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. Author manuscript; available in PMC: 2008 Nov 21.
Published in final edited form as: J Am Chem Soc. 2006 Mar 8;128(9):2885–2892. doi: 10.1021/ja0572912

Total Synthesis and Evaluation of [Ψ[CH2NH]Tpg4]Vancomycin Aglycon: Reengineering Vancomycin for Dual D-Ala-D-Ala and D-Ala-D-Lac Binding

Brendan M Crowley 1, Dale L Boger 1,*
PMCID: PMC2586002  NIHMSID: NIHMS63177  PMID: 16506767

Abstract

An effective synthesis of [Ψ[CH2NH]Tpg4]vancomycin aglycon (5) is detailed in which the residue 4 amide carbonyl of vancomycin aglycon has been replaced with a methylene. This removal of a single atom was conducted to enhance binding to D-Ala-D-Lac countering resistance endowed to bacteria that remodel their D-Ala-D-Ala peptidoglycan cell wall precursor by a similar single atom change (ester O for amide NH). Key elements of the approach include a synthesis of the modified vancomycin ABCD ring system featuring a reductive amination coupling of residues 4 and 5 for installation of the deep-seated amide modification, the first of two diaryl ether closures for formation of the modified CD ring system (76%, 2.5–3:1 kinetic atropodiastereoselectivity), a Suzuki coupling for installation of the hindered AB biaryl bond (90%) on which the atropisomer stereochemistry could be thermally adjusted, and a macrolactamization closure of the AB ring system (70%). Subsequent DE ring system introduction enlisted a room temperature aromatic nucleophilic substitution reaction for formation of the remaining diaryl ether (86%, 6–7:1 kinetic atropodiastereoselectivity) completing the carbon skeleton of 5. Consistent with expectations and relative to the vancomycin aglycon, 5 exhibited a 40-fold increase in affinity for D-Ala-D-Lac (Ka = 5.2 × 103 M−1) and a 35-fold reduction in affinity for D-Ala-D-Ala (Ka = 4.8 × 103 M−1) providing a glycopeptide analogue with balanced, dual binding characteristics. Beautifully, 5 exhibited antimicrobial activity (MIC = 31 μg/mL) against a VanA resistant organism that remodels its D-Ala-D-Ala cell wall precursor to D-Ala-D-Lac upon glycopeptide antibiotic challenge displaying a potency that reflects these binding characteristics.

The most common strains of enterococci resistant to vancomycin (1), VanA and VanB, possess an inducible resistance pathway in which the terminal dipeptide of the cell wall peptidoglycan precursor is modified from D-Ala-D-Ala to D-Ala-D-Lac.1 Binding of the antibiotic to this modified ligand is reduced 1000-fold leading to a 1000-fold drop in antimicrobial activity.1d,k In a recent disclosure,2 we provided the first experimental study on the origin of this loss in binding affinity, partitioning the effect into lost H-bond and repulsive lone pair contributions, Figure 1. Thus, the binding affinity of vancomycin for 3, which incorporates a methylene (CH2) in place of the linking amide NH of Ac2-L-Lys-D-Ala-D-Ala, was compared with that of Ac2-L-Lys-D-Ala-D-Ala (2) and Ac2-L-Lys-D-Ala-D-Lac (4). The vancomycin affinity for 3 was approximately 10-fold less than that of 2, but 100-fold greater than that of 4. This indicated that the reduced binding affinity of 4 (4.1 kcal/mol) may be attributed to both the loss of a key H-bond and a destabilizing lone pair/lone pair interaction introduced with the ester oxygen of 4 (2.6 kcal/mol) with the latter, not the H-bond, being responsible for the greater share (100-fold) of the 1000-fold binding reduction. These observations have significant ramifications on the reengineering of vancomycin to bind D-Ala-D-Lac suggesting that the design could focus principally on removing the destabilizing lone pair interaction rather than reintroduction of a H-bond and that this may be sufficient to compensate for two of the three orders of magnitude in binding affinity lost with D-Ala-D-Lac. Thus, synthesis of a vancomycin analogue with removal of the residue 4 carbonyl and its destabilizing lone pair interaction could potentially restore much of the binding affinity of the antibiotic for the modified ligand. At present, such a deep-seated change in the molecule can only be achieved by total synthesis.3 Having developed a convergent and efficient synthesis of the vancomycin aglycon4 and having extended its effectiveness in the synthesis of the teicoplanin5 and ristocetin aglycons,6 we embarked on the total synthesis of [Ψ[CH2NH]Tpg4]vancomycin aglycon (5) in which the residue 4 carbonyl has been replaced with a methylene. Through such efforts, we were able to establish the effect this structural modification has on the binding affinity for both D-Ala-D-Ala and D-Ala-D-Lac and probe its impact on the antimicrobial activity of the analogue.

Figure 1.

Figure 1

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Binding constants of D-Ala-D-Ala, D-Ala-D-Lac, and a peptide analogue with vancomycin.

Challenges and Synthetic Plan for [Ψ[CH2NH]Tpg4]Vancomycin Aglycon

The desired analogue 5 was anticipated to be prepared by a route analogous to that developed for vancomycin,4 with notable modifications. Thus, two aromatic nucleophilic substitution reactions with formation of the biaryl ethers would be enlisted for CD and DE macrocyclization, a key macrolactamization reaction would be employed for cyclization of the AB ring system, and the defined order of CD, AB, and DE ring closures was expected to permit sequential control of the atropisomer stereochemistry of each of the newly formed ring systems or their immediate precursors, Figure 2. Thus, in addition to any kinetic diastereoselection that may be achieved in the ring closures, this order was expected to permit the recycling of any undesired atropisomer for each newly introduced ring system by thermal equilibration providing a predictable control of the stereochemistry and dependably funneling all synthetic material into one of eight possible atropdiastereomers. Key to recognition of this preferential order of ring closures was our establishment of the thermodynamic parameters of atropisomerism for the individual vancomycin ring systems: DE ring system7 (Ea = 18.7 kcal/mol) < AB biaryl precursor4 (Ea = 25.1 kcal/mol) < CD ring system7 (Ea = 30.4 kcal/mol). Thus, the molecule was to be assembled by coupling the modified and fully functionalized ABCD ring system 27 with the E ring tripeptide 28 followed by a diastereoselective aromatic nucleophilic substitution reaction for closure of the 16-membered DE ring system with formation of the biaryl ether linkage. Notably, the activating nitro substituent additionally serves as the precursor functionality for aryl chloride introduction and the analogous vancomycin ring closures4,8–10 were effected with preferential formation of the natural (P)-atropisomer. The E ring tripeptide 28 would be derived in the manner described for vancomycin4 except that the E ring subunit would be prepared enlisting an improved route developed during our more recent total synthesis of the ristocetin aglycon6 employing an α-hydroxypinanone11 as the chiral auxiliary for a diastereoselective aldol addition. The most significant deviations rest with the required modifications in the preparation of the ABCD subunit which house the modified amide and include the use of a reductive amination coupling of residues 4 and 5 (D and B rings) with protection of the newly generated amine as a methyl carbamate and an experimentally-derived altered order to the assembly of the BCD tripeptide. A relatively small and robust amine protecting group was chosen to avoid the introduction of unfavorable steric interactions that may affect the CD macrocyclic ring closure and that would be stable throughout the synthesis, yet still be compatible with a final stage global deprotection. CD macrocyclization enlisting a key aromatic nucleophilic substitution reaction for formation of 16-membered biaryl ether followed by Suzuki coupling of the A ring subunit and AB macrolactamization was anticipated to complete the preparation of the modified ABCD ring system 27 enlisting a ring closure order that would permit the sequential and selective thermal adjustment of the CD and AB ring system atropisomer stereochemistry. Key unknown features of the approach include the feasibility of conducting the critical CD ring closure enlisting the residue 4 protected amine versus amide, the resulting unknown atropisomer stereochemical issues (kinetic and thermodynamic diastereoselectivity), and the impact the deep-seated structural change would have on the conformational features of the CD or ABCD ring systems and those of the final molecule. Finally, the subtle choices of a nitrile as a precursor to the residue 3 side chain carboxamide permits a final stage amide deprotection yet conveys stability to any projected thermal atropisomer equilibrations in its presence,7f and the use of a MEM protected hydroxymethyl precursor (vs a methyl ester) to the C-terminus carboxylic acid enhances the rate of the projected AB macrolactamization4 and precludes inadvertent epimerization throughout the synthesis.

Figure 2.

Figure 2

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Plan for [Ψ[CH2NH]Tpg4]vancomycin aglycon.

Synthesis of the BCD Tripeptide

The B and D subunits 6 and 7 were prepared following previously optimized procedures.6,7 Oxidation of alcohol 712 (2.0 equiv of Dess–Martin periodinane, CH2Cl2, 0–25 °C, 1 h, 100%) was followed by immediate reductive amination coupling of the sensitive aldehyde 8 with 613 (1.1 equiv, CH3OH, 3Å MS, 0 °C, 45 min; 3.0 equiv of AcOH, 3.0 equiv of NaBH3CN, −20 °C, 2 d) to afford amine 9 in good yield (75%) and excellent diastereoselectivity (12:1), Scheme 1. Shorter reaction times (14–20 h) at higher temperatures (−15 to −5 °C) led to substandard selectivities (4:1 to 9:1) and the use of less NaBH3CN (1.5–2.0 equiv) or lower temperatures (−30 °C) led to incomplete reactions. Longer reaction times (3–8 d) led to only marginal increases in yield (82% after 8 d) and roughly equal diastereoselectivities. Initial efforts to prepare 9 directly by displacement of the mesylate derived from alcohol 7 were ineffective as were attempts to conduct the reductive amination with the BC dipeptide and 8. Amine protection of 9 as the methyl carbamate (10 equiv of MeOCOCl, 10 equiv of K2CO3, THF, 0–25 °C, 18 h, 85%) followed by benzyl ether deprotection14 (Raney Ni, CH3OH, 0 °C, 5 h, 98%) and saponification (3.0 equiv of LiOH, THF–H2O, 0 °C, 6 h, 100%) provided 12. Unexpectedly, the order of these latter two deprotections proved important. Saponification of 1015 under a variety of conditions (LiOH, THF–H2O or t-BuOH–H2O, –10 to 0 °C; LiOOH, THF–H2O; Me3SnOH, 1,2-dichloroethane, 70 °C) led to variable amounts of an epimer (5–20%) that was difficult to separate from the product. In contrast, benzyl ether deprotection of 10 followed by saponification of 11 reduced the amount of epimer (0–3%) presumably due to preferential deprotonation of the phenols such that subsequent Cα deprotonation at residue 5 was less facile.15 Coupling of 12 with 1316 (3.0 equiv of DEPBT,17 3.0 equiv of NaHCO3, DMF, 0–25 °C, 8 h) gave tripeptide 14 in good yield (70%) and excellent diastereoselectivity (14:1). A range of other more conventional coupling reagents (EDCI–HOAt, HATU, FDPP) also provided good conversions (65–80%), but suffered from considerable competitive racemization.

Scheme 1.

Scheme 1

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Synthesis of the ABCD Ring System

This set the stage for a detailed examination of one of the critical reactions in the approach to 5 entailing the cyclization of 14. After considerable optimization (Supporting Information Tables S1 and S2), cyclization of 14 (20 equiv of K2CO3, 20 equiv of CaCO3, 3 wt equiv of 3Å MS, 12 mM THF, 75 °C bath temp, 12 h) afforded 15 in good yield (54%) and good atropodiastereoselectivity (2.5:1, 15 (54%) and 16 (22%)) even when conducted on a large scale (2.7 g), Scheme 2. The inclusion of CaCO3 in the reaction mixture is critical and serves to trap the liberated fluoride arising from the aromatic nucleophilic substitution as an insoluble byproduct (CaF2) preventing TBS ether deprotection and a subsequent competitive base-catalyzed retro aldol reaction of the free alcohol. Nearly comparable results were obtained by promoting the ring closure of 15 with the stronger base t-BuOK (1.0 equiv, THF, −20 °C, 18 h) providing 15 and its atropisomer 16 in 57% and 19% (3:1 atropodiastereoselectivity), respectively, under remarkably mild reaction conditions (−20 °C, THF). However, the use of t-BuOK proved more sensitive to the reaction parameters, suffered competitive racemization if excess base was employed, and proved more difficult to implement on a large scale than the reaction enlisting K2CO3/CaCO3. The cyclization of 14 represents a considerable improvement over the analogous ring closure reaction enlisted in our original synthesis of vancomycin (50–65%, 1:1 atropisomers vs 76–87%, 2.5–3:1 atropisomers) where both the overall conversion and atropodiastereoselectivity were lower illustrating that the closure of 14 may benefit from the increased conformational flexibility of the cyclization substrate.18 Unlike the vancomycin CD ring system in which the atropisomers could be thermally equilibrated at 120–140 °C permitting the recycling and productive use of the unnatural atropisomer, the atropisomers 15 and 16 could not be thermally interconverted even at temperatures as high as 210–230 °C, Scheme 3.

Scheme 2.

Scheme 2

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Scheme 3.

Scheme 3

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Reduction of the nitro group (Raney Ni, 0 °C, CH3OH, 1 h) followed by diazotization (1.3 equiv of HBF4, 1.3 equiv of t-BuONO, CH3CN, 0 °C, 30 min) and Sandmeyer substitution (50 equiv of CuCl, 60 equiv of CuCl2, H2O, 0–25 °C, 1 h, 70% from 15) cleanly provided 17 without loss of the atropisomer stereochemistry inherent in starting 15. The unnatural atropisomer 16 was also subjected to these conditions to cleanly give 18 (75%) (Scheme 3). The stereochemical assignments of these two compounds and their relationship as atropisomers (vs epimers) were established by 2D ROESY 1H–1H NMR experiments and confirmed chemically by their reductive dechlorination (H2, 10%, Pd/C) to afford the identical product 19 (Scheme 3). Diagnostic NOE crosspeaks for 17 were observed between C5a6-H/C36-H (s), C5a6-H/C26-H (s), C5b6-H/C6b6-H (s), C55-H/C4b5-H (s), C36-H/C26-H (s), C25-H/C4a5-H (w), C25-H/C4b5-H (w), C24-H/C1b4-H (s), C24-H/C4b4-H (s), C1a4-H/C1b4-H (s), C54-OH/C64-OMe (s), and C55-H/C65-OMe (m) and no NOE crosspeaks were observed between C5b6-H/C36-H and C5b6-H/C26-H. Diagnostic NOE crosspeaks for 18 were observed between C5b6-H/C6b6-H (s), C5b6-H/C36-H (s), C5b6-H/C26-H (s), C55-H/C4b5-H (s), C36-H/C26-H (s), C26/N16-H (w), C25-H/C4a5-H (m), C25-H/C4b5-H (m), C24-H/C1b4-H (s), C1b4-H/C4a5-H (w), C1b4-H/C4b5-H (w), C1a4-H/C4b4-H (s), C1b4-H/C1a4-H (s), C4b4-H/C54-OH (m), C54-OH/C64-OMe (w), C55-H/C65-OMe (w), C24-H/N14-H (w) and no NOE crosspeaks were observed between C5a6-H/C36-H and C5a6-H/C26-H.

Suzuki coupling of 17 with the hindered A ring boronic acid 204 (0.3 equiv of Pd2(dba)3, 1.5 equiv of (o-tol)3P, toluene–CH3OH–1 N aq Na2CO3 10:3:1, 80 °C, 30 min) proceeded in excellent yield (90%) under remarkably effective conditions4 given the steric constraints of the substrate 20 providing a separable 1:1.3 mixture of atropisomers (21:22) slightly favoring the unnatural configuration. Thermal equilibration of isolated 22 was carried out initially employing our reported conditions for vancomycin (o-dichlorobenzene, 120 °C, 18 h, 81% recovery of material)7 to afford a 1:1.1 separable mixture permitting the recycling of this unnatural atropisomer. An examination of the parameters for this isomerization (k = 0.12 h−1, t1/2 = 5.9 h at 120 °C and k = 0.36 h−1, t1/2 = 1.8 h at 135 °C) revealed that it proceeds with an energy of activation (Ea) of 25.6 kcal/mol (ΔH‡ = 24.8 kcal/mol, ΔS‡ = −0.26 e.u., ΔG‡ = 24.9 kcal/mol) essentially indistinguishable from that observed with the authentic vancomycin AB biaryl system, but it does not result in the analogues 3:1 thermodynamic preference for the natural atropisomer. However, the unusual and unexpected atropisomer stability of the CD ring system allowed us to improve on the recycling conditions. Heating the mixture in a microwave reactor at an elevated temperature (210 °C, o-dichlorobenzene) shortened the reaction time significantly (5 min vs 18 h) and slightly improved the recovery of material (88% vs 81%). This improvement impacted the efficiency of the recycling of 22 by allowing multiple equilibrations to be run in a single day rather than over the course of a week. Silyl ether deprotection of 21 (1.2 equiv of Bu4NF, THF, 0 °C, 10 min) followed by N-Cbz removal (H2, 10% Pd/C, 1% Cl3CCO2H–CH3OH, 15 min, 95%) and methyl ester hydrolysis (1.0 equiv of LiOH, THF–H2O, 0 °C, 1 h, 96%) gave amino acid 25. Notably, N-Cbz removal in the absence of Cl3CCO2H5 was much slower (11 h) and these conditions led to competitive chloride reduction.19 Macrolactamization with closure of the AB ring system was effected by treatment of 25 with PyBOP (3.0 equiv, 6.0 equiv of NaHCO3, 0.001 M CH2Cl2–DMF 5:1, 0–25 °C, 12 h) to afford the fully functionalized bicyclic ABCD ring system 26 in good yield (70%) with only trace amounts of competitive epimerization (<3%). Alternative coupling reagents (EDCI and HOAt or HOBt, HATU) and reaction conditions (10–100% DMF–CH2Cl2, 3–5 equiv of Na2CO3, −5 to 0 °C) led to lower conversions (30–52%) or required extended reaction times (3 d). N-Boc deprotection (HCO2H–CHCl3 1:1, 10 h, 84%) gave the free amine 27 for coupling with the E ring tripeptide. Confirmation of the atropisomer stereochemistry and amide conformational assignments for 26 were established by 2D ROESY 1H–1H NMR. Diagnostic NOE crosspeaks for 26 were observed between C54-OH/C4b4-H (s), C54-OH/C64-OMe (s), N17-H/C4a5-H (s), N17-H/C25-H (s), N17-H/C36-H (m), N17-H/C26-H (m), C5a6-H/C36-H (s), C5a6-H/C26-H (s), C5b6-H/N16-H (m), C36-OH/N16-H (s), C5b6-H/C36-OH (m), C6b6-H/C5b6-H (s), C6b6-H/C4a4-H (w), N14-H/C4b4-H (m), N14-H/C4a4-H (w), C4b5-H/C55-H (s), C26-H/C4a5-H, C4b5-H/C1b4-H (m), C4a5-H/C67-H (w), C4a5-H/C25-H (s), C55-H/C65-OMe (s), C47-H/C27-H (s), C47-H/C1b7-H (s), C47-H/C1a7-H (w), C47-H/C5b7-OMe (s), C47-H/C67-H (w), C67-H/C25-H (w), C67-H/C5b7-OMe (s), C67-H/C5a7-OMe (s), C25-H/C36-H (m), C25-H/C26-H (s), C36-H/C26-H (m), C17-(MEM-CH2)1/C1a7-H (s), C17-(MEM-CH2)1/C17-(MEM-CH2)2 (s), C27-H/C1b7-H (s), C27-H/C1a7-H (s) and no NOE crosspeaks were observed between C5b6-H/C36-H, C5b6/C26-H, C26-H/C36-OH, N16-H/N17-H, N16-H/C25-H, and N16-H/C4a5-H. Most important in this spectroscopic assessment was not only the expected confirmation of the CD and AB atropisomer stereochemistry, but also the establishment of a vancomycin-like conformation for 26 bearing a cis amide linking the residues 5 and 6 (strong diagnostic C25-H/C26-H NOE) maintaining the spatial relationships and orientations of the AB ring system (strong diagnostic C25-H/C4a5-H and C26-H/C4a5-H NOEs) and CD ring systems (diagnostic C6b6-H/C4a4-H NOE). Although this might be considered unusual on the surface, even the natural atropisomer of the isolated AB ring system of vancomycin, without the surrounding CD ring system, adopts a conformation incorporating this cis amide structure illustrating that it is the confines of the AB ring system, not that of the CD ring system, that defines this key cis amide conformational preference.4 The lack of discernable NOEs to the methyl carbamate protecting the amine of the modified amide established that it extends out and away from the ABCD ring system binding pocket.

Synthesis of the Full Carbon Skeleton

Coupling of 27 and 28 (2.0 equiv of DEPBT,17 2.2 equiv of NaHCO3, THF, 0–25 °C, 14 h, 73%) afforded 29 with excellent diastereoselectivity (12:1) arising from little competitive racemization, Scheme 4. These conditions were utilized based on our experience with the teicoplanin5 and ristocetin6 aglycons and are superior to those originally reported for vancomycin4 (EDCI) in terms of diastereoselectivity (12:1 vs 3:1). Closure of the DE ring system with formation of the key biaryl ether was accomplished by treatment of 29 with CsF (10 equiv, 20 equiv of CaCO3,20 3Å MS, DMF, 25 °C, 17 h) to afford 30 in good yield (74%) and good atropodiastereoselectivity (6–7:1). Notably, the closure of 30 was conducted under milder conditions than those originally disclosed for vancomycin4,7–10 (DMF vs DMSO at 25 °C with added 3Å MS and CaCO3) and approaches the kinetic atropisomer diastereoselectivity observed in our efforts4 (8:1), while surpassing that detailed in the related Evans10 efforts (5:1), and contrasts the closure detailed by Nicolaou21 (1:3) where the unnatural atropisomer predominated with an alternative substrate and method of ring closure. Thus, consistent with the adoption of a vancomycin-like conformation by 26, the amide modification in the ABCD ring system of 29 had little impact on the ease or diastereoselectivity of the DE ring closure. Reduction of the nitro group22 (H2, 10% Pd/C, THF, 8 h, 94%) followed by diazotization of the resulting amine 32 (1.2 equiv of HBF4, 1.2 equiv of t-BuONO, CH3CN, 0 °C, 20 min) and Sandmeyer substitution (50 equiv of CuCl, 60 equiv of CuCl2, H2O, 0–25 °C, 1 h, 55%) gave 33, which embodies the full carbon skeleton of 5.

Scheme 4.

Scheme 4

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Completion of the Synthesis

With the full carbon skeleton in hand, attention was directed towards completion of the synthesis, Scheme 4. TBS ether protection of the secondary alcohols (65 equiv of CF3CONMeTBS, CH3CN, 55 °C, 22 h; aq citric acid, 25 °C, 13 h, 96%) followed by MEM ether deprotection of 34 (12 equiv of B-bromocatecholborane (BCB), CH2Cl2, 0 °C, 2 h; 5.1 equiv of Boc2O, 6.0 equiv of NaHCO3, dioxane–H2O 2:1, 0–25 °C, 2.5 h, 80%) and two-step oxidation of the resulting primary alcohol 35 (4.0 equiv of Dess–Martin periodinane, CH2Cl2, 0 °C, 15 min then 25 °C, 1 h; 9.0 equiv of 80% aq NaClO2, 7.0 equiv of NaH2PO4·H2O, t-BuOH/2-methyl-2-butene 4:1, 25 °C, 20 min, 82%) provided the carboxylic acid 36. Hydrolysis of the residue 3 nitrile with formation of the carboxamide 37 (40 equiv of 30% aq H2O2, 8.0 equiv of 10% aq K2CO3, DMSO, 25 °C, 3.5 h, 87%)4 set the stage for a final global deprotection.23 In a final key reaction, 37 was treated with AlBr3 (35 equiv, EtSH, 25 °C, 5 h, 80%) to afford 5 arising from the remarkable deprotection of four aryl methyl ethers, the two TBS ethers, the N-terminus Boc group, and the critical residue 4 methyl carbamate.

Assessment of [Ψ[CH2NH]Tpg4]Vancomycin Aglycon

A subtle element in the design of 5 and choice of simply removing the residue 4 carbonyl rests with the projected properties of the molecule. In principle, one might consider reengineering the capabilities of a reverse H-bond into the vancomycin structure removing the destabilizing lone pair interaction with D-Ala-D-Lac and reinstating the lost H-bond. Such opportunities include amidine derivatives (e.g. [Ψ[C(=NH)NH]Tpg4]vancomycin aglycon 39, Figure 3) and ongoing efforts have provided a thioamide intermediate we intend to use to access such derivatives.18 Significantly, such derivatives should enhance D-Ala-D-Lac binding approaching the level of affinity observed with vancomycin and D-Ala-D-Ala. However, these derivatives would also accordingly reduce binding to D-Ala-D-Ala. Consequently, they would be expected to gain antimicrobial activity against constituitively resistant bacteria endowed with a D-Ala-D-Lac peptidoglycan cell wall precursor (e.g. VanD), but be inactive against sensitive and inducibly resistant bacteria (VanA and VanB) that maintain or at least start with a D-Ala-D-Ala peptidoglycan cell wall precursor. The closest modified vancomycins that would be expected to reproduce the binding results observed in Figure 1 are those that replace the amide bond linking residues 4 and 5 with a methylene (CH2CH2) or ethylene (CH=CH) linker. Such derivatives, by analogy with the results in Figure 1, would be expected to enhance D-Ala-D-Lac affinity 100-fold missing only the contribution to binding derived from the H-bond. However, they might be expected to pay an additional penalty for binding D-Ala-D-Ala derived from not only the loss of the H-bond (10-fold), but also for not satisfying a ligand H-bond donor (10-fold?, for a cumulation 100-fold reduction?). As such, the derivatives might be expected to be marginally active against sensitive and inducibly resistant bacteria growing with a D-Ala-D-Ala peptidoglycan cell wall precursor (10 to 100-fold loss in activity), and more active against constituitively resistant bacteria (10-fold loss in activity) bearing the endowed D-Ala-D-Lac precursor. Consequently, and while we intend to examine such vancomycin analogues, they were not the first to be targeted for synthesis.

Figure 3.

Figure 3

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Potential modifications.

The targeted analogue 5 incorporating an amine in the linkage of residue 4 with residue 5 not only removes the offending carbonyl and the destabilizing lone pair interaction with D-Ala-D-Lac, but it maintains a local polar environment (protonated amine) that may better accommodate the binding of an electronegative group or atom (NH of D-Ala-D-Ala amide or O of D-Ala-D-Lac ester). Intuitively, we anticipated that while this might not bind D-Ala-D-Lac quite as well as derivatives such as 40, it would be better than 40 at binding D-Ala-D-Ala. In the best case, 5 might bind D-Ala-D-Ala and D-Ala-D-Lac with equal affinities making it effective for the treatment of sensitive or resistant bacteria regardless of the structure of the peptidoglycan cell wall precursor. Notably, this dual binding results from a reduction in the affinity for D-Ala-D-Ala and an enhancement in the affinity for D-Ala-D-Lac such that the affinity for either splits that observed with vancomycin. Accordingly, while the spectrum of antimicrobial activity increases relative to vancomycin to include sensitive and resistant bacteria, the potency of the derivative would be anticipated to be reduced 30–40 fold.

The results of our assessment of 5 alongside vancomycin (1) and its aglycon 38 are compiled in Table 1. An additional analogue 41, derived from N-Boc deprotection of the synthetic intermediate 33 (eq. 1), was also examined that bears the methoxycarbonyl protecting group on the residue 4/5 linking amine. The binding affinity of 5 for Ac2-L-Lys-D-Ala-D-Ala (2) and Ac2-L-Lys-D-Ala-D-Lac (4) was essentially equivalent (4.8 vs 5.2 × 103 M−1, respectively) with the D-Ala-D-Lac binding being slightly better. Impressively, this represented the anticipated results relative to the vancomycin aglycon where the enhancement for binding D-Ala-D-Lac is 43-fold (5.2 × 103 vs 1.2 × 102 M−1) and the reduction in binding affinity for D-Ala-D-Ala is 37-fold (4.8 × 103 vs 1.7 × 105 M−1). In addition, the comparison of 5 with 41 seems to reflect the intuitive expectations of the impact of the polar amine (protonated) versus its carbamate derivative where the binding affinity for D-Ala-D-Ala with 5 versus 41 increases 3-fold (4.8 vs 1.6 × 103 M−1) while the impact on D-Ala-D-Lac is a more marginal 1.2-fold increase in affinity (5.2 vs 4.1 × 103 M−1). Although there are additional structural features in the comparison of 5 and 41 that might impact the absolute affinities measured, in both instances the binding increases with the free amine 5 and it is with 5 that the dual binding is balanced.

Table 1.

Binding and Antimicrobial Properties

Compound Ka (M−1), 2a Ka (M−1), 4b Ka2/Ka4 VanAc, MIC (μg/mL)d
1, vancomycin 2.0 × 105 1.8 × 102 1100 >500 (2000)e
38, vancomycin aglycon 1.7 × 105 1.2 × 102 1400 >500 (640)e
5 4.8 × 103 5.2 × 103 0.92 31
41 1.6 × 103 4.1 ×103 0.40 31
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a

Ac2-L-Lys-D-Ala-D-Ala.

b

Ac2-L-Lys-D-Ala-D-Lac.

c

Enterococcus faecalis (VanA, BM4166).

d

Vancomycin and vancomycin aglycon exhibit MICs of 1–2.5 μg/mL against wild type E. faecalis.

e

Taken from ref. 25.

graphic file with name nihms63177e1.jpg (1)

The four compounds were compared in an antimicrobial assay against VanA Enterococcus faecalis (BM4166) that is inducibly resistant to treatment by glycopeptide antibiotics including vancomycin and teicoplanin, Table 1. It is the most difficult of the resistant organisms to treat (vs VanB) and characteristic of such organisms, they grow unchallenged enlisting a D-Ala-D-Ala peptidoglycan cell wall precursor, but switch to D-Ala-D-Lac upon glycopeptide treatment. As such, it represents a superb test of whether 5 and related dual D-Ala-D-Ala/D-Lac binding antibiotics might prove useful in the treatment of resistant bacteria. Beautifully, 5 as well as 41 exhibited MICs of 31 μg/mL being roughly 40-fold more potent than vancomycin or its aglycon (MICs = 2000 and 640 μg/mL) correlating well with the ca. 40-fold increase in binding affinity for D-Ala-D-Lac. Moreover, this potency is roughly 30-fold weaker than that observed with vancomycin and its aglycon against sensitive E. faecalis (MICs = 1–2.5 μg/mL) correlating with the 35 to 40-fold loss in binding affinity for D-Ala-D-Ala. These results suggest that regardless of the peptidoglycan cell wall precursor utilized by the organism, it remains equally sensitive to treatment by 5 and 41.

Conclusions

The modification of the dipeptide terminus of peptidoglycan cell wall precursors from D-Ala-D-Ala to D-Ala-D-Lac in resistant bacteria reduces the binding affinity of vancomycin for the ligand by 1000-fold leading to a 1000-fold loss in biological activity. We had shown that a modified peptide ligand possessing a methylene in place of the lactate oxygen restores 100-fold of this binding affinity by removal of a destabilizing lone pair interaction. This suggested that removal of the residue 4 carbonyl in the vancomycin aglycon would produce an analogue with enhanced affinity for D-Ala-D-Lac and might restore much of the biological activity of the molecule that is lost with resistant bacteria. Moreover and among the range of potential modifications that could be envisioned, that entailing the simple removal of the residue 4 carbonyl providing 5 was anticipated to bind D-Ala-D-Ala and D-Ala-D-Lac with similar affinities providing an analogue that might be equivalently effective against sensitive (D-Ala-D-Ala) and resistant (D-Ala-D-Lac) bacteria. Consequently, we extended our efforts on the preparation of glycopeptide antibiotics to a total synthesis of the [Ψ[CH2NH]Tpg4]vancomycin aglycon (5) in which the residue 4 carbonyl has been replaced with a methylene. Consistent with expectations and relative to the vancomycin aglycon, 5 exhibited a 40-fold increase in affinity for D-Ala-D-Lac (Ka = 5.2 × 103 M−1) and a corresponding 35-fold reduction in affinity for D-Ala-D-Ala (Ka = 4.8 × 103 M−1) providing a molecule with balanced, dual binding characteristics. Beautifully, 5 exhibited antimicrobial activity against a VanA resistant organism that remodels its D-Ala-D-Ala peptidoglycan cell wall precursor to D-Ala-D-Lac upon glycopeptide challenge displaying a potency that reflects these binding characteristics.

Supplementary Material

1si20051221_03. Supporting Information.

Full experimental details are provided. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgments

We gratefully acknowledge the financial support of the National Institutes of Health (CA 41101) and the Skaggs Institute for Chemical Biology. We wish to thank Drs. M. Lall, D. Horne, J. J. McAtee, O. Rogel, and C. C. McComas for initiating and contributing to studies on the preparation of the modified CD and ABCD ring systems and Professor Julius Rebek for his discussions, interest, and encouragement throughout the work. BMC is a Skaggs Fellow and recipient of Bristol-Myers Squibb (2004–2005) and Fletcher Jones Foundation fellowships (2003–2004).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1si20051221_03. Supporting Information.

Full experimental details are provided. This material is available free of charge via the Internet at http://pubs.acs.org.

NIHMS63177-supplement-1si20051221_03.pdf (146.4KB, pdf)

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