Total Synthesis of [Ψ[C(=NH)NH]Tpg⁴]Vancomycin and its (4-Chlorobiphenyl)methyl Derivative: Impact of Peripheral Modifications on Vancomycin Analogs Redesigned for Dual D-Ala-D-Ala and D-Ala-D-Lac Binding

Abstract

The total synthesis of two key analogs of vancomycin containing single atom exchanges in the binding pocket are disclosed (residue 4 amidine and thioamide) as well as their peripherally modified (4-chlorobiphenyl)methyl (CBP) derivatives. Their assessment indicate that combined pocket amidine and CBP peripherally modified analogs exhibit a remarkable spectrum of antimicrobial activity (VSSA, MRSA, VanA and VanB VRE) and impressive potencies (MIC = 0.06–0.005 μg/mL) against both vancomycin sensitive and resistant bacteria, and likely benefit from two independent and synergistic mechanisms of action. Like vancomycin, such analogs are likely to display especially durable antibiotic activity not prone to rapidly acquired clinical resistance.

Vancomycin (1) is the key member of the glycopeptide antibiotics that are the most important class of drugs used against resistant bacterial infections.¹ Although it was disclosed in 1956² and introduced into the clinic in 1958, the structure of vancomycin was established only 25–30 years later (Figure 1).³ After more than 50 years of clinical use and with the even more widespread use of glycopeptide antibiotics for livestock (avoparcin), vancomycin-resistant pathogens have slowly emerged worldwide. This was first restricted to vancomycin-resistant Enterococci (VRE)⁴ but more recently includes vancomycin-resistant Staphylococcus aureus (VRSA).⁵ This has intensified interest in the development of alternative treatments for resistant pathogens that display the remarkable durability of vancomycin, including new derivatives of the glycopeptide antibiotics.^1,6,7

Figure 1.

Structure of vancomycin (1), its (4-chlorobiphenyl)-methyl derivative 4, and targeted synthetic analogs.

Recently, we described studies on the binding pocket redesign of vancomycin in efforts to directly address the molecular basis of bacterial resistance.⁸ Vancomycin inhibits bacterial cell wall synthesis by binding the precursor peptidoglycan peptide terminus D-Ala-D-Ala, inhibiting transpeptidase-catalyzed cell wall cross-linking and maturation.⁹ In the clinically resistant phenotypes (VanA and VanB), synthesis of the precursor lipid intermediates I and II continue, but resistant bacteria sense the antibiotic challenge¹⁰ and conduct a late stage remodeling of their peptidoglycan termini from D-Ala-D-Ala to D-Ala-D-Lac¹¹ to avoid the antibiotic action. The binding affinity of vancomycin for the altered ligand¹¹ is reduced 1000-fold,¹² resulting in a corresponding 1000-fold loss in antimicrobial activity.

These studies also revealed that the redesign of the vancomycin binding pocket for use against vancomycin-resistant bacteria must target compounds that not only establish binding to D-Ala-D-Lac, but that also maintain D-Ala-D-Ala binding. Subsequent to a validation in which [Ψ[CH₂NH]Tpg⁴]-vancomycin aglycon (10)¹³ displayed such dual binding properties that reinstated activity against VanA VRE, the total synthesis of [Ψ[C(=NH)NH]Tpg⁴]vancomycin aglycon (9)¹⁴ was reported in efforts that improved the dual binding affinities and antimicrobial activity (Figure 2). Amidine 9 displayed balanced binding affinity for both target ligands within 2-fold that of vancomycin aglycon for D-Ala-D-Ala and exhibited effective antimicrobial activity against VanA VRE, being equipotent to the activity that vancomycin displays against sensitive bacterial strains. Not only did this represent replacement of a single atom in the antibiotic aglycon (O→NH) to counter a complementary exchange in the cell wall precursors of resistant bacteria (NH→O), but the modified antibiotic maintained the ability to bind D-Ala-D-Ala in the unaltered peptidoglycan. The extension of these studies to the total synthesis of [Ψ[C(=NH)NH]Tpg⁴]vancomycin (3) with introduction of the L-vancosaminyl-1,2-D-glucosyl disaccharide, representing a binding pocket analog of vancomycin itself containing this single atom change is detailed herein. Although the attached carbohydrate in vancomycin does not alter in vitro antimicrobial activity or influence target D-Ala-D-Ala or D-Ala-D-Lac binding, it impacts in vivo activity, increasing water solubility, influencing pharmacokinetic and distribution properties and contributing a potential second mechanism of action.

Figure 2.

Synthetic analogs of vancomycin aglycon (7) that contain key modifications to the binding pocket.

Because of their structural complexity, essentially all analogs of the glycopeptide antibiotics consist of semisynthetic derivatives of the natural products.^1,6 The most significant of the modifications introduce peripheral hydrophobic groups and the most widely studied entails 4-chlorobiphenyl substitution of a peripheral carbohydrate.⁶ This has been explored in a variety of glycopeptide antibiotics and at range of positions, most notably in oritavancin,¹⁵ the N-(4-chlorobiphenyl)methyl derivative of chloroeremomycin, and with vancomycin itself (4, CBP-vancomycin).¹⁶ Studies on their mechanism of action have shown that the chlorobiphenyl side chain promotes antibiotic dimerization and membrane anchoring and establishes antimicrobial activity against vancomycin-resistant organisms despite a lack of improved binding with D-Ala-D-Ala or D-Ala-D-Lac.¹⁷ It is possible such semisynthetic changes to vancomycin also avoid bacterial sensing of the antibiotic challenge and this may account for their VanB VRE activity (like teicoplanin).¹⁰ Alternatively, they may entail a second mechanism of action. The direct inhibition of transglycosylases mediated by the modified carbohydrate has been implicated as a second mechanism by which the lipophilic glycopeptides with impaired D-Ala-D-Ala binding properties exhibit antimicrobial activity.¹⁸ Others, including telavancin, have been shown to function both through the traditional mechanism of inhibition of cell wall synthesis by binding D-Ala-D-Ala and also through the disruption of bacterial membrane integrity, a mechanism typically not observed for glycopeptides.¹⁹ Regardless of the origin of the effects, such derivatives often increase antibiotic potency as much as 100-fold. While increasing bacterial sensitivity to the antibiotics, VanA vancomycin-resistant bacterial strains (MIC = ca. 10 μg/mL) remain 1000-fold less sensitive than susceptible strains (MIC = ca. 0.01 μg/mL).

Given the distinct origins of their impact on the antimicrobial activity of vancomycin, we expected that incorporation of such peripheral hydrophobic modifications into the structure of a binding pocket modified vancomycin would further increase their antimicrobial activity against not only sensitive, but also vancomycin-resistant bacteria to truly remarkable potencies. Aside from the merits of such analogs as new therapeutics, their increased potencies would have the additional impact of reducing the amount of material needed for preclinical exploration. Although this conceivably could be demonstrated by chlorobiphenyl substitution of the synthetic aglycon 9, the most definitive assessment of the dual impact would be a direct comparison of chlorobiphenyl vancomycin (4) with 6, wherein a change in a single atom in the binding pocket was introduced, despite the synthetic challenges this poses. Herein, we report not only the total synthesis of [Ψ[C(=NH)NH]Tpg⁴]vancomycin (3) from synthetic [Ψ[C(=S)NH]Tpg⁴]vancomycin aglycon (8),¹⁴ but also its (4-chlorobiphenyl)methyl derivative 6. This was accomplished by using two sequential enzymatic glycosylations to first provide [Ψ[C(=S)NH]Tpg⁴]vancomycin (2) followed by a Ag(I)-promoted²⁰ conversion of the thioamide to an amidine. Additional single-step introduction of the (4-chlorobiphenyl)methyl group into [Ψ[C(=S)NH]Tpg⁴]vancomycin (2) to provide 5 followed by Ag(I)-promoted conversion of amidine introduction afforded 6. In addition to the opportunity to assess [Ψ[C(=NH)NH]Tpg⁴]vancomycin (3) and the impact of combining the vancomycin pocket redesign with a key peripheral modification, the approach was designed to shed light on the role of the chlorobiphenyl modification through examination of [Ψ[C(=S)NH]Tpg⁴]vancomycin (2) and its (4-chlorobiphenyl)methyl derivative, which fail to effectively bind D-Ala-D-Ala or D-Ala-D-Lac.

The two sequential glycosylations of synthetic 8¹⁴ were conducted with the purified recombinant glycosyltransferases²¹ GtfE and GtfD and the synthetic glycosyl donors (UDP-glucose²² for GtfE and UDP-vancosamine²³ for GtfD) under recently described conditions²³ to provide the pseudoaglycon 13 (75%, HPLC conversion 86–92%) and [Ψ[ C(=S)NH]Tpg⁴]-vancomycin (2, 87%, HPLC conversion >95%) (Scheme 1).²⁴ Direct conversion of thioamide 2 to the corresponding amidine (10 equiv AgOAc, sat. NH₃–MeOH, 25 °C, 7 h, 37% unoptimized)²⁰ provided [Ψ[C(=NH)NH]Tpg⁴]vancomycin (3).²⁴ Significantly, the latter reaction was implemented without competitive deglycosylation and the entire sequence (conversion of 8 to 3) was conducted without protecting groups.

Scheme 1.

Subsequent introduction of the chlorobiphenyl group into [Ψ[C(=S)NH]Tpg⁴]vancomycin (2) by selective reductive amination (1.5 equiv 4-(4-chlorophenyl)benzaldehyde, 5 equiv i-Pr₂NEt, DMF, 70 °C, 2 h; NaCNBH₃, 70 °C, 5 h) provided 5 (57%) without observation of competitive reactions of either the thioamide (reduction) or the N-terminal free amine (reductive amination), using conditions modified from those disclosed for chlorobiphenyl vancomycin itself.²⁵ Direct AgOAc-promoted (10 equiv, sat. NH₃–MeOH, 25 °C, 7 h) conversion of the thioamide to the amidine provided 6 (45%, unoptimized), the chlorobiphenyl derivative of [Ψ[C(=NH)NH]Tpg⁴]vancomycin (3), without the need for intervening protecting groups throughout the 4-step sequence. By design, the final reaction introducing the amidine was conducted effectively on fully functionalized substrates (2 and 5), lacking protecting groups and incorporating the vancomycin disaccharide.

The antimicrobial activity of the compounds was evaluated against a panel of Gram-positive bacteria that include vancomycin-sensitive S. aureus (VSSA), methicillin-resistant S. aureus (MRSA), and both VanA (E. faecalis and E. faecium) and VanB (E. faecalis) vancomycin-resistant Enterococci (VRE) of which VanA is the most stringent of the resistant organisms (Figure 3). In line with reports of its impact, introduction of the (4-chlorobiphenyl)methyl group into vancomycin (4 vs 1) results in 100-fold improvements in the activity against VanA and VanB VRE and 20-fold improvements against VSSA and MRSA in the strains examined. Like the aglycon thioamide 8, vancomycin thioamide (2) was ineffective as an antimicrobial agent (>32 μg/mL). Notably, this behavior is derived from a single atom exchange in the binding pocket (O→S) and is analogous to that of other analogs bearing altered binding pockets incapable of binding D-Ala-D-Ala.^18,26 However, introduction of the (4-chlorobiphenyl)methyl group into vancomycin thioamide (2) with 5 reinstates impressive and equally potent activity (MIC = 2–4 μg/mL) against all vancomycin sensitive and resistant strains despite its inability to bind the primary cell wall target. It is unlikely such effective activity can be achieved simply by the effects of antibiotic membrane anchoring or dimerization. Rather, it likely reflects potent antimicrobial activity derived from a second mechanism of action impacting cell wall synthesis unrelated to D-Ala-D-Ala/D-Ala-D-Lac binding such as transglycosylase inhibition.¹⁸ Importantly, the vancomycin amidine 3, like the aglycon amidine 9, was found to exhibit potent activity against VanA resistant bacteria (MIC = 0.5 μg/mL), reinstating activity equal to the potency observed with vancomycin against sensitive bacteria. Most significantly, introduction of the chlorobiphenyl group into vancomycin amidine (3) with 6 resulted in a compound with a remarkable spectrum of activity and impressive potencies. Not only does 6 match the activity that chlorobiphenyl vancomycin (4) displays against vancomycin-sensitive bacteria (VSSA and MRSA), but it also exhibits this extraordinary potency against VanA and VanB vancomycin-resistant bacteria. In fact, the activity of 6 against the most stringent of the resistant bacteria, VanA VRE, was nearly 10-fold better than the potency it displays against the sensitive bacteria. Because of the insights derived from examination of the thioamides 2 and 5, this behavior of 6 likely represents a spectrum of activity and potency derived from bacterial cell wall synthesis inhibition through two independent mechanisms, suggesting resistance is less likely to emerge.

Figure 3.

In vitro antibacterial activity.

Herein we detailed the completion of the total syntheses of [Ψ[C(=S)NH]Tpg⁴]vancomycin (2) and [Ψ[C(=NH)NH]Tpg⁴]-vancomycin (3), two fully adorned analogs of vancomycin that contain a single atom exchange in the binding pocket as well their corresponding chlorobiphenyl derivatives 5 and 6. By design, the sequential enzyme-catalyzed glycosylation reactions of the thioamide aglycon, the final amidine introduction, and the intermediate reductive amination used for the chlorobiphenyl introduction were conducted without the need of protecting groups, establishing the foundation and providing the methodology for potential semi-synthetic or biosynthetic preparations of such glycopeptide analogs. In line with expectations based on the behavior of the corresponding aglycons and in stark contrast to one another, the vancomycin amidine reestablishes potent antimicrobial activity against VanA VRE, whereas vancomycin thioamide is inactive even against vancomycin-sensitive bacteria. Introduction of a peripheral chlorobiphenyl modification converts vancomycin thioamide into an effective antimicrobial agent active against vancomycin sensitive and resistant bacteria (MIC = 2–4 μg/mL) even though it is not capable of effective D-Ala-D-Ala/D-Ala-D-Lac binding and converts the vancomycin amidine into a compound with a remarkable spectrum of activity and truly impressive potencies (MIC = 0.06–0.005 μg/mL) that are likely derived from cell wall biosynthesis inhibition through two independent mechanisms. In addition to indicating that such peripheral and pocket synthetic modifications are synergistic, such analogs are likely to display durable antibiotic activity⁸ and may be less prone to rapidly acquired clinical resistance.