Chemoenzymatic Synthesis of Glycosylated Macrolactam Analogues of the Macrolide Antibiotic YC-17

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DOI:10.1002/adsc.201500250 Chemoenzymatic Synthesis of Glycosylated Macrolactam Analogues of the Macrolide Antibiotic YC-17

Pramod B. Shinde,a,e Hong-Se Oh,b Hyemin Choi,c Kris Rathwell,a Yeon Hee Ban,a Eun Ji Kim,a Inho Yang,a Dong Gun Lee,c David H. Sherman,d Han-Young Kang,b,*and YeoJoon Yoona,* a Department of Chemistry andNano Science,Ewha Womans University,Seoul 120-750, Republic of Korea Fax: (+82)-2-3277-3419;phone:(+ 82)-2-3277-4446;e-mail:[email protected] b Department of Chemistry,Chungbuk National University,Cheongju 361-763, Republic of Korea Fax: (+82)-43-267-2279;phone:(+ 82)-43-261-2305;e-mail:[email protected] c School of Life Sciences,BK21Plus KNU Creative BioResearch Group,College of Natural Sciences,Kyungpook National University,Daehak-ro 80, Buk-gu, Daegu 702-701,Republic of Korea d Department of Medicinal Chemistry,Life Science Institute,DepartmentofChemistry,and Department of Microbiology &Immunology, University of Michigan, Ann Arbor, Michigan 48109, USA e Present address:Institute of Bioinformatics and Biotechnology (IBB), Savitribai Phule Pune University (formerly University of Pune), Pune 411-007, India

Received:March 12, 2015; Revised:June 15, 2015;Published online:August 19, 2015

Supporting information for this article is availableonthe WWW under http://dx.doi.org/10.1002/adsc.201500250.

Abstract: YC-17 is a12-membered ring macrolide sugars for subsequent glycosylation. Some YC-17 antibiotic producedfrom Streptomyces venezuelae macrolactam analogues were active against erythro- ATCC 15439 andiscomposed of the polyketide mac- mycin-resistant bacterial pathogens and displayed rolactone10-deoxymethynolide appendedwith d- improved metabolic stability in vitro.The enhanced desosamine.Inorder to develop structurally diverse therapeutic potentialdemonstrated by these glycosy- macrolactam analoguesofYC-17 with improved lated macrolactam analogues reveals the uniquepo- therapeutic potential, acombined approach involv- tential of chemoenzymatic synthesis in antibiotic ing chemical synthesis and engineered cell-based bio- drug discoveryand development. transformation was employed. Eight new antibacterial macrolactam analogues of YC-17 were generated by supplying anovelchemically synthesized macro- Keywords: antibacterial activity;glycosyltransferase; lactam aglycone to S. venezuelae mutantsharboring macrolide glycosides; Streptomyces venezuelae;YC- plasmids capable of synthesizing several unnatural 17

Introduction lide esterases of resistantpathogens.While analogue generation via macrolactone structural modification is Macrocyclic polyketides are ahighly diverse and clini- necessary to increase the stability or efficacy of these cally important class of natural products that include clinically important pharmaceuticals,manyofthe antibiotics (erythromycin),[1] immunosuppressants next-generationstructures remainsusceptible to hy- (FK506),[2] antiparasitics (avermectin),[3] antifungal drolysisbythe macrolide esterases producedbysome agents (amphotericin B),[4] andantitumor agents antibiotic-resistant microbial pathogens.[6] Conse- (epothilone)[5] (Figure 1A). Their structures are char- quently,the need for bioactive molecules with newor acterizedbyamacrolactone and are often decorated improved pharmacologicalproperties that can toler- by one or more deoxyand amino sugar moieties. ate these conditions has further increasedinterest in While the macrolactone is an important pharmaco- generating novelmacrolides.[7] Forexample,semisyn- phore for the observed biological activity,many of thetic derivatives of erythromycin with modified agly- these largering systems are susceptible to degrada- cone structures,such as clarithromycin,[8] azithromy- tion by the acidicpHofthe stomach,esterases in the cin,[9] and telithromycin,[10] displayincreasedstability blood plasma, various hepatic enzymes,orthe macro- under acidic conditions (Figure 1B),[11] andthe recent-

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Figure 1. Representative structures of natural macrolides (A)and their semisynthetic derivatives (B).

2698 asc.wiley-vch.de 2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Adv.Synth. Catal. 2015, 357,2697 –2711 FULL PAPERS Chemoenzymatic Synthesis of Glycosylated Macrolactam Analogues ly approved anticancer agent ixabepilone (Figure 1B), Pik PKS generates both the 12- and14-membered asemisynthetic lactam analogue of epothilone B, was macrolactone rings of 10-dml andnarbonolide,and developed to increase metabolic stability against es- the glycosyltransferase DesVII, along with its auxili- terasecleavage.[12] In addition to modifying the mac- ary partner DesVIII, transfer TDP-d-desosamine rolide aglycone,modificationofthe sugar moieties onto the aforementioned aglyconestogiveYC-17 and can also significantly alter biological activity profiles, narbomycin, respectively.Following glycosylation, and several chemical and enzymatic synthetic ap- narbomycin is converted to pikromycin by the action proachesfor the diversification of macrolides have of PikC cytochrome P450 monooxygenase.This P450 been employed.[13] also converts narbomycin to neopikromycin and nova- Since the synthesis of azithromycin,[9] manyazalides pikromycin, and YC-17 to methymycin, neomethymy- have been reported containing nitrogen atoms at- cin, and novamethymycin.[19] Due to inherent flexibili- tachedtoC-9, C-10, C-11, or C-12 (lactone carbonyl ty,DesVII/DesVIII can transfer arange of structural- designated as C-1), and afew macrolactam analogues ly diverse TDP-sugars,demonstrating their potential of erythromycin Ahave been synthesized(Fig- for the structural diversification of macrolide antibiot- ure 1B).[14] However, macrolactam analogues that ics both in vitro and in vivo.[17] bear structurally diverse sugar moieties have not been Theengineered strains of S. venezuelae capable of explored, and we were motivatedtoaccess and test generating desosamine andarange of alternative the biological activity and selected pharmacological sugar moieties produced newanalogueswhich exhib- properties of this newclass of molecules. ited improved in vitro antibacterial activitiesand met- Thesynthesis of 12-membered macrolides bearing abolic stabilities.Tothe best of our knowledge,this is asingle sugar group is generally more facile than the the first synthesis of diverselyglycosylated macrolac- synthesisof14-membered macrolides that contain tam analogues for amacrolide antibiotic, andthe first two sugar moieties.Inaddition,12-membered mono- report of DesVII/DesVIII recognizing and processing glycosylated macrolides still displaysignifican biologi- alactam for glycosylation. By utilizing chemical syn- cal activities. Forexample,incontrast to erythromy- thesis to supply an unnatural functional group in com- cin, clarithromycin, and roxithromycin, methymycin bination with flexible biological machinery,novel possesses a12-membered ring macrolactone,10-deox- macrolide analogues with increased therapeutic po- ymethynolide (10-dml) which is appended to asingle tential were generated. amino sugar,desosamine.Despite these chemical differences,methymycin (Figure 1A) retainsantibiotic activity against Gram-positive bacteria similar to the Results and Discussion larger polyglycosylated macrolideantibiotics.[15] Thus, YC-17(Figure 1A) produced from Streptomyces vene- Synthesis of Aza-(10-deoxymethynolide)(1) zuelae ATCC 15439 was selectedfor its relative ease of synthesis,[16] and alactam analogue of this macro- Theretrosynthetic analysisofAZDM 1 is shown in lide possessing diverse sugar moieties was envisioned. Scheme S1 (Supporting Information). Analogous to However, pathwayengineering using apolyketide our previously reported synthesis of 10-dml, AZDM synthase (PKS) to replace the oxygeninthe lactone 1 could be synthesizedfrom key amine 2 and known moiety of the aglycone core with nitrogen is not avail- carboxylic acid 3[16] using EDC coupling and ring clos- able,and both the chemical synthesis of novelsugar ing metathesis(RCM) as the key steps.Our synthesis moieties andthe subsequent glycosylationwith the of AZDM 1 is summarized in Scheme 1. Thedesired corresponding aglycone are impractical. Therefore, amine 2 was preparedinastereoselective mannerin we have employed acombined approach involving four steps starting from propanal. Crotylationof chemical synthesis and biotransformation to efficient- propanal was successfully performed to provide alco- ly generate aseries of macrolactam glycosides.First, hol 4 according to the previously reported proce- amacrolactam analogue of the YC-17 aglycone 10- dure.[20] Mesylationofalcohol 4 followed by success- dml, aza-(10-dml) (AZDM; 1), was chemically syn- ful nucleophilicsubstitution with sodium azide and re- thesized, andanengineered strain of S. venezuelae ductionofthe resultant azide with LiAlH4 afforded with its substrate-flexible glycosyltransferase was em- amine 2.This key amine wassuccessfully coupled ployed to assemble the newcompounds.[7,17] with carboxylicacid 3[16] utilizing EDC to afford 6. The S. venezuelae YJ028 mutant strain[18] in which Theprimary TBS ether was selectively cleaved yield- the entire biosynthetic gene cluster encoding the pik- ing alcohol 7,which in turn was oxidized with DMP romycin (Pik) PKS anddesosamine biosyntheticen- to yield aldehyde 8.Grignard reactionofaldehyde 8 zymes were deleted was chosen as the biotransforma- with vinylmagnesium bromidefollowed by DMPoxi- tion host, and different deoxy sugar biosynthetic gene dation producedvinyl ketone 9.RCM with the cassettes and the genesencoding DesVII/DesVIII second generation Grubbs catalyst successfully pro- were expressed. In the wild-type S. venezuelae strain, vided protectedmacrolactam 10 (see Supporting In-

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Scheme 1. Synthesis of AZDM 1. formation for detailed synthetic methodsand Fig- vose biosynthetic genes with desVII/desVIII), and ure S1 to Figure S16 for 1Hand 13CNMR spectra of pLOLV2 (expressing TDP-l-olivose andTDP-l-digi- the synthetic intermediates). Thedesired AZDM toxose biosynthetic genes with desVII/desVIII)(Sup- 1 was finally obtained upon cleavage of the TBS porting Information,Table S1). Mutant strains were ether with TBAF.The structure of the synthesized cultured in 50 mL of SCM liquid medium at 308Cfor macrolactam 1 was fullyconfirmed by 1D (1Hand 48 hunder appropriate antibiotic selection. AZDM 13 1 C) and2D(COSY, HSQC,and HMBC) NMRanal- 1 (5 mgLÀ )was added into eachgrowing medium ysis aided by MS data (Supporting Information, Fig- and then incubated for an additional 48 h. Organic ex- ure S17 to Figure S22). tracts of the culture broths were preparedand subjected to HPLC-ESI-MSanalysis using apreviously described method.[21] TheHPLCchromatograms were Production and HPLC-ESI-MS/MS Analysis of monitored for common characteristicfragmentions AZDM Glycosides 11–18 arising from AZDM 1 at m/z=296,278,260 and, thus,peaks for the glycosylated AZDMswere identi- In order to glycosylate AZDM 1,four previously con- fied (Figure 2). structed expression plasmids[21] were independently Themutant YJ028/pLRHM2expressing l-rham- introduced to the mutant strain S. venezuelae YJ028 nose biosynthetic genesproduced l-rhamnosyl- to carry out boththe biosynthesis of the unnatural AZDM (11)(Figure 3A) and d-quinovosyl-AZDM TDP-deoxy sugars andthe subsequent glycosylation (12)(Figure 3A) at relatively lowlevels.Inthe of these sugars with the exogeneously supplied HPLC-ESI-MSanalysis,glycosides 11 and 12 were AZDM (1). These plasmids include pDDSS (express- eluted at 15.2 min and 16.5 min, respectively (Fig- ing TDP-d-desosamine biosynthetic genes with glyco- ure 2A), andbothofthese compoundsshowed similar syltransferase genes desVII/desVIII), pLRHM2(ex- mass spectra with their proton adduct ions appearing pressing TDP-l-rhamnose biosynthetic genes with at m/z =442. MS/MS analysisofthese compounds desVII/desVIII), pDQNV (expressingTDP-d-quino- producedthe characteristic fragmentation pattern

2700 asc.wiley-vch.de 2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Adv.Synth. Catal. 2015, 357,2697 –2711 FULL PAPERS Chemoenzymatic Synthesis of Glycosylated Macrolactam Analogues

and m/z=158,respectively,confirming that TDP-N- dedimethyl-N-acetyl-d-desosamine and d-desosamine were transferred to their respective AZDM glycosides.The structures of glycosides 13 and 16 were pre- dicted based on the fragmentation pattern according to the difference between the parent and daughter peakscorresponding to the loss of the neutral sugar (Supporting Information,Figure S25toFigure S28). TDP-3-keto-4,6-dideoxy-d-glucose may be converted to TDP-3-O-demethyl-d-chalcose by the probable action of the S. venezuelae pathwayindependent 3-ketoreductase (Sv 3-KR;Figure 3B).[23] Subsequent glycosylation reactionofAZDM 1 with sugars TDP-3- keto-4,6-dideoxy-d-glucose,TDP-N-dedimethyl-N- acetyl-d-desosamine,and TDP-3-O-demethyl-d-chalcose yielded glycosides 16, 14,and 13,respectively. Thedetection of atrace amount of glycoside 14 containing TDP-N-dedimethyl-N-acetyl-d-desosaminein Figure 2. HPLC-ESI-MS extracted ion chromatograms this S. venezuelae YJ028/pDDSS strainsupports the (m/z=296) of cultures of recombinant S. venezuelae strains. previously reported observation which suggests N- [24] Compounds 11 ([M +H]+ =442and 12 ([M +H]+ =442) de- acetylation as aself-protecting reaction. Mutant tected from YJ028/pLRHM2 (A). Compound 12 ([M+ strain YJ028/pLOLV2 showed three peaksarising H]+ =442) detected from YJ028/pDQNV(B). Compounds from AZDM glycosidesatretention times of 16.5, 12 ([M+ H]+ = 442), 13 ([M+ H]+ = 426), 14 ([M+ H]+ = 16.7, and 17.7 min(Figure 2D). Thesedisplayed + + 467), 15 ([M+ H] = 453), and 16 ([M +H] =424)detected proton adduct ions at m/z 442,426, and 426,respec- + = from YJ028/pDDSS (C). Compounds 12 ([M +H] =442), tively,and were identified as theirrespective macro- ([M H]+ 426), and ([M H]+ 426) detected from 17 + = 18 + = lactam glycosides:macrolactam glycoside 12, d-olivo- YJ028/pLOLV2 (D). All chromatograms are drawn to the same scale. syl-AZDM (17), and l-olivosyl-AZDM(18)(Fig- ure S29 andFigure S30). Detection of glycoside 17 possessing TDP-d-olivose suggested the action of Sv 4-KR on the intermediate sugar TDP-4-keto-2,6-di- with adifference between the parent anddaughter deoxy-d-glucose (Figure 3B). Further hydroxylation peak corresponding to the loss of the neutral sugar of the AZDM glycosidescatalyzed by the PikC P450 (Supporting Information, Figure S23 and Figure S24). of the S. venezuelae mutant strains was not detected. Macrolactam glycoside 12 may have arisen as aresult This is most likely due to the inability of the PikC of apreviously observed S. venezuelae pathwayinde- P450 hydroxylase to accept the macrolactam ring with pendent 4-ketoreductase (Sv 4-KR).[22] Sv 4-KR can altered sugars lacking an N,N-dimethylamino group, reduce TDP-4-keto-6-deoxy-d-glucose,abiosynthetic which is required for active site anchoring.[25] intermediate of TDP-l-rhamnose,toTDP-d-quinovose,which couldthen be appended to AZDM (1)by DesVII/DesVIII to afford 12 (Figure 3B). As expect- Structural Characterization of AZDM Glycosides 11– ed, macrolactam glycoside 12 wasalso producedby 13, 16, and 17 mutant YJ028/pDQNVharboring the d-quinovose biosynthetic genes (Figure 2B). HPLC-ESI-MS analy- Glycosides 11–18 were obtained from theirrespective sis of the organic extract of the mutant YJ028/pDDSS culturebroths.These broths were processed[21] and possessing d-desosamine biosynthetic genes showed subjected to repeated chromatographicseparation af- five peaks at retention times 16.5, 18.0, 19.7, 23.2,and fording pure compounds 11 (1.4 mg from 2.4 Lculture 23.7 min with proton adduct ions m/z=442,426, 467, of YJ028/pLRHM2), 12 (0.3mgfrom 1.8 Lcultureof 453, and424, respectively (Figure 2C), which were YJ028/pDQNV), 13 (1.2mgfrom 2.4 Lcultureof identifiedasmacrolactam glycoside 12,3-O-demeth- YJ028/pDDSS), 16 (1.8 mg from 2.4 Lculture of yl-d-chalcosyl-AZDM (13), N-dedimethyl-N-acetyl-d- YJ028/pDDSS), and 17 (0.8 mg from 3.0 Lcultureof desosaminyl-AZDM (14), d-desosaminyl-AZDM YJ028/pLOLV2). Macrolactam glycosides 14, 15,and (15), and 3-keto-4,6-dideoxy-d-glucosyl-AZDM(16), 18 were only detected in trace amountsinsufficient to respectively (Figure 3A). In addition to the common obtain NMRspectroscopic data. Thestructures of the fragment ions arising from AZDM,the proton adduct isolated glycosides 11–13, 16,and 17 were determined ions m/z= 467 and m/z =453 of macrolactam glyco- using 1D (1Hand 13C) and 2D (COSY,HSQC, sides 14 and 15 displayed afragmention at m/z=172 HMBC,and NOESY) NMR, MS/MS,and HR-ESI-

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Figure 3. Structures of macrolactam glycosides 11–18 (A)and proposed biosynthesis of the different deoxy sugars directed by the plasmids (B). Thefunctions of the proteinscarrying out each step are showninbrackets:Nucleotidyl transferase (NT), 4,6-dehydratase (4,6-DH), 2,3-dehydratase (2,3-DH), aminotransferase (AT), deaminase (DA), epimerase (EP), 4-ketoreductase (4-KR), N-methyltransferase(N-MT), 3-ketoreductase (3-KR), S. venezuelae pathway independent reductase (SvRed).

MS data. Thestereochemistry and conformation of NOE correlations (Supporting Information, Fig- the attached sugars in the isolated macrolactam glyco- ure S31 to Figure S61).[21] sides were confirmed on the basis of coupling con- Thesodium adduct ion observedatm/z= 464.2614 stant values(Supporting Information, TableS2) and (calcd.:464.2624) in the HR-ESI-MS data of AZDM

2702 asc.wiley-vch.de 2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Adv.Synth. Catal. 2015, 357,2697 –2711 FULL PAPERS Chemoenzymatic Synthesis of Glycosylated Macrolactam Analogues glycoside 11 suggested amolecular formula of HR-ESI-MS and 13CNMR data. TheHR-ESI-MS ex- 13 + C23H39NO7.The CNMR and HSQC spectra (Sup- hibited apeak at m/z= 464.2627 [M+ Na] (calcd.: porting Information, Figure S32 and Figure S34) indi- 464.2624) and the 13CNMR spectrum(Supporting In- cated the presence of 23 carbons in the macrolactam formation, Figure S38) showed the presenceofthe 23 glycoside:aketone at dC =207.6 (C-7), an ester at carbons requiredfor the AZDM glycoside.The NMR dC = 177.4 (C-1), twoolefiniccarbons at dC =149.7 (C- data (Supporting Information, Table S2) for 12 were 9) and dC =126.5 (C-8), an anomericcarbon at dC = quite similarwith that of 11 exceptfor the expected 105.1 (C-1’), five oxygenated methine carbons at dC = difference in the chemical shift values for the sugar 91.2 (C-3), 73.7 (C-4’), 72.5 (C-2’), 72.4 (C-3’), and and C-3position. Theanomeric proton appeared as 70.4 (C-5’), four methine carbons at dC = 51.9 (C-11), adoublet (J= 8.1 Hz) at dH =4.27 (H-1’)whereas the 46.4 (C-6), 46.1 (C-2), and 39.6 (C-10), three methyl- anomeric proton in 11 was broad singletatdH = 4.67 ene carbons at dC =35.3 (C-5), 34.3 (C-4), and 27.2 (H-1’); all other sugar protons exhibited an axial ori- (C-12), and six methyl carbons at dC =17.9 (C-15), entation as deduced from the large coupling constant 17.6 (C-6’), 17.7 (C-14), 17.7 (C-16), 11.5 (C-13), and values. Theaxial orientations were also supported by 10.0 (C-17). The 1HNMR spectrum (Supporting In- the NOE correlations (Supporting Information, Fig- formation, Figure S31) displayed the signalscharacter- ure S42) between H-1’,H-3’,and H-5 (Supporting In- istic of these macrolide glycosides such as twoolefinic formation, Figure S61) indicating that the sugar in 12 [21] protonsatdH = 6.72 (H-9)and 6.44 (H-8), an anome- was d-quinovose Thestructure of 12 was supported ric proton at dH =4.67 (H-1’), amethine proton at by well assigned COSY, HSQC,and HMBCdata dH =3.97 (H-11), five oxygenated methine protonsat (Supporting Information, Figures S39, S40, and S41). dH =3.92 (H-2’), 3.71 (H-5’), 3.59 (H-3’), 3.48 (H-3), AZDM glycoside 13 possessed amolecular formula + and 3.40 (H-4’), and six methyl group signals at dH = of C23H39NO6 assigned from the [M+ Na] ion ob- 1.24 (H-6’), 1.24 (H-14), 1.23 (H-16), 1.07 (H-17), 1.00 served at m/z= 448.2673 in the HR-ESI-MS. 1H- and (H-15), and0.93 (H-13). TheCOSY spectrum(Sup- 13CNMR data (Supporting Information,Table S2) porting Information, Figure S33) showedthe presence showedittobeanalogoustothose of macrolactam 12 of three spin systems including one for the deoxy with 23 carbon centers.Careful comparison of the sugar (Supporting Information, Figure S61). All of 1D- and 2D-NMR data of 13 with those of 12 indicat- these spinsystems were connected with the help of ed the presence of only four oxygenatedcarbons in HMBC correlations (Supporting Information, Fig- the sugar moiety (including the anomericcarbon) at ure S61). Theattachment of the sugar moiety to the dC = 105.4, 77.5, 72.5, and 68.8;and one additional C-3 position of AZDM was confirmed using the methylene group at dC =42.2 (dH =1.90 and1.27, H- HMBC spectrum(Supporting Information, Fig- 4’)suggesting that the appended sugar moiety in 13 ure S35). Correlationswere observedinthe HMBC was adideoxyhexose instead of the 6-deoxyhexose in spectrumbetween the anomeric proton at dH = 4.67 11 and 12.Furtheranalysis of the COSYand HMBC (H-1’)and the carbon at dC =91.2 (C-3) as wellasbe- data (Supporting Information, Figure S45 and Fig- tween the oxygenated methine proton at dH = 3.48 ure S47) of 13 revealedthat the additional methylene (H-3) and the anomeric carbon at dC =105.1 (C-1) group waspresentatthe C-4’ positionand that the confirming the point of attachment. Similarly,the po- sugar moiety was 3-O-demethyl-d-chalcose due to the sitions of all methyl groups were determined on the NOE correlations and the large coupling constant basis of theirHMBC correlation to their respective valuesfor all the sugar protons(Supporting Informa- neighboring carbon centers (Supporting Information, tion, Figure S48 and Figure S61). Figure S61). Themain difference between AZDM Themolecular formulaofmacrolactam glycoside [21a] glycoside 11 and its lactone counterpart was the 16 was deduced to be C23H37NO6 on the basis of the shift upfield for the C-11 (dC =51.9 for 11,74.6 for l- sodium adduct ion observed at m/z=446.2510 in the rhamnosyl-10-dml) and H-11 (dH =3.97 for 11,4.96 HR-ESI-MS.The NMRdata (Supporting Informa- for l-rhamnosyl-10-dml) peaks. Thesugar in 11 was tion, TableS2) of 16 were very similar to those of 13 confirmed as l-rhamnose on the basis of the small except for the presence of an additional ketone coupling constant valuesfor H-1 (br s), H-2 (br s), moiety giving risetothe signal at dC = 206.9. The indicating their equatorial orientation,and the large HMBC spectrum(Supporting Information, Fig- coupling constant valuesofH-3(d, J=9.5Hz), H-4 ure S53) showed correlations between protons H-2’ (t, J=9.5Hz), H-5 (dq, J=9.5, 7Hz), indicating their (dH =3.96) and H-4’ (dH =2.44) andthe carbonyl peak [21] axial orientation. This configuration was also sup- at dC =206.9 establishing it as C-3’ (Supporting Infor- ported by the coupling constantbetween C-1 andH- mation, Figure S61). Thelarge coupling constants of 1 1 ( JC,H =169 Hz) and other literature citations re- all the protons in the attachedsugar moiety,support- porting similar sugar moieties.[26] ed by the NOE correlations (Supporting Information,

Themolecular formula for macrolactam glycoside Figure S54) between proton signalsatdH = 4.39 (d, 12 wasdeducedtobeC23H39NO7 with the help of J=8.0 Hz,H-1’)and dH = 3.70 (m, H-5’)(Supporting

Adv.Synth. Catal. 2015, 357,2697 –2711 2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim asc.wiley-vch.de 2703 Pramod B. Shindeetal. FULL PAPERS

Information, Figure S61), suggested that the append- for each gene cassette is expected to be similar. ed sugar was 3-keto-4,6-dideoxy-glucose in the d-con- Therefore,the difference in the rate of production for formation. macrolactam glycosidesislikelydue to the varying TheHR-ESI-MS of AZDM glycoside 17 displayed catalytic efficiency of the glycosyltransferase with re- asodium adduct ion peak at m/z=448.2672 (calcd.: spect to each substrate ratherthan adifference in the

448.2675) indicating C23H39NO6 as its molecular for- mutant strain growthbehavior or difference in sugar mula. Analysisofthe 1H- and 13CNMR data (Sup- gene cassette expression levels. porting Information, Table S2) suggested 17 was also an AZDM glycoside having adideoxyhexose moiety. In the COSY spectrum(Supporting Information, Fig- Assessment of in vitro Antibacterial Activityand ure S57), the anomericproton signal at dH = 4.47 (d, Stability against Macrolide Esterase J=9.5, 1.5 Hz, H-1’)was coupled to the methylene protonsatdH =2.17 (ddd, J= 12.0, 5.5, 1.5Hz, H-2’a) Isolated compounds were evaluated for in vitro anti- and dH = 1.43 (m, H-2’b), which showed further cou- bacterial activities against erythromycin-susceptible pling to the oxygenated methine proton signal at dH = Enterococcus faecium ATCC 19434 and Staphylococ- 3.48 (ddd, J=12.0,9.0, 5.5 Hz, H-3’). This indicated cus aureus ATCC 25923 as wellasagainst erythromy- that the sugar was 2,4-dideoxyhexose (Supporting In- cin-resistant clinical isolates E. faecium P00558 and S. formation, Figure S61). Thelarge coupling constant aureus P00740 (Supporting Information, Table S4). valuesofall the sugar protonsidentified it as d-oli- Although the activitiesofmost of the AZDM glyco- vose and the NOE correlations (Supporting Informa- sides against the tested strains were similar to those tion, Figure S60) between H-1’ (dH =4.47), H-3’ (dH = of theirmacrolactone counterparts (Supporting Infor- [21a] 3.48), andH-5’ (dH =3.17) (Supporting Information, mation, TableS5), AZDM glycosides 11 and 12 ex- Figure S61) further supported this identification.[21] hibited four times higher activity against erythromy- Through the production of the macrolactam glyco- cin-susceptible pathogens when comparedwith eryth- sides and analysis of the DesVII/DesVIII-catalyzed romycin andwere active against erythromycin-resist- glycosylations in this study,afew novelobservations ant pathogenicstrains.AZDM glycosides 11 (MIC, were made.First, although previouswork demonstrat- 7.5–30 mmol) and 12 (7.5–30 mmol) containing 6-deox- ed that DesVII/DesVIII catalyzes the transfer of vari- yhexose l-rhamnose and d-quinovose,respectively, ous sugars to unnatural macrolactones and evenacy- displayed better activitiesagainst all strains tested clic aglycones,[17,27] these results demonstrate for the than those glycosides containing dideoxyhexose moi- first time that the macrolideglycosyltransferase eties (13, 16,and 17)(30–120 mmol) supporting the DesVII/DesVIII can recognize and process amacro- previousobservation that the additional hydroxy lactam aglycone providing aset of new glycosylated group of 11 and 12 may positively influence their anti- macrolactams.Second,eventhough l-digitoxosyl-nar- bacterial activities.[21] bonolide, d-boivinosyl-narbonolide,and d-boivinosyl- Because the resistancemechanisms of E. faecium 10-dmlwere previouslyisolated from amutant strain P00558 and S. aureus P00740 are not known,we carrying plasmid pLOLV2,[21] AZDM glycosides con- tested the susceptibility of erythromycin, l-rhamno- taining l-digitoxose and d-boivinose were not detect- syl-10-dml, and AZDM glycoside 11 to the well- ed in this presentstudy.Thirdly,tothe best of our known macrolideesterase EreB[29] isolated from the knowledge,this is the first report of the isolation of erythromycin-resistant Escherichia coli to investigate aglycoside containing TDP-3-keto-4,6-dideoxy-d-glu- the potential advantage of the macrolactam pharma- cose,confirming it as abiosynthetic intermediateof cophore for the treatment of macrolide-resistant TDP-d-desosamine.Additionally,inapreviousstudy, pathogens.The ereB gene (GenBank Accession No. a desV deletion S. venezuelae mutant was found to P05789) was synthesized, overexpressed, and purified produce 3-O-demethyl d-chalcosylmethymycin in- from E. coli BL21(DE3)pLysS.During HPLC-ESI- stead of the expected methymycin analogue possess- MS analysis (Figure 4A) of the reactionmixture con- ing TDP-3-keto-4,6-dideoxy-d-glucose.[28] These final taining erythromycinand EreB,itwas observedthat two observations suggest that the glycosylation effi- erythromycin (retention time 29.5 min) was initially ciency of DesVII/DesVIII towards different sugar converted partially to its linearform (retention time donors varies with the structure of the aglycone (Sup- 22 min).[6] Additionally,at1hincubation, erythromy- porting Information, TableS3). However, the intracel- cin was completely converted to the inactivation lular concentration of each sugar donor and the exact product (Figure 4D and E). TheMSofboth the sub- glycosylation efficiency towards different sugar strate and product were similar to the previouslyre- donors and acceptors cannot be measured in this in ported spectra.[6] When l-rhamnosyl-10-dml and vivo system. Nevertheless,because the same expres- AZDM glycoside 11 were separately incubated with sion plasmid and cloning strategywere used to ex- EreB and subjected to subsequent HPLC-ESI-MS press each sugar gene cassette,the level of expression analysis (Figure 4B and C), we observed that both

2704 asc.wiley-vch.de 2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Adv.Synth. Catal. 2015, 357,2697 –2711 FULL PAPERS Chemoenzymatic Synthesis of Glycosylated Macrolactam Analogues

Figure 4. Erythromycin esteraseassay.HPLC-ESI-MS total ion chromatograms of erythromycin (A), l-rhamnosyl-10-dml (B), and l-rhamnosyl-AZDM (11)(C)after incubationwith esterase. Each chromatogram in (A), (B), and (C)isdrawn to the same scale:linear erythromycin (^), erythromycin (*), l-rhamnosyl-10-dml (~), and l-rhamnosyl-AZDM (11)(!). Mass spectra of erythromycin (D), and linear erythromycin (E). compounds were not hydrolyzed by EreB even after drug almost completely disappearing after a2hincu- 24 hincubation. The m/z values corresponding to the bation, andgivingrise to its degradation product 5- hydrolyzed productsofl-rhamnosyl-10-dml (m/z= desosaminyl-erythronolide Alacking the cladinose 297 and 443)and AZDM glycoside 11 (m/z=296 and sugar (Figure 5A).[30] Theerythromycin peak eluted at 442) were not observed on the extracted ion chroma- 29.5 min and showedthe [M +H]+ ion at m/z=734 togram (EIC) (Supporting Information, Figure S62). and the peak due to desosaminyl-erythronolide A It is plausible that EreB may be an esterase specific eluted at 33 min anddisplayed [M+ Na]+ and[M+ to 14-membered macrolides andmay not be able to H]+ ions at m/z= 562 and 540,respectively(Figure 5D recognize 12-membered macrolides (l-rhamnosyl-10- and E). Thedifference between the proton adduct dml and AZDM glycoside 11)assubstrates. Never- ions of erythromycin and 5-desosaminyl-erythronolide theless, these results demonstrate that macrolactam Awas accounted for by the loss of the mass due to glycosides such as AZDM glycoside 11 are not sus- cladinose as expected. Macrolactam 11 and l-rhamno- ceptibletothe microbialesterases that can degrade syl-10-dml, however, were intact after2hincubation erythromycin, and, although the resistance mecha- in SGF (Figure 5B andC), and aglycone ion peaksre- nisms of E. faecium P00558 and S. aureus P00740 are sulted from the loss of the rhamnose moietyfrom the not known,these macrolactam glycosides are still l-rhamnosyl-10-dml (m/z=297 for the 10-dml [M + active against erythromycin-resistant pathogens as dis- H]+)and AZDM glycoside 11 (m/z =296 for the playedbytheir antibacterial activity (Supporting In- AZDM [M+ H]+)werenot found on the EIC (Sup- formation, Table S4). porting Information, Figure S63). Cladinose is known to be cleaved directlyfrom erythromycin under acidic conditions giving rise to its degradationproducts[30] Assessment of in vitro Stability in Simulated Gastric and the 6-hydroxy group also plays an importantrole Fluid and LiverMicrosomes in the acid degradation of erythromycinbyforming aspiroketal ring.Hence,clarithromycin, the 6-me- Since AZDM glycoside 11 and its macrolactone deriv- thoxy derivativeoferythromycin, wasfound to be ative (l-rhamnosyl-10-dml)[21a] displayed stronger an- stable in acidic conditions.[31] In contrast, the glycosi- tibacterial activity than erythromycin, they were sub- dic linkagesofAZDM glycoside 11 and l-rhamnosyl- jected to simulated gastric fluid (SGF) and liver mi- 10-dmlseem to be more stable in SGF,and should crosomal metabolic stability assayswith the aim of allow bothAZDM glycoside 11 and l-rhamnosyl-10- findingthe differences in gastric and metabolic stabili- dml to survive the acidicenvironment of the stomach. ties among macrolactam glycosides,macrolactone gly- Since a b-glycosidic bond is comparatively more cosides, anderythromycin. When erythromycinwas stable than an a-glycosidic bond,[32] the better stability incubatedwith SGF,HPLC-ESI-MSanalysisofthe of 11 and l-rhamnosyl-10-dml over erythromycin reactionmixture indicated partial degradationofer- might have resulted from the combination of the pres- ythromycin immediatelyafter incubation, with the ence of a b-glycosidic linkage andthe absence of cor-

Adv.Synth. Catal. 2015, 357,2697 –2711 2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim asc.wiley-vch.de 2705 Pramod B. Shindeetal. FULL PAPERS

Figure 5. Simulated gastric fluid (SGF)assay.HPLC-ESI-MS total ion chromatograms of erythromycin (A), l-rhamnosyl-10- dml (B), and l-rhamnosyl-AZDM (11)(C)after incubation with SGF.Each chromatogramin(A), (B), and (C)isdrawn to the same scale:erythromycin (*), 5-desosaminyl-erythronolide A(^), l-rhamnosyl-10-dml (~), and l-rhamnosyl-AZDM (11)(!). Mass spectra of erythromycin (D), and 5-desosaminyl-erythronolide A(E). responding 6-hydroxy group.Erythromycinhas erythromycin and hydroxylated l-rhamnosyl-10-dml, ashort elimination half-life as it undergoes rapid me- respectively (Figure 6A, B, D, andE)after incubation tabolism,primarily in the liver,tothe inactive N-des- for 2h.HPLC-ESI-MSanalysis of the reactionmix- methyl-erythromycin, which is immediately eliminat- ture containing erythromycinexhibited two major ed throughbile.[33] Other macrolide antibiotics are peaks(Figure 6A): the peak eluted at 29.5 min was also metabolized to descladinosyl, N-desmethyl, and identifiedaserythromycin from its [M+ H]+ ion at hydroxylderivatives by various hepatic hydrolytic and m/z= 734, and the other peak, which eluted at 27 min, oxidative enzymes.[34] To ascertain the metabolic pat- displayed an [M+ Na]+ ion at m/z=742 and an [M + tern, erythromycin, l-rhamnosyl-10-dml, and AZDM H]+ ion at m/z =720 (Figure 6D). This newmetabolite glycoside 11 were incubated with rat liver microsomes was identifiedasN-desmethyl-erythromycin on the and NADPH. Consequently,erythromycinand l- basis of the 14 Da observed difference to that of er- rhamnosyl-10-dml were metabolized to N-desmethyl- ythromycin. This is further supported by the presence

Figure 6. Liver microsomal assay.HPLC-ESI-MS total ion chromatograms of erythromycin (A), l-rhamnosyl-10-dml (B), and l-rhamnosyl-AZDM (11)(C)after incubation with liver microsomes.Each chromatogram in (A), (B), and (C)isdrawn to the same scale: N-desmethyl-erythromycin (^), erythromycin (*), hydroxylated l-rhamnosyl-10-dml ( o ), l-rhamnosyl-

10-dml (~), and l-rhamnosyl-AZDM (11)(!). Mass spectra of N-desmethyl-erythromycin (D)and hydroxylated l-rhamnosyl-10-dml (E).

2706 asc.wiley-vch.de 2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Adv.Synth. Catal. 2015, 357,2697 –2711 FULL PAPERS Chemoenzymatic Synthesis of Glycosylated Macrolactam Analogues of the [N-desmethyl-desosamine]+ ion peak at m/z= macrolactam glycosides. To the best of our knowl- 144 since the [desosamine]+ ion peak appears at m/z edge,this is the first example of aset of diversely gly- = 158 –a14Dadifference.Similarly,HPLC-ESI-MS cosylated macrolactam analogues of amacrolideanti- analysis of the assay with l-rhamnsoyl-10-dml also biotic, and to the best of our knowledge,this is also showedtwo peaks afterincubationwith rat liver mi- the first example of DesVII/DesVIII desosaminylgly- crosomes (Figure 6B). Thepeakeluted at 22.7 minex- cosyltransferase accepting and glycosylating amacro- + + hibited the characteristic ions [M +Na] ,[M+ NH4] , lactam to generate an array of new macrolactam gly- + and [M +H rhamnose H2O] at m/z =465, 460,and cosides. Some of the novelAZDM glycosidesdisplay 279, respectivelyÀ ,confirÀming l-rhamnosyl-10-dml as improved antibacterial activitiescompared to erythro- the structure (Supporting Information, Figure S64). In mycin against botherythromycin-susceptible ander- contrast, the peak eluted at 15 min showed[M+Na]+, ythromycin-resistant pathogens.Moreover, these new + [M+H rhamnose H2O] and [M + H rhamnose 2 antibiotics are resistanttodegradationbythe bacteri- + À À À À H2O] ions at m/z=481,295,and 277 (Figure 6E). al macrolide esterases that degrade erythromycin. Thedifference of 16 Da between the corresponding TheAZDM glycosides describedhere also show im- ions with those of l-rhamnsoyl-10-dml indicated that proved in vitro stability in SGF relativetoerythromy- the metabolite at 15 min is its hydroxylated analogue. cin andshow greater stability than botherythromycin Careful comparison of bothspectra from l-rhamno- and the corresponding macrolactone glycosides in rat syl-10-dml and its hydroxylated analogue revealedthe liver microsome assays.These results demonstrate the + shift in fragment ions [M +H rhamnose H2O] and successful applicationcombiningchemical synthesis + À À [M+H rhamnose 2H2O] from m/z= 279 to m/z= with inherently flexible biosynthetic enzymes to gen- 295 andÀm/z=261Àto m/z= 277, respectively,suggest- erate arange of novel glycosylated macrolactam ana- ing that hydroxylation occurred on the aglycone core logues of YC-17. Aseries of assaysdemonstrated that and not on the rhamnose sugar. Since it has been re- some of these new molecules have enhancedpharma- ported that clarithromycinismetabolized to 16-hy- cological properties and support this approach as droxy-clarithromycin,[35] the obtained hydroxylated apowerful strategyfor structural diversification to metabolite of l-rhamnosyl-10-dml is likelyfunctional- create novel macrolactam glycosideswith potent anti- ized at the C-12 position. However, AZDM glycoside biotic activity for drug discovery and development 11 was found to be stablefor the durationofthe 2h programs. incubationwith rat liver microsomes (Figure 6C). The stability of AZDM glycoside 11 was also supported by the absenceofdesmethylated (m/z=428 and281) Experimental Section or hydroxylated (m/z=480,294, and 276)product ion peaksonthe EIC (Supporting Information, Fig- General Procedures ure S65). This result suggests that AZDM glycosides, NMR spectra were recorded on Bruker Avance 900 MHz, such as compound 11,would ideally survive first-pass Varian INOVA 500 MHz,and BrukerAvance III 400 MHz metabolism and remain in the body longer than eryth- spectrometers.Chemical shifts are given in ppm using tetra- romycin and related macrolactone glycosides. Thus, methylsilane (TMS) as an internal reference.CD3OD and the AZDM glycosides mayexert aprolonged antibi- CDCl3 were used as NMR solvent and the chemical shift otic actionsimilar to azithromycin.[34] Overallitcan valueswere corrected to 3.31/49.1 ppm and 7.27/77.0 ppm be concluded that AZDM glycosidesare more stable for 1H/13Cnuclei, respectively. Mnova software from Mestre- in gastric acid than erythromycin, are more metabol- lab Research S.L. wasemployed for NMR data processing. ically stable than both erythromycinand macrolac- HPLC-ESI-MS/MS using an analytical reversed-phase tone glycosides,and cannot be hydrolyzed by erythro- Nova-Pak C18 column (Waters,Milford, MA, USA;150 3.9 mm, 4.0 mm) were recorded on aWaters/Micromass mycin esterase.These results with the low cytotoxicity Quattromicro MS interface consisting of aWaters 2695 sep- of compounds 11 and 12 against human cell line HEK arationmodule connected directly to aMicromass Quattro 293 (IC50 >50 mmol,bothcompounds, Supporting In- micro MS.The HR-ESI-MSdata were obtained using formation) suggest the potential usability of these aWaters SYNAPT G2-S massspectrometercoupled with compounds. UPLC.HPLC purification was done using preparative Spherisorb S5 ODS2 (Waters, 25020mm, 5 mm) and semipreparative Watchers 120 ODS-BP (25010mm, 5 mm) col- Conclusions umns on an Acme 9000 HPLC system (YL Instrument Co. Ltd.,Korea) consisting of an SP930D gradientpump cou- Thedevelopment of amacrolactam aglycone based pled with aUV730D UV detector set to 220 nm. on YC-17 by synthetic chemistry combinedwith the cell-basedgeneration of unnatural deoxyhexose sugars and aglycone biotransformation has enabled the production of adiverse set of biologically active

Adv.Synth. Catal. 2015, 357,2697 –2711 2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim asc.wiley-vch.de 2707 Pramod B. Shindeetal. FULL PAPERS

Synthesis of Aza-(10-deoxymethynolide)(1) same additive concentrations (solution B) at 20 to 70% solution Bfor 25 min, to 90% solutionBfor 15 min, maintained Thechemical synthesis of the synthetic intermediates as at 90% solutionBfor 9min, and then to 20% solution Bfor well as compound 1 are described in the SupportingInfor- another 11 min for column re-equilibration. mation.

Bacterial Strains, Culture Conditions, and Genetic Isolation and Identification of AZDM Glycosides Manipulation Thewhole culture broth (2.4 L) of strain S. venezuelae S. venezuelae mutant strainswere propagated on SPAagar YJ028/pLRHM2 was centrifuged, and the supernatant was (1 gyeast extract, 1gbeef extract, 2gtryptose,10gglucose, subjected to solvent-solvent partitionwith ethyl acetate.The trace amountofferroussulfate,and 15 gagar per L).[36] obtainedextract was evaporated, and the resultant brown Standard protocols were followedfor protoplast preparation residuewas fractionated by reversed-phase semipreparative and transformation of S. venezuelae.[36] To select the trans- HPLC employinggradient elution (40% aqueousmethanol formantsofS. venezuelae,R2YE agar plates[36] supplement- to 60 min, to 100% aqueousmethanol for 61–72 min, to ed with thiostreptonwere employed. S. venezuelae ATCC 40% aqueous methanol for 72–82 min) with aflow rate of [37] [38] 1 15439 and Streptomyces antibioticus ATCC 11891 were 2mLminÀ .The fraction containing AZDM glycoside 11 used for obtaining genomic DNA. Routine sub-cloning was was purified by reversed-phase semipreparative HPLC em- done using plasmid Litmus28 (New England Biolabs) and ploying 18% aqueous acetonitrile as mobile phase with 1 Escherichia coli DH5a (Invitrogen). Forexpressing the aflow rate of 2mLminÀ to afford the pure compound as genes involved in the biosynthesis of deoxy sugars and their an amorphous white solid (yield:1.4 mg). transfer, ahigh-copy-number E. coli-Streptomyces shuttle Theresidue obtained from the whole culture broth (1.8 L) vector, pSE34,[39] containingthe constitutive ermE* promot- of strain S. venezuelae YJ028/pDQNVwas fractionatedby er[40] and athiostrepton resistance marker,[41] was used. reversed-phase semipreparative HPLCemploying gradient elution (44% aqueous methanolto37min, to 100% aqueous methanol for 38–48 min, to 44% aqueous methanol for 49– Construction of Plasmids and Mutants 1 59 min) with aflow rate of 2mLminÀ .The fractioncontain- Construction of pDDSS, pDQNV,pLRAM2, and pLOLV2 ing AZDM glycoside 12 was further purified by reversed- was previously described.[21] These plasmids were separately phase semipreparative HPLC using 20% aqueous acetoni- [18] 1 introduced into S. venezuelae YJ028 and the resulting re- trile as mobile phase with aflow rate of 2mLminÀ to yield combinants were used for in vivo glycosylationofthe syn- the pure compound (0.3 mg). Thewhole culture broth thesized AZDM 1.Details regardingthe construction of (2.4 L) of strain S. venezuelae YJ028/pDDSS was processed plasmids are described in the Supporting Information(see similarly to obtain abrown extract. This brown residue was also Table S1 for combinedgene organizations). fractionated by reversed-phase semipreparative HPLC employing gradientelution (50% aqueousmethanol to 25 min, to 60% aqueous methanolfor 35–45 min, to 50% aqueous Production and HPLC-ESI-MS/MS Analysis of 1 AZDM Glycosides methanol for 46–55 min) with aflow rate of 2mLminÀ .The fractioncontainingAZDM glycoside 16 was further purified Therecombinants YJ028/pDDSS,YJ028/pDQNV,YJ028/ by reversed-phase semipreparative HPLC employing28% pLRAM2, and YJ028/pLOLV2 were cultured in SGGP aqueousacetonitrile as mobile phase with aflow rateof 1 liquid medium (Tryptone 4g,yeast extract 4g,MgSO4 0.5 g, 2mLminÀ to yield the pure compound as an amorphous glucose 10 g, glycine 2g,and potassium phosphate buffer white solid (yield:1.8 mg). 0.01 mol per 1L)[36] containing the antibiotic thiostreptonin Similarly AZDM glycoside 13 was obtained by purifying 1 aconcentration of 25 mgmLÀ at 308Cfor 24 h. This seed the fractioncontainingthe target compound on reversed- culture(5mL) was used to inoculate 50 mL of SCM phase semipreparative HPLC employing 28%aqueousace- 1 medium (1.5% soluble starch, 2.0% soytone,0.01% CaCl2, tonitrile as mobile phase with aflow rate of 2mLminÀ to 0.15% yeast extract, and 1.05% MOPS,pH7.2)[26] in 250- furnish the pure compound (yield:1.2 mg). mL baffled Erlenmeyer flasks to which antibiotic thiostrep- Theresidue obtainedfrom the whole culture broth (3.0 L) 1 ton (25 mgmLÀ )was added. This was initially incubated at of strain YJ028/pLOLV2 was grossly separated by reversed- 308Cfor 48 hfollowed by feeding of the synthesized phase open column chromatography using gradient elution 1 AZDM 1 in aconcentration of 5 mgmLÀ and then further startingfrom 50% aqueousmethanol to 100% aqueous incubated for 48 h. Each grown culturewas centrifuged methanol. Obtained fractions were analyzed by HPLC-ESI- (5,000 g for 10 min) and the supernatant was passed MS/MS for the presence of the macrolactam glycosides and through an OASIS HLB (Waters,Milford, MA) SPE the fractioncontaining AZDMglycoside 17 was further sep- column previously conditionedwith 5mLmethanol and arated by reversed-phase semipreparative HPLC employing 5mLwater. Thecolumn was washed with 5mL20% (vol/ 23% aqueousacetonitrile as mobile phase with aflow rate 1 vol) methanol and then air-dried for ca. 30 s. Thecolumn of 2mLminÀ to obtain the pure compound (yield:0.8 mg). was eluted three times with 1mL0.5% (vol/vol) methanolic l-Rhamnosyl-AZDM (11): amorphous white powder; 1H- acetic acid and the organic extracts were concentrated and 13CNMR, see Table S2 (SupportingInformation); + + under vacuum.HPLC-ESI-MS analysis was performed with (+)-ESI-MS: m/z =459 [M+ NH4] ,442 [M+H] ,406 [M+ 1 + + aflow rate of 250 mLminÀ using agradient of 5mmolam- H 2H2O] ,296 [M+H sugar] ,278 [M+H sugar À + À + À À moniumacetate–0.05% (vol/vol) acetic acid in water (solu- H2O] ,260 [M+H sugar 2H2O] ;HR-ESI-MS: m/z= + À À tion A) and 80% (vol/vol) aqueous acetonitrile with the 464.2614 [M+ Na] (calcd. for C23H39NO7Na:464.2624).

2708 asc.wiley-vch.de 2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Adv.Synth. Catal. 2015, 357,2697 –2711 FULL PAPERS Chemoenzymatic Synthesis of Glycosylated Macrolactam Analogues

d-Quinovosyl-AZDM (12): amorphous white powder; 1H- resuspended in lysis buffer (50 mmol sodiumphosphate and 13CNMR, see Table S2 (SupportingInformation); buffer, pH 8.0, containing300 mmol NaCl and10mmol imi- + + (+)-ESI-MS: m/z =459 [M+ NH4] ,442 [M+H] ,296 [M+ dazole), and then lysed by sonication. Clarified cell lysate + + H sugar] ,278 [M+H sugar H2O] ,260 [M+ H su- was passed through aNi-NTAsuperflow column(Qiagen). garÀ 2H O]+;HR-ESI-MS:À m/z =À464.2627 [M +Na]+ (calcdÀ . After washingthe column with washing buffer (50 mmol À 2 for C23H39NO7Na:464.2624). sodium phosphate buffer, pH 8.0, containing 300 mmolNaCl 3-O-Demethyl-d-chalcosyl-AZDM (13): amorphous white and 20 mmol imidazole), N-His6-tagged recombinant pro- powder; 1H- and 13CNMR, see Table S2 (Supporting Infor- teins were eluted with elution buffer (50 mmol sodium phos- mation); (+)-ESI-MS: m/z 448 [M+Na]+,443.0 [M+ phate buffer, pH 8.0, containing300 mmol NaCl and + + + NH4] ,426 [M+H] ,408 [M+ H H2O] ,296 [M+ 500 mmol imidazole). Thepurified protein was subjected to + À+ H sugar] ,278 [M+H sugar H2O] ,260 [M+ H su- 13% SDS-PAGEand visualized with Coomassie blue stain. garÀ 2H O]+; (+)-HR-ESI-MÀ S: Àm/z =448.2673 [M+ Na]À + Theresultant protein was dialyzed against 50 mmol HEPES À 2 (calcd.for C23H39NO6Na:448.2675). (pH 7.5) containing 150 mmol NaCl and 2% (v/v) glycerol, N-Dedimethyl-N-acetyl-d-desosaminyl-AZDM (14): and then stored at 808Cbefore use in the in vitro reaction. (+)-ESI-MS: m/z =489 [M+ Na]+,467 [M+H]+,296 [M+ 8 mgoferythromycinÀ , l-rhamnosyl-10-dml, and AZDM gly- + + + H sugar] ,278 [M+H sugar H2O] ,172 [sugar] . coside 11 were transferred to separate 2-mL glass vials. Àd-Desosaminyl-AZDÀM (15):À(+)-ESI-MS: m/z =475 [M + 100 mLphosphate buffer were added into each glass vial, Na]+,453 [M+H]+,296 [M+H sugar]+,278 [M+ H su- and the reaction was started by adding 2 mmol of EreB. + + À À gar H2O] ,158 [sugar] . These reactionmixtures were incubated at 378Cinashaking 3-KÀ eto-4,6-dideoxy-d-glucosyl-AZDM (16): amorphous water bath (150 rpm) for 0, 1, 2, and 24 h. Thereactions white powder; 1H- and 13CNMR, see Table S2 (Supporting were stopped by adding 150 mLcold acetonitrile.The reac- Information);(+)-ESI-MS: m/z =446 [M +Na]+,441 [M+ tion mixtures were centrifuged (3,000 g for 10 min) at 48C, + + + NH4] ,424 [M+H] ,296 [M+H sugar] ,278 [M+ H su- the supernatant was separated,and then HPLC-ESI-MS/MS + À +; À gar H2O] ,260 [M+ H sugar 2H2O] (+)-HR-ESI-MS: analysis was performed. This experimentwas carried out in À À+ À m/z= 446.2510 [M+Na] (calcd. for C23H37NO6Na: triplicateform. 446.2519). d-Olivosyl-AZDM (17): amorphous white powder; 1H- and 13CNMR, see Table S2 (SupportingInformation); Stability Study in SimulatedGastricFluid(SGF) + + (+)-ESI-MS: m/z=448 [M+Na] ,443 [M+NH4] ,426 + + + AZDMglycoside 11 and l-rhamnosyl-10-dml were selected [M+H] ,296 [M +H sugar] ,278 [M+ H sugar H2O] , for determiningthe stability in SGF.Erythromycin was also À + À À 260 [M H sugar 2H2O] ;HR-ESI-MS: m/z= 448.2672 subjected to stability assay as acontrol. TheSGF was pre- À+ À À [M+Na] (calcd. for C23H39NO6Na:448.2675). pared followingthe specifications in the United States Phar- l-Olivosyl-AZDM (18): amorphous white powder; macopeia (NaCl 2g,pepsin 3.2 g, and HCl 7mL, per 1L). (+)-ESI-MS: m/z =448 [M+ Na]+,426 [M+H]+,296 [M+ + + Three stock solutionswere prepared by dissolving 0.25 mg H sugar] ,278 [M+H sugar H2O] ,260 [M+ H su- of each compound in 1mLofmethanol. 200 mLaliquots of garÀ 2H O]+. À À À À 2 each compound were transferred to 2-mL vials and after evaporating methanol, 1mLSGF was added into each vial. Antibacterial Activity Assay Aliquots (100 mL) from these solutions were incubated in six glass vials of 2-mL capacity at 37 Cinashaking water bath Theerythromycin-resistant strains Enterococcus faecium 8 (120 rpm) for 0, 30, 60, and 120 min.[42] Thewhole experi- P00558and Staphylococcus aureus P00740 were clinically ment was performed in triplicateand reactionmixtures isolatedaspreviously described.[21] Theerythromycin-sus- were analyzed by HPLC-ESI-MS/MS using above men- ceptible strains E. faecium ATCC 19434 and S. aureus tionedconditions to see degradation profiles. ATCC 25923 were obtained from the American Type Cul- ture Collection (ATCC) (Manassas,VA, USA). Thedetails of the antibacterial assay,performed in triplicate, have been Metabolic Stability Study with Rat LiverMicrosomes described previously.[21] 1 Similarly,stock solutions of 2 mgmLÀ were prepared by sep- Stability Study with Erythromycin Esterase arately dissolving AZDM glycoside 11, l-rhamnosyl-10-dml, and erythromycininDMSO. 20 mLDMSO solutions of each DNAencoding ereB from E. coli was synthesized by Bion- compound were transferred to 2-mL glass vials and 655 mL eer (Republic of Korea). This DNAfragment was digested phosphate buffer wasadded followed by the additionof with NdeIand BamHI and then clonedinto pET15b (Nova- 500 mgrat liver microsomal proteins(Sigma–Aldrich, USA). gen, Germany) containing an N-terminal His6-tag. Theex- 100 mLaliquotsofeach reaction mixturewere transferred pression plasmidwas introduced into E. coli BL21(DE3)- into six glass vials of 2-mL capacity and were preincubated pLysS (Novagen)for overexpression under the T7 promoter. at 378Cinashaking water bath (150 rpm) for 5min. 500 mg Cells were grown in LB medium supplemented with of NADPH were added into each vial and the reaction mix- 1 1 50 mgmLÀ ampicillin and 25 mgmLÀ chloramphenicol. Each tures were further incubated for 0, 30, 60, and 120 min. The liter of culture was inoculatedwith 10 mL of overnight start- reactionswere terminated by adding150 mLcold acetoni- er culture. Theculturewas grown at 378Ctoan optical den- trile.[43] Thereaction mixtures were centrifuged (3,000 g for sity (OD600)of0.6,and then expression was induced for 3h 10 min) at 48Cfor 10 min and the supernatant wasanalyzed with 0.1 mmolisopropyl-b-d-thiogalactopyranoside (IPTG). by HPLC-ESI-MS/MS. This experiment was carried out in Cells were harvested by centrifugation(6,000 g for 10 min), triplicate.

Adv.Synth. Catal. 2015, 357,2697 –2711 2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim asc.wiley-vch.de 2709 Pramod B. Shindeetal. FULL PAPERS

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