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Rapid access to glycopeptide antibiotic precursor peptides coupled with cytochrome P450-mediated catalysis: towards a biomimetic synthesis of glycopeptide antibiotics† Clara Brieke, Veronika Kratzig, Kristina Haslinger, Andreas Winkler and Max J. Cryle* Understanding the mechanisms underpinning glycopeptide antibiotic biosynthesis is key to the future ability to reinvent these compounds. For effective in vitro characterization of the crucial later steps of the biosynthesis, facile access to a wide range of substrate peptides as their Coenzyme A (CoA) conjugates is essential. Here we report the development of a rapid route to glycopeptide precursor CoA conjugates that affords both high yields and excellent purities. This synthesis route is applicable to the synthesis of peptide CoA-conjugates containing racemization-prone arylglycine residues: such residues are hallmarks of non-ribosomal peptide synthesis and have previously been inaccessible to peptide synthesis using Fmoc-type chemistry. We have applied this route to generate glycopeptide precursor peptides in their carrier protein-bound form as substrates to explore the specificity of the first oxygenase enzyme from

Received 21st November 2014, Accepted 3rd December 2014 DOI: 10.1039/c4ob02452d www.rsc.org/obc

vancomycin biosynthesis (OxyBvan). Our results indicate that OxyBvan is a highly promiscuous catalyst for phenolic coupling of diverse glycopeptide precursors that accepts multiple carrier protein substrates, even on carrier protein domains from alternate glycopeptide biosynthetic machineries. These results represent the first important steps in the development of an in vitro biomimetic synthesis of modified glycopeptide aglycones.

Introduction The glycopeptide antibiotics are a unique class of complex natural products that remain in clinical use against Grampositive bacteria – including multi-drug resistant strains – despite their deployment over fifty years ago.1 The growing emergence of bacterial resistance to these compounds, which include vancomycin and teicoplanin, highlights the need for novel active derivatives.2,3 Despite impressive advances in total syntheses and modification strategies,4–9 the development of new compounds is hampered by the structural complexity of these glycopeptides: they possess a rigid aglycone structure with multiple stereogenic centers. Their rigid structure is essential for their antibiotic activity and is provided by the crosslinking of multiple aromatic amino acid side chain residues. In nature, these crosslinking steps are performed by

Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany. E-mail: [email protected] † Electronic supplementary information (ESI) available: Peptide characterization; cloning primers; CD-spectra and melting curves; alignment of PCP-domains; characterization of Sfp-catalyzed PCP-loading; results of in vitro turnovers with MS-fragmentation analysis. See DOI: 10.1039/c4ob02452d

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Cytochrome P450 (Oxy) enzymes that act on a non-ribosomally assembled precursor heptapeptide (Fig. 1).10,11 The utilization of Oxy enzymes as biocatalysts in a biomimetic synthesis approach – in which the aglycone assembly naturally performed by non-ribosomal peptide synthetases (NRPS) is replaced by solid phase peptide synthesis (SPPS) – would open new possibilities to overcome difficulties in the chemical synthesis of these compounds. Such a synthesis route would also serve to diversify glycopeptide aglycone structures from those available from semi-synthesis.12,13 Several Oxy enzymes have been structurally characterized10,11 and the order and role of the Oxys have been assigned for balhimycin14–16 and teicoplanin-like A47934,17 but the selectivity mechanisms controlling these crucial biosynthetic steps remain undetermined. In order to investigate this complex system, peptide-based probes are essential tools: one demonstration is the in vitro characterization of the first crosslinking reaction to form the aglycone C-O-D ring using OxyB from the vancomycin gene cluster (OxyBvan). Here, Robinson et al. showed that precursor peptides bound to peptidyl carrier protein domains (PCP or T-domains) of the respective NRPS are the true substrates of OxyB.18–20 As the synthesis of these peptides was non-trivial, an SPPS route based on Alloc-chemistry was utilized; reported yields of the isolated hexa- and heptapeptides prior to conver-

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Results and discussion

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Synthesis of peptidyl-CoA conjugates for glycopeptide antibiotic peptides including arylglycine residues

Fig. 1 (A) Aglycone maturation in glycopeptide biosynthesis: an NRPSderived precursor peptide (domain labels: C-condensation; A-adenylation; T-thiolation/peptidyl carrier protein; X-domain of unknown function; TE-thioesterase) is modified by several P450s that catalyze the crosslinking of aromatic amino acid side chains. (B) Overview of the different structural types of glycopeptide antibiotics.

sion to CoA-conjugates exceed 50% in only one case and more typically vary from 3–13%.18,21 We have recently developed an efficient SPPS protocol based upon Fmoc-chemistry that allows the synthesis of arylglycine-rich peptides in a manner preventing epimerization of these residues: glycopeptide antibiotics are the perfect test case for this methodology given that teicoplanin-type glycopeptides contain five such residues.22 We have now extended this methodology to establish a rapid route to the generation of precursor peptides covalently linked to PCP domains via their natural phosphopantetheine linkers. This methodology is of general relevance to the investigation of non-ribosomal peptide synthetase systems, many of which contain arylglycine residues that have previously prevented access to these types of essential substrates. With greatly improved access to diverse peptide substrates, we have examined – for the first time – the ability of OxyBvan to accept substrates that include alternate peptides from multiple glycopeptide types (I, II, III/IV, V; Fig. 1B) and the effect of using PCP domains from alternative producer NRPS systems. Our results indicate that OxyBvan is a highly effective in vitro catalyst for the installation of the C-O-D ring in multiple glycopeptide classes. In combination with our rapid Fmoc-based synthesis route, these represent important steps towards a biomimetic synthesis for the production of new, modified glycopeptide antibiotic aglycones using a reduced set of Oxy enzyme biocatalysts.

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The aglycone structures found for the different classes of glycopeptide antibiotics are closely related; major differences are restricted to the side chains of residues one, two and three of the peptide (see Fig. 1B and 2B).4 Given this, we wanted to conduct a thorough assessment of the tolerance of OxyBvan for substitutions at these positions to examine its potential as a versatile biocatalyst. As hexapeptides have been shown to be substrates for OxyBvan and since the β-hydroxylation of tyrosine on position 6 is not required for OxyB activity18,19 we focused on a basic set of hexapeptides ( peptides 1–5, Fig. 2B) mimicking biosynthetic intermediates of five types of glycopeptides. With our previous development of an Fmoc-route enabling the synthesis of arylglycine-containing peptides without racemization and far more rapidly than previously possible, we wished to adapt this to develop an equally rapid route to produce these peptides as their coenzyme A (CoA) thioester conjugates. The synthesis of such CoA thioester conjugates enables these to be enzymatically loaded onto purified apo-PCP domains to yield their peptidyl-form using the promiscuous transferase Sfp.23,24 This technique to obtain loaded-PCP substrates has been widely exploited as the most efficient way to obtain PCP-substrates – as cargo small molecules such as chromophores, crosslinkable units or even peptides have been employed25 – to investigate specific steps of NRPS-biosynthesis without the need to reconstitute the entire biosynthetic apparatus in vitro.23 With several options for the synthesis of thioesters via Fmoc chemistry available, we settled – after initial trials – upon the resin developed by Dawson26 and optimized the reported protocols for this resin to generate glycopeptide-type peptides. Using our reported Fmoc-conditions22 SPPS was performed on Dawson resin (1, Fig. 2A) followed by two-step activation of the Dawson linker (2→3, Fig. 2A): the conditions for activation of the Dawson linker had to be carefully optimized due to the inherent risks of modification of unprotected, reactive moieties of the peptide by the activating agent p-nitrophenylchloroformate and the base treatment required to complete the activation of the linker. Displacement of the protected peptide from the resin using 4-mercaptophenyl-acetic acid (MPAA) then directly generated the thioester functionality.27 The direct conversion of the protected peptide to the thioester allows the synthesis of peptide thioesters without modification of the N-terminus (e.g. methylation, acetylation) as no activation of the unprotected peptide is required. MPAA was chosen to generate the initial thioester for several reasons: increased solubility of the product peptide thioesters in aqueous solution and a reduction in toxicity compared to thiophenol, whilst maintaining a highly reactive thioester to enable rapid thioester exchange with CoA.27 After TFA treatment to globally deprotect the peptide, the crude peptide thioesters ( peptides 1–5-MPAA) were converted with CoA under controlled basic conditions ( pH 8.3) to the desired

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Fig. 2 (A) Synthesis route using epimerization-free peptide assembly on Dawson resin with Fmoc-chemistry utilized for the respective hexapeptide substrates ( peptides 1–5-T) of OxyBvan turnover assays. Peptide displacement from activated resin as MPAA thioesters is followed by conversion to CoA conjugate peptides 1–5-CoA. (B) Structures of the hexapeptides synthesized in this study that are representatives of all five classes of glycopeptide antibiotics (differing residues are depicted in gray).

conjugate for attachment to the PCP domain; control of the pH was critical to prevent significant hydrolysis at higher pH. Initial syntheses affording the vancomycin-like peptide 1-CoA revealed that whilst the isolation of the desired thioester peptide was possible, the formation of oxidized MPAA species occurred to significant levels during resin displacement. This necessitated undesirable and time-consuming HPLC purification, which was not appropriate for the route we had envisaged (see Fig. S1†). By performing both resin displacement and CoA conversion under reducing conditions (addition of tributylphosphine and tris(2-carboxylethyl)phosphine (TCEP) respectively) the purification workload of the CoA-peptide syntheses could be dramatically minimized through the use of a final, single solid-phase extraction (SPE) step: this purification method is simple, fast and cost effective and requires no significant instrumentation. With this optimized route in hand we then completed the syntheses of five representative hexapeptideCoA conjugates from glycopeptide classes I (vancomycin-type), II (actinoidin-type), III/IV (teicoplanin-type) and V (kistamycintype), together with the recently published pekiskomycin glycopeptide antibiotic.2,4,28 The yields of all peptide conjugates were high (∼50%) and the purities of these conjugates using

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SPE purification alone was over 83% in all cases (Fig. 3, additionally see ESI†). With the incorporation of arylglycine residues being a hallmark of non-ribosomal peptide synthesis,29 this development of a highly effective route to the synthesis of peptide CoA conjugates ready for PCP-loading has great potential to access a wide range of peptidyl-CoA conjugates. The use of Fmoc-chemistry in SPPS takes full advantage of the wide range of Fmoc-protected building blocks reported to enable the synthesis of peptides bearing functional groups including those appropriate for bioorthogonal chemistry,30 FRET,31 photoreactive crosslinking32 as well as labels for EPR/NMR.33 The high yield and simple purification reduces the cost and instrumental requirements for the synthesis of such peptides, which are important considerations as multiple peptides are often required to investigate specific aspects of NRPS-function in vitro. PCP-loading and the effect of alternate PCP-domains on the efficiency of OxyBvan catalyzed oxidation Oxidation of carrier protein bound substrates by Cytochromes P450 (P450s) has proven to be a common motif in natural product biosynthesis,10,11 even allowing astonishing selectivity

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Fig. 3 Analytical reverse-phased HPLC traces of SPE-purified CoA-peptides: (A) peptide-1-CoA (yield: 37 mg, 22.8 µmol, 46%; purity: 91%); (B) peptide-2-CoA (yield: 42 mg, 26.4 µmol, 53%; purity: 91%); (C) peptide-3-CoA (yield: 36 mg, 21.3 µmol, 42%; purity: 83%); (D) peptide-4-CoA (yield: 37 mg, 21.9 µmol, 44%; purity: 96%); (E) peptide5-CoA (yield: 56 mg, 32.4 µmol, 65%; purity: 92%). Purities were calculated from peak integration (detection at λ = 260 nm) employing a 5% cut-off of peak area (for peptide-4-CoA a 3% cut-off was used); according to LC-MS analysis no other CoA species could be detected – thus, no other species were present that could interfere with the PCP loading reaction. HPLC-gradient: 0–2 min 95% solvent A, 2–25 min up to 25% solvent B, flow rate 1 mL min−1 (see Experimental section).

amongst multiple, highly similar PCP domains.34,35 As the essential role of the PCP in substrate peptide presentation to OxyBvan has been demonstrated,20 we wished to assess the acceptance of substrates bound to the excised PCP domains from module 7 (PCP7) of the NRPS machineries of multiple glycopeptide antibiotics. These domains are significantly different in sequence to the other PCP-domains within glycopeptide NRPS-systems36 and are of great interest due to their interactions with multiple NRPS-domains in cis as well as external oxygenase enzymes in trans. To determine whether the correct peptide/PCP pair is required for effective oxidation to monocyclic peptides, we chose PCP constructs from the NRPS machineries of vancomycin-like A82846B (cep, type I),37 teicoplanin-like A47934 (sta, type IV)38 and complestatin (com, type V).39 Investigating the effect of altering the peptide/PCP pair on the efficiency of OxyBvan catalysis was important, as a high specificity for “matched” peptide/PCP pairs by OxyBvan would not only require the expression and purification of multiple PCP domains (corresponding to the natural NRPS system for each desired peptide), it would also greatly restrict the

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potential use of the Oxy enzymes as biocatalysts for the transformation of peptidyl-PCP substrates. As excised PCP domains can be troublesome due to low protein yields and a tendency to aggregate,35 PCPs were expressed as fusion proteins with N-terminal thioredoxin or GB1 (IgG binding B1 domain).40 The isolated proteins (GB1-PCP7cep, Trx-PCP7sta and Trx-PCP7com) were soluble and folded as confirmed by CD measurements (Fig. S12 and S13†). Additionally the yields of all fusion proteins were dramatically improved (1.1–2.5 µmol L−1 culture) over those of the isolated vancomycin PCP7 domain (180 nmol L−1 culture): this is of key importance as PCP domains are needed to support peptide turnover in stoichiometric amounts relative to peptide. Loading of peptides 1–5-CoA onto a conserved serine residue on the apo-PCP domains was then assessed using a mutant of the promiscuous phosphopantetheinyl transferase Sfp.23 Due to initial problems encountered with the loading of very hydrophobic peptides onto PCP domains, we utilized a modified form of Sfp (the R4-4 mutant).41 This mutant has, in our hands, proven a more reliable tool in ensuring loading of all peptides onto PCP-domains. This mutant also has the added benefit of shorter reaction times over the original wild type Sfp enzyme. MALDI-MS analysis of the loading mixtures revealed that all CoA conjugates and PCP fusion proteins were suitable substrates for Sfp loading using the R4-4 mutant (see ESI†). Generally, oxidation reactions catalyzed by P450s require redox partner proteins to shuttle electrons from NAD(P)H to the P450. We concentrated on bacterial P450-redox systems that have the advantage of using less expensive NADH as electron source rather than other eukaryotic/prokaryotic redox partners: we determined that efficient electron transfer was maintained using the [2Fe–2S] ferredoxin HaPux and flavindependent reductase HaPuR from Rhodopseudomonas palustris HaA2.42 Quenching the turnover assays using methylhydrazine yielded a mixture of hydrolyzed and methylhydrazide peptides, which were analyzed using HPLC-MS to determine the yield of monocyclic products ( peptides 6–10, Fig. 4A and S16†). Hydrolysis of peptides using basic conditions alone caused protein precipitation and aggregation, which resulted in concomitant aggregation of the cleaved, hydrophobic peptides in solution. Cleavage with methylhydrazine, whilst leading to the formation of two regioisomers of the peptide hydrazide and small proportions of hydrolyzed peptides, was successful in preventing protein precipitation and loss of peptide from solution. Thus, we persisted with methyl hydrazine cleavage as the preferred method for the cleavage of highly hydrophobic peptides from their PCP-domains to enable sufficient signal for MS analysis of the OxyB turnover reactions ( performed on nanomolar scale per reaction). Initially, we compared the oxidation efficiency of vancomycin-type peptide 1 bound to the three different PCP domains by OxyBvan (Fig. 4B). Previously, OxyBvan has been reported to accept substrate peptides with high efficiency on peptidylPCP6van and -PCP7van domains and we wished to test other PCP domains.19 By using the fusion construct GB1-PCP7cep we

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Fig. 4 (A) Conversion of peptides 1–5-T attached to either GB1-PCP7cep, Trx-PCP7sta or Trx-PCP7com to the monocyclic product peptides 6–10 via OxyBvan oxidation using the following reagents (1) OxyBvan, NADH, HaPuX, HaPuR, glucose, alcohol-dehydrogenase, air, Hepes buffer pH 7, 30 °C; (2) methylhydrazine, 15 min, SPE-purification. (B) Comparative time courses of product formation ( peptide 6) from substrate peptide 1 attached to different PCP domains. (C) Comparison of crosslinking efficiency of OxyBvan for the different glycopeptide-type peptides 1–5-T attached to three different PCP domains using conditions as shown in (A); incubation time: 1 h. All turnover reactions were performed in triplicate.

obtained a 92% yield of monocyclic peptide product 6 after 1 h, revealing that OxyBvan accepts substrates bound to PCP domains from alternative producer NRPS systems and that the crosslinking reaction is not disturbed by the presence of a protein fusion partner. The oxidation of peptide 1-T loaded onto Trx-PCP7com was also well supported, whilst Trx-PCP7sta resulted in reduced turnover. OxyBvan is able to catalyze efficient oxidation of peptides corresponding to all glycopeptide types Anticipating some substrate flexibility based on in vivo experiments that have been performed on the balhimycin system,16 we continued to test the ability of OxyBvan to accept different representative glycopeptide substrates 2–5-T (Fig. 4), obtaining oxidized products 7–10 in varying yields for each combination. These experiments demonstrate that OxyBvan tolerates amino acid residues on positions one to three that vary in size as well in polarity (see Fig. 1B). Comparing yields of peptide 7 and peptide 8, it can be seen that a hydrophobic residue on position three appears to promote OxyBvan turnover. The oxidation of the kistamicin-like peptide 5 demonstrates that even different stereochemistry of the amino acid residue at position three and bulky residues at position two of the peptide are tolerated, identifying OxyBvan as a versatile biocatalyst for the formation of different glycopeptide antibiotics. This is especially noteworthy as recent turnover studies with OxyBtei from the teicoplanin producing NRPS systems have revealed that OxyBtei has a higher degree of specificity for the peptide structure.43 This represents a highly unexpected difference

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between vancomycin- and teicoplanin-type systems with regards to the C-O-D cyclization reactions. Regarding PCP selectivity, peptides bound to Trx-PCP7sta were typically poorer substrates for OxyBvan compared to GB1PCP7cep and Trx-PCP7com; the latter afforded consistently the best turnover results. These results are quite remarkable considering the high sequence identity between PCP7cep and PCP7sta (84%) and the fact that PCP7com has much lower identity and is more distantly related to the vancomycin gene cluster (Fig. S15†). As all three fusion proteins are highly stable (see Fig. S13 and S14†), these differences cannot be explained by altered protein stabilities. Instead, minor alterations in the PCP structure – as have been postulated for other NRPS systems34 – may well be the cause of these differences in acceptance of alternate PCP-substrates by OxyBvan. In general, the selectivity of OxyBvan for matched peptide/ PCP-pairs is low, with the most versatile PCP domain for peptide presentation being that from complestatin biosynthesis, PCP7com. This reduction in selectivity for the peptide/ PCP-pair is comparable to the reduced selectivity observed in aminoacyl-PCP/P450 systems such as OxyD from balhimycin biosynthesis44 and P450sky from skyllamycin biosynthesis,34,35,45 where the chief determinant of substrate binding and oxidation by the P450 in question is the PCPdomain. This mechanism of substrate selectivity stems from the mechanism of the non-ribosomal peptide synthesis itself: in both the case of aminoacyl-PCP oxidation (OxyD, P450sky) and peptidyl-PCP oxidation (OxyBvan) the selectivity of the NRPS adenylation domains (responsible for amino acid selec-

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tion) and condensation domains (responsible for peptide synthesis) is high.46 As these domains represent the chief control mechanisms ensuring fidelity in NRPS-biosynthesis, downstream processes that require loaded PCP-domains as substrates can rely upon the selectivity of the A/C domains to ensure that the PCP in question is correctly loaded. This has the consequence that downstream enzymes are only required to ensure binding to the correct PCP-domain. Such mechanisms have been observed in investigations of glycopeptide antibiotics in vivo, where alternate peptides have acted as substrates for Oxy enzymes.47–49 In this work, we have shown that the peptide selectivity of OxyBvan is far more relaxed than previously believed and OxyBvan is capable of supporting oxidation of precursor peptides corresponding to five types of glycopeptide antibiotics. Given that not all in vivo complementation experiments performed with Oxy enzymes have been able to successfully reconstitute crosslinking activity,16,17 the broad spectrum of activity displayed by OxyBvan indicates that this enzyme is one example of an Oxy that is an appropriate biocatalyst for oxidative glycopeptide antibiotic transformation in vitro.

Experimental General methods All chemicals and solvents were obtained from commercial suppliers (Sigma-Aldrich, Munich-DE; VWR, Darmstadt-DE) and used without further purification. Dawson Dbz AM resin (100–200 mesh, 0.42 mmol g−1), activators and protected amino acids were obtained from Merck Novabiochem (Darmstadt-DE). (D)- and (L)-4-hydroxyphenylglycine and were obtained from Sigma-Aldrich. Alcohol-dehydrogenase (274 U mg−1) was obtained from Sorachim (Lausanne-CH); NADH was purchased from Gerbu Biotechnik GmbH (Heidelberg-DE). Solid phase extraction purifications were performed using Strata-X SPE columns (Phenomenex, Aschaffenburg-DE). HPLC analysis and purifications were carried out using a High Performance Liquid Chromatograph/Mass Spectrometer LCMS-2020 (ESI, operating both in positive and negative mode) equipped with a SPD-M20A Prominence Photo Diode Array Detector in preparative mode and a SPD-20A Prominence Photo Diode Array Detector in analytical mode, all from Shimadzu (Duisburg-DE). For analytical analyses the solvent delivery module LC-20AD was used; for preparative purifications two LC-20AP units were used. Analytical separations were performed on Waters XBridge BEH300 Prep C18 columns (5 or 10 µm, 4.6 × 250 mm). Preparative separations were performed on a Waters XSelect CSH130 C18 column (5 µm, 10 × 150 mm, peptides 1–5-CoA) at a flow rate of 10 mL min−1 and on a Waters XBridge BEH300 Prep C18column (5 µm, 19 × 150 mm, peptides 1–5-MPAA) at a flow rate of 20 mL min−1 (Eschborn-DE). The solvents used were water + 0.1% formic acid (solvent A) and HPLC-grade acetonitrile + 0.1% formic acid (solvent B). Electrospray Ionisation Mass Spectrometry (ESI-MS) and ESI-MS/MS measurements were performed with HPLC-purified

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samples on a Bruker maXis ultra-high resolution time-of-flight (TOF) mass spectrometer (Bremen-DE). Samples were analyzed in positive mode at a capillary voltage of 4.75 kV and a drying temperature of 100 °C. MS spectra of intact species were acquired in standard operation mode and subsequently species of interest were fragmented by collision induced dissociation (CID). Matrix-assisted laser desorption/ionization (MALDI-MS) measurements were performed on a Shimadzu Biotech Axima Performance TOF mass spectrometer using sinapinic acid as the matrix. Nuclear magnetic resonance spectra and high-resolution mass spectra were recorded at the University of Heidelberg on a Bruker DRX 400 spectrometer and a Bruker Apex Qe hybrid 9.4 FT-ICR spectrometer, respectively. All protein purification steps were performed at 4 °C, with buffers filtered through a 0.2 µm filter and degassed prior to use. The determination of protein concentration was performed using 280 nm absorption and calculated extinction coefficients for the corresponding protein constructs.

Solid phase peptide synthesis The solid phase peptide synthesis of 1–5 was performed on a Tribute UV peptide synthesizer (Protein Technologies, USA) on a 50 µM scale. Fmoc-L- and D-hydroxyphenylglycine were synthesized according to a previous report.22 Solid phase peptide synthesis was performed on Fmoc-protected Dawson Dbz AM resin using a Fmoc-based strategy with 1% DBU for Fmocdeprotection and COMU/triethylamine as the activator/activating base as previously described.22

Resin activation and peptide displacement using MPAA Resin activation was performed online during peptide synthesis in a two-step process: in the first step, the resin was washed with DCM before p-nitrophenylchloroformate (50 mg, 5 equivalents) was added in 5 mL of DCM. After agitation for 40 minutes, the resin was washed with DCM (4 times) and DMF (3 times) before DIPEA (44 µL, 5 equivalents) was added in 3 mL of DMF. This was left to react for 15 min before the resin was washed with DMF (3 times) and DCM (3 times) and dried under a stream of nitrogen. For thiol displacement of the peptide the resin was transferred into a tube and swollen in 3 mL of dry DMF (15 minutes). 4-Mercaptophenylacetic acid (84 mg, 10 equivalents) and tributylphosphine (200 µL, 1.2 mmol) were added to the resin that was maintained under an argon atmosphere: the reaction was left to react for 24 hours with continuous agitation. The DMF solution was then separated from the resin via filtration, removed in vacuo and the remaining residue dissolved in 5 mL of 95 : 2.5 : 2.5 TFA–H2O–TIPS with gentle shaking for two hours. The solution was concentrated under a stream of nitrogen to ∼1 mL and the peptide precipitated via addition of cold diethyl ether. The peptide was collected via centrifugation, dissolved in 50% acetonitrile/50% water, analyzed via analytical HPLC-MS and freeze-dried.

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Activated peptide thioester displacement with CoA The initial MPAA peptide from 50 µmol scale synthesis was dissolved in ∼ 6 mL 50 mM potassium phosphate buffer pH 8.3 (H2O–ACN 2 : 1) with 20 mM TCEP added. CoA (35 mg, 0.875 equivalents) was then added and the reaction gently agitated at room temperature for 1–2 h. Following this, the reaction was diluted with 50 mM KPi buffer ( pH 7.0) until the ACN concentration was 5% or lower. The conjugate was purified using four Strata-X SPE tubes (200 mg resin/3 mL tubes) according to the following protocol ( per column): 1. activation with methanol (5 mL), 2. equilibration with 50 mM KPi buffer ( pH 7.0, 3 mL), 3. loading the reaction sample, 4. washing with 50 mM KPi buffer ( pH 7.0, 3 mL), 5. washing with 5% methanol in 50 mM KPi buffer ( pH 7.0, 3 mL), 6. elution of peptide-CoA in 25–30% ACN in water ( pH 5–6, 3 mL). The elution fraction was collected and freeze-dried (for characterization see ESI†). Cloning, expression and characterization of OxyBvan and PCP domains Construct cloning. Cloning of OxyBvan was performed as previously described.43 PCP-domains as predicted by Interpro50 (secondary structure prediction by Jpred)51 were obtained as synthetic genes codon-optimized for expression in E. coli from Eurofins Genomics (Ebersberg-DE). These were amplified using specific primers (Table S2†) to introduce the unique restriction sites NcoI (5′) and XhoI (3′) which allowed cloning of the constructs into modified E. coli expression plasmids where the expression of all constructs is under control of a T7 promoter.40 PCP7cep was expressed as an N-terminal fusion of IgG binding B1 domain of Streptococcus (GB1), with an N-terminal hexahistidine tag followed by GB1 and a TEV cleavage site together with a C-terminal Strep-II tag. PCP7sta and PCP7com were expressed as N-terminal fusions of thioredoxin A (Trx) followed by a TEV cleavage site and a C-terminal hexahistidine tag. Expression and purification of OxyBvan, GB1-PCP7cep, Trx-PCP7sta and Trx-PCP7com. Expression and purification of OxyBvan was performed as previously reported.43 PCP7 constructs were expressed with either GB1 (PCP7cep) or Trx (PCP7sta and PCP7com) as N-terminal fusion partner.40 BL21 (DE3)gold E. coli cells transformed with PCP7 constructs were grown overnight at 37 °C in LB + kanamycin (50 mg L−1) to provide a starter culture for expression. 2 × 2.5 L of LB + kanamycin (50 mg L−1) was inoculated with 1% (v/v) of overnight culture and grown at 37 °C to an absorbance (600 nm) of 0.6 before the temperature was reduced to 18 °C. 20 minutes later expression was induced using IPTG (0.25 mM for PCP7cep and PCP7sta, 0.1 mM for PCP7com). After overnight growth at 18 °C, cells were collected by centrifugation at 4 °C (5000g), resuspended in lysis buffer (50 mM Tris.HCl pH 8.0, 50 mM NaCl, 10 mM imidazole) including protease inhibitor cocktail (Sigma Aldrich, Munich, Germany) and lysed by three passes through a fluidizer (Microfluidics, Newton-USA). The lysis solution was clarified by centrifugation (50 000g) and the soluble fraction

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was incubated at 4 °C for 90 min with 5 mL of Ni2+-NTA resin pre-equilibrated with 2 × 10 column bed volumes (CV) of wash buffer (50 mM Tris pH 8.0, 300 mM NaCl, 10 mM imidazole). The resin was separated from the non-bound fraction by centrifugation at 1000g before being transferred into a column format and washed with 10 CV of wash buffer. The proteins were eluted by gravity flow with 3 CV of elution buffer (50 mM Tris pH 8.0, 300 mM NaCl, 300 mM imidazole). SDS-PAGE analysis of Trx-PCP7com revealed >95% purity of the protein in the elution fractions, which were pooled, dialyzed against storage buffer (50 mM Tris pH 7.4, 150 mM NaCl) overnight and concentrated using centrifugal concentrators with a 10 000 MW cut-off (Sartorius Stedim Biotech, Göttingen-DE). Aliquots were flash frozen in liquid nitrogen and stored at −80 °C. The Ni2+NTA elution fractions of Trx-PCP7sta and GB1-PCP7cep were pooled and concentrated. Trx-PCP7sta was further purified by size exclusion chromatography on a Superose-12 column (GE Healthcare, Munich-DE) connected to an Äkta FPLC using exchange buffer (50 mM Tris·HCl pH 7.4, 100 mM NaCl). The elution fractions were analyzed by SDS-PAGE, appropriate fractions pooled and concentrated using centrifugal concentrators with a 10 000 MW cut-off. Aliquots were flash frozen in liquid nitrogen and stored at −80 °C. GB1-PCP7cep carrying a C-terminal Strep-II tag was subjected to StrepTrap affinity chromatography. Therefore, the Ni2+-NTA elution fraction of GB1PCP7cep was diluted 1 : 10 in low salt buffer (20 mM Tris pH 8.0, 50 mM NaCl) in order to reduce the concentration of imidazole before being loaded onto three sequentially connected 5 ml StrepTrap HP columns (GE Healthcare, Munich-DE) connected to an Äkta FPLC system. The column was washed with 3 CV SA buffer (100 mM Tris.HCl pH 8.0, 150 mM NaCl) and the protein eluted using 100% SB buffer (100 mM Tris. HCl pH 8.0, 150 mM NaCl, 2 mM desthiobiotin; 3 CV). The fractions were analyzed by SDS-PAGE, appropriate fractions pooled, concentrated and subjected to gel filtration on a Superose-12 column (GE Healthcare, Munich-DE) connected to an Äkta FPLC as described for Trx-PCP7sta. The elution fractions were analyzed by SDS-PAGE, appropriate fractions pooled, concentrated using centrifugal concentrators with a 10 000 MW cut-off, flash frozen in aliquots in liquid nitrogen and stored at −80 °C. Final protein yield was 18 mg (1.1 µmol) L−1 culture for GB1-PCP7cep, 48 mg (2.1 µmol) L−1 culture for Trx-PCP7sta and 60 mg (2.5 µmol) L−1 culture for Trx-PCP7com. The identity of the purified proteins was confirmed by MALDI-MS peptide map fingerprinting of a tryptic digest of excised SDS-PAGE protein bands (see also Fig. S12†). CD spectra and melting temperature determination of GB1PCP7cep, Trx-PCP7sta and Trx-PCP7com. Samples were exchanged against 50 mM sodium phosphate buffer pH 7.4 by dialysis and the protein concentration was set to 30 µM for CD measurements. The wavelength dependent ellipticity was recorded at 20 °C using a J-810 CD Spectropolarimeter (Jasco, Gross-Umstadt-DE) from λ = 205 to 260 nm in a quartz-glass cuvette with a path length of 0.1 cm (Hellma). Melting curves of the proteins were recorded as the change in ellipticity at 222 nm from 20 °C to 95 °C with a rate of 1 °C min−1.

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Subsequently, the samples were incubated at 95 °C for 1 min and refolding curves of the proteins were recorded from 95 °C to 20 °C with a rate of 1° min−1. The data was analyzed by calculating the mean residue ellipticity [θ]mrw,λ in [deg cm2 dmol−1] using:

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½θmrw;λ ¼

M ðN  1Þθλ 10dc

were M is the molecular mass (in Da) and N is the number of amino acids of the protein. θλ is the observed ellipticity (in degrees) at wavelength λ, d the optical path length (in cm), and c the concentration of the sample (in g mL−1). M and N are 18853.0 Da and 173 aa for GB1-PCP7cep, 22934.1 Da and 214 aa for Trx-PCP7sta and 23484.6 Da and 218 aa for Trx-PCP7com. The data was further analyzed using GraphPad Prism 5 software. Normal CD spectra were smoothed using a 2nd order polynomial and 4 neighbors on each side to average. Melting curve data was analyzed using the Boltzmann sigmoidal nonlinear fit and melting temperatures (Tm) were obtained. These were Tm = 64.88 °C for GB1-PCP7cep, Tm = 55.35 °C for Trx-PCP7sta and Tm = 53.95 °C for Trx-PCP7com. PCP loading and OxyB-catalyzed turnover PCP loading using Sfp. PCP loading was performed in Hepes buffer (50 mM, 50 mM NaCl, pH 7.0) by incubating peptide-CoA thioesters (120 µM) and the respective apo-PCP (60 µM), in the presence of R4-4 Sfp41 (6 µM) and MgCl2 (10 mM) for 1 h at 30 °C. After completion, excess peptide and MgCl2 were removed by four sequential concentration/dilution steps in Hepes buffer (50 mM, 50 mM NaCl, pH 7.0) using Amicon centrifugal concentrators (MW cut-off, 10 kDa). The resulting peptide-PCP solution was adjusted to a concentration of 50 µM and directly used for turnover studies (for characterization see Table S3†). OxyBvan turnover assays. A Hepes buffer solution (50 mM, 50 mM NaCl, pH 7.0) containing peptide-PCP (50 µM), OxyBvan (2 µM), HaPuX (5 µM),42 HaPuR (1 µM)42 and NADH (2 mM) was incubated for 1 hour at 30 °C with gentle shaking. To supply an NADH regeneration system, glucose (0.33% w/v) and alcohol-dehydrogenase (9 units mL−1) were added. The reaction was halted by the addition of 1/13 volume of a methylhydrazine solution (1 : 1 in water) and incubated for further 15 minutes, generating a mixture of hydrolyzed peptides and methylhydrazide peptides (two isomers present). Cleaved peptides were separated from the proteins by solid phase extraction (Phenomenex (Aschaffenburg-DE), Strata-X columns, 30 mg/1 mL). Columns were washed with acetonitrile and methanol (each 1.5 mL), equilibrated with water (1.5 mL), washed with 10% methanol (v/v) in water (1 mL) and the product peptides eluted with acetonitrile + 0.05% TFA (500 µL) following loading of the reaction onto the equilibrated columns. After removing the solvent in vacuo, the remaining solid was re-dissolved in acetonitrile–water 1 : 1 (100 µL), filtered and analyzed by analytical reversed-phase HPLC-MS using single ion monitoring (Waters XBridge BEH300 Prep C18 column, 4.6 × 250 mm, particle size: 5 µm; gradient:

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0–4 min 95% solvent A, 4–4.5 min up to 15% solvent B, 4.5–25 min up to 50% solvent B, flow rate 1 mL min−1); conversion yields were calculated by comparing the ratio of the peak area of monocyclised peptides to the peak area of all peptide species present. Methylhydrazine products were isolated and analyzed using ESI-MS/MS (see ESI†).

Conclusions We report a methodology to rapidly and efficiently produce arylglycine-containing peptide thioester intermediates appropriate for carrier protein loading in non-ribosomal peptide synthetase machineries. Given that arylglycine residues are a common feature of such peptides this route will dramatically expand the general ability to investigate non-ribosomal peptide synthesis in vitro. To demonstrate the potential implications of rapid synthesis of diverse peptide structures, we have investigated the first aromatic crosslinking step catalyzed by the vancomycin enzyme OxyB. Through these experiments, we have shown that OxyBvan catalyzes C-O-D ring formation efficiently on peptides representative of five glycopeptide types and tolerates different amino acid compositions and stereochemistries in the N-terminal portion of the peptide. Additionally, the selectivity of OxyBvan for oxidation of matched peptide/PCP-pairs is low, with the 7th PCP domain from complestatin biosynthesis found to be an excellent peptide delivery platform to enable their aromatic crosslinking by OxyBvan. This combination of an Fmoc-based SPPS-route to produce peptides conjugates that can be efficiently loaded onto PCP domains together with the wide acceptance of substrate peptides by OxyBvan passes significant barriers in the development a biomimetic synthesis of glycopeptide antibiotics.

Abbreviations of solvents and chemicals used in this study ACN Acetonitrile CoA Coenzyme A COMU (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate DCM Dichloromethane DIPEA Diisopropylethylamine DMF Dimethylformamide IPTG Isopropyl-β-D-thiogalactopyranoside KPi Potassium phosphate MPAA Mercaptophenylacetic acid TCEP Tris(2-carboxyethyl)phosphine TFA Trifluoroacetic acid TIPS Triisopropylsilane

Acknowledgements We thank A. Koch (MPI-Hd) for assistance with protein expression; I. Bravic and M. Schröter (MPI-Hd) for assistance

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with solid phase peptide synthesis; M. Müller (MPI-Hd) for MALDI-MS measurements and S. Bell (University of Adelaide) for assistance with redox proteins. M.J.C. is grateful for the support of the Deutsche Forschungsgemeinschaft (EmmyNoether Program, CR 392/1-1) and the Deutsche Akademischer Austausch Dienst (Group of Eight Australia – Germany Joint Research Co-operation Scheme, grant 56265933).

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Rapid access to glycopeptide antibiotic precursor peptides coupled with cytochrome P450-mediated catalysis: towards a biomimetic synthesis of glycopeptide antibiotics.

Understanding the mechanisms underpinning glycopeptide antibiotic biosynthesis is key to the future ability to reinvent these compounds. For effective...
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