University of Groningen
Chemical Modification of Peptide Antibiotics
de Vries, Reinder
DOI:
10.33612/diss.171585325
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2021
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de Vries, R. (2021). Chemical Modification of Peptide Antibiotics. University of Groningen.
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Chapter 5
Cu(II)-Catalyzed β-Silylation of Dehydroalanine Residues in
Peptides and Proteins
In this chapter the Cu(II)-catalyzed β-silylation of dehydroalanine (Dha) residues in peptides and proteins is described. This approach allowed for efficient silylation of naturally occurring Dha residues in various Ribosomally synthesized and Post-translationally modified Peptides (RiPPs). Modification of a Dha residue that was chemically introduced onto Small Ubiquitin-like Modifier (SUMO), a 12.5 kDa protein, demonstrated the generality of the method. NMR studies on the modified peptide thiostrepton demonstrated the high chemo- and site-selectivity of this approach.
Published as: R. H. de Vries, G. Roelfes, Chem. Commun. 2020, 56, 11058–11061
Chapter 5
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5.1 Introduction
The introduction of silylated amino acids into biologically active peptides is an attractive approach in medicinal chemistry to overcome some of the poor pharmacological properties associated with such substances.[1] The substitution of carbon residues for silicon isosteres hampers the recognition by proteolytic enzymes and thus improves resistance against degradation.[2–5] Additionally, silyl groups greatly increase the lipophilicity of peptides, which can be an important factor for enhancing cellular uptake.[6]
Methods for the silylation of amino acids and peptides have shown to be effective, yet often require harsh conditions or multiple synthetic steps.[7–11] Currently,
a mild approach that is suitable for the chemical incorporation of silyl groups onto large peptides and proteins is lacking despite the growing popularity of such compounds in medicine.[12] Here we report the rapid and selective Cu(II)-catalyzed
β-silylation of dehydroalanine (Dha) residues in peptides and proteins (Scheme 5.1).
Scheme 5.1: Cu(II)-catalyzed β-silylation of Dha (blue) in peptides and proteins using
Suginome’s Reagent (PinBSiMe2Ph).
Dehydroalanines are α,β-unsaturated amino acids[13] which occur naturally in
ribosomally synthesized and post-translationally modified peptides (RiPPs)[14] and can
also be introduced chemically into other peptides and proteins.[15] They are uniquely
reactive as electrophiles, which makes them attractive chemical handles for late-stage modification of such complex natural products.[16–27] Cu(II)-catalyzed β-silyl conjugate
additions to α,β-unsaturated carbonyl substrates using PinBSiMe2Ph (also known as
Suginome’s Reagent) have been performed at room temperature, in aqueous media and open to the air.[28,29] Therefore, we envisioned this method to be an excellent
starting point for exploring the silylation of Dha in peptides and proteins.
5.2 Results and Discussion
First, the β-silylation of Dha acceptors in the thiopeptide thiostrepton was
investigated (Figure 5.1A). Due to the poor water solubility of thiostrepton
2,2,2-trifluoroethanol (TFE) was used as a co-solvent in our initial screening conditions. Using 10 equivalents of PinBSiMe2Ph and 1 mol% CuSO4, >95 % conversion
N H H N O O peptide/protein N H H N O O SiMe2Ph PinBSiMe2Ph Cu(II)
of thiostrepton was achieved within 1 hour, which was accompanied by the rapid formation of the singly and doubly silylated peptide as detected with LC-MS (Figure 5.1B). Splitting of the peaks of both the singly and doubly silylated peptide indicated a mixture of diastereomers (Figure 5.1B). In contrast to previous studies,[29] efficient
silylation was achieved without the use of 4-picoline as a Brønsted base. Since the base was not required for the reaction and the presence of amine bases is known to be detrimental for the stability of thiostrepton in solution[30], the base was omitted.
1 mol% CuSO4, TFE/H2O 1:1, r.t. 1 hour 10 eq. PinBSiMe2Ph thiostrepton(1 mM) 2 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 SiMe2Ph SiMe2Ph A) B) C)
Figure 5.1: A) β-silylation of thiostrepton. B) LC-MS UV (280 nm) chromatogram of the
crude reaction mixture after 1 hour (*single silylation, **double silylation). C) LC-MS UV (280 nm) chromatograms of purified 2a and 2b.
To demonstrate the chemo- and site-selectivity of this approach for the different Dha residues within thiostrepton, the doubly modified products (2a and 2b) were isolated using preparative HPLC and the purified products (Figure 5.1C) were characterized via HRMS and 2D NMR spectroscopy (Figure 5.2). An authentic sample of unmodified thiostrepton was prepared and 1H NMR, 1H-1H COSY NMR and 1H-1H NOESY NMR were recorded for thiostrepton. The data of unmodified thiostrepton was
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used to assign the characteristic methylene signals of the different dehydroamino acids. Then, 1H NMR, 1H-1H TOCSY NMR and 1H-1H NOESY NMR were recorded for 2a
and 2b. The 1H NMR spectra of thiostrepton and 2a and 2b were compared to
determine which residues were modified in products 2. The methylene signals of Dha3 (purple) and Dhb8 (yellow) are conserved in 2a and the signals of Dha16 (blue) and
Dha17 (green) have disappeared (Figure 5.2A). Also, the appearance of new
α-hydrogens (red box) can be observed. This is consistent with a double β-silylation at the tail region of thiostrepton. Upon inspection of the spin systems of the new α-protons (4.39 ppm and 4.28 ppm) in TOCSY NMR, they show crosspeaks with diastereotopic β-carbon signals (1.51/1.19 ppm and 1.33/1.11 ppm, respectively) and N-H signals (6.19 ppm and 7.28/7.17 ppm, respectively) (Figure 5.2B). This in accordance with silylation at the β-carbons of the Dha residues.
A) B) C) D) S N NH H N O O NH2 O SiMe2Ph SiMe2Ph 1H-1H-TOCSY S N NH H N O O NH2 O SiMe2Ph SiMe2Ph 1H-1H-NOESY
Figure 5.2: A) Stacked 1H NMR spectra of thiostrepton (top) and product 2a (bottom),
showing the region between 4.0 ppm and 7.0 ppm. B) Zoom of TOCSY NMR of product 2a, showing the key correlations. C) Stacked NMR spectra of product thiostrepton (top) and 2b (bottom), showing the region between 4.0 ppm and 7.0 ppm. D) Zoom of NOESY NMR of product 2b, showing a key correlation.
For product 2b similar signals and crosspeaks were observed in 1H and TOCSY
NMR (Figure 5.2C). Additionally, crosspeaks can be observed in the NOESY NMR
spectrum of 2b between the α-proton of the sub terminal silylated amino acid (3.98 ppm) with the N-H signal of the terminal silylated amino acid (6.17 ppm) (Figure 5.2D). This proves that the modified amino acids are next to each other, which is consistent with double β-silylation of the tail region of thiostrepton. Unfortunately, it was difficult to resolve the different diastereomers and even with the NMR techniques used they could ultimately not be assigned.
Next, the scope of the reaction on different Dha-containing peptides was studied, starting with the thiopeptide nosiheptide (Figure 5.3A). Similar conditions were used, except a higher catalyst loading (2 eq.) and addition of 10 equivalents 4-picoline were necessary to achieve efficient silylation. In the analysis of the crude product via LC-MS 80 % conversion of nosiheptide to its singly silylated derivative was detected after 1 hour of reaction time (Figure 5.3B). Modification of nosiheptide that lacks the terminal Dha, which is present as an impurity in the commercially available nosiheptide, as well as any double modification, was not observed. This indicates that, similar to thiostrepton, the reaction is completely selective for the terminal Dha over the internal Dhb. CuSO4,4-picoline TFE/H2O 1:1, r.t. PinBSiMe2Ph 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 SiMe2Ph
Figure 5.3: A) β-silylation of nosiheptide. B) LC-MS UV (280 nm) of the crude reaction
mixture after 1 hour (*single silylation).
Nisin Z, a member of the lanthipeptide family of RiPPs, was also investigated as a substrate (Figure 5.4A). In contrast to thiopeptides, nisin Z has a high aqueous
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solubility, avoiding the need for TFE as a co-solvent and allowing us to conduct the silylation in pure water. It was found that also here the addition of 4-picoline was necessary to achieve efficient silylation. After 1 hour of reaction at room temperature the mixture was analyzed by LC-MS and the mass spectrum of the crude product containing the nisin species was deconvoluted (Figure 5.4B). A small amount of nisin Z was found, as well as singly and doubly silylated nisin Z products (including the known degradation products of nisin due to water addition (+H2O) and cleavage of the two
C-terminal amino acids (-CTerm.))[31] (Figure 5.4B). In contrast to thiostrepton, which
has a Dha(3) that is buried in a macrocycle and a Dhb(8) without carbonyl substituent, the dehydroamino acids in nisin Z are all reactive and accessible resulting in a mixture of regioisomers that could not be resolved individually using UPLC-MS.
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 NH O HN O OH NH2 NH2 O H2N PinBSiMe2Ph CuSO4,4-picoline H2O nisin Z A) B)
Figure 5.4: A) β-silylation of the lantipeptide nisin Z. B) Deconvolution result of the raw
mass spectrum of the crude reaction mixture after 1 hour (*single silylation, **double silylation).
The scope of the reaction was extended to proteins. For this purpose, we performed the reaction on the 12.5 kDa protein Small Ubiquitin-like Modifier (SUMO). SUMO_G98Dha, a mutant with a Dha that was chemically introduced near the C-terminus, was subjected to the silylation conditions (Figure 5.5A) and after 1 hour at room temperature the reaction mixture was analyzed by LC-MS. Upon deconvolution of the mass spectra the singly silylated protein was observed, together with a small amount of unreacted SUMO_G98Dha (Figure 5.5B).
Figure 5.5: A) Silylation of SUMO_G98Dha. B) Deconvolution result of the raw mass
spectrum of the crude reaction mixture after 1 hour (*single silylation, small peak at 12796 Da is an unidentified minor byproduct).
5.3 Conclusion
In conclusion, the Cu(II)-catalyzed β-silylation of Dha residues is a straightforward, fast and selective method for the silylation of bioactive peptides and proteins. The reaction is robust and efficient in aqueous media both with and without added co-solvent, enabling the silylation of a variety of natural products. Moreover, the modification of chemically incorporated Dha residues demonstrates that the method is generally applicable in the silylation of peptides and proteins.
SUMO_G98Dha CuSO4, 4-picoline H2O, r.t. PinBSiMe2Ph SiMe2Ph A) B)
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5.4 Experimental
General remarksChemicals were purchased from Sigma-Aldrich, Acros Organics and TCI Europe, and used without further purification. PinBSiMe2Ph (purchased from TCI Europe) has to be stored under
Ar atmosphere in the freezer and is preferably used as soon as possible after first opening of the container. Thiostrepton, nosiheptide and nisin Z were purchased from CalBioChem, Carbosynth Ltd. and Handary, respectively. SUMO_G98Dha was prepared following reported literature procedures.[25]1H NMR, 1H-1H COSY NMR, 1H-1H TOCSY NMR and 1H-1H NOESY NMR
spectra were recorded on a Brüker Ascend 600 operating at 600 MHz. Chemical shifts in 1H
NMR spectra were internally referenced to solvent signals (CDCl3 at δH = 7.26 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 Waters Acquity UPLC BEH C8 1.7 μm (2.1 mm x 150 mm) column, 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 H2O (solvent A) and 0.1 % FA
in 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) unless mentioned otherwise. For nisin Z and SUMO_G98Dha the mass spectra were deconvoluted using the MagTran algorithm (version 1.03).[32] High-resolution mass spectrometry was performed on a LTQ
Orbitrap XL spectrometer (ESI+). 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 60 % B to 10 % B over 40 minutes (total runtime 55 minutes).
β-silylation of thiostrepton: In a typical procedure, 1.7 mg (1 μmol) thiostrepton was dissolved
in 0.5 mL TFE. Then, 495 µL ddH2O and 5 µL 2 mM (0.01 µmol) CuSO4 (aq.) were added,
followed by 2.7 µL (10 µmol) PinBSiMe2Ph and 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. >95% conversion of thiostrepton was observed, as well as the formation of singly (m/z = 1801 [M+H]+) and doubly (m/z = 1937 [M+H]+) silylated thiostrepton
Preparative scale and purification: 25 mg (15 μmol) thiostrepton was dissolved in 3.5 mL TFE.
Then, 3.425 mL ddH2O was added, followed by 75 µL 2 mM (0.15 µmol) CuSO4 (aq.). Finally, 40
µL (0.15 mmol) PinBSiMe2Ph was added and the mixture was stirred at r.t. for 1 hour. The
mixture was extracted with 10 mL DCM and the organic phase was washed with 5 mL ddH2O,
dried over MgSO4 and the solvent was evaporated. The residue was redissolved in 3.5 mL TFE,
after which 3.5 mL ddH2O/ACN 1:1 was added. The clear solution was filtered over a microfilter
(0.45 μm). Individual diastereomers of doubly silylated thiostrepton (2) were isolated using
preparative HPLC. Analysis of the fractions by LC-MS, followed by lyophilization of the combined pure fractions resulted in isolation of pure 2a and 2b as white brittle solids, which were
identified using HRMS (calcd. C88H110N19O18S5Si2 [M+H]+: 1936.641, found: 1936.645 (2a) and
1936.648 (2b) (Figure 5.6). NMR samples were prepared by dissolving approximately 1 mg of
peptide in 500 µL CDCl3.
Figure 5.6: HRMS spectra of 2a (top) and 2b (bottom).
β-silylation of nosiheptide: In a typical procedure, 1.2 mg (1 μmol) nosiheptide was dissolved in
500 µL TFE. 450 µL H2O and 50 µL 40 mM (2 µmol) CuSO4 (aq.) were added, followed by 2.7 µL
(10 µmol) PinBSiMe2Ph and 1 µL (10 µmol) 4-picoline. After stirring 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. 80 % conversion of nosiheptide was observed based on UV peak integration (not including nosiheptide – Dha, which was already present in the commercially available nosiheptide as an impurity), as well as the formation of singly silylated nosiheptide (m/z = 1358 [M+H]+) as major product. 1930 1932 1934 1936 1938 1940 1942 1944 1946 1948 1950 m/z 0 10 20 30 40 50 60 70 80 90 1000 1936.64181937.6455 1938.6423 1939.6404 1940.6416 1941.6391 1943.5916 1945.8337 1931.0810 1930 1932 1934 1936 1938 1940 1942 1944 1946 1948 1950 m/z 0 10 20 30 40 50 60 70 80 90 1000 1937.6503 1936.6481 1938.6505 1939.6514 1940.6465 1941.6433 1949.5031 1934.4347 1931.6875 1935.6270 1943.6456 1948.1328
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β-silylation of nisin Z: In a typical procedure, 3.3 mg (1 μmol) nisin Z was dissolved in 975 µL
H2O. 25 µL 40 mM (1 µmol) CuSO4 (aq.) were added, followed by 28 µL (100 µmol) PinBSiMe2Ph
and 1 µL (10 µmol) 4-picoline. After stirring 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. Upon deconvolution of the raw mass data a small amount of nisin Z (Mcalc = 3331 Da,
Mobs = 3331 Da) and singly and doubly silylated nisin Z products (including the known and well
documented water addition (+H2O) and cleavage of the two C-terminal amino acids (-CTerm.))
were observed.[31] The following major products were identified: nisin Z – Cterm. + 1 mod. (M calc
= 3268 Da, Mobs = 3268 Da), nisin Z + H2O + 1 mod. (Mcalc = 3484 Da, Mobs = 3484 Da), nisin Z + 2
mod. (Mcalc = 3602 Da, Mobs = 3604 Da), nisin Z + 2 H2O + 2 mod. (Mcalc = 3638 Da, Mobs = 3637
Da) (see Figure 5.4).
β-silylation of SUMO_G98Dha: 40 µL 312 µM (12.5 nmol) SUMO_G98Dha in 50 mM PBS
(pH=7.0) was added to 85 µL 5.9 mM (0.5 µmol) 4-picoline in ddH2O. 3.2 µL 40 mM (0.125 µmol)
CuSO4 (aq.) was added, followed by 1.4 µL (5 µmol) PinBSiMe2Ph and the mixture was stirred at
r.t. for 1 hour. 50 µL reaction mixture was diluted with 200 µL ddH2O/ACN 1:1 and analyzed
directly by LC-MS. Besides starting material (Mcalc = 12516 Da, Mobs = 12519 Da), also the
Appendix: NMR spectra
Figure S1: 1H NMR spectrum of 2a.
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Figure S3: 1H-1H NOESY NMR spectrum of 2a.
Figure S5: 1H-1H TOCSY NMR spectrum of 2b.
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Chapter 5
