prepared by dissolving approximately 1 mg of peptide in 500 µL CDCl3/MeOD 3:1.
Figure 4.9: HRMS spectrum of 3 (top) and the calculated HRMS spectrum simulation (bottom). β-borylation of nosiheptide: In a typical procedure, 0.6 mg (0.5 μmol) nosiheptide was dissolved in 250 µL TFE. 275 µL 2 mM (0.55 µmol) CuSO4 (aq.) was added, followed by 2.3 mg
1772 1773 1774 1775 1776 1777 1778 1779 1780 1781 1782 1783 1784 1785 1786 1787 1788 m/z 0 10 20 30 40 50 60 70 80 90 1000 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 1778.525 1779.526 1780.525 1777.527 1781.525 1782.523 1772.523 1776.518 1783.524 1775.495 1784.563 1774.535 1786.0521787.013 1788.516 1772.185 1778.527 1779.530 1777.530 1780.534 1781.526 1782.529 1776.534 1783.533 1784.525 1785.528 1786.532 1787.533
Chapter 4
92
(25 µmol) B2(OH)4 and 0.5 µL (5 µmol) 4-picoline. The mixture was stirred at r.t. for 1 hour. 50
µL of the reaction mixture was diluted with 200 µL H2O/ACN 1:1, filtered over a microfilter (0.45
µm) and analyzed directly by LC-MS. 94 % conversion of nosiheptide to the singly borylated nosiheptide 4 (m/z = 1268 [M+H]+) was observed based on UV peak integration (not including
nosiheptide – Dha, which was already present in the commercially available nosiheptide as an impurity).
Preparative scale and purification of borylated nosiheptide: 18 mg (15 μmol) nosiheptide was dissolved in 3.5 mL TFE and 2.6 mg (16.5 µmol) CuSO4 in 3.5 mL H2O was added. 67 mg (0.75
mmol) B2(OH)4 was added, followed by 15 µL (0.15 mmol) 4-picoline and the mixture was
stirred at r.t. for 2 hours. Then, the mixture was filtered over a plug of Celite, 2 mL H2O/ACN 1:1
was added and finally the solution was filtered over a microfilter (0.45 μm). The obtained clear filtrate was purified using preparative HPLC. Analysis of the fractions by LC-MS, followed by lyophilization of the combined pure fractions resulted in isolation of pure 4 as a yellow brittle solid, which was identified using HRMS (calcd. C53H50BNaN13O14S6 (double methyl boronic ester
due to MeOH in eluent) [M+Na]+: 1318.191, found: 1318.192, Figure 4.10).
Purification of nosiheptide: For MIC assays, commercially available nosiheptide was also purified using preparative HPLC. 15 mg nosiheptide was dissolved in 5.5 mL TFE, after which 4.5 mL H2O/ACN 1:1 was added. The turbid solution was filtered over Celite twice before it was
filtered over a microfilter (0.45 µm). The obtained clear filtrate was purified using preparative HPLC. Analysis of the fractions by LC-MS, followed by lyophilization of the combined pure fractions resulted in isolation of pure nosiheptide as a yellow brittle solid, which was identified using HRMS (calcd. C53H50BNaN13O14S6 [M+Na]+: 1244.137, found: 1244.138).
Figure 4.10: HRMS spectrum of 4 (top) and the calculated HRMS spectrum simulation (bottom).
1312 1314 1316 1318 1320 1322 1324 1326 1328 1330 1332 m/z 0 10 20 30 40 50 60 70 80 90 1000 10 20 30 40 50 60 70 80 90 100 R el at iv e A bundanc e 1318.192 1319.194 1320.193 1317.195 1321.190 1322.187 1326.157 1327.160 1324.180 1329.155 1313.147 1312.142 1315.1701316.167 1330.6831332.128 1318.191 1319.194 1320.187 1317.195 1321.190 1322.193 1323.186 1325.182 1326.185 1328.181 1329.184 1331.188
β-borylation of nisin Z: 3.3 mg (1 µmol) nisin Z and 9 mg (100 µmol) B2(OH)4 were dissolved in
975 µL ddH2O. 25 µL 40 mM (1 µmol) CuSO4 (aq.) was added, followed by 1 µL (10 µmol)
4-picoline and the mixture was stirred at r.t. for 1 hour. The mixture was purified by NAP-10 desalting column (GE Healthcare). The peptide fraction was analyzed directly by LC-MS and the obtained mass spectrum was deconvoluted. Full conversion to triply borylated nisin Z was observed, as well as the doubly borylated product for nisin Z where the two C-terminal residues were lost (nisin Z – Cterm.), a well-documented hydrolysis side reaction of nisin.[47] For both products the loss of 1-4 H2O was also observed, which is the result of condensation reactions
between the boronic acids and –OH and –NH2 residues that are known to happen in peptides
and proteins.[43–46] The identity of the products was confirmed by obtaining pure samples using
preparative HPLC and subjecting these samples to HRMS, followed by deconvolution of the mass spectrum. HRMS calcd. C141H232B3N41O41S7 (triply borylated nisin Z – 3H2O) 3412.572,
found 3412.565 (Figure 4.11); C132H221B2N39O39S7 (doubly borylated nisin Z – Cterm.) 3222.473,
found 3222.470 (Figure 4.12).
Figure 4.11: Deconvoluted HRMS spectrum of triply borylated nisin Z – 3 H2O (top) and the
calculated HRMS spectrum simulation (bottom).
3409 3410 3411 3412 3413 3414 3415 3416 3417 3418 3419 3420 Mass 3409.5576 3410.5464 3411.5598 B3412.5649 B3413.5703 B3414.5674 3415.5642 3416.5696 3417.5417 3418.5635 3419.5605 3409 3410 3411 3412 3413 3414 3415 3416 3417 3418 3419 3420 m/z 0 10 20 30 40 50 60 70 80 90 1000 3413.5682 3414.5715 3412.5718 3411.5684 3415.5749 3416.5673 3410.5721 3417.5707 3418.5740 3419.5665 3409.5757
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Figure 4.12: Deconvoluted HRMS spectrum of doubly borylated nisin Z – Cterm. (top) and the calculated HRMS spectrum simulation (bottom).
β-borylation of SUMO_G98Dha: 20 µL 885 µM (17.7 nmol) SUMO_G98Dha in 50 mM PBS (pH=7.0) was added to 157 µL 4.5 mM (0.71 µmol) 4-picoline in ddH2O. 4.5 µL 40 mM (0.177
µmol) CuSO4 (aq.) was added, followed by 0.6 mg (7.1 µmol) B2(OH)4 and the mixture was
stirred at r.t. for 1 hour. The mixture was purified by a PD MiniTrap G-25 column (GE Healthcare) and the eluted fraction was concentrated to 45 µM using a Vivaspin Turbo 4 spin filter (5000 MWCO, Sartorius). The protein solution was analyzed by HRMS and the mass spectrum was deconvoluted (see Figure 4.5)
Boronate-triol coupling of borylated SUMO_G98Dha: To further confirm the presence of boronic acid in the modified protein, the borylated protein was purified by preparative HPLC and 75 µL of the obtained protein fraction was reacted with 5 µL 40 mM [N-Bn-TAAD]+Br- in
H2O/ACN 1:1 and 3 µL 1M NaHCO3 (aq.). After shaking for 3 hours at room temperature the
mixture was analyzed by LC-MS and the obtained mass spectrum was deconvoluted, showing the borylated protein and its boronate-triol complex (see Figure 4.8).
Biological activity assays
Preparation of stock solutions of antimicrobial agents: Thiostrepton, nosiheptide and their borylated variants were dissolved and diluted in DMSO to a concentration of 640 µg/mL using UV absorbance at 280 nm and known extinction coefficients for thiostrepton (ε = 27000 M-1cm-1)[18] and nosiheptide (ε = 39000 M-1cm-1),[18] and stored at -20 °C. Before use, they were
3220 3221 3222 3223 3224 3225 3226 3227 3228 3229 Mass 3220.4529 3221.4783 3222.4695 C3223.4778 C3224.4690 C3225.4773 3226.4771 3227.4683 3228.4680 3221 3222 3223 3224 3225 3226 3227 3228 3229 3230 m/z 0 10 20 30 40 50 60 70 80 90 1000 3223.4768 3224.4802 3222.4734 3221.4771 3225.4835 3226.4760 3227.4793 3220.4807 3228.4827 3229.4751
diluted 20 fold in Mueller Hinton Broth 2 (CAMHB; cation-adjusted, Sigma-Aldrich). Vancomycin, which was used for the quality controls, was dissolved in MilliQ to a concentration of 256 mg/mL, stored at -20 °C and diluted in CAMHB to a final concentration of 256 µg/mL before use.
Strains and growth conditions: The MIC values of thiostrepton, nosiheptide and their borylated 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). The incubation temperature was 37 °C for all steps.
DMSO as a solvent: As the antimicrobial agents were diluted from stock solutions 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[52], which is outlined in
short here.
First, 50 µL and 100 µL of sterile CAMHB is added to columns 2-11 and 12, respectively, of a 96-well plate. 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.[53]
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 tested strain is considered the MIC value. All compounds were tested three times in triplicates.
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Water solubility assays
To compare the water solubility of the borylated (3 and 4) and unmodified (thiostrepton and nosiheptide) peptides, approximately 1 mg peptide was suspended in 2 mL aqueous medium with the assistance of 30 seconds of sonication. The homogeneous suspensions were stirred vigorously (1000 rpm) at r.t. for 24 hours in order to ensure saturation and reaching equilibrium. Aliquots were then filtered over a microfilter (0.45 µm). From the clear filtrates, samples of known volume were taken in duplicate for each peptide and freezedried. The obtained solids were redissolved in a known amount of DMSO, resulting in clear solutions. The concentrations of these solutions were determined by measuring the UV absorbance at 280 nm (in duplicate for each sample) and using known extinction coefficients for thiostrepton (ε = 27000 M-1cm-1) and nosiheptide (ε = 39000 M-1cm-1).[18] From these concentrations the water
solubility in µg/mL was calculated by taking into account the dilution factors involved in the preparation of the DMSO samples and the molecular weights of the different peptides.
Chemical mutagenesis of thiostrepton via oxidation of 3: To 34 µL of a 1 mM solution (34 nmol) of 3 in H2O/ACN 1:1 was added 34 µL THF, followed by 34 µL 10 mM (0.34 µmol)
NaBO3.4H2O (aq.). The resulting clear solution was stirred at r.t. for 30 minutes. The reaction
mixture was analyzed directly by LC-MS and full conversion to the peptide containing the Ser-Ser motif was observed. HRMS showed formation of the product (calcd. C72H90N19O20S5
[M+H]+: 1700.521, found: 1700.521).
Fluorescence turn-on labeling of 3 with Alizarin Red S (ARS): To 3 GC vials fitted with 250 µL inserts was added 10 µL 10 mM (0.1 µmol) Alizarin Red S (aq.). To vial 1 was added 20 µL DMSO, to vial 2 20 µL 0.5 mM (10 nmol) thiostrepton in DMSO and to vial 3 was added 20 µL 0.5 mM (10 nmol) 3 in DMSO. To all vials was added 200 µL ddH2O and the resulting
fluorescence was observed under a UV lamp at 365 nm. It was found that vials 1 and 2 containing the DMSO and unmodified thiostrepton controls, respectively, did not show significant fluorescence. In vial 3, which contained the borylated thiostrepton 3, a significant increase in fluorescence was observed due to labeling with Alizarin Red S.
Reversible boronate-triol coupling of 3 with N-Bn-TAAD: 36.2 µL 40 mM [N-Bn-TAAD]+Br- in
H2O/ACN 1:1 was added to 58 µL 1.0 mM 3 in H2O/ACN 1:1. Then, 5.8 µL 0.1 M NaHCO3 (aq.)
was added and the mixture was stirred at r.t. overnight. The mixture was analyzed by LC-MS and high (>90 % by UV peak integration) conversion to singly (m/z = 1012.4 [M-H2O+2H]2+) and
doubly (m/z = 1063.4 [M+2H]2+) labeled peptide was observed. 1 µL 6M HCl (aq.) was added and the mixture was stirred at r.t. for 3 hours, after which the mixture was again analyzed by LC-MS. Almost complete hydrolysis of the boronate-triol complexes to the peptide with free boronic acids was observed (m/z = 1720.7 [M-2H2O+H]+), along with a byproduct with m/z =
1750 that could not be identified. The mixture was again basified using 15 µL 1M NaHCO3 (aq.)
and stirred at r.t. for 24 hours and analyzed by LC-MS. Again good conversion (67 % based on UV peak integration) to the singly and doubly labeled peptide was observed. The lower conversion compared to the initial coupling is attributed to dilution of the reaction mixture and increase of the ionic strength due to the addition of acid and base. Also, the byproduct observed at m/z = 1750 under the acidic conditions had disappeared almost completely. These results show that the boronate-triol coupling is indeed reversible under pH control. Also, both 3 and the triol coupling products appear to have good stability after stirring at room temperature in aqueous base and acid for a total of 48 hours.
Appendix: NMR spectra
Chapter 4
98
Figure S2: 1H-1H-TOCSY NMR spectrum of 3.
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