Guanidiniocarbonyl-Pyrrole (GCP) C- and N-Terminus Modifications of Vancomycin

Abstract

A new vancomycin C-terminus modification is detailed that improves antimicrobial activity especially against vancomycin-resistant organisms. Incorporation of a C-terminus cationic guanidiniocarbonyl-pyrrole (GCP) group reinstates activity against vancomycin-resistant bacteria and further improves activity against sensitive organisms by a mechanism independent of D-Ala-D-Ala binding. The functional effects of the added GCP group is apparent in the behavior of 8a, which exhibited pronounced improvements in activity against vancomycin-resistant bacteria relative to vancomycin (ca. 100-fold) and improved activity against sensitive organisms (ca. 10-fold) where the series (8a–d) displayed a dependence on the linker length (potency: n = 2>3>4>5) most evident against vancomycin-sensitive organisms. When combined with an added CBP peripheral modification, the activity against vancomycin-resistant organisms synergistically improved as much 3000-fold relative to vancomycin, ca. 30-fold or better relative to 8a, and as much as 10-fold relative to CBP-vancomycin. The beneficial effects are observed with introduction at the C-terminus, but not N-terminus, and a focused SAR indicates they are structure (e.g., linker length) as well as site specific. These observations along with mechanistic studies are consistent with targeting a specific feature in the bacterial cell wall versus a nonspecific role attributable to a cationic modification, especially given its modest pK_a (7–8).

Graphical Abstract

INTRODUCTION

In recent studies, we reported two C-terminus peripheral modifications on vancomycin that increase the antimicrobial potency by introducing a new additional mechanism of action (bacteria cell envelope permeabilization) independent of D-Ala-D-Ala/D-Lac binding.¹ These modifications, a trimethylammonium salt^2–5 and later a more effective persistently protonated guanidinium cation (Figure 1),^6–8 could be combined with additional binding pocket modifications that convey dual D-Ala-D-Ala/D-Ala-D-Lac binding to directly overcome the intrinsic molecular basis of vancomycin resistance and/or peripheral carbohydrate modifications (e.g., (4-chlorobiphenyl)methyl, CBP) that inhibit transglycosylase and cell wall biosynthesis independent of D-Ala-D-Ala/D-Ala-D-Lac binding.¹ This provided durable, potent antibiotics expressing multiple independent synergistic mechanisms of action that are active against both vancomycin-sensitive and vancomycin-resistant Gram-positive pathogens.¹ These added C-terminus modifications were found to be structure and site specific^2–6 and not only increased potency and antibiotic durability against raising resistance, but the select members in the series examined also exhibited improved pharmacokinetic (PK), physical (e.g., solubility) and pharmacological properties, and displayed effective in vivo efficacy against a multidrug and vancomycin-resistant S. aureus strain (VRS-2, MRSA/VRSA) without introducing acute liabilities.^1,2,5–8 Mechanistic studies have suggested that a binding interaction with membrane or cell wall embedded teichoic acid⁶ may be responsible for or contributing to the added functional activity of induced cell envelope permeability without membrane depolarization or cell lysis.^1,2,5–8

Figure 1.

Structure of vancomycin and key analogues.

The improvement in the properties of vancomycin and its derivatives by a simple C-terminus modification, which likely interacts with the negatively charged phosphate backbone of teichoic acid, led us to explore incorporation of an additional modification, Schmuck’s cationic guanidiniocarbonyl-pyrrole (GCP) that has been shown to further improve guanidine binding to oxyanions including carboxylates and phosphates in water, Figure 2.^9–11 In addition to an intramolecular H-bond that fixes the guanidinium orientation and conformation, GCP forms a H-bonding network with oxyanions consisting of four H-bonds that together with the ionic interaction leads to strong binding even in aqueous solution. The potential use of GCP in place of a C-terminus guanidine itself was addressed herein with the preparation of C- and N-terminus GCP modifications to both vancomycin (1) and (4-chlorobiphenyl)methylvancomycin (CBP-vancomycin, 2) and their evaluation alongside vancomycin, CBP-vancomycin (2), G3-vancomycin⁶ (3), and G3,CBP-vancomycin⁶ (4). Like the C-terminus guanidinium modifications, GCP incorporation was found to improve antimicrobial activity especially against vancomycin-resistant organisms, act synergistically with a vancosamine CBP modification and independent of D-Ala-D-Ala/D-Lac binding, and functionally induce membrane permeability through introduction of an additional independent mechanism of action. In addition to such attributes and with the added mechanism of action, the durability of such compounds against raising resistance can be anticipated to be further and substantially improved.

Figure 2.

Structure of GCP and our past modifications.

RESULTS

Synthesis.

The GCP core 5, obtained from 5-carbomethoxypyrrole-2 carboxylic acid by a procedure reported by Schmuck,¹⁰ was activated with propylphosphonic anhydride (T3P) and coupled (1.5–3 equiv T3P, 3–9 equiv NMM (N-methylmorpholine), 18 h, 25 °C, DMF) with a systematic series of commercially available mono-Boc protected diamines (H₂N(CH₂)_nNHBoc, n = 2–5), providing the corresponding amides 6a–d (51–74%), Scheme 1. Subsequent Boc deprotection by treatment with trifluoroacetic acid (TFA, CH₃CN (1:1), 25 °C, 2 h) afforded the amines 7a–d as their TFA salts that were coupled with the vancomycin C-terminus carboxylic acid with T3P (for a–c) or HBTU (hexafluorophosphate benzotriazole tetramethyl uranium) for d (NMM, DMF, 25 °C, 2–18 h) to provide the first set of target compounds 8a–d (44–51%). Coupling of the same four free amines 7a–d with CBP-vancomycin (2, 10 equiv T3P, 30 equiv NMM, DMF, 25 °C, 21 h) provided a second set of target compounds 9a–d (39–50%) that additionally incorporates the vancosamine CBP modification. Notably, the couplings were conducted with excess 7a–d (10 equiv) with both vancomycin (1) and CBP-vancomycin (2) without need for protecting groups for the less reactive, more hindered amines found in the vancosamine and NMe-Leu resides.

Scheme 1.

Synthesis of GCP C-terminus modified vancomycins and CBP-vancomycins

Complementary to the two-step, one pot reductive amination under basic conditions used for selective functionalization of the vancosamine primary amine via intermediate imine formation, a mildly acidic reductive amination procedure was used for selective modification at the N-terminus N-methylleucine residue of vancomycin and CBP-vancomycin.^3,12 The mildly acidic reductive amination conditions (1% AcOH, pH 4–5) enables selective tertiary amine formation at the N-methylleucine terminus by promoting iminium formation and allowing NaCNBH₃ reduction even with the required excess aldehyde needed to drive the reaction to completion without competing reaction at the vancosamine residue.³ Reductive amination at the N-methylleucine amine was conducted with a systematic series of Boc-protected aminoaldehydes (BocNH(CH₂)_nCHO,¹³ where n = 1–3) in a 1% acetic acid/DMF solution, employing sodium cyanoborohydride (NaCNBH₃, 50 equiv) as the reducing agent, Scheme 2. The reactions were conducted at 40 °C for 6 h, followed by room temperature for 18 h (with an exception for 10c, where 50 °C for 24 h was applied), to provide the tertiary amine intermediates 10a–c (39–56%) for vancomycin and 11a–c (41–54%) for CBP-vancomycin. The enlisted stoichiometry of the aldehyde varied (n = 1, 25 equiv; n = 2, 15–25 equiv; n = 3, 10 equiv), reflecting optimization for reaction efficiency. Subsequent amine Boc deprotection was carried out with TFA in acetonitrile (1:1). Confirmation of the selective N-terminus modification was achieved with 10a through use of the TOCSY-defined spin system of the newly introduced alkyl chain and its subsequent NOESY correlation with the adjacent N-methyl substituent of the terminal N-methylleucine (δ 3.10 and 2.71 CH₂’s with δ 2.08 for NMe, Supporting Information).

Scheme 2.

Synthesis of GCP-modified N-terminus vancomycins and CBP-vancomycins

The NBoc deprotections of 10a–c and 11a–c were followed by coupling with 15 (1.7 equiv) in anhydrous DMF at 40 °C for 20–28 h in the presence of iPr₂NEt as base, adjusting the pH to 7. Following RP-HPLC purification, Boc deprotection (TFA/CH₃CN, 1:1) delivered the products 12a–c (33–42%) and 13a–c (18–41%). The activated ester derived from the GCP core (15) was freshly synthesized by coupling 5 with PyOxyma¹⁴ (1-cyano-2-ethoxy-2-oxoethylidene-aminooxy)-tri-1-pyrrolidinophosphonium hexafluorophosphate) in DMF in the presence of base (iPr₂NEt) followed by in-situ Boc protection of guanidine, and finally purification. Numerous variations and variables for the final GCP introduction without NBoc protection were explored including different coupling reagents and activated esters as well as their stoichiometry, reaction solvent or temperature, and these alternatives were not successful. The utilization of a Boc-protected activated ester 15 for the GCP introduction proved critical in these amide couplings.

Antimicrobial Activity.

The antimicrobial activity of the compounds was established in a standard broth microdilution assay¹⁵ and minimum inhibitory concentrations (MIC) of these derivatives against a representative panel of vancomycin-sensitive and vancomycin-resistant Gram-positive organisms are summarized in Figure 3. The first GCP series 8a–d with a C-terminus modification exhibited improved activity relative to vancomycin and displayed a smooth trend of diminished potency as the linking chain length increased (potency: n = 2 > 3 > 4 > 5). The most potent of these was 8a and it was found to be ca. 10-fold more potent than vancomycin against the vancomycin-sensitive and ca. 100-fold more active against the vancomycin-resistant organisms tested, indicating a beneficial role for the C-terminus modification independent of D-Ala-D-Ala/D-Lac targeting. The compound 8a was also essentially equipotent with G3-vancomycin (3) against the vancomycin-sensitive organisms tested, but 2–8 fold less active against the vancomycin-resistant organisms examined. Notably, the behavior of C-terminus GCP modification mirrors that of the guanidine modification where G3 (H₂NC(=NH)NH(CH₂)₃NH-) was among the most potent in a series that also displays a potency that is dependent on the linker length (potency: (CH₂)_n where n = 3,4 > 2,5,6).⁶ The second series of C-terminus GCP-modified analogues 9a–d, which are combined with the additional peripherally CBP-modified vancosamine, displayed more potent activity. This is most easily observed by comparing the activity against the VanA E. faecium strain (column 1, Figure 3) where the potency was found to be improved by as much 3000-fold relative to vancomycin, ca. 30-fold or better relative to 8a, and >10-fold relative to CBP-vancomycin (2). However, in general, the improvement in potency was more muted relative to CBP-vancomycin (2) especially against vancomycin-sensitive organisms where the compounds likely act through three versus two independent mechanisms of action.

Figure 3.

Antimicrobial activity of C-terminus GCP-modified vancomycins.

In our previous studies exploring the timethylammonium salt modifications and the persistently protonated guanidinium cations, their introduction at the N-terminus,^3,6 (vs C-terminus) had little or no beneficial effect. In a similar fashion, the first GCP series 12a–c examined that directly interrogates its impact when it is introduced at this site resulted in a loss in activity against vancomycin-sensitive organisms (1.2–10 fold) and no detected activity against the vancomycin-resistant pathogens at the concentrations examined (Figure 4). The same impact was observed with the second series 13a–c with compounds that also contain the vancosamine CBP group where a significant loss in potency was observed relative to CBP-vancomycin (2) and the detrimental effect was more evident. At best, this reduction in potency was 2–10 fold (for 13b) and often even larger for the remaining compounds in the series (Figure 4).

Figure 4.

Antimicrobial activity of N-terminus GCP-modified vancomycins.

The GCP C-Terminus Modification Induces Bacteria Cell Permeabilization: An Added Synergistic Mechanism of Action.

To establish whether the mechanism of action introduced by the peripheral C-terminus modification is the same as that demonstrated earlier in our work for the trimethylammonium salt and guanidinium cation,^2–7 the representative compound 9a was examined in a permeability assay and conducted alongside 1 and 2 (Figure 5) with a vancomycin-resistant enterococci (VRE) strain. While no permeabilization was induced by either vancomycin or CBP-vancomycin, a strong induced cell envelop permeabilization was observed with GCP,CBP-vancomycin 9a in accordance with our prior observations made with both the analogous trimethylammonium salt and guanidinium cation modifications.^2,5–8 Although limited to the examination of 9a, the results suggest incorporation of the C-terminus GCP group for the 8a–d and 9a–d series increases antimicrobial activity by introducing a mechanism of action independent of D-Ala-Ala/D-Lac binding and synergistic with the CBP modification that functionally induces cell permeability and further impacts the cell wall integrity.

Figure 5.

Examination of cell membrane permeability in VanA VRE (E. faecium BAA-2317) induced by compounds 1 and 2 (no induced permeability) versus 9a (induced permeability) at 10 μM (added at 5 min). Cell envelop permeability is indicated by an increase in fluorescence resulting from propidium iodide (PI) influx following compound administration.

DISCUSSION AND CONCLUSIONS

Vancomycin displays broad spectrum antimicrobial activity against Gram-positive pathogens,¹⁶ where its activity against the ESKAPE pathogens Enterococcus faecalis/faecium and S. aureus is of special importance. It is the most widely recognized antibiotic used for treatment of refractory Gram-positive bacterial infections including methicillin-resistant S. aureus (MRSA). Its use and that of related glycopeptide antibiotics¹⁶ or the clinically approved semisynthetic derivatives^17–19 have steadily increased over the past decades.²⁰ Although it possesses an unusually durable mechanism of action,¹⁶ resistance is now common in enterococci (VRE) and is emerging in S. aureus (VRSA).^21,22 These developments along with the rise in antibiotic resistance in general pose a public health crisis, requiring the development of new antibiotics.

Complementary to efforts to directly overcome the molecular basis of vancomycin resistance²³ through redesign of the binding pocket,²⁴ permitting dual D-Ala-D-Ala/D-Lac binding, we have reported the incorporation of peripheral modifications that synergistically improve potency, add independent mechanisms of action, and further enhance their durability against raising resistance.⁷ We disclosed two previous C-terminus modifications, incorporating a trimethylammonium salt and later an even more effective guanidinium cation,^2–6 that were found to be effective and synergistic with key pocket modifications as well as a well-established CBP vancosamine modification that imparts direct transglycosylase inhibition.^25–28 The latter two result in cell wall biosynthesis inhibition but do so by two distinct mechanisms, only one of which is dependent on D-Ala-D-Ala/D-Lac binding. The C-terminus modifications we introduced induce cell envelop permeability, reducing cell wall/membrane integrity, without cell lysis or depolarization, and its functional activity is also independent of D-Ala-D-Ala/D-Lac binding.^2–6 When combined, these modifications provide vancomycin analogs with up to three independent mechanisms of action, only one of which is dependent on the original ligand binding.⁷ Notably, the nature of the trimethylammonium cation effect that we described (specific for the trimethylammonium salt) is distinct from the long chain quaternary ammonium salt effects²⁹ for which the trimethylammonium salt is ineffective, but it and the alternative protonated guanidinium cation may be related to the independent efforts with C-terminus Arg modifications described by Wender and Cegelski.^30–33 The later discoveries have since been extended by Wender and Cegelski ³⁴ and by others in creative ways.^35–37

Herein we disclose a third effective and attractive C-terminus modification, incorporation of a cationic guanidiniocarbonyl-pyrrole (GCP), first described by Schmuck and shown to improve guanidine binding to oxyanions including carboxylates and phosphates in water.¹⁰ Like the observations made with the prior trimethylammonium salt and guanidinium modifications, the beneficial effects observed with the GCP introduction at the C-terminus, but not N-terminus, indicate it is both structure (e.g., linker length) and site specific.^2–6 This is consistent with its targeting a specific feature in the bacterial cell wall as opposed to a nonspecific role attributable to a cationic modification, especially given its more modest pK_a (7–8 vs 13, Figure 2). For the prior guanidinium modification, we have also shown that it does not impact ligand binding affinity and that added lipoteichoic acid reduced and eliminated the antimicrobial effect of the guanidinium modification and prevented the induced cell envelop permeability.⁶ Combined, these and other studies implicated cell wall embedded teichoic acid³⁸ and its phosphate backbone as a likely binding target for the modifications and responsible for the added synergistic mechanism of action. The functional effects of this added synergistic mechanism of action imparted by the C-terminus GCP modification is apparent in the behavior of 8a, which exhibited pronounced improvements in activity against vancomycin-resistant bacteria relative to vancomycin (ca. 100-fold) and improved activity against vancomycin-sensitive organisms ca. 10-fold where the series (8a–d) also displayed a clear smooth dependence on the linker length (potency: n = 2 > 3 > 4 > 5) most evident against the vancomycin-sensitive organisms examined. When combined with the added CBP peripheral modification, the activity of the series 9a–d against vancomycin-resistant organisms was found to be improved by as much 3000-fold relative to vancomycin, ca. 30-fold or better relative to 8a, and as much as 10-fold relative to CBP-vancomycin (2). Combined, these observations are consistent with an effect that is synergistic with and independent of the effects derived from vancomycin pocket modifications that impart D-Ala-D-Ala/Lac binding as well as direct transglycosylase inhibition introduced with the CBP sugar modification. As such, the GCP group serves as an effective and unique vancomycin C-terminus modification, complementary to those previously disclosed.

EXPERIMENTAL

General Methods

All reagents and solvents were used as supplied without further purification unless otherwise noted. Preparative TLC (PTLC) and column chromatography were conducted using Millipore SiO₂ 60 F₂₅₄ PTLC (0.5 mm) plates. ¹H and ¹³C{1H} NMR spectra were obtained on a Bruker Avance III HD 600 MHz spectrometer equipped with either a 5 mm QCI or 5 mm CPDCH probe. Chemical shifts (δ) are reported in parts per million (ppm). Abbreviations used to designate multiplicities are: s = singlet, d = doublet, t = triplet, q = quartet, qu = quintet, m = multiplet. Coupling constants (J) are reported in Hertz (Hz). Mass spectrometry analysis was performed by direct sample injection on an Agilent G1969A ESI-TOF mass spectrometer. Structural assignments were made with additional information from TOCSY/NOSEY experiments. Analytical and preparative reverse-phase HPLC was performed using a Waters HPLC. In vitro antimicrobial activity was determined on samples established to be ≥95% pure by RP-HPLC under conditions detailed in each experimental procedure.

Compound 6a.

A solution of 5 (138 mg, 0.59 mmol, 1 equiv) and tert-butyl (2-aminoethyl)carbamate (188 μL, 1.18 mmol, 2 equiv) in anhydrous DMF (1.5 mL) was treated with N-methylmorpholine (NMM, 0.196 mL, 1.77 mmol, 3 equiv) and T3P (50% wt in EtOAc, 0.518 mL, 0.88 mmol, 1.5 equiv). The reaction mixture was stirred for 18 h at room temperature under argon. The solvent was concentrated and the residue was purified by semi-preparative reverse-phase HPLC (Waters XBridge^® Prep 5μm OBD^™ C18–19 × 150 mm, MeCN/H₂O (0.1% HCOOH) = 35/65–55/45 gradient over 12 min, flow rate = 30 mL/min, λ = 278 nm, t_R = 4.5 min) to provide 6a (142 mg, 71%) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 6.86 (d, J = 4.2 Hz, 1H), 6.79 (d, J = 3.6 Hz, 1H), 3.43 (t, J = 6.1 Hz, 2H), 3.34–3.32 (m, 4H), 3.26 (t, J = 6.2 Hz, 2H), 1.42 (s, 9H); ¹³C{1H} NMR (150 MHz, CD₃OD) δ 161.4, 161.1, 157.3, 130.0, 124.8, 115.1, 111.2 (2C), 78.8, 39.5, 39.4, 27.3 (3C); HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₁₄H₂₃N₆O₄⁺ 339.1775; Found 339.1773.

Compound 6b.

A solution of 5 (149 mg, 0.64 mmol, 1 equiv) and tert-butyl (3-aminopropyl)carbamate (224 mg, 1.28 mmol, 2 equiv) in anhydrous DMF (2 mL) was treated with NMM (0.707 mL, 6.43 mmol, 10 equiv) and T3P (50% wt in EtOAc, 1.9 mL, 3.22 mmol, 5 equiv). The reaction mixture was stirred for 22 h at room temperature under argon. The solvent was concentrated and the residue was purified by semi-preparative reverse-phase HPLC (Waters XBridge^® Prep 5μm OBD^™ C18–19 × 150 mm, MeCN/H₂O (0.1% HCOOH) = 35/65–55/45 gradient over 12 min, flow rate = 30 mL/min, λ = 278 nm, t_R = 5.1 min) to provide 6b (168 mg, 74%) as a white solid. ¹H NMR (600 MHz, CD₂Cl₂) δ 10.26 (pyrrole-NH, 1H), 7.73 (NH, 1H), 6.92–6.91 (m, 1H), 6.82–6.76 (m, 1H), 5.99–5.64 (m, 2H), 5.11–4.97 (m, 1H), 3.48 (q, J = 6.1 Hz, 2H), 3.31–3.21 (m, 2H), 1.79–1.66 (m, 2H), 1.49 (s, 9H); ¹³C{1H} NMR (150 MHz, CD₂Cl₂) δ 160.7, 160.5, 159.6, 159.4, 157.4, 129.4, 125.2, 115.4, 110.6, 79.8, 36.9, 35.6, 29.8, 28.0 (3C); HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₁₅H₂₅N₆O₄⁺ 353.1932; Found 353.1933.

Compound 6c.

A solution of 5 (46.7 mg, 0.20 mmol, 1 equiv) and tert-butyl (4-aminobutyl)carbamate (0.072 μL, 0.40 mmol, 2 equiv) in anhydrous DMF (1.2 mL) was treated with NMM (0.133 mL, 1.21 mmol, 6 equiv) and T3P (50% wt in EtOAc, 0.377 mL, 2.42 mmol, 2 equiv). The reaction mixture was stirred for 48 h at room temperature under argon. The solvent was concentrated and the residue was purified by semi-preparative reverse-phase HPLC (Waters XBridge^® Prep 5μm OBD^™ C18–19 × 150 mm, MeCN/H₂O (0.1% HCOOH) = 25/75–45/55 gradient over 12 min, flow rate = 30 mL/min, λ = 254 nm, t_R = 7.6 min) to provide 6c (37.5 mg, 51%) as a white solid. ¹H NMR (600 MHz, CD₂Cl₂) δ 10.17–9.99 (pyrrole-NH, 1H), 6.76 (d, J = 3.9 Hz, 1H), 6.71–6.66 (m, 1H), 6.60–6.56 (m, 1H), 5.11–4.97 (m, 1H), 3.34 (q, J = 6.3 Hz, 2H), 3.06 (q, J = 6.0 Hz, 2H), 3.03–2.98 (m, 1H), 1.81–1.62 (m, 5H), 1.55–1.50 (m, 2H), 1.49–1.45 (m, 2H), 1.34 (s, 9H); ¹³C{1H} NMR (150 MHz, CD₂Cl₂) δ 160.7, 159.8, 156.3, 156.0, 130.0, 124.6, 115.2, 109.8, 79.0, 40.0, 39.8, 39.2, 29.6, 28.1 (3C), 25.9; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₁₆H₂₇N₆O₄⁺ 367.2088; Found 367.2085.

Compound 6d.

A solution of 5 (100 mg, 0.43 mmol, 1 equiv) and tert-butyl (5-aminopentyl)carbamate (0.180 μL, 0.86 mmol, 2 equiv) in anhydrous DMF (4 mL) was treated with NMM (0.284 mL, 2.58 mmol, 6 equiv) and T3P (50% wt in EtOAc, 0.506 mL, 0.86 mmol, 2 equiv). The reaction mixture was stirred for 48 h at room temperature under argon. The solvent was concentrated and the residue was purified by semi-preparative reverse-phase HPLC (Waters XBridge^® Prep 5μm OBD^™ C18–19 × 150 mm, MeCN/H₂O (0.1% HCOOH) = 45/55–65/35 gradient over 12 min, flow rate = 30 mL/min, λ = 258 nm, t_R = 6.5 min) to provide 6d (37.5 mg, 51%) as a white solid. ¹H NMR (600 MHz, CD₂Cl₂) δ 10.09–9.92 (pyrrole-NH, 1H), 6.89 (dd, J = 2.8, 1.1 Hz, 1H), 6.63–6.59 (m, 1H), 6.27–6.19 (m, 1H), 4.72–4.61 (m, 1H), 3.42 (q, J = 6.8 Hz, 2H), 3.12 (t, J = 6.5 Hz, 2H), 1.79–1.70 (m, 2H), 1.63 (qu, J = 7.2 Hz, 2H), 1.53 (qu, J = 7.2 Hz, 2H), 1.44 (s, 9H), 1.42–1.39 (m, 2H); ¹H NMR (600 MHz, CD₃OD)* δ 6.86 (d, J = 3.9 Hz, 1H), 6.80 (d, J = 3.9 Hz, 1H), 3.32 (qu, J = 2.4 Hz, 2H), 3.05 (t, J = 6.9 Hz, 2H), 1.62 (qu, J = 7.3 Hz, 2H), 1.52 (qu, J = 6.9 Hz, 2H), 1.43 (s, 9H), 1.41–1.37 (m, 2H);¹³C{1H} NMR (150 MHz, CD₃OD) δ 161.2, 161.1, 157.2, 130.2, 124.8, 115.1 (2C), 111.2, 78.4, 39.8, 38.9, 29.2, 28.8, 27.4 (3C), 23.8; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₁₇H₂₉N₆O₄⁺ 381.2245; Found 381.2244.

*Exchangeable protons disappeared

General Procedure for 7a-7d (Boc Removal):

Compound 6a-6d were subjected to Boc deprotection by adding acetonitrile and trifluoroacetic acid (CH₃CN:TFA, 1:1, 0.4 mL:0.4 mL) and the resulting solution stirred at room temperature (25 °C) for 2 h. Concentration under nitrogen gave the 7a-7d TFA salts, which were used freshly for their corresponding coupling reactions.

Compound 8a.

A solution of vancomycin hydrochloride (13.0 mg, 9.0 μmol) and 7a (21.5 mg, 90.2 μmol, 10 equiv) in anhydrous DMF (600 μL) was treated with NMM (29.7 μL, 271 μmol, 30 equiv), followed by T3P (50% wt in EtOAc, 54.2 μL, 90.2 μmol, 10 equiv). The reaction mixture was stirred for 20 h at room temperature under argon. Direct purification by semi-preparative reverse-phase HPLC (Phenomenex Luna^® C18–100 × 30 mm, MeCN/H₂O (0.07% TFA) = 05/95–60/40 gradient over 30 min, flow rate = 15 mL/min, λ = 254 nm, t_R = 4.6 min) afforded 8a tris-TFA salt (8.7 mg, 51%) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 9.03 (br s, 1H), 8.66 (br s, 1H), 8.31 (br s, 1H), 7.73–6.63 (m, 4H), 7.24 (br s, 1H), 7.06 (s, 1H), 6.83–6.78 (m, 3H), 6.43 (s, 1H), 6.37 (s, 1H), 5.98–5.89 (m, 1H), 5.55–5.43 (m, 3H), 5.38 (br s, 1H), 5.31 (br s, 1H), 4.71 (br s, 1H) 4.62 (br s, 1H), 4.32 (br s, 1H), 4.25 (br s, 1H), 4.07 (br s, 1H), 3.86 (br s, 1H), 3.73–3.63 (m, 2H), 3.55–3.50 (m, 2H), 3.48 (s, 1H), 3.25 (s, 1H), 2.92–2.88 (m, 1H), 2.78 (s, 3H), 2.08 (dd, J = 13.8, 4.3 Hz, 1H), 1.96–1.94 (m, 1H), 1.90–1.84 (m, 1H), 1.74–1.63 (m, 2H), 1.53 (s, 3H), 1.21 (d, J = 5.6 Hz, 3H), 1.02–0.88 (m, 6H); HRMS (ESI-TOF) m/z: [M + 2H]²⁺ Calcd for C₇₅H₈₉Cl₂N₁₅O₂₅²⁺ 834.7760; Found 834.7763.

Compound 8b.

A solution of vancomycin hydrochloride (6.2 mg, 4.3 μmol) and 7b (10.9 mg, 43.2 μmol, 10 equiv) in anhydrous DMF (500 μL) was treated with NMM (14 μL, 130 μmol, 30 equiv), followed by T3P (50% wt in EtOAc, 50.8 μL, 86.4 μmol, 10 equiv). The reaction mixture was stirred for 20 h at room temperature under argon. Direct purification by semi-preparative reverse-phase HPLC (Phenomenex Luna^® C18–100 × 30 mm, MeCN/H₂O (0.07% TFA) = 05/95–50/50 gradient over 30 min, flow rate = 15 mL/min, λ = 254 nm, t_R = 5.1 min) afforded 8b tris-TFA salt (4.2 mg, 51%) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 7.68 (br s, 3H), 7.60 (d, J = 8.1 Hz, 2H), 7.18 (br s, 1H), 7.04 (s, 1H), 6.74 (br s, 1H), 6.43 (d, J = 2.1 Hz, 1H), 6.39 (d, J = 2.0 Hz, 1H), 5.96–5.80 (m, 1H), 5.53–5.40 (m, 2H), 5.33 (d, J = 6.2 Hz, 2H), 5.27 (s, 1H), 4.74–4.68 (m, 2H), 4.28 (br s, 1H), 4.16 (s, 1H), 4.04 (t, J = 6.9 Hz, 1H), 3.93–3.75 (m, 3H), 3.36–3.58 (m, 2H), 3.41 (t, J = 1.6 Hz, 1H), 3.39–3.35 (m, 1H), 3.18 (t, J = 1.6 Hz, 1H), 2.91 (d, J = 16.2 Hz, 1H), 2.76 (s, 3H), 2.05 (dd, J = 13.3, 4.1 Hz, 1H), 1.92 (d, J = 12.9 Hz, 1H), 1.67–1.64 (m, 3H), 1.52 (br s, 3H), 1.17 (d, J = 5.0 Hz, 3H), 1.09–1.01 (m, 2H), 1.00–0.89 (m, 6H); HRMS (ESI-TOF) m/z: [M + 2H]²⁺ Calcd for C₇₆H₉₁Cl₂N₁₅O₂₅²⁺ 841.7838; Found 841.7835.

Compound 8c.

A solution of vancomycin hydrochloride (5.8 mg, 4.3 μmol) and 7c (10.8 mg, 40.5 μmol, 10 equiv) in anhydrous DMF (600 μL) was treated with NMM (13 μL, 122 μmol, 30 equiv), followed by T3P (50% wt in EtOAc, 47.6 μL, 81.0 μmol, 10 equiv). The reaction mixture was stirred for 20 h at room temperature under argon. Direct purification by semi-preparative reverse-phase HPLC (Phenomenex Luna^® C18–100 × 30 mm, MeCN/H₂O (0.07% TFA) = 05/95–50/50 gradient over 30 min, flow rate = 15 mL/min, λ = 254 nm, t_R = 5.1 min) afforded 8c tris-TFA salt (3.4 mg, 44%) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 7.68 (br s, 3H), 7.60 (d, J = 9.6 Hz, 2H), 7.18 (br s, 1H), 7.04 (s, 1H), 6.73 (br s, 1H), 6.43 (s, 1H), 6.38 (s, 1H), 6.01–5.80 (m, 1H), 5.56–5.39 (m, 2H), 5.33 (d, J = 6.4 Hz, 2H), 5.27 (s, 1H), 4.70 (br s, 2H), 4.27 (br s, 1H), 4.16 (s, 1H), 4.04 (t, J = 6.9 Hz, 1H), 3.92 (br s, 1H), 3.85–3.78 (m, 2H), 3.63–3.60 (m, 2H), 3.42–3.34 (m, 2H), 3.18 (t, J = 1.6 Hz, 1H), 2.92 (d, J = 15.2 Hz, 1H), 2.76 (s, 3H), 2.06 (dd, J = 13.2, 4.2 Hz, 1H), 1.92 (d, J = 12.6 Hz, 1H), 1.71–1.62 (m, 3H), 1.52 (br s, 3H), 1.17 (d, J = 5.9 Hz, 3H), 1.09–1.00 (m, 2H), 0.99–0.90 (m, 6H); HRMS (ESI-TOF) m/z: [M + 2H]²⁺ Calcd for C₇₇H₉₃Cl₂N₁₅O₂₅²⁺ 848.7916; Found 848.7917.

Compound 8d.

A solution of vancomycin hydrochloride (6.3 mg, 4.3 μmol) and 7d (12.2 mg, 43.5 μmol, 10 equiv) in anhydrous DMF (600 μL) was treated with NMM (14.4 μL, 131 μmol, 30 equiv), followed by HBTU (16.5 mg, 43.5 μmol, 10 equiv). The reaction mixture was stirred for 48 h at room temperature under argon.* Direct purification by semi-preparative reverse-phase HPLC (Phenomenex Luna^® C18–100 × 30 mm, MeCN/H₂O (0.07% TFA) = 05/95–50/50 gradient over 30 min, flow rate = 15 mL/min, λ = 254 nm, t_R = 17.2 min) afforded 8d tris-TFA salt (3.7 mg, 44%) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 9.02 (br s, 1H), 8.60 (br s, 1H), 7.18 (br s, 1H), 7.71 (d, J = 12.2 Hz, 2H), 7.64 (d, J = 6.6 Hz, 1H), 7.23 (br s, 1H), 7.08 (s, 1H), 6.87 (d, J = 3.9 Hz, 1H), 6.80 (d, J = 3.9 Hz, 1H), 6.44 (s, 1H), 6.41 (s, 1H), 5.48 (br s, 1H), 5.54–5.48 (m, 2H), 5.37 (d, J = 17.7 Hz, 2H), 5.31(s, 1H), 5.10 (s, 1H), 4.71–4.66 (m, 2H), 4.31 (br s, 1H), 4.21 (s, 1H), 4.07 (t, J = 6.9 Hz, 1H), 3.94–3.91 (m, 1H), 3.87 (s, 3H), 3.82–3.76 (m, 1H), 3.75–3.62 (m, 2H), 3.44 (s, 1H), 3.40–3.36 (m, 2H), 3.21 (s, 1H), 2.93 (d, J = 14.4 Hz, 1H), 2.78 (s, 3H), 2.34–2.28 (m, 1H), 2.08 (dd, J = 13.2, 4.0 Hz, 1H), 1.94 (d, J = 13.0 Hz, 1H), 1.67–1.60 (m, 7H), 1.52 (br s, 3H), 1.46 (qu, J = 7.4 Hz, 3H), 1.21 (d, J = 6.1 Hz, 3H), 1.05–0.88 (m, 8H); HRMS (ESI-TOF) m/z: [M + 2H]²⁺ Calcd for C₇₈H₉₅Cl₂N₁₅O₂₅²⁺ 855.7995; Found 855.7991.

* T3P gave negligible amounts of product when either 10 or 20 equiv were used.

Compound 9a.

A solution of CBP-vancomycin bis-TFA salt (20.5 mg, 11.9 μmol) and 7a (28.4 mg, 119.2 μmol, 10 equiv) in anhydrous DMF (600 μL) was treated with NMM (39.3 μL, 358 μmol, 30 equiv), followed by T3P (50% wt in EtOAc, 70 μL, 119.2 μmol, 10 equiv). The reaction mixture was stirred for 22 h at room temperature under argon. Direct purification by semi-preparative reverse-phase HPLC (Phenomenex Luna^® C18–100 × 30 mm, MeCN/H₂O (0.07% TFA) = 05/95–50/50 gradient over 30 min, flow rate = 15 mL/min, λ = 254 nm, t_R = 10.2 min) afforded 9a tris-TFA salt (12.4 mg, 49%) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 9.03 (br s, 1H), 8.65 (br s, 1H), 7.76 (s, 1H), 7.71 (d, J = 7.6 Hz, 3H), 7.64 (d, J = 8.4 Hz, 4H), 7.57 (d, J = 7.9 Hz, 3H), 7.47 (d, J = 8.3 Hz, 3H), 7.27 (br s, 1H), 7.06 (s, 1H), 6.82–6.77 (m, 3H), 6.43 (s, 1H), 6.37 (s, 1H), 5.86 (br s, 1H), 5.48 (br s, 2H), 5.38–5.32 (m, 2H), 4.71 (br s, 2H), 4.66 (br s, 1H), 4.26 (s, 1H), 4.18 (d, J = 12.4 Hz, 1H), 4.14–4.06 (m, 2H), 3.87 (s, 3H), 3.81–3.76 (m, 1H), 3.67–3.63 (m, 2H), 3.55–3.50 (m, 2H), 3.48 (s, 1H), 3.00 (s, 3H), 2.92–2.90 (m, 1H), 2.78 (s, 2H), 2.21–2.19 (m, 2H), 2.08–2.03 (m, 1H), 1.76–1.63 (m, 6H), 1.28 (d, J = 5.4 Hz, 3H), 1.00–0.92 (m, 6H); HRMS (ESI-TOF) m/z: [M + 2H]²⁺ Calcd for C₈₈H₉₈Cl₃N₁₅O₂₅²⁺ 934.7956; Found 934.7961.

Compound 9b.

A solution of CBP-vancomycin bis-TFA salt (12.3 mg, 7.1 μmol) and 7b (18.0 mg, 71.3 μmol, 10 equiv) in anhydrous DMF (300 μL) was treated with NMM (23.5 μL, 214 μmol, 30 equiv), followed by T3P (50% wt in EtOAc, 42 μL, 71.3 μmol, 10 equiv). The reaction mixture was stirred for 22 h at room temperature under argon. Direct purification by semi-preparative reverse-phase HPLC (Phenomenex Luna^® C18–100 × 30 mm, MeCN/H₂O (0.07% TFA) = 05/95–50/50 gradient over 30 min, flow rate = 15 mL/min, λ = 254 nm, t_R = 8.4 min) afforded 9b tris-TFA salt (6.3 mg, 42%) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 9.01 (br s, 1H), 8.72 (br s, 1H), 7.72 (d, J = 8.2 Hz, 4H), 7.64 (d, J = 8.5 Hz, 4H), 7.57 (d, J = 7.9 Hz, 2H), 7.48 (d, J = 8.4 Hz, 3H), 7.23 (br s, 1H), 7.07 (s, 1H), 6.80–6.75 (m, 1H), 6.46 (d, J = 2.1 Hz, 1H), 6.41 (s, 1H), 5.87 (br s, 1H), 5.50 (br s, 1H), 5.37 (br s, 2H), 5.30 (br s, 1H), 4.75 (br s, 2H), 4.29 (br s, 1H), 4.18 (d, J = 12.4 Hz, 2H), 4.12 (br s, 1H), 4.06 (t, J = 7.0 Hz, 1H), 3.86 (t, J = 8.8 Hz, 1H), 3.82–3.76 (m, 1H), 3.68–3.61 (m, 2H), 3.44–3.38 (m, 2H), 3.21 (t, J = 1.5 Hz, 1H), 3.01 (s, 1H), 2.94 (d, J = 14.2 Hz, 1H), 2.97 (s, 3H), 2.07 (dd, J = 13.3, 4.6 Hz, 1H), 2.04 (d, J = 13.0 Hz, 1H), 1.81 (br s, 1H), 1.76–1.65 (m, 5H), 1.31 (s, 1H), 1.27 (br s, 3H), 1.06–0.90 (m, 6H); HRMS (ESI-TOF) m/z: [M + 2H]²⁺ Calcd for C₈₉H₁₀₀Cl₃N₁₅O₂₅²⁺ 941.8034; Found 941.8032.

Compound 9c.

A solution of CBP-vancomycin bis-TFA salt (10.3 mg, 6.0 μmol) and 7c (16.0 mg, 60.0 μmol, 10 equiv) in anhydrous DMF (400 μL) was treated with NMM (19.8 μL, 180 μmol, 30 equiv), followed by T3P (50% wt in EtOAc, 35.4 μL, 60.0 μmol, 10 equiv). The reaction mixture was stirred for 22 h at room temperature under argon. Direct purification by semi-preparative reverse-phase HPLC (Phenomenex Luna^® C18–100 × 30 mm, MeCN/H₂O (0.07% TFA) = 05/95–50/50 gradient over 30 min, flow rate = 15 mL/min, λ = 254 nm, t_R = 8.4 min) afforded 9c tris-TFA salt (4.9 mg, 39%) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 9.00 (br s, 1H), 8.71 (br s, 1H), 7.72 (d, J = 8.1 Hz, 4H), 7.64 (d, J = 8.4 Hz, 4H), 7.57 (d, J = 8.1 Hz, 2H), 7.48 (d, J = 8.3 Hz, 3H), 7.24 (br s, 1H), 7.07 (s, 1H), 6.78–6.75 (m, 1H), 6.45 (d, J = 1.9 Hz, 1H), 6.42 (s, 1H), 5.97 (br s, 1H), 5.59–5.46 (m, 2H), 5.39–5.34 (m, 2H), 5.30 (br s, 1H), 4.74 (br s, 2H), 4.29 (br s, 1H), 4.18 (d, J = 12.0 Hz, 2H), 4.14 (br s, 1H), 4.06 (t, J = 6.5 Hz, 1H), 3.68–3.62 (m, 3H), 3.44–3.42 (m, 1H), 3.21 (t, J = 1.5 Hz, 1H), 3.00 (s, 1H), 2.94 (d, J = 14.6 Hz, 1H), 2.79 (s, 3H), 2.22–2.17 (m, 3H), 2.04 (d, J = 11.8 Hz, 1H), 1.87 (br s, 1H), 1.76–1.65 (m, 5H), 1.34–1.31 (m, 1H), 1.26 (br s, 4H), 1.04–0.92 (m, 6H); HRMS (ESI-TOF) m/z: [M + 2H]²⁺ Calcd for C₉₀H₁₀₂Cl₃N₁₅O₂₅²⁺ 948.8113; Found 948.8117.

Compound 9d.

A solution of CBP-vancomycin bis-TFA salt (9.7 mg, 5.6 μmol) and 7d (15.9 mg, 56.7 μmol, 10 equiv) in anhydrous DMF (300 μL) was treated with NMM (18.7 μL, 170 μmol, 30 equiv), followed by T3P (50% wt in EtOAc, 33.4 μL, 56.7 μmol, 10 equiv). The reaction mixture was stirred for 22 h at room temperature under argon. Direct purification by semi-preparative reverse-phase HPLC (Phenomenex Luna^® C18–100 × 30 mm, MeCN/H₂O (0.07% TFA) = 05/95–50/50 gradient over 30 min, flow rate = 15 mL/min, λ = 254 nm, t_R = 10.2 min) afforded 9d tris-TFA salt (6.0 mg, 50%) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 9.02 (br s, 1H), 8.61 (br s, 1H), 8.16 (br s, 1H), 7.75 (s, 1H), 7.72 (d, J = 8.2 Hz, 4H), 7.64 (d, J = 8.4 Hz, 4H), 7.57 (d, J = 8.1 Hz, 3H), 7.48 (d, J = 8.4 Hz, 3H), 7.26 (br s, 1H), 7.08 (s, 1H), 6.86 (d, J = 3.9 Hz, 2H), 6.80 (d, J = 3.9 Hz, 2H), 6.44 (s, 1H), 6.41 (s, 1H), 5.82 (br s, 1H), 5.56–5.48 (m, 2H), 5.40–5.36 (m, 2H), 5.32 (br s, 1H), 4.71–4.67 (m, 1H), 4.22 (br s, 1H), 4.19–4.15 (m, 1H), 4.11–4.06 (m, 1H), 3.87 (d, J = 4.2 Hz, 6H), 3.78 (br s, 1H), 3.63 (br s, 2H), 3.21–3.19 (m, 1H), 3.01 (s, 2H), 2.96–2.91 (m, 1H), 2.78 (s, 2H), 2.21–2.18 (m, 1H), 2.04 (d, J = 12.8 Hz, 1H), 1.72–1.60 (m, 12H), 1.48–1.42 (m, 4H), 1.30 (br s, 2H), 1.27 (d, J = 6.0 Hz, 4H), 1.06–0.96 (m, 6H); HRMS (ESI-TOF) m/z: [M + 2H]²⁺ Calcd for C₉₁H₁₀₄Cl₃N₁₅O₂₅²⁺ 955.8191; Found 955.8196.

Compound 10a.

Vancomycin hydrochloride (52.6 mg, 36.3 μmol) and tert-butyl (2-oxoethyl)carbamate (145 mg, 908 μmol, 25 equiv) were dissolved in a mixture of 1% AcOH in DMF (v:v, 0.8/7.2 mL). NaBH₃CN in THF (1.0 M, 1.815 mL, 1.815 mmol, 50 equiv) was then added to the solution and the mixture was stirred at 40 °C (heating block) for 6 h, followed by stirring for 18 h at room temperature under argon. The mixture was concentrated under nitrogen and purified by semi-preparative reverse-phase HPLC (Waters XBridge^® Prep 5μm OBD^™ C18–19 × 150 mm, MeCN/H₂O (0.07% TFA) = 15/85–35/65 gradient over 12 min, flow rate = 30 mL/min, λ = 220 nm, t_R = 10.2 min) to afford 10a bis-TFA salt (32.4 mg, 56%) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 7.56 (t, J = 12.2 Hz, 2H), 7.46–7.40 (m, 2H), 7.16 (d, J = 8.4 Hz, 1H), 6.90 (s, 1H), 6.85 (d, J = 8.7 Hz, 1H), 6.70 (d, J = 8.4 Hz, 1H), 6.43 (d, J = 1.9 Hz, 1H), 6.29 (d, J = 2.1 Hz, 1H), 5.62 (br s, 1H), 5.35–5.28 (m, 3H), 5.25–5.21 (m, 2H), 4.58 (s, 1H), 4.52 (s, 1H), 4.43 (br s, 1H), 4.02 (s, 1H), 3.73 (br s, 2H), 3.63 (dd, J = 11.5, 4.4 Hz, 1H), 3.55–3.48 (m, 1H), 3.45–3.40 (m, 2H), 3.32 (s, 1H), 3.27 (br s, 4H), 2.94–2.91 (m, 1H), 2.86–2.83 (m, 1H), 2.61 (d, J = 14.5 Hz, 1H), 2.58–2.52 (m, 2H), 2.31 (s, 3H), 1.91 (s, 1H), 1.62–1.58 (m, 1H), 1.56–1.52 (m, 1H), 1.41–1.38 (m, 2H), 1.36–1.34 (m, 3H), 1.32 (s, 9H), 1.31 (br s, 3H), 1.12 (d, J = 6.4 Hz, 3H), 0.82 (t, J = 5.1 Hz, 6H); HRMS (ESI-TOF) m/z: [M + 2H]²⁺ Calcd for C₇₃H₉₀Cl₂N₁₀O₂₆²⁺ 796.2697; Found 796.2700.

Compound 10b.

Vancomycin hydrochloride (61.9 mg, 42.7 μmol) and tert-Butyl (3-oxopropyl)carbamate (111 mg, 641 μmol, 15 equiv) were dissolved in a mixture of 1% AcOH in DMF (v:v, 0.5/4.5 mL). Then NaBH₃CN in THF (1.0 M, 2.138 mL, 2.138 mmol, 50 equiv) was then added to the solution and the mixture was stirred at 40 °C (heating block) for 6 h, followed by stirring for 18 h at room temperature under argon. The mixture was concentrated under nitrogen and purified by semi-preparative reverse-phase HPLC (Waters XBridge^® Prep 5μm OBD^™ C18–19 × 150 mm, MeCN/H₂O (0.07% TFA) = 05/95–95/05 gradient over 12 min, flow rate = 30 mL/min, λ = 220 nm, t_R = 3.6 min) to afford 10b bis-TFA salt (26.7 mg, 39%) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 7.72–7.68 (m, 3H), 7.62 (s, 1H), 7.19 (br s, 1H), 7.08 (s, 1H), 6.74 (br s, 1H), 6.46 (t, J = 1.8 Hz, 1H), 6.43 (s, 1H), 6.01 (br s, 1H), 5.54–5.48 (m, 2H), 5.38 (d, , J = 10.1 Hz, 2H), 5.30 (s, 1H), 4.73 (br s, 3H), 4.19 (s, 1H), 3.88–3.83 (m, 2H), 3.65 (br s, 2H), 3.25–3.22 (m, 2H), 3.19–3.15 (m, 1H), 3.07 (s, 1H), 2.99–2.90 (m, 3H), 2.08 (d, J = 8.2 Hz, 2H), 1.95 (d, J = 11.2 Hz, 2H), 1.68–1.61 (m, 1H), 1.59–1.51 (m, 2H), 1.44 (s, 9H), 1.36–1.29 (m, 2H), 1.19 (s, 4H), 0.98 (t, J = 5.2 Hz, 4H); HRMS (ESI-TOF) m/z: [M + 2H]²⁺ Calcd for C₇₄H₉₂Cl₂N₁₀O₂₆²⁺ 803.2775; Found 803.2779.

Compound 10c.

Vancomycin hydrochloride (49.9 mg, 34.4 μmol) and tert-butyl (4-oxobutyl)carbamate (64.5 mg, 344 μmol, 10 equiv) were dissolved in a mixture of 1% AcOH in DMF (v:v, 0.2/1.8 mL). NaBH₃CN in THF (1.0 M, 1.722 mL, 1.722 mmol, 50 equiv) was then added to the solution and the mixture was stirred at 50 °C (heating block) for 24 h under argon. The mixture was concentrated under nitrogen and purified by semi-preparative reverse-phase HPLC (Waters XBridge^® Prep 5μm OBD^™ C18–19 × 150 mm, MeCN/H₂O (0.07% TFA) = 10/90–25/75 gradient over 12 min, flow rate = 30 mL/min, λ = 223 nm, t_R = 4.5 min) to afford 10c bis-TFA salt (28.4 mg, 51%) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 7.81–7.72 (m, 2H), 7.69 (s, 1H), 7.60 (d, J = 6.9 Hz, 1H), 7.16 (d, J = 5.6 Hz, 1H), 7.06 (s, 1H), 6.64 (br s, 1H), 6.47 (br s, 1H), 6.43 (s, 1H), 6.04 (br s, 1H), 5.58 (br s, 1H), 5.51 (br s, 1H), 5.38 (d, J = 6.7 Hz, 2H), 5.28 (s, 1H), 4.76 (br s, 2H), 4.66 (s, 1H), 4.27 (br s, 1H), 4.16 (s, 1H), 4.03 (t, J = 5.5 Hz, 1H), 3.88–3.84 (m, 2H), 3.65 (br s, 2H), 3.44–3.40 (m, 1H), 2.95 (d, J = 13.1 Hz, 1H), 2.79 (s, 3H), 2.08 (dd, J = 11.4, 3.4 Hz, 1H), 1.95 (d, J = 11.4 Hz, 1H), 1.85–1.80 (m, 1H), 1.71–1.65 (m, 2H), 1.57 (s, 3H), 1.19 (s, 3H), 0.95 (s, 6H); HRMS (ESI-TOF) m/z: [M + 2H]²⁺ Calcd for C₇₅H₉₄Cl₂N₁₀O₂₆²⁺ 810.2853; Found 810.2852.

Compound 11a.

CBP-Vancomycin bis-TFA salt (57.2 mg, 34.6 μmol) and tert-butyl (2-oxoethyl)carbamate (138 mg, 867 μmol, 25 equiv) were dissolved in a mixture of 1% AcOH in DMF (v:v, 0.5/4.5 mL). NaBH₃CN in THF (1.0 M, 1.733 mL, 1.733 mmol, 50 equiv) was then added to the solution and the mixture was stirred at 40 °C (heating block) for 6 h, followed by stirring for 18 h at room temperature under argon. The mixture was concentrated under nitrogen and purified by semi-preparative reverse-phase HPLC (Waters XBridge^® Prep 5μm OBD^™ C18–19 × 150 mm, MeCN/H₂O (0.07% TFA) = 25/75–45/55 gradient over 12 min, flow rate = 30 mL/min, λ = 226 nm, t_R = 7.1 min) to afford 11a bis-TFA salt (33.8 mg, 54%) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 7.72 (d, J = 9.9 Hz, 3H), 7.68 (d, J = 8.6 Hz, 1H), 7.64 (d, J = 8.4 Hz, 4H), 7.57 (d, J = 7.9 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 7.30 (br s, 1H), 7.06 (s, 1H), 6.82 (br s, 1H), 6.45 (d, J = 8.8 Hz, 2H), 6.17 (br s, 1H), 5.81 (br s, 2H), 5.57–5.47 (m, 2H), 5.40 (s, 1H), 5.37–5.33 (m, 1H), 4.68 (br s, 1H), 4.18 (d, J = 11.9 Hz, 2H), 4.10 (d, J = 11.6 Hz, 1H), 3.94–3.84 (m, 2H), 3.81–3.74 (m, 2H), 3.69–3.61 (m, 3H), 3.56 (t, J = 5.8 Hz, 2H), 3.44 (t, J = 1.6 Hz, 1H), 3.21 (t, J = 1.6 Hz, 1H), 3.17 (t, J = 5.8 Hz, 1H), 3.10 (d, J = 12.7 Hz, 1H), 2.20 (dd, J = 15.1, 6.3 Hz, 1H), 2.04 (d, J = 12.6 Hz, 1H), 1.68 (s, 3H), 1.54–1.51 (m, 6H), 1.45 (br s, 9H), 1.27 (d, J = 5.9 Hz, 3H), 1.01–0.94 (m, 6H); HRMS (ESI-TOF) m/z: [M + 2H]²⁺ Calcd for C₈₆H₉₉Cl₃N₁₀O₂₆²⁺ 896.2893; Found 896.2890.

Compound 11b.

CBP-Vancomycin bis-TFA salt (64.7 mg, 39.2 μmol) and tert-butyl (3-oxopropyl)carbamate (102 mg, 589 μmol, 15 equiv) were dissolved in a mixture of 1% AcOH in DMF (v:v, 0.4/3.6 mL). NaBH₃CN in THF (1.0 M, 1.962 mL, 1.962 mmol, 50 equiv) was then added to the solution and the mixture was stirred at 40 °C (heating block) for 6 h, followed by stirring for 18 h at room temperature under argon. The mixture was concentrated under nitrogen and purified by semi-preparative reverse-phase HPLC (Waters XBridge^® Prep 5μm OBD^™ C18–19 × 150 mm, MeCN/H₂O (0.07% TFA) = 25/75–45/55 gradient over 12 min, flow rate = 30 mL/min, λ = 220 nm, t_R = 6.7 min) to afford 11b bis-TFA salt (28.7 mg, 41%) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 7.74 (br s, 1H), 7.71 (d, J = 6.6 Hz, 3H), 7.64 (d, J = 7.0 Hz, 4H), 7.57 (d, J = 6.2 Hz, 2H), 7.46 (d, J = 7.0 Hz, 2H), 7.26 (br s, 2H), 7.07 (s, 1H), 6.79 (br s, 1H), 6.50–6.42 (m, 2H), 5.86 (br s, 1H), 5.56–5.54 (m, 1H), 5.50 (s, 2H), 5.39 (d, J = 11.4 Hz, 2H), 5.33 (br s, 1H), 4.73 (br s, 2H), 4.18 (d, J = 10.2 Hz, 2H), 4.12 (d, J = 9.3 Hz, 1H), 3.87 (t, J = 6.4 Hz, 1H), 3.79–3.75 (m, 1H), 3.68–3.52 (m, 4H), 3.42 (br s, 1H), 3.21–3.11 (m, 3H), 2.93–2.79 (m 3H), 2.23–2.17 (m, 1H), 2.08–2.02 (m, 1H), 1.91–1.87 (m, 3H), 1.72–1.68 (m, 4H), 1.44 (br s, 9H), 1.28 (d, J = 5.0 Hz, 3H), 1.02–0.94 (m, 6H); HRMS (ESI-TOF) m/z: [M + 2H]²⁺ Calcd for C₈₇H₁₀₁Cl₃N₁₀O₂₆²⁺ 903.2971; Found 903.2974.

Compound 11c.

CBP-Vancomycin bis-TFA salt (56.2 mg, 34.0 μmol) and tert-butyl (4-oxobutyl)carbamate (63.8 mg, 341 μmol, 10 equiv) were dissolved in a mixture of 1% AcOH in DMF (v:v, 0.2/1.8 mL). NaBH₃CN in THF (1.0 M, 1.704 mL, 1.704 mmol, 50 equiv) was then added to the solution and the mixture was stirred at 50 °C (heating block) for 24 h under argon. The mixture was concentrated under nitrogen and purified by semi-preparative reverse-phase HPLC (Waters XBridge^® Prep 5μm OBD^™ C18–19 × 150 mm, MeCN/H₂O (0.07% TFA) = 15/85–35/65 gradient over 12 min, flow rate = 30 mL/min, λ = 220 nm, t_R = 9.3 min) to afford 11c bis-TFA salt (32.3 mg, 52%) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 8.98 (br s, 1H), 7.75 (br s, 1H), 7.72 (d, J = 7.0 Hz, 4H), 7.64 (d, J = 7.0 Hz, 4H), 7.57 (d, J = 6.8 Hz, 2H), 7.48 (d, J = 7.2 Hz, 3H), 7.21 (br s, 1H), 7.08 (s, 1H), 6.72 (br s, 1H), 6.45 (d, J = 1.5 Hz, 1H), 6.43 (d, J = 1.4 Hz, 1H), 6.06 (br s, 1H), 5.63 (br s, 1H), 5.52 (s, 1H), 5.39 (d, J = 13.4 Hz, 2H), 5.29 (s, 1H), 4.72 (br s, 2H), 4.28 (br s, 1H), 4.18 (d, J = 7.1 Hz, 2H), 4.16 (br s, 1H), 4.05 (t, J = 5.4 Hz, 1H), 3.86 (t, J = 6.8 Hz, 2H), 3.66 (br s, 2H), 3.62 (s, 2H), 3.57 (t, J = 5.1 Hz, 1H), 3.44 (br s, 1H), 3.07 (t, J = 5.7 Hz, 1H), 2.98–2.93 (m, 2H), 2.79 (s, 3H), 2.20 (dd, J = 11.3, 3.4 Hz, 1H), 2.05 (d, J = 11.2 Hz, 1H), 1.87 (s, 1H), 1.78–1.72 (m, 4H), 1.69–1.67 (m, 2H), 1.63 (t, J = 6.7 Hz, 1H), 1.54 (br s, 3H), 1.44 (s, 9H), 1.25 (d, J = 4.8 Hz, 3H), 0.94 (br s, 6H); HRMS (ESI-TOF) m/z: [M + 2H]²⁺ Calcd for C₈₈H₁₀₃Cl₃N₁₀O₂₆²⁺ 910.3050; Found 910.3052.

General Procedure for Boc Removal of Compounds 10a-c and 11a-c:

Compound 10a-c and 11a-c were subjected to Boc deprotection by adding acetonitrile and trifluoroacetic acid (CH₃CN:TFA, 1:1, 0.2 mL:0.2 mL) and stirred at room temperature (25 °C) for 2 h. The mixtures were concentrated under nitrogen to give the 10a-c and 11a-c TFA salts, which were used directly in their corresponding coupling reactions.

Compound 12a.

A solution of Boc-deprotected compound 10a (11.9 mg, 8.0 μmol) in anhydrous DMF (50 μL) was treated with iPr₂NEt (10 μL, pH = 7) and added to a solution of 15 (5.9 mg, 14.0 μmol, 1.7 equiv) in anhydrous DMF (450 μL) in an oven-dried half-dram vial. The reaction mixture was stirred for 24 h at 40 °C (heating block) under argon. Direct purification was performed by semi-preparative reverse-phase HPLC (Waters XBridge^® Prep 5μm OBD^™ C18–19 × 150 mm, MeCN/H₂O (0.07% TFA) = 35/65–55/45 gradient over 12 min, flow rate = 30 mL/min, λ = 220 nm, t_R = 2.1 min) to afford Boc-protected 12a. Subsequent Boc deprotection was carried out by adding acetonitrile and trifluoroacetic acid (CH₃CN:TFA, 1:1, 0.15 mL:0.15 mL) and stirring at room temperature (25 °C) for 2 h, yielding 12a tris-TFA salt (5.8 mg, 41%) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 9.01 (br s, 1H), 8.73 (br s, 1H), 7.76–7.75 (m, 1H), 7.71 (br s, 1H), 7.68 (br s, 1H), 7.64 (d, J = 8.7 Hz, 1H), 7.61–7.60 (m, 1H), 7.27 (d, J = 8.8 Hz, 1H), 7.06 (d, J = 2.4 Hz, 1H), 6.98 (br s, 1H), 6.82 (d, J = 8.6 Hz, 1H), 6,48 (d, J = 2.3 Hz, 1H), 6.43 (d, J = 2.3 Hz, 1H), 5.82 (br s, 1H), 5.48 (d, J = 7.3 Hz, 2H), 5.35 (br s, 2H), 5.30 (d, J = 3.5 Hz, 1H), 4.79 (d, J = 6.2 Hz, 1H), 4.68 (br s, 1H), 4.37 (br s, 1H), 4.19 (s, 1H), 3.98–3.60 (m, 2H), 3.59–3.45 (m, 1H), 3.44–3.37 (m, 1H), 3.37 (s, 1H), 2.96 (d, J = 14.8 Hz, 1H), 2.85–2.75 (m, 1H), 2.50 (s, 2H), 2.33–2.29 (m, 1H), 2.23–2.19 (m, 1H), 2.08–2.01 (m, 1H), 1.72–1.57 (m, 4H), 1.52–1.48 (m, 1H), 1.28–1.21 (m, 2H), 0.99 (t, J = 6.9 Hz, 8H); HRMS (ESI-TOF) m/z: [M + 2H]²⁺ Calcd for C₇₅H₈₈Cl₂N₁₄O₂₆²⁺ 835.2680; Found 835.2687.

Compound 12b.

A solution of Boc-deprotected compound 10b (5.7 mg, 3.8 μmol) in anhydrous DMF (50 μL) was treated with iPr₂NEt (7 μL, pH = 7) and added to a solution of 15 (2.8 mg, 6.6 μmol, 1.6 equiv) in anhydrous DMF (250 μL) in an oven-dried half-dram vial. The reaction mixture was stirred for 24 h at 40 °C (heating block) under argon. Direct purification was performed by semi-preparative reverse-phase HPLC (Waters XBridge^® Prep 5μm OBD^™ C18–19 × 150 mm, MeCN/H₂O (0.07% TFA) = 10/90–35/65 gradient over 12 min, flow rate = 30 mL/min, λ = 220 nm, t_R = 4.8 min) to afford Boc-protected 12b. Subsequent Boc deprotection was carried out by adding acetonitrile and trifluoroacetic acid (CH₃CN:TFA, 1:1, 0.15 mL:0.15 mL) and stirring at room temperature (25 °C) for 2 h, yielding 12b tris-TFA salt (2.8 mg, 42%) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 8.95 (br s, 1H), 8.11 (s, 1H), 8.00 (s, 3H), 7.69 (s, 1H), 7.61 (d, J = 8.3 Hz, 2H), 7.30 (d, J = 8.8 Hz, 1H), 7.05 (s, 1H), 6.81 (br s, 1H), 6.46 (d, J = 2.3 Hz, 1H), 6.44 (s, 1H), 5.74 (br s, 1H), 5.39 (s, 1H), 5.34 (s, 1H), 4.18 (br s, 1H), 4.15–4.10 (m, 1H), 3.48 (s, 1H), 3.44 (qu, J = 1.6 Hz, 3H), 3.21 (qu, J = 1.6 Hz, 3H), 3.01 (s, 9H), 2.88 (d, J = 0.7 Hz, 8H), 2.45 (br s, 3H), 2.30 (t, J = 7.5 Hz, 1H), 2.22 (t, J = 7.6 Hz, 1H), 2.08 (s, 1H), 2.05 (s, 1H), 2.01 (s, 1H), 1.88 (s, 1H), 1.73 (br s, 1H), 1.62 (br s, 1H), 1.60–1.48 (m, 1H), 1.37 (br s, 2H), 1.30 (br s, 1H), 1.24 (br s, 2H), 1.00–0.94 (m, 8H); HRMS (ESI-TOF) m/z: [M + 2H]²⁺ Calcd for C₇₆H₉₀Cl₂N₁₄O₂₆²⁺ 842.2758; Found 842.2753.

Compound 12c.

A solution of Boc-deprotected compound 10c (7.2 mg, 4.7 μmol) in anhydrous DMF (50 μL) was treated with iPr₂NEt (7 μL, pH = 7) and added to a solution of 15 (3.5 mg, 8.3 μmol, 1.7 equiv) in anhydrous DMF (350 μL) in an oven-dried half-dram vial. The reaction mixture was stirred for 24 h at 40 °C (heating block) under argon. Direct purification was performed by semi-preparative reverse-phase HPLC (Waters XBridge^® Prep 5μm OBD^™ C18–19 × 150 mm, MeCN/H₂O (0.07% TFA) = 45/55–65/35 gradient over 12 min, flow rate = 30 mL/min, λ = 220, t_R = 2.2 min) to afford Boc-protected 12c. Subsequent Boc deprotection was carried out by adding acetonitrile and trifluoroacetic acid (CH₃CN:TFA, 1:1, 0.15 mL:0.15 mL) and stirring at room temperature (25 °C) for 2 h, yielding 12c tris-TFA salt (2.8 mg, 33%) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 8.93 (br s, 1H), 8.73 (br s, 1H), 7.72–7.66 (m, 2H), 7.60 (d, J = 8.6 Hz, 1H), 7.24 (d, J = 8.6 Hz, 1H), 7.07 (s, 1H), 6.74 (br s, 1H), 6.46 (d, J = 2.3 Hz, 1H), 6.43 (d, J = 2.3 Hz, 1H), 5.35 (br s, 2H), 5.31 (br s, 1H), 4.32 (br s, 1H), 4.18 (s, 1H), 4.03 (t, J = 6.9 Hz, 1H), 3.75 (qu, J = 6.6 Hz, 2H), 3.66–3.56 (m, 1H), 3.44 (qu, J = 1.6 Hz, 1H), 3.25 (q, J = 7.4 Hz, 1H), 3.21 (qu, J = 1.6 Hz, 1H), 2.78 (s, 4H), 1.87 (br s, 1H), 1.68 (br s, 1H), 1.40–1.38 (m, 6H), 1.38 (s, 2H), 1.37 (s, 1H), 1.31 (br s, 1H), 1.02–0.91 (m, 6H); HRMS (ESI-TOF) m/z: [M + 2H]²⁺ Calcd for C₇₇H₉₂Cl₂N₁₄O₂₆²⁺ 849.2836; Found 849.2840.

Compound 13a.

A solution of Boc-deprotected compound 11a (5.2 mg, 2.8 μmol) in anhydrous DMF (50 μL) was treated with iPr₂NEt (7 μL, pH = 7) and added to a solution of 15 (2.7 mg, 6.5 μmol, 2.2 equiv) in anhydrous DMF (300 μL) in an oven-dried half-dram vial. The reaction mixture was stirred for 24 h at 40 °C (heating block) under argon. Direct purification was performed by semi-preparative reverse-phase HPLC (Waters XBridge^® Prep 5μm OBD^™ C18–19 × 150 mm, MeCN/H₂O (0.07% TFA) = 15/85–35/65 gradient over 12 min, flow rate = 30 mL/min, λ = 220, t_R = 1.6 min) to afford Boc-protected 13a. Subsequent Boc deprotection was carried out by adding acetonitrile and trifluoroacetic acid (CH₃CN:TFA, 1:1, 0.15 mL:0.15 mL) and stirring at room temperature (25 °C) for 2 h, yielding 13a tris-TFA salt (2.4 mg, 41%) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 7.99 (br s, 3H), 7.76 (br s, 1H), 7.71 (d, J = 7.6 Hz, 2H), 7.64 (d, J = 8.4 Hz, 3H), 7.56 (d, J = 6.1 Hz, 2H), 7.48 (d, J = 8.2 Hz, 3H), 7.28 (d, J = 8.4 Hz, 1H), 7.03 (br s, 1H), 6.97 (br s, 1H), 6.89 (d, J = 3.9 Hz, 1H), 6.82 (d, J = 3.9 Hz, 1H), 6.40 (br s, 1H), 5.81 (br s, 1H), 5.52–5.47 (m, 2H), 5.39 ( br s, 2H), 5.31 (br s, 1H), 5.19 (s, 1H), 4.60 (br s, 4H), 4.16 (br s, 2H), 3.93–3.89 (m, 4H), 3.78–3.85 (m, 1H), 3.67–3.64 (m, 3H), 3.45 (q, J = 1.6 Hz, 2H), 3.37 (s, 2H), 2.44 (br s, 2H), 2.23–2.18 (m, 1H), 2.07–2.01 (m, 2H), 1.96 (s, 1H), 1.88 (s, 1H), 1.73–1.61 (m, 4H), 1.41–1.37 (m, 3H), 1.28 (d, J = 6.2 Hz, 3H), 1.26–1.20 (m, 2H), 1.01–0.96 (m, 6H), 0.92 (t, J = 6.8 Hz, 2H); HRMS (ESI-TOF) m/z: [M + 2H]²⁺ Calcd for C₈₈H₉₉Cl₃N₁₄O₂₆²⁺ 936.2955; Found 936.2951.

Compound 13b.

A solution of Boc-deprotected compound 11b (5.4 mg, 3.2 μmol) in anhydrous DMF (50 μL) was treated with iPr₂NEt (6 μL, pH = 7) and added to a solution of 15 (2.3 mg, 5.4 μmol, 1.7 equiv) in anhydrous DMF (300 μL) in an oven-dried half-dram vial. The reaction mixture was stirred for 28.5 h at 40 °C (heating block) under argon. Direct purification was performed by semi-preparative reverse-phase HPLC (Waters XBridge^® Prep 5μm OBD^™ C18–19 × 150 mm, MeCN/H₂O (0.07% TFA) = 25/75–45/55 gradient over 12 min, flow rate = 30 mL/min, λ = 220, t_R = 5.6 min) to afford Boc-protected 13b. Subsequent Boc deprotection was carried out by adding acetonitrile and trifluoroacetic acid (CH₃CN:TFA, 1:1, 0.15 mL:0.15 mL) and stirring at room temperature (25 °C) for 2 h, yielding 13b tris-TFA salt (1.8 mg, 28%) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 7.73–7.66 (m, 4H), 7.63 (d, J = 7.9 Hz, 3H), 7.60–7.52 (m, 3H), 7.47 (d, J = 7.9 Hz, 3H), 7.35–7.26 (m, 1H), 7.09–6.96 (m, 1H), 6.77 (d, J = 4.1 Hz, 1H), 6.56 (s, 1H), 6.47–6.40 (m, 1H), 5.84–5.66 (m, 2H), 5.51–5.47 (m, 2H), 5.40–5.34 (m, 3H), 4.69 (s, 1H), 4.64 (s, 1H), 4.59 (s, 3H), 4.42–4.34 (m, 1H), 4.25–4.05 (m, 4H), 3.90–3.81 (m, 5H), 3.78–3.74 (m, 2H), 3.67–3.54 (m, 5H), 3.44 (s, 2H), 3.21 (s, 2H), 3.01 (s, 1H), 2.88 (m, 1H), 2.77–2.74 (m, 1H), 2.69–2.66 (m, 1H), 2.61–2.59 (m, 1H), 2.48–2.38 (m, 3H), 2.22–2.10 (m, 1H), 2.08–1.99 (m, 1H), 1.88–1.69 (m, 3H), 1.63–1.58 (m, 3H), 1.37 (t, J = 7.0 Hz, 1H), 1.32–1.27 (m, 4H), 0.93–0.79 (m, 6H); HRMS (ESI-TOF) m/z: [M + 2H]²⁺ Calcd for C₈₉H₁₀₁Cl₃N₁₄O₂₆²⁺ 942.2955; Found 942.2958.

Compound 13c.

A solution of Boc-deprotected compound 11c (5.2 mg, 3.0 μmol) anhydrous DMF (50 μL) was treated with iPr₂NEt (5 μL, pH = 7) and added to a solution of 15 (2.2 mg, 5.2 μmol, 1.7 equiv) in anhydrous DMF (300 μL) in an oven-dried half-dram vial. The reaction mixture was stirred for 28.5 h at 40 °C (heating block) under argon. Direct purification was performed by semi-preparative reverse-phase HPLC (Waters XBridge^® Prep 5μm OBD^™ C18–19 × 150 mm, MeCN/H₂O (0.07% TFA) = 35/65–55/45 gradient over 12 min, flow rate = 30 mL/min, λ = 282, t_R = 4.8 min) to afford Boc-protected 13c. Subsequent Boc deprotection was carried out by adding acetonitrile and trifluoroacetic acid (CH₃CN:TFA, 1:1, 0.15 mL:0.15 mL) and stirring at room temperature (25 °C) for 2 h, yielding 13c tris-TFA salt (1.1 mg, 18%) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 7.82 (br s, 1H), 7.72 (s, 1H), 7.61 (d, J = 7.0 Hz, 6H), 7.50–7.41 (m, 6H), 7.29 (br s, 1H), 7.08 (s, 1H), 7.03 (br s, 1H), 6.99 (dd, J = 6.5 Hz, 1H), 6.92 (d, J = 4.0 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 6.75 (d, J = 3.9 Hz, 1H), 6.57 (br s, 1H), 6.40 (d, J = 2.1 Hz, 1H), 5.74 (s, 1H), 5.47 (br s, 2H), 5.39 (br s, 2H), 4.59 (br s, 3H), 4.33 (qu, J = 7.2 Hz, 1H), 4.15 (br s, 1H), 3.85–3.83 (m, 2H), 3.78–3.75 (m, 2H), 3.70–3.64 (m, 2H), 3.56–3.53 (m, 2H), 3.44 (t, J = 1.4 Hz, 3H), 3.24 (br s, 2H), 3.20 (t, J = 1.7 Hz, 3H), 3.01 (s, 1H), 2.88 (s, 1H), 2.03–1.89 (m, 4H), 2.79 (s, 3H), 1.67–1.55 (m, 4H), 1.55–1.40 (m, 4H), 1.36 (t, J = 7.1 Hz, 4H), 1.30 (br s, 3H), 1.28–1.23 (m, 5H), 1.19 (d, J = 6.9 Hz, 1H), 1.01 (d, J = 4.8 Hz, 4H), 0.96–0.89 (m, 5H); HRMS (ESI-TOF) m/z: [M + 2H]²⁺ Calcd for C₉₀H₁₀₃Cl₃N₁₄O₂₆²⁺ 949.3033; Found 949.3028.

Compound 15.

Compound 5 (22.2 mg, 95.8 μmol) and iPr₂NEt (17 μL, 1.01 equiv) in anhydrous DMF (I mL) were added to an oven-dried scintillation vial. PyOxim (60.6 mg, 115 μmol,1.2 equiv) was added, and the reaction mixture was stirred at 40 °C (heating block) for 2 h under argon. The reaction was monitored by LCMS and TLC. When complete, in-situ Boc protection was conducted by adding Boc anhydride (35 μL, 152.5 μmol, 2.2 equiv) to the reaction mixture. The reaction mixture was stirred at 40 °C (heating block) for 17 h under Argon. The crude yellow reaction mixture was filtered through a SiO₂ plug atop a cotton plug where impurities are removed with 10–40% EtOAc in hexanes. Following elution with MeOH, the remaining mixture was purified using PTLC (10% MeOH in CH₂Cl₂) to provide the 15 as a light-yellow solid (4.5 mg, 11%). ¹H NMR (600 MHz, CDCl₃) δ 9.91 (br s, 1H), 7.12 (dd, J = 6.0, 1.9 Hz, 1H), 6.89 (d, J = 5.9, 2.1 Hz, 1H), 4.44 (q, J = 10.7 Hz, 2H), 3.87 (s, 3H), 1.50 (s, 9H), 1.37 (t, J = 10.7 Hz, 3H); HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₁₇H₂₁N₆O₇⁺ 421.1466; Found 421.1465.

In vitro Antimicrobial Assay.

¹⁵ One day before experiments were run, fresh cultures of vancomycin-sensitive Staphlococcus aureus (VSSA strain ATCC 25923), methicillin and oxacillin-resistant Staphlococcus aureus (MRSA strain ATCC 43300), vancomycin-resistant Enterococcus faecalis (VanA VRE, ATCC BAA-2573), Enterococcus faecium (VanA VRE, ATCC BAA-2317), and vancomycin-resistant Enterococcus faecalis (VanB VRE, strain TX-2516) were grown. Selected compounds were also assessed for activity against Escherichia coli (ATCC 25922), Acinetobacter baumannii (ATCC BAA-1710), Pseudomonas aeruginosa (ATCC 15442), Klebsiella pneumoniae (ATCC 700603). The bacteria were inoculated and grown in an orbital shaker at 37 °C in 100% Mueller-Hinton broth (VSSA and MRSA), 100% brain-heart infusion broth (sensitive enterococci, VanA and VanB VRE, A. baumannii and K. pneumoniae) or 100% Luria broth (E. coli and P. aeruginosa). After 24 h, the bacterial stock solutions were serial diluted with the culture medium (10% Mueller-Hinton broth for VSSA and MRSA, 10% brain-heart infusion broth for sensitive enterococci, VanA and VanB VRE, A. baumannii and K. pneumoniae or 10% Luria Broth for E. coli and P. aeruginosa, containing 0.002% Tween-80) to achieve a turbidity equivalent to a 1:100 dilution of a 0.5 M McFarland solution. This diluted bacterial stock solution was then inoculated in a 96-well flat-bottom non-treated microtiter plate (Corning 3370), supplemented with serial diluted aliquots of the antibiotic solution in DMSO (4 μL), to achieve a total assay volume of 0.1 mL. The plate was then incubated at 37 °C for 18 h, after which minimal inhibitory concentrations (MICs) were determined by monitoring the cell growth (observed as a pellet) in the wells. The lowest concentration of antibiotic (in μg/mL) capable of eliminating cell growth in the wells is the reported MIC value. The reported MIC values for the vancomycin analogues were determined against vancomycin as a standard in the first well.

Permeability Assay.

³⁹ One day before experiments were run, cultures of vancomycin-resistant Enterococcus faecium (VanA VRE, ATCC BAA-2317) were inoculated and grown in an orbital shaker at 37 °C in 100% brain-heart infusion broth for 12 h. The above bacterial solution was subjected to a subculture to obtain fresh mid log phase bacterial cells (incubation time = 6 h). The bacterial suspension was diluted to a total volume of 7 mL with OD₆₀₀ = 0.6. After the cultured bacteria was harvested (3000 rpm, 4 °C, 20 min), the white bacterial precipitate was washed and resuspended in 5 mM glucose and 5 mM HEPES buffer (1:1, 5.00 mL, pH = 7.2). This bacterial suspension (130 μL) was charged in a 96-well black plate with a clear bottom (Corning 3651). Propidium iodide (PI, 10 μL, 150 μM DMSO solution) was added to the above suspension and the fluorescence was monitored at 25 °C for 5 min at 20 or 30 s intervals using a microplate reader (Molecular Devices^®, Max Gemini EX) at an excitation wavelength of 535 nm and an emission wavelength of 617 nm. The antibiotic solution (10 μL, 150 μM buffer solution) was added to the cell suspension and the fluorescence was monitored at 25 °C for an additional 15 min.

Supplementary Material

ASSOCIATED CONTENT

Supporting Information

Copies of the NMR characterization of the compounds and table of compound purities (Table S1) (pdf).

Data Availability

The data underlying this study is available in the published article and its Supporting Information.