doi.org/10.26434/chemrxiv.12809603.v1

Platform to Discover Protease-Activated Antibiotics and Application to Siderophore-Antibiotic Conjugates

Jonathan Boyce, Bobo Dang, Beatrice Ary, Quinn Edmondson, Charles Craik, William F. DeGrado, Ian Seiple

Submitted date: 14/08/2020 • Posted date: 17/08/2020 Licence: CC BY-NC-ND 4.0 Citation information: Boyce, Jonathan; Dang, Bobo; Ary, Beatrice; Edmondson, Quinn; Craik, Charles; DeGrado, William F.; et al. (2020): Platform to Discover Protease-Activated Antibiotics and Application to Siderophore-Antibiotic Conjugates. ChemRxiv. Preprint. https://doi.org/10.26434/chemrxiv.12809603.v1

Here we present a platform for discovery of protease-activated prodrugs and apply it to antibiotics that target Gram-negative bacteria. Because cleavable linkersfor prodrugs had not been developed for bacterial proteases, we used substrate phage to discover substrates for proteases found in the bacterial periplasm. Rather than focusing on a single protease, we used a periplasmic extract to find sequences with the greatest susceptibility to the endogenous mixture of periplasmic proteases. Using a fluorescence assay, candidate sequences were evaluated to identify substrates that release native amine-containing payloads without an attached peptide “scar”. We next designed conjugates consisting of: 1) an N-terminal siderophore to facilitate uptake; 2) a protease-cleavable linker; 3) an amine-containing antibiotic. Using this strategy, we converted daptomycin – which by itself is active only against Gram-positive bacteria – into an antibiotic capable of targeting Gram-negative Acinetobacter species. We similarly demonstrated siderophorefacilitated delivery of oxazolidinone and macrolide antibiotics into a number of Gram-negative species. These results illustrate this platform’s utility for development of protease-activated prodrugs, including Trojan horse antibiotics.

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Manuscript_SAC platform_Boyce et al.pdf (4.07 MiB) view on ChemRxiv download file

Supporting Information_Boyce et al.pdf (7.85 MiB) view on ChemRxiv download file Platform to discover protease-activated antibiotics and application to siderophore-antibiotic conjugates Jonathan H. Boyce,1,2 Bobo Dang,3,* Beatrice Ary,1 Quinn Edmondson,1 Charles S. Craik,1 William F. DeGrado,1,2,* and Ian B. Seiple1,2,*

1. Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California 94158, United States 2. Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California 94158, United States 3 Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, 18 Shilongshan Road, Hangzhou 310024, Zhejiang Province, China.

Abstract.

Here we present a platform for discovery of protease-activated prodrugs and apply it to antibiotics that target Gram-negative bacteria. Because cleavable linkers for prodrugs had not been developed for bacterial proteases, we used substrate phage to discover substrates for proteases found in the bacterial periplasm. Rather than focusing on a single protease, we used a periplasmic extract to find sequences with the greatest susceptibility to the endogenous mixture of periplasmic proteases. Using a fluorescence assay, candidate sequences were evaluated to identify substrates that release native amine-containing payloads without an attached peptide “scar”. We next designed conjugates consisting of: 1) an N-terminal siderophore to facilitate uptake; 2) a protease-cleavable linker; 3) an amine-containing antibiotic. Using this strategy, we converted daptomycin – which by itself is active only against Gram-positive bacteria – into an antibiotic capable of targeting Gram-negative Acinetobacter species. We similarly demonstrated siderophore- facilitated delivery of oxazolidinone and macrolide antibiotics into a number of Gram-negative species. These results illustrate this platform’s utility for development of protease-activated prodrugs, including Trojan horse antibiotics.

Introduction.

The well-recognized term, “ESKAPEE” (previously ESKAPE),1 encompasses the names of seven species of clinically relevant pathogens (E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, Enterobacter sp., and E. coli) that are associated with resistance to commonly prescribed antibiotics and are largely responsible for the world’s nosocomial infections.2 Five of these pathogens are Gram-negative species, whose outer membrane and associated resistance-nodulation-cell division (RND) efflux pumps render them resistant to many classes of antibiotics.3 Indeed, the outer membrane shields the bacteria from molecules that are unable to pass through porins,4 providing an effective barrier to many molecules that would otherwise be effective antibiotics against these pathogens.5 Gram-negative therapies can be delivered by siderophore-mediated antibiotic delivery using Nature’s Trojan- horse approach.6-8 Siderophores are small-molecule chelators that are produced by bacteria to sequester Fe(III),9 which is an essential nutrient required for bacterial growth and virulence.10 In the case of Gram-negative pathogens, outer membrane proteins (e.g. TonB-dependent transporters)11 bind to iron-chelated siderophores and provide opportunities for facilitated transport.12,13 Owing to the promiscuity of their transport systems, bacteria also use siderophores in warfare against other microbes.12,14 For example, Streptomycetes produce albomycins (Figure 1), which are natural siderophore-antibiotic conjugates (SACs) and highly effective antibiotics against Gram-negative Enterobacteriaceae.15 Albomycins are recognized by siderophore uptake machinery, transported into the cytoplasm, and activated by peptidase N, which cleaves the N-terminal serine-amide bond (Figure 1) and releases the t-RNA Figure 1. Albomycins, natural siderophore-antibiotic synthetase inhibitor (blue) to bind to its target.16-19 Here we extend this strategy conjugates with peptidase-cleavable linkers. by developing an unbiased platform for the discovery of linkers that are cleaved by periplasmic proteases,20 which demonstrates that this platform can produce SACs with both broad and narrow spectra of activity. There are two categories of SACs, depending on the type of linker they possess: non-cleavable and cleavable. There has been significant progress in the development of non-cleavable SACs,21,22b,23 with the first siderophore–β-lactam conjugate recently approved by the FDA.24 However, their use is often limited to periplasmic-targeting antibiotics (e.g. daptomycin, vancomycin, and b-lactams). The few examples of cytoplasmic-targeting, non-cleavable SACs may be less effective than the parent antibiotic for two reasons:25,26b- d,27-34 1) the conjugate may not pass through the inner membrane to reach the cytoplasm, or 2) the bulky siderophore component may interfere with binding to the target.12,25b,35 Therefore, cleavable linkers are traditionally thought to be required for SAC compatibility with cytoplasmic-targeting antibiotics.36 The majority of Gram-positive antibiotics are cytoplasmic-targeting and may require a cleavable linker if they were to be converted into SACs for Gram-negative pathogens.12,36,37 Despite extensive work over the last thirty years, only a few cleavable linker strategies have been developed for SACs and a number of challenges remain.22,25-27,35,36,38-40 Despite optimization for hydrolytic stability,35 ester linkers for SACs (e.g. A, Figure 2) are 1 susceptible to premature cleavage prior to bacterial-cell entry.25,26c-d,27,35,40 SACs with disulfide (e.g. B) and trimethyl-lock linkers based on reduction- (e.g. C) and esterase-triggered cleavage mechanisms were less active than the parent antibiotic.26,39 Recent work by Nolan demonstrated siderophore degradation by the cytoplasmic hydrolase IroD in two uropathogenic strains with a non-cleavable linker.38 Miller and coworkers developed the β-lactam linker D that releases a Gram-positive antibiotic upon ring-opening of the b- lactam ring and subsequent fragmentation.22a Importantly, with few exceptions,22a,27,40 cleavable SACs incorporate a DNA-gyrase- inhibiting fluoroquinolone antibiotic, ciprofloxacin or norfloxacin,41,42 which are already active against Gram-negative pathogens but become active only upon hydrolytic release of the parent drug. These SACs provide a method to study siderophore-mediated antibiotic delivery, but do not expand the arsenal of Gram-negative antibiotics.25,26,35,36,38,39 The lack of cleavable linkers for bacterial proteases is a central reason behind the lack of antibiotic diversity associated with SACs.36 A protease-cleavable SAC can be classified as a protease-activated prodrug, which upon proteolytic cleavage of an inactivating peptide leads to the release of an active drug.43,44 Peptide linkers developed for this activation mechanism in cancer therapy are undergoing clinical trials, one approved by the FDA.45-48 Most proteolytically activated prodrugs are optimized for cleavage by mammalian proteases, including matrix metalloproteinases (MMPs),49-51 lysosomal proteases52-55 (e.g. cathepsins and legumain),46,56- 58 and serine proteases (e.g. kallikreins),59,60 which are upregulated in disease (e.g. cancer, neurodegenerative disorders, inflammation, etc.).43,45,61,62 Proteolytically activated prodrugs with cleavable linkers are well-established and include antibody-drug conjugates,63-69 antibody-antibiotic conjugates,70,71 peptide-drug conjugates,72 macromolecular prodrugs,43,73 and protease-activatable photosensitizers,74,75 with the cathepsin B-sensitive valyl-citrulline (Val-Cit) linker being the most successful and widely known.66,68 Although most protease-activated prodrugs target cancer, antibody-antibiotic conjugates are undergoing clinical trials for intracellular bacterial infections associated with difficult-to-treat persisters.70,71 However, current therapies are limited to the treatment of intracellular S. aureus, a Gram-positive pathogen, and the linker is optimized for cleavage by mammalian proteases. Linkers have not

Figure 2. A. Selected cleavable linkers that have previously been used for SACs. B. Concept for SACs that contain a linker that can be cleaved by periplasmic proteases. C. Workflow for the development of protease-cleavable SACs. been developed for bacterial proteases, which may lead to pathogen-specific therapeutics that conserve the microbiota and broad- spectrum antibiotics that target proteases conserved across multiple species. Several technologies have been developed to screen large libraries for protease-substrate profiling,76 some of which have been successfully applied to prodrug development, such as synthetic combinatorial libraries of fluorogenic substrates,77,78 positional scanning of synthetic combinatorial libraries,79,80 substrate phage libraries,81,82 multiplex substrate profiling by mass spectrometry,76,83- 85 and others.77,86-90 Given the enormous substrate diversity of fully randomized peptide libraries, substrate phage display81 provides an unbiased selection tool to discover cleavable linkers for SACs, which can lead to a manageable set of highly specific substrates for a given protease. In contrast to substrate phage display, phage display has been used in vitro and in vivo to design targeted-peptide conjugates.82 In these efforts, it is common to use mixtures of proteins or whole cells for selection of high-affinity binders.91 However, substrate phage display has not been applied to prodrug development,82 and its use for profiling complex biological mixtures is limited.92 Our work takes full-advantage of substrate phage display to thoroughly explore the substrate specificity of protease mixtures in the bacterial periplasm for the production of protease-cleavable SACs. 2 In designing protease-activated prodrugs for bacteria-specific proteases, there may be advantages to targeting multiple proteases over an individual protease, as more than one may be responsible for activation of a conjugate in vivo.46,93,94 The importance of this concept was brought to light when the in-vivo deletion of cathepsin B resulted in drug release from the combined activity of several proteases with overlapping substrate specificity.95,96 In fact, it might be advantageous to target multiple proteases rather than one individually to minimize the possibility of resistance when designing antibiotic prodrugs. Thus, we screened broadly for peptides that are cleaved by the multiple proteases present in an unfractionated periplasmic extract. Moreover, the convenience of isolating the periplasmic extract enhances throughput by eliminating the need for expression and purification of specific proteases.

Results.

Substrate Phage Display Leads to WSPKYM– M13 RFG and WSWC–KWASG as Substrates for Phage Periplasmic Cleavage. To discover efficient peptide substrates using the method of substrate periplasmic extract H N 2 H2N 85 COOH COOH phage, we built a random hexapeptide library protease cleavage 1) amplification 6-mer peptide 6-mer peptide 6-mer peptide 6-mer peptide 6-mer peptide 6-mer peptide genetically fused to the pIII gene of M13. A (in E. coli)

AviTag AviTag AviTag AviTag AviTag AviTag AviTag AviTag 2) enzymatic phagemid vector allows monovalent display of the biotin biotin biotin biotin biotin biotin biotin biotin biotinylation strepavidin strepavidin strepavidin strepavidin strepavidin strepavidin strepavidin strepavidin corresponding protein on the tip of the phage. A Original Library (107 unique peptides) 3) immobilization on streptavidin GGS spacer was incorporated at each end of the Repeat Process 3x randomized peptide to enhance flexibility. An AviTagâ sequence was also incorporated at the N- terminus for biotinylation of the displayed peptides. The biotin is used to immobilize the phage library on a streptavidin-coated surface, periplasmic extract and a protease can then cleave at favorable protease cleavage H2N H N H2N 2 4th Round of Selection 6-mer peptide 6-mer peptide 6-mer peptide 6-mer peptide peptide sequences. Proteolysis releases the H2N 1) amplification (in E. coli) phage, which are then amplified and sequenced to AviTag AviTag AviTag AviTag biotin biotin biotin biotin determine the favorable substrates for the 2) phagemid isolation strepavidin strepavidin strepavidin strepavidin protease of interest. 3) Next-Generation Sequencing Library Containing Enriched Sequences The process of “biopanning” entails the 4) Sequences ranked by enrichment factor following steps: 1) enzymatic biotinylation of the Optimization WSWC KWASG â 97,98 for cleavage-site AviTag sequence, 2) immobilization of the WCKWAS analysis by LC/MS WSWCKWASG periplasmic extract biotinylated library on streptavidin 96-well plates, protease cleavage PKYMRF +Trp–Ser at N-term WSPKYMRFG WSPKYM RFG 3) cleavage of the immobilized library by +Gly at C-term 18 h at 37 oC incubation with the periplasmic extract of E. coli Enriched Sequences Synthesized Sequences Verification of Cleavage Site by LC/MS K12 MG1655 at 37 oC, 4) amplification of the eluted phage using E. coli TG-1 cells, and 5) Figure 3. Substrate phage display leads to optimal sequences for cleavage in the periplasmic extract of E. coli K12 MG1655. isolation and purification of phage for the next round of selection. The periplasmic extract used in panning was obtained by osmotic shock of E. coli K12 MG1655.99 We carried out four rounds of selection (Figure 3), with the stringency being increased with each succeeding round by reducing the amount of extract and decreasing the incubation time. The phagemid from the input library and final round of biopanning were isolated, barcoded, and submitted for Next Generation Sequencing.100 Sequences for further characterization were ranked based on the extent of enrichment relative to the original library (Table 1). Sequence Reads Initial reads Enrichment Factor Six highly enriched sequences (SKNQSLGG, SGSDSSVG, SNHADVHG, SKSEMLSG, KNQSLG 10652 0.5 21304 SWCKWASG, and SPKYMRFG) were synthesized with flanking amino acids to mimic GSDSSV 9239 0.5 18478 the GGS spacers in the phage library. A tryptophan was added to the N-terminus NHADVH 8138 0.5 16276 to facilitate detection by HPLC. Each peptide was found to be cleaved to varying KSEMLS 7742 0.5 15484 extents following treatment with periplasmic extract for 18 h at 37 oC. The WCKWAS 15307 1 15307 cleavage sites and extent of proteolysis were evaluated by LC/MS (Table S5), which PKYMRF 13192 1 13192 revealed that the sequences WSPKYM–RFG (dash – indicates site of cleavage) and Table 1. Highly enriched sequences found through WSWC–KWASG may be optimal linkers for cleavable SACs.100 In addition to their substrate phage display. promising cleavage profiles described in Table S5, the presence of these sequences

in the original library contributed to their selection as potential linker candidates. In protease substrate nomenclature, the Cys and Met residues at the C-terminal side of the cleavage site are designated as the P1 positions, while the Lys and Arg residues at the N-terminal side are designated as the P1’ positions. Although the residues on the P’ side are sometimes important for efficient cleavage,101 this is not always the case.102 3

WSPKYM conjugates are efficiently cleaved without a P’ peptide. With the candidate sequences WSPKYM–RFG and WSWC– KWASG in-hand, we asked whether the residues on the C-terminal P’ sequence were required for proteolysis. The lack of a P’ peptide sequence is considered to be advantageous for prodrugs in which a drug is linked to the P1 residue via an amide bond, releasing the parent drug without an appended peptide “scar” that might adversely affect its biological activity (Figure 4A). To probe this question, we used a solid-phase method to synthesize fluorescent substrates in which 7-amino-4-carbamoylmethylcoumarin (ACC) was coupled directly to the P1 Cys or Met residue as an antibiotic surrogate (See Supporting Information).105,106 We were indeed pleased to find that peptide 1, which contains the WSPKYM-coumarin sequence was efficiently cleaved by treatment with a periplasmic extract of E. o coli K12 (100 µg/mL total protein) at 37 C (t1/2 = 5.3 ± 0.1 hr). On the other hand, peptide 2 (WSWC-coumarin) was cleaved 25-fold less rapidly under these conditions (Figure 4B). Thus, WSPKYM was determined to be more suitable than WSWC for the development of cleavable SACs.

Design and synthesis of SACs that incorporate daptomycin, an oxazolidinone, and solithromycin. To explore the versatility of SACs, we selected three structurally and mechanistically diverse antibiotics that act on targets in either the periplasm or the cytoplasm. Each antibiotic has an amine, which can be unmasked upon proteolysis of the WSPKYM linker. The lipopeptide daptomycin (4) interacts with the cytoplasmic membrane in Gram-positive bacteria, leading to increased membrane permeability and membrane depolarization.105a-c However, it is ineffective against Gram-negative bacteria and challenging to functionalize without loss of potency.105d Nevertheless, the Miller group has shown that daptomycin can gain activity in Gram-negative species if conjugated to a siderophore with a non-cleavable linker.22b,23 Here, we examine the use of a protease- cleavable linker. We also chose two ribosomal protein synthesis inhibitors, amino- oxazolidinone 5 and solithromycin 6, as examples of antibiotics that must gain access to the cytoplasm to be active.22a Since both oxazolidinones and macrolides bind deep within the large ribosomal subunit in fairly occluded binding sites, siderophore conjugates without cleavable linkers have been met with limited success, potentially due to interference of the linkers with binding.12,21,37,106,107 Our strategy would avoid this complication by enabling release of the parent antibiotics. For attachment to the N-terminal side of the linker, we sought a siderophore that was synthetically accessible, had a low molecular Figure 4. Evaluation of cleavage in periplasmic extract using weight, and was compatible with a variety of bacterial siderophore linker–fluorophore conjugates. Continuous fluorescent assay of uptake systems. The bis-catecholate, azotochelin-like108 siderophore the cleavage of antibiotic–fluorophore conjugates by E. coli K12 extract. 25 µL of extract (Total Protein: 200 ug/mL) was diluted (Miller Siderophore, Scheme 1)22 was selected due to its ease of into assay buffer containing 25 µL of cleavage substrate (25 µM). synthesis and its ability to carry large cargo (e.g. daptomycin) into A. Cleavage of the amide bond between the peptide and the 7- 22,23 baumannii, E. coli, and P. aeruginosa. We used a modified version amino-4-carbamoylmethylcoumarin releases the free coumarin of Miller’s protocol to access siderophore 10, which has acid-labile derivative, which is brightly fluorescent. The time course for Ac- ketal protecting groups that can be removed concomitantly with tert- WSPKYM-ACC (1) is well described by a pseudo-first order butyl and tert-butoxycarbonyl (Boc) protecting groups on the amino process with a half-life of 5.3 ± 0.1 hr. acid sidechains.100 We developed a modular synthetic route that enables the facile incorporation of a variety of linkers, antibiotics, and siderophores (Scheme 1). Gram-scale linker assembly and siderophore attachment were accomplished by solid-phase synthesis to provide the partially protected intermediate 3 in 50% overall yield, and the antibiotic was then coupled to the C-terminus in solution. Following acidolytic deprotection, the final SACs (7 - 9) were obtained in 12-53% yield over two to four steps. Several aspects of our route merit further discussion. The majority of the synthesis proceeds on solid phase, simplifying purification and facilitating parallel synthesis of analogues. Antibiotics are directly attached in the penultimate step, enabling rapid access to the final antibiotic conjugates in only two steps from intermediate 3.100 The synthesis requires only a single HPLC purification following the coupling step, and the final products are purified by trituration. Daptomycin and solithromycin are commercially available and the oxazolidinones were synthesized following the protocols of Miller22a and Rafai Far.109a

4 Scheme 1. Modular synthetic platform for SAC synthesis. A. siderophore WSPKYM antibiotic

Siderophore: Antibiotic:

NH NH2 2 O N N O O O Me O N H H H N N N HO N N Me N N OR2 NH N N OH H H O F O O O OR1 CONH2 O NH O O Me Me O O O HO C HN O H 2 eperezolid N N Me HO C O OMe 1 N N 2 O R O Me H H O Me NMe2 OR2 O NH O O O HO NH Me O O Me HO CO2H H H O O O O Me N H N N N Miller Siderophore N N O Me N H H NH2 O O Ph Me O F Me F 10 R1, R2 = O Me O Ph CO2H NH2 1 2 10a R , R = −H daptomycin (4) eperezolid-NH2 (5) solithromycin (6) B. OtBu

OtBu 1. antibiotic O O O O i i H H H H BuOCOCl, Pr2EtN FmocHN 1. SPPS N N N N N or EDC, HOBt O O N N N OH H H siderophore WSPKYM antibiotic Ph O O O O O O 2. AcOH O 2. TFA, TIPS-H, EDT, H2O Ph NH SMe SMe O O Ph NHBoc Ph 3

Table 2. Conjugates synthesized in this work.a

Compound N-terminal C-terminal Designation linker Number Substitution Substitution

1 Ac-WSPKYM-ACC Acetyl WSPKYM ACC 2 Ac-WSWC-ACC Acetyl WSWC ACC 7 L-Linker Daptomycin Conjugate Miller Siderophore WSPKYM daptomycin (4)

8 L-Linker Eperezolid-NH2 Conjugate Miller Siderophore WSPKYM eperezolid-NH2 (5) 9 L-Linker Solithromycin Conjugate Miller Siderophore WSPKYM solithromycin (6)

11 Conjugate Without Antibiotic, Acid Miller Siderophore WSPKYM free acid (-OH)

12 Conjugate Without Antibiotic, Ester Miller Siderophore WSPKYM methyl ester (-OMe)

13 D-Linker Daptomycin Conjugate Miller Siderophore wspkym (D-linker) daptomycin (4)

14 D-Linker Eperezolid-NH2 Conjugate Miller Siderophore wspkym (D-linker) eperezolid-NH2 (5)

15 Conjugate With Inactive Enantiomer Miller Siderophore WSPKYM ent-eperezolid-NH2

16 D-Linker Solithromycin Conjugate Miller Siderophore wspkym (D-linker) solithromycin (6)

17 Conjugate With WSWC Linker Miller Siderophore WSWC eperezolid-NH2 (5)

18 Conjugate Without Siderophore Acetyl WSPKYM eperezolid-NH2 (5) a For detailed synthetic methods, see Supporting Information.

We also synthesized several compounds to probe the mechanism of action of 7, 8, and 9 using modifications of our existing protocol (11-18, see Table 2). These included conjugates with D-amino acid linkers (e.g. 13, 14, and 16)110 and conjugates that lack an antibiotic or contain an inactive enantiomer of the antibiotic (e.g. 11, 12, and 15). To compare the effectiveness of conjugates containing a WSWC linker, which did not cleave effectively in our fluorogenic assay (Figure 4B), we synthesized WSWC conjugate 17. We also synthesized a siderophore-free conjugate (18) to determine the dependence of activity on the siderophore.

Determination of the antibacterial activity of SACs 7-9 and iron-dependent activity. The minimum inhibitory concentrations (MICs) of conjugates 7-9 were evaluated according to the standard CLSI antimicrobial susceptibility testing guidelines in Meuller-Hinton-II (MH-II) broth with dipyridyl to sequester iron from the media and promote siderophore-mediated transport (Tables 3-5, S1).100,111-117 Controls that lacked a siderophore did not show activity-dependence on dipyridyl concentration, while the siderophore conjugate became increasingly active at higher levels of dipyridyl (Table S3A-C). This phenomenon can be explained by the enhanced expression of outer-membrane transport proteins for siderophore uptake in iron-deficient media.109b The absence of dipyridyl from the growth

5 medium dramatically attenuated siderophore-conjugate activities without influencing the MIC of the free antibiotic.100 These results correlate well with expected growth-inhibitory activity of SACs.

We included 15 bacterial strains in our assay (14 Gram-negative and one Gram-positive), and have highlighted selected activities below (for full-activity tables and strain details, see Supporting Information). Two genetically modified strains of E. coli were included: a DsurA strain that is deficient in outer-membrane proteins and has increased permeability,113a and a DbamBDtolC mutant, which has a deficient BamACDE outer-membrane-assembly complex and lacks the TolC-transport protein.113a,b This strain is widely used because it is defective in small-molecule efflux.

Antibacterial activity of daptomycin-conjugate 7 (Table 3). Daptomycin is used to treat Gram-positive infections, but it lacks activity against Gram-negative species. Therefore, we were gratified to find that the L-linker daptomycin conjugate 7 showed species-specific activity against Acinetobacter species and E. coli, with MIC values in the 1 to 10 µM range, while daptomycin itself was inactive against these species (MIC > 48 µM). We also measured the activity of 7 against a panel of pathogenic Gram-negative organisms, and found that high activity was observed only for Acinetobacter species and E. coli. These findings indicate that this approach has the potential to produce Gram-negative antibiotics with relatively narrow-spectrum activity for precision antibiotics. Moreover, as expected, 7 was inactive against S. aureus, suggesting that it was not proteolytically activated by this Gram-positive bacterium. To confirm periplasmic proteolysis of 7 and determine if significant cleavage occurred by a secreted protease, we evaluated the activity of the D-linker daptomycin conjugate 13 (in which the chirality of the amino acids in the linker were reversed). Compound 13 was 2 to 10-fold less active than 7 against E. coli, Acinetobacter baumannii, and Acinetobacter nosocomialis. However, 13 did not entirely lose activity against these species, suggesting that the uncleaved conjugate might have some low level of intrinsic activity against these organisms. Finally, we found that derivatives of 7 lacking the daptomycin payload (11 and 12) were essentially inactive, showing only weak activity against the compromised DbamBDtolC E. coli mutant. Taken together, these data indicate that we have successfully introduced Gram-negative activity into a derivative of daptomycin, and the enhanced activity of 7 relative to 13 is consistent with our guiding hypothesis of stereospecific proteolytic activation. It is also clear that proteolytic activation likely occurred in the periplasm, rather than by an extracellular protease in the medium, given that daptomycin has no activity against Acinetobacter baumannii and Acinetobacter nosocomialis. These results are also of interest with respect to the mechanism of action of daptomycin. It has been previously reported that the target for daptomycin (4) may be absent in Gram-negative species due to the differing membrane compositions between Gram- positive and Gram-negative bacteria.118 Given that 7 is active against Acinetobacter and E. coli, it would appear that daptomycin is able to act on the cytoplasmic membranes of these Gram-negative species once they gain access. Also, our finding that daptomycin itself is equipotent against S. aureus Newman and in the outer membrane-compromised E. coli ∆surA is consistent with this conclusion.

Table 3. Antibacterial activity (MIC in µM) of daptomycin SAC 7 and derivatives thereof.a tolC tolC surA surA ∆ ∆ E. coli wild type knockout Newman S. aureus bamB E. coli K12 pathogenic ∆ Gram-positive E. coli A. baumannii baumannii A. efflux knockout outer-membrane

Daptomycin Conjugates and Controls nosocomialis A. multidrug resistant Daptomcyin (4) >39 >39 >39 >39 0.6 0.6 L -Linker Daptomycin Conjugate (7) 11 11 5 1 >21 >21 D -Linker Daptomycin Conjugate (13) >23 23 23 11 23 >23 Conjugate Without Antibiotic, Acid (11) >48 48 >24 >48 >48 >48 Conjugate Without Antibiotic, Ester (12) >48 24 >24 ND 48 >48 aFor strain descriptions, see Supporting Information.

Activity of oxazolidinone conjugate 8 in E. coli (Table 4). The oxazolidinone class of antibiotics are active against Gram-positive bacteria, but members of this class lack activity against Gram-negative bacteria, due to the presence of endogenous efflux pumps. Nevertheless, mutants of E. coli such as DbamBDtolC are susceptible to oxazolidinones because these bacterial strains have disruptions in their efflux systems. This strain is susceptible to eperezolid (Table S1), but the corresponding amine variant, eperezolid-NH2 (5) (Table 4), has only minimal activity against S. aureus Newman. The low activity of 5 is likely a result of its inability to diffuse through the cytoplasmic membrane rather than intrinsic inactivity against its ribosomal target. We therefore asked whether conjugate 8 could deliver 5 to a DbamBDtolC strain of E. coli. We were pleased to discover an MIC of 1 µM of 8 for this mutant; the corresponding derivative with an all-D linker showed strongly decreased activity with an MIC of 20 µM. Additionally, conjugate 8 displayed no activity in a cell-free translation assay at 38 µM (Figure 5), indicating that the intact conjugate is not capable of directly inhibiting the ribosome. These findings suggest that potent inhibition of bacterial growth requires enzymatic cleavage of the linker. Supporting this suggestion, 34% cleavage of 8 to the parent antibiotic eperezolid-NH2 occurred after 11–hours of incubation with bacterial periplasmic extract (Table S2). Finally, as expected, conjugate 8 was not active in wild-type strains with functional endogenous efflux pumps (Table S1).

6 The cleavage of 8 by periplasmic extracts might seem inconsistent with the finding that the corresponding eperezolid-NH2 (5) has only minimal activity (40 µM) against both E. coli DsurA and S. aureus. This finding indicates that the cytoplasmic membrane of both Gram-positive and Gram-negative bacteria provide a barrier for the diffusion of 5 into the cytoplasm. There are two possible explanations for the potent activity of 8 given the lack of activity of 5: Facilitated transport leads to large differences in the concentrations of molecules in the periplasm versus the cytoplasm, so siderophore-mediated transport into the periplasm could lead to accumulation of 8 and the corresponding cleavage product 5, enhancing its effective concentration and hence potency. The alternate possibility is that 8 is actively transported to the cytoplasm where it is activated by a cytoplasmic protease.119 We also synthesized a number of additional control Table 4. Antibacterial activity (MIC in µM) and in vitro evaluation of molecules to probe the antibacterial mechanism of 8, eperezolid-NH conjugate 8 and derivatives thereof.a including conjugate 17 with the WSWC linker, which was 2 found to be inefficiently cleaved in the fluorescent assay. Not tolC tolC surA surA ∆

surprisingly, this analogue had only weak (MIC = 37 µM) ∆

activity, as did compounds 11, 12, and 15 that lacked an active E. coli knockout Newman S. aureus Eperezolid bamB ∆ Gram-positive E. coli antibiotic payload. Similarly, the conjugate 18 without a efflux knockout Conjugates and Controls outer-membrane siderophore was inactive. However, as was the case for some Eperezolid-NH2 (5) >171 43 43 of the daptomycin analogues, a number of the compounds L -Linker Eperezolid-NH2 Conjugate (8) 1 38 >38 lacking the antibiotic payload retained a modicum of activity D -Linker Eperezolid-NH2 Conjugate (14) 19 >38 >38 (10 - 20 µM), so long as the C-terminus was not negatively Conjugate Without Antibiotic, Acid (11) 48 >48 >48 charged. This activity was greatest for the derivative with the Conjugate Without Antibiotic, Ester (12) 24 48 >48 inactive antibiotic 15 (MIC = 9 µM). We suggest that this Conjugate With Inactive Enantiomer (15) 9 38 >38 activity might occur by a mechanism similar to many non- Conjugate With WSWC Linker (17) 37 ND ND helical proline-containing cationic antimicrobial peptides.120 Conjugate Without Siderophore (18) >77 ND >77 % Eperezolid Release From 8 In Extract 34 ± 1.5 ND ND The linker WSPKYM has two aromatic residues known to a interact favorably with membranes, a hydrophobic Met and a For strain descriptions and extract cleavage procedure, see Supporting Information. cationic Lys residue. Also, as is the case for most cationic antimicrobial peptides the introduction of a C-terminal carboxylate decreases antimicrobial activity. Thus, it might be interesting to use the siderophore strategy to deliver short antimicrobial peptides to the cytoplasmic membrane, although such studies are beyond the scope of this work.

Table 5. Antimicrobial activity (MIC in µM) of solithromycin conjugate 9 and derivatives thereof.a tolC tolC surA surA ∆ ∆ E. coli S. typhi knockout wild type wild type pathogenic bamB pathogenic pathogenic pathogenic S. enterica pathogenic E. coli K12 E. coli DCO E. aerogenes Gram-positive ∆ E. coli efflux knockout K. pneumoniae pneumoniae K. outer-membrane A. nosocomialis nosocomialis A. multidrug resistant

Solithromycin Conjugates and Controls S. aureus Newman Solithromycin (6) 5 1 1 9 9 1 1 5 2 1 L -Linker Solithromycin Conjugate (9) 7 7 7 7 13 >27 >27 3 7 0.4 D -Linker Solithromycin Conjugate (16) >27 >27 >27 >27 >27 27 >27 3 13 0.8 Conjugate Without Antibiotic, Acid (11) >48 >48 ND >48 ND >48 >48 >48 >48 48 Conjugate Without Antibiotic, ester (12) ND >48 ND ND ND 48 >48 >48 ND 24 aFor strain descriptions and extract cleavage procedure, see Supporting Information.

Proteolysis of conjugate 9 restores the activity of solithromycin in three ESKAPEE pathogens (E. coli, E. aerogenes, and K. pneumoniae) and in A. nosocomialis (Table 5). One frequently used strategy to evaluate cleavable SACs involves the use of antibiotic payloads that are already active against Gram-negative pathogens but rendered inactive due to the overall bulk of the SAC.25,26,35,36,38,39 The successful release of the parent drug can then be conveniently monitored by the emergence of antibacterial activity. We adopted this strategy to examine the release of solithromycin from the L-linker SAC 9 and its D-linker analogue 16. The D-linker analogue was entirely inactive (MIC > 20 µM) against A. nosocomialis, S. typhi, S. enterica, E. aerogenes, and K. pneumoniae. In contrast to 16, the L-linker analogue 9 showed robust activity, which is consistent with proteolytic activation in these pathogenic strains.

7 When compared to the parent drug, the potencies of 9 ranged from In Vitro Translation slightly greater than solithromycin to 6-fold less potent, indicating broad 1.2 specificity to the activation mechanism. Exclusion of DP from the growth medium resulted in loss of activity for solithromycin conjugate 9, which 1.0 shows that the activation is not the result of proteolytic cleavage associated with an enzyme in the extracellular medium. If 9 were cleaved in the 0.8 medium, this would result in the release of solithromycin (6), which is active in these strains (Table S3B). 0.6 We unexpectedly observed activity of the D-linker variant 16 against E. coli, which surprisingly had similar activity to its L-linker analogue 9. These 0.4 results could be explained by the active transport of 16 into the cytoplasm Relative Fluorescence and direct ribosomal inhibition by the conjugate. To investigate this 0.2 possibility, we conducted an in vitro translation assay with E. coli ribosomes (Figure 5), which showed that solithromycin conjugates 9 and 16 inhibit

0.0 translation in E. coli comparable to the free antibiotic at a concentration of 2 10 µM. These data indicate that cleavage of these conjugates should not Control -Linker -Linker -Linker L L D Control be required for activity in E. coli. Conjugate 8 (6) 2 Solithromycin Solithromycin Solithromycin Solithromycin The lack of D-linker activity in Gram-negative species other than E. coli is Eperezolid-NHConjugate 8 Conjugate 9 Conjugate 16 currently under investigation, but might simply reflect differences in the Figure 5. In vitro translation shows the ability of transport systems among Gram-negative species. Conjugates 9 and 16 may conjugates-Linker Solithromycin 8 (38 Conjugate µM), 9 9 (10 µM), and 16 (10 µM) to -Linker Eperezolid-NHL -Linker Solithromycin Conjugate 16 12,13 L inhibit the 70SD E. coli ribosome. not be transported to the cytoplasm in the pathogenic strains, thereby requiring linker cleavage for activity. It is anticipated that siderophore transport would be required for solithromycin-conjugate uptake into the cytoplasm due to the inability of 9 and 16 to passively diffuse through the E. coli inner membrane since they are not active against the compromised E. coli ∆surA. Structural differences between ribosomes of various species may also contribute to activity variations. Taken together, our results provide strong support for the proteolysis of 9 in five pathogenic strains but also show that a cleavable solithromycin conjugate is not required for activity in E. coli.

Conclusions.

The strategy developed here should be broadly applicable for discovery of protease-activated peptide prodrugs for a variety of applications. Here, we focused on delivering antibiotics by designing protease-cleavable siderophore conjugates. By targeting bacterial periplasmic proteases broadly, we were able to design conjugates that act against a broad (or narrow) spectrum of Gram- negative bacteria, illustrating the potential of this approach. Our results provide strong support for the overall mechanism of proteolytic release of the antibiotic from conjugates 7 – 9. Although we have not yet identified the proteases responsible for activity against our substrates, we purposefully avoided targeting a single protease to decrease the chances of resistance arising from mutants of a single protein. Moreover, the use of chemically stable amide linkers provides an advantage to targeting proteases over esterases and b-lactamases by avoiding the need for esters and b-lactams, which are chemically more labile. Importantly, the modular design and facile synthetic route provides opportunity for rapid variation and evaluation of the siderophore, linker, and antibiotic. This has led to the discovery of cleavable conjugates with activity against several clinically relevant Gram-negative pathogens. Throughout the course of this work, we made a number of unexpected discoveries with impacts that extend beyond the scope of protease-cleavable prodrugs. Our daptomycin conjugate 7 completely lacks the Gram-positive activity of daptomycin and has gained Gram-negative activity, effectively “flipping” the spectrum of activity of this potent antibiotic. We found that conjugates with D- linkers, which are unlikely to be cleaved proteolytically, have moderate activity against several strains of Gram-negative bacteria. Perhaps the most unexpected results are the activities of the solithromycin conjugates 9 and 16 in a cell-free translation assay, which indicate that these large (MW > 2000) conjugates may directly inhibit the ribosome in E. coli. These results are extremely surprising in the context of solithromycin–ribosome structural data,121 and may provide the basis for new macrolide-peptide-hybrid antibiotics. In summary, this work provides a robust methodology for selection and screening of Trojan-horse prodrugs applied to the persistent and growing problem of antibacterial resistance. Using phage display, one can rapidly screen vast peptide libraries, and by varying the selection strategy one can screen for linkers with desired characteristics. For example, by using periplasmic extracts from different species of bacteria in succeeding selections, one can assure broad activity over the desired range of bacteria. Alternatively, negative selection could be incorporated to select against cleavage of serum proteases or beneficial members of the microbiome. Thus, the potential for fine-tuning the protocol for future practical applications is substantial.

Acknowledgements.

We thank Adam Cotton, Peter Rowheder, Dr. Sam Ivry, and Dr. Matthew Ravalin for helpful discussions. We thank Bruk Mensa for helpful discussions and for providing P. aeruginosa ATCC 10145, S. typhi, S. aureus Newman, E. coli K12 MG1655, E. coli BW25113 8 ∆tolC, E. coli BW25113 ∆surA. We thank Neha Prasad for helpful discussions and for providing K. pneumoniae MGH 78578, E. cloacae ATCC13047, E. aerogenes ATCC 13048, P. aeruginosa PA01, and P. aeruginosa PA14, and S. enterica 14028s. We thank Jenna Pellegrino for providing E. coli BW25113 ∆bamB∆tolC and for helpful discussions on in vitro translation assays. We thank Professor Joanne Engel for a generous gift of A. nosocomialis M2. J.H.B. was supported by the Natural Institutes of Health under the Ruth L. Kirschstein National Research Service Award 5T32HL007731-27 from the National Heart Lung and Blood Institute (NHLBI). Q.E. was supported by the National Science Foundation Graduate Research Fellowship Program under Grant no. 1650113. B.A. was supported by the National Institutes of Health under Grant no. T32 Al 0605357. This project was supported by the David and Lucile Packard Foundation (I.B.S.) and the National Institutes of Health under Grant no. R35 GM122603 (W.F.D.).

Author Information.

Corresponding Authors [email protected] [email protected] [email protected]

References

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14 (114) Dubowchik, G. M.; Firestone, R. A. Cathepsin B-sensitive dipeptide prodrugs. 1. A model study of structural requirements for efficient release of doxorubicin. Bioorg. Med. Chem. Lett. 1998, 1, 3341–3346. (115) Mikkelsen, H.; McMullan, R.; Filloux, A. The Pseudomonas aeruginosa reference strain PA14 displays increased virulence due to a mutation in ladS. PLoS One. 2011, 6, e29113. (116) Knight, D. B.; Rudin, S. D.; Bonomo, R. A.; Rather, P. N. Acinetobacter nosocomialis: Defining the role of efflux pumps in resistance to antimicrobial therapy, surface motility, and biofilm formation. Front. Microbiol. 2018, 9, 1902. (117) Chen, T. L.; Lee, Y. T.; Kuo, S. C.; Yang, S. P.; Fung, C. P.; Lee, S. D. Rapid identification of Acinetobacter baumannii, Acinetobacter nosocomialis and Acinetobacter pittii with a multiplex PCR assay. J. Med. Microbiol. 2014, 63, 1154–1159. (118) Randall, C. P.; Mariner, K. R.; Chopra, I.; O'Neill, A. J. The target of daptomycin is absent from Escherichia coli and other gram- negative pathogens. Antimicrob. Agents Chemother. 2013, 75, 637–639. (119) (a) Miller, C. G. Peptidases and Proteases of Escherichia Coli and Salmonella Typhimurium. Annu Rev Microbiol. 1975, 29, 485– 504. (b) Lazdunsk, A. M. Peptidases and proteases of escherichia coli and salmonella typhimurium. FEMS Microbiol. Rev. 1989, 63, 265–276. (120) (a) Koehbach, J.; Craik, D. J. The vast structural diversity of antimicrobial peptides. Trends Pharmacol. Sci. 2019, 40, 517–528. (b) Lai, P. K., Tresnak, D. T., Hackel, B. J. Identification and elucidation of proline-rich antimicrobial peptides with enhanced potency and delivery. Biotechnol. Bioeng. 2019, 116, 2439–2450. (c) Li, W. F.; Ma, G. X.; Zhou, X. X. Apidaecin-type peptides: biodiversity, structure- function relationships and mode of action. Peptides 2006, 27, 2350–2359. (121) PDB ID: 4WWW

15 Manuscript_SAC platform_Boyce et al.pdf (4.07 MiB) view on ChemRxiv download file

Supporting Information

Platform to discover protease-activated antibiotics and application to siderophore-antibiotic conjugates Jonathan H. Boyce*, Bobo Dang**, Beatrice Ary*, Quinn Edmondson*, Charles S. Craik*, William F. DeGrado*, Ian B. Seiple.*

*Department of Pharmaceutical Chemistry and Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California 94158, United States

**Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, 18 Shilongshan Road, Hangzhou 310024, Zhejiang Province, China.

Email: [email protected], [email protected], [email protected]

Table of Contents

I. SUPPLEMENTARY TABLES and FIGURES…….……………………..……….…...……S2

II. GENERAL INFORMATION……………………………..……………..……….…...……S10

III. EXPERIMENTAL PROCEDURES AND COMPOUND CHARCTERIZATION…………………………………………………..…………………….S12

IV. MIC, PERIPLASMIC CLEAVAGE ASSAYS, AND CELL-FREE TRANSLATION………..……………………...…………………………………………….…S61

V. SUBSTRATE PHAGE DISPLAY…………………………………………………………S66

VI. REFERENCES CITED…………..…………………...………………...………….……....S70

VII. SELECT NMR SPECTRA…………………...……………………..…………………….S72

VIII. HPLC TRACES FOR CLEAVAGE IN PERIPLASMIC EXTRACT…...... S103

S1 I: Supplementary Tables

Full MIC Table Minimum Inhibitory Concentration Gram-Negative Strains

(µM) c d b b tolC tolC surA ∆ ∆ M2 M2 PA01 PA14 E. coli 14028s S. typhi MG1655 MG1655 knockout wild type wild type Newman S. aureus pathogenic pathogenic pathogenic E. cloacae pathogenic bamB BW25113 pathogenic E. coli K12 1797 ATCC 78578 MGH S. enterica ATCC13047 ATCC13047 E. coli DCO ATCC 13048 13048 ATCC 10145 ATCC ∆ E. aerogenes Gram-positive A. baumannii baumannii A. P. aeruginosa P. aeruginosa P. aeruginosa efflux-knockout E. coli K. pneumoniae K. outer-membrane A. nosocomialis nosocomialis A. multidrug resistant multidrug resistant multidrug resistant

Conjugates and Controls multidrug resistant multidrug resistant Daptomcyin (4) >39 >39 >39 >39 >39 >39 >39 >39 >39 >39 >39 >39 >39 0.6 0.6 L -Linker Daptomycin Conjugate (7) 11 21 11 5 1 >21 >21 >21 >21 >21 >21 21 >21 >21 >21 D -Linker Daptomycin Conjugate (13) >23 >23 23 23 11 NDa NDa NDa NDa NDa NDa NDa >23 23 >23 Conjugate Without Antibiotic, Acid (11) >48 >48 48 >24 >48 >48 NDa >48 NDa >48 >48 >48 >48 >48 >48 Conjugate Without Antibiotic, Ester (12) >48 NDa 24 >24 NDa NDa NDa >48 NDa NDa >48 >24 >48 48 >48

a a a Eperezolid-NH2 (5) >171 >171 >171 >171 >171 ND ND >171 ND >171 >171 >171 >171 48 48 Ent -Eperezolid-NH2 >171 >171 >171 >171 >171 >171 NDa >171 NDa >171 >171 >171 >171 >171 >171 Eperezolid-OH 170 >170 5 >170 >170 NDa NDa >170 NDa >170 >170 >170 >170 3 5 Ent -Eperezolid-OH >170 >170 >170 >170 >170 NDa NDa >170 NDa >170 >170 >170 >170 170 >170 a a a a L -Linker Eperezolid-NH2 Conjugate (8) >38 >38 1 >38 >38 ND ND >38 ND ND >38 >38 >38 38 >38 D -Linker Eperezolid Conjugate (14) >38 >38 19 >38 >38 >38 NDa >38 NDa >38 >38 >38 >38 >38 >38 Conjugate With Inactive Enantiomer (15) >38 >38 9 >19 >38 NDa NDa >38 NDa NDa >38 >38 >38 38 >38 Conjugate With WSWC Linker (17) >47 NDa 37 NDa NDa NDa NDa NDa NDa NDa NDa NDa NDa NDa NDa e Conjugate Without Siderophore (18) >77 NDa >77 NDa NDa NDa NDa >77 NDa NDa >77 NDa NDa NDa >77

Solithromycin (6) 4 2 1 5 5 9 9 1 1 9 19 38 19 1 1 L -Linker Solithromycin Conjugate (9) 3 7 0.4 >27 7 7 13 7 7 >27 >27 >27 >27 >27 >27 L -Linker Solithromycin Conjugate Epimer (S27A)f 3 13 0.8 >27 >27 13 13 13 13 27 >27 >27 >27 24 >27 D -Linker Solithromycin Conjugate (16) 3 13 0.8 >27 >27 >27 >27 >27 >27 >27 >27 >27 >27 27 >27 D -Linker Solithromycin Conjugate Epimer (S27B)g 3 7 0.8 >27 >27 13 >27 >27 >27 >27 >27 >27 >27 13 >27 Table S1. MICs for conjugates and controls in 14 Gram-negative strains and one Gram-positive strain. All MICs were evaluated in biological duplicate and technical triplicate. All MIC assays were conducted in Meuller-Hinton-II (MH-II) broth with DP (600 µM for P. aeruginosa and 200 µM for all other strains). aND = Not Determined. bE. coli ∆bamB∆tolC is an efflux knockout and lacks the lipopeptide BamB, which results in a functional, albeit less efficient, BamACDE protein complex for outer-membrane protein (OMP) assembly. cSewell, A.; Dunmire, J.; Rowe, T.; Bouhenni, R. Mol Vis. 2014, 20, 1182–1191. dE. coli ∆surA is an outer-membrane knockout that lacks the chaperone SurA, which compromises the primary pathway of OMP assembly. eEvaluated in E. coli ∆tolC. f,gDuring the coupling reaction to attach solithromycin to the WSPKYM linker, racemization resulted in an epimerized adduct for both the L-linker and D-linker conjugates, S27A and S27B respectively. In addition to differences in activity between the L- and D-linker solithromycin conjugates 9 and 16, the activities of the diastereomers S27A and S27B further supported that proteolysis of solithromycin conjugate 9 was responsible for its activity in five pathogenic strains. abbreviations: DP=dipyridyl (iron scavenger), Ent-eperezolid = enantiomer of eperezolid (inactive molecule).

S2

Potential for Scarless Cleavage in the Bacterial Periplasm

L -Linker % Eperezolid-NH (5) Eperezolid-NH 2 2 release in Periplasmic e Conjugate (8) %Error Extractd [MIC (µM)]c Strainb E. coli K12 MG1655 >38 3.8 ± 0.4 E. coli DCO >38 6.8 ± 1.3 E. coli ∆bamB∆tolC BW25113 1 33.9 ± 1.5 A. baumannii ATCC 1797 >19 30 ± 8.4 A. nosocomialis M2 >38 3.1 ± 2.1 a P. aeruginosa PA01 >38 NDa ND a P. aeruginosa ATCC 10145 >38 NDa ND a E. cloacae ATCC 13047 NDa NDa ND a S. enterica 14028s NDa NDa ND a E. aerogenes ATCC 13048 NDa NDa ND Table S2. aND = Not Determined. bThe periplasmic extract was isolated by osmostic shock from each strain grown in LB media and in MH-II media. cThe MICs d o were evaluated in biological triplicate. The release of eperezolid-NH2 was monitored by HPLC after 11 h of incubating substrate and periplasmic extract at 37 C with mixing at 1050 rpm. The antibiotic peak was confirmed by MS and by retention time comparison to a standard at a concentration corresponding to the theoretical yield of eperezolid-NH2 release. The following equation was used to calculate the % yield: area of antibiotic peak/area of standard, which was then averaged over two biological replicates. eThe error was calculated from the following equation: standard deviation/sqrt(# of replicates). Please refer to HPLC data on pages S123-126 for analysis of scarless eperezolid-NH2 release in the periplasmic extract of five strains listed in Table S2.

For analysis of scarless daptomycin release from conjugate 7 in the periplasmic extract of all 10 strains listed in Table S2, please refer to HPLC data on pages S103-S122.

Discussion of Table S2: The MICs for conjugate 8 are compared to the % eperezolid-NH2 release in periplasmic extract. A measurable % yield confirms the potential for scarless antibiotic release in the bacterial periplasm of the corresponding strain. The % yield (% scarless cleavage) in periplasmic extract may not reflect the extent of non-scarless cleavage or the amount of antibiotic released in the periplasm of the live bacterial cell. The best yield for the scarless antibiotic release of eperezolid-NH2 (5) from conjugate 8 was observed in the extract for E. coli ∆bamB∆tolC, in which the conjugate was most active. Eperezolid-NH2 is an efflux substrate in Gram-negative bacteria, which may rationalize the low correlations of cleavage and activity for 8 in wild-type strains.

S3

Oxazolidinones As Efflux Substrates in Gram-Negative Bacteria, and Eperezolid-OH Permeates the OM, Unlike Eperezolid-NH2

Amide Conjugate Ester Conjugate

O O HO HO HN HO HO HN

H OTFA amide bond N H ester bond N OTFA N NH 3 N NH O N 3 O O NH O N H O O NH HO O S H O HO O S OH H O N O OH H O N O OH N N H O N OH N N H N O O H N H N O O H N N O N O OH O F OH N F O N O

NH NH Miller Siderophore O Miller Siderophore WSPKYM WSPKYM O eperezolid amide eperezolid ester 8 S1

Amide Conjugate Components Ester Conjugate Components

O O alcohol amine HO HO HO HN HO HN O O OTFA HO OTFA H2N H H N N N N N NH N NH 3 N 3 N O N O N O NH O NH O H O O H O O S O S F HO F HO N O N O OH H O OH H O O N O N OH N N OH N N OH OH H N H N NH O H NH O H O O O O OH OH Miller Siderophore Miller Siderophore eperezolid amine eperezolid alcohol WSPKYM WSPKYM 11 5 11 S2

Figure S1. Comparison of the cleaved components resulting from proteolysis of eperezolid amide conjugate 8 and hydrolysis of eperezolid ester conjugate S1.

Discussion of Figure S1. Although some Gram-positive antibiotics are not active in E. coli because they cannot penetrate the outer membrane, many are small enough to pass through porins but are prone to efflux as evidenced by their activity against efflux-pump knockouts.S1 Indeed, many oxazolidinones, like the alcohol analog of 5, eperezolid-OH (S2), are known efflux substrates in Gram-negative pathogens,S2 and are only active in efflux-knockouts of E. coli (Table S1). Eperezolid-NH2 (5) is also an efflux substrate as evidenced by the exclusive activity of conjugate 8 in E. coli ∆bamB∆tolC. Conjugate S1 required rapid assay evaluation following its synthesis due to its tendency to hydrolyze in solution.

S4

Siderophore-Mediated Transport Is Supported by MIC Data at Variable Concentrations of Dipyridyl (DP) Minimum Inhibitory Concentration tolC tolC tolC tolC

(µM) ∆ ∆ ∆

bamB bamB bamB no DP DP no ∆ ∆ ∆ 129 µM DP 129 µM DP 200 µM

SACs (Depend On Fe-concentration) E. coli E. coli E. coli L -Linker Eperezolid Amide Conjugate (8) 19 5 1 a Eperezolid Ester Conjugate (S34) ND 5 0.6 a Conjugate With Inactive Enantiomer (15) >38 ND 9

Controls (not dependent on Fe-conc.) polymyxin B 0.4 0.7 0.4 Eperezolid-OH 5 5 5 Ent-Eperezolid-OH >170 >170 >170

Eperezolid-NH2 (5) >171 >171 >171 Ent-Eperezolid-NH2 >171 >171 >171 Table S3A. aND = Not Determined. The MICs of siderophore conjugates 8, 15, and S1 at different concentrations of DP are consistent with siderophore-mediated transport. A concentration of 200 µM DP proved optimal for siderophore transport of all three conjugates. Although not shown in this Table, 600 µM DP was determined to be optimal for siderophore uptake in P. aeruginosa; other species did not grow at this concentration. Each MIC is the result of at least two biological replicates, and each replicate was evaluated in triplicate. abbreviations: DP=dipyridyl (iron scavenger), Ent-eperezolid = enantiomer of eperezolid (inactive molecule).

Discussion of Table S3A: The MICs of the controls do not vary significantly with DP concentration, while the siderophore conjugates become more active with increasing levels of DP. This phenomenon can be explained by the enhanced expression of outer-membrane transport proteins for siderophore uptake in iron- deficient media.

Upon hydrolysis, the ester conjugate S1 releases eperezolid-OH (S2). The amide conjugate 8 releases eperezolid-NH2 (5) following proteolysis. The similar MICs obtained for both ester conjugate S1 and amide conjugate 8 in E. coli ∆bamB∆tolC suggest that they may proceed by a similar mechanism of action via conjugate uptake and proteolytic cleavage. For consistent replicates, the MICs of conjugate S1 required immediate evaluation after solubilizing in DMSO due to its hydrolytic instability.

S5

A. A. S. typhi wild type pathogenic

Solithromycin pathogenic pathogenic S. enterica pathogenic pathogenic E. coli K12 E. aerogenes nosocomialis nosocomialis

Conjugates and Controls pneumoniae K. 200 µM Dipyridyl L -Linker Solithromycin Conjugate (9) 7 7 7 7 13 3 D -Linker Solithromycin Conjugate (16) >27 >27 >27 >27 >27 3 Solithromycin (6) 5 1 1 9 9 5 0 µM Dipyridyl L -Linker Solithromycin Conjugate (9) >27 >27 >27 >27 >27 >27 Solithromycin (6) 5 1 1 19 28 5 Table S3B. To determine that premature cleavage was not largely responsible for the activity of solithromycin conjugate 9 in pathogenic A. nosocomialis, S. typhi, S. enterica, E. aerogenes, and K. pneumoniae, we tested the MIC of the L-linker conjugate 9 at 0 µM DP to prevent siderophore transport, which revealed that the conjugate was not active at this DP concentration. This result confirmed that premature cleavage was not largely responsible for the activity of conjugate 9, as release of solithromycin in the extracellular medium would result in some activity. The equal activities of the L- and D-linker solithromycin conjugates 9 and 16 in E. coli suggest the possibility of bacterial-growth inhibition without cleavage. To test this hypothesis, we showed that conjugate 9 and its D-linker analogue 16 inhibit in-vitro translation of the E. coli ribosome (Table 5 in manuscript). The L- and D-linker solithromycin conjugates 9 and 16 may also inhibit translation in the five pathogenic strains if they had equal access to the target. This possibility is supported by solithromycin’s ability to inhibit the growth of these pathogenic strains. In the five pathogenic strains, periplasmic cleavage of 9 may be responsible for its activity for the following reasons: 1) Since the D-linker solithromycin conjugate 16 is not active in the pathogenic strains, it may not be reaching its ribosomal target in the cytoplasm. 2) Proteolytic cleavage is responsible for the activity in these strains based on large-activity differences between the D- and L-linker conjugates. 3) The D- and L-linker conjugates 9 and 16 may not passively diffuse through the inner membrane due to low activity in E. coli ∆surA and S. aureus Newman (Table S1). There may be a cooperative effect between DP and solithromycin (6) in E. aerogenes (~2-fold activity increase at 200 µM DP) and K. pneumoniae (~3-fold activity increase at 200 µM DP).

MG1655 MG1655 wild type MG1655 wild type MG1655 wild type MG1655 wild type MG1655 wild type 0 µM DP MG1655 wild type MG1655 wild type MG1655 wild type MG1655 wild type 10 µM DP 20 µM DP 40 µM DP 80 µM DP E. coli K12 K12 coli E. K12 coli E. K12 coli E. K12 coli E. K12 coli E. K12 coli E. K12 coli E. K12 coli E. K12 coli E. 120 µM DP 160 µM DP 200 µM DP 250 µM DP SACs (Depend On Fe-concentration) L -Linker Solithromycin Conjugate (9) >27 >27 >27 >27 >27 >27 2 0.8 0.8 D -Linker Solithromycin Conjugate (16) 27 27 27 27 13 13 0.8 0.8 0.8 Controls (not dependent on Fe-conc.) solitromycin 5 10 5 5 5 5 5 5 5 Table S3C. Siderophore transport is activated between 120-160 µM DP in E. coli K12. MICs at various DP concentrations show no cooperativity between solithromycin and DP in this strain. An optimal concentration of DP for siderophore uptake was determined to be 200 µM for the solithromycin conjugate. The equal activities of 9 and 16 in E. coli suggest that the conjugate is inhibiting translation without cleavage. However, we cannot rule-out the possibility of cleavage in E. coli despite the ability of the whole conjugate to inhibit translation (Table 5 of manuscript), and the proteolysis of 9 is required for its activity in several pathogenic strains (see Table S3B).

S6

Oxazolidinone Conjugate Activities in E. coli ∆bamB∆tolC May be Explained by Linker Proteolysis in the Cytoplasm

MIC (µM) MIC (µM) MIC (µM)

tolC tolC tolC tolC surA surA ∆ ∆ ∆ ∆ E. coli E. coli E. coli BW2113 BW2113 knockout Newman BW25113 BW25113 BW25113 BW25113 S. aureus bamB bamB Combination of the Conjugate bamB ∆ ∆ ∆ E. coli Gram-positive efflux-knockout efflux-knockout efflux-knockout Conjugate Components outer-membrane Conjugates Components for S34 in Equal Concentrations Siderophore-Linker-OH (11) 48 >48 >48 L -Linker Eperezolid Ester Conjugate (S1) 0.6 Siderophore-Linker-OH (11) + Eperezolid-OH (S2) 2 Eperezolid-OH (S2) 5 3 5 L -Linker Eperezolid Amide Conjugate (8) 1 Eperezolid-NH2 (5) >163 41 41

Table S4. The combination of cytoplasmic cleavage and synergy or coalism may explain the activity of oxazolidinone conjugates in E. coli ∆bamB∆tolC.

Discussion of Table S4. Synergy or coalism between the cleaved conjugate components following cytoplasmic transport and intracellular hydrolysis may explain the enhanced activity of ester conjugate S1, which is significantly more active than its cleaved components (eperezolid-OH (S2) and Sideropore-Linker-OH 11, >8-fold and 80-fold, respectively) in E. coli ∆bamB∆tolC. To investigate the possibility of synergy or coalism, we combined eperezolid-OH and the siderophore- linker component 11 in equimolar quantities, which led to a 2-fold improvement in activity (MIC = 2 µM). However, the improvement remains >3-fold less active than conjugate S1 (0.6 µM vs. 2 µM), and the activity is similar to that of eperezolid-OH (S2) in an outer-membrane knockout of E. coli (MIC = 3µM), suggesting that cytoplasmic transport and proteolysis may be responsible for the enhanced activity of S1 (MIC = 0.6 µM). Indeed, cytoplasmic uptake would enable eperezolid- OH to bypass the outer- and inner-membrane barriers, which may explain why the combination of component 11 and eperezolid-OH (S2) does not reach the full potential of conjugate S1. Conjugate transport of the related analogue 8 to the cytoplasm is also a likely possibility in E. coli ∆bamB∆tolC. In the event of periplasmic cleavage of 8, the passive diffusion of the cleaved eperezolid-NH2 (5) through the E. coli cytoplasmic membrane may result in low activities because eperezolid-NH2 (5) is not active in a mutant of E. coli with a compromised outer membrane (E. coli ∆surA BW2113) or in Gram-positive strains that lack an outer membrane (MIC = 41 µM). Therefore, proteolysis in the cytoplasm may be responsible for the high activity of conjugate 8 (MIC = 1 µM).

Proteolysis of conjugate 8 would be necessary to inhibit bacterial growth based on its inability to inhibit translation in E. coli at 38 µM (Figure 5 of manuscript).

S7

Cleavage-Site Analysis For Enriched Sequences Identified from Substrate Phage Display

Recovered Starting Synthesized Sequence P-Side (Major) P-Side (Minor) P' side (Major) P' side (Minor) Major Fragment Material WSKNQSLGG W NDa SKNQSLGG NDa 50% NDa WSGSDSSVG W NDa SGSDSSVG NDa 50% NDa WSNHADVHG NH2-WSNHA NDa SNHADVHG NDa 30% SNHADVHG WSKSEMLSG NDa NDa MLSG SKSEMLSG 20% MLSG-OH WSWCKWASG WSWC NDa KWASG NDa 3% WSWC and KWASG a WSPKYMRFG ND WSPKYM RFG YMRFG and MRFG 30% RFG Table S5. Cleavage-site analysis of sequences from substrate phage display. The synthesized sequences (25 µM) were incubated with E. coli K12 periplasmic extract (Total Protein: 100 µg/mL) at 37 oC for 18 h. Determination of the cleavage site was revealed by LC/MS analysis.

S8

Growth Curves Reveal That Solithromycin Conjugate 9 Delays the Growth of P. aeruginosa ATCC 10145, a Multi-Drug Resistant Strain

Conjugate 8 (37.6 µM vs. no compound) Conjugate 9 (26.9 µM vs. no compound) 0.5 0.5

0.45 0.45

0.4 0.4

0.35 0.35 0.3 0.3 0.25 0.25 OD600 0.2 OD600 0.2

0.15 0.15

0.1 0.1

0.05 0.05 0 0 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 Time (h) Time (h)

37.6 uM 37.6 uM 37.6 uM no compound no compound no compound 26.9 uM 26.9 uM 26.9 uM no compound no compound no compound

Figure S2. The delayed growth caused by solithromycin conjugate 9 may be due to an iron-withholding effect at higher concentrations of conjugate, siderophore- conjugate out-competition with endogenous siderophores (this strain is known to produce the pyoverdine siderophore), or through expression of orthogonal outer-membrane-protein receptors for siderophore-mediated transport. All growth curves were conducted in sterilized 96-well plates and monitored at OD600 over 16 h in 50% reduced MH-II broth containing 600 µM DP.

S9

II. General Information

A. Instrumentation and Methods All reactions were carried out in oven-dried glassware under an argon atmosphere unless otherwise noted. Proton nuclear magnetic resonance (1H NMR) spectra and carbon nuclear magnetic resonance (13C NMR) spectra were recorded on 400 MHz Bruker Avance III HD 2-channel instrument NMR spectrometers at 23 °C or 50 oC. Proton chemical shifts are expressed in parts per million (ppm, δ scale) and are referenced to residual protium in the NMR solvent (CHC13: δ 7.26). 13C NMR were recorded at 100 MHz at ambient temperature with the same solvents unless

1 otherwise stated. Chemical shifts are reported in parts per million relative to CDCl3 ( H, δ 7.26; 13 1 13 13 C, δ 77.0) or CD3OD ( H, δ 3.31, 4.78; C, δ 49.3). All C NMR spectra were recorded with complete proton decoupling. Analytical thin-layer chromatography (TLC) was performed using glass plates pre-coated with silica gel (0.25-mm, 60-Å pore size, 230−400 mesh, SILICYCLE INC) impregnated with a fluorescent indicator (254 nm). TLC plates were visualized by exposure to ultraviolet light (UV), and then were stained by submersion in a basic aqueous solution of potassium permanganate or with an acidic ethanolic solution of anisaldehyde, followed by brief heating. An Acquity UPLC BEH C18 1.7 µm column was used for analytical UPLC-MS. Preparative HPLC purification was performed on a Waters Prep HPLC or Varian ProStar HPLC using a C4 or C18 column. Analytical reverse-phase HPLC analyses were performed on an Agilent 1100 series HPLC system using a Phenomenex Kinetex 2.6 µm C18, 50 ´ 2.1 mm column. Flow rates were controlled at 0.35 mL/min. Peptide detection was based on UV absorption at 220 nm, while conjugate detection (following siderophore attachment) was based on UV absorption at 254nm. Mass spectrometry data for conjugates and peptides were obtained using Shimadzu AXIMA Performance MALDI-TOF spectrometer in a reflectron mode with α-Cyano-4- hydroxycinnamic acid as the matrix. Liquid chromatography/MS experiments were conducted on a Shimadzu HPLC with an Applied Biosystems 3200 QTRAP LC/MS/MS system. High resolution mass spectra were obtained using a Waters Acquity UPLC (Ultra Performance Liquid Chromatography) with a binary solvent manager, SQ mass spectrometer, Waters 2996 PDA (Photo-Diode Array) detector, and Evaporative Light Scattering Detector (ELSD).

S10 B. Reagents and Solvents

CH2Cl2, DMF, THF, ethyl ether, and acetonitrile to be used in anhydrous reaction mixtures were dried by passage through activated alumina columns immediately prior to use. Hexanes used were ≥85% n-hexane. Other commercial solvents and reagents were used as received, unless otherwise noted. Fmoc-protected amino acids were purchased from GL Biochem. 2-(6-Chloro-1H- benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU), trifluoroacetic acid (TFA) were purchased from Chem-Impex International and Aldrich. 4-Methylpiperidine was purchased from Acros Organics. Rink Amide-ChemMatrix resin (0.5 mmol/g loading) was purchased from Biotage. The oxazolidinones eperezolid-NH2 (5) and eperezolid-OH (S2) were prepared by the protocols of Miller and Rafai Far.S3,S4 All other reagents and relevant catalysts were purchased from Sigma-Aldrich, Acros, Alfa Aesar, and Strem Chemicals.

Solid-phase peptide synthesis (SPPS)

All peptides were synthesized on 3.7 mmol scale using a CHEMGLASS® glass 500-mL medium- porosity sintered glass solid phase peptide synthesis vessel GL-32 or at 20 µmol scale using a Biotage Syro II parallel peptide synthesizer. 2-Chlorotrityl resin (Chem-Impex, 1.0-2.0 mmol/g loading) or rink amide-ChemMatrix® resin (Biotage, 0.5 mmol/g loading) were used for the synthesis. A typical SPPS reaction cycle includes Fmoc deprotection, washing, coupling, and post- coupling washing steps. On the peptide synthesizer, the deprotection was carried-out for 5 minutes at 23 ºC with 20% 4-methylpiperidine in dimethylformamide (DMF). On the peptide synthesizer, a standard double coupling was accomplished in 8 min at 23 ºC with 5 equivalents of Fmoc- protected amino acids, 4.98 equivalents of HCTU, and 10 equivalents of DIEA (relative to the amino groups on resin) in DMF at a final concentration of 0.125 M amino acids. Chromatographic separations for 8-mer peptide syntheses were obtained using a linear gradient of 1-61% acetonitrile (with 0.08% TFA) in water (with 0.1% TFA) over 10 min, 1-41% acetonitrile (with 0.08% TFA) in water (with 0.1% TFA) over 20 min, or 1-41% acetonitrile (with 0.08% TFA) in water (with 0.1% TFA) over 40 min with column at room temperature. The synthesis on larger scales (3.7 mmol) are described herein (Section III).

S11

III. Experimental Procedures and Compound Characterization

A. Siderophore Synthesis

1) Ph Ph Cl Cl 165 oC, 1h OH 2) LiOH O Ph H2O/THF OH reflux, 9h O Ph

O OMe 92% over 2 steps O OH methyl 2,3-dihydroxybenzoate S3 S3: Method A (purification by column chromatography): To a flame-dried 100-mL round- bottom flask containing methyl 2,3-dihydroxybenzoate (500 mg, 2.97 mmol, 1.0 equiv) under argon was added dichlorodiphenylmethane (0.86 mL, 4.46 mmol, 1.5 equiv). The mixture was then heated to 165 oC for 1h. After cooling to 23 ºC, the mixture was diluted with EtOAc (8

mL). The organic layers were washed with NaHCO3 (5 mL), H2O (5 mL), brine (5 mL), dried

over MgSO4, and concentrated under reduced pressure to provide crude product (767 mg), which was used directly in the next reaction without further purification. The crude product (767 mg,

2.31 mmol) was dissolved in THF/H2O (1:1, 60 mL). Lithium hydroxide monohydrate (1.32 g, 31.5 mmol, 13.6 equiv) was added in a single portion, and the mixture was heated to reflux for 24

h. After cooling to 23 ºC, the reaction was neutralized with 10% AcOH/H2O (50mL) until the solution reached pH 4. The solution was extracted into EtOAc (3 x 100 mL), and the combined

organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. The crude solid was purified by column chromatography (10 - 30% EtOAc/hexanes, then 95% EtOAc/AcOH) to provide 867.6 mg (92% over 2 steps) of S3 as a white solid. Spectroscopic data for S3 were found to be identical with those reported in the literature.S5,S6

S3: Method B (purification by triteration, no column chromatography): To a 100-mL round- bottom flask containing methyl-2,3-dihydroxybenzoate (8.0 g, 47.6 mmol, 1.0 equiv) under argon was added dichlorodiphenylmethane (14 mL, 71.4 mmol, 1.5 equiv). The mixture was then heated to 165 oC for 1h, cooled to 23 ºC, and diluted with EtOAc (400 mL). The solution was washed

with NaHCO3 (1x120 mL), H2O (1x120 mL), and brine (1x120 mL). The organic layer was

S12 concentrated under reduced pressure [note: did not dry over MgSO4 or filter] to provide a crude residue (15.8 g, 47.6 mmol). In a 1-L round-bottom flask containing the crude residue was added

THF/H2O (1:1, 400 mL). Lithium hydroxide monohydrate (21.0 g, 500 mmol, 10.5 equiv) was added. The mixture was heated to 100 oC for 9 h, cooled to r.t., and concentrated under reduced pressure. To the aqueous mixture was added 1M HCl (400 mL) and EtOAc (600 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (2 x 400 mL). The combined

organic layers were washed with H2O (1x200 mL) and brine (1x200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude material was transferred to a 125-

mL round-bottom flask in CH2Cl2 and concentrated to a volume of ~50 mL. A white solid began to precipitate in the flask and then hexanes (10 mL) was added. The mother liquor was carefully decanted into a 250-mL round-bottom flask so as not to disturb the solid, and the product was

washed with 1:1 hexanes/CH2Cl2 (1x25 mL). The wash was carefully decanted into the mother liquor so as to avoid decantation of solid product. The first crop of crystals provided 6.88 g (45 % over 2 steps) of product S3 as a white crystalline solid. [Note: the pure product is best solvated in a 1:1 mixture of CH2Cl2/methanol; it is less soluble in CH2Cl2, EtOAc, or methanol]. A second crop was afforded by concentrating the mother liquor under reduced pressure to ~5 mL. Spectroscopic data for S3 were found to be identical with those reported in the literature.S5,S6

S3: Method C (purification by potassium salt formation, no column chromatography): To a 100-mL round-bottom flask containing methyl-2,3-dihydroxybenzoate (3.30 g, 19.6 mmol, 1.0 equiv) under argon was added dichlorodiphenylmethane (5.50 mL, 28.6 mmol, 1.5 equiv). The mixture was then heated to 165 oC for 1h. After cooling to 23 ºC, the mixture was diluted with

EtOAc (100 mL), washed with NaHCO3 (1x30 mL), H2O (1x30 mL), brine (1x30 mL), dried over

MgSO4, and concentrated under reduced pressure to provide crude product (6.52 g), which was used directly in the next reaction. The crude product S3 (6.52 g) was dissolved in THF/H2O (1:1, 610 mL), lithium hydroxide monohydrate (13.5 g) was added, and the mixture was heated to reflux

for 24 h. After cooling to 23 ºC, the mixture was neutralized with 10% AcOH/H2O (733 mL)

to pH=4 and extracted into EtOAc (3 x 1 L). The combined organic layers were dried over MgSO4,

filtered, and concentrated under reduced pressure. The crude product was dissolved in Et2O (1000

mL), and pure product (226.9 mg) was collected as a solid precipitate. The Et2O solution was extracted with aqueous saturated K2CO3 (200 mL), which led to the formation of a potassium salt

S13 precipitate. The Et2O layer was concentrated under reduced pressure to a volume of 200 mL, and the white solid K-salt was stirred for 24h, filtered, and washed with Et2O. The aqueous layer was

extracted with EtOAc (3x250 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated, and the resulting K-salt was combined with the filtered K-salt. To a suspension of the product potassium salt in EtOAc (250 mL) was added 1M HCl (250 mL), and the aqueous layer was extracted with EtOAc (2x250 mL). The combined organic layers were washed with

H2O (250 mL) and brine (250 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to provide 4.25 g (67% over 2 steps) of product S3 as the pure free-acid. Spectroscopic S5,S6 data for S3 were found to be identical with those reported in the literature. S3: Rf = 0.28 (40 1 % EtOAc/hexanes); H NMR (400 MHz, CDCl3) δ 11.9 (br s, 1H), 7.74 – 7.61 (m, 4H), 7.52 (dd, J = 8.2, 1.2 Hz, 1H), 7.46 – 7.36 (m, 6H), 7.10 (dd, J = 7.7, 1.2 Hz, 1H), 6.91 (t, J = 8.0 Hz, 1H).

13 C NMR (100 MHz, CDCl3) δ 170.4, 149.0, 148.5, 139.7, 129.5, 128.5, 126.5, 123.4, 121.4, + 118.4, 113.3, 112.2. HR-MS: m/z Calcd. for C20H14O4 [M+H ]:319.0892, Found 319.1050.

O Cl Ph (1.3 equiv) O pyridine (1.26 equiv) OH O O Ph CH2Cl2 Ph 99% O Benzyl Phenyl Carbonate: To a 1-L flame-dried, round-bottom flask containing a solution of

benzyl alcohol (15.5 mL, 150 mmol, 1.0 equiv) in CH2Cl2 (210 mL) under argon was added pyridine (15.0 mL, 189 mmol, 1.26 equiv). The flask was immersed in an ice-water bath and stirred for 10 min. A solution of phenyl chloroformate (24.5 mL, 195 mmol, 1.3 equiv) in

CH2Cl2 (115 mL) was prepared in a 250-mL flame-dried, round-bottom flask, which was transferred via syringe in a dropwise manner to the solution of benzyl alcohol. The mixture was then warmed to 23 ºC, and monitored by TLC (2.5% EtOAc/pentane). The reaction was complete

after 4 h. The mixture was transferred to a 2-L separatory funnel, diluted with CH2Cl2 (500 mL), and washed with water (300 mL), 5% NaOH (300 mL), 1M HCl (300 mL), and brine (300 mL).

The organic layer was then dried over Na2SO4 and concentrated under reduced pressure. Purification by column chromatography (silica gel, 2.5% EtOAc/pentane) afforded 33.9 g (99%) of benzyl phenyl carbonate as a clear oil. Spectroscopic data for benzyl phenyl carbonate were found to be identical with those reported in the literature.S7

S14 O O Ph Ph O (1.2 equiv) NH2 Cbz NH2 H2N N EtOH H 80 oC, 7.5 h S4 59% S4: To a 1-L round-bottom flask containing a solution of 1,4-diaminobutane (6.50 mL, 65.0 mmol, 1.0 equiv) in EtOH (54.0 mL) under argon was added a solution of benzyl phenyl carbonate (15.0 mL, 78.0 mmol, 1.2 equiv) in ethanol (18.0 mL) via syringe over 20 minutes. Following the addition of benzyl phenyl carbonate, a white solid precipitated after 5 minutes. The mixture was then heated to 80 oC for 7.5 h. After cooling to 23 ºC, additional white solid precipitated. The white solid was filtered, washed with EtOH (2x60 mL), and discarded. [Note: The white solid is bis-Cbz-protected-diaminobutane]. The EtOH filtrate was concentrated under reduced pressure,

diluted in CH2Cl2 (300 mL), and washed with 1 M HCl (300 mL, note: a white colloidal dispersion

formed). The aqueous layer was extracted with CH2Cl2 (5x100 mL). The combined CH2Cl2 layers (a white colloidal dispersion) were discarded. The aqueous layer was then basified with 15%

NaOH until pH 14, extracted with CH2Cl2 (2x300 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to provide 8.53 g (59%) of the primary amine S4 as an oil

1 (suspended with white crystalline solid). Rf = 0.34 (20% MeOH/CH2Cl2 with 3% NH4OH); H

NMR (400 MHz, CD3OD) δ 7.46 – 7.23 (m, 5H), 5.06 (s, 2H), 3.12 (t, J = 6.6 Hz, 2H), 2.62 (t, J 13 = 6.7 Hz, 2H), 1.64 – 1.35 (m, 4H). C NMR (100 MHz, CD3OD) δ 157.5, 137.1, 128.0, 127.5, + 127.4, 65.9, 40.8, 40.2, 29.6, 26.9. HR-MS: m/z Calcd. for C12H18N2O2 [M+H ]:223.1368, Found 223.1530.

O Br O (1 equiv) O Et N (3 equiv) H Cbz NH 3 Cbz N N 2 N O H THF, r.t., 24 h H S4 S5 71%

S5: To a 500-mL flame-dried, round-bottom flask under argon containing compound S4 (3.65 g, 16.4 mmol, 1.0 equiv) was added anhydrous THF (55 mL) and triethylamine (6.8 mL, 49.3 mmol, 3.0 equiv), followed by dropwise addition of methyl bromoacetate over 10 minutes. The reaction

was monitored by TLC (10% MeOH/CH2Cl2). After 24 h, the reaction was complete, and the triethylammonium salt was filtered. The filtrate was concentrated under reduced pressure to

S15 provide the crude product as a clear oil. Upon transferring to a 50-mL round-bottom flask in

CH2Cl2, a white emulsion formed. Purification by column chromatography (2.5-5%

MeOH/CH2Cl2) provided 3.42 g (71%) of methyl ester S5. S5: Rf = 0.31 (5% MeOH/CH2Cl2); 1 H NMR (400 MHz, CDCl3) δ 7.39 – 7.26 (m, 5H), 5.19 – 4.99 (m, 3H), 3.73 (s, 3H), 3.46 (s, 2H), 3.26 – 3.16 (m, 2H), 2.78 – 2.63 (m, 2H), 2.47 (br s, 2H), 1.67 – 1.50 (m, 4H). 13C NMR (100

MHz, CDCl3) δ 171.8, 156.5, 136.6, 128.5, 128.1, 66.6, 52.0, 50.0, 48.8, 40.7, 27.5, 26.5. HR- + MS: m/z Calcd. for C15H22N2O4 [M+H ]:295.1580, Found 295.1765.

O Ph SOCl2 (4.5 equiv) O Ph NEt3 (cat) O Ph O Ph o CHCl3, 70 C, 6 h HO O Cl O S3 I1 used immediately for preparation of S6

Intermediate I1 (for preparation of compound S6 and S7): Acid S3 (1.38 g, 4.35 mmol, 1.0 equiv) was added to a 500-mL flame-dried, round-bottom flask. The flask was purged with argon,

and acid S3 was suspended in CDCl3 (22 mL). Triethylamine (21 µL, 3.5mol%) was then added, which resulted in a red solution. The temperature of an oil bath was stabilized between 45-50 oC, and the mixture was stirred for 10 minutes at this temperature. Thionyl chloride (1.43 mL, 19.6 mmol, 4.5 equiv) was added dropwise over the course of 30 min. The mixture was then heated to 70 oC and stirred for 6 h. The reaction was monitored by 1H-NMR. Upon completion, the mixture was cooled to 23 ºC, and concentrated under reduced pressure using a high vacuum manifold. A water bath was used to warm the flask to ensure that all solvent had evaporated to provide a yellow oil. After backfilling with argon, the crude acid chloride I1 was used immediately for the preparation of S6 (vide infra) without further purification.

S16 O Ph Ph O Ph O Ph I1 O Cl O O (1.5 equiv) H O Cbz N O NEt3 (1.5 equiv) N O Cbz N H N O S5 THF, 50 oC, 9 h H S6 98%

S6: To a 250-mL flame-dried, round-bottom flask containing a solution of amine S5 (839 mg, 2.85 mmol, 1.0 equiv) in dry THF (12.2 mL) under argon was added triethylamine (0.61 mL, 4.36 mmol, 1.53 equiv). Acid chloride I1 (1.46 g, 4.35 mmol, 1.53 equiv) was then added in three portions as a solution in THF (24.4 mL; portion 1: 15 mL, rinse portion 2: 5 mL, and rinse portion 3: 4.4 mL). Following the addition of the I1, the reaction began to smoke and a white milky suspension formed. The mixture was then heated to 50 oC. After 9 h, triethylamine (0.1 mL, 0.7 mmol, 0.25 equiv) was added, and the reaction was stirred for an additional 30 min. The mixture

was then cooled to 23 ºC, and CH2Cl2 (20 mL) and 1M HCl (20 mL) were added. The layers were separated, and the aqueous layer was extracted with CH2Cl2 (2x20 mL). The combined organic

layers were washed with H2O (1x30 mL) [note: Brine was avoided to prevent bad emulsion.], dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified

by column chromatography (silica gel, 1-5% MeOH/CH2Cl2) to provide 1.66 g (98%) of amide

S6. [note: During the purification, impurities were eluted in pure CH2Cl2, and the product was 1 eluted in 2 % MeOH/CH2Cl2.]. S6: Rf = 0.32 (4% MeOH/CH2Cl2); H NMR (400 MHz, CDCl3; ~1.8:1 mixture of rotamers as determined by 1H NMR analysis) δ 7.61 – 7.49 (m, 4H), 7.41 – 7.27 (m, 11H), 6.96 – 6.81 (m, 3H), 5.07 (d, J = 8.0 Hz, 2H), 4.92 (t, J = 6.1, 5.6 Hz, 0.4H)**, 4.47 (t, J = 6.0 Hz, 0.6H)*, 4.22 (s, 1.23H)*, 3.97 (s, 0.77H)**, 3.77 (s, 1.9H)*, 3.58 (t, J = 7.0 Hz, 1H), 3.46 (s, 1.1H)**, 3.30 – 3.18 (m, 2H), 2.81 (q, J = 6.6 Hz, 1H), 1.75 – 1.55 (m, 2H), 1.47 – 1.22 (m, 2H), 1.05 (p, J = 7.1 Hz, 1H).

*denotes major rotamer ** denotes minor rotamer

13 1 C NMR (100 MHz, CDCl3; ~1.8:1 mixture of rotamers as determined by H NMR analysis) δ 169.5, 169.5, 167.8, 156.5, 156.3, 147.2, 143.2, 143.1, 139.7, 139.6, 136.6, 136.6, 129.3, 129.3, 128.5, 128.5, 128.3, 128.3, 128.2, 128.1, 128.1, 126.6, 126.4, 122.3, 122.2, 121.0, 120.9, 117.6,

S17 117.5, 117.4, 109.9, 109.8, 66.6, 52.2, 52.1, 50.4, 49.6, 46.9, 46.2, 40.7, 40.2, 29.7, 27.1, 26.7, + 25.4, 24.3. HR-MS: m/z Calcd. for C35H34N2O7 [M+H ]:595.2366, Found 595.2504.

SOCl2 (4.5 equiv) O Ph O Ph NEt3 (cat) o CHCl3, 70 C O Ph O Ph

HO O Cl O S3 I1 used immediately for preparation of S7

Ph O Ph Ph O Ph Ph Ph O O Ph O O Ph O Cl O I1 (2 equiv) O NEt (2 equiv) O 10% Pd/C (0.6 equiv) 3 O O O o O H2, MeOH, 1.5 h O THF, 50 C, 9 h N O N O Cbz N H N O N 93% over 2 steps H N O H 2 O S7 S6 I2 O Ph used immediately Ph

Intermediate I2 (for preparation of compound S7): To a 500-mL flame-dried, round-bottom flask containing a solution of compound S6 (1.62 g, 2.72 mmol) in anhydrous MeOH (27 mL) was added 10% Pd/C (174 mg, 1.63 mmol). The flask was fitted with a 3-way valve-adapter to

evacuate and purge the system with H2 gas. After purging the system with H2 (3x), the mixture was stirred at 23 ºC for 1.5 h, at which point the reaction was complete as determined by TLC analysis (50% EtOAc/hexanes). The mixture was filtered through celite, washed with MeOH, and concentrated under reduced pressure to provide 1.24 g of the crude intermediate I2, which was used immediately without further purification in the next reaction. I2: HR-MS: m/z Calcd. for + C27H28N2O5 [M+H ]:461.1998, Found 461.2154.

S7: To a 250-mL flame-dried, round-bottom flask containing a solution of the crude intermediate I2 (1.02 g, 2.22 mmol, 1.0 equiv) in THF (9.5 mL) under argon was added TEA (0.61 mL). To this was added the acid chloride I1 (1.46 g, 4.35 mmol, 1.96 equiv, prepared as described above) in three portions as a solution in THF (19 mL; portion 1: 11.7 mL, rinse portion 2: 3.9 mL, and rinse portion 3: 3.4 mL). The reaction was placed in a preheated oil bath at 50 oC and stirred for 9 h at this temperature. The mixture was then cooled to 23 ºC, and TLC analysis (55% EtOAc/hexanes) suggested that the reaction was complete. The reaction was then diluted with

CH2Cl2 (20 mL) and 1 M HCl (20 mL), and the layers the were separated. The aqueous layer

S18 was extracted with CH2Cl2 (2 x 20 mL). The combined organic layers were washed with H2O

(1x20 mL) and brine (1 x 20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. Purification by column chromatography (silica gel, 20-60% EtOAc/hexanes; note: The product began eluting at 50% EtOAc/hexanes, and the product was fully-eluted at 60% EtOAc/hexanes) provided 771 mg (46% over 2 steps) of compound S7 and an additional 805 mg of crude S7. [note: Further purification of the recovered 805 mg of crude S7 was not necessary, which allowed for saponification (described below) and subsequent purification to provide the

1 ketal-protected siderophore 10 as a pure product]. S7: Rf = 0.25 (55% EtOAc/hexanes); H NMR 1 (400 MHz, CD3OD, ~2:1 mixture of rotamers as determined by H NMR analysis) δ 7.72 – 6.34 (m, 26H), 4.22 (s, 1.2H)*, 4.00 (m, 0.5H)**, 3.94 (s, 0.2H)**, 3.72 – 3.48 (m, 0.94H), 3.65 (s, 2.1H)*, 3.39 (s, 0.9H)**, 3.06 (t, J = 6.7 Hz, 1.44H), 1.85 – 1.70 (m, 1.1H), 1.55 – 1.33 (m, 1.5H), 1.31 – 1.08 (m, 1.6H).

*denotes major rotamer ** denotes minor rotamer

13 1 C NMR (100 MHz, CD3OD; ~2:1 mixture of rotamers as determined by H NMR analysis) δ 169.6, 169.4, 168.8, 168.6, 164.7, 164.5, 147.5, 147.3, 147.2, 144.9, 144.8, 143.0, 142.7, 139.7, 139.5, 139.3, 139.2, 129.4, 129.3, 129.2, 129.1, 129.0, 128.9, 128.2, 128.1, 128.0, 128.0, 128.0, 127.9, 127.7, 126.1, 126.0, 126.0, 125.9, 125.9, 125.8, 122.2, 122.1, 121.9, 121.8, 121.3, 120.3, 120.1, 118.2, 117.5, 117.3, 117.1, 115.9, 115.8, 111.4, 111.4, 109.7, 109.6, 51.2, 51.2, 50.0, 49.9, + 46.3, 39.0, 38.5, 26.6, 26.0, 25.3, 24.2. HR-MS: m/z Calcd. for C47H40N2O8 [M+H ]:761.2785, Found 761.2534.

S19 Ph Ph O Ph O Ph O O LiOH (11 equiv) O O O O H2O/THF (1:1) O O r.t., 4 h N N H N O N O H 73% H O O O Ph S7 O Ph 10 Ph Ph Siderophore Component

Siderophore Component 10: To a 20-mL scintillation vial containing a solution of methyl ester

S7 in THF/H2O (1:1, 11.6 mL) was added lithium hydroxide monohydrate (261 mg, 6.21 mmol, 11 equiv). The mixture was stirred at 23 ºC for 4 h. The reaction was complete as determined by

TLC analysis (10% MeOH/CH2Cl2). The mixture was then acidified with 1 M HCl (4 mL), stirred

for 2 min, and extracted with CH2Cl2 (3x4 mL). The combined organic layers were dried over

Na2SO4, filtered, and concentrated under reduced pressure. Purification by column chromatography (silica gel, 55% EtOAc/hexanes (to remove impurity); then 2-10%

MeOH/CH2Cl2) provided 320 mg (73%) of diphenylketal-protected siderophore 10. Siderophore 1 Component 10: Rf = 0.52 (10% MeOH/CH2Cl2); H NMR (400 MHz, CD3OD, ~1.9:1 mixture of rotamers as determined by 1H NMR analysis) δ 7.87 – 6.58 (m, 26H), 4.18 (s, 1.3H)*, 3.94 (s, 0.7H)**, 3.61 (t, J = 6.0 Hz, 0.7H), 3.47 (q, J = 6.1 Hz, 0.7H), 3.23 (t, J = 7.6 Hz, 1.3H), 3.00 (q, J = 6.5 Hz, 1.3H), 1.71 (t, J = 3.0 Hz, 1.4H), 1.42 (p, J = 8.2 Hz, 1.3H), 1.10 (p, J = 7.2 Hz, 1.3H).

*denotes major rotamer ** denotes minor rotamer

13 1 C NMR (100 MHz, CD3OD; ~1.9:1 mixture of rotamers as determined by H NMR analysis) δ 170.7, 170.6, 168.8, 168.5, 164.6, 164.4, 164.4, 147.5, 147.5, 147.3, 147.2, 145.0, 144.8, 143.0, 142.8, 139.7, 139.5, 139.3, 139.2, 129.5, 129.3, 129.1, 129.0, 128.3, 128.2, 128.1, 126.1, 126.0, 126.0, 125.9, 122.2, 122.2, 122.0, 121.9, 121.5, 120.3, 120.2, 118.2, 117.6, 117.5, 117.3, 116.0, 115.9, 111.5, 111.4, 109.7, 109.6, 53.5, 50.1, 49.9, 46.8, 46.2, 39.2, 38.8, 38.7, 26.7, 26.1, 25.4, + 24.3. HR-MS: m/z Calcd. for C46H38N2O8 [M+H ]:747.2628, Found 747.2713.

S20 B. Linker Synthesis [amino acid sequences: WSPKYM and WSWC]

Resin Loading onto 2-Chlorotrityl Resin: For large-scale peptide synthesis (>3 mmol), the amino acids were manually loaded onto 2-chlorotrityl chloride resin (1-2 mmol/g, Chem-Impex). For small-scale peptide synthesis (<0.1 mmol), the P1 amino acid was purchased preloaded onto 2-chlorotrityl chloride resin. For manual loading, 2-chlorotrityl chloride resin (>4.2 g) and amino acid (>3.0 mmol) were dried on high vacuum overnight before use in 20-mL scintillation vials. Only anhydrous reagents were used.

DIEA (1.1 equiv), CH Cl , S 2 2 Cl Cl S OH r.t., 24 h Cl O O 62 % Fmoc NH 4.0-gram scale O Fmoc NH 1.0 – 2.0 mmol/g (0.5 equiv) meq=0.809 mmol/g 2-chlorotrityl chloride resin Fmoc-L-Met-OH Fmoc-L-Met-2-chlorotrityl resin S8 General Resin-Loading Procedure (Gram-Scale): Fmoc-L-methionine-2-chlorotrityl resin S8: To a 500-mL flame-dried, round-bottom flask was added dry Fmoc-L-methionine (1.11 g, 3.0 mmol). The flask was purged with argon, and the amino acid was then suspended in anhydrous CH2Cl2 (40 mL). The flask was placed in a sonicator for 5 min to assist with solubilizing the amino acid. (note: A uniform solution did not form until DIEA was added). Dry 2-chlorotrityl chloride resin (4.0 g, ~6.0 mmol, 1.0 – 2.0 mmol/g) was added in a single portion, and the sides of the flask were rinsed with additional anhydrous CH2Cl2 (20 mL). The addition of anhydrous DIEA (1.2 mL, 6.90 mmol) resulted in a dark uniform solution, and the mixture was mixed vigorously on a Mistral Multi-Shaker® for 24 h. The resin

was then filtered through a 50-mL solid phase peptide synthesis vessel and washed with CH2Cl2 (3x20 mL). Resin capping was achieved by adding a 2.5% solution of MeOH (0.8 mL) and DIEA

(0.8 mL) in CH2Cl2 (30.4 mL), and the solid phase peptide synthesis vessel was rotated for 30 min

TM on a Fisherbrand nutator. The resin was then washed with CH2Cl2 (3x20 mL), dried on high vacuum to provide 4.61 g (3.73 mmol, 0.809 mmol/g) of S8 as a violet-colored resin in 62% yield.

S21 General Procedure for Quantification of Resin Loading: To three, 20-mL scintillation vials containing Fmoc-L-methionine-2-chlorotrityl resin S8 (10 mg) was added a solution of 30% piperidine in DMF (0.5 mL). The vials were capped, mixed, and allowed to stand for 30 min. Absolute ethanol (19.5 mL) was added to each of the three vials, which were then shaken, and the resin was allowed to settle for 5 min. The absorbance was measured at 300nm using a Thermo Scientific NanoDrop® ND-1000 spectrophotometer. The following equation was used to calculate the resin loading from absorbance: Resin Loading (mmol/g) = [3.05 x (absorbance average)/10 mg] x 10 The x10 factor is due to the small path length of the nanodrop; 3.05 was calculated on a 1.0 cm quartz cuvette.

Vial 1: abs300nm: 0.292 nm, 0.285 nm, 0.285 nm, 0.298 nm, 0.291 nm absavg: 0.290 nm 3.05x0.290/10.1 mg x10=0.876 mmol/g.

Vial 2: abs300nm: 0.227 nm, 0.234 nm, 0.238 nm, 0.224 nm, 0.221 nm absavg: 0.229 nm 3.05x0.229/10 mg x10=0.698 mmol/g.

Vial 3: abs300nm:0.290 nm, 0.272 nm, 0.273 nm, 0.282 nm, 0.281 nm absavg: 0.280 nm 3.05x0.280/10m g x10=0.854 mmol/g. Resin loading for Fmoc-L-methionine-2-chlorotrityl resin = 0.809 mmol/g (Loading Average)

DIEA (1.1 equiv), S CH Cl , Cl 2 2 Cl OH S r.t., 24 h Cl O O 61 % NH O Fmoc 4.0-gram scale Fmoc NH 1.0 – 2.0 mmol/g (0.5 equiv) meq=0.766 mmol/g 2-chlorotrityl chloride resin Fmoc-D-Met-OH Fmoc-D-Met-2-chlorotrityl resin S9 Fmoc-D-methionine-2-chlorotrityl resin S9 (4.76 g, 3.64 mmol, 0.766 mmol/g) was provided in 61% yield in the same manner described above for the preparation of Fmoc-L-methionine-2- chlorotrityl resin S8 from Fmoc-D-methionine (1.11 g, 3.00 mmol), 2-chlorotrityl chloride resin

(4.0 g, ~6.00 mmol, 1 – 2 mmol/g), DIEA (1.2 mL, 6.9 mmol), and CH2Cl2 (60 mL).

S22 DIEA (1.1 equiv), Trt S CH2Cl2, Cl OH Trt Cl r.t., 24 h S Cl O O 47% Fmoc NH 4.0-gram scale O Fmoc NH 1.0 – 2.0 mmol/g (0.5 equiv) 0.511mmol/g 2-chlorotrityl chloride resin Fmoc-L-Cys(Trt)-OH Fmoc-L-Cys(Trt)-2-chlorotrityl resin S10 Fmoc-L-Cys(Trt)-2-chlorotrityl resin S10 (5.48 g, 2.8 mmol, 0.511 mmol/g) was provided in 47% yield in the same manner described above for the preparation of Fmoc-L-methionine-2- chlorotrityl resin S8 from Fmoc-L-Cys(Trt)-OH (1.8 g, 3.0 mmol), 2-chlorotrityl chloride resin

(4.0 g, ~6.00 mmol, 1 – 2 mmol/g), DIEA (1.2 mL, 6.9 mmol), and CH2Cl2 (60 mL).

General Procedure for Linker Synthesis on 2-Chlorotrityl Resin (Gram-Scale):

Nitrogen flow was used to mix the beads (see picture above). When vacuum was applied to remove solvent from the vessel, the system was purged with argon using an argon balloon and a 24/40 septum that was fitted on top of the vessel to prevent undesired oxidation. For the following coupling reactions and Fmoc-deprotections described below, all stock solutions of 4- methylpiperidine, amino acids, HCTU, and DIEA were prepared in DMF directly before use.

S23 SPPS Cl couplings: S Amino Acid (6.0 equiv, 500 mM) HCTU (5.97 equiv, 500 mM) O DIEA (12.0 equiv, 500 mM) r.t., 1 h O O Cl H NH S Fmoc deprotections: Boc 20% 4-methylpiperidine Boc N H O N N O r.t., 10 min t-Bu O O 4.6-g scale O O Fmoc NH H NH HN N meq=0.809 mmol/g N Fmoc-L-Met-2-chlorotrityl resin Fmoc O O S8 t-Bu Fmoc-L-W(Boc)S(t-Bu)PK(Boc)Y(t-Bu)M-2-chlorotrityl resin S11

Fmoc-L-(W(Boc)S(t-Bu)PK(Boc)Y(t-Bu)M)-2-chlorotrityl resin S11: To a CHEMGLASS® 500-mL medium frit solid phase peptide synthesis vessel (see photo) was added Fmoc-L- methionine-2-chlorotrityl resin S8 (4.62 g, 3.73 mmol, 0.809 mmol/g). The resin was swelled for

5 min in CH2Cl2 (100 mL). For each Fmoc-deprotection, 20% 4-methylpiperidine was added, followed by nitrogen mixing for 10 min (2x100 mL). After each Fmoc-deprotection, the resin was

washed with DMF (4x100 mL, mixed with nitrogen for 3 min/wash) and CH2Cl2 (3 x 100 mL, allowed to sit for 3 min/wash without mixing). The coupling of each amino acid was accomplished by sequentially adding the following 500 mM stock solutions in DMF: amino acid (44.8 mL, 22.4 mmol, 6.0 equiv), HCTU (44.7 mL, 22.3 mmol, 5.98 equiv), followed by DIEA (89.6 mL, 44.8 mmol, 12.0 equiv). The reagents were mixed by applying steady nitrogen pressure for 1 h. In the case of serine coupling to proline’s secondary amine, the coupling procedure was repeated. [note: Analytical HPLC and LC/MS analysis of Fmoc-L-S(t-Bu)PK(Boc)Y(t-Bu)M-OH revealed that the coupling of Fmoc-L-S(t-Bu)-OH to proline’s secondary amine of the peptide PK(Boc)Y(t-Bu)M- 2-chlorotrityl resin was incomplete after a single coupling step]. After each coupling was

complete, the solvent was removed and the resin was washed with DMF (3x100 mL) and CH2Cl2 (3x100 mL). The reaction progress was evaluated after each step of the synthesis by removing a

small aliquot of the resin (~20 mg) and mixing with AcOH/TFE/CH2Cl2 (1:1:3) with 2.5% 1,2- ethanedithiol (EDT) for 15 minutes on a nutator. The filtrate was collected and concentrated under reduced pressure, followed by 1H-NMR and analytical HPLC analysis. The final coupling reaction provided 7.04 g of Fmoc-L-W(Boc)S(t-Bu)PK(Boc)Y(t-Bu)M-2-chlorotrityl resin S11, which can stored for more than a year at -20 oC without decomposition or methionine oxidation.

S24 Amino Acid Stocks (500 mM): Fmoc-Tyr(t-Bu)-OH (11.3 g) in DMF (49.3 mL, 500 mM), Fmoc-Lys(Boc)-OH (11.5 g) in DMF (49.3 mL, 500 mM), Fmoc-Pro-OH (8.31 g) in DMF (49.3 mL, 500 mM), Fmoc-Ser(tBu)-OH (9.45 g) in DMF (49.3 mL, 500 mM), Fmoc-Trp(Boc)-OH (13.0 g) in DMF (49.3 mL, 500 mM), HCTU Stock (500 mM): HCTU (10.2 g) in DMF (49.1 mL, 500 mM) DIEA Stock (500 mM): DIEA (8.6 mL) in DMF (90 mL)

SPPS Cl couplings: S Amino Acid (6.0 equiv, 500 mM) HCTU (5.97 equiv, 500 mM) O DIEA (12.0 equiv, 500 mM) r.t., 1 h O O Cl Fmoc deprotections: Boc H NH S Boc N H O 20% 4-methylpiperidine N N O r.t., 10 min t-Bu O O 2.0-g scale O O Fmoc NH H NH HN N N meq=0.766 mmol/g Fmoc O Fmoc-D-Met-2-chlorotrityl resin O t-Bu S9 Fmoc-D-W(Boc)S(t-Bu)PK(Boc)Y(t-Bu)M-2-chlorotrityl resin S12 Fmoc-D-W(Boc)S(t-Bu)PK(Boc)Y(t-Bu)M-2-chlorotrityl resin S12 (2.93 g) was afforded from Fmoc-D-methionine-2-chlorotrityl resin S9 (2.0 g, 1.53 mmol, 0.766 mmol/g) in the same manner described above for the preparation of Fmoc-L-W(Boc)S(t-Bu)PK(Boc)Y(t-Bu)M-2-chlorotrityl resin S11.

Boc SPPS N couplings: Amino Acid (6.0 equiv, 500 mM) Cl HCTU (5.97 equiv, 500 mM) Trt DIEA (12.0 equiv, 500 mM) S O r.t., 1 h HN O N Fmoc NH Boc Fmoc deprotections: O O 20% 4-methylpiperidine NH HN H Fmoc r.t., 10 min O N O t-Bu 0.511mmol/g O 5.19-g scale O Fmoc-L-Cys(Trt)-2-chlorotrityl resin S S10 Trt Cl

Fmoc-L-W(Boc)S(t-Bu)W(Boc)C(Trt)-2-chlorotrityl resin S13 Fmoc-L-W(Boc)S(t-Bu)W(Boc)C(Trt)-2-chlorotrityl resin S13 (5.53 g) was afforded from Fmoc-L-Cys(Trt)-2-chlorotrityl resin S10 (5.19 g, 4.68 mmol, 0.511 mmol/g) in the same manner described above for the preparation of Fmoc-L-W(Boc)S(t-Bu)PK(Boc)Y(t-Bu)M-2-chlorotrityl resin S11.

S25 C. Conjugate Synthesis (On Solid Phase): General Procedure for Siderophore Coupling and Resin Cleavage (detailed for compound 3):

1) 20% 4-MePip DMF, 2x10 min, r.t.

2) Ph O Ph O

O O O N H N O H Cl O 10 (2.0 equiv) S O Ph O Ph Ph S O HCTU (1.97 equiv), O OH DIEA (4 equiv), Ph O Boc O O DMF, r.t., 3 h NH H NH Boc O O N N H H NH Boc O 3) AcOH, TFE, EDT N Boc N H N DCM r.t., 15 min N O t-Bu t-Bu O O O O O Fmoc H NH O O NH N N H N N N H N Ph O O O H N O O Ph O O t-Bu t-Bu S11 3 Protected-Miller Siderophore-L-WSPKYM-OH Common Intermediate for Antibiotic Conjugation Synthesis of Protected-Miller Siderophore-L-WSPKYM-OH, compound 3: Step 1 (Fmoc-Deprotection): Fmoc-L-W(Boc)S(t-Bu)PK(Boc)Y(t-Bu)M-2-chlorotrityl resin S11 (470 mg) was Fmoc-deprotected by the procedure described in Section IIIB [General Procedure for Linker Synthesis on 2-Chlorotrityl Resin] to provide L-W(Boc)S(t-Bu)PK(Boc)Y(t- Bu)M-2-chlorotrityl resin (448.2 mg). The product resin was washed with DMF and swelled in

CH2Cl2, dried under high vacuum, and immediately used in Step 2 (vide infra).

Step 2 (Siderophore Coupling): To a 1-dram (3.7 mL) scintillation vial containing L-W(Boc)S(t- Bu)PK(Boc)Y(t-Bu)M-2-chlorotrityl resin (99 mg, 0.080 mmol, 0.809 mmol/g) was added the following solutions in DMF: siderophore component 10 (256 uL, 0.128 mmol, 500 mM in DMF), HCTU (253 uL, 0.126 mmol, 500 mM in DMF), and DIEA (512 uL, 0.256 mmol, 500 mM in DMF). The vial was capped, and the mixture was shaken vigorously on a Mistral Multi-Shaker® for 3 h. The resin was transferred via pipet (with DMF) to a 50-mL solid phase peptide synthesis vessel. The product resin was washed with DMF (3x6 mL) and CH2Cl2 (3x6 mL) and immediately used in the next step.

Step 3 (Resin Cleavage, Synthesis of Protected-Miller Siderophore-L-WSPKYM-OH, Compound 3): To the 50-mL solid phase peptide synthesis vessel containing the protected-Miller Siderophore-L-WSPKYM-2-chlorotrityl resin (0.080 mmol, isolated in Step 2) was added the

following cocktail: AcOH/TFE/CH2Cl2/EDT (2 mL/2 mL/6 mL/300 µL). The reaction vessel was then capped and mixed on a nutator for 15 min. The mixture was filtered, and the filtrate was

S26 concentrated under reduced pressure. The product was azeotropped with benzene (3x10 mL), sonicating for 10 seconds after adding benzene. [notes: Sonication of the product as a solution in benzene led to the rapid formation of a dry, white solid upon evaporation. Azeotroping with benzene was necessary to remove residual AcOH, which accelerates the oxidation of sulfides in air.S8 Exposure to oxygen was minimized by backfilling the rotary evaporator with argon when releasing the vacuum. [note: Compound 3 oxidizes slowly in air and will oxidize more rapidly in the presence of AcOH.] Compound 3 was afforded as a white solid in 50% yield over 13 steps (74.8 mg, 40.4 µmol). The product was sufficiently pure by analytical HPLC and NMR analysis, and was used in subsequent coupling reactions without further purification.

If further purification is needed in the event of oxidation, then purification by column

chromatography (silica gel, 2-16% MeOH/CH2Cl2) provided 62.1 mg (42% over 13 steps) of product 3 as a white solid. [note: The use of AcOH for column chromatography was avoided to minimize further oxidation of product. However, this resulted in significant product dragging with some product remaining on the column. In the case of acids 3 and S14-S16 it was preferred to take the crude material forward without further purification. Removal of any residual oxidized product could be purified following coupling to the antibiotic (vide infra).] Protected-Miller 1 Siderophore-L-WSPKYM-OH 3: Rf = 0.27 (10% MeOH/CH2Cl2); H NMR (400 MHz,

CD3OD) δ 8.12 – 7.74 (m, 1H), 7.70 – 6.45 (m, 35H), 4.73 – 3.78 (m, 7H), 3.78 – 3.35 (m, 5H), 3.27 – 2.04 (m, 11H), 2.03 – 1.76 (m, 5H), 1.76 – 1.46 (m, 12H), 1.37 (s, 13H), 1.29 – 1.12 (m, 13 12H), 1.12 – 0.97 (m, 12H). C NMR (100 MHz, CD3OD) δ 168.3, 164.4, 156.9, 153.9, 149.5, 147.5, 147.3, 144.8, 142.9, 139.6, 139.3, 139.2, 135.3, 130.4, 129.7, 129.4, 129.2, 129.1, 128.2, 128.2, 128.1, 126.1, 126.0, 125.8, 123.9, 122.0, 121.5, 120.3, 118.7, 118.1, 117.6, 117.3, 115.8, 115.0, 111.5, 109.6, 83.6, 78.4, 78.0, 73.8, 53.5, 27.9, 27.5, 27.1, 26.5. MALDI-TOF (reflectron + mode): m/z Calcd. for C103H122N10NaO20S [M-H ]:1849.86, Found 1849.16.

S27 Protected-Miller Siderophore-L-WSPKYM-OH; 3 (crude product) 680 580 480 380

Abs 280 180 80 -20 0 5 10 15 20 25 30 Time (min)

Protected-Miller Siderophore-L-WSPKYM-OH; 3 (purified product) 2480

1980

1480 Abs 980

480

-20 0 5 10 15 20 25 30 Time (min)

S28 1) 20% 4-MePip DMF, 2x10 min, r.t.

2) Ph O Ph O

O O O N H N O H Cl O 10 (2.0 equiv) S O Ph O Ph Ph S O HCTU (1.97 equiv), O OH DIEA (4 equiv), Ph O Boc O O DMF, r.t., 3 h NH H NH Boc O O N N H H NH Boc O 3) AcOH, TFE, EDT N Boc N H N DCM r.t., 15 min N O t-Bu t-Bu O O O O O Fmoc H NH O O NH N N H N N N H N Ph O O O H N O O Ph O O t-Bu t-Bu S12 S14 Protected-Miller Siderophore-D-WSPKYM-OH Common Intermediate for D-Linker Conjugation Protected Siderophore-D-WSPKYM-OH, compound S14 (61.2 mg, 47% over 13 steps) was provided by Fmoc-deprotection of S12 (0.070 mmol), coupling to the siderophore component 10, and cleavage from resin as described above in the General Procedure for Siderophore Coupling 1 and Resin Cleavage. S14: Rf = 0.44 (10% MeOH/CH2Cl2); H NMR (400 MHz, CD3OD) δ 8.39 – 6.47 (m, 35H), 4.72 – 4.49 (m, 3H), 4.44 – 3.74 (m, 3H), 3.73 – 3.39 (m, 5H), 3.27 – 2.75 (m, 9H), 2.71 – 2.39 (m, 2H), 2.33 – 1.77 (m, 9H), 1.77 – 1.50 (m, 12H), 1.50 – 1.20 (m, 26H), 1.16 – 0.88 (m, 10H). 13C NMR (100 MHz, MeOD) δ 173.3, 173.2, 172.8, 172.7, 172.6, 172.0, 171.8, 170.1, 168.9, 168.5, 164.3, 164.3, 157.0, 153.8, 149.6, 147.5, 147.2, 147.1, 145.0, 144.8, 142.9, 139.7, 139.3, 139.2, 129.6, 129.5, 129.2, 129.1, 128.3, 128.2, 128.0, 127.9, 126.1, 126.0, 125.8, 124.1, 123.8, 122.3, 122.0, 121.5, 120.3, 118.2, 117.6, 117.3, 115.8, 114.8, 111.5, 109.6, 83.6, 78.4, 78.0, 73.7, 62.3, 61.4, 60.6, 54.5, 54.5, 54.4, 54.1, 53.0, 51.4, 50.0, 48.6, 41.9, 39.8, 39.1, 38.7, 36.6, 31.4, 31.2, 30.9, 30.6, 29.8, 29.4, 29.2, 27.9, 27.5, 27.1, 26.7, 26.4, 26.3, 26.1, 25.3,

24.7, 24.1, 23.1, 13.9. MALDI-TOF (linear mode): m/z Calcd. for C103H122N10NaO20S + [M+Na ]:1873.85, Found 1873.88.

Protected Miller Siderophore -D-WSPKYM-OH; S14 680

580 (crude product) 480 380 280

Abs 180 80 -20 0 5 10 15 20 25 30 Time (min)

S29 1) 20% 4-MePip DMF, 2x10 min, r.t.

2) Ph O Ph O Boc O O O Boc N N H N O H N O 10 (2.0 equiv) O Ph Ph HCTU (1.97 equiv), HN O O DIEA (4 equiv), NH N HN Fmoc Boc DMF, r.t., 3 h O O HN NH N Boc HN H O O N O 3) AcOH, TFE, EDT O O HN H O N O t-Bu DCM r.t., 15 min O N O O Ph Ph O t-Bu O S OH O Trt Cl Ph S Trt O Ph S13 S15 Fmoc-L-W(Boc)S(t-Bu)W(Boc)C(Trt)-2-chlorotrityl resin Protected-Miller Siderophore-L-WSWC-OH Common Intermediate for Antibiotic Conjugation Protected-Miller Siderophore-L-WSWC-OH, compound S15 (26.4 mg, 36% over 9 steps) was provided by Fmoc deprotection of S13 (0.041 mmol), coupling to the siderophore component 10, and cleavage from resin as described above in the General Procedure for Siderophore Coupling 1 and Resin Cleavage. S15: Rf = 0.11 (5% MeOH/CH2Cl2); H NMR (400 MHz, CDCl3) δ 8.38 – 13 6.40 (m, 50H), 5.20 – 1.95 (m, 18H), 1.80 – 0.65 (m, 31H). C NMR (100 MHz, CDCl3) δ 149.6, 149.5, 149.5, 147.2, 146.9, 145.3, 144.7, 144.4, 144.3, 144.0, 143.0, 139.5, 139.0, 138.9, 135.5, 129.7, 129.5, 128.5, 128.3, 128.0, 127.9, 127.8, 127.7, 127.3, 126.8, 126.5, 126.4, 124.5, 122.7, 122.5, 122.2, 115.2, 115.1, 110.0, 100.0, 83.4, 74.0, 29.7, 28.2, 27.3, 27.1, 27.1, 27.1. MALDI- + TOF (linear mode): m/z Calcd. for C107H106N8NaO17S [M+Na ]:1829.73, Found 1829.49; m/z + Calcd. for C107H106KN8O17S [M+K ]:1845.70, Found 1845.76.

Protected-Miller Siderophore-L-WSWC-OH; conjugate S15 680

580

480

380

Abs 280

180

80

-20 0 5 10 15 20 25 30 Time (min)

S30 Cl S S O OH 1) Ac2O, pyridine O Boc O O Boc O H NH DMF, 30 min, r.t. H NH N N H N Boc N H Boc O N O N t-Bu 2) AcOH, TFE, EDT t-Bu DCM r.t., 15 min O O O O O NH H O NH Ac H N N N H2N N H N O 73% over 13 steps O O O t-Bu S11 t-Bu S16 Protected Ac-L-WSPKYM-OH Protected Ac-L-WSPKYM-OH, compound S16:

To a 50-mL solid phase peptide synthesis vessel containing the Fmoc-deprotected resin S11 (484

mg, 0.391 mmol, 0.809 mmol/g) was added CH2Cl2 (6 mL) to swell the resin for 2 min. The solvent removed, and DMF (11 mL), Ac2O (2.3 mL), and DIEA (2.3 mL) were added. The vessel was capped, and the mixture was rotated on a nutator for 15 min. The contents were drained, and the resin was resubjected to the reaction conditions and rotated for 15 min. Following solvent

removal, the was washed with DMF (3X6 mL), CH2Cl2 (1x6 mL), MeOH (3x6 mL), and CH2Cl2 (3x6 mL). The resin was dried under high vacuum and immediately used in the next step (resin cleavage). Cleavage from resin wash achieved as described above for Step 3 in the General Procedure for Siderophore Coupling and Resin Cleavage. After drying on high vacuum, the white solid was washed with hexane to remove residual ethanedithiol providing 335 mg (287 mmol, 73% yield) of the N-acetylated compound S16. The crude product was sufficiently pure by 1H NMR and LC analysis. [Use of the crude sample in subsequent steps showed no reduction in yield when compared to that of the purified sample (see below).]

If oxidation product was present as determined by LC/MS analysis, the product was further purified by column chromatography (silica gel, 2-14% MeOH/CH2Cl2; 20% MeOH/CH2Cl2) to provide 200 mg (44%). [note: The use of AcOH for column chromatography was avoided to minimize further oxidation of product. However, this resulted in significant product dragging with some product remaining on the column, which may explain the reduced yield upon chromatographic purification. In the case of acids 3 and S14-S16, it was preferred to take the crude material forward without further purification. Any undesired oxidized product could be removed following coupling to the antibiotic (vide infra).] S16: Rf = 0.35 (10% MeOH/CH2Cl2); 1 H NMR (400 MHz, CD3OD) δ 8.42 – 6.79 (m, 11H), 4.80 – 4.04 (m, 5H), 3.74 – 2.76 (m, 10H), 2.61 – 2.39 (m, 2H), 2.32 – 0.96 (m, 53H). 13C NMR (100 MHz, MeOD) δ 174.4, 173.0, 173.0,

S31 172.7, 172.3, 172.2, 171.8, 171.7, 171.5, 171.1, 170.1, 156.9, 153.9, 149.6, 135.3, 132.2, 132.1, 130.5, 130.4, 129.7, 129.6, 124.2, 124.1, 123.9, 123.8, 122.3, 118.8, 116.2, 116.2, 116.0, 114.8, 83.6, 83.4, 79.2, 78.4, 78.0, 73.7, 73.5, 62.4, 61.4, 60.9, 60.4, 54.5, 54.1, 53.7, 53.5, 53.1, 53.0, 52.1, 51.7, 46.8, 39.9, 36.6, 31.2, 29.8, 29.2, 29.0, 28.0, 27.6, 27.2, 26.4, 26.4, 24.8, 23.2, 23.0,

22.2, 21.4, 14.0. MALDI-TOF (Reflectron mode): m/z Calcd. for C59H88N8NaO14S [M+Na+]:1187.60, Found 1187.57.

Protected Ac-L-WSPKYM-OH; S16 (crude product)

980 880 780 680 580 480 Abs 380 280 180 80 -20 0 5 10 15 20 25 30 Time (min)

Protected Ac-L-WSPKYM-OH; S16 (purified product) 2480

1980

1480 Abs 980

480

-20 0 5 10 15 20 25 30 Time (min)

S32 D. Conjugate Synthesis (Coupling to Antibiotics):

O O N Boc N N O O Ph H2N H S N N F O O OH O S Ph 5 (2.0 equiv) Boc NH O O NH Boc O O EDC (1.5 equiv) HN H H H NH O N O N HOBt (2 equiv) O N N Boc N H H O O Ph NH O N N DIEA (3 equiv) Ph N O O N N t-Bu DMF, r.t., 4 h O H O O N N O O O N F O O H O N H O NH t-Bu O N N 55% O NH Ph t-Bu O O H N Ph O O O Ph O O Ph t-Bu 3 S17 Common Intermediate to L-Linker Conjugates Protected-Miller Siderophore-L-WSPKYM-Eperezolid General Procedure for Eperezolid-NH2 Coupling: Protected-Miller Siderophore-L-WSPKYM-Eperezolid, compound S17: To a 5-mL round- bottom flask was added the protected-Miller Siderophore-L-WSPKYM-OH 3 (20 mg, 0.011 mmol, 1.0 equiv), eperezolid-NH2 5 (8.5 mg, 0.022 mmol, 2.0 equiv), EDC (3.1 mg, 0.016 mmol,

1.45 equiv), and HOBt (4.1 mg, 0.022 mmol, 80 wt% in H2O, 2.0 equiv). DMF (0.11 mL) was then added, followed by DIEA (5.6 µL, 0.032 mmol. 2.91 equiv). The mixture was sonicated until all of the solid dissolved and then was stirred for 4 h. The mixture was concentrated under reduced pressure, and purification by column chromatography (silica gel, 1-10% MeOH/CH2Cl2) provided 1 13.3 mg (55%) of product S17 as a white solid. S17: Rf = 0.29 (5% MeOH/CH2Cl2); H NMR

(300 MHz, CD3OD) δ 8.22 – 6.34 (m, 38H), 4.79 – 3.39 (m, 24H), 3.21 – 2.86 (m, 11H), 2.26 – 1.84 (m, 11H), 1.73 – 1.52 (m, 11H), 1.49 – 0.97 (m, 37H). LC/MS: m/z Calcd. for 2+ + C121H146FN15O23S [M+2/2 ]:1114.5, Found 1114.6.

O O N N N O O Ph H2N H S N F O Ph OH (2.0 equiv) O O Boc NH Boc O O H NH EDC (1.5 equiv) N N N Boc H HOBt (2 equiv) O N O DIEA (3 equiv) Boc S t-Bu NH DMF, r.t., 4 h O HN H O O H O O O N O N N H NH O H N N N 88% NH N O Ph Ph Ph N O O N N O O H N O O H O O N O O N O Ph O O N F t-Bu H O t-Bu O O NH Ph t-Bu 3 O Ph S18 O Common Intermediate to L-Linker Conjugates Protected Ent-Eperezolid Conjugate (Inactive Antibiotic) Protected-Miller Siderophore-L-WSPKYM-ent-Eperezolid, compound S18 (20.2 mg, 88% yield) was provided from the coupling of the common intermediate 3 (19.0 mg, 10.3 µmol, 1.0

equiv) and ent-eperezolid-NH2 (8.07 mg, 20.5 µmol, 2.0 equiv) using the procedure described

1 above for the preparation of compound S17. S18: Rf = 0.15 (5% MeOH/CH2Cl2); H NMR (400

MHz, CD3OD) δ 8.13 – 6.38 (m, 38H), 4.83 – 3.39 (m, 24H), 3.25 – 2.82 (m, 12H), 2.63 – 1.51 13 (m, 27H), 1.51 – 0.98 (m, 34H). C NMR (100 MHz, CD3OD) δ 172.9, 172.8, 172.6, 172.1, 172.1,

S33 171.7, 167.2, 157.0, 155.1, 154.2, 151.8, 149.6, 147.5, 147.3, 147.1, 144.8, 139.9, 139.6, 139.3, 139.2, 129.6, 129.4, 129.2, 129.1, 128.2, 128.2, 128.0, 126.1, 126.0, 125.8, 124.2, 123.9, 122.3, 122.2, 122.0, 121.4, 119.4, 118.2, 117.6, 117.3, 115.8, 114.1, 111.5, 111.4, 109.6, 107.1, 106.9, 78.4, 78.1, 73.9, 73.7, 73.5, 72.1, 53.4, 53.0, 50.6, 50.2, 48.3, 44.6, 42.0, 41.9, 41.8, 40.7, 38.7, 36.5, 31.0, 29.7, 29.2, 27.9, 27.5, 27.1, 26.4, 26.3, 23.1, 21.1, 13.9. MALDI-TOF (linear mode): + m/z Calcd. for C121H144FKN15O23S [M+K ]:2264.99, Found 2265.16; m/z Calcd. for + C121H144FN15NaO23S [M+Na ]:2249.02, Found 2249.05.

Protected-Miller Siderophore-L-WSPKYM-ent-Eperezolid; S18

1380

1180

980

780

Abs 580

380

180

-20 0 5 10 15 20 25 30 Time (min)

O O N N N O H2N H N Boc F N 5 (2.0 equiv) O Boc S O Boc NH EDC (1.5 equiv) N O S O OH HOBt (2 equiv) Boc NH HN HN H O N DIEA (3 equiv) HN H H O O NH O N O O H DMF, r.t., 4 h O O N O N N N O O H Ph Ph N NH N O O O Ph Ph N O O N N O N H O 79% O O H O t-Bu O N O t-Bu O N N F O t-Bu H O Ph O O NH O Ph t-Bu Ph O O S14 Ph S19 Common Intermediate to D-Linker Conjugates Protected D-Linker Eperezolid Conjugate Protected-Miller Siderophore-D-WSPKYM-Eperezolid, compound S19 (18.1 mg, 79% yield) was provided from the coupling of protected-Miller Siderophore-D-WSPKYM-OH S14 (19.0 mg,

10.3 µmol, 1.0 equiv) and eperezolid-NH2 5 (8.07 mg, 20.5 µmol, 2.0 equiv) using the procedure described above for the preparation of protected conjugate S17. S19: Rf = 0.14 (5% 1 MeOH/CH2Cl2); H NMR (400 MHz, CD3OD) δ 8.10 – 6.37 (m, 38H), 4.80 – 4.70 (m, 1H), 4.64 – 4.50 (m, 3H), 4.42 – 3.37 (m, 20H), 3.24 – 2.80 (m, 12H), 2.68 – 2.34 (m, 2H), 2.22 – 1.78 (m, 12H), 1.73 – 1.49 (m, 13H), 1.49 – 1.19 (m, 25H), 1.15 – 0.97 (m, 9H). 13C NMR (100 MHz,

S34 MeOD) δ 191.7, 172.6, 167.2, 157.0, 156.6, 155.1, 154.2, 154.0, 147.5, 139.6, 139.2, 129.6, 129.4, 129.1, 128.2, 128.2, 128.0, 126.1, 126.0, 125.8, 123.9, 121.4, 119.5, 118.2, 117.6, 117.3, 115.8, 114.8, 114.0, 111.5, 109.6, 107.1, 83.6, 78.1, 73.7, 72.1, 63.0, 61.2, 54.3, 53.0, 50.6, 50.2, 41.8, 40.7, 40.0, 38.6, 36.5, 31.0, 29.7, 27.9, 27.5, 27.1, 26.4, 26.3, 21.1, 13.9. MALDI-TOF (linear + mode): m/z Calcd. for C121H144FKN15O23S [M+K ]:2264.99, Found 2264.87; m/z Calcd. for + C121H144FN15NaO23S [M+Na ]:2249.02, Found 2249.47.

Protected-Miller Siderophore-D-WSPKYM-Eperezolid; S19 780

680

580

480

380 Abs

280

180

80

-20 0 5 10 15 20 25 30 Time (min)

Boc Boc N O O N N N N O H2N H N F O O 5 (2.0 equiv) O HN HN O NH O N HN NH N Boc EDC (1.5 equiv) HN Boc O O O O HOBt (2 equiv) O O HN H O O N HN H O N O O N O N O F N O DIEA (3 equiv) Ph O H Ph Ph t-Bu Ph t-Bu O DMF, r.t., 3 h O N OH HN O O N N O S Ph S Ph O Trt 51% Trt O O O Ph Ph S15 S20 Common Intermediate for Antibiotic Conjugation Protected-Miller Siderophore-L-WSWC-Eperezolid Conjugate Protected-Miller Siderophore-L-WSWC-Eperezolid, compound S20 (11.7 mg, 51% yield) was prepared from S15 (19.0 mg, 10.5 µmol, 1.0 equiv) and eperezolid-NH2 5 (8.1 mg, 21 µmol, 2.0 equiv) by the procedure described above for the preparation of compound S17. However, in this case, the reaction was only stirred for 3 h, and purification by column chromatography (silica 1 gel, 0.5-5% MeOH/CH2Cl2) required a slower gradient. S20: Rf = 0.43 (10% MeOH/CH2Cl2); H

NMR (400 MHz, CDCl3) δ 8.13 – 7.27 (m, 31H), 7.25 – 6.54 (m, 19H), 5.25 – 2.54 (m, 33H), 2.04

S35 – 0.67 (m, 34H). MALDI-TOF (linear mode): m/z Calcd. for C125H128FKN13O20S + + [M+Na ]:2264.99, Found 2264.87; m/z Calcd. for C121H144FN15NaO23S [M+Na ]:2204.90, Found 2205.77.

Protected-Miller Siderophore-L-WSWC-Eperezolid; 2480 conjugate S20

1980

1480 Abs 980

480

-20 0 5 10 15 20 25 30 Time (min)

O O N H O N S O O N N N N N O S H2N H O OH N F F HN 5 (2.0 equiv) O Boc O O H NH Boc O O N Boc N H EDC (1.5 equiv) H NH N O HOBt (2 equiv) N Boc N H t-Bu N O DIEA (3 equiv) t-Bu O O H O NH DMF, r.t., 4 h O Ac O O N N Ac H NH H N N N O 94% H N O O O t-Bu S26 t-Bu S21 Protected Ac-L-WSPKYM-OH Protected Ac-L-WSPKYM-Eperezolid Conjugate Protected Ac-L-WSPKYM-Eperezolid, compound S21 (21.2 mg, 94% yield) was prepared from S16 (17.0 mg, 14.6 µmol, 1.0 equiv) and eperezolid-NH2 5 (11.5 mg, 29.2 µmol, 2.0 equiv) by the procedure described above for the preparation of compound S17. S21: Rf = 0.41 (10% 1 MeOH/CH2Cl2); H NMR (400 MHz, CD3OD) δ 8.47 – 7.73 (m, 5H), 7.74 – 6.73 (m, 12H), 4.85 – 3.96 (m, 10H), 3.83 – 3.45 (m, 11H), 3.29 – 2.86 (m, 10H), 2.21 – 1.78 (m, 15H), 1.77 – 1.24 13 (m, 33H), 1.17 (d, J = 2.7 Hz, 9H). C NMR (100 MHz, CD3OD) δ 173.1, 173.0, 172.6, 172.1, 171.7, 170.1, 167.2, 157.0, 156.6, 155.1, 154.2, 154.0, 149.6, 149.6, 135.8, 135.8, 135.3, 133.9, 133.8, 132.0, 131.9, 130.4, 129.7, 129.6, 127.0, 124.2, 124.1, 123.9, 122.3, 119.5, 118.8, 116.0,

S36 114.8, 114.0, 107.1, 106.9, 83.6, 78.4, 78.1, 73.7, 73.5, 72.1, 61.4, 60.9, 53.4, 53.1, 52.4, 50.6, 50.2, 44.6, 41.9, 41.8, 40.7, 39.8, 36.5, 31.1, 29.7, 29.2, 29.0, 27.9, 27.5, 27.1, 26.4, 26.3, 24.8,

22.9, 21.2, 21.1, 13.9. MALDI-TOF (Reflectron mode): m/z Calcd. for C77H110FN13NaO17S [M+Na+]:1562.77, Found 1562.48.

Ac-L-WSPKYM-Eperezolid, conjugate S21

2480

1980

1480

Abs 980

480

-20 0 5 10 15 20 25 30 Time (min)

N N

N NH2

O O H3C CH3 N OMe F O F N O O H3C O O H3C HO O Ph S H3C O O O Boc O O O O OH CH CH3 Boc O O O O Ph OH O O N OTFA N 3 OH CH CH F CH3 O N OTFA N 3 3 O H CH NH 6 (2.0 equiv) MeO O H 3 Boc O O O HN MeO O H NH O Boc N O HN N H NH H3C O Boc N Boc N H EDC (1.5 equiv) Ph CH O NH H C O Ph N S 3 Ph H 3 N HOBt (2 equiv) O + Ph N S CH3 O t-Bu N O O NH DIEA (4 equiv) O O O H N O O O H O NH O N H O NH O N O O H O H DMF, r.t., 4 h O N N O O N N N N O N N Ph O t-Bu N N H N N O H N O H O N t-Bu N N N O O O H O N Ph H O Ph O O O Ph O t-Bu Ph O Ph O t-Bu 3 S22A S22B t-Bu Common Intermediate to L-Linker Conjugates Protected-Miller Siderophore L-WSPKYM-Solithromycin Epimerized Protected-Miller Siderophore-L-WSPKYM-Solithromycin 16% (29% brsm) 12% (21% brsm) Protected-Miller Siderophore-L-WSPKYM-Solithromycin, Compound S22A: To a 5-mL round bottom flask containing compound 3 (26.9 mg, 15.0 µmol, 1.0 equiv) was added solithromycin 6 (24.5 mg, 29.0 µmol, 2.0 equiv), HOBt (5.6 mg, 29.0 µmol, 2.0 equiv), EDC (4.2 mg, 22.0 µmol, 1.50 equiv), DMF (0.15 mL), and DIEA (10.0 µL, 58.0 µmol, 3.0 equiv). The mixture was placed under argon and was stirred for 4 h. Dilution of the mixture with 70 %

CH3CN/H2O (5 mL), followed by purification by prep HPLC (C18 column, 5-60% CH3CN/H2O

with 0.1% TFA for 40 min, 60-80% CH3CN/H2O with 0.1% TFA for 30 min, and 80-95%

CH3CN/H2O with 0.1% TFA for 5 min; Note: A C18 column is preferred over a C4 column for separation of these compounds.) afforded product S22A (6.5 mg, 16% yield, 29% brsm) as a white

solid. [note: Product S22A elutes at ~70% CH3CN/H2O with 0.1% TFA]. The diastereomer S22B (4.8 mg, 12% yield, 21% brsm) was also isolated, which may have resulted from racemization during coupling, along with the recovered starting material 3 (12 mg, 45% yield).

S37 F O O S22A: MALDI-TOF (Linear mode): m/z Calcd. for Boc O O OH O O N OTFA N CH3 CH3 CH3 H + MeO O O HN 146 185 16 29 O Boc N C H FN NaO S [M+Na ]:2701.31, Found 2701.51; H NH H3C Ph N CH3 O Ph S O N + O NH O O H O H m/z Calcd. for C146H185FKN16O29S [M+K ]:2717.29, O O N O N N N t-Bu N N N O H O N H O Ph O Found 2717.42. Ph O t-Bu S22A Protected-Miller Siderophore L-WSPKYM-Solithromycin 16% (29% brsm) Protected-Miller Siderophore-L-WSPKYM-Solithromycin, conjugate S22A 480 380 280

Abs 180 80 -20 0 5 10 15 20 25 30 Time (min)

F O O S22B: MALDI-TOF (Linear mode): m/z Calcd. Boc O O OH O O N OTFA N CH3 CH3 CH3 H + MeO O O HN 146 185 16 29 O Boc N for C H FN NaO S [M+Na ]:2701.31, Found H NH H3C Ph N CH3 O Ph S O N O NH O O H O H 2701.35; m/z Calcd. for C146H185FKN16O29S O O N O N N N t-Bu N N N O H O N H O + Ph O [M+K ]:2717.29, Found 2717.03. Ph O S22B t-Bu Epimerized Protected-Miller Siderophore-L-WSPKYM-Solithromycin 12% (21% brsm) Epimerized Protected-Miller Siderophore-L-WSPKYM- Solithromycin; Conjugate S22B

980

780

580

Abs 380

180

-20 0 5 10 15 20 25 30 Time (min)

S38 N N

N NH2

O O H3C CH3 N O OMe F F N H3C O O O H3C HO O H3C O O O Boc Boc OTFA O O O Boc O O OH CH OTFA OH O O O O N N CH3 3 O N CH3 CH3 N CH N CH S F H 3 H 3 Boc MeO O MeO O O NH O HN O HN 6 (2.0 equiv) O Boc N O Boc N NH H C NH OH Ph H 3 H H3C O N CH3 O Ph N CH3 O HN HN H EDC (1.5 equiv) Ph S O Ph S O N N O O NH O N O HOBt (2 equiv) NH N O H O H O NH N O O O O H O O O H O H Ph Ph DIEA (4 equiv) O O N O O N N O N O N O O N H O O DMF, r.t., 3 h N N N N t-Bu N N H N t-Bu N N H N t-Bu t-Bu O O N O O N H O H O O Ph Ph Ph O O O Ph S23A O Ph S23B O Ph S14 t-Bu t-Bu Protected-Miller Siderophore-D-WSPKYM-Solithromycin Epimerized Protected-Miller Siderophore-D-WSPKYM-Solithromycin Common Intermediate to D-Linker Conjugates

13% yield (19%brsm) 12% yield (18% brsm) Protected-Miller Siderophore-D-WSPKYM-Solithromycin, compound S23A (5.2 mg, 13% yield, 19% brsm) was prepared from S14 (27 mg, 14.6 µmol, 1.0 equiv), solithromycin 6 (24.6 mg, 29.2 µmol, 2.0 equiv), HOBt (5.6 mg, 29.2 µmol, 2.0 equiv), EDC (4.2 mg, 21.9 µmol, 1.5 equiv), DMF (0.15 mL), and DIEA (10.0 µL, 58.3 µmol, 4.0 equiv) as described above for the preparation of solithromycin L-linker conjugate S22A. After 3 h of reaction time, the mixture was concentrated under reduced pressure, and the residue was dissolved in 70% CH3CN/H2O and

purified by prep HPLC (5 to 95% CH3CN/H2O with 0.1% TFA). [note: Product S23A elutes at

~70% CH3CN/H2O]. Product S23B (4.8 mg, 12% yield, 18% brsm) was also isolated, which may have resulted from the racemization of S23A, along with recovered starting material S24 (9.3 mg, 34% yield). F 1 O O S23A: H NMR (400 MHz, CD3OD) δ 9.70 – 9.47 (m, Boc OTFA O O OH O O N CH3 CH3 N CH H 3 MeO O O HN O Boc N 1H), 8.65 – 6.56 (m, 45H), 4.71 – 3.38 (m, 24H), 3.22 H NH H3C Ph N CH3 O Ph S O N O NH O O H O H – 2.72 (m, 15H), 2.68 – 2.30 (m, 5H), 2.29 – 0.59 (m, O O N O N N N t-Bu N N N O H O N H O Ph O 89H). MALDI-TOF (Linear mode): m/z Calcd. for Ph S23A O t-Bu + C146H185FN16NaO29S [M+Na ]:2701.31, Found Protected-Miller Siderophore-D-WSPKYM-Solithromycin

13% yield (19%brsm) 2701.55; m/z Calcd. for C146H185FKN16O29S [M+K+]:2717.29, Found 2717.88.

Protected-Miller Siderophore-D-WSPKYM-Solithromycin; 1380 conjugate S23A 1180 980 780

Abs 580 380 180 -20 0 5 10 15 20 25 30 Time (min)

S39

F 1 O O S23B: H NMR (400 MHz, CD3OD) δ 8.45 – 6.64 Boc O O OTFA OH O O N CH3 CH3 N CH H 3 MeO O O HN (m, 46H), 4.71 – 3.83 (m, 4H), 3.78 – 3.33 (m, 7H), O Boc N H NH H3C Ph N CH3 O Ph S O N O NH O O H O H 3.23 – 1.79 (m, 32H), 1.79 – 0.65 (m, 89H). O O N O N N N t-Bu N N N O H O N H O Ph O MALDI-TOF (Linear mode): m/z Calcd. for Ph S23B O t-Bu + C146H185FN16NaO29S [M+Na ]:2701.31, Found Epimerized Protected-Miller Siderophore-D-WSPKYM-Solithromycin 12% yield (18% brsm) 2701.78; m/z Calcd. for C146H185FKN16O29S [M+K+]:2717.29, Found 2717.42.

Epimerized Protected-Miller Siderophore-D-WSPKYM- Solithromycin, conjugate S23B 580 480 380 280 Abs 180 80 -20 0 5 10 15 20 25 30 Time (min)

S40 Method A

1) O Cl O (1.1 equiv)

DIEA (1.1 equiv) THF, 0 oC, 1 h Boc O OH N 2) H2N O O H H H O N N N N Boc H O O NH O Ph HN HO2C OH O O O HN O H H O O NH CO H N N Ph O 2 N N CO H H O O NH 2 O O S O HN O O O H H H2NOC H N N O N O N HN H t-Bu N N H O O 4 H N N O H O O (5 equiv) NH Ph S NH2 O O H O o O N N DIEA (50 equiv), DMF, 0 C to r.t., 5 h H O Ph OH O O N HN O Ph H O O NH O t-Bu HO C Boc O 33% over 2 steps NH 2 H NH O O N N H Ph H HO2C NH Boc O O N N CONH2 HO t-Bu H O N N O O H O O O H O N N O O NH H N H N CO H N N H O 2 N O Ph H O O H N NH O O Ph O O O O S24 HO t-Bu Method B 3 NH2 Common Intermediate to L-Linker Conjugates 1) O Cl O (1.1 equiv) Protected-Miller Siderophore-L-WSPKYM-Daptomycin

DIEA (1.1 equiv) THF, 0 oC, 1 h

O OH 2) H2N O O H H O N N N H O O HO C HN OH O O 2 O H H HN CO H N N O 2 N H NH CO2H O O HN O O H H O H2NOC N N N HN H O O 4 (5 equiv) NH2 o NaHCO3 (16 equiv), DMF, 0 C to r.t., 2 h

30% over 2 steps Protected-Miller Siderophore-L-WSPKYM-Daptomycin, Compound S24, Method A: To a flame-dried 5-mL round-bottom flask was added compound 3 (9.3 mg, 5.0 µmol, 1.0 equiv). The flask was purged with argon, and THF (0.1 mL) was added. The solution was cooled to 0 oC, and a 1 M solution of DIEA in THF (6 µL, 6 µmol, 1.2 equiv) was added, followed by a 1 M solution of isobutyl chloroformate in THF (6uL, 6 µmol, 1.2 equiv). The mixture was stirred at 0 oC for 1 h. The solution containing the resulting mixed anhydride product of compound 3 was transferred into a suspension of daptomycin (vide infra). To a 5-mL flame-dried, round-bottom flask was added daptomycin (40.7 mg, 25.1 µmol, 5.02 equiv). The flask was purged with argon, and DIEA (44 µL, 0.251 mmol, 50.2 equiv) was added, followed by DMF (0.1 mL) to form a suspension. The suspension was cooled to 0 oC in a dry ice/acetone bath. The solution of the 3-derived mixed anhydride in THF was then added via syringe to the suspension of daptomycin over the course of 5 min. Additional DMF (0.2 mL) was used for transfer. The mixture went from suspension to clear solution 5 min after the mixed anhydride had been added. The mixture was stirred for 5 h and was warmed slowly to 23 ºC in the ice-water bath. The mixture was diluted with 3:1

DMSO/H2O and purified by HPLC (5 to 95% CH3CN/H2O with 0.1% TFA). [note: The product

eluted at ~80% CH3CN/H2O]. The product S24 (5.7 mg, 33% yield) was provided as a white solid

S41 following lyophilization. [note: The same yield was accomplished when allowing the mixture to stir for 18 h at 4 oC]. Protected-Miller Siderophore-L-WSPKYM-Daptomycin, Compound S24, Method B: The 3- derived mixed anhydride (2.9 µmol) was prepared from compound 3 (5.4 mg, 2.9 µmol, 1 equiv), a 1 M solution of DIEA in THF (4.0 µL, 4.0 µmol, 1.38 equiv), and a 1 M solution of isobutylchloroformate in THF (4.0 µL, 4.0 µmol, 1.38 equiv) by the procedure described above in

Method A. A 160 mM stock solution of sodium bicarbonate (39 mg) in MilliQ H2O (3 mL) was prepared. To a 5-mL round-bottom flask containing daptomycin 4 (14.2 mg, 8.76 µmol, 3 equiv)

under argon was added an aqueous solution of NaHCO3 (0.29 mL, 160 mM, 16 equiv). The solution was cooled to 0 oC, and the S3-derived mixed anhydride was then added dropwise as a solution in THF (0.1 mL) over the course of 2 min. Additional THF (0.2 mL) was used for the transfer. The ice bath was removed, and the reaction was stirred at 23 ºC for 2 h. The mixture

was concentrated under reduced pressure and purified by HPLC (5 to 95% CH3CN/H2O with 0.1%

TFA). [note: The product eluted at 80% CH3CN/H2O with 0.1%TFA]. The protected conjugate S24 (3.0 mg) was isolated in 30% yield. S24: MALDI-TOF (Linear Mode): m/z Calcd. for

+ + C175H221N27NaO45S [M+Na ]:3475.55, Found 3474.75. LC/MS (positive mode): m/z Calcd. for 3+ + C175H224N27O45S [M+3H /3]:1152.2, Found 1152.8. Analytical HPLC Conditions: 2-50%

CH3CN/H2O (2 min); 50-90% CH3CN/H2O (8 min); 90-95% CH3CN/H2O (10 min); 95%

CH3CN/H2O (5 min); 2% CH3CN/H2O (5 min).

Protected-Miller Siderophore-L-WSPKYM-Daptomycin; S24 Abs

0 5 10 15 20 25 30 Time (min)

S42 Boc N O 1) H N Boc O Cl O (1.1 equiv) Ph S O NH Ph O O OH DIEA (1.1 equiv) Ph O O NH Ph N O THF, 0 oC, 45 min O O NH Boc O O O S H NH H N N O N O Boc H t-Bu O O N N H N 2) OH H2N t-Bu H N N O NH O H O O O H O O O NH H O O H O N H O N N O N N N N O H Ph N H O H O HO2C HN O N HN OH O O O O O N O H H HN Ph H O O CO H N N t-Bu HO C Ph O O O 2 N NH 2 CO H H O O t-Bu NH 2 O O Ph H HO C NH O HN O 2 O H H H2NOC N CONH O N N 2 HO N HN H H O N N O O H O O O 4 H O N N S14 H N CO H NH (5 equiv) H O 2 N O Common Intermediate to D-Linker Conjugates 2 H DIEA (22.5 equiv), DMF, r.t., 5 h NH O O O HO NH2 S25 Protected-Miller Siderophore-D-WSPKYM-Daptomycin Protected-Miller Siderophore-D-WSPKYM-Daptomycin, Compound S25: To a flame-dried, 5-mL round-bottom flask containing common intermediate S14 (12.0 mg, 6.48 µmol, 1 equiv) under argon was added THF (0.13 mL). The solution was cooled to 0 oC and a 1 M stock solution of DIEA in THF (7.13 µL, 7.13 µmol, 1.1 equiv) was added, followed by the addition of a 1 M stock solution of isobutyl chloroformate in THF (7.13 µL, 7.13 µmol, 1.1 equiv). The mixture was stirred at 0 oC for 1 h. Before the mixed anhydride formation was complete, daptomycin 4 (54.0 mg, 33.3 µmol, 5.14 equiv) was added to a separate flame-dried, 5-mL round-bottom flask. The flask was purged with argon, and DIEA (29 µL, 0.166 mmol, 22.5 equiv) was added, followed by DMF (0.1 mL) to form a suspension. After the reaction was complete, the solution of the S14- derived mixed anhydride (6.48 µmol) was transferred in THF (0.1 mL) to the suspension of daptomycin in DMF via syringe over the course of 1 min. Additional THF (0.1 mL) was used to transfer remaining mixed anhydride. The mixture went from suspension to clear solution over the course of 3h. The mixture was stirred for a total of 5 h at 23 ºC before purification by HPLC (5 to

95%CH3CN/H2O with 0.1%TFA) [Note: The product elutes at ~80% CH3CN/H2O]. The product S25 was isolated in 17% yield (3.9 mg) as a white solid following lyophilization. S25: 1H NMR

(400 MHz, CD3OD) δ 8.21 – 6.36 (m, 60H), 4.78 – 3.38 (m, 37H), 3.20 – 2.28 (m, 33H), 2.28 – 3+ + 0.50 (m, 92H). HRMS: m/z Calcd. for C175H224N27O45S [M+3H /3]:1152.1936, Found

4+ + 1152.2644. m/z Calcd. for C175H225N27O45S [M+4H /4]:864.3970, Found 864.3387.

Protected-Miller Siderophore-D-WSPKYM-Daptomycin; S25 280 180

Abs 80 -20 0 5 10 15 20 25 30 Time (min)

S43 E. Conjugate Synthesis (Global Deprotection to Final Conjugate):

General Procedure for Global Deprotection: TFA Source: (Aldrich ReagentPlus 99%) [note: Depending on the source of TFA, due to the presence of trace metals, distillation may be necessary to avoid excessive sulfide oxidation during global deprotections of these conjugates. TFA (Aldrich ReagentPlus 99%) did not lead to excessive sulfide oxidation under an argon atmosphere and did not require distillation.] TFA (4 mL) was added to a 20-mL scintillation vial under argon. The TFA was degassed by passing a stream of argon through a thin needle (22 G) into the liquid for 10 min. To a separate, 20-mL scintillation vial under argon was added EDT (50 µL), triisopropylsilane

(TIPS-H, 25 µL), H2O (50 µL), and degassed TFA (1.88 mL). To a separate, 20-mL scintillation vial containing the protected conjugate under argon was added the deprotection cocktail

(EDT/TIPS-H/H2O/TFA) via syringe. (note: The syringe was flushed with argon 3x prior to the transfer.) The mixture was stirred at 23 ºC for 1.5–2 h. The mixture was then concentrated under reduced pressure and azeotropped with benzene (3x2 mL), sonicating for 5 seconds after adding benzene. [note: Sonication in benzene led to the rapid formation of a dry, white solid upon evaporation. Azeotroping with benzene was necessary to remove residual TFA, which accelerates the oxidation of the conjugates in air. Exposure to oxygen was minimized by backfilling the rotary evaporator with argon when releasing the vacuum].

O Ph S OH O Ph OH S O OH OH NH Boc O O H NH TFA, O OTFA N Boc N H NH O O O H2O, TIPS–H, EDT H NH N N t-Bu 2 h, r.t. H3N H N OH O O O N H O NH 85% N N O O Ph O N O NH O O H N H O N N HO Ph O O O H N O t-Bu HO HO

3 11 Common Intermediate to D-Linker Conjugates Miller Siderophore WSPKYM Miller Siderophore L-Linker Conjugate (no antibiotic) Miller Siderophore L-Linker Conjugate (no antibiotic), Conjugate 11: Conjugate 11 (6.9 mg, 85% yield) was provided from compound 3 (11.4 mg, 6.15 µmol) by the General Procedure for Global Deprotection described above. Purification of conjugate 11 was accomplished by a wash strategy. The crude, solid product was washed with ether (3x2 mL), sonicated, and filtered through

S44 a cotton plug. The ether washes, which only contained impurities, were discarded. The crude product was then washed with H2O (3x2 mL), 10% CH3CN/H2O (2x1 mL), and 40% CH3CN/H2O (4x1 mL). Each solvent system was filtered through a cotton plug into a separate flask. The washes were concentrated under reduced pressure and analyzed by analytical HPLC and LC/MS. From the

40% CH3CN/H2O was isolated 6.9 mg (85%) of conjugate 11 as a white solid. An additional 2.9

mg (impure) of 11 was isolated from the 10% CH3CN/H2O wash. 11: MALDI-TOF (Reflectron + mode): m/z Calcd. for C59H74N10O16S [M+H ]:1211.50, Found 1211.60.

Miller Siderophore-L-WSPKYM-OH (no antibiotic); conjugate 11 1780

1580

1380

1180

980

Abs 780

580

380

180

-20 0 5 10 15 20 25 30 Time (min)

Boc N H N OTFA O Boc S O NH S O H3N HN H H TFA, O HN H O O N O N N H H H2O, TIPS–H, EDT HO OH N O N NH N O H N Ph Ph N O O N N 2 h, r.t. NH N O O O H O O O N N O O N O N N O H O O N F O N O H O 96% HO N N F t-Bu O H O O NH OH Ph t-Bu OH NH O O OH Ph O S17 8

Protected-Miller Siderophore-L-WSPKYM-eperezolid Miller Siderophore WSPKYM eperezolid Miller Siderophore-L-WSPKYM-eperezolid Miller Siderophore-L-WSPKYM-eperezolid, Conjugate 8: Conjugate 8 (9.7 mg, 96% yield) was provided from compound S17 (13.3 mg, 5.97 µmol) by the General Procedure for Global Deprotection described above. Purification of conjugate 7 was accomplished by a wash strategy.

S45 The crude, solid product was washed with ether (3x1 mL), sonicated, and filtered through a cotton plug. The ether washes, which only contained impurities, were discarded. The crude product was

then washed with H2O (3x1 mL), 40% CH3CN/H2O (2x1 mL), and 80% CH3CN/H2O (2x1 mL).

Each solvent system was filtered through a cotton plug into a separate flask. The 40% CH3CN/H2O and 80% CH3CN/H2O washes provided 8.6 mg (96%) of eperezolid conjugate 8 as a white solid. 8: + MALDI-TOF (Reflectron mode): m/z Calcd. for C77H96FN15O19S [M+H ]:1586.67, Found + 1586.83.; m/z Calcd. for C77H96FN15NaO19S [M+Na ]:1608.66, Found 1608.86.

Miller Siderophore-L-WSPKYM-Eperezolid, conjugate 8 480 430 380 330 280 230 Abs 180 130 80 30 -20 0 5 10 15 20 25 30

Time (min)

Boc H N N OTFA O O Boc S S NH TFA, H3N O O HN H H H O, TIPS–H, EDT HN H H 2 N O O N O N N HO OH O N N H 2 h, r.t. H O NH N O NH O N Ph Ph N O O N N N O O N N O O H O O H O O N 77% O N O O N O HO N N F N H O F H O t-Bu O OH O NH OH NH Ph t-Bu OH O O Ph O S18 15 Miller Siderophore Protected-Miller Siderophore-L-WSPKYM-ent-eperezolid WSPKYM ent-eperezolid

Miller Siderophore-L-WSPKYM-ent-eperezolid (inactive antibiotic) Miller Siderophore-L-WSPKYM-ent-eperezolid (inactive antibiotic), Conjugate 15: Conjugate 15 (11.8 mg, 77% yield) was provided from compound S18 (20.2 mg, 9.07 µmol) by the General Procedure for Global Deprotection described above. Purification of conjugate 15 was accomplished by a wash strategy. The crude, solid product was washed with ether (3x1 mL),

S46 sonicated, and filtered through a cotton plug. The ether washes, which only contained impurities,

were discarded. The crude product was then washed with H2O (3x1 mL), 40% CH3CN/H2O (2x1

mL), and 80% CH3CN/H2O (2x1 mL). Each solvent system was filtered through a cotton plug into a separate flask. The 40% CH3CN/H2O and 80% CH3CN/H2O washes provided 11.8 mg (77%) of ent-eperezolid conjugate 15 as a white solid. 15: MALDI-TOF (Reflectron mode): m/z Calcd. + for C77H96FN15O19S [M+H ]:1586.67, Found 1586.86.; m/z Calcd. for C77H96FN15NaO19S [M+Na+]:1608.66, Found 1608.86.

Miller Siderophore-L-WSPKYM-ent-Eperezolid; Conugate 15 1980

1780

1580

1380

1180

980 Abs 780

580

380

180

-20 0 5 10 15 20 25 30 Time (min)

Boc H N N OTFA O O Boc S S NH H3N O O HN H H HN H H N O O N O N N TFA, HO OH O N N H H O NH N O H O, TIPS–H, EDT NH O N Ph Ph N O O N N 2 N O O N N O O H O O H O O N 2 h, r.t. O N O O N O HO N N F N H O F H O t-Bu O 69% OH O NH OH NH Ph t-Bu OH O O Ph O S19 14 Protected-Miller Siderophore-D-WSPKYM-eperezolid Miller Siderophore D-WSPKYM eperezolid

Miller Siderophore-D-WSPKYM-eperezolid Miller Siderophore-D-WSPKYM-eperezolid, Conjugate 14: Conjugate 14 (9.5 mg, 69% yield) was provided from compound S19 (18.1 mg, 8.13 µmol) by the General Procedure for Global Deprotection described above. Purification of conjugate 14 was accomplished by a wash strategy.

S47 The crude, solid product was washed with ether (3x1 mL), sonicated, and filtered through a cotton plug. The ether washes, which only contained impurities, were discarded. The crude product was

then washed with H2O (3x1 mL), 50% CH3CN/H2O (2x1 mL), and 80% CH3CN/H2O (2x1 mL).

Each solvent system was filtered through a cotton plug into a separate flask. The 50% CH3CN/H2O

and 80% CH3CN/H2O washes provided 9.5 mg (69%) of D-linker conjugate 14 as a white solid. + 14: MALDI-TOF (Reflectron mode): m/z Calcd. for C77H96FN15O19S [M+H ]: 1586.67, Found + 1586.73.; m/z Calcd. for C77H96FN15NaO19S [M+Na ]: 1608.66, Found 1608.75.

Miller Siderophore-D-WSPKYM-Eperezolid; conjugate 14 680

580

480

380 Abs 280

180

80

-20 0 5 10 15 20 25 30 Time (min)

Boc H N N

O O HN TFA, HN NH O N NH O NH HN Boc O H2O, TIPS–H, EDT HN O O O O O HN H 2 h, r.t. OH O HN H O N O HO N HO O N O F N O N O F N Ph Ph H H t-Bu O O HN N O 66% HN N O O N N OH N N Ph S HS Trt O O O O O OH Ph

S20 S26

Protected-Miller Siderophore-L-WSWC-Eperezolid Conjugate Miller Siderophore L-WSWC eperezolid Miller Siderophore-L-WSWC-Eperezolid Miller Siderophore-L-WSWC-eperezolid, Conjugate S26: Conjugate S26 (3.3 mg, 66% yield) was provided from compound S20 (8.1 mg, 3.7 µmol) by the General Procedure for Global Deprotection described above. Purification of conjugate S26 was accomplished by a wash strategy

S48 followed by HPLC. The crude, solid product was washed with ether (3x2 mL), sonicated, and filtered through a cotton plug. The ether washes, which only contained impurities, were discarded.

The crude product was then washed with 20% CH3CN/H2O (1x1 mL), 65% CH3CN/H2O (2x1

mL), 80% CH3CN/H2O (1x2 mL), and DMSO (1x 0.5 mL). Each solvent system was filtered through a cotton plug into a separate flask. The majority of the product was isolated in the DMSO wash with a minor impurity (~5:1 as determined by analytical HPLC analysis). Further purification by prep HPLC (5 to 95% CH3CN/H2O with 0.1 % TFA, C4 column) provided 3.3 mg – (66%) of S26 as a white solid. S26: HRMS: m/z Calcd. for C66H74FN13O16S [M-2H ]:1353.4936, Found 1353.4269.

Miller Siderophore-L-WSWC-Eperezolid; Conjugate S26 580

480

380

280 Abs

180

80

-20 0 5 10 15 20 25 30 Time (min)

S49 F F O O O O Boc OTFA O O OH O OTFA O O O CH3 CH3 H OH O N N O CH3 CH3 CH3 N N H CH3 MeO O H O HN OTFA MeO O Boc N HO HN O NH TFA, OH N H H3C NH H C Ph N CH3 O H 3 3 Ph S H O, TIPS–H, EDT N CH3 O O 2 S O N 2 h, r.t. N O NH NH O O H O H O H O O N O O H O O N O O N N N 84% HO N N t-Bu N N N N O H O N N N H N H O OH O N H OTFA Ph H O O OH Ph S22A O 9 OH t-Bu Protected-Miller Siderophore-L-WSPKYM-Solithromycin Miller Siderophore WSPKYM solithromycin

Miller Siderophore-L-WSPKYM-Solithromycin Miller Siderophore-L-WSPKYM-Solithromycin, Conjugate 9: Conjugate 9 was provided from compound S22A (3.6 mg, 1.3 µmol) by the General Procedure for Global Deprotection described above. Purification of conjugate 9 was accomplished by a wash strategy. The crude, solid product was washed with ether (3x2 mL), sonicated, and filtered through a cotton plug. The ether washes, which only contained impurities, were discarded. The crude product was then washed with H2O (1x0.5 mL), 10% CH3CN/H2O (2x1 mL), 40% CH3CN/H2O (2x1 mL), and DMSO (1x0.5 mL). Each solvent system was filtered through a cotton plug into a separate flask.

The 40% CH3CN/H2O wash provided 2.7 mg (84%) of conjugate 9 as a white solid following lyophilization. Before testing in bacteria, the compound was purified by HPLC (5 to 95%

CH3CN/H2O with 0.1 % TFA, C4 column) to remove trace oxidized product providing 2.1 mg (66%) of conjugate 9 before testing in bacteria. 9: MALDI-TOF (Reflectron mode): m/z Calcd.

+ for C102H137FN16O25S [M+H ]:2037.96, Found 2038.57.

Miller Siderophore-L-WSPKYM-Solithromycin, conjugate 9 235

185

135 Abs 85

35

-15 0 5 10 15 20 25 30 Time (min)

S50 F F O O O O Boc OTFA O O OH O OTFA O O O O N CH3 CH3 H OH CH CH N CH O N N 3 3 H 3 CH3 MeO O OTFA H O HN MeO O O Boc N TFA, HO HN NH H C OH N H 3 H NH3 H C Ph CH3 O H O, TIPS–H, EDT 3 Ph N S 2 N CH3 O O S O N 2 h, r.t. NH N O H O NH O O H O O O O H O O N O H O N O N N N 96% HO N N t-Bu N N N N O H O N N N H N H O OH H O N H Ph O O OH Ph O OH OTFA S22B t-Bu S27A Protected-Miller Siderophore-L-WSPKYM-Solithromycin Epimerized Miller Siderophore-L-WSPKYM-Solithromycin Epimerized Azotochelin-L-WSPKYM-Solithromycin, Conjugate S27A: Conjugate S27A (0.6 mg, 96% yield) was provided from compound S22B (0.7 mg, 0.26 µmol) by the General Procedure for Global Deprotection described above. Purification of conjugate S27A was accomplished by a wash strategy. The crude, solid product was washed with ether (3x2 mL), sonicated, and filtered through a cotton plug. The ether washes, which only contained impurities,

were discarded. The crude product was then washed with H2O (1x0.5mL), 10% CH3CN/H2O (2x1

mL), 40% CH3CN/H2O (2x1 mL), 80% CH3CN/H2O (2x1 mL), and DMSO (1 x 0.5 mL). Each solvent system was filtered through a cotton plug into a separate flask. The DMSO wash provided 0.6 mg (96%) of conjugate S27A as a white solid following concentration under reduced pressure. + S31A: MALDI-TOF (Reflectron mode): m/z Calcd. for C102H137FN16O25S [M+H ]:2037.96, + Found 2038.29. m/z Calcd. for C102H137FN16NaO25S [M+Na ]:2059.95, Found 2060.39.

Epimerized Miller Siderophore-L-WSPKYM-Solithromycin; S27A 430

380

330

280

230

Abs 180

130

80

30

-20 0 5 10 15 20 25 30 Time (min)

S51 F F O O O O Boc OTFA O O OH O OTFA O O O N N CH3 CH3 H OH O CH O N CH3 CH3 H 3 N CH MeO O TFA, H 3 O HN MeO O O Boc N HO HN OTFA NH H C H2O, TIPS–H, EDT N Ph H 3 OH NH Ph N S CH3 O 2 h, r.t. H 3 H3C O N S CH3 O N O O NH N O O H O H 95% O NH O O N O O H O H O N N O O N N HO N N t-Bu N N H N N O O N N N N H O OH H O N Ph H O O O OH Ph OH t-Bu S23A 16 Protected-Miller Siderophore-D-WSPKYM-Solithromycin Miller Siderophore D-WSPKYM solithromycin

Miller Siderophore-D-WSPKYM-Solithromycin Miller Siderophore-D-WSPKYM-Solithromycin, Conjugate 16: D-Linker conjugate 16 (4.2 mg, 95% yield) was provided from compound S23A (5.2 mg, 1.9 µmol) by the General Procedure for Global Deprotection described above. Purification of conjugate 16 was accomplished by a wash strategy. The crude, solid product was washed with ether (3x2 mL), sonicated, and filtered through a cotton plug. The ether washes, which only contained impurities, were discarded. The crude product was then washed with H2O (1x0.5 mL), 10% CH3CN/H2O (2x1 mL), 40%

CH3CN/H2O (2x1 mL), 80% CH3CN/H2O (2x1 mL), and DMSO (1 x 0.5 mL). Each solvent system was filtered through a cotton plug into a separate flask. The CH3CN/H2O washes provided 4.2 mg (95%) of conjugate 16 as a white solid following concentration under reduced pressure.

The product was purified by HPLC (5 to 95% CH3CN/H2O with 0.1 % TFA, C4 column) to remove trace oxidized product providing 4.3 mg (97%) of conjugate 16 for testing in bacteria. [It should be noted that the amount of product isolated from prep HPLC was roughly identical in quantity and purity to that isolated from the wash method.] 16: MALDI-TOF (Reflectron mode): m/z + Calcd. for C102H137FN16O25S [M+H ]:2037.96, Found 2038.07. m/z Calcd. for + C102H137FN16NaO25S [M+Na ]:2059.95, Found 2060.14.

Miller Siderophore-D-WSPKYM-Solithromycin; conjugate 16 340 290 240 190

Abs 140 90 40 -10 0 5 10 15 20 25 30 Time (min)

S52 F F O O O O Boc OTFA O O O OTFA O O OH CH CH H OH O O N N 3 3 O N N CH3 CH3 CH3 CH H OTFA H 3 MeO O MeO O O HN HO HN O Boc N TFA, OH N H NH H3C NH H C Ph CH H O, TIPS–H, EDT H 3 3 Ph N S 3 O 2 N CH3 O O S O N 2 h, r.t. N O NH O NH O O H O H O O H O H O O N O O N O N N HO N N 96% N N t-Bu N N H N N N OTFA O O N N H O H O OH H N H Ph O O OH Ph O OH t-Bu S23B S27B Epimerized Protected-Miller Siderophore-D-WSPKYM-Solithromycin Epimerized Miller Siderophore-D-WSPKYM-Solithromycin Epimerized Miller Siderophore-D-WSPKYM-Solithromycin, Conjugate S27B: Conjugate S31B (4.1 mg, 96% yield) was provided from compound S23B (4.8 mg, 1.8 µmol) by the General Procedure for Global Deprotection described above. Purification of conjugate S27B was accomplished by a wash strategy. The crude, solid product was washed with ether (3x2 mL), sonicated, and filtered through a cotton plug. The ether washes, which only contained impurities,

were discarded. The crude product was then washed with H2O (1x0.5 mL), 10% CH3CN/H2O

(2x1 mL), 40% CH3CN/H2O (2x1 mL), 80% CH3CN/H2O (2x1 mL), and DMSO (1 x 0.5 mL).

Each solvent system was filtered through a cotton plug into a separate flask. The CH3CN/H2O washes provided 4.1 mg (96%) of conjugate S27B as a white solid following concentration under reduced pressure. The product was purified by HPLC to remove trace oxidized product providing 4.1 mg (96%) of conjugate S27B for testing in bacteria. [note: The amount of product isolated from prep HPLC was roughly identical in quantity and purity to that isolated from the wash purification method]. S27B: MALDI-TOF (Reflectron mode): m/z Calcd. for + C102H137FN16O25S [M+H ]:2037.96, Found 2038.24. m/z Calcd. for C102H137FN16NaO25S [M+Na+]:2059.95, Found 2060.30.

Epimerized Miller Siderophore-D-WSPKYM-Solithromycin, S27B 1580 1380 1180 980 780 Abs 580 380 180 -20 0 5 10 15 20 25 30 Time (min)

S53 Boc NH N

H OTFA H N N Boc O O NH3 Ph NH O O HO O NH Ph O O NH N N HO O O O O S O S OH N H O O N H O TFA, t-Bu N N H N N H H2O, TIPS–H, EDT H N H N N N O H O O H O 2 h, r.t.; NH NH O O H O O O H O NaHCO3 O N N O N N H H HO OH N HN O O N HN H O O Ph H O O NaO C t-Bu HO C NH 2 NH 2 HO O O O H NaO2C NH Ph H HO2C NH O O N CONH2 HO N CONH2 HO H H O N O N O N O H O O N O H O H O N N H O N N H H N N H O CO2Na N H O CO2H N O O NH H NH H O O O O O NaO O HO NH2 NH2

S24 7 Miller Siderophore Protected-Miller Siderophore-L-WSPKYM-daptomycin WSPKYM daptomycin Miller Siderophore-L-WSPKYM-daptomycin Miller Siderophore-L-WSPKYM-daptomycin, conjugate 7: Conjugate 7 (5.0 mg, 1.7 µmol, crude) was provided from compound S24 (5.8 mg, 1.7 µmol) by the General Procedure for Global Deprotection described above. Purification of conjugate 7 was accomplished by a wash strategy. [Note: Due to the solubility properties of our compound, HPLC purification was not practical using the standard solvent system (CH3CN/H2O with 0.1% TFA). Thus, a wash purification of this product was implemented, which provided the more soluble sodium salt. Such strategies have been discuss previously for compounds involving daptomycin].S9 A 1-L stock solution of aqueous 5 mM NaHCO3 was prepared from anhydrous NaHCO3 (420.1 mg, 5 mmol) in MilliQ water (1 L). To a 1-dram (3.7-mL) vial containing the crude conjugate 7 (5.0 mg, 1.7 µmol) was added a 0.005

M aqueous solution of NaHCO3 (1.7 mL, 8.5 µmol). The crude product was extracted with CH2Cl2

(3x1 mL), and the aqueous layer was collected, along with a precipitate from the CH2Cl2 extracts. The aqueous layer and the precipitate were combined in the original 1-dram vial and lyophilized. Purification was accomplished by the following wash protocol: 1) The solid was

washed with CH3CN (1 mL) and decanted. The acetonitrile contained mostly impurities. 2) The

solid was then washed with CH3CN/H2O (5:1, 1 mL) and decanted. The decanted wash was concentrated under reduced pressure and analyzed by LC/MS to reveal mostly product with minor impurities. An additional wash of this material with CH3CN/H2O (5:1, 1 mL) provided a pure sample of conjugate 7 (1.0 mg), which was used for testing in bacteria. 3) The solid was then

washed with CH3CN/H2O (1:1, 1 mL) and decanted to provide 1.3 mg of pure conjugate 7 after concentrating under reduced pressure, and this material was used for testing in bacteria. Total product isolated: 2.3 mg (64% yield). Conjugate 7: LC/MS (positive mode): m/z Calcd. for

S54 2+ + 2+ C131H175N27O41S [M+2H /2]:1407.6, Found 1407.3. HRMS: m/z Calcd. for C131H175N27O41S [M+2H+/2]:1407.6091, Found 1407.0343.

Miller Siderophore-L-WSPKYM-Daptomycin; conjugate 7 Abs

0 5 10 15 20 25 30 Time (min)

Boc NH N

H OTFA H N N Boc O NH3 O Ph NH O O HO O NH Ph O O NH N N HO O O O O S S O N H O H OH O N O N N H t-Bu N N H H N N H N N NH O H O O H O O O H NH N O O O H O O N O N N H H TFA, HO OH N HN O N HN H O O Ph O O O HO C t-Bu H H2O, TIPS–H, EDT NH 2 HO2C HO O O NH 2 h, r.t. H HO2C NH Ph H O HO C NH N CONH O O 2 2 HO N CONH2 HO H O N N O O H 97% H O O H O N N O O H O N N H O N N N H H H O CO2H N CO H N O H O 2 N O NH H NH H O O O O O HO O HO NH2 NH2 S25 13

Protected-Miller Siderophore-D-WSPKYM-daptomycin Miller Siderophore D-WSPKYM daptomycin Miller Siderophore-D-WSPKYM-daptomycin Miller Siderophore-D-WSPKYM-daptomycin, conjugate 13: Conjugate 13 (0.9 mg, 97% yield) was provided from compound S25 (1.1 mg, 0.32 µmol) by the General Procedure for Global Deprotection described above. Purification of conjugate 13 was accomplished by a wash strategy. The crude, solid product was washed with ether (3x2 mL), sonicated, and filtered through a cotton plug. The ether washes, which only contained impurities, were discarded. The crude product was then washed with H2O (1x0.5 mL), 20% CH3CN/H2O (1x1 mL), 50% CH3CN/H2O (1x1 mL),

80% CH3CN/H2O (1x1 mL), and DMSO (1 x 0.5 mL). Each solvent system was filtered through a cotton plug into a separate flask. The DMSO wash provided 0.9 mg (97%) of conjugate 13 as a

S55 solid following lyophilization. 13: MALDI-TOF (Reflectron mode, negative): m/z Calcd. for + C131H173N27O41S [M-H ]:2811.20, Found 2811.41 m/z Calcd. for C131H173N27NaO41S [M+Na- + 2+ + H ]:2834.19, Found 2834.47. HRMS: m/z Calcd. for C131H175N27O41S [M+2H /2]:1407.6091,

3+ + Found 1407.0880. m/z Calcd. for C131H176N27O41S [M+3H /3]:938.7418, Found 938.4005.

Miller Siderophore-D-WSPKYM-Daptomycin; conjugate 13 880 780 680 580 480

Abs 380 280 180 80 -20 0 5 10 15 20 25 30 Time (min)

O O Ph OH Ph S S S O Ph OH O Ph OMe OH OMe O O OTFA NH MeI NH O Boc O O Boc O O NH O O H NH NaHCO3 H NH H NH N N H N N H N Boc DMF Boc O H3N H N O N OH t-Bu t-Bu N O O O O O O O O O O O O N H O NH N H NH O N H O NH N N N N Ph N Ph N O H N O H N HO H N O O O O Ph O Ph O O HO HO t-Bu t-Bu 3 I3 12

Common Intermediate to L-Linker Conjugates Protected-Miller Siderophore-L-WSPKYM-OMe Miller Siderophore WSPKYM OMe Miller Siderophore-L-WSPKYM-OMe (no antibiotic) Miller Siderophore-L-WSPKYM-OMe (no antibiotic), 12: To a flame-dried, 5-mL round-

bottom flask containing common intermediate 3 (11.0 mg, 5.94 µmol) was added NaHCO3 (10

mg, 119 µmol) and DMF (0.59 mL). CH3I (23.7 uL, 380 µmol) was added, and the mixture was stirred for 2.5 h. Additional MeI (30 uL, 480 µmol) was added, and the mixture was stirred for an additional 1.5 h, at which point TLC analysis (6% MeOH/CH2Cl2 with 4% AcOH) suggested completion. The mixture was concentrated under reduced pressure at 40 oC to provide 11.2 mg of a mixture containing protected intermediate I3 and an undesired bis-methylated product. [note: Methylations of Boc-protected lysines may occur under these conditions leading to bis- methylation of compound 3].S10 The flask containing I3 was purged with argon. The General Procedure for Global Deprotection described above followed by HPLC purification (5 to 95%

S56 CH3CN/H2O with 0.1% TFA, 20 min) provided conjugate 12 (1.7 mg, 23% yield over 2 steps). A bis-methylated product (3.8 mg, 52%) was also isolated. 12: MALDI-TOF (Reflectron mode) + m/z Calcd. for C60H76N10O16S [M+H ]:1225.52, Found 1226.19.

Miller Siderophore-L-WSPKYM-OMe (no antibiotic); 40 conjugate 12 35 30 25 20

Abs 15 10 5 0 -5 0 5 10 15 20 25 30 Time (min)

O O O H O N N H N N O N O N N N S O S F O HN HN F OTFA Boc O O O O H NH TFA, H NH N N H N H N Boc O H2O, TIPS–H, EDT 3 H N N OH t-Bu 2 h, r.t. O O O O NH 75% O O Ac H Ac H NH N N N N H N H N O O O S16 HO 18 t-Bu Ac Protected Ac-L-WSPKYM-eperezolid WSPKYM eperezolid

Ac-L-WSPKYM-eperezolid (no siderophore) Ac-L-WSPKYM-eperezolid (no siderophore), conjugate 18: Conjugate 18 (13.9 mg, 75% yield) was provided from compound S16 (21.2 mg, 13.8 µmol) by the General Procedure for Global Deprotection described above. Purification of conjugate 18 was accomplished by a wash strategy. The crude, solid product was washed with ether (3x2 mL), sonicated, and filtered through a cotton plug. The ether washes, which only contained impurities, were discarded. The crude

product was then washed with H2O (3x2 mL), 20% CH3CN/H2O (2x1 mL), and 80% CH3CN/H2O

(4x1 mL), and 50% CH3CN/H2O (4x1 mL). Each solvent system was filtered through a cotton plug into a separate flask and concentrated under reduced pressure. The 50% CH3CN/H2O and

S57 80% CH3CN/H2O washes provided 13.9 mg (75%) of acetylated eperezolid conjugate 18. 18: + MALDI-TOF (Reflectron mode): m/z Calcd. for C59H78FN13O13S [M+H ]:1228.55, Found + 1228.29; m/z Calcd. for C59H78FN13NaO13S [M+Na ]:1250.54, Found 1250.29. Product retention time: 12.19 min. Initial peak is DMSO.

Ac-L-WSPKYM-Eperezolid (no siderophore); conjugate 18 2480

1980

1480

Abs 980

480

-20 0 5 10 15 20 25 30 Time (min)

O HO HO HN

H N OTFA Miller Siderophore-L-WSPKYM-Eperezolid Ester, N NH3 O NH N O O H HO O S O compound S1: Compound S1 was prepared in a similar OH H O N O OH N N H N O O H N O N manner described for the preparation of eperezolid

OH O F N O amide conjugate 8, but in this case using a catalytic

NH Miller Siderophore WSPKYM O amount of DMAP for esterification with eperezolid-OH eperezolid ester S1 (S2). LC/MS (positive mode): m/z Calcd. for + 2+ + C79H96F4N14O22S [M+H ]: 1587.7, Found 1587.8. Cald. for C77H97FN14O20S [M+2H /2]: 794.3, Found 794.4.

S58 F. Synthesis of ACC Conjugates: The 7-Amino-4-carbamoylmethylcoumarin (ACC)S11,S12 conjugates, Ac-L-WSWC-ACC 2 and Ac-L-WSPKYM-ACC 1, were prepared from Fmoc-7-Aminocoumarin-4-Acetic Acid-Rink Amide AM resin (Fmocs-ACC resin) by the following general procedure: (described below for the case of conjugate 2)

H N Ac-L-WSWC-ACC (2): To a 50-mL solid phase peptide synthesis vessel containing Fmoc-ACC resin (319.1 mg, 47.9 µmol) was added HN O NH Ac NH O O HN H O HO N O DMF (8 mL), and the resin was swelled by mixing with nitrogen for O HN HS O 15 min. Fmoc-deprotection on resin proceeded under the standard 2 NH2 O conditions described below. The stock solutions of Fmoc-Cys(Trt)- WSWC ACC Ac-L-WSWC-ACC OH (2.6 mL, 300 mM), HATU (2.6 mL, 300 mM), and 2,4,6-collidine (1.3 mL, 600 mM) were added to the ACC resin. The resin was then mixed with nitrogen for 12 h. The process was repeated. After loading the first amino acid, the resin was capped with a 1.2

M solution of Ac2O in DMF (6.4 mL), which was mixed with the resin for 15 min. The resin was then washed with DMF (4x6 mL). Following the first amino acid loading onto ACC resin, the remaining amino acids were coupled by the general procedure described below. Stock Solutions: Fmoc-Cys(Trt)-OH (300 mM, 504.7 mg) in DMF (2.87 mL), HATU (300 mM, 680.8 mg) in DMF (6 mL), 2,4,6-collidine (600 mM, 0.48 mL) in DMF (6 mL). General Procedure for Fmoc Deprotection on ACC resin: Fmoc deprotection was achieved by mixing 40% 4-methylpiperidine (10 mL) with the ACC resin for 3 min, followed by mixing 20% 4-methylpiperidine for 10 min. The resin was then washed with DMF (4x6 mL), with mixing for 3 min each. Stock solutions preparation: Fmoc-W(Boc)-OH (1.8654 g, 11.8 mL DMF, 300 mM), Fmoc- S(tBu)-OH (679.1 mg in 5.9 mL DMF, 300 mM), HATU (3.4 g in 30 mL DMF, 300 mM), NMM (2.1 mL in 16 mL DMF, 1.2 M), 2,4,6-collidine (1.27 mL in 16 mL DMF, 600 mM), Ac2O (1.58 mL in 14 mL DMF, 1.2 M). General Procedure for Amino Acid Coupling on ACC resin: Coupling 1: To the Fmoc-deprotected resin was added a 300 mM solution of amino acid in DMF (2.6 mL), a 300 mM solution of HATU in DMF (2.6 mL), and a 1.2 M solution of N- methylmorpholine in DMF (1.3 mL). The contents were mixed for 20 min with nitrogen, and the solvent was removed.

S59 Coupling 2: To the resin was added a 300 mM solution of amino acid in DMF (2.6 mL), a 300 mM solution of HATU in DMF (2.6 mL), and a 600 mM solution of 2,4,6-collidine in DMF (1.3 mL). The contents were mixed for 30 min with nitrogen, and the solvent was removed.

Acetylation of the N-Terminus: To the peptide resin was added a 1.2 M solution of Ac2O in DMF (6.4 mL). The contents were mixed with nitrogen for 15 min, and the solvent was

removed. The resin was then washed with DMF (4x6 mL) and CH2Cl2 (3x6 mL).

Resin Cleavage and Deprotection: The resin was washed with MeOH (3x8 mL) and CH2Cl2 (3x8 mL). After drying the resin under reduced pressure for 30 min, the 50-mL solid phase peptide

vessel was then purged with argon. EDT (200 µL), H2O (160 µL), TIPS-H (160 µL), and TFA (6.1 mL) were then added in sequential order to the dry resin. Without removing the argon balloon and septa, the vessel was then shaken with a Mistral Multi-Shaker® for 1.2h. Upon completion, the TFA solution was filtered into a round-bottom flask, and the resin was washed with

Et2O/hexanes (1:1, 180 mL). The Et2O/hexanes filtrate was combined with the TFA filtrate. The solution was cooled to 0 oC, and the precipitated solid was filtered, washed with chilled

Et2O/Hexanes (1:1, 2x5 mL), and purified by HPLC (5 to 95% CH3CN/H2O with 0.1 % TFA, 50 minutes) to provide 1.9 mg of ACC conjugate 2. Conjugate 2: LC/MS (positive mode): m/z + Calcd. for C41H42N8O9S [M+H ]:823.3, Found 823.6.

Ac-L-WSWC-ACC, conjugate 2

S60

H N OTFA Ac-L-WSPKYM-ACC (1): Conjugate 1 was prepared by the S H N 3 O H O HN O N H general procedure described above. HRMS: m/z Calcd. for Ac NH O O N N O H O HO N O + N H O C52H64N10O12S [M+H ]: 1053.4426, Found 1053.4425. m/z OH NH2 1 + + O Calcd. for C52H64N10O12NaS [M+Na ]: 1075.4318, Found

WSPKYM ACC 1075.4221. Ac-L-WSPKYM-ACC

Ac-L-WSPKYM-ACC; conjugate 1 680 580 480 380

Abs 280 180 80 -20 0 5 10 15 20 25 30 Time (min)

IV. MIC AND PERIPLASMIC CLEAVAGE ASSAYS

A. Antibiotic Susceptibility Testing by the Broth Microdilution Method. Strains: E. coli DCO was purchased from the Coli Genetic Stock Center (CGSC), and A. baumannii ATCC 1797 was purchased from ATCC. The remaining strains were obtained as gifts.

Broth and Agar Preparation: Mueller-Hinton II (MH-II, cation-adjusted) broth was prepared

from solid MH-II (22 g) and MilliQ H2O (1 L), which was autoclaved in a presterilized 1-L glass bottle. MH-II agar plates were prepared from autoclaved MH-II (22 g), Bacto agar (17 g), and MilliQ water (1 L). The bacterial strains were obtained from glycerol stocks by streaking onto the MH-II agar plates (no antibiotics were introduced into the MH-II agar).

Preparation of sterilized CaCl2*2H2O Stock Solutions (only for daptomycin-conjugates): In the case of daptomycin-containing conjugates, a stock solution of CaCl2 (50mg/mL) was prepared

from CaCl2*2H2O (20 mL, 66.2 mg/mL). The solution was filter-sterilized utilizing a Steriflip

S61 Vacuum Filtration System with a Millipore Express PLUS Membrane (0.22 µm). In the case of

daptomycin conjugates, the CaCl2*2H2O solution (100 µL) was added to 48.3-mL aliquots of autoclaved MH-II broth to reach a final concentration of 100 µg/mL Ca2Cl in MH-II.

Preparation of sterilized 2,2'-dipyridyl (DP) and PBS stocks: A sterile stock solution of 2,2'- bipyridyl solution (40 mL, 1 mg/mL) was prepared in a 50-mL Falcon tube and then was filter sterilized utilizing a Steriflip Vacuum Filtration System with a Millipore Express PLUS Membrane (0.22 µm). A sterile 1X PBS stock solution [10X PBS (10mL), and MilliQ WATER (90mL)] was prepared and filter sterilized with an Olympus Plastics 250-mL vacuum-driven filter system with a PES membrane (0.22 µm). A 200 µM DP solution in autoclaved MH-II media was prepared from 1 mg/mL DP (1.56 mL) in MH-II (48.4 mL). A 600 µM DP solution in autoclaved MH-II media was prepared from 1 mg/mL DP (4.68 mL) in MH-II (45.3 mL) were prepared 24 h before use. [Note: Avoided the use of bovine serum albumin (BSA) in the PBS stock solution for siderophore conjugates, which may inhibit catechol-type siderophore mediated iron-transport].S13

Bacterial Culture Preparation: All autoclaved and sterilized solutions were transferred using sterilized Sarstedt Serological Pipettes (10-mL, 25-mL, and 50-mL), which are attached to an Eppendorf Easypet3. The autoclaved MH-II media contained DP (600 µM, 200 µM, 160 µM, 129µM, or without DP). The culture tubes were then placed bottom-side-up in an incubator set at 37 oC without shaking for 18-20 h. A sterilized pipet tip was gently touched on the surface of a single colony and added to autoclaved MH-II broth (5 mL) in a sterilized 12-mL culture tube. The cultures was incubated for 18 h at 37 oC with shaking at 250 rpm. The cultures were diluted 200- fold into autoclaved MH-II broth (4-mL) with 2,2'-dipyridyl (DP) and incubated at 37 oC with

shaking at 250 rpm for 3-5 h, or until an optical density at 600 nm (OD600) between 0.2 and 0.6 was achieved. An aliquot of the culture (1 mL) was transferred to a sterilized culture tube and diluted with PBS until an OD600 of 0.13. The cultures (11.5 µL) were then diluted into MH-II media (15-mL) to a concentration of 1x105 cells/mL. The diluted cultures (90 µL/well) were then added to substrate (10 µL at the following concentrations: 640 µg/mL, 320 µg/mL, 160 µg/mL, 80 µg/mL, 40 µg/mL, 20 µg/mL, and 10 µg/mL) in sterilized 96-well plates to achieve a final substrate concentration of 64 µg/mL, 32 µg/mL, 16 µg/mL, 8 µg/mL, 4 µg/mL, 2 µg/mL, and 1 µg/mL. [note: Polymyxin B was tested at the following final concentrations (µg/mL): 8, 4, 2, 1, 0.5, 0.25,

S62 0.125.] The 96-well plates were then sealed with sterilized gas-permeable covers. The plates were incubated at 37 oC with shaking at 225 rpm for 16-20h. Each well condition was prepared in triplicate. The turbidity of each well was examined and the MIC was counted as the lowest

concentration that lacked turbidity. The OD600 was also measured.

B. Procedure for Isolating Periplasmic Extract: Stock Preparations [20% sucrose/30mM Tris, 50 mM Tris, 50 mM Tris with 0.01% Tween]: A filter-sterilized, 1-L aqueous solution of 20% sucrose/30mM Tris base was prepared with sucrose (200 g) and Tris base (3.63 g). [Note: EDTA was not used in this procedure to avoid the elimination of metalloproteases from the resulting extract]. A 50-mL aqueous solution of 50 mM Tris base was prepared from solid Tris base (340 mg), along with a separate 50 mM Tris solution containing 0.01% Tween 20 (note: Tween prevents coagulation of proteins).

Osmotic Shock Procedure (without EDTA): An overnight culture on a MH-II plate (prepared as described above) at 37 oC for 18 h. Added a single colony to 100 mL of MH-II media and a single colony to 100 mL of LB media in a sterile 2.5-L Erlenmeyer flask. The flask was incubated with shaking until an OD600 of 0.5 was reached (~5-6 hours). Centrifuged the cultures at 4150 rpm for 40 min at 4 oC or at 15,000 rpm for 10 min at 4 oC. Poured-off the supernatant into bleach, so as not to disturb the pelleted cells. The filter-sterilized 20% sucrose/30mM Tris base (50 mL) was added to the cell pellets after removal of supernatant using a sterile 50-mL GeneMate serological pipet. The pellets were suspended with the pipet tip and subsequently vortexed to achieve a relatively homogenous suspension and mixed for 10 min at 4 oC. The suspension was then centrifuged at 4150 rpm for 30 min at 4 oC or at 15,000 rpm for 10 min at 4 oC. The supernatant was decanted-off so as not to disturb the pellets. Filter-sterilized cold water (50 mL) was added and vortexed to provide a homogeneous suspension, which was allowed to shake for 10 minutes at 4 oC. The suspension was then centrifuged at 4150 rpm for 30 min at 4 oC or at 15,000 rpm for 10 min at 4 oC. The supernatant (periplasmic extract) was filtered through a 500-mL vacuum filter (PES: 0.22 µm), ensuring that the filtrate remained at 4 oC by keeping the plastic filtration container on ice. The filtrate was then concentrated in an Amicon Ultra-15 Centrifugal Filter Ultracel -10K (15-mL) by centrifuging at 3600 rpm for 15 min at 4 oC. The process was repeated until the periplasmic extract reached a volume of 0.5-2 mL and a total protein concentration <2.5

S63 mg/mL. A nanodrop was used to determine the protein concentrations. Depending on the concentration, the periplasmic extract may be diluted with aqueous Tris solution (pH=8). The periplasmic extract were then aliquoted (200 µL/Eppendorf tube) into 1.5-mL Eppendorf tubes and flash-frozen with liquid nitrogen. [Note: To avoid reduction in protease activity, glycerol was not added to the periplasmic extract prior to freezing. All experiments were conducted with a fresh 200 µL aliquot of periplasmic extract to avoid inconsistencies after multiple freeze-thaw cycles. Before use, the periplasmic extract was thawed slowly at 4 oC.]

Total Protein/Periplasmic Extract (Evaluated concentration at OD 280 nm using NanodropTM): E. coli K12 MG1655 (from LB media): 1.522 mg/mL; subsequent preparation: 745 µg/mL E. coli K12 MG1655 (from MH-II media): 1.979 mg/mL A. nosocomialis M2 (from LB media): 548 µg/mL A. nosocomialis M2 (from MH-II media): 545 µg/mL A. baumannii ATCC 1797 (from LB media): 1.006 mg/mL A. baumannii ATCC 1797 (from MH-II media): 565 µg/mL E. coli DCO (from LB media): 1.581 mg/mL E. coli DCO (from MH-II media): 1.906 mg/mL E. coli BW25113 ∆bamB∆tolC (from LB media): 924 µg/mL E. coli BW25113 ∆bamB∆tolC (from MH-II media): 919 µg/mL P. aeruginosa PA01 (from LB media): 2.401 mg/mL P. aeruginosa PA01 (from MH-II media): 1.769 mg/mL P. aeruginosa ATCC10145 (from LB media): 2.002 mg/mL P. aeruginosa ATCC10145 (from MH-II media): 1.447 mg/mL S. enterica 14028s (from LB media): 939 µg/mL S. enterica 14028s (from MH-II media): 1.257 µg/mL E. cloacae ATCC 13047 (from LB media): 917 µg/mL E. cloacae ATCC 13047 (from MH-II media): 1.549 mg/mL E. aerogenes ATCC 13048 (from LB media): 2.471 µg/mL E. aerogenes ATCC 13048 (from MH-II media): 1.456 µg/mL K. pneumoniae MGH78578 (from LB media): low protein yield <50 µg/mL K. pneumoniae MGH78578 (from MH-II media): low protein yield <50 µg/mL

S64

C. ACC Cleavage in Periplasmic Extract: Spectroscopic Evaluation of Scarless Linkers. [Note: The assay and all stock solutions were prepared at 4 oC.] A 50 µM solution of substrates 1 and 2 were prepared from Tris base solution (299.3 µL) and a 1 mM substrate stock solution (15.8 µL) in DMSO. [Note: Relevant calculation: Quantity of substrate stock = 50 µM (315 µL)/1000 µM]. Each 50 µM solution of 1 and 2 (25 µL) was then transferred to a 96-well plate (opaque, 200 µL/well volume) in triplicate over two rows. As a control, each substrate was mixed in triplicate with a 50 mM Tris solution (25 µL) instead of periplasmic extract. A 200 µg/mL solution of E. coli K12 MG1655 periplasmic extract was then prepared by adding the 50 mM Tris base solution with 0.01% Tween (130.3 µL) to periplasmic extract of E. coli K12 MG1655 (19.7 µL, 1.522 mg/mL), which had been slowly thawed at 4 oC over 30 min. The 200 µg/mL solution of E. coli K12 MG1655 periplasmic extract (25 µL) was then transferred to the 96-well plate (opaque, 200 µL/well volume) containing 1 and 2 resulting in a maximum volume of 50 µL/well. The final concentrations of periplasmic extract and substrate were 100 µg/mL and 25 µM, respectively. The plate was sealed with a transparent polyolefin silicone film (ThermofisherTM NucTM Sealing Tape, 12-565-513) and placed in a plate reader set to 37 oC with shaking at 225 rpm for 8 h. ACC excitation wavelengths: 300-410 nm at 5 nm intervals. ACC excitation maximum: 355 nm. ACC emission wavelengths: 410-500 nm at 5 nm intervals. ACC Emission

maximum is 460 nm. See Figure 4 for data. The data for 1 were fit to the equation F(t) = (F0 –

Plateau)*exp(-k*t)+Plateau, in which F(t) and F0 are the fluorescent intensities observed at time t and at t=0, respectively. K is the pseudo-first order rate constant. T is time in hr. Plateau is the fluorescence intensity extrapolated to infinite time. The data were analyzed by the method of non-

linear least squares using the program GraphPad Prism.

D. Scarless Cleavage of Eperezolid-NH2 (5) and Daptomycin (4) in Periplasmic Extract. The periplasmic extract [25 µL, 400 µg/mL total protein for E. coli K12 MG1655 and A. nosocomialis; see total protein list above in section B for other strains] was mixed with the daptomycin conjugate 7 (25 µL, 117.6 µM) or the eperezolid conjugate 8 (25 µL, 117.6 µM) in a 1.5-mL Eppendorf tube. The reaction mixture was mixed at 1050 rpm in a table-top incubator set at 37 oC for 11 h. After 11 h, 25 µL was removed and placed in a 400-µL glass insert fitted in a

mass-spec vial, which was followed by the addition of MilliQ H2O (25 µL) and 1 M HCl (2 µL).

S65 The reaction mixture was analyzed by HPLC (54 minutes, 0 to 95% CH3CN/H2O with 0.1 % TFA,

254 nm). The % scarless cleavage of eperezolid-NH2 (5) was evaluated by the following equation: ( ) % �������� �������� = � � , � � . where 58.8 µM is the maximum amount of product that could be produced in the reaction mixture.

E. In Vitro Translation. The ability of conjugates to inhibit the 70S E. coli ribosome was tested in vitro using the PURExpress, In-Vitro Protein Synthesis Kit (NEB), murine RNAse inhibitor (NEB), and 7.5 ng/µl of template DNA encoding the fluorescent protein mEGFP. The volumes of each component in the reaction mixture were scaled down from the NEB protocol for a final reaction volume of 4 µL. Analogs were screened at a concentration of 10 µM (conjugate 8 at 38 µM), with a final concentration of 1.9% DMSO. All reactions were performed in triplicate. Translation reactions were carried out at 37 oC for 1 hour. To assist in the transfer of reactions to 96-well half-area NBS microplates (Corning 3993) for final measurements, the volume was increased to 50 µL by adding

buffer C (20 mM Tris-HCl pH 7.5, 60 mM NH4Cl, 6 mM MgCl2, 0.5 mM EDTA). mEGFP was excited at 485 nm; its emission was recorded at 535 nm. For comparison of analog activities across multiple initial screens, fluorescence readouts were normalized to the blank.

V. Substrate Phage Display

A. Phage Library Construction: Phage library construction. All phages were propagated in the E. coli TG1 strain (Lucigen). E. coli CJ236 was purchased from Lucigen. The recombinantly expressed BirA enzyme used for biotinylating AviTag was a gift from the Wells lab at UCSF. Phagemid vector pCES1 was obtained from the Craik lab at UCSF. Pierce streptavidin-coated high-capacity plates were purchased from ThermoFisher ScientificTM. The AviTag-displaying M13 phage libraries were constructed in the phagemid vector pCES1. The AviTag-TEV sequence was first fused to the N-terminus of the pIII protein in the pCES1 vector using ApaL1 and Not1 restriction sites affording the protein sequence as MGLNDIFEAQKIEWHEGGSENLYFQGGSAAAHHHHHHGAAEQKLISEEDLNG-. Single- stranded M13 DNA was purified using the M13 DNA kit from Qiagen. For library construction,

S66 we followed the protocol by Chen et al.S12 Phage libraries were constructed using an oligonucleotide that encoded the reverse complement of the protein sequence – IEWHEGGSXXXXXXGGSAAAHHH- using the primer 5’- TGATGATGATGTGCGGCCGCACTACCACCMNNMNNMNNMNNMNNMNNGCTGCCG CCTTCATGCCATTCAAT-3’ for the AviTag-XXXXXX- library (codon M = A, C; N = A, C, T, G). Double-stranded DNA was generated according to Sidhu et al.S12,S13 and electroporated into E. coli TG1. A library size of 4´109 was generated and amplified for 15 h at 37 °C in 250 mL 2xYT media containing AMP (30 mg/mL) and Kan (15 mg/mL). For phage biotinylation, purified phages (4 ´ 1012 cfu) were resuspended in 10 mM Tris (0.5 mL, pH 8.0) and concentrated in a 30 kDa MWCO tube; this process was repeated 3x to ensure sufficient buffer exchange. After buffer exchange, we added water (40 µL), 2´ biotinylation buffer (80 uL, composition of biotinylation buffer: 0.1 M Tris, 10 mM MgCl2, 2 mM Biotin, pH 8.0) to wash the 30 kDa MWCO tube and transfer the remaining solution to a 1.5-mL Eppendorf tube. Additional water (20 µL) and 2´ biotinylation buffer (20 µL) was used to complete the transfer to the Eppendorf tube. We then added 0.1 M ATP (~6 uL) and BirA enzyme (2 µL, 3U/µL) to the phage solution (~200 µL). The mixture was vortexed and cooled to 4 °C overnight for biotinylation. An anti-biotin western blot was then performed to confirm that phages were biotinylated.

B. Substrate phage selection experiments.

AviTag AviTag XXXXXX Phage XXXXXX Phage Binding Biotin Biotin Washing Streptavidin

Amplify

XXX Phage Cleavage NGS Four rounds of substrate selection were conducted (Figure 3 of manuscript). In round one, the streptavidin-coated ELISA plate was blocked with 2% BSA for 30 min and washed three times with phosphate-buffered saline containing 0.1 % Tween 20 (PBST) buffer. The plate was then coated with ~ 109 phages (one well) by gently shaking the phages for 2 hours. The wells were then washed with PBST (6x), and then the wells were washed with PBST (0.3 mL, 3 ´ 20 min) while incubating at 37 oC.

S67

For the first round of cleavage selection, periplasmic extract (50 µL) was added to one well and shaken at 37 ˚C for 3 hours. Cleaved phages were amplified overnight in 2xYT (30 mL) at 37 ˚C with shaking. The second, third, and fourth rounds of selection were performed by repeating the procedure above with 25 uL extract + 25 uL PBS added to each well, with reduced cleavage times: 2 hours, 1 hour, and 30 minutes, respectively. The output phage titer is ~ 3´108 cfu for each round of selection. The final output phages were sequenced by Next Generation Sequencing.

Note: Output reads are the number of total reads of the same peptide sequence in the output library. Initial reads are the number of the total reads of the same peptide sequence in the initial naive library. Ranking is based on the ratio of output reads over initial reads. Read number 0.5 means the specific sequence was not found in the sequencing-result file. Sequences colored in red were selected for further validation.

K12 periplasmic extract selection results (Enrichment factor > 5000) Sequence Output Reads Initial reads Enrichment factor KNQSLG 10652 0.5 21304 GSDSSV 9239 0.5 18478 NHADVH 8138 0.5 16276 YSDSET 7764 0.5 15528 KSEMLS 7742 0.5 15484 WCKWAS 15307 1 15307 NKWNPS 7651 0.5 15302 SCYQCQ 7591 0.5 15182 TFATCK 7405 0.5 14810 ALWIYR 6805 0.5 13610 PKYMRF 13192 1 13192 RFQPPV 6467 0.5 12934 RVGLGA 6412 0.5 12824 MYPPTW 6396 0.5 12792 PGSQSL 11285 1 11285 QSFSID 5616 0.5 11232 VSPNRH 5371 0.5 10742 RTVYYP 5056 0.5 10112 YFQYHP 4856 0.5 9712

S68 KNYVFQ 4385 0.5 8770 KHPSRR 4073 0.5 8146 NTHPAN 4030 0.5 8060 YTTEAF 3980 0.5 7960 SREASN 3912 0.5 7824 HLMFNC 3676 0.5 7352 WRPHPT 3622 0.5 7244 SLGFSK 3529 0.5 7058 LEIPRF 3448 0.5 6896 QNKPSV 3407 0.5 6814 DVNWSG 3187 0.5 6374 NLKREL 3185 0.5 6370 STLHQS 3028 0.5 6056 YKEYQT 2938 0.5 5876 SHNLNY 2851 0.5 5702 PCNTNR 2826 0.5 5652 NNAQNQ 2761 0.5 5522 VSYSPR 2755 0.5 5510 FTADRC 2705 0.5 5410 LESEPG 2681 0.5 5362 LEPRSP 2581 0.5 5162 NRHART 2561 0.5 5122

Sequences selected for validation. The peptides were synthesized as WSXXXXXXG, where XXXXXX represents the specific hexapeptide. Trp was added at the N-terminus to help the peptide bind with the reverse-phase HPLC column. The N-terminus is a free amine, while the C-terminus is a carboxylamide. From K12 selection KNQSLG GSDSSV NHADVH KSEMLS WCKWAS PKYMRF

S69

WSPKYMRFG cleavage with K12 extract. Cleavage Conditions: 50 mM Tris (pH 8.0), peptide (0.2 mg/mL), extract 0.1 mg/mL (total protein concentration based on OD 280), 37 °C, 18 hours. Cleaved peptide 2 is WSPKYM.

See Table S5 for cleavage-site analysis of all six sequences.

VI. References Cited S1. Liu, A.; Tran, L.; Becket, E.; Lee, K.; Chinn, L.; Park, E.; Tran, K.; Miller, J. H. Antimicrob Agents Chemother. 2010, 54, 1393–1403. S2. Schumacher, A.; Trittler, R.; Bohnert, J. A.; Kümmerer, K.; Pagès, J. M.; Kern, W. V. J. Antimicrob. Chemother. 2007, 59, 1261–1264. S3. Liu, R.; Miller, P. A.; Vakulenko, S. B.; Stewart, N. K.; Boggess, W. C.; Miller, M. J. J. Med. Chem. 2018, 61, 3845. S4. Delorme, D.; Houghton, T.; Lafontaine, Y.; Tanaka, K.; Deitrick, E.; Kang, T.; Rafai Far, A. Int. Patent Application PCT/IB2006/004233, December 6, 2006. S5. Baco, E.; Hoegy, F.; Schalk, I. J.; Mislin, G. L. A. Org. Biomol. Chem. 2014, 12, 749. S6. Abergel, R. J.; Rees, J.; Strong, R. K. WO 2018/063638 A1, August 28, 2017.

S70 S7. Xu, J.-X.; Wu, X.-F; org. lett. 2018, 20, 5938. S8. Norris, K.; Halstrøm, J.; Brunfeldt, K. Acta. Chem. Scand. 1971, 25, 945. S9. Ghosh, M.; Miller, P. A.; Möllman, U.; Claypool, W. D.; Schroeder, V. A.; Wolter, W. R.; Suckow, M.; Yu, H.; Li, S.; Huang, W.; Zajicek, J.; Miller, M. J. J. Med. Chem. 2017, 60, 4577. S10. Malkov, A. V.; Vrankova, K.; Cerny, M.; Kocovsky, P. J. Org. Chem. 2009. 74, 8425. S11. Harris, J. L.; Backes, B. J.; Leonetti, F.; Mahrus, S.; Ellman, J. A.; Craik, C. S. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 7754. S12. Maly, D. J.; Leonetti, F.; Backes, B. J.; Dauber, D. S.; Harris, J. L.; Craik, C. S.; Ellman, J. A. J. Org. Chem., 2002, 67, 910. S13. Wilhelm, S. W.; MacAuley, K.; Trick, C. G. limnol. oceanogr. 1998, 43, 992–997. S14. Chen, G. & Sidhu, S. S. Methods Mol Biol 2014, 1131, 113. S15. Sidhu, S. S., Lowman, H.B., Cunningham, B. C. & Wells, J. A. Methods Enzymol 2000, 328, 333.

S71 VII: Select NMR Spectra

O Ph

O Ph

O OH

S3

S72

O Ph

O Ph O OH

S3

S73

Cbz NH N 2 H S4

S74

Cbz NH2 N H S4

S75

O H Cbz N N O H S5

S76

O H Cbz N N O H S5

S77

Ph O Ph O

O O

Cbz N N O H S6

S78

Ph O Ph O

O O Cbz N N O H S6

S79

Ph O Ph O

O O O N N O H

O S7 O Ph Ph

S80

Ph O Ph O

O O O N N O H

O S7 O Ph Ph

S81

Ph

O Ph O

O O O

N H N O H O O Ph 10 Ph Siderophore Component

S82

Ph O Ph O O O O N H N O H O O Ph 10 Ph Siderophore Component

S83

O Ph S O Ph OH O NH Boc O O H NH N Boc N H N O t-Bu O O O N H O NH N Ph N O O H N O Ph O O t-Bu

3

S84

O Ph S O Ph OH O NH Boc O O H NH N Boc N H N O t-Bu O O O N H O NH N Ph N O O H N O Ph O O t-Bu

3

S85

O Ph S

O Ph OH O NH Boc O O H NH N Boc N H N O t-Bu O O O N H O NH N Ph N O O H N O Ph O O t-Bu S14

S86

O Ph S

O Ph OH O NH Boc O O H NH N Boc N H N O t-Bu O O O N H O NH N Ph N O O H N O Ph O O t-Bu

S14

S87

Boc

N

O HN O HN NH N Boc O O O HN H O N O O N O Ph Ph t-Bu O OH O Ph S Trt O Ph

S15

S88

Boc N

O HN O HN NH N Boc O O O HN H O N O O N O Ph Ph t-Bu O OH O Ph S Trt O Ph S15

S89

S OH

Boc O O H NH N Boc N H N O t-Bu O O O Ac H NH N N H N O O t-Bu S16

S90

S

OH

Boc O O H NH N Boc N H N O t-Bu O O O Ac H NH N N H N O O

t-Bu S16

S91

Boc N O S Boc NH O HN H H O N O N O H N NH N O Ph Ph N O O N N O O H O N O O N O N H O F t-Bu O O NH Ph t-Bu O O Ph S17

S92

Boc N O S Boc NH O HN H H O N O N O H N NH N O Ph Ph N O O N N O O H O N O O N O N H O F t-Bu O O NH Ph t-Bu O Ph S18 O

S93

Boc N O S Boc NH O HN H H O N O N O H N NH N O Ph Ph N O O N N O O H O N O O N O N H O F t-Bu O O NH Ph t-Bu O Ph S18 O

S94

Boc N O S Boc NH O HN H H O N O N O H N NH N O Ph Ph N O O N N O O H O N O O N O N H O F t-Bu O O NH Ph t-Bu O Ph O S19

S95

Boc N O S Boc NH O HN H H O N O N O H N NH N O Ph Ph N O O N N O O H O N O O N O N H O F t-Bu O O NH Ph t-Bu O O Ph S19

S96

Boc

N

O HN O N HN NH Boc O O O O HN H O N O O N O F N Ph Ph H t-Bu O HN N O O N N Ph S Trt O O O Ph

S20

S97

O O N H N O N N S O HN F

Boc O O H NH N Boc N H N O t-Bu O O O Ac H NH N N H N O O t-Bu S21

S98

O O N H N O N N S O HN F

Boc O O H NH N Boc N H N O t-Bu O O O Ac H NH N N H N O O t-Bu S21

S99

F O O Boc OTFA O O OH O O N CH3 CH3 N CH H 3 MeO O O HN O Boc N H NH H3C Ph N CH3 O Ph S O N O NH O O H O H O O N O N N N t-Bu N N N O H O N H O Ph O Ph S23A O t-Bu

S100

F O O Boc O O OTFA OH O O N CH3 CH3 N CH H 3 MeO O O HN O Boc N H NH H3C Ph N CH3 O Ph S O N O NH O O H O H O O N O N N N t-Bu N N N O H O N H O Ph O Ph S23B O t-Bu

S101

Boc N

H N Boc O NH Ph O Ph O O NH N O O O S O N H O t-Bu N N H H N N NH O H O O O H O O N N H O N HN Ph O O O t-Bu H HO2C O NH Ph H O HO C NH O 2 N CONH2 HO H O N N O O H O H O N N H N CO H H O 2 N O NH H O O O HO S25 NH2

S102

VIII: HPLC TRACES FOR CLEAVAGE IN PERIPLASMIC EXTRACT

Cleavage Analysis of Daptomycin Conjugate 7 (58.8 µM) in Periplasmic Extract:

DAD1 D, Sig=254,8 Ref=800,50 (JONBOYCE\19072350.D) DAD1 D, Sig=254,8 Ref=800,50 (JONBOYCE\19072363.D) Norm. periplasmic extract derived from 700 A. nosocomialis grown from LB media. daptomycin

600

500

400

300

Assay Conditions: daptomycin conjugate 7 (58.8 µM) + periplasmic extract of A. nosocomialis (400 µg/mL) at 37 200 Peaks overlapping with daptomycin oC for 11 h.

100

0

14 16 18 20 22 24 26 28 30 min S103

periplasmic extract derived from Peaks overlapping with daptomycin A. nosocomialis grown from LB media.

Assay Conditions: daptomycin conjugate 7 (58.8 µM) + periplasmic extract of A. nosocomialis (400 µg/mL) at 37 oC for 24 h.

daptomycin

S104

DAD1 D, Sig=254,8 Ref=800,50 (JONBOYCE\19072351.D) DAD1 D, Sig=254,8 Ref=800,50 (JONBOYCE\19072363.D) Norm. periplasmic extract derived from 700 A. nosocomialis grown from MH-II media.

daptomycin 600

500

400

300 Assay Conditions: daptomycin conjugate 7 (58.8 µM) + periplasmic extract of A. nosocomialis (400 µg/mL) at 37 oC for 11 h. Peak overlaps with daptomycin

200

100

0

10 15 20 25 30 min S105

DAD1 D, Sig=254,8 Ref=800,50 (JONBOYCE\19092602.D) DAD1 D, Sig=254,8 Ref=800,50 (JONBOYCE\19092613.D) mAU periplasmic extract derived from A. baumannii grown from LB media.

150

daptomycin 125

daptomycin conjugate 7 (58.8 µM) + periplasmic extract of A. baumannii (503 µg/mL) at 37 oC for 11 h.

100

Peak overlaps with daptomycin 75

50

25

0

-25

0 10 20 30 40 50 min S106

DAD1 D, Sig=254,8 Ref=800,50 (JONBOYCE\19092602.D) DAD1 D, Sig=254,8 Ref=800,50 (JONBOYCE\19092612.D) Norm. periplasmic extract derived from A. baumannii grown from MH-II media. daptomycin 120

100

80 daptomycin conjugate 7 (58.8 µM) + periplasmic extract of A. baumannii (283 µg/mL) at 37 oC for 11 h.

Peak overlaps with daptomycin

60

40

20

0

-20

-40

0 10 20 30 40 50 min S107

DAD1 D, Sig=254,8 Ref=800,50 (JONBOYCE\19092602.D) DAD1 D, Sig=254,8 Ref=800,50 (JONBOYCE\19092621.D) Norm. periplasmic extract derived from P. aeruginosa PA01 grown from LB media. daptomycin

120

100

daptomycin conjugate 7 (58.8 µM) + periplasmic extract 80 of P. aeruginosa PA01 (1.2 mg/mL) at 37 oC for 11 h.

60

40 daptomycin release not apparent

20

0

-20

-40 \

0 10 20 30 40 50 min

S108

periplasmic extract derived from P. aeruginosa PA01 grown from MH-II daptomycin conjugate 7 (58.8 µM) + periplasmic extract media. of P. aeruginosa PA01 (885 µg/mL) at 37 oC for 11 h.

daptomycin release not apparent

daptomycin

S109

DAD1 D, Sig=254,8 Ref=800,50 (JONBOYCE\19092602.D) DAD1 D, Sig=254,8 Ref=800,50 (JONBOYCE\19092625.D) mAU periplasmic extract derived from P. aeruginosa ATCC 10145 grown from LB media.

500

400

300

daptomycin conjugate 7 (58.8 µM) + periplasmic 200 extract of P. aeruginosa ATCC 10145 (1.0 mg/mL) at 37 oC for 11 h.

daptomycin

100

daptomycin release not apparent

0

0 10 20 30 40 50 min

S110

DAD1 D, Sig=254,8 Ref=800,50 (JONBOYCE\19092602.D) DAD1 D, Sig=254,8 Ref=800,50 (JONBOYCE\19092626.D) mAU periplasmic extract derived from 2250 P. aeruginosa ATCC 10145 grown from MH-II media.

2000

1750

1500

1250

daptomycin conjugate 7 (58.8 µM) + periplasmic extract of P. aeruginosa ATCC 10145 (724 1000 µg/mL) at 37 oC for 11 h.

750

500 daptomycin release not apparent

250 Daptomycin

0

0 10 20 30 40 50 min

S111

periplasmic extract derived from E. aerogenes grown from LB media.

daptomycin release not apparent

daptomycin conjugate 7 (58.8 µM) + periplasmic extract of E. aerogenes (1.24 mg/mL) at 37 oC for 11 h.

daptomycin

S112

periplasmic extract derived from E. aerogenes grown from MH-II media.

daptomycin release not apparent

daptomycin conjugate 7 (58.8 µM) + periplasmic o extract of E. aerogenes (728 µg/mL) at 37 C for 11 h.

daptomycin

S113

DAD1 D, Sig=254,8 Ref=800,50 (JONBOYCE\19072359.D) DAD1 D, Sig=254,8 Ref=800,50 (JONBOYCE\19072363.D) Norm. periplasmic extract derived from 700 E. coli K12 MG1655 grown from LB media.

600

500

daptomycin

400

300

daptomycin conjugate 7 (58.8 µM) + periplasmic extract of E. coli K12 MG1655 (400 µg/mL) at 37 oC for 11 h.

200

100

0

12.5 15 17.5 20 22.5 25 27.5 30 32.5min

S114

periplasmic extract derived from E. coli MG1655 grown from MH-II media.

daptomycin conjugate 7 (58.8 µM) + periplasmic extract of E. coli K12 MG1655 (400 µg/mL) at 37 oC daptomycin release not apparent for 11 h.

daptomycin

daptomycin

S115

periplasmic extract derived from E. cloacae grown from LB media.

daptomycin conjugate 7 (58.8 µM) + periplasmic extract of E. cloacae (459 µg/mL) at 37 oC for 11 h. daptomycin release not apparent

daptomycin

S116

periplasmic extract derived from E. cloacae grown from MH-II media.

daptomycin release not apparent

daptomycin conjugate 7 (58.8 µM) + periplasmic o extract of E. cloacae (775 µg/mL) at 37 C for 11 h.

daptomycin

S117

periplasmic extract derived from E. coli ∆bamB∆tolC grown from LB media.

daptomycin release not apparent

daptomycin conjugate 7 (58.8 µM) + periplasmic extract of E. coli ∆bamB∆tolC (462 µg/mL) at 37 oC for 11 h.

daptomycin

S118

periplasmic extract derived from E. coli ∆bamB∆tolC grown from MH-II media.

daptomycin conjugate 7 (58.8 µM) + periplasmic extract daptomycin release not apparent of E. coli ∆bamB∆tolC (460 µg/mL) at 37 oC for 11 h.

daptomycin

S119

periplasmic extract derived from S. enterica grown from LB media.

daptomycin conjugate 7 (58.8 µM) + periplasmic extract daptomycin release not apparent of S. enterica (470 µg/mL) at 37 oC for 11 h.

daptomycin

S120

periplasmic extract derived from S. enterica grown from MH-II media.

daptomycin release not apparent daptomycin conjugate 7 (58.8 µM) + periplasmic extract of S. enterica (629 µg/mL) at 37 oC for 11 h.

daptomycin

S121

periplasmic extract derived from E. coli DCO grown from MH-II media. ‘ daptomycin release not apparent daptomycin conjugate 7 (58.8 µM) + periplasmic extract of E. coli DCO (953 µg/mL) at 37 oC for 11 h.

daptomycin

S122

Cleavage Analysis of Oxazolidinone Conjugate 8 (58.8 µM) in Periplasmic Extract. The yields represent the amount of eperezolid-NH2 (5) released in each reaction divided by the maximal amount of 5 that can be produced in each reaction, which was determined by the peak areas. The release of eperezolid-NH2 (5) from conjugate 8 was confirmed by Mass Spec and via comparison of overlapping retention times with synthesized eperezolid-NH2 (5) (see below).

S123

periplasmic extract derived from A. nosocomialis grown from LB media. eperezolid conjugate 8 (58.8 µM) + periplasmic extract of A. nosocomialis (400 µg/mL) at 37 oC for 11 h.

Scarless eperezolid release (~1%)

periplasmic extract derived from A. nosocomialis grown from MH-II media.

eperezolid conjugate 8 (58.8 µM) + periplasmic extract of A. nosocomialis (400 µg/mL) at 37 oC for 11 h.

Scarless eperezolid release (5.2%)

S124

periplasmic extract derived from Scarless eperezolid release E. coli K12 MG1655 grown from LB media.

eperezolid conjugate 8 (58.8 µM) + periplasmic extract of E. coli K12 MG1655 (400 µg/mL) at 37 oC for 24 h.

S125

periplasmic extract derived from E. coli K12 MG1655 grown from LB media.

eperezolid conjugate 8 (58.8 µM) + periplasmic extract of E. coli K12 MG1655 (400 µg/mL) at 37 oC for 11 h.

Scarless eperezolid release (4.2%)

periplasmic extract derived from E. coli K12 MG1655 grown from MH-II media.

eperezolid conjugate 8 (58.8 µM) + periplasmic extract of E. coli K12 MG1655 (400 µg/mL) at 37 oC for 11 h. Scarless eperezolid release (3.4%)

Note: Cleavage of solithromycin from solithromycin conugate 9 was difficult to measure, which may be due to the rapid elution of solithromycin from the column under the acidic conditions required for cleavage analysis. S126

Supporting Information_Boyce et al.pdf (7.85 MiB) view on ChemRxiv download file