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.


Introduction
Rising rates of infection by drug-resistantGram-negative bacteria are becoming ag lobalc hallenge for public health systems and the economy. [1] The outer leaflet of the Gram-negative bacterialc ell envelope acts as an efficient permeability barrier and harbours major surfacec omponents,s uch as lipopolysaccharide (LPS), that interact with both innate and adaptive immune systems. [2] In general,t he LPS architecture includes the O-antigenic polysaccharide, the oligosaccharide core region and the lipid Ad omain. [3] Lipid Ai sc omposed of an acylated bisphosphorylated b-(1!6)-linked glucosamine disaccharide and is am ajor signalling molecule eliciting innate immune responses. [4] The lipid Ai su sually extendedb y3deoxy-d-manno-oct-2-ulosonic acid (keto-deoxyoctosonate, Kdo) residues,w hich in severalb acterial strains are partially replaced by the Kdo-isosterica nd acid-stable 3-hydroxy deriva-tive d-glycero-d-talo-oct-2-ulosonic acid (keto-octosonic acid, Ko). [5] The pronounced negative charge imparted by the phosphate and carboxylate groups in LPS serves as target for potent cationic antimicrobialp eptides (CAMPs)a nd lipopeptides such as polymyxin B(PmB)o rcolistin.
These antibiotics are used as last-resort treatments against multidrug-resistantb acteria. [6,7] Many bacteria, however,d evelop resistance mechanismst hat counteract the action of CAMPs and polymyxins through covalentm odification of the Kdo/Ko and lipid Aw ith positively charged appendices such as phosphoethanolamine, 2-amino-2-deoxy-sugars or 4-amino-4deoxy-l-arabinose (Ara4N). [8] For example, both the esterlinked and the glycosidically linked phosphate groups of the Burkholderia multivorans lipid Am ay be substituted by Ara4N residues (Scheme 1). [9] Ara4N substitution is implicated as a major causeo fa ntibiotic resistance in many bacteria, such as Burkholderia cenocepacia, Klebsiella pneumoniae, Pseudomonas aeruginosa, Yersinia pestis and Yersinia enterocolitica, Proteus mirabilis and Salmonella enterica serovarT yphimurium. [6a, 10] Inhibition of the biosynthetic pathwayi nvolvedi nt he formation of the activated sugar and blockingo ft he Ara4N transfer reaction, whichi sc atalysed by the ArnT transferase, should thus restorethe antimicrobial efficacy of PmB andC AMPs.
As shown by Raetz, a-configured undecaprenyl-phosphate Ara4N (UndP-Ara4N) serves as the donors ubstrate for ArnT,a membrane-embedded lipid-to-lipid glycosyl transferase. [11] ArnT from S. enterica serovar Typhimurium and from B. cenocepacia have been partly characterized [12][13][14][15] and a2 .8 crystal structure of ArnT from Cupriavidus metallidurans,i ncluding a cocrystal structure obtainedw ithU ndP as the ligand, has been 4-Amino-4-deoxy-l-arabinopyranose (Ara4N) residues have been linked to antibiotic resistance due to reduction of the negative chargei nt he lipid Aa nd 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 as eries of anomeric phosphodiester-linked lipid Ara4N derivativesc ontaining linear aliphatic chains as well as E-a nd Z-configured monoterpene units. Coupling reactions were based on sugar-derived H-phosphonates, followed by oxidation and globald eprotection.T he enzymatic Ara4N transfer was performed in vitro with crude membranes from ad eep-rough mutant from Escherichia coli as acceptor. Product formation was detected by TLC and LC-ESI-QTOF mass spectrometry.O ut of seven analogues tested, only the a-neryl derivativew as accepted by the Burkholderia cenocepacia ArnT protein, leading to substitution of the Kdo 2 -lipid Aa cceptor and thus affording evidence that ArnT is an inverting glycosyl transferase that requires the Z-configured double bond next to the anomericp hosphate moiety.T his approachp rovides an easily accessible donor substrate forb iochemical studies relating to modificationso fb acterial LPS that modulate antibiotic resistance and immune recognition.
reported. [16] Unfortunately,t he bindings ite interactions with the ArnT portion were modelled on the basis of docking studies that used the incorrect anomeric configuration of the Ara4N residue, displaying the sugar part in the inverted 1 C 4 chair conformation. [16] Thus, the underlying ArnT reaction mechanism and detailedk nowledge relating to binding of donor and acceptors ubstrates are still lacking. It is also unknown whether as ingle ArnT enzymea cts as am ultifunctional transferase( substituting both lipid Aa nd the inner core sugars)o rw hether separate Ara4N transferases are neededf or glycosylation at O-8 of Kdo ando fK o.
Only minor amountso ft he native Ara4N-P-undecaprenyl donor (0.2 mg) had previously been isolatedf rom bacteria. [11] Thus, chemical syntheses of Ara4N donors tructures and potential acceptors ubstrates are neededf or study of the underlying enzymatic mechanisms and transport/translocation processes, in particular in order to obtain X-ray structures with the liganded sugar and binding data in the presence of substrates and substrate analogues,t hereby enabling the rational design of efficient enzyme inhibitors. Inhibitors of the enzymatic formylation of Ara4N-UDP (catalysedb yA rnB) with activity in the high micromolar range had previously been prepared, but were not sufficiently activet oc onfer PmB sensitivity. [17] We report here the chemical synthesis of seven phosphodiester-activated4 -amino-4-deoxy-l-arabinose derivatives, the development of an assay to monitor the in vitro transfer reaction catalysed by ArnT from B. cenocepacia,and the product characterization by LC-ESI-QTOF mass spectrometry.

Synthesis of Ara4N enzyme donors
For the synthesis of the Ara4N donor,t he precursor material was readilyp repared in multigram quantities from methyl b-d-xylopyranoside (1,S cheme 2). [18] A4 -azido group was chosen  as the amino group precursor,a nd the 2-O-a nd 3-O-positions  were protected with at etraisopropyl-disiloxane-1,3-diyl (TIPDS) group insteado fb enzylg roups, to avoid any need for hydrogenolytic deblockings teps that would not be compatible with the prenyl double bonds. For the coupling steps we relied on the preparation of the anomeric H-phosphonates that are frequently used for the assembly of phosphodiester-linked glycans because these are more stable than intermediate phosphoramidites. [19] We had previously optimized the formation of the axial b-anomericp roduct needed for the connection of Ara4N H-phosphonate to the reducing end of Burkholderia lipid A. [20] Structural evidence for the a-l configuration of the Und-P-Ara4N donor substrate leading to the b configurationo f Ara4N residues when linked to lipid Aa sw ell as to Kdo and Ko suggested that the a-configured derivatives should serve as the primaryt argets for the enzymatic transfer. [21] To achieve preferred formation of the equatorial H-phosphonate in the reactionw ith salicyl chlorophosphite, slow addition of reagent over al ong time period was essential to allow for ak inetically controlled reaction at the more nucleophilic equatorial hydroxy group (Scheme2,T able 1). [22,23] To achieves ignificant enrichmento ft he a-isomer 5,t he optimum addition rates should be in ar ange between 0.1 and 0.2 equivalents of the chlorophosphite reagent per hour (entries 5a nd 6).
Next, the formation of the phosphodiester derivative was elaborated throught he use of octan-1-ol (6)a nd the b-anomeric H-phosphonate 4 in model reactions. Activation of the Hphosphonate 4 was first attempted with pivaloyl chloride in pyridine followed by oxidation with aqueousi odine. [24] In addition to product 7,amajor by-product corresponding to an Npyridinium glycoside was isolated.T op revent this side reaction, sterically hindered 2,6-dimethylpyridine as abase with enhanced nucleophilicity was then used throughout the coupling reactions. In this way,p roduct mixtures of 7 and ring-opened derivative 8 werei solated in combined yields of 40-42 %. Extending the reaction time for the coupling step, however,l ed to increased formation of the ring-opened form. To increase the stability of the anomeric phosphodiester,t he products were converted into their triethylammonium salts. The formation of the phosphodiester was confirmedt hroughi ts 31 PNMR Scheme1.Ara4N-modified lipid As tructure of B. multivorans.  29 Si signal at À15.9 ppm, whereas the H-3 and H-2 protonso fc ompound 7 at 4.32 and 4.03 ppm, respectively,s howed connectivity to 29 Si NMR signals at À8.5 and À9.0 ppm. Because global deprotection would include removal of the silyl group anyway, the mixture of 7 and 8 was directly employed for the ensuing deprotection steps. Removal of the TIPDS group was achieved by treatment with triethylaminet rihydrofluoride (TREAT) followed by removalo ft he fluoride ions with calcium carbonate. Staudinger reduction of the 4-azido group with trimethylphosphine in the presence of aqueous THF and NaOH gave phosphodiester 9 as its sodium salt after final purification on Bio-GelP 2i n 59 %y ield over two steps.
Coupling between H-phosphonate 4 and geraniol (Scheme3), however,g ave additional by-products, leadingt o mixtures that required multiple chromatographic purification steps in order to providet he target derivative. Severals ide reactions, most probably involving mixed anhydrides and Hpyrophosphonatesa sr eactive intermediates,h ave previously been reported in pivaloyl-chloride-mediated H-phosphonate diesters ynthesis. [25] We explored the activation pathway of Hphosphonate 4 in the absence of the alcoholicc omponent by use of 31 PNMR monitoringa nd isolation of the main product after iodine-mediated oxidation. H-Phosphonate 4 reactedw ith pivaloyl chloride in CDCl 3 in the presenceo f2 ,6-lutidinew ithin 15 min, accompanied by the appearance of two 31 PNMR signals (at À6.50 and À8.04 ppm) that did not change over a periodo f2ha tr oomt emperature. Upon oxidation, am ain signala tÀ6.05 ppm wass een, and gradually additional minor upfield-shifted signals appeared. As the main product, the acyl phosphonate 12 wast hen isolatedb yc hromatography.T he structure assignment of 12 was based on the significantly downfield-shifted C=Os ignal at 218.7 ppm, with J C,P = 154.6 Hz;t his is in close agreement with published NMR data for acyl H-phosphonates. [26] The structure was also confirmed by LC-ESI-MS data that revealed am olecular ion of m/z 566.2488, thus suggesting as tructure related to ester 14 but with loss of one oxygen atom (Scheme 3).
Formation of this by-product might be explained in terms of phosphite formation induced by 2,6-lutidine followed by an Arbusov reaction with the acyl chloride. Since the isolated yields of the phosphodiester derivatives were modest,adifferent activation and oxidation protocolw as then tested with Hphosphonate 5 and eicosan-1-ol (18,S cheme 4).
Coupling of H-phosphonate 5 was performed with chlorotripyrrolidinophosphonium hexafluorophosphate( PyTP) followed by conversion into the silylated phosphite triester with bis(trimethylsilyl)acetamide (BSA) and subsequento xidation with camphorsulfonic acid (CSO). [27,28] This approach, however,p roduced the diester derivative 19 only in ap oory ield. Compound 19 wasf inally converted into the deprotected derivative 20.I nv iew of theser esults, we again opted for the pivaloyl chloride activation for the synthesis of the a-geranyl and a-neryl derivatives 23 and 24,r espectively.S imilarly to the outcome for the b-analogues 13 and 15,t he condensation step also led to formation of minor amountso fO -a nd C-linked pivaloyl derivatives 21 and 22.R emoval of theseb y-productsw as effectively accomplished upon deprotection of the mixture and azide reduction, which eventually afforded the diester derivatives 25 and 26 in their pure states after chromatography on BioGel P-2. Enzymatic assay for the ArnT-catalysed reaction ArnT from B. cenocepacia was expressed in Escherichia coli as a FLAG-His 10 -taggedp rotein as previously described, and crude membranes werep repared. [14a] The presence of ArnT in the membranes was confirmed by SDS-PAGE followed by Coomassie BrilliantB lue (CBB) staininga nd western blot with use of anti FLAG antibodies ( Figure 1).
Prior to testing of the substrates for the enzymatic reaction, am odified assay based on the commercial deep rough type LPS containing lipid Aa nd the a-(2!4)-linked Kdo disaccharide, termedK LA, was developed. The assay should be compatible with the phenol/water extraction conditions used for LPS isolation and the hydrolytic lability of phosphodiester-linked 4aminoarabinosyl substituents. [29] After extensive exploration of conditions and isolation protocolsw ef ocused on monitoring of the glycosyl transfer reaction by preparative TLC, [30] which allowed for visual inspection of product formation,a nd subsequent structurala nalysiso fp roducts by LC-ESI-QTOF mass spectrometry.F urther,t his approach also allows for removal of residual surfactants and extractionm edia arising from the celllysing procedure and LPS isolation.T he donor substrates were treated with KLA in the presence of ArnT for 17 ha t3 08C. The samples were concentrated, dissolved in chloroform/methanol 4:1, spotted in 5 mLp ortionso nto prewashed silica gel 60 TLC plates and developed with CHCl 3 /pyridine/88% HCOOH/water 50:50:16:5 ( Figure 2). The lipid fractions were recoveredf rom the plates through as ingle acidic Bligh-Dyer extraction followed by centrifugation and treatment with at wo-phase Bligh-Dyer extraction.T hey were then subjected to LC-ESI-QTOF analysis.
The glycosyl acceptorK LA couldb ed etected in an R f range of % 0.24-0.28 in all experiments. Formation of an ew com-pounda tR f % 0.15 and reduction of the intensity of acceptor substrate KLA was observed in the assay with the a-neryl derivative 26 ( Figure 2, lane a), whereas none of the other substrates showed product formation.
These resultsw ere confirmed by LC-ESI-MS analysis of lipid fractionse xtracted from the TLC plates after using the anomeric neryl derivatives 26 and 17 and the a-geranyl derivative 25 as substrates. These selected donors should clarify the dependence of the ArnT reaction with respectt ot he anomeric configuration of the arabinosyl unit, as well as to the stereochemistry of the monoterpene unit. To achieve sufficient concentration of products,a ssays were runi nd uplicatei nt he cases of compounds 17 and 25 and were repeated seven times in that of compound 26.T LC-extracted samples were subjected to C5 reversed-phaseH PLC separation and eventually measured in the positive mode with the ESI-QTOF instrument.H PLC profiles and spectra are presented in Figure 3. Lipid extracts from reactions with 17 and 25 gave only mass data corresponding to the unreacted acceptorK LA substrate, with monoisotopic m/z values of 1119.674 for the doubly charged molecular ion [M+ +2H + ] 2 + and 1128.187 for the ammoniuma dduct [M+ +NH 4 + + +H + ] 2 + (Figure 3.) The formation of the Ara4N-KLA in the reactionw ith 26 was evident from the appearance of peaks at higher molecular mass (1185.705)c orresponding to the transfer of one Ara4N residue. In three assays avery minor peak (3-5.5 %) was detected at m/z 1250.740, indicative of the transfer of as econd Ara4N residue.F or quantification of the Ara4N-KLA derivative formed in the reactionw ith 26,t he areas of the ion chromatogramsf rom the first four isotopic peaks of KLA and product were summed and compared, leading to an approximate range of 40 to 65 %c onversion of 26.T he attachment site of the Ara4N residue couldn ot be determined. Literature data for lipid Am odification in the presence of the Kdo core, however, suggest preferential formation of the phosphodiester-linked Ara4N at the 4' position of lipid A. [11,31]   The ArnT reactionw as thus only productivew itht he a-neryl derivative 26,a ss hown by TLC and LC-ESI-MS analysis. This was evidencet hat the enzyme is specific for the a-anomeric configuration of the l-arabinosyl unit and the Z configuration of the lipid part. Dependenceo fg lycosyl transferases on lipid chain length and double bond configurationh as previously been reported for related pyrophosphate substrate derivatives. The lipid carrier specificity of the N-glycosylation enzyme PglB from Campylobacter jejuni has been studied, for example, revealing that the enzyme is not active with C10 andC 15 lipid appendages but readily transferss ubstrates with four prenyl units, provided that the two proximal units are cis-configured. [32] In contrast,t he membrane-associated UDP GlcNAc transferaseM urG involved in peptidoglycan synthesis hasa more relaxed acceptors ubstrate specificity,w ithaneryl derivative having previously been observed to be accepted by the glycosyltransferase. [33] As imilar preference wasr eported for a galactofuranosyl transferase from Mycobacteriumt uberculosis that accepted a C-linked neryl derivative as substrate. [34] The synthetic a-neryl donors ubstrate for ArnT reactions should be useful for study not only of the modification of the phosphate groups in the lipid Ad omain, but also of Ara4N gly-coside formationi nt he core regiono fs everal Enterobacteriaceae. It remains to be determined whether chain elongation including three or four prenyl units will be neededf or the donor substrates for optimum enzymatic transfer.

Conclusion
A4 -amino-4-deoxy-l-arabinosyl transferasef rom B. cenocepacia was successfully expressed and used in ac rude membrane preparation to test the substrate specificities of as mall library of lipid/phosphate-linked 4-amino-4-deoxy-l-arabinosyl derivatives. The phosphodiester derivatives were obtained in modest yields from 2,3-O-TIPDS-protected 4-azido-substituted H-phosphonate derivatives by pivaloyl-chloride-promotedc oupling steps followedb yo xidation. Formation of by-products in the activation step could be analysed, and azide reduction and final purification steps were optimised to give the deprotected donor substrates. Glycosyl transfer reactions were monitored by TLC, followed by structuralc haracterization of the TLC extracts. The ArnT reactionw as only productive with the a-neryl derivative, as shown by LC-ESI-MS analysis; this was therefore evidencethat the enzymeiss pecific for the a-anomeric config-  uration of the arabinosyl unit and the Z configurationo ft he lipid part. As imple synthetic donors ubstrate for testing enzymatic pathways involved in antibiotic resistancem echanisms and crystallographic studies is therefore now availablew ithout the necessity to incorporate oligoprenyl lipid chains.

Experimental Section
General methods:A ll reactions were carried out in oven-dried glassware. Solvents and reagents were purchased from commercial suppliers and used as provided without further purification unless stated otherwise. Solvents (THF,t oluene, CH 2 Cl 2 )w ere dried over activated molecular sieves (4 ). 2,6-Lutidine was distilled over CaH 2 and stored over CaH 2 .C ation-exchange resin DOWEX 50 H + was regenerated by consecutive washing with HCl (3 m), water and dry MeOH. Concentration of organic solutions was performed under reduced pressure at < 40 8C. Optical rotation was measured with an Anton Paar MCP100 Polarimeter at 20 8C. Reactions were followed by TLC with Merck plates:g enerally on 5 10 cm, layer thickness 0.25 mm, silica gel 60F 254 ,o ra lternatively on HPTLC plates with 2.5 cm concentration zone (Merck). Spots were visualized with UV (254 nm) and/or by anisaldehyde/H 2 SO 4 staining. Preparative chromatography was performed either with silica gel (0.040-0.063 mm) or with af lash-purification system (Interchim, PuriFlash 4125). NMR spectra were recorded with aB ruker Avance III 600 instrument ( 1 Ha t6 00 MHz, 13 Ca t1 51 MHz, 31 Pa t2 43 MHz, 29 Si at 119.2 MHz) and use of standard Bruker NMR software. Chemical shifts are given in ppm downfield from SiMe 4 using the residual peak of CDCl 3 (7.26 for 1 Ha nd 77.00 for 13 C), CD 3 OD (3.31 for 1 H and 49.86 for 13 C) or D 2 O( 0.00 for 1 H, external calibration to 2,2dimethyl-2-silapentane-5-sulfonic acid), 67.40 for 13 C( external calibration to 1,4-dioxane in D 2 O), and orthophosphoric acid (d = 0) for 31 P. HRMS ESI-TOF data were obtained with aW aters Micromass Q-TOF Ultima Global instrument.
Optimization of anomeric H-phosphonate formation (compounds 4a nd 5):C ompounds 4 and 5 [4-azido-4-deoxy-2,3-O-(tetraisopropyldisiloxane-1,3-diyl)-b-a nd -a-l-arabinopyranose hydrogen phosphonate, triethylammonium salts] were essentially prepared according to the literature and gave matching NMR data and optical rotation values. [20] To enrich compound 5,h owever,t he following conditions were used:d ry pyridine (13 equiv) was added to salicyl chlorophosphite (3,4equiv) in dry THF (2.52 mL mmol À1 3)a t08Ca nd the mixture was stirred for 1h at RT.T he solution was transferred into as yringe and added at ar ate of 0.1 equiv h À1 to as olution of 2 (54 mg, 0.129 mmol) in dry THF (3.5 mL). After complete addition of the activated salicyl phosphite pyridinium salt, aqueous ammonium bicarbonate solution (1 m,1 .1 mL) was added and the mixture was stirred for an additional 30 min and then diluted with EtOAc (25 mL) and washed with aqueous ammonium bicarbonate buffer (0.3 m,1 5mL). The aqueous layer was reextracted with dichloromethane (2 20 mL) and the combined organic layers were filtered over cotton. To luene (5 mL) was added and volatiles were evaporated under reduced pressure prior to chromatographic separation according to the literature. [20] NMR analysis of the crude product showed a 5/4 mixture (6.1:1).
General Procedure Af or phosphodiester synthesis: H-Phosphonate 4 or 5 was co-evaporated with dry toluene (4 10 mL) and dried under high vacuum overnight. The acceptor alcohol (4 or 5equiv) was pre-dried over molecular sieves (4 )a nd added at RT under Ar to as olution of the H-phosphonate in dry 2,6-lutidine (1.0-2.4 mL/0.1 mmol). The solution was stirred for 10 min, pivaloyl chloride (2-4.5 equiv) was then added dropwise at RT,a nd the re-action was monitored by 31 PNMR, by transferring aliquots (50 mL) of the reaction mixture into an NMR tube and adding CDCl 3 (0.4 mL). The reaction mixture was cooled to 0 8C, as olution of iodine (2 %, 1.5 equiv) in 2,6-lutidine/water (50:1, 1.0 mL/0.1 mmol) was added dropwise, and the mixture was stirred at 0 8Cf or 1h. The solution was then diluted with chloroform followed by addition of 1:17 %a q. Na 2 S 2 O 3 /2 m triethylammonium bicarbonate (TEAB) buffer.T he aqueous phase was extracted with chloroform and the combined organic phases were filtered over cotton, concentrated and co-evaporated with toluene (10 mL) under reduced pressure. The product was purified by two to four consecutive chromatographic purification steps on silica gel [chloroform containing Et 3 N( 1%)!chloroform/MeOH 9:1+ Et 3 N( 1%)].T os tabilize the phosphate bridge, purified compounds and pooled fractions were dissolved in chloroform (14 mL) and washed with TEAB buffer (0.25 m,8mL). The organic phase was dried over cotton and concentrated to give the stable phosphodiester derivative.
General Procedure Bf or TIPDS removal:T riethylamine trihydrofluoride (3 equiv) was added at 0 8Ct oasolution of phosphodiester in dry THF (2.5 mL mmol À1 )i nascrew-top Teflon flask and the mixture was stirred at RT under Ar until complete conversion. CaCO 3 (6 equiv) and dry THF (2.5 mL mmol À1 )w ere then added. The suspension was stirred at RT for 1hand centrifuged at 1972 g for 15 min. The liquid phase was recovered and lyophilised overnight to give the crude intermediate product that was directly used for the next reaction.
General Procedure Cf or azide reduction:P Me 3 (4 equiv) was added at RT under Ar to asolution of the crude intermediate product in THF/0.1 m NaOH (1:2) and the mixture was stirred until full conversion. The solution was diluted with water and washed with chloroform. The combined organic phases were twice re-extracted with water and stripped with Ar until the solution became clear. The aqueous phase was lyophilised and the residue was purified by gel chromatography (BioGel P-2, eluent aq. EtOH, 5%)a nd lyophilised overnight to afford the pure target phosphodiester derivative.
Octyl 1-O-[4-azido-4-deoxy-2,3-O-(tetraisopropyldisiloxane-1,3diyl)-b-l-arabinopyranosyl] phosphate, triethylammonium salt (7): H-Phosphonate 4 (46 mg, 0.079 mmol) was co-evaporated with dry toluene (4 10 mL) and dried under high vacuum overnight. Octan-1-ol 6 (25 mL, 0.159 mmol) was pre-dried over molecular sieves (4 )a nd added to as olution of 4 in dry 2,6-lutidine (1.9 mL). This was stirred for 10 min at RT under Ar.P ivaloyl chloride (44 mL, 0.357 mmol) was then added dropwise at RT and the reaction mixture was stirred for 46 h. The solution was cooled to 0 8Ca nd as olution (2 %) of iodine (30.2 mg, 0.119 mmol) in 2,6-lutidine/water (50:1, 0.8 mL) was added dropwise and stirred at 0 8C for 1h.T he solution was diluted with chloroform (50 mL) followed by addition of am ixture of aq. Na 2 S 2 O 3 (7 %)/TEAB buffer (2 m;1 :1, 20 mL). The aqueous phase was extracted with chloroform (2 50 mL) and the combined organic phases were filtered over cotton, concentrated and co-evaporated with toluene (7 mL) under reduced pressure. The product was purified by flash chromatography on silica gel (chloroform containing 1% Et 3 N!9:1c hloroform/ MeOH + 1% Et 3 N). Product-containing fractions were pooled and concentrated. The residue was dissolved in chloroform (15 mL) and washed with TEAB buffer (0.25 m,8mL). The organic phase was dried over cotton and concentrated to give ap roduct fraction (23. 8 1-ol (65.3 mL, 0.415 mmol) was pre-dried over molecular sieves (4 )and added at RT under Ar to asolution of 5 in dry 2,6-lutidine (2.5 mL). The mixture was stirred for 10 min and treated with pivaloyl chloride (57.4 mL, 0.466 mmol) for 22.5 ha tRT.T he reaction mixture was cooled to 0 8C, as olution (2 %) of iodine (39.5 mg, 0.155 mmol) in 2,6-lutidine/water (50:1, 1.10 mL) was added dropwise, and stirring was continued at 0 8Cf or 1h.W orkup and purification as described in General Procedure Aa fforded 10 (  ture was stirred. After 2.25 h, as econd aliquot (50 mL) of the reaction mixture was transferred into an NMR tube and diluted with CDCl 3 (0.4 mL). The NMR spectrum showed total conversion of the starting material into an intermediate product. After 2.25 h, the solution was cooled to 0 8C, as olution (2 %) of iodine (23.9 mg, 0.094 mmol) in 2,6-lutidine/water (50:1, 0.64 mL) was added dropwise, and the mixture was stirred at 0 8Cf or 1h.A tt his point, an NMR sample was withdrawn from the reaction mixture;i ts howed completion of the reaction. The solution was diluted with chloroform (40 mL) followed by addition of am ixture of 7% aq. Na 2 S 2 O 3 / 2 m TEAB buffer (1:1, 16 mL). The aqueous phase was extracted with chloroform (2 40 mL) and the combined organic phases were filtered over cotton, concentrated and co-evaporated with toluene (3 mL) under reduced pressure. The product was purified by flash chromatography on silica gel (chloroform containing 1% Et 3 N!chloroform/MeOH 9:1+ +1% Et 3 N). Product-containing fractions were pooled and concentrated. The residue was dissolved in chloroform (7 mL) and washed with TEAB buffer (0.25 m,4mL). The organic phase was dried over cotton and concentrated to give a fraction of pure 12 (10.7 mg). 1    Expression and membrane preparation of ArnT:A rnT-FLAG-His 10 recombinant protein was expressed as previously reported. [14a, 35] pFT1 plasmid encoding the B. cenocepacia arnT-FLAG-His 10 was introduced into E. coli DH5a by CaCl 2 transformation. For protein expression, cells were grown in lysogeny broth (LB, 1L)m edium supplemented with ampicillin (100 mgmL À1 )a t3 78Cw ith shaking until A 600 reached 0.5-0.6 and were induced with l-arabinose (0.2 %, w/v)f or 4h.C ells were harvested by centrifugation (10 min at 11 270 g and 4 8C) and stored at À20 8C. All subsequent steps were carried out at 4 8C. Bacterial pellets were resuspended in Tris·HCl (pH 8.0, 50 mm)w ith protease inhibitors and lysed at 25 000 psi with ac ell disruptor (Constant Systems, Kennesaw,G A). Cell debris was pelleted by centrifugation at 9590 g for 15 min at 4 8C, and aliquots of the supernatant were centrifuged at 42 220 g for 1h to collect total membranes. One pellet containing crude membranes was resuspended in Tris·HCl (pH 8.0, 50 mm). Proteins were separated by SDS-PAGE (12 %) and transferred to an itrocellulose membrane. Immunoblots were probed with anti-FLAG mouse monoclonal antibodies (Sigma) for 1h.I RDye 800CW Goat anti-Mouse IgG (LI-COR) was used as secondary antibody.R eacting bands were detected with an Odyssey infrared imaging system (Li-cor Bioscience, Lincoln, NE). Protein concentration was determined by the Bradford assay (Bio-Rad).
Assay conditions to detect ArnT activity:A rnT from B. cenocepacia was assayed in ar eaction mixture (100 mL) consisting of MES buffer (pH 6.5, 50 mm), Triton X-100 (0.2 %) and Kdo 2 -lipid A( KLA, 30 mm)a sa cceptor.C rude membranes from ArnT expression (1 mg mL À1 )w ere used as the source of l-Ara4N transferase;s ynthesized a-a nd b-Ara4N-lipid phosphodiester derivatives (150 mm) were used as donor.R eaction mixtures were incubated at 30 8Cf or 17 h. The samples were concentrated with as peed vacuum concentrator for 40 min, dissolved in CHCl 3 /MeOH (4:1, 2 30 mL), and spotted (5 mLp ortions) onto prewashed silica gel 60 TLC plates (prewashing of the plates in CHCl 3 /pyridine/88 %f ormic acid/water (50:50:16:5) for 1h with shaking at 180 rpm before drying overnight). Reaction products were separated in CHCl 3 /pyridine/88 % formic acid/water (50:50:16:5), and the plates were dried for 2hat room temperature. The bands corresponding to the KLA or modified KLA products could be seen transiently as white zones while the TLC plates were drying at RT and subsequently by staining of a lane cut from the plate with anisaldehyde/H 2 SO 4 .T LC areas containing the putative KLA and l-Ara4N-modified KLA derivatives were removed with ac lean knife and the lipids were extracted from the chips with an acidic single-phase Bligh-Dyer mixture (304 mL) containing CHCl 3 /MeOH/0.1 m HCl (1:2:0.8). After vortexing of the mixture and sonication for 40 min, solids were removed by centrifugation at 11 000 g for 10 min and the supernatant was transformed into atwo-phase Bligh-Dyer system by adding chloroform (80 mL) and water (80 mL). After vortexing and centrifugation of the samples at 2000 g for 10 min, the lower phase containing the lipids was withdrawn and dried with as peed vacuum concentrator for 15 min at RT.F or each synthetic Ara4N-lipid phosphodiester donor three separate assays were carried out, followed by dissolution in CHCl 3 /MeOH (4:1, 3 50 mL). The combined solutions were concentrated in one vial and stored at 4 8Cp rior to mass spectrometric analysis.