A Natural Lipotrisaccharide and Its Derivatives Selectively Lyse

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A Natural Lipotrisaccharide and Its Derivatives Selectively Lyse Streptococcus pneumoniae via Interaction with Cell Membrane Bo Liu, Xue Liu, Jingren Zhang, and Gang Liu ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00008 • Publication Date (Web): 06 Mar 2017 Downloaded from http://pubs.acs.org on March 7, 2017

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Table of Contents Graphics:

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A Natural Lipotrisaccharide and Its Derivatives Selectively Lyse Streptococcus pneumoniae via Interaction with Cell Membrane

Bo Liu,†,,§ Xue Liu,‡,§ Jing-Ren Zhang,‡,* Gang Liu†,*

†Center

for Life Sciences & Department of Pharmacology and Pharmaceutical Sciences, Beijing,

100084, People’s Republic of China;

‡ Center

for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing,

100084, People’s Republic of China;

Beijing

§These

Institute of Petrochemical Technology, Beijing, 102607, People’s Republic of China.

authors equally contribute to this study.

*Corresponding authors. E-mail: (J.R.Z.) [email protected]. Tel.: +86 10 62795892. Email: (G.L.) [email protected]. Tel.: +86 10 62797740.

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Abstract: A natural lipotrisaccharide (NP000778, 1a), a new triglycosidic tri-O-substituted glycolipid isolated from the Morinda citrifolia plant and its chemical derivatives were identified to be active against major Gram-positive pathogens, particularly Streptococcus pneumoniae. Additional evidence indicated that 1a and its synthetic derivatives exerted their bactericidal activities against Streptococcus pneumoniae by selectively targeting the bacterial membrane, leading to the rapid lysis of the pneumococci. Efficient synthesis of 1a and its derivatives was performed using an application of the intramolecular aglycon delivery (IAD) reaction to establish its structure-activity relationships (SARs). SAR analysis indicated that trisaccharide glycolipid compounds showed good selectivity and high potency against Streptococcus pneumoniae. These compounds contain a linear chain with a chain length from C3 to C9 at 2’-position and 4’position, as well as a 2-methyl butyryl group at 3’-position, without an aza substitution in the lipid chain. This is the first lipotrisaccharide identified with potent bactericidal activity via interaction with cell membrane. The results reported herein offer a valuable guideline for the design of glycolipid derivatives that selectively target pathogenic bacteria.

Keywords: natural lipotrisaccharide, Morinda citrifolia plant, Streptococcus pneumoniae, cell membrane, drug-resistant pathogen

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Introduction Antimicrobial drugs have been the mainstay of bacterial infection treatment since the advent of penicillin.1-4 However, the wide use of antimicrobials has led to the emergence of drug-resistant bacterial pathogens, which has gradually eroded the overwhelming power of these medications in the last decades.5 Among the most extensively drug-resistant bacterial pathogens are Acinetobacter baumannii, Enterobacter species, Enterococcus faecium, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae.6 These pathogens not only cause devastating disease, but also represent the socalled “superbugs” that are extensively resistant to currently available therapeutics.7 The pace of antimicrobial discovery has lagged far behind the clinical need to combat bacterial diseases caused by drug-resistant pathogens. Only a few new antimicrobial drugs have been approved for clinical use in the last 40 years.8 Thus, novel antimicrobials are urgently needed worldwide. Lipooligosaccharides, a type of glycolipid found in some bacteria, are truncated structures (no Oantigen) of bacterial lipopolysaccharide (LPS).9 Recent studies have suggested that both natural and synthetic lipooligosaccharides are potential drug candidates, having demonstrated antiviral activity by inhibiting virus-cell binding and fusion10, and may also be associated with the induction of virulence in some bacteria.11-13 In addition, a few glycolipids and their mimics have been found to possess potent antimicrobial activity, 14-16 an observation that motivated us to search for an active glycolipid that can be utilized for resistant bacteria. Herein, we report that a natural lipotrisaccharide (NP000778, 1a, Figure 1), originally from the Morinda citrifolia plant,17

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and its chemical derivatives possess selective antibacterial activities against major Gram-positive pathogens, particularly Streptococcus pneumoniae. Subsequent investigations have indicated that this type of compound inhibits S. pneumoniae growth by rapid cell lysis via interaction with cell membrane. Structurally, 1a, a trisaccharide template compound, is composed of either a trehalose (2a-2g), a disaccharide [6–O-(6-O-(-D-mannopyranosyl)-β-D-glucopyranose] scaffold (3), or a monosaccharide template (4) pharmacophore structure (Figure 1). All of these scaffoldanchoring variant aliphatic chains were synthesized to further outline the structure activity relationships (SARs).

Figure 1. Chemical structures of lipotrisaccharide 1a and its synthetic derivatives

Results and Discussion The antibacterial activities of 1a were screened against bacteria that represent major drug resistant pathogens: Escherichia coli (E. coli), Enterococcus faecalis (E. faecalis), Staphylococcus aureus (S. aureus), and Streptococcus pneumoniae (S. pneumoniae). While this compound exhibited no antibacterial activity against E. coli (data not shown), it showed selective

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inhibition againstS. pneumoniae growth under the concentrations tested thus far (Table 2). To explore the antibacterial mechanism and summarize their SARs, the natural product 1a and its derivatives were selected as templates for retrosynthetic analysis (Figure 2).

Figure 2. Retorsynthesis for the natural lipotrisaccharide (1a) and its derivatives (2, 3, 4).

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Chemistry Derivatives 2a-2j (Figure 1) were directly synthesized from the key intermediate 8, which was derived from the chemical linkage of building block 518, 19 and 620 (Table 1 and Scheme 1). The final product 1a was obtained by β-mannnosylation with acceptor 8 with donor 721, 22 (Scheme 2). The intermediates 5 and 6 for synthesis of 8 and 7 were prepared from commercially available β-D-glucosepentaacetate, 1,2:5,6-Di-O-isopropylidene-α-D-glucose, and mannose via 5, 6 and 6 steps, respectively, in yields from 36%-48% (see supporting information).

Table 1. Tested conditions for formation of intramolecular 1,1-,-glycosidic bonda Entry

Donor/TfOMe/DTBMPb

DDQ

Solvent

Temp

Yield c(%)

1

1.0/1.0/1.1

Commercial

DCE

45°C

16

2

2.0/2.0/2.2

Commercial

DCE

45°C

25

3

2.0/2.0/2.2

Commercial

DCM

reflux

trace

4

2.0/2.0/2.2

Fresh Crystal

DCE

45°C

54

5

2.0/2.0/2.2

Fresh Crystal

DCE

W, 10 min

42

6

2.0/2.0/2.2

Fresh Crystal

DCE

W, 15 min

52

7

2.0/2.0/2.2

Fresh Crystal

DCE

W, 20 min

44

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aReaction

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conditions: all of the reactions were performed in 3 mL of solvent in the argon

atmosphere. bequivalent ratio. c Isolated yield.

The challenging 1,1-α,α linkage of trehalose to prepare protected 10 (an intermediate for synthesis of 8, Table 1) was achieved by condensing 1-O-DMB glucoside 5 and thioglycoside 6 through an optimized Bertozzi’s convergent synthesis method or intramolecular aglycon delivery (IAD) technique.18, 19 As indicated in Table 1, by using two-fold increases in the amount of donor 5 and catalyst (TfOMe/DTBMP), intermediate 10 was obtained at a slightly higher yield (25%, entry 2) than 1.0 equivalent consumption (entry 1), whereas only trace desired product was observed in the reflux conditions of dichloromethane (entry 3). Critically, when fresh crystalized DDQ was used, the yield was significantly increased from 16% (entry 1-3) to 54% (entry 4) in 1,2-Dichloroethane (DCE) for a 24-h reaction at 45 °C. Microwave conditions in DCE further promoted the glycosylation progress, enabling shortening of the reaction time from 24 hours to 15 minutes (entries 5, 6, and 7).

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Scheme 1. Synthetic Procedures for Compound 8a

aa)

R1COOH, DMAP, EDCI, r.t, 84%-90% or R1Br, NaH, DMF, 0 °C -r.t, 81%; b) PdCl2, DCM:MeOH=2:1, 67%-85%; c) R2COOH, DMAP, EDCI, 80%-93% or R2Br, NaH, DMF, 0 °Cr.t, 77%;d) 16% CF3COOH, 8% MeOH, DCM, 0°C-r.t., 70%-89%; e) Trt-Cl, DMAP, Py, 81%87%; f) R3COOH, DMAP, EDCI, 85%-89% or R3Br, NaH, DMF, 0 °C -r.t, 84%; g) FeCl3.6H2O, DCM, 0.5h, 79%-86%. Note*: While the R2= 2-methylbutyl, a diastereoisomeric mixture of compounds 1d-f, 1j, 2a-d, and 2g, 3 and 4 were afforded R:S=1:1, and synthetizing the compound 1h,1i,2e,2f, R2 represents the benzyl formate.

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Scheme 2. Synthetic Procedures for Compound 18a

aa)

phthalic anhydride, 1.5eq DBU, 1.5eq 4Å MS; b) i, 5eq DTBMP, 2.5eq Tf2O 1.5eq -78°C, 15 min, ii, acceptor, -78°C to 0°C, 1 h, 75%-83%; c) 3atm, H2, 20% Pd(OH)2, 7h, 84%-89%; The lipodisaccharide intermediate 8 was prepared from 10 with respective acylations of 2’OH, 3’-OH, and 4’-OH, whereas the 3’-OH and 4’,6’-diol of 10 were previously selectively protected by the allyl and 4,6-benzylidene groups (Scheme 1). After R1 was assembled at the 2’position of 10 to yield 11, the 3’-position was then deprotected by removal of the allyl group with PdCl2 treatment to give 12, followed by acylation of the 3’-OH to yield 13. The benzylidene

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group of 13 was subsequently removed, resulting in a diol 14. The 6’-OH of 14 was selectively protected by a Trt group to afford 15. Alternatively, we tried tert-butyldiphenylsilcyl protection instead of the Trt group, but the protected compound was unstable. Then the 4’-OH was firstly acylated with R3 (compound 16), and then 8 was finally prepared after removal of the Trt group under Lewis acidic conditions (FeCl3.6H2O), Although the stereoselective construction of β-mannopyranoside in 1a continues to pose a significant challenge, several protocols have been developed to address this synthetic limitation.21-24 Crich and co-workers found the promotion of 4,6-O-benzylidene mannopyranoses for the construction of β-mannosidic linkages.25-31 Moreover, the one-pot glycosylation method with anomeric hydroxy sugars has been proven to be an efficient method to prepare βmannopyranosides.21 Therefore, further glycosylation of 8 (acceptor) in our route by a benzylidene protected triflate oxocarbenium ion 17 (donor), which was previously prepared by anomeric hydroxyl mannose 7, stereoselectively afforded a fully protected compound 18 (Scheme 2). All benzyl protected groups of 18 were simultaneously removed by H 2 in the presence of 20% Pd(OH)2/C and purified by a prepared HPLC to give the final compound 1a-1j. Direct removal of the benzyl protected groups of 8 led to a number of derivatives of 2a-2g. Alternatively, the fatty chains were also anchored via ether bonds (1b) by NaH promoted condensation instead of EDCI/DMAP acylation conditions to investigate the role of the acyl group (see supporting information). Scheme 3. Synthetic Procedures for Compound 4a

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a(a)

PhCH(OMe)2, CSA, CH3CN, 81% (b) nBu2SnO, MeOH, reflux, then Allyl-Br, DMF, 58%, (c) undecylic acid, EDCI, DMAP, 86%; (d) PdCl2, DCM:MeOH=2:1, 79%; (e) 2-Methylbutyric acid, EDCI, DMAP, 79%; (f) CF3COOH, DCM, MeOH, 72%; (g) Trt-Cl, DMAP, Py, 84%; (h) Heptanoic acid, EDCI, DMAP, 85%; (i) FeCl3•6H2O, DCM, 93% The synthesis of compound 4 began with methyl α-glucopyranside 19 in which 4,6-diol was regioselectively furnished by benzylidene to create 20 (Scheme 3). The 3-OH of 20 was selectively masked with an allyl group by treatment with nBu2SnO in the presence of allyl bromide. Next, a free 2-OH was acylated by undecylic acid to gain 22. Removal of the allyl group of 22 followed by the introduction of the 2-methylbutyric ester on 3-position provided 23. The benzylidene group was then deprotected in the presence of trifluoroacetic acid, the Trt (trityl) group was subsequently and regioselectively equipped with 6-OH and followed with a heptanoic acid esterification on 4-position that afforded 24. The Trt (trityl) group was finally removed to Scheme 4. Synthetic Procedures for Compound 3a

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a

a), phthalic anhydride, 1.5eq DBU, 1.5eq 4 Å MS; b)i, 5eq DTBMP, 2.5eq Tf2O 1.5eq -78°C, 15 min; ii, acceptor, -78°C to 0°C, 1 h, 89%; c) 3atm, H2, 20% Pd(OH)2, 7 h, 85%; give the desired 4 under Lewis acid conditions (FeCl 3•6H2O) in DCM. A stereoselective glycosylation of building block 7 with compound 4 by the procedure of βmannoside 18 as described above afforded the disaccharide intermediate 25. Removal of the 4,6O-benzal and Bn of 25 using hydrogenolysis with the help of Pd(OH)2 gave compound 3 (Scheme 4). Collectively, all of the derivatives of 1, 2, 3 and 4 in Table 2 were prepared accordingly, where R1, R2, and R3, respectively, represent different aliphatic alkyls anchored on either a monosaccharide, disaccharide or trisaccharide template via acylation or an ether bond. Antibacterial activity screening The antimicrobial activities of 1a and its derivatives were determined against three major Gram-positive pathogens: E. faecalis, S. aureus, and S. pneumoniae. Although several compounds showed inhibitory activities against E. faecalis and S. aureus (1j, 2a-2c), S.

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pneumoniae was particularly susceptible to most of the compounds when observing the MIC values under the tested conditions (Table 2). All of the compounds discussed (1a-1j, 2a-2g, 3 and 4) in this manuscript exhibited a regular pattern of structure-dependent antimicrobial activity. The potency and selectivity against S. pneumoniae was dependent on the chain length and the form of the aliphatic chain, the position of the fatty-acid residues, and the motif of the saccharide core structure. The natural product 1a and its diastereoisomer 1c at R2 (3’- position) exhibited almost equal activity (MIC=6.25~12.5 μg/mL). Replacing the acyl bonds of the lipid chains at the 2’, 3’, and 4’ positions simultaneously with three ether bonds occurring in compound 1b led to the same level of antibacterial activity (MIC=5~10 μg/mL) as 1a, although it had reduced selectivity for S. aureus (MIC=16 μg/mL). Repositioning of R1 and R3 of 1a created compound 1e that demonstrated improved potency with MIC values ranging from 2.5~5 μg/mL against S. pneumoniae without changing its selectivity to S. aureus (MIC>64 μg/mL) and E. faecalis (MIC>64 μg/mL). Permutation of the 4’-O-acyl group with the 2’-O-acyl group (1a, MIC=6.25~12.5 μg/mL) and straightening the terminal isopropyl group of the branch lipid chain to a linear chain could also improve their potency (1f and 1g MIC=2.5~5 μg/mL). The antibacterial activity against S. pneumoniae was slightly reduced when the 2-methyl butyryl group at 3’-OH was omitted (compounds 1h, MIC=6.25~12.5 μg/mL and 1i, MIC=12.5~25 μg/mL). Furthermore, in comparison to compound 1a, the elongated acyl linear chain C11 (1g) of R3 or C11 (1f) of R1 also enhanced the potency against S. pneumoniae while still retaining the

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selectivity to S. aureus and E. faecalis. However, further C16 acyl chain replacement of R1 (1j, MIC >64 μg/mL) dramatically reduced the activity against S. pneumoniae and instead had a slight bactericidal effect on S. aureus (MIC=32 μg/mL) and E. faecalis (MIC=16 μg/mL). These data might imply that an even longer aliphatic chain plays a role in selectively disturbing the function of S. aureus and E. faecalis. Reduced selectivity for S. pneumoniae was observed for trehalose displayed compounds 2a (MIC=32 μg/mL for both S. aureus and E. faecalis), 2b and 2c (MIC=16 μg/mL for S. aureus and E. faecalis), and 2g (MIC=64 μg/mL for S. aureus) by removing the mannose moiety of the natural product 1a, although these compounds showed the same level of activity against S. pneumoniae (MIC=5~10 μg/mL). Similar to compounds 1h and 1i, decreased potency was observed with trehalose derivatives 2e-2f (MIC=12.5~25 μg/mL), when the 2-methylbutyl group (R2 at 3’ position) was omitted. Compound 4 (MIC>64 μg/mL) presented a significant decrease in both selectivity and activity against S. pneumoniae. Interestingly, compound 3 (MIC=6.25~12.5 μg/mL), a β-mannosidic disaccharide derivative, which displayed acyl linear lipid chains at C11 (2’-position) and C7 (3’-position), maintained both antibacterial activity and selectivity, suggesting that the β-mannosidic disaccharide template is necessary for both the potency and selectivity of the compounds. Another interesting finding in this study was that 2a appeared to have lower selectivity toward S. pneumoniae than 3, although both 2a and 3 were anchored exactly to the same R1, R2, and R3 substituent. This result further confirmed that the mannosidic disaccharide was the pharmacophore core structure for selective targeting the cell membrane of S. pneumoniae (see below for the biological mechanism

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studies). We summarized the SARs findings from this study and thus provided general pharmacophore information for this class of compounds against S. pneumoniae in Figure 3. Table 2. The MIC values of synthetic lipooligosaccharides against major multi-drug resistant Gram-positive pathogens Compound

R1

R2

R3

S. pneumoniae

S. aureus

E. faecalis

g/mL(M) 1aa

1ba

1ca

1da

1ea

1fa

6.25~12.5

>64

>64

(7.0-14.0)

(>70)

(>70)

5~10

16

>64

(5.9-11.8)

(18.8)

(>70)

6.25~12.5

>64

>64

(7.0-14.0)

(>70)

(>70)

2.5~5

>64

>64

(2.8-5.6)

(>70)

(>70)

2.5~5

>64

>64

(2.8-5.6)

(>70)

(>70)

2.5~5

>64

>64

(2.8-5.6)

(>70)

(>70)

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1ga

1ha

1ia

H

H

1ja

2ab

2.5~5

>64

(2.8-5.6)

(>70)

6.25-12.5

>64

(7.7-15.5)

(>70)

(>70)

12.5-25

>64

>64

(15.5-31.0)

(>70)

(>70)

>64

32

16

(>70)

33.3

16.6

32

32

(8.5-17.1)

5~10 (6.9-13.7)

2cb

5~10 (6.9-13.7)

2db

2eb

(43.9)

16 (21.9)

16

(43.9)

32 (43.9)

16

(21.9)

(21.9)

>64

>64

(13.7-27.4)

(>70)

(>70)

12.5~25

>64

>64

(14.4-28.7)

(>70)

(>70)

10~20

H

(>70)

>64

6.25-12.5

2bb

>64

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2fb

H

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12.5~25

>64

>64

(19.4-38.7)

(>70)

(>70)

2gb

6.25~12.5 (9.7-19.3)

3cc

(>70)

>64

>64

(>70)

(>70)

>64

>64

>64

(>100)

(>100)

(>100)

(8.4-16.8)

Van

0.125-0.25 (0.09-0.17)

a=

, b=

, c=

64

(>70)

6.25-12.5

4dd

64

1-2 (0.69-0.14)

0.5-1 (0.34-0.68)

, d=

e= value of minimum inhibitory concentration (MIC), Van=vancomycin

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Figure 3. Schematic representation of the generic rule of the Lipooligosaccharides in selective inhibition of S. pneumoniae growth.

Selectivity test Table 3. Antibacterial activity of compound 1f against extensive drug resistant Gramnegative bacteria. Species

Straina

MIC μg/mL(M)

K. pneumoniae

TH4079

>64(>70)

P. aeruginosa

TH4089

>64(>70)

A. baumannii

TH4100

>64(>70)

V. cholerae N16961

ATCC39315

>64(>70)

aStrain

TH4079 (K. pneumoniae), TH4089 (P. aeruginosa), TH4100 (A. baumannii) were human isolates.

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Our subsequent work focused on compound 1f, one of the most potent compounds against S. pneumoniae. We initially tested its activity against several extensively drug resistant Gramnegative pathogens: Klebsiella pneumoniae (K. pneumoniae), Pseudomonas aeruginosa (P. aeruginosa), Acinetobacter baumannii (A. baumannii) and Vibrio cholera (V. cholera). None of these bacteria were susceptible to 1f (MIC > 64 g/mL) (Table 3). Because 1f is not effective against E. faecalis and S. aureus, we tested whether it had antibacterial activity against bacteria in the genus Streptococcus by measuring the MICs against Streptococcus agalactiae (S. agalactiae), Streptococcus gordonii (S. gordonii), Streptococcus mitis (S. mitis), Streptococcus oralis (S. oralis), Streptococcus pyogenes (S. pyogenes), and Streptococcus sanguis (S. sanguis). Although the MIC values differed among these species, 1f was active against all six Streptococcus species tested thus far (Table 4). S. mitis, S. oralis, and S. pyogenes showed relatively higher susceptibility to 1f (MIC=2.5-5 g/mL), whereas the S. sanguis strain ST146 displayed the lower susceptibility (MIC=20-40 g/mL). This result suggests that 1f targeted the same or similar structure/function in the microorganisms of the Streptococcus genus. Table 4. Antibacterial activity of compound 1f against selected Streptococcus species. Species

Straina

MIC μg/mL (M)

S. agalactiae

ST4265

5.0-10.0 (5.6-11.2)

S. gordonii

ATCC49818

10.0-20

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(11.2-22.4) S. mitis

ATCC49456

2.5-5 (2.8-5.6)

S. oralis

ST137

2.5-5 (2.8-5.6)

S. pyogenes

ST4285

2.5-5 (2.8-5.6)

S. sanguis

NCTC7863

20-40 (22.4-44.8)

aStrains

ST137 (S. oralis), ST4265 (S. agalactiae), and ST4285 (S. sanguis) were human

isolates. We then determined the susceptibility of five multi-drug resistant S. pneumoniae clinical isolates to 1f. Multi-drug resistance is defined as being resistant to at least one agent in three or more clinical antimicrobial categories.32 All of the strains were susceptible to 1f with the same MIC value (1.25-2.5 μg/L) (Table 5). This experiment further indicated that 1f targets an essential structure or function of S. pneumoniae that is completely different from those targeted by existing antimicrobials. Table 5. Antibacterial activity of compound 1f against representative multidrug-resistant S. pneumoniae isolates. Straina

Drug of resistanceb

MIC g/mL (M)

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ST2582

PEN, CRO, ERY, TCY, SXT, DA

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1.25-2.5 (1.4-2.8)

ST2784

ERY, TCY, SXT, DA

1.25-2.5 (1.4-2.8)

ST2863

PEN, ERY, TCY, SXT, DA

1.25-2.5 (1.4-2.8)

ST2882

AMX, ERY, CHL, SXT, DA

1.25-2.5 (1.4-2.8)

ST2889

PEN, ERY, CHL, SXT, DA

1.25-2.5 (1.4-2.8)

a

All S. pneumoniae strains used in this experiment were isolated from the blood or cerebrospinal fluid (CSF) of human patients (JRZ, unpublished). Drug susceptibility was determined using Vitek 2 AST-GP68 cards (bioMe´rieux, France) according to the performance standards for antimicrobial susceptibility testing from the 25 th informational supplement (CLSI document M100-S25, 2015) of the Clinical and Laboratory Standards Institute (clsi.org). bAMX, amoxicillin; CHL, chloramphenicol; CRO, ceftriaxone; DA, clindamycin; ERY, erythromycin; PEN, penicillin; SXT, sulfamethoxazole; TCY, tetracycline. Hemolysis tests To ascertain the mode of anti-pneumococcal activity, we investigated in vitro erythrocyte hemolysis toxicity of selected compounds using rabbit blood according to Cyprotex’s in vitro toxic hemolysis assay (Figure 4). Compared with 1% Triton-100 (positive control), compounds 1b, 1j, 2b, 2c, 2e, 2f, 3 and 4 did not show detectable hemolysis at 100 μg/mL, whereas compounds 1a, 1c-1i, 2a and 2d led to lysis of less than 15% rabbit erythrocytes at a final concentration of 100 μg/mL.

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The data revealed that compound 2g is toxic to rabbit erythrocytes. This compound consists of a nitrogen atom-containing lipid chain and is structurally different from the other tested compounds. This result suggests that the lipid chain contributes to the toxicity of 2g, and the corresponding nitrogen atom is critical for the selectivity of 1a and its derivatives for erythrocytes and bacterial cells.

Figure 4. In vitro cytotoxicity (hemolysis) of compounds 1a and its representative derivatives.a aRabbit

erythrocytes were suspended in PBS to 2×108 per mL and incubated with 1% Triton X100 or each of the compounds at a concentration of 100 μg/mL for 45 minutes at 37°C. Hemolysis was determined by measuring the absorbance of the heme in the supernatant at OD540. The hemolysis values of the compounds were normalized to that of 1% Triton X-100 (100% hemolysis). Kinetics of bacterial death We next determined whether the antibacterial activity of 1f is dose-dependent by treating pneumococci with various concentrations of the compound. The result showed that 1f reduced the density of pneumococcal cultures in a dose-dependent manner (Figure 5A). This observation

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was confirmed by the loss of bacterial viability after the treatment with 1f (Figure 5B). The cultures with the highest concentration of the compound (50 μg/mL) showed the most dramatic reduction in bacterial viability, resulting in an undetectable level of viable bacteria. As positive controls, vancomycin and DOC (deoxycholate) showed typical features of bacteriolytic molecules. Vancomycin is a bactericidal drug that lyses S. pneumoniae;2, 33 DOC also induces rapid autolysis of S. pneumoniae.34 Compound 1f lysed the pneumococci even faster than DOC and vancomycin (Figure 6A). At a dose of 10-fold MIC, treatment with vancomycin reduced viable cells of the culture by 4 orders of magnitude in 6 hours, whereas a similar dose of 1f achieved undetectable levels (reduced more than 8 orders of magnitude) of bacteria in 30 minutes (Figure 6B). The kinetics of bacterial death revealed that 1f is a bactericidal compound that lyses pneumococci in a dose-dependent fashion.

Figure 5. Dose-dependent bactericidal activity of compound 1f against S. pneumoniae D39.a aLog-phase

S. pneumoniae D39 was incubated with different concentrations of compound 1f for 30 minutes. Cell turbidity (A) and viability (B) were monitored before and after the 0.5-hour treatment by OD620 and CFU measurement, respectively.

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Figure 6. Kinetics of the compound 1f activity in killing S. pneumoniae. a a

Log-phase S. pneumoniae D39 was incubated in the presence of compound 1f (30 μg/mL), vancomycin (5 μg/mL), or DOC (200 μg/mL). Cell turbidity (A) and viability (B) were monitored for 6 hours by OD620 and CFU measurement, respectively. Exploration of the antibacterial mechanism The impact of compound 1f on the cell membrane of S. pneumoniae was determined using a cell membrane permeabilization assay with the fluorescent probe DiSC3(5), a cytoplasmic membrane potential sensitive dye.35 The fluorescence of DiSC3(5) is self-quenched once it binds to the intact cytoplasmic membrane of bacteria; the fluorescence of DiSC3(5) increases as the membrane is depolarized by membrane-active agents.35 Compound 1f was added to DiSC3(5) treated bacterial cells at concentrations of 40 μg/mL, 20 μg/mL, 10 μg/mL, and 5 μg/mL. Vancomycin (a cell wall synthesis inhibitor) and Triton X-100 were used as negative and positive controls, respectively. As shown in Figure 7, one minute after the addition of the reagents, bacterial cells treated with 1f and Triton X-100 showed significantly increased

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fluorescence, whereas cells treated with vancomycin did not. This result indicated that 1f is able to perturb the cell membrane.

Figure 7. Permeabilization of bacterial cell membrane by compound 1f. a a

Log-phase S. pneumoniae D39 cells were incubated in the presence of 0.4 μM DiSC3(5) for self-quenching, followed by treatment with 100 mM KCl to equilibrate the cytoplasmic and external K+ concentration. Compound 1f was added to final concentrations of 5 μg/mL, 10 μg/mL, 20 μg/mL and 40 μg/mL. Vancomycin (5 μg/mL) and Triton X-100 (4%, v/v) were added as negative and positive controls, respectively.

Summary and Conclusion In conclusion, this study represents the first report of natural and synthetic lipotrisaccharides with bactericidal activity via interaction with the cell membrane. The natural lipotrisaccharide 1a and some of its synthetic derivatives were found to be highly potent against S. pneumoniae, which is a major cause of bacterial pneumonia, meningitis, and otitis media in humans. Extensive drug resistance has substantially limited the options for clinical treatment of pneumococcal disease, leaving vancomycin as a last resort for treatment in certain cases of

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pneumococcal infection.33 As compound 1a and some of its synthetic derivatives are effective against S. pneumoniae clinical isolates that are resistant to the vast majority of the currently available drugs, our results strongly suggest that these compounds target a novel bacterial structure or function, which is different from those of existing antimicrobials. Although the parent compound 1a is only effective against S. pneumoniae, several of its derivatives (e.g., 1j, 2a, 2b and 2c) showed moderate antibacterial activities against E. faecalis and S. aureus, although still not active against gram-negative bacteria. But this result still suggests that further chemical modifications of compound 1a may broaden their spectrum of activity against Grampositive bacteria. Thus, the lipotrisaccharides identified in this work have opened a new venue for future development of novel antimicrobials for treating infections caused by S. pneumoniae and other drug resistant pathogens. Antimicrobials with broad spectrums have contributed to the selection-based rise of drug resistant bacteria and antibiotic-induced diseases due to adverse impacts on host microbiota (e.g., antibiotic-induced colitis).36, 37 Thus, antimicrobials with narrow spectrums have been proposed as alternatives for specifically targeting pathogens and minimizing the impact of treatment on indigenous microbes. Along this line, the lipotrisaccharides discussed in this study provide insightful clues for future development of novel antimicrobials with limited spectrums of activity. With the improvements in clinical diagnosis of bacterial infections in terms of pathogen identification, drug resistance profiling, and diagnosis time, the antimicrobials with focused

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spectrums of activity may be advantageous over those with broad spectrums of activity in future treatment of microbial infections. This study has developed an efficient synthetic route for natural lipotrisaccharide 1a and its derivatives through the application of an IAD reaction to establish the trehaloside compounds. The natural product 1a and its derivatives showed the SAR of the saccharide motifs and the lipophilic moieties against S. pneumoniae. The potency and selectivity in killing S. pneumoniae were dependent on the lipid chain length and linear form, the position of the fatty-acid residues and the motif of the saccharide core structure. Collectively, a mannose-glucose disaccharide core structure is a basic template for both potency and selectivity of the compounds against S. pneumoniae but not trehalose. A trehalose disaccharide (compound 2a-2c) moiety, which displays the same R1, R2, and R3 lipid chains, reduced the selectivity to S. pneumoniae compared with trisaccharide derivatives (compounds 1e-1f), along with further significant decreases in the selectivity and activity by compound 4. The chiral molecules (1a and 1c) of the lipid chain displayed the same antibacterial activity but the racemic compound 1d had better activity. A nitrogen-containing lipid chain (2g) appeared toxic to rabbit erythrocytes, and an alkyl lipid chain derivative 1b, instead of the corresponding acylated 1a, provided the same level of antibacterial activity but had lower selectivity for S. aureus. Linear and longer lipid chains of 2’position and 4’-positions (R1 and R3, 1f and 1g) could improve the activity, but with a limited extension of the carbon number (e.g., 1j). Clearly, omitting the R2 acyl chain (1h and 1i) reduced the antibacterial ability. Therefore, the original full trisaccharide guaranteed good selectivity and

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the highest potency against S. pneumoniae when it contained the proper acyl lipid chains, i.e., the C3-C9 chains of R1 and R3 (2’-position), and the 2-methyl butyryl group of R2 (3’-position), without aza substitution in the chain.

Experimental Section All of the other commercial materials were used without further purification as received unless otherwise noted. Dry DCM and DCE were distilled from calcium hydride. Dry THF was distilled from sodium hydride. All glycosylated reactions were carried out under anhydrous conditions with freshly distilled solvents, unless otherwise noted. Reactions were monitored by analytical thin-layer chromatography on silica gel GF254 pre-coated on glass plates, preparative TLC on silica gel and the silica gel for column chromatography were phased from Qingdao Haiyang Chemical and Special Silica Gel Co., Ltd. All of the final products were purified by preparative HPLC with an ELSD monitor. Spots were detected under UV (254 nm) and/or by staining with 10% (volume fraction) H2SO4/ethanol. Proton nuclear magnetic resonance (1H NMR) and carbon nuclear magnetic resonance (13C NMR) spectroscopy were performed on 300 M, 400 M, 500 M and 600 M spectrometers. 2,3,4,6-O-Benzyl-4’,6’-di-O-benzylidene-3’-O-allyl-D-trehalose (10). A mixture of (1.38 g, 2 mmol) of 519 and (352 mg, 1 mmol) of 620 was azeotropically dried with toluene for three times under reduced pressure. To the residue were added 0.5 g of 4Å MS and 20 mL of dry DCM. The mixture was cooled to 0°C, and (449 mg,2.0 mmol) of DDQ (freshly recrystallized from

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benzene) was added and stirred for 30 min under an argon atmosphere. The reaction was allowed to warm up to r.t. and was left stirring for 4.5 h until TLC analysis showed the reaction was completed. The reaction was therefore quenched with 30 mL of freshly prepared ascorbate buffer (0.7 g of ascorbic acid, 1.06 g of citric acid, and 0.92 g of NaOH in 100 mL of H2O), diluted with 30 mL EtOAc, then stirred vigorously for 15 min, during which time the brown solution turned into lemon yellow. The reaction mixture was filtered through celite, and the organic layer was washed with saturated NaHCO3 and saturated NaCl and dried over Na2SO4. The solution was then concentrated under reduced pressure and the residue dried azeotropically with toluene (3), after which 451.7 mg (2 mmol) of DTBMP, 0.5 g of 4Å MS, and 25 mL of dry DCE were added. The mixture was cooled to 0°C and (226 L, 2 mol) of MeOTf was added. The reaction mixture was stirred for 15 min at 0°C and heated to 45°C overnight (or microwaved for 15 min). The salmon-colored reaction mixture was cooled to 0°C and quenched with 4mL of Et 3N, turning green. After being diluted with EtOAc (30 mL) and saturated NaHCO 3, the residue was stirred for 15 min at r.t. and filtered through celite. The grey-green organic layer was washed with H2O and saturated NaCl and then dried over Na 2SO4. The residue was examined under flash chromatography by using of 30:0, 30:1, 25:1 toluene/ EtOAc to yield colorless oil (888.2 mg, 1.07 mmol, 54%) of 10. [α]25D +174.8, 1H NMR (500 MHz, CDCl3): = = 7.47-7.49 (m, 2H, arom H), 7.24-7.40 (m, 21H, arom H), 7.13-7.15 (m, 2H, arom H), 5.92- 6.00 (m, 1H, arom Hallyl), 5.54 (s, 1H, CHPh), 5.30 (d, 1H, 9.6Hz, 1-H), 5.16-5.20 (m, 3H, arom H-allyl, 1’-H), 4.98 (m, 1H), 4.85 (d, 1H, 10.8Hz, CH2Ph), 4.83 (d, 1H, 10.8Hz, CH2Ph), 4.69-4.75 (m, 2H), 4.62 (d,

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1H, 10.8Hz, CH2-Ph), 4.45-4.51 (m, 3H), 4.13- 4.25 (m, 3H), 4.08-4.11 (m, 1H), 4.00 (dd, 1H, 9. 6Hz, 9.6Hz, 3’-H), 3.82 (dd, 1H, 9.6Hz, 9.6Hz, 4’-H), 3.70- 3.76 (m, 4H), 3.63-3.68 (m, 2H), 3.60 (dd, 1H, 9.6Hz, 9.6Hz, 3’-H).

13C

NMR (126 MHz, CDCl3) = 138.78, 138.17, 137.93,

137.49, 134.97, 128.94, 128.56, 128.42, 128.40, 128.21, 128.08, 128.00, 127.92, 127.86, 127.84, 127.80, 127.73, 127.61, 126.13, 117.13, 101.24 (CHPh), 95.27 (1’-C), 93.74 (1-C), 82.26, 81.89, 79.34, 78.54, 77.65, 75.63, 75.25, 73.84, 73.43, 73.32, 71.88, 70.76, 68.99, 68.31, 63.17. HRMS(ESI+) m/z 831.3719 [M+H]+ (Calcd. for C50H55O11, 831.3712). General procedure of acylation of 2’-position of ,-trehaloside 10 to prepare 11. To a solution of compound 8 (1 mmol), DMAP (0.5 mmol), EDCI (2 mmol) in DCM 20 mL, were stirred at room temperature. After the reaction was completed, the organic layer was washed with H2O and saturated NaCl and then dried over Na2SO4, then concentrated under reduced pressure. Chromatographic purification afforded the 2-O-alkylated compounds (11-A to 11-E); see Supporting Information for details. General procedure of deprotection of 3’-O-Allyl of 11 to prepare 12. To a solution of compound 11 (1 mmol) in 20 mL, a mixed solvent of DCM/MeOH=1:9 (v:v), 0.1 mmol PdCl2 was added, and then stirred at room temperature. After the reaction was completed by TLC analysis, the reaction was filtered through a short silica gel filter and was washed with DCM/MeOH=4:1 (v:v) mixed solvent. After being concentrated under reduced pressure, the residue was dissolved in 20 mL of DCM and washed with saturated NaCl and dried over

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Na2SO4. Removal of the solvent under a reduced pressure and then flash chromatography by silica gel GF254 offered the compounds 12 (12A to 12E); see Supporting Information for details. General procedure of acylation of 3’-position to prepare compound 13. DMAP (0.5 mmol) and EDCI (2 mmol) was introduced into a solution of compound 12 (1 mmol), in DCM 20 mL and stirred at room temperature. After the reaction was completed, the organic layer was washed with H2O, saturated NaCl and then dried over Na2SO4. After being concentrated under reduced pressure, the residue was purified by chromatography to afford the 3’-O-acylation intermediate (13A-13E); see Supporting Information for details. General procedure of deproduction of 4,6-O-benzylidene group to prepare compound 14. To a solution of compound 13 (1 mmol) and 1.6 mL MeOH in 20 mL DCM, stirred for 3 min at 0°C, then 3.2 mL of TFA was added dropwise, the reaction temperature was allowed to rise up to r.t., and after the TLC analysis of the reaction, it was washed with saturated NaHCO 3 and brine and then dried over Na2SO4. Chromatographic purification afforded the colorless syrup compound 14 (14A-14E); see Supporting Information for details. General procedure of selective protection of 6’-position to prepare compound 15. A solution of compound 14 (1 mmol), DMAP (0.5 mmol) and Trt-Cl (2 mmol) in dry pyridine was stirred at 80°C for 3 hours. After the reaction was completed according to the TLC, the solvent was concentrated under reduced pressure; redissolved with DCM; and washed with brine; dried over Na2SO4; and then was chromatographically purified to afford the colorless syrup compound 15 (15A-15E); see Supporting Information for details.

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General procedure of acylation of 4’-positon to prepare compound 16. DMAP (5 mmol) and EDCI (10 mmol) was added to a solution of compound 12 (1 mmol) in 20 mL DCM under reflux stirring conditions. After the reaction was completed by TLC detection, the organic layer was washed with H2O and saturated. NaCl and then dried over Na2SO4. The filtrate was then concentrated under reduced pressure chromatographic purification, which created the 4’-Oacylation intermediate 16 (16A-16E); see Supporting Information for details. General procedure of deprotection of 6’-Trt to prepare compound 17. FeCl3•6H2O (3 mmol, 810.9 mg) was mixed into a solution of compound 16 (1 mmol) in 25 mL DCM, then stirred for another 0.5 hour until the reaction was detected to be completed by TLC. The product was washed with 25 mL H2O and extracted with DCM (2×25 mL); the organic layer was dried over Na2SO4 and concentrated under reduced pressure; the residue was purified by chromatography and created the 6’-OH intermediate 17 (17A-17E). See Supporting Information for details. General procedure of glycosylation of 6’-OH with mannose 7 to prepare 18. To a mixture of phthalic anhydride (1.1 mmol), 4 Å MS, donor 722 (1.0 mmol) and DBU (1.2 mmol) in 15 mL DCM were added in the argon atmosphere, then was stirred for 15 min. Next, the mixture was transferred to -78°C, the solution of DTBMP (2.2 mmol, 451.7 mg) in 5 mL DCM and Tf2O (2.2 mmol, 370 μL) by a syringe, and stirred for 15 min at -78°C. Furthermore, the solution of acceptor 6 (1.1 mmol) in 3 mL dry DCM was stirred at -78°C for 30 min, then the temperature was allowed to rise up to 0°C in 1 hour. Et3N quenched the reaction and was then filtered

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through celite. The organic layer was washed with saturated NaHCO 3 and saturated. NaCl and then dried over Na2SO4. The reaction mixture in toluene was subjected to flash chromatography with the use of petroleum ether:EtOAc=8:1, 6:1 to create the intermediate 18 (18A-18E); see Supporting Information for details. General procedure of global deprotection of benzyl group to prepare 1, 2, 3 and 4. Compound 8 or 18 (0.15 mmol) was dissolved in a mixed solvent of MeOH/CHCl2 (3:1, 12 mL). Pd(OH)2/C (0.25 mmol, 35 mg) was suspended in the solution. The mixture was then stirred under 3 atm H2 for 7 h. The catalyst was filtered off, and the residue was purified through preparative HPLC with an ELSD monitor, and compounds 1a-j, 2a-g, 3 and 4 were obtained after freeze drying. See supporting information for details. Natural lipotrisaccharide 1a. Amorphous powder. [α]25D +176.8 (c=0.94 CHCl3). 1H NMR (400 MHz, MeOD): 5.65 (dd, 1H, J =10.0Hz, 3’-H), 5.36 (d, 1H, J=3.6Hz, 1’-H), 5.21 (dd, 1H, J=10.0Hz, 4’-H), 5.11 (d, 1H, J=3.6Hz, 1’-H), 5.03 (m, 1H, 2’-H), 4.46 (m, 2H, 1’’-H), 4.02 (m, 1H), 3.92 (d, 1H, J=3.6Hz, 2-H), 3.67-3.82 (m, 5H), 3.52- 3.61 (m, 4H), 3.44 (dd, 1H, J=9.6Hz, J=3.6Hz, 2-H), 3.36 (dd, 1H, J=9.6Hz, 4-H), 3.20 (m, 1H), 2.27-2.39 (m, 5H, CHCO, CH2CO), 1.42-1.59 (m, 6H, CH2CH2CO, CHCH2CO), 1.19-1.31 (m, 12H, CH2), 1.09-1.10 (d, 3H, 7.2Hz CH(CH3)CHCO), 0.90-0.93 (m,15H, CH3).

13C

NMR (101 MHz, MeOD) : =175.63 (CO),

172.75 (CO), 172.62 (CO), 101.16 (1’’-C), 95.05 (1’-C), 91.58 (1-C), 76.94, 73.79, 73.13, 73.09, 71.53, 70.78, 70.27, 70.16, 70.01, 68.91, 68.58, 67.53, 67.06, 61.40, 61.07, 40.96, 38.79, 38.16,

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33.72, 33.36, 29.42, 29.01, 28.84, 27.74, 27.56, 27.05, 26.19, 24.29, 22.30, 21.63, 21.48, 21.45, 15.64, 10.69. HRMS(ESI+):m/z 891.4567 (Calcd. for C36H65O18Na, 891.4560) Lipotrisaccharide deritive 1b. Amorphous powder. [α]25D +130 (c=0.20, CHCl3), 1H NMR (400 MHz, MeOD): =5.25(d, 1H, J=3.2Hz, 1-H), 5.09(d, 1H, J=3.2Hz, 1’-H), 4.57 (brs, 1H, 1’’-H), 4.30(dd, 1H, J=10.0Hz, J=10.0Hz, 3-H) 4.10-4.13 (m, 2H), 3.86-3.90 (m, 2H), 3.803.85(m, 3H), 3.68-3.78 (m, 4H), 3.25-3.68 (m, 4H), 3.48-3.51 (m, 3H), 3.28-3.39 (m, 4H), 3.213.25 (m, 1H), 1.46-1.68 (m, 6H, CH2-O), 1.31 (s, 24H, CH2CH2-O), 1.14-1.21 (m, 1H, CHCH2O), 0.92-0.96 (m, 12H, CH3). 13C NMR (101 MHz, MeOD) δ 100.87 (1’’-C), 94.54 (1’C), 91.81 (1-C), 80.98, 80.49, 78.32, 78.06, 76.94, 73.86, 73.12, 72.70, 72.56, 71.83, 71.04, 70.86, 70.60, 70.21, 68.27, 67.04, 61.41, 61.11, 35.78, 31.67, 31.63, 30.14, 29.98, 29.37, 29.06, 28.99, 26.13, 25.94, 22.33, 22.29, 15.82, 13.03, 10.49.HRMS (ESI+) m/z 849.5186 [M+Na]+ (Calcd. for C41H78O16Na, 849.5182). Lipotrisaccharide deritive 1c. Amorphous powder. [α]25D +122.8 (c=0.67, CHCl3). 1H NMR (400 MHz, MeOD): =5.65 (dd, 1H, J=10.0Hz, 10.0Hz, 3’-H), 5.35 (brs, 1H, 1-H), 5.20 (dd, 1H, J=10.0Hz, J=10.0Hz, 4’-H), 5.10 (d, 1H, J=3.6Hz, 1’-H), 5.04 (m, 1H, 2’-H), 4.46 (m, 2H, 1’’-H), 4.02 (m, 1H), 3.93 (d, 1H, J=3.6Hz, 2-H), 3.66-3.80 (m, 5H), 3.54- 3.63 (m, 4H), 3.46 (m, 1H), 3.38 (d, 1H, J=9.6Hz, 4-H), 3.22 (m, 1H), 2.29-2.39 (m,5H, CHCO, CH2CO) 1.421.59 (m, 6H, CH2CH2CO), 1.31 (m, 8H, CH2), 1.20-1.26 (m, 4H, CH2), 1.09-1.11 (m, 3H), 0.920.95 (m, 15H, CH3).13C NMR (101 MHz, MeOD): =175.63 (CO), 172.75 (CO), 172.62 (CO),

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101.16 (1’’-C), 95.05 (1’-C), 91.58 (1-C), 76.94, 73.79, 73.13 73.09, 71.53, 70.78, 70.27, 70.16, 70.01, 68.91, 68.58, 67.53, 67.06, 61.40, 61.07, 40.96, 38.79, 38.16,33.72, 33.36, 29.42, 29.01, 28.84, 27.74, 27.56, 27.05, 26.19, 24.29, 22.30, 21.63, 21.48, 21.45, 15.64, 10.69. HRMS (ESI+) m/z 891.4567 [M+Na]+ (Calcd. for C36H65O18Na, 891.4560). Lipotrisaccharide deritive 1d. Amorphous powder. [α]25D +146 (c=0.59, CHCl3) 1H NMR (400 MHz, MeOD): =5.61 (dd, 1H, J=10.0Hz, 3’-H), 5.33 (d, 1H, 3.2Hz, 1-H), 5.19 (dd, 1H, J=10.0Hz, 4’-H), 5.09 (d, 1H, J=3.6Hz, 1’-H), 5.01 (m, 1H, 2’-H), 4.42 (m, 2H), 4.00 (d, 1H, J=10.0Hz, 3’’-H), 3.91 (m, 1H), 3.86 (d, 1H, J=10.0Hz), 3.64- 3.79 (m, 4H), 3.53-3.61 (m, 3H), 3.50 (dd, 1H, J=9.6Hz, J=3.6Hz, 2-H), 3.41 (m, 1H), 3.35 (d, 1H, J=9.6Hz, 4-H), 3.15-3.19 (m, 1H), 2.17-2.42 (m, 5H, CHCO, CH2CO), 1.49-1.64 (m, 6H, CH2CH2CO), 1.29 (brs, 8H, CH2), 1.16-1.22 (m, 4H, CH2, (CH3)2CH), 1.06-1.08 (m, 3H, CH3), 0.85-0.90 (m, 15H, CH3). 13C NMR (101 MHz, MeOD) δ 175.63 (CO), 172.75 (CO), 172.62 (CO), 101.16 (1’’-C), 95.05 (1’-C), 91.58 (1-C), 76.94, 73.79, 73.13, 73.09, 71.53, 70.78, 70.27, 70.16, 70.01, 68.91, 68.58, 67.53, 67.06, 61.40, 61.07, 40.96, 38.79, 38.16, 33.72, 33.36, 29.42, 29.01, 28.84, 27.74, 27.56, 27.05, 26.19, 24.29, 22.30, 21.63, 21.48, 21.45, 15.64, 10.69. HRMS (ESI+) m/z 891.4568 [M+Na]+ (Calcd. for C36H65O18Na, 891.4560). Lipotrisaccharide deritive 1e. Amorphous powder. [α]25D +90.9 (c=0.44, CHCl3). 1HNMR (400 MHz, MeOD): 5.63 (dd, 1H, J=10.0Hz, J=10.0Hz, 3’-H), 5.35 (brs, 1H, 1-H), 5.21 (dd, 1H, J=9.6Hz, J=9.6Hz, 4’-H), 5.11 (d, 1H, J=3.6Hz, 1’-H), 5.03 (m, 1H, 2’-H), 4.45 (m, 2H), 4.00

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(d, 1H, J=10.4Hz, 3’’-H), 3.93 (m, 1H), 3.88 (d, 1H, J=9.6Hz), 3.80 (m, 1H), 3.76 (m, 1H), 3.73 (m, 1H), 3.66-3.71 (m, 2H), 3.61-3.63 (m, 1H), 3.57 (d, 1H, J=9.6Hz), 3.53 (dd, 1H, J=9.6Hz, J=3.6Hz, 2-H), 3.43 (m, 1H), 3.37 (d, 1H, J=9.6Hz,4-H), 3.18-3.21 (m, 1H), 2.24-2.43 (m, 5H, CHCO, CH2CO), 1.43-1.67 (m, 6H, CH2CH2CO), 1.31 (m, 8H, CH2), 1.17-1.23 (m, 4H, CH2), 1.08-1.11 (m, 3H, CH2(CH3)CHCO), 0.87-0.91 (m, 15H, CH3). 13C NMR (101 MHz, MeOD) δ 175.36 (CO), 172.60 (CO), 172.41 (CO), 101.00 (1’’ -C), 94.88 (1’ -C), 91.44 (1-C), 76.79, 73.63, 72.97, 71.37, 70.61, 70.18, 70.01, 69.84, 68.80, 68.32, 67.38, 66.91, 61.25, 60.92, 40.91, 38.64, 38.00, 33.57, 33.22, 29.25, 29.19, 28.85, 28.67, 27.57, 27.40, 26.89, 25.95, 24.16, 22.11, 21.46, 21.31, 21.30, 15.48, 10.52. HRMS (ESI+) m/z 891.4567 [M+Na]+ (Calcd. for C36H65O18Na, 891.4560). Lipotrisaccharide deritive 1f. Amorphous powder. [α]25D +90.9(c=0.44, CHCl3). 1H NMR 5.63 (dd, 1H, J=9.6Hz, J=9.6Hz, 3’-H), 5.35 (brs, 1H, 1-H), 5.21 (dd, 1H, J=9.6Hz, J=9.6Hz, 4’H), 5.10 (d, 1H, J=3.6Hz, 1’-H), 5.02-5.04 (m, 1H, 2’-H), 4.42-4.46 (m, 2H, 1’’-H, 6’a-H), 4.00 (d, 1H, J=9.6Hz, 3’’-H), 3.92 (m, 2H, 6b’-H), 3.78 (dd, 1H, 9.6Hz, 3-H), 3.69-3.73 (m, 2H, 5-H), 3.63-3.67(m, 2H, 5’-H, 6’’a-H), 3.60(dd, 1H, 2’’-H), 3.55 (dd, 1H, 9.6Hz, 4’’-H), , 3.51 (dd, 1H, J=9.6Hz, J=3.6Hz, 2-H), 3.43 (dd, 1H, 10.6Hz, 3.6Hz, 6a-H), 3.35-3.37 (m, 1H, 4-H), 3.15-3.21 (m, 1H, 5’’-H), 2.23-2.42 (m, 5H, CHCO, CH2CO), 1.45-1.76 (m, 6H, CH2CH2CO), 1.31 (m, 20H, CH2), 1.08-1.10 (m, 3H, CH2(CH3)CHCO), 0.87-0.93 (m, 9H, CH3). 13C NMR (101 MHz, MeOD,diastereomers) δ 175.36 (CO, 3’-OOC), 174.80 (CO, 2’-OOC), 172.41 (CO, 4’-

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OOC), 101.00 (1’’ -C), 94.88 (1’-C), 91.44 (1-C), 76.79 (5’-C), 73.63 (5-C), 72.97 (2-C), 71,49 (2’-C), 71.37 (4-C), 71.14 (3’-C) 70.61 (2’-C), 70.18 (6’a-C), 69.84 (4’-C), 68.80 (2’’-C), 68.72 (3’-C), 67.38 (4’’-C), 61.25 (3-C), 60.92 (5’-C), 40.91 (CH2(CH3)CHCO), 38.64 (CH2CO), 38.00 (CH2CO), 33.57, 33.22, 29.25, 29.19, 28.85, 28.67, 27.57, 27.40, 26.89, 25.95, 24.16, 22.11, 21.46, 21.31, 21.30, 15.48, 10.52. HRMS (ESI+) m/z 891.4567 [M+Na]+ (Calcd. for C42H72O18Na, 891.4560). Lipotrisaccharide deritive 1g. Amorphous powder. [α]25D +176(c=0.17, CHCl3).1H NMR (400 MHz, MeOD, diastereomers): 5.61 (dd, 1H, J=9.6Hz, J=9.6Hz, 3’-H), 5.33 (d, 1H, J=3.6Hz, 1-H), 5.18 (dd, 1H, J=9.6Hz, J=9.6Hz, 4’-H), 5.08 (d, 1H, J=3.6Hz, 1’-H), 5.01 (m, 1H, 2’-H), 4.42 (s, 1H), 4.40-4.44 (m, 1H), 3.99 (d, 1H, J=9.6Hz, 3’’-H), 3.90 (m, 1H), 3.85 (dd, 1H, J=11.4Hz, J=2.8Hz, 6a’-H), 3.77 (dd, 1H, J=9.6Hz, J=9.6Hz), 3.69-3.72 (m, 2H), 3.64-3.66 (m, 2H), 3.57-3.60 (m, 1H), 3.56 (dd, 1H, J=9.6Hz, J=9.6Hz, 4’’-H), 3.50 (dd, 1H, J=9.6Hz, J=3.6Hz, 2-H), 3.41(m, 1H), 3.34 (m, 1H) , 3.15-3.18 (m, 1H), 2.19-2.37 (m, 5H, CHCO, CH2CO), 1.40-1.62 (m, 6H, CH2CH2CO), 1.29-1.33 (m, 20H, CH2), 1.06-1.08 (m, 3H, CH2(CH3)CHCO), 0.86-0.92 (m, 9H, CH3).

13C

NMR (101 MHz, MeOD, diastereomers):

=175.47 (CO), 172.80 (CO), 172.72 (CO), 101.32 (1’’ -C), 95.22 (1’ -C), 91.21 (1 -C), 76.70, 73.78, 73.12, 73.08, 71.51, 70.91, 70.33, 70.13, 69.99, 68.93, 68.58, 68.47, 67.55, 60.90, 65.12, 61.27, 60.84, 40.95, 33.17, 31.59, 31.38, 30.87, 30.64, 30.23, 29.37, 29.09, 28.43, 25.80, 23.72,

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21.92, 18.85, 15.71, 15.28, 12.97, 10.14 HRMS(ESI+) m/z 891.4555 [M+Na]+ (Calcd. for C42H72O18Na, 891.4560). Lipotrisaccharide deritive 1h. Amorphous powder. [α]25D +97 (c=1.0, CHCl3). 1H NMR (400 MHz, MeOD): =5.29(d, 1H, J=3.2Hz, 1-H), 5.09 (d, 1H, J=3.2Hz, 1’-H), 4.99 (dd, 1H, J=9.6Hz, J=9.6Hz, 3’-H), 4.80 (dd, 1H, J=10.0Hz, J=3.6Hz, 2’-H), 4.46 (brs, 1H, 1’’-H), 4.294.33 (m, 2H), 4.14 (dd, 1H, J=9.6Hz, 3’-H), 3.86-3.98 (m, 3H), 3.69- 3.74 (m, 3H),3.58-3.68 (m, 3H) 3.52 (dd, 1H, J=9.6Hz, J=3.2Hz, 2-H), 3.35-3.48 (m, 2H), 3.17-3.25 (m, 1H), 2.28-2.45 (m, 4H, CH2CO), 1.57-1.67 (m, 4H, CH2CH2CO), 1.31 (m, 20H, CH2), 0.90-0.93 (m, 6H, CH3). 13C NMR (101 MHz, MeOD): =173.67 (CO), 173.22 (CO), 101.08 (1’’ -C), 94.59 (1’ -C), 91.43 (1C), 76.90, 73.58, 72.90, 72.70, 71.78, 70.67, 69.76, 68.94, 68.18, 67.99, 66.62, 61.26, 60.63, 47.66, 47.17, 46.96, 33.69, 33.44, 31.66, 31.25, 29.30, 29.24, 29.05, 28.84, 28.52, 24.56, 24.41, 22.32, 22.16, 13.02, 12.98. HRMS (ESI+) m/z 807.3986 [M+Na]+ (Calcd. for C36H64O18Na, 807.3985). Lipotrisaccharide deritive 1i. Amorphous powder. [α]25D +176 (c=0.17, CHCl3). 1H NMR (400 MHz, MeOD): =5.31 (d, 1H, 3.6Hz, 1-H), 5.09 (d, 1H, J=3.6Hz, 1’-H), 4.98 (dd, 1H, J=9.6Hz, J=9.6Hz, 3’-H), 4.81 (dd, 1H, J=9.6Hz, J=3.6Hz, 2’-H), 4.47 (brs, 1H, 1’’-H), 4.294.33 (m, 1H), 4.15 (dd, 1H, J=9.6Hz, J=9.6Hz, 3’-H), 3.93-3.97 (m, 2H), 3.88 (m, 1H, NCH210.4Hz), 3.70-3.75 (m, 4H), 3.57-3.62 (m, 3H), 3.52 (dd, 1H, J=9.6Hz, 3.2Hz, 2-H), 3.43 (dd, 1H, J=9.6Hz, J=3.2Hz, 6a-H), 3.39 (dd, 1H, J=9.6Hz, J=9.6Hz, 4’’-H), 3.18-3.21 (m, 1H),

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2.37-2.43 (m, 4H, CH2CO), 1.51-1.66 (m, 2H, CH2CH2CO), 1.32-1.35 (m, 8H, CH2) , 1.19-1.27 (m, 4H, CH2, (CH3)2CH), 0.89-0.93 (m, 12H, CH3).

13C

NMR (101 MHz, MeOD,

diastereomers): =173.45 (CO), 173.26 (CO), 101.22 (1’’ -C), 94.60 (1’-C), 91.43 (1-C), 76.90, 73.78, 73.12, 72.90, 72.64, 71.50, 70.83, 70.78, 69.99, 69.68, 69.30, 68.66, 67.93, 67.04, 61.54, 60.61, 47.18, 46.74, 38.73, 37.82, 33.70, 33.61, 29.49, 29.08, 28.88, 27.74, 27.56, 27.11, 24.62, 22.26, 21.64, 21.51, 21.50. HRMS(ESI+) m/z 807.3988 [M+Na]+ (Calcd. for C36H64O18Na, 807.3985). Lipotrisaccharide deritive 1j. Amorphous powder. [α]25D = +68.0 (c=2.94, CHCl3) .1H NMR (400 MHz, MeOD, diastereomers): =5.61 (dd, 1H, J=9.6Hz, J=9.6Hz, 3’-H), 5.35 (brs, 1H, 1’-H), 5.20 (dd, 1H, J=9.6Hz, J=9.6Hz, 4’-H), 5.11 (d, 1H, J=3.2Hz, 1-H), 5.03 (m, 1H, 2’H), 4.45 (m, 2H), 3.54-4.01 (m, 10H), 3.33-3.45 (m, 3H), 3.20 (brs, 1H), 2.32 (m, 5 H, CH2CO, CHCO), 1.58 (m, 6H, CH2CHCO, CH2CH2CO), 1.30 (m, 30H, CH2), 1.09 (m, 3H, CH2(CH3)CH), 0.91 (m, 9H,CH3). 13C NMR (101 MHz, MeOD, diastereomers): =175.63 (CO), 172.82 (CO), 172.58 (CO), 101.38 (1’’-C), 95.04 (1’-C), 91.52 (1-C), 76.91, 73.76, 73.12, 73.08, 71.50, 70.76, 70.34, 70.26, 70.13, 69.99, 68.98, 68.92, 68.57, 68.46, 67.55, 67.02, 61.36, 61.06, 48.27, 47.84, 47.20, 46.59, 41.06, 40.94, 33.65, 33.31, 31.58, 31.09, 29.38, 29.37, 29.33, 29.18, 29.08, 29.02, 28.85, 28.55, 26.21, 26.12, 22.34, 22.14, 15.70, 15.66, 13.08, 13.01, 10.72. HRMS (ESI+) m/z 961.5341 [M+Na]+ (Calcd. for C46H82O19Na, 961.5343).

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Trehaloside deritive 2a. Amorphous powder. LC-ELSD: [α]25D +88.6 (c=0.42, CHCl3). 1H NMR (400 MHz, MeOD): =5.63 (dd, 1H, J=9.6Hz, J=9.6Hz, 3’-H), 5.37 (brs, 1H, 1’-H), 5.16 (dd, 1H, J=9.6Hz, J=9.6Hz, 4’-H), 5.12 (d, 1H, J=3.6Hz, 1-H), 5.02 (m, 1H, 2’-H), 4.26 (dd, 1H, J=9.6Hz, J=2.8Hz, 2-H), 3.72-3.81 (m, 1H), 3.64-3.68 (m, 3H), 3.53 (m, 2H), 3.36 (m, 2H), 2.26-2.40 (m, 5H, CH2CO, CHCO), 1.38-1.65 (m, 6H, CH2CH2CO), 1.31 (m, 20H, CH2), 1.081.10 (m, 3H, CH2(CH3)CHCO), 0.87-0.92 (m, 9H, CH3).

13C

NMR (101 MHz, MeOD) δ=

175.76 (CO), 175.63 (CO), 172.76 (CO), 172.51 (CO), 172.47 (CO), 94.86 (1’-C), 91.48 (1-C), 73.16, 73.05, 71.54, 70.37, 70.30, 70.14, 70.08, 70.00, 69.94, 68.53, 68.43, 61.06, 60.18, 41.06, 40.94, 33.56, 33.39, 31.64, 31.20, 29.27, 29.17, 29.03, 29.00, 28.84, 28.48, 26.21, 26.07, 24.37, 24.35, 24.33, 24.28,22.31, 22.09, 15.65, 15.57, 13.02, 12.94, 10.67. HRMS (ESI+) m/z 729.4035 [M+Na]+ (Calcd. for C35H62O14Na, 729.4032). Trehaloside deritive 2b. Amorphous powder. [α]25D +93.5 (c=0.21, CHCl3). 1H NMR (400 MHz, MeOD, diastereomers): =5.63 (dd, 1H, J=9.6Hz, J=9.6Hz, 3’-H), 5.37 (brs, 1H, 1-H), 5.16(dd, 1H, J=9.6Hz, J=9.6Hz, 4’-H), 5.12 (brs, 1H, 1’-H), 5.02 (dd, 1H, J=9.6Hz, J=3.2Hz, 2’H), 4.26 (d, 1H, J=9.6Hz, J=2.8Hz, 2-H), 3.73-3.81 (m, 2H) , 3.64-3.68 (m, 3H) , 3.51-3.56 (m, 2H), 3.36 (m, 1H), 2.24-2.40 (m, 5H, CH2CO, CHCO), 1.40-1.65 (m, 6H, CH2CH2CO), 1.31 (m, 20H, CH2), 1.09-1.13 (m, 3H, CH2(CH3)CHCO), 0.87-0.92 (m, 9H, CH3). 13C NMR (101 MHz, MeOD, diastereomers): =176.06 (CO), 175.66 (CO), 174.12 (CO), 172.56 (CO), 172.40 (CO), 94.73 (1’-C), 91.42 (1-C), 73.17, 73.05, 71.54, 70.37, 70.30, 70.21, 70.14, 70.08, 70.01, 69.93,

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68.54, 68.44, 61.06, 60.22, 47.61, 47.18, 41.06, 40.72, 33.42, 33.08, 31.55, 31.22, 29.27, 28.98, 28.80, 26.02, 24.62, 24.14, 22.37, 22.19, 15.85, 15.46, 12.69, 10.67. HRMS(ESI+) m/z 729.4033 [M+Na]+ (Calcd. for C35H62O14Na, 729.4032). Trehaloside deritive 2c. Amorphous powder. [α]25D +174.6 (c=5.9, CHCl3). 1H NMR (400 MHz, MeOD, diastero): =5.63 (dd, 1H, 10.0Hz, 10.0Hz, 3’-H), 5.37 (m, 1H, 1-H), 5.16 (dd, 1H, J=10.0Hz, 4’-H) , 5.12 (d, 1H, J=3.6Hz, 1’-H), 5.02 (d, 1H, J=10.0Hz, 2’-H), 4.25 (m, 1H, 2-H), 3.79 (dd, 1H, J=10.0Hz, J=10.0Hz, 3-H), 3.72-3.75 (m, 1H), 3.64-3.69 (m, 3H) , 3.50-3.57 (m, 2H), 3.36 (dd, 1H, J=10.0Hz, J=10.0Hz, 4-H), 2.22-2.41 (m, 5H, CH2CO, CHCO), 1.38-1.67 (m, 6H, CH2CH2CO), 1.31 (m, 10H, CH2), 1.17-1.23 (m, 2H, (CH3)2CH), 1.08-1.11 (m, 3H, CH2(CH3)CH), 0.87-0.91 (m, 15H, CH3).

13C

NMR (101 MHz, MeOD, diastero): =175.61

(CO), 172.72 (CO), 172.50 (CO), 172.46 (CO), 94.85 (1’-C), 91.47 (1’-C), 73.16,73.06, 71.54, 70.36, 70.29, 70.13, 70.08, 70.06, 70.00, 69.94, 68.50, 68.41, 61.05, 60.13, 60.10, 41.07, 40.94, 38.80, 38.13, 33.54, 29.39, 29.01, 28.80, 27.73, 27.56, 27.06, 26.22, 26.07, 24.38, 22.20, 22.15, 21.62, 21.46, 15.68, 15.58, 10.67. HRMS (ESI+) m/z 729.4033 [M+Na]+ (Calcd. for C35H62O14Na, 729.4032). Trehaloside deritive 2d. Amorphous powder. [α]25D = +110.0 (c=3.2, CHCl3). 1H NMR (400 MHz, MeOD, diasteromers): =5.60 (dd, 1H, J=9.6Hz, J=9.6Hz, 3’-H), 5.35 (brs, 1H, 1-H), 5.14 (dd, 1H, J=9.6Hz, J=9.6Hz, 4’-H), 5.10 (d, 1H, 3.6Hz, 1’-H), 4.99 (dd, 1H, 9.6Hz, 3.6Hz, 2’-H), 4.22 (m, 1H, 2-H), 3.70-3.79 (m, 2H), 3.62-3.67 (m, 3H), 3.48-3.55 (m, 2H), 3.30-3.36

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(m, 1H), 2.18-2.40 (m, 5H, CH2CO, CHCO), 1.38-1.67 (m, 6H, CH2CH2CO), 1.29 (m, 10H, CH2), 1.16- 1.22 (m, 4H, (CH3)2CH,CH2), 1.06-1.09 (m, 3H, CH2(CH3)CHCO), 0.85-0.91 (m, 15H, CH3). 13C NMR (101 MHz, MeOD, diastereomers) δ = 175.51 (CO), 172.82 (CO), 172.55 (CO), 172.56 (CO), 94.95 (1’-C), 91.57 (1-C), 73.26, 73.16, 71.54, 70.36, 70.29, 70.13, 70.08, 70.08, 70.01, 69.96, 68.52, 68.41, 61.06, 60.15, 60.12, 41.09, 40.96, 38.82, 38.16, 33.56, 29.40, 29.02, 28.84, 27.75, 27.59, 27.07, 26.23, 26.09, 24.34, 22.22, 22.16, 21.63, 21.45, 15.67, 15.58, 10.69. HRMS(ESI+) m/z 729.4037 [M+Na]+ (Calcd for C35H62O14Na: 729.4032). Trehaloside deritive 2e. Amorphous powder. [α]25D +135.1 (c=0.37, CHCl3). 1H NMR (400 MHz, MeOD): =5.32 (d, 1H, J=3.6Hz, 1-H), 5.10 (d, 1H, J=3.6Hz, 1’-H), 4.92 (dd, 1H, J=10.0Hz, 10.0Hz, 4’-H), 4.80 (dd, 1H, J=10.0Hz, J=3.6Hz, 2’-H), 4.11-4.18 (m, 2H), 3.69-3.74 (m, 3H), 3.59-3.66 (m, 2H), 3.50- 3.55 (m, 2H) , 3.36-3.40 (m, 1H), 2.37-2.45 (m, 4H, CH2CO), 1.63-1.67 (m, 4H, CH2CH2CO), 1.30 (m, 52H, CH2), 0.90-0.93 (m, 6H, CH3). 13C NMR (101 MHz, MeOD) δ= 173.48 (CO), 173.28 (CO), 94.49 (1’-C), 91.42 (1’-C), 73.22, 72.81, 72.68, 71.53, 70.97,70.34, 70.02, 68.62, 60.95, 60.70, 33.71, 33.48, 31.67, 29.39, 29.36, 29.25, 29.21, 29.07, 29.04, 28.85, 28.81, 24.61, 24.43, 22.33, 13.03. HRMS (ESI+) m/z 869.5966 [M+Na]+ (Calcd. for C46H86O13Na: 869.5961). Trehaloside deritive 2f. Amorphous powder. [α]25D +106.6 (c=0.8, CHCl3) 1H NMR (400 MHz, MeOD) δ = 5.32 (brs, 1H, 1-H), 5.10 (brs, 1H, 1’-H), 4.91 (dd, 1H, J=10.0Hz, J=10.0Hz, 4’-H), 4.80 (dd, 1H, J=10.0Hz, J=2.8Hz, 2’-H), 4.09-4.18 (m, 2H), 3.66-3.73 (m, 3H), 3.59-3.62

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(m, 2H), 3.50-3.54 (m, 2H), 3.38 (dd, 1H, J=10.0Hz, J=10.0Hz, 4-H), 2.37-2.43 (m, 4H, CH2CO), 1.43-1.75 (m, 6H, CH2CH2CO, CH3CH), 1.31-1.33 (m, 8H, CH2) , 1.18-1.26 (m, 4H, (CH3)2CHCH2), 0.88-0.92 (m, 12H, CH3). 13C NMR (101 MHz, MeOD) δ = 173.45(CO), 173.27 (CO), 94.48 (1’-C), 91.40 (1-C), 73.22, 72.82, 72.70, 71.52, 70.97, 70.33, 70.00, 68.62, 60.94, 60.70, 38.81, 38.09, 33.71, 33.63, 29.45, 29.06, 28.81, 27.74, 27.57, 27.08, 24.61, 22.26, 21.62, 21.50. HRMS(ESI+) m/z 645.3461 [M+Na]+ (Calcd. for C30H54O13Na: 645.3457). Trehaloside deritive 2g. Amorphous powder. [α]25D +94.6 (c=1.9, CH3Cl). 1H NMR (400 MHz, MeOD, diastereomers): =5.49 (m, 1H, 4’-H), 5.28 (brs, 1H, 1-H), 5.06 (brs, 1H, 1’-H), 4.49 (m, 1H), 4.25 (m, 2H), 3.52-3.78 (m, 7H), 3.35 (m, 1H), 3.21 (m, 2H), 2.92 (s, 6H, (CH3)2N), 2.32-2.56 (m, 3H, CH2CO), 2.06 (m, 2H, N-CH2), 1.49-1.67 (m, 6H, CH2CH2CO, NCH2CH2), 1.30 (m, 14H, CH2), 1.15 (m, 3H, CH2(CH3)CH2CO), 0.91 (m, 6H, CH3CH2).

13C

NMR (101 MHz, MeOD, diastereomers): =176.03 (CO),173.01 (CO), 171.93 (CO), 94.94 (1’C), 91.58 (1-C), 73.27, 73.07, 71.78, 71.74, 71.50, 70.41, 70.22, 70.13, 70.07, 68.59, 68.49, 62.79, 60.80, 56.75, 47.83, 47.41, 46.57, 42.15, 41.12, 41.05, 33.44, 33.41, 31.66, 29.99, 29.29, 29.19, 29.04, 29.02, 28.85, 26.45, 26.30, 24.38, 24.30, 22.33, 19.53, 15.86, 15.62, 13.05, 10.69, 10.57. HRMS (ESI+) m/z 646.3542 [M+Na]+ (Calcd. for C29H53NO13Na, 646.3538). Lipodisaccharide deritive 3. Amorphous powder. [α]25D +85.0 (c=1.8 CHCl3). 1H NMR (400 MHz, MeOD, diastereomers): =5.48 (dd, 1H, J=9.6Hz, J=9.6Hz, 3’-H), 5.17-5.22 (dd, 1H, J=9.6Hz, J=9.6Hz, 4’-H), 4.95 (brs, 1H, 1’-H), 4.86-4.91 (m, 1H), 4.45 (brs, 1H), 3.96-4.05 (m,

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2H), 3.87-3.93 (m, 2H), 3.73 (dd, 1H, J=12.0Hz, J=5.6Hz, 6a-H), 3.56-3.64 (m, 2H), 3.42-3.43 (m, 1H), 3.33 (s, 3H, OCH3), 3.18-3.22 (m, 1H), 2.26-2.40 (m, 5H, CH2CO, CHCO), 1.40-1.65 (m, 6H, CH2CH2CO), 1.31 (m, 20H, CH2), 1.07-1.10 (m, 3H, CH2(CH3)CH), 0.87-0.93 (m, 9H, CH3). 13C NMR (101 MHz, MeOD, diastereomers): =175.65 (CO), 172.80 (CO), 172.56 (CO), 172.51 (CO), 101.33 (1’-H), 96.58 (1-C), 76.97, 73.83, 70.83, 70.79, 69.83, 68.56, 68.51, 68.40, 68.29, 67.54, 67.08, 61.54, 54.61, 48.09, 47.67, 47.17, 41.17, 40.94, 33.47, 33.37, 31.64, 31.20, 29.37, 29.25, 29.15, 29.02, 28.93, 28.67, 28.52, 26.21, 26.10, 24.45, 24.35, 22.31, 22.12, 15.72, 15.61, 13.03, 12.99, 10.66,10.62. HRMS (ESI+) m/z 743.4192 [M+Na]+ (Calcd. for C36H64O14Na, 743.4188).

Lipomonosaccharide deritive 4. Amorphous powder. [α]25D +63.4 (c=2.9 CHCl3). 1H NMR (400 MHz, CDCl3, diastereomers): =5.59 (dd, 1H, J=9.6Hz, J=9.6Hz, 3’-H), 5.03 (dd, 1H, J=9.6Hz, J=9.6Hz, 3’-H), 4.98 (m, 1H, 1-H), 4.8 (dd, 1H, J=9.6Hz, J=2.0Hz, 2-H), 3.77 (dd, 1H, J=10.6Hz, J=2.4Hz, 6a-H), 3.70 (m, 1H), 3.57 (dd, 1H, J=11.2Hz, J=3.6Hz, 6b-H), 3.40 (s, 3H, OCH3), 2.23-2.37 (m, 5H, CH2CO, CHCO) , 1.56-1.61 (m, 6H, CH2CH2CO), 1.25 (m, 20H, CH2), 1.06-1.08 (m, 3H, CH2(CH3)CH) 0.84-0.89 (m, 9H, CH3). 13C NMR (100 MHz, CDCl3, diastereomers): δ = 175.42 (CO), 175.32 (CO), 173.48 (CO), 172.97 (CO), 96.88 (1-C), 96.86 (1-C), 71.03, 70.97, 69.42, 69.37, 69.02, 69.00, 68.75, 68.67, 61.07, 55.42, 41.19, 41.06, 34.02, 33.97, 33.95, 31.87, 31.34, 29.53, 29.43, 29.28, 29.20, 29.03, 28.77, 26.49, 26.42, 24.76, 24.74,

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24.70, 24.68, 22.66, 22.41, 16.64, 16.63, 14.09, 13.97, 11.60, 11.55. HRMS (ESI+) m/z 581.3846 [M+Na]+ (Calcd. for C30H54O9Na, 581.3849). Bacterial strains and growth conditions. S. pneumoniae and E. faecalis were grown in Todd-Hewitt broth supplemented with 5% yeast extract (THY) at 37 °C. A. baumannii, K. pneumoniae, P. aeruginosa, S. aureus, and V. cholerae were cultured in Mueller-Hinton broth (MHB) with aeration. Antibacterial activity. MIC (Minimum Inhibitory Concentration) was determined using a micro-dilution method according to guidelines described by the Clinical and Laboratory Standards Institute (CLSI) with certain modifications in 96-well plates (200 L/well), as described below. Bacteria were cultured to mid-log phase and diluted to a final concentration 106 CFU/mL. Compounds were dissolved in DMSO at 20 mg/mL as stock solutions and serially diluted with desirable culture medium. The bacterial cultures and antimicrobial solutions were mixed in a 1:1 volume ratio. The final concentration of DMSO is less than 1%. Growth of the bacterial culture was periodically detected by spectrophotometry for 24 hr, in terms of culture turbidity. MIC was recorded as the lowest concentration of each compound that completely inhibited bacterial growth. Vancomycin and DMSO were included as positive and negative controls, respectively. In vitro hemolysis assay. The hemolysis assay was carried out as described.38 Fresh rabbit blood was washed in sterile PBS by centrifugation for 10 min at 2000 rpm, and resuspended in PBS to approximately 2×10 8

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cells/mL. Suspensions of blood cells and each compound were mixed at a 1:1 volume ratio in a 96-well plate and incubated at 37°C for 45 min. At the end of incubation, the mixtures were centrifuged for 10 min at 2000 rpm; the absorbance values (OD 540) of the supernatants (100 μL) were measured by spectrophotometry. Triton X-100 was used at concentrations of 0.01, 0.1, and 1% (v/v) as a positive control of hemolysis. Permeabilization of bacterial cell membrane. The permeabilization assay was carried out as described35. Log-phase cultures were washed in PBS by centrifugation and resuspended in PBS to an optical density of 0.1 at 620 nm. The cell suspension was incubated with 0.4 M DiSC3(5) (dipropylthiacarbocyanine) (Sigma, Shanghai, China), a membrane potential sensitive cyanine dye, until a stable reduction in fluorescence was reached as a result of DiSC3(5) uptake and quenching in the cell due to an intact membrane potential. After the intracellular and external K + concentrations were equilibrated with 100 mM KCl, the samples were placed into wells of a 96-well flat-bottom fluorescence microtiter plate followed by the addition of different concentrations of drug. Fluorescence was monitored using a fluorescence spectrophotometer at an excitation wavelength of 622 nm and an emission wavelength of 670 nm. The induction of fluorescence, which results from the disruption of the cytoplasmic membrane by different concentrations of compounds was recorded. The background was subtracted using a control that contained only the cells and dye. ASSOCIATED CONTENT

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Supporting Information.

The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.

The synthesis procedure and spectra data of intermediates. NMR spectrum of all final compounds were attached (PDF) AUTHOR INFORMATION Corresponding Author: Jing-Ren Zhang, E-mail: (J.R.Z.) [email protected]. Tel.: +86 10 62795892. Gang Liu E-mail: (G.L.) [email protected]. Tel.: +86 10 62797740. Present Addresses Jing-Ren Zhang Center for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing, China Gang Liu Center for Life Sciences & Department of Pharmacology and Pharmaceutical Sciences; Author Contribution

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Bo Liu§ and Xue Liu§ contributed equally to this work.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Bo Liu‡ and Xue Liu‡ contributed equally.

Funding Sources the National Natural Science Foundation of China (No. 81273364) and National 863 Program of China (No. 2012AA020303)

ACKNOWLEDGMENT This research was supported by the grants from the National Natural Science Foundation of China (No. 81273364) and National 863 Program of China (No. 2012AA020303) for support. ABBREVIATIONS A. baumannii, Acinetobacter baumannii; AMX, amoxicillin; Bu4NI, tetrabutylammonium iodide; Bn, benzyl; CHL, chloramphenicol; CFU, clonal formation unit; CRO, ceftriaxone; DA, clindamycin;

DBU,

dichloromethane;

1,8-Diazabicycloundec-7-ene;

DDQ,

diisopropylethylamine;

DCE,

1,2-dichloroethane;

2,3-dichloro-5,6-dicyano-1,4-benzoquinone;

-DMB

glucoside,

-dimethoxybenzyl

DIPEA,

glucoside;

DCM, N,N-

DMAP,

4-

dimethylaminopyridine; DMBOH, 3,4-dimethoxybenzyl alcohol; DOC, deoxycholate; DMSO,

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dimethyl

sulfoxide;

DTBMP,

2,6-di-tert-butyl-4-methylpyridine;

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EDCI,

1-ethyl-3-(3-

dimethyllaminopropyl) carbodiimide hydrochloride; E. coli, Escherichia coli; E. faecalis, Enterococcus faecalis; ELSD, evaporative light scattering detector; ERY, erythromycin; HPLC, high performance liquid chromatography; IAD, intramolecular aglycon delivery; K. pneumoniae, Klebsiella pneumoniae; LPS, lipopolyosaccharide; MIC, minimum inhibitory concentration; MALDI-TOF/TOF, matrix-assisted laser desorption/ ionization time of flight mass spectrometry; NMR, nuclear magnetic resonance; OD, optical density; PBS, Phosphate Buffered Saline; PEN, penicillin; PTP1B, protein tyrosine phosphatase 1B; P. aeruginosa, Pseudomonas aeruginosa; SAR, structure-activity relationship; S. aureus, Staphylococcus aureus; S. mitis, Streptococcus mitis; S. oralis, Streptococcus oralis; S. pneumoniae, Streptococcus pneumoniae; S. pyogenes, Streptococcus pyogenes; S. agalactiae, Streptococcus agalactiae; S. sanguis, Streptococcus pyogenes; SXT, sulfamethoxazole; TCL, thin Layer chromatography; TCY, tetracycline; TfOMe,

trifluoromethanesulfonate;

Tf2O,

trifluoromethanesulfonic

anhydride;

THF,

tetrahydrofuran; Trt, trityl; UV, ultraviolet; Van, vancomycin; THF, tetrahydrofuran REFERENCES (1) Fischbach, M. A., and Walsh, C. T. (2009) Antibiotics for emerging pathogens, Science 325, 1089-1093. (2) Moreillon, P., Markiewicz, Z., Nachman, S., and Tomasz, A. (1990) Two bactericidal targets for penicillin in pneumococci: autolysis-dependent and autolysis-independent killing mechanisms, Antimicrob Agents Chemother 34, 33-39. (3) Chopra, I. (2002) New developments in tetracycline antibiotics: glycylcyclines and

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