Synthetic Phosphodiester-Linked 4-Amino-4-deoxy-l-arabinose Derivatives Demonstrate that ArnT is an Inverting Aminoarabinosyl Transferase

Abstract

4-Amino-4-deoxy-l-arabinopyranose (Ara4N) residues have been linked to antibiotic resistance due to reduction of the negative charge in the lipid A and core regions of the bacterial lipopolysaccharide (LPS). To study the enzymatic transfer of Ara4N onto lipid A, which is catalysed by the ArnT transferase, we chemically synthesised a series of anomeric phosphodiester-linked lipid Ara4N derivatives containing linear aliphatic chains as well as E- and Z-configured monoterpene units. Coupling reactions were based on sugar-derived H-phosphonates, followed by oxidation and global deprotection. The enzymatic Ara4N transfer was performed in vitro with crude membranes from a deep-rough mutant from Escherichia coli as acceptor. Product formation was detected by TLC and LC-ESI-QTOF mass spectrometry. Out of seven analogues tested, only the α-neryl derivative was accepted by the Burkholderia cenocepacia ArnT protein, leading to substitution of the Kdo₂ -lipid A acceptor and thus affording evidence that ArnT is an inverting glycosyl transferase that requires the Z-configured double bond next to the anomeric phosphate moiety. This approach provides an easily accessible donor substrate for biochemical studies relating to modifications of bacterial LPS that modulate antibiotic resistance and immune recognition.

Keywords: Burkholderia; carbohydrates; glycolipids; glycosyl transferases; lipopolysaccharide.

Scheme 1

Ara4N‐modified lipid A structure of B. multivorans.

Scheme 2

Synthesis of anomeric H‐phosphonates and octyl phosphodiester derivatives 9 and 11. a) Pyridine, THF, RT, then 1 m aq. NH₄HCO₃; b) pivaloyl chloride, 2,6‐lutidine, RT, then aq. I₂, 0 °C; c) triethylamine trihydrofluoride (TREAT), THF, RT, then CaCO₃, MeOH, RT, then Me₃P, THF, aq. NaOH.

Scheme 3

Synthesis of β‐geranyl and β‐neryl derivatives 16 and 17, respectively. a) PivCl, 2,6‐lutidine, RT, then aq. I₂, 0 °C; b) ROH, PivCl, 2,6‐lutidine, RT; aq. I₂, 0 °C; c) TREAT, THF, RT; CaCO₃, MeOH, RT, then Me₃P, THF, aq. NaOH.

Scheme 4

Synthesis of α‐geranyl and α‐neryl derivatives. a) PyTP, BSA, then CSO; b) TREAT, THF, RT; then CaCO₃, MeOH, RT; then DTT; c) ROH, PivCl, 2,6‐lutidine, RT; aq. I₂, 0 °C; d) TREAT, THF, RT; CaCO₃, MeOH, RT, then Me₃P, THF, aq. NaOH.

Figure 1

ArnT from B. cenocepacia expressed in E. coli membranes. A) ArnT‐FLAG‐His₁₀ (71.8 kDa) in total cell lysate and after total membrane preparation was analysed by SDS‐PAGE followed by CBB staining. B) Immunodetection of ArnT‐FLAG‐His₁₀ in total membrane with anti‐FLAG antibodies. CM: crude membrane.

Figure 2

In vitro detection of B. cenocepacia ArnT activity with α‐Ara4N‐neryl phosphate 26 by TLC. ArnT from B. cenocepacia was assayed with use of KLA (30 μm) as acceptor, crude membranes from ArnT expression (1 mg mL⁻¹) as source of l‐Ara4N transferase and 150 μm synthetic donor substrates (lane a: 26, lane b: 17, lane c: 25, lane d: 16, lane e: 11, lane f: 9, lane g: 20. Acceptor KLA is visible as spot on the right‐hand lane on each TLC plate.). Lane h shows a control assay containing 26 but lacking acceptor KLA. Reaction mixtures were incubated at 30 °C for 17 h. Spots were stained with anisaldehyde/H₂SO₄.

Figure 3

In vitro detection of B. cenocepacia ArnT activity with α‐Ara4N‐neryl phosphate 26 by LC‐ESI‐MS. A) LC‐MS chromatograms of purified KLA derivatives from assays containing 1) α‐Ara4N‐neryl phosphate 26, 2) β‐Ara4N‐neryl phosphate 17 and 3) α‐Ara4N‐geranyl phosphate 25 as donors. KLA (EIC= 1119.674, 1120.175, 1120.677, 1128.187, 1128.688, 1129.190), KLA+1 Ara4N (EIC=1185.203, 1185.704, 1186.206, 1193.716, 1194.217, 1194.719) and KLA+2 Ara4N (EIC=1250.733, 1251.235, 1251.737,1252.238). B) MS spectra of purified KLA derivatives from assays containing 1) α‐Ara4N‐neryl phosphate 26, 2) β‐Ara4N‐neryl phosphate 17 and 3) α‐Ara4N‐geranyl phosphate 25 as donors. KLA: calcd for C₁₁₀H₂₀₂N₂O₃₉P₂+2 H²⁺ [M+2 H]²⁺: 1119.675. KLA+1 Ara4N: calcd for C₁₁₅H₂₁₁N₃O₄₂P₂+2 H²⁺ [M+2 H]²⁺: 1185.20. KLA+2 Ara4N: calcd for C₁₂₀H₂₂₀N₄O₄₅P₂+2 H²⁺ [M+2 H]²⁺: 1250.733.