Precursor Manipulation in Glycopeptide Antibiotic ... - ACS Publications

Feb 12, 2018 - University, Clayton, Victoria 3800, Australia. §. Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, ...
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Article Cite This: J. Org. Chem. 2018, 83, 7206−7214

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Precursor Manipulation in Glycopeptide Antibiotic Biosynthesis: Are β‑Amino Acids Compatible with the Oxidative Cyclization Cascade? Melanie Schoppet,†,‡,§ Julien Tailhades,†,‡ Ketav Kulkarni,‡ and Max J. Cryle*,†,‡,§ †

EMBL Australia and ‡The Monash Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia § Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany

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S Supporting Information *

ABSTRACT: Natural products such as the glycopeptide antibiotics (GPAs, including vancomycin and teicoplanin) are of great pharmaceutical importance due to their use against Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus. GPAs are assembled in a complex process based on nonribosomal peptide synthesis and latestage, multistep cross-linking of the linear heptapeptide performed by cytochrome P450 monooxygenases. These P450 enzymes demonstrate varying degrees of substrate selectivity toward the linear peptide precursor, with limited information available about their tolerance regarding modifications to amino acid residues within the essential antibiotic core of the GPA. In order to test the acceptance of altered residues by the P450catalyzed cyclization cascade, we have explored the use of β-amino acids in both variable and highly conserved positions within GPA peptides. Our results indicate that the incorporation of β-amino acids at the C-terminus of the peptide leads to a dramatic reduction in the efficiency of peptide cyclization by the P450s during GPA biosynthesis, whereas replacement of residue 3 is well tolerated by the same enzymes. These results show that maintaining the C-terminal 3,5-dihydroxyphenylglycine residue is of key importance to maintain the efficiency of this complex and essential enzymatic cross-linking process.

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INTRODUCTION Nature is one of the largest reservoirs of compounds used in the pharmaceutical industry, with natural products having a broad range of applications and playing a prominent role in drug discovery.1,2 However, given that many natural products are difficult to isolate or are only produced in low quantities, it is important to identify synthetic routes as alternatives to their host-based production. Additionally, the emergence of bacterial strains resistant to our most common antibiotics has resulted in an even greater need to synthesize new bioactive derivatives of such compounds.3 However, due to the chemical complexity of many important natural products, their chemical synthesis is often highly challenging and not scalable for production.4,5 Glycopeptide antibiotics (GPAs) represent one very important group of natural products that include key compounds vancomycin and teicoplanin, which are both used as last resort antibiotics against methicillin-resistant Staphylococcus aureus (MRSA).6,7 The mechanism of GPA action is based on interactions between the GPA and the D-Ala-D-Ala motif found in the essential bacterial cell wall precursor lipid II (Figure 1), specifically from a series of key hydrogen bonding interactions between the peptide backbone of the GPA and the lipid II terminal dipeptide (D-Ala-D-Ala). The highly crosslinked nature of the aromatic side chains of the GPA peptide core serves to deliver conformational stability, allowing the peptide backbone to form the requisite interactions; thus, aromatic cross-linking of the peptide is key to GPA activity.6,7 © 2018 American Chemical Society

Like many peptide-based antibiotics, GPAs are produced by a large, modular enzymatic peptide assembly line known as a nonribosomal peptide synthetase (NRPS), which separates peptide synthesis from the ribosome (Figure 1A).8,9 Typically, linear NRPS enzyme machineries can be divided into modules that are responsible for amino acid incorporation into the growing peptide. Each module can be further subdivided into different catalytically active domains that each has a specific function to play during peptide synthesis.8 Essential domains present in every module are adenylation (A) domains, peptidyl carrier (PCP) domains, and condensation (C) domains. The A domains perform the selection of specific amino acids and activate them by adenylation. Subsequently, the activated amino acid is loaded onto a neighboring PCP domain via a thioester bond to the post-translationally added phosphopantetheinyl PCP group for incorporation into the growing peptide. The C domains are then responsible for the formation of an amide bond between the PCP-loaded amino acid and the growing PCP-bound peptide. In the final module of an NRPS assembly line, a thioesterase (TE) domain is typically required to release the peptide from the machinery through processes including hydrolysis or macrocyclization.8 In addition to the essential Special Issue: Synthesis of Antibiotics and Related Molecules Received: February 12, 2018 Published: April 30, 2018 7206

DOI: 10.1021/acs.joc.8b00418 J. Org. Chem. 2018, 83, 7206−7214

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Figure 1. Schematic representation of GPA biosynthesis and mechanism of antibiotic activity. (A) NRPS-mediated biosynthesis of the linear heptapeptide teicoplanin precursor peptide, showing the seven NRPS modules along with the amino acids activated by each adenylation domain. (B) Peptide maturation catalyzed by cytochrome P450 (Oxy) enzymes, which occurs while the peptide is attached to the PCP domain of the final NRPS module and is mediated by the unique X domain (M7). (C) Following thioesterase-mediated cleavage of the completely cross-linked GPA peptide, further modifications to the GPA are performed, such as glycosylation and acylation, as shown here for teicoplanin A2-1. (D) Mechanism of antibiotic action of GPAs, in which the GPA binds to the terminal D-Ala-D-Ala motif of the bacterial lipid II cell wall precursor (here a simplified version is depicted), thus preventing bacterial cell wall biosynthesis.

determined that the X domain, present in the final module of the GPA NRPS, plays an essential role in the activity of the Oxy enzymes, where it acts as a recruitment platform for these P450 enzymes during peptide maturation.18 The discovery of the role of the X domain has allowed several of the GPA cyclization steps to be examined in detail, although the effects of altering the heptapeptide backbone on Oxy-catalyzed activity have not been investigated so far in vitro.18−23 In NRPS biosynthesis, one important consequence of the disconnection of peptide production from the ribosome is the ease and commonality with which NRPS machineries incorporate nonproteinogenic amino acids, such as phenylglycine (Phg) derivatives, into such NRPs: the large number of amino acid residues identified to date result in significant diversity of these bioactive compounds.24 Phenylglycine residues are unique α-amino acids, with the direct linkage of the aromatic ring to the α-carbon leading to difficulties during synthesis of NRPs bearing these residues because of problems associated with steric bulk and their tendency to epimerize even under relatively mild reaction conditions. GPA peptides constantly contain (S)-3,5-dihydroxyphenylglycine (L-Dpg) residues at the C-terminus (position 7) of the heptapeptide, and some GPAs contain an additional L-Dpg residue in the variable N-terminal portion (position 3) of the peptide; these residues are highly prone to epimerization during solid-phase peptide synthesis (SPPS).25,26 Epimerization of

core domains, epimerization (E) domains that alter the stereochemistry of specific amino acids within nonribosomal peptides (NRPs) are found within many NRPS modules.8 In the case of GPA biosynthesis, the formation of the linear peptide is followed by several maturation events in order to create the final biologically active compound that can then be used as an antibiotic (Figure 1B,C).6,7,9 One vital step is accomplished by P450 monooxygenases (Oxy enzymes, specifically OxyA/B/C/E), which are responsible for the introduction of specific oxidative cross-links into the linear peptide (Figure 1B).9−14 These linkages (either aryl (AB, performed by OxyC) or phenolic ether bridges (C-O-D, D-O-E, optional F-O-G ring in teicoplanin-type GPAs, performed by OxyB, OxyA, and OxyE, respectively)) between the side chains of aromatic residues within the peptide are essential for the activity of GPAs (Figure 1D).6,7,9 These cross-links also present some of the most significant synthetic challenges to overcome in the chemical synthesis of GPAs.4 The need to understand this process has become more pressing as bacteria become increasingly resistant to common antibiotics, including GPAs. In spite of the challenges, total synthesis has delivered some impressive examples of modified GPAs able to overcome resistant bacterial phenotypes: the stumbling block lies in their production in high yields for pharmaceutical use.4,5,15−17 Given the importance of the GPA cross-linking cascade, our group has concentrated on understanding this process. We have 7207

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Figure 2. Actinoidin- and teicoplanin-like CoA heptapeptides synthesized in this study (1−7). Peptide sequences 3−7 contain β-amino acids in position 3 or 7 of the peptide; these residues are depicted in blue, with the additional methylene group highlighted in pink.

and the synthetic difficulties associated with this amino acid position, we aimed to explore the use of β-amino acids, which are not prone to racemization and extend the linear peptide by only a methylene group, at either position 3 or 7 of GPA peptides to ascertain both their utility in synthesis as well as their potential acceptance from a biosynthetic standpoint. βAmino acids have been shown to be compatible with NRPSmediated biosynthesis and redesign in other systems,30 but the acceptance of such residues in GPA biosynthesis has yet to be explored. Given that transformations of natural GPAs into semisynthetic compounds often rely on changes of the Cterminus of the peptide, we sought to investigate the extent to which modifications at this position could be incorporated into GPA biosynthesis pathways by elucidating the selectivity of the Oxy enzyme cascade for changes to the last residue of the GPA precursor peptide.

these residues is problematic because the Oxy enzymes are stereoselective and have even been shown to be inhibited by peptide substrates bearing a D-Dpg residue in position 7 of the peptide.21 In previous studies, replacement of both Dpg residues with (S)-4-hydroxyphenylglycine (Hpg) residues has overcome this issue, as the peptide epimers produced in this case can be separated by HPLC.19,21,22 However, the disadvantage of switching the terminal Dpg residue for a Hpg residue is that it alters the size of the conserved GPA AB ring upon cross-linking, and in vivo studies have shown that, while cross-linked by the Oxy enzymes, this modified GPA has no antibiotic activity.27 Previous studies have also investigated the potential to alter the C-terminus of the GPA peptide either by substituting the Dpg residue or by elongating the GPA peptide.28,29 Weist et al. showed that the introduction of different phenylglycine derivatives in position 7 of the peptide influenced both bicyclic and tricyclic ring formation, although some modifications were tolerated by the Oxy enzymes based on in vivo experiments.29 These studies observed that AB ring formation can only be maintained when the modified Phg residue bears at least one hydroxyl or methoxy substituent in the 3-position of the aromatic ring.29 Furthermore, Butz et al. demonstrated in an impressive piece of re-engineering that the GPA producing NRPS machinery could be extended to generate an octapeptide, but the ability of the Oxy cascade to cyclize this extended peptide was minimal: some OxyB activity could be observed to generate the initial C-O-D ring, whereas the second and third reactions mediated by OxyA and OxyC were completely abolished.28 Given the limited number of examples exploring the acceptance of C-terminally altered GPA peptides

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RESULTS AND DISCUSSION With the Oxy enzymes occupying such an important role in GPA biosynthesis, determining their selectivity is crucial to understanding the potential redesign capacity of GPA biosynthesis. As the majority of GPA diversity is found in the Nterminal residues of the peptide (first and third residues, specifically),6 in this study, we focused on exploring the alteration of the C-terminal seventh residue in two template peptide sequences (teicoplanin- and actinoidin-type GPA peptides) and comparing the effects of substituting this highly conserved Dpg residue with alterations to the third residue of teicoplanin-type GPAs. The correct stereochemistry of the Cterminal residue has already been shown to be extremely important for the final peptide maturation by the Oxy enzymes: 7208

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The Journal of Organic Chemistry a change from L- to D-configuration of this residue decreases the activity of the Oxy enzymes to a significant extent.21 By switching the C-terminal residue to a β-amino acid in this study, we aimed to determine the effect of this relatively small alteration within the peptide backbone at a crucial position of the GPA peptide while at the same time overcoming any potential issues of epimerization. Furthermore, accessing a teicoplanin-type peptide in which the third (Dpg) residue had been substituted for a β-amino acid would also allow us to compare the acceptance of such residues in both the highly conserved (seventh) as well as variable positions (third) within the GPA peptide. To this end, we synthesized seven different peptides to determine their acceptance by OxyB and OxyA enzymes from various GPA biosynthetic pathways. Specifically, these included control GPA peptides based on an actinoidin sequence (peptides either not chlorinated 1 or chlorinated 2 at residues 2/6 of the peptide), as well as with three actinoidintype and two teicoplanin-type heptapeptides bearing β-amino acids at the C-terminus or position 3 of these peptides (Figure 2). In the β-amino-acid-containing peptides, we introduced (3R)-3-amino-3-(3,5-dihydroxyphenyl)propanoic acid (β-3,5Dpg, peptides 3 and 4) and (3R)-3-amino-3-(3hydroxyphenyl)propanoic acid (β-3-Hpg, peptide 5) in position 7 of the linear actinoidin backbone, as well as (3R)-3-amino-3(4-hydroxyphenyl)propanoic acid (β-4-Hpg, peptide 6) in position 7 of the linear teicoplanin backbone. As a probe to explore the selectivity of OxyB and OxyA toward alterations in the N-terminal portion of the peptide, we synthesized a further teicoplanin-like peptide possessing a β-3,5-Dpg residue in position 3 of the substrate (peptide 7). We also sought to test the effect of tyrosine chlorination within the β-3,5-Dpgcontaining peptide on Oxy activity and to compare this with the data for the actinoidin- and teicoplanin-type peptides.19,23 All peptides were synthesized using Fmoc-based SPPS (see Experimental Section).26 The resultant peptides were cleaved from the resin and converted into their corresponding CoA peptides.25 These thioester peptides were then loaded using the promiscuous phosphopantetheinyl transferase (Sfp R4-4 mutant)31 onto the peptidyl carrier protein domain of a didomain construct excised from the teicoplanin NRPS (PCPXtei) that also contains the X domain essential for Oxy recruitment.18 An analysis of the acceptance of these peptides by the GPA Oxy enzymes was accomplished by first screening various OxyB enzymes to select the homologue with the highest activity in introducing the initial C-O-D cross-link for each peptide. Subsequently, several OxyA homologues were screened in order to identify the OxyA homologue with the best activity in introducing the D-O-E cross-link (Scheme 1). For the chlorinated peptide analogues, the OxyB/OxyA pair established for the nonchlorinated peptides was then used to test the cyclization of the chlorinated analogues. In initial experiments, all nonchlorinated heptapeptides were tested, and the cyclization activity of the GPA Oxy enzymes was compared to their activity against the standard actinoidin peptide sequence. The results of OxyB homologue screening on actinoidin peptide 1 showed that the balhimycin (OxyBbal) and vancomycin homologue (OxyBvan)32−34 were the most active enzymes in catalyzing the formation of the monocyclic C-O-D peptide, with yields of 91% and 86%, respectively (Table 1 and Figure 3A). Thus, OxyBbal was selected for further experiments to generate the monocyclic compound for subsequent OxyA homologue screening. When comparing the different OxyA enzymes (Figure 3B), it was clear that all OxyA

Scheme 1. Workflow of the GPA Peptide Cyclization Reactions Explored in This Study (Example for Peptides 3 and 4)a

CoA heptapeptides were first loaded onto the PCP-X construct by the promiscuous phosphopantetheinyl transferase Sfp (R4-4 mutant). Subsequently, formation of bicyclic heptapeptide was mediated by the actions of the GPA biosynthetic P450 enzymes OxyB (shown in violet) and OxyA (shown in yellow). Cleavage of the peptides from the PCP domain was then accomplished using a methylamine solution to isolate the final compound as the methylamide peptide for analysis by liquid chromatography−mass spectrometry. a

homologues tested were active against the actinoidin peptide sequence 1, but that the ristomycin homologue (OxyAris) was the most effective, yielding 29% bicyclic product in this case (Table 1). We then performed OxyB homologue screening for peptide 3 bearing a β-3,5-Dpg residue in position 7 of the peptide (Figure 3C). All OxyB homologues tested were active and introduced the C-O-D cross-link, albeit with reduced yield of formation. One observable trend was that homologues displaying high initial activity levels with actinoidin peptides maintained ∼50% of activity against the actinoidin peptide with 7209

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Table 1. Analysis of Oxy-Mediated Cyclization of Peptides 1−7 Synthesized in This Study (Triplicate Experiments, Results Shown in %) substrate 1

2

OxyB

Van Bal Tei Ris Cep Dbv12 StaH

86 91 38 13 84 85 44

± ± ± ± ± ± ±

6 1 15 2 9 5 16

OxyA

Tei Ris Cep Dbv14 StaF Pek

18 29 5 9 6 8

± ± ± ± ± ±

2 3 1 1 1 1

52 ± 3

36 ± 1

3

4

5

6

7

47 ± 1 45 ± 1 10 ± 2 2±1 46 ± 1 42 ± 2 2±1

51 ± 1

54 ± 1

54 ± 2

65 ± 2 65 ± 2

13 ± 1

0%

0% 0% 0% 0% 0% 0%

1 5 1 2 1 3

± ± ± ± ± ±

1 3 1 1 1 2

31 41 10 11 34 6

± ± ± ± ± ±

2 4 2 2 2 2

Figure 3. Comparison of OxyB and OxyA activity against different heptapeptide substrates. (A) OxyB screening of structurally different peptides, showing the yield of monocyclic peptide formation for the two most active homologues. (B) OxyA screening of structurally different peptides, showing the yield of bicyclic peptide formation using the best performing OxyA homologue. (C) Summary of structural elements varying within the different substrate peptides.

the β-3,5-Dpg residue (OxyBvan, OxyB from chloroeremomycin biosynthesis (OxyBcep), OxyB from A40926 (Dbv12), and balhimycin biosynthesis (OxyBbal)), whereas homologues with lower initial activity against the actinoidin template peptide displayed very low activity levels with peptide 3 containing β3,5-Dpg (including OxyB homologues from teicoplanin biosynthesis (OxyBtei), OxyBris and OxyB from A47934 biosynthesis (StaH)). No OxyA activity was observed for any homologue against the β-amino-acid-containing heptapeptide 3, which combined with the OxyB results indicates that the GPA cyclization cascade is highly sensitive to changes in the Cterminal portion of the peptide backbone of the GPA precursor

(Table 1). These results complement those of in vivo experiments described by Butz et al., which showed that the elongation of the GPA peptide by one amino acid led to very low levels of OxyB activity and no observable OxyA activity against these longer octapeptide substrates.28 As alterations to the side chain of the C-terminal residue of the natural peptide substrate of the GPA cyclization cascade have been shown to be supported by experiments performed both in vitro18,23 and in vivo,29 we also investigated what influence the incorporation of different β-amino acids at the Cterminal residue had on Oxy activity. To this end, we synthesized heptapeptide 5 bearing a β-3-Hpg residue in 7210

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homologues displaying levels of activity against 7 significantly higher than those of 3−6 (up to 41%). This indicate that the structure of the GPA peptide displays degrees of flexibility in the N-terminal portion due to the tolerance of the Oxy enzymes for these positions, but this is not reflected in the specificity that these enzymes display for the structure of the Cterminus of the peptide.

position 7 of the peptide and performed turnover experiments using OxyBvan along with subsequent OxyA homologue screening (Figure 3C). The results show the same trend as for the β-3,5-Dpg-containing peptide 3, which is tolerated by OxyB to a similar extent (54% monocyclization) and shows no formation of bicyclic product with any OxyA homologue. In vivo experiments from the Süssmuth group have shown that an α-3-Hpg-containing peptide is able to be fully cyclized by the GPA cyclization cascade,29 which again demonstrates that the addition of a methylene group within the peptide backbone of the GPA precursor is deleterious to Oxy activity on such peptide substrates. Previous experiments have shown that a 4Hpg residue in position 7 of the normal-length GPA peptide is well tolerated by the GPA cyclization cascade, which has been widely explored with in vitro experiments using OxyA and OxyB enzymes.18−23,35 Given these results, we also decided to synthesize a teicoplanin-like heptapeptide 6 possessing a β-4Hpg residue in the last position of the peptide sequence (Figure 3C). Initial cyclization of 6 by OxyBvan yielded 54% monocyclic product, which is comparable to the other β-amino-acidcontaining peptides 3 and 5. 6 was also shown to be a viable substrate for OxyA-mediated cyclization for all homologues tested, albeit with very low yields of bicyclic product detected (1−5%). In order to explore how the presence of chlorotyrosine residues in position 2/6 of the peptide influences the Oxy cascade with these altered peptides, peptides 2 (3,5-Dpg in position 7) and 4 (β-3,5-Dpg in position 7) were synthesized with chlorinated tyrosine residues at positions 2 and 6 (Figure 3C). Previous experiments have demonstrated that higher yields of cross-linked products can be achieved using chlorinated peptide substrates for the Oxy enzymes but only if the X domain is present in these assays.19,25,32 Comparison of the acceptance of both actinoidin 1 and the dichloroactinoidin peptide 2 shows that the formation of the monocyclic product decreases: conversion of linear compound 2 to the monocyclic molecule is around one-half as efficient when using OxyBbal (Table 1). However, compound 2 yields a slightly higher amount of bicyclic product (∼25% more) than 1 when using OxyAris. The second chlorinated compound we tested was the β-amino-acid-containing heptapeptide 4 possessing a β-3,5-Dpg residue in position 7 of the peptide. Oxy-catalyzed turnover experiments yield 51% monocyclic and 13% bicyclic product, showing that the most important role of peptide chlorination in the GPA cyclization cascade is in maintaining OxyA-mediated D-O-E ring formation. This mirrors results obtained for other GPA peptides and demonstrates the highest level of bicyclic product for any peptide bearing a β-amino acid at position 7 tested in this study.19,25 With the low level of Oxy-mediated cross-linking of peptides 3−6 bearing C-terminal β-amino acid residues, we next investigated the selectivity of these enzymes toward peptide 7 in which a β-3,5-Dpg residue was used to replace the standard 3,5-Dpg residue found in this position of teicoplanin-type GPAs (Figure 3C). Given that this modification lies outside the highly conservedand synthetically most challengingAB and C-OD ring-containing portion of the molecule, we hypothesized that 7 would be an acceptable substrate for OxyB enzymes. The results of the cyclization assay of 7 performed with OxyBvan and OxyBbal showed that 7 was indeed a good substrate for both OxyB homologues, with 65% of the initial peptide converted into the monocyclic form. In subsequent OxyA screening for introduction of the D-O-E ring, we observed several

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CONCLUSION Our experiments demonstrate the exquisite tuning of the GPA cyclization cascade that has occurred in order to synthesize these complex antibiotics: even small alterations in structure such as an additional methylene group at a crucial position within the peptide backbone appear sufficient to reduce or even eliminate Oxy-mediated cyclization. Based on these results, it appears that maintenance of the natural C-terminus within GPA precursor peptides is crucial, whereas alteration of the peptide backbone extension at position 3 is tolerated by the Oxy enzymes. These results serve to act as initial “guidelines” within which biosynthetic re-engineering of GPAs can operate and indicate that the challenge of synthesizing GPAs is not merely a problem faced by chemical synthesis but also by the biocatalysts that naturally produce these fascinating molecules.

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EXPERIMENTAL SECTION

General Information. All chemicals were commercially available: 2-chlorotrityl chloride resin (0.8 mmol/g, Biochem), DCM (Chemsupply), hydrazine monohydrate 64−65% (Sigma-Aldrich), MeOH (Scharlau), N,N′-diisopropylethylamine (DIEA) (Sigma-Aldrich), Fmoc-amino acids (Merck), COMU (Merck), triethylamine (SigmaAldrich), 2,6-lutidine (Sigma-Aldrich), DBU (Sigma-Aldrich), DMF (Ajax Finechem), TFA (Sigma-Aldrich), triisopropylsilane (TIS) (Sigma-Aldrich), urea (Sigma-Aldrich), NaH2PO4 (Sigma-Aldrich), NaNO2 (Sigma-Aldrich), coenzyme A (Affymetrix). Peptide PCP Turnovers. Commercial 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) (Sigma-Aldrich), NaCl (SigmaAldrich), MgCl2 (Sigma-Aldrich), glucose (Sigma-Aldrich), glucose dehydrogenase (Sorachim), and NADH (Sigma-Aldrich) were used. Peptide analysis, purification, and turnover analyses were performed on a HPLC-MS system from Shimadzu (LCMS-2020, ESI operating in positive and negative mode). UV spectra were recorded via a SPD-20A Prominence photodiode array detector in analytical mode and via a SPD-M20A Prominence photodiode array detector in preparative mode. Solvents employed were water 0.1% formic acid (FA) and acetonitrile (ACN) + 0.1% FA for analytical measurements and water + 0.1% trifluoroacetic acid (TFA) and acetonitrile + 0.1% TFA for preparative runs. Turnover analyses were performed using a Waters XBridgePeptide BEH C18 column, 300 Å, 3.5 μm, 4.6 mm × 250 mm employing a gradient of 5−95% ACN + 0.1% FA in 30 min. Crude peptides were purified using a preparative HPLC Waters XBridge Peptide BEH C18 OBD prep column, 300 Å, 5 μm, 19 mm × 150 mm employing a gradient of 10−40% ACN + 0.1% FA or 15−45% ACN + 0.1% FA in 30 min. NMR Analysis. 1H NMR analysis spectra were recorded on a Bruker Avance III 600 MHz. Solvent was CD3CN/D2O (20:80, v/v′). HRMS Analysis. HRMS was performed on an Agilent 6220 Accurate Mass LC-TOF system with an Agilent 1200 series HPLC. Peptide Synthesis: Resin Preparation. Peptide synthesis was performed manually by SPPS (scale 0.05 mmol). 2-Chlorotrityl chloride resin (200 mg) was swelled in DCM (8 mL, 30 min), washed with DMF (3×), and incubated with 5% hydrazine solution in DMF (6 mL, 2 × 30 min). The resin was washed with DMF (3×), and a solution of DMF/TEA/MeOH (7:2:1) (4 mL, 15 min) was added. For CoA peptide 6, a modified synthetic procedure was adopted (see Supporting Information Figure S2). Amino Acid Coupling and Fmoc Deprotection. The first Fmoc amino acid (0.06 mmol) was coupled using COMU (0.06 mmol) and 7211

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Article

The Journal of Organic Chemistry 2,6-lutidine (0.06 mmol, 0.12 M) overnight. In the second step, unreacted hydrazine groups were capped with Boc−glycine−OH (0.15 mmol) activated with COMU (0.15 mmol) and 2,6-lutidine (0.15 mmol, 0.12 M) for 1 h. Fmoc removal was performed using 1% DBU solution (3 mL, 3 × 30 s) in DMF followed by Fmoc- or Boc-amino acid coupling (0.15 mmol) with COMU (0.15 mmol) and 2,6-lutidine (0.15 mmol, 0.12 M) for 40 min. The final amino acid added was always Boc-protected. Peptide Cleavage. Cleavage of the hydrazide peptide from resin and removal of protecting groups (tBu and Boc) were accomplished using TFA/TIS/H2O (95:2.5:2.5 v/v′/v″, 5 mL) for 1.5 h with shaking at room temperature. The resin was removed by filtration and washed with TFA (2×). Subsequently, the filtrate was concentrated under a N2 stream to ∼1 mL and precipitated with ice cold diethyl ether (∼8 mL), followed by centrifugation in a flame-resistant centrifuge. Crude peptide then was purified using preparative RPHPLC, with 8−17% yields of the purified hydrazide peptide. CoA Peptide Formation. The purified hydrazide peptide was dissolved in buffer 1 containing urea (6 M) and NaH2PO4 (0.2 M), pH 3 (obtained via addition of HCl) to a final concentration of 5 mM. The solution was cooled to −15 °C using a salt/ice bath. Subsequently, 0.5 M NaNO2 (0.95 equiv) was added, and the mixture was stirred for 10 min. Coenzyme A (1.2 equiv) was dissolved in buffer 1 and added to the reaction. The pH was adjusted to 6.5 using KH2PO4/K2HPO4 buffer (6:94 v/v 1 M, pH 8.0). The reaction mixture was stirred on ice for 30 min and monitored by HPLC-MS. The final product was purified using preparative RP-HPLC, gradient 10−40% ACN or 15−45% ACN in 30 min. Expression and Purification of PCP-Xtei Protein. The expression vector pET-GB1-PCP-Xtei was transformed into Escherichia coli BL21(DE3). For expression, 10 L LB medium supplemented with kanamycin (50 μg/mL) was used. Inoculation was performed using 1/ 100 of an overnight preculture, and the main culture was incubated at 37 °C (170 rpm) until an OD600 nm of 0.6 was reached. Protein expression was induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG, 0.1 mM final concentration), and incubation continued overnight at 18 °C. The cells were harvested via centrifugation (7550 rcf, 10 min, 4 °C), and the pellet was resuspended in 15 mL of lysis buffer (50 mM Tris-HCl, pH 7.3, 50 mM NaCl, 10 mM imidazole, protease inhibitor (Sigma)) per 2 L of initial culture. For purification, the cells were thawed and lysed via sonication (Consonic), and the lysate was clarified via centrifugation (38 420 rcf, 40 min, 4 °C). The supernatant was used for NiNTA purification in batch mode (NiNTA buffer A: 50 mM Tris-HCl, pH 7.4, 300 mM NaCl, 10 mM imidazole; NiNTA buffer B: 50 mM TrisHCl, pH 7.4, 300 mM NaCl, 300 mM imidazole). Fractions containing the desired protein were combined and concentrated. In a second purification step, Streptactin affinity purification was performed using an Ä kta purification system (GE Health Care, StrepTrap 6 mL) (buffer A: 100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA; buffer B: 100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 2 mM desthiobiotin). The fractions containing the desired protein were pooled, concentrated, flash frozen, and stored at −80 °C. OxyA and OxyB Expression and Purification. All P450 enzymes (expression vectors pET28 or pET151d) were transformed into E. coli KRX cells and expressed in LB medium (10 L) supplemented with the respective antibiotic. Inoculation was performed using 1/100 of the culture volume with an overnight culture, with subsequent growth taking place at 37 °C (120 rpm) until an OD600 nm of 0.40−0.45 was reached. Subsequently, δ-aminolevulinic acid (100 μg/L) was added, and protein expression was induced through addition of 0.1% (w/v) rhamnose and 0.1 mM IPTG (final concentration); incubation continued overnight at 18 °C (90 rpm). Harvesting of the cells, lysis, clarification, and NiNTA purification were performed as described for PCP-Xtei. Dialysis was performed into anion exchange chromatography (AEX) buffer A (20 mM Tris-HCl, pH 8.0, 50 mM NaCl) overnight, followed by further purification using using anion exchange chromatography (GE Healthcare, ResourceQ 6 mL) with an Ä kta system. The protein was initially loaded, and any unbound sample was washed from the column using 5xCV of AEX buffer A.

Bound protein was then eluted by running a gradient from 0 to 50% AEX buffer B (20xCV, 20 mM Tris-HCl, pH 8.0, 1 M NaCl). All fractions containing the protein were combined. As a final purification step, size exclusion chromatography was performed using an Ä kta system equipped with a 320 mL Superose 12 column (GE Healthcare) (50 mM Tris-HCl, 7.4, 100 mM NaCl). Turnover Assay. Peptide turnover assays were performed following a previously established protocol.18 Briefly, the CoA peptide was loaded onto PCP-Xtei for 1 h at 30 °C using the transferase Sfp R4-4 in HEPES buffer (50 mM, pH 7.0), MgCl2 (10 mM), NaCl (50 mM), PCP-Xtei (40 μM), CoA peptide (80 μM), and Sfp R4-4 (4 μM). For the P450-catalyzed cross-linking reactions (total volume 105 μL), OxyB (0.5 μM), OxyA (1 μM), PuR36 (0.66 μM), PuxB A105 V36 mutant (2.5 μM), glucose (0.33%), glucose dehydrogenase (0.033 mg/mL), PCP-bound peptide (33 μM), and NADH (2 mM) were combined and incubated for 1 h at 30 °C at 300 rpm. The cross-linked peptide was subsequently cleaved from the PCP by adding 40% methylamine solution in water (0.5 M) and incubating for 15 min at room temperature. After cleavage, the pH was adjusted to pH ∼7.0 with 0.1% formic acid in water. Purification was performed using solidphase extraction (SPE) columns (Strata-X-SPE cartridges) that had been activated with 1 mL of MeOH and equilibrated with 1 mL of H2O. The neutralized solution (1 mL) was applied to the SPE column via gravity flow, washed with 5% MeOH (1 mL), and eluted with 1% formic acid in MeOH (500 μL). The sample was dried using a concentrator (Eppendorf) and dissolved again in 50 μL of ACN/H2O (1:1) prior to analysis by HPLC-MS. Compound Characterization. Actinoidin Hydrazide (1a): white solid, 4.9 mg, 9% yield was isolated with a purity >85%; ESI m/z [M + H]+ calcd for C59H60N9O14 1118.43; found 1118.35; ESI m/z [M − H]− calcd for C59H58N9O14 1116.41; found 1116.35. Actinoidin CoA (1): white solid, 1.54 mg, 18% yield was isolated with a purity >90%; 1H NMR (600 MHz, CD3CN) δ 8.52 (s, 1H), 8.22 (s, 1H), 7.14−7.08 (m, 5H), 6.97−6.92 (m, 4H), 6.92−6.87 (m, 2H), 6.78−6.74 (m, 2H), 6.74−6.70 (m, 4H), 6.69−6.65 (m, 3H), 6.64−6.58 (m, 3H), 6.50 (d, J = 8.4 Hz, 2H), 6.47−6.43 (m, 1H), 6.34−6.29 (m, 1H), 6.28−6.25 (m, 2H), 6.23−6.19 (m, 1H), 6.03 (d, J = 5.7 Hz, 1H), 5.35−5.30 (m, 1H), 5.29 (s, 1H), 5.19 (s, 1H), 5.14 (s, 1H), 4.89 (s, 1H), 4.79−4.74 (m, 1H), 4.73−4.70 (m, 1H), 4.53−4.48 (m, 1H), 4.47−4.39 (m, 2H), 4.39−4.36 (m, 1H), 4.35−4.33 (m, 1H), 4.14 (bs, 1H), 3.91 (s, 1H), 3.79−3.73 (m, 1H), 3.46−3.40 (m, 1H), 3.27−3.10 (m, 5H), 2.95−2.82 (m, 4H), 2.78−2.71 (m, 2H), 2.69− 2.59 (m, 4H), 0.83 (s, 3H), 0.65 (s, 3H); HRMS (ESI-TOF) m/z [M − 2H]2− calcd for C80H89N14O30P3S 925.2407; found 925.2403. Actinoidin-(Cl)-hydrazide (2a): white solid, 9.2 mg, 15% yield was isolated with a purity >85%; ESI m/z [M + H]+ calcd for C59H58Cl2N9O14 1186.35; found 1186.35; ESI m/z [M − H]− calcd for C59H56Cl2N9O14 1184.33; found 1184.45. Actinoidin-(Cl)-CoA (2): white solid, 1.84 mg, 19% yield was isolated with a purity >90%; 1H NMR (600 MHz, CD3CN) δ 8.43 (s, 1H), 8.12 (s, 1H), 7.14−7.08 (m, 7H), 6.99−6.85 (m, 10H), 6.78− 6.72 (m, 3H), 6.72−6.68 (m, 3H), 6.68−6.63 (m, 4H), 6.31 (s, 2H), 6.25 (s, 1H), 6.00 (d, J = 6.2 Hz, 1H), 5.36−5.29 (m, 3H), 5.19−5.14 (m, 2H), 5.13 (d, J = 6.8 Hz, 2H), 4.91 (s, 1H), 4.13−4.09 (m, 1H), 3.92−3.88 (m, 1H), 3.78−3.72 (m, 1H), 3.45−3.40 (m, 1H), 3.26− 3.22 (m, 2H), 3.20−3.16 (m, 2H), 3.14−3.11 (m, 1H), 2.94−2.87 (m, 3H), 2.87−2.81 (m, 3H), 2.72−2.63 (m, 5H), 0.80 (s, 3H), 0.61 (s, 3H); HRMS (ESI-TOF) m/z [M − 2H] 2 − calcd for C80H87Cl2N14O30P3S 959.2017; found 959.2007. Actinoidin-7-(β-3,5-Dpg)-hydrazide (3a): white solid, 4.5 mg, 8% yield was isolated with a purity >85%; ESI m/z [M + H]+ calcd for C60H62N9O14 1132.44; found 1132.45; ESI m/z [M − H]− calcd for C60H60N9O14 1130.43; found 1130.35. Actinoidin-7-(β-3,5-Dpg)-CoA (3): white solid, 0.8 mg, 14% yield, was isolated with a purity >90%; 1H NMR (600 MHz, CD3CN) δ 8.47 (s, 1H), 8.16 (s, 1H), 7.16−7.06 (m, 6H), 6.94 (d, J = 8.3 Hz, 4H), 6.91−6.86 (m, 3H), 6.80−6.59 (m, 10H), 6.51 (d, J = 8.1 Hz, 2H), 6.25−6.20 (m, 2H), 6.16−6.12 (m, 1H), 6.01 (d, J = 5.8 Hz, 1H), 5.36−5.30 (m, 2H), 5.20 (s, 1H), 5.13 (s, 1H), 5.08−5.02 (m, 1H), 4.11 (s, 1H), 3.92 (s, 1H), 3.81−3.73 (m, 1H), 3.47−3.40 (m, 1H), 7212

DOI: 10.1021/acs.joc.8b00418 J. Org. Chem. 2018, 83, 7206−7214

The Journal of Organic Chemistry

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3.34−3.23 (m, 2H), 3.15−3.08 (m, 2H), 2.93−2.87 (m, 1H), 2.85− 2.77 (m, 3H), 2.75−2.69 (m, 2H), 2.69−2.54 (m, 5H), 0.85−0.77 (m, 3H), 0.64 (s, 3H); HRMS (ESI-TOF) m/z [M − 2H]2− calcd for C81H91N14O30P3S 932.2485; found 932.2443. Actinoidin-(Cl)-7-(β-3,5-Dpg)-hydrazide (4a): white solid, 7.8 mg, 13% yield was isolated with a purity >85%; ESI m/z [M + H]+ calcd for C60H60Cl2N9O14 1200.36; found 1200.40; ESI m/z [M − H]− calcd for C60H58Cl2N9O14 1198.35; found 1198.55. Actinoidin-(Cl)-7-(β-3,5-Dpg)-CoA (4): white solid, 0.3 mg, 4% yield was isolated with a purity >90%; 1H NMR (600 MHz, CD3CN) δ 8.52 (s, 1H), 8.21 (s, 1H), 7.13−7.07 (m, 5H), 6.94−6.88 (m, 5H), 6.89−6.84 (m, 2H), 6.77−6.60 (m, 10H), 6.24−6.21 (m, 2H), 6.15− 6.11 (m, 1H), 6.04−6.00 (m, 1H), 5.35−5.31 (m, 1H), 5.19−5.15 (m, 1H), 5.12−5.08 (m, 1H), 5.08−5.03 (m, 1H), 4.92−4.89 (m, 1H), 4.78−4.69 (m, 2H), 4.13 (s, 2H), 3.93 (s, 1H), 3.80−3.73 (m, 1H), 3.47−3.42 (m, 1H), 3.34−3.25 (m, 3H), 3.17−3.09 (m, 2H), 2.95− 2.89 (m, 2H), 2.87−2.78 (m, 4H), 2.69−2.62 (m, 4H), 2.60−2.53 (m, 2H), 2.27−2.22 (m, 2H), 2.14−2.10 (m, 2H), 0.83 (s, 3H), 0.65 (s, 3H); HRMS (ESI-TOF) m/z [M − 2H] 2 − calcd for C81H89Cl2N14O30P3S 966.2095; found 966.2065. Actinoidin-7-(β-3-Hpg)-hydrazide (5a): white solid, 10 mg, 17% yield was isolated with a purity >85%; ESI m/z [M + H]+ calcd for C60H62N9O13 1116.45; found 1116.45; ESI m/z [M − H]− calcd for C60H60N9O13 1114.43; found 1116.40. Actinoidin-7-(β-3-Hpg)-CoA (5): white solid, 0.59 mg, 10% yield was isolated with a purity >90%; 1H NMR (600 MHz, CD3CN) δ 8.51 (s, 1H), 8.23 (s, 1H), 7.21−7.12 (m, 3H), 7.10−7.00 (m, 5H), 7.00− 6.91 (m, 3H), 6.83−6.70 (m, 6H), 6.70−6.61 (m, 5H), 6.61−6.50 (m, 5H), 6.47−6.41 (m, 2H), 6.03 (d, J = 5.9 Hz, 1H), 5.35−5.31 (m, 1H), 5.22 (s, 1H), 5.14−5.08 (m, 1H), 5.06 (d, J = 7.1 Hz, 1H), 5.00− 4.95 (m, 1H), 4.85 (d, J = 10.8 Hz, 1H), 4.76−4.68 (m, 1H), 4.13− 4.08 (m, 1H), 3.95−3.89 (m, 1H), 3.80−3.74 (m, 1H), 3.47−3.40 (m, 1H), 3.35−3.26 (m, 1H), 3.21−3.04 (m, 2H), 3.02−2.76 (m, 6H), 2.75−2.65 (m, 2H), 2.65−2.50 (m, 3H), 2.47−2.42 (m, 1H), 2.41− 2.33 (m, 2H), 2.31−2.25 (m, 2H), 0.84 (s, 3H), 0.65 (s, 3H); HRMS (ESI-TOF) m/z [M − 2H]2− calcd for C81H91N14O29P3S 924.2510; found 924.2484. Teicoplanin-7-(β-4-Hpg)-CoA (6): white solid, 1.7 mg, 23% yield was isolated with a purity >90%; 1H NMR (600 MHz, CD3CN) δ 8.55 (s, 1H), 8.26−8.22 (m, 1H), 7.20−7.16 (m, 1H), 7.12−7.06 (m, 2H), 7.04−6.99 (m, 4H), 6.93 (d, J = 8.0 Hz, 2H), 6.78 (t, J = 8.1 Hz, 4H), 6.73−6.61 (m, 10H), 6.55 (d, J = 7.5 Hz, 2H), 6.50 (d, J = 7.7 Hz, 2H), 6.05 (d, J = 4.5 Hz, 1H), 5.28 (s, 1H), 5.13 (s, 1H), 5.09 (t, J = 6.5 Hz, 2H), 4.91 (s, 1H), 4.79−4.72 (m, 3H), 4.14 (s, 2H), 3.93 (s, 2H), 3.80−3.74 (m, 2H), 3.49−3.41 (m, 2H), 3.33−3.25 (m, 3H), 3.24−3.19 (m, 1H), 3.16−3.07 (m, 2H), 2.92−2.84 (m, 3H), 2.82− 2.74 (m, 5H), 2.74−2.67 (m, 2H), 2.62−2.53 (m, 2H), 2.29−2.19 (m, 4H), 2.17−2.10 (m, 2H), 0.83 (s, 3H), 0.65 (s, 3H); HRMS (ESITOF) m/z [M − 2H]2− calcd for C80H89N14O30P3S 925.2407; found 925.2399. Actinoidin-3-(β-3,5-Dpg)-hydrazide (7a): white solid, 3.4 mg, 14% yield was isolated with a purity >85%; ESI m/z [M + H]+ calcd for C59H60N9O16 1150.42; found 1150.65; ESI m/z [M − H]− calcd for C59H58N9O16 1148.40; found 1148.60. Actinoidin-7-(β-3,5-Dpg)-CoA (7): white solid, 0.49 mg, 20% yield was isolated with a purity >90%; 1H NMR (600 MHz, CD3CN) δ 8.55−8.48 (m, 1H), 8.24−8.15 (m, 1H), 7.34−7.30 (m, 2H), 7.14− 7.08 (m, 2H), 6.98 (d, J = 8.5 Hz, 1H), 6.94 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.1 Hz, 2H), 6.76−6.70 (m, 4H), 6.67 (d, J = 8.4 Hz, 2H), 6.64 (d, J = 8.4 Hz, 1H), 6.61 (dd, J = 7.7, 5.0 Hz, 3H), 6.50 (d, J = 8.1 Hz, 1H), 6.46 (d, J = 8.1 Hz, 1H), 6.29−6.25 (m, 2H), 6.22 (s, 1H), 6.09− 6.06 (m, 2H), 6.04 (d, J = 5.7 Hz, 1H), 5.29 (s, 1H), 5.16 (s, 1H), 5.14 (d, J = 4.3 Hz, 2H), 4.92−4.88 (m, 1H), 4.17−4.13 (m, 2H), 3.91 (s, 1H), 3.78−3.74 (m, 1H), 3.47−3.43 (m, 1H), 3.26−3.20 (m, 2H), 3.21−3.15 (m, 2H), 3.15−3.10 (m, 1H), 2.96−2.86 (m, 3H), 2.85− 2.81 (m, 1H), 2.74 (s, 1H), 2.73−2.69 (m, 1H), 2.69−2.66 (m, 1H), 2.65−2.61 (m, 1H), 2.61−2.54 (m, 2H), 2.50 (dd, J = 13.2, 8.0 Hz, 2H), 2.22−2.11 (m, 3H), 0.82 (s, 3H), 0.64 (s, 3H); HRMS (ESITOF) m/z [M − 2H]2− calcd for C80H89N14O32P3S 941.2356; found 941.2325.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.8b00418. Synthesis of CoA peptides, MS (ESI) data for peptide hydrazide intermediates, HPLC traces and 1H NMR spectra for CoA peptides, and calculation of turnover results (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Max J. Cryle: 0000-0002-9739-6157 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank G. Stier (BZH-Heidelberg) for fusion protein vectors, S. Bell (University of Adelaide) for redox proteins, J. Yin (University of Chicago) for the R4-4 Sfp expression plasmid, A. Kirchberg (Monash) for assistance in protein preparation, and M. Peschke (MPI) for assistance with turnover assays. This work was supported by the Deutsche Forschungsgemeinschaft (Emmy−Noether Program, CR 392/1-1 (M.J.C.)), Monash University and EMBL Australia (M.J.C.). This research was supported under Australian Research Council’s Discovery Projects funding scheme (Project Number DP170102220) to M.J.C. M.J.C. would also like to acknowledge the support of the National Health and Medical Research Council through the provision of a Career Development Fellowship (APP1140619).

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REFERENCES

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DOI: 10.1021/acs.joc.8b00418 J. Org. Chem. 2018, 83, 7206−7214